Identification of Substituted Naphthotriazolediones as Novel

Article pubs.acs.org/jmc

Identification of Substituted Naphthotriazolediones as Novel Tryptophan 2,3-Dioxygenase (TDO) Inhibitors through StructureBased Virtual Screening Jian-Sung Wu,† Shu-Yu Lin,†,§ Fang-Yu Liao,†,§ Wen-Chi Hsiao,† Lung-Chun Lee,† Yi-Hui Peng,† Chia-Ling Hsieh,† Mine-Hsine Wu,† Jen-Shin Song,† Andrew Yueh,† Chun-Hwa Chen,† Shiu-Hwa Yeh,† Chia-Yeh Liu,‡ Shu-Yi Lin,‡ Teng-Kuang Yeh,† John T.-A. Hsu,† Chuan Shih,† Shau-Hua Ueng,*,† Ming-Shiu Hung,*,† and Su-Ying Wu*,† †

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC ‡ Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC S Supporting Information *

ABSTRACT: A structure-based virtual screening strategy, comprising homology modeling, ligand−support binding site optimization, virtual screening, and structure clustering analysis, was developed and used to identify novel tryptophan 2,3dioxygenase (TDO) inhibitors. Compound 1 (IC50 = 711 nM), selected by virtual screening, showed inhibitory activity toward TDO and was subjected to structural modifications and molecular docking studies. This resulted in the identification of a potent TDO selective inhibitor (11e, IC50 = 30 nM), making it a potential compound for further investigation as a cancer therapeutic and other TDO-related targeted therapy.

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INTRODUCTION Tryptophan (Trp), an essential amino acid, is not only used in protein synthesis but also catabolized to produce bioactive metabolites including kynurenine, kynurenic acid, quinolinic acid, and the coenzyme NAD+ via the kynurenine pathway (Figure S1 in Supporting Information). The first and rate limiting step of this pathway is carried out by two hemecontaining enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which differ in their tissue distribution and regulation.1−3 More than 90% of total tryptophan is catabolized in the liver where in mammals TDO is mainly expressed to regulate systemic tryptophan levels. In contrast, IDO has a broad distribution in extrahepatic tissues and contributes to the balance between immune tolerance and attack.1−3 In addition to the liver, TDO is also expressed in the brain, where it regulates the production of neuroactive tryptophan metabolites and influences 5-hydroxytryptamine levels to modulate neurogenesis and anxiety-like behavior.4,5 TDO also functions in controlling growth of © 2015 American Chemical Society

pathogens and alloantigen-induced T cell activation by alteration of local tryptophan levels.6 Recent studies implicate TDO in tumor progression and suggest its inhibition may be a potential therapeutic strategy in cancer immunotherapy.7,8 Screening of human brain tumor cell lines reveals that TDO is responsible for the constitutive catabolism of tryptophan in gliomas, and its protein levels in human brain tumor specimens increase with malignancy.7 TDO is also detected in a significant proportion of human cancers, including hepatocarcinoma, melanoma, and bladder cancer.8 Several studies provide strong evidence for the immunosuppressive role of TDO in tumors. Presence of TDO induces tumor tolerance in the host’s immune system by depleting tryptophan levels and producing bioactive metabolites. Depletion of tryptophan reduces T cell proliferation,7,8 whereas TDO-derived kynurenines suppress antitumor immune responses and promote tumor cell survival Received: June 16, 2015 Published: September 8, 2015 7807

DOI: 10.1021/acs.jmedchem.5b00921 J. Med. Chem. 2015, 58, 7807−7819

Journal of Medicinal Chemistry

Article

conformation, in order to interact with substrates or inhibitors. Since the ligand-bound form of DmTDO is not available, loop 1 and loop 2 (sequence identity and similarity of 1oop 1 and loop2 between XcTDO and hTDO are 71.43% and 80.95%, respectively) were rebuilt based on the Xanthomonas campestris TDO (XcTDO) structure, which was solved as the structure of XcTDO bound with the substrate, L-Trp. Since both structural biology and kinetic studies showed that TDO forms a tetramer15,17,18 when exhibiting activity and binding to the substrate, the tetrameric hTDO model was constructed from the monomeric hTDO model by applying crystallographic symmetry and was subsequently subjected to energy minimization using the CHARM force field in the software package Discovery Studio. The tetrameric hTDO model was further refined by the ligand−support homology modeling method,21,22 in which the information on a known ligand is utilized to construct and optimize the binding site. This method aims to provide more accurate insights into the binding site upon ligand binding, particularly when the structure of the protein bound with a ligand is unavailable. In this study, (E)-6-fluoro-3-[2-(1H-tetrazol-5-yl)vinyl]-1H-indole (LM10),13 a known TDO inhibitor, was docked into the Trpbinding site of the constructed hTDO model and the subsequent energy minimization was performed to refine the conformations of the surrounding residues upon the ligand binding. Finally, the model of hTDO containing heme group was validated by a Ramachandran plot using the PROCHECK23 program. The Ramachandran plot showed that 88.5% of residues were in the most favored regions and 10.7% of residues were in the allowed regions, indicating the model to be of good quality. The stereochemical qualities of the model (i.e., the side chain conformations, bond lengths and bond angles, and hydrogen bond geometry) were also analyzed by PROCHECK, and the results showed all stereochemical parameters of the model to be comparable with or better than well refined structures at a similar resolution. Furthermore, potential errors of the model were checked by the ProSA2003 program,24,25 which evaluated the quality of the model by calculating an energy plot in which the calculated energies were plotted as a function of amino acid sequence. Generally, positive values indicate a problematic or an unusual region of the model. Figure S2 in the Supporting Information shows that all of the residue energies of the model were negative with the exception of few residues in the N-terminal part, confirming the quality of the constructed ligand-bound hTDO model. Virtual Screening and Analogues Search. The constructed ligand-bound hTDO model was then used to identify novel TDO inhibitors by virtual screening a chemical database. The Specs database, a commercially available database containing 202 874 compounds, was first filtered to exclude compounds that do not follow Oprea’s leadlikeness criteria26 (150 < MW < 450, −3.5 < ClogP < 4.5, 0 < number of rings < 4, 0 < rotational bonds < 10, 0 < donor < 5, 0 < acceptor < 8). Next, 102 442 compounds in the filtered database were docked to the hTDO model using the GOLD Suite (CCDC, version 5.2)27 and ranked by the heme scoring function implemented in ChemScore.28 The top-ranked 200 compounds were then filtered to exclude redundant compounds by structural clustering analysis using Discovery Studio (version 2.5, Accelrys, Inc.). The 30 remaining compounds with high docking score and bearing structurally diverse scaffolds were experimentally evaluated for their ability to inhibit hTDO.

