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

- IDO1酶抑制作用：IDO5L（化合物5l）作为IDO1的竞争性抑制剂，IC50为76 nM。对色氨酸2,3-双加氧酶（TDO）具有选择性，在浓度高达10 μM时对TDO活性无显著抑制 [1]
- 抑制细胞内色氨酸分解代谢：在IFN-γ刺激的HeLa细胞（表达IDO1）中，IDO5L抑制色氨酸向犬尿氨酸的转化。在1 μM浓度下，与未处理细胞相比，犬尿氨酸水平降低80%，且不影响细胞活力 [1]
IDO5L 或化合物 5l 是一种强 IDO 抑制剂 (HeLa IC50=19 nM)[1]。在 HeLa 细胞测试中，IDO5L 是 IDO1 最强大的抑制剂之一 (IC50=19 nM)[2]。

- 小鼠黑色素瘤模型中的抗肿瘤疗效：在携带B16F10黑色素瘤的C57BL/6小鼠中，IDO5L（50 mg/kg，腹腔注射，每日两次）给药14天显著抑制肿瘤生长，与溶媒处理对照组相比，肿瘤体积减少45%。此效应与肿瘤内色氨酸水平升高（表明IDO1被抑制）和CD8+ T细胞浸润增加相关 [1]
- 药效动力学活性：在小鼠中，单次腹腔注射IDO5L（50 mg/kg）后4小时，血浆犬尿氨酸/色氨酸比值降低60%，证实其在体内对IDO1的抑制作用 [1]
- PET成像潜力：IDO5L的放射性标记形式[(18)F]IDO5L在小鼠IDO1表达肿瘤中显示特异性蓄积。注射后1小时，肿瘤与肌肉摄取比为3.2，当与过量未标记IDO5L共同给药时，该比值降低60%，表明对IDO1的特异性结合 [2]
当对患有 GM-CSF 分泌 B16 黑色素瘤肿瘤的小鼠进行 IDO5L 测试时，结果显示 IDO 的药效学抑制作用，如血浆犬尿氨酸水平降低 (> 50%) 和剂量依赖性功效所示。根据初步口服药代动力学研究，IDO5L 被迅速清除（t1/2<0.5 h），口服给药不是体内研究的合适给药方法。在此期间，测得的 IDO5L 血浆暴露量 (2.5 μM) 高于我们计算的小鼠蛋白结合调整后的 B16 细胞 IC50（PBadjIC50 = 1.0 μM，鼠细胞 B16 IC50 = 46 nM）。值得注意的是，4 小时后，随着 IDO5L 暴露水平从小鼠 PBadjIC50 以下 1.0 μM 降至 0.1 μM，犬尿氨酸水平恢复至基线[1]。

体外生物学。[1]
IDO酶测定。带有N端His标签的人IDO在大肠杆菌中表达并纯化至均一性。IDO催化色氨酸吲哚核吡咯环的氧化裂解，产生N'-甲酰基犬尿氨酸。如文献所述，在室温下使用20 nM IDO和2 mM D-Trp，在50 mM磷酸钾缓冲液（pH 6.5）中存在20 mM抗坏血酸、3.5µM亚甲基蓝和0.2 mg/mL过氧化氢酶的情况下进行测定。通过连续跟踪由于N'-甲酰基犬尿氨酸的形成而在321nm处的吸光度增加来记录初始反应速率。为了确定抑制模式，在几种抑制剂浓度下测定了D-Trp的Km和Vmax值。Ki值使用描述竞争性抑制剂行为的以下方程式确定。未观察到对Vmax的影响，但Km与抑制剂浓度呈线性关系。该曲线表明竞争性抑制。Ki值通过以下方程的线性回归确定：Km，eff=Km（1+[I]/Ki）。有关通过吸收光谱和Soret峰分析确定配体与IDO结合动力学的更多详细信息，请参阅以下参考文献，Sono，M.，Taniguchi，T.，Watanabe，Y.和Hayaishi，O.吲哚胺2,3-双加氧酶；色氨酸与铁、亚铁和共结合酶结合的平衡研究。J.生物学。化学。(1980), 255, 1339-1345. [1]

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抑制模式——结合动力学。[1]
对于抑制模式分析，最终的D-Trp浓度在0.6 mM至30 mM之间变化。通过非线性回归分析（Graphpad Prism），这些反应的初始反应速率符合Michaelis Mention方程。通过Km与[抑制剂]曲线的线性回归确定竞争性Ki，使得Ki=-（x截距）。例如，如图1所示，化合物1被确定为IDO相对于底物D-rp的竞争性抑制剂。

