Discovery of secondary sulphonamides as IDO1 inhibitors with potent antitumour effects in vivo

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) as a key rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism plays an important role in tumour immune escape. Herein, a variety of secondary sulphonamides were synthesised and evaluated in the HeLa cell-based IDO1/kynurenine assay, leading to the identification of new IDO1 inhibitors. Among them, compounds 5d, 5l and 8g exhibited the strongest inhibitory effect with significantly improved activity over the hit compound BS-1. The in vitro results showed that these compounds could restore the T cell proliferation and inhibit the differentiation of naïve CD4+ T cell into highly immunosuppressive FoxP3+ regulatory T (Treg) cell without affecting the viability of HeLa cells and the expression of IDO1 protein. Importantly, the pharmacodynamic assay showed that compound 5d possessed potent antitumour effect in both CT26 and B16F1 tumours bearing immunocompetent mice but not in immunodeficient mice. Functionally, subsequent experiments demonstrated that compound 5d could effectively inhibit tumour cell proliferation, induce apoptosis, up-regulate the expression of IFN-γ and granzyme B, and suppress FoxP3+ Treg cell differentiation, thereby activate the immune system. Thus, compound 5d could be a potential and efficacious agent for further evaluation.


Introduction
With the increasing recognition of the interaction between the immune system and tumour cells, immunotherapy has gained tremendous attention in the field of cancer research 1,2 . The recent clinical successes of immune checkpoint inhibitors and chimeric antigen receptor T cell (CAR-T) therapy have dramatically changed the landscape of the cancer treatment [3][4][5][6] . To date, the US Food and Drug Administration (FDA) has approved six immune checkpoint inhibitors blocking cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1), and the second CAR-T cell therapy for the treatment of solid and hematological malignancies. However, their limited response rate and immune-related adverse effects call for a need to develop other immunotherapy options to solve those problems [5][6][7] . Indoleamine 2,3-dioxygenase 1 (IDO1) is a haem-containing enzyme that catalyses the initial and rate-limiting step in the catabolism of the essential amino acid tryptophan along the kynurenine pathway 8 . IDO1 plays an important role in the regulation of local inflammation and immune tolerance 9 . In cancer, aberrant activation of IDO1 results in suppression of antitumour immunity. The depletion of tryptophan and the accumulation of kynurenine and its downstream metabolites in the tumour microenvironment have been demonstrated to induce effector T cell anergy and enhance regulatory T (Treg) cell function, which eventually lead to an immunosuppressive milieu that helps tumour cells evade detection and killing by the immune system 10 . Tryptophan depletion has been implicated in the activation of general control non-derepressible 2 (GCN2) and suppression of the mechanistic target of rapamycin kinase (mTOR) pathways 11,12 . Furthermore, kynurenine and other tryptophan metabolites can activate the aryl hydrocarbon receptor (AHR) 13 . Indeed, high expression of IDO1 in tumour cells has been shown to correlate with worse clinical prognosis in patients with a variety of cancers, including colorectal, ovarian, endometrial, and hepatocellular carcinomas [14][15][16][17][18] . Moreover, preclinical studies have demonstrated that IDO inhibitors can effectively restore antitumour immunity and have the potential to synergize with chemotherapy, radiotherapy, and other immunotherapies, including anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies [19][20][21][22] . Hence, these findings have motivated interest in developing IDO1 inhibitors for cancer immunotherapy.
So far, a number of IDO1 inhibitors have been identified from high-throughput screening 23,24 , structure-based design [25][26][27] , and natural product screening 28,29 . Several of them, including, navoximod, epacadostat, PF-06840003, linrodostat, LY-3381916 and KHK2455 (structure not disclosed), have entered clinical development as monotherapy and in combination with other immune CONTACT Wenjie Guo guowj@nju.edu; Qiang Xu molpharm@163.com therapies or chemotherapy (Figure 1) 30 . However, the FDA has not yet approved IDO inhibitors for the treatment of malignancies to date. We therefore need to continue to identify novel IDO1 inhibitors.
In recent years, we have been working on the development of novel IDO1 inhibitors [31][32][33][34] . From our previous study on the interactions between IDO1 and imidazole derivatives, it has been found that metal chelation, hydrogen bonding, and hydrophobic interactions between haem and the ligand contribute a large proportion to the binding affinity of the inhibitor 31 . Furthermore, the human IDO1 crystal structures in complex with the ligand Amg-1 (PDB ID: 4PK5) revealed that IDO1 is characterised by a small and highly lipophilic active site including two pockets (A and B), implying that the hydrophobic interaction may account for a large part of protein-ligand nonbonding interactions ( Figure 2). In addition, Muller's team demonstrated that as the charge on the coordinating atom decreased, the binding affinity to the haem iron increased, which would be possible to improve IDO1 inhibitory activity 35 . These findings provide directions for improving the binding affinity of novel IDO1 inhibitors.
In this study, we screened our in-house compound library using the HeLa cell-based IDO1/kynurenine assay, and found that the secondary sulphonamide compound BS-1 showed inhibitory activity against IDO1 with an IC 50 value of 48.42 mM. As well known, bezenesulfonamide is a common pharmacophore that is the basis of several groups of drugs, such as sulpha antibiotics, thiazide diuretics and nonsteroidal antiinflammatory drug (NSAID) celecoxib 36,37 . Moreover, the sulphonyl is a heme-binding function that coordinates to the haem iron of IDO1 38 . Therefore, the hit compound BS-1 was further modified, as well as a variety of secondary sulphonamides were synthesised and evaluated in the HeLa cellbased IDO1/kynurenine assay. The resulting compounds 5d, 5l and 8g showed high inhibitory activity without affecting the cell viability and the IDO1 protein expression. Subsequent in vitro and in vivo experiments demonstrated that compound 5d could exert potent antitumour effects by activating the mouse immune system.

