Unexpected Transformations during Pyrroloiminoquinone Biosynthesis.

Pyrroloiminoquinone-containing natural products have long been known for their biological activities. They are derived from tryptophan, but their biosynthetic pathways have remained elusive. Studies on the biosynthetic gene cluster (BGC) that produces the ammosamides revealed that the first step is attachment of Trp to the C-terminus of a scaffold peptide in an ATP- and tRNA-dependent manner catalyzed by a PEptide Aminoacyl-tRNA Ligase (PEARL). The indole of Trp is then oxidized to a hydroxyquinone. We previously proposed a chemically plausible and streamlined pathway for converting this intermediate to the ammosamides using additional enzymes encoded in the BGC. In this study, we report the activity of four additional enzymes from two gene clusters, which show that the previously proposed pathway is incorrect and that Nature's route toward pyrroloiminoquinones is much more complicated. We demonstrate that, surprisingly, amino groups in pyrroloiminoquinones are derived from (at least) three different sources, glycine, asparagine, and leucine, all introduced in a tRNA-dependent manner. We also show that an FAD-dependent putative glycine oxidase (Amm14) is required for the process that incorporates the nitrogens from glycine and leucine and that a quinone reductase is required for the incorporation of asparagine. Additionally, we provide the first insights into the evolutionary origin of the PEARLs as well as related enzymes, such as the glutamyl-tRNA-dependent dehydratases involved in the biosynthesis of lanthipeptides and thiopeptides. These enzymes appear to all have descended from the ATP-GRASP protein family.

A Divergent Synthesis of Numerous Pyrroloiminoquinone Alkaloids Identifies Promising Antiprotozoal Agents.

On the basis of a streamlined route to the pyrroloiminoquinone (PIQ) core, we made 16 natural products spread across four classes of biosynthetically related alkaloid natural products, and multiple structural analogs, all in ≤8 steps longest linear sequence (LLS). The strategy features a Larock indole synthesis as the key operation in a five-step synthesis of a key methoxy-PIQ intermediate. Critically, this compound was readily diverged via selective methylation of either (or both) of the imine-like or pyrrole nitrogens, which then permitted further divergence by either O-demethylation to o-quinone natural products or displacement of the methoxy group with a range of amine nucleophiles. Based on a single, early report of their potential utility against the malaria parasite, we assayed these compounds against several strains of Plasmodium falciparum, as well as two species of the related protozoan parasite Babesia. In combination with evaluations of their human cytotoxicity, we identified several compounds with potent (low-nM IC50) antimalarial and antibabesial activities that are much less toxic toward mammalian cells and are therefore promising lead compounds for antiprotozoal drug discovery.

Semisynthetic Studies Establish a Role for Conjugate Halide Exchange in the Formation of Chlorinated Pyrroloiminoquinones and Related Alkaloids.

Two novel pyrroloiminoquinone alkaloids, 6-chlorodamirone A and 6-bromodamirone A, have been identified for the first time from the marine sponge Latrunculia sp. (order: Poecilosclerida: family Latrunculiidae), sourced from Western Australia. Alongside these new compounds, seven previously known metabolites were also isolated. Despite being obtained in submilligram quantities, the structures of these natural products were successfully elucidated using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. To confirm the structures of these newly discovered alkaloids, a semisynthetic approach was employed starting from the more abundant metabolite, damirone A, additionally, single crystal X-ray crystallography was used to validate our structural proposals. The semisynthetic studies suggest that the chlorinated alkaloids are likely formed through a nonenzymatic conjugate halide substitution reaction rather than an enzymatic process. This reactivity parallels that observed in related metabolites, such as the caulibugulones B and C. Furthermore, a biomimetic cascade reaction was attempted to synthesize the spirodienone moiety characteristic of the discorhabdin alkaloids, inspired by the nucleophilic substitution observed in the tricyclic damirone A system. Albeit unsuccessful, these findings provide valuable insight into the reactivity of halogenated pyrroloiminoquinones under various conditions.

