CYP1-Activation and Anticancer Properties of Synthetic Methoxylated Resveratrol Analogues.

Naturally occurring stilbenoids, such as the (E)-stilbenoid resveratrol and the (Z)-stilbenoid combretastatin A4, have been considered as promising lead compounds for the development of anticancer drugs. The antitumour properties of stilbenoids are known to be modulated by cytochrome P450 enzymes CYP1A1 and CYP1B1, which contribute to extrahepatic phase I xenobiotic and drug metabolism. Thirty-four methyl ether analogues of resveratrol were synthesised, and their anticancer properties were assessed, using the MTT cell proliferation assay on a panel of human breast cell lines. Breast tumour cell lines that express CYP1 were significantly more strongly affected by the resveratrol analogues than the cell lines that did not have CYP1 activity. Metabolism studies using isolated CYP1 enzymes provided further evidence that (E)-stilbenoids can be substrates for these enzymes. Structures of metabolic products were confirmed by comparison with synthetic standards and LC-MS co-elution studies. The most promising stilbenoid was (E)-4,3',4',5'-tetramethoxystilbene (DMU212). The compound itself showed low to moderate cytotoxicity, but upon CYP1-catalysed dealkylation, some highly cytotoxic metabolites were formed. Thus, DMU212 selectively affects proliferation of cells that express CYP1 enzymes.

Synthesis and antitubercular activity of novel 4-arylalkyl substituted thio-, oxy- and sulfoxy-quinoline analogues targeting the cytochrome bc1 complex.

A library of 4-substituted quinolines was synthesised based on the structural features of the privileged 4-(benzylthio)-6-methoxy-2-methylquinoline scaffold. Quinoline-based chemical probes have proven to be effective anti-tuberculosis agents with the ability of inhibiting components of Mycobacterium tuberculosis (MTB) respiratory chain including the b subunit of the cytochrome bc1 complex. Novel 4-(arylalkyl)-thio, -oxy and sulfoxy-quinoline analogues were tested for their ability to inhibit the growth of MTB H37Rv and QcrB mutant strains, and the compounds mode of action was investigated. Members of the 4-subtituted thio- and sulfoxyquinoline series exhibited significant growth inhibitory activity in the high nanomolar range against wild-type MTB and induced depletion of intracellular ATP. These probes also showed reduced potency in the QcrB T313I mutant strain, thus indicating the cytochrome bc1 oxidase complex as the molecular target. Interestingly, new 4-(quinolin-2-yl)oxy-quinoline 4i was more selective for the QcrB T313I strain compared to the wild-type strain.

Aminobenzofuran-containing analogues of proximicins exhibit higher antiproliferative activity against human UG-87 glioblastoma cells compared to temozolomide.

A new series of proximicin analogues containing a benzofuran moiety as the replacement of the di-furan scaffold of the parent compound were synthesised and evaluated for their anti-proliferative activities against human glioblastoma cells U-87 MG. Proximicins A, B, and C are secondary metabolites produced by Verrucosispora Fiedleri MG-37, a Gram-positive actinomycete isolated from deep-sea sediment. Proximicins exhibit significant cytotoxic and apoptotic effects in a number of tumour cell lines, although further investigations on these natural products biological activity are hampered by the challenging synthesis of their constitutive di-furan unit. Therefore, the easily-synthesisable benzofuran ring was elected as a replacement of the di-furan platform, and a library of proximicin analogues was prepared in which different substituents were introduced at both the N-terminus and C-terminus of the benzofuran core unit. The novel compounds were tested against U-87 MG, as it was previously found that proximicins targeted this cancerous cell line, and the human healthy cell line WI-38. Temozolomide, the chemotherapeutic agent of choice for the treatment of glioblastoma, was used as a control. Analysis of growth inhibitory concentration values revealed that a number of furan-benzofuran-containing proximicin analogues, including 23(16) (IC50 U-87 MG = 6.54 µg mL-1) exhibited higher antiproliferative activity against glioblastoma cells compared to both proximicins A-C and temozolomide (IC50 U-87 MG = 29.19 µg mL-1) in U-87 MG.

C2-linked alkynyl poly-ethylene glycol(PEG) adenosine conjugates as water-soluble adenosine receptor agonists.

