Synthesis and Structure–Activity Relationships of 5′-Aryl-14-alkoxypyridomorphinans: Identification of a μ Opioid Receptor Agonist/δ Opioid Receptor Antagonist Ligand with Systemic Antinociceptive Activity and Diminished Opioid Side Effects

Abstract

We previously identified a pyridomorphinan (6, SRI-22138) possessing a 4-chlorophenyl substituent at the 5′-position on the pyridine and a 3-phenylpropoxy at the 14-position of the morphinan as a mixed μ opioid receptor (MOR) agonist and δ/κ opioid receptor (DOR/KOR) antagonist with potent antinociceptive activity and diminished tolerance and dependence in rodents. Structural variations at the 5′- and 14-positions of this molecule gave insights into the structure–activity relationships for binding and functional activity. Subtle structural changes exerted significant influence, particularly on the ability of the compounds to function as agonists at the MOR. In vivo evaluation identified compound 20 (SRI-39067) as a MOR agonist/DOR antagonist that produced systemically active potent antinociceptive activity in tail-flick assay in mice, with diminished tolerance, dependence/withdrawal, reward liability, and respiratory depression versus morphine. These results support the hypothesis that mixed MOR agonist/DOR antagonist ligands may emerge as novel opioid analgesics with reduced side effects.

Graphical Abstract

INTRODUCTION

For relief of moderate to severe acute pain, opioids remain the mainstay of treatment. However, the use of opioid analgesics is associated with severe limiting side effects including abuse liability, respiratory depression, constipation, and development of tolerance and dependence.^1–7 Therefore, considerable effort has been expended on the discovery of novel opioid analgesics that are as efficacious as the prototypic opioid analgesic morphine but possessing reduced side effects. Opioid drugs exert their analgesic effects through binding and activation of the three opioid receptor subtypes, namely, the μ opioid receptor (MOR), the δ opioid receptor (DOR), and the κ opioid receptor (KOR).^8,9 Several lines of evidence suggest that there are physical and/or functional interactions between these G-protein coupled receptors, and molecules that interact with multiple receptors may prove advantageous in the search for opioid ligands with diminished side effect profiles.^10–15 Considerable evidence from gene knockout/knockdown studies and studies using pharmacological probes suggests that activation of the MOR with simultaneous inhibition of the DOR leads to MOR-mediated analgesic activity with diminished development of tolerance, dependence, and potentially other side effects.^13,15–19 Based on these observations, we^20,21 and others^13,22–28 pursued the development of mixed function ligands possessing a MOR agonist/DOR antagonist profile of activity using peptidic, peptidomimetic, and nonpeptide opioid templates. Exemplary MOR agonist/DOR antagonist ligands evaluated in animal models are shown in Figure 1.

Figure 1.

Structures of MOR agonist/DOR antagonist ligands 1–6 and the MOR-DOR antagonist 7.

We previously synthesized and evaluated a series of 14-alkoxypyridomorphinans possessing a 4-(chlorophenyl) group at the 5′-position on the pyridine ring that led to the identification of compound 6 as a ligand possessing a balanced binding affinity profile at the MOR and DOR with agonist activity at the MOR and potent antagonist activity at the DOR.²¹ Pharmacological evaluation with this compound demonstrated antinociceptive activity comparable to morphine in the mouse warm water tail-flick assay. Moreover, on repeated administration, compound 6 displayed diminished development of analgesic tolerance compared to morphine. These studies with compound 6 were performed by intracerebroventricular (icv) administration because of its poor blood–brain barrier penetration capacity. The current effort was undertaken to expand the structure–activity relationship (SAR) in the pyridomorphinan scaffold of this prototype compound 6 and to identify improved ligands with the mixed MOR agonist/DOR antagonist profile and acceptable absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic (PK) properties to enable proof-of-concept studies with systemic administration.

RESULTS AND DISCUSSION

Compound Design.

The design principle that led to the identification of compound 6 was that the nonselective dual MOR/DOR antagonist pyridomophinan 7 (Figure 1) possessing a C-14 hydroxyl group could be transformed to a MOR agonist with retention of the DOR antagonist profile by installation of a 3-(phenylpropyl) group on the C-14 oxygen atom.²¹ In our previous DOR antagonist-focused effort, we had demonstrated that a number of compounds related to 7, possessing various aryl and heteroaryl groups at the 5′-position, displayed a dual MOR/DOR antagonist profile similar to that of 7.^20,29–31 Therefore, we pursued the installation of a 3-(phenylpropyl) group on C-14 oxygen on a series of pyridomorphinans possessing substituent variations on the phenyl group at the 5′-position (compounds 8–29, Table 1) and a few heteroaryl variants in place of the phenyl group at the 5′-position (compounds 30–42, Table 2). In addition, we pursued targeted changes to the alkyl group on the oxygen at C-14 using 6 and its deschloro analogue 8 as the templates. This study was intended to explore the consequence of installation of a substituent other than a 3-(phenylpropyl) group (compounds 43–52, Table 3) on opioid receptor binding and functional activity.

Table 1.

Binding Affinity, Functional Activity, and In Vitro ADME Data for 5′-Aryl-14-(phenylpropoxy) Compounds 8–29

compd	R	Binding K i(nM)a			[35S]GTPγS Couplingb						Log Dc	MLM t½ (min)d
		MOR	DOR	KOR	MOR Agonist EC50 (nM)	MOR Agonist Emax (%)	DOR Antagonist IC50 (nM)	DOR Antagonist Imax (%)	KOR Agonist EC50 (nM)	KOR Agonist Emax (%)
6 e		7.55 ± 1.23	2.1 ± 0.2	60.9 ± 31	8.20 ± 4.78	47.5 ± 18.7	4.88 ± 2.75	67.0 ± 16.4	12.6 ± 5.5	10.9 ± 1.7	0.393	9.72
8		23.1 ± 12.9	19.3 ± 9.8	132 ± 18	1.27 ± 0.07	105 ± 3	0.93 ± 0.07	98.3 ± 0.9	>3333	(19.1)f	0.868	8.96
9		138 ± 22	20.9 ± 1.8	75.9 ± 10.2	26.6 ± 15.0	78.3 ± 2.7	< 0.51	90.7 ± 5.7	5.52 ± 0.96	34.3 ± 1.2	3.64	9.64
10		2.33 ± 0.14	3.42 ± 0.29	6.69 ± 0.39	117 ± 27	37 ± 4	34.5 ± 18.5	101 ± 4	56.6 ± 11.3	15.3 ± 0.9	0.428	10.4
11		171 ± 4	53.7 ± 8.1	35.9 ± 7.5	130 ± 61	68.0 ± 5.3	3.20 ± 0.62	103 ± 4	NDg	NDg	2.84	10.4
12		370 ± 13	7.52 ± 0.20	71.2 ± 4.8	NDg	NDg	NDg	NDg	NDg	NDg	2.92	8.08
13		235 ± 20	97.2 ± 14.4	152 ± 18	5.53 ± 0.70	92.0 ± 1.7	14.3 ± 6.3	92.6 ± 4.1	NDg	NDg	2.68	6.48
14		24.4 ± 5.0	26.8 ± 4.7	17.6 ± 2.9	20.6 ± 10.0	111 ± 4	< 0.169	105 ± 9	NCh	NCh	3.39	7.09
15		191 ± 13	34.4 ± 4.5	102 ± 6.2	2.31± 0.41	115 ± 3	3.63 ± 1.1	104 ± 0.6	NDg	NDg	2.44	7.86
16		29.9 ± 0.6	3.78 ± 0.61	129 ± 46	25 ± 5	58 ± 3	28.2 ± 20.7	136 ± 7	NCh	NCh	4.52	8.27
17		135 ± 29	64.1 ± 8.9	175 ± 44	141 ± 78	122 ± 6	2.48 ± 0.50	81.3 ± 6.9	NDg	NDg	3.73	8.28
18		249 ± 27	93.4 ± 2.3	134 ± 15	3.40 ± 1.19	101 ± 1	51.6 ± 31.0	74.3 ± 1.2	NDg	NDg	3.08	8.27
19		61.9 ± 4.3	29.7 ± 9.4	126 ± 7	7.18 ± 2.13	79.0 ± 1.0	0.79 ± 0.07	94.3 ± 1.9	NCh	NCh	2.01	8.65
20		91.3 ± 14.1	48.0 ± 8.9	103 ± 16	13.0 ± 1.2	78.7 ± 2.0	2.40 ± 0.28	85.7 ± 1.2	NCh	NCh	2.58	10.5
21		148 ± 13	30.6 ± 9.1	140 ± 24	23.5 ± 6.5	81.0 ± 1.2	22.0 ± 2.7	92.3 ± 0.7	2.02 ± 0.64	20.2 ± 0.2	1.93	9.03
22		12.9 ± 0.8	8.37 ± 0.33	18.1 ± 3.2	1.12 ± 0.04	104 ± 5	1.28 ± 0.14	90.3 ± 10.5	NDg	NDg	3.73	7.36
23		6.97 ± 0.70	69.6 ± 5.1	7.96 ± 1.65	233 ± 24	94.7 ± 0.3	12.3 ± 3.9	82.7 ± 1.7	NCh	NCh	1.93	9.78
24		15.8 ± 1.6	68.9 ± 7.9	11.9 ± 2.4	1.50 ± 0.19	98.0 ± 11.1	3.76 ± 1.54	99.3 ± 3.8	NCh	NCh	3.38	7.10
25		4.70 ± 0.58	53.1 ± 4.6	6.18 ± 1.88	2.10 ± 0.26	116 ± 3	2.53 ± 0.99	96.3 ± 2.7	NCh	NCh	1.87	7.83
26		56.4 ± 5.6	14.0 ± 3.4	22.7 ± 10.0	28.9 ± 16.5	57.0 ± 2.1	11.7 ± 0.4	74.0 ± 1.0	NCh	NCh	3.65	12.7
27		141 ± 62	> 3333	13.5 ± 2.9	NDg	NDg	NDg	NDg	NDg	NDg	2.52	10.3
28		337 ± 80	> 3333	608 ± 343	NDg	NDg	NDg	NDg	NDg	NDg	2.60	14.0
29		159 ± 37	394 ± 338	15.1 ± 3.9	NDg	NDg	NDg	NDg	NDg	NDg	3.15	11.0
DAMGO					25.5 ± 1.4	100
Naloxone		59.5 ± 2.9	98.1 ± 3.0				153 ± 6	100
U50,488				31.9 ± 9.0					35.8 ± 2.0	100

^aBinding affinities determined by competitive displacement of [³H]diprenorphine in membrane preparations from CHO cells expressing the human MOR, DOR, or KOR.

^b[³⁵S]GTPγS binding for functional activity performed using the same membrane preparations. MOR and KOR agonist efficacy expressed as percent stimulation versus standard agonists DAMGO and U50,4888, respectively. DOR antagonist potency (IC₅₀) and efficacy (I_max) determined versus the standard agonist SNC80 (100 nM). DOR antagonist efficacy (I_max) calculated as the percent of the reference antagonist naloxone. KOR efficacy in parenthesis is the maximum stimulation produced at 10 μM. All values expressed as mean ± SEM for N = 3–4 independent experiments.

^cThe log D measured at pH 7.4.

^dIn vitro mouse liver microsomal half-life values in minutes.

^eData for compound 6 included for comparison.

^fPercent maximum stimulation at 10 μM.

^gND = Not determined.

^hNC = Not converged (no agonist activity detected).

Table 2.

Binding Affinity, Functional Activity, and In Vitro ADME Data for 5′-Heteroaryl-14-(phenylpropoxy) Compounds 30–42

compd	R	Binding K i(nM)a			[35S]GTPγS Couplingb						log Dc	MLM t½ (min)d
		MOR	DOR	KOR	MOR Agonist EC50 (nM)	MOR Agonist Emax (%)	DOR Antagonist IC50 (nM)	DOR Antagonist Imax (%)	KOR Agonist EC50 (nM)	KOR Agonist Emax (%)
30		14.7 ± 1.5	< 0.169	13.8 ± 2.6	1.61 ± 0.28	84.7 ± 8.7	0.98 ± 0.12	101 ± 0.3	NCe	NCe	3.30	7.07
31		105 ± 1	6.52 ± 0.28	19.2 ± 4.3	526 ± 83	37.7 ± 4.6	0.51 ± 0.05	99.3 ± 0.7	NDf	NDf	2.32	6.25
32		82.7 ± 2.5	30.6 ± 7.9	27.8 ± 0.8	>3333	(30.2 ± 5.3)g	0.67 ± 0.03	93.3 ± 0.7	NCe	NCe	3.67	8.22
33		47.1 ± 2.1	6.43 ± 1.49	15.8 ± 1.8	56.3 ± 26.5	49.3 ± 8.7	18.3 ± 0.6	91.0 ± 2.0	NCe	NCe	3.53	11.1
34		1.05 ± 0.37	63.5 ± 51.2	<0.51	1.88 ± 0.18	81.0 ± 4.0	0.39 ± 0.03	86.7 ± 0.9	NCe	NCe	1.74	9.60
35		10.7 ± 0.6	3.50 ± 1.14	6.62 ± 2.88	2.69± 0.37	93.5 ± 3.5	0.70 ± 0.08	89.3 ± 0.3	NCe	NCe	4.32	13.4
36		66.2 ± 4.7	22.1 ± 7.8	30.8 ± 8.5	>3333	(37.3 ± 5.1)g	0.71 ± 0.02	92.0 ± 2.1	NCe	NCe	3.30	10.1
37		47.9 ± 5.8	5.18 ± 0.37	24.8 ± 1.3	>3333	(29.3 ± 5.9)g	0.87 ± 0.14	92.3 ± 0.3	NCe	NCc	2.00	26.8
38		88.7 ± 1.4	40.1 ± 8.8	19.8 ± 0.9	>3333	(40.8 ± 3.8)g	1.27 ± 0.15	96.7 ± 0.7	NCe	NCe	3.55	8.72
39		121 ± 66	257 ± 211	29.8 ± 21.3	3.29 ± 0.82	115 ± 3	0.62 ± 0.08	92.3 ± 1.2	NCe	NCe	0.707	7.00
40		3.79 ± 1.63	7.96 ± 2.22	8.40 ± 5.25	659 ± 484	44 ± 2	19.6 ± 8.7	106 ± 6	NCe	NCe	0.943	21.7
41		99.3 ± 17.8	64.5 ± 3.3	23.1 ± 4.0	3.46 ± 1.19	74.7 ± 3.4	0.68 ± 0.05	92.0 ± 3.1	NDf	NDf	2.11	18.5
42		3.18 ± 0.07	8.12 ± 1.97	14.8 ± 4.8	17.5 ± 6.1	70.0 ± 2.5	< 0.17	101 ± 3	NCe	NCe	2.85	6.35
DAMGO					25.5 ± 1.4	100
Naloxone		59.5 ± 2.9	98.1 ± 3.0				153 ± 6	100
U50,488				31.9 ± 9.0					35.8 ± 2.0	100

^aSee corresponding footnotes in Table 1.

^bSee corresponding footnotes in Table 1.

^cSee corresponding footnotes in Table 1.

^dSee corresponding footnotes in Table 1.

^eNC = not converged (no agonist activity detected).

^fND = not determined.

^gPercent maximum stimulation at 10 μM.

Table 3.

Binding Affinity, Functional Activity, and In Vitro ADME Data for 5′-Aryl-14-alkoxy Compounds 43–52^a

compd	R	R1	Binding K i (nM)a			35S-GTPγS Couplingb						log Dc	MLM t½ (min)d
			MOR	DOR	KOR	MOR Agonist EC50 (nM)	MOR Agonist Emax (%)	DOR Antagonist IC50 (nM)	DOR Antagonist Imax (%)	KOR Agonist EC50 (nM)	KOR Agonist Emax (%)
43			5.37 ± 0.51	5.24 ± 1.65	10.3 ± 5.7	NCe	NCe	4.69 ± 2.45	16.7 ± 1.2	NDf	NDf	1.73	12.2
44			819 ± 96	172 ± 85	21.3 ± 2.3	NCe	NCe	25.4 ± 21	102 ± 3.1	NDf	NDf	2.72	160
45			799 ± 131	32.5 ± 12.6	31 ± 4.6	NCe	NCe	10.8 ± 9	101 ± 0.6	NDf	NDf	2.79	9.40
46			3.96 ± 0.43	5.89 ± 3.70	5.3 ± 1.4	>3333	64 ± 9.8	< 0.51	93 ± 1	NCe	NCe	0.0434	8.60
47			3.82 ± 0.06	29.2 ± 9.2	21.9 ± 5.6	NCe	NCe	< 0.51	96 ± 2	NDf	NDf	0.818	8.88
48			70.9 ± 3.4	11.0 ± 0.8	60.2 ± 20.3	93.9 ± 9.1	60.0 ± 1.5	63.8 ± 40.3	100	NCe	NCe	2.38	81.1
49			95.3 ± 8.2	12.9 ± 2.1	57.9 ± 3.8	23.6 ± 1.4	66.0 ± 4.6	28.7 ± 11.9	100	34.5 ± 14.2	9.54 ± 0.19	3.27	27.6
50			5.99 ± 0.53	64.5 ± 28.9	24.8 ± 7.3	9.12 ± 4.93	68.3 ± 1.2	< 0.17	96.3 ± 2.7	NDf	NDf	3.37	6.49
51			92.1 ± 4.1	14.6 ± 0.2	57.1 ± 12.4	24.8 ± 2.7	24.3 ± 2.6	66.6 ± 16.5	100	NDf	NDf	2.33	16.8
52			499 ± 47	7.48 ± 0.97	252 ± 20	NDf	NDf	NDf	NDf	NDf	NDf	2.60	6.67
DAMGO						25.5 ± 1.4	100
Naloxone			59.5 ± 2.9	98.1 ± 3.0				153 ± 6	100
U50,488					31.9 ± 9.0					35.8 ± 2.0	100

^aSee corresponding footnotes in Table 1.

^bSee corresponding footnotes in Table 1.

^cSee corresponding footnotes in Table 1.

^dSee corresponding footnotes in Table 1.

^eNC = not converged (no agonist activity detected).

^fND = not determined.

Synthesis.

Several synthetic approaches were utilized for the preparation of the desired target compounds. The original synthetic route developed for the preparation of compound 6 was adopted for the preparation of the 5′-aryl target compounds 8–10 and the 5′-heteroaryl target compounds 36 and 38, as depicted in Scheme 1. The starting materials 54–58 were synthesized by annulation of the pyridine ring on the C-ring of naltrexone (53) by condensation with substituted malondialdehydes and ammonium acetate in acetic acid. Dialkylation of the phenol and the tertiary alcohol groups with phenylpropyl bromide followed by selective removal of the phenypropyl group from the phenolic oxygen using BBr₃ yielded the desired target compounds.

Scheme 1. Synthesis of Pyridomorphinans 8–10, 36, and 38^a.

^aReagents and conditions: (a) RCH(CHO)₂, AcONH₄, AcOH, reflux, 16 h; (b) NaH, Ph(CH₂)₃Br, DMF, 0 °C to rt, 4 h; (c) BBr₃, CH₂Cl₂, −78 to 0 °C, 1 h.

We also developed an alternative, more versatile approach utilizing the 3-bromopyridine 60, prepared by dialkylation of the previously described 59,³⁰ as the key starting material. Suzuki coupling of 60 with arylboronic acids or esters, followed by deprotection of the phenol, as depicted in Scheme 2, gave the desired target compounds 11–18, 22–29, 31–34, 37, and 39–41. Similarly, coupling of 60 with pyrrole using Pd₂(dba)₃ as the catalyst in the presence of Cs₂CO₃ and P(t-Bu)₃ in toluene followed by 3-O-dealkylation with BBr₃ gave 30 (Table 2).

Scheme 2. Synthesis of Pyridomorphinans 11–18, 22–29, 31–34, 37, and 39–41^a.

^aReagents and conditions: (a) NaH, Ph(CH₂)₃Br, DMF, 0 °C to rt, 4 h; (b) RB(OH)₂ or RBpin, Pd(PPh₃)₄, K₂CO₃, 10:1 DMF/H₂O, mw, 100 °C, 1 h; (c) BBr₃, CH₂Cl₂, −78 to 0 °C, 1 h.

For the preparation of the targets containing a methoxy group (19–21 and 35), intermediates carrying benzyl as the phenolic protecting group were utilized to facilitate selective deprotection under reductive or acidic conditions (Scheme 3). The benzyl-protected bromopyridine 85 was accessible from 59 via benzylation of the phenolic OH followed by phenypropylation of the 14-OH group.

Scheme 3. Synthesis of Pyridomorphinans 19–21 and 35^a.

^aReagents and conditions: (a) RBpin, Pd(PPh₃)₄, K₂CO₃, 10:1 DMF/H₂O, mw, 100 °C, 1 h; (b) TFA, reflux, 1.5 h or H₂, 10% Pd/C, 1:1 CH₂Cl₂/MeOH, 20 h.

The oxadiazolyl compound 42 was prepared from the carbethoxypyridine 90 via conversion to hydrazide and cyclization to oxadiazole with triethyl orthoacetate, followed by etherification of the 14-OH and deprotection of the phenol, as shown in Scheme 4.

Scheme 4. Synthesis of Oxadiazolyl Pyridomorphinan 42^a.

^aReagents and conditions: (a) N₂H₄·H₂O, EtOH, reflux, 16 h; (b) CH₃C(OEt)₃, AcOH, 150 °C, 3 h; (c) NaH, Ph(CH₂)₃Br, DMF, 0 °C to rt, 4 h; (d) H₂, 10% Pd/C, 1:1 CH₂Cl₂/MeOH, 20 h.

For the synthesis of the target compound 43 possessing a pendant 4-pyridyl group in the 14-O-alkyl substituent, we initially attempted dialkylation of 7 or monoalkylation of 92 with 3-(4-pyridylpropyl)bromide and related alkylating agents. However, all our attempts under a variety of reaction conditions failed to yield the desired alkylated intermediates, possibly because of base-promoted elimination reaction predominating over nucleophilic substitution reaction. This difficulty was overcome by a stepwise derivatization involving 14-O-allylation, Heck coupling with 4-bromopyridine, and saturating the double bond, followed by acid-catalyzed deprotection of the 3-O-benzyl group, as shown in Scheme 5.

Scheme 5. Synthesis of 14-(3(4-Pyridyl)propoxy)pyridomorphinan 43^a.

^aReagents and conditions: (a) BnBr, K₂CO₃, Me₂CO, reflux, 3 h; (b) CH₂═CHCH₂Br, NaH, DMF, 0 °C to rt, 4 h; (c) 4-bromopyridine hydrochloride, Pd(PPh₃)₄, Pd(OAc)₂, K₂CO₃, 130 °C, 1 h; (d) H₂, 10% Pd/C, 1:1 CH₂Cl₂/MeOH, 20 h; (e) TFA, reflux, 1.5 h.

A dialkylation and selective mono-dealkylation sequence (Scheme 6) was successful for the preparation of the 2-phenoxyethoxy (44) and the 2-quinolinylmethoxy (45) target compounds using 2-phenoxyethyl triflate and 2-(chloromomethyl)quinoline as the alkylating agent, respectively.

Scheme 6. Synthesis of 14-Alkoxypyridomorphinans 44 and 45^a.

^aReagents and conditions: (a) PhOCH₂CH₂OSO₂CF₃, PMP, CH₃NO₂, 50 °C, 1 h; (b) BBr₃, CH₂Cl₂, −78 to 0 °C, 1 h; (c) 2-(chloromethyl)quinoline, NaH, DMF, 0 °C to rt, 4 h.

The allylation–Heck coupling route, shown in Scheme 5, for the preparation of 43 was adapted for the synthesis of the 3-(3-pyridylpropoxy) compound 46 and the 3-(4-pyridylpropoxy) compound 47 (Table 3) using 5′-phenylpyridomorphinan 54²⁹ as the starting material. In the preparation of these compounds, saturation of the double bond and removal of the phenolic-O benzyl-protecting group were accomplished by catalytic hydrogenation in a single step. Dialkylation of 54 with 4-(fluorophenyl)propyl bromide followed by treatment with BBr₃ to selectively remove the alkyl group from the phenolic oxygen delivered the desired target compound 48 (Table 3). The cyclohexylpropoxy (49) and the 4-phenylbutoxy (51) compounds were synthesized as depicted in Scheme 7.

Scheme 7. Synthesis of 14-Alkoxypyridomorphinans 49 and 51^a.

^aReagents and conditions: (a) BnBr, K₂CO₃, Me₂CO, reflux, 3 h; (b) 3-cyclohexylpropyl bromide or 4-phenylbutyl bromide, NaH, DMF, 0 °C to rt, 4 h; (c) BBr₃, CH₂Cl₂, −78 °C to rt (for 49) or TFA, 70 °C, 2 h (for 51).

A synthetic sequence similar to that described in Scheme 5 was deployed for the preparation of compound 50bold using compound 98 as the starting material. Thus, allylation of 98 and palladium-catalyzed coupling of the allyl derivative with 4-bromo-3,6-dihydro-2H-pyran gave the benzyl-protected diene intermediate which was subjected to catalytic hydrogenation to saturate the double bonds with simultaneous removal of the benzyl-protecting group to obtain the desired compound 50. Similarly, catalytic hydrogenation of the 14-O-allyl derivative of 98 gave the propoxy compound 52 (Table 3).

In Vitro Pharmacology and ADME Evaluation.

The binding affinity of the compounds at the MOR, DOR, and KOR was evaluated using well-established radioligand displacement assays. Membranes prepared from Chinese hamster ovary (CHO) cells stably expressing human MOR, DOR, or KOR and [³H]diprenorphine as the radioligand were used in determining the binding affinities.^32,33 The functional activity evaluations were performed using the guanosine-5′-O-(3[³⁵S]thio-triphosphate) ([³⁵S]-GTPγS) coupling assay using membranes from the same cells. For MOR agonist experiments, [d-Ala²,Me-Phe,Gly-ol⁵]enkephalin (DAMGO) was used as the reference agonist and the E_max values were calculated relative to DAMGO as the full agonist (E_max 100%). For DOR antagonist evaluation, SNC80 was used as the agonist ligand to determine the ability of the test compounds to inhibit SNC80-mediated stimulation of [³⁵S]-GTPγS binding. KOR agonist evaluations were performed similar to MOR agonist experiments with (±)-U50,488 as the standard agonist ligand (E_max 100%). Because the goal of our effort was to identify compounds with agonist activity at the MOR and antagonist activity at the DOR, the compounds were evaluated first as agonists at the MOR and antagonists at the DOR. Only selected compounds that were of interest based on binding and functional profiles at the MOR and DOR were then evaluated at the KOR to determine whether they displayed undesirable agonist activity.

All compounds were also evaluated via in vitro ADME assays to determine aqueous solubility, log D, and mouse liver and human liver microsomal stability. Mouse liver microsomal stability and log D data are presented along with binding affinity and functional activity data of the compounds in Tables 1–3. Human liver microsomal stability, solubility, and calculated physicochemical properties of the target compounds are given in Table S1 (Supporting Information).

Structure–Activity Relationships.

