Characterization and Quantitation of a Sulfoconjugated Metabolite for Detection of Methyltestosterone Misuse and Direct Identification by LC-MS.

Methyltestosterone (MT) is one of the most frequently misused anabolic androgenic steroids detected in doping control analysis. The metabolism of MT in humans leads to several phase І metabolites and their corresponding phase Ⅱ conjugates. Previous studies have postulated the 3α-sulfoconjugate of 17α-methyl-5ß-androstane-3α,17ß-diol (S2) as principal sulfate metabolite of MT, with a detection window exceeding 10 days. However, a final direct and unambiguous confirmation of the structure of this metabolite is missing until now. In this study, we established an approach to detect and identify S2, using intact analysis by liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) without complex sample pretreatment. An in vitro study yielded the LC-MS/MS reference retention times of all 3-sulfated 17-methylandrostane-3,17-diol diastereomers, allowing for accurate structure assignment of potentially detected metabolites. In an in vivo excretion study with a single healthy male volunteer, the presence of the metabolite S2 was confirmed after a single oral dose of 10mg MT. The reference standard was chemically synthesized, characterized by accurate mass mass spectrometry (MS) and nuclear magnetic resonance (NMR), and quantified by quantitative qNMR. Thus, this study finally provides accurate structure information on the S2 metabolite and a direct analytical method for detection of MT misuse. The availability of the reference material is expected to be of benefit for further evaluation and subsequent analytical method validation in anti-doping research.

Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation.

The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4'-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase Ⅱ metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings.

Determination of Total Sennosides and Sennosides A, B, and A1 in Senna Leaflets, Pods, and Tablets by Two-Dimensional qNMR.

In the present work, a two-dimensional qNMR method for the determination of sennosides was established. Using band-selective HSQC and the cross correlations of the characteristic 10-10' bonds, we quantified the total amount of the value-determining dianthranoids in five minutes, thus, rendering the method not only fast, but also specific and stability indicating. The validation of the method revealed excellent accuracy (recovery rates of 98.5 to 103%), precision (RSD values of 3.1%), and repeatability (2.2%) and demonstrated the potential of 2D qNMR in the quality control of medicinal plants. In a second method, the use of 2D qNMR for the single analysis of sennosides A, B, and A1 was evaluated with acceptable measurement times (31 min), accuracy (93.8%), and repeatability (5.4% and 5.6%) for the two major purgatives sennoside A and B. However, the precision for sennoside B and A1 was not satisfactory, mainly due to the low resolution of the HSQC signals of the two compounds.

Antimicrobial Prenylated Isoflavones from the Leaves of the Amazonian Medicinal Plant Vatairea guianensis Aubl.

Vatairea guianenis Aubl. (Fabaceae) is an Amazonian medicinal plant species traditionally used for treating skin diseases. In an initial screening, a V. guianensis leaf extract and its subextracts showed antibacterial and antifungal activities. The EtOAc subextract was selected for chemical workup and afforded five known (1-4 and 8) and six undescribed isoflavones, vatairenones C-H (5-7 and 9-11). All isoflavones are prenylated in position C-8, displaying either chain-prenylated (1-7) or ring-closed forms (8-11). The most bioactive compound (3) exhibited in vitro activity against clinically relevant bacteria and fungi with IC50 values ranging from 6.8 to 26.9 µM. Due to its broad antimicrobial activity and low general toxicity, compound 3 is a potential lead compound for structural modifications. The results of the present study support the ethnomedicinal use of V. guianensis in the treatment of dermatological disorders. 1H NMR spectra of some of the isolated compounds showed intricate signal patterns, which might explain repeated errors in assigning the correct structure of the isoflavonoid B-ring in the literature and which we resolved by higher order spectra simulations.

Insights into polyamine metabolism: homospermidine is double-oxidized in two discrete steps by a single copper-containing amine oxidase in pyrrolizidine alkaloid biosynthesis.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

Cytotoxic constituents and a new hydroxycinnamic acid derivative from Leontodon saxatilis (Asteraceae, Cichorieae).

