Design and optimization of 1H-1,2,3-triazole-4-carboxamides as novel, potent, and selective inverse agonists and antagonists of PXR

Abstract

Pregnane X receptor (PXR) is a key regulator of drug metabolism. Many drugs bind to and activate PXR, causing adverse drug responses. This suggests that PXR inhibitors have therapeutic value, but potent PXR inhibitors have so far been lacking. Herein, we report the structural optimization of a series of 1H-1,2,3-triazole-4-carboxamides compounds that led to the discovery of compound 85 as a selective and the most potent inverse agonist and antagonist of PXR, with low nanomolar IC₅₀ values for binding and cellular activity. Importantly, compound 89, a close analog of 85, is a selective and pure antagonist with low nanomolar IC₅₀ values for binding and cellular activity. This study has provided novel, selective, and most potent PXR inhibitors (a dual inverse agonist/antagonist and a pure antagonist) for use in basic research and future clinical studies and also shed light on how to reduce the binding affinity of a compound to PXR.

Graphical Abstract

INTRODUCTION

Pregnane X receptor (PXR; also known as SXR, NR1I2), which was discovered in 1998 and belongs to the human nuclear receptor subfamily 1 group I (NR1I), is highly expressed in liver and intestine¹. PXR plays an important role in the absorption, distribution, metabolism, and elimination of xenobiotics and endobiotics, and it maintains metabolizing homeostasis^1–4. PXR also plays a key role in other physiologic and pathophysiologic processes, such as glucose and lipid homeostasis, inflammation, bone metabolism, oxidative stress, and cancer^{5, 6}.

PXR was originally identified as a master transcription factor regulating the expression of key genes that encode members of the phase I and phase II metabolic enzymes and drug transporters^7–9. PXR is the main regulator of the CYP3A4 gene¹⁰, which encodes cytochrome P450 3A4, the most abundant hepatic and intestinal phase I enzyme, which is responsible for metabolizing more than 50% of clinically used drugs, along with many other xenobiotics and endobiotics, in humans¹¹. In the absence of ligands, PXR is located in the cytoplasm and forms a complex with cytoplasmic constitutive active/androstane receptor retention protein (CCRP) and the chaperone protein heat-shock protein (Hsp)90¹². Once a ligand binds to PXR, PXR forms a heterodimer with retinoid X receptor (RXR)α and activates target gene transcription^{3, 4}.

PXR shares a common domain structure with other members of the nuclear receptor superfamily: an activation function 1 (AF-1), a DNA-binding domain (DBD), a hinge region, a ligand-binding domain (LBD), and an activation function 2 (AF-2)⁵. The highly conserved DBD is at the N-terminus and binds to short stretches of DNA in the target promoters. The LBD is at the C-terminus of PXR and serves as the docking site for ligands. Upon ligand binding, PXR changes its conformation in the AF-2 helix, interacts with accessory proteins, and regulates the expression of target genes¹³. The flexible, large, and relatively hydrophobic ligand-binding pocket of the PXR LBD enables binding of a wide range of hydrophobic ligands^{7, 14}. Since Watkins et al.¹⁵ (2001) first showed the crystal structure of the LBD with the PXR agonist SR12813, 49 structures of the LBD (as of 07/27/2022), encompassing a diverse set of agonists or apo structures, have been deposited in the Protein Data Bank (PDB). These structures exhibit a wide range of possible binding modes and molecular interactions¹⁶. However, no crystal structure of the PXR LBD in complex with an antagonist or inverse agonist has yet been reported (notes: both antagonist and inverse agonist are inhibitors. An inverse agonist is usually also an antagonist, but an antagonist might not be an inverse agonist).

Although PXR agonists have been extensively investigated and may cause adverse drug–drug or diet–drug interactions during drug therapy¹⁷, PXR activation might nevertheless be beneficial. For example, rifampicin (a potent human PXR [hPXR] agonist) has been shown to have an immune-suppressive effect in humans^{18, 19}. PXR may also be a novel target for treating inflammatory bowel disease²⁰. The antibiotic rifaximin (Xifaxan) (a gut-specific agonist of PXR) was approved by the FDA in May 2015 for treating this condition. In experimental models of colitis, rifaximin antagonized the effects of tumor necrosis factor alpha (TNF-α) on intestinal epithelial cells by activating PXR²¹.

PXR activation enhances drug metabolism, decreases drug efficacy, causes drug–drug interactions, and potentially leads to treatment failure⁶. One approach to avoiding or decreasing PXR activation by drug candidates is to design or modify compounds that have reduced or no PXR activating function for each drug target of interest. Although this is a tedious, resource-intensive process that often results in new drug candidates with altered chemical properties, multiple pharmaceutical companies (AstraZeneca, Bayer, Bristol-Myers Squibb, GlaxoSmithKline, Sanofi, and Xenon) have published the results of their efforts to design drug candidates with reduced potential for PXR activation¹⁶. Recently, Schneider et al. published a description of their novel inhibitors of B-Raf, based on dabrafenib, that do not bind or activate PXR²².

Instead of designing or modifying compounds for each drug target, the undesired effect of drug-induced PXR activation may be countered by developing potent and selective PXR inhibitors (antagonists and/or inverse agonists) and co-administering them as co-drugs with the PXR-activating drug. Developing PXR inhibitors has been an active area of research. Compounds that have other known bioactivities, such as metformin²³, ketoconazole^{24, 25}, and sulforaphane²⁶, have been reported to be PXR inhibitors. However, they failed to demonstrate in vivo activity in humans^{27, 28}, possibly because of toxicity and/or insufficient potency. Recently, several antagonists and inverse agonists of PXR with novel chemical scaffolds, low toxicity, and high selectivity have been reported, such as SJC2²⁹ and SPA70³⁰. So far, however, no PXR antagonist has been approved for clinical use, and more potent PXR antagonists might be needed for successful clinical studies. Figure 1 shows representative reported PXR agonists and antagonists.

Figure 1.

Structures of representative reported hPXR agonists (rifampicin, rifaximin, SR12813, and SJB7) and antagonists (ketoconazole, SPA70, sulforaphane, and metformin).

We recently reported the creation of a library of triazole analogs that function as PXR antagonists, identified by high-throughput screening (HTS)³¹. These antagonists include compounds with a sulfonyl linkage (such as 1-substituted-phenyl-4-substituted-phenylsulfonyl-5-methyl-1H-1,2,3-triazole) and compounds with a carbonyl amide linkage (such as 1-substituted-phenyl-4-substituted-phenylaminocarbonyl-1H-1,2,3-triazole). One of the lead compounds (with a sulfonyl linkage), 4-((4-(tert-butyl)phenyl)sulfonyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole (SPA70), is a selective antagonist of hPXR that has low toxicity^{30, 31}. Accordingly, we designed and evaluated a library of SPA70 analogs, leading to compounds with similar chemical structures but different cellular activities³². However, derivatization of SPA70 did not yield analogs with substantially more potent PXR inhibiting activity. In the present work, we combined what we learned from the optimization of SPA70 (which has a sulfonyl linkage³²) with the discovery of triazole analogs that have a carbonyl amide linkage³¹, with the goal of improving potency while maintaining high selectivity and low toxicity, as we achieved during the optimization of SPA70. Our medicinal chemistry effort led to selective and highly potent PXR inhibitors with low toxicity and low nanomolar IC₅₀ values in receptor binding and cell-based assays. Among these inhibitors are a dual antagonist and inverse agonist, compound 85 (SJPYT-306), and a pure antagonist, compound 89 (SJPYT-310), of hPXR (Figures 2 and 3).

Figure 2.

Design of and structural optimization strategies for 1H-1,2,3-triazole-4-carboxamides as novel modulators of hPXR with improved potency.

Figure 3.

Dose–response curves for a dual inverse agonist and antagonist (compound 85) and a pure antagonist (compound 89). The hPXR time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay was used to determine binding affinities (Binding). HepG2 hPXR-CYP3A4-luciferase stable cells were used to test the compounds in agonistic mode (Agonist) (a compound was tested alone) or antagonistic mode (Antagonist) (a compound was tested in the presence of 5 μM rifampicin). % Activity refers to % activation (for agonistic activity) or % inhibition (for the antagonistic activity or competitive binding). The same HepG2 cells were also used to evaluate compound cytotoxicity (% Cytotoxicity) as a control for the agonistic and antagonistic assays.

RESULTS AND DISCUSSION

Design and Initial Structure–Activity Relation (SAR) Study and Optimization of Compound 1

In our previous publication, we identified, by HTS, 1,4,5-substituted 1,2,3-triazole analogs as candidate hPXR antagonists. The triazole analogs can be divided into two series: those with a sulfonyl linkage and those with a carbonyl amide linkage³¹. Because, among the triazole analogs, SPA70 (with a sulfonyl linkage) is the most selective and potent hPXR antagonist/inverse agonist with low toxicity, we focused on the analogs with a sulfonyl linkage in our previous lead optimization³². However, SPA70-based lead optimization yielded no analog with substantially improved potency³². The amide functional group plays a critical role in many biologically active molecules, including many clinically approved drugs³³. Therefore, we replaced the sulfonyl linkage in SPA70 with a carbonyl amide linkage to generate a series of 1H-1,2,3-triazole-4-carboxamides analogs as exemplified by compound 1 (Figure 2). Compound 1 showed moderate binding (IC₅₀ = 1.2 μM) and very weak antagonist activity (IC₅₀ = 34 μM) (Table 1). No crystal structure of the PXR LBD complexed with an antagonist has yet been reported—this is one of the main challenges in PXR antagonist development. However, we have reported an hPXR LBD co-crystal structure with SJB7 (PDB code 5X0R), an hPXR agonist with a chemical structure almost identical to that of SPA70³⁰. As an initial step in the structure-guided design, PDB 5X0R was used for molecular docking. The docking experiments suggested that the tert-butyl moiety of compound 1 occupied a hydrophobic pocket (Figure 4B). The 2,5-dimethoxyphenyl portion of compound 1 is oriented similarly to that of SPA70 (Figure 4A), while failing to make significant contacts with residues of the AF-2 helix. The lack of these interactions between a ligand and regions of the AF-2 helix fails to maintain the AF-2 helix in the active orientation favorable for coactivator recruitment to the LBD, thereby disabling the ligand to activate hPXR. Repositioning of the tert-butyl substituent from the 4-position to the 3-position resulted in compound 2 displaying improved binding (IC₅₀ = 0.65 μM), inverse agonistic (IC₅₀ = 0.48 μM), and antagonistic (IC₅₀ = 4.1 μM) activities (Figure 2 and Table 1). Docking results indicate that compound 2 interacts with the hPXR LBD in a similar manner as SPA70 (Figure 4C).

Table 1.

Structures and hPXR activities of compounds 1–17

Compound	R1	Bindinga (IC50/μM) (%inhibition at 10 μM)	Agonistb (EC50/μM) (%activation at 10 μM)	Inverse agonistc (IC50/μM) (%inhibition at 10 μM)	Antagonistd (IC50/μM) (%inhibition at 10 μM)
T0901317		0.026 ± 0.003 (100 ± 1)%	NTe	NT	NT
Rifampicin		NT	1.17 ± 0.1 (100 ± 2)%	NA	NT
SPA70		0.21 ± 0.04 (87 ± 3)%	NAf	0.022 ± 0.004 (100 ± 2)%	0.23 ± 0.02 (100 ± 1)%
1		1.2 ± 0.4 (76 ± 7)%	NA	NA	34 ± 18 (58 ± 9)%
2		0.65 ± 0.3 (78 ± 2)%	NA	0.48 ± 0.1 (102 ± 11)%	4.1 ± 2 (92 ± 8)%
3		2.97 ± 0.1 (62 ± 4)%	0.74 ± 0.1 (35 ± 2)%	NA	30 ± 2 (37 ± 7)%
4		NA	25 ± 5 (33 ± 2)%	NA	NA
5		10.9 ± 0.4 (52 ± 5)%	3.5 ± 0.9 (30 ± 3)%	NA	NA
6		16.1 ± 0.5 (49 ± 1)%	NA	NA	NA
7		1.0 ± 0.6 (74 ± 8)%	NA	3.0 ± 0.8 (123 ± 18)%	4.8 ± 0.4 (82 ± 6)%
8		10.5 ± 2 (49 ± 4)%	NA	NA	NA
9		2.7 ± 0.5 (64 ± 4)%	NA	8.8 ± 1 (64 ± 8)%	21 ± 3 (56 ± 4)%
10		9.4 ± 4 (50 ± 4)%	NA	26 ± 6 (36 ± 4)%	NA
11		NA	NA	NA	NA
12		NA	NA	NA	NA
13		1.4 ± 0.7 (24 ± 6)%	NA	NA	NA
14		0.21 ± 0.06 (65 ± 0.5)%	NA	0.049 ± 0.003 (108 ± 5)%	0.15 ± 0.03 (107 ± 3)%
15		NA	NA	NA	NA
16		NA	10.2 ± 2 (34 ± 6)%	NA	NA
17		12.6 ± 3 (36 ± 3)%	NA	NA	NA

^aT0901317 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the hPXR binding assay;

^brifampicin at 10 μM as 100% activation and DMSO (0.3%) as 0% activation for the cell-based hPXR agonistic assay;

^cSPA70 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the cell-based hPXR inverse agonistic assay;

^dSPA70 at 10 μM with rifampicin (5 μM) as 100% inhibition and rifampicin alone (5 μM) as 0% inhibition for the cell-based hPXR antagonistic assay;

^eNT: not tested;

^fNA: No IC₅₀ value could be determined experimentally within the concentration range tested (the highest concentration tested was 30 μM).

Figure 4.

Docking poses of SPA70 (A), compound 1 (B), and compound 2 (C) in the ligand binding site of hPXR LBD (PDB code: 5X0R). Ligands are displayed as sticks with carbon atoms in olive green, oxygen atoms in red, nitrogen atoms in blue and sulfur atoms in yellow. Representative residues of hPXR LBD are represented as sticks and surface (light blue). The AF-2 helix is illustrated as cartoon (raspberry red).

SAR Studies at the R¹ Position

To better understand the SAR, we prepared compound 3 with a 2-(tert-butyl)phenyl group added to the amido nitrogen in the general scaffold of compound 1 (Table 1). The binding and cellular activities of compounds 1–3 indicated that the 3-tert-butyl, as in compound 2, was critical for retaining the PXR binding and inverse agonistic/antagonistic potencies of analogs, suggesting that an optimal angle and distance between the tert-butyl moiety and the carbonyl amide linkage was required. To better understand the SAR of the 3-position, the tert-butyl moiety in compound 2 (binding IC₅₀ = 0.65 μM, inverse agonistic IC₅₀ = 0.48 μM, antagonistic IC₅₀ = 4.1 μM) was replaced with various substituents in compounds 4–7. Compound 4, with a hydrogen atom, had no measurable PXR binding activity (although marginal agonistic activity was detected); compound 5, with a methyl group, and compound 6, with an ethyl group, had very weak hPXR binding activity. Compound 7, with an isopropyl substitution, had hPXR binding affinity (IC₅₀ = 1.0 μM) that was higher than that of compounds 4–6, and it retained PXR inverse agonistic and antagonistic activities (inverse agonistic IC₅₀ = 3.0 μM, antagonistic IC₅₀ = 4.8 μM), although these activities were less potent than those of compound 2, demonstrating that the isopropyl group was tolerated for retaining PXR inverse agonistic and antagonistic activities. Compounds 4–7 showed weaker PXR binding and cellular inverse agonistic and antagonistic activities when compared with compound 2, indicating that substituents smaller than a tert-butyl group were detrimental to PXR binding and cellular inverse agonistic and antagonistic activities.

We further explored the SAR of the 3-position by replacing the 3-tert-butyl group in compound 2 with various nitrogen substituents to generate compounds 8–13. The PXR binding activities of compounds 8–13 were weaker or had been lost, and almost all nitrogen-substituted compounds lost their PXR cellular activities. Compounds 9 (pyrrolidin-1-yl, binding IC₅₀ = 2.7 μM), 10 (piperidin-1-yl, binding IC₅₀ = 9.4 μM), and 13 (1H-pyrrol-1-yl, binding IC₅₀ = 1.4 μM) showed weaker binding activities with substituents larger than a tert-butyl group, indicating that larger substituents would lead to weaker binding activities. It was noteworthy that compounds 11 and 12, with a hydrophilic morpholino group and a 4-methylpiperazin-1-yl group, respectively, at the 3-position, exhibited no binding affinity or cellular activity, which demonstrated that the 3-position must be hydrophobic to retain activity.

Interestingly, compound 14, with 3,5-di-tert-butylphenyl substituents at the R¹ position in the general scaffold of compound 1, exhibited a binding affinity (binding IC₅₀ = 0.21 μM) that was higher than that of compound 2 (binding IC₅₀ = 0.65 μM). Compound 14 also displayed strong inverse agonistic (IC₅₀ = 0.019 μM) and antagonistic (IC₅₀ = 0.15 μM) activities. When we modified the R1 group or the 3-(tert-butyl)phenyl group of compound 2 by introducing different rings, as in compounds 15 (R¹ = 2-(tert-butyl)pyridin-4-yl), 16 (R¹ = 1-methyl-1H-benzo[d]imidazol-2-yl), and 17 (R¹ = 2,6-diethoxypyrimidin-4-yl), the resulting compounds had only marginal binding and cellular activities or had lost them completely. These findings demonstrated that the 3-tert-butyl substituent was beneficial to maintaining optimal hPXR binding activity and that there might be an additional hydrophobic site on hPXR that interacted with the hydrophobic 5-tert-butyl group in compound 14 (Figure 5). Therefore, the 3-tert-butyl group on the right phenyl ring of compound 2 had to be retained. The hydrophobic substituents at the 5-position of the right phenyl ring of compound 2 were explored in the next step of the SAR investigation.

Figure 5.

Presumed important hydrophobic pockets in the PXR LBD that interact with the hydrophobic substituents at the 3- and 5-positions of the right phenyl ring of compound 2 and its analogs.

SAR of the R² Position

After exploring various substituents on the 3-(tert-butyl) moiety and determining that the 3-tert-butyl group on the right phenyl ring of compound 2 had to be retained, we focused on optimizing the hydrophobic pocket around the 5-position. We first introduced different groups (amino or substituted amino groups and hydroxy or substituted hydroxy groups) at the R² position to probe the effect of bulkiness and polarity on compound activity (Table 2). Compounds with the amino group (compound 18) or its derivatives (compounds 19–22) and compounds with the hydroxyl group (compound 23) or its derivatives (compounds 24 and 25) all retained the inverse agonistic and antagonistic activities seen in compound 2. Both the amino group (as in compound 18) and the hydroxyl group (as in compound 23) reduced hPXR binding; however, introducing alkyl groups into the amino group (as in compounds 19–22) and the hydroxyl group (as in compounds 24 and 25) substantially improved both the binding and cellular activity. In addition, at the R² position, we introduced bromine (in compound 26) and a cyano group (in compound 27), both of which improved hPXR cellular activity. Interestingly, however, the maximal binding activity at a concentration of 10 μM decreased (Table 2) relative to that of compound 2 (Table 1). We also synthesized analogs with relatively large substituent groups. Among these, the compound with the benzene ring substituent (compound 28) had potent cellular activity; however, we could not determine the hPXR binding IC₅₀ for this compound because of assay interference at high compound concentrations. Introducing a pinacol boronate group (in compound 29) or a hydrophilic tetrazolium group (in compound 30) reduced binding. Together, these data suggest that the R² position has a relatively large hydrophobic pocket.

Table 2.

Structures and hPXR activities of compounds 18–30

Compound	R2	Bindinga (IC50/μM) (%inhibition at 10 μM)	Agonistb (EC50/μM) (%activation at 10 μM)	Inverse agonistc (IC50/μM) (%inhibition at 10 μM)	Antagonistd (IC50/μM) (%inhibition at 10 μM)
T0901317		0.026 ± 0.003 (100 ± 1)%	NTe	NT	NT
Rifampicin		NT	1.17 ± 0.1 (100 ± 2)%	NA	NT
SPA70		0.21 ± 0.04 (87 ± 3)%	NAf	0.022 ± 0.004 (100 ± 2)%	0.23 ± 0.02 (100 ± 1)%
18		3.0 ± 0.05 (64 ± 2)%	NA	2.3 ± 0.23 (87 ± 1)%	3.9 ± 0.2 (93 ± 6)%
19		0.33 ± 0.1 (77 ± 6)%	NA	0.12 ± 0.02 (126 ± 4)%	0.41 ± 0.1 (109 ± 6)%
20		0.27 ± 0.04 (70 ± 2)%	NA	0.11 ± 0.02 (120 ± 5)%	0.34 ± 0.04 (110 ± 3)%
21		0.45 ± 0.1 (76 ± 1)%	NA	0.039 ± 0.01 (110 ± 12)%	0.13 ± 0.03 (109 ± 5)%
22		0.50 ± 0.1 (76 ± 2)%	NA	0.15 ± 0.05 (89 ± 7)%	0.62 ± 0.3 (82 ± 17)%
23		2.8 ± 0.8 (82 ± 4)%	NA	0.34 ± 0.04 (103 ± 10)%	2.0 ± 0.3 (104 ± 3)%
24		0.70 ± 0.2 (84 ± 8)%	NA	0.12 ± 0.03 (114 ± 3)%	0.50 ± 0.2 (108 ± 5)%
25		0.63 ± 0.1 (70 ± 2)%	NA	0.054 ± 0.007 (113 ± 1)%	0.21 ± 0.09 (107 ± 3)%
26		0.085 ± 0.01 (22 ± 4)%	NA	0.14 ± 0.02 (139 ± 7)%	0.64 ± 0.03 (107 ± 2)%
27		0.26 ± 0.04 (43 ± 1)%	NA	0.12 ± 0.02 (138 ± 27)%	0.47 ± 0.06 (109 ± 1)%
28		NAg	NA	0.014 ± 0.001 (139 ± 16)%	0.12 ± 0.03 (109 ± 2)%
29		2.4 ± 0.5 (67 ± 1)%	NA	0.33 ± 0.1 (138 ± 19)%	1.6 ± 0.2 (104 ± 2)%
30		2.4 ± 0.4 (75 ± 2)%	NA	21.5 ± 6 (65 ± 12)%	NA

^aT0901317 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the hPXR binding assay;

^brifampicin at 10 μM as 100% activation and DMSO (0.3%) as 0% activation for the cell-based hPXR agonistic assay;

^cSPA70 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the cell-based hPXR inverse agonistic assay;

^dSPA70 at 10 μM with rifampicin (5 μM) as 100% inhibition and rifampicin alone (5 μM) as 0% inhibition for the cell-based hPXR antagonistic assay;

^eNT: not tested;

^fNA: No IC₅₀ value could be determined experimentally within the concentration range tested (the highest concentration tested was 30 μM).