Binding Affinity of 5′-Aryl Compounds at the MOR.

The data for the group of compounds possessing structural variations on the 5′-phenyl ring are presented in Table 1. Most of the 5′-aryl compounds displayed only modest binding affinity at the MOR. The lead compound 6 possessing the 4-chlorophenyl substituent displayed single-digit nanomolar binding affinity at the MOR (K_i = 7.55 nM). Among compounds with other substituent groups on the phenyl ring, only three compounds, the 4-fluorophenyl compound 10, the 2-(dimethylamino)phenyl compound 23, and the 4-(dimethylamino)phenyl compound 25, showed improved binding affinity at the MOR with K_i values of 2.33, 6.97, and 4.70 nM, respectively. Compounds that displayed moderate binding affinity with the K_i value in the range of 10–100 nM include the unsubstituted (8), 3-methyl (14) 2-methyl-4-fluoro (16), 2-methoxy (19), 3-methoxy (20), 3-hydroxy (22), and 3-dimethylamino (24) phenyl derivatives. In contrast to the 4-fluoro compound with high affinity, the difluoro compounds 11 and 12 displayed a marked reduction in binding affinity. Other substituents that also had a deleterious effect on MOR affinity include 4-bromo (9), 2-methyl (13), 2,6-dimethyl (18), 4-methyl (15), 2-cyclopropyl (17), 4-methoxy (21), and 2-, 3-, or 4-acetylamino (compounds 27, 28, and 29) groups.

Binding Affinity of 5′-Aryl Compounds at the DOR.

At the DOR, the structure of the ligands had significant effects on the binding affinities. Removal of the chlorine at the para-position on the phenyl ring in the lead compound 6 yielded compound 8 that displayed nearly 10-fold reduction in affinity. Replacement with other substituents such as bromine (compound 9), methyl (compound 15), methoxy (compound 21), dimethylamino (compound 25), or acetylamino (compound 29) all led to moderate to significant reductions in affinity at the DOR. Although the 4-fluoro compound 10 maintained high affinity, the 3,4-difluoro (12) and the 2,4-difluoro (11) compounds displayed ~3- and ~25-fold reductions in affinity, respectively. Substitution at the 2-position of the phenyl ring, in general, appears to result in significant reductions in binding affinity at the DOR, as exemplified by the data on the 2-methyl, 2-cyclopropyl, 2,6-dimethyl, 2-methoxy, 2-dimethylamino, and 2-acetylamino compounds 13, 17, 18, 19, 23, and 27, respectively. Among ortho-, meta-, and para-substituted isomers, the ortho-methyl compound 13 shows lower affinity (DOR K_i = 97.2 nM) than the meta- and para-methyl compounds 14 and 15 (DOR K_i = 26.8 and 34.4 nM, respectively). In contrast, the methoxy (19–21) and dimethylamino (23–25) positional isomers did not display significant differences in their binding affinity at the DOR. The acetylamino substituent at the ortho-, meta-, or para-position (compounds 27, 28, and 29) proved detrimental to binding at the DOR.

Binding Affinity of 5′-Aryl Compounds at the KOR.

Most of the 5′-aryl compounds displayed weak binding affinity at the KOR. Exceptions include the 4-fluoro (10), 2-dimethylamino (23), and 4-dimethylamino (25) compounds that display KOR binding K_i values of <10 nM. Significant binding affinity at the KOR was also displayed by compounds possessing 3-dimethylamino (24, K_i = 11.9 nM), 2-acetylamino (27, K_i = 13.5 nM), 4-acetylamino (29, K_i = 15.1 nM), and 3-OH (22, K_i = 18.1 nM) groups.

Functional Activity of 5′-Aryl Compounds at the MOR.

The evaluated 5′-arylpyridomorphinans displayed a partial to full agonist profile with efficacy (E_max) ranging from 37 to 122%. A wide range of potencies were also displayed by these ligands (EC₅₀ ranging from 1.12 to 233 nM). Surprisingly, the functional potency of the compounds did not always reflect the binding affinity of the compounds. Except for compounds 10, 17, and 23, all of the phenyl-substituted compounds displayed much stronger functional potency compared to their binding affinity. For example, the MOR affinity of 4-methyl (15) and 2,6-dimethyl (18) compounds are in the 200 nM range but are >70-fold more potent as functional full agonists with EC₅₀ < 4 nM. Interestingly, among the dimethylamino isomers, although the ortho-substituted compound 23 displayed greatly diminished agonist potency (EC₅₀ = 233 nM), the meta- and para-substituted compounds 24 and 25 emerged as highly potent, full agonists with EC₅₀ values of 1.50 and 2.10 nM, respectively. These findings suggest that these ligands have high intrinsic efficacy at the MOR, enabling high potency functional activity even with modest binding affinity.

Functional Activity of 5′-Aryl Compounds at the DOR.

At the DOR, most of these compounds consistently displayed an antagonist profile of activity with high potency and efficacy (I_max > 70%). Sub-nanomolar antagonist IC₅₀ was displayed by the unsubstituted (8), 4-bromo (9), 3-methyl (14), and 2-methoxy (19) compounds. The presence of a 4-fluoro (10), 2-methyl (13), 2,6-dimethyl (18), or 4-methoxy (21) group tended to diminish DOR antagonist potency. The 2-(dimethylamino)methyl compound 26 also displayed moderate potency and efficacy as an antagonist.

Functional Activity of 5′-Aryl Compounds at the KOR.

Only compounds that emerged as promising MOR agonist/DOR antagonists were evaluated for potential agonist activity at the KOR. The evaluated compounds were found to be devoid of significant agonist efficacy at the KOR. Most of the compounds did not produce detectable activation. These are designated as nonconverged (NC) in Table 1. The 4-bromo compound 9 and the 4-methoxy compound 21 displayed agonist potencies of 5.52 and 2.02 nM, respectively. These compounds, however, displayed <35% efficacy, indicating a partial agonist profile at the KOR. Because these compounds displayed strong to moderate KOR binding affinity but little agonist activity, they likely act as KOR antagonists. However, this was not explicitly tested.

Binding Affinity of 5′-Heteroaryl Compounds at the MOR.

Among 5′-heterocyclic analogues, high affinity binding (K_i < 10 nM) was displayed by compounds possessing 2-methyl-4-pyridyl (34), 2-methoxy-4-pyridyl (35), 1-methyl-4-pyrazolyl (40), and 2-(5-methyl-1,3,4-oxadiazolyl) (42) substituents. All three pyridyl isomers (31–33) displayed modest to weak binding affinity. The introduction of a methoxy (35) or methyl (34) group at the 2-position of the 4-pyridyl moiety led to significant (4-fold and 45-fold, respectively) improvements in binding affinity. Whereas the N-methyl-4-pyrazolyl compound 40 displayed high affinity (K_i = 3.79 nM), the N-unsubstituted pyrazole 39 displayed significantly decreased affinity (K_i = 121 nM). This >30-fold reduction in binding affinity could be due to the hydrogen-bond donor feature present in the latter.

Binding Affinity of 5′-Heteroaryl Compounds at the DOR.

Among the 5′-heteroaryl compounds, the 1-pyrrolyl compound 30 displayed sub-nanomolar binding affinity at the DOR. The 2-pyridyl (31) and 4-pyridyl (33) isomers displayed higher affinity (DOR K_i < 10 nM) than the 3-pyridyl isomer (32, DOR K_i = 30.6 nM). Among the 5-membered heterocyclic compounds, the 1-methyl-4-pyrazolyl compound 40 and the 2-(5-methyl-1,3,4-oxadiazolyl) compound 42 displayed a DOR binding affinity of <10 nM. Similar to the profile at the MOR, the N-unsubstituted pyrazole 39 displayed significantly decreased affinity in comparison to the N-methyl congener 34 (K_i value of 257 nM for 39 vs 7.96 nM for 40).

Binding Affinity of 5′-Heteroaryl Compounds at the KOR.

Most of the compounds displayed moderate binding affinity with K_i values in the 10–30 nM range. The 2-methyl-4-pyridyl compound (34) and the 1-methyl-4-pyrazolyl compound (40) that displayed high affinity at the MOR also displayed high affinity at the KOR.

Functional Activity of 5′-Heteroaryl Compounds at the MOR.

More disparate results were seen in the agonist functional activity in this series of compounds. The 1-pyrrolyl compound 30 displayed high potency (EC₅₀ 1.61 nM) and efficacy (E_max 84.7%). However, some of the compounds, for example, compounds 32 and 36–38, displayed very weak potency as agonists. The low potency of these compounds prevented full evaluation of their efficacy with fully saturated concentration–response curves. For these compounds, the efficacy listed in parenthesis in Table 2 is the maximum stimulation produced at 10 μM concentration in the GTPγS assay, which was <50%. Among isomeric pyridyl compounds, only the 4-pyridyl isomer 33 displayed moderate potency and efficacy. Interestingly, introduction of a methyl group (compound 34) or a methoxy group (compound 35) at the 2-position of the 4-pyridyl ring gave ligands with high potency (EC₅₀ < 3 nM) and efficacy (E_max > 80%).

Functional Activity of 5′-Heteroaryl Compounds at the DOR.

All of the compounds displayed high antagonist potency (IC₅₀ < 1.0–2.0 nM) and efficacy (I_max > 85%) except for the 4-pyridyl compound 33 and the N-methylpyrazolyl compound 40 that displayed moderate potencies of 18.3 and 19.6 nM, respectively.

Functional Activity of 5′-Heteroaryl Compounds at the KOR.

Similar to the 5′-aryl compounds, all of the evaluated 5′-heteroaryl compounds displayed NC dose–response curves, indicating their nonagonist profile at the KOR. Similar to the 5′-aryl series, these compounds likely act as KOR antagonists.

Binding Affinity and Functional Activity of 14-Alkoxy Compounds.

Conversion of the pendant phenyl group in the 14-O-substituent of 6 to a 4-pyridyl group gave compound 43 that retained high affinity binding at the MOR, DOR, and KOR. However, the functional activity at the MOR was drastically altered to a nonagonist profile. Moreover, the antagonist efficacy at the DOR was also greatly diminished (I_max 16.7%). Exchanging an oxygen atom for the benzylic methylene group of the propyl chain of 6 led to compound 44 that displayed greatly diminished binding at the MOR and DOR. The drastic reduction of binding affinity at the MOR could be attributed to favorable hydrophobic interactions sustained by the methylene group of the C₆H₅CH₂ substituent and unfavorable repulsion encountered by the electron-rich oxygen atom of the C₆H₅O group. The fused benzene ring of the quinolinylmethyl substituent present in 45 could potentially mimic the phenyl group of the phenylpropyl substituent of 6. However, this compound while retaining moderate binding potency at the DOR and KOR, lost affinity at the MOR. Interestingly, in the functional assay, these three compounds 43–45 failed to function as agonists at the MOR. On the 5′-phenylpyridomorphinan template, the 14-O-3-(3-pyridylpropyl) compound 46 and the 14-O-3-(4-pyridylpropyl) compound 47 displayed moderate to high binding affinity at all three receptors. These two compounds also lost agonist potency and efficacy at the MOR, while retaining potent and efficacious antagonist activity at the DOR. The introduction of a fluorine atom at the para-position on the phenyl ring of 8 gave compound 48 that displayed diminished MOR binding and agonist potency. Saturating the pendant phenyl to a cyclohexyl group (compound 49) also led to a reduction in binding affinity and agonist activity at the MOR. Interestingly, replacement of the cyclohexyl group with a 4-pyranyl group gave a compound (50) that displayed single-digit nanomolar binding and agonist potency but moderate efficacy at the MOR. Despite its weak binding affinity at the DOR, in the functional assay, the compound displayed potent DOR antagonist activity. Because, in principle, antagonist activity should rely on intrinsic efficacy, this disparity may reflect allosteric binding for this ligand, producing strong functional activity but weak orthosteric competition. Extension of the linker chain length from propyl to butyl (compound 51) also had a deleterious effect on MOR binding and MOR agonist activity. Removal of the pendant aryl group altogether (compound 52) also resulted in poor binding affinity at both the MOR and KOR. Thus, the nature of the alkoxy substituent has a significant influence on binding and functional activity, and subtle changes in the structure lead to drastic changes in binding and activation of the MOR.

ADME Properties.

In vitro ADME screens were used to profile lead compounds for drug-like properties and to help prioritize potent compounds for in vivo proof-of-concept studies. Our strategy focused on profiling compounds for aqueous solubility and lipophilicity (log D) as these properties are important predictors of drug bioavailability.^34,35 As our ideal lead candidate profile includes central nervous system (CNS) drug exposure, we sought leads with physicochemical properties, indicative of good brain permeability/CNS exposure.^36,37 Additionally, we screened compounds for metabolic stability using mouse and human liver microsomes to help predict drug exposure to rank the lead compounds. For the majority of compounds screened, aqueous solubility was moderate to high (Table S1). In an effort to identify leads with good permeability, we prioritized compounds with log D in the range of 0–3.^38–40 With only a few exceptions, all compounds screened fell within this range (Tables 1–3).

Most of the compounds screened were rapidly metabolized in mouse liver microsome (MLM) preparations, with t_1/2 values falling less than 30 min, with the exception of compounds 44 (t_1/2 = 160 min) possessing an oxygen in place of the benzylic methylene group and 48 (t_1/2 = 81 min) possessing a fluorine at the para-position of the phenyl ring of the phenylpropyl group. Oxidative N-dealkylation of the cyclopropylmethyl (CPM) group at N17 and the metabolic susceptibility of benzylic methylene and the aryl ring of the 14-O-substituent could potentially be contributing to the observed poor MLM stability of most of the compounds. Rapid metabolism in MLM suggests poor exposure in vivo and influenced our decision to dose intravenous (iv) or subcutaneous (sc) for in vivo studies. Overall, compounds were significantly more stable in human liver microsome (HLM) preparations (Table S1). Species differences in microsomal stability are not uncommon; additional studies are needed to determine the mechanism of this apparent species difference.

Molecular Modeling.

In an effort to gain insights into the SAR, we performed molecular docking studies using the X-ray crystal structures of the active state of the MOR (PDB ID 5C1M)⁴¹ and the inactive state of the DOR and KOR (PDB IDs 4N6H and 4DJH, respectively).^42,43 Because the binding affinities of most of the ligands spanned a relatively narrow range, the differences in the docking scores, in general, were subtle. Nevertheless, the type and complementarity of interactions between the ligand and the receptor residues seen in the docking poses gave useful insights. The general binding mode of the ligands is illustrated using the docking pose of compounds 8 and 46 at the MOR (Figure 2). The epoxymorphinan core binds to the bottom of the orthosteric pocket defined by residues W293^6.48, H297^6.52, D147^3.32, and M151^3.36. The CPM group forms hydrophobic contact with W293^6.48, while the basic nitrogen N17, which is protonated at physiological pH, forms a salt bridge interaction with D147^3.32. The phenolic hydroxyl forms a hydrogen bond with H297^6.52 either directly or through a network of two water molecules. These interactions are shown by all ligands and are conserved in all three receptor subtypes.

Figure 2.

Docking poses at the MOR (PDB ID 5C1M). (A) Compound 8 and (B) compound 46. Compounds are colored green. Hydrogen bond, salt bridge, hydrophobic contact, and π–π stacking are indicated by black, cyan, yellow, and dark green dashed lines, respectively. All residue numbers are based on the MOR crystal structure 5C1M. Extracellular side is facing top.

The aryl/heteroaryl substituent at the 5′-position on the pyridine ring, on the other hand, plays a role in influencing binding selectivity at the MOR, DOR, and KOR. This is due to the extracellular region that these substituents occupy, which varies in volume and the nature of the residues among the three receptor subtypes. As shown in the docking pose of compound 8 at the MOR (Figure 2A), the phenyl group at the 5′-position forms π–π stacking interaction with W318^7.34 and a potential π–cation interaction with K233^5.40 and with the subtype variant K303^6.58 residue.^41,44,45 Besides selectivity, such interactions may also influence conformational changes that play a role in the functional state of the receptor. At the DOR, the volume of this extracellular region is even larger than in the MOR and it is capable of accommodating a phenyl ring in its favorable orientation. Indeed, in the docking pose of compound 8 at the DOR (Figure 3A), the phenyl group adopts an orientation that positions it near W284^6.58 and K214^5.40 which could form π–π stacking and π–cation interaction. In contrast, when docked to the KOR (Figure 3B) in which the volume of this region is smaller than that of the MOR, the phenyl group comes in close unfavorable contact with glutamate E297^6.58 (corresponding to K303^6.58 in the MOR), thus providing a possible explanation for the five- to sevenfold lower affinity at the KOR compared to its affinity at the MOR and DOR.

Figure 3.

Docking poses of compound 8 at the (A) DOR (PDB ID 4N6H) and (B) KOR (PDB ID 4DJH). Compounds are colored green. Hydrogen bond, salt bridge, hydrophobic contact, π–π stacking, and π–cation interaction are indicated by black, cyan, yellow, dark green, and purple dashed lines, respectively. Extracellular side is facing top. W274^6.48, H278^6.52, and D128^3.32 in the DOR and W287^6.48, H291^6.52, and D138^3.32 in the KOR correspond to conserved residues W293^6.48, H297^6.52, and D147^3.32 in the MOR.

The substituent extending from the oxygen at the 14-position binds to a hydrophobic region in all three receptor subtypes. A group such as the phenylpropoxy at the C-14 has the appropriate size and hydrophobicity to fit into this hydrophobic region and interact with residues such as I144^3.29 in the MOR. As discussed, compounds with some of the substituent variations at C-14 did not effectively stimulate the MOR. For example, in contrast to the MOR agonist profile of compound 8, its 3-pyridyl analogue 46 displayed neither efficacious agonism nor antagonism at the MOR. A plausible explanation is provided by the docking result with 46, which indicates that the pendant pyridyl ring of 46 binds in a different orientation than the phenyl ring of 8. Instead of the hydrophobic contact that the phenyl ring of 8 has with residues such as I144^3.29 (Figure 2A), the pyridyl ring adopts an orientation that forms a polar hydrogen bond interaction with N127^2.63 (Figure 2B). Thus, the new pattern of interaction displayed by 46 in this middle region of TM2/TM3 might affect the conformational change in a manner that is different from that induced by a typical agonist of the MOR. Moreover, the specific interaction brought about by the pendant group of the substituent on C-14 might prevent the ligand from going deeper into the pocket, thus reducing its ability to interact with residues at the bottom of the orthosteric site, which plays a key role in the conformational switch needed for the activation of the receptor.^41,46

Antinociceptive Studies in Mice.

The antinociceptive potency and efficacy of select compounds were evaluated in male CD1 mice using the 55 °C warm water tail-flick assay. Previous studies with compound 6 were performed via icv administration. In the current study, we selected 16 compounds that displayed promising in vitro binding and functional activity profiles and evaluated them following icv or iv administration. Tail withdrawal latencies were assessed at various time points for up to 5 h post dose and were compared to vehicle. Because of variability in aqueous solubility of the test compounds, three different vehicles were used: saline (icv and iv), 100% DMSO (icv), and 10:10:80 DMSO/Tween 80/saline (icv, iv). When 100% DMSO was used as a vehicle for icv injections, the tail-flick latency was significantly increased over saline or 10:10:80 for the first hour of evaluation (p = 0.001 = 0.05); data collected with 100% DMSO were excluded based upon solubility limitations and confounding effects on behavior. No statistically significant differences were seen among vehicles for iv administration (p = 0.97); therefore, data points were combined. The observed antinociceptive profile of the evaluated compounds is presented in Table 4. Compounds 9, 16, 21, 48, and 50 showed poor antinociceptive activity by the icv route of administration and were not advanced to systemic evaluation. All other compounds were evaluated after iv dosing.

Table 4.

Antinociceptive Activity of Selected Compounds in Warm Water Tail-Flick Assay^a

compd	route	dose	MPE ± SEM (n)	time of effect onset (min)	time of peak effect (min)	duration of effect (min)
9	icv	10 nmol	75 ± 22 (5)	10	30	10–45
14	iv	10 mg/kg	100 ± 0 (4)	15	45	15–180
15	iv	10 mg/kg	100 ± 0 (5)	15	15	15–240
16	icv	10 nmol	65 ± 18 (5)	10	10	10
19	iv	10 mg/kg	100 ± 0 (4)	15	15	15–240
20	iv	10 mg/kg	100 ± 0 (5)	15	30–180	15–300
21	icv	10 nmol	86 ± 14 (5)	10	10	15–300
25	iv	10 mg/kg	100 ± 0 (4)	15	15	15–300
30	iv	10 mg/kg	99 ± 0.5 (5)	15	45	15–180
34	iv	10 mg/kg	100 ± 0 (5)	15	30	15–120
39	iv	10 mg/kg	75 ± 14 (5)	15	15	15–60
41	iv	10 mg/kg	100 ± 0 (4)	15	15	15–120
42	iv	10 mg/kg	100 ± 0 (5)	15	30	15–180
48	icv	10 nmol	80 ± 13 (5)	15	15	15–45
49	iv	10 mg/kg	100 ± 0 (3)	30	60	30–180
50	icv	10 nmol	77 ± 22 (4)	10	45	10–60

^aTail-flick latencies were determined as described in the Experimental Section. The time of effect onset is listed when the antinociceptive effect was first observed to be >30%. Duration of the effect is given as the total time for which antinociception remained above 30% MPE.

Based on a maximum possible effect (MPE) duration of >2 h and lack of respiratory depression, aggression, and related side effects via qualitative observation, two compounds 20 and 42 were chosen for further evaluation. These two compounds produced dose-dependent antinociception following iv administration of 1, 3, and 10 mg/kg doses. Maximal effects were observed following all three doses of 20 starting 15 min after injections that persisted through 90 min. The highest dose tested, 10 mg/kg, retained >50% antinociceptive effects that were significantly higher than vehicle-treated values for 4 h after administration (Figure 4A). Administration of 42 dose-dependently mitigated heat evoked nociception beginning 15 min after injection. In contrast to 20, which displayed a threshold antinociceptive effect (100 ± 0%) for 15–180 min, the maximal peak effect (80 ± 20%) of 42 occurred at 45 min (Figure 4B). Relative to morphine (10 mg/kg, iv),⁴⁷ compound 20 produced potent, maximal antinociception despite modest MOR/DOR binding affinity (91 and 48 nM, respectively, Table 1). One potential explanation is that although 20 had modest binding affinity, it showed a strong in vitro functional potency and efficacy (MOR agonist EC₅₀ = 13 nM and DOR antagonist IC₅₀ = 2.4 nM, Table 1). This difference suggests that 20 has strong intrinsic efficacy, whereby modest binding could still produce potent, efficient receptor activation.⁴⁸ In addition, blood–brain barrier permeability, P-gp efflux, and favorable PK properties could underlie the potency and efficacy of 20 in producing antinociception and for its ability to produce antinociception by systemic administration compared with compound 6.

Figure 4.

Antinociceptive dose– and time–response curves for 20 (panel A) and 42 (panel B) in the 55 °C warm water tail-flick assay following iv administration to naïve male CD-1 mice. Compounds were evaluated in a minimum of two groups with the tester blinded to treatment (sample sizes per group noted within the graph). Two-way repeated measure (RM) ANOVA Bonferroni post hoc was used for statistical analysis.

Because compound 20 showed a longer duration and maximal antinociception compared to 42 and morphine (10 mg/kg) following iv administration, additional in vivo characterization was pursued with compound 20 (designated SRI-39067). We evaluated compound 20 by sc administration of 3.2, 10, 18, and 32 mg/kg doses as compared to morphine. Importantly, compound 20 produced significant dose-dependent antinociception following sc administration (F(36,190) = 4.436, p < 0.0001) with a longer duration than morphine^49,50 (Figure 5). The calculated A₅₀ value at the 60 min time point after administration was 20.8 mg/kg, and the A₉₀ was 21.1 mg/kg (Figure 5B). Compound 20 was also evaluated after oral administration at doses of 1, 10, and 32 mg/kg. The 10 mg/kg dose produced a ≤16% effect over 5 h, while the 32 mg/kg resulted in a 100% effect in 1/6 animals and <30% in 5/6 animals; there was no significant difference between the doses (two-way ANOVA interaction p = 0.18). The time of peak effect was 90 min (Figure 5B).

Figure 5.

Antinociceptive dose– and time–response curve for morphine sulfate [(A) sc] and compound 20 administered subcutaneously [sc: panel (B)] and oral gavage [po: panel (C)] in the 55 °C warm water tail-flick assay using naïve male CD1 mice. Compound 20 was evaluated in a minimum of two groups for a total of n = 5–7 per condition. Two-way RM ANVOA Bonferroni post hoc was used for statistical analysis.

We next evaluated the sensitivity of antinociceptive effects of 20 to naloxone. Although the compound displayed potent antinociceptive effects in naïve mice (Figure 5), in mice pretreated with naloxone (10 mg/kg, ip; t = −10 min), the antinociceptive effects of compound 20 (32 mg/kg, sc) were completely blocked (Figure 6). The vehicle controls in the presence or absence of naloxone did not display any significant differences in tail-flick latency across the duration of evaluation. These data suggest that compound 20 elicits antinociceptive effects via its agonist activity at opioid receptors in vivo.

Figure 6.

Blockade of antinociceptive efficacy of compound 20 by pretreatment with naloxone. Pharmacological blockade of opioid receptors with the nonselective antagonist naloxone (10 mg/kg ip) prevented the antinociception observed after sc administration of 20 alone (32 mg/kg, sc) over a duration of 4 h, as in Figure 5A (n = 5–6 male CD1 mice per treatment). Data represented as the mean ± SEM of areas under the curve for each condition. One-way ANOVA Bonferroni post hoc was used for statistical analysis. n.s. = not statistically significant; ****p < 0.0001.

Antinociceptive Tolerance.

Antinociceptive dose response curves for compound 20 and morphine sulfate were generated using male CD1 mice in the warm water tail-flick assay on day 1 of dosing. On three subsequent days, animals were injected (sc) with the A₉₀ dose of either compound 20 (21 mg/kg, determined in Figure 5A) or morphine sulfate⁵¹ (10 mg/kg) twice a day (9 a.m. and 5 p.m.). The antinociceptive dose response curves were again generated on day 5 (Figure 7). Whereas the repeated administration of morphine produced a 3.3-fold right-ward shift of ED₅₀ from 6.4 to 21.7 mg/kg, compound 20 displayed only 1.2-fold shift in ED₅₀ from 20.8 to 25.4 mg/kg. This suggests that compound 20 produced significantly less tolerance development than morphine (Figure 7).

Figure 7.

Repeated administration of compound 20 produces less antinociceptive tolerance. Dose response curves were generated in naïve male CD1 mice on day 1. Subsequently, mice were dosed twice a day with 21 mg/kg 20, the calculated A₉₀, or 10 mg/kg morphine sulfate for 4 days. On day 5, the dose response curve was regenerated from data collected 45 min after drug administration. Nonlinear regression. ED₅₀ values were compared between days 1 and 5 to determine the development of antinociceptive tolerance (95% confidence interval). N = 5–8/group.

Physical Dependence and Precipitated Withdrawal.