Chemistry
Melting points were determined on a RDCSY-I capillary apparatus and were uncorrected. Allmaterials used were commercially  available and used as supplied. HG/T2354-92 silica gel 60 F254 sheets were used for analytical thin-layer chromatography (TLC). Column chromatography was performed on silica gel (300-400 mesh). 1 H NMR spectra were recorded on a Bruker AV-300 spectrometer. Chemical shifts (d) were given in parts per million (ppm) relative to the solvent peak. J values are in Hz. Chemical shifts are expressed in ppm downfield from internal standard TMS. Mass spectra (MS) were measured using a Thermo Scientific iCAP RQ ICP-MS. All the reagents and solvents were reagent grade and were used without further purification unless otherwise specified.
2.1.1. General preparation of compounds 3a-i To a solution of substituted aniline (0.97 mmol) in DCM (15 ml) was added triethylamine (1.22 mmol) 39 . A solution of 4-acrylamidobenzenesulfonyl chloride (0.81 mmol) in DCM (10 ml) was added dropwise to the mixture at 0 C. The reaction was stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure and the crude product was recrystallization to afford target compounds 3a-i.

General preparation of compounds 4a-f
To a solution of compounds 3 (0.68 mmol) in ethanol (15 ml) was added hydrochloric acid (1 ml) 39 . Then the mixture was stirred at 70 C for 12 h. After the reaction was completed, the solvent was evaporated under reduced pressure. Water was added and the pH was adjusted to 7-8 with saturated NaHCO 3 solution. The aqueous phase was extracted with EtOAc (3 Â 30 ml). The combined organic layers were washed with water, brine, and dried. The solvent was removed in vacuo and the crude product was recrystallization to afford target compounds 4a-f.
2.1.5. General preparation of 4-acrylamidobenzenesulfonyl chloride 7 N-phenylacrylamide (0.031 mol) was added slowly to sulphuryl chloride (15 ml) at 0 C for more than 0.5 h. The reaction was stirred at 70 C for 1 h. After the reaction was completed, the mixture was poured into ice water slowly to quench the reaction. The mixture was stirred at rt for 1 h and the solid was filtered-off and washed with water (2 Â 50 ml). The crude solid was dissolved in CH 2 Cl 2 (50 ml), the organic layer was washed with brine, and the solvent was evaporated under reduced pressure to afford 4-acrylamidobenzenesulfonyl chloride as off-white solid (Yield 72.8%).

General preparation of compounds 8a-j
To a solution of substituted aniline or heterocyclic amine (0.98 mmol) in DCM (15 ml) was added pyridine (1.22 mmol). A solution of 4-acrylamidobenzenesulfonyl chloride (0.81 mmol) in DCM (10 ml) was added dropwise to the mixture at 0 C. The reaction was stirred at rt for 4 h. The solvent was evaporated under reduced pressure and the crude product was recrystallization to afford target compounds 8a-j.

