Structure-activity relationship and biological investigation of SR18292 (16), a suppressor of glucagon-induced glucose production

Abstract

Despite a myriad of available pharmacotherapies for the treatment of Type-2 diabetes (T2D), challenges still exist in achieving glycemic control. Several novel glucose-lowering strategies are currently under clinical investigation highlighting the need for more robust treatments. Previously, we have shown that suppressing PGC1α activity with a small molecule (SR18292, 16) can reduce glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Despite structural similarities in 16 to known β-blockers, detailed SAR studies described herein have led to the identification of analogs lacking β-adrenergic activity that still maintain the ability to suppress glucagon-induced glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Hence, these compounds exert their biological effects in a mechanism that does not include adrenergic signaling. These probe molecules may lead to a new therapeutic approach to treat T2D either as a single agent or in combination therapy.

Graphical Abstract

INTRODUCTION

The epidemic prevalence of Type 2 Diabetes requires the development of new anti-diabetic drugs to ameliorate hyperglycemia. Targeting the liver is an attractive approach, as uncontrolled hepatic glucose production (HGP) is a main contributor to the hyperglycemia observed in T2D and is a result of the reduced ability of insulin to suppress HGP^1–3. Commonly used anti-diabetic drugs currently include metformin, sulfonylureas, thiazolidinediones (TZDs), incretin mimetics, DPP4 antagonists⁴, and sodium-glucose cotransporter 2 (SGLT2) inhibitors⁵, where each drug targets a different regulatory component of glucose homeostasis. Importantly, several studies have demonstrated that increased hepatic gluconeogenesis, rather than glycogenolysis, is the primary reason for the elevated HGP, and the subsequent hyperglycemia, in T2D patients^{6, 7}. Thus, targeting components within the HGP process, and specifically gluconeogenesis, is considered a useful way to normalize blood glucose concentrations. Accordingly, the first line drug for T2D treatment is the biguanide metformin, which reduces blood glucose concentration primarily by suppressing gluconeogenesis and HGP^8–10. This highlights the possibility that new drugs that will also target gluconeogenic components might also serve as anti-diabetic agents.

The transcription coactivator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) has been shown to significantly control hepatic gluconeogenesis by promoting expression of critical enzymes in the gluconeogenic pathway^11–13. Modulating the acetylation status of PGC-1α can potently affect its gluconeogenic activity. Manipulations that augment PGC-1α lysine acetylation have been shown to inhibit its pro-gluconeogenic activity and reduce HGP, ameliorating diabetic symptoms^14–17. We have previously designed a high throughput AlphaLisa screen to discover small molecules that induce PGC-1α acetylation with the goal that hits from this screen will ultimately suppress HGP¹⁸. We identified a set of small molecules that can induce PGC-1α acetylation, suppress expression of gluconeogenic genes and reduce glucose secretion from cultured primary hepatocytes¹⁸. We further showed that an analog of a single hit from this screen, 16, can potently improve whole body insulin sensitivity. This is achieved by specifically improving the liver’s response to insulin without changing glucose uptake. Although the direct target of 16 is still not known, and its inhibitory effect on PGC-1α is probably indirect, its specificity toward suppression of HGP makes it a promising chemical scaffold that can potentially be used as an anti-diabetic drug.

Here, we performed a structure-activity relationship (SAR) study of 16 in order to dissect in more detail the structural elements required to elicit biological activity. The conclusions from this study will help design probes that can be further used to find a direct target for 16. In addition, 16 contains a pharmacophore similar to that of several β-adrenergic receptor (β-AdR) antagonists and is especially related to pindolol^{19, 20}. However, most commercial β-blockers are secondary amino alcohols, wherein 16 contains a tertiary amine (Figure 1). This likely reduces some of the effects at the β-adrenergic receptor, but not at the molecule’s direct target. We counter-screened 16 in a panel of ~50 GPCR’s, ion channels, and transporters and identified only a few off-target effects²¹. Not surprisingly, 16 had reasonable binding to β-adrenergic receptors (β1 K_i=0.80 μM; β2 K_i = 1.3 μM), but also weak affinity for 5HT1a (K_i=2.1 μM). We show here that the anti-diabetic effects of 16, both in vitro and in vivo, can be uncoupled from its β-AdR antagonist effects, suggesting that 16 improves diabetic symptoms in a mechanism that does not involve inhibition of adrenergic signaling. Moreover, we identified one analog, 38, with excellent bioactivity lacking β-AdR activity that can potentially be used for the potential treatment of T2D. Key modifications are shown in red (Figure 1).

Figure 1.

SAR study of 16 led to 38

RESULTS AND DISCUSSION

Most analogs could be synthesized following the general protocol as outlined in Scheme 1a. Selected analogs were made as described in Schemes 1b and 1c. Reductive amination of commercially available aldehydes 1 with the corresponding amines 2 afforded secondary amines 3.

Scheme 1. Synthesis of 16 analogs.

Reaction conditions: (a) NaBH₄, MeOH, 0°C->rt; (b) K₂CO₃, KI, CH₃CN, 80°C; (c) K₂CO₃, DMF, 130°C; (d) MeI, NaH, DMF, 0°C->rt; (e) 1. BOC₂O, 4-DMAP, CH₃CN, rt; 2. K₂CO₃, MeOH, rt; (f) Toluene, 130°C; (g) Et₂NH, Toluene, 150°C; (h) 1,3-dibromopropane, NaH, DMF, 0°C->rt; (i) i-PrOH, 120°C.

Treatment with 2-(chloromethyl)oxirane 4 in the presence of K₂CO₃ gave tertiary amines 5. Ring opening with phenols or amines provided α-amino alcohol final products (16-31, 33-37). O-alkylation with iodomethane afforded ethers 32 and 38. Boc protection of 4-hydroxyindole 8, followed by O-alkylation led to oxirane intermediate 10. Ring opening by secondary amines 3 gave α-amino alcohols 11, which were methylated and deprotected to provide the desired ether compounds 39-41. Finally, Boc-protected 4-hydroxyindole 9 was O-alkylated to provide bromide 13, which could then be treated with amine 14 to give amine 15. Deprotection of the indole gave 42.