In our ongoing research for the discovery of new constituents with antimyeloma activity, we investigated 15 compounds present in the aerial parts of Leontodon saxatilis for their cytotoxic potential against NCI-H929, U266, and OPM2 cell lines. One of the isolated compounds displayed a new natural product and was identified as 5-feruloyl-2α-hydroxyquinic acid after LC-MS and NMR experiments. Of the remaining compounds, cichoric acid and three flavone glycosides, apigenin 4'-O-ß-d-glucoside, luteolin 7-O-ß-d-glucoside and luteolin 4'-O-ß-d-glucoside, showed moderate cytotoxic activity, whereas the effects of two aglyones apigenin and luteolin were more pronounced. Though the cytotoxic potential of the two aglycones (against other cell lines) was reported in various studies, our work moreover showed that cooccurrence of these two compounds with similar components of lower activity led to comparable results and at the same time minimized the damage of healthy fibroblast cells. Thus, our work could be a starting point for additional studies on the synergistic effect of similar components against myeloma cell lines.

Prediction of the sweetening effect of Siraitia grosvenorii (luo han guo) fruits by two-dimensional quantitative NMR.

During the last decade, dried fruits of Siraitia grosvenorii (luo han guo), also known as monk fruit, have become popular food ingredients. Luo han guo extracts, which are promoted as non-caloric natural sweeteners, are now incorporated into dietary supplements, soft drinks, and energy shakes. The compounds responsible for the sweetening effect are glycosylated cucurbitane-type triterpenoids, the so-called mogrosides. However, of the more than 40 known mogroside compounds, only 11-α-hydroxy-mogrosides exhibit a sweetening effect, whereas the other triterpenoids are non- or bitter-tasting. We have used two-dimensional quantitative NMR to determine selectively the content of 11-α-hydroxy-mogrosides in these dried fruits and thus to predict their sweetening potential. Homonuclear (H,H COSY) and heteronuclear (HSQC) methods were developed, validated, and compared. Both techniques were found suitable for the quality control of luo han guo fruits and extracts, the COSY experiment being advantageous with regard to accuracy, precision, and limit of quantification.

Biological Activities of Two Major Copaiba Diterpenoids and Their Semi-synthetic Derivatives.

The oleoresin of Copaifera reticulata Ducke, Fabaceae, is a traditional Brazilian remedy used for a wide range of applications. Commonly named copaiba, the oleoresin has been found to exhibit strong antimicrobial effects in our previous study, which could be attributed to some of its diterpenoid constituents. In order to find new biological activities and to eventually enhance the before observed effects, (-)-polyalthic acid (1) and kaurenoic acid (2), together with eight prepared semi-synthetic derivatives (1a-1c and 2a-2e) were evaluated for their cytotoxic, antibacterial and antifungal properties. Regarding the gram-positive bacteria Enterococcus faecium and methicillin-resistant Staphylococcus aureus, we found that both the exocylic methylene group and the carboxyl group were crucial for the activity against these two clinically relevant bacterial strains. Investigation of the antifungal activity, in contrast, showed that the carboxyl group is unnecessary for the effect against the dermatophytes Trichophyton rubrum and Cryptococcus neoformans, indicated by low micromolar IC50 values for both (-)-polyalthic acid diethylamide (1a) as well as (-)-polyalthic acid methyl ester (1b). Apart from studying the biological activity, the structure of one semi-synthetic derivative, compound 1c, is being reported for the first time. During the course of the structure elucidation of the new compound, we discovered inconsistencies regarding the stereochemistry of polyalthic acid and its stereoisomers, which we clarified in the present work. Graphical Abstract.

Two-dimensional qNMR of anthraquinones in Frangula alnus (Rhamnus frangula) using surrogate standards and delay time adaption.

In the absence of adequate reference material, a problem often encountered in natural product chemistry, we investigated the use of surrogate standards in two-dimensional qNMR for the quantification of anthraquinones in the bark of alder buckthorn (Frangula alnus). Using the integrals of cross signals in the HSQC spectrum obtained from commercial standards rutin and duroquinone and adapting the delays for the 1JCH coupling, we quantified the total amount of anthraquinones and anthraquinone glucosides, as well as the content of the value-determining glucofrangulins and frangulins. Thereby, duroquinone was used as an external standard to establish the calibration curve for the methylated anthraquinone scaffold, whereas calibration curves for the glycosides were obtained using the anomeric proton signals of the rutinose disaccharide. The method was validated for accuracy, precision, specificity, linearity and limit of quantitation and shows clear advantages over the method of the European Pharmacopeia, especially in terms of specificity and meaningfulness of the results. Apart from being a useful alternative in the quality control of alder buckthorn, the presented approach demonstrates, moreover, the versatility of sophisticated 2D measurements in quantitative NMR.

