Research ArticleExperimental Studies

Structure-activity Relationship of Indomethacin Derivatives as IDO1 Inhibitors

TOHRU OBATA, SARA SHIRATANI, TOMOMI NADA, YAYOI KASAYA, MITSUHIRO ARISAWA, SATOSHI SHUTO and MOTOHIRO TANAKA
Anticancer Research May 2021, 41 (5) 2287-2296; DOI: https://doi.org/10.21873/anticanres.15004

Abstract

Background/Aim: Indoleamine 2,3-dioxygenase (IDO) is regarded as an important molecular target for cancer immune therapy. This study aimed to examine the IDO1 inhibitory activity of newly synthesized indomethacin derivatives to develop an IDO1 inhibitor. Materials and Methods: The inhibitory effects of indole-containing compounds against recombinant human IDO1 (rhIDO1) were evaluated. Results: While some drugs including those with an indole scaffold could inhibit rhIDO1, simple indole compounds were inactive. A total of 27 indomethacin derivatives, including 18 newly synthesized derivatives, were evaluated. Numerous derivatives showed enhanced IDO1 inhibitory activity. The functional group at the 3-position had a strong effect on IDO1 inhibitory activity. The IDO1 inhibitory activity was not directly correlated with tumor cell cytotoxicity. Conclusion: We report the finding of novel IDO1 inhibitors and the structure-activity relationship based on indomethacin derivatives. Our findings will be beneficial for the development of IDO1 inhibitors for cancer immune therapy.

Key Words:

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyzes the oxidative cleavage of L-tryptophan (L-Trp) to L-formylkynurenine. This step is the first and rate-limiting step in the L-Trp catabolic pathway (1). Nicotinamide adenine dinucleotide (NAD+), which is required as a cofactor in many biochemical processes, is supplied by the action of this kynurenine pathway in the absence of any dietary niacin intake (2). The kynurenine pathway of L-Trp is a major route for NAD+ biosynthesis. IDO1 is expressed in normal immune system tissues and in many tumors, and is thought to play an important role in tumorigenesis by facilitating tumor immune escape (3, 4). Low tryptophan levels can cause serious damage to T cells, such as cell cycle arrest and cell death, and kynurenine metabolites can inhibit T cell proliferation and differentiation to regulatory T cells (5). The poor prognosis seen in patients with ovarian and colorectal cancers is thought to correlate with high IDO expression levels (6, 7). Therefore, IDO is regarded as an important molecular target for the development of chemotherapeutic inhibitors for the treatment of tumors. Some drugs that target IDO, such as 1-methyl-tryptophane, navoximod (NLG919), NLG802, epacadostat (INCB 024360), and STB001, have been evaluated in clinical trials (2, 8-10). Here, we focused on IDO inhibitors that contained the indole scaffold in their molecular structure. One of the major drugs that contains the indole scaffold is indomethacin, which belongs to the class of non-steroidal anti-inflammatory drugs (NSAIDs). Indomethacin exerts its effects by suppressing prostaglandin production via the inhibition of cyclooxygenase (COX) in the arachidonic acid cascade (11).

In this study, we report the discovery of novel IDO1 inhibitors and the structure-activity relationships among indomethacin derivatives. Several known IDO inhibitor regents (e.g., methyltryptophan and norharman) including those with the indole scaffold were explored for their inhibitory effects against recombinant human IDO1 (rhIDO1) in in vitro experiments. Indomethacin derivatives, previously synthesized in other studies (12, 13), that showed enhancement of doxorubicin antitumor activity in combination with a COX inhibitor, were utilized to identify new potent IDO inhibitors. The IDO1 inhibitory activity of newly synthesized indomethacin derivatives was also evaluated.

Materials and Methods

Chemicals. 1-Methyl-L-tryptophan (1-L-MT), 1-methyl-D-tryptophan (1-D-MT), and indomethacin (INM) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Norharman (Nor) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Celecoxib (CBX) and 3-indolemethanol (3-IM) were purchased from, Cayman Chemical (Ann Arbor, MI, USA). Etodolac (Eto), oncrasin 1 (ONC-1), 3-indolepropionic acid (3-IPA), 3-indolebutyric acid (3-IBA), 3-indoleethanol (3-IE), 1-methylindole (1-MI), and indol-1-yl-acetic acid (1-IAA) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Nimesulide (NIM), acemetacin (Ace), indole, 3-indoleacetic acid (3-IAA), and other special grade chemicals and reagents were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). A fraction of indomethacin derivatives was synthesized according to previous reports (12, 13). Another ten indomethacin derivatives including a benzoyl group (compounds 8, 9, 11-17, 19) and eight indomethacin derivatives including a benzyl group (compounds 20-27) (asterisk in Table I), were newly synthesized according to the conventional method. The preparation and chemical properties of the 18 newly synthesized indomethacin derivatives are shown in the following section. All compounds were dissolved in dimethyl sulfoxide (DMSO).

View this table:
Table I.

Structure of indomethacin derivatives with IDO1 inhibitory effect.

Synthesis and chemical properties. 1H-NMR spectra were recorded in CDCl3 at 25°C unless otherwise noted, at 400 or 500 MHz, with TMS as an internal standard. 13C-NMR spectra were recorded in CDCl3 at 25°C unless otherwise noted, at 400 or 500 MHz on JOEL (Tokyo, Japan) JNM-AL-400, JMM-ECX-400P and JMM-ECA-500. Electrospray mass spectra were recorded in positive mode on JEOL JMS-700TZ, JMS-HX110, JMS-FABmate and JMS-T100GCv (Thermo Fisher Scientific, Waltham, MA, USA). Elemental analysis was performed to confirm ≥95% sample purity (within ±0.4% of the calculated value on J-SCIENCE LAB (Kyoto, Japan) MICRO CORDER JM10.

