Chemical Research on Antitumor Isoquinoline Marine Natural Products and Related Compounds.

The biologically active, naturally occurring 1,2,3,4-tetrahydroisoquinoline-quinone (THIQ) family members isolated from Actinomycetes and marine organisms have been studied thoroughly over the past five decades. Among them, marine natural products along with their reduced compounds, such as renieramycins and ecteinascidins, have attracted interest due to their fantastic structures and meager availability in nature as well as their potent antitumor profiles. As part of our search for new anticancer metabolites through the isolation and characterization of anticancer THIQ compounds from Thai marine animals, we have developed a fascinating THIQ natural product chemistry and medicinal chemistry based on knowledge of the chemistry of saframycin antibiotics as well as their isolation, characterization, transformation, partial synthesis, and total synthesis. This review mainly presents our contributions during 1999-2019 to the field of research on biologically active renieramycin along with ecteinascidin marine natural products.

Enantioseparation of ß-carboline, tetrahydroisoquinoline and benzazepine analogues of pharmaceutical importance: Utilization of chiral stationary phases based on polysaccharides and sulfonic acid modified Cinchonaalkaloids in high-performance liquid and subcritical fluid chromatography.

High-performance liquid chromatographic (HPLC) and subcritical fluid chromatographic (SFC) separations of the enantiomers of structurally diverse, basic ß-carboline, tetrahydroisoquinoline and benzazepine analogues of pharmacological interest were performed applying chiral stationary phases (CSPs) based on (i) neutral polysaccharides- and (ii) zwitterionic sulfonic acid derivatives of Cinchona alkaloids. The aim of this work was to reveal the influence of structural peculiarities on the enantiorecognition on both types of CSP through the investigation of the effects of the composition of the bulk solvent, the structures of the chiral analytes (SAs) and chiral selectors (SOs) on retention and stereoselectivity. As a general tendency, valid for all polysaccharide SOs studied, the increase of the concentration of the apolar component in the mobile phase (n-hexane for LC or liquid CO2 for SFC) was found to significantly increase retention, which in most cases, was accompanied with increased selectivity and resolution. In a way, similar behaviour was registered for the zwitterionic SOs. In polar ionic mode employing eluent systems composed of methanol and acetonitrile with organic acid and base additives, moderate increases in retention factor, selectivity and resolution were observed with increasing acetonitrile content. However, under SFC conditions, an extremely high increase in retention was observed with increased CO2 content, while selectivity and resolution changed only slightly. Thermodynamic parameters derived from temperature dependence studies revealed that separations are controlled by enthalpy.

Synergistic Cytotoxicity of Renieramycin M and Doxorubicin in MCF-7 Breast Cancer Cells.

Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp., with nanomolar IC50s against several cancer cell lines. Our goal is to evaluate its combination effects with doxorubicin (DOX) in estrogen receptor positive MCF-7 breast cancer cells. MCF-7 cells were treated simultaneously or sequentially with various combination ratios of RM and DOX for 72 h. Cell viability was determined using the MTT assay. Synergism or antagonism was determined using curve-shift analysis, combination index method and isobologram analysis. Synergism was observed with pharmacologically achievable concentrations of DOX when administered simultaneously, but not sequentially. The IC95 values of RM and DOX after combination were reduced by up to four-fold and eight-fold, respectively. To gain insights on the mechanism of synergy, real-time profiling, cell cycle analysis, apoptosis assays, and transcriptome analysis were conducted. The combination treatment displayed a similar profile with DNA-damaging agents and induced a greater and faster cell killing. The combination treatment also showed an increase in apoptosis. DOX induced S and G2/M arrest while RM did not induce significant changes in the cell cycle. DNA replication and repair genes were downregulated commonly by RM and DOX. p53 signaling and cell cycle checkpoints were regulated by DOX while ErbB/PI3K-Akt, integrin and focal adhesion signaling were regulated by RM upon combination. Genes involved in cytochrome C release and interferon gamma signaling were regulated specifically in the combination treatment. This study serves as a basis for in vivo studies and provides a rationale for using RM in combination with other anticancer drugs.

