Absolute Configuration and Conformational Analysis of Brevipolides, Bioactive 5,6-Dihydro-α-pyrones from <i>Hyptis brevipes</i>

The (6′<i>S</i>)-configuration of brevipolides A–J (<b>1</b>–<b>10</b>), isolated from <i>Hyptis brevipes</i>, was established by X-ray diffraction analysis of <b>9</b> in conjunction with Mosher’s ester analysis of the tetrahydro derivative <b>11</b> obtained from both geometric isomers <b>8</b> and <b>9</b> as well as by chemical correlations. The structure of the new brevipolide J (<b>10</b>) was characterized through NMR and MS data as having the same 6-heptyl-5,6-dihydro-2<i>H</i>-pyran-2-one framework possessing the cyclopropane moiety of all brevipolides but substituted by an isoferuloyl group instead of the <i>p</i>-methoxycinnamoyl moiety found in <b>8</b> and <b>9</b>. Conformational analysis of these cytotoxic 6-heptyl-5,6-dihydro-α-pyrones was carried out on compound <b>9</b> by application of a protocol based on comparison between experimental and DFT-calculated vicinal ¹H–¹H NMR coupling constants. Molecular modeling was used to correlate minimum energy conformers and observed electronic circular dichroism transitions for the isomeric series of brevipolides. Compounds <b>7</b>–<b>10</b> exhibited moderate activity (ED₅₀ 0.3–8.0 μg/mL) against a variety of tumor cell lines

Studies of (−)-Pironetin Binding to α‑Tubulin: Conformation, Docking, and Molecular Dynamics

A comprehensive conformational analysis for the anticancer agent pironetin (<b>1</b>) was achieved by molecular modeling using density functional theory calculations at the B3PW91/DGTZVP level in combination with calculated and experimental ¹H–¹H coupling constants comparison. Two solvent-dependent conformational families (<i>L</i> and <i>M</i>) were revealed for the optimum conformations. Docking studies of the pironetin–tubulin complex determined a quantitative model for the hydrogen-bond interactions of pironetin through the αAsn249, αAsn258, and αLys352 amino groups in α-tubulin, which supported the formation of a covalent adduct between the αLys352 and the β carbon atom of the α,β-unsaturated lactone. Saturation-transfer difference NMR spectroscopy confirmed that pironetin binds to tubulin, while molecular dynamics exposed a distortion of the tubulin secondary structure at the H8 and H10 α-helices as well as at the S9 β-sheet, where αLys352 is located. A large structural perturbation in the M-loop geometry between the αIle274 and αLeu285 residues, an essential region for molecular recognition between α–α and β–β units of protofilaments, was also identified and provided a rationale for the pironetin inhibitory activity

Studies of (−)-Pironetin Binding to α‑Tubulin: Conformation, Docking, and Molecular Dynamics

A comprehensive conformational analysis for the anticancer agent pironetin (<b>1</b>) was achieved by molecular modeling using density functional theory calculations at the B3PW91/DGTZVP level in combination with calculated and experimental ¹H–¹H coupling constants comparison. Two solvent-dependent conformational families (<i>L</i> and <i>M</i>) were revealed for the optimum conformations. Docking studies of the pironetin–tubulin complex determined a quantitative model for the hydrogen-bond interactions of pironetin through the αAsn249, αAsn258, and αLys352 amino groups in α-tubulin, which supported the formation of a covalent adduct between the αLys352 and the β carbon atom of the α,β-unsaturated lactone. Saturation-transfer difference NMR spectroscopy confirmed that pironetin binds to tubulin, while molecular dynamics exposed a distortion of the tubulin secondary structure at the H8 and H10 α-helices as well as at the S9 β-sheet, where αLys352 is located. A large structural perturbation in the M-loop geometry between the αIle274 and αLeu285 residues, an essential region for molecular recognition between α–α and β–β units of protofilaments, was also identified and provided a rationale for the pironetin inhibitory activity

Absolute Configuration of Acremoxanthone C, a Potent Calmodulin Inhibitor from <i>Purpureocillium lilacinum</i>

Bioassay-guided fractionation of an extract prepared from the culture medium and mycelium of <i>Purpureocillium lilacinum</i> allowed the isolation of two calmodulin (CaM) inhibitors, namely, acremoxanthone C (<b>1</b>) and acremonidin A (<b>2</b>). The absolute configuration of <b>1</b> was established as 2<i>R</i>, 3<i>R</i>, 1′<i>S</i>, 11′<i>S</i>, and 14′<i>R</i> through extensive NMR spectroscopy and molecular modeling calculations at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and experimental specific rotation, ³<i>J</i>_C,H, and ³<i>J</i>_H,H values. Compounds <b>1</b> and <b>2</b> bind to the human calmodulin (<i>h</i>CaM) biosensor <i>h</i>CaM M124C-<i>mBBr</i>, with dissociation constants (<i>K</i>_d) of 18.25 and 19.40 nM, respectively, 70-fold higher than that of chlorpromazine (<i>K</i>_d = 1.24 μM), used as positive control. Docking analysis using AutoDock 4.2 predicted that <b>1</b> and <b>2</b> bind to CaM at a similar site to that which KAR-2 binds, which is unusual. Furthermore, a novel, sensible, and specific fluorescent biosensor of <i>h</i>CaM, i<i>.</i>e<i>., h</i>CaM T110C-<i>mBBr</i>, was constructed; this device is labeled at a site where classical inhibitors do not interact and was successfully applied to measure the interaction of <b>1</b> with CaM. This is the first report of xanthone–anthraquinone heterodimers in species of <i>Paecilomyces</i> or <i>Purpureocillium</i> genera

