Applications of inverse-detected 2D nmr methods are reviewed. Sequences presently available for direct (one-bond) heteronuclear correlation via / (HMQC and others) are discussed individually followed by examples of applications to natural products presented by class. Correlations of heteronuclear pairs other than 'H-13C and *H-15N are briefly considered. Optimization of parameters necessary in the acquisition of inverse-detected heteronuclear chemical shift correlation data are considered, followed by an illustration using the degradation product of 1-bromomaaliol. Methods for performing HMQC-COSY (RELAY) and HMQC-TOCSY experiments are considered with examples. Various ways of utilizing HMQC-TOCSY data are discussed followed by an illustration of the assembly of the protonated portion of the carbon skeleton of the degradation product of 1-bromomaaliol by varying the mixing times in a series of HMQC-TOCSY experiments. Long-range proton-detected heteronuclear chemical shift correlation experiments (HMBC) are presented with extensive examples of the application of these experiments to a diverse assortment of natural products, which are presented by class of compounds. A practical example of the application of the HMBC experiment to the degradation product of 1-bromomaaliol follows. The relatively new HMQC-NOESY experiment is presented followed by a discussion of the relatively few examples of this experiment presently contained in the literature. Finally, examples of proton-detected one-dimensional analogues of 2D nmr experiments are described; these include SELINCOR, selective one-dimensional HMQC-TOCSY, and SIMBA (selective inverse multiple bond analysis).

From the alkaloidal fractions of the West African plant Cryptolepis sanguinolenta (Asclepiadaceae), two alkaloids were purified: one was identified as the known indoloquinoline alkaloid cryptolepine [1], A second, novel alkaloid was shown to have an empirical formula of C34H24N4O based on exact mass measurement. Through the concerted application of a series of homonuclear and inverse-detected 2D nmr experiments, the structure of the second alkaloid was established as a spiro-nonacyclic alkaloid, cryptospirolepine. One portion of the structure of cryptospirolepine [2] may be biogenetically derived from cryptolepine [1],Cryptolepis sanguinolenta (Lindl.) Schlechter (Asclepiadaceae), a shrub indigenous to West Africa, has long been employed by Ghanaian traditional healers in the treatment of various fevers, including malaria (1). A root decoction has been used in the clinical therapy both of malaria and of urinary and upper respiratory tract infections by Oku Ampofo at the Centre for Scientific Research into Plant Medicine in Ghana since 1974 (1). The indoloquinoline alkaloid cryptolepine (5-methyl-5H-indolo-[3,2-¿}quinoline) [1] was first isolated from extracts of the roots of Cryptolepis triangularis N. E.Br., a species native to the Belgian Congo, by Clinquart in 1929 (2). Shortly thereafter, the alkaloid was again isolated from the same species by Delvaux (3). Quite paradoxically, cryptolepine had been synthesized some 20 years prior by Fichter and coworkers (4-6). Cryptolepine was isolated from a Nigerian sample of Cr. sanguinolenta in 1951 by Gellert et al. (7). Almost 30 years later, the alkaloid was isolated from a

Identification of degradants of pharmaceuticals is a necessary challenge of the drug development process following the subjection of candidate molecules to a variety of physico-chemical stresses. It would be desirable to be able to conduct such studies on a minimal amount of material. As a prototypical study, the isolation and identification of degradants of a sample of the complex indoloquinoline alkaloid, cryptospirolepine, was undertaken after prolonged storage in DMSO solution using a combination of cryogenic NMR probe technology and CASE (Computer-Assisted Structure Elucidation) programs. None of the starting alkaloid remained after storage; a chromatogram of the DMSO solution demonstrated the presence of >25 components in the mixture. The two most abundant degradation products were identified as the known alkaloid cryptolepinone (~35%) and an unprecedented rearrangement product, DP-2, (~16%).

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Numerous indoloquinoline alkaloid structures have been identified from extracts of the West African plant Cryptolepis sanguinolenta. Recently, through the use of 2D NMR methods and cryogenic NMR probe technology in conjunction with computer-assisted structure elucidation (CASE) methods, the structures of some chemical degradation products of this family of alkaloids have also been reported. We now report the characterization of a novel indoloquinoline dimeric alkaloid, quindolinocryptotackieine, through the extensive utilization of CASE methods. The NMR data presented here were collected over a decade earlier before the elucidation of the structure was possible, since manual analysis did not present a conclusive structure, whereas CASE produced a series of structures from which the structure could be verified. The original mass spectrometric (MS) data collected for the sample were problematic. Contemporary MS data were instead recollected from remaining small quantities of this alkaloid using modern instrumentation. The re-collected data gave a usable molecular ion and several key fragment ions that were diagnostically useful.

A variety of imidazo[4,5-c]pyridines (3-deazapurines) were synthesized. With use of these aglycons as pentosyl acceptors, the corresponding ribonucleosides and 2'-deoxyribonucleosides were prepared by an enzymatic method involving transfer of the pentosyl moiety from appropriate pyrimidine nucleosides. With most of the imidazo[4,5-c]pyridines, the products obtained from the enzyme-catalyzed reactions were pentosylated exclusively in the 1-position. However, some 3-pentosylation occurred with aglycons that had H or N3 in the 4-position. In addition to the 2'-deoxy congener of the ribonucleoside of 4-amino-1H-imidazo[4,5-c]pyridine, the 5'-deoxy and 2',5'-dideoxy congeners were synthesized. All of the aglycons and their nucleosides were tested for toxicity to mammalian cells in culture. None were markedly cytotoxic. These compounds were also evaluated for their ability to inhibit lymphocyte-mediated cytolysis in vitro. 3-Deazaadenosine (23) and its 2'-deoxy congener (38) were the most potent inhibitors (ED50 = 20 microM). In addition to these two in vitro tests, in vivo inhibition of the inflammatory response in the rat carregeenan pleurisy model was determined. 3-Deazaadenosine (23) was the most potent compound (ED50 = 3 mg/kg) in this in vivo test.

The relatively rare alkaloid cryptolepine, 5‐methyl‐5H‐indolo[3,2‐b]quinoline, was isolated from Cryptolepis sanguinolenta (Lindl.) Schlecter (Asclepiadaceae). Unequivocal proton and carbon nmr assignments in dimethyl sulfoxide are reported based on two‐dimensional nmr methods, including COSY, inverse‐detected direct (HMQC), and long‐range (HMBC) correlations. Several of the assignments were confirmed using one‐dimensional SIMBA spectra ‐ a new, selective, one‐dimensional analogue of the proton‐detected long‐range heteronuclear shift correlation experiment (HMBC). The SIMBA experiment was also used in an attempt to observe a two‐bond coupling from the H11 proton to C10a at several optimizations.

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