key: cord-0696173-4hlr5wk5
authors: Chen, Yingying; Wang, Xinchen; Ma, Xinyuan; Liang, Shuobin; Gao, Qianqian; Tretyakova, Elena V.; Zhang, Yongmin; Zhou, Demin; Xiao, Sulong
title: Facial Synthesis and Bioevaluation of Well-Defined OEGylated Betulinic Acid-Cyclodextrin Conjugates for Inhibition of Influenza Infection
date: 2022-02-09
journal: Molecules
DOI: 10.3390/molecules27041163
sha: b1631ccd462badf96770793a6279a26da24b6843
doc_id: 696173
cord_uid: 4hlr5wk5

Betulinic acid (BA) and its derivatives exhibit a variety of biological activities, especially their anti-HIV-1 activity, but generally have only modest inhibitory potency against influenza virus. The entry of influenza virus into host cells can be competitively inhibited by multivalent derivatives targeting hemagglutinin. In this study, a series of hexa-, hepta- and octavalent BA derivatives based on α-, β- and γ-cyclodextrin scaffolds, respectively, with varying lengths of flexible oligo(ethylene glycol) linkers was designed and synthesized using a microwave-assisted copper-catalyzed 1,3-dipolar cycloaddition reaction. The generated BA-cyclodextrin conjugates were tested for their in vitro activity against influenza A/WSN/33 (H1N1) virus and cytotoxicity. Among the tested compounds, 58, 80 and 82 showed slight cytotoxicity to Madin-Darby canine kidney cells with viabilities ranging from 64 to 68% at a high concentration of 100 μM. Four conjugates 51 and 69–71 showed significant inhibitory effects on influenza infection with half maximal inhibitory concentration values of 5.20, 9.82, 7.48 and 7.59 μM, respectively. The structure-activity relationships of multivalent BA-cyclodextrin conjugates were discussed, highlighting that multivalent BA derivatives may be potential antiviral agents against influenza infection.

Influenza viruses are widespread human respiratory pathogens that can cause serious infections with significant morbidity and mortality [1] . Recently, coinfection of influenza virus with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported [2] , highlighting that the prevention and treatment of influenza will be more important than ever. Due to the lack of activity against influenza B and the widespread resistance of M2 ion channel inhibitors among circulating influenza strains, the antiviral drugs currently recommended for the treatment of influenza are limited to neuraminidase (NA) [3] and polymerase acidic protein (PA) inhibitors [4] . Although variants resistant to NA and PA inhibitors are much less than M2 inhibitors, the high variability of influenza viruses, such as seasonal H1N1 viruses carrying the H275Y, H275Y and I38T mutations [5] and the H7N9 virus carrying the R294K mutation [6] , enables the rapid evolution of antiviral resistance to drugs, underscoring the urgent need for the development of new anti-influenza drugs.

