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.

Department of Biotechnology University of Chemistry and Technology Prague 16628 Prague Czech Republic

Department of Chemistry of Natural Compounds University of Chemistry and Technology Prague 16628 Prague Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University and University Hospital in Olomouc 77900 Olomouc Czech Republic

Zobrazit více v PubMed

Sousa J.L.C., Freire C.S.R., Silvestre A.J.D., Silva A.M.S. Recent developments in the functionalization of betulinic acid and its natural analogues: A route to new bioactive compounds. Molecules. 2019;24:355. doi: 10.3390/molecules24020355. PubMed DOI PMC

Zuco V., Supino R., Righetti S.C., Cleris L., Marchesi E., Gambacorti-Passerini C., Formelli F. Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett. 2002;175:17–25. doi: 10.1016/S0304-3835(01)00718-2. PubMed DOI

Pisha E., Chai H., Lee I.S., Chagwedera T.E., Farnsworth N.R., Cordell G.A., Beecher C.W.W., Fong H.H.S., Kinghorn A.D., Brown D.M., et al. Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat. Med. 1995;1:1046–1051. doi: 10.1038/nm1095-1046. PubMed DOI

Noda Y., Kaiya T., Kohda K., Kawazoe Y. Enhanced cytotoxicity of some triterpenes toward leukemia L1210 cells cultured in low pH media: Possibility of a New Mode of Cell Killing. Chem. Pharm. Bull. 1997;45:1665–1670. doi: 10.1248/cpb.45.1665. PubMed DOI

Fujioka T., Kashiwada Y., Kilkuskie R.E., Cosentino L.M., Ballas L.M., Jiang J.B., Janzen W.P., Chen I.-S., Lee K.-H. Anti-AIDS agents, 11. Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids. J. Nat. Prod. 1994;57:243–247. doi: 10.1021/np50104a008. PubMed DOI

Kodr D., Rumlová M., Zimmermann T., Džubák P., Drašar P., Jurášek M. Antitumor and anti-HIV derivatives of betulinic acid. Chem. Listy. 2020;114:658–667.

Fulda S., Friesen C., Los M., Scaffidi C., Mier W., Benedict M., Nunez G., Krammer P.H., Peter M.E., Debatin K.M. Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res. 1997;57:4956–4964. PubMed

Fulda S., Scaffidi C., Susin S.A., Krammer P.H., Kroemer G., Peter M.E., Debatin K.M. Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J. Biol. Chem. 1998;273:33942–33948. doi: 10.1074/jbc.273.51.33942. PubMed DOI

Liu W.K., Ho J.C.K., Cheung F.W.K., Liu B.P.L., Ye W.C., Che C.T. Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line. Eur. J. Pharmacol. 2004;498:71–78. doi: 10.1016/j.ejphar.2004.07.103. PubMed DOI

Fulda S., Jeremias I., Steiner H.H., Pietsch T., Debatin K.M. Betulinic acid: A new cytotoxic agent against malignant brain-tumor cells. Int. J. Cancer. 1999;82:435–441. doi: 10.1002/(SICI)1097-0215(19990730)82:3<435::AID-IJC18>3.0.CO;2-1. PubMed DOI

Mullauer F.B., van Bloois L., Daalhuisen J.B., Ten Brink M.S., Storm G., Medema J.P., Schiffelers R.M., Kessler J.H. Betulinic acid delivered in liposomes reduces growth of human lung and colon cancers in mice without causing systemic toxicity. Anti-Cancer Drug. 2011;22:223–233. doi: 10.1097/CAD.0b013e3283421035. PubMed DOI

Takada Y., Aggarwal B.B. Betulinic acid suppresses carcinogen-induced NF-kappa B activation through inhibition of I kappa B alpha kinase and p65 phosphorylation: Abrogation of cyclooxygenase-2 and matrix metalloprotease-9. J. Immunol. 2003;171:3278–3286. doi: 10.4049/jimmunol.171.6.3278. PubMed DOI

