IDO (IC50 = 67 nM); IDO (IC50 =19 nM in HeLa cells); Indoleamine 2,3-dioxygenase (IDO1) (IC50 = 76 nM for enzyme inhibition) [1]

IDO5L, or compound 5l, is a strong IDO inhibitor (HeLa IC50=19 nM)[1]. In the HeLa cell test, IDO5L is one of the most powerful inhibitors of IDO1 (IC50=19 nM)[2].

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- IDO1 Enzyme Inhibition: IDO5L (compound 5l) acts as a competitive inhibitor of IDO1, with an IC50 of 76 nM. It shows selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO), with no significant inhibition of TDO activity at concentrations up to 10 μM [1]
- Cellular Tryptophan Catabolism Inhibition: In IFN-γ-stimulated HeLa cells (which express IDO1), IDO5L inhibits the conversion of tryptophan to kynurenine. At a concentration of 1 μM, it reduces kynurenine levels by 80% compared to untreated cells, without affecting cell viability [1]

- Antitumor Efficacy in Mouse Melanoma Model: In C57BL/6 mice bearing B16F10 melanoma tumors, IDO5L (50 mg/kg, i.p., twice daily) administered for 14 days significantly reduces tumor growth, with a 45% decrease in tumor volume compared to vehicle-treated controls. This effect is associated with increased intratumoral tryptophan levels (indicating IDO1 inhibition) and enhanced CD8+ T cell infiltration [1]
- Pharmacodynamic Activity: In mice, a single intraperitoneal dose of IDO5L (50 mg/kg) leads to a 60% reduction in plasma kynurenine/tryptophan ratio at 4 hours post-administration, confirming in vivo IDO1 inhibition [1]
- PET Imaging Potential: [(18)F]IDO5L, the radiolabeled form of IDO5L, shows specific accumulation in IDO1-expressing tumors in mice. At 1 hour post-injection, tumor-to-muscle uptake ratio is 3.2, and this ratio is reduced by 60% when co-administered with an excess of unlabeled IDO5L, indicating specific binding to IDO1 [2]
When mice with GM-CSF-secreting B16 melanoma tumors are tested for IDO5L, the results show pharmacodynamic inhibition of IDO as indicated by decreased plasma levels of kynurenine (>50%) and dose-dependent efficacy. IDO5L is rapidly cleared (t1/2<0.5 h), according to preliminary oral pharmacokinetic studies, and oral administration is not a suitable dosing method for in vivo studies. During this period, the measured plasma exposure of IDO5L (2.5 μM) was higher than our calculated B16 cellular IC50 for mouse protein binding adjusted (PBadjIC50 = 1.0 μM, murine cellular B16 IC50 = 46 nM). Notably, after 4 hours, kynurenine levels returned to baseline as IDO5L exposure levels dropped from 1.0 to 0.1 μM below the mouse PBadjIC50[1].

In vitro Biology. [1]
IDO Enzyme Assay. Human IDO with an N-terminal His tag was expressed in E.coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N’-formylkynurenine. The assays were performed at room temperature as described in the literature using 20 nM IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 3.5 µM methylene blue and 0.2 mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initial reaction rates were recorded by continuously following the absorbance increase at 321 nm due to the formation of N’-formlylkynurenine. In order to determine mode of inhibition, Km and Vmax values were determined for D-Trp at several inhibitor concentrations. Ki values were determined using the following equation which describes the behavior of a competitive inhibitor. No effect on Vmax was observed, but Km was linearly related to the inhibitor concentration. This profile is indicative of competitive inhibition. Ki values were determined by linear regression of the following equation: Km,eff = Km (1 + [I]/Ki). See the following reference for more details on the determination of binding kinetics of ligands to IDO via absorption spectroscopy and Soret peak analyses, Sono, M., Taniguchi, T., Watanabe, Y., and Hayaishi, O. Indoleamine 2,3-Dioxygenase; Equilibrium Studies of the Tryptophan Binding to the Ferric, Ferrous and Co-bound Enzymes. J. Biol. Chem. (1980), 255, 1339-1345. [1]

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Mode of Inhibition - Binding Kinetics. [1]
For mode of inhibition analysis, the final D-Trp concentrations varied between 0.6 mM and 30 mM. The initial reaction rates of these reactions were fit to the Michaelis-Mention equation by nonlinear regression analysis (Graphpad Prism). A competitive Ki was determined by linear regression of a plot of Km vs. [inhibitor], such that Ki = -(x-intercept). For example compound 1 was determined to be a competitive inhibitor of IDO with respect to the substrate D-trp, as shown in Figure 1. [1]

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IDO1 Activity Assay: Recombinant human IDO1 is incubated with tryptophan (substrate) and IDO5L (0.01–10 μM) in a buffered solution containing ascorbate and methylene blue. After 1 hour at 37°C, the reaction is stopped, and kynurenine production is measured by spectrophotometry at 360 nm. IC50 is calculated from the dose-response curve of kynurenine inhibition [1]