^g28: At high concentrations, compound 28 displayed assay interference, affecting measurement at the absorption wavelength in the binding experiment, and no value could be obtained at 10 μM.

SAR of the R³ Position

After exploring the SAR at the 5-position, we examined the SAR at the 4-position on the right phenyl ring of compound 2. Similar to the exploration strategy applied to the 5-position, amino or substituted amino groups and a hydroxy group or alkoxy groups were assessed at the 4-position (Table 3).

Table 3.

Structures and hPXR activities of compounds with general scaffold 3

Compound	R3	Bindinga (IC50/μM) (%inhibition at 10 μM)	Agonistb (EC50/μM) (%activation at 10 μM)	Inverse agonistc (IC50/μM) (%inhibition at 10 μM)	Antagonistd (IC50/μM) (%inhibition at 10 μM)
T0901317		0.026 ± 0.003 (100 ± 1)%	NTe	NT	NT
Rifampicin		NT	1.17 ± 0.1 (100 ± 2)%	NA	NT
SPA70		0.21 ± 0.04 (87 ± 3)%	NAf	0.022 ± 0.004 (100 ± 2)%	0.23 ± 0.02 (100 ± 1)%
2	H	0.65 ± 0.3 (78 ± 2)%	NA	0.48 ± 0.1 (102 ± 11)%	4.1 ± 2 (92 ± 8)%
31		4.1 ± 1.4 (86 ± 3)%	0.28 ± 0.09 (33 ± 7)%	NA	NA
32		1.8 ± 0.5 (65 ± 7)%	2.0 ± 0.2 (50 ± 14)%	NA	NA
33		0.75 ± 0.2 (92 ± 3)%	0.3 ± 0.04 (28 ± 18)%	NA	14.1 ± 3.7 (68 ± 4)%
34		0.47 ± 0.1 (78 ± 1)%	0.57 ± 0.04 (47 ± 4)%	NA	7.5 ± 0.9 (44 ± 5)%
35		0.73 ± 0.08 (84 ± 2)%	NA	NA	3.9 ± 0.4 (64 ± 4)%
36		4.1 ± 0.48 (69 ± 1)%	NA	0.21 ± 0.08 (138 ± 24)%	0.86 ± 0.1 (108 ± 5)%
37		3.0 ± 0.30 (66 ± 1)%	NA	0.34 ± 0.1 (95 ± 26)%	1.7 ± 0.3 (107 ± 6)%
38		NA	NA	3.2 ± 0.3 (73 ± 1)%	7.3 ± 0.9 (60 ± 5)%

^aT0901317 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the hPXR binding assay;

^brifampicin at 10 μM as 100% activation and DMSO (0.3%) as 0% activation for the cell-based hPXR agonistic assay;

^cSPA70 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the cell-based hPXR inverse agonistic assay;

^dSPA70 at 10 μM with rifampicin (5 μM) as 100% inhibition and rifampicin alone (5 μM) as 0% inhibition for the cell-based hPXR antagonistic assay;

^eNT: not tested;

^fNA: No IC₅₀ value could be determined experimentally within the concentration range tested (the highest concentration tested was 30 μM).

Introducing an amino modification (in compound 31, hPXR binding IC₅₀ = 4.1 μM, agonistic EC₅₀ = 0.28 μM) or an acetylamino modification (in compound 32, hPXR binding IC₅₀ = 1.8 μM, agonistic EC₅₀ = 2.0 μM) at the 4-position reduced the binding activity and resulted in analogs that were weak hPXR agonists, as compared to compound 2 (hPXR binding IC₅₀ = 0.65 μM, no agonistic activity, inverse agonistic IC₅₀ = 0.48 μM, antagonistic IC₅₀ = 4.1 μM). Analogs with a methylamino modification (compound 33) or a dimethylamino modification (compound 34) retained hPXR binding affinity, but they were partial agonists and partial antagonists of hPXR. Introducing a butylamino group at the 4-position created compound 35 as an hPXR antagonist with binding affinity similar to that of the parental compound 2 (hPXR binding IC₅₀ = 0.73 μM, no agonistic activity, no inverse agonistic activity, antagonistic IC₅₀ = 3.9 μM). Hydroxylation or alkoxylation at the 4-position generated compounds 36, 37, and 38, which had reduced hPXR binding affinity when compared to compound 2. Compound 38 is almost inactive, and no binding activity could be detected. The activities of compounds 31–38 suggest that introducing amino and hydroxyl groups or their derivatives at the R³ position does not improve the PXR binding and cellular activities of the analogs.

SAR of the R⁴ Position

After exploring the SAR at the 5-position and 4-position of the right phenyl ring of compound 2, we focused on studying the SAR at the 6-position (the R⁴ position on general scaffold 4 in Table 4) of the right phenyl ring of compound 2 by introducing amino and hydroxyl groups or their derivatives.

Table 4.

Structures and hPXR activities of compounds with general scaffold 4

Compound	R4	Bindinga (IC50/μM) (%inhibition at 10 μM)	Agonistb (EC50/μM) (%activation at 10 μM)	Inverse agonistc (IC50/μM) (%inhibition at 10 μM)	Antagonistd (IC50/μM) (%inhibition at 10 μM)
T0901317		0.026 ± 0.003 (100 ± 1)%	NTe	NT	NT
Rifampicin		NT	1.17 ± 0.1 (100 ± 2)%	NA	NT
SPA70		0.21 ± 0.04 (87 ± 3)%	NAf	0.022 ± 0.004 (100 ± 2)%	0.23 ± 0.02 (100 ± 1)%
2	H	0.65 ± 0.3 (78 ± 2)%	NA	0.48 ± 0.1 (102 ± 11)%	4.1 ± 2 (92 ± 8)%
39		NA	0.90 ± 0.2 (25 ± 7)%	NA	NA
40		3.2 ± 2.3 (60 ± 9)%	0.99 ± 0.22 (60 ± 13)%	NA	NA
41		0.36 ± 0.03 (75 ± 4)%	0.15 ± 0.02 (63 ± 7)%	NA	12 ± 4 (24 ± 6)%
42		2.3 ± 0.4 (84 ± 3)%	NA	NA	NA
43		1.1 ± 0.3 (64 ± 7)%	NA	NA	1.8 ± 0.3 (80 ± 1)%
44		0.17 ± 0.04 (73 ± 3)%	NA	0.077 ± 0.002 (118 ± 2)%	0.46 ± 0.07 (105 ± 4)%
45		0.027 ± 0.006 (79 ± 2)%	NA	0.055 ± 0.01 (114 ± 13)%	0.28 ± 0.03 (101 ± 4)%
46		0.022 ± 0.01 (90 ± 3)%	NA	0.024 ± 0.006 (89 ± 9)%	0.24 ± 0.08 (101 ± 2)%
47		0.039 ± 0.01 (86 ± 1)%	NA	0.043 ± 0.003 (109 ± 5)%	0.21 ± 0.012 (103 ± 2)%
48		0.027 ± 0.007 (73 ± 3)%	NA	0.044 ± 0.009 (116 ± 6)%	0.24 ± 0.03 (101 ± 5)%
49		0.58 ± 0.2 (53 ± 0.4)%	NA	0.44 ± 0.1 (83 ± 12)%	2.2 ± 0.03 (72 ± 6)%
50		1.3 ± 0.23 (48 ± 5)%	NA	NA	1.3 ± 0.1 (49 ± 6)%
51		1.2 ± 0.3 (78 ± 2)%	NA	5.1 ± 1.9 (157 ± 18)%	3.4 ± 0.01 (93 ± 4)%
52		0.28 ± 0.09 (87 ± 1)%	NA	1.8 ± 0.1 (113 ± 11)%	1.7 ± 0.04 (105 ± 3)%
53		31 ± 5 (47 ± 3)%	NA	8.9 ± 1 (137 ± 12)%	7.2 ± 0.8 (112 ± 3)%
54		0.54 ± 0.07 (68 ± 5)%	NA	1.4 ± 0.4 (81 ± 7)%	4.6 ± 1 (91 ± 4)%
55		6.5 ± 0.6 (49 ± 4)%	NA	4.7 ± 3 (86 ± 3%)	12 ± 0.8 (73 ± 3%)
56		NA	NA	10 ± 3 (127 ± 7)%	7 ± 0.9 (109 ± 4)%

^aT0901317 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the hPXR binding assay;

^brifampicin at 10 μM as 100% activation and DMSO (0.3%) as 0% activation for the cell-based hPXR agonistic assay;

^cSPA70 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the cell-based hPXR inverse agonistic assay;

^dSPA70 at 10 μM with rifampicin (5 μM) as 100% inhibition and rifampicin alone (5 μM) as 0% inhibition for the cell-based hPXR antagonistic assay;

^eNT: not tested;

^fNA: No IC₅₀ value could be determined experimentally within the concentration range tested (the highest concentration tested was 30 μM).

Amino, acetylamino, and (tert-butoxycarbonyl)amino groups (in compounds 39, 40, and 41, respectively) were the first to be investigated at the R⁴ position. However, these modifications either almost abolished the hPXR activities (as in compound 39) or resulted in analogs with hPXR agonistic activities (compound 40 and 41).

Next, we focused on using the hydroxy group and its derivatives to explore further the SAR of the R⁴ position. All analogs with a hydroxy or substituted hydroxy group at the R⁴ position lacked hPXR agonistic activity. Instead, we observed improved hPXR binding and improved hPXR inverse agonistic and antagonistic activities among these analogs. With a hydroxy group at the R⁴ position, compound 42 had reduced hPXR binding (IC₅₀ = 2.3 μM) and no detectable cellular activities, possibly because it lacked cell permeability. However, when the hydroxy group at the R⁴ position was replaced with a more lipophilic or bulky group, such as a methoxy group (in compound 43; binding IC₅₀ = 1.1 μM, antagonistic IC₅₀ = 1.8 μM), an ethoxy group (in compound 44; binding IC₅₀ = 0.17 μM, inverse agonistic IC₅₀ = 0.077 μM, antagonistic IC₅₀ = 0.46 μM), a propoxy group (in compound 45; binding IC₅₀ = 0.027 μM, inverse agonistic IC₅₀ = 0.055 μM, antagonistic IC₅₀ = 0.28 μM), or a butoxy group (in compound 46; binding IC₅₀ = 0.022 μM, inverse agonistic IC₅₀ = 0.024 μM, antagonistic IC₅₀ = 0.24 μM), the resulting analogs had improved hPXR binding and cellular activities, with compound 46 being the most potent in this respect. With a butoxy group at the R⁴ position, the hPXR binding potency of compound 46 was 105 fold higher than that of compound 42, which has a hydroxy group at the R⁴ position. Compared to the EC₅₀ and IC₅₀ values of the parental compound 2, which has a proton at the R⁴ position, the potency of compound 46 was increased 30 fold, 20 fold, and 17 fold in terms of hPXR binding, inverse agonistic activity, and antagonistic activity, respectively. Further lipophilic modifications of the hydroxy group in compound 42, as in compounds 47 (with a pentyloxy group), 48 (with a hexyloxy group), and 49 (with an octyloxy group), did not further increase the hPXR binding, inverse agonistic activity, or antagonistic activity of the analogs, although the hPXR activities were generally maintained.

Although a butoxy group at the R⁴ position resulted in an analog (compound 46) with the most potent hPXR binding, inverse agonistic activity, and antagonistic activity, the respective Log P and cLogP values for compound 46 were 5.77 and 5.31 (as predicted by ChemDraw version 17.1.0.105), suggesting that compound 46 was too lipophilic to be a good drug-like molecule³⁴. To reduce the lipophilicity of the compound while retaining the alkoxy substituent feature at the R⁴ position, we introduced alkoxy substituents with terminal hydrophilic groups, such as a terminal cyano group (in compound 50), a terminal hydroxy group (in compound 51), terminal dimethylamino groups (in compounds 52 and 53), terminal morpholino groups (in compounds 54 and 55), and a terminal 4-methylpiperazin-1-yl group (in compound 56). However, compounds with those hydrophilic modifications did not retain the optimal hPXR activity observed in compound 46. These data suggest that the binding pocket around this end of the molecule is a hydrophobic pocket that can accommodate molecules with a certain side chain length. For instance, compounds 52 and 54 have shorter side chains and bind stronger than 53 and 55, respectively.

The SAR exploration of the amide scaffold at positions 3, 4, 5, and 6 of the right phenyl ring led to compound 46, the most potent hPXR dual inverse agonist and antagonist with the highest binding affinity so far (hPXR binding IC₅₀ = 0.022 μM, no agonistic activity, inverse agonistic IC₅₀ = 0.024 μM, antagonistic IC₅₀ = 0.24 μM). Compared to the initial lead compound 2, compound 46 had 30-fold increased hPXR binding activity, 20-fold increased inverse agonistic activity, and 17-fold increased antagonistic activity (hPXR binding IC₅₀ = 0.65 μM, no agonistic activity, inverse agonistic IC₅₀ = 0.48 μM, antagonistic IC₅₀ = 4.1 μM) (Figure 6). Compound 46 was, therefore, chosen as the new lead compound for substituent optimization at the left phenyl ring of the amide scaffold.

Figure 6.

Right ring optimization of the amide scaffold created compound 46, the most potent hPXR inverse agonist and antagonist discovered. Compound 46 was chosen as the new lead compound for further optimization, focusing on its left phenyl ring (the fold change was calculated by dividing the activity of compound 2 [IC₅₀ or EC₅₀ value] by the corresponding activity of compound 46).

SAR of the R⁵ Position

After exploring the SAR of the amide scaffold at its right phenyl ring, we focused on optimizing the left phenyl ring SAR, using compound 46 as the new initial lead compound (Table 5).

Table 5.

Structures and hPXR activities of compounds with general scaffold 5

Compound	R5	Bindinga (IC50/μM) (%inhibition at 10 μM)	Agonistb (EC50/μM) (%activation at 10 μM)	Inverse agonistc (IC50/μM) (%inhibition at 10 μM)	Antagonistd (IC50/μM) (%inhibition at 10 μM)
T0901317		0.026 ± 0.003 (100 ± 1)%	NTe	NT	NT
Rifampicin		NT	1.17 ± 0.1 (100 ± 2)%	NA	NT
SPA70		0.21 ± 0.04 (87 ± 3)%	NAf	0.022 ± 0.004 (100 ± 2)%	0.23 ± 0.02 (100 ± 1)%
46		0.022 ± 0.01 (90 ± 3)%	NA	0.024 ± 0.006 (89 ± 9)%	0.24 ± 0.08 (101 ± 2)%
57		0.022 ± 0.004 (88 ± 2)%	NA	NA	0.3 ± 0.1 (76 ± 5)%
58		0.016 ± 0.003 (94 ± 2)%	NA	NA	0.12 ± 0.02 (88 ± 2)%
59		0.04 ± 0.009 (94 ± 3)%	NA	0.0069 ± 0.001 (61 ± 4)%	0.094 ± 0.009 (95 ± 2)%
60		0.059 ± 0.01 (86 ± 2)%	NA	NA	0.70 ± 0.06 (74 ± 5)%
61		0.070 ± 0.009 (82 ± 2)%	NA	NA	0.46 ± 0.06 (83 ± 5)%
62		0.014 ± 0.003 (95 ± 2)%	NA	NA	0.074 ± 0.02 (84 ± 2)%
63		0.053 ± 0.04 (18 ± 1)%	1.48 ± 0.2 (48 ± 1)%	NA	NA
64		0.016 ± 0.001 (40 ± 8)%	NA	NA	NA
65		1.31 ± 0.06 (28 ± 8)%	NA	NA	NA
66		0.12 ± 0.002 (10 ± 0.4)%	1.68 ± 0.3 (16 ± 6)%	NA	NA
67		0.12 ± 0.006 (38 ± 5)%	2.41 ± 0.6 (42 ± 8)%	NA	NA
68		7.6 ± 2 (75 ± 5)%	4.3 ± 1 (44 ± 4)%	NA	NA
69		NA	NA	NA	NA
70		0.028 ± 0.004 (86 ± 1)%	0.48 ± 0.2 (22 ± 6)%	NA	0.40 ± 0.08 (68 ± 2)%
71		1.1 ± 1 (64 ± 2)%	NA	NA	0.44 ± 0.1 (32 ± 10)%
72		0.53 ± 0.02 (14 ± 12)%	3.7 ± 0.7 (36 ± 5)%	NA	NA
73		NA	NA	NA	NA
74		0.33 ± 0.2 (75 ± 5)%	1.0 ± 0.09 (83 ± 4)%	NA	NA
75		0.44 ± 0.3 (54 ± 11)%	4.1 ± 1 (86 ± 7)%	NA	NA
76		0.093 ± 0.005 (75 ± 3)%	0.24 ± 0.09 (45 ± 2)%	NA	1.44 ± 0.2 (26 ± 3)%
77		NA	4.4 ± 0.08 (26 ± 4)%	NA	NA
78		NA	2.8 ± 0.5 (32 ± 4)%	NA	NA
79		NA	4.6 ± 1 (43 ± 8)%	NA	NA

^aT0901317 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the hPXR binding assay;

^brifampicin at 10 μM as 100% activation and DMSO (0.3%) as 0% activation for the cell-based hPXR agonistic assay;

^cSPA70 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the cell-based hPXR inverse agonistic assay;

^dSPA70 at 10 μM with rifampicin (5 μM) as 100% inhibition and rifampicin alone (5 μM) as 0% inhibition for the cell-based hPXR antagonistic assay;

^eNT: not tested;

^fNA: No IC₅₀ value could be determined experimentally within the concentration range tested (the highest concentration tested was 30 μM).

First, we replaced the methoxy group at the 5-position of the 2,5-dimethoxyphenyl with halogens (57–59) or trifluoromethyl (60), trifluoromethoxy (61), or methyl (62) groups. Like compound 46, all of these analogs lacked hPXR agonistic activity, and except for compound 59, they were pure hPXR antagonists with no hPXR inverse agonistic activity. Like compound 46, compound 59 was a dual hPXR inverse agonist and antagonist. By comparing the activities of these analogs, we found that the compounds with a substituted chloro group (compound 58, hPXR binding IC₅₀ = 0.016 μM, hPXR antagonistic IC₅₀ = 0.12 μM) or a methyl group (compound 62, hPXR binding IC₅₀ = 0.014 μM, hPXR antagonistic IC₅₀ = 0.074 μM) had improved binding and antagonistic activities when compared to compound 46 (hPXR binding IC₅₀ = 0.022 μM, hPXR inverse agonistic IC₅₀ = 0.024 μM, hPXR antagonistic IC₅₀ = 0.24 μM). Compound 59, with a substituted bromo group (hPXR binding IC₅₀ = 0.04 μM, hPXR inverse agonistic IC₅₀ = 0.0069 μM, hPXR antagonistic IC₅₀ = 0.094 μM), retained potent binding activity with improved inverse agonistic and antagonistic activities. Analogs with strong electron-withdrawing groups, such as a trifluoromethyl group (in compound 60, hPXR binding IC₅₀ = 0.059 μM, hPXR antagonistic IC₅₀ = 0.70 μM) or a trifluoromethoxy group (in compound 61, hPXR binding IC₅₀ = 0.070 μM, PXR antagonistic IC₅₀ = 0.46 μM), also displayed potent hPXR binding and antagonistic activities, although these activities were not as potent as those of compounds 57–59 and 62. These data suggest that modification of the 5-methoxy group is well tolerated and that analogs with such modifications generally retain potent PXR antagonistic activities.

Next, we modified the methoxy group at the 2-position of the 2,5-dimethoxyphenyl of compound 46. We used halogens (in compounds 63–65) and a methyl group (in compound 66) to replace the methoxy group. The halogen and methyl groups reduced the hPXR binding activity of the analogs, with the maximal hPXR binding activity observed being less than 50% of that of the control T0901317 at a concentration of 10 μM. In terms of cellular activity, compounds 63 and 66 were very weak hPXR agonists, with marginal maximal activity less than 50% of that of the control compound rifampicin at 10 μM. No detectable hPXR cellular activity was observed for compounds 64 and 65. These data suggest that the methoxy group at the 2-position of the 2,5-dimethoxyphenyl is crucial for the hPXR binding and cellular activities of the compounds. If both of the 2,5-position methoxy groups were removed, as in compound 67, the result was a weak hPXR agonist with low hPXR binding activity (38%) and low agonistic activity (42%) at the concentration of 10 μM. Replacing the left phenyl ring with a pyridin3-yl ring (in compound 68) greatly reduced the hPXR activity, and replacing the left phenyl ring with a pyridin4-yl ring (in compound 69) abolished the hPXR activity.

We next examined the effect on the activity of the compound with only one methoxy group on the left phenyl ring. With only the 2-methoxy group present, compound 70 retained high hPXR binding activity (IC₅₀ = 0.028 μM) but only partial hPXR agonistic and antagonistic activities, indicating that the 5-methoxy in the 2,5-dimethoxyphenyl is important for maintaining hPXR inverse agonistic and antagonistic activities. Replacing the 2-methoxy phenyl ring, as present in compound 70, with a 2-methoxy-pyridin-3-yl ring (in compound 71) further reduced the hPXR binding activity, indicating that a pyridinyl substituent was not tolerable in this position. Compared with compound 70, compound 72, with a 3-methoxy group, had hPXR binding activity that was lower by a factor of 19, with very low maximal hPXR binding, and compound 73, with a 4-methoxy group, had no hPXR binding activity at all. Data derived from these modified compounds indicate that the 2-methoxy group is critical to optimal hPXR activity. On the basis of retaining the 2-position methoxy group, moving the 5-position methoxy group to the 3-position (in compound 74), the 4-position (in compound 75), or the 6-position (in compound 76) resulted in analogs that had weaker hPXR binding activities but had gained hPXR agonistic activities. Compound 76, with the 2,6-dimethoxy groups, retained high hPXR binding activity and behaved as a partial agonist and a partial antagonist of hPXR. These data indicate that the 5-methoxy group is critical for maintaining the hPXR inverse agonistic and antagonistic activities of analogs.

Other dimethoxy substitution patterns, such as 3,4-dimethoxy (in compound 77), 3,5-dimethoxy (in compound 78), and modified 3,4-dimethoxy in the form of a 1,3-dioxole ring (in compound 79), resulted in analogs with no detectable hPXR binding and only marginal cellular activities.

