Toward "E-Ring-Free" Lamellarin Analogues: Synthesis and Preliminary Biological Evaluation.

Since the discovery of anticancer properties of a naturally occurring hexacyclic marine alkaloid Lamellarin D, the attempts have been made to prepare its synthetic analogues and elucidate the effects of each structural component on their activity profile. While F-ring-free, A-ring-free and B-ring-open lamellarins are known, E-ring-free analogues have never been investigated. In this work, we developed a facile and straightforward synthetic method toward E-ring-free lamellarin analogues based on the [3+2]-cycloaddition. For the first time, we prepared several pentacyclic lamellarin analogues without E-ring in their structure and assessed their cytotoxicity in a panel of cancer cell lines in comparison with several hexacyclic lamellarins. E-ring-free lamellarins were devoid of cytotoxicity due to their poor solubility in cellular environment.

Allylpolyalkoxybenzene Inhibitors of Galactonolactone Oxidase from Trypanosoma cruzi.

Inhibition of biosynthetic pathways of compounds essential for Trypanosoma cruzi is considered as one of the possible action mechanisms of drugs against Chagas disease. Here, we investigated the inhibition of galactonolactone oxidase from T. cruzi (TcGAL), which catalyzes the final step in the synthesis of vitamin C, an antioxidant that T. cruzi is unable to assimilate from outside and must synthesize itself, and identified allylbenzenes from plant sources as a new class of TcGAL inhibitors. Natural APABs (apiol, dillapiol, etc.) inhibited TcGAL with IC50 = 20-130 µM. The non-competitive mechanism of TcGAL inhibition by apiol was established. Conjugation of APABs with triphenylphosphonium, which ensures selective delivery of biologically active substances to the mitochondria, increased the efficiency and/or the maximum percentage of TcGAL inhibition compared to nonmodified APABs.

Application of Pluronics for Enhancing Aqueous Solubility of Lipophilic Microtubule Destabilizing Compounds on the Sea Urchin Embryo Model.

In screening, the dilution of DMSO stock solution of a lipophilic molecule with an assay medium often causes compound precipitation. To overcome the issue, the application of Pluronics as cosolvents was examined using a phenotypic sea urchin embryo assay that allows for the quick and facile evaluation of the antiproliferative effect together with systemic toxicity. Maximum tolerated concentration values for Pluronics L121, P123, and F127 were 1.4 µM, 8.6 µM, and 39.7 µM, respectively, and correlated directly with their hydrophilicity. Pluronics L121 and P123 suppressed cleavage and blastomeres retained the round shape, unlike hydrophilic Pluronic F127, which induced fertilization envelope creasing and embryo deformation that could be associated with the interaction of hydrophilic PEO units with mucopolysaccharides at the surface of sea urchin embryos. The toxicity of P123, but not of L121 and F127, was temperature-dependent and markedly increased at lower temperatures. CMC values obtained at different temperatures confirmed that the toxic effect of P123 was associated with both unimers and micelles, whereas F127 toxicity was related mainly to micelles. Evaluation using phenotypic sea urchin embryo assay revealed that potent microtubule destabilizers, namely albendazole, diarylisoxazole, and two chalcones, retained antimitotic activity after the dilution of their DMSO or 2-pyrrolidone stock solutions with 1.25% w/v Pluronic P123 or 5% w/v Pluronic F127. It was suggested that Pluronic P123 and Pluronic F127 could be used as cosolvents to improve the solubility of lipophilic molecules in aqueous medium.

Synthesis and biological evaluation of indolylglyoxylamide bisphosphonates, antimitotic microtubule-targeting derivatives of indibulin with improved aqueous solubility.

