New Inhibitors of Indoleamine 2,3-Dioxygenase 1: Molecular Modeling

Article pubs.acs.org/jmc

New Inhibitors of Indoleamine 2,3-Dioxygenase 1: Molecular Modeling Studies, Synthesis, and Biological Evaluation Antonio Coluccia,† Sara Passacantilli,† Valeria Famiglini,† Manuela Sabatino,‡ Alexandros Patsilinakos,‡,⊥ Rino Ragno,‡,⊥ Carmela Mazzoccoli,§ Lorenza Sisinni,§ Alato Okuno,∥ Osamu Takikawa,∥ Romano Silvestri,† and Giuseppe La Regina*,† †

Istituto Pasteur − Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco and ‡Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Rome, Italy § Laboratorio di Ricerca Pre-Clinica e Traslazionale, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Centro di Riferimento Oncologico della Basilicata (CROB), Via Padre Pio 1, I-85028 Rionero in Vulture, Italy ∥ National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology 35 Gengo, Morioka, Obu, Aichi 474-8511, Japan ⊥ Alchemical Dynamics s.r.l., Piazzale Aldo Moro 5, I-00185 Rome, Italy S Supporting Information *

ABSTRACT: Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive target for anticancer therapy. Herein, we report a virtual screening study which led to the identification of compound 5 as a new IDO1 inhibitor. In order to improve the biological activity of the identified hit, arylthioindoles 6−30 were synthesized and tested. Among these, derivative 21 exhibited an IC50 value of 7 μM, being the most active compound of the series. Furthermore, compounds 5 and 21 induced a dose-dependent growth inhibition in IDO1 expressing cancer cell lines HTC116 and HT29. Three-dimensional quantitative structure−activity relationship studies were carried out in order to rationalize obtained results and suggest new chemical modifications.

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INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO1) is a monomeric hemecontaining enzyme that catalyzes the rate-limiting step of the Ltryptophan (L-Trp) catabolism via the kynurenine pathway.1 The reaction, that takes place through the cleavage of the 2,3double bond of the tryptophan indole ring, is catalyzed independently by IDO1, tryptophan 2,3-dioxygenase (TDO), and indoleamine 2,3-dioxygenase 2 (IDO2) in mammals.2 Despite this, these enzymes show differences in tissue expression and substrate specificity.3 Trp IDO1-mediated catabolism causes general control nonderepressible 2 (GCN2) kinase activation that acts as a molecular sensor in T cells leading to cycle arrest, anergy, and increased sensitivity to apoptosis.4,5 Furthermore, it has been demonstrated that some Trp metabolites, such as Nformylkynurenine, suppress T cell functions.6,7 Indeed, © 2016 American Chemical Society

kynurenine activates the aryl hydrocarbon receptor (AhR), which modulates immune response and IDO1 activity.8 Thus, IDO1 Trp metabolism modifies the interactions between adaptive immune cells leading to immune modulatory signals that direct T cells toward tolerance. Nowadays, IDO1-mediated immune tolerance is widely accepted as one of the most important mechanisms evolved by tumors to escape the immune surveillance.9 Indeed, many human tumors constitutively express IDO1,10 such as breast,11 prostate,12 lung13 and colon cancers,14 as well as neuroblastoma and melanoma.15 It has also been demonstrated that IDO1 expression is induced by tumor necrosis factor alpha (TNF-α) and other inflammatory mediators. Therefore, IDO1 might be Received: May 10, 2016 Published: October 3, 2016 9760

DOI: 10.1021/acs.jmedchem.6b00718 J. Med. Chem. 2016, 59, 9760−9773

Journal of Medicinal Chemistry

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the only reported crystallographic data about Trp binding are about TDO2-Trp complex (pdb code: 2NW8).35 Despite the fact that IDO1 and TDO2 share only 10% overall sequence identity, key active site residues are conserved.36,37 Accordingly, we superimposed the two crystal structures (data not shown) obtaining the binding coordinates for Trp in IDO1. The complex was studied by means of molecular dynamics, occupying the sixth iron atom valence with an oxygen molecule and applying a restraint to the heme and to the species coordinating the iron atom (His346 and oxygen molecule) (Figure 1). L-Tpr was stabilized into the catalytic pocket by

secondarily induced as a consequence of the initial host inflammatory response to the tumor.16 Furthermore, increased IDO1 expression correlates with different tumor progression parameters and shorter patient survival.17 All this evidence confirms that IDO1 inhibition might enhance the efficacy of pharmacological cancer therapy. Indeed, preclinical studies in mouse tumor models showed that IDO1 inhibitors have a synergistic effect when used in combination with some anticancer drugs,18 such as cyclophosphamide, doxorubicin, paclitaxel, and cisplatin. Moreover, this positive effect was immune-mediated, because it fails when the tumor grows in immune-deficient hosts.19 Hence, IDO1 may facilitate survival, growth, invasion, and metastasis of malignant cells expressing tumor-associated antigens. Therefore, IDO1 inhibition is a potential breakthrough approach to cancer therapy. A number of IDO1 inhibitors have been identified, and among these, many compounds are substrate analogues (L-Trp, herein just Trp) or known heme binders.20 To the best of our knowledge, only a small number of compounds have entered clinical trials.21 Among these, 1methyl-DL-tryptophan (DL-1MT) (1), developed by NewLink Genetics, was the first one (Chart 1).22,23 Preclinical/clinical Chart 1. Chemical Structures of IDO1 Inhibitors 1−4

Figure 1. PLANTS proposed binding modes of L-Trp (yellow) and L1MT (purple). L-Trp pose suggested by molecular dynamics simulations is reported as cyan. Heme is reported as a green stick, iron as a silver sphere, and oxygen molecule as a red stick. IDO1 structure is showed as a cartoon.

polar interactions between the Trp carboxyl moiety and Arg231 and the Trp amine group with a carboxyl group of the heme; Tshaped contacts between indole core and Phe163 phenyl ring were also observed (Figure 1S, Supporting Information). From molecular dynamic trajectory, we got a representative frame, which was used to carry out docking studies. Thus, we ran docking studies for L-Trp and L-1MT. The PLANTS proposed binding mode for L-Trp well fitted the reaction mechanism proposed for its catabolism,37 and also L1MT poses were consistent (Figure 1). These promising results prompted the studies of the training set. Considering the indole moiety as an IDO1 privileged scaffold, we managed our screening library by retaining only derivatives bearing an indole core (about 2500 molecules). The docking poses were sorted according to the ChemPLP score,38 and the best 500 structures were visually analyzed within the active site. During the visual inspection, we monitored the RMSD between the indole moiety of L-Trp and screened compounds. Only derivatives showing RMSD values below or equal to 1 were retained, and the best 15 compounds were selected for biological assay. Among the tested compounds, derivative 5 (Table 1) showed the best biological activity exhibiting a strong inhibition of IDO1 activity (80% at 100 μM) and an IC50 value of 17.5 μM (Figure 2S, Supporting Information). Binding mode analysis of compound 5 docked conformation revealed a good superimposition with that of the above derived

studies helped to clarify the different mechanisms of action of the two enantiomers. Indeed, while 1-methyl-L-tryptophan acts as IDO1 competitive inhibitor,3 1-methyl-D-tryptophan modifies the Trp carriage across cell membranes24 and/or alters cellular signals downstream of IDO1 pathway by targeting some Trp-sensitive immunoregulatory kinases.25 NewLink Genetics also identified an imidazole derivative NLG919 (structure not disclosed), which entered a phase I clinical trial for the treatment of recurrent advanced solid tumors.26,27 Incyte developed oxadiazole 2 (IC50 = 71.8 ± 17.5 nM) as reversible and competitive inhibitor, which is currently being evaluated in clinical trials (ClinicalTrials.gov identifiers: NCT02862457, NCT02178722 and NCT02166905).28−30 Amgen reported derivative 3 (IC50 = 3 μM) as competitive inhibitor showing a good selectivity over IDO2 and TDO (80and 30-fold, respectively).31 Very recently, X-ray studies clarified the binding mode of compound 3,32 while earlier, only 4-phenylimidazole (4), a weak IDO1 inhibitor (IC50 = 143 μM), was crystallized with the enzyme.33 In order to identify new IDO1 inhibitors, we carried out a structure-based virtual screening (VS) study using the experimental IDO1/4 complex (pdb code: 2D0T)33 and, as main filter, the PLANTS34 proposed docking poses. However, 9761



