Marina N. Semenova

Novel derivatives of 1,3,4-oxadiazoles are potent mitostatic agents featuring strong microtubule depolymerizing activity in the sea urchin embryo and cell culture assays.

A series of novel 1,3,4-oxadiazole derivatives based on structural and electronic overlap with combretastatins have been designed and synthesized. Initially, we tested all new compounds in vivo using the phenotypic sea urchin embryo assay to yield a number of agents with anti-proliferative, anti-mitotic, and microtubule destabilizing activities. The experimental data led to identification of 1,3,4-oxadiazole derivatives with isothiazole (5-8) and phenyl (9-12) pharmacophores featuring activity profiles comparable to that of combretastatins, podophyllotoxin and nocodazole. Cytotoxic effects of the two lead molecules, namely 6 and 12, were further confirmed and evaluated by conventional assays with the A549 human cancer cell line including cell proliferation, cell cycle arrest at the G2/M phase, cellular microtubule distribution, and finally in vitro microtubule assembly with purified tubulin. The modeling results using 3D similarity (ROCS) and docking (FRED) correlated well with the observed activity of the molecules. Docking data suggested that the most potent molecules are likely to target the colchicine binding site.

Sea urchin embryo as a model organism for the rapid functional screening of tubulin modulators

Identification of antimitotic molecules that affect tubulin dynamics is a multistep procedure. It includes in vitro tubulin polymerization assay, studies of a cell cycle effect, and general cytotoxicity assessment. To simplify this lengthy screening protocol, we have introduced and validated an assay system based on the sea urchin embryos. The proposed two-step procedure involves the fertilized egg test for mitotic arrest and the behavioral assessment of a free-swimming blastula. In order to validate the assay, we have analyzed the effect of a panel of known antiproliferative agents on the sea urchin embryo. For all tubulin destabilizing drugs, we observed rapid spinning and lack of forward movement of an embryo. Both effects are likely to result from the in vivo microtubule disassembly caused by test molecules. Notably, the described assay yields rapid information on antiproliferative, antimitotic, cytotoxic, and tubulin destabilizing activities of the molecules along with their solubility and permeability potential. Moreover, measured potencies of the test articles correlated well with the reported values in both in vitro and cell based assays.

Polyalkoxybenzenes from Plants. 5. Parsley Seed Extract in Synthesis of Azapodophyllotoxins Featuring Strong Tubulin Destabilizing Activity in the Sea Urchin Embryo and Cell Culture Assays

A series of 4-azapodophyllotoxin derivatives with modified rings B and E have been synthesized using allylpolyalkoxybenzenes from parsley seed oil. The targeted molecules were evaluated in vivo in a phenotypic sea urchin embryo assay for antimitotic and tubulin destabilizing activity. The most active compounds identified by the in vivo sea urchin embryo assay featured myristicin-derived ring E. These molecules were determined to be more potent than podophyllotoxin. Cytotoxic effects of selected molecules were further confirmed and evaluated by conventional assays with A549 and Jurkat human leukemic T-cell lines including cell growth inhibition, cell cycle arrest, cellular microtubule disruption, and induction of apoptosis. The ring B modification yielded 6-OMe substituted molecule as the most active compound. Finally, in Jurkat cells, compound induced caspase-dependent apoptosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway.

Polyalkoxy Substituted 4H‑Chromenes: Synthesis by Domino Reaction and Anticancer Activity

A series of 4H-chromenes containing various modifications in the ring B and polyalkoxy substituents in the ring E has been synthesized by Knoevenagel-Michael-hetero-Thorpe-Ziegler three-component domino reaction with the overall yield of 45-82%. The targeted molecules were evaluated in a phenotypic sea urchin embryo assay for antimitotic and microtubule destabilizing activity. The most active compounds 5{1,5} and 5{5,5} featured sesamol-derived ring B and m-methoxyphenyl or m-methoxymethylenedioxyphenyl ring E. Compounds 5{3,1}, 5{1,2}, 5{5,4}, 5{1,5}, and 5{5,5} exhibited strong cytotoxicity in the NCI60 human tumor cell line anticancer drug screen. Surprisingly, cell growth inhibition caused by these agents was more pronounced in the multidrug resistant NCI/ADR-RES cells than the parent OVCAR-8 cell line. The results suggest that polyalkoxy substited 4H-chromenes may prove to be advantageous for further design as anticancer agents.

Triphenylphosphonium Cations of the Diterpenoid Isosteviol: Synthesis and Antimitotic Activity in a Sea Urchin Embryo Model.

