Nodulisporic Acid Research Articles

Indole Diterpenoid Synthetic Studies. Construction of the Heptacyclic Core of (‐)‐Nodulisporic Acid D (I).

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Indole Diterpenoid Synthetic Studies. Construction of the Heptacyclic Core of (−)-Nodulisporic Acid D

[structure: see text] Construction of the heptacyclic core of (-)-nodulisporic acid D, a representative member of a recently discovered class of architecturally complex, ectoparasiticidal indole alkaloids, has been achieved. The modular synthetic strategy comprises an expedient, stereocontrolled synthesis of a tricyclic western hemisphere, in conjunction with union of an eastern hemisphere, exploiting the 2-substituted indole synthetic protocol introduced and developed in our laboratory.

Indole Diterpenoid Synthetic Studies. The Total Synthesis of (+)-Nodulisporic Acid F

[structure: see text] A stereocontrolled total synthesis of (+)-nodulisporic acid F, the simplest member of a family of novel ectoparasiticidal agents, has been achieved. Highlights of the effective modular synthetic strategy include anionic union of a tricyclic lactone with o-toluidine via our 2-substituted indole synthetic protocol, an optimized C-ring construction protocol, and a late-stage installation of the alpha,beta-unsaturated carboxylic acid side chain via the B-alkyl Suzuki-Miyaura cross-coupling tactic.

Nodulisporic acids D-F: structure, biological activities, and biogenetic relationships.

Nodulisporic acids D, E, and F are the newest members of a family of nontremorogenic indole-diterpenoids that are potent, orally bioavailable, antiflea agents derived from a fungus belonging to the genus Nodulisporium. The four members of the D series are each devoid of an isoprene residue that is present at C-26 in the three nodulisporic acids described originally (the A series). Nodulisporic acid E (11a) has a simpler structure, which lacks not only the isoprene residue at C-26 but also two that form the A/B rings. Nodulisporic acid F is the simplest of all nodulisporic acids and is devoid of all three isoprene residues of the indole unit; as such, it represents the earliest biosynthetic intermediate in this series. A biogenetic grid based on mutation studies is proposed that encompasses all the known nodulisporic acids. Structure-activity relationships of the known natural nodulisporic acids have been elucidated. Within a series the most active compound possesses a dienoic acid chain, and overall, the end product of the biogenetic grid, i.e., nodulisporic acid A, exhibits the most potent antiflea activity. Additionally, the stereochemistries of C-3' ' and C-4' ' of nodulisporic acid D(2) and therefore of nodulisporic acids A(2), B(2), and C(2) have been assigned.

Nodulisporic Acid B, B1, and B2: A Series of 1′‐Deoxy‐nodulisporic Acids from Nodulisporium sp.

Abstract During the re-isolation of the lead compound nodulisporic acid A ( 1a ) and targeted chemical screening for related compounds, we discovered a series of 1′-deoxy congeners named herein nodulisporic acids B ( 1b ), B 1 ( 2b ), and B 2 ( 3b ). In comparison with nodulisporic acid A, these compounds were less active and were chemically unstable resulting into formation of Δ 23 dehydro derivatives. Therefore, these compounds were stabilized and isolated as sodium salts and methyl ester. Nodulisporic acid B is 100-fold less active than nodulisporic acid A against fleas. The isolation, structure elucidation, and biological activities of these compounds are described.

Nodulisporic acids C, C1, and C2: a series of D-ring-opened nodulisporic acids from the fungus Nodulisporium sp.

Nodulisporic acids C (4a), C1 (5a), and C2 (6a), a series of D-ring-opened nodulisporic acids, were isolated from fermentations of a mutant culture of Nodulisporium sp. MF5954. Nodulisporic acid C, the most potent of the three, showed good activity against fleas with an LD90 of 10 micro g/mL. These compounds tend to be unstable in the free acid form and were isolated as stable sodium salts.

Nodulisporic acid side-Chain modifications: access to the 2″, 3″, 4″, and 6″ registers

Efficient routes to the 2″, 3″, 4″, and 6″ registers of the nodulisporic acid side chain are disclosed.

VNTR polymorphism in the ITS1 region (rDNA) in isolates of a Nodulisporium species

Variable Number of Tandem Repeats (VNTR) polymorphism has been identified in the ITS1 region of the rDNA in 13 strains belonging to a species of Nodulisporium producing the novel indole diterpene nodulisporic acid. The number of tandem repeats found in these isolates varies from 2, 4 or 5 repetitions, and produces three length morphs in the ITS1 rDNA, spanning a length difference of 217, 308 and 352 nucleotides. Repeated units are rather variable, and they encompass three tandem repeated motifs of 11 nucleotides. In all the individuals, the variants are positioned in the same physical order, following the distribution of the most parsimonious tree. This suggests that this sequence have been enlarged by slipped-strand mispairing occurrences.