Unified Synthesis and Biological Evaluation of Makaluvamine J and Its Analogs.

Makaluvamine J, a pyrroloiminoquinone alkaloid of marine sponge origin, and its analogs were synthesized and assessed for their potential to develop as a novel and selective growth inhibitor targeting human pancreatic cancer PANC-1 cells. Ts-damirone B, a common precursor featuring a pyrroloiminoquinone core structure, was synthesized through Bartoli indole synthesis and IBX-mediated oxidation. Late-stage diversification at N-5 and N-9 yielded makaluvamine J and several analogs. A structure-activity relationship (SAR) analysis highlighted the significance of the lipophilic side chain at N-9 for the growth inhibitory activity of PANC-1 cells. The modest alkyl group at N-5 was found to improve selectivity against other cancer cells. Among the prepared analogs, the tryptamine analog 24 showed potent and selective cytotoxicity (IC50 = 0.029 µM, selective index = 13.1), exceeding those of natural products.

Current Perspectives on Pyrroloiminoquinones: Distribution, Biosynthesis and Drug Discovery Potential.

Pyrroloiminoquinones are a group of cytotoxic alkaloids most commonly isolated from marine sponges. Structurally, they are based on a tricyclic pyrrolo[4,3,2-de]quinoline core and encompass marine natural products such as makaluvamines, tsitsikammamines and discorhabdins. These diverse compounds are known to exhibit a broad spectrum of biological activities including anticancer, antiplasmodial, antimicrobial, antifungal and antiviral activities as well as the inhibition of several key cellular enzymes. The resurgence of interest in pyrroloiminoquinones and the convoluted understanding regarding their biological activities have prompted this review. Herein, we provided a concise summary of key findings and recent developments pertaining to their structural diversity, distribution, biogenesis, and their potential as chemical probes for drug development, including a discussion of promising synthetic analogs.

IDO1 and TDO inhibitory evaluation of analogues of the marine pyrroloiminoquinone alkaloids: Wakayin and Tsitsikammamines.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophane 2,3-dioxygenase (TDO) are two heme-containing enzymes which catalyze the conversion of tryptophan to N-formylkynurenine. Both enzymes are well establish therapeutic targets as important factors in the tumor immune evasion mechanism. A number of analogues of the marine pyrroloquinoline alkaloids tsitsikammamines or wakayin have been synthesized, two of them were synthesized using an original method to build the bispyrroloquinone framework. All the derivatives were evaluated in a cellular assay for their capacity to inhibit the enzymes. Six compounds have shown a significant potency on HEK 293-EBNA cell lines expressing hIDO1 or hTDO.

Unlocking the Diversity of Pyrroloiminoquinones Produced by Latrunculid Sponge Species.

Sponges of the Latrunculiidae family produce bioactive pyrroloiminoquinone alkaloids including makaluvamines, discorhabdins, and tsitsikammamines. The aim of this study was to use LC-ESI-MS/MS-driven molecular networking to characterize the pyrroloiminoquinone secondary metabolites produced by six latrunculid species. These are Tsitsikamma favus, Tsitsikamma pedunculata, Cyclacanthia bellae, and Latrunculia apicalis as well as the recently discovered species, Tsitsikamma nguni and Tsitsikamma michaeli. Organic extracts of 43 sponges were analyzed, revealing distinct species-specific chemical profiles. More than 200 known and unknown putative pyrroloiminoquinones and related compounds were detected, including unprecedented makaluvamine-discorhabdin adducts and hydroxylated discorhabdin I derivatives. The chemical profiles of the new species T. nguni closely resembled those of the known T. favus (chemotype I), but with a higher abundance of tsitsikammamines vs. discorhabdins. T. michaeli sponges displayed two distinct chemical profiles, either producing mostly the same discorhabdins as T. favus (chemotype I) or non- or monobrominated, hydroxylated discorhabdins. C. bellae and L. apicalis produced similar pyrroloiminoquinone chemistry to one another, characterized by sulfur-containing discorhabdins and related adducts and oligomers. This study highlights the variability of pyrroloiminoquinone production by latrunculid species, identifies novel isolation targets, and offers fundamental insights into the collision-induced dissociation of pyrroloiminoquinones.

Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges.

The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.

A Novel Marine Natural Product Derived Pyrroloiminoquinone with Potent Activity against Skin Cancer Cells.

Non-melanoma skin cancer is one of the major ailments in the United States. Effective drugs that can cure skin cancers are limited. Moreover, the available drugs have toxic side effects. Therefore, skin cancer drugs with less toxic side effects are urgently needed. To achieve this goal, we focused our work on identifying potent lead compounds from marine natural products. Five lead compounds identified from a class of pyrroloiminoquinone natural products were evaluated for their ability to selectively kill squamous cell carcinoma (SCC13) skin cancer cells using an MTT assay. The toxicity of these compounds was also evaluated against the normal human keratinocyte HaCaT cell line. The most potent compound identified from these studies, C278 was further evaluated for its ability to inhibit cancer cell migration and invasion using a wound-healing assay and a trans-well migration assay, respectively. To investigate the molecular mechanism of cell death, the expression of apoptotic and autophagy proteins was studied in C278 treated cells compared to untreated cells using western blot. Our results showed that all five compounds effectively killed the SCC13 cells, with compound C278 being the most effective. Compound C278 was more effective in killing the SCC13 cells compared to HaCaT cells with a two-fold selectivity. The migration and the invasion of the SCC13 cells were also inhibited upon treatment with compound C278. The expression of pro-apoptotic and autophagy proteins with concomitant downregulation in the expression of survival proteins were observed in C278 treated cells. In summary, the marine natural product analog compound C278 showed promising anticancer activity against human skin cancer cells and holds potential to be developed as an effective anticancer agent to combat skin cancer.

Macrophilones from the Marine Hydroid Macrorhynchia philippina Can Inhibit ERK Cascade Signaling.

Six new macrophilone-type pyrroloiminoquines were isolated and identified from an extract of the marine hydroid Macrorhynchia philippina. The proton-deficient and heteroatom-rich structures of macrophilones B-G (2-7) were elucidated by spectroscopic analysis and comparison of their data with those of the previously reported metabolite macrophilone A (1). Compounds 1-7 are the first pyrroloiminoquines to be reported from a hydroid. The macrophilones were shown to inhibit the enzymatic conjugation of SUMO to peptide substrates, and macrophilones A (1) and C (3) exhibit potent and selective cytotoxic properties in the NCI-60 anticancer screen. Bioinformatic analysis revealed a close association of the cytotoxicity profiles of 1 and 3 with two known B-Raf kinase inhibitory drugs. While compounds 1 and 3 showed no kinase inhibitory activity, they resulted in a dramatic decrease in cellular protein levels of selected components of the ERK signal cascade. As such, the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads that target the ERK signal transduction pathway.

Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites.

Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

Another Look at Pyrroloiminoquinone Alkaloids-Perspectives on Their Therapeutic Potential from Known Structures and Semisynthetic Analogues.

This study began with the goal of identifying constituents from Zyzzya fuliginosa extracts that showed selectivity in our primary cytotoxicity screen against the PANC-1 tumor cell line. During the course of this project, which focused on six Z. fuliginosa samples collected from various regions of the Indo-Pacific, known compounds were obtained consisting of nine makaluvamine and three damirone analogues. Four new acetylated derivatives were also prepared. High-accuracy electrospray ionization mass spectrometry (HAESI-MS) m/z ions produced through MS² runs were obtained and interpreted to provide a rapid way for dereplicating isomers containing a pyrrolo[4,3,2-de]quinoline core. In vitro human pancreas/duct epithelioid carcinoma (PANC-1) cell line IC50 data was obtained for 16 compounds and two therapeutic standards. These results along with data gleaned from the literature provided useful structure activity relationship conclusions. Three structural motifs proved to be important in maximizing potency against PANC-1: (i) conjugation within the core of the ABC-ring; (ii) the presence of a positive charge in the C-ring; and (iii) inclusion of a 4-ethyl phenol or 4-ethyl phenol acetate substituent off the B-ring. Two compounds, makaluvamine J (9) and 15-O-acetyl makaluvamine J (15), contained all three of these frameworks and exhibited the best potency with IC50 values of 54 nM and 81 nM, respectively. These two most potent analogs were then tested against the OVCAR-5 cell line and the presence of the acetyl group increased the potency 14-fold from that of 9 whose IC50 = 120 nM vs. that of 15 having IC50 = 8.6 nM.