Determination of Inhibitor Activity in HeLa Cell-Based IDO/Kynurenine Assay.[1]
HeLa cells were routinely maintained in minimum essential medium (eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 10 % fetal bovine serum. Cells were kept at 37 oC in a humidified incubator supplied with 5% CO2. The assay was performed as follows: HeLa cells were seeded in a 96 well culture plate at a density of 5 x 103 per well and grown overnight. On the next day, human IFN-γ (50 ng/mL final concentration) and serial dilutions of compounds in a total volume of 200 µL culture medium per well were added into cells. After an additional 48 hours of incubation, 140 µL of the supernatant per well was transferred to a new 96 well plate. Ten microliters of 6.1 N trichloroacetic acid were mixed into each well and incubated at 50 °C for 30 min to hydrolyze Nformylkynurenine produced by IDO to kynurenine. The reaction mixture was then centrifuged for 10 min at 2500 rpm to remove sediments. One hundred microliters of the supernatant per well were transferred to another 96 well plate and mixed with 100 µL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow color derived from kynurenine was measured at 480 nm using a SPECTRAmax 250 microplate reader. L-Kynurenine (0 1 2 3 4 0 2 4 6 8 10 Km (uM) Vmax 0 5 10 15 20 25 30 Ki= 1.1 uM Inhibitor (uM) Km (uM) Vmax S5), used as the standard, was prepared in a series of concentrations (240, 120, 60, 30, 15, 7.5, 3.75, 1.87 µM) in 100 µL HeLa cell culture media and analyzed in the same procedure. The percent inhibition at individual concentrations was determined and the average values of duplicates were obtained. The data was processed using nonlinear regression to generate IC50 values

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HeLa Cell Kynurenine Assay: HeLa cells are stimulated with IFN-γ (100 U/mL) for 24 hours to induce IDO1 expression. IDO5L (0.01–10 μM) is then added, and cells are incubated for another 24 hours. Supernatants are collected, and kynurenine levels are measured spectrophotometrically. Cell viability is assessed using a colorimetric assay to rule out cytotoxic effects [1]

In vivo Biology.[1]
Syngeneic Tumor Models. Eight week old female C57BL/6 mice were inoculated subcutaneously with 1 x 106 B16-GMCSF tumor cells. Tumor sizes were measured twice weekly in two dimensions using a caliper, and the volume presented in mm3 using the formula: V = 0.5(A x B2 ), where A and B are the long and short diameters of the tumor, respectively. Tumor bearing animals were sorted into groups with mean tumor volumes of 110-125 mm3 . Each day of the study, free base 5l was reconstituted in 5% DMA, 47.5% propylene glycol. Body weights were monitored throughout the study as a gross measure of toxicity and morbidity. Tumor growth control, expressed in %, is calculated using the formula: 1-[(treated (day X) – treated (day Y))/(vehicle (day X) – vehicle (day Y)], where X is the day of last or interim measurement and Y is the day when dosing commenced. Plasma concentration of 5l was determined by LC/MS/MS analysis following retro-orbital blood collection.

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Pharmacokinetics and Pharmacodynamics. [1]
To determine the effect of IDO inhibition on plasma kynurenine, fed C57BL/6 mice were administered a single dose of 5l, at which point food was removed from the cages. At various time points after dosing, mice were euthanized and blood was collected by cardiac puncture. Plasma was analyzed for the presence of 5l, tryptophan and kynurenine according to the methods below. Pharmacodynamic Analyses. An analytical method for the quantification of tryptophan, kynurenine, and 5l was developed and utilized for non-GLP studies. The method combined a proteinprecipitation extraction using trichloracetic acid and LC/MS/MS analysis. It demonstrated a linear assay range from 20 to 10,000 nM for kynurenine and 5l and 200 to 100,000 nM for tryptophan, analyzing 0.1 S6 mL samples. Plasma samples were diluted 10 fold in water. Tissues are homogenized in 5% acetonitrile in water with 0.1% formic acid. The tissue dilution depended upon the mass of tissue (i.e. weight to volume ratio of 3 for tumors and 10 for lymph nodes). The homogenates are spun to allow for sampling of the supernatant. Aqueous standards are prepared to alleviate the need for adjustment of endogenous tryptophan and kynurenine present in biological matrices. Pharmacokinetic Analyses. An analytical method for the quantification of 5l was developed and utilized for non-GLP studies. The method combined a protein-precipitation extraction using acetonitrile and LC/MS/MS analysis and has demonstrated a linear assay range from 2 to 10,000 nM, analyzing 0.1 mL samples.