[5] and the H7N9 virus carrying the R294K mutation, [6] enables the rapid evolution of 46 antiviral resistance to drugs, underscoring the urgent need for the development of new 47 anti-influenza drugs. 48 The entry of influenza virus into host cells is a six-step dynamic process, [7] which 49 represents an attractive target for antiviral therapy. Influenza viruses attach to host cells 50 by binding the globular head of hemagglutinin (HA), a homotrimeric type I membrane 51 glycoprotein expressed on the virion surface, to sialylated host cells. The interaction be-52 tween HA and sialic acid is usually weak with an association constant of 10 3 M -1 . [8] How-53 ever, the interactions between multiple HA trimers on the viral surface (~ 600-1200 mole-54 cules per virus particle) and sialic acid-terminated glycoproteins and glycolipids on the 55 cell surface (~ 50-200 residues per 100 nm 2 ) substantially increase through multivalent 56 effects. [9] To this end, linear polymers, [10] dendritic polymers, [11] and nanoparticles [12] 57 have been used as different display systems for highly potent influenza virus inhibitors. 58 We have previously reported the synthesis of multivalent pentacyclic triterpene conju-59 gates [13] and found that three heptavalent pentacyclic triterpene derivatives 1-3 display 60 broad-spectrum anti-influenza virus activity with half maximal inhibitory concentration 61 (IC50) values in the 1.60-18.74 μM range ( Figure 1 ). Two years later, Li et al demonstrated 62 the synthesis of a series of random glycyrrhetinic acid/oligo(ethylene glycol) (OEG)-ap-63 pended norbomene copolymers 4 as potential nanocarriers for drug delivery. [14] In an-64 other study, Yang et al reported the synthesis of PEGylated oleanolic acid-functionalized 65 human serum albumin conjugates 5-7 and their potential use as anti-infective agents. [15] 66 These results rationalize the construction of multivalent pentacyclic triterpenes as poten-67 tial inhibitors to block the replication of influenza viruses. (1) (2) (3) (4) (5) (6) (7) . 70 Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid found in several 71 species of plants, notably Betula pubescens, commonly known as white birch. Owing to its 72 unusual multiple biological effects, BA has garnered attention from researchers in the sci-73 entific community and pharmaceutical industry in recent years. [16] The remarkable anti-74 HIV-1 potency of BA derivatives, such as bevirimat and BMS-955176, is one of their most 75 important properties. [17, 18] The interesting anti-HIV-1 properties of BA derivatives led 76 to the examination of their anti-influenza activity. Hong et al. [19] reported that BA shows 77 weak anti-influenza activity against A/PR/8/34 virus (10 μM: ~30%). Antiviral-guided iso-78 lation of the leafstalk extract of Schefflera heptaphylla led to the identification of two 3-epi-79 betulinic acid derivatives with anti-influenza A (H1N1) virus activity. [20] Betulinic alde-80 hyde isolated from Alnus japonica, which is used in folk remedies for influenza, exhibits 81 Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid found in several species of plants, notably Betula pubescens, commonly known as white birch. Owing to its unusual multiple biological effects, BA has garnered attention from researchers in the scientific community and pharmaceutical industry in recent years [16] . The remarkable anti-HIV-1 potency of BA derivatives, such as bevirimat and BMS-955176, is one of their most important properties [17, 18] . The interesting anti-HIV-1 properties of BA derivatives led to the examination of their anti-influenza activity. Hong et al. [19] . reported that BA shows weak anti-influenza activity against A/PR/8/34 virus (10 µM:~30%). Antiviralguided isolation of the leafstalk extract of Schefflera heptaphylla led to the identification of two 3-epi-betulinic acid derivatives with anti-influenza A (H1N1) virus activity [20] . Betulinic aldehyde isolated from Alnus japonica, which is used in folk remedies for influenza, exhibits certain anti-influenza effects against avian influenza KBNP-0028 (H9N2) virus with an EC 50 value of 28.4 µM [21] . Simple modifications of BA at position C-3 or C-28 provide compounds with significant activities against influenza A virus [22, 23] . However, the very poor water solubility of these compounds hampers their further devel-Molecules 2022, 27, 1163 3 of 23 opment in vivo and inspires more research on better hydrophilic derivatives with potential pharmaceutical applications.

Based on these literature results of the antiviral activities of lupane-type triterpenoids and our interest in the development of natural products as potential anti-influenza agents [13, 24, 25] , it was valuable to design and synthesize a variety of multivalent triterpenoid conjugates to disclose the relationship between their structure and activity. In our recent study, we found that one multivalent BA-α-cyclodextrin (CD) conjugate, CYY1-11, showed good anti-influenza activity (IC 50 = 5.20 µM) against A/WSN/33 virus [25] . In the present work, we further describe the synthesis of a range of well-defined hexa-, hepta-and octavalent BA derivatives based on α-, βand γ-CD scaffolds, respectively, with varying OEG chains (0, 1, 2, 4, 6 and 8 OEG units) as linkers via click chemistry. A total of 36 BA-CD conjugates, including compound CYY1-11 (named 51 in this manuscript), were examined to determine their anti-influenza activity against A/WSN/33 virus, and four conjugates, 51 and 69-71, showed significant antiviral activities. The structure-activity relationships (SAR) of multivalent BA-CD conjugates were discussed to explore potential therapeutic agents for influenza infection.

The multivalent presentation of bioactive molecules to polymers, such as poly(ethylene glycol) (PEG), has aroused extensive interest and been widely applied in many different fields [26, 27] , especially in drug delivery systems [28] . Difficulties in loading a quantitative amount of drugs at a specific position of polymeric carriers, such as polymethyl methacrylate, make drug delivery systems hard to work with. To pursue our research interests in natural products with significant anti-influenza activity, we planned to synthesize a series of multivalent BA derivatives based on CD scaffolds linked by a variable length OEG chain via click chemistry. Three natural CD scaffolds and six OEG linkers were selected because of their beneficial effects on the grafted ligands, such as water solubility and good biocompatibility and immune compatibility [29] .

