Synthesis of N-Heterocyclic Analogues of 28- O Methyl Betulinate, and Their Antibacterial and Antifungal Properties

: The paper presents the results on the one-pot pyridine quaternization using betulinic 28-O -methyl ester ( 1 ) and Tempo + Br 3- cation followed by reduction of the resulting salt ( 2 ) to 1,2,5,6-tetrahydropyridine derivative ( 3 ). The structures of new compounds are confirmed by means of 1D and 2D-NMR spectroscopy, as well as MALDI TOF/TOF spectrometry. The derivatives 2 and 3 are active against S. aureus at the minimum inhibitory concentration (MIC) of 4 μg/mL and 16 μg/mL , correspondingly.


Introduction
N-Heterocyclic compounds represent a large class of organic molecules, many of which are biologically active substances, thus widely applied in medicine [1]. Various nitrogen heterocycles are involved in the numerous biochemical processes occurring in living cells. A perfect example is group B vitamins (B1, B2, B6, folic acid, etc.) and some coenzymes. In studies of N-heterocycles, quaternary ammonium compounds and their reduction products-1,2,5,6-tetrahydropyridines-are of particular interest as they can act as membrane-active substances exhibiting antitumor and antimicrobial properties [2,3].
Currently, the synthesis of compounds that selectively act upon tumor cells or microorganisms is of significant interest. One approach to solve this problem is the creation of molecules that can cause a general loss of structural organization and integrity of the cytoplasmic membrane in bacteria. Moreover, an important factor is the ability of the synthesized compounds to selectively bind to nucleic acids and cleave them, as well as to accumulate in the mitochondria of tumor cells and cause their death by cell apoptosis. In world practice, a direction in which natural compounds are used as the initial synthetic matrix owing to their low toxicity and high biocompatibility is widely developed. Among them, lupane triterpenoids are considered to be one of the most promising compounds, as they contain a hydrophobic carbocyclic backbone, which possesses a high affinity to lipid membranes [4,5]. The synthesis of their ammonium derivatives is a promising task for the development of targeted agents against bacterial infections and malignant neoplasms. Usually, the reactions proceed in several stages, involving preparation and isolation of intermediate products-halogen or amino derivatives, the corresponding modification of which provides quaternary ammonium salts [6,7]. The development of new one-pot methods and approaches to the synthesis of target derivatives would not only create effective and quite simple methods for their preparation, but also significantly enrich the range of quaternized ammonium analogues of natural compounds.
In continuation of our research on the synthesis of pyridinium analogue of natural compounds, we prepared a quaternized pyridine derivative of betulinic acid C28-methyl ester (1) via the one-pot method developed by us [8,9], which avoids the additional stage of halogenation and increases the yield of the target product. Thus, in the presence of Tempo + Br3 -cation (Scheme 1), in situ allylic bromination of substrate 1 at the C-30 position of the triterpenoid takes place. Subsequent quaternization provides a pyridine derivative 2 with a yield of 96%. The reaction proceeds under mild conditions, and the method is distinguished by tolerance to the functional groups of the initial natural molecule. It should be noted that the reaction pathway significantly depends on the position of the double bond in the molecule (Scheme 2). Thus, the reaction with an internal double bond goes via electrophilic addition (Path B) [10]. In the case of compound 1, the reaction can go via an electrophilic addition or substitution, but if this path were realized, a brominated product with a pyridine moiety attached to the C-20 quaternary atom would be observed. However, this product was not found. Therefore, we suggest that for lupane-type triterpenes with the isopropenyl group, the radical allylic bromination is realized on the first steps of the reaction (path A). Further, terpene bromide, obtained, in situ, the N-alkylate pyridine molecule. Probably, in our reaction, Tempo + Br3 -acts as a carrier of Br2, although in some cases, it can also work as an oxidizing agent [10]. The radical nature of the process may also be indicated by the use of azobisisobutyronitrile (AIBN) as the initiator in the lupane triterpene bromination by the N-bromosuccinimide (NBS) [6]. Furthermore, to show the possibility of further transformations of pyridine analogue, we synthesized tetrahydropyridine derivative 3 via the reduction of 2 by NaBH4 [11]. The formation of N-heterocycle 3 proceeded in dry methanol for five hours at room temperature with a yield of 85%.
The structures of the synthesized compounds were identified by means of MALDI TOF/TOF mass spectrometry (MS), as well as one-( 1 H and 13 C) and two-dimensional (COSY HH, NOESY, HSQC, HMBC) NMR spectroscopy. Owing to the presence of a pyridinium fragment in the triterpene molecule at the C-30 position, methylene AB protons resonate as two doublets at 5.57 and 5.69 ppm, whereas the signal of the corresponding carbon atom is shifted to 66.4 ppm. Furthermore, the HMBC spectra exhibit cross-peaks of the C-30 atom with the pyridine ring protons in the ortho-position. Moreover, the NOESY spectra show the cross-peaks of the same protons with protons at C-30 and C-29. In compound 3, the C-30 atom resonates at 63.00 ppm. The signals of the double-bond protons H-3` and H-4` are shifted to the upfield to 5.68 ppm and 5.74 ppm, respectively. They correlate with resonance lines of carbon atoms at 125.7 and 125.2 ppm, respectively, in the HSQC spectrum and with signals of protons at C-2' and C-5' atoms in the COSY HH spectrum.
The results of primary screening on the antibacterial and antifungal activity of the initial methyl ester 1, its quaternized salt 2, and the 1,2,5,6-tetrahydropyridine derivative 3 are presented in Table  1. The initial compound 1 showed weak antimicrobial activity against all tested bacterial strains and fungi. The compounds 2 and 3 exhibited antimicrobial activity, inhibiting growth and reproduction of gram-positive Staphylococcus aureus bacteria in the range of 81.7%-88.0%. The compounds 2 and 3 showed moderate inhibitory activity towards the gram-negative bacteria and proved to be inactive against fungi.
The derivatives 2 and 3 were selected for further testing. Following the CLSI [12] recommendations, the minimum inhibitory concentration (MIC) was examined, calculating the lowest concentration at which the complete inhibition of bacteria or fungi was registered ( Table 2). The complete inhibition of growth was revealed at ≤20% of growth (or >80% inhibition), while the exposures were chosen only if the next maximum concentration exhibited complete inhibition of 80%-100%. Colistin and vancomycin were used as positive standards for the bacterial inhibition of gram-negative and gram-positive bacteria, respectively. Fluconazole was used as a positive standard for the inhibition of C. albicans and C. neoformans fungi. The compounds 2 and 3 were active against S. aureus at the MIC of 4 μg/mL and 16 μg/mL, correspondingly. It can be concluded that the introduction of the pyridine fragment while preserving the ester group at the C-28 position and the hydroxyl function at the C-3 atom of betulinic acid provides an increase in the antibacterial effect towards the S. aureus bacteria, which is comparable with the level of inhibitory activity of the comparison drug, Vancomycin (Table 2). Table 1. Percentage growth inhibition of bacterial strains and fungi by compounds 1-3 at a concentration of 32 µg/mL.