Side‐Chain Homologation of Nodulisporic Acid: Synthesis of Potent New Dienyl Derivatives.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Nodulisporic acid B, B1, and B2: a series of 1′-deoxy-nodulisporic acids from Nodulisporium sp.

During the re-isolation of the lead compound nodulisporic acid A (1a) and targeted chemical screening for related compounds, we discovered a series of 1′-deoxy congeners named herein nodulisporic acids B (1b), B1 (2b), and B2 (3b). In comparison with nodulisporic acid A, these compounds were less active and were chemically unstable resulting into formation of Δ23 dehydro derivatives. Therefore, these compounds were stabilized and isolated as sodium salts and methyl ester. Nodulisporic acid B is 100-fold less active than nodulisporic acid A against fleas. The isolation, structure elucidation, and biological activities of these compounds are described.

Nodulisporic acid: its chemistry and biology.

The discovery of the natural product nodulisporic acid A (NsA A) and its potent, systemic insecticidal activity at Merck Research Laboratories in 1992 stimulated intense scientific scrutiny. Interest in this new class of indole diterpenes led to extensive delineation of its properties, both chemical and biological. Synthetic modification of NsA A served to identify its pharmacologically permissive and non-permissive regions, produced semisynthetic derivatives with enhanced adulticidal flea efficacy (both in vitro and in vivo), and provided useful tools to support biological studies. Early observations in rodent models showed a wide therapeutic index for NsA A and detailed mechanism of action investigations demonstrated that it selectively modulated an invertebrate specific glutamate-gated ion channel for which no mammalian homolog exists. Consistent with these mechanistic conclusions, dogs treated orally with either NsA A or closely related amide analogs (15 mg/kg dosages) showed no apparent toxicity, and measurement of systemic flea efficacy in these animals demonstrated that prolonged antiparasitic activity was attained, up to 14 days subsequent to treatment with a single p.o. dose for fleas or up to 4 weeks for ticks. The extended flea efficacy was correlated to both the in vivo pharmacokinetic profile of a given analog and its intrinsic in vitro potency against fleas. In addition, studies directed towards the total synthesis of NsA A have been reported.

Biosynthesis of nodulisporic acid A: precursor studies.

Nodulisporic acid A (NAA) is an indole-diterpene natural product produced by an indeterminate species of the endophytic fungus Nodulisporium. NAA (Figure 1) is structurally related to the paspaline class of fungal metabolites. The biosynthetic origin proposed for these alkaloids involves the acetate/mevalonic acid pathway leading to geranylgeranyl pyrophosphate (GGPP). GGPP is then proposed to condense with tryptophan to form the basic indole-diterpene core. A washed cell procedure was devised to incorporate labeled precursors into NAA by a mutant Nodulisporium culture designated MF6244. Incorporation of 2-(13)C-acetate and 2-(13)C-mevalonolactone into NAA was found to occur in the classical mevalonic acid pattern. In addition to the four mevalonic acid units that form the eastern side of the molecule, three additional isoprenylations occur to form the western and southern regions of NAA. Contrary to published reports on related compounds, incubations of Nodulisporium MF6244 with (14)C- and (13)C-tryptophan showed no incorporation of label into NAA. However, high levels of incorporation into NAA were obtained with known tryptophan precursors (14)C-, (13)C-, and (15)N-anthranilic acid and (14)C- and (13)C-ribose. A novel pathway for the biosynthesis of NAA is presented.

Side-chain homologation of nodulisporic acid: synthesis of potent new dienyl derivatives

A series of new, diene-modified nodulisporic acid analogues ( 2) bearing diverse functionality at the 3″- and 4″-sites was efficiently prepared from the 3″-aldehyde 3. Biological evaluation of these synthetic nodulisporic acid analogues for systemic flea efficacy identified potent compounds and further clarified the structural requirements for ectoparasite activity.

ChemInform Abstract: Nodulisporic Acid A (I) Synthetic Studies. Part 1. Overall Strategy and Construction of a Western Hemisphere Subtarget (II).

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Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits.

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.

COMPARISON OF NODULISPORIC ACID ANALOGS IN A LUCILIA SERICATA IN VITRO ASSAY AND A CTENOCEPHALIDES FELIS MEMBRANE FEEDING SYSTEM

A medicinal chemistry program on the nodulisporic acid chemical class, guided by an artificial membrane flea-feeding assay, has recently identified permissive and nonpermissive regions of the pharmacophore for exploitation against fleas. This pathway was validated when several promising compounds from this program were administered orally to dogs at 15.0 mg/kg and found to have >90% flea activity for 2 wk. To determine if a surrogate insect assay would have provided the same guidance, a nodulisporic acid analog series was examined in both a Lucilia sericata larval assay and an artificial membrane flea-feeding assay using Ctenocephalides felis. Results from both insect assays were concordant in that even subtle chemical modification or substitution to the left-hand side of the nodulisporic acid pharmacophore resulted in substantial loss of insecticidal activity. Both assays were also in general agreement that the only modifications to the pharmacophore that did not result in loss of activity occurred to the C-8 side chain on the right-hand side of the molecule. Although there was good agreement between the 2 assays on the general regions of the pharmacophore, there was variance on individual compounds in the mono- and disubstituted amide series from the C-8 side chain. For example, the L. sericata assay showed several analogs from this subclass to possess similar activity to the parent acid, whereas the membrane assay indicated superior activity against fleas relative to the same parent. Consequently, although there was substantial general agreement between the assays, it was concluded that finer optimization of a lead compound should be done against the target parasite, even if it is ex vivo, as early as possible in a medicinal chemistry program.

