VEGFR-2 Kinase Research Articles

Discovery of acyl ureas as highly selective small molecule CSF1R kinase inhibitors

Based on the structure of an early lead identified in Deciphera’s proprietary compound collection of switch control kinase inhibitors and using a combination of medicinal chemistry guided structure activity relationships and structure-based drug design, a novel series of potent acyl urea-based CSF1R inhibitors was identified displaying high selectivity for CSF1R versus the other members of the Type III receptor tyrosine kinase (RTK) family members (KIT, PDGFR-α, PDGFR-β, and FLT3), VEGFR2 and MET. Based on in vitro biology, in vitro ADME and in vivo PK/PD studies, compound 10 was selected as an advanced lead for Deciphera’s CSF1R research program.

Modified Benzoxazole-Based VEGFR-2 Inhibitors and Apoptosis Inducers: Design, Synthesis, and Anti-Proliferative Evaluation.

This work is one of our efforts to discover potent anticancer agents. We modified the most promising derivative of our previous work concerned with the development of VEGFR-2 inhibitor candidates. Thirteen new compounds based on benzoxazole moiety were synthesized and evaluated against three human cancer cell lines, namely, breast cancer (MCF-7), colorectal carcinoma (HCT116), and hepatocellular carcinoma (HepG2). The synthesized compounds were also evaluated against VEGFR-2 kinase activity. The biological testing fallouts showed that compound 8d was more potent than standard sorafenib. Such compound showed IC50 values of 3.43, 2.79, and 2.43 µM against the aforementioned cancer cell lines, respectively, compared to IC50 values of 4.21, 5.30, and 3.40 µM reported for sorafenib. Compound 8d also was found to exert exceptional VEGFR-2 inhibition activity with an IC50 value of 0.0554 μM compared to sorafenib (0.0782 μM). In addition, compound 8h revealed excellent cytotoxic effects with IC50 values of 3.53, 2.94, and 2.76 µM against experienced cell lines, respectively. Furthermore, compounds 8a and 8e were found to inhibit VEGFR-2 kinase activity with IC50 values of 0.0579 and 0.0741 μM, exceeding that of sorafenib. Compound 8d showed a significant apoptotic effect and arrested the HepG2 cells at the pre-G1 phase. In addition, it exerted a significant inhibition for TNF-α (90.54%) and of IL-6 (92.19%) compared to dexamethasone (93.15%). The molecular docking studies showed that the binding pattern of the new compounds to VEGFR-2 kinase was similar to that of sorafenib.

HR-LCMS assisted phytochemical screening and an assessment of anticancer activity of Sargassum Squarrossum and Dictyota Dichotoma using in vitro and molecular docking approaches

The research in the area of marine bioactives have substantially improved in the last few decades. The identified marine algae namely Sargassum squarrossum and Dictyota dichotoma were subjected to phytochemical profiling using the most sophisticated High-resolution-LCMS, 1H NMR and revealed the presence of many bioactive compounds.In vitro MTT tests on lung and breast cancer cells revealed that the DCM fractions of Sargassum squarrossum and Dictyota dichotoma have anticancer characteristics, and they also demonstrated significant dose-dependant cytotoxicity on tumour cell lines. In MCF7 cells, the IC50 values for Sargassum squarrossum and Dictyota dichotoma were 139.51 µg/ml and 241.89 µg/ml whereas in A549 the cell line, the IC 50 values for the DCM fractions of Sargassum squarrossum and Dictyota dichotoma were 95.37 µg/ml and 92.09 µg/ml, respectively. Sargassum squarrossum shows potent anticancer activity in MCF7 and A549 whereas Dictyota dichotoma has potent anticancer activity in A549 and moderate activity in MCF7 cell lines.The most prevalent enzymes VEGFR-2 and AXL tyrosine kinase, contribute to the development of cancer. A computational method was used to confirm the anticancer effects of the newly identified compounds targeting the above enzymes. According to the molecular docking studies, most of the detected compounds from marine algae were selectively interacting with AXL and VEGFR receptors with good binding affinity scores. The current work demonstrated the phytochemical assessment of marine algae and showed the ability of marine natural products to provide unique metabolites with significant biological activity.

Synthesis, characterization, chemical and biological activities of 4-(4-methoxyphenethyl)-5- benzyl-2-hydroxy-2H-1,2,4-triazole-3(4H)-one phthalocyanine derivatives

Different properties of phthalocyanine compounds can be measured both theoretically and experimentally. In this study, tetra substituted phthalocyanines (H2 (2) and CuII(3) were performed using 4-(4-(4-methoxyphenethyl)-3-benzyl-5-oxo-4,5-dihydro-1,2,4 triazol-1-yl)phthalonitrile (1). The structures of all these original compounds synthesized were elucidated using distinctive of spectroscopic techniques.Theoretical comparison of the chemical and biological activities of phthalocyanine molecules and its copper metal complex has been made. Chemical activities were compared with Gaussian software program and biological activities were compared with molecular docking calculations. Used proteins for biological activity are the crystal structure of estrogen receptor protein (from the breast cancer), ID: 1A52, crystal structure of VEGFR kinase protein (from liver cancer), ID: 3WZE, crystal structure of MLK4 kinase (colon cancer) ID: 4UYA, and crystal structure of an allosteric Eya2 phosphatase inhibitor protein (from lung cancer), ID: 5ZMA. After, the interactions between molecules and proteins were determined using the Protein-Ligand Interaction Profiler (PLIP) server.

