Systematic optimization of fragment TLX ligands towards agonism and inverse agonism

Emily C Hank; Loris Knümann; Úrsula López-García; Arthur Kardanov; Vasily Morozov; Georg Höfner; Daniel Merk

doi:10.1021/acs.jmedchem.5c02718

. Author manuscript; available in PMC: 2026 Mar 5.

Published before final editing as: J Med Chem. 2026 Jan 28;69(3):2831–2849. doi: 10.1021/acs.jmedchem.5c02718

Systematic optimization of fragment TLX ligands towards agonism and inverse agonism

Emily C Hank ¹, Loris Knümann ¹, Úrsula López-García ¹, Arthur Kardanov ¹, Vasily Morozov ¹, Georg Höfner ¹, Daniel Merk ^1,^*

PMCID: PMC7618817 EMSID: EMS212277 PMID: 41603564

Abstract

The transcription factor tailless homologue (TLX, NR2E1) maintains persistence of neural stem cells (NSC) in a proliferating, undifferentiated state thereby controlling NSC homeostasis and enabling neurogenesis. TLX is responsive to small molecule ligands offering potential access to new neuroprotective treatments, but TLX ligands are very rare. Here, we used a drug fragment screening hit as lead to develop TLX modulators and identified substructures tuning activity between agonism and inverse agonism. Structural optimization provided potent TLX activating and inhibiting fragment ligands with validated binding and favorable ligand efficiency for structural extension.

Introduction

The human tailless homologue receptor (TLX, NR2E1) is an orphan nuclear receptor (NR) with a peculiar repressor function in neural stem cells (NSC) and neural progenitors (NP), enabling their proliferation and self-renewal.^1,2 It binds co-repressors such as atrophin³ and lysine-specific demethylase 1 (LSD1)⁴ and recruits histone deacetylases⁵ to suppress transcription of key cell cycle and proliferation regulators, such as p21 and the phosphatase and tensin homologue (pten).^3,5,6 This ability of TLX to control the expression of tumor suppressor genes suggests a critical role in the regulation of NSC proliferation, cell cycle progression and differentiation.^2,5,7 Accordingly, studies in mouse embryos have demonstrated that TLX-deficiency leads to premature NSC differentiation and cell cycle alterations.^8,9 TLX-expressing cells isolated from adult mouse brains have been demonstrated to multiply, regenerate and differentiate in vitro while these abilities were lacking in cells isolated from TLX-null brains, but could be rescued by exogenous TLX expression.² Moreover, isolated TLX-positive murine cells in culture can yield NSC in both activated or quiescent states while loss of TLX increases the amount of non-proliferating NSC, supporting the NR’s involvement in NSC activation.⁶

TLX is also critical in brain development since NR2E1-deletion in mice reduced dendritic length and arborization¹³ and caused thinning of the neocortical layers¹⁴ as well as an overall shrinkage of cerebral structures, including the limbic system, olfactory bulbs^14–16 and forebrain¹⁷. Phenotypic observations of TLX germline deletion models included aggressive and violent behavior^8,16,18,19, hyperactivity^20,21 and decreased anxiety and fear conditioning^8,21,22, potentially caused by impaired memory function.^8,20,22 Conversely, animal models of TLX-overexpression have demonstrated improved NSC self-renewal^23,24 and enhanced neurogenesis after stroke²⁵, along with increased anterior brain mass and elevated learning and memory capacity.²³ Outside the brain, TLX expression has been detected in retinal progenitor cells, where loss of TLX was found to cause retinal and optic nerve deficiencies and affect vision._16,26,27

These lines of evidence support therapeutic potential of TLX in the context of neurodegenerative diseases by stimulating neurogenesis and counteracting neuroinflammation^23,28–32, in retinal dystrophies³, and in mental disorders.^19,33,34 However, effects of pharmacological TLX modulation have not been evaluated due to a lack of potent, selective and well-characterized TLX ligands.³² The receptor’s druggability was first explored in 2014 by a DSF-based fragment screening for binders of the TLX ligand binding domain (LBD), which led to the identification of three ligands, including ccrp2 (1, Chart 1, K_d = 0.65 µM).¹⁰ A few further TLX ligand scaffolds have been discovered since (reviewed by Lewandowski et al. ³⁵), but high-quality chemical tools for target validation of TLX are still very rare. We have identified the fragment-like TLX agonist 2 (EC₅₀ = 20 µM) by drug fragment screening.^11,36 Extension of 2 and preliminary optimization offered access to the TLX agonists 3-5 displaying submicromolar potencies^11,12, underscoring the potential of 2 as lead for TLX modulator development. The SAR of the fragment-like 2 has not been systematically studied, however. Here, we report extensive SAR elucidation of the TLX ligand chemotype of 2 and marked optimization on fragment level via systematic structural modification. Interestingly, removal of a key polar feature in the heterocyclic motif inverted activity and offered access to inverse TLX agonists.

Results & Discussion

Aiming to increase potency (i.e., improve the EC₅₀ value) and efficacy (i.e., enhance repression as observed by lower remaining reporter activity) of the fragment hit 2, we commenced SAR elucidation by testing importance of the methylimidazole motif and by scanning for opportunities for structural extension (Table 1). Removal of the methyl group (6) enhanced potency by 5-fold and improved efficacy while further removal of the distal nitrogen in the pyrrole analogue 7 was not favored suggesting importance of the polar feature. The imidazole regiochemistry of 2 was preferred as evident from diminished TLX agonism of the isomers 8-10. Replacement of the methylimidazole (2) by a 1-methyl-1H-pyrazole-4-yl motif (11) was tolerated while the alternative pyrazole regiochemistries of 12-15 decreased TLX agonism, further pointing to a key role of polarity distribution in this ring. Removal of the methyl substituent from the preferred pyrazole (11) in 16 was tolerated and enhanced efficacy while introduction of an additional methyl group in 5-position (17) improved potency thus indicating that the scaffold could be expanded in this region. Indeed, extension of the original imidazole (2) to the corresponding benzimidazole (18) was accompanied by an almost tenfold improvement in potency. The indole-1-yl derivative 19 failed to activate TLX while the inverted analogue 20 retained TLX agonism providing further evidence that the distal nitrogen was critical.

Table 1. Impact of modifications on the methylimidazole.

ID	EC₅₀ (TLX)^a	efficacy (remain. act.)^a
2	20±3 μM	0.44±0.04
6	4.4±0.9 μM	0.28±0.05
7	15±7 μM	0.2±0.2
8	inactive (100 μM)	-
9	2.8±0.9	0.81±0.05
10	> 30 μM	0.70±0.06 at 100 μM
11	10±4 μM	0.4±0.1
12	> 30 μM	0.74±0.09 at 100 μM
13	> 30 μM	0.48±0.09 at 100 μM
14	> 30 μM	0.29±0.05 at 100 μM
15	> 30 μM	0.6±0.1 at 100 μM
16	> 10 μM	0.15±0.02 at 100 μM
17	4±2 μM	0.5±0.1
18	2.4±0.9 μM	0.27±0.09
19	inactive (30 μM)	-
20	9.1±0.7 μM	0.20±0.05

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TLX modulation was determined in a hybrid reporter gene assay measuring the TLX-mediated repression of VP16-induced reporter activity (Figure S1). Remaining activity (remain. act.) refers to the reporter activity vs. DMSO (0.1%) control. Data are the mean±S.E.M., n≥3. All compounds were counterscreened to exclude non-specific effects on VP16 activity. Data for 2 from ³⁶.

Based on these results, we continued SAR elucidation of 2 by exploring pyridine as alternative polar heterocycle to replace the imidazole (Table 2). The methylimidazole of 2 was mimicked well by a 2-methyl-4-pyridyl motif (21) and a 6-methyl-3-pyridyl motif (22) with the former being slightly more active. In both cases, the removal of the methyl group (23, 24) had no major impact on TLX agonism while double methylation (25, 26) boosted potency. Replacement of the (mono)methyl substituents by an acetyl substituent (27, 28) was tolerated and might be a valuable modification to improve polarity. Enhancing polarity within the ring by incorporation of a second nitrogen atom in the pyrimidin-5-yl derivative 29 was favored while the hydrophobic dimethylphenyl analogue 30 of the most active dimethylpyridyl derivative 25 interestingly displayed an inverted activity profile and acted as weak inverse TLX agonist.

Table 2. Replacement of the methylimidazole.

ID	EC₅₀ (TLX)^a	efficacy (fold act.)/ (remain. act.)^a
2	20±3 μM	0.44±0.04
21	7±2 μM	0.28±0.07
22	16±4 μM	0.37±0.07
23	13±4 μM	0.2±0.1
24	10±2 μM	0.35±0.06
25	1.6±0.8 μM	0.31±0.08
26	6±2 μM	0.24±0.09
27	5±2 μM	0.30±0.05
28	5±2 μM	0.25±0.09
29	2.1±0.5 μM	0.40±0.06
30	inverse agonist >30 μM	2.2±0.2 at 50 μM

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TLX modulation was determined in a hybrid reporter gene assay measuring the TLX-mediated repression of VP16-induced reporter activity (Figure S1). Maximum fold reporter activation (fold act.) or remaining activity (remain. act.) refer to the reporter activity vs. DMSO (0.1%) control. Data are the mean±S.E.M., n≥3. All compounds were counterscreened to exclude non-specific effects on VP16 activity. Data for 2 from ³⁶.

These informative initial insights supported an SAR model for TLX agonism of the scaffold in which a polar (nitrogen) atom seemed critical in the distal region (i.e., meta/para to the trifluoromethylaniline core) but was not tolerated in other positions. Removal of this polar center disrupted activity or resulted in inverse agonism. Hydrophobic substituents surrounding the polar center were mostly favored and could valuably enhance potency. Especially installation of an additional aromatic ring (18 vs. 2) markedly increased TLX agonism prompting us to explore further two-ring systems in this region (Table 3).

Table 3. SAR of bicyclic motifs.

ID	EC₅₀ (TLX)^a	efficacy (remain. act.)^a
2	20±3 μM	0.44±0.04
18	2.4±0.9 μM	0.27±0.09
20	9.1±0.7 μM	0.19±0.05
31	18±1 μM	0.2±0.1
32	1.1±0.1 μM	0.44±0.03
33	3.0±0.2 μM	0.18±0.05
34	1.4±0.4 μM	0.27±0.07
35	2.3±0.7 μM	0.27±0.07
36	Toxic ≥30 μM	0.84±0.06 at 10 μM
37	inactive (10 μM)	-
38	3.7±0.6 μM	0.32±0.04
39	0.7±0.2 μM	0.27±0.06
40	9.1±0.7 μM	0.29±0.06
41	3.5±0.8 μM	0.23±0.08

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Comparing the isomeric N-methylindol-3-yl (20), N-methylindol-4-yl (31) and N-methylindol-5-yl (32) derivatives revealed a strong preference in terms of potency for the indol-5-yl (32) regiochemistry that was also evident - though less pronounced - in the demethylated analogues (33, 34). The 2,3-dihydroindole 35 retained a similar TLX agonist profile as 34 indicating that aromaticity was not critical, but evaluation of the corresponding benzofuran (36), benzothiophene (37) and imidazopyridine (38) derivatives revealed a marked preference for nitrogen-containing motifs.

Structural fusion of the preferred pyridine (21/25) and N-methylindol-5-yl (32) motifs by incorporating an additional nitrogen atom in 7-position of the indole (39) yielded the first fragment TLX agonist with sub-micromolar potency. Introduction of an additional nitrogen atom in 2-position of the N-methylindol-5-yl motif (40) and inversion of 39 to the pyrrolo[2,3-b]pyridine-3-yl analogue 41 resembling the geometry of 18 were less favored.

After substantially improving the TLX agonist profile of the fragment hit 2 via modification of the methylimidazole region, we moved our attention to the trifluoromethylaniline core of the scaffold. Using the favored 2-methylpyridin-4-yl substituent (21) as fixed motif in 3-position we varied the substitution pattern on the aniline motif (Table 4). Removal of the trifluoromethyl group (42) was detrimental in terms of TLX activation efficacy and shifting it from 5- (21) to 4-position (43) diminished TLX agonist potency highlighting importance of the 5-substituent for potent and efficient TLX activation. Alternative groups in 5-position pointed to a preference for electron withdrawing groups with the 5-methyl (44) and 5-methoxy (45) derivatives displaying reduced potency and efficacy, while the trifluoromethoxy (46), cyano (47) and chloro (48) substituents were more favored. Nevertheless, the original trifluoromethyl group (21) remained superior. 4,5-Dichlorosubstitution (49) retained a similar activity profile as 21 suggesting that also larger motifs might be tolerated. Therefore, we tested incorporation of a small heterocycle to mimic the 4,5-disubstitution and indeed observed enhanced TLX agonism of the indole analogue 50 while the regioisomer 51 was inactive. The aniline core motif presented an overall rather strict SAR with few opportunities for improvement and only the indole motif of 50 emerged as a potential alternative to the original trifluoromethyl substituent of 21. The fused compound 52 combining the indole of 50 and the favored 1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl substituent of the most active agonist 39 displayed potent TLX agonism thus supporting suitability of the alternative core scaffold for combination with other preferred modifications. Nevertheless, 52 failed to outmatch the most active agonist 39 bearing the original trifluoromethylaniline core.

Table 4. SAR of the trifluoromethylaniline core.

ID	EC₅₀ (TLX)^a	efficacy (remain. act.)^a
21	7±2 μM	0.28±0.07
42	0.7±0.3 μM	0.76±0.03
43	> 30 μM	0.39±0.04 at 50 μM
44	> 30 μM	0.44±0.09 at 30 μM
45	15±4 μM	0.56±0.05
46	13.6±0.6 μM	0.24±0.03
47	6±2 μM	0.4±0.1
48	10±7 μM	0.44±0.08
49	9.6±0.7 μM	0.31±0.04
50	3.8±0.7 μM	0.26±0.06
51	inactive (50 μM)	-
52	1.4±0.3 μM	0.50±0.03

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Systematic SAR evaluation of the fragment-like TLX modulator scaffold of 2 enabled substantial improvement in potency via modification or replacement of the methylimidazole residue. The resulting optimized fragment TLX ligands may serve as valuable leads for structural extention. Additionally, the structure-activity data revealed that removal of the distal polar center which appeared critical for agonism could invert activity to modest TLX inhibition. Ligands blocking the repressor activity of TLX, i.e., inverse agonists, are very rare but would be valuable as complementary chemical tools to study the receptor’s biological roles. Therefore, we set out to explore the SAR around 30 for molecular determinants driving the observed activity switch (Table 5).

Table 5. Preliminary SAR of inverse TLX agonists.

ID	TLX modulation IC₅₀/EC₅₀^a	efficacy (fold act.)/ (remain. act.)^a
30	inverse agonist IC₅₀ >30 μM	2.2±0.2 at 50 μM
53	inverse agonist IC₅₀ 25±3 μM	1.9±0.1
54	inverse agonist IC₅₀ 16±3 μM	2.5±0.1
55	inverse agonist IC₅₀ > 30 μM	2.0±0.5 at 30 μM
56	inverse agonist IC₅₀ 10±2 μM	2.0±0.2
57	inverse agonist IC₅₀ >30 μM	2.2±0.5 at 30 μM
58	inverse agonist IC₅₀ >30 μM	2.1±0.5 at 50 μM
59	inactive (10 μM)	-
60	inactive (10 μM)	-
61	inverse agonist IC₅₀ 19±3 μM	1.66±0.09
62	inverse agonist IC₅₀ > 30 μM	1.8±0.4 at 30 μM
63	inverse agonist IC₅₀ 32±2 μM	2.3±0.1
64	inverse agonist IC₅₀ 39±7 μM	1.7±0.1
65	inverse agonist IC₅₀ 2.9±0.8 μM	1.81±0.07
66	inverse agonist IC₅₀ 2.9±0.9 μM	2.3±0.1
67	agonist EC50 6±1 μM	0.32±0.08

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The methylation pattern of 30 turned out to be critical for inverse agonism (53-55) with a methyl substituent being favored in 3-position (54) of the phenyl motif over the 2- (53) and especially the 4-position (55). Double methylation in 2-/3-positions (56) was superior to the 3,4- (57) and 3,5- (30) dimethyl analogues indicating an overall preference for 3-substitution. Bulkier hydrophobic groups (ethyl (58), tert-butyl (59)), an acetyl substituent (60) and halogen atoms (fluorine (61), chlorine (62)) were not tolerated. The 3-trifluoromethyl (63) and trifluoromethoxy (64) analogues retained inverse TLX agonism but with reduced potency. Only a 3-methoxy substituent (65) and a 3-dimethylamino motif (66) achieved improved inverse TLX agonism. The more polar 3-hydroxy substituent (67) inverted the activity profile back to TLX agonism.

