Synthesis and biological evaluation of new berberine derivatives as cancer immunotherapy agents through targeting IDO1

To discover small-molecule cancer immunotherapy candidates through targeting Indoleamine 2,3dioxygenase 1 (IDO1), twentyefive new berberine (BBR) derivatives defined with substituents on position 3 or 9 were synthesized and examined for repression of IFN-g-induced IDO1 promoter activities. Structureeactivity relationship (SAR) indicated that large volume groups at the 9-position might be beneficial for potency. Among them, compounds 2f, 2i, 2n, 2o and 8b exhibited increased activities, with inhibition rate of 71e90% compared with BBR. Their effects on IDO1 expression were further confirmed by protein level as well. Furthermore, compounds 2i and 2n exhibited anticancer activity by enhancing the specific lysis of NK cells to A549 through IDO1, but not cytotoxicity. Preliminary mechanism revealed that both of them inhibited IFN-g-induced IDO1 expression through activating AMPK and subsequent inhibition of STAT1 phosphorylation. Therefore, compounds 2i and 2n have been selected as IDO1 modulators for small-molecule cancer immunotherapy for next investigation. © 2017 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).


Introduction
Over the past few years, the cancer immunotherapy has made great progress, which showns significant efficacy in human solid tumors, with several drugs approved by the FDA, such as programmed cell death protein 1 (PD-1) antibody Keytruda ® and Opdivo ® [1,2]. Clinical benefit of these antibodies as single agent, however, has been limited to some factors such as a subset of patients, high cost, difficulty to generate a decent amount and efficacy not for all tumor types. These limitations call for the development of rational combination strategies or small-molecule therapeutics aiming to extend therapeutic benefit to a broader range of patients [3]. Meanwhile, as promising immunotherapy candidates, Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors including Epacadostat and Indoximod have already entered clinic trial. Especially, combination of small-molecule IDO inhibitors with PD-1 antibodies could significantly improve the objective response rate (ORR) in specific tumor treatment [4]. Phase I/II results showed that Epacadostat and Keytruda combination demonstrated an ORR of 53% (10 out of 19 patients) and a disease control rate of 74% (15 out of 19 patients) across multiple malignancies [5]. Since it is a single-chain catalytic enzyme with a well-defined biochemistry [6], IDO1 is considered to be an attractive target for small-molecule immunotherapeutics development. Therefore, it is a promising therapeutic strategy to develop small-molecule IDO1 inhibitors or modulators and provide new components for cancer immunotherapy combination.
A number of pathways are related to IDO1 expression [7], and IFN-g is the major inducer of IDO1 expression. In an effort to discover and explore novel small-molecule immunotherapeutics through targeting IDO1, the high-throughput screening model on inhibition of IFN-g-induced IDO1 promoter activity was then established in our laboratory. Given its inherent sensitivity, large signal dynamics, and simple set up, the reporter assay platform has been used as a high-throughput homogenous assay for screening IDO1 modulators in this study. Then, a library of natural products constructed in our lab was screened for their IDO1 regulating ability. Luckily, berberine (BBR, Fig. 1), as a Traditional Chinese Medicine used in China for decades against diarrhea, exhibited a moderate potency with inhibition rate of 17% at 10 mM [8e12]. The unique isoquinoline skeleton of BBR evokes our great interest to carry out structure-activity relationship (SAR) so as to discover small-molecule cancer-immunotherapy candidates targeting IDO1. Therefore, a series of novel BBR derivatives, including esters, amides and sulfonates on positions 3 and 9 as depicted in Fig. 1, were prepared and evaluated for their IDO1 effects, as well as primary mode of mechanism of the representative compounds.

Synthesis
A total of twenty-five new BBR derivatives were designed and semi-synthesized as displayed in Schemes 1e3, taking commercial available BBR, palmatine (PMT) or jatrorrhizine (JTH) as the starting material respectively. As shown in Scheme 1, BBR esters 2aeq and sulfonate 3a were obtained by esterification and sulfonation of compound 1 using previously reported methods [13,14]. The key intermediate 4 was prepared with 2,4-dimethoxyaniline as the nucleophilic reagent as well as the solvent. Another key intermediate 5 with a free amine group was then acquired, and HCl/CH 3 OH was selected to remove 2,4-dimethoxybenzyl in 80% yield. The desired products 6aec were obtained by amidation with corresponding acyl chloride using pyridine as the base with the yields of 33e37%.
As described in Scheme 2, compounds 8a and 8b were prepared through de-methylation and esterification from PMT using the procedures reported previously in overall yields of 25e33%. Similarly, as depicted in Scheme 3, the desired products 9a and 9b were obtained via esterification of JTH by 33e34% yields. All the final products were purified via flash column chromatography using CH 3 OH/CH 2 Cl 2 as the gradient eluent (see Table 1).

