Synthesis of erythrodiol C-ring derivatives and their activity against Chlamydia trachomatis.

To develop new potential agents against Chlamydia trachomatis among oleanane type triterpenoids the synthesis, spectral and X-ray analysis as well as antimicrobial screening of C-12 oxygen and nitrogen derivatives of erythrodiol is presented. The reduction of methyl 3ß-acetoxy-12-oxo-oleanoate with LiAlH4 led to isomeric erythrodiol 12ß- and 12α-hydroxy-derivatives, their stereochemistry with respect to the position of hydroxyl-group at C-12 was determined based on the multiplets splitting patterns, the magnitude of the spin-spin interaction, and NOESY interactions. Methyl 3ß-acetoxy-12-oxo-oleanoate was transformed to 12E-hydroxyimino- and 12E-methoxyimino-derivatives by the interaction with NH2OH∙HCl or CH3ONH2∙HCl, respectively. By Beckmann rearrangement with SOCl2 in dioxane 12E-oxime was converted to C-lactame and its following reduction with LiAlH4 in THF or dioxane led to erythrodiol C-azepanone or C-azepane derivatives. The structure 3-O,12-N-bis-acetyl-derivative of C-azepane-erythrodiol was confirmed by the single crystal X-ray analysis. Erythrodiol 12ß-hydroxy- and C-azepane derivatives were found to be lead compounds with significant activity against C. trachomatis with MIC 1.56 and 3.125 µg/mL. Molecular docking was employed to suggest potential binding interaction, the tested compounds are likely to act as Cdu1 protein inhibitors while 12ß-hydroxy-erythrodiol exhibited the highest affinity towards this respective target protein. These results indicated that C-ring oxygen and nitrogen erythrodiol derivatives might be considered for further research in the design of antibacterial agents against Chlamydia trachomatis.

The Introduction of Hydrazone, Hydrazide, or Azepane Moieties to the Triterpenoid Core Enhances an Activity Against M. tuberculosis.

BACKGROUND: Triterpenoids exhibit a wide spectrum of antimicrobial activity. OBJECTIVE: The objective of this study was to synthesize a series of nitrogen derivatives based on lupane, oleanane, and ursane triterpenoids with high antitubercular activity. METHODS: Isonicotinoylhydrazones were prepared via the reaction of 3-oxotriterpenic acids or betulonic aldehyde with isoniazid (INH) in yields of 54-72%. N-Acylation of betulonic or azepanobetulinic acids led to lupane C28 hydrazides and dihydrazides. The derivatives were evaluated for their in vitro antimycobacterial activities against Mycobacterium tuberculosis (MTB) H37RV and single-drug resistance (SDR)-TB in the National Institute of Allergy and Infectious Diseases, USA. Molecular docking was performed to evaluate the possible binding modes of investigated compounds in the active site of Diterpene synthase (Rv3378c). RESULTS: The obtained compounds are represented by C3 or C28 conjugates with hydrazine hydrate or INH. Some compounds demonstrated from high minimum inhibitory concentration (MIC ≤ 10 µg/mL) to excellent (MICs from 0.19 to 1.25 µg/mL) activity against MTB H37RV. Two lupane conjugates with INH were the leading compounds against MTB H37RV and some SDR-strains with MICs ranged from 0.19 to 1.70 µg/mL. Molecular docking of active compounds to diterpene synthase showed that these moieties accommodate the active site of the enzyme. CONCLUSION: It was revealed that the conjugation of lupanes with INH at C3 is more effective than at C28 and the lupane skeleton is preferable among oleanane and ursane types. The replacement of native hexacarbocyclic A ring to seven-member azepane ring is favorably for inhibition of both MTB H37RV and SDR-strains. These data could possibly mean that the antitubercular activity against INH-resistant strains (INH-R) came from both triterpenoid and isoniazid parts of the hybrid molecules. Azepanobetulin showed the highest activity against both INH-R strains in comparison with other triterpenoids and INH. Thus, the introduction of hydrazone, hydrazide (dihydrazide), or azepane moieties into the triterpenoid core is a promising way for the development of new anti-tubercular agents.

Antimycobacterial activity of azepanobetulin and its derivative: In vitro, in vivo, ADMET and docking studies.

The antimycobacterial investigation of azepanobetulin and its amide derivative was performed. Both compounds showed increased in vitro antibacterial activity on the H37Rv MTB strain in aerobic and anaerobic conditions. Basing on differences between MIC and IC50 values a predominant bactericidal effect for amide in contrast to azepanobetulin with a bacteriostatic antibacterial mechanism is defined. Both compounds showed a strong antibacterial effect against resistant MTB strains with amide derivative being slightly more active. Amide derivative also showed a higher antibacterial potency against non-tuberculous mycobacterial strains (M. avium, M. abscessus). Molecular docking studies showed that the inhibition of tuberculosinyl adenosine transferase (Rv3378c) could constitute an antimycobacterial mechanism of action for these triterpenic azepane derivatives. The pharmacokinetic profile was evaluated by ADMET studies and azepanobetulin showing the better results was evaluated by in vivo experiments. This compound has demonstrated a statistically significant antimycobacterial activity compared to control, but inferior to isoniazid. Our findings show that pentacyclic triterpene derivatives holding a seven-membered azepane A-ring are the promising template for the development of new agents with high antibacterial potential against M. tuberculosis H37Rv, non-tuberculous mycobacterial and drug- resistant strains.

