Synthesis of fusidane triterpenoid Mannich bases as potential antibacterial and antitumor agents

Abstract Mannich bases (8 examples) were synthesized via aminomethylation of fusidane propargyl esters. In vitro antimicrobial screening against key ESKAPE pathogens showed that the fusidic acid based Mannich products exhibit a high antimicrobial effect against Gram-positive bacteria Staphylococcus aureus and the fungus Cryptococcus neoformans. Moreover, the cytotoxic effect of fusidic acid and its analogs, which showed high antibacterial activity, was determined by MTT assay on cancer HepG2, HCT-116, SH-SY5Y, MCF-7, A549 and conditionally normal cells HEK293. A remarkable cytotoxic activity of fusidic acid propargyl ester and its aminomethylene derivatives against cancer and nontumoral HEK293 cells with IC50 values within 4.2–25 µM was found. GRAPHICAL ABSTRACT


Introduction
Natural compounds, as a source of therapeutic agents, have been used to treat a wide range of ailments for thousands of years (Romano and Tatonetti 2019), especially cancers and infectious diseases (Harvey et al. 2015;Melander et al. 2020).Due to unique physical and chemical properties, significant structural diversity and wide spectrum of pharmacological activity natural products and their derivatives accounted for 32% of all small molecule drugs approved from January 1981 to September 2019 (Newman and Cragg 2020).Currently, they are still considered as promising molecular targets for drug development research owing to the discovery of new potential therapeutic possibilities (Huang and Zhang 2022).Among natural compounds, fusidane terpenoids belonging to a small group of 29-nor protostane triterpenes are of particular interest.They are produced by various types of fungi (Hanson 2008), and generally exhibit antimicrobial properties.In the series of fusidanes, a special place is occupied by fusidic acid (FA), which is used in clinical practice as an antibiotic, both in systemic and local therapy of staphylococcal infections (Zhao et al. 2013).Since the discovery of FA, many of its derivatives showing a variety of biological activities have been obtained.For example, FA analogs containing terminal amino groups exhibited an antitumor effect and the chemosensitizing activity towards the cancer cell lines as well (Guo et al. 2019;Ni et al. 2019).A number of esters and amides of fusidane triterpenoids were synthesized, which demonstrated an antiplasmoidal effect (Kaur et al. 2015;Espinoza-Moraga et al. 2017;Kaur et al. 2018).Moreover, FA analogs were obtained that had antimycobacterial (Dziwornu et al. 2019;Njoroge et al. 2019;Singh et al. 2020;Strydom et al. 2020), anti-inflammatory (Wu et al. 2018), fungicidal activity (Cao et al. 2020), as well as a number of derivatives that exhibited an antibacterial effect comparable to that of FA and have a longer period of resistance development (Garcia Chavez et al. 2021).
The classical Mannich reaction is a three-component condensation of structurally distinct substrates containing at least one active hydrogen atom (X-H), an aldehyde moiety, and an amine reactant, which provides a set of compounds known as Mannich bases (Mannich and Kr€ osche 1912;Blicke 2011).Numerous Mannich bases possess biological activity, for example, they exhibit antitumor (Csuk et al. 2013;Roman 2015), antibacterial (Tamilvendan et al. 2012;Savaliya et al. 2013), antimycobacterial (Das et al. 2010;Ji et al. 2011), antifungal (Gul et al. 2002;Rossignol et al. 2013), antimalarial (Li et al. 2003;Singh et al. 2011;Roy et al. 2013), antiviral (Mazzei et al. 2008), anticonvulsant (Gul et al. 2004;Byrtus et al. 2011;Kami nski et al. 2013) properties, as well as other activities (Roman 2015).It should be noted that the copper-catalyzed reaction of terminal alkynes as CH-acids with aldehydes and amines (A 3 reaction) provides propargylamines.This reaction is widely used in chemistry and pharmacology for the synthesis of new materials and compound libraries for high-throughput drug screening (Volkova et al. 2021).Taking into account these data, in the framework of our research on the synthesis of biologically active fusidanes (Salimova et al. 2018(Salimova et al. , 2019(Salimova et al. , 2020;;Salimova and Parfenova 2021) we obtained a series of new aminopropargyl analogs of FA and screened the antibacterial and antitumor activity of the synthesized compounds.

