Tyrosine kinase inhibitors: from rational design to clinical trials.

Protein kinases play a crucial role in signal transduction as well as in cellular proliferation, differentiation, and various regulatory mechanisms. The inhibition of growth related kinases, especially tyrosine kinases, might provide new therapies for diseases such as cancer. The progress made in the crystallization of protein kinases has confirmed that the ATP-binding domain of tyrosine kinases is an attractive target for drug design. Three successful examples of drug design at Novartis using a tyrosine kinase as a molecular target are described. PKI166, a pyrrolo[2,3,-d]pyrimidine derivative, is a dual inhibitor of both the EGFR and the ErbB2 kinases. The compound entered clinical trials in 1999, based on its favorable preclinical profile: potent inhibition of EGF-mediated signalling in cells, in vivo antitumor activity in several EGFR overexpressing xenograft tumor models in nude mice, long-lasting inhibition of EGF-stimulated EGFR autophosphorylation in tumor tissue, good oral bioavailability in animals, and no prohibitive in vitro and in vivo toxicity findings. The anilino-phthalazine derivative PTK787/ZK222584 (Phase I, co-developed by Schering AG, Berlin) is a potent and selective inhibitor of both the KDR and Flt-1 kinases with interesting anti-angiogenic and pharmacokinetic properties (orally bioavailable). STI571 (Glivec, Gleevec), a phenylamino-pyrimidine derivative, is a potent inhibitor of the Abl tyrosine kinase, which is present in 95% of patients with chronic myelogenous leukemia (CML). The compound specifically inhibits proliferation of v-Abl and Bcr-Abl expressing cells (including cells from CML patients) and shows anti-tumor activity as a single agent in animal models at well-tolerated doses. Pharmacologically relevant concentrations are achieved in the plasma of animals (oral administration). Promising data from phase I and II clinical trials in CML patients (98% haematological response rate in Phase I) support the fact that the STI571 represents a new treatment modality for CML. In addition, potent inhibition of the PDGFR and c-Kit tyrosine kinases also indicates its possible clinical use in solid tumors.

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.

Molecular approaches to receptors as targets for drug discovery.

Many receptors have been selected as viable drug discovery targets. One particular class of receptors that have received much interest and so far relatively good success are the receptor protein tyrosine kinases (RPTKs). Typically, RPTKs are activated following the binding of the peptide growth factor ligand to its receptor. The RPTKs play crucial roles in signal transduction pathways that regulate a number of cellular functions, such as cell differentiation and proliferation, both under normal physiological conditions as well as in a variety of pathological disorders. A variety of different tumour types have been shown to have dysfunctional RPTKs, either as a result of excess production of the growth factor, the receptor or both, or via mutations in the RPTKs structure. Irrespective of the cause, this leads to the over-activity of the particular RPTK system and in turn to the aberrant and inappropriate cellular signalling within the tumour cell. RPTKs are attractive targets in the search for therapeutic agents, not only against cancers but also against many other disease indications. Although an ever-increasing number of RPTKs have been selected as viable molecular targets for drug discovery programmes, four examples will be covered in this article. These are the epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R), fibroblast growth factor receptor (FGR-R) and vascular endothelial growth factor receptor (VEGF-R), with the main emphasis of interest being on their role in oncology.

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma.

We determined whether down-regulation of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of a novel EGF-R tyrosine kinase inhibitor (PKI166) alone or in combination with gemcitabine (administered i.p.) can inhibit growth and metastasis of human pancreatic carcinoma cells implanted into the pancreas of nude mice. Therapy beginning 7 days after orthotopic injection of L3.6pl human pancreatic cancer cells reduced the volume of pancreatic tumors by 59% in mice treated with gemcitabine only, by 45% in those treated with PKI166 only, and by 85% in those given both drugs. The combination therapy also significantly inhibited lymph node and liver metastasis, which led to a significant increase in overall survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with significant reduction in tumor cell production of VEGF and IL-8, which in turn correlated with a significant decrease in microvessel density and an increase in apoptotic endothelial cells. Collectively, our results demonstrate that oral administration of an EGF-R tyrosine kinase inhibitor decreased growth and metastasis of human pancreatic cancer growing orthotopically in nude mice and increased survival. The therapeutic effects were mediated in part by inhibition of tumor-induced angiogenesis attributable to a decrease in production of proangiogenic molecules by tumor cells and increased apoptosis of tumor-associated endothelial cells.

