Synthesis, Molecular Pharmacology, and Structure–Activity Relationships of 3-(Indanoyl)indoles as Selective Cannabinoid Type 2 Receptor Antagonists

Abstract

Synthetic indole cannabinoids characterized by a 2′,2′-dimethylindan-5′-oyl group at the indole C3 position constitute a new class of ligands possessing high affinity for human CB₂ receptors at a nanomolar concentration and a good selectivity index. Starting from the neutral antagonist 4, the effects of indole core modification on the pharmacodynamic profile of the ligands were investigated. Several N1 side chains afforded potent and CB₂-selective neutral antagonists, notably derivatives 26 (R¹ = n-propyl, R² = H) and 35 (R¹ = 4-pentynyl, R² = H). Addition of a methyl group at C2 improved the selectivity for the CB₂ receptor. Moreover, C2 indole substitution may control the CB₂ activity as shown by the functionality switch in 35 (antagonist) and 49 (R¹ = 4-pentynyl, R² = CH₃, partial agonist).

Graphical Abstract

INTRODUCTION

The endocannabinoid system (ECS) is implicated in a large spectrum of physiological and pathological processes, especially ones related to the central and peripheral nervous systems.^1-10 Part of the complex intercellular communicating system are two types of heptahelical G-protein coupled receptors identified as cannabinoid receptor type 1 (CB₁) and cannabinoid receptor type 2 (CB₂).^11-14 CB₁ is expressed primarily in the central nervous system^14,15 but can also be found in locations outside the brain.^16-19 CB₂, which is sometimes called the peripheral cannabinoid receptor, is expressed mainly in the cells associated with the immune system.^11,14 CB₂ receptors are also found in low concentrations in activated microglia and infiltrated macrophages in the brain,^20,21 gastrointestinal tract tissues and other cells such as vascular endothelial cells, cardiomyocytes, and bone cells.²²

Small-molecule modulators of CB₂ receptor activity are of interest for therapeutic indications including neurological disorders, pain, and inflammation. CB₂ agonists have potential utility for treating neurological disorders such as reperfusion brain injury;³³ amyotrophic lateral³⁴ and multiple sclerosis;^35,36 and Alzheimer’s,^37,38 Parkinson’s,^37,38 and Huntington’s diseases.^37-39 CB₂ agonists also exhibit anti-inflammatory effects and antinociceptive effects in acute pain, persistent inflammatory pain, postoperative pain, cancer pain, and neuropathic pain,^6,40-42 and they may aid in cardioprotection and treatment of gliomas.^20,43-45 Interestingly, CB₂ inverse agonists are also of interest for modulating inflammatory nociception.^46,47 This seemingly contradictory potential for agonists and inverse agonists in similar indications stems from the ability of the CB₂ receptors to mediate different processes depending on the conditions: recruitment of immune cells or interference with the action of other chemoattractants.⁴⁶ Neutral antagonists would similarly be of great interest in this regard. CB₂ neutral antagonists are relatively rare⁴⁸ and as such have received less attention. However, there is some evidence that CB₂ neutral antagonists may be promising in the treatment of obesity-associated inflammation, insulin resistance, and nonalcoholic fatty liver disease.⁴⁹ Selective CB₂ receptor modulators are needed across the full spectrum of functional activities for therapeutic targeting of the ECS.

Medicinal chemistry efforts targeting the ECS have been complicated by psychoactivity of CB₁-receptor agonists. These side effects are associated with drugs of abuse. Many CB₁ agonists developed in academic and pharmaceutical research labs have been hijacked for abuse in the recreational drug market.^23-26 The illicit demand for cannabinoid agonists has been met with regulatory controls on their supply, which incidentally also restricts legitimate pharmaceutical research.²⁷ CB₂-selective ligands are attractive for chemotherapeutic development due in part to the absence of known psychoactive side effects,^31,32 but many compounds designed to target CB₂ receptors⁵⁰ have nonetheless ended up as Schedule I controlled substances,⁵¹ presumably due to (off-target) CB₁ interactions.

While marijuana-like psychoactivity is a primary factor behind the potential for abuse of many ECS modulators, ease of synthesis is an auxiliary supply-side factor behind the illicit production and distribution. For example, the simple two-step synthesis of JWH-018 (Scheme 1)²⁸ increases its potential for abuse.^29,52 Many 1,3-disubstituted indoles like JWH-018 can be made from indole and two simple electrophiles (e.g., acyl and alkyl halides), and many similarly have emerged as drugs of abuse.⁵³ Conversely, less synthetically accessible cannabinoid ligands are less subject to abuse. Such compounds enable one to explore the chemotherapeutic potential of ECS modulators without the same risk of fueling the recreational drug market.

Scheme 1. Synthesis of JWH-018^a.

^aReagents and conditions: (a) MeMgBr, Et₂O/THF, 0 °C, then 1-naphthoyl Cl, reflux. (b) n-Pentyl Cl, KOH, DMSO, 80 °C.

There is a need for novel ECS modulators, particularly ones having low potential for abuse. To this end, we designed ligands that incorporate 2,2-dimethylindane into the known indole cannabinoid framework. 2,2-Dimethylindanes blend the benzenoid ring system of the traditional Huffman-(JWH)-type compounds, with the bulky cycloalkane motifs of influential⁵⁴ CB₂-selective indole cannabinoids from Abbott.³⁰ 2,2-Dimethylindane carboxylates can be crafted using recent synthetic methodologies,^55,56 but they are less accessible than the commercially available building blocks needed to prepare many indole cannabinoid drugs of abuse. We expect our designs to support development of novel ECS modulators specifically for legitimate academic and pharmaceutical research endeavors and without risk of “significant diversion”⁵² to illicit markets.

Indanoylindoles have not been examined previously as cannabinoid ligands. The first two indanoylindoles we considered—GBD-002 and GBD-003, Figure 1—indeed exhibit high affinities for both CB receptors, with GBD-003 showing selectivity for CB₂ (Figure 1). Functional assays showed that GBD-002, like JWH-018, is a full agonist, whereas positional isomer GBD-003 is a neutral antagonist (Table 2, vide infra). Considering that CB₂ antagonists are promising but rare, we focused on preparing a series of indanoylindoles based on GBD-003 for preliminary molecular pharmacology and structure–activity relationship (SAR) studies. Several CB₂-selective neutral antagonists are described herein.

Figure 1.

Rational design of a novel class of potent cannabinoids.

Table 2.

Calculated Binding Free Energies (kcal/mol) Compared to Experimental Binding Free Energy (kcal/mol)

	rCB1				rCB2
ligand	Ki (nM)	ΔGPBa	ΔGexpb	ΔGcorrc	Ki (nM)	ΔGPBa	ΔGexpb	ΔGcorrc	ΔΔGexpe	ΔΔGcorra,d
GBD-002	1.46	−41.92	−12.13	−12.65	1.36	−40.79	−12.17	−12.28	0.04	0.37
GBD-003	38.80	−34.00	−10.17	−10.07	4.90	−35.81	−11.41	−10.66	1.23	0.59
GBD-005	2559.00	−27.79	−7.68	−8.04	130.00	−31.10	−9.45	−9.12	1.78	1.08
GBD-013	152.00	−32.25	−9.36	−9.50	15.60	−35.26	−10.72	−10.48	1.36	0.98
GBD-014	325.00	−31.22	−8.91	−9.16	24.00	−35.93	−10.46	−10.70	1.55	1.54
GBD-029	1904.00	−27.89	−7.85	−8.07	62.50	−34.65	−9.89	−10.28	2.04	2.21
			rmsdf	0.23			rmsdf	0.43

^aCalculated binding free energies obtained from the MM-PBSA calculations without any empirical correction.

^bThe experimental binding free energy was converted from the experimental K_i using the thermodynamic equation ΔG_exp = −RT ln (K_i).

^cCalculated binding free energies after empirical correction using eq 1.

^dΔΔG_corr is the corrected binding free-energy difference [ΔG(CB₁R) − ΔG(CB₂R)].

^eΔΔG_exp = −RT ln (K_i (CB₁R)/K_i(CB₂R)) = ΔG_exp(CB₁R) − ΔG_exp(CB₂R).

^fThe root-mean-square deviation (RMSD) of the corrected ΔG binding energy values versus the experimental ΔG binding energy values.

RESULTS AND DISCUSSION

Chemistry.

We prepared indanoylindoles from carboxylates 10 and 21 (Scheme 2). The synthesis of GBD-002 is outlined in Scheme 3. Starting from methyl isobutyrate (5), α-substitution with 2-fluorobenzyl bromide generates ester 6 (81%), which on reduction with DIBAL-H is converted to alcohol 7 in 88% yield. The alkyl iodide precursor (9) of indane 10 is obtained through a modified Finkelstein reaction in 81% overall yield. The two-step sequence involves mesylation of the 1° alcohol (7), followed by nucleophilic displacement with NaI-saturated N-methylpyrrolidone (NMP) to provide iodide 9.

Scheme 2. Overview of the Synthetic Approach.

Scheme 3. Synthesis of Benzoate 10, the Precursor to 3-(Indan-4-oyl)indole GBD-002^a.

^aReagents and conditions: (a) 2-fluorobenzyl bromide, LDA, THF, −78 °C to rt, 5 h, 81%. (b) DIBAL-H, THF, −78 °C to rt, 1 h, 88%. (c) MsCl, Et₃N, CH₂Cl₂, 0 °C, 1 h, 91%. (d) NaI, NMP, 140 °C, 8 h, 89%. (e) (i) t-BuLi, n-pentane-Et₂O (4:1 by vol), −78 °C, 15 min; (ii) THF, −78 °C; (iii) −78 °C to rt to −78 °C; (iv) ClCO₂Et, −78 °C (15 min) to rt (15 min).

Indane-4-carboxylate 10 is prepared by treating 9 with tert-butyllithium (t-BuLi), followed by ethyl chloroformate under carefully controlled conditions.⁵⁷ This transformation is envisioned to proceed by a five-step, one-pot process, starting with lithium–iodine exchange and lithiation ortho to the fluoro substituent to produce a dilithio intermediate. Release of lithium fluoride generates a benzyne-tethered alkyllithium that undergoes 5-exo-dig cyclization to the 4-lithiated indane, which is then trapped with ethyl chloroformate to produce 10.

Meanwhile, our lab was developing a synthesis of diyne monoester 16 (Scheme 4).⁵⁵ Dimedone (12) is condensed with triflic anhydride and reacted with the lithium enolate of ethyl acetate for a Claisen-type tandem addition/fragmentation to generate β-keto ester 14, which is then dehydrated to give 16. Rhodium-catalyzed cyclotrimerization of 16 with TMS-acetylene provides a mixture of regioisomers (17 and 18), which can be hydrolyzed to 10 and then to 11. Acid 11 is converted to the acid chloride and then coupled with N-pentylindole to complete the synthesis of GBD-002 (3).

Scheme 4. Synthesis of the 3-(Indan-4-oyl)indole GBD-002^a.

^aReagents and conditions: (a) Tf₂O, pyridine, CH₂Cl₂, −78 °C to rt, 20 min, 97%. (b) (i) lithio ethyl acetate, THF, −78 °C, 20 min; (ii) LHMDS, 0 °C (10 min) to rt (1 h), 63%. (c) Tf₂O, aq. LiOH, hexanes, 0 °C to rt, 30 min, 88%. (d) LHMDS, THF, 0 °C to rt, 1 h, 63%. (e) (i) [Rh(cod)₂]BF₄, (±)-BINAP, H₂, CH₂Cl₂, rt, 1 h; (ii) TMS-acetylene, CH₂Cl₂, rt, 1 h, 90%. (f) KI, TMSCl, H₂O, CH₃CN, 60 °C, 12 h, 68%. (g) KOH, H₂O, EtOH, rt, 8 h, 95%. (h) (i) SOCl₂, DMF, CH₂Cl₂, rt, 8 h; (ii) N-pentylindole, HFIP, rt, 24 h, 68%.

The synthesis of 3-(indan-5-oyl)indoles also begins with dimedone (12, Scheme 5) and builds on prior synthetic methodologies from the Dudley lab.^56,58 Vinylogous acyl triflate 13 is reduced with DIBAL-H to produce hydroxycycloalkenyl triflate 19, which undergoes tandem fragmentation/olefination to furnish dienyne 20. Rhodium-catalyzed cycloisomerization of 20, followed by oxidation with DDQ, provides 21 in a one-pot process. Saponification of 21 gives acid 22, which is converted to the acid chloride for Friedel–Crafts acylation of indole and 2-methylindole (Scheme 4, R² = H, Me). The resulting 3-acylindoles (23 and 24) were then N-alkylated to produce a small collection of indanoylindoles, designated as GBD-003 thru GBD-031.

