N-(3-Hydroxyphenyl)-3,8-diazabicyclooctanes as Opioid Receptors Probes. 1. Investigation of the Phenolic Hydroxyl Group

Abstract

N-(3-Hydroxyphenyl)-3,8-diazabicyclooctanes represent a novel class of synthetic opioids with potent activity and a distinct pharmacological profile. The prototype of this class, atoxifent, exhibits strong opioid receptor activity while minimizing severe respiratory depression, distinguishing it from fentanyl. To gain deeper insight into ligand-receptor interactions and the factors influencing functional activity, we systematically investigated the role of the phenolic hydroxyl group. Our approach focused on (1) assessing hydrogen bonding interactions with opioid receptors, (2) modulating ionization via pKa adjustments, and (3) exploring bioisosteric replacements. In vitro assay showed that 3-amino (11), 3-cyclopropyl sulfonamide (12), and 3-carboxamido (13) derivatives retained high MOR agonist activity. Notably, 13 displayed approximately 2.4 times greater in vitro metabolic stability than atoxifent. In vivo antinociceptive studies showed that 11, 12, and 13 act as partial agonists. These findings offer valuable insight into how N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes interact with opioid receptors.

Graphical Abstract

1. Introduction

The quest for safer and more effective analgesics has driven innovation in opioid pharmacology, marked by significant breakthroughs in structural design. A pivotal advancement occurred in 1960 with the introduction of 4-anilido-1-phenethylpiperidine compounds, led by fentanyl (Figure 1), which redefined pain management benchmarks [1]. However, the groundwork for such advancements had been laid earlier in the late 1950s, when a Swiss chemical company synthesized 2-benzylbenzimidazoles, a structurally novel analgesic class [2–5]. Among these, etonitazene stood out, demonstrating antinociceptive potency approximately three times greater than morphine in rodent studies [6]. Although no benzimidazole derivatives reached the market as analgesics, this research demonstrated for the first time that high morphine-like activity could be achieved with structures lacking the complex phenanthrene skeleton of traditional opium alkaloids [2, 6].

Figure 1:

Structures of fentanyl, etonitazene, bromadol, atoxifent, morphine, cyclazocine, and methadone.

In the 1970s, Upjohn and other pharmaceutical companies sought non-addictive opioid analgesics with morphine-like potency but without respiratory depression or dependency [7]. While no commercially viable drugs emerged, the research uncovered potent, selective opioid receptor agonists that advanced understanding of opioid mechanisms. One such compound, BDPC/bromadol, showed an analgesic potency about 500 times that of morphine and around three times that of fentanyl. However, none of these substances underwent clinical trials or commercial development, though some appeared on the illicit market [8, 9].

This ongoing exploration of synthetic opioids underscores the dual challenge posed by these compounds: their potential for therapeutic innovation and their contribution to a growing public health crisis. The rise of illicit synthetic opioids has exacerbated the opioid epidemic, demanding not only effective public health interventions but also advancements in the development of safer opioid therapeutics [10]. According to recent CDC data, opioid overdose deaths in 2021 surpassed 100,000 for the first time, marking a devastating milestone driven largely by the escalating impact of synthetic opioids like illicitly manufactured fentanyl [11]. This surge persisted despite widespread public awareness campaigns, policies aimed at curbing non-prescription opioid access, and efforts to mitigate the risks of misuse.

In the pursuit of more effective rescue agents for synthetic opioid overdoses, our research unexpectedly led to the discovery of a novel chemotype of potent synthetic opioids. Specifically, we found that conformationally constraining a piperazine ring into a 3,8-diazabicyclooctane ring transforms a mu-opioid receptor (MOR) antagonist into a potent MOR agonist [12]. Atoxifent, the prototype of this class, demonstrated strong in vitro agonist activity and, in mice, produced prolonged antinociception reversible by naltrexone. Repeated dosing of atoxifent induced antinociceptive tolerance and withdrawal effects comparable to fentanyl. However, in rats, atoxifent did not cause profound respiratory depression typically associated with fentanyl-induced fatalities, highlighting its potential as a safer therapeutic option.

The identification of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes as a structural class of highly potent synthetic opioid opens a new avenue for chemical investigation of opioid receptor pharmacology. It also raises significant questions as to their mechanism of action with opioid receptors compared to other classes of opioids. The present work aims to investigate the role of phenolic hydroxyl group in the bioactivity of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes.

Classically, the phenolic hydroxyl group in opioids is thought to be essential for tight binding to the MORs [13]. Specifically, the phenolic hydroxyl group of the opioid forms a water-mediated bridge to H6.52, enhancing affinity and efficacy [14–16]. Consequently, substitutions of the phenolic hydroxyl group that disrupt hydrogen bonding generally result in lower affinity MOR ligands. For example, codeine, heroin, and 3-deoxymorphine which have 3-methyl ether, 3-acetate ester, and 3-H substitutions, respectively, have MOR affinities substantially lower than those of their 3-hydroxy congeners [13, 17, 18].

While many classical opioids feature a phenolic hydroxyl group, several potent analgesics exhibit strong receptor activity even in its absence. For example, fentanyl retains high MOR affinity despite lacking a phenolic hydroxyl group [19] and its phenolic derivatives show no improvement in receptor activity [20]. Similarly, methadone, bromadol, and etonitazene maintain potent opioid activity without the phenolic hydroxyl group, [2–5, 21, 22] demonstrating that different structural modifications can interact with MORs through alternative binding mechanisms. This adaptability is further evident in benzomorphan-based opioids like cyclazocine, where bioisosteric modifications of the phenolic hydroxyl group, such as amino and carboxamido, were introduced to slow metabolism while preserving receptor affinity and prolonging duration of action [23–27]. These findings emphasize that while the phenolic hydroxyl group is crucial in classical opioids, certain classes of MOR agonists utilize different molecular interactions to engage with the receptor.

As N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes represents a new class of synthetic opioids, the role of the phenolic hydroxyl group in opioid receptor interaction remains poorly understood. Our previous work demonstrated that moving the phenolic hydroxyl group from the 3-position to the 2-position maintains comparable activity [12]. In contrast, removing the phenolic hydroxyl group results in a 300-fold loss of activity, emphasizing the critical role of the phenolic hydroxyl group in receptor functional activity [12]. These findings highlight the need for a deeper understanding of the molecular interactions mediated by the phenolic hydroxyl group within this novel scaffold.

To address this, we prepared a small series of analogues (1 – 20) to help elucidate the role of the phenolic hydroxyl group in producing MOR agonist activity. Our approach focused on the following points: (1) evaluating the role of hydrogen bonding interactions with opioid receptors, (2) assessing the influence of ionization through pKa modulation, and (3) exploring bioisosteric replacements to determine their effects on receptor activity, intrinsic efficacy, and in vitro metabolic stability. Our results are described below.

2. Results and Discussion

2.1. Chemistry

Compounds 1 – 20 were synthesized to assess how the phenolic hydroxyl group in N-(3-hydroxyphenyl)-3,8-diazabicyclooctane influences functional activity at MORs. Structural modifications, including homologation of the phenyl ring and hydroxyl group, were introduced to probe potential hydrogen bonding interactions. Additionally, chloro and fluoro substituents were introduced to assess how changes in pKa influence receptor activity. Furthermore, we evaluated the effects of various phenolic hydroxyl group substitutions, including nitro, amino, cyclopropyl sulfonamide, and carboxamido groups, to better understand their impact on receptor interaction. All compounds were synthesized and evaluated for functional activity at MORs (Table 1). Select compounds were further analyzed for functional selectivity of downstream signaling pathway, activity at delta (DORs), kappa (KORs), and nociceptin (NORs) receptors (Table 2), in vitro metabolism (Table 3), and in vivo effects (Figure 5).

Table 1.

Evaluation of functional activity of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes at MORs using Eurofins DiscoverX HitHunter^® and PathHunter^® Assays.

Compound	R	MOR cAMP			β-Arrestin Recruitment
		pEC50±SEMa	EC50 (nM)	%Emax ± SEMa	pEC50 ± SEMa	EC50 (nM)	%Emax ± SEMa
DAMGO	N/A	9.5 ± 0.1	0.32	102.9 ± 1.1	7.1 ± 0.1	75	102.3 ± 0.2
Morphine	N/A	8.1 ± 0.1	7.9	101.6 ± 0.2	6.4 ± 0.1	360	33.4 ± 1.8
Fentanyl	N/A	9.7 ± 0.1	0.20	102.4 ± 0.3	7.3 ± 0.1	54	68.1 ± 2.0
Buprenorphine	N/A	8.8 ± 0.1	1.6	93.4 ± 3.3	< 5.0	> 10,000	N/C
Etonitazene	N/A	10.5 ± 0.2	0.032	101.0 ± 0.4	8.5 ± 0.1	3.0	94.4 ± 5.9
Bromadol	N/A	11.5 ± 0.2	0.0032	100.4 ± 0.6	9.1 ± 0.1	0.86	107.3 ± 2.1
Atoxifent		9.5 ± 0.1	0.32	99.2 ± 0.7	< 5.0	> 10,000	0
1		6.6 ± 0.1	250	58.7 ± 5.8	< 5.0	> 10,000	0
2		7.4 ± 0.2	40	45.5 ± 4.4	N/D	N/D	N/D
3		7.7 ± 0.1	20	102.1 ± 1.1	N/D	N/D	N/D
4		6.6 ± 0.1	250	107.8 ± 0.9	N/D	N/D	N/D
5		7.2 ± 0.1	63	102.6 ± 0.5	N/D	N/D	N/D
6		7.7 ± 0.1	20	99.6 ± 1.1	N/D	N/D	N/D
7		8.0 ± 0.1	10	94.9 ± 1.8	N/D	N/D	N/D
8		9.1 ± 0.2	0.79	99.9 ± 0.6	< 5.0	> 10,000	0
9		7.9 ± 0.2	13	102.6 ± 1.0	< 5.0	> 10,000	[16.3 ± 3.2]
10		8.0 ± 0.1	10	100.6 ± 0.4	< 5.0	> 10,000	0
11		9.6 ± 0.1	0.25	103.6 ± 2.4	< 5.0	> 10,000	0
12		9.3 ± 0.1	0.50	98.8 ± 0.4	7.6 ± 0.1	25	4.3 ± 0.2
13		9.6 ± 0.1	0.25	101.8 ± 0.3	7.8 ± 0.1	16	7.5 ± 1.3
14		8.5 ± 0.1	3.2	101.6 ± 0.6	6.2 ± 0.1	700	26.5 ± 2.4
15		< 5.0	> 10,000	[92.7 ± 3.3]	N/D	N/D	N/D
16		8.4 ± 0.2	4.0	60.9 ± 3.9	< 5.0	> 10,000	0
17		8.6 ± 0.1	2.5	76.9 ± 6.2	< 5.0	> 10,000	0
18		8.0 ± 0.2	10	100.2 ± 0.5	< 5.0	> 10,000	[10.7 ± 1.3]
19		8.6 ± 0.1	2.5	101.2 ± 0.6	< 5.0	> 10,000	0
20		6.5 ± 0.1	320	100.0 ± 0.1	N/D	N/D	N/D

^aValues are expressed as the mean ± SEM of at least three independent experiments; N/A = Not applicable; N/D = Not determined; N/C = Not calculable; Data in square brackets are max response observed in the test dose range.

Table 2.

Evaluation of functional activity of selected N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes at DORs, KORs, and NORs using Eurofins DiscoverX HitHunter^® Assays.

	DOR cAMP			KOR cAMP			NOR cAMP
Compound	pEC50±SEMa	EC50 (nM)	Emax ± SEM (%)a	pEC50± SEMa	EC50 (nM)	Emax ± SEM (%)a	pEC50± SEMa	EC50 (nM)	Emax ± SEM (%)a
Morphine	6.3 ± 0.1	500	74.5 ± 4.7	6.5 ± 0.1	320	84.6 ± 3.8	< 5.0	> 10,000	[13.8 ± 5.8]
Fentanyl	6.8 ± 0.1	160	87.5 ± 1.5	6.1 ± 0.1	790	89.7 ± 3.7	< 5.0	> 10,000	[47.0 ± 8.1]
Buprenorphine	9.1 ± 0.2	0.79	65.5 ± 7.8	8.5 ± 0.1	3.2	66.3 ± 6.7	7.3 ± 0.1	50	74.7 ± 4.8
DADLE	10.4 ± 0.1	0.04	87.1 ± 1.8	N/D	N/D	N/D	N/D	N/D	N/D
U50,488H	N/D	N/D	N/D	9.6 ± 0.1	0.25	99.7 ± 0.9	N/D	N/D	N/D
N/OFQ	< 5.0	> 10,000	0	< 5.0	> 10,000	[74.3 ± 3.6]	10.0 ± 0.1	0.10	100.5 ± 0.3
Atoxifent	8.8 ± 0.1	1.6	69.3 ± 7.7	< 5.0	> 10,000	0	< 5.0	> 10,000	[17.4 ± 1.1]
11	8.8 ± 0.1	1.6	71.1 ± 3.6	< 5.0	> 10,000	0	< 5.0	> 10,000	0
12	8.8 ± 0.1	1.6	71.0 ± 4.7	< 5.0	> 10,000	0	< 5.0	> 10,000	0
13	8.0 ± 0.2	10	81.3 ± 3.0	< 5.0	> 10,000	0	< 5.0	> 10,000	0
16	7.8 ± 0.3	16	34.8 ± 3.0	N/D	N/D	N/D	< 5.0	> 10,000	0
17	8.3 ± 0.2	5.0	58.0 ± 2.8	N/D	N/D	N/D	< 5.0	> 10,000	0
18	6.8 ± 0.1	160	56.2 ± 6.2	N/D	N/D	N/D	7.6 ± 0.5	25	41.9 ± 19.3
19	7.9 ± 0.3	13	71.4 ± 1.8	N/D	N/D	N/D	8.5 ± 0.1	3.2	19.5 ± 3.4

^aValues are expressed as the mean ± SEM of at least three independent experiments; N/D = Not determined; Data in square brackets are max response observed in the test dose range

Table 3:

Evaluation of in vitro metabolic stability in human liver microsomes (HLM) and mouse liver microsomes (MLM) of selected N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes.

