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Irofulven is a semi-synthetic derivative of Illudin S, a toxic sesquiterpene isolated from the mushroom Omphalotus illudens. Irofulven has displayed significant antitumor activity in various clinical trials but displayed a limited therapeutic index. A new derivative of irofulven was prepared by reacting hydroxyurea with irofulven under acidic conditions. Acetylation of this new compound with acetic anhydride produced a second derivative. Both of these new derivatives displayed significant antitumor activity in vitro and in vivo comparable to or exceeding that of irofulven.

Illudin S and M (1, 2) are highly toxic sesquiterpenes found in the basidiomycete Omphalotus illudens. Illudins have a low therapeutic index, but acylfulvene derivatives display potent in vivo antitumor activity against a variety of multidrug resistant tumors. The lead acylfulvene (4), irofulven (5), in a randomized phase IIB clinical trial significantly increased overall survival in patients with metastatic hormone-refractory prostate cancer who failed prior treatment with two different standard chemotherapeutic regimens. Irofulven is unique, as the primary allylic hydroxyl group can undergo displacement with a variety of nucleophiles to produce analogues that retain key functional groups required for biological activity including the reactive cyclopropylmethyl carbinol and alpha,beta-unsaturated ketone. As described here, we synthesized a variety of urea, carbamate, and sulfonamide derivatives that retain key functional groups and display potent biological activity toward target solid tumor cells in vitro but are relatively nontoxic toward a nontarget B-cell derived cell line.

Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway.The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents.Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA.These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

The novel agent Irofulven (HMAF, NSC 683863) has demonstrated significant antitumor activity against solid tumors in various xenograft models and human clinical trials. The antitumor potential of combining irofulven with 72 different anti-metabolite, enzyme inhibiting, and miscellaneous agents was investigated in this study. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with these different agents. Irofulven in combination with select anti-metabolites, notably cytidine or adenine-derived agents, displayed strong synergistic activity in both in vitro and in vivo studies. Agents demonstrating strong synergistic interaction with irofulven included gemcitabine, cyclocytidine, cytarabine, fludarabine phosphate, cladribine, and 5-fluorouracil. Other anti-metabolites, enzyme inhibitors, and a variety of miscellaneous agents failed to interact beneficially when administered in combination with irofulven. The therapeutic activity of irofulven is enhanced considerably when irofulven is combined with select anti-metabolite agents, and further clinical evaluation of these combinations is warranted. The synergistic interaction with these combinations may stem from a variety of actions including inhibition of the nucleotide excision repair (NER) pathway, topoisomerase I activity, and caspase-dependent and independent induction of apoptosis.

(Hydroxymethyl)acylfulvene (HMAF, irofulven; 4), a third-generation derivative of a natural product extracted from the mushroom Omphalotus illudens, is selectively toxic towards certain forms of malignant tumors. Conversion of HMAF and cognates to stable aromatic derivatives is triggered by thiol attack in vitro and in vivo. Quantum-chemical methods predict well the structure for several functionalized derivatives of irofulven as compared to known X-ray crystallographic structures. Computational reaction profiles for thiol attack and aromatic rearrangement of irofulven and illudin S, a toxin from which irofulven is derived, provide insight into HMAF's selectivity and toxicity. Methods used include hybrid density-functional theory (HDFT), HartreeFock (HF), and MollerPlesset second-order perturbation theory (MP2). Solvent effects have been explored by means of the new continuum-solvation method, COSab, presented in an accompanying paper.

Bicyclic and tricyclic analogues of anticancer sesquiterpene illudin S have been synthesized. These contain a spiro-cyclobutane instead of spiro-cyclopropane structure. The cytotoxicity of the former is less than that of the corresponding cyclopropane-containing compounds.