ATP site-directed competitive and irreversible inhibitors of protein kinases.

Several tyrosine and serine/threonine protein kinases have emerged in the last few years as attractive targets in the search for new therapeutic agents being applicable in many different disease indications. Initially, inhibition of these protein kinases by ATP site-directed inhibitors was considered less prone to success, but medicinal chemists from both academia and industry have been able to impart potency and selectivity to a limited number of scaffolds by modulating and fine-tuning the interactions of the modified template with the ATP binding site of the selected kinase. The chemical templates that have been used in the synthesis of ATP site-directed protein kinase inhibitors are reviewed with emphasis on the kinase inhibitors that have entered or are about to enter clinical trials. Examples have been selected to illustrate how structure-based design approaches and new methods to increase compound diversity have had an impact on this area of research.

Strategies toward the design of novel and selective protein tyrosine kinase inhibitors.

Protein tyrosine kinases play a fundamental role in signal transduction pathways. Deregulated tyrosine kinase activity has been observed in many proliferative diseases (e.g., cancer, psoriasis, restenosis, etc.). Tyrosine kinases are, therefore, attractive targets for the design of new therapeutic agents against cancer. We have built up a pharmacophore model of the ATP-binding site of the epidermal growth factor receptor (EGFR) kinase and used it for the rational design of kinase inhibitors. Several examples of the successful use of this model are presented in this review. Amongst these, 4-substituted-pyrrolo[2,3-d]pyrimidines, a new class of highly potent and selective inhibitors of the EGFR kinase, have been identified and further optimized. The most active derivatives inhibited the EGFR tyrosine kinase with IC50 values between 1 and 5 nM. In EGF-dependent cellular systems, tyrosine phosphorylation, as well as c-fos mRNA expression, was inhibited with similar IC50 values. Further successful application of this pharmacophore model led to the identification and optimization of phenylamino-pyrazolo[4,3-d]pyrimidines and substituted isoflavones and quinolones, other classes of potent, selective, and ATP competitive EGFR kinase inhibitors with IC50 values in the low nanomolar range. Structure-activity relationships of both classes are discussed.

Use of a pharmacophore model for the design of EGFR tyrosine kinase inhibitors: isoflavones and 3-phenyl-4(1H)-quinolones.

Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGFR protein tyrosine kinase together with published X-ray crystal data of quercetin (2) in complex with the Hck tyrosine kinase and of deschloroflavopiridol (3b) in complex with CDK2, a putative binding mode of the isoflavone genistein (1) was proposed. Then, based on literature data suggesting that a salicylic acid function, which is represented by the 5-hydroxy-4-keto motif in 1, could serve as a pharmacophore replacement of a pyrimidine ring, superposition of 1 onto the potent EGFR tyrosine kinase inhibitor 4-(3'-chlorophenylamino)-6, 7-dimethoxyquinazoline (4) led to 3'-chloro-5,7-dihydroxyisoflavone (6) as a target structure which in fact was 10 times more potent than 1. The putative binding mode of 6 suggests a sulfur-aromatic interaction of the m-chlorophenyl moiety with Cys 773 in the "sugar pocket" of the EGFR kinase model. Replacement of the oxygen in the chromenone ring of 6 by a nitrogen atom further improved the inhibitory activity against the EGFR kinase. With IC50 values of 38 and 8 nM, respectively, the quinolones 11 and 12 were the most potent compounds of the series. N-Alkylation of 11 did not further improve enzyme inhibitory activity but led to derivatives with cellular activity in the lower micromolar range.