A series of 12 novel polyethylene-glycol(PEG)-alkynyl C2-adenosine(ADN) conjugates were synthesized using a robust Sonogashira coupling protocol and characterized by NMR spectroscopy and mass spectrometry analysis. The ADN-PEG conjugates showed null to moderate toxicity in murine macrophages and 12c was active against Mycobacterium aurum growth (MIC = 62.5 mg/L). The conjugates were not active against Mycobacterium bovis BCG. Conjugates 10b and 11b exhibited high water solubility with solubility values of 1.22 and 1.18 mg/ml, respectively, in phosphate buffer solutions at pH 6.8. Further, 10b and 11b induced a significant increase in cAMP accumulation in RAW264.7 cells comparable with that induced by adenosine. Analogues 10c, 11c and 12c were docked to the A1 , A2A , A2B and A3 adenosine receptors (ARs) using crystal-structures and homology models. ADN-PEG-conjugates bearing chains with up to five ethyleneoxy units could be well accommodated within the binding sites of A1 , A2A and A3 ARs. Docking studies showed that compound 10b and 11b were the best A2A receptor binders of the series, whereas 12c was the best binder for A1 AR. In summary, introduction of hydrophilic PEG substituents at the C2 of adenine ring significantly improved water solubility and did not affect AR binding properties of the ADN-PEG conjugates.

Mycobactin Analogues with Excellent Pharmacokinetic Profile Demonstrate Potent Antitubercular Specific Activity and Exceptional Efflux Pump Inhibition.

In this study, we have designed and synthesized pyrazoline analogues that partially mimic the structure of mycobactin, to address the requirement of novel therapeutics to tackle the emerging global challenge of antimicrobial resistance (AMR). Our investigation resulted in the identification of novel lead compounds 44 and 49 as potential mycobactin biosynthesis inhibitors against mycobacteria. Moreover, candidates efficiently eradicated intracellularly surviving mycobacteria. Thermofluorimetric analysis and molecular dynamics simulations suggested that compounds 44 and 49 bind to salicyl-AMP ligase (MbtA), a key enzyme in the mycobactin biosynthetic pathway. To the best of our knowledge, these are the first rationally designed mycobactin inhibitors to demonstrate an excellent in vivo pharmacokinetic profile. In addition, these compounds also exhibited more potent whole-cell efflux pump inhibition than known efflux pump inhibitors verapamil and chlorpromazine. Results from this study pave the way for the development of 3-(2-hydroxyphenyl)-5-(aryl)-pyrazolines as a new weapon against superbug-associated AMR challenges.

Novel C-3-(N-alkyl-aryl)-aminomethyl rifamycin SV derivatives exhibit activity against rifampicin-resistant Mycobacterium tuberculosis RpoBS522L strain and display a different binding mode at the RNAP ß-subunit site compared to rifampicin.

Antimicrobial resistance is a main concern in tuberculosis treatment and is often associated with the emergence of Mycobacterium tuberculosis strains resistant to rifampicin (RIF), which is one of the cornerstones of tuberculosis chemotherapy. In this study, aminoalkyl-aromatic ring tails were appended to the C3 position of rifamycin core to assess the role of C3 substitutions to the anti-mycobacterial activity of the rifamycin antibiotics. The typical hydrazone unit of RIF was replaced by an amino-alkyl linkage to connect the aromatic ring tails with the rifamycin naphthoquinone core. Eight novel C3-(N-alkyl-aryl)-aminoalkyl analogues of rifamycin SV were synthesised and screened in vitro against wild-type HR37Rv and "hypervirulent" HN-878 strains, and a panel of rifampicin-resistant M. tuberculosis clinical isolates carrying mutations at the 522, 531 and 455 positions of the rpoB gene (RpoBS522L, RpoBS531L and RpoBH455D strains). The analogues exhibited anti-tubercular activity against H37Rv and HN-878 at submicromolar or nanomolar concentrations, and against clinical H37Rv isolates bearing the S522L mutations at low micromolar concentration. Benzylamine moiety-including analogue 8 was as active as rifampicin against HN-878 with a MIC90 value of 0.02 µM, whereas 14 and 15, which included tryptamine and para-methyl-sulfonylbenzylamine C3-substituents, respectively, showed higher anti-tubercular activity (MIC90 = 3 µM) compared to rifampicin against the S522L mutated H37Rv strain. Detailed in silico analysis of different RNAP molecular systems predicted a distinct, possibly novel, binding mode for the new rifamycin analogues. These were found to occupy a different space in the binding pockets of both wild type and mutated RNAP proteins compared to that of rifampicin. Moreover, the molecular modelling experiments investigated the ability of the novel analogues aromatic tails to establish key interactions at the RNAP binding site. These interesting findings might pave the way for generating rifamycin analogues that can overcome anti-microbial resistance in M. tuberculosis.

