C-Terminal Decarboxylation of Proline-Derived Building Blocks for Protein-Binding Peptides.

Using a set of conformationally restricted Proline-derived Modules (ProMs), our group has recently succeeded in developing inhibitors for the enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain, which is a key mediator of cell migration and plays an important role in tumor metastasis. While these (formally) pentapeptidic compounds show nanomolecular binding affinities towards EVH1, their drug-like properties and cell permea-bility need to be further optimized before they can be clinically tested as therapeutic agents against metastasis. In this study, we sought to improve these properties by removing the C-terminal carboxylic acid function of our peptoids, either by late-stage decarboxylation or by direct synthesis. For late-stage decarboxy-lation of ProM-like systems, a method for reductive halo decarboxylation was optimized and applied to several proline-derived substrates. In this way, a series of new decarboxy ProMs suitable as building blocks for decarboxy EVH1 inhibitors were obtained. In addition, we incorporated decarboxy-ProM-1 into the penta-peptide-like compound Ac[2-Cl-F][ProM-2][Decarb-ProM-1], which showed similar affinity towards EVH1 as the methyl ester derivative (Ac[2-Cl-F][ProM-2][ProM1]OMe). However, despite better calculated drug-like properties, this compound did not inhibit chemotaxis in a cellular assay.

A pH-Sensitive Double Chromophore Fluorescent Dye for Live-Tracking of Lipophagy.

Lipid droplet (LD) degradation provides metabolic energy and important building blocks for various cellular processes. The two major LD degradation pathways include autophagy (lipophagy), which involves delivery of LDs to autolysosomes, and lipolysis, which is mediated by lipases. While abnormalities in LD degradation are associated with various pathological disorders, our understanding of lipophagy is still rudimentary. In this study, we describe the development of a lipophilic dye containing two fluorophores, one of which is pH-sensitive and the other pH-stable. We further demonstrate that this "Lipo-Fluddy" can be used to visualize and quantify lipophagy in living cells, in an easily applicable and protein label-free approach. After estimating the ability of compound candidates to penetrate LDs, we synthesized several BODIPY and (pH-switchable) rhodol dyes, whose fluorescence properties (incl. their photophysical compatibility) were analyzed. Of three Lipo-Fluddy dyes synthesized, one exhibited the desired properties and allowed observation of lipophagy by fluorescence microscopy. Also, this dye proved to be non-toxic and suitable for the examination of various cell lines. Moreover, a method was developed to quantify the lipophagy process using flow cytometry, which could be applied in the future in the identification of lipophagy-related genes or in the screening of potential drugs against lipophagy-related diseases.

A first-in-class ß-glucuronidase responsive conjugate for selective dual targeted and photodynamic therapy of bladder cancer.

In this report, we present a novel prodrug strategy that can significantly improve the efficiency and selectivity of combined therapy for bladder cancer. Our approach involved the synthesis of a conjugate based on a chlorin-e6 photosensitizer and a derivative of the tyrosine kinase inhibitor cabozantinib, linked by a ß-glucuronidase-responsive linker. Upon activation by ß-glucuronidase, which is overproduced in various tumors and localized in lysosomes, this conjugate released both therapeutic modules within targeted cells. This activation was accompanied by the recovery of its fluorescence and the generation of reactive oxygen species. Investigation of photodynamic and dark toxicity in vitro revealed that the novel conjugate had an excellent safety profile and was able to inhibit tumor cells proliferation at submicromolar concentrations. Additionally, combined therapy effects were also observed in 3D models of tumor growth, demonstrating synergistic suppression through the activation of both photodynamic and targeted therapy.

Design of an aryne-platform for the synthesis of non-racemic heterocyclic allocolchicinoids.

A four-step semisynthetic approach towards a highly versatile allocolchicine-related chiral aryne intermediate starting from naturally occurring colchicine was developed, and some of its synthetic transformations were studied. The in situ generated benzyne intermediate afforded a number of non-racemic heterocyclic allocolchicinoids, which were shown to exhibit potent cytotoxicity towards COLO 357, OSA and Raji cells. The proposed methodology is attractive for the synthesis of libraries of new cytotoxic tubulin inhibitors.

