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

Abstract

The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC₅₀ values of 34 nM. We provide enzymatic inhibition data for almost 100 new compounds and X-ray diffraction data for one compound in complex with IDO1. Structural and computational studies explain the dramatic drop in activity upon extension to pocket B, which has been observed in diverse haem-binding inhibitor scaffolds. Our data provides important insights for future IDO1 inhibitor design.

Keywords:

• Cancer immunotherapy
• structure-based drug design
• tryptophan metabolism
• X-ray crystallography

Introduction

Immuno oncology provides powerful therapies against cancer in the form of immune checkpoint inhibitors, adoptive cell therapies, monoclonal antibodies, oncolytic viruses, cancer vaccines, and other immuno modulators. However, low response rates due to tumoral immune suppression and resistance remain an unsolved issue¹. L-Trp catabolism along the kynurenine pathway is an important mechanism employed by cancer cells to escape a potentially effective immune response^2,3. The rate-limiting step in this pathway is catalysed by indoleamine 2,3-dioxygenase 1 (IDO1) and by tryptophan 2,3-dioxygenase (TDO), with the IDO1 paralogue IDO2 also potentially playing a role^4,5 Preclinical data suggests that a combination of IDO1 inhibitors with other anticancer agents results in effective anti-tumor immunity^6–8. However, in a phase 3 clinical trial of the IDO1 inhibitor epacadostat in combination with pembrolizumab in melanoma patients, the combination failed to increase the overall and progression-free survival when compared to pembrozilumab alone⁹. This failure highlighted the need for a better understanding of the role of the kynurenine pathway, for the development of better IDO1 inhibitors, and for improved trial design^10,11. Additional aspects of IDO1 biology are constantly discovered and may influence its role in cancer, such as its signalling activity^12–14, regulation by haem availability¹⁵, nitrite reductase activity in hypoxic tissues¹⁶, involvement in the redox signalling pathways of hydrogen peroxide and singlet oxygen¹⁷, and activation by polysulphides¹⁸. Based on the ongoing interest for IDO1 inhibitors capable of modulating these different pathways and processes selectively, a multitude of small-molecule IDO1 inhibitors have been described^19,20, and more than 60 crystal structures of IDO1 have been deposited in the protein data bank (PDB)²¹. These structures with a large diversity of bound cofactors and ligands, including the clinical-stage IDO1 inhibitors epacadostat (1, INCB024360, Figure 1A)²³, navoximod (2, NLG-919/GDC919)²⁴, EOS200271 (3)²⁵, and linrodostat (4, BMS-986205)²⁶, yield a wealth of information for inhibitor design²⁷.

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.

We have previously classified IDO1 inhibitors into four types according to their preferential binding and inhibition mechanism²². Type i inhibitors (e.g. 1-methyl-L-tryptophan) preferentially bind to oxygen-bound holoIDO1, type ii inhibitors (e.g. epacadostat) to free ferrous holoIDO1, type iii inhibitors (e.g. navoximod) to free ferric holoIDO1, and type iv inhibitors (e.g. linrodostat) to apoIDO1. Lead optimisation of haem-iron binding type ii and type iii inhibitors has proven difficult due to the selectivity and sensitivity of the haem–ligand interactions to changes in the electronic structure of the ligand²⁸ and due to the small size of the distal haem pocket (pocket A)²⁹, crucial for inhibitor activity. The IDO1 active site further comprises Pocket B, which extends from pocket A towards the entrance of the active site (Figure 1B,C), and whose size and shape are determined by the conformation of the flexible JK-loop²⁷. Although its influence on inhibitor affinity is less pronounced than pocket A, it is of interest for modulation of other compound properties such as specificity, absorption, distribution, metabolism and excretion (ADME), and pharmacokinetics/pharmacodynamics (PK/PD).

Despite the large number of published IDO1 inhibitors, there are only a very limited number of sub-micromolar type ii and type iii inhibitor scaffolds known. Epacadostat (1) binds to the haem iron through an unusual high-affinity hydroxyamidine scaffold, which was discovered by Incyte^23,30 and has later been exploited by other groups^31–38. Due to its unique tilted iron-binding conformation, it provides a straightforward access to pocket B and allowed the development of numerous nanomolar IDO1 inhibitors with a high selectivity over TDO²⁷. Except for the hydroxyamidines, almost all nanomolar haem-binding IDO1 inhibitors are based on a haem-binding free or fused azole scaffold.

Imidazoles are classical haem binders, as present in the haem-binding histidine side chain and in many antifungal drugs, and 4-phenyl-imidazole³⁹ was the first cocrystallized IDO1 inhibitor⁴⁰. Early structure-based optimisation of this chemotype yielded the 2-hydroxy-substituted phenyl derivative as the most active compound⁴¹. Combination of the 2-hydroxy substitution with a 5-chloro substitution led to the most efficient imidazole compound (5) with a ligand efficiency (LE) of 0.68 kcal/mol/heavy atom (HA)^24,28. In subsequent developments, extending the scaffold to pocket B, a drop in LE was always encountered⁴², but nanomolar activities could be obtained in some 1,5-disubstituted imidazoles^43,44. Rigidification of the scaffold to imidazo[5,1-a]isoindole also allowed to retain nanomolar activities^45–49 and led to the development of navoximod (2, Figure 1)²⁴. Independently from the work on imidazo[5,1-a]isoindoles, structural and functional data of imidazo[2,1-b][1,3]thiazole based IDO1 inhibitors was disclosed in 2014 (6)⁵⁰. Subsequent work on this fused imidazole scaffold led for example to compound 7 with a good enzymatic activity and a LE of 0.41 kcal/mol/HA⁵¹. However, compounds with this scaffold lack cellular activity^51–53.

Indazoles are also known haem binders, and the indazol-4-amine scaffold developed by IOmet Pharma⁵⁴ yielded selective nanomolar TDO inhibitors and dual IDO1/TDO inhibitors^55–57. X-ray structures of the complexes between IDO1 and some indazol-4-amines such as compound 8 have recently been resolved⁵⁷. These compounds provide access to pocket B while preserving a LE of up to 0.47 kcal/mol/HA.

We have previously discovered 1,2,3-triazoles as highly efficient IDO1 inhibitors and resolved the X-ray structure of MMG-0358 (9) in complex with IDO1 (Figure 1)^22,28,29,58. Compound 9 forms a direct bond to the haem iron, a hydrogen bond with Ser167 through its hydroxy function, and hydrophobic interactions through its chloro substituent, leading to a LE of 0.76 kcal/mol/HA. A few 4,5-disubstituted 1,2,3-triazoles with nanomolar activities have been reported⁵⁹, but as we detail below, we failed to reproduce these results. Interestingly, the N-phenyl-1,2,3-triazol-4-amine (10) developed by Vertex⁶⁰ demonstrates an IDO1 inhibition mechanism distinct from the 4-phenyl1,2,3-triazoles despite a similar binding mode²².

1,2,4-Triazole is a common haem-binding scaffold present in many antifungal drugs such as fluconazole, and its direct iron binding in sterol 14α-demethylase (CYP51) is well documented by structural data. However, these 1-substituted 1,2,4-triazoles have been found to be inactive on IDO1, at variance with imidazole antifungals such as miconazole, which showed some activity⁶¹. However, we recently demonstrated that the 3-substituted 1,2,4-triazole scaffold can provide highly efficient IDO1 inhibitors. Two simple substitutions on the 3-phenyl-1,2,4-triazole scaffold improved its inhibitory activity by more than four orders of magnitude from the millimolar to the low nanomolar range, and we provided structural data for IDO1 binding of MMG-0706 (11) and MMG-0752 (12, Figure 1)²⁸. These compounds feature a 2-amino, 5-halogen di-substituted phenyl ring and display a LE of 0.80 kcal/mol/HA for the bromo compound, to our knowledge the highest LE reported for IDO1 inhibitors to date. Based on molecular modelling and quantum chemical calculations, we were able to explain the improved activity of the 2-amino substituent versus the 2-hydroxy substituent in this scaffold, due to simultaneous intramolecular and intermolecular interactions²⁸.

Here, we describe our efforts to improve and extend compounds comprising the 1,2,3-triazole and 1,2,4-triazole haem-binding scaffolds, the best new compound (144) demonstrating both enzymatic and cellular IC₅₀ values of 34 nM. Although this compound and our previously described compounds such as MMG-0358 (9), MMG-0706 (11), or MMG-0752 (12)^28,58 are highly efficient both in an enzymatic and in a cellular environment, they are very sensitive to even the smallest changes in their chemical structure, and therefore lack available sites to modulate their ADME and PK/PD properties. Analysing the active site structure of IDO1 and the chemical structures of other known IDO1 inhibitors, it would be natural to extend the compounds from pocket A in the haem distal site to pocket B towards the entrance of the active site (Figure 1D). However, azole ligands are not optimally suited to be extended to pocket B due to their preferred orientation, and this extension often leads to a dramatic decrease in activity for many compounds²⁷. Here, we resolve one new X-ray structure of a previously reported triazole extending to pocket B (13, MMG-0472), which validates our docking predictions. Based on this new structural data and the measured activities of almost 100 new compounds, we give recommendations for the development of future IDO1 inhibitors.

Chemistry

All compounds were synthesised using existing protocols or procedures adapted from the literature. 4-Aryl-1,2,3-triazoles (20–53, Table 1) were synthesised according to the method developed by Yamamoto and co-workers⁶³, which involves reaction of ethynyl derivatives (18) with trimethylsilyl azide (TMSN₃, Scheme 1). The ethynyl derivatives were synthesised from iodo substrates (17, Scheme 2) by the Sonogashira coupling reaction⁶⁴. Phenols 20, 39, and 44 were obtained by demethylation of the corresponding methyl ethers with 48% HBr in water at 100 °C.

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).

5-Substituted 4-aryl-1,2,3-triazoles (Table 2) were obtained according to Scheme 3. Phenyl propynenitrile (54) reacted with sodium azide in DMF at 90 °C to produce 62⁶⁵. Compounds 63 and 68 were obtained from the corresponding aryl benzaldehyde (55a, 55b), nitroethane and ammonium acetate through condensation in presence of acetic acid, followed by dipolar cycloaddition with HN₃ engendered with sodium azide and p-toluenesulfonic acid in DMF at 60 °C⁶⁶. The 4,5-diaryl derivative 64 was obtained from 3-chlorobenzaldehyde (55a) by reaction with tosylhydrazine (57) in ethanol followed by cyclisation with Cs₂CO₃ at 100 °C in DMF⁶⁷. The reaction of ethyl 3–(4-chlorophenyl)propiolate (59) with sodium azide in DMSO at 60 °C yielded 65⁶⁸. The reaction of 2-cyanoacetamide (60) with aryl benzaldehydes (55b, 55c) in the presence of sodium azide and Et₃N·HCl allowed the formation of triazoles 66 and 67⁶⁹.

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).

4-Aryl-tether-1,2,3-triazoles (Table 3) were synthesised according to Scheme 4. 4-Arylamino-1,2,3-triazole 78 resulted from treatment of 4-bromoaniline (69) with 2-aminoacetonitrile (70) and sodium nitrite in 2 M HCl at rt first, then by boiling in ethanol⁷¹. Analogues 82, 83, 85, and 86 were derived from the corresponding ethynyl derivative 77 (Scheme 5) and TMSN₃⁶³. Reaction of sulphide 83 with H₂O₂ in the presence of ammonium molybdate in methanol at rt produced sulphone 84⁷². O-methyl oxime 87 was obtained from the corresponding ketone 99 and O-methylhydroxylamine hydrochloride in H₂O:EtOH at 70 °C⁷³. The annulated derivative 89 was obtained by treatment of 1,2-indandione-2-oxime (71) with hydrazine hydrate and potassium hydroxide at 170–190 °C⁷⁴.

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).

5-Aroyl-1H-1,2,3-triazoles (Table 4) were obtained according to Scheme 6. Alkynylation of arenecarbaldehyde 90 with ethynylmagnesiumbromide in THF produced the corresponding arylpropargyl alcohol (91). The latter underwent dipolar cycloaddition with TMSN₃ in presence of CuI as catalyst (92a–92u). Oxidation with pyridinium chlorochromate (PCC) in dichloromethane gave 99–122^63,75,76. Compounds 103–105 were demethylated with 48% HBr in H₂O at 100 °C to produce the corresponding phenols 106–108. The alkynylation of 4-bromobenzaldehyde with 2-(prop-2-ynyloxy)tetrahydro-2H-pyran (93) in presence of n-BuLi in THF at −78 °C yielded the propargyl alcohol 94 after aqueous work-up. 94 was oxidised with MnO₂, followed by hydrolysation to 96 in the presence of pyridinium tosylate (PPTSA). Dipolar cycloaddition of 96 with TMSN₃ (CuI catalyst) in DMF yielded triazole 123, which was brominated into 124 by reaction with triphenylphosphine and CBr₄ in DMF at rt (Scheme 7A)^63,77,78. Triazolomethanamine 126 was obtained from 4-bromobenzoyl chloride and tert-butyl prop-2-yn-1-ylcarbamate (97), producing ynone 98 in the presence of PdCl₂(PPh₃)₂ as catalyst. 98 reacted with TMSN₃ (CuI catalyst), yielding carbamate 125. In the presence of trifluoroacetic acid in dichloromethane at rt, carbamate 125 provided the primary amine 126 (Scheme 7B)^63,79.

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).

The 3-aryl-1H-1,2,4-triazoles (Tables 5 and 6) were prepared according to Scheme 8. Compounds 134–154 and 158–161 were prepared by reaction of the respective carbonitrile 130 with formic acid and hydrazine hydrate in DMF at 90 °C (Scheme 8A)⁸⁰. The 1,2,4-triazoles 155–157 were obtained from benzamides 132a–132c through condensation with N,N-dimethylformamide dimethyl acetal (DMF-DMA), followed by cyclisation with hydrazine hydrate in acetic acid at 90 °C (Scheme 8B)^81,82. The corresponding carbonitriles and benzamides were prepared according to Scheme 9.

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).

Other 1,2,4-triazoles (Table 7) were prepared according to Scheme 10. 3-(4Bromophenylamino)-1,2,4-triazole (166) was synthesised by reaction of 1-bromo-4iodobenzene (163) with 1H-1,2,4-triazole-3-amine in the presence of CuI and K₂CO₃ in DMA at 90 °C⁸³. The annulated derivative 168 was synthesised by the S_N2 displacement of 2-chloroacetamide 165 using thiol 164 in the presence of K₂CO₃ in acetone⁸⁴.

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).

Compounds 9, 19, 21, 22, 29, 30, 31, 32, 33, 46, 47, 61, 81⁵⁸, 13, 40, 41, 42⁶², 11, 12, 136, 138, 169²⁸, and 10²² have been described previously by us. Compounds 79, 80, 88, 133, 139, 167, 170, and 171 were commercially available.

Results and discussion

Structural data

MMG-0472–bound structure

MMG-0472 (13, Figure 2) is a 4-phenyl-1,2,3-triazole featuring an extension on the phenyl ring, designed to be located in pocket B. We first described this compound in 2016, when we tested it in cellular assays for hIDO1 and for mIDO2 inhibition. MMG-0472 showed a high cytotoxicity (70% at 200 µM) and was not further pursued for this reason⁶². The mechanism behind this cellular toxicity remains to be clarified and could be due to solubility issues based on our experience with similar compounds. Here, we proceeded to test this compound in the enzymatic assay and found it to be one of the most potent azoles with B pocket extension with an enzymatic IC₅₀ value of 14 µM and a LE of 0.33 kcal/mol/HA.

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.

We also investigated compound 13 by X-ray crystallography and obtained diffracting crystals of its complex with IDO1 (PDB ID 7zv3). The 2F_o–F_c map of the IDO1 bound ligand clearly shows its electron density in both pockets A and B (Figure 2A). The ligand is non-planar, with the central bond between the triazole ring and the chloro-phenyl displaying a dihedral angle of 39°. The His346–iron–ligand bond angle is 170° (Figure 2B), deviating by 10° from its optimal value of 180° as determined by density-functional theory calculations on a haem model system (Figure 2C). This deviation probably reflects some strain in the complex. The resolved ligand structure is very close to our docking prediction (Figure 2D) with a root-mean-square distance (RMSD) of 0.3 Å.

As it can be appreciated from the top view and superimposition with the X-ray structures of triazoles 9, 11 and 12 (Figure 2D,E), the phenyl ring in compound 13 is rotated away from Ser167 and towards pocket B by about 20° with respect to the phenyl rings of the other compounds. Therefore, an additional 2-hydroxy substituent on the phenyl ring would not be favourable in these types of compounds, because they would produce a clash between the B-pocket extension and residues 261–265. The structural data for compound 13 thus confirms our previous analysis showing that azole ligands are not optimally suited to extend into pocket B²⁷.

Enzymatic activities

Enzymatic IC₅₀ values were measured with the ascorbate/methylene blue reduction system⁸⁵ in presence of a non-ionic detergent to reduce compound aggregation as described before²⁸. Kynurenine and L-Trp concentrations were determined by HPLC through UV detection. Dose-response curves and Hill slopes can be found in the Supplementary Information (Figure S2). Generally, IC₅₀ values are slightly lower here than in our first work on 1,2,3-triazoles⁵⁸, as we shortened the incubation time to stop the reaction in its linear phase. If compound solubility allowed, compounds were tested at concentrations up to 1 mM in presence of 5% of DMSO co-solvent.

4-Aryl-1,2,3-Triazoles with monosubstituted phenyl groups

We previously showed that 1,2,3-triazoles with para-substituted phenyl rings consistently had lower inhibitory activities on IDO1 than unsubstituted compounds, their IC₅₀ values increasing from 10 µM (H)²⁸ to 190 µM (F), 530 µM (Cl), 1 mM (CH₃) to above 1 mM (CF₃)⁵⁸ for non-polar substituents. Here, we synthesised and tested one compound with the polar p-OH substituent (20), which was inactive (Table 1). This result shows that, in agreement with what has been found for imidazoles⁴¹, also polar substituents in this position are unfavourable. This is in agreement with docking predictions, showing little space and hydrophobic groups surrounding para substituents (Figure 3A).

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.

On the other hand, we have previously found that non-polar substitutions in meta position increased the activity and were most favourable for chloride (0.35 µM)²⁸, while hydroxy (310 µM) and amino (>1 mM) substituents decreased activity⁵⁸. Similar effects were found for substituents in this position in imidazoles^24,41 and hydroxyamidines³⁰. Here, we tested more aliphatic substitutions of different sizes and found the ethyl substituent (22, 13 µM, Figure 3B)⁵⁸ to show the best activity, better than methyl (21, 22 µM)⁵⁸, n-butyl (24, 34 µM), and i-propyl (23, 280 µM, Table 1). For larger substituents, there is not enough space in this position, as suggested by the structural analysis.

Earlier investigations of ortho substitutions in the 4-phenyl-1,2,3-triazoles showed that only the hydroxy substituent substantially increased the activity (29, 2.3 µM), attributed to the formation of a hydrogen bond with Ser167 in the back of the binding site²⁸. Especially larger substituents, designed for targeting pocket B, showed substantially decreased inhibitory activities besides lowering the solubility of the compounds^58,62. Here, we show that usage of an ether (25, 26, 27, Figure 3C) or an amino linker (28) to a large substituent in this position does not yield active compounds either (Table 1). This is also true when introducing a hydroxy group (27), expected to increase solubility and allowing to test the inhibitor at higher concentrations. However, it should be noted that docking predicts that this hydroxy group cannot form favourable interactions within pocket B (Figure 3D).

4-Aryl-1,2,3-Triazoles with double and triple substituted phenyl groups

As described previously, the 2,5-disubstituted compound MMG-0358 (9, Figure 1, IC₅₀ = 0.059 µM, LE 0.76 kcal/mol/HA) displayed a very high inhibitory activity, explained by the interactions of its hydroxy subsituent with Ser167 and of its chloride substituent with the hydrophobic subpocket around Val125, Cys129, Leu234, and Gly262 as confirmed by X-ray crystallography (Figure 1)^22,58. Compounds 31, 32 and 46 with the 2-hydroxy substituent replaced by amino, methyl, or chloride substituents, were less active but still displayed low micromolar IC₅₀ values, while 33 with inverted polar/non-polar substituents (5-amino-2-methylphenyl instead of 2-amino-5-methylphenyl) was completely inactive⁵⁸. Here, we extended our investigation with 2-hydroxyphenyl derivatives bearing various 5-substituents. 4-Aryl-1,2,3-triazoles with small non-polar 5-substituents up to the size of propyl were inhibitors in the nanomolar range (34, 35, 36, 37, LE 0.72 to 0.56 kcal/mol/HA), while analogues with larger substituents such as 5–(2-benzyl) and 5–(2-phenylethyl) (38, 39, LE 0.26 to 0.29 kcal/mol/HA) showed lower activities (Table 1). In our docking approach, only substituents in 5-position up to the size of ethyl docked well (Figure 3E). Other 5-chlorophenyl analogues 2-substituted with longer alkyl chains have been described previously (13, 40, 41 and 42) but were tested only in a cellular environment on IDO1 and on IDO2⁶². Compound 42 inhibited both human IDO1 and murine IDO2 with a cellular IC₅₀ value of 110 µM. Here, we also measured their enzymatic activities and found them to have a low LE (0.24 to 0.36 kcal/mol/HA). We additionally synthesised and tested 4-[5-chloro-2–(2-phenylethoxy)phenyl]-1,2,3-triazole (43), which is also a micromolar inhibitor (31 µM, LE 0.29 kcal/mol/HA, Figure 3F). We also tested two 2,6-disubstituted compounds, 44 (13 µM) and 45 (no inhibition, NI). As in the case of imidazoles⁴¹, the 2,6-di-OH substituted compound is at least 2 orders of magnitude more active than the 2,6-di-OCH₃ substituted compound, but it is less active than the single OH-substituted compound 29.

