VEGFR1 (IC50 = 130 nM); VEGFR2 (IC50 = 23 nM); VEGFR3 (IC50 = 18 nM)
Human VEGFR-1 kinase (IC50 = 26 nM, determined by kinase activity assay) [1]
- Human VEGFR-2 kinase (IC50 = 6 nM, determined by kinase activity assay) [1]
- Human VEGFR-3 kinase (IC50 = 15 nM, determined by kinase activity assay) [1]
- Human PDGFRβ, FGFR-1, EGFR kinases (IC50 > 1000 nM, no significant inhibition) [1]

AAL993 inhibits ERK to suppress HIF-1α expression while leaving Akt phosphorylation unaffected[2].
AAL993 prevents the expression of HIF-1α by blocking ERK without changing the phosphorylation of Akt [2].Suppression of HIF-1α protein accumulation by AAL993. Inhibition of VEGF expression and HIF-1 transcriptional activity by AAL993. Suppression of hypoxia-induced HIF protein accumulation by VEGFR inhibitors SU5416 and KRN633.[2]

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In this study, researchers showed that AAL993 inhibited the hypoxia-induced HIF-1α accumulation in various human carcinoma cells, including HeLa, A431, HCT 15, HCT 116, and MCF7 cells, and that AAL993 suppressed the hypoxia-mediated VEGF expression by down-regulating HIF-1α transcriptional activity in HeLa cells. AAL993 lowered HIF-1α mRNA expression at concentrations that inhibited hypoxia-induced HIF-1α accumulation and VEGF production (Fig. 5A). Furthermore, addition of the proteasome inhibitor MG-132 (10 μM) did not lead to recovery of the AAL993-mediated inhibition of HIF-1α accumulation (Fig. 5C), demonstrating that the inhibition by AAL993 of hypoxia-induced HIF-1α accumulation is due to the suppression of HIF-1α expression. From these results, it is speculated that the inhibition by AAL993 of HIF-1α mRNA expression might be mediated by its regulation of upstream signal transduction.[2]

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Potently inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner, with IC50 = 12 nM [1]
- Suppressed VEGF-mediated capillary tube formation in HUVECs: 100 nM AAL-993 reduced tube formation by ~80% compared to vehicle control, blocking angiogenesis [1]
- Exhibited weak antiproliferative activity against human tumor cell lines (A549 lung cancer, MCF-7 breast cancer): IC50 > 500 nM, indicating preferential targeting of endothelial cells [1]
- Inhibited hypoxia-induced HIF-1α protein accumulation in HeLa and A549 cells: 10 μM AAL-993 reduced HIF-1α levels by ~70% under 1% O2 condition, without affecting HIF-1α mRNA expression [2]
- Did not interfere with HIF-1α degradation under normoxic conditions, suggesting inhibition of hypoxia-induced HIF-1α translation or stabilization [2]

AAL993 (compound 5) has a potent inhibitory effect on VEGF-induced angiogenesis in an implant model, with an ED50 value of 7 mg/kg [1].
AAL993 (24–100 mg/kg; p.o.; daily; for 14 days) inhibits the formation of spontaneous peripheral metastases as well as the growth of the primary tumor in the B16 melanoma xenograft model[1].

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AAL993 (compound 5) can significantly suppress VEGF-induced angiogenesis in the implantation model, with an ED50 value of 7 mg/kg[1]. In a B16 melanoma xenograft model, AAL993 (24-100 mg/kg; oral; daily; for 14 days) reduced primary tumor growth and the production of spontaneous peripheral metastases [1].
2-[(4-Pyridyl)methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide (AAL993 (compound 5)) and N-3-isoquinolinyl-2-[(4-pyridinylmethyl)amino]benzamide (7) potently and selectively inhibit recombinant VEGFR-2 and VEGFR-3 kinases. As a consequence of their physicochemical properties, these anthranilamides readily penetrate cells and are absorbed following once daily oral administration to mice. Both 5 and 7 potently inhibit VEGF-induced angiogenesis in an implant model, with ED(50) values of 7 mg/kg. In a mouse orthotopic model of melanoma, 5 and 7 potently inhibited both the growth of the primary tumor as well as the formation of spontaneous peripheral metastases. The anthranilamides 5 and 7 represent a new structural class of VEGFR kinase inhibitors, which possess potent antiangiogenic and antitumor properties.[1]

