Design, synthesis, and biological evaluation of novel benzo[6,7]indolo[3,4-c]isoquinolines as anticancer agents with topoisomerase I inhibition.

A novel series of benzo[6,7]indolo[3,4-c]isoquinolines 3a-3f was designed by scaffold hopping of topoisomerase I inhibitor benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-ones (BBPIs), which were developed by structural modification of the natural marine product lamellarin. The unconventional pentacycle was constructed by Bischler-Napieralski-type condensation of amide 11 and subsequent intramolecular Heck reaction. In vitro anticancer activity of the synthesized benzo[6,7]indolo[3,4-c]isoquinolines was evaluated on a panel of 39 human cancer cell lines (JFCR39). Among the compounds tested, N-(3-morpholinopropyl) derivative 3e showed the most potent antiproliferative activity, with a mean GI50 value of 39 nM. This compound inhibited topoisomerase I activity by stabilizing the enzyme-DNA complex.

Identification of Azalamellarin N as a Pyroptosis Inhibitor.

Pyroptosis is a form of regulated cell death that promotes inflammation; it attracts much attention because its dysregulation leads to various inflammatory diseases. To help explore the precise mechanisms by which pyroptosis is regulated, in this study, we searched for chemical compounds that inhibit pyroptosis. From our original compound library, we identified azalamellarin N (AZL-N), a hexacyclic pyrrole alkaloid, as an inhibitor of pyroptosis induced by R837 (also called imiquimod), which is an agonist of the intracellular multiprotein complex nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. However, whereas the effect of AZL-N on R837-induced pyroptosis was relatively weak, AZL-N strongly inhibited pyroptosis induced by extracellular ATP or nigericin, which are different types of NLRP3 inflammasome agonists. This was in contrast with the results that MCC950, a well-established NLRP3 inhibitor, consistently inhibited pyroptosis irrespective of the type of stimulus. We also found that AZL-N inhibited activation of caspase-1 and apoptosis-associated speck-like proteins containing a caspase activation and recruitment domain (ASC), which are components of the NLRP3 inflammasome. Analysis of the structure-activity relationship revealed that a lactam ring of AZL-N, which has been shown to contribute to the strong binding of AZL-N to its known target protein kinases, is required for its inhibitory effects on pyroptosis. These results suggest that AZL-N inhibits pyroptosis by targeting molecule(s), which may be protein kinase(s), that act upstream of NLRP3 inflammasome activation, rather than by directly targeting the components of the NLRP3 inflammasome. Further identification and analysis of target molecule(s) of AZL-N will shed light on the regulatory mechanisms of pyroptosis, particularly those depending on proinflammatory stimuli.

Synthesis and structure-activity relationship study of aldose reductase inhibiting marine alkaloid lukianol A and its derivatives.

Lukianol A (1a) and its six derivatives 1b-1g, in which each hydroxyl groups of 1a was individually modified, were synthesized via the common intermediate 7a, which was obtained by condensation of the styryl carbazate 10 with p-hydroxyphenylpyruvic acid and subsequent [3,3]-sigmatropic rearrangement. The synthesized lukianol derivatives were evaluated for their ability to inhibit human aldose reductase. 4'-O-methyl (1b) and 4'-dehydroxy (1g) derivatives showed the same level of inhibitory activity as 1a (IC50 2.2 µm), indicating that the 4'-OH is irrelevant for the activity. In contrast, methylation of the hydroxyl group at the 4″'-position (1d) resulted in the loss of activity at a concentration of 10 µm, and masking the hydroxyl group at the 4″-position (1e) caused a 9-fold decrease in activity compared with that of 1b, suggesting that the 4″-OH is an essential group, and the 4″'-OH is required for higher activity.

Unique Mode of Antiviral Action of a Marine Alkaloid against Ebola Virus and SARS-CoV-2.