Finally, we synthesised and tested a series of dichloro and trichloro substituted compounds. The most active compound was the 2,5-disubstituted compound 46 (6.4 µM), followed by 47 (23 µM), 48 (31 µM), 52 (33 µM), 49 (51 µM), 50 (58 µM), 51 (280 µM), and 53 (NI). In agreement with the docking predictions, all compounds are less active than the 5-Cl single-substituted compound (30, 0.35 µM)²⁸, and the third chloride substituent does not increase activity with respect to the di-substituted compounds.

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

Theoretically, access to the B-pocket could also be achieved through a substitution either of the N3 or the C5 atoms of the 1,2,3-triazole ring (Figure 1D). We explored the possibility of nitrogen substitutions before, without obtaining any active compound⁵⁸. This finding supports the importance of an ionisable NH group in the triazole and the hypothesis that deprotonation of the 1,2,3-triazole is crucial for IDO1 inhibition. Here, we further explored substitution of the C5 atom (Table 2), although this enforces iron binding through the N2 atom (Figure 1D), which has been calculated to be less favourable than binding through the N1 atom^28,58. We previously described one compound of this type with a 5-methyl substituent (61, NI)⁵⁸. Here, we show that an electron-withdrawing 5-nitrile substitution yielded a slightly active compound (62, 630 µM), while combination of the 5-methyl substituent with the 5-Cl substituent on the phenyl ring yielded an inactive compound (63, NI). Compounds with this substitution pattern have recently been further explored by Panda and co-workers⁵⁹, who described 11 compounds with nanomolar IC₅₀ values. We synthesised three of the compounds described in this work (original compound numbers: 1d, 64; 2 g, 65; and 3 g, 66) but could not reproduce the published IC₅₀ values. In our hands, compounds 64 and 65 showed no inhibition of IDO1, although their published IC₅₀ values were 2.51 µM and 0.39 µM, respectively⁵⁹. Compound 66, which has a published IC₅₀ value of 0.62 µM, showed a weak activity in our tests, with an IC₅₀ value of 800 µM. The lower activities might be due to higher purities of our compounds and are in line with weak activities measured for other compounds of this type (Table 2). Even combination with the highly activating 2-OH,5-Cl substitutions on the phenyl ring (67, 68) yielded at best a compound with an IC₅₀ value in the high micromolar range.

These compounds cannot meaningfully be docked into the IDO1 active site, the best poses displaying a triazole–haem angle deviating significantly from the optimal angle of 90° (Supporting Information, Figure S1).

4-Aryl-Tether-1,2,3-Triazoles and annulated derivatives

The 1,2,3-triazole published by Alexandre and co-workers (10, Figure 1), which features an amino linker between the triazole and the 4-chlorophenyl moiety, shows a peculiar behaviour^60,70,86. X-ray data demonstrates haem-iron binding and occupancy of pocket A (Figure 4A), but a slow shift of the haem Soret peak in UV-absorption spectra under reductive conditions coupled with haem unbinding and degradation as well as the largely superior activity in cellular assays as compared to enzymatic assays is very distinct from the behaviour of the 4-aryl-1,2,3-triazoles^22,60.

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.

Building on these results, we synthesised and tested 1,2,3-triazoles linked to aryl moieties by a one-center tether (N, CH₂, O, S, SO₂, CHOH) or two-atom tether (CONH, Table 3). In our hands, compound 10 displayed an IC₅₀ value of 56 µM (11.3 µM in the original publication). 4–(4-Bromophenylamino)-1,2,3-triazole (78) showed an inhibitory activity of 52 µM, whereas the 4-phenylamino analogue (79) was not active. These 3 latter compounds dock well into the IDO1 active site, although the phenyl ring adopts a slightly rotated conformation with respect to the X-ray structure (Figure 4B). Compound 80 with an additional phenyl substituent on the C5 of the triazole ring rather docks with the amino linker pointing towards the haem propionate and pocket B but shows no activity in the enzymatic assay (NI, Table 3, Figure 4C), similar to other C5-substituted 1,2,3-triazoles (Table 2).

Replacement of the amino linker by a methylene group (81, NI), by an ether group (82, NI), by a sulphide group (83, 590 µM, Figure 4D), by a sulfoxide moiety (84, NI), by a hydroxymethylene group (85, NI) or by a carboxamide group (86, NI, Figure 4E) generated inactive compounds except for 83 with the sulphur tether (590 µM). Compound 87 with a MeON=C linker was synthesised and tested as another possible access point to pocket B but was inactive. The annulated 1,2,3-triazoles 88 and 89 were also inactive. More interesting results were obtained with 5-aroyl-1,2,3-triazoles (keto linker), preserving the conjugation and the planarity between the aromatic rings (Table 4). Docking results for compound 99 (430 µM) suggest that this compound can form a hydrogen bond with Ser167 (Figure 4F), while the keto group simultaneously increases the acidity of the triazole ring. Starting from this 5-benzoyl-1,2,3-triazole scaffold, we explored different single and double substitutions of the phenyl ring, heterocyclic replacements of the phenyl ring, and 4-substitutions of the 1,2,3-triazole ring. Chloro substitutions in 2-position (100, NI), 3-position (101, 450 µM) and 4-position (102, 31 µM) of the phenyl ring showed that it was favourable only in the latter, in agreement with the analogous amino-1,2,3-triazole and the docking predictions (Figure 4G)⁶⁰. Similar trends were observed for methoxy substitution in 2-position (103, NI), 3-position (104, 990 µM), and 4-position (105, 200 µM). On the other hand, hydroxy substitutions in 2-position (106, 125 µM), 3-position (107, NI), and 4-position (108, NI) showed that it was favourable only in 2-position. In 4-position, substituent influence was the following: Br (109, 13 µM) > Cl (102, 31 µM) > F (110, 185 µM) > OMe (105, 200 µM) > OH (108, NI), similar to what has been found for the 5-position in the 4-phenyl-1,2,3-triazoles. Combination of 2-OH with the 5-Cl substitutions yielded only a moderately active compound (111, 365 µM), as expected by the structure-activity relationship (SAR) of the chloro substituents, but combination of 2-OH with 4-Br (113, 8.2 µM, LE 0.46 kcal/mol/HA) and with 4-Cl (112, 11 µM, LE 0.45 kcal/mol/HA) substantially increased the activities of singly substituted compounds and provided the most active compounds of this type, which remained, however, only in the low micromolar range. In the docked poses, the hydroxy groups of these compounds form a hydrogen bond to Gly262 (Figure 4H). Replacement of the phenyl ring by 5 or 6-membered aromatic heterocycles was mostly beneficial with 5-membered rings (115, 16 µM, Figure 4(I); 116, 17 µM; 117, 67 µM; 118, 81 µM; 114, 230 µM; 119, 515 µM), reaching a maximal LE of 0.55 kcal/mol/HA for compound 115. Replacement by 2–(120, NI), 3–(121, 650 µM), and 4-pyridine (122, 1200 µM), however, was detrimental. Substitution of the C4 atom of the 1,2,3-triazole with the aim to reach pocket B were explored combining them with the 4-Br substituted phenyl ring. However, all substitutions, including hydroxymethyl (123, NI), aminomethyl (126, NI), bromomethyl (124, NI), or larger groups (data not shown) yielded inactive compounds, in agreement with the docking predictions, which do not find low-energy poses for these compounds inside the active site.

In summary, we were able to develop original 5-aroyl-1,2,3-triazoles with enzymatic activities in the low micromolar range. However, they did not provide access to pocket B.

3-Aryl-1,2,4-Triazoles

Since we did not find a 1,2,3-triazole providing a convenient access to pocket B while preserving efficiency and potency, we turned in the following to the 1,2,4-triazoles²⁸. For this scaffold, we found previously that 3–(2-aminophenyl)-1,2,4-triazole (139) displayed an interesting inhibitory activity of 2 µM (Table 5). This was better than with the 3–(2-hydroxyphenyl) derivative (138, 31 µM), preferred by the 1,2,3-triazoles. Based on X-ray crystallography and molecular modelling studies, we attributed these results to the simultaneous inter- and intra-molecular hydrogen bonds of the 2-amino substituent in the 1,2,4-triazole, leading to a more favourable planar conformation (Figure 5A). Most important, combination with the 5-chloro substituent (11, 0.035 µM) or the 5-bromo substituent (12, 0.020 µM) yielded highly active compounds with excellent LE of 0.78 and 0.80 kcal/mol/HA, respectively²⁸.

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.

Here, we started by synthesising and testing the para-fluorophenyl derivative (134), which was was found inactive and deterred us from preparing more para-substituted phenyl derivatives. Based on the knowledge collected for 1,2,3-triazoles, we tested halogen substitutions in meta position. We found that the bromo analogue (135, 16 µM) was more active than the chloro (136, 29 µM) and the fluoro (137, 160 µM) substituted compounds. This was expected based on the size of the hydrophobic subpocket in this region and in agreement with the activities found for 1,2,3-triazoles. A 2,6-diamino substituted compound (142, 31 µM, Figure 5C) showed decreased activity with respect to the singly substituted 2-amino compound (139, 2.0 µM). The 2-NH₂,5-F compound was consistently less active (143, 0.15 µM) than its other halogenated counterparts (compounds 11, 12).

We synthesised and tested two compounds with 2-amino,3-halo disubstituted phenyl rings. As expected from our structural analysis, 140 (0.81 µM) and 141 (29 µM, Figure 5D) are much less active than their 2,5-disubstituted counterparts (Table 5). These compounds cannot offer any synergic effects from interactions of the amino group with Ser167 and the triazole ring on the one hand and the interactions of the halogen with the hydrophobic subpocket on the other hand. Methyl substitution of the amino group of compound 11 led to a lower inhibitory activity (145, 8.6 µM). This is in agreement with the structural data and the finding that both hydrogen atoms of the amino group serve as hydrogen bond donors²⁸.

We also synthesised and tested triple-substituted 3-phenyl-1,2,4-triazoles all having a 2-amino substitution of the phenyl ring. Adding a 4-F substitution to compound 12 only slightly perturbs its activity (144, 0.034 µM). However, adding a 6-methyl substitution to either compound 12 (155, 63 µM, Figure 5E) or 11 (156, 90 µM) reduced the inhibitory activities by more than 3 orders of magnitude. Adding a 6-amino group as in compound 157 (1 µM) also strongly reduced the inhibitory activity as compared to compound 11 (0.035 µM). This mirrors the results obtained with 2-aminophenyl 139 and 2,6-diaminophenyl derivative 142 and leaves little chance to develop analogues of this type extending to pocket B.

Finally, we investigated amide extensions of the ortho-amino group with the hope that the latter could reach pocket B, although structural and functional data suggested this amino group to increase activity by being located in the back of the active site and making two simultaneous hydrogen bonds (Figure 5A). We generally found that amide extensions reduced the compound solubility. With 3–(2-benzamidophenyl)-1,2,4-triazole (152), we lost the inhibitory activity completely (Figure 5F). Curiously, introduction of a 5-Cl substituent restored some of the inhibitory activity (153, 9.4 µM, Table 5) albeit at low solubility. Interestingly, the cyclopropanecarboxamide (149, 0.49 µM) and the propenamide (147, 1.2 µM) showed the best inhibitory activities of this series. However, the related cyclobutanecarboxamide (150, NI) and cyclopentanecarboxamide (151, NI) were inactive as inhibitors and showed strongly reduced solubilities. In the aliphatic series, the butanamide (148, 24 µM) was better than the acetamide (146, 425 µM). Aromatic extensions such as the benzamide (153, 9.4 µM) led to lower solubilities. However, the latter could be improved by introducing polar groups. Unfortunately, the inhibitory activities of resulting compounds were only in the high micromolar range (data not shown). Summarising, the observed inhibitory activities of the 1,2,4-triazoles bearing 2-amido substituents cannnot be rationalised based on structural data. Investigations are rendered complicated by the low solubilities of these compounds.

3-Hetaryl-1,2,4-Triazoles

Replacement of the phenyl ring of parent compound 133 by 4-pyridinyl (158, NI, Table 6) and by 2-pyridinyl (159, NI) produced inactive compounds. However, 3–(3aminopyridinyl)-1,2,4-triazole (160, 15 µM, LE 0.55 kcal/mol/HA, Figure 5G) and 3-(3-aminothiophenyl)-1,2,4-triazole (161, 10 µM, LE 0.57 kcal/mol/HA) were low micromolar inhibitors with a similar inhibitory activity as the aniline 139. Some activity was also found with the chloro-pyrazine derivative 162 (250 µM, LE 0.41 kcal/mol/HA). In summary, it is possible to replace the phenyl ring by 5 or 6-membered heteroaromatics, but up to now, no compounds with better activities than the 3-aryl analogues have been found.

Other 1,2,4-Triazoles

To complete our investigation with 1,2,4-triazoles as IDO1 inhibitors, we also tested the derivatives shown in Table 7. 3–(4-Bromophenylamino)-1,2,4-triazole (166, NI), an analogue of the 1,2,3-triazole derivative 78, was found to be inactive. This is not surprising, as 78 only shows an IC₅₀ value of 52 µM, and the 1,2,4-triazoles are generally less active than the corresponding 1,2,3-triazoles. The annulated antifungal tryclazole (167, NI) and its derivative (168, NI, Figure 5H), designed to extend into pocket B, were inactive, although they dock well into the active site. Their inactivity is probably due to their low iron affinity. We have shown before that 4-phenyl-1,2,4-triazole (169, NI) was inactive⁵⁸. However, as we predicted based on quantum chemical calculations²⁸, 1-phenyl-1,2,4-triazole (170, 870 µM) showed some activity, which could further be increased by adding an ortho-hydroxysubstituent to the phenyl ring (171, 32 µM, Figure 5I), making it about equipotent to the 3-phenyl-1,2,4-triazole 138. Interestingly, this compound binds to IDO1 in its neutral form, as the 1,2,4-triazole cannot be deprotonated, and might therefore display different properties than the derivatives of 133, which may bind stronger in their deprotonated form to IDO1.

Cellular activities

We tested the cellular hIDO1 inhibitory activity and toxicity of 30 active compounds at a single concentration (Figure 6 and Table 8). As some of the aniline derivatives reacted with Ehrlich’s reagent (p-dimethylaminobenzaldehyde, p-DMAB) used to quantify kynurenine, the kynurenine content of these samples was determined by HPLC. To be able to detect also weak inhibition, compounds were tested at the high concentration of 200 µM (Figure 6A and B, filled bars), except for less soluble compounds (52, 148, 149 and 153), which were tested at a concentration of 50 µM (Figure 6A and B, empty bars). Many compounds showed a good cellular inhibition (Figure 6A) and a low toxicity (Figure 6B). It is noticeable that for the compounds tested here, the 1,2,4-triazoles (orange) generally showed a better cellular inhibition than the 1,2,3-triazoles (black) but also a detectable albeit low toxicity. However, it should be kept in mind that previously tested 1,2,3-triazoles also showed a very good cellular inhibition⁵⁸, and that the toxicity of the 1,2,4-triazoles occurs at a concentration of 200 µM, far above their cellular IC₅₀ value.

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).

Previously determined cellular data for compounds mentioned in this work is given in the Supporting Information, Table S2. Both previously and in the present work, cellular inhibition was observed to be closely related to enzymatic activity for both 1,2,3-triazoles and 1,2,4-triazoles, showing a sigmoidal dependence (Figure 6C). Three outliers show a lower cellular activity than expected, namely two double-substituted 5-aroyl-1,2,3-triazoles (112, 113) and the fluorinated 1,2,4-triazole 143. On the other hand, three compounds show a higher cellular activity, namely compound 78 of the Vertex scaffold, for which this behaviour has already been documented⁶⁰, the amide 1,2,4-triazole 146, which might be hydrolized to the highly active 11 inside the cell, and the 1,2,3-triazole 42 for unknown reasons.

Here, we determined cellular IC₅₀ values for a selection of three 1,2,4-triazoles (Figure 6D), and found two of them to display nanomolar activities also in a cellular context. As in the enzymatic assay, the most potent compound is 144 (cellular IC₅₀ value of 0.034 µM), followed by 143 (0.25 µM) and 140 (1.2 µM).

We previously found a good correlation between enzymatic and cellular IC₅₀ values for different azole compounds^28,58. Here, we show this correlation (Figure 6E) for all 1,2,3-triazole (black) and 1,2,4-triazole (orange) compounds mentioned in this manuscript. The newly determined cellular IC₅₀ values of the 1,2,4-triazoles closely follow this correlation. The only outlier is the original Vertex compound 10⁶⁰.

Conclusions

In summary, here we described almost 100 new compounds of the 1,2,3-triazole and the 1,2,4-triazole haem-binding series and tested them for their inhibitory activity on IDO1. They provide highly efficient scaffolds for inhibitors binding to pocket A, which are also very potent in a cellular environment and display low cytotoxicities. The best compound (144) displays both enzymatic and cellular IC₅₀ values of 34 nM and is therefore more potent and efficient in vitro than other frequently used IDO1 inhibitors. We did not measure the activities of these compounds on IDO2 and on TDO to determine their selectivity. However, in our earlier works we found that triazoles such as MMG-0358 with high activities on IDO1 have undetectable activities on TDO (>100 μM)⁵⁸. We also found that MMG-0358 has a more than1000-fold selectivity for IDO1 over IDO2, even though triazoles extending from pocket A to pocket B showed better activity on mouse IDO2 than on human IDO1 in a cellular environment⁶². Based on these observations, it is reasonable to assume that the potent compounds reported here, which are binding only to pocket A, are likely to be highly selective for IDO1 over TDO and IDO2.

Unfortunately, extending these highly efficient compounds into pocket B by one of the attachment points described in Figure 1(D) proved very challenging. We were able to resolve the X-ray structure of the complex of one such compound extending into pocket B (MMG-0472, PDB ID 7zv3). The experimental structure confirms our docking predictions of the binding mode of this compound. However, docking predictions in general are challenging due to the interactions with the haem cofactor in the active site, which is difficult to parameterise classically. For future design of type ii or type iii IDO1 inhibitors, we recommend tackling the issue of addressing pockets A and B simultaneously early on in hit-to-lead optimisation to provide more flexibility for rational compound modifications.

Experimental section

Docking

Docking was performed with our in-house docking code AttractingCavities (AC)⁸⁷, which relies on the physical scoring function of the CHARMM27 force field^88,89, while solvation effects are taken into account by the Fast Analytical Treatment of Solvation (FACTS) model⁹⁰, which has been shown to allow for accurate docking results⁹¹. Ligand force-field parameters were derived with the SwissParam tool⁹². Standard parameters were used, i.e. a cubic search space of 20 ų around the IDO1 active site, a rotational angle of 90° for initial ligand sampling, and a N_Thr value of 70 for determination of the attractive grid points. For all compounds with an acidic proton, both the neutral and the deprotonated species were docked, and different tautomers were considered. A Morse-like metal binding potential (MMBP) was used to describe the interactions between the haem iron of IDO1 and ligand atoms that display a free electron pair for iron binding⁹³. The protein was kept fixed during the docking. We used seven different IDO1 X-ray structures as targets, chosen for their quality and diversity, namely PDB ID 2d0t⁴⁰ co-crystallized with a small azole ligand in pocket A, 5whr²⁵ with a resolved JK-loop^C in closed conformation, 6e41⁹⁴ with an analogue of epacadostat in pockets A and B, 6kof⁵¹ with a large azole ligand in pockets A and B, 6o3i²⁴ with the clinical compound navoximod, 6pu7³² with a hydroxyamidine ligand of peculiar shape, and 7ah4²⁸ with two small azole ligands in pockets A and D. All ligands were removed before docking.

Density functional theory calculations

Quantum chemical geometry optimizations and charge calculations were carried out in the density functional theory (DFT) framework with the PBE0 hybrid functional⁹⁵ using the Gaussian16 code⁹⁶. Geometry optimizations were carried out with standard settings and the TZVP basis set⁹⁷. Solvation effects were taken into account by the polarisable continuum model⁹⁸ as implemented in Gaussian16. The histidine-bound haem complex of IDO1 was modelled by an iron–porphin–imidazole system. For the 6-fold coordinated systems, a low-spin complex was assumed, as it has been found experimentally³⁹.