Indibulin (D-24851) derivatives with bisphosphonate fragment connected to the N1 atom of imidazole ring were synthesized by alkylation of (indolyl-3)methylglyoxylates with ethylenebisphosphonate. Biological evaluation of targeted compounds 4a-d using the phenotypic sea urchin embryo assay provided evidence that replacing of p-chlorobenzene ring in indibulin by bisphosphonate group did not eliminate antimitotic microtubule destabilizing activity. The most active molecule, tetraacid 5a, at physiological pH formed tetrasodium salt 6a with aqueous solubility value of at least 10 mg/mL. Molecule 5a was more potent in the sea urchin embryo assay than the parent indibulin. This compound also exhibited pronounced cytotoxicity against A549 lung carcinoma and A375 melanoma cell lines.

Computational modeling and target synthesis of monomethoxy-substituted o-diphenylisoxazoles with unexpectedly high antimitotic microtubule destabilizing activity.

The ability of monomethoxy-substituted o-diphenylisoxazoles 2a-d to interact with the colchicine site of tubulin was predicted using computational modeling, docking studies, and calculation of binding affinity. The respective molecules were synthesized in high yields by three steps reaction using easily available benzaldehydes, acetophenones, and arylnitromethanes as starting material. The calculated antitubulin effect was confirmed in vivo in a sea urchin embryo model. Compounds 2a and 2c showed high antimitotic microtubule destabilizing activity compared to that of CA4. Isoxazole 2a also exhibited significant cytotoxicity against human cancer cells in NCI60 screen. For the first time, isoxazole-linked CA4 derivatives 2a and 2c with only one methoxy substituent were identified as potent antimitotic microtubule destabilizing agents. These molecules could be considered as promising structures for further optimization.

Synthesis and cytotoxicity of novel simplified eleutherobin analogues as potential antitumour agents.

Mixed simplified structures containing the paclitaxel and eleutherobin pharmacophore moieties were analyzed using molecular docking techniques and synthesized based on adamantane and 8-oxabicyclo[3.2.1]octane scaffolds. The crucial role of substituents' stereochemistry in biological activity is discussed. At micromolar concentrations the selected analogues interfered with tubulin dynamics in vitro and in a living organism. Furthermore, new compounds were cytotoxic against human tumour cell lines. The simplified eleutherobin analogues may be considered as prototypes of a new class of antitumour agents.

Antioxidant Activity of Natural Allylpolyalkoxybenzene Plant Essential Oil Constituents.

Free-radical-scavenging capacity antioxidant and membrane-protective properties of natural and related synthetic allylpolyalkoxybenzenes with different numbers of alkoxy/methoxy groups in the aromatic ring were evaluated using several in vitro models. These included the DPPH assay, inhibition of lipid peroxidation products accumulation, inhibition of H2O2-induced hemolysis, and oxidation of oxyhemoglobin. A synthetic protocol for the synthesis of natural nothoapiol (9) from a parsley seed metabolite, apiol (7), was developed. A structure-activity relationship study revealed that both the methylenedioxy fragment and methoxy groups in the aromatic ring are favorable for antioxidant activity. Hydroxyapiol (14), containing a hydroxy group in the aromatic core, was identified as the most potent compound. The pentaalkoxy-substituted nothoapiol (9) showed antioxidant activity in mouse brain homogenates, whereas in mouse erythrocytes it exhibited a marked pro-oxidant effect. Despite their low free-radical-scavenging capacity, allylpolyalkoxybenzenes can contribute to the total antioxidant potencies of plant essential oils.

Mitochondriotropic and Cardioprotective Effects of Triphenylphosphonium-Conjugated Derivatives of the Diterpenoid Isosteviol.