Quality control of Aloe vera (Aloe barbadensis) and Aloe ferox using band-selective quantitative heteronuclear single quantum correlation spectroscopy (bs-qHSQC).

In the present study, band-selective quantitative heteronuclear single quantum correlation spectroscopy (bs-qHSQC) was applied for the quality control of the two Aloe species present in the European Pharmacopeia. After development and validation of a complete spectral range (csr-) qHSQC assay, a specific pulse program with selective excitation was applied and the measuring time was reduced from 135 to 32 min, while maintaining the same resolution. This bs-qHSQC method (method I) showed slightly higher RSD values compared to the csr-qHSQC method (maximum RSD of 2.80%), but better recovery rates in comparison to the assay of the Pharmacopeia (97.3% for Aloe vera and 96.6% for Aloe ferox). Apart from quantifying the total anthranoid content, the method moreover allows the quantitation of aloin among other aloin derivatives, such as 7-hydroxyaloin, as well as the differentiation of the two investigated species. Additionally, a second bs-qHSQC method (method II) for the fast (4 min) determination of the aloin A/B ratio was developed and compared to 13C qNMR measurements. Showing the same results in much less analysis time, the latter approach contributes to a general problem in natural product chemistry, the co-occurrence of structurally similar compounds and their analysis in complex matrices, e.g. plant extracts.

Lignans and sesquiterpene lactones from Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae).

Four undescribed lignans and two undescribed sesquiterpenic acids, together with three known compounds (hypochoeroside C, hypochoeroside D, and 5-O-caffeoylshikimic acid) were isolated from the roots of Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae). The lignans were identified as 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranosyl-2'-O-methacrylate, (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one, and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid. The two sesquiterpenic acids were identified as the ring open precursors of hypochoerosides C and D. Structures were elucidated using NMR and HRMS. Absolute configurations of (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid were determined using electronic circular dichroism (ECD) spectroscopy. 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside was evaluated for its anti-proliferative activity against myeloma cell lines MM1S, U266, and NCI-H929 and showed cytotoxicity at 100 mM against MM1S strain. No neurotoxicity was observed for major compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, and hypochoeroside D in a fluorescence assay measuring neurite outgrowth in dorsal root ganglion (DRG) neurons. Additionally, compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, hypochoeroside D, and hypochoerosidic acid D were quantified in unstressed and drought-stressed plants using HPLC-DAD. Drought-stressed plants were found to contain lower concentrations of the lignan 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside and sesquiterpene lactone hypochoeroside C.

Axitinib: A Photoswitchable Approved Tyrosine Kinase Inhibitor.

The goal of photopharmacology is to develop photoswitchable enzyme modulators as tunable (pro-)drugs that can be spatially and temporally controlled by light. In this context, the tyrosine kinase inhibitor axitinib, which contains a photosensitive stilbene-like moiety that allows for E/Z isomerization, is of interest. Axitinib is an approved drug that targets the vascular endothelial growth factor receptor 2 (VEGFR2) and is licensed for second-line therapy of renal cell carcinoma. The photoinduced E/Z isomerization of axitinib has been investigated to explore if its inhibitory effect can be turned "on" and "off", as triggered by light. Under controlled light conditions, (Z)-axitinib is 43 times less active than that of the E isomer in an VEGFR2 assay. Furthermore, it was proven that kinase activity in human umbilical vein cells (HUVECs) was decreased by (E)-axitinib, but only weakly affected by (Z)-axitinib. By irradiating (Z)-axitinib in vitro with UV light (λ=385 nm), it is possible to switch it almost quantitatively into the E isomer and to completely restore the biological activity of (E)-axitinib. However, switching the biological activity off from (E)- to (Z)-axitinib was not possible in aqueous solution due to a competing irreversible [2+2]-photocycloaddition, which yielded a biologically inactive axitinib dimer.

Quantification of diterpene acids in Copaiba oleoresin by UHPLC-ELSD and heteronuclear two-dimensional qNMR.