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Table 1. Inhibition of IDO1 Activity by New Compounds 5−30a

compd

R1

R2

R3

Z

X

IDO1 IC50 (μM)b

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 230 331

H H H H H COOMe H H H H H H H H H H H CH3 H H H H H H H H

5-Cl 5-Cl 5-Cl 5-Cl 5-Cl 5-Cl 5-Cl 5-Cl 5-Cl 5-Cl 5-Cl 5-F 5-Br 5-I 5-OEt 5-Cl 5-Cl 5-Cl 5-Cl 5-Cl H H 4-Cl 6-Cl 7-Cl 6,7-Cl2

2-OMe 2-OMe 2-OMe 2-OMe H 2-OMe 3-OMe 4-OMe 2-Me 3-Me 4-Me 2-OMe 2-OMe 2-OMe 2-OMe 2,4-(OMe)2 3,5-(OMe)2 3,5-(OMe)2 3,5-(Me)2 3,4,5-(OMe)3 3,5-(OMe)2 3,5-(OMe)2 3,5-(OMe)2 3,5-(OMe)2 3,5-(OMe)2 3,5-(OMe)2

CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH N CH CH CH CH

S SO2 CO CH2 S S S S S S S S S S S S S S S S S S S S S S

17.5 >200 >200 63 >200 >200 21 16 21 100 >200 32 24 45 >200 >50 7 45 20 40 25 >200 52 15 >200 90 0.0718 3

a The reaction mixture contained potassium phosphate buffer, ascorbic acid, methylene blue, catalase, human recombinant IDO1,40,41 L-Trp, and testing materials diluted in DMSO. bThe IC50 values were the means of more than two independent experiment sets calculated by linear interpolation.

including Mcl1, Bcl-xL, and β-catenin expression. Finally, threedimensional (3-D) quantitative structure−activity relationship (QSAR) models were derived to get insights on 3-D SARs of the newly synthesized derivatives.

bound L-Trp (Figure 2). Hydrophobic interactions with Phe163 and Leu234 stabilized the indole moiety, while the phenyl ring established π−cation interactions with Arg231. Further biological characterization highlighted compound 5 as inactive against TDO2, confirming the higher substrate specificity of the enzyme (Figure 3S, Supporting Information).3 In addition, biochemical studies disclosed that compound 5 proved to be an uncompetitive inhibitor of IDO1 (Figure 4S, Supporting Information). This has been already seen; indeed compounds able to bind the IDO1 catalytic pocket show or seem to act as uncompetitive inhibitors.20,33,39 Our experiments suggested that compound 5 does not bind to the binding site for L-Trp in the catalytic center, rather it binds the far or distal site of the center, and the binding inhibits the catalytic activity in a concentration-dependent manner. Starting with this promising data, we designed, prepared, and tested a new set of derivatives (compounds 6−30) in order to identify the main pharmacophore features of this new class of IDO1 inhibitors. Effects of the most active compounds 5 and 21 on growth inhibition and cell cycle progression/distribution in HTC116 and HT29 cancer cell lines were also evaluated,

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CHEMISTRY Compounds 5,42 10,43 23,42 and 24,42 were prepared as previously reported. Oxidation with m-chloroperbenzoic acid in chloroform at 25 °C for 2 h of 5 gave the corresponding sulphone derivative 6 (Scheme 1a). Microwave-assisted reaction of 5-chloro-1H-indole (35) with 2-methoxybenzoyl chloride (51) in the presence of aluminum chloride in 1,2dichloroethane at 110 °C (150 W) for 2 min provided ketone 7, that was reduced to the corresponding methylene derivative 8 with lithium aluminum hydride in boiling tetrahydrofuran for 4 h under Ar stream (Scheme 1b). Reaction of 4-Cl (33), 5-F (34), 5-Cl (35), 5-Br (36), 5-I (36), 5-OEt42 (38), 7-aza (32), and 2-Me-5Cl (39) indoles with H (43), 2-Me (44), 3-Me (45), 4-Me (46), 2-OMe (47), 3-OMe (48), 4-OMe (49) benzenethiols or 1,2-bis(3,5dimethoxyphenyl)disulfide43 (54) in the presence of copper(I) 9762



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position 2′ (5, IC50 = 17.5 μM and 13, IC50 = 21 μM) to either 3′ (11, IC50 = 21 μM and 14, IC50 = 100 μM) or 4′ (12, IC50 = 16 μM and 15, IC50 > 200 μM). Complete loss of activity was also obtained by introducing an ester function at position 2 of the indole moiety to give compound 10 (IC50 > 200 μM). Then, we investigated the effect of the chlorine atom at position 5 of the indole central core. Compared to compound 5 (IC50 = 17.5 μM), the substitution with a different halogen, such as fluorine (16, IC50 = 32 μM), bromine (17, IC50 = 24 μM) and iodine (18, IC50 = 45 μM), did not produce a remarkable effect on IDO1 inhibition, while the presence of a more sterically hindered ethoxy group gave rise to an active compound (19, IC50 > 200 μM). In addition, we studied the influence of a second and third group on the phenyl ring. Compared to the 2′-OMe derivative (5, IC50 = 17.5 μM), the introduction of a second methoxy group at position 4′ (20, IC50 > 50 μM) did not produce any improvement of the activity. On the contrary, 3′,5′-(OMe)2 substitution pattern provided the most active compound 21 (IC50 = 7 μM), being about 3-fold more active than 3′-OMe derivative 11 (IC50 = 21 μM). Furthermore, the same compound was 3- and 6-fold more active than 3,5-(Me)2 (23, IC50 = 20 μM) and 3′,4′,5′-(OMe)3 (24, IC50 = 40 μM) and 2Me-3′,5′-(OMe)2 (22, IC50 = 45 μM) derivatives, respectively. Finally, we explored the role of the halogen atom at positions 4−7 of the indole moiety. Compared to compound 21 (IC50 = 7 μM), while the substitution of C7 with a nitrogen atom (26) and the presence of a chlorine atom at position 7 (29) provided inactive compounds (IC50 > 200 μM), 5-H (25, IC50 = 25 μM) and 6-Cl derivatives (28, IC50 = 15 μM) showed moderate inhibition of IDO1 activity. A further loss of activity was also observed when the chlorine atom was either moved to position 4 (27, IC50 = 52 μM) or doubled at positions 6 and 7 (30, IC50 = 90 μM). Growth Inhibition of HTC116 and HT29 Cancer Cell Lines. HCT116 and HT29 are two human colon carcinoma cell lines with a constitutive IDO1 expression.45 These cell lines showed a cell-autonomous mechanism by which IDO1 tryptophan catabolites directly promote cancer cell proliferation.55 The two most active derivatives 5 and 21 were evaluated for growth inhibition using HT29 and HCT116 by means of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Both compounds induced a dosedependent growth inhibition on each treated cell line even if HT29 cells proved to be more sensitive. Indeed, the viability of HCT116 and HT29 was around 40% at 20 μM and 10 μM, respectively, for compound 5 after 24 h treatment (Figure 3A). Compound 21 reduced cell viability around 40% in HCT116 and HT29 at 10 μM and 2.5 μM, respectively, thus proving to be more potent than compound 5 at lower doses. Cell viability in treated cells is compared to an untreated control (Figure 3B). It is important to note that in our previous studies,42 derivative 5 did not provide any effect (IC50 > 2500 μM) on growth of MCF-7 human breast carcinoma cells. So, the abovereported enzymatic results together with data about cell proliferation of HCT116 (expressing IDO1), HT29 (expressing IDO1), and MCF7 (not-expressing IDO1) cancer cell lines lead us to exclude off target phenomena for studied compounds. Effects on Cell Cycle Progression/Distribution in HCT116 and HT29 Cancer Cell Lines. Compounds 5 and 21 showed also an important effect on the cell cycle progression in HCT116 and HT29 cancer cell lines, exhibiting