Melzig M.F., Bormann H. Betulinic acid inhibits aminopeptidase N activity. Planta Med. 1998;64:655–657. doi: 10.1055/s-2006-957542. PubMed DOI

Kwon H.J., Shim J.S., Kim J.H., Cho H.Y., Yum Y.N., Kim S.H., Yu J. Betulinic acid inhibits growth factor-induced in vitro angiogenesis via the modulation of mitochondrial function in endothelial cells. Jpn. J. Cancer Res. 2002;93:417–425. doi: 10.1111/j.1349-7006.2002.tb01273.x. PubMed DOI PMC

Kashiwada Y., Nagao T., Hashimoto A., Ikeshiro Y., Okabe H., Cosentino L.M., Lee K.-H. Anti-AIDS agents 38. Anti-HIV activity of 3-O-acyl ursolic acid derivatives. J. Nat. Prod. 2000;63:1619–1622. doi: 10.1021/np990633v. PubMed DOI

Sundquist W.I., Krausslich H.G. HIV-1 Assembly, budding, and maturation. Cold Spring Harb. Perspect. Med. 2012;2:a006924. doi: 10.1101/cshperspect.a006924. PubMed DOI PMC

Smith P.F., Ogundele A., Forrest A., Wilton J., Salzwedel K., Doto J., Allaway G.P., Martin D.E. 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. Antimicrob. Agents Chemother. 2007;51:3574–3581. doi: 10.1128/AAC.00152-07. PubMed DOI PMC

Martin D.E., Blum R., Wilton J., Doto J., Galbraith H., Burgess G.L., Smith P.C., Ballow C. Safety and pharmacokinetics of bevirimat (PA-457), a novel inhibitor of human immunodeficiency virus maturation, in healthy volunteers. Antimicrob. Agents Chemother. 2007;51:3063. doi: 10.1128/AAC.01391-06. PubMed DOI PMC

Martin D.E., Blum R., Doto J., Galbraith H., Ballow C. Multiple-Dose Pharmacokinetics and safety of bevirimat, a novel inhibitor of HIV maturation, in healthy volunteers. Clin. Pharmacokinet. 2007;46:589–598. doi: 10.2165/00003088-200746070-00004. PubMed DOI

Margot N.A., Gibbs C.S., Miller M.D. Phenotypic susceptibility to bevirimat in isolates from HIV-1-infected patients without prior exposure to bevirimat. Antimicrob. Agents Chemother. 2010;54:2345–2353. doi: 10.1128/AAC.01784-09. PubMed DOI PMC

Zhao Y., Gu Q., Morris-Natschke S.L., Chen C.-H., Lee K.-H. Incorporation of privileged structures into bevirimat can improve activity against wild-type and bevirimat-resistant HIV-1. J. Med. Chem. 2016;59:9262–9268. doi: 10.1021/acs.jmedchem.6b00461. PubMed DOI PMC

Zhao Y., Chen C.-H., Morris-Natschke S.L., Lee K.-H. Design, synthesis, and structure activity relationship analysis of new betulinic acid derivatives as potent HIV inhibitors. Eur. J. Med. Chem. 2021;215:113287. doi: 10.1016/j.ejmech.2021.113287. PubMed DOI PMC

Mukherjee R., Jaggi M., Rajendran P., Siddiqui M.J.A., Srivastava S.K., Vardhan A., Burman A.C. Betulinic acid and its derivatives as anti-angiogenic agents. Bioorg. Med. Chem. Lett. 2004;14:2181–2184. doi: 10.1016/j.bmcl.2004.02.044. PubMed DOI

Kim J.Y., Koo H.M., Kim D.S.H.L. Development of C-20 modified betulinic acid derivatives as antitumor agents. Bioorg. Med. Chem. Lett. 2001;11:2405–2408. doi: 10.1016/S0960-894X(01)00460-7. PubMed DOI

Chowdhury A.R., Mandal S., Mittra B., Sharma S., Mukhopadhyay S., Majumder H.K. Betulinic acid, a potent inhibitor of eukaryotic topoisomerase I: Identification of the inhibitory step, the major functional group responsible and development of more potent derivatives. Med. Sci. Monit. 2002;8:BR254–BR265. PubMed