With this strict SAR for inverse TLX agonists based on 30, we next explored scaffold-hopping to bi- and heterocyclic motifs (Table 6). The activities observed for 53-67 indicated that only hydrophobic groups maintained inverse agonist potency and that substitution was favored in 3-position. In line with this preferred geometry, the α-naphthyl (68) but not the β-naphthyl (69) derivative acted as inverse TLX agonist, and also the (di)methylthiophene analogues 70-72 mimicking the favored 3-tolyl derivative 54 displayed robust inhibition of TLX activity - even with improved efficacy. Replacement of the methyl group by an acetyl motif (73, 74), which had been tolerated in the agonist series (27, 28), inverted the activity profile to rather potent TLX activators. The same effect was evident for other polar substituents such as the ester (75) and amide (76, 77) analogues which exhibited TLX agonism, and also the incorporation of a nitrogen atom in the thiazoles 78-80 inverted or disrupted activity thus corroborating the hypothesis that polarity was not tolerated for inverse agonist activity.

Table 6. Hetero- and bicyclic motifs in inverse agonists.

ID	TLX modulation IC₅₀/EC₅₀^a	efficacy (fold act.)/ (remain. act.)^a
68	inverse agonist IC₅₀ 12±1 μM	3.1±0.1
69	inactive (10 μM)	-
70	inverse agonist IC₅₀ 15±5 μM	3.1±0.4
71	inverse agonist IC₅₀ 12±3 μM	3.0±0.2
72	inverse agonist IC₅₀ 16±5 μM	3.1±0.4
73	agonist EC₅₀ 4±2 μM	0.27±0.07
74	agonist EC₅₀ 1.5±0.5 μM	0.19±0.06
75	agonist EC₅₀ 4±2 μM	0.41±0.09
76	agonist EC₅₀ 2.8±0.5 μm	0.44±0.04
77	agonist EC₅₀ 5±1 μM	0.52±0.04
78	agonist EC₅₀ 33±9 μM	0.5±0.2
79	inactive (30 μM)	-
80	agonist	0.6±0.1 at 100 μM

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Systematic SAR analysis of the scaffold of 2 revealed key features for bidirectional TLX modulation (Figure 1). The methylimidazol could be replaced by various mono- and bicyclic aromatic systems wherein 1-2 distal polar centers were essential for potent TLX agonism and methyl or acetyl substituents on the aromatic system were favored. Replacement by purely hydrophobic aromatic systems could invert the activity profile to inverse agonism, but inverse agonists displayed a more strict SAR. Modifications in the 5-trifluoromethylaniline core were less productive. The substituent in 5-position turned out to be essential but the trifluormethyl group could be replaced without full loss of activity with a preference for electron withdrawing motifs. Substitution in 4-position was tolerated but only an additional ring (i.e., indole) enhanced potency.

Within the series of fragment-like TLX modulators derived from 2, the 2,6-dimethylpyridin-4-yl (25), 2-acetylpyridin-4-yl (27) and 1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl (39) derivatives evolved as the most favored agonists with low micromolar to sub-micromolar potencies (Table 7, dose-response curves in Figure S2). Favorable ligand efficiency (LE) and lipophilic ligand efficiency (LLE) as well as sufficient aqueous solubility support their suitability as leads for structural extension. Additionally, the 3-methoxyphenyl (65) and 3,5-dimethylthiophen-2-yl (72) residues emerged as motifs to obtain inverse agonists (dose response curves in Figure S2). Though slightly less potent and more hydrophobic, these fragments are also highly valuable starting points for medicinal chemistry as TLX inhibitors are very rare.

Table 7. Features of the most favored fragment TLX agonists (25, 27, 39) and inverse agonists (65, 72).


	25	27	39	65	72
TLX^a	EC₅₀ 1.6±0.8 μM (0.31±0.08 rem.)	EC₅₀ 5±2 μM (0.30±0.05 rem.)	EC₅₀ 0.7±0.2 μM (0.27±0.06 rem.)	IC₅₀ 2.9±0.8 μM (1.81±0.07 fold)	IC₅₀ 16±5 μM (3.1±0.4 fold)
DSF ^b	ΔT_m -(1.6±0.4)°C	ΔT_m -(1.2±0.4)°C	ΔT_m -(2.1±0.4)°C	ΔT_m 5±1°C	ΔT_m 4±1°C
ITC^c	n.d.	n.d.	K_^d 1.1±0.1	n.d.	n.d.
logP^d	3.6	2.8	3.3	3.9	4.3
LE^e	0.42	0.37	0.40	0.40	0.37
LLE^e	2.18	2.53	2.81	1.60	0.54
aq. sol. [mg/L]^f	68±16	15.7±0.4	12±1	39.3±0.6	12±2

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From Gal4-TLX hybrid assay (Figures S1; dose-response curves in Figure S2).

Difference of the melting points T_m between compound-treated TLX LBD (2 µM) and TLX LBD (2 µM) in the DMSO control.

K_d value (mean±SD, n=2) from isothermal titration calorimetry (ITC). Data in Figure 2c,d. n.d. – not determined.

LogP was calculated with the AlogPS³⁷ resource. ^e Ligand efficiency (LE) and lipophilic ligand efficiency (LLE) were calculated according to ref. ³⁸.

Thermodynamic aqueous solubility was determined by UV-metric quantification from the supernatant of oversaturated compound solutions, n = 3.

For validation of direct TLX modulation, we employed two orthogonal binding assays using the recombinant TLX LBD. Differential scanning fluorimetry (DSF, Figure 2a) revealed effects of all favored fragments on thermal stability of TLX. As observed previously for the TLX agonists ccrp2 (1)¹⁰ and the lead 2³⁶ as well as for other nuclear receptors³⁹, the TLX agonists 25, 27 and 39 diminished the melting temperature of the protein. Interestingly, the inverse agonists 65 and 72 mediated the opposite effect and caused a positive thermal shift of the TLX LBD indicating that agonism and inverse agonism induced different structural/conformational effects and might differently affect dimerization. The absolute effects on the melting temperature aligned with the respective rank-order of potency for agonists and inverse agonists adding further confidence to the binding results.

(a) DSF assay. The reference agonist ccrp2¹⁰ was used at 100 µM, the fragments at 300 µM with 2 µM TLX. N=3; * p<0.05, ** p<0.01, *** p<0.001 (one-way ANOVA with Dunnett’s multiple comparisons test). (b) Affinity-selection-mass-spectrometry (ASMS, Figure S3). The test compounds **25, 27** and 39 were used at 1 µM with 35 µM TLX LBD. 65 and 72 were used at 10 µM with 50 µM TLX LBD. N = 9 (intact), N=3 (inactivated); * p<0.05, ** p<0.01, *** p<0.001 (t-test). (c,d) Isothermal titration calorimetry (ITC) of the 39-TLX LBD interaction (K_d 1.1±0.1 µM). Representative buffer-TLX LBD (top), 39-buffer (middle) and 39-TLX LBD titrations (bottom) are shown in (c) and the corresponding heats and fit are shown in (d).

Additionally, we validated binding of the fragments by affinity-selection-mass-spectrometry (ASMS) as second binding assay (Figure 2b; schematic method illustration in Figure S3). TLX LBD protein (35-50 µM) was incubated with the agonists (1 µM) or inverse agonists (10 µM), separated from unbound ligands by size-exclusion chromatography (SEC), and denaturated to release the bound ligands for quantification by LC-MS/MS. The same procedure was repeated with heat inactivated protein to determine unspecific binding. The ASMS assay confirmed robust specific binding of 25, 27, 39, 65 and 72 to the TLX LBD validating direct TLX modulation. As additional orthogonal evidence for binding of the scaffold, we studied the interaction of the most potent representative 39 with the TLX LBD by isothermal titration calorimetry (ITC; Figure 2c,d) which revealed a direct 1:1 binding with a K_d value of 1.1±0.1 µM aligning well with the compound’s in vitro potency.

To further investigate the suitability of the most active fragment TLX modulators 25, 39 and 65 as leads, we determined their selectivity in a representative panel of nuclear receptors covering the NR1, NR2, NR4 and NR5 families (Figure 3). 25 and 39 displayed no pronounced off-target activity at the 30 µM test concentration with moderate RORα inhibition and weak activation of RXRα, PPARγ and PXR, while 65 mediated strong (~75-fold) RXRα activation. These off-targets will have to be considered in further optimization but given the typically higher promiscuity of fragments⁴⁰, 25, 39 and 65 nevertheless displayed generally favorable selectivity profiles to serve as leads for structural extension to more potent TLX modulators. Additionally, the pronounced RXR agonism of 65 despite the absence of an acidic motif may be worth further investigation for the design of selective RXR ligands and dual TLX/RXR modulators.

Selectivity profiling of the most active fragment TLX modulators **25, 39** and 65 in a representative panel of nuclear receptors in uniform Gal4-hybrid reporter gene assays. Reference ligands (where available) are reported in the Experimental section. Data are the mean±S.E.M.; n=3.

As phenotypic effects of pharmacological TLX modulation are elusive and validated cellular models to determine TLX modulator effects are lacking, we employed a reporter system to test engagement of native human TLX in cellular context by the most active fragment TLX ligands. We used a reporter for the human nuclear receptor response element DR1 which binds several nuclear receptor dimers including RXR:PPAR and RXR:RXR⁴¹, and can be activated, for example, by the PPARγ agonist pioglitazone (Fig. 4a). Co-transfection of the DR1 reporter and an expression construct for full-length human TLX in HEK293T cells resulted in a markedly reduced reporter signal compared to cells transfected only with the DR1 reporter (Fig. 4a) indicating that the repressor TLX could inhibit DR1 activity. In absence of TLX, the ligands 25, 27, 39 and 72 had no effect on pioglitazone-induced DR1 activity (Figure 2b). However, when TLX was exogenously expressed, the TLX agonists 25, 27 and 39 robustly suppressed DR1 activation and the inverse TLX agonist 72 enhanced pioglitazone-induced DR1 activity. These results indicated that the TLX agonists enhanced and the inverse agonist blocked the receptor’s repressor activity supporting functional effects of the fragments in cellular context.

(a) Exogenous TLX expression markedly diminished baseline and pioglitazone-induced (PIO) DR1 activity in HEK293T cells. (b) The TLX modulators **25, 27, 39** and 72 had no relevant effect on pioglitazone-induced DR1 activity in absence of exogenous TLX expression. (c) With exogenous TLX expression, the TLX agonists **25, 27** and 39 markedly enhanced repression of pioglitazone-induced DR1 activity while the inverse agonist 72 mediated de-repression. Data are the mean±SD normalized DR1 reporter activity [RLU]; n=6; ** p<0.01, *** p<0.001 (ANOVA with Bonferroni’s multiple comparisons test). Fragment 65 was excluded from these experiments for its RXR agonist activity that would cause DR1 activation.

Conclusion

The orphan nuclear receptor TLX is a promising target with a prominent role in neuronal health and regeneration. Its peculiar repressor function and the lack of high-quality structural data have impeded ligand discovery, however, and TLX is still highly understudied. Chemical tools with different, orthogonal TLX modulation effects are urgently needed to explore and validate therapeutic potential of the nuclear receptor. Using a moderately active screening hit as lead, we identified fragment-like TLX modulators with agonistic or inverse agonistic activity and orthogonally validated binding. Our results establish a preliminary SAR model for the bidirectional TLX modulation profile of the scaffold and form a strong basis for further optimization. Albeit their binding site in the TLX LBD and molecular activation/repression mechanisms remain to be elucidated, the new TLX modulators are an important contribution to the collection of ligands for this understudied orphan receptor. Further optimization of the fragments by structural extension is warranted.

Chemistry

Suzuki-Miyaura type coupling reactions under XPhos Pd G3 catalysis were performed to prepare compounds 8-12, 14-17, 20-41 and 56-80 either directly from 3-bromo-5-(trifluoromethyl)aniline (SM1) and the respective heteroaryl boronic acid or Bpin ester (SM43-66) or via the Bpin ester intermediate 81, prepared from SM1 by borylation⁴², which was reacted with the respective heteroaryl bromides (SM7-SM42, Scheme 1). Compounds 6, 13, 18 and 19 were generated from SM1 via Ullman-type coupling with imidazole (SM3), 4-methyl-1H-pyrazole (SM4), benzimidazole (SM5) or indole (SM6), respectively. Synthesis of compound 5 was achieved as described in the literature⁴³, from 5-(trifluoromethyl)benzene-1,3-diamine (SM67) and 2,5-dimethoxytetrahydrofuran (SM68). Starting materials described by SM numbers were commercially available.

Scheme 1 — ^a Reagents & Conditions: (a) FeCl₃ (1 mol%), cat. HCl, H₂O/DMF = 1:1, 60°C, 1 h, 29%;⁴³ (b) CuI (10 mol%), N,N’-Dimethyl-1,2-cyclohexanediamine (20 mol%), sat. aq. K₂CO₃, 100°C, air, 48 h, 6-40%; (c) B₂Pin₂ (1.1 eq.), Pd(dppf)Cl₂ (2.5 mol%), KOAc (3 eq.), 1,4-dioxane, N₂, 90°C, overnight, 93%;⁴² (d) XPhosPdG3 (10 mol%), K₃PO₄, dioxane/H₂O, N₂, 100 °C, overnight, 14-98%.

For the variation of the core aniline substitution pattern, 2-methylpyridine-4-boronic acid (SM69) was coupled with the aryl bromides SM70-79 in Suzuki-Miyaura type reactions to yield compounds 42-51 (Scheme 2). Compound 52 was prepared by the same strategy from SM78 and 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (SM80, Scheme 2).

Scheme 2 — ^a Reagents & Conditions: (a) XPhosPdG3 (10 mol%), K₃PO₄, dioxane/H₂O, N₂, 100 °C, overnight, 14-70%.

Experimental Procedures

Chemistry

General

All chemicals were of reagent grade, purchased from commercial sources (e.g., Sigma-Aldrich, TCI, BLDpharm) and used without further purification unless otherwise specified. All reactions were conducted under nitrogen (N₂) or argon atmosphere and in absolute solvents purchased from Sigma-Aldrich. Other solvents, especially for work-up procedures, were of reagent grade or purified by distillation (iso-hexane, cyclohexane, ethyl acetate, EtOH). Reactions were monitored by thin layer chromatography (TLC) on TLC Silica gel 60 F254 coated aluminum sheets by Merck and visualized under ultraviolet light (254 nm). Purification by column chromatography was performed on a puriFlash® XS520Plus system (Advion, Ithaca, NY, USA) using high performance spherical silica columns (SIHP, 30 or 50 µm) by Interchim and a gradient of iso-hexane or cyclohexane to ethyl acetate, reversed-phase (RP) column chromatography was performed on a puriFlash® 5.250 system (Advion) using C18HP columns (SIHP, 15 µm) by Interchim and a gradient of H₂O with 10% MeCN to 100% MeCN (HPLC gradient grade). Mass spectra were obtained on a puriFlash®-CMS system (Advion) using atmospheric pressure chemical ionization (APCI). High-resolution mass spectra (HRMS) were obtained with a Thermo Finnigan LTQ FT instrument for electron impact ionization (EI). NMR spectra were recorded on Bruker Avance III HD 400 MHz or 500 MHz spectrometers equipped with a CryoProbeTM Prodigy broadband probe (Bruker). Chemical shifts are reported in δ values (ppm) relative to residual protium signals in the NMR solvent (¹H-NMR: Acetone-d₆: δ = 2.05 ppm; DMSO-d₆: δ = 2.50 ppm; MeOD-d₄: δ = 3.31 ppm; CD₂Cl₂: δ = 5.32 ppm, ¹³C-NMR: Acetone-d₆: δ = 206.26, 29.84 ppm; DMSO-d₆: δ = 39.52 ppm; MeOD-d₄:δ = 49.0 ppm; CD₂Cl₂: δ = 53.84 ppm) and coupling constants (J) in hertz (Hz). The splitting of signals is indicated by s for singlet, d for doublet, t for triplet, q for quartet, dd for doublet of a doublet and m for multiplet. The purity of the compounds was determined by ¹H NMR (qHNMR) according to the method described by Pauli et al.⁴⁴ with internal calibration. To ensure accurate determination of peak area ratio, the qHNMR measurements were conducted under conditions allowing for complete relaxation. Maleic acid (LOT#BCCH2407, purity 99.85%) was used as internal standard in DMSO-d₆. All compounds for biological testing had a purity >95% according to quantitative ¹H NMR (qHNMR).

