Discovery of Indoleamine 2,3-Dioxygenase 1 (IDO-1) Inhibitors Based on Ortho-Naphthaquinone-Containing Natural Product

There is great interest in developing small molecules agents capable of reversing tumor immune escape to restore the body’s immune system. As an immunosuppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO-1) is considered a promising target for oncology immunotherapy. Currently, none of IDO-1 inhibitors have been launched for clinical practice yet. Thus, the discovery of new IDO-1 inhibitors is still in great demand. Herein, a series of diverse ortho-naphthaquinone containing natural product derivatives were synthesized as novel IDO-1 inhibitors. Among them, 1-ene-3-ketone-17-hydroxyl derivative 12 exhibited significantly improved enzymatic and cellular inhibitory activity against IDO-1 when compared to initial lead compounds. Besides, the molecular docking study disclosed that the two most potent compounds 11 and 12 have more interactions within the binding pocket of IDO-1 via hydrogen-bonding, which may account for their higher IDO-1 inhibitory activity.


Introduction
Although immune checkpoint blockades such as anti-PD-L1 (programmed cell death-ligand 1), anti-PD-1 (programmed cell death protein 1) [1], and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) [2] have demonstrated attractive therapeutic effects in multiple clinical trials, this new modality often suffers from a low response rate at least due to the immune escape developed by tumors [3].Therefore, there is great interest in developing small molecule agents capable of reversing tumor immune escape to restore the body's immune system.
Indoleamine 2,3-dioxygenase 1 (IDO-1) is a monomeric heme-containing enzyme found in nonhepatic human tissues [4].It catalyzes the oxidative cleavage of the pyrrole ring of L-tryptophan (L-Trp) in the first and rate-limiting step of the kynurenine pathway to produce N-formylkynurenine [5].This reaction not only leads to a local decrease of L-Trp concentration, but also generates a variety of catabolic products, which both account for the immunosuppressive effects of IDO-1 [6][7][8][9].Thus, IDO-1 is considered as one of the key factors in the process of immune evasion in tumor microenvironment [10].Overexpression of IDO-1 has been observed in a number of human malignancies, including ovarian, colorectal, and pancreatic cancers [11].A growing body of clinical evidence indicated that the high expression of IDO-1 in tumors is directly correlated with a low survival rate [12].In addition, IDO-1 inhibitors have demonstrated a significant cooperative effect with chemotherapy, radiotherapy, or cancer vaccines in preclinical models of cancer [13][14][15][16].Accordingly, inhibition of IDO-1 would be a very useful strategy for the treatment of cancer.In the past few years, intense efforts have been devoted to developing IDO-1 inhibitors for the immuno-oncology therapy [17].A number of structurally diverse natural products and synthetic IDO-1 inhibitors have been reported.Several IDO-1 inhibitors entered clinical trials such as indoximod (1) [13,18], epacadostat (2) [3], and NLG919 (3) (Figure 1) [19].However, none of them have been launched for clinical practice yet.Thus, the discovery of new IDO-1 inhibitors is still in great demanded.
Natural products have long served as valuable starting points for drug discovery due to their unique molecular frameworks and novel mechanisms of actions.Danshen, a well-known traditional Chinese medicine (TCM) herb derived from the dried root or rhizome of Salvia miltiorrhiza Bunge, has long been used in Asian countries for multiple therapeutic remedies including cardiovascular and cerebrovascular disorders as well as inflammatory diseases [20][21][22].Tanshinones such as Tan-IIA (6) (Figure 1), a group of lipophilic furano-o-naphthaquinone diterpenes isolated exclusively from this TCM herb, have demonstrated various pharmacological activities, such as cardio-protection, antibacterial, anti-inflammatory, antioxidant, anti-platelet aggregation, and anticancer properties [23,24].
Molecules 2019, 24, x 2 of 11 of catabolic products, which both account for the immunosuppressive effects of IDO-1 [6][7][8][9].Thus, IDO-1 is considered as one of the key factors in the process of immune evasion in tumor microenvironment [10].Overexpression of IDO-1 has been observed in a number of human malignancies, including ovarian, colorectal, and pancreatic cancers [11].A growing body of clinical evidence indicated that the high expression of IDO-1 in tumors is directly correlated with a low survival rate [12].In addition, IDO-1 inhibitors have demonstrated a significant cooperative effect with chemotherapy, radiotherapy, or cancer vaccines in preclinical models of cancer [13][14][15][16].
Accordingly, inhibition of IDO-1 would be a very useful strategy for the treatment of cancer.In the past few years, intense efforts have been devoted to developing IDO-1 inhibitors for the immunooncology therapy [17].A number of structurally diverse natural products and synthetic IDO-1 inhibitors have been reported.Several IDO-1 inhibitors entered clinical trials such as indoximod (1) [13,18], epacadostat (2) [3], and NLG919 (3) (Figure 1) [19].However, none of them have been launched for clinical practice yet.Thus, the discovery of new IDO-1 inhibitors is still in great demanded.
Natural products have long served as valuable starting points for drug discovery due to their unique molecular frameworks and novel mechanisms of actions.Danshen, a well-known traditional Chinese medicine (TCM) herb derived from the dried root or rhizome of Salvia miltiorrhiza Bunge, has long been used in Asian countries for multiple therapeutic remedies including cardiovascular and cerebrovascular disorders as well as inflammatory diseases [20][21][22].Tanshinones such as Tan-IIA (6) (Figure 1), a group of lipophilic furano-o-naphthaquinone diterpenes isolated exclusively from this TCM herb, have demonstrated various pharmacological activities, such as cardio-protection, antibacterial, anti-inflammatory, antioxidant, anti-platelet aggregation, and anticancer properties [23,24].As part of our drug discovery program towards identification of lead compounds from natural products bearing o-naphthaquinone scaffold [25][26][27][28][29][30][31], we screened our in-house compound library derived from o-naphthaquinones to pursue novel IDO-1 inhibitors, and found acyloxy derivatives of As part of our drug discovery program towards identification of lead compounds from natural products bearing o-naphthaquinone scaffold [25][26][27][28][29][30][31], we screened our in-house compound library derived from o-naphthaquinones to pursue novel IDO-1 inhibitors, and found acyloxy derivatives of 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three o-naphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26][27][28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).   1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7h
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7b
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7i
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7c
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone 7j Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.
As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.
As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.
6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.
As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone 7k Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7e
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7l
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7m
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