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IDO1活性测定：重组人IDO1与色氨酸（底物）和IDO5L（0.01–10 μM）在含抗坏血酸和亚甲蓝的缓冲液中孵育。37°C孵育1小时后终止反应，通过360 nm处的分光光度法测量犬尿氨酸生成量。从犬尿氨酸抑制的剂量-反应曲线计算IC50 [1]

基于IDO/犬尿氨酸测定HeLa细胞抑制剂活性。[1]
HeLa细胞常规维持在含有2 mM L-谷氨酰胺的最低必需培养基（鹰）中，Earle的BSS被调节为含有1.5 g/L碳酸氢钠、0.1 mM非必需氨基酸、1 mM丙酮酸钠和10%胎牛血清。将细胞保持在37℃的加湿培养箱中，培养箱中装有5%的二氧化碳。测定方法如下：将HeLa细胞以每孔5 x 103的密度接种在96孔培养板中，并生长过夜。第二天，将人IFN-γ（终浓度为50 ng/mL）和化合物的系列稀释液加入细胞中，每孔总体积为200µL培养基。再孵育48小时后，将每孔140µL上清液转移到新的96孔板上。将10微升6.1 N三氯乙酸混合到每个孔中，在50°C下孵育30分钟，以将IDO产生的Nformylkynurenine水解为犬尿氨酸。然后将反应混合物在2500rpm下离心10分钟以去除沉淀物。将每孔100微升上清液转移到另一个96孔板上，并与100µL 2%（w/v）对二甲氨基苯甲醛的乙酸溶液混合。使用SPECTRAmax 250酶标仪在480nm下测量犬尿氨酸的黄色。在100µL HeLa细胞培养基中以一系列浓度（240、120、60、30、15、7.5、3.75、1.87µM）制备用作标准品的L-犬尿氨酸（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抑制剂（uM)。

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HeLa细胞犬尿氨酸实验：HeLa细胞用IFN-γ（100 U/mL）刺激24小时以诱导IDO1表达。随后加入IDO5L（0.01–10 μM），细胞再孵育24小时。收集上清液，分光光度法测量犬尿氨酸水平。采用比色法评估细胞活力以排除细胞毒性 [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 of IDO5L after intraperitoneal administration (50 mg/kg) is 2.3 hours [1]
- Biodistribution of Radiolabeled Form: [(18)F]IDO5L shows rapid clearance from blood, with 80% of the injected dose eliminated via the kidneys within 2 hours. It exhibits good tumor penetration, with peak tumor uptake at 1 hour post-injection [2]

- No significant toxicity is observed in mice treated with IDO5L (50 mg/kg, i.p., twice daily for 14 days). There are no changes in body weight, blood cell counts, or serum levels of ALT, AST, or creatinine compared to controls [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, it increases local tryptophan levels, reversing immunosuppression in the tumor microenvironment and enhancing T cell-mediated antitumor immunity [1]
- Dual Utility: IDO5L has potential as both a therapeutic agent (for cancer immunotherapy) and a diagnostic tool via its radiolabeled form [(18)F]IDO5L, which enables non-invasive PET imaging of IDO1 expression in tumors [1,2]
A hydroxyamidine chemotype has been discovered as a key pharmacophore in novel inhibitors of indoleamine 2,3-dioxygenase (IDO). Optimization led to the identification of 5l, which is a potent (HeLa IC50 = 19 nM) competitive inhibitor of IDO. Testing of 5l in mice demonstrated pharmacodynamic inhibition of IDO, as measured by decreased kynurenine levels (>50%) in plasma and dose dependent efficacy in mice bearing GM-CSF-secreting B16 melanoma tumors.[1]

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To synthesize (18) F-labeled positron emission tomography (PET) ligands, reliable labeling techniques inserting (18) F into a target molecule are necessary. The (18) F-fluorobenzene moiety has been widely utilized in the synthesis of (18) F-labeled compounds. The present study utilized [(18) F]-labeled aniline as intermediate in [(18) F]-radiolabeling chemistry for the facile radiosynthesis of 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide ([(18) F]IDO5L) as indoleamine 2,3-dioxygenase 1 (IDO1) targeted tracer. IDO5L is a highly potent inhibitor of IDO1 with low nanomolar IC50 . [(18) F]IDO5L was synthesized via coupling [(18) F]3-chloro-4-fluoroaniline with carboximidamidoyl chloride as a potential PET probe for imaging IDO1 expression. Under the optimized labeling conditions, chemically and radiochemically pure (>98%) [(18) F]IDO5L was obtained with specific radioactivity ranging from 11 to 15 GBq/µmol at the end of synthesis within ~90 min, and the decay-corrected radiochemical yield was 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

109.56

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (9.20 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (9.20 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (9.20 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2% DMSO+corn oil: 5mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。