The Mycobactin Biosynthesis Pathway: A Prospective Therapeutic Target in the Battle against Tuberculosis.

The alarming rise in drug-resistant clinical cases of tuberculosis (TB) has necessitated the rapid development of newer chemotherapeutic agents with novel mechanisms of action. The mycobactin biosynthesis pathway, conserved only among the mycolata family of actinobacteria, a group of intracellularly surviving bacterial pathogens that includes Mycobacterium tuberculosis, generates a salicyl-capped peptide mycobactin under iron-stress conditions in host macrophages to support the iron demands of the pathogen. This in vivo essentiality makes this less explored mycobactin biosynthesis pathway a promising endogenous target for novel lead-compounds discovery. In this Perspective, we have provided an up-to-date account of drug discovery efforts targeting selected enzymes (MbtI, MbtA, MbtM, and PPTase) from the mbt gene cluster (mbtA-mbtN). Furthermore, a succinct discussion on non-specific mycobactin biosynthesis inhibitors and the Trojan horse approach adopted to impair iron metabolism in mycobacteria has also been included in this Perspective.

Synthesis and Biological Evaluation of a Novel C8-Pyrrolobenzodiazepine (PBD) Adenosine Conjugate. A Study on the Role of the PBD Ring in the Biological Activity of PBD-Conjugates.

Here we sought to evaluate the contribution of the PBD unit to the biological activity of PBD-conjugates and, to this end, an adenosine nucleoside was attached to the PBD A-ring C8 position. A convergent approach was successfully adopted for the synthesis of a novel C8-linked pyrrolo(2,1-c)(1,4)benzodiazepine(PBD)-adenosine(ADN) hybrid. The PBD and adenosine (ADN) moieties were synthesized separately and then linked through a pentynyl linker. To our knowledge, this is the first report of a PBD connected to a nucleoside. Surprisingly, the compound showed no cytotoxicity against murine cells and was inactive against Mycobacterium aurum and M. bovis strains and did not bind to guanine-containing DNA sequences, as shown by DNase I footprinting experiments. Molecular dynamics simulations revealed that the PBD-ADN conjugate was poorly accommodated in the DNA minor groove of two DNA sequences containing the AGA-PBD binding motif, with the adenosine moiety of the ligand preventing the covalent binding of the PBD unit to the guanine amino group of the DNA duplex. These interesting findings shed further light on the ability of the substituents attached at the C8 position of PBDs to affect and modulate the biological and biophysical properties of PBD hybrids.

2-furyl(phenyl)methanol isolated from Atractilis gummifera rhizome exhibits anti-leishmanial activity.

We report for the first time the isolation of 2-furyl(phenyl)methanol (5) from the chloroform extracts of the Atractylis gummifera roots. A. gummifera is a thistle belonging to the Asteraceae family that produces the ent-kaurane diterpenoid glycoside atractyloside (ATR). ATR (1) was isolated and chemically modified to obtain its aglycone atractyligenin (2) and the methylated derivatives ATR-OMe (3) and genine-OMe (4). The compounds 1-5 were structurally characterised and evaluated against the intracellular amastigote, cultured within macrophages, and the extracellular promastigote of Leishmania donovani, the protozoan parasite responsible for the highly infective disease visceral leishmaniasis, which is fatal if untreated. The 2-furyl(phenyl)methanol 5 exhibited notable activity against the promastigote.

Integrated Target-Based and Phenotypic Screening Approaches for the Identification of Anti-Tubercular Agents That Bind to the Mycobacterial Adenylating Enzyme MbtA.