To determine the ability of the 16 analogs to suppress glucose secretion we used isolated mouse primary hepatocytes. Upon fasting, elevated secretion of glucagon from pancreatic α cells stimulates HGP to maintain normal blood glucose concentration when nutrients are limited^{3, 22}. To induce secretion of glucose from cultured primary hepatocytes we stimulated them with glucagon, which mimics the fasting response, and used pyruvate and lactate as substrates for glucagon-induced gluconeogenesis. As previously reported¹⁸, 16 suppresses the glucagon-induced glucose secretion to the media by ~58% when pyruvate and lactate are used as substrates (Table 1, 16). In addition, as predicted by its molecular structure, 16 was also able to suppress lipolysis in cultured adipocytes by ~26%, as measured by glycerol release to the medium, although to a much lesser extent when compared to propranolol, a potent antagonist of β-AdR (Table 1, Pro). While we do not know the direct target of these compounds, the functional phenotypic assays described herein are fully capable of driving SAR towards compounds that suppress glucagon-induced glucose production (SGIGP). Initial studies began with examining the western portion of the molecule. Moving the 4-methyl group in 16 around the ring had little effect on SGIGP or SNIL (17-18). Attempts to replace the 4-methyl group with alternate substituents similarly had little effect on SGIGP (19-22,24), however there was an increasing trend in inhibiting lipolysis relative to the parent (16). A very bulky substituent at the 4-position (23) did not have much effect on SGIGP, however it did seem to reduce effects on lipolysis. Interestingly, a secondary amine (R¹=H, 25) had little effect on glucose production, but the greatest effect on lipolysis inhibition almost rivaling propranolol.

Table 1.

Suppression of glucagon-induced glucose production in hepatocytes and norepinephrine (NE)-induced lipolysis in adipocytes by the different compounds. The difference between glucagon- or NE-stimulated cells and non-stimulated cells is considered as 100% suppression. Data are shown as mean ±SEM, n=3–6/group. For both SGIGP and SNIL compound concentration is 1μM.

We next turned our attention to the other substituent on the nitrogen atom in the linker (Table 2). Decreasing the size of the substituent from t-butyl (16) to isopropyl (35) to cyclopropyl was not beneficial with regards to inhibiting glucagon induced glucose release from hepatocytes. Removing the group altogether (37) reduced activity even further. While 36 had reduced inhibition on lipolysis, it came with reduced activity on glucose release as well.

Table 2.

Suppression of glucagon-induced glucose production and norepinephrine (NE)-induced lipolysis is shown as % suppression of the glucagon/NE effects. The basal non-stimulated state is considered as 100% suppression. Data are shown as mean ±SEM, n=3–6/group. For both SGIGP and SNIL compound concentration is 1μM.

#	R2	SGIGP	SNIL
16	t-Bu	57.7±6.07%	25.8±5.02%
35	i-Pr	59.5±9.31%	51.6±2.38%
36	cyclopropyl	37.8±5.55%	11.6±3.28%
37	H	34.5±3.73%	40.6±0.64%

Modifications to the Eastern portion of the molecule are highlighted in Table 3. Attaching the molecule to the 5-position of the indole ring as in 26 did little more than increase inhibition of lipolysis. Replacement of indole with simple phenyl rings (27, 28) were detrimental to activity in suppressing glucose secretion, and the naphthyl analog (29) completely ablated activity. A pyridine ring substitution (30) was moderately tolerated, as was N-acetylated phenol 31, but neither were as active as 16. N-methyl indole derivative 32 retained similar activity to 16 indicating the NH was not required as a hydrogen bond donor for activity. Attempts to replace the ether link at C4 of the indole with NH were marginally successful (33-34), though analogs were less active than 16.

Table 3.

Suppression of glucagon-induced glucose production and norepinephrine (NE)-induced lipolysis is shown as % suppression of the glucagon/NE effects. The basal, non-stimulated state, is considered as 100% suppression. Data are shown as mean ±SEM, n=3–6/group. For both SGIGP and SNIL compound concentration is 1μM.

During the synthesis of N-methylindole analog 32, the bis-methylated analog 38 could be isolated as a major by-product in the presence of excess methylating reagent (Figure 1). The O-methyl ether retained activity of the parent 16 on inhibiting glucagon stimulated glucose secretion, but nearly completely abolished inhibition of lipolysis. This is perhaps not surprising given the preference for a free amino alcohol pharmacophore for β-adrenergic activity^{23, 24}.

Scatter plot analysis of the data collected to date indicated little to no correlation between the ability of a compound to reduce glucose secretion in hepatocytes and suppress lipolysis in adipocytes (Figure 2). Importantly, the ability of two compounds (38 and 23) to suppress lipolysis was largely lost while still retaining the ability to reduce glucose secretion from hepatocytes, similar to the parent compound (16) (Figure 2).

Figure 2.

The data from Tables 1, 2, 3 presented as a scatter plot correlation between SNIL and SGIGP.

A more detailed analysis of the β-AdR antagonistic effect of 16 clearly shows that it is a weak antagonist compared to a classical β-AdR antagonist like propranolol (Figure 3A). In accordance with this, 16 does not reduce the phosphorylation of hormone-sensitive lipase (HSL) in fat tissue isolated from fasted mice that have been treated with 16 (Figure 3B). Phosphorylation of HSL is the major molecular pathway by which adrenergic signaling promotes lipolysis from adipose tissue²⁵ and the lack of change in HSL phosphorylation supports the idea that 16 does not act as a β-AdR antagonist in vivo. Moreover, inhibition of adrenergic signaling in the liver is expected to result in inhibition of glycogenolysis and accumulation of liver glycogen²⁶. While their blood glucose concentration is significantly lower, fasted mice that have been treated with 16 do not show increased accumulation of liver glycogen compared to vehicle-treated mice (Figures 3C and D), providing further support that 16 does not act as a β-AdR antagonist in vivo.

Figure 3.

(A) Fully differentiated cultured brown adipocytes were treated with either 16 or propranolol at the indicated dose for 30 min followed by norepinephrine (NE) stimulation (1 μM) for 90 min. Media was collected and glycerol levels were measured. For each dose n=3;***, P<0.001; two-way ANOVA (B) HSL phosphorylation level is not altered in epidydimal white adipose tissue collected from mice fed HFD for 2 months and treated with 16 (50mg/kg). (C) Fasting Blood glucose (6 hrs fast) and (D) hepatic glycogen levels in HFD mice treated with 16. n=4/5, Vehicle/16; **, P<0.01; two tailed t-test.

To better compare the β-AdR antagonistic effect of 16 and its analogs 38 and 23 we generated a dose response curve and showed that 38 and 23 lose β-AdR antagonistic activity in cultured adipocytes in a wide range of concentrations (Figure 4A). Over expression of PGC-1α in hepatocytes is sufficient to promote glucose release (Fig. 4B), even without glucagon stimulation, highlighting its important contribution to this process. 38 was able to inhibit the PGC-1α-driven glucose release, similar to 16, implying that both analogs inhibit glucose release through a mechanism that involves inhibition of PGC-1α activity. Importantly, 38 and 23 retain their ability to reduce fasting blood glucose in diabetic mice (Figure 4C) providing additional evidence that the β-AdR antagonistic effect of 16 is uncoupled from its anti-diabetic effects. Moreover, like 16, mice that have been treated with 23 do not show reduced phosphorylation of HSL in fat tissue or accumulation of liver glycogen (Figure 4D and 4E) which is consistent with no β-AdR antagonism in vivo.