Figure 2. Proposed binding mode of compound 5 (white). L-1MT (purple) is reported as a comparison. Heme is reported as a green stick, iron as a silver sphere, and oxygen molecule as a red stick. IDO1 structure is showed as a cartoon.

iodide in DMSO at 110 °C for 20 h under Ar stream provided 3-arylthioindoles 9, 11−19, 22, 26, and 27 (Scheme 1b). Derivatives 20, 21, 25, and 29 were prepared by treating H (31), 5-Cl (35), or 7-Cl (41) indoles with 1,2-bis(2,4dimethoxyphenyl) (53) or 1,2-bis(3,5-dimethoxyphenyl)43 (54) disulfides in the presence of sodium hydride in anhydrous N,N-dimethylformamide at 110 °C for 12 h under Ar stream. Addition of an aqueous solution of iodine/potassium iodide to an ethanol mixture of 6-Cl (37) or 6,7-Cl244 (42) indoles and 3,5-dimethoxybenzenthiol43 (50) at 25 °C for 1 h gave compounds 28 and 30, respectively. 1,2-bis(2,4-Dimethoxyphenyl)disulfide (53) was prepared starting from 2,4-dimethoxyaniline (52), that was treated with sodium nitrite and 37% hydrogen chloride aqueous solution in water at −5 °C for 20 min and then with potassium ethyl xantogenate in the same solvent at 65 °C for 30 min to obtain the corresponding O-ethyl carbonodithioate (Scheme 1c). The latter was converted into the disulfide by hydrolysis with 3 N sodium hydroxide aqueous solution in ethanol at 65 °C for 2 h and oxidation with iodine in the same solvent at 25 °C for 10 min.

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RESULTS AND DISCUSSION Inhibition of IDO1 Activity. With the aim to identify the structural requirements needed for IDO1 inhibition, we carried out some SAR studies. The importance of the sulfur bridge was first investigated. Compared with sulfur derivative 5 (IC50 = 17.5 μM), the oxidation to sulphone (6) or the substitution with a ketone group (7) gave rise to loss of activity (IC50 > 200 μM), whereas the replacement with a methylene bridge caused a 4-fold reduction of activity (8, IC50 = 63 μM). Next, we evaluated the importance of a single substitution on the phenyl ring. While 2′-H derivative 9 proved to be inactive (9, IC50 > 200 μM), methoxy shifting from position 2′ (5, IC50 = 17.5 μM) to either 3′ (11, IC50 = 21 μM) or 4′ (12, IC50 = 16 μM) did not have a remarkable effect on enzyme inhibition. Compared to the methoxy counterpart (5, 11, and 12), the presence of a methyl group on the phenyl ring gave rise to a progressive loss of activity by moving the substituent from 9763



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Scheme 1. Synthesis of Compounds 6−9, 11−22, and 25−30a

a

9, 11−22, and 25−30: see Table 1. 31: R1 = R2 = H, Z = CH. 32: R1 = R2 = H, Z = N. 33: R1 = H, R2 = 4-Cl, Z = CH. 34: R1 = H, R2 = 5-F, Z = CH. 35: R1 = H, R2 = 5-Cl, Z = CH. 36: R1 = H, R2 = 5-Br, Z = CH. 37: R1 = H, R2 = 5-I, Z = CH. 38: R1 = H, R2 = 5-OEt, Z = CH. 39: R1 = Me, R2 = 5-Cl, Z = CH. 40: R1 = H, R2 = 6-Cl, Z = CH. 41: R1 = H, R2 = 7-Cl, Z = CH. 42: R1 = H, R2 = 6,7-Cl2, Z = CH. Reagents and reaction conditions: (a) m-chloroperbenzoic acid, chloroform, 25 °C, 2 h, 55%; (b) 51, anhydrous aluminum chloride, 1,2-dichloroethane, closed vessel, 110 °C, 75 W, 2 min, 25%; (c) lithium aluminum hydride, anhydrous tetrahydrofuran, reflux temperature, 4 h, Ar stream, 37%; (d) (9, 11−19) appropriate benzenethiol or (22, 26, 27) disulfide, copper(I) iodide, DMSO, 110 °C, 20 h, Ar stream, 5−52%; (e) (20, 21, 25, 29) appropriate disulfide, sodium hydride, anhydrous N,N-dimethylformamide, 60 °C, 12 h, Ar stream, 2−41%; (f) (28, 30) 50, iodine/potassium iodide, aqueous ethanol, 25 °C, 1 h, 8−25%; (g) sodium nitrite, 37% hydrogen chloride aqueous solution, water, −5 °C, 20 min, used as crude product; (h) potassium ethyl xantogenate, water, 60 °C, 30 min, used as crude product; (i) 3 N sodium hydroxide aqueous solution, ethanol, 65 °C, 2 h, used as crude product; (j) iodine, ethanol, 25 °C, 10 min, 4%.

Figure 3. Growth inhibition of HTC116 and HT29 cancer cell lines by compounds 5 and 21. (A) Dose-dependent effect of 5 treatment on cell viability of HCT116 and HT29. (B) Dose-dependent effect of 21 treatment on cell viability of HCT116 and HT29. Cells were exposed for 24 h to the indicated concentrations of 5 and 21 and viability determined by MTT assay. Cell viability is expressed as the percentage (%) of untreated cells. (*)(#) P < 0.05; (**) P < 0.01.

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a specific cell cycle block in G2/M phase. HCT116 and HT29 cell cultures treated for 24 h with 20 μM and 10 μM of 5 and with 10 μM and 2.5 μM of 21, respectively, were incubated with propyl iodide to analyze their DNA content in flow cytometry assays compared to vehicle control (0.1% DMSO) (Figure 4). Both inhibitors arrested cell cycle progression at

Figure 5. Cell cycle analysis of HCT116 (A) and HT29 (B) cells treated with 0.1% DMSO or 20, 10, or 2.5 μM 5 or 21 for 24 h and harvested after a further 24 h recovery in drug-free medium. Histograms show the fold change vs DMSO of cells with G0/G1, S, and G2/M DNA content expressed as mean values ± SD calculated from three independent experiments. (*) P < 0.05 vs cell cycle.

Figure 4. Cell cycle analysis of HCT116 (A) and HT29 (B) cells treated with 0.1% DMSO or 20, 10, or 2.5 μM 5 or 21 for 24 h. Histograms show the fold change vs DMSO of cells with G0/G1, S and G2/M DNA content expressed as mean values ± SD calculated from three independent experiments. (*) P < 0.05.

elucidated). These data confirm the important role of IDO1 inhibition in proliferation and survival of cancer cells. 3-D QSAR Studies. Application of various alignment rules produced 3200 (400 alignments × 8 probes) initial 3-D QSAR models by means of the 3-D QSAutogrid/R procedure.46 The best preliminary models (highest q2s) resulted in those obtained by obfit derived atom-by-atom alignment (Figure 7). For the latter, 124,000 pretreated models were generated by the VPO procedure (Table 2). This procedure led to eight optimized models endowed by q2 values ranging from 0.25 to 0.36 (Table 1S, Supporting Information). The latter bestpretreated models were subjected to a SA variable selection to obtain 8 final models endowed with r2 and q2 values ranging from 0.83−0.88 and 0.15−0.60, respectively. All generated models were obtained using a discrete template flexibility, obtaining quasi 4-D QSARs.47 Among the final models, the ones based on probes C (steric information) and HD (electrostatic and H-bonding information) were selected (Table 2 and Table 1S, Supporting Information) for the subsequent model’s graphical interpretations. The probe A derived model, although displaying high r2 and q2 values, was not selected as it is completely overlapped to that resulting with C probe. In general comparative molecular field analysis (CoMFA) derived 3-D QSAR is analyzed by means of the so-called CoMFA contour maps in which each grid point is the result of the corresponding PLS coefficient (PLSCoeff) by the MIF

indicated concentrations in HCT116 and HT29 human carcinoma cell lines. The significant increase in the proportion of cells in G2/M phase is also reflected in abundant rounded cells observed under the light microscope in both cases. This indicated that IDO1 inhibitors 5 and 21 exerted a selective action specifically at mitosis process. After a 24 h exposure to 5 and 21 and subsequent incubation with drug-free medium for 24 h, HCT116 and HT29 cell lines treated showed a recovery of the cell cycle (Figure 5), confirming a reversible inhibition. Moreover, 5 and 21 induced a reversible cell cycle arrest at concentrations of 20, 10, and 2.5 μM in HCT116 and HT29 cells. Effects on Mcl1, Bcl-xL, and β-catenin Expression. In order to establish a correlation between cell cycle arrest and cell death, we assessed the protein expression of two pro-survival factors, Bcl-xL and Mcl1, by Western Blotting. As shown in Figure 6, treatment with compounds 5 (20 μM in HCT116 and 10 μM in HT29) and 21 (10 μM in HCT116 and 2.5 μM in HT29) resulted in a significant decrease of Bcl-xL and Mcl1 content in both colon cancer cell lines. Moreover, the exposure to both compounds at the indicated doses affected the treated cells level of β-catenin, a protein involved in cell adhesion mechanisms, causing decreased expression (mechanism not 9765