Compound 8 (0.0566 g, 0.145 mmol, 58%, a white solid) was prepared from corresponding benzyl group (0.120 g, 0.250 mmol). mp 186°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ8.20 (d, 1H, J=7.6 Hz, indole-aromatic), 7.63 (d, 1H, J=7.0 Hz, indole-aromatic), 7.43-7.37 (m, 2H, aromatic), 7.25-7.21 (m, 2H, aromatic), 7.15-6.98 (m, 7H, aromatic), 3.57 (s, 2H, indole-CH2); 13C-NMR (100 MHz, CDCl3) δ177.3, 167.4, 137.8, 136.7, 135.6, 132.1, 131.5, 130.9, 130.3, 130.0, 129.7, 129.4, 128.2, 127.9, 126.3, 125.6, 124.1, 119.0, 115.4, 114.5, 30.6; LRMS (EI) m/z 389.1 (M+); HRMS (EI) calcd for C23H16ClNO3: 389.0819 (M+), found: 389.0814; Elemental analysis calcd (%) for C23H16ClNO3: C, 70.86; H, 4.14; N, 3.59. found: C, 70.78; H, 4.20; N, 3.60.

Compound 9 (0.0788 g, 0.202 mmol, quant, a white solid) was prepared from corresponding benzyl compound (0.0969 g, 0.200 mmol), mp 167°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.91-7.89 (m, 1H, indole-aromatic), 7.70-7.68 (m, 1H, indole-aromatic), 7.43-7.36 (m, 4H, aromatic), 7.27-7.10 (m, 1H, aromatic), 3.72 (s, 2H, indole-CH2); 13C-NMR (125 MHz, CDCl3) δ177.8, 168.4, 138.1, 137.0, 136.7, 134.0, 132.1, 131.2, 130.0, 129.3, 129.1, 128.2, 128.2, 127.9, 125.2, 123.6, 119.3, 114.4, 113.2, 30.7; LRMS (ESI) m/z 523.07 [(M+Na)+]; HRMS (ESI) calcd for C23H16ClNO3Na: 412.0711 [(M+Na)+], found: 412.0711; Elemental analysis calcd (%) for C23H16ClNO3: C, 70.84; H, 4.17; N, 3.58. found: C, 70.47; H, 4.24; N, 3.58.

In order to prepare compound 11, to a solution of corresponding compound with 4-bromophenyl function (0.0717 g, 0.140 mmol) and anisole (440 μl, 0.410 mmol) in dichloromethane (0.700 ml), the solution of AlCl3 (0.0310 g, 0.230 mmol) in nitromethane (0.600 ml) was added dropwise at 0°C. After stirred for 5 h at the same temperature, the mixture was poured into ice-cooled water and acidified to pH 1 with 1 M HCl. The organic layer was separated, washed with H2O and brine, dried over Na2SO4, filtered, and concentrated. The crude residue was subjected to column chromatography (hexane/AcOEt=3:1) to give compound 11 as a white powder (0.0520 g, 0.120 mmol, 86%). mp 208-208.5°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.79-7.76 (m, 1H, indole-aromatic), 7.70-7.67 (m, 1H, indole-aromatic), 7.38-7.31 (m, 6H, aromatic), 7.27-7.16 (m, 5H, aromatic), 3.71 (s, 2H, indole-CH2); 13C-NMR (100 MHz, CDCl3) δ168.9, 138.3, 137.0, 134.0, 131.4, 131.4, 131.3, 130.0, 129.2, 128.3, 128.2, 127.5, 125.0, 123.5, 119.3, 114.3, 113.0, 30.6; LRMS (EI) m/z 433.05 (M+); HRMS (EI) calcd for C23H16BrNO3: 433.0314 (M+), found: 433.0309; Elemental analysis calcd (%) for C23H16BrNO3: C, 63.61; H, 3.71; N, 3.23. found: C, 63.20; H, 3.77; N, 3.21.

Compound 12 (0.0222 g, 0.0595 mmol, 63%, a white solid) was prepared from corresponding benzyl compound (0.0439 g, 0.0950 mmol), mp 197°C (AcOEt); 1H-NMR (500 MHz, CDCl3) δ7.79-7.76 (m, 1H, indole-aromatic), 7.71-7.67 (m, 1H, indole-aromatic), 7.57-7.54 (dd, 2H, J=8.9, 3.4 Hz, aromatic), 7.36-7.32 (m, 2H, indole-aromatic), 7.29-7.26 (m, 2H, aromatic), 7.23-7.12 (m, 3H, aromatic), 6.90-6.86 (m, 2H, aromatic), 3.73 (s, 2H, indole-CH2); 13C-NMR (125 MHz, CDCl3) δ177.4, 168.6, 166.1, 164.0, 138.3, 137.0, 132.7, 132.6, 131.3, 129.9, 129.1, 128.3, 128.1, 124.9, 123.3, 119.3, 115.4, 115.3, 114.2, 112.7, 30.7; LRMS (EI) m/z 373.12 (M+); HRMS (EI) calcd for C23H16FNO3: 373.1114 (M+), found: 373.1111; Elemental analysis calcd (%) for C23H16FNO3: C, 73.99; H, 4.32; N, 3.75. found: C, 73.99; H, 4.40; N, 3.78.

