Discovery of Anticancer Agents of Diverse Natural Origin.

Recent progress is described in an ongoing collaborative multidisciplinary research project directed towards the purification, structural characterization, chemical modification, and biological evaluation of new potential natural product anticancer agents obtained from a diverse group of organisms, comprising tropical plants, aquatic and terrestrial cyanobacteria, and filamentous fungi. Information is provided on how these organisms are collected and processed. The types of bioassays are indicated in which initial extracts, chromatographic fractions, and purified isolated compounds of these acquisitions are tested. Several promising biologically active lead compounds from each major organism class investigated are described, and these may be seen to be representative of a very wide chemical diversity.

Spiroscytalin, a new tetramic acid and other metabolites of mixed biogenesis from Scytalidium cuboideum.

Spiroscytalin (1), a new tetramic acid that possesses an uncommon spiro-ring fusion between a polyketide-derived octalin ring system and a 2,4-pyrrolidinedione, along with two known compounds, leporin B (2) and purpactin A (3), were isolated from a solid phase culture of the fungus Scytalidium cuboideum (MSX 68345). The molecular connectivity of 1-3 was determined using NMR spectroscopy and mass spectrometry. The relative configurations of 1 and 2 were determined by NOESY experiments. The absolute configuration of 1 was determined by electronic circular dichroism (ECD) via a combination of experimental measurements and computational calculations. While leporin B was known, it displayed activities that had not been reported previously, including cytotoxicity against three human tumor cell lines and antibacterial activity against Candida albicans and Staphylococcus aureus.

Biosynthetically Distinct Cytotoxic Polyketides from Setophoma terrestris.

Sixteen polyketides belonging to diverse structural classes, including monomeric/dimeric tetrahydroxanthones and resorcylic acid lactones, were isolated from an organic extract of a fungal culture Setophoma terrestris (MSX45109) using bioactivity-directed fractionation as part of a search for anticancer leads from filamentous fungi. Of these, six were new: penicillixanthone B (5), blennolide H (6), 11-deoxy blennolide D (7), blennolide I (9), blennolide J (10), and pyrenomycin (16). The known compounds were: secalonic acid A (1), secalonic acid E (2), secalonic acid G (3), penicillixanthone A (4), paecilin B (8), aigialomycin A (11), hypothemycin (12), dihydrohypothemycin (13), pyrenochaetic acid C (14), and nidulalin B (15). The structures were elucidated using a set of spectroscopic and spectrometric techniques; the absolute configurations of compounds 1-10 were determined using ECD spectroscopy combined with time-dependent density functional theory (TDDFT) calculations, while a modified Mosher's ester method was used for compound 16. The cytotoxic activities of compounds (1-15) were evaluated using the MDA-MB-435 (melanoma) and SW-620 (colon) cancer cell lines. Compounds 1, 4, and 12 were the most potent with IC50 values ranging from 0.16 to 2.14 µM. When tested against a panel of bacteria and fungi, compounds 3 and 5 showed promising activity against the Gram-positive bacterium Micrococcus luteus with MIC values of 5 and 15 µg/mL, respectively.

Sorbicillinoid analogs with cytotoxic and selective anti-Aspergillus activities from Scytalidium album.

As part of an ongoing project to explore filamentous fungi for anticancer and antibiotic leads, 11 compounds were isolated and identified from an organic extract of the fungus Scytalidium album (MSX51631) using bioactivity-directed fractionation against human cancer cell lines. Of these, eight compounds were a series of sorbicillinoid analogs (1-8), of which four were new (scalbucillin A (2), scalbucillin B (3), scalbucillin C (6) and scalbucillin D (8)), two were phthalides (9-10) and one was naphthalenone (11). Compounds (1-11) were tested in the MDA-MB-435 (melanoma) and SW-620 (colon) cancer cell lines. Compound 1 was the most potent with IC50 values of 1.5 and 0.5 µM, followed by compound 5 with IC50 values of 2.3 and 2.5 µM at 72 h. Compound 1 showed a 48-h IC50 value of 3.1 µM when tested against the lymphocytic leukemia cell line OSU-CLL, while the nearly identical compound 5 had almost no activity in this assay. Compounds 1 and 5 showed selective and equipotent activity against Aspergillus niger with minimum IC values of 0.05 and 0.04 µg ml(-1) (0.20 and 0.16 µM), respectively. The in vitro hemolytic activity against sheep erythrocytes of compounds 1 and 5 was investigated and were found to provoke 10% hemolysis at 52.5 and 45.0 µg ml(-1), respectively, indicative of a promising safety factor.