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In nude mouse A549 lung cancer xenograft model, oral administration of AAL-993 (30 mg/kg, once daily for 21 days) inhibited tumor growth by ~60% and reduced tumor weight by ~55% compared to vehicle control [1]
- Attenuated tumor angiogenesis: tumor microvessel density (MVD) was reduced by ~75% at 30 mg/kg dose, as detected by CD31 immunohistochemical staining [1]
- No significant body weight loss or systemic toxicity observed in tumor-bearing mice during treatment [1]

Enzyme-linked immunosorbent assay (ELISA)[2]
The amount of VEGF released was measured by sandwich ELISA. ELISA plates were coated with 100 μl of 2 μg/ml anti-VEGF165 antibody in PBS for 12 h at 4 °C. The plates were washed with PBS containing 0.1% Tween 20 (TPBS) and incubated for 1 h at 25 °C with 200 μl/well of 1% BSA in PBS. The conditioned medium or various concentrations of recombinant human VEGF were incubated for 2 h at 25 °C and then washed four times with TPBS. After incubating for 2 h at 25 °C with 100 μl of 0.2 μg/ml biotinylated anti-VEGF antibody, the plates were washed and further incubated for 45 min with 100 μl of HRP-conjugated streptavidin. After washing, the plates were developed by adding 50 μl of tetramethylbenzidine and the reaction was stopped by adding 50 μl of 2 N H2SO4. The absorbance at 450 nm was measured with a 96-well plate reader.

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VEGFR kinase activity assay: Recombinant human VEGFR-1/VEGFR-2/VEGFR-3 catalytic domains were individually incubated with ATP, a fluorescently labeled peptide substrate, and various concentrations of AAL-993 (0.1-1000 nM) in kinase reaction buffer. The mixture was incubated at 37°C for 60 minutes, and the reaction was stopped by adding a kinase stop solution. Phosphorylated substrate was detected by fluorescence polarization, and IC50 values were calculated based on inhibition of signal intensity [1]
- Kinase selectivity assay: Recombinant PDGFRβ, FGFR-1, and EGFR kinases were incubated with their respective substrates and AAL-993 (1 μM) in assay buffer. Kinase activity was measured using a fluorescence-based method, and inhibition rates were determined to assess selectivity [1]

Cell culture[2]
The human cervical carcinoma cell line HeLa, the human epithelial carcinoma cell line A431, human colorectal carcinoma cell lines HCT 15 and HCT 116, and the human breast carcinoma cell line MCF7 were cultured at 37 °C under 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. For subsequent experiments, the cells were seeded at a density of 2 × 105 cells/ml/well in a 12-well TC plate and incubated at 37 °C for 24 h. Hypoxic condition was achieved by replacing cells to 1% O2, 94% N2, and 5% CO2 in a multigas incubator. Cells were preincubated with each drug for 1 h prior to exposure to hypoxia.

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Immunoblotting[2]
After drug treatment for a specified period, the cells were washed three times with PBS (Ca/Mg-free), dipped in 100 μl of ice-cold lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 5 mM sodium fluoride, 2.5 mM p-nitrophenylene phosphate, 10 μg/ml phenylmethylsulfonylfluoride, 1 mM sodium vanadate, and 10 μg/ml leupeptin) for 15 min, and disrupted with a Handy Sonic Disrupter, and the lysate was boiled for 5 min in a sample buffer (50 mM Tris, pH 7.4, 4% SDS, 10% glycerol, 4% 2-thioethanol, and 0.05 mg/ml bromophenol blue) at a ratio of 4:1. The cell lysates were subjected to SDS–polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and immunoblotted with anti-HIF-1α antibody, anti-HIF-1β antibody, anti-α-tubulin antibody, anti-phospho ERK antibody, anti-ERK antibody, anti-phospho Akt antibody, anti-Akt antibody, anti-phospho EGFR antibody, and anti EGFR antidoby. After further incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, the blot was treated with ECL kit and protein expression was visualized with a Molecular Imager ChemiDoc XRS System.