Physical dependence was determined using a precipitated withdrawal approach.⁵² Mice were dosed for 4 days with vehicle, morphine sulfate(10 mg/kg, ip), or compound 20 (21 mg/kg = A₉₀, sc). Prior to naloxone administration, mice were evaluated for evidence of spontaneous withdrawal over 30 min as a baseline (BL); no symptoms of spontaneous withdrawal were observed. Naloxone (10 mg/kg ip) was then administered. Body weight, fecal output, urination, and stereotypical withdrawal behaviors (i.e., jumping, paw tremors, wet-dog shakes, backward steps, etc.) were recorded. Changes in the number of steps and wet-dog shakes were not observed in this cohort and thus not included in overall calculations. Within all treatment groups, the number of animals displaying diarrhea and jumping behavior was increased. Both morphine and compound 20 significantly increased the urinary output over vehicle, whereas only compound 20 significantly increased the number of fecal pellets excreted (Figure 8A,B). Although urination and diarrhea are important features of precipitated opioid withdrawal, the literature suggests that jumping behaviors elicited by opioid antagonists are the most robust somatic predictor of physical dependence.^53,54 Mice dosed with morphine jumped significantly more than vehicle or compound 20-exposed mice; compound 20 did not elicit a significant increase when compared with vehicle treatment, suggesting that at least some markers of dependence and withdrawal are reduced for 20 (Figure 8C). Calculation of the overall withdrawal score was performed and then normalized to vehicle conditions. Under these conditions, both morphine and compound 20 induced significant physical dependence and withdrawal compared to vehicle (Figure 8D; p = 0.002 and p = 0.05, respectively, one-way ANOVA F = 7.786, Tukey post hoc, outliers identified n = 2 compound 20, n = 2 vehicle, n = 0 morphine).

Figure 8.

Physical dependence of mice injected repeatedly with A₉₀ doses of 20 (red) or morphine sulfate (10 mg/kg ip; yellow). Mice were dosed with compound 20, as in Figure 7. On day 5, 4 h after the final dose of the drug or vehicle (white), mice were evaluated for behaviors indicative of withdrawal (spontaneous) for 30 min; no behaviors indicating spontaneous withdrawal were observed. Mice were then dosed with the opioid antagonist naloxone (10 mg/kg, ip) to precipitate withdrawal. Behaviors were reassessed for 30 min post dosing. Behaviors included are the amount of urine expelled onto a filter paper (A), number of fecal pellets (B), and number of jumps (C); the overall withdrawal scores were calculated then normalized to vehicle controls (D). One-way ANOVA/behavior Tukey post hoc was used for statistical analysis. n.s. = not statistically significant; *p < 0.05, **p < 0.01, and ***p < 0.001; N = 6–10 included/group.

Respiratory Depression.

Respiratory depression was assessed using whole body plethysmography.⁵⁵ Morphine significantly reduced the respiratory rate at 40 min and 45 min of observation (p = 0.01 and 0.02, respectively), whereas the tidal volume was reduced at 42 and 45 min of data collection (p = 0.04 and 0.008, respectively). Morphine-exposed mice did present with a significant reduction in minute ventilation as compared to saline control mice during a full duration of 5% CO₂ challenge from 41 to 45 min (p = 0.0004 to 0.004). In contrast, mice receiving compound 20 did not show a reduction in any respiratory measures across the duration of the experiment. However, the tidal volume appeared to increase with escalating doses (up to 32 mg/kg) during the 5% CO₂ challenge (Figures 9 and S1 in the Supporting Information). These data suggest that compound 20 at >A₉₀ dose does not induce respiratory depression acutely.

Figure 9.

Respiratory measures of naïve mice and mice injected acutely with morphine sulfate (top, yellow) or 20 (bottom, red). An acute bolus of morphine sulfate (10 mg/kg) reduced the respiratory rate, followed by tidal volume and minute ventilation, indicating the induction of respiratory depression as measured by whole body plethysmograph (n = 6–8) in the first 45 min after drug administration. Mice receiving compound 20 (32 mg/kg) did not deviate from the BL respiratory rate, tidal volume, or minute ventilation nor were those values different from vehicle-treated mice, suggesting a lack of respiratory depression over the 45 min observation period. Two-way RM ANOVA Bonferroni post hoc was used for statistical analysis. *p < 0.05; ***p < 0.001.

Conditioned Place Preference/Aversion.

Conditioned place preference (CPP) is defined as a >50 s increase over BL values. Conditioned place aversion (CPA) is defined as a decrease from the BL greater than 50 s. Neutral responses occurred when test values were within 50 s of the BL. The number of mice exhibiting CPP or CPA for saline, vehicle, morphine in saline, morphine in vehicle, and compound 20 is shown in Figure 10A–E. Comparison of difference scores between treatments for animals showing CPA, CPP, or no preferences showed no difference between treatment groups (Figure 10F). Because difference scores capture before and after differences that may be masked by time in neutral chambers when using a three-chamber system, the total amount of time per chamber was also determined (Figure 10G). These data suggest that animals treated with morphine in vehicle and compound 20 spend equal amounts of time in each of the three chambers after conditioning, whereas morphine- and vehicle-paired mice spend significantly more time in the drug-paired and counter-paired chamber, respectively (n = 10–15/group, **p < 0.01). Together, these results indicate that compound 20 induced CPP in fewer animals than morphine and that the preference occurs to a lesser degree.

Figure 10.

Reward liability as measured by CPP/CPA of naïve male CD1 and male CD1 mice injected repeatedly with compound 20 or morphine sulfate. The same approximate numbers of mice displayed CPP, CPA, and neutrality after saline (A) or the vehicle of 10% DMSO, 10% Tween 80, and 80% saline (B). Morphine prepared in saline (C) or vehicle (D) induced significant place preference (F), regardless of preparation; ^p = 0.05, *p = 0.01 (one-way ANOVA Bonferroni post hoc). Five days of administration of compound 20 led to even numbers of CPA and CPP (E), but the degree of aversion or preference was not different from saline or vehicle. These findings were not confounded by the use of a three-chamber system (G). (One-way ANOVA Bonferroni post hoc.) N = 10–15/condition.

CONCLUSIONS

In this present effort, the influence of various aryl and heteroaryl substituents at the 5′-position of the 14-phenylpropoxy-pyridomorphinan scaffold on opioid receptor binding, functional activity, and ADME properties was explored. Subtle changes in the 5′-phenyl group had significant influence on binding affinity, agonist potency, and efficacy at the MOR. The binding affinity at the MOR was not always reflective of the agonist potency and efficacy at the MOR. For example, compounds 13, 15, and 18 displayed poor binding affinity but displayed good agonist potency and efficacy at the MOR, suggesting that these compounds have high intrinsic efficacy. At the DOR, most of the 5′-aryl compounds retained antagonist potency and efficacy. Potent DOR antagonist activity was also displayed by most of the compounds bearing a 5′-heteroaryl group. However, compounds such as 32 and 36–38 possessing a six-membered heteroaryl ring lost their ability to function as agonists at the MOR. Similarly, replacement of the pendant phenyl ring of the 14-O-phenylpropyl group by pyridine rings (43, 46, and 47) also led to loss of agonist activity at the MOR. Thus, in this pyridomorphinan series, MOR activation was more sensitive to the nature of the substituents than DOR inhibition. None of the compounds evaluated produced any significant agonist activity at the KOR.

Using in vivo antinociception and side effect profiling, we narrowed the selection of MOR agonist/DOR antagonist compounds to one 5′-aryl compound 20 and one 5′-heteroaryl compound 42. Of these two compounds, the 5′-(3-methoxyphenyl) compound 20 displayed potent antinociceptive activity by the systemic, sc route of administration. Of significant interest is our finding that unlike morphine, compound 20 displayed diminished propensity to produce analgesic tolerance on repeated administration and produced no apparent respiratory depression and rewarding effect. These results encourage further pursuit of ligands possessing mixed MOR agonist/DOR antagonist activity in the efforts toward identification of novel opioid analgesics with diminished side effects.

EXPERIMENTAL SECTION

Chemical Synthesis.

General Methods.

All solvents and reagents were used as purchased without further purification. Unless otherwise stated, reactions were carried out under a nitrogen atmosphere. Reaction conditions and yields were not optimized. The progress of all reactions was monitored by thin-layer chromatography (TLC) on precoated silica gel (60F254) aluminum plates (0.25 mm) from E. Merck and visualized using UV light (254 nm). Microwave reactions were performed using CEM Discover LabMate system with Intelligent Technology for Focused Microwave Synthesizer (Explorer 48) or Biotage Initiator Robot 8 microwave synthesizer. Purification of compounds was performed on an Isco Teledyne Combiflash Rf200 with four channels to carry out sequential purification. Universal RediSep solid sample loading prepacked cartridges (5.0 g silica) were used to absorb the crude product and purified on 12 g silica RediSep Rf Gold silica (20–40 μm spherical silica) columns using appropriate solvent gradients. Melting points were determined in open capillary tubes with a Thomas-Hoover melting point apparatus or SRS OptiMelt automated melting point system and are uncorrected. Highresolution mass spectrometry (HRMS) analysis was performed with an Agilent 1100 LC–MS TOF instrument using electrospray ionization (ESI). ¹H NMR spectra were recorded at 400 MHz on an Agilent/Varian MR-400 spectrometer, and ¹³C NMR spectra were recorded on an Agilent/Varian MR-400 spectrometer or on a Bruker AVANCE III-HD 600 or 850 MHZ spectrometer. The chemical shifts (δ) are reported in parts per million (ppm) and referenced according to the deuterated solvent for ¹H spectra (CDCl₃, 7.26, DMSO-d₆, 2.50, or TMS 0.0) and ¹³C spectra (CDCl₃, 77.2 or DMSO-d₆, 39.5). Purity of final compounds was checked by analytical HPLC using an Agilent 1100 LC system equipped with a Phenomenex Kinetex C18 column (5 μm, 4.6 × 150 mm) and a diode array detector (DAD) using the solvent system as follows: solvent A: H₂O/0.1% trifluoroacetic acid and solvent B: CH₃CN/0.1% trifluoroacetic acid, 0–95% B over 22 min, flow rate 1 mL/min, λ 254 nm and λ 280 nm (system 1) or using a Waters HPLC system equipped with a Sunfire C18 column (5 μm, 4.6 × 150 mm) and a Waters 2998 photodiode array detector using the solvent system: solvent A: H₂O/0.1% formic acid and solvent B: CH₃CN/0.1% formic acid, 10–90% B over 20 min, flow rate 2 mL/min, λ 254 nm (system 2) or using an Agilent 1200 LC system equipped with a Phenomenex Kinetex Phenyl–Hexyl column (2.6 μm, 4.6 × 50 mm) and a DAD using the solvent system: solvent A: H₂O/0.1% formic acid and solvent B: CH₃CN/0.1% formic acid, 0–95% B over 4.5 min, flow rate 2 mL/min, λ 254 nm (system 3). On the basis of NMR, HPLC–DAD, and HRMS (mass error less than 5 ppm), all final compounds were ≥95% pure.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-phenyl-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (8).

Step 1.

To a stirred solution of (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-11-phenyl-5,6,7,8,9,13b-hexahydro-8aH-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline-1,8a-diol 54²⁹ (0.5 g, 1.0 mmol) in dimethylformamide (DMF) (10 mL) was added sodium hydride (0.2 g, 6.0 mmol, 60% dispersion in mineral oil) at 0 °C. After the mixture was stirred for 40 min, 3-phenylpropyl bromide (0.4 g, 2.2 mmol) was added dropwise. The reaction mixture was allowed to come to room temperature and stirred for 4 h. Excess of sodium hydride was decomposed with drops of ice-cold water, and the mixture was then diluted with water and extracted with CHCl₃ (3 × 20 mL). Organic layers were dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The solvent was removed under reduced pressure. The residue was purified by chromatography over a column of silica gel using hexanes/EtOAc (40:60) as the eluent to obtain (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-11-phenyl-1,8a-bis(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (0.24 g, 34%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.79 (d, J = 2.2 Hz, 1H), 7.72 (s, 1H), 7.64 (dq, J = 6.4, 1.3 Hz, 2H), 7.52–7.41 (m, 2H), 7.44–7.34 (m, 1H), 7.30–6.98 (m, 8H), 6.91 (dt, J = 8.2, 1.4 Hz, 2H), 6.66 (dd, J = 8.2, 1.2 Hz, 1H), 6.60 (d, J = 8.3 Hz, 1H), 5.73 (d, J = 1.1 Hz, 4H), 5.42 (d, J = 1.1 Hz, 1H), 4.43 (td, J = 5.2, 1.4 Hz, 1H), 4.00–3.88 (m, 1H), 3.65 (t, J = 7.2 Hz, 2H), 3.38 (tdd, J = 6.5, 5.1, 1.2 Hz, 1H), 3.30–3.21 (m, 1H), 3.14 (d, J = 18.6 Hz, 1H), 2.99 (d, J = 16.9 Hz, 1H), 2.70–2.44 (m, 5H), 2.35 (td, J = 16.9, 16.0, 8.1 Hz, 3H), 2.15 (t, J = 11.2 Hz, 1H), 1.92–1.78 (m, 2H), 1.75–1.54 (m, 3H), 1.48 (d, J = 11.5 Hz, 1H), 0.73 (s, 1H), 0.42 (d, J = 7.7 Hz, 2H), 0.11 (d, J = 10.3 Hz, 1H), 0.06 (s, 1H); ESI MS m/z: 689.4 [M + H]⁺.

Step 2.

A solution of the abovementioned intermediate (0.22 g, 0.3 mmol) in anhydrous CH₂Cl₂ (10 mL) was cooled to −78 °C. Boron tribromide (1.7 mL, 1.7 mmol, 1 M in CH₂Cl₂) was added dropwise, and the mixture was stirred for 1 h at 0 °C. The mixture was then allowed to come to room temperature, and the reaction was quenched by the addition of drops of ice-cold water. The mixture was diluted with water and extracted with CHCl₃ (3 × 20 mL). Organic layers were dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with CHCl₃/MeOH (95:5) to obtain 0.06 g (32%) of the desired product 8 as a white solid; mp 110–112 °C; TLC (7.5% MeOH/CH₂Cl₂): R_f = 0.40; ¹H NMR (400 MHz, CDCl₃): δ 8.75 (dd, J = 2.3, 0.8 Hz, 1H), 7.52–7.36 (m, 7H), 7.17–7.10 (m, 2H), 7.10–7.04 (m, 1H), 7.00–6.95 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 3.71 (dt, J = 8.1, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.18 (m, 2H), 2.84 (d, J = 16.6 Hz, 1H), 2.79–2.67 (m, 2H), 2.58–2.41 (m, 5H), 2.38–2.26 (m, 2H), 1.80–1.64 (m, 3H), 0.88–0.71 (m, 1H), 0.61–0.33 (m, 2H), 0.20–0.03 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 152.4, 146.8, 143.5, 142.3, 139.0, 137.5, 136.3, 135.8, 131.1, 131.0, 129.2, 128.5, 128.3, 128.2, 127.3, 126.1, 125.6, 119.1, 116.9, 91.2, 59.9, 59.4, 55.6, 47.9, 44.7, 32.5, 31.6, 31.5, 30.6, 23.6, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₈H₃₉N₂O₃ [M + H]⁺: 571.29552; found, 571.29556; HPLC (system 2) t_R = 6.29 min, purity = 100%.

(4bS,8R,8aS,13bR)-11-(4-Bromophenyl)-7-(cyclopropylmethyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (9).

Step 1.

Following the procedure described in step 1 for the preparation of 8, the 4-bromophenyl compound 55³¹ (0.6 g, 1.1 mmol) was reacted with 3-phenylpropyl bromide (0.7 g, 3.4 mmol) in the presence of sodium hydride (0.3 g, 6.8 mmol, 60% dispersion in mineral oil) to obtain (4bS,8R,8aS,13bR)-11-(4-bromophenyl)-7-(cyclopropylmethyl)-1,8a-bis(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (0.3 g, 35%). ¹H NMR (400 MHz, CDCl₃): δ 8.75 (d, J = 2.1 Hz, 1H), 7.60–7.55 (m, 2H), 7.40–7.34 (m, 3H), 7.24–7.18 (m, 2H), 7.18–7.06 (m, 6H), 6.98 (d, J = 7.4 Hz, 2H), 6.64 (dd, J = 8.3, 2.4 Hz, 1H), 6.59–6.53 (m, 1H), 5.58 (d, J = 2.9 Hz, 1H), 4.15–4.06 (m, 1H), 4.03–3.95 (m, 1H), 3.72 (d, J = 7.1 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.28–3.19 (m, 2H), 2.84 (d, J = 16.5 Hz, 1H), 2.73 (q, J = 9.4, 7.2 Hz, 4H), 2.57 (d, J = 16.5 Hz, 1H), 2.52–2.42 (m, 4H), 2.38–2.25 (m, 2H), 2.02–1.93 (m, 2H), 1.72 (s, 3H), 0.81 (s, 1H), 0.55–0.45 (m, 2H), 0.13 (s, 2H); ESI MS m/z: 767.3 [M + H]⁺.

Step 2.

The abovementioned intermediate (0.3 g, 0.4 mmol) was reacted with boron tribromide (2.4 mL, 2.4 mmol, 1 M in CH₂Cl₂), as described in step 2 for the preparation of 8, to give the desired product 9 (0.08 g, 30%) as a white solid; mp 120–121 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 8.73 (d, J = 2.1 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.40–7.34 (m, 3H), 7.16–7.06 (m, 3H), 6.99–6.95 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 3.76–3.68 (m, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.28–3.18 (m, 2H), 2.88–2.80 (m, 1H), 2.80–2.66 (m, 2H), 2.59–2.51 (m, 1H), 2.51–2.40 (m, 4H), 2.37–2.25 (m, 2H), 1.77–1.67 (m, 4H), 0.84–0.76 (m, 1H), 0.53–0.45 (m, 2H), 0.14–0.11 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 152.9, 146.4, 143.5, 142.3, 139.0, 136.4, 135.6, 135.1, 132.3, 131.1, 131.1, 128.8, 128.5, 128.2, 126.0, 125.7, 122.7, 119.1, 116.9, 91.0, 59.9, 59.4, 55.5, 47.9, 44.6, 32.5, 31.6, 31.4, 30.6, 29.8, 23.5, 9.4, 4.2; HRMS (ESI) m/z calcd for C₃₈H₃₈BrN₂O₃ [M + H]⁺: 649.20603; found, 649.20460; HPLC (system 1) t_R = 15.86 min, purity = 98.6%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(4-fluorophenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (10).

Step 1.

The 4-fluorophenyl compound 56³¹ (1.0 g, 2.1 mmol) was reacted with 3-phenylpropyl bromide (1.3 g, 1.0 mmol) in the presence of sodium hydride (0.5 g, 12.8 mmol, 60% dispersion in mineral oil), as described in step 1 for the preparation of 8, to obtain (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-11-(4-fluorophenyl)-1,8a-bis(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (0.84 g, 56%). ¹H NMR (400 MHz, CDCl₃): δ 8.74 (d, J = 2.2 Hz, 1H), 7.49–7.42 (m, 2H), 7.36 (d, J = 2.2 Hz, 1H), 7.24–7.17 (m, 2H), 7.16–7.07 (m, 8H), 7.01–6.94 (m, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.58 (s, 1H), 4.11 (dt, J = 9.6, 6.3 Hz, 1H), 3.99 (dt, J = 9.6, 6.3 Hz, 1H), 3.71 (dt, J = 8.1, 5.9 Hz, 1H), 3.65 (d, J = 5.9 Hz, 1H), 3.29–3.18 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.75–2.67 (m, 4H), 2.56 (d, J = 16.4 Hz, 1H), 2.51–2.41 (m, 4H), 2.37–2.24 (m, 2H), 2.01–1.92 (m, 2H), 1.78–1.64 (m, 3H), 0.80 (q, J = 6.6 Hz, 1H), 0.49 (dtd, J = 7.8, 4.9, 3.5 Hz, 2H), 0.18–0.07 (m, 2H); ESI MS m/z: 707.3 [M + H]⁺.

Step 2.

The abovementioned intermediate (0.4 g, 0.6 mmol) was reacted with boron tribromide (3.4 mL, 3.4 mmol, 1 M in CH₂Cl₂), as described in step 2 for the preparation of 8, to give 0.03 g (9%) of the title compound 10 as a yellow solid; mp 160–161 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.69 (d, J = 2.2 Hz, 1H), 7.48–7.41 (m, 2H), 7.36 (d, J = 2.2 Hz, 1H), 7.18–7.05 (m, 5H), 6.97 (d, J = 6.6 Hz, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.61 (s, 1H), 3.72 (dt, J = 8.3, 6.0 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.18 (m, 2H), 2.83 (d, J = 16.6 Hz, 1H), 2.78–2.65 (m, 2H), 2.58–2.40 (m, 5H), 2.37–2.25 (m, 2H), 1.77–1.65 (m, 4H), 0.81 (dd, J = 12.5, 6.7 Hz, 1H), 0.49 (m, 2H), 0.20–0.06 (m, J = 5.4, 4.9 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 163.9 (d, J_C–F = 248 Hz, 1C), 152.4, 146.5, 143.3, 142.2, 138.7, 135.6, 135.3, 133.5, 130.9 (d, J_{C–C–C–F} = 9 Hz, 1C), 128.9, 128.8, 128.4, 128.1, 126.0, 125.5, 119.0, 116.6, 116.1 (d, J_C–C–F = 22 Hz, 1C), 91.1, 59.7, 59.3, 55.4, 47.8, 44.5, 32.4, 31.5, 31.3, 30.4, 29.7, 23.4, 9.3, 4.1, 3.5; HRMS (ESI) m/z calcd for C₃₈H₃₈FN₂O₃ [M + H]⁺: 589.28610; found, 589.28665; HPLC (system 2) t_R = 5.58 min, purity = 97.7%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2,4-difluorophenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (11).

Step 1.

(4bS,8R,8aS,13bR)-11-Bromo-7-(cyclopropylmethyl)-5,6,7,8,9,13b-hexahydro-8aH-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline-1,8a-diol (59)³⁰ (0.30 g, 0.7 mmol) in DMF (10 mL) was added to sodium hydride (0.16 g, 4 mmol, 60% dispersion in mineral oil) at 0–5 °C. After the mixture was stirred for 40 min, 3-phenylpropyl bromide (0.29 g, 1.5 mmol) was added dropwise. The reaction mixture was allowed to come to room temperature and stirred for 4 h. Excess of sodium hydride was decomposed with drops of ice-cold water, and the mixture was then diluted with water and extracted with CHCl₃ (3 × 20 mL). Organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The solvent was removed under reduced pressure. The residue was purified by chromatography over a column of silica gel using hexanes/EtOAc (40:60) as the eluent to obtain (4bS,8R,8aS,13bR)-11-bromo-7-(cyclopropylmethyl)-1,8a-bis(3-phenylpropoxy)-6,7,8,8a,9,13b-hexa-hydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (60) (0.14 g, 31%). ¹H NMR (400 MHz, CDCl₃): δ 7.35–7.08 (m, 11H), 7.14–7.02 (m, 2H), 6.75–6.58 (m, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.08 (d, J = 1.4 Hz, 1H), 3.97 (dddt, J = 60.4, 32.4, 9.2, 6.3 Hz, 4H), 3.05 (d, J = 18.5 Hz, 1H), 2.84–2.57 (m, 5H), 2.56–1.84 (m, 11H), 1.65 (dd, J = 9.8, 3.1 Hz, 1H), 1.57 (dddd, J = 22.5, 13.1, 6.4, 2.8 Hz, 1H), 0.84 (s, 1H), 0.61–0.44 (m, 2H), 0.22–0.05 (m, 2H); ESI MS m/z: 691.2 [M + H]⁺.

Step 2.

Under an argon atmosphere, the abovementioned intermediate 60 (0.25 g, 0.4 mmol), 2-(2,4-difluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.17 g, 0.7 mmol), potassium carbonate (0.15 mg, 1.1 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.04 mmol) were added to 5.5 mL of 10:1 DMF/H₂O. The resulting mixture was heated in a microwave at 100 °C for 1 h. The mixture was allowed to cool down to room temperature, and water was added. The aqueous layer was extracted with ethyl acetate (3 × 20 mL). Organic layers were dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with CHCl₃/MeOH (95:5) to obtain (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-11-(2,4-difluorophenyl)-1,8a-bis(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolone (61) (0.21 g, 84%). ¹H NMR (400 MHz, CDCl₃): δ 8.68 (q, J = 1.2, 0.8 Hz, 1H), 7.72–7.64 (m, 1H), 7.50–7.44 (m, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.33 (td, J = 8.6, 6.3 Hz, 1H), 7.24–7.19 (m, 2H), 7.18–7.10 (m, 4H), 7.03–6.97 (m, 2H), 6.97–6.88 (m, 2H), 6.65 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.58 (s, 1H), 4.11 (dt, J = 9.6, 6.3 Hz, 1H), 4.00 (dt, J = 9.6, 6.4 Hz, 1H), 3.73 (dt, J = 8.2, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.18 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.79–2.65 (m, 4H), 2.57 (d, J = 16.5 Hz, 1H), 2.52–2.41 (m, 4H), 2.32 (ddd, J = 13.6, 10.8, 7.5 Hz, 2H), 2.04–1.92 (m, 2H), 1.79–1.66 (m, 3H), 0.87–0.75 (m, 1H), 0.56–0.44 (m, 2H), 0.19–0.06 (m, 2H); ESI MS m/z: 725.3 [M + H]⁺.

Step 3.

The abovementioned intermediate 61 (0.21 g, 0.3 mmol) was reacted with boron tribromide (1.8 mL, 1.8 mmol, 1 M in CH₂Cl₂), as described in step 2 for the preparation of 8, to obtain 0.08 g (43%) of the title compound 11 as a pale red solid; mp 210–211 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.80; ¹H NMR (400 MHz, CDCl₃): δ 8.63 (s, 1H), 7.41 (s, 1H), 7.32 (td, J = 8.6, 6.3 Hz, 1H), 7.18–7.12 (m, 2H), 7.12–7.06 (m, 1H), 7.02–6.98 (m, 2H), 6.98–6.88 (m, 2H), 6.68 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 5.60 (s, 1H), 3.73 (dt, J = 8.1, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.30–3.18 (m, 2H), 2.83 (d, J = 16.6 Hz, 1H), 2.79–2.65 (m, 2H), 2.55 (d, J = 16.4 Hz, 1H), 2.51–2.40 (m, 5H), 2.38–2.26 (m, 2H), 1.79–1.64 (m, 3H), 0.85–0.76 (m, 1H), 0.50 (tt, J = 7.5, 4.1 Hz, 2H), 0.16–0.12 (m, 2H); ¹⁹F NMR (376 MHz, CDCl₃): δ −109.50 (p, J = 7.8 Hz), −113.00 (q, J = 8.9 Hz); HRMS (ESI) m/z calcd for C₃₈H₃₇F₂N₂O₃ [M + H]⁺: 607.27668; found, 607.27678; HPLC (system 2) t_R = 7.22 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(3,4-difluorophen-yl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (12).

Step 1.