Nature as a remarkable chemist: a personal story of the discovery and development of Taxol.

The development of a new anticancer drug with a novel structure and unique mechanism of action is an important event, especially when the drug plays a clear role in improving the outcome for cancer patients. No drug fits this description better than Taxol. However, during the early phases of its development, there was little interest in the drug, particularly in the medical community. The story of Taxol is long and fascinating, and includes many examples in which the drug could have been dropped, resulting in its antitumor activity never being available to patients. It was 21 years between the original landmark paper on the isolation and structural determination of Taxol and its approval in 1992 by the FDA for its use in the treatment of ovarian cancer.

Waol A, trans-dihydrowaol A, and cis-dihydrowaol A: polyketide-derived γ-lactones from a Volutella species.

An organic extract of a filamentous fungus (MSX 58801), identified as a Volutella sp. (Hypocreales, Ascomycota), displayed moderate cytotoxic activity against NCI-H460 human large cell lung carcinoma. Bioactivity-directed fractionation led to the isolation of three γ-lactones having the furo[3,4-b]pyran-5-one bicyclic ring system [waol A (1), trans-dihydrowaol A (2), and cis-dihydrowaol A (3)]. The structures were elucidated using a set of spectroscopic and spectrometric techniques; the absolute configuration of 2 was established via a modified Mosher's ester method. Compounds 1 and 2 were evaluated for cytotoxicity against a human cancer cell panel.

Peptaibols, tetramic acid derivatives, isocoumarins, and sesquiterpenes from a Bionectria sp. (MSX 47401).

An extract of the filamentous fungus Bionectria sp. (MSX 47401) showed both promising cytotoxic activity (>90% inhibition of H460 cell growth at 20 µg/mL) and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). A bioactivity-directed fractionation study yielded one new peptaibol (1) and one new tetramic acid derivative (2), and the fungus biosynthesized diverse secondary metabolites with mannose-derived units. Five known compounds were also isolated: clonostachin (3), virgineone (4), virgineone aglycone (5), AGI-7 (6), and 5,6-dihydroxybisabolol (7). Compounds 5 and 7 have not been described previously from natural sources. Compound 1 represents the second member of the peptaibol structural class that contains an ester-linked sugar alcohol (mannitol) instead of an amide-linked amino alcohol, and peptaibols and tetramic acid derivatives have not been isolated previously from the same fungus. The structures of the new compounds were elucidated primarily by high-field NMR (950 and 700 MHz), HRESIMS/MS, and chemical degradations (Marfey's analysis). All compounds (except 6) were examined for antibacterial and antifungal activities. Compounds 2, 4, and 5 showed antimicrobial activity against S. aureus and several MRSA isolates.

Benzoquinones and terphenyl compounds as phosphodiesterase-4B inhibitors from a fungus of the order Chaetothyriales (MSX 47445).

Three bioactive compounds were isolated from an organic extract of an ascomycete fungus of the order Chaetothyriales (MSX 47445) using bioactivity-directed fractionation as part of a search for anticancer leads from filamentous fungi. Of these, two were benzoquinones [betulinan A (1) and betulinan C (3)], and the third was a terphenyl compound, BTH-II0204-207:A (2). The structures were elucidated using a set of spectroscopic and spectrometric techniques; the structure of the new compound (3) was confirmed via single-crystal X-ray diffraction. Compounds 1-3 were evaluated for cytotoxicity against a human cancer cell panel, for antimicrobial activity against Staphylococcus aureus and Candida albicans, and for phosphodiesterase (PDE4B2) inhibitory activities. The putative binding mode of 1-3 with PDE4B2 was examined using a validated docking protocol, and the binding and enzyme inhibitory activities were correlated.

