Design and Synthesis of Indoleamine 2,3-Dioxygenase 1 Inhibitors and Evaluation of Their Use as Anti-Tumor Agents

Indoleamine 2,3-dioxygenase (IDO) 1 is the key enzyme for regulating tryptophan metabolism and is an important target for interrupting tumor immune escape. In this study, we designed four series of compounds as potential IDO1 inhibitors by attaching various fragments or ligands to indole or phenylimidazole scaffolds to improve binding to IDO1. The compounds were synthesized and their inhibitory activities against IDO1 and tryptophan 2,3-dioxygenase were evaluated. The cytotoxicities of the compounds against two tumor cell lines were also determined. Two compounds with a phenylimidazole scaffold (DX-03-12 and DX-03-13) showed potent IDO1 inhibition with IC50 values of 0.3–0.5 μM. These two IDO1 inhibitors showed low cell cytotoxicity, which indicated that they may exert their anti-tumor effect via immune modulation. Compound DX-03-12 was investigated further by determining the in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that DX-03-12 had satisfactory properties in mice, with rapid absorption, moderate plasma clearance (∼36% of hepatic blood flow), acceptable half-life (∼4.6 h), and high oral bioavailability (∼96%). Daily oral administration of 60 mg/kg of compound DX-03-12 decreased tumor growth by 72.2% after 19 days in a mouse melanoma cell B16-F10 xenograft model compared with the untreated control. Moreover, there was no obvious weight loss in DX-03-12-treated mice. In conclusion, compound DX-03-12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.

IDO1 is an important enzyme in tryptophan metabolism and contributes to creating peripheral immune tolerance by degrading l-tryptophan, which suppresses the function of T cells, and by producing N-formylkynurenine, which inhibits T cells [8]. In addition, IDO1 can also trigger other activators of anti-tumor immunity. Notably, IDO1 is overexpressed in a range of cancer tissues [4][5][6]. Thus, blocking the activity of IDO1 is a potential strategy for tumor immunotherapy.
The crystal structures of recombinant human IDO1 have been resolved [9,10], and biochemical studies have revealed that the heme iron in the active site of IDO1 binding and forms adducts with exogenous ligands including O 2 − , NO, CO, H 2 S, and CN − [9,[11][12][13][14]. IDO1 tolerates various substrates, whereas TDO has strict substrate specificity. This difference can be used to develop selective IDO1 inhibitors [8].
The crystal structures of recombinant human IDO1 have been resolved [9,10], and biochemical studies have revealed that the heme iron in the active site of IDO1 binding and forms adducts with exogenous ligands including O2 − , NO, CO, H2S, and CN − [9,[11][12][13][14]. IDO1 tolerates various substrates, whereas TDO has strict substrate specificity. This difference can be used to develop selective IDO1 inhibitors [8].
During the development of anti-tumor drugs, potent IDO1 inhibitors were discovered, and intensive efforts have been made to develop potent IDO1/TDO inhibitors with various structural scaffolds. Some typical examples of IDO1/TDO inhibitors are shown in Figure 1 [1,[15][16][17][18][19][20][21]. The indole and phenylimidazole scaffolds have produced several potent IDO1 inhibitors. For example, IDO1 inhibitors indoximod (pathway IC50 = 450 nM) and PF-0684003 (IDO1 IC50 = 410 nM) are indole derivatives [13], and GDC-0919 (IDO1 IC50 = 13 nM) and AMCL-17g (IDO1 IC50 = 77 nM) are phenylimidazole derivatives [20]. In this study, in the early stages of searching for new IDO1 inhibitors, we used indole and phenylimidazole as the core scaffolds. Four series of compounds were designed with different strategies. The compounds were synthesized and the 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 Strategy for the Four Compound Series
Four series of compounds were designed ( Figure 2) to explore the structure-activity relationship (SAR). The SAR information was used for further optimization. Indole is derived from tryptophan and phenylimidazole is an IDO1 inhibitor [18]. Thus, tryptophan and phenylimidazole fit into the active site of IDO1, and they were selected as the core scaffolds for designing the inhibitors. As we mentioned in the introduction, several exogenous ligands including hydroxylamine, O2 − , NO, CO, H2S, and CN − were able to bind with the heme iron in the active site of IDO1 [9,[11][12][13][14]. We attached various fragments and ligands to these scaffolds to strengthen the binding to the active site of IDO1. In this study, in the early stages of searching for new IDO1 inhibitors, we used indole and phenylimidazole as the core scaffolds. Four series of compounds were designed with different strategies. The compounds were synthesized and the 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.
In series 1, several ligands, such as hydroxylamine and SO, that interact strongly with heme iron were linked to the double bond of the indole scaffold. We hoped to observe a synergistic effect between the ligand and the indole ring in the IDO1 binding. Series 2 was based on compound PF-0684003 (Figure 1), which is an IDO1 inhibitor developed by Pfizer. The structure of PF-0684003 cocrystalized with IDO1 revealed that there was no interaction between PF-0684003 and the heme iron, which is usually observed between other IDO1 inhibitors and IDO1 [13]. Thus, several potential heme binding ligands, such as hydroxylamine and SO, were added to the structure of PF-0684003 to enhance the inhibitory activity.
Phenylimidazole is a weak IDO1 inhibitor. Crystal structures have revealed that the imidazole ring binds with the heme iron [18,19]. Thus, in series 3 and 4, we used phenylimidazole as the scaffold, and we attached fragments to either the imidazole ring (series 3) or the phenyl ring (series 4). These fragments were intended to improve the binding in the active site of IDO1 through various interactions, such as hydrogen bonds and π-π interactions.

