Tyrosine Kinase Inhibitors. 17. Irreversible Inhibitors of the Epidermal Growth Factor Receptor:  4-(Phenylamino)quinazoline- and 4-(Phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides Bearing Additional Solubilizing Functions

Abstract

4-Anilinoquinazoline- and 4-anilinopyrido[3,2-d]pyrimidine-6-acrylamides substituted with solubilizing 7-alkylamine or 7-alkoxyamine side chains were prepared by reaction of the corresponding 6-amines with acrylic acid or acrylic acid anhydrides. In the pyrido[3,2-d]pyrimidine series, the intermediate 6-amino-7-alkylamines were prepared from 7-bromo-6-fluoropyrido[3,2-d]pyrimidine via Stille coupling with the appropriate stannane under palladium(0) catalysis. This proved a versatile method for the introduction of cationic solubilizing side chains. The compounds were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Quinazoline analogues with 7-alkoxyamine solubilizing groups were potent irreversible inhibitors of the isolated EGFR enzyme, with IC(50[app]) values from 2 to 4 nM, and potently inhibited both EGFR and erbB2 autophosphorylation in cells. 7-Alkylamino- and 7-alkoxyaminopyrido[3,2-d]pyrimidines were also irreversible inhibitors with equal or superior potency against the isolated enzyme but were less effective in the cellular autophosphorylation assays. Both quinazoline- and pyrido[3,2-d]pyrimidine-6-acrylamides bound at the ATP site alkylating cysteine 773, as shown by electrospray ionization mass spectrometry, and had similar rates of absorptive and secretory transport in Caco-2 cells. A comparison of two 7-propoxymorpholide analogues showed that the pyrido[3,2-d]pyrimidine-6-acrylamide had greater amide instability and higher acrylamide reactivity, being converted to glutathione adducts in cells more rapidly than the corresponding quinazoline. This difference may contribute to the observed lower cellular potency of the pyrido[3,2-d]pyrimidine-6-acrylamides. Selected compounds showed high in vivo activity against A431 xenografts on oral dosing, with the quinazolines being superior to the pyrido[3,2-d]pyrimidines. Overall, the quinazolines proved superior to previous analogues in terms of aqueous solubility, potency, and in vivo antitumor activity, and one example (CI 1033) has been selected for clinical evaluation.

Phosphoproteomic analysis of Her2/neu signaling and inhibition

“…In this study, we obtained quantitative measurements on 462 proteins in Her2-transfected cells and, by simultaneously comparing three conditions, measured the effect of a Her2-targeted TKI. PD168393 is a preclinical compound used in the design of CI-1033, a TKI that is currently in clinical trials (30); therefore, this approach can be applied to drugs that are in clinical use or development to understand their effects on cellular networks. The identified phosphoproteins included many known Her2 and EGFR signaling proteins, as well as multiple previously unidentified Her2 signaling proteins, which should significantly advance the understanding of Her2.…”

The Development of HKI‐272 and Related Compounds for the Treatment of Cancer

“…Some representative compounds are shown in Table 1 along with some compounds lacking a water-solubilizing moiety for comparison. The ParkeDavis group took a similar approach, but in their case, they decided to reserve the 6-position for the Michael acceptor, usually an acrylamide, and to incorporate the water-solubilizing substituent on the 7-position of the quinazoline [30]. Ultimately, this approach led to their clinical candidate 8 (CI-1033) [31,32].…”

A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin

“…Moreover, EGFR was constitutively phosphorylated on tyrosine residues as shown by reciprocal EGFR and phosphotyrosine immunoprecipitations followed by immunoblotting (Figure 6a, top panels). In order to determine whether H55a#1 cells were strictly dependent on the EGFR pathway, we performed growth assays in the presence of CI-1033 (formerly PD183805), a small molecule kinase inhibitor speci®c for ErbB family receptors and with no activity against other tyrosine kinases (Fry et al, 1994(Fry et al, , 1998Smaill et al, 2000). We have previously shown that this inhibitor blocks the growth of SUM-149PT cells and two breast cancer cell lines with ErbB-2 ampli®cations, but had no e ect on cells which do not express activated ErbB kinases (Rao et al, 2000).…”