Design, Synthesis and Biological Evaluation of Phenyl Urea Derivatives as IDO1 Inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing intracellular enzyme that catalyzes the first and rate-determining step of tryptophan metabolism and is an important immunotherapeutic target for the treatment of cancer. In this study, we designed and synthesized a new series of compounds as potential IDO1 inhibitors. These compounds were then evaluated for inhibitory activity against IDO1 and tryptophan 2,3-dioxygenase (TDO). Among them, the three phenyl urea derivatives i12, i23, i24 as showed potent IDO1 inhibition, with IC50 values of 0.1–0.6 μM and no compound exhibited TDO inhibitory activity. Using molecular docking, we predicted the binding mode of compound i12 within IDO1. Compound i12 was further investigated by determining its in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that compound i12 had satisfactory properties in mice, with moderate plasma clearance (22.45 mL/min/kg), acceptable half-life (11.2 h) and high oral bioavailability (87.4%). Compound i12 orally administered at 15 mg/kg daily showed tumor growth inhibition (TGI) of 40.5% in a B16F10 subcutaneous xenograft model and 30 mg/kg daily showed TGI of 34.3% in a PAN02 subcutaneous xenograft model. In addition, the body weight of i12-treated mice showed no obvious reduction compared with the control group. Overall, compound i12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.


Introduction
The tryptophan/kynurenine pathway plays an important role in cancer immunotherapy [1]. Activation of this pathway promotes the degradation of tryptophan and leads to the formation of kynurenine and other bioactive metabolites, such as 3-hydroxykynurenine and 3-hydroxyanthranilic acid. A local depletion of tryptophan induces T cell cycle arrest and the accumulation of tryptophan metabolites converses naïve T cells into regulatory T cells and induces T cell apoptosis. These exert a local immunosuppressive effect which can lead to tumor progression and recurrence [2][3][4]. Indoleamine 2, 3-dioxygenase 1 (IDO1, EC 1. 13.11.52) catalyzes the initial and rate-limiting step in the catabolism of tryptophan along the kynurenine pathway [5]. Cancer cells and a variety of immune cells in the tumor microenvironment are shown to overexpress IDO1, which is often associated with worse response to anticancer therapies and decreased survival of cancer patients [6,7]. Inhibition of Inhibition of IDO1 was shown to increase the therapeutic efficacy of cancer vaccines, immune checkpoint inhibitors, or chemotherapy in multiple clinical mouse models [8][9][10]. On this basis, several IDO1 inhibitors have been developed and are currently under clinical development ( Figure  1) [  In this study, a new series of compounds were designed based on the phenyl urea scaffold in order to search for new IDO1 inhibitors. The compounds were synthesized and their IDO1/TDO inhibitory activities were determined. The in vivo pharmacokinetic profile and anti-tumor efficacy of a potent IDO1 inhibitor were evaluated to explore its potential as an anti-tumor agent.

Design Strategies of the Compounds
It was disclosed that the compound BMS-E30 showed potent IDO1 inhibitory activity (IC50 = 0.7 nM) in the IDO1 kynurenine assay with human IDO1/HEK 293 cells (Scheme 1) [11], however, its enzyme inhibitory activity was relatively weak with an IC50 of 8.569 μM ( Table 1). The lack of hemecoordinating element distinguished compound BMS-E30 from other IDO1 inhibitors in the literature. We considered that BMS-E30 effectively inhibited IDO1 by targeting its apo-form subsequent to the disclosure of the IDO1/BMS-978587 crystal structure (6AZV) because the structures of BMS-E30 and BMS-978587 were similar (Scheme 1) [12]. Considering the two flexible chains of diisobutylamino group in compound BMS-E30, a new series of compounds were designed by replacing the diisobutylamino group with a 3,5-dimethylpiperidinyl group for reducing the entropy loss of binding IDO1. The peripheral phenyl urea group was also modified to explore the structure-activity relationship (SAR) which was used for further optimization to obtain better IDO1 inhibitors. The flexible disobutylamino group was replaced with the rigid 3, 5-dimethylpiperidinyl group to optimize the space steric effect of substituent and NH of the phenyl urea group was further replaced with CH to test whether the phenyl urea group is essential to IDO1 potency.