Mitochondria play a crucial role in the cell fate; in particular, reducing the accumulation of calcium in the mitochondrial matrix offers cardioprotection. This affect is achieved by a mild depolarization of the mitochondrial membrane potential, which prevents the assembly and opening of the mitochondrial permeability transition pore. For this reason, mitochondria are an attractive target for pharmacological interventions that prevent ischaemia/reperfusion injury. Isosteviol is a diterpenoid created from the acid hydrolysis of Steviarebaudiana Bertoni (fam. Asteraceae) glycosides that has shown protective effects against ischaemia/reperfusion injury, which are likely mediated through the activation of mitochondrial adenosine tri-phosphate (ATP)-sensitive potassium (mitoKATP) channels. Some triphenylphosphonium (triPP)-conjugated derivatives of isosteviol have been developed, and to evaluate the possible pharmacological benefits that result from these synthetic modifications, in this study, the mitochondriotropic properties of isosteviol and several triPP-conjugates were investigated in rat cardiac mitochondria and in the rat heart cell line H9c2. This study's main findings highlight the ability of isosteviol to depolarize the mitochondrial membrane potential and reduce calcium uptake by the mitochondria, which are typical functions of mitochondrial potassium channel openings. Moreover, triPP-conjugated derivatives showed a similar behavior to isosteviol but at lower concentrations, indicative of their improved uptake into the mitochondrial matrix. Finally, the cardioprotective property of a selected triPP-conjugated derivative was demonstrated in an in vivo model of acute myocardial infarct.

Facile Synthesis of Natural Alkoxynaphthalene Analogues from Plant Alkoxybenzenes.

Analogues of the bioactive natural alkoxynaphthalene pycnanthulignene D were synthesized by an efficient method. The starting plant allylalkoxybenzenes (1) are easily available from the plant essential oils of sassafras, dill, and parsley. The target 1-arylalkoxynaphthalenes (5) exhibited antiproliferative activity in a phenotypic sea urchin embryo assay.

Efficient Synthesis of Glaziovianin A Isoflavone Series from Dill and Parsley Extracts and Their in Vitro/in Vivo Antimitotic Activity.

A concise six-step protocol for the synthesis of isoflavone glaziovianin A (GVA) and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. The reaction sequence involved an efficient conversion of the key intermediate epoxides 7 into the respective ß-ketoaldehydes 8 followed by their Cu(I)-mediated cyclization into the target series 9. The biological activity of GVA and its derivatives was evaluated using a panel of seven human cancer cell lines and an in vivo sea urchin embryo assay. Both screening platforms confirmed the antimitotic effect of the parent GVA (9cg) and its alkoxy derivatives. Structure-activity relationship studies suggested that compounds 9cd and 9cf substituted with trimethoxy- and dillapiol-derived B-rings, respectively, were less active than the parent 9cg. Of the evaluated human cancer cell lines, the A375 melanoma cell line was the most sensitive to the tested molecules. Notably, the target compounds were not cytotoxic against human peripheral blood mononuclear cells up to 10 µM concentration. Phenotypic readouts from the sea urchin assay unequivocally suggest a direct microtubule-destabilizing effect of isoflavones 9cg, 9cd, and 9cf.

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.

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.

Synthesis and antiproliferative activity of conformationally restricted 1,2,3-triazole analogues of combretastatins in the sea urchin embryo model and against human cancer cell lines.

A series of 1,5-diaryl- and 4,5-diaryl-1,2,3-triazole derivatives of combretastatin A4 were synthesized and evaluated as antimitotic microtubule destabilizing agents using the sea urchin embryo model. Structure-activity relationship studies identified compounds substituted with 3,4,5-trimethoxyphenyl and 3,4-methylenedioxy-5-methoxyphenyl ring A and 4-methoxyphenyl ring B as potent antiproliferative agents with high cytotoxicity against a panel of human cancer cell lines including multi-drug resistant cells. 4,5-Diaryl-1,2,3-triazoles (C-C geometry) were found to be considerably more active than the respective 1,5-diaryl-1,2,3-triazoles (N-C geometry). Compound 10ad' induced G2/M cell cycle arrest and apoptosis in human T-leukemia Jurkat cells via caspase 2/3/9 activation and downregulation of the antiapoptotic protein XIAP. A mitotic catastrophe has been evaluated as another possible cell death mode.

Toxicity of organic solvents and surfactants to the sea urchin embryos.