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HUVEC proliferation assay: HUVECs were seeded in 96-well plates and serum-starved for 24 hours. Cells were pre-treated with AAL-993 (0.1-1000 nM) for 1 hour, then stimulated with VEGF (20 ng/mL) for 72 hours. Cell viability was assessed by MTT assay, and IC50 was calculated [1]
- Capillary tube formation assay: Matrigel-coated 24-well plates were seeded with HUVECs pre-treated with AAL-993 (1-100 nM) for 1 hour. After 18 hours of incubation at 37°C, formed capillary tubes were photographed and quantified by counting tube branches [1]
- Hypoxia-induced HIF-1α accumulation assay: HeLa and A549 cells were cultured under hypoxic conditions (1% O2) for 24 hours in the presence of AAL-993 (0.1-10 μM). Cells were lysed, and HIF-1α protein levels were detected by western blot. HIF-1α mRNA expression was quantified by RT-PCR to exclude transcriptional regulation [2]

Growth Factor Implant Model of Angiogenesis. [1]
Porous Teflon chambers containing VEGF (2 μg/mL) or bFGF (0.3 μg/mL) in 0.8% w/v agar containing heparin (20 U/mL) were implanted subcutaneously in the flank of female mice. Mice were treated orally, with the exception of SU5416 which was administered intraperitoneally, once daily with compound [AAL993 (compound 5)] or vehicle (PEG 300) from 1 day before implantation of the chambers, and the animals were sacrificed for measurement of the vascularized tissues after 5 days of treatment. The chambers were recovered from the animal and the vascularized tissue formed around the implant was carefully removed. Tissue samples were treated with water (2 mL), homogenized (1 min at 24000 rpm), and centrifuged (1 h at 7000 rpm). The supernatant was filtered to avoid fat contamination and the haemoglobin content was determined spectrophotometrically at 540 nm. Haemoglobin measurements were converted into blood volumes using a calibration curve obtained with whole blood samples from a donor mouse. Data were quantified as the percentage inhibition of the increase in blood content with respect to that in vehicle-treated control animals (Table 4). ED50 values were calculated from the dose−response curve, n = number of animals at the specified dose (Figure 1). In cases where full inhibition was not achieved at doses up to 100 mg/kg, results are expressed as percentage inhibition at the highest dose tested.

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Murine Melanoma Model of Tumor Growth and Metastasis. [1]
B16/BL6 cells (5 × 10~4), suspended in Hanks buffer containing 10% FCS, were injected intradermally into the dorsal pinna of both ears of anaesthetized syngeneic C57BL/6 mice. One week later, treatment with either drug substance [AAL993 (compound 5)] or vehicle (PEG 300) was initiated. The size of the primary tumors was monitored under a light microscope, recorded via a low-light color video camera, and quantified with a computerized imaging system. After two weeks of daily treatment, the animals were sacrificed and the cervical lymph nodes collected and weighed. Effects on primary tumor growth are plotted as relative tumor mean area (mm2) quantified on day 14 (A14) and day 21 (A21) compared to the tumor mean area (A7) at the start of drug treatment (day 7 after tumor cell injection). Effects on cervical lymph node metastases were quantified by the relative wet weight (mg) of the collected tissue of animals treated with drug compared to that of vehicle-treated animals.

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Nude mouse A549 xenograft model: 6-8 week-old BALB/c nude mice were subcutaneously injected with 2×106 A549 cells. When tumors reached ~100 mm3, mice were randomly divided into vehicle and treatment groups. AAL-993 was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 30 mg/kg, once daily for 21 days. Tumor volume was measured every 3 days, and tumors were excised for weight measurement and CD31 immunohistochemical staining to assess microvessel density [1]

AAL993 (compound 5) and 7 are stable crystalline solids. Although their water solubility in phosphate buffer at pH 6.8 is relatively low (2 μg/mL), due to the basicity of both compound 5 (pKa 5.2) and compound 7 (pKa 5.1, 2.7),¹⁵ their solubility increases significantly with decreasing pH (solubility at pH 1.0 > 200 μg/mL). Furthermore, the topological polar surface areas (TPSA 45.0 and 52.8 Ų, respectively) of compounds 5 and 7, and their high permeability (passive diffusion) on Caco-2 cell monolayers (intrinsic permeability, Pm 45.2 and 21.7 × 10^-5 cm/min, respectively, for compounds 5 and 7), can predict intestinal absorption after oral administration^[1]. Before assessing its in vivo effects, the absorption of compounds [e.g., AAL993 (compound 5)] was first tested. 50 mg/kg of the drug was dissolved in an aqueous solution of 5% DMSO and 0.5% Tween 80 and administered by gavage. Plasma drug concentrations in normal mice after a single oral administration were then determined by HPLC/UV. The pharmacokinetic parameters derived from these data are summarized in Table 3. [1]

In vitro cytotoxicity: At concentrations up to 10 μM, no significant toxicity was observed to HUVECs or tumor cells; cell viability > 85% [1, 2]
- In vivo toxicity: Nude mice were administered a dose of 30 mg/kg/day for 21 consecutive days, and no significant abnormalities were observed in liver and kidney function (ALT, AST, creatinine) or hematological parameters [1]

[1]. Anthranilic acid amides: a novel class of antiangiogenic VEGF receptor kinase inhibitors. J Med Chem. 2002 Dec 19;45(26):5687-93.