Lamellarin α 20-sulfate is a cell-impenetrable marine alkaloid that can suppress infection that is mediated by the envelope glycoprotein of human immunodeficiency virus type 1. We explored the antiviral action and mechanisms of this alkaloid against emerging enveloped RNA viruses that use endocytosis for infection. The alkaloid inhibited the infection of retroviral vectors that had been pseudotyped with the envelope glycoprotein of Ebola virus and SARS-CoV-2. The antiviral effects of lamellarin were independent of the retrovirus Gag-Pol proteins. Interestingly, although heparin and dextran sulfate suppressed the cell attachment of vector particles, lamellarin did not. In silico structural analyses of the trimeric glycoprotein of the Ebola virus disclosed that the principal lamellarin-binding site is confined to a previously unappreciated cavity near the NPC1-binding site and fusion loop, whereas those for heparin and dextran sulfate were dispersed across the attachment and fusion subunits of the glycoproteins. Notably, lamellarin binding to this cavity was augmented under conditions where the pH was 5.0. These results suggest that the final action of the alkaloid against Ebola virus is specific to events following endocytosis, possibly during conformational glycoprotein changes in the acidic environment of endosomes. Our findings highlight the unique biological and physicochemical features of lamellarin α 20-sulfate and should lead to the further use of broadly reactive antivirals to explore the structural mechanisms of virus replication.

Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod.

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga Tricleocarpa jejuensis.

Our previous studies have found that (±)-(E)-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga Tricleocarpa jejuensis showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.

Concise synthesis and in vitro anticancer activity of benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-ones (BBPIs), topoisomerase I inhibitors based on the marine alkaloid lamellarin.

Benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-ones (BBPIs) are potent anticancer compounds having unique BBPIs ring system designed on the basis of the marine natural product lamellarin D. In this study, we describe an alternative synthesis of a 2-demethoxy series of BBPIs, employing van Leusen pyrrole synthesis and an intramolecular Heck reaction as the key reactions. Cytotoxicity of the derivatives against several cancer and normal cell lines is reported.

Synthesis and evaluation of azalamellarin N and its A-ring-modified analogues as non-covalent inhibitors of the EGFR T790M/L858R mutant.

Azalamellarin N, a synthetic lactam congener of the marine natural product lamellarin N, and its A-ring-modified analogues were synthesized and evaluated as potent and non-covalent inhibitors of the drug-resistant epidermal growth factor receptor T790M/L858R mutant. An in vitro tyrosine kinase assay indicated that the inhibitory activities of the synthetic azalamellarin analogues were higher than those of the corresponding lamellarins. The azalamellarin analogue bearing two 3-(dimethylamino)propoxy groups at C20- and C21-positions exhibited the highest activity and selectivity against the mutant kinase [IC50 (T790M/L858R) = 1.7 nM; IC50 (WT) = 4.6 nM]. The inhibitory activity was attributed to the hydrogen bonding interaction between the lactam NH group of the B-ring and carbonyl group of a methionine residue.

Algicidal hydroxylated C18 unsaturated fatty acids from the red alga Tricleocarpa jejuensis: Identification, synthesis and biological activity.

Bioassay-guided separation of a methanol extract of Tricleocarpa jejuensis by monitoring algicidal activity against the red tide phytoplankton Chattonella antiqua led to the isolation of an active fraction consisting of a mixture of four isomeric compounds. The active compounds were identified as (E)-9-hydroxyoctadec-10-enoic acid (1), (E)-10-hydroxyoctadec-8-enoic acid (2), (E)-11-hydroxyoctadec-12-enoic acid (3) and (E)-12-hydroxyoctadec-10-enoic acid (4) by NMR, IR and mass spectral data. The structures were confirmed by comparison of the NMR and MS data with those of authentic samples of 1-4 obtained by unambiguous syntheses. Synthesized hydroxy acids 1-4 and related compounds were assessed for algicidal activity against C. antiqua and it was found that all of 1-4 had high activity (>80% mortality at 24 h) at a concentration of 20 µg/mL. A structure-activity relationship study using 11 related compounds revealed that the presence of the hydroxyl group is important for the activity and the double bond may be replaced with a triple bond.

Lamellarin alkaloids: Isolation, synthesis, and biological activity.

Lamellarins are marine alkaloids containing fused 14-phenyl-6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinoline or non-fused 3,4-diarylpyrrole-2-carboxylate ring systems. To date, more than 50 lamellarins have been isolated from a variety of marine organisms, such as mollusks, tunicates, and sponges. Many of them, especially fused type I lamellarins, exhibit impressive biological activity, such as potent cytotoxicity, topoisomerase I inhibition, protein kinases inhibition, and anti-HIV-1 activity. Due to their useful biological activity and limited availability from natural sources, a number of synthetic methods have been developed. In this chapter, we present an updated and comprehensive review on lamellarin alkaloids summarizing their isolation, synthesis, and biological activity.