Figure 4.

(A) Fully differentiated cultured brown adipocytes were treated with 16, 23 or 38 at the indicated dose for 30 min followed by Norepinephrine (NE) stimulation (1 μM) for 90 min. Media was collected and glycerol levels were measured. For each dose n=3; **, P<0.01, ***, P<0.001; two-way ANOVA (B) Overexpression of PGC-1α using adenoviral vectors promotes glucose release from primary hepatocytes. 16 and 38 (10μM) are able to inhibit the PGC-1α-driven glucose release . (C) fasting blood glucose (overnight fast) of ob/ob mice treated with 16, 38 or 23 (25mg/kg). (D) Liver glycogen levels of ob/ob mice treated with 16 or 23 (25mg/kg); *, P<0.05, **, P<0.01, one-way ANOVA (E) HSL phosphorylation is not altered in brown adipose tissue of ob/ob mice treated with 16 or 23 (25mg/kg)

Following up on 38, we were curious to characterize the simple O-methyl ether analog of 16 given that N-methylation of the indole was not productive (32, Table 3). Surprisingly, this compound had more than 2-fold improvement in suppressing glucagon stimulated glucose production relative to 38 as well as 16, while retaining little to no activity on inhibiting lipolysis (39, Table 4). Further investigation of the Western portion of the molecule exhibited similar SAR as in the 16 series with the best substitution as a cyclopentylmethyl group (41) exhibiting a similar suppression of SGIGP relative to 16 with no effect on lipolysis. The analog lacking the O-methyl ether (42) altogether was considerably less potent emphasizing the importance of this substituent for activity. Nonetheless, analogs like 38 and 41 highlight the ability to completely dissociate the ability to suppress glucose secretion from β-adrenergic activity.

Table 4.

Suppression of glucagon-induced glucose production and norepinephrine (NE)-induced lipolysis is shown as % suppression of the glucagon/NE effects. The basal, non-stimulated state, is considered as 100% suppression. Data are shown as mean ±SEM, n=3–6/group. For both SGIGP and SNIL compound concentration is 1μM.

#	Compound	SGIGP	SNIL
Pro	-	-	76.4±5.71%
16		57.7±6.07%	25.8±5.02%
38		56.8±5.26%	3.22±1.12%
39		135±9.90%	−8.50±5.83%
40		22.7±3.26%	14.6±3.77%
41		66.4±2.50%	1.00±1.64%
42		28.3±4.05%	9.91±6.97%

CONCLUSION

Here we describe the synthesis and structure-activity relationship of 16 and its analogs. Starting with a weak β-adrenergic receptor scaffold, we were able to modify specific portions of the molecule to optimize anti-gluconeogenic potential as well as minimize β-adrenergic antagonist activity as measured via lipolysis both in vitro and in vivo. Reducing the β-adrenergic activity of 16 is important, as β-blockers are commonly used to treat hypertension and related heart problems, which can complicate the therapeutic usage of this small molecule. Ablation of β-adrenergic activity was accomplished by O-methylation of the secondary alcohol, a known requirement for β-blocking efficacy. Our exploration uncoupled the anti-gluconeogenic effect of 16 from its β-AdR blocking effect and generated useful probes that can be further used to understand 16’s mechanism of action. These studies are currently underway now that selective inhibitors like 38 have been identified and will be reported in due course.

EXPERIMENTAL SECTION

Chemistry.

All solvents and chemicals were reagent grade. Unless otherwise mentioned, all reagents and solvents were purchased from commercial vendors and used as received. Flash column chromatography was carried out on a Teledyne ISCO CombiFlash Rf system using prepacked columns. Solvents used include hexane, ethyl acetate (EtOAc), dichloromethane and methanol. Purity and characterization of compounds were established by a combination of HPLC, TLC, mass spectrometry, and NMR analyses. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance DPX-400 (400 MHz), a Bruker Ultrashield 500 Plus and Avance III 600 (600 MHz) spectrometer and were determined in chloroform-d or DMSO-d⁶ with solvent peaks as the internal reference. Chemical shifts are reported in ppm relative to the reference signal, and coupling constant (J) values are reported in hertz (Hz). Thin layer chromatography (TLC) was performed on EMD precoated silica gel 60 F254 plates, and spots were visualized with UV light or iodine staining. Low resolution mass spectra were obtained using a Thermo Scientific ultimate 3000/ LCQ Fleet system (ESI). High resolution mass spectra were obtained using a Thermo Scientific EXACTIVE system (ESI). All compounds containing a stereogenic center are racemic. All test compounds were greater than 95% pure as determined by Agilent 1100 series HPLC using a Supelco Discovery HS C18 10cm × 2.1mm, 5μm column or Agilent 1260 Infinity II using an Agilent ZORBAX SB C18 250mm × 4.6 mm, 5 μm column.

General Procedure for the synthesis of 3a-3d, 3f-3k:

To a room temperature solution of aldehyde 1 (1 eq.) in MeOH was added amine 2 (1.2 eq.). The solution was stirred at room temperature for 1 hour, cooled to 0 °C, and then treated with NaBH₄ (1.5 eq.) in one portion. The reaction mixture was allowed to warm to room temperature overnight with stirring. The reaction was quenched with water, then was concentrated. The residue was dissolved in HCl (1N), washed with Et₂O. The water phase was basified with NaOH (1N) until pH>10, extracted with DCM, washed with brine, dried (Na₂SO₄) and concentrated in vacuo to give the desired secondary amine 3 which was used for the next step without any further purification.

General Procedure for the synthesis of 5a-k:

To a solution of amine 3 (1 eq.) in acetonitrile was added 2-(chloromethyl)oxirane 4 (3 eq.), K₂CO₃ (3 eq.) and KI (3 eq.). The mixture was heated to 80 °C for 16 h, cooled, and filtered, washing with ethyl acetate. The filtrate was concentrated in vacuo and purified by flash chromatography on silica gel (EtOAc/PE) to afford amine 5 as colorless oil.

