Structure-based optimization of type III indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Figures & data

Figure 1. (A) Examples of clinical and other potent IDO1 inhibitors. (B) and (C) Rotated views of the binding pockets A and B in IDO1-active site (PDB ID 6r63²²; ligand MMG-0358). (D) Main lead optimisation strategies pursued in this work. The red arrow denotes a preferentially hydrogen-bonding substituent, the green arrow a hydrophobic substituent, and the blue arrows potential access points to pocket B. As demonstrated before, the acidic hydrogens on the triazole rings are crucial for activity and therefore cannot provide access to pocket B.

Scheme 1. General synthesis of 4-aryl-1,2,3-triazoles. Reagents and conditions: (a) TMSA, PdCl₂(PPh₃)₂, Et₃N, CuI, dioxane, 45 °C, 5 h. (b) KF, MeOH, rt, 3 h, overall yield 48-85% for 2 steps. (c) TMSN₃, CuI, DMF:MeOH (9:1), 100 °C, 10–12 h, yield 44–85%.

Scheme 2. Synthesis of aryl iodides. Reagents and conditions: (a) aq. NH₃, KI, I₂, DMF, rt, 1 h, yield 50%. (b) K₂CO₃, DMF, 80 °C, overnight, yield 50–95%. (c) Cs₂CO₃, DMF, 100 °C, 24 h, yield 60%. (d) PhCH₂P(Br)(Ph)₃, [(CH₃)₃Si]₂NNa, THF, rt. (e) N₂H₄·H₂O, FeCl₃·6H₂O, EtOH, 100 °C, 24 h, overall yield 90% for 2 steps.

Table 1. 4-Aryl-1,2,3-triazoles with substituted phenyl groups.

Compound	R2	R3	R4	R5	R6	IC50 [$\mu$M] (SD^a)	LE^b [kcal/mol/HA]
19						10 (1)^28	0.62
20			–OH			$>$1000
21						22^58	0.53
22						13^58	0.51
23						280 (20)	0.35
24						34 (–)	0.41
25						$>$500
26						$>$100
27						$>$500
28						$>$250
29	–OH					2.3 (0.8)^28	0.64
30				–Cl		0.35 (0.04)^28	0.73
9	–OH			–Cl		0.059 (0.003)^28	0.76
31	–			–Cl		1.5 (0.2)^28	0.61
32	–			–Cl		4.3^28	0.56
33	–			–		$>$1000^28
34	–OH			–F		0.14 (0.01)	0.72
35	–OH					0.55 (0.05)	0.66
36	–OH					0.25 (0.02)	0.64
37	–OH					0.75 (0.03)	0.56
38	–OH					88 (–)	0.29
39	–OH					160 (–)	0.26
40^62				–Cl		9.5 (0.8)	0.36
13^62				–Cl		15 (1)	0.33
41^62				–Cl		49 (9)	0.29
42^62				–Cl		260 (50)	0.24
43				–Cl		31 (0.4)	0.29
44	–OH				–OH	13 (3)	0.51
45	–				–OCH3	$>$1000
46	–Cl			–Cl		6.4^58	0.54
47	–Cl	–Cl				23^58	0.49
48	–Cl		–Cl			31 (0.09)	0.47
49		–Cl	–Cl			51 (4)	0.45
50		–Cl		–Cl		58 (7)	0.44
51	–Cl				–Cl	280 (20)	0.37
52	–Cl		–Cl	–Cl		33 (0.5)	0.44
53	–Cl		–Cl		–Cl	$>$500

^a Standard Deviation (SD). ^bLigand Efficiency (LE).

Scheme 3. Synthesis of 5-substituted 4-aryl-(1H)-1,2,3-triazoles. Reagents and conditions: (a) NaN₃, DMF, 90 °C, 3 h, yield 75%. (b) Nitroethane, NH₄OAc, AcOH, reflux, 2 h. (c) NaN₃, pTsOH, DMF, 60 °C, 14 h, overall yield 65–70% for 2 steps. (d) EtOH, rt, 2 h (e) Cs₂CO₃, DMF, 100 °C, 4 h, overall yield 70% for 2 steps. (f) NaN₃, DMSO, 60 °C, 6 h, yield 60%. (g) NaN₃, Et₃N·HCl, DMF, 70 °C, 10 h, yield 55–65%.

Table 2. 5-Substituted 4-Aryl-1,2,3-Triazoles.

