Isolation and Characterization of Aphidicolin Derivatives from Tolypocladium inflatum.

Inflatin G (1), a new aphidicolin analogue, together with seven known compounds inflatin A (2), inflatin B (3), aphidicolin (4), aphidicolin-17-monoacetate (5), gulypyrone A (6), pyridoxatin rotamers A (7) and B (8), were isolated from the ascomycete fungus Tolypocladium inflatum. Their structures were determined through NMR analyses and the circular dichroism data of the in situ formed [Rh2(OCOCF3)4] complexes. Compounds 1, 4, 5, 7, and 8 showed modest cytotoxicity against four human cancer cell lines A549, CNE1-MP1, A375, and MCF-7.

Structural basis for inhibition of DNA replication by aphidicolin.

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.

Isolation and characterization of aphidicolin and chlamydosporol derivatives from Tolypocladium inflatum.

Six new secondary metabolites including two aphidicolin analogues, inflatins A (1) and B (2), and four chlamydosporol derivatives, inflatins C-F (3-6), have been isolated from the crude extract of Tolypocladium inflatum. The structures of 1-6 were determined mainly by NMR experiments, and 4 and 5 were further confirmed by X-ray crystallography. The absolute configurations of C-16 in 1 and C-5 in 3 were deduced via the circular dichroism data of the in situ formed [Rh2(OCOCF3)4] complexes, whereas that of 4 was assigned by X-ray crystallography using Cu Kα radiation. Compounds 1 and 2 showed modest cytotoxicity against a panel of eight human tumor cell lines.

Metabolites from the fungus Phoma sp. 7210, associated with Aizoon canariense.

A new metabolite, 3,16-diketoaphidicolan (1), was isolated together with four known compounds: aphidicolin (2), 17-acetyl-aphidicolin (3), (+)-eupenoxide (4), and phomoxanthone A (5) from the endophytic fungus Phoma sp. The structure of the new compound 1 was determined by spectroscopic methods (mainly extensive 1D and 2D NMR experiments and by mass spectral measurements) and confirmed by X-ray crystallography. Its absolute configuration was assigned by means of the solid-state CD/TDDFT approach comparing the solid-state CD spectrum with the TDDFT-calculated one on the X-ray geometry.

In vitro antiviral activity of aphidicolin and its derivates. Synergistic effects of aphidicolin with other antiviral drugs.

Twenty derivatives of aphidicolin were tested against HSV (herpes simplex virus), HCMV (human cytomegalovirus) and adenovirus in vitro. In addition, the antiviral activity of aphidicolin (CAS 38966-21-1) in combination with aciclovir (CAS 59277-89-3) or cidofovir (CAS 113852-37-2) against HSV was determined. The antiviral effects were evaluated using plaque reduction assay in Vero cells or human Foreskin Fibroblasts (HFF) for HSV and HCMV, respectively. Combination indexes were calculated using the method of Chou and Talalay. Two derivatives (K14254 and K14266) that are considered to be prodrugs of aphidicolin were shown to inhibit HCMV and HSV replication comparably to aphidicolin. None of the tested substances inhibited adenovirus replication. Aphidicolin acted synergistically with aciclovir in a 1:1 molar ratio and with cidofovir in different ratios. Aphidicolin and its two antiviral active derivatives might represent useful additional tools for antiviral therapy of HSV and HCMV infections, especially in combination with clinically used drugs.

Treatment of drug-resistant human neuroblastoma cells with cyclodextrin inclusion complexes of aphidicolin.

Treatment failure in most neuroblastoma (NB) patients is related to primary and/or acquired resistance to conventional chemotherapeutic agents. Aphidicolin (APH), a tetracyclic diterpene, exhibits specific cytotoxic action against NB cells. The purpose of this study was to compare antitumoral efficacy of APH in parental NB cell lines and cell subclones that exhibit drug resistance to vincristine (VCR), doxorubicin (DOX) and cisplatin. Due to poor solubility of APH in water, gamma-cyclodextrin (gamma-CD) inclusion complexes of APH were used for systemic treatment of xenotransplanted parental and VCR-resistant UKF-NB-3 tumours. APH and its gamma-CD inclusion complexes inhibited growth of parental and drug-resistant NB cells at equimolar doses in vitro. Growth of VCR-sensitive and -resistant NB tumors was inhibited at equal doses in a dose-dependent fashion in vivo. These results indicate that the specific cytotoxic activity of APH against NB cells in vitro and in vivo is independent of cellular mechanisms facilitating drug resistance to conventional chemotherapeutic drugs. Hence, taking into account our previous findings that APH acts synergistically with VCR and DOX, APH might be an additive tool for the therapy of NB and is suitable for evaluation in clinical studies of NB treatment protocols.