Biological assays
The main bioassays performed in the study, including HeLa cellbased IDO1/kynurenine assay, Western blot analysis, T cell proliferation and Treg cell differentiation experiments, were carried out as described previously 32 .

Cellular thermal shift assay
HeLa cells were seeded in 6-well culture plates at a density of 2 Â 10 5 per well. On the next day, human IFN-c (100 ng/mL) were added and incubated for 24 h, and then the cells were treated with 5d for 2 h. At the end of incubation, cells collected and subjected to CETSA assay. Briefly, incubated cells were equally divided into 10 parts, each part was heated for 3 min under different temperature (43,46,49,52, 55, 58, 61, 64, 67, and 70 C), and then the heated cells were kept at À80 C for 12 h, then at room temperature for 5 min, and the process repeated one more time. After that, cell lysates were extracted by centrifugation at 20,000 Â g, 20 min. Levels of IDO1 protein were assessed by Western blot analysis.

2.2.2.
In vivo antitumour activity assay BALB/c mice (6-8 weeks old, 18-22 g) were supplied by Model Animal Research Centre of Nanjing University (Nanjing, China). Mice were maintained under standard specific-pathogen-free (SPF) conditions (21 ± 2 C and 12-h light: dark cycle). Animal welfare and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals (The Ministry of Science and Technology of China, 2006) and the related ethical regulations of our university. All efforts were made to minimise animals' suffering and to reduce the number of animals used. Mouse colon cancer cells (CT26) and melanoma cells (B16F1) were cultured and collected by centrifugation (1000 rpm, 5 min) and washed twice with ice-cold PBS. Then cells were diluted to 1 Â 10 7 /ml and 1 Â 10 6 CT26/B16F1 cells (in 0.1 ml PBS) were injected subcutaneously into the right flanks of mice. All mice formed tumours three days after injection. Then mice were randomly distributed into five groups (n ¼ 10/8) according to tumour volumes. 5d (3.125, 6.5, 12.5 mg/kg) were administered (i.g) to each group respectively every day. 5-FU (25 mg/kg) and Cis-platinum (1 mg/kg) were administered (i.p) every three days. Tumour length and tumour width were measured with a Vernier calliper every three days. Tumour volumes were measured and calculated using the equation volume ¼ a Â b 2 /2, where "a" is the maximal width and "b" is maximal orthogonal width. After the administration were completed, mice were weighed, euthanized, and tumours were removed and the weight were taken.
Eight-week-old male BALB/c nude mice, weighing 18-22 g, were supplied by Model Animal Research Centre of Nanjing University (Nanjing, China). 1 Â 10 7 /ml and 1 Â 10 6 CT26 cells (in 0.1 ml PBS) were injected subcutaneously into the right flanks of mice. After CT26 xenograft BALB/c nude mouse model was established, all mice were randomly divided into three groups (n ¼ 8). 5d (12.5 mg/kg) were administered (i.g) every day and 5-FU (25 mg/kg) awere administered (i.p) every three days. The following experimental procedures were similar to the described above.

Immunohistochemistry analysis
For immunohistochemistry staining, the sections were deparaffinized, rehydrated, and washed in 1% PBS-Tween 20, and then treated with 2% hydrogen peroxide, blocked with 3% goat serum (Life Technology, 16210-064) and incubated for 2 h at room temperature with specific primary antibodies. Then the slides were incubated with streptavidin-HRP (Shanghai Gene Company, GK500705) for 40 min, then stained with DAB (Shanghai Gene Company, GK500705) substrate and counter-stained with haematoxylin. Images were acquired by microscopy (Olympus IX51).

Molecular modelling
In order to consider the flexibility of both ligand and protein, the induced fit docking (IFD) protocol in Schr€ odinger was employed.
In IFD calculations, the ligands were first docked into the rigid receptor using softened energy function in Glide. By default, a maximum 20 poses per ligand were retained. Then, the protein degrees of freedom for each complex were sampled and the protein-ligand complexes were minimised. The protein structure in each pose now reflected an induced fit to the ligand structure and conformation. The best protein-ligand complex was then identified based on the predicted binding affinities of the docked ligand. Here, the residues within 5 Å of each of the 20 ligand poses were subjected to a conformational search and energy minimizations, and the residues outside this range were fixed. Finally, the minimised ligand was rigorously redocked into the induced-fit protein structure using Glide XP scoring mode, and metal constraints can be applied to both Glide docking stages in IFD protocol. The choice of the best-docked structure for each ligand was made using a model energy score that combines the energy grid score, the binding affinity predicted by GlideScore, and the internal strain energy for the model potential used to direct the conformational-search algorithm.