Further SAR Exploration of the Butoxy Group

Because of the possible hydrophobic pocket around the 5-position (Figure 5) and the effect whereby a butoxy group in the 6-position of R⁴ improved compound potency, as in compound 46 (Figure 6), we hypothesized that there was more space near the R⁴ that could be used to improve compound potency. To test this hypothesis, we introduced branched alkoxy groups at the butoxy site (Table 6). First, we created compound 80 by introducing a methyl group at the 1-position of the butoxy in compound 46. Compared to compound 2, compound 80 had greatly improved activities: hPXR binding IC₅₀ = 0.0062 μM for 80 vs. 0.65 μM for 2 (a 105-fold increase); hPXR inverse agonistic IC₅₀ = 0.0016 μM for 80 vs 0.48 μM for 2 (a 300-fold increase); and antagonistic activity IC₅₀ = 0.014 μM for 80 vs 4.1 μM for 2 (a 293-fold increase). It is possible that the hydrophobic pocket occupied by the ortho-substitution at this R⁴ position led to a conformation of the amide scaffold that was favorable to binding hPXR and that introducing a methyl group further strengthened this effect. To investigate whether the 1-position at the alkoxy group was the best site for branching, we created two compounds by introducing a methyl group at the 2-position of the butyl of compound 46 to create compound 81 and at the 3-position of the butyl of compound 46 to create compound 82. Both of these compounds were hPXR dual inverse agonists and antagonists without agonistic activity. Compound 81 (binding IC₅₀ = 0.007 μM, inverse agonistic IC₅₀ = 0.013 μM, antagonistic IC₅₀ = 0.11 μM) had hPXR binding and inverse agonistic and antagonistic activities that were slightly weaker than those of compound 80 but were still better than those of compound 46. Compound 82 (binding IC₅₀ = 0.012 μM, inverse agonistic IC₅₀ = 0.042 μM, antagonistic IC₅₀ = 0.25 μM) had hPXR binding and inverse agonistic and antagonistic activities that were even weaker than those of compound 81. These data suggest that there is more space in the hydrophobic pocket near the R⁴ position and that the enhancing effect of the modifications weakens as the methyl group moves away from the right phenyl ring.

Table 6.

Structures and hPXR activities of compounds with general scaffold 6

Compound	R5	R4	Bindinga (IC50/μM) (%inhibition at 10 μM)	Agonistb (EC50/μM) (%activation at 10 μM)	Inverse agonistc (IC50/μM) (%inhibition at 10 μM)	Antagonistd (IC50/μM) (%inhibition at 10 μM)
T0901317			0.026 ± 0.003 (100 ± 1)%	NTe	NT	NT
Rifampicin			NT	1.17 ± 0.1 (100 ± 2)%	NA	NT
SPA70			0.21 ± 0.04 (87 ± 3)%	NAf	0.022 ± 0.004 (100 ± 2)%	0.23 ± 0.02 (100 ± 1)%
46			0.022 ± 0.01 (90 ± 3)%	NA	0.024 ± 0.006 (89 ± 9)%	0.24 ± 0.08 (101 ± 2)%
80			0.0062 ± 0.002 (90 ± 2)%	NA	0.0016 ± 0.0005 (117 ± 6)%	0.014 ± 0.002 (107 ± 3)%
81			0.007 ± 0.001 (86 ± 4)%	NA	0.013 ± 0.002 (67 ± 4)%	0.11 ± 0.02 (94 ± 1)%
82			0.012 ± 0.004 (82 ± 3)%	NA	0.042 ± 0.006 (94 ± 4)%	0.25 ± 0.02 (98 ± 3)%
83			0.02 ± 0.007 (92 ± 1)%	NA	NA	0.015 ± 0.0005 (88 ± 2)%
84			0.016 ± 0.005 (91 ± 3)%	NA	NA	0.0069 ± 0.002 (83 ± 1)%
85			0.006 ± 0.001 (87 ± 1)%	NA	0.001 ± 0.0001 (125 ± 12)%	0.014 ± 0.001 (106 ± 1)%
86			0.026 ± 0.0084 (81 ± 3)%	NA	0.014 ± 0.001 (91 ± 3)%	0.15 ± 0.01 (97 ± 2)%
87			0.007 ± 0.003 (93 ± 3)%	NA	NA	0.018 ± 0.002 (87 ± 2)%
88			0.015 ± 0.007 (86 ± 5)%	NA	NA	0.12 ± 0.007 (76 ± 2)%
89			0.004 ± 0.001 (90 ± 3)%	NA	NA	0.009 ± 0.001 (72 ± 1)%
90			0.011 ± 0.004 (87 ± 2)%	0.0081 ± 0.002 (21 ± 5)%	NA	0.076 ± 0.005 (57 ± 1)%

^aT0901317 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the hPXR binding assay;

^brifampicin at 10 μM as 100% activation and DMSO (0.3%) as 0% activation for the cell-based hPXR agonistic assay;

^cSPA70 at 10 μM as 100% inhibition and DMSO (0.3%) as 0% inhibition for the cell-based hPXR inverse agonistic assay;

^dSPA70 at 10 μM with rifampicin (5 μM) as 100% inhibition and rifampicin alone (5 μM) as 0% inhibition for the cell-based hPXR antagonistic assay;

^eNT: not tested;

^fNA: No IC₅₀ value could be determined experimentally within the concentration range tested (the highest concentration tested was 30 μM).

Because compounds 58 (hPXR binding IC₅₀ = 0.016 μM, antagonistic IC₅₀ = 0.12 μM) and 62 (hPXR binding IC₅₀ = 0.014 μM, antagonistic IC₅₀ = 0.074 μM) had very strong hPXR binding activity and showed only hPXR antagonistic activity, we synthesized the corresponding derivatives 83 and 84 with a 2-methoxy-5-chlorophenyl group and a 2-methoxy-5-methylphenyl group at the R⁵ site, respectively. Compared with the corresponding compounds 58 and 62, compounds 83 (binding IC₅₀ = 0.02 μM, antagonistic IC₅₀ = 0.015 μM) and 84 (binding IC₅₀ = 0.016 μM, agonistic IC₅₀ = 0.0077 μM, antagonistic IC₅₀ = 0.0069 μM) showed no enhanced binding but were 10 fold as potent in terms of their hPXR antagonistic activities.

As compounds 80, 83, and 84 were racemic mixtures, we synthesized the corresponding individual chiral compounds 85–90 through the Mitsunobu reaction. We found that the “S” chiral compounds (85, 87, and 89) displayed activities higher than those of the racemic mixtures (80, 83, and 84) and the corresponding “R” chiral compounds (86, 88, and 90). Compounds 85 (binding IC₅₀ = 0.0060 μM, inverse agonist IC₅₀ = 0.001 μM, antagonist IC₅₀ = 0.014 μM) and 89 (binding IC₅₀ = 0.004 μM, antagonistic IC₅₀ = 0.009 μM) are, respectively, the most potent hPXR inverse agonist/antagonist and the most potent pure antagonist without agonistic activity derived from these efforts.

Molecular Docking

To understand the mode of action of the 1H-1,2,3-triazole-4-carboxamides analogs of SPA70 described in this paper, molecular docking experiments were performed using the representative compounds 85 and 89 (Figure 7).

Figure 7.

Molecular docking studies of the representative compounds 85 (A) and 89 (B). Compounds are depicted as sticks (olive green) and residues of the hPXR LBD are shown as light blue sticks and surface (PDB code: 5X0R).

In similar manner to that seen in SPA70 and compound 2, the 3-tert-butyl moiety on the phenyl ring is tightly packed in a hydrophobic pocket. The aryl group is also seen occupying another hydrophobic cavity, which could be the reason for their improved potency. As expected from antagonists, these compounds fail to stabilize the AF-2 helix in the active orientation needed to recruit the coactivator (e.g. SRC-1). The lack of significant interactions between the compounds and the AF-2 residues as illustrated by the docking studies support this notion of antagonism. The estimated distance between 85/89 and L428 (within the AF-2 helix) is approximately 4 Å. Similar observations can be made with 14, 36 and 46 (Figure S1 in Supporting Information), which together with 1, 2, 85 and 89, represent all five points of scaffold modifications (R¹-R⁵).

Even though the compounds in this study are molecules that occupy the ligand binding pocket, NR can also be regulated in an allosteric manner, whereby ligand binding is known to cause structural changes of the receptor³⁵. As a future direction, allosteric regulation of PXR activity could be considered in the development of new modulators, particularly when the stabilization of the AF-2 helix in the active orientation can be altered.

Evaluation of the Nuclear Receptor Selectivity of Compounds

We have previously described selective hPXR antagonists with a sulfonyl linkage^{30, 32}. To determine whether the most potent lead compounds based on the amide scaffold (with a carbonyl amide instead of a sulfonyl linkage), i.e., compounds 85 and 89, retained the receptor selectivity seen in the sulfonyl analogs, we carried out similar receptor selectivity assays with two other members of the human NR1I subfamily, namely human constitutive androstane receptor (hCAR; NR1I3) and human vitamin D(3) receptor (VDR; NR1I1), and three other nuclear receptors, namely mouse PXR (mPXR), human farnesoid X receptor (hFXR) and human liver X receptor α (hLXRα). Similar to the previously discovered sulfone scaffold analogs, the much more potent compounds 85 and 89 were selective for hPXR without affecting the activity of mPXR, hCAR, hFXR, hLXRα, or hVDR (Figure 8), and they showed only marginal cytotoxicity in the cell models used (HepG2 and HEK293), and in hepatoma cells Hepa 1–6 and HepaRG (Figure S2 in Supporting Information). Therefore, our efforts have produced more potent, but similarly selective, hPXR inhibitors with low toxicity (Figures 3, 8 and S2 in Supporting Information). Whether metabolites of compounds 85 and 89 will be generated and cause non-specific activity remains to be further investigated.

Figure 8.

The representative compounds 85 and 89 do not activate or inhibit mPXR, hCAR, hFXR, hLXRα, or hVDR. The control compounds in these studies were pregnenolone 16α-carbonitrile (PCN, a mPXR agonist), 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO, an hCAR agonist), GW4064 (an hFXR agonist), T0901317 (an hLXRα agonist), and 1α,25-dihydroxyvitamin D3 (1,25(OH)VD3, an hVDR agonist^{30, 32}. % Activity refers to % activation (for testing agonistic activity of 85 or 89, tested alone) or % inhibition (for the antagonistic activity of 85 or 89, tested in the presence of an agonist as indicated).

Chemistry

The synthesis of SPA70 amide derivatives is illustrated in Schemes 1–6. In Scheme 1, compound 93 was prepared by cyclizing ethyl acetoacetate with 2-azido-1,4-dimethoxybenzene (compound 92), which was generated from commercially available 2,5-dimethoxyaniline (compound 91) via diazotization³². Compound 93 was then coupled with the corresponding aniline in the presence of EDCI and HOBt/DMAP to give compounds 1–17³⁶.

Scheme 1.

Synthesis of Compounds 1–17

Reagents and conditions: (a) concentrated HCl, NaNO₂, H₂O, 0°C, 15 min; (b) NaN₃, H₂O, room temperature, 2 h; (c) ethyl acetoacetate, MeONa, MeOH, 60°C, overnight; (d) corresponding anilines for compounds 1–16, EDCI, HOBt, DIEA, DMF, room temperature, overnight; (e) 2,6-diethoxypyrimidin-4-amine for compound 17, EDCI, DMAP, DMF, room temperature, overnight.

Scheme 6.

Synthesis of Compounds 80–90

Reagents and conditions: (a) for 80–84, corresponding halogenated compounds, Cs₂CO₃, DMF, 60°C, overnight; (b) for 85, 87, and 89, (R)-pentan-2-ol, PPh₃, DIAD, DCM, room temperature, overnight; (c) for 86, 88, and 90, (S)-pentan-2-ol, PPh₃, DIAD, DCM, room temperature, overnight; (d) 2-amino-4-(tert-butyl)phenol, EDCI, HOBt, DIEA, DMF, room temperature, overnight.

The synthesis of the R² derivatives of compounds 18–30 are depicted in Scheme 2. Key intermediate compounds 18 and 23 were first prepared by condensing compound 93 with 5-(tert-butyl)benzene-1,3-diamine (compound 94a) or 3-amino-5-(tert-butyl)phenol (compound 94b). The intermediate compounds 18 and 23 were then subjected to S_N2 nucleophilic substitutions with the corresponding alkyl halides to give compounds 19–22, 24, and 25³⁷. Compound 93 was condensed with 3-bromo-5-(tert-butyl)aniline (compound 95) to yield the desired product, compound 26. Palladium-catalyzed cross-coupling between compound 26 and Zn(CN)₂ gave compound 27³⁸, which was cyclized with NaN₃ to yield compound 30³⁹. A Suzuki cross-coupling between compound 26 and phenylboronic acid in the presence of the Pd(Ph₃P)₄ catalyst yielded compound 28⁴⁰. Miyaura borylation of the aryl halide 26 gave the pinacol boronic acid ester, compound 29⁴¹.

Scheme 2.

Synthesis of Compounds 18–30

Reagents and conditions: (a) EDCI, HOBt, DIEA, DMF, room temperature, overnight; (b) corresponding halogenated compounds, Cs₂CO₃, DMF, 60°C, overnight; (c) Zn(CN)₂, Pd(Ph₃P)₄, DMF, 100°C, 8 h; (d) phenylboronic acid, K₂CO₃, Pd(Ph₃P)₄, DMF/water, 100°C, 8 h; (e) bis(pinacolato)diboron, CH₃COOK, Pd(dppf)Cl₂, DMF, 100°C, 8 h; (f) NaN₃, NH₄Cl, DMSO, 100°C, 18 h.

The synthesis of compounds 31–38 is summarized in Scheme 3. Compound 93 was condensed with N-(4-amino-2-(tert-butyl)phenyl)acetamide (compound 96) or 4-amino-2-(tert-butyl)phenol (compound 97) to yield the desired compounds 32 and 36. Compound 31 was prepared by acidic hydrolysis of compound 32⁴². Compound 31 and 36 were then subjected to S_N2 nucleophilic reactions with the corresponding alkyl halides to give compounds 33, 35, 37, and 38. Compound 34 was synthesized by reductive amination of compound 31⁴³.

Scheme 3.

Synthesis of Compounds 31–38

Reagents and conditions: (a) EDCI, HOBt, DIEA, DMF, room temperature, overnight; (b) HCl, EtOH, 120°C, overnight; (c) Corresponding halogenated compounds, Cs₂CO₃, DMF, 60°C, overnight; (d) NaBH₃CN, HCHO (aq.), MeOH, room temperature, overnight.

To prepare compound 39, 4-(tert-butyl)-2-nitroaniline (compound 98) was first protected by the Boc group at its amino group then its nitro group was reduced by using Pd/C in a hydrogen atmosphere to generate tert-butyl (2-amino-4-(tert-butyl)phenyl)carbamate (compound 100)⁴⁴. Amidation of the carboxylic acid 93 by the aniline compound 100 generated the 6-position Boc-protected aniline compound 41, which was then converted to compound 39 after the Boc protecting group was removed. To prepare compound 40, the 4-(tert-butyl)aniline (compound 101) was first acetylated to form N-(4-(tert-butyl)phenyl)acetamide (102), then this was converted to compound 104 via nitration and reduction⁴⁵. Compound 94 was coupled with compound 93 to generate compound 19. Compound 93 was a common starting material for compounds 39–56. Amide formations of compound 93 with 2-amino-4-(tert-butyl)phenol and 5-(tert-butyl)-2-methoxyaniline yielded the corresponding compounds 42 and 43. Alkylation of compound 42 with the respective alkyl halides generated the corresponding compounds 44–56 via an S_N2 nucleophilic reaction³⁷. The synthesis of compounds 39–56 is summarized in Scheme 4.

Scheme 4.

Synthesis of Compounds 39–56

Reagents and conditions: (a) Boc₂O, DMAP, DIEA, DCM, room temperature, overnight; (b) H₂, Pd/C, room temperature, overnight; (c) EDCI, HOBt, DIEA, DMF, room temperature, overnight; (d) TFA, DCM, room temperature, 1 h; (e) acetyl chloride, TEA, DCM, 0°C to room temperature, 2 h; (f) KNO₃, 98% H₂SO₄, 0°C to room temperature, overnight; (g) Corresponding halogenated compounds, Cs₂CO₃, DMF, 60°C, overnight.

The preparation of compounds 57–79 is summarized in Scheme 5. Substituted aniline compounds 105a–v were first converted to the corresponding azide compounds 106a–w through diazotization and nucleophilic substitution. Azide compounds 100a–w were cyclized with ethyl acetoacetate then hydrolyzed to obtain the corresponding acids (107a–v)³². Compound 109 was prepared by alkylating 4-(tert-butyl)-2-nitrophenol (compound 108) with 1-iodobutane, then the nitro group in compound 109 was reduced to an amino group to generate 2-butoxy-5-(tert-butyl)aniline (compound 110) in the presence of a catalytic amount of Pd/C under H₂⁴⁶. The acid compounds (107a–v) were condensed with compound 110 to yield compounds 57–79.

Scheme 5.

Synthesis of Compounds 57–79

Reagents and conditions: (a) concentrated HCl, NaNO₂, H₂O, 0°C, 15 min; (b) NaN₃, H₂O, room temperature, 2 h; (c) ethyl acetoacetate, MeONa, MeOH, 60°C, overnight; (d) EDCI, HOBt, DIEA, DMF, room temperature, overnight; (e) K₂CO₃, DMF, 1-iodobutane, 90°C, 2 h; (f) H₂, Pd/C, room temperature, overnight; (g) 2-amino-4-(tert-butyl)phenol, EDCI, HOBt, DIEA, DMF, room temperature, overnight; (h) 2-bromopentane, Cs₂CO₃, DMF, 60°C, overnight.

Compounds 107b and 107f were amidated with 2-amino-4-(tert-butyl)phenol to generate compounds 111 and 112, respectively. Compounds 42, 111, and 112 were alkylated with the respective alkyl halides to generate the corresponding compounds 80–84 via an S_N2 nucleophilic reaction. Compounds 42, 111, and 112 were coupled with the respective (R)-pentan-2-ol and (S)-pentan-2-ol to generate the corresponding compounds 85–90 via a Mitsunobu reaction⁴⁷.

CONCLUSION

To investigate potential hPXR dual inverse agonists and pure PXR antagonists based on the amide scaffold, we started with compound 2 (hPXR binding IC₅₀ = 0.65 μM, inverse agonistic IC₅₀ = 0.48 μM, antagonistic IC₅₀ = 4.1 μM) as the lead compound and explored the effect of substituents at the 3-, 4-, 5-, and 6-positions of its right phenyl ring. We found that compound 46 (hPXR binding IC₅₀ = 0.22 μM, inverse agonistic IC₅₀ = 0.024 μM, antagonistic IC₅₀ = 0.24 μM) with a 3-tert-butyl-5-butoxy substitution pattern at the right phenyl ring had greatly improved hPXR activities (binding activity increased 30 fold; inverse agonistic activity increased 20 fold; antagonistic activity increased 17 fold) when compared with compound 2 (Figure 9). Compound 46 was then adopted as the new lead compound for further SAR exploration, which led to compound 85, a dual hPXR inverse agonist and antagonist with hPXR binding activity increased 108 fold, inverse agonistic activity increased 480 fold, and antagonistic activity increased 293 fold relative to that of compound 2. Compound 89 is the most potent pure hPXR antagonist discovered, with hPXR binding activity increased 163 fold and antagonistic activity increased 456 fold relative to that of compound 2. Compound 89 does not exhibit the hPXR inverse agonistic activity observed with compounds 2, 46, and 85. Additionally, compounds 85 and 89 are selective for hPXR without toxicity. Our study has, therefore, produced potent and selective lead compounds for use in basic research and preclinical studies. Our studies also shed light on how to reduce the binding affinity of a compound to hPXR, to reduce the liability of PXR-activating compounds in inducing adverse effects such as drug-drug interactions.

Figure 9.

SAR optimization of the amide scaffold to produce potent hPXR dual inverse agonists and antagonists, as well as pure hPXR antagonists (the fold increase in activity was calculated by dividing the activity of compound 2 by the corresponding activity of compound 46, 85, or 89).

EXPERIMENTAL SECTION

General methods and syntheses.

Organic reagents were purchased from commercial suppliers unless otherwise noted and were used without further purification. All solvents were analytical or reagent grade, and the solvents were dried using the Glass Contour Solvent System from SG Water USA. All reactions with water-sensitive and/or air-sensitive starting materials were carried out in pre-dried glassware under an argon atmosphere, using standard procedures. Flash column chromatography was performed using Biotage Isolera Flash Systems and Biotage SNAP Ultra or Biotage SNAP Ultra C18 columns. All reactions and compound purities were monitored by UPLC-MS, using a Waters Acquity UPLC MS system with a C18 column in a 2-min gradient [H₂O + 0.1% formic acid (FA) → acetonitrile (ACN) + 0.1% FA], detectors of PDA (215–400 nm) and ELSD, and an Acquity SQD ESI-positive mass spectrometer (Waters Corporation, Milford, MA). High-resolution mass spectra were determined by using a Waters Acquity UPLC system with a C18 column (H₂O + 0.1% FA → ACN + 0.1% FA gradient over 2.5 min) and a Xevo G2Q-TOF ESI-positive mass spectrometer in resolution mode. Compounds were internally normalized to leucine–enkephalin lock solution, with a calculated error of <3 ppm. All final compounds used for SAR studies had purity of 95% or greater by HPLC. All NMR spectra were recorded on a Bruker 400 MHz or 500 MHz spectrometer in the solvents indicated, and spectra were processed using MestReNova (14.1.0) (Supporting Information). The chemical shift values are expressed in parts per million (ppm) relative to tetramethylsilane as the internal standard. Coupling constants (J) are reported in hertz (Hz).

Synthesis Procedures for Compounds 1–17 (Scheme 1)

1-(2,5-Dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 93). Hydrochloric acid (37%, 30 mL) was added to a solution of 2,5-dimethoxyaniline (compound 91, 7.66 g, 50 mmol) in water (50 mL) with stirring at 0°C. A solution of sodium nitrite (3.45 g, 50.0 mmol) in water (10 mL) was added dropwise to the mixture at 0°C. After the mixture had been stirred for 15 min, hexane (50 mL) was added, then a solution of NaN₃ (3.58 g, 55.0 mmol) in water (10 mL) was added dropwise at 0°C. The reaction mixture was stirred at 0°C for 15 min and then kept at ambient temperature for 1.5 h. The mixture was extracted with hexane (150 mL), and the organic layer was washed with water (2 × 50 mL) and once with brine (50 mL). The organic layer was dried with MgSO₄, filtered, and concentrated under reduced pressure to provide an oil (crude compound 92). The oil was used directly for the next step without further purification. CH₃OH (100 mL), ethyl acetoacetate (7.65 mL, 60 mmol) and CH₃ONa (10.8 g, 200 mmol) were added to the oil and the reaction mixture was stirred at 60°C overnight. The mixture was evaporated to remove the solvent, then NaOH (8 g, 200 mmol) and water (100 mL) were added to the residue and stirred at ambient temperature for 3 h. The pH of the mixture was adjusted to 4 with hydrochloric acid (37%), then the mixture was extracted with ethyl acetate (2 × 200 mL). The combined organic phase was dried with MgSO₄, filtered, and concentrated under reduced pressure to provide 1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 93) as a brown solid (7.4 g, 56% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.10 (s, 1H), 7.26 (d, J = 9.2 Hz, 1H), 7.20 (dd, J = 9.1, 3.1 Hz, 1H), 7.11 (d, J = 3.0 Hz, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 2.31 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.56, 153.04, 147.62, 140.31, 135.76, 123.71, 117.32, 113.93, 113.82, 56.27, 55.81, 9.06.