Screening and Biological Effects of Marine Pyrroloiminoquinone Alkaloids: Potential Inhibitors of the HIF-1α/p300 Interaction.

Inhibition of the hypoxia-inducible factor 1α (HIF-1α) pathway by disrupting its association with the transcriptional coactivator p300 inhibits angiogenesis and tumor development. Development of HIF-1α/p300 inhibitors has been hampered by preclinical toxicity; therefore, we aimed to identify novel HIF-1α/p300 inhibitors. Using a cell-free assay designed to test compounds that block HIF-1α/p300 binding, 170 298 crude natural product extracts and prefractionated samples were screened, identifying 25 active extracts. One of these extracts, originating from the marine sponge Latrunculia sp., afforded six pyrroloiminoquinone alkaloids that were identified as positive hits (IC50 values: 1-35 µM). Luciferase assays confirmed inhibition of HIF-1α transcriptional activity by discorhabdin B (1) and its dimer (2), 3-dihydrodiscorhabdin C (3), makaluvamine F (5), discorhabdin H (8), discorhabdin L (9), and discorhabdin W (11) in HCT 116 colon cancer cells (0.1-10 µM, p < 0.05). Except for 11, all of these compounds also reduced HIF-1α transcriptional activity in LNCaP prostate cancer cells (0.1-10 µM, p < 0.05). These effects occurred at noncytotoxic concentrations (<50% cell death) under hypoxic conditions. At the downstream HIF-1α target level, compound 8 (0.5 µM) significantly decreased VEGF secretion in LNCaP cells (p < 0.05). In COLO 205 colon cancer cells no activity was shown in the luciferase or cytotoxicity assays. Pyrroloiminoquinone alkaloids are a novel class of HIF-1α inhibitors, which interrupt the protein-protein interaction between HIF-1α and p300 and consequently reduce HIF-related transcription.

Evaluation of the Antioxidant Activity of the Marine Pyrroloiminoquinone Makaluvamines.

Makaluvamines are pyrroloiminoquinones isolated from Zyzzya sponges. Until now, they have been described as topoisomerase II inhibitors with cytotoxic effects in diverse tumor cell lines. In the present work, seven makaluvamines were tested in several antioxidant assays in primary cortical neurons and neuroblastoma cells. Among the alkaloids studied, makaluvamine J was the most active in all the assays. This compound was able to reduce the mitochondrial damage elicited by the well-known stressor H2O2. The antioxidant properties of makaluvamine J are related to an improvement of the endogenous antioxidant defenses of glutathione and catalase. SHSY5Y assays proved that this compound acts as a Nrf2 activator leading to an improvement of antioxidant defenses. A low concentration of 10 nM is able to reduce the reactive oxygen species release and maintain a correct mitochondrial function. Based on these results, non-substituted nitrogen in the pyrrole plus the presence of a p-hydroxystyryl without a double bond seems to be the most active structure with a complete antioxidant effect in neuronal cells.

Resonant-convergent PCM response theory for the calculation of second harmonic generation in makaluvamines A-V: pyrroloiminoquinone marine natural products from poriferans of genus Zyzzya.

