The synthesis and structure-activity relationship study of a series of compounds with heterocycles in place of the cis double bond in combretastatin A-4 (CA-4) are described. Substituted tosylmethyl isocyanides were found to be the key intermediates in construction of the heterocycles. Cytotoxicities of the heterocycle-based CA-4 analogues were evaluated against NCI-H460 and HCT-15 cancer cell lines. 3-Amino-4-methoxyphenyl and N-methyl-indol-5-yl were the best replacements for the 3-hydroxy-4-methoxyphenyl in CA-4. 4,5-Disubstituted imidazole was found to be the best for the replacement of the cis double bond in CA-4. Medicinal chemistry efforts led to the discovery of compounds 24h and 25f that were found to be 32 and 82% bioavailable, respectively, in rat. Evaluation of 24h and 25f against murine M5076 reticulum sarcoma in mice revealed that both compounds were orally efficacious with an increase in life span of 38.5 and 40.5%, respectively.

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HL-K6-3512) as well as by grants (HL-02212 and HL-16346) from the National Institutes of Health. A Fulbright Travelling Fellowship to C. G. C. is gratefully acknowledged.

Fibrinogen displays a regulation of considerable physiological significance by lowering the Ca2+ requirement for the conversion of the fibrin-stabilizing factor (Factor XIII) zymogen to the range of concentrations of this ion found in plasma. Fibrinogen modulates both Ca2+-dependent steps in the complex process of zymogen activation, involving the heterologous dissociation of subunits of the thrombin-modified zymogen (Factor XIII') species (a'2 a'2) and the unmasking b2 of iodoacetamide titratable sites during generation of transamidating activity (a'2 --a*2). It is interesting that a thrombinindependent pathway of zymogen activation (a2b2 X a -A), b2 which we found to operate at Ca2+ concentrations above 50 mM, is not affected by the presence of fibrinogen. Regulation by fibrinogen thus appears to be specific for controlling only the physiological pathway of zymogen conversion.Fibrinogen has always been characterized as the plasma protein that serves as the precursor for the fibrin network formed during the clotting of blood. Accordingly, only the reaction of fibrinogen with thrombin (1-3) and that of fibrin with fibrinoligase (coagulation Factor XIIIa; refs. 4-6) were thought to be of physiological importance. Our recent work reveals that the fibrinogen molecule displays a hitherto unrecognized regulatory property of considerable physiological significance. It will be shown in the present paper that fibrinogen greatly enhances the generation of fibrinoligase activity and lowers the Ca2+ requirement for this process. As such, modulation by fibrinogen is somewhat reminiscent of the biochemical functions of troponin C in muscle (7) and of the various Ca2+-dependent regulatory proteins in other tissues (see refs. 7-11).Fibrinoligase, a transamidase that is responsible for the covalent fusion of fibrin molecules by 'y-glutamyl-elysine bridges The fibrin-stabilizing factor zymogen (Factor XIII) and the pure b subunits were isolated from outdated human plasma by published procedures (24). These purified materials were stored at 40 in 50 mM Tris1HCl, pH 7.5/1 mM EDTA. Concentrations of the zymogen and the b protein were calculated on the basis of E "m = 13.8 at 280 nm (25). The functional molarity of Factor XIII was measured by titration with iodoacetamide as described by Curtis et al. (14). The molarity of thrombin was determined from pre-steadystate kinetics for the hydrolysis of p-nitrophenylguanidobenzoate (26).Factor XIII zymogen was hydrolytically converted from the a2b2 native state to the a'2b2 one (Factor XIII') at 25°, pH 7.5, in solutions of 0.15 ml containing 10MM zymogen and 0.17,gM thrombin, as well as 50 mM Tris-HCl and 0.5 mM EDTA. After a reaction time of 30 min, sufficient amount of hirudin (Sigma, lot 27B-2330) was added to provide a 2-fold excess of hirudin over the clotting units of thrombin, in terms of the thrombinneutralizing potency of the hirudin preparation as specified by the supplier. Total conversion of the a subunits to a' was verified by sodium dodecyl sulfate disc gel polyacryl...

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (CIg) to promote the uptake of ' 25 1-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM) . The uptake of g-Ltx* by PM was enhanced by CIg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake . Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37°C after particle uptake removed <15% of the radioactivity incorporated by the monolayers . However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by >75% . Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex.Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed CIg-dependent particle uptake . Phagocytosis of g-Ltx* by PM in the presence of CIg and heparin was confirmed by electron microscopy . Finally, g-Ltx* could also be effectively opsonized with CIg at 37°C before their addition to the monolayers . These studies suggest that the recognition of g-Ltx* in the presence of CIg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements . Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells.The process of phagocytosis by vertebrate phagocytes is markedly enhanced by humoral recognition factors called opsonins . Immune opsonins are antibody or complement proteins that interact with foreign antigens and with receptors on the surface of phagocytic cells (26, 36) . There also exist opsonins that enhance the uptake of colloidal material by macrophages of the reticuloendothelial system that are not immune proteins (34). One such nonimmune opsonin has been purified from rat (1,2,6,22) and human serum (4) and was originally designated a-2-macroglobulin on account of its electrophoretic mobility and size (2,22) . More recent studies have demonstrated that this nonimmune opsonin isolated from human serum is identical to cold-insoluble globulin (CIg) or fibronectin (5), a high molecular weight adhesive glycoprotein, a form of which is also found on the surface of many cell types (10,38) .Up until now, quantitation of the opsonic activity of CIg has depended largely on a liver slice assay utilizing radiolabeled,