Chemistry
The synthetic pathways for the target compounds were shown in Scheme 1 and 2. N-acetyl secondary sulphonamides 3 were prepared by the condensation reaction of substituted 4-acetamidobenzenesulfonyl chloride 1 with various amines 2. Deacetylation of compounds 3 with concentrated HCl in ethanol gave amino secondary sulphonamides hydrochloride salts, and followed by neutralisation with saturated NaHCO 3 solution to afford the corresponding amino secondary sulphonamides 4. Finally, the compounds 5 were prepared via the condensation reaction of compounds 4 with acryloyl chloride (Scheme 1).
Alternatively, N-phenylacrylamide 6 was synthesised by condensation of aniline with acryloyl chloride in the presence of triethylamine, and followed by chlorosulfonation with chlorosulfonic acid to give 4-acetamidobenzenesulfonyl chloride 7. Subsequently,

Effect of secondary sulphonamides on IDO1 activity
It is well known that IDO1 is broadly expressed in human cancers and is induced by pro-inflammatory cytokines, such as interferon gamma (IFN-c) and tumour necrosis factor alpha (TNF-a) 41 . Here we utilised a human cervical cancer cell line HeLa that endogenously expresses IDO1 upon IFN-c treatment to evaluate the IDO1 inhibitory activity of the synthesised compounds by measuring kynurenine secreted into the media. This cell-based assay, as a rapid and simple method for determination IDO1/kynurenine, is closely related to the potency of the enzyme assay and has been widely used to identify various IDO1 inhibitors 19,23,29,42 . In addition, cytotoxic compounds can reduce cell viability, which will decrease the IDO1 levels and then lower the conversion of tryptophan to kynurenine. Thus, we also employed this cellular assay to detect false positive IDO1 inhibitors and cytotoxic compounds by measuring HeLa cell viability. Clinical candidate navoximod was used as an assay control. The results were shown in Table 1 and 2.
Our group had already synthesised a series of N-acetyl secondary sulphonamides as the analogs of compound BS-1 and evaluated their inhibitory activity 43 . To our surprise, although most of these compounds had weak inhibitory activity against IDO1, N-(4-(N-(4-methoxybenzyl)sulfamoyl)phenyl)acetamide (HeLa IC 50 : 5.88 mM) showed good inhibitory activity, and its potency was remarkably increased by 8.4-fold as compared with the hit BS-1. Inspired by N-(4-(N-(4-methoxybenzyl)sulfamoyl)phenyl)acetamide, we further synthesised the analogues 3a-i bearing an electrondonor group on the benzyl group, but they displayed only weak activity. Subsequently, we tried to remove the acetyl group of Nacetyl secondary sulphonamides, and found that all the obtained aniline compounds 4a-f had no improvement in activity. These results indicated that a free amino group on the phenyl ring with strong electron-donating ability appeared to be unfavourable for the inhibitory activity of these compounds. To this end, we tried to introduce an acryloyl group instead of the acetyl group to the amine group. As expected, compounds 5a-e bearing an acetyl group showed moderate to good inhibitory activity. Especially, compound 5d (HeLa IC 50 : 6.26 mM) exhibited significantly stronger activity than the hit compound BS-1 (HeLa IC 50 : 49.42 mM) and its corresponding acetyl compound 4e (HeLa IC 50 > 50 mM).
We then further optimised the inhibitory activity of N-acryloyl secondary sulphonamides by modifying the substituents, including n, R 2 and R 3 , while retaining the acryloyl group at R 1 . As shown in Table 2, most of N-acryloyl secondary sulphonamides displayed good inhibitory activity against IDO1. Among them, benzylamine derivative 5l and aniline derivative 8g exhibited potent activity with IC 50 values of 6.64 mM and 3.76 mM, which were significantly increased by 7.4-fold and 13.1-fold as compared with the hit BS-1, respectively. It is worth noting that the compounds 5d, 5g, 5l, and 8d with a substituent at 3-position on aniline or benzylamine had higher activity than those compounds 5c, 5f, 5k and 8c with a substituent at 4-position (5c vs 5d, 5f vs 5g, 5k vs 5l, 8c vs 8d). However, compound 5i (IC 50 : 39.53 mM) with a fluoro substituent   , which showed similar inhibitory activity with compound 5a without substituent on aniline (IC 50 : 22.36 mM), likely due to the similar size of the fluorine and hydrogen atoms, Interestingly, compound 8g containing a mchlorine and a p-fluorine atoms showed the strongest potency (IC 50 : 3.76 mM), while compound 5m with m,p-di-chlorine atoms had only moderate activity (IC 50 : 37.27 mM). It might be duo to the large volume of the 4-substituent on aniline or benzylamine that cause the bad clashes with the neighbouring residues in active site of IDO1. In addition, we also tried to replace benzene or benzyl rings with aromatic heterocycles (8 h-8j), including pyrimidine and isoxazole, but they showed only weak activity.
Taken together, compounds 5d, 5l and 8g showed good inhibitory activity in the HeLa cell-based IDO1/kynurenine assay. Furthermore, the viability of HeLa cells was evaluated by the MTT method at the end of the assay. The readouts showed that most of the synthesised compounds did not affect cell viability under the experimental conditions (data not shown), indicating that IDO1 inhibitory activity of these compounds is not mediated by their cytotoxicity.