N-(4-(tert-Butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 1). 4-(tert-butyl)aniline (0.358 g, 2.400 mmol) was added to a solution of 1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 93, 0.526 g, 2 mmol), EDCI (0.460 g, 2.400 mmol), HOBt (wetted with not less than 20% by weight of water, 0.405 g, 2.400 mmol), and DIEA (0.661 ml, 4.00 mmol) in DMF (5 mL). The resulting mixture was stirred at room temperature overnight, then it was diluted with water (100 mL) and extracted with EtOAc (50 mL × 2). The combined organic phase was washed with saturated aq. NaHCO₃, water, and brine, then dried with MgSO₄ and concentrated. The residue was purified by flash chromatography (0% to 100% EtOAc in hexane) to give compound 1 as a white solid (625 mg, 79% yield, 98.80% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.04 (s, 1H), 7.69–7.56 (m, 2H), 7.43–7.36 (m, 2H), 7.09 (dd, J = 9.2, 3.0 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 3.82 (d, J = 1.1 Hz, 3H), 3.77 (d, J = 1.0 Hz, 3H), 2.52 (d, J = 0.9 Hz, 3H), 1.33 (d, J = 0.9 Hz, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.31, 153.70, 148.04, 147.20, 139.41, 137.95, 135.16, 125.88, 124.36, 119.61, 117.49, 113.82, 113.34, 56.34, 56.00, 34.41, 31.40, 9.22. ESI-TOF HRMS: m/z 395.2078 (C₂₂H₂₆N₄O₃ + H⁺ requires 395.2077).

N-(3-(tert-Butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 2). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-(tert-butyl)aniline, to give a white solid (653 mg, 83% yield, 98.99% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.08 (s, 1H), 7.69 (t, J = 2.0 Hz, 1H), 7.61 (ddd, J = 8.0, 2.2, 1.0 Hz, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.18 (ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.2 Hz, 1H), 6.96 (d, J = 3.0 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.53 (s, 3H), 1.35 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.37, 153.71, 152.30, 148.04, 139.41, 137.98, 137.57, 128.71, 124.36, 121.33, 117.49, 117.07, 117.00, 113.83, 113.34, 56.34, 56.01, 34.81, 31.34, 9.24. ESI-TOF HRMS: m/z 395.2079 (C₂₂H₂₆N₄O₃ + H⁺ requires 395.2077).

N-(2-(tert-Butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 3). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 2-(tert-butyl)aniline, to give a white solid (326 mg, 83% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 9.71 (s, 1H), 7.51 (dd, J = 7.5, 1.9 Hz, 1H), 7.45 (dd, J = 7.6, 1.9 Hz, 1H), 7.32–7.16 (m, 5H), 3.78 (s, 3H), 3.77 (s, 3H), 2.39 (s, 3H), 1.43 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.52, 153.07, 147.66, 144.10, 138.72, 137.48, 135.27, 128.87, 126.55, 126.37, 126.21, 123.69, 117.42, 114.02, 113.84, 56.28, 55.83, 34.59, 30.60, 8.69. ESI-TOF HRMS: m/z 395.2074 (C₂₂H₂₆N₄O₃ + H⁺ requires 395.2077).

1-(2,5-Dimethoxyphenyl)-5-methyl-N-phenyl-1H-1,2,3-triazole-4-carboxamide (compound 4). This compound was synthesized by using a procedure similar to that described for compound 1, employing 93 and aniline, to give a white solid (456 mg, 67% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.08 (s, 1H), 7.75–7.68 (m, 2H), 7.41–7.35 (m, 2H), 7.17–7.07 (m, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 2.9 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.52 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.41, 153.71, 148.03, 139.54, 137.86, 137.80, 129.08, 124.33, 124.25, 119.81, 117.51, 113.82, 113.35, 56.34, 56.01, 9.22. ESI-TOF HRMS: m/z 339.1453 (C₁₈H₁₈N₄O₃ + H⁺ requires 339.1452).

1-(2,5-Dimethoxyphenyl)-5-methyl-N-(m-tolyl)-1H-1,2,3-triazole-4-carboxamide (compound 5). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and m-toluidine, to give a white solid (562 mg, 80% yield, 99.00% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.05 (s, 1H), 7.61 (d, J = 2.2 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.30–7.21 (m, 1H), 7.09 (ddd, J = 9.1, 3.0, 1.1 Hz, 1H), 7.03 (dd, J = 9.2, 1.1 Hz, 1H), 6.98–6.93 (m, 2H), 3.82 (d, J = 1.0 Hz, 3H), 3.76 (d, J = 1.1 Hz, 3H), 2.52 (d, J = 1.0 Hz, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.35, 153.71, 148.03, 139.52, 139.01, 137.87, 137.72, 128.89, 125.07, 124.32, 120.44, 117.52, 116.87, 113.80, 113.34, 56.35, 56.01, 21.56, 9.23. ESI-TOF HRMS: m/z 353.1602 (C₁₉H₂₀N₄O₃ + H⁺ requires 353.1608).

1-(2,5-Dimethoxyphenyl)-N-(3-ethylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 6). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-ethylaniline, to give a white solid (209.9 mg, 57% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 7.76 (t, J = 1.9 Hz, 1H), 7.70–7.64 (m, 1H), 7.31–7.19 (m, 3H), 7.15 (d, J = 3.0 Hz, 1H), 6.95 (dt, J = 7.7, 1.2 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 2.61 (q, J = 7.6 Hz, 2H), 2.39 (s, 3H), 1.20 (t, J = 7.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.41, 153.06, 147.69, 144.08, 138.93, 138.58, 137.59, 128.41, 123.72, 123.15, 119.77, 117.81, 117.35, 113.99, 113.84, 56.28, 55.84, 28.28, 15.47, 8.77. ESI-TOF HRMS: m/z 367.1762 (C₂₀H₂₂N₄O₃ + H⁺ requires 367.1765).

1-(2,5-Dimethoxyphenyl)-N-(3-isopropylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 7). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-isopropylaniline, to give a white solid (305.8 mg, 80% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 7.80 (t, J = 2.0 Hz, 1H), 7.69 (ddd, J = 8.1, 2.2, 1.1 Hz, 1H), 7.27 (dd, J = 8.5, 6.8 Hz, 2H), 7.24–7.19 (m, 1H), 7.15 (d, J = 3.0 Hz, 1H), 6.98 (dt, J = 7.7, 1.4 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 2.87 (p, J = 6.9 Hz, 1H), 2.40 (s, 3H), 1.22 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.41, 153.07, 148.80, 147.68, 138.93, 138.59, 137.61, 128.39, 123.74, 121.71, 118.40, 117.91, 117.34, 113.99, 113.82, 56.27, 55.82, 33.50, 23.82, 8.77. ESI-TOF HRMS: m/z 381.1924 (C₂₁H₂₄N₄O₃ + H⁺ requires 381.1921).

1-(2,5-Dimethoxyphenyl)-N-(3-(dimethylamino)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 8). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and N¹,N¹-dimethylbenzene-1,3-diamine, to give a light brown solid (301.5 mg, 79% yield, 96.68% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.18 (s, 1H), 7.32 (t, J = 2.2 Hz, 1H), 7.29–7.24 (m, 2H), 7.21 (dd, J = 9.1, 3.1 Hz, 1H), 7.16–7.09 (m, 2H), 6.49 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 2.90 (s, 6H), 2.39 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.31, 153.06, 150.76, 147.68, 139.33, 138.83, 137.70, 128.86, 123.74, 117.33, 113.99, 113.83, 108.47, 108.17, 104.54, 56.28, 55.83, 40.10, 8.78. ESI-TOF HRMS: m/z 382.1877 (C₂₀H₂₃N₅O₃ + H⁺ requires 382.1874).

1-(2,5-Dimethoxyphenyl)-5-methyl-N-(3-(pyrrolidin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxamide (compound 9). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-(pyrrolidin-1-yl)aniline, to give a white solid (341.0 mg, 84% yield, 96.55% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.03 (s, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.06 (dd, J = 9.1, 2.9 Hz, 2H), 7.00 (d, J = 9.2 Hz, 1H), 6.98–6.92 (m, 2H), 6.40–6.33 (m, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.37–3.29 (m, 4H), 2.55–2.45 (m, 3H), 2.02–1.95 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 159.32, 153.67, 148.51, 148.01, 139.32, 138.78, 138.11, 129.58, 124.35, 117.40, 113.86, 113.35, 108.01, 107.09, 103.08, 56.32, 55.99, 47.82, 25.45, 9.24. ESI-TOF HRMS: m/z 408.2026 (C₂₂H₂₅N₅O₃ + H⁺ requires 408.2030).

1-(2,5-Dimethoxyphenyl)-5-methyl-N-(3-(piperidin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxamide (compound 10). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-(piperidin-1-yl)aniline, to give a light brown solid (316 mg, 75% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.03 (s, 1H), 7.46 (s, 1H), 7.23 (t, J = 8.1 Hz, 1H), 7.08 (dq, J = 5.9, 2.9 Hz, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.74 (s, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.21 (t, J = 5.4 Hz, 4H), 2.52 (s, 3H), 1.77–1.66 (m, 4H), 1.59 (q, J = 5.8 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 159.36, 153.70, 148.04, 139.36, 138.68, 138.02, 129.46, 124.36, 117.49, 113.81, 113.34, 112.46, 110.42, 107.68, 56.34, 56.01, 50.51, 25.78, 24.32, 9.25. ESI-TOF HRMS: m/z 422.2190 (C₂₃H₂₇N₅O₃ + H⁺ requires 422.2187).

1-(2,5-Dimethoxyphenyl)-5-methyl-N-(3-morpholinophenyl)-1H-1,2,3-triazole-4-carboxamide (compound 11). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-morpholinoaniline, to give a white solid (248.2 mg, 59% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 7.54 (t, J = 2.2 Hz, 1H), 7.40–7.35 (m, 1H), 7.28 (d, J = 9.2 Hz, 1H), 7.24–7.16 (m, 2H), 7.15 (d, J = 3.0 Hz, 1H), 6.70 (ddd, J = 8.3, 2.5, 0.9 Hz, 1H), 3.78 (s, 3H), 3.77–3.73 (m, 7H), 3.13–3.08 (m, 4H), 2.38 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.38, 153.06, 151.37, 147.68, 139.40, 138.92, 137.59, 128.96, 123.71, 117.36, 113.99, 113.84, 111.29, 110.73, 107.12, 66.06, 56.29, 55.84, 48.48, 8.78. ESI-TOF HRMS: m/z 424.1982 (C₂₂H₂₅N₅O₄ + H⁺ requires 424.1980).

1-(2,5-Dimethoxyphenyl)-5-methyl-N-(3-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxamide (compound 12). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-(4-methylpiperazin-1-yl)aniline, to give a light brown solid (348.5 mg, 80% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.23 (s, 1H), 7.53 (t, J = 2.2 Hz, 1H), 7.35 (dd, J = 7.9, 1.8 Hz, 1H), 7.28 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.0 Hz, 1H), 7.19–7.13 (m, 2H), 6.69 (dd, J = 8.3, 2.4 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.13 (t, J = 4.8 Hz, 4H), 2.46 (t, J = 4.9 Hz, 4H), 2.39 (s, 3H), 2.22 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.35, 153.06, 151.28, 147.68, 139.35, 138.89, 137.61, 128.91, 123.72, 117.35, 113.99, 113.84, 110.93, 110.90, 107.35, 56.29, 55.84, 54.56, 48.06, 45.73, 8.78. ESI-TOF HRMS: m/z 437.2295 (C₂₃H₂₈N₆O₃ + H⁺ requires 437.2296).

N-(3-(1H-Pyrrol-1-yl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 13). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-(1H-pyrrol-1-yl)aniline, to give a white solid (352.2 mg, 69% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.63 (s, 1H), 8.15 (t, J = 2.1 Hz, 1H), 7.84 (dd, J = 8.4, 1.6 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H), 7.31 (q, J = 3.8, 2.9 Hz, 3H), 7.27 (d, J = 9.1 Hz, 1H), 7.21 (dd, J = 9.1, 3.0 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 6.29 (t, J = 2.2 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 2.42 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.68, 153.08, 147.67, 140.15, 139.86, 139.27, 137.41, 129.81, 123.69, 118.86, 117.34, 117.09, 114.63, 114.01, 113.80, 111.39, 110.48, 56.26, 55.81, 8.80. ESI-TOF HRMS: m/z 404.1716 (C₂₂H₂₁N₅O₃ + H⁺ requires 404.1717).

N-(3,5-Di-tert-butylphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 14). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3,5-di-tert-butylaniline, to give a white solid (177.2 mg, 79% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.08 (s, 1H), 7.59 (d, J = 1.7 Hz, 2H), 7.22 (t, J = 1.7 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.96 (d, J = 2.9 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.53 (s, 3H), 1.36 (s, 18H). ¹³C NMR (101 MHz, CDCl₃) δ 159.32, 153.71, 151.71, 148.05, 139.29, 137.26, 124.40, 118.43, 117.48, 114.38, 113.84, 113.35, 56.34, 56.01, 35.00, 31.47, 9.28. ESI-TOF HRMS: m/z 451.2695 (C₂₆H₃₄N₄O₃ + H⁺ requires 451.2704).

N-(2-(tert-Butyl)pyridin-4-yl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 15). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 2-(tert-butyl)pyridine-4-amine, to give a light brown solid (122.4 mg, 47% yield, 98.62% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.20 (s, 1H), 8.52 (d, J = 5.5 Hz, 1H), 7.67 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 5.6, 2.0 Hz, 1H), 7.10 (dd, J = 9.1, 3.0 Hz, 1H), 7.04 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.52 (s, 3H), 1.40 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 170.51, 159.86, 153.73, 149.36, 147.95, 145.31, 140.11, 137.40, 124.10, 117.60, 113.80, 113.34, 110.92, 109.15, 56.34, 56.02, 37.47, 30.12, 9.25. ESI-TOF HRMS: m/z 396.2038 (C₂₁H₂₅N₅O₃ + H⁺ requires 396.2030).

1-(2,5-dimethoxyphenyl)-5-methyl-N-(1-methyl-1H-benzo[d]imidazol-2-yl)-1H-1,2,3-triazole-4-carboxamide (compound 16). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 1-methyl-1H-benzo[d]imidazol-2-amine, to give a light brown solid (261 mg, 67% yield, 98.46% purity). ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.34 (m, 1H), 7.33–7.21 (m, 3H), 7.05 (dd, J = 9.1, 2.9 Hz, 1H), 7.00 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 2.9 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.73 (s, 3H), 2.56 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 153.69, 148.22, 142.01, 139.04, 130.49, 125.52, 125.11, 123.22, 123.19, 117.15, 113.99, 113.52, 111.77, 109.94, 109.18, 56.47, 55.99, 28.77, 10.04. ESI-TOF HRMS: m/z 393.1671 (C₂₀H₂₀N₆O₃ + H⁺ requires 393.1670).

N-(2,6-diethoxypyrimidin-4-yl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 17). To a solution of 1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 93, 100 mg, 0.380 mmol) in DMF (5 mL) were added 2,6-diethoxypyrimidin-4-amine (139 mg, 0.760 mmol), EDCI (146 mg, 0.760 mmol), and N,N-dimethylpyridin-4-amine (46.4 mg, 0.380 mmol). The reaction mixture was stirred at room temperature overnight then diluted with water (50 mL) and extracted with EtOAc (50 mL × 2). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried with anhydrous Na₂SO₄, and concentrated. The residue was purified by flash chromatography (0% to 100% acetonitrile in water) to give compound 17 as white solid (93.8 mg, 58% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.53 (s, 1H), 7.31 (s, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.2 Hz, 1H), 6.94 (d, J = 3.0 Hz, 1H), 4.40 (dq, J = 10.2, 7.0 Hz, 4H), 3.82 (s, 3H), 3.76 (s, 3H), 2.50 (s, 3H), 1.40 (dt, J = 13.8, 7.1 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 172.97, 164.23, 160.03, 158.47, 153.72, 147.94, 140.32, 137.22, 124.15, 117.62, 113.74, 113.36, 88.68, 63.40, 62.77, 56.35, 56.02, 14.51, 14.45, 9.26. ESI-TOF HRMS: m/z 429.1880 (C₂₀H₂₄N₆O₅ + H⁺ requires 429.1881).

Synthesis Procedures for Compounds 18–30 (Scheme 2)

N-(3-Amino-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 18). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 5-(tert-butyl)benzene-1,3-diamine (compound 94a), to give a light brown solid (1.33 g, 65% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.00 (s, 1H), 7.25 (t, J = 2.0 Hz, 1H), 7.09 (dd, J = 9.2, 3.0 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.92 (t, J = 1.8 Hz, 1H), 6.53 (t, J = 1.8 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.51 (s, 3H), 1.30 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.32, 153.70, 153.36, 148.04, 146.57, 139.37, 138.47, 138.02, 124.36, 117.50, 113.81, 113.34, 108.67, 107.63, 104.16, 56.34, 56.01, 34.70, 31.25, 9.23. ESI-TOF HRMS: m/z 410.2190 (C₂₂H₂₇N₅O₃ + H⁺ requires 410.2187).

N-(3-(tert-Butyl)-5-(methylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 19) and N-(3-(tert-butyl)-5-(dimethylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 20). To a solution of N-(3-amino-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (0.205 g, 0.5 mmol) in DMF (5 mL) at room temperature were added iodomethane (0.300 mL, 0.600 mmol) and Cs₂CO₃ (0.195 g, 0.600 mmol). The suspension was stirred overnight then poured into water (20 mL) and extracted with EtOAc (25 mL × 2). The EtOAc layer was washed with water, dried with anhydrous Na₂SO₄, and concentrated. The residue was purified by silica gel chromatography (0% to 100% EtOAc in hexane) to give product N-(3-(tert-butyl)-5-(methylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 19) as a white solid (102.5 mg, 48% yield, 100% purity) or N-(3-(tert-butyl)-5-(dimethylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 20) as a white solid (79.2 mg, 34% yield, 99.28% purity). Compound 19: ¹H NMR (400 MHz, CDCl₃) δ 9.02 (s, 1H), 7.11 (t, J = 2.0 Hz, 1H), 7.10–7.07 (m, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.97–6.94 (m, 2H), 6.46 (t, J = 1.9 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.89 (s, 3H), 2.52 (s, 3H), 1.32 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.30, 153.70, 153.08, 149.59, 148.06, 139.29, 138.60, 138.11, 124.40, 117.49, 113.82, 113.34, 106.59, 106.41, 101.20, 56.34, 56.01, 34.79, 31.31, 31.03, 9.26. ESI-TOF HRMS: m/z 424.2346 (C₂₃H₂₉N₅O₃ + H⁺ requires 424.2343). Compound 20: ¹H NMR (400 MHz, CDCl₃) δ 9.04 (s, 1H), 7.17 (s, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.06–7.00 (m, 2H), 6.96 (d, J = 3.0 Hz, 1H), 6.58 (s, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.00 (s, 6H), 2.53 (s, 3H), 1.35 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.30, 153.70, 152.80, 151.07, 148.06, 139.24, 138.44, 138.17, 124.41, 117.48, 113.83, 113.35, 106.21, 105.95, 101.75, 56.34, 56.02, 40.89, 35.01, 31.41, 9.28. ESI-TOF HRMS: m/z 438.2501 (C₂₄H₃₁N₅O₃ + H⁺ requires 438.2500).

N-(3-(tert-Butyl)-5-(butylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 21) and N-(3-(tert-butyl)-5-(dibutylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 22). Those compounds were synthesized by using a procedure similar to that described for compounds 19 and 20, employing compound 18 and 1-iodobutane, to give compound 21 as a white solid (89.6 mg, 48% yield, 99.52% purity) and compound 22 as a white solid (45.9 mg, 22% yield, 100% purity). Compound 21: ¹H NMR (400 MHz, CDCl₃) δ 8.98 (s, 1H), 7.12–7.00 (m, 3H), 6.95 (d, J = 3.0 Hz, 1H), 6.92 (t, J = 2.1 Hz, 1H), 6.48 (dd, J = 2.4, 1.6 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.34–3.26 (m, 4H), 2.53 (s, 3H), 1.65–1.56 (m, 4H), 1.45–1.36 (m, 4H), 1.34 (s, 9H), 0.98 (t, J = 7.3 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 159.19, 153.70, 152.68, 148.60, 148.08, 139.19, 138.55, 138.29, 124.45, 117.44, 113.84, 113.35, 105.49, 104.77, 100.83, 56.34, 56.01, 50.96, 34.96, 31.40, 29.56, 20.43, 14.07, 9.28. ESI-TOF HRMS: m/z 466.2807 (C₂₆H₃₅N₅O₃ + H⁺ requires 466.2813). Compound 22: ¹H NMR (400 MHz, CDCl₃) δ 9.00 (s, 1H), 7.13–7.06 (m, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.92 (t, J = 1.7 Hz, 1H), 6.44 (t, J = 1.9 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.16 (t, J = 7.1 Hz, 2H), 2.52 (s, 3H), 1.68–1.56 (m, 2H), 1.52–1.40 (m, 2H), 1.31 (s, 9H), 0.97 (t, J = 7.3 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) δ 159.27, 153.70, 153.05, 148.87, 148.06, 139.27, 138.57, 138.13, 124.41, 117.48, 113.82, 113.34, 106.59, 106.34, 101.39, 56.34, 56.01, 43.89, 34.77, 31.71, 31.31, 20.33, 13.96, 9.25. ESI-TOF HRMS: m/z 522.3427 (C₃₀H₄₃N₅O₃ + H⁺ requires 522.3439).

N-(3-(tert-Butyl)-5-hydroxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 23). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-amino-5-(tert-butyl)phenol (compound 94b), to give a white solid (892.4 mg, 55% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 7.57 (t, J = 2.1 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.06 – 6.98 (m, 2H), 6.95 (d, J = 2.9 Hz, 1H), 6.71 (dd, J = 2.3, 1.6 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 2.51 (s, 3H), 1.29 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.66, 156.61, 153.79, 153.69, 148.05, 139.72, 138.11, 137.83, 124.20, 117.62, 113.79, 113.35, 109.22, 109.06, 104.77, 56.34, 56.02, 34.79, 31.22, 9.29. ESI-TOF HRMS: m/z 411.2022 (C₂₂H₂₆N₄O₄ + H⁺ requires 411.2027).