Synthesis, docking study, and structure-activity relationship of novel niflumic acid derivatives acting as anticancer agents by inhibiting VEGFR or EGFR tyrosine kinase activities

A new series of niflumic acid (NF) derivatives were synthesized by esterification of (NF) to give ester compound 1, which was treated with hydrazine hydrate to produce (NF) hydrazide 2. Hydrazine-1-carboxamide compounds (3A–C), and hydrazine-1-carbothioamide derivatives (4A–D) were synthesized by treatment of (NF) hydrazide with phenyl isocyanate, and phenyl isothiocyanate derivatives, respectively. The cyclization of (4B–D) and (3B) was achieved using NaOH solution to produce 1,2,4-triazole derivatives (5A–C) and 6, respectively. The prepared compounds were characterized using IR, 1HNMR, 13CNMR, and MS (ESI) spectroscopy. A molecular docking study was performed to evaluate the binding affinity of the synthesized compounds against EGFR and VEGFR kinase domains which revealed that compounds 3B, and 4A had the best binding energy (-7.87, and -7.33 kcal/mol, respectively) against VEGFR, while compound 5A had the best binding energy (-7.95 kcal/mol) against EGFR. The biological investigation results indicated that all the tested compounds caused cell killing in the two cancer cell lines (Hep G2 and A549) studied, with compound 4C being the most cytotoxic, as well as being cancer selective. Additionally, compound 4C-treated Hep G2 cells were arrested at the S and G2/M cell cycle phases. Cytotoxicity of compound 4C was attributed to apoptosis as determined by flow cytometry and qRT-PCR results of the apoptosis markers p53, BAX, and caspase-3. Finally, compound 4C inhibited VEGFR kinase activity, while compound 5B inhibited EGFR kinase activity. In conclusion, the novel (NF) derivatives are potent anticancer agents, inhibiting cell proliferation by inhibiting EGFR and VEGFR tyrosine kinase enzymes.

Molecular Docking and In Vitro Anticancer Screening of Synthesized Arylthiazole linked 2H-indol-2-one Derivatives as VEGFR-2 Kinase Inhibitors.

Indoline-2,3-dione comprises a leading course group of heterocycles endowed with appealing biological actions, including anticancer activity. There are significant justifications for exploring the anticancer activity of Schiff base derivatives of isatin as a vast number of reports have documented remarkable antiproliferative action of isatin nucleus against various cancer cell lines. A series of arylthiazole linked 2H-indol-2-one derivatives (5a-t) was designed and synthesized as potential VEGFR-2 kinase inhibitors keeping the essential pharmacophoric features of standard drugs, like sunitinib, sorafenib, nintedanib, etc. They were evaluated for their in vitro anticancer activity. The aim of this study was to investigate and assess the anticancer potential of isatin-containing compounds along with their kinase inhibition activity. The title compounds were synthesized by reacting substituted isatins with para-substituted arylthiazoles using appropriate reaction conditions. Selected synthesized derivatives went under preliminary screening against a panel of 60 cancer cell lines at NCI, the USA, for single-dose and five dose assays. Molecular docking was performed to explore the binding and interactions with the active sites of the VEGFR-2 receptor (PDB Id: 3VHE). Derivatives 5a, 5b, 5c, 5d, 5g, 5h, and 5m were assessed for in vitro inhibition potency against Human VEGFR-2 using ELISA (Enzyme- Linked Immunosorbent Assay) kit. All the target compounds were determined against human colon cancer cell line SW480 (colorectal adenocarcinoma cells). Cellular apoptosis/necrosis was determined by flow cytometry using annexin V-FITC. DNA content of the cells was analyzed by flow cytometry and the cycle distribution was quantified. Compounds 5a and 5g exhibited noteworthy inhibition during a five-dose assay against a panel of 60 cell lines with MID GI50 values of 1.69 and 1.54 μM, respectively. Also, both the lead compounds 5a and 5g demonstrated promising VEGFR-2 inhibitory activity with IC50 values of 5.43±0.95 and 9.63±1.32 μM, respectively. The aforesaid potent compounds were found effective against SW480 (colorectal adenocarcinoma cells) with IC50 values of 31.44 μM and 106.91 μM, respectively. Compound 5a was found to arrest the cell cycle at the G2/M phase, increasing apoptotic cell death. The docking study also supported VEGFR-2 inhibitory activity as both compounds 5a and 5g displayed promising binding and interactions with the active sites of VEGFR-2 receptor (PDB: 3VHE) with docking scores - 9.355 and -7.758, respectively. All the compounds obeyed Lipinski's rule of five. Indoline-2,3-dione and thiazole have huge potential to be considered a steer combination approach for developing promising kinase inhibitors as cancer therapeutics.

