Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Ancistrocladinium A Induces Apoptosis in Proteasome Inhibitor-Resistant Multiple Myeloma Cells: A Promising Therapeutic Agent Candidate.

The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.

Atroposelective Total Synthesis of the Fourfold ortho-Substituted Naphthyltetrahydroisoquinoline Biaryl O,N-Dimethylhamatine.

A stereoselective total synthesis of O,N-dimethylhamatine, an analogue of an axially chiral naphthylisoquinoline natural biaryl product from tropical Ancistrocladus lianas, is reported. The route features a late-stage atropo-diastereoselective biaryl bond formation. Generation of this especially challenging, sterically hindered tetra-ortho-substituted array was achieved by using Nolan's (IPr*NHC)PdCinCl pre-catalyst under mild Negishi coupling conditions. Discussion is offered regarding the selectivity obtained experimentally and predicted from DFT calculations on the key biaryl coupling step that leads to the desired M-diastereomer.

Discovery of Pyrrolo[3,2- d]pyrimidin-4-one Derivatives as a New Class of Potent and Cell-Active Inhibitors of P300/CBP-Associated Factor Bromodomain.

Herein, we report the discovery of a series of new P300/CBP-associated factor (PCAF) bromodomain (BRD) inhibitors, which were obtained through a hit discovery process and subsequent structure-based optimization and structure-activity relationship analyses toward a retrieved hit compound (12). Among these inhibitors, ( R, R)-36n is the most potent one with an IC50 of 7 nM in homogeneous time-resolved fluorescence assay and a KD of 78 nM in isothermal titration calorimetry assay. This compound also exhibited activity against GCN5 and FALZ, but weak or no activity against other 29 BRD proteins and 422 kinases, indicating considerable selectivity. X-ray cocrystal structure analysis revealed the molecular interaction mode and the precise stereochemistry required for bioactivity. Cellular activity, preliminary RNA-seq analysis, and pharmacokinetic properties were also examined for this compound. Collectively, this study provides a versatile tool molecule to explore molecular mechanisms of PCAF BRD regulation and also offers a new lead compound for drug discovery targeting PCAF.

Effects of the novel polyphenol conjugate DPP-23 on head and neck squamous cell carcinoma cells in vitro.

Despite partial advances in therapy for patients suffering from head and neck squamous cell carcinomas (HNSCC), prognosis still remains poor with minimal improvement in survival for over the last several decades. Some agents found are known to cause cancer cell death in vitro by promoting cellular reactive oxygen species (ROS) accumulation. This is particularly of interest as some cancer cells are more sensitive to ROS than normal cells. It could be shown that the novel polyphenol conjugate (E)-3-(3',5'-Dimethoxyphenyl)-1-(2'-methoxyphenyl)prop-2-en-1-one (DPP-23) offers antitumor effects by the selective generation of ROS without an indication of toxicity in normal tissues in vitro and in vivo. In order to further evaluate the role of DPP-23 as a potential agent in head and neck oncology, the present study investigated its cytotoxic effects on well-established HNSCC cell lines such as HLaC 78 and FaDu, as well as primary human bone marrow stem cells (hBMSCs) and human peripheral blood lymphocytes in vitro. As DPP-23 is not commercially available, it was synthesized via a 'cold' procedure of the Claisen-Schmidt condensation. Following cell treatment with DPP-23 for 24 h, viability and apoptosis were measured via a MTT assay and the Annexin V-propidium iodide test. The results suggest a dose-dependent cytotoxicity in the tested HNSCC tumor cell lines, as well as in hBMSC and lymphocytes. In contrast to previous findings, these preliminary results indicate that the cytotoxic effects of DPP-23 in benign cells may be notably greater than previously suspected. This may indicate a limitation for in the feasibility, or at least of the systemic application, of DPP-23 for patients with HNSCC.

Ancistrocyclinones A and B, unprecedented pentacyclic N,C-coupled naphthylisoquinoline alkaloids, from the Chinese liana Ancistrocladus tectorius.

