Betulinic Acid Decorated with Polar Groups and Blue Emitting BODIPY Dye: Synthesis, Cytotoxicity, Cell-Cycle Analysis and Anti-HIV Profiling

Betulinic acid (BA) is a potent triterpene, which has shown promising potential in cancer and HIV-1 treatment. Here, we report a synthesis and biological evaluation of 17 new compounds, including BODIPY labelled analogues derived from BA. The analogues terminated by amino moiety showed increased cytotoxicity (e.g., BA had on CCRF-CEM IC50 > 50 μM, amine 3 IC50 0.21 and amine 14 IC50 0.29). The cell-cycle arrest was evaluated and did not show general features for all the tested compounds. A fluorescence microscopy study of six derivatives revealed that only 4 and 6 were detected in living cells. These compounds were colocalized with the endoplasmic reticulum and mitochondria, indicating possible targets in these organelles. The study of anti-HIV-1 activity showed that 8, 10, 16, 17 and 18 have had IC50i > 10 μM. Only completely processed p24 CA was identified in the viruses formed in the presence of compounds 4 and 12. In the cases of 2, 8, 9, 10, 16, 17 and 18, we identified not fully processed p24 CA and p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat.


Introduction
Betulinic acid (BA) is a natural pentacyclic triterpene of the lupane type ( Figure 1). Despite its low solubility in aqueous solutions, this substance is gaining attention with its wide range of interesting biological activity. BA is often derivatized to increase solubility, enhance the therapeutic effect, and target the drug to the specific site of action [1]. BA shows a significant degree of selectivity for cytotoxicity against a variety of tumour cells mboxciteB2-biomedicines-1332342,B3-biomedicines-1332342,B4-biomedicines-1332342 and activity against HIV-1 [5]. There are several possible mechanisms of action of BA (reviewed in [6]), which provide an advantage in the development of resistance to one of the mechanisms and may thus find application in the treatment of tumours resistant to current chemotherapeutics [6]. One is the direct action of BA on the mitochondrial membrane, leading to an increase of outer membrane permeability, its depolarization and release of cytochrome c into the cytosol. It is then responsible for triggering apoptosis [7]. Among BA has been shown to have anti-HIV-1 activity in the past. Although the test results were not groundbreaking, and the effect was observed only at relatively high concentrations [5] This discovery inevitably led to the synthesis of several other analogues. One of the derivatives with strong anti-HIV-1 activity was 3-O-(3,3-dimethylsuccinyl) betulinic acid, known as bevirimat (Figure 1, BT) [15]. BT acts as an inhibitor of HIV-1 particle maturation. Inhibition of viral particle maturation appears to be a critical point of therapeutic intervention. During the maturation phase, the viral protease cleaves the Gag polyprotein while releasing the individual structural proteins. The final step is the cleavage of p25 CA-SP1 to a functional p24 CA protein. Inhibition of the last step of maturation results in virus particles with aberrantly formed mature cores that are incapable of further infection [16]. BT advanced to the second phase of clinical testing [17][18][19], during which virus reduction was observed in only 40-50% of patients. The remainder of the patients developed resistance due to natural polymorphic variation in the Gag polyprotein [20]. With this result, the clinical studies were terminated.
Given the important features of BA mentioned above, it is no surprise that many research groups addressed it. Hundreds of derivatives have been prepared over the last few decades. However, with derivatization, for example, the expected effect disappeared, resistance developed rapidly, or toxicity to normal cells increased dramatically. For anti-HIV derivatives, several so-called "privileged structures" (Figure 1), structural motifs that can be the basis for the design of an effective drug, were found [21,22]. BA is most often chemically modified at C-3 and C-28 positions. Addition to the double bond between carbon atoms C-20 and C-30 usually does not significantly enhance activity, on the contrary, the activity often disappears. This finding generally applies to both anti-cancer and anti-HIV effects [23][24][25]. Recent works have confirmed that the presence of an extra amine BA has been shown to have anti-HIV-1 activity in the past. Although the test results were not groundbreaking, and the effect was observed only at relatively high concentrations [5] This discovery inevitably led to the synthesis of several other analogues. One of the derivatives with strong anti-HIV-1 activity was 3-O-(3,3-dimethylsuccinyl) betulinic acid, known as bevirimat (Figure 1, BT) [15]. BT acts as an inhibitor of HIV-1 particle maturation. Inhibition of viral particle maturation appears to be a critical point of therapeutic intervention. During the maturation phase, the viral protease cleaves the Gag polyprotein while releasing the individual structural proteins. The final step is the cleavage of p25 CA-SP1 to a functional p24 CA protein. Inhibition of the last step of maturation results in virus particles with aberrantly formed mature cores that are incapable of further infection [16]. BT advanced to the second phase of clinical testing [17][18][19], during which virus reduction was observed in only 40-50% of patients. The remainder of the patients developed resistance due to natural polymorphic variation in the Gag polyprotein [20]. With this result, the clinical studies were terminated.
Given the important features of BA mentioned above, it is no surprise that many research groups addressed it. Hundreds of derivatives have been prepared over the last few decades. However, with derivatization, for example, the expected effect disappeared, resistance developed rapidly, or toxicity to normal cells increased dramatically. For anti-HIV derivatives, several so-called "privileged structures" (Figure 1), structural motifs that can be the basis for the design of an effective drug, were found [21,22]. BA is most often chemically modified at C-3 and C-28 positions. Addition to the double bond between carbon atoms C-20 and C-30 usually does not significantly enhance activity, on the contrary, the activity often disappears. This finding generally applies to both anti-cancer and anti-HIV effects [23][24][25]. Recent works have confirmed that the presence of an extra amine group introduced by conjugation into a BA molecule can significantly increase antitumour potency [26,27].
This work presents the preparation and biological evaluation of new analogues of BA and BT containing an amino group. In the past, fluorescent analogues of BA labelled with green-emitting BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) [28,29] and redemitting Rhodamine B [30] were synthesized to study its localization and trafficking in living cells. In this work, we synthesized and studied new derivatives of BA and BT labelled at C-3 and C-28 positions using a small blue-emitting BODIPY dye.