Divergent total synthesis of the revised structures of marine anti-cancer meroterpenoids (+)-dysiherbols A-E.

We report here a concise and divergent enantioselective total synthesis of the revised structures of marine anti-cancer sesquiterpene hydroquinone meroterpenoids (+)-dysiherbols A-E (6-10) using dimethyl predysiherbol 14 as a key common intermediate. Two different improved syntheses of dimethyl predysiherbol 14 were elaborated, one starting from Wieland-Miescher ketone derivative 21, which is regio- and diastereoselectively α-benzylated prior to establishing the 6/6/5/6-fused tetracyclic core structure through intramolecular Heck reaction. The second approach exploits an enantioselective 1,4-addition and a Au-catalyzed double cyclization to build-up the core ring system. (+)-Dysiherbol A (6) was prepared from dimethyl predysiherbol 14via direct cyclization, while (+)-dysiherbol E (10) was synthesized through allylic oxidation and subsequent cyclization of 14. Epoxidation of 14 afforded allylic alcohol 45 or unexpectedly rearranged homoallylic alcohol 44. By inverting the configuration of the hydroxy groups, exploiting a reversible 1,2-methyl shift and selectively trapping one of the intermediate carbenium ions through oxy-cyclization, we succeeded to complete the total synthesis of (+)-dysiherbols B-D (7-9). The total synthesis of (+)-dysiherbols A-E (6-10) was accomplished in a divergent manner starting from dimethyl predysiherbol 14, which led to the revision of their originally proposed structures.

Isotope-labeled ergothioneine clarifies the mechanism of reaction with singlet oxygen.

Recently we have uncovered a non-enzymatic multi-step cycle for the regeneration of ergothioneine (ET), after reaction with noxious singlet oxygen (1O2), by glutathione (GSH). When living cells were loaded with ET labeled with deuterium and N-15 atoms (D5-ET) and exposed to light in the presence of a photosensitizer, no loss of deuterium at position 5 of the imidazole ring was observed, in contradiction to our previous mechanistic proposal. Therefore, it was necessary to reexamine the in vitro products of ET and 1O2 by liquid chromatography coupled to high resolution mass spectrometry. Pure 1O2 was generated by thermolysis at 37 °C of the endoperoxide DHPNO2. The use of D5-ET enabled us to revise and extend the reaction scheme. On the main pathway, 1O2 attacks the imidazole ring, and the hydroperoxide intermediates are reduced rapidly by ET or GSH via different mechanisms. The intramolecular water elimination from the 5-hydroperoxide described previously is slower and not a part of the cycle. On another side path, 1O2 attacks the sulfur of ET to form a sulfine (S-oxide). The reduction of the sulfine also allows for the complete regeneration of ET. Experiments with methanol instead of water as solvent revealed that, in the absence of GSH, ET was attacked 6 times more frequently at the ring than at the sulfur. In the presence of 1 mM GSH or higher, both side paths were abandoned. ET efficiently captures 1O2 with its ring and can then be regenerated to a large extent by GSH, without enzyme involvement.

An organometallic analogue of combretastatin A-4 and its apoptosis-inducing effects on lymphoma, leukemia and other tumor cells in vitro.

Hexacarbonyl[1,3-dimethoxy-5-((4'-methoxyphenyl)ethynyl)benzene]dicobalt (NAHO27), an organometallic analogue of combretastatin A-4, has been synthesized and its activity against lymphoma, leukemia, breast cancer and melanoma cells has been investigated. It was shown that NAHO27 specifically induces apoptosis in BJAB lymphoma and Nalm-6 leukemia cells at low micromolar concentration and does not affect normal leukocytes in vitro. It also proved to be active against vincristine and daunorubicin resistant leukemia cell lines with p-glycoprotein-caused multidrug resistance and showed a pronounced (550%) synergistic effect when co-applied with vincristine at very low concentrations. Mechanistic investigations revealed NAHO27 to induce apoptosis via the mitochondrial (intrinsic) pathway as reflected by the processing of caspases 3 and 9, the involvement of Bcl-2 and smac/DIABLO, and the reduction of mitochondrial membrane potential. Gene expression analysis and protein expression analysis via western blot showed an upregulation of the proapoptotic protein harakiri by 9%.

