Stereoselective Oxidation of α-Copaene, a Fire Ant Repellent Sesquiterpene from the Essential Oil of Dipterocarpus turbinatus.

Imported fire ants are significant agricultural pests. Repellents can be used to prevent foraging fire ants from entering sensitive areas, including electrical equipment, nursing homes, and hospitals. Bioassay-guided fractionation of the essential oil extracted from gurjun balsam (Dipterocarpus turbinatus) resulted in the identification of (-)-α-copaene (1) as the repellent constituent with a minimum repellent effective dose (MRED) of 15.6 µg/g against both red imported fire ants (Solenopsis invicta) and hybrid imported fire ants (Solenopsis invicta × Solenopsis richteri). Stereoselective oxidation of 1 via autoxidation and chemical methods produced (-)-5R-hydroperoxy-α-copaene (2), (+)-3S-hydroperoxycopa-4-ene (3), (-)-α-copaene oxide (4), (+)-ß-copaen-4α-ol (5), copaenediol (6), and copaene ketol (7). Reduction of 2 and 3 with triphenylphosphine afforded (-)-5R-hydroxy-α-copaene (2a) and (+)-3S-hydroxycopa-4-ene (3a), respectively, which led to the structural revision of copa-3-en-2α-ol and copa-2-en-4-ol as 2a and 3, respectively. The configurational assignment of compound 4 in the literature was also clarified by the detailed analysis of 2D NMR spectroscopic data. Compounds 2-7 showed repellency with MREDs ranging from 3.9 to 15.6 µg/g against hybrid and red imported fire ants, indicating that chemical modification can enhance the repellent effect of (-)-α-copaene.

Rapid preparation and proton NMR fingerprinting of polysaccharides from Radix Astragali.

The purity, content, and structure of the polysaccharides prepared from a specific medicinal plant are the fundamental basis to interpret the observed biological activities. An ultrafiltration-based method has been developed for rapid preparation of total and fractional polysaccharides from Radix Astragali in high yield and purity. This method involves extraction of plant material by hot water, treatment with Sevag reagent, and ultrafiltration using molecular weight cutoff concentrators. The prepared polysaccharides were assessed by 1H NMR spectroscopy, providing general purity, fingerprinting, and structural information. This method may be used to efficiently screen polysaccharides in plants.

Repellency of Carvacrol, Thymol, and Their Acetates against Imported Fire Ants.

In the United States, imported fire ants are commonly referred to as red imported fire ants (Solenopsis invicta Buren), black imported fire ants (S. richteri Forel), and hybrid imported fire ants (S. invicta × S. richteri). They are significant pests, and their control heavily relies on synthetic insecticides. The extensive use of insecticides has led to public concern about their potential negative effects on human health and the well-being of wildlife and the environment. As an alternative, plant-derived natural compounds, particularly essential oils (EOs) and their main constituents, show promise as safe and environmentally friendly products for controlling fire ants. Repellants are useful in managing fire ants, and plant-derived natural repellants may serve as a safer and more environmentally friendly option. This study investigates the repellency of EO-derived compounds carvacrol, thymol, and their acetates against imported fire ant workers. The results revealed that carvacrol, a GRAS compound (Generally Recognized As Safe), was the most potent repellent against S. invicta, S. richteri, and their hybrid, with minimum repellent effective doses (MREDs) of 0.98 µg/g, 7.80 µg/g, and 0.98 µg/g, respectively. Thymol also exhibited strong repellency, with MREDs of 31.25 µg/g, 31.25 µg/g, and 7.8 µg/g, respectively. Furthermore, thyme-red essential oil, characterized by a thymol chemotype containing 48.8% thymol and 5.1% carvacrol, was found to effectively repel the hybrid ants with an MRED of 15.6 µg/g. In contrast, thyme essential oil, characterized by a linalool chemotype lacking thymol and carvacrol, did not exhibit any repellent effect, even at the highest tested dose of 125 µg/g. This study provides the first evidence of the potent repellency of carvacrol and thymol against imported fire ant workers, indicating their potential as promising repellents for fire ant control.