Compound 13 (0.0353 g, 0.0916 mmol, quant, a white powder) was prepared from corresponding benzyl compound (0.0439 g, 0.0900 mmol). mp 205°C (AcOEt); 1H-NMR (500 MHz, CDCl3) δ7.69 (d, 1H, J=6.9 Hz, aromatic), 7.60-7.58 (m, 3H, aromatic), 7.34-7.32 (m, 2H, aromatic), 7.30-7.18 (m, 5H, aromatic), 6.75 (d, 2H, J=8.9 Hz, aromatic), 3.79 (s, 3H, OCH3), 3.76 (s, 2H, indole-CH2); 13C-NMR (125 MHz, CDCl3) δ172.5, 168.4, 163.3, 137.8, 136.5, 132.4, 131.4, 129.3, 129.2, 128.3, 127.8, 126.5, 124.2, 122.5, 119.7, 114.1, 113.3, 113.0, 55.6, 30.4; LRMS (EI) m/z 385.07 (M+); HRMS (EI) calcd for C24H19NO4: 385.1314 (M+), found: 385.1313; Elemental analysis calcd (%) for C24H19NO4: C, 74.79; H, 4.97; N, 3.63. found: C, 74.70; H, 5.04; N, 3.62.

Compound 14 (0.0686 g, 0.193 mmol, 97%, a white powder) was prepared from corresponding benzyl compound (0.0881 g, 0.200 mmol). mp 171-171.5°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ8.07-7.67 (m, 2H, indole-aromatic), 7.54 (d, 2H, J=8.0 Hz, aromatic), 7.46-7.12 (m, 10H, aromatic), 3.73 (s, 2H, indole-CH2); 13C-NMR (100 MHz, CDCl3) δ177.6, 169.8, 138.6, 137.0, 135.1, 132.6, 131.5, 130.0, 129.9, 129.2, 128.2, 128.1, 128.0, 124.8, 123.2, 119.2, 114.2, 112.6, 30.8; LRMS (ESI) m/z 378.11 [(M+Na)+]; HRMS (ESI) calcd for C23H17NO3Na: 378.1101 [(M+Na)+], found: 378.1095; Elemental analysis calcd (%) for C23H17NO3: C, 77.73; H, 4.82; N, 3.94. found: C, 77.71; H, 4.91; N, 3.92.

Compound 15 (0.0375 g, 0.0999 mmol, quant, white column crystal) was prepared from corresponding benzyl compound (0.0470 g, 0.101 mmol) as described in general procedure B. mp 235-235.5°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ8.33 (d, 1H, J=8.0 Hz, aromatic), 7.52-7.50 (m, 1H, aromatic), 7.43-7.41 (m, 2H, aromatic), 7.39-7.31 (m, 4H, aromatic), 7.26-7.22 (m, 5H, aromatic); 13C-NMR (100 MHz, CDCl3) δ169.0, 146.0, 140.1, 136.5, 132.5, 131.4, 130.7, 130.7, 129.2, 128.8, 127.8, 127.2, 125.0, 124.2, 123.1, 112.9, 109.1; LRMS (EI) m/z 375.1 (M+); HRMS (EI) calcd for C22H14ClNO2: 375.0662 (M+), found: 375.0668; Elemental analysis calcd (%) for C22H14ClNO3: C, 70.31; H, 3.75; N, 3.73. found: C, 70.58; H, 3.90; N, 3.78. Compound 16 (0.0476 g, 0.118 mmol, 84%, yellow amorphous solid) was prepared from corresponding benzyl compound (0.0697 g, 0.141 mmol) as described in general procedure B. mp 158°C; 1H-NMR (500 MHz, CDCl3) δ7.73-7.71 (m, 1H, indole-aromatic), 7.65-7.63 (m, 1H, indole-aromatic), 7.45 (d, 2H, J=8.6 Hz, aromatic), 7.33-7.29 (m, 2 H, aromatic), 7.21-7.18 (m, 7H, aromatic), 3.07 (t, 2H, J=8.1 Hz, CH2CO2Bn), 2.65 (t, 2H, J=8.1 Hz, indole-CH2); 13C-NMR (125 MHz, CDCl3) δ177.9, 168.8, 138.7, 137.1, 136.8, 133.8, 131.9, 131.3, 129.8, 29.0, 128.8, 128.4, 128.3, 127.9, 124.8, 123.2, 119.0, 118.9, 114.3, 34.0, 19.6; LRMS (EI) m/z 403 (M+); HRMS (EI) calcd for C24H18ClNO3: 403.0975 (M+), found: 403.0977; Elemental analysis calcd (%) for C24H18ClNO3: C, 71.38; H, 4.49; N, 3.47. found: C, 71.13; H, 4.69; N, 3.43.

In order to prepare compound 17, a solution of corresponding acetal compound (0.0601 g, 0.139 mmol), TsOH × H2O (4.00 mg, 0.0210 mmol) in acetone aq. (96 v/v %, 1.17 ml) is stirred at 60°C for 1 day. Then AcOEt was added, and the mixture was washed with sat. NaHCO3 aq., brine. The organic layer was dried over Na2SO4, filtered, concentrated and the residue was purified by silica gel column chromatography (hexane/AcOEt=10:1) to afford compound 17 with ketone (0.0525 g, 0.135 mmol, 97%) as white plate crystal. mp 165°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.77-7.75 (m, 1H, aromatic), 7.52-7.49 (m, 1H, aromatic), 7.46 (d, 3H, J=8.5 Hz), 7.32-7.30 (m, 2H, aromatic), 7.20-7.16 (m, 6H, aromatic), 3.76 (s, 2H, indole-CH2), 2.14 (s, 3H, COCH3); 13C-NMR (100 MHz, CDCl3) δ206.0, 168.6, 138.7, 137.8, 137.0, 133.5, 131.6, 131.3, 129.7, 129.3, 128.4, 128.3, 128.1, 124.9, 123.4, 119.1, 114.2, 114.1, 40.0, 29.5; LRMS (EI) m/z 387 (M+); HRMS (EI) calcd for C24H18ClNO2: 387.1026 (M+), found: 387.1021; Elemental analysis calcd (%) for C24H18ClNO2: C, 74.32; H, 4.68; N, 3.61. found: C, 74.00; H, 4.72; N, 3.62.