Synthesis of Selected Compounds
Although the compounds were classified into four series for examining the SAR, the structural diversity meant that they required a variety of synthetic routes. We describe representative synthetic schemes for four compounds. Syntheses for all the compounds are included in the Experimental section. Synthesis of DX-03-12.
2-Iodoimidazole was substituted with trifluoromethylbenzyl bromide to give compound 12. Compound 12 underwent a Suzuki-Miyaura coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr 3 to obtain DX-04-02. Scheme 3 shows the synthesis of DX-03-12. First, the imidazole starting material was protected with trityl chloride to avoid various side reactions. Compound 6 was obtained by the Suzuki-Miyaura reaction with a boronic acid [22]. The methyl group was removed by BBr3 to expose the hydroxyl group in compound 7. Compound 8 was prepared in parallel by melting 3chloropropylamine and isobenzofuran-1,3-dione together. Compounds 7 and 8 were subjected to an SN2 nucleophilic reaction to obtain compound 9 under strong basic conditions. This step had a low yield, which was explained by the high steric hindrance of the large molecules and the weak nucleophilicity of compound 7. The amine protecting group was removed with hydrazine hydrate to obtain compound 10. Compound 10 was reacted with trifluoromethanesulfonylchloride under basic conditions to give compound 11. Finally, DX-03-12 was obtained by removing the protecting group on the imidazole with acetic acid.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl group was removed with BBr3 to obtain DX-04-02.

In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve commercially available compounds were purchased (see Section 3.1). In total, we screened 50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor, and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in this study. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table  1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table  1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table  1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table  1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table  1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table  1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table  1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured ( Table 1). The series 1 compounds contained heme binding fragments including CO, NO, CN, H 2 S, imidazole, and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC 50 of 11.1 µM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron.
Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines. lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines. Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines. Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines. Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. 16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines. Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. 16, and DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1 inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 μM. In addition, cell cytotoxicity screening showed that most of the compounds were not toxic to mammalian cancer cell lines. Generally speaking, these molecules have a rather low molecular weight, and the rather small moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was observed for this series of compounds. Interestingly, for these four active compounds, they all contain the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to the double bond of the indole scaffold, while still maintaining the binding with the heme iron. Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines. The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines. The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines. The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines. The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study. Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However, the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that these PF-0684003 derivatives were not toxic to cancer cell lines.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a previous study.  (Table 4). Most compounds in this series showed marginal IDO1 inhibition, but compound DX-04-05 showed moderate IDO1 inhibition. However, compound DX-04-06, in which the hydroxyl group was methylated, showed decreased IDO1 inhibition. Most compounds in this series were toxic to the cancer cell lines H460 and MCF7 with an IC 50 of around 10 −5 M, which indicates the compounds also interrupt the function of other targets in the cells. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10 showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11, DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3-2.4 μM. Interestingly, these three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors. In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1. The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine (DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13, the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported IDO1 inhibitors [17,19]. The terminal fragments, such as sulfamide and urea, are important to maintain the binding [17,19]. In series 4, the design strategy was to attach fragments to the imidazole ring instead of the phenyl ring in series 3. However, this strategy does not maintain the IDO1 inhibitory activity. In addition, the free hydroxyl group in series 4 appears to be important for forming a hydrogen bond with an amino acid residue, such as Ser167 [7], in the active site; methylating this hydroxyl group decreased IDO1 inhibition (DX-04-05 versus DX-04-06).
In summary, based on the SAR study, compounds DX-03-12 and DX-03-13 showed potent IDO1 inhibition activity, and compound DX-03-13 has some structure similarity as previous published patent (WO2011/056652Al) [23]. Thus, the most potent IDO1 inhibitor DX-03-12 was used as the lead compound for further study.