N-(3-(tert-Butyl)-5-methoxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 24). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 23 and iodomethane, to give a white solid (118.2 mg, 70% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.09 (s, 1H), 7.41 (t, J = 2.1 Hz, 1H), 7.16 (t, J = 1.7 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.2 Hz, 1H), 6.96 (d, J = 3.0 Hz, 1H), 6.75 (dd, J = 2.4, 1.6 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H), 2.52 (s, 3H), 1.33 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.98, 159.37, 153.71, 153.49, 148.03, 139.41, 138.62, 137.96, 124.33, 117.50, 113.83, 113.34, 109.37, 108.59, 101.80, 56.34, 56.01, 55.36, 34.90, 31.27, 9.26. ESI-TOF HRMS: m/z 425.2173 (C₂₃H₂₈N₄O₄ + H⁺ requires 425.2184).

N-(3-Butoxy-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 25). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 23 and 1-iodobutane, to give a white solid (125.9 mg, 67% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.07 (s, 1H), 7.40 (t, J = 2.1 Hz, 1H), 7.14 (t, J = 1.8 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.96 (d, J = 2.9 Hz, 1H), 6.74 (dd, J = 2.3, 1.6 Hz, 1H), 4.01 (t, J = 6.5 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 2.52 (s, 3H), 1.84–1.72 (m, 2H), 1.57–1.45 (m, 2H), 1.33 (s, 9H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.57, 159.34, 153.71, 153.41, 148.04, 139.38, 138.53, 137.99, 124.35, 117.50, 113.83, 113.34, 109.18, 109.07, 102.38, 67.73, 56.34, 56.02, 34.89, 31.46, 31.28, 19.31, 13.92, 9.26. ESI-TOF HRMS: m/z 467.2658 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

N-(3-Bromo-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 26). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 3-bromo-5-(tert-butyl)aniline (compound 95), to give a white solid (17.2 g, 61% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.50 (s, 1H), 8.09 (t, J = 1.8 Hz, 1H), 7.90 (t, J = 1.8 Hz, 1H), 7.30–7.26 (m, 2H), 7.24–7.21 (m, 1H), 7.13 (d, J = 3.0 Hz, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.22 (s, 3H), 2.39 (s, 3H), 1.30 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.69, 153.67, 153.19, 147.79, 139.95, 139.22, 137.34, 123.82, 123.23, 121.24, 119.87, 117.49, 116.58, 114.06, 56.43, 55.93, 34.75, 30.84, 8.76. ESI-TOF HRMS: m/z 473.1186 (C₂₂H₂₅BrN₄O₃ + H⁺ requires 473.1183).

N-(3-(tert-Butyl)-5-cyanophenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 27). Under N₂, zinc cyanide (0.322 g, 2.75 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.122 g, 0.106 mmol) were added to a degassed solution of N-(3-bromo-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 26, 1 g, 2.113 mmol) in DMF (10 mL). The reaction mixture was stirred at 100°C for 8 h. After dilution with EtOAc (100 mL), the suspension was filtered over celite and concentrated. The residue was purified by flash chromatography (0% to 100% EtOAc in hexane) to give compound 27 as a white solid (726 mg, 82% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 8.07 (dd, J = 2.1, 1.4 Hz, 1H), 7.79 (t, J = 1.9 Hz, 1H), 7.43 (t, J = 1.6 Hz, 1H), 7.10 (dd, J = 9.1, 3.0 Hz, 1H), 7.04 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.52 (s, 3H), 1.36 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.55, 153.88, 153.72, 148.09, 139.82, 138.47, 137.45, 124.95, 124.40, 121.17, 120.19, 118.92, 117.58, 113.97, 113.55, 112.89, 56.42, 56.02, 35.02, 30.99, 9.12. ESI-TOF HRMS: m/z 420.2025 (C₂₃H₂₅N₅O₃ + H⁺ requires 420.2030).

N-(5-(tert-Butyl)-[1,1’-biphenyl]-3-yl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 28). Under N₂, K₂CO₃ (58.4 mg, 0.423 mmol), phenylboronic acid (30.9 mg, 0.254 mmol), and palladium-tetrakis(triphenylphosphine) (12.2 mg, 10.56 μmol) were added to a degassed solution of N-(3-bromo-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 26, 100 mg, 0.211 mmol) in DMF (10 mL) and water (5 mL). The reaction mixture was stirred at 100°C for 8 h. After cooling and dilution with EtOAc (100 mL), the suspension was filtered through celite and concentrated. The residue was purified by silica gel chromatography (0% to 100% EtOAc in hexane) to give compound 28 as a white solid (79 mg, 80% yield, 98.23% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.15 (s, 1H), 7.89 (t, J = 1.8 Hz, 1H), 7.68 (t, J = 1.9 Hz, 1H), 7.66 (d, J = 1.4 Hz, 1H), 7.65–7.62 (m, 1H), 7.48–7.39 (m, 3H), 7.38–7.32 (m, 1H), 7.10 (dd, J = 9.1, 2.9 Hz, 1H), 7.04 (d, J = 9.1 Hz, 1H), 6.97 (d, J = 3.0 Hz, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 2.54 (s, 3H), 1.42 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.45, 153.88, 152.72, 148.18, 141.94, 141.50, 139.36, 138.06, 138.03, 128.62, 127.37, 127.25, 124.63, 120.41, 117.52, 116.10, 116.01, 113.99, 113.57, 56.43, 56.02, 34.97, 31.38, 9.17. ESI-TOF HRMS: m/z 471.2395 (C₂₈H₃₀N₄O₃ + H⁺ requires 471.2391).

N-(3-(tert-Butyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 29). Under N₂, CH₃COOK (2.073 g, 21.13 mmol) and 4,4,4’,4’,5,5,5’,5’-octamethyl-2,2’-bi(1,3,2-dioxaborolane) (4.02 g, 15.84 mmol), Pd(dppf)Cl₂ (0.386 g, 0.528 mmol) was added to a degassed solution of N-(3-bromo-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 26, 5 g, 10.56 mmol) in DMF (30 mL). The reaction mixture was stirred at 100°C for 8 h. The reaction mixture was cooled to room temperature and diluted with EtOAc (100 mL). The suspension was then filtered through celite and concentrated. The residue was purified by silica gel chromatography (0% to 100% EtOAc in hexane) to give product 29 as a white solid (5.1 g, 93% yield, 95.10% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.08 (s, 1H), 8.03 (t, J = 2.1 Hz, 1H), 7.82 (dd, J = 2.2, 0.9 Hz, 1H), 7.62 (dd, J = 1.9, 0.9 Hz, 1H), 7.08 (dd, J = 9.1, 3.0 Hz, 1H), 7.02 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 2.9 Hz, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 2.52 (s, 3H), 1.38 (s, 9H), 1.35 (s, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 159.38, 153.86, 151.63, 148.16, 139.20, 138.05, 137.24, 127.40, 124.66, 123.35, 120.18, 117.50, 113.95, 113.57, 83.76, 56.42, 56.01, 34.83, 31.39, 24.87, 9.14. ESI-TOF HRMS: m/z 521.2922 (C₂₈H₃₇BN₄O₅ + H⁺ requires 521.2930).

N-(3-(tert-Butyl)-5-(1H-tetrazol-5-yl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 30). A mixture of N-(3-(tert-butyl)-5-cyanophenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 27, 300 mg, 0.715 mmol), sodium azide (186 mg, 2.86 mmol), and ammonium chloride (153 mg, 2.86 mmol) was stirred in 10 mL of DMSO at 100°C for 18 h. The mixture was cooled to room temperature and extracted with EtOAc (100 mL). The organic layer was washed with brine, dried with anhydrous Na₂SO₄, and concentrated. The crude product was purified by flash chromatography (0% to 100% EtOAc in hexane) to provide compound 30 as a white solid (135.2 mg, 41% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.31 (s, 1H), 8.36 (s, 1H), 7.99 (s, 1H), 7.70 (s, 1H), 7.14–6.98 (m, 2H), 7.00–6.90 (m, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 2.48 (s, 3H), 1.34 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 160.04, 154.04, 153.86, 148.05, 139.91, 137.95, 137.48, 124.24, 121.17, 120.21, 117.66, 116.27, 113.95, 113.53, 56.40, 56.03, 35.08, 31.09, 9.19. ESI-TOF HRMS: m/z 463.2203 (C₂₃H₂₆N₈O₃ + H⁺ requires 463.2201).

Synthesis Procedures for Compounds 31–38 (Scheme 3)

N-(4-Acetamido-3-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 32). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and N-(4-amino-2-(tert-butyl)phenyl)acetamide (compound 96), to give a white solid (341.0 mg, 75% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.44 (s, 1H), 9.25 (s, 1H), 7.64 (h, J = 2.4 Hz, 2H), 7.33 (d, J = 9.4 Hz, 1H), 7.28 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.0 Hz, 1H), 7.15 (d, J = 3.0 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 2.38 (s, 3H), 2.04 (s, 3H), 1.31 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.98, 159.35, 153.05, 147.69, 141.79, 138.95, 137.51, 136.80, 136.01, 126.50, 123.69, 123.42, 118.67, 117.39, 113.99, 113.85, 56.30, 55.85, 34.39, 30.80, 23.11, 8.77. ESI-TOF HRMS: m/z 452.2288 (C₂₄H₂₉N₅O₄ + H⁺ requires 452.2293).

N-(4-Amino-3-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 31). To a solution of N-(4-acetamido-3-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 32, 0.135 g, 0.3 mmol) in water (10 mL) and EtOH (5 mL) was added 12 M hydrochloric acid (2 mL). The mixture was stirred at 80°C overnight then cooled to room temperature, whereupon its pH was adjusted to 8 with saturated NaHCO₃ solution. The mixture was then extracted with EtOAc (100 mL). The organic phase was washed with saturated NaHCO₃ solution (50 mL), water (50 mL), and brine (50 mL). The organic phase was then dried with anhydrous MgSO₄, filtered, and concentrated. The residue was purified by flash chromatography (0% to 100% EtOAc in hexane) to provide compound 31 as a white solid (78.5 mg, 64% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 9.99 (s, 1H), 7.28 (d, J = 9.1 Hz, 1H), 7.22 (dt, J = 6.2, 3.4 Hz, 2H), 7.13 (d, J = 2.9 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.89 (dd, J = 8.5, 2.2 Hz, 1H), 4.77 (s, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 2.37 (s, 3H), 1.33 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.01, 153.04, 147.70, 146.01, 138.63, 137.73, 136.78, 128.04, 125.90, 123.76, 117.33, 114.01, 113.84, 109.01, 108.91, 56.29, 55.84, 33.58, 29.37, 8.73. ESI-TOF HRMS: m/z 410.2183 (C₂₂H₂₇N₅O₃ + H⁺ requires 410.2187).

N-(3-(tert-Butyl)-4-(methylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 33). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 31 and iodomethane, to give a white solid (142.1 mg, 67% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.01 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.12–7.06 (m, 2H), 7.05–6.99 (m, 2H), 6.95 (d, J = 3.0 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 2.95 (s, 3H), 2.52 (s, 3H), 1.41 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.22, 153.70, 148.06, 147.99, 139.26, 138.15, 137.08, 129.46, 126.58, 124.42, 117.46, 113.84, 113.35, 107.89, 102.74, 56.34, 56.00, 33.88, 31.20, 29.98, 9.25. ESI-TOF HRMS: m/z 424.2343 (C₂₃H₂₉N₅O₃ + H⁺ requires 424.2343).

N-(3-(tert-Butyl)-4-(dimethylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 34). Compound 31 (0.205 g, 0.5 mmol), formaldehyde (0.150 g, 5.00 mmol), and NaBH₃CN (0.094 g, 1.500 mmol) were added to MeOH (5 mL). The reaction mixture was stirred at room temperature for 12 h then diluted with water (30 mL) and extracted with EtOAc (25 mL × 2). The combined EtOAc layers were washed with water (30 mL), dried with anhydrous Na₂SO₄, and concentrated. The residue was purified by flash chromatography (0% to 100% acetonitrile in water) to give product 34 as a white solid (143.2 mg, 65% yield, 99.62% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.02 (s, 1H), 7.77 (d, J = 2.3 Hz, 1H), 7.39 (dd, J = 8.6, 2.3 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 2.9 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 2.64 (s, 6H), 2.52 (s, 3H), 1.44 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.28, 155.72, 153.72, 148.04, 143.39, 139.37, 137.95, 136.66, 127.25, 124.36, 117.50, 117.01, 116.81, 113.83, 113.35, 56.34, 56.01, 47.11, 35.22, 30.94, 9.24. ESI-TOF HRMS: m/z 438.2498 (C₂₄H₃₁N₅O₃ + H⁺ requires 438.2500).

N-(3-(tert-Butyl)-4-(butylamino)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 35). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 31 and 1-iodobutane, to give compound 35 as a white solid (148.9 mg, 64% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 8.98 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.11–7.05 (m, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.99 (dd, J = 8.4, 2.3 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.22 (t, J = 6.9 Hz, 2H), 2.52 (s, 3H), 1.84–1.61 (m, 2H), 1.56–1.47 (m, 2H), 1.42 (s, 9H), 1.00 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.20, 153.70, 148.07, 147.09, 139.24, 138.16, 137.00, 129.24, 126.65, 124.43, 117.47, 113.83, 113.34, 107.81, 103.13, 56.34, 56.01, 44.13, 33.89, 31.70, 30.05, 20.60, 13.97, 9.25. ESI-TOF HRMS: m/z 466.2810 (C₂₆H₃₅N₅O₃ + H⁺ requires 466.2813).

N-(3-(tert-Butyl)-4-hydroxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 36). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 4-amino-2-(tert-butyl)phenol (compound 97), to give a white solid (579.2 mg, 47% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.14 (s, 1H), 9.17 (s, 1H), 7.61 (d, J = 2.6 Hz, 1H), 7.52 (dd, J = 8.5, 2.5 Hz, 1H), 7.27 (d, J = 9.2 Hz, 1H), 7.21 (dd, J = 9.1, 3.1 Hz, 1H), 7.13 (d, J = 3.0 Hz, 1H), 6.73 (d, J = 8.6 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 2.37 (s, 3H), 1.36 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.92, 153.05, 152.16, 147.69, 138.45, 137.80, 135.13, 129.89, 123.80, 119.83, 119.29, 117.30, 115.62, 113.99, 113.83, 56.28, 55.84, 34.39, 29.30, 8.74. ESI-TOF HRMS: m/z 411.2021 (C₂₂H₂₆N₄O₄ + H⁺ requires 411.2027).

N-(3-(tert-Butyl)-4-methoxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 37). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 36 and iodomethane, to give a white solid (84.2 mg, 82% yield, 118% purity). ¹H NMR (400 MHz, CDCl₃) δ 8.96 (s, 1H), 7.66 (dd, J = 8.7, 2.7 Hz, 1H), 7.45 (d, J = 2.7 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H), 2.52 (s, 3H), 1.40 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.21, 155.44, 153.70, 148.06, 139.20, 138.97, 138.07, 130.44, 124.42, 119.30, 118.77, 117.46, 113.83, 113.34, 111.94, 56.34, 56.01, 55.36, 34.94, 29.67, 9.22. ESI-TOF HRMS: m/z 425.2190 (C₂₃H₂₈N₄O₄ + H⁺ requires 425.2184).

N-(4-Butoxy-3-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 38). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 36 and iodobutane, to give a white solid (138.6 mg, 74% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 8.95 (s, 1H), 7.64 (dd, J = 8.7, 2.7 Hz, 1H), 7.45 (d, J = 2.7 Hz, 1H), 7.09 (dd, J = 9.1, 3.0 Hz, 1H), 7.03 (d, J = 9.2 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 2.52 (s, 3H), 1.84 (dq, J = 8.6, 6.5 Hz, 2H), 1.59–1.51 (m, 2H), 1.41 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.18, 154.82, 153.70, 148.07, 139.17, 138.68, 138.10, 130.12, 124.43, 119.34, 118.73, 117.46, 113.83, 113.34, 112.05, 67.76, 56.34, 56.01, 34.98, 31.62, 29.74, 19.63, 13.90, 9.22. ESI-TOF HRMS: m/z 467.2645 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

Synthesis Procedures for Compounds 39–56 (Scheme 4)

tert-Butyl (2-amino-4-(tert-butyl)phenyl)carbamate (compound 100). To a solution of 4-(tert-butyl)-2-nitroaniline (compound 98, 5 g, 25.7 mmol), N,N-dimethylpyridin-4-amine (0.314 g, 2.57 mmol), and DIEA (6.65 g, 51.5 mmol) in DCM (100 mL) was added Boc₂O (6.18 g, 28.3 mmol). The reaction mixture was stirred at room temperature overnight then evaporated to afford the crude product. Water (100 mL) was added to the residue, then this was extracted with EtOAc (200 mL). The organic phase was washed with saturated NH₄Cl solution then dried with MgSO₄ and concentrated to give compound 99 as a yellow solid (5.88 g, 78% yield). To a solution of tert-butyl (4-(tert-butyl)-2-nitrophenyl)carbamate (compound 99, 5.88 g, 19.98 mmol) in 100 mL of MeOH was added Pd/C (1 g, 10%) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under an H₂ balloon at room temperature for 12 h then filtered. The filtrate was concentrated and purified by flash chromatography to obtain compound 100 as a brown solid (3.22 g, 61% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.0 Hz, 1H), 6.83–6.78 (m, 2H), 6.13 (s, 1H), 1.51 (s, 9H), 1.27 (s, 9H).¹³C NMR (101 MHz, CDCl₃) δ 154.02, 149.63, 139.64, 124.67, 121.99, 116.80, 114.84, 80.43, 34.32, 31.31, 28.34.

tert-Butyl (4-(tert-butyl)-2-(1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenyl)carbamate (compound 41). This compound was synthesized by using a procedure similar to that described for compound 1, employing compounds 93 and 100, to give a white solid (891.2 mg, 87% yield, 99.60% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.23 (s, 1H), 7.63–7.56 (m, 2H), 7.26 (dd, J = 8.6, 2.2 Hz, 1H), 7.10 (dd, J = 9.1, 3.0 Hz, 2H), 7.04 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 2.52 (s, 3H), 1.52 (s, 9H), 1.32 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 160.30, 153.88, 153.72, 148.34, 148.04, 139.65, 137.68, 128.81, 128.60, 124.33(2C), 123.57, 121.61, 117.49, 113.85, 113.35, 80.55, 56.36, 56.00, 34.47, 31.32, 28.36, 9.27. ESI-TOF HRMS: m/z 510.2720 (C₂₇H₃₅N₅O₅ + H⁺ requires 510.2711).

N-(2-Amino-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 39). A solution of tert-butyl (4-(tert-butyl)-2-(1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenyl)carbamate (compound 41, 200 mg, 0.392 mmol) in DCM (10 mL) was added TFA (10 mL, 0.392 mmol) in an ice bath. The solution was stirred at room temperature for 1 h then diluted with water (20 mL) and extracted with EtOAc (25 mL × 2). The combined EtOAc layers were washed with water (30 mL), dried with anhydrous Na₂SO₄, and concentrated. The residue was purified by flash chromatography (0% to 100% EA in hexane) to give compound 39 as a light brown solid (117.5 mg, 79% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 8.98 (s, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.16–7.06 (m, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.80 (dd, J = 8.3, 2.0 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 2.51 (s, 3H), 1.30 (s, 9H).¹³C NMR (101 MHz, CDCl₃) δ 159.71, 153.71, 148.05, 142.68, 142.66, 139.43, 138.14, 137.75, 124.40, 124.08, 123.47, 122.09, 117.85, 117.46, 113.84, 113.36, 56.36, 56.01, 34.06, 31.50, 9.22. ESI-TOF HRMS: m/z 410.2181 (C₂₂H₂₇N₅O₃ + H⁺ requires 410.2187).

N-(4-(tert-Butyl)-2-nitrophenyl)acetamide (compound 103). A solution of 4-(tert-butyl)aniline (compound 101, 15 mL, 94 mmol) and TEA (19.69 mL, 141 mmol) in DCM (300 mL) was cooled to 0°C then acetyl chloride (7.37 mL, 104 mmol) was added dropwise. The resulting solution was stirred for 2 h, allowing it to warm to room temperature. The reaction mixture was then diluted with water and DCM. The organic phase was washed with 1 N HCl and brine, dried with anhydrous Na₂SO₄, filtered, and concentrated to give the crude compound 102 as a white solid. This solid was used without purification for the next step. H₂SO₄ (98%, 100 mL) was cooled to 0°C then crude compound 102 was added. KNO₃ (10.56 g, 104 mmol) was added (in several portions) to the resulting solution, and the mixture was allowed to warm to room temperature over 2 h then stirred at room temperature overnight. The reaction mixture was then poured over ice and extracted three times with EtOAc (200 mL). The combined organic phase was washed with saturated aq. NaHCO₃, water, and brine then dried with anhydrous MgSO₄, filtered, and concentrated. The residue was purified by flash chromatography (0% to 100% EA in hexane) to provide N-(4-(tert-butyl)-2-nitrophenyl)acetamide (compound 103) as a yellow solid (12.04 g, 54% yield [in two steps]). ¹H NMR (400 MHz, DMSO-d₆) δ 10.62 (s, 1H), 8.29 (d, J = 2.3 Hz, 1H), 8.19 (dd, J = 8.5, 2.3 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 2.50 (s, 3H), 1.75 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.40, 148.05, 142.38, 130.91, 128.72, 125.23, 120.98, 34.39, 30.65, 23.24.

N-(2-Amino-4-(tert-butyl)phenyl)acetamide (compound 104). To a solution of N-(4-(tert-butyl)-2-nitrophenyl)acetamide (12.4 g, 52.5 mmol) in MeOH (100 mL) was added Pd/C (1 g, 10%) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under an H₂ balloon at room temperature for 24 h then filtered. The filtrate was concentrated and purified by flash chromatography (0% to 100% EA in hexane) to obtain compound 104 as a brown solid (3.22 g, 61% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 9.06 (s, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H), 6.56 (dd, J = 8.3, 2.2 Hz, 1H), 4.73 (s, 2H), 2.01 (s, 3H), 1.22 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.13, 148.12, 141.38, 124.94, 121.09, 113.20, 112.81, 33.90, 31.19, 23.18.

N-(2-Acetamido-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 40). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and N-(2-amino-4-(tert-butyl)phenyl)acetamide (compound 104), to give a white solid (3.65 g, 81% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.24 (s, 1H), 8.56 (s, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 2.2 Hz, 1H), 7.28 (dd, J = 8.5, 2.3 Hz, 1H), 7.09 (dd, J = 9.1, 2.9 Hz, 1H), 7.04 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 2.50 (s, 3H), 2.16 (s, 3H), 1.31 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 167.10, 158.55, 151.84, 147.23, 146.11, 137.85, 135.51, 126.85, 126.53, 123.68, 122.32, 121.90, 119.42, 115.67, 111.96, 111.49, 54.48, 54.13, 32.65, 29.40, 22.33, 7.38. ESI-TOF HRMS: m/z 452.2290 (C₂₄H₂₉N₅O₄ + H⁺ requires 452.2293).

