Synthesis and structure–activity relationship of 6-arylureido-3-pyrrol-2-ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors

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Abstract

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure–activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.

Graphical abstract

A series of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives were synthesized and identified as potent inhibitors of the vascular endothelial growth factor receptor and platelet-derived growth factor receptor families of receptor tyrosine kinases.

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Introduction

Angiogenesis refers to the formation of new blood vessels from the endothelium of preexisting vasculature.1 Pathological angiogenesis has been implicated in a wide array of vascular hyperproliferative disorders, most notably cancer,1 rheumatoid arthritis,2 diabetic retinopathy,3 endometriosis,4 age-related macular degeneration,5 and ocular neovascularization.6 Solid tumors, in particular, are dependant on angiogenesis for growth beyond a certain critical size via the induction of new capillaries sprouting from existing blood vessels, thereby securing a supply of nutrients and oxygen and enabling waste removal.1, 7 In addition, angiogenesis also promotes metastasis.8

Receptor tyrosine kinases (RTKs) represent a large family of membrane-bound enzymes that play key roles in tumor growth, survival, and metastasis. RTK activity is tightly regulated in normal cells, but aberrant RTK activation, in particular that of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR), has been linked to the development and progression of variety of human cancers.9 The VEGFR family comprises FLT1 (VEGFR1), KDR (VEGFR2), and FLT4 (VEGFR3), which play pivotal roles in angiogenesis and contribute to tumor progression through their ability to mediate tumor angiogenesis and lymphangiogenesis, and to enhance vascular permeability.10 The PDGFR family comprises PDGFRα, PDGFRβ, colony stimulating factor 1 receptor (CSF1R), c-KIT, and FLT3, which promote tumor cell growth and metastasis through modification of the tumor microenvironment.11 Additionally, mutants of FLT3 and c-KIT are directly associated with proliferation of acute myeloid leukemia blast cells12 and gastrointestinal stromal tumor cells,13 respectively.

Inhibition of RTK pathways has become an important approach for the discovery of new anticancer drugs.14 The approval of bevacizumab,15 an antibody against VEGF, by the United States Food and Drug Administration (FDA) for the treatment of first-line metastatic colorectal cancer in combination with chemotherapy,16 has promoted greater interest in this field. Several selective KDR inhibitors, including SU5416,17 PTK787,18 CP-547632,19 SU6668,20 and ZD6474,21 are in late-stage clinical trials. Although VEGFR and PDGFR are compelling cancer targets individually, tumors are capable of secreting multiple angiogenic factors, and they depend on these factors at different stages of progression.22 As a result of the complex and redundant cellular signaling network associated with RTKs, broad-acting and multitargeted RTK inhibitors may be more advantageous than selective agents, owing to their ability to block multiple signaling pathways associated with tumor survival.23 This is evidenced by the FDA’s recent approval of multitargeted kinase inhibitors such as imatinib [inhibits Abelson tyrosine kinase; (Abl), c-KIT protein (CD117), and PDGFR; approved for chronic myelogenous leukemia,24 gastrointestinal stromal tumors, and a number of other malignancies], sunitinib [inhibits KDR, PDGFR2, PDGFRβ, c-KIT and FLT3; approved for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors],25 sorafenib [inhibits Raf kinase, KDR, PDGFRβ, and c-KIT; approved for the treatment of advanced renal cell carcinoma],26 and lapatinib [inhibits EGFR, and ErbB-2; approved for advanced metastatic breast cancer in conjugation with chemotherapy].27 These agents demonstrate clinical benefits with manageable side effects.

The activity of inhibitors having a urea moiety,28 especially the diphenyl urea motif,29 against kinases such as VEGFR, PDGFR, Tie-2, Raf, and p38 has been described. These candidates, which include sorafenib,26 PD-173074,28(b), 28(c) CP-547632,19 KRN633,28c and ABT-86929a, either have been approved by the FDA or are in late-stage clinical trials (Fig. 1). Similarly, there are many reports on oxindole derivatives as potential RTK inhibitors;30 these include sunitinib,25, 30(a) SU5416,17, 30(b) and SU666820, 30(c) (Fig. 1). However, structure–activity relationship (SAR) studies on ureido-substituted oxindoles against VEGFR and PDGFR have been less explored.31 As part of our efforts toward the discovery and biological evaluation of new anticancer agents,32 we report here the synthesis and SAR studies of a series of 6-ureido/thioureido-substituted indolin-2-ones as potent multitargeted RTK inhibitors. This study has led to the identification of potential kinase inhibitors for members of both the VEGFR and PDGFR families.

Section snippets

Chemistry

The 5-, 6-, and 7-arylureido-substituted 3-arylmethylidene-2-oxindole derivatives 1354 were prepared according to a general method (Scheme 1). 5-Amino-2-oxindole (6) is commercially available, whereas a direct reduction of 7-nitro-2-oxindole led to 7-amino-2-oxindole (7) in high yield. Hydrogenation followed by cyclization of 2,4-dinitrophenyl acetic acid (8) afforded 6-amino-2-oxindole (9) in moderate yield. Reaction of compounds 6, 7, and 9 with aryl isocyanates afforded the corresponding

In vitro kinase activity

All prepared compounds were screened for inhibitory activity against a panel of RTKs that included c-KIT, FLT3, KDR and VEGFR3. We first estimated the percent inhibition of each kinase in the presence of 0.1 μM of each compound (Table 1). Derivatives 13 and 14, each containing a 5-arylureido substituent in conjunction with a 3-phenylmethylidene group on the indolin-2-one ring, exhibited poor inhibition of all kinases tested. Replacement of the phenylmethylidene group with pyrrol-2-ylmethylidene

Conclusion

We developed a new series of 6-arylureido-3-pyrrol-2-yl-indolin-2-one derivatives as multitargeted RTK inhibitors that potently inhibit members of both the VEGFR and PDGFR families, each of which plays a major role in angiogenesis. SAR studies of these compounds revealed that the arylureido moiety at the 6-position plays a key role in kinase inhibition. Incorporation of a pyrrolylmethylidene moiety at the 3-position generated a series of compounds with potent kinase inhibitory activity and

Gemera; experimental procedures

Melting points were measured using open capillaries on an electrothermal apparatus and were uncorrected. NMR spectra were recorded on Bruker AMX series spectrometers at 298 K. Mass spectra were recorded on a Finnigan MAT TSQ-7000 mass spectrometer. Elemental analyses for C, H, N and S were carried out on a Heraeus VariaEL-III elemental analyzer. The thin layer chromatographic analyses were performed using precoated silica gel plates (60 F254, Merck), and the spots were examined under UV light.

Acknowledgment

This study was supported by the research grant of Ministry of Economic Affairs, project number 98-EC-17-A-02-04-0624.

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