Illudin S is a natural sesquiterpene drug with strong anti-tumour activity. Inside cells, unstable active metabolites of illudin cause the formation of as yet poorly characterised DNA lesions. In order to identify factors involved in their repair, we have performed a detailed genetic survey of repair-defective mutants for responses to the drug. We show that 90% of illudin's lethal effects in human fibroblasts can be prevented by an active nucleotide excision repair (NER) system. Core NER enzymes XPA, XPF, XPG, and TFIIH are essential for recovery. However, the presence of global NER initiators XPC, HR23A/HR23B and XPE is not required, whereas survival, repair and recovery from transcription inhibition critically depend on CSA, CSB and UVS, the factors specific for transcription-coupled NER. Base excision repair and non-homologous end-joining of DNA breaks do not play a major role in the processing of illudin lesions. However, active RAD18 is required for optimal cell survival, indicating that the lesions also block replication forks, eliciting post-replication-repair-like responses. However, the translesion-polymerase DNA pol eta is not involved. We conclude that illudin-induced lesions are exceptional in that they appear to be ignored by all of the known global repair systems, and can only be repaired when trapped in stalled replication or transcription complexes. We show that the semisynthetic illudin derivative hydroxymethylacylfulvene (HMAF, Irofulven), currently under clinical trial for anti-tumour therapy, acts via the same mechanism.

Purpose: Irofulven (6-hydroxymethylacylfulvene, MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a toxin present in the Omphalotus mushroom. Irofulven has demonstrated activity against a broad range of solid tumors in both xenograft models and human trials. The potential application of administering irofulven in combination with aziridine-containing chemotherapeutic agents was evaluated in this study. Methods: Human lung carcinoma MV522 cells and BALB/c athymic mice bearing the human lung carcinoma MV522 xenograft were used to evaluate the activity of irofulven in combination with aziridine-containing drugs. Results: Irofulven in combination with either thiotepa or mitomycin C demonstrated a strong synergistic (supraadditive) activity both in vitro and in vivo, that exceeded results obtained with monotherapy at the same or higher doses of these agents. The majority of xenograft-bearing animals that received subtoxic doses of irofulven, and either thiotepa or mitomycin C, demonstrated a complete cure. In contrast, there was no detectable synergistic activity between irofulven and other aziridine-containing drugs, including AZQ and thiotepa metabolites such as TEPA or AZD. Conclusions: These results indicate that the therapeutic activity of irofulven is enhanced when combined with mitomycin C or thiotepa, and further evaluation of these combinations is therefore warranted.

Many analogues of the antitumor agent irofulven have been readily prepared by replacing the allylic hydroxyl with a variety of nucleophiles. Analogues of acylfulvene (the precursor to irofulven) were also prepared by Michael reaction with acrolein. The toxicity of the analogues was determined, as well as preclinical antitumor activity. Several analogues exhibited good activity in mouse xenografts. Structural requirements for activity are discussed.

The primary hydroxyl group in hydroxymethylacylfulvene, a potent antitumor drug, is readily replaced by thiols including cysteine, N-acetylcysteine, homocysteine, and glutathione. Best yields are obtained when reaction is carried out in the presence of dilute sulfuric acid. A variety of sulfur-containing analogues have been prepared, and their toxicity to tumor cells was examined.

Tritiated derivatives of the toxic sesquiterpene illudin S (1) have been prepared by fermentation of Omphalotus illudens in the presence of [3H]-sodium acetate. [3H]-illudin S was converted to antitumour [3H]-acylfulvene (4) by treatment with dilute sulfuric acid. Antitumor [14C]-hydroxymethylacylfulvene (5) was best prepared by reacting acylfulvene with [14C]-paraformaldehyde in dilute sulfuric acid. © 1998 John Wiley & Sons, Ltd.