Pharmacological evaluation
2.2.1. SAR for down-regulated IFN-g-induced IDO1 promoter activity Thus, we screened all the newly-synthesized BBR analogues for their IDO1 regulating abilities by IDO1 promoter reporter assay. A549 cells were transfected with a pGL4-IDO1-luc vector and the inhibition rates of the BBR analogues on IFN-g-induced IDO1 promoter activities are shown in Fig. 2.
First, SAR analysis was focused on the influence of substitutions on position 9 of ring D, and 17 new ester derivatives (2aeq) were prepared and tested. Compounds 2aed with different substituted benzene and heterocycle were examined for their abilities to inhibit IDO1 promoter activity, and their activities almost vanished compared with BBR. Then, three analogues 2eeg with alkyl chain attached were synthesized and screened, and compound 2f bearing 2-ethyl butyrate gave the most potent activity with the inhibitory rate of 90%. Compounds 2hek with cyclic groups attached were tested, and 2,2,3,3-tetramethylcyclopropane-1-carboxylate substituted analogue 2i showed satisfactory inhibitory activity on IDO1 activity. It seems that tertiary carbon or quaternary carbon substituent with large volume might be beneficial for the inhibitory ability. Based on these results, the replacement of bridged-ring derivatives (2leo) at position 9 were created and tested. Surprisingly, compounds 2n and 2o gave ideal activity with inhibitory rate of 71% and 89% respectively, which indicated that large-volume rigid structure might be favourable for the ability. The combination of aromatic ring and alkyl chain (compounds 2p and 2q) on position 9 resulted in an obvious drop of inhibitory activity. Moreover, the inhibitory activity of compounds 3a and 6aec reduced significantly or lost completely, while the ester linker fragment was switched to sulfonate or amide. Further SAR study was conducted for the substituents on position 3 of ring A according to the above SAR analysis. The methylenedioxy was opened while two analogues 8aeb were created, and compound 8b gave an satisfactory activity with inhibitory rate of 72% which indicated that ring A might not be essential for the activity maintaining. Moving the adamantate group from ring D to position 3 of ring A, compound 9b gave acceptable potencies with inhibition rate of 55%.
Based on the screening results, compounds 2f, 2i, 2n, 2o and 8b exhibited the potent activities for modulating IFN-g-induced IDO1 promoter. In addition, as compounds 2c, 2e, 2q, 6a and 8a possess different types of structure, all of them were selected as representative compounds to investigate their inhibitory effects on protein expression level of IDO1.

BBR analogues inhibited IFN-g-induced IDO1 expression by protein level
In order to further confirm the regulating effect of BBR analogues, we examined whether they could block protein expression level of IDO1 or not. A549 cells were pre-treated with 10 mM of the indicated compounds for 2 h and then treated with IFN-g for 24 h. IDO1 expression level in total cell lysate was detected by western blot. As shown in Fig. 3, treatment with compounds 2f, 2i, 2n, 2o and 8b significantly reduced IFN-g-induced IDO1 expression, which was consistent with the results of dual-luciferase reporter screening model.

Effects of BBR analogues on A549 cell viability
To exclude the BBR analogues-induced IDO1 down-regulation resulted from the potential cytotoxicity, the cytotoxic effects of the ten representative compounds on A549 cells were evaluated by MTT assay. As displayed in Fig. 4, the results revealed that compounds 2i, 2n, 6a and 8a showed no cytotoxic activities in A549 cells, much lower than that of other BBR analogues.

Effects of BBR analogues on killing activity of human NK cells toward A549 cells
To determine whether BBR analogues could enhances the specific lysis of NK cells to A549 cells, A549 cells were pre- treated with the indicated compounds with or without IFN-g for 16 h. Cells were washed and then NK cells were co-cultured with A549 cells at 10:1, NK killing activity was assessed by LDH assay (Fig. 5A) and cell impedance assay (Fig. 5B), respectively. As shown in Fig. 5A, compounds 2i, 2n and 8b significantly enhanced the specific lysis of NK cells to A549 cells. In consideration of the cytotoxic effects of the three compounds, we choose compounds 2i and 2n to assess its effects on killing activity of human NK cells toward A549 cells by cell impedance assay. 1-Methyl-D-tryptophan (1-MT) was used as a positive IDO1 inhibitor. As shown in Fig. 5B, significant reduction in the cell index of A549 cells was observed following compounds 2i and 2n treatment compared with BBR, and compound 2i exhibited stronger activity than 1-MT.