and motility. Systemic blockade by a TDO inhibitor restores the ability of mice to reject TDO expressing tumors.8 These studies suggest a substantial role for TDO in immune tolerance and tumor progression, and inhibition of TDO can reactivate the immune system to break tumor-induced immune resistance. To date, only a few classes of TDO inhibitors have been reported, such as tryptophan analogues,9 β-carboline derivatives,10 fluoropyridylvinylindoles,11,12 tetrazoylvinylindoles,8,13 and derivatives of natural products,14 and most of them show weak inhibition of TDO activity. The above-mentioned inhibitors were identified using high throughput screening (HTS) or synthetic modifications based on the Trp structure. The crystal structures of two bacterial TDOs (Xanthomonas campestris TDO and Ralstonia metallidurans TDO)15,16 and one eukaryotic TDO (Drosophila melanogaster TDO)17 have been reported, and the structures were solved in the presence of heme with or without L-Trp analogues. The human TDO structure was recently determined by Meng et al.18 in apo form without heme binding. These structures have given insights into the molecular mechanisms of substrate binding and catalysis and, as such, can be used to design novel TDO inhibitors. Here, we report a structure-based virtual screening approach to identify novel TDO inhibitors and further demonstrate the utility of the hit compounds in generating more potent TDO inhibitors. A homology modeling study was first performed to construct a ligand-bound TDO model, and this model was applied in structure-based virtual screening on a filtered chemical database containing 102 442 compounds. Compound 1 was selected from the virtual screening, and enzymatic evaluation showed inhibitory activity against TDO and hence was used as the starting point for structural modification. This resulted in the identification of 11e as a potent TDO inhibitor (IC50 = 30 nM). Moreover, 11e was 21-fold more selective toward TDO when compared against IDO, making it a potential for further development as a therapeutic TDO-related target.

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RESULTS AND DISCUSSION Homology Modeling of Human TDO. A homology modeling study was performed to obtain a model of ligandbound human TDO for use in structure-based virtual screening. In general, homology models built from templates with over 50% sequence identity are regarded as useful models in the prediction of detailed protein−ligand interactions.19 The sequence identity and similarity between human TDO and Drosophila melanogaster TDO (DmTDO) are 58.7% and 78.2%, respectively, suggesting that the crystal structure of DmTDO (PDB code 4HKA) could be used as a template for homology modeling studies. Five structure models of human TDO (hTDO) were generated using the MODELLER20 program in Discovery Studio, and the total energy derived from probability density functions (PDFs) was calculated for each model. The model with the lowest energy value, which indicates the highest similarity with the template, was selected as the initial model. The initial model was then subjected to modification to allow the binding of substrates or inhibitors. The substrate-binding site of TDO is surrounded by loop 1 (the loop connecting αhelix F and G), loop 2 (the loop connecting α-helix O and P), and the distal plane of heme. It has been reported15,17 that loop 1 and loop 2 induced conformational changes upon the substrate or inhibitor binding in which loop 1 would move away to accommodate substrates or inhibitors and loop 2 is likely to adopt a closed conformation instead of an open 7808



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Figure 1. Workflow of homology modeling virtual screening.

Scheme 1a

a

Reagents and conditions: (a) NaN3, DMSO, rt, overnight; (b) EtOAc, reflux, overnight (2−22%).

(Scheme 1).30 The commercially available, monosubstituted benzyl bromides 5a−e were used to synthesize the corresponding benzyl azides 6a−e in a neutral nucleophilic substitution reaction. The 1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione moieties were constructed by 1,3-cycloaddition of azide 6a−e and 1,4-naphthoquinone 7 to synthesize 4a−e in yields of 2−22%. Commercially available benzyl chloride 8a and benzyl bromides 8b and 8c were reacted with sodium azide to give the corresponding benzyl azides 9a−c in high yields (Scheme 2). Compound 9a was then coupled with the diethylamine in the presence of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) to afford the amide 10a. Compound 10a was used to synthesize the desired 1Hnaphtho[2,3-d][1,2,3]triazole-4,9-diones 11a by 1,3-cycloaddition with 1,4-naphthoquinone 7. Under the same conditions as shown in Scheme 1, 9a−c were converted to the desired derivatives 12a−c. Amides 11b−j were prepared by amide bond formation of acids 12a−c with a variety of amines.

Among them, 1, ranked 10th out of 102 442 screened compounds, showed the inhibition toward TDO in a dose dependent manner, and the IC50 value was determined to 711 nM for TDO inhibition (Figure S3 in the Supporting Information). Compound 1 contains a 1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione core, and since no naphthotriazoledione compounds have been reported as TDO inhibitors, the structure novelty of 1 prompted us to further investigate its potential for TDO inhibition and was subjected to further structure−activity relationship (SAR) studies. A flowchart depicting the various stages of virtual screening, including homology modeling, ligand−support binding site optimization, model validation, structure-based virtual screening, and clustering analysis, is summarized in Figure 1. Chemistry. Compounds 1, 4f, and 4g were purchased from Specs. Compounds 2 and 3 were synthesized as previously reported (see Supporting Information).29 Compounds 4a−e and 12a−c were prepared using a modified literature method 7809



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Scheme 2a

a

Reagents and conditions: (a) NaN3, DMSO; (b) Et2NH, HBTU, CH2Cl2, rt (100%); (c) 1,4-naphthoquinone, EtOAc, reflux (4−49%); (d) various amines, HBTU, CH2Cl2, rt (12−95%).

loop 2, αF, and the distal plane of the heme and formed hydrophobic contacts with Phe140, Leu147, Thr342, and the heme. The interactions of core structures and phenyl group with the heme and surrounding residues are important for the potency as evidenced by synthesizing and testing compounds 2 and 3 (Figure 3). In 2, the removal of the benzyl group resulted in the loss of the hydrophobic interactions with Phe140, Leu147, Thr342, and the heme and consequently decreased the activity (IC50 > 10 000 nM). Modification of the core naphtho[2,3-d]triazole structure (i.e., compound 3) led to the loss of coordinate binding with the iron in heme and complete loss of TDO inhibition (IC50 > 10 000 nM). On the basis of the docking study of 1, a variety of analogs were either purchased or synthesized and their biological activities were evaluated. As shown in Table 1, N-benzyl substituted 1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione derivatives 4a−g showed poor or retained inhibitory activity against TDO, regardless of whether a halogen or methyl group was introduced at the ortho-, meta-, or para-position of the benzyl moiety. However, when a carboxyl group was introduced at meta- or para- position of the benzyl ring (compounds 12a and 12b), these correspondingly exhibited an improvement in TDO inhibitory activity compared to 1. Further introducing the N,N′-diethylamide group at the meta-position of the benzyl ring 11a resulted in a 8-fold increase in potency. Modification of 11a by replacing the N,N′-diethylamide group with the Nmethylpiperazinylamide on the benzyl group led to a further 3fold improvement in activity for 11e (IC50 = 30 nM) compared with 11a (IC50 = 90 nM). On the basis of the structure of 11e,

Structure−Activity Relationship (SAR) Studies. To further explore structure−activity relationship and identify more active compounds, 1 was docked into the active site of hTDO. The docking model (Figure 2) proposed that 1 was

Figure 2. Docking position of 1 in the active site of TDO.

bound in the Trp-binding site and its naphthotriazoledione core was in a position almost perpendicular to the heme plane. The triazole ring coordinated with the iron atom in heme, and the quinone part of 1 made strong hydrophobic interactions with His76, Ser150, and Ser151 in the active site and Tyr42′ and Tyr45′ in the N-terminal segment of the adjacent monomer. The phenyl group of 1 occupied the pocket between

Figure 3. Chemical structures of compounds 1, 2, and 3. 7810



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Table 1. TDO and IDO Inhibition of 1 and Analogues

IC50 (nM)

a

compd

R1

R2

R3

TDO inhibition

IDO inhibition

selectivity ratioa

1 4a 4b 4c 4d 4e 4f 4g 12a 12b

H CH3 H H H H H H H H

Cl H H H Br H H CH3 COOH H

H H F CH3 H Br Cl H H COOH

711 ± 27.0 561 ± 30.7 7380 ± 914.1 1088 ± 185.9 768 ± 97.5 1586 ± 192.7 6460 ± 807 1745 ± 217.7 167 ± 16.0 312 ± 9.6

767 ± 26.3 808.9 ± 67.9 2587 ± 198.5 1770 ± 254.0 3163 ± 443.7 >10000 >10000 4612 ± 507.6 1242 ± 64.6 2323 ± 7.5

1.1 1.4 0.4 1.6 4.1

2.6 7.4 7.4

Selectivity ratio is calculated as (IC50 value of IDO)/(IC50 value of TDO).