Synthesis of Selected Compounds
In Scheme 2, nitration of 4-fluorobenzaldehyde was performed with nitric acid and sulfuric acid to produce compound a [13]. Under basic conditions, compound b was obtained through a nucleophilic aromatic substitution reaction with 3,5-dimethylpiperidine. An aldehyde group addition reaction was performed in the presence of (trifluoromethyl)trimethylsilane to obtain compound c [14], which was oxidized to compound d with Dess-Martin periodinane. Compound e was prepared by a Horner-Wadsworth-Emmons reaction reaction of d with triethyl In this study, a new series of compounds were designed based on the phenyl urea scaffold in order to search for new IDO1 inhibitors. The compounds were synthesized and their IDO1/TDO inhibitory activities were determined. The in vivo pharmacokinetic profile and anti-tumor efficacy of a potent IDO1 inhibitor were evaluated to explore its potential as an anti-tumor agent.

Design Strategies of the Compounds
It was disclosed that the compound BMS-E30 showed potent IDO1 inhibitory activity (IC 50 = 0.7 nM) in the IDO1 kynurenine assay with human IDO1/HEK 293 cells (Scheme 1) [11], however, its enzyme inhibitory activity was relatively weak with an IC 50 of 8.569 µM ( Table 1). The lack of heme-coordinating element distinguished compound BMS-E30 from other IDO1 inhibitors in the literature. We considered that BMS-E30 effectively inhibited IDO1 by targeting its apo-form subsequent to the disclosure of the IDO1/BMS-978587 crystal structure (6AZV) because the structures of BMS-E30 and BMS-978587 were similar (Scheme 1) [12]. Considering the two flexible chains of diisobutylamino group in compound BMS-E30, a new series of compounds were designed by replacing the diisobutylamino group with a 3,5-dimethylpiperidinyl group for reducing the entropy loss of binding IDO1. The peripheral phenyl urea group was also modified to explore the structure-activity relationship (SAR) which was used for further optimization to obtain better IDO1 inhibitors. Inhibition of IDO1 was shown to increase the therapeutic efficacy of cancer vaccines, immune checkpoint inhibitors, or chemotherapy in multiple clinical mouse models [8][9][10]. On this basis, several IDO1 inhibitors have been developed and are currently under clinical development ( Figure  1) [1].  In this study, a new series of compounds were designed based on the phenyl urea scaffold in order to search for new IDO1 inhibitors. The compounds were synthesized and their IDO1/TDO inhibitory activities were determined. The in vivo pharmacokinetic profile and anti-tumor efficacy of a potent IDO1 inhibitor were evaluated to explore its potential as an anti-tumor agent.

Design Strategies of the Compounds
It was disclosed that the compound BMS-E30 showed potent IDO1 inhibitory activity (IC50 = 0.7 nM) in the IDO1 kynurenine assay with human IDO1/HEK 293 cells (Scheme 1) [11], however, its enzyme inhibitory activity was relatively weak with an IC50 of 8.569 μM ( Table 1). The lack of hemecoordinating element distinguished compound BMS-E30 from other IDO1 inhibitors in the literature. We considered that BMS-E30 effectively inhibited IDO1 by targeting its apo-form subsequent to the disclosure of the IDO1/BMS-978587 crystal structure (6AZV) because the structures of BMS-E30 and BMS-978587 were similar (Scheme 1) [12]. Considering the two flexible chains of diisobutylamino group in compound BMS-E30, a new series of compounds were designed by replacing the diisobutylamino group with a 3,5-dimethylpiperidinyl group for reducing the entropy loss of binding IDO1. The peripheral phenyl urea group was also modified to explore the structure-activity relationship (SAR) which was used for further optimization to obtain better IDO1 inhibitors.