Purpose: Acylfulvene derivatives demonstrate marked efficacy in xenograft carcinoma models as compared with the parent illudin compounds. To elucidate the increased therapeutic efficacy of acylfulvene analogs, we compared them with the illudin compounds in terms of their in vitro cytotoxicity, cellular accumulation and DNA incorporation. Methods: The cytotoxicity of various acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabelled acylfulvene analog was prepared and used for cellular accumulation and DNA incorporation studies. Results: The prototype acylfulvene analog retained selective histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines. In vitro killing of tumor cells by acylfulvene required up to a 30-fold increase in molecules per cell, as compared with illudin S, indicating that acylfulvene was less toxic on a cellular level. At equitoxic concentrations, acylfulvene incorporation into genomic tumor cell DNA was equivalent to illudin S suggesting that cellular metabolism has a role in acylfulvene cytotoxicity. Analysis of cellular accumulation of acylfulvene into tumor cells revealed a markedly higher Vmax for tumor cells, and a lower Vd for diffusion accumulation into other cells. Conclusions: The combination of higher Vmax and lower Vd may explain the increased in vivo efficacy of acylfulvene.

Brassinolide and related brassinosteroids are a novel group of steroids which appear to be ubiquitous in plants. There is compelling evidence, particularly from recent genetic studies, that these steroids are essential for normal plant growth and development. Synthesis of brassinosteroids and aspects of their biochemistry are reviewed.

Hydroxymethylacylfulvene (HMAF) is a semisynthetic derivative of the toxic sesquiterpene illudin S. HMAF (also designated MGI-114) has potent antitumor properties and is currently in clinical trials. It reacts with thiols at neutral and acidic pH forming novel products in which the primary allylic hydroxyl is displaced by thiol. These derivatives retain antitumor activity.

Illudins are novel low molecular weight natural products cytotoxic to human tumor cells in vitro. Illudin-derived analogs are effective against experimental human cancers nonresponsive to conventional anticancer agents. It is not known why some illudin analogs are more efficacious in vitro and in vivo than other analogs. Therefore, the in vitro cytotoxicity of the parent compound illudin S towards tumor cells was characterized using radiolabeled drug. Two cell lines sensitive at nanomolar concentrations using only a 15-min exposure period displayed a saturable, energy-dependent accumulation of illudins with relatively low K(m) and high Vmax values. A nonsensitive cell line, requiring millimolar concentrations to achieve in vitro toxicity, showed minimal illudin uptake with higher K(m) and lower Vmax values. No release of radioactivity could be demonstrated from tumor cells, indicating that there was no efflux of illudin S (or metabolites) from these cells. The number of intracellular illudin S molecules required to kill 50% of cells of different tumor cell lines varied from 78000 to 1114000 molecules per cell and was correlated with the 2-h IC50 value determined using a colony-forming assay. Illudin S was cytotoxic to a variety of multidrug-resistant tumor cell lines regardless of whether resistance was mediated by gp170/mdrl, gp180/MRP, GSHTR-pi, topoisomerase I, topoisomerase II, increased DNA repair capacity, or alterations in intracellular thiol content. Information obtained in this study could be used to design clinical phase I trials and to develop analogs with improved therapeutic indexes.

Brassinosteroids are widely distributed plant compounds that modulate cell elongation and division, but little is known about the mechanism of action of these plant growth regulators. To investigate brassinosteroids as signals influencing plant growth and development, we identified a brassinosteroid-insensitive mutant in Arabidopsis thaliana (L.) Henyh. ecotype Columbia. The mutant, termed bri1, did not respond to brassinosteroids in hypocotyl elongation and primary root inhibition assays, but it did retain sensitivity to auxins, cytokinins, ethylene, abscisic acid, and gibberellins. The bri1 mutant showed multiple deficiencies in developmental pathways that could not be rescued by brassinosteroid treatment, including a severely dwarfed stature; dark green, thickened leaves; male sterility; reduced apical dominance; and de-etiolation of darkgrown seedlings. Genetic analysis suggests that the Bri1 phenotype is caused by a recessive mutation in a single gene with pleiotropic effects that maps 1.6 centimorgans from the cleaved, amplified, polymorphic sequence marker DHS1 on the bottom of chromosome IV. The multiple and dramatic effects of mutation of the BRI1 locus on development suggests that the BRI1 gene may play a critical role in brassinosteroid perception or signal transduction.