Dauricine combined with clindamycin inhibits severe pneumonia co-infected by influenza virus H5N1 and Streptococcus pneumoniae in vitro and in vivo through NF-κB signaling pathway.

Dauricine, isolated from Menispermum dauricum, has been widely used for treatment of various diseases, including cardiac ischemia and inflammation-related diseases. However, little is known regarding to the effect of dauricine on severe pneumonia. Therefore, the aim was to investigate the effect of dauricine on severe pneumonia and its mechanism during progress. Herein, H5N1 and Streptococcus pneumoniae (D39) were conducted to induce severe pneumonia in both BEAS-2B cells and mice. In vitro, dauricine reversed the protein and mRNA expressions of TNF-α, IL-6 and IL-1ß, examined by ELISA and qRT-PCR assay, respectively. In addition, the nuclear translocation of NF-κB/p65 and the phosphorylation expressions of IκBα and IKKα/ß, examined by western blotting, were dose-dependently dropped by dauricine. However, dauricine had no significant effect on MAPKs, including JNK, ERK and p38. In vivo, dauricine significantly decreased MPO activity, the lung wet/dry weight ratio, the protein and mRNA expression of TNF-α, IL-6 and IL-1ß, the expressions of NF-κB/p65, and attenuated the lung histological alterations. Besides, compared to dauricine alone, combined with clindamycin had more remarkably effects on severe pneumonia in vitro. Overall, the results suggested that dauricine, a relatively drug that targets NF-κB, in combination with clindamycin, maybe a novel therapeutic strategy for severe pneumonia.

6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid attenuates colon carcinogenesis via blockade of IL-6 mediated signals.

In this study, we investigated the in vivo antiproliferative activity of 6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (M1) in dimethylhydrazine (DMH) induced colorectal carcinoma (CRC) using albino Wistar rats. M1 was administered to DMH induced CRC rats at 10 and 25 mg/kg doses for 15 days. Various physiological, oxidative parameters, histopathology, ELISA, gene and protein expression studies were conducted to evaluate the anti-CRC potential of M1. The histopathology and biochemical tests indicated the protective action of M1 in DMH-induced colon cancer. ELISA confirms that M1 reduced the increased concentration of IL-6 more prominently than those of IL-2 and COX-2. Gene expression analysis revealed that M1 attenuated the increased mRNA over-expression of IL-6, JAK2 and STAT3. The result obtained from quantitative western blot analysis demonstrated that the CRC condition was produced by the IL-6 induced activation/phosphorylation of JAK2 and STAT3 and further down-regulated with M1 treatment. This evidence was supported well with the application of data-based mathematical modeling. Applying the fitted model, we predicted the quantitative behavior of STAT3 populations not accessible to experimental measurement. Later, 1H NMR based serum metabolic profiling was carried out using rat sera to investigate the impact of M1 on CRC-induced metabolic alterations. M1 showed its ability to restore the perturbed metabolites in CRC condition. Altogether, our study provided the first time evidence that M1 exhibits anti-CRC potential through the blockade of IL-6/JAK2/STAT3 oncogenic signaling.

Synthesis and Detailed Examination of Spectral Properties of (S) and (R)-Higenamine 4'-O-ß-d-Glucoside and HPLC Analytical Conditions to Distinguish the Diastereomers.

Higenamine is a tetrahydroisoquinoline present in several plants that has ß-adrenergic receptor agonist activity. Study of the biosynthesis of higenamine has shown the participation of norcoclaurine synthase, which controls the stereochemistry to construct the (S)-isomer. However, when isolated from nature, higenamine is found as the racemate, or even the (R)-isomer. We recently reported the isolation of higenamine 4'-O-ß-d-glucoside. Herein, its (R)- and (S)-isomers were synthesized and compared to precisely determine the stereochemistry of the isolate. Owing to their similar spectral properties, determination of the stereochemistry based on NMR data was considered inappropriate. Therefore, a high-performance liquid chromatography method was established to separate the isomers, and natural higenamine 4'-O-ß-d-glucoside was determined to be a mixture of isomers.