The bromo compound 60 (0.22 g, 0.3 mmol) was reacted with 2-(3,4-difluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.15 g, 0.6 mmol) in the presence of potassium carbonate (0.13 g, 1.0 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.03 mmol), as described in step 2 for the preparation of 11, to give (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-11-(3,4-difluorophenyl)-1,8a-bis(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolone (62) (0.12 g, 54%). ¹H NMR (400 MHz, CDCl₃): δ 8.72 (d, J = 2.2 Hz, 1H), 7.67 (ddd, J = 12.0, 8.3, 1.4 Hz, 1H), 7.50–7.43 (m, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.32–7.27 (m, 1H), 7.25–7.06 (m, 8H), 7.01–6.96 (m, 2H), 6.64 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.58 (s, 1H), 4.11 (dt, J = 9.6, 6.4 Hz, 1H), 3.99 (dt, J = 9.6, 6.3 Hz, 1H), 3.76–3.68 (m, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.18 (m, 2H), 2.98–2.79 (m, 2H), 2.79–2.66 (m, 4H), 2.56 (d, J = 16.5 Hz, 1H), 2.51–2.41 (m, 3H), 2.38–2.24 (m, 2H), 2.03–1.92 (m, 2H), 1.72 (td, J = 14.0, 5.7 Hz, 3H), 0.81 (s, 1H), 0.50 (td, J = 8.0, 4.8 Hz, 2H), 0.17–0.08 (m, 2H); ESI MS m/z: 725.3 [M + H]⁺.

Step 2.

The abovementioned intermediate 62 (0.21 g, 0.3 mmol) was reacted with boron tribromide (1.0 mL, 1.0 mmol, 1 M in CH₂Cl₂), as described in step 2 for the preparation of 8, to give 0.07 g (64%) of the desired compound 12 as a pale red solid; mp 181–182 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.67 (d, J = 2.2 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.32–7.27 (m, 1H), 7.25–7.18 (m, 2H), 7.17–7.06 (m, 3H), 6.98 (d, J = 6.7 Hz, 2H), 6.68 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.60 (s, 1H), 3.77–3.68 (m, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.17 (m, 2H), 2.83 (d, J = 16.6 Hz, 1H), 2.79–2.65 (m, 2H), 2.54 (d, J = 16.6 Hz, 1H), 2.46 (dt, J = 15.6, 6.8 Hz, 4H), 2.39–2.24 (m, 3H), 1.79–1.63 (m, 3H), 0.80 (p, J = 7.2, 6.8 Hz, 1H), 0.56–0.44 (m, 2H), 0.13 (d, J = 2.8 Hz, 2H); ¹⁹F NMR (376 MHz, CDCl₃): δ −136.56 (ddd, J = 21.2, 11.0, 6.6 Hz), −138.36 (dt, J = 18.8, 9.3 Hz); HRMS (ESI) m/z calcd for C₃₈H₃₇F₂N₂O₃ [M + H]⁺: 607.27668; found, 607.27660; HPLC (system 2) t_R = 7.27 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(o-tolyl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (13).

Step 1.

The bromo compound 60 (0.22 g, 0.3 mmol; 0.25 g, 0.4 mmol) was reacted with 4,4,5,5-tetramethyl-2-(o-tolyl)-1,3,2-dioxaborolane (0.08 g, 0.4 mmol) in the presence of potassium carbonate (0.15 g, 1.1 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.04 mmol), as described in step 2 for the preparation of 11, to give (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-1,8a-bis(3-phenylpropoxy)-11-(o-tolyl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (63) (0.22 g, 88%). ¹H NMR (400 MHz, CDCl₃): δ 8.55 (dd, J = 2.2, 0.7 Hz, 1H), 7.26–7.19 (m, 6H), 7.13–7.08 (m, 2H), 7.05–7.00 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.60 (s, 1H), 4.14 (dt, J = 9.7, 6.3 Hz, 1H), 4.03 (dt, J = 9.7, 6.4 Hz, 1H), 3.75 (dt, J = 8.3, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.31–3.20 (m, 2H), 2.86–2.65 (m, 6H), 2.62–2.41 (m, 6H), 2.37–2.26 (m, 2H), 2.18 (s, 3H), 2.05–1.94 (m, 2H), 1.78–1.67 (m, 3H), 1.62 (d, J = 5.7 Hz, 3H), 0.86–0.76 (m, 1H), 0.50 (dtd, J = 7.8, 4.5, 3.2 Hz, 2H), 0.15–0.11 (m, 2H); ESI MS m/z: 703.4 [M + H]⁺.

Step 2.

The abovementioned intermediate 63 (0.22 g, 0.3 mmol) was reacted with boron tribromide (1.9 mL, 1.9 mmol, 1 M in CH₂Cl₂) to give 0.06 g (30%) of the desired compound 13 as a white solid; mp 110–111 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.52 (d, J = 2.0 Hz, 1H), 7.31–7.26 (m, 1H), 7.26–7.20 (m, 3H), 7.17 (dd, J = 8.1, 6.5 Hz, 2H), 7.14–7.08 (m, 2H), 7.04–7.00 (m, 2H), 6.69 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 3.75 (dt, J = 8.3, 5.8 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.32–3.19 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.73 (td, J = 13.1, 12.0, 6.0 Hz, 2H), 2.56 (d, J = 16.4 Hz, 1H), 2.52–2.40 (m, 4H), 2.32 (dt, J = 11.8, 6.5 Hz, 2H), 2.19 (s, 3H), 1.98 (s, 1H), 1.78–1.66 (m, 3H), 0.87–0.74 (m, 1H), 0.50 (dq, J = 7.9, 4.0 Hz, 2H), 0.13 (t, J = 3.0 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 152.0, 148.4, 143.6, 142.4, 138.9, 138.0, 137.8, 137.2, 135.8, 131.2, 130.7, 130.5, 130.0, 128.5, 128.3, 128.3, 126.2, 125.7, 119.1, 116.8, 91.4, 59.8, 59.7, 55.6, 48.0, 44.7, 32.9, 31.9, 31.5, 30.6, 29.8, 23.5, 20.5, 9.4, 4.3, 3.6; HRMS (ESI) m/z calcd for C₃₉H₄₁N₂O₃ [M + H]⁺: 585.31117; found, 585.31002; HPLC (system 1) t_R = 15.17 min, purity 99.0%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(m-tolyl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (14).

This compound was prepared by the two-step procedure described for the preparation of 11. In step 1, the bromo compound 60 was reacted with 4,4,5,5-tetramethyl-2-(m-tolyl)-1,3,2-dioxaborolane to obtain intermediate 64. Yield: 34%. ¹H NMR (400 MHz, CDCl₃): δ 8.81–8.76 (m, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.36–7.29 (m, 3H), 7.24–7.18 (m, 3H), 7.16–7.08 (m, 6H), 7.01–6.96 (m, 2H), 6.64 (dd, J = 8.2, 1.0 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 5.59 (d, J = 1.1 Hz, 1H), 4.16–4.06 (m, 1H), 4.00 (dt, J = 9.9, 6.4 Hz, 1H), 3.76–3.60 (m, 2H), 3.30–3.19 (m, 2H), 2.84 (d, J = 16.5 Hz, 1H), 2.78–2.67 (m, 4H), 2.57 (d, J = 16.5 Hz, 1H), 2.52–2.42 (m, 4H), 2.41 (s, 3H), 2.37–2.28 (m, 2H), 2.01–1.91 (m, 2H), 1.78–1.65 (m, 3H), 0.85–0.71 (m, 1H), 0.49 (ddd, J = 8.0, 4.7, 3.6 Hz, 2H), 0.17–0.06 (m, 2H); ESI MS m/z: 703.3 [M + H]⁺. In step 2, this intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain the title compound 14. Yield: 67%; white solid; mp 78–80 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 8.70 (d, J = 2.1 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H), 7.35–7.26 (m, 3H), 7.23–7.18 (m, 1H), 7.17–7.11 (m, 2H), 7.11–7.05 (m, 1H), 7.02–6.96 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.61 (s, 1H), 3.72 (dd, J = 10.2, 4.1 Hz, 1H), 3.64 (d, J = 5.8 Hz, 1H), 3.30–3.13 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.79–2.65 (m, 2H), 2.58–2.42 (m, 6H), 2.40 (s, 3H), 2.36–2.26 (m, 2H), 1.71 (dq, J = 20.9, 6.4, 4.7 Hz, 3H), 0.86–0.76 (m, 1H), 0.56–0.36 (m, 2H), 0.13 (s, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 152.3, 146.7, 143.5, 142.4, 139.0, 138.8, 137.5, 136.5, 135.9, 131.1, 130.9, 129.1, 128.5, 128.3, 128.2, 128.0, 126.0, 125.6, 124.4, 119.1, 116.9, 100.1, 91.1, 59.9, 59.4, 55.6, 47.9, 44.7, 32.5, 31.6, 31.5, 29.8, 23.5, 21.6, 9.4, 4.2; HRMS (ESI) m/z calcd for C₃₉H₄₁N₂O₃ [M + H]⁺: 585.31117; found, 585.31153; HPLC (system 1) t_R = 15.30 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(p-tolyl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (15).

This compound was prepared following the same procedure described for 11. The bromo compound 60 was reacted with 4,4,5,5-tetramethyl-2-(p-tolyl)-1,3,2-dioxaborolane to obtain intermediate 65. Yield: 76%. ¹H NMR (400 MHz, CDCl₃): δ 8.79 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 8.5, 2.0 Hz, 3H), 7.28–7.18 (m, 4H), 7.17–7.05 (m, 6H), 7.01–6.96 (m, 2H), 6.65 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H), 4.11 (dt, J = 9.7, 6.4 Hz, 1H), 4.00 (dt, J = 9.7, 6.4 Hz, 1H), 3.71 (dt, J = 8.4, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.30–3.16 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.79–2.67 (m, 4H), 2.57 (d, J = 16.4 Hz, 1H), 2.52–2.42 (m, 4H), 2.40 (s, 3H), 2.31 (ddd, J = 17.7, 13.0, 6.4 Hz, 2H), 2.03–1.92 (m, 2H), 1.77–1.68 (m, 3H), 0.86–0.75 (m, 1H), 0.50 (dtd, J = 7.8, 5.1, 3.5 Hz, 2H), 0.18–0.07 (m, 2H); ESI MS m/z: 703.4 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain the desired compound 15. Yield: 19%; white solid; mp 185–186 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.74 (d, J = 2.2 Hz, 1H), 7.44–7.38 (m, 3H), 7.24 (s, 1H), 7.14 (dd, J = 8.0, 6.2 Hz, 2H), 7.11–7.04 (m, 1H), 7.01–6.95 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 3.71 (q, J = 6.5 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.23 (dd, J = 16.1, 7.8 Hz, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.79–2.66 (m, 2H), 2.58–2.41 (m, 6H), 2.40 (s, 3H), 2.36–2.24 (m, 2H), 1.70 (s, 4H), 0.80 (d, J = 9.6 Hz, 1H), 0.49 (dd, J = 8.1, 3.5 Hz, 2H), 0.12 (d, J = 3.0 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 152.0, 146.5, 143.5, 142.3, 139.1, 138.2, 136.2, 135.6, 134.5, 131.1, 130.9, 129.9, 128.5, 128.2, 127.1, 125.6, 119.1, 117.0, 91.0, 59.8, 59.4, 55.6, 47.9, 44.7, 32.5, 31.6, 30.5, 29.8, 23.5, 21.3, 14.3, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₉H₄₁N₂O₃ [M + H]⁺: 585.31117; found, 585.31098; HPLC (system 1) t_R = 15.16 min, purity = 97.1%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(4-fluoro-2-methylphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (16).

This compound was prepared by the same procedure described for 11. The bromo compound 60 was reacted with 2-(4-fluoro-2-methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane to obtain 66. Yield: 78%. ¹H NMR (400 MHz, CDCl₃): δ 8.50 (dd, J = 2.2, 0.7 Hz, 1H), 7.71–7.64 (m, 1H), 7.58–7.52 (m, 1H), 7.50–7.43 (m, 1H), 7.25–7.19 (m, 2H), 7.18–7.08 (m, 6H), 7.07–6.99 (m, 3H), 6.98–6.88 (m, 2H), 6.66 (d, J = 8.2 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H), 4.13 (dt, J = 9.7, 6.3 Hz, 1H), 4.02 (dt, J = 9.7, 6.4 Hz, 1H), 3.75 (dt, J = 8.3, 5.8 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.31–3.19 (m, 2H), 2.81 (d, J = 16.4 Hz, 1H), 2.78–2.70 (m, 3H), 2.57 (d, J = 16.3 Hz, 1H), 2.46 (dt, J = 15.3, 6.9 Hz, 4H), 2.37–2.26 (m, 2H), 2.16 (s, 2H), 2.04–1.94 (m, 2H), 1.77–1.67 (m, 3H), 0.81 (q, J = 6.7 Hz, 1H), 0.50 (dtd, J = 7.8, 4.5, 3.2 Hz, 2H), 0.13 (dq, J = 5.0, 1.3 Hz, 2H); ESI MS m/z: 721.4 [M + H]⁺. Phenolic-O-dealkylation of this intermediate with boron tribromide in CH₂Cl₂ gave 16 as a white solid in 44% yield. mp 106–107 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 8.49 (dd, J = 2.2, 0.8 Hz, 1H), 7.22–7.15 (m, 3H), 7.14–7.05 (m, 2H), 7.04–6.99 (m, 2H), 6.99–6.89 (m, 2H), 6.68 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 5.27 (s, 1H), 3.76 (dt, J = 8.3, 5.8 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.31–3.18 (m, 2H), 2.86–2.66 (m, 3H), 2.55 (d, J = 16.5 Hz, 1H), 2.45 (tdd, J = 9.0, 6.1, 3.1 Hz, 4H), 2.37–2.26 (m, 2H), 2.17 (s, 3H), 1.79–1.69 (m, 2H), 1.25 (s, 1H), 0.90–0.74 (m, 1H), 0.50 (dtd, J = 7.6, 4.4, 3.2 Hz, 2H), 0.13 (td, J = 3.1, 2.4, 1.3 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 152.3, 148.5, 143.5, 142.4, 138.9, 138.1, 136.3, 133.8, 131.6, 131.5, 131.1, 130.6, 128.5, 128.3, 126.2, 125.7, 119.2, 117.3, 117.2, 116.8, 113.1, 113.0, 91.3, 59.8, 59.7, 55.6, 48.0, 44.7, 32.9, 31.9, 31.4, 29.9, 23.5, 20.7, 4.3, 3.6; HRMS (ESI) m/z calcd for C₃₉H₄₀FN₂O₃ [M + H]⁺: 603.30175; found, 603.30150; HPLC (system 2) t_R = 7.40 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2-cyclopropylphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (17).

This compound was prepared by the same procedure described for 11. The bromo compound 60 was reacted with 2-(2-cyclopropylphenyl)-boronic acid to obtain 67. Yield: 73%. ¹H NMR (400 MHz, CDCl₃): δ 8.67 (dd, J = 2.2, 0.7 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.29 (dd, J = 7.5, 1.5 Hz, 1H), 7.25–7.07 (m, 9H), 7.03–6.99 (m, 2H), 6.92 (dd, J = 7.8, 1.3 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 4.14 (dt, J = 9.7, 6.3 Hz, 1H), 4.02 (dt, J = 9.7, 6.4 Hz, 1H), 3.75 (dt, J = 8.3, 5.8 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.31–3.18 (m, 2H), 2.83 (d, J = 16.4 Hz, 1H), 2.73 (tt, J = 12.1, 7.2 Hz, 4H), 2.58 (d, J = 16.4 Hz, 1H), 2.52–2.39 (m, 4H), 2.37–2.25 (m, 2H), 2.05–1.94 (m, 2H), 1.72 (qd, J = 6.5, 6.0, 3.8 Hz, 4H), 1.26 (s, 1H), 0.87–0.77 (m, 1H), 0.75–0.60 (m, 4H), 0.56–0.44 (m, 2H), 0.13 (ddt, J = 4.0, 2.8, 1.6 Hz, 2H); ESI MS m/z: 729.5 [M + H]⁺. Reaction of this intermediate with boron tribromide in CH₂Cl₂ gave the desired compound 17. Yield: 53%; white solid; mp 114–115 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.80; ¹H NMR (400 MHz, CDCl₃): δ 8.66 (dd, J = 2.1, 0.7 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H), 7.29 (td, J = 7.6, 1.6 Hz, 1H), 7.22–7.14 (m, 3H), 7.14–7.08 (m, 2H), 7.04–6.99 (m, 2H), 6.92 (dd, J = 7.8, 1.2 Hz, 1H), 6.68 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.63 (s, 1H), 3.76 (dt, J = 8.3, 5.8 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.31–3.19 (m, 2H), 2.84 (d, J = 16.5 Hz, 1H), 2.79–2.68 (m, 2H), 2.56 (d, J = 16.4 Hz, 1H), 2.51–2.39 (m, 4H), 2.37–2.27 (m, 2H), 1.75–1.67 (m, 6H), 0.77–0.71 (m, 2H), 0.69–0.64 (m, 2H), 0.54–0.45 (m, 2H), 0.13 (ddt, J = 3.7, 2.5, 1.4 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 151.9, 148.8, 143.6, 142.4, 141.3, 138.9, 138.3, 138.2, 137.2, 131.2, 130.4, 129.9, 128.5, 128.3, 126.2, 125.7, 125.6, 124.0, 119.1, 116.7, 91.5, 59.8, 59.8, 55.6, 48.0, 44.7, 32.9, 32.0, 31.5, 30.7, 29.9, 23.5, 13.6, 10.0, 9.7, 9.4, 4.3, 3.5; HRMS (ESI) m/z calcd for C₄₁H₄₃N₂O₃ [M + H]⁺: 611.32682; found, 611.32525; HPLC (system 2) t_R = 8.06 min, purity = 98.5%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2,6-dimethylphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (18).

This compound was prepared by the same procedure described for 11. The bromo compound 60 was reacted with 2-(2,6-dimethylphenyl)-boronic acid to obtain 68. Yield: 70%. ¹H NMR (400 MHz, CDCl₃): δ 8.38 (d, J = 2.0 Hz, 1H), 7.25–7.11 (m, 8H), 7.11–7.03 (m, 5H), 6.68 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.60 (s, 1H), 4.14 (dt, J = 9.7, 6.4 Hz, 1H), 4.03 (dt, J = 9.7, 6.4 Hz, 1H), 3.75 (dt, J = 8.3, 6.0 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.30–3.20 (m, 2H), 2.84–2.66 (m, 5H), 2.59 (d, J = 16.3 Hz, 1H), 2.51–2.41 (m, 4H), 2.37–2.26 (m, 2H), 2.05–1.96 (m, 5H), 1.88 (s, 3H), 1.69 (ddt, J = 9.7, 5.6, 2.3 Hz, 3H), 1.26 (s, 1H), 0.83 (tt, J = 13.1, 7.0 Hz, 1H), 0.56–0.44 (m, 2H), 0.13 (ddd, J = 4.7, 2.7, 1.6 Hz, 2H); ESI MS m/z: 717.4 [M + H]⁺. Reaction of this intermediate with boron tribromide in CH₂Cl₂ gave 18 as a white solid. Yield: 34%; mp 111–112 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.36 (dd, J = 2.1, 0.8 Hz, 1H), 7.24–7.03 (m, 10H), 6.70 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 8.1 Hz, 1H), 5.63 (s, 1H), 3.77 (dt, J = 8.3, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.32–3.18 (m, 2H), 2.86–2.66 (m, 3H), 2.57 (d, J = 16.3 Hz, 1H), 2.52–2.39 (m, 4H), 2.37–2.27 (m, 2H), 2.01 (s, 3H), 1.88 (s, 3H), 1.74–1.64 (m, 3H), 0.81 (s, 1H), 0.50 (dd, J = 8.5, 5.4 Hz, 2H), 0.13 (d, J = 3.6 Hz, 2H); 13C NMR (151 MHz, CDCl₃): δ 152.1, 148.5, 143.7, 142.4, 139.0, 138.2, 137.5, 136.6, 136.4, 130.9, 128.5, 128.3, 128.0, 127.7, 127.6, 126.1, 125.7, 119.1, 116.8, 91.4, 60.0, 59.8, 55.6, 48.0, 44.7, 33.1, 32.2, 31.5, 29.9, 21.3, 21.0, 9.4, 4.3, 3.6; HRMS (ESI) m/z calcd for C₄₀H₄₃N₂O₃ [M + H]⁺: 599.32682; found, 599.32604; HPLC (system 1) t_R = 15.56 min, purity 96.2%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2-methoxyphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (19).

Step 1.

To a stirred solution of (4bS,8R,8aS,13bR)-11-bromo-7-(cyclopropylmethyl)-5,6,7,8,9,13b-hexahydro-8aH-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline-1,8a-diol (59)³⁰ (1.59 g, 3.5 mmol) in acetone (50 mL) were added potassium carbonate (1.45 g, 10.5 mmol) and benzyl bromide (1.4 mL, 4.2 mmol). The reaction mixture was heated at reflux for 3 h. The reaction mixture was cooled to room temperature and filtered. The inorganic solids were washed several times with acetone (3 × 40 mL). The solvent was removed under reduced pressure. The residue was purified by chromatography over a column of silica gel using CHCl₃/MeOH (95:5) as the eluent to obtain (4bS,8R,8aS,13bR)-1-(benzyloxy)-11-bromo-7-(cyclopropylmethyl)-5,6,7,8,9,13b-hexahydro-8aH-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-8a-ol (0.66 g, 35%). ¹H NMR (400 MHz, CDCl₃): δ 8.63 (dd, J = 2.3, 0.9 Hz, 1H), 7.50 (d, J = 1.7 Hz, 1H), 7.32–7.21 (m, 5H), 6.69 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.49 (s, 1H), 5.19–5.07 (m, 2H), 4.91 (s, 1H), 3.28 (d, J = 6.5 Hz, 1H), 3.14 (d, J = 18.7 Hz, 1H), 2.74 (q, J = 5.3, 4.5 Hz, 1H), 2.70 (d, J = 2.6 Hz, 1H), 2.68–2.61 (m, 1H), 2.56 (d, J = 16.2 Hz, 1H), 2.48–2.36 (m, 3H), 2.36–2.27 (m, 1H), 1.81 (d, J = 10.8 Hz, 1H), 0.88 (ddt, J = 9.2, 7.5, 2.9 Hz, 1H), 0.62–0.50 (m, 2H), 0.19–0.08 (m, 2H); ESI MS m/z 545.1 [M + H]⁺.

Step 2.

The abovementioned intermediate (2.0 g, 3.7 mmol) was reacted with 3-phenylpropyl bromide (2.2 g, 11.0 mmol) in the presence of sodium hydride (0.6 g, 14.7 mmol, 60% dispersion in mineral oil) to obtain (4bS,8R,8aS,13bR)-1-(benzyloxy)-11-bromo-7-(cyclopropylmethyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (85) (0.78 g, 32%). ¹H NMR (400 MHz, CDCl₃): δ 8.65 (d, J = 2.2 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.29 (tt, J = 6.2, 3.0 Hz, 2H), 7.26–7.20 (m, 5H), 7.17–7.12 (m, 1H), 7.04–6.99 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 8.2 Hz, 1H), 5.51 (s, 1H), 5.19–5.05 (m, 2H), 3.69 (dt, J = 8.0, 6.0 Hz, 1H), 3.61 (d, J = 5.8 Hz, 1H), 3.24–3.14 (m, 2H), 2.78–2.62 (m, 3H), 2.54–2.46 (m, 3H), 2.41 (td, J = 12.0, 6.0 Hz, 2H), 2.34–2.21 (m, 2H), 1.75 (dtd, J = 10.4, 7.4, 4.4 Hz, 2H), 1.66 (dd, J = 11.6, 3.4 Hz, 1H), 0.84–0.72 (m, 1H), 0.48 (dq, J = 7.5, 4.2 Hz, 2H), 0.16–0.06 (m, 2H); ESI MS m/z: 663.2 [M + H]⁺.

Step 3.

Under an atmosphere of argon, the abovementioned intermediate 85 (0.20 g, 0.3 mmol), 2-(2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.07 g, 0.3 mmol), potassium carbonate (0.13 g, 0.9 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.03 mmol) were added to 10:1 DMF/H₂O (5.5 mL). The resulting mixture was heated in a microwave at 100 °C for 1 h. The mixture was then allowed to cool to room temperature, and the mixture was diluted with water. The aqueous layer was extracted with ethyl acetate (3 × 20 mL). Organic layers were dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with CHCl₃/MeOH (95:5) to obtain (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-(2-methoxyphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline 86 (0.17 g, 81%). ¹H NMR (400 MHz, CDCl₃): δ 8.75 (d, J = 2.1 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.38–7.27 (m, 4H), 7.26–7.20 (m, 4H), 7.15 (td, J = 6.8, 1.2 Hz, 2H), 7.11–7.06 (m, 1H), 7.05–6.96 (m, 4H), 6.68–6.64 (m, 1H), 6.52 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 5.24–5.10 (m, 2H), 3.74 (s, 3H), 3.73–3.69 (m, 1H), 3.64 (d, J = 5.9 Hz, 1H), 3.30–3.16 (m, 2H), 2.81 (d, J = 16.4 Hz, 1H), 2.72 (td, J = 11.8, 10.6, 5.9 Hz, 2H), 2.45 (q, J = 10.7, 10.0 Hz, 4H), 2.29 (ddd, J = 18.2, 12.7, 6.5 Hz, 2H), 1.77–1.67 (m, 3H), 0.85–0.76 (m, 1H), 0.49 (dq, J = 7.7, 4.0 Hz, 2H), 0.13–0.11 (m, 2H); ESI MS m/z: 691.4 [M + H]⁺.

Step 4.

To a solution of the abovementioned intermediate 86 (0.16 g 0.2 mmol) in a mixture of CH₂Cl₂ (7 mL) and MeOH (7 mL) under an argon atmosphere was added 10% palladium(II) carbon (16.0 mg, 10 wt %). The reaction mixture was evacuated under vacuum and flushed with hydrogen (H₂; 3 cycles) and was continued to stir under a H₂ atmosphere at room temperature for 20 h. The reaction mixture was filtered through a pad of celite followed by rinsing with EtOH. The solvent was removed under reduced pressure. The residue was purified by chromatography over a column of silica gel using CHCl₃/MeOH (95:5) as the eluent to obtain the title compound 19 (0.1 g). Yield: 68%; white solid; mp 200–201 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.80; ¹H NMR (400 MHz, CDCl₃): δ 8.69 (dd, J = 2.1, 0.7 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.35 (ddd, J = 8.2, 7.4, 1.7 Hz, 1H), 7.23 (dd, J = 7.5, 1.8 Hz, 1H), 7.18–7.12 (m, 2H), 7.11–7.06 (m, 1H), 7.04–6.95 (m, 4H), 6.68 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 3.73 (s, 3H), 3.65 (d, J = 5.8 Hz, 1H), 3.31–3.18 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.79–2.66 (m, 2H), 2.55 (d, J = 16.5 Hz, 1H), 2.46 (td, J = 10.1, 9.4, 5.6 Hz, 5H), 2.39–2.26 (m, 3H), 1.78–1.66 (m, 3H), 0.81 (p, J = 7.0, 6.3 Hz, 1H), 0.53–0.46 (m, 2H), 0.14–0.12 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 156.7, 151.7, 148.7, 143.6, 142.4, 139.0, 138.2, 133.9, 131.2, 130.8, 130.3, 129.7, 128.5, 128.2, 126.7, 125.6, 121.1, 119.0, 116.8, 111.3, 91.3, 59.8, 59.6, 55.6, 47.9, 44.7, 32.7, 32.1, 31.7, 31.5, 30.6, 29.8, 23.5, 9.4, 4.3, 3.6; HRMS (ESI) m/z calcd for C₃₉H₄₁N₂O₄ [M + H]⁺: 601.30608; found, 601.30498; HPLC (system 1) t_R = 14.56 min, purity = 97.8%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(3-methoxyphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (20).

