Sea Urchin Embryo Model As a Reliable in Vivo Phenotypic Screen to Characterize Selective Antimitotic Molecules. Comparative evaluation of Combretapyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles as Tubulin-Binding Agents

Abstract

A series of both novel and reported combretastatin analogues, including diarylpyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles, were synthesized via improved protocols to evaluate their antimitotic antitubulin activity using in vivo sea urchin embryo assay and a panel of human cancer cells. A systematic comparative structure–activity relationship studies of these compounds were conducted. Pyrazoles 1i and 1p, isoxazole 3a, and triazole 7b were found to be the most potent antimitotics across all tested compounds causing cleavage alteration of the sea urchin embryo at 1, 0.25, 1, and 0.5 nM, respectively. These agents exhibited comparable cytotoxicity against human cancer cells. Structure–activity relationship studies revealed that compounds substituted with 3,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl ring B displayed the highest activity. 3-Hydroxy group in the ring B was essential for the antiproliferative activity in the diarylisoxazole series, whereas it was not required for potency of diarylpyrazoles. Isoxazoles 3 with 3,4,5-trimethoxy-substituted ring A and 3-hydroxy-4-methoxy-substituted ring B were more active than the respective pyrazoles 1. Of the azoles substituted with the same set of other aryl pharmacophores, diarylpyrazoles 1, 4,5-diarylisoxazoles 3, and 4,5-diaryl-1,2,3-triazoles 7 displayed similar strongest antimitotic antitubulin effect followed by 3,4-diarylisoxazoles 5, 1,5-diaryl-1,2,3-triazoles 8, and pyrroles 10 that showed the lowest activity. Introduction of the amino group into the heterocyclic core decreased the antimitotic antitubulin effect of pyrazoles, triazoles, and to a lesser degree of 4,5-diarylisoxazoles, whereas potency of the respective 3,4-diarylisoxazoles was increased.

Effective Synthesis of 3,4‐Diaryl‐isoxazole‐5‐carboxamides and their Antiproliferative Properties

“…In our previous work 3,4‐diaryl isoxazoles were identified as antimitotics with microtubule destabilizing mode of action . Therefore, the targeted 3,4‐diaryl‐isoxazole‐5‐carboxamides 6c–f , h , i were evaluated for antimitotic microtubule destabilizing activity using a phenotypic sea urchin embryo assay , . Antimitotic properties of a tested molecule were estimated by the ability to disrupt cleavage – quick successive embryonic cell divisions, whereas specific alteration of embryo motility, spinning on the bottom of the culture vessel instead of forward swimming, indicated microtubule destabilizing mechanism of action.…”

“…The sea urchin embryo assay was conducted as described previously , . Fertilized eggs and hatched blastulae were exposed to 2‐fold decreasing concentrations of compounds.…”

“…4,5‐Diarylisoxazole scaffold can be found in a wide variety of non steroidal anti‐inflammatory drugs (NSAIDs),, inhibitors of platelet aggregation, tubulin polymerization inhibitors,, estrogen receptor modulators, antiviral, anti‐diabetic, anti‐convulsant, and other agents . Polyoxygenated 4,5‐diarylisoxazoles with 3‐carboxamide groups (Figure , A) were reported to inhibit heat shock protein Hsp90, a chaperone that assists folding of various proteins including those implicated in malignancy.…”

A Survey on Tubulin and Arginine Methyltransferase Families Sheds Light on P. lividus Embryo as Model System for Antiproliferative Drug Development

“…P. lividus embryo is a widely recognized model for several research programs spanning from cell proliferation assays [100] and differentiation patterns to toxicological studies [56,57,61,101,102,103,104,105,106,107,108], and also in poor-resource settings. Moreover, the sea urchin embryo has been also intensively used in order to select antimitotic compounds (and glean insights into the biological mechanism) from libraries of 1,3,4-oxadiazole derivatives [43], combretastatin derivatives [44,45] and analogues [109,110] and also 3-amino-thieno[2,3-b]pyridines [111].…”

Preparation of Ring-Methoxylated Arylnitromethanes by the Victor Meyer Reaction

“…[1][2][3][4][5] In our previous work, nitrostilbenes prepared from ANMs were utilized as starting materials for the synthesis of 3,4-diaryl-pyrroles and -isoxazoles. 6,7 The Victor Meyer reaction, [8][9][10][11][12][13][14][15][16][17][18][19] which involves the reaction of an alkyl halide with silver nitrite in a nonpolar solvent, e.g., diethyl ether or benzene, has been the method of choice for the preparation of ANMs since the 19th century. 1,11 In 1955, Kornblum reported that while p-nitrobenzyl bromide and benzyl bromide reacted with silver nitrite to form arylnitromethanes, the corresponding reaction of pmethoxybenzyl bromide gave predominantly a nitrite ester (55%) and small amount of p-methoxyphenylnitromethane (26%).…”