Nanogel-mediated drug delivery system for anticancer agent: pH stimuli responsive poly(ethylene glycol/acrylic acid) nanogel prepared by gamma irradiation

The popularity of nanogel as nano drug carrier lies in its adjustable physical properties, and the ability to encapsulate drug particles with improved properties is being developed to meet the diverse pH-sensitive nanogel for anticancer agent. Monitoring pH has been identified as an important diagnostic element during the treatment process. A pH-sensitive nanogel consisting of (PEG/PMAc) in the ratio of (50:50%) hasbeen cross-linkedby γ-irradiation techniques at an irradiation dose of 5 kGy. Compound 4 and its nanogel 5 were synthesized and assessed for their anticancer effects against HepG2, A549, MCF-7 and HCT-116 as dual VEGFR-2 and EGFR tyrosine kinases inhibitors. The molecular design was performed to investigate the binding mode of compound 4 with VEGFR-2 and EGFR receptors. Our compound 5 in nanogel showed enhanced anticancer activities against the four tested cancer cell lines and also showed higher inhibition activities against VEGFR-2 and EGFRT790M kinases than the derivative 4. Finally, our derivative 4 showed good in silico calculated ADMET profile. It was expected to show good GIT absorption in human, lower CNS side effects, no hepatotoxic actions and higher acute and oral chronic toxic doses in comparing to sorafenib and erlotinib. The obtained results showed that, our compound could be useful as a template for future design, optimization, adaptation and investigation to produce more potent and selective dual VEGFR-2/EGFRT790M inhibitors with higher anticancer activity.

New thiophene, thienopyridine and thiazoline-based derivatives: Design, synthesis and biological evaluation as antiproliferative agents and multitargeting kinase inhibitors

Multitargeting kinase inhibitors recently proved to be a profitable approach for conquering cancer proliferation. The current study represents the design and synthesis of new thiophene, thienopyridine, and thiazoline-based derivatives 4-14a,b. All the target compounds were examined in vitro against three cancer cell lines; the liver (HepG-2), breast (MCF-7), and colon (HCT-116) where the thiophene-based compounds 5a-c, demonstrated the most potent activity. Furthermore, the latter derivatives revealed a safety profile against WI-38 normal cell line of selectivity indices ranging from 4.43 to 17.44. In vitro enzyme assay of 5a-c revealed that the carbohydrazide analog 5c has the most promising multitargeting inhibiting activity against Pim-1, VEGFR-2, and EGFRWT enzymes of IC50 values; 0.037 ± 0.02, 0.95 ± 0.24, and 0.16 ± 0.05 µM, respectively. As it was the most potent analog, 5c was further subjected to cell cycle and apoptosis analysis. The results indicated that it induced preG1 arrest and an apoptotic effect in the early and late stages. Moreover, further apoptosis studies were carried out for 5c to evaluate its proapoptotic potential. Interestingly, 5c enhanced the levels of Bax/Bcl-2 ratio, p53, and active caspase 3 by 18, 6.4, and 24 folds, respectively compared to the untreated cells. The antimicrobial evaluation showed that only compounds 3 and 5a produced broad-spectrum potency, while 5b and 5c exhibited outstanding antifungal effects. Finally, a molecular docking study was carried out to discover the probable interactions of compound 5c with the active sites of Pim-1, VEGFR-2, and EGFRWT kinases.

A phase 1b study (STELLAR-002) of XL092 administered in combination with nivolumab (NIVO) with or without ipilimumab (IPI) or bempegaldesleukin (BEMPEG) in patients (pts) with advanced solid tumors.