In this study, we present the quantitation of eight diterpene acids in the oleoresin of Copaifera reticulata Ducke by UHPLC-ELSD and quantitative HSQC (heteronuclear single quantum correlation spectroscopy). UHPLC was performed using reversed phase material and external calibration and showed RSD values of ≤ 3% (repeatability) and ≤ 4% (precision), and mean recovery rates of 91.2-104.8%. LOQs were determined with 10 and 20 µg/mL, and LODs with 4 and 8 µg/mL, respectively. For the qHSQC method, calibration curves of eight different NMR cross-peaks (furylic, endo- and exocyclic methine signals, exocyclic methylene and methyl signals) were established and normalized with dimethyl terephthalate, which served as internal standard. This approach allowed the direct quantification of four major and one minor diterpene, whereas simple calculation procedures led to the contents of the remaining minor compounds. Comparison with the results of the UHPLC assay showed good agreement for seven of the eight diterpene acids. In terms of precision, the qHSQC method was advantageous for the quantification of the three main compounds, whereas UHPLC-ELSD was superior in the determination of the minor components. In contrast to previous reports, kolavenic acid was identified as a major diterpene acid in the oleoresin of Copaifera reticulata, with amounts of 4.0 ± 0.3%.

N-7-Guanine Adduct of the Active Monoepoxide of Prodrug Treosulfan: First Synthesis, Characterization, and Decomposition Profile Under Physiological Conditions.

(2S,3S)-1,2:3,4-diepoxybutane (DEB) cross-links DNA guanines by forming the intermediate epoxy-adduct ((2'S,3'S)-N-7-(3',4'-epoxy-2'-hydroxybut-1'-yl)guanine [EHBG]). This process is presently considered a primary mechanism for the action of treosulfan (TREO), the prodrug that transforms to DEB via the monoepoxide intermediate (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate (EBDM). In this article, the N-7-guanine adduct of EBDM ((2'S,3'S)-N-7-(2'3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)guanine [HMSBG]) was synthesized for the first time, and its stability was investigated at physiological in vitro conditions. To synthesize HMSBG, EBDM, formed in-situ from TREO, was treated with guanosine in glacial acetic acid at 60°C followed by ribose cleavage in 1 M HCl at 80°C. HMSBG was stable during the synthesis, which showed that a ß-hydroxy group protects the sulfonate moiety against hydrolysis in acid environment. At pH 7.2 and 37°C, HMSBG exclusively underwent first-order epoxidation to EHBG with a half-life of 5.0 h. EHBG further decomposed to trihydroxybutyl-guanine, chlorodihydroxybutyl-guanine (major products), phosphodihydroxy-guanine, and a structural isomer (minor products). The isomeric derivative was identified as guanine with a fused 7-membered ring, which provided a new insight into the EHBG stability. To conclude, the exclusive conversion of HMSBG to EHBG indicates that EBDM might contribute to DNA cross-linking independently from DEB and play a more important role in the TREO action than expected before.

Detoxification of Trimethylamine N-Oxide by the Mitochondrial Amidoxime Reducing Component mARC.

Although known for years, the toxic effects of trimethylamine N-oxide (TMAO), a physiological metabolite, were just recently discovered and are currently under investigation. It is known that elevated TMAO plasma levels correlate with an elevated risk for cardiovascular disease (CVD). Even though there is a general consensus about the existence of a causal relationship between TMAO and CVD, the underlying mechanisms are not fully understood. TMAO is an oxidation product of the hepatic flavin-containing monooxygenases (FMO), mainly of isoform 3, and it is conceivable that humans also have an enzyme reversing this toxification by reducing TMAO to its precursor trimethylamine (TMA). All prokaryotic enzymes that use TMAO as a substrate have molybdenum-containing cofactors in common. Such molybdenum-containing enzymes also exist in mammals, with the so-called mitochondrial amidoxime reducing component (mARC) representing the most recently discovered mammalian molybdenum enzyme. The enzyme has been found to exist in two isoforms, mARC1 and mARC2, both being capable of reducing a variety of N-oxygenated compounds, including nonphysiological N-oxides. To investigate whether the two isoforms of this enzyme are able to reduce and detoxify TMAO, we developed a suitable analytical method and tested TMAO reduction with a recombinant enzyme system. We found that one of the two recombinant human mARC proteins, namely, hmARC1, reduces TMAO to TMA. The N-reductive activity is relatively low and identified via the kinetic parameters with Km = (30.4 ± 9.8) mM and Vmax = (100.5 ± 12.2) nmol/(mg protein·min). Nevertheless, the ubiquitous tissue expression of hmARC1 allows a continuous reduction of TMAO whereas the counter-reaction, the production of TMAO through FMO3, can take place only in the liver where FMO3 is expressed. TMAO reduction in porcine liver subfractions showed the characteristic enrichment of N-reductive activity in the outer mitochondrial membrane. TMAO reduction was also found in human cell cultures. These findings indicate the role of hmARC1 in the metabolomic pathway of TMAO, which might contribute to the prevention of CVD. This also hints at a physiological function of the molybdenum enzyme, which remains mainly unknown to date.