2-Methyl-N-(4-methylbenzyl)-N-(oxiran-2-ylmethyl)propan-2-amine (5a), colorless oil, Rf = 0.3 (PE:EA=10:1). ¹H NMR (600 MHz, CDCl₃) δ 1.17 (s, 9H), 2.33 (s, 3H), 3.60 (d, J = 14.8 Hz, 1H), 3.83 (d, J = 14.8 Hz, 1H), 7.09 (t, J = 3.0 Hz, 1H), 7.10 (d, J = 7.8 Hz, 2H), 7.28 (d, J = 7.9 Hz, 2H). ¹³C NMR (600 MHz, CDCl₃) δ139.51, 136.04, 128.86, 128.17, 54.87, 54.26, 52.83, 52.45, 47.37, 27.53, 21.19. ESI (M+H)⁺ = 234.

2-Methyl-N-(3-methylbenzyl)-N-(oxiran-2-ylmethyl)propan-2-amine (5b), colorless oil, Rf = 0.3 (PE:EA=10:1). ¹H NMR (600 MHz, CDCl₃) δ 7.23 – 7.18 (m, 1H), 7.04 (d, J = 7.0 Hz, 1H), 3.87 (d, J = 14.8 Hz, 1H), 3.62 (d, J = 14.7 Hz, 1H), 2.91 (dd, J = 14.5, 4.8 Hz, 1H), 2.81 – 2.77 (m, 1H), 2.51 – 2.47 (m, 1H), 2.36 (s, 1H), 2.13 (dd, J = 4.9, 2.7 Hz, 1H), 1.20 (s, 3H). ¹³C NMR (126 MHz, CDCl₃ ) δ 142.47, 137.60, 128.92, 128.03, 127.30, 125.36, 54.85, 54.47, 52.79, 52.36, 47.31, 27.49, 21.54. ESI (M+H)⁺ = 234.

General Procedure for the synthesis of 16–24, 26–31, 33–36:

To a solution of amine 5 (1 eq.) in DMF was added phenol or amine 6 (3 eq.) and K₂CO₃ (3 eq.). The mixture was heated to 130 °C for 12 h and then cooled. The reaction mixture was diluted with ethyl acetate, washed with water. Then the organic phase was concentrated and dissolved in DCM, washed with NaOH (1N), brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM:EA) or by reverse-phase preparative HPLC to give the desired product 16–24, 26–31, 33–36.

1-((1H-Indol-4-yl)oxy)-3-(tert-butyl(4-methylbe zyl)amino)propan-2-ol (16), off-white solid, Rf = 0.3 (DCM:EA = 3:1). ¹H NMR (600 MHz, CDCl₃) δ 8.23 (s, 1H), 7.27 (d, J = 9.5 Hz, 2H), 7.15 (d, J = 9.3 Hz, 2H), 7.10 (t, J = 3.6 Hz, 1H), 7.08 (t, J = 9.4 Hz, 1H), 7.01 (d, J = 9.8 Hz, 1H), 6.64 (td, J = 2.6, 0.9 Hz,1H), 6.42 (d, J = 9.1 Hz, 1H), 3.96 (dd, J = 11.5, 6.1Hz, 1H), 3.93 (dd, J = 11.5, 6.1Hz, 1H), 3.88 (d, J = 17.2 Hz, 1H), 3.64 (dd, J = 16.7, 6.2 Hz, 1H), 3.62 (d, J = 17.4 Hz, 1H), 2.92 (dd, J = 16.4, 10.6 Hz, 1H), 2.85 (dd, J =16.4, 6.0 Hz, 1H), 2.36(s, 3H),1.24 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 152.67, 138.90, 137.40, 136.59, 129.33, 128.39, 122.80, 122.63, 118.85, 104.62, 100.78, 100.06, 70.43, 67.54, 55.86, 55.59, 54.11, 27.60, 21.23. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₃H₃₁N₂O₂⁺ 367.2380; found 367.2398.

1-((1H-Indol-4-yl)oxy)-3-(tert-butyl(3-methylbenzyl)amino)propan-2-ol (17), off-white solid, Rf = 0.3 (DCM:EA = 3:1). ¹H NMR (600 MHz, CDCl₃) δ 8.24 (s, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 7.17 (d, J = 7.2Hz, 1H), 7.08 (t, J = 7.1Hz, 1H), 7.06 (d, 7.1Hz, 1H), 7.05 (d, 7.1Hz, 1H), 6.62 (s, 1H), 6.41 (d, J = 6.4 Hz, 1H), 3.95 (m, J = 3.9Hz, 2H), 3.86 (d, J = 3.9Hz, 1H), 3.65 (dd, J = 3.6Hz, 1H), 3.62 (d, J = 3.62Hz, 1H), 2.91 (dd, J = 2.9Hz, 1H), 2.85 (dd, J = 2.9Hz, 1H), 2.36(s, 3H),1.22 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 152.64, 142.01, 138.14, 137.38, 129.11, 128.51, 127.81, 125.47, 122.76, 122.66, 118.81, 104.65, 100.72, 99.98, 70.39, 67.57, 55.88, 54.26, 29.83, 27.58, 21.58. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₃H₃₁N₂O₂⁺ 367.2380; found 367.2394.

1-((1H-Indol-5-yl)oxy)-3-(tert-butyl(4-methylbe zyl)amino)propan-2-ol (26), off-white solid, Rf = 0.3 (DCM:EA = 3:1). ¹H NMR (500 MHz, CDCl₃) δ 8.15 (s, 1H), 7.25 – 7.21 (m, 3H), 7.15 (t, J = 2.8 Hz, 1H), 7.12 (d, J = 7.7 Hz, 2H), 7.01 (d, J = 2.4 Hz, 1H), 6.81 (dd, J = 8.8, 2.4 Hz, 1H), 6.45 (t, J = 2.7 Hz, 1H), 3.86 (d, J = 14.4 Hz, 1H), 3.81 (d, J = 5.1 Hz, 2H), 3.61 – 3.52 (m, 2H), 2.88 – 2.73 (m, 2H), 2.33 (s, 3H), 1.20 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 153.49, 138.89, 136.53, 131.19, 129.30, 128.30, 124.96, 112.89, 111.67, 103.59, 102.41, 71.32, 67.45, 55.83, 55.53, 54.00, 27.58, 21.22. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₃H₃₁N₂O₂⁺ 367.2380; found 367.2394.

1-(tert-Butyl(4-methylbenzyl)amino)-3-phenoxypropan-2-ol (27), off-white solid, Rf = 0.3 (DCM:EA = 3:1). ¹H NMR (600 MHz, Chloroform-d) δ 10.84 (s, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.26 (t, J = 7.9 Hz, 2 H), 7.14 (d, J = 7.4 Hz, 2H), 6.95 (t, J = 7.3 Hz, 1H), 6.74 (d, J = 7.8 Hz, 2H), 4.69 (d, J = 12.1 Hz, 1H), 3.75 (d, J = 12.2 Hz, 1H), 3.68 (d, J = 9.2 Hz, 1H), 3.58 (t, J = 8.4 Hz, 1H), 3.31 (d, J = 12.9 Hz, 2H), 3.27 – 3.17 (m, 1H), 2.30 (s, 3H), 1.58 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ 158.29, 140.54, 132.05, 130.05, 129.63, 125.58, 121.24, 114.34, 69.61, 65.43, 64.87, 55.00, 25.33, 21.41. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₁H₃₀NO₂⁺ 328.2271; found 328.2283.