Scheme 5. Synthesis of 3-(Indan-5-oyl)indoles^a.

^aReagents and conditions: (a) Tf₂O, pyridine, CH₂Cl₂, −78 °C to rt, 20 min, 97%. (b) DIBAL-H, THF, −78 °C, 1–2 h, 90%. (c) Ethyl-E-4-(diethoxyphosphoryl)but-2-enoate, LHMDS, THF, −78 °C (15 min) to rt to 60 °C (1 h), 82%. (d) (i) [Rh(nbd)Cl]₂, AgSbF₆, CH₂Cl₂, rt, 1 h; (ii) DDQ, rt, 2 h, 85%. (e) KOH, H₂O, EtOH, rt, 8 h, 96%. (f) (i) SOCl₂, DMF, CH₂Cl₂, rt, 8 h; (ii) indole or 2-methylindole, HFIP, rt, 24 h, 53–58%. (g) Alkyl halide, NaH, DMSO, rt, 8 h.

We routinely produce gram quantities of indane-5-carboxylate 21 as a synthetic building block for other projects in the lab, and thus, it serves for us as a readily available and convenient starting point for the synthesis of various indanoylindoles. However, the four-step synthesis effectively ensures that indole cannabinoids derived from 21 will not be cost-competitive with ones that have been co-opted by the recreational drug market (e.g., JWH-018) and thus are unlikely to be subject to widespread abuse as recreational drugs.

Receptor Binding Affinity and SAR.

The indanoylindoles thus prepared are high-affinity cannabinoid ligands. Initially, a screen to detect compounds with affinity for human CB₁ (hCB₁ and human CB₂ (hCB₂) receptors was conducted by determining the ability of a relatively high concentration (1 μM) of each compound to displace a 0.2 nM concentration of the well-characterized nonselective CB₁/CB₂ cannabinoid radioligand [³H]CP-55,940 from its binding site (Table 1). Under these conditions, the concentration of a compound producing 50% displacement would approximate the affinity (e.g., K_i) for the receptor being examined, with higher displacement associated with greater affinity for the CB receptors. Compounds producing less than 50% displacement are predicted to exhibit low affinity (less than 1 μM). Selected compounds showing promising affinity and/or selectivity were selected for determination of K_i and selectivity index (SI) (Figure 2) and/or were screened for functional activity (vide infra; Table 3).

Table 1.

Screen of (Dimethylindanoyl)indole Derivatives for Binding to hCB₁ and/or hCB₂ Receptors

			% displacement of [3H]CP-55,940 (0.2 nM) (1 μM of each compound)
compound	R1	R2	hCB1	hCB2
GBD-002 (3)a,b	Pentyl	H	85.7 ± 5.2	94.1 ± 2.3
GBD-003 (4)a,b	pentyl	H	93.4 ± 5.0	99.8 ± 0.9
GBD-001 (23)	H	H	9.4 ± 3.1	22.2 ± 6.0
GBD-004 (25)	methyl	H	10.8 ± 1.4	23.9 ± 13.0
GBD-005 (26)a,b	propyl	H	47.4 ± 0.3	90.5 ± 2.5
GBD-006 (27)b	butyl	H	82.7 ± 0.6	92.9 ± 1.3
GBD-007 (28)b	hexyl	H	79.1 ± 3.0	83.6 ± 4.9
GBD-008 (29)	isobutyl	H	42.2 ± 4.9	83.1 ± 2.4
GBD-009 (30)b	benzyl	H	69.2 ± 2.3	84.6 ± 3.3
GBD-010 (31)b	4-fluorobenzyl	H	88.0 ± 1.4	92.3 ± 2.5
GBD-011 (32)b	E-2-pentenyl	H	84.2 ± 1.2	86.9 ± 3.9
GBD-012 (33)b	Z-2-pentenyl	H	60.7 ± 4.8	86.4 ± 5.0
GBD-013 (34)a,b	4-pentenyl	H	80.0 ± 3.8	95.2 ± 2.9
GBD-014 (35)a,b	4-pentynyl	H	71.0 ± 6.4	92.8 ± 1.6
GBD-015 (36)	2-pentynyl	H	55.6 ± 5.7	65.9 ± 2.8
GBD-016 (37)	allyl	H	16.0 ± 5.3	60.6 ± 5.4
GBD-017 (38)	propargyl	H	33.4 ± 3.3	56.0 ± 3.6
GBD-018 (39)	CH2CO2CH2CH3	H	19.4 ± 7.7	18.0 ± 10.9
GBD-020 (40)	propyl	methyl	44.4 ± 1.9	73.9 ± 3.4
GBD-021 (41)	butyl	methyl	43.5 ± 0.6	85.4 ± 3.9
GBD-022 (42)	pentyl	methyl	53.8 ± 0.8	83.6 ± 3.1
GBD-023 (43)	hexyl	methyl	26.2 ± 1.6	55.0 ± 5.9
GBD-024 (44)	benzyl	methyl	18.1 ± 13.3	51.2 ± 11.1
GBD-025 (45)	4-fluorobenzyl	methyl	38.4 ± 5.0	48.8 ± 0.0
GBD-026 (46)	E-2-pentenyl	methyl	35.1 ± 3.3	73.7 ± 3.8
GBD-027 (47)	Z-2-pentenyl	methyl	43.0 ± 2.0	82.9 ± 3.4
GBD-028 (48)	4-pentenyl	methyl	38.6 ± 1.8	86.4 ± 3.3
GBD-029 (49)a,b	4-pentynyl	methyl	41.9 ± 2.2	93.1 ± 3.6
GBD-030 (50)	2-pentynyl	methyl	16.4 ± 3.1	30.2 ± 2.7
GBD-031 (51)	propargyl	methyl	51.9 ± 0.8	63.0 ± 6.8

^aThis compound was selected for determination of K_i.

^bThis compound was selected for screening of functional activity.

Figure 2.

Affinities (K_i) of selected derivatives for hCB₁ and hCB₂ receptors.

Table 3.

[³⁵S]-GTPγS Binding Assays of Selected and Reference Compounds for Efficacy at the hCB₂ Cannabinoid Receptors

compound	[35S]-GTPγS specific binding (control = 100%)a
CP-55,950	131 ± 6**
AM-630	67 ± 4**
GBD-002 (3)	134 ± 5*
GBD-003 (4)	108 ± 9
GBD-005 (26)	99 ± 3
GBD-006 (27)	102 ± 4
GBD-007 (28)	102 ± 9
GBD-009 (30)	87 ± 8
GBD-010 (31)	90 ± 3
GBD-011 (32)	81 ± 2**
GBD-012 (33)	106 ± 7
GBD-013 (34)	92 ± 2*
GBD-014 (35)	96 ± 5
GBD-029 (49)	121 ± 8

^aThe results are expressed as the percentages of stimulation of [³⁵S]-GTPγS binding (the basal value set at 100%) obtained for a concentration of ligands of 10 μM. Data are the mean ± standard error of the mean of three experiments. Statistical significance from 100% (basal) as assessed by one-sample t-test

^*P < 0.05

^**P < 0.01.

GBD-002 and GBD-003 produced almost 100% displacement of the radioligand from both hCB₁ and ¹CB₂ receptors in initial binding screens (Table 1) and (as predicted from these initial screens) were subsequently determined to exhibit high affinity for cannabinoid receptors (CBRs) (Figure 2). Specifically, GBD-002 exhibited a K_i (hCB₁) of 1.46 ± 0.49 nM and a K_i (hCB₂) of 1.36 ± 0.52 nM; however, with an SI of only 1.1 (nonselective). The positional isomer GBD-003 is also a high-affinity ligand for both receptors (hCB₁ K_i = 38.8 ± 19.8 nM and hCB₂ K_i = 4.9 ± 0.2 nM), with improved selectivity for CB₂ (SI = 7.9). Having determined that GBD-003 binds to CB₂ receptors in a relatively selective manner, we investigated the broader series of indanoylindoles for binding to CB₁ and CB₂ receptors (Table 1). The N1-pentyl chain was first replaced with different lipophilic alkyl moieties including chains based on other JWH cannabinoids^{28,30,50,53,59} and novel N-alkyl groups among reported indole cannabinoids such as Z-2-pentenyl, 2-pentynyl, 4-pentynyl, and propargyl. The analogue lacking the N-substituent (23) showed little affinity for either receptor. A similar activity profile was found when the H was substituted with a methyl group (25). Replacement with longer straight alkyl chains afforded compounds with good to excellent CBR affinities, consistent with previously reported links between chain length and receptor binding in other synthetic indole cannabinoids.⁵⁹ Compounds 26 (propyl), 27 (butyl), and 4 (pentyl) exhibited increasing CBR affinity with increasing chain length until lower affinity was observed for the 6-carbon (28, hexyl) variant.

Introduction of an isobutyl substituent (29) led to poor affinity for CB₁ (42% displacement) and good affinity and selectivity for CB₂ (83% displacement). Comparison of isobutyl 29 with its unbranched isomer (27, n-butyl) and desmethyl analogue (26, n-propyl) suggests that this branching disfavors CB₁ receptor binding and enhances CB₂ selectivity.

Compounds 30 (benzyl) and 31 (4-fluorobenzyl) bearing aromatic rings in the N-alkyl substituents displayed CBR affinities comparable to those of the aliphatic counterparts 27 (butyl) and 28 (hexyl) but less than that of 4 (pentyl), suggesting some steric tolerance of the receptors. Selectivity for binding to either CB₁ or CB₂ receptors was modest at best in these benzyl variants.

Replacement of the alkyl chain with a more hydrophilic group has a profound effect on the pharmacology of the ligands. Compound 39, punctuated with an ester group of a similar length [−CH₂C(O)OCH₂CH₃] as compared with n-pentyl, showed a complete loss of affinity and selectivity (only 19 and 18% displacement at CB₁ and CB₂, respectively). This observation is consistent with the results reported by Hynes et al.,⁶⁰ Frost et al.,³⁰ and Longworth et al.⁶¹ for fenchyl amide indoles, cyclopropanecarbonyl indoles, and adamantyl amide indoles, respectively, in which polar N1 side chains generally cause a decrease in CB₂ receptor binding relative to the n-pentyl analogue.

Another set of compounds was synthesized to evaluate the effect of unsaturation in the alkyl chain, starting with unsaturated versions of the three- and five-carbon straight alkyl chains that appear to provide good CB₂ affinity and selectivity. Addition of double bonds to the pentyl chain yielded compounds 32–34, bearing the E-2-pentenyl, Z-2-pentenyl, and 4-pentenyl moieties, respectively. Although less potent than the n-pentyl analogue 4, the pentenyl compounds still retained good receptor affinities, particularly at CB₂ (Table 1). The Z-isomer (33) was more selective than the E-isomer (32), and terminal alkene isomer 34 was more potent and CB₂-selective. The alkynyl analogues 35 (4-pentynyl) and 36 (2-pentynyl) showed slightly lower affinities than the alkenyl analogues, although terminal alkyne 35 showed promising selectivity for CB₂. In the three-carbon unsaturated series, the allyl (37) and propargyl (38) analogues exhibited modest CB₂ affinities displacing only 61 and 56% of [³H]-CP-55,940, respectively, compared to the corresponding saturated counterpart 26 (propyl, 90% displacement).

We next examined N-substitution in the 2-methylindole series, inspired by the potent CBR ligand WIN-55,212-2. The 2-methylindoles generally showed lower affinity for both receptors but better CB₂ selectivity compared to the analogues devoid of the additional substituent. Like the trend in the indole series, CB₂ binding increased with the length of the N1-alkyl chain up to 5 carbons (40–43), whereas CB₁ affinity was largely unaffected by these modifications in chain length. Aung et al.⁵⁹ reported that the presence of a methyl group at C2 of the N1-propyl analogue enhanced CB₂ selectivity (JWH-072, SI = 6 vs JWH-015, SI = 24). However, in this indanoylindole scaffold, 2-methylation has a negative impact (26 vs 40, Table 1). Neither of the N-arylmethyl variants (44 and 45) offered any apparent advantages in these initial assays, nor did the 2-pentynyl (50) or propargyl (51) variants. On the other hand, the pentenyl derivatives (46–48) showed an increase in CB₂ affinity without a substantive effect on CB₁ affinity, leading to enhanced selectivity, and the 4-pentynyl side chain of 49 showed good affinity and selectivity for CB₂.