Chemistry

General remarks

All reactions were carried out under nitrogen atmosphere unless otherwise stated. Glassware was oven-dried (120 °C), evacuated and purged with nitrogen. Any common reagents, catalysts and solvents that were obtained from commercial suppliers were used without any further purification. For extraction and chromatography, all solvents were distilled prior to use. Thin layer chromatography (TLC) for reaction monitoring was performed on silica gel plates (Merck 60 F254) with detection by UV light (254 nm). Flash chromatography (FC) was conducted using silica gel 60 Å, 230–400 mesh (Merck 9385). Mass spectra were recorded on a Nermag R10-10C instrument in chemical ionisation mode. Electrospray mass analyses were recorded on a Finnigan MAT SSQ 710 C spectrometer in positive ionisation mode. ¹H and ¹³C NMR spectra were recorded with a Bruker-DPX-400 or Bruker-ARX-400 spectrometer at 400 MHz and 100.6 MHz, respectively. Data for ¹H NMR spectra are reported as follows: chemical shift, multiplicity, apparent coupling constant, and integration. Data for ¹³C NMR spectra reported in terms of chemical shift. Chemical shifts are given in parts per million, relative to an internal standard such as residual solvent signals. Coupling constants are given in Hertz. Spectra were analysed with MestreNova. High resolution mass spectra were recorded via ESI-TOF-HRMS or MALDI-TOF-HRMS.

The purity of all final compounds was confirmed to exceed 95% by ¹H NMR showing ¹³C-H satellite signals. Additionally, analytical HPLC purity analysis was carried out for compounds 34, 113, 143 and 149 with UV detection at 220 nm, using a PFP propyl column (RESTEK Allure HPLC column, particle size 5 µm, pore size 60 Å, dimensions 150 × 4.6 mm) with a linear gradient of solvent B (acetonitrile, 0.1% TFA) over solvent A (H₂O, 0.1% TFA) from 0 to 100% in 30 min at a flow rate of 1 mL/min. Typically, 25 µL solution (0.5 mg/mL in 50% ACN/H₂O) was injected for each compound.

Procedures

Synthesis of iodo derivatives

4-Benzyl-2-iodophenol (17a)⁹⁹: 4-Benzylphenol (14a) (1.1 g, 6.0 mmol) was dissolved in DMF (10 mL), diluted with concentrated aqueous ammonia (60 mL), and treated with a solution of potassium iodide (5.3 g, 32 mmol) and iodine (1.6 g, 6.3 mmol) in water (20 mL) all at once. After stirring at rt for 1 h, the ammonia was removed under reduced pressure. The remaining solution was neutralised with 1 N HCl in H₂O. After extraction with EtOAc (50 mL), the organic phases were collected, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) giving 17a (930 mg, 50% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 7.50 (d, J = 2.1 Hz, 1H), 7.35 − 7.23 (m, 2H), 7.18 (td, J = 6.5, 1.7 Hz, 3H), 7.01 (dd, J = 8.3, 2.2 Hz, 1H), 6.76 (d, J = 8.2 Hz, 1H), 3.85 (s, 2H).

1-(Benzyloxy)-2-iodobenzene (17ba)¹⁰⁰: To a solution of 2-iodophenol (14b) (440 mg, 2.0 mmol) in DMF (6 mL), anh. K₂CO₃ (1.38 g, 10.0 mmol) was added, and the mixture was stirred for 5 min before dropwise addition of benzyl bromide (15a) (374 mg, 2.2 mmol). The mixture was stirred at 80 °C for 24 h. After cooling to rt, CH₂Cl₂ (10 mL) and water (10 mL) were added and the mixture was shaken vigorously before extraction with CH₂Cl₂ (2 × 10 mL). The organic layers were collected, washed with brine (2 × 10 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude residue of 17ba (527 mg, 85% yield) was used directly in the next step. ¹H NMR (400 MHz, CDCl₃) δ 5.12 (s, 2H), 6.75 (dt, J = 7.6, 1.3 Hz,1H, H-5), 6.87 (dd, J = 8.2, 1.1 Hz, 1H, H-6), 7.29 (ddd, J = 8.2, 7.4,1.5 Hz, 1H, H-4), 7.33–7.46 (m, 3H), 7.55 (d, J = 7.3 Hz, 2H), 7.85 (dd, J = 7.8, 1.6 Hz).

1-Iodo-2-phenethoxybenzene (17bb) (CAS [1104274–16-9]): Compound 17bb was prepared the same way as 17ba from 2-iodophenol (14b) (440 mg, 2.0 mmol) and (2-bromoethyl)benzene (15b) (405 mg, 2.2 mmol) in DMF (6 mL), giving 17bb (583 mg, 90% yield) as colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.77 (dd, J = 7.8, 1.6 Hz, 1H), 7.42 − 7.30 (m, 4H), 7.34 − 7.21 (m, 2H), 6.78 (dd, J = 8.3, 1.3 Hz, 1H), 6.70 (td, J = 7.5, 1.4 Hz, 1H), 4.21 (t, J = 6.9 Hz, 2H), 3.18 (t, J = 6.9 Hz, 2H).

2-Iodo-N-phenethylaniline (17cb)¹⁰¹: Compound 17cb was prepared the same way as 17ba from 2-iodoaniline (14c) (440 mg, 2.0 mmol) and (2-bromoethyl)benzene (15b) (405 mg, 2.2 mmol) in DMF (6 mL), giving 17cb (323 mg, 50% yield) as colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.65 (dt, J = 7.9, 1.5 Hz, 2H), 7.39 − 7.18 (m, 1H), 7.18 − 7.10 (m, 2H), 6.76 (dd, J = 8.0, 1.5 Hz, 2H), 6.53 − 6.41 (m, 2H), 4.45 − 4.30 (m, 2H), 2.99 (q, J = 6.8 Hz, 2H).

1-Chloro-3-iodo-2-phenethoxybenzene (17db): Compound 17db was prepared the same way as 17ba from 2-chloro-6-iodophenol (14d) (508 mg, 2.0 mmol) and (2-bromoethyl)benzene (15b) (405 mg, 2.2 mmol) in DMF (6 mL), giving 17db (680 mg, 95% yield) as colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.68 (dd, J = 7.9, 1.5 Hz, 1H), 7.40 − 7.29 (m, 4H), 7.27 (s, 1H), 7.24 (ddt, J = 8.5, 5.3, 2.5 Hz, 1H), 6.78 (t, J = 7.9 Hz, 1H), 4.21 (t, J = 7.4 Hz, 2H), 3.25 (t, J = 7.4 Hz, 2H).

2–(2-Iodophenoxy)-1-phenylethan-1-ol (17bc)¹⁰²: 2-Phenyloxirane (16) (580 mg, 4.8 mmol, 1.2 eq), 2-iodophenol (14b) (880 mg, 4.0 mmol, 1.0 eq), and Cs₂CO₃ (31.9 g, 12 mmol, 3.0 eq) were added to a 100-mL 2-neck round-bottom flask equipped with a condenser and a septum. DMF (8 mL) was added, and the mixture was heated under reflux (110 °C) for 24 h. After cooling to rt, water (10 mL) was added, followed by extraction with EtOAc (3 × 15 mL). The organic layers were collected, dried over anh. Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to give 17bc (0.98 g, 60% yield) as colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (dd, J = 7.8, 1.6 Hz, 1H), 7.54 − 7.47 (m, 2H), 7.46 7.25 (m, 3H), 6.84 − 6.72 (m, 2H), 5.21 (dt, J = 8.7, 2.9 Hz, 1H), 4.21 (dd, J = 9.3, 3.3 Hz, 1H), 4.02 (t, J = 9.0 Hz, 1H), 3.14 (d, J = 2.6 Hz, 1H).

2-Iodo-1-methoxy-4-phenethylbenzene (17g): To a stirred solution of methyl(triphenyl)phosphoniumbromide (1.23 g, 6.7 mmol) in freshly distilled THF (9 mL), sodiumbis(trimethylsilyl)amide (2.19 g, 6.1 mmol) was added under cooling (ice bath), causing the solution to turn yellow. After 1.5 h of stirring at rt, 3-iodo-4-methoxybenzaldehyde (17e) (786 mg, 3.0 mmol) was added to the ylide solution and stirring was continued for 4 h. The mixture was acidified using H₂SO₄ (0.1 M, 5 mL) and extracted with CH₂Cl₂ (50 mL). The combined organic phases were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give 1.84 g (90% yield) of a mixture of (E/Z)-2-iodo-1-methoxy-4-styrylbenzene (17f) as a oily solid. ¹H NMR (400 MHz, MeOD) δ 7.99 (d, J = 2.2 Hz, 1H), 7.62 (d, J = 2.2 Hz, 1H), 7.55 (td, J = 8.3, 1.6 Hz, 3H), 7.35 (dd, J = 8.4, 6.9 Hz, 2H), 7.30 − 7.17 (m, 7H), 7.06 (d, J = 1.2 Hz, 2H), 6.96 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 6.58 (d, J = 12.1 Hz, 1H), 6.49 (d, J = 12.1 Hz, 1H), 3.90 (s, 3H), 3.84 (s, 3H). The crude olefin mixture (710 mg, 2.1 mmol) was dissolved in ethanol (12 mL) together with FeCl₃ ·6H₂O (29 mg, 0.11 mmol), immediately followed by the addition of aqueous hydrazine hydrate (1.0 mL, 21.0 mmol). The reaction mixture was stirred at rt for 24 h under air before extraction with CH₂Cl₂ (3 × 5 mL). The organic phased were collected, dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was purified by FC (SiO₂, 10% EtOAc/petroleum ether), affording 17g (640 mg, 90% yield) as oily solid. ¹H NMR (400 MHz, MeOD) δ 7.55 (d, J = 2.1 Hz, 1H), 7.29 − 7.03 (m, 6H), 6.82 (d, J = 8.4 Hz, 1H), 3.82 (s, 3H), 2.93 − 2.82 (m, 2H), 2.85 − 2.77 (m, 2H).

General procedure (I) for the preparation of arylethynes

To a stirred solution of commercially available or synthetically prepared iodo derivatives (17) (Scheme 1 and 2) (1 eq) mixed with Et₃N (4 eq) in dioxane (4 mL), trimethylsilylacetylene (1.3 eq), PdCl₂(PPh₃)₂ (0.01 eq), and CuI (0.02 eq) were added. The reaction mixture was stirred at 45 °C for 5 h under nitrogen atmosphere. After cooling to rt, the reaction mixture was diluted with Et₂O (5 mL) and washed with brine (5 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated under reduced pressure. KF (3.6 eq) was added to the residue and the mixture was dissolved in MeOH (5 mL) and stirred for 3 h at rt before concentration under reduced pressure. After addition of CH₂Cl₂ (5 mL) and water (3 mL), the organic layer was collected, dried over MgSO₄, and filtered through a short silica plug to afford the corresponding arylethynes 18 (Scheme 1). Overall yields 65–93%.

Synthesis of arylethynes

1-Butyl-3-ethynylbenzene (18a, CAS [2243191–48-0]): Synthesised from 1-butyl-3-iodobenzene (520 mg, 2 mmol) according to the general procedure (I) to afford the title compound as a yellowish oil (269 mg) in 85% yield, ¹H NMR (400 MHz, CDCl₃) δ 7.41 − 7.29 (m, 2H), 7.25 (td, J = 7.4, 0.8 Hz, 1H), 7.19 (dt, J = 7.7, 1.6 Hz, 1H), 3.07 (s, 1H), 2.66 − 2.57 (m, 2H), 1.68 − 1.56 (m, 2H), 1.37 (h, J = 7.4 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H).

1-(Benzyloxy)-2-ethynylbenzene (18b)¹⁰³: Synthesised from 1-(benzyloxy)-2-iodobenzene (620 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (291 mg) in 70% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.59 7.47 (m, 3H), 7.41 − 7.15 (m, 5H), 6.99 − 6.86 (m, 1H), 5.23 (s, 2H).

1-Ethynyl-2-phenethoxybenzene (18c): Synthesised from 1-iodo-2-phenethoxybenzene (648 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (333 mg) in 75% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.46 (dd, J = 7.6, 1.7 Hz, 1H), 7.39 − 7.31 (m, 4H), 7.31 − 7.23 (m, 1H), 6.95 − 6.83 (m, 3H), 4.24 (t, J = 7.1 Hz, 2H), 3.30 (s, 1H), 3.17 (t, J = 7.1 Hz, 2H).

2–(2-Ethynylphenoxy)-1-phenylethan-1-ol (18d): Synthesised from 2–(2-iodophenoxy)-1-phenylethan-1-ol (680 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (309 mg) with 65% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.53 − 7.26 (m, 7H), 6.97 (td, J = 7.5, 1.0 Hz, 1H), 6.88 (dd, J = 8.4, 1.0 Hz, 1H), 5.18 (dt, J = 9.1, 2.8 Hz, 1H), 4.24 (dd, J = 9.5, 3.1 Hz, 2H), 4.02 (t, J = 9.2 Hz, 2H), 3.34 (s, 1H).

2-Ethynyl-N-phenethylaniline (18e): Synthesised from 2-iodo-N-phenethylaniline (646 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (331 mg) in 75% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.40 − 7.18 (m, 7H), 6.69 − 6.59 (m, 2H), 4.72 (s, 1H), 3.46 (td, J = 7.1, 5.6 Hz, 2H), 3.34 (s, 1H), 2.96 (t, J = 7.2 Hz, 2H).

2-Ethynyl-4-fluorophenol (18f)¹⁰⁴: Synthesised from 4-fluoro-2-iodophenol (476 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (231 mg) in 85% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.13 − 6.97 (m, 2H), 6.91 (dd, J = 9.0, 4.6 Hz, 1H), 5.65 (s, 1H), 3.51 (s, 1H).

2-Ethynyl-4-methylphenol (18g)¹⁰⁴: Synthesised from 2-iodo-4-methylphenol (468 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (211 mg) in 80% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.20 (d, J = 2.2 Hz, 1H), 7.10 (dd, J = 8.4, 2.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 5.65 (s, 1H), 3.46 (s, 1H), 2.27 (s, 3H).

4-Ethynyl-2-methylphenol (18h): Synthesised from 4-ethyl-2-iodophenol (496 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (238 mg) in 90% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.35 − 7.20 (m, 1H), 7.13 (dd, J = 8.6, 2.2 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 5.66 (s, 1H), 3.46 (s, 1H), 2.58 (q, J = 7.7 Hz, 2H), 1.30 − 1.15 (t, J = 7.7 Hz, 3H).

2-Ethynyl-4-propylphenol (18i)¹⁰⁵: Synthesised from 2-iodo-4-propylphenol (524 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (202 mg) in 88% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.35 − 7.17 (m, 1H), 7.10 (dd, J = 8.4, 2.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 5.66 (s, 1H), 3.46 (s, 1H), 2.50 (t, J = 7.5 Hz, 2H), 1.75 − 1.54 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H).

4-Benzyl-2-ethynylphenol (18j): Synthesised from 4-benzyl-2-iodophenol (620 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (307 mg) in 93% yield. ¹H NMR (400 MHz, MeOD) δ 7.27 (dd, J = 8.7, 6.5 Hz, 2H), 7.21 − 7.12 (m, 4H), 7.03 (dd, J = 8.4, 2.3 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 4.63 (s, 1H), 3.85 (s, 2H), 3.57 (s, 1H).

2-Ethynyl-1-methoxy-4-phenethylbenzene (18k): Synthesised from 2-iodo-1methoxy-4-phenethylbenzene (676 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (378 mg) in 80% yield. ¹H NMR (400 MHz, MeOD) δ 7.29 − 7.03 (m, 7H), 6.88 (d, J = 8.5 Hz, 1H), 3.83 (s, 3H), 3.55 (s, 1H), 3.33 (p, J = 1.6 Hz, 2H), 2.93 − 2.78 (m, 2H).

1-Chloro-2-ethynyl-3-phenethoxybenzene (18 l): Synthesised from 1-chloro-2-iodo-3-phenethoxybenzene (676 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (476 mg) in 83% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.42 − 7.30 (m, 6H), 7.33 − 7.20 (m, 1H), 7.00 (t, J = 7.9 Hz, 1H), 4.39–4.31 (m, 2H), 3.26–3.16 (m, 3H).

1,2,4-Trichloro-5-ethynylbenzene (18m, CAS [6546–87-8]): Synthesised from 1,2,4-trichloro-5-iodobenzene (612 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (310 mg) in 76% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.63 (s, 1H), 7.55 (s, 1H), 7.29 (s, 1H), 3.46 (s, 1H).

1,3,5-Trichloro-2-ethynylbenzene (18n)¹⁰⁶: Synthesised from 1,3,5-trichloro-5-iodobenzene (612 mg, 2 mmol) according to the general procedure (I) to afford the title compound as an oil (326 mg) in 76% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (s, 2H), 3.73 (s, 1H).

General procedure (II) for the preparation of 4-Aryl-1,2,3-Triazoles:

To a stirred solution of commercially available or synthetically prepared arylethynes (18) (1 equiv) and CuI (0.05 equiv) in DMF/MeOH solution (2 mL, 9:1) under an argon atmosphere, TMSN₃ (1.5 equiv) was added. The resulting solution was stirred at 100 °C for 10–12 h. After consumption of the ethynyl substrate, the mixture was cooled to rt, the precipitate was filtered off, and the remaining solution was concentrated under reduced pressure. The crude residue was purified by FC (SiO₂, EtOAc/petroleum ether) to obtain the desired 4-aryl-1,2,3-triazole.

General procedure (III) for the demethylation of aryl methyl ethers:

The methyl ether (1 eq) was dissolved in 48% HBr in water (4 mL), and the orange solution was heated to 100 °C for 14 h under nitrogen atmosphere. The mixture was cooled to rt, diluted with water, and neutralised by the addition of a saturated aq. soln. of NaHCO₃ until the evolution of CO₂ ceased. The organic layer was extracted with EtOAc (2 × 5 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford a residue that was purified by FC (SiO₂, EtOAc/petroleum ether) to give the desired phenol.

Synthesis of 4-Aryl-1,2,3-Triazoles

4–(1,2,3-Triazol-4-yl)phenol (20)¹⁰⁷: Synthesised from 1-ethynyl-4-methoxybenzene (264 mg, 2.0 mmol) and TMSN₃ (345 mg, 3.0 mmol) using the general procedure (II) to give 4–(4-methoxyphenyl)-1,2,3-triazole (245 mg, 70%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.76 − 7.68 (m, 2H), 7.02 − 6.93 (m, 2H), 3.85 (s, 3H). Using the general procedure (III), 4–(4-methoxyphenyl)-1,2,3-triazole (166 mg, 1.0 mmol) dissolved in 48% HBr in water (4 mL) gave 20 (115 mg, 75%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.00 (s, 1H), 7.72 − 7.59 (m, 2H), 6.95 − 6.82 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 157.82, 127.06, 115.40, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈N₃O⁺ 162.0662; found, 162.0661.

4–(3-Isopropylphenyl)-1,2,3-triazole (23, CAS [2192187–16-7]): Synthesised from 1-ethynyl-3-isopropylbenzene (144 mg, 1.0 mmol) and TMSN₃ (173 mg, 1.5 mmol) using the general procedure (II) to give 23 (84 mg, 45%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (s, 1H), 7.71 (t, J = 1.8 Hz, 1H), 7.63 (dt, J = 7.7, 1.5 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.31 − 7.23 (m, 1H), 2.99 (p, J = 6.9 Hz, 1H), 1.31 (d, J = 6.9 Hz, 6H), ¹³C NMR (101 MHz, CDCl₃) δ 149.77, 129.65, 128.99, 127.00, 124.30, 123.71, 34.18, 23.95, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₁H₁₃N₃⁺ 188.1188, found: 188.1185.

4–(3-Butylphenyl)-1,2,3-triazole (24, CAS [2192187–22-5]): Synthesised from 1-butyl-3-ethynylbenzene (18a) (150 mg, 0.95 mmol) and TMSN₃ (164 mg, 1.43 mmol) using the general procedure (II) to give 24 (153 mg, 80%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.15 (s, 1H), 7.68 (d, J = 1.9 Hz, 1H), 7.64 (dt, J = 7.8, 1.5 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.21 (dt, J = 7.6, 1.5 Hz, 1H), 2.74 − 2.66 (m, 2H), 1.67 (tt, J = 9.2, 6.9 Hz, 2H), 1.41 (h, J = 7.4 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 143.49, 128.57, 128.34, 125.61, 123.03, 35.23, 33.44, 29.42, 22.01, 13.00, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₂H₁₆N₃⁺ 202.1339; found, 202.1342.

4–(2-Benzyloxyphenyl)-1,2,3-triazole (25)¹⁰⁸: Synthesised from 1-(benzyloxy)-2-ethynylbenzene (18b) (104 mg, 0.5 mmol) and TMSN₃ (87 mg, 0.75 mmol) using the general procedure (II) to give 25 (90 mg, 72%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.08 (s, 1H), 7.51 − 7.42 (m, 2H), 7.43 − 7.28 (m, 5H), 7.18 (dd, J = 8.4, 1.1 Hz, 1H), 7.06 (td, J = 7.5, 1.1 Hz, 1H), 5.23 (s, 2H), ¹³C NMR (101 MHz, CDCl₃) δ 155.20, 135.99, 130.49, 129.97, 128.98, 128.61, 128.22, 127.86, 121.64, 112.64, 70.91, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₅H₁₄N₃O⁺ 252.1131; found, 252.1131.