H 7g
Molecules 2019, 24, x 3 of 11 6 possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. using machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further performed structural optimization based on the acyloxy derivatives to explore the chemical space of o-naphthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical synthesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel IDO-1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone H As outlined in Scheme 1, further hydrolysis of compound 7a with K 2 CO 3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 • C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H 2 O for 1.5 h to directly produce the ∆ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.

Biological Evaluation
All the synthesized derivatives were evaluated for their IDO-1 inhibitory activities using epacadostat as the positive control agent.The potent compounds were further assayed for their cellular activity in HEK 293 cells over-expressing human IDO-1.As shown in Table 2, a series of acyloxylated derivatives 7a-m was first evaluated.Compared to 6, acyclic acyloxylated derivatives 7a-b and 7e-f displayed significantly improved IDO-1 inhibitory activity with IC50 values ranging from 2 to 8 µM.However, installation of aryl or heteroaryl group at C-15 of the furan ring (7c-d) resulted in loss of the activity with IC50 value greater than 20 µM.Among cyclic acyloxylated derivatives 7g-m, furan compound 7k possessed the most potent activity against IDO-1 with an IC50 value of 4.60 µM.Most of the six-membered cyclic acyloxylated derivatives 7h-i exhibited relatively weak activity with IC50 values around 20 µM except pyrazine compound 7m.Hydrolysis of the acyloxyl into the hydroxyl (compound 8) displayed comparable inhibitory activity to that of precursor 7a.Compound 9 with 1-ene possessed 61.2% inhibitory rate at 20 µM, which is less potent than that reported by literature [32].Compound 10 with hydroxyl group at the allylic position recovered the activity with an IC50 value of 3.10 µM.Compound 11 with 1-ene-3-ketone moiety exhibited significantly improved IDO-1 inhibitory activity with an IC50 value of 0.84 µM, and 17hydroxyl derivative 12 also possessed more potent activity with 0.37 µM IC50 value.By contrast, both 1-ketone-2-ene compound 14 and its 17-hydroxyl analogue 15 demonstrated weak activities with IC50 values around 20 µM.

