Design, synthesis, and X-ray crystal structures of 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors.

To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structures (2.0 and 1.4A, respectively) with mouse DHFR and NADPH as well as 1b with human DHFR (1.5A) were determined. The E- and Z-isomers adopt different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2-13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-diamino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8-C9 unsaturated compounds 2-7 and catalytic reduction gave the saturated 8-13. Homologation of the C9-methyl analog maintains DHFR inhibitory activity. In addition, inhibition of EGFR and PDGFR-beta were discovered for saturated C9-homologated analogs 9 and 10 that were absent in the saturated C9-methyl analogs.

Design, synthesis and biological evaluation of substituted pyrrolo[2,3-d]pyrimidines as multiple receptor tyrosine kinase inhibitors and antiangiogenic agents.

Direct and indirect involvement of receptor tyrosine kinases (RTKs) in tumor growth and metastasis makes them ideal targets for anticancer therapy. A paradigm shift from inhibition of single RTK to inhibition of multiple RTKs has been recently demonstrated. We designed and synthesized eight N(4)-phenylsubstituted-6-(2-phenylethylsubstituted)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as homologated series of our previously published RTK inhibitors. We reasoned that increased flexibility of the side chain, which determines potency and selectivity, would improve the spectrum of RTK inhibition. These compounds were synthesized using a bis-electrophilic cyclization to afford substituted pyrrolo[2,3-d]pyrimidines followed by chlorination and substitution at the 4-position with various anilines. Five additional compounds of this series were previously reported by Gangjee et al.(1) with activities against IGFR only. Their synthesis, characterization and biological activities against a variety of other RTKs are reported in this study for the first time. The biological evaluation, in whole cell assays, showed several analogs had remarkable inhibitory activity against epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet-derived growth factor receptor-beta (PDGFR-beta), the growth of A431 cells in culture, and in the chicken embryo chorioallantoic membrane (CAM) angiogenesis assay. The inhibitory data against the RTKs in this study demonstrate that variation of the 6-ethylaryl substituents as well as the N(4)-phenyl substituents of these analogs does indeed control both the potency and specificity of inhibitory activity against RTKs. In addition, homologation of the chain length of the 6-substituent from a methylene to an ethyl increases the spectrum of RTK inhibition. New multi-RTK inhibitors (8, 12) and potent inhibitors of angiogenesis (15, 19) were identified with the best compound, N(4)-(3-trifluromethylphenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (15), with an IC(50) value of 30nM in the CAM angiogenesis inhibition assay.

Laboratory diet profoundly alters gene expression and confounds genomic analysis in mouse liver and lung.

Nutritional studies in laboratory animals have long shown that various dietary components can contribute to altered gene expression and metabolism, but diet alone has not been considered in whole animal genomic studies. In this study, global gene expression changes in mice fed either a non-purified chow or a purified diet were investigated and background metal levels in the two diets were measured by ICP-MS. C57BL/6J mice were raised for 5 weeks on either the cereal-based, non-purified LRD-5001 diet or the purified, casein-based AIN-76A diet, as part of a larger study examining the effects of low dose arsenic (As) in the diet or drinking water. Affymetrix Mouse Whole Genome 430 2.0 microarrays were used to assess gene expression changes in the liver and lung. Microarray analysis revealed that animals fed the LRD-5001 diet displayed a significantly higher hepatic expression of Phase I and II metabolism genes as well as other metabolic genes. The LRD-5001 diet masked the As-induced gene expression changes that were clearly seen in the animals fed the AIN-76A diet when each dietary group was exposed to 100 ppb As in drinking water. Trace metal analysis revealed that the LRD-5001 diet contained a mixture of inorganic and organic As at a total concentration of 390 ppb, while the AIN-76A diet contained approximately 20 ppb. These findings indicate that the use of non-purified diets may profoundly alter observable patterns of change induced by arsenic and, likely, by other experimental treatments, particularly, altering gene and protein expression.

Exposure to arsenic at levels found inU.S. drinking water modifies expression in the mouse lung.

