Pre-clinical evaluation of a novel class of anti-cancer agents, the Pyrrolo-1, 5-benzoxazepines.

Microtubules are currently ranked one of the most validated targets for chemotherapy; with clinical use of microtubule targeting agents (MTAs) extending beyond half a century. Recent research has focused on the development of novel MTAs to combat drug resistance and drug associated toxicities. Of particular interest are compounds structurally different to those currently used within the clinic. The pyrrolo-1, 5-benzoxazepines (PBOXs) are a structurally distinct novel group of anti-cancer agents, some of which target tubulin. Herein, we review the chemistry, mechanism of action, preclinical development of the PBOXs and comparisons with clinically relevant chemotherapeutics. The PBOXs induce a range of cellular responses including; cell cycle arrest, apoptosis, autophagy, anti-vascular and anti-angiogenic effects. The apoptotic potential of the PBOXs extends across a wide spectrum of cancer-derived cell lines, by targeting tubulin and multiple molecular pathways frequently deregulated in human cancers. Extensive experimental data suggest that combining the PBOXs with established chemotherapeutics or radiation is therapeutically advantageous. Pre-clinical highlights of the PBOXs include; cancer specificity and improved therapeutic efficacy as compared to some current first line therapeutics.

Polypharmacology of dopamine receptor ligands.

Most neurological diseases have a multifactorial nature and the number of molecular mechanisms discovered as underpinning these diseases is continuously evolving. The old concept of developing selective agents for a single target does not fit with the medical need of most neurological diseases. The development of designed multiple ligands holds great promises and appears as the next step in drug development for the treatment of these multifactorial diseases. Dopamine and its five receptor subtypes are intimately involved in numerous neurological disorders. Dopamine receptor ligands display a high degree of cross interactions with many other targets including G-protein coupled receptors, transporters, enzymes and ion channels. For brain disorders like Parkinsons disease, schizophrenia and depression the dopaminergic system, being intertwined with many other signaling systems, plays a key role in pathogenesis and therapy. The concept of designed multiple ligands and polypharmacology, which perfectly meets the therapeutic needs for these brain disorders, is herein discussed as a general ligand-based concept while focusing on dopaminergic agents and receptor subtypes in particular.

PBOX-15, a novel microtubule targeting agent, induces apoptosis, upregulates death receptors, and potentiates TRAIL-mediated apoptosis in multiple myeloma cells.

BACKGROUND: In recent years, much progress has been made in the treatment of multiple myeloma. However, a major limitation of existing chemotherapeutic drugs is the eventual emergence of resistance; hence, the development of novel agents with new mechanisms of action is pertinent. Here, we describe the activity and mechanism of action of pyrrolo-1,5-benzoxazepine-15 (PBOX-15), a novel microtubule-targeting agent, in multiple myeloma cells. METHODS: The anti-myeloma activity of PBOX-15 was assessed using NCI-H929, KMS11, RPMI8226, and U266 cell lines, and primary myeloma cells. Cell cycle distribution, apoptosis, cytochrome c release, and mitochondrial inner membrane depolarisation were analysed by flow cytometry; gene expression analysis was carried out using TaqMan Low Density Arrays; and expression of caspase-8 and Bcl-2 family of proteins was assessed by western blot analysis. RESULTS: Pyrrolo-1,5-benzoxazepine-15 induced apoptosis in ex vivo myeloma cells and in myeloma cell lines. Death receptor genes were upregulated in both NCI-H929 and U266 cell lines, which displayed the highest and lowest apoptotic responses, respectively, following treatment with PBOX-15. The largest increase was detected for the death receptor 5 (DR5) gene, and cotreatment of both cell lines with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), the DR5 ligand, potentiated the apoptotic response. In NCI-H929 cells, PBOX-15-induced apoptosis was shown to be caspase-8 dependent, with independent activation of extrinsic and intrinsic apoptotic pathways. A caspase-8-dependent decrease in expression of Bim(EL) preceded downregulation of other Bcl-2 proteins (Bid, Bcl-2, Mcl-1) in PBOX-15-treated NCI-H929 cells. CONCLUSION: PBOX-15 induces apoptosis and potentiates TRAIL-induced cell death in multiple myeloma cells. Thus, PBOX-15 represents a promising agent, with a distinct mechanism of action, for the treatment of this malignancy.

Novel pyrrolo-1,5-benzoxazepine compounds display significant activity against resistant chronic myeloid leukaemia cells in vitro, in ex vivo patient samples and in vivo.