As described above, PEGs and OEGs are ubiquitously used in the pharmaceutical industry and biomedical research for the modification of proteins, peptides or nonpeptides. Therefore, OEGs were selected as the linkers between the BA pharmacophore and CD scaffold. Designed bifunctional amino alkyne linkers of different lengths (1, 2, 4, 6 and 8 OEG units) 26-30 were prepared from commercially available 2-azidoethanol 20, di(ethylene glycol) 8, tetra(ethylene glycol) 9, hexa(ethylene glycol) 10 or octa(ethylene glycol) 11 in 27-64% yields over four steps according to conventional methods (Scheme 1) [30, 31] . Compound 31 could be accessed by synthesis from the cheaper precursor betulin, as reported previously [25, 32] . Subsequent activation of the carboxylic acid by using TBTU/DIPEA in THF gave compound 32 in 83% yield, which was then subjected to aminolysis with commercially available propargylamine or bifunctional aminoalkyne linkers 26-30 to afford BA derivatives 33-38 bearing a terminal alkynyl group at the C-28 position (Scheme 2), and their structures were characterized with 1 H and 13 C NMR (Supplementary Materials). 1 H NMR spectra of 33-38 showed one proton of the terminal alkynyl group at δ 2.41~2.43 ppm, while 13 C NMR spectra displayed two carbons of the terminal alkynyl group at δ 71.06 and 80.14 ppm for 33 [33] and δ 74.49~74.65 and 79.44~79.58 ppm for 34-38.

Multiazide-substituted CD scaffolds 48-50 were synthesized in 58-71% yields over three steps according to the methods described elsewhere (Scheme 3) [34, 35] . The coppercatalyzed 1,3-dipolar cycloaddition between each terminal alkyne-modified BA, 33-38, and each multiazide-appended CD scaffold, 48-50, was performed at 100 • C in the presence of sodium ascorbate and a copper sulfate catalytic system in THF/H 2 O (1:1, v/v) under mi- Multiazide-substituted CD scaffolds 48-50 were synthesized in 58-71% yields over 139 three steps according to the methods described elsewhere (Scheme 3). [34, 35] The copper-140 catalyzed 1,3-dipolar cycloaddition between each terminal alkyne-modified BA, 33-38, 141 and each multiazide-appended CD scaffold, 48-50, was performed at 100 °C in the pres-142 ence of sodium ascorbate and a copper sulfate catalytic system in THF/H2O (1:1, v/v) under 143 microwave irradiation to yield a series of acetyl-protected BA-CD conjugates, 51-68, in 144 42-55% yields, followed by a deacetylation reaction under Zemblén transesterification 145 conditions to afford the desired homomultivalent conjugates 69-86 in good to excellent 146 yields. Multiazide-substituted CD scaffolds 48-50 were synthesized in 58-71% yields over 139 three steps according to the methods described elsewhere (Scheme 3). [34, 35] The copper-140 catalyzed 1,3-dipolar cycloaddition between each terminal alkyne-modified BA, 33-38, 141 and each multiazide-appended CD scaffold, 48-50, was performed at 100 °C in the pres-142 ence of sodium ascorbate and a copper sulfate catalytic system in THF/H2O (1:1, v/v) under 143 microwave irradiation to yield a series of acetyl-protected BA-CD conjugates, 51-68, in 144 42-55% yields, followed by a deacetylation reaction under Zemblén transesterification 145 conditions to afford the desired homomultivalent conjugates 69-86 in good to excellent 146 yields.

The structures of synthesized multivalent BA-CD conjugates 51-86 were characterized by NMR spectroscopy and MALDI-TOF mass spectrometry (Supplementary Materials). Except for the signals of the linker, the 1 H and 13 C NMR spectra of 51-68 are similar to each other; therefore, only the assignment of conjugate 64 is discussed in detail as an example. The 2D 1 H-13 C HSQC spectrum of conjugate 64 is shown in Figure 2 , and inspection of it led to the assignment of most of the peaks. In the low-field region, the signal at δ H 7.75 ppm (Supplementary Materials), according to the 1 H-13 C correlation spectrum, was assigned to triazolyl-CH. The proton of CONH at δ H 6.13 ppm was easily identified, as there was no correlation in the 2D NMR spectrum. As a C 7 -symmetric macromolecular triazole adduct, conjugate 64 showed only one set of characteristic anomeric resonances [δ H 5.49 ppm (β-CD-H 1 )]. Likewise, the other protons H 2-6 of the β-CD scaffold were also assigned based on the HSQC spectrum. Two sets of peaks were clearly observed at 3.62-3.60 and 3.53-3.46 ppm (overlap with β-CD-H 4 and NHCH 2 ), which were assigned to the eleven OCH 2 protons of the OEG group. Based on the literature data [36] , major 1 The 1 H and 13 C NMR signals of conjugates 69-86 were assigned based on precursors 51-65. As expected, in the majority of cases, the de-O-acetylation of the CD scaffold caused an upfield shift in CD-H 2 and CD-H 3 peaks of~1.3 and~1.5 ppm, respectively, but a downfield shift in both CD-C 2 and CD-C 3 peaks of~2.7-3.3 ppm. For example, for conjugate 70, β-CD-H 2 and β-CD-H 3 were observed as a triplet and broad doublet at δ 3.45 and 3.86 ppm, respectively, which is upfield compared to the corresponding signals in conjugate 52 (δ 4.77 and 5.32 ppm, respectively).