In order to prepare compound 19, a mixture of corresponding compound 10 (20.0 mg, 0.0513 mmol), HOBt × NH3 (12.0 mg, 0.0789 mmol), EDC × HCl (12.0 mg, 0.0626 mmol) in DMF (0.513 ml) was stirred for 1 day at room temperature. To the mixture AcOEt and 1 M HCl was added, the organic phase was washed with sat. NaHCO3 aq., brine, then dried over Na2SO4, filtered, concentrated and the residue was purified by silica gel column chromatography (hexane/AcOEt=1:1) to afford compound 19 (0.0171 g, 0.0440 mmol, 88%) as yellow plate crystal. mp 224°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.70-7.64 (m, 2H, aromatic), 7.50 (d, 2H, J=8.8 Hz, aromatic), 7.35-7.33 (m, 2H, aromatic), 7.26-7.20 (m, 7H, aromatic), 5.63 (br, 2H, CONH2), 3.66 (s, 2H, indole-CH2); 13C-NMR (100 MHz, CDCl3) δ168.7, 139.1, 138.4, 137.1, 133.3, 131.4, 131.1, 129.7, 129.0, 128.6, 128.5, 128.4, 125.2, 123.6, 119.2, 114.3, 113.9, 32.2; LRMS (EI) m/z 388 (M+); HRMS (EI) calcd for C23H17ClN2O2: 388.0979 (M+), found: 388.0993; Elemental analysis calcd (%) for C23H17ClN2O2: C, 71.04; H, 4.41; N, 7.20. found: C, 71.22; H, 4.42; N, 7.20.

In order to prepare compound 20, A solution of corresponding benzyl compound (0.0588 g, 0.126 mmol) in DMSO (2.50 ml) and 15% NaOH aq. (1.26 ml) was stirred at room temperature for 1 h. Then the mixture was added 1 M HCl, extracted with AcOEt, washed with brine, dried over Na2SO4, filtered, and concentrated. The crude residue was subjected to column chromatography (hexane/AcOEt=15:1) to give deprotected compound 20 (0.0383 g, 0.102 mmol, 81%) as a white powder. mp 188°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.71-7.70 (m, 1H, aromatic), 7.38-7.33 (m, 6H, aromatic), 7.20-7.04 (m, 5H, aromatic), 6.55 (d, 2H, J=8.0 Hz, aromatic), 5.28 (s, 2H, N-CH2Ph), 3.75 (s, 2H, indole-CH2); 13C-NMR (100 MHz, CDCl3) δ178.1, 139.7, 136.6, 135.4, 131.7, 130.5, 130.3, 129.3, 128.7, 128.6, 128.3, 127.8, 127.2, 127.2, 122.6, 120.3, 119.2, 110.3, 106.0, 45.5, 30.9; LRMS (EI) m/z 375.05 (M+); HRMS (EI) calcd for C23H18ClNO2: 375.1026 (M+), found:375.1024; Elemental analysis calcd (%) for C23H18NO2: C, 73.50; H, 4.83; N, 3.73. found: C, 73.10; H, 4.98; N, 3.74. Compound 21 (0.0368 g, 0.098 mmol, quant., a white powder) was prepared from corresponding benzyl compound (0.0457 g, 0.0980 mmol) as described for the preparation of compound 20: mp 176°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.70-7.68 (m, 1H, aromatic), 7.42-7.41 (m, 2H, aromatic), 7.37-7.35 (m, 3H, aromatic), 7.22-7.13 (m, 5H, aromatic), 6.98 (s, 1H, aromatic), 6.78 (d, 1H, J=7.2 Hz, aromatic), 5.20 (s, 2H, N-CH2Ph), 3.73 (s, 2H, indole-CH2); 13C-NMR (100 MHz, CDCl3) δ177.9, 140.1, 139.6, 136.6, 134.6 130.6, 130.5, 130.0, 128.7, 128.6, 127.8, 127.4, 126.2, 126.0, 124.8, 124.2, 122.6, 120.3, 119.2; LRMS (EI) m/z 375.05 (M+); HRMS (EI) calcd for C23H18ClNO2: 375.1026 (M+), found: 375.1022; Elemental analysis calcd (%) for C23H18NO2: C, 73.50; H, 4.83; N, 3.73. found: C, 73.77; H, 4.94; N, 3.75.

In order to prepare compound 22, a solution of corresponding benzyl compound (1.60 mg, 3.40 μmol) in DMSO (68.0 μml) and 15% NaOH aq. (34.0 μml) was stirred at room temperature for 3 h. Then the mixture was added 1 M HCl, extracted with AcOEt, washed with brine, dried over Na2SO4, filtered, and concentrated. The crude residue was subjected to column chromatography (hexane/AcOEt=10:1) to give deprotected compound 22 (1.10 mg, 3.10 μmol, 90%) as a white column crystal. mp 208°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.69-7.67 (m, 1 H, aromatic), 7.41 (d, 2 H, J=3.2 Hz, aromatic), 7.35 (d, 2 H, J=3.0 Hz, aromatic), 7.20-7.16 (m, 6 H, aromatic), 6.87 (d, 2 H, J=8.2 Hz, aromatic), 5.20 (s, 2 H, N-CH2Ph), 3.72 (s, 2 H, indole-CH2); 13C-NMR (125 MHz, CDCl3) δ139.6, 136.6, 136.5, 132.9, 130.6, 130.5, 128.8, 128.7, 128.6, 127.8, 127.4, 122.5, 120.3, 119.2, 110.3, 105.9, 47.1, 21.0; LRMS (EI) m/z 375.17 (M+); HRMS (EI) calcd for C23H18ClNO2: 375.1026 (M+), found: 375.1025; Elemental analysis calcd (%) for C23H18NO2: C, 73.50; H, 4.83; N, 3.73. found: C, 73.64; H, 4.89; N, 3.76.