Biological Evaluation
All the synthesized derivatives were evaluated for their IDO-1 inhibitory activities using epacadostat as the positive control agent.The potent compounds were further assayed for their cellular activity in HEK 293 cells over-expressing human IDO-1.As shown in Table 2, a series of acyloxylated derivatives 7a-m was first evaluated.Compared to 6, acyclic acyloxylated derivatives 7a-b and 7e-f displayed significantly improved IDO-1 inhibitory activity with IC 50 values ranging from 2 to 8 µM.However, installation of aryl or heteroaryl group at C-15 of the furan ring (7c-d) resulted in loss of the activity with IC 50 value greater than 20 µM.Among cyclic acyloxylated derivatives 7g-m, furan compound 7k possessed the most potent activity against IDO-1 with an IC 50 value of 4.60 µM.Most of the six-membered cyclic acyloxylated derivatives 7h-i exhibited relatively weak activity with IC 50 values around 20 µM except pyrazine compound 7m.Hydrolysis of the acyloxyl into the hydroxyl (compound 8) displayed comparable inhibitory activity to that of precursor 7a.Compound 9 with 1-ene possessed 61.2% inhibitory rate at 20 µM, which is less potent than that reported by literature [32].Compound 10 with hydroxyl group at the allylic position recovered the activity with an IC 50 value of 3.10 µM.Compound 11 with 1-ene-3-ketone moiety exhibited significantly improved IDO-1 inhibitory activity with an IC 50 value of 0.84 µM, and 17-hydroxyl derivative 12 also possessed more potent activity with 0.37 µM IC 50 value.By contrast, both 1-ketone-2-ene compound 14 and its 17-hydroxyl analogue 15 demonstrated weak activities with IC 50 values around 20 µM.Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound 12 exhibited the

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound 12 exhibited the

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse acyloxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 45%-95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded 1-hydroxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the presence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% yield.To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was performed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly produce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead of the 1-ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, but also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds 11 and 12 were produced by further oxidation of 10 although it was not observed in the reaction.Alternatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of compound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound 12 exhibited the  1) [32].Inspired by these results, we further rmed structural optimization based on the acyloxy derivatives to explore the chemical space of hthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical esis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel 1 inhibitors.

sults and Discussion
hemistry Following our previously established acyloxylation procedure [26 -28], a series of diverse xy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded droxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the nce of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% .To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was rmed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly uce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead  1) [32].Inspired by these results, we further rmed structural optimization based on the acyloxy derivatives to explore the chemical space of hthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical esis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel 1 inhibitors.

sults and Discussion
hemistry Following our previously established acyloxylation procedure [26 -28], a series of diverse xy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in 95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded roxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the nce of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% .To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was rmed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly uce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead g machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further rmed structural optimization based on the acyloxy derivatives to explore the chemical space of phthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical hesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel -1 inhibitors.

sults and Discussion
hemistry Following our previously established acyloxylation procedure [26 -28], a series of diverse xy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in -95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded droxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the ence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% .To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was rmed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly uce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead g machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further ormed structural optimization based on the acyloxy derivatives to explore the chemical space of phthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical hesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel -1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse oxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in -95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded droxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the ence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% .To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was ormed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly uce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35] in 81% yield as sole product, instead g machine-learning-based virtual screening (Figure 1) [32].Inspired by these results, we further ormed structural optimization based on the acyloxy derivatives to explore the chemical space of phthaquinone scaffold for IDO-1 inhibition.Herein, we disclosed our effort on the chemical hesis, biological evaluation, and molecular docking of o-naphthaquinone derivatives as novel -1 inhibitors.

Chemistry
Following our previously established acyloxylation procedure [26 -28], a series of diverse oxy derivatives 7a-m [27] were readily accessed by action of different carboxylic acids with 6 in -95% yields (Table 1).As outlined in Scheme 1, further hydrolysis of compound 7a with K2CO3 in methanol afforded droxyl product 8 [33] in 99% yield, which further underwent an elimination reaction in the ence of pyridinium p-toluenesulfonate (PPTS) at 110 °C to give the 1-ene analogue 9 [33] in 85% .To introduce a hydroxyl group to the 3-position of the A-ring, an allylic oxidation was ormed by treatment of 9 with selenium dioxide in refluxing 1,4-dioxane/H2O for 1.5 h to directly uce the Δ 1 -3-ketone (1-ene-3-ketone) derivative 11 [34,35]  Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound 12 exhibited the Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound 12 exhibited the Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound 12 exhibited the Compounds 9-14 were further assayed for their activity to inhibit IDO-1 in HEK 293-hIDO-1 cells.Compared to their enzymatic activity, most of them displayed less potent inhibitory activity against IDO-1 probably due to their poor permeability.Among them, compound 12 exhibited the most potent cellular inhibitory activity with an IC 50 value of 3.85 µM.The calculated cLogP values of 11 and 12 with Chemdraw were 3.90 and 2.36, respectively, which may account for their difference on the cellular potency.

