A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure–activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.

Key words: Hedysarum theinum Krasnob., Fabaceae, 8-hydroxydiadzein, 6″-O-acetylononin, (-)-catechin, (-)-epicatechin, protocatechoic acid.In continuation of research on the chemical composition of Hedysarum theinum Krasnob.[1], which has valuable medicinal properties, we studied the chemical composition of the low-molecular-weight fractions of the alcohol extract of roots of this plant.Roots of H. theinum were extracted successively multiple times with EtOAc and ethanol. The chemical composition of the EtOAc extract has been reported by us [1]. Alcohol extracts have previously been separated into fractions of monomeric and oligomeric compounds by dissolving the dried extract in water and then extracting successively with organic solvents, e.g., diethylether, EtOAc, and BuOH [2]. However, in our instance the use of this method led to formation of emulsions and a precipitate during the extraction by diethylether or CHCl 3 . This complicated the separation process. In order to avoid this step, which caused losses, we separated the dried ethanol extract by extraction in a Soxhlet apparatus successively by CHCl 3 , EtOAc, acetone, and ethanol. HPLC analysis showed the presence of low-molecular-weight compounds in the CHCl 3 and EtOAc fractions of the alcohol extract and practically none in the acetone and ethanol fractions.Column chromatography of the CHCl 3 fraction over silica gel and preparative TLC on silica gel isolated medicarpin (1), raspberry ketone (2), vestitol (3), and rhododendrol (4).The EtOAc fraction was separated by chromatography over polyamide into water-soluble compounds (water eluent) and aglycons (MeOH eluent). Preparative separation of the aglycon fraction using column adsorption chromatography over silica gel with subsequent rechromatography over reversed-phase sorbent (Diasorb C16T) isolated vestitol (3), formononetin (5), protocatechoic acid (6), the isoflavonoids 8-hydroxydiadzein (7) and 6″-O-acetylononin (8), and (-)-catechin (9) and (-)-epicatechin (10). The structures of 1-10 were proved by spectral analysis including PMR, 13 C NMR, IR, and UV spectroscopy in addition to GC-MS. The resulting spectra were similar to those reported in the literature.

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Oligomeric proanthocyanidines from Hedysarum theinum roots that were cleaved by benzylmercaptan were shown to be heterogeneous mixtures consisting of prodelphinine and procyanidine structural units with the former dominating. Fractionation of oligomeric proanthocyanidines on polyamide and MCI gel CHP20P sorbent isolated fractions containing primarily mixtures of di-and trimeric, tri-and tetrameric, or tetra-and pentameric proanthocyanidines. Analysis by mass spectrometry (MALDI-TOF) showed that fractions of trimers and tetramers contained more proanthocyanidines with a single A-type bond than fractions of dimers and trimers.In continuation of research on the chemical composition of the rare and endangered plant Hedysarum theinum Krasnob.[1], which possesses valuable medicinal properties, we turned our attention to the fact that a significant part of the substances extracted from roots of this plant are oligomeric proanthocyanidines (oPA, condensed tannins) (Fig. 1). Thus, it was noted previously that the yield of extracted substances was about 15-20% after exhaustive methanol extraction of the roots [2]. Spectrophotometric determination of oPA in the methanol extract by reaction with vanillin/HCl gave an oPA content at the 90-95% level [2]. Despite this inflated value, the results indicate that oPA are one of the main extractable substances from roots of H. theinum.A literature search shows that rather homogeneous oPA could be separated preparatively into pure components of diand trimeric [3] and sometimes tetra-and pentameric proanthocyanidines [4]. Heterogeneous oPA typically have a mediumlength oPA chain and a ratio of structural units that can be established, for example, by depolymerization under acidic conditions in the presence of nucleophiles such as benzylmercaptan [5,6], thiophenol [4], cysteamine [7], or phloroglucinol [8]. The C-8-C-4 bond is cleaved during the reaction at a random position of the oPA. The nucleophile substitutes at the C-4 position of the resulting "upper" fragment. Only the terminal unit is obtained in an unsubstituted form upon full cleavage of the oPA. The middle units give 4-substituted derivatives. The ratio of substituted and unsubstituted products enables the average degree of oPA polymerization to be found. Furthermore, an advantage of this approach is the preservation of information about the stereochemistry of the C-2 and C-3 centers of the oPA units. The analytical method for oPA has been described in detail [9,10].

Isoflavonoids (-)-medicarpin, (-)-vestitol, and formononetin and butylphenols raspberry ketone and rhododendrol were isolated for the first time from the ethylacetate extract of Hedysarum thienum roots by column chromatography. GC-MS showed that the ethylacetate extract contained fatty acids, the principal ones being palmitic, linoleic, oleic, behenic, and lignocerinic.Hedysarum theinum is a perennial herbaceous plant with a thick reddish-brown root, because of which it is popularly called red root. This is a rare high-mountain subalpine species with a dispersed Central Asia-South Siberia distribution (Altai, Mongolia, Jugarskii Alatau). It is found most frequently in regions with a humid climate in the lower part of the high-mountain belt in subalpine and alpine meadows, rocky slopes, and forest glades in cedar-broadleaf woods [1]. In habitats with optimal ecological conditions that have not been greatly affected by civilization (collection, grazing), H. theinum is the dominant and subdominant species.The decoction or tincture of red root, which has a whole range of unique biologically active properties, has been used since long ago in Altai as a systemic, tonic, and anti-inflammatory agent. Modern folk medicine considers red root to possess immunostimulant properties and to cleanse actively the vascular system, which facilitates restoration of all body functions [2].The chemical composition of the root and aerial part of H. theinum has not previously been studied in detail. The yield of extracted substances has been reported for extraction of roots by various solvents [3]. The methanol extract reacts with vanillin and HCl to give a characteristic reaction for oligomeric proanthocyanidines [4].We studied previously the composition of successive extracts (hexane, ether, ethylacetate) of H. theinum roots using GC-MS and HPLC [5]. They contained fatty acids, triterpenes, and phenols. However, compounds of the different classes were not well separated.Herein we report results from the extraction of H. theinum roots by ethylacetate and the chemical composition of the extract. The yield of extract after six-fold extraction was 1.0%. The following results were obtained after separation of the extract by column chromatography over silica gel.The nonpolar part of the ethylacetate extract (fraction 1, see Experimental) was methylated by diazomethane. The products were subsequently analyzed by GC-MS. The principal components of fraction 1 were the methyl esters of palmitic (16:0), linoleic (18:2), oleic (18:1), behenic (22:0), and lignocerinic (24:0) acids (Table 1). According to GC-MS, the ethylacetate extract contained only one triterpene, stigmast-4-en-3-one, whereas the neutral part of the hexane extract contained at least 12 compounds in addition to fatty acids, 8 of which were identified as triterpenes [5].