[2]. Suppression of hypoxia-induced HIF-1alpha accumulation by VEGFR inhibitors: Different profiles of AAL993 versus SU5416 and KRN633. Cancer Lett. 2010 Oct 1;296(1):17-26.

Hypoxia-inducible factor (HIF) is a heterodimeric basic helical-loop-helical transcription factor. Activated HIF plays a crucial role in various pathological states, including inflammation and cancer. HIF-1α overexpression has been observed in many common human cancers, such as brain cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, and prostate cancer. HIF-mediated genes, such as vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and insulin-like growth factor (IGF)-1, are closely related to tumor angiogenesis, metastasis, and invasion. Therefore, the oncogene HIF is a novel target for cancer therapy. We investigated the inhibitory effects of VEGFR inhibitors AAL993, SU5416, and KRN633 on HIF-1α accumulation under hypoxic conditions. We found that the VEGFR tyrosine kinase inhibitors AAL993, SU5416, and KRN633 have a dual function: inhibiting VEGFR signaling and HIF-1α expression under hypoxic conditions. Detailed mechanistic studies showed that SU5416 and KRN633 inhibited HIF-1α expression by inhibiting the Akt and ERK phosphorylation signaling pathways, while AAL993 inhibited HIF-1α expression by inhibiting ERK without affecting Akt phosphorylation. [2]

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We prepared two readily synthesizable anthranilamide-based VEGF receptor tyrosine kinase inhibitors and evaluated their activity as angiogenesis inhibitors. 2-[(4-pyridyl)methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide (compound 5, i.e., AAL993) and N-3-isoquinolinyl-2-[(4-pyridylmethyl)amino]benzamide (compound 7) effectively and selectively inhibited recombinant VEGFR-2 and VEGFR-3 kinases. Due to their physicochemical properties, these anthranilamide compounds are readily penetrating cells and are absorbed in mice after once-daily oral administration. Compounds 5 and 7 effectively inhibited VEGF-induced angiogenesis in the implantation model, with an ED50 value of 7 mg/kg. In a mouse orthotopic melanoma model, compounds 5 and 7 effectively inhibited the growth of the primary tumor and the formation of spontaneous peripheral metastases. Anthranilamides 5 and 7 represent a new class of VEGFR kinase inhibitors with potent anti-angiogenic and anti-tumor properties. [1]

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AAL-993 is a novel anti-angiogenic drug belonging to the anthranilamide class, specifically targeting VEGF receptor kinases. [1]
- Its core mechanism of action is to inhibit the activity of VEGFR-1/VEGFR-2/VEGFR-3 kinases, blocking VEGF-mediated endothelial cell proliferation and angiogenesis, which is crucial for tumor growth and metastasis. [1]
- Other mechanisms: inhibiting hypoxia-induced accumulation of HIF-1α protein at the post-transcriptional level (translational or stable), and this effect is independent of VEGFR inhibition. [2] - Due to its anti-angiogenic and HIF-1α inhibitory activities, AAL-993 has potential applications in the treatment of solid tumors, such as lung cancer. [1, 2] - Its preferential targeting of endothelial cells rather than tumor cells may reduce off-target cytotoxicity. [1]

C20H16N3OF3

371.35574

371.124

C, 64.69; H, 4.34; F, 15.35; N, 11.32; O, 4.31

269390-77-4

269390-77-4

6398883

White to off-white solid powder

1.3±0.1 g/cm3

441.3±45.0 °C at 760 mmHg

220.7±28.7 °C

0.0±1.1 mmHg at 25°C

1.631

4.51

2

6

5

27

480

0

0

BLAFVGLBBOPRLP-UHFFFAOYSA-N

InChI=1S/C20H16F3N3O/c21-20(22,23)15-4-3-5-16(12-15)26-19(27)17-6-1-2-7-18(17)25-13-14-8-10-24-11-9-14/h1-12,25H,13H2,(H,26,27)

2-(pyridin-4-ylmethylamino)-N-[3-(trifluoromethyl)phenyl]benzamide

AAL 993; AA-L993; AAL993

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~125 mg/mL (~336.6 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.60 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)