Step 1.

The bromo compound 85 (0.20 g, 0.3 mmol) was reacted with 2-(3-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.07 g, 0.3 mmol) in the presence of potassium carbonate (0.13 g, 0.9 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.03 mmol), as described in step 3 for the preparation of 19, to give 87 (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-(3-methoxyphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (87) (0.05 g, 26%). ¹H NMR (400 MHz, CDCl₃): δ 8.82 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 2.3 Hz, 1H), 7.40–7.35 (m, 1H), 7.32–7.28 (m, 2H), 7.25–7.20 (m, 4H), 7.17–7.12 (m, 3H), 7.12–7.09 (m, 1H), 7.09–7.05 (m, 1H), 7.01–6.96 (m, 2H), 6.93 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 6.66 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 5.20–5.10 (m, 2H), 3.84 (s, 3H), 3.75–3.68 (m, 1H), 3.65 (d, J = 5.9 Hz, 1H), 3.22 (d, J = 6.5 Hz, 2H), 2.77–2.70 (m, 2H), 2.55 (d, J = 16.8 Hz, 1H), 2.49–2.33 (m, 6H), 1.76–1.68 (m, 3H), 0.80 (s, 1H), 0.50–0.47 (m, 2H), 0.11 (d, J = 7.0 Hz, 2H); ESI MS m/z: 691.3 [M + H]⁺.

Step 2.

A solution of the abovementioned intermediate 87 (0.05 g, 0.08 mmol) in 2,2,2-trifluoroacetic acid (5 mL) was heated at reflux for 1.5 h. The mixture was cooled to room temperature, and 2,2,2-trifluoroacetic acid was removed under reduced pressure. The residue was dissolved in water and neutralized with aqueous NH₄OH. The resulting suspension was extracted with EtOAc (3 × 10 mL) and washed with water (10 mL). The extract was dried, and the solvent was removed under reduced pressure. The residue was purified by chromatography over a column of silica gel using CHCl₃/MeOH (95:5) as the eluent to obtain 25.0 mg (54%) of the desired product 20 as a white solid. mp 107–108 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.69 (s, 1H), 7.35 (dd, J = 17.9, 9.8 Hz, 2H), 7.15–7.02 (m, 4H), 7.01–6.90 (m, 4H), 6.69 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 5.60 (s, 1H), 3.83 (s, 3H), 3.67 (d, J = 20.8 Hz, 2H), 3.24 (dq, J = 11.4, 5.3, 4.6 Hz, 2H), 2.90–2.63 (m, 3H), 2.57–2.28 (m, 8H), 1.71 (d, J = 19.5 Hz, 3H), 0.81 (s, 1H), 0.50 (s, 2H), 0.13 (s, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 160.2, 146.8, 143.5, 142.3, 139.0, 136.2, 135.9, 131.1, 130.2, 128.5, 128.2, 125.6, 119.7, 119.1, 116.9, 113.7, 113.1, 91.1, 59.8, 59.4, 55.5, 47.9, 44.7, 33.6, 32.5, 32.1, 31.5, 29.8, 29.8, 29.5, 23.3, 22.8, 18.0, 14.3, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₉H₄₁N₂O₄ [M + H]⁺: 601.30608; found, 601.30498; HPLC (system 1) t_R = 14.85 min, purity = 98.2%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(4-methoxyphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (21).

Step 1.

The bromo compound 85 (0.20 g, 0.3 mmol) was reacted with 2-(4-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.07 g, 0.3 mmol) in the presence of potassium carbonate (0.13 g, 0.9 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.03 mmol), as described in step 3 for the preparation of 19, to obtain (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-(4-methoxyphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (88) (0.16 g, 78%). ¹H NMR (400 MHz, CDCl₃): δ 8.79 (dd, J = 2.3, 0.7 Hz, 1H), 7.50–7.44 (m, 2H), 7.38 (d, J = 2.1 Hz, 1H), 7.31–7.27 (m, 2H), 7.25–7.19 (m, 3H), 7.17–7.11 (m, 2H), 7.11–7.05 (m, 1H), 7.01–6.96 (m, 4H), 6.66 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 5.21–5.09 (m, 2H), 3.85 (s, 3H), 3.74–3.67 (m, 1H), 3.64 (d, J = 5.8 Hz, 1H), 3.22 (dd, J = 16.7, 11.0 Hz, 2H), 2.82 (d, J = 16.5 Hz, 1H), 2.78–2.65 (m, 2H), 2.58–2.40 (m, 5H), 2.36–2.23 (m, 2H), 1.72 (ddd, J = 23.9, 12.6, 6.9 Hz, 3H), 0.85–0.75 (m, 1H), 0.49 (dq, J = 7.8, 4.3 Hz, 2H), 0.17–0.09 (m, 2H); ESI MS m/z: 691.3 [M + H]⁺.

Step 2.

The abovementioned intermediate 88 (0.16 g, 0.2 mmol) was debenzylated with 10% palladium(II) carbon (16.0 mg, 10 wt %) under a H₂ atmosphere, as described in step 4 in the preparation of 19, to obtain 0.05 g (33%) of the desired compound 21 as a white solid. mp 100–101 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.61 (s, 1H), 7.39–7.30 (m, 3H), 7.10 (dq, J = 14.2, 7.3 Hz, 3H), 7.00–6.91 (m, 4H), 6.69 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.59 (s, 1H), 3.84 (s, 3H), 3.74–3.59 (m, 2H), 3.32–3.15 (m, 2H), 2.82–2.66 (m, 3H), 2.46 (q, J = 13.1, 10.5 Hz, 6H), 2.38–2.22 (m, 2H), 1.83–1.62 (m, 3H), 0.80 (s, 1H), 0.54–0.43 (m, 2H), 0.13 (d, J = 0.9 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 159.9, 151.7, 146.3, 143.5, 142.3, 135.9, 135.3, 131.2,130.9, 129.8, 128.5, 128.3, 128.2, 126.0, 125.6, 119.1, 116.9, 114.6, 91.1, 59.9, 59.4, 55.6, 55.5, 47.9, 44.7, 33.6, 32.5, 32.1, 31.6, 29.9, 29.5, 23.3, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₉H₄₁N₂O₄ [M + H]⁺: 601.30608; found, 601.30432; HPLC (system 1) t_R = 14.54 min, purity = 99%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(3-hydroxyphenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (22).

This compound was prepared by the same procedure described for 11. The bromo compound 60 was reacted with 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol to obtain 69. Yield: 63%. ¹H NMR (400 MHz, CDCl₃): δ 8.84 (d, J = 2.1 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.17–7.03 (m, 8H), 7.02–6.94 (m, 6H), 6.62 (d, J = 8.2 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.63 (s, 1H), 4.05 (dt, J = 9.5, 6.2 Hz, 1H), 3.93 (dt, J = 9.6, 6.4 Hz, 1H), 3.75–3.60 (m, 2H), 3.30–3.16 (m, 2H), 2.84–2.67 (m, 3H), 2.60–2.39 (m, 7H), 2.31 (ddd, J = 19.7, 12.3, 7.5 Hz, 2H), 1.89–1.66 (m, 5H), 0.84–0.71 (m, 1H), 0.50 (ddt, J = 7.9, 5.3, 2.7 Hz, 2H), 0.14–0.12 (m, 2H); ESI MS m/z: 705.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 22 as a white solid. Yield: 58%; white solid; mp 155–157 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.40; ¹H NMR (400 MHz, CDCl₃): δ 8.49 (s, 1H), 7.23 (s, 1H), 7.10–6.95 (m, 4H), 6.95–6.86 (m, 2H), 6.77 (t, J = 5.9 Hz, 2H), 6.63 (t, J = 5.8 Hz, 2H), 6.34 (s, 1H), 5.55 (s, 1H), 3.65 (dt, J = 10.8, 5.8 Hz, 2H), 3.27–3.16 (m, 2H), 2.83–2.60 (m, 4H), 2.55–2.25 (m, 8H), 1.73–1.56 (m, 3H), 0.77 (p, J = 6.6 Hz, 1H), 0.49 (dp, J = 9.5, 5.1 Hz, 2H), 0.27–0.03 (m, 2H); ¹³C NMR (214 MHz, CDCl₃): δ 157.5, 151.5, 144.6, 143.4, 142.2, 139.9, 137.1, 136.4, 135.6, 131.7, 130.9, 129.9, 128.4, 128.2, 125.6, 125.5, 125.1, 119.3, 118.1, 117.6, 116.1, 113.8, 89.5, 59.8, 59.5, 55.6, 47.8, 44.7, 32.4, 31.5, 31.4, 30.4, 23.6, 9.4, 3.7; HRMS (ESI) m/z calcd for C₃₈H₃₉N₂O₄ [M + H]⁺: 587.29043; found, 587.28972; HPLC (system 1) t_R = 13.61 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2-(dimethylamino)phenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (23).

As described for the preparation of 11, the bromo compound 60 was reacted with N,N-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline to obtain 70. Yield: 34%. ¹H NMR (400 MHz, CDCl₃): δ 8.76 (dd, J = 2.2, 0.7 Hz, 1H), 7.51 (d, J = 2.1 Hz, 1H), 7.31–7.27 (m, 1H), 7.26–7.09 (m, 10H), 7.06–7.01 (m, 3H), 6.99 (dd, J = 7.4, 1.2 Hz, 1H), 6.66 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 5.58 (s, 1H), 4.12 (dt, J = 9.8, 6.3 Hz, 1H), 4.02 (dt, J = 9.7, 6.3 Hz, 1H), 3.73 (dt, J = 8.3, 6.1 Hz, 1H), 3.64 (d, J = 5.8 Hz, 1H), 3.31–3.17 (m, 2H), 2.82 (d, J = 16.4 Hz, 1H), 2.77–2.68 (m, 4H), 2.59–2.53 (m, 1H), 2.49–2.45 (m, 3H), 2.44 (s, 6H), 2.36–2.27 (m, 2H), 2.03–1.93 (m, 2H), 1.70 (t, J = 7.7 Hz, 3H), 0.85–0.75 (m, 1H), 0.49 (td, J = 7.9, 4.7 Hz, 2H), 0.13 (s, 2H); ESI MS m/z: 732.4 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 23 as a white solid. Yield: 43%. mp 102–104 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 8.75 (d, J = 1.9 Hz, 1H), 7.53 (d, J = 2.1 Hz, 1H), 7.29 (ddd, J = 8.1, 7.3, 1.7 Hz, 1H), 7.21–7.15 (m, 2H), 7.12 (ddd, J = 5.9, 4.2, 2.4 Hz, 2H), 7.07–6.98 (m, 4H), 6.68 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 3.74 (dt, J = 8.1, 6.0 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.32–3.19 (m, 2H), 2.83 (d, J = 16.4 Hz, 1H), 2.79–2.66 (m, 2H), 2.54 (d, J = 16.4 Hz, 1H), 2.48 (d, J = 8.3 Hz, 3H), 2.45 (s, 6H), 2.37–2.29 (m, 2H), 1.69 (ddd, J = 11.3, 8.4, 4.7 Hz, 3H), 1.26 (s, 2H), 0.80 (q, J = 6.6 Hz, 1H), 0.56–0.42 (m, 2H), 0.13 (dd, J = 4.6, 1.4 Hz, 2H); ¹³C NMR (214 MHz, CDCl₃): δ 151.8, 151.6, 148.3, 143.6, 142.5, 138.8, 137.2, 137.1, 131.6, 131.2, 130.6, 129.1, 128.5, 128.3, 126.2, 125.7, 122.2, 119.0, 118.3, 116.6, 91.7, 59.9, 59.8, 55.6, 48.0, 44.7, 43.7, 32.9, 32.0, 30.7, 29.9, 29.5, 23.5, 9.4, 4.3, 3.6; HRMS (ESI) m/z calcd for C₄₀H₄₄N₃O₃ [M + H]⁺: 614.33772; found, 614.33649; HPLC (system 1) t_R = 13.73 min, purity = 95.7%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(3-(dimethylamino)phenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (24).

Following the procedure for the preparation of 11, the bromo compound 60 was reacted with N,N-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline to obtain 71 in 65% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.80 (d, J = 2.2 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.33–7.28 (m, 1H), 7.25–7.18 (m, 2H), 7.17–7.05 (m, 6H), 7.01–6.96 (m, 2H), 6.84 (ddd, J = 7.6, 1.7, 0.9 Hz, 1H), 6.80 (t, J = 2.1 Hz, 1H), 6.76 (ddd, J = 8.4, 2.6, 0.9 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H), 4.11 (dt, J = 9.7, 6.4 Hz, 1H), 4.00 (dt, J = 9.6, 6.4 Hz, 1H), 3.71 (dt, J = 8.0, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.19 (m, 2H), 2.97 (s, 6H), 2.85 (d, J = 16.5 Hz, 1H), 2.77–2.68 (m, 4H), 2.58 (d, J = 16.6 Hz, 1H), 2.47 (ddd, J = 12.5, 6.8, 3.3 Hz, 4H), 2.37–2.25 (m, 2H), 2.04–1.93 (m, 2H), 1.76–1.67 (m, 3H), 0.82 (q, J = 6.4 Hz, 1H), 0.54–0.44 (m, 2H), 0.20–0.05 (m, 2H); ESI MS m/z: 732.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 24 as a white solid. Yield: 63%. mp 140–142 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.77 (dd, J = 2.3, 1.0 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.17–7.12 (m, 2H), 7.11–7.05 (m, 1H), 7.01–6.96 (m, 2H), 6.84 (ddd, J = 7.5, 1.7, 0.8 Hz, 1H), 6.81–6.74 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 3.72 (dt, J = 8.1, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.30–3.18 (m, 2H), 2.98 (s, 6H), 2.85 (d, J = 16.6 Hz, 1H), 2.79–2.67 (m, 2H), 2.56 (d, J = 16.6 Hz, 1H), 2.51–2.41 (m, 4H), 2.37–2.26 (m, 2H), 1.76–1.68 (m, 4H), 0.80 (t, J = 6.4 Hz, 1H), 0.54–0.42 (m, 2H), 0.13 (ddt, J = 3.8, 2.6, 1.2 Hz, 2H); 13C NMR (214 MHz, CDCl₃): δ 152.2, 151.1, 147.1, 143.5, 142.4, 138.8, 138.6, 137.4, 136.0, 131.2, 130.8, 129.8, 128.6, 128.5, 128.3, 126.2, 125.6, 119.0, 116.7, 115.7, 112.4, 111.4, 91.5, 59.9, 59.4, 55.6, 47.9, 44.7, 40.7, 32.6, 31.7, 30.6, 29.9, 23.6, 9.4, 4.2; HRMS (ESI) m/z calcd for C₄₀H₄₄N₃O₃ [M + H]⁺: 614.33772; found, 614.33714; HPLC (system 1) t_R = 12.66 min, purity = 98.8%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(4-(dimethylamino)phenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (25).

This compound was prepared by the same procedure described for 11. The bromo compound 60 was reacted with N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline to obtain 72. Yield: 61%. ¹H NMR (400 MHz, CDCl₃): δ 8.77 (dd, J = 2.3, 0.7 Hz, 1H), 7.46–7.40 (m, 2H), 7.36 (d, J = 2.2 Hz, 1H), 7.25–7.18 (m, 2H), 7.17–7.05 (m, 7H), 7.01–6.97 (m, 2H), 6.81–6.76 (m, 2H), 6.64 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 8.1 Hz, 1H), 5.58 (s, 1H), 4.11 (dt, J = 9.6, 6.4 Hz, 1H), 4.00 (dt, J = 9.6, 6.4 Hz, 1H), 3.74–3.61 (m, 2H), 3.30–3.16 (m, 2H), 3.00 (s, 6H), 2.87–2.78 (m, 1H), 2.78–2.66 (m, 5H), 2.56 (d, J = 16.4 Hz, 1H), 2.52–2.41 (m, 5H), 2.38–2.25 (m, 2H), 2.04–1.92 (m, 2H), 0.80 (q, J = 6.6 Hz, 1H), 0.56–0.43 (m, 2H), 0.19–0.07 (m, 2H); ESI MS m/z: 732.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 25. Yield: 26%; pale yellow solid; mp 128–130 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.73 (d, J = 2.0 Hz, 1H), 7.45–7.38 (m, 2H), 7.37 (d, J = 2.2 Hz, 1H), 7.18–7.07 (m, 3H), 7.00–6.96 (m, 2H), 6.80–6.76 (m, 2H), 6.66 (dd, J = 8.1, 0.9 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 3.70 (dt, J = 8.1, 6.0 Hz, 1H), 3.64 (d, J = 5.8 Hz, 1H), 3.29–3.17 (m, 2H), 3.00 (s, 6H), 2.86–2.66 (m, 3H), 2.58–2.40 (m, 6H), 2.37–2.25 (m, 2H), 1.80–1.64 (m, 3H), 0.87–0.77 (m, 1H), 0.55–0.44 (m, 2H), 0.17–0.09 (m, 2H); ¹³C NMR (214 MHz, CDCl₃): δ 150.9, 150.6, 146.2, 143.5, 142.4, 138.9, 136.3, 134.6, 131.2, 130.7, 128.6, 128.3, 128.2, 127.9, 126.1, 125.6, 125.1, 119.0, 116.7, 112.9, 91.5, 91.5, 59.9, 59.4, 55.6, 47.9, 44.7, 40.6, 32.5, 31.6, 31.5, 30.6, 23.6, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₄₀H₄₄N₃O₃ [M + H]⁺: 614.33772; found, 614.33632; HPLC (system 1) t_R = 13.06 min, purity = 94.5%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2-((dimethylamino)methyl)phenyl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (26).

The bromo compound 60 was reacted with NN-dimethyl-1-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenyl)methanamine, as described in step 2 for the preparation of 11, to obtain 73 in 59% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 2.2, 0.7 Hz, 1H), 7.49 (dd, J = 7.5, 1.4 Hz, 1H), 7.42 (d, J = 2.1 Hz, 1H), 7.35 (td, J = 7.5, 1.5 Hz, 1H), 7.29 (td, J = 7.5, 1.5 Hz, 1H), 7.26–7.23 (m, 1H), 7.23–7.19 (m, 1H), 7.19–7.14 (m, 5H), 7.14–7.09 (m, 2H), 7.07–7.03 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.60 (s, 1H), 4.13 (dt, J = 9.7, 6.3 Hz, 1H), 4.03 (dt, J = 9.6, 6.4 Hz, 1H), 3.77–3.69 (m, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.30–3.19 (m, 4H), 2.83 (d, J = 16.4 Hz, 1H), 2.78–2.67 (m, 4H), 2.57 (d, J = 16.4 Hz, 1H), 2.52–2.42 (m, 4H), 2.33 (dd, J = 12.6, 6.8 Hz, 2H), 2.08 (s, 6H), 2.05–1.96 (m, 2H), 1.72 (td, J = 12.7, 11.7, 5.4 Hz, 3H), 0.85–0.75 (m, 1H), 0.53–0.43 (m, 2H), 0.13 (td, J = 3.2, 1.3 Hz, 2H); ESI MS m/z: 746.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 26 as a pale brown solid in 34% yield. mp 120–121 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 8.52 (s, 1H), 7.60–7.48 (m, 4H), 7.37 (d, J = 7.4 Hz, 1H), 7.20 (dd, J = 12.4, 4.3 Hz, 3H), 7.10 (d, J = 7.5 Hz, 3H), 6.67 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 4.98 (d, J = 13.6 Hz, 1H), 4.86 (d, J = 13.8 Hz, 1H), 3.66 (dd, J = 24.6, 6.6 Hz, 2H), 3.33–3.13 (m, 2H), 2.85 (d, J = 16.8 Hz, 1H), 2.72 (d, J = 19.2 Hz, 5H), 2.56 (d, J = 8.1 Hz, 6H), 2.44 (dt, J = 18.8, 6.1 Hz, 2H), 2.32 (dd, J = 12.9, 7.4 Hz, 2H), 1.81–1.74 (m, 2H), 1.68 (d, J = 11.3 Hz, 1H), 0.82–0.71 (m, 1H), 0.51–0.45 (m, 2H), 0.12–0.09 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 153.4, 148.4, 143.3, 142.5, 141.8, 139.3, 138.4, 135.6, 134.6, 132.0, 130.9, 128.6, 128.3, 127.0, 125.6, 119.3, 117.2, 90.3, 59.8, 58.3, 55.5, 47.8, 45.5, 45.1, 44.6, 32.7, 31.9, 31.3, 30.4, 29.8, 29.5, 23.5, 22.8, 14.3, 9.3, 4.1, 3.7; HRMS (ESI) m/z calcd for C₄₁H₄₆N₃O₃ [M + H]⁺: 628.35337; found, 628.35178; HPLC (system 1) t_R = 16.33 min, purity = 95.7%.

N-(2-((4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-1-hydroxy-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-11-yl)phenyl)-acetamide (27).

This compound was prepared by the same procedure described for 11. Reaction of the bromo compound 60 with N-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-acetamide gave 74 in 62% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.60 (d, J = 2.2 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.39 (td, J = 8.3, 7.8, 1.7 Hz, 1H), 7.29 (d, J = 2.1 Hz, 1H), 7.25–7.08 (m, 11H), 7.02–6.98 (m, 2H), 6.77 (d, J = 6.2 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 5.60 (s, 1H), 4.13 (dt, J = 9.5, 6.3 Hz, 1H), 4.00 (dt, J = 9.5, 6.4 Hz, 1H), 3.73 (q, J = 6.5 Hz, 1H), 3.67 (d, J = 5.8 Hz, 1H), 3.28–3.18 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.79–2.68 (m, 4H), 2.57 (d, J = 16.4 Hz, 1H), 2.51–2.41 (m, 4H), 2.37–2.26 (m, 2H), 2.05–1.96 (m, 2H), 1.84 (d, J = 1.2 Hz, 3H), 1.71 (d, J = 12.1 Hz, 2H), 0.85–0.75 (m, 1H), 0.57–0.44 (m, 2H), 0.13 (dq, J = 5.0, 1.4 Hz, 2H); ESI MS m/z: 746.4 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 27 in 47% yield as a pale yellow solid. mp 214–215 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ ¹H NMR (400 MHz, DMSO-d₆): δ 9.40 (s, 1H), 9.06 (s, 1H), 8.50 (d, J = 2.1 Hz, 1H), 7.49 (dd, J = 8.4, 1.5 Hz, 2H), 7.37 (td, J = 8.0, 7.4, 2.0 Hz, 1H), 7.32–7.23 (m, 2H), 7.21–7.14 (m, 2H), 7.13–7.07 (m, 1H), 7.05–7.00 (m, 2H), 6.51 (s, 2H), 5.37 (s, 1H), 3.64 (t, J = 7.0 Hz, 2H), 3.28 (t, J = 7.5 Hz, 1H), 3.12 (d, J = 18.5 Hz, 1H), 2.98 (d, J = 16.7 Hz, 1H), 2.71–2.63 (m, 1H), 2.61–2.52 (m, 1H), 2.46–2.34 (m, 5H), 2.29 (dd, J = 12.6, 6.8 Hz, 1H), 2.22–2.13 (m, 1H), 1.85 (s, 3H), 1.63 (p, J = 7.2 Hz, 2H), 1.47 (d, J = 11.5 Hz, 1H), 0.78–0.66 (m, 1H), 0.51–0.36 (m, 2H), 0.17–0.00 (m, 2H); ¹³C NMR (214 MHz, DMSO): δ 168.8, 152.0, 147.2, 143.5, 142.0, 139.3, 137.2, 135.2, 134.2, 132.5, 131.0, 130.5, 130.3, 128.4, 128.2, 128.1, 127.3, 125.9, 125.5, 125.1, 118.5, 117.0, 89.9, 76.4, 59.0, 58.9, 54.6, 47.1,44.0, 31.9, 31.4, 30.7, 30.5, 23.0, 22.8, 9.1, 3.9; HRMS (ESI) m/z calcd for C₄₀H₄₂N₃O₄ [M + H]⁺: 628.31698; found, 628.31533; HPLC (system 1) t_R = 13.29 min, purity = 99.5%.

N-(3-((4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-1-hydroxy-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-11-yl)phenyl)-acetamide (28).

The bromo compound 60 was reacted with N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide, as described in step 2 for the preparation of 11, to obtain 75 in 76% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.67 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.23–7.05 (m, 11H), 7.01–6.95 (m, 2H), 6.62 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.60 (s, 1H), 4.03 (t, J = 8.0 Hz, 1H), 3.90 (d, J = 6.6 Hz, 1H), 3.70 (dt, J = 8.1, 6.0 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.17 (m, 2H), 2.78–2.65 (m, 5H), 2.46 (h, J = 6.7, 5.9 Hz, 5H), 2.37–2.28 (m, 2H), 2.23 (s, 3H), 1.90 (d, J = 7.5 Hz, 2H), 1.77–1.65 (m, 3H), 0.86–0.76 (m, 1H), 0.50 (ddt, J = 8.1, 4.7, 2.4 Hz, 2H), 0.15–0.12 (m, 2H); ESI MS m/z: 746.4 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 28 in 70% yield as a white solid. mp 165–166 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 8.40 (s, 1H), 8.13 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.25–6.99 (m, 6H), 6.93 (d, J = 7.3 Hz, 2H), 6.69 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.56 (s, 1H), 3.74–3.54 (m, 2H), 3.31–2.92 (m, 3H), 2.68 (q, J = 17.8, 16.4 Hz, 3H), 2.51–2.23 (m, 7H), 2.19 (s, 3H), 1.80–1.56 (m, 3H), 0.78 (s, 1H), 0.48 (d, J = 7.8 Hz, 2H), 0.18–0.04 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 169.1, 151.9, 145.6, 143.6, 142.3, 139.4, 138.4, 135.3, 131.2, 128.5, 128.2, 127.4, 125.8, 125.6, 120.4, 119.3, 117.4, 90.4, 76.5, 59.8, 59.4, 55.6, 47.8, 44.6, 32.5, 32.1, 31.5, 31.3, 30.5, 29.8, 24.7, 23.5, 9.4, 4.2, 3.7; HRMS (ESI) m/z calcd for C₄₀H₄₂N₃O₄ [M + H]⁺: 628.31698; found, 628.31612; HPLC (system 1) t_R = 13.31 min, purity = 96.5%.

