Tumor Growth In Xenograft Model Research Articles

Targeting Vacuolar H+-ATPases as a New Strategy against Cancer

Growing evidence suggests a key role of tumor acidic microenvironment in cancer development, progression, and metastasis. As a consequence, the need for compounds that specifically target the mechanism(s) responsible for the low pH of tumors is increasing. Among the key regulators of the tumor acidic microenvironment, vacuolar H(+)-ATPases (V-ATPases) play an important role. These proteins cover a number of functions in a variety of normal as well as tumor cells, in which they pump ions across the membranes. We discuss here some recent results showing that a molecular inhibition of V-ATPases by small interfering RNA in vivo as well as a pharmacologic inhibition through proton pump inhibitors led to tumor cytotoxicity and marked inhibition of human tumor growth in xenograft models. These results propose V-ATPases as a key target for new strategies in cancer treatment.

Peroxisome Proliferator-Activated Receptor-γ in Lung Cancer: Defining Specific Versus “Off-Target― Effectors

A large number of studies have indicated that thiazolidinediones (TZDs) such as rosiglitazone and pioglitazone inhibit tumor growth, progression, and metastasis. These agents are specific agonists for the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) but also engage other pathways. In lung cancer, these agents have been shown to induce apoptosis and inhibit tumor growth in xenograft models. Retrospective studies have indicated a significant decrease in lung cancer risk in patients using these agents, suggesting that TZDs may be chemopreventive for lung cancer. However, emerging data suggest that chronic use of these agents is associated with increased risk of adverse cardiovascular events. It is therefore critical to determine the relative contributions of PPARγ-dependent versus PPARγ-independent pathways in mediating both the anti-tumorigenic effects and the cardiovascular effects of TZDs. This review examines these pathways with a specific focus on the role of TZDs and PPARγ in lung cancer.

Targeting endothelial and tumor cells with semaphorins

Neuropilins (NRP) are receptors for the class 3 semaphorin (SEMA3) family of axon guidance molecules and the vascular endothelial growth factor (VEGF) family of angiogenesis factors. Although the seminal studies on SEMA3s and NRPs first showed them to be mediators of axon guidance, it has become very apparent that these proteins play an important role in vascular and tumor biology as well. Neuronal guidance and angiogenesis are regulated similarly at the molecular level. For example, SEMA3s not only repel neurons and collapse axon growth cones, but have similar effects on endothelial cells and tumor cells. Preclinical studies indicate that SEMA3F is a potent inhibitor of tumor angiogenesis and metastasis. In addition, neutralizing antibodies to NRP1 enhance the effects of anti-VEGF antibodies in suppressing tumor growth in xenograft models. This article reviews NRP and SEMA3 structural interactions and their role in developmental angiogenesis, tumor angiogenesis and metastasis based on cell culture, zebrafish and murine studies.

384 POSTER Targeting integrin alpha5beta1 on multiple tumour-associated cell types inhibits tumour growth in xenograft models

384 POSTER Targeting integrin alpha5beta1 on multiple tumour-associated cell types inhibits tumour growth in xenograft models

Novel phase II study design of MORAb-003, a monoclonal antibody against folate receptor alpha in platinum-sensitive ovarian cancer in first relapse

5583 Background: Folate receptor alpha (FRA) is over-expressed in the majority of epithelial ovarian cancers (EOC) but largely absent from normal tissue. MORAb-003 (M3) is a humanized monoclonal antibody to FRA. Binding of M3 to FRA blocks phosphorylation by Lyn kinase; mediates FRA-positive tumor cell killing; and suppresses tumor growth in xenograft models. High-dose M3 is non-toxic in monkeys. A phase 1 study showed no significant AEs and suggestions of efficacy in platinum-resistant EOC. This phase 2 efficacy study will determine the efficacy of M3 in platinum-sensitive EOC in first relapse, either as a single agent (SA) in asymptomatic relapse, or in combination with platinum and taxane (P/T) in symptomatic relapse, and to maintain a second response. Methods: Subjects with EOC in first relapse are eligible. For CA125 elevation without symptoms (± measurable disease) the subject receives SA M3 until progression. For symptomatic relapse the subject receives P/T and M3. Subjects with CR or PR receive SA M3 maintenance therapy with the objective of prolonging the second remission longer than the first. Endpoints include CA125 and CT scans. ORR and PFS will be determined for each arm. Results: To date, 15 subjects have been treated, 8 in combination therapy and 7 on single-agent M3. No significant drug-related adverse events have been reported. All subjects treated with the combination of M3 and P/T for symptomatic relapse who have reached at least the first evaluation point have achieved a normalization of CA125 and reduction of tumor size (CR or PR) by CT scan using RECIST criteria, including 3 of 3 subjects with first remissions of 6 to 9 months. Early responses have been observed in the SA M3 treatment arm. Conclusions: This first phase 2 study will determine the efficacy of M3, either as SA or in combination with P/T chemotherapy to achieve a meaningful response rate in EOC in first relapse, and to maintain the second remission longer than the subject’s own first remission. These data will be used to define the most effective phase of disease in which to use M3, and to focus a pivotal study on the area of greatest efficacy. The early results continue to demonstrate activity of M3 in EOC. No significant financial relationships to disclose.