Moreover, as presented in Table 2, the positions of the aromatic substituents on the N1-benzyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione affected TDO inhibition; molecules bearing meta-amide substituents were generally more active than those bearing para-amide substituents. To investigate the difference at the molecular level, a docking study of 11h (paraamide substituent) was carried out and compared with that of 11e (meta-amide substituent). It was found that moving the amide substituent from the meta to para position results in a steric clash with Phe140 of TDO (Figure S4a). To avoid this and accommodate the para-amide substituent, the phenyl group of 11h is required to undergo a conformational change to shift away from Phe140. This moves 11h away from the hydrophobic pocket, decreasing the edge to edge π−π interactions with Phe72 and hydrophobic interactions with Phe140 (Figure S4b). This might explain the weaker inhibition of TDO by para-amide substituted N1-benzyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione 11h, compared with the meta-amide substituted 11e. In the structural modification process, it is found that initial hit 1 and its analogs displayed comparable inhibition against both of TDO and IDO (Table 1), but inhibition of TDO was significantly improved by introducing rigid tertiary amides on the para- and meta-positions of the N-benzyl group (Table 2). The most potent compound, 11e, exhibited a 21-fold increase in inhibitory activity toward TDO when compared to IDO. A molecular docking study of 11e with IDO protein was performed and compared with that of TDO to give insights into the observed selectivity of 11e (Figure 5a and Figure 5b). It revealed that 11e was unable to interact with Arg231 of the IDO (the corresponding residue Arg144 in TDO) whereas 11e formed the extensive interactions, including the hydrogen binding interactions, with Arg144 of the TDO protein. As shown in our docking study and also observed in the published ligand-bound IDO structures,31 Arg231 moved away from the binding site to form the strong cation−π interactions with the side chain of Phe227. That the interactions of the guanidium group of Arg231 with the side chain of Phe227 are important for the protein activity and structure stabilization of IDO protein was previously proven in a mutagenesis study.31 In

a variety of rigid amide moieties on benzyl group were synthesized. Their chemical structures and biological activities are shown in Table 2. Compounds 11c, 11d, and 11e bearing 4-acetyl-1-piperazinyl, 4-morpholinylphenyl, and 4-methyl-1piperazinyl groups, respectively, exhibited potent TDO inhibitory activities. Moreover, the meta-amide substituted N1-benzyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-diones (11c, 11d, and 11e) displayed more potent inhibition toward TDO compared with para-amide substituted N1-benzyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-diones (11f, 11g, and 11h). Compounds 11i and 11j bearing 4-methoxy-1-piperidinyl and 4methylene N,N′-diethylamide groups, respectively, displayed comparable activities with other para-amide substituted analogs. As revealed in the SAR studies, 11e exhibited the most potent inhibition against TDO. To understand the improved potency of 11e at the molecular level, the molecular docking studies of 11e in the active site of TDO were performed and the docking model was compared with that of 1 (Figure 4a and Figure 4b) to elucidate the differences in binding interactions between compounds 1 and 11e. The core structure and the phenyl group of 11e superimposed well with that of 1 and made similar coordination and hydrophobic interactions with the heme group and surrounding residues. The most significant difference between these two structures is the extended Nmethylpiperazinylamide group of 11e, which formed an additional hydrogen bonding interaction with Arg144 and extra hydrophobic interactions with the surrounding residues. Arg144 is strictly conserved through eukaryotic and prokaryotic TDOs family, and it has been reported that mutation of Arg144 to Ala has resulted in the significant decrease of TDO enzymatic activity.17,18 Our docking study revealed that the carbonyl moiety of 11e formed hydrogen bonding interactions with Arg144 and contributed to the inhibition against TDO, consistent with studies examining the role of Arg144 in modulating TDO.17,18 Furthermore, the 4-methyl-1-piperazinyl group extended into the area between helix F, helix O, and loop 2 and made additional hydrophobic interactions with Met335, Leu336, Lys339, and Ala340, which would also contribute to the improved potency of 11e. 7811



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Table 2. TDO and IDO Inhibition of 11a−j

a

Selectivity ratio is calculated as (IC50 value of IDO)/(IC50 value of TDO).

contrast, in the structure of TDO, the cation−π interactions of Arg144 with the Phe residue are absent, and Arg144 is located

toward the binding site to interact with the substrates or inhibitors. 7812



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Figure 5. Proposed binding modes of 11e in the active site of (a) TDO and (b) IDO. Figure 4. (a) Docking position of 11e (cyan) in the active site of TDO. The red dotted line is the potential H-bond between Arg144 and 11e. (b) Docking position of 11e in the active site of TDO and its superimposition with 1 (gray).

redox-cycling compounds with activation concentrations (AC50) of >50 μM. Moreover, protein tyrosine phosphatase 1B (PTP1B) which is efficiently inhibited by quinones through the redox or covalent modification35−37 is known to detect redox-cycling compounds. Therefore, PTP1B inhibition assay38,39 was performed with our compounds by Eurofins Panlabs, Ltd. Compound 1 and compound 11e at 10 μM showed inhibition of 11% and 20%, respectively, suggesting both compounds have no inhibition against PTP1B with IC50 value of >10 μM and they are unlikely to interfere with the assay by redox cycling or alkylation. To further investigate whether our compounds are promiscuous compounds with activity to multiple unrelated biological targets, several inhibition or binding experiments of compounds 4a and 11e to a variety of protein or receptors were performed. The results showed that neither compound 11e nor compound 4a showed any activity or binding to different types of proteins or receptors (Table 3 and Figures S6−S13 in the Supporting Information). EGFR (epidermal growth factor receptor), a transmembrane tyrosine kinase, is activated by ligand-induced dimerization and plays critical roles in regulating cell proliferation, differentiation, and migration. Its abnormal activation is associated with a variety of human cancers.40 μOpioid receptor is classified as the class A family of G-proteincoupled receptor, and it is the main target of the pain control drug, morphine.41 The FMS-like tyrosine kinase 3 (FLT3) is a member of the class III membrane-bound receptor tyrosine kinase (RTK) family and has a crucial role in normal

Evaluation of PAINS-Liable Property. The compounds reported in this study have been filtered for pan assay interference compounds (PAINS).32 Our compounds containing the quinones substructures have been flagged as PAINSliable compounds as detected by the PAINS filters.32 Several studies were performed to further investigate the PAINS-liable properties of our compounds. First, to ensure that our compounds did not interfere with biological assays, compounds 1, 4a, 11e and water (Figure S5) with addition of different concentrations of kynurenine in the absence of TDO protein were measured at OD 480 nm. The results showed that all of the test compounds did not interfere with the signal detection. Second, for compounds containing quinones substructures, the promiscuity is likely due to redox cycling or alkylation. To evaluate the redox-cycling ability of our compounds, a horseradish peroxidase−phenol red (HRP-PR) H2O2 detection assay was performed.33 Redox-cycling compounds at 50 μM could generate significant levels (≥50%) of H2O2 in the presence of strong reducing agents, which would react with a number of proteins and therefore interfere with the biological assays.34 The incubation of 50 μM of the initial compound, 1, with 0.8 mM dithiothreitol (DTT) produced 11% of H2O2, and the incubation of 50 μM of the most active compound, 11e, produced 26% of H2O2, showing both compounds are non7813



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binds to TDO and makes coordination with iron of heme group.

