A series of substituted 4-anilinoquinazolines and related compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptor (Flt and KDR) tyrosine kinase activity. Enzyme screening indicated that a narrow structure-activity relationship (SAR) existed for the bicyclic ring system, with quinazolines, quinolines, and cinnolines having activity and with quinazolines and quinolines generally being preferred. Substitution of the aniline was investigated and clearly indicated that small lipophilic substituents such as halogens or methyl were preferred at the C-4' position. Small substituents such as hydrogen and fluorine are preferred at the C-2' position. Introduction of a hydroxyl group at the meta position of the aniline produced the most potent inhibitors of Flt and KDR tyrosine kinases activity with IC(50) values in the nanomolar range (e.g. 10, 12, 13, 16, and 18). Investigation of the quinazoline C-6 and C-7 positions indicates that a large range of substituents are tolerated at C-7, whereas variation at the C-6 is more restricted. At C-7, neutral, basic, and heteroaromatic side chains led to very potent compounds, as illustrated by the methoxyethoxy derivative 13 (IC(50) < 2 nM). Our inhibitors proved to be very selective inhibitors of Flt and KDR tyrosine kinase activity when compared to that associated with the FGF receptor (50- to 3800-fold). Observed enzyme profiles translated well with respect to potency and selectivity for inhibition of growth factor stimulated proliferation of human umbilical vein endothelial cells (HUVECs). Oral administration of selected compounds to mice produced total plasma levels 6 h after dosing of between 3 and 49 microM. In vivo efficacy was demonstrated in a rat uterine oedema assay where significant activity was achieved at 60 mg/kg with the meta hydroxy anilinoquinazoline 10. Inhibition of growth of human tumors in athymic mice has also been demonstrated: compound 34 inhibited the growth of established Calu-6 lung carcinoma xenograft by 75% (P < 0.001, one tailed t-test) following daily oral administration of 100 mg/kg for 21 days.

We have previously shown that 4-anilinoquinazolines can be potent inhibitors of vascular endothelial growth factor (VEGF) receptor (Flt-1 and KDR) tyrosine kinase activity. A novel subseries of 4-anilinoquinazolines that possess basic side chains at the C-7 position of the quinazoline nucleus have been synthesized. This subseries contains potent, nanomolar inhibitors of KDR (median IC(50) 0.02 microM, range 0.001-0.04 microM), which are comparatively less potent vs Flt-1 tyrosine kinase (median IC(50) 0.55 microM, range 0.02-1.6 microM). The compounds also retain some inhibitory activity against the tyrosine kinase associated to the endothelial growth factor receptor (EGFR) (median IC(50) 0.2 microM, range 0.075-0.8 microM) but demonstrate selectivity vs that associated to the FGF receptor 1 (median IC(50) 2.5 microM, range 0.9-19 microM). This selectivity profile is also evident in a growth factor-stimulated human endothelial cell (HUVEC) proliferation assay (i.e., inhibition of VEGF > EGF > FGF), with inhibition of VEGF-induced proliferation being achieved at nanomolar concentrations (median IC(50) 0.06 microM). Further examination of compound 2 (ZD6474) in recombinant enzyme assays revealed excellent selectivity for the inhibition of KDR tyrosine kinase (IC(50) 0.04 microM) vs the kinase activity of erbB2, MEK, CDK-2, Tie-2, IGFR-1R, PDK, PDGFRbeta, and AKT (IC(50) range: 1.1 to >100 microM). Anilinoquinazolines possessing basic C-7 side chains exhibited markedly improved aqueous solubility over previously described anilinoquinazolines possessing neutral C-7 side chains (up to 500-fold improvement at pH 7.4). In addition, aqueous solubility of the neutral fraction present at pH 7.4 of the basic subseries of anilinoquinazoline proved to be higher than that of the neutral analogue 1 (ZD4190). Oral administration of representative compounds to mice (50 mg/kg) produced plasma levels between 0.2 and 3 microM at 24 h after dosing. Our development candidate 2 demonstrated a very attractive in vitro profile combined with excellent solubility (330 microM at pH 7.4) and good oral bioavailability in rat and dog (> 80 and > 50%, respectively). This compound demonstrated highly significant, dose-dependent, antitumor activity in athymic mice. Once daily oral administration of 100 mg/kg of compound 2 for 21 days inhibited the growth of established Calu-6 lung carcinoma xenografts by 79% (P < 0.001, Mann Whitney rank sum test), and substantial inhibition (36%, P < 0.02) was evident with 12.5 mg/kg/day.

Insertion of new wall during the growth of Bacillus licheniformis occurred discretely and probably at the sites of incipient cross wall formation.

Novel 3-substituted quinuclidine inhibitors of cholesterol biosynthesis are reported. Compounds were optimized against oxidosqualene cyclase-lanosterol synthase (OSC) inhibition in vivo, rather than by the conventional optimization of structure-activity relationship information based on in vitro OSC inhibition. Thus, examination of HPLC lipid profiles from orally dosed rats showed cholesterol biosynthetic intermediates and whether cholesterol levels were reduced. A new substituted quinuclidine pharmacophore 18a-c was rapidly found for the inhibition of OSC, and the most promising inhibitors were validated by the confirmation of potent OSC inhibition. Compound 16 gave an IC50 value of 83 +/- 11 nM for human and an IC50 value of 124 +/- 14 nM, for rat, coupled with oral and selective inhibition of cholesterol biosynthesis derived from OSC inhibition (rat, ED50 = 1.3 +/- 0.7 mg/kg, n = 5; marmoset, 15 mg/kg dose, n = 3, caused complete inhibition). These 3-substituted quinuclidines, which were derived from a quinuclidine series previously known to inhibit cholesterol biosynthesis at the squalene synthase step, may afford a novel series of hypocholesterolemic agents acting by the inhibition of OSC.

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