Compound	R1	R2	R4	R5	IC50 [$\mu$M] (SD^a)	LE^b [kcal/mol/HA]
61					$>$1000^58
62					630 (70)	0.34
63				–Cl	$>$1000
64^59				–Cl	$>$50
65^59			–Cl		$>$1000
66^59			–Cl		800	0.28
67		–OH		–Cl	$>$1000
68		–OH		–Cl	220 (7)	0.36

^a Standard Deviation (SD). ^bLigand Efficiency (LE).

Scheme 4. Aryl-tether-1,2,3-triazoles and annulated derivatives. Reagents and conditions: (a) NaNO₂, 2M HCl, CH₃COONa, 0 °C-rt, 1 h. (b) EtOH, reflux, overnight, overall yield 70% for 2 steps. (c) TMSN₃, CuI, DMF:MeOH (9:1), 100 °C, 10–12 h, yield 50–70%. (d) H₂O₂, (NH₄)₂MoO₄, MeOH, 0 °C- rt, 14 h, yield 80%. (e) CH₃ONH₂·HCl, NaOAc, H₂O:EtOH, 70 °C, rt, 15 h, yield 55%. (f) N₂H₄·H₂O, KOH, diethylene glycol, 170–190 °C, 6 h, yield 40%.

Scheme 5. Synthesis of ethynes. Reagents and conditions: (a) NaOH, DMSO, rt, 4 h. (b) n-BuLi, Et₂O, -78 °C-rt, 2h, overall yield 40% for 2 steps. (c) n-BuLi, THF, -78 °C-rt, overnight. (d) KF, MeOH, rt, 4 h, overall yield 85% for 2 steps. (e) propiolic acid, DCC, DMAP, Et₂O, rt, 18 h, yield 90%.

Table 3. 4-Aryl-tether-1,2,3-triazoles and annulated derivatives.

Compound	A	R1	R4	IC50 [$\mu$M] (SD^a)	LE^b [kcal/mol/HA]
10^60	–NH–		–Cl	57 (20)	0.44
78^70	–NH–		–Br	52 (8)	0.45
79	–NH–			$>$100
80	–NH–	–phenyl		$>$250
81	–CH2–			$>$1000^58
82	–O–			$>$1000
83	–S–			590 (20)	0.37
84	–SO2–			$>$1000
85	–CHOH–			$>$1000
86				$>$1000
87				$>$1000
88				$>$100
89				$>$1000

^a Standard Deviation (SD). ^bLigand Efficiency (LE).

Scheme 6. Synthesis of 5-aroyl and 5-hetaroyl-1,2,3-triazoles. Reagents and conditions: (a) ethynylmagnesium bromide, THF, 3 °C-rt, 1 h. (b) TMSN₃, CuI, DMF:MeOH (9:1), 100 °C, 10–12 h, overall yield 55–85% for 2 steps. (c) PCC, DCM, rt, 2 h, yield 35–73%. (d) 48% HBr in H₂O, 100 °C, 14 h, yield 45–50%.

Scheme 7. Synthesis of 4-substituted-5-aroyl-(1H)-1,2,3-triazoles. Reagents and conditions: (a) 4-Bromobenzaldehyde, n-BuLi, -78 °C to -38 °C, THF, 3 h, yield 88%. (b) MnO₂, DCM, rt, 5 h, yield 97%. (c) PPTSA, EtOH, 50 °C, 4 h, yield 75%. (d) TMSN₃, CuI, DMF/MeOH (9:1), 100 °C, 10–12 h, yield 50%. (e) CBr₄, PPh₃, DMF, rt, 4 h, yield 85%. (f) 4-Bromobenzoyl chloride, PdCl₂(PPh₃)₂, CuI, Et₃N,THF, rt,1 h, yield 90%. (g) TMSN₃, CuI, DMF/MeOH (9:1), 10 °C, 10-12 h, yield 45%. (h) TFA, DCM, rt, overnight, yield 75%.

Table 4. 5-Aroyl and 5-Hetaroyl-1,2,3-Triazoles.