Cannabinoid Receptor Type II Ligands from Sandalwood Oil and Synthetic α-Santalol Derivatives.

Bioassay-guided fractionation of the essential oil of Santalum album led to the identification of α-santalol (1) and ß-santalol (2) as new chemotypes of cannabinoid receptor type II (CB2) ligands with Ki values of 10.49 and 8.19 µM, respectively. Nine structurally new α-santalol derivatives (4a-4h and 5) were synthesized to identify more selective and potent CB2 ligands. Compound 4e with a piperazine structural moiety demonstrated a Ki value of 0.99 µM against CB2 receptor and did not show binding activity against cannabinoid receptor type I (CB1) at 10 µM. Compounds 1, 2, and 4e increased intracellular calcium influx in SH-SY5Y human neuroblastoma cells that were attenuated by CB2 antagonism or inverse agonism, supporting the results that these compounds are CB2 agonists. Molecular docking showed that 1 and 4e had similar binding poses, exhibiting a unique interaction with Thr114 within the CB2 receptor, and that the piperazine structural moiety is required for the binding affinity of 4e. A 200 ns molecular dynamics simulation of CB2 complexed with 4e confirmed the stability of the complex. This structural insight lays a foundation to further design and synthesize more potent and selective α-santalol-based CB2 ligands for drug discovery.

Matricaria chamomilla Essential Oils: Repellency and Toxicity against Imported Fire Ants (Hymenoptera: Formicidae).

Matricaria chamomilla flower essential oils (EOs) blue Egyptian (EO-1), chamomile German CO2 (EO-2), and chamomile German (EO-3) and the pure compound α-bisabolol were evaluated against red imported fire ants (RIFA), Solenopsis invicta Buren, black imported fire ants, S. richteri Forel (BIFA), and hybrid imported fire ants (HIFA) for their repellency and toxicity. A series of serial dilutions were tested starting from 125 µg/g until the failure of the treatment. Based on the amount of sand removed, EO-1 showed significant repellency at dosages of 7.8, 7.8, and 31.25 µg/g against RIFA, BIFA, and HIFA, respectively. EO-3 was repellent at 3.9, 7.8, and 31.25 µg/g against BIFA, RIFA, and HIFA, whereas α-bisabolol was active at 7.8, 7.8, and 31.25 µg/g against BIFA, HIFA, and RIFA, respectively. DEET (N, N-diethyl-meta-toluamide) was active at 31.25 µg/g. Toxicity of EOs and α-bisabolol was mild to moderate. For EO-1, LC50 values were 93.6 and 188.11 µg/g against RIFA and BIFA; 98.11 and 138.4 µg/g for EO-2; and 142.92 and 202.49 µg/g for EO-3, respectively. The LC50 of α-bisabolol was 159.23 µg/g against RIFA. In conclusion, M. chamomilla EOs and α-bisabolol offer great potential to be developed as imported fire ant repellents.

Thiol-Chromene "Click" Reaction-Activated Chemiluminescent Probe for Thiol Detection In Vitro and In Vivo.

Biothiols are biomolecules found in a higher content in cancer cells compared to normal cells, marking them useful cancer biomarkers. Chemiluminescence is widely used in biological imaging due to its excellent sensitivity and signal-to-noise ratio (SNR). In this study, we designed and prepared a chemiluminescent probe that is activated by a thiol-chromene "click" nucleophilic reaction. This probe is initially chemiluminescent but turned off and releases extremely strong chemiluminescence in the presence of thiols. It has high selectivity to thiol compared with other analytes. Real-time imaging of mice tumor sites showed significant chemiluminescence after the probe was injected, and the chemiluminescence of osteosarcoma tissues was also significantly stronger than that in adjacent tissues. We conclude that this chemiluminescent probe has potential to detect thiol, diagnose cancer, especially in its early stages, and aid in the development of related cancer drugs.