In order to prepare compound 23, a solution of corresponding benzyl compound (0.0925 g, 0.200 mmol) in DMSO (4.00 ml) and 15% NaOH aq. (2.00 ml) was stirred for 4 h. The reaction was quenched with 1 M HCl aq., then the organic compound was extracted with diethylether, washed with brine, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by column chromatography to give compound 23 (0.0744 g, 0.200 mmol, quant.) as a colorless plate crystal. mp 183°C (AcOEt); 1H-NMR (500 MHz, CDCl3) δ7.68-7.66 (m, 1H, aromatic), 7.40-7.36 (m, 5H, aromatic), 7.22-7.16 (m, 3H, aromatic), 6.86 (d, 2H, J=9.0 Hz, aromatic) 6.75 (d, 2H, J=9.0 Hz, aromatic), 5.16 (s, 2H, NCH2Ph), 3.74 (s, 3H, OCH3), 3.71 (s, 2H, CH2CO); 13C-NMR (125 MHz, CDCl3) δ178.1, 158.6, 139.7, 136.6, 1309, 130.6, 130.1, 128.5, 128.5, 127.7, 127.2, 122.3, 120.0, 119.1, 114.0, 110.5, 105.5, 55.2, 47.2, 30.9; LRMS (EI) m/z 461.21 (M+); HRMS (EI) calcd for C24H21NO3: 461.1991 (M+), found: 461.1991; Elemental analysis calcd (%) for C24H21NO3: C, 77.61; H, 5.70; N, 3.77. found: C, 77.10; H, 5.76; N, 3.62.

Compound 24 (1.20 mg, 3.00 μmmol, quant., white granular crystal) was prepared from corresponding benzyl compound (1.60 mg, 3.00 μmmol) as described in the preparation of compound 23. mp 196°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.67-7.65 (m, 1H, aromatic), 7.40-7.38 (m, 5H, aromatic), 7.26-7.20 (m, 1H, aromatic), 7.19-7.14(m, 2H, aromatic), 6.70 (d, 1H, J=8.8 Hz, aromatic), 6.49 (dd, 1H, J=8.8, 2.0 Hz, aromatic), 6.42 (d, 1H, J=2.0 Hz, aromatic), 5.18 (s, 2H, NCH2Ph), 3.81 (s, 3H, OCH3), 3.71 (s, 2H, CH2CO), 3.69 (s, 3H, OCH3); 13C-NMR (100 MHz, CDCl3) δ178.1, 158.6, 139.7, 136.6, 130.9, 130.6, 130.1, 128.5, 128.5, 127.7 127.2, 122.3, 120.0, 119.1, 114,0, 110.5, 105.5, 55.8, 55.2, 47.2, 30.9; LRMS (EI) m/z 401.15 (M+); HRMS (EI) calcd for C25H23NO4: 401.1627 (M+), found: 401.1621; Elemental analysis calcd (%) for C25H23NO4 × 0.2 H2O: C, 74.13; H, 5.82; N, 3.46. found: C, 74.07; H, 6.00; N, 3.12.

In order to prepare compound 25, boron tribromide (1.0 M in dichloromethane, 0.312 ml, 0.312 mmol) was added to a stirred solution of corresponding benzyl compound (0.0600 g, 0.120 mmol) in 3.47 ml of dichloromethane in ice-ethanol bath. After stirred for 8 h in ambient temp., water was added and the mixture was extracted with dichloromethane. The organic layer was dried over Na2SO4, concentrated under reduced pressure and the resulting crude was purified by column chromatography (Hexane/AcOEt=2:1) to afford compound 25 (0.0442 g, 0.118 mmol, 99%) as white granular crystal. mp 169°C (CHCl3); 1H-NMR (400 MHz, CDCl3) δ7.72-7.70 (m, 1H, aromatic), 7.63-7.61 (m, 1H, aromatic), 7.54-7.52 (m, 1H, aromatic), 7.40-7.36 (m 4H, aromatic), 7.19-7.14 (m, 3H, aromatic), 6.68 (d, 1H, J=8.4 Hz, aromatic), 6.40 (d, 1H, J=8.4 Hz, aromatic), 6.31 (s, 1H, aromatic), 5.10 (s, 2H, NCH2Ph), 4.23-4.20 (m, 2H, OH), 3.71 (s, 2H, CH2CO); 13C-NMR (100 MHz, CDCl3) δ178.3, 149.0 149.0, 148.0, 139.7, 136.8, 131.0, 130.6, 128.5, 127.8, 122.3, 1201, 119.0, 118.3, 111.2, 110.5, 109.3, 109.3, 105.7, 55.7, 30.9; LRMS (EI) m/z 373.12 (M+); HRMS (EI) calcd for C23H19NO4: 373.1314 (M+), found: 373.1315; Elemental analysis calcd (%) for C23H19NO4 × 0.7 H2O: C, 71.56; H, 5.33; N, 3.63. found: C, 71.47; H, 5.05; N, 3.41.