1,4-Dihydroindeno[1,2-c]pyrazoles with Acetylenic Side Chains as Novel and Potent Multitargeted Receptor Tyrosine Kinase Inhibitors with Low Affinity for the hERG Ion Channel

The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.

CNTO 859, a humanized anti‐tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models

Thromboembolic complications are frequently associated with advanced cancer. Interestingly, one of the major initiators of blood coagulation, tissue factor (TF), is reported to be overexpressed in several tumor types and can be found on both tumor cells and tumor vasculature. Although the exact mechanisms have yet to be elucidated, TF expressed on tumor cells can trigger intracellular signaling events through various pathways that can lead to tumor angiogenesis, proliferation, and metastasis. There exists preclinical evidence that disruption of TF dependent signaling can effectively inhibit tumor cell migration, metastasis, and angiogenesis. Here, we report for the first time that an antibody to tissue factor can also prevent tumor growth in vivo. Prophylactic administration of CNTO 859, a humanized anti-human TF antibody, was shown to inhibit experimental lung metastasis of MDA-MB-231 human breast carcinoma cells by over 99% compared to a control antibody. Furthermore, therapeutic doses of CNTO 859 were shown to reduce tumor incidence and growth of orthotopically implanted MDA-MB-231 cells.

Spotlight

Ray et al., pp. 13–23 Vanadium, a metallic element, is an essential component of some enzymes. Several organisms, including chicken and rat, are known to require small amounts of vanadium for growth and reproduction. In recent years, growing evidence has pointed to a protective role of vanadium in cancer development. A suppressive function of the micronutrient was uncovered for cancers of liver, lung, and the gastrointestinal tract. Ray and colleagues have now added breast cancer to this list. When vanadium in trace amounts was added to the drinking water of Sprague-Dawley rats, the incidence of chemically induced mammary adenocarcinomas was significantly reduced. While 90% of the control-treated rats developed palpable tumors after treatment with a carcinogen (DMBA), this only occurred in 50% of rats that were fed drinking water supplemented with vanadium. The authors further demonstrate that vanadium treatment reduces cell proliferation of the human MCF7 breast cancer cell line and is associated with the induction of p53-dependent apoptosis in rat mammary tissue. These results underline the potential benefit of vanadium added to vitamin and mineral preparations. Whether a vanadium supplement should be included in the therapeutic regimen of women with breast cancer should be investigated further. Incidence of palpable mammary tumors in rats treated with the DMBA carcinogen. One group (triangles) was fed drinking water supplemented with vanadium immediately after carcinogen treatment for 35 weeks, while the control group (circles) was fed regular drinking water. Rectangles indicate p values < 0.001, crosses mark p values < 0.05. Cantor et al., pp. 24–32 Several suspected tumor suppressors, including FHIT, WWOX, FRA3B and RASSF1A, are frequently silenced in lung cancer. Importantly, the expression of these genes is commonly suppressed at the level of epigenetic modifications, such as DNA methylation and histone deacetylation. These two epigenetic modifications are under the control of enzymes, DNA methylases and histone deacetylases that can be reversibly inhibited with small molecules. Reactivating the expression of such silenced tumor suppressor genes has shown therapeutic potential in hematopoietic malignancies, but no information is available on the feasibility of this approach for solid tumors, such as lung cancer. Cantor and colleagues report that conditional expression of the suspected tumor suppressors FHIT and WWOX in lung cancer cell lines using an inducible expression system is associated with inhibition of tumor growth in xenograft models. They also show that treatment of mice carrying xenograft H1299 tumors (a lung cancer cell line lacking expression of FHIT and WWOX) with an HDAC inhibitor (trichostatin A) and a DNA methylase inhibitor (5-aza-2-deoxycytidine) suppressed the growth of small tumors, but not large tumors. This inhibition of tumor growth was associated with a reactivation of expression of FHIT and WWOX and other tumor suppressor genes. While other mechanisms in addition to tumor suppressor reactivation could also play a role in the therapeutic effects of these agents, in particular induction of apoptosis via cell cycle abnormalities, these studies demonstrate the potential usefulness of these small molecule combinations for the treatment of solid tumors. Khatami et al., pp. 171–175 The number of men diagnosed with early-stage prostate cancer has dramatically increased due to a simple blood test that measures circulating prostate-specific antigen (PSA). Because of the slow growth of prostate tumors and because of the significant complications associated with treatment, men diagnosed with early-stage prostate cancer often choose to postpone active treatment. However, there are no reliable markers that can identify those patients in whom surveillance would be suitable. It is generally accepted that an average surveillance of 2 years does not reduce the chance of cure by radical prostatectomy. The study by Khatami and colleagues questions whether this is always an appropriate approach. In Sweden 10,000 men were invited to regular PSA screening tests. More than 600 were diagnosed with prostate cancer during a 10-year period, and less than half of them chose surveillance as an initial treatment option. The authors studied the risk of PSA recurrence in those patients who received radical prostatectomy after initial surveillance. They discovered that the risk of relapse was significantly associated with the PSA doubling time (PSADT) before operation. Men with a short PSADT (< 4 years) had a high risk of relapse while men with a longer PSADT were completely protected. The authors conclude that men with early-stage prostate carcinoma and a PSADT of > 4 years have an excellent chance of cure by prostatectomy after initial surveillance. However, other patients have a greater risk of disease progression and should be informed about this risk in discussions of treatment options.