Chemistry of Renieramycins. 17. A New Generation of Renieramycins: Hydroquinone 5-O-Monoester Analogues of Renieramycin M as Potential Cytotoxic Agents against Non-Small-Cell Lung Cancer Cells.

A series of hydroquinone 5-O-monoester analogues of renieramycin M were semisynthesized via bishydroquinonerenieramycin M (5) prepared from renieramycin M (1), a major cytotoxic bistetrahydroisoquinolinequinone alkaloid isolated from the Thai blue sponge Xestospongia sp. All 20 hydroquinone 5-O-monoester analogues possessed cytotoxicity with IC50 values in nanomolar concentrations against the H292 and H460 human non-small-cell lung cancer (NSCLC) cell lines. The improved cytotoxicity toward the NSCLC cell lines was observed from the 5-O-monoester analogues such as 5-O-acetyl ester 6a and 5-O-propanoyl ester 7e, which exhibited 8- and 10-fold increased cytotoxicity toward the H292 NSCLC cell line (IC50 3.0 and 2.3 nM, respectively), relative to 1 (IC50 24 nM). Thus, the hydroquinone 5-O-monoester analogues are a new generation of the renieramycins to be further developed as potential marine-derived drug candidates for lung cancer treatment.

Antiproliferative effect of isolated isoquinoline alkaloid from Mucuna pruriens seeds in hepatic carcinoma cells.

The present study was undertaken to investigate the antiproliferative action of isolated M1 (6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) from Mucuna pruriens seeds using human hepatic carcinoma cell line (Huh-7 cells). Initially, docking studies was performed to find out the binding affinities of M1 to caspase-3 and 8 enzymes. Later, cytotoxic action of M1 was measured by cell growth inhibition (MTT), followed by caspase-3 and 8 enzymes assay colorimetrically. Our results collectively suggested that M1 had strong binding affinity to caspase-8 in molecular modelling. M1 possessed antiproliferative activity on Huh-7 cells (EC50 = 13.97 µM) and also inhibited the action of caspase-8 enzyme, signified process of apoptosis. M1 was active against Huh-7 cells that may be useful for future hepatic cancer treatment.

High-performance liquid chromatographic enantioseparation of cationic 1,2,3,4-tetrahydroisoquinoline analogs on Cinchona alkaloid-based zwitterionic chiral stationary phases.

The stereoisomers of 1,2,3,4-tetrahydroisoquinoline analogs were resolved for the first time by applying a polar ionic mobile phase on a quinine or a quinidine moiety fused with a chiral sulfonic acid-type chiral selector immobilized on silica [Chiralpak ZWIX(+)™ and Chiralpak ZWIX(-)™]. The effects of the nature and concentrations of the mobile phase components and additives and temperature on the retention and enantioseparation on the investigated chiral columns were studied. Experiments were performed in the temperature range 10-50 °C. Thermodynamic parameters were calculated from plots of ln α versus 1/T. The separations were generally enthalpy-controlled, but entropy-controlled separation was also observed below 30 °C. The enantiomer elution order was determined in some cases and was observed to be opposite on the ZWIX(+)™ and ZWIX(-)™ columns. Our results contribute to a better understanding of the enantiorecognition mechanism of chiral bases with chiral zwitterionic selectors.

Detection of a novel neurotoxic metabolite of Parkinson's disease-related neurotoxin, 1-benzyl-1,2,3,4- tetrahydroisoquinoline.