Pharmacokinetic Study of DX-03-12
The in vivo pharmacokinetic properties of compound DX-03-12 were evaluated in male ICR mice. DX-03-12 was administered orally either as a bolus at a dose of 30 mg/kg or intravenously at a dose of 3 mg/kg. The plasma concentration-time profiles are shown in Figure 3. The pharmacokinetic parameters were determined by non-compartmental analysis ( Table 5). After intravenous administration (3 mg/kg), compound DX-03-12 showed a moderate clearance of 33.3 mL/min/kg, which was approximately 36% of mouse hepatic blood flow (90 mL/min/kg). The volume of distribution in the steady state was 10 L/kg, suggesting that there was an extensive distribution of compound DX-03-12 in tissues. Compound DX-03-12 was rapidly absorbed after oral administration of 30 mg/kg. The plasma concentration was quantifiable at the first sampling time point (5 min) and remained detectable at 24 h. The maximum plasma concentration of compound DX-03-12 of 2963 ng/mL was observed 1 h post-dose. Compound DX-03-12 was eliminated relatively slowly with a half-life of 4.6 h. The oral bioavailability of compound DX-03-12 was calculated to be 96%.
The pharmacokinetic study revealed that compound DX-03-12 showed rapid absorption and almost complete oral bioavailability in mice. After oral administration of 30 mg/kg in mice, it exhibited a long half-life (4.6 h) due to moderate plasma clearance. The pharmacokinetic properties of compound DX-03-12 in mice indicate that it has the potential to be a once-a-day drug.

In Vivo Anti-Tumor Efficacy Study of Compound DX-03-12
To determine whether the compound was effective against tumors in vivo, the anti-tumor effect of DX-03-12 was evaluated in a B16F10 subcutaneous xenograft model in syngeneic mice (Figure 4). Oral and intraperitoneal (i.p.) administration (30 or 60 mg/kg/day) of DX-03-12 significantly decreased the growth of melanoma B16F10 xenograft tumors in a dose-dependent manner. DX-03-12

In Vivo Anti-Tumor Efficacy Study of Compound DX-03-12
To determine whether the compound was effective against tumors in vivo, the anti-tumor effect of DX-03-12 was evaluated in a B16F10 subcutaneous xenograft model in syngeneic mice (Figure 4). Oral and intraperitoneal (i.p.) administration (30 or 60 mg/kg/day) of DX-03-12 significantly decreased the growth of melanoma B16F10 xenograft tumors in a dose-dependent manner. DX-03-12 oral treatment resulted in a 72.2% decrease in tumor weight and i.p. treatment resulted in a 72.3% decrease at a dose of 60 mg/kg compared with the control tumor after 19 days treatment. DX-03-12 had no obvious effect on the body weight of the mice and the peripheral white blood cells, which indicated that DX-03-12 had low toxicity in mice. In this study, the classic first line anti-cancer drug cyclophosphamide (CTX) was used as the reference drug. Figure 3. Mean plasma concentration-time profile in male ICR mice after administration of a single oral (30 mg/kg) or intravenous (3 mg/kg) dose of compound DX-03-12. PO stands for per os, which means oral administration of drug; iv stands for intravenous administration of drug.