Role of the ABCG2 drug transporter in the resistance and oral bioavailability of a potent cyclin-dependent kinase/Aurora kinase inhibitor

Cell cycle kinase inhibitors have advanced into clinical trials in oncology. One such molecule, JNJ-7706621, is a broad-spectrum inhibitor of the cyclin-dependent kinases and Aurora kinases that mediate G(2)-M arrest and inhibits tumor growth in xenograft models. To determine the putative mechanisms of resistance to JNJ-7706621 that might be encountered in the clinic, the human epithelial cervical carcinoma cell line (HeLa) was exposed to incrementally increasing concentrations of JNJ-7706621. The resulting resistant cell population, designated HeLa-6621, was 16-fold resistant to JNJ-7706621, cross-resistant to mitoxantrone (15-fold) and topotecan (6-fold), and exhibited reduced intracellular drug accumulation of JNJ-7706621. ABCG2 was highly overexpressed at both the mRNA ( approximately 163-fold) and protein levels. The functional role of ABCG2 in mediating resistance to JNJ-7706621 was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C, restored the sensitivity of HeLa-6621 cells to JNJ-7706621 and to mitoxantrone; (b) human embryonic kidney-293 cells transfected with ABCG2 were resistant to both JNJ-7706621 and mitoxantrone; and (c) resistant cells that were removed from the drug for 12 weeks and reverted to susceptibility to JNJ-7706621 showed near-normal ABCG2 RNA levels. ABCG2 is likely to limit the bioavailability of JNJ-7706621 because oral administration of JNJ-7706621 to Bcrp (the murine homologue of ABCG2) knockout mice resulted in an increase in the plasma concentration of JNJ-7706621 compared with wild-type mice. These findings indicate that ABCG2 mediates the resistance to JNJ-7706621 and alters the absorption of the compound following administration.

Targeting the Lymphotoxin-β Receptor with Agonist Antibodies as a Potential Cancer Therapy

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LT beta R activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LT beta R with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

Targeting Brn-3b in breast cancer therapy

The Brn-3b POU domain transcription factor is elevated in a significant proportion of breast cancers and in neuroblastoma tumours, where it is associated with increased proliferation, anchorage-independent growth, faster and larger tumour growth in xenograft models, resistance to growth inhibitory stimuli and increased migratory potential. These effects are associated with the ability of Brn-3b to regulate specific genes associated with these processes. Reducing Brn-3b can reverse many of these effects, suggesting that it may be possible to alter the growth and behaviour of tumour cells by abrogating Brn-3b in these cancers. This review discusses the effect of altering Brn-3b in these cancer cells and possible approaches to targeting Brn-3b as a strategy for therapy in treatment of breast cancers.

