Cascade Transformation of the Ansamycin Benzoquinone Core into Benzoxazole Influencing Anticancer Activity and Selectivity.

The metal-free cascade transformation of geldanamycin benzoquinone core is proposed at relatively mild conditions. This approach yields new benzoxazole ansamycin antibiotics and enables their functionalization in an atom-economic manner, irrespective of the type of amine used. The analysis of the heterocyclization course reveals the dependence of its rate on the nature of the para-substituent within the benzylamine moiety (EDG/EWG) and the strength of the base. The reduction of the ansamycin core enables an increase in anticancer potency and selectivity.

Regioselective approach to colchiceine tropolone ring functionalization at C(9) and C(10) yielding new anticancer hybrid derivatives containing heterocyclic structural motifs.

The influence of base type, temperature, and solvent on regioselective C(9)/C(10) "click" modifications within the tropolone ring of colchiceine (2) is investigated. New ether derivatives of 2, bearing alkyne, azide, vinyl, or halide aryl groups enable assembly of the alkaloid part with heterocycles or important biomolecules such as saccharides, geldanamycin or AZT into hybrid scaffolds by dipolar cycloaddition (CuAAC) or Heck reaction. Compared to colchicine (1) or colchiceine (2), ether congeners, as e.g. 3e [IC50s(3e) ∼ 0.9 nM], show improved or similar anticancer effects, whereby the bulkiness of the substituents and the substitution pattern of the tropolone proved to be essential. Biological studies reveal that expanding the ether arms by terminal basic heterocycles as quinoline or pyridine, decreases the toxicity in HDF cells at high anticancer potency (IC50s ∼ 1-2 nM). Docking of ether and hybrid derivatives into the colchicine pocket of αGTP/ß tubulin dimers reveals a relationship between the favourable binding mode and the attractive anticancer potency.

An Approach to Modify 14-Membered Lactone Macrolide Antibiotic Scaffolds.

A ketolide derivative with (12R)-configuration was obtained via a novel ketene acetal in acidic conditions. The structure of this atypical ß-keto ketene acetal intermediate within the macrocyclic system has been determined by NMR and X-ray methods. The use of basic conditions at an elevated temperature yielded new, doubly α,ß-unsaturated ketone macrolide derivatives with (4E)-configuration as two conformational isomers of folded-in or folded-out conformations.

Anticancer activity and toxicity of new quaternary ammonium geldanamycin derivative salts and their mixtures with potentiators.

Geldanamycin (GDM) has been modified by different type neutral/acidic/basic substituents (1-7) and by quinuclidine motif (8), transformed into ammonium salts (9-13) at C(17). These compounds have been characterised by spectroscopic and x-ray methods. Derivative 8 shows better potency than GDM in MCF-7, MDA-MB-231, A549 and HeLa (IC50s = 0.09-1.06 µM). Transformation of 8 into salts 9-13 reduces toxicity (by 11-fold) at attractive potency, e.g. MCF-7 cell line (IC50∼2 µM). Our studies show that higher water solubility contributes to lower toxicity of salts than GDM in healthy CCD39Lu and HDF cells. The use of 13 mixtures with potentiators PEI and DOX enhanced anticancer effects from IC50∼2 µM to IC50∼0.5 µM in SKBR-3, SKOV-3, and PC-3 cancer cells, relative to 13. Docking studies showed that complexes between quinuclidine-bearing 8-13 and Hsp90 are stabilised by extra hydrophobic interactions between the C(17)-arms and K58 or Y61 of Hsp90.

Synthesis, structure and anticancer activity of new geldanamycin amine analogs containing C(17)- or C(20)- flexible and rigid arms as well as closed or open ansa-bridges.

The nucleophilic attack of amines at C(17) or C(17)/C(20) positions of geldanamycin's (GDM) benzoquinone, via initial 1,4-Michael conjugate addition mechanism, yield new analogs with closed or open ansa-bridges (1-31), respectively. X-ray structures of analogs 22 and 24 reveals an unexpected arrangement of the ansa-bridge in solid (conformer B), that is located between those of conformers A, prevailing in solution (trans-lactam), and C, crucial at binding to Hsp90 (cis-lactam). The structure of a new-type conformer B allows to better understand the molecular recognition mechanism between the GDM analogs and the target Hsp90. Combined analysis of: anticancer test results (SKBR-3, SKOV-3, PC-3, U-87, A-549) and those performed in normal cells (HDF), KD values and docking modes at Hsp90 as well as clogP parameters, reveals that the rigid C(17)-arm (piperidyl, cyclohexyl) with a H-bond acceptor as carbonyl group together with a lipophilicity clogP∼3 favor high potency of analogs, even up to IC50 ∼0.08 µM, at improved selectivity (SIHDF > 30), when compared to GDM. The most active 25 show higher anticancer potency than 17-AAG (in SKOV-3 and A-549) as well as reblastatin (in SKBR-3 and SKOV-3). Opening of the ansa-bridge within GDM analogs, at the best case, decreases activity (IC50∼2 µM) and toxicity in HDF cells (SIHDF∼2-3), relative to GDM.