General procedure A (Suzuki-coupling)

A 50-100 mL Schlenk flask was charged with a stir bar, the boronic acid/pinacol ester (1.0 eq.) and potassium phosphate (3.0 eq.). The flask was evacuated and backfilled with nitrogen three times. The previously de-gassed (3 freeze thaw-cycles) solvent mixture (dioxane/water = 85:15), together with the Pd catalyst (10 mol%) and the heteroaryl bromide (1.5-2.0 eq.) was added and the reaction mixture was stirred at 100°C under reflux and nitrogen atmosphere. After reaction control by TLX indicated completion of the reaction, the solution was cooled down to room temperature and the solvents were evaporated. The residue was taken up in EtOAc and washed with water and brine. The organic layer was dried over Na₂SO₄, concentrated and purified by flash column chromatography give the respective compound.

General procedure B (Ullmann coupling)

A 20 mL reaction tube was charged with the heterocycle (1.0 eq.), the aryl bromide (1.5-2.0 eq.), CuI (10 mol%) and N,N-dimethyl-1,2-diamino cyclohexane (20 mol%). 5 mL of sat. K₂CO₃ in water were added and the reaction mixture was stirred for 24-48h at 100°C open to air. After cooling down to room temperature, the mixture was filtered over celite and eluted with EtOAc. The phases were separated and the aqueous layer was extracted two more times with EtOAc. The combined organic layers were washed with brine and dried over Na₂SO₄ and the crude product was purified by flash column chromatography to yield the desired compounds.

3-(1H-Imidazol-1-yl)-5-(trifluoromethyl)aniline (6)

Preparation according to general procedure B from imidazole (SM3, 217 mg, 3.15 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 566 µL, 4.00 mmol, 1.3 eq.). Further purification by RP column chromatography yielded a colorless solid (43 mg, 6%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.11 (s, 1H), 7.54 (s, 1H), 7.14 (s, 1H), 7.01 – 6.96 (m, 2H), 6.95 – 6.89 (m, 1H). ¹³C NMR (101 MHz, MeOD-d₄) δ 152.21, 139.92, 136.95, 134.02 (q, J = 32.2 Hz), 130.26, 125.29 (q, J = 271.7 Hz), 119.71, 110.69 (q, J = 4.0 Hz), 110.42, 106.30 (q, J = 3.8 Hz). HRMS (GC/EI+): m/z calculated 227.0665 for C₁₀H₈F₃N₃, found 227.0664 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.3%.

3-(1H-Pyrrol-1-yl)-5-(trifluoromethyl)aniline (7)

Preparation according a the literature procedure⁴³ from 2,5-dimethoxytetrahydrofuran (SM67, 333 µL, 2.54 mmol, 1.0 eq.) and 5-(trifluoromethyl)benzene-1,3-diamine (SM66, 447 mg (2.54 mmol, 1.0 eq.) in water/DMF = 1:1. Purification by flash column chromatography and by RP column chromatography yielded an orange crystalline solid (166 mg, 29%). ¹H NMR (400 MHz, DMSO) δ 7.29 (t, J = 2.2 Hz, 2H), 6.97 – 6.89 (m, 2H), 6.77 – 6.71 (m, 1H), 6.25 (t, J = 2.2 Hz, 2H), 5.79 (s, 2H).¹³C NMR (101 MHz, DMSO-d₆) δ 150.73, 141.37, 131.02 (q, J = 31.4 Hz), 124.15 (q, J = 272.2 Hz), 119.00, 110.60, 107.38, 106.78 (q, J = 4.0 Hz), 102.76 (q, J = 3.9 Hz). HRMS (GC/EI+): m/z calculated 226.0712 for C₁₀H₈F₃N₃, found 226.0712 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 99.9%.

3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)aniline (81)

This intermediate was prepared as described in previous literature⁴² with dry dioxane instead of DMSO as solvent. Purification by flash column chromatography and lyophilization yielded the compound as a yellowish crystalline solid (93%). ¹H NMR (400 MHz, DMSO-d₆) δ = 7.19 – 7.14 (m, 1H), 7.02 – 6.97 (m, 1H), 6.96 – 6.90 (m, 1H), 5.59 (s, 2H), 1.28 (s, 12H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 149.04, 129.40 (d, J = 30.5 Hz), 125.88, 123.31, 123.17, 116.80, 111.89, 83.87, 24.66. MS (APCI+): m/z 287.6 ([M+H]⁺).

3-(2-Methyl-1H-imidazol-5-yl)-5-(trifluoromethyl)aniline (8)

Preparation according to general procedure A from 81 (144 mg, 0.50 mmol, 1.0 eq.) and 5-bromo-2-methyl-1H-imidazole (SM7, 121 mg, 0.75 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a yellowish oil (35 mg, 29 %). ¹H NMR (400 MHz, Acetone-d₆) δ 7.44 – 7.27 (m, 3H), 6.80 – 6.75 (m, 1H), 5.00 (s, 2H), 2.35 (s, 3H). ¹³C NMR (126 MHz, Acetone) δ 150.13, 145.44, 140.74, 138.16, 131.77 (q, J = 31.1 Hz), 125.87 (q, J = 270.8 Hz), 114.25, 112.99, 110.28, 108.73, 14.18. HRMS (GC/EI+): m/z calculated 241.0821 for C₁₁H₁₀F₃N₃, found 241.0822 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.4%.

3-(1,2-Dimethyl-1H-imidazol-5-yl)-5-(trifluoromethyl)aniline (9)

Preparation according to general procedure A from 81 (144 mg, 0.50 mmol, 1.0 eq.) and 5-bromo-1,2-dimethyl-1H-imidazole (SM8, 135 mg, 0.75 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a yellowish oil (35 mg, 27 %). ¹H NMR (400 MHz, DMSO-d₆) δ 6.86 (s, 1H), 6.84 – 6.81 (m, 2H), 6.79 – 6.74 (m, 1H), 5.70 (s, 2H), 3.51 (s, 3H), 2.33 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 150.77, 147.76, 134.48, 133.11, 133.46 – 132.40 (m), 125.78, 125.68 (q, J = 273.0 Hz), 118.62, 114.23 (q, J = 4.1 Hz), 111.37 – 111.09 (m), 31.82, 13.09. HRMS (GC/EI+): m/z calculated 255.0978 for C₁₂H₁₂F₃N₃, found 255.0978 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.1%.

3-(1,2-Dimethyl-1H-imidazol-4-yl)-5-(trifluoromethyl)aniline (10)

Preparation according to general procedure A from 81 (149 mg, 0.52 mmol, 1.0 eq.) and 4-bromo-1,2-dimethyl-1H-imidazole (SM9, 102 mg, 0.56 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (39 mg, 29%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.47 (s, 1H), 7.20 – 7.15 (m, 1H), 7.11 – 7.06 (m, 1H), 6.66 – 6.60 (m, 1H), 5.49 (s, 2H), 3.56 (s, 3H), 2.30 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.49, 144.90, 137.26, 136.32, 129.91 (q, J = 30.5 Hz), 124.69 (d, J = 272.2 Hz), 117.62, 112.43, 107.82 – 107.56 (m), 107.25 – 106.94 (m), 32.53, 12.50. HRMS (GC/EI+): m/z calculated 255.0978 for C₁₂H₁₂F₃N₃, found 255.0976 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.9%.

3-(1-Methyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)aniline (11)

Preparation according to general procedure A from 81 (140 mg, 0.49 mmol, 1.0 eq.) and 4-bromo-1-methyl-1H-pyrazole (SM10, 76 µL, 0.73 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a pale-yellow solid (53 mg, 46%). ¹H NMR (400 MHz, DMSO-d₆) δ = 8.11 (s, 1H), 7.78 (s, 1H), 6.95 (s, 2H), 6.68 (s, 1H), 5.54 (s, 2H), 3.85 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ = 149.77, 136.00, 134.18, 130.38 (q, J = 30.6 Hz), 128.13, 127.92 – 121.29 (m), 121.28, 113.18, 108.56 (q, J = 4.1 Hz), 107.43 (q, J = 4.0 Hz), 38.66. HRMS (GC/EI+): m/z calculated 241.0821 for C₁₁H₁₀F₃N₃, found 241.0821 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.0%.

3-(1-Methyl-1H-pyrazol-3-yl)-5-(trifluoromethyl)aniline (12)

Preparation according to general procedure A from 81 (159 mg, 0.55 mmol, 1.0 eq.) and 3-bromo-1-methyl-1H-pyrazole (SM11, 85 µL, 0.83 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless solid (90 mg, 68%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.72 (d, J = 2.3 Hz, 1H), 7.30 – 7.23 (m, 1H), 7.18 – 7.14 (m, 1H), 6.79 – 6.73 (m, 1H), 6.63 (d, J = 2.3 Hz, 1H), 5.60 (s, 2H), 3.87 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 149.70, 149.22, 134.97, 132.41, 130.13 (q, J = 30.7 Hz), 124.57 (q, J = 272.3 Hz), 113.38, 108.46 (q, J = 4.1 Hz), 102.59, 38.68. HRMS (GC/EI+): m/z calculated 241.0821 for C₁₁H₁₀F₃N₃, found 241.0820 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.4%.

3-(4-Methyl-1H-pyrazol-1-yl)-5-(trifluoromethyl)aniline (13)

The compound was prepared according to general procedure B from 4-methyl-1H-pyrazole (SM4, 63 mg, 1.50 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 283 µL, 2.00 mmol, 1.3 eq.) to yield a colorless solid (107 mg, 30%). ¹H NMR (400 MHz, DMSO) δ 8.25 – 8.22 (m, 1H), 7.56 – 7.52 (m, 1H), 7.25 – 7.22 (m, 1H), 7.15 – 7.12 (m, 1H), 6.75 – 6.72 (m, 1H), 5.83 (s, 2H), 2.08 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 150.61, 141.75, 141.13, 130.82 (q, J = 32.0 Hz), 126.20, 127.92 – 119.91 (m), 117.88, 107.13 – 106.86 (m), 105.90, 101.22 – 100.93 (m), 8.71. HRMS (GC/EI+): m/z calculated 241.0821 for C₁₁H₁₀F₃N₃, found 241.0820 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.2%.

3-(5-Methyl-1H-pyrazol-3-yl)-5-(trifluoromethyl)aniline (14)

Preparation according to general procedure A from 81 (162 mg, 0.56 mmol, 1.0 eq.) and 3-bromo-5-methyl-1H-pyrazole (SM12, 143 mg, 0.87 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless solid (58 mg, 43%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.21 (s, 1H), 7.16 (s, 1H), 6.80 – 6.75 (m, 1H), 6.38 (s, 1H), 2.25 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 149.20, 148.49, 141.06, 134.86, 130.13 (q, J = 30.9 Hz), 124.53 (q, J = 272.3 Hz), 113.75, 108.84 (d, J = 3.7 Hz), 108.73 (d, J = 4.1 Hz), 101.30, 10.98. HRMS (GC/EI+): m/z calculated 241.0821 for C₁₁H₁₀F₃N₃, found 241.0821 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.9%.

3-(1,5-Dimethyl-1H-pyrazol-3-yl)-5-(trifluoromethyl)aniline (15)

Preparation according to general procedure A from 81 (145 mg, 0.50 mmol, 1.0 eq.) and 3-bromo-1,5-dimethyl-1H-pyrazole (SM13, 98 mg, 0.56 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (73 mg, 57%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.20 (s, 1H), 7.11 (s, 1H), 6.74 (s, 1H), 6.42 (s, 1H), 5.58 (s, 2H), 3.75 (s, 3H), 2.27 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 149.66, 147.56, 139.99, 135.16, 130.08 (q, J = 30.7 Hz), 124.58 (q, J = 272.3 Hz), 113.23, 108.30 (q, J = 4.0 Hz), 102.25, 36.06, 10.77. HRMS (GC/EI+): m/z calculated 255.0978 for C₁₂H₁₂F₃N₃, found 255.0978 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.1%.

3-(1H-Pyrazol-4-yl)-5-(trifluoromethyl)aniline (16)

Preparation according to general procedure A from 1H-pyrazol-4-ylboronic acid (SM44, 68 mg, 0.58 mmol, 1.05 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 80 µL, 0.55 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (28 mg, 22%). ¹H NMR (400 MHz, MeOD-d₄) δ 7.94 (s, 2H), 7.12 – 7.05 (m, 2H), 6.82 – 6.77 (m, 1H). ¹³C NMR (101 MHz, MeOD-d₄) δ 150.51, 135.72, 133.00 (q, J = 31.5 Hz), 125.90 (q, J = 271.4 Hz), 122.98, 115.75, 111.82 (q, J = 3.9 Hz), 110.06 (q, J = 3.9 Hz). HRMS (GC/EI+): m/z calculated 227.0665 for C₁₀H₈F₃N₃, found 227.0664 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.0%.

3-(1,5-Dimethyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)aniline (17)

Preparation according to general procedure A from 81 (149 mg, 0.52 mmol, 1.0 eq.) and 4-bromo-1,5-dimethyl-1H-pyrazole (SM14, 128 mg, 0.71 mmol, 1.4 eq.). Further purification by RP column chromatography yielded a colorless solid (90 mg, 68%). ¹H NMR (400 MHz, DMSO) δ 7.53 (s, 1H), 6.86 – 6.80 (m, 1H), 6.77 – 6.70 (m, 2H), 5.59 (s, 2H), 3.77 (s, 3H), 2.35 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.75, 136.24, 135.33, 135.08, 130.17 (q, J = 30.7 Hz), 124.55 (q, J = 272.3 Hz), 118.97, 115.46, 110.44 – 110.19 (m), 107.32 – 107.05 (m), 36.33, 10.14. MS (APCI+): m/z 255.5 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.5%.

3-(1H-Benzo[d]imidazol-1-yl)-5-(trifluoromethyl)aniline (18)

Preparation according to general procedure B from benzimidazole (SM5, 241 mg, 2.02 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 354 µL, 2.50 mmol, 1.2 eq.). Tetrabutylammonium bromide (32 mg, 0.10 mmol, 5 mol%) was added for better solubility. Further purification by RP column chromatography yielded a colorless solid (166 mg, 30%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.56 (s, 1H), 7.80 – 7.74 (m, 1H), 7.64 – 7.58 (m, 1H), 7.40 – 7.28 (m, 2H), 7.11 – 7.07 (m, 1H), 7.06 – 7.01 (m, 1H), 6.99 – 6.93 (m, 1H), 6.03 (s, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 151.13, 143.83, 143.17, 137.47, 132.83, 131.36 (q, J = 31.6 Hz), 123.97 (q, J = 272.4 Hz), 123.55, 122.58, 120.04, 111.24, 110.74, 108.73 (d, J = 3.9 Hz), 106.28 (d, J = 4.0 Hz). HRMS (GC/EI+): m/z calculated 277.0821 for C₁₄H₁₀F₃N₃, found 277.0820 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.4%.

3-(1H-Indol-1-yl)-5-(trifluoromethyl)aniline (19)

Preparation according to general procedure B from indole (SM6, 245 mg, 2.01 mmol, 1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 443 µL, 3.00 mmol, 1.5 eq.). Tetrabutylammonium bromide (30 mg, 0.10 mmol, 5 mol%) was added for better solubility. Further purification by RP column chromatography yielded a yellow oil (232 mg, 40¹H NMR (400 MHz, DMSO) δ 7.69 – 7.61 (m, 2H), 7.56 (dt, J = 8.3, 0.9 Hz, 1H), 7.26 – 7.19 (m, 1H), 7.18 – 7.09 (m, 1H), 7.06 – 7.00 (m, 1H), 6.91 – 6.89 (m, 1H), 6.88 – 6.85 (m, 1H), 6.69 (dd, J = 3.3, 0.8 Hz, 1H), 5.94 (s, 2H).¹³C NMR (126 MHz, DMSO-d₆) δ 150.94, 140.55, 134.89, 131.10 (q, J = 31.3 Hz), 129.18, 128.29, 124.10 (q, J = 272.4 Hz), 122.42, 121.04, 120.42, 111.58, 110.43, 107.59 (q, J = 3.9 Hz), 106.45 (q, J = 4.0 Hz), 103.78. HRMS (GC/EI+): m/z calculated 276.0869 for C₁₅H₁₁F₃N₂, found 276.0866 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.2%.