1-((1H-Indol-5-yl)amino)-3-(tert-butyl(4-methylbenzyl)amino)propan-2-ol (33), off-white solid, Rf = 0.2 (DCM:EA = 3:1). ¹H NMR (500 MHz, CDCl₃) δ 7.97 (s, 1H), 7.24 (s, 1H), 7.22 (s, 1H), 7.14 (t, J = 8.0 Hz, 3H), 7.08 (t, J = 2.8 Hz, 1H), 6.76 (d, J = 2.2 Hz, 1H), 6.54 (dd, J = 8.6, 2.2 Hz, 1H), 6.38 – 6.37 (m, 1H), 3.85 (d, J = 14.2 Hz, 1H), 3.55 (d, J = 14.3 Hz, 2H), 3.42 (tt, J = 7.8, 3.1 Hz, 1H), 3.13 (dd, J = 11.9, 3.8 Hz, 1H), 2.92 – 2.82 (m, 2H), 2.61 (dd, J = 13.6, 4.1 Hz, 1H), 2.34 (s, 3H), 1.20 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 142.77, 138.75, 136.60, 130.20, 129.33, 128.83, 128.35, 124.38, 112.66, 111.59, 102.45, 101.82, 67.15, 55.83, 55.51, 54.54, 49.01, 27.59, 21.22. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₃H₃₂N₃O⁺ 366.2540; found 366.2555.

1-((1H-Indol-7-yl)amino)-3-(tert-butyl(4-methylbenzyl)amino)propan-2-ol (34), off-white solid, Rf = 0.2 (DCM:EA = 3:1). ¹H NMR (500 MHz, CDCl₃) δ 8.58 (s, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.13 (dd, J = 14.9, 7.8 Hz, 3H), 7.01 (d, J = 2.7 Hz, 1H), 6.95 (t, J = 7.7 Hz, 1H), 6.47 – 6.45 (m, 1H), 6.36 (d, J = 7.6 Hz, 1H), 3.81 (d, J = 14.2 Hz, 2H), 3.58 (d, J = 14.1 Hz, 1H), 3.47 (d, J = 3.4 Hz, 1H), 3.23 (dd, J = 12.6, 3.1 Hz, 1H), 2.95 – 2.84 (m, 2H), 2.61 (dd, J = 13.6, 4.3 Hz, 1H), 2.36 (s, 3H), 1.22 (d, J = 1.4 Hz, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 138.59, 136.77, 134.47, 129.38, 128.57, 128.53, 126.95, 123.51, 120.47, 111.77, 104.56, 103.00, 67.48, 55.97, 55.59, 53.77, 48.13, 27.58, 21.27. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₃H₃₂N₃O⁺ 366.2540; found 366.2555.

1-(tert-Butyl(4-methylbenzyl)amino)-3-((1-methyl-1H-indol-4-yl)oxy)propan-2-ol (32). To a solution of 16 (1 eq.) in THF was cooled to 0 °C, and then added with NaH (1.5 eq.) in one portion. The reaction mixture was stirred at 0 °C for 1 h before added with MeI (1 eq.). Then the mixture was allowed to warm to room temperature overnight with stirring. The reaction was quenched with saturated aqueous NH₄Cl solution and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate solution, brine, dried (Na₂SO₄) and concentrated in vacuo. The crude residue was purified on silica gel (DCM/EA) to give the desired product 32 as an off-white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.25 – 7.23 (m, 1H), 7.15 – 7.08 (m, 3H), 6.97 – 6.92 (m, 2H), 6.56 (d, J = 3.1 Hz, 1H), 6.41 (d, J = 7.7 Hz, 1H), 3.97 – 3.88 (m, 2H), 3.85 (d, J = 14.4 Hz, 1H), 3.77 (s, 3H), 3.62 (td, J = 9.7, 5.0 Hz, 2H), 2.86 (qd, J = 14.0, 7.0 Hz, 2H), 2.35 (s, 3H), 1.22 (s, 10H). ¹³C NMR (126 MHz, CDCl₃) δ 152.70, 138.95, 138.35, 136.55, 129.32, 128.37, 127.26, 122.39, 119.27, 102.85, 100.50, 98.47, 70.46, 67.55, 55.85, 55.57, 54.19, 33.14, 27.59, 21.23. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₄H₃₃N₂O₂⁺ 381.2537; found 381.2551.

N-(tert-Butyl)-2-methoxy-3-((1-methyl-1H-indol-4-yl)oxy)-N-(4-methylbenzyl)propan-1-amine (38). To a solution of 16 (1 eq.) in THF was cooled to 0 °C, and then added with NaH (3 eq.) in one portion. The reaction mixture was stirred at 0 °C for 1 h before added with MeI (3 eq.). Then the mixture was allowed to warm to room temperature overnight with stirring. The reaction was quenched with saturated aqueous NH₄Cl solution and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate solution, brine, dried (Na₂SO₄) and concentrated in vacuo. The crude residue was purified on silica gel (DCM/EA) to give the desired product 38 as an off-white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.42 – 7.37 (m, 2H), 7.27 – 7.21 (m, 3H), 7.09 – 7.05 (m, 2H), 6.69 (dd, J = 3.1, 0.8 Hz, 1H), 6.49 (d, J = 7.7 Hz, 1H), 4.26 (dd, J = 10.0, 3.3 Hz, 1H), 3.97 (dd, J = 10.1, 6.0 Hz, 1H), 3.89 (s, 3H), 3.88 – 3.85 (m, 2H), 3.53 (s, 3H), 3.50 (ddt, J = 8.5, 5.6, 2.7 Hz, 1H), 3.02 (dd, J = 13.9, 8.2 Hz, 1H), 2.90 (dd, J = 14.0, 5.2 Hz, 1H), 2.47 (s, 3H), 1.28 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 152.93, 140.07, 138.33, 135.98, 128.96, 128.29, 127.10, 122.35, 119.41, 102.58, 100.39, 98.72, 80.07, 69.16, 58.37, 55.72, 55.65, 52.62, 33.13, 27.41, 21.20. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₅H₃₅N₂O₂⁺ 395.2693; found 395.2708.