The mechanisms of action of drinking water arsenic in the lung and the threshold for biologic effects remain controversial. Our study utilizes Affymetrix 22,690 transcript oligonucleotide microarrays to assess the long-term effects of increasing doses of drinking water arsenic on expression levels in the mouse lung. Mice were exposed at levels commonly found in contaminated drinking water wells in the United States (0, 0.1, 1 ppb), as well as the 50 ppb former maximum contaminant level, for 5 weeks. The expression profiles revealed modification of a number of important signaling pathways, many with corroborating evidence of arsenic responsiveness. We observed statistically significant expression changes for transcripts involved in angiogenesis, lipid metabolism, oxygen transport, apoptosis, cell cycle, and immune response. Validation by reverse transcription-PCR and immunoblot assays confirmed expression changes for a subset of transcripts. These data identify arsenic-modified signaling pathways that will help guide investigations into mechanisms of arsenic's health effects and clarify the threshold for biologic effects and potential disease risk.

A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization.

BACKGROUND: Endothelial dysfunction (ED) is functionally characterized by decreased vasorelaxation, increased thrombosis, increased inflammation, and altered angiogenic potential, has been intimately associated with the progression and severity of cardiovascular disease. Patients with compromised cardiac function oftentimes have a state of chronic mild decreased oxygen at the level of the vasculature and organs, which has been shown to exacerbate ED. Hypoxia inducible factor (HIF) is a transcription factor complex shown to be the master regulator of the cellular response to decreased oxygen levels and many HIF target genes have been shown to be associated with ED. METHODS: Human endothelial and aortic smooth muscle cells were exposed either to A) normoxia (21% O2) for three weeks, or to B) mild decreased oxygen (15% O2) for three weeks to mimic blood oxygen levels in patients with heart failure, or to C) mild decreased oxygen for two weeks followed by one week of normoxia ("memory" treatment). Levels of HIF signaling genes (HIF-1alpha, HIF-2alpha, VEGF, BNIP3, GLUT-1, PAI-1 and iNOS) were measured both at the protein and mRNA levels. RESULTS: It was found that chronic exposure to mild decreased oxygen resulted in significantly increased HIF signaling. There was also a "memory" of HIF-1alpha and HIF target gene induction when oxygen levels were normalized for one week, and this "memory" could be interrupted by adding a small molecule HIF inhibitor to the last week of normalized oxygen. Finally, levels of ubiquitylated HIF-1alpha were reduced in response to chronic mild decreased oxygen and were not full restored after oxygen normalization. CONCLUSION: These data suggest that HIF signaling may be contributing to the pathogenesis of endothelial dysfunction and that normalization of oxygen levels may not be enough to reduce vascular stress.

Mutant p53 facilitates pro-angiogenic, hyperproliferative phenotype in response to chronic relative hypoxia.

There is much controversy in the literature regarding the role of p53 status response on hypoxia inducible factor (HIF) signaling in response to chronic relative hypoxia (CRH). The goal of this paper was to methodically examine this response in isogenically matched tumor cells. We report that p53-mutant (MUT) cells, versus p53-wild-type (WT) cells, showed decreased apoptosis, increased cell proliferation with higher basal HIF-1alpha levels in response to CRH. In addition, we found increased HIF-mediated transactivation and increased VEGF release with decreased HIF-1alpha/p53 and HIF-1alpha/MDM-2 partnering in p53-MUT versus p53-WT cells in response to CRH.

Assessment of angiogenic properties of biomaterials using the chicken embryo chorioallantoic membrane assay.

The angiogenic potential of a biomaterial is a critical factor for successful graft intake in tissue engineering. We developed a modified, rapid and reproducible chicken embryo chorioallantoic membrane (CAM) assay to evaluate the ability of biomaterials in inducing blood vessel density. Five biomaterials including one-layer porcine small intestinal submucosa (SIS), two-layer SIS, four-layer vacuum pressed (VP) SIS, polyglycolic acid (PGA) and PGA modified with poly(lactic-co-glycolic acid) (PLGA) were analyzed. A circular section (1.2 mm diameter) of each biomaterial was placed near a group of blood vessels in the CAM. Blood vessels around the biomaterials were captured with black and white images at 96 h post implantation; and the images were subjected to densitometry evaluation. One-layer SIS induced a significant increase in blood vessel density as compared to the cellulose nitrate negative control, and had the greatest increase in blood vessel density as compared to four-layer VP SIS, PGA, or PLGA modified PGA. Although two-layer SIS has enhanced physical structure for surgical manipulation, its induction in blood vessel density was significantly lower than the one-layer SIS. Stripping the SIS proteins or incubating one-layer SIS with neutralizing antibodies against basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) resulted in decreased angiogenesis. Consistent with results obtained from bladder augmentation animal models, these results confirmed that angiogenic growth factors were present in SIS and affected the angiogenic potential of biomaterials. These data also demonstrated that the CAM assay can be used to ascertain methodically the angiogenic potential of biomaterials.