Two unique pentacyclic N,C-coupled naphthylisoquinolines, the ancistrocyclinones A (5) and B (6), were discovered in the Chinese liana Ancistrocladus tectorius. Furthermore, six known, likewise N,C-coupled alkaloids, viz., ancistrocladinium A (7a) and its mono- and bisphenolic analogs 8a and 9a were isolated, along with their atropo-diastereomers 7b, 8b, and 9b. The stereostructures of 5 and 6 were determined by HRESIMS, 1D and 2D NMR, oxidative degradation, and ECD calculations. The pentacyclic ancistrocyclinones A (5) and B (6) are structurally similar to berberine alkaloids - yet arising from a most different biosynthetic pathway: they are apparently formed by N,C-coupling of their polyketide-derived molecular halves, followed by oxidative cyclo-condensation. Biomimetic conversion of the co-occurring 4'-O-demethylancistrocladinium A (8a) to ancistrocyclinone A (5) via a quinoid intermediate supported the postulated pathway. Ancistrocyclinone A (5) was found to significantly inhibit the viability of drug-sensitive human leukemia (CCRF-CEM) and multidrug-resistant tumor cells (CEM/ADR5000) with comparable efficacies.

Jozilebomines A and B, Naphthylisoquinoline Dimers from the Congolese Liana Ancistrocladus ileboensis, with Antiausterity Activities against the PANC-1 Human Pancreatic Cancer Cell Line.

Two new naphthylisoquinoline dimers, jozilebomines A (1a) and B (1b), were isolated from the roots of the Congolese plant Ancistrocladus ileboensis, along with the known dimer jozimine A2 (2). These compounds are Dioncophyllaceae-type metabolites, i.e., lacking oxygen functions at C-6 and with an R-configuration at C-3 in their tetrahydroisoquinoline moieties. The dimers 1a and 1b consist of two 7,1'-coupled naphthylisoquinoline monomers linked through an unprecedented 3',6″-coupling in the binaphthalene core and not, as in 2, via the C-3-positions of the two naphthalene units. Thus, different from the C2-symmetric jozimine A2 (2), the new jozilebomines are constitutionally unsymmetric. The central biaryl axis of each of the three dimers is rotationally hindered, so that 1a, 1b, and 2 possess three consecutive chiral axes. The two jozilebomines have identical constitutions and the same absolute configurations at all four stereogenic centers, but differ from each other in their axial chirality. Their structural elucidation was achieved by HRESIMS, 1D and 2D NMR, oxidative degradation, and experimental and calculated ECD data. They exhibited distinct and specific antiplasmodial activities. All dimers showed potent cytotoxicity against HeLa human cervical cancer cells and preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions. Furthermore, these dimers significantly inhibited the colony formation of PANC-1 cells, even when exposed to noncytotoxic concentration for a short time. Jozilebomines A (1a) and B (1b) and jozimine A2 (2) represent novel potential candidates for future drug development against pancreatic cancer.

Antileukemic ancistrobenomine B and related 5,1'-coupled naphthylisoquinoline alkaloids from the Chinese liana Ancistrocladus tectorius.

A striking feature of the metabolite pattern of the Southeast Asian liana Ancistrocladus tectorius (Ancistrocladaceae) is the predominance of 5,1'-coupled naphthylisoquinoline alkaloids. About 20 alkaloids of this coupling type have so far been discovered in this plant species. Here, we report on the isolation of four new 5,1'-linked naphthylisoquinolines from the twigs and stems of A. tectorius. Two of them, the ancistrobenomines B (5) and C (6), belong to the very rare group of alkaloids with a fully dehydrogenated isoquinoline portion. Likewise unusual for naphthylisoquinoline alkaloids is the presence of a hydroxymethylene group at C-3. Within the large class of meanwhile ca. 180 such natural products, this structural peculiarity had so far been known only from two other representatives isolated from the Malaysian species A. benomensis, and from one single naphthalene-devoid 3-hydroxymethyleneisoquinoline from A. tectorius. Seven further 5,1'-linked alkaloids, previously isolated from related Asian and African Ancistrocladus species, have now been identified for the first time in A. tectorius. Their structural elucidation was achieved by spectroscopic analysis including HRESIMS, 1D and 2D NMR, and by chemical (oxidative degradation) and chiroptical (electronic circular dichroism) methods. Ancistrobenomine B (5) exhibited moderate effects against Plasmodium falciparum and Trypanosoma brucei rhodesiense in vitro, and it was found to display strong cytotoxic activities against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000.