Compound	Aryl	R1	IC50 [$\mu$M] (SD^a)	LE^b [kcal/mol/HA]
99			430 (60)	0.35
100			$>$1000	0.20
101			450 (40)	0.33
102			31 (6)	0.44
103			$>$1000
104			920 (200)	0.28
105			200 (20)	0.34
106			130 (30)	0.38
107			$>$1000
108			$>$1000
109			13 (0.8)	0.48
110			190 (10)	0.36
111			370 (30)	0.31
112			11 (0.2)	0.45
113			8.2 (1)	0.46
114			230 (20)	0.41
115			16 (0.7)	0.55
116			17 (1)	0.54
117			68 (3)	0.47
118			82 (6)	0.46
119			510 (100)	0.37
120			$>$1000
121			650 (100)	0.33
122			$>$1000
123		–CH2OH	$>$1000
124		–CH2Br	$>$1000
126		–CH2NH2	$>$1000

^a Standard Deviation (SD). ^bLigand Efficiency (LE).

Scheme 8. Synthesis of 3-aryl-1H-1,2,4-triazoles. Reagents and conditions: (a) Formic acid, N₂H₄·H₂O, DMF, 90 °C, 12 h, yield 20–45%. (b) N,N-dimethyl formamide-dimethyl acetal (DMF-DMA), reflux, 1 h. (c) N₂H₄·H₂O, AcOH, 90 °C, 1.5 h, overall yield 70-80% for 2 steps. (d) SnCl₂·2H₂O, EtOH, 70 °C, 1 h, yield 40–66%.

Scheme 9. Synthesis of aryl carbonitriles and aryl carboxamides. Reagents and conditions: (a) t-BuOK, dimethyl oxalate, N,N-dimethyl acetamide, 140 °C, 5 h, yield 70%. (b) pyridine, DMAP, rt, 3 h, yield 75–85%. (c) HNO₃, H₂SO₄, rt, 3 h (d) (COCl)₂, NH₄OH, DMF, DCM, reflux, overall yield 70–75% for 2 steps.

Table 5. 3-Aryl-1,2,4-Triazoles.

Compound	R2	R3	R4	R5	R6	IC50 [$\mu$M] (SD^a)	LE^b [kcal/mol/HA]
133						$>$1000^28
134			–F			$>$1000
135				–Br		16 (1)	0.55
136				–Cl		31 (4)^28	0.51
137				–F		160 (20)	0.43
138	–OH					31 (1)^28	0.51
139	–NH2					2.0 (0.7)^28	0.65
140	–NH2	–F				0.81 (0.2)	0.64
141	–NH2	–Cl				29 (3)	0.48
142	–NH2				–NH2	31 (7)	0.47
12	–NH2			–Br		0.020 (0.001)^28	0.80
11	–NH2			–Cl		0.035 (0.004)^28	0.78
143	–NH2			–F		0.15 (0.006)	0.72
144	–NH2		–F	–Br		0.034 (0.004)	0.73
145				–Cl		8.7 (3)	0.49
146				–Cl		420 (90)	0.29
147				–Cl		1.2 (0.04)	0.47
148				–Cl		25 (7)	0.35
149				–Cl		0.49 (0.05)	0.48
150				–Cl		$>$100
151				–Cl		$>$100
152						$>$100
153				–Cl		9.3 (0.3)	0.33
154		–Cl				$>$1000
155	–NH2			–Br	–CH3	63 (10)	0.41
156	–NH2			–Cl	–CH3	96 (3)	0.39
157	–NH2			–Cl	–NH2	1.0 (0.02)	0.55

^a Standard Deviation (SD). ^bLigand Efficiency (LE).

Table 6. 3-Hetaryl-1,2,4-triazoles.

Compound	Aryl	IC50 [$\mu$M] (SD^a)	LE^b [kcal/mol/HA]
158		$>$1000
159		$>$1000
160		16 (7)	0.55
161		10 (2)	0.57
162		250 (10)	0.41

^a Standard Deviation (SD). ^bLigand Efficiency (LE).

Scheme 10. Synthesis of other 1,2,4-triazoles. Reagents and conditions: (a) K₂CO₃, CuI, N,N-dimethyl acetamide, 90 °C, 48 h, yield 39%. (b) K₂CO₃, acetone, reflux, 9h, yield 60%.

Table 7. Other 1,2,4-triazoles.

Compound	Structure	IC50 [$\mu$M] (SD^a)	LE^b [kcal/mol/HA]
166		$>$1000
167		$>$1000
168		$>$1000
169		$>$1000
170		870	0.38
171		27	0.52

^a Standard Deviation (SD). ^bLigand Efficiency (LE).