Chemical Synthesis
Aluminium silica gel sheets for detection in UV light (TLC Silica gel 60 F254, Merck, Darmstadt, Denmark) were used for thin-layer chromatography (TLC), subsequent visualization was proceeded by a diluted solution of sulfuric acid in methanol and plates were heated. Silica gel (30-60 µm, SiliTech, MP Biomedicals, Costa Mesa, CA, USA) was used for column chromatography. NMR Spectra were recorded by Agilent-MR DDR2 (Santa Clara, CA, USA). HRMS were measured by LTQ ORBITRAP VELOS with HESI+/HESIionization (Thermo Scientific, Waltham, MA, USA). For microwave synthesis, an Initiator Classic 355,301 (Biotage, Uppsala, Sweden) was used.
The solvents for column chromatography and reactions were purchased from PENTA (Praha, Czech Republic) and were used without further distillation.

Cell Lines
All cells (if not indicated otherwise) were purchased from the American Tissue Culture Collection (ATCC; Manassas, VA, USA). The highly chemosensitive CCRF-CEM line is derived from T lymphoblastic leukaemia, K562 represent cells of chronic myelogenous leukaemia. Colorectal adenocarcinoma HCT116 cell line and its p53 gene knockout counterpart (HCT116p53−/−, Horizon Discovery Ltd., Cambridge, UK) were used as models to assess the impact of p53 deficiency on cell line sensitivity. A549 cells are derived from lung adenocarcinoma and U2OS from human osteosarcoma. CEM-DNR and K562-Tax are well-characterized daunorubicin and paclitaxel-resistant sublines of CCRF-CEM and K562. The CEM-DNR resistant cells overexpress the P-glycoprotein and LRP protein, the K562-Tax overexpress P-glycoprotein but is losing the expression of LRP, which is present at parental K562 cell line. P-glycoprotein belongs to the ABC transporters' family and is involved in the primary and acquired multidrug resistance phenomenon by the efflux of toxic compounds, LRP protein is involved in the lysosomal degradation. MRC-5 and BJ cell lines were used as a non-tumour control and represent human fibroblasts. The cells were maintained in Nunc/Corning 80 cm 2 plastic tissue culture flasks and cultured in cell culture medium according to ATCC or Horizon recommendations (DMEM/RPMI 1640 with 5 g/L-glucose, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 10% fetal calf serum, and NaHCO 3 ).

MTS Assay
To perform the cytotoxicity MTS assay, cell suspensions were prepared and diluted according to the cell type and the expected target cell density (25,000-35,000 cells/mL) based on cell growth characteristics. Cells were added by an automatic pipettor (30 µL) into 384 well microtiter plates. All tested compounds were dissolved in 100% DMSO and four-fold dilutions of the intended test concentration were added in 0.15 µL aliquots at time zero to the microtiter plate wells by the echo-acoustic liquid handler Echo550 (Labcyte, San Jose, CA, USA). The experiments were performed in technical duplicates and at least three biological replicates. The cells were incubated with the tested compounds for 72 h at 37 • C, in a 5% CO 2 atmosphere at 99% humidity. At the end of the incubation period, the cells were assayed by using the MTS test. Aliquots (5 µL) of the MTS stock solution were pipetted into each well and incubated for an additional 1-4 h. After this incubation period, the optical density (OD) was measured at 490 nm with an Envision microplate reader (Perkin Elmer, Waltham, Massachusetts, USA). Tumour cell survival (TCS) was calculated using the following equation: TCS = (OD drug-exposed well /mean OD control wells ) × 100%. The IC 50 value, the drug concentration that is lethal to 50% of the tumour cells, was calculated from the appropriate dose-response curves in Dotmatics software (The Old Monastery, Windhill, Bishop s Stortford, Herts, UK).

Cell Cycle and Apoptosis Analysis
CCRF-CEM cells were seeded in 6-well plates at a density of 1 × 106/well. After 24 h, compounds at concentrations corresponding to 1× or 5 × IC 50 were added to the wells and incubated for 24 h. Cells were then harvested, washed with cold 1 × PBS and fixed in ice-cold 70% ethanol. Fixed cells were incubated overnight at −20 • C, washed in hypotonic citrate buffer, treated with RNase (50 µg mL −1 ) and incubated with propidium iodide for 15 min. DNA content was analysed using Becton Dickinson flow cytometer and cell cycle data were analysed in the program ModFitLT (Verity, Carrollton, TX, USA). Apoptosis was measured in a logarithmic model expressing the percentage of the particles with propidium content lower than cells in G0/G1 phase (<G1) of the cell cycle. The mitotic marker pH3Ser10 antibody (Sigma) and secondary anti-mouse-FITC antibody (Sigma) were used for labelling and subsequent flow cytometry analysis of ethanol-fixed CCRF-CEM cells.