A comparative toxicity of widely applied organic solvents (methanol, ethanol, n-propanol, i-propanol, n-butanol, 2-butanol, i-butanol, t-butanol, 3-methoxy-3-methylbutanol-1 (MMB), ethylene glycol, diethylene glycol, 2-methoxyethanol, 2-ethoxyethanol, glycerol, ethyl acetate, acetonitrile, benzene, dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, 2-pyrrolidone, and N-methyl-2-pyrrolidone) and surfactants (PEG 300, PEG 6000, Tween 20, Tween 80, miramistin, and Cremophor EL) was studied using a sea urchin embryo model. Sea urchin embryo morphological alterations caused by the tested chemicals were described. The tested molecules affected P. lividus embryo development in a concentration-dependent manner. The observed phenotypic anomalies ranged from developmental delay and retardation of plutei growth to formation of aberrant blastules and gastrules, cleavage alteration/arrest, and embryo mortality. Discernible morphological defects were found after embryo exposure with common pharmaceutical ingredients, such as glycerol, Tween 80, and Cremophor EL. In general, solvents were less toxic than surfactants. PEG 6000 PEG 300, DMSO, ethanol, and methanol were identified as the most tolerable compounds with minimum effective concentration (MEC) values of 3.0-7.92 mg/mL. Previously reported MEC value of Pluronic F127 (4.0 mg/mL) fell within the same concentration range. Toxic effects of methanol, ethanol, DMSO, 2-methoxyethanol, 2-ethoxyethanol, Tween 20, and Tween 80 on P. lividus embryos correlated well with their toxicity obtained using other cell and animal models. The sea urchin embryos could be considered as an appropriate test system for toxicity assessment of solvents and surfactants for their further application as solubilizers of hydrophobic molecules in conventional in vitro cell-based assays and in vivo mammalian models. Nevertheless, to avoid adverse effect of a solubilizing agent in ecotoxicological and biological experiments, the preliminary assessment of its toxicity on a chosen test model would be beneficial.

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.

Triphenylphosphine Derivatives of Allylbenzenes Express Antitumor and Adjuvant Activity When Solubilized with Cyclodextrin-Based Formulations.

Allylbenzenes (apiol, dillapiol, myristicin and allyltetramethoxybenzene) are individual components of plant essential oils that demonstrate antitumor activity and can enhance the antitumor activity of cytotoxic drugs, such as paclitaxel, doxorubicin, cisplatin, etc. Triphenylphosphine (PPh3) derivatives of allylbenzenes are two to three orders of magnitude more potent than original allylbenzenes in terms of IC50. The inhibition of efflux pumps has been reported for allylbenzenes, and the PPh3 moiety is deemed to be responsible for preferential mitochondrial accumulation and the depolarization of mitochondrial membranes. However, due to poor solubility, the practical use of these substances has never been an option. Here, we show that this problem can be solved by using a complex formation with cyclodextrin (CD-based molecular containers) and polyanionic heparin, stabilizing the positive charge of the PPh3 cation. Such containers can solubilize both allylbenzenes and their PPh3 derivatives up to 0.4 mM concentration. Furthermore, we have observed that solubilized PPh3 derivatives indeed work as adjuvants, increasing the antitumor activity of paclitaxel against adenocarcinomic human alveolar basal epithelial cells (A549) by an order of magnitude (in terms of IC50) in addition to being quite powerful cytostatics themselves (IC50 in the range 1-10 µM). Even more importantly, CD-solubilized PPh3 derivatives show pronounced selectivity, being highly toxic for the A549 tumor cell line and minimally toxic for HEK293T non-tumor cells, red blood cells and sea urchin embryos. Indeed, in many cancers, the mitochondrial membrane is more prone to depolarization compared to normal cells, which probably explains the observed selectivity of our compounds, since PPh3 derivatives are known to act as mitochondria-targeting agents. According to the MTT test, 100 µM solution of PPh3 derivatives of allylbenzenes causes the death of up to 85% of A549 cancer cells, while for HEK293T non-cancer cells, only 15-20% of the cells died. The hemolytic index of the studied substances did not exceed 1%, and the thrombogenicity index was < 1.5%. Thus, this study outlines the experimental foundation for developing combined cytostatic medications, where effectiveness and selectivity are achieved through decreased concentration of the primary ingredient and the inclusion of adjuvants, which are safe or practically harmless substances.