N-(4-((4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-1-hydroxy-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-11-yl)phenyl)-acetamide (29).

Reaction of the bromo compound 60 with N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide gave 76 in 61% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.78–8.72 (m, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.49–7.43 (m, 2H), 7.38 (d, J = 2.2 Hz, 1H), 7.23–7.17 (m, 2H), 7.16–7.05 (m, 6H), 7.00–6.95 (m, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.58 (s, 1H), 4.10 (ddd, J = 9.4, 6.9, 5.8 Hz, 1H), 3.98 (ddd, J = 9.2, 6.9, 5.9 Hz, 1H), 3.71 (dt, J = 8.1, 5.9 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.28–3.19 (m, 2H), 2.83 (d, J = 16.5 Hz, 1H), 2.78–2.66 (m, 4H), 2.56 (d, J = 16.5 Hz, 1H), 2.46 (td, J = 9.2, 8.5, 5.9 Hz, 4H), 2.36–2.25 (m, 2H), 2.21 (s, 3H), 2.02–1.92 (m, 2H), 1.62 (dd, J = 7.5, 6.5 Hz, 4H), 0.85–0.74 (m, 1H), 0.49 (dtd, J = 7.9, 4.9, 3.5 Hz, 2H), 0.16–0.09 (m, 2H); ESI MS m/z: 746.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 29 in 72% yield as a white solid. mp 173–174 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.40 (s, 1H), 8.13 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.25–6.99 (m, 6H), 6.93 (d, J = 7.3 Hz, 2H), 6.69 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.56 (s, 1H), 3.74–3.54 (m, 2H), 3.31–2.92 (m, 3H), 2.68 (q, J = 17.8, 16.4 Hz, 3H), 2.51–2.23 (m, 7H), 2.19 (s, 3H), 1.80–1.56 (m, 3H), 0.78 (s, 1H), 0.48 (d, J = 7.8 Hz, 2H), 0.18–0.04 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 169.1, 151.9, 145.6, 143.6, 142.3, 139.4, 138.4, 135.3, 131.2, 128.5, 128.2, 127.4, 125.8, 125.6, 120.4, 119.3, 117.4, 90.4, 76.5, 59.8, 59.4, 55.6, 47.8, 44.6, 32.5, 32.1, 31.5, 31.3, 30.5, 29.8, 24.7, 23.5, 9.4, 4.2, 3.7; HRMS (ESI) m/z calcd for C₄₀H₄₂N₃O₄ [M + H]⁺: 628.31698; found, 628.31682; HPLC (system 1) t_R = 13.05 min, purity = 98.1%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(1H-pyrrol-1-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (30).

A mixture of the bromo compound 60 (0.25 g, 0.4 mmol), 1H-pyrrole (28 μL, 0.4 mmol), cesium carbonate (0.2 g, 0.6 mmol), Pd₂(dba)₃ (9.9 mg, 10.8 μmol), and tri-tert-butylphosphine (10.8 μL, 10.8 μmol) in toluene (5 mL) was heated at 100 °C for 15 h under a nitrogen atmosphere. The mixture was allowed to cool down to room temperature and diluted with water. The aqueous layer was extracted with ethyl acetate (3 × 20 mL). Organic layers were dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with CHCl₃/MeOH (95:5) to obtain (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-1,8a-bis(3-phenylpropoxy)-11-(1H-pyrrol-1-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (0.17 g, 70%). ¹H NMR (400 MHz, CDCl₃): δ 8.68 (dd, J = 2.6, 0.7 Hz, 1H), 7.24–7.09 (m, 8H), 7.03–6.98 (m, 3H), 6.64 (dd, J = 8.1, 5.3 Hz, 1H), 6.58–6.53 (m, 1H), 6.38–6.35 (m, 1H), 5.56 (s, 1H), 4.09 (dt, J = 9.6, 6.4 Hz, 1H), 3.99 (dt, J = 9.7, 6.4 Hz, 1H), 3.74–3.67 (m, 1H), 3.64 (t, J = 5.8 Hz, 1H), 3.29–3.16 (m, 2H), 2.86–2.64 (m, 5H), 2.58–2.42 (m, 4H), 2.31 (ddd, J = 15.7, 12.2, 7.6 Hz, 2H), 2.03–1.90 (m, 2H), 1.80–1.64 (m, 3H), 1.57 (d, J = 2.1 Hz, 4H), 0.80 (t, J = 6.3 Hz, 1H), 0.54–0.43 (m, 2H), 0.16–0.09 (m, 2H); ESI MS m/z: 678.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 30 in 32% yield as a white solid. mp 116–118 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.49 (d, J = 2.5 Hz, 1H), 7.17–7.05 (m, 4H), 7.01–6.96 (m, 2H), 6.91 (t, J = 2.2 Hz, 2H), 6.71 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 8.1 Hz, 1H), 6.37–6.27 (m, 2H), 5.58 (s, 1H), 3.74–3.58 (m, 2H), 3.28–3.13 (m, 2H), 2.82–2.63 (m, 3H), 2.55–2.38 (m, 5H), 2.38–2.23 (m, 2H), 1.79–1.62 (m, 3H), 0.87–0.69 (m, 1H), 0.55–0.41 (m, 2H), 0.18 to −0.04 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 150.5, 143.5, 142.2, 139.6, 139.3, 136.2, 128.5, 128.5, 128.3, 128.2, 127.9, 125.7, 125.7, 119.2, 119.0, 117.3, 111.5, 90.4, 76.5, 59.8, 59.4, 55.5, 47.9, 44.6, 32.4, 31.5, 31.4, 30.5, 23.5, 9.4, 4.2; HRMS (ESI) m/z calcd for C₃₆H₃₈N₃O₃ [M + H]⁺: 560.29077; found, 560.2902; HPLC (system 1) t_R = 14.85 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(pyridin-2-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (31).

The bromo compound 60 was reacted with pyridin-2-ylboronic acid, as described in step 2 for the preparation of 11, to obtain 77 in 29% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.62 (dd, J = 2.2, 0.8 Hz, 1H), 7.39 (d, J = 2.2 Hz, 1H), 7.23 (ddt, J = 6.4, 5.5, 1.1 Hz, 4H), 7.19–7.10 (m, 5H), 7.04–6.96 (m, 2H), 6.63 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.48 (s, 1H), 4.07 (dt, J = 9.6, 6.4 Hz, 1H), 3.98 (dt, J = 9.6, 6.3 Hz, 1H), 3.69 (dt, J = 7.9, 6.0 Hz, 1H), 3.62 (d, J = 5.9 Hz, 1H), 3.25–3.15 (m, 2H), 2.81–2.58 (m, 6H), 2.55–2.38 (m, 6H), 2.29 (ddd, J = 19.3, 12.4, 7.6 Hz, 2H), 2.03–1.92 (m, 2H), 1.85–1.61 (m, 4H), 0.85–0.72 (m, 1H), 0.55–0.43 (m, 2H), 0.11 (dq, J = 5.0, 1.3 Hz, 2H); ESI MS m/z: 691.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 31 in 26% yield as a white solid. mp 184–185 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 9.05 (d, J = 2.1 Hz, 1H), 8.71–8.66 (m, 1H), 7.96 (d, J = 2.1 Hz, 1H), 7.78–7.68 (m, 2H), 7.26 (ddd, J = 7.2, 4.8, 1.3 Hz, 1H), 7.17–7.11 (m, 2H), 7.10–7.03 (m, 1H), 7.02–6.97 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 3.70 (dt, J = 8.0, 6.1 Hz, 1H), 3.64 (d, J = 5.8 Hz, 1H), 3.29–3.17 (m, 2H), 2.88 (d, J = 16.6 Hz, 1H), 2.72 (ddt, J = 17.1, 11.9, 5.2 Hz, 2H), 2.57–2.40 (m, 6H), 2.31 (ddd, J = 17.5, 12.8, 6.5 Hz, 2H), 1.71 (ddt, J = 18.1, 11.7, 5.5 Hz, 3H), 0.83–0.74 (m, 1H), 0.57–0.42 (m, 2H), 0.17–0.06 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 154.4, 154.0, 150.2, 146.4, 143.5, 142.4, 139.0, 137.1, 135.9, 134.4, 131.2, 131.1, 128.6, 128.2, 126.0, 125.6, 123.0, 120.8, 119.1, 116.9, 91.0, 59.9, 59.4, 55.6, 47.9, 44.6, 32.5, 31.6, 31.3, 30.5, 29.8, 23.5, 9.4, 4.2; HRMS (ESI) m/z calcd for C₃₇H₃₈N₃O₃ [M + H]⁺: 572.29077; found, 572.29036; HPLC (system 2) t_R = 5.76 min, purity = 95.5%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(pyridin-3-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (32).

Following the procedure for the preparation of 11, the bromo compound 60 was reacted 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine to obtain 78 in 82% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.78 (dd, J = 2.3, 0.7 Hz, 1H), 8.76 (dd, J = 2.4, 0.9 Hz, 1H), 8.63 (dd, J = 4.8, 1.6 Hz, 1H), 7.80 (ddd, J = 7.9, 2.4, 1.6 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.38 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 7.24–7.18 (m, 2H), 7.18–7.06 (m, 6H), 7.01–6.97 (m, 2H), 6.65 (d, J = 8.2 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H), 4.11 (dt, J = 9.6, 6.4 Hz, 1H), 3.99 (dt, J = 9.7, 6.4 Hz, 1H), 3.73 (dt, J = 8.0, 6.0 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.31–3.19 (m, 2H), 2.86 (d, J = 16.5 Hz, 1H), 2.78–2.69 (m, 4H), 2.59 (d, J = 16.5 Hz, 1H), 2.53–2.41 (m, 4H), 2.39–2.26 (m, 2H), 2.03–1.92 (m, 2H), 1.73 (td, J = 13.6, 6.0 Hz, 3H), 0.85–0.77 (m, 1H), 0.65–0.56 (m, 2H), 0.49 (tt, J = 8.8, 5.0 Hz, 2H); ESI MS m/z: 690.4 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 32 in 17% yield as a white solid. mp 219–220 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.40; ¹H NMR (400 MHz, CDCl₃): δ 8.92 (s, 1H), 8.85 (d, J = 2.2 Hz, 1H), 8.64 (dd, J = 4.8, 1.6 Hz, 1H), 7.83 (ddd, J = 7.9, 2.4, 1.7 Hz, 1H), 7.45 (d, J = 2.2 Hz, 1H), 7.39 (ddd, J = 7.9, 4.8, 0.8 Hz, 1H), 7.17–7.11 (m, 2H), 7.11–7.05 (m, 1H), 7.01–6.96 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.64 (s, 1H), 3.73 (dt, J = 8.1, 5.9 Hz, 1H), 3.66 (d, J = 5.9 Hz, 1H), 3.30–3.19 (m, 2H), 2.87 (d, J = 16.6 Hz, 1H), 2.80–2.67 (m, 2H), 2.58 (d, J = 16.5 Hz, 1H), 2.47 (dt, J = 12.9, 6.8 Hz, 4H), 2.38–2.26 (m, 2H), 1.79–1.65 (m, 4H), 0.80 (q, J = 6.7 Hz, 1H), 0.58–0.42 (m, 2H), 0.23–0.07 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 153.7, 149.2, 148.2, 146.7, 143.6, 142.3, 139.2, 135.6, 134.6, 133.2, 132.9, 131.4, 131.0, 128.5, 128.3, 125.8, 125.7, 123.9, 119.2, 117.1, 91.0, 59.8, 59.4, 55.5, 47.9, 44.6, 32.5, 31.6, 31.5, 30.6, 23.5, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₇H₃₈N₃O₃ [M + H]⁺: 572.29077; found, 572.28940; HPLC (system 2) t_R = 4.58 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(pyridin-4-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (33).

A procedure similar to that employed for the preparation of 11 was used. Reaction of the bromo compound 60 with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine gave 79 in 55% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.83 (d, J = 2.2 Hz, 1H), 8.70–8.66 (m, 2H), 7.46 (d, J = 2.2 Hz, 1H), 7.44–7.40 (m, 2H), 7.24–7.17 (m, 2H), 7.17–7.05 (m, 6H), 7.01–6.94 (m, 2H), 6.65 (d, J = 8.2 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H), 4.10 (dt, J = 9.6, 6.4 Hz, 1H), 3.99 (dt, J = 9.6, 6.4 Hz, 1H), 3.73 (q, J = 6.3 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.30–3.18 (m, 2H), 2.86 (d, J = 16.6 Hz, 1H), 2.80–2.66 (m, 4H), 2.58 (d, J = 16.5 Hz, 1H), 2.54–2.41 (m, 4H), 2.38–2.26 (m, 1H), 2.03–1.92 (m, 2H), 1.79–1.70 (m, 2H), 1.25 (s, 2H), 0.81 (d, J = 7.3 Hz, 1H), 0.54–0.46 (m, 2H), 0.13 (d, J = 3.8 Hz, 2H); ESI MS m/z: 690.4 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 33 in 30% yield as a white solid. mp 233–234 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.86 (d, J = 2.0 Hz, 1H), 8.65–8.60 (m, 2H), 7.86 (d, J = 2.2 Hz, 1H), 7.73–7.69 (m, 2H), 7.11–7.05 (m, 2H), 7.04–6.98 (m, 1H), 6.96–6.87 (m, 2H), 6.56 (s, 2H), 5.47 (s, 1H), 3.81 (s, 1H), 3.75 (dt, J = 8.5, 5.9 Hz, 1H), 3.40–3.33 (m, 1H), 3.29–3.22 (m, 1H), 3.08 (d, J = 17.0 Hz, 1H), 2.79–2.67 (m, 2H), 2.62–2.30 (m, 8H), 1.81–1.59 (m, 3H), 0.92–0.77 (m, 1H), 0.61–0.42 (m, 2H), 0.26–0.10 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 154.5, 150.6, 146.5, 144.9, 143.5, 142.2, 139.0, 135.8, 133.4, 131.5, 131.0, 128.5, 128.3, 126.0, 125.7, 121.7, 119.3, 117.0, 90.9, 59.8, 59.5, 55.5, 47.9, 44.6, 32.5, 31.6, 31.5, 30.6, 23.5, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₇H₃₈N₃O₃ [M + H]⁺: 572.29077; found, 572.28991; HPLC (system 2) t_R = 3.81 min, purity 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2-methylpyridin-4-yl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (34).

This compound was prepared by the same procedure described for 11. The bromo compound 60 was reacted with 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine to obtain 80 in 88% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.80 (dd, J = 2.3, 0.7 Hz, 1H), 8.56 (dd, J = 5.2, 0.8 Hz, 1H), 7.44 (d, J = 2.2 Hz, 1H), 7.27 (d, J = 1.5 Hz, 1H), 7.23–7.18 (m, 3H), 7.17–7.06 (m, 7H), 7.00–6.95 (m, 2H), 6.65 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H), 4.11 (dt, J = 9.6, 6.3 Hz, 1H), 3.99 (dt, J = 9.6, 6.4 Hz, 1H), 3.73 (dt, J = 8.2, 5.9 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.29–3.17 (m, 2H), 2.85 (d, J = 16.6 Hz, 1H), 2.80–2.66 (m, 5H), 2.62 (s, 3H), 2.58 (d, J = 16.5 Hz, 1H), 2.51–2.41 (m, 5H), 2.39–2.25 (m, 2H), 2.02–1.92 (m, 2H), 0.80 (dd, J = 11.9, 6.8 Hz, 1H), 0.57–0.43 (m, 2H), 0.19–0.08 (m, 2H); ESI MS m/z; 704.3 [M + H]⁺. This intermediate was reacted with boron tribromide in CH₂Cl₂ to obtain 34 in 24% yield as a white solid. mp 198–200 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.40; ¹H NMR (400 MHz, CDCl₃): δ 8.79 (d, J = 2.2 Hz, 1H), 8.56 (dd, J = 5.3, 0.8 Hz, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.30–7.27 (m, 1H), 7.23 (dd, J = 5.4, 1.7 Hz, 1H), 7.17–7.07 (m, 3H), 6.99–6.95 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.62 (s, 1H), 3.73 (dt, J = 8.1, 5.9 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.24 (dt, J = 12.7, 5.6 Hz, 2H), 2.86 (d, J = 16.6 Hz, 1H), 2.79–2.66 (m, 2H), 2.62 (s, 3H), 2.56 (d, J = 16.5 Hz, 1H), 2.51–2.42 (m, 4H), 2.32 (ddd, J = 13.5, 10.8, 7.5 Hz, 2H), 1.78–1.67 (m, 4H), 0.86–0.74 (m, 1H), 0.55–0.44 (m, 2H), 0.17–0.09 (m, 2H); ¹³C NMR (214 MHz, CDCl₃): δ 159.4, 154.3, 150.0, 146.6, 143.5, 142.3, 138.9, 135.8, 133.7, 131.4, 131.0, 128.5, 128.5, 128.4, 128.3, 126.1, 125.7, 121.3, 119.2, 118.9, 117.0, 91.1, 59.9, 59.5, 55.6, 47.9, 44.6, 32.5, 31.6, 31.5, 30.6, 24.7, 23.6, 9.4, 4.2; HRMS (ESI) m/z calcd for C₃₈H₄₀N₃O₃ [M + H]⁺: 586.30642; found, 586.30626; HPLC (system 1) t_R = 11.48 min, purity 96%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(2-methoxypyridin-4-yl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (35).

This compound was prepared using a procedure similar to that described for the preparation of 19. The bromo compound 85 was reacted with 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine to obtain 89 in 97% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.83 (d, J = 2.2 Hz, 1H), 8.23 (dd, J = 5.4, 0.7 Hz, 1H), 7.33–7.28 (m, 2H), 7.25–7.20 (m, 3H), 7.18–7.12 (m, 2H), 7.11–7.06 (m, 1H), 7.04 (dd, J = 5.4, 1.5 Hz, 1H), 7.01–6.96 (m, 2H), 6.90 (dd, J = 1.6, 0.7 Hz, 1H), 6.67 (d, J = 8.2 Hz, 1H), 6.53 (d, J = 8.2 Hz, 1H), 5.61 (s, 1H), 5.20–5.08 (m, 2H), 3.98 (s, 3H), 3.76–3.67 (m, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.28–3.18 (m, 2H), 2.84 (d, J = 16.6 Hz, 1H), 2.73 (td, J = 13.0, 11.9, 5.9 Hz, 2H), 2.55 (d, J = 16.5 Hz, 1H), 2.46 (dt, J = 13.9, 6.8 Hz, 4H), 2.37–2.23 (m, 2H), 1.79–1.66 (m, 4H), 0.86–0.74 (m, 1H), 0.49 (pd, J = 8.9, 7.9, 4.1 Hz, 2H), 0.18–0.04 (m, 2H); ESI MS m/z: 692.3 [M + H]⁺. This intermediate was debenzylated by hydrogenation in the presence of 10% palladium(II) carbon, as in step 4 for the preparation of 19, to obtain the title compound 35 in 63% yield as a white solid. mp 120–122 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.76 (dq, J = 10.2, 3.0 Hz, 1H), 8.21 (dp, J = 4.7, 2.4 Hz, 1H), 7.42 (dd, J = 5.8, 3.0 Hz, 1H), 7.18–7.04 (m, 3H), 7.04–6.94 (m, 3H), 6.87 (dt, J = 4.1, 2.0 Hz, 1H), 6.67 (dt, J = 8.5, 3.0 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.61 (s, 1H), 3.98 (s, 3H), 3.72 (q, J = 6.6 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.15 (m, 2H), 2.84 (dd, J = 16.5, 3.8 Hz, 1H), 2.72 (tt, J = 16.1, 8.9 Hz, 2H), 2.54 (d, J = 15.8 Hz, 1H), 2.45 (dt, J = 12.8, 7.0 Hz, 4H), 2.38–2.23 (m, 2H), 1.71 (dq, J = 20.8, 12.6, 9.7 Hz, 3H), 0.87–0.71 (m, 1H), 0.55–0.42 (m, 2H), 0.12 (h, J = 5.5 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 165.0, 154.3, 147.8, 147.6, 146.4, 143.6, 142.2, 139.1, 135.8, 133.4, 131.3, 131.0, 128.5, 128.2, 125.9, 125.7, 119.2, 117.1, 115.1, 108.7, 90.8, 59.8, 59.4, 55.5, 53.8, 47.9, 44.6, 32.5, 31.5, 31.5, 30.6, 23.5, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₈H₄₀N₃O₄ [M + H]⁺: 602.30133; found, 602.30102; HPLC (system 1) t_R = 14.03 min, purity = 99.2%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(pyrimidin-4-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (36).

This compound was prepared by the two-step procedure similar to that described for the preparation of 8. Thus, reaction of 57³¹ with 3-phenylpropyl bromide in the presence of sodium hydride gave (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-1,8a-bis(3-phenylpropoxy)-11-(pyrimidin-4-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline in 31% yield. ¹H NMR (400 MHz, CDCl₃): δ 9.28 (d, J = 1.3 Hz, 1H), 9.16 (d, J = 1.6 Hz, 1H), 8.80 (d, J = 5.3 Hz, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.70 (dd, J = 5.4, 1.4 Hz, 1H), 7.23–7.05 (m, 7H), 7.02–6.96 (m, 2H), 6.64 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.59 (s, 1H), 4.09 (dt, J = 9.6, 6.3 Hz, 1H), 3.98 (dt, J = 9.6, 6.4 Hz, 1H), 3.72 (dt, J = 8.4, 6.1 Hz, 1H), 3.66 (d, J = 5.9 Hz, 1H), 3.30–3.19 (m, 2H), 2.92 (d, J = 16.6 Hz, 1H), 2.80–2.66 (m, 4H), 2.58 (d, J = 16.6 Hz, 1H), 2.52–2.41 (m, 4H), 2.38–2.24 (m, 2H), 2.01–1.90 (m, 2H), 1.78–1.67 (m, 3H), 1.62–1.58 (m, 1H), 0.85–0.74 (m, 1H), 0.56–0.43 (m, 2H), 0.19–0.07 (m, 2H); ESI MS m/z: 691.3 [M + H]⁺. This intermediate was then reacted with boron tribromide in CH₂Cl₂ to give 36. Yield: 41%; white solid; mp 244–245 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.40; ¹H NMR (400 MHz, CDCl₃): δ 9.29 (d, J = 1.6 Hz, 2H), 9.06 (s, 1H), 8.92 (d, J = 5.4 Hz, 1H), 8.24 (d, J = 2.1 Hz, 1H), 8.15 (dd, J = 5.4, 1.4 Hz, 1H), 7.14–7.07 (m, 2H), 7.06–6.99 (m, 1H), 6.95–6.89 (m, 2H), 6.51 (s, 2H), 5.42 (s, 1H), 3.65 (td, J = 8.3, 7.1, 4.6 Hz, 2H), 3.30–3.22 (m, 1H), 3.09 (dd, J = 32.2, 17.7 Hz, 2H), 2.68 (dd, J = 10.9, 5.1 Hz, 1H), 2.58 (td, J = 12.2, 5.2 Hz, 1H), 2.47–2.27 (m, 6H), 2.18 (td, J = 11.9, 3.6 Hz, 1H), 1.60 (h, J = 6.5 Hz, 2H), 1.49 (d, J = 11.7 Hz, 1H), 0.81–0.69 (m, 1H), 0.51–0.38 (m, 2H), 0.17–0.02 (m, 2H); ¹³C NMR (214 MHz, DMSO): δ 160.5, 158.9, 158.4, 155.8, 146.4, 143.4, 141.8, 139.5, 135.9, 131.2, 131.2, 130.8, 128.2, 128.1, 128.1, 125.5, 125.0, 118.8, 117.8, 117.1, 89.5, 76.3, 59.0, 54.7, 47.1, 44.0, 31.7, 31.2, 30.7, 30.5, 9.2, 3.9, 3.4; HRMS (ESI) m/z calcd for C₃₆H₃₇N₄O₃ [M + H]⁺: 573.28602; found, 573.28417; HPLC (system 2) t_R = 4.99 min, purity 96.4%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(pyrimidin-5-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (37).

This compound was prepared using the same procedure described for 11. The bromo compound 60 (0.25 g, 0.4 mmol) was reacted with 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (0.15 g, 0.7 mmol) in the presence of potassium carbonate (0.15 g, 1.1 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.04 mmol) to give 0.18 g (71%) of 81. ¹H NMR (400 MHz, CDCl₃): δ 9.24 (s, 1H), 8.86 (s, 2H), 8.77 (dd, J = 2.2, 0.7 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.24–7.06 (m, 7H), 7.02–6.97 (m, 2H), 6.65 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H), 4.11 (dt, J = 9.7, 6.4 Hz, 1H), 3.99 (dt, J = 9.6, 6.4 Hz, 1H), 3.74 (dt, J = 7.9, 6.1 Hz, 1H), 3.67 (d, J = 5.8 Hz, 1H), 3.29–3.21 (m, 2H), 2.88 (d, J = 16.6 Hz, 1H), 2.80–2.66 (m, 4H), 2.59 (d, J = 16.6 Hz, 1H), 2.52–2.41 (m, 4H), 2.38–2.25 (m, 2H), 2.01–1.92 (m, 2H), 1.79–1.67 (m, 3H), 1.31 (d, J = 4.7 Hz, 1H), 0.80 (q, J = 6.8 Hz, 1H), 0.55–0.45 (m, 2H), 0.14 (dd, J = 2.9, 1.6 Hz, 2H); ESI MS m/z: 691.4 [M + H]⁺. This intermediate (0.18 g, 0.3 mmol) was reacted with boron tribromide (1.5 mL, 1.5 mmol, 1 M in CH₂Cl₂) to obtain 0.04 g (26%) of 37 as a pale yellow solid. mp 153–154 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 9.27 (s, 1H), 9.10 (s, 1H), 9.01 (s, 2H), 7.49 (s, 1H), 7.19–7.04 (m, 3H), 6.98 (d, J = 7.3 Hz, 2H), 6.68 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.64 (s, 1H), 3.79–3.59 (m, 2H), 3.31–3.19 (m, 2H), 2.89 (d, J = 16.6 Hz, 1H), 2.73 (d, J = 10.2 Hz, 2H), 2.61 (d, J = 16.7 Hz, 1H), 2.48 (t, J = 7.7 Hz, 4H), 2.33 (d, J = 12.7 Hz, 2H), 2.17 (s, 1H), 1.80–1.62 (m, 3H), 0.80 (s, 1H), 0.49 (d, J = 8.1 Hz, 2H), 0.21–0.06 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 158.1, 155.1, 146.5, 143.5, 142.2, 139.1, 135.5, 131.9, 131.2, 130.9, 129.6, 128.5, 128.3, 125.7, 119.4, 117.1, 90.8, 59.8, 59.5, 55.5, 48.0, 44.6, 32.5, 31.6, 30.7, 29.8, 29.5, 23.5, 14.3, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₆H₃₇N₄O₃ [M + H]⁺: 573.28602; found, 573.28623; HPLC(system 2) t_R = 4.83 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(pyrazin-2-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (38).