In Vivo Anti-Tumor Efficacy Study of Compound DX-03-12
To determine whether the compound was effective against tumors in vivo, the anti-tumor effect of DX-03-12 was evaluated in a B16F10 subcutaneous xenograft model in syngeneic mice (Figure 4). Oral and intraperitoneal (i.p.) administration (30 or 60 mg/kg/day) of DX-03-12 significantly decreased the growth of melanoma B16F10 xenograft tumors in a dose-dependent manner. DX-03-12 oral treatment resulted in a 72.2% decrease in tumor weight and i.p. treatment resulted in a 72.3% decrease at a dose of 60 mg/kg compared with the control tumor after 19 days treatment. DX-03-12 had no obvious effect on the body weight of the mice and the peripheral white blood cells, which indicated that DX-03-12 had low toxicity in mice. In this study, the classic first line anti-cancer drug cyclophosphamide (CTX) was used as the reference drug. Mice bearing B16F10 xenografts were dosed orally or i.p. with vehicle (0.5% methylcellulose), CTX (Cyclophosphamide, orally, 60 mg/kg twice a week), DX-03-12 (i.p., 30 mg/kg/day), DX-03-12 (i.p., 60 mg/kg/day), DX-03-12 (orally, 30 mg/kg/day), or DX-03-12 (orally, 60 mg/kg/day). Results are expressed as the mean ± SD (n = 5-7 for each group). * p < 0.05 and *** p < 0.001 versus vehicle. (C) The body weight from each group after the treatment. There is no obvious body weight difference among all of the groups. (D) The white blood cells detected in the blood of all groups of animals. There is also no obvious WBC difference among all of the groups.

Chemistry
Reagents and solvents were obtained from commercial suppliers and used as received. 1 H-NMR spectra were obtained on an NMR spectrometer (Mercury, Varian, San Diego, CA, USA; 400 MHz). 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. All of the synthesized compounds have the purity over than 95%.

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 ICR mice were used in the single dose pharmacokinetic studies. Compound DX-03-12 was prepared as 3 mg/mL suspension with 0.5% CMC (containing 0.3% Tween 80) for oral use and was formulated as 3 mg/mL solution with 5% DMSO in saline for intravenous injection. Twenty mice were divided into two groups, 10 in each 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 DX-03-12. 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 (15 µL) were precipitated by five volumes of acetonitrile. The supernatant were analyzed by liquid chromatography/tandem mass spectrometry (Agilent Technologies, Santa Clara, CA, USA) with a Zobax C18 column (50 mm × 2.1 mm, 3.5 µm). Compound detection was performed with the mass spectrometer in MRM (multiple reaction monitoring, Agilent Technologies, Santa Clara, CA, USA) negative ionization mode. The selected reaction monitoring transitions were m/z 348.5→160 for compound DX-03-12. 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 day 0 of the experiment, 1 × 10 6 cells were injected subcutaneously into mice, and treatment was initiated at day 1 following the mice enrolled randomly in control and experimental groups [27]. For control group, 0.5% CMCNa was orally administered every day. Compound DX-03-12 was dissolved in 0.5% CMC for oral treatment or dissolved in saline for intraperitoneal treatment. When the mice were sacrificed, 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 a one-way ANOVA model.

Conclusions
We designed four series of IDO1 inhibitors using indole and phenylimidazole as the scaffolds. The design strategies were based on the mechanism of IDO1, focusing on the interaction between the inhibitor and the heme iron. The four series of compounds were synthesized and their IDO1 and TDO inhibition was evaluated. Most compounds showed marginal IDO1 inhibition, but eight compounds showed moderate IDO1 inhibition. In particular, DX-03-12 and DX-03-13 showed IDO1 inhibition with IC 50 s of 0.3 and 0.5 µM and low cell cytotoxicities against two cancer cell lines. A pharmacokinetic study showed that DX-03-12 had satisfactory properties, with rapid absorption, moderate plasma clearance, acceptable half-life, and high oral bioavailability. The in vivo anti-tumor efficacy in a B16F10 subcutaneous xenograft mouse model showed that DX-03-12 orally administered at a dose of 60 mg/kg inhibited tumor growth by 72.2% compared with the control tumors. In summary, IDO1 inhibitor DX-03-12 exhibits promising drug like properties, good pharmacokinetic properties, low cellular toxicity, and impressive in vivo anti-tumor efficacy.