Illudin analogs are cytotoxic to a variety of multidrug resistant cell lines, and display an unusual toxicity towards DNA helicase-deficient cell lines. Earlier illudin analogs demonstrated efficacy in several xenograft models, including a metastatic MV522 lung cancer model, resistant to conventional anticancer agents. These illudin analogs prolonged life span as compared to conventional agents, but did not induce complete remission of primary tumors. In vitro screening studies identified a semisynthetic derivative, hydroxymethylacylfulvene (HMAF, MGI-114), with increased selective cytotoxicity towards carcinoma cells. The HMAF analog was markedly effective in the experimental MV 522 metastasizing lung carcinoma xenograft system, a model refractory to treatment with existing anticancer agents. Treatment with paclitaxel, doxorubicin, or cisplatin failed to significantly inhibit primary tumor growth or prolong life span of MV522 tumor-bearing animals. Treatment with mitomycin C at the LD20 increased life span in surviving animals up to 61% (p = 0.04). Treatment with HMAF induced primary tumor regression in all animals and increased life span greater than 150% (p0.001). Thus, administration of HMAF inhibited development of lung metastasis in a model refractory to treatment with conventional anticancer agents. These results support further evaluation of HMAF as a therapeutic agent for treatment of solid tumors such as adenocarcinoma of the lung.

Acylfulvene, derived from the sesquiterpene illudin S by treatment with acid (reverse Prins reaction), is far less reactive to thiols than illudin S. However, it is reduced readily to an aromatic product, in the same way as illudin S. This may explain its greatly improved therapeutic index compared to that of the parent compound.

Nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that mediate the transcriptional activities of steroids, retinoids, and thyroid hormones. A growing number of related proteins have been identified that possess the structural features of hormone receptors, but that lack known ligands. Known as orphan receptors, these proteins represents targets for novel signaling molecules. We have isolated a mammalian orphan receptor that forms a heterodimeric complex with the retinoid X receptor. A screen of candidate ligands identified farnesol and related metabolites as effective activators of this complex. Farnesol metabolites are generated intracellularly and are required for the synthesis of cholesterol, bile acids, steroids, retinoids, and farnesylated proteins. Intermediary metabolites have been recognized as transcriptional regulators in bacteria and yeast. Our results now suggest that metabolite-controlled intracellular signaling systems are utilized by higher organisms.

Illudins, novel natural products with a structure unrelated to any other known chemical, display potent in vitro and in vivo anti-cancer activity against even multi-drug resistant tumors, and are metabolically activated to an unstable intermediate that binds to DNA. The DNA damage produced by illudins, however, appears to differ from that of other known DNA damaging toxins. The sensitivity pattern of the various UV-sensitive cell lines differs from previously studied DNA cross-linking agents. Normally, the ERCC1- (excision repair cross complementing) and ERCC4-deficient cell lines are most sensitive to DNA cross-linking agents, with ERCC2-, ERCC3- and ERCC5-deficient cell lines having minimal sensitivity. With illudins the pattern is reversed, with ERCC2 and ERCC3 being the most sensitive. The sensitivity to illudins in complementation groups 1 through 3 is due to a deficiency of the ERCC1-3 gene products, as cellular drug accumulation studies revealed no differences in transport capacity or total drug accumulation. Also, a transgenic cell line in which ERCC2 activity was expressed through an expression vector regained its relative resistance to the illudins. The EM9 cell line, which displays sensitivity to monoadduct producing chemicals, was not sensitive. Thus, excision repair is involved in repair of illudin-induced damage and, unlike other anti-cancer agents, the involvement of ERCC2 and ERCC3 helicases is critical for repair to occur. The requirement for ERCC2 and ERCC3, combined with the finding that ERCC1 but not ERCC2 is upregulated in drug-resistant tumors, may explain the efficacy of illudins against drug-resistant tumors. The inhibition of DNA synthesis in cells within minutes after exposure to illudins at nanomolar concentrations may be related to the finding that the ERCC3 gene product is actually the p89 helicase component of the BTF2 (TFII) basic transcription factor and the high sensitivity of ERCC3-deficient cells to illudins.