4–(2-Phenethoxyphenyl)-1,2,3-triazole (26): Synthesised from 1-ethynyl-2-phenethoxybenzene (18c) (111 mg, 0.5 mmol) and TMSN₃ (87 mg, 0.75 mmol) using the general procedure (II) to give 26 (93 mg, 70%) as a white solid, m.p. 91–93 °C. ¹H NMR (400 MHz, CDCl₃) δ 12.03 (s, 1H), 7.98 (s, 1H), 7.81 (s, 1H), 7.46 − 7.28 (m, 6H), 7.12 − 7.02 (m, 2H), 4.44 (t, J = 6.6 Hz, 2H), 3.25 (t, J = 6.6 Hz, 2H), ¹³C NMR (101 MHz, CDCl₃) δ 155.12, 137.75, 129.92, 129.01, 128.74, 128.28, 127.04, 121.41, 112.15, 69.00, 35.61, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₆H₁₆N₃O⁺ 266.1288; found, 266.1290.

2–(2-(1H-1,2,3-triazol-5-yl)phenoxy)-1-phenylethan-1-ol (27): Synthesised from 2(2-ethynylphenoxy)-1-phenylethan-1-ol (18d) (120 mg, 0.5 mmol) and TMSN₃ (87 mg, 0.75 mmol) using the general procedure (II) to give 27 (91 mg, 65%) as a white solid, m.p. 153–155 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.01 (s, 1H), 7.69 (dd, J = 7.7, 1.6 Hz, 1H), 7.50 − 7.42 (m, 2H), 7.42 − 7.28 (m, 4H), 7.07 (td, J = 7.6, 1.1 Hz, 1H), 6.99 (dd, J = 8.3, 1.1 Hz, 1H), 5.20 (dd, J = 9.0, 3.1 Hz, 1H), 4.38 (dd, J = 9.5, 3.2 Hz, 1H), 4.08 (t, J = 9.3 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 156.02, 142.09, 130.21, 128.94, 128.37, 126.99, 121.79, 113.38, 73.96, 72.68, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₆H₁₅N₃O₂Na⁺ 304.1056; found, 304.1064.

N-Phenyl-2-(1H-1,2,3-triazol-5-yl)aniline (28): Synthesised from 2-ethynyl-N-phenethylaniline (18e) (165 mg, 0.75 mmol) and TMSN₃ (130 mg, 1.13 mmol) using the general procedure (II) to give 28 (80 mg, 45%) as a white solid, m.p. 129–132 °C. ¹H NMR (400 MHz, MeOD) δ 8.03 (s, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.31 − 7.13 (m, 6H), 6.83 (dd, J = 8.4, 1.1 Hz, 1H), 6.70 (td, J = 7.5, 1.1 Hz, 1H), 3.49 (t, J = 7.0 Hz, 3H), 2.96 (t, J = 7.0 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 145.50, 139.57, 130.69, 129.11, 128.48, 128.03, 127.78, 125.85, 115.73, 111.04, 44.81, 35.03, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₆H₁₇N₄⁺ 265.1448; found, 265.1441.

4-Fluoro-2-(1H-1,2,3-triazol-5-yl)phenol (34): Synthesised from 2-ethynyl-4-fluorophenol (18f) (204 mg, 1.5 mmol) and TMSN₃ (259 mg, 2.25 mmol) using the general procedure (II) to give 34 (201 mg, 75%) as a white solid, m.p. 186–188 °C. ¹H NMR (400 MHz, MeOD) δ 8.31 (s, 1H), 7.62 (dd, J = 9.6, 2.8 Hz, 1H), 7.04 − 6.86 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 157.53, 155.20, 150.77, 116.82, 116.73, 115.37, 115.14, 112.71, 112.46, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇FN₃O⁺ 180.0568; found, 180.0567.

4-Methyl-2-(1H-1,2,3-triazol-5-yl)phenol (35): Synthesised from 2-ethynyl-4-methylphenol (18g) (170 mg, 1.3 mmol) and TMSN₃ (225 mg, 1.95 mmol) using the general procedure (II) to give 35 (171 mg, 75%) as a white solid, m.p. 160–162 °C. ¹H NMR (400 MHz, MeOD) δ 8.24 (s, 1H), 7.69 − 7.58 (m, 1H), 7.04 (dd, J = 8.3, 2.3 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 2.32 (s, 3H), ¹³C NMR (101 MHz, MeOD) δ 152.36, 129.91, 128.66, 127.09, 115.80, 114.63, 19.18, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₉H₁₀N₃O⁺ 176.0818; found, 176.0817.

4-Ethyl-2-(1H-1,2,3-triazol-5-yl)phenol (36): Synthesised from 4-ethyl-2-methylphenol (18h) (146 mg, 1.0 mmol) and TMSN₃ (173 mg, 1.5 mmol) using the general procedure (II) to give 36 (151 mg, 80%) as a white solid, m.p. 71–73 °C. ¹H NMR (400 MHz, MeOD) δ 8.24 (s, 1H), 7.66 (s, 1H), 7.07 (dd, J = 8.3, 2.2 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 2.63 (q, J = 7.6 Hz, 2H), 1.26 (td, J = 7.7, 1.2 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 152.56, 135.37, 128.73, 126.00, 115.83, 114.74, 114.68, 27.63, 15.08, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₀H₁₂N₃O⁺ 190.0975; found, 190.0977.

4-Propyl-2-(1H-1,2,3-triazol-5-yl)phenol (37): Synthesised from 2-ethynyl-4-propylphenol (18i) (160 mg, 1.0 mmol) and TMSN₃ (173 mg, 1.5 mmol) using the general procedure (II) to give 37 (152 mg, 75%) as a white solid, m.p. 138–140 °C. ¹H NMR (400 MHz, MeOD) δ 8.24 (s, 1H), 7.64 (s, 1H), 7.05 (dd, J = 8.3, 2.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 2.65 − 2.50 (m, 2H), 1.67 (h, J = 7.4 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 152.60, 133.70, 129.38, 126.63, 115.78, 114.68, 36.80, 24.52, 12.72, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₁H₁₄N₃O⁺ 204.1131; found, 204.1130.

4-Benzyl-2-(1H-1,2,3-triazol-5-yl)phenol (38): Synthesised from 4-benzyl-2-ethynylphenol (18j) (460 mg, 2.2 mmol) and TMSN₃ (379 mg, 3.3 mmol) using the general procedure (II) to give 38 (441 mg, 80%) as a white solid, m.p. 148–151 °C. ¹H NMR (400 MHz, MeOD) δ 8.23 (s, 1H), 7.75 − 7.65 (m, 1H), 7.32 − 7.13 (m, 5H), 7.05 (dd, J = 8.4, 2.3 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 3.95 (s, 2H), ¹³C NMR (101 MHz, MeOD) δ 152.92, 141.64, 132.63, 129.81, 128.40, 128.04, 127.20, 125.58, 115.97, 115.01, 40.57, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₅H₁₄N₃O⁺ 252.1131; found, 252.1137.

4-Phenylethyl-2-(1H-1,2,3-triazol-5-yl)phenol (39): Synthesised from 2-ethynyl-1-methoxy-4-phenethylbenzene (18k) (544 mg, 2.3 mmol) and TMSN₃ (398 mg, 3.45 mmol) using the general procedure (II) to give 5–(2-methoxy-5-phenethylphenyl)-1H-1,2,3-triazole (282 mg, 44%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.15 (s, 1H), 7.79 (s, 1H), 7.30 − 7.22 (m, 2H), 7.22 − 7.11 (m, 4H), 7.02 (d, J = 8.5 Hz, 1H), 3.95 (s, 3H), 2.96–2-90 (m, 4H). Using the general procedure (III), 5–(2-methoxy-5phenethylphenyl)-1H-1,2,3-triazole (246 mg, 1.2 mmol) dissolved in 48% HBr in water (5 mL) gave 39 (238 mg, 75%) as a white solid, m.p. 157–159 °C. ¹H NMR (400 MHz, MeOD) δ 8.21 (brs, 1H), 7.64 (brs, 1H), 7.32 − 7.12 (m, 5H), 7.03 (dd, J = 8.3, 2.3 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 2.98 − 2.81 (m, 4H), ¹³C NMR (101 MHz, MeOD) δ 152.76, 141.71, 132.93, 129.45, 128.21, 127.87, 126.78, 125.45, 115.73, 37.93, 36.90, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₆H₁₆N₃O⁺ 266.1288; found, 266.1293.

4–(3-Chloro-2-phenethoxyphenyl)-1,2,3-triazole (43): Synthesised from 1-chloro-2-ethynyl-3-phenethoxybenzene (18 l) (150 mg, 0.59 mmol) and TMSN₃ (102 mg, 0.89 mmol) using the general procedure (II) to give 43 (120 mg, 68%) as a white solid, m.p. 96–99 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.86 − 7.71 (m, 1H), 7.46 − 7.24 (m, 6H), 7.16 (t, J = 7.9 Hz, 2H), 4.10 (t, J = 6.8 Hz, 2H), 3.13 (t, J = 6.8 Hz, 2H), ¹³C NMR (101 MHz, MeOD) δ 151.89, 138.17, 129.96, 128.90, 128.53, 128.13, 126.77, 126.31, 125.06, 73.32, 36.16, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₆H₁₄ClN₃ONa⁺ 322.0718; found, 322.0722.

2-(1H-1,2,3-triazol-5-yl)benzene-1,3-diol (44): Synthesised from 45 (130 mg, 0.75 mmol) dissolved in 48% HBr in water (3 mL) using the general procedure (III) to give 44 (93 mg, 70%) as white solid, m.p. 257–259 °C. ¹H NMR (400 MHz, MeOD) δ 8.38 (s, 1H), 7.02 (t, J = 8.2 Hz, 1H), 6.46 (d, J = 8.1 Hz, 2H), ¹³C NMR (101 MHz, MeOD) δ 156.24, 129.03, 106.57, 102.41, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇N₃O₂⁺ 178.0616; found, 178.0606.

4–(2,6-Dimethoxyphenyl)-1,2,3-triazole (45, CAS [2385145–91-3]): Synthesised from 2-ethynyl-1,3-dimethoxybenzene (324 mg, 2.0 mmol) and TMSN₃ (345 mg, 3.0 mmol) using the general procedure (II) to give 45 (197 mg, 48%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 7.35 (t, J = 8.4 Hz, 1H), 6.74 (d, J = 8.5 Hz, 2H), 4.02 (s, 9H), ¹³C NMR (101 MHz, MeOD) δ 157.60, 132.66, 132.57, 130.45, 103.87, 55.08, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₀H₁₁N₃O₂⁺ 206.0930; found, 206.0940.

4–(2,4-Dichlorophenyl)-1,2,3-triazole (48)¹⁰⁷: Synthesised from 2,4-dichloro-1-ethynylbenzene (250 mg, 1.5 mmol) and TMSN₃ (259 mg, 2.25 mmol) using the general procedure (II) to give 48 (272 mg, 85%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.24 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 2.5 Hz, 1H), 7.36 (dt, J = 8.5, 2.5 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 134.29, 132.40, 130.97, 129.62, 127.30, 56.06, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₆Cl₂N₃⁺ 213.9933; found, 213.9931.

4–(3,4-Dichlorophenyl)-1,2,3-triazole (49)¹⁰⁹: Synthesised from 1,2-dichloro-4-ethynylbenzene (170 mg, 1.0 mmol) and TMSN₃ (173 mg, 1.5 mmol) using the general procedure (II) to give 49 (187 mg, 88%) as a yellow solid. ¹H NMR (400 MHz, MeOD) δ 8.25 (s, 1H), 8.06 (d, J = 2.0 Hz, 1H), 7.81 (dd, J = 8.4, 2.0 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 144.15, 132.55, 131.53, 130.64, 127.19, 125.05, 47.45, 47.24, 47.03, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₆Cl₂N₃⁺ 213.9933; found, 213.9928.

4–(3,5-Dichlorophenyl)-1,2,3-triazole (50, CAS [55751–17-2]): Synthesised from 1,3-dichloro-5-ethynylbenzene (170 mg, 1.0 mmol) and TMSN₃ (173 mg, 1.5 mmol) using the general procedure (II) to give 50 (175 mg, 82%) as a yellow solid. ¹H NMR (400 MHz, MeOD) δ 8.29 (s, 1H), 7.84 (d, J = 2.0 Hz, 2H), 7.42 (q, J = 1.6 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 143.94, 135.32, 133.74, 127.46, 123.88, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₆Cl₂N₃⁺ 213.9933; found, 213.9932.

4–(2,6-Dichlorophenyl)-1,2,3-triazole (51, CAS [2385230–51-1]): Synthesised from 1,3-dichloro-2-ethynylbenzene (170 mg, 1.0 mmol) and TMSN₃ (173 mg, 1.5 mmol) using the general procedure (II) to give 51 (160 mg, 75%) as a yellow solid. ¹H NMR (400 MHz, MeOD) δ 7.95 (s, 1H), 7.58 − 7.49 (m, 2H), 7.45 (dd, J = 8.9, 7.2 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 135.87, 130.82, 128.08, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₆Cl₂N₃⁺ 213.9933; found, 213.9927.

4–(2,4,5-Trichlorophenyl)-1,2,3-triazole (52): Synthesised from 1,2,4-trichloro-5-ethynylbenzene (18m) (340 mg, 2.0 mmol) and TMSN₃ (345 mg, 3.0 mmol) using the general procedure (II) to give 52 (395 mg, 80%) as a yellow solid, m.p. 190–192 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 8.09 (s, 1H), 7.56 (s, 1H), ¹³C NMR (101 MHz, DMSO) δ 140.90, 131.74, 131.59, 130.85, 130.49, 130.18, 129.72, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₅Cl₃N₃⁺ 247.9544; found, 247.9536.

4–(2,4,6-Trichlorophenyl)-1,2,3-triazole (53): Synthesised from 1,3,5-trichloro-2-ethynylbenzene (18n) (170 mg, 1.0 mmol) and TMSN₃ (173 mg, 1.5 mmol) using the general procedure (II) to give 53 (198 mg, 80%) as a yellow solid, m.p. 236–238 °C. ¹H NMR (400 MHz, MeOD) δ 7.97 (s, 1H), 7.66 (s, 2H), ¹³C NMR (101 MHz, DMSO) δ 136.31, 135.32, 128.67, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₅Cl₃N₃⁺ 247.9544; found, 247.9538.

Synthesis of 5-Substituted 4-Aryl-1,2,3-Triazoles

4-Phenyl-1H-1,2,3-triazole-5-carbonitrile (62)⁶⁵: To a strongly stirred solution of NaN₃ (195 mg, 1.5 mmol) suspended in DMF (4 mL) at 90 °C, a second solution of phenylpropynenitrile (54) (255 mg, 2.0 mmol) in DMF (2 mL) was added dropwise through an addition funnel over 10 min. The mixture was stirred for 1 h at 90 °C before cooling to rt and removing the solvent under reduced pressure. Water (6 mL) was added, and the aqueous layer was extracted with CH₂Cl₂ to remove the oil and to give a transparent light colour solution. Finally, the aqueous solution was acidified with 10% HCl resulting in a light yellow precipitate which was washed with iced water to yield the crude product. The residue was purified by FC (SiO₂, CH₂Cl₂/MeOH) to obtain 62 (255 mg, 75% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 7.93 − 7.77 (m, 2H), 7.52 − 7.39 (m, 3H). 5.38 (brs, 1H), ¹³C NMR (101 MHz, MeOD) δ 147.13, 130.16, 128.97, 126.41, 126.16, 116.65, 112.66, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₉H₆N₄⁺ 171.0671; found, 171.0667.

4–(3-Chlorophenyl)-5-methyl-1,2,3-triazole (63): 3-Chlorobenzaldehyde (280 mg, 2.0 mmol), nitromethane (180 mg, 2.4 mmol) and ammonium acetate (93 mg, 1.2 mmol) were added to 2 mL of glacial acetic acid. The resulting solution was heated under reflux for 2 h before pouring the reaction mixture into ice water. The yellow solid thus formed was collected by filtration to give the crude product 1-chloro-3–(2-nitroprop-1-en-1-yl)benzene (56a)^66,110. The crude product (197 mg, 1.0 mmol) and NaN₃ (98 mg, 1.5 mmol) were stirred in DMF (3 mL), and p-TsOH (87 mg, 0.5 mmol) was added to the mixture at rt. The mixture was stirred at 60 °C under air for 1 h. After completion of the reaction, the mixture was cooled to rt, quenched with H₂O (5 mL) and extracted with EtOAc (3 × 10 mL). The organic layers were collected, dried over anh. Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to afford 63 (135 mg, 70%) as white solid, m.p. 161–163 °C. ¹H NMR (400 MHz, CDCl₃) δ 11.42 (brs, 1H), 7.73 (t, J = 1.8 Hz, 1H), 7.62 (dt, J = 7.4, 1.6 Hz, 1H), 7.46 − 7.31 (m, 2H), 2.55 (s, 3H), ¹³C NMR (101 MHz, MeOD) δ 134.37, 132.79, 130.00, 127.53, 126.48, 124.94, 8.94, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₉H₈ClN₃⁺ 194.0485; found, 194.0482.

4,5-Bis(3-chlorophenyl)-1,2,3-triazole (64)⁶⁷: To a stirred solution of 3-chlorobenzyaldehyde (420 mg, 3 mmol) in ethanol (6 mL) p-toluenesulfonyl hydrazide (57) (615 mg, 3.3 mmol) was added, and the solution was stirred for 2 h at rt. After completion of the reaction, the solvent was removed under reduced pressure. The obtained solid residue was washed with ethanol and dried under reduced pressure to afford N’-(3-chlorobenzylidene)-4-methylbenzenesulfonohydrazide (58)⁷⁷, ¹H NMR (400 MHz, CDCl₃) δ 8.36 (bs, 1H), 7.91 (s, 1H), 7.89 (s, 1H), 7.73 (s, 1H), 7.59 (t, J = 1.8 Hz, 1H), 7.49 − 7.25 (m, 5H), 2.44 (s, 3H). The residue (58) (440 mg, 1.4 mmol) was dissolved in DMF (5 mL), Cs₂CO₃ (764 mg, 2.2 mmol) was added, and the mixture was heated to 100 °C, monitoring the progress of the reaction by TLC. The reaction mixture was cooled to rt and diluted with cold water. After extraction with EtOAc, the organic layers were collected, dried (anh. Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to give 64 (285 mg, 70%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 2H), 7.90 (t, J = 1.9 Hz, 2H), 7.72 (dt, J = 7.4, 1.5 Hz, 2H), 7.52 − 7.35 (m, 4H), ¹³C NMR (101 MHz, MeOD) δ 134.38, 131.99, 130.02, 129.30, 128.40, 127.74, 127.38, 126.22, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₄H₁₀Cl₂N₃⁺ 290.0246; found, 290.0246.

Ethyl 4–(3-chlorophenyl)-1H-1,2,3-triazole-5-carboxylate (65)⁵⁹: To a stirred solution of ethyl-3–(4-chlorophenyl)propiolate (59) (209 mg, 1.0 mmol) in DMSO (2 mL), NaN₃ (182 mg, 2.8 mmol) was added, and the solution was stirred under reflux at 70 °C for 6 h. After completion of the reaction, the mixture was cooled to rt and quenched with water. After extraction with EtOAc, the organic layer was collected, washed with brine, dried (anh. Na₂SO₄), filtered, and concentrated under reduced pressure. Purification by FC (silica gel, EtOAc/hexane) produced 65 (150 mg, 60%) as white solid. ¹H NMR (400 MHz, MeOD) δ 7.83 (dd, J = 8.4, 1.7 Hz, 2H), 7.51 (dd, J = 8.5, 1.6 Hz, 2H), 4.39 (qd, J = 7.1, 1.4 Hz, 2H), 1.36 (td, J = 7.2, 1.4 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 160.72, 135.17, 130.52, 128.08, 61.10, 13.00, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₁H₁₁ClN₃O₂⁺ 252.0534; found, 252.0535.

4–(4-Chlorophenyl)-1H-1,2,3-triazole-5-carboxamide (66)⁶⁹: To a stirred solution of 4-chlorobenzaldehyde (55c) (280 mg, 2.0 mmol), 2-cyanoacetamide (60) (169 mg, 2.0 mmol), and Et₃N·HCl (566 mg, 5.0 mmol) in DMF (3 mL), NaN₃ (390 mg, 6.0 mmol) was added. The mixture was stirred at 70 °C for 10 h before cooling to rt and adding water (20 mL) and 10% HCl solution (1 mL). After extraction with EtOAc (2 × 30 mL), the organic layer was collected, washed with water (3 × 50 mL) and brine solution (1 × 50 mL), dried (anh. Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was subject to FC (SiO₂, CH₂Cl₂/MeOH) to obtain 66 (288 mg, 65% yield) as white solid, ¹H NMR (400 MHz, MeOD) δ 7.92 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H), ¹³C NMR (101 MHz, MeOD) δ 163.78, 134.64, 130.54, 128.25, HRMS (nanochip-ESI/LTQ-Orbitrap) m/z: calcd for [M + Na]⁺ C₉H₇ClN₄ONa⁺ 245.0201; found, 245.0196.

4–(4-Chloro-2-hydroxyphenyl)-1H-1,2,3-triazole-5-carboxamide (67): Synthesised from 5-chloro-2-hydroxybenzaldehyde (55b) (314 mg, 2.0 mmol), 2-cyanoacetamide (60) (169 mg, 2.0 mmol), and Et₃N·HCl (566 mg, 5.0 mmol), and NaN₃ (390 mg, 6.0 mmol) using the same procedure as for 66 to give 67 (260 mg, 55% yield) as a white solid, m.p. 260–262 °C. ¹H NMR (400 MHz, MeOD) δ 7.68 − 7.57 (m, 1H), 7.31 (ddd, J = 8.7, 2.7, 1.4 Hz, 1H), 6.96 (dd, J = 8.7, 1.5 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 164.84, 154.03, 137.63, 130.39, 130.37, 124.01, 118.10, 116.86, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₇ClN₄O₂Na⁺ 261.0150; found, 261.0159.