Chemistry
The alkynyl fragment was introduced into the FA (1) molecule by propargylation of the carboxyl group by the reaction of the latter with 3-bromoprop-1-yne in dry acetone at reflux in the presence of K 2 CO 3 .The resulting propargyl ester FA 2 was oxidized by CrO 3 in glacial acetic acid, as a result of which the 3,11-dioxo derivative of propargylfusidate 3 was isolated in 90% yield (Scheme 1) (Salimova and Parfenova 2022).
The acetylene fragment introduced into the triterpene caused a shift of the carboxyl carbon atom signal in the 13 C NMR spectrum to a highfield, compared to FA.Moreover, two signals of triple bond carbons at d 74.9 and 77.6 ppm appeared in the spectrum. 1NMR spectrum of compound 2 exhibited two doublets at 4.60 and 4. 71 ppm corresponded to methylene protons of the propargyl group.
Compound 2 was reacted with secondary amines (morpholine, pyrrolidine, N-methylpiperazine and piperidine) and formaldehyde under Mannich reaction conditions catalyzed by CuCl.As a result of aminomethylation, Mannich bases 4-7 were isolated in 75-85% yields (Scheme 2).In the 1 H NMR spectra of the obtained compounds, a broadened singlet appeared in the range of 3.21-3.44ppm was assigned to the CH 2group of the aminopropargyl function.The signal of 3' atom in the 13 NMR spectra of compounds 4-7 shifted to a lowfield (d ¼ 81.5-82.5 ppm) compared to the starting compound (d ¼ 77.6 ppm) and became as a resonance line of quaternary atom (according to DEPT spectra).As follows from the NOESY spectra, under the conditions of the aminomethylation reaction the configuration of the C 3 atom of ring A changes from a to b.
During the primary screening, the antimicrobial activity of compounds 2, 4-7 against S. aureus (MRSA) was found, while analogs 8-11 containing keto-groups was inactive.Mannich bases 4-7 additionally inhibited the growth of the fungal culture C. neoformans (Table S1).Minimum inhibitory concentrations (MIC) were determined for the active compounds and cytotoxic effect was studied using the human embryonic kidney cell line HEK293 ( 50 ).
It was found that Mannich bases 4, 6, and 7 showed high antibacterial activity, inhibiting the growth and reproduction of more than 90% of Gram-positive bacteria S. aureus (MRSA) at a concentration of 0.37-0.38mM, which is comparable to the activity of the native antibiotic fusidic acid.Derivative 6, which contained an N-methylpiperazine fragment in its structure, had the highest activity.The results of antimicrobial screening also showed that Mannich bases 4-7 exhibited a high fungicidal activity that was not found in FA.The highest antifungal activity was exhibited by compound 4 containing a morpholine fragment.The compound inhibited the growth of C. neoformans at a concentration of 0.38 mM.Nevertheless, biological tests revealed the high cytotoxicity of the FA aminomethylene derivatives.The highest toxicity against conditionally normal human cells HEK293 was shown by compound 6, which caused the complete death of the cell line at a concentration of 0.37 mM (Table S2).The obtained data on the cytotoxicity of the synthesized compounds suggested that compounds 2, 4-7 may exhibit an anticancer effect.Based on this conclusion, we studied the cytotoxic properties of FA Mannich bases towards the cell lines of tumor origin.

Cytotoxic activity
In the follow-up experiments, concentration-dependent cytotoxic activity of the FA 1, its propargyl ester 2, as well as Mannich bases 4-7 was evaluated in the five cancer cell lines (HepG2, HCT-116, SH-SY5Y, MCF-7, A549), as well as in the conditionally normal cells (HEK293) by MTT assay.Compounds 2, 4-7 were found to possess the potent cytotoxic activity against cancer and non-tumoral HEK293 cell lines with IC 50 values within 4.2 -25 mM, indicating no selectivity towards malignant cells.As follows from Table S3, compounds could be arranged according to their ability to inhibit cell survival in the following order: 5 > 6 > 7 > 4 > 2. Among the synthesized compounds, FA propargyl ether derivative with pyrrolidinemethylene substituent 5 displayed a pronounced cytotoxicity towards HEK293, HepG2, HCT-116 and A549 cells with a mean IC 50 values below 10 mM and close to that of reference compound Fluorouracil.Mannich bases with fragments of morpholine 4, N-methylpiperazine 6 and piperidine 7 were less potent, although still active, with IC 50 values in a range of 6 -19 mM, dependently on cell line.These results highlight the potential of Mannich bases with a pyrrolidine substituent in the molecule 5 as a prototype for a therapeutic agent based on its obvious cytotoxic effect.

Experimental
All experimental procedures are described in the supplementary materials.

Conclusion
As a result, fusidic acid Mannich bases (8 examples) were obtained by the Cu-catalyzed reaction of FA propargyl ester with secondary amines (morpholine, pyrrolidine, N-methylpiperazine and piperidine) and formaldehyde.The studies showed that the introduction of propargyl aminomethylene fragments into the molecules provides high antibacterial activity against methicillin-resistant S. aureus, and also pronounced fungicidal and antitumor effect, which is absent in the native antibiotic.The FA derivative containing an N-methylpiperazine fragment had the greatest antimicrobial effect, inhibiting the reproduction of S. aureus bacteria (MRSA) at MIC 0.37 mM, which is comparable to the activity of the original antibiotic.The Mannich base containing a morpholine substituent exhibited the highest fungicidal activity, causing complete death of the C. neoformans fungal culture at MIC 0.38 mM.In vitro cytotoxic assay have demonstrated the ability of FA propargyl ester and Mannich bases to inhibit cell viability of human cancer cells HepG2, HCT-116, SH-SY5Y, MCF-7, A549 and confirmed cytotoxic properties against HEK293 cells.