Compound 26 (0.0655 g, 0.0152 mmol, quant., a yellow granular crystal) was prepared from corresponding benzyl compound (0.0793 g, 0.0152 mmol) as described in the preparation of compound 23. mp 146°C (AcOEt); 1H-NMR (400 MHz, CDCl3) δ7.58 (d, 1H, 7.2 Hz, aromatic), 7.34-7.20 (m, 5H, aromatic), 7.19-7.07 (m, 3H, aromatic), 6.03 (s, 2H, aromatic), 5.10 (s, 2H, NCH2Ph), 3.70 (s, 3H, p-OCH3), 3.64 (s,2H, CH2CO), 3.56 (s, 6H, m-OCH3); 13C-NMR (125 MHz, CDCl3) δ178.5, 177.3, 153.2, 140.0, 136.8, 136.7, 133.7, 130.8, 130.5, 128.5, 128.5, 127.8, 122.3, 120.0, 119.0, 110.3, 106.0, 103.0, 60.7, 55.8, 47.6, 30.8, 25.5; LRMS (EI) m/z 431.26 (M+); HRMS (EI) calcd for C26H25NO5: 431.1733 (M+), found: 431.1729; Elemental analysis calcd (%) for C26H25NO5 × 0.8 H2O: C, 70.03; H, 6.01; N, 3.14. found: C, 70.34; H, 6.35; N, 2.68.

In order to prepare compound 27, boron tribromide (1.0 M in dichloromethane, 0.492 ml, 0.492 mmol) was added to a stirred solution of compound 26 (0.708 g, 0.164 mmol) in 5.47 ml of dichloromethane in ice-ethanol bath. After stirred for 8 h in ambient temperature, water was added and the mixture was extracted with dichloromethane. The organic layer was dried over Na2SO4, concentrated under reduced pressure and the resulting crude was purified by column chromatography (Hexane/AcOEt=2:1) to afford the desired compound 27 (0.0589 g, 0.151 mmol, 95%) as white amorphous solid. mp 205-206°C; 1H-NMR (400 MHz, CDCl3) δ7.65 (d, 2H, J=6.8 Hz, aromatic), 7.42 (m, 4H, aromatic), 7.24-7.16 (m, 3H, aromatic), 6.24 (s, 1H, aromatic), 5.99 (s, 1H, aromatic), 5.13 (s, 2H, NCH2Ph), (s, 2H, CH2CO); 13C-NMR (100 MHz, CDCl3) δ178.0, 176.7, 167.8, 147.0, 144.0, 1397, 136.8, 132.4, 131.2, 131.1, 131.0, 130.0, 128.8, 122.4, 120.1, 119.0, 110.5, 106.7, 105.8, 101.1, 56.0, 28.9; LRMS (EI) m/z 389.2 (M+); HRMS (EI) calcd for C23H19NO5: 389.1263 M+), found:389.1265; Elemental analysis calcd (%) for C23H19NO5 × 0.4 H2O: C, 69.65; H, 5.03; N, 3.53. found: C, 69.71; H, 5.22; N, 3.25.

Cytotoxicity assay in human tumor cells. The human glioblastoma tumor cell line T98G, which has been used in previous studies (12, 13), was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell line was maintained at 37°C in an atmosphere of 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA). The cell cytotoxicity assay was performed as previously described (14, 15). Briefly, the inhibitory effects of the drugs on the growth of tumor cells were examined using a colorimetric assay involving 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Aliquots (180 μl) of an exponentially growing cell suspension (2,000 cells/198 μl/well) were incubated with 2 μl of various concentrations of drugs diluted with DMSO. After exposure to the drugs for 72 h, 20 μl of MTT solution (3 mg/ml) was added to each well, and the cell cultures were further incubated at 37°C for 4 h. After removal of the medium, the formazan crystals formed were dissolved in 200 μl of DMSO. The absorbance of each well was measured at 570 nm using a microplate reader (MTP-800AFC; CORONA Electric, Hitachinaka, Japan). The inhibition ratio was calculated using the following formula: IR (%)=(1 − T/C) ×100, where C is the mean optical density of the control group and T is that of the treatment group. The half-maximal inhibitory concentration (IC50) value was defined as the concentration of the drug required to induce a 50% reduction in growth relative to that of the control. The IC50 value was determined by a graphical correlation of the dose-response curve with at least three concentration points and was and was indicated average value from some independent assays.

IDO inhibition assay. His tagged rhIDO1 was purchased from BPS Bioscience (San Diego, CA, USA) and the assay was performed according to previous reports (16, 17). Briefly, the 100 μl reaction mixture contained 2 μl of assay sample dissolved in DMSO, 2 mM L-Trp, 20 mM ascorbic acid, 10 μM methylene blue, 2,250 U/ml bovine liver catalase, and 2 μg/ml rhIDO1 in 10 mM MES (pH 6.8). The reaction mixture was incubated at 37°C for 2 h after which 20 μl of 30% (w/v) trichloroacetic acid was added and further incubated at 50°C for 15 min. After centrifugation at 15,000 × g for 15 min at 25°C, 75 μl of the supernatant was transferred to a new microplate and 2% 4-(dimethylamino)benzaldehyde in acetic acid was added to each well for colorimetric measurement. The absorbance of each well was measured at 450 nm on a microplate reader. The inhibition ratios and IC50 values were calculated according to the formula shown above in the description of the cytotoxicity assay.

