New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models.

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Selective inhibition of ICAM-1 and E-selectin expression in human endothelial cells. 2. Aryl modifications of 4-(aryloxy)thieno[2,3-c]pyridines with fine-tuning at C-2 carbamides.

The elevated expression of cell adhesion molecules (CAMs) on the lumenal surface of vascular endothelial cells is a critical early event in the complex inflammatory process. The adhesive interactions of these CAMs that include E-selectin, ICAM-1, and VCAM-1 with their counter-receptors on leukocytes, such as integrins of the alpha(L)beta(2) family, result in migration of the leukocytes to the site of inflammation and cause tissue injury. Pharmaceutical agents that could suppress the induced expression of one or more of these cell adhesion molecules would provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A-205804 (1), a potent and selective inhibitor of the induced expression of E-selectin and ICAM-1 over VCAM-1, was further modified with emphasis at the C-4 and C-2 positions to identify a more potent drug candidate with a good pharmacokinetic profile and physical properties. Replacement of the C-4 sulfur linkage in 1 with an oxygen atom eliminated one of the two major metabolites for this lead molecule. The para-position of the 4-phenoxy group of the thieno[2,3-c]pyridine lead is found to be very critical for a higher in vitro potency and selectivity of E-selectin and ICAM-1 over VCAM-1 expression. This position is presumably close to the solvent-accessible region of the target protein-inhibitor complex. An attempt to install a water-solubilizing group at the para-position of the phenoxy group to increase the aqueous solubility of this lead series through various linkages failed to provide an ideal inhibitor. Only small substituents such as fluorine are tolerated at the meta- and ortho-positions of the 4-phenoxy to retain a good in vitro potency. Bromo, trifluoromethyl, pyrazol-1-yl, and imidazol-1-yl are among the better substituents at the para-position. With fine-tuning at the C-2 position we discovered a series of very potent (IC(50) < 5 nM for ICAM-1) and selective (>200-fold vs VCAM-1) inhibitors with a good pharmacokinetic profile. Demonstrated efficacy in a rat rheumatoid arthritis model and in a mice asthma model with selected compounds is also reported.

Discovery of inhibitors of cell adhesion molecule expression in human endothelial cells. 1. Selective inhibition of ICAM-1 and E-selectin expression.

A critical early event in the inflammatory cascade is the induced expression of cell adhesion molecules on the lumenal surface of vascular endothelial cells. These adhesion molecules include E-selectin, ICAM-1, and VCAM-1, which serve to recruit circulating leukocytes to the site of the inflammation. These adhesive interactions allow the leukocytes to firmly adhere to and cross the vascular endothelium and migrate to the site of tissue injury. Pharmaceutical agents which would prevent the induced expression of one or more of the cell adhesion molecules on the endothelium might be expected to provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A thieno[2,3-d]pyrimidine, A-155918, was identified from a whole-cell high-throughput assay for compounds which inhibited the tumor necrosis factor-alpha (TNFalpha)-induced expression of E-selectin, ICAM-1, or VCAM-1 on human vascular endothelial cells. Traditional medicinal chemistry methods were applied to this low-micromolar inhibitor, resulting in the 2,4-disubstituted thieno[2,3-c]pyridine A-205804, a potent and selective lead inhibitor of E-selectin and ICAM-1 expression (IC(50) = 20 and 25 nM, respectively). The relative position of the nitrogen atom in the thienopyridine isomer was shown to be critical for activity, as was a small amide 2-substituent.

Studies on an immunosuppressive macrolactam, ascomycin: synthesis of a C-33 hydroxyl derivative.

Ascomycin 2, a close analogue of the immunosuppressant FK506 1, was modified to incorporate a hydroxyl group at the C-33 position. This increased the aqueous solubility of ascomycin by a hundred-fold at pH 7.4 and by approximately 300-fold at pH 6.5. Ascomycin 3 also exhibited an excellent immunosuppressive activity in vitro, as tested in a human mixed lymphocyte proliferation (HuMLR) assay, and in vivo using a rat popliteal lymph node (rPLN) hyperplasia assay.

Discovery of ascomycin analogs with potent topical but weak systemic activity for treatment of inflammatory skin diseases.

Drug therapy for the major inflammatory skin diseases, which include atopic dermatitis, psoriasis and allergic contact dermatitis, is often inadequate due to poor efficacy, toxicity, or both. Much research has focused on the macrolactam T cell inhibitors as a promising new class of agents for immunotherapy, and medicinal chemistry efforts to design novel ascomycin analogs have produced clinically promising agents. A synthetic program to modify the ascomycin nucleus to alter its physicochemical properties and promote systemic clearance is described. A biologic screening strategy to identify analogs with reduced systemic activity and rapid pharmacokinetic elimination led to identification of the clinical candidate, ABT-281. A swine contact hypersensitivity model was used as a stringent indicator of skin penetration as human doses of topical corticosteroids produced inhibition only in the 50% range and ED50 values were 100-fold less potent than in rat. Also, cyclosporine was confirmed to be topically inactive in swine, as seen in human. ABT-281 had topical potency equal to tacrolimus (FK506) despite a severalfold lower potency for inhibiting swine T cells in vitro, consistent with superior skin penetration. ABT-281 was found to have a shorter duration of action after i.v. dosing in monkeys using an ex vivo whole blood IL-2 production assay. Systemic potency was reduced by 30-fold or more in rat popliteal lymph node hyperplasia and contact hypersensitivity assays. Following i.v. or i.p. administration in the swine contact hypersensitivity model, ABT-281 was 19- and 61-fold less potent, respectively, than FK506. Pharmacokinetic studies showed that ABT-281 had a shorter half life and higher rate of clearance than FK506 in all three species. The potent topical activity and reduced systemic exposure of ABT-281 may thus provide both efficacy and a greater margin of safety for topical therapy of skin diseases.

Effects of medium alterations on in vitro development of Brugia malayi larvae in cultured mosquito thoraces.

A recent study showed that 1-day-old, intracellularly lodged larvae of Brugia species develop in vitro to the infective third-stage larvae (L3) in excised thoraces of susceptible mosquitoes in the diphasic insect tissue culture medium containing a nutrient agar base overlaid with a 1:1 mixture of Schneider's Drosophila medium and Grace's insect cell culture medium supplemented with 20% fetal bovine serum (FBS) and antimicrobial agents. In the present investigation, the diphasic culture medium was used to evaluate the effects of medium alterations on the development of 1-day-old, intracellularly lodged larvae of subperiodic Brugia malayi in excised thoraces of Aedes aegypti to the L3. One-day-old larvae developed to the L3 in medium without nutrient agar base, at pH 7.0 and pH 7.5, in Hanks' balanced salt solution (HBSS) and in HBSS supplemented with bovine albumin fraction-V (BAF-V). These larvae also developed in the absence of FBS in the overlay medium, in overlay medium containing 5-20% FBS, in medium components obtained from different sources, in serum free Sf-900 (GIBCO) medium, and when FBS is replaced by BAF-V in the overlay medium. The percentage of L3 was not increased substantially in infected excised thoraces of mosquitoes when nutrient supplements, such as folic acid, p-aminobenzoic acid, glucose, lipid concentrate, hemin, or reduced glutathione, were added to the overlay medium containing BAF-V. These results suggested that 1-day-old, intracellularly lodged larvae developed to the L3 in infected excised thoraces of mosquitoes at almost the same rate as in intact mosquito, when excised thoraces were maintained alive under optimal conditions in a culture medium.