Table 3. Summary of Activity of 4a and 11e toward TDO and Other Proteins

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IC50 (nM) protein/receptora

11e

4a

TDO inhibition EGFR inhibition μ-opioid receptor binding FLT3 inhibition CXCR4 binding Aurora A inhibition dengue NS2B/NS3 inhibition mitochondrial complex I inhibition catalase inhibition

30 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000

561 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000

CONCLUSION In this study, N1-benzyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9diones were identified by structure-based virtual screening as novel TDO inhibitors. Subsequent structural modification led to the identification of a potent TDO inhibitor, 11e, which exhibited a 24-fold improvement in TDO inhibition compared to the initial hit 1. Comparison of the docking model of TDO in complex with 11e and that of TDO in complex with 1 revealed that additional hydrogen bonding interactions with the conserved Arg144 residue along with the hydrophobic interactions provided by the N-methylpiperazinylamide with the surrounding residues contributed to the improved potency of 11e. Until now, limited TDO inhibitors have been reported and 11e, displaying excellent potency in the low nanomolar range, ranks among the most potent TDO inhibitors reported so far. By modification of the substituents on the phenyl ring, compounds exhibited selectivity for TDO over IDO. The most active compound, 11e, showed almost 21-fold more potent inhibition of TDO than IDO. The docking studies revealed that 11e was unable to interact with Arg231 in IDO (the corresponding residue Arg144 in TDO) whereas 11e formed the extensive interactions with Arg144 in TDO protein, which would be the reason for the selectivity. Arg144 is important for the binding of substrates or inhibitors and plays a role in TDO selectivity. On the basis of the above results, designing compounds to interact with Arg144 in human TDO would be an effective approach to improve the potency as well as selectivity of TDO inhibitors. In conclusion, a novel family of TDO inhibitors has been identified through the use of structure-based drug design, which is a combination of homology modeling, structure-based virtual screening, molecular docking studies, and structure-guided lead optimization. To the best of our knowledge, this study is the first to identify novel TDO inhibitors by structure-based virtual screening, and compound 11e is one of the most potent TDO inhibitors ever identified. It is currently undergoing additional evaluation to further assess its potential as a cancer therapeutic.

a

Table abbreviations: TDO, tryptophan 2,3-dioxygenase; EGFR, epidermal growth factor receptor; FLT3, FMS-like tyrosine kinase 3; CXCR4, C-X-C chemokine receptor type 4.

hematopoiesis.42 CXCR4 is a G-protein-coupled seven-transmembrane receptor and is broadly expressed in a variety of human tissues, particularly in the immune and central nervous systems.43 Aurora A is a serine/threonine kinase, which regulates mitotic progression, centrosome maturation, and spindle assembly.44 Dengue NS3 protease plays an important role in the dengue virus replication, and it requires the cofactor, NS2B, to increase the proteolytic activity and protein stability. The NS2B/NS3 protease has been extensively studied for the development of anti-dengue virus inhibitor.45 Mitochondrial complex I (also named as NADH:ubiquinone oxidoreductase or NADH oxidase/coenzyme Q reductase) is the enzyme that catalyzes the transfer of two electrons from NADH to ubiquinone and also serves as a proton pump to transport four protons from the mitochondrial matrix to the intermembrane space. Dysfunction of complex I has resulted in a variety of neurodegenerative disorders.46 Catalase is an oxidoreductase responsible for the decomposition of hydrogen peroxide to water and oxygen. It plays an important role in preventing oxidative damage to cells and tissues by hydrogen peroxide.47 All of the above-mentioned proteins present diverse classes of proteins and have different biological functions. Compound 11e and compound 4a showed activity toward TDO but have no effects on all other tested proteins (or receptors) as summarized in Table 3. As mentioned above, the active compounds reported in this study, compounds 11e and 4a as the examples, are unlikely to be PAINS candidates. Furthermore, the UV−visible spectra study was performed to evaluate the binding of compound 11e to TDO. TDO is a heme-containing protein, and the unique UV absorption properties of porphyrins serve as a powerful tool in the studies of heme−proteins.48 Absorbance spectra of heme group is highly sensitive to the changes in the polarity of heme surroundings upon the ligand/substrate binding, which usually results in well-pronounced changes in the spectral properties of the heme.49 Therefore, the changes in the UV−vis spectra of TDO caused by the TDO−ligand interaction could be utilized to evaluate the binding of compounds to TDO. The UV−vis spectra of ferric TDO were measured in the presence and absence of compound 11e (Figure S14). The absorption spectrum of ferric TDO in the absence of compound 11e exhibits a Soret signal at 407 nm, similar to the previous publications.48 The Soret signal shifts to 413 nm in the presence of compound 11e, demonstrating that compound 11e

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EXPERIMENTAL SECTION

Homology Modeling Study of Human TDO. The model study of hTDO was performed using the program MODELLER implemented in Discovery Studio (version 2.5, Accelrys, Inc.). The X-ray structure of DmTDO structure (PDB code 4HKA)17 was chosen as a template protein to construct the hTDO model. All of the parameters were set to default values. Five models were generated and scored by probability density function (PDF) total energy.20 The model with the lowest energy value, which indicates the highest similarity with the template, was selected. This model was further modified to accommodate substrates or inhibitors by replacing the two flexible loops (loop 1, residues 147−157; loop 2, residues 337−347) in hTDO homology model with the corresponding residues (loop 1, residues 119−129; loop 2, residues 249−259) in the XcTDO crystal structure (PDB code 2NW8).15 The tetrameric hTDO model, which was constructed from the monomeric hTDO model by using crystallographic symmetry of DmTDO crystal structure, was then subjected to energy minimization using the program Smart-Minimizer in Discovery Studio with a 0.01 minimizing rms gradient and 2000 minimizing steps. Ligand−Support Homology Modeling Study. The tetramer model of hTDO was prepared for binding site optimization by adding hydrogen atoms in SYBYL-X suite (version 1.2, Tripos).50 The known TDO inhibitor, LM10, was docked into the Trp-binding site, and the 7814