Naturally occurring low-molecular weight compounds with a chemical structure like that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, such as 1-benzyl-1,2,3,4-tetrahydroisoquinoline(1BnTIQ), are candidates for the endogenous neurotoxins that cause Parkinson's disease (PD). 1BnTIQ is an endogenous amine in human CSF and increases in the CSF of patients with PD. It inhibits complex Iand elicits PD-like behavioral abnormalities in monkey and mouse. In this study, we searched metabolites of 1BnTIQ by rat liver S9 using liquid chromatography-tandem mass spectrometry, and identified a dehydrated metabolite, 1-benzyl-3,4-dihydroisoquinoline (1BnDIQ). 1BnDIQ was identified by corresponding mass spectra and precursor ion scans in authentic and complete enzyme samples. Multiple reaction monitoring analysis showed microsome-dependent 1BnDIQ production. We previously reported that 1BnDIQ is more toxic than 1BnTIQ in cytotoxicity study in SH-SY5Y neuroblastoma cells. In addition, 1BnTIQ is reported to pass through the blood-brain barrier of the rat brain, and 1BnDIQ is supposed to be more lipophilic than 1BnTIQ. 1BnDIQ may easily reach the brain, and it might contribute to PD-related neurotoxicity.

Hyalachelins A-C, unusual siderophores isolated from the terrestrial myxobacterium Hyalangium minutum.

Three new siderophores, termed hyalachelins A-C (1-3), were isolated from the terrestrial myxobacterium Hyalangium minutum. Their structures were determined by 2D NMR and HR-MS/MS experiments, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and circular dichroism experiments. Hyalachelins are unusual catecholate-type siderophores that bear a 3,7,8-trihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Their iron chelating activities were evaluated in a CAS assay showing EC50 values of ∼30 µM.

Ultra-performance LC separation and quadrupole time-of-flight MS identification of major alkaloids in Plumula Nelumbinis.

INTRODUCTION: As an essential medicine and tea source in many countries, Plumula Nelumbinis potentially exerts its major biological activities through its alkaloids. However, the activities of Plumula Nelumbinis are not fully understood due to the lack of studies on its chemical components. OBJECTIVE: To establish an ultra-performance liquid chromatography combined with diode-array detector (UPLC/DAD) method, coupled to an electrospray ionisation with quadrupole time-of-flight mass spectrometry (ESI/QTOF/MS) method, for the separation and identification of Plumula Nelumbinis alkaloids. METHODS: The eluant from an UPLC separation of an ethanol extract of Plumula Nelumbinis was directly infused into an ESI/QTOF/MS system. Both positive and negative ion modes of ESI with low and high collision energy (CE) were used to obtain sufficient MS information. RESULTS: Twenty-one alkaloids were tentatively identified based on their chromatographic characteristics, UV spectra, exact mass, MS fragments and literature reports. They consist of six bis-1-benzyltetrahydroisoquinoline, eleven benzyltetrahydroisoquinoline (including two glycoalkaloids and two quaternary ammoniums), two aporphine, one proaporphine and one indole alkaloids. Eleven were identified in Plumula Nelumbinis for the first time and seven were first reported in Nelumbo nucifera Gaertn. Five compounds, namely norcoclaurine-4'-O-glucoside, norcoclaurine-6-O-glucoside, isolotusine, 6-demethyl-4-demethylN-methylcoclaurine and N-norisoliensinine, were characterised and proposed as new compounds. CONCLUSION: The established UPLC/DAD - ESI/QTOF/MS method is efficient for systematic identification of the alkaloids in Plumula Nelumbinis extract.

New isoquinolinequinone alkaloids from the South China Sea nudibranch Jorunna funebris and its possible sponge-prey Xestospongia sp.

Two new renieramycin-type bistetrahydroisoquinolinequinone alkaloids, fennebricins A (1) and B (5), and one new isoquinolinequinone alkaloid, N-formyl-1,2-dihydrorenierol (7), were isolated from the skin of the South China Sea nudibranch Jorunna funebris and its possible sponge-prey Xestospongia sp., together with eight known metabolites, including three bistetrahydroisoquinolinequinones (2-4) and five isoquinolinequinones (8-12). Their structures were elucidated by analysis of spectroscopic data including 1D and 2D NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS) and by comparison with data for related known compounds. All the metabolites except for 7 occurred simultaneously in the two animals, supporting recent ecological studies that the nudibranch J. funebris preys on the sponge of the genus Xestospongia.

Trabectedin and plitidepsin: drugs from the sea that strike the tumor microenvironment.