BrDU Incorporation Analysis
Cells were cultivated as in the method above and pulse-labelled with 10 µM 5-bromo-2-deoxyuridine (BrDU) for 30 min before collection to the test tubes. The cells were washed with cold 1 × PBS and fixed in ice-cold 70% ethanol. Before analysis, they were washed with 1 × PBS and incubated in 2M HCl for 30 min at room temperature. Following neutralization with 0.1M Na 2 B 4 O 7 (borax), the cells were washed with 0.5% Tween-20 and 1% BSA in 1 × PBS. The cell pellets were stained using a primary anti-BrdU antibody (Exbio, Vestec, Czech Republic) for 30 min at room temperature and a secondary anti-mouse-FITC antibody (Sigma). The samples were then incubated with propidium iodide (0.1 mg mL −1 ), treated with RNase A (0.5 mg mL −1 ) for 1 h at room temperature in the dark and analysed as above.

BrU Incorporation Analysis
Cells were cultured, treated as above, pulse-labelled with 1 mM 5-bromouridine (BrU) for 30 min and fixed in 1% buffered paraformaldehyde with 0.05% NP-40 at room temperature for 15 min. Following overnight incubation at 4 • C, they were washed with 1% glycine in 1 × PBS, washed with 1 × PBS again and stained with primary anti-BrdU antibody cross-reacting to BrU (Exbio) for 30 min and secondary anti-mouse-FITC antibody (Sigma). The analysis was performed similarly to the BrDU analysis.

Fluorescent Microscopy
U2OS cell line (ATCC, USA) was transduced with premade lentiviral particles (Vectalis-TaKaRa, Japan) with sequences that express fluorescent protein tag mCherry targeted to specific subcellular locations. All cell lines were prepared according to the vendor's instructions. The U2OS-Nuc cell line was prepared by using rLV.EF1.mCherry-Nuc-9 (cat. n. 0023VCT), containing a NLS sequence that imports protein into the nucleus. The U2OS-ER cell line was transduced by rLV.EF1.mCherry-ER-9 (cat. n. 0025VCT), which contains a calreticulin signal sequence and a KDEL sequence that associates protein with the endoplasmic reticulum. The U2OS-GA cell line was transduced by rLV.EF1.mCherry-Golgi-9 (cat. n. 0022VCT), containing a human GT precursor, a protein localized in Golgi Apparatus. The U2OS-Mito cell line was prepared by using rLV.EF1.mCherry-Mito-9 (cat. n. 0024VCT), containing a mitochondrial targeting sequence.
U2OS cells with fluorescent fusion proteins (density 1.0 × 103 per well) were seeded into 384 CellCarrier plates (Perkin Elmer, Waltham, MA, USA) and pre-incubated for 24 h at 37 • C and 5% CO 2 . The attached cells were treated with tested compounds in concentration 10 µM for 1 h and subsequently rinsed with fresh media. The live-cell imaging was performed by Cell Voyager CV7000 (Yokogawa, Tokyo, Japan) spinning disc confocal microscopy system at 37 • C in a 5% CO 2 atmosphere. Live cells were monitored by a 60 × water immersion objective. The fluorescent signal was excited by lasers (405 nm and 561 nm) and the emission was filtered by bandpass filters (BP 445/45 and BP 595/20). All images were post-processed, and Pearson's and Mander's coefficients were calculated using the JACoP plugin in Image-J software.
HEK-293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (Sigma) and 1% L-glutamine (Sigma) at 37 • C under 5% CO 2 . A day before transfection, cells were plated at 3 × 105 cells per well. The following day, cells were transfected with the appropriate vectors using polyethylenimine (PEI, 1 mg/mL) at a 2:1 PEI:DNA ratio. Four hours post-transfection, the culture medium was replaced with fresh DMEM, containing various concentrations of tested compounds, solubilized in DMSO. At 48 h post-transfection, the culture media containing released virions were harvested, filtered through 0.45-µm pores membrane and used for immunochemical quantification and characterization by ELISA and Western blot using rabbit anti-HIV-1 CA antibody.

Single-Round Infectivity Assay
The infectivity was determined similarly as described earlier [33][34][35]. Briefly, 48 h post-transfection, the culture media from HEK 293 cells transfected with psPAX2, pWPXLd-GFP and pHEF-VSV-G vectors at a ratio 1:1:1 in the presence of tested compounds were collected and filtered through a 0.45-µm filter. HIV-1 CA content was determined by ELISA [33]. The freshly seeded HEK 293 cells were infected with ELISA-normalized amounts of VSV-G pseudotyped HIV-1 particles and incubated for 48 h. The cells were fixed with 2% paraformaldehyde and transferred to a FACS tube. Quantification of GFPpositive cells was performed using a BD FACS Aria III flow cytometer (BD Life Sciences, San Jose, CA, USA).

Western Blot
At 48 h post-transfection, 100 µL aliquots of virus-containing culture media were combined with 20 µL of PLB (6×) and the samples were analysed by Western blot using rabbit anti-HIV-1 CA (in house production). Proteins were resolved by reducing SDS-PAGE (12%) and blotted onto a nitrocellulose membrane. The antigen-antibody complexes were detected by Clarity™ Western ECL Substrate (Biorad, Hercules, CA, USA) and visualized using the FUSION 7S system (Vilber Lourmat, Marne-la-Vallée, France).