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best conformation was used to optimize side chain conformation in the active site, defined as a region within 20 Å of the iron atom in heme group. A systematic search of side chain conformation was performed by the side chain refinement protocol in Discovery Studio using a CHARMm based energy minimization method. Structure Validation. The quality of the final hTDO model was checked with PROCHECK23 and ProSa2003.24,25 A Ramachandran plot generated by PROCHECK with a hypothetical resolution of 2.7 Å revealed that 88.5% of the amino acid residues are in the most favorable region, 10.7% in additional allowed region, and 0.2% in generously allowed region. The quality of the protein fold was further inspected with ProSa2003. This software shows the energetic architecture of protein folds as a function of amino acid sequence position. Virtual Screening. Specs (www.specs.net) database containing 202 874 compounds was first filtered by leadlike properties26 (150 < MW < 450, −3.5 < ClogP < 4.5, 0 < number of rings < 4, 0 < rotational bonds < 10, 0 < donor < 5, 0 < acceptor < 8) and led to a total of 102 442 compounds. The 3D coordinates of each molecule in the filtered database were then generated by the CONCORD module of SYBYL for the following virtual screening. The virtual screening was performed by the GOLD Suite software package (version 5.2, CCDC, Cambridge, U.K.). The Chemscore of heme scoring function with metal parameters derived from statistics obtained from the PDB database was used. The region within a radius of 15 Å centered on the iron atom of heme was defined as the active site for docking study. Ligand-Dependent Genetic Algorithm parameter setting was used to automatically calculate an optimal number of operations for a given compound based on the number of rotatable bonds, ligand flexibility, the volume of protein binding site, and the number of water molecules used during docking procedure. The search efficiency, a parameter to optimize the predictive accuracy of the results and maximize the speed of docking, was set to 10%, and the default parameter settings for library screening were used for virtual screening except the early termination option was set to off. The top 200 compounds ranked by Chemscore were selected for further study. Structural Clustering Analysis. The structural clustering analysis was performed using the cluster ligands protocol in Discovery Studio 2.5 to exclude redundant compounds. The relocation clustering algorithm was employed to classify molecules by number of clusters as requested. The default properties setting FCFP_6 was applied to compute the dissimilarity between two compound structures, and the number of clusters was set to 30. General Methods. All commercial chemicals and solvents were reagent grade and used without further purification unless otherwise stated. All reactions were carried out under an atmosphere of dry nitrogen or argon and were monitored by TLC, using Merck 60 F254 silica gel glass-backed plates; compounds were detected visually under UV irradiation (254 nm) or by spraying with phosphomolybdic acid reagent (Aldrich). Flash column chromatography was carried out using silica gel (Merck Kieselgel 60, no. 9385, 230−400 mesh ASTM) or Teledyne Isco CombiFlash automated flash chromatography systems. 1 H and 13C NMR spectra were obtained with a Varian Mercury 300 or Varian Mercury 400 spectrometer, and the chemical shifts (δ) were recorded in ppm and reported relative to TMS or the solvent signal. Multiplicities are recorded with the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. High-resolution mass spectra (HRMS) were measured with a VARIAN 901-MS electrospray ionization (ESI) or by using JEOL JMS-700 fast atom bombardment (FAB) mass spectrometer. Purity of the final compounds was determined with an Hitachi 2000 series HPLC system using a C18 column (Agilent ZORBAX Eclipse XDB-C18 5 mm, 4.6 mm × 150 mm), operating at 25 °C. Elution was carried out using CH3CN as mobile phase A and using water containing 0.1% formic acid + 10 mmol NH4OAc as mobile phase B. Elution conditions were the following: 0 min, 10% phase A + 90% phase B; at 45 min, 90% phase A + 10% phase B; at 50 min, 10% phase A + 90% phase B; at 60 min, 10% phase A + 90% phase B. The flow rate of the mobile phase was 0.5 mL/min, and the injection volume of the sample was 5 μL. Signals were detected at 254 nm. The purity of all tested compounds is >95%

except for compounds 2 (90%), 4a (92.5%), 11c (93%), and 11f (94%). General Procedure A for the Synthesis of Compounds 1 and 4a−e. Sodium azide (1.5 equiv) was added to the solution of the required benzyl bromide (1 equiv) in DMSO (3−6 mL). The reaction mixture was stirred at room temperature or 55−70 °C. To the resulting mixture was added 30 mL of water, and the water phase was extracted with ethyl acetate (30 mL × 2). The combined organic layer was dried (MgSO4), and the solution of resulting azido compound was used directly for the next step. 1,4-Naphthoquinone (1−2 equiv) was added to the solution of azido compound in 60 mL of ethyl acetate, and the reaction mixture was refluxed for 18 h. The solvent was removed in vacuo to give a solid. The solid was purified to give the desired product. 1-(3-Chlorobenzyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9dione (1). Following general procedure A, sodium azide (0.290 g, 4.50 mmol) was reacted with 3-chlorobenzyl bromide (0.400 mL, 3.00 mmol) in 3 mL of DMSO for 16 h. The resulting azido compound was reacted with 1,4-naphthoquinone (0.840 g, 5.30 mmol). After workup, the crude mixture was purified by automated flash chromatograph (SiO2, gradient hexane/ethyl acetate = 100:0, then hexane/ethyl acetate = 50:50) to give the desired product 1 as a yellow solid (70.0 mg, 7%, purity 98.6%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.24 (d, 1 H), 7.88−7.79 (m, 2 H), 7.50 (s, 1 H), 7.42−7.39 (m, 1 H), 7.32−7.30 (m, 2 H), 5.99 (s, 2 H). HRMS (FAB) m/z calcd for C17H10ClN3O2 (M)+: 323.0462, found 323.0461. 1-(2-Methylbenzyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9dione (4a). Following general procedure A, sodium azide (0.390 g, 6.00 mmol) was reacted with 2-methylbenzyl bromide (0.540 mL, 4.00 mmol) in 4 mL of DMSO for 17 h. The resulting azido compound was reacted with 1,4-naphthoquinone (1.90 g, 12.0 mmol). After workup, the crude mixture was purified by automated flash chromatography (SiO2, gradient hexane/ethyl acetate = 95:5, then hexane/ethyl acetate = 60:40) to give the desired product 4a as a brown solid (28.0 mg, 2%, purity 92.5%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.22 (d, 1 H), 7.87−7.78 (m, 2 H), 7.24−7.23 (m, 2 H), 7.19−7.15 (m, 2 H), 6.06 (s, 2 H), 2.56 (s, 3 H). HRMS (FAB) m/z calcd for C18H13N3O2 (M)+: 303.1008, found 303.1015. 1-(4-Fluorobenzyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9dione (4b). Following general procedure A, sodium azide (0.550 g, 8.40 mmol) was reacted with 4-fluorobenzyl bromide (0.700 mL, 5.60 mmol) in 6 mL of DMSO for 1 h at 70 °C. The resulting azido compound was reacted with 1,4-naphthoquinone (1.07 g, 6.70 mmol). After workup, the crude mixture was washed with ethanol and dichloromethane and filtered to give the desired product 4b as a palebrown solid (70.0 mg, 4%, purity 98.3%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.23 (d, 1 H), 7.87−7.80 (m, 2 H), 7.54 (dd, 2 H), 7.05 (dd, 2 H), 5.99 (s, 2 H). HRMS (FAB) m/z calcd for C17H10FN3O2 (M): 307.0757, found 307.0759. 1-(4-Methylbenzyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9dione (4c). Following general procedure A, sodium azide (0.439 g, 6.80 mmol) was reacted with 4-methylbenzyl bromide (0.833 g, 4.50 mmol) in 5 mL of DMSO for 17 h at 55 °C. The resulting azido compound (0.600 g, 4.00 mmol) was reacted with 1,4-naphthoquinone (1.27 g, 8.00 mmol). After workup, the crude mixture was purified by automated flash chromatography (SiO2, gradient hexane/ ethyl acetate = 100:0, then hexane/ethyl acetate = 50:50) to give the desired product 4c as a yellow solid (80.0 mg, 7%, purity 97.9%). 1H NMR (CDCl3, 300 MHz) δ 8.33 (d, 1 H), 8.22 (d, 1 H), 7.87−7.76 (m, 2 H), 7.41 (d, 2 H), 7.16 (d, 2 H), 5.98 (s, 2 H), 2.31 (s, 3 H). HRMS (ESI) m/z calcd for C18H13N3O2 (M + H): 304.1086, found 304.1080. 1-(3-Bromobenzyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9dione (4d). Following general procedure A, sodium azide (0.488 g, 7.50 mmol) was reacted with 3-bromobenzyl bromide (1.25 g, 5.00 mmol) in 5 mL of DMSO for 17 h at 60 °C. The resulting azido compound (1.00 g, 4.70 mmol) was reacted with 1,4-naphthoquinone (1.18 g, 7.00 mmol). After workup, the crude mixture was purified by automated flash chromatography (SiO2, gradient hexane/ethyl acetate = 100:0, then hexane/ethyl acetate = 50:50) to give the desired 7815