The Spirocyclic Imine from a Marine Benthic Dinoflagellate, Portimine, Is a Potent Anti-Human Immunodeficiency Virus Type 1 Therapeutic Lead Compound.

In this study, we aimed to find chemicals from lower sea animals with defensive effects against human immunodeficiency virus type 1 (HIV-1). A library of marine natural products consisting of 80 compounds was screened for activity against HIV-1 infection using a luciferase-encoding HIV-1 vector. We identified five compounds that decreased luciferase activity in the vector-inoculated cells. In particular, portimine, isolated from the benthic dinoflagellate Vulcanodinium rugosum, exhibited significant anti-HIV-1 activity. Portimine inhibited viral infection with an 50% inhibitory concentration (IC50) value of 4.1 nM and had no cytotoxic effect on the host cells at concentrations less than 200 nM. Portimine also inhibited vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV-1 vector infection. This result suggested that portimine mainly targeted HIV-1 Gag or Pol protein. To analyse which replication steps portimine affects, luciferase sequences were amplified by semi-quantitative PCR in total DNA. This analysis revealed that portimine inhibits HIV-1 vector infection before or at the reverse transcription step. Portimine has also been shown to have a direct effect on reverse transcriptase using an in vitro reverse transcriptase assay. Portimine efficiently inhibited HIV-1 replication and is a potent lead compound for developing novel therapeutic drugs against HIV-1-induced diseases.

Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one scaffold.

A new class of topoisomerase I inhibitors containing the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (abbreviated as BBPI) ring system have been developed based on structure-activity relationship studies of the cytotoxic marine alkaloid lamellarin D. The pentacyclic BBPI scaffold was constructed from N-tert-butoxycarbonylpyrrole by sequential and regioselective functionalization of the pyrrole core using directed lithiation, conventional electrophilic substitution, and palladium-catalyzed cross-coupling reactions. Further N-alkylation of the scaffold followed by selective deprotection of the O-isopropyl group produced a range of N-substituted BBPI derivatives. The BBPIs thus prepared exhibited potent topoisomerase I inhibitory activity in DNA relaxation assays. The activities of BBPIs were higher than those of lamellarin D and camptothecin; they showed potent and selective antiproliferative activity in the panel of 39 human cancer cell lines established by Japanese Foundation for Cancer Research. COMPARE analyses indicated that the inhibition patterns of the BBPIs correlated well with those of the known topoisomerase I inhibitors such as SN-38 and TAS-103. The water-soluble valine ester derivative exhibited antitumor activity in vivo against murine colon carcinoma colon 26. The activity was comparable to that of the approved anticancer agent irinotecan.

Design, synthesis, and evaluation of A-ring-modified lamellarin N analogues as noncovalent inhibitors of the EGFR T790M/L858R mutant.

A series of A-ring-modified lamellarin N analogues were designed, synthesized, and evaluated as potential noncovalent inhibitors of the EGFR T790M/L858R mutant, a causal factor in the drug-resistant non-small cell lung cancer. Several water-soluble ammonium- or guanidinium-tethered analogues exhibited good kinase inhibitory activities. The most promising analogue, 14f, displayed an excellent inhibitory profile against the T790M/L858R mutant [IC50 (WT) = 31.8 nM; IC50 (T790M/L858R) = 8.9 nM]. The effects of A-ring-substituents on activity were rationalized by docking studies.

Synthesis, resolution, and biological evaluation of atropisomeric (aR)- and (aS)-16-methyllamellarins N: unique effects of the axial chirality on the selectivity of protein kinases inhibition.

The total synthesis of the optically active (aR)- and (aS)-16-methyllamellarins N (3a and 3b) was achieved via resolution on HPLC chiral stationary phase. The kinase inhibitory activities of both enantiomers were evaluated on eight protein kinases relevant to cancer and neurodegenerative diseases (CDK1/cyclin B, CDK2/cyclin A, CDK5/p25, GSK-3α/ß, PIM1, DYRK1A, CLK3, and CK1). Isomer (aR)-3b exhibited potent but nonselective inhibition on all protein kinases except CK1, while (aS)-3a selectively inhibited only GSK-3α/ß, PIM1, and DYRK1A. The different inhibition profiles of (aS)-3a and (aR)-3b were elucidated by docking simulation studies. Although parental lamellarin N (2) inhibited the action of topoisomerase I, both (aS)-3a and (aR)-3b showed no inhibition of this enzyme. The phenotypic cytotoxic activities of 2, (aS)-3a, and (aR)-3b on three cancer cell lines (HeLa, SH-SY5Y, and IMR32) changed according to their topoisomerase I and protein kinase inhibitory activities.