Antileishmanial activities of aphidicolin and its semisynthetic derivatives.

Aphidicolin and a series of semisynthetic aphidicolan derivatives have been identified in in vitro tests as novel drugs with antiparasitic potential. All compounds have been tested against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major and against intracellular amastigotes of L. donovani in murine macrophages. The compounds showed antileishmanial activity at concentrations in the microgram range (50% effective concentration [EC(50)] = 0.02 to 1.83 microg/ml). The most active derivative (aphidicolin-17-glycinate hydrochloride) had EC(50)s of 0. 2 microg/ml against extracellular and 0.02 microg/ml against intracellular L. donovani parasites. To validate the pharmacological potential of tested drugs, pharmacological safety was determined by testing all compounds against two neoplastic cell lines (squamous carcinoma [KB] and melanoma [SK-Mel]) and against murine bone marrow-derived macrophages as host cells. With minor exceptions only for macrophages, tested aphidicolans did not show significant cytotoxicity (EC(50) > 25.0 microg/ml). Structure-activity relationships of these aphidicolan derivatives are discussed.

New approach to aphidicolin and total asymmetric synthesis of unnatural (11R)-(-)-8-epi-11-hydroxyaphidicolin by tandem transannular Diels-Alder/aldol reactions.

The 8-epiaphidicolane skeleton (3) was formed in one key reaction by highly diastereoselective tandem transannular Diels-Alder (TADA)-aldol reactions from the trans-trans-cis trienic macrocycle (4). The unnatural derivative (11R)-(-)-8-epi-11-hydroxyaphidicolin (2) was thus constructed, and an original solution to the C16 functionalization problem of many aphidicolin (1) syntheses is presented.

Cytotoxicity of aphidicolin and its derivatives against neuroblastoma cells in vitro: synergism with doxorubicin and vincristine.

Disseminated neuroblastoma diseases are still indicated by a poor outcome despite treatment regimens including radiation therapy and high-dose chemotherapy with stem cell rescue. Therefore, new substances and treatment regimens are of interest. Aphidicolin (APH), a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, has a specific toxicity for neuroblastoma cells. Furthermore, it was shown to enhance the effects of X-ray radiation and chemotherapy on malignant cells. To find new substances, 20 APH derivatives were tested for their anti-neuroblastoma efficacy in vitro in UKF-NB-2 cells. Five derivatives had antitumoral activity in neuroblastoma cells. A relationship between the structure and the antitumoral efficacy showed that the hydroxyl groups at C-3 and C-18 are essential for the antitumoral effects. Furthermore, antitumoral effects of APH in combination with doxorubicin and vincristine, both part of commonly used treatment regimens for disseminated neuroblastoma diseases, were tested in the neuroblastoma cell line UKF-NB-2. APH was found to act synergistically with vincristine and synergistically to additive with doxorubicin depending on the molecular ratio of the substances in combination. This may offer the chance to use APH and its derivatives as additional tools in the treatment of neuroblastomas.

Total asymmetric synthesis of the aphidicolin derivative (11R)-(-)-8-epi-11-hydroxyaphidicolin using tandem transannular Diels-Alder/aldol reactions.

[reaction: see text] Aphidicolin unnatural derivative (2) was synthesized using a new tandem transannular Diels-Alder/aldol methodology. The 8-epi-aphidicolane skeleton is constructed in a highly diastereoselective manner and converted into (11R)-(-)-8-epi-11-hydroxyaphidicolin (2). An efficient method for the difficult C16 funtionalization is presented.

Aphidicolin glycinate inhibits human neuroblastoma cell growth in vivo.

Aphidicolin is a fungal derived tetracyclic diterpene antibiotic. It is selectively toxic for neuroblastoma (NB) cells in vitro but has no significant effects on the viability of normal human cells and a variety of other tumor entities. We evaluated the antitumoral effects of the water soluble ester aphidicolin glycinate (AphiG) on established human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. Furthermore, we explored the efficacy of direct intraneoplastic and systemic delivery of AphiG. Systemic administration of AphiG (60 mg/kg intraperitoneally, twice per day on 10 consecutive days) significantly suppressed tumor growth but was not able to induce any cures. In contrast, intratumoral AphiG injections (60 or 40 mg/kg/twice a day for 4 days) induced complete tumor regression. Two weeks after the end of treatment no tumor cells were microscopically detectable. Animals were free of tumor for more than 90 days. Histologic examination of inner organs and bone marrow did not reveal any apparent toxic effects of AphiG. These data strongly indicate that AphiG deserves further evaluation as a specific treatment for neuroblastoma.

Enhancement of melphalan activity by inhibition of DNA polymerase-alpha and DNA polymerase-beta.