Bildziukevich U., Rarova L., Janovska L., Saman D., Wimmer Z. Enhancing effect of cystamine in its amides with betulinic acid as antimicrobial and antitumor agent in vitro. Steroids. 2019;148:91–98. doi: 10.1016/j.steroids.2019.04.004. PubMed DOI

Bildziukevich U., Vida N., Rárová L., Kolář M., Šaman D., Havlíček L., Drašar P., Wimmer Z. Polyamine derivatives of betulinic acid and beta-sitosterol: A comparative investigation. Steroids. 2015;100:27–35. doi: 10.1016/j.steroids.2015.04.005. PubMed DOI

Brandes B., Hoenke S., Fischer L., Csuk R. Design, synthesis and cytotoxicity of BODIPY-FL labelled triterpenoids. Eur. J. Med. Chem. 2020;185:111858. doi: 10.1016/j.ejmech.2019.111858. PubMed DOI

Krajčovičová S., Staňková J., Džubák P., Hajdúch M., Soural M., Urban M. A synthetic approach for the rapid preparation of BODIPY conjugates and their use in imaging of cellular drug uptake and distribution. Chem. Eur. J. 2018;24:4957–4966. doi: 10.1002/chem.201706093. PubMed DOI

Sommerwerk S., Heller L., Kerzig C., Kramell A.E., Csuk R. Rhodamine B conjugates of triterpenoic acids are cytotoxic mitocans even at nanomolar concentrations. Eur. J. Med. Chem. 2017;127:1–9. doi: 10.1016/j.ejmech.2016.12.040. PubMed DOI

Pal A., Ganguly A., Chowdhuri S., Yousuf M., Ghosh A., Barui A.K., Kotcherlakota R., Adhikari S., Banerjee R. Bis-arylidene oxindole-betulinic acid conjugate: A fluorescent cancer cell detector with potent anticancer activity. ACS Med. Chem. Lett. 2015;6:612–616. doi: 10.1021/acsmedchemlett.5b00095. PubMed DOI PMC

Rumlová M., Křížová I., Keprová A., Hadravová R., Doležal M., Strohalmová K., Pichová I., Hájek M., Ruml T. HIV-1 protease-induced apoptosis. Retrovirology. 2014;11:37. doi: 10.1186/1742-4690-11-37. PubMed DOI PMC

Dostálková A., Kaufman F., Křížová I., Kultová A., Strohalmová K., Hadravová R., Ruml T., Rumlová M. Mutations in the basic region of the Mason-Pfizer monkey virus nucleocapsid protein affect reverse transcription, genomic RNA packaging, and the virus assembly site. J. Virol. 2018;92:e00106-18. doi: 10.1128/JVI.00106-18. PubMed DOI PMC

Křížová I., Hadravová R., Štokrová J., Günterová J., Doležal M., Ruml T., Rumlová M., Pichová I. The G-patch domain of Mason-Pfizer monkey virus is a part of reverse transcriptase. J. Virol. 2012;86:1988. doi: 10.1128/JVI.06638-11. PubMed DOI PMC

Strohalmová-Bohmová K., Spiwok V., Lepšík M., Hadravová R., Křížová I., Ulbrich P., Pichová I., Bednárová L., Ruml T., Rumlová M. Role of Mason-Pfizer monkey virus CA-NC spacer peptide-like domain in assembly of immature particles. J. Virol. 2014;88:14148. doi: 10.1128/JVI.02286-14. PubMed DOI PMC

Goud T.V., Tutar A., Biellmann J.-F. Synthesis of 8-heteroatom-substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes (BODIPY) Tetrahedron. 2006;62:5084–5091. doi: 10.1016/j.tet.2006.03.036. DOI