960. Forced Expression of the Heat Shock Protein H11 Causes Growth Arrest and Apoptosis of Melanoma Cells through TAK1 Activation and Inhibits Tumor Growth in Xenograft Models

The newly cloned small heat shock protein H11 evidences cell-type specific silencing by aberrant DNA methylation in 5/9 melanoma cell lines. In these cells, the de-methylating agent Aza-C causes H11 overload and triggers growth arrest and apoptosis. Apoptosis is blocked by antisense, but not sense, H11 oligonucleotides, indicating that it is induced by H11. Aza-C treatment did not induce H11 expression nor trigger growth arrest/apoptosis in cultured normal melanocytes. To identify the mechanism of growth arrest/apoptosis induced by H11 overload, melanoma cells were stably transfected with H11 under the control of a tetracycline sensitive promoter and examined for altered gene expression and cell survival before and after treatment with Doxycycline (Dox). Dox (2 |[mu]|g/ml) caused a time-dependent increase in H11 expression, reaching maximal levels on day 3 post treatment. H11 overload was accompanied by growth arrest (first seen on days 1-2 post Dox), activation of caspases-9 and-3, seen as early as day 1 post Dox and a time-dependent increase in the% TUNEL+ (apoptotic) cells (70-85% on days 3-4 post Dox). Immunoprecipitation/immunoblotting and immunocomplex PK assays indicated that H11 bound and activated TAK1. TAK1 did not bind and was not activated by the H11 mutant, W51C which is dominant negative for apoptosis. Caspase activation was inhibited with the dominant negative TAK1 mutant K63W or the p38MAPK pharmacologic inhibitor SB203580, indicating that apoptosis is caused by H11-mediated activation of the TAK1/p38MAPK pathway. H11-mediated growth arrest was also caused by activated TAK1, through phosphorylation of b-catenin and inhibition of MITF and CDK2, which are required for melanoma cell proliferation. H11 did not bind nor activate TAK1 in normal melanocytes. Studies of the highly tumorigenic A2058 and LM melanoma cultures in a mouse xenograft model, indicated that tumor growth was significantly (p<0.001) reduced by treatment with Aza-C (5mg/kg) given by ip injection on days 6 and 15 after initiation of the xenografts (3 doses at 3hr intervals). Decreased tumor growth was due to H11 induced apoptosis as evidenced by H11/TUNEL co-localization in residual tumor cells. The data suggest that H11 is a promising target for chemogene therapy of melanoma and identify additional members of the H11-mediated pro-apoptotic cascade.

Thienopyrimidine Ureas as Novel and Potent Multitargeted Receptor Tyrosine Kinase Inhibitors

A series of novel thienopyrimidine-based receptor tyrosine kinase inhibitors has been discovered. Investigation of structure-activity relationships at the 5- and 6-positions of the thienopyrimidine nucleus led to a series of N,N'-diaryl ureas that potently inhibit all of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases. A kinase insert domain-containing receptor (KDR) homology model suggests that these compounds bind to the "inactive conformation" of the enzyme with the urea portion extending into the back hydrophobic pocket adjacent to the adenosine 5'-triphosphate (ATP) binding site. A number of compounds have been identified as displaying excellent in vivo potency. In particular, compounds 28 and 76 possess favorable pharmacokinetic (PK) profiles and demonstrate potent antitumor efficacy against the HT1080 human fibrosarcoma xenograft tumor growth model (tumor growth inhibition (TGI) = 75% at 25 mg/kg.day, per os (po)).

Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer

Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.

Androgens, lipogenesis and prostate cancer

Both experimental and epidemiological data indicate that androgens are among the main factors controlling the development, maintenance and progression of prostate cancer. Identifying the genes that are regulated by androgens represents a major step towards the elucidation of the mechanisms underlying the impact of androgens on prostate cancer cell biology and is an attractive approach to find novel targets for prostate cancer therapy. Among the genes that have been identified thus far, several genes encode lipogenic enzymes. Studies aimed at the elucidation of the mechanisms underlying androgen regulation of lipogenic genes revealed that androgens coordinately stimulate the expression of these genes through interference with the molecular mechanism controlling activation of sterol regulatory element-binding proteins (SREBPs), lipogenic transcription factors governing cellular lipid homeostasis. The resulting increase in lipogenesis serves the synthesis of key membrane components (phospholipids, cholesterol) and is a major hallmark of cancer cells. Pharmacologic inhibition of lipogenesis or RNA-interference-mediated down-regulation of key lipogenic genes induces apoptosis in cancer cell lines and reduces tumor growth in xenograft models. While increased lipogenesis is already found in the earliest stages of cancer development (PIN) and initially is androgen-responsive it persists or re-emerges with the development of androgen-independent cancer, indicating that lipogenesis is a fundamental aspect of prostate cancer cell biology and is a potential target for chemoprevention and for antineoplastic therapy in advanced prostate cancer.

Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction of tumor growth.