Compound	HLM t1/2 (min)	MLM t1/2 (min)
Morphine	> 145.0	> 145.0
Fentanyl	53.4	4.6
Atoxifent	38.0	1.7
8	26.6	3.3
11	36.5	3.5
12	35.6	6.4
13	92.7	14.2
16	13.0	2.1
17	58.7	5.8

Figure 5.

Nociceptive dose-response curves for buprenorphine (ED₅₀ = 0.6441 mg/kg, E_max = 50.12, n = 6, R² = 0.8235), atoxifent (ED₅₀ = 0.9323 mg/kg, E_max = 91.71, n = 6, R² = 0.8834), 11 (ED₅₀ = 0.8494 mg/kg, E_max = 43.86, n = 6, R² = 0.5160), 12 (ED₅₀ = 2.804 mg/kg, E_max = 33.45, n = 5, R² = 0.5893), and 13 (ED₅₀ = 0.6581 mg/kg, E_max = 31.91, n = 6 R² = 0.6040). Statistical significance using log ED₅₀ comparison: P value = 0.4143, F(DFn, DFd) = 0.9910 (4, 154). Statistical significance for E_max comparison across all groups: P value = 0.0388, F(4, 154) = 2.592. Statistical significance of E_max comparing only 11 – 13 and buprenorphine: P = 0.1654, F(3, 122) = 1.725. Points represent Mean (± SEM) percent maximum possible effect (% MPE) for vehicle and five cumulative doses of agonist.

Synthesis of 1 – 10 and 20 are shown in Scheme 1. Key intermediate 21 was prepared as described previously [12]. Coupling of 21 with commercially available 1-bromo-4-(methoxymethoxy)benzene and 1-bromo-3,5-bis(ethoxymethoxy)benzene afforded 22a and 22b respectively in good yields. Compounds 22a and 22b were then treated with 48% HBr under microwave conditions to afford 1 and 2, respectively. Commercially available 23a – d were treated with (chloromethoxy)ethane under basic conditions to give 24a – d respectively in excellent yields. Buchwald-Hartwig coupling of 21 with 24a – d provided 25a – d in moderate yields [28–31]. Treatment of 25a – d with 48% HBr under microwave conditions yielded 3, 6, 7, and 8 respectively. In addition, 4, 5, 9, and 20 were obtained in low to moderate yields through Buchwald-Hartwig coupling of intermediate 21 with commercially available 2-(3-bromophenyl)propan-2-ol, 3-(3-bromophenyl)oxetan-3-ol, 1-bromo-3-(difluoromethyl)benzene, and 2-(3-bromophenyl)oxazole, respectively. Coupling of 21 under basic conditions with commercially available aryl fluoride produced 10 [28–31].

Scheme 1:

Synthesis of 1 – 10 and 20.^a

^aReagents and Conditions: (a) Appropriately substituted aryl bromide, Pd(OAc)₂, XPhos, NaOtBu, 1,4-dioxane, 100°C, overnight, 20 – 73%; (b) 48% HBr, methanol, MW, 65°C, 20 minutes, 83 – 99%; (c) Appropriate phenol or alcohol, (chloromethoxy)ethane, DIPEA, DCM, rt, overnight, 84 – 99%; (d) Appropriately substituted aryl fluoride, potassium carbonate, anhydrous DMSO, 85°C, overnight, 17%.

Synthesis of 13 – 19 are shown in Scheme 2. Coupling of 21 with commercially available aryl fluoride under basic conditions yielded 26a – c in low to moderate yields. Hydrolysis of 26a – c using 30% (w/w) hydrogen peroxide solution provided amides 13, 14, and 15, respectively in moderate yields. Commercially available phenols 27a and 27b were treated with (chloromethoxy)ethane under basic conditions to afford intermediates 28a and 28b in good yields. Buchwald-Hartwig coupling of 21 with 28a and 28b, produced 29a and 29b, respectively in low to moderate yields [28–31]. Subsequent hydrolysis of 29a and 29b with 30% (w/w) hydrogen peroxide solution yielded 30a and 30b in good yields, which were then treated with 48% HBr under microwave conditions to give amides 16 and 17, respectively in good yields. Additionally, Buchwald-Hartwig coupling of 21 with commercially available aryl bromides afforded 31a and 31b in moderate to excellent yields [28–31]. Hydrolysis of 31a and 31b using 30% (w/w) hydrogen peroxide solution provided amides 18 and 19, respectively in excellent yields.

Scheme 2:

Synthesis of 13 – 19.^a

^aReagents and Conditions: (a) Appropriate aryl fluoride, potassium carbonate, anhydrous DMSO, 85°C, overnight, 20 – 25%; (b) 30% (w/w) Hydrogen peroxide solution, potassium carbonate, anhydrous DMSO, rt, 2 hours, 30 – 83%; (c) Appropriate phenol, (chloromethoxy)ethane, DIPEA, DCM, rt, overnight, 70 – 71%; (d) Intermediate 28a or 28b, Pd(OAc)₂, XPhos, NaOtBu, 1,4-dioxane, 100°C, overnight, 15 – 59%; (e) 48% HBr, methanol, MW, 65°C, 20 minutes, 54 – 68%; (f) Appropriate aryl bromide, Pd₂(dba)₃, (±)-BINAP, cesium carbonate, toluene, 100°C, overnight, 42 – 85%.

Synthesis of 11 and 12 are shown in Scheme 3. Atoxifent was coupled with 5-amino-6-chloropyrazine-2,3-dicarbonitrile in the presence of cesium carbonate to afford 11 [32]. Sulfonylation of 11 with cyclopropanesulfonylchloride under basic conditions gave 12 in low yield.

Scheme 3:

Synthesis of 11 and 12.^a

^aReagents and Conditions (a) 5-Amino-6-chloropyrazine-2,3-dicarbonitrile, cesium carbonate, DMSO, 80°C, 10 minutes, 62%; (b) Acetic acid, zinc dust, 80°C, 10 minutes; (c) Cyclopropanesulfonylchloride, DIPEA, acetonitrile, rt, 3 hours, 34%.

2.2. Functional Assessment of N-(3-Hydroxyphenyl)-3,8-diazabicyclooctanes at MORs

Compounds 1 – 20 were characterized at MORs using the commercially available Eurofins DiscoverX HitHunter^® cAMP Assay (Table 1) [33–35]. Moving the OH to the 4-position (1) led to a 700-fold reduction in potency and lower intrinsic activity compared to atoxifent (1: pEC₅₀, %E_max = 6.6, 58.7 vs atoxifent: pEC₅₀, %E_max = 9.5, 99.2). The reduced potency and efficacy of 1 are likely attributed to its inability to interact with the receptor site in the same manner as atoxifent.

To better understand the impact of hydrogen bonding interactions, we introduced a phenolic hydroxyl group at the 5-position (2). We aimed to improve hydrogen bonding within the receptor pocket by leveraging the rotational flexibility around the aryl-nitrogen axis. The 5-position was chosen over the 2- or 4-position, as substituting the phenolic hydroxyl group at those sites would lead to a catechol moiety [36]. However, this modification resulted in a partial agonist with reduced activity (2: pEC₅₀, %E_max = 7.4, 45.5 vs atoxifent: pEC₅₀, %E_max = 9.5, 99.2). This decrease in potency and intrinsic efficacy could be attributed to steric clashes of 2 at the receptor site. Additionally, differences in pKa of the phenolic hydroxyl group between 2 and atoxifent could affect the compound’s ionization, potentially altering receptor interactions (Supporting Table 1: 2 pKa = 9.01 and 10.76, atoxifent pKa = 9.81).

To explore the role of the hydrogen bond of atoxifent, we explored the effect of homologation between the aromatic ring and the phenolic hydroxyl group (3). The goal was to increase the distance between the phenyl ring and the hydroxyl group, allowing the hydroxyl group to extend further into the receptor pocket. However, this led to a 40-fold reduction in activity (3: pEC₅₀ = 7.7 vs atoxifent: pEC₅₀ = 9.5). Next, we constrained the benzyl alcohol moiety by introducing a gem-dimethyl group (4),[37] but this modification led to a 12-fold decrease in activity compared to 3 (4: pEC₅₀ = 6.6 vs 3: pEC₅₀ = 7.7). In pursuit of a better alternative, we substituted the gem-dimethyl group with an oxetane group (5), a contemporary bioisostere of the gem-dimethyl group [38]. This modification was better tolerated than 4 (5: pEC₅₀ = 7.2 vs 4: pEC₅₀ = 6.6) likely due to the oxetane moiety’s ability to act as a hydrogen bond acceptor, which contributed to increased activity. Overall, homologation between the phenyl ring and the OH group led to reduced activity at MORs.

To further understand the reduced activity and efficacy of 2 compared to atoxifent, we examined whether ionization changes resulting from pKa modulation were affecting receptor interactions. To do this, we replaced the phenolic hydroxyl group in 2 with a chlorine atom (6). This modification resulted in a full agonist with similar potency (6: pEC₅₀, %E_max = 7.7, 99.6 vs 2: pEC₅₀, %E_max = 7.4, 45.5). The improved efficacy may be due to the difference in pKa of the phenolic hydroxyl group between 6 and 2 (Supporting Table 1: 6 pKa = 8.70, 2 pKa = 9.01 and 10.76). To further investigate this result, we replaced the chlorine atom with a methyl group (7), which is comparable in size to chlorine but increases the pKa of the phenolic hydroxyl group (Supporting Table 1: 7 pKa = 9.89, 6 pKa = 8.70) [39]. Intriguingly, this modification resulted in comparable activity and efficacy to 6 (7: pEC₅₀ = 8.0, %E_max = 94.9 vs 6: pEC₅₀, %E_max = 7.7, 99.6). These findings indicate that receptor activity is not solely determined by ionization changes from pKa modulation; instead, factors like the sterics of the substituent group may also contribute. To investigate this, we introduced a fluorine atom (8) at the 5-position, given its atomic size is similar to hydrogen [40]. Compound 8 was better tolerated than 6 and 7 likely due to fluorine’s smaller atomic radius compared to chlorine atom and methyl group (8: pEC₅₀ = 9.1 vs 6: pEC₅₀ = 7.7 vs 7: pEC₅₀ = 8.0). Collectively, these results suggest that receptor activity is governed not only by ionization changes from pKa modulation but also by the sterics of the substituent group.

Next, we explored alternative substitutions for the phenolic hydroxyl group. By replacing the phenolic hydroxyl group with a difluoromethyl group (9), a bioisostere that preserves hydrogen bonding while introducing greater lipophilicity (Supporting Table 1: 9 logP = 5.30, atoxifent logP = 3.94) [41–43]. This modification resulted in a 60-fold decrease in potency (9: pEC₅₀ =7.9 vs atoxifent: pEC₅₀ = 9.5). We then explored the electronic effects on activity by replacing the phenolic hydroxyl group with a nitro group (10). This modification exhibited reduced activity compared to atoxifent (10: pEC₅₀ = 8.0 vs atoxifent: pEC₅₀ = 9.5). This is likely due to the reduced electron density on the aromatic ring, which may facilitate interaction with the receptor through electrostatic interactions with oppositely charged amino acid residues in the receptor’s binding pocket. Next, we evaluated the potential of a 3-NH₂ group (11) as a bioisostere for the phenolic hydroxyl group in this scaffold. To our delight, 11 is well tolerated at MORs (11: pEC₅₀ = 9.6). We then assessed the effect of the cyclopropyl sulfonamide moiety (12) at the MORs, as it acts as a nonclassical bioisostere of phenol [44]. Interestingly, 12 exhibited comparable activity to atoxifent (12: pEC₅₀ = 9.3 vs atoxifent: pEC₅₀ = 9.5). These modifications indicate that although electrostatic interactions are tolerated, hydrogen bonding interactions are more favorable for receptor activity.

Given the important role of hydrogen bond interactions, we evaluated the carboxamido group (13), a phenol bioisostere that retains the ability to engage in hydrogen bonding interaction [45, 46]. This modification was well tolerated (13: pEC₅₀ = 9.6 vs atoxifent: pEC₅₀ = 9.5) and is consistent with the classical opioid SAR [26]. Encouraged by this outcome, we investigated the impact of moving the carboxamido group to assess how 2-carboxamido (14) and 4-carboxamido (15) substitution influences receptor interactions. Moving the carboxamido to the 2-position resulted in nearly a 14-fold decrease in activity (14: pEC₅₀ = 8.5 vs 13: pEC₅₀ = 9.6). This decrease in activity is likely to result from unfavorable steric effects of 2-carboxamido at the receptor. Additionally, substituting the carboxamido group at the 4-position led to a complete loss of potency at MORs (15: pEC₅₀ <5.0 vs 13: pEC₅₀ = 9.6), which could be attributed to the steric effects of 4-carboxamido group at the receptor.