Cell viability assays are widely used to assess potential compound-induced toxicity. Measurement of intracellular ATP levels using ATP/luminescence readouts, such as the CellTiter-Glo reagent, is one of the most conventional and commonly used methods. Here, we evaluated the cytotoxicity of BA-CD conjugates 51-86 in MDCK cells before determining the anti-influenza virus activity. Culture medium containing 1% DMSO was used as a vehicle control. No significant effects on cell viability were observed with most of the multivalent BA-CD conjugates at a concentration of 100 µM, except for three conjugates 58, 80 and 82, which were slightly cytotoxic, with a MDCK cell viability of less than 70% (68%, 64% and 67%, respectively) ( Figure S1 ). However, parental compound 31 possessed strong cytotoxicity towards host MDCK cells with a viability of 8.5% at the same concentration, which may due to its better cell permeability [37] , encompassing the role of membrane damage in BA induced apoptosis.

Next, we employed the cytopathic effect (CPE) reduction assay to investigate the anti-influenza activity of the multivalent BA derivatives. Except for three conjugates 58, 80 and 82 with weak cytotoxicity, the other 33 conjugates were evaluated. SAR analysis suggested that the α-CD scaffold-based conjugates exhibited higher antiviral activity against A/WSN/33 virus than the other two CD scaffold-based conjugates (e.g., 51 vs. 52 and 53, 57 vs. 59, and 75 vs. 76 and 77) ( Figure 3 ). One of the most likely reasons was that steric hindrance caused by the multiple crowded BAs inhibited the interaction between the ligand and the target protein. An exception was conjugate 81, for which approximately 1.5-fold decreases in activity was observed compared with that of conjugate 83. In general, the linker between BA and the CD scaffold had no obvious effect on antiviral activity. Seven conjugates 51, 57, 69-71, 75 and 78 exhibited an inhibition rate against influenza virus A/WSN/33 (H1N1) of over 50% at a concentration of 20 µM. Further viral yield reduction studies with A/WSN/33 virus showed that they displayed dose-dependent inhibition of influenza virus replication (Table 1) . Among them, conjugates 57, 75 and 78 only showed weak anti-influenza activity with IC 50 over 10 µM, therefore the 50% cytotoxic concentration, CC 50 , values in MDCK cells were not further determined. The other four conjugates 51 and 69-71 showed potent antiviral activities with IC 50 values falling within the low micromolar range (IC 50 : 5.20-9.82 µM). More specifically, hexavalent conjugate 51 (IC 50 of 5.20 µM) showed the highest activity and was at least 20-42 times more active than its parent compound BA (50 µg/mL: 27.6% [38] , EC 50 > 219.0 µM [39] ). In addition, the CC 50 was not determined for 51 because the dose-response curve was not achieved at the highest concentration tested (200 µM), displaying over 38.4-fold selectivity. Detailed studies on the biological activities of 51 have been described by Chen et al. [25] . The CC 50 values of conjugates 69-71 in MDCK cell were also not determined because they had a value over 100 µM, in other words they were also not cytotoxic. These results indicated that the grafting of multiple BAs onto the primary face of CD scaffolds was an effective strategy for enhancing the anti-influenza activity of BAs.