Effect of 5d, 5 l, 8f and 8g on IDO1 protein expression
To exclude the possibility that these secondary sulphonamides inhibit IDO1 activity by down-regulating IDO1 expression, we utilised Western blot to measure the expression of IDO1 protein in HeLa cells treated with different compounds. As depicted in Figure 3, compared with the blank group, the expression of IDO1 protein was significantly induced in IFN-c-treated HeLa cells. Evidently, all tested compounds 5d, 5l, 8f and 8g did not influence IDO1 expression as compared with the control group. In addition, we further confirmed that treatment of HeLa cells with different concentrations of compound 5d (3, 10, 30 mM) for 24 h or 48 h also did not affect IDO1 expression. The results demonstrated that these compounds blocked the kynurenine pathway by affecting IDO1 enzyme activity rather than its expression and/or HeLa cell viability.

Compounds 5d and 8g could effectively restore T cell proliferation
T lymphocytes are exceedingly sensitive to low tryptophan levels. Overactivation of the kynurenine pathway by high IDO1 expression leads to local tryptophan depletion, which can suppress T cell proliferation by activating the GCN2 kinase pathway and eventually result in T cell anergy 44 . Here, to determine whether secondary sulphonamides could rescue IDO1-mediated blockade of T cell proliferation, we used a co-culture of IDO1-expressing B16F1 melanoma cells with naïve CD4 þ T cells to mimic the tumour microenvironment. Given that melanoma is a highly immunogenic tumour and IDO1 constitutively expresses in melanoma cells 40 , B16F1 murine melanoma cell lines that express IDO1 were then used to co-culture with T cells. Naïve CD4 þ T cells were isolated from spleens of C57BL/6 mice. As shown in Figure 4, compounds 5d, 8g and navoximod displayed a significant augmentation on T cell activity stimulated with B16F1 cells. Especially, compound 5d showed better enhancement of T cell proliferation than navoximod. These findings demonstrated that compounds 5d and 8g could reverse the suppression of T lymphocytes caused by IDO1.

Compounds 5d and 8g could effectively inhibit FoxP3 1 Treg cell differentiation
Besides causing local tryptophan depletion, high IDO1 expression can also accumulate metabolites of kynurenine pathway. The elevated metabolites can not only induce effector T cell death directly through their cytotoxicity, but also promote the differentiation of naïve CD4 þ T cells into CD4 þ FoxP3 þ Treg cells by activating the AHR pathway 10 . As a highly immunosuppressive subset of CD4 þ T cells, FoxP3 þ Treg cells are critical to the maintenance of immune homeostasis and are involved in tumour immune escape, thereby contributing to tumour development and progression 45 . Therefore, we also utilised co-culture of B16F1 cells with naïve CD4 þ T cells to test whether the synthesised compounds could suppress IDO1 mediated Treg cell differentiation. As illustrated in Figure 5, when the naïve CD4 þ T cells were co-cultured with B16F1 cells under Con A stimulation for 48 h, the number of the FoxP3 þ Treg cells increased approximately 4 times compared with naïve T cells cultured alone, indicating that IDO1expressing B16F1 cells could significantly stimulate naïve T cell differentiation into FoxP3 þ Treg cells. However, treatment with 5d, 8g and navoximod could significantly reduce FoxP3 þ Treg cell differentiation. Especially, compound 5d displayed the best effect in this test, and its FoxP3 þ Treg cell number returned to the initial level. These data evidenced that compounds 5d and 8g could effectively suppress the differentiation of naïve CD4 þ T cell into FoxP3 þ Treg cells, which would help reverse IDO1-mediated immune suppression.