Given the potent MOR activity and efficacy observed with 13, we sought to further investigate the likely bioactive orientation of the carboxamido group by introducing conformational constraint. We aimed to use intramolecular hydrogen bonds to stabilize the conformation of the ligand, as intramolecular hydrogen bonds can reduce both conformational and translational entropy [47]. To this end, we introduced a phenolic hydroxyl group at the 4-position (16), facilitating a hydrogen bond interaction with the carbonyl group of the carboxamide, as shown in conformation A (Figure 2). This modification resulted in partial agonist activity compared to 13 (16: pEC₅₀, %E_max = 8.4, 60.9 vs 13: pEC₅₀, %E_max = 9.6, 101.8). Next, we examined the conformation arising between the carbonyl group of the carboxamide and the 2-position phenolic hydroxyl group (17), as depicted in conformation B (Figure 2). This modification also resulted in partial agonist activity compared to 13 (17: pEC₅₀, %Emax = 8.6, 76.9 vs 13: pEC₅₀, %E_max = 9.6, 101.8). The shift in biological activity from a full agonist (13) to a partial agonist (16 and 17) could be due to the likelihood of the hydroxyl group being ionized in solution (Supporting Table 1: 16 pKa = 8.78, 17 pKa = 8.51). These results suggest that the incorporation of a phenolic hydroxyl group to induce conformational constraint in an attempt to identify the bioactive conformation was a suboptimal approach.

Figure 2:

Possible conformation A and B of carboxamido group.

In pursuit of an alternative constraint motif, we incorporated a fluorine atom adjacent to the 3-carboxamido group. The introduction of a fluorine atom at the 4-positon (18) allowed us to explore the conformation formed between the hydrogen atom of the carboxamide and the fluorine atom, as illustrated in conformation C (Figure 3). This modification resulted in lower potency, but comparable efficacy compared to 13 (18: pEC₅₀, %E_max = 8.0, 100.2 vs 13: pEC₅₀, %E_max = 9.6, 101.8). To further study the alternative conformation, we added a fluorine atom at the 2-position (19) to explore the conformation formed between the hydrogen atom of the carboxamide and the fluorine atom, as shown in conformation D (Figure 3). This modification also led to lower potency, but comparable efficacy compared to 13 (19: pEC₅₀, %E_max = 8.6, 101.2 vs 13: pEC₅₀, %E_max = 9.6, 101.8). The higher potency of 19 (pEC₅₀ = 8.6) relative to 18 (pEC₅₀ = 8.0) suggests that conformation D more closely represents the bioactive conformation of the 3-carboxamido derivative (13).

Figure 3:

Possible conformations C and D of carboxamido group.

Subsequently, we investigated the effect of replacing the 3-carboxamido group (13) with an oxazole ring 20 [48]. Given our earlier observations on the important role of strong hydrogen bonding interactions and the role of conformation of the carboxamido group on receptor interaction, we anticipated that the oxazole ring would not possess optimal receptor interactions. As expected, this modification resulted in a significant loss of activity (20: pEC₅₀ = 6.5 vs 13: pEC₅₀ = 9.6).

To understand the downstream signaling pathway of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes, we evaluated selected analogues for their ability to recruit β-arrestin2. This was assessed via MOR activation using the previously described enzyme fragment complementation (EFC) assay (Table 1) [33]. Analogues were chosen for β-arrestin-2 recruitment analysis based upon the activity seen in the G-protein mediated cAMP assay. All analogues with potencies below 10 nM, along with 1, which demonstrated partial agonist activity, were included in the evaluation.

Similar to atoxifent, 1, 8, 10, 11, 16, 17, and 19 did not recruit β-arrestin2 (pEC₅₀, %E_max = <5.0, 0). However, 9 (pEC₅₀ < 5.0, %E_max = 16.3), 12 (pEC₅₀ = 7.6, %E_max = 4.3), 13 (pEC₅₀ = 7.8, %E_max = 7.5), 14 (pEC₅₀ = 6.2, %E_max = 26.5), and 18 (pEC₅₀ < 5.0, %E_max = 10.7) were found to weakly recruit β-arrestin2. The differences in β-arrestin2 recruitment profiles for 9, 12 – 14, and 18 compared to atoxifent may be due to differential receptor binding. However, assuming 9, 12 – 14, and 18 adopt binding modes comparable to atoxifent due to their structural similarity, it is possible that these analogs induce distinct receptor conformations that weakly promote arrestin-mediated signaling [49, 50].

Based on these findings, the role of the phenolic hydroxyl group in influencing the MOR activity of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes is summarized in Figure 4. The position of the phenolic hydroxyl group is important for optimal receptor interaction, as evidenced by the reduced potency of the 4-phenol derivative (1) and 3-homologated derivatives (3 – 5). Hydrogen bond acceptors are less favorable, as indicated by the lower potency of 3-nitro (10) and 3-oxazole (20), whereas hydrogen bond donors at the 3-position are more favorable for MOR activity, as demonstrated by the potent activity of 11 – 13. Ionization via pKa modulation influences receptor activity but does not fully explain the observed effects. Steric factors also appear to contribute, with the 5-position favoring smaller substituents, as seen by the comparable activity of the 5-fluoro derivative (8) and atoxifent. Bioisosteric replacements of the phenolic hydroxyl group, including 3-amino (11), 3-sulfonamide (12), and 3-carboxamido (13), are well tolerated, though 12 and 13 show weak β-arrestin2 recruitment.

Figure 4.

Role of the phenolic hydroxyl group in the bioactivity of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes.

Given the potent G-protein-mediated cAMP activity and no or weak β-arrestin2 recruitment, we screened 11 – 13 for activity at delta opioid receptors (DORs), kappa opioid receptors (KORs), and nociceptin receptors (NORs) (Table 2). We found that 11 – 13 were found to possess potent partial agonist activity at DORs comparable to atoxifent (atoxifent: pEC₅₀ = 8.8, %E_max = 69.3, 11: pEC₅₀ = 8.8, %E_max = 71.1, 12: pEC₅₀ = 8.8, %E_max = 71.0, 13: pEC₅₀ = 8.0, %E_max = 81.3). At KORs, 11 – 13 showed no agonist activity (pEC₅₀ < 5.0), similar to atoxifent. In contrast, opioid agonists such as morphine, fentanyl, and buprenorphine exhibited activity at KORs. Studies suggest that the absence of KOR activation may offer a more favorable therapeutic window, minimize dysphoric side effects while maintaining potent analgesic efficacy [51]. Moreover, similar to atoxifent, 11 – 13 displayed no significant agonist activity at NORs.

Additionally, 16 – 19 were also screened at DORs and NORs due to their high potency in the G-protein-mediated cAMP assay and lack of β-arrestin2 recruitment. Interestingly, 16 results in reduced potency and efficacy at DORs compared to atoxifent (atoxifent: pEC₅₀ = 8.8, %E_max = 69.3, 16: pEC₅₀ = 7.8, %E_max = 34.8). But 17 results in comparable potency and efficacy at DORs to atoxifent (atoxifent: pEC₅₀ = 8.8, %E_max = 69.3, 17: pEC₅₀ = 8.3, %E_max = 58.0). In contrast, 18 displays lower potency but comparable efficacy to atoxifent (atoxifent: pEC₅₀ = 8.8, %E_max = 69.3, 18: pEC₅₀ = 6.8, %E_max = 56.2). However, 19 exhibits lower potency but similar efficacy compared to atoxifent (atoxifent: pEC₅₀ = 8.8, %E_max = 69.3, 19: pEC₅₀ = 7.9, %E_max = 71.4). Like atoxifent, 16 and 17 showed no agonist activity at NORs. In contrast, 18 and 19 demonstrated partial agonist activity at NORs (18: pEC₅₀ = 7.6, %E_max = 41.9, 19: pEC₅₀ = 8.5, %E_max = 19.5).

2.3. Evaluation of In Vitro Metabolism

To further understand the pharmacokinetic properties of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes we tested the in vitro metabolic stability of selected analogues in human liver microsomes (HLM) and mouse liver microsomes (MLM) (Table 3). Morphine (HLM t_1/2 > 145.0 minutes), fentanyl (HLM t_1/2 = 53.4 minutes), and atoxifent (HLM t_1/2 = 38.0 minutes) demonstrated good in vitro metabolic stability, with morphine exhibiting the highest stability, followed by fentanyl and atoxifent. Given the potent and G-protein selective activity of 8 observed through G-protein mediated cAMP and β-arrestin2 recruitment assays, we wanted to assess its in vitro metabolic stability. However, we found that 8 (HLM t_1/2 = 26.6 minutes) has a lower metabolic stability profile than atoxifent. We then evaluated the in vitro metabolic stability profile for 11 (HLM t_1/2 = 36.5 minutes) and 12 (HLM t_1/2 = 35.6 minutes) both of which displayed a metabolic profile in human liver microsomes similar to atoxifent.

Next, we assessed the in vitro metabolic stability of 13 because of its potent activity in G-protein mediated cAMP assay. Interestingly, 13 (HLM t_1/2 = 92.7 minutes) demonstrated a significant improvement in in vitro metabolic stability compared to atoxifent. This is likely due to the structural modification from a 3-phenol to a 3-carboxamido group. Previous studies have reported that phenols are prone to O-glucuronidation [52], and replacing the phenol with a carboxamido group helps prolong the ligand’s duration of action while maintaining its affinity for MORs [53]. Subsequently, we were interested in evaluating the in vitro metabolic stability of 16 and 17, given their partial agonist activity observed in the G-protein mediated cAMP assay and G-protein selective signaling observed through β-arrestin2 assays. We found that 16 demonstrated poor in vitro metabolic stability, with an HLM t_1/2 of 13.0 minutes. However, 17 exhibited good in vitro metabolic stability, with an HLM t_1/2 of 58.7 minutes, which is more than atoxifent and comparable to fentanyl. This result is particularly intriguing as 13 exhibits significantly greater in vitro metabolic stability compared to 16 and 17, which contains an adjacent hydroxyl group to the 3-carboxamido group.

Interestingly, in MLM, we observed that 8 (MLM t_1/2 = 3.3 minutes) exhibited higher in vitro metabolic stability than atoxifent (MLM t_1/2 = 1.7 minutes). In contrast, 11 (MLM t_1/2 = 3.5 minutes) and 12 (MLM t_1/2 = 6.4 minutes) displayed an in vitro metabolic stability profile superior to atoxifent. Similarly, 13 (MLM t_1/2 = 14.2 minutes) showed greater MLM metabolic stability compared to atoxifent. Moreover, 16 (MLM t_1/2 = 2.1 minutes) demonstrated comparable in vitro MLM metabolic stability than atoxifent, which differed from its HLM in vitro metabolic stability profile. Additionally, the MLM half-life of 17 (MLM t_1/2 = 5.8 minutes) was greater than atoxifent and comparable to fentanyl (MLM t_1/2 = 4.6 minutes). The differences in liver microsomal stability between human and mouse profiles are likely attributed to variations in enzyme composition and expression levels, which influence the metabolic rate in each species [54]. Overall, the replacement of 3-phenol to 3-carboxamido group (13), resulted in a significant improvement of in vitro metabolic stability.

2.4. In Vivo Pharmacology of Selected N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes

Given their high potency, selectivity, and promising in vitro metabolic stability, 11 – 13 were selected for in vivo evaluation. Their supraspinal analgesic effects were assessed using the hot plate assay following intraperitoneal (i.p.) administration in B6129/F1 hybrid mice (Figure 5). The B6129/F1 strain, a hybrid of C57/BL6 and 129S inbred mouse strains was selected to improve hybrid vigor, leading to more broadly applicable experimental outcomes while reducing the influence of founder effects [55].

The antinociceptive effects of 11 – 13, buprenorphine, and atoxifent were evaluated and nonlinear regression (variable slope, four parameters) was used to calculate ED₅₀ and E_max values. Notably, we found that 11 and 13 exhibited comparable ED₅₀ values to atoxifent (11: 0.8494 mg/kg; 13: 0.6581 mg/kg; atoxifent: 0.9323 mg/kg) but achieved lower maximal possible effect (%MPE) at the highest dose (i.e., E_max) (11: 44%, 13: 32%, atoxifent: 92%), indicating reduced in vivo efficacy. In contrast, 12 demonstrated higher ED₅₀ and reduced E_max compared to atoxifent (12: ED₅₀, E_max = 2.804 mg/kg, 33% vs atoxifent: ED₅₀, E_max = 0.9323 mg/kg, 92%). These findings are notable because 11 – 13 functioned as full agonists in vitro, but they demonstrate partial agonist activity in vivo similar to the pharmacological profile of buprenorphine (ED₅₀, E_max = 0.6441 mg/kg, 50%). Statistical analysis via log ED₅₀ comparison revealed no significant difference in the ED₅₀ values between 11 – 13, atoxifent, and buprenorphine (P value = 0.4143, F(4, 154) = 0.9910). The E_max values of 11 – 13, buprenorphine, and atoxifent showed a significant difference in highest %MPE (P value = 0.0388, F(4, 154) = 2.592). However, comparison of 11 – 13 with buprenorphine alone revealed no significant difference (P = 0.1654, F(3, 122) = 1.725), indicating comparable antinociceptive efficacy in vivo. Overall, these findings are relevant because, despite their strong in vitro efficacy, 11 – 13 exhibit reduced in vivo efficacy compared to atoxifent.