(+)-Ascosalitoxin and Vermelhotin, a Calmodulin Inhibitor, from an Endophytic Fungus Isolated from <i>Hintonia latiflora</i>

Chemical investigation of the endophytic MEXU 26343, isolated from the medicinal plant <i>Hintonia latiflora</i>, yielded the known polyketide vermelhotin (<b>1</b>) and a new salicylic aldehyde derivative, namely, 9<i>S</i>,11<i>R</i>-(+)-ascosalitoxin (<b>2</b>). The structure and absolute configuration of the new compound were established through extensive NMR spectroscopy and molecular modeling calculations at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and experimental optical rotation values. In addition, chemical transformations of <b>2</b> yielded suitable derivatives for NOESY and ¹H–¹H NMR coupling constant analyses, which reinforce the stereochemical assignment. The potential affinity of <b>1</b> and <b>2</b> with (Ca²⁺)₄-<i>h</i>CaM in solution was measured using the fluorescent biosensor <i>h</i>CaM M124C-<i>mBBr</i>. The results showed that <b>1</b> bound to the protein with a dissociation constant (<i>K</i>_d) of 0.25 ± 0.04 μM, close to that of chlorpromazine (<i>K</i>_d = 0.64 ± 0.03 μM), a classical CaM inhibitor. The stoichiometry ratio of <b>1</b> to (Ca²⁺)₄-<i>h</i>CaM was 1:4, similar to other well-known CaM ligands

Absolute Configuration of Menthene Derivatives by Vibrational Circular Dichroism

The aerial parts of <i>Ageratina glabrata</i> afforded (−)-(3<i>S</i>,4<i>R</i>,5<i>R</i>,6<i>S</i>)-3,5,6-trihydroxy-1-menthene 3-<i>O</i>-β-d-glucopyranoside (<b>1</b>) and (−)-(3<i>S</i>,4<i>S</i>,6<i>R</i>)-3,6-dihydroxy-1-menthene 3-<i>O</i>-β-d-glucopyranoside (<b>3</b>). Acid hydrolysis of <b>1</b> yielded (+)-(1<i>R</i>,4<i>S</i>,5<i>R</i>,6<i>R</i>)-1,5,6-trihydroxy-2<i>-</i>menthene (<b>5</b>) and (+)-(1<i>S</i>,4<i>S</i>,5<i>R</i>,6<i>R</i>)-1,5,6-trihydroxy-2-menthene (<b>6</b>), while hydrolysis of <b>3</b> yielded (+)-(3<i>S</i>,4<i>S</i>,6<i>R</i>)-3,6-dihydroxy-1<i>-</i>menthene (<b>10</b>), (+)-(1<i>R</i>,4<i>S</i>,6<i>R</i>)-1,6-dihydroxy-2<i>-</i>menthene (<b>11</b>), and (+)-(1<i>S</i>,4<i>S</i>,6<i>R</i>)-1,6-dihydroxy-2<i>-</i>menthene (<b>12</b>). The structures of the new compounds <b>1</b>, <b>2</b>, <b>5</b>–<b>9</b>, and <b>11</b> were defined by 1D and 2D NMR experiments, while the absolute configurations of the series of compounds were determined by comparison of the experimental vibrational circular dichroism (VCD) spectra of the 1,6-acetonide 5-acetate derived from <b>6</b> and of the 1,6-acetonide derived from <b>12</b> with their DFT-calculated spectra. In addition, Flack and Hooft X-ray parameters of <b>10</b> permitted the same conclusion. The results further led to the absolute configuration reassignment of <b>10</b> isolated from <i>Brickellia rosmarinifolia</i>, <i>Mikania saltensis</i>, <i>Ligularia muliensis</i>, <i>L. sagitta</i>, and <i>Lindera strychnifolia</i>, as well as of <b>11</b> from <i>Cacalia tangutica</i>, as <i>ent</i>-<b>11</b>