Results

IDO1 inhibition of existing inhibitors. Since the IDO1 catalytic reaction requires L-Trp as a substrate, some IDO1 inhibitors have a similar molecular structure that allows them to act as competitive inhibitors (Figure 1) (10). One of the major drugs that contains the indole skeleton is indomethacin, which is categorized as an NSAID. Three known IDO1 inhibitors and six COX inhibitors were assessed for their inhibitory activity against rhIDO1 (Figure 2). As a result, under conditions where the known IDO1 inhibitors could moderately inhibit rhIDO1, the COX inhibitors showed potent inhibitory activity. However, nimesulide which contains neither an indole moiety nor a pyrrole group, had only weak IDO1 inhibitory activity. Among the drugs with a strong inhibitory activity, celecoxib has a pyrrole ring, but the structure of other COX inhibitors (acemetacin and oncrasin 1) was very similar and included an indole skeleton. The base structure of indole and several substituted indole compounds are shown in Figure 3. Unfortunately, the indole skeleton alone or simple indole compounds (substituted at the 1- or 3-position of the indole ring) had no effect on rhIDO1 activity. Only 3-IM and 1-IAA showed weak inhibition. In contrast, COX inhibitors with an indole scaffold, such as indomethacin, acemetacin, and oncrasin 1, exhibited potent inhibitory activity against rhIDO1 that was similar to known IDO1 inhibitors.

Figure 1.
Figure 1.

Structure of IDO1 inhibitors and COX inhibitors (NSAIDs).

Figure 2.
Figure 2.

Inhibitory activity of IDO1 inhibitors and COX inhibitors towards rhIDO1. IDO1- and COX-inhibitors are illustrated as closed and opened bars, respectively. All compounds were evaluated at 0.5 mM. Inhibitory activity is shown as inhibition ratio (%) versus control (no compound).

Figure 3.
Figure 3.

Inhibitory activity of simple indole compounds towards rhIDO1. All compounds were evaluated at 0.5 mM. Inhibitory activity is shown as inhibition ratio (%) versus control (no compound).

Structure-activity relationship among indomethacin derivatives. Since indomethacin and its related NSAIDs (acemetacin and oncrasin 1) were among the compounds with an indole scaffold that showed potent activity as IDO1 inhibitors, 27 indomethacin derivatives were synthesized to evaluate their IDO1 inhibitory activity and direct cell cytotoxicity (Figure 4 and Table I). Seven indomethacin derivatives (compounds 1-7), including indomethacin, had a 4-chlorobenzoyl group at the 1-position and lacked the phenyl group at the 2-position. Twelve indomethacin derivatives (compounds 8-19) were substituted at the 2-position with a phenyl group and had a variety of benzoyl groups at the 1-position. Eight indomethacin derivatives (compounds 20-27) were substituted at the 2-position with a phenyl group and had a variety of benzyl group at the 1-position. Eight compounds, including indomethacin, were synthesized as previously described (12, 13). Eighteen indomethacin derivatives were newly synthesized for this study, and their chemical properties are shown in Materials and Methods.

Figure 4.
Figure 4.

Inhibitory activity of 27 indomethacin derivatives towards rhIDO1. The tested derivatives were tested at two concentration (0.1 and 0.5 mM) in the rhIDO1 enzymatical reaction.

As a first step in their evaluation, their cytotoxicity towards human glioblastoma T98G cell line was examined. Only three indomethacin derivatives (compounds 16, 17, and 19) showed moderate cytotoxicity (IC50 values of 18.4, 49.6, and 30.8 μM, respectively) whereas the other indomethacin derivatives did not have any antitumor activity. The inhibitory effect of these indomethacin derivatives against rhIDO1 varied considerably (Table I and Figure 4). In our experimental assay system, the IC50 value of indomethacin for IDO1 inhibition was 0.20 mM, and many of the indomethacin derivatives were found to be more potent IDO1 inhibitors than the lead compound (indomethacin) in this series. The inhibitory effect of these indomethacin derivatives was dose-dependent (Figure 4). Figure 5 shows the comparison of IC50 values for IDO1 inhibition among all the tested indomethacin derivatives. Almost all compounds indicated potent inhibitory activity against rhIDO1, but compounds 15 and 17 had very weak activity and the IC50 value of compounds 2 and 17 was over 1 mM. Compound 18 with a phenyl group at the 2-position and benzyl acetate at the 3-position was essentially inactive towards rhIDO1. To evaluate whether the functional group on benzene ring affects IDO1 inhibitory activity, the methoxy group on the benzene ring was substituted. When the methoxy group at the 5-position, including indomethacin, was moved to the 6-position (compound 2), the inhibitory activity was remarkably reduced. However, a shift to the 7-position or removal of the methoxy group maintained inhibitory activity (compounds 3 and 4 showed IC50 values of 0.22 and 0.34 mM, respectively). When the functional group at the 2-position was changed to methyl, ethyl, or phenyl, the phenyl group-substituted compound 10 exhibited stronger inhibitory activity than compounds 4 and 5 with methyl and ethyl substitutions, respectively. When 4-chlorobenzoyl at the 1-position was substituted with other functional groups (compounds 8-14), substitution with 4-florobenzoyl and unsubstituted benzoyl (compounds 12 and 14) exhibited weak inhibitory activity. With the exception of carbonic acid, substitution of the 3-position with a ketone, ester, or amide (compounds 17, 18, and 19, respectively) showed a tendency to decrease IDO1 inhibitory activity. Substitution with long carbon chain (compounds 15, 10, and 16) tended to increase inhibitory activity. Indomethacin derivatives with a benzyl group (compounds 20, 21, and 22) at the 1-position had a greater inhibitory effect than the corresponding derivatives with a benzoyl group (compounds 8, 9, and 10). 2-chlorobenzyl and 3-chlorobenzyl derivatives (compounds 20 and 21, respectively) had the most potent inhibitory activity among all the tested indomethacin derivatives, with the IC50 values being decreased five-fold compared to that of indomethacin (0.027 and 0.038 mM, respectively).

Figure 5.
Figure 5.

Comparison of the inhibitory activity of all the tested indomethacin derivatives towards rhIDO1. IC50 value was plotted as –pIC50, and that of compound 2 and 18 were over 1 mM.