Kim D., Ma D., Kim M., Jung Y., Kim N.H., Lee C., Cho S.W., Park S., Huh Y., Jung J., et al. Fluorescent labeling of protein using blue-emitting 8-amino-BODIPY derivatives. J. Fluoresc. 2017;27:2231–2238. doi: 10.1007/s10895-017-2164-5. PubMed DOI

Chang Y.-T., Alamudi S.H., Satapathy R., Su D. Background-free fluorescent probes for live cell imaging. WO2017078623A1. US Patent. 2017

Qian K., Bori I.D., Chen C.-H., Huang L., Lee K.-H. Anti-AIDS agents 90. Novel C-28 modified bevirimat analogues as potent HIV maturation inhibitors. J. Med. Chem. 2012;55:8128–8136. doi: 10.1021/jm301040s. PubMed DOI PMC

Staudinger H., Meyer J. Über neue organische Phosphorverbindungen III. Phosphinmethylenderivate und Phosphinimine. Helv. Chim. Acta. 1919;2:635–646. doi: 10.1002/hlca.19190020164. DOI

Neises B., Steglich W. Simple method for the esterification of carboxylic acids. Angew. Chem. Int. Ed. 1978;17:522–524. doi: 10.1002/anie.197805221. DOI

Kotsantis P., Silva L.M., Irmscher S., Jones R.M., Folkes L., Gromak N., Petermann E. Increased global transcription activity as a mechanism of replication stress in cancer. Nat. Commun. 2016;7:13087. doi: 10.1038/ncomms13087. PubMed DOI PMC

Fantin V.R., St-Pierre J., Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell. 2006;9:425–434. doi: 10.1016/j.ccr.2006.04.023. PubMed DOI

Zielonka J., Joseph J., Sikora A., Hardy M., Ouari O., Vasquez-Vivar J., Cheng G., Lopez M., Kalyanaraman B. Mitochondria-targeted triphenylphosphonium-based compounds: Syntheses, mechanisms of action, and therapeutic and diagnostic applications. Chem. Rev. 2017;117:10043–10120. doi: 10.1021/acs.chemrev.7b00042. PubMed DOI PMC

Evers M., Poujade C., Soler F., Ribeill Y., James C., Lelievre Y., Gueguen J.C., Reisdorf D., Morize I., Pauwels R., et al. Betulinic acid derivatives: A new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action. J. Med. Chem. 1996;39:1056–1068. doi: 10.1021/jm950670t. PubMed DOI

Kashiwada Y., Hashimoto F., Cosentino L.M., Chen C.H., Garrett P.E., Lee K.H. Betulinic acid and dihydrobetulinic acid derivatives as potent anti-HIV agents. J. Med. Chem. 1996;39:1016–1017. doi: 10.1021/jm950922q. PubMed DOI

Soler F., Poujade C., Evers M., Carry J.C., Henin Y., Bousseau A., Huet T., Pauwels R., DeClercq E., Mayaux J.F., et al. Betulinic acid derivatives: A new class of specific inhibitors of human immunodeficiency virus type 1 entry. J. Med. Chem. 1996;39:1069–1083. doi: 10.1021/jm950669u. PubMed DOI

Li F., Goila-Gaur R., Salzwedel K., Kilgore N.R., Reddick M., Matallana C., Castillo A., Zoumplis D., Martin D.E., Orenstein J.M., et al. PA-457: A potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing. Proc. Natl. Acad. Sci. USA. 2003;100:13555–13560. doi: 10.1073/pnas.2234683100. PubMed DOI PMC

Schur F.K.M., Obr M., Hagen W.J.H., Wan W., Jakobi A.J., Kirkpatrick J.M., Sachse C., Krausslich H.G., Briggs J.A.G. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation. Science. 2016;353:506–508. doi: 10.1126/science.aaf9620. PubMed DOI

Adamson C.S., Sakalian M., Salzwedel K., Freed E.O. Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV-1 maturation inhibitor bevirimat. Retrovirology. 2010;7:1–8. doi: 10.1186/1742-4690-7-36. PubMed DOI PMC