Molecular Targets of Cannabinoids Associated with Depression.

Novel therapeutic strategies are needed to address depression, a major neurological disorder affecting hundreds of millions of people worldwide. Cannabinoids and their synthetic derivatives have demonstrated numerous neurological activities and may have the potential to be developed into new treatments for depression. This review highlights cannabinoid (CB) receptors, monoamine oxidase (MAO), N-methyl-D-aspartate (NMDA) receptor, gammaaminobutyric acid (GABA) receptor, and cholecystokinin (CCK) receptor as key molecular targets of cannabinoids that are associated with depression. The anti-depressant activity of cannabinoids and their binding modes with cannabinoid receptors are discussed, providing insights into rational design and discovery of new cannabinoids or cannabimimetic agents with improved druggable properties.

Synthesis and Antifungal Activity Evaluation of Phloeodictine Analogues.

The phloeodictine-based 6-hydroxy-2,3,4,6-tetrahydropyrrolo[1,2-a]pyrimidinium structural moiety with an n-tetradecyl side chain at C-6 has been demonstrated to be a new antifungal template. Thirty-four new synthetic analogues with modifications of the bicyclic tetrahydropyrrolopyrimidinium skeleton and the N-1 side chain have been prepared and evaluated for in vitro antifungal activities against the clinically important fungal pathogens including Cryptococcus neoformans ATCC 90113, Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, and Aspergillus fumigatus ATCC 90906. Nineteen compounds (5, 21-31, 34-38, 44, and 48) showed antifungal activities against the aforementioned five fungal pathogens with minimum inhibitory concentrations (MICs) in the range 0.88-10 µM, and all were fungicidal with minimum fungicidal concentrations (MFCs) similar to the respective MIC values. Compounds 24, 36, and 48 were especially active against C. neoformans ATCC 90113 with MIC/MFC values of 1.0/1.0, 1.6/1.6, and 1.3/2.0 µM but exhibited low cytotoxicity with an IC50 > 40 µM against the mammalian Vero cells. The structure and antifungal activity relationship indicates that synthetic modifications of the phloeodictines can afford analogues with potent antifungal activity and reduced cytotoxicity, necessitating further preclinical studies of this new class of antifungal compounds.

Cytotoxic Diterpenoids from Euphorbia fischeriana.

Five new diterpenoids, named euphorfischerins A-E, were isolated from the roots of Euphorbia fischeriana. Their chemical structures and absolute configurations were determined by interpretation of NMR, HR-ESI-MS, ECD and X-ray diffraction data. Euphorfischerin A showed cytotoxicity against the human cancer cell lines HeLa, H460 and Namalwa with IC50 values of 4.6, 11.5 and 16.4 µM, respectively, while euphorfischerin B gave comparable IC50 values of 9.5, 17.4 and 13.3 µM against the three cancer cell lines, respectively.

Identification of Antifungal Bisphosphocholines from Medicinal Gentiana Species.

Gentiana species including G. crassicaulis, G. macrophylla, G. dahurica, and G. straminea are used in traditional Chinese medicine as "Qinjiao" for the treatment of rheumatism, hepatitis, and pain. Four antifungal bisphosphocholines [irlbacholine (2) and three new analogues, gentianalines A-C (1, 3, and 4)] were identified from G. crassicaulis by a bioassay-guided fractionation and structure elucidation approach. Subsequent chemical analysis of 56 "Qinjiao" samples (45 from G. crassicaulis, five from G. macrophylla, three from G. dahurica, and three from G. straminea) showed that bisphosphocholines were present in all four Gentiana species, with irlbacholine as the major compound ranging from 2.0 to 6.2 mg per gram of dried material. Irlbacholine exhibited potent in vitro antifungal activity against Cryptococcus neoformans, Aspergillus fumigatus, Candida albicans, and Candida glabrata with minimum inhibitory concentration (MIC) values of 0.63, 1.25, 10.0, and 5.0 µg/mL, respectively. Identification of the bisphosphocholines, a rare class of antifungal natural products, in these medicinal plants provides scientific evidence to complement their medicinal use. The bisphosphocholines carrying a long aliphatic chain possess amphiphilic molecule-like properties with a tendency of retention in both normal and reversed-phase silica gel column chromatography and thereby may be neglected in natural products discovery. This report may stimulate interest in this class of compounds, which warrant the further study of other biological activities as well.

