Sulfonylbenzoyl-nitrostyrenes: potential bisubstrate type inhibitors of the EGF-receptor tyrosine protein kinase.

The synthesis and biological activities of a series of sulfonylbenzoyl-nitrostyrene derivatives, a novel class of selective bisubstrate type inhibitors of the EGF-receptor tyrosine protein kinase, are described. The most potent derivatives inhibited the EGF-R tyrosine kinase, using angiotensin II as exogenous substrate, with IC50 values of less than or equal to 1 microM. No inhibition of the v-abl tyrosine kinase or the serine/threonine kinases PKC and PK-A was observed. In addition, active derivatives (compounds 5 and 12) effectively blocked the autophosphorylation of the EGF-R in vitro. Starting from the acids 5, 7, and 9, a series of esters, amides, and peptides was synthesized with the aim of increasing cellular penetration. Amides 14-18 showed potent antiproliferative effects using the EGF-dependent Balb/MK mouse epidermal keratinocyte cell line. Additionally, with the amide 14 inhibition of EGF-R autophosphorylation was demonstrated in the A431 cell line. CAMM studies using a computer-generated model for the transition state of the gamma-phosphoryl transfer from ATP to a tyrosine moiety and fitting experiments using the highly potent derivative 7 (IC50 value = 54 nM) support the hypothesis that the sulfonylbenzoyl group mimics a diphosphate moiety in the transition state. These results demonstrate that the rational design of tyrosine kinase inhibitors, using the inhibitory nitrostyrene moiety as a tyrosine mimic together with the sulfonylbenzoyl moiety as a diphosphate mimic, leads to highly potent and selective multisubstrate type inhibitors.

Use of the sulfo-phospo-vanillin reaction in a routine method for determining total lipids in human cerebrospinal fluid.

Adult human cerebrospinal fluid (CSF) was assayed for total lipid levels by the sulfo-phospho-vanillin color reaction. Lipids in a 1-2 ml sample of CSF are extracted with a 2:1 chlofoform-methanol mixture and the solvent extract, after equilibration with 0.74 percent potassium chloride, is evaporated to dryness. The lipid in the residue is estimated by the sulfo-phospho-vanillin reaction. The extraction process is essentially quantitative for lipid mixtures normally found in CF. The technique has an average coefficient of variation of 1.5 percent and the recovery of added lipids is nearly quantitative. It can be used as a rapid routine method for estimating CSF total lipids and several samples can be processed simultaneously. Estimations of total lipid levels using the sulfo-phospho-vanillin reaction agree quite well with the results obtained by densitometric quantitation of charred lipids. A preliminary analysis of 22 samples of adult human CSF containing normal levels of total protein gave an average total lipid level of 0.78 (+/- 0.07 SEM) mg/dl. Total lipid levels in CSF did not significantly correlate with age. However lipid levels in CSF show a significant positive correlation with CSF total protein levels.