was not determined for 51 because the dose-response curve was not achieved at the high-219 est concentration tested (200 μM), displaying over 38.4-fold selectivity. Detailed studies 220 on the biological activities of 51 have been described by Chen et al. [25] The CC50 values of 221 conjugates 69-71 in MDCK cell were also not determined because they had a value over 222 100 μM, in other words they were also not cytotoxic. These results indicated that the graft-223 ing of multiple BAs onto the primary face of CD scaffolds was an effective strategy for 224 enhancing the anti-influenza activity of BAs. During the last two decades, BA and its derivatives have attracted special interest due to their remarkable anti-HIV-1 activity with three derivatives (bevirimat [40] , BMS-955176 [41] and GSK-2838232 [42] ) entering clinical trials. In recent years, an increasing number of studies with regard to their potential applications against other viruses have been performed [43] [44] [45] . As a class of anti-HIV-1 agents with new mechanisms of action (entry [17] and maturation [41] ), however, the anti-influenza mechanism of action of BA and its derivatives has not yet been clearly elucidated. The primary study of the antiviral mechanism of 51 based on surface plasmon resonance assay indicated that multivalent BA derivatives can bind specifically with influenza HA protein with K D value of 1.50 µM [25] , thus blocking influenza virus entry into host cells. Compared to 51, some conjugates, such as 81 and 83, showed relative weak binding affinity to influenza HA protein with K D values over 10 µM ( Figure S2 ), which agreed well with their anti-influenza activities. Further efforts to uncover the function of the HA protein binding domain of multivalent BA-CD conjugates and to investigate how the structural features contribute to the binding domain will provide important insights into the multivalent binding mechanism and help to guide the design of more effective multivalent pentacyclic triterpene derivatives targeting this important protein. The NMR spectra were obtained on Bruker 400 and 600 MHz spectrometers (Bruker Daltonics., Billerica, MA, USA). The value of chemical shifts (δ) are given in ppm and coupling constants (J) in hertz (Hz). High-resolution electrospray mass spectra (HRMS) and MALDI-TOF-MS were recorded by a Bruker APEX IV FT_MS (7.0 T) mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) and an AB Sciex TOF/TOF™ 72115 mass spectrometer (AB Sciex, Redwood City, CA, USA), respectively. The reaction progress and chromatography fractions were monitored by analytical thin-layer chromatography (TLC) on 0.25 mm thickness E. Merck pre-coated plates of silica gel 60 F 254 . The spots were visualized by immersion of the TLC plate in an appropriate solution followed by heating with a hot gun. The following staining solutions were applied: ninhydrin staining solution [ninhydrin (10.0 g) and ethanol (300 mL)], cerium molybdate staining solution [Ce(NH 4 ) 2 (NO 3 ) 6 (0.5 g, 0.9 mmol), (NH 4 ) 6 [30, 31] , BA derivatives 32-33 [33] and hexavalent BA-α-CD conjugate 51 [25] were synthesized by literature methods, and the data were consistent with those published.

Na 2 CO 3 (0.68 mmol, 2.0 equiv.) was added to a solution of 1-benzotriazolyl 3βhydroxy-lup-20(29)-en-28-oate (32) (0.34 mmol, 1.0 equiv.) and terminal propargylated OEG-tethered amine (26-30) (0.41 mmol, 1.2 equiv.) in DMF (4 mL), and the mixture was stirred at room temperature for 24 h. After completion of the reaction, as indicated by TLC, the solvent was evaporated under reduced pressure, and the obtained residue was extracted with CH 2 Cl 2 (10 mL × 3), dried with anhydrous Na 2 SO 4 , filtered, and evaporated. The pure product was obtained by column chromatography performed on silica gel. 13 13 N-(3,6,9 ,12-Tetraoxapentadec-14-yn-1-yl)-3β-hydroxy-lup-20(29)en-28-amide (36) Prepared from 32 and 3,6,9,12-tetraoxapentadec-14-yn-1-amine (28) 13 