Chemistry
The synthesis of fluorescent labels was based on 8-thiomethyl BODIPY (BODIPY-SMe; Figure 2), which was prepared in our laboratory, according to the procedure previously described in the literature [36]. The thiomethyl group is reactive towards amines. After this reaction, secondary amines are formed with significant fluorescence characterized by emission in the blue region of the spectrum. For the preparation of betulinic acid conjugates, a carboxy-terminated derivative (BODIPY-CO 2 H, Figure 2) was prepared by reaction of BODIPY-SMe and β-alanine [37] and an amino-terminated derivative by reaction with 3-azidopropan-1-amine [38] and reduction of azide (BODIPY-N 3 , Figure 2) to amine (BODIPY-NH 2 , Figure 2) by catalytic hydrogenation.
(PEI, 1 mg/mL) at a 2:1 PEI:DNA ratio. Four hours post-transfection, the culture medium was replaced with fresh DMEM, containing various concentrations of tested compounds, solubilized in DMSO. At 48 h post-transfection, the culture media containing released virions were harvested, filtered through 0.45-μm pores membrane and used for immunochemical quantification and characterization by ELISA and Western blot using rabbit anti-HIV-1 CA antibody.

Single-Round Infectivity Assay
The infectivity was determined similarly as described earlier [33][34][35]. Briefly, 48 h post-transfection, the culture media from HEK 293 cells transfected with psPAX2, pWPXLd-GFP and pHEF-VSV-G vectors at a ratio 1:1:1 in the presence of tested compounds were collected and filtered through a 0.45-μm filter. HIV-1 CA content was determined by ELISA [33]. The freshly seeded HEK 293 cells were infected with ELISA-normalized amounts of VSV-G pseudotyped HIV-1 particles and incubated for 48 h. The cells were fixed with 2% paraformaldehyde and transferred to a FACS tube. Quantification of GFP-positive cells was performed using a BD FACS Aria III flow cytometer (BD Life Sciences, San Jose, CA, USA).

Western Blot
At 48 h post-transfection, 100 μL aliquots of virus-containing culture media were combined with 20 μL of PLB (6×) and the samples were analysed by Western blot using rabbit anti-HIV-1 CA (in house production). Proteins were resolved by reducing SDS-PAGE (12%) and blotted onto a nitrocellulose membrane. The antigen-antibody complexes were detected by Clarity™ Western ECL Substrate (Biorad, Hercules, CA, USA) and visualized using the FUSION 7S system (Vilber Lourmat, Marne-la-Vallée, France).

Chemistry
The synthesis of fluorescent labels was based on 8-thiomethyl BODIPY (BODIPY-SMe; Figure 2), which was prepared in our laboratory, according to the procedure previously described in the literature [36]. The thiomethyl group is reactive towards amines. After this reaction, secondary amines are formed with significant fluorescence characterized by emission in the blue region of the spectrum. For the preparation of betulinic acid conjugates, a carboxy-terminated derivative (BODIPY-CO2H, Figure 2) was prepared by reaction of BODIPY-SMe and β-alanine [37] and an amino-terminated derivative by reaction with 3-azidopropan-1-amine [38] and reduction of azide (BODIPY-N3, Figure 2) to amine (BODIPY-NH2, Figure 2) by catalytic hydrogenation. Betulinoyl azidopropylamide (N-(3-azidopropyl)-3β-hydroxylup-20(29)-en-28-amide) 1 was prepared by reacting BA with 3-azidopropan-1-amine using carbodiimide Betulinoyl azidopropylamide (N-(3-azidopropyl)-3β-hydroxylup-20(29)-en-28-amide) 1 was prepared by reacting BA with 3-azidopropan-1-amine using carbodiimide chemistry ( Figure 3A). The reaction was catalysed by EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in the presence of 4-DMAP (4-dimethylaminopyridine) and HOBt (1-hydroxybenzotriazole). The bevirimat derivative 2 was prepared from compound 1 by reaction with 2,2-dimethylsuccinic anhydride according to a protocol reported in the literature [39]. By Staudinger reduction [40] catalysed by triphenylphosphine in aqueous THF, the azido group of compound 1 was reduced to amino derivative 3. In an effort to reduce derivative 2 by the same method, a non-separable mixture of products was obtained. By the reaction of BA with BODIPY-NH 2 catalysed by DCC (N,N -dicyclohexylcarbodiimide) in the presence of 4-DMAP, derivative 4 was obtained. This reaction proceeded without difficulty in good yield ( Figure 3A). Azide 1 was further conjugated at the C-3 position with BODIPY-CO 2 H by Steglich esterification [41] to produce derivative 5. The azide group of derivative 5 was reduced by Staudinger reduction to amine 6. When attempting to modify compound 4 with dimethylsuccinic anhydride under the conditions used to prepare derivative 2, degradation of the fluorescent label occurred, probably due to too high a temperature. Therefore, another synthetic procedure using a tert-butoxycarbonyl protecting group (Boc) on the terminal amino group was chosen for the synthesis of other "aminopropyl" derivatives ( Figure 3B). The N-Boc-1,3-diaminopropane linker was conjugated to BA to give compound 7, which could already be used to prepare the bevirimat derivative 8. The protecting group was removed in an acidic environment to give amine 9. From compound 9, a fluorescently labelled derivative of bevirimat was prepared by the reaction with BODIPY-SMe. Analogous to the synthetic procedure shown in Figure 3B, a series of substances with a piperazine linker at position C-28 was prepared (Figure 4). The introduction of the piperazine motif was chosen on the basis of promising results for the so-called privileged structures published previously [21]. The exception was that the C-3 hydroxyl was first acetylated to produce compound 11, and in the next step, the carboxyl group was activated to reactive chloride. After condensation with 1-(2-N-Boc-aminoethyl) piperazine, Analogous to the synthetic procedure shown in Figure 3B, a series of substances with a piperazine linker at position C-28 was prepared ( Figure 4). The introduction of the piperazine motif was chosen on the basis of promising results for the so-called privileged structures published previously [21]. The exception was that the C-3 hydroxyl was first acetylated to produce compound 11, and in the next step, the carboxyl group was activated to reactive chloride. After condensation with 1-(2-N-Boc-aminoethyl) piperazine, tertiary amide 12 was obtained. Deacetylation of 12 occurred in a relatively low yield; however, part of the starting material was recovered during the separation of the reaction mixture.
Biomedicines 2021, 9, x FOR PEER REVIEW 15 of 23 tertiary amide 12 was obtained. Deacetylation of 12 occurred in a relatively low yield; however, part of the starting material was recovered during the separation of the reaction mixture. Experimental details of the preparation of substances are described in Section 2.1. and the NMR, HRMS spectra (Figures S1-S51) and photochemical properties ( Figure S52 and Table S1) of the substances are shown in the Supplementary Material.