Novel 5-substituted, 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors with antiangiogenic and antitumor activity.

Recent evidence suggests that combination therapy of cancer with receptor tyrosine kinase (RTK) inhibitors, which are usually cytostatic, with conventional chemotherapeutic agents, which are usually cytotoxic, provide an improved treatment option. We have designed, synthesized, and evaluated a series of novel 2,4-diamino-5-substituted furo[2,3-d]pyrimidines with RTK and dihydrofolate reductase (DHFR) inhibitory activity in single molecules, as potential cytostatic and cytotoxic agents with antitumor activity. These compounds were synthesized from 2,4-diamino-5-chloromethyl furo[2,3-d]pyrimidine and aryl methyl ketones using the Wittig reaction to afford the C-8-C-9 unsaturated analogs followed by catalytic reduction to the corresponding saturated compounds. The saturated and unsaturated C-8-C-9 bridged compounds were evaluated as inhibitors of vascular endothelial growth factor receptor (VEGFR-2, Flk, KDR), epidermal growth factor receptor, and platelet-derived growth factor receptor-beta (PDGFR-beta). Selected analogs were also evaluated as antiangiogenic agents in the chicken embryo chorioallantoic membrane (CAM) assay. The compounds were also evaluated as inhibitors of human (h) DHFR and Toxoplasma gondii (tg) DHFR. In each evaluation, a known standard compound was used as a comparison. Of the compounds evaluated, compound 32 was as potent as the standard compounds against VEGFR-2 and PDGFR-beta, showing dual inhibitory activity against RTK. This analog was also highly effective in the CAM assay. A second analog 18 also demonstrated dual VEGFR-2 and PDGFR-beta inhibitory activity as well as potent antiangiogenic activity in the CAM assay. Four additional analogs were also effective against PDGFR-beta and in the CAM assay. An unsaturated C-8-C-9 moiety was necessary for RTK inhibitory activity. Compound 32 also showed inhibitory activity against hDHFR and tgDHFR, illustrating the multitarget inhibitory potential of these analogs. The biological activity of these analogs also suggests the necessity of an unsaturated C-8-C-9 bridge for dual RTK and DHFR inhibitory activity. Compounds 18 and 32 were also evaluated in a B16 melanoma mouse model and were found to be more active as antitumor agents than methotrexate. In addition, both 18 and 32 were also active in decreasing lung metastases in a mouse model of B16 melanomas.

Role of HIF signaling on tumorigenesis in response to chronic low-dose arsenic administration.

Trivalent inorganic arsenic (arsenite, arsenic trioxide, As(III)) is a primary contaminant of groundwater supplies worldwide. As(III), marketed as trisenox, is also an FDA-approved agent to treat cancer It has been previously shown by our laboratory that As(III) administered at doses lower than a therapeutic anticancer dose results in an increase in tumor formation and blood vessel density of tumors. In this work it was found that chronic administration of As(III) approaching the EPA action level of 10 ppb, given in the drinking water of mice 5 weeks prior to B16-F10 melanoma implantation, increased the growth rate of primary tumors and the number of metastases to the lung. Further, levels of arsenic in the tumor and lung were found to be much greater than those in the blood and similar to pro-angiogenic As(III) doses. Levels of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) surrounding the blood vessels in the tumors of the As(III)-treated mice were also found to be increased. Exposure of isolated B16-F10 tumor cells to chronic (3 or 7 day) but not acute (4 h) low-dose As(III) was found to increase HIF-1alpha expression and secretion of VEGF. Finally, coadministration of an inhibitor of HIF (YC-1) or a VEGFR-2 kinase inhibitor (SU5416) was found to antagonize the pro-angiogenic effects of low-dose As(III). Together, these results suggest that chronic exposure to low-dose As(III) could stimulate growth of tumors through a HIF-dependent stimulation of angiogenesis.