Specific Interactions between Rifamycin Antibiotics and Water Influencing Ability To Overcome Natural Cell Barriers and the Range of Antibacterial Potency.

Rifamycins are a group of macrocyclic antibiotics mainly used for the treatment of various bacterial infections including tuberculosis. Spectroscopic studies of rifamycins evidence the formation of temperature- and solvent-dependent equilibria between A-, B-, and C-type conformers in solutions. The B- and C-type conformers of rifamycin antibiotics are exclusively formed in the presence of water molecules. A- and B-type conformers exhibit a hydrophilic and "open" ansa-bridge nature whereas the C-type conformer is more lipophilic due to the presence of a "closed" ansa-bridge structure. The involvement of the lactam moiety of the ansa-bridge in intramolecular H-bonds within rifapentine and rifampicin implicates the formation of a more hydrophilic A-type conformer. In contrast to rifampicin and rifapentine, for rifabutin and rifaximin, the "free" lactam group enhances conformational flexibility of the ansa-bridge, thereby enabling interconversion between A- and C-type conformers. In turn, an equilibrium between A- and C-type conformers for rifamycins improves their adaptation to the changing nature of bacteria cell membranes, especially those of Gram-negative strains and/or to efflux pump systems.

Synthesis, docking and antibacterial studies of more potent amine and hydrazone rifamycin congeners than rifampicin.

New rifamycin congeners (1-33) with incorporated amine and hydrazone substituents leading to lipophilic and/or basic nature and altered rigidity of modified C(3) arm were synthesized and structurally characterized in detail. NMR spectroscopic studies at different temperatures indicate two types of structures of rifamycin congeners that are realized in solution: zwitterionic and non-ionic forms in dependence of the basicity of modified C(3) arm. The presence of rifamycin congeners in these two possible forms has a significant impact on the physico-chemical parameters such as lipophilicity (clogP) and water solubility and different binding mode of the C(3) arm of antibiotic at RNAP binding pocket (molecular target) leading to different antibacterial potency. The highest antibacterial potency against S. aureus (including MRSA and MLSB strains) and S. epidermidis strains, even higher than reference rifampicin (Rif) and rifaximin (Rifx) antibiotics, was found for rifamycin congeners bearing at the C(3) arm relatively rigid and basic substituents (bipiperidine and guanidine groups). These modifications provide favorable docking mode and excellent water solubility resulting in high potency (MICs 0.0078 µg/mL what gives ∼ 8.5 nM), irrespective whether rifamycin congener is a tertiary amine (15) or hydrazone (29). In turn, for a higher antibacterial potency of rifamycin congeners against E. faecalis strain (MICs 0.5 µg/mL that is 0.6 µM) as compared to Rif and Rifx, the most crucial factors are: bulkiness and the lipophilic character of the end of the C(3) rebuilt arm.

The mechanism of activation of amidobenzylidene ruthenium chelates - latent catalysts of olefin metathesis.

Amidobenzylidene ruthenium chelates - latent catalysts of olefin metathesis can be easily activated by the addition of Brønsted or Lewis acids. Their activation in the presence of hydrogen chloride involves the formation of catalytically active trans-dichloro carbamatobenzylidene ruthenium chelates.

Synthesis, Antibacterial, and Anticancer Evaluation of Novel Spiramycin-Like Conjugates Containing C(5) Triazole Arm.

Huisgen cycloaddition allowed obtaining of novel triazole-bridged antibiotics (6-16) with the reconstructed C(5) arm of spiramycin. (1)H-(1)H NOESY couplings indicated the structure of novel derivatives in solution and demonstrated that the rebuilt C(5) arm is slightly differently oriented relative to the aglycone part if compared to that of spiramycin (1). Combined analysis of biological data together with experimentally determined lipophilicity (clogP) and solubility show the importance of the chemical nature of the newly introduced triazole C(5) arm in the presence of attractive antibacterial and anticancer potency. The most cytotoxic active triazole conjugates having a hydrophobic and bulky C(5) arm showed higher selectivity toward cancer cell lines (HeLa, KB, MCF-7, Hep-G2, and U87) relative to HDF normal cells than that of the parent spiramycin. Our studies have demonstrated that the aldehyde group is not crucial for the presence of interesting antibacterial [MIC(S. pneumoniae) ∼ 1.2 µM] and anticancer [IC50(HepG2) ∼ 6 µM] properties of 16-membered lactone macrolides based on spiramycin's aglycone.

