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.

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.

Synthetic Makaluvamine Analogs Decrease c-Kit Expression and Are Cytotoxic to Neuroendocrine Tumor Cells.

In an effort to discover viable systemic chemotherapeutic agents for neuroendocrine tumors (NETs), we screened a small library of 18 drug-like compounds obtained from the Velu lab against pulmonary (H727) and thyroid (MZ-CRC-1 and TT) neuroendocrine tumor-derived cell lines. Two potent lead compounds (DHN-II-84 and DHN-III-14) identified from this screening were found to be analogs of the natural product makaluvamine. We further characterized the antitumor activities of these two compounds using pulmonary (H727), thyroid (MZ-CRC-1) and pancreatic (BON) neuroendocrine tumor cell lines. Flow cytometry showed a dose-dependent increase in apoptosis in all cell lines. Induction of apoptosis with these compounds was also supported by the decrease in myeloid cell leukemia-1 (MCL-1) and X-chromosome linked inhibitor of apoptosis (XIAP) detected by Western blot. Compound treatment decreased NET markers chromogranin A (CgA) and achaete-scute homolog 1 (ASCL1) in a dose-dependent manner. Moreover, the gene expression analysis showed that the compound treatment reduced c-Kit proto-oncogene expression in the NET cell lines. Induction of apoptosis could also have been caused by the inhibition of c-Kit expression, in addition to the known mechanisms such as damage of DNA by topoisomerase II inhibition for this class of compounds. In summary, makaluvamine analogs DHN-II-84 and DHN-III-14 induced apoptosis, decreased neuroendocrine tumor markers, and showed promising antitumor activity in pulmonary, thyroid, and pancreatic NET cell lines, and hold potential to be developed as an effective treatment to combat neuroendocrine tumors.

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.

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.

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.

Development and validation of an HPLC-MS/MS analytical method for quantitative analysis of TCBA-TPQ, a novel anticancer makaluvamine analog, and application in a pharmacokinetic study in rats.

We have recently designed and synthesized several novel iminoquinone anticancer agents that have entered preclinical development for the treatment of human cancers. Herein we developed and validated a quantitative HPLC-MS/MS analytical method for one of the lead novel anticancer makaluvamine analog, TCBA-TPQ, and conducted a pharmacokinetic study in laboratory rats. Our results indicated that the HPLC-MS/MS method was precise, accurate, and specific. Using this method, we carried out in vitro and in vivo evaluations of the pharmacological properties of TCBA-TPQ and plasma pharmacokinetics in rats. Our results provide a basis for future preclinical and clinical development of this promising anticancer marine analog.

Antibacterial and Antibiofilm Activities of Makaluvamine Analogs.

Streptococcus mutans is a key etiological agent in the formation of dental caries. The major virulence factor is its ability to form biofilms. Inhibition of S. mutans biofilms offers therapeutic prospects for the treatment and the prevention of dental caries. In this study, 14 analogs of makaluvamine, a marine alkaloid, were evaluated for their antibacterial activity against S. mutans and for their ability to inhibit S. mutans biofilm formation. All analogs contained the tricyclic pyrroloiminoquinone core of makaluvamines. The structural variations of the analogs are on the amino substituents at the 7-position of the ring and the inclusion of a tosyl group on the pyrrole ring N of the makaluvamine core. The makaluvamine analogs displayed biofilm inhibition with IC50 values ranging from 0.4 µM to 88 µM. Further, the observed bactericidal activity of the majority of the analogs was found to be consistent with the anti-biofilm activity, leading to the conclusion that the anti-biofilm activity of these analogs stems from their ability to kill S. mutans. However, three of the most potent N-tosyl analogs showed biofilm IC50 values at least an order of magnitude lower than that of bactericidal activity, indicating that the biofilm activity of these analogs is more selective and perhaps independent of bactericidal activity.

Development and validation of an HPLC-MS/MS analytical method for quantitative analysis of TCBA-TPQ, a novel anticancer makaluvamine analog, and application in a pharmacokinetic study in rats / 中国天然药物

We have recently designed and synthesized several novel iminoquinone anticancer agents that have entered preclinical development for the treatment of human cancers. Herein we developed and validated a quantitative HPLC-MS/MS analytical method for one of the lead novel anticancer makaluvamine analog, TCBA-TPQ, and conducted a pharmacokinetic study in laboratory rats. Our results indicated that the HPLC-MS/MS method was precise, accurate, and specific. Using this method, we carried out in vitro and in vivo evaluations of the pharmacological properties of TCBA-TPQ and plasma pharmacokinetics in rats. Our results provide a basis for future preclinical and clinical development of this promising anticancer marine analog.