Figure 2. (A) Detail of X-ray structure of compound MMG-0472 (13, orange) bound to IDO1 (PDB ID 7zv3). The 2F_o−F_c map of the active site is contoured at 1.0 σ. (B) Same Xray structure, highlighting the His346–iron–ligand bond angle of 170°. (C) DFT-optimized model of MMG-0472 binding to haem. The imidazole–iron–ligand bond angle of 180° is highlighted. (D) Superposition of the X-ray structure of MMG-0472 (orange) and its binding pose predicted by docking (cyan). The RMSD between the structures is 0.3 Å. (E) Top view of MMG-0472 X-ray structure, showing the passage from pocket A to pocket B in-between residues Phe163 on the one side and residues 261–265 (magenta) on the other side. (F) Same view, superimposed with X-ray structures of compounds 9, 11 and 12 (PDB ID 6r63, 7ah5, 7ah6)^22,28.

Figure 3. Binding poses of 4-aryl-1,2,3-triazoles predicted by docking. (A) Compound 20: non-optimal pose, the imidazole-iron-triazole bond angle deviates from 180° due to limited space for the p-OH substituent. (B) Compound 22: the m-ethyl substituent is of good size for the hydrophobic subpocket between Val125, Cys129, Leu234 and Gly262. (C) Compound 26: inactive compound with larger ortho substituent using an ether linker. (D) Compound 27: no favourable interactions between hydroxy group and pocket B. (E) Compound 36. (F) Compound 43.

Figure 4. Tethered 1,2,3-triazoles. (A) X-ray structure of compound 10 (PDB ID 6f0a)⁶⁰. (B) Superposition of X-ray structure and of docking pose of 10. Docking poses of (C) compound 80; (D) compound 83; (E) compound 86; (F) compound 99; (G) compound 102; (H) compound 112; (I) compound 115. Hydrogen bonds are shown in orange.

Figure 5. 1,2,4-Triazoles. (A) X-ray structure of compound 11 (PDB ID 7ah5)²⁸. (B) Superposition of X-ray structure and of docking pose of compound 11. Docking poses of (C) compound 141; (D) compound 142; (E) compound 152; (F) compound 155; (G) compound 160; (H) compound 168; (I) compound 171. Hydrogen bonds are displayed in orange.

Figure 6. Cellular inhibition and comparison to enzymatic inhibition. (A) Cellular inhibition of kynurenine production at a single compound concentration. Data for 1,2,3-triazole compounds is shown in black, for 1,2,4-triazoles in orange. Values measured at a compound concentration of 200 µM are given in filled bars, values measured at 50 µM in empty bars. (B) Cellular toxicity under the same conditions and using the same colour code as part (A). (C) Cellular inhibition as a function of enzymatic activity (Act = RTlog(IC₅₀)). The dashed line is a sigmoidal fit to all data points except for the marked outliers. Compounds measured at a concentration of 50 µM are marked by empty symbols. (D) Cellular dose-response curves of compounds 140, 143, and 144 measured in this work. (E) Correlation between cellular activity and enzymatic activity for compounds mentioned in this manuscript. Colour code as in part (A). Filled symbols denote the newly determined data shown in part (D).

Table 8. Cellular inhibition and toxicity data determined in this work.

Compound	Conc. [$\mu$M]	Inh. [%]	Tox. [%]	Cell. IC50 [$\mu$M] (SD^a)
48	200	63 (8)	0 (0)
49	200	65 (7)	0 (0)
52	50	44 (20)	7.9 (1)
78	200	97 (1)	0 (0)
102	200	37 (7)	0 (0)
105	200	22 (3)	0 (0)
106	200	12 (3)	0 (0)
109	200	55 (10)	0 (0)
110	200	12 (5)	0 (0)
111	200	20 (3)	0 (0)
112	200	32 (6)	0 (0)
113	200	36 (8)	0 (0)
114	200	20 (0.6)	0 (0)
115	200	73 (5)	0 (0)
116	200	62 (7)	0 (0)
117	200	23 (3)	0 (0)
118	200	17 (7)	0 (0)
119	200	4.8 (2)	0 (0)
121	200	3.9 (6)	0 (0)
140	200	100 (0)	17 (3)	0.81 (0.2)
141	200	82 (7)	13 (4)
142	200	56 (6)	11 (0.4)
143	200	86 (3)	18 (0.5)	0.15 (0.06)
144	200	100 (0)	17 (2)	0.034 (0.004)
145	200	93 (3)	0 (0)
146	200	89 (4)	0 (0)
147	200	100 (0)	16 (1)
148	50	93 (1)	17 (2)
149	50	100 (0)	19 (2)
153	50	87 (4)	5.9 (8)

^a Standard Deviation (SD).

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