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A novel analytical method has been developed for direct quantification of intracellular nucleoside triphosphates (NTPs). Lysates of human peripheral blood mononuclear cells (PBMCs) were extracted by protein precipitation, and the filtered extracts were analyzed by weak anion exchange liquid chromatography (WAX-LC) coupled to detection by mass spectrometry (MS). Compared with ion pairing (IP)-LC/MS/MS, the only MS-compatible direct detection method for NTPs currently available, the new method completely avoids the usage of ion-pairing reagents and has a shorter analytical time of only 2 min. The method was validated and is being used to determine the amount of the triphosphate metabolite of D-D4FC (DPC817), an investigational HIV nucleoside reverse transcriptase inhibitor (NRTI), in human PBMC samples from clinical studies. By using a PE Sciex API 4000 triple quadrupole instrument operating in positive ion MRM mode, the method was able to achieve a lower limit of quantitation (LLOQ) of 5 fmol/10(6) cells in samples containing 3 x 10(6) lysed cells (6 fmol on-column). With minor adaptation, the method described here may be suitable for analyzing other NTPs. This paper also provides a discussion of the unique retention characteristics of WAX-LC, the principles of which may prove to be valuable for designing other forms of directly coupled ion-exchange (IX)-LC/MS methods suited for high sensitivity quantitative analysis.

The acetogenins are a class of potent bioactive compounds found in various plant species in the Annonaceae.* 1 Motivated by their promising potential as new antitumor drugs,1 we have now investigated the bioactive leaves of Rollinia mucosa (Jacq.) Baill. (Annonaceae). Some initial results have been reported elsewhere.2 Herein, we report the isolation, absolute structure determination, and bioactivity of mucocin (1, Figure 1). The previously known acetogenins are usually characterized (among other features) by their bearing one to three tetrahydrofuran (THF) rings.1 1 is the first annonaceous acetogenin to be reported that bears a hydroxylated tetrahydropyran (THP) ring along with a THF ring. This finding adds a new skeletal type to the family of annonaceous acetogenins.The isolation of 1 was guided by the brine shrimp lethality test (BST)3 45using repetitive open column and HPLC chromatography to separate the partitioned ethanol extract.2,4 The molecular formula of 1 was determined as CsvHeeOg by HRFABMS (MH+, m/z found 639.4828, caled 639.4836). A DEPT experiment revealed the presence of two methyl, 10 methine, and two quaternary carbons (some methylene carbon peaks overlapped at <5 29.5). The planar structure was established by means of COSY, EIMS, and EIMS of its TMS derivative (la, Figure 1). The cross peak corresponding to H-20/23 was successfully observed in the double-relayed COSY spectrum of 1. The existence of the methylated ,/9-unsaturated y-lactone with a 4-OH was confirmed by comparing the and 13C NMR data (Table 1) with those of known annonaceous acetogenins.1The relative stereochemistry of 1 was established as follows. Applying Bom's rule,5 the <3 value of H-16 indicated a three relationship at C-15/16. The configurational assignment of the THF ring as trans was suggested by the close match of the ,3C NMR data (from C-12 to C-16) with those of synthetic model 3 Purdue University. * Missouri Botanical Garden.(1) For recent reviews on annonaceous acetogenins, see: (a) Rupprecht,

Two new sesquiterpenes, 10 alpha-hydroxyamorphan-4-en-3-one (1) and 4 alpha-methylcadinane-4 alpha-methyl-1 alpha,2 alpha,10 alpha-triol (2), together with four known compounds, sesquiterpenes 10 alpha-hydroxycadinan-4-en-3-one (3) and alpha-cadinol (4), diterpene ferruginol and lignan helioxanthin, were isolated from the whole plant of Taiwania cryptomerioides under bioassay-guided fractionations. The structures of 1 and 2 were elucidated mainly by the NMR spectroscopic analyses. Bioactivities of the isolated compounds against brine shrimp, yellow fever mosquito larvae, and human tumor cells are reported; compound 4 was the most bioactive, showing selectivity for the human colon tumor cell line (HT-29).

The Annonaceous, acetogenins are a class of natural products that have excellent anticancer, antiinfective, and pesticidal properties. 1 While the pace of discovering unknown acetogenins is showing an upward trend, 1 most of the newly reported acetogenins, however, belong to several classical types usually having unsubstituted tetrahydrofuran (THF) rings. 1 Mucocin, the first tetrahydropyran (THP) ring-bearing acetogenin, was recently discovered in the bioactive leaf extracts of Rollinia mucosa (Jacq.) Baill. (Annonaceae) 2 and has, as well, a nonadjacent THF ring. We have now isolated another closely related acetogenin, muconin (1, Figure 1), that bears an unsubstituted THP ring along with an adjacent THF ring. 1 is the second THP-bearing acetogenin to be reported and further indicates the existence of a novel acetogenin type with THP ring(s) as the most salient structural feature. In addition, mucoxin (2, Figure 1) is also reported here as the first acetogenin that bears a hydroxylated THF ring. 3 Both 1 and 2 were isolated 4 from the previously described leaf extract 5 by activity-directed open column fractionation using the brine shrimp lethality test 1 (BST) and, at later stages, by 1 H NMR-monitored purification using repetitive normal, and reversed-phase HPLC. The molecular formulas of 1 and 2 4 apparently suggested that they were C 37 acetogenins bearing two THF rings and three hydroxyls. The retention time of 2 on normal-phase HPLC, however, was much shorter than those typically observed for this type of acetogenins. Although the 1 H NMR spectra of 1 and 2 (Table 1) 6 showed the diagnostic peaks of the 2,4-disubstituted R, -unsaturated γ-lactone terminal (1 bears a 4-OH), which is common to most acetogenins, 1 they also presented certain salient features. For 1, the unusual δ values (3.16 and 3.31) of two protons were reminiscent of the THP oxymethines in mucocin. 2 For 2, the presence of a well-defined pseudo triplet (1 H, δ 3.74, J ) 3 Hz) was never encountered in our previous experiences with acetogenins. The above peaks were not lost to D 2 O exchanges.The presence of a THP ring in 1 was indicated by an intensive NOESY cross peak at δ 3.16/3.31 (later assigned as H-13/17) and the lack of the same cross peak in its regular, single-relayed, and double-relayed COSY spectra. The coexistence of a THF ring in 1 was required by its degree of unsaturation and was suggested by the 1 H and 13 C NMR data (Table 1). It was then deduced from the COSY spectrum that the THP ring was flanked on one side by a hydroxyl (OH-12) and, on the other, by a THF ring that was itself hydroxyl flanked. The position of the OH-12 was not proposed on the THP ring because no long-range 1 H-1 H coupling was observed between H-12 and additional oxygenated moieties, and the neighboring methylene protons of OH-12 did not show a split in their δ values. 7 The above NMR-based structural hypothesis was ambiguous in the sequential placement of the THF/THP system, and the absolute coordinates of oxygenation were determined by the EIMS o...