TPS4600 Background: XL092 is a novel oral inhibitor of receptor tyrosine kinases, including MET, VEGFR2, and TAM kinases (AXL, MER), which are implicated in tumor growth, metastasis, angiogenesis, and immune suppression of the tumor microenvironment. XL092 has a relatively short half-life (̃21h) to support convenient daily dosing and help manage tolerability. Preclinical studies of XL092 with an anti‒PD-1 immune checkpoint inhibitor (ICI) demonstrated antitumor activity in tumor models, and BEMPEG (IL-2 pathway agonist) showed synergy with anti‒PD-L1 and anti‒CTLA-4 agents. This phase 1b trial will evaluate the safety and clinical activity of XL092 alone and in combination with NIVO (anti‒PD-1 mAb) ±IPI (anti‒CTLA-4 mAb) or ±BEMPEG in pts with advanced solid tumors including genitourinary cancers. Presented here is the study design. Methods: This multicenter phase 1b, open-label study (NCT05176483) will enroll pts with unresectable advanced or metastatic solid tumors in dose-escalation and expansion stages. In the dose-escalation stage, ̃36 pts will be enrolled in three XL092 combination therapy cohorts using a rolling 6 design. Cohort A: XL092 (starting dose [SD] 100 mg PO QD) + NIVO (360 mg IV Q3W); Cohort B: XL092 (SD 80 mg PO QD) + NIVO (3 mg/kg IV Q3W × 4, then 480 mg IV Q4W) + IPI (1 mg/kg Q3W × 4); Cohort C: XL092 (SD 100 mg PO QD) + NIVO (360 mg IV Q3W) + BEMPEG (0.006 mg/kg IV Q3W). The primary objective of the dose-escalation stage is to determine the recommended doses of XL092 with the NIVO regimens to be used in the expansion stage. The expansion stage will include cohorts of advanced genitourinary tumors: Cohort 1, clear-cell renal cell carcinoma (ccRCC), no prior systemic therapy; Cohort 2, ccRCC, 1 prior ICI combination regimen; Cohort 3, metastatic castration-resistant prostate cancer (mCRPC), 1 prior novel-hormonal therapy; Cohort 4, urothelial carcinoma (UC), 1 prior platinum-based regimen, ICI-naïve; Cohort 5, UC, ≤2 prior systemic regimens, ICI-experienced; Cohort 6, non-ccRCC, no prior systemic therapy. In each cohort, pts will be randomized to one of the following treatments (based on tumor cohort): single-agent XL092 (Cohorts 2‒6); XL092+NIVO (Cohorts 1‒6); NIVO+IPI (Cohort 1); XL092+NIVO+IPI (Cohorts 1, 3, 6); NIVO+BEMPEG (Cohort 1), XL092+NIVO+BEMPEG (Cohort 1, 2, 4‒6). Thirty pts will be enrolled in each single-agent XL092 arm and 40 pts in each combination therapy arm. Expansion stage objectives are to assess preliminary efficacy, safety, and pharmacokinetics of XL092 alone or in combination in each tumor-specific cohort. Primary efficacy endpoints include objective response rate by investigator per RECIST v1.1 and progression-free survival by blinded independent radiology committee per Prostate Working Group 3 criteria (mCRPC cohort only). The study is currently enrolling pts. Clinical trial information: NCT05176483.

A multicenter, open-label, phase I/II study of FN-1501 in patients with advanced solid tumors and acute myeloid leukemia.

e15083 Background: FN-1501, a potent inhibitor of receptor FMS-like tyrosine kinase 3 (FLT3) and CDK4/6, KIT, PDGFR, VEGFR2, ALK and RET tyrosine kinase proteins, has demonstrated significant in vivo anti-tumor activity in a broad range of solid tumor and leukemia xenograft models. FLT3 mutations have an established role as a therapeutic target in Acute Myeloid Leukemia (AML), where the gene plays a critical role in the growth, differentiation, and survival of hematopoietic cells. An open-label, Phase I/II study ( NCT03690154 ) is evaluating the safety and PK profile of FN-1501 as monotherapy in patients (pts) with advanced solid tumors and relapsed, refractory (R/R) AML. Methods: Pts received FN-1501 IV thrice weekly for 2 weeks followed by 1 week off treatment in 21-day cycles. Dose escalation follows a 3+3 design. Primary objectives include determination of maximum tolerated dose (MTD), safety, and recommended phase 2 dose (RP2D). Secondary objectives include pharmacokinetics (PK) and preliminary anti-tumor activity. Exploratory objectives include the relationship between pharmacogenetic mutations (e.g., FLT3, TP53, KRAS, NRAS), safety, efficacy and pharmacodynamic effects of FN-1501. Dose expansion at RP2D was designed to further explore safety and efficacy. Results: As of Dec 3, 2021 data cut-off (DCO), 47 pts with advanced solid tumors (N = 46) or AML (N = 1) were enrolled at doses ranging from 2.5 to 226 mg. The median age was 65 (range 30-92) with 57% female and 43% male. The median prior lines of treatment were 5 (range 1-12). Forty pts with median exposure of 9.5 cycles (range 1-18 cycles) were evaluable for dose limiting toxicity (DLT). Treatment-related adverse events (TRAEs) were reported in 64% of pts. The most common treatment-emergent adverse events (TEAEs) defined as those occurring in ≥ 20% of patients primarily consisted of reversible grade 1-2 fatigue (34%), nausea (32%) and diarrhea (26%). The most common grade ≥ 3 events occurring in ≥ 5% of pts consisted of diarrhea and hyponatremia. Dose escalation was discontinued due to DLTs of grade 3 thrombocytopenia (N = 1) and grade 3 infusion related reaction (N = 1) in 2 pts in the 226 mg dose group (2nd DLT reported after DCO). At the time of DCO, 33 pts were evaluable for disease response showing 1 with partial response (PR) (47% target lesion shrinkage), 15 with stable disease (SD) and 17 with progression of disease (PD). The PR lasted > 4 months (mts) in a patient with endometrial carcinoma (ca) at the 40 mg dose level. The longest treatment exposures were recorded in 6 pts with SD of 2.6 to > 12 mts (thymoma [1]; ovarian ca [2]; renal cell ca [1]; laryngeal ca [1] and intestinal adeno ca [1]) at doses ranging from 15 mg to 170 mg. Conclusions: FN-1501 IV has shown reasonable safety, tolerability, and preliminary activity against solid tumors up to 170 mg. Dose escalation was terminated based on 2 DLTs occurring at the 226 mg dose level. Clinical trial information: NCT03690154.