Cytotoxicity on a Panel of Cell Lines
The in vitro cytotoxicity of derivatives was assessed using MTS assay on the normal human foreskin and lung fibroblasts BJ and MRC-5 and cancer cell lines of a different histogenetic type (Table 1)  Experimental details of the preparation of substances are described in Section 2.1. and the NMR, HRMS spectra (Figures S1-S51) and photochemical properties ( Figure S52 and Table S1) of the substances are shown in the Supplementary Material.

Cytotoxicity on a Panel of Cell Lines
The in vitro cytotoxicity of derivatives was assessed using MTS assay on the normal human foreskin and lung fibroblasts BJ and MRC-5 and cancer cell lines of a different histogenetic type (Table 1)

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives ( Table 2).

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives (Table 2).

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives (Table 2).

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives (Table 2).

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives (Table 2).

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives (Table 2).

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives (Table 2). CCRF-CEM cell line, but comparable activity was observed in all tested cell lines, including non-tumour lines.

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cycle profile of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivatives (Table 2). ing non-tumour lines.

Cell Cycle, Apoptosis and DNA/RNA Synthesis
To reveal cytostatic effects, we examined proliferation markers and cell cyc of the sensitive CCRF-CEM cell line following a 24 h incubation with the derivat ble 2).

Live Cells Imaging
The group of six derivatives of BA and BODIPY was studied on the U2OS-Nuc cell line with the nucleus labelled by fluorescein protein mCherry. The functionalized BODIPY dyes (BODIPY-CO 2 H and BODIPY-NH 2 ), as well as precursor BODIPY-SMe, were used as a control. All fluorescent microscopic images of this pilot experiment are shown in Figure S53. To achieve a better specificity of the staining, we have focused on the short incubation with the fluorescent conjugates. After short incubation (1 h), conjugates 4 and 6 out of this group of derivatives were localized in living cells, but only with the weak signal in the nucleus of the studied cell line ( Figure 5B-Pearson's and Mander's coefficients). The functionalized BODIPY dyes were not detected in the U2OS-Nuc cell line and thus it is highly possible that cellular uptake of conjugates 4 and 6 is due to their groups on BA residue. Other studied derivatives of BA and BODIPY were not detected in living cells under our experimental conditions; however, it is possible that the signal can be observed at later intervals. BODIPY-SMe is reactive due to the 8-thiomethyl group and it was predicted to penetrate cell compartments; this was confirmed by fluorescent microscopy.
To further study the cellular localization of conjugates 4 and 6, we decided to continue with fluorescent microscopy on cell lines with fluorescently labelled structures of mitochondria, endoplasmic reticulum, and Golgi apparatus, which are the most published targets of BA [29,30]. The results of these colocalization experiments are shown in Figure 5. Both conjugates demonstrated presence in multiple cellular structures. Pearson's coefficient ( Figure 5B To further study the cellular localization of conjugates 4 and 6, we decided to continue with fluorescent microscopy on cell lines with fluorescently labelled structures of mitochondria, endoplasmic reticulum, and Golgi apparatus, which are the most published targets of BA [29,30]. The results of these colocalization experiments are shown in Figure  5. Both conjugates demonstrated presence in multiple cellular structures. Pearson's coef- To conclude our results from fluorescent microscopy study of six derivatives of BA and BODIPY, only conjugates 4 and 6 are detected in living cells under our experimental conditions (1 h following the treatment). Furthermore, we were able to almost perfectly colocalize both conjugates with cellular structures as endoplasmic reticulum and mitochondria, which is in agreement with data published in the past [21]. Compound 4 has, in its structure, fluorophore attached to the carboxyl group at the C-28 position, thus it is more similar to the pristine structure of BA and has low cytotoxicity (close to free BA). Conversely, compound 6 contains a conjugated amine at the C-28 position and the fluorophore is attached to the hydroxyl group at the C-3 position of BA. Its cytotoxicity is markedly more pronounced than in the case of substance 4. It is clear that the "polar head" of the molecule is responsible for cytotoxicity. Moreover, this moiety can be used for the intracellular targeted delivery, or organelle/mitochondrion targeting, as is described by the previous research works [43,44]. As the localization of both compounds is similar, it is likely that the direct target remained unchanged, but the effect of compound 6 was potentiated by the presence of free amine moiety in the molecule. The localization of the compounds in lipid rich compartments (mitochondria, endoplasmic reticulum) can also be explained by the lipid character of the BA and its analogues. The calculated values of lipophilicity (logP) of the substances are close to BA (Table S2). The acidity constants (pKa) are indicative and their reproducibility is difficult because, in comparison with BA, the compounds described here are mostly in the form of amide or ester derivatives.