Design and synthesis of a tetracyclic tripeptide mimetic frozen in a polyproline type II (PP2) helix conformation.

A synthesis of the new tetracyclic scaffold ProM-19, which represents a XPP tripeptide unit frozen in a PPII helix conformation, was developed. As a key building block, N-Boc-protected ethyl (1S,3S,4R)-2-azabicyclo[2.2.1]hept-5-ene-2-carboxylate was prepared through a diastereoselective aza-Diels-Alder reaction and subsequent hydrogenolytic removal of the chiral N-1-phenylethyl substituent under temporary protection of the double bond through dihydroxylation and reconstitution by Corey-Winter olefination. The target compound Boc-[ProM-19]-OMe was then prepared via subsequent peptide coupling and Ru-catalyzed ring-closing metathesis steps employing (S)-N-Boc-allylgylcine and cis-5-vinyl-proline methyl ester as additional building blocks. In addition, Ac-[2-Cl-Phe]-[Pro]-[ProM-19]-OMe was prepared by solution phase peptide synthesis as a potential ligand for the ena-VASP EVH1 domain.

Phenanthroindolizidine Alkaloids Isolated from Tylophora ovata as Potent Inhibitors of Inflammation, Spheroid Growth, and Invasion of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), representing the most aggressive form of breast cancer with currently no targeted therapy available, is characterized by an inflammatory and hypoxic tumor microenvironment. To date, a broad spectrum of anti-tumor activities has been reported for phenanthroindolizidine alkaloids (PAs), however, their mode of action in TNBC remains elusive. Thus, we investigated six naturally occurring PAs extracted from the plant Tylophora ovata: O-methyltylophorinidine (1) and its five derivatives tylophorinidine (2), tylophoridicine E (3), 2-demethoxytylophorine (4), tylophoridicine D (5), and anhydrodehydrotylophorinidine (6). In comparison to natural (1) and for more-in depth studies, we also utilized a sample of synthetic O-methyltylophorinidine (1s). Our results indicate a remarkably effective blockade of nuclear factor kappa B (NFκB) within 2 h for compounds (1) and (1s) (IC50 = 17.1 ± 2.0 nM and 3.3 ± 0.2 nM) that is different from its effect on cell viability within 24 h (IC50 = 13.6 ± 0.4 nM and 4.2 ± 1 nM). Furthermore, NFκB inhibition data for the additional five analogues indicate a structure-activity relationship (SAR). Mechanistically, NFκB is significantly blocked through the stabilization of its inhibitor protein kappa B alpha (IκBα) under normoxic as well as hypoxic conditions. To better mimic the TNBC microenvironment in vitro, we established a 3D co-culture by combining the human TNBC cell line MDA-MB-231 with primary murine cancer-associated fibroblasts (CAF) and type I collagen. Compound (1) demonstrates superiority against the therapeutic gold standard paclitaxel by diminishing spheroid growth by 40% at 100 nM. The anti-proliferative effect of (1s) is distinct from paclitaxel in that it arrests the cell cycle at the G0/G1 state, thereby mediating a time-dependent delay in cell cycle progression. Furthermore, (1s) inhibited invasion of TNBC monoculture spheroids into a matrigel®-based environment at 10 nM. In conclusion, PAs serve as promising agents with presumably multiple target sites to combat inflammatory and hypoxia-driven cancer, such as TNBC, with a different mode of action than the currently applied chemotherapeutic drugs.

Synthetic α-Helical Peptides as Potential Inhibitors of the ACE2 SARS-CoV-2 Interaction.

