Discovery of Antitubulin Agents with Antiangiogenic Activity as Single Entities with Multitarget Chemotherapy Potential

Abstract

Antiangiogenic agents (AA) are cytostatic, and their utility in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of vascular endothelial growth factor receptor-2 (VEGFR2) inhibitors with antitubulin agents have been particularly successful. We have discovered a novel, potentially important analogue, that combines potent VEGFR2 inhibitory activity (comparable to that of sunitinib) with potent antitubulin activity (comparable to that of combretastatin A-4 (CA)) in a single molecule, with GI₅₀ values of 10^–7 M across the entire NCI 60 tumor cell panel. It potently inhibited tubulin assembly and circumvented the most clinically relevant tumor resistance mechanisms (P-glycoprotein and β-III tubulin expression) to antimicrotubule agents. The compound is freely water-soluble as its HCl salt and afforded excellent antitumor activity in vivo, superior to docetaxel, sunitinib, or Temozolomide, without any toxicity.

Antiangiogenic agents (AA) have defined a new paradigm for cancer treatment.¹ The principal mediator of angiogenesis is the vascular endothelial growth factor (VEGF) and its receptor tyrosine kinase (RTK) VEGFR2.² Rapid regrowth of vasculature in tumors after the removal of VEGFR2 inhibitors from the treatment regimen attests to their cytostatic mechanism.³ VEGFR2 inhibitors are known to cause regression of impaired tumor blood vessels but transiently normalize the surviving vessels, which have less leakiness and are more like normal blood vessels. Such treatment therefore can transiently increase blood flow in the surviving vasculature.⁴ Administering a cytotoxic agent during this transient tumor vasculature normalization provides improved drug delivery to the tumor, dependent on the surviving vasculature. However, administering separate antiangiogenic and cytotoxic agents may miss the timing window. Single entities with both antiangiogenic and cytotoxic components should allow the cytotoxicity to be manifested even at low concentrations in the tumor as soon as the antiangiogenic effect and vasculature normalization occur. Hence, these agents might not need the cytotoxic component to be as potent as conventional cytotoxic drugs and as such could perhaps avoid or reduce dose-limiting toxicities associated with conventional chemotherapy. Additionally, such single agents could circumvent pharmacokinetic problems of multiple agents, avoid drug–drug interactions, and be used at lower doses to minimize toxicities and tumor cell resistance.

It has been our long-standing interest to design single entities with multiple targets.⁵⁻⁸ We chose inhibition of tubulin as the mechanism for the cytotoxic effect because antitubulin agents are some of the most effective drugs in cancer chemotherapy.⁹ Additionally, evidence for the remarkable clinical success of VEGFR2 inhibitors in combination with antitubulin agents is abundant in the literature.¹⁰ Structural similarities of antitubulin¹¹ agents and RTK inhibitors¹² previously reported from our laboratory provided the rationale to incorporate structural features of both antitubulin activity and RTK inhibitory activity in single molecules to explore the possibility of providing dual inhibitory activities in single compounds. We¹¹ previously reported the design, synthesis, and antitumor activity of pyrrolo[3,2-d]pyrimidine (1; Figure 1), which inhibits tubulin assembly. Substituted 7-benzyl pyrrolo[2,3-d]pyrimidines with general structure 2 (Figure 1) were reported as antiangiogenic, antimetastatic, and antitumor agents.⁹ Substituents on the benzyl group and the location of the benzyl group dictate RTK inhibitory activity in pyrrolo[2,3-d]pyrimidines. Hence, having determined the antitubulin effects of 1, it was of interest to engineer RTK inhibitory activity without loss of antitubulin activity by incorporating the 7-benzyl group onto the pyrrolo[3,2-d]pyrimidine scaffold. The results of this hybrid design afforded compound 3 (Figure 1). Compounds 4 and 5 were designed to evaluate the importance of the 4′-OCH₃ and the 4-NCH₃ moieties, respectively. Compound 6 was designed by incorporating a 2′-OCH₃ group to explore the effect of substituents at this position. The antimitotic activity of 3 also prompted its evaluation against cell lines which overexpress the multidrug resistance protein P-glycoprotein (Pgp) and the β-tubulin isoform βIII. Tumors with either of these proteins are associated with significant resistance to antitubulin agents, especially with resistance to paclitaxel.¹³

Figure 1.

Hybrid design from lead compounds.

Glossary

Abbreviations

AA

antiangiogenic agents

VEGFR2

vascular endothelial growth factor receptor-2

RTK

receptor tyrosine kinase

Pgp

P-glycoprotein

CAM

chicken chorioallantoic membrane

CA4P

combretastatin A-4 phosphate

BLBCs

basal-like breast cancer cells

Supporting Information Available

Tumor cell inhibitory activity (NCI) GI₅₀ (nM) of 3·HCl, and the Experimental Section. This material is available free of charge via the Internet at http://pubs.acs.org.

This work was supported, in part, by NIH Grants CA136944 (to A.G.), CA114021 (to A.G.), the Duquesne University Adrian Van Kaam Chair in Scholarly Excellence (to A.G.), and NSF equipment grant CHE 0614785 (for the NMR).

The authors declare no competing financial interest.

Funding Statement

National Institutes of Health, United States