Guided by preliminary screening data from Table 1, we chose a small set of ligands and determined their binding affinities (K_i) for both CB receptors to determine an SI, as outlined in Figure 2. Data for JWH-018 were reported previously⁵⁹ and are provided for reference in Figure 1. The N-pentyl indanoylindoles (3 and 4) were the most potent, and N-(4-pentynyl)-2-methylindole 49 was the most CB₂-selective, having an hCB₂ K_i of 62.5 ± 10.0 nM and SI = 30.5.

Molecular Modeling.

The six compounds highlighted in Figure 2 were also examined computationally in combined molecular docking/molecular dynamics (MD) simulations (Figure 3), which correlated with experimentally measured binding affinities (K_i, cf. Table 2 and Figure S1). The good correlation between experimental and computational data suggests that this computational protocol provides the opportunity to test future designs in silico.

Figure 3.

Binding modes of selected derivatives in CB₁ (A1–F1) and CB₂ (A2–F2) receptors. (A1) GBD-002 (3), CB₁; (A2) GBD-002 (3), CB₂; (B1) GBD-003 (4), CB₁; (B2) GBD-003 (4), CB₂; (C1) GBD-005 (26), CB₁; (C2) GBD-005 (26), CB₂; (D1) GBD-013 (34), CB₁; (D2) GBD-013 (34), CB₂; (E1) GBD-014 (35), CB₁; (E2) GBD-014 (35), CB₂; (F1) GBD-029 (49), CB₁; (F2) GBD-029 (49), CB₂. Original crystal structures of CB₁R (5XR8) and CB₂R (5ZTY) were obtained from the Protein Data Bank for modeling. Pivotal to the difference in binding modes of these compounds between CB₁R and CB₂R are the residue changes L193/I110 and L481/V261 (in CB₁R and CB₂R, respectively). These substitutions allow for the central indole scaffold of the GBD compounds to rotate, allowing for the dimethylindane moiety and its connecting ketone to engage in hydrogen bonding with S285. Modifications to the aliphatic chain such as truncation (C1-2) and dehydrogenation (D1-2, E1-2) reduce hydrophobic interactions with the nearby hydrophobic pockets in CB₁R and CB₂R. The resulting indole moiety rotation due to the residue changes between CB₁R and CB₂R also allows for substitutions (F1–F2) on the indole moiety with CB2R that are otherwise unavailable in CB₁R.

Binding of GBD-002 with CB₁R showed a strong hydrogen bond with the nearby S505 via the carbonyl oxygen and π−π stacking interactions between the indole ring with F200 and the indane ring with F268 (Figure 3A1). In the CB₂R model, the indane ring interacted similar to the CB₁ receptor in the lipophilic pocket formed by F183, S285, and F87 resulting in the retention of hydrogen bond (Figure 3A2). Meanwhile, the nonconserved residues V261 and I110 (substituted with L482 and L193 in the CB₁ receptor, respectively) allowed more room for the indole core to relax and rotate, changing the binding angle in the central transmembrane binding site. The indane ring of the positional isomer GBD-003 induced a steric strain in CB₁R, shifting the moiety closer to the ECL2 domain (Figure 3B1). The shift rotated the carbonyl group away from S505 which makes hydrogen bonding unfavorable. In CB₂R however, the loss in affinity was not observed as I110 and V261 still allowed strong π−π stacking interactions with F183 and hydrogen bonds with S285 (Figure 3B2).

A >60-fold reduction in CB₁R affinity was observed in GBD-005, with the propyl tail being too short to occupy the binding pocket delimited by I271, L276, and Y275 and create effective van der Waals interactions (Figure 3C1). The truncation of the lipophilic tail is not as deleterious within CB₂R as the scaffold is still able to retain the hydrogen bond (Figure 3C2). The greater loss of CB₁ binding affinity translates into greater CB₂ selectivity. GBD-013 and GBD-014 have similar binding modes to GBD-003. Although the three compounds have N-substituents with similar lengths, the double bond in GBD-013 weakens the hydrophobic interactions in CB₂R (Figure 3D2) and more so in CB₁R (Figure 3D1), resulting in a 10-fold selectivity. Hydrophobic interactions of the 5 C chain with the hydrophobic channel in CB₁R further reduced with the substitution of a triple bond (GBD-014, Figure 3E1,E2).

2-Methylation of the indole core (GBD-029) positioned the ligand such that the additional methyl group clashes with L193 in CB₁R (Figure 3F1). This crowding disrupts the preferred binding mode and substantially weakens the binding affinity. In CB₂R (Figure 3F2), on the other hand, the steric constraint is less prominent as the rotation of the indole ring places the methyl group in an open space between transmembrane helices 5 and 6, thereby limiting the steric strain between the ligand and protein, affording a higher potency and selectivity against CB2R.

The experimental and computational binding data are compiled in Table 2 and depicted graphically in Figure S1. Experimental binding affinities (K_i) were converted into free energies of binding (ΔG_exp), and absolute magnitudes of raw calculated protein binding energies (ΔG_PB) were adjusted using a standard correction (ΔG_corr) to align with the experimental data using eq 1

$$\Delta G_{\text{corr}}=0.3248\times \Delta G_{\text{PB}}+1.0477$$ (1)

The difference between the CB₁R and CB₂R binding energies defines selectivity as measured experimentally (ΔΔG_exp) and estimated computationally (ΔΔG_corr). We see good correlation (R² = 0.94) between the calculated (ΔG_PB) and measured (ΔG_exp) binding energies (Figure S1, top), suggesting that SARs can be used to estimate binding affinities (and selectivities, Figure S1, bottom) with a reasonable degree of confidence.

CB₂R Functional Activity.

Several compounds in the indole series and 49, the most selective in the 2-methylindole series, were subjected to in vitro pharmacological evaluation in a [³⁵S]GTPγS binding assay to evaluate the intrinsic functional activity. The CBR-mediated G-protein activities of the selected compounds at CB₂R as well as reference ligands CP-55,940 (CB₂ full agonist) and AM-620 (CB₂ inverse agonist) were measured by examining the ability of a single, high (near receptor-saturating) 10 μM concentration to modulate [³⁵S]GTPγS binding in hCB2-transfected CHO-cells as previously described.⁶²

The molecular geometry of the indanoyl moiety has a striking effect on hCB₂R-mediated G-protein activation (Table 3). Consistent with other indole cannabinoids, GBD-002 increased [³⁵S]GTPγS binding, indicative of the full agonist property (134 ± 5% basal). When the indane point of attachment was moved from C4 (GBD-002) to C5 (GBD-003), the indanoylindane evoked little if any agonist response (108 ± 9% basal) compared to the reference full agonist CP-55,940 (131 ± 6% basal). This lack of significant agonist activity suggests that GBD-003 may act as a neutral hCB₂R antagonist. Co-incubation of GBD-003 in CHO-hCB2 membranes produced a concentration-dependent reduction in [³⁵S]GTPγS binding elicited by CP-55,940 (Figure 4). The ability to reverse the response of a full agonist (CP-55,940, IC₅₀ = 1.8 μM, K_b = 9.7 nM) indicates that GBD-003 is best characterized as a CB₂R antagonist.

Figure 4.

GBD-003 acts as an antagonist at CHO-hCB2 receptors, producing concentration-dependent reduction of G-protein activation produced by the hCB2 receptor agonist CP-55,940.

Other indanoylindoles examined are best broadly characterized functionally as neutral antagonists or perhaps weak inverse agonists. The N-alkyl derivatives (26–28), similar to the results from receptor binding studies, suggest that the length of the alkyl chain does not influence functional activity. Arylmethyl derivatives (30, benzyl and 31, p-fluorobenzyl) reduced [³⁵S]GTPγS binding to 87 ± 8% and 90 ± 3% of basal, respectively, consistent with weak inverse agonism. Two of the N-pentenyl isomers (32 and 34) behaved as weak inverse agonists (81 ± 2% basal and 92 ± 2% basal, respectively), whereas Z-isomer 33 registered as a weak agonist/antagonist (106 ± 7% basal) in our screening, and N-pentynyl 35 insignificantly altered the basal binding (96 ± 5% basal), consistent with an antagonist profile. Interestingly, the addition of a methyl group to C2 of 35 to produce compound 49 shifted the CB₂R activity from the antagonist to the agonist (121 ± 8% basal).

CONCLUSIONS

We identify (2,2-dimethylindanoyl)indoles as a promising new class of indole cannabinoids comprising potent and CB₂-selective ligands and presumably having lower potential for abuse as recreational drugs compared to other indole cannabinoids. Similar to the reference compound JWH-018, GBD-002 (3) and GBD-003 (4) displayed excellent affinities for both CB receptors. The latter (GBD-003) is moderately selective for CB₂ and, in contrast to most indole cannabinoid ligands, can be characterized as a neutral antagonist. We developed a preliminary SAR profile for these new (2,2-dimethylindanoyl)indoles. The observed patterns on affinities and selectivity in both the indole and methylindole series are generally in accordance with a number of previously reported SAR investigations of indole-derived cannabinoids, with the major exception of their unusual antagonist functional activity. The N-alkyl substituent provides opportunities to alter affinity and selectivity while broadly retaining antagonist activity. Other changes to the indanoylindole core structure had larger effects on functional activity, with GBD-029 (2-methylindole) and GBD-002 (indanoyl positional isomer) exhibiting agonist activity. The indanoylindole derivatives reported here provide a new template for future development of CB₂-selective neutral antagonists.

EXPERIMENTAL SECTION

Chemistry.

All the chemicals were used as received unless otherwise stated. Diethyl ether (Et₂O), tetrahydrofuran (THF), methylene chloride (DCM), and toluene were dried over a column of molecular sieves under argon. All reactions were carried out under an inert nitrogen atmosphere unless otherwise stated. Crude products were purified in a Biotage Isolera One Flash Purification System using Biotage prepacked cartridges (50 μm irregular silica). Yields refer to isolated material following silica gel chromatography. The purity of the compounds tested was determined by quantitative ¹H NMR using the absolute internal calibrant method (see the Supporting Information). ¹H NMR and ¹³C NMR spectra were recorded on a JEOL 400 MHz spectrometer using CDCl₃ and dimethyl sulfoxide-d₆ (DMSO-d₆) as the deuterated solvent [≥99.8 atom % D, contains 0.03% (v/v) TMS]. The chemical shifts (δ) were reported in parts per million (ppm) relative to the internal standard TMS. High-resolution mass spectroscopy (HRMS) data were obtained on a UHR-TOF maXis 4G instrument (Bruker Daltonics, Bremen, Germany) using electrospray ionization (ESI). Melting points (mp) were taken in open capillaries on a Stuart melting point apparatus SMP11 and are uncorrected.

Methyl 3-(2-Fluorophenyl)-2,2-dimethylpropanoate (6).⁶³

A mixture of diisopropylamine (3.0 mL, 21.5 mmol) and THF (98 mL) was first cooled and stirred at −78 °C for 15 min. Next, n-BuLi (1.6 M in hexanes, 13.5 mL, 21.5 mmol) was added dropwise via a syringe. The mixture was stirred for another 15 min at −78 °C, 25 min at 0 °C, and 15 min at −78 °C. Methyl isobutyrate (2.2 mL, 19.6 mmol) was then added at the same temperature. After 30 min, 2-fluorobenzyl bromide (2.7 mL, 21.5 mmol) was finally added. The mixture was stirred at −78 °C for 1 h and then at ambient temperature for 5 h. After the reaction, the yellow solution was extracted with ether (3 × 50 mL). The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. Purification by automatic flash column chromatography on silica gel (gradient elution from 2 to 20% EtOAc-hexanes) afforded a colorless, viscous liquid (84% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.19 (s, 6H), 2.92 (s, 2H), 3.67 (s, 3H), 6.98–7.06 (m, 2H), 7.07–7.13 (m, 1H), 7.16–7.23 (m, 1H). ¹⁹FNMR (376 MHz, CDCl₃): δ −115.90. ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 24.71, 38.64, 38.65, 43.64, 51.80, 115.21 (d, J_C–F = 20 Hz), 123.64 (d, J_C–F = 4 Hz), 124.88 (d, J_C–F = 16 Hz), 128.30 (d, J_C–F = 8 Hz), 132.21 (d, J_C–F = 5 Hz), 161.46 (d, J_C–F = 244 Hz), 177.79. HRMS (ESI, m/z): for C₁₂H₁₆FO₂ [M + H]⁺ calcd, 211.1129; found, 211.1124.