Discussion

Cancer is mainly treated with surgery, radiation, and chemotherapy, and recently, the use of cancer immunotherapy to strengthen the immune system of patients with various types of tumors has received attention. Various types of cancer immunotherapy have been adopted, such as immune checkpoint inhibitors, cancer vaccines, monoclonal antibodies, and T cell transfer. It has been widely recognized that cancer immunotherapy as well as traditional chemotherapy are beneficial and can effectively overcome tumor-induced immunosuppression (18). Several immune checkpoint inhibitors, such as pembrolizumab and nivolumab, have been recently developed and utilized in the clinical treatment of many patients with various types of tumors. These treatments are specific monoclonal antibodies that interfere with the programmed cell death protein 1 (PD-1)/PD-L1 pathway in the immune system (19). The action of IDO leads to a depletion of tryptophan and the production of kynurenine metabolites. As a result, T- and NK-cells were damaged, leading to cell cycle arrest or cell death. Furthermore, T cell differentiation to mature regulatory T cells was inhibited by kynurenine metabolites. As a result of this immunosuppression, tumors can escape from the immune system. In support of this idea, high IDO1 expression levels are associated with a poor prognosis in many cancers (6, 7). As a result, IDO inhibitors are believed to have excellent therapeutic potential, and many drugs, such as indoximod, epacadostat, and navoximod have been developed for cancer immunotherapy (2, 20).

In this study, the potential inhibitory activities of compounds that had the indole scaffold were evaluated using an in vitro rhIDO1 enzyme reaction, in order to identify new IDO1 inhibitors. Many COX inhibitors, including indomethacin, have an indole scaffold within their molecular structure, and it was found that their IDO1 inhibition activity was as potent as that of existing IDO1 inhibitors (Figure 2). However, nimesulide which belongs to the NSAIDs, had only weak IDO1 inhibitory activity, and contained neither an indole moiety nor a pyrrole group. Although there are a few reports suggesting that COX-2 inhibitors can suppress interferon-γ via the IDO pathway (21, 22), it is a novel finding that indomethacin, a COX-2 inhibitor, directly inhibits rhIDO1. However, the indole skeleton alone is not sufficient for IDO1 inhibitory activity because the simple indole structure was found to have very weak activity (Figure 3). Rather, 3-IE enhanced rhIDO1 activity and acted as an activator for IDO enzyme. This result agrees with previous studies, which reported that 3-IE enhances the IDO activity via binding to an accessory binding site located near its catalytic site (23, 24). In order to develop more potent candidates, we selected indomethacin as a lead compound and explored the structure-activity relationship of existing and newly synthesized compounds (Figure 6). Among the 27 indomethacin derivatives tested, two compounds with 2-chlorobenzyl or 3-chlorobenzyl at the 1-position and a phenyl group at the 2-position (compounds 20 or 21, respectively) showed the most potent inhibitory activity against rhIDO1, with their inhibitory activity being enhanced more than five-fold compared to the lead compound, indomethacin (Table I). However, compound 18, which had a benzyl acetate at the 3-position was essentially inactive towards rhIDO1. Based on this, substitution of the functional group at the 3-position seems to have a strong effect on IDO1 inhibition. At this position, it is thought that indole-containing compounds interact with the iron atom in the heme group of IDO1, and IDO1 then cleaves the covalent bond between the 2- and 3-position, suggesting that this region of the indole ring is recognized by the IDO1 active site (23). Therefore, based on the results of this study, other positions in the indole scaffold seem to have a little influence on the IDO1 enzymatic activity. Furthermore, the tested indomethacin derivatives had weak cell cytotoxicity and this is in agreement with previous studies (12, 13), that showed enhancement of doxorubicin antitumor activity in combination with indomethacin derivative. The IDO1 inhibitory activity is not directly correlated with cell cytotoxicity, because the indomethacin derivatives with the highest degree of cytotoxicity (compounds 16, 17, and 19) and the most potent IDO1 inhibitors (compounds 20 and 21) were different. IDO1 protein, which is expressed in tumor cells and in mature dendritic cells located in lymphoid organs (4), can lead to the inhibition of T cell proliferation and cell cycle arrest resulting in T cells not attacking the targeted tumor cells. Therefore, activation of T cells is required for any IDO inhibitor to exert its anti-tumor activity. IDO inhibition itself does not seem to demonstrate direct cell cytotoxicity under single cell culture conditions without T cell. Docking analysis between indomethacin derivatives and IDO1 enzyme, and analysis of the quantitative structure-activity relationship should be employed to support these conclusions. Furthermore, the IDO1 inhibitory activity of indomethacin derivatives should be confirmed in in vivo conditions in the presence of mature T cells. Finally, the findings reported by this study will be beneficial for the development of IDO1 inhibitors for cancer immune therapy.

Figure 6.
Figure 6.

Structure-activity relationship based on indomethacin derivatives.

Acknowledgements

The Authors would like to thank Editage (www.editage.com) for English language editing.

Footnotes

  • Conflicts of Interest

    All Authors declare no competing financial interests in relation to this study.

  • Authors’ Contributions

    T.O., and M.T. designed the study; T.N., Y.K., M.A. and S.S. synthesized and analyzed chemicals.

  • Received April 2, 2021.
  • Revision received April 14, 2021.
  • Accepted April 16, 2021.

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Structure-activity Relationship of Indomethacin Derivatives as IDO1 Inhibitors
TOHRU OBATA, SARA SHIRATANI, TOMOMI NADA, YAYOI KASAYA, MITSUHIRO ARISAWA, SATOSHI SHUTO, MOTOHIRO TANAKA
Anticancer Research May 2021, 41 (5) 2287-2296; DOI: 10.21873/anticanres.15004
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