28-Homobrassinolide has been synthesized from stigmasterol in an overall yield of 21%. The biological activity of 28-homobrassinolide, brassinolide, 24-epibrassinolide, 22 S ,23 S ,24-epibrassinolide, and 22 S ,23 S ,28-homobrassinolide have been compared in the soybean epicotyl elongation assay. All the steroids except 22 S ,23 S ,28-homobrassinolide exhibited a similar biological activity, but the latter compound was substantially less active. There appears to be a correlation between biological activity and overall dimensions of the steroidal side chain.

We examined the effects of brassinosteroids on Arabidopsis thaliana (L.) Henyh. ecotype Columbia in order to develop a model system for studying gene regulation by plant steroids. Submicromolar concentrations of two brassinosteroids, brassinolide and 24-epibrassinolide, stimulated elongation of Arabidopsis peduncles and inhibited root elongation, respectively. Furthermore, brassinolide altered the abundance of specific in vitro translatable mRNAs from peduncles and whole plants of Arabidopsis. Root elongation in the auxin-insensitive Arabidopsis mutant axr1 was inhibited by 24-epibrassinolide but not by 2,4-D, indicating an independent mode of action for these growth regulators in this physiological response.

Oogonium development in female strains of the water mould Achlya is initiated in nature by a steroid mixture of oogoniol, dehydro-oogoniol and thei

Methylene acetal of an epimer of illudin S ( 5 ) was obtained by treating illudin S with a large excess of paraformaldehyde in 1N H 2 SO 4 solution. X-ray crystallographic analysis showed that the configuration at the allylic carbon has been inverted. This acetal was found to be less toxic and showed a higher therapeutic index than illudin S.

Hydroxymethylacylfulvene (HMAF) is a semisynthetic derivative of the toxic sesquiterpene illudin S. HMAF (also designated MGI-114) has potent antitumor properties and is currently in clinical trials. It reacts with thiols at neutral and acidic pH forming novel products in which the primary allylic hydroxyl is displaced by thiol. These derivatives retain antitumor activity.

Illudin S and M are extremely toxic sesqiuterpenes produced by Omphalotus illudens. At low pH they behave as functional alkylating agents, but at physiological pH they do not react with oxygen or nitrogen nucleophiles. Illudins react spontaneowly with sulfur nucleophiles, glutathioone or cysteine, at or slightly below pH 7, and toxicity to HL 60 cell can be modulated by altering glutathione levels in cells

We have studied the effect of brassinolide (BR), a plant steroidal lactone, on the expression of auxin-regulated genes in soybean (Glycine max L. cv Williams 82) epicotyls. BR caused up to 4-fold increases in epicotyl length during extended assays at 10(-7)m, in the absence of added auxin. Structurally related steroids failed to induce elongation or to alter the BR effect. Northern blot analysis, using sequences corresponding to auxin-regulated genes as probes, has shown that the molecular mechanism of BR-induced elongation is likely to differ from that of auxin-induced elongation in this system. BR does not rapidly induce members of the GH, SAUR, or JCW auxin-inducible gene families before the onset of elongation. BR enhances SAUR and GH1 transcripts after 18 h but has no effect on JCW1 or GH3 transcripts at any time examined. We have shown by two-dimensional gel analysis of in vitro translated mRNA that a submicromolar concentration of BR alters the pattern of gene expression in elongating soybean epicotyls.