Iron is essential for the pathogenicity and virulence of Mycobacterium tuberculosis, which synthesises salicyl-capped siderophores (mycobactins) to acquire this element from the host. MbtA is the adenylating enzyme that catalyses the initial reaction of mycobactin biosynthesis and is solely expressed by mycobacteria. A 3200-member library comprised of lead-like, structurally diverse compounds was screened against M. tuberculosis for whole-cell inhibitory activity. A set of 846 compounds that inhibited the tubercle bacilli growth were then tested for their ability to bind to MbtA using a fluorescence-based thermal shift assay and NMR-based Water-LOGSY and saturation transfer difference (STD) experiments. We identified an attractive hit molecule, 5-hydroxyindol-3-ethylamino-(2-nitro-4-trifluoromethyl)benzene (5), that bound with high affinity to MbtA and produced a MIC90 value of 13 µm. The ligand was docked into the MbtA crystal structure and displayed an excellent fit within the MbtA active pocket, adopting a binding mode different from that of the established MbtA inhibitor Sal-AMS.

Activity of DNA-targeted C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates against aerobically and hypoxically grown Mycobacterium tuberculosis under acidic and neutral conditions.

Mycobacterium tuberculosis (Mtb) is the aetiological agent of tuberculosis, the leading cause of death worldwide from a single infectious agent. Mtb is a highly adaptable human pathogen that might enter a dormant non-replicating (NR), drug-tolerant stage. Reactivation of dormant Mtb can lead to active disease. Antibiotic treatments of active and latent tuberculosis are long, complex and may fail to fully eradicate the infection. Therefore, it is imperative to identify novel compounds with new mechanisms of action active against NR bacilli. Dormant Mtb habitat is mostly thought to be the pH-neutral and hypoxic caseous granuloma. We have used the Wayne culture model to reproduce this environment and tested the activities of two DNA-targeted agents, C8-linked-pyrrolobenzodiazepine(PBD)-polyamide conjugates 1 and 2, against Mtb grown in aerobic and hypoxic conditions in both acidic and pH-neutral media. PBD 2 showed growth inhibitory activity at 5.1 µg/ml against 19-day-old hypoxic NR Mtb cultures with 1.8 log10 CFU reduction on day 21 at pH 7.3. PBD 2 was particularly effective against 5-day-old aerobic cells at pH 7.3, with CFU reduction (>6.8 log10) on day 21 at 5.1 µg/ml being identical to that of rifampin at 8 µg/ml. PBD 2 qualifies as a promising lead against aerobic and NR Mtb.

DNA sequence-selective C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates show anti-tubercular-specific activities.

New chemotherapeutic agents with novel mechanisms of action are in urgent need to combat the tuberculosis pandemic. A library of 12 C8-linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD)-heterocyclic polyamide conjugates (1-12) was evaluated for anti-tubercular activity and DNA sequence selectivity. The PBD conjugates were screened against slow-growing Mycobacterium bovis Bacillus Calmette-Guérin and M. tuberculosis H37Rv, and fast-growing Escherichia coli, Pseudomonas putida and Rhodococcus sp. RHA1 bacteria. DNase I footprinting and DNA thermal denaturation experiments were used to determine the molecules' DNA recognition properties. The PBD conjugates were highly selective for the mycobacterial strains and exhibited significant growth inhibitory activity against the pathogenic M. tuberculosis H37Rv, with compound 4 showing MIC values (MIC=0.08 mg l-1) similar to those of rifampin and isoniazid. DNase I footprinting results showed that the PBD conjugates with three heterocyclic moieties had enhanced sequence selectivity and produced larger footprints, with distinct cleavage patterns compared with the two-heterocyclic chain PBD conjugates. DNA melting experiments indicated a covalent binding of the PBD conjugates to two AT-rich DNA-duplexes containing either a central GGATCC or GTATAC sequence, and showed that the polyamide chains affect the interactions of the molecules with DNA. The PBD-C8 conjugates tested in this study have a remarkable anti-mycobacterial activity and can be further developed as DNA-targeted anti-tubercular drugs.

Synthesis, anti-mycobacterial activity and DNA sequence-selectivity of a library of biaryl-motifs containing polyamides.

The alarming rise of extensively drug-resistant tuberculosis (XDR-TB) strains, compel the development of new molecules with novel modes of action to control this world health emergency. Distamycin analogues containing N-terminal biaryl-motifs 2(1-5)(1-7) were synthesised using a solution-phase approach and evaluated for their anti-mycobacterial activity and DNA-sequence selectivity. Thiophene dimer motif-containing polyamide 2(2,6) exhibited 10-fold higher inhibitory activity against Mycobacterium tuberculosis compared to distamycin and library member 2(5,7) showed high binding affinity for the 5'-ACATAT-3' sequence.