Chemometrics-Assisted Identification of Anti-Inflammatory Compounds from the Green Alga Klebsormidium flaccidum var. zivo.

The green alga Klebsormidium flaccidum var. zivo is a rich source of proteins, polyphenols, and bioactive small-molecule compounds. An approach involving chromatographic fractionation, anti-inflammatory activity testing, ultrahigh performance liquid chromatography-mass spectrometry profiling, chemometric analysis, and subsequent MS-oriented isolation was employed to rapidly identify its small-molecule anti-inflammatory compounds including hydroxylated fatty acids, chlorophyll-derived pheophorbides, carotenoids, and glycoglycerolipids. Pheophorbide a, which decreased intracellular nitric oxide production by inhibiting inducible nitric oxide synthase, was the most potent compound identified with an IC50 value of 0.24 µM in lipopolysaccharides-induced macrophages. It also inhibited nuclear factor kappaB activation with an IC50 value of 32.1 µM in phorbol 12-myristate 13-acetate-induced chondrocytes. Compared to conventional bioassay-guided fractionation, this approach is more efficient for rapid identification of multiple chemical classes of bioactive compounds from a complex natural product mixture.

Puupehenone, a Marine-Sponge-Derived Sesquiterpene Quinone, Potentiates the Antifungal Drug Caspofungin by Disrupting Hsp90 Activity and the Cell Wall Integrity Pathway.

The cell wall-targeting echinocandin antifungals, although potent and well tolerated, are inadequate in treating fungal infections due to their narrow spectrum of activity and their propensity to induce pathogen resistance. A promising strategy to overcome these drawbacks is to combine echinocandins with a molecule that improves their activity and also disrupts drug adaptation pathways. In this study, we show that puupehenone (PUUP), a marine-sponge-derived sesquiterpene quinone, potentiates the echinocandin drug caspofungin (CAS) in CAS-resistant fungal pathogens. We have conducted RNA sequencing (RNA-seq) analysis, followed by genetic and molecular studies, to elucidate PUUP's CAS-potentiating mechanism. We found that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2 (Mpk1), the terminal mitogen-activated protein (MAP) kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its cochaperone Cdc37. Thus, we show that PUUP potentiates CAS activity by a previously undescribed mechanism which involves a disruption of Hsp90 activity and the CWI pathway. Given the requirement of the Hsp90-Cdc37 complex in Slt2 activation, we suggest that inhibitors of this complex would disrupt the CWI pathway and synergize with echinocandins. Therefore, the identification of PUUP's CAS-potentiating mechanism has important implications in the development of new antifungal combination therapies.IMPORTANCE Fungal infections cause more fatalities worldwide each year than malaria or tuberculosis. Currently available antifungal drugs have various limitations, including host toxicity, narrow spectrum of activity, and pathogen resistance. Combining these drugs with small molecules that can overcome these limitations is a useful strategy for extending their clinical use. We have investigated the molecular mechanism by which a marine-derived compound potentiates the activity of the antifungal echinocandin caspofungin. Our findings revealed a mechanism, different from previously reported caspofungin potentiators, in which potentiation is achieved by the disruption of Hsp90 activity and signaling through the cell wall integrity pathway, processes that play important roles in the adaptation to caspofungin in fungal pathogens. Given the importance of stress adaptation in the development of echinocandin resistance, this work will serve as a starting point in the development of new combination therapies that will likely be more effective and less prone to pathogen resistance.