3-(2-Fluorophenyl)-2,2-dimethylpropan-1-ol (7).⁶³

A mixture of 6 (2.0 g, 9.5 mmol) and THF (43 mL) was first cooled and stirred at −78 °C for 15 min. At the same temperature, DIBAL-H (1.0 M in toluene, 20.9 mL, 20.9 mmol) was then added dropwise. After stirring for 1 h, the mixture was warmed to ambient temperature and ether (21 mL) was added. The solution was quenched in an ice bath with water (0.8 mL), then 15% NaOH (0.8 mL), and finally water (2.1 mL). The resulting heterogeneous mixture was removed from the bath, dried over MgSO₄, filtered, and concentrated under reduced pressure. Purification by automatic flash column chromatography on silica gel (gradient elution from 5 to 40% EtOAc-hexanes) afforded a white, waxy solid (71% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.90 (s, 6H), 1.69 (br s, 1H), 2.63 (s, 2H), 3.31 (d, J = 8 Hz, 2H), 6.99–7.08 (m, 2H), 7.15–7.22 (m, 2H). ¹⁹FNMR (376 MHz, CDCl₃): δ −116.17. ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 24.03, 36.92, 36.93, 37.03, 71.02, 115.25 (d, J_C-F = 23 Hz), 123.67 (d, J_C–F = 3 Hz), 125.77 (d, J_C–F = 16 Hz), 127.96 (d, J_C–F = 8 Hz), 133.13 (d, J_C–F = 5 Hz), 161.68 (d, J_C–F = 242 Hz). HRMS (ESI, m/z): for C₁₁H₁₄FO [M − H]⁻ calcd, 181.1034; found, 181.1030.

3-(2-Fluorophenyl)-2,2-dimethylpropyl Methanesulfonate (8).⁶³

Triethylamine (4.6 mL, 32.9 mmol) was added to a solution of 7 (4.0 g, 22.0 mmol) and dichloromethane (73 mL). The mixture was cooled to 0 °C, and then, methanesulfonyl chloride (2.1 mL, 26.3 mmol) was added. After stirring for 1 h, the mixture was warmed to ambient temperature and diluted with dichloromethane. The mixture was successively washed with cold water, chilled 10% HCl, saturated NaHCO₃, and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure, producing a colorless, viscous liquid (91% yield). The product was used immediately in the next step without purification. ¹H NMR (400 MHz, CDCl₃): δ 1.00 (s, 6H), 2.68 (s, 2H), 3.03 (s, 3H), 3.92 (s, 2H), 7.01–7.11 (m, 2H), 7.13–7.25 (m, 2H). ¹⁹FNMR (376 MHz, CDCl₃): δ −115.22. ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 23.84, 35.82, 37.00, 37.11, 76.86, 115.39 (d, J_C–F = 23 Hz), 123.75 (d, J_C–F = 4 Hz), 124.39 (d, J_C–F = 16 Hz), 128.38 (d, J_C–F = 8 Hz), 132.80 (d, J_C–F = 4 Hz), 161.43 (d, J_C–F = 244 Hz).

1-Fluoro-2-(3-iodo-2,2-dimethylpropyl)benzene (9).⁶³

NaI (16.9 g, 113.0 mmol) was added to a solution of 8 (5.9 g, 22.6 mmol) and NMP (45 mL). The mixture was heated to 140 °C overnight. After cooling to ambient temperature, the orange mixture was diluted with water, followed by extraction with pentane. The organic layer was then successively washed with saturated Na_S2O₃, saturated CuSO₄, water, and brine. Purification of the crude product by automatic flash column chromatography on silica gel (gradient elution from 2 to 20% EtOAc-hexanes) afforded a colorless, viscous liquid (89% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.05 (s, 6H), 2.72 (s, 2H), 3.20 (s, 2H), 6.99–7.10 (m, 2H), 7.19–7.28 (m, 2H). ¹⁹FNMR (376 MHz, CDCl₃): δ −114.86. ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 24.14, 26.79, 26.80, 35.28, 38.66, 115.33 (d, J_C–F = 23 Hz), 123.64 (d, J_C–F = 4 Hz), 125.25 (d, J_C–F = 15 Hz), 128.22 (d, J_C–F = 8 Hz), 132.44 (d, J_C–F = 5 Hz), 161.43 (d, J_C–F = 244 Hz).

Ethyl 2,2-Dimethyl-2,3-dihydro-1H-indene-4-carboxylate (10).⁵⁷

Method 1:

Compound 9 (4.5 g, 15.5 mmol) was first dissolved in 155 mL of anhydrous n-pentane-diethyl ether solution (4:1 by volume). The solution was cooled to −78 °C, after which t-BuLi (1.9 M solution in pentane, 26.1 mL, 49.5 mmol) was added dropwise. The solution was stirred at the same temperature for 15 min, and then, dry THF (7.8 mL) was added, turning the blue solution to a yellow, heterogeneous mixture. Next, the reaction mixture was warmed to room temperature (rt), stirred for 30 min, and cooled back to −78 °C. Ethyl chloroformate (15.3 mL, 154.7 mmol) was added dropwise, and the mixture was stirred for 15 min before warming up to rt and stirring for another 15 min. The mixture was quenched with water and extracted with diethyl ether (3 × 100 mL). The organic layer pool was dried over Na₂SO₄ and concentrated under reduced pressure, producing a brown, viscous liquid which was used in the next step without purification and characterization.

Method 2:

A mixture of TMS-esters 17 and 18 (20.3 mg, 0.07 mmol), KI (58.0 mg, 0.35 mmol), H₂O (6 μL, 0.35 mmol), and acetonitrile (0.7 mL) was mixed in a vial. Trimethylsilyl chloride (44 μL, 0.35 mmol) was then added dropwise, and the mixture was stirred at 60 °C. After 12 h, the mixture was quenched with saturated NaHCO₃ and extracted with ethyl acetate. The organic layer pool was washed brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by automatic flash column chromatography on silica gel (isocratic elution: 5% EtOAc-hexanes) to afford 10 as a colorless, viscous liquid (10.4 mg, 68% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.15 (s, 6H), 1.39 (t, 3H), 2.74 (s, 2H), 3.10 (s, 2H), 4.35 (q, 2H), 7.19 (t, 1H), 7.33 (d, 1H), 7.81 (d, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.38, 28.85, 39.52, 47.27, 48.79, 60.56, 126.08, 127.04, 127.86, 128.86, 145.10, 145.95, 167.25. HRMS (ESI, m/z): for C₁₄H₉O₂ [M + H]⁺ calcd, 219.1375; found, 219.1380.

2,2-Dimethyl-2,3-dihydro-1H-indene-4-carboxylic acid (11).

Ester 10 (crude from method 1 or purified from method 2) was first dissolved in ethanol (0.2 M). KOH (2.0 M solution in H₂O, 3.0 eq) was then added, and the mixture was stirred overnight at ambient temperature. The mixture was then cooled in an ice bath and acidified with concentrated HCl until pH 1. The solids were filtered and washed with cold water. The resulting white powder (Method 1: 17% over two steps, Method 2: 95%) was used in the next step without further purification. ¹H NMR (400 MHz, CDCl₃): δ 1.16 (s, 6H), 2.76 (s, 2H), 3.16 (s, 2H), 7.23 (t, 1H), 7.39 (d, 1H), 7.90 (d, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.95, 39.69, 47.34, 49.04, 125.90, 126.36, 128.81, 129.99, 145.42, 147.17, 172.84. HRMS (ESI, m/z): for C₁₂H₁₃O₂ [M − H]⁻ calcd, 189.0921; found, 189.0914. mp decomposes at 277 °C.

(2,2-dimethyl-2,3-dihydro-1H-inden-4-yl) (1-pentyl-1H-indol-3-yl)methanone (GBD-002, 3).⁶⁴

A mixture containing 11 (164.8 mg, 0.87 mmol) and dichloromethane (5.1 mL) was cooled to 0 °C. Thionyl chloride (0.16 mL, 2.2 mmol) was added dropwise, followed by a catalytic amount of DMF (7 μL, 0.09 mmol). After stirring at ambient temperature for 8 h, the mixture was concentrated under reduced pressure and redissolved in 0.6 mL of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). The solution was added dropwise to a mixture of N-pentylindole (486.6 mg, 2.6 mmol) and 0.6 mL of HFIP. The mixture was stirred at ambient temperature for 24 h and concentrated under reduced pressure. Purification by automatic flash column chromatography on silica gel (gradient elution from 5 to 40% EtOAc-hexanes) afforded a colorless oil (68% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.88 (t, 3H), 1.13 (s, 6H), 1.26–1.40 (m, 4H), 1.86 (quint, 2H), 2.78 (s, 2H), 2.91 (s, 2H), 4.12 (t, 2H), 7.19–7.24 (m, 1H), 7.29–7.35 (m, 3H), 7.36–7.42 (m, 2H), 7.46 (s, 1H), 8.39–8.44 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 13.91, 22.21, 28.69, 28.97, 29.56, 40.03, 47.05, 47.14, 47.53, 109.83, 116.34, 122.60, 122.79, 123.41, 125.68, 126.09, 126.56, 127.05, 136.86, 137.32, 137.61, 142.78, 144.92, 191.88. HRMS (ESI, m/z): for C₂₅H₃₀NO [M + H]⁺ calcd, 360.2322; found, 360.2317. Purity (qNMR), 100.4%.

5,5-Dimethyl-3-oxocyclohex-1-en-1-yl trifluoromethanesulfonate, VAT (13).⁵⁶

Pyridine (11.9 mL, 146.8 mmol) was added to a suspension of dimedone (10.3 g, 73.4 mmol) in dry dichloromethane (367 mL) at ambient temperature. The mixture was cooled to −78 °C and stirred for 10 min. Triflic anhydride (13.6 mL, 80.7 mmol) was then added slowly. After 20 min, the mixture was removed from the bath and warmed to ambient temperature. The mixture was quenched with 1 M HCl after complete consumption of the starting material and extracted with diethyl ether (3 × 50 mL). The organic layer pool was washed with saturated Na₂CO₃ and brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by automatic flash column chromatography on silica gel (gradient elution from 2% to 20% EtOAc-hexanes) to afford VAT as a colorless, viscous liquid (19.3 g, 97% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.14 (s, 6H), 2.32 (s, 2H), 2.55 (s, 2H), 6.08 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.03, 33.42, 42.37, 50.58, 116.84, 118.38, 166.09, 197.55.

Ethyl 5,5-Dimethyl-3-oxooct-7-ynoate (14).

LHMDS (1.0 M solution in THF, 9.1 mL, 9.1 mmol) was first cooled to −78 °C for 10 min. Ethyl acetate (0.9 mL, 9.1 mmol) was then added, and the mixture was stirred for 15 min. In a separate flask, a mixture of 13 (2.1 g, 7.6 mmol) in THF (30 mL) was also cooled to −78 °C. To this mixture, the ethyl lithioacetate solution was added slowly. After 20 min, the mixture was warmed to 0 °C, and then, LHMDS (1.0 M solution in THF, 22.7 mL, 22.7 mmol) was added dropwise over a period of 20 min. The resulting red solution was warmed to ambient temperature and stirred for 1 h. Saturated NH₄Cl was added, and the mixture was extracted with ether (3 × 20 mL). The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by automatic flash column chromatography on silica gel (isocratic elution: 5% EtOAc-hexanes) to afford the β-ketoester as a yellow, viscous liquid (1.0 g, 63% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.08 (s, 6H), 1.26 (t, 3H), 1.99 (t, 1H), 2.26 (d, 2H), 2.57 (s, 2H), 3.41 (s, 2H), 4.17 (q, 2H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.09, 26.93, 31.14, 33.42, 50.95, 51.55, 61.32, 70.54, 81.90, 167.12, 201.92.

Ethyl (Z)-5,5-Dimethyl-3-(((trifluoromethyl)sulfonyl)oxy)oct-2-en-7-ynoate (15).