Enhanced bioactivity of silybin B methylation products.

Flavonolignans from milk thistle (Silybum marianum) have been investigated for their cellular modulatory properties, including cancer chemoprevention and hepatoprotection, as an extract (silymarin), as partially purified mixtures (silibinin and isosilibinin), and as pure compounds (a series of seven isomers). One challenge with the use of these compounds in vivo is their relatively short half-life due to conjugation, particularly glucuronidation. In an attempt to generate analogues with improved in vivo properties, particularly reduced metabolic liability, a semi-synthetic series was prepared in which the hydroxy groups of silybin B were alkylated. A total of five methylated analogues of silybin B were synthesized using standard alkylation conditions (dimethyl sulfate and potassium carbonate in acetone), purified using preparative HPLC, and elucidated via spectroscopy and spectrometry. Of the five, one was monomethylated (3), one was dimethylated (4), two were trimethylated (2 and 6), and one was tetramethylated (5). The relative potency of all compounds was determined in a 72 h growth-inhibition assay against a panel of three prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a human hepatoma cell line (Huh7.5.1) and compared to natural silybin B. Compounds also were evaluated for inhibition of both cytochrome P450 2C9 (CYP2C9) activity in human liver microsomes and hepatitis C virus infection in Huh7.5.1 cells. The monomethyl and dimethyl analogues were shown to have enhanced activity in terms of cytotoxicity, CYP2C9 inhibitory potency, and antiviral activity (up to 6-fold increased potency) compared to the parent compound, silybin B. In total, these data suggested that methylation of flavonolignans can increase bioactivity.

Use of the hollow fiber assay for the discovery of novel anticancer agents from fungi.

The hollow fiber assay (HFA) is a drug discovery tool to aid investigators in the prioritization of lead compounds identified by in vitro testing for further development in animal models of disease. In the HFA, cells are cultured in hollow fibers containing pores of a diameter (500 kDa) large enough for proteins and other macromolecules to enter, but too small for the cells to escape. The fibers are filled with cells, sealed and placed in the peritoneal cavity of immunodeficient mice. The mice undergo a predetermined treatment regimen after which the fibers are retrieved and the cells evaluated for activity of a target relevant to the disease modeled. The HFA combines advantages of both in vitro and in vivo assay systems. It uses the same cell lines used in culture systems, is a rapid assay, and requires fewer animals and less test substance than conventional xenograft systems. Like traditional in vivo assays, the test substance is evaluated in a live animal, which affords an initial assessment of associated toxicity and pharmacokinetic properties of the test substance.

Cytotoxic epipolythiodioxopiperazine alkaloids from filamentous fungi of the Bionectriaceae.

Bioactivity-directed fractionation of the organic extracts of two filamentous fungi of the Bionectriaceae, strains MSX 64546 and MSX 59553 from the Mycosynthetix library, led to the isolation of a new dimeric epipolythiodioxopiperazine alkaloid, verticillin H (1), along with six related analogs, Sch 52900 (2), verticillin A (3), gliocladicillin C (4), Sch 52901 (5), 11'-deoxyverticillin A (6) and gliocladicillin A (7). The structures of compounds 1-7 were determined by extensive NMR and HRMS analyses, as well as by comparisons to the literature. All compounds (1-7) were evaluated for cytotoxicity against a panel of human cancer cell lines, displaying IC(50) values ranging from 1.2 µM to 10 nM. Compounds 1-5 were examined for activity in the NF-κB assay, where compounds 2 and 3 revealed activity in the sub-micromolar range. Additionally, compounds 1, 3 and 4 were tested for EGFR inhibition using an enzymatic assay, while compound 3 was examined against an overexpressing EGFR(+ve) cancer cell line.

Peptaibols from two unidentified fungi of the order Hypocreales with cytotoxic, antibiotic, and anthelmintic activities.