Illudin M and illudin S, antitumor sesquiterpenes from Omphalotus illudens, have been found to react with thiols in a pH-dependent manner. The optimum pH values for reaction of illudin M with methyl thioglycolate, cysteine, and glutathione were 5.8, 5.6, and 6.1, respectively, and pseudo-first-order rate constants at 25 degrees C (10-fold excess of thiol) were 44 x 10(-3), 11.5 x 10(-3), and 11.3 x 10(-3) min-1. In all cases, thiol added to the alpha, beta-unsaturated ketone giving an unstable intermediate. Subsequent loss of the tertiary hydroxyl and opening of the cyclopropane ring afforded a stable aromatic product. The toxicity of illudin S to HL60 cells was increased by lowering glutathione levels in the cells and vice versa. General toxicity and antitumor activity of illudins are discussed in the light of these results.

Acylfulvenes, a class of semisynthetic analogs of Illudin S, show high toxicity toward prostate cancer cells. Here we probe the effect of changes in hydrophilic character of the analogs.

Stereoselective synthesis of (-)-irofulven has been achieved by cycloaddition of (R)-5-chloro-5-methyl-2-cyclopentenone to the 1,3-dipolar intermediate from 1-acetyl-1-(diazoacetyl)cyclopropane. The enantiomer, (+)-irofulven, was prepared in a similar way starting with (S)-5-chloro-5-methyl-2-cyclopentenone. (+)-Irofulven was 5 to 6 times less toxic than (-)-irofulven to adenocarcinoma (MV 522) cells.

Chemical investigation on the flowers of Parthenium hysterophorus has resulted in the isolation of four new pseudoguaianolides, hysterones A-D along with the known compounds, parthenin, coronopilin, 2beta-hydroxycoronopilin and tetraneurin-A. The structures of the new compounds were established by interpretation of their spectral (1D and 2D NMR) data. The X-ray crystallographic analysis of hysterones A and C was also carried out.

Reaction of antitumor agent irofulven (1) with zinc and acetic acid yielded several new indene derivatives (6, 7, 8, 10) as well as the known indene (9). These all have greatly reduced toxicity to human leukemia (HL60) cells compared to irofulven.

Three sesquiterpenes, illudosone hemiacetal (1a), isoomphadione (2) and illudiolone (3) were isolated from the liquid culture extract of Omphalotus illudens. Their structures were elucidated by spectroscopic techniques as well as by X-ray crystallographic analysis.

Reduction of (+/-)-5-chloro-5-methyl-2-cyclopentenone with BH(3).THF and catalytic (R)-2-methyl-CBS-oxazaborolidine gave readily separable diastereomers with high ee values. Oxidation of the diastereomers furnished the enantiomers of the chloromethylcyclopentenone.

A new sesquiterpene, omphadiol (4), has been isolated from cultures of Omphalotus illudens. Several known compounds, including illudosin (1), were also obtained. Structures were determined using MS, NMR, and X-ray techniques.

Purpose: The acylfulvenes are a class of antitumor agents derived from the fungal toxin illudin S. One acylfulvene derivative, MGI 114 (HMAF), demonstrates marked efficacy in xenograft carcinoma models when compared to the parent acylfulvene or related illudin compounds. The maximum tolerated dose (MTD) of the two analogs in animals, however, is similar. To help elucidate the basis of the increased therapeutic efficacy of MGI 114, we determined the in vitro cytotoxicity, cellular accumulation and DNA incorporation of this drug and compared the results with those from the parent acylfulvene analog. Methods: The cytotoxicity of acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabeled MGI 114 was used for cellular accumulation and DNA incorporation studies. Results: MGI 114 retained relative histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines previously noted with the parent acylfulvene compound. Markedly fewer intracellular molecules of MGI 114 were required to kill human tumor cells in vitro as compared to the parent acylfulvene, indicating that MGI 114 was markedly more toxic on a cellular level. At equitoxic concentrations, however, the incorporation of MGI 114 into genomic tumor cell DNA was equivalent to that of acylfulvene. Analysis of cellular accumulation of MGI 114 into tumor cells revealed a lower Vmax for tumor cells, and a markedly lower Vd for diffusion accumulation as compared to acylfulvene. Conclusions: The addition of a single methylhydroxyl group to acylfulvene to produce MGI 114 results in a marked increase in cytotoxicity in vitro towards tumor cells as demonstrated by the reduction in IC50 values. There was a corresponding decrease in the number of intracellular molecules of MGI 114 required to kill tumor cells, but no quantitative alteration in covalent binding of the drugs to DNA at equitoxic concentrations. This indicates that cellular metabolism plays a role in the in vitro cytotoxicity of MGI 114. The equivalent incorporation into genomic DNA at equitoxic doses suggests that DNA damage produced by acylfulvene and MGI 114 is equivalent in regard to cellular toxicity and ability to repair DNA. This increased cellular toxicity, together with the decrease in diffusion rate, may explain the increased therapeutic efficacy of MGI 114 as compared to the parent acylfulvene analog.