A mixture of 14 (0.97 g, 4.6 mmol) and hexanes (23 mL) was cooled in an ice bath. Saturated aqueous LiOH (1.5 mL/mmol β-ketoester) was added, and the resulting suspension was stirred for 5 min. Triflic anhydride (1.9 mL, 11.5 mmol) was then added slowly. The mixture was removed from the bath and stirred for 30 min. After the reaction, the mixture was diluted with water and extracted with ether (3 × 10 mL). The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by automatic flash column chromatography on silica gel (isocratic elution: 5% EtOAc-hexanes) to afford the triflate as a light-yellow, viscous liquid (1.39 g, 88% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.09 (s, 6H), 1.32 (t, 3H), 2.09 (t, 1H), 2.16 (d, 2H), 2.40 (s, 2H), 4.26 (q, 2H), 5.84 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.00, 26.72, 31.61, 34.82, 45.00, 61.33, 71.51, 80.85, 115.01, 116.70, 119.88, 156.43, 162.40.

Ethyl 5,5-Dimethylocta-2,7-diynoate (16).

To a mixture of 15 (1.2 g, 3.5 mmol) in THF (17 mL) cooled at 0 °C, LHMDS (1.0 M solution in THF, 3.8 mL, 3.8 mmol) was added. The solution was warmed to ambient temperature and stirred for 1 h. After the reaction, saturated NH₄Cl was added, and the mixture was extracted with ether (3 × 10 mL). The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by automatic flash column chromatography on silica gel (gradient elution from 2 to 20% EtOAc-hexanes) to afford the diyne as a light-yellow oil (0.42 g, 63% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.11 (s, 6H), 1.31 (t, 3H), 2.03 (t, 1H), 2.22 (s, 2H), 2.37 (s, 2H), 4.22 (q, 2H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.04, 26.39, 30.87, 31.13, 34.09, 61.83, 70.76, 75.14, 81.29, 86.70, 153.72.

Ethyl 2,2-Dimethyl-5-(trimethylsilyl)-2,3-dihydro-1H-indene-4-carboxylate (17/18).

In a vial, [Rh(COD)₂]BF₄ (16.2 mg, 0.04 mmol), (±)-BINAP (24.6 mg, 0.04 mmol), and CH₂Cl₂ (7.9 mL) were stirred for 1 h at ambient temperature under a H₂ atmosphere. The mixture was concentrated and redissolved in CH₂Cl₂ (2.0 mL). TMS-acetylene (0.25 mL, 1.7 mmol) was added, followed by a solution of diyne 16 (152.0 mg, 0.79 mmol) in CH₂Cl₂ (7.9 mL). After stirring for 1 h at ambient temperature, the mixture was concentrated and purified by automatic flash column chromatography on silica gel (isocratic elution: 2% EtOAc-hexanes) to afford a mixture of TMS-esters as a colorless, viscous liquid (206 mg, 90% yield). ¹H NMR (400 MHz, CD₃CO): ortho isomer: δ 0.29 (s, 9H), 1.14 (s, 6H), 1.40 (t, 3H), 2.73 (s, 2H), 2.91 (s, 2H), 4.36 (q, 2H), 7.25 (d, 1H), 7.42 (d, 1H). Meta isomer: δ 0.28 (s, 9H), 1.15 (s, 6H), 1.40 (t, 3H), 2.74 (s, 2H), 3.09 (s, 2H), 4.36 (q, 2H), 7.47 (d, 1H), 7.95 (d, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 1.38, 15.37, 15.45, 29.90, 29.96, 40.42, 40.43, 48.28, 48.49, 49.30, 49.99, 61.58, 61.90, 127.60, 127.67, 133.91, 134.32, 134.65, 135.22, 138.57, 139.19, 144.47, 145.51, 146.68, 147.62, 168.62, 170.96. HRMS (ESI, m/z): for C₁₇H₂₇O₂Si [M + H]⁺ calcd, 291.1768; found, 291.1775.

3-Hydroxy-5,5-dimethyl-cyclohex-1-en-1-yl Trifluoromethane Sulfonate, VHAT (19).⁵⁸

A mixture of 13 (19.3 g, 70.9 mmol) and dry THF (322 mL) was cooled to −78 °C. DIBAL-H (1.0 M solution in toluene, 92.2 mL, 92.2 mmol) was added slowly, and the resulting mixture was stirred for 10 min. The mixture was warmed to ambient temperature until complete consumption of the starting material and then diluted with ether (~160 mL) before cooling in an ice bath. Water (3.7 mL) was added slowly, followed by 15% aqueous NaOH (3.7 mL) and water (9.2 mL). The mixture was removed from the bath and dried over MgSO₄. After 15 min of stirring, the mixture was filtered. The filtrate was concentrated under reduced pressure and finally purified by automatic flash column chromatography on silica gel (gradient elution from 5% to 40% EtOAc-hexanes) to afford VHAT as a colorless, viscous liquid (14.5 g, 75% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.99 (s, 3H), 1.09 (s, 3H), 1.33 (dd, 1H), 1.83 (dd, 1H), 1.96 (broad, s, 1H), 2.01 (d, 1H), 2.27 (d, 1H), 4.43–4.46 (m, 1H), 5.80 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 25.95, 30.55, 32.30, 41.08, 44.17, 65.57, 116.91, 120.35, 149.82.

Ethyl-E-4-(diethoxyphosphoryl)but-2-enoate.⁶⁵

Triethyl phosphite (10.0 g, 38.8 mmol) and ethyl 4-bromocrotonate (7.9 g, 46.6 mmol) were combined in a pressure flask and heated to 140 °C overnight. The mixture was cooled to ambient temperature, transferred to a flask, and distilled until complete removal of ethyl bromide. The crude product was a brown, viscous liquid and used in the next step without purification. The ¹H NMR is consistent with the literature report.

Ethyl (2E, 4E)-7,7-Dimethyldeca-2,4-dien-9-ynoate (20).⁵⁶

A mixture of 19 (5.3 g, 19.5 mmol), ethyl-E-4-(diethoxyphosphoryl)-but-2-enoate (7.3 g, 29.2 mmol), and dry THF (97 mL) was cooled to −78 °C. LHMDS (1.0 M solution in THF, 42.8 mL, 42.8 mmol) was added slowly, and the resulting mixture was stirred for 15 min. The mixture was warmed to ambient temperature and then at 60 °C for 1 h or until complete consumption of the starting material. After cooling to ambient temperature, 1 M HCl (~97 mL) was added, and the mixture was extracted with ethyl acetate (3 × 25 mL). The organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by automatic flash column chromatography on silica gel (gradient elution from 2 to 20% EtOAc-hexanes) to afford dienyne as a light-yellow, viscous liquid (3.2 g, 75% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.10 (s, 6H), 1.30 (t, 3H), 2.03 (t, 1H), 2.08 (d, 2H), 2.19 (d, 2H), 4.21 (q, 2H), 5.82 (d, 1H) 6.10–6.16 (m, 1H), 6.22 (dd, 1H), 7.28 (dd, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.30, 26.66, 31.51, 34.31, 44.43, 60.19, 70.32, 81.99, 119.83, 131.13, 140.44, 144.57, 167.19.

Ethyl 2,2-Dimethyl-2,3-dihydro-1H-indene-5-carboxylate (21).⁵⁶

To a mixture of 20 (3.2 g, 14.6 mmol) in dry dichloromethane (291 mL), di-μ-chlorobis(norbornadiene)dirhodium [Rh(nbd)Cl]₂ (0.14 g, 0.29 mmol) was added, followed by silver hexafluoroantimonate (AgSbF₆, 0.43 g, 1.24 mmol). The resulting mixture was stirred at ambient temperature for 1 h. DDQ (4.1 g, 17.5 mmol) was added, and stirring was continued until complete oxidation (1–2 h). After the reaction, the brown mixture was filtered in basic alumina. The filtrate was concentrated under reduced pressure and purified by automatic flash column chromatography on silica gel (gradient elution from 1 to 10% EtOAc-hexanes) to afford the ester as a colorless, viscous liquid (2.7 g, 85% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.15 (s, 6H), 1.38 (t, 3H), 2.75 (s, 4H), 4.35 (q, 2H), 7.21 (d, 1H), 7.81–7.86 (m, 2H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.38, 28.60, 40.51, 47.31, 47.74, 60.70, 124.53, 125.79, 127.88, 128.52, 143.84, 149.27, 167.09.

2,2-Dimethyl-2,3-dihydro-1H-indene-5-carboxylic acid (22).

Compound 21 (3.7 g, 17.1 mmol) was first dissolved in ethanol (82 mL). Aqueous KOH (9.1 mL) was then added, and the mixture was stirred overnight at ambient temperature. After the reaction, the mixture was neutralized with 3 M HCl. The solids were filtered and washed with cold water. The crude product was white, fluffy crystals (2.8 g, 85%) and used in the next step without further purification. ¹H NMR (400 MHz, CDCl₃): δ 1.16 (s, 6H), 2.77 (s, 4H), 7.25 (d, 1H), 7.89–7.93 (m, 2H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.69, 40.65, 47.36, 47.93, 124.80, 126.56, 127.40, 128.73, 144.16, 150.62, 172.73 HRMS (ESI, m/z): for C₁₂H₁₃O₂ [M − H]⁻ calcd, 189.0921; found, 189.0915. mp 158–160 °C.

General Procedure for the Synthesis of Indoles 23 and 24.⁶⁴

A mixture containing 22 (500.0 mg, 2.6 mmol) and dichloromethane (15.5 mL) was cooled to 0 °C. Thionyl chloride (0.4 mL, 5.3 mmol) was added dropwise, followed by a catalytic amount of DMF (0.02 mL, 0.3 mmol). After stirring at ambient temperature for 24 h, the mixture was concentrated under reduced pressure and redissolved in 1.5 mL of 1,1,1,3,3,3-HFIP. The acid chloride solution was added dropwise to a mixture of indole (2.8 mmol) and 2.0 mL of HFIP. The mixture was stirred at ambient temperature for 24 h, diluted with DCM, concentrated under reduced pressure, and purified by automatic flash column chromatography on silica gel (gradient elution from 1 to 10% MeOH─CH₂Cl₂).

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1H-indol-3-yl)-methanone (GBD-001, 23).

Obtained as white, powdery crystals (58% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 1.15 (s, 6H), 2.77 (overlapping s, 4H), 7.19–7.27 (m, 2H), 7.31 (d, 1H), 7.50–7.53 (m, 1H), 7.55–7.58 (m, 1H), 7.59 (d, 1H), 7.93 (s, 1H), 8.22–8.25 (m, 1H), 12.00 ppm (s, 1H). ¹³C{¹H}NMR (100 MHz, DMSO-d₆): δ 28.45 40.12, 46.94, 47.10, 112.20, 115.13, 121.50, 121.75, 123.01, 124.40, 124.72, 126.36, 126.99, 135.40, 136.66, 138.84, 143.34, 146.69, 190.08. HRMS (ESI, m/z): for C₂₀H₁₈NO [M − H]⁻ calcd, 288.1394; found, 288.1394. mp 224–225 °C. Purity (qNMR), 100.1%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1H-indol-3-yl)methanone (GBD-019, 24).

Obtained as white, powdery crystals (53% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 1.13 (s, 6H), 2.72 (s, 2H), 2.76 (s, 2H), 7.00 (t, 1H), 7.10 (t, 1H), 7.27 (d, 1H), 7.24 (d, 1H), 7.36 (d, 1H), 7.38 (d, 1H), 7.43 (s, 1H), 11.87 (s, 1H). ¹³C{¹H}NMR (100 MHz, DMSO-d₆): δ 14.13, 28.30, 40.21, 46.83, 47.07, 111.18, 112.75, 120.01, 120.74, 121.67, 124.35, 124.51, 126.77, 127.35, 134.92, 139.72, 143.27, 143.80, 146.77, 191.84. HRMS (ESI, m/z): for C₂₁H₂₂NO [M + H]⁺ calcd, 304.1696; found, 304.1636. mp 239–241 °C.

General Procedure for the Synthesis of 4, 26–34, 36, 37, and 39.