During viral cell entry, the spike protein of SARS-CoV-2 binds to the α1-helix motif of human angiotensin-converting enzyme 2 (ACE2). Thus, alpha-helical peptides mimicking this motif may serve as inhibitors of viral cell entry. For this purpose, we employed the rigidified diproline-derived module ProM-5 to induce α-helicity in short peptide sequences inspired by the ACE2 α1-helix. Starting with Ac-QAKTFLDKFNHEAEDLFYQ-NH2 as a relevant section of α1, a series of peptides, N-capped with either Ac-ßHAsp-[ProM-5] or Ac-ßHAsp-PP, were prepared and their α-helicities were investigated. While ProM-5 clearly showed a pronounced effect, an even increased degree of helicity (up to 63 %) was observed in sequences in which non-binding amino acids were replaced by alanine. The binding affinities of the peptides towards the spike protein, as determined by means of microscale thermophoresis (MST), revealed only a subtle influence of the α-helical content and, noteworthy, led to the identification of an Ac-ßHAsp-PP-capped peptide displaying a very strong binding affinity (KD =62 nM).

Synthesis and Biological Evaluation of Water-Soluble Esterase-Activated CO-Releasing Molecules Targeting Mitochondria.

Due to the beneficial effects of carbon monoxide as a cell-protective and anti-inflammatory agent, CO-releasing molecules (CORMs) offer some promising potential applications in medicine. In this context, we synthesized a set of acyloxy-cyclohexadiene-Fe(CO)3 complexes, all displaying a N-methyl-pyridinium triflate moiety in the ester side chain, as mitochondria-targeting esterase-triggered CORM prodrugs. Whereas the compounds in which the acyloxy substituent is attached to the 2-position of the diene-Fe(CO)3 unit (A series) spontaneously release CO upon dissolution in phosphate buffer, which remarkably is partly suppressed in the presence of porcine liver esterase (PLE), the 1-substituted isomers (B series) show the expected PLE-induced release of CO (up to 3 equiv.). The biological activity of Mito-CORMs 2/3-B and their isophorone-derived analogs 2/3-A', which also displayed PLE-induced CO release, was assessed by using human umbilical vein endothelial cells (HUVEC). Whereas Mito-CORMs 2/3-B were not cytotoxic up to 500 µM (MTT assay), Mito-CORMs 2/3-A' caused significant toxicity at concentrations above 50 µM. The anti-inflammatory potential of both Mito-CORM variants was demonstrated by concentration-dependent down-regulation of the pro-inflammatory markers VCAM-1, ICAM-1 and CXCL1 as well as induction of HO-1 in TNFα-stimulated human umbilical vein endothelial cells (HUVECs; western blotting and qPCR). Energy phenotyping by seahorse real-time cell metabolic analysis, revealed opposing shifts of metabolic potentials in cells treated either with Mito-CORMs 2/3-B (increased mitochondrial respiration and glycolytic activity) or Mito-CORMs 2/3-A' (suppressed mitochondrial respiration and increased glycolytic activity). Thus, the Mito-CORMs represent valuable tools for the safe and targeted delivery of CO to mitochondria as a subcellular compartment to induce positive anti-inflammatory effects with only minor shifts in cellular energy metabolism. Also, due to their water solubility, these compounds provide a promising starting point for further pharmacological studies.

B-nor-methylene Colchicinoid PT-100 Selectively Induces Apoptosis in Multidrug-Resistant Human Cancer Cells via an Intrinsic Pathway in a Caspase-Independent Manner.

Colchicine, the main active alkaloid from Colchicum autumnale L., is a potent tubulin binder and represents an interesting lead structure for the development of potential anticancer chemotherapeutics. We report on the synthesis and investigation of potentially reactive colchicinoids and their surprising biological activities. In particular, the previously undescribed colchicinoid PT-100, a B-ring contracted 6-exo-methylene colchicinoid, exhibits extraordinarily high antiproliferative and apoptosis-inducing effects on various types of cancer cell lines like acute lymphoblastic leukemia (Nalm6), acute myeloid leukemia (HL-60), Burkitt-like lymphoma (BJAB), human melanoma (MelHO), and human breast adenocarcinoma (MCF7) cells at low nanomolar concentrations. Apoptosis induction proved to be especially high in multidrug-resistant Nalm6-derived cancer cell lines, while healthy human leukocytes and hepatocytes were not affected by the concentration range studied. Furthermore, caspase-independent initiation of apoptosis via an intrinsic pathway was observed. PT-100 also shows strong synergistic effects in combination with vincristine on BJAB and Nalm6 cells. Cocrystallization of PT-100 with tubulin dimers revealed its (noncovalent) binding to the colchicine-binding site of ß-tubulin at the interface to the α-subunit. A pronounced effect of PT-100 on the cytoskeleton morphology was shown by fluorescence microscopy. While the reactivity of PT-100 as a weak Michael acceptor toward thiols was chemically proven, it remains unclear whether this contributes to the remarkable biological properties of this unusual colchicinoid.