16-Membered Macrolide Lactone Derivatives Bearing a Triazole-Functionalized Arm at the Aglycone C13 Position as Antibacterial and Anticancer Agents.

A series of new C13-triazole-bridged and C13-ether leucomycin analogues with a reduced aldehyde group were synthesized. Derivatives with the highest antibacterial [MIC values (S. epidermidis, S. pneumoniae): ∼2-4 µg mL(-1) ; 2.55-5.09 µm] and cytotoxic [IC50 values (HeLa, KB, MCF-7, A549, HepG2 cells): ∼1.35-3.70 µm] potencies were those with the best aqueous solubility and bearing a saccharide-triazole arm at the C13 position of the aglycone. These derivatives preferentially bind at the ribosomal tunnel and show the most attractive selectivity indexes [SI; calculated relative to the human dermal fibroblast (HDF) cell line], even higher than that of the reference compound cytarabine. Results of molecular docking studies of this type of macrolide antibiotics at the ribosomal tunnel, together with experimentally determined lipophilicity and aqueous solubility values, as well as biological assay data revealed the importance of the introduced functional group at the aglycone C13 arm to the future design of anticancer and antibacterial drug candidates. Our results clearly indicate that the high antibacterial and anticancer activities of these types of macrolides do not necessarily depend on the presence of the aldehyde group at the aglycone lactone ring.

Synthesis, structure and antimicrobial evaluation of a new gossypol triazole conjugates functionalized with aliphatic chains and benzyloxy groups.

Synthetic limitations in the copper-catalyzed azide alkyne cycloaddition (CuAAC) on gossypol's skeleton functionalized with alkyne (2) or azide (3) groups have been indicated. Modified approach to the synthesis of new gossypol-triazole conjugates yielded new compounds (24-31) being potential fungicides. Spectroscopic studies of triazole conjugates 24-31 have revealed their structures in solution, i.e., the presence of enamine-enamine tautomeric forms and π-π stacking intramolecular interactions between triazole arms. Biological evaluation of the new gossypol-triazole conjugates revealed the potency of 30 and 31 derivatives, having triazole-benzyloxy moieties, comparable with that of miconazole against Fusarium oxysporum. The results of HPLC evaluation of ergosterol content in different fungi strains upon treatment of gossypol and its derivatives enabled to propose a mechanism of antifungal activity of these compounds.

Structure-activity relationship studies of new rifamycins containing l-amino acid esters as inhibitors of bacterial RNA polymerases.

New rifamycins (1-12) combined with different l-amino acids, containing methyl, ethyl, tert-butyl and benzyl groups at the ester part, via amine linkage, were synthesized and their structures in solution were determined by spectroscopic FT-IR and 1D and 2D NMR methods as well as visualized by DFT calculations. Two types of rifamycin structures were detected in solution: a zwitterionic one with the transferred proton from O(8)H phenol to secondary N(38) atom and a pseudocyclic structure stabilized via formation of intramolecular H-bond within the protonated basic C(3)-substituent. The presence of these rifamycins' structures influenced physico-chemical (logP, solubility) parameters and antibacterial properties. The bulkiness at the ester substituent of new rifamycins containing aromatic l-amino acids was found to be an important factor, besides the solubility, to achieve relatively high antibacterial activity against reference S. epidermidis and reference S. aureus and MRSA strains (MICs 0.016-0.063 µg/mL), comparable to that of rifampicin. SAR for the novel derivatives was discussed in view of the calculated structures of rifamycin-RNAP complexes.

The effect of complexation of 3-formylrifamycin SV macrocyclic ether derivatives with metal cations and small nitrogen-containing organic molecules on antibacterial activity against S. aureus and S. epidermidis.

Spectroscopic studies of ether rifamycins (1-9) have shown that all these compounds tend to be zwitterions with different localizations of intramolecularly transferred proton, which influences their solubility and logP values. According to ESI MS studies, rifamycins 3 and 4 form complexes with Li(+) or Na(+), while the other ones (7-9) coordinate small organic molecules, which can be further replaced by Na(+) cation. Biological assays revealed that the use of 7-9 in the form of complexes with small organic molecules improves their antibacterial potency as a result of changed: logP, solubility and binding mode with bacterial RNA polymerases.