NaH (27.6 mg, 0.69 mmol) and DMSO (1.0 mL) were mixed thoroughly in a round-bottom flask. In a separate pear-shaped flask, 23 (100 mg, 0.35 mmol) and DMSO (1.5 mL) were heated to ~40 °C to form a solution and then added slowly to the NaH suspension. After stirring at ambient temperature for 30 min, the alkyl halide (0.42 mmol) was added. The resulting mixture was stirred overnight at ambient temperature. The reaction mixture was quenched with water and extracted with ethyl acetate (2 × 10 mL). The organic layer was washed with brine, dried over Na₂SO₄, concentrated under reduced pressure, and purified by automatic flash column chromatography on silica gel (gradient elution from 5 to 40% EtOAc-hexanes).

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-pentyl-1H-indol-3-yl)methanone (GBD-003, 4).

Obtained as white crystals (88% yield). ¹H NMR (400 MHz, CDCl₃) δ 0.89 (t, 3H), 1.19 (s, 6H), 1.26–140 (m, 4H), 1.87 (quintet, 2H), 2.79 (s, 4H), 4.15 (t, 2H), 7.22–7.27 (m, 1H), 7.28–7.35 (m, 2H), 7.36–7.41 (m, 1H), 7.57–7.62 (m, 2H), 7.64 (s, 1H), 8.39–8.45 (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃) δ 13.93, 22.22, 28.72, 28.98, 29.57, 40.48, 47.10, 47.52, 47.43, 109.77, 115.67, 122.40, 122.80, 123.31, 124.27, 125.15, 127.30, 127.45, 136.68, 136.75, 139.21, 143.88, 147.37, 191.20. HRMS (ESI, m/z) for C₂₅H₃₀NO [M + H]⁺ calcd, 360.2322, found 360.2318. mp 62–64 °C. Purity (qNMR), 99.9%.

(2,2-dimethyl-2,3-dihydro-1H-inden-5-yl) (1-propyl-1H-indol-3-yl)methanone (GBD-003, 26).

Obtained as white, powdery crystals (83% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.95 (t, 3H), 1.19 (s, 6H), 1.91 (sextet, 2H), 2.79 (s, 4H), 4.13 (t, 2H), 7.23–7.27 (m, 1H), 7.29–7.35 (m, 2H), 7.36–7.42 (m, 1H), 7.58–7.62 (m, 2H), 7.64 (s, 1H), 8.39–8.45 (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 11.42, 23.21, 28.73, 40.48, 47.52, 47.73, 48.74, 109.79, 115.67, 122.42, 122.82, 123.32, 124.26, 125.13, 127.28, 127.46, 136.76, 136.78, 139.21, 143.90, 147.36, 191.22. HRMS (ESI, m/z): for C₂₃H₂₆NO [M + H]⁺ calcd, 332.2009; found, 332.2006. mp 91–93 °C. Purity (qNMR), 99.2%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-butyl-1H-indol-3-yl)methanone (GBD-006, 27).

Obtained as white, powdery crystals (85% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.93 (t, 3H), 1.19 (s, 6H), 1.36 (sextet, 2H), 1.86 (quintet, 2H), 2.80 (s, 4H), 4.17 (t, 2H), 7.23–7.27 (m, 1H), 7.29–7.35 (m, 2H), 7.36–7.41 (m, 1H), 7.58–7.62 (m, 2H), 7.64 (s, 1H), 8.39–8.44 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃) δ 13.63, 20.09, 28.73, 31.92, 40.48, 46.86, 47.52, 47.73, 109.77, 15.69, 122.41, 122.82, 123.31, 124.27, 125.14, 127.28, 127.45, 136.68, 136.76, 139.21, 143.90, 147.37, 191.20. HRMS (ESI, m/z): for C₂₄H₂₈NO [M + H]⁺ calcd, 346.2165; found, 346.2163. mp 70–72 °C. Purity (qNMR), 100.1%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-hexyl-1H-indol-3-yl)methanone (GBD-007, 28).

Obtained as white, powdery crystals (82% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.87 (t, 3H), 1.19 (s, 6H), 1.31 (m, 6H), 1.87 (quintet, 2H), 2.80 (s, 2H), 4.15 (t, 2H), 7.23–7.27 (m, 1H), 7.29–7.36 (m, 2H), 7.37–7.41 (m, 1H), 7.57–7.62 (m, 2H), 7.64 (s, 1H), 8.39–8.44 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 13.97, 22.50, 26.54, 28.73, 29.83, 31.28, 40.48, 47.12, 47.53, 47.74, 109.77, 115.68, 122.41, 122.82, 123.31, 124.27, 125.15, 127.30, 127.47, 136.67, 136.75, 139.23, 143.88, 147.36, 191.20. HRMS (ESI, m/z): for C₂₆H₃₂NO [M + H]⁺ calcd, 374.2478; found, 374.2477. mp 61–63 °C. Purity (qNMR), 99.7%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-isobutyl-1H-indol-3-yl)methanone (GBD-008, 29).

Obtained as yellowish oil (53% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.94 (d, 6H), 1.18 (s, 6H), 2.23 (m, 1H), 2.79 (s, 4H), 3.94 (d, 2H), 7.22–7.26 (m, 1H), 7.28–7.33 (m, 2H), 7.34–7.39 (m, 1H), 7.54–7.61 (m, 2H), 7.64 (s, 1H), 8.39–8.44 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 20.19, 28.72, 29.15, 40.46, 47.51, 47.71, 54.68, 109.97, 115.54, 122.37, 122.76, 123.29, 124.25, 125.16, 127.29, 127.40, 137.00, 137.16, 139.18, 143.89, 147.38, 191.23. HRMS (ESI, m/z): for C₂₄H₂₈NO [M + H]⁺ calcd, 346.2165; found, 346.2161. Purity (qNMR), 98.5%.

(1-Benzyl-1H-indol-3-yl) (2,2-dimethyl-2,3-dihydro-1H-inden-5-yl)methanone (GBD-009, 30).

Obtained as white, powdery crystals (86% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.17 (s, 6H), 2.78 (s, 4H), 5.37 (s, 2H), 7.11–7.15 (m, 2H), 7.21–7.35 (m, 7H), 7.58–7.62 (app. d, 1H), 7.64 (s, 2H), 8.42–8.46 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.71, 40.47, 47.50, 47.71, 50.74, 110.14, 116.25, 122.64, 122.82, 123.63, 124.31, 125.16, 126.78, 127.31, 127.52, 128.10, 128.98, 135.92, 136.96, 137.02, 139.01, 143.91, 147.52, 191.24. HRMS (ESI, m/z): for C₂₇H₂₆NO [M + H]⁺ calcd, 380.2009; found, 380.2005. mp 111–114 °C. Purity (qNMR), 99.6%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(4-fluorobenzyl)-1H-indol-3-yl)methanone (GBD-010, 31).

Obtained as white, powdery crystals (90% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 2.78 (s, 4H), 5.34 (s, 2H), 6.98–7.04 (m, 2H), 7.09–7.14 (m, 2H), 7.24 (m, 1H), 7.28–7.35 (m, 3H), 7.58–7.65 (m, 3H), 8.40–8.43 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.70, 40.49, 47.50, 47.71, 50.09, 110.02, 115.86, 116.06, 116.40, 122.72, 122.90, 123.72, 124.33, 125.15, 127.31, 127.55, 131.64, 128.50, 128.58, 136.68, 136.87, 138.95, 143.96, 147.62, 163.65, 191.20. ¹⁹FNMR (376 MHz, CDCl₃): δ −113.65. HRMS (ESI, m/z): for C₂₇H₂₅FNO [M + H]⁺ calcd, 398.1915; found, 398.1911. mp 124–126 °C. Purity (qNMR), 100.5%.

(E)-(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(pent-2-en-1-yl)-1H-indol-3-yl)methanone (GBD-011, 32).

Obtained as white, powdery crystals (88% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.98 (t, 3H), 1.18 (s, 6H), 2.06 (quint, 2H), 2.79 (s, 4H), 4.69–4.74 (app. d, 2H), 5.56–5.65 (m, 1H, J = 15.3 Hz, 6.0 Hz, 1.4 Hz), 5.71–5.80 (m, 1H, J = 15.4 Hz, 6.2 Hz, 1.4 Hz), 7.22–7.27 (m, 1H), 7.29–7.35 (m, 2H), 7.36–7.42 (m, 1H), 7.58–7.62 (m, 2H), 7.65 (s, 1H), 8.39–8.44 (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 13.21, 25.18, 28.72, 40.48, 47.52, 47.72, 48.94, 110.05, 115.86, 122.46, 122.70, 122.77, 123.33, 124.28, 125.14, 127.30, 127.52, 136.47, 136.85, 137.27, 139.17, 143.87, 147.38, 191.20. HRMS (ESI, m/z): for C₂₅H₂₈NO [M + H]⁺ calcd, 358.2165; found, 358.2163. mp 80–82 °C. Purity (qNMR), 99.1%.

(Z)-(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(pent-2-en-1-yl)-1H-indol-3-yl)methanone (GBD-012, 33).

Obtained as white, powdery crystals (68% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.07 (t, 3H), 1.17 (s, 6H), 2.24 (quint, 2H), 2.78 (s, 4H), 4.78 (app. d, 2H), 5.51–5.59 (m, 1H, J = 10.8 Hz, 6.8 Hz, 0.8 Hz), 5.68–5.76 (m, 1H, J = 10.7 Hz, 7.4 Hz, 0.8 Hz), 7.22–7.26 (m, 1H), 7.29–7.34 (m, 2H), 7.35–7.40 (m, 1H), 7.56–7.62 (m, 2H), 7.63 (s, 1H), 8.37–8.43 (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.00, 20.96, 28.72, 40.48, 43.79, 47.52, 47.73, 109.83, 115.91, 122.54, 122.58, 122.82, 123.36, 124.28, 125.14, 127.29, 127.60, 136.18, 136.83, 136.85, 139.17, 143.87, 147.38, 191.18. HRMS (ESI, m/z): for C₂₅H₂₈NO [M + H]⁺ calcd, 358.2165; found, 358.2162. mp 70–71 °C. Purity (qNMR), 99.5%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(pent-4-en-1-yl)-1H-indol-3-yl)methanone (GBD-013, 34).

Obtained as light-yellow, powdery crystals (74% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 1.97 (quint, 2H), 2.09 (q, 2H), 2.79 (s, 4H), 4.16 (t, 2H), 5.01–5.04 (m 1H), 5.05–5.09 (m, 1H), 5.72–5.84 (ddt, 1H), 7.22–7.26 (m, 1H), 7.29–7.34 (m, 2H), 7.35–7.40 (m, 1H), 7.56–7.61 (m, 2H), 7.63 (s, 1H), 8.39–8.44 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.72, 28.77, 30.67, 40.48, 46.24, 47.53, 47.73, 109.75, 115.80, 116.17, 122.46, 122.86, 123.37, 124.28, 125.12, 127.26, 127.48, 136.69, 136.71, 136.79, 139.18, 143.88, 147.39, 191.18. HRMS (ESI, m/z): for C₂₅H₂₈NO [M + H]⁺ calcd, 358.2165; found, 358.2162. mp 78–79 °C. Purity (qNMR), 99.3%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(pent-2-yn-1-yl)-1H-indol-3-yl)methanone (GBD-015, 36).

Obtained as light-yellow, powdery crystals (55% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.13 (t, 3H), 1.19 (s, 6H), 2.18–2.25 (qt, 2H), 2.79 (s, 4H), 4.88 (t, 2H), 7.23–7.27 (m, 1H), 7.31–7.38 (m, 2H), 7.42–7.48 (m, 1H), 7.60–7.68 (m, 2H), 7.77 (s, 1H), 8.39–8.45 (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.37, 13.58, 28.71, 37.02, 40.49, 47.53, 47.73, 71.93, 88.93, 109.79, 116.00, 122.70, 122.88, 123.53, 124.31, 125.15, 127.33, 127.63, 136.07, 136.48, 139.05, 143.85, 147.47, 191.18. HRMS (ESI, m/z): for C₂₅H₂₆NO [M + H]⁺ calcd, 356.2009; found, 332.2007. mp 98–99 °C. Purity (qNMR), 96.8%.

(1-Allyl-1H-indol-3-yl) (2,2-dimethyl-2,3-dihydro-1H-inden-5-yl)-methanone (GBD-016, 37).

