Carlos M. Cerda-García-Rojas

Application of molecular mechanics in the total stereochemical elucidation of spicigerolide, a cytotoxic 6-tetraacetyloxyheptenyl-5,6-dihydro-α-pyrone from Hyptis spicigera☆

Abstract Bioactivity-directed fractionation of the crude extract prepared from the medicinal Mexican plant Hyptis spicigera (Lamiaceae) tested on KB cells led to the isolation of spicigerolide (1). The structure for this novel cytotoxic compound was elucidated as 6R-[3S,4S,5S,6S-tetraacetyloxy-1Z-heptenyl]-5,6-dihydro-2H-pyran-2-one. The relative stereochemistry of this flexible molecule was determined by a combination of molecular mechanics calculations and 1H–1H coupling constant data, while the absolute configuration was established according to CD measurements. The MM/3JH–H calculations, as applied to 1, was validated with model linear compounds prepared from l -rhamnose: 2,3,4,5-tetra-O-acetyl-6-deoxy- l -mannose (5) and tetra-O-acetyl-1,6-dideoxy- l -mannitol (8). Both compounds possess the same stereochemistry predicted to be present in the acyclic moiety of spicigerolide (1) but lacking the stereochemical influence of the chiral pyrone.

Conformational Behavior and Absolute Stereostructure of Two Phytotoxic Nonenolides from the Fungus Phoma herbarum

Abstract Bioactivity-directed fractionation of extracts from the fungus Phoma herbarum led to the isolation of two new phytotoxic nonenolides: (7S,8S,9R)-7,8-dihydroxy-9-propyl-5-nonen-9-olide (1) and (2R,7S,8S,9R)-2,7,8-trihydroxy-9-propyl-5-nonen-9-olide (2), which were named herbarumins I and II, respectively. The stereostructures were elucidated by spectroscopic methods and a combination of molecular modeling, NOESY and 1H–1H coupling constant data, which revealed that in CDCl3 solution, 1 exists in one preferred conformation, while 2 exhibits a conformational equilibrium. Compounds 1 and 2 caused significant inhibition of radicle growth of seedlings of Amaranthus hypochondriacus.

A PC program for calculation of dihedral angles from 1H NMR data

Abstract The program ALTONA, written in BASIC, calculates plots of H-C-C-H dihedral angles from proton-proton NMR vicinal coupling constants using an empirically generalized Karplus-type equation, which takes into account the electronegativity and the orientation of the substituents attached to the considered H-C-C-H fragment. Generation of a plot for the fragment results after introduction of the atomic symbols of the substituents (C, H, O, N, S, P, F, Cl, Br, and/or I) and their orientation (+ or −). ALTONA is compiled in Turbo BASIC for PC compatible computers and is included in the present paper as ALTONA.EXE on disk.

Changes in the concentration of trigonelline in a semi-arid leguminous plant (Prosopis laevigata) induced by an arbuscular mycorrhizal fungus during the presymbiotic phase

Abstract. An in vitro presymbiotic system between mesquite [Prosopis laevigata (Willd.) M.C. Johnst], a semi-arid leguminous plant, and pregerminated spores of Gigaspora rosea Nicol. & Schenck was established. After characteristic hyphal branching, high performance liquid chromatographic analyses of methanol extracts from P. laevigata roots revealed a concentration change in one ultraviolet-detectable product. This product was identified by nuclear magnetic resonance and mass spectrometry as trigonelline, a pyridine alkaloid. The concentration of trigonelline was constant in the aerial parts of the plant with or without G. rosea, but its concentration in the roots increased 1.8-fold when G. rosea was present. Trigonelline may be a regulatory factor during early signal events in the establishment of the arbuscular mycorrhizal symbiosis in P. laevigata.

Calmodulin inhibitors from the fungus Emericella sp.

Two new xanthones identified as 15-chlorotajixanthone hydrate (1) and 14-methoxytajixanthone (2) were isolated from an Emericella sp. strain 25379 along with shamixanthone (3) and tajixanthone hydrate (4). The stereostructures of 1 and 2 were elucidated by spectroscopic and molecular modeling methods. The absolute configuration at the stereogenic centers of 1 was established according to CD measurements. In the case of 2, however, the absolute configuration at C-20 and C-25 was designated as S and R, respectively, by Mosher ester methodology. Thereafter, the configuration at C-14 and C-15 of 2 was established as S and S, respectively by comparing the optical rotation and (1)H-(1)H coupling constant experimental values with those obtained through molecular modeling calculations at DFT B3LYP/DGDZVP level of theory for diasteroisomers 2a-2d. The activation of the calmodulin-sensitive cAMP phosphodiesterase (PDE1) was inhibited in the presence of 1-4 in a concentration-dependent manner. The effect of compounds 2 (IC(50)=5.54 microM) and 4 (IC(50)=5.62 microM) was comparable with that of chlorpromazine (CPZ; IC(50)=7.26 microM), a well known CaM inhibitor used as a positive control. The inhibition mechanism of both compounds was competitive with respect to CaM according to a kinetic study. A docking analysis with 2 and 4 using the AutoDock 4.0 program revealed that they interacted with CaM in the same pocket as trifluoropiperazine (TFP).