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product 4d as a yellow solid (0.380 g, 22%, purity 99.5%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.23 (d, 1 H), 7.88−7.79 (m, 2 H), 7.66 (t, 1 H), 7.48−7.44 (m, 2 H), 7.23 (d, 1 H), 5.98 (s, 2 H). 1-(4-Bromobenzyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9dione (4e). Following general procedure A, sodium azide (0.488 g, 7.50 mmol) was reacted with 4-bromobenzyl bromide (1.25 g, 5.00 mmol) in 5 mL of DMSO for 18 h at 60 °C. The resulting azido compound (0.930 g, 4.40 mmol) was reacted with 1,4-naphthoquinone (1.05 g, 6.60 mmol). After workup, the crude mixture was purified by automated flash chromatography (SiO2, gradient hexane/ ethyl acetate = 100:0, then hexane/ethyl acetate = 50:50) to give the desired product 4e as a yellow solid (30.0 mg, 2%, purity 96.9%). 1H NMR (CDCl3, 400 MHz) δ 8.33 (d, 1 H), 8.22 (d, 1 H), 7.87−7.81 (m, 2 H), 7.49 (dd, 2 H), 7.40 (dd, 2 H), 5.96 (s, 2 H). 3-(Azidomethyl)benzoic Acid (9a). Sodium azide (0.330 g, 7.50 mmol) was added to a solution of 3-(chloromethyl)benzoic acid (0.850 g, 5.00 mmol) in DMSO (5 mL), and the reaction mixture was stirred for 5 h at room temperature. The reaction mixture was quenched by adding water and extracted with ethyl ether. The organic layers were dried over MgSO4, filtered, concentrated, and dried under high vacuum to give the desired product 9a as a white solid which was used directly for next step without further purification. 1H NMR (CDCl3, 400 MHz) δ 8.11−8.07 (m, 2 H), 7.59 (d, 1 H), 7.52 (t, 1 H), 4.44 (s, 2 H). 3-(Azidomethyl)-N,N-diethylbenzamide (10a). A mixture of 9a (0.870 g, 4.90 mmol), HBTU (1.86 g, 4.90 mmol), and diethylamine (0.600 mL, 5.90 mmol) in 18 mL of dichloromethane was stirred for 19 h at room temperature. The resulting mixture was poured into saturated NaHCO3, and it was extracted with dichloromethane. The organic layers were washed with saturated NaHCO3 and water, dried over MgSO4, filtered, concentrated, and dried under high vacuum to give the desired product 10a as a gray gum which was used directly for next step without further purification. 1H NMR (CDCl3, 400 MHz) δ 8.28−8.10 (m, 1.5 H), 7.76−7.33 (m, 2.5 H), 4.52 (s, 1 H), 4.37 (s, 1 H), 3.54 (br s, 2 H), 3.25 (br s, 2 H), 1.25 (s, 3 H), 1.11 (s, 3 H). 3-[(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-1yl)methyl]-N,N-diethylbenzamide (11a). Following general procedure A, 10a (1.14 g, 4.90 mmol) was reacted with 1,4naphthoquinone (0.780 g, 4.90 mmol). After workup, the crude mixture was purified by automated flash chromatography (SiO2, gradient hexane/ethyl acetate = 100:0, then hexane/ethyl acetate = 0:100) to give the desired product 11a as a red-brown solid (65.0 mg, 3%, purity 98.1%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.21 (d, 1 H), 7.87−7.79 (m, 2 H), 7.58−7.52 (m, 2 H), 7.42−7.27 (m, 2 H), 6.03 (s, 2 H), 3.52 (br s, 2 H), 3.18 (br s, 2 H), 1.24 (br s, 3 H), 1.08 (br s, 3 H). HRMS (ESI) m/z calcd for C22H20N4O3 (M + Na): 411.1433, found 411.1428. 3-[(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-1yl)methyl]benzoic Acid (12a). Following general procedure A, sodium azide (5.17 g, 80.0 mmol) was reacted with 3-(chloromethyl)benzoic acid (9.04 g, 53.0 mmol) in 50 mL of DMSO for 5 h. The resulting azido compound 9a (2.66 g, 15.0 mmol) was reacted with 1,4-naphthoquinone (3.56 g, 22.5 mmol). After cooling, the mixture was filtered and washed with ethanol to give the desired product 12a as a yellow-brown solid (2.27 g, 45%, purity 99.0%). 1H NMR (DMSO-d6, 400 MHz) δ 13.12 (br s, 1 H), 8.21−8.15 (m, 2 H), 8.03 (s, 1 H), 7.98−7.90 (m, 3 H), 7.64 (d, 1 H), 7.51 (t, 1 H), 6.11 (s, 2 H). HRMS (FAB) m/z calcd for C18H11N3O4 (M): 333.0750, found 333.0750. 4-[(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-1yl)methyl]benzoic Acid (12b). Following general procedure A, sodium azide (0.330 g, 7.50 mmol) was reacted with (4-bromoethyl)benzoic acid (1.08 g, 5.00 mmol) in 5 mL of DMSO for 4 h. The resulting azido compound 9b was reacted with 1,4-naphthoquinone (0.950 g, 6.00 mmol). After cooling, the mixture was filtered and washed with ethanol to give the desired product 12b as a white solid (0.340 g, 20%, purity 99.5%). 1H NMR (DMSO-d6, 300 MHz) δ 8.21 (d, 1 H), 8.15 (d, 1 H), 7.97−7.91 (m, 4 H), 7.48 (d, 2 H), 6.12 (s, 2 H).