N-(5-(tert-Butyl)-2-hydroxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 42). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 2-amino-4-(tert-butyl)phenol, to give a white solid (181.8 mg, 56% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 10.01 (s, 1H), 9.61 (s, 1H), 8.33 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.0 Hz, 1H), 7.16 (d, J = 3.0 Hz, 1H), 6.99 (dd, J = 8.4, 2.4 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H), 1.27 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.45, 153.06, 147.65, 144.15, 141.51, 138.80, 137.30, 125.64, 123.61, 120.65, 117.44, 116.80, 114.28, 113.98, 113.89, 56.32, 55.85, 33.91, 31.39, 8.72. ESI-TOF HRMS: m/z 411.2025 (C₂₂H₂₆N₄O₄ + H⁺ requires 411.2027).

N-(5-(tert-Butyl)-2-methoxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 43). This compound was synthesized by using a procedure similar to that described for compound 1, employing compound 93 and 5-(tert-butyl)-2-methoxyaniline, to give a white solid (355.9 mg, 84% yield, 95.16% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 9.56 (s, 1H), 8.41 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.23 (dd, J = 9.1, 3.0 Hz, 1H), 7.17–7.09 (m, 2H), 7.03 (d, J = 8.6 Hz, 1H), 5.75 (s, 1H), 3.91 (s, 3H), 3.78 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H), 1.29 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.47, 153.06, 147.65, 146.31, 142.91, 138.90, 137.20, 126.36, 123.58, 120.65, 117.44, 116.78, 113.99, 113.91, 110.48, 56.33, 56.04, 55.85, 34.00, 31.31, 8.72. ESI-TOF HRMS: m/z 425.2189 (C₂₃H₂₈N₄O₄ + H⁺ requires 425.2184).

N-(5-(tert-Butyl)-2-ethoxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 44). This compound was synthesized by using a procedure similar to that described for compound 19, employing 42 and iodoethane, to give a white solid (87.6 mg, 82% yield, 100% purity). ¹H NMR (500 MHz, DMSO-d₆) δ 9.61 (s, 1H), 8.44 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.23 (dd, J = 9.2, 3.1 Hz, 1H), 7.16 (d, J = 3.0 Hz, 1H), 7.10 (dd, J = 8.6, 2.4 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 4.16 (q, J = 6.9 Hz, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H), 1.41 (t, J = 6.9 Hz, 3H), 1.29 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 158.91, 153.55, 148.12, 145.79, 143.44, 139.40, 137.76, 127.18, 124.07, 121.04, 117.95, 116.96, 114.46, 114.40, 112.11, 64.77, 56.83, 56.34, 34.52, 31.81, 15.22, 9.24. ESI-TOF HRMS: m/z 439.2329 (C₂₄H₃₀N₄O₄ + H⁺ requires 439.2340).

N-(5-(tert-Butyl)-2-propoxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 45). This compound was synthesized by using a procedure similar to that described for compound 19, employing 42 and 1-iodopropane, to give a white solid (99.4 mg, 90% yield, 100% purity). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.46 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.1 Hz, 1H), 7.17 (d, J = 3.1 Hz, 1H), 7.10 (dd, J = 8.6, 2.4 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.07 (t, J = 6.3 Hz, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H), 1.81 (h, J = 7.4 Hz, 2H), 1.29 (s, 9H), 1.07 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 158.89, 153.55, 148.10, 145.82, 143.43, 139.39, 137.77, 127.25, 124.06, 120.97, 117.98, 116.76, 114.44, 114.38, 111.99, 70.39, 56.82, 56.34, 34.53, 31.82, 22.62, 10.91, 9.23. ESI-TOF HRMS: m/z 453.2500 (C₂₅H₃₂N₄O₄ + H⁺ requires 453.2496).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 46). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 2 and 1-iodobutane, to give a white solid (391.8 mg, 84% yield, 98.02% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 9.65 (s, 1H), 8.46 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.1 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.10 (t, J = 6.3 Hz, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H), 1.78 (dq, J = 8.4, 6.4 Hz, 2H), 1.61–1.47 (m, 2H), 1.29 (s, 9H), 0.96 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.40, 153.06, 147.61, 145.34, 142.91, 138.87, 137.27, 126.74, 123.58, 120.44, 117.48, 116.27, 113.94, 113.88, 111.44, 68.08, 56.31, 55.84, 34.01, 31.30, 30.75, 18.65, 13.65, 8.71. ESI-TOF HRMS: m/z 467.2645 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

N-(5-(tert-Butyl)-2-(pentyloxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (47). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 1-iodopentane, to give a white solid (97.3 mg, 83% yield, 100% purity). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.45 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.23 (dd, J = 9.2, 3.1 Hz, 1H), 7.16 (d, J = 3.1 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.10 (t, J = 6.3 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 2.41 (s, 3H), 1.84–1.75 (m, 2H), 1.56–1.46 (m, 2H), 1.38 (dt, J = 14.9, 7.3 Hz, 2H), 1.29 (s, 9H), 0.91 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 158.91, 153.55, 148.12, 145.87, 143.43, 139.36, 137.75, 127.26, 124.07, 120.97, 117.97, 116.77, 114.46, 114.38, 112.00, 68.90, 56.82, 56.34, 34.53, 31.81, 28.91, 28.12, 22.38, 14.39, 9.22. ESI-TOF HRMS: m/z 481.2825 (C₂₇H₃₆N₄O₄ + H⁺ requires 481.2809).

N-(5-(tert-Butyl)-2-(hexyloxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 48). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 1-bromohexane, to give a colorless oil (105.2 mg, 87% yield, 100% purity). ¹H NMR (500 MHz, DMSO-d₆) δ 9.65 (s, 1H), 8.45 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.23 (dd, J = 9.2, 3.0 Hz, 1H), 7.15 (d, J = 3.0 Hz, 1H), 7.09 (dd, J = 8.5, 2.4 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.09 (t, J = 6.3 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 2.41 (s, 3H), 1.82–1.73 (m, 2H), 1.52 (tt, J = 9.5, 6.1 Hz, 2H), 1.37–1.30 (m, 4H), 1.29 (s, 9H), 0.86 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 158.92, 153.55, 148.12, 145.88, 143.42, 139.35, 137.75, 127.25, 124.08, 120.98, 117.95, 116.80, 114.46, 114.39, 112.00, 68.92, 56.82, 56.34, 34.52, 31.82, 31.47, 29.18, 25.60, 22.53, 14.34, 9.22. ESI-TOF HRMS: m/z 495.2966 (C₂₈H₃₈N₄O₄ + H⁺ requires 495.2966).

N-(5-(tert-Butyl)-2-(octyloxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 49). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 1-iodooctane, to give a colorless oil (158.2 mg, 61% yield, 100% purity). ¹H NMR (400 MHz, DMSO-d₆) δ 9.65 (s, 1H), 8.45 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.0 Hz, 1H), 7.13 (d, J = 3.0 Hz, 1H), 7.08 (dd, J = 8.5, 2.4 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 4.09 (t, J = 6.3 Hz, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 2.41 (s, 3H), 1.85–1.71 (m, 2H), 1.51 (p, J = 7.1 Hz, 2H), 1.28 (s, 17H), 0.84–0.74 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.41, 153.05, 147.61, 145.33, 142.92, 138.82, 137.25, 126.77, 123.60, 120.43, 117.43, 116.26, 113.90, 113.88, 111.49, 68.41, 56.30, 55.82, 34.01, 31.29, 31.14, 28.69, 28.62, 25.42, 22.03, 13.84, 8.70. ESI-TOF HRMS: m/z 523.3279 (C₃₀H₄₂N₄O₄ + H⁺ requires 523.3279).

N-(5-(tert-Butyl)-2-(cyanomethoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 50). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 2-bromoacetonitrile, to give a white solid (81.9 mg, 74% yield, 100% purity). ¹H NMR (500 MHz, DMSO-d₆) δ 9.60 (s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.25–7.18 (m, 3H), 5.31 (s, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 2.40 (s, 3H), 1.30 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 159.21, 153.55, 148.14, 145.58, 144.81, 139.53, 137.60, 127.33, 124.06, 121.72, 119.03, 117.95, 117.09, 114.50, 114.40, 112.90, 56.83, 56.35, 54.87, 34.66, 31.70, 9.24. ESI-TOF HRMS: m/z 450.2132 (C₂₄H₂₇N₅O₄ + H⁺ requires 450.2136).

N-(5-(tert-Butyl)-2-(4-hydroxybutoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 51). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 4-bromobutan-1-ol, to give a white solid (329.5 mg, 30% yield, 99.20% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.80 (s, 1H), 8.64 (d, J = 2.3 Hz, 1H), 7.08 (ddd, J = 8.5, 5.1, 2.7 Hz, 2H), 7.02 (d, J = 9.2 Hz, 1H), 6.96 (d, J = 3.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.13 (t, J = 5.9 Hz, 2H), 3.81 (d, J = 3.4 Hz, 5H), 3.75 (s, 3H), 2.54 (s, 3H), 2.07–1.96 (m, 2H), 1.90 (dt, J = 8.3, 6.3 Hz, 2H), 1.36 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 159.22, 153.68, 148.00, 145.58, 144.05, 139.25, 138.42, 127.54, 124.37, 120.17, 117.55, 116.95, 113.74, 113.30, 110.65, 68.83, 62.35, 56.34, 56.02, 34.48, 31.59, 29.72, 25.51, 9.30. ESI-TOF HRMS: m/z 483.2599 (C₂₆H₃₄N₄O₅ + H⁺ requires 483.2602).

N-(5-(tert-Butyl)-2-(2-(dimethylamino)ethoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 52). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42, 2.2 eq Cs₂CO_3, and 2-chloro-N,N-dimethylethan-1-amine hydrochloride, to give a white solid (67.1 mg, 57% yield, 97.97% purity). ¹H NMR (500 MHz, DMSO-d₆) δ 9.92 (s, 1H), 8.42 (d, J = 2.3 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.2, 3.1 Hz, 1H), 7.16 (d, J = 3.0 Hz, 1H), 7.11 (dd, J = 8.6, 2.4 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 4.20 (t, J = 5.5 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 2.68 (t, J = 5.5 Hz, 2H), 2.40 (s, 3H), 2.27 (s, 6H), 1.29 (s, 9H).¹³C NMR (126 MHz, CDCl₃) δ 159.47, 153.68, 148.03, 145.36, 144.50, 139.15, 138.46, 127.78, 124.44, 120.33, 117.48, 117.45, 113.75, 113.30, 111.35, 67.29, 58.01, 56.33, 56.01, 45.77, 34.50, 31.56, 9.30. ESI-TOF HRMS: m/z 482.2776 (C₂₆H₃₅N₅O₄ + H⁺ requires 482.2762).

N-(5-(tert-Butyl)-2-(3-(dimethylamino)propoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 53). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42, 2.2 eq Cs₂CO₃, and 3-bromo-N,N-dimethylpropan-1-amine hydrobromide, to give a white solid (88.9 mg, 73% yield, 100% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.78 (s, 1H), 8.62 (d, J = 2.4 Hz, 1H), 7.11–7.05 (m, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 4.15 (t, J = 6.1 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 2.89–2.74 (m, 2H), 2.53 (s, 3H), 2.44 (s, 6H), 2.32–2.12 (m, 2H), 1.35 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 159.27, 153.69, 148.04, 145.35, 144.25, 139.15, 138.45, 127.50, 124.43, 120.19, 117.37, 116.98, 113.88, 113.30, 110.73, 66.69, 56.32, 56.23, 56.01, 45.02, 34.49, 31.57, 26.95, 9.26. ESI-TOF HRMS: m/z 496.2916 (C₂₇H₃₇N₅O₄ + H⁺ requires 496.2918).

N-(5-(tert-Butyl)-2-(2-morpholinoethoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 54). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42, 4-(2-chloroethyl)morpholine hydrochloride, and 2.2 eq Cs₂CO₃ to give a white solid (88.5 mg, 69% yield, 100% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.75 (s, 1H), 8.62 (d, J = 2.4 Hz, 1H), 7.08 (ddd, J = 8.6, 4.7, 2.7 Hz, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.1 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 4.30 (s, 2H), 3.82 (s, 3H), 3.76 (s, 8H), 3.01 (s, 2H), 2.90–2.68 (m, 4H), 2.53 (s, 3H), 1.35 (s, 9H). ^13C NMR (126 MHz, CDCl₃) δ 159.39, 153.69, 148.03, 145.25, 144.61, 139.18, 138.34, 127.59, 124.40, 120.30, 117.49, 117.37, 113.75, 113.32, 111.03, 66.82, 66.52, 57.62, 56.34, 56.02, 53.96, 34.51, 31.56, 9.28. ESI-TOF HRMS: m/z 524.2872 (C₂₈H₃₇N₅O₅ + H⁺ requires 524.2867).

N-(5-(tert-Butyl)-2-(3-morpholinopropoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 55). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42, 2.2 eq Cs₂CO₃, and 4-(3-bromopropyl)morpholine hydrobromide, to give a white solid (92.9 mg, 71% yield, 100% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.78 (s, 1H), 8.63 (d, J = 2.4 Hz, 1H), 7.08 (ddd, J = 8.5, 7.7, 2.7 Hz, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.1 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 4.16 (t, J = 6.1 Hz, 2H), 3.82 (s, 3H), 3.80–3.63 (m, 7H), 2.53 (s, 9H), 2.11 (s, 2H), 1.35 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 159.31, 153.69, 148.01, 145.39, 144.25, 139.14, 138.40, 127.54, 124.45, 120.16, 117.38, 117.04, 113.85, 113.30, 110.75, 66.71, 56.33, 56.01, 55.46, 53.53, 34.49, 31.57, 26.14, 9.27. ESI-TOF HRMS: m/z 538.3035 (C₂₉H₃₉N₅O₅ + H⁺ requires 538.3024).

N-(5-(tert-Butyl)-2-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 56). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42, 3.2 eq Cs₂CO₃, and 1-(2-bromoethyl)-4-methylpiperazine dihydrobromide (108 mg, 0.292 mmol), to give a white solid (51.9 mg, 40% yield, 98.11% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.78 (s, 1H), 8.61 (d, J = 2.3 Hz, 1H), 7.08 (ddd, J = 8.5, 7.3, 2.7 Hz, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.94 (d, J = 3.0 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 4.21 (t, J = 5.6 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 2.91 (t, J = 5.6 Hz, 2H), 2.88–2.62 (m, 8H), 2.53 (s, 3H), 2.41 (s, 3H), 1.35 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 159.44, 153.69, 147.99, 145.36, 144.56, 139.28, 138.39, 127.92, 124.41, 120.37, 117.47, 117.36, 113.86, 113.28, 111.64, 66.73, 56.73, 56.30, 56.02, 54.47, 52.41, 45.15, 34.51, 31.56, 9.30. ESI-TOF HRMS: m/z 537.3182 (C₂₉H₄₀N₆O₄ + H⁺ requires 537.3184).

Synthesis Procedures for Compounds 57–79 (Scheme 5)

Compounds 107a–v were synthesized by using a procedure similar to that described for compound 93, employing the corresponding aromatic amines.

1-(5-Fluoro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107a). Brown solid (1.45 g, 29% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.15 (s, 1H), 7.58–7.45 (m, 2H), 7.36 (dd, J = 10.2, 4.7 Hz, 1H), 3.79 (s, 3H), 2.32 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.47, 156.56, 154.19, 150.40, 150.38, 140.47, 135.83, 123.71, 123.61, 118.73, 118.51, 115.96, 115.70, 114.10, 114.02, 56.50, 9.01.

1-(5-Chloro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107b). Brown solid (2.11 g, 39% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (dd, J = 8.9, 2.7 Hz, 1H), 7.67 (d, J = 2.6 Hz, 1H), 7.37 (d, J = 8.9 Hz, 1H), 3.81 (s, 3H), 2.32 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.47, 152.75, 140.52, 135.85, 131.99, 128.24, 124.29, 124.17, 114.55, 56.45, 9.02.

1-(5-Bromo-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107c). Brown solid (1.33 g, 29% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.16 (s, 1H), 7.82 (dd, J = 9.0, 2.5 Hz, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.31 (d, J = 9.0 Hz, 1H), 3.80 (s, 3H), 2.32 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.46, 153.17, 140.53, 135.83, 134.89, 130.92, 124.61, 115.00, 111.41, 56.41, 9.03.

1-(2-Methoxy-5-(trifluoromethyl)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107d). Brown solid (1.03 g, 17% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.17 (s, 1H), 8.05–8.01 (m, 1H), 7.99–7.95 (m, 1H), 7.54 (d, J = 8.8 Hz, 1H), 3.90 (s, 3H), 2.33 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.46, 156.61, 140.69, 135.93, 129.65, 129.62, 127.76, 126.07, 126.04, 126.00, 125.96, 125.06, 123.77, 122.36, 121.95, 121.62, 121.29, 120.96, 113.81, 56.72, 9.03.

1-(2-Methoxy-5-(trifluoromethoxy)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107e). White solid (2.48 g, 78.1% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.27–13.11 (m, 1H), 7.79–7.59 (m, 2H), 7.48–7.40 (m, 1H), 3.84 (s, 3H), 2.33 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.46, 152.88, 140.95, 140.93, 140.90, 140.88, 140.53, 135.93, 125.27, 123.82, 122.11, 121.34, 118.79, 114.11, 56.58, 9.01.

1-(2-Methoxy-5-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107f). Brown solid (1.54 g, 31% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.09 (s, 1H), 7.43 (ddd, J = 8.5, 2.3, 0.8 Hz, 1H), 7.30–7.26 (m, 1H), 7.22 (d, J = 8.5 Hz, 1H), 3.76 (s, 3H), 2.32 (s, 3H), 2.30 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.57, 151.39, 140.24, 135.76, 132.47, 130.16, 128.58, 123.10, 112.68, 55.96, 19.67, 9.07.

1-(2-Fluoro-5-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107g). Brown solid (788 mg, 16% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.51 (t, J = 9.3 Hz, 1H), 7.27 (ddd, J = 12.9, 7.6, 3.4 Hz, 2H), 3.81 (s, 3H), 2.41 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.92, 155.83, 155.81, 151.29, 148.87, 139.43, 123.08, 122.94, 118.07, 118.00, 117.63, 117.42, 113.59, 56.12, 8.91.

1-(2-Chloro-5-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107h). Brown solid (1.44 g, 27% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.22 (s, 1H), 7.69 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 3.0 Hz, 1H), 7.28 (dd, J = 9.0, 3.0 Hz, 1H), 3.83 (s, 3H), 2.35 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.37, 158.78, 140.18, 135.97, 133.05, 130.90, 121.35, 118.51, 115.08, 56.09, 9.04.

1-(2-Bromo-5-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107i). Brown solid (1.69 g, 27% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.23 (s, 1H), 7.81 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 3.0 Hz, 1H), 7.21 (dd, J = 9.0, 3.0 Hz, 1H), 3.82 (s, 3H), 2.34 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.40, 159.37, 139.98, 135.96, 134.79, 133.87, 118.82, 115.34, 110.60, 56.05, 9.16.

1-(5-Methoxy-2-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107j). Brown oil (0.89 g, 18% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 10.23 (s, 1H), 7.77 (d, J = 2.7 Hz, 1H), 7.14 (dd, J = 8.3, 0.8 Hz, 1H), 6.63 (dd, J = 8.4, 2.7 Hz, 1H), 3.71 (s, 3H), 2.43 (s, 3H), 2.20 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.31, 158.47, 140.64, 134.50, 132.16, 131.16, 126.99, 117.07, 112.41, 55.69, 16.29, 9.42.

5-Methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107k). Brown solid (1.84 g, 45% yield).¹H NMR (400 MHz, DMSO-d₆) δ 13.14 (s, 1H), 7.72–7.57 (m, 5H), 2.51 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.56, 138.96, 136.48, 135.27, 130.01, 129.68, 125.44, 9.69.

5-Methyl-1-(pyridin-3-yl)-1H-1,2,3-triazole-4-carboxylic acid (compound 107l). Brown solid (1.10 g, 27%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (d, J = 2.5 Hz, 1H), 8.81 (dd, J = 4.8, 1.5 Hz, 1H), 8.15 (ddd, J = 8.1, 2.6, 1.5 Hz, 1H), 7.71 (dd, J = 8.2, 4.8 Hz, 1H), 2.53 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.42, 150.94, 145.95, 139.62, 136.69, 133.25, 132.16, 124.44, 9.53.

5-Methyl-1-(pyridin-4-yl)-1H-1,2,3-triazole-4-carboxylic acid (compound 107m). Brown solid (1.49 g, 37%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.93–8.77 (m, 2H), 7.82–7.69 (m, 2H), 2.62 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.34, 151.41, 142.21, 139.28, 137.11, 119.12, 9.81.

1-(2-Methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107n). Brown solid (1.79 g, 38% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.62 (ddd, J = 8.4, 7.5, 1.7 Hz, 1H), 7.47 (dd, J = 7.8, 1.7 Hz, 1H), 7.33 (dd, J = 8.5, 1.2 Hz, 1H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 3.80 (s, 3H), 2.30 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.57, 153.59, 140.33, 135.80, 132.28, 128.42, 123.43, 120.89, 112.83, 55.95, 9.04.

1-(3-Methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107o). Brown solid (1.93 g, 41% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.54 (td, J = 7.9, 0.8 Hz, 1H), 7.22–7.15 (m, 3H), 3.83 (s, 3H), 2.50 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.55, 159.88, 139.02, 136.41, 136.26, 130.50, 117.49, 115.81, 111.16, 55.63, 9.69.

1-(4-Methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107p). Brown solid (4.32 g, 19% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.59–7.50 (m, 2H), 7.19–7.10 (m, 2H), 3.85 (s, 3H), 2.46 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.63, 160.13, 138.97, 136.32, 128.07, 126.95, 114.70, 55.58, 9.62.

1-(2,3-Dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107q). Brown solid (1.85 g, 35% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.14 (s, 1H), 7.37 (dd, J = 8.4, 1.6 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.08 (dd, J = 7.8, 1.6 Hz, 1H), 3.92 (s, 3H), 3.59 (s, 3H), 2.33 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.52, 153.04, 143.86, 140.13, 135.79, 128.68, 124.40, 119.54, 115.38, 61.04, 56.19, 9.17.

1-(2,4-Dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107r). Brown solid (2.09 g, 40% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.38 (d, J = 8.7 Hz, 1H), 6.83 (d, J = 2.6 Hz, 1H), 6.72 (dd, J = 8.7, 2.5 Hz, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 2.28 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.60, 162.14, 154.78, 140.46, 135.65, 129.14, 116.48, 105.50, 99.37, 56.05, 55.72, 9.04.