Lu W.X., Salzwedel K., Wang D., Chakravarty S., Freed E.O., Wild C.T., Li F. A single polymorphism in HIV-1 subtype C SP1 is sufficient to confer natural resistance to the maturation inhibitor bevirimat. Antimicrob. Agents Chemother. 2011;55:3324–3329. doi: 10.1128/AAC.01435-10. PubMed DOI PMC

Van Baelen K., Salzwedel K., Rondelez E., Van Eygen V., De Vos S., Verheyen A., Steegen K., Verlinden Y., Allaway G.P., Stuyver L.J. Susceptibility of human immunodeficiency virus type 1 to the maturation inhibitor bevirimat is modulated by baseline polymorphisms in Gag spacer peptide 1. Antimicrob. Agents Chemother. 2009;53:2185–2188. doi: 10.1128/AAC.01650-08. PubMed DOI PMC

Coric P., Turcaud S., Souquet F., Briant L., Gay B., Royer J., Chazal N., Bouaziz S. Synthesis and biological evaluation of a new derivative of bevirimat that targets the Gag CA-SP1 cleavage site. Eur. J. Med. Chem. 2013;62:453–465. doi: 10.1016/j.ejmech.2013.01.013. PubMed DOI

Wang D., Lu W.X., Li F. Pharmacological intervention of HIV-1 maturation. Acta Pharm. Sin. B. 2015;5:493–499. doi: 10.1016/j.apsb.2015.05.004. PubMed DOI PMC

Gu M., Zhao P., Zhang S.Y., Fan S.J., Yang L., Tong Q.C., Ji G., Huan C. Betulinic acid alleviates endoplasmic reticulum stress-mediated nonalcoholic fatty liver disease through activation of farnesoid X receptors in mice. Brit. J. Pharmacol. 2019;176:847–863. doi: 10.1111/bph.14570. PubMed DOI PMC

Ye Y.Q., Zhang T., Yuan H.Q., Li D.F., Lou H.X., Fan P.H. Mitochondria-targeted lupane triterpenoid derivatives and their selective apoptosis-inducing anticancer mechanisms. J. Med. Chem. 2017;60:6353–6363. doi: 10.1021/acs.jmedchem.7b00679. PubMed DOI

Dubinin M.V., Semenova A.A., Ilzorkina A.I., Mikheeva I.B., Yashin V.A., Penkov N.V., Vydrina V.A., Ishmuratov G.Y., Sharapov V.A., Khoroshavina E.I., et al. Effect of betulin and betulonic acid on isolated rat liver mitochondria and liposomes. Biochim. Biophys. Acta-Biomembr. 2020;1862 doi: 10.1016/j.bbamem.2020.183383. PubMed DOI

Dubinin M.V., Semenova A.A., Nedopekina D.A., Davletshin E.V., Spivak A.Y., Belosludtsev K.N. Effect of F16-betulin conjugate on mitochondrial membranes and its role in cell death initiation. Membranes. 2021;11:352. doi: 10.3390/membranes11050352. PubMed DOI PMC

Dubinin M.V., Semenova A.A., Ilzorkina A.I., Penkov N.V., Nedopekina D.A., Sharapov V.A., Khoroshavina E.I., Davletshin E.V., Belosludtseva N.V., Spivak A.Y., et al. Mitochondria-targeted prooxidant effects of betulinic acid conjugated with delocalized lipophilic cation F16. Free Radic. Bio. Med. 2021;168:55–69. doi: 10.1016/j.freeradbiomed.2021.03.036. PubMed DOI

Wang X., Lu X.C., Zhu R.L., Zhang K.X., Li S., Chen Z.J., Li L.X. Betulinic acid induces apoptosis in differentiated PC12 cells via ROS-mediated mitochondrial pathway. Neurochem. Res. 2017;42:1130–1140. doi: 10.1007/s11064-016-2147-y. PubMed DOI

Terpenes and Terpenoids Conjugated with BODIPYs: An Overview of Biological and Chemical Properties

Plant Secondary Metabolites Used for the Treatment of Diseases and Drug Development