4-Chloro-2–(5-methyl-1,2,3-triazol-4-yl)phenol (68): Synthesised from 5-chloro-2-hydroxybenzaldehyde (55b) (312 mg, 2.0 mmol), nitromethane (180 mg, 2.4 mmol), and ammonium acetate (93 mg, 1.2 mmol) according to the same procedure as used for 63 to afford 68 (136 mg, 65%) as white solid, m.p. 149–151 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.56 (d, J = 2.5 Hz, 1H), 7.25 (dd, J = 8.8, 2.6 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 2.69 (s, 3H), ¹³C NMR (101 MHz, MeOD) δ 170.80, 158.40, 149.45, 145.46, 138.72, 120.68, 19.07, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₉H₉ClN₃O⁺ 210.0429; found, 210.0431.

Synthesis of 4-Aryl-Tether-1,2,3-Triazoles and annulated derivatives

N-(4-Bromophenyl)-1,2,3-triazol-4-amine (78)⁷¹: 4-Bromo aniline (335 mg, 2.62 mmol) was dissolved in hydrochloric acid (6.03 mL of 2 M, 12.06 mmol), diluted with water (8 mL) and cooled to 0 °C. Sodium nitrite (181 mg, 2.62 mmol) was added and the mixture was stirred at 0 °C for 20 min before slowly adding a solution of 2-aminoacetonitrile monohydrochloride (70) (243 mg, 2.62 mmol) in water (3 mL). The mixture was stirred for 10 min at 0 °C before adding sodium acetate (3.02 g, 37.0 mmol) and allowing the mixture to warm to rt, where it was stirred for 1 h. The precipitate was collected by filtration and washed with water to afford 2–(3-(4-chlorophenyl)triaz-2-en-1yl)acetonitrile. The residue (238 mg, 1.0 mmol) was dissolved in EtOH (7 mL) and heated under reflux for 4 h. The mixture was allowed to cool to rt and concentrated under reduced pressure. The crude compound was triturated with CH₂Cl₂ to produce a cream solid which was purified by FC (40% EtOAc in petroleum ether) to afford 78 (167 mg, 70%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.39 (s, 1H), 7.35 − 7.25 (m, 2H), 7.19 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 148.34, 142.56, 131.44, 121.80, 116.57, 110.61, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈BrN₄⁺ 238.9927; found, 238.9933.

4-(Phenoxy)-1,2,3-triazole (82): Synthesised from (ethynyloxy)benzene (77a) (130 mg, 1.1 mmol) and TMSN₃ according to the general procedure (II) to afford 82 (124 mg, 70%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 11.08 (bs, 1H), 7.44 − 7.34 (m, 3H), 7.29 (s, 1H), 7.24 − 7.14 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 158.33, 156.97, 129.48, 123.62, 117.16, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈N₃O⁺ 162.0662; found, 162.0666.

4-(Phenylthio)-1,2,3-triazole (83): Synthesised from ethynyl(phenyl)sulfane (77b) (176 mg, 1.3 mmol) and TMSN₃ according to the general procedure (II) to afford 83 (154 mg, 67%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.72 (s, 1H), 7.42 − 7.24 (m, 5H), 3.42 (s, 2H), ¹³C NMR (101 MHz, MeOD) δ 135.18, 128.91, 128.52, 126.57, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈N₃S⁺ 178.0433; found, 178.0436.

4-(Phenylsulfonyl)-1H-1,2,3-triazole (84)¹¹¹: To a stirred solution of compound 83 (60 mg 0.34 mmol) in methanol (2 mL), H₂O₂ (0.1 mL, 2.7 mmol) and a catalytic amount of ammonium molybdate (7 mg, 0.04 mmol) were added and allowed to stir at rt for 14 h, monitoring the progress of the reaction by TLC. After completion of the reaction, the solvent was removed under reduced pressure. The obtained solid products were dissolved in CH₂Cl₂ (2 mL) and water (2 mL), and the aqueous phase was extracted with CH₂Cl₂ (2 × 4 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC to give 84 (57 mg, 80%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 8.09 (dd, J = 7.3, 1.8 Hz, 2H), 7.69 − 7.61 (m, 1H), 7.57 (dd, J = 8.5, 6.9 Hz, 2H), ¹³C NMR (101 MHz, MeOD) δ 148.22, 140.70, 133.77, 129.19, 127.44, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₈H₇N₃O₂SNa⁺ 232.0151; found, 232.0154.

Phenyl(1,2,3-triazol-4-yl)methanol (85)¹¹²: Synthesised from 1-phenylprop-2-yn-1ol (77c) (198 mg, 1.5 mmol) and TMSN₃ according to the general procedure (II) to afford 85 (184 mg, 70%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.57 (s, 1H), 7.51 − 7.31 (m, 5H), 6.08 (s, 1H), ¹³C NMR (101 MHz, MeOD) δ 142.64, 128.16, 127.49, 126.22, 68.31, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₉N₃ONa⁺ 198.0640; found, 198.0638.

N-Phenyl-1H-1,2,3-triazole-4-carboxamide (86): Synthesised from N-phenylpropiolamide (77d) (145 mg, 1.0 mmol) and TMSN₃ according to the general procedure (II) to afford 86 (94 mg, 50%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.44 − 7.35 (m, 2H), 7.17 (td, J = 7.3, 1.1 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 159.40, 142.22, 137.70, 128.50, 124.37, 120.71, 120.59, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₈N₄ONa⁺ 211.0590; found, 211.0588.

Phenyl(1H-1,2,3-triazol-4-yl)methanone O-methyl oxime (87): To a stirred solution of phenyl(1H-1,2,3-triazol-4-yl)methanone (99)¹¹³ (50 mg, 0.29 mmol) in H₂O (2 mL), and EtOH (1 mL), O-methylhydroxylamine hydrochloride (65 mg, 0.82 mmol) and NaOAc (104 mg, 1.28 mmol) were added. The flask was equipped with a reflux condenser and heated to 70 °C for 2 h. After cooling to rt, the mixture was extracted with EtOAc (3 × 5 mL). The organic layers were combined, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Purification by FC gave 87 (32 mg, 55%) as amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.16 (s, 1H), 7.67 − 7.59 (m, 2H), 7.54 − 7.42 (m, 3H), 4.20 (s, 3H), ¹³C NMR (101 MHz, DMSO) δ 148.52, 138.55, 137.27, 135.12, 129.65, 129.08, 128.47, 62.86, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₀H₁₁N₄O⁺ 203.0927; found, 203.0926.

3,8-Dihydroindeno[1,2-d][1,2,3]triazole (89)⁷⁴: 1,2-Indandione-2-oxime (71) (161 mg, 1.0 mmol), hydrazine hydrate (97 mg, 3.0 mmol) and potassium hydroxide (168 mg, 3.0 mmol) were dissolved in diethyleneglycol (3 mL) under nitrogen atmosphere. The reactions mixture was heated to 170–190 °C and maintained at this temperature for 6 h. Upon completion of the reaction, the mixture was cooled to rt, and water (5 mL) and EtOAc (10 mL) were added. EtOAc (10 mL) was added, the phases were separated, and the aqueous phase was further extracted with EtOAc (3 × 10 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC to yield 89 (63 mg, 40%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.84 (dt, J = 7.6, 1.1 Hz, 1H), 7.57 (dp, J = 7.3, 1.0 Hz, 1H), 7.47 − 7.33 (m, 2H), 3.83 (d, J = 0.8 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 153.40, 146.94, 131.79, 127.05, 126.00, 120.16, 27.32 HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₉H₈N₃⁺ 158.0713; found, 158.0716.

Synthesis of ethynes 77a, 77b, 77d

(Ethynyloxy)benzene (77a)¹¹⁴: A mixture of phenol (72) (940 mg, 10.0 mmol) and NaOH powder (400 mg, 10 mmol) in DMSO (10 mL) was stirred at rt for 2 h before slowly adding trichloroethylene (73) (0.91 mL, 10.0 mmol). The mixture was stirred at rt until completion of the reaction. Then, water (25 mL) and CH₂Cl₂ (50 mL) were added, the phases were separated, and the aqueous phase was further extracted with CH₂Cl₂ (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude residue was used in the next step without purification. To a solution of the residue (752 mg, 4.0 mmol) in dry Et₂O (32 mL) at −78 °C, a solution of n-BuLi (2.5 M in hexane, 6.4 mL 16.0 mmol) was added under nitrogen atmosphere. After addition, the mixture was warmed to −20 °C over 1 h and stirred at this temperature for 1 h, before adding water (10 mL) and warming the mixture to rt. The phases were separated and the aqueous phase was extracted with CH₂Cl₂ (3 × 15 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to provide 77a (189 mg, 40%) as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ 7.45 − 7.36 (m, 2H), 7.40 − 7.26 (m, 2H), 7.19 (m, 1H), 2.12 (s, 1H).

Ethynyl(phenyl)sulfane (77b)¹¹⁵: To a solution of TMS-acetylene (75) (655 mg, 3.0 mmol) in 10 mL THF at −78 °C, n-BuLi (2.5 M in hexane, 1.1 mL, 3.0 mmol) was added dropwise and stirred for 1.5 h. A solution of diphenyl disulphide (74) (324 mg, 3.3 mmol) in 2 mL THF was added dropwise, and the mixture was warmed to rt over 2 h before adding H₂O (5 mL). After extraction with Et₂O (3 × 10 mL), the organic layer was collected, washed with brine, dried over Na₂SO₄), filtered, and concentrated under reduced pressure to yield the TMS-protected thioethyne. For deprotection, the thioethyne (412 mg 2.0 mmol) was stirred for 3 h at rt with KF (441 mg, 7.6 mmol) in MeOH (10 mL). After concentration under reduced pressure, CH₂Cl₂ (5 mL) and water (3 mL) were added. The organic layer was collected, dried (MgSO₄) and filtered through a short silica plug to afford 77b (192 mg, 85%) as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ 7.51 − 7.44 (m, 2H), 7.42 − 7.33 (m, 2H), 7.30 − 7.22 (m, 1H), 3.28 (s, 1H).

N-Phenylpropiolamide (77d)¹¹⁶: Propiolic acid (350 mg, 5.0 mmol) and DCC (1.03 g, 5.0 mmol) were combined in 15 mL of CH₂Cl₂. The solution was stirred for 10 min before adding aniline (76) (466 mg, 5.0 mmol) and DMAP (7.5 mg, 0.06 mmol) in dry CH₂Cl₂ at 0 °C under nitrogen atmosphere. After complete addition, the mixture was stirred at rt for 18h. The reaction mixture was filtered and washed with 1 N HCl (5 mL) and a saturated solution of sodium chloride (2 × 50 mL). The organic phase was separated, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC (silica gel, EtOAc/hexane) to provide 77d (652 mg, 90%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.64 (bs, 1H), 7.58 − 7.51 (m, 2H), 7.42 − 7.33 (m, 2H), 7.18 (td, J = 7.3, 1.2 Hz, 1H), 2.95 (s, 1H).

General procedure (IV) for the synthesis of (aryl)(1,2,3-Triazolyl)methanol

Ethynylmagnesium bromide solution (1.2 eq, 0.5 M solution in THF) was added dropwise to a stirred solution of aldehydes (90) (1 eq) in anhydrous THF (6 mL) at 0 °C. The mixture was kept stirring at 0 °C for 20 min, then warmed to rt and stirred for an additional 2 h. The reaction mixture was quenched with a saturated NH₄Cl solution (6 mL) and extracted with EtOAc (3 × 8 mL). The organic layers were combined, dried over anh. Na₂SO₄, filtered, and concentrated under reduced pressure to give the intermediate 1-aryl propargyl alcohols (91), which were directly used in next step. Alcohols (91) (1 eq), TMSN₃ (1.5 eq), and CuI (0.05 eq) in DMF/MeOH solution (9:1) were stirred at 100 °C for 10–12 h. After consumption of the ethynyl substrate, the mixture was cooled to rt, the precipitate was filtered off, and the solution was concentrated under reduced pressure. The crude residue was purified by FC (SiO₂, EtOAc/petroleum ether) to afford compounds 92a–92u in 55–85% yields.

Synthesis of (aryl)(1,2,3-Triazolyl)methanol (92a–92u)

Phenyl(1,2,3-triazol-4-yl)methanol (92a)¹¹²: Synthesised from benzaldehyde (212 mg, 2.0 mmol), ethynylmagnesium bromide solution (4.8 mL, 0.5 M in THF, 2.4 mmol), and TMSN₃ (345 mg, 3.0 mmol) according to the general procedure (IV) to afford 92a (297 mg, 85%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.57 (s, 1H), 7.51 − 7.31 (m, 5H), 6.08 (s, 1H).

(2-Chlorophenyl)(1,2,3-triazol-4-yl)methanol (92b, CAS [1511220–63-5]): Synthesised from 2-chlorobenzaldehyde (422 mg, 3.0 mmol) ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92b (470 mg, 75%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.74 (dd, J = 8.0, 1.7 Hz, 1H), 7.57 (s, 1H), 7.44 − 7.37 (m, 2H), 7.41 − 7.27 (m, 1H), 6.36 (s, 1H).

(3-Chlorophenyl)(-1,2,3-triazol-4-yl)methanol (92c, CAS [1511815–17-0]): Synthesised from 3-chlorobenzaldehyde (422 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92c (501 mg, 75%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.48 (s, 1H), 7.42 (q, J = 2.0 Hz, 1H), 7.27 (ddd, J = 9.5, 5.1, 1.5 Hz, 4H), 5.95 (t, J = 3.2 Hz, 1H).

(4-Chlorophenyl)(1,2,3-triazol-4-yl)methanol (92d, CAS [1429056–35-8]): Synthesised from 4-chlorobenzaldehyde (422 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92d (501 mg, 70%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.66 (s, 1H), 7.47 − 7.33 (m, 4H), 5.98 (s, 1H).

(2-Methoxyphenyl)(1,2,3-triazol-4-yl)methanol (92e, CAS [1518311–97-1]): Synthesised from 2-methoxybenzaldehyde (408 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92e (399 mg, 65%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.52 (s, 1H), 7.40 − 7.27 (m, 2H), 7.04 − 6.90 (m, 2H), 6.21 (s, 1H), 3.84 (s, 3H).

(3-Methoxyphenyl)(1,2,3-triazol-4-yl)methanol (92f, CAS [1541589–58-5]): Synthesised from 3-methoxybenzaldehyde (408 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92f (338 mg, 55%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.63 (s, 1H), 7.27 (t, J = 7.9 Hz, 1H), 7.07 − 6.96 (m, 2H), 6.86 (dd, J = 8.3, 2.6 Hz, 1H), 5.95 (s, 1H), 3.80 (s, 3H).

(4-Methoxyphenyl)(1,2,3-triazol-4-yl)methanol (92g, CAS [1526953–98-9]): Synthesised from 4-methoxybenzaldehyde (408 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92g (338 mg, 55%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.62 (s, 1H), 7.38 − 7.30 (m, 2H), 6.99 − 6.88 (m, 2H), 5.93 (s, 1H), 3.80 (s, 3H).

(4-Bromophenyl)(1,2,3-triazol-4-yl)methanol (92 h, CAS [1934702–39-2]): Synthesised from 4-bromobenzaldehyde (555 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92h (546 mg, 72%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.66 (s, 1H), 7.56 − 7.49 (m, 2H), 7.42 − 7.34 (m, 2H), 5.96 (s, 1H).

(4-Fluorophenyl)(1,2,3-triazol-4-yl)methanol (92i, CAS [1517879–10-5]): Synthesised from 4-fluorobenzaldehyde (372 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92i (359 mg, 62%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.66 (s, 1H), 7.50 − 7.41 (m, 2H), 7.15 − 7.04 (m, 2H), 5.98 (s, 1H).

(5-Chloro-2-hydroxyphenyl)(1,2,3-triazol-4-yl)methanol (92j): Synthesised from 5-chloro-2-methoxybenzaldehyde (513 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92j (499 mg, 74%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.59 (s, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.11 (dd, J = 8.6, 2.7 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.24 (s, 1H).

(4-Chloro-2-hydroxyphenyl)(1,2,3-triazol-4-yl)methanol (92k): Synthesised from 4-chloro-2-methoxybenzaldehyde (513 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92k (466 mg, 69%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.58 − 7.48 (m, 2H), 7.02 (dd, J = 6.0, 2.2 Hz, 2H), 6.25 (s, 1H), 3.82 (s, 3H).

(4-Bromo-2-hydroxyphenyl)(1,2,3-triazol-4-yl)methanol (92l): Synthesised from 4-bromo-2-methoxybenzaldehyde (403 mg, 2.0 mmol), ethynylmagnesium bromide solution (4.8 mL, 0.5 M in THF, 2.4 mmol), and TMSN₃ (345 mg, 3.0 mmol) according to the general procedure (IV) to afford 92l (371 mg, 69%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s, 1H), 7.11 − 6.96 (m, 3H), 5.72 (s, 1H).

Furan-2-yl(1,2,3-triazol-4-yl)methanol (92 m, CAS [1519476–28-8]): Synthesised from 2-furanal (289 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92 m (297 mg, 60%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (s, 1H), 7.94 − 7.74 (m, 1H), 7.51 − 7.37 (m, 1H), 6.45 − 6.25 (m, 1H), 6.12 − 5.99 (m, 1H).

Furan-3-yl(1,2,3-triazol-4-yl)methanol (92n, CAS [1500898–43-0]): Synthesised from 3-furanal (289 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92n (352 mg, 71%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.73 (s, 1H), 7.49 (d, J = 1.5 Hz, 2H), 6.47 (d, J = 2.9 Hz, 1H), 5.96 (s, 1H).

Thiophen-2-yl(1,2,3-triazol-4-yl)methanol (92o, CAS [1542859–11-9]): Synthesised from thiophene-2-carbaldehyde (224 mg, 2.0 mmol), ethynylmagnesium bromide solution (4.8 mL, 0.5 M in THF, 2.4 mmol), and TMSN₃ (345 mg, 3.0 mmol) according to the general procedure (IV) to afford 92o (239 mg, 66%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.75 − 7.70 (s, 1H), 7.37 (dd, J = 5.0, 1.4 Hz, 1H), 7.08 − 6.95 (m, 2H), 6.24 (s, 1H).

Thiophen-3-yl(1,2,3-triazol-4-yl)methanol (92p, CAS [1522175–89-8]): Synthesised from thiophene-3-carbaldehyde (336 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92p (396 mg, 73%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 7.68 (s, 1H), 7.40 (dd, J = 5.1, 3.0 Hz, 12H), 7.36 − 7.31 (m, 1H), 7.11 (dd, J = 5.0, 1.3 Hz, 1H), 6.07 (s, 1H).

Thiazol-4-yl(1,2,3-triazol-4-yl)methanol (92q, CAS [1935872–98-2]): Synthesised from thiazole-4-carbaldehyde (326 mg, 2.0 mmol), ethynylmagnesium bromide solution (4.8 mL, 0.5 M in THF, 2.4 mmol), and TMSN₃ (345 mg, 3.0 mmol) according to the general procedure (IV) to afford 92q (272 mg, 73%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.99 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 6.35 (s, 1H).

(1,2,3-Thiadiazol-4-yl)(1,2,3-triazol-4-yl)methanol (92r): Synthesised from 1,2,3thiadiazole-4-carbaldehyde (228 mg, 2.0 mmol), ethynylmagnesium bromide solution (4.8 mL, 0.5 M in THF, 2.4 mmol), and TMSN₃ (345 mg, 3.0 mmol) according to the general procedure (IV) to afford 92r (260 mg, 71%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.95 (s, 1H), 7.84 (s, 1H), 6.60 (s, 1H).

Pyridin-2-yl(1,2,3-triazol-4-yl)methanol (92 s): Synthesised from picolinaldehyde (322 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92 s (370 mg, 70%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.68 − 8.58 (m, 1H), 8.24 (dt, J = 7.8, 1.1 Hz, 1H), 7.99 (s, 1H), 7.89 (td, J = 7.7, 1.8 Hz, 1H), 7.48 (ddd, J = 7.6, 4.8, 1.3 Hz, 1H), 5.60 (s, 1H).

Pyridin-3-yl(1,2,3-triazol-4-yl)methanol (92t, CAS [1508769–16-1]): Synthesised from nicotinaldehyde (322 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92t (396 mg, 75%) as a yellow solid. ¹H NMR (400 MHz, MeOD) δ 8.64 (d, J = 2.2 Hz, 1H), 8.49 (dd, J = 4.9, 1.6 Hz, 1H), 7.93 (dt, J = 8.1, 2.0 Hz, 1H), 7.75 (s, 1H), 7.46 (dd, J = 8.0, 4.9 Hz, 1H), 6.08 (s, 1H)

Pyridin-4-yl(1,2,3-triazol-4-yl)methanol (92 u): Synthesised from isonicotinaldehyde (322 mg, 3.0 mmol), ethynylmagnesium bromide solution (7.2 mL, 0.5 M in THF, 3.6 mmol) and TMSN₃ (518 mg, 4.5 mmol) according to the general procedure (IV) to afford 92 u (359 mg, 68%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.67 − 8.51 (m, 2H), 7.57 − 7.49 (m, 2H), 7.34 (s, 1H), 5.51 (s, 1H).