In Vitro Biological Evaluation
We prepared the compounds using the synthetic scheme in Section 2.2 and these compounds were screened in vitro for their IDO1 and TDO inhibitory activities. The reported IDO1 inhibitor epacadostat was used as the reference compound in this study [15].
As shown in Table 1, compounds g1-g3 showed no IDO1 inhibitory activity. When the carboxyl group in compounds i1-i3 was exposed by hydrolysis, IDO1 inhibitory activity appeared. The enzymatic results of compounds g1-g3 and i1-i3 indicated that the carboxyl group plays a critical role in the binding activity. In addition, the replacement of phenyl ring with a cyclohexyl group (compound i4) or an n-hexyl group (compound i5) resulted in loss of inhibition. This suggested that phenyl ring is important for the binding activity.

In Vitro Biological Evaluation
We prepared the compounds using the synthetic scheme in Section 2.2 and these compounds were screened in vitro for their IDO1 and TDO inhibitory activities. The reported IDO1 inhibitor epacadostat was used as the reference compound in this study [15].
As shown in Table 1, compounds g1-g3 showed no IDO1 inhibitory activity. When the carboxyl group in compounds i1-i3 was exposed by hydrolysis, IDO1 inhibitory activity appeared. The enzymatic results of compounds g1-g3 and i1-i3 indicated that the carboxyl group plays a critical role in the binding activity. In addition, the replacement of phenyl ring with a cyclohexyl group (compound i4) or an n-hexyl group (compound i5) resulted in loss of inhibition. This suggested that phenyl ring is important for the binding activity.
At the para-position, substitution with other halogens such as fluorine (i18, 5.475 μM) and bromine (i19, 4.077 μM) had similar activity as chlorine (i3, 5.687 μM). In addition, small para-alkyl substituents (i2, 8.613 μM; i20, 9.975 μM) produced inhibition against IDO1, whereas substitution with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the parasubstituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 μM; i23, 0.415 μM; i24, 0.157 μM) and the p-NO2 derivative (i24) was 55-fold more potent than compound BMS-E30.
At the para-position, substitution with other halogens such as fluorine (i18, 5.475 μM) and bromine (i19, 4.077 μM) had similar activity as chlorine (i3, 5.687 μM). In addition, small para-alkyl substituents (i2, 8.613 μM; i20, 9.975 μM) produced inhibition against IDO1, whereas substitution with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the parasubstituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 μM; i23, 0.415 μM; i24, 0.157 μM) and the p-NO2 derivative (i24) was 55-fold more potent than compound BMS-E30.
The o,p-disubstituted phenyl urea derivatives, bearing o,p-difluoro (i16) or o,p-dichloro (i17) substituents, were also explored and had no inhibitory activities. These results demonstrated a strong preference for para-substitution.
At the para-position, substitution with other halogens such as fluorine (i18, 5.475 µM) and bromine (i19, 4.077 µM) had similar activity as chlorine (i3, 5.687 µM). In addition, small para-alkyl substituents (i2, 8.613 µM; i20, 9.975 µM) produced inhibition against IDO1, whereas substitution with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the para-substituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 µM; i23, 0.415 µM; i24, 0.157 µM) and the p-NO 2 derivative (i24) was 55-fold more potent than compound BMS-E30.
Finally, replacement of proximal NH of the urea with methylene was also investigated. The observation that phenylacetamide derivatives j1, j2, j3 had no inhibitory activity was suggestive that the proximal NH was crucial to IDO1 potency. As shown in Tables 1 and 2, none of the synthesized compounds showed inhibitory activity against TDO, which demonstrated that the phenyl urea derivatives were selective IDO1 inhibitors.
In summary, compound i24 showed the most potent IDO1 inhibitory activity based on the SAR study, however, it contains a nitro group which is considered to be easily metabolized and toxic in vivo [16][17][18], so the second most potent IDO1 inhibitor i12 was used as the lead compound for further study. with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the parasubstituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 μM; i23, 0.415 μM; i24, 0.157 μM) and the p-NO2 derivative (i24) was 55-fold more potent than compound BMS-E30. substituents (i2, 8.613 μM; i20, 9.975 μM) produced inhibition against IDO1, whereas substitution with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the parasubstituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 μM; i23, 0.415 μM; i24, 0.157 μM) and the p-NO2 derivative (i24) was 55-fold more potent than compound BMS-E30. with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the parasubstituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 μM; i23, 0.415 μM; i24, 0.157 μM) and the p-NO2 derivative (i24) was 55-fold more potent than compound BMS-E30.
with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the parasubstituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 μM; i23, 0.415 μM; i24, 0.157 μM) and the p-NO2 derivative (i24) was 55-fold more potent than compound BMS-E30.               with an isopropyl group (i21) led to the loss of activity. This result suggested that the size of the parasubstituent on the benzene ring cannot be too large. Further optimization revealed that substitution with electron withdrawing groups at the para-position was beneficial for the activity (i12, 0.331 μM; i23, 0.415 μM; i24, 0.157 μM) and the p-NO2 derivative (i24) was 55-fold more potent than compound BMS-E30.