Obtained as yellowish oil (81% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 2.79 (s, 4H), 4.76 (m, 2H), 5.10–5.17 (m, 1H), 5.23–5.28 (m, 1H), 5.94–6.04 (m, 1H), 7.22–7.26 (m, 1H), 7.29–7.38 (m, 3H), 7.57–7.61 (m, 2H), 7.64 (s, 1H), 8.41–8.45 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.67, 40.43, 47.46, 47.66, 49.33, 109.97, 116.01, 118.39, 122.50, 122.74, 123.42, 124.25, 125.04, 127.23, 127.39, 132.07, 136.60, 136.80, 139.04, 143.87, 147.40, 191.17. HRMS (ESI, m/z): for C₂₃H₂₄NO [M + H]⁺ calcd, 330.1852; found, 330.1848. Purity (qNMR), 98.3%.

Ethyl 2-(3-(2,2-Dimethyl-2,3-dihydro-1H-indene-5-carbonyl)-1H-indol-1-yl)acetate (GBD-018, 39).

Obtained as yellowish oil (28% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 1.26 (t, 3H), 27.8, (s, 4H), 4.22 (q, 2H), 4.86 (s, 2H), 7.22–7.36 (m, 4H), 7.59–7.63 (m, 2H), 7.65 (s, 1H), 8.40–8.45 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 14.10, 28.69, 40.47, 47.48, 47.70, 48.15, 62.11, 109.20, 116.80, 122.76, 122.91, 123.85, 124.32, 125.11, 127.21, 127.34, 137.10, 137.32, 138.89, 143.90, 147.60, 167.49, 191.28. HRMS (ESI, m/z): for C₂₄H₂₆NO₃ [M + H]⁺ calcd, 376.1907; found, 376.1904. Purity (qNMR), 96.5%.

(2,2-dimethyl-2,3-dihydro-1H-inden-5-yl) (1-methyl-1H-indol-3-yl)methanone (GBD-004, 25).

NaH (14.2 mg, 0.36 mmol) and DMSO (1.0 mL) were mixed thoroughly in a round-bottom flask. In a separate pear-shaped flask, 23 (51.4 mg, 0.18 mmol) and DMSO (1.5 mL) were heated to ~40 °C to form a solution and then added slowly to the NaH suspension. After stirring at ambient temperature for 30 min, (chloromethyl)trimethylsilane (0.03 mL, 0.21 mmol) was added. The resulting mixture was stirred overnight at ambient temperature. The reaction was quenched with water and extracted with ethyl acetate (2 × 5 mL). The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. Purification by automatic flash column chromatography on silica gel (gradient elution from 5 to 40% EtOAc-hexanes) afforded white, powdery crystals (64% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 2.79, (s, 4H), 3.85 (s, 3H), 7.22–7.26 (m, 1H), 7.31–7.39 (m, 3H), 7.56 (s, 1H), 7.57–7.62 (app. d, 1H), 7.63 (s, 1H), 8.40–8.44 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃) δ 28.71, 33.53, 40.48, 47.51, 47.71, 109.53, 115.75, 122.51, 122.74, 123.47, 124.28, 125.06, 127.03, 127.28, 137.46, 137.61, 139.15, 143.86, 147.36, 191.18. HRMS (ESI, m/z): for C₂₁H₂₂NO [M + H]⁺ calcd, 304.1696; found, 304.1693. mp 92–95 °C. Purity (qNMR), 99.9%.

General Procedure for the Synthesis of 35 and 38.

To a mixture of 23 (100 mg, 0.35 mmol) and DMSO (2.0 mL) was added KOH (38.8 mg, 0.69 mmol). After stirring at ambient temperature for 1 h, the alkyl halide (0.52 mmol) was added. The resulting mixture was stirred for 2 h at ambient temperature. The reaction was quenched with water and extracted with ethyl acetate (2 × 10 mL). The organic layer was washed with brine, dried over Na₂SO₄, concentrated under reduced pressure, and purified by automatic flash column chromatography on silica gel (gradient elution from 5 to 40% EtOAc-hexanes).

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(pent-4-yn-1-yl)-1H-indol-3-yl)methanone (GBD-014, 35).

Obtained as light-yellow, powdery crystals (79% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 2.03–2.12 (m, 3H), 2.17–2.22 (td, 2H, J = 6.4 Hz, 2.5 Hz), 2.79, (s, 4H), 4.34 (t, 2H), 7.22–7.28 (m, 1H), 7.29–7.36 (m, 2H), 7.39–7.45 (m, 1H), 7.58–7.63 (d, 1H), 7.65 (m, 2H), 8.39–8.46 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 15.64, 28.06, 28.71, 40.49, 45.29, 47.53, 47.73, 70.21, 82.30, 109.68, 115.84, 122.55, 122.91, 123.48, 124.32, 125.20, 127.33, 127.53, 136.59, 136.97, 139.06, 143.88, 147.47, 191.15. HRMS (ESI, m/z): for C₂₅H₂₆NO [M + H]⁺ calcd, 356.2009; found, 356.2006. mp 84–85 °C. Purity (qNMR), 100.4%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(prop-2-yn-1-yl)-1H-indol-3-yl)methanone (GBD-017, 38).

Obtained as light-yellow oil (66% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.19 (s, 6H), 2.49 (t, 1H), 2.80 (s, 4H), 4.93 (d, 2H), 7.23–7.28 (m, 1H), 7.32–7.41 (m, 2H), 7.43–7.49 (m, 1H), 7.59–7.67 (m, 2H), 7.74 (s, 1H), 8.39–8.45 ppm (m, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 28.71, 36.53, 40.50, 47.50, 47.73, 75.02, 76.29, 109.64, 116.47, 122.87, 122.97, 123.76, 124.34, 125.14, 127.33, 127.56, 135.81, 136.41, 138.89, 143.96, 147.63, 191.21. HRMS (ESI, m/z): for C₂₃H₂₂NO [M + H]⁺ calcd, 328.1696; found, 328.1695. Purity (qNMR), 100.0%.

General Procedure for the Synthesis of 40–48 and 50.

NaH (26.4 mg, 0.66 mmol) and DMSO (1.0 mL) were mixed thoroughly in a round-bottom flask. In a separate pear-shaped flask, 24 (100 mg, 0.33 mmol) and DMSO (1.5 mL) were heated to ~40 °C to form a solution and then added slowly to the NaH suspension. After stirring at ambient temperature for 30 min, the alkyl halide (0.40 mmol) was added. The resulting mixture was stirred overnight at ambient temperature. The reaction mixture was quenched with water and extracted with ethyl acetate (2 × 10 mL). The organic layer was washed with brine, dried over Na₂SO₄, concentrated under reduced pressure, and purified by automatic flash column chromatography on silica gel (gradient elution from 5 to 40% EtOAc-hexanes).

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-propyl-1H-indol-3-yl)methanone (GBD-020, 40).

Obtained as yellowish oil (72% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.01 (t, 3H), 1.17 (s, 6H), 1.85 (sextet, 2H), 2.59 (s, 3H), 2.75 (s, 2H), 2.78 (s, 2H), 4.11 (t, 2H), 7.05 (app. t, 1H), 7.15–7.21 (m, 2H), 7.33 (app. t, 2H), 7.53 (app. d, 1H), 7.60 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 11.48, 12.43, 22.99, 28.64, 40.50, 44.84, 47.40, 47.74, 109.36, 113.92, 121.02, 121.06, 121.70, 124.24, 125.41, 127.28, 127.86, 135.83, 139.56, 143.70, 143.79, 147.83, 193.28. HRMS (ESI, m/z): for C₂₄H₂₈NO [M + H]⁺ calcd, 346.2165; found, 346.2162. Purity (qNMR), 98.8%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-butyl-2-methyl-1H-indol-3-yl)methanone (GBD-021, 41).

Obtained as yellowish oil (71% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.98 (t, 3H), 1.16 (s, 6H), 1.43 (sextet, 2H), 1.78 (quintet, 2H), 2.59 (s, 3H), 2.74 (s, 2H), 2.78 (s, 2H), 4.13 (t, 2H), 7.04 (app. t, 1H), 7.15–7.21 (m, 2H), 7.32 (dd, 2H), 7.52 (app. d, 1H), 7.59 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.42, 13.83, 20.31, 28.65, 31.81, 40.52, 43.15, 47.41, 47.76, 109.33, 113.94, 121.02, 121.09, 121.70, 124.25, 125.42, 127.31, 127.88, 135.79, 139.58, 143.72, 143.77, 147.84, 193.28. HRMS (ESI, m/z): for C₂₅H₃₀NO [M + H]⁺ calcd, 360.2322; found, 360.2318. Purity (qNMR), 97.3%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-pentyl-1H-indol-3-yl)methanone (GBD-022, 42).

Obtained as yellowish oil (74% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.92 (t, 3H), 1.17 (s, 6H), 1.34–1.43 (m, 4H), 1.80 (quintet, 2H), 2.59 (s, 3H), 2.75 (s, 2H), 2.78 (s, 2H), 4.12 (t, 2H), 7.05 (m, 1H), 7.15–7.21 (m, 2H), 7.33 (app. t, 2H), 7.53 (app. d, 1H), 7.61 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.42, 13.95, 22.41, 28.65, 29.13, 29.43, 40.51, 43.34, 47.41, 47.75, 109.33, 113.92, 121.02, 121.08, 121.71, 124.24, 125.41, 127.31, 127.86, 135.78, 139.59, 143.70, 143.75, 147.82, 193.26. HRMS (ESI, m/z): for C₂₆H₃₂NO [M + H]⁺ calcd, 374.2478; found, 374.2477. Purity (qNMR), 96.2%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-hexyl-2-methyl-1H-indol-3-yl)methanone (GBD-023, 43).

Obtained as yellowish oil (62% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.89 (t, 3H), 1.17 (s, 6H), 1.28–1.44 (m, 6H), 1.79 (quintet, 2H), 2.59 (s, 3H), 2.74 (s, 2H), 2.78 (s, 2H), 4.12 (t, 2H), 7.04 (m, 1H), 7.15–7.21 (m, 2H), 7.32 (app. t, 2H), 7.52 (app. d, 1H), 7.59 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.44, 14.00, 22.55, 26.72, 28.65, 29.70, 31.47, 40.52, 43.39, 47.42, 47.76, 109.32, 113.93, 121.02, 121.09, 121.71, 124.25, 125.43, 127.32, 127.87, 135.78, 139.60, 143.71, 143.75, 147.83, 193.28. HRMS (ESI, m/z): for C₂₇H₃₄NO [M + H]⁺ calcd, 388.2635; found, 388.2632. Purity (qNMR), 98.1%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-benzyl-2-methyl-1H-indol-3-yl)methanone (GBD-024, 44).

Obtained as yellowish oil (56% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 2.54 (s, 3H), 2.76 (s, 2H), 2.79 (s, 2H), 5.40 (s, 2H), 7.01–7.11 (m, 3H), 7.13–7.23 (m, 2H), 7.24–7.33 (m, 4H), 7.39–7.43 (app. d, 1H), 7.54–7.58 (app. d, 1H), 7.63 (br s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.48, 28.65, 40.53, 46.62, 47.42, 47.77, 109.54, 114.45, 121.16, 121.36, 122.14, 124.30, 125.47, 125.99, 127.31, 127.69, 127.96, 128.97, 136.28, 136.31, 139.39, 143.79, 143.95, 148.06, 193.34. HRMS (ESI, m/z): for C₂₈H₂₈NO [M + H]⁺ calcd, 394.2165; found, 394.2162. Purity (qNMR), 97.4%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (1-(4-fluorobenzyl)-2-methyl-1H-indol-3-yl)methanone (GBD-025, 45).

Obtained as yellowish oil (52% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (s, 6H), 2.53 (s, 3H), 2.76 (s, 2H), 2.79 (s, 2H), 5.36 (s, 2H), 6.98–7.02 (m, 4H), 7.06–7.11 (m, 1H), 7.14–7.27 (m, 3H), 7.39–7.43 (app. d, 1H), 7.54–7.57 (app. d, 1H), 7.63 (br s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.44, 28.64, 40.54, 45.98, 47.41, 47.77, 109.40, 114.59, 115.83, 116.04, 121.23, 121.45, 122.24, 124.32, 125.47, 127.32, 127.96, 127.72, 127.64, 132.01, 136.14, 139.29, 143.86, 143.83, 148.16, 163.41, 193.29. ¹⁹FNMR (376 MHz, CDCl₃): δ −114.40. HRMS (ESI, m/z): for C₂₈H₂₇FNO [M + H]⁺ calcd, 412.2071; found, 412.2068. Purity (qNMR), 96.8%.

(E)-(2,2-dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-(pent-2-en-1-yl)-1H-indol-3-yl)methanone (GBD-026, 46).