Compound 5d could bind to IDO1 protein in HeLa cells
The cellular thermal shift assay (CETSA) is usually used to identify compounds and their potential targets. CESTA can detect the target engagement of drugs in a cellular context based on the biophysical principle of ligand-induced thermal stabilisation of target proteins 46 . To this end, we further employed the CETSA assay to detect whether compound 5d could directly bind to the target protein IDO1 in HeLa cells. As presented in Figure 6, treatment with 5d could improve the thermal stability of IDO1 protein in HeLa cells as compared with the vehicle control, and still maintained the protein stability at 55 C. These results suggested that compound 5d could enter HeLa cells and then directly bind to IDO1 protein.

Compound 5d could effectively inhibit colon carcinoma growth in immunocompetent mice, but not in immunodeficient mice
Given that it was the most prominent inhibitor in vitro, we chose compound 5d for further pharmacodynamic evaluation in vivo. Bowel cancer is the third leading cause of cancer in the world, thus we first evaluated the efficacy of compound 5d on the growth of murine CT26 colon carcinoma xenograft model in immunocompetent BALB/c mice. Fluorouracil (5-FU) is one of the most commonly used drugs to different cancers including bowel cancer, and was used as the positive control. The CT26 tumour-  Based on the B16F1-T cell co-culture system, use MTT assay to test T cells proliferation. The Navoximod and compounds were added to the system at the concentration of 1.5 and 18 lM. Data represent mean ± SEM (n ¼ 3).
bearing mice were treated with 5d (3.125, 6.25, 12.5 mg/kg, i.g, every day) or 5-FU (25 mg/kg, i.p, every other day). Tumour volumes were measured every two days for 14 days. As outlined in Figure 7, treatment of 5d significantly suppressed the growth of tumour in a dose-dependent manner as compared with the vehicle group. Especially, 5d reduced tumour volume by 35.9% and average tumour weight by 37.5% at the dose of 12.5 mg/kg, which were comparable to that of 5-FU. Moreover, no significant change in body weight of the mice was observed in administration groups compared with the vehicle group during the 14day treatment.
Then, in order to clarify whether the in vivo antitumour efficacy of compound 5d was related to the mouse immune system, we repeated the above experiment in immunodeficient nude mice. Briefly, BALB/c nude mice bearing established CT26 colon carcinoma xenograft were treated with compound 5d and 5-FU for 10 days. As introduced in Figure 8, administration of 5d did not block tumour growth as compared with the vehicle group, while classical cytotoxics 5-FU significantly suppressed tumour growth. Taken together, these results implied that the in vivo antitumour efficacy of compound 5d was achieved by activating mouse immune system rather than its cytotoxicity.

Compound 5d could inhibit the growth of tumour cells by curbing proliferation and inducing apoptosis in CT26 tumour tissues
In order to further observe morphological changes in tumour tissues after administration of compound 5d, the tumour tissue sections obtained from the BALB/c mice bearing CT26 colorectal tumours were stained with haematoxylin-eosin (H&E) to highlight different tissue structures, such as the nucleus and cytoplasm. As illustrated in Figure 9(A), the number of tumour cells in 5-FUtreated group was significantly reduced as compared with the vehicle group. Similarly, administration of 5d decreased the number of tumour cells, as well as led to the morphological changes including cell shrinkage and chromatin condensation in a dosedependent manner. In addition, it was clearly observed from the enlarged local image that there are more blood vessels in the compound 5d group, and their morphology appears more normal, suggesting that compound 5d might contribute to tumour vascular normalisation, thereby promoting antitumour effect of the immune system 47 .  Then, to understand the mechanism of tumour cell death, we detected the level of proliferating cell nuclear antigen (PCNA) protein and tumour cell apoptosis by immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) experiments, respectively. PCNA is known as a molecular marker for assessing cell proliferation status, and TUNEL assay is the most widely used method to identify apoptotic cells. The results of IHC analysis showed that the expression of PCNA protein in the 5-FU group was significantly reduced as compared with the vehicle group. The similar results were observed in the 5d treatment group and showed a doseresponse relationship (Figure 9(B)). In addition, the results of TUNEL assay showed that the tumour cells had less apoptosis in the vehicle group, while the apoptosis increased in the 5-FU group. Likewise, treatment with 5d also significantly induced the tumour cell apoptosis in a dose-dependent manner (Figure 9(C)). These data revealed that compound 5d displayed in vivo antitumour efficacy by inhibiting tumour cell proliferation and inducing apoptosis.
To verify the above conclusions, we further performed H&E and IHC staining on the nude mouse CT26 tumour tissues. As depicted in Figure 10(A), the tumour cells in the 5d-treated group remained intact morphology and were arranged in an orderly manner as that in the vehicle-treated group, while the results of 5-FU treatment were just the opposite. Moreover, the results of TUNEL assay showed that administration of 5d had no influence on the expression of PCNA (Figure 10(B)). These findings further supported the above conclusions.