{4-[(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol1-yl)methyl]phenyl}acetic Acid (12c). Following general procedure A, sodium azide (0.430 g, 6.60 mmol) was reacted with 4(bromomethyl)phenylacetic acid (1.01 g, 4.40 mmol) in 5 mL of DMSO and stirred at 60 °C for 5 h. The resulting azido compound 9c in 60 mL of ethyl acetate was reacted with 1,4-naphthoquinone (0.840 g, 5.30 mmol). After cooling, the resulting precipitate was filtered off and washed with 20 mL of ethanol to give the desired product 12c as a white solid (0.220 g, 14%, purity 99.8%). 1H NMR (DMSO-d6, 300 MHz) δ 8.20−8.15 (m, 2 H), 7.97−7.92 (m, 2 H), 7.36 (d, 2 H), 7.26 (d, 2 H), 6.01 (s, 2H), 3.55(s, 2H). HRMS (FAB) m/z calcd for C19H13N3O4 (M): 347.0906, found 347.0905. General Procedure B for the Synthesis of Compound 11b−j. A mixture of 12a−c (1 equiv), O-(benzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate (HBTU, 1 equiv), and diethylamine (3 equiv) in 5−10 mL of dichloromethane was stirred at room temperature for 18 h. The resulting mixture was poured into 50 mL of saturated NaHCO3 and extracted by CH2Cl2 (25 mL × 2). The combined organic layer was washed with 50 mL of water and 50 mL of brine, dried (MgSO4), and concentrated in vacuum. The residue was washed with 30 mL of hexane/ethyl acetate 1:1 to give the desired product. 4-[(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-1yl)methyl]-N,N-diethylbenzamide (11b). Following general procedure B, 12b (0.130 g, 0.400 mmol), HBTU (0.150 g, 0.400 mmol), and diethylamine (0.120 mL, 1.20 mmol) in 7 mL of dichloromethane were reacted to give 11b as a yellow solid (0.130 g, 85%, purity 99.6%). 1H NMR (CDCl3, 300 MHz) δ 8.34 (d, 1 H), 8.23 (d, 1 H), 7.88−7.79 (m, 2 H), 7.54 (d, 2 H), 7.36 (d, 2 H), 6.03 (s, 2 H), 3.60− 3.40 (br s, 2 H), 3.30−3.17 (br s, 2 H), 1.30−1.17 (br s, 3 H), 1.17− 1.03 (br s, 3 H). HRMS (ESI) m/z calcd for C18H13N3O2 (M + Na)+: 411.1433, found 411.1428. 1-{3-[(4-Acetyl-1-piperazinyl)carbonyl]benzyl}-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (11c). Following general procedure B, 12a (0.200 g, 0.600 mmol), HBTU (0.250 g, 0.600 mmol), and 1acetylpiperazine (0.110 g, 0.900 mmol) in 12 mL of dichloromethane were reacted to give 11c as a yellow solid (30.0 mg, 12%, purity 93.0%). 1H NMR (CDCl3, 300 MHz) δ 8.34 (d, 1 H), 8.21 (d, 1 H), 7.88−7.79 (m, 2 H), 7.61−7.58 (m, 2 H), 7.47−7.39 (m, 2 H), 6.04 (s, 2 H), 3.85−3.30 (m, 8 H), 2.13 (br s, 3 H). HRMS (ESI) m/z calcd for C24H21N5O4 (M): 443.1594, found 443.1602. 1-[3-(4-Morpholinylcarbonyl)benzyl]-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (11d). Following general procedure B, 12a (0.330 g, 1.00 mmol), HBTU (0.420 g, 1.10 mmol), and morpholine (0.180 mL, 2.00 mmol) in 12 mL of dichloromethane were reacted to give 11d as a yellow solid (0.380 g, 93%, purity 99.8%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.21 (d, 1 H), 7.88−7.79 (m, 2 H), 7.59−7.57 (m, 2 H), 7.45−7.38 (m, 2 H), 6.04 (s, 2 H), 3.82−3.57 (m, 6 H), 3.39 (br s, 2 H). HRMS (ESI) m/z calcd for C22H18N4O4 (M): 402.1328, found 402.1333. 1-{3-[(4-Methyl-1-piperazinyl)carbonyl]benzyl}-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (11e). Following general procedure B, 12a (0.200 g, 0.600 mmol), HBTU (0.250 g, 0.600 mmol), and 1methypiperazine (0.100 mL, 0.900 mmol) in 12 mL of dichloromethane were reacted to give 11e as an orange solid (0.200 g, 79%, purity 98.6%). 1H NMR (CDCl3, 300 MHz) δ 8.33 (d, 1 H), 8.21 (d, 1 H), 7.89−7.79 (m, 2 H), 7.61−7.58 (m, 2 H), 7.46−7.38 (m, 2 H), 6.04 (s, 2 H), 3.96 (br s, 2 H), 3.62 (br s, 2 H), 2.76 (br s, 4 H), 2.54 (s, 3 H). HRMS (ESI) m/z calcd for C23H21N5O3 (M): 415.1644, found 415.1640. 1-{4-[(4-Acetylpiperazin-1-yl)carbonyl]benzyl}-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (11f). Following general procedure B, 12b (80.0 mg, 0.240 mmol), HBTU (100 mg, 0.260 mmol), and 1acetylpiperidine (46.0 mg, 0.360 mmol) in 5 mL of dichloromethane were reacted to give 11f as a yellow solid (101 mg, 95%, purity 94.0%). 1 H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.23 (d, 1 H), 7.88− 7.80 (m, 2 H), 7.58 (d, 2 H), 7.41 (d, 2 H), 6.05 (s, 2 H), 3.83−3.28 (m, 8 H), 2.12 (br s, 3 H). HRMS (ESI) m/z calcd for C24H21N5O4 (M + Na): 466.1491, found 466.1496. 7816



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was centrifuged for 10 min at 2500 rpm, and an amount of 100 μL of the supernatant per well was mixed with 100 μL of 2% (w/v) pdimethylaminobenzaldehyde in acetic acid. The reactions were measured at OD 480 nm. The method in IDO enzyme assay was performed as described previously.53 Redox Cycling Assay. H2O2 generated in the reaction was measured using catalase activity colorimetric/fluorometric assay kit (BioVision, USA) according to the manufacturer’s instructions and Soares et al.34 with minor modification. Briefly, compounds at 50 μM were incubated with 0.8 mM dithiothreitol for 15 min at room temperature. The reaction was terminated by the stop solution, and a developer mix was then added to the mixture for 10 min. The signal was measured by the spectrophotometer at the absorbance of 560 nm.55 PTP1B Assay. PTP1B inhibition assay was performed with our compounds by Eurofins Panlabs, Ltd. All reaction was carried out in black Polystyrol 96-well plates with flat bottoms. 10 μM compound is preincubated with 170 μM/mL PTP1B for 15 min at 37 °C in modified HEPES buffer, pH 7.2. Then 10 μM 6,8-difluoro-4methylumbelliferyl phosphate (DiFMUP) is added and reacted for another 1h. The amount of 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU) formed is read at 358 nm (excitation)/450 nm (emission) in a fluorescense plate reader. UV−Visible Spectra. UV−vis spectra were recorded on NanoDrop 2000 (Thermo Fisher Scientific Inc.) with 50 μM TDO (50 mM Tris buffer (pH 8.0), 200 mM NaCl, 10 mM imidazol, and 10% DMSO) mixed with 2 mM compound 11e.