tert-Butyl 4-hydroxy-1H-indole-1-carboxylate (9). To a solution of 4-Hydroxyindole (8, 1 eq.) in acetonitrile was added di-tert-butyl dicarbonate (3 eq.) and DMAP (0.1 eq.). The solution was aged at room temperature for 1 hour, and then concentrated in vacuo. Solid potassium carbonate (5 eq.) was added to a solution of the crude residue in methanol and the mixture was stirred at room temperature for 3 hours. The reaction mixture was acidified with acetic acid and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by flash column chromatography (hexane/ethyl acetate=90:10) to obtain tert-butyl 4-hydroxy-1H-indole-1-carboxylate 9 as a colorless solid. ¹H NMR (600 MHz, CDCl₃ ) δ 7.74 (d, J = 7.3 Hz, 1H), 7.53 (d, J = 3.3 Hz, 1H), 7.16 (t, J = 8.1 Hz, 1H), 6.70 – 6.65 (m, 2H), 1.68 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ 150.10, 148.89, 137.01, 125.30, 124.82, 119.82, 108.38, 107.93, 103.74, 84.04, 28.31. EI (M+H)⁺ = 234.

tert-Butyl 4-(oxiran-2-ylmethoxy)-1H-indole-1-carboxylate (10). To a solution of 9 (1 eq.) in acetonitrile was added 2-(chloromethyl)oxirane 4 (3 eq.), K₂CO₃ (3 eq.) and KI (3 eq.). The mixture was heated to 70 °C for 16 h, cooled, and filtered, washing with ethyl acetate. The filtrate was concentrated in vacuo and purified by flash chromatography on silica gel (EtOAc/hexanes) to afford tert-butyl 4-(oxiran-2-ylmethoxy)-1H-indole-1-carboxylate 10 as a colorless solid. ¹H NMR (600 MHz, CDCl₃) δ 7.78 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 3.5 Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 6.73 (d, J = 3.7 Hz, 1H), 6.66 (d, J = 7.9 Hz, 1H), 4.35 (dd, J = 11.1, 3.1 Hz, 1H), 4.09 (dd, J = 11.1, 5.6 Hz, 1H), 3.43 (ddt, J = 5.7, 4.0, 3.0 Hz, 1H), 2.94 – 2.92 (m, 1H), 2.80 (dd, J = 4.9, 2.7 Hz, 1H), 1.67 (s, 9H). ¹³C NMR (151 MHz,CDCl₃ ) δ 151.86, 149.93, 136.71, 125.09, 124.63, 121.15, 108.99, 104.42, 104.29, 83.82, 69.09, 50.35, 44.88, 28.29. EI (M+H)⁺ = 290.

General Procedure for the synthesis of 11 or 25:

A solution of 10 (1 eq.) and amine 3 (1 eq.) in toluene was heated to 130 °C overnight. The solution was cooled, and concentrated in vacuo to purify on silica gel (DCM/EA) to afford the title compound.

tert-Butyl 4-(3-(tert-butyl(4-methylbenzyl)amino)-2-hydroxypropoxy)-1H-indole-1-carboxylate (11a), off-white solid, Rf = 0.3 (DCM:EA=3:1). ¹H NMR (600 MHz, CDCl₃) δ 7.72 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 3.8 Hz, 1H), 7.21 (d, J = 7.7 Hz, 2H), 7.16 (t, J = 8.1 Hz, 1H), 7.11 (d, J = 7.7 Hz, 2H), 6.64 (d, J = 3.8 Hz, 1H), 6.51 (d, J = 8.0 Hz, 1H), 3.84 (m, 3H), 3.57 (d, J = 14.4 Hz, 2H), 2.85 (m, 1H), 2.77 (dd, J = 13.7, 4.9 Hz, 1H), 2.32 (s, 3H), 1.65 (s, 9H), 1.19 (s, 11H). ¹³C NMR (126 MHz, CDCl₃) δ 152.22, 149.96, 138.74, 136.59, 129.31, 128.37, 125.09, 124.32, 121.05, 108.50, 104.49, 104.22, 83.69, 70.56, 67.42, 55.84, 55.56, 53.86, 28.29, 27.55, 21.20. ESI (M+H)⁺ = 467.

tert-Butyl 4-(3-(tert-butyl(2-methylbenzyl)amino)-2-hydroxypropoxy)-1H-indole-1-carboxylate (11b), off-white solid, Rf = 0.3 (DCM:EA=3:1). ¹H NMR (600 MHz, CDCl₃) δ 7.72 (d, J = 7.0 Hz, 1H), 7.47 (d, J = 3.1 Hz, 1H), 7.40 −7.38 (m, 1H), 7.17–7.13 (m, 4H), 6.63 (d, J = 3.7 Hz, 1H), 6.49 (d, J = 8.0 Hz, 1H), 3.91 (d, J = 14.1 Hz, 1H), 3.81 (dd, J = 4.8, 3.0 Hz, 2H), 3.62 (d, J = 14.1 Hz, 1H), 3.32 (dt, J = 10.0, 5.0 Hz, 1H), 2.86 (dd, J = 13.8, 8.6 Hz, 1H), 2.78 (dd, J = 13.9, 5.1 Hz, 1H), 2.38 (s, 3H), 1.66 (s, 9H), 1.22 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 152.21, 150.02, 138.98, 136.45, 130.62, 129.60, 127.19, 126.10, 125.13, 124.39, 121.06, 108.54, 104.48, 104.25, 70.54, 67.94, 56.15, 53.40, 53.20, 28.34, 27.25, 19.54. ESI (M+H)⁺ = 467.

tert-Butyl 4-(3-(tert-butyl(4-methylbenzyl)amino)-2-hydroxypropoxy)-1H-indole-1-carboxylate (25), off-white solid, Rf = 0.3 (DCM:MeOH=10:1). ¹H NMR (600 MHz, DMSO-d⁶) δ 11.07 (s, 1H), 7.20 (s, 1H), 6.99 (d, J = 7.8 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 7.21 (t, J = 7.2 Hz, 2H), 6.44 (s, 1H), 4.00–4.06 (m, 2H), 3.87–3.93 (m, 1H), 2.74 (dd, J = 11.2, 6.5 Hz, 1H), 2.64 (dd, J = 11.2, 6.5 Hz, 1H), 1.19 (s, 11H). ¹³C NMR (126 MHz, DMSO-d⁶) δ 152.09, 137.33, 123.43, 121.74, 118.42, 104.80, 99.93, 98.38, 70.57, 68.87, 50.01, 45.38, 28.62. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₁₅H₂₂N₂O₂⁺ 263.1754; found 263.1763.