1-(2,6-Dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107s). Brown solid (412.3 mg, 8% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 7.58 (t, J = 8.5 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 3.75 (s, 6H), 2.22 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 163.04, 156.08, 141.02, 136.01, 133.05, 112.27, 105.27, 56.75, 9.11.

1-(3,4-Dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107t). Brown solid (2.31 g, 44% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.19 (d, J = 2.2 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 8.5, 2.2 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 2.47 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.61, 149.80, 149.04, 139.13, 136.15, 127.99, 117.88, 111.58, 109.53, 55.89, 55.74, 9.62.

1-(3,5-Dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107u). Brown solid (1.85 g, 35% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 6.78 (d, J = 2.2 Hz, 2H), 6.74 (t, J = 2.2 Hz, 1H), 3.82 (s, 6H), 2.51 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.53, 160.83, 139.06, 136.77, 136.35, 103.94, 101.64, 55.75, 9.70.

1-(Benzo[d][1,3]dioxol-5-yl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107v). Brown solid (1.99 g, 40% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.24 (d, J = 2.0 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 7.06 (dd, J = 8.3, 2.1 Hz, 1H), 6.18 (s, 2H), 2.46 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.56, 148.47, 147.90, 139.18, 136.15, 128.92, 119.68, 108.40, 106.75, 102.30, 9.59.

1-Butoxy-4-(tert-butyl)-2-nitrobenzene (compound 109). To a solution of 4-(tert-butyl)-2-nitrophenol (compound 108, 9.76 g, 50 mmol) and K₂CO₃ (7.60 g, 55.0 mmol) in DMF (100 mL) was added 1-iodobutane (6.26 mL, 55.0 mmol). The resulting mixture was stirred for 2 h at 90°C then concentrated, diluted with water (100 mL), and extracted with EtOAc (100 mL × 2). The organic phase was washed with saturated aq. Na₂CO₃, water, and brine then dried with anhydrous MgSO₄, filtered, and concentrated. The residue was purified by silica gel chromatography (0% to 100% EtOAc in hexane) to give compound 109 as a light brown oil (11.91 g, 95% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, J = 2.5 Hz, 1H), 7.51 (ddd, J = 8.8, 2.5, 0.6 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 1.80 (dq, J = 8.0, 6.4 Hz, 2H), 1.58 – 1.44 (m, 2H), 1.31 (d, J = 0.6 Hz, 9H), 1.00 – 0.92 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 150.28, 143.49, 139.54, 131.00, 122.30, 114.21, 69.38, 34.29, 31.18, 31.05, 19.10, 13.75.

2-Butoxy-5-(tert-butyl)aniline (110). To a solution of 1-butoxy-4-(tert-butyl)-2-nitrobenzene (compound 109, 11.91 g, 47.4 mmol) in MeOH (300 mL) was added Pd/C (1 g, 10%) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under an H₂ balloon at room temperature for 24 h then filtered and concentrated. The resulting residue, a brown oil (10.5 g, 100% yield), was used without purification for the next step. ¹H NMR (400 MHz, DMSO-d₆) δ 6.70–6.63 (m, 2H), 6.49 (dd, J = 8.3, 2.4 Hz, 1H), 4.50 (s, 2H), 3.89 (t, J = 6.4 Hz, 2H), 1.68 (dq, J = 8.4, 6.4 Hz, 2H), 1.49–1.38 (m, 2H), 1.20 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 143.57, 142.99, 136.98, 112.72, 111.43, 111.28, 67.42, 33.62, 31.40, 30.98, 18.78, 13.73.

The following compounds (57–79) were prepared from the corresponding acids (107a–v) and 2-butoxy-5-(tert-butyl)aniline (compound 110) by a method similar to that used to prepare compound 1.

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(5-fluoro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 57). White solid (339.5 mg, 75% yield, 98.40% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 8.65 (d,J = 2.4 Hz, 1H), 7.28–7.21 (m, 1H), 7.18 (dd, J = 7.8, 3.1 Hz, 1H), 7.09–7.01 (m, 2H), 6.86 (d, J = 8.6 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.78 (s, 3H), 2.54 (s, 3H), 1.87 (dq, J = 7.9, 6.4 Hz, 2H), 1.64–1.53 (m, 2H), 1.36 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.24, 157.43, 155.02, 150.43, 150.40, 145.71, 143.78, 139.09, 138.57, 127.47, 124.57, 124.47, 120.19, 120.15, 118.47, 118.25, 117.01, 116.02, 115.77, 113.12, 113.04, 110.62, 68.51, 56.36, 34.44, 31.59, 31.31, 19.25, 13.87, 9.25. ESI-TOF HRMS: m/z 455.2453 (C₂₅H₃₁FN₄O₃ + H⁺ requires 455.2453).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(5-chloro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 58). White solid (378.9 mg, 80% yield, 99.32% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 8.65 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 8.9, 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 7.03 (d, J = 8.9 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.80 (s, 3H), 2.54 (s, 3H), 1.88 (dq, J = 8.4, 6.5 Hz, 2H), 1.66–1.53 (m, 2H), 1.36 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.22, 152.73, 145.71, 143.78, 139.06, 138.59, 131.82, 128.55, 127.46, 125.88, 124.98, 120.20, 117.02, 113.29, 110.61, 68.52, 56.22, 34.45, 31.59, 31.32, 19.26, 13.88, 9.25. ESI-TOF HRMS: m/z 471.2154 (C₂₅H₃₁ClN₄O₃ + H⁺ requires 471.2158).

1-(5-Bromo-2-methoxyphenyl)-N-(2-butoxy-5-(tert-butyl)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 59). White solid (436.8 mg, 85% yield, 99.00% purity).¹H NMR (400 MHz, CDCl₃) δ 9.80 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 7.64 (dd, J = 8.9, 2.5 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.80 (s, 3H), 2.54 (s, 3H), 1.88 (dq, J = 8.4, 6.5 Hz, 2H), 1.66–1.54 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.22, 153.24, 145.72, 143.79, 139.06, 138.58, 134.76, 131.36, 127.46, 125.32, 120.18, 117.04, 113.71, 112.62, 110.60, 68.52, 56.18, 34.45, 31.59, 31.32, 19.25, 13.88, 9.26. ESI-TOF HRMS: m/z 515.1662 (C₂₅H₃₁BrN₄O₃ + H⁺ requires 515.1653).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2-methoxy-5-(trifluoromethyl)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 60). White solid (357.2 mg, 71% yield, 98.37% purity).¹H NMR (400 MHz, CDCl₃) δ 9.82 (s, 1H), 8.65 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 8.8, 2.3 Hz, 1H), 7.72 (d, J = 2.3 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.08 (dd, J = 8.5, 2.4 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 2.55 (s, 3H), 1.88 (dq, J = 8.3, 6.5 Hz, 2H), 1.66–1.55 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.15, 156.43, 145.73, 143.80, 139.13, 138.71, 129.40, 129.36, 129.32, 129.28, 127.51, 127.43, 126.18, 126.14, 126.10, 126.07, 124.81, 124.51, 124.18, 123.84, 123.50, 123.17, 122.11, 120.25, 117.02, 112.42, 110.62, 68.52, 56.34, 34.44, 31.58, 31.31, 19.25, 13.86, 9.25. ESI-TOF HRMS: m/z 505.2417 (C₂₆H₃₁F₃N₄O₃ + H⁺ requires 505.2421).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2-methoxy-5-(trifluoromethoxy)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 61). White solid (422.9 mg, 81% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.82 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 7.42 (ddt, J = 9.2, 3.0, 1.0 Hz, 1H), 7.35 (dd, J = 3.0, 1.0 Hz, 1H), 7.14–7.06 (m, 2H), 6.86 (d, J = 8.6 Hz, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.82 (s, 3H), 2.55 (s, 3H), 1.94–1.82 (m, 2H), 1.68–1.55 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.16, 152.69, 145.71, 143.79, 142.20, 142.18, 142.15, 142.13, 139.10, 138.65, 127.45, 124.80, 124.64, 121.96, 121.74, 120.23, 119.18, 117.01, 112.91, 110.63, 68.51, 56.31, 34.43, 31.56, 31.31, 19.24, 13.84, 9.24. ESI-TOF HRMS: m/z 521.2375 (C₂₆H₃₁F₃N₄O₄ + H⁺ requires 521.2370).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2-methoxy-5-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 62). White solid (369.8 mg, 82% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 8.67 (d, J = 2.4 Hz, 1H), 7.31 (dd, J = 8.4, 2.3 Hz, 1H), 7.20 (d, J = 2.2 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.76 (s, 3H), 2.53 (s, 3H), 2.36 (s, 3H), 1.88 (dq, J = 8.1, 6.5 Hz, 2H), 1.66–1.55 (m, 2H), 1.36 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.51, 151.78, 145.72, 143.76, 138.98, 138.43, 132.33, 130.77, 128.83, 127.59, 123.91, 120.07, 117.01, 112.08, 110.59, 68.51, 55.88, 34.45, 31.60, 31.33, 20.26, 19.26, 13.89, 9.27. ESI-TOF HRMS: m/z 451.2706 (C₂₆H₃₄N₄O₃ + H⁺ requires 451.2704).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2-fluoro-5-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 63). White solid (349.6 mg, 77% yield, 99.53% purity).¹H NMR (400 MHz, CDCl₃) δ 9.79 (s, 1H), 8.63 (d, J = 2.4 Hz, 1H), 7.24 (t, J = 9.2 Hz, 1H), 7.13–7.05 (m, 2H), 7.01 (dd, J = 5.7, 3.1 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 3.85 (s, 3H), 2.63 (d, J = 1.7 Hz, 3H), 1.88 (dq, J = 8.4, 6.5 Hz, 2H), 1.64–1.57 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.99, 156.23, 156.21, 151.57, 149.12, 145.72, 143.82, 138.85, 138.75, 127.40, 123.56, 123.42, 120.25, 118.01, 117.93, 117.57, 117.36, 117.07, 112.87, 110.60, 68.52, 56.11, 34.45, 31.58, 31.30, 19.24, 13.86, 9.14. ESI-TOF HRMS: m/z 455.2456 (C₂₅H₃₁FN₄O₃ + H⁺ requires 455.2453).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2-chloro-5-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 64). White solid (372.1 mg, 79% yield, 100% purity).¹H NMR (400 MHz, CDCl₃) δ 9.82 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.13–7.05 (m, 2H), 6.97 (d, J = 3.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.09 (t, J = 6.4 Hz, 2H), 3.83 (s, 3H), 2.58 (s, 3H), 1.88 (dq, J = 8.4, 6.5 Hz, 2H), 1.60 (h, J = 7.4 Hz, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.07, 159.02, 145.71, 143.80, 138.74, 138.69, 133.64, 131.06, 127.43, 122.72, 120.25, 118.13, 117.03, 114.38, 110.62, 68.54, 55.97, 34.45, 31.60, 31.32, 19.26, 13.89, 9.28. ESI-TOF HRMS: m/z 471.2163 (C₂₅H₃₁ClN₄O₃ + H⁺ requires 471.2158).

1-(2-Bromo-5-methoxyphenyl)-N-(2-butoxy-5-(tert-butyl)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 65). White solid (402.8 mg, 78% yield, 100% purity).¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 7.02 (dd, J = 8.9, 2.9 Hz, 1H), 6.96 (d, J = 2.9 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.82 (s, 3H), 2.57 (s, 3H), 1.88 (dq, J = 8.5, 6.5 Hz, 2H), 1.66–1.54 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.68, 159.08, 145.70, 143.79, 138.70, 138.55, 135.36, 134.11, 127.44, 120.24, 118.41, 117.01, 114.73, 111.47, 110.63, 68.54, 55.94, 34.45, 31.60, 31.32, 19.27, 13.90, 9.41. ESI-TOF HRMS: m/z 515.1655 (C₂₅H₃₁BrN₄O₃ + H⁺ requires 515.1653).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(5-methoxy-2-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 66). White solid (153.2 mg, 68% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.82 (s, 1H), 8.65 (d, J = 2.4 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 7.06 (ddd, J = 16.1, 8.5, 2.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 2.7 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 3.82 (s, 3H), 2.52 (s, 3H), 1.98 (s, 3H), 1.93–1.83 (m, 2H), 1.67–1.54 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.27, 158.41, 145.73, 143.80, 138.71, 137.85, 134.91, 132.03, 127.46, 127.11, 120.20, 117.05, 116.83, 112.49, 110.60, 68.54, 55.66, 34.45, 31.59, 31.31, 19.26, 16.29, 13.88, 9.25. ESI-TOF HRMS: m/z 510.2720 (C₂₆H₃₄N₄O₃ + H⁺ requires 510.2711).

N-(2-Butoxy-5-(tert-butyl)phenyl)-5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamide (compound 67). White solid (321.8 mg, 79% yield, 98.57% purity).¹H NMR (400 MHz, CDCl₃) δ 9.82 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 7.62–7.55 (m, 3H), 7.52–7.48 (m, 2H), 7.08 (dd, J = 8.5, 2.4 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 2.70 (s, 3H), 1.89 (dq, J = 8.3, 6.5 Hz, 2H), 1.65–1.56 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.21, 145.73, 143.81, 139.24, 136.93, 135.67, 129.96, 129.67, 127.43, 125.29, 120.23, 117.05, 110.62, 68.53, 34.45, 31.59, 31.30, 19.24, 13.87, 9.91. ESI-TOF HRMS: m/z 407.2436 (C₂₄H₃₀N₄O₂ + H⁺ requires 407.2442).

N-(2-Butoxy-5-(tert-butyl)phenyl)-5-methyl-1-(pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamide (compound 68). White solid (360.7 mg, 88% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.78 (s, 1H), 8.80 (dd, J = 6.8, 3.7 Hz, 2H), 8.62 (d, J = 2.4 Hz, 1H), 7.86 (ddd, J = 8.2, 2.6, 1.5 Hz, 1H), 7.54 (dd, J = 8.2, 4.8 Hz, 1H), 7.06 (dd, J = 8.5, 2.4 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 4.08 (d, J = 6.4 Hz, 2H), 2.73 (s, 3H), 1.86 (dq, J = 8.2, 6.5 Hz, 2H), 1.57 (h, J = 7.4 Hz, 2H), 1.34 (s, 9H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.77, 151.03, 145.87, 145.69, 143.81, 139.56, 137.20, 132.57, 132.50, 127.26, 124.18, 120.41, 117.02, 110.66, 68.52, 34.44, 31.58, 31.28, 19.23, 13.86, 9.80. ESI-TOF HRMS: m/z 408.2386 (C₂₃H₂₉N₅O₂ + H⁺ requires 408.2394).

N-(2-Butoxy-5-(tert-butyl)phenyl)-5-methyl-1-(pyridin-4-yl)-1H-1,2,3-triazole-4-carboxamide (compound 69). White solid (382.8 mg, 94% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.78 (s, 1H), 8.86 (d, J = 5.6 Hz, 2H), 8.61 (d, J = 2.4 Hz, 1H), 7.59–7.48 (m, 2H), 7.08 (dd, J = 8.5, 2.4 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 2.82 (s, 3H), 1.97–1.75 (m, 2H), 1.58 (h, J = 7.5 Hz, 2H), 1.35 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.64, 151.63, 145.70, 143.84, 142.68, 139.93, 136.77, 127.20, 120.48, 118.51, 117.04, 110.65, 68.52, 34.44, 31.57, 31.27, 19.23, 13.86, 10.18. ESI-TOF HRMS: m/z 408.2396 (C₂₃H₂₉N₅O₂ + H⁺ requires 408.2394).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 70). White solid (352.2 mg, 81% yield, 99.43% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.84 (s, 1H), 8.67 (d, J = 2.4 Hz, 1H), 7.52 (ddd, J = 8.3, 7.6, 1.7 Hz, 1H), 7.38 (dd, J = 7.8, 1.7 Hz, 1H), 7.18–7.04 (m, 3H), 6.86 (d, J = 8.6 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.80 (s, 3H), 2.53 (s, 3H), 1.92–1.82 (m, 2H), 1.67–1.54 (m, 2H), 1.37 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.47, 154.02, 145.72, 143.76, 139.02, 138.47, 132.02, 128.49, 127.57, 124.27, 121.04, 120.10, 117.02, 112.19, 110.59, 68.51, 55.84, 34.45, 31.61, 31.33, 19.26, 13.89, 9.27. ESI-TOF HRMS: m/z 437.2547 (C₂₅H₃₂N₄O₃ + H⁺ requires 437.2547).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2-methoxypyridin-3-yl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 71). White solid (355.8 mg, 81% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.80 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 8.38 (dd, J = 5.0, 1.8 Hz, 1H), 7.75 (dd, J = 7.6, 1.8 Hz, 1H), 7.12 (dd, J = 7.6, 5.0 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.98 (s, 3H), 2.56 (s, 3H), 1.94–1.81 (m, 2H), 1.67–1.50 (m, 2H), 1.36 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.16, 158.21, 149.37, 145.71, 143.80, 138.98, 138.76, 137.06, 127.44, 120.19, 119.35, 117.10, 117.05, 110.58, 68.50, 54.14, 34.45, 31.58, 31.31, 19.24, 13.87, 9.30. ESI-TOF HRMS: m/z 438.2503 (C₂₄H₃₁N₅O₃ + H⁺ requires 438.2500).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(3-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 72). White solid (337.6 mg, 77% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 8.63 (d, J = 2.4 Hz, 1H), 7.50–7.45 (m, 1H), 7.12–7.03 (m, 4H), 6.86 (d, J = 8.5 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 2.71 (s, 3H), 1.88 (dq, J = 8.4, 6.5 Hz, 2H), 1.67–1.53 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 160.46, 159.19, 145.72, 143.81, 139.22, 136.95, 136.61, 130.35, 127.43, 120.22, 117.27, 117.04, 115.81, 111.09, 110.60, 68.52, 55.66, 34.45, 31.59, 31.30, 19.24, 13.87, 9.95. ESI-TOF HRMS: m/z 437.2541 (C₂₅H₃₂N₄O₃ + H⁺ requires 437.2547).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(4-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 73). White solid (315.7 mg, 72% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 7.42–7.36 (m, 2H), 7.07 (dq, J = 8.2, 3.4, 2.8 Hz, 3H), 6.86 (d, J = 8.5 Hz, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.89 (s, 3H), 2.66 (s, 3H), 1.88 (dq, J = 8.2, 6.5 Hz, 2H), 1.64–1.53 (m, 2H), 1.36 (s, 9H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 160.64, 159.29, 145.72, 143.80, 139.04, 137.04, 128.50, 127.47, 126.70, 120.18, 117.03, 114.76, 110.61, 68.52, 55.67, 34.44, 31.59, 31.30, 19.24, 13.87, 9.81. ESI-TOF HRMS: m/z 437.2543 (C₂₅H₃₂N₄O₃ + H⁺ requires 437.2547).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2,3-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 74). White solid (401.8 mg, 86% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 8.67 (d, J = 2.4 Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 7.11 (dd, J = 8.4, 1.5 Hz, 1H), 7.06 (dd, J = 8.5, 2.4 Hz, 1H), 6.98 (dd, J = 7.9, 1.6 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.92 (s, 3H), 3.64 (s, 3H), 2.55 (s, 3H), 1.88 (dq, J = 8.5, 6.6 Hz, 2H), 1.65–1.53 (m, 2H), 1.35 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.37, 153.49, 145.68, 144.38, 143.78, 139.07, 138.47, 129.50, 127.53, 124.36, 120.12, 119.65, 117.02, 114.46, 110.61, 68.53, 61.49, 56.19, 34.44, 31.59, 31.31, 19.25, 13.88, 9.33. ESI-TOF HRMS: m/z 467.2657 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2,4-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 75). White solid (379.5 mg, 81% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.82 (s, 1H), 8.65 (t, J = 1.9 Hz, 1H), 7.33–7.21 (m, 1H), 7.06 (dd, J = 8.6, 2.4 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.67 – 6.54 (m, 2H), 4.08 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.78 (s, 3H), 2.50 (d, J = 1.0 Hz, 3H), 1.88 (p, J = 6.7 Hz, 2H), 1.59 (h, J = 7.4 Hz, 2H), 1.36 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.52, 159.54, 155.17, 145.72, 143.75, 139.15, 138.37, 129.12, 127.59, 120.03, 117.48, 117.03, 110.56, 104.81, 99.46, 68.50, 55.83, 55.74, 34.44, 31.60, 31.32, 19.25, 13.88, 9.24. ESI-TOF HRMS: m/z 467.2656 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(2,6-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 76). White solid (375.2 mg, 80% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.85 (s, 1H), 8.67 (d, J = 2.4 Hz, 1H), 7.45 (t, J = 8.5 Hz, 1H), 7.06 (dd, J = 8.5, 2.4 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 6.71 (s, 1H), 6.69 (s, 1H), 4.08 (t, J = 6.5 Hz, 2H), 3.77 (s, 6H), 2.45 (s, 3H), 1.88 (dq, J = 8.5, 6.5 Hz, 2H), 1.66–1.54 (m, 2H), 1.36 (d, J = 0.6 Hz, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.65, 156.28, 145.74, 143.72, 139.50, 138.17, 132.09, 127.69, 119.91, 117.01, 112.86, 110.50, 104.28, 68.49, 56.16, 34.44, 31.60, 31.34, 19.26, 13.89, 8.87. ESI-TOF HRMS: m/z 467.2639 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(3,4-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 77). White solid (382.7 mg, 82% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.80 (s, 1H), 8.63 (d, J = 2.4 Hz, 1H), 7.06 (dd, J = 8.5, 2.4 Hz, 1H), 7.02–6.97 (m, 3H), 6.85 (d, J = 8.5 Hz, 1H), 4.07 (t, J = 6.5 Hz, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 2.67 (s, 3H), 1.87 (dq, J = 8.4, 6.5 Hz, 2H), 1.65–1.52 (m, 2H), 1.35 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.21, 150.27, 149.68, 145.70, 143.79, 139.05, 137.09, 128.53, 127.45, 120.20, 117.62, 116.99, 111.01, 110.62, 109.03, 68.50, 56.24, 56.21, 34.44, 31.58, 31.29, 19.23, 13.87, 9.87. ESI-TOF HRMS: m/z 467.2656 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

N-(2-Butoxy-5-(tert-butyl)phenyl)-1-(3,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (78). White solid (363.4 mg, 78% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 8.65 (d, J = 2.4 Hz, 1H), 7.05 (dd, J = 8.5, 2.4 Hz, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.60 (dd, J = 7.7, 2.2 Hz, 3H), 4.07 (t, J = 6.4 Hz, 2H), 3.81 (s, 6H), 2.71 (s, 3H), 1.87 (dq, J = 8.2, 6.5 Hz, 2H), 1.58 (h, J = 7.4 Hz, 2H), 1.35 (s, 9H), 1.00 (t, J = 7.4 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) δ 161.33, 159.11, 145.68, 143.75, 139.16, 137.05, 136.93, 127.45, 120.21, 116.96, 110.63, 103.68, 101.67, 68.49, 55.69, 34.43, 31.58, 31.31, 19.25, 13.88, 9.95.ESI-TOF HRMS: m/z 467.2657 (C₂₆H₃₄N₄O₄ + H⁺ requires 467.2653).