Biological Screening
The antimicrobial screening was performed by CO-ADD (The Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (United Kingdom) and The University of Queensland (Australia).

Antimicrobial Assay
The bacteria were cultured in cation-adjusted Mueller-Hinton broth (CAMHB) at 37 °C overnight followed by a series of dilutions, and then added to each well of the compound-containing plates, giving a cell density of 5 × 10 5 CFU/mL and a total volume of 50 mL. All the plates were covered and incubated at 37 °C for 18 h without shaking.

Antifungal Assay
Fungi strains were cultured for three days on yeast extract-peptone dextrose (YPD) agar at 30 °C. A yeast suspension of 1 × 10 6 to 5 × 10 6 CFU/mL (as determined by OD530) was prepared from five colonies. The suspension was subsequently diluted and added to each well of the compoundcontaining plates, giving a final cell density of fungi suspension of 2.5 × 10 3 CFU/mL and a total volume of 50 mL. All plates were covered and incubated at 35 °C for 24 h without shaking.
The inhibition of bacterial growth was determined by the measuring of absorbance at 600 nm (OD600), using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well, using negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. The significance of the inhibition values was determined by modified Z-scores, calculated using the median and MAD of the samples (no controls) on the same plate. Samples with an inhibition value above 80% and a Z-score above 2.5 for either replicate (n = 2 on different plates) were classed as active. Samples with inhibition values between 50% and 80% and a Z-score above 2.5 for either replicate (n = 2 on different plates) were classed as partially active.

Hit Confirmation
The percentage of growth inhibition was calculated for each well, using negative control (media only) and positive control (bacteria without inhibitors) on the same plate. The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by inhibition of ≥80%. Also, the maximal percentage of growth inhibition is reported as DMax, indicating any compounds with partial activity. Hits were classified by MIC ≤16 μg/mL in either replicate (n = 2 on different plates).

Conclusions
Thus, we demonstrated an example of pyridine quaternization using betulinic acid 28-O-methyl ester and Tempo + Br3 -cation, followed by reduction of the resulting salt to 1,2,5,6-tetrahydropyridine derivative. The prepared quaternized and reduced pyridine derivatives of betulinic acid exhibited high antibacterial activity against gram-positive Staphylococcus aureus bacteria compared with the initial compound.