Anti-HIV Activity
Bevirimat (3-O-(3 ,3 -dimethylsuccinyl) betulinic acid) and its derivatives were shown to be maturation inhibitors of HIV-1 [45][46][47]. By binding to the CA-SP1 region of HIV-1 Gag polyprotein, bevirimat prevents HIV-1 protease-mediated release of C-terminal part of CA from a spacer peptide 1 (SP1) [48]. This results in a block of the final step of virus maturation and subsequently abolishes HIV-1 infectivity. An atomic model of HIV-1 CA-SP1 suggested that this inhibitor stabilizes the CA-SP1 structure, thus preventing the proteolytic cleavage [49]. Although bevirimat is a potent inhibitor of HIV-1 maturation, its clinical development was discontinued in 2010 due to the bevirimat resistance caused by Gag SP1 natural polymorphism (Q6, V7 and T8) [50][51][52]. However, bevirimat derivatives with modification at the C-28 position seem to overcome the problem with HIV-1 resistance [53,54]. Here, using VSV-G pseudotyped HIV-1 particles, we tested the effect of 17 BA derivatives on HIV-1 maturation and infectivity. The 50% cytotoxic concentration (IC 50 ) of the compounds was first evaluated by Resazurin assay. Two of the tested compounds, 3 and 14, were highly toxic to HEK 293 cells at a concentration lower than 5 µM and significant cytotoxicity was also found for compound 6 (IC 50 12 µM) ( Table 3). tion signal was again detected in the U2OS-GA cell line. BODIPY-SMe was used based on the data from the pilot experiment as a positive control with perfect colocalization in all studied cell lines. Images with entire microscopic fields are shown in Figures S54-S56. To conclude our results from fluorescent microscopy study of six derivatives of BA and BODIPY, only conjugates 4 and 6 are detected in living cells under our experimental conditions (1 h following the treatment). Furthermore, we were able to almost perfectly colocalize both conjugates with cellular structures as endoplasmic reticulum and mitochondria, which is in agreement with data published in the past [21]. Compound 4 has, in its structure, fluorophore attached to the carboxyl group at the C-28 position, thus it is more similar to the pristine structure of BA and has low cytotoxicity (close to free BA). Conversely, compound 6 contains a conjugated amine at the C-28 position and the fluorophore is attached to the hydroxyl group at the C-3 position of BA. Its cytotoxicity is markedly more pronounced than in the case of substance 4. It is clear that the "polar head" of the molecule is responsible for cytotoxicity. Moreover, this moiety can be used for the intracellular targeted delivery, or organelle/mitochondrion targeting, as is described by the previous research works [43,44]. As the localization of both compounds is similar, it is likely that the direct target remained unchanged, but the effect of compound 6 was potentiated by the presence of free amine moiety in the molecule. The localization of the compounds in lipid rich compartments (mitochondria, endoplasmic reticulum) can also be explained by the lipid character of the BA and its analogues. The calculated values of lipophilicity (logP) of the substances are close to BA (Table S2). The acidity constants (pKa) are indicative and their reproducibility is difficult because, in comparison with BA, the compounds described here are mostly in the form of amide or ester derivatives.

Anti-HIV Activity
Bevirimat (3-O-(3´,3´-dimethylsuccinyl) betulinic acid) and its derivatives were shown to be maturation inhibitors of HIV-1 [45][46][47]. By binding to the CA-SP1 region of HIV-1 Gag polyprotein, bevirimat prevents HIV-1 protease-mediated release of C-terminal part of CA from a spacer peptide 1 (SP1) [48]. This results in a block of the final step of virus maturation and subsequently abolishes HIV-1 infectivity. An atomic model of HIV-1 CA-SP1 suggested that this inhibitor stabilizes the CA-SP1 structure, thus preventing the proteolytic cleavage [49]. Although bevirimat is a potent inhibitor of HIV-1 maturation, its clinical development was discontinued in 2010 due to the bevirimat resistance caused by Gag SP1 natural polymorphism (Q6, V7 and T8) [50][51][52]. However, bevirimat derivatives with modification at the C-28 position seem to overcome the problem with HIV-1 resistance [53,54]. Here, using VSV-G pseudotyped HIV-1 particles, we tested the effect of 17 BA derivatives on HIV-1 maturation and infectivity. The 50% cytotoxic concentration (IC50) of the compounds was first evaluated by Resazurin assay. Two of the tested compounds, 3 and 14, were highly toxic to HEK 293 cells at a concentration lower than 5 μM and significant cytotoxicity was also found for compound 6 (IC50 12 μM) ( Table  3). tion signal was again detected in the U2OS-GA cell line. BODIPY-SMe was used based on the data from the pilot experiment as a positive control with perfect colocalization in all studied cell lines. Images with entire microscopic fields are shown in Figures S54-S56. To conclude our results from fluorescent microscopy study of six derivatives of BA and BODIPY, only conjugates 4 and 6 are detected in living cells under our experimental conditions (1 h following the treatment). Furthermore, we were able to almost perfectly colocalize both conjugates with cellular structures as endoplasmic reticulum and mitochondria, which is in agreement with data published in the past [21]. Compound 4 has, in its structure, fluorophore attached to the carboxyl group at the C-28 position, thus it is more similar to the pristine structure of BA and has low cytotoxicity (close to free BA). Conversely, compound 6 contains a conjugated amine at the C-28 position and the fluorophore is attached to the hydroxyl group at the C-3 position of BA. Its cytotoxicity is markedly more pronounced than in the case of substance 4. It is clear that the "polar head" of the molecule is responsible for cytotoxicity. Moreover, this moiety can be used for the intracellular targeted delivery, or organelle/mitochondrion targeting, as is described by the previous research works [43,44]. As the localization of both compounds is similar, it is likely that the direct target remained unchanged, but the effect of compound 6 was potentiated by the presence of free amine moiety in the molecule. The localization of the compounds in lipid rich compartments (mitochondria, endoplasmic reticulum) can also be explained by the lipid character of the BA and its analogues. The calculated values of lipophilicity (logP) of the substances are close to BA (Table S2). The acidity constants (pKa) are indicative and their reproducibility is difficult because, in comparison with BA, the compounds described here are mostly in the form of amide or ester derivatives.