Our previous studies exploring melphalan resistance in the human rhabdomyosarcoma xenograft TE-671 MR revealed elevation of DNA polymerase-alpha and DNA polymerase-beta. The present study evaluated the alteration of melphalan activity in TE-671 (melphalan-sensitive) and TE-671 MR (melphalan-resistant) subcutaneous xenografts in nude mice after DNA polymerase-alpha was inhibited using aphidicolin glycinate (AG) and DNA polymerase-beta was inhibited using dideoxycytidine (DDC). Administration of AG or DDC did not produce toxicity or demonstrate antineoplastic activity when given alone. AG (90 mg/m2) enhanced the activity of melphalan against TE-671, with growth delays increasing by 8.4, 15.8, and 21.2 days over the regimen with melphalan only. AG (180 mg/m2) only modestly increased melphalan activity against TE-671 MR, with the growth delays increasing from 9.6 and 12.1 days using melphalan alone to 12.1 and 14.5 days using melphalan plus AG. AG (180 mg/m2) plus melphalan (the dose lethal to 10% of animals) produced greater weight loss compared with melphalan alone, whereas DDC plus melphalan produced no additional toxicity. DDC modestly enhanced the activity of melphalan plus AG against TE-671 MR. AG plus O6-benzylguanine did not increase the activity of 1,3-bis(2-chloroethyl)-1-nitrosourea against TE-671 or TE-671 MR. AG (90 mg/m2 and 180 mg/m2) inhibited DNA polymerase-alpha to 80% and 72% of control in TE-671 and 64% and 37% in TE-671 MR, and DDC inhibited DNA polymerase-beta to 59% in TE-671 and 48% in TE-671 MR. These results suggest a role for AG-mediated enhancement of melphalan activity, particularly in the treatment of newly diagnosed, melphalan-sensitive tumors.

Antitumor activity and biochemical effects of aphidicolin glycinate (NSC 303812) alone and in combination with cisplatin in vivo.

Aphidicolin, an inhibitor of DNA polymerases alpha and delta, is cytotoxic in vitro against tumor cells. The poor solubility of aphidicolin has led to the development of aphidicolin glycinate (AG; NSC 303812), a water soluble ester currently in early clinical trials. The antitumor activity of AG was investigated in a series of transplantable murine tumors in vivo. The drug demonstrated activity against the i.p. implanted B16 melanoma, producing maximum increased life spans of 75% following i.p. administration every 3 h for three doses on days 1-9. Treatment schedules involving both single injections per day on days 1-9 and multiple injections per day on days 1, 5, and 9 were less effective, indicating that this antitumor activity is schedule dependent. Similarly, greater activity was observed against the i.p. M5076 sarcoma when three daily injections were given on days 1-9 (57% increased life span) than with a single injection either on days 1-9 (36% increased life span) or on days 1, 5, 9, and 13 (inactive). Further scheduling studies in the s.c. M5076 sarcoma model showed that a 7-day infusion was superior to both a 24-h infusion and a 7-day course of three bolus treatments per day. On the assumption that DNA polymerase inhibition is the basis for this antitumor activity, inhibition of DNA synthesis in BALB/c x DBA/2 F1 mice was investigated by measuring incorporation of [3H]thymidine (20 microCi, i.v.) into DNA of spleen and jejunum. At 2 h after administration of AG, inhibition of DNA synthesis was dose dependent (median inhibitory dose, 60 mg/kg in both tissues) and was > 99% at 300 mg/kg. The inhibition was rapid in onset; AG (100 mg/kg i.p.) produced maximal (> 98%) inhibition in both tissues at 30 min. Recovery occurred in the intestine within 16 h; in spleen recovery was delayed to 24 h, and was followed by a rebound incorporation at 48 h (203%). A comparison of the inhibition of thymidine incorporation in tumor cells (B16 melanoma and P388 leukemia) and normal jejunum revealed no significant differences in the extent of inhibition or the rapidity of recovery in these tissues. The rapid recovery of DNA synthesis inhibition supports the use of prolonged infusion schedules in clinical trials, but the lack of evidence of selectivity for tumor cells suggests that AG may be of limited therapeutic value as a single agent. Thus, we evaluated AG in combination with cisplatin in an in vivo model of cisplatin refractory human ovarian cancer.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of inhibitors of bacteriophage T4 DNA polymerase.

Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotides, aphidicolin and pyrophosphate analogs, but with lower sensitivities than other members of the B family DNA polymerases. The nucleotides N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAdATP) inhibited T4 DNA polymerase with competitive Ki values of 0.82 and 0.54 microM with respect to dGTP and dATP, respectively. The same compounds were more potent inhibitors in truncated assays lacking the competitor dNTP, displaying apparent Ki values of 0.001 and 0.0016 microM, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. Each of the non-substrate derivatives, BuPdGDP and BuPdGMPCH2PP, inhibited T4 DNA polymerase with similar potencies in both the truncated and variable competitor assays. These results indicate that BuPdGTP inhibits T4 DNA polymerase by distinct mechanisms depending upon the assay conditions. Reversible competitive inhibition predominates in the presence of dGTP, and incorporation in the absence of dGTP leads to potent inhibition by the modified primer:template. The implications of these findings for the use of these inhibitors in the study of B family DNA polymerases is discussed.

Isosteres of the DNA polymerase inhibitor aphidicolin as potential antiviral agents against human herpes viruses.

A variety of isosteres of the DNA polymerase inhibitor aphidicolin were synthesized as potential antiherpes agents. Modeling studies indicated that the bicyclooctane C, D rings of aphidicolin could be replaced by an aromatic moiety while maintaining the spatial arrangement of the hydroxyl group equivalent to the essential C18 hydroxyl group of aphidicolin. Of the racemic isosteres synthesized only 13, the compound with the greatest structural similarity to aphidicolin, showed any significant antiviral activity in primary assays. An enantioselective synthesis of the compound was carried out and the 4aS isomer 36 was shown to account for the observed antiviral activity noted against herpes simplex virus 1 and human cytomegalovirus.

Mass spectrometric identification and analysis of some aphidicolin metabolites in cancer patients.

We used gas chromatography/mass spectrometry to identify and quantitate some aphidicolin metabolites in plasma and urine of patients receiving aphidicolin-17-glycinate. The major metabolite found in plasma was 3-ketoaphidicolin, present at about one thent the concentrations of aphidicolin. 3-Ketoaphidicolin undergoes dehydroxymethylation to give 18-nor-3-ketoaphidicolin. This metabolite was also found in plasma and its concentration reached a maximum of 1% of aphidicolin. Small amounts of aphidicolin and 3-ketoaphidicolin were found free in urine but almost all of the drug was conjugated to glucuronic acid, as shown by mass spectrometric analysis of urine extracts and by enzymatic digestion with beta-glucoronidase followed by gas chromatographic/mass spectrometric analysis.

Activity of aphidicolin glycinate alone or in combination with cisplatin in a murine ovarian tumor resistant to cisplatin.

Aphidicolin, a reversible inhibitor of DNA polymerase alpha and delta, has recently been reported to reverse the resistance to cisplatin (DDP) of an ovarian cancer cell line. We investigated the pharmacokinetics of aphidicolin in mice and examined its activity either alone or in combination with DDP in the DDP-sensitive M5076 (M5) murine reticular cell sarcoma as well as in a DDP-resistant subline (M5/DDP). The drug was cleared from plasma very rapidly (clearance, 41.6 ml min-1 kg-1), showing a half-life of 15 min. Aphidicolin concentrations in the tumor were approximately 50% of those found in plasma at steady state. Using several dose schedules and continuous infusions we failed to detect significant antitumor activity for aphidicolin glycinate. Potentiation of the activity of DDP by aphidicolin glycinate was moderate in mice bearing M5 tumor as well as in those bearing M5/DDP tumor. These data do not support the possible clinical use of aphidicolin in combination with DDP. However, further studies should be carried out in different tumor models before this possibility is conclusively ruled out.

Phase I and clinical pharmacological evaluation of aphidicolin glycinate.

The toxicity profile and the pharmacokinetics of aphidicolin glycinate, a water-soluble analogue of aphidicolin, have been evaluated in two consecutive phase I clinical studies. In the first study, aphidicolin glycinate was given by 1-hour infusion for 5 consecutive days, every 3 weeks (daily x 5 study); in the second study, which was planned on the basis of the pharmacokinetic information obtained in the previous study, the drug was given by 24-hour continuous infusion. Treatment was repeated every 3 weeks. In the daily x 5 study, the daily dose was escalated from 12 mg/m2 to the maximum tolerated dose of 2250 mg/m2. Local toxicity was dose limiting. Elimination half-life was 2 +/- 0.2 hours (mean +/- SE) with aphidicolin being undetectable 6-8 hours after the end of the infusion. In the 24-hour continuous-infusion study, the dose was escalated from 435 mg/m2 to the maximum tolerated dose of 4500 mg/m2. Local toxicity was dose limiting, while other toxic effects were absent. The experimentally determined concentrations at the steady state were in agreement with those predicted on the basis of the available pharmacokinetic data. The targeted concentration at the steady state of 3 micrograms/mL was achieved at doses greater than or equal to 3000 mg/m2. Twenty-four-hour continuous infusion is the recommended schedule for clinical evaluations of aphidicolin glycinate as the synchronizing agent or in combination with cisplatin.