Dioncophyllines C2, D2, and F and Related Naphthylisoquinoline Alkaloids from the Congolese Liana Ancistrocladus ileboensis with Potent Activities against Plasmodium falciparum and against Multiple Myeloma and Leukemia Cell Lines.

Dioncophylline F (1), the first 5,8'-coupled dioncophyllaceous alkaloid (i.e., lacking an oxygen function at C-6 and possessing an R-configuration at C-3), was isolated from the recently described Congolese liana Ancistrocladus ileboensis. Two further, likewise Dioncophyllaceae-type, alkaloids, the dioncophyllines C2 (2) and D2 (3), were identified, along with the Ancistrocladaceae-type compound ancistrocladisine B (4), which is oxygenated at C-6 and S-configured at C-3. The structures of the new compounds were determined by spectroscopic, chemical, and chiroptical methods. The stereostructure of 1 was further confirmed by total synthesis. As a consequence of the lack of a methyl group ortho to their biaryl axes, both dioncophylline F (1) and the 7,8'-coupled dioncophylline D2 (3) occur as pairs of configurationally semistable and, thus, slowly interconverting atropo-diastereomers, whereas dioncophylline C2 (2), with its 5,1'-linkage, is configurationally stable at the axis. Eight further known naphthylisoquinolines were isolated from A. ileboensis, among them dioncophylline A (P-10), its 4'-O-demethyl analogue P-11, and 5'-O-methyldioncophylline D (7), which were found to display strong cytotoxic activities against multiple myeloma INA-6 cells (P-10 even stronger than the standard drug melphalan) and against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000. Moreover, the dioncophyllines 1, 3, and 7 showed high-and specific-activities against the malaria parasite Plasmodium falciparum.

Directed Synthesis of All Four Pure Stereoisomers of the N,C-Coupled Naphthylisoquinoline Alkaloid Ancistrocladinium A.

The first preparation of the N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A and its likewise naturally occurring minor atropisomer, in an atropisomerically pure form, is described. The synthesis succeeded by resolution of the already rotationally hindered, and thus atropo-diastereomeric acetamide precursors, which were then, without major loss of stereochemical information, cyclized to the respective target molecules. The strategy was applied to the first synthesis of the regioisomeric product ancistrocladinium D, likewise in a stereochemically pure form.

Ancistectorine D, a naphthylisoquinoline alkaloid with antiprotozoal and antileukemic activities, and further 5,8'- and 7,1'-linked metabolites from the Chinese liana Ancistrocladus tectorius.

From the twigs and stems of the Chinese liana Ancistrocladus tectorius (Ancistrocladaceae), two new 5,8'-coupled naphthylisoquinolines, ancistectorine D (5) and its 6-O-demethyl derivative 6, were isolated, along with two new 7,1'-linked alkaloids, 6-O-methylancistectorine B1 (7) and ancistectorine B2 (8). Two further compounds, ancistroealaine A (4) and 6-O-demethyl-8-O-methyl-7-epi-ancistrobrevine D (10), already known from related Asian and African Ancistrocladus species, were discovered for the first time in A. tectorius. The structure elucidation was achieved by spectroscopic analysis including HRESIMS, 1D and 2D NMR, and by chemical (oxidative degradation) and chiroptical (circular dichroism) methods. Chemotaxonomically remarkable, 5,8'-coupled naphthylisoquinolines have as yet been found quite rarely in Asian Ancistrocladus species, where only two examples have so far been detected, while alkaloids with this coupling type represent the by far the largest group of constituents in African taxa. Ancistectorine D (5) shows promising activities against the protozoan parasites Trypanosoma cruzi and Leishmania donovani, and it was likewise found to display strong cytotoxic activities against human leukemia (CCRF-CEM) and multidrug-resistant tumor cells (CEM/ADR5000).