Anti-HIV Activity
Bevirimat (3-O-(3´,3´-dimethylsuccinyl) betulinic acid) and its derivatives were shown to be maturation inhibitors of HIV-1 [45][46][47]. By binding to the CA-SP1 region of HIV-1 Gag polyprotein, bevirimat prevents HIV-1 protease-mediated release of C-terminal part of CA from a spacer peptide 1 (SP1) [48]. This results in a block of the final step of virus maturation and subsequently abolishes HIV-1 infectivity. An atomic model of HIV-1 CA-SP1 suggested that this inhibitor stabilizes the CA-SP1 structure, thus preventing the proteolytic cleavage [49]. Although bevirimat is a potent inhibitor of HIV-1 maturation, its clinical development was discontinued in 2010 due to the bevirimat resistance caused by Gag SP1 natural polymorphism (Q6, V7 and T8) [50][51][52]. However, bevirimat derivatives with modification at the C-28 position seem to overcome the problem with HIV-1 resistance [53,54]. Here, using VSV-G pseudotyped HIV-1 particles, we tested the effect of 17 BA derivatives on HIV-1 maturation and infectivity. The 50% cytotoxic concentration (IC50) of the compounds was first evaluated by Resazurin assay. Two of the tested compounds, 3 and 14, were highly toxic to HEK 293 cells at a concentration lower than 5 μM and significant cytotoxicity was also found for compound 6 (IC50 12 μM) ( Table  3). presented in multiple cellular structures, the obtained 6 almost perfectly label mitochondria and endoplasm tion signal was again detected in the U2OS-GA cell l on the data from the pilot experiment as a positive co all studied cell lines. Images with entire microscopic f To conclude our results from fluorescent micros and BODIPY, only conjugates 4 and 6 are detected in conditions (1 h following the treatment). Furthermor colocalize both conjugates with cellular structures as chondria, which is in agreement with data published i its structure, fluorophore attached to the carboxyl gr more similar to the pristine structure of BA and has Conversely, compound 6 contains a conjugated amine ophore is attached to the hydroxyl group at the C-3 pos edly more pronounced than in the case of substance 4 the molecule is responsible for cytotoxicity. Moreov intracellular targeted delivery, or organelle/mitochon the previous research works [43,44]. As the localizatio likely that the direct target remained unchanged, but t tiated by the presence of free amine moiety in the mo pounds in lipid rich compartments (mitochondria, e explained by the lipid character of the BA and its anal ophilicity (logP) of the substances are close to BA (Ta are indicative and their reproducibility is difficult be compounds described here are mostly in the form of a