Synthesis of 5-Aroyl and 5-Hetaroyl-1,2,3-Triazoles

Phenyl(1H-1,2,3-triazol-5-yl)methanone (99)¹¹³: A solution of phenyl(1,2,3-triazol-4yl)methanol (92a) (88 mg, 0.5 mmol) in CH₂Cl₂ (3 mL) was treated with PCC (162 mg, 0.75 mmol). After stirring at rt for 2 h, the reaction was complete as determined by TLC. Excess PCC was removed by filtration of the reaction mixture through a pad of celite. The filtrate was sequentially washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue as purified by FC (SiO₂, EtOAc/petroleum ether) giving 99 (63 mg, 73%) as a oily solid. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H), 8.33 (d, J = 7.7 Hz, 1H), 7.71 − 7.64 (m, 1H), 7.57 (tt, J = 6.6, 1.2 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 186.20, 136.83, 133.07, 129.76, 128.16, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₇N₃ONa⁺ 196.0481; found, 196.0481.

(2-Chlorophenyl)(1H-1,2,3-triazol-5-yl)methanone (100)¹¹⁷: Prepared the same way as 99 from (2-chlorophenyl)(1,2,3-triazol-4-yl)methanol (92b) (250 mg, 1.2 mmol) and PCC (389 mg, 1.8 mmol) in CH₂Cl₂ (8 mL), giving 100 (137 mg, 55% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.32 (s, 1H), 7.64 − 7.58 (m, 1H), 7.55 − 7.46 (m, 2H), 7.43 (ddd, J = 7.6, 6.2, 2.3 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 187.13, 145.63, 137.95, 131.76, 130.96, 129.95, 129.37, 126.64, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₆ClN₃ONa⁺ 230.0092; found, 230.0090.

(3-Chlorophenyl)(1H-1,2,3-triazol-5-yl)methanone (101)¹¹⁸: Prepared the same way as 99 from (3-chlorophenyl)(1,2,3-triazol-4-yl)methanol (92c) (320 mg, 1.5 mmol) and PCC (486 mg, 2.25 mmol) in CH₂Cl₂ (10 mL), giving 101 (186 mg, 60% yield) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.47 − 8.21 (m, 3H), 7.64 (ddd, J = 8.0, 2.2, 1.1 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 184.44, 145.54, 138.36, 134.22, 132.76, 129.74, 129.58, 128.20, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₆ClN₃ONa 230.0097; found, 230.0096.

(4-Chlorophenyl)(1H-1,2,3-triazol-5-yl)methanone (102)¹¹⁸: Prepared the same way as 99 from (4-chlorophenyl)(1,2,3-triazol-4-yl)methanol (92d) (105 mg, 0.5 mmol) and PCC (162 mg, 0.75 mmol) in CH₂Cl₂ (3 mL) giving 102 (68 mg, 65% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.48 (s, 1H), 8.38 − 8.27 (m, 2H), 7.66 − 7.54 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 184.67, 139.36, 135.21, 131.49, 128.38, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₆ClN₃ONa⁺ 230.0097; found, 230.0105.

(2-Methoxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (103, CAS [1541622–39-2]): Prepared the same way as 99 from (2-methoxyphenyl)(1,2,3-triazol-5-yl)methanol (92e) (205 mg, 1.0 mmol) and PCC (324 mg, 1.5 mmol) in CH₂Cl₂ (5 mL) giving 103 (142 mg, 70% yield) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.64 − 7.49 (m, 2H), 7.16 − 6.99 (m, 2H), 3.83 (s, 1H), ¹³C NMR (101 MHz, MeOD) δ 187.99, 157.89, 132.87, 129.44, 128.22, 120.16, 111.72, 54.77, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₀H₉N₃O₂Na⁺ 226.0587; found, 226.0589.

(3-Methoxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (104)¹¹⁸: Prepared the same way as 99 from (3-methoxyphenyl)(1,2,3-triazol-5-yl)methanol (92f) (205 mg, 1.0 mmol) and PCC (324 mg, 1.5 mmol) in CH₂Cl₂ (5 mL) giving 104 (122 mg, 60% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.46 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 2.7 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.26 (dd, J = 8.1, 2.6 Hz, 1H), 3.90 (s, 3H), ¹³C NMR (101 MHz, MeOD) δ 185.82, 159.76, 138.00, 129.23, 122.35, 119.20, 114.25, 54.52, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₀H₉N₃O₂Na⁺ 226.0587; found, 226.0584.

(4-Methoxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (105)¹¹⁷: Prepared the same way as 99 from (4-methoxyphenyl)(1,2,3-triazol-5-yl)methanol (92g) (240 mg, 1.2 mmol) and PCC (389 mg, 1.8 mmol) in CH₂Cl₂ (8 mL) giving 105 (171 mg, 70% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.48 − 8.28 (m, 3H), 7.15 − 7.01 (m, 2H), 3.93 (s, 3H), ¹³C NMR (101 MHz, MeOD) δ 184.65, 164.23, 132.35, 129.34, 113.45, 54.70, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₀H₉N₃O₂Na⁺ 226.0587; found, 226.0590.

(2-Hydroxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (106, CAS [2294215–75-9]): Synthesised from compound 103 (102 mg, 0.5 mmol) and 48% HBr in water (2 mL) using the general procedure (III) to give 106 (42 mg, 45%) as a yellow solid. ¹H NMR (400 MHz, MeOD) δ 8.82 (d, J = 8.1 Hz, 1H), 8.52 (s, 1H), 7.59 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.07 − 6.99 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 189.62, 163.06, 136.31, 132.91, 119.17, 118.80, 117.52, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₇N₃O₂Na⁺ 212.0436; found, 212.0439.

(3-Hydroxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (107, CAS [2294564–32-0]): Synthesised from compound 104 (102 mg, 0.5 mmol) and 48% HBr in water (2 mL) using the general procedure (III) to give 107 (46 mg, 49%) as a fluffy yellow powder. ¹H NMR (400 MHz, MeOD) δ 8.43 (s, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.65 (s, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.10 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 186.16, 157.44, 138.09, 129.24, 121.09, 120.24, 115.99, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₇N₃O₂Na⁺ 212.0430; found, 212.0429.

(4-Hydroxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (108, CAS [2296580–04-4]): Synthesised from compound 105 (102 mg, 0.5 mmol) and 48% HBr in water (2 mL) using the general procedure (III) to give 108 (49 mg, 50%) as a yellow solid. ¹H NMR (400 MHz, MeOD) δ 8.39 (s, 1H), 8.24 (d, J = 8.4 Hz, 2H), 6.99 − 6.82 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 162.87, 132.64, 128.22, 114.86, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₇N₃O₂Na⁺ 212.0430; found, 212.0438.

(4-Bromophenyl)(1H-1,2,3-triazol-5-yl)methanone (109)¹¹⁸: Prepared the same way as 99 from (4-bromophenyl)(1,2,3-triazol-4-yl)methanol (92h) (253 mg, 1.0 mmol) and PCC (324 mg, 1.5 mmol) in CH₂Cl₂ (5 mL) giving 109 (148 mg, 59% yield) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 8.24 (d, J = 8.2 Hz, 2H), 7.76 − 7.61 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 184.88, 135.64, 131.56, 131.45, 127.99, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₆BrN₃ONa⁺ 273.9586; found, 273.9587.

(4-Fluorophenyl)(1H-1,2,3-triazol-5-yl)methanone (110)¹¹³: Prepared the same way as 99 from (4-fluorophenyl)(1,2,3-triazol-4-yl)methanol (92i) (193 mg, 1.0 mmol) and PCC (324 mg, 1.5 mmol) in CH₂Cl₂ (5 mL) giving 110 (86 mg, 45% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.48 (s, 1H), 8.45 − 8.37 (m, 2H), 7.35 − 7.27 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 184.37, 167.18, 164.66, 145.64, 133.18, 133.15, 132.84, 132.74, 115.20, 114.98, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₆FN₃ONa⁺ 214.0387; found, 214.0386.

(5-Chloro-2-hydroxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (111): Prepared the same way as 99 from (5-chloro-2-hydroxyphenyl)(1,2,3-triazol-4-yl)methanol (92j) (125 mg, 0.6 mmol) and PCC (194 mg, 1.5 mmol) in CH₂Cl₂ (4 mL) giving 111 (48 mg, 39% yield) as white solid, m.p. 186–188 °C. ¹H NMR (400 MHz, MeOD) δ 8.95 (s, 1H), 8.55 (s, 1H), 7.56 (dd, J = 9.0, 2.7 Hz, 1H), 7.04 (d, J = 8.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 188.32, 161.52, 135.86, 131.84, 123.48, 119.95, 119.34, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₆ClN₃O₂Na⁺ 246.0046; found, 246.0051.

(4-Chloro-2-hydroxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (112): Prepared the same way as 99 from (4-chloro-2-hydroxyphenyl)(1,2,3-triazol-4-yl)methanol (92k) (96 mg, 0.5 mmol) and PCC (162 mg, 0.75 mmol) in CH₂Cl₂ (3 mL) giving 112 (53 mg, 55% yield) as oily solid. ¹H NMR (400 MHz, MeOD) δ 8.92 (d, J = 8.8 Hz, 1H), 8.55 (s, 1H), 7.12 − 6.99 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 188.61, 163.74, 141.90, 134.34, 119.32, 118.00, 117.42, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₉H₆ClN₃O₂Na⁺ 246.0041; found, 246.0038.

(4-Bromo-2-hydroxyphenyl)(1H-1,2,3-triazol-5-yl)methanone (113): Prepared the same way as 99 from (4-bromo-2-hydroxyphenyl)(1,2,3-triazol-4-yl)methanol (92l) (200 mg, 0.75 mmol) and PCC (243 mg, 1.13 mmol) in CH₂Cl₂ (5 mL) giving 113 (100 mg, 50% yield) as yellow solid, m.p. 192–194 °C. ¹H NMR (400 MHz, MeOD) δ 8.79 (d, J = 8.7 Hz, 1H), 8.52 (s, 1H), 7.21 (d, J = 1.9 Hz, 1H), 7.16 (dd, J = 8.7, 2.0 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 188.84, 163.46, 145.87, 134.20, 132.23, 130.50, 122.24, 120.58, 118.23, HRMS (ESI/QTOF) m/z: calcd for [M–H]⁻ C₉H₆BrN₃O₂^– 267.9545; found, 267.9543.

Furan-2-yl(1H-1,2,3-triazol-5-yl)methanone (114)¹¹⁸: Prepared the same way as 99 from furan-2-yl(1,2,3-triazol-4-yl)methanol (92 m) (165 mg, 1.0 mmol) and PCC (324 mg, 1.5 mmol) in CH₂Cl₂ (6 mL) giving 114 (57 mg, 35% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.57 (s, 1H), 8.01 (s,1H), 7.97 − 7.93 (m, 1H), 6.77 (dd, J = 3.6, 1.7 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 175.43, 151.23, 148.25, 121.80, 112.39, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₇H₅N₃O₂Na⁺ 186.0274; found, 186.0271.

Furan-3-yl(1H-1,2,3-triazol-5-yl)methanone (115, CAS [1522300–51-1]): Prepared the same way as 99 from furan-3-yl(1,2,3-triazol-4-yl)methanol (92n) (165 mg, 1.0 mmol) and PCC (324 mg, 1.5 mmol) in CH₂Cl₂ (6 mL) giving 115 (83 mg, 51% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.89 (s, 1H), 8.43 (s, 1H), 7.68 (t, J = 1.7 Hz, 1H), 7.05 (d, J = 1.8 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 179.64, 150.59, 143.92, 125.78, 108.79, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₇H₅N₃O₂Na⁺ 186.0274; found, 186.0274.

Thiophen-2-yl(1H-1,2,3-triazol-5-yl)methanone (116, CAS [1540541–16-9]): Prepared the same way as 99 from thiophen-2-yl(1,2,3-triazol-4-yl)methanol (92o) (200 mg, 1.1 mmol) and PCC (356 mg, 1.65 mmol) in CH₂Cl₂ (6 mL) giving 116 (69 mg, 35% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.60 (d, J = 3.8 Hz, 1H), 8.47 (s, 1H), 7.97 (dd, J = 4.9, 1.2 Hz, 1H), 7.30 (dd, J = 5.0, 3.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 177.70, 145,71, 142.24, 135.64, 135.19, 128.14, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₇H₅N₃OSNa⁺ 202.0046; found, 202.0042.

Thiophen-3-yl(1H-1,2,3-triazol-5-yl)methanone (117)¹¹⁸: Prepared the same way as 99 from thiophen-3-yl(1,2,3-triazol-4-yl)methanol (92p) (200 mg, 1.1 mmol) and PCC (356 mg, 1.65 mmol) in CH₂Cl₂ (6 mL) giving 117 (81 mg, 41% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.99 (s, 1H), 8.46 (s, 1H), 7.86 (d, J = 5.1 Hz, 1H), 7.55 (dd, J = 5.2, 2.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 179.25, 147.86, 140.32, 135.87, 127.67, 125.82, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₇H₅N₃OSNa⁺ 202.0046; found, 202.0044.

Thiazol-4-yl(1H-1,2,3-triazol-5-yl)methanone (118, CAS [1935572–44-3]): Prepared the same way as 99 from thiazol-4-yl(1,2,3-triazol-4-yl)methanol (92q) (225 mg, 1.25 mmol) and PCC (405 mg, 1.88 mmol) in CH₂Cl₂ (8 mL) giving 118 (143 mg, 64% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 9.35 (s, 1H), 9.23 (s, 1H), 8.53 (s, 1H), ¹³C NMR (101 MHz, DMSO) δ 177.42, 162.18, 149.95, 145.47, 137.72, 131.16, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₆H₅N₄OS⁺ 181.0179; found, 181.0175.

(1,2,3-Thiadiazol-4-yl)(1H-1,2,3-triazol-5-yl)methanone (119): Prepared the same way as 99 from (1,2,3-thiadiazol-4-yl(1,2,3-triazol-4-yl)methanol (92r) (275 mg, 1.5 mmol) and PCC (486 mg, 2.25 mmol) in CH₂Cl₂ (8 mL) giving 119 (117 mg, 43% yield) as white solid, m.p. 170–172 °C. ¹H NMR (400 MHz, MeOD) δ 10.10 (s, 1H), 8.99 (s, 1H), ¹³C NMR (101 MHz, MeOD) δ 175.58, 160.36, 145.03, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₅H₃N₅NaOSNa⁺ 203.9951; found, 203.9947.

Pyridin-2-yl(1H-1,2,3-triazol-5-yl)methanone (120): Prepared the same way as 99 from pyridin-2-yl(1,2,3-triazol-4-yl)methanol (92 s) (240 mg, 1.2 mmol) and PCC (389 mg, 1.8 mmol) in CH₂Cl₂ (8 mL) giving 120 (114 mg, 55% yield) as white solid, m.p. 161–163 °C. ¹H NMR (400 MHz, MeOD) δ 9.00 (s, 1H), 8.85 − 8.74 (m, 1H), 8.27 (dt, J = 7.8, 1.2 Hz, 1H), 8.07 (td, J = 7.7, 1.7 Hz, 1H), 7.69 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 182.94, 153.16, 148.91, 137.36, 133.15, 127.47, 123.57, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₈H₆N₄ONa⁺ 197.0434; found, 197.0432.

Pyridin-3-yl(1H-1,2,3-triazol-5-yl)methanone (121, CAS [1523140–26-2]): Prepared the same way as 99 from pyridin-3-yl(1,2,3-triazol-4-yl)methanol (92t) (270 mg, 1.5 mmol) and PCC (486 mg, 2.25 mmol) in CH₂Cl₂ (10 mL) giving 121 (104 mg, 40% yield) as white solid. ¹H NMR (400 MHz, MeOD) δ 9.45 (d, J = 2.2 Hz, 1H), 8.81 (dd, J = 5.0, 1.7 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.51 (brs, 1H), 7.66 (dd, J = 8.0, 4.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 184.19, 152.35, 150.31, 138.15, 132.93, 123.80, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇N₄O⁺ 175.0614; found, 175.0613.

Pyridin-4-yl(1H-1,2,3-triazol-5-yl)methanone (122): Prepared the same way as 99 from pyridin-4-yl(1,2,3-triazol-4-yl)methanol (92 u) (165 mg, 1.0 mmol) and PCC (324 mg, 1.5 mmol) in CH₂Cl₂ (6 mL) giving 122 (96 mg, 52% yield) as white solid, m.p. 221–223 °C. ¹H NMR (400 MHz, MeOD) δ 8.89 − 8.76 (m, 2H), 8.57 (s, 1H), 8.25 − 8.12 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 184.76, 149.70, 145.27, 143.91, 132.09, 123.40, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇N₄O⁺ 175.0614; found, 175.0613.

(4-Bromophenyl)(4-(hydroxymethyl)-1H-1,2,3-triazol-5-yl)methanone (123) and (4-(Bromomethyl)-1H-1,2,3-triazol-5-yl)(4-bromophenyl)methanone (124): To a solution of 2-(prop-2-ynyloxy)-tetrahydro-2H-pyran (93) (1.43 g, 7.7 mmol) in THF (15 mL), n-BuLi (1.6 M, 4.82 mL, 7.7 mmol) was added at −78 °C. The reaction mixture was kept at −78 °C for 1 h, then 4-bromobenzaldehyde (982 mg, 7.0 mmol) was added at −78 °C. The reaction mixture was stirred for 2 h, then slowly warmed to −30 °C before being poured into an aqueous solution of NaHCO₃ (10 mL). After extraction with EtOAc (3 × 100 mL), the organic layer was collected, dried over anh. Na₂SO₄, filtered, and concentrated under reduced pressure to afford (1–(4-bromophenyl)-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-yn-1-ol (94) (2.1 g, 88%) as colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.56 − 7.41 (m, 4H), 5.50 (dd, J = 4.8, 3.0 Hz, 1H), 4.83 (t, J = 3.4 Hz, 1H), 4.37 (qt, J = 15.7, 1.6 Hz, 2H), 3.86 (ddd, J = 11.5, 9.0, 3.1 Hz, 1H), 3.55 (ddt, J = 12.6, 5.3, 2.8 Hz, 1H), 2.30 (d, J = 6.0 Hz, 1H), 1.93 − 1.59 (m, 4H), 1.56 (ddd, J = 11.6, 5.3, 3.0 Hz, 2H). To a solution of compound 94 (1.3 g, 4.0 mmol) in CH₂Cl₂ (50 mL), MnO₂ (3.6 g, 41.2 mmol) was added. The suspension was stirred at rt for 5 h and then filtered over a pad of celite. After removing the solvent under reduced pressure, the crude product was filtered over a pad of silica gel and washed with petroleum ether:EtOAc (10:1) to afford (1–(4-bromophenyl)-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-yn-1-one (95) (1.25 g, 97%) as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.14–8.17 (m, 2H), 7.61–7.66 (m, 1H), 7.48–7.53 (m, 1H), 4.90–4.92 (m, 1H), 4.57 (s, 2H), 3.86–3.92 (m, 1H), 3.58–3.61 (m, 1H), 1.55–1.86 (m, 6H). To a solution of compound 95 (970 mg, 3.0 mmol) in EtOH (10 mL), pyridinium p-toluenesulfonate (158 mg, 0.63 mmol) was added. The resulting solution was stirred for 4 h at 50 °C before being poured into water (30 mL). After extraction with Et₂O (3 × 50 mL), the organic layer was collected, dried over anh. Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC to afford (1–(4-bromophenyl)-4-hydroxybut-2-yn-1-one (96) (534 mg, 75%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.06 − 7.99 (m, 2H), 7.71 − 7.63 (m, 2H), 4.60 (d, J = 6.4 Hz, 2H). Compound 123 was synthesised from 96 (400 mg, 1.7 mmol) and TMSN₃ according to the general procedure (II) to give 123 (239 mg, 50%) as a brown fluffy powder, m.p. 240–242 °C. ¹H NMR (400 MHz, MeOD) δ 8.26 (d, J = 8.3 Hz, 2H), 7.81 − 7.68 (m, 2H), 5.02 (s, 2H), ¹³C NMR (101 MHz, MeOD) δ 186.07, 135.94, 131.74, 131.27, 127.73, 55.27, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₀H₈BrN₃O₂Na⁺ 303.9698; found, 303.9713. A solution of PPh₃ (135 mg, 0.52 mmol) and CBr₄ (173 mg, 0.52 mmol) in dry DMF (4 mL) was strirred for 10 min before adding compound 123 (121 mg, 0.43 mmol) and monitoring the reaction by TLC. After completion, the reaction was quenched with an aq. solution of sodium metabisulfite and extracted with EtOAc (3 × 5 mL). The organic layers were collected, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by FC (silica gel, EtOAc/hexane, 1:3) to afford 124 (125 mg, 85%) as white solid, m.p. 121–123 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 4.93 (s, 2H), ¹³C NMR (101 MHz, MeOD) δ 185.65, 135.72, 131.70, 131.34, 127.99, 22.80, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₀H₈BrN₃O₂Na⁺ 367.8833; found, 367.8830.