Axially Chiral Dimeric Naphthalene and Naphthoquinone Metabolites, from Root Cultures of the West African Liana Triphyophyllum peltatum.

Root cultures of the West African liana Triphyophyllum peltatum were initiated from stem explants of in vitro cultivated shoots. From these organ cultures, three new binaphthalenes, one binaphthoquinone, and two (bi)naphthalene glucosides were isolated, with substitution patterns related to those of the naphthylisoquinoline alkaloids, which are the "normal" main metabolites of T. peltatum. The structures of the diglucoside dioncoquinoside A (1) and of the axially chiral biaryls triphyoquinols A1 (3), A2 (4), and B (5), triphyoquinoside A (6), and triphyoquinone A (7) were elucidated by spectroscopic analysis (HRESIMS, 1D and 2D NMR) and by application of electronic circular dichroism (ECD) spectroscopy in combination with the exciton chirality method and quantum-chemical ECD calculations. The root cultures likewise produced the known alkaloids dioncophylline A (8), 5'-O-demethyldioncophylline A (9), dioncopeltine A (10), habropetaline A (11), and 5'-O-methyldioncophylline D (12a/b), the naphthalene glucoside plumbaside A (2), and the naphthoquinones plumbagin (13), droserone (14), and 8-hydroxydroserone (15).

First Atroposelective Total Synthesis of Enantiomerically Pure Ancistrocladidine and Ancistrotectorine.

The first regio- and stereoselective total synthesis of the axially chiral 7,3'-coupled naphthylisoquinoline alkaloids ancistrocladidine (1) and ancistrotectorine (2) has been described. Both possess a 7,3'-coupled axis, which before now, was difficult to attain synthetically. Moreover, target 2 has a sensitive relative cis-array of the two methyl groups at C1 and C3 in the tetrahydroisoquinoline part. The key step in the chosen strategy was the construction of the biaryl axis in accordance with the "lactone method": the two molecular halves, which were activated in an "inverse-halogenated" form, were prefixed by an ester bridge, followed by intramolecular coupling, and atroposelective cleavage of the lactone auxiliary bridge delivered the desired biaryl scaffold.

Ancistrotectoquinones A and B, the First Quinoid Naphthylisoquinoline Alkaloids, from the Chinese Liana Ancistrocladus tectorius.

From the leaves and stems of Ancistrocladus tectorius (Ancistrocladaceae) from the Chinese island Hainan, two novel-type 7,3'-coupled naphthylisoquinolines, named ancistrotectoquinones A (4) and B (5), were isolated. They are the first alkaloids with a 1,4-naphthoquinone portion coupled to an isoquinoline moiety. Due to the lowered degree of steric hindrance next to the biaryl axis and for electronic reasons, 4 and 5 occur as pairs of configurationally semi-stable, and, thus slowly interconverting atropo-diastereomers. The Gibbs free energies of activation between the two atropisomers of ancistrotectoquinone A (4a/b) were determined by measuring the time-dependent decrease of diastereomeric purity of freshly separated samples enriched with the M- or P-atropisomer and, computationally, by DFT calculations. The absolute configurations at the biaryl axes of the atropo-diastereomers of 4a/b and 5a/b were assigned by online LC-CD analysis. The stereostructure of 4a/b was further confirmed by its semi-synthesis from the likewise 7,3'-coupled 'normal' naphthylisoquinoline alkaloid ancistrotectorine (6), by biomimetic oxidation with Fremy's salt.