3-(1-Methyl-1H-indol-3-yl)-5-(trifluoromethyl)aniline (20)

Preparation according to general procedure A from 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (SM43, 137 mg, 0.51 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 81 µL, 0.56 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a yellow oily solid (96 mg, 64%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.27 – 7.12 (m, 3H), 7.02 (s, 1H), 6.72 (s, 1H), 5.62 (s, 2H), 3.83 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.88, 137.25, 136.99, 130.26 (q, J = 30.7 Hz), 128.15, 125.12, 124.69 (q, J = 272.1 Hz), 121.63, 119.89, 119.14, 114.76, 114.04, 110.35, 109.72 (q, J = 3.4 Hz), 106.63 (q, J = 3.8 Hz), 32.57. HRMS (GC/EI+): m/z calculated 290.1025 for C₁₆H₁₃F₃N₂, found 290.1025 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.0%.

3-(2-Methylpyridin-4-yl)-5-(trifluoromethyl)aniline (21)

Preparation according to general procedure A from 3-bromo-5-(trifluoromethyl)aniline (SM1, 106 µL, 0.75 mmol, 1.5 eq.) and 2-methylpyridine-4-boronic acid (SM45, 68.5 mg, 0.5 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (43 mg, 34%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J = 5.3 Hz, 1H), 7.51 (s, 1H), 7.41 (d, J = 5.3 Hz, 1H), 7.17 – 7.13 (m, 1H), 7.12 – 7.08 (m, 1H), 6.95 – 6.91 (m, 1H), 5.77 (s, 2H), 2.53 (s, 3H). ¹³C NMR (126 MHz, Acetone-d₆) δ 160.04, 150.96, 150.67, 148.38, 141.25, 132.65 (q, J = 31.5 Hz), 125.54 (q, J = 271.5 Hz), 121.59, 119.44, 116.66, 112.05 (q, J = 4.1 Hz), 111.49 (q, J = 3.8 Hz), 24.63. HRMS (GC/EI+): m/z calculated 252.0869 for C₁₃H₁₁F₃N₂, found 252.0869 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.5%.

3-(6-Methylpyridin-3-yl)-5-(trifluoromethyl)aniline (22)

Preparation according to general procedure A from 81 (155 mg, 0.54 mmol, 1.0 eq.) and 5-bromo-2-methylpyridine (SM15, 105 mg, 0.61 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (93 mg, 69%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (d, J = 2.4 Hz, 1H), 7.90 (dd, J = 8.1, 2.5 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.10 – 7.05 (m, 1H), 7.05 – 7.00 (m, 1H), 6.90 – 6.85 (m, 1H), 5.72 (s, 2H), 2.50 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.38, 150.08, 146.73, 138.98, 134.41, 132.21, 130.64 (q, J = 30.9 Hz), 124.42 (q, J = 272.2 Hz), 123.17, 115.03, 109.81 (q, J = 4.1 Hz), 108.96 (q, J = 3.9 Hz), 23.68. HRMS (GC/EI+): m/z calculated 252.0869 for C₁₃H₁₁F₃N₂, found 252.0870 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.0%.

3-(Pyridin-4-yl)-5-(trifluoromethyl)aniline (23)

Preparation according to general procedure A from 4-pyridylboronic acid (SM47, 63 mg, 0.50 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 74 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (50 mg, 42%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 – 8.61 (m, 2H), 7.66 – 7.60 (m, 2H), 7.20 – 7.15 (m, 1H), 7.14 – 7.10 (m, 1H), 6.98 – 6.92 (m, 1H), 5.82 (s, 2H¹³C NMR (101 MHz, DMSO) δ 150.24, 150.23, 146.57, 139.10, 130.77 (q, J = 30.9 Hz), 124.33 (q, J = 272.2 Hz), 121.28, 115.15, 110.13 (q, J = 4.1 Hz), 109.88 (q, J = 4.1 Hz). HRMS (GC/EI+): m/z calculated 238.0712 for C₁₂H₉F₃N₂, found 238.0713 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.8%.

3-(Pyridin-3-yl)-5-(trifluoromethyl)aniline (24)

Preparation according to general procedure A from 3-pyridylboronic acid (SM48,111 mg, 0.90 mmol, 1.2 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1,106 µL, 0.75 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (174 mg, 98%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (d, J = 2.4 Hz, 1H), 8.59 (dd, J = 4.8, 1.6 Hz, 1H), 8.05 – 7.98 (m, 1H), 7.52 – 7.44 (m, 1H), 7.12 – 7.08 (m, 1H), 7.08 – 7.04 (m, 1H), 6.94 – 6.88 (m, 1H), 5.75 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 150.13, 148.92, 147.55, 138.94, 135.11, 134.22, 131.28 – 130.29 (m), 123.89, 115.32, 110.34 – 109.83 (m), 109.55 – 108.96 (m). HRMS (GC/EI+): m/z calculated 238.0712 for C₁₂H₉F₃N₂, found 238.0711 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.7%.

3-(2,6-Dimethylpyridin-4-yl)-5-(trifluoromethyl)aniline (25)

Preparation according to general procedure A from 81 (140 mg, 0.49 mmol, 1.0 eq.) and 4-bromo-2,6-dimethylpyridine (SM16, 97 mg, 0.52 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (28 mg, 22%). ¹H NMR (400 MHz, MeOD-d₄) δ 7.30 (s, 2H), 7.18 – 7.14 (m, 1H), 7.14 – 7.10 (m, 1H), 7.01 – 6.96 (m, 1H), 2.55 (s, 6H). ¹³C NMR (126 MHz, MeOD-d₄) δ 159.30, 151.05, 150.71, 141.26, 133.24 (q, J = 31.6 Hz), 125.73 (q, J = 271.4 Hz), 119.83, 117.07, 112.69 (q, J = 4.0 Hz), 112.27 (q, J = 3.9 Hz), 23.78. HRMS (GC/EI+): m/z calculated 266.1025 for C₁₄H₁₃F₃N₂, found 266.1027 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.3%.

3-(5,6-Dimethylpyridin-3-yl)-5-(trifluoromethyl)aniline (26)

Preparation according to general procedure A from 81 (146 mg, 0.51 mmol, 1.0 eq.) and 5-bromo-2,3-dimethylpyridine (SM17, 73 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (48 mg, 35%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J = 2.3 Hz, 1H), 7.76 (d, J = 2.3 Hz, 1H), 7.10 – 7.06 (m, 1H), 7.05 – 7.01 (m, 1H), 6.90 – 6.84 (m, 1H), 5.71 (s, 2H), 2.45 (s, 3H), 2.31 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 156.28, 150.05, 143.84, 139.06, 134.94, 132.68, 131.34, 130.62 (q, J = 31.6 Hz), 126.15 – 122.93 (m), 115.01, 109.81 (d, J = 4.0 Hz), 108.90 (d, J = 3.9 Hz), 22.01, 18.57. HRMS (GC/EI+): m/z calculated 266.1025 for C₁₄H₁₃F₃N₂, found 266.1025 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.8%.

1-(4-(3-Amino-5-(trifluoromethyl)phenyl)pyridin-2-yl)ethan-1-one (27)

Preparation according to general procedure A from 81 (153 mg, 0.53 mmol, 1.1 eq.) and 1-(4-bromopyridin-2-yl)ethanone (SM18, 100 mg, 0.49 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a pale-yellow solid (53 mg, 39%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.78 (dd, J = 5.0, 0.8 Hz, 1H), 8.12 (dd, J = 1.9, 0.8 Hz, 1H), 7.95 (dd, J = 5.1, 1.9 Hz, 1H), 7.28 – 7.23 (m, 1H), 7.21 – 7.16 (m, 1H), 7.00 – 6.95 (m, 1H), 5.84 (s, 2H), 2.68 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 208.84, 163.18, 159.83, 159.53, 157.14, 147.80, 140.36 (q, J = 31.2 Hz), 134.30, 137.98 – 129.49 (m), 127.63, 124.63, 119.94 (q, J = 3.9 Hz), 119.30 (q, J = 4.0 Hz), 35.28. HRMS (GC/EI+): m/z calculated 280.0818 for C₁₄H₁₁F₃N₂O, found 280.0819 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.5%.

1-(5-(3-Amino-5-(trifluoromethyl)phenyl)pyridin-3-yl)ethan-1-one (28)

Preparation according to general procedure A from 81 (144 mg, 0.50 mmol, 1.0 eq.) and 3-acetyl-5-bromopyridine (SM19, 71 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (59 mg, 42%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.11 (d, J = 2.0 Hz, 1H), 9.05 (d, J = 2.3 Hz, 1H), 8.42 (t, J = 2.2 Hz, 1H), 7.20 – 7.15 (m, 2H), 6.97 – 6.91 (m, 1H), 5.79 (s, 2H), 2.70 (s, 3H¹³C NMR (101 MHz, DMSO) δ 197.48, 151.25, 150.22, 148.63, 138.02, 135.07, 133.37, 132.02, 130.83 (q, J = 31.5 Hz), 124.38 (q, J = 272.6 Hz), 115.50, 110.25 (q, J = 4.1 Hz), 109.63 (q, J = 3.8 Hz), 27.19. HRMS (GC/EI+): m/z calculated 280.0818 for C₁₄H₁₁F₃N₂O, found 280.0818 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.3%.

3-(Pyrimidin-5-yl)-5-(trifluoromethyl)aniline (29)

Preparation according to general procedure A from pyrimidine-5-boronic acid (SM49, 63 mg, 0.51 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 74 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (54 mg, 45%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 9.07 (s, 2H), 7.19 – 7.15 (m, 1H), 7.14 – 7.11 (m, 1H), 6.98 – 6.93 (m, 1H), 5.80 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 157.62, 154.78, 150.23, 135.68, 132.88, 130.90 (q, J = 31.1 Hz), 128.47 – 120.08 (m), 115.24, 110.11 (q, J = 3.9 Hz), 109.86 (q, J = 3.9 Hz). HRMS (GC/EI+): m/z calculated 239.0665 for C₁₁H₈F₃N₃, found 239.0665 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 99.0%.

3’,5’-Dimethyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (30)

Preparation according to general procedure A from 3,5-dimethylbenzeneboronic acid (SM50, 79 mg, 0.53 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 71 µL, 0.50 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a yellowish solid (102 mg, 77%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.20 (s, 2H), 7.09 – 7.03 (m, 1H), 7.01 (s, 1H), 6.99 – 6.96 (m, 1H), 6.86 – 6.81 (m, 1H), 5.66 (s, 2H), 2.32 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.81, 142.16, 139.52, 137.95, 130.31 (q, J = 30.8 Hz), 129.27, 124.53 (q, J = 272.5 Hz), 124.41, 115.32, 109.95 (q, J = 3.8 Hz), 108.50 (q, J = 4.0 Hz), 20.94, 20.92. HRMS (GC/EI+): m/z calculated 265.1073 for C₁₅H₁₄F₃N, found 265.1076 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.2%.

3-(1-Methyl-1H-indol-4-yl)-5-(trifluoromethyl)aniline (31)

Preparation according to general procedure A from 81 (195 mg, 0.68 mmol, 1.1 eq.) and 4-bromo-1-methyl-1H-indole (SM20, 132 mg, 0.62 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless oily solid (108 mg, 61%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.46 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 3.1 Hz, 1H), 7.28 – 7.20 (m, 1H), 7.15 – 7.12 (m, 1H), 7.10 (dd, J = 7.3, 0.9 Hz, 1H), 7.02 – 6.96 (m, 1H), 6.91 – 6.84 (m, 1H), 6.50 (dd, J = 3.2, 0.9 Hz, 1H), 3.83 (s, 3H). ¹H NMR (400 MHz, DMSO) δ 7.46 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 3.1 Hz, 1H), 7.28 – 7.20 (m, 1H), 7.15 – 7.12 (m, 1H), 7.10 (dd, J = 7.3, 0.9 Hz, 1H), 7.02 – 6.96 (m, 1H), 6.91 – 6.84 (m, 1H), 6.50 (dd, J = 3.2, 0.9 Hz, 1H), 5.70 (s, 2H), 3.83 (s, 3H). HRMS (GC/EI+): m/z calculated 290.1025 for C₁₆H₁₃F₃N₂, found 290.1026 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.1%.

3-(1-Methyl-1H-indol-5-yl)-5-(trifluoromethyl)aniline (32)

Preparation according to general procedure A from 3-bromo-5-(trifluoromethyl)aniline (SM1, 212 µL, 1.50 mmol, 1.5 eq.) and 1-methylindole-5-boronic acid (SM51, 175 mg, 1.0 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless oil (104 mg, 36%). ¹H NMR (400 MHz, MeOD-d₄) δ 7.77 – 7.72 (m, 1H), 7.42 – 7.32 (m, 2H), 7.17 – 7.13 (m, 2H), 7.12 (d, J = 3.1 Hz, 1H), 6.90 – 6.84 (m, 1H), 6.46 (dd, J = 3.1, 0.7 Hz, 1H), 3.75 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 150.17, 145.82, 138.03, 132.82, 133.23 – 132.06 (m), 130.86, 130.47, 126.06 (q, J = 271.4 Hz), 121.78, 119.96, 117.79, 113.64 (q, J = 4.0 Hz), 110.50, 110.00 (q, J = 3.8 Hz), 102.18, 32.86. HRMS (GC/EI+): m/z calculated 290.1025 for C₁₆H₁₃F₃N₂, found 290.1029 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.0%.

3-(1H-Indol-4-yl)-5-(trifluoromethyl)aniline (33)

Preparation according to general procedure A from 81 (147 mg, 0.51 mmol, 1.0 eq.) and 4-bromo-1H-indole (SM21, 65 µL, 0.56 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless oily solid (84 mg, 60%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.28 (s, 1H), 7.46 – 7.39 (m, 2H), 7.20 – 7.15 (m, 1H), 7.15 – 7.13 (m, 1H), 7.06 (dd, J = 7.3, 1.0 Hz, 1H), 7.02 – 6.99 (m, 1H), 6.88 – 6.83 (m, 1H), 6.55 – 6.49 (m, 1H), 5.69 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.77, 142.61, 136.38, 132.21, 130.03 (q, J = 31.1 Hz), 125.96, 125.37, 128.84 – 120.06 (m), 121.30, 118.31, 116.90, 111.64 – 111.28 (m), 111.22, 108.37 – 107.85 (m), 100.03. HRMS (GC/EI+): m/z calculated 276.0869 for C₁₅H₁₁F₃N₂, found 276.0868 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.4%.

3-(1H-Indol-5-yl)-5-(trifluoromethyl)aniline (34)

Preparation according to general procedure A from 81 (150 mg, 0.52 mmol, 1.0 eq.) and 5-bromoindole (SM22, 160 mg, 0.82 mmol, 1.6 eq.). Further purification by RP column chromatography yielded a yellow solid (79 mg, 55%). ¹H NMR (400 MHz, MeOD-d₄) δ 7.76 (dd, J = 1.8, 0.8 Hz, 1H), 7.44 (dt, J = 8.4, 0.9 Hz, 1H), 7.34 (dd, J = 8.5, 1.8 Hz, 1H), 7.26 (d, J = 3.1 Hz, 1H), 7.19 – 7.16 (m, 1H), 7.14 – 7.11 (m, 1H), 6.89 – 6.83 (m, 1H), 6.50 (dd, J = 3.1, 0.9 Hz, 1H). ¹³C NMR (101 MHz, MeOD-d₄) δ 150.21, 146.05, 137.49, 132.81 (d, J = 3.5 Hz), 133.16 – 132.10 (m), 129.97, 126.45, 130.33 – 121.88 (m), 121.73, 119.62, 117.82, 113.93 – 113.41 (m), 112.46, 110.11 – 109.68 (m), 102.83. HRMS (GC/EI+): m/z calculated 276.0869 for C₁₅H₁₁F₃N₂, found 276.0869 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.8%.