CuSO 4 ·5H 2 O (0.10 mmol, 1.0 equiv.) and sodium L-ascorbate (1.1 equiv. per mol azide) were added to a solution of multiazide-substituted α-, β-and γ-CD (48-50) (0.10 mmol, 1.0 equiv.) and terminal propargylated OEG-tethered BA derivatives (33-38) (1.1 equiv. per mol azide) in THF-H 2 O (10 mL, v/v 1:1). The reaction vessel was placed in a vigorously stirred CEM Discover SP microwave reactor (100 • C, 50 W) and heated for 1 h. After the reaction mixture was extracted with CH 2 Cl 2 (10 mL × 3), it was washed with water and brine and dried with anhydrous Na 2 SO 4 . The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography. 13 13 13 13 38.31, 37.72, 37.17, 34.39, 33.60, 29.65, 29.39, 29.27, 27.98, 27.39, 25.59, 20.90, 20.77, 20.64, 19.46, 18.29, 16.15, 16.12, 15.38, 14. 42.45, 40.73, 38.84, 38.71, 38.32, 37.73, 37.18, 34.40, 33.60, 31.89, 30.89, 29.74, 29.66, 29.62, 29.58, 29.52, 29.48, 29.44, 29.40, 29.32, 29.28, 29.22, 27.99, 27.40, 25.61, 20.92, 20.73, 20.68, 19.47, 18.30, 16.16, 16.12, 15.38, 14. 42.43, 40.72, 38.83, 38.69, 38.31, 37.72, 37.17, 34.39, 33.59, 30.88, 29.39, 27.98, 27.39, 25.59, 20.90, 20.76, 20.64, 19.46, 18.28, 16.15, 16.11, 15.37, 14. (3,6,9,12,15,18,21,24- Octaoxaheptacos-26yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-2,3di-O-acetyl-β-CD (67) Prepared from 38 and 49 according to general procedure B, the residue was purified by flash chromatography (eluent: CH 2 Cl 2 :CH 3 OH = 10:1) to afford 67 as a white foam with a yield of 64%. R f = 0.34 (CH 2 Cl 2 :CH 3 OH = 10:1); 1 The per-O-acetylated multivalent BA-CD conjugates (51-68) were dissolved in CH 3 OH (~5 mL per 100 mg of conjugate). CH 3 ONa (0.1 eq per mol of acetate, 30% in CH 3 OH) was added, and the solution was stirred at room temperature for 6 h. The solution was neutralized with Amberlite IR-120 H + resin and filtered, the solvent was evaporated under reduced pressure, and the crude product was purified by short RP column chromatography (eluted by CH 3 OH) to afford the desired products. 

Data was analyzed using GraphPad Prism 8.3.0 (GraphPad Software Inc., San Diego, CA, USA). The half maximal inhibitory concentration (IC 50 ) and cytotoxicity concentration for 50% cell death (CC 50 ) was calculated using a non-linear regression dose response curve. Selectivity Index (SI) was calculated as the rate of CC 50 to IC 50 .

In summary, an efficient and conventional method is presented here for the synthesis of multivalent BA derivatives by using α-, βand γ-CD as scaffolds with different biocompatible OEG linker structures. Our approach involves the construction of two building blocks: terminal propargylated OEG-tethered BAs and multiazide substituted per-O-acetylated CDs in the first stage, followed by a regioselective 1,3-dipolar cycloaddition reaction and a subsequent de-O-acetylation reaction to provide the desired multivalent BA-CD conjugates. This general strategy is particularly suitable for the rapid assembly of structurally well-defined multivalent compound libraries based on CD scaffolds. Due to the numerous free hydroxyl groups at the CD scaffold and the OEG linker, the generated BA-CD derivatives are expected to display high solubility and good compatibility in biological environments. No obvious cytotoxicity to MDCK cells was observed for these conjugates at concentrations up to 100 µM. Further in vitro testing showed that four conjugates, 51 and 69-71, were potent against A/WSN/33 (H1N1) virus with IC 50 values below 10 µM. The work presented herein demonstrated that multivalent BA derivatives have the potential to fight viral infection.

The following are available online, Figure S1 : Cytotoxicity of multivalent BA-CD conjugates to MDCK cells; Figure S2 : Binding sensorgrams for conjugates 81 and 83 interaction with influenza HA protein; Selected NMR, ESI-HRMS or MALDI-TOF MS spectra.

Author Contributions: Y.C. and Q.G. synthesized and characterization of the conjugates; X.W. and S.L. carried out the in vitro anti-influenza activity and cytotoxicity experiments; E.V.T. and Y.Z. discussed the results and assisted in writing the manuscript; X.M. analyzed the NMR data; D.Z. and S.X. supervised the project and wrote the paper. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: Not applicable.

Data Availability Statement: The data of this study is contained within the article or supplementary materials. The data presented in this study are available in this manuscript.

94 (s, each 24H, 16 × CH 3 ), 0.92 (s, 48H, 16 × CH 3 ), 0.81, 0.72 (s, each 24H, 16 × CH 3 ), 0.66 (s, 8H); 13 C NMR (150 MHz, CDCl 3 /CD 3 OD = 1:1 v/v): δ 177

-Propyn-1-yloxy)ethyl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-α-CD (72) Prepared from 54 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

(m, 12H), 1.65-0.85 (m, other aliphatic ring protons), 1.65, 0.95 (s, each 18H, 12 × CH 3 ), 0.92 (s, 36H, 12 × CH 3 ), 0.81, 0.72 (s, each 18H, 12 × CH 3 ), 0.66 (d, 6H, J = 9

-Propyn-1-yloxy)ethyl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-β-CD (73) Prepared from 55 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

(m, 7H), 3.23 (t, 7H, J = 9

-Propyn-1-yloxy)ethyl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-γ-CD (74) Prepared from 56 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