Synthesis of side chain truncated 3"-aldehyde, 3"-carboxylic acid, and 1"-aldehyde from nodulisporic acid A.

An efficient synthesis of the truncated 3"-aldehyde (3) from nodulisporic acid A (1) under mild conditions is described. Further oxidation of 3 to 3"-carboxylic acid (4) and its subsequent oxidative degradation produced 1"-aldehyde (5). These new derivatives are versatile intermediates for the preparation of new, side chain modified derivatives of nodulisporic acid A. [reaction: see text]

Development of a mouse model to determine the systemic activity of potential flea-control compounds

Probe studies were performed to determine if the cat flea ( Ctenocephalides felis), the most common ectoparasite of companion animals, will feed on laboratory mice and, if so, to incorporate this into a small animal assay to detect systemically active compounds. Consequently, a protocol was developed which incorporated acepromazine maleate to temporarily sedate various strains of mice and allow fleas a window of time to feed undisturbed. For validation of the model, CD-1 mice were dosed per os with seven known insecticides at 30, 10 and 1 mg/kg. Mice were sedated with 0.0125 ml acepromazine maleate intraperitoneally, and infested with fleas. After 2 h, fleas were removed, one-third were examined immediately to confirm the occurrence of feeding, and 77% were found to have ingested a blood meal. The remaining fleas were incubated for 24 h to determine mortality. Nitenpyram, the active ingredient in Capstar™, was highly active (>94%) at 1 mg/kg. Selamectin, the active ingredient in Revolution™, was very active (86%) at 10 mg/kg, but inactive at 1 mg/kg. Fipronil, the active ingredient of Frontline ® Topspot™, was very active (83%) at 30 mg/kg, moderately active (54%) at 10 mg/kg and inactive at 1 mg/kg. Cythioate, the active ingredient in Proban ®, and nodulisporic acid, a recently discovered oral insecticide, were moderately active (64 and 55%, respectively) at 10 mg/kg, but both were inactive at 1 mg/kg. Lufenuron and ivermectin exhibited no efficacy at any level tested. These findings suggest that this mouse model can effectively identify systemic flea-control leads and, subsequently, reduce the use of large animals in research.

Biogeography and relatedness of Nodulisporium strains producing nodulisporic acid

Nodulisporic acid, a novel indole terpene with insecticidal properties, was first isolated from a fermentation broth of an endophytic Nodulisporium sp. Following an extensive culture screening effort, fermentations of 12 other strains of Nodulisporium also yielded nodulisporic acid. These strains came from a variety of environmental substrata collected from seven tropical regions in four continents. Cultural characteristics and microscopic features show that all the nodulisporic acid-producing Nodulisporium strains are morphologically very similar. AP-PCR and sequencing of the rDNA region consisting of the two internal transcribed spacers and the 5.8S gene revealed that the isolates were distributed into three groups, according to the length of the ITS1. The two groups with the longest sequences were not distinguishable, based on nucleotide divergence data calculated from the common region of ITS1. The group of isolates with shorter sequences showed lower homology with the other groups in the ITS1 region, but those strains could not be distinguished from the other groups, according to ITS2 sequences. These data suggest that the nodulisporic acid producing isolates are very closely related and may constitute a single species, although divided into populations showing some degree of genetic differentiation. Comparison of the sequences obtained in this work with sequences from other xylariaceous fungi with Nodulisporium-type anamorphs failed in determining the teleomorph of the nodulisporic acid-producing Nodulisporium species. However, it revealed that these isolates constitute a monophyletic group, clearly different from other tropical and temperate isolates of Nodulisporium not able to produce nodulisporic acid.

Nodulisporic acid A synthetic studies. 1. Overall strategy and construction of a western hemisphere subtarget.

In this, the first of two Letters, we outline our overall strategy for the construction of (+)-nodulisporic acid A (1), a representative member of a new class of indole diterpenes. In addition, we describe the efficient assembly of (-)-6, an advanced western hemisphere subtarget, comprising the ABC rings of (+)-nodulisporic acid A (1). The synthesis proceeded in 9% overall yield (longest linear sequence, 11 steps), exploiting a Shibasaki-Mori tandem transmetalation-cyclization to construct ring B. [reaction: see text]