3-(Indolin-5-yl)-5-(trifluoromethyl)aniline (35)

Preparation according to general procedure A from 81 (144 mg, 0.50 mmol, 1.0 eq.) and 5-bromoindoline (SM23, 116 mg, 0.58 mmol, 1.2 eq.). Further purification by RP column chromatography yielded a colorless solid (53 mg, 38%). ¹H NMR (400 MHz, MeOD-d₄) δ 7.35 – 7.31 (m, 1H), 7.25 – 7.20 (m, 1H), 7.06 – 7.03 (m, 1H), 7.03 – 7.00 (m, 1H), 6.83 – 6.80 (m, 1H), 6.70 (d, J = 8.1 Hz, 1H), 3.51 (t, J = 8.4 Hz, 2H), 3.04 (t, J = 8.3 Hz, 2H). ¹³C NMR (101 MHz, MeOD-d₄) δ 153.14, 150.22, 144.92, 132.67 (q, J = 31.3 Hz), 132.53, 131.89, 127.17, 126.03 (q, J = 271.3 Hz), 124.07, 116.91, 112.81 (q, J = 4.1 Hz), 111.10, 109.87 (q, J = 3.7 Hz), 48.28, 30.68. HRMS (GC/EI+): m/z calculated 278.1025 for C₁₅H₁₃F₃N₂, found 278.1027 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.1%.

3-(Benzofuran-5-yl)-5-(trifluoromethyl)aniline (36)

Preparation according to general procedure A from 81 (159 mg, 0.55 mmol, 1.0 eq.) and 5-bromobenzufurane (SM24, 76 µL, 0.61 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless oil (87 mg, 57%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.04 (d, J = 2.2 Hz, 1H), 7.87 (dd, J = 2.0, 0.6 Hz, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.53 (dd, J = 8.6, 1.9 Hz, 1H), 7.13 – 7.07 (m, 1H), 7.05 – 6.98 (m, 2H), 6.87 – 6.82 (m, 1H), 5.68 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 154.11, 149.92, 146.78, 142.40, 134.92, 130.39 (q, J = 30.8 Hz), 127.89, 124.54 (q, J = 272.1 Hz), 123.40, 119.41, 115.62, 111.59, 110.25 (q, J = 3.7 Hz), 108.25 (q, J = 4.0 Hz), 107.02. HRMS (GC/EI+): m/z calculated 277.0709 for C₁₅H₁₀F₃NO, found 277.0710 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.2%.

3-(Benzo[b]thiophen-5-yl)-5-(trifluoromethyl)aniline (37)

Preparation according to general procedure A from 81 (143 mg, 0.50 mmol, 1.0 eq.) and 5-bromobenzo[b]thiophene (SM25, 144 mg, 0.68 mmol, 1.4 eq.). Further purification by RP column chromatography yielded a colorless oil (104 mg, 72%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.05 – 8.00 (m, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.63 – 7.52 (m, 2H), 7.41 (d, J = 5.5 Hz, 1H), 7.21 – 7.17 (m, 1H), 7.17 – 7.14 (m, 1H), 6.96 – 6.90 (m, 1H). ¹³C NMR (101 MHz, MeOD-d₄) δ 150.58, 144.42, 141.77, 140.63, 138.10, 132.93 (q, J = 31.5 Hz), 128.33, 125.94 (q, J = 271.6 Hz), 125.17, 124.54, 123.72, 122.80, 117.72, 113.43 (q, J = 4.0 Hz), 110.78 (q, J = 3.9 Hz). HRMS (GC/EI+): m/z calculated 293.0481 for C₁₅H₁₀F₃NS, found 293.0485 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.0%.

3-(Imidazo[1,2-a]pyridin-7-yl)-5-(trifluoromethyl)aniline (38)

Preparation according to general procedure A from 81 (145 mg, 0.50 mmol, 1.0 eq.) and 7-bromoimidazo[1,2-a]pyridine (SM26, 123 mg, 0.62 mmol, 1.2 eq.). Further purification by RP column chromatography yielded a colorless solid (61 mg, 44%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.62 (d, J = 7.1 Hz, 1H), 7.98 (s, 1H), 7.81 – 7.76 (m, 1H), 7.62 (s, 1H), 7.23 – 7.18 (m, 2H), 7.15 (s, 1H), 6.92 – 6.87 (m, 1H), 5.73 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 150.05, 144.76, 139.83, 135.40, 134.10, 130.62 (q, J = 30.9 Hz), 127.08, 124.43 (q, J = 272.4 Hz), 114.96, 113.41, 113.05, 111.18, 109.81 (q, J = 4.0 Hz), 109.24 (q, J = 3.3 Hz). HRMS (GC/EI+): m/z calculated 277.0821 for C₁₄H₁₀F₃N₃, found 277.0821 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.6%.

3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-5-(trifluoromethyl)aniline (39)

Preparation according to general procedure A from 81 (193 mg, 0.67 mmol, 1 eq.) and 5-bromo-1-methyl-1H-pyrrolo[2,3-b]pyridine (SM27, 150 mg, 0.69 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (123 mg, 63%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J = 2.2 Hz, 1H), 8.17 (d, J = 2.2 Hz, 1H), 7.57 (d, J = 3.4 Hz, 1H), 7.14 – 7.08 (m, 1H), 7.08 – 7.03 (m, 1H), 6.88 – 6.83 (m, 1H), 6.52 (d, J = 3.4 Hz, 1H), 5.69 (s, 2H), 3.85 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 149.98, 147.20, 141.14, 140.75, 131.02, 130.52 (q, J = 30.8 Hz), 127.63, 126.51, 124.54 (q, J = 272.2 Hz), 119.93, 115.48, 110.21 (q, J = 4.0 Hz), 108.20 (q, J = 3.9 Hz), 99.28, 30.97. HRMS (GC/EI+): m/z calculated 291.0978 for C₁₅H₁₂F₃N₃, found 291.0980 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.0%.

3-(1-Methyl-1H-indazol-5-yl)-5-(trifluoromethyl)aniline (40)

Preparation according to general procedure A from 81 (165 mg, 0.58 mmol, 1.0 eq.) and 5-bromo-1-methyl-1H-indazole (SM28, 124 mg, 0.58 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (101 mg, 60%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (d, J = 1.0 Hz, 1H), 7.99 – 7.96 (m, 1H), 7.72 (dt, J = 8.8, 0.9 Hz, 1H), 7.65 (dd, J = 8.8, 1.7 Hz, 1H), 7.15 – 7.10 (m, 1H), 7.08 – 7.02 (m, 1H), 6.86 – 6.81 (m, 1H), 5.67 (s, 2H), 4.07 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 149.93, 142.44, 139.28, 132.93, 132.19, 130.41 (q, J = 30.7 Hz), 125.42, 124.57 (q, J = 272.4 Hz), 124.06, 118.60, 115.49, 110.24 – 110.07 (m), 110.15, 108.13 (q, J = 3.9 Hz), 35.46. HRMS (GC/EI+): m/z calculated 291.0978 for C₁₅H₁₂F₃N₃, found 291.0979 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.7%.

3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-(trifluoromethyl)aniline (41)

Preparation according to general procedure A from 81 (144 mg, 0.5 mmol, 1.0 eq.) and 3-bromo-1-methyl-1H-pyrrolo[2,3-b]pyridine (SM29, 163 mg, 0.75 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless solid (55 mg, 38%). ¹H NMR (400 MHz, DMSO) δ 8.34 (dd, J = 4.6, 1.5 Hz, 1H), 8.27 (dd, J = 8.0, 1.6 Hz, 1H), 7.98 (s, 1H), 7.25 – 7.18 (m, 2H), 7.07 – 7.02 (m, 0H), 6.77 – 6.71 (m, 1H), 5.64 (s, 2H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.97, 147.87, 142.90, 136.24, 130.42 (q, J = 30.7 Hz), 128.13, 127.65, 126.40 – 122.87 (m), 117.40, 116.18, 114.53, 112.43, 109.53 – 109.19 (m), 107.08 – 106.78 (m), 30.96. HRMS (GC/EI+): m/z calculated 291.0978 for C₁₅H₁₂F₃N₃, found 291.0976 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.0%.

3-(2-Methylpyridin-4-yl)aniline (42)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 83 mg, 0.61 mmol, 1.2 eq.) and 3-bromoaniline (SM70, 54 µL, 0.50 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (32 mg, 34%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.38 (d, J = 5.4 Hz, 1H), 7.51 – 7.49 (m, 1H), 7.44 – 7.41 (m, 1H), 7.25 – 7.16 (m, 1H), 7.07 – 7.02 (m, 1H), 7.02 – 6.98 (m, 1H), 6.83 – 6.75 (m, 1H), 2.56 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 159.54, 151.72, 149.80, 149.58, 139.87, 130.88, 122.60, 120.30, 117.51, 117.29, 114.54, 23.80. HRMS (FIA/ESI+): m/z calculated 184.0995 for C₁₂H₁₃N₂, found 185.1072 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.4%.

3-(2-Methylpyridin-4-yl)-4-(trifluoromethyl)aniline (43)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 83 mg, 0.60 mmol, 1.2 eq.) and 3-bromo-4-(trifluoromethyl)aniline (SM71, 73 µL, 0.50 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (53 mg, 42%). ¹H NMR (400 MHz, DMSO) δ 8.45 (dd, J = 5.1, 0.9 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.16 (s, 1H), 7.12 – 7.05 (m, 1H), 6.67 (dd, J = 8.4, 2.3 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 5.96 (s, 2H), 2.50 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 157.46, 151.80, 148.36, 139.44, 127.56 (q, J = 5.2 Hz), 125.07 (q, J = 271.2 Hz), 122.77, 120.76, 115.28, 112.76 (q, J = 30.1 Hz), 112.15, 23.98. HRMS (FIA/ESI+): m/z calculated 252.0869 for C₁₃H₁₁F₃N₂, found 253.0945 ([M+H]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 98.5%.

3-Methyl-5-(2-methylpyridin-4-yl)aniline (44)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 115 mg, 0.84 mmol, 1.2 eq.) and 3-bromo-5-methylaniline (SM72, 87 µL, 0.70 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (98 mg, 70%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.43 (d, J = 5.2 Hz, 1H), 7.44 – 7.39 (m, 1H), 7.32 (dd, J = 5.2, 2.1 Hz, 1H), 6.74 – 6.71 (m, 1H), 6.73 – 6.67 (m, 1H), 6.50 – 6.44 (m, 1H), 5.15 (s, 2H), 2.22 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.27, 149.30, 149.24, 148.30, 138.71, 138.05, 120.27, 118.25, 115.29, 115.17, 109.33, 24.11, 21.22. HRMS (GC/EI+): m/z calculated 198.1151 for C₁₃H₁₄N₂, found 198.1152 ([M]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 97.5%.

3-Methoxy-5-(2-methylpyridin-4-yl)aniline (45)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 83 mg, 0.60 mmol, 1.2 eq.) and 3-bromo-4-methoxyaniline (SM73, 69 µL, 0.50 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a brown oily solid (35 mg, 32%). ¹H NMR (500 MHz, MeOD-d₄) δ 8.35 (d, J = 5.3 Hz, 1H), 7.44 (s, 1H), 7.37 (d, J = 5.3 Hz, 1H), 6.65 – 6.60 (m, 1H), 6.56 – 6.52 (m, 1H), 6.41 – 6.36 (m, 1H), 3.78 (s, 3H), 2.54 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 162.48, 159.28, 151.35, 150.78, 149.31, 140.65, 122.38, 120.11, 107.44, 103.31, 102.41, 55.49, 23.61. HRMS (FIA/ESI+): m/z calculated 214.1101 for C₁₃H₁₄N₂O, found 215.1177 ([M+H]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 98.2%.

3-(2-Methylpyridin-4-yl)-5-(trifluoromethoxy)aniline (46)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 83 mg, 0.60 mmol, 1.2 eq.) and 3-bromo-5-(trifluoromethoxy)aniline (SM74, 76 µL, 0.51 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (70 mg, 52%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.42 (d, J = 5.3 Hz, 1H), 7.52 – 7.47 (m, 1H), 7.43 – 7.40 (m, 1H), 6.98 – 6.93 (m, 1H), 6.82 – 6.76 (m, 1H), 6.67 – 6.61 (m, 1H), 2.58 (s, 3H¹³C NMR (101 MHz, MeOD) δ 159.90, 152.16 (q, J = 1.9 Hz), 152.10, 150.37, 149.85, 141.59, 122.63, 121.98 (q, J = 255.2 Hz), 120.30, 112.56, 108.46, 108.23, 23.83. HRMS (GC/EI): m/z calculated 268.0818 for C₁₃H₁₁F₃N₂O, found 268.0817 ([M]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 95.2%.

3-Amino-5-(2-methylpyridin-4-yl)benzonitrile (47)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 85 mg, 0.62 mmol, 1.2 eq.) and 3-amino-5-benzonitrile (SM75, 59 µL, 0.50 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (33 mg, 31%). ¹H NMR (500 MHz, MeOD-d₄) δ 8.44 (d, J = 5.3 Hz, 1H), 7.52 (s, 1H), 7.44 (dd, J = 5.3, 2.0 Hz, 1H), 7.25 – 7.23 (m, 1H), 7.23 – 7.22 (m, 1H), 7.01 – 6.97 (m, 1H), 2.59 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 160.03, 151.36, 149.95, 149.69, 141.33, 122.66, 120.27, 119.98, 119.49, 118.36, 118.04, 114.59, 23.85. HRMS (FIA/ESI+): m/z calculated 209.0947 for C₁₃H₁₁N₃, found 210.1024 ([M+H]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 95.1%.

3-Chloro-5-(2-methylpyridin-4-yl)aniline (48)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 81 mg, 0.59 mmol, 1.2 eq.) and 3-amino-5-chloroaniline (SM76, 60 µL, 0.49 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (50 mg, 46%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.40 (d, J = 5.4 Hz, 1H), 7.48 (s, 1H), 7.40 (ddd, J = 5.4, 1.9, 0.7 Hz, 1H), 6.93 – 6.91 (m, 1H), 6.90 – 6.89 (m, 1H), 6.76 – 6.75 (m, 1H), 2.57 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 159.80, 151.62, 150.46, 149.77, 141.47, 136.55, 122.61, 120.26, 116.37, 115.97, 112.54, 23.83. HRMS (FIA/ESI+): m/z calculated 218.0605 for C₁₂H₁₁ClN₂, found 219.0682 ([M+H]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 97.9%.

3,4-Dichloro-5-(2-methylpyridin-4-yl)aniline (49)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 70 mg, 0.51 mmol, 1.0 eq.) and 3-bromo-4,5-dichloroaniline (SM77, 70 µL, 0.51 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a yellow solid (18 mg, 14%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.44 (d, J = 5.3 Hz, 1H), 7.33 – 7.28 (m, 1H), 7.24 (dd, J = 5.3, 1.7 Hz, 1H), 6.88 (d, J = 2.6 Hz, 1H), 6.57 (d, J = 2.7 Hz, 1H), 2.58 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 159.26, 150.62, 149.45, 149.14, 141.33, 134.80, 125.35, 123.03, 117.65, 116.94, 116.33, 23.80. HRMS (GC/EI): m/z calculated 252.0216 for C₁₂H₁₀Cl₂N₂, found 252.0216 ([M]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 97.8%.

4-(2-Methylpyridin-4-yl)-1H-indol-6-amine (50)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 83 mg, 0.60 mmol, 1.0 eq.) and 4-bromo-1H-indol-6-amine (SM78, 110 mg, 0.50 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a brown solid (39 mg, 35%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.52 (d, J = 5.2 Hz, 1H), 7.62 – 7.61 (m, 1H), 7.56 (dd, J = 5.1, 1.4 Hz, 1H), 7.19 (d, J = 3.2 Hz, 1H), 6.89 – 6.84 (m, 1H), 6.79 (d, J = 1.9 Hz, 1H), 6.54 (dd, J = 3.3, 1.0 Hz, 1H), 2.65 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 159.42, 151.61, 149.82, 144.72, 139.67, 131.75, 124.66, 124.04, 121.94, 119.92, 111.29, 101.14, 98.65, 24.38. HRMS (GC/EI): m/z calculated 223.1103 for C₁₄H₁₃N₃, found 223.1103 ([M]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 97.9%.