Polymeric Micelles Formulation of Combretastatin Derivatives with Enhanced Solubility, Cytostatic Activity and Selectivity against Cancer Cells.

Combretastatin derivatives is a promising class of antitumor agents, tubulin assembly inhibitors. However, due to poor solubility and insufficient selectivity to tumor cells, we believe, their therapeutic potential has not been fully realized yet. This paper describes polymeric micelles based on chitosan (a polycation that causes pH and thermosensitivity of micelles) and fatty acids (stearic, lipoic, oleic and mercaptoundecanoic), which were used as a carrier for a range of combretastatin derivatives and reference organic compounds, demonstrating otherwise impossible delivery to tumor cells, at the same time substantially reduced penetration into normal cells. Polymers containing sulfur atoms in hydrophobic tails form micelles with a zeta potential of about 30 mV, which increases to 40-45 mV when cytostatics are loaded. Polymers with tails of oleic and stearic acids form poorly charged micelles. The use of polymeric 400 nm micelles provides the dissolution of hydrophobic potential drug molecules. Micelles could significantly increase the selectivity of cytostatics against tumors, which has been shown using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Fourier transform infrared (FTIR) spectroscopy, flow cytometry and fluorescence microscopy. Atomic force microscopy presented the difference between the unloaded micelles and those loaded with the drug: the size of the former was 30 nm on average, while the latter had a "disc-like" shape and a size of about 450 nm. The loading of drugs into the core of micelles was confirmed by UV and fluorescence spectroscopy methods; shifts of absorption and emission maxima into the long-wavelength region by tens of nm was observed. With FTIR spectroscopy, a high interaction efficiency of micelles with the drug on cells was demonstrated, but at the same time, selective absorption was observed: micellar cytostatics penetrate into A549 cancer cells 1.5-2 times better than the simple form of the drugs. Moreover, in normal HEK293T, the penetration of the drug is reduced. The proposed mechanism for reducing the accumulation of drugs in normal cells is the adsorption of micelles on the cell surface and the preservation of cytostatics to penetrate inside the cells. At the same time, in cancer cells, due to the structural features of the micelles, they penetrate inside, merging with the membrane and releasing the drug by pH- and glutathione-sensitive mechanisms. From a methodological point of view, we have proposed a powerful approach to the observation of micelles using a flow cytometer, which, in addition, allows us to quantify the cells that have absorbed/adsorbed cytostatic fluorophore and distinguish between specific and non-specific binding. Thus, we present polymeric micelles as drug delivery systems in tumors using the example of combretastatin derivatives and model fluorophore-cytostatic rhodamine 6G.

Synthesis and comparative evaluation of 4-oxa- and 4-aza-podophyllotoxins as antiproliferative microtubule destabilizing agents.

A series of novel 4-oxa-podophyllotoxin derivatives 7 featuring the intact lactone ring D and various substituents in rings B and E has been synthesized and evaluated in a phenotypic sea urchin embryo assay along with the representative 4-aza-analogs 5 for their antimitotic and microtubule destabilizing activity. The most active compounds exhibited myristicin-derived or a 3',5'-dimethoxy substitution pattern in the ring E and a 6-methoxy moiety replacing the methylenedioxy ring A. Compounds 5xb, 5xe, 5yb, 7xa, 7xb, and 7xc showed potent antiproliferative effects in the NCI60 cytotoxicity screen. Notably, growth of the multi-drug resistant NCI/ADR-RES cells was more affected by these agents than the parent OVCAR-8 cell line. Although generally 4-oxa-podophyllotoxins were less potent than the respective 4-aza-derivatives in these assays, stability of the former series towards oxidation may prove to be of interest for the development of anticancer agents with in vivo activity.

Plant Alkylbenzenes and Terpenoids in the Form of Cyclodextrin Inclusion Complexes as Antibacterial Agents and Levofloxacin Synergists.