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H2O2, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H2O2 (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days−1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H2O2 had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H2O2, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H2O2, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H2O2 with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H2O2 in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.

Structure, Conformation and Absolute Configuration of New Antifeedant Dolabellanes from Trichilia trifolia

Abstract The structures of three new dolabellane diterpenoids, (1R,3E,7Z,11S,12S)-dolabella-3,7,18-trien-17-oic acid (1), (1R,3E,6R,7Z,11S,12S)-dolabella-3,7,18-trien-6,17-olide (2) and (1R,3S,4R,7Z,11S,12S)-3-hydroxydolabella-7,18-dien-4,17-olide (3), isolated from the wood of Trichilia trifolia, were elucidated by 1D and 2D NMR spectroscopy. The stereochemistry of 1 and 2 was confirmed by X-ray diffraction analysis, while that of 3 was ascertained from NOESY data. Comparison between experimental and calculated 1H–1H vicinal coupling constants and the analysis of molecular mechanics structures revealed that the 11-membered ring of 1 and 2 exists in a conformational equilibrium in solution, while in 3 this ring possesses a more rigid structure. The absolute configuration of 3 was established from its Cotton effects. Dolabellanes 1–3 caused significant feeding reduction by the rice weevil Sitophilus oryzae.

Vibrational Circular Dichroism of Africanane and Lippifoliane Sesquiterpenes from Lippia integrifolia

The agreement between theoretical and experimental vibrational circular dichroism curves of (4R,9R,10R)-(+)-african-1(5)-ene-2,6-dione (1) and (4R,9S,10R)-lippifoli-1(6)-en-5-one (2), isolated from the widely used plant Lippia integrifolia, allowed the determination of the conformation and absolute configuration of 1 and confirmed both structural features for 2. Molecular modeling of 1 and 2 was carried out by means of a systematic and a Monte Carlo search protocol followed by geometry optimization employing density functional theory calculations with the B3LYP/6-31G(d), B3LYP/DGDZVP, and/or B3PW91/DGDZVP2 functionals/basis sets. Validation of the minimum energy conformations for both tricyclic substances was achieved by comparison of the experimental and theoretical vicinal (1)H-(1)H NMR coupling constants obtained by DFT-GIAO calculations.

Vicinal 1H–1H NMR Coupling Constants from Density Functional Theory as Reliable Tools for Stereochemical Analysis of Highly Flexible Multichiral Center Molecules

A protocol for stereochemical analysis, based on the systematic comparison between theoretical and experimental vicinal (1)H-(1)H NMR coupling constants, was developed and applied to a series of flexible compounds (1-8) derived from the 6-heptenyl-5,6-dihydro-2H-pyran-2-one framework. The method included a broad conformational search, followed by geometry optimization at the DFT B3LYP/DGDZVP level, calculation of the vibrational frequencies, thermochemical parameters, magnetic shielding tensors, and the total NMR spin-spin coupling constants. Three scaling factors, depending on the carbon atom hybridizations, were found for the (1)H-C-C-(1)H vicinal coupling constants: f((sp3)-(sp3)) = 0.910, f((sp3)-(sp2)) = 0.929, and f((sp2)-(sp2))= 0.977. A remarkable correlation between the theoretical (J(pre)) and experimental (1)H-(1)H NMR (J(exp)) coupling constants for spicigerolide (1), a cytotoxic natural product, and some of its synthetic stereoisomers (2-4) demonstrated the predictive value of this approach for the stereochemical assignment of highly flexible compounds containing multiple chiral centers. The stereochemistry of two natural 6-heptenyl-5,6-dihydro-2H-pyran-2-ones (14 and 15) containing diverse functional groups in the heptenyl side chain was also analyzed by application of this combined theoretical and experimental approach, confirming its reliability. Additionally, a geometrical analysis for the conformations of 1-8 revealed that weak hydrogen bonds substantially guide the conformational behavior of the tetraacyloxy-6-heptenyl-2H-pyran-2-ones.

Absolute configuration of 7,8-seco-7,8-oxacassane diterpenoids from Acacia schaffneri.

Chemical investigations of Acacia schaffneri led to the isolation of the new diterpenoid (5S,7R,8R,9R,10S)-(-)-7,8-seco-7,8-oxacassa-13,15-diene-7,17-diol (1), together with the known (5S,7R,8R,9R,10S)-(-)-7,8-seco-7,8-oxacassa-13,15-dien-7-ol-17-al (2) and (5S,7R,8R,9R,10S)-(-)-7,8-seco-7,8-oxacassa-13,15-dien-7-ol (3). Compounds 2 and 3 were analyzed by single-crystal X-ray diffraction, while the structure of 1 was determined by 1D and 2D NMR experiments and by chemical correlation with 2. Oxidation of 3 afforded conformationally restricted (5S,8R,9R,10S)-(-)-8-hydroxy-7,8-seco-cassa-13,15-dien-7-oic acid ε-lactone (4), which was studied by vibrational circular dichroism spectroscopy. Comparison of the experimental VCD spectrum of 4 with the DFT//B3PW91/DGDZVP2 calculated spectrum assigned for the first time the absolute configuration of these seco-oxacassane diterpenes.