6-(2-Methylpyridin-4-yl)-1H-indol-4-amine (51)

Preparation according to general procedure A from 2-methylpyridine-4-boronic acid (SM69, 863 mg, 0.63 mmol, 1.0 eq.) and 6-bromo-1H-indol-4-amine (SM79, 114 mg, 0.51 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a brown solid (52 mg, 46%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.33 (d, J = 5.3 Hz, 1H), 7.56 – 7.52 (m, 1H), 7.49 – 7.44 (m, 1H), 7.20 – 7.19 (m, 1H), 7.19 – 7.19 (m, 1H), 6.74 (d, J = 1.5 Hz, 1H), 6.55 (dd, J = 3.2, 1.0 Hz, 1H), 2.55 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 159.14, 153.09, 149.28, 141.67, 139.01, 133.23, 125.27, 122.52, 120.39, 120.30, 103.55, 102.21, 99.62, 23.82. HRMS (GC/EI): m/z calculated 223.1103 for C₁₄H₁₃N₃, found 223.1102 ([M]⁺). qHNMR (400 MHz, DMSO-d₆) purity = 98.3%.

4-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indol-6-amine (52)

Preparation according to general procedure A from 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (SM80, 156 mg, 0.57 mmol, 1.2 eq.) and bromo-1H-indol-6-amine (SM78, 105 mg, 0.48 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (46 mg, 37%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.64 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.11 (d, J = 2.1 Hz, 1H), 7.54 (d, J = 3.4 Hz, 1H), 7.08 – 7.02 (m, 1H), 6.62 – 6.57 (m, 1H), 6.56 – 6.47 (m, 2H), 6.34 – 6.28 (m, 1H), 4.81 (s, 2H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 146.67, 144.09, 142.35, 138.09, 131.28, 130.50, 129.13, 127.40, 122.44, 119.84, 118.13, 109.71, 99.86, 99.06, 94.43, 30.94. HRMS (DEP/EI+): m/z calculated 262.1213 for C₁₆H₁₄N₄, found 262.1224 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.7%.

2’-Methyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (53)

Preparation according to general procedure A from 2-tolylboronic acid (SM52, 87 mg, 0.61 mmol, 1.2 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 72 µL, 0.51 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless oil (60 mg, 47%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.31 – 7.21 (m, 3H), 7.19 – 7.15 (m, 1H), 6.87 – 6.82 (m, 1H), 6.77 – 6.71 (m, 1H), 6.69 – 6.64 (m, 1H), 5.65 (s, 2H), 2.21 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.38, 142.89, 140.68, 134.53, 130.33, 129.69 (q, J = 30.8 Hz), 129.13, 127.57, 125.94, 124.49 (q, J = 272.6 Hz), 117.71, 112.03 (q, J = 4.2 Hz), 108.16 (q, J = 4.1 Hz), 19.98. HRMS (GC/EI+): m/z calculated 251.0916 for C₁₄H₁₂F₃N, found 251.0918 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.7%.

3’-Methyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (54)

Preparation according to general procedure A from 3-tolylboronic acid (SM53, 70 mg, 0.50 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 74 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a yellow oil (29 mg, 23%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.44 – 7.40 (m, 1H), 7.40 – 7.31 (m, 2H), 7.23 – 7.16 (m, 1H), 7.09 – 7.03 (m, 1H), 7.01 – 6.95 (m, 1H), 6.88 – 6.81 (m, 1H), 5.67 (s, 2H), 2.37 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.95, 142.12, 139.60, 138.17, 130.40 (q, J = 30.6 Hz), 128.87, 128.54, 127.29, 128.79 – 120.36 (m), 123.76, 115.33, 109.96 (d, J = 4.2 Hz), 108.58 (d, J = 3.8 Hz), 21.06. HRMS (GC/EI+): m/z calculated 251.0916 for C₁₄H₁₂F₃N, found 251.0917 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.8%.

4’-Methyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (55)

Preparation according to general procedure A from 4-tolylboronic acid (SM54, 71 mg, 0.50 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 74 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (49 mg, 39%). ¹H NMR (400 MHz, DMSO) δ 7.49 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 7.7 Hz, 2H), 7.08 – 7.02 (m, 1H), 7.00 – 6.94 (m, 1H), 6.85 – 6.80 (m, 1H), 5.66 (s, 2H), 2.34 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 149.94, 141.88, 137.24, 136.71, 130.39 (q, J = 30.9 Hz), 129.54, 126.45, 124.53 (q, J = 272.5 Hz), 115.03, 109.70 (q, J = 3.8 Hz), 108.37 (q, J = 3.8 Hz), 20.66. HRMS (GC/EI+): m/z calculated 251.0916 for C₁₄H₁₂F₃N, found 251.0916 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.4%.

2’,3’-Dimethyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (56)

Preparation according to general procedure A from 81 (146 mg, 0.51 mmol, 1.0 eq.) and 1-bromo-2,3-dimethylbenzene (SM30, 90 µL, 0.66 mmol, 1.3 eq.). Further purification by RP column chromatography yielded a colorless solid (80 mg, 60%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.17 (d, J = 7.2 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.99 (dd, J = 7.5, 1.6 Hz, 1H), 6.86 – 6.81 (m, 1H), 6.72 – 6.66 (m, 1H), 6.64 – 6.59 (m, 1H), 5.65 (s, 2H), 2.28 (s, 3H), 2.08 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.32, 143.57, 141.00, 136.89, 133.20, 129.59 (q, J = 30.8 Hz), 128.97, 126.92, 125.30, 124.50 (q, J = 272.5 Hz), 117.86, 112.22 (q, J = 3.6 Hz), 108.02 (q, J = 3.3 Hz), 20.27, 16.54. HRMS (GC/EI+): m/z calculated 265.1073 for C₁₅H₁₄F₃N, found 265.1073 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.6%.

3’,4’-Dimethyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (57)

Preparation according to general procedure A from 3,4-dimethylphenylboronic acid (SM55, 70 µL, 0.50 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 74 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a yellow oil (68 mg, 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.38 (d, J = 2.0 Hz, 1H), 7.31 (dd, J = 7.8, 2.0 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 7.08 – 7.02 (m, 1H), 6.99 – 6.95 (m, 1H), 6.85 – 6.79 (m, 1H), 5.64 (s, 2H), 2.28 (s, 3H), 2.25 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 149.88, 142.00, 137.07, 136.76, 135.99, 130.33 (q, J = 30.7 Hz), 130.03, 127.63, 124.56 (q, J = 272.6 Hz), 123.88, 115.04, 109.71 (q, J = 4.0 Hz), 108.30 (q, J = 3.9 Hz), 19.45, 19.03. HRMS (GC/EI+): m/z calculated 265.1073 for C₁₅H₁₄F₃N, found 265.1073 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.7%.

3’-Ethyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (58)

Preparation according to general procedure A from 3-ethylphenylboronic acid (SM56, 100 mg, 0.65 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 96 µL, 0.68 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a yellowish oily solid (148 mg, 86%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.45 (t, J = 1.8 Hz, 1H), 7.42 (dt, J = 7.7, 1.7 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.24 (dt, J = 7.3, 1.6 Hz, 1H), 7.22 – 7.19 (m, 1H), 7.18 – 7.12 (m, 1H), 7.01 – 6.95 (m, 1H), 6.00 (s, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 147.61, 144.57, 142.32, 139.32, 130.43 (q, J = 31.0 Hz), 128.97, 127.49, 126.20, 124.37 (q, J = 273.0 Hz), 124.07, 116.82, 111.81 (q, J = 3.9 Hz), 110.01 (q, J = 3.3 Hz), 28.19, 15.70. HRMS (GC/EI+): m/z calculated 265.1073 for C₁₅H₁₄F₃N, found 265.1077 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.1%.

3’-(Tert-butyl)-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (59)

Preparation according to general procedure A from 3-tert-butylphenylboronic acid (SM57, 196 mg, 1.1 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 141 µL, 1.0 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a light-yellow oil (251 mg, 86%). ¹H NMR (400 MHz, CD₂Cl₂) δ 7.58 (dt, J = 2.6, 1.0 Hz, 1H), 7.46 – 7.36 (m, 3H), 7.19 (tq, J = 1.5, 0.7 Hz, 1H), 7.06 (ddd, J = 2.2, 1.6, 0.6 Hz, 1H), 6.88 (ddt, J = 2.2, 1.6, 0.7 Hz, 1H), 4.02 (s, 2H), 1.37 (s, 9H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 152.31, 147.96, 144.18, 140.23, 132.13 (q, J = 31.4 Hz), 128.94, 125.41, 124.79 (q, J = 272.2 Hz), 124.63, 124.57, 117.02, 114.07 (q, J = 3.9 Hz), 110.22 (q, J = 3.9 Hz), 35.13, 31.52. HRMS (GC/EI+): m/z calculated 293.1386 for C₁₇H₁₈F₃N, found 293.1387 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.6%.

1-(3’-Amino-5’-(trifluoromethyl)-[1,1’-biphenyl]-3-yl)ethan-1-one (60)

Preparation according to general procedure A from 81 (145 mg, 0.50 mmol, 1.0 eq.) and 3’-bromoacetophenone (SM57, 80 µL, 0.60 mmol, 1.2 eq.). Further purification by RP column chromatography yielded a colorless solid (113 mg, 80%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.12 (t, J = 1.8 Hz, 1H), 8.01 – 7.94 (m, 1H), 7.91 – 7.84 (m, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.17 – 7.11 (m, 1H), 7.10 – 7.05 (m, 1H), 6.92 – 6.87 (m, 1H), 5.74 (s, 2H), 2.65 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 197.92, 150.09, 141.12, 139.99, 137.43, 131.31, 130.58 (q, J = 31.0 Hz), 129.43, 127.69, 126.10, 124.45 (q, J = 272.4 Hz), 115.37, 110.00 (q, J = 3.5 Hz), 109.03 (q, J = 4.2 Hz), 26.88. HRMS (GC/EI+): m/z calculated 279.0866 for C₁₅H₁₂F₃NO, found 279.0868 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.9%.

3’-Fluoro-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (61)

Preparation according to general procedure A from 3-fluorophenylboronic acid (SM58, 87 mg, 0.63 mmol, 1.2 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 74 µL, 0.52 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless oil (67 mg, 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.55 – 7.40 (m, 3H), 7.26 – 7.17 (m, 1H), 7.11 – 7.07 (m, 1H), 7.06 – 7.01 (m, 1H), 6.91 – 6.85 (m, 1H), 5.71 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.60 (d, J = 243.6 Hz), 150.01, 142.08 (d, J = 7.8 Hz), 140.61 (d, J = 2.3 Hz), 130.90 (d, J = 8.6 Hz), 130.54 (q, J = 30.9 Hz), 124.42 (q, J = 272.5 Hz), 122.76 (d, J = 2.6 Hz), 115.35, 114.59 (d, J = 21.0 Hz), 113.42 (d, J = 22.1 Hz), 110.05 (q, J = 4.0 Hz), 109.15 (q, J = 3.6 Hz). HRMS (GC/EI+): m/z calculated 255.0666 for C₁₃H₉F₄N, found 255.0669 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.3%.

3’-Chloro-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (62)

Preparation according to general procedure A from 3-chlorophenylboronic acid (SM59, 87 mg, 0.56 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 72 µL, 0.51 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a yellowish solid (89 mg, 64%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.65 (t, J = 1.9 Hz, 1H), 7.58 (dt, J = 7.5, 1.6 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.45 (dt, J = 8.1, 1.6 Hz, 1H), 7.11 – 7.06 (m, 1H), 7.06 – 7.01 (m, 1H), 6.91 – 6.86 (m, 1H), 5.72 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 150.04, 141.75, 140.42, 133.71, 130.80, 130.57 (q, J = 31.1 Hz), 127.72, 126.42, 125.40, 124.40 (q, J = 272.4 Hz), 115.36, 110.03 (q, J = 3.3 Hz), 109.18 (q, J = 3.8 Hz). HRMS (GC/EI+): m/z calculated 271.0370 for C₁₃H₉ClF₃N, found 271.0376 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.0%.

3’,5-Bis(trifluoromethyl)-[1,1’-biphenyl]-3-amine (63)

Preparation according to general procedure A from 3-(trifluoromethyl)phenylboronic acid (SM60, 139 mg, 0.73 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 94 µL, 0.67 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (128 mg, 63%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, J = 7.7 Hz, 1H), 7.91 – 7.88 (m, 1H), 7.72 (dt, J = 15.4, 7.7 Hz, 2H), 7.17 – 7.12 (m, 1H), 7.11 – 7.06 (m, 1H), 6.94 – 6.88 (m, 1H), 5.75 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 150.13, 140.64, 140.36, 130.84, 130.66 (q, J = 30.8 Hz), 130.11, 129.58 (q, J = 31.3 Hz), 124.85 (q, J = 271.6 Hz), 124.48 (q, J = 3.7 Hz), 124.17 (q, J = 272.8 Hz), 123.08 (q, J = 3.6 Hz), 115.44, 110.10 (q, J = 3.8 Hz), 109.30 (q, J = 3.5 Hz). HRMS (GC/EI+): m/z calculated 305.0634 for C₁₄H₉F₆N, found 305.0639 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.0%.

3’-(Trifluoromethoxy)-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (64)

Preparation according to general procedure A from 3-trifluoromethoxyphenylboronic acid (SM61, 227 mg, 1.1 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 141 µL, 1.0 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (206 mg, 64%). ¹H NMR (400 MHz, CD₂Cl₂) δ 7.53 (dt, J = 7.8, 1.5 Hz, 1H), 7.49 (td, J = 7.8, 0.6 Hz, 1H), 7.43 (ddp, J = 2.5, 1.5, 0.9 Hz, 1H), 7.26 (ddq, J = 7.8, 2.5, 1.2 Hz, 1H), 7.17 (tt, J = 1.5, 0.8 Hz, 1H), 7.04 (ddd, J = 2.2, 1.6, 0.6 Hz, 1H), 6.92 (ddq, J = 2.2, 1.3, 0.7 Hz, 1H), 4.06 (s, 2H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 150.03 (q, J = 1.9 Hz), 148.19, 142.67, 141.96, 132.45 (q, J = 31.8 Hz), 130.71, 126.02, 124.61 (q, J = 272.5 Hz), 120.96 (q, J = 257.1 Hz), 120.62, 120.12, 116.77, 113.86 (q, J = 3.9 Hz), 111.02 (q, J = 3.8 Hz). HRMS (GC/EI+): m/z calculated 321.0583 for C₁₄H₁₉F₆NO, found 321.0582 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 99.4%.

3’-Methoxy-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (65)

Preparation according to general procedure A from 3-methoxyphenylboronic acid (SM62, 167 mg, 1.1 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 141 µL, 1.0 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a light-yellow oily solid (224 mg, 84%). ¹H NMR (400 MHz, CD₂Cl₂) δ 7.36 (ddd, J = 8.2, 7.6, 0.4 Hz, 1H), 7.19 (tt, J = 1.5, 0.8 Hz, 1H), 7.15 (ddd, J = 7.7, 1.7, 1.0 Hz, 1H), 7.09 (dd, J = 2.6, 1.7 Hz, 1H), 7.05 (ddt, J = 2.2, 1.6, 0.6 Hz, 1H), 6.92 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 6.89 (ddt, J = 2.3, 1.6, 0.7 Hz, 1H), 4.02 (s, 2H), 3.85 (s, 3H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 160.50, 147.98, 143.40, 141.93, 132.16 (q, J = 31.5 Hz), 130.25, 124.74 (q, J = 272.2 Hz), 119.79, 116.89, 113.98 (q, J = 3.8 Hz), 113.72, 113.09, 110.48 (q, J = 3.8 Hz), 55.72. HRMS (GC/EI+): m/z calculated 267.0866 for C₁₄H₁₂F₃NO, found 267.0864 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 99.8%.