Lenvatinib for the treatment of thymic epithelial tumors (TETs): A real-life multicenter experience.

8585 Background: TETs are rare malignancies of the anterior mediastinum being thymoma (T) B3 and thymic carcinoma (TC) the most aggressive subtypes. There is no standard treatment after platinum-based chemotherapy in refractory or metastatic setting. A phase 2 trial has reported clinical benefit for lenvatinib 24mg (objective response rate [ORR] of 38%), a novel multi-targeted inhibitor of VEGFR, FGFR, RET, c-Kit, and other kinases; significant toxicity grade 3 hypertension was 64%. No real-life data exists. Methods: We selected patients (pts) under lenvatinib as a second-line or beyond for refractory TETs from 8 International centers from France (belonging to the nationwide network RYTHMIC) and United States. We analyzed epidemiologic, clinical and pathological characteristics of patients with TET’s. The toxicity was evaluated according to CTCAE v4, with a local evaluation of efficacy and we assessed toxicity profile and survival outcomes. Results: From March 2020 to December 2021, 29 pts were enrolled. Median age at diagnosis was 49 (24-71), 51.7% were women, 6/29 (20.7%) reported auto-immune disorders (AIDs). TC was the most frequent subtype (n=18, 62.1%), followed by B3 and B2. Lenvatinib was used as a second line for 52% of pts, mainly starting from 14 mg/daily (n=20, 69%) and one pts with concomitant pembrolizumab. The ORR was 17% (95%CI 3.0-32.0) with partial responses only seen in TC, and the disease control rate was 76% (95%CI 59.0-92.0). Response was observed with the dose of 24mg in 3 pts and 14mg in 2 pts, with a median follow-up period of 5 months (m) (95%CI 3.2-6.7), PFS at 6 and 12 m was 64% and 30%, respectively. Toxicity is summarized in table 1. Dose de-escalations were needed in 27.5% of pts. Conclusions: We confirm the activity of lenvatinib in pts with advanced or metastatic T and TC, despite the use of lower doses than the phase 2 study.[Table: see text]

A phase II trial of pembrolizumab and cabozantinib in patients (pts) with recurrent metastatic head and neck squamous cell carcinoma (RMHNSCC).

6008 Background: Pembrolizumab (pembro) is an immune checkpoint inhibitor (ICI) approved for treating pts with recurrent/metastatic (RM) HNSCC. Cabozantinib (cabo) is a multiple receptor tyrosine kinase inhibitor (TKI) targeting MET and VEGFR2 shown to reduce tumor growth, metastasis, and angiogenesis and has immuno-modulatory properties. Methods: This is a phase II, open label multi-center, single arm trial evaluating the tolerability and clinical benefit of pembro administered at 200 mg every 3 weeks and cabo 40 mg daily for pts with RM HNSCC who have not received prior ICI. It had a lead-in safety cohort allowing reduction of cabo dose to 20mg daily. Eligible pts had RM HNSCC, deemed incurable, with a tumor PD-L1 CPS > 1, RECIST 1.1 measurable disease, a life expectancy of > 3 months, an Eastern Cooperative Group (ECOG) Performance Status (PS) of 0-1. Enrollment was initiated in March of 2019. We estimated that the ORR will improve to 35% with pembro+ cabo (from a historic of 18%) with a significance level of 0.05 and 80% power. Results: A total of 47 pts were screened, 13 screen failures [cavitation on scan (4), inability to swallow pills (2), other exclusions (7)]; 34 pts were enrolled, 32 were dosed and 31 evaluable (at least one follow-up scan). Pts had cancers of the oropharynx (21, 65%), nasopharynx (6, 19%), larynx/hypopharynx (4, 13%) and oral cavity (1,3%). 32 patients received cabo at 40 mg daily; 13 patients (41%) were dose reduced to 20mg daily [mucositis, increased liver function (LFT) tests, diarrhea]; males (n=29, 90%), median age 63 years (range 53-67); presence of distant metastases (34, 100%); prior radiation (29, 90%), chemotherapy (14, 43%); ECOG PS =0 (16, 50%), 1 (16,50%); HPV/p16 positive (17, 53%), negative (5, 16%), not applicable (10, 29%); the most frequent adverse event (AE) (all grades) was fatigue (16,50.0%), grade 3 or 4 treatment-related AE were increased LFTs, hyponatremia (3, 9.3% each). With a median follow up of 12.7 months (mos) (range 6.9- 20.5 mos), a RECIST 1.1 overall response rate ORR= 45.2% (CR=0; PR=14, 45.2%; SD =14, 45.2%; PD=3.0, 10%) with an overall clinical benefit of 90.4% were observed; The 1-yr OS was 67.7% (95% CI, 42.9%-83.6%; median 22.3 mos) and 1-yr PFS was 51.8% (95% CI, 28.8%-70.7%; median 14.6 mos). Conclusions: This phase II trial of pembro + cabo met its primary endpoint of ORR. The regimen is well-tolerated with very encouraging clinical activity in RM HNSCC and warrants further exploration in this disease. The study was supported by a grant from Exelixis to NFS. Clinical trial information: NCT03468218.