As part of an ongoing investigation of filamentous fungi for anticancer leads, an active culture was identified from the Mycosynthetix library (MSX 70741, of the order Hypocreales, Ascomycota). The fungal extract exhibited cytotoxic activity against the H460 (human nonsmall cell lung carcinoma) cell line, and bioactivity-directed fractionation yielded peptaibols 1-12 and harzianums A (13) and B (14). Structure elucidation of 1-12 was facilitated by high-resolution MS/MS using higher-energy collisional dissociation and by high field NMR (950 MHz). The absolute configuration was determined by Marfey's analysis of the individual amino acids; the time required for such analysis was decreased via the development of a 10-min ultra performance liquid chromatography method. The isolated peptaibols (1-12), along with three other peptaibols isolated and elucidated from a different fungus (MSX 57715) of the same order (15-17), were examined for activity in a suite of biological assays, including those for cytotoxic, antibacterial, and anthelmintic activities.

Cytotoxic xanthone-anthraquinone heterodimers from an unidentified fungus of the order Hypocreales (MSX 17022).

Two new xanthone-anthraquinone heterodimers, acremoxanthone C (5) and acremoxanthone D (2), have been isolated from an extract of an unidentified fungus of the order Hypocreales (MSX 17022) by bioactivity-directed fractionation as part of a search for anticancer leads from filamentous fungi. Two known related compounds, acremonidin A (4) and acremonidin C (3) were also isolated, as was a known benzophenone, moniliphenone (1). The structures of these isolates were determined via extensive use of spectroscopic and spectrometric tools in conjunction with comparisons to the literature. All compounds (1-5) were evaluated against a suite of biological assays, including those for cytotoxicity, inhibition of the 20S proteasome, mitochondrial transmembrane potential and nuclear factor-κB.

Obionin B: An o-pyranonaphthoquinone decaketide from an unidentified fungus (MSX 63619) from the Order Pleosporales.

A fungal extract (MSX 63619), from the Mycosynthetix library of over 50,000 fungi, displayed promising cytotoxicity against a human tumor cell panel. Bioactivity-directed fractionation led to the isolation of an o-pyranonaphthoquinone decaketide, which we termed obionin B (1). The structure of 1 was deduced via spectroscopic and spectrometric techniques. The IC(50) value of 1 was moderate, ranging from 3 to 13 µM, depending on the cell line tested.

Cyclodepsipeptides, sesquiterpenoids, and other cytotoxic metabolites from the filamentous fungus Trichothecium sp. (MSX 51320).

Two new cyclodepsipeptides (1 and 2), two new sesquiterpenoids (3 and 4), and the known compounds guangomide A (5), roseotoxin S, and three simple trichothecenes were isolated from the cytotoxic organic extract of a terrestrial filamentous fungus, Trichothecium sp. The structures were determined using NMR spectroscopy and mass spectrometry. Absolute configurations of the cyclodepsipeptides were established by employing chiral HPLC, while the relative configurations of 3 and 4 were determined via NOESY data. The isolation of guangomide A was of particular interest, since it was reported previously from a marine-derived fungus.

Resorcylic acid lactones with cytotoxic and NF-κB inhibitory activities and their structure-activity relationships.

As part of our ongoing investigation of filamentous fungi for anticancer leads, an active fungal extract was identified from the Mycosynthetix library (MSX 63935; related to Phoma sp.). The initial extract exhibited cytotoxic activity against the H460 (human non-small cell lung carcinoma) and SF268 (human astrocytoma) cell lines and was selected for further study. Bioactivity-directed fractionation yielded resorcylic acid lactones (RALs) 1 (a new natural product) and 3 (a new compound) and the known RALs zeaenol (2), (5E)-7-oxozeaenol (4), (5Z)-7-oxozeaenol (5), and LL-Z1640-1 (6). Reduction of (5E)-7-oxozeaenol (4) with sodium borohydride produced 3, which allowed assignment of the absolute configuration of 3. Other known resorcylic acid lactones (7-12) were purchased and assayed in parallel for cytotoxicity with isolated 1-6 to investigate structure-activity relationships in the series. Moreover, the isolated compounds (1-6) were examined for activity in a suite of biological assays, including antibacterial, mitochondria transmembrane potential, and NF-κB. In the latter assay, compounds 1 and 5 displayed sub-micromolar activities that were on par with the positive control, and as such, these compounds may serve as a lead scaffold for future medicinal chemistry studies.