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account that in GOLPE/GRID 3-D QSARs it is possible to describe for each training set molecule the linked activity contribution plot,49 in this report the average MIF values (Aver) are used in place of the StDevs to define the average activity contribution plot (AACP, PLSCoeff × Aver). In particular, the probe C derived model AACP shows three main polyhedrons (A, B and C, Figure 8) mainly associated with the most sterically variable parts of the arylthioindole scaffold. Big cyan polyhedrons, associated with positive average activity contribution, are situated along C5-indole substituent (Figure 8, A area) and phenyl meta position (Figure 8, B area). On the other hand, a big yellow polyhedron runs along the second phenyl ring meta position and its close to the ortho one. Other minor polyhedrons are also surrounding the aligned training set, and among these, of particular relevance, are two yellows identified by the D′, D″ ,and E letters in Figure 8. The D′ and D″ areas are related to chlorine substituents of compounds 28 and 30 (Figure 7S, Supporting Information), while area E to the para methoxy group of derivative 12 (Figure 8S, Supporting Information). The AACP relative to HD probe based model, as depicted in Figure 8, shows overlapping information with those of probe C thus highlighting that sterical features are the most representative to explain SARs. Nevertheless, at a deeper inspection, the intrinsic electrostatic nature of probe HD raised some differences among the two AACPs. In particular, comparing the meshed (HD probe) with the surface (C probe) style polyhedrons in Figure 8, some of the formers are closer to the overlapped training set structures suggesting the following: in the indole position C-5, an electron-rich substituent is favorable for the activity (F letter in Figure 8 and Figure 5S, Supporting Information); the quasi-complete coinciding C and HD polyhedrons around phenyl meta positions confirm the exclusive sterical nature of the substitution in those sites (Figure 8, letter G and H); two small polyhedrons depicted by letter I in Figure 8 are overlapping the indole C-6 and C-7 positions, and these positions are substituted by chlorine atoms in compound 28 (6chloro mono substituted) and 30 (6,7-dichloro substituted), from the latter activity contribution plot analysis (Figure 7S, Supporting Information), the 7-chloro is negatively influencing the activity (Figure 7SA, Supporting Information), while the 6chloro is related to some favorable influence. Regarding the molecular modeling studies, analysis of the 3D QSAR models suggested a list of preliminary SARs to apply at the phenylthioindole scaffold and is potentially useful for the lead optimization (Figure 9). In particular, the phenylthio aromatic ring should only be substituted in one meta position, and small substituents could be inserted in the para position; either ortho or double meta substitutions have negative effects on the bioactivity. Regarding the indole substitution pattern,

Figure 6. Protein expression levels of the antiapoptotic factors, Mcl1 and Bcl-xL, and β-catenin assayed by Western blotting in DMSO and 5 and 21 treated cells for 24 h; β-actin served as loading control. Graph bars show the average of data resulting from densitometric analysis of three independent blots. (*) P < 0.05 vs DMSO treated.

Figure 7. Best alignment obtained by obfit.

associated standard deviation (StDev).48 Nevertheless, these default CoMFA plots do not allow a precise interpretation of the 3-D structure activity relationships, because StDevs are always positive values, and consequently the PLSCoeff × StDev product (CoMFA standard) is not able to indicate areas associated with increase or decrease of activity. Taking into

Table 2. 3-D QSAR Results with the VPO and Simulated Annealing probe

grid spacinga

cutoffb

zeroingc

min stdd

r2e

q2f

SDEPg

qYS2h

SDEPYSi

qSA2j

SDEPSAk

C HD

1.7 1.1

5 3

0.01 0.009

0.05 0.045

0.88 0.83

0.25 0.29

0.26 0.25

−1.00 −0.57

0.41 0.37

0.60 0.49

0.19 0.21

a

Spacing between the grid nodes. bCutoff of very low or very high energy value. cZeroing of very low data points. dMinimum starndard deviation value. eCoventional square-correlation coefficient. fCross validation coefficient. gStandard deviation error of prediction. hAverage cross validation coefficient using leave-one-out (LOO) method obtained after Y-scrambling process, using 100 iterations. iStandard deviation error of prediction obtained after Y-scrambling process using 100 iterations. jCross validation coefficient obtained after simulated annealing. kStandard deviation error of prediction after simulated annealing. 9766



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Figure 8. Average activity contribution plots. The training set is shown. The average activity contribution plot displayed in mesh is derived from HD probe (2 PCs), the surfaced average activity contribution plot is derived from C probe, considering 3 PCs (cyan surfaces and meshes indicated positive values, yellow surfaces and meshes indicate negative values). Blue letters refer to HD probe, while black ones to the C probe.

Figure 9. Graphical representation of SARs derived from 3-D QSAR analysis.

metabolism, excretion, and toxicity (ADMET) of a candidate chemical lead(s) are critical and establish benchmarks against which compounds synthesized during lead optimization can be evaluated. Several tools are available to profile compound ADMET properties using in silico calculations. Here compounds 5 and 21 were evaluated by means of web servers and specialized programs. Tables 4S−6S (Supporting Information), report the admetSAR,50 Molinspiration,51 and Volsurf+52 ADMET calculated properties. In general, either 5 or 21 showed no violation of both the Lipinski53 and Veber54 indications, suggesting that compounds of this class could lead to an orally active drug. Other calculated properties indicated that compounds 5 and 21 can be considered as lead compounds to deserve further optimization. In particular,

substituents at position 4 are not tolerated (table activity); 3-D QSAR analysis indicated that bulky and electron-withdrawing substituents at position 5 are correlated with a positive activity contribution; positions 6 and 7 have complementary significance, and this can be gathered from the electrostatic nature of the attached substituents. In particular, electronwithdrawing groups are associated with an increase of activity if positioned at C-6, while a negative effect is obtained at position 7. Finally concerning the sulfur bridge, the only S → CH2 isosteric replacement seems to destroy the quantitative correlation, although more data should be generated for a more accurate analysis. Application ADMET in Silico Profiling. Assessments of the pharmacological properties of absorption, distribution, 9767