To confirm the antinociceptive effects of 11, 12, and 13 were mediated by opioid receptors, antagonist studies were conducted (Figure 6). Pretreatment with naloxone (10 mg/kg) was found to antagonize the antinociceptive effects of atoxifent (10 mg/kg). Similarly, the antinociceptive effects of 11 (10 mg/kg), 12 (10 mg/kg), and 13 (10 mg/kg) were antagonized by naloxone. These results confirm that the antinociceptive effects of 11 – 13 are dependent upon MOR activation.

Figure 6.

Effect of naloxone (NLX) pre-treatment on nociceptive behavior in hot plate test. (A) Experimental design and (B) Antagonist effect of NLX on atoxifent (Atox) and 11 – 13. Data expressed as mean ± SEM n = 6 for each group.

The observed differences between in vitro and in vivo efficacy for 11 – 13 may be due to pharmacokinetic factors such as distribution and clearance. Solubility also appears to play a role, as 13 was readily soluble, while 11, 12, and buprenorphine were poorly soluble, potentially affecting their exposure, receptor engagement, and overall pharmacodynamic response. Another possible explanation for the difference in in vitro versus in vivo efficacy could be the difference in receptor reserves between the two systems. Ligands with low intrinsic efficacy can appear as full agonists in systems with high receptor reserve, such as in vitro assays that overexpress receptors [56, 57]. In addition, the in vitro G-protein cAMP accumulation assay measures the second messenger cAMP, which is considered a relatively amplified system and may further contribute to the Emax seen in vitro [58]. In contrast, in vivo systems with endogenous MOR expression have lower receptor reserves [58, 59]. As a result, even full receptor occupancy by the ligand may elicit a limited signaling response, resulting in partial agonism [59].

The submaximal antinociceptive efficacy of 11 – 13 is of particular interest, as it is similar to the in vivo pharmacological profile of buprenorphine, a clinically approved partial agonist used in the management of pain and opioid use disorder [60–63]. Buprenorphine provides effective analgesia while exhibiting a ceiling effect on respiratory depression, which contributes to its improved safety profile [61, 64]. Similarly, the in vivo partial agonist profile of 11 – 13 suggests their potential as valuable probes for designing opioid receptor ligands with reduced adverse effects.

3. Conclusions

Through the synthesis and evaluation of compounds 1 – 20, we gained valuable insights into the role of the phenolic hydroxyl group in the bioactivity of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes. The results demonstrate that hydroxyl substitution at the 3-position of the phenyl ring is optimal for MOR activity. Additionally, a hydrogen bond donor at the 3-position is favorable for activity. This suggests that the phenolic hydroxyl group in N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes engages the receptor in a manner analogous to traditional opioids [13, 65, 66]. However, our findings indicate that modulating the pKa of N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes affects receptor activity. Although pKa modulation alone does not fully account for the observed effects. Other contributing factors, such as steric effects, also play a significant role in influencing activity.

Interestingly, the phenolic hydroxyl group in the N-(3-hydroxyphenyl)-3,8-diazabicyclooctane scaffold can be replaced with bioisosteric substitutions. The most potent of these is the 3-carboxamido derivative (13), whose bioactive conformation was identified. The effective bioisosteric replacement is noteworthy, as similar substitutions in the 4,5-epoxymorphinan class, including morphine and naltrexone, have led to reduced activity compared to the parent compound [67–69]. Although atoxifent and 11 – 13 show similar MOR activity, bioisosteric modifications impact β-arrestin2 recruitment, in vitro metabolic stability, and in vivo antinociceptive effects. Collectively, these findings offer valuable insights that improve our understanding of the role of the phenolic hydroxyl group within the N-(3-hydroxyphenyl)-3,8-diazabicyclooctane framework.

4. Experimental Section

4.1. Safety Statement.

Work with highly potent MOR agonists carries the substantial risk of respiratory depression. The risks associated with these compounds can be mitigated by (1) the proper use of personal protective equipment; (2) working a well-ventilated fume hood; (3) online training on how to spot an opioid overdose and administer Narcan^®; and (4) never working with these substances without observation of other research group members.

4.2. General Methods for Chemistry.

Reactions were performed in oven-dried glassware under normal atmosphere (unless otherwise specified). All chemical reagents were purchased from commercial suppliers and used without further purification. Anhydrous solvents were either purchased from commercial suppliers or obtained from a solvent purification system in which solvent was passed through two columns of activated alumina under nitrogen. Reactions were monitored by thin-layer chromatography (TLC) on Supelco TLC Silica gel 60 F₂₅₄ 20 × 20 cm silica gel plates (Sigma Aldrich) and visualized by UV (254 nm). Flash column chromatography was performed on a CombiFlash NextGen 300+ purification system (Teledyne Isco) using prepacked RediSepRf silica columns. Melting points were determined on a Thermo 1001D Mel-Temp Electrothermal Melting Point Apparatus. ¹H and ¹³C NMR were recorded on a Bruker 400 MHz spectrometer, 500 MHz Varian spectrometer, and/or Bruker 600 MHz spectrometer. Chemical shifts are reported in ppm and referenced with respect to the residual solvent: CDCl₃ at 7.26 ppm, MeOD at 3.31 ppm, DMSO-d6 at 2.50 ppm (¹H NMR); CDCl₃ at 77.16 ppm, MeOD at 49.00 ppm, DMSO-d6 at 39.52 ppm (¹³C NMR). In other cases, CDCl₃ or DMSO-d6 containing tetramethylsilane (TMS) were utilized. Coupling constants (J) are reported in Hz. High-resolution mass spectra (HRMS) were obtained on an Agilent 6230 time-of-flight mass spectrometer with an electrospray ion source in positive mode. Compounds selected for biological testing were identified as ≥ 95% pure by HPLC on an Agilent 1260 Infinity II with diode array detection at 214 nm. A Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 μm) with a 5-minute gradient mobile phase of 10 – 100% acetonitrile / 0.1% TFA in water (method A) and an Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) with a 5-minute gradient mobile phase of 10 – 100% acetonitrile / 0.1% H₃PO₄ in water (method B) were utilized.

4.2.1. 8-(4-(Methoxymethoxy)phenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (22a).

A mixture of 21 [12] (300.0 mg, 1.302 mmol), 1-bromo-4-(methoxymethoxy)benzene (311.0 mg, 1.433 mmol), Pd(OAc)₂ (29.2 mg, 0.236 mmol), XPhos (124.2 mg, 1.413 mmol), and sodium tert-butoxide (187.7 mg, 1.954 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 22a as a yellow oil (348 mg, 73%). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.5 Hz, 2H), 7.18 (d, J = 7.2 Hz, 3H), 6.96 – 6.88 (m, 2H), 6.77 – 6.67 (m, 2H), 5.08 (s, 2H), 4.09 – 4.03 (m, 2H), 3.48 (s, 3H), 2.63 (t, J = 7.6 Hz, 2H), 2.55 (dd, J = 10.8, 2.6 Hz, 2H), 2.44 (d, J = 10.5 Hz, 2H), 2.25 (t, J = 7.0 Hz, 2H), 2.03 – 1.86 (m, 4H), 1.73 (p, J = 7.2 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 149.4, 143.0, 128.5, 128.3, 125.7, 117.9, 116.5, 95.5, 56.6, 56.4, 55.8, 55.5, 33.3, 28.5, 27.2.

4.2.2. 4-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenol (1).

A mixture of 22a (348 mg, 0.950 mmol) and 48% HBr (0.22 mL, 1.899 mmol) in MeOH (6 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 1 as a colorless oil (300 mg, 98%). ¹H NMR (400 MHz, MeOD) δ 7.30 – 7.11 (m, 5H), 6.78 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 4.08 – 4.02 (m, 2H), 2.68 – 2.58 (m, 4H), 2.43 (d, J = 10.7 Hz, 2H), 2.26 (t, J = 7.1 Hz, 2H), 1.94 (m, J = 29.3, 5.5 Hz, 4H), 1.75 (m, J = 7.3 Hz, 2H); ¹³C NMR (101 MHz, MeOD) δ 149.8, 142.1, 140.8, 128.1, 127.9, 125.3, 117.4, 115.6, 57.1, 56.5, 55.6, 48.3, 48.0, 47.8, 47.6, 47.4, 47.2, 47.0, 33.0, 28.1, 26.9. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₁H₂₆N₂O, 323.2118; found, 323.2132.

4.2.3. 8-(3,5-Bis(ethoxymethoxy)phenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (22b).

A mixture of 21 [12] (300.0 mg, 1.302 mmol), 1-bromo-3,5-bis(ethoxymethoxy)benzene[70] (437.2 mg, 1.433 mmol), Pd(OAc)₂ (29.2 mg, 0.130 mmol), XPhos (62.1 mg, 0.130 mmol), and sodium tert-butoxide (187.7 mg, 1.954 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 22b as a yellow oil (350 mg, 59%). ¹H NMR (400 MHz, CDCl₃) δ 7.31 – 7.23 (m, 3H), 7.21 – 7.12 (m, 3H), 6.14 (d, J = 1.4 Hz, 2H), 5.16 (s, 4H), 4.08 (d, J = 4.2 Hz, 2H), 3.77 – 3.66 (m, 4H), 2.63 (t, J = 7.6 Hz, 2H), 2.53 (dd, J = 10.8, 2.5 Hz, 2H), 2.47 – 2.40 (m, 2H), 2.24 (t, J = 7.0 Hz, 2H), 2.06 – 1.86 (m, 4H), 1.78 – 1.66 (m, 2H), 1.22 (t, J = 7.0 Hz, 6H); 13C NMR (101 MHz, CDCl3) 162.5, 159.6, 142.1, 128.5, 128.3, 126.4, 97.5, 93.3, 68.1, 64.1, 55.6, 55.2, 33.3, 28.0, 27.3, 15.1.

4.2.4. 5-(3-(3-Phenylpropyl))-3,8-diazabicyclo[3.2.1]octan-8-yl)benzene-1,3-diol (2).

A mixture of 22b (200 mg, 0.440 mmol) and 48% HBr (0.099 mL, 0.880 mmol) in MeOH (5 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 2 as a colorless oil (133 mg, 89). ¹H NMR (600 MHz, MeOD) δ 7.23 (t, J = 7.6 Hz, 2H), 7.18 – 7.09 (m, 3H), 5.84 (d, J = 1.9 Hz, 2H), 5.72 (t, J = 2.0 Hz, 1H), 4.04 (dd, J = 4.8, 2.5 Hz, 2H), 2.64 – 2.50 (m, 4H), 2.45 – 2.38 (m, 2H), 2.23 (t, J = 7.2 Hz, 2H), 2.02 – 1.84 (m, 4H), 1.72 (h, J = 8.3 Hz, 2H); ¹³C NMR (151 MHz, MeOD) δ 158.9, 149.1, 142.1, 128.1, 127.9, 125.3, 94.8, 92.9, 56.7, 55.6, 54.8, 48.0, 47.9, 47.8, 47.6, 47.5, 47.3, 47.2, 32.9, 28.0, 27.2. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₁H₂₆N₂O₂, 339.2067; found, 339.2066.

4.2.5. 1-Bromo-3-((ethoxymethoxy)methyl)benzene (24a).

A mixture of 23a (1.0 g, 5.347 mmol), (chloromethoxy)ethane (0.607 mg, 6.416 mmol), and DIPEA (1.9 mL, 10.693 mmol) in DCM (8 mL) was stirred overnight. Upon completion the reaction mixture was quenched with water and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and brine (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 24a as an off-white solid (1.20 g, 92%), mp: 52 – 53 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.52 (d, J = 1.9 Hz, 1H), 7.45 – 7.37 (m, 1H), 7.27 (d, J = 7.4 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 4.76 (s, 2H), 4.57 (s, 2H), 3.64 (q, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 139.2, 132.2, 130.4, 129.5, 123.7, 92.4, 69.3, 63.9, 15.2.

4.2.6. 8-(3-((Ethoxymethoxy)methyl)phenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (25a).

A mixture of 21 [12] (250.0 mg, 1.085 mmol), 24a (292.6 mg, 1.194 mmol), Pd(OAc)₂ (24.4 mg, 0.109 mmol), XPhos (103.5 mg, 0.217 mmol), and sodium tert-butoxide (156.5 mg, 1.628 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 25a as a yellow oil (338.0 mg, 79%). ¹H NMR (400 MHz, CDCl₃) δ 7.31 – 7.22 (m, 2H), 7.17 (dd, J = 7.4, 5.2 Hz, 4H), 6.77 (t, J = 2.1 Hz, 1H), 6.73 – 6.65 (m, 2H), 4.75 (s, 2H), 4.54 (s, 2H), 4.17 (m, J = 4.4, 2.2 Hz, 2H), 3.72 – 3.60 (m, 2H), 2.67 – 2.60 (m, 2H), 2.55 (dd, J = 10.8, 2.6 Hz, 2H), 2.44 (dd, J = 10.7, 1.6 Hz, 2H), 2.23 (t, J = 7.0 Hz, 2H), 2.01 (q, J = 6.1 Hz, 2H), 1.93 (m, J = 10.1, 4.8 Hz, 2H), 1.78 – 1.66 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 147.4, 142.5, 139.2, 129.5, 128.5, 128.3, 125.7, 116.8, 114.7, 114.6, 94.3, 69.6, 63.4, 56.6, 55.5, 55.2, 33.3, 28.5, 28.0, 15.2.

4.2.7. (3-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)methanol (3).