BACKGROUND: Imatinib is a direct and potent inhibitor of the constitutively active tyrosine kinase, breakpoint cluster region-Abelson (Bcr-Abl), which is central to the pathogenesis of chronic myeloid leukaemia (CML) patients. As such, imatinib has become the front-line treatment for CML patients. However, the recent emergence of imatinib resistance, commonly associated with point mutations within the kinase domain, has led to the search for alternative drug treatments and combination therapies for CML. METHODS: In this report, we analyse the effects of representative members of the novel pro-apoptotic microtubule depolymerising pyrrolo-1,5-benzoxazepines or PBOX compounds on chemotherapy-refractory CML cells using a series of Bcr-Abl mutant cell lines, clinical ex vivo patient samples and an in vivo mouse model. RESULTS: The PBOX compounds potently reduce cell viability in cells expressing the E225K and H396P mutants as well as the highly resistant T315I mutant. The PBOX compounds also induce apoptosis in primary CML samples including those resistant to imatinib. We also show for the first time, the in vivo efficacy of the pro-apoptotic PBOX compound, PBOX-6, in a CML mouse model of the T315I Bcr-Abl mutant. CONCLUSION: Results from this study highlight the potential of these novel series of PBOX compounds as an effective therapy against CML.

Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network.

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.

Caspase-activated DNase (CAD)-independent oligonucleosomal DNA fragmentation in chronic myeloid leukaemia cells; a requirement for serine protease and Mn2+-dependent acidic endonuclease activity.

We have previously reported that the pro-apoptotic pyrrolobenzoxazepine, PBOX-6, induces apoptosis in chronic myelogenous leukaemia (CML) cells which is accompanied by oligonucleosomal DNA fragmentation. In this study we show that PBOX-6-induced oligonucleosomal DNA fragmentation occurs in the absence of caspase and CAD activation in CML cells. Dissection of the signalling pathway has revealed that induction of apoptosis requires the upstream activation of a trypsin-like serine protease that promotes the phosphorylation and inactivation of anti-apoptotic Bcl-2. In addition, in this system chymotrypsin-like serine proteases are dispensable for high molecular weight DNA fragmentation, however are required for the activation of a relatively small manganese-dependent acidic endonuclease that is responsible for oligonucleosomal fragmentation of DNA. Furthermore, we demonstrate mitochondrial involvement during PBOX-6-induced apoptosis and suggest the existence of unidentified mitochondrial effectors of apoptosis.

Characterization of the 1H-cyclopentapyrimidine-2,4(1H,3H)-dione derivative (S)-CPW399 as a novel, potent, and subtype-selective AMPA receptor full agonist with partial desensitization properties.

(S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound 2b appears to be a weakly desensitizing, full agonist at AMPA receptors and therefore represents a new pharmacological tool to investigate the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitization.

Pyrrolo-1,5-benzoxazepines: a new class of apoptotic agents.

Some members of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) potently induce apoptosis in a number of human cancerous cell lines including HL-60 cells and the drug-resistant chronic myelogenous leukaemia cell line, K562. The apoptotic induction seems to be independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR), which binds these PBOXs with high affinity, due to a lack of correlation between their affinities for the receptor and their apoptotic potencies and their high apoptotic activity in PBR-deficient cells. PBOX-6, a potent member of the series, induces a transient activation of c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which correlates with induction of apoptosis. Expression of a cytoplasmic inhibitor of the JNK signal transduction pathway, Jip-1, prevents JNK activity and significantly reduces the extent of apoptosis induced by PBOX-6. This demonstrates the requirement for JNK in the cellular response to this apoptotic agent. In addition, PBOX-6 activates caspase-3-like proteases in K562 and HL-60 cells. The caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk), blocks caspase-3-like protease activity in both cell types but only prevents PBOX-6-induced apoptosis in HL-60 cells, suggesting that the requirement for caspase-3-like proteases in the apoptotic pathway is dependent on the cell type.

The stereoselective targeting of a specific enzyme-substrate complex is the molecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors.

The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (+/-)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3'-azido-3'-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J. Med. Chem. 42, 4462-4470). The (+/-)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis. Only one enantiomer, (R)-(-)-PPO464, displayed antiviral activity against both the wild type and the K103N mutant HIV-1 RT and was found to interact exclusively with the reaction intermediate formed by RT complexed with both the DNA and the nucleotide substrates. Being the first compound of its class to display this behavior, (R)-(-)-PPO464 is the representative of a novel generation of nonnucleoside inhibitors. (R)-(-)-PPO464 showed significant synergism when tested in combination with other RT inhibitors and efficiently inhibited viral replication when tested against the laboratory strain HIV-1 IIIB or against either wild type or multidrug-resistant clinical isolates. Pharmacokinetic studies in mice and rats showed a more favorable profile for (R)-(-)-PPO464 than for the corresponding racemate. (R)-(-)-PPO464 was also found to easily cross the blood-brain barrier. The coadministration of the HIV-1 protease inhibitor ritonavir increased the bioavailability of (R)-(-)-PPO464, having little effect on its plasma and brain elimination rates.