The first-order hyperpolarizability, ß, has been calculated for a group of marine natural products, the makaluvamines. These compounds possess a common cationic pyrroloiminoquinone structure that is substituted to varying degrees. Calculations at the MP2 level indicate that makaluvamines possessing phenolic side chains conjugated with the pyrroloiminoquinone moiety display large ß values, while breaking this conjugation leads to a dramatic decrease in the calculated hyperpolarizability. This is consistent with a charge-transfer donor-π-acceptor (D-π-A) structure type, characteristic of nonlinear optical chromophores. Dynamic hyperpolarizabilities calculated using resonance-convergent time-dependent density functional theory coupled to polarizable continuum model (PCM) solvation suggest that significant resonance enhancement effects can be expected for incident radiation with wavelengths around 800 nm. The results of the current work suggest that the pyrroloiminoquinone moiety represents a potentially useful new chromophore subunit, in particular for the development of molecular probes for biological imaging. The introduction of solvent-solute interactions in the theory is conventionally made in a density matrix formalism, and the present work will provide detailed account of the approximations that need to be introduced in wave function theory and our program implementation. The program implementation as such is achieved by a mere combination of existing modules from previous developments, and it is here only briefly reviewed.

Marine pyrroloiminoquinone alkaloids.

Recent reports on the synthetic studies of marine pyrroloiminoquinone alakloids and their analogs are reviewed.

[Synthetic Studies on Complex Natural Products Based on Development of a Novel Synthetic Method for Heteroaromatic Skeleton].

Among my recent work on the syntheses of complex natural products based on the development of a novel synthetic method for the heteroaromatic skeleton, this article primarily deals with the total syntheses of (+)-CC-1065, isobatzeline A/B, and batzeline A. These syntheses were accomplished via a novel indole synthesis utilizing a ring expansion reaction of benzocyclobutenone oxime sulfonate as the key step. The 1,2-dihydro-3H-pyrrolo[3,2-e]indole segments of (+)-CC-1065 were rapidly constructed via a two-directional double-ring expansion strategy. Highly substituted pyrrolidine-fused common 5-chloro-2-methylthioindoles of isobatzeline A/B and batzeline A were constructed using a ring expansion reaction of benzocyclobutenone oxime sulfonate with NaSMe and a benzyne-mediated cyclization/functionalization reaction.

Synthesis of the marine pyrroloiminoquinone alkaloids, discorhabdins.

Many natural products with biologically interesting structures have been isolated from marine animals and plants such as sponges, corals, worms, etc. Some of them are discorhabdin alkaloids. The discorhabdin alkaloids (discorhabdin A-X), isolated from marine sponges, have a unique structure with azacarbocyclic spirocyclohexanone and pyrroloiminoquinone units. Due to their prominent potent antitumor activity, discorhabdins have attracted considerable attention. Many studies have been reported toward the synthesis of discorhabdins. We have accomplished the first total synthesis of discorhabdin A (1), having the strongest activity in vitro among discorhabdins in 2003. In 2009, we have also accomplished the first total synthesis of prianosin B (2), having the 16,17-dehydropyrroloiminoquinone moiety, by a novel dehydrogenation reaction with a catalytic amount of NaN(3). These synthetic studies, as well as syntheses of the discorhabdins by various chemists to-date, are reviewed here.

13C NMR discrimination of regioisomeric bispyrroloquinone/bispyrroloiminoquinone ring systems.

The structural assignment of bispyrroloquinone and bispyrroloiminoquinone regioisomers was achieved using (13)C NMR spectral data. In the case of bispyrroloiminoquinones, the carbonyl group in the regioisomer possessing a nitrogen atom in both alpha-positions was systematically less deshielded than the carbonyl group in the other regioisomer. In the case of bispyrroloquinones, the most deshielded carbonyl group in the regioisomer with a nitrogen atom in both alpha-positions was more deshielded than the same carbonyl group in the other regioisomer.

alpha-halogenoacrylic derivatives of antitumor agents.

In this review article we have reported a series of hybrid compounds characterized by the presence of a alpha-halogenocryloyl alkylating moiety of low chemical reactivity, linked to known antitumor agents or their active moieties. Among them, brostallicin (PNU-166196), was selected for clinical development and is now undergoing Phase II studies in patients with advanced or metastatic soft tissue sarcoma.