Regio- and Stereoselective Functionalization of 16-Membered Lactone Aglycone of Spiramycin via Cascade Strategy.

Functionalization of 16-membered aglycone of spiramycin with the use of intramolecular cascade strategy yielded access to novel types of diastereopure bicyclic spiramycin derivatives containing tetrahydrofuran ring. Experimental results shows that a specific sequence of regio- and stereoselective transformations, based on the intramolecular transesterification, E1cB tandem eliminations, 1,2-addition to carbonyl, and 1,6-conjugate addition at the spiramycin aglycone, proceeds with the inversion of absolute configuration at C(5) stereogenic center. Performed cascade and multistep transformations have opened new possibilities in divergent modifications, not only spiramycin but also the whole family of leucomycin type antibiotics having a similar structure of 16-membered aglycone lactone ring.

Regioselective long-range proton transfer in new rifamycin antibiotics: a process in which crown ethers act as stronger Brønsted bases than amines.

Water-mediated proton transfer in six new derivatives of 3-formylrifamycin SV that contain crown, aza-crown, and benzo-crown ether rings were investigated by FTIR and NMR spectroscopy. (1)H-(1)H COSY couplings provide evidence for the formation of zwitterionic structures of the aza-crown and crown ether derivatives of rifamycin, in which a proton from one of the phenolic groups is transferred to tertiary and secondary nitrogen atoms. The increased intensity of the continuous absorption in the mid-infrared region together with the NMR data indicate proton transfer from the phenol group of the rifamycin core to the cavity of the benzo-crown ether ring. This proton transfer is achieved by formation of hydronium (H3O(+)) or Zundel ions (H5O2(+)), which form intermolecular hydrogen bonds with the oxygen atoms of the crown ether. DFT calculations are in agreement with the spectroscopic data and allow visualization of the structures of all new rifamycin derivatives, characterized by different intramolecular protonation sites.

Structure and evaluation of antibacterial and antitubercular properties of new basic and heterocyclic 3-formylrifamycin SV derivatives obtained via 'click chemistry' approach.

Thirty four novel derivatives of 3-formylrifamycin SV were synthesized via reductive alkylation and copper(I)-catalysed azide-alkyne cycloaddition. According to the obtained results, 'click chemistry' can be successfully applied for modification of structurally complex antibiotics such as rifamycins, with the formation of desired 1,2,3-triazole products. However, when azide-alkyne cycloaddition on 3-formylrifamycin SV derivatives demanded higher amount of catalyst, lower temperature and longer reaction time because of the high volatility of substrates, an unexpected intramolecular condensation with the formation of 3,4-dihydrobenzo[g]quinazoline heterocyclic system took place. Structures of new derivatives in solution were determined using one- and two-dimensional NMR methods and FT-IR spectroscopy. Computational DFT and PM6 methods were employed to correlate their conformation and acid-base properties to biological activity and establish SAR of the novel compounds. Microbiological, physico-chemical (logP, solubility) and structural studies of newly synthesised rifamycins indicated that for the presence of relatively high antibacterial (MIC ~0.01 nmol/mL) and antitubercular (MIC ~0.006 nmol/mL) activities, a rigid and basic substituent at C(3) arm, containing a protonated nitrogen atom "open" toward intermolecular interactions, is required.

13C and 15N CP/MAS, 1H-15N SCT CP/MAS and FTIR spectroscopy as tools for qualitative detection of the presence of zwitterionic and non-ionic forms of ansa-macrolide 3-formylrifamycin SV and its derivatives in solid state.