N³,N³-Dimethyl-5’-(trifluoromethyl)-[1,1’-biphenyl]-3,3’-diamine (66)

Preparation according to general procedure A from 3-(N,N-dimethylamino)phenylboronic acid (SM63, 182 mg, 1.1 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 141 µL, 1.0 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a light-yellow oil (221 mg, 79%). ¹H NMR (500 MHz, CD₂Cl₂) δ 7.28 (ddd, J = 8.2, 7.3, 0.7 Hz, 1H), 7.19 (td, J = 1.5, 0.7 Hz, 1H), 7.06 (dt, J = 2.1, 1.0 Hz, 1H), 6.90 – 6.85 (m, 3H), 6.75 (ddd, J = 8.3, 2.5, 1.0 Hz, 1H), 4.00 (s, 2H), 2.99 (s, 6H). ¹³C NMR (126 MHz, CD₂Cl₂) δ 151.51, 147.86, 144.58, 141.34, 131.98 (q, J = 31.6 Hz), 129.81, 124.81 (q, J = 272.3 Hz), 117.05, 115.63, 114.11 (q, J = 4.1 Hz), 112.49, 111.45, 110.17 (q, J = 3.8 Hz), 40.83. HRMS (GC/EI+): m/z calculated 280.1182 for C₁₅H₁₅F₃N₂, found 280.1187 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 99.0%.

3’-Amino-5’-(trifluoromethyl)-[1,1’-biphenyl]-3-ol (67)

Preparation according to general procedure A from 3-hydroxyphenylboronic acid (SM64, 152 mg, 1.1 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 141 µL, 1.0 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (166 mg, 66 ¹H NMR (500 MHz, CD₂Cl₂) δ 7.31 (t, J = 7.8 Hz, 1H), 7.20 – 7.16 (m, 1H), 7.15 – 7.12 (m, 1H), 7.06 – 7.01 (m, 2H), 6.90 – 6.87 (m, 1H), 6.87 – 6.82 (m, 1H), 5.16 (s, 1H), 4.02 (s, 2H). ¹³C NMR (126 MHz, CD₂Cl₂) δ 156.53, 147.96, 143.06, 142.16, 132.18 (q, J = 31.8 Hz), 130.49, 124.71 (q, J = 272.2 Hz), 119.86, 116.82, 115.19, 114.31, 113.95 (q, J = 3.9 Hz), 110.57 (q, J = 3.8 Hz). HRMS (GC/EI+): m/z calculated 253.0709 for C₁₃H₁₀F₃NO, found 253.0708 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.6%.

3-(Naphthalen-2-yl)-5-(trifluoromethyl)aniline (68)

Preparation according to general procedure A from 3-naphthaleneboronic acid (SM65, 95 mg, 0.55 mmol, 1.1 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 71 µL, 0.50 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (103 mg, 71%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 1H), 8.06 – 7.90 (m, 3H), 7.77 (dd, J = 8.5, 1.9 Hz, 1H), 7.61 – 7.49 (m, 2H), 7.26 – 7.21 (m, 1H), 7.19 – 7.15 (m, 1H), 6.93 – 6.87 (m, 1H), 5.74 (s, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 150.05, 141.81, 136.92, 133.23, 132.44, 130.54 (q, J = 30.8 Hz), 128.53, 128.25, 127.49, 126.50, 126.31, 125.36, 124.91, 124.55 (q, J = 272.2 Hz), 115.57, 110.22 (q, J = 4.1 Hz), 108.73 (q, J = 3.9 Hz). HRMS (GC/EI+): m/z calculated 287.0916 for C₁₇H₁₂F₃N, found 287.0920 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.0%.

3-(Naphthalen-1-yl)-5-(trifluoromethyl)aniline (69)

Preparation according to general procedure A from 81 (146 mg, 0.51 mmol, 1.0 eq.) and 1-bromonapthalene (SM32, 85 µL, 0.61 mmol, 1.2 eq.). Further purification by RP column chromatography yielded a colorless oily solid (79 mg, 54%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.02 – 7.98 (m, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.82 – 7.77 (m, 1H), 7.60 – 7.49 (m, 3H), 7.43 (dd, J = 7.0, 1.3 Hz, 1H), 6.98 – 6.93 (m, 1H), 6.92 – 6.88 (m, 1H), 6.83 – 6.78 (m, 1H), 5.76 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.57, 141.64, 138.83, 133.37, 130.64, 129.97 (q, J = 30.8 Hz), 128.40, 127.92, 126.59, 126.50, 126.00, 125.53, 125.04, 124.50 (q, J = 273.7 Hz), 118.42, 112.69 (q, J = 4.1 Hz), 108.63 (q, J = 3.6 Hz). HRMS (GC/EI+): m/z calculated 287.0916 for C₁₇H₁₂F₃N, found 287.0920 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.5%.

3-(5-Methylthiophen-2-yl)-5-(trifluoromethyl)aniline (70)

Preparation according to general procedure A from 5-methylthiphene-2-boronic acid (SM66, 71 mg, 0.5 mmol, 1.0 eq.) and 3-bromo-5-(trifluoromethyl)aniline (SM1, 74 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a pale-yellow solid (18 mg, 14%). ¹H NMR (400 MHz, MeOD) δ 7.16 (d, J = 3.6 Hz, 1H), 7.08 – 7.06 (m, 1H), 7.03 – 7.00 (m, 1H), 6.83 – 6.78 (m, 1H), 6.76 – 6.74 (m, 1H), 2.49 (s, 3H). ¹³C NMR (101 MHz, MeOD-d₄) δ 150.64, 142.12, 141.13, 137.61, 132.99 (q, J = 31.4 Hz), 127.43, 127.90 – 124.08 (m), 124.70, 115.30, 111.39 – 111.17 (m), 110.68 – 110.40 (m), 15.25. HRMS (GC/EI+): m/z calculated 257.0481 for C₁₂H₁₀F₃NS, found 257.0484 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 98.0%.

3-(5-Methylthiophen-3-yl)-5-(trifluoromethyl)aniline (71)

Preparation according to general procedure A from 81 (147 mg, 0.51 mmol, 1.0 eq.) and 4-bromo-2-methylthiophene (SM33, 86 µL, 0.77 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless solid (57 mg, 44%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.59 (d, J = 1.6 Hz, 1H), 7.18 (s, 1H), 7.16 – 7.10 (m, 2H), 6.85 (s, 1H), 5.52 (s, 2H), 2.48 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 147.53, 140.40, 140.06, 137.05, 130.45 (q, J = 30.9 Hz), 124.42, 124.37 (q, J = 272.1 Hz), 119.73, 115.78, 111.18, 109.73, 15.04. HRMS (GC/EI+): m/z calculated 257.0481 for C₁₂H₁₀F₃NS, found 257.0481 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.0%.

3-(3,5-Dimethylthiophen-2-yl)-5-(trifluoromethyl)aniline (72)

Preparation according to general procedure A from 81 (144 mg, 0.5 mmol, 1.0 eq.) and 2-bromo-3,5-dimethyl thiophene (SM34, 149 mg, 101 µL, 0.75 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless oily solid (103 mg, 76%). ¹H NMR (400 MHz, DMSO-d₆) δ 6.89 – 6.83 (m, 1H), 6.81 – 6.76 (m, 1H), 6.76 – 6.70 (m, 1H), 6.67 (s, 1H), 5.72 (s, 2H), 2.40 (s, 3H), 2.20 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.81, 137.44, 135.82, 133.53, 133.21, 130.15 (q, J = 31.1 Hz), 130.08, 128.46 – 120.11 (m), 116.45, 111.19 – 110.91 (m), 108.29 – 107.99 (m), 14.86, 14.79. HRMS (GC/EI+): m/z calculated 271.0637 for C₁₃H₁₂F₃NS, found 271.0641 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.8%.

1-(5-(3-Amino-5-(trifluoromethyl)phenyl)thiophen-2-yl)ethan-1-one (73)

Preparation according to general procedure A from 81 (150 mg, 0.52 mmol, 1.0 eq.) and 2-acetyl-5-bromothiophene (SM35, 66 µL, 0.53 mmol, 1.1 eq.). Further purification by RP column chromatography yielded a colorless solid (70 mg, 49%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, J = 3.9 Hz, 1H), 7.64 (d, J = 4.0 Hz, 1H), 7.15 (s, 2H), 6.88 (s, 1H), 5.83 (s, 2H), 2.54 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 190.68, 150.44, 150.23, 142.99, 135.05, 134.26, 130.85 (q, J = 31.4 Hz), 125.59, 124.17 (q, J = 272.4 Hz), 114.01, 110.16 – 109.87 (m), 109.03 – 108.75 (m), 26.39. HRMS (GC/EI+): m/z calculated 285.0430 for C₁₃H₁₀F₃NOS, found 285.0433 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.5%.

1-(4-(3-Amino-5-(trifluoromethyl)phenyl)thiophen-2-yl)ethan-1-one (74)

Preparation according to general procedure A from 81 (144 mg, 0.5 mmol, 1.0 eq.) and 2-acetyl-4-bromo thiophene (SM36, 159 mg, 99 µL, 0.75 mmol, 1.5 eq.). Further purification by flash column yielded a yellowish solid (136 mg, 96%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.33 (d, J = 1.5 Hz, 1H), 8.26 (d, J = 1.5 Hz, 1H), 7.22 – 7.18 (m, 1H), 7.18 – 7.14 (m, 1H), 6.87 – 6.81 (m, 1H), 5.67 (s, 2H), 2.61 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 191.08, 149.93, 144.77, 141.79, 135.81, 132.34, 130.56 (q, J = 31.1 Hz), 129.88, 128.78 – 120.10 (m), 114.41, 109.87 – 109.45 (m), 109.15 – 108.81 (m), 26.70. HRMS (GC/EI+): m/z calculated 285.0430 for C₁₃H₁₀F₃NOS, found 285.0431 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.7%.

Methyl 5-(3-amino-5-(trifluoromethyl)phenyl)thiophene-2-carboxylate (75)

Preparation according to general procedure A from 81 (144 mg, 0.5 mmol, 1.0 eq.) and methyl 5-bromothiophene 2-carboxylate (SM37, 167 mg, 100 µL, 0.75 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless solid (112 mg, 74%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.79 (d, J = 4.0 Hz, 1H), 7.59 (d, J = 4.0 Hz, 1H), 7.15 – 7.10 (m, 2H), 6.91 – 6.85 (m, 1H), 5.83 (s, 2H), 3.84 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.68, 150.24, 149.47, 134.77, 134.09, 131.55, 130.88 (q, J = 31.0 Hz), 125.26, 128.31 – 119.62 (m), 113.95, 110.19 – 109.76 (m), 109.09 – 108.73 (m), 52.35. HRMS (GC/EI+): m/z calculated 301.0379 for C₁₃H₁₀F₃NO₂S, found 301.0383 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.7%.

5-(3-Amino-5-(trifluoromethyl)phenyl)thiophene-2-carboxamide (76)

Preparation according to general procedure A from 81 (149 mg, 0.52 mmol, 1.1 eq.) and 5-bromo-2-thiophenecarboxamide (SM38, 100 mg, 0.47 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (84 mg, 63%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (s, 1H), 7.73 (d, J = 3.9 Hz, 1H), 7.51 (d, J = 3.9 Hz, 1H), 7.45 (s, 1H), 7.08 (s, 2H), 6.84 (s, 1H), 5.79 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.66, 150.17, 146.75, 139.57, 134.74, 130.76 (q, J = 31.1 Hz), 129.74, 124.89, 124.25 (q, J = 272.4 Hz), 113.80, 109.79 – 109.23 (m), 108.93 – 108.39 (m). HRMS (GC/EI+): m/z calculated 286.0382 for C₁₂H₉F₃N₂OS, found 286.0384 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 97.0%.

N-(5-(3-Amino-5-(trifluoromethyl)phenyl)thiophen-2-yl)acetamide (77)

Preparation according to general procedure A from 81 (160 mg, 0.56 mmol, 1.2 eq.) and N-(5-bromothiophen-2-yl)acetamide (SM39, 110 mg, 0.48 mmol, 1.0 eq.). Further purification by RP column chromatography yielded a colorless solid (62 mg, 43%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.23 (d, J = 4.0 Hz, 1H), 7.01 – 6.98 (m, 1H), 6.97 – 6.94 (m, 1H), 6.72 – 6.67 (m, 1H), 6.60 (d, J = 4.0 Hz, 1H), 5.66 (s, 2H), 2.08 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.46, 149.97, 139.92, 135.96, 132.11, 130.52 (q, J = 30.9 Hz), 124.42 (q, J = 271.0 Hz), 121.19, 112.71, 111.24, 107.71, 107.68, 22.53. HRMS (GC/EI+): m/z calculated 300.0539 for C₁₃H₁₁F₃N₂OS, found 300.0543 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.9%.

3-(5-Methylthiazol-2-yl)-5-(trifluoromethyl)aniline (78)

Preparation according to general procedure A from 81 (160 mg, 0.56 mmol, 1.0 eq.) and 2-bromo-5-methylthiazole (SM40, 79 µL, 0.75 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless solid (38 mg, 29%). ¹H NMR (400 MHz, MeOD-d₄) δ 7.55 – 7.50 (m, 1H), 7.36 – 7.31 (m, 2H), 6.99 – 6.95 (m, 1H), 2.53 (d, J = 1.2 Hz, 3H). ¹³C NMR (126 MHz, MeOD-d₄) δ 167.36, 149.88, 142.18, 136.66, 136.27, 133.36 (q, J = 32.0 Hz), 125.45 (q, J = 271.6 Hz), 116.51, 113.58 (q, J = 3.9 Hz), 112.52 (q, J = 4.0 Hz), 11.82. HRMS (GC/EI+): m/z calculated 258.0433 for C₁₁H₉F₃N₂S, found 258.0436 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.9%.

3-(4-Methylthiazol-2-yl)-5-(trifluoromethyl)aniline (79)

Preparation according to general procedure A from 81 (144 mg, 0.5 mmol, 1.0 eq.) and 2-bromo-4-methylthiazole (SM41, 135 mg, 82 µL, 0.75 mmol, 1.5 eq.). Further purification by flash column chromatography yielded a colorless solid (82 mg, 64%). ¹H NMR (400 MHz, Acetone-d₆) δ 7.53 – 7.48 (m, 1H), 7.47 – 7.41 (m, 1H), 7.26 – 7.16 (m, 1H), 7.06 – 7.01 (m, 1H), 5.38 (s, 2H), 2.49 – 2.39 (m, 3H). ¹³C NMR (101 MHz, Acetone-d₆) δ 166.73, 154.88, 150.93, 136.40, 133.09 – 131.98 (m), 129.54 – 121.08 (m), 115.51, 115.17, 112.40 – 111.97 (m), 111.28 – 110.82 (m), 17.25. HRMS (GC/EI+): m/z calculated 258.0433 for C₁₁H₉F₃N₂S, found 258.0435 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 95.5%.

3-(Thiazol-2-yl)-5-(trifluoromethyl)aniline (80)

Preparation according to general procedure A from 81 (143 mg, 0.50 mmol, 1.0 eq.) and 2-bromothiazole (SM42, 68 µL, 0.75 mmol, 1.5 eq.). Further purification by RP column chromatography yielded a colorless solid (27 mg, 22%). ¹H NMR (400 MHz, MeOD-d₄) δ 7.87 (d, J = 3.3 Hz, 1H), 7.62 (d, J = 3.3 Hz, 1H), 7.45 – 7.38 (m, 2H), 7.01 – 6.98 (m, 1H). ¹³C NMR (126 MHz, MeOD-d₄) δ 169.25, 151.24, 144.49, 136.05, 133.36 (q, J = 31.9 Hz), 125.53 (q, J = 271.6 Hz), 121.15, 116.15, 113.11 (q, J = 3.8 Hz), 111.88 (q, J = 4.1 Hz). HRMS (GC/EI+): m/z calculated 244.0277 for C₁₀H₇F₃N₂S, found 244.0277 ([M]^•+). qHNMR (400 MHz, DMSO-d₆) purity = 96.2%.