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recorded with a Bruker Avance (400 MHz) spectrometer in the indicated solvent. Carbon NMR (13C NMR) were recorded with a Varian Mercury (75 MHz) spectrometer in the indicated solvent. Fid files were processed by MestreLab Research SL MestreReNova 6.2.1− 769 software. Chemical shifts are expressed in δ units (ppm) from tetramethylsilane. Elemental analyses of biologically evaluated compounds were found to be within ±0.4% of the theoretical values, and their purity was found to be >95% by high-pressure liquid chromatography (HPLC). The HPLC system used (Thermo Fisher Scientific Inc. Dionex UltiMate 3000) consisted of a SR-3000 solvent rack, a LPG-3400SD quaternary analytical pump, a TCC-3000SD column compartment, a DAD-3000 diode array detector, and an analytical manual injection valve with a 20 μL loop. Samples were dissolved in acetonitrile (1 mg/mL). HPLC analysis was performed by using a Thermo Fisher Scientific Inc. Acclaim 120 C18 column (5 μm, 4.6 mm × 250 mm), at 25 ± 1 °C with an appropriate solvent gradient (acetonitrile/water), flow rate of 1.0 mL/min and signal detector at 206, 230, 254, and 365 nm. Chromatographic data were acquired and processed by Thermo Fisher Scientific Inc. Chromeleon 6.80 SR15 Build 4656 software. 5-Chloro-3-((2-methoxyphenyl)sulfonyl)-1H-indole (6). A mixture of 5 (0.10 g, 0.34 mmol) and m-chloroperbenzoic acid (0.15 g, 0.68 mmol) in chloroform (10.0 mL) was stirred at 25 °C for 2 h and diluted with water. Layers were separated, and the organic one was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, ethyl acetate:n-hexane = 7:3 as eluent) to furnish 6 (0.06 g, 55%) as a solid, mp 235−238 °C (from ethanol). 1H NMR (CDCl3): δ 3.80 (s, 3H), 6.90 (d, J = 8.7 Hz, 1H), 7.11−7.13 (m, 1H), 7.26−7.35 (m, 2H), 7.50−7.52 (m, 1H), 7.94−7.96 (m, 2H), 8.22 (d, J = 7.6 Hz, 1H), 8.71 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3348 cm−1. Anal. calcd for C15H12ClNO3S (321.78): C, 55.99; H, 3.76; Cl, 11.02; N, 4.35; S, 9.96; found: C, 55.97; H, 3.73; Cl, 10.99; N, 4.33; S, 9.92. (5-Chloro-1H-indol-3-yl)(2-methoxyphenyl)methanone (7). A mixture of 35 (0.50 g, 3.3 mmol), 51 (0.56 g, 0.44 mL, 3.4 mmol) and anhydrous aluminum chloride (0.44 g, 3.3 mmol) in anhydrous 1,2,-dichloroethane (2.0 mL) was placed into the microwave cavity and highly stirred. A starting microwave irradiation of 75 W was used, with the temperature being ramped up from 25 to 110 °C. Once 110 °C was reached, taking about 1 min, the reaction mixture was held at this temperature for 2 min, then diluted with water and extracted with chloroform. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, n-hexane:ethyl acetate = 3:2 as eluent) to furnish 7 (0.24 g, 25%) as a solid, mp 235−237 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.72 (s, 3H), 7.05 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.25−7.32 (m, 2H), 7.46−7.52 (m, 2H), 7.70 (s, 1H), 8.12 (s, 1H), 12.12 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 1593, 3320 cm−1. Anal. calcd for C16H12ClNO2 (285.73): C, 67.26; H, 4.23; Cl, 12.41; N, 4.90; found: C, 67.20; H, 4.21; Cl, 12.45; N, 4.88. 5-Chloro-3-(2-methoxybenzyl)-1H-indole (8). A solution of 7 (40.0 mg, 0.14 mmol) in anhydrous tetrahydrofuran (2.0 mL) was added dropwise to a cold suspension of lithium aluminum hydride (10.6 mg, 0.28 mmol) in the same solvent (2.0 mL) under Ar stream. The mixture was heated at reflux for 4 h, cooled, cautiously quenched on crushed ice, and extracted with ethyl acetate. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, nhexane:acetone = 4:1 as eluent) to furnish 8 (14.0 mg, 37%) as an oil. 1 H NMR (CDCl3): δ 3.88 (s, 3H), 4.06 (s, 2H), 6.85−6.91 (m, 2H), 6.97 (s, 1H), 7.10−7.14 (m, 2H), 7−18−7.27 (m, 2H), 7.57 (s, 1H), 7.95 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3422 cm−1. Anal. calcd for C16H14ClNO (271.74): C, 70.72; H, 5.19; Cl, 13.05; N, 5.15; found: C, 70.71; H, 5.21; Cl, 13.00; N, 5.13. General Procedure for the Preparation of Compounds 9 and 11−19. 5-Chloro-3-(phenylthio)-1H-indole (9). To a mixture of 35 (0.25 g, 1.65 mmol) and 43 (0.18 g, 0.17 mL, 1.63 mmol) in DMSO (1.6 mL), copper(I) iodide (15.7 mg, 0.08 mmol) was added. The

from Tables 4S and 6S (Supporting Information), the predicted metabolic and toxicity features indicated ameliorable cytochrome interaction profile and a low level of toxicity. The most relevant drawback is the Volsurf computed metabolic stability stating that some modifications are needed to increase this feature.

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CONCLUSIONS By screening an in house library of 2500 indole-based compounds arylthioindole 5 was identified as a new IDO1 inhibitor. SAR studies allowed us to identify the main pharmacophore groups of this new class of IDO1 inhibitors, leading to derivative 21 as the most active compound of the series. In particular, SAR data highlighted that (a) the sulfur bridge is detrimental for enzyme inhibition, its oxidation to sulphone, or substitution with either ketone or methylene groups brought by inactive compounds; (b) the introduction of halogens on the indole ring is allowed, with the 5-Cl substituted compound being the best one; and (c) the presence of 3,5(OMe)2 substitution pattern on the phenyl ring gave rise to the most interesting compound. Cellular assays in IDO1 expressing cancer cells confirmed antiproliferative effects of this class of compounds, excluding off target phenomena. In attempt to rationalize obtained biological results and to improve enzyme inhibition, 3D-QSAR studies were carried out. These promising results prompt us for further development of this new class of compounds as new and selective IDO1 inhibitors.

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EXPERIMENTAL SECTION

Chemistry. All reagents and solvents were handled according to material safety data sheet of the supplier and were used as purchased without further purification. 1H-Indole (31), 7-azaindole (32), 4chloro-1H-indole (33), 5-fluoro-1H-indole (34), 5-chloro-1H-indole (35), 5-bromo-1H-indole (36), 5-iodo-1H-indole (37), 2-methyl-5chloro-1H-indole (39), 6-chloro-1H-indole (40), 7-chloro-1H-indole (41), benzenethiol (43), 2-methylbenzenethiol (44), 3-methylbenzenethiol (45), 4-methylbenzenethiol (46), 2-methoxybenzenethiol (47), 3-methoxybenzenethiol (48), 4-methoxybenzenethiol (49), 2methoxybenzoyl chloride (51), and 2,4-dimethoxyaniline (52) were commercially available. Microwave-assisted reactions were performed on a CEM Discover SP single-mode reactor equipped with Explorer 72 autosampler, controlling the instrument settings by PC-running CEM Synergy 1.60 software. Closed vessel experiments were carried out in capped microwave-dedicated vials (10 mL) with cylindrical stirring bar (length 8 mm, diameter 3 mm). Stirring, temperature, irradiation power, maximum pressure (Pmax), PowerMAX (simultaneous coolingwhile-heating), ActiVent (simultaneous venting-while-heating), and ramp and hold times were set as indicated. Reaction temperature was monitored by an external fiber optic temperature sensor. After completion of the reaction, the mixture was cooled to 25 °C via air-jet cooling. Organic solutions were dried over anhydrous sodium sulfate. Evaporation of the solvents was carried out on a Büchi Rotavapor R210 equipped with a Büchi V-850 vacuum controller and a Büchi V700 vacuum pump. Column chromatography was performed on columns packed with silica gel from Macherey−Nagel (70−230 mesh). Silica gel thin-layer chromatography (TLC) cards from Macherey−Nagel (silica gel precoated aluminum cards with fluorescent indicator visualizable at 254 nm) were used for TLC. Developed plates were visualized with a Spectroline ENF 260C/FE UV apparatus. Melting points (mp) were determined on a Stuart Scientific SMP1 apparatus and are uncorrected. Infrared spectra (IR) were recorded on a PerkinElmer Spectrum 100 FT-IR spectrophotometer equipped with universal attenuated total reflectance accessory and IR data acquired and processed by PerkinElmer Spectrum 10.03.00.0069 software. Band position and absorption ranges are given in cm−1. Proton nuclear magnetic resonance (1H NMR) spectra were 9768