This compound was prepared by the procedure similar to that described for the preparation of 8. Reaction of 58³¹ with 3-phenylpropyl bromide in the presence of sodium hydride gave (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-1,8a-bis(3-phenylpropoxy)-11-(pyrazin-2-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline in 28% yield. ¹H NMR (400 MHz, CDCl₃): δ 9.16 (d, J = 2.2 Hz, 1H), 8.99 (d, J = 1.6 Hz, 1H), 8.65 (dd, J = 2.5, 1.6 Hz, 1H), 8.56 (d, J = 2.5 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.22–7.05 (m, 8H), 7.02–6.97 (m, 2H), 6.64 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.60 (s, 1H), 4.10 (dt, J = 9.7, 6.3 Hz, 1H), 3.99 (dt, J = 9.7, 6.3 Hz, 1H), 3.76–3.68 (m, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.30–3.22 (m, 2H), 2.98–2.86 (m, 1H), 2.78–2.68 (m, 4H), 2.59 (d, J = 16.6 Hz, 1H), 2.52–2.42 (m, 4H), 2.38–2.24 (m, 2H), 2.02–1.90 (m, 2H), 1.72 (td, J = 12.5, 11.3, 5.4 Hz, 3H), 0.85–0.75 (m, 1H), 0.56–0.44 (m, 2H), 0.19–0.08 (m, 2H); ESI MS m/z: 691.3 [M + H]⁺. This intermediate (0.35 g, 0.5 mmol) was reacted with boron tribromide (3.0 mL, 3.0 mmol, 1 M in CH₂Cl₂) to give 0.11 g (37%) of 38 as a white solid. mp 137–138 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 9.31 (d, J = 2.2 Hz, 1H), 9.11 (d, J = 1.6 Hz, 1H), 8.66 (dd, J = 2.5, 1.5 Hz, 1H), 8.57 (d, J = 2.5 Hz, 1H), 7.98 (d, J = 2.1 Hz, 1H), 7.20–7.11 (m, 2H), 7.12–7.04 (m, 1H), 7.03–6.96 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.69 (s, 1H), 5.65 (s, 1H), 3.72 (dt, J = 8.1, 6.1 Hz, 1H), 3.66 (d, J = 5.8 Hz, 1H), 3.31–3.18 (m, 2H), 2.92 (d, J = 16.6 Hz, 1H), 2.58 (d, J = 16.6 Hz, 1H), 2.53–2.40 (m, 4H), 2.38–2.26 (m, 2H), 1.79–1.61 (m, 5H), 0.79 (q, J = 6.5 Hz, 1H), 0.56–0.43 (m, 2H), 0.19–0.06 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 153.7, 149.2, 148.2, 146.7, 143.6, 142.3, 139.2, 135.6, 134.6, 133.2, 132.9, 131.4, 131.0, 128.5, 128.3, 125.8, 125.7, 123.9, 119.2, 117.1, 91.0, 59.8, 59.4, 55.5, 47.9, 44.6, 32.5, 31.6, 31.5, 30.6, 23.5, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₆H₃₇N₄O₃ [M + H]⁺: 573.28602; found, 573.28543; HPLC (system 2) t_R = 5.19 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-phenylpropoxy)-11-(1H-pyrazol-4-yl)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (39).

This compound was prepared using the procedure described for 11. Reaction of tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate with the bromo compound 60 gave the pyrazole intermediate 82 directly in 30% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.73 (d, J = 2.2 Hz, 1H), 7.84 (d, J = 0.6 Hz, 2H), 7.34–7.32 (m, 1H), 7.22–7.16 (m, 2H), 7.15–7.06 (m, 6H), 7.00–6.96 (m, 2H), 6.67–6.62 (m, 1H), 6.55 (d, J = 8.1 Hz, 1H), 5.56 (s, 1H), 4.10 (dt, J = 9.7, 6.3 Hz, 1H), 3.98 (dt, J = 9.6, 6.4 Hz, 1H), 3.75–3.67 (m, 1H), 3.64 (d, J = 5.8 Hz, 1H), 3.28–3.18 (m, 2H), 2.84–2.65 (m, 5H), 2.57–2.41 (m, 5H), 2.37–2.25 (m, 2H), 2.01–1.89 (m, 2H), 1.79–1.65 (m, 3H), 0.85–0.76 (m, 1H), 0.54–0.44 (m, 2H), 0.14–0.12 (m, 2H); ESI MS m/z: 679.4 [M + H]⁺. This intermediate was then reacted with boron tribromide in CH₂Cl₂ to give 39. Yield: 50%; white solid; mp 223–224 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, DMSO-d₆): δ 13.06 (s, 1H), 9.00 (s, 1H), 8.79 (d, J = 2.1 Hz, 1H), 8.28–8.24 (m, 1H), 7.96 (d, J = 1.8 Hz, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.15–7.09 (m, 2H), 7.08–7.03 (m, 1H), 6.96–6.91 (m, 2H), 6.50 (s, 2H), 5.34 (s, 1H), 3.68–3.59 (m, 2H), 3.23 (q, J = 6.9 Hz, 1H), 3.11 (d, J = 18.5 Hz, 1H), 2.89 (d, J = 16.8 Hz, 1H), 2.67 (dd, J = 11.4, 5.0 Hz, 1H), 2.61–2.52 (m, 1H), 2.48–2.41 (m, 1H), 2.41–2.27 (m, 5H), 2.17 (td, J = 11.8, 3.6 Hz, 1H), 1.61 (dq, J = 13.8, 6.9 Hz, 2H), 1.46 (d, J = 11.3 Hz, 1H), 0.73 (dd, J = 11.9, 5.9 Hz, 1H), 0.51–0.36 (m, 2H), 0.09 (m, 2H); ¹³C NMR (151 MHz, DMSO): δ 150.6, 144.7, 143.4, 141.9, 139.4, 136.5, 133.2, 131.0, 130.8, 128.4, 128.2, 128.1, 126.1, 125.5, 125.0, 118.4, 117.6, 117.0, 89.9, 76.3, 59.0, 58.7, 54.7, 46.9, 44.0, 31.7, 31.3, 30.7, 30.5, 22.9, 9.2, 3.8; HRMS (ESI) m/z calcd for C₃₅H₃₇N₄O₃ [M + H]⁺: 561.28602; found, 561.28488; HPLC (system 1) t_R = 12.06 min, purity = 99.6%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(1-methyl-1H-pyrazol-4-yl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (40).

This compound was prepared according to the procedure described for the preparation of 11. The bromo compound 60 was reacted with 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole to obtain 83 in 53% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.68 (d, J = 2.1 Hz, 1H), 7.71 (s, 1H), 7.60 (s, 1H), 7.29 (d, J = 2.1 Hz, 1H), 7.24–7.18 (m, 2H), 7.18–7.06 (m, 6H), 7.01–6.96 (m, 2H), 6.63 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 8.2 Hz, 1H), 5.55 (s, 1H), 4.10 (dt, J = 9.6, 6.4 Hz, 1H), 4.02–3.97 (m, 1H), 3.95 (s, 3H), 3.70 (dt, J = 8.0, 5.9 Hz, 1H), 3.64 (d, J = 5.8 Hz, 1H), 3.28–3.17 (m, 2H), 2.82–2.67 (m, 5H), 2.56–2.41 (m, 5H), 2.38–2.24 (m, 2H), 2.02–1.92 (m, 2H), 1.79–1.69 (m, 2H), 1.26 (s, 1H), 0.85–0.75 (m, 1H), 0.56–0.44 (m, 2H), 0.12 (dq, J = 4.4, 1.3 Hz, 2H); ESI MS m/z: 693.3 [M + H]⁺. This intermediate was then reacted with boron tribromide in CH₂Cl₂ to obtain 40 in 37% yield as a pale yellow solid. mp 189–190 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.54 (d, J = 9.5 Hz, 1H), 7.68 (d, J = 3.9 Hz, 1H), 7.50 (d, J = 9.1 Hz, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.18–7.04 (m, 3H), 6.97 (d, J = 7.3 Hz, 2H), 6.68 (dd, J = 8.1, 2.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.57 (s, 1H), 3.92 (d, J = 3.5 Hz, 3H), 3.69 (q, J = 6.6 Hz, 1H), 3.62 (d, J = 5.8 Hz, 1H), 3.28–3.15 (m, 2H), 2.81–2.62 (m, 3H), 2.45 (dt, J = 12.9, 6.9 Hz, 6H), 2.36–2.26 (m, 2H), 1.80–1.61 (m, 3H), 0.86–0.72 (m, 1H), 0.48 (dp, J = 9.6, 5.0 Hz, 2H), 0.18–0.02 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 150.9, 144.5, 143.7, 142.3, 139.6, 136.8, 133.6, 131.3, 131.0, 128.5, 128.2, 128.0, 127.2, 125.6, 125.4, 119.1, 119.0, 117.4, 90.4, 76.5, 59.8, 59.3, 55.5, 47.7, 44.6, 39.2, 32.4, 31.5, 31.1, 30.5, 23.5, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₆H₃₉N₄O₃ [M + H]⁺: 575.30167; found, 575.30177; HPLC (system 2) t_R = 4.99 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(3-methylisoxazol-4-yl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (41).

This compound was prepared using a procedure similar to that described for the preparation of 11. The bromo compound 60 was reacted with 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole to obtain 84 in 30% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.60 (d, J = 2.2 Hz, 1H), 8.40 (s, 1H), 7.24–7.08 (m, 9H), 7.01–6.96 (m, 2H), 6.65 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.56 (s, 1H), 4.11 (dt, J = 9.6, 6.4 Hz, 1H), 3.99 (dt, J = 9.7, 6.4 Hz, 1H), 3.74 (dt, J = 8.1, 5.9 Hz, 1H), 3.65 (d, J = 5.9 Hz, 1H), 3.30–3.17 (m, 2H), 2.82 (d, J = 16.5 Hz, 1H), 2.78–2.66 (m, 4H), 2.55 (d, J = 16.5 Hz, 1H), 2.45 (ddt, J = 9.8, 5.9, 3.3 Hz, 4H), 2.37–2.25 (m, 5H), 2.02–1.92 (m, 2H), 1.72 (td, J = 14.3, 6.5 Hz, 3H), 0.80 (q, J = 6.6 Hz, 1H), 0.50 (tt, J = 7.4, 4.0 Hz, 2H), 0.13 (dq, J = 4.0, 1.2 Hz, 2H); ESI MS m/z: 694.4 [M + H]⁺. This intermediate was then reacted with boron tribromide in CH₂Cl₂ to obtain 41 in 40% yield as a white solid. mp 191–192 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.60 (d, J = 2.1 Hz, 1H), 8.44 (s, 1H), 7.26–7.24 (m, 1H), 7.19–7.09 (m, 3H), 7.02–6.95 (m, 2H), 6.67 (dd, J = 8.1, 0.7 Hz, 1H), 6.60–6.55 (m, 1H), 5.59 (s, 1H), 3.74 (dt, J = 8.1, 5.8 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.29–3.17 (m, 2H), 2.86–2.66 (m, 3H), 2.54 (d, J = 16.6 Hz, 1H), 2.45 (dt, J = 11.6, 6.3 Hz, 4H), 2.37–2.28 (m, 5H), 1.80–1.65 (m, 4H), 0.87–0.69 (m, 1H), 0.49 (dp, J = 9.2, 4.9 Hz, 2H), 0.12 (t, J = 5.8 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 157.4, 155.7, 153.3, 147.1, 143.4, 142.3, 138.9, 136.5, 131.4, 131.0, 128.5, 128.3, 126.1, 125.8, 125.2, 119.3, 117.5, 116.9, 91.0, 59.8, 59.6, 55.5, 47.9, 44.6, 32.6, 31.7, 31.5, 30.6, 23.5, 10.9, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₆H₃₈N₃O₃ [M + H]⁺: 576.28568; found, 576.28601; HPLC (system 2) t_R = 6.28 min, purity = 96.8%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-(5-methyl-1,3,4-oxadiazol-2-yl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (42).

Step 1.

A mixture of 3-O-benzylnaltrexone⁵⁶ (10 g, 23.2 mmol), ethyl 2-formyl-3-oxopropanoate (4.0 g, 27.8 mmol), and ammonium acetate (5.4 g, 69.5 mmol) in glacial acetic acid (45 mL) was stirred under reflux at 110 °C for 18 h. After cooling, the reaction mixture was concentrated under reduced pressure, the residue was suspended in water (40 mL), and the pH of the mixture was adjusted to 7 with concentrated aqueous NH₄OH. The resulting suspension was extracted with CHCl₃ (3 × 80 mL) and washed with water (160 mL). The extract was dried, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography over a column of silica using hexanes/EtOAc (40:60) as the eluent to yield 90 (0.9 g, 7.3%). ¹H NMR (400 MHz, CDCl₃): δ 9.16 (d, J = 2.1 Hz, 1H), 7.98 (d, J = 2.0 Hz, 1H), 7.32–7.28 (m, 2H), 7.25–7.24 (m, 1H), 7.24–7.21 (m, 2H), 6.68 (d, J = 8.1 Hz, 1H), 6.56 (dd, J = 8.2, 0.8 Hz, 1H), 5.56 (s, 1H), 5.17–5.07 (m, 2H), 4.38 (qd, J = 7.2, 0.7 Hz, 2H), 3.34 (s, 1H), 3.16 (d, J = 18.7 Hz, 1H), 2.81–2.64 (m, 4H), 2.57–2.16 (m, 5H), 1.88–1.79 (m, 1H), 1.38 (t, J = 7.1 Hz, 3H), 0.90 (s, 1H), 0.61–0.53 (m, 2H), 0.17 (d, J = 5.1 Hz, 2H). ESI MS m/z: 539.2 [M + H]⁺.

Step 2.

A mixture of the abovementioned intermediate 90 (0.9 g, 1.7 mmol) and hydrazine hydrate (1.1 g, 16.9 mmol) in ethanol (10 mL) was heated at 80 °C for overnight. The solvent was evaporated to obtain (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-8a-hydroxy-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline-11-carbohydrazide ESI MS m/z: 525.2 [M + H]⁺. The crude product was used as such in the next step.

Step 3.

The abovementioned intermediate (0.9 g, 1.7 mmol) and 1,1,1-triethoxyethane (0.8 g, 5.1 mmol) in acetic acid (15 mL) were heated at 150 °C for 3 h. The reaction mixture was cooled down to room temperature, and the solvent was removed under vacuum. The residue was neutralized with NH₄OH, extracted with CHCl₃ (3 × 80 mL), and washed with water (20 mL). The extract was dried, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography over a column of silica using hexanes/EtOAc (40:60) as the eluent to yield 91 (0.6 g, 61%). ¹H NMR (400 MHz, CDCl₃): δ 9.17 (d, J = 1.8 Hz, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.34–7.28 (m, 2H), 7.27–7.19 (m, 3H), 6.70 (dd, J = 8.2, 1.2 Hz, 1H), 6.57 (dd, J = 8.2, 1.0 Hz, 1H), 5.58 (d, J = 0.9 Hz, 1H), 5.31–4.98 (m, 2H), 3.17 (d, J = 18.8 Hz, 1H), 2.78 (d, J = 15.3 Hz, 3H), 2.63 (s, 3H), 2.41 (d, J = 33.9 Hz, 2H), 1.85 (d, J = 12.2 Hz, 1H), 1.59 (s, 4H), 0.92 (s, 1H), 0.59 (s, 2H), 0.19 (s, 2H). ESI MS m/z: 549.2 [M + H]⁺.

Step 4.

The abovementioned intermediate 91 (0.5 g, 0.9 mmol) was reacted with 3-phenylpropyl bromide (0.5 g, 2.7 mmol) in the presence of sodium hydride (0.15 g, 3.7 mmol, 60% dispersion in mineral oil) to obtain (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-(5-methyl-1,3,4-oxadiazol-2-yl)-8a-(3-phenylpropoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (0.23 g, 38%). ¹H NMR (400 MHz, CDCl₃): δ 9.15 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 2.1 Hz, 1H), 7.33–7.28 (m, 2H), 7.24 (dtt, J = 7.2, 5.0, 1.9 Hz, 3H), 7.20–7.15 (m, 2H), 7.13–7.05 (m, 1H), 7.03–6.97 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 8.1 Hz, 1H), 5.58 (s, 1H), 5.16–5.03 (m, 2H), 3.76–3.57 (m, 2H), 3.26–3.11 (m, 2H), 2.99–2.82 (m, 1H), 2.78–2.21 (m, 11H), 1.77–1.65 (m, 2H), 1.63 (s, 2H), 0.85–0.73 (m, 1H), 0.49 (dq, J = 8.5, 4.4 Hz, 2H), 0.12 (p, J = 1.9 Hz, 2H); ESI MS m/z: 667.3 [M + H]⁺.

Step 5.

The abovementioned intermediate (0.23 g, 0.3 mmol) was debenzylated with 10% palladium(II) carbon (23 mg, 10 wt %) under a H₂ atmosphere to give 0.13 g (63%) of the desired compound 42 as a white solid. mp 128–130 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.60; ¹H NMR (400 MHz, CDCl₃): δ 9.09 (dt, J = 4.7, 2.2 Hz, 1H), 7.95 (s, 1H), 7.17 (ddd, J = 7.5, 6.4, 1.4 Hz, 2H), 7.12–7.06 (m, 1H), 7.03–6.97 (m, 2H), 6.67 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.57 (d, J = 1.8 Hz, 1H), 3.72 (dt, J = 8.3, 6.1 Hz, 1H), 3.65 (d, J = 5.8 Hz, 1H), 3.30–3.18 (m, 2H), 2.87 (dd, J = 16.7, 2.0 Hz, 1H), 2.79–2.66 (m, 2H), 2.64 (s, 3H), 2.57–2.39 (m, 5H), 2.39–2.21 (m, 2H), 1.70 (tdd, J = 11.7, 9.2, 5.0 Hz, 3H), 0.84–0.72 (m, 1H), 0.58–0.42 (m, 2H), 0.18–0.05 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 164.3, 162.7, 156.9, 145.8, 142.2, 135.4, 131.8, 131.8, 130.8, 128.5, 128.3, 125.7, 119.8, 119.4, 117.3, 90.6, 76.5, 59.8, 59.5, 55.4, 47.9, 47.9, 44.6, 32.5, 31.6, 31.3, 30.5, 23.5, 11.3, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₅H₃₇N₄O₄ [M + H]⁺: 577.28093; found, 577.27998; HPLC (system 1) t_R = 13.17 min, purity 98.6%.

(4bS,8R,8aS,13bR)-11-(4-Chlorophenyl)-7-(cyclopropylmethyl)-8a-(3-(pyridin-4-yl)propoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (43).

Step 1.

Compound 7²⁹ (2 g, 4.1 mmol) was reacted with benzyl bromide (0.6 mL, 4.9 mmol) in the presence of potassium carbonate (1.1 g, 8.2 mmol) to obtain 92 (1.3 g, 56%). ¹H NMR (400 MHz, CDCl₃): δ 8.38 (td, J = 7.0, 6.6, 2.2 Hz, 1H), 7.85–7.76 (m, 1H), 7.30 (dq, J = 6.1, 3.6, 3.2 Hz, 3H), 7.21 (dd, J = 16.3, 8.6 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 7.01–6.88 (m, 2H), 5.77 (d, J = 6.4 Hz, 2H), 4.79 (d, J = 43.5 Hz, 2H), 3.81 (dt, J = 27.1, 6.1 Hz, 2H), 3.05 (t, J = 6.0 Hz, 1H), 2.88 (t, J = 6.2 Hz, 3H), 2.76 (t, J = 5.1 Hz, 2H), 1.68 (dp, J = 40.9, 5.6 Hz, 5H), 1.48 (d, J = 6.3 Hz, 1H); ESI MS m/z: 577.2 [M + H]⁺.

Step 2.

The abovementioned intermediate 92 (7.0 g, 12.1 mmol) was reacted with 3-bromoprop-1-ene (2.9 g, 24.3 mmol) in the presence of sodium hydride (2.9 g, 72.8 mmol, 60% dispersion in mineral oil) to obtain 93 (3.6 g, 48%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.79 (d, J = 2.3 Hz, 1H), 7.77–7.66 (m, 2H), 7.53 (dt, J = 9.0, 1.6 Hz, 1H), 7.25 (ddt, J = 5.1, 4.1, 1.3 Hz, 2H), 6.73 (dd, J = 8.2, 0.9 Hz, 1H), 6.60 (d, J = 8.2 Hz, 1H), 5.41 (s, 1H), 5.10–4.83 (m, 2H), 4.16 (dd, J = 13.0, 5.0 Hz, 1H), 3.87 (dd, J = 12.7, 5.1 Hz, 1H), 3.74–3.64 (m, 1H), 3.46–3.33 (m, 1H), 3.39–3.19 (m, 2H), 3.32 (s, 9H), 3.21–2.96 (m, 1H), 2.66 (dd, J = 11.8, 4.2 Hz, 1H), 2.61–2.44 (m, 3H), 2.42–2.30 (m, 2H), 2.14 (dd, J = 12.5, 8.9 Hz, 1H), 1.47 (d, J = 12.0 Hz, 1H), 0.47 (t, J = 7.8 Hz, 1H), 0.11 (s, 1H).

Step 3.

A mixture of the abovementioned intermediate 93 (0.28 g, 0.5 mmol), 4-bromopyridine hydrochloride (0.09 g, 0.54 mmol), potassium carbonate (0.14 g, 1.0 mmol), tetrakis(triphenylphosphine)palladium(0) (0.005 g, 0.005 mmol), and diacetoxypalladium (0.001 g, 0.005 mmol) in DMF (8 mL) was heated at 130 °C for 1 h under an atmosphere of argon. The mixture was allowed to cool down to room temperature and was diluted with water. The aqueous layer was extracted with ethyl acetate (3 × 20 mL). Organic layers were dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with CHCl₃/MeOH (95:5) to obtain 0.19 g (59%) of 94. The crude product was used as such in the next step.

Step 4.

The abovementioned intermediate 94 (0.16 g, 0.2 mmol) was treated with 10% palladium(II) carbon (16.0 mg, 10 wt %) under a H₂ atmosphere at 40 psi to give 95 which was used for the next step without purification.

Step 5.

The abovementioned intermediate 95 (0.15 g, 0.2 mmol) was refluxed in 2,2,2-trifluoroacetic acid (3 mL). The mixture was cooled to room temperature, and the acid was removed under reduced pressure. The residue was dissolved in water and neutralized with aqueous NH₄OH. The resulting suspension was extracted with EtOAc (3 × 10 mL) and washed with water (10 mL). The extract was dried, and the solvent was removed under reduced pressure. The residue was purified by chromatography over a column of silica gel using CHCl₃/MeOH (95:5) as the eluent to obtain 45 mg (35%) of 43 as a white solid. mp 150–154 °C; TLC (7.5% MeOH/CHCl₃): R_f = 0.63; ¹H NMR (400 MHz, DMSO-d₆): δ 9.02 (s, 1H), 8.80 (d, J = 2.3 Hz, 1H), 8.33–8.22 (m, 2H), 7.76–7.65 (m, 3H), 7.62–7.42 (m, 2H), 6.95–6.88 (m, 2H), 6.49 (s, 2H), 5.36 (s, 1H), 3.61 (dd, J = 10.0, 5.6 Hz, 2H), 3.29–3.20 (m, 1H), 3.10 (d, J = 18.5 Hz, 1H), 2.97 (d, J = 16.9 Hz, 1H), 2.64 (dt, J = 16.6, 8.4 Hz, 1H), 2.59–2.23 (m, 6H), 2.15 (dd, J = 12.9, 9.4 Hz, 1H), 1.71–1.53 (m, 1H), 1.61 (s, 1H), 1.46 (d, J = 11.8 Hz, 1H), 0.82 (s, 1H), 0.42 (d, J = 7.9 Hz, 3H), 0.09 (t, J = 13.4 Hz, 3H); HRMS (ESI) m/z calcd for C₃₇H₃₇ClN₃O₃ [M + H]⁺: 606.25180; found, 606.24893; HPLC (system 2) t_R = 3.73 min, purity 96.2%.

(4bS,8R,8aS,13bR)-11-(4-Chlorophenyl)-7-(cyclopropylmethyl)-8a-(2-phenoxyethoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (44).

To a solution of 2-phenoxyethanol (2.0 g, 14.5 mmol) in CH₂Cl₂ (200 mL) and anhydrous triethylamine (2.2 g, 21.7 mmol) cooled to −30 °C under argon was added slowly trifluoromethanesulfonic anhydride (4.90 g, 17.37 mmol). The solution was stirred at −30 °C for 1 h. The solution was diluted with ethyl acetate and washed with 20% HCl, saturated aqueous NaHCO₃, and brine. Organic layers were collected and dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure to obtain a red viscous oil. The crude 2-phenoxyethyl trifluoromethanesulfonate thus obtained (0.41 g, 1.5 mmol) was added to a solution of 7²⁹ (0.3 g, 0.8 mmol) in nitromethane (2.5 mL). To the reaction mixture was added 1,2,2,6,6-pentamethylpiperidine (0.19 g, 1.2 mmol), and the reaction mixture was heated at 50 °C for 1.5 h. The solvent was removed under reduced pressure. The residue was treated with 10% HCl and then neutralized with aqueous NH₄OH. The mixture was diluted with water and extracted with CHCl₃ (3 × 20 mL). Organic layers were dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with CHCl₃/MeOH (95:5) to obtain 0.35 g (81%) of 96. ¹H NMR (400 MHz, DMSO-d₆): δ 9.51 (d, J = 2.0 Hz, 1H), 8.74 (d, J = 1.9 Hz, 1H), 7.94–7.84 (m, 2H), 7.74–7.64 (m, 2H), 7.30–7.18 (m, 4H), 7.00–6.80 (m, 8H), 6.78 (d, J = 8.3 Hz, 1H), 6.40 (s, 1H), 5.41 (td, J = 9.5, 8.9, 4.7 Hz, 1H), 5.29 (d, J = 14.2 Hz, 1H), 5.18 (s, 1H), 4.70 (s, 1H), 4.76–4.60 (m, 1H), 4.38–4.20 (m, 2H), 4.23–4.10 (m, 1H), 4.16 (s, 2H), 3.39 (d, J = 6.2 Hz, 1H), 3.19 (d, J = 19.0 Hz, 1H), 3.03 (d, J = 17.0 Hz, 1H), 2.82–2.65 (m, 3H), 2.43 (d, J = 6.5 Hz, 3H), 2.25–2.13 (m, 1H), 1.70 (d, J = 12.4 Hz, 1H), 0.51 (dd, J = 8.8, 6.7 Hz, 2H), 0.16 (d, J = 4.9 Hz, 2H); ESI MS m/z: 727.3 [M + H]⁺. Phenolic-O-delalkylation of this intermediate (0.20 g, 0.3 mmol) with boron tribromide (0.35 g, 1.4 mmol) and the usual workup gave 0.1 g (59%) of 44 as a white solid. mp 156–158 °C; TLC (7.5% MeOH/CH₂Cl₂): R_f = 0.14; ¹H NMR (400 MHz, DMSO-d₆): δ 9.52 (d, J = 1.9 Hz, 1H), 9.39 (s, 1H), 8.73 (d, J = 1.9 Hz, 1H), 7.96–7.84 (m, 2H), 7.74–7.62 (m, 2H), 7.33–7.22 (m, 2H), 6.99–6.88 (m, 3H), 6.61 (q, J = 8.1 Hz, 2H), 6.34 (s, 1H), 5.44 (ddd, J = 13.7, 9.2, 4.0 Hz, 1H), 5.30 (d, J = 14.1 Hz, 1H), 5.14 (s, 1H), 4.79–4.63 (m, 1H), 3.48–3.32 (m, 2H), 3.19–3.07 (m, 1H), 3.01 (d, J = 17.0 Hz, 1H), 2.75 (d, J = 16.5 Hz, 2H), 2.65 (dd, J = 18.8, 6.4 Hz, 1H), 2.41 (d, J = 6.6 Hz, 3H), 2.25–2.13 (m, 1H), 1.68 (d, J = 11.9 Hz, 1H), 0.49 (q, J = 9.4, 8.6 Hz, 2H), 0.15 (d, J = 4.9 Hz, 2H); HRMS (ESI) m/z calcd for C₃₇H₃₆ClN₂O₄ [M + H]⁺: 607.23581; found, 607.23475; HPLC (system 2) t_R = 3.74 min, purity 96.8%.