BBR analogues inhibited IFN-g-induced STAT1 transcription activity by inhibiting STAT1 phosphorylation and activating AMPK
IFN-g-induced IDO1 expression involves the activation of signal transducer and activator or transcription 1 (STAT1), which can be activated via tyrosine phosphorylation by Janus kinases 1 (JAK1). Activated STAT1 homodimerizes and translocates into the nucleus, where it binds to and activates IFN-g-responsive specific promoters of IDO1 [15,16]. Considering that STAT1 plays a critical role in IFN-ginduced IDO1 expression, preliminary mechanism study was carried out to verify if the ten selected compounds work through STAT1 pathway. STAT1 phosphorylation at residue Tyr701 is required for acting as an active transcription factor. Therefore, we asked whether BBR analogues interfered with STAT1 phosphorylation. The expression level of STAT1 and phosphorylation of STAT1 Tyr701 were detected by western blot. As shown in Fig. 6, stimulation of cells with IFN-g alone resulted in a rapid increase in Tyr701 phosphorylation of STAT1, and this increase was remarkably inhibited by the treatment of compounds 2e, 2f, 2i, 2n, 2o and 8b respectively.

Conclusions
Taking IDO1 as the target, a series of novel BBR derivatives including esters, amides and sulfonates on different positions were designed, prepared and examined for their activity for suppression of IFN-g-induced IDO1 promoter expression. SAR analysis indicated that large volume substituent at the 9-position might be beneficial for enhancing the potency. Among them, compounds 2f, 2i, 2n, 2o and 8b exhibited increased potency with inhibitory rate of 71e90% compared with BBR. Their activities were further confirmed by protein level. Furthermore, compounds 2i and 2n exhibited their anticancer activity by enhancing the specific lysis of NK cells to A549 cells, as through targeting IDO1, but not cytotoxicity. Preliminary mechanism revealed that compounds 2i and 2n inhibited IFN-g-induced IDO1 expression through activating AMPK and subsequent inhibition of STAT1 phosphorylation. Thus, the results provided powerful information on further strategic optimization, and compounds 2i and 2n have been selected as promising IDO1 modulators for cancer immunotherapy for next investigation.

General
Melting point (mp) was obtained with CXM-300 melting point apparatus and uncorrected. The 1 H NMR spectra was performed on a Varian Inova 500 or 600 MHz spectrometer (Varian, San Francisco, CA) and 13 C NMR on a Bruker Avance III 500 or 600 spectrometer with Me 4 Si as the internal standard, all the samples were dissolved in DMSO-d 6 before testing. ESI high-resolution mass spectra (HRMS) was recorded on an Autospec UItima-TOF mass spectrometer (Micromass UK Ltd, Manchester, UK). Flash chromatography was performed on CombiflashRf 200 (Teledyne, Nebraska, USA), particle size 0.038 mm. Antibodies against IDO1, STAT1, phospho-STAT1 (Thr701), AMPKa, phospho-AMPK (Thr172) and phospho-ACC (Ser79) were purchased from Cell Signaling Tech-

Chemistry
4.2.1. General synthesis procedure for synthesis of compounds 2aeq and 3a BBR (3.71 g, 10 mmol) was heated at 195e210 C for 10e15 min under vacuum (30e40 mmHg) to afford the black oil, which was acidified with ethanol/concentrated HCl (95:5). The solvent was removed by evaporation, the residue was collected and then purified by flash chromatography over silica gel using CH 2 Cl 2 /CH 3 OH as the gradient eluent, affording the title compound 1 (2.85 g, 80%) as a yellow solid.
To a stirred solution of 1 (100 mg, 0.28 mmol) in anhydrous CH 3 CN, triethylamine (175 mL, 1.26 mmol) was added and heated to 70 C. Then the RCOCl/RSO 2 Cl (1.1e1.2 eq) was added and stirred for 5e6 h. The mixture was cooled to precipitate completely, filtrated and washed with CH 2 Cl 2 to afford compounds 2aeq and 3a.