A Macrocyclic Dimeric Diterpene with a <i>C₂</i> Symmetry Axis

An unprecedented macrocyclic dimeric diterpene containing a <i>C₂</i> symmetry axis was isolated from <i>Acacia schaffneri</i>. This compound, named schaffnerine, was characterized as (5<i>S</i>,7<i>S</i>,8<i>R</i>,­9<i>R</i>,10<i>S</i>,­17<i>S</i>,5′<i>S</i>,7′<i>S</i>,­8′<i>R</i>,9′<i>R</i>,10′<i>S</i>,­17′<i>S</i>)-7,8:7,17′:16,­17:17,7′:7′,­8′:16′,17′-hexaepoxy-7,8-<i>seco</i>-7′,8′-<i>seco</i>-dicassa-13,13′-diene (<b>1</b>) from its spectroscopic data. Comparison of its experimental vibrational circular dichroism spectrum with that calculated using density functional theory, at the B3LYP/DGDZVP level, assigned its preferred conformation and absolute configuration. The latter was confirmed by evaluation of the Flack and Hooft parameters obtained after single-crystal X-ray diffraction analysis

Methodology for the Absolute Configuration Determination of Epoxythymols Using the Constituents of <i>Ageratina glabrata</i>

A methodology to determine the enantiomeric excess and the absolute configuration (AC) of natural epoxythymols was developed and tested using five constituents of <i>Ageratina glabrata</i>. The methodology is based on enantiomeric purity determination employing 1,1′-bi-2-naphthol (BINOL) as a chiral solvating agent combined with vibrational circular dichroism (VCD) measurements and calculations. The conformational searching included an extensive Monte Carlo protocol that considered the rotational barriers to cover the whole conformational spaces. (+)-(8<i>S</i>)-10-Benzoyloxy-6-hydroxy-8,9-epoxythymol isobutyrate (<b>1</b>), (+)-(8<i>S</i>)-10-acetoxy-6-methoxy-8,9-epoxythymol isobutyrate (<b>4</b>), and (+)-(8<i>S</i>)-10-benzoyloxy-6-methoxy-8,9-epoxythymol isobutyrate (<b>5</b>) were isolated as enantiomerically pure constituents, while 10-isobutyryloxy-8,9-epoxythymol isobutyrate (<b>2</b>) was obtained as a 75:25 (8<i>S</i>)/(8<i>R</i>) scalemic mixture. In the case of 10-benzoyloxy-8,9-epoxythymol isobutyrate (<b>3</b>), the BINOL methodology revealed a 56:44 scalemic mixture and the VCD measurement was beyond the limit of sensitivity since the enantiomeric excess is only 12%. The racemization process of epoxythymol derivatives was studied using compound <b>1</b> and allowed the clarification of some stereochemical aspects of epoxythymol derivatives since their ACs have been scarcely analyzed and a particular behavior in their specific rotations was detected. In more than 30 oxygenated thymol derivatives, including some epoxythymols, the reported specific rotation values fluctuate from −1.6 to +1.4 passing through zero, suggesting the presence of scalemic and close to racemic mixtures, since enantiomerically pure natural constituents showed positive or negative specific rotations greater than 10 units

Absolute Configuration of (13<i>R</i>)- and (13<i>S</i>)‑Labdane Diterpenes Coexisting in <i>Ageratina jocotepecana</i>

Chemical investigation of the hexanes extracts of <i>Ageratina jocotepecana</i> afforded (−)-(5<i>S</i>,9<i>S</i>,10<i>S</i>,13<i>S</i>)-labd-7-en-15-oic acid (<b>1</b>), methyl (−)-(5<i>S</i>,9<i>S</i>,10<i>S</i>,13<i>S</i>)-labd-7-en-15-oate (<b>2</b>), (+)-(5<i>S</i>,8<i>R</i>,9<i>R</i>,10<i>S</i>,13<i>R</i>)-8-hydroxylabdan-15-oic acid (<b>3</b>), and (−)-(5<i>S</i>,9<i>S</i>,10<i>S</i>,13<i>Z</i>)-labda-7,13-dien-15-oic acid (<b>5</b>). The coexistence of (13<i>R</i>)- and (13<i>S</i>)-labdanes in this member of the Asteraceae family was demonstrated by vibration circular dichroism measurements of ester <b>2</b> and methyl (+)-(5<i>S</i>,8<i>R</i>,9<i>R</i>,10<i>S</i>,13<i>R</i>)-8-hydroxylabdan-15-oate (<b>4</b>) in comparison to the DFT B3LYP/DGDZVP-calculated spectra. In addition, transformation of <b>1</b> and <b>3</b> with HClO₄ in MeOH yielded epimeric methyl (+)-(5<i>S</i>,10<i>S</i>,13<i>S</i>)-labd-8-en-15-oate (<b>6</b>) and methyl (+)-(5<i>S</i>,10<i>S</i>,13<i>R</i>)-labd-8-en-15-oate (<b>7</b>), respectively, confirming the presence of C-13 epimers in this plant. Diterpene <b>1</b> showed remarkable antibacterial activity against <i>Bacillus subtilis</i> (MIC 0.15 mg/mL) and <i>Staphylococcus aureus</i> (MIC 0.78 mg/mL), while diterpene <b>3</b> exhibited moderate activities against the same organisms