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Novel and potent tacrine-related hetero- and homobivalent ligands for acetylcholinesterase and butyrylcholinesterase.

Based upon synthetic and biochemical results, a novel and potent tacrine analogue and heterobivalent analogues of tacrine, were designed. The role played by the amino groups of homo- and heterobivalent ligands in the interaction with the peripheral and catalytic sites of AChE and BuChE were investigated. The syntheses of these materials together with the results of AChE/BuChE inhibition assays are detailed.

Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor bcr-abl.

Expression of the transforming oncogene bcr-abl in chronic myelogenous leukemia (CML) cells is reported to confer resistance against apoptosis induced by many chemotherapeutic agents such as etoposide, ara-C, and staurosporine. In the present study some members of a series of novel pyrrolo-1,5-benzoxazepines potently induce apoptosis, as shown by cell shrinkage, chromatin condensation, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage, in three CML cell lines, K562, KYO.1, and LAMA 84. Induction of apoptosis by a representative member of this series, PBOX-6, was not accompanied by either the down-regulation of Bcr-Abl or by the attenuation of its protein tyrosine kinase activity up to 24 h after treatment, when approximately 50% of the cells had undergone apoptosis. These results suggest that down-regulation of Bcr-Abl is not part of the upstream apoptotic death program activated by PBOX-6. By characterizing the mechanism in which this novel agent executes apoptosis, this study has revealed that PBOX-6 caused activation of caspase 3-like proteases in only two of the three CML cell lines. In addition, inhibition of caspase 3-like protease activity using the inhibitor z-DEVD-fmk blocked caspase 3-like protease activity but did not prevent the induction of apoptosis, suggesting that caspase 3-like proteases are not essential in the mechanism by which PBOX-6 induces apoptosis in CML cells. In conclusion, this study demonstrates that PBOX-6 can bypass Bcr-Abl-mediated suppression of apoptosis, suggesting an important potential use of these compounds in the treatment of CML.

Non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis and biological evaluation of novel quinoxalinylethylpyridylthioureas as potent antiviral agents.

New heterocyclic derivatives of ethylpyridylthiourea, quinoxalinylethylpyridylthiourea (QXPT) and analogues, inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevented HIV-1 cytopathogenicity in T4 lymphocytes. Several of these novel non-nucleoside RT inhibitors, with a substituted pyrroloquinoxalinone heteroaromatic skeleton, showed inhibitory activity against wild-type RT as well as against mutant RTs containing the single amino acid substitutions L1001, K103N, V106A, Y1811 and Y188L that was much greater than other non-nucleoside inhibitors such as nevirapine. Maximum potency in enzymatic assays was achieved with a fluoropyrroloquinoxaline skeleton linked to the ethylpyridylthiourea moiety (FQXPT). In cell-based assays on different cell lines and on human monocyte-macrophages, 6-FQXPT exhibited EC50 values in the nanomolar range, with a promising selectivity index. Moreover, 6-FQXPT showed synergistic antiviral activity with zidovudine.

Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents.

Some, but not all, of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) induce apoptosis as shown by cell shrinkage, chromatin condensation, and DNA fragmentation in three human cell lines, HL-60 promyelocytic, Jurkat T lymphoma, and Hut-78 s.c. lymphoma cells. This chemical selectivity, together with the lack of apoptotic activity against rat Leydig cells, argues against a general cell poisoning effect. PBOX-6, a potent member of the series, caused activation of a member of the caspase-3 family of proteases. In addition, the caspase-3-like inhibitor z-DEVD-fmk, but not the caspase-1-like inhibitor z-YVAD-fmk prevented PBOX-6-induced apoptosis, suggesting that caspase 3-like proteases are involved in the mechanism by which PBOX compounds induce apoptosis. The release of cytochrome c into the cytosol in HL-60 cells in response to PBOX-6 suggests that this cellular response may be important in the mechanism by which PBOX-6 induces apoptosis. However, reactive oxygen intermediates do not play a key role in PBOX-6-induced apoptosis because neither the free radical scavenger TEMPO nor the antioxidant N-acetylcysteine had any effect on PBOX-6-induced apoptosis. The apoptotic induction seems independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR) that binds these pyrrolobenzoxazepines with high affinity, due to the lack of correlation between their affinities for the receptor and their apoptotic potencies, their high apoptotic activity in PBR-deficient cells such as Jurkats, and their lack of apoptotic induction in PBR-rich rat Leydig cells. These PBOXs also can overcome nuclear factor-kappaB-mediated resistance to apoptosis. This suggests an important potential use of these compounds in drug-resistant cancers.