A mixture of 25a (338.0 mg, 0.857 mmol) and 48% HBr (0.099 mL, 0.880 mmol) in MeOH (5 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 3 as a colorless oil (285.0 mg, 99%). ¹H NMR (400 MHz, CDCl₃) δ 7.25 (d, J = 7.3 Hz, 2H), 7.22 – 7.12 (m, 4H), 6.78 (t, J = 2.0 Hz, 1H), 6.73 – 6.64 (m, 2H), 4.60 (s, 2H), 4.17 (dd, J = 4.5, 2.3 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.55 (dd, J = 10.9, 2.6 Hz, 2H), 2.47 – 2.40 (m, 2H), 2.23 (t, J = 7.0 Hz, 2H), 2.07 – 1.88 (m, 4H), 1.72 (dt, J = 14.1, 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 147.5, 142.4, 142.3, 129.6, 128.5, 128.3, 125.7, 115.8, 114.7, 113.8, 65.8, 56.6, 55.5, 55.2, 33.3, 28.5, 28.1. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₈N₂O, 337.2274; found, 337.2290.

4.2.8. 2-(3-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)propan-2-ol (4).

A mixture of 21 [12] (250.0 mg, 1.085 mmol), 2-(3-bromophenyl)propan-2-ol (256.8 mg, 1.194 mmol), Pd(OAc)₂ (24.4 mg, 0.109 mmol), XPhos (103.5 mg, 0.217 mmol), and sodium tert-butoxide (156.5 mg, 1.628 mmol) in 1,4-Dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 4 as a yellow oil (338.0 mg, 79%). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (dd, J = 8.1, 7.0 Hz, 2H), 7.17 (dd, J = 9.0, 7.3 Hz, 4H), 6.97 (t, J = 2.1 Hz, 1H), 6.81 – 6.74 (m, 1H), 6.65 (dd, J = 8.2, 2.4 Hz, 1H), 4.19 (dt, J = 4.4, 2.3 Hz, 2H), 2.68 – 2.53 (m, 4H), 2.45 (dd, J = 10.7, 1.5 Hz, 2H), 2.25 (t, J = 7.0 Hz, 2H), 2.02 (q, J = 5.8 Hz, 2H), 1.94 (dd, J = 9.0, 4.4 Hz, 2H), 1.79 – 1.67 (m, 3H), 1.56 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 150.5, 147.3, 142.5, 129.2, 128.5, 128.3, 125.7, 113.6, 113.5, 111.5, 72.7, 56.6, 55.6, 55.3, 33.3, 31.7, 28.5, 28.0. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₄H₃₂N₂O, 365.2587; found, 365.2598.

4.2.9. 3-(3-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)oxetan-3-ol (5).

A mixture of 21 [12] (250.0 mg, 1.085 mmol), 3-(3-bromophenyl)oxetan-3-ol (273.5 mg, 1.194 mmol), Pd(OAc)₂ (24.4 mg, 0.109 mmol), XPhos (51.7 mg, 0.109 mmol), and sodium tert-butoxide (156.5 mg, 1.628 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 5 as a yellow oil (80.0 mg, 20%). ¹H NMR (400 MHz, CDCl₃) δ 7.31 – 7.26 (m, 2H), 7.25 – 7.21 (m, 1H), 7.17 (dt, J = 6.3, 1.5 Hz, 3H), 6.96 (t, J = 2.1 Hz, 1H), 6.89 (dd, J = 7.6, 1.7 Hz, 1H), 6.73 (dd, J = 8.3, 2.4 Hz, 1H), 4.92 (d, J = 6.8 Hz, 2H), 4.87 (d, J = 7.0 Hz, 2H), 4.20 (dd, J = 4.6, 2.4 Hz, 2H), 2.72 – 2.53 (m, 4H), 2.47 – 2.40 (m, 2H), 2.24 (t, J = 7.0 Hz, 2H), 2.08 – 1.89 (m, 4H), 1.79 – 1.67 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 147.6, 143.7, 142.4, 129.8, 128.5, 128.3, 125.7, 114.9, 113.2, 111.3, 85.5, 76.1, 56.6, 55.5, 55.2, 33.3, 28.5, 28.1. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₄H₃₀N₂O₂, 379.2380; found, 379.2371.

4.2.10. 1-Bromo-3-chloro-5-(ethoxymethoxy)benzene (24b).

A mixture of 23b (1.0 g, 4.820 mmol), (chloromethoxy)ethane (0.547 g, 5.785 mmol), and DIPEA (0.493 mL, 5.785 mmol) in DCM (6 mL) was stirred overnight. Upon completion the reaction mixture was quenched with water and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and brine (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 24b as an off-white solid (1.2 g, 94%), mp: 68 – 69 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.15 (q, J = 1.6 Hz, 1H), 7.11 (q, J = 1.6 Hz, 1H), 7.00 (q, J = 1.7 Hz, 1H), 5.19 (d, J = 1.3 Hz, 2H), 3.71 (qd, J = 7.1, 1.4 Hz, 2H), 1.23 (td, J = 7.1, 1.4 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 158.4, 137.2, 135.7, 114.9, 113.5, 104.7, 93.7, 65.2, 15.0.

4.2.11. 8-(3-Chloro-5-(ethoxymethoxy)phenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (25b).

A mixture of 21 [12] (300.0 mg, 1.302 mmol), 24b (345.8 mg, 1.302 mmol), Pd(OAc)₂ (29.2 mg, 0.130 mmol), XPhos (62.1 mg, 0.130 mmol), and sodium tert-butoxide (187.7 mg, 1.954 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with dichloromethane (DCM) (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 25b as a yellow oil (130 mg, 24%). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.9 Hz, 2H), 7.21 – 7.06 (m, 3H), 6.55 (p, J = 1.8 Hz, 1H), 6.49 – 6.30 (m, 2H), 5.17 (d, J = 15.3 Hz, 2H), 4.16 – 4.04 (m, 2H), 3.72 (p, J = 7.0 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.54 (dt, J = 11.0, 2.8 Hz, 2H), 2.42 (dd, J = 15.8, 10.6 Hz, 2H), 2.24 (t, J = 7.0 Hz, 2H), 2.06 – 1.89 (m, 3H), 1.78 – 1.66 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 159.3, 149.0, 130.1, 128.5, 128.3, 128.3, 125.7, 108.0, 103.8, 102.3, 93.2, 64.3, 64.1, 56.5, 55.6, 33.3, 28.5, 28.1, 15.1.

4.2.12. 3-Chloro-5-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenol (6).

A mixture of 25b (130.0 mg, 0.313 mmol) and 48% HBr (0.070 mL, 0.627 mmol) in MeOH (5 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 6 as a colorless oil (108.0 mg, 97%). ¹H NMR (400 MHz, CDCl₃) δ 7.30 – 7.22 (m, 2H), 7.21 – 7.12 (m, 3H), 6.30 (t, J = 1.9 Hz, 1H), 6.16 (t, J = 1.9 Hz, 1H), 6.09 (t, J = 2.2 Hz, 1H), 4.02 (dd, J = 4.5, 2.4 Hz, 2H), 2.65 – 2.49 (m, 4H), 2.43 – 2.38 (m, 1H), 2.23 (t, J = 7.1 Hz, 2H), 2.05 – 1.86 (m, 4H), 1.72 (p, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 157.8, 148.9, 142.3, 135.6, 128.5, 128.3, 125.8, 107.9, 105.1, 100.9, 56.8, 55.5, 54.9, 33.4, 28.3, 27.9. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₁H₂₅ClN₂O, 357.1728; found, 357.1725.

4.2.13. 1-Bromo-3-(ethoxymethoxy)-5-methylbenzene (24c).

A mixture of 23c (1.0 g, 5.347 mmol), (chloromethoxy)ethane (0.607 g, 6.416 mmol), and DIPEA (1.1 mL, 6.416 mmol) in DCM (6 mL) was stirred overnight. Upon completion the reaction mixture was quenched with water and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and brine (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 24c as an off-white solid (1.3 g, 99%), mp: 56 – 57 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.02 (d, J = 2.2 Hz, 1H), 6.97 (s, 1H), 6.78 (d, J = 2.1 Hz, 1H), 5.18 (s, 2H), 3.71 (q, J = 7.1 Hz, 2H), 2.29 (s, 3H), 1.23 (t, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 158.1, 141.1, 125.6, 122.4, 116.6, 115.8, 93.2, 64.4, 21.3, 15.1.

4.2.14. 8-(3-(Ethoxymethoxy)-5-methylphenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (25c).

A mixture of 21 [12] (770.0 mg, 3.343 mmol), 24c (819.3 mg, 3.343 mmol), Pd(OAc)₂ (75.0 mg, 0.334 mmol), XPhos (159.4 mg, 0.130 mmol), and sodium tert-butoxide (481.9 mg, 5.014 mmol) in 1,4-dioxane (12 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 25c as a clear oil (490.0 mg, 37%). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.5 Hz, 2H), 7.21 – 7.14 (m, 3H), 6.30 – 6.22 (m, 3H), 5.18 (s, 2H), 4.17 – 4.07 (m, 2H), 3.72 (q, J = 7.0 Hz, 2H), 2.63 (t, J = 7.7 Hz, 2H), 2.53 (dd, J = 10.8, 2.5 Hz, 2H), 2.47 – 2.40 (m, 2H), 2.24 (d, J = 5.7 Hz, 4H), 2.01 – 1.87 (m, 4H), 1.72 (p, J = 7.2 Hz, 2H), 1.26 – 1.20 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.7, 148.3, 142.5, 140.1, 128.52, 128.3, 125.7, 110.0, 105.7, 101.0, 93.2, 64.1, 56.6, 55.5, 55.2, 33.3, 28.5, 28.0, 22.0, 15.2.

4.2.15. 3-Methyl-5-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenol (7).

A mixture of 25c (490.0 mg, 1.288 mmol) and 48% HBr (0.289 mL, 2.575 mmol) in MeOH (8 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 7 as a colorless oil (360.0 mg, 83%). ¹H NMR (400 MHz, CDCl₃) δ 7.25 (d, J = 6.8 Hz, 2H), 7.16 (d, J = 7.5 Hz, 3H), 6.18 (s, 1H), 6.06 (d, J = 2.3 Hz, 1H), 6.00 (s, 1H), 4.09 – 4.03 (m, 2H), 2.66 – 2.50 (m, 4H), 2.45 (s, 1H), 2.24 (t, J = 7.1 Hz, 2H), 2.21 (s, 3H), 2.05 – 1.86 (m, 4H), 1.73 (p, J = 7.3 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 156.9, 148.4, 142.3, 140.5, 128.5, 128.3, 125.7, 109.0, 105.8, 99.9, 56.8, 55.5, 55.1, 33.4, 28.3, 27.9, 21.9. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₈N₂O, 337.2274; found, 337.2291.

4.2.16. 1-Bromo-3-(ethoxymethoxy)-5-fluorobenzene (24d).

A mixture of 23d (1.0 g, 5.236 mmol), (chloromethoxy)ethane (0.594 g, 6.283 mmol), and DIPEA (1.1 mL, 6.283 mmol) in DCM (6 mL) was stirred overnight. Upon completion the reaction mixture was quenched with water and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and brine (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 24d as a colorless oil (1.1 g, 84%). ¹H NMR (400 MHz, CDCl₃) δ 7.00 (dt, J = 2.4, 1.4 Hz, 1H), 6.89 (dt, J = 8.0, 2.0 Hz, 1H), 6.73 (dt, J = 10.5, 2.2 Hz, 1H), 5.19 (s, 2H), 3.71 (q, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 115.7, 115.6, 112.6, 112.3, 103.3, 103.0, 93.3, 64.7, 15.0.

4.2.17. 8-(3-(Ethoxymethoxy)-5-fluorophenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (25d).

A mixture of 21 [12] (300 mg, 1.302 mmol), 24d (324.4 mg, 1.302 mmol), Pd(OAc)₂ (29.2 mg, 0.130 mmol), XPhos (62.1 mg, 0.130 mmol), and sodium tert-butoxide (187.7 mg, 1.954 mmol) in 1,4-dioxane (8 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with dichloromethane (DCM) (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 25d as a yellow oil (75.0 mg, 14%). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.5 Hz, 2H), 7.21 – 7.13 (m, 3H), 6.15 (m, J = 24.7, 14.2, 2.3 Hz, 3H), 5.16 (s, 2H), 4.09 – 4.03 (m, 2H), 3.71 (q, J = 7.0 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.53 (dd, J = 11.0, 2.5 Hz, 2H), 2.41 (d, J = 10.6 Hz, 2H), 2.24 (t, J = 7.0 Hz, 2H), 2.06 – 1.87 (m, 4H), 1.72 (p, J = 7.2 Hz, 2H), 1.22 (t, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 166.0, 163.6, 159.7, 159.6, 149.0, 148.9, 142.4, 128.5, 128.3, 125.7, 99.0, 99.0, 96.2, 95.9, 93.2, 92.7, 92.5, 64.3, 56.6, 55.6, 55.0, 33.3, 28.5, 28.1, 15.2.

4.2.18. 3-Fluoro-5-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenol (8).