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reaction mixture was heated at 110 °C for 20 h under Ar stream, cooled, diluted with water, and extracted with ethyl acetate. Organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, n-hexane:ethyl acetate = 3:1 as eluent) to furnish 9 (0.16 g, 39%) as a solid, mp 112−115 °C (from ethanol), lit. 112.5−113.5 °C.55 5-Chloro-3-((3-methoxyphenyl)thio)-1H-indole (11). Synthesized as 9, starting from 35 and 48. Slurry, yield 35%. 1H NMR (DMSO-d6): δ 3.63 (s, 3H), 6.54−6.56 (m, 2H), 6.66 (d, J = 8.2 Hz, 1H), 7.10− 7.14 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.33 (s, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.85 (s, 1H), 11.90 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3413 cm−1. Anal. calcd for C15H12ClNOS (289.78): C, 62.17; H, 4.17; Cl, 12.23; N, 4.83; S, 11.06; found: C, 62.15; H, 4.15; Cl, 12.21; N, 4.85; S, 11.05. 5-Chloro-3-((4-methoxyphenyl)thio)-1H-indole (12). Synthesized as 9, starting from 35 and 49. Solid, yield 9%, mp 95−98 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.67 (s, 3H), 6.81−6.84 (m, 2H), 6.70−6.71 (m, 2H), 7.15−7.17 (m, 1H), 7.34−7.36 (m, 1H), 7.46− 7.48 (m, 1H), 7.82 (s, 1H), 11.79 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3401 cm−1. Anal. calcd for C15H12ClNOS (289.78): C, 62.17; H, 4.17; Cl, 12.23; N, 4.83; S, 11.06; found: C, 62.18; H, 4.15; Cl, 12.20; N, 4.85; S, 11.03. 5-Chloro-3-(o-tolylthio)-1H-indole (13). Synthesized as 9, starting from 35 and 44. Solid, yield 16%, mp 78−80 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.40 (s, 3H), 6.51−6.60 (m, 1H), 6.90−7.01 (m, 2H), 7.23−7.25 (m, 2H), 7.28 (s, 1H), 7.51−7.54 (m, 1H), 7.83 (s, 1H), 11.84 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3396 cm−1. Anal. calcd for C15H12ClNS (273.78): C, 65.81; H, 4.42; Cl, 12.95; N, 5.12; S, 11.71; found: C, 65.79; H, 4.40; Cl, 12.93; N, 5.10; S, 11.69. 5-Chloro-3-(m-tolylthio)-1H-indole (14). Synthesized as 9, starting from 35 and 45. Solid, yield 7%, mp 100−103 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.18 (s, 3H), 6.75 (d, J = 7.1 Hz, 1H), 6.98−7.01 (m, 2H), 7.08 (t, J = 7.9 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.84 (s, 1H), 11.87 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3396 cm−1. Anal. calcd for C15H12ClNS (273.78): C, 65.81; H, 4.42; Cl, 12.95; N, 5.12; S, 11.71; found: C, 65.79; H, 4.40; Cl, 12.93; N, 5.12; S, 11.68. 5-Chloro-3-(p-tolylthio)-1H-indole (15). Synthesized as 9, starting from 35 and 46. Solid, yield 10%, mp 120−122 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.19 (s, 3H), 6.93−7.04 (m, 4H), 7.17 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.83 (s, 1H), 11.84 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3400 cm−1. Anal. calcd for C15H12ClNS (273.78): C, 65.81; H, 4.42; Cl, 12.95; N, 5.12; S, 11.71; found: C, 65.78; H, 4.39; Cl, 12.98; N, 5.12; S, 11.75. 5-Fluoro-3-((2-methoxyphenyl)thio)-1H-indole (16). Synthesized as 9, starting from 34 and 47. Slurry, yield 52%. 1H NMR (DMSO-d6): δ 3.87 (s, 3H), 6.40−6.45 (m, 1H), 6.74−6.76 (m, 1H), 6.98−7.04 (m, 4H), 7.51−7.53 (m, 1H), 7.78 (s, 1H), 11.81 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3404 cm−1. Anal. calcd for C15H12FNOS (273.33): C, 65.92; H, 4.43; F, 6.95; N, 5.12; S, 11.73; found: C, 65.92; H, 4.41; F, 6.98; N, 5.10; S, 11.74. 5-Bromo-3-((2-methoxyphenyl)thio)-1H-indole (17). Synthesized as 9, starting from 36 and 47. Solid, yield 8%, mp 165−167 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.88 (s, 3H), 6.37 (d, J = 7.5 Hz, 1H), 6.70 (t, J = 7.0 Hz, 1H), 6.97−7.05 (m, 2H), 7.29 (d, J = 8.1 Hz, 1H), 7.42 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 11.91 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3432 cm−1. Anal. calcd for C15H12BrNOS (334.23): C, 53.90; H, 3.62; Br, 23.91; N, 4.19; S, 9.59; found: C, 53.92; H, 3.60; Br, 23.89; N, 4.21; S, 9.60. 5-Iodo-3-((2-methoxyphenyl)thio)-1H-indole (18). Synthesized as 9, starting from 37 and 47. Solid, yield 43%, mp 170−172 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.88 (s, 3H), 6.37 (d, J = 8.2 Hz, 1H), 6.70 (t, J = 6.9 Hz, 1H), 6.97−7.05 (m, 2H), 7.35−7.45 (m, 2H), 7.62 (s, 1H), 7.73 (s, 1H), 11.87 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3429 cm−1. Anal. calcd for C15H12INOS (381.23): C, 47.26; H, 3.17; I, 33.29; N, 3.67; S, 8.41; found: C, 47.22; H, 3.15; I, 33.31; N, 3.67; S, 8.40.

5-Ethoxy-3-((2-methoxyphenyl)thio)-1H-indole (19). Synthesized as 9, starting from 3842 and 47. Solid, yield 30%, mp 105−108 °C (from ethanol). 1H NMR (DMSO-d6): δ 1.26 (t, J = 6.7 Hz, 3H), 3.86−3.93 (m, 5H), 6.40−6.42 (m, 1H), 6.68−6.82 (m, 3H), 6.95− 7.02 (m, 2H), 7.35−7.39 (m, 1H), 7.62 (s, 1H), 11.52 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3269 cm−1. Anal. calcd for C17H17NO2S (299.39): C, 68.20; H, 5.72; N, 4.68; S, 10.71; found: C, 68.18; H, 5.74; N, 4.70; S, 10.69. General Procedure for the Preparation of Compounds 20, 21, 25, and 29. 5-Chloro-3-((2,4-dimethoxyphenyl)thio)-1H-indole (20). To a mixture of sodium hydride (0.05 g, 1.1 mmol; 60% in mineral oil) in anhydrous N,N-dimethylformamide (2.0 mL), 35 was added (0.150. g, 0.96 mmol) under Ar stream, and the reaction mixture was stirred at 25 °C for 10 min. 53 (0.36 g, 1.1 mmol) was added, and the reaction mixture was stirred at 60 °C for 12 h under the same conditions, cooled, diluted with water, and extracted with ethyl acetate. Organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, n-hexane:ethyl acetate = 2:1 as eluent) to furnish 20 (0.02 g, 6%) as a solid, mp 130−135 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.62 (s, 3H), 3.75 (s, 3H), 6.49−6.56 (m, 2H), 7.11−7.14 (m, 2H), 7.24−7.26 (m, 1H), 7.39−7.41 (m, 1H), 7.59− 7.61 (m, 1H), 11.74 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3337 cm−1. Anal. calcd for C16H14ClNO2S (319.80): C, 60.09; H, 4.41; Cl, 11.08; N, 4.38; S, 10.02; found: C, 60.05; H, 4.44; Cl, 11.08; N, 4.41; S, 10.05. 5-Chloro-3-((3,5-dimethoxyphenyl)thio)-1H-indole (21). Synthesized as 20, starting from 35 and 54.43 Solid, yield 19%, mp 110−113 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.62 (s, 6H), 6.12 (s, 2H), 6.24 (s, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.33 (s, 1H), 5.51 (d, J = 8.4 Hz, 1H), 7.84 (s, 1H), 11.90 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3349 cm−1. Anal. calcd for C16H14ClNO2S (319.80): C, 60.09; H, 4.41; Cl, 11.08; N, 4.38; S, 10.02; found: C, 60.07; H, 4.39; Cl, 11.05; N, 4.41; S, 10.00. 3-((3,5-Dimethoxyphenyl)thio)-1H-indole (25). Synthesized as 20, starting from 31 and 54.43 Solid, yield 41%, mp 125−128 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.51 (s, 6H), 6.14 (s, 2H), 6.22 (s, 1H), 7.07−7.10 (m, 1H), 7.17−7.21 (m, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 11.70 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3393 cm−1. Anal. calcd for C16H15NO2S (285.36): C, 67.34; H, 5.30; N, 4.91; S, 11.23; found: C, 67.36; H, 5.32; N, 4.89; S, 11.21. 7-Chloro-3-((3,5-dimethoxyphenyl)thio)-1H-indole (29). Synthesized as 20, starting from 41 and 54.43 Solid, yield 2%, mp 105−108 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.61 (s, 6H), 6.14 (s, 2H), 6.23 (s, 1H), 7.09 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.83 (s, 1H), 12.11 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3294 cm−1. Anal. calcd for C16H14ClNO2S (319.80): C, 60.09; H, 4.41; Cl, 11.08; N, 4.38; S, 10.02; found: C, 60.11; H, 4.39; Cl, 11.06; N, 4.38; S, 10.04. General Procedure for the Preparation of Compounds 22, 26, and 27. 5-Chloro-3-((3,5-dimethoxyphenyl)thio)-2-methyl-1Hindole (22). To a mixture of 39 (0.25 g, 1.50 mmol) and 5443 (0.51 g, 1.50 mmol) in DMSO (1.51 mL), copper(I) iodide (28.7 mg, 0.15 mmol) was added. The reaction mixture was heated at 110 °C for 20 h under Ar stream, cooled, diluted with water, and extracted with ethyl acetate. Organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, n-hexane:ethyl acetate = 2:1 as eluent) to furnish 22 (0.20 g, 40%), mp 135−140 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.43 (s, 3H), 3.67(s, 6H), 6.04 (s, 2H), 6.22 (s, 1H), 7.10 (dd, J = 6.5 and 8.5 Hz, 1H), 7.24 (s, 1H), 7.40 (d, J = 8.6 Hz, 1H), 11.88 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3353 cm−1. Anal. calcd for C17H16ClNO2S (333.83): C, 61.16; H, 4.83; Cl, 10.62; N, 4.20; S, 9.60; found: C, 61.14; H, 4.81; Cl, 10.60; N, 4.18; S, 9.63. 3-((3,5-Dimethoxyphenyl)thio)-1H-pyrrolo[2,3-b]pyridine (26). Synthesized as 22, starting from 32 and 54.43 Solid, yield 5%, mp 155−158 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.61 (s, 6H), 6.14 (s, 2H), 6.23 (s, 1H), 7.13−7.15 (m, 1H), 7.79 (d, J = 7.4 Hz, 9769