Algicidal sesquiterpene hydroquinones from the brown alga Dictyopteris undulata.

Bioassay-guided fractionation of a methanol extract of the brown alga, Dictyopteris undulata, led to the isolation of a novel sesquiterpene hydroquinone named zonarenone, together with seven known sesquiterpene hydroquinones, zonarol, isozonarol, yahazunol, zonaroic acid, chromazonarol, isochromazonarol, and 2-(3,7,11-trimethyl-2,6,10-dodecatrienyl)hydroquinone. The structure of zonarenone was elucidated on the basis of spectroscopic information. The isolated compounds, excepting zonaroic acid, showed moderate to high cell lysis activity against the red tide microalgal species, Heterosigma akashiwo and Heterocapsa circularisquama, at a concentration of 1 µg/mL.

Algicidal activity of glycerolipids from brown alga Ishige sinicola toward red tide microalgae.

Bioassay-guided fractionation of a methanol extract of the brown alga, Ishige sinicola, led to the isolation of five algicidal compounds. Their structures were determined to be α-monoglycerides of eicosa-5Z,8Z,11Z,14Z-tetraenoic (arachidonic) acid, octadeca-6Z,9Z,12Z,15Z-tetraenoic acid, linoleic acid and oleic acid, and 1-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-sn-glycerol on the basis of spectroscopic data and a comparison with the data in the literature. These glycerolipids showed moderate-to-high cell lysis activity against the red tide microalgal species, Heterosigma akashiwo, Karenia mikimotoi and Alexandrium catenella, at a concentration of 20 µg/mL.

Synthesis, structure-activity relationships, and mechanism of action of anti-HIV-1 lamellarin α 20-sulfate analogues.

Lamellarin α and six different types of lamellarin α 20-sulfate analogues were synthesized and their structure-activity relationships were investigated using a single round HIV-1 vector infection assay. All lamellarin sulfates having pentacyclic lamellarin core exhibited anti-HIV-1 activity at a 10 µM concentration range regardless of the number and position of the sulfate group. On the other hand, non-sulfated lamellarin α and ring-opened lamellarin sulfate analogues did not affect HIV-1 vector infection in similar concentrations. The lamellarin sulfates utilized in this study did not exhibit unfavorable cytotoxic effect under the concentrations tested (IC(50)>100 µM). Confocal laser scanning microscopic analysis indicated that hydrophilic lamellarin sulfates were hardly incorporated in the cell. HIV-1 Env-mediated cell-cell fusion was suppressed by lamellarin sulfates. These results suggested that lamellarin sulfates have a novel anti-HIV-1 activity besides the previously reported integrase activity inhibition, possibly at a viral entry step of HIV-1 replication.

Design and synthesis of lamellarin D analogues targeting topoisomerase I.

A general synthetic route to rationally designed lamellarin D analogues, 1-dearyllamellarin D (1) and 1-substituted 1-dearyllamellarin D (2), has been developed. The key pentacyclic intermediate 22 was prepared by palladium-catalyzed direct arylation of 12, which in turn was synthesized via C-2-selective lithiation of 15 followed by palladium-catalyzed cross-coupling as the key reactions. Compound 22 was converted to a wide range of C-1-substituted analogues 2 via regioselective electrophilic substitution and palladium-catalyzed cross-coupling reactions.

Synthetic approach to telomerase inhibitor dictyodendrin B: synthesis of the pyrrolo[2,3-c]carbazole core.

The core structure of the telomerase inhibitor, dictyodendrin B, was synthesized by using the palladium-catalyzed cross-coupling reaction of 3-aryl-1-(2-arylethyl)-4-hydroxy-2,5-bismethoxycarbonylpyrrole triflate with 7-alkoxyindole-3-boronate as the key step.