A mixture of 25d (70.0 mg, 0.176 mmol) and 48% HBr (0.039 mL, 0.351 mmol) in MeOH (5 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 8 as a colorless oil (108.0 mg, 97%). ¹H NMR (400 MHz, CDCl₃) δ 7.25 (d, J = 7.1 Hz, 2H), 7.21 – 7.08 (m, 3H), 6.04 (dt, J = 12.0, 2.2 Hz, 1H), 5.98 (d, J = 2.2 Hz, 1H), 5.89 (dt, J = 9.9, 2.1 Hz, 1H), 4.02 (dd, J = 4.7, 2.4 Hz, 2H), 2.61 (t, J = 7.6 Hz, 2H), 2.54 (dd, J = 11.0, 2.5 Hz, 2H), 2.45 – 2.35 (m, 2H), 2.24 (t, J = 7.2 Hz, 2H), 2.06 – 1.87 (m, 4H), 1.73 (p, J = 7.3 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 163.7, 157.9 (d, J = 14.5 Hz), 149.1 (d, J = 13.1 Hz), 142.2, 128.5, 128.3, 125.8, 98.1, 95.1 (d, J = 25.9 Hz), 92.4 (d, J = 25.3 Hz), 56.8, 55.6, 54.9, 33.4, 28.3, 28.0. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₁H₂₅FN₂O, 341.2024; found, 341.2029.

4.2.19. 8-(3-(Difluoromethyl)phenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (9).

A mixture of 21 [12] (250.0 mg, 1.085 mmol), 1-bromo-3-(difluoromethyl)benzene (224.7 mg, 1.085mmol), Pd(OAc)₂ (24.4 mg, 0.109 mmol), XPhos (51.7 mg, 0.109 mmol), and sodium tert-butoxide (156.5 mg, 1.628 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with dichloromethane (DCM) (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 9 as a light-yellow oil (120.0 mg, 31%). ¹H NMR (400 MHz, CDCl₃) δ 7.35 – 7.07 (m, 7H), 6.99 – 6.79 (m, 3H), 3.35 (d, J = 8.1 Hz, 3H), 3.14 – 2.89 (m, 2H), 2.69 (t, J = 7.7 Hz, 2H), 2.43 (d, J = 15.2 Hz, 2H), 2.03 – 1.55 (m, 7H); ¹³C NMR (101 MHz, CDCl₃) δ 151.4, 142.2, 129.4, 128.4, 128.3, 125.8, 115.3, 114.7, 109.9, 58.5, 53.0, 51.5, 33.6, 30.3, 26.1. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₆F₂N₂, 357.2137; found, 357.2147

4.2.20. 8-(3-Nitrophenyl)-3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octane (10).

A mixture of 21 [12] (100 mg, 0.434 mmol), 1-fluoro-3-nitrobenzene (73.5 mg, 0.521 mmol), potassium carbonate (120.0 mg, 0.868 mmol) in anhydrous DMSO (5 mL) was stirred at 85°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 30 mL) and brine (30 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 10 as a yellow oil (48 mg, 35%). ¹H NMR (400 MHz, CDCl₃) δ 7.57 (dd, J = 8.6, 1.9 Hz, 2H), 7.38 – 7.26 (m, 3H), 7.23 – 7.13 (m, 3H), 7.06 – 6.99 (m, 1H), 3.42 – 3.34 (m, 4H), 3.15 – 3.01 (m, 2H), 2.74 – 2.59 (m, 2H), 2.48 – 2.40 (m, 2H), 2.07 – 1.91 (m, 2H), 1.85 (p, J = 7.5 Hz, 2H), 1.72 (t, J = 6.8 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 151.8, 149.3, 142.2, 129.5, 128.4, 128.3, 125.8, 118.6, 112.1, 107.4, 58.4, 53.2, 51.6, 33.6, 30.3, 28.5, 26.1. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₁H₂₅N₃O₂, 352.2020; found, 352.2025.

4.2.21. 3-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)aniline (11).

A mixture of atoxifent [12] (230 mg, 0.713 mmol) in anhydrous DMSO (4 mL) and anhydrous cesium carbonate powder (929.6 mg, 2.853 mmol) was heated at 80°C with vigorous stirring. To the mixture was added a solution of 5-amino-6-chloropyrazine-2,3-dicarbonitrile (192.1 mg, 1.070 mmol) in DMSO (1.0 ml) over 10 minutes. The reaction mixture was cooled to room temperature and treated with acetic acid (6.0 mL) and zinc (466.3 mg, 7.133 mmol). The resulting mixture was heated at 80°C for 10 minutes. The reaction mixture was cooled to room temperature, quenched with water, and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 30 mL) and brine (30 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 11 as a yellow oil (143.0 mg, 62%). ¹H NMR (600 MHz, CDCl₃) δ 7.32 – 7.29 (m, 1H), 7.26 (d, J = 6.7 Hz, 3H), 7.18 (q, J = 5.5 Hz, 1H), 7.14 – 7.10 (m, 2H), 7.05 (d, J = 7.6 Hz, 1H), 6.89 (dd, J = 8.2, 2.9 Hz, 1H), 5.72 (s, 2H), 4.27 (d, J = 5.0 Hz, 2H), 3.02 (d, J = 11.2 Hz, 2H), 2.67 (d, J = 11.2 Hz, 2H), 2.61 (q, J = 5.3 Hz, 2H), 2.52 (t, J = 7.7 Hz, 2H), 2.19 (d, J = 7.8 Hz, 2H), 2.06 – 2.00 (m, 2H), 1.91 – 1.83 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 157.4, 148.3, 141.9, 130.5, 128.5, 128.3, 125.8, 107.9, 105.0, 102.7, 56.8, 55.3, 54.6, 33.2, 27.7, 27.6. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₁H₂₇N₃, 322.2278; found, 322.2276.

4.2.22. N-(3-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)cyclopropane-sulfonamide (12).

A mixture of 11 (60 mg, 0.187 mmol), cyclopropanesulfonylchloride (26.2 mg, 0.187 mmol), and DIPEA (0.033 mL, 0.187 mmol) in acetonitrile (4 mL) stirred at 85°C overnight. Upon completion the reaction mixture was cooled to room temperature, quenched with water, and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and brine (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 12 as a brown oil (27.0 mg, 34%). ¹H NMR (400 MHz, CDCl₃) δ 7.31 – 7.13 (m, 6H), 7.03 (t, J = 8.0 Hz, 1H), 6.33 (dd, J = 8.3, 2.1 Hz, 1H), 6.26 (t, J = 2.3 Hz, 1H), 6.17 (dd, J = 8.0, 2.2 Hz, 1H), 4.12 – 4.06 (m, 2H), 2.63 (q, J = 6.2 Hz, 3H), 2.54 (dd, J = 10.8, 2.6 Hz, 2H), 2.48 – 2.34 (m, 2H), 2.33 – 2.18 (m, 2H), 2.08 – 1.92 (m, 3H), 1.95 – 1.80 (m, 1H), 1.73 (h, J = 7.1 Hz, 2H), 1.36 – 1.10 (m, 4H); ¹³C NMR (151 MHz, CDCl₃) δ 151.1, 148.5, 142.3, 130.3, 128.5, 128.3, 125.7, 113.9, 110.1, 109.0, 56.5, 55.6, 54.9, 33.3, 28.4, 28.1, 27.7, 6.2. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₄H₃₁N₃O₂S, 426.2210; found, 426.2221.

4.2.23. 3-(3-(3-Phenylpropyl))-3,8-diazabicyclo[3.2.1]octan-8-yl)benzonitrile (26a).

A mixture of 21 [12] (273.0 mg, 1.185 mmol), 3-fluorobenzonitrile (172.2 mg, 1.422 mmol), and potassium carbonate (327.6 mg, 2.370 mmol), in anhydrous DMSO (4 mL) was heated in microwave reactor at 180°C (200W) for 1 hour. The reaction mixture was cooled to room temperature, quenched with water, and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 26a as a light-yellow oil (80.0 mg, 20%). ¹H NMR (400 MHz, CDCl₃) δ 7.31 – 7.26 (m, 2H), 7.25 – 7.13 (m, 4H), 7.03 – 6.91 (m, 3H), 3.45 – 3.25 (m, 4H), 3.02 (dd, J = 10.7, 2.4 Hz, 2H), 2.69 (t, J = 7.7 Hz, 2H), 2.46 – 2.38 (m, 2H), 1.95 (dt, J = 6.7, 2.8 Hz, 2H), 1.83 (m, J = 9.3, 4.9 Hz, 2H), 1.69 (d, J = 7.4 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 151.2, 142.2, 129.7, 128.4, 128.3, 125.8, 120.8, 119.7, 117.3, 116.0, 112.9, 58.4, 53.0, 51.6, 33.6, 30.3, 26.1.

4.2.24. 3-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (13).

A mixture of 26a (145.0 mg, 0.437 mmol), potassium carbonate (120.9 mg, 0.875 mmol), in anhydrous DMSO (4 mL) was cooled over an ice bath. 30% Hydrogen peroxide solution was added dropwise (0.153 mL, 1.969 mmol), and the reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was quenched with water and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing DCM/MeOH gradient to afford 13 as a colorless oil (50 mg, 30%). ¹H NMR (600 MHz, CDCl₃) δ 7.30 – 7.22 (m, 4H), 7.21 – 7.14 (m, 3H), 6.99 (ddt, J = 7.6, 1.6, 0.7 Hz, 1H), 6.90 (ddd, J = 8.4, 2.6, 0.8 Hz, 1H), 6.06 (s, 1H), 5.67 (s, 1H), 4.22 (dq, J = 4.4, 2.0 Hz, 2H), 2.66 – 2.60 (m, 2H), 2.56 (dd, J = 11.0, 2.5 Hz, 2H), 2.42 (dd, J = 10.8, 1.6 Hz, 2H), 2.23 (t, J = 7.0 Hz, 2H), 2.03 (dd, J = 6.8, 5.3 Hz, 2H), 1.95 (dd, J = 8.0, 4.1 Hz, 2H), 1.76 – 1.68 (m, 2H); ¹³C NMR (151 MHz, CDCl₃) δ 170.0, 147.5, 142.4, 134.6, 129.6, 128.5, 128.3, 125.7, 118.7, 115.1, 114.6, 56.6, 55.5, 55.0, 33.3, 28.5, 28.1. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₇N₂O, 350.2227; found, 350.2222.

4.2.25. 2-(3-(3-Phenylpropyl))-3,8-diazabicyclo[3.2.1]octan-8-yl)benzonitrile (26b).

A mixture of 21 [12] (300.0 mg, 1.302 mmol), 2-fluorobenzonitrile (189.3 mg, 1.563 mmol), and potassium carbonate (360.0 mg, 2.605 mmol), in anhydrous DMSO (5 mL) was heated in microwave reactor at 180°C (200W) for 1 hour. The reaction mixture was cooled to room temperature, quenched with water, and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 26b as a yellow oil (110.0 mg, 26%). ¹H NMR (400 MHz, CDCl₃) δ 7.49 (dd, J = 7.7, 1.7 Hz, 1H), 7.35 (m, J = 8.8, 7.3, 1.7 Hz, 1H), 7.30 – 7.23 (m, 2H), 7.18 (d, J = 7.4 Hz, 3H), 6.89 – 6.76 (m, 2H), 4.30 (m, J = 4.4, 2.1 Hz, 2H), 2.73 (dd, J = 10.9, 2.8 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H), 2.48 (dd, J = 10.8, 1.6 Hz, 2H), 2.34 (t, J = 7.0 Hz, 2H), 1.99 (t, J = 6.2 Hz, 2H), 1.87 (dd, J = 8.2, 4.2 Hz, 2H), 1.82 – 1.71 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 152.4, 142.4, 135.1, 133.6, 128.5, 128.3, 125.7, 119.7, 119.1, 116.7, 101.6, 58.8, 58.0, 56.3, 33.4, 28.5, 27.5.

4.2.26. 2-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (14).

A mixture of 26b (78.0 mg, 0.235 mmol), potassium carbonate (65 mg, 0.471 mmol), in anhydrous DMSO (4 mL) cooled over an ice bath. 30% Hydrogen peroxide solution (0.082 mL, 1.059 mmol) was added dropwise, and the reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was quenched with water and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing DCM/MeOH gradient to afford 14 as a colorless oil (30 mg, 37%). ¹H NMR (400 MHz, CDCl₃) δ 8.27 – 8.21 (m, 1H), 7.92 (dd, J = 7.7, 1.7 Hz, 1H), 7.37 – 7.22 (m, 3H), 7.22 – 7.14 (m, 3H), 7.04 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.04 – 5.99 (m, 1H), 3.78 (dt, J = 4.2, 2.3 Hz, 2H), 2.79 (dd, J = 11.0, 2.9 Hz, 2H), 2.71 – 2.59 (m, 2H), 2.42 – 2.31 (m, 4H), 1.95 (dd, J = 7.5, 4.9 Hz, 2H), 1.85 – 1.70 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 170.3, 149.4, 142.3, 131.8, 131.8, 128.5, 128.3, 126.1, 125.8, 122.2, 117.5, 61.7, 59.4, 56.2, 33.3, 28.6, 27.3. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₇N₃O, 350.2227; found, 350.2219.

4.2.27. 4-(3-(3-Phenylpropyl))-3,8-diazabicyclo[3.2.1]octan-8-yl)benzonitrile (26c).