Predicted Binding Mode of Compound i12 with IDO1
The molecular docking study was performed to investigate the binding mode of compound i12 with IDO1 (6AZV) by using CDOCKER protocol integrated in Accelrys Discovery Studio [12,19]. As shown in Figure 2, the carboxylic group in compound i12 forms hydrogen bonds with the backbone amide of Ala-264 and with His-346, which is supposed to make critical contributions to the binding since the carboxylic group is an essential pharmacophore as SAR demonstrated.
The phenyl urea group in compound i12 binds via edge-to-face π-interaction with Tyr126 and hydrogen bonds with Ser167, which is also important for potency and is consistent with the SAR results. The peripheral phenyl ring was placed into the hydrophobic pocket where it is suited to extend a small para-substituent, which explained the loss of activity of compounds bearing bulky substituents or substituents at the orthoand meta-positions. Particularly, electron-withdrawing groups at the para-position of the benzene ring were beneficial to the phenyl urea group as hydrogen bond donors, providing a rationale for 100-fold improvement in IDO1 inhibitory activity observed when compound i12 is compared to compound i1.
The molecular docking study was performed to investigate the binding mode of compound i12 with IDO1 (6AZV) by using CDOCKER protocol integrated in Accelrys Discovery Studio [12,19]. As shown in Figure 2, the carboxylic group in compound i12 forms hydrogen bonds with the backbone amide of Ala-264 and with His-346, which is supposed to make critical contributions to the binding since the carboxylic group is an essential pharmacophore as SAR demonstrated.

Pharmacokinetic Study of Compound i12
Compound i12 was further evaluated in in vivo pharmacokinetic study using male C57BL/6 mice. The plasma concentration-time profiles are shown in Figure 3 and the pharmacokinetic parameters were determined by non-compartmental analysis (Table 3). when compound i12 is compared to compound i1.

Pharmacokinetic Study of Compound i12
Compound i12 was further evaluated in in vivo pharmacokinetic study using male C57BL/6 mice. The plasma concentration-time profiles are shown in Figure 3 and the pharmacokinetic parameters were determined by non-compartmental analysis (Table 3).    When intravenously administered at a dose of 3 mg/kg, compound i12 had moderate clearance of 22.45 mL/min/kg and an extensive distribution in tissues with V ss value of 15.80 L/kg. After an oral administration of 30 mg/kg, compound i12 was absorbed with T max value of 2 h and C max value of 2702 ng/mL. Compound i12 was eliminated slowly with a half-time of 11.2 h and showed reasonable oral exposure with AUC(0-∞) value of 11,523 h·ng/mL. The oral bioavailability of compound i12 was determined to be 87.4%, supporting a further evaluation of its in vivo efficacy.