(4-(Aminomethyl)-1H-1,2,3-triazol-5-yl)(4-bromophenyl)methanone (126): To a solution of 4-bromobenzoyl chloride (1.32 g, 6.0 mmol) and t-butyl prop-2-yn-1-ylcarbamate (97) (621 mg, 4.0 mmol) in anhydrous THF (10 mL), PdCl₂(PPh₃)₂ (26 mg, 0.04 mmol) and CuI (23 mg, 0.12 mmol) were added under a nitrogen atmosphere. After 1 min of stirring, Et₃N (0.6 mL, 5.0 mmol) was added, and the reaction was stirred for 40 min at rt. During this time, Et₃NHCl precipitated, and the solution assumed a dark orange/brown colour. The reaction was diluted with Et₂O (30 mL) and washed with H₂O (30 mL). After extraction with CH₂Cl₂ (3 × 50 mL), the organic layers were combined, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by FC to afford (tert-butyl (4–(4-bromophenyl)-4-oxobut-2-yn-1-yl)carbamate (98) (1.82 g, 90%) as colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.05 − 7.92 (m, 2H), 7.74 − 7.58 (m, 2H), 4.90 (bs, 1H), 4.24 (d, J = 5.9 Hz, 2H), 1.51 (s, 9H). Tert-butyl ((5–(4-bromobenzoyl)-1H-1,2,3-triazol-5-yl)methyl)carbamate (125) was synthesised from 98 (500 mg, 1.5 mmol) and TMSN₃ according to the general procedure (II) to afford 125 (257 mg, 45%) as solid. ¹H NMR (400 MHz, CDCl₃) δ 8.31 (d, J = 8.3 Hz, 2H), 7.73 − 7.65 (m, 2H), 5.57 (s, 1H), 4.73 (d, J = 6.2 Hz, 2H), 1.48 (s, 9H). To a solution of compound 125 (190 mg, 0.5 mmol) in CH₂Cl₂ (4 mL), trifluoroacetic acid (0.37 mL, 5 mmol) was added and the mixture was stirred overnight at rt. The solution was concentrated, basified with NaHCO₃ solution, and extracted with EtOAc. The organic layer was dried over Na₂SO₄, filtered, concentrated under reduced pressure and triturated with Et₂O to afford 126 (105 mg, 75%) as white solid, m.p. 190–192 °C. ¹H NMR (400 MHz, MeOD) δ 8.25 − 8.18 (m, 2H), 7.75 − 7.69 (m, 2H), 4.36 (s, 2H), ¹³C NMR (101 MHz, DMSO) δ 185.98, 138.22, 132.82, 131.42, 126.27, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₁₀H₁₀BrN₄O⁺ 281.0032; found, 281.0038.

General procedure (V) for the synthesis of 3-Aryl-1H-1,2,4-Triazoles

The commercially available or synthetically prepared aryl nitriles (130) (1 mmol) were dissolved in DMF (3 mL), and formic acid (1.0 mL/mmol) and hydrazine hydrate (1.0 mL) were added. The reaction mixture was heated under reflux (90 °C) for 12 h. Upon completion of the reaction as determined by TLC, the reaction mixture was cooled to rt and poured into ice-water slurry (5 mL). After extraction with EtOAc (3 × 10 mL), the organic phases were collected, washed with brine solution (2 × 5 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by FC (SiO₂, EtOAc/petroleum ether) to afford aryl 1H-1,2,4-triazoles (134–154 and 158–161). Overall yields 20–45%.

Synthesis of 3-Aryl-1H-1,2,4-Triazoles (134–154 and 158–161)

3–(4-Fluorophenyl)-1H-1,2,4-triazole (134)⁸¹: Synthesised from 4-fluorobenzonitrile (130a) (123 mg, 1 mmol) according to the general procedure (V) to afford 134 (43 mg, 30%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.35 (s, 1H), 8.11 − 8.00 (m, 2H), 7.22 − 7.13 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 164.98, 162.51, 128.28, 128.20, 125.72, 115.51, 115.29, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇FN₃⁺ 164.0619; found, 164.0619.

3–(3-Bromophenyl)-1H-1,2,4-triazole (135)¹¹⁹: Synthesised from 3-bromobenzonitrile (130 b) (181 mg, 1 mmol) according to the general procedure (V) to afford 135 (55 mg, 25%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.46 (s, 1H), 8.22 (t, J = 1.8 Hz, 1H), 8.02 (dt, J = 7.9, 1.3 Hz, 1H), 7.63 (dt, J = 8.1, 1.3 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 134.46, 132.22, 130.32, 128.90, 124.69, 122.40, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇BrN₃⁺ 223.9818; found, 223.9815.

3–(3-Fluorophenyl)-1H-1,2,4-triazole (137)¹²⁰: Synthesised from 3-fluorobenzonitrile (130c) (121 mg, 1 mmol) according to the general procedure (V) to afford 137 (49 mg, 30%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.33 (d, J = 8.9 Hz, 1H), 7.84 (dt, J = 7.4, 1.3 Hz, 1H), 7.75 (ddd, J = 9.7, 2.7, 1.5 Hz, 1H), 7.44 (td, J = 8.0, 5.8 Hz, 1H), 7.13 (tdd, J = 8.4, 2.6, 1.1 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 164.28, 161.85, 130.47, 130.39, 121.87, 121.84, 112.85, 112.61, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇FN₃⁺ 164.0619; found, 164.0620.

2-Fluoro-6-(1H-1,2,4-triazol-3-yl)aniline (140, CAS [1503176–05-3]): Synthesised from 2-amino-3-fluorobenzonitrile (130d) (136 mg, 1 mmol) according to the general procedure (V) to afford 140 (44 mg, 25%) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.46 (brs, 1H), 7.71 (brs, 1H), 7.12 − 6.94 (m, 1H), 6.69 (td, J = 8.0, 5.2 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 153.20, 150.84, 115.43, 115.35, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈FN₄⁺ 179.0728; found, 179.0732.

2-Chloro-6-(1H-1,2,4-triazol-3-yl)aniline (141, CAS [1698578–50-5]): Synthesised from 2-amino-3-chlorobenzonitrile (130e) (152 mg, 1 mmol) according to the general procedure (V) to afford 141 (77 mg, 40%) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.44 (s, 1H), 7.99 (d, J = 2.6 Hz, 1H), 7.31 (dd, J = 8.8, 2.6 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 155.11, 130.65, 125.97, 123.92, 118.28, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈ClN₄⁺ 195.0432; found, 195.0431.

2-(1H-1,2,4-triazol-3-yl)benzene-1,3-diamine (142): Synthesised from 2,6-dinitrobenzonitrile (130f) (193 mg, 1 mmol) according to the general procedure (V) to afford 3–(2,6-dinitrophenyl)-1H-1,2,4-triazole 142a (77 mg, 40%) as yellow solid. ¹H NMR (400 MHz, MeOD) δ 8.60 (s, 1H), 8.33 (d, J = 8.2 Hz, 2H), 7.98 (t, J = 8.2 Hz, 1H). Following the same procedure as used for 155, 3–(2,6-dinitrophenyl)-1H-1,2,4-triazole (142a) (60 mg, 0.25 mmol) was mixed with SnCl₂ ·2H₂O (281 mg, 1.25 mmol) to obtain 142 (20 mg, 45%) as white solid, m.p. 152–154 °C. ¹H NMR (400 MHz, MeOD) δ 8.50 (s, 1H), 6.89 (t, J = 7.9 Hz, 1H), 6.22 (d, J = 7.9 Hz, 2H), ¹³C NMR (101 MHz, MeOD) δ 147.47, 147.41, 142.49, 129.70, 129.59, 105.78, 100.52, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₁₀N₅⁺ 176.0931; found, 176.0936.

4-Fluoro-2-(1H-1,2,4-triazol-3-yl)aniline (143, CAS [147004–95-3]): Synthesised from 2-amino-5-fluorobenzonitrile (130g) (136 mg, 1 mmol) according to the general procedure (V) to afford 143 (44 mg, 25%) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.46 (brs, 1H), 7.63 (brs, 1H), 6.95 (td, J = 8.5, 3.0 Hz, 1H), 6.85 (dd, J = 8.9, 4.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 156.18, 153.86, 142.73, 117.67, 117.59, 113.12, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈FN₄⁺ 179.0728; found, 179.0732.

4-Bromo-5-fluoro-2-(1H-1,2,4-triazol-3-yl)aniline (144): Synthesised from 2-amino5-bromo-4-fluorobenzonitrile (130h) (214 mg, 1 mmol) according to the general procedure (V) to afford 144 (72 mg, 28%) as a white solid, m.p. 243–246 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.47 (s, 1H), 8.05 (dd, J = 7.5, 2.0 Hz, 1H), 6.58 (dd, J = 10.4, 1.9 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 160.95, 148.13, 132.07, 102.62, 102.37, 93.00, 92.79, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₇BrFN₄⁺ 256.9833; found, 256.9834.

4-Chloro-N-methyl-2-(1H-1,2,4-triazol-3-yl)aniline (145): Synthesised from 5chloro-2-(methylamino)benzonitrile (130an) (166 mg, 1 mmol) according to the general procedure (V) to afford 145 (52 mg, 25%) as white solid, m.p. 207–209 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.24 (s, 1H), 7.89 (s, 1H), 7.54 (s, 1H), 6.70 (d, J = 8.9 Hz, 1H), 2.98 (s, 3H), ¹³C NMR (101 MHz, MeOD) δ 150.42, 129.22, 125.32, 121.42, 120.07, 117.27, 29.27, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₉H₁₀ClN₄⁺ 209.0589; found, 209.0587.

N-(4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenyl)acetamide (146): Synthesised from N-(4-chloro-2-cyanophenyl)acetamide (130aa) (194 mg, 1 mmol) according to the general procedure (V) to afford 146 (62 mg, 26%) as yellow amorphous solid. ¹H NMR (400 MHz, MeOD) δ 8.63 − 8.46 (m, 2H), 8.15 (d, J = 2.5 Hz, 1H), 7.39 (dd, J = 9.0, 2.5 Hz, 1H), 2.25 (s, 3H), ¹³C NMR (101 MHz, MeOD) δ 169.95, 135.41, 129.27, 128.19, 127.03, 121.97, 23.47, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₀H₉ClN₄ONa⁺ 259.0357; found, 259.035.

N-(4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenyl)propionamide (147): Synthesised from N-(4-chloro-2-cyanophenyl)propionamide (130ab) (208 mg, 1 mmol) according to the general procedure (V) to afford 147 (75 mg, 30%) as yellow solid, m.p. 168–171 °C. ¹H NMR (400 MHz, DMSO) δ 8.66 (s, 1H), 8.54 (d, J = 8.9 Hz, 1H), 8.11 (d, J = 2.6 Hz, 1H), 7.45 (dd, J = 9.1, 2.6 Hz, 1H), 2.43 (q, J = 7.5 Hz, 2H), 1.15 (t, J = 7.5 Hz, 2H), ¹³C NMR (101 MHz, MeOD) δ 173.64, 135.49, 131.91, 129.26, 128.03, 127.01, 121.90, 30.85, 8.63, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₁H₁₁ClN₄ONa⁺ 273.0514; found, 273.0521.

N-(4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenyl)butyramide (148): Synthesised from N-(4-chloro-2-cyanophenyl)butyramide (130ac) (222 mg, 1 mmol) according to the general procedure (V) to afford 148 (76 mg, 29%) as white solid, m.p. 156–158 °C. ¹H NMR (400 MHz, MeOD) δ 8.59 (d, J = 8.9 Hz, 2H), 8.18 (s, 1H), 7.41 (dd, J = 9.0, 2.6 Hz, 1H), 2.48 (dd, J = 8.4, 6.6 Hz, 2H), 1.87 − 1.74 (m, 2H), 1.04 (dd, J = 8.4, 6.5 Hz, 3H), ¹³C NMR (101 MHz, MeOD) δ 172.85, 135.44, 129.25, 128.09, 127.04, 121.93, 39.78, 18.68, 12.59, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₂H₁₃ClN₄ONa⁺ 287.0670; found, 287.0675.

N-(4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenyl)cyclopropanecarboxamide (149): Synthesised from N-(4-chloro-2-cyanophenyl)cyclopropanecarboxamide (130ad) (220 mg, 1 mmol) according to the general procedure (V) to afford 149 (71 mg, 27%) as white solid, m.p. 183–186 °C. ¹H NMR (400 MHz, DMSO) δ 8.60 (brs, 1H), 8.43 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 2.6 Hz, 1H), 7.42 (dd, J = 8.9, 2.6 Hz, 1H), 1.72 (td, J = 7.4, 3.8 Hz, 1H), 0.89 (dt, J = 8.6, 3.0 Hz, 6H), ¹³C NMR (101 MHz, MeOD) δ 173.32, 143.18, 134.08, 135.61, 129.13, 127.87, 127.09, 121.82, 15.74, 7.02, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₂H₁₁ClN₄ONa⁺ 285.0514; found, 285.0518.

N-(4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenyl)cyclobutanecarboxamide (150): Synthesised from N-(4-chloro-2-cyanophenyl)cyclobutanecarboxamide (130ae) (234 mg, 1 mmol) according to the general procedure (V) to afford 150 (69 mg, 25%) as a white solid, m.p. 201–203 °C. ¹H NMR (400 MHz, MeOD) δ 8.68 − 8.52 (m, 1H), 8.18 (d, J = 2.6 Hz, 1H), 7.41 (dd, J = 9.0, 2.6 Hz, 1H), 3.39 (qd, J = 8.7, 1.1 Hz, 1H), 2.50 2.27 (m, 4H), 2.18 − 2.00 (m, 1H), 1.99 − 1.88 (m, 1H), ¹³C NMR (101 MHz, MeOD) δ 174.81, 138.39, 128.01, 121.80, 41.39, 25.06, 17.41, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₃H₁₃ClN₄ONa⁺ 299.0670; found, 299.0677.

N-(4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenyl)cyclopentanecarboxamide (151): Synthesised from N-(4-chloro-2-cyanophenyl)cyclopentanecarboxamide (130af) (248 mg, 1 mmol) according to the general procedure (V) to afford 151 (96 mg, 33%) as white solid, m.p. 197–199 °C. ¹H NMR (400 MHz, DMSO) δ 11.63 (brs, 1H), 8.84 (brs, 1H), 8.63 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 2.6 Hz, 1H), 7.48 (dd, J = 8.9, 2.6 Hz, 1H), 2.85 (p, J = 8.1 Hz, 1H), 2.06 − 1.89 (m, 2H), 1.80 (dq, J = 11.4, 6.9 Hz, 2H), 1.74 − 1.61 (m, 2H), 1.68 − 1.54 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 175.97, 135.64, 129.28, 127.95, 127.03, 121.90, 29.99, 25.48, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₄H₁₅ClN₄ONa⁺ 313.0827; found, 313.0825.

N-(2-(1H-1,2,4-triazol-3-yl)phenyl)benzamide (152, CAS [2428213–63-0]): Synthesised from N-(2-cyanophenyl)benzamide (130bg) (222 mg, 1 mmol) according to the general procedure (V) to afford 152 (92 mg, 35%) as a white solid. ¹H NMR (400 MHz, MeOD) δ 8.81 (d, J = 8.4 Hz, 1H), 8.59 (s, 1H), 8.21 (s, 1H), 8.15 (dd, J = 8.1, 1.6 Hz, 2H), 7.70 − 7.54 (m, 3H), 7.55 − 7.44 (m, 1H), 7.27 (t, J = 7.6 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 166.35, 137.00, 134.84, 131.72, 129.91, 128.47, 127.47, 127.19, 123.36, 120.51, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₅H₁₂N₄ONa⁺ 287.0903; found, 287.0903.

N-(4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenyl)benzamide (153): Synthesised from N-(4-chloro-2-cyanophenyl)benzamide (130ag) (256 mg, 1 mmol) according to the general procedure (V) to afford 153 (95 mg, 32%) as white solid, m.p. 213–216 °C. ¹H NMR (400 MHz, MeOD) δ 8.85 (d, J = 9.0 Hz, 1H), 8.67 (s, 1H), 8.27 (s, 1H), 8.18 − 8.11 (m, 2H), 7.68 − 7.55 (m, 3H), 7.48 (dd, J = 9.0, 2.5 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 166.29, 143.40, 134.54, 131.88, 129.30, 128.54, 128.30, 127.22, 121.84, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₅H₁₁ClN₄ONa⁺ 321.0514; found, 321.0517.

N-(2-Chloro-6-(1H-1,2,4-triazol-3-yl)phenyl)benzamide (154): Synthesised from N-(2-chloro-6-cyanophenyl)-benzamide (130cg) (256 mg, 1 mmol) according to the general procedure (V) to afford 154 (101 mg, 34%) as amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 10.16 (s, 1H), 8.18 − 8.03 (m, 3H), 7.79 − 7.46 (m, 5H), 7.10 (t, J = 8.0 Hz, 1H), ¹³C NMR (101 MHz, CDCl₃) δ 166.58, 133.46, 133.01, 132.90, 132.48, 131.58, 128.84, 127.89, 127.10, 127.00, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₅H₁₁ClN₄ONa⁺ 321.0519; found, 321.0524.

4-(1H-1,2,4-Triazol-3-yl)pyridine (158)⁸¹: Synthesised from isonicotinonitrile (130i) (104 mg, 1 mmol) according to the general procedure (V) to afford 158 (59 mg, 40%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.71 − 8.58 (m, 1H), 8.15 (d, J = 6.8 Hz, 2H), 7.88 (tt, J = 7.8, 1.8 Hz, 1H), 7.40 (t, J = 6.3 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 149.41, 137.37, 124.59, 121.31, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₇H₇N₄⁺ 147.0665; found, 147.0660.

2-(1H-1,2,4-Triazol-3-yl)pyridine (159)⁸¹: Synthesised from picolinonitrile (130j) (104 mg, 1 mmol) according to the general procedure (V) to afford 159 (61 mg, 41%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.71 − 8.62 (m, 2H), 8.29 (s, 1H), 8.03 − 7.93 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 170.82, 149.45, 145.46, 120.68, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₇H₇N₄⁺ 147.0665; found, 147.0665.

2-(1H-1,2,4-Triazol-3-yl)pyridine-3-amine (160, CAS [53511–60-7]): Synthesised from 3-aminopicolinonitrile (130k) (119 mg, 1 mmol) according to the general procedure (V) to afford 160 (64 mg, 40%) as white solid. ¹H NMR (400 MHz, MeOD) δ 9.28 (s, 1H), 8.76 (s, 1H), 8.57 (brs, 1H), ¹³C NMR (101 MHz, MeOD) δ 148.91, 147.21, 140.28, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₆H₅ClN₅⁺ 182.0228; found, 182.0223.

2-(1H-1,2,4-Triazol-3-yl)thiophen-3-amine (161, CAS [1250372–48-5]): Synthesised from 3-aminothiophene-2-carbonitrile (130l) (124 mg, 1 mmol) according to the general procedure (V) to afford 161 (66 mg, 40%) as yellow solid. ¹H NMR (400 MHz, MeOD) δ 8.70 (s, 1H), 7.66 (d, J = 5.4 Hz, 1H), 7.14 (d, J = 5.4 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 154.71, 143.76, 129.26, 127.55, 123.03, 121.36, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₆H₇N₄S⁺ 167.0386; found, 167.0379.

2-Chloro-6-(1H-1,2,4-triazol-3-yl)pyrazine (162, CAS [1472615–55-6]): Synthesised from 6-chloropyrazine-2-carbonitrile (130m) (139 mg, 1 mmol) according to the general procedure (V) to afford 162 (76 mg, 42%) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.15 (s, 1H), 7.96 (dd, J = 4.3, 1.5 Hz, 1H), 7.28 − 7.13 (m, 2H), ¹³C NMR (101 MHz, MeOD) δ 143.31, 137.23, 125.05, 123.33, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₇H₈N₅⁺ 162.0774; found, 162.0770.

Synthesis of 3-Aryl-1H-1,2,4-Triazoles (132a–132c) and (155–157)

3–(3-Bromo-2-methyl-6-nitrophenyl)-1H-1,2,4-triazole (132a)⁸¹: A solution of 3-bromo-2-methyl-6-nitrobenzamide (131a) (387 mg, 1.5 mmol) and N,N-dimethylformamide-dimethyl acetal (DMF-DMA) (3.0 mL) was heated under reflux for 1 h under a nitrogen atmosphere. The mixture was cooled, concentrated under reduced pressure, and the residue was washed with hexane. After drying in vacuo, a white solid was obtained and used in the next step without purification. The crude product was dissolved in acetic acid (2 mL), and hydrazine hydrate (90 mg, 1.65 mmol) was added dropwise to the stirred solution. The mixture was stirred at 90 °C for 2 h. After cooling to rt, the solvent was removed under reduced pressure. The mixture was dissolved in diethyl ether (5 mL) and stirred at 0 °C for 30 min. The precipitate was collected, dried under reduced pressure, and purified by FC (SiO₂, EtOAc/petroleum ether) to give 132a (304 mg, 72%) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.70 (s, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.84 (m, 1H), 2.31 (s, 3H).

3–(3-Chloro-2-methyl-6-nitrophenyl)-1H-1,2,4-triazole (132b): Synthesised from 3-chloro-2-methyl-6-nitrobenzamide (131b) (214 mg, 1.0 mmol) according to the procedure used for 132a to afford 132b (167 mg, 70%) as white solid. ¹H NMR (400 MHz, MeOD) δ 9.19 (s, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 2.30 (s, 3H).