Head-to-Head Comparison of Selected Extra- and Intracellular CO-Releasing Molecules on Their CO-Releasing and Anti-Inflammatory Properties.

Over the past decade, a variety of carbon monoxide releasing molecules (CORMs) have been developed and tested. Some CORMs spontaneously release CO once in solution, while others require a trigger mechanism to release the bound CO from its molecular complex. The modulation of biological systems by CORMs depends largely on the spatiotemporal release of CO, which likely differs among the different types of CORMs. In spontaneously releasing CORMs, CO is released extracellularly and crosses the cell membrane to interact with intracellular targets. Other CORMs can directly release CO intracellularly, which may be a more efficient method to modulate biological systems. In the present study, we compared the efficacy of extracellular and intracellular CO-releasing CORMs that either release CO spontaneously or require an enzymatic trigger. The efficacy of such CORMs to modulate HO-1 and VCAM-1 expression in TNF-α-stimulated human umbilical vein endothelial cells (HUVEC) was evaluated.

ET-CORM Mediated Vasorelaxation of Small Mesenteric Arteries: Involvement of Kv7 Potassium Channels.

Although the vasoactive properties of carbon monoxide (CO) have been extensively studied, the mechanism by which CO mediates vasodilation is not completely understood. Through-out published studies on CO mediated vasodilation there is inconsistency on the type of K+-channels that are activated by CO releasing molecules (CORMs). Since the vasorelaxation properties of enzyme triggered CORMs (ET-CORMs) have not been studied thus far, we first assessed if ET-CORMs can mediate vasodilation of small mesenteric arteries and subsequently addressed the role of soluble guanylate cyclase (sGC) and that of K-channels herein. To this end, 3 different types of ET-CORMs that either contain acetate (rac-1 and rac-4) or pivalate (rac-8) as ester functionality, were tested ex vivo on methoxamine pre-contracted small rat mesenteric arteries in a myograph setting. Pre-contracted mesenteric arteries strongly dilated upon treatment with both types of acetate containing ET-CORMs (rac-1 and rac-4), while treatment with the pivalate containing ET-CORM (rac-8) resulted in no vasodilation. Pre-treatment of mesenteric arteries with the sGC inhibitor ODQ abolished rac-4 mediated vasodilation, similar as for the known sGC activator SNP. Likewise, rac-4 mediated vasodilation did not occur in KCL pretreated mesenteric arteries. Although mesenteric arteries abundantly expressed a variety of K+-channels only Kv7 channels were found to be of functional relevance for rac-4 mediated vasodilation. In conclusion the current results identified Kv7 channels as the main channel by which rac-4 mediates vasodilation. In keeping with the central role of Kv7 in the control of vascular tone and peripheral resistance these promising ex-vivo data warrant further in vivo studies, particularly in models of primary hypertension or cardiac diseases, to assess the potential use of ET-CORMs in these diseases.

Total Synthesis of (+)-Erogorgiaene and the Pseudopterosin A-F Aglycone via Enantioselective Cobalt-Catalyzed Hydrovinylation.

Due to their pronounced bioactivity and limited availability from natural resources, metabolites of the soft coral Pseudopterogorgia elisabethae, such as erogorgiaene and the pseudopterosines, represent important target molecules for chemical synthesis. We have now developed a particularly short and efficient route towards these marine diterpenes exploiting an operationally convenient enantioselective cobalt-catalyzed hydrovinylation as the chirogenic step. Other noteworthy C-C bond forming transformations include diastereoselective Lewis acid-mediated cyclizations, a Suzuki coupling and a carbonyl ene reaction. Starting from 4-methyl-styrene the anti-tubercular agent (+)-erogorgiaene (>98 % ee) was prepared in only 7 steps with 46 % overall yield. In addition, the synthesis of the pseudopterosin A aglycone was achieved in 12 steps with 30 % overall yield and, surprisingly, was found to exhibit a similar anti-inflammatory activity (inhibition of LPS-induced NF-κB activation) as a natural mixture of pseudopterosins A-D or iso-pseudopterosin A, prepared by ß-D-xylosylation of the synthetic aglycone.