The prevailing paradigm states that cancer cells acquire multiple genetic mutations in oncogenes or tumor suppressor genes whose respective activation/up-regulation or loss of function serve to impart aberrant properties, such as hyperproliferation or inhibition of cell death. However, a tumor is now considered as an organ-like structure, a complex system composed of multiple cell types (e.g., tumor cells, inflammatory cells, endothelial cells, fibroblasts, etc.) all embedded in an inflammatory stroma. All these components influence each other in a complex and dynamic cross-talk, leading to tumor cell survival and progression. As the microenvironment has such a crucial role in tumor pathophysiology, it represents an attractive target for cancer therapy. In this review, we describe the mechanism of action of trabectedin and plitidepsin as an example of how these specific drugs of marine origin elicit their antitumor activity not only by targeting tumor cells but also the tumor microenvironment.

High-performance liquid chromatographic enantioseparation of naphthol-substituted tetrahydroisoquinolines on polysaccharide-based chiral stationary phases.

The stereoisomers of 15 naphthol-substituted tetrahydroisoquinoline analogs were separated on chiral stationary phases containing the chiral selectors cellulose tris-(3,5-dimethylphenyl carbamate) (Cellulose 1), cellulose tris-(3-chloro-4-methylphenyl carbamate) (Cellulose 2), cellulose tris-(4-methylbenzoate) (Cellulose 3) and cellulose tris-(4-chloro-3-methylphenyl carbamate) (Cellulose 4). Experiments were performed in normal-phase mode with n-heptane(n-hexane)-alcohol-diethylamine mobile phases in the temperature range 5-40 °C. Thermodynamic parameters were calculated from plots of lnk or lnα vs 1/T. On the Cellulose 1 column, Δ(ΔH°) ranged from -6.8 to 5.9 kJ/mol, Δ(ΔS°) from -16.7 to 21.0 J/mol/K and the Gibbs energy, -Δ(ΔG°) from 0.2 to 1.5 kJ/mol; on the Cellulose 2 column, Δ(ΔH°) ranged from -7.8 to 2.5 kJ/mol, Δ(ΔS°) from -16.1 to 13.2 J/mol/K and - Δ(ΔG°) from 0.7 to 3.0 kJ/mol. Both enthalpy- and entropy-controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature.

Two new alkaloids from Corydalis humosa.

Two new alkaloids, named 1,1-dimethyl-6-methoxy-7-hydroxyl-1,2,3,4-tetrahydroisoquinoline (1) and (1R)-(4-hydroxybenzyl)-7-hydroxyl-8-O-ß-d-glucopyranosyl-1,2,3,4-tetrahydroisoquinoline (2), together with 11 known compounds (3-13), were isolated from the tubers of Corydalis humosa. Their structures were elucidated on the basis of their spectroscopic and chemical evidence.

Dauricine can inhibit the activity of proliferation of urinary tract tumor cells.

OBJECTIVE: To explore the anti-tumor effects of asiatic moonseed rhizome extraction-dauricine on bladder cancer EJ cell strain, prostate cancer PC-3Mcell strain and primary cell culture system. METHODS: The main effective component-phenolic alkaloids ofMenispermum dauricum was extracted and separated from asiatic moonseed rhizome by chemical method. MTT method was used to detect dauricine anti-tumor effect. RESULTS: Dauricine had an obvious proliferation inhibition effect on the main tumor cells in urinary system. The minimum drug sensitivity concentration was between 3.81-5.15 µg/mL, and the inhibition ratio increased with the increase of concentration. CONCLUSIONS: Dauricine, the main effective component extracted from asiatic moonseed rhizome, had a good inhibition effect on tumor cells in urinary system. At the same time, Dauricine has certain inhibition effects on the primary cultured tumor cell.