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1H), 7.91 (s, 1H), 8.30−8.31 (m, 1H), 12.27 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3336 cm−1. Anal. calcd for C15H14N2O2S (286.35): C, 62.92; H, 4.93; N, 9.78; S, 11.20; found: C, 62.90; H, 4.92; N, 9.77; S, 11.21. 4-Chloro-3-((3,5-dimethoxyphenyl)thio)-1H-indole (27). Synthesized as 22, starting from 33 and 54.43 Solid, yield 16%, mp 160−162 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.61 (s, 6H), 6.08−6.09 (m, 2H), 6.20−6.22 (m, 1H), 7.03−7.07 (m, 1H), 7.14−7.16 (m, 1H), 7.44−7.49 (m, 1H), 7.77−7.81 (m, 1H), 12.02 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3371 cm−1. Anal. calcd for C16H14ClNO2S (319.80): C, 60.09; H, 4.41; Cl, 11.08; N, 4.38; S, 10.02; found: C, 60.11; H, 4.39; Cl, 11.11; N, 4.38; S, 10.00. General Procedure for the Preparation of Compounds 28 and 30. 6-Chloro-3-((3,5-dimethoxyphenyl)thio)-1H-indole (28). To a mixture of 40 (75.6 mg, 0.50 mmol) and 5043 (98.7 mg, 0.58 mmol) in ethanol (5.0 mL), a solution of iodine (0.12 g, 0.49 mmol) and potassium iodide (0.35 g, 2.32 mmol) in water (1.33 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 1 h, diluted with water, and extracted with ethyl acetate. Organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, n-hexane:ethyl acetate = 7:1 as eluent) to furnish 28 (38.0 mg, 25%) as an oil. 1H NMR (DMSO-d6): δ 3.60 (s, 6H), 6.11 (s, 2H), 6.22 (s, 1H), 7.09 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.53 (s, 1H), 7.80 (s, 1H), 11.83 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3413 cm−1. Anal. calcd for C16H14ClNO2S (319.80): C, 60.09; H, 4.41; Cl, 11.08; N, 4.38; S, 10.02; found: C, 60.07; H, 4.40; Cl, 11.07; N, 4.40; S, 10.03. 6,7-Dichloro-3-((3,5-dimethoxyphenyl)thio)-1H-indole (30). Synthesized as 28, starting from 4244 and 50.43 Oil, yield 8%. 1H NMR (DMSO-d6): δ 3.61 (s, 6H), 6.13 (s, 2H), 6.24 (s, 1H), 7.27−7.29 (m, 1H), 7.34−7.36 (m, 1H), 7.89 (s, 1H), 12.31 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν 3382 cm−1. Anal. calcd for C16H13Cl2NO2S (354.25): C, 54.25; H, 3.70; Cl, 20.01; N, 3.95; S, 9.05; found: C, 54.27; H, 3.72; Cl, 20.00; N, 3.95; S, 9.03. 1,2-Bis(2,4-dimethoxyphenyl)disulfide (53). A mixture of 52 (5.0 g, 0.03 mol), 37% hydrogen chloride aqueous solution (5.9 mL), and crushed ice (8.0 g) was cooled to −5 °C. A solution of sodium nitrite (2.25 g, 30 mmol) in water (12.0 mL) was added dropwise over 10 min. The reaction mixture was stirred in the same conditions for 10 min and added dropwise to a hot solution (65 °C) of potassium ethyl xantogenate (10.46 g, 60 mmol) in water (31.0 mL). The mixture was stirred in the same conditions for 30 min, cooled, and extracted with ethyl acetate. Organic layer was washed with brine, dried, and filtered to give S-(2,4-dimethoxyphenyl) O-ethyl carbonodithioate, that was used without further purification. A mixture of the latter and 3 N sodium hydroxide aqueous solution (91.0 mL) in ethanol (85.0 mL) was heated to 65 °C for 2 h, cooled, and made acidic with 37% hydrogen chloride aqueous solution (pH ≈ 2−3) while stirring. A saturated solution of iodine in ethanol was added dropwise until a persistent violet coloration was observed. The reaction mixture was stirred at 25 °C for 10 min and diluted with ethyl acetate and a saturated aqueous solution of sodium thiosulfate. The layers were separated, and the organic one was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica gel, n-hexane:ethyl acetate = 3:1 as eluent) to furnish 53 (0.40 g, 4%) as a solid, mp 115−117 °C (from ligroin), lit. 117 °C.56,57 Enzymatic Assay. The standard assay mixture (200 μL) contained 50 mM of potassium phosphate buffer (pH = 6.5), 10 mM ascorbate, 10 μM methylene blue, 100 μg/mL catalase, 200 μM L-tryptophan, and 10 μg/mL of rhIDO1. At 37 °C, the reaction was started by the addition of the substrate, terminated after 60 min by adding 40 μL of 30% (w/v) trichloroacetic acid, and further incubated at 50 °C for 15 min to hydrolyze N-formylkynurenine produced by indole-amine 2,3dioxygenase to kynurenine. After centrifugation at 1500g for 5 min at 20 °C, kynurenine in the supernatant was measured by HPLC with a reversed phase column (4.6 mm × 15 cm) of TSK-100Z. The mobile phase was 10 mM ammonium acetate containing 10% (w/v) methanol, and kynurenine was detected by absorbance at 360 nm.