Design, synthesis and anticancer activity of novel 2-arylbenzimidazole/2-thiopyrimidines and 2-thioquinazolin-4(3H)-ones conjugates as targeted RAF and VEGFR-2 kinases inhibitors

In the current study, series of 2-arylbenzimidazole-thiopyrimidine and -thioquinazolin-4(3H)-ones conjugates 12a-d, 13a,b and 14a-l have been synthesized. All the synthesized compounds were tested in vitro for their anticancer activities against a panel of cancer cell lines at NCI - US and their growth inhibition (GI) % were determined at 10 µM. Compounds 14c and 14g-i were selected to be screened at the five dose assay and were found to exhibit GI50 values 1.1–30.0 µM. The benzimidazole-quinazolinone derivative 14c, in particular, showed potent anticancer activity against the tested cancer cell lines (GI50 of 1.3–4.2 µM). In addition, compounds 12a,b, 13a, 14a-e, 14g, 14i and 14j were selected to be tested against some cancer cell lines using MTT assay and the benzimidazole-quinazolinone 14g was found to have potent anticancer activities against melanoma (Mel-501 and A-375), breast (MCF-7), colon (HCT-116), prostate (PC-3), lung (A-549) and pancreas (Paca-2) cancer cell lines reporting IC50 values ranging between 0.1 and 6.2 µM. Moreover, the synthesized hybrids were tested in vitro on kinases; BRAF (wt), BRAF (V600E), CRAF and VEGFR-2. The benzimidazole-quinazolinone derivatives 14f,g revealed potent RAF kinases inhibitory activities on BRAF (wt), BRAF (V600E) and CRAF showing IC50 values 0.002–0.1 µM, whereas, the benzimidazole-quinazolinone derivatives 14i and 14k showed moderate VEGFR-2 inhibitory activity (IC50 = 20.60 and 6.14 µM, respectively). Moreover, the representative compounds 14g and 14i caused cell cycle arrest of A-375 melanoma cell line at G2/M phase and were found to induce late apoptosis. CRAF in the DFG-out inactive conformation homology modeling was first reported in this study and molecular docking studies on BRAF, CRAF and VEGFR-2 were also performed to investigate the binding modes of the target compounds and their interactions with the key amino acids; BRAF (Glu500, Cys531 and Asp593), CRAF (Glu393, Cys424 and Asp486) and VEGFR-2 (Glu885, Cys919 and Asp1046).

The Crystal Structure of 2-Amino-4-(2,3-Dichlorophenyl)-6-Methoxy-4H-Benzo[h]chromene-3-Carbonitrile: Antitumor and Tyrosine Kinase Receptor Inhibition Mechanism Studies

The target compound, 2-amino-4-(2,3-dichlorophenyl)-6-methoxy-4H-benzo[h]chromene -3-carbonitrile (4), was synthesized via the reaction of 4-methoxynaphthalen-1-ol (1), 2,3-dichlorobenzaldehyde (2), and malononitrile (3) in an ethanolic piperidine solution under microwave irradiation. The synthesized β-enaminonitrile derivative (4) was characterized by spectral data and X-ray diffraction. The in vitro anti-proliferative profile was conducted against five cancer cell lines and was assessed for compound 4, which revealed strong and selective cytotoxic potency. This derivative showed promising inhibition efficacy against the EGFR and VEGFR-2 kinases in comparison to Sorafenib as a reference inhibitor. Lastly, the docking analysis into the EGFR and VEGFR-2 active sites was performed to clarify our biological findings.

Pharmacokinetic Exposures Associated With Oral Administration of Sorafenib in Dogs With Spontaneous Tumors.

Sorafenib is a multi-kinase small molecule inhibitor that targets serine/threonine and tyrosine kinases including the RAF kinase family, VEGFR-2, and PDGFR. The aim of this study was to evaluate the systemic pharmacokinetics of a previously defined tolerable oral dose of sorafenib in tumor-bearing dogs. Six client-owned dogs with a cytologic or histologic diagnosis of cancer were enrolled in this open-label, tolerability study. Dogs were administered sorafenib at an intended dose of 3 mg/kg and serum samples were obtained for analysis of sorafenib serum concentrations at 0, 1, 2, 6, 12, 24, 48, 72, 96, and 168 h post-drug administration. Median time to peak serum sorafenib concentration occurred at 4 h (range 2–12 h) resulting in an average serum concentration of 54.9 ± 33.5 ng/mL (118.2 ± 72.1 nM). Mean sorafenib levels declined by over 70% relative to peak serum concentrations by 24 h in all dogs, suggesting the value of at least twice daily administration. Doses of 3 mg/kg were well-tolerated and no patients in the study experienced adverse events that were attributable to sorafenib. Future trials in dogs with cancer are recommended at this dosing schedule to assess the effect of sorafenib administration on anti-tumor efficacy signals and relevant pharmacodynamic target modulation in vivo.