A mixture of 21 [12] (300 mg, 1.302 mmol), 4-fluorobenzonitrile (189.3 mg, 1.563 mmol), and potassium carbonate (360.0 mg, 2.605 mmol), in anhydrous DMSO (5 mL) was heated in microwave reactor at 180°C (200W) for 1 hour. The reaction mixture was cooled to room temperature, quenched with water, and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 26c a yellow oil (250.0 mg, 58%). ¹H NMR (400 MHz, CDCl₃) δ 7.48 – 7.39 (m, 2H), 7.27 (dd, J = 8.6, 6.7 Hz, 2H), 7.23 – 7.13 (m, 3H), 6.73 – 6.65 (m, 2H), 4.22 (dd, J = 4.7, 2.4 Hz, 2H), 2.67 – 2.54 (m, 4H), 2.39 – 2.32 (m, 2H), 2.25 (t, J = 7.0 Hz, 2H), 2.07 (dd, J = 7.3, 4.7 Hz, 2H), 1.96 (dd, J = 7.8, 4.2 Hz, 2H), 1.79 – 1.67 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 152.4, 142.4, 135.2, 133.6, 128.6, 128.4, 125.7, 119.7, 119.1, 116.7, 101.6, 58.8, 58.0, 56.3, 33.3, 28.6, 27.4.

4.2.28. 4-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (15).

A mixture of 26c (200.0 mg, 0.603 mmol), potassium carbonate (166.8 mg, 1.207 mmol), in anhydrous DMSO (4 mL) was cooled over an ice bath. 30% Hydrogen peroxide solution (0.210 mL, 2.715 mmol) was added dropwise, and the reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was quenched with water and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing DCM/MeOH gradient to afford 15 as a colorless oil (150 mg, 71%). ¹H NMR (400 MHz, CDCl₃) δ 7.72 – 7.64 (m, 2H), 7.31 – 7.23 (m, 2H), 7.17 (td, J = 5.2, 2.8 Hz, 3H), 6.76 – 6.68 (m, 2H), 5.74 (s, 2H), 4.23 (dd, J = 4.5, 2.4 Hz, 2H), 2.66 – 2.52 (m, 5H), 2.40 (dd, J = 10.9, 1.6 Hz, 2H), 2.23 (d, J = 14.0 Hz, 1H), 2.11 – 1.91 (m, 4H), 1.78 – 1.66 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 169.2, 149.8, 142.3, 129.4, 128.5, 128.3, 125.7, 120.7, 114.1, 56.5, 55.2, 55.1, 33.3, 28.4, 28.1. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₇N₃O, 350.2227; found, 350.2216.

4.2.29. 5-Bromo-2-(ethoxymethoxy)benzonitrile (28a).

A mixture of 27a (1.0 g, 5.050 mmol), (chloromethoxy)ethane (0.573 mg, 6.060 mmol), and DIPEA (1.8 mL, 10.100 mmol) in DCM (8 mL) was stirred overnight. Upon completion the reaction mixture was quenched with water and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and brine (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 28a as an off-white solid (0.900 g, 70%), mp: 142 – 143 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J = 2.5 Hz, 1H), 7.61 (dd, J = 9.0, 2.5 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 5.32 (s, 2H), 3.77 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 158.4, 137.2, 135.7, 116.7, 114.9, 113.5, 104.7, 93.7, 65.2, 15.0.

4.2.30. 2-(Ethoxymethoxy)-5-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzonitrile (29a).

A mixture of 21 [12] (500.0 mg, 2.171 mmol), 28a (611.5 mg, 2.388 mmol), Pd(OAc)₂ (48.7 mg, 0.217 mmol), XPhos (103.5 mg, 0.0.217 mmol), and sodium tert-butoxide (312.9 mg, 3.256 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 29a as a colorless oil (515 mg, 58%). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.5 Hz, 2H), 7.21 – 7.15 (m, 2H), 7.11 (d, J = 9.0 Hz, 1H), 6.95 – 6.86 (m, 2H), 5.22 (s, 2H), 4.04 (dd, J = 4.5, 2.4 Hz, 2H), 3.79 (q, J = 7.1 Hz, 2H), 3.48 (s, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.56 (dd, J = 11.0, 2.6 Hz, 2H), 2.39 (dd, J = 10.8, 1.5 Hz, 2H), 2.25 (t, J = 7.0 Hz, 2H), 2.07 – 1.86 (m, 3H), 1.79 – 1.67 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl3) δ 151.0, 142.6, 142.3, 128.5, 128.3, 125.7, 121.5, 119.0, 117.5, 117.0, 103.9, 94.4, 67.9, 56.2, 56.4, 55.0, 33.3, 28.3, 27.8, 15.2.

4.2.31. 2-(Ethoxymethoxy)-5-(3-(3-phenylpropyl))-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (30a).

A mixture of 29a (515.0 mg, 1.270 mmol), potassium carbonate (351.0 mg, 2.540 mmol), in anhydrous DMSO (6 mL) cooled over an ice bath. 30% Hydrogen peroxide solution (0.443 mL, 5.715 mmol) was added dropwise, and the reaction mixture was stirred for 2 hours. The reaction mixture was quenched with water and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing DCM/MeOH gradient to afford 30a as a colorless oil (281.0 mg, 52%). ¹H NMR (400 MHz, CDCl₃) δ 7.79 (s, 1H), 7.60 (d, J = 3.2 Hz, 1H), 7.27 (dd, J = 8.0, 6.9 Hz, 2H), 7.21 – 7.12 (m, 3H), 7.09 (d, J = 9.0 Hz, 1H), 6.86 (dd, J = 9.0, 3.2 Hz, 1H), 5.70 (s, 1H), 5.29 (d, J = 8.1 Hz, 2H), 4.16 (d, J = 4.5 Hz, 2H), 3.75 (q, J = 7.1 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.56 (dd, J = 10.9, 2.6 Hz, 2H), 2.46 – 2.39 (m, 2H), 2.24 (t, J = 7.0 Hz, 2H), 2.06 – 1.96 (m, 2H), 1.96 – 1.89 (m, 1H), 1.78 – 1.66 (m, 2H), 1.24 (t, J = 7.0 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 172.0, 153.8, 142.5, 139.1, 128.5, 128.3, 125.7, 121.8, 119.7, 118.6, 114.0, 92.3, 58.7, 58.4, 56.4, 63.5, 33.4, 28.6, 27.6, 15.2.

4.2.32. 2-Hydroxy-5-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (16).

A mixture of 30a (281.0 mg, 0.663 mmol) and 48% HBr (0.149 mL, 1.327 mmol) in MeOH (5 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 16 as yellow oil (132 mg, 54%). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (d, J = 7.7 Hz, 2H), 7.16 (dd, J = 27.5, 7.6 Hz, 4H), 6.97 (d, J = 7.8 Hz, 1H), 6.75 (t, J = 7.9 Hz, 1H), 6.61 (s, 1H), 5.95 (s, 1H), 4.05 (s, 2H), 2.76 – 2.58 (m, 4H), 2.48 (d, J = 10.4 Hz, 2H), 2.34 (q, J = 7.5 Hz, 2H), 2.06 – 1.83 (m, 4H), 1.77 (p, J = 7.2 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 172.1, 153.8, 142.5, 139.1, 128.5, 128.3, 125.7, 121.8, 119.7, 118.6, 114.1, 58.7, 58.4, 56.4, 33.4, 28.5, 27.6 HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₇N₃O₂, 366.2176; found, 366.2178.

4.2.33. 3-Bromo-2-(ethoxymethoxy)benzonitrile (28b).

A mixture of 27b (1.0 g, 5.050 mmol), (chloromethoxy)ethane (0.573 mg, 6.060 mmol), and DIPEA (1.8 mL, 10.100 mmol) in DCM (8 mL) stirred overnight. Upon completion the reaction mixture was quenched with water and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and brine (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 28b as an off-white solid (0.924 g, 71%), mp: 135 – 136 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (dd, J = 8.0, 1.6 Hz, 1H), 7.56 (dd, J = 7.7, 1.6 Hz, 1H), 7.09 (t, J = 7.9 Hz, 1H), 5.34 (s, 2H), 4.01 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 156.6, 138.3, 132.8, 125.6, 118.1, 115.7, 109.9, 98.7, 66.5, 14.9.

4.2.34. 2-(Ethoxymethoxy)-3-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzonitrile (29b).

A mixture of 21 [12] (350.0 mg, 1.519 mmol), 28b (428.0 mg, 1.671 mmol), Pd(OAc)₂ (34.0 mg, 0.152 mmol), XPhos (103.5 mg, 0.217 mmol), and sodium tert-butoxide (219.0 mg, 2.279 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 29b as a colorless oil (91.0 mg, 15%). ¹H NMR (400 MHz, CDCl₃) δ 7.32 – 7.25 (m, 1H), 7.22 – 7.14 (m, 3H), 7.10 – 6.98 (m, 3H), 5.30 (d, J = 5.1 Hz, 3H), 4.10 (s, 2H), 4.03 – 3.85 (m, 2H), 2.72 – 2.61 (m, 4H), 2.41 – 2.27 (m, 4H), 1.97 (q, J = 6.2 Hz, 2H), 1.78 (m, J = 21.9, 8.8, 6.1 Hz, 4H), 1.27 (t, J = 7.1 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 153.8, 142.5, 139.1, 128.5, 128.3, 125.7, 121.8, 119.7, 118.7, 114.0, 115.8, 92.3, 58.7, 58.4, 56.4, 63.5, 33.4, 28.6, 27.6, 15.2.

4.2.35. 2-(Ethoxymethoxy)-3-(3-(3-phenylpropyl))-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (30b).

A mixture of 29b (90.0 mg, 0.222 mmol), potassium carbonate (61.3 mg, 0.444 mmol), in anhydrous DMSO (6 mL) was cooled over an ice bath. 30% Hydrogen peroxide solution (0.077 mL, 0.999 mmol) was added dropwise, and the reaction mixture was stirred for 2 hours. The reaction mixture was quenched with water and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing DCM/MeOH gradient to afford 30b as a colorless oil (85.0 mg, 90%). ¹H NMR (500 MHz, CDCl₃) δ 7.75 (s, 1H), 7.58 (dd, J = 7.8, 1.7 Hz, 1H), 7.29 (dd, J = 8.2, 6.9 Hz, 2H), 7.20 (d, J = 7.8 Hz, 3H), 7.07 (t, J = 7.9 Hz, 1H), 7.00 (dd, J = 8.1, 1.7 Hz, 1H), 5.37 (s, 2H), 4.03 (s, 1H), 3.69 (q, J = 7.0 Hz, 2H), 2.99 (s, 3H), 2.76 – 2.64 (m, 4H), 2.41 – 2.31 (m, 4H), 1.97 (q, J = 6.2 Hz, 2H), 1.87 – 1.73 (m, 3H), 1.18 (t, J = 7.0 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 172.0, 153.8, 142.5, 139.1, 128.5, 128.3, 125.7, 121.8, 119.7, 118.7, 114.0, 92.3, 58.7, 58.4, 56.4, 63.5, 33.4, 28.6, 27.6, 15.2.

4.2.36. 2-Hydroxy-3-(3-(3-phenylpropyl))-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (17).

A mixture of 30b (85.0 mg, 0.201 mmol) and 48% HBr (0.045 mL, 0.401 mmol) in MeOH (5 mL) was heated in microwave reactor for 15 minutes at 65°C (200W). Upon completion, the reaction mixture was cooled to room temperature and neutralized using NaHCO₃ to pH 9, quenched with water, and extracted with EtOAc (3 × 20 mL). The combined organic layer was washed vigorously with water (3 × 20 mL) and saturated NaCl (15 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified using column chromatography utilizing DCM/MeOH gradient to afford 17 as a yellow oil (50.0 mg, 68%). ¹H NMR (600 MHz, CDCl₃) δ 7.31 – 7.24 (m, 2H), 7.24 – 7.15 (m, 3H), 7.12 (dd, J = 8.0, 1.4 Hz, 1H), 6.98 (dd, J = 7.9, 1.3 Hz, 1H), 6.76 (t, J = 7.9 Hz, 1H), 6.64 (s, 1H), 5.76 (s, 1H), 4.05 (dt, J = 5.0, 2.3 Hz, 2H), 2.72 (dd, J = 10.8, 2.9 Hz, 2H), 2.67 (t, J = 7.7 Hz, 2H), 2.50 – 2.45 (m, 2H), 2.35 (t, J = 7.0 Hz, 2H), 1.99 (dd, J = 7.2, 5.1 Hz, 2H), 1.92 (dd, J = 8.2, 4.2 Hz, 2H), 1.81 – 1.72 (m, 2H); ¹³C NMR (151 MHz, CDCl₃) δ 172.0, 153.8, 142.5, 139.1, 128.5, 128.3, 125.7, 121.8, 119.7, 118.6, 114.0, 58.7, 58.4, 56.4, 33.3, 28.6, 27.6. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₇N₃O₂, 366.2176; found, 366.2165.

4.2.37. 2-Fluoro-5-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzonitrile (31a).

A mixture of 21 [12] (250.0 mg, 1.085 mmol), 5-bromo-2-fluorobenzonitrile (217.1 mg, 1.085 mmol), Pd₂(dba)₃ (99.4 mg, 0.109 mmol), (±)-BINAP (67.6 mg, 0.109 mmol), and cesium carbonate (530.4 mg, 1.628 mmol) in toluene (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 31a as yellow oil (160.5 mg, 42%). ¹H NMR (400 MHz, CDCl₃) δ 7.80 – 7.70 (m, 1H), 7.18 (dd, J = 7.9, 6.1 Hz, 3H), 7.03 (t, J = 8.7 Hz, 1H), 6.91 (dt, J = 9.1, 3.7 Hz, 1H), 6.86 (dd, J = 5.0, 3.1 Hz, 1H), 4.07 (s, 2H), 3.49 (d, J = 5.2 Hz, 2H), 2.68 – 2.52 (m, 4H), 2.37 (d, J = 10.7 Hz, 2H), 2.24 (t, J = 7.0 Hz, 2H), 2.04 (dd, J = 7.4, 4.6 Hz, 2H), 1.94 (dd, J = 7.9, 4.2 Hz, 2H), 1.73 (p, J = 7.2 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 152.2, 142.4, 141.7, 135.5, 133.6, 128.5, 128.2, 119.8, 119.1, 116.7, 101.6, 58.8, 58.0, 56.3, 33.3, 27.9, 26.8.