Hydroxymethylacylfulvene (HMAF, MGI 114) is derived from the sesquiterpene illudin S by treatment with dilute sulfuric acid and excess paraformaldehyde. It is less cytotoxic than illudin S but exhibits much greater selectivity in toxicity to malignant cells compared to normal cells. HMAF is believed to undergo bioreductive activation in vivo. It is now being tested in human clinical phase II trials against solid tumors.

Illudins are novel compounds from which a potent class of antitumor agents, called acylfulvenes, have been synthesized. The model illudin, illudin S, has marked in vitro and in vivo toxicity but displays a poor therapeutic index. The toxicity of illudin S is believed to involve a combination of enzymatic reduction and chemical reaction. Enzymatic reduction by a cytosolic NADPH-dependent enzyme produces an aromatic metabolite, as does reaction with thiols. Acylfulvene is formed from illudin S by reverse Prins reaction. Acylfulvene is 100-fold less toxic in vitro and in vivo than illudin S but possesses marked antitumor efficacy in vivo, thus displaying opposite properties from illudin S. For this reason we investigated the in vitro metabolism of acylfulvene. Incubation of acylfulvene with NADPH and rat liver cytosol yielded two metabolites. One metabolite, the aromatic product, is similar to that obtained with illudin S in this in vitro system and was anticipated. The other metabolite, the hydroxylated product, was not expected and no corresponding metabolite for illudin S could be detected. The production of this hydroxylated metabolite from acylfulvene may explain, in part, the increased antitumor activity of novel acylfulvenes as compared with the illudins.

Illudins are a novel class of agents with a chemical structure entirely different from current chemotherapeutic agents. A new semisynthetic derivative, MGI 114 (NSC 683,863, 6-hydroxymethyl-acylfulvene, HMAF), is markedly effective in a variety of lung, breast and colon carcinoma xenograft models. This analogue, MGI 114, is currently in phase I human clinical trials, and is scheduled for two different phase II trials. To determine if MGI 114 could be effective in vivo against mdr tumour cells, we generated an mdr1/gp170-positive clone of the metastatic MV522 human lung carcinoma line by transfecting a eukaryotic expression vector containing the cDNA encoding for the human gp170 protein. This MV522/mdr1 daughter line retained the metastatic ability of parental cells. The parental MV522 xenograft is mildly responsive in vivo to mitomycin C and paclitaxel, as evidenced by partial tumour growth inhibition and a small increase in life span, whereas MV522/mdr1 xenografts were resistant to these agents. In contrast to mitomycin C and paclitaxel, MGI 114 produced xenograft tumour regressions in 32 of 32 animals and completely eliminated tumours in more than 30% of MV522/mdr1 tumour-bearing mice. Thus, MGI 114 should be effective in vivo against mdr1/gp170-positive tumours.

Reaction of the fungal sesquiterpene illudin S with excess paraformaldehyde in dilute H2SO4 gives (hydroxymethyl)acylfulvene. The primary allylic hydroxyl thus formed can undergo very facile replacement by a variety of nucleophiles. (Hydroxymethyl)acylfulvene (MGI.114) was more toxic than a precursor, acylfulvene, but less toxic than the parent compound illudin S to HL 60 cells.