Novel antiproliferative agents bearing substituted thieno[2,3-d]pyrimidine scaffold as dual VEGFR-2 and BRAF kinases inhibitors and apoptosis inducers; design, synthesis and molecular docking

A series of novel thieno[2,3-d]pyrimidine derivatives was designed and synthesized based on multitarget directed drug design strategy. All the newly synthesized compounds were evaluated for their antiproliferative activity in the National Cancer Institute (NCI) against a panel of 60 tumor cell lines. Compounds 4a and 4b showed a significant antiproliferative activity at 10 µM dose, and were accordingly evaluated at five dose concentrations. They showed potent and broad-spectrum antiproliferative activity, with GI50 values in the micromolar range of 1.44–6.93 µM and 1.66–5.82 µM, respectively. They also showed TGI values in the cytostatic range of 3.49–97.3 µM and 3.33–77.3 µM respectively. These two compounds potently inhibited VEGFR-2 with IC50 = 0.111 ± 0.006 and 0.049 ± 0.003 µM, BRAFV600E with IC50 = 0.089 ± 0.005 and 0.063 ± 0.003 µM and BRAFWT IC50 = 0.071 ± 0.004 and 0.05 ± 0.003 µM, respectively in comparison to sorafenib IC50 values of 0.031 ± 0.002, 0.035 ± 0.002 and 0.021 ± 0.001 µM against VEGFR-2, BRAFV600E and BRAFWT, respectively. Compounds 4a and 4b showed also potent down-regulation of total VEGFR-2 and phosphorylated VEGFR-2. In addition, the HUVECs migratory potential was greatly reduced resulting in significantly disrupted wound healing patterns after treatment with compounds 4a and 4b for 72 h. Furthermore, Compounds 4a and 4b induced apoptosis by 22.82- and 25.81-fold increase in the total apoptosis percentage in breast cancer MCF7 cell line. This apoptotic activity was supported by an increase in the level of apoptotic caspase-9 by 6.17- and 9.07-fold, respectively. Moreover, the cell cycle analysis showed that compounds 4a and 4b arrested the cell cycle mainly in the G1 and G1/S phases, respectively. The molecular modeling studies were performed to assess the binding pattern and affinity of derivatives 4a and 4b toward the VEGFR-2 and BRAF active sites.

FOXM1 Promotes Intestinal Vascular Endothelial Cell Proliferation and Protects Neonatal Mice against Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a life‐threatening gastrointestinal illness affecting mostly premature infants characterized by intestinal inflammation and necrosis. While several factors such as abnormal intestinal colonization and an immature immune response have been shown to play a role in NEC, we previously found that defective intestinal microvascular development promotes NEC development. FOXM1 is a protein expressed in cells undergoing proliferation and a member of the Forkhead box family, a master regulator of cell cycle progression. FoxM1 has been shown to mediate endothelial cell proliferation under stress conditions. Here we hypothesize that promoting FOXM1 expression protects neonatal mice against intestinal injury in an experimental NEC model by promoting endothelial cell proliferation. To test our hypothesis, we first examined FOXM1 mRNA expression during NEC development in a neonatal mouse model. Second, we investigated the role of FOXM1 in NEC development using FOXM1 transgenic and knockout animals. Finally, we examined in vitro whether FOXM1 can protect endothelial cell proliferation under stress condition. We found that FOXM1 expression was significantly decreased in the intestine of pups exposed to the NEC model for 24 and 48 hours compared to dam‐fed littermate controls. Neonatal mice that lack of FOXM1 in endothelial cells showed higher mortality rate and more severe intestinal injury when exposed to the experimental NEC model. While their 3‐day survival remained unchanged, pups with endothelial‐cell specific overexpression of FOXM1 had decreased incidence of severe intestinal injury. Furthermore, when cultured in vitro, intestinal endothelial cells obtained from pups overexpressing FOXM1 showed increased proliferation, which was unaffected by VEGFR2 inhibition using VEGFR2 kinase inhibitor Ki‐8751. In conclusion, FOXM1 protects against neonatal NEC by promoting endothelial cell proliferation.