Enantioselective Total Synthesis and Structural Revision of Dysiherbol†A.

A 12-step total synthesis of the natural product dysiherbol A, a strongly anti-inflammatory and anti-tumor avarane meroterpene isolated from the marine sponge Dysidea sp., was elaborated. As key steps, the synthesis features an enantioselective Cu-catalyzed 1,4-addition/enolate-trapping opening move, an Au-catalyzed double cyclization to build up the tetracyclic core-carbon skeleton, and a late installation of the C5-bridgehead methyl group via proton-induced cyclopropane opening associated with spontaneous cyclic ether formation. The obtained pentacyclic compound (corresponding to an anhydride of the originally suggested structure for dysiherbol A) showed identical spectroscopic data as the natural product, but an opposite molecular rotation. CD-spectroscopic measurements finally confirmed that both the constitution and the absolute configuration of the originally proposed structure of (+)-dysiherbol A need to be revised.

ItaCORMs: conjugation with a CO-releasing unit greatly enhances the anti-inflammatory activity of itaconates.

Endogenous itaconate as well as the gasotransmitter CO have recently been described as powerful anti-inflammatory and immunomodulating agents. However, each of the two agents comes along with a major drawback: Whereas itaconates only exert beneficial effects at high concentrations above 100 µM, the uncontrolled application of CO has strong toxic effects. To solve these problems, we designed hybrid prodrugs, i.e. itaconates that are conjugated with an esterase-triggered CO-releasing acyloxycyclohexadiene-Fe(CO)3 unit (ItaCORMs). Here, we describe the synthesis of different ItaCORMs and demonstrate their anti-inflammatory potency in cellular assays of primary murine immune cells in the low µmolar range (<10 µM). Thus, ItaCORMs represent a promising new class of hybrid compounds with high clinical potential as anti-inflammatory agents.

Enantioselective Cleavage of Cyclobutanols Through Ir-Catalyzed C-C Bond Activation: Mechanistic and Synthetic Aspects.

The Ir-catalyzed conversion of prochiral tert-cyclobutanols to ß-methyl-substituted ketones proceeds under comparably mild conditions in toluene (45-110 °C) and is particularly suited for the enantioselective desymmetrization of ß-oxy-substituted substrates to give products with a quaternary chirality center with up to 95 % ee using DTBM-SegPhos as a chiral ligand. Deuteration experiments and kinetic isotope effect measurements revealed major mechanistic differences to related RhI -catalyzed transformations. Supported by DFT calculations we propose the initial formation of an IrIII hydride intermediate, which then undergoes a ß-C elimination (C-C bond activation) prior to reductive C-H elimination. The computational model also allows the prediction of the stereochemical outcome. The Ir-catalyzed cyclobutanol cleavage is broadly applicable but fails for substrates bearing strongly coordinating groups. The method is of particular value for the stereo-controlled synthesis of substituted chromanes related to the tocopherols and other natural products.

Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells.

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.

Colchicine Alkaloids and Synthetic Analogues: Current Progress and Perspectives.

Colchicine, the main alkaloid of Colchicum autumnale, is one of the most famous natural molecules. Although colchicine belongs to the oldest drugs (in use since 1500 BC), its pharmacological potential as a lead structure is not yet fully exploited. This review is devoted to the synthesis and structure-activity relationships (SAR) of colchicine alkaloids and their analogues with modified A, B, and C rings, as well as hybrid compounds derived from colchicinoids including prodrugs, conjugates, and delivery systems. The systematization of a vast amount of information presented to date will create a paradigm for future studies of colchicinoids for neoplastic and various other diseases.