In vitro characterization

Hybrid Reporter Gene Assays

Hybrid reporter gene assays. TLX modulation was determined in a Gal4 hybrid reporter gene assay in HEK293T cells (German Collection of Microorganisms and Cell Culture GmbH, DSMZ) using pFR-Luc (Stratagene, La Jolla, CA, USA; reporter), pRL-SV40 (Promega, Madison, WI, USA; internal control), pECE-SV40-Gal4-VP16⁴⁵ (#71728, Addgene, Watertown, MA), and pFA-CMV-hTLX-LBD³⁶, coding for the hinge region and ligand binding domain of the canonical isoform of human TLX. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), high glucose supplemented with 10% fetal calf serum (FCS), sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C and 5% CO2 and seeded in 96-well plates (3×104 cells/well). After 24 h, medium was changed to Opti-MEM without supplements and cells were transiently transfected using Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Five hours after transfection, cells were incubated with the test compounds in Opti-MEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL) and 0.1% DMSO for 16 h before luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s protocol on a Tecan Spark luminometer (Tecan Deutschland GmbH, Crailsheim, Germany). Firefly luminescence was divided by Renilla luminescence and multiplied by 1000 resulting in relative light units (RLU) to normalize for transfection efficiency and cell growth. Fold activation was obtained by dividing the mean RLU of test compound by the mean RLU of the untreated control. All samples were tested in at least three biologically independent experiments in duplicates. For dose-response curve fitting and calculation of EC₅₀ values and remaining activity (= “bottom”), the equation “[Agonist] vs. response -- Variable slope (four parameters)” was used in GraphPad Prism (version 7.00, GraphPad Software, La Jolla, CA, USA). Selectivity profiling was performed with pFA-CMV-THRα-LBD⁴⁶, pFA-CMV-RARα-LBD⁴⁷, pFA-CMV-PPARγ-LBD⁴⁸, pFA-CMV-RORα-LBD⁴⁹, pFA-CMV-CAR-LBD⁵⁰, pFA-CMV-PXR-LBD⁵⁰, pFA-CMV-LXRα-LBD⁵⁰, pFA-CMV-HNF4α-LBD⁵¹, pFA-CMV-hRXRα-LBD⁵², pFA-CMV-TR2-LBD⁴⁸, pFA-CMV-PNR-LBD⁵³, pFA-CMV-Nur77-LBD⁵⁴, and pFA-CMV-SF1-LBD⁵⁵. The following reference ligands were used: T3 (1 µM, THRα), Tretinoin (1 µM, RARα), Pioglitazone (1 µM, PPARγ), SR1001 (1 µM, revERBα and RORα), SR12813 (1 µM, PXR), CITCO (10 µM, CAR), T0901317 (1 µM, LXRα), Bexarotene (1 µM, RXRα), PR3 (1 µM, PNR) and compound 29 from Vietor et al.⁵⁶ (0.3 µM, Nur77).

DR1 activity assay

The repression of DR1 activity by TLX was determined in a reporter gene assay in transiently transfected HEK293T cells using a firefly luciferase reporter for the human DR1 response element (#1015, Addgene, gift from Bruce Spiegelman)⁵⁷ and pRL-SV40 (Promega) as internal control. To observe TLX effects on DR1 activity, a construct for constitutive expression (CMV promoter) of full-length human TLX³⁶ was co-transfected. Cell culture, seeding, transient transfection, and test compound treatment were performed as described for hybrid reporter gene assays. Cells were treated with 0.1% DMSO as negative control, pioglitazone (1 µM) to induce DR1 activity, or with pioglitazone (1 µM) and a TLX modulator (25, 27, 39 (10 µM each) or 72 (50 µM)). After 16 h incubation, luciferase activity was assayed as described for reporter gene assays, and firefly activity was normalized to renilla activity to obtain RLU. All samples were tested in at least six biologically independent experiments in duplicates.

Affinity Selection Mass Spectrometry (ASMS)

A mixture containing compounds 25, 27 and 39 (1 µM final concentration each) and ccrp2¹⁰ (1 µM) as reference, was incubated (at 4 °C for 1h) with TLX LBD protein (35 µM) in buffer (50 mM Tris, 200 mM NaCl, pH 7.4) containing 1% (v/v) DMSO in a total volume of 55 µL (N=9). Compounds 65 and 72 were tested at 10 µM for sufficient detection with 50 µM TLX LBD to maintain excess of the protein. In parallel, this procedure was performed for denatured (82 °C, 30 min) TLX LBD (35 µM or 50 µM) under otherwise identical conditions (N=3). After equilibration of the PD SpinTrap G-25 columns containing Sephadex G-25 medium (Cytiva, Little Chalfont, United Kingdom) according to the manufacturer’s instructions, 50 µL of each binding sample were transferred onto an SEC column followed by centrifugation (800 g, 2 min) to generate the eluates E1. In the same way the pure compound mixture (1 µM in buffer with 1% DMSO) without TLX incubation was subjected to SEC as further negative control (N=3). Next, aliquots of 40 µL of each E1 eluate were diluted to 400 µL with 0.1% HCOOH in H₂O and 0.1% HCOOH in MeCN (90:10, v/v) containing 100 nM Terfenadine as internal standard for quantification by LC-MS. In the same way samples containing 10 nM test compounds and 100 nM Terfenadine, as well as blanks, were generated by dilution with 40 µL of pure buffer as LC-ESI-MS/MS control. Subsequently, the generated samples were sonicated for 5 min, vortexed and finally centrifuged (15.000 g, 5 min). After transfer of the samples to a 96 deep well plate LC-ESI-MS/MS was performed with an API5500 QTrap mass spectrometer (Sciex, Darmstadt, Germany) coupled to an Agilent 1260 infinity HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a SIL-20AHT autosampler (Shimadzu, Duisburg, Germany) and a rack changer II (Shimadzu, Duisburg, Germany) controlled by the Analyst software 1.6.3. For chromatography, a Triart C18 column (3 µm, 50 mm x 2 mm, YMC-Europe, protected with a 0.5 µm and 0.2 µm frit) as stationary phase in combination with 0.1 % (v/v) HCOOH in H₂O (A) and 0.1 % (v/v) HCOOH in MeCN (B) as mobile phase under gradient elution (0-2 min: 90%A/10%B, 2-4 min: 90%A/10%B → 5%A/95%B, 4-6 min: 5%A/95%B, 6-7 min: 5%A/95%B → 90%A/10%B, 7-10 min: 90%A/10%B) was used at a flow rate of 400 µL/min, injecting 50 µL per sample. MS data acquisition was performed under positive ESI conditions in the MRM mode recording 25 at m/z 267 → 250, 27 at m/z 281 → 263, 39 at m/z 292 → 252, 65 at m/z 268 → 139, 72 at m/z 272 → 255 and Terfenadine at m/z 472 → 436 from 4.7 to 7.0 min. Quantification of the test compounds was based on area ratios of analyte vs internal standard.

Differential Scanning Fluorimetry (DSF)

2 µM of recombinant TLX LBD protein in buffer (15 mM HEPES pH 7.5, 150 mM NaCl) supplemented with SYPRO orange dye (1:1000 dilution) and compounds 25, 27, 39, 65 and 72 (300 µM, solubilized with DMSO at a final concentration of 2%) or DMSO (2%) were added to a white 96-well PCR plate to a final volume of 20 µL. The experiment was conducted on a QuantStudio 1 PCR system (Applied Biosystems, Waltham, CA, USA) heating from 25°C to 90°C with a heating rate of 0.015°C s^-1. ccrp2¹⁰ (100 µM) served as reference ligand. Each sample was tested in at least three replicates. Amplification plots were analyzed by using a Boltzmann sigmoidal fit in GraphPad Prism (version 7.00, GraphPad Software, La Jolla, CA, USA) to obtain melting points (T_m). Δ Tm corresponds to Δ Tm = Tm (TLX LBD with compound) − Tm (TLX LBD untreated). Statistical significance was evaluated with a one-way ANOVA with Dunnett’s multiple comparisons test in GraphPad Prism (version 7.00, GraphPad Software, La Jolla, CA, USA).

Determination of Thermodynamic Aqueous Solubility

The aqueous solubility of 25, 27, 39, 65 and 72 was assessed by mixing 0.5-1 mg of each test compound with an appropriate volume of water for a theoretical concentration of 2 mM to obtain an oversaturated mixture. The mixture was agitated in a VWR Thermal Shake lite (VWR International GmbH, Darmstadt, Germany) for 48 h at 600 rpm and a constant temperature of 25 °C. The supersaturated mixtures were subsequently centrifuged at 16.400 g for 15 min (25 °C). Part of the supernatant was taken off for quantification by ultraviolet (UV) absorbance at 254 nm (for 39, 65 and 72) or 260 nm (for 25 and 27) with external calibration. The external calibration samples contained 1% DMSO and the test samples were spiked with DMSO to 1% concentration right before the measurement. Absorbance was measured with a Tecan Spark luminometer using a Quartz 96-well plate (Tecan Deutschland GmbH, Crailsheim, Germany). The solubility test was repeated in three independent experiments.

Isothermal titration calorimetry (ITC)

ITC experiments were conducted on an Affinity ITC instrument (TA Instruments, New Castle, DE) at 25 °C with a stirring rate of 75 rpm. TLX LBD protein (60 µM) in buffer (20 mM Tris pH 7.5, 150 mM NaCl, mM TCEP, 5% glycerol) containing 3% DMSO was titrated with compound 39 (300 μM in the same buffer containing 3% DMSO) in 21 injections (1x 1 µL, 20x 4 μL) with an injection interval of 120 s. As control experiments, the compound was titrated to the buffer, and the buffer was titrated to the TLX LBD protein under otherwise identical conditions. The heats of the compound-TLX LBD titrations were corrected using the heats of the compound-buffer titrations. Results were analyzed using NanoAnalyze software (version 3.11.0, TA Instruments, New Castle, DE) with independent binding model.

Supplementary Material

Supporting info.

EMS212277-supplement-Supporting_info_.pdf^{(12.5MB, pdf)}

Acknowledgements

This research was funded by the European Union (ERC, NeuRoPROBE, 101040355). Views and opinions expressed are however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. pECE-SV40-Gal4-VP16 was a gift from Lea Sistonen (Addgene plasmid #71728).

Abbreviations Used

ASMS: affinity-selection-mass-spectrometry
DSF: differential scanning fluorimetry
ITC: isothermal titration calorimetry
LBD: ligand binding domain
LE: ligand efficiency
LLE: lipophilic ligand efficiency
LSD1: lysine-specific demethylase 1
NP: neural progenitors
NR: nuclear receptor
NSC: neural stem cells
pten: phosphatase and tensin homologue
ROR: retinoic acid receptor-related orphan receptor
RXR: retinoic X receptor
SEC: size-exclusion chromatography
TLX: tailless homologue.

Footnotes

Notes

The authors declare a competing interest: E.H. and D.M. are inventors of PCT/EP2025/063165 claiming compounds described in this study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting info.

EMS212277-supplement-Supporting_info_.pdf^{(12.5MB, pdf)}

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PERMALINK

Systematic optimization of fragment TLX ligands towards agonism and inverse agonism

Emily C Hank

Loris Knümann

Úrsula López-García

Arthur Kardanov

Vasily Morozov

Georg Höfner

Daniel Merk

Abstract

Introduction

Results & Discussion

Table 1. Impact of modifications on the methylimidazole.

Table 2. Replacement of the methylimidazole.

Table 3. SAR of bicyclic motifs.

Table 4. SAR of the trifluoromethylaniline core.

Table 5. Preliminary SAR of inverse TLX agonists.

Table 6. Hetero- and bicyclic motifs in inverse agonists.

Figure 1. Summarized SAR for fragment TLX modulators.

Table 7. Features of the most favored fragment TLX agonists (25, 27, 39) and inverse agonists (65, 72).

Figure 2. Validation of direct binding of 25, 27, 39, 65 and 72 to the recombinant TLX LBD.

Figure 3.

Figure 4. Effects of the TLX agonists 25, 27, 39 and the inverse agonist 72 on DR1 activity in HEK293T cells in absence and presence of TLX.

Conclusion

Chemistry

Scheme 1. Synthesis of 6-41 and 53-80.a.

Scheme 2. Synthesis of 42-52.a.

Experimental Procedures

Chemistry

General

General procedure A (Suzuki-coupling)

General procedure B (Ullmann coupling)

3-(1H-Imidazol-1-yl)-5-(trifluoromethyl)aniline (6)

3-(1H-Pyrrol-1-yl)-5-(trifluoromethyl)aniline (7)

3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)aniline (81)

3-(2-Methyl-1H-imidazol-5-yl)-5-(trifluoromethyl)aniline (8)

3-(1,2-Dimethyl-1H-imidazol-5-yl)-5-(trifluoromethyl)aniline (9)

3-(1,2-Dimethyl-1H-imidazol-4-yl)-5-(trifluoromethyl)aniline (10)

3-(1-Methyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)aniline (11)

3-(1-Methyl-1H-pyrazol-3-yl)-5-(trifluoromethyl)aniline (12)

3-(4-Methyl-1H-pyrazol-1-yl)-5-(trifluoromethyl)aniline (13)

3-(5-Methyl-1H-pyrazol-3-yl)-5-(trifluoromethyl)aniline (14)

3-(1,5-Dimethyl-1H-pyrazol-3-yl)-5-(trifluoromethyl)aniline (15)

3-(1H-Pyrazol-4-yl)-5-(trifluoromethyl)aniline (16)

3-(1,5-Dimethyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)aniline (17)

3-(1H-Benzo[d]imidazol-1-yl)-5-(trifluoromethyl)aniline (18)

3-(1H-Indol-1-yl)-5-(trifluoromethyl)aniline (19)

3-(1-Methyl-1H-indol-3-yl)-5-(trifluoromethyl)aniline (20)

3-(2-Methylpyridin-4-yl)-5-(trifluoromethyl)aniline (21)

3-(6-Methylpyridin-3-yl)-5-(trifluoromethyl)aniline (22)

3-(Pyridin-4-yl)-5-(trifluoromethyl)aniline (23)

3-(Pyridin-3-yl)-5-(trifluoromethyl)aniline (24)

3-(2,6-Dimethylpyridin-4-yl)-5-(trifluoromethyl)aniline (25)

3-(5,6-Dimethylpyridin-3-yl)-5-(trifluoromethyl)aniline (26)

1-(4-(3-Amino-5-(trifluoromethyl)phenyl)pyridin-2-yl)ethan-1-one (27)

1-(5-(3-Amino-5-(trifluoromethyl)phenyl)pyridin-3-yl)ethan-1-one (28)

3-(Pyrimidin-5-yl)-5-(trifluoromethyl)aniline (29)

3’,5’-Dimethyl-5-(trifluoromethyl)-[1,1’-biphenyl]-3-amine (30)

3-(1-Methyl-1H-indol-4-yl)-5-(trifluoromethyl)aniline (31)

3-(1-Methyl-1H-indol-5-yl)-5-(trifluoromethyl)aniline (32)

3-(1H-Indol-4-yl)-5-(trifluoromethyl)aniline (33)

3-(1H-Indol-5-yl)-5-(trifluoromethyl)aniline (34)

3-(Indolin-5-yl)-5-(trifluoromethyl)aniline (35)

3-(Benzofuran-5-yl)-5-(trifluoromethyl)aniline (36)

3-(Benzo[b]thiophen-5-yl)-5-(trifluoromethyl)aniline (37)

3-(Imidazo[1,2-a]pyridin-7-yl)-5-(trifluoromethyl)aniline (38)

3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-5-(trifluoromethyl)aniline (39)

3-(1-Methyl-1H-indazol-5-yl)-5-(trifluoromethyl)aniline (40)

3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-(trifluoromethyl)aniline (41)

3-(2-Methylpyridin-4-yl)aniline (42)

3-(2-Methylpyridin-4-yl)-4-(trifluoromethyl)aniline (43)

3-Methyl-5-(2-methylpyridin-4-yl)aniline (44)

3-Methoxy-5-(2-methylpyridin-4-yl)aniline (45)

3-(2-Methylpyridin-4-yl)-5-(trifluoromethoxy)aniline (46)

3-Amino-5-(2-methylpyridin-4-yl)benzonitrile (47)

3-Chloro-5-(2-methylpyridin-4-yl)aniline (48)

3,4-Dichloro-5-(2-methylpyridin-4-yl)aniline (49)

4-(2-Methylpyridin-4-yl)-1H-indol-6-amine (50)

6-(2-Methylpyridin-4-yl)-1H-indol-4-amine (51)

4-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indol-6-amine (52)

Scheme 1. Synthesis of 6-41 and 53-80.^a.

Scheme 2. Synthesis of 42-52.^a.

N³,N³-Dimethyl-5’-(trifluoromethyl)-[1,1’-biphenyl]-3,3’-diamine (66)