Abstract 489: Glutathione Regulates Endothelial Cell Function And Vegfr2 Signaling

Under physiological and pathological conditions angiogenesis progresses in an idealized redox window. Mice heterozygous for the Glutamate cysteine ligase modifier (GCLm) subunit knockout have augmented ischemic remodeling. During occlusion, the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that the cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling. To test this hypothesis, we first performed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype control (WT). Outgrowth area in the Het group was significantly higher than WT and KO at day 7 of outgrowth (2.15 fold, n=4, P<0.001). MAECs were isolated and relative migration rates were determined using under-agarose migration assays. Het MAECs were 1.25 fold faster than WT (n=4 p<0.001) while KO MAECs were slower, 0.90 fold (n=3, p<0.05). Cell proliferation assays are currently underway. Concurrently, we inhibited glutathione reductase in primary human aortic endothelial cells using 2-AAPA. 2-AAPA (75 μm) alone didn’t activate VEGFR2 (determined by western blot for pY1175 VEGFR2). 2-AAPA in combination with H 2 O 2 (100 μM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2-AAPA with H 2 O 2 induced a significant increase in pY416 proto-oncogene c-Src (Src) 9.5 fold (p<0.01, n=6), This effect was attenuated by the addition of DTT. Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2-AAPA + H 2 O 2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2-AAPA + H 2 O 2 mediated activation of Src. Combined these data suggest Src mediates the 2-AAPA + H 2 O 2 activation of VEGFR2. Glutathione S-transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 μM) abrogated the effects of 2-AAPA + H 2 O 2 on VEGFR2 and experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.

Glutathione Regulates Endothelial Cell Function and VEGFR2 Signaling

Under physiological and pathological conditions angiogenesis progresses in an idealized window of reactive oxygen species. Mice heterozygous for the Glutamate cysteine ligase modifier subunit knockout have augmented ischemic remodeling. During occlusion the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling.MethodsTo test this hypothesis, we first preformed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype controls (WT). VEGF stimulated chemotaxis was determined using the under‐agarose migration assay. Primary human aortic endothelial cells (HAEC) were purchased from Lonza and cultured following suppliers’ recomendations. Phospho‐Y1175 VEGFR2/total VEGFR2 and phospho‐Y416 proto‐oncogene c‐Src (Src)/total Src were determined by western blotting.ResultsOutgrowth area in the Het group was significantly larger than WT and KO at day 7 (2.15 fold, n=4, P<0.001). Het MAECs were 1.25 fold faster (n=4 p<0.001) while KO MAECs were 0.90 fold slower than WT (n=3, p<0.05) migrating towards VEGF‐A. Concurrently, we inhibited glutathione reductase in HAECs using 2‐acetylamino‐3‐[4‐(2‐acetylamino‐2‐carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2‐AAPA). 2‐AAPA (75 µM) alone didn’t significantly activate VEGFR2. However, 2‐AAPA (75 µM) in combination with H2O2 (100 µM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2‐AAPA both with and without H2O2 induced a significant increase in active Src, 5.3 and 9.0 fold (p<0.05, p<0.01, n=6) respectively. This effect was attenuated by the addition of Dithiothreitol (1mM). Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2‐AAPA + H2O2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2‐AAPA + H2O2 mediated activation of Src. Glutathione S‐transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 µM) abrogated the effects of 2‐AAPA + H2O2 on VEGFR2.ConclusionCombined these data suggest Src mediates the 2‐AAPA + H2O2 activation of VEGFR2. And that glutathionylation is necessary for this effect, co‐immunoprecipitation experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.

Synergistic Anti-Angiogenic Effect of Combined VEGFR Kinase Inhibitors, Lenvatinib, and Regorafenib: A Therapeutic Potential for Breast Cancer.

Background: Breast cancer currently affects more than two million women worldwide, and its incidence is steadily increasing. One of the most essential factors of invasion and metastasis of breast cancer cells is angiogenesis and non-angiogenic vascularization. Lenvatinib and Regorafenib share the same anti-angiogenic effect by inhibiting vascular endothelial growth factor receptors (VEGFRs subtypes 1 to 3) and have been approved for treating different types of cancer. Methods: We investigated Lenvatinib and Regorafenib effects on a well-established in-vitro model of breast cancer using MCF-7 (estrogen, progesterone receptor-positive, and HER2-negative), MDA-MB-231 (triple negative), as well as Human Umbilical Vascular Endothelial Cell line (HUVEC) cell lines. We performed the cell viability assay on four groups of cells, which included a control group, a Lenvatinib treated only group, a Regorafenib treated only group, and a group treated with a combination of both drugs at 24, 48, and 72 h. Data were analyzed as means ± standard deviation, and the drug–drug interactions with Compusyn software. Cellular migration assay, tube formation assay, and Western blots were conducted to determine the functional and the protein expression of downstream signals such as Caspase-9, anti-apoptotic Survivin, P-ERK, and total-ERK in the control and treatment groups. Results: MCF-7 cells showed a reduction in cell survival rates with higher dosing and longer incubation periods with each drug and with the combination of drugs. A synergistic interaction was identified (CI < 1) with both drugs on MCF7 at different dose combinations and at a higher dose in MDA-MB-231 cells. Furthermore, there was a marked decrease in the anti-angiogenic effect of both drugs in tube formation assay using MDA-MB-231 cells and survivin protein expression in MCF-7, and those antitumor markers showed a better outcome in drug combination than the use of each drug alone. Conclusion: Our result is the first to report the synergistic anti-angiogenic potential of combination therapy of Lenvatinib and Regorafenib. Therefore, it shows their therapeutic potential in breast cancer, including the aggressive types. Further studies are warranted to confirm and explore this therapeutic approach.