Compound 5d facilitated immune system rejuvenation in CT26 tumour-bearing mice
To investigate the effect of compound 5d on the immune function in CT26 tumour-bearing mice, we carried out the IHC experiment to detect the expression of IFN-c and granzyme B in the tumour tissues. IFN-c is a key cytokine secreted primarily by activated T cells and NK cells, which is essential for both innate and adaptive immunity. Therefore, IFN-c is an important sign of rejuvenation of the immune system. Moreover, cytotoxic T lymphocytes (CTLs) is the key effector cells of the adaptive antitumour immune response. Granzyme B is the primary mediator of apoptosis by CTLs and NK cells. It can initiate the processing of caspases and apoptosis when it is released from activated CTLs 48 . As detailed in Figure 11, compared with the vehicle, administration of compound 5d increased the expression of IFN-c in a dosedependent manner in the CT26 tumour tissues, suggesting that 5d treatment could facilitate T cell activation and the rejuvenation of the immune system in the tumour microenvironment. Similarly, all treatments of 5d elevated granzyme B levels, which was consistent with the results of the TUNEL assay described above.
Furthermore, we also detected the content of FoxP3 þ Treg cells in the CT26 tumour-bearing mouse spleens by fluorescence activated cell sorting (FACS) assay ( Figure 12). The results showed that 5d treatment decreased the number of FoxP3 þ Treg cells in a dose-dependent manner. Especially, the amount in the high dose group was reduced 2.4-fold compared with the vehicle group. Taken together, these results revealed that the compound 5d Figure 8. Compound 5d did not suppress the growth of CT26 tumour in nude mice. 1 Â 10 6 CT26 cells were transplanted subcutaneously into the armpit of immunodeficient BALB/c nude mice. Three days after transplantation, mice were randomly allocated to vehicle control or treatment groups (n ¼ 8). Drugs were administered on days 1-9. (A) Tumour volume and mouse body weight (B) were recorded. After sacrifice, solid tumours were separated and weighed (C, D). Data represent mean ± SEM, n ¼ 8, Ã p < 0.05, ÃÃ p < 0.01 vs vehicle.
could promote T cell activation, decrease FoxP3 þ Treg cell population and heighten the function of CTLs.

Compound 5d could effectively suppress melanoma growth in immunocompetent mice
In addition, we also utilised a B16F1 melanoma mouse xenograft model to observe the efficacy of compound 5d on the other tumours. Malignant melanoma is a highly invasive tumour and its immunotherapy has been receiving attention in recent years. As mentioned above, B16F1 is a murine melanoma cell line that expresses IDO1. The mouse xenograft model was established by subcutaneously implanting B16F1 melanoma cells into BALB/c mice and treated with 5d (3.125, 6.25, 12.5 mg/kg, i.g, every day) or the positive control Cis-platinum (1 mg/kg, i.p, every other day).
Tumour volumes were measured every two days for 11 days. As shown in Figure 13, treatment with 5d significantly suppressed the growth of tumour in a dose-dependent manner both in tumour volume and in tumour weight as compared with the vehicle group without significant body weight loss. Especially at high dose, the average volume of tumour was reduced by 34.3% after 5d treatment, which were comparable to Cis-platinum.
Analogously, we also performed H&E, IHC and TUNEL experiments on the B16F1 melanoma tissues (Figure 14 and 15). These results indicated that compound 5d could effectively contribute to the morphological changes of B16F1melanoma cells, reduce the number of tumour cells by inhibiting proliferation and inducing apoptosis, and facilitate tumour vascular normalisation. Importantly, compound 5d also promoted T cell activation by upregulating IFN-c expression, and elevated CTLs function by stimulating granzyme B secretion. These results reaffirm that compound 5d could inhibit tumour growth by activating the mouse immune system.