9 Hz), 2.05 (d, 8H, J = 12.5 Hz), 1.85-1.75 (m, 16H), 1.63-0.84 (m, other aliphatic ring protons

12 (dd, 6H, J = 11.0, 5.2 Hz), 3.06 (dt, 6H, J = 10.9, 4.3 Hz), 2.48 (dt, 6H, J = 12.5, 3.2 Hz), 2.08 (d, 6H

42H, overlap with H 2 O), 4.18 (m, 7H), 3.87 (t, 7H, J = 9.1 Hz), 3.63-3.56 (m, 28H), 3.50-3.42 (m, 21H), 3.23 (t, 7H, J = 10

-Propyn-1-yloxy)ethoxy)ethyl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-γ-CD (77) Prepared from 59 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

600 MHz, CDCl 3 /CD 3 OD = 1:1 v/v): δ 7.93 (s, 8H), 5.18 (d, 8H, J = 2.8 Hz

CDCl 3 /CD 3 OD = 1:1 v/v): δ 178

12-Tetraoxapentadec-14-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-α-CD (78) Prepared from 60 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

(m, 30H), 3.14 (dd, 6H, J = 10

CDCl 3 /CD 3 OD = 1:1 v/v): δ 178

12-Tetraoxapentadec-14-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-β-CD (79) Prepared from 61 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

09 (dt, 7H, J = 11.0, 4.1 Hz), 2.49 (t, 7H, J = 11.6 Hz), 2.09 (d, 7H

12-Tetraoxapentadec-14-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-γ-CD (80) Prepared from 62 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

26 (s, 8H), 3.15 (dd, 8H, J = 10.4, 5.8 Hz), 3.09 (dt, 8H, J = 11.0, 4.3 Hz), 2.48 (m, 8H), 2.08 (d, 8H, J = 13

(m, 144H), 3.29-3.22 (m, 6H), 3.12 (dd, 6H, J = 10.3, 5.8 Hz), 3.06 (dt, 6H, J = 13.5, 6.7 Hz), 2.46 (dt, 6H, J = 12.6, 3.2 Hz), 2.08-2.05 (m, 6H), 1.93-1.76 (m, 12H), 1.66-0.86 (m, other aliphatic ring protons), 1.66, 0.97 (s, each 18H, 12 × CH 3 ), 0.93 (s, 36H, 12 × CH 3 ), 0.82, 0.73 (s, each 18H, 12 × CH 3 ), 0.67 (d, 6H, J = 9.2 Hz); 13 C NMR (150 MHz

18-Hexaoxaheneicos-20-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-β-CD (82) Prepared from 64 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

(m, 14H), 1.66-0.86 (m, other aliphatic ring protons), 1.66, 0.97 (s, each 21H, 14 × CH 3 ), 0.93 (s, 42H, 14 × CH 3 ), 0.82, 0.73 (s, each 21H, 14 × CH 3 ), 0.67 (d, 7H, J = 9

18-Hexaoxaheneicos-20-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-γ-CD (83) Prepared from 65 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

24-Octaoxaheptacos-26-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-α-CD (84) Prepared from 66 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

(m, 28H), 3.51 (t, 2H, J = 5.3 Hz), 3.45-3.39 (m, 2H), 3.25 (m, 1H), 3.12 (dd, 1H, J = 10

24-Octaoxaheptacos-26-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-β-CD (85) Prepared from 67 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol

(m, 28H), 3.51 (t, 2H, J = 5.2 Hz), 3.47-3.39 (m, 2H), 3.24 (m, 1H), 3.12 (dd, 1H, J = 10

24-Octaoxaheptacos-26-yn-1-yl)-3β-hydroxy-lup-20(29)-en-28-oyl]-aminomethyl-1H-1,2,3-triazol-1-yl]-γ-CD (86) Prepared from 68 according to general procedure C, the residue was purified by RP flash chromatography (eluent: methanol) to afford

22 (s, 1H), 3.11 (dd, 1H, J = 10.2, 6.1 Hz), 3.05 (dt, 1H, J = 11.0, 4.3 Hz), 2.42 (dt, 1H

Cytotoxicity Test The cytotoxicity of the synthesized BA-CD conjugates was evaluated with the CellTiter-Glo luminescent cell viability assay kit. Briefly, 10,000 MDCK cells in DMEM supplemented temperature for 10 min, the luminescence intensity was measured by an Infinite M2000 PRO™ instrument