Pyridine Alkaloids in the Venom of Imported Fire Ants.

Venomous imported fire ants cause significant medical problems. Alkaloids are an important component of imported fire ant venom. Piperidine and piperideine alkaloids have been identified in fire ant venom. In this study, we studied the venom alkaloids of the red imported fire ant, Solenopsis invicta Buren, the black imported fire ant, Solenopsis richteri Forel, and the hybrid, S. invicta × S. richteri. Pyridine alkaloids were detected the first time in fire ants using solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (SPME-GC-MS). The thermal desorption process was manipulated to facilitate the isolation and identification of pyridine alkaloids that were coeluted with piperidine or piperideine alkaloids in GC. After SPME extraction of ant venom, we conducted a series of consecutive GC-MS injections, each with a partial desorption. Hidden pyridine alkaloid peaks were revealed after the overlapping piperidine or piperidiene alkaloid peaks had been desorbed. Using this approach, 10 2-methyl-6-alkyl (or alkenyl)pyridines (1-10) were found the first time in the venom of imported fire ants. Structures of three pyridine alkaloids were confirmed by synthesis, including 2-methyl-6-undecylpyridine (1), 2-methyl-6-tridecylpyridine (7), and 2-methyl-6-pentadecylpyridine (10). We also developed a silica gel column chromatography method to separate the pyridine alkaloids from other alkaloids. Using column chromatography and GC-MS with single ion monitoring at 107 m/z, five pyridine alkaloids were quantified for both workers and female alates of S. invicta and S. richteri.

Quantitative determination and pharmacokinetic study of fusaricidin A in mice plasma and tissues using ultra-high performance liquid chromatography-tandem mass spectrometry.

Fusaricidins are a family of cyclic lipodepsipeptides that convey antifungal and antibacterial activity. Fusaricidin A (FA) is one of the Fusaricidins major compounds and it is showing promising activity against fungi and bacteria. In the present study, a fast and sensitive ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the analysis of FA in mice plasma, liver, kidney and brain tissues. The instrument was operated in positive electrospray ionization mode. Multiple reaction monitoring (MRM) mode was performed with ion pairs of m/z: 883.5→256.3, 883.5→197.2 and 883.5→72.1 for FA. The method was validated for linearity, repeatability, accuracy, stability, limits of detection (LOD) and limits of quantification (LOQ). The LOD and LOQ were 0.01 and 0.05 ng/mL for plasma and tissues, respectively. The calibration curve (10-200 ng/mL) was linear ( r2 = 0.99). Precision and accuracy values were found to be < 10% (within acceptable limit). The pharmacokinetic and tissue distribution characteristics of FA were determined in plasma, liver, kidney and brain of CD1 mice after I.V. administration of a single dose of 15 mg/kg body weight. Highest plasma concentration (Cmax) was calculated to be 4169.97 ± 50 ng/mL with a tmax of 0.08 h. The plasma clearance rate of FA was 397.6 ± 203 mL/h with a t1/2 of 2.2 ± 0.5 h and apparent volume of distribution during the terminal phase (Vz) of 979.2 ± 318 mL. The highest tissue concentration (Cmax) was found in the liver (219 ± 14 ng/mg) at a tmax of 0.08 h followed by the kidneys (38.6 ± 16 ng/mg) at tmax of 0.2 h. FA was poorly distributed to the brain with a Cmax of 0.45 ± 0.2 ng/mg and a tmax of 0.08 h. The method for quantitative analysis and pharmacokinetic data provided will support the development of various formulation approaches and therapeutic application for future clinical studies.