General Procedure for the synthesis of 12:

To a solution of 11 (1 eq.) in THF cooled to 0 °C, was added NaH (1.5 eq.) in one portion. The reaction mixture was stirred at 0 °C for 1 h and then MeI (1.5 eq.) was added dropwise. The mixture was allowed to warm to room temperature overnight with stirring. The reaction was quenched with saturated aqueous NH₄Cl solution and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate solution, brine, dried (Na₂SO₄) and concentrated in vacuo. The crude residue was purified on silica (DCM/EA) gel to give the desired product 12.

tert-Butyl 4-(3-(tert-butyl(4-methylbenzyl)amino)-2-methoxypropoxy)-1H-indole-1-carboxylate (12a), off-white solid, Rf = 0.4 (DCM:EA=3:1). ¹H NMR (600 MHz, CDCl₃) δ 7.74 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 3.7 Hz, 1H), 7.24 (d, J = 7.7 Hz, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.08 (d, J = 7.7 Hz, 2H), 6.65 (d, J = 3.7 Hz, 1H), 6.48 (d, J = 7.9 Hz, 1H), 4.11 – 3.77 (m, 2H), 3.76 – 3.67 (m, 2H), 3.39 (s, 3H), 3.32 (dt, J = 6.1, 3.4, 3.0 Hz, 1H), 2.89 – 2.74 (m, 2H), 2.33 (s, 3H), 1.68 (s, 9H), 1.14 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ δ 152.47, 150.05, 139.90, 136.58, 136.09, 128.98, 128.33, 125.08, 124.21, 121.16, 108.29, 104.72, 104.20, 83.66, 79.99, 69.23, 58.36, 55.80, 55.67, 52.31, 28.33, 27.39, 21.19. ESI (M+H)⁺ = 481.

tert-Butyl 4-(3-(tert-butyl(2-methylbenzyl)amino)-2-methoxypropoxy)-1H-indole-1-carboxylate (12b), off-white solid, Rf = 0.4 (DCM:EA=3:1). ¹H NMR (600 MHz, CDCl₃) δ 7.72 (d, J = 6.8 Hz, 1H), 7.50–7.47 (m, 1H), 7.46 (d, J = 3.2 Hz, 1H), 7.18 (t, J = 8.1 Hz, 1H), 7.15–7.12 (m, 2H), 7.11 (dd, J = 8.8, 5.1 Hz, 1H), 6.64 (d, J = 3.7 Hz, 1H), 6.50 (d, J = 7.9 Hz, 1H), 4.06 (dd, J = 10.0, 3.0 Hz, 1H), 3.82 (d, J = 14.6 Hz, 1H), 3.78 (dd, J = 10.0, 5.8 Hz, 1H), 3.65 (d, J = 14.7 Hz, 1H), 3.29 (s, 3H), 3.07–3.03 (m, 1H), 2.90 (dd, J = 13.9, 8.5 Hz, 1H), 2.71 (dd, J = 13.9, 5.0 Hz, 1H), 2.35 (s, 3H), 1.66 (s, 9H), 1.14 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 152.47, 150.04, 140.04, 136.35, 130.17, 129.59, 126.71, 125.83, 125.08, 124.24, 121.16, 108.33, 104.71, 104.18, 83.68, 69.11, 58.30, 55.92, 53.65, 51.94, 29.84, 28.33, 27.04, 19.46. ESI (M+H)⁺ = 481.

General Procedure for the synthesis of 39–41:

To a room temperature solution of 12 (1 eq.) in i-PrOH was added Et₂NH (20 eq.). The reaction mixture was sealed and heated to 150 °C overnight. The mixture was concentrated in vacuo and purified on silica gel (DCM/EA) to give the desired product 39-41.

3-((1H-Indol-4-yl)oxy)-N-(tert-butyl)-2-methoxy-N-(4-methylbenzyl)propan-1-amine (39), white solid, Rf = 0.3 (DCM:EA = 3:1). ¹H NMR (500 MHz, CDCl₃) δ 8.12 (s, 1H), 7.24 (s, 1H), 7.10 −7.04 (m, 4H), 7.00 (d, J = 8.2 Hz, 1H), 6.61 (t, J = 2.3 Hz, 1H), 6.34 (d, J = 7.6 Hz, 1H), 4.11 (dd, J = 10.0, 3.2 Hz, 1H), 3.81 (dd, J = 10.0, 6.0 Hz, 1H), 3.76–3.68 (m, 2H), 3.39 (s, 3H), 3.37–3.34 (m, 1H), 2.87 (dd, J = 13.9, 8.2 Hz, 1H), 2.75 (dd, J = 13.9, 5.1 Hz, 1H), 2.32 (s, 3H), 1.13 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 152.96, 140.06, 137.38, 136.03, 128.98, 128.33, 122.85, 122.41, 119.00, 104.31, 100.72, 100.42, 80.10, 69.17, 58.40, 55.75, 55.67, 52.61, 27.42, 21.21. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₄H₃₃N₂O₂⁺ 381.2537; found 381.2540.

1-((1H-Indol-4-yl)oxy)-3-((4-methylbenzyl)amino)propan-2-ol (37). To a room temperature solution of 11d (1 eq.) in i-PrOH was added Et₂NH (20 eq.). The reaction mixture was sealed and heated to 150 °C overnight. The mixture was concentrated in vacuo and purified on silica gel (DCM/EA) to give 1-((1H-indol-4-yl)oxy)-3-((4-methylbenzyl)amino)propan-2-ol (37) as a colorless solid. ¹H NMR (400 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.23 (d, J = 7.9 Hz, 2H), 7.15 (d, J = 7.8 Hz, 2H), 7.12 – 7.06 (m, 2H), 7.03 (d, J = 8.2 Hz, 1H), 6.65 – 6.60 (m, 1H), 6.51 (d, J = 7.5 Hz, 1H), 4.24 – 4.16 (m, 1H), 4.17 – 4.08 (m, 2H), 3.88 – 3.77 (m, 2H), 3.01 – 2.82 (m, 2H), 2.62 (s, 2H), 2.34 (s, 3H). ¹³C NMR (126 MHz, Chloroform-d) δ 152.44, 137.45, 137.05, 136.83, 129.29, 128.25, 122.85, 122.84, 118.85, 104.93, 100.96, 99.95, 70.75, 68.69, 53.71, 51.44, 21.23. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₁₉H₂₃N₂O₂⁺ 311.1754; found 311.1767.

tert-Butyl 4-(3-bromopropoxy)-1H-indole-1-carboxylate (13). To a solution of 9 (1 eq.) in THF cooled to 0 °C, was added NaH (1.5 eq.) in one portion. The reaction mixture was stirred at 0 °C for 1 h before and then 1,3-dibromopropane (1.5 eq.) was added dropwise. The mixture was allowed to warm to room temperature overnight with stirring. The reaction was quenched with saturated aqueous NH₄Cl solution and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate solution, brine, dried (Na₂SO₄) and concentrated in vacuo. The crude residue was purified on silica gel to give the desired product 13 as a colorless solid. ¹H NMR (600 MHz, CDCl₃) δ 7.77 (d, J = 7.2 Hz, 1H), 7.51 (d, J = 3.1 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 6.69 (d, J = 2.7 Hz, 1H), 6.68 (s, 1H), 4.25 (t, J = 5.8 Hz, 2H), 3.66 (t, J = 6.5 Hz, 2H), 2.40 (p, J = 6.2 Hz, 2H), 1.68 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 152.03, 149.96, 136.68, 125.18, 124.57, 121.11, 108.71, 104.29, 104.22, 83.81, 65.69, 32.61, 30.18, 28.31. EI (M+H)⁺ = 354.