Although steroid hormones are important for animal development, the physiological role of plant steroids is unknown. The Arabidopsis DET2 gene encodes a protein that shares significant sequence identity with mammalian steroid 5 alpha-reductases. A mutation of glutamate 204, which is absolutely required for the activity of human steroid reductase, abolishes the in vivo activity of DET2 and leads to defects in light-regulated development that can be ameliorated by application of a plant steroid, brassinolide. Thus, DET2 may encode a reductase in the brassinolide biosynthetic pathway, and brassinosteroids may constitute a distinct class of phytohormones with an important role in light-regulated development of higher plants.

Irofulven (NSC 683863), a semi-synthetic analog of the fungal product illudin S, that has been evaluated in a variety of phase I and II clinical trials. Promising results were noted in clinical trials against a variety of solid cancers including colon, sarcoma, prostate, liver, ovarian and pancreas. Irofulven in a randomized phase IIB trial increased median overall lifespan in patients with metastatic taxane-refractory prostate cancer [HR = 0.69]. As irofulven was the first member of the acylfulvene class of agents to be created, we synthesized several hundred analogs for further testing. Modifications were specifically made to 7 sites of the irofulven molecule including the cyclopropane ring, the chiral center, the carbonyl group, the tertiary hydroxyl site, and several nucleophilic sites. Modifications at several sites completely eliminated both the cytotoxicity and antitumor activity of the analog. Analogs with modifications at one site retained cytotoxic activity, as well as in vivo toxicity in rodents, but surprisingly eliminated antitumor activity in xenograft models. Analogs in which the tertiary hydroxyl group of irofulven was replaced with specific functional groups were noted to have an increase in tumor selectivity as noted by both cell culture and xenograft studies. Specific functional groups noted to be favorable for increased activity included carbamate, sulfonamide, and urea moieties. Additional derivatives of each of these specific functional groups were synthesized and tested in a variety of cell culture and xenograft studies including multidrug resistant models. These studies identify molecular sites critical for the antitumor activity of acylfulvenes, as well as identify promising new analogs for further investigation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 751.

Illudins are potent natural products derived from Omphalotus illudens and related fungi. The chemical structure of illudins differs from that of other conventional chemotherapeutic agents. While illudins are toxic to most tumor cells after prolonged exposure (greater than or equal to 48 hr), with shorter exposure times (less than or equal to 2 hr), they show selective toxicity for human myelocytic leukemia and epidermoid, lung, ovarian, and breast carcinoma cells of various species of origin. The apparent histologic specificity of illudin S toxicity is based on an energy-dependent transport mechanism present in sensitive cells, but absent in cells relatively resistant to illudin S. For human myeloid leukemia HL60 cells, the Michaelis constant was 4.2 microM and the maximum velocity was 12.2 pmol/minute per 10 million cells or 730 pmol/hour per 10 million cells. The energy-dependent transport mechanism was detected in other mammalian tumor cells.

Methylene acetal of an epimer of illudin S ( 5 ) was obtained by treating illudin S with a large excess of paraformaldehyde in 1N H 2 SO 4 solution. X-ray crystallographic analysis showed that the configuration at the allylic carbon has been inverted. This acetal was found to be less toxic and showed a higher therapeutic index than illudin S.

(±)-Hydroxymethylacylfulvene is synthesized in 14 steps from 4-hydroxy-5-methyl-2-cyclopenten-1-one and 1-acetyl-1-(diazoacetyl)cyclopropane in 15% overall yield.

Brassinolide has been synthesized from stigmasterol in an overall yield of 7%. The key step in the synthesis is aldol condensation of 2α,3α-isopropylidenedioxy-6-oxo-23,24-dinor-5α-cholan-22-al with 3-isopropylbut-2-enolide carried out at – 78 °C, which gives a product with 22R,23R stereochemistry in high yield. Catalytic hydrogenation of this product is highly stereoselective leading to the desired 24S stereochemistry in an intermediate which is readily transformed into brassinolide.