Molecular modelling studies
Based on their outstanding performance in biological activity, we performed computational studies to elucidate the interactions of compounds 5d with IDO1. Since the high flexibility of the side chain and backbone of IDO1 protein, the classical semi-flexible docking method often poses a problem for IDO1 docking, whose algorithm relies on a rigid protein structure. Thus, the induced fit docking (IFD) method was utilised to overcome the difficulty mentioned above. The cocrystallization of Amg-1 in complex with human IDO1 protein (PDB ID: 4PK5) was employed as the template in the following docking calculations because of its relatively  high resolution and more structural information at pocket B. As shown in Figure 16, The benzsulfamide moiety situated deeply in pocket A and their benzene ring was interacted with Phe163 through p-p interaction, while the benzylamine moiety extended towards pocket B formed by Phe163, Phe226, Arg231, Leu234 and Ile354. Specifically, one oxygen atom of the sulphonyl group bond directly to the haem iron of IDO1 while the other oxygen atom formed a hydrogen bond with the main chain NH group of  . Compound 5d suppressed melanoma growth in mice. 1 Â 10 6 melanoma cells were transplanted subcutaneously into the armpit of the BALB/c mice. Three days after transplantation, mice were randomly allocated to vehicle control or treatment groups (n ¼ 6). Drugs were administered on days 1-11. (A) Tumour volume and mouse body weight (B) were recorded. After sacrifice, solid tumours were separated and weighed (C, D). Data represent mean ± SEM, n ¼ 6, Ã p < 0.05, ÃÃ p < 0.01 vs vehicle.
Ala264. Acryloyl groups located at the top of pocket A, and ethenyl moiety of acryloyl group extended towards a small hydrophobic subpocket formed by Met88, Leu124, Cys129, Leu234 and Gly262. This computational study finds that the occupation of the small hydrophobic subpocket in pocket A may a key factor affecting IDO1 inhibitory activity. These data could be useful for further optimisation.

Conclusions
In this study, the hit compound BS-1 was found as an IDO1 inhibitor in-house compound library. After structural optimisation, a series of sulphonamide derivatives were designed, synthesised, and evaluated in the HeLa cell-based IDO1/kynurenine assay, resulting in the identification of secondary sulphonamides as novel IDO1 inhibitors. Among them, the compounds 5d, 5l and 8g displayed the strongest inhibitory effect, and their activities were significantly improved over the hit compound BS-1. The in vitro results showed that these compounds could bind to IDO1 protein in HeLa cells without affecting the cell viability and the IDO1 protein expression under experimental conditions, indicating that these compounds blocked the kynurenine pathway by influencing IDO1 enzyme activity rather than IDO1 expression and/or cell viability. In addition, these compounds could promote the T cell proliferation and suppress the differentiation of naïve CD4 þ T cell into FoxP3 þ Treg cells, suggesting that these sulphonamides could reverse IDO1-mediated immunosuppression.
Based on its excellent performance in vitro, compound 5d was selected for further evaluating the pharmacodynamic effects in three types of tumour-bearing mouse models, including two immunocompetent mouse xenografts and one immunodeficient mouse xenograft. These in vivo experiments demonstrated that treatment with 5d could effectively inhibit the growth of murine CT26 coloreatic carcinoma xenograft tumour in a dose-dependent manner in immunocompetent mice, but not in immunodeficient mice, which implied that the in vivo efficacy of compound 5d was closely associated with the mouse immune system. Similarly, 5d also significantly blocked the growth of B16F1 melanoma in immunocompetent mice. Functionally, biochemical experiment and pathological analysis were utilised to detect these tumour tissues. The results from two immunocompetent mouse models indicated that compound 5d could induce the morphological changes of both CT26 and B16F1 tumour cells, decrease the number of tumour cells by suppressing proliferation and inducing apoptosis. Importantly, compound 5d could contribute to T cell  activation by stimulating IFN-c expression, improve CTLs function by inducing granzyme B secretion, and inhibit FoxP3 þ Treg cell differentiation, which would help facilitate the rejuvenation of the immune system. These promising results motivate us to further develop this new type of IDO1 inhibitors.