4.2.38. 2-Fluoro-5-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (18).

A mixture of 31a (160.5 mg, 0.458 mmol), potassium carbonate (126.6 mg, 0.916 mmol), in anhydrous DMSO (6 mL) cooled over an ice bath. 30% Hydrogen peroxide solution (0.160 mL, 2.060 mmol) was added dropwise, and the reaction mixture was stirred for 2 hours. The reaction mixture was quenched with water and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing DCM/MeOH gradient to afford 18 as a yellow oil (140.0 mg, 83%). ¹H NMR (400 MHz, CDCl₃) δ 7.48 (dd, J = 6.5, 3.2 Hz, 1H), 7.29 (t, J = 7.5 Hz, 2H), 7.23 – 7.15 (m, 3H), 7.00 (dd, J = 11.3, 8.9 Hz, 1H), 6.87 (dt, J = 8.9, 3.7 Hz, 1H), 6.78 (d, J = 12.5 Hz, 1H), 6.56 (s, 1H), 4.21 – 4.10 (m, 2H), 2.69 – 2.51 (m, 4H), 2.43 (d, J = 10.7 Hz, 2H), 2.26 (t, J = 7.0 Hz, 2H), 2.10 – 1.90 (m, 4H), 1.75 (p, J = 7.2 Hz, 2H); ¹³C NMR (101 MHz, CDCl3) δ 165.7 (d, J = 3.1 Hz), 153.6 (d, J = 238.9 Hz), 144.3, 142.3, 128.5, 128.3, 125.7, 120.3 (d, J = 12.4 Hz), 120.3 (d, J = 8.4 Hz), 117.6, 116.9 (d, J = 26.1 Hz), 56.6, 56.0, 55.0, 33.3, 28.4, 28.0. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₆FN₃O, 368.2133; found, 368.2157.

4.2.39. 2-Fluoro-3-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzonitrile (31b).

A mixture of 21 [12] (300.0 mg, 1.302 mmol), 3-bromo-2-fluorobenzonitrile (260.5 mg, 1.302 mmol), Pd₂(dba)₃ (119.3 mg, 0.130 mmol), (±)-BINAP (81.1 mg, 0.130 mmol), and cesium carbonate (636.5 mg, 1.628 mmol) in toluene (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 31b as a yellow oil (386.0 mg, 85%). ¹H NMR (400 MHz, CDCl₃) δ 7.39 – 7.16 (m, 5H), 7.03 (m, J = 25.3, 7.0, 4.0 Hz, 3H), 4.20 (d, J = 4.7 Hz, 2H), 2.71 – 2.62 (m, 4H), 2.41 (d, J = 10.7 Hz, 2H), 2.31 (t, J = 7.0 Hz, 2H), 2.04 (t, J = 6.1 Hz, 2H), 1.94 (dd, J = 7.9, 4.0 Hz, 2H), 1.77 (p, J = 7.2 Hz, 2H), 1.64 – 1.50 (m, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 142.3, 128.5, 128.3, 125.8, 125.1, 122.6, 121.9, 57.5, 57.4, 56.5, 56.4, 33.3, 28.4, 27.7.

4.2.40. 2-Fluoro-3-(3-(3-phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)benzamide (19).

A mixture of 31b (386.0 mg, 1.105 mmol), potassium carbonate (305.3 mg, 2.209 mmol), in anhydrous DMSO (8 mL) cooled over an ice bath. 30% Hydrogen peroxide solution (0.385 mL, 4.971 mmol) was added dropwise, and the reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was quenched with water and extracted with EtOAc (3 × 15 mL). The combined organic layer was washed vigorously with water (3 × 15 mL) and brine (10 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude product was subjected to column chromatography utilizing DCM/MeOH gradient to afford 19 as a light-yellow oil (310.0 mg, 76%). ¹H NMR (600 MHz, CDCl₃) δ 7.31 – 7.24 (m, 2H), 7.22 – 7.15 (m, 3H), 7.12 (dd, J = 8.0, 1.4 Hz, 1H), 6.98 (dd, J = 7.9, 1.3 Hz, 1H), 6.76 (t, J = 7.9 Hz, 1H), 6.64 (s, 1H), 5.76 (s, 1H), 4.05 (dt, J = 5.0, 2.3 Hz, 2H), 2.72 (dd, J = 10.8, 2.9 Hz, 2H), 2.67 (t, J = 7.7 Hz, 2H), 2.50 – 2.45 (m, 2H), 2.35 (t, J = 7.0 Hz, 2H), 1.99 (dd, J = 7.2, 5.1 Hz, 2H), 1.93 (h, J = 9.1 Hz, 2H), 1.81 – 1.72 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 172.1, 153.8, 142.5, 139.1, 128.5, 128.3, 125.7, 121.8, 119.7, 118.6, 114.1, 58.7, 58.4, 56.4, 33.4, 28.5, 27.6. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₂H₂₆FN₃O, 368.2133; found, 368.2146.

4.2.41. 2-(3-(3-(3-Phenylpropyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)oxazole (20).

A mixture of 21 [12] (300.0 mg, 1.302 mmol), 2-(3-bromo-phenyl)-oxazole (291.8 mg, 1.302 mmol), Pd(OAc)₂ (29.2 mg, 0.0.130 mmol), XPhos (62.1 mg, 0.130 mmol), and sodium tert-butoxide (187.7 mg, 1.954 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C overnight. The reaction mixture was cooled to room temperature, quenched with water, and extracted with DCM (3 × 20 mL). The combined organics were washed with brine, dried over Na₂SO₄, concentrated, and subjected to column chromatography utilizing hexanes/EtOAc gradient to afford 20 as a colorless oil (81.0 mg, 17%). ¹H NMR (400 MHz, CDCl₃) δ 7.67 (s, 1H), 7.46 (t, J = 2.0 Hz, 1H), 7.32 – 7.13 (m, 8H), 6.85 (dd, J = 8.2, 2.5 Hz, 1H), 4.25 (dd, J = 4.4, 2.3 Hz, 2H), 2.67 – 2.53 (m, 4H), 2.49 – 2.42 (m, 2H), 2.23 (t, J = 7.0 Hz, 2H), 2.11 – 1.91 (m, 5H), 1.72 (p, J = 7.3 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 162.6, 147.5, 142.4, 138.3, 129.9, 128.5, 128.3, 125.7, 117.5, 115.3, 113.0, 56.6, 55.5, 55.1, 33.3, 28.5, 28.1. HRMS (ESI) m/z for [M + H]⁺ calcd for C₂₄H₂₇N₃O, 374.2227; found, 374.2230.

4.2.42. General procedure for oxalate salt formation.

Oxalic acid (1 equiv) was added to a solution of the appropriate free base in MeOH. Once in solution, the MeOH was removed in vacuo and Et₂O was added to the crude residue. The resulting precipitate was collected by filtration and dried under vacuum to afford the corresponding oxalate salt.

4.3. Biological Methods.

4.3.1. Cell Lines and Cell Culture.

The cAMP Hunter^™ CHO-K1 stably expressing the human μ opioid receptor (OPRM1), κ opioid receptor (OPRK1), the human δ opioid receptor (OPRD1), and nociceptin receptor (OPRL1) were purchased from Eurofins DiscoverX (Fremont, CA) and maintained in F-12 media supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY), 1% penicillin/streptomycin/ʟ-glutamine (Life Technologies), and 800 μg/mL Geneticin (Mirus Bio, Madison, WI). The PathHunter^™ CHO-K1 stably expressing the human μ opioid receptor β-arrestin-2 EFC cell line was purchased from Eurofins DiscoverX (Fremont, CA) and maintained in F-12 media with 10% fetal bovine serum (Life Technologies, Grand Island, NY), 1% penicillin/streptomycin/ʟ-glutamine (Life Technologies), 800 μg/mL Geneticin (Mirus Bio, Madison, WI), and additional 300 μg/mL Hygromycin B (Mirus Bio). All cells were grown at 37 °C and 5% CO₂ in a humidified incubator.

4.3.2. Forskolin-induced cAMP accumulation assay.

Assays were performed as previously described [34]. Briefly, cells were plated in a 384-well white tissue culture plate (10,000 cells/well) and incubated overnight at 37°C. Compounds were first dissolved in DMSO to form stock solutions (5 mM concentration), and then 9 doses of 100X solutions were prepared by serial dilution with DMSO. Then 5X solutions were prepared by diluting 100X solutions with assay buffer consisting of Hank’s Buffered Salt Solution, HEPES, and forskolin. In the agonist assays, cells were treated with compounds (at 1X final concentration with 20 μM final forskolin concentration) and incubated for 30 min at 37°C. The HitHunter cAMP Assay for Small Molecules by Eurofins DiscoverX (Fremont, CA, USA) was then used according to manufacturer’s directions and the BioTek Synergy H1 hybrid and Cytation 5 plate readers (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (BioTek, Winooski, VT, USA) were used to quantify luminescence. Data were blank subtracted with vehicle control followed by normalization to the forskolin control and were then analyzed in GraphPad Prism 10.2.2 (GraphPad, LaJolla, CA, USA) using nonlinear regression.

4.3.3. β-Arrestin2 recruitment assay.

Assays proceeded as previously described [71]. Briefly, 5,000 cells/well of the PathHunter CHO-K1 OPRM1 β-arrestin-2 cell line was plated in 384-well white tissue culture microplates and incubated overnight at 37°C. 10 doses of 100X compound solutions were prepared by serial dilution with DMSO from the 5 mM DMSO stock solutions. 5X working solutions were then prepared by further dilution with assay buffer consisting of Hank’s Buffered Salt Solution and HEPES. Cells were incubated with compounds (at 1X final concentration) at 37°C for 90 min. The PathHunter Detection Kit by Eurofins DiscoverX (Fremont, CA, USA) was then used according to the manufacturer’s directions, and the BioTek Synergy H1 hybrid and Cytation 5 plate readers (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (BioTek, Winooski, VT, USA) were used to quantify luminescence. Data were blank subtracted with vehicle control followed by normalization to the maximum response of DAMGO and were then analyzed in GraphPad Prism 10.2.2 (GraphPad, LaJolla, CA, USA) using nonlinear regression.

4.3.4. Animals.

B6129/F1 strain mice were bred in-house, generated from parent strains C57BL/6Ntac crossed with 129S6/SvEvTac from Taconic (Germantown, NY). At the time of testing, mice were aged 6–12 weeks, weighing 20–40 g. Mice were housed in cages of 1–5 mice of the same sex, exposed to a 12 h light/dark cycle, and given free access to food and water. All animal experiments were approved by the University of Kentucky Institutional Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

4.3.5. Drugs for in Vivo Experiments.

Buprenorphine hydrochloride (National Institute on Drug Abuse Drug Supply Program) was dissolved in 10% Captisol^® aqueous solution. Compound 13 was dissolved in 0.9% NaCl saline and compounds 11, 12, and atoxifent were dissolved in 10% Captisol^® aqueous solution. All compounds were administered via i.p. injection. Doses were expressed as free base weight.

4.3.6. Hot Plate Test (HPT) in mice.

Supraspinal analgesia was measured using the hot plate test (HPT) following the procedures described by Woolfe and Macdonald [72]. Briefly, a timer was started when the mouse first touched the hotplate and was stopped with the lick of one paw, either front or hind, measuring paw lick latency. Baseline latencies were first recorded after mice had acclimated for at least 1 h in the experiment room. Then, mice were injected intraperitoneally (i.p.) with vehicle (10% Captisol aq. solution except for 13 saline was used) followed by increasing cumulative doses of agonist and tested over a 1hr period in the hotplate apparatus, with 10 min between injections. The hotplate analgesia meter (Columbus Instruments, Columbus, OH) was maintained at a constant temperature of 55 ± 0.1 °C and a maximum cutoff time of 30 s was set to prevent permanent tissue damage.

4.3.7. Data Analysis.

Statistical analysis was performed in GraphPad Prism 10.2.3 using Nonlinear Regression analyses to estimate ED₅₀.

Supplementary Material

Appendix A. Supplementary Data

Highlights.

• N-(3-hydroxyphenyl)-3,8-diazabicyclooctanes are an emerging opioid class.

• H-bond donors at 3-position are favorable for MOR activity.

• Amino (11), sulfonamide (12), and carboxamide (13) substitutions are tolerated.

• Carboxamide 13 shows 2.4-fold greater in vitro metabolic stability than atoxifent.

• N-(3-Hydroxyphenyl)-3,8-diazabicyclooctanes 11 – 13 are partial agonists in vivo.

List of Abbreviations

% MPE

percentage maximum possible effect

AUC

area under the curve

cAMP

cyclic adenosine monophosphate

DOR

delta opioid receptor

EC₅₀

half maximal effective concentration

E_max

maximal efficacy

GPCRs

G protein-coupled receptors

KOR

kappa opioid receptor

MOR

mu opioid receptor

EOM

Ethoxymethoxy

pEC₅₀

Negative logarithm of half maximal effective concentration

HLM

human liver microsomes

MLM

mouse liver microsomes

i.p.

intraperitoneal

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.