Anti-HIV Activity
Bevirimat (3-O-(3´,3´-dimethylsuccinyl) betulin shown to be maturation inhibitors of HIV-1 [45][46][47]. B HIV-1 Gag polyprotein, bevirimat prevents HIV-1 pro nal part of CA from a spacer peptide 1 (SP1) [48]. Thi of virus maturation and subsequently abolishes HIV HIV-1 CA-SP1 suggested that this inhibitor stabilizes ing the proteolytic cleavage [49]. Although bevirimat ration, its clinical development was discontinued in 2 caused by Gag SP1 natural polymorphism (Q6, V7 an derivatives with modification at the C-28 position se HIV-1 resistance [53,54]. Here, using VSV-G pseudoty effect of 17 BA derivatives on HIV-1 maturation and centration (IC50) of the compounds was first evaluat tested compounds, 3 and 14, were highly toxic to HEK than 5 μM and significant cytotoxicity was also found 3). ), HEK 293 cells were transfected with the lentiviral vectors and treated with the tested compounds. The cells producing HIV-1 particles in the presence or absence of DMSO (at a final concentration of 1%) were used as controls. At 48 h post-transfection, the content of HIV-1 capsid (CA) protein from the culture media was quantified by ELISA and normalized amounts of VSV-G pseudotyped HIV-1 viruses were used to infect fresh HEK 293 cells. HIV-1 infectivity was determined 48 h later by quantification of GFP-producing cells by flow cytometry. The 50% infection inhibition (IC 50i ) was defined as the concentration of the compound that reduced the HIV-1 infectivity by 50% compared to the untreated controls.
Apart from these three cytotoxic compounds, 14 fewer toxic compounds were used in the HIV-1 single-round infectivity assay. HIV-1 particles pseudotyped with VSV glycoproteins were produced in HEK 293 cells in the presence of tested compounds. At 48 h post-transfection, the content of HIV-1 capsid (CA) protein from the culture media was quantified by ELISA and normalized amounts of VSV-G pseudotyped HIV-1 viruses were used to infect fresh HEK 293 cells. At 48 h post-infection, the HIV-1 infectivity was determined by quantification of GFP-producing cells by flow cytometry. The 50% infection inhibition (IC 50i ) was defined as the concentration of the compound that reduced the HIV-1 infectivity by 50% compared to the untreated controls ( Table 3). The compounds 1, 7, 13, 15 and 5 did not exhibit any potent anti-HIV-1 activity (data not shown). Conversely, compounds 2, 4, 9 and 12 inhibited anti-HIV-1 activity with IC 50i from 11.7 to 44.1 µM. The compounds 8, 10, 16, 17 and 18 inhibited HIV-1 with IC 50i below 10 µM (Table 3). To analyse whether these bevirimat derivatives also act as maturation inhibitors of CA-SP1 cleavage, the HIV-1 virions released from the HEK 293 cells treated with the selected compounds (2, 4, 8, 9, 10, 12, 16, 17 and 18) were analysed by Western blot using anti-HIV-1 CA antibody ( Figure 6).
Apart from these three cytotoxic compounds, 14 fewer toxic compounds were used in the HIV-1 single-round infectivity assay. HIV-1 particles pseudotyped with VSV glycoproteins were produced in HEK 293 cells in the presence of tested compounds. At 48 h post-transfection, the content of HIV-1 capsid (CA) protein from the culture media was quantified by ELISA and normalized amounts of VSV-G pseudotyped HIV-1 viruses were used to infect fresh HEK 293 cells. At 48 h post-infection, the HIV-1 infectivity was determined by quantification of GFP-producing cells by flow cytometry. The 50% infection inhibition (IC50i) was defined as the concentration of the compound that reduced the HIV-1 infectivity by 50% compared to the untreated controls ( Table 3). The compounds 1, 7, 13, 15 and 5 did not exhibit any potent anti-HIV-1 activity (data not shown). Conversely, compounds 2, 4, 9 and 12 inhibited anti-HIV-1 activity with IC50i from 11.7 to 44.1 μM. The compounds 8, 10, 16, 17 and 18 inhibited HIV-1 with IC50i below 10 μM (Table 3). To analyse whether these bevirimat derivatives also act as maturation inhibitors of CA-SP1 cleavage, the HIV-1 virions released from the HEK 293 cells treated with the selected compounds (2, 4, 8, 9, 10, 12, 16, 17 and 18) were analysed by Western blot using anti-HIV-1 CA antibody ( Figure 6).  Figure S57).
Only completely processed p24 CA of molecular weight of 24 kDa was identified in the viruses formed in the presence of compounds 4 and 12. However, in the samples treated with compounds 2, 8, 9, 10, 16, 17 and 18, we identified not only fully processed p24 CA, but also p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat, i.e., the block of the final step of HIV-1 maturation.

Conclusions
This study describes the synthesis and biological evaluation of 17 betulinic acid derivatives. The biological profiling revealed that BA derivatives 3 and 14 with modification at C-28 show increased cytotoxicity. However, the cytotoxicity was not specifically directed against cancer cell lines and was not associated with cell cycle arrest. The most effective compounds with sub-micromolar IC50 values 3 and 14 possess a hydroxyl group at C-3, whereas structures with a succinyl hemiester group displayed medium cytotoxicity or were inactive. The study introduced six original structures with BODIPY moiety linked to the lupane skeleton. BODIPY conjugates 4, 5, 15 and 18 showed low or no cytotoxic activity. In contrast, BODIPY derivative 6 induced strong and derivative 10 medium cytotoxicity in the entire cell line panel, although they do not share any similar substituents at positions C-3 and C-28. The cellular localization of BODIPY conjugates was further  Figure S57).
Only completely processed p24 CA of molecular weight of 24 kDa was identified in the viruses formed in the presence of compounds 4 and 12. However, in the samples treated with compounds 2, 8, 9, 10, 16, 17 and 18, we identified not only fully processed p24 CA, but also p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat, i.e., the block of the final step of HIV-1 maturation.

Conclusions
This study describes the synthesis and biological evaluation of 17 betulinic acid derivatives. The biological profiling revealed that BA derivatives 3 and 14 with modification at C-28 show increased cytotoxicity. However, the cytotoxicity was not specifically directed against cancer cell lines and was not associated with cell cycle arrest. The most effective compounds with sub-micromolar IC 50 values 3 and 14 possess a hydroxyl group at C-3, whereas structures with a succinyl hemiester group displayed medium cytotoxicity or were inactive. The study introduced six original structures with BODIPY moiety linked to the lupane skeleton. BODIPY conjugates 4, 5, 15 and 18 showed low or no cytotoxic activity. In contrast, BODIPY derivative 6 induced strong and derivative 10 medium cytotoxicity in the entire cell line panel, although they do not share any similar substituents at positions C-3 and C-28. The cellular localization of BODIPY conjugates was further studied in U2OS cells using fluorescent microscopy. Fluorescent derivatives 4 and 6 colocalized with endoplasmic reticulum and mitochondria, which is in agreement with previous studies showing interaction with the processes and proteins localized in these organelles [55,56]. Uncoupling of the mitochondrial respiration, followed by radical burst and mitochondrial membrane disruption, is one of the well-described effects of betulin and betulinic acid [57][58][59][60]. Thus, we believe that reliable tools to study the derivatives of BA on living cells were established. The anti-HIV-1 activity showed that compounds 2, 8, 9, 10, 16 and 18 with IC 50i lower than 10 µM did not fully process the p24 CA and p25 CA-SP1 proteins, suggesting a similar mechanism of inhibition as described for bevirimat.  Figure S57: Effect of selected tested compounds on CA-SP1 processing of HIV-1 Gag polyprotein (a duplicate of western blot showed in Figure 5. in the article).