General synthesis procedure for synthesis of compounds 6aec
The solution of BBR (7.4 g, 20 mmol) in 2,4dimethoxybenzylamine (15 mL, 78 mmol) was stirred at 120 C for 6e8 h. The mixture was cooled to room temperature and washed with acetone (3 Â 50 mL) to remove the remaining amine. The reside was purified by flash chromatography over silica gel using CH 2 Cl 2 /CH 3 OH (96.5:3.5) as the gradient eluent to afford compound 4 (3.5 g, 37%). Mp: 239e240 C (Dec.); 1  3. General synthesis procedure for synthesis of compounds 8a and 8b PMT (3.87 g, 10 mmol) was heated at 195e210 C for 10e15 min under vacuum (30e40 mmHg) to afford the black oil, which was acidified with ethanol/concentrated HCl (95:5). The solvent was removed by evaporation, the residue was collected and then purified by flash chromatography over silica gel using CH 2 Cl 2 /CH 3 OH as the gradient eluent, affording the title compound 7 (3.2 g, 86%) as a yellow solid.
To a stirred solution of compound 7 (100 mg, 0.29 mmol) in anhydrous CH 3

Cell culture and western blot analysis
A549 human lung cancer cell lines were procured from ATCC. The cells were cultured in DME/F-12 (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Hyclone, UT, USA), 100 U/ml penicillin, and 100 mg/mL streptomycin sulfate, and incubated at 37 C in a humidified atmosphere with 5% CO 2 . Actived NK cells were purchased from Stemcell Technologies (Vancouver, BC, Canada).
Western blot was performed as described previously [23]. Briefly, A549 cells were washed with PBS and lysed in M2 lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM b-glycerophosphate, 5 mM EGTA, 1 mM sodium pyrophoshate, 5 mM NaF, 1 mM Na 3 VO 4 , 0.5% Triton X-100, and 1 mM DTT) supplemented with protease inhibitor cocktail (Sigma P8340). Proteins were separated by SDS-PAGE and were electrically transferred to a PVDF membrane. The membrane was probed with the appropriate primary antibody and with a HRP-conjugated secondary antibody. Blots were visualized by Tanon 5200 system (Tanon, Shanghai, China).

MTT assay
The effect of the indicated compounds on the cell viability of A549 cells was evaluated using the MTT assay [24]. Briefly, Cells were seeded (5 Â 10 3 /well) into 96-well plate and the investigated compounds were added at indicated concentrations for 48 h. Next, MTT solution at a concentration of 5 mg/mL was added to each well. After subsequent 4 h, the culture medium was removed and formazan crystals were dissolved with 150 mL DMSO. Finally, the absorbance was measured at 570 nm using a microplate reader (Multiskan FC, Thermo, USA).

IDO1 promoter activity assay
A549 cells were co-transfected with pGL4-IDO1-luc with the pRL-CMV plasmid using the Vigofect transfection reagent (Beijing, China) as instructed by the manufacturers. After 24 h of transfection, cells were pretreated with the indicated compounds (10 mmol/L) for 2 h and then stimulated with or without IFN-g (10 ng/mL) for 24 h. Following IFN-g treatment, the cells were lysed, and the luciferase activity was determined using the luciferase reporter assay system (Promega, Madison, CA, USA) according to manufacturer's protocols. The luciferase activity values were normalized to the expression of the Renilla luciferase, and presented as the percentages of luciferase activity.

LDH release assay
Cytotoxicity of human NK cells against A549 cells was assessed with LDH release assay, as previously described [25]. Briefly, A549 cells were freshly plated at 5 Â 10 3 /well in 96-well plates and pretreated with the indicated compounds (10 mmol/L) for 2 h and then stimulated with IFN-g (10 ng/mL) for 16 h. A549 cells were washed and then were cocultured with NK cells at 1:10 in triplicate in RPMI 1640 plus 10% FBS. Four hours later, cytotoxicity assay was conducted using non-radioactive lactate dehydrogenase (LDH) release using a cytotoxicity detection kit (CytoTox 96, Promega, Madison, WI, USA) as the manufacturer's instructions. Spontaneous release and maximum release were determined by incubating target cells without effector cells in medium alone or in 0.5% NP40, respectively. The percent cytotoxicity was calculated as follows: (experimental release-spontaneous release)/(maximum releasespontaneous release) Â 100%.

Cell impedance assay
A549 cells were freshly plated at 5 Â 10 3 /well in E16 plates (Roche Diagnostics, Basel, Switzerland). The plate was connected to an xCELLigence RTCA SP instrument (Roche Diagnostics) within a humidified cell culture incubator. After 4 h incubation and A549 cells were treated with the indicated compounds (10 mmol/L) with IFN-g (10 ng/mL). After 16 h, A549 cells were washed and cocultured with NK cells at 1:10 in triplicate in RPMI 1640 plus 10% FBS. Data was analyzed using the RTCA Software 1.2 program (Roche Diagnostics). All data is presented as the mean normalized cellular index ± SEM over time.