Molecular Docking Study
To understand the binding modes of our compounds for IDO-1, compounds 11, 12, and 14 were docked into the binding pocket of an IDO-1 X-ray crystal structure (PDB code 5WHR).All of them could interact with residues Phe226, Thr379, Phe163, Tyr126, and Gly378 within hydrophobic pockets (Figure S17).The ortho-diketone moiety of 11 interacted with heme Fe 2+ through two coordination bonds, and its 3-ketone had an additional hydrogen-bond interaction with Thr379 (Figure 2a).For compound 12, the ortho-diketone moiety not only formed two coordination bonds with heme Fe 2+ (Figure 2b), but also had a hydrogen-bond interaction with the NH of Ala264 (Figure S17b).Besides, the 17-hydroxyl group of 12 also interacted with residue Ser167 via a hydrogen-bond (Figure S17b).The 1-ketone group of 14 formed only one coordination bond with Fe 2+ of heme (Figure 2c).These docking results suggested that compounds 12 and 11 have more interactions with IDO-1 than 14, which may account for their more potent IDO-1 inhibitory activity.
Molecules 2019, 24, x 6 of 11 most potent cellular inhibitory activity with an IC50 value of 3.85 µM.The calculated cLogP values of 11 and 12 with Chemdraw were 3.90 and 2.36, respectively, which may account for their difference on the cellular potency.

6
possessing moderate IDO-1 inhibitory activity.When our project was ongoing, three onaphthaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al.
-1 inhibitory activity.When our project was ongoing, three othaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. machine-learning-based virtual screening (Figure

e 1 -
ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, lso gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds d 12 were produced by further oxidation of 10 although it was not observed in the reaction.natively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of ound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone H 54.5 ± 2.3% -7j les 2019, 24, x 3 of 11 ssessing moderate IDO-1 inhibitory activity.When our project was ongoing, three othaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al. machine-learning-based virtual screening (Figure

e 1 -
ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, lso gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds d 12 were produced by further oxidation of 10 although it was not observed in the reaction.natively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of ound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone H 60.4 ± 3.9% -7k ules 2019, 24, x 3 of 11 ssessing moderate IDO-1 inhibitory activity.When our project was ongoing, three othaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al.

e 1 -
ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, lso gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds nd 12 were produced by further oxidation of 10 although it was not observed in the reaction.rnatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of pound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone H 4.60 ± 2.81 -7l ules 2019, 24, x 3 of 11 ossessing moderate IDO-1 inhibitory activity.When our project was ongoing, three othaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al.

e 1 -
ene-3-hydroxyl derivative 10.Interestingly, prolonged reaction time not only provided 11, also gave rise to 17-hydroxyl enone derivative 12 in 28% yield.It was believed that compounds nd 12 were produced by further oxidation of 10 although it was not observed in the reaction.rnatively, reduction of 11 with NaBH4 provided allylic alcohol 10.In addition, treatment of pound 8 with 2-iodoxybenzoic acid in a mixture solvent of toluene and DMSO produced 1-ketone H 5.71 ± 0.47 -7m ules 2019, 24, x 3 of 11 ossessing moderate IDO-1 inhibitory activity.When our project was ongoing, three othaquinone derivatives 4, 5, 9 were also identified as IDO-1 inhibitors by Wang and Xu et al.
± 8.6% -Epacadostat 0.086 ± 0.009 0.023 ± 0.003 a The values are the mean ± SE of two independent experiments; b Not determined.

Figure 2 .
Figure 2. Docking models of compounds 11, 12, and 14 bound to IDO-1.(a) Computational modeling at the active site of IDO-1 for 11; (b) computational modeling at the active site of IDO-1 for 12; (c) computational modeling at the active site of IDO-1 for 14.

Figure 2 .
Figure 2. Docking models of compounds 11, 12, and 14 bound to IDO-1.(a) Computational modeling at the active site of IDO-1 for 11; (b) computational modeling at the active site of IDO-1 for 12; (c) computational modeling at the active site of IDO-1 for 14.

Table 2 .
The IDO-1 inhibitory activity of o-naphthaquinone derivatives in enzyme-and cell-based assays.

Table 2 .
The IDO-1 inhibitory activity of o-naphthaquinone derivatives in enzyme-and cell-based assays.

IC 50 (µM) or Inhibitory Rate at 20 µM a Cellular IC 50 (µM) a
a The values are the mean ± SE of two independent experiments; b Not determined.

12 exhibited the
a The values are the mean ± SE of two independent experiments; b Not determined.

12 exhibited the
a The values are the mean ± SE of two independent experiments; b Not determined.

12 exhibited the
a The values are the mean ± SE of two independent experiments; b Not determined.

12 exhibited the
a The values are the mean ± SE of two independent experiments; b Not determined.

12 exhibited the
a The values are the mean ± SE of two independent experiments; b Not determined.