1-(Benzo[d][1,3]dioxol-5-yl)-N-(2-butoxy-5-(tert-butyl)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 79). White solid (374.9 mg, 83% yield, 100% purity). ¹H NMR (400 MHz, CDCl₃) δ 9.80 (s, 1H), 8.64 (d, J = 2.7 Hz, 1H), 7.13–7.01 (m, 1H), 6.91 (s, 3H), 6.85 (d, J = 8.5 Hz, 1H), 6.06 (s, 2H), 4.07 (t, J = 6.5 Hz, 2H), 2.66 (s, 3H), 1.88 (h, J = 8.1, 6.9 Hz, 2H), 1.59 (p, J = 7.3 Hz, 2H), 1.35 (s, 9H), 1.00 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.15, 148.96, 148.50, 145.69, 143.76, 139.00, 137.09, 129.33, 127.44, 120.22, 119.27, 116.99, 110.63, 108.46, 106.58, 102.33, 68.50, 34.44, 31.59, 31.30, 19.25, 13.88, 9.82. ESI-TOF HRMS: m/z 451.2339 (C₂₅H₃₀N₄O₄ + H⁺ requires 451.2340).

Synthesis Procedures for Compounds 80–92 (Scheme 6)

N-(5-(tert-Butyl)-2-hydroxyphenyl)-1-(5-chloro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 111). To a solution of 1-(5-chloro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107b, 324 mg, 1.210 mmol) in DMF (5 mL) was added 2-amino-4-(tert-butyl)phenol (200 mg, 1.210 mmol), HOBt, 80% (307 mg, 1.816 mmol), and EDCI (348 mg, 1.816 mmol), followed by N-ethyl-N-isopropylpropan-2-amine (626 mg, 4.84 mmol). The suspension was stirred at room temperature overnight, then the reaction mixture was then diluted with water (50 mL) and extracted with EtOAc (50 mL × 2). The EtOAc layer was washed with water, dried with anhydrous Na₂SO₄, and concentrated. The residue was purified by silica gel chromatography (0% to 100% acetonitrile in water) to give product 111 as a white solid (351.9 mg, 70% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 10.04 (s, 1H), 9.62 (s, 1H), 8.34 (d, J = 2.4 Hz, 1H), 7.73 (dq, J = 5.0, 2.7 Hz, 2H), 7.43–7.37 (m, 1H), 7.00 (dd, J = 8.4, 2.4 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 3.84 (s, 3H), 2.43 (s, 3H), 1.28 (s, 9H). ¹³C NMR (126 MHz, DMSO-d6) δ 157.80, 152.22, 143.64, 140.95, 138.48, 136.82, 131.55, 127.71, 125.04, 123.67, 123.64, 120.16, 116.30, 114.02, 113.74, 55.92, 33.36, 30.83, 8.13.

N-(5-(tert-Butyl)-2-hydroxyphenyl)-1-(2-methoxy-5-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 112). To a solution of 1-(2-methoxy-5-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (compound 107f, 299 mg, 1.210 mmol) in DMF (5 mL) were added 2-amino-4-(tert-butyl)phenol (200 mg, 1.210 mmol), HOBt, 80% (307 mg, 1.816 mmol), and EDCI (348 mg, 1.816 mmol), followed by N-ethyl-N-isopropylpropan-2-amine (626 mg, 4.84 mmol). The suspension was stirred at room temperature overnight, then the reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL × 2). The EtOAc layer was washed with water, dried with anhydrous Na₂SO₄, and concentrated. The residue was purified by silica gel chromatography (0% to 100% acetonitrile in water) to give product 112 as a white solid (300.8 mg, 63% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 10.02 (s, 1H), 9.61 (s, 1H), 8.33 (d, J = 2.4 Hz, 1H), 7.45 (ddd, J = 8.5, 2.3, 0.8 Hz, 1H), 7.32 (dd, J = 2.1, 0.8 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 6.98 (dd, J = 8.5, 2.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H), 2.39 (s, 3H), 2.34 (s, 3H), 1.27 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.91, 150.85, 143.58, 140.93, 138.17, 136.74, 132.04, 129.63, 128.02, 125.07, 122.43, 120.08, 116.22, 113.70, 112.16, 55.42, 33.35, 30.83, 19.13, 8.16.

N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 80). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 2-bromopentane, to give a white solid (98.8 mg, 84% yield, 99.69% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.85 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 9.1, 3.0 Hz, 1H), 7.06 (dd, J = 8.6, 2.5 Hz, 1H), 7.03 (d, J = 9.2 Hz, 1H), 6.96 (d, J = 3.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.43 (h, J = 6.1 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 2.54 (s, 3H), 1.93–1.82 (m, 1H), 1.70–1.60 (m, 1H), 1.57–1.45 (m, 2H), 1.37 (d, J = 6.1 Hz, 3H), 1.36 (s, 9H), 0.96 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.40, 153.68, 148.02, 144.71, 143.80, 139.06, 138.54, 128.48, 124.52, 120.10, 117.43, 117.07, 113.74, 113.31, 112.37, 75.36, 56.34, 56.00, 38.68, 34.46, 31.60, 19.92, 18.79, 14.14, 9.32. ESI-TOF HRMS: m/z 481.2809 (C₂₇H₃₆N₄O₄ + H⁺ requires 481.2809).

N-(5-(tert-Butyl)-2-(2-methylbutoxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 81). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 1-bromo-2-methylbutane, to give a white solid (94.2 mg, 81% yield, 100% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.88 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 7.10–7.05 (m, 2H), 7.02 (d, J = 9.1 Hz, 1H), 6.97 (d, J = 3.0 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 3.95 (dd, J = 8.9, 5.6 Hz, 1H), 3.87 (dd, J = 8.8, 6.4 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 2.54 (s, 3H), 2.04–1.94 (m, 1H), 1.71–1.61 (m, 1H), 1.45–1.37 (m, 1H), 1.36 (s, 9H), 1.13 (d, J = 6.8 Hz, 3H), 0.98 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.43, 153.67, 148.01, 145.77, 143.73, 139.04, 138.49, 127.57, 124.49, 120.03, 117.48, 116.90, 113.72, 113.29, 110.36, 73.37, 56.32, 56.00, 34.83, 34.46, 31.60, 26.20, 16.78, 11.42, 9.30. ESI-TOF HRMS: m/z 481.2812 (C₂₇H₃₆N₄O₄ + H⁺ requires 481.2809).

N-(5-(tert-Butyl)-2-(isopentyloxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 82). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 42 and 1-bromo-3-methylbutane, to give a white solid (88.1 mg, 75% yield, 100% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.81 (s, 1H), 8.65 (d, J = 2.4 Hz, 1H), 7.10–7.06 (m, 2H), 7.03 (d, J = 9.1 Hz, 1H), 6.97 (d, J = 3.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.10 (t, J = 6.6 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 2.54 (s, 3H), 1.96 (dp, J = 13.4, 6.7 Hz, 1H), 1.80 (q, J = 6.7 Hz, 2H), 1.36 (s, 9H), 1.00 (d, J = 6.7 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 159.44, 153.68, 148.04, 145.72, 143.75, 139.08, 138.48, 127.51, 124.50, 120.07, 117.45, 117.03, 113.76, 113.31, 110.45, 67.26, 56.34, 56.00, 38.00, 34.45, 31.60, 25.23, 22.69, 9.31. ESI-TOF HRMS: m/z 481.2798 (C₂₇H₃₆N₄O₄ + H⁺ requires 481.2809).

N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(5-chloro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 83). This compound was synthesized by using a procedure similar to that described for compound 19, employing compound 111 and 2-bromopentane, to give a white solid (81.9 mg, 70% yield, 100% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.83 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 7.51 (dd, J = 8.9, 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 7.08–7.02 (m, 2H), 6.86 (d, J = 8.6 Hz, 1H), 4.43 (h, J = 6.1 Hz, 1H), 3.81 (s, 3H), 2.53 (s, 3H), 1.94–1.79 (m, 1H), 1.69–1.62 (m, 1H), 1.56–1.47 (m, 2H), 1.37 (d, J = 6.2 Hz, 3H), 1.36 (s, 9H), 0.96 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.21, 152.73, 144.72, 143.82, 139.05, 138.64, 131.81, 128.58, 128.39, 125.90, 125.00, 120.21, 117.08, 113.25, 112.40, 75.40, 56.23, 38.68, 34.46, 31.59, 19.92, 18.78, 14.14, 9.28. ESI-TOF HRMS: m/z 485.2309 (C₂₆H₃₃ClN₄O₃ + H⁺ requires 485.2314).

N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(2-methoxy-5-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 84). This compound was synthesized by using a procedure similar to that described for compound 23, employing compound 112 and 2-bromopentane, to give a white solid (89.8 mg, 75% yield, 99.35% purity). ¹H NMR (500 MHz, CDCl₃) δ 9.85 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 7.32 (ddd, J = 8.5, 2.2, 0.9 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 7.05 (dd, J = 8.5, 2.4 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 4.43 (h, J = 6.1 Hz, 1H), 3.78 (s, 3H), 2.52 (s, 3H), 2.37 (s, 3H), 1.93–1.83 (m, 1H), 1.68–1.63 (m, 1H), 1.58–1.45 (m, 2H), 1.37 (d, J = 6.1 Hz, 3H), 1.36 (s, 9H), 0.96 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.50, 151.78, 144.72, 143.79, 138.97, 138.48, 132.29, 130.78, 128.86, 128.51, 123.93, 120.07, 117.07, 112.39, 112.04, 75.38, 55.89, 38.68, 34.46, 31.60, 20.28, 19.91, 18.79, 14.15, 9.30. ESI-TOF HRMS: m/z 465.2865 (C₂₇H₃₆N₄O₃ + H⁺ requires 465.2860).

(S)-N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 85). N-(5-(tert-Butyl)-2-hydroxyphenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 42, 100 mg, 0.244 mmol), (R)-pentan-2-ol (39.7 μL, 0.365 mmol), and PPh₃ (83 mg, 0.317 mmol) were added to dry DCM (10 mL) in a flame-dried 25-mL round-bottom flask charged with a stir bar under an inert atmosphere. To the solution was added diisopropyl azodicarboxylate (DIAD, 72.0 μL, 0.365 mmol). The reaction mixture was stirred at room temperature overnight then quenched by adding sodium hydroxide solution (0.5 N, 10 mL) and extracted with EtOAc (25 mL × 2). The combined organic layers were washed with water and brine, dried over anhydrous MgSO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0% to 100% EtOAc in hexane) to give product 85 as a white solid (83.2 mg, 71% yield, 95.86% purity, %ee = 94.96%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.49 (d, J = 2.3 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.1 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 4.53 (h, J = 6.1 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H), 1.81–1.57 (m, 2H), 1.54–1.39 (m, 2H), 1.32–1.28 (m, 12H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 158.84, 153.56, 148.10, 144.71, 143.47, 139.38, 137.79, 128.14, 124.07, 120.96, 117.98, 116.74, 114.44, 114.38, 113.54, 75.17, 56.82, 56.34, 38.60, 34.54, 31.81, 20.12, 18.51, 14.44, 9.23. ESI-TOF HRMS: m/z 481.2804 (C₂₇H₃₆N₄O₄ + H⁺ requires 481.2809).

(R)-N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(2,5-dimethoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 86). This compound was synthesized by using a procedure similar to that described for compound 85, employing compound 42 and (S)-pentan-2-ol, to give a white solid (92.1 mg, 79% yield, 96.58% purity, %ee = 100%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.48 (d, J = 2.3 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.1, 3.1 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 4.53 (h, J = 6.0 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H), 1.79–1.70 (m, 1H), 1.67–1.59 (m, 1H), 1.53–1.40 (m, 2H), 1.30 (d, J = 6.1 Hz, 3H), 1.29 (s, 9H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.77, 152.48, 147.02, 143.64, 142.39, 138.30, 136.71, 127.05, 122.99, 119.88, 116.90, 115.66, 113.36, 113.30, 112.46, 74.09, 55.74, 55.26, 37.52, 33.46, 30.73, 19.04, 17.43, 13.36, 8.15. ESI-TOF HRMS: m/z 481.2806 (C27H36N4O4 + H⁺ requires 481.2809).

(S)-N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(5-chloro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 87). This compound was synthesized by using a procedure similar to that described for compound 85, employing compound 111 and (R)-pentan-2-ol, to give a white solid (88.6 mg, 76% yield, 95.75% purity, %ee = 96.94%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 7.73 (dq, J = 5.3, 2.7 Hz, 2H), 7.43–7.38 (m, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.04 (d, J = 8.6 Hz, 1H), 4.53 (h, J = 6.1 Hz, 1H), 3.83 (s, 3H), 2.42 (s, 3H), 1.78–1.70 (m, 1H), 1.68–1.58 (m, 1H), 1.54–1.39 (m, 2H), 1.30 (d, J = 6.1 Hz, 3H), 1.29 (s, 9H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.66, 152.18, 143.67, 142.39, 138.53, 136.77, 131.55, 127.70, 126.99, 123.63, 123.58, 119.94, 115.71, 114.03, 112.45, 74.08, 55.92, 37.53, 33.46, 30.73, 19.03, 17.43, 13.36, 8.10. ESI-TOF HRMS: m/z 485.2314 (C₂₆H₃₃ClN₄O₃ + H⁺ requires 485.2314).

(R)-N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(5-chloro-2-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 88). This compound was synthesized by using a procedure similar to that described for compound 85, employing compound 111 and (S)-pentan-2-ol, to give a white solid (70.3 mg, 60% yield, 96.24% purity, %ee = 100%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.48 (d, J = 2.3 Hz, 1H), 7.73 (dq, J = 5.3, 2.7 Hz, 2H), 7.42–7.37 (m, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 4.53 (h, J = 6.1 Hz, 1H), 3.83 (s, 3H), 2.42 (s, 3H), 1.79–1.69 (m, 1H), 1.67–1.57 (m, 1H), 1.54–1.40 (m, 2H), 1.30 (d, J = 6.1 Hz, 3H), 1.29 (s, 9H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.66, 152.18, 143.67, 142.39, 138.53, 136.77, 131.55, 127.70, 126.99, 123.63, 123.58, 119.94, 115.71, 114.03, 112.45, 74.08, 55.92, 37.53, 33.46, 30.73, 19.03, 17.43, 13.36, 8.10. ESI-TOF HRMS: m/z 485.2312 (C₂₆H₃₃ClN₄O₃ + H⁺ requires 485.2314).

(S)-N-(5-(tert-Butyl)-2-(pentan-2-yloxy)phenyl)-1-(2-methoxy-5-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 89). This compound was synthesized by using a procedure similar to that described for compound 85, employing compound 112 and (R)-pentan-2-ol, to give a white solid (36.8 mg, 62% yield, 97.09% purity, %ee = 97.1%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.48 (d, J = 2.3 Hz, 1H), 7.45 (ddd, J = 8.5, 2.2, 0.8 Hz, 1H), 7.35–7.31 (m, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 4.52 (h, J = 6.1 Hz, 1H), 3.78 (s, 3H), 2.39 (s, 3H), 2.34 (s, 3H), 1.74 (ddt, J = 13.5, 9.7, 6.1 Hz, 1H), 1.67–1.58 (m, 1H), 1.47 (ddt, J = 16.5, 13.2, 7.2 Hz, 2H), 1.30 (d, J = 6.1 Hz, 3H), 1.29 (s, 9H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.80, 150.83, 143.66, 142.40, 138.22, 136.74, 132.04, 129.63, 128.03, 127.07, 122.40, 119.87, 115.69, 112.48, 112.16, 74.12, 55.43, 37.53, 33.47, 30.74, 19.13, 19.04, 17.43, 13.37, 8.15. ESI-TOF HRMS: m/z 485. 465.2855 (C₂₇H₃₆N₄O₃ + H⁺ requires 465.2860).

(R)-N-(5-(tert-butyl)-2-(pentan-2-yloxy)phenyl)-1-(2-methoxy-5-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (compound 90). This compound was synthesized by using a procedure similar to that described for compound 85, employing compound 112 and (S)-pentan-2-ol, to give a white solid (16.8 mg, 71% yield, 97.36% purity, %ee = 96.42%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.48 (d, J = 2.3 Hz, 1H), 7.45 (ddd, J = 8.5, 2.2, 0.8 Hz, 1H), 7.34–7.31 (m, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 4.52 (h, J = 6.1 Hz, 1H), 3.78 (s, 3H), 2.39 (s, 3H), 2.34 (s, 0H), 1.80–1.69 (m, 1H), 1.68–1.56 (m, 1H), 1.54–1.40 (m, 2H), 1.30 (d, J = 6.1 Hz, 3H), 1.29 (s, 9H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.80, 150.83, 143.66, 142.40, 138.22, 136.74, 132.04, 129.63, 128.03, 127.07, 122.40, 119.87, 115.68, 112.48, 112.16, 74.12, 55.43, 37.53, 33.46, 30.73, 19.13, 19.04, 17.43, 13.37, 8.15. ESI-TOF HRMS: m/z 485. 465.2847 (C₂₇H₃₆N₄O₃ + H⁺ requires 465.2860).

Biology

Lipofectamine 3000 reagent, GeneBLAzer^® NR-UAS-bla HEK 293T cells for VDR, FXR and LXRα, LiveBLAzer^™ FRET – B/G Loading Kit with CCF4-AM and all tissue culture reagents were purchased from Invitrogen (Carlsbad CA). HEK293 (ATCC CRL-1573), human hepatocellular carcinoma HepG2 (ATCC CRL-10741) and mouse hepatoma Hepa 1–6 (ATCC CRL-1830) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Human hepatoma HepaRG 5F cells (cat. no MTOX1010–1VL) were purchased from Sigma (St. Louis, MO) and maintained as previously described⁴⁸. All cell lines have been authenticated by short tandem repeat DNA profiling and tested negative for mycoplasma contamination. DMEM (Dulbecco’s Modified Eagle Medium), phenol red–free DMEM, Tb-anti-GST (terbium-anti–glutathione S-transferase), GST hPXR-LBD, Tris (pH 7.5, 1 M), and dithiothreitol (DTT, 1 M) were purchased from Thermo Fisher Scientific (Atlanta, GA). MgCl₂ (1 M) was purchased from Boston BioProducts (Ashland, MA). Rifampicin, PCN, CITCO, 1α,25-Dihydroxyvitamin D3, GW4064 and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). T0901317 was purchased from Cayman Chemical (Ann Arbor, MI). Staurosporine was purchased from LC Laboratories (Woburn, MA). Steadylite HTS reagent and 384-well white tissue culture–treated plates were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). The 384-well black low-volume assay plates were purchased from Corning Inc. (Tewksbury, MA). BODIPY FL vindoline was synthesized in-house as described previously⁴⁹. CellTiter-Glo Luminescent Cell Viability Assay reagent was purchased from Promega (Madison, WI). Fetal bovine serum (FBS) and charcoal/dextran-treated FBS were purchased from HyClone Laboratories (Logan, UT).

TR-FRET PXR Competitive Binding Assays; hPXR, hCAR and mPXR Luciferase Reporter Assays; Cytotoxicity Assays; and hVDR, hLXRα and hFXR Transactivation Assays.

TR-FRET PXR competitive binding assays, hPXR luciferase reporter assay using HepG2 hPXR-CYP3A4-luciferase stable cells, hCAR luciferase reporter assay using HepG2 hCAR-CYP2B6-luciferase stable cells and mPXR luciferase reporter assay have been previously described³². hVDR, hLXRα and hFXR transactivation assays, and HepG2, HEK293, Hepa 1–6, and HepaRG cytotoxicity assays were performed according to previously published protocols^{30, 32}. In the luciferase reporter and transactivation assays, a compound was tested in either an agonistic mode (tested alone) or an antagonistic mode (tested in the presence of an agonist as indicated). In the cytotoxicity assays, 2,500 and 5,000 cells were used for 1-day and 3-day assay, respectively. Staurosporine (56 μM) was used as the positive cytotoxicity control (100% cytotoxicity). Data are presented as the means and SDs from three independent experiments.

Dose–Response Data Analysis.

In all of the assays (i.e., the luciferase reporter, cytotoxicity, NR transactivation and TR-FRET PXR competitive binding assays), compounds were tested in quadruplicate at least three times. For dose–response data analysis, the relative assay signals (% Activity, refers to % activation or % inhibition as indicated from the luciferase reporter, NR transactivation and PXR TR-FRET competitive binding assays), and the relative cytotoxicity (% cytotoxicity from the cytotoxicity assays) for each chemical at the corresponding concentrations were plotted using GraphPad PRISM software (version 9.1.0) (GraphPad Software, San Diego, CA) to generate the dose–response curves and to derive IC₅₀ or EC₅₀ values (if applicable) via the built-in sigmoidal dose–response fitting equation.

Computational Docking Studies.

The chemical structures of compounds were prepared using the LigPrep module from Schrödinger (Maestro Version 12.3.013, MMshare Version 4.9.013, Release 2020–1, Platform Linux-x86_64). The structures were converted to 3D format, hydrogen atoms were added, and ionization states at pH 7 were generated⁵⁰ before docking to the hPXR LBD (PDB code: 5X0R), using the GLIDE module^{51, 52}. Chain B was processed using the Protein Preparation Wizard in the Maestro suite to assign the correct hetero atom bond order, remove water molecules, and add the protonation state of the protein⁵⁰. The docking experiments were conducted using the Ligand Docking protocol (SP-mode) in the Maestro suit, with the grid set to encompass the ligand binding site of the hPXR LBD. The resulting poses were ranked based on scores provided by GLIDE.

Abbreviations

AF-2

activation function 2

BSA

bovine serum albumin

CITCO

6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime

CYP

cytochrome P450

DBD

DNA-binding domain

DIAD

Diisopropyl azodicarboxylate

DMEM

Dulbecco’s Modified Eagle Medium

hCAR

human constitutive androstane receptor

hFXR

human farnesoid X receptor

hLXRα

human liver X receptor α

hPXR

human PXR

HTS

high-throughput screening

LBD

ligand binding domain

LC-MS

liquid chromatography–mass spectrometry

mPXR

mouse PXR

NR

nuclear receptor

PCN

pregnenolone 16α-carbonitrile

PXR

pregnane X receptor

RXR

retinoid X receptor

SAR

structure–activity relation

SRC-1

steroid receptor coactivator 1

TR-FRET

time-resolved fluorescence resonance energy transfer

VDR

vitamin D receptor