A series of novel triphenylphosphonium (TPP) cations of the diterpenoid isosteviol (1, 16-oxo-ent-beyeran-19-oic acid) have been synthesized and evaluated in an in vivo phenotypic sea urchin embryo assay for antimitotic activity. The TPP moiety was applied as a carrier to provide selective accumulation of a connected compound into mitochondria. When applied to fertilized eggs, the targeted isosteviol TPP conjugates induced mitotic arrest with the formation of aberrant multipolar mitotic spindles, whereas both isosteviol and the methyltriphenylphosphonium cation were inactive. The structure-activity relationship study revealed the essential role of the TPP group for the realization of the isosteviol effect, while the chemical structure and the length of the linker only slightly influenced the antimitotic potency. The results obtained using the sea urchin embryo model suggested that TPP conjugates of isosteviol induced mitotic spindle defects and mitotic arrest presumably by affecting mitochondrial DNA. Since targeting mitochondria is considered as an encouraging strategy for cancer therapy, TPP-isosteviol conjugates may represent promising candidates for further design as anticancer agents.

cis-Restricted 3-Aminopyrazole Analogues of Combretastatins: Synthesis from Plant Polyalkoxybenzenes and Biological Evaluation in the Cytotoxicity and Phenotypic Sea Urchin Embryo Assays

We have synthesized a series of novel cis-restricted 4,5-polyalkoxydiaryl-3-aminopyrazole analogues of combretastatins via short synthetic sequences using building blocks isolated from dill and parsley seed extracts. The resulting compounds were tested in vivo in the phenotypic sea urchin embryo assay to reveal their antimitotic and antitubulin effects. The most potent aminopyrazole, 14a, altered embryonic cell division at 10 nM concentration, exhibiting microtubule-destabilizing properties. Compounds 12a and 14a displayed pronounced cytotoxicity in the NCI60 anticancer drug screen, with the ability to inhibit growth of multi-drug-resistant cancer cells.

Research Article: In vivo Evaluation of Indolyl Glyoxamides in the Phenotypic Sea Urchin Embryo Assay

We have devised a ‘one‐pot’ phenotypic in vivo assay for the rapid evaluation of potential tubulin inhibitors using the sea urchin embryo model. An effect of a small molecule on two specific developmental stages of sea urchin embryo, namely: (i) fertilized egg test for antimitotic activity and (ii) behavioral monitoring of a free‐swimming blastulae for changes in the embryo swimming pattern could be quantified by a threshold concentration resulting in respective abnormalities. Derivatives of the clinical candidate D‐24851 featured good correlation between activity in tubulin polymerization assay and our in vivo data. Importantly, we demonstrated that in these series, the N‐substitution of indole is non‐essential to attain profound in vitro and cellular effects.

Application of plant allylpolyalkoxybenzenes in synthesis of antimitotic phenstatin analogues

Phenstatin and its derivatives with the modified ring A have been synthesized, using plant allylpolyalkoxybenzenes as a starting material. The targeted molecules were evaluated in a phenotypic sea urchin embryo assay for antiproliferative activity. It was found that phenstatin ring A modifications yielded antimitotic compounds. The most effective myristicin derivative 7d (combretastatin A-2 analogue) was determined to be ca. 10 times more potent than phenstatin, displaying antimitotic tubulin-destabilizing activity at the same concentration range as combretastatins. In contrast to combretastatins, 7d featured the steric stability with potential for further design as anticancer agent.

Comparative in vivo evaluation of polyalkoxy substituted 4H-chromenes and oxa-podophyllotoxins as microtubule destabilizing agents in the phenotypic sea urchin embryo assay.

A series of polyalkoxy substituted 7-hydroxy- and 7-methoxy-4-aryl-4H-chromenes were evaluated using the sea urchin embryo model to yield several compounds exhibiting potent antimitotic microtubule destabilizing activity. Data obtained by the assay were further confirmed in the NCI60 human cancer cell screen. The replacement of methylenedioxy ring A and lactone ring D in podophyllotoxin analogues by 7-methoxy, 2-NH2, and 3-CN groups in 4-aryl-4H-chromenes resulted in potent antimitotic microtubule destabilizing agents. Feasible synthesis and high yields render 7-methoxy-4H-chromenes to be a promising series for further anticancer drug development.

A Synthetic Derivative of Plant Allylpolyalkoxybenzenes Induces Selective Loss of Motile Cilia in Sea Urchin Embryos

Polyalkoxybenzenes are plant components displaying a wide range of biological activities. In these studies, we synthesized apiol and dillapiol isoxazoline analogues of combretastatins and evaluated their effect on sea urchin embryos. We have shown that p-methoxyphenyl isoxazoline caused sea urchin embryo immobilization due to the selective excision of motile cilia, whereas long immotile sensory cilia of apical tuft remained intact. This effect was completely reversed by washing the embryos. The compound did not alter cell division, blastulae hatching, and larval morphogenesis. In our hands, the molecule would serve as a convenient tool for in vivo studying morphogenetic processes in the sea urchin embryo. We anticipate that both the assay and the described derivative could be used for studies in ciliary function in embryogenesis.