3-((1H-Indol-4-yl)oxy)-N-(tert-butyl)-N-(4-methylbenzyl)propan-1-amine (42). To a solution of 13 (1 eq.) in i-PrOH was added amine 14 (1.5eq.). The mixture was sealed and heated to 120 °C overnight and then cooled and concentrated in vacuo and purified on silica gel to afford 3-((1H-indol-4-yl)oxy)-N-(tert-butyl)-N-(4-methylbenzyl)propan-1-amine (42) as an off-white solid. ¹H NMR (600 MHz, CDCl₃) δ 8.10 (s, 1H), 7.34 (d, J = 7.8 Hz, 2H), 7.16 – 7.07 (m, 4H), 6.99 (d, J = 8.2 Hz, 1H), 6.65 (t, J = 2.8 Hz, 1H), 6.44 (d, J = 7.7 Hz, 1H), 4.01 (t, J = 6.1 Hz, 2H), 3.73 (s, 2H), 2.85 (t, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.85 – 1.80 (m, 2H), 1.18 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 158.72, 152.64, 137.40, 133.79, 129.60, 122.77, 122.66, 118.83, 114.01, 104.66, 100.77, 100.00, 70.45, 67.45, 55.87, 55.32, 55.16, 53.92, 27.58. HRMS (ESI⁺), m/z: [M + H]⁺, calcd. for C₂₃H₃₁N₂O⁺ 351.2431; found 351.2431.

Animal procedures.

All mice were purchased from Jackson laboratories and housed under a 12 h light/12 h dark cycle at 22 °C. Before handling, mice were acclimated for at least 1 week in our animal facility. For drug administration, compounds were re-suspended in a 10% DMSO/10% Tween80/80% PBS solution at a final concentration of 10 mg/ml and injected via I.P at 5μl/g body weight. Compounds were injected for a total of 3 times. For overnight fast, food was removed after the second injection and on the following morning (~9am) a third injection was administered and blood glucose was measured 3hrs after the last injection. For 6hrs fast, the third injection was given in the morning (~9am) and mice were fasted for 6hrs following the last injection before blood glucose was measured. Glycemia was measured by tail bleed using a glucometer (OneTouch). For all experiments, age- and body weight- matched animals were used. For protein extracts and biochemistry studies, tissues were removed following each experiment and snap frozen in liquid nitrogen. All studies were performed according to protocols approved by Beth Israel Deaconess Medical Center’s Animal Care and Use Committee.

Glucose production assay.

Primary hepatocytes were isolated from 8- to 12-week-old male C57BL/6 mice by perfusion with liver digest medium (Invitrogen, 17703–034) followed by 70 μm mesh filtration. Percoll (Sigma, P7828) gradient centrifugation allowed primary hepatocytes isolation from other cell types and debris. Cells were seeded in plating medium (DMEM with 10% FBS, 2mM sodium pyruvate, 1% penicillin/streptomycin, 1μM dexamethasone, and 100nM insulin). After 4 h of seeding, the medium was changed and incubated in maintenance medium (DMEM with 0.2% BSA, 2mM sodium pyruvate, 1% penicillin/streptomycin, 0.1μM dexamethasone, and 1nM insulin). The following day (day 1) hepatocytes were treated overnight with the indicated compounds at 1μM. On day 2, media was changed to glucose production media (glucose free DMEM with 0.2% BSA, 20mM sodium pyruvate, 2mM sodium lactate, 1% penicillin/streptomycin, 4mM glutamine, sodium bicarbonate) supplemented with glucagon (200nM) and fresh compounds. After 4hrs of incubation, media was collected and glucose level was measured using a glucose assay kit from Eton Bioscience Inc.

Lipolysis assay.

Immortalized brown adipocytes were allowed to differentiate for 5–7 days in differentiating medium (DMEM with 10% FBS, 1μM Rosiglitazone, 0.5mM IBMX, 5μM Dexamethasone, 20nM insulin, 1nM T3). Upon differentiation, medium was changed to DMEM containing 2% BSA and cells were immediately treated with the indicated compounds for 30 min and then stimulated with norepinephrine (1μM) for additional 90 min. Medium was collected and glycerol levels secreted to the medium were measured using free glycerol reagent (Sigma, F-6428).

Liver glycogen measurement.

~50mg of pulverized liver were homogenized in 6% perchloric acid and homogenate was centrifuged for 10min at x13,000g at 4°C and neutralized with KHCO₃. Supernatant was subjected to amyloglucosidase digestion (0.5 mg/ml in 0.2M acetate, pH=4.8) for 1hr at 37°C. following digestion, glucose concentration was measured using glucose measurement kit (Eton bioscience Inc.). Glucose levels of the pre amyloglucosidase digested samples was subtracted to determine glycogen levels.

ABBREVIATIONS

T2D

Type 2 Diabetes

HGP

hepatic glucose production

SAR

structure-activity relationship

PGC-1α

peroxisome proliferator-activated receptor gamma coactivator 1-alpha

β-AdR

β-adrenergic receptor

TZDs

thiazolidinediones

SGLT2

sodium-glucose cotransporter 2

5HT1a

5-hydroxytryptamine receptor 1a

SGIGP

suppression of glucagon-induced glucose production

SNIL

Suppression of norepinephrine-induced lipolysis

MeOH

methanol

EtOH

ethanol

CH₃CN

acetonitrile

Boc

t-butoxycarbonyl

DMF

Dimethylformamide

4-DMAP

4-Dimethylaminopyridine

TFA

Trifluoroacetic acid

DCM

dichloromethane

DIPEA

N,N-Diisopropylethylamine

NE

norepinephrine

SGIGP

suppression of glucagon-induced glucose production

SNIL

suppression of norepinephrine-induced lipolysis

HSL

hormone-sensitive lipase

HFD

high fat diet