1-[4-(Morpholin-4-ylcarbonyl)benzyl]-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (11g). Following general procedure B, 12b (40.0 mg, 0.120 mmol), HBTU (50.0 mg, 0.130 mmol), and morpholine (20.0 μL, 2.30 mmol) in 2 mL of dichloromethane were reacted to give 11g as a yellow solid (0.350 g, 73%, purity 98.3%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.23 (d, 1 H), 7.88−7.79 (m, 2 H), 7.57 (d, 2 H), 7.40 (d, 2 H), 6.04 (s, 2 H), 3.81−3.50 (m, 6 H), 3.38 (br s, 2 H). HRMS (ESI) m/z calcd for C22H18N4O4 (M + Na): 425.1226, found 425.1233. 1-{4-[(4-Methylpiperazin-1-yl)carbonyl]benzyl}-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (11h). Following general procedure B, 12b (80.0 mg, 0.240 mmol), HBTU (0.100 g, 0.260 mmol), and 1methylpiperazine (40.0 μL, 0.360 mmol) in 5 mL of dichloromethane were reacted to give 11h as a yellow solid (70.0 mg, 69%, purity 97.1%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.23 (d, 1 H), 7.89−7.81 (m, 2 H), 7.58 (d, 2 H), 7.41 (d, 2 H), 6.05 (s, 2 H), 3.88 (br s, 4 H), 3.00−2.71 (m, 7 H). HRMS (ESI) m/z calcd for C23H21N5O3 (M + H): 416.1723, found 416.1718. 1-{4-[(4-Methoxypiperidin-1-yl)carbonyl]benzyl}-1Hnaphtho[2,3-d][1,2,3]triazole-4,9-dione (11i). Following general procedure B, 12b (80.0 mg, 0.240 mmol), HBTU (0.100 g, 0.260 mmol), and 4-methoxypiperidine (40.0 μL, 0.360 mmol) in 5 mL of dichloromethane were reacted to give 11i as a pale-yellow solid (40.0 mg, 35%, purity 98.4%). 1H NMR (CDCl3, 400 MHz) δ 8.34 (d, 1 H), 8.23 (d, 1 H), 7.88−7.80 (m, 2 H), 7.55 (d, 2 H), 7.39 (d, 2 H), 6.04 (s, 2 H), 3.98 (br s, 1 H), 3.58−3.40 (m, 3 H), 3.35 (s, 3 H), 3.16 (br s, 1 H), 1.98−1.63 (m, 4 H). HRMS (ESI) m/z calcd for C24H22N4O4 (M + H): 431.1719, found 431.1716. 2-{4-[(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-1-yl)methyl]phenyl}-N,N-diethylacetamide (11j). Following general procedure B, 12c (0.150 g, 0.400 mmol), HBTU (0.190 g, 0.500 mmol), and diethylamine (0.120 mL, 1.20 mmol) in 8 mL of dichloromethane were reacted to give 11j as a yellow solid (0.150 g, 86%, purity 99.4%). 1H NMR (acetone-d6, 300 MHz) δ 8.28−8.23 (m, 2 H), 7.99−7.91 (m, 2 H), 7.43 (d, 2 H), 7.29 (d, 2 H), 6.08 (s, 2 H), 3.68(s, 2H), 3.41−3.28 (m, 4 H), 1.12−1.00 (m, 6 H). HRMS (ESI) m/z calcd for C23H22N4O3 (M + Na): 425.1590, found 425.1578. Construction, Expression, and Purification of TDO and IDO Proteins. The coding region of human TDO2 gene encoding the human TDO protein was cloned into the pET-14b vector in frame with the His tag at the N terminus. Expression and purification of TDO protein were performed according to Paglino et al.51 and Structural Genomics Consortium et al.52 In brief, this TDO2 containing construct was transformed to Escherichia coli BL21(DE3) cells. The transformants were grown at 30 °C in TB medium containing 100 μg/mL ampicillin with sequential addition of 0.5 mM δ-aminolaevulinic acid and 0.5 mM isopropyl-β-D-thiogalactoside to induce protein expression. Cells after growth for an additional 20 h at 22 °C were then harvested by centrifugation and resuspended in buffer A (50 mM sodium phosphate, pH 8.0, 500 mM NaCl, 0.5 mM TCEP, and protease−inhibitor cocktail). After sonication to disrupt the cell membrane, the lysate was centrifuged at 14 500 rpm for 30 min at 4 °C. The supernatant was then filtered through a 0.22 μm filter and loaded onto a Ni affinity column (HisTrap FF, Amersham) preequilibrated with buffer A. The column was then washed with 10, 20, and 30 mM imidazole in buffer A, and His-tagged TDO protein was eluted with buffer A containing 250 mM imidazole. TDO protein was concentrated by centrifugation in a Centriprep filter (Amicon Inc.) and the buffer was exchanged to Tris-HCl (pH 8.0) during the process of concentration. TDO protein purified was stored at −80 °C until use. The method in construction, expression, and purification of human IDO protein was performed as described previously.53 TDO and IDO Enzyme Assays. TDO enzyme assay was performed as previously described.51,54 In brief, compounds and recombinant human TDO protein in the assay buffer (50 mM potassium phosphate buffer (pH 8.0), 20 mM ascorbic acid, 10 μM methylene blue, 100 μg/mL catalase, and 200 μM L-tryptophan) in a total volume of 100 μL were incubated at 37 °C for 1 h. The reactions were then incubated with 20 μL of 30% of TCA at 60 °C for 15 min to hydrolyze N-formylkynurenine to kynurenine. The reaction mixture

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00921. Proposed catalytic mechanism of TDO/IDO; quality of the hTDO model; DmTDO structure and XcTDO structure evaluated by ProSa2003; dose−response inhibition of 1 and 11e; structural alignment and docking pose of 11h and 11e; spectrophotometry analysis of 1, 4a, and 11e; inhibition of 4a and 11e to a variety of proteins; UV−vis spectra of ferric TDO with 11e; synthesis of 2 and 3; spectra data and HPLC purity data for 2 and 3 (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*S.-H.U.: phone, +886-37-246-166, extension 35791; fax, + 886-37-586-456; e-mail, [email protected]. *M.-S.H.: phone, +886-37-246-166, extension 35721; fax, + 886-37-586-456; e-mail, [email protected]. *S.-Y.W.: phone, +886-37-246-166, extension 35713; fax, + 886-37-586-456; e-mail,: [email protected]. Author Contributions §

S.-Y.L. and F.-Y.L. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Grant support was from National Health Research Institute, Taiwan, ROC (Grant BP-103-PP-04), and National Science Council (Grant NSC-102-2325-B-400-001 for S.-Y.W.). 7817



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ABBREVIATIONS USED TDO, tryptophan 2,3-dioxygenase; IDO, indoleamine 2,3dioxygenase; Trp, tryptophan; hTDO, human tryptophan 2,3dioxygenase; DmTDO, Drosophila melanogaster tryptophan 2,3dioxygenase; XcTDO, Xanthomonas campestris tryptophan 2,3dioxygenase; PDF, probability density function; SAR, structure−activity relationship; HEM, heme

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