In Vivo Anti-Tumor Efficacy Study of Compound i12
Compound i12 was tested for its in vivo antitumor activities in melanoma and pancreatic cancer xenograft models (Figures 4 and 5). In a mouse B16F10 subcutaneous xenograft model, compound i12 orally administered at 15 mg/kg daily demonstrated obvious in vivo anti-tumor activity and showed tumor growth inhibition (TGI) of 40.5%. In addition, the body weight of i12-treated mice showed no significant changes compared with the control group. The classic first line anti-cancer drug cyclophosphamide (CTX) was used as the reference drug.
Compound i12 was tested for its in vivo antitumor activities in melanoma and pancreatic cancer xenograft models (Figures 4 and 5). In a mouse B16F10 subcutaneous xenograft model, compound i12 orally administered at 15 mg/kg daily demonstrated obvious in vivo anti-tumor activity and showed tumor growth inhibition (TGI) of 40.5%. In addition, the body weight of i12-treated mice showed no significant changes compared with the control group. The classic first line anti-cancer drug cyclophosphamide (CTX) was used as the reference drug. We also evaluated compound i12 in a PAN02 pancreatic cancer xenograft model, in which compound i12 oral treatment resulted in a 34.3% decrease in tumor weight at a dose of 30 mg/kg daily compared with the control group.

General Information
Reagents and solvents were obtained from commercial suppliers and used as received. 1 H-NMR spectra were obtained on a 400 MHz Mercury NMR spectrometer (Varian, San Diego, CA, USA). The control group mice bearing PAN02 pancreatic cancer xenografts were dosed orally with vehicle (0.5% sodium salt of carboxymethyl cellulose, CMC-Na); the CTX group were administered CTX intraperitoneally at the dose of 60 mg/kg; the treated group were administered i12 orally at the dose of 10, 30 or 100 mg/kg. ** p < 0.01 and *** p < 0.001 versus vehicle. (B) The body weight of each group after the treatment. There is no obvious body weight difference among any of the i12-treated groups.
We also evaluated compound i12 in a PAN02 pancreatic cancer xenograft model, in which compound i12 oral treatment resulted in a 34.3% decrease in tumor weight at a dose of 30 mg/kg daily compared with the control group.

General Information
Reagents and solvents were obtained from commercial suppliers and used as received. 1 H-NMR spectra were obtained on a 400 MHz Mercury NMR spectrometer (Varian, San Diego, CA, USA). Electrospray ionization (ESI) mass spectra and high-resolution mass spectroscopy (HRMS) were performed with a liquid chromatograph/mass selective detector time-of-flight mass spectrometer (LC/MSD TOF, Agilent Technologies, Santa Clara, CA, USA). Silica gel column chromatography was performed with silica gel 60G (Qingdao Haiyang Chemical, Qingdao, China). Purity was determined using HPLC, LC/MS and NMR spectroscopy (Supplementary Materials). All of the synthesized compounds have purities over 95%.

General Procedure B for the Synthesis of i1-i24 and j1-j3
To a solution of compound g1-g24 or h1-h3 (1 equiv.) in tetrahydrofuran (4 volumes), methanol (1 volume) and water (1 volume) was added sodium hydroxide (3 equiv.). The resulting mixture was stirred at room temperature until the starting material disappeared in TLC. Part of the tetrahydrofuran and methanol was removed in vacuo and the crude was diluted with water (2 volumes) and the pH was adjusted to ca. 4 using 1 N HCl solution. The aqueous phase was then extracted with ethyl acetate (15 v × 3) and the combined organic extracts were washed with saturated NaCl solution, dried over anhydrous Na 2 SO 4 and concentrated. The residue was purified by column chromatography (silica gel, DCM/MeOH = 30:1, v/v) to afford the products i1-i24 or j1-j3.   Reaction of compound f and p-tolyl isocyanate following the general procedure A afforded compound g2 (white solid, 86.1% yield). 1

The Enzyme Assay for IDO1 and TDO Inhibition
Recombinant human IDO1 and TDO were expressed and purified according to the reported procedures [20]. The assay was performed according to the literature: A standard reaction mixture (100 µL) containing 100 mM potassium phosphate buffer (pH 6.5), 40 mM ascorbic acid (neutralized with NaOH), 200 µg/mL catalase, 20 µM methylene blue and 0.05 µM rhIDO1 or rhTDO was added to the solution containing the substrate L-tryptophan and the test sample at a determined concentration. The reaction was carried out at 37 • C for 45 min and stopped by adding 20 µL of 30% (w/v) trichloroacetic acid. After heating at 65 • C for 15 min, 100 µL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid was added to each well. The yellow pigment derived from kynurenine was measured at 492 nm using a SYNERGY-H1 microplate reader (Biotek Instruments, Inc., Winooski, VT, USA). IC 50 was analyzed using the GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) [