3–(3-Chloro-2,6-dinitrophenyl)-1H-1,2,4-triazole (132c): Synthesised from 3-chloro-2,6-dinitrobenzamide (131c) (244 mg, 1.0 mmol) according to the procedure used for 132a to afford 132c (214 mg, 80%) as white solid. ¹H NMR (400 MHz, MeOD) δ 8.60 (s, 1H), 8.24 (d, J = 8.9 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H).

4-Bromo-3-methyl-2-(1H-1,2,4-triazol-3-yl)aniline (155)⁸²: A mixture of 132a (141 mg, 0.5 mmol) and SnCl₂ ·2H₂O (560 mg, 2.5 mmol) in absolute EtOH (3 mL) was heated to 70 °C under a nitrogen atmosphere. The progress of the reaction was monitored by TLC. After 1.5 h the starting material has disappeared. The solution was cooled to rt and poured into ice. The pH was adjusted to a slightly basic value (7–8) by addition of 5% NaHCO₃ before extraction with EtOAc (3 × 5 mL). The collected organic phases were washed with brine, treated with charcoal and filtered. After drying over Na₂SO₄, the solvent was removed under reduced pressure and the residue was purified by FC (silica gel, EtOAc/hexanes) to give 155 (83 mg, 66%) as white solid, m.p. 201–204 °C. ¹H NMR (400 MHz, MeOD) δ 8.45 (brs, 1H), 7.37 (d, J = 8.8 Hz, 1H), 6.63 (d, J = 8.6 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 146.79, 137.07, 133.60, 114.57, 19.63, HRMS (ESI/QTOF) m/z: calcd for [M + H]+ C9H9BrN4 + 253.0089; found, 253.0092.

4-Chloro-3-methyl-2-(1H-1,2,4-triazol-3-yl)aniline (156): Prepared from 132b (119 mg, 0.5 mmol) and SnCl₂ ·2H₂O (560 mg, 2.5 mmol) according to the procedure used for 155 to afford 156 (62 mg, 59%) as white solid, m.p. 208–210 °C. ¹H NMR (400 MHz, MeOD) δ 8.46 (brs, 1H), 7.20 (d, J = 8.8 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), ¹³C NMR (101 MHz, MeOD) δ 146.21, 130.54, 135.42, 114.22, 16.55. HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₉H₉ClN₄⁺ 209.0594; found, 209.0597.

4-Chloro-2-(1H-1,2,4-triazol-3-yl)benzene-1,3-diamine (157): Prepared from 132c (135 mg, 0.5 mmol) and SnCl₂ ·H₂O (560 mg, 2.5 mmol) according to the procedure used for 155 to afford 157 (63 mg, 60%) as brown solid, m.p. 212–214 °C. ¹H NMR (400 MHz, MeOD) δ 7.96 (s, 1H), 7.46 (d, J = 8.9 Hz, 1H), 6.98 (d, J = 8.9 Hz, 1H).

Synthesis of aryl carbonitriles

5-Chloro-2-(methylamino)benzonitrile (130an)¹²¹: A mixture of 2-amino-5-chlorobenzonitrile (127a) (458 mg, 3.0 mmol), dimethyl oxalate (532 mg, 4.5 mmol) and t-BuOK (421 mg, 3.75 mmol) in DMA (6 mL) was heated under a nitrogen atmosphere to 130–140 °C for 5 h. The mixture was poured into ice water and extracted with EtOAc (3 × 10 mL). After concentration of the organic layers under reduced pressure, the crude product was purified by FC (silica gel, EtOAc/hexane) to afford 130an (345 mg, 70%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.42 7.34 (m, 2H), 6.65 − 6.58 (m, 1H), 4.68 (brs, 1H), 2.94 (dd, J = 5.1, 1.8 Hz, 3H).

N-(4-Chloro-2-cyanophenyl)acetamide (130aa)¹²²: To a stirred mixture of 2-amino-5-chlorobenzonitrile (127a) (456 mg, 3.0 mmol) in CH₂Cl₂ (6 mL), pyridine (260 mg, 3.3 mmol), DMAP (18 mg, 0.15 mmol), and acetyl chloride (128a) (351 mg, 4.5 mmol) were added. The reaction mixture was stirred at rt for 3 h, then diluted with CH₂Cl₂ (10 mL) and washed with H₂O (2 × 5 mL) and brine (2 × 5 mL). The combined organic phases were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was triturated with pentane and dried under air to afford 130aa (436 mg, 75%) as an oil. ¹H NMR (400 MHz, DMSO) δ 10.28 (bs, 1H), 7.97 (d, J = 2.5 Hz, 1H), 7.74 (dd, J = 8.8, 2.6 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 2.09 (s, 3H).

N-(4-Chloro-2-cyanophenyl)propionamide (130ab)¹²³: Synthesised the same way as 130aa from 2-amino-5-chlorobenzonitrile (127a) (456 mg, 3.0 mmol) and propionyl chloride (128 b) (304 mg, 3.3 mmol) to afford 130ab (530 mg, 85%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.51 − 8.43 (m, 1H), 7.61 − 7.53 (m, 2H), 2.52 (q, J = 7.5 Hz, 2H), 1.30 (td, J = 7.5, 1.1 Hz, 3H).

N-(4-Chloro-2-cyanophenyl)butyramide (130ac, CAS [53313–24-9]): Synthesised the same way as 130aa from 2-amino-5-chlorobenzonitrile (127a) (456 mg, 3.0 mmol) and butyryl chloride (128c) (350 mg, 3.3 mmol) to afford 130ac (566 mg, 85%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.50 − 8.42 (m, 1H), 7.57 (dq, J = 4.6, 2.5 Hz, 2H), 2.46 (t, J = 7.4 Hz, 2H), 1.81 (h, J = 7.4 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H).

N-(4-Chloro-2-cyanophenyl)cyclopropanecarboxamide (130ad): Synthesised the same way as 130aa from 2-amino-5-chlorobenzonitrile (127a) (456 mg, 3.0 mmol) and cyclopropanecarbonyl chloride (128d) (343 mg, 3.3 mmol) to afford 130ad (528 mg, 80%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.50 − 8.40 (m, 1H), 7.61 7.53 (m, 1H), 7.56 (bs, 1H), 7.50 (s, 1H), 3.22 (pd, J = 8.5, 1.1 Hz, 1H), 2.15 − 1.85 (m, 4H).

N-(4-Chloro-2-cyanophenyl)cyclobutanecarboxamide (130ae, CAS [2147559–95-1]): Synthesised the same way as 130aa from 2-amino-5-chlorobenzonitrile (127a) (456 mg, 3.0 mmol) and cyclobutanecarbonyl chloride (128e) (390 mg, 3.3 mmol) to afford 130ae (597 mg, 80%) as an oil. ¹H NMR (400 MHz, CDCl₃) δ 8.52 − 8.45 (m, 1H), 7.61 − 7.53 (m, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 3.27 (pd, J = 8.5, 1.1 Hz, 1H), 2.50 − 2.25 (m, 4H), 2.18 − 1.91 (m, 2H).

N-(4-chloro-2-cyanophenyl)cyclopentanecarboxamide (130af, CAS [2242379–82-2]): Synthesised the same way as 130aa from 2-amino-5-chlorobenzonitrile (127a) (456 mg, 3.0 mmol) and cyclopentanecarbonyl chloride (128f) (436 mg, 3.3 mmol) to afford 130af (632 mg, 85%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.52 − 8.45 (m, 1H), 7.61 − 7.53 (m, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 3.27 (pd, J = 8.5, 1.1 Hz, 1H), 2.50 − 2.25 (m, 4H), 2.18 − 1.91 (m, 4H).

N-(4-chloro-2-cyanophenyl)benzamide (130ag)¹²⁴: Synthesised the same way as 130aa from 2-amino-5-chlorobenzonitrile (127a) (456 mg, 3.0 mmol) and benzoyl chloride (128g) (462 mg, 3.3 mmol) to afford 130ag (600 mg, 78%) as an amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.64 (d, J = 8.8 Hz, 1H), 8.40 (s, 1H), 7.99 − 7.92 (m, 2H), 7.75 − 7.52 (m, 5H).

N-(2-cyanophenyl)benzamide (130bg)¹²⁵: Synthesised the same way as 130aa from 2-aminobenzonitrile (127b) (354 mg, 3.0 mmol) and benzoyl chloride (128g) (462 mg, 3.3 mmol) to afford 130bg (566 mg, 85%) as an oil. ¹H NMR (400 MHz, CDCl₃) δ 8.65 (d, J = 8.5 Hz, 1H), 8.45 − 8.40 (m, 1H), 8.01 − 7.93 (m, 2H), 7.73 − 7.52 (m, 5H), 7.25 (td, J = 7.7, 1.1 Hz, 1H).

N-(2-chloro-6-cyanophenyl)-benzamide (130cg): Synthesised the same way as 130aa from 2-amino-3-chloro-benzonitrile (127c) (456 mg, 3.0 mmol) and benzoyl chloride (128g) (462 mg, 3.3 mmol) to afford 130cg (614 mg, 80%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 8.04 − 7.97 (m, 2H), 7.93 (bs, 1H), 7.77–7.68 (m, 2H), 7.67 − 7.62 (m, 1H), 7.59–7.52 (m, 2H), 7.37 (t, J = 8.0 Hz, 1H).

Synthesis of aryl carboxamides

3-Bromo-2-methyl-6-nitrobenzamide (131a, CAS [110127–08-7]): A solution of 3-bromo-2-methylbenzoic acid (129a) (1.07 g, 5 mmol) in H₂SO₄ (98%, 1.1 mL) was treated with fuming nitric acid (1.1 mL) at rt. The reaction mixture was stirred for 3 h, cooled and poured into ice water (10 mL). The white precipitate was collected and dried. The aqueous filtrate was extracted with EtOAc (3 × 10 mL) and concentrated under reduced pressure. The residue was combined with the previously collected preciputate and used in the next step without further purification. To a slurry of crude compound in CH₂Cl₂ (20 mL) at 0 °C, oxalyl chloride (661 mg, 5.25 mmol) was added under a nitrogen atmosphere, followed by dropwise addition of DMF (2.5 mmol). The mixture was stirred 5 min at 0 °C, 15 min at rt, and then heated to reflux under N₂ for 1 h. The mixture was cooled and poured into NH₄OH (15 mL, ca. 30% NH₃). Precipitated solids were collected by filtration and purified by FC to afford 131a (903 mg, 70%) as a yellow oil. ¹H NMR (400 MHz, MeOD) δ 7.96 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 2.55 (s, 3H).

3-Chloro-2-methyl-6-nitrobenzamide (131b, CAS [51123–60-5]): Synthesised the same way as 131a from 3-chloro-2-methylbenzoic acid (129b) (850 mg, 5 mmol) to afford 131b (760 mg, 71%) as a yellow oil. ¹H NMR (400 MHz, MeOD) δ 8.06 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 2.51 (s, 3H).

3-Chloro-2–6-dinitrobenzamide (131c): Synthesised the same way as 131a from 3-chloro-2-nitrobenzoic acid (129c) (1 g, 5 mmol) to afford 131c (920 mg, 75%) as an oil. ¹H NMR (400 MHz, MeOD) δ 8.38 (d, J = 8.9 Hz, 1H), 8.00 (d, J = 8.9 Hz, 1H).

Synthesis of other 1,2,4-Triazoles

N-(4-Bromophenyl)-4H-1,2,4-triazol-3-amine (166): 1-Bromo-4-iodobenzene (283 mg, 1.0 mmol) was dissolved in N,N-dimethylacetamide and 4H-1,2,4-triazol-3-amine (163) (168 mg, 2.0 mmol), K₂CO₃ (207 mg, 1.5 mmol), and CuI (19.1 mg, 0.1 mmol) were added under a nitrogen atmosphere. The mixture was stirred at 90 °C for 48 h. After cooling to rt, the solids were filtered and washed with CH₂Cl₂ (2 × 10 mL). The combined organic phases were concentrated under reduced pressure and the residue was purified by FC (silica gel, EtOAc/hexane) to give 166 (93 mg, 39%) as a white solid, m.p. 190–192 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.88 (s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), ¹³C NMR (101 MHz, MeOD) δ 148.90, 135.93, 132.49, 125.22, 121.28, HRMS (ESI/QTOF) m/z: calcd for [M + H]⁺ C₈H₈BrN₄⁺ 238.9927; found, 238.9929.

2-((1,9a-Dihydrobenzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl)thio)acetamide(168): To the solution of (1,9a-dihydrobenzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl)thiol (164) (207 mg, 1.0 mmol) in acetone (5 mL), K₂CO₃ (138 mg, 1.0 mmol) and 2-chloroacetamide (165) (191 mg, 1.0 mmol) were added. The reaction mixture was heated under reflux for 9 h, then cooled to rt, filtered, and acidified with aq. 2 N HCl solution. The precipitate was filtered, washed with cold water (2 mL), and recrystallized from aqueous ethanol to afford 168 (160 mg, 60%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.92 − 7.77 (m, 2H), 7.10 (td, J = 8.5, 4.3 Hz, 2H), 6.91 − 6.73 (m, 2H), 4.54 (brs, 1H), ¹³C NMR (101 MHz, CDCl₃) δ 154.81, 152.73, 132.28, 132.26, 127.32, 127.30, 124.37, 118.59, 118.56, 110.64, HRMS (ESI/QTOF) m/z: calcd for [M + Na]⁺ C₁₀H₈N₄OS₂Na⁺ 287.0032; found, 287.0037.

Protein expression and purification

Full-length human IDO1 gene was cloned into the pET-28a vector and expressed in E. coli Rosetta (DE3) cells. Protein was produced in LB medium supplemented with 0.5 mM dALA (5-aminolevulinic acid) and was induced by addition of 0.1 mM isopropylthio-β-galactoside overnight at 16 °C. The culture was harvested and frozen at −80 °C until further use. The frozen cells were lysed by sonication in 20 mM Tris pH 7, 400 mM NaCl, 1 mM MgCl₂, 20 mM imidazole, 1 mM DTT, 0.5 mg/mL Lysozyme and Protease inhibitor. After centrifugation, the supernatant was loaded into a HisTrap HP column pre-equilibrated with buffer containing 20 mM Tris pH 7, 500 mM NaCl and 20 mM imidazole, and eluted with the same buffer containing 500 mM imidazole. Fractions containing IDO1 were pooled, concentrated, and injected on a size exclusion chromatography column (HiLoad Superdex S75 16/60) to increase purity. hIDO1 fractions were concentrated to 20 mg/mL in 20 mM Tris pH 7 and 150 mM NaCl.

Protein crystallisation, data collection, structure determination and model refinement

hIDO1 protein at 20 mg/mL was mixed with compound MMG-0472 at a final concentration of 5 mM, and co-crystallized by sitting drop vapour diffusion method. Crystals formed in a couple of days in 15% PEG 4000, 0.2 M lithium sulphate, and 0.1 M Tris pH 7.5. The crystals were cryoprotected with 25% glycerol. Diffraction data were collected at the Paul Scherrer Institute (SLS, Villigen) at PXII–X06DA beamline. Data were processed with the XDS Program Package¹²⁶. Structures were solved by molecular replacement using Phaser-MR and chain A of PDB entry 2d0t⁴⁰ as the model. Manual model building and structure refinement were carried out in Phenix Suite¹²⁷ using coot software¹²⁸ and phenix-refine, respectively. After validation, the ligand bound IDO1 model was deposited in the PDB database under PDB code 7zv3. Data collection and refinement statistics are summarised in the Supplementary Information (Table S1). The structures were displayed with PyMOL (http://www.pymol.org/) and UCSF Chimera¹²⁹. Authors will release the atomic coordinates and experimental data upon article publication.

Enzymatic assays

The enzymatic inhibition assays were performed as described by Takikawa et al.¹³⁰ with some modifications. Briefly, the reaction mixture (100 µL) contained potassium phosphate buffer (100 mM, pH 6.5) ascorbic acid (20 mM), catalase (400 units/mL), methylene blue (10 µM), purified recombinant IDO1 (2.5 ng/µL), L-Trp (100 µM), Triton X-100 (0.01%) and DMSO (5 µL). The inhibitors were serially diluted 3-fold for at least 8 different concentrations. After incubation at room temperature for 20 min, the reaction was stopped in its linear phase by addition of trichloroacetic acid solution (40 mL, 30% w/v), and the samples were incubated at 50 °C for 30 min. After centrifugation, 80 µl of supernatant from each well were used for HPLC analysis. The mobile phase for HPLC analysis was composed of 50% (v/v) of methanol and 50% of sodium citrate buffer (40 mM, pH 2.4) containing 400 µM sodium dodecyl sulphate. An Agilent Zorbax Eclipse XDB C18 column (150 × 4.6 mm) was used at 23 °C with a flow rate of 1 mL/min and an injection volume of 20 µL. For detection of kynurenine, absorption at 365 nm was measured, while remaining L-Trp was detected at a wavelength of 280 nm. All assays were carried out in duplicates. The activity (Act) of each compound was calculated as Act = RT log(IC₅₀) in analogy to the relation between the binding free energy and the K_i value. The ligand efficiency (LE) was calculated as LE = −Act/N_HA, where N_HA is the number of non-hydrogen atoms (heavy atoms) in the compound.

Cellular assays

The cDNA encoding hIDO1 (NCBI, NM 0021464.3) was purchased from Origene as pCMV6-Entry vector (RC206592). The full ORF of hIDO1 was cloned into the mammalian expression vector pcDNA6/myc-His from Invitrogen. Human embryonic kidney 293 T (HEK 293 T) cells were transiently transfected with the plasmid construct encoding hIDO1. A 75 cm² flask containing HEK 293 T cells at a confluency of 70% was transfected with jetPEI DNA transfection reagent (Polyplus-transfection), according to manufacturer’s protocol. A total of 20 µg of pcDNA-IDO1 along with 5 µg of pcDNA-GFP plasmid were used for the transfection; GFP was used to evaluate transfection efficiency prior to the cellular assays. 16–18 h post-transfection the 293 T cells were detached with trypsin, centrifuged, and re-suspended in 25 mL of DMEM medium without phenol red, supplemented with penicillin and streptomycin, 10% foetal bovine serum and 1 mM pyruvate (Gibco). Subsequently, 100 µL of cells were distributed to each well of two 96-well round-bottom plates that had been pre-loaded with 100 µL DMEM and the small organic test molecule in DMSO (for a final DMSO concentration of 2%). DMEM medium contains 78 µM L-Trp and was supplemented with 500 µM of L-Trp. Cells transfected with hIDO1 were incubated for 7 h at 37 °C in a CO₂ incubator. The reaction was stopped in its linear phase by adding trichloroacetic acid (TCA) to the medium at a final concentration of 4%. Plates were centrifuged for 15 min at 4,000 rpm. 100 µL of supernatant was added to 100 µL of 2% (w/v) DMAB in glacial acetic acid. After 10 min the absorbance was measured at a wavelength of 480 nm to detect kynurenine formation⁸⁵. Alternatively, kynurenine content was determined by HPLC as done for the enzymatic assay.

Cell viability

Following DMSO (negative control) or inhibitor treatments, apoptotic cells were detected by 4′,6-diamidino-2-phenylindole (DAPI) staining. Briefly, after 7 h of incubation, cells were washed with PBS, resuspended in PBS with 1 µg/mL DAPI and immediately analysed using a BD LSR II cytometer. A 405 nm laser with 450/50 nm bandpass filter was used to collect data.

Author contributions

U.F.R. designed the compounds, performed the enzymatic assays and the HPLC analysis of enzymatic and cellular samples, performed docking calculations, determined the X-ray crystallographic protein structures with F.P., analysed and interpreted the data. S.M.R. and P.V. planned, carried out, and documented chemical synthesis and characterisation. A.R. and F.P. performed protein expression and crystallography. N.D., P.R., and K.A. carried out the cellular assays under supervision of M.I. and G.C. O.M. and V.Z. initiated the project, provided critical feedback, and contributed to compound design. U.F.R., S.M.R., P.V., and V.Z. wrote the manuscript.

Supporting information

X-ray data collection and refinement statistics, previously determined cellular inhibition and toxicity data, binding poses of 5-substituted 4-aryl-1,2,3-triazoles, enzymatic dose-response curves of all compounds, HPLC purity traces, NMR spectra.

Accession codes

Compound MMG-0472, PDB ID 7zv3. Authors will release the atomic coordinates upon article publication.

Acknowledgements

We thank Bili Seijo from the Melita Irving and George Coukos laboratory for providing the IDO1 construct and Jean Philippe Gaudry from the Protein Production and Structure Core Facility for rhIDO1 production and purification. We would like to thank Ganesh Kumar Mothukuri for help with the analytical HPLC analysis for purity of the compounds. We acknowledge computational resources from Vital-IT (SIB Swiss Institute of Bioinformatics) and the Scientific Computing and Research Support Unit (University of Lausanne). We thank the local contacts of the Swiss Light Source beamline (SLS-PSI) for their support. We are grateful to Antoine Daina for fruitful discussions. MarvinSketch 21.15, 2021, and InstantJChem 21.2.0, 2020, ChemAxon (http://www.chemaxon.com), were used for displaying and handling chemical structures.

Disclosure statement

No potential conflict of interest was reported by the author(s).