BACPTDP: a water-soluble camptothecin pro-drug with enhanced activity in hypoxic/acidic tumors.

UNLABELLED: Hypoxia is a common feature of solid tumors. Up-regulation of hypoxia-inducing factor-1 (HIF-1) occurs in the majority of primary malignant tumors and in two-thirds of metastases, while most normal tissues are negative. HIF-1 induces the glycolytic phenotype, which creates an acidic extracellular microenvironment and associated pH gradient such that drugs that are weak acids are selectively taken up and retained in acidic tumors. 7-Butyl-10-amino-camptothecin (BACPT) is a prime example of an agent that can exploit the tumor pH gradient for enhanced selectivity. PURPOSE: This study profiles the antitumor activity of BACPT in vitro and its water-soluble dipeptide ester, BACPTDP, in vivo. METHODS: Antitumor activity was evaluated by proliferation assays in cancer cell lines and in murine xenograft models for human neuroblastoma (IMR-32), colon (HT29), ovarian (SK-OV-3), pancreatic (Panc-1), glioma (SF-295) and non-small-cell lung (NCI-H460) cancers. RESULTS: BACPT had superior antiproliferative activity compared to established drugs in monolayer cultures of human neuroblastoma and pancreatic tumor cell lines and in 3-dimensional histocultures of colon and primary ovarian cancer. Antitumor activity of BACPTDP was comparable to irinotecan in IMR-32, HT29, SF-295 and NCI-H460 xenografts, significantly greater in SK-OV-3 and in Panc-1 where complete regressions were observed. Combination of BACPT with gemcitabine produced additive to synergistic interactions in Panc-1 cells that were independent of drug ratio and optimal when gemcitabine was administered 24 h prior to BACPT. CONCLUSIONS: BACPTDP is a water-soluble camptothecin pro-drug that spontaneously generates the lipid-soluble active agent, BACPT. This topoisomerase inhibitor exploits solid tumor physiology for improved selectivity and activity against multiple tumor types with particular promise for use in treating pediatric neuroblastoma and pancreatic carcinoma.

Thielavin B methyl ester: a cytotoxic benzoate trimer from an unidentified fungus (MSX 55526) from the Order Sordariales.

As part of our ongoing investigation of filamentous fungi for anticancer leads, an active fungal extract was identified from the Mycosynthetix library (MSX 55526; from the Order Sordariales). Bioactivity-directed fractionation yielded the known ergosterol peroxide (2) and 5α,8α-epidioxyergosta-6,9(11),22-trien-3ß-ol(3), and a new benzoate trimer, termed thielavin B methyl ester (1). The structure elucidation of 1 was facilitated by the use of HRMS coupled to an APPI (atmospheric pressure photoionization) source. Compound 1 proved to be moderately active against a panel of three cancer cell lines.

Dereplication of macrocyclic trichothecenes from extracts of filamentous fungi through UV and NMR profiles.

Macrocyclic trichothecenes (MTs), which have potent cytotoxicity, have been isolated from many different fungal species. These compounds were evaluated clinically by the US National Cancer Institute in the 1970s and 1980s. However, they have yet to be advanced into viable drugs because of severe side effects. Our team is investigating a diverse library of filamentous fungi for new anticancer leads. To avoid reisolating MTs through bioactivity-directed fractionation studies, a protocol for their facile dereplication was developed. The method uses readily available photodiode array detectors to identify one of two types of characteristic UV spectra for these compounds. In addition, diagnostic signals can be observed in the (1)H-NMR spectra, particularly for the epoxide and conjugated diene moieties, even at the level of a crude extract. Using these techniques in a complementary manner, MTs can be dereplicated rapidly.