(13)C, (15)N CP/MAS, including (1)H-(13)C and (1)H-(15)N short contact time CP/MAS experiments, and FTIR methods were applied for detailed structural characterization of ansa-macrolides as 3-formylrifamycin SV (1) and its derivatives (2-6) in crystal and in powder forms. Although HPLC chromatograms for 2/CH3 OH and 2/CH3 CCl3 were the same for rifampicin crystals dissolved in respective solvents, the UV-vis data recorded for them were different in 300-375 nm region. Detailed solid state (13)C and (15)N CP/MAS NMR and FTIR studies revealed that rifampicin (2), in contrast to 3-formylrifamycin SV (1) and its amino derivatives (3-6), can occur in pure non-ionic or zwitterionic forms in crystal and in pure these forms or a mixture of them in a powder. Multinuclear CP/MAS and FTIR studies demonstrated also that 3-6 derivatives were present exclusively in pure zwitterionic forms, both in powder and in crystal. On the basis of the solid state NMR and FTIR studies, two conformers of 3-formylrifamycin SV were detected in powder form due to the different orientations of carbonyl group of amide moiety. The PM6 molecular modeling at the semi-empirical level of theory, allowed visualization the most energetically favorable non-ionic and zwitterionic forms of 1-6 antibiotics, strongly stabilized via intramolecular H-bonds. FTIR studies indicated that the originally adopted forms of these type antibiotics in crystal or in powder are stable in standard laboratory conditions in time. The results presented point to the fact that because of a possible presence of two forms of rifampicin (compound 2), quantification of the content of this antibiotic in relevant pharmaceuticals needs caution.

A new model of binding of rifampicin and its amino analogues as zwitterions to bacterial RNA polymerase.

Seven new benzyl (3-9) and four new phenethyl (10-13) amino analogues of ansa-macrolide rifampicin (1) were synthesized using the optimised method of reductive amination. Structures of 3-13 in solution were determined by 1D and 2D NMR and FT-IR methods whereas the energetically most favoured conformation of amino analogues was calculated with the use of the PM5 method. Spectroscopic and semi-empirical studies revealed the presence of zwitterionic forms of all 3-13 analogues in solutions containing water traces. (1)H-(15)N HSQC and (1)H-(15)N HMBC in combination with (1)H-(1)H COSY and (1)H-(13)C HMBC two dimensional spectroscopic methods unambiguously evidenced that the presence of the zwitterionic form of ansa-macrolides was a consequence of proton transfer from the O(8)-H phenolic group to the secondary amine moiety within 3-13 structures. (1)H-(1)H NOESY studies indicated two different orientations of the substituent introduced at the C(3) position for benzyl and phenethyl amino analogues of rifampicin and their similar conformation within the ansa-bridges in solution. FT-IR studies of the deprotonation of molecule and comparison of these data with those for indicated 3-13 C(8)=O double bond character after formation of zwitterions in solution. Results of an antibacterial test against Gram-(-) and Gram-(+) strains were compared with detailed structural information on new analogues of 3-13 to indicate some structure-activity relationships. Molecular recognition studies of 1 and 12 inhibitors at the binding site of bacterial RNA polymerase (RNAP) as zwitterions revealed key intermolecular interactions and led to the proposition of a new model of RNAP inhibition, which explains significant differences in antibacterial properties of rifampicin and its analogues.

Redetermination of rifampicin pentahydrate revealing a zwitterionic form of the antibiotic.

Rifampicin belongs to the family of naphthalenic ansamycin antibiotics. The first crystal structure of rifampicin in the form of the pentahydrate was reported in 1975 [Gadret, Goursolle, Leger & Colleter (1975). Acta Cryst. B 31, 1454-1462] with the rifampicin molecule assumed to be neutral. Redetermination of this crystal structure now shows that one of the phenol -OH groups is deprotonated, with the proton transferred to a piperazine N atom, confirming earlier spectroscopic results that indicated a zwitterionic form for the molecule, namely (2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)-21-acetyloxy-6,9,17,19-tetrahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-[(E)-N-(4-methylpiperazin-4-ium-1-yl)formimidoyl]-1,11-dioxo-1,2-dihydro-2,7-(epoxypentadeca[1,11,13]trienimino)naphtho[2,1-b]furan-5-olate pentahydrate, C(43)H(58)N(4)O(12)·5H(2)O. The molecular structure of this antibiotic is stabilized by a system of four intramolecular O-H···O and N-H···N hydrogen bonds. Four of the symmetry-independent water molecules are arranged via hydrogen bonds into helical chains extending along [100], whereas the fifth water molecule forms only one hydrogen bond, to the amide group O atom. The rifampicin molecules interact via O-H···O hydrogen bonds, generating chains along [001]. Rifampicin pentahydrate is isostructural with recently reported rifampicin trihydrate methanol disolvate.

Intramolecular proton transfer impact on antibacterial properties of ansamycin antibiotic rifampicin and its new amino analogues.

Intramolecular proton transfer in rifampicin (1) and its analogues 2-9 with the formation of zwitterions has been indicated by multinuclear NMR and crystallographic studies. Biological tests of 1-9 in combination with the analysis of ligand-protein interactions have revealed the relationship between the protonation site and extremely high antibacterial activity.