Pharmacokinetic Studies
The animal Care and Welfare Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences approved all animal care, housing, and laboratory procedures. Male C57BL/6 mice were used in the single dose pharmacokinetic studies. Compound i12 was prepared as a 3 mg/mL suspension with 0.5% CMC for oral use and was formulated as a 3 mg/mL solution with 10% DMSO in 20% HP-β-CD for intravenous injection. Sixteen mice were divided into two groups, 10 in the oral group and six in the intravenous group. After fasting 12 h with free access to water, mice were treated with a 3 mg/kg i.v. or 30 mg/kg oral dose of compound i12. Blood samples (50 µL) were collected at 5, 15, 30 min, 1, 2, 4, 6, 8, 12 and 24 h after oral administration and 2, 5, 15, 30 min, 1, 2, 4, 6, 8, 12 and 24 h after intravenous injection. After centrifugation, the plasma samples (20 µL) were precipitated by four volumes of acetonitrile. The supernatant were analyzed by liquid chromatography/tandem mass spectrometry with a Zorbax C18 column (50 mm × 2.1 mm, 3.5 µm). Compound detection was performed with the mass spectrometer in positive ionization mode by t-SIM: m/z 489.209 for compound i12. The pharmacokinetic parameters were calculated with WinNonlin software V6.3 using non-compartmental analysis (Pharsight Corporation, Mountain View, CA, USA).

In Vivo Studies
The mouse melanoma cells B16F10 were cultured and harvested in saline at 6 × 10 6 cells/0.2 mL volume. Cells (0.2 mL) were injected subcutaneously into male C57BL/6 mice at day 0 of the experiment, and treatment was initiated at day 1 following the mice enrolled randomly in control and experimental groups. For control group, 0.5% CMCNa was orally administered every day. The CTX group were administered CTX intraperitoneally at the dose of 100 mg/kg. Compound i12 was dissolved in 0.5% CMC-Na for oral treatment. After 17 days, the mice were sacrificed and the tumors were stripped and weighted. The tumor growth inhibition (TGI) was calculated as TGI = (1 − tumor weight treatment /tumor weight vehicle ) × 100%. The statistical analysis was performed with GraphPad Prism 8.0 software and the significance level was evaluated with one-way ANOVA model [22].
The mouse pancreatic cancer cells PAN02 were cultured and harvested in saline at 6 × 10 6 cells/0.2 mL volume. Cells (0.2 mL) were injected subcutaneously into male C57BL/6 mice at day 0 of the experiment, and treatment was initiated at day 1 following the mice enrolled randomly in control and experimental groups. For control group, 0.5% CMCNa was orally administered every day.
The CTX group were administered CTX intraperitoneally at the dose of 60 mg/kg Compound i12 was dissolved in 0.5% CMC-Na for oral treatment. After 15 days, the mice were sacrificed and the tumors were stripped and weighted. The tumor growth inhibition (TGI) was calculated as TGI = (1 − tumor weight treatment /tumor weight vehicle ) × 100%. The statistical analysis was performed with GraphPad Prism 8.0 software and the significance level was evaluated with one-way ANOVA model [22].

Conclusions
In summary, we designed a new series of phenyl urea derivatives as IDO1 inhibitors through a ring formation strategy. Systematic SAR led to the discovery of the promising anticancer compound i12 with favourable drug-like properties. Compound i12 had potent IDO1 inhibitory activity with the IC 50 of 0.331 µM and exhibited a satisfactory PK profile with moderate plasma clearance (22.45 mL/min/kg) and high oral bioavailability (87.4%). In addition, Compound i12 orally administered at 15 mg/kg daily showed a TGI of 40.5% in a B16F10 subcutaneous xenograft model and at 30 mg/kg daily showed a TGI of 34.3% in a PAN02 subcutaneous xenograft model. Overall, compound i12 is a promising anti-tumor agent with the potent in vitro enzymatic activity, good pharmacokinetic properties and satisfied in vivo anti-tumor efficacy.