An extended pyrrolobenzodiazepine-polyamide conjugate with selectivity for a DNA sequence containing the ICB2 transcription factor binding site.

The binding of nuclear factor Y (NF-Y) to inverted CCAAT boxes (ICBs) within the promoter region of DNA topoisomerase IIα results in control of cell differentiation and cell cycle progression. Thus, NF-Y inhibitory small molecules could be employed to inhibit the replication of cancer cells. A library of pyrrolobenzodiazepine (PBD) C8-conjugates consisting of one PBD unit attached to tri-heterocyclic polyamide fragments was designed and synthesized. The DNA-binding affinity and sequence selectivity of each compound were evaluated in DNA thermal denaturation and DNase I footprinting assays, and the ability to inhibit binding of NF-Y to ICB1 and ICB2 was studied using an electrophoretic mobility shift assay (EMSA). 3a was found to be a potent inhibitor of NF-Y binding, exhibiting a 10-fold selectivity for an ICB2 site compared to an ICB1-containing sequence, and showing low nanomolar cytotoxicity toward human tumor cell lines. Molecular modeling and computational studies have provided details of the covalent attachment process that leads to formation of the PBD-DNA adduct, and have allowed the preference of 3a for ICB2 to be rationalized.

Structural characterization and antimicrobial evaluation of atractyloside, atractyligenin, and 15-didehydroatractyligenin methyl ester.

We report the first complete structure elucidation of the ent-kaurane diterpenoid glycoside atractyloside (1) by means of NMR and X-ray diffractometry techniques. Extensive one- and two-dimensional NMR experiments were employed to assign the proton and carbon signals of 1, and crystallography experiments established the configurations of all stereogenic centers. Furthermore, we present a novel semisynthetic route for the preparation of the highly cytotoxic aglycone derivative of 1, 15-didehydroatractyligenin methyl ester (3). All compounds were tested for their antibiotic activity against Enterococcus faecalis, Escherichia coli, and several strains of Staphylococcus aureus, including fluoroquinolone-resistant (SA1199B) and two epidemic MRSA (EMRSA-15 and -16) strains. Compound 3 exhibited moderate activity against all of the Staph. aureus strains with an MIC value of 128 mg/L.

Efficient synthesis and biological evaluation of proximicins A, B and C.

A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using cellular methods. Proximicin B induced apoptosis in both Hodgkin's lymphoma and T-cell leukemia cell lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carcinoma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy activity against antibiotic-resistant Gram-positive cocci.

Novel C8-linked pyrrolobenzodiazepine (PBD)-heterocycle conjugates that recognize DNA sequences containing an inverted CCAAT box.

A series of novel DNA-interactive C8-linked pyrrolobenzodiazepine (PBD)-heterocycle polyamide conjugates has been synthesised to explore structure/sequence-selectivity relationships. One conjugate (2d) has a greater selectivity and DNA binding affinity for inverted CCAAT sequences within the Topoisomerase IIα promoter than the known C8-bis-pyrrole PBD conjugate GWL-78 (1b).

Efficient solid-phase synthesis of a library of distamycin analogs containing novel biaryl motifs on SynPhase Lanterns.

Distamycin is a naturally occurring antibiotic that binds to AT-rich sequences in the minor groove of DNA in a noncovalent manner. It continues to be of interest as a "building block" for more-complex small-molecule ligands capable of targeting specific DNA sequences for gene regulation purposes (i.e., transcription factor inhibitors). We report here a convenient and efficient synthesis of a library of 72 novel analogs (3a-f) of the parent distamycin on SynPhase Lanterns. To investigate structure-activity relationships including DNA-binding affinity and sequence-selectivity, two previously unexplored points of diversification have been introduced into the distamycin structure by replacing one of its pyrrole rings with novel biaryl motifs. The key aspects of the synthetic approach include the development of an efficient protocol for preparation of the heterocyclic polyamide chain, optimization of the Suzuki-Miyaura cross-coupling reaction and application of a split-and-mix technique based on radiofrequency encoding. In addition, a series of biaryl carboxamide derivatives (4a-f) has been synthesized utilizing the title library diversity reagents.