Pyrroloquinoxaline derivatives as high-affinity and selective 5-HT(3) receptor agonists: synthesis, further structure-activity relationships, and biological studies.

The synthesis, pharmacological evaluation, and structure-activity relationships (SARs) of a series of novel pyrroloquinoxalines and heteroaromatic-related derivatives are described. The new pyrroloquinoxaline-related ligands were tested in rat cortex, a tissue expressing high density of 5-HT(3) receptors, and on NG108-15 cells and exhibited IC(50) values in the low nanomolar or subnanomolar range, as measured by the inhibition of [(3)H]zacopride binding. The SAR studies detailed herein delineated a number of structural features required for improving affinity. Some of the ligands were employed as "molecular yardsticks" to probe the spatial dimensions of the lipophilic pockets L1, L2, and L3 in the 5-HT(3) receptor cleft, while the 7-OH pyrroloquinoxaline analogue was designed to investigate hydrogen bonding with a putative receptor site H1 possibly interacting with the serotonin hydroxy group. The most active pyrroloquinoxaline derivatives showed subnanomolar affinity for the 5-HT(3) receptor. In functional studies ([(14)C]guanidinium accumulation test in NG108-15 hybrid cells, in vitro) most of the tested compounds showed clear-cut 5-HT(3) agonist properties, while some others were found to be partial agonists. Several heteroaromatic systems, bearing N-substituted piperazine moieties, have been explored with respect to 5-HT(3) affinity, and novel structural leads for the development of potent and selective central 5-HT(3) receptor agonists have been identified. Preliminary pharmacokinetic studies indicate that these compounds easily cross the blood-brain barrier (BBB) after systemic administration with a brain/plasma ratio between 2 and 20, unless they bear a highly hydrophilic group on the piperazine ring. None of the tested compounds showed in vivo anxiolytic-like activity, but potential analgesic-like properties have been possibly disclosed for this new class of 5-HT(3) receptor agonists.

Pyrrolobenzoxazepinone derivatives as non-nucleoside HIV-1 RT inhibitors: further structure-activity relationship studies and identification of more potent broad-spectrum HIV-1 RT inhibitors with antiviral activity.

Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (+/-)-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)- one (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis and biological evaluation of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridooxazepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized compounds (PBOs 13a-d and PPOs 13i-k) displayed higher inhibitory activity against wild-type RT and clinically relevant mutant RTs containing the single amino acid substitutions L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentrations in the brain. The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of analogues such as 13b-d, 13i, and 13j, in combination with nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance to known NNRTI.

Thiazolothiazepine inhibitors of HIV-1 integrase.

A series of thiazolothiazepines were prepared and tested against purified human immunodeficiency virus type-1 integrase (HIV-1 IN) and viral replication. Structure-activity studies reveal that the compounds possessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups are in general more potent against purified IN than those containing only one carbonyl group. Substitution with electron-donating or -withdrawing groups did not enhance nor abolish potency against purified IN. By contrast, compounds with a naphthalene ring system showed enhanced potency, suggesting that a hydrophobic pocket in the IN active site might accommodate an aromatic system rather than a halogen. The position of sulfur in the thiazole ring appears important for potency against IN, as its replacement with an oxygen or carbon abolished activity. Further extension of the thiazole ring diminished potency. Compounds 1, 19, and 20 showed antiviral activity and inhibited IN within similar concentrations. These compounds inhibited IN when Mn(2+) or Mg(2+) was used as cofactor. None of these compounds showed detectable activities against HIV-1 reverse transcriptase, protease, virus attachment, or nucleocapsid protein zinc fingers. Therefore, thiazolothiazepines are potentially important lead compounds for development as inhibitors of IN and HIV replication.

Preparation and biological properties of biotinylated PhTX derivatives.

We report the synthesis of several highly functionalized biotinylated philanthotoxin (PhTX) analogues (7, 8, 10, 13-16) designed on the basis of earlier structure-activity relationship studies. Despite the extensive modifications, the binding to nicotinic acetylcholine receptor (nAChR) is in the low micromolar range according to an inhibition assay using 3H-thienylcyclohexyl-piperidine (TCP). A patch clamp functional assay gave comparable results. Compounds exemplified by 16, which consists of a biotinylated ligand linked to a bifunctional photoaffinity probe (BPP), represent a new type of probe which should find use in photo-crosslinking studies of ligand receptor interactions.