When the cells had grown to approximately 70-80% confluence, the media were removed, and the cells were infected with influenza A/WSN/33 virus at a multiplicity of infection (MOI) of 0.2 in DMEM (with 1% FBS and 2 µg/mL TPCK-treated trypsin) containing the corresponding concentration of test samples with two-fold serial dilution

All plates were incubated at 37 • C and 5% CO 2 for 36 h, and cell viability was determined using CellTiter-Glo reagent

Influenza epidemiology-past, present, and future

SARS-CoV-2 and influenza virus co-infection

Antivirals targeting the neuraminidase. Cold Spring Harb

The first cap-dependent endonuclease inhibitor for the treatment of influenza

Global transmission of oseltamivir-resistant influenza

Emerging antiviral resistance

Entry of influenza A virus: Host factors and antiviral targets

Quantification of multivalent interactions between sialic acid and influenza A virus spike proteins by single-molecule force spectroscopy

Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors

Polyacrylamides bearing pendant α-sialoside groups strongly inhibit agglutination of erythrocytes by influenza virus: The strong inhibition reflects enhanced binding through cooperative polyvalent interactions

Novel dendritic α-sialosides: Synthesis of glycodendrimers based on a 3,3'-iminobis(propylamine) core

Synthesis of multivalent sialyllactosamine-carrying glyco-nanoparticles with high affinity to the human influenza virus hemagglutinin

Pentacyclic triterpenes grafted on CD cores to interfere with influenza virus entry: A dramatic multivalent effect

Natural triterpenoid-and oligo(ethylene glycol)-pendant-containing block and random copolymers: Aggregation and pH-controlled release

Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated neomucin for influenza virus capture and entry inhibition

Betulin and its derivatives as novel compounds with different pharmacological effects

Triterpene derivatives that block entry of human immunodeficiency virus type 1 into cells

Betulinic acid and dihydrobetulinic acid derivatives as potent anti-HIV agents

Anti-influenza activity of betulinic acid from Zizyphus jujuba on influenza A/PR/8 virus

Antiviral triterpenoids from the medicinal plant Schefflera heptaphylla

An anti-influenza component of the bark of Alnus japonica

Lupane triterpenes and derivatives with antiviral activity

Synthesis of triterpenoid acylates: Effective reproduction inhibitors of influenza A (H1N1) and papilloma viruses

Discovery of pentacyclic triterpenoids as potential entry inhibitors of influenza viruses

Synthesis of a hexavalent betulinic acid derivative as a hemagglutinin-targeted influenza virus entry inhibitor

Well-defined multivalent ligands for hepatocytes targeting via asialoglycoprotein receptor

Designing multivalent probes for tunable superselective targeting

Polymer-drug conjugate therapeutics: Advances, insights and prospects

PEGylation, successful approach to drug delivery

Enzymatic transhalogenation of dendritic RGD peptide constructs with the fluorinase

Structure-based tetravalent zanamivir with potent inhibitory activity against drug-resistant influenza viruses

A concise semi-synthetic approach to betulinic acid from betulin

Synthesis and anti-HCV entry activity studies of β-cyclodextrin-pentacyclic triterpene conjugates

Selective halogenation at primary positions of cyclomaltooligosaccharides and a synthesis of per-3,6-anhydro cyclomaltooligosaccharides

Amino acid derivatives of β-cyclodextrin

Computer-assisted structure elucidation: Application of CISOC-SES to the resonance assignment and structure generation of betulinic acid

Betulinic acid-induced cytotoxicity in human breast tumor cell lines MCF-7 and T47D and its modification by tocopherol

Cytotoxic and antiviral triterpenoids from the mangrove plant Sonneratia paracaseolaris

Antiviral activity of acyl derivatives of betulin and betulinic and dihydroquinopimaric acids

Phase I and II study of the safety, virologic effect, and pharmacokinetics/pharmacodynamics of single-dose 3-O-(3 ,3 -dimethylsuccinyl)betulinic acid (bevirimat) against human immunodeficiency virus infection

Identification and characterization of BMS-955176, a second-generation HIV-1 maturation inhibitor with improved potency, antiviral spectrum, and gag polymorphic coverage

A phase IIa study evaluating safety, pharmacokinetics, and antiviral activity of GSK2838232, a novel, second-generation maturation inhibitor, in participants with human immunodeficiency virus type 1 infection

Olive-derived triterpenes suppress SARS COV-2 main protease: A promising scaffold for future therapeutics

Betulinic acid exhibits antiviral effects against dengue virus infection

Betulinic acid exerts cytoprotective activity on Zika virus-infected neural progenitor cells

The authors declare no conflict of interest.Sample Availability: Samples of the compounds 69-86 are available from the authors.