We have found through ex vivo and in vivo angiogenesis models that the adrenomedullin gene-related peptide, proadrenomedullin NH2-terminal 20 peptide (PAMP), exhibits a potent angiogenic potential at femtomolar concentrations, whereas classic angiogenic factors such as vascular endothelial growth factor and adrenomedullin mediate a comparable effect at nanomolar concentrations. We found that human microvascular endothelial cells express PAMP receptors and respond to exogenous addition of PAMP by increasing migration and cord formation. Exposure of endothelial cells to PAMP increases gene expression of other angiogenic factors such as adrenomedullin, vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor C. In addition, the peptide fragment PAMP(12-20) inhibits tumor cell-induced angiogenesis in vivo and reduces tumor growth in xenograft models. Together, our data demonstrate PAMP to be an extremely potent angiogenic factor and implicate this peptide as an attractive molecular target for angiogenesis-based antitumor therapy.

The Brn-3b Transcription Factor Regulates the Growth, Behavior, and Invasiveness of Human Neuroblastoma Cells in Vitro and in Vivo

Neuroblastomas are the second most common solid tumor in children but the molecular mechanisms underlying the initiation and progression of this disease are poorly understood. We previously showed that the Brn-3b transcription factor is highly expressed in actively proliferating neuroblastoma cells but is significantly decreased when these cells are induced to differentiate. In this study, we analyzed the effects of manipulating Brn-3b levels in the human neuroblastoma cell line, IMR-32 and showed that constitutive overexpression of Brn-3b consistently increased cellular growth and proliferation in monolayer as well as in an anchorage-independent manner compared with controls whereas stably decreasing Brn-3b can reduce the rate of growth of these cells. Cells with high Brn-3b also fail to respond to growth inhibitory retinoic acid, as they continue to proliferate. Moreover, Brn-3b levels significantly modified tumor growth in vivo with elevated Brn-3b resulting in faster tumor growth in xenograft models whereas decreasing Brn-3b resulted in slower growth compared with controls. Interestingly, elevated Brn-3b levels also enhances the invasive capacity of these neuroblastoma cells with significantly larger numbers of migrating cells observed in overexpressing clones compared with controls. Because invasion and metastasis influence morbidity and mortality in neuroblastoma and so significantly affect the course and outcome of neuroblastomas, this finding is very important. Our results therefore suggest that Brn-3b transcription factor contributes to proliferation of neuroblastoma cells in vivo and in vitro but may also influence progression and/or invasion during tumorigenesis. It is possible that decreasing Brn-3b levels may reverse some effects on growth and proliferation of these cells.

Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. III. In vitro and in vivo antitumor activities.

We have discovered seven novel 12-membered macrolides, pladienolides A to G, from Streptomyces platensis Mer-11107, with pladienolide B the most potently inhibiting hypoxia induced-VEGF expression and proliferation of the U251 cancer cell line. A growth inhibitory study using a 39-cell line drug-screening panel demonstrated that pladienolide B has strong antitumor activities in vitro. A COMPARE analysis reveals that it has a unique antitumor spectrum that sets it apart from anticancer drugs currently in clinical use. This result suggests that pladienolide B has a novel mechanism of action. A series of xenograft studies were conducted to evaluate the in vivo potency of pladienolides. Pladienolide B extensively inhibited tumor growth in xenograft models. In the most sensitive model, using BSY-1 xenografts, tumors were completely regressed by administration of pladienolide B. For the reason of their novel mechanism of action and excellent in vivo efficacy, pladienolides appear to have major potential for use in cancer treatment.

Optimization of 4-phenylamino-3-quinolinecarbonitriles as potent inhibitors of Src kinase activity.

Subsequent to the discovery of 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (1a) as an inhibitor of Src kinase activity (IC(50) = 30 nM), several additional analogues were prepared. Optimization of the C-4 anilino group of 1a led to 1c, which contains a 2,4-dichloro-5-methoxy-substituted aniline. Replacement of the methoxy group at C-7 of 1c with a 3-(morpholin-4-yl)propoxy group provided 2c, resulting in increased inhibition of both Src kinase activity and Src-mediated cell proliferation. Analogues of 2c with other trisubstituted anilines at C-4 were also potent Src inhibitors, and the propoxy group of 2c was preferred over ethoxy, butoxy, or pentoxy. Replacement of the morpholine group of 2c with a 4-methylpiperazine group provided 31a, which had an IC(50) of 1.2 nM in the Src enzymatic assay, an IC(50) of 100 nM for the inhibition of Src-dependent cell proliferation and was selective for Src over non-Src family kinases. Compound 31a, which had higher 1 and 4 h plasma levels than 2c, effectively inhibited tumor growth in xenograft models.