Obtained as yellowish oil (78% yield). ¹H NMR (400 MHz, CDCl₃): δ 0.94 (t, 3H), 1.17 (s, 6H), 2.02 (quint, 2H), 2.56 (s, 3H), 2.75 (s, 2H), 2.78 (s, 2H), 4.69–4.73 (m, 2H), 5.48–5.58 (m, 2H), 7.03–7.09 (m, 1H), 7.15–7.22 (m, 2H), 7.31 (dd, 1H), 7.37 (dd, 1H), 7.54 (d, 1H), 7.61 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.32, 13.19, 25.07, 28.62, 40.49, 44.92, 47.38, 47.73, 109.41, 114.02, 121.02, 121.09, 121.79, 122.51, 124.23, 125.41, 127.24, 127.87, 135.38, 135.84, 139.51, 143.69, 143.88, 147.85, 193.27. HRMS (ESI, m/z): for C₂₆H₃₀NO [M + H]⁺ calcd, 372.2322; found, 372.2318. Purity (qNMR), 95.9%.

(Z)-(2,2-dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-(pent-2-en-1-yl)-1H-indol-3-yl)methanone (GBD-027, 47).

Obtained as white, powdery crystals (73% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.11 (t, 3H), 1.17 (s, 6H), 2.29 (quint, 2H), 2.58 (s, 3H), 2.75 (s, 2H), 2.78 (s, 2H), 4.78–4.83 (m, 2H), 5.32–5.40 (m, 1H), 5.57–5.65 (m, 1H), 7.04–7.09 (m, 1H), 7.16–7.22 (m, 2H), 7.30 (dd, 1H), 7.36 (dd, 1H), 7.53 (d, 1H), 7.61 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.39, 14.03, 21.07, 28.65, 40.52, 40.63, 47.41, 47.76, 109.24, 114.15, 121.12, 121.16, 121.81, 123.38, 124.26, 125.44, 127.37, 127.91, 134.90, 135.76, 139.50, 143.67, 143.73, 147.91, 193.26. HRMS (ESI, m/z) for C₂₆H₃₀NO [M + H]⁺: calcd, 372.2322; found, 372.2322. mp 65–66 °C. Purity (qNMR), 100.5%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-(pent-4-en-1-yl)-1H-indol-3-yl)methanone (GBD-028, 48).

Obtained as yellowish oil (48% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.17 (s, 6H), 1.90 (quintet, 2H), 2.18 (q, 2H), 2.58 (s, 3H), 2.74 (s, 2H), 2.78 (s, 2H), 4.14 (t, 2H), 5.03–5.14 (m, 2H), 5.78–5.90 (ddt, 1H), 7.02–7.08 (m, 1H), 7.15–7.21 (m, 2H), 7.29–7.36 (app. t, 2H), 7.53 (app. d, 1H), 7.60 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.41, 28.58, 28.64, 30.90, 40.50, 42.68, 47.41, 47.75, 109.27, 114.04, 115.95, 121.08, 121.11, 121.76, 124.25, 125.41, 127.31, 127.87, 135.75, 136.94, 139.52, 143.64, 143.72, 147.87, 193.25. HRMS (ESI, m/z): for C₂₆H₃₀NO [M + H]⁺ calcd, 372.2322; found, 372.2319. Purity (qNMR), 99.7%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-(pent-2-yn-1-yl)-1H-indol-3-yl)methanone (GBD-030, 50).

Obtained as yellowish oil (22% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.09 (t, 3H), 1.17 (s, 6H), 2.15 (qt, 2H), 2.62 (s, 3H), 2.75 (s, 2H), 2.78 (s, 2H), 4.84 (t, 2H), 7.05–7.11 (m, 1H), 7.17–7.25 (m, 2H), 7.36–7.42 (m, 2H), 7.54 (app. d, 1H), 7.62 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.29, 12.33, 13.59, 28.64, 33.04, 40.51, 47.39, 47.75, 72.65, 86.73, 109.33, 114.42, 121.08, 121.31, 122.02, 124.27, 125.46, 127.26, 127.94, 135.57, 139.36, 143.38, 143.74, 148.01, 193.23. HRMS (ESI, m/z): for C₂₆H₂₈NO [M + H]⁺ calcd, 370.2165; found, 370.2163. Purity (qNMR), 99.4%.

General Procedure for the Synthesis of 49 and 51.

To a mixture of 24 (100 mg, 0.33 mmol) and DMSO (2.0 mL) was added KOH (37.0 mg, 0.66 mmol). After stirring at ambient temperature for 1 h, the alkyl halide (0.49 mmol) was added. The resulting mixture was stirred for 2 h at ambient temperature. The reaction mixture was quenched with water and extracted with ethyl acetate (2 × 10 mL). The organic layer was washed with brine, dried over Na₂SO₄, concentrated under reduced pressure, and purified by automatic flash column chromatography on silica gel (gradient elution from 5 to 40% EtOAc-hexanes).

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-(pent-4-yn-1-yl)-1H-indol-3-yl)methanone (GBD-029, 49).

Obtained as yellowish oil (63% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.17 (s, 6H), 2.02 (quintet, 2H), 2.10 (t, 1H), 2.26–2.32 (td, 2H), 2.61 (s, 3H), 2.75 (s, 2H), 2.78 (s, 2H), 4.29 (t, 2H), 7.03–7.09 (m, 1H), 7.16–7.22 (m, 2H), 7.33–7.40 (m, 2H), 7.53 (app. d, 1H), 7.60 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.37, 15.95, 28.17, 28.62, 40.49, 41.83, 47.38, 47.73, 69.89, 82.75, 109.23, 114.16, 121.10, 121.17, 121.88, 124.25, 125.41, 127.29, 127.87, 135.79, 139.42, 143.65, 143.73, 147.94, 193.23. HRMS (ESI, m/z): for C₂₆H₂₈NO [M + H]⁺ calcd, 370.2165; found, 370.2163. Purity (qNMR), 100.5%.

(2,2-Dimethyl-2,3-dihydro-1H-inden-5-yl) (2-methyl-1-(prop-2-yn-1-yl)-1H-indol-3-yl)methanone (GBD-031, 51).

Obtained as colorless oil (32% yield). ¹H NMR (400 MHz, CDCl₃): δ 1.17 (s, 6H), 2.33 (t, 1H), 2.63 (s, 3H), 2.75 (s, 2H), 2.79 (s, 2H), 4.89 (d, 2H), 7.07–7.11 (m, 1H), 7.18–7.25 (m, 2H), 7.36–7.43 (m, 2H), 7.54 (app. d, 1H), 7.62 (s, 1H). ¹³C{¹H}NMR (100 MHz, CDCl₃): δ 12.25, 28.65, 32.61, 40.53, 47.40, 47.77, 73.17, 77.22, 109.12, 114.80, 121.23, 121.55, 122.27, 124.32, 125.50, 127.30, 127.99, 135.50, 139.17, 143.01, 143.81, 148.21, 193.22. HRMS (ESI, m/z): for C₂₄H₂₄NO [M+ H]⁺ calcd, 342.1852; found, 342.1849. Purity (qNMR), 97.2%.

Pharmacology.

CP-55,940, rimonabant, and AM-630 were obtained from Tocris Bioscience (Bristol, UK). All drugs were prepared as a stock solution in 100% DMSO at a concentration of 100 mM, divided into aliquots, and maintained at −4 °C until use. [³H]CP-55,950 (168 Ci/mmol) and [³⁵S]GTPγS (1250 Ci/mmol) were purchased from Perkin Elmer (Boston, MA). All other reagents were obtained from Fisher Scientific Inc. (Pittsburgh, PA).

Competition Receptor Binding.

Increasing concentrations of the GBD compounds were incubated with 0.2 nM of the nonselective CB₁/CB₂ agonist [³H]CP-55,940 in a final volume of 1 mL of the binding buffer (50 mM Tris, 0.05% bovine serum albumin, 5 mM of MgCl₂, pH 7.4) as described previously.⁶⁶ Each binding assay contained 100 or 50 μg of membrane protein prepared from CHO-hCB1 or CHO-hCB₂ cells, respectively. Reactions were incubated for 90 min at rt. Nonspecific binding was defined as binding observed in the presence of 10 μM of the nonselective CB₁/CB₂ ligand WIN-55,212-2. Reactions were terminated by rapid vacuum filtration through Whatman GF/B glass fiber filters, followed by three washes with the ice-cold binding buffer. Filters were then immediately placed into scintillation vials with 4 mL of the scintiverse™ BD cocktail scintillation fluid (Fisher Scientific, Fair Lawn, NJ). Samples were incubated overnight in the scintillation fluid, and vortexed and bound reactivity was determined by employing a liquid scintillation spectrophotometer (Tri Carb 2100 TR Liquid Scintillation Analyzer, Packard Instrument Company, Meriden, CT).

[³⁵S]GTPγS Binding.

All [³⁵S]GTPγS assays were conducted as described previously⁶² in a buffer containing 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), 100 mM NaCl, 10 mM MgCl₂, 0.05% BSA, and 20 units/L of adenosine deaminase. Assays were performed in triplicate in a final volume of 1 mL with all reactions containing 0.1 nM [³⁵S]GTPγS and an experimental drug or drug vehicle and 50 or 25 μg of membrane protein prepared from CHO-hCB₂ cells, respectively. Nonspecific binding was defined by inclusion of 10 μM of nonradiolabeled GTPγS. Reactions were incubated at 30 °C for 30 min and terminated by rapid vacuum filtration through Whatman GF/B glass fiber filters and followed by four 1 mL washes with the ice-cold filtration buffer (20 mM HEPES, pH 7.4, 0.05% BSA). Filters were then immediately placed into scintillation vials with 4 mL of the scintiverse™ BD cocktail scintillation fluid (Fisher Scientific, Fair Lawn, NJ). Samples were incubated overnight in the scintillation fluid, and vortexed and bound reactivity was determined by employing a liquid scintillation spectrophotometer (Tri Carb 2100 TR Liquid Scintillation Analyzer, Packard Instrument Company, Meriden, CT).

Data Analysis.

Curve fitting was conducted utilizing GraphPad Prism v6.0b (GraphPad Software, Inc.; San Diego, CA). Nonlinear regression for one-site competition was used to determine the IC₅₀ for competition receptor binding. The Cheng-Prusoff equation was employed to convert the experimental IC₅₀ values obtained from competition receptor binding experiments to K_i values (a quantitative measure of receptor affinity). Statistical significance of values from 100% (basal) in the [³⁵S]GTPγS assay was assessed by the one-sample t-test (*P < 0.05 and **P < 0.01).

Molecular Modeling.

To elucidate the binding modes of the GBD series of compounds, a hybrid docking/MD simulation approach was utilized. This methodology has been previously used accurately and precisely to estimate the binding affinity and selectivity of compounds toward the cannabinoid receptors.⁶⁷ In summary, the selected ligands (Figure 2) GBD-002 and GBD-003 had their lowest energy conformation estimated using OpenEye OMEGA.⁶⁸ These ligands were then prepared with Autodock Tools to the PDBQT format for use with Autodock Vina.^69,70 CB₁ and CB₂ receptor structures (PDB ID: 5XR8 for CB₁R and 5ZTY for CB₂R) were used for the initial docking, and the grid center was placed upon the original crystal structure ligand.^71,72 The resulting binding poses (Figure 3A1,A2,B1,B2) then had their atomic charges calculated using the AM1-BCC method in the Antechamber module of AMBER16^73,74 and were subsequently energy-minimized using the SANDER module of AMBER16.⁷³ The energy-minimized binding structures then had their binding free energy estimated using the MMPBSA.py⁷⁵ module of AMBER16.⁷³ The rest of the GBD series of compounds were then modeled using PyMol⁷⁶ to revise the structure of GBD-003 within either CB₁R or CB₂R as the initial binding structure. The initial binding structures of these compounds were similarly energy-minimized and had their binding free energy estimated by using the same MMPBSA.py module.

ABBREVIATIONS

ECS

endocannabinoid system

HFIP

1,1,1,3,3,3-hexafluoroisopropanol

BINAP

(2,2′-bis(diphenylphosphino)-1,1′-binaphthyl)

SI

selectivity index

nbd

norbornadiene

n-BuLi

n-butyllithium

t-BuLi

tert-butyllithium

MsCl

mesyl chloride

Tf₂O

triflic anhydride