Allylpolyalkoxybenzenes (APABs) and terpenoids from plant essential oils exhibit a range of remarkable biological effects, including analgesic, antibacterial, anti-inflammatory, antioxidant, and others. Synergistic activity with antibiotics of different classes has been reported, with inhibition of P-glycoprotein and impairment of bacterial cell membrane claimed as probable mechanisms. Clearly, a more detailed understanding of APABs' biological activity could help in the development of improved therapeutic options for a range of diseases. However, APABs' poor solubility in water solutions has been a limiting factor for such research. Here, we found that complex formation with ß-cyclodextrins (CD) is an efficient way to transform the APABs into a water-soluble form. Using a combination of spectroscopic (FTIR, NMR, UV) methods, we have estimated the binding constants, loading capacity, and the functional groups of both APABs and monoterpenes involved in complex formation with CD: ethylene, aromatic, methoxy and hydroxy groups. In the presence of a molar excess of CD (up to 5 fold) it was possible to achieve the complete dissolution of APABs and terpenoids in an aqueous medium (at 90-98% encapsulation) higher by 10-1000 times. Further, we have demonstrated that CD-APABs, if used in combination with levofloxacin (Lev), can be antagonistic, indifferent, additive, or synergistic, mostly depending on the concentration ratio: at high Lev concentration with the addition of APAB is typically neutral or even antagonistic; while at a Lev concentration below MIC, the addition of CD-APAB is either additive or synergistic (according to FICI criteria). An over three-fold increase in Lev antibacterial activity was observed in combination with eugenol (EG), as per the growth inhibition diameter measurement in agar. Interestingly, a synergistic effect could be observed with both Gram-positive and Gram-negative bacteria. So, obviously, the APAB-CD and terpenoid-CD mechanism of action is not limited to their interaction with the bacterial membrane, which has been shown earlier for CDs. Further research may open new prospects for the development of adjuvants to improve the therapeutic regimens with existing, as well as with new anti-infective drugs.

Synthesis, Screening and Characterization of Novel Potent Arp2/3 Inhibitory Compounds Analogous to CK-666.

Branched actin networks polymerized by the Actin-related protein 2 and 3 (Arp2/3) complex play key roles in force generation and membrane remodeling. These networks are particularly important for cell migration, where they drive membrane protrusions of lamellipodia. Several Arp2/3 inhibitory compounds have been identified. Among them, the most widely used is CK-666 (2-Fluoro-N-[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide), whose mode of action is to prevent Arp2/3 from reaching its active conformation. Here 74 compounds structurally related to CK-666 were screened using a variety of assays. The primary screen involved EdU (5-ethynyl-2'-deoxyuridine) incorporation in untransformed MCF10A cells. The resulting nine positive hits were all blocking lamellipodial protrusions and cell migration in B16-F1 melanoma cells in secondary screens, showing that cell cycle progression can be a useful read-out of Arp2/3 activity. Selected compounds were also characterized on sea urchin embryos, where Arp2/3 inhibition yields specific phenotypes such as the lack of triradiate spicules and inhibition of archenteron elongation. Several compounds were filtered out due to their toxicity in cell cultures or on sea urchin development. Two CK-666 analogs, 59 (N-{2-[5-(Benzyloxy)-2-methyl-1H-indol-3-yl] ethyl}-3-bromobenzamide) and 69 (2,4-Dichloro-N-[2-(7-chloro-2-methyl-1H-indol-3-yl) ethyl]-5-[(dimethylamino) sulfonyl] benzamide), were active in all assays and significantly more efficient in vivo than CK-666. These best hits with increased in vivo potency were, however, slightly less efficient in vitro than CK-666 in the classical pyrene-actin assay. Induced-fit docking of selected compounds and their possible metabolites revealed interaction with Arp2/3 that suppresses Arp2/3 activation. The data obtained in our screening validated the applicability of original assays for Arp2/3 activity. Several previously unexplored CK-666 structural analogs were found to suppress Arp2/3 activation, and two of them were identified as Arp2/3 inhibitors with improved in vivo efficiency.