Synthesis and cholinesterase inhibiting potential of A-ring azepano- and 3-amino-3,4-seco-triterpenoids.

In this study, a series of A-ring azepano- and 3-amino-3,4-seco-derivatives were synthesized from betulin, oleanolic, ursolic and glycyrrhetinic acids aiming to develop new cholinesterase inhibitors. Azepanobetulin, azepanoerythrodiol and azepanouvaol were modified to give amide and tosyl derivatives, while azepano-anhydrobetulines and azepano-glycyrrhetols were obtained for the first time. Oleanane and ursane type 3-amino-3,4-seco-4(23)-en triterpenic alcohols were synthesized by reducing the corresponding 2-cyano-derivatives accessible from Beckmann type 2 rearrangements. The compounds were screened in colorimetric Ellman's assays to determine their ability to act as inhibitors for the enzymes acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum). While most of these compounds were only moderate inhibitors for AChE, several of them were shown to be inhibitors for BChE acting as mixed-type inhibitors. Azepanobetulin 1, its C28-amide derivatives 7 and 8, azepano-11-deoxo-glycyrrhetol 12 and azepanouvaol 18 held inhibition constants Ki ranging between 0.21 ± 0.06 to 0.68 ± 0.19 µM. Thus, they were approximately 4 to 10 times more active than standard galantamine hydrobromide. For all of the compounds reasonably high docking scores for BChE were obtained being in good agreement with the experimental results from the enzymatic studies. As a result, A-ring azepano-triterpenoids were found to be new scaffolds for the development of BChE inhibitors.

Synthesis and antimycobacterial activity of triterpeni≿ A-ring azepanes.

A series of A-ring azepanones and azepanes derived from betulonic, oleanonic and ursonic acids was synthesized and evaluated for their in vitro antimycobacterial activities against M. tuberculosis (MTB) H37Rv and SDR-TB in the National Institute of Allergy and Infectious Diseases. Triterpenic A-azepano-28-hydroxy-derivatives were synthesized by the reduction with LiAlH4 of triterpenic azepanones available from the Beckmann rearrangement of the corresponding C3-oximes. Modification of azepanes at NH-group and atoms С12, C20, C28 and C29 of triterpenic core led to the derivatives with oxo, epoxy, aminopropyl, oximino and acyl substituents. The primary assay of tested triterpenoids against MTB H37Rv demonstrated their MIC values ranged from 3.125 to >200 µM. Ursane type A-azepano-28-cinnamoates were the most active being 2 and 4 times more efficient than the initial 28-hydroxy-derivative. The follow-up testing revealed A-azepano-28-cinnamoyloxybetulin as a leader compound with MIC 2 and MBC 4 µM against MTB H37Rv and MICs 4, 1 and 1 µM against INH, RIF and OFX resistant strains, respectively. Five oleanane and ursane azepanes pronounced better activity than isoniazid against INH-R1 and rifampicin against INH-R2 strains. This work opens a new direction in the design and synthesis of new antitubercular agents basing on azepanotriterpenoids.

Adipose stromal cells stimulate angiogenesis via promoting progenitor cell differentiation, secretion of angiogenic factors, and enhancing vessel maturation.

Adipose-derived stromal cells (ASCs) are suggested to be potent candidates for cell therapy of ischemic conditions due to their ability to stimulate blood vessel growth. ASCs produce many angiogenic and anti-apoptotic growth factors, and their secretion is significantly enhanced by hypoxia. Utilizing a Matrigel implant model, we showed that hypoxia-treated ASCs stimulated angiogenesis as well as maturation of the newly formed blood vessels in vivo. To elucidate mechanisms of ASC angiogenic action, we used a co-culture model of ASCs with cells isolated from early postnatal hearts (cardiomyocyte fraction, CMF). CMF contained mature cardiomyocytes, endothelial cells, and progenitor cells. On the second day of culture CMF cells formed spontaneously beating colonies with CD31+ capillary-like structures outgrowing from those cell aggregates. However, these vessel-like structures were not stable, and disassembled within next 5 days. Co-culturing of CMF with ASCs resulted in the formation of stable and branched CD31+ vessel-like structures. Using immunomagnetic depletion of CMF from vascular cells as well as incubation of CMF with mitomycin C-treated ASCs, we showed that in co-culture ASCs enhance blood vessel growth not only by production of paracrine-acting factors but also by promoting the endothelial differentiation of cardiac progenitor cells. All these mechanisms of actions could be beneficial for the stimulation of angiogenesis in ischemic tissues by ASCs administration.