Thalidomide has demonstrated clinical activity in various malignancies affecting immunomodulatory and angiogenesis pathways. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort. We recently designed and synthesized a new class of compounds, consisting of both tetrafluorinated thalidomide analogs (Gu973 and Gu998) and tetrafluorobenzamides (Gu1029 and Gu992). In this study, we demonstrate the anti-angiogenic properties of these newly synthesized compounds. We examined the specific anti-angiogenic characteristics in vitro using rat aortic rings with carboxyamidotriazole as a positive control. Additionally, further in vitro efficacy was evaluated using HUVECs and PC3 cells treated with 5μM and 10μM doses of each compound. All compounds were seen to reduce microvessel outgrowth in rat aortic rings as well as inhibit HUVECs to a greater extent, at lower concentrations than previously tested thalidomide analogs. The anti-angiogenic properties of the compounds was also examined in vivo in fli1:EGFP zebrafish embryos, where all compounds were seen to inhibit the extent of outgrowth of newly developing blood vessels. In addition, Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos, while Gu998 and Gu992 showed no difference. The compounds anti-tumor effects were also explored in vivo using the human prostate cancer PC3 xenograft model. All four compounds were also screened in vivo in chicken embryos to investigate their teratogenic potential. This study establishes these novel thalidomide analogs as a promising immunomodulatory class with anti-cancer effects that warrant further development to characterize their mechanisms of action.

A general synthesis protocol for the generation of tri- and tetrasubstituted 5-carbamoylhydantoins is described. Starting from barbituric acids and following bromination and reaction with primary amines, 5-aminobarbituric acids 3a-s and 8 were prepared. Compounds 3 and 8 were subjected to different conditions of a base-catalyzed rearrangement reaction to yield the 1,5,5-trisubstituted hydantoins 4a-s and the 1,3,5,5-tetrasubstituted hydantoin 5c, respectively. Alkylation of 4a-s afforded 1,3,5,5-tetrasubstituted hydantoins 5a-h. Mechanisms that explain the transformation of corresponding aminobarbituric acids to hydantoins 4a-s and 5c were discussed in terms of the formation of ring-opened intermediates. Aminobarbituric acids 3a-s unsubstituted at position 3 underwent a ring contraction via intermediate isocyanates which were trapped by the amino function. A different mechanism involving a carbamate intermediate was concluded for conversion of the 1,3,5,5-tetrasubstituted aminobarbituric acid 8.

The development of novel thalidomide derivatives as immunomodulatory and anti-angiogenic agents has revived over the last two decades. Herein we report the design and synthesis of three chemotypes of barbituric acids derived from the thalidomide structure: phthalimido-, tetrafluorophthalimido-, and tetrafluorobenzamidobarbituric acids. The latter were obtained by a new tandem reaction, including a ring opening and a decarboxylation of the fluorine-activated phthalamic acid intermediates. Thirty compounds of the three chemotypes were evaluated for their anti-angiogenic properties in an ex vivo assay by measuring the decrease in microvessel outgrowth in rat aortic ring explants. Tetrafluorination of the phthalimide moiety in tetrafluorophthalimidobarbituric acids was essential, as all of the nonfluorinated counterparts lost anti-angiogenic activity. An opening of the five-membered ring and the accompanying increased conformational freedom, in case of the corresponding tetrafluorobenzamidobarbituric acids, was well tolerated. Their activity was retained, although their molecular structures differ in torsional flexibility and possible hydrogen-bond networking, as revealed by comparative X-ray crystallographic analyses.

No abstract

The introduction of fluorine into bioactive molecules is a matter of importance in medicinal chemistry. In this study, representatives of various chemical entities of fluoroaromatic compounds were synthesized. Depending on the reaction conditions, either tetrafluorophthalimides or ammonium tetrafluorophthalamates are accessible from tetrafluorophthalic anhydride and primary amines. Tetrafluorophthalamic acids undergo thermal decarboxylation to yield tetrafluorobenzamides. These could be successfully converted upon treatment with primary amines, in the course of an aromatic nucleophilic substitution, to 2,3,5-trifluorobenzamides with respective amino substituents at the 4-position. The five structure types were characterized by means of spectroscopic and crystallographic methods. The synthesized compounds were evaluated as inhibitors of angiogenesis by measuring microvessel outgrowth in a rat aortic ring assay. The biological activity was maintained throughout these different polyfluorinated chemotypes.