Quantification of the total amount of black cohosh cycloartanoids by integration of one specific 1H NMR signal.

Quantitative analysis is an important field in the quality control of medicinal plants, aiming to determine the amount of pharmacologically active constituents in complex matrices. Often biological effects of herbal drugs are not restricted to single compounds, but are rather caused by a number of often biogenetically related plant metabolites. Depending on the complexity of the analyzed plant extract, conflicts between accuracy, such as total content assays using photometric or colorimetric methods, and comprehensiveness, e.g. quantification of one or a few lead compounds can occur. In this study, we present a qHNMR approach determining the total amount of cycloartanoids in black cohosh (Actaea racemosa) rhizomes. Perdeuterated methanol containing 1,2,4,5-tetrachloro-3-nitrobenzene as an internal standard was used for extraction. Amounts of cycloartanoids were then measured by integrating 1H NMR signals of all cycloartenoids' H-19 exo protons. Due to their unusually low chemical shifts, these signals are well separated from all remaining signals in crude extracts. Thus, accurate (recovery rates of 99.5-102.5%) and precise (relative standard deviations below 2.5%) quantification of cycloartanoids was accomplished. To the best of our knowledge, this is the first example of a quantification of the total amount of a pharmacologically relevant compound class by integration of one 1H NMR signal characteristic for all members of this particular compound class. Additionally, we propose a new term and unit for the evaluation of medicinal plants and herbal medicinal products: the "specific partial amount of substance" of pharmacologically active constituents, indicated in mmol/g.

A Dual Topoisomerase Inhibitor of Intense Pro-Apoptotic and Antileukemic Nature for Cancer Treatment.

Classic cytotoxic drugs remain indispensable instruments in antitumor therapy due to their effectiveness and a more prevalent insensitivity toward tumor resistance mechanisms. Herein we describe the favorable properties of 6-(N,N-dimethyl-2-aminoethoxy)-11-(3,4,5-trimethoxyphenyl)pyrido[3,4-c][1,9]phenanthroline (P8-D6), a powerful inducer of apoptosis caused by an equipotent inhibition of human topoisomerase I and II activities. A broad-spectrum effect against human tumor cell lines at nanomolar concentrations, as well as strong antileukemic effects, were shown to be superior to those of marketed topoisomerase-targeting drugs and dual topoisomerase inhibitors in clinical trials. The facile four-step synthesis, advantageous drugability properties, and initial in vivo data encourage the application of P8-D6 in appropriate animal tumor models and further drug development.

11-Substituted Benzo[c]phenanthridines: New Structures and Insight into Their Mode of Antiproliferative Action.

The synthesis of various new structures of a library of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines (BP) and 11-substituted 6-aminobenzo[c]phenanthridines (BP-D) is presented. These structures, further synthetic modifications, and the preparation of follow-up products which delivered about 40 new derivatives are described. Their potential as antiproliferative drug candidates was investigated by comparison of NCI 60 developmental therapeutics program (DTP) human tumor cell line screening data based on the results of in vitro tumor cell growth inhibition, including about 40 hitherto unpublished compound test results with up to 60 cancer cell lines. NCI-COMPARE studies helped to suggest the modes of action of the highly active antiproliferative drugs. These findings are supported by in vitro biological investigations showing either inhibition of tubulin polymerization and depolymerization or topoisomerase inhibition. Together with physicochemical parameters of the drug candidates, structure-activity relationships are critically discussed. Tubulin interaction or inhibition of topoisomerase I and IIα/ß activity are two rationales that can explain the antiproliferative activity observed in the NCI 60 DTP human tumor cell line screen. However, it can also be reasonably assumed that these compounds address several targets, thus prohibiting the identification of simple structure-activity relationships. The new structures described herein are thought to act as so-called multitarget drugs, thus being of special interest in the area of multidrug resistance.