Hypotensive and cardio-chronotropic constituents of Tinospora crispa and mechanisms of action on the cardiovascular system in anesthetized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for the control of blood pressure. We previously found that an extract of Tinospora crispa stems decreased the mean arterial blood pressure (MAP) with a transient decrease, followed by an increase in the heart rate (HR) in rats. AIM OF THE STUDY: To identify the active components of the Tinospora crispa extract and investigate the mechanisms of action on blood pressure and heart rate in anesthetized rats. MATERIALS AND METHODS: The active components of Tinospora crispa extract were separated by column chromatography and a preparative HPLC. The effects and mechanisms of the active compounds on blood pressure and heart rate were studied in anesthetized, normal and reserpinized rats in vivo. RESULTS: 5 active compounds: adenosine, uridine, salsolinol, higenamine and tyramine were isolated. Adenosine decreased MAP and HR and this effect was inhibited by DMPX (A(2A) adenosine receptor antagonist). Uridine increased MAP and decreased HR and this was inhibited by suramin but not by DMPX. Salsolinol decreased the MAP and HR and this was inhibited by phentolamine but not by ICI-118,551 (ß(2)-adrenoceptor antagonist) or atropine. In reserpinized rats, salsolinol had a hypertensive effect that was inhibited by prazosin and phentolamine, but not by atenolol, and caused an increase in HR that was inhibited by atenolol, but not by prazosin or phentolamine. Higenamine decreased the MAP with an increase in HR. The hypotensive effect was inhibited by ICI-118,551 or atenolol, whereas the increase in HR was not inhibited by ICI-118,551. Atenolol inhibited the increase in HR at a small dosage of higenamine but potentiated it at a higher dosage. In reserpinized rats, a small dosage of higenamine tended to potentiate the effect but at a higher dosage it caused inhibition. ICI-118,551 significantly inhibited this hypotensive effect. Tyramine caused an increase in MAP and HR and these effects almost disappeared in reserpinized rats. CONCLUSIONS: The results demonstrate that these 5 compounds from Tinospora crispa acted in concert on the cardiovascular system of anesthetized rats. Salsolinol, tyramine and higenamine acted via the adrenoreceptors, whereas uridine and adenosine acted via the purinergic adenosine A(2) and P(2) receptors to decrease blood pressure with a transient decrease of HR followed by an increase.

Inhibitory effects of a bisbenzylisoquinline alkaloid dauricine on HERG potassium channels.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Menispermum dauricum have been widely used for the treatment of inflammation, allergy and arrhythmia in China for a long time. Dauricine (Dau), a bisbenzylisoquinline alkaloid from Menispermum dauricum, mainly contributes to the anti-arrhythmic effect and has received pharmacological attention. Dau can prolong the action potential duration (APD), which has been attributed to its ability to modulate Ca(2+) and several K(+) channels. However, its effects on human-ether-a-go-go-related gene (HERG) channels are unknown. AIM OF THE STUDY: The effects of Dau on HERG channels were investigated. MATERIALS AND METHODS: Whole-cell patch-clamp technique was used to record HERG current (I(HERG)) carried by recombinant HERG channels expressed in HEK293 cells. RESULTS: Dau inhibited I(HERG) in a concentration-dependent manner with an IC(50) of 3.5 µM. Development of block and washout were fast. The inhibitory action of Dau was contingent on channel gating, showing significant voltage and time dependence. Dau inhibited I(HERG) in the open and inactivated states, but not in the closed states. The activation curve was shifted in a negative direction. CONCLUSIONS: Dau inhibits HERG encoded potassium channels and this action might be a molecular mechanism for the previously reported APD prolongation with this drug.

Liquid chromatographic resolution of 1-aryl-1,2,3,4-tetrahydroisoquinolines on a chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid.

A liquid chromatographic chiral stationary phase (CSP) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied for the first time to the resolution of biologically important 1-aryl-1,2,3,4-tetrahydroisoquinolines. The unusual resolution of cyclic secondary amino compounds on a chiral crown ether-based CSP was quite successful with the use of a mixture of methanol-acetonitrile-triethylamine at a ratio of 30/70/0.5 (v/v/v) as a mobile phase. From the chromatographic behaviours for the resolution of seven 1-aryl-1,2,3,4-tetrahydroisoquinolines, the steric bulkiness of the 1-phenyl ring at the chiral center of analytes was concluded to play an important role in the chiral recognition.