Identification of fusaricidins from the antifungal microbial strain Paenibacillus sp. MS2379 using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Paenibacillus sp. MS2379 is a highly efficient microbial strain producing fusaricidins, a class of lipopeptides that have demonstrated strong antifungal activities against a broad array of fungal pathogens. An integrated approach combining chromatographic fractionation, UHPLC-QTOF-MS analysis, and NMR spectroscopic interpretation was employed to characterize antifungal metabolites produced by this microbial strain, resulting in the identification of 48 fusaricidins including 30 cyclic and 18 open-chain species. In this regard, UHPLC-QTOF-MS played a vital role in determining structures of 28 new fusaricidins through peptide fragment analysis. The structural determination of the new fusaricidins by the high-resolution mass spectrometry was validated by follow-up isolation and NMR spectroscopic analysis of representative compounds. It is worth noting that novel fusaricidins with amino acid residues of serine and γ-aminobutyric acid were identified, which is of great biosynthetic significance for this biologically important class of compounds. The present study again illustrates the power of UHPLC-QTOF-MS for structural identification of lipopeptides, and the structural diversity of the identified fusaricidins makes this microbial strain unique as a potential biocontrol agent.

Synthesis and Anti-Inflammatory Activities of Phloroglucinol-Based Derivatives.

The natural product phloroglucinol-based derivatives representing monoacyl-, diacyl-, dimeric acyl-, alkylated monoacyl-, and the nitrogen-containing alkylated monoacylphloro- glucinols were synthesized and evaluated for inhibitory activities against the inflammatory mediators such as inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-κB). The diacylphloroglucinol compound 2 and the alkylated acylphloroglucinol compound 4 inhibited iNOS with IC50 values of 19.0 and 19.5 µM, respectively, and NF-κB with IC50 values of 34.0 and 37.5 µM, respectively. These compounds may serve as leads for the synthesis of more potent anti-inflammatory compounds for future drug discovery.

Anti-Leishmanial and Cytotoxic Activities of a Series of Maleimides: Synthesis, Biological Evaluation and Structure-Activity Relationship.

In the present study, 45 maleimides have been synthesized and evaluated for anti-leishmanial activities against L. donovani in vitro and cytotoxicity toward THP1 cells. All compounds exhibited obvious anti-leishmanial activities. Among the tested compounds, there were 10 maleimides with superior anti-leishmanial activities to standard drug amphotericin B, and 32 maleimides with superior anti-leishmanial activities to standard drug pentamidine, especially compounds 16 (IC50 < 0.0128 µg/mL) and 42 (IC50 < 0.0128 µg/mL), which showed extraordinary efficacy in an in vitro test and low cytotoxicities (CC50 > 10 µg/mL). The anti-leishmanial activities of 16 and 42 were 10 times better than that of amphotericin B. The structure and activity relationship (SAR) studies revealed that 3,4-non-substituted maleimides displayed the strongest anti-leishmanial activities compared to those for 3-methyl-maleimides and 3,4-dichloro-maleimides. 3,4-dichloro-maleimides were the least cytotoxic compared to 3-methyl-maleimides and 3,4-non-substituted maleimides. The results show that several of the reported compounds are promising leads for potential anti-leishmanial drug development.

Bioactive Penicipyrrodiether A, an Adduct of GKK1032 Analogue and Phenol A Derivative, from a Marine-Sourced Fungus Penicillium sp. ZZ380.

Penicipyrrodiether A, an adduct of GKK1032 analogue and phenol A derivative, was isolated from a culture of marine-associated fungus Penicillium sp. ZZ380 and represents the first example of this type of fungal metabolite. Its structure was elucidated by extensive spectroscopic analyses, including 1D- and 2D-NMR, HRESIMS, MS/MS, and electronic circular dichroism calculation as well as single-crystal X-ray diffraction. Penicipyrrodiether A showed antibacterial activity in inhibiting the growth of methicillin-resistant Staphylococcus aureus with a MIC value of 5.0 µg/mL. Its plausible pathway for biosynthesis has been proposed.

Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics.

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol ( Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acetyl-3-butanoylchloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1- p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 µg/mL, respectively.

Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box.

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.