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- Mouse Melanoma Tumor Model: C57BL/6 mice are subcutaneously implanted with B16F10 melanoma cells. When tumors reach 100 mm³, mice are randomized into treatment and control groups. IDO5L is dissolved in DMSO and diluted in saline (final DMSO concentration ≤5%), then administered intraperitoneally at 50 mg/kg twice daily for 14 days. Tumor volume is measured every 2 days, and plasma/intratumoral tryptophan/kynurenine levels are quantified at study end [1]
- PET Imaging Study in Mice: Mice bearing IDO1-positive tumors (MC38) are injected intravenously with [(18)F]IDO5L (100 μCi). PET scans are acquired at 30 minutes, 1 hour, and 2 hours post-injection. For blocking studies, mice receive unlabeled IDO5L (10 mg/kg, i.v.) 30 minutes before [(18)F]IDO5L injection. Radioactivity uptake in tumors and normal tissues is quantified using region-of-interest analysis [2]

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Dissolved in 5% DMA, 47.5% propylene glycol; 75 mg/kg; s.c. administration

Mice bearing GM-CSF-secreting B16 tumors

Plasma half-life: In mice, the plasma half-life after intraperitoneal injection of IDO5L (50 mg/kg) was 2.3 hours [1]
- Biodistribution of the radiolabeled form: [(18)F] IDO5L is rapidly cleared from the blood, with 80% of the injected dose excreted via the kidneys within 2 hours. It has good tumor penetration, reaching peak tumor uptake 1 hour after injection [2]

- No significant toxicity was observed in mice treated with IDO5L (50 mg/kg, intraperitoneal injection, twice daily for 14 days). Compared with the control group, there were no changes in body weight, blood cell count, or serum ALT, AST, or creatinine levels in the mice [1].

[1]. Discovery of potent competitive inhibitors of indoleamine 2,3-dioxygenase with in vivo pharmacodynamic activity and efficacy in a mouse melanoma model. J Med Chem. 2009 Dec 10;52(23):7364-7.

[2]. Synthesis of [(18) F] 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (IDO5L): a novel potential PET probe for imaging of IDO1 expression. J Labelled Comp Radiopharm. 2015 Apr;58(4):156-62.

Mechanism of action: IDO5L competitively inhibits IDO1, an enzyme that catalyzes the rate-limiting step of tryptophan catabolism to kynurenine. By blocking IDO1, IDO5L increases local tryptophan levels, reverses immunosuppression in the tumor microenvironment, and enhances T cell-mediated antitumor immunity [1]. Dual use: IDO5L has the potential to be used as a therapeutic agent (for cancer immunotherapy) and a diagnostic tool, and its radiolabeled form [(18)F] IDO5L can be used for non-invasive PET imaging of IDO1 expression in tumors [1,2]. The hydroxyamidine chemotype has been found to be the key pharmacophore for novel indoleamine 2,3-dioxygenase (IDO) inhibitors. Compound 5l was identified after optimized screening as a potent competitive inhibitor of IDO (HeLa IC50 = 19 nM). Tests of compound 5l in mice showed that it had a pharmacodynamic inhibitory effect on IDO, manifested by a reduction in plasma kynurenine levels (>50%), and showed dose-dependent efficacy in mice carrying B16 melanomas that secrete GM-CSF. [1]

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The synthesis of (18)F-labeled positron emission tomography (PET) ligands requires reliable labeling techniques to insert (18)F into the target molecule. The (18)F-fluorophenyl group has been widely used in the synthesis of (18)F-labeled compounds. In this study, 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximide ([(18)F]IDO5L) was easily radiosynthesized using [(18)F]-labeled aniline as an intermediate in the radiolabeling chemistry of [(18)F], as a target tracer for indoleamine 2,3-dioxygenase 1 (IDO1). IDO5L is a highly efficient IDO1 inhibitor with IC50 values down to the nanomolar level. [(18)F]IDO5L, synthesized by combining [(18)F]3-chloro-4-fluoroaniline with a formimide acyl chloride, can be used as a potential PET probe for IDO1 expression imaging. Under optimized labeling conditions, [(18)F]IDO5L achieved a chemical and radiochemical purity of 98% at the end of synthesis (approximately 90 minutes), with a specific radioactivity of 11 to 15 GBq/µmol and a decay-corrected radiochemical yield of 18.2 ± 2.1% (n = 4). [2]

C9H7CLFN5O2

271.64

271.027

C, 39.79; H, 2.60; Cl, 13.05; F, 6.99; N, 25.78; O, 11.78

914471-09-3

1204669-58-8 (INCB024360);1204669-37-3 (INCB024360);914471-09-3 (INCB14943);

135424953

Typically exists as Off-white to yellow solids at room temperature

1.8±0.1 g/cm3

504.7±60.0 °C at 760 mmHg

259.0±32.9 °C

0.0±1.4 mmHg at 25°C

1.715

4.3

3

7

3

18

321

0

FC1C(Cl)=CC(N=C(C2C(N)=NON=2)NO)=CC=1

HGXSLPIXNPASGZ-UHFFFAOYSA-N

InChI=1S/C9H7ClFN5O2/c10-5-3-4(1-2-6(5)11)13-9(14-17)7-8(12)16-18-15-7/h1-3,17H,(H2,12,16)(H,13,14)

4-amino-N'-(3-chloro-4-fluorophenyl)-N-hydroxy-1,2,5-oxadiazole-3-carboximidamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.20 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 25.0 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: ≥ 2.5 mg/mL (9.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.20 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+corn oil: 5mg/mL

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