All determinations were carried out in triplicate. The presented data are average values. The compounds inhibitory activity against TDO2 was tested in the same way. Cell Cultures. HT29 and HCT116 cells were grown in DMEM containing 4.5 g/L of glucose, supplemented with 10% FBS, 100 units/mL of penicillin and streptomycin, and 2 mM glutamine. Cell Vitality Assays. HCT116 and HT29 vitality cells were determined using the MTT colorimetric assay. HCT116 and HT29 cells were seeded into 24-well culture plates, and after 24 h of growth to allow attachment of cells to the wells, test compounds were added at 5−80 μM and 2.5−80 μM. After 24 h of growth and removal of the culture medium, 500 μL/well of PBS containing 500 μM MTT was added. Cell cultures were further incubated at 37 °C for 3 h in the dark. The solutions were then gently aspirated from each well, and the formazan crystals within the cells were dissolved in propan-2-ol and 0.04 N HCl (200 μL). Optical densities were red at 550 nm using a Multiskan Spectrum Thermo Electron Corporation reader. The results were expressed as % relative to vehicle-treated control (0.1% DMSO). Three independent measurements were performed in triplicate for each assay. Flow Cytometric Analysis. Cell samples were analyzed in a Coulter Epics XL cytofluorimeter (Beckman Coulter) equipped with EXPO 32 ADC software. At least 10000 cells per sample were acquired and analyzed using BD CellQuest Pro Software. Western Blotting Analysis. Cultured cells were lysed in 150 mM NaCl, 0,1% SDS, 5 mM EDTA, 1% sodium deoxycholate, 1% Triton X-100, 50 mM Tris, pH 7.4 and protease inhibitor cocktail and centrifuged at 12,000g for 15 min at 4 °C to remove cellular debris. The protein concentration was measured using the Bradford assay (bio-Rad). The proteins were separated by 12% SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Bio-Rad). After blocking with 5% skim milk for 1 h, the membranes were incubated with Mcl1 (1:1000; Cell Signaling Technology), Bcl-xL (1:1000; Cell Signaling Technology), β-catenin (1:500; BD Biosciences), and β-actin (1:5000; SIGMA) overnight at 4 °C. After incubation with corresponding suited 1:2.500 horseradish peroxidaseconjugated secondary antibody (1:2500; Cell Signaling Technology). The signals were developed using the enhanced chemiluminescence kit (ClarityTM Western ECL Substrate, Bio-Rad) and analyzed with the ChemiDoc Imaging System XRS+ (Bio-Rad) and with the Image Lab 4.1 software. Dynamics Simulation and Molecular Docking Studies. All molecular modeling studies were performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 12. The IDO1 structure was downloaded from the PDB data bank (pdb code: 2D0T).33 The Trp binding coordinates were obtained by TDO/Trp crystal structure (pdb code: 2NW8).35 Molecular dynamics was carried out by Amber suite.58 Heme parameters were from the Bryce group database.59,60 The minimized structure was solvated in a periodic octahedron simulation box using TIP3P water molecules, providing a minimum of 10 Å of water between the protein surface and any periodic box edge. Ions were added to neutralize the charge of the total system. The water molecules and Na+ ions were energy-minimized, keeping the coordinates of the complex fixed (1000 cycle), and then the whole system was minimized (5000 cycle). Following minimization, the entire system was heated to 298 K (2 ns). The production simulation (20 ns) was conducted at 298 K with constant pressure and periodic boundary condition. During heating and production, a restraint of 10 kcal mol−1 Å−2 was applied to heme, O2, and His335. Production was carried out on GeForce gtx780 gpu. Trajectories analyses were carried out by cpptraj program.61 Molecular dynamics snapshots were obtained by computing the average for the latest 3000 steps, and then the representative step was selected as the one with the lowest RMSD versus computed average. Because in-house library compounds were featured by a good accordance with the requirements of the Lipinsky’s rules, no prefilter was applied. All compounds were in mol2 format, and the geometry was optimized by minimization keeping all the heavy atoms fixed until an RMSD gradient of 0.05 kcal mol−1 Å−1 was reached. Docking simulations were performed with PLANTS,34 using a 12 Å radius grid sphere. The gridcenter was obtained as the 9770



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centroid of the cocrystallized inhibitor coordinates. 3D-QSAR models are described in the Supporting Information. The images in the manuscript were generated by PyMOL.62 Training Set. Among the new synthesized indole derivatives, 15 were used as the training set to develop the final 3-D QSAR models. Inactive compounds were not included in the training set. Compound 8 was also excluded because the trials it was included in did not lead to any statistically significant model. Molecular Alignment Rules. The training set molecules were drawn in SMILES format63−66 and subsequently subjected to an extensive alignment procedure. To systematically inspect the most suitable molecular alignment, different molecules among the training set (Table 2S, Supporting Information) were selected as templates using the following criteria: the most active (21), the least active (14), the heaviest (18), the largest (13), and the most flexible (24). Attempts to select either the longest (14) or the least flexible (13) molecules as template lead to reference molecules that were already selected by other criteria. For the molecular alignment Surflex-sim,67 Align-it,68−70 Sheap,71 Shape-it,68−70 Kcombu,72 and Obfit73 were used to effectively align the training set to afford a total of 400 different alignments. The molecular alignments were carried out on the whole template conformational ensemble generated by balloon.74 Balloon. Default settings were used except for the following: the number of generated conformations was set to 500; the maximum iteration for conjugate gradient structure optimization was set to 1000, and 500 maximum number of n generations was used. This program produced an ensamble of atom-by-atom least-squared aligned conformations. Surflex-Sim. Default values were used except that dynamic ring search was turned on; only one conformation was saved, and the max depth of recurrence was set 100. Shaep. All the default parameters were used. Shape-It. Only the number of additional iterations in the simulating annealing optimization step was changed in the default settings. Align-It. All the default parameters were used. Fkcombu. The volume overlap and a flexible procedure were used. Obfit. This was used as an atom-by-atom fitting procedure using a minimal common substructure (MCS) SMART string generated by an in house python script using the RDKit library.75 3-D QSAR Models. To generate the models, the 3-D QSAutogrid/R procedure was applied as previously described.46 Initial models were built on the above 400 described alignments and 8 atom probes. The best eight models were then optimized by a variable pretreatment optimization (VPO) procedure by varying the grid step, cutoff, zeroing, and minimum standard deviation values (Table 3S, Supporting Information). Among all the combinations, the bestpretreated models (highest q2 values) were selected and optimized by a simulated annealing (SA) variable selection algorithm. For the final models, lack of chance correlation was checked by Y-scrambling procedure.46 The training set biological activity was converted into pIC50 with the −log(IC50) formula, expressing IC50 in molar concentration.

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3-D QSAR results with the VPO, simulated annealing, and Y-scrambling, and 2-D and 3-D structures, features, and their values of the selected template molecules, grid optimization, VPO analysis parameters, admetSAR data, calculated drug likeness properties, and Volsurf calculated ADMET properties (PDF) Compound data (CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: +39 06 4991 3404. E-mail: giuseppe.laregina@ uniroma1.it. Fax: +39 06 4991 3993. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from Progetti di Ricerca di Università 2015, Sapienza Università di Roma (C26A15RT82 and C26A15J3BB). Authors are grateful to Dr. Andrea Brancale (Cardiff University, U.K.) for helpful discussion and suggestions.

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ABBREVIATIONS USED IDO1, indoleamine 2,3-dioxygenase 1; L-Trp, L-tryptophan; TDO2, tryptophan 2,3-dioxygenase 2; IDO2, indoleamine 2,3dioxygenase 2; VS, virtual screening; SAR, structure−activity relationship

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REFERENCES

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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.6b00718. 13 C NMR and MS data of 7, 9, 13, 19, 22, and 25, snapshot of IDO1/L-Trp complex trajectory, effect of compound 5 on IDO1 activity, effect of compound 5 on TDO2 activity, inhibition of IDO1 activity by compound 5, average activity contribution plots (C and HD probes), C probe average activity contribution plots (compounds 12 and 30), C probe average activity contribution plots (compounds 28 and 30), C probe average activity contribution plots (compound 12), average activity contribution plots (C and HD probes), 9771



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New Inhibitors of Indoleamine 2,3-Dioxygenase 1: Molecular Modeling

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