(4bS,8R,8aS,13bR)-11-(4-Chlorophenyl)-7-(cyclopropylmethyl)-8a-(quinolin-2-ylmethoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (45).

Step 1.

Compound 7²⁹ (0.29 g, 0.6 mmol) was reacted with 2-(chloromethyl)quinoline (0.22 g, 1.3 mmol) in the presence of sodium hydride (0.14 g, 3.4 mmol, 60% dispersion in mineral oil) to obtain 0.2 g (46%) of 97. ¹H NMR (400 MHz, DMSO-d₆): δ 9.59 (s, 1H), 9.52 (s, 1H), 9.39 (s, 1H), 8.74 (s, 1H), 8.29 (d, J = 1.0 Hz, 1H), 8.00–7.91 (m, 1H), 7.94–7.84 (m, 2H), 7.77–7.60 (m, 2H), 7.27 (dd, J = 8.5, 7.4 Hz, 2H), 7.06–6.84 (m, 4H), 6.62 (p, J = 8.0 Hz, 2H), 6.34 (s, 1H), 5.44 (dd, J = 13.1, 8.5 Hz, 1H), 5.31 (d, J = 14.6 Hz, 1H), 5.14 (s, 1H), 4.74 (s, 1H), 3.49–3.20 (m, 3H), 3.18–3.07 (m, 1H), 3.02 (dd, J = 18.2, 9.1 Hz, 1H), 2.75 (d, J = 16.6 Hz, 2H), 2.71–2.61 (m, 1H), 2.65–2.49 (m, 1H), 2.51–2.32 (m, 4H), 2.21 (d, J = 11.3 Hz, 1H), 1.68 (d, J = 12.3 Hz, 1H), 0.92 (s, 1H), 0.50 (t, J = 7.9 Hz, 2H), 0.15 (d, J = 5.0 Hz, 2H); ESI MS m/z: 769.3 [M + H]⁺.

Step 2.

The abovementioned intermediate 97 (0.28 g, 0.4 mmol) was reacted with boron tribromide (0.55 g, 2.2 mmol), and following the usual workup and purification, 0.01 g (5%) of 45 was obtained as a white solid. mp 152–154 °C; TLC (7.5% MeOH/CHCl₃): R_f = 0.37; ¹H NMR (400 MHz, DMSO-d₆): δ 9.05 (s, 1H), 8.75 (d, J = 2.2 Hz, 1H), 8.14 (d, J = 8.6 Hz, 1H), 7.83 (dd, J = 10.2, 8.5 Hz, 2H), 7.76–7.55 (m, 2H), 7.53–7.43 (m, 4H), 6.58–6.47 (m, 2H), 5.42 (d, J = 1.2 Hz, 1H), 4.94 (d, J = 12.6 Hz, 1H), 4.57 (d, J = 12.6 Hz, 1H), 3.90 (d, J = 5.8 Hz, 1H), 3.30–3.09 (m, 3H), 2.80 (dd, J = 11.5, 4.9 Hz, 1H), 2.72–2.31 (m, 6H), 2.31–2.19 (m, 1H), 1.56 (d, J = 11.1 Hz, 1H), 0.84 (s, 1H), 0.49–0.38 (m, 1H), 0.42 (s, 1H), 0.17–0.03 (m, 2H); HRMS (ESI) m/z calcd for C₃₉H₃₅ClN₃O₃ [M + H]⁺: 629.23937; found, 629.23736; HPLC (system 3) t_R = 2.26 min, purity 95.9%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-phenyl-8a-(3-(pyridin-3-yl)propoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (46).

This compound was prepared by a procedure similar to that employed for the preparation of 43.

Step 1.

Compound 54²⁹ (5 g, 11.1 mmol) was reacted with benzyl bromide (1.6 mL, 13.3 mmol) in the presence of potassium carbonate (3.1 g, 22.1 mmol) to obtain 1.7 g (29%) of 98. ¹H NMR (400 MHz, CDCl₃): δ 8.85–8.76 (m, 1H), 7.53 (d, J = 7.2 Hz, 3H), 7.45 (t, J = 7.7 Hz, 2H), 7.41–7.35 (m, 1H), 7.33–7.28 (m, 2H), 7.27–7.20 (m, 3H), 6.69 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H), 5.62 (s, 1H), 5.23–5.09 (m, 2H), 4.95 (s, 1H), 3.32 (d, J = 6.4 Hz, 1H), 3.16 (d, J = 18.7 Hz, 1H), 2.85–2.63 (m, 4H), 2.52–2.30 (m, 4H), 1.85 (d, J = 12.6 Hz, 1H), 0.99–0.83 (m, 1H), 0.64–0.50 (m, 2H), 0.25–0.14 (m, 2H); ESI MS m/z: 543.3 [M + H]⁺.

Step 2.

The abovementioned intermediate 98 (1.5 g, 2.8 mmol) was reacted with 3-bromoprop-1-ene (1.0 mL, 11.1 mmol) in the presence of sodium hydride (0.7 g, 16.6 mmol, 60% dispersion in mineral oil) to obtain (4bS,8R,8aS,13bR)-8a-(allyloxy)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-phenyl-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (1.6 g, 99%). ¹H NMR (400 MHz, CDCl₃): δ 8.81 (d, J = 2.1 Hz, 1H), 7.53 (d, J = 7.6 Hz, 2H), 7.48–7.42 (m, 3H), 7.40–7.29 (m, 2H), 7.27–7.16 (m, 3H), 6.68 (dd, J = 8.2, 1.5 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 5.85–5.71 (m, 1H), 5.65 (s, 1H), 5.24–5.09 (m, 2H), 5.05 (dt, J = 17.2, 1.9 Hz, 1H), 4.96 (dd, J = 10.4, 2.0 Hz, 1H), 4.31–4.22 (m, 1H), 3.95–3.86 (m, 1H), 3.71 (d, J = 5.7 Hz, 1H), 3.24 (d, J = 18.5 Hz, 1H), 2.95–2.88 (m, 1H), 2.86 (d, J = 4.1 Hz, 1H), 2.73 (tt, J = 12.0, 5.1 Hz, 2H), 2.60 (d, J = 16.5 Hz, 1H), 2.49 (dt, J = 19.0, 6.5 Hz, 2H), 2.41–2.26 (m, 2H), 1.79–1.63 (m, 1H), 0.91 (q, J = 6.6 Hz, 1H), 0.55 (p, J = 9.1 Hz, 2H), 0.18 (d, J = 4.8 Hz, 2H); ESI MS m/z: 583.3 [M + H]⁺.

Step 3.

The abovementioned intermediate (0.4 g, 0.7 mmol) was reacted with 3-bromopyridine (0.11 g, 0.7 mmol), potassium carbonate (0.1 g, 0.7 mmol), tetrakis(triphenylphosphine)palladium(0) (0.008 g, 0.007 mmol), and diacetoxypalladium (0.002 g, 0.007 mmol) in DMF (8 mL) at 130 °C for 1 h. Workup of the reaction mixture and purification by column chromatography on silica gel eluting with CHCl₃/MeOH (95:5) gave 0.10 g (22%) of (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-phenyl-8a-(((E)-3-(pyridin-3-yl)allyl)oxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline. ¹H NMR (400 MHz, DMSO-d₆): δ 8.84–8.72 (m, 2H), 8.38–8.27 (m, 2H), 8.24–8.15 (m, 1H), 7.75–7.57 (m, 5H), 7.60–7.35 (m, 5H), 7.29–7.20 (m, 9H), 7.24–7.10 (m, 1H), 6.99 (dd, J = 7.9, 4.7 Hz, 1H), 6.80–6.71 (m, 1H), 6.62 (dd, J = 8.2, 6.4 Hz, 1H), 6.56–6.48 (m, 1H), 5.53 (d, J = 3.1 Hz, 1H), 5.45 (s, 1H), 5.08–4.90 (m, 3H), 4.66–4.54 (m, 1H), 3.87 (d, J = 5.8 Hz, 1H), 3.80 (s, 1H), 3.25–3.12 (m, 3H), 3.08–2.91 (m, 2H), 2.69 (d, J = 12.7 Hz, 1H), 2.56 (s, 1H), 2.53 (d, J = 7.9 Hz, 1H), 2.45–2.25 (m, 2H), 2.26–2.14 (m, 1H), 1.65–1.43 (m, 2H), 0.95 (s, 1H), 0.78 (s, 1H), 0.66 (d, J = 6.3 Hz, 1H), 0.50–0.37 (m, 2H), 0.11 (td, J = 9.2, 8.8, 4.6 Hz, 1H), 0.07 (s, 3H).

Step 4.

The abovementioned intermediate (0.26 g, 0.4 mmol) was treated with 10% palladium(II) carbon (50.0 mg, 10 wt %) under a H₂ atmosphere at 40 psi to give 0.06 g (25%) of 46 as a white solid. mp 126–130 °C; TLC (7.5% MeOH/CHCl₃): R_f = 0.47; ¹H NMR (400 MHz, DMSO-d₆): δ 9.02 (s, 1H), 8.78 (d, J = 2.2 Hz, 1H), 8.32–8.23 (m, 3H), 8.19 (d, J = 2.2 Hz, 1H), 7.71 (d, J = 2.2 Hz, 1H), 7.72–7.61 (m, 2H), 7.54–7.43 (m, 2H), 7.48–7.29 (m, 3H), 7.12 (ddd, J = 7.7, 4.7, 0.9 Hz, 1H), 6.48 (d, J = 7.7 Hz, 2H), 5.37 (s, 1H), 3.62 (t, J = 7.4 Hz, 2H), 3.10 (d, J = 18.5 Hz, 1H), 2.99 (d, J = 16.9 Hz, 1H), 2.65 (dd, J = 11.2, 5.1 Hz, 1H), 2.54 (td, J = 12.0, 5.0 Hz, 1H), 2.49–2.22 (m, 6H), 2.21–2.10 (m, 1H), 1.61 (dp, J = 13.4, 6.8 Hz, 2H), 1.45 (t, J = 11.4 Hz, 1H), 0.51–0.33 (m, 2H), 0.10 (dd, J = 10.1, 4.7 Hz, 1H); HRMS (ESI) m/z calcd for C₃₇H₃₈N₃O₃ [M + H]⁺: 572.29077; found, 572.29104; HPLC (system 2) t_R = 3.51 min, purity 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-phenyl-8a-(3-(pyridin-4-yl)propoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (47).

The allyloxy intermediate obtained in step 2 in the preparation of 46 (0.4 g, 0.7 mmol) was reacted with 4-bromopyridine hydrochloride (0.13 g, 0.8 mmol) in the presence of potassium carbonate (0.29 g, 2.0 mmol), tetrakis(triphenylphosphine)palladium(0) (0.008 g, 0.007 mmol), and diacetoxypalladium (0.002 g, 0.007 mmol) in DMF (8 mL) to obtain 0.12 g (27%) of (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-phenyl-8a-(((E)-3-(pyridin-4-yl)allyl)oxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline. This intermediate (0.12 g, 0.2 mmol) was treated with 10% palladium(II) carbon (20.0 mg, 10 wt %) under a H₂ atmosphere at 40 psi to obtain 0.06 g (54%) of 47 as a white solid. mp 136–138 °C; TLC (7.5% MeOH/CHCl₃): R_f = 0.30; ¹H NMR (400 MHz, DMSO-d₆): δ 9.02 (s, 1H), 8.80 (d, J = 2.2 Hz, 1H), 8.30 (d, J = 1.1 Hz, 1H), 8.29–8.22 (m, 2H), 7.71 (d, J = 2.2 Hz, 1H), 7.70–7.62 (m, 2H), 7.47 (t, J = 7.5 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), 6.92 (d, J = 5.6 Hz, 2H), 6.49 (s, 2H), 5.36 (s, 1H), 3.63 (d, J = 6.1 Hz, 2H), 3.11 (d, J = 18.5 Hz, 1H), 2.98 (d, J = 16.9 Hz, 1H), 2.65 (d, J = 11.6 Hz, 2H), 2.54 (dd, J = 12.2, 5.0 Hz, 1H), 2.46–2.23 (m, 5H), 2.16 (dd, J = 12.9, 9.4 Hz, 1H), 1.61 (dt, J = 14.5, 7.4 Hz, 2H), 1.46 (d, J = 11.9 Hz, 1H), 1.21 (s, 1H), 0.42 (d, J = 8.2 Hz, 2H), 0.11 (d, J = 10.9 Hz, 1H), 0.05 (s, 1H); HRMS (ESI) m/z calcd for C₃₇H₃₈N₃O₃ [M + H]⁺: 572.29077; found, 572.29104; HPLC (system 2) t_R = 2.97 min, purity 97%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-8a-(3-(4-fluorophenyl)propoxy)-11-phenyl-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (48).

Step 1.

Compound 54²⁹ (0.55 g, 1.22 mmol) was dissolved in anhydrous DMF (8 mL) under an argon atmosphere, and the mixture was cooled to 0 °C. Sodium hydride (0.292 g, 7.29 mmol) was added, and the mixture was stirred at 0 °C for 5 min. The reaction mixture was allowed to warm to room temperature and stirred at room temperature for 20 min. The reaction mixture was cooled again to 0 °C, and 1-(3-bromopropyl)-4-fluorobenzene (1.055 g, 4.86 mmol) was added. Ice bath was removed, and the reaction mixture was allowed to attain room temperature and stirred for another 5 h. The reaction mixture was then quenched by adding ice-cold water, and the mixture was extracted with CH₂Cl₂ (3 × 60 mL) and washed with brine (30 mL). The organic layer was dried over anhydrous Na₂SO₄ and filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel using hexanes/EtOAc as the eluent to obtain 0.175 g (19.9%) of (4bS,8R,8aS,13bR)-7-(cyclopropylmethyl)-1,8a-bis(3-(4-fluorophenyl)propoxy)-11-phenyl-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline. ESI-MS m/z: 725.3 [M + H]⁺.

Step 2.

A solution of the abovementioned intermediate (0. 170 g, 0.23 mmol) in anhydrous CH₂Cl₂ (12 mL) was cooled to −78 °C and treated dropwise with 1 M solution of boron tribromide (2.35 mL, 2.35 mmol). The mixture was allowed to warm up to 0 °C and stirred for 1 h. Workup of the reaction mixture gave 19 mg (14%) of 48 as a light brown waxy solid. TLC (10% MeOH/CH₂Cl₂): R_f = 0.25; ¹H NMR (400 MHz, CD₃OD): δ 8.76–8.74 (m, 1H), 7.72 (d, J = 2.2 Hz, 1H), 7.62 (d, J = 1.5 Hz, 1H), 7.60 (t, J = 1.4 Hz, 1H), 7.51–7.46 (m, 2H), 7.45–7.40 (m, 1H), 6.92 (dd, J = 8.6, 5.5 Hz, 2H), 6.83–6.77 (m, 2H), 6.59 (s, 2H), 5.49 (d, J = 9.5 Hz, 1H), 3.73 (d, J = 8.0 Hz, 1H), 3.07 (d, J = 17.6 Hz, 2H), 2.77–2.67 (m, 3H), 2.59 (d, J = 17.0 Hz, 2H), 2.42 (t, J = 7.7 Hz, 3H), 1.74 (d, J = 8.3 Hz, 4H), 1.25 (t, J = 7.2 Hz, 2H), 0.89 (d, J = 18.6 Hz, 2H), 0.55 (s, 2H), 0.22 (s, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 161.9, 160.3, 150.0, 146.7, 143.5, 137.8, 137.3, 135.8, 134.7, 129.8, 129.7, 128.4, 127.2, 119.2, 116.9, 114.9, 114.8, 111.4, 91.1, 76.9, 59.8, 59.1, 55.5, 44.6, 31.6, 31.4, 29.8, 9.4, 4.2, 3.6; HRMS (ESI) m/z calcd for C₃₈H₃₈FN₂O₃ [M + H]⁺: 589.2861; found, 589.28675; HPLC (system 2) t_R = 7.11 min, purity = 100%.

(4bS,8R,8aS,13bR)-8a-(3-Cyclohexylpropoxy)-7-(cyclopropylmethyl)-11-phenyl-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (49).

The benzyl compound 98, described in step 1 in the preparation of 46 (0.580 g, 1.07 mmol), was reacted with (3-bromopropyl)cyclohexane (0.438 g, 2.14 mmol) in anhydrous DMF (8 mL) in the presence of sodium hydride (0.128 g, 3.21 mmol). Workup and purification, as described in step 1 in the preparation of 48, gave 0.40 g (56%) of (4bS,8R,8aS,13bR)-1-(benzyloxy)-8a-(3-cyclohexylpropoxy)-7-(cyclopropylmethyl)-11-phenyl-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline (99). ¹H NMR (400 MHz, CDCl₃): δ 8.84–8.77 (m, 1H), 7.58–7.49 (m, 1H), 7.50–7.33 (m, 3H), 7.34–7.15 (m, 4H), 6.66 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 5.60 (s, 1H), 5.23–5.07 (m, 1H), 3.67 (dd, J = 14.6, 7.1 Hz, 1H), 3.27–3.16 (m, 1H), 2.86 (d, J = 16.4 Hz, 1H), 2.79–2.62 (m, 1H), 2.60–2.22 (m, 3H), 1.63–1.45 (m, 4H), 1.54 (m, 7H), 1.38 (s, 1H), 1.10–1.02 (m, 4H), 0.98–0.85 (m, 1H), 0.90 (s, 1H), 0.73–0.62 (m, 1H), 0.53 (m, 1H), 0.21–0.02 (m, 6H), 0.02 to −0.14 (m, 4H); ESI-MS m/z: 667.4 [M + H]⁺. Reaction of this intermediate (0.36 g, 0.54 mmol) with boron tribromide (5.40 mL, 5.40 mmol), as described in step 2 in the preparation of 48, gave 0.02 g (7%) of 49 as a light yellow solid. mp 148–149 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.27; ¹H NMR (400 MHz, CD₃OD): δ 8.73 (d, J = 2.1 Hz, 1H), 7.77 (s, 1H), 7.63–7.59 (m, 2H), 7.50–7.44 (m, 2H), 7.43–7.38 (m, 1H), 6.63 (s, 2H), 3.69 (dt, J = 8.6, 5.5 Hz, 1H), 3.38 (d, J = 19.6 Hz, 2H), 3.17 (d, J = 17.2 Hz, 1H), 2.93 (s, 1H), 2.78–2.67 (m, 3H), 2.62 (d, J = 17.1 Hz, 2H), 1.76 (s, 2H), 1.52 (s, 3H), 1.42 (d, J = 9.0 Hz, 4H), 1.04 (m, 5H), 0.95–0.80 (m, 4H), 0.72 (m, 1H), 0.68–0.54 (m, 4H), 0.36 (m, 2H); HRMS (ESI) m/z calcd for C₃₈H₄₅N₂O₃ [M + H]⁺: 577.34247; found, 577.34135; HPLC (system 2) t_R = 7.89 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-phenyl-8a-(3-(tetrahydro-2H-pyran-4-yl)propoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (50).

Step 1.

Compound 98 (1.20 g, 2.21 mmol) was dissolved in anhydrous DMF (10 mL) and cooled at 0 °C. Sodium hydride (0.35 g, 8.85 mmol) was added, and the reaction mixture was warmed up to room temperature and stirred for 15 min. Then, 3-bromoprop-1-ene (0.60 mL, 6.63 mmol) was added dropwise, and the reaction mixture was allowed to stir at rt for 3 h. Then, water (250 mL) was added, and the reaction mixture was extracted with EtOAc (3 × 100 mL). The organic extract was then washed with saturated NaCl solution and dried over anhydrous Na₂SO₄. The solvent was removed, and the crude product was purified by flash chromatography using hexanes/EtOAc as the eluent to obtain 1.1 g (85%) of (4bS,8R,8aS,13bR)-8a-(allyloxy)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-phenyl-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline. ¹H NMR (400 MHz, CDCl₃): δ 8.81 (dd, J = 2.3, 0.7 Hz, 1H), 7.56–7.52 (m, 2H), 7.48–7.43 (m, 3H), 7.41–7.37 (m, 1H), 7.31–7.28 (m, 2H), 7.23–7.21 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.53 (dd, J = 8.1, 0.8 Hz, 1H), 5.80–5.73 (m, 1H), 5.63 (s, 1H), 5.21–5.11 (m, 3H), 5.04 (dd, J = 17.3, 1.8 Hz, 1H), 4.95 (dd, J = 10.4, 1.7 Hz, 1H), 4.26 (ddd, J = 10.8, 3.4, 1.7 Hz, 1H), 3.91–3.86 (m, 1H), 3.70 (d, J = 5.8 Hz, 1H), 3.24 (d, J = 18.5 Hz, 1H), 2.87 (d, J = 16.5 Hz, 1H), 2.77–2.70 (m, 2H), 2.64–2.58 (m, 1H), 2.52–2.45 (m, 2H), 2.38–2.28 (m, 2H), 1.72–1.68 (m, 1H), 0.95–0.88 (m, 1H), 0.57–0.51 (m, 2H), 0.17 (m, 2H); ESI-MS m/z: 583.3 [M + H]⁺.

Step 2.

The abovementioned intermediate (1.10 g, 1.89 mmol), tetrakis triphenylphosphine palladium(0) (0.22 g, 0.19 mmol), diacetoxypalladium (0.04 g, 0.19 mmol), potassium carbonate (0.65 g, 4.72 mmol), and 4-bromo-3,6-dihydro-2H-pyran (0.46 g, 2.83 mmol) were added to anhydrous DMF (15 mL) at room temperature. The reaction mixture was then degassed with argon for 5 min, and then, the mixture was heated at 110 °C for 3 h. The reaction mixture was then filtered through a pad of celite and washed with EtOAc several times. Water was added, and the mixture was extracted with EtOAc and dried over anhydrous Na₂SO₄. Removal of the solvent under reduced pressure and purification by flash chromatography using CHCl₃/MeOH as the eluent yielded 0.4 g (32%) of (4bS,8R,8aS,13bR)-1-(benzyloxy)-7-(cyclopropylmethyl)-11-phenyl-8a-(((E)-3-(tetrahydro-2H-pyran-4-yl)allyl)oxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinoline. ESI-MS m/z: 665.3 [M + H]⁺.

Step 3.

The abovementioned intermediate (0.40 g, 0.60 mmol) was dissolved in MeOH (10 mL). 10% palladium on carbon (35.6 mg, 0.03 mmol) was added, and the reaction mixture was purged with hydrogen gas three times. The reaction vessel was fitted with a hydrogen gas-filled balloon, and the mixture was vigorously stirred at room temperature for 2 h. Workup of the reaction mixture gave 13 mg (3.8%) of 50 as a colorless oil. TLC (10% MeOH/CH₂Cl₂): R_f = 0.50; ¹H NMR (400 MHz, CDCl₃): δ 8.81 (s, 1H), 7.56 (d, J = 1.7 Hz, 1H), 7.54–7.41 (m, 5H), 6.79 (d, J = 8.1 Hz, 1H), 6.68 (d, J = 8.1 Hz, 1H), 5.68 (s, 1H), 4.35 (d, J = 6.3 Hz, 1H), 3.80 (d, J = 11.7 Hz, 2H), 3.63 (d, J = 6.9 Hz, 1H), 3.52 (dd, J = 13.5, 7.7 Hz, 1H), 3.35 (d, J = 19.5 Hz, 1H), 3.21 (dtd, J = 9.7, 6.9, 6.1, 3.4 Hz, 3H), 3.14–3.04 (m, 4H), 2.95 (d, J = 12.5 Hz, 2H), 2.77 (d, J = 16.5 Hz, 2H), 1.88 (d, J = 13.5 Hz, 1H), 1.62–1.53 (m, 2H), 1.38–1.30 (m, 3H), 1.18 (s, 1H), 1.00 (dq, J = 11.9, 6.2, 5.0 Hz, 2H), 0.89–0.79 (m, 3H), 0.70 (ddd, J = 18.0, 8.8, 5.1 Hz, 2H), 0.52–0.42 (m, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 150.6, 147.1, 143.7, 140.7, 137.6, 136.3, 135.9, 129.5, 129.1, 127.2, 120.5, 120.0, 118.9, 110.2, 89.2, 76.2, 68.1, 63.8, 58.3, 56.4, 46.7, 46.3, 34.6, 33.3, 33.1, 31.5, 29.9, 28.1, 25.9, 25.0, 5.9, 5.8, 3.2; HRMS (ESI) m/z calcd for C₃₇H₄₃N₂O₄ [M + H]⁺: 579.3217; found, 579.3208; HPLC (system 1) t_R = 13.49 min, purity = 99.7%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-phenyl-8a-(4-phenylbutoxy)-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (51).

Step 1.

Compound 98 (1.05 g, 1.94 mmol) was reacted with sodium hydride (0.31 g, 7.74 mmol) and (4-bromobutyl)benzene (0.83 g, 3.87 mmol) in anhydrous DMF (10 mL). Workup and purification, as described in step 1 in the preparation of 48, gave 100. Yield: 0.4 g (30.6%). ¹H NMR (400 MHz, CDCl₃): δ 8.79–8.77 (m, 1H), 7.47–7.45 (m, 2H), 7.44–7.42 (m, 2H), 7.40–7.36 (m, 2H), 7.31–7.28 (m, 2H), 7.22 (t, J = 1.3 Hz, 1H), 7.21 (dd, J = 2.2, 0.9 Hz, 1H), 7.17 (d, J = 1.7 Hz, 1H), 7.16–7.15 (m, 1H), 7.14–7.13 (m, 1H), 7.09 (d, J = 7.2 Hz, 1H), 7.02–6.99 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.51 (s, 1H), 5.61 (s, 1H), 5.19 (d, J = 11.9 Hz, 1H), 5.12 (d, J = 12.1 Hz, 1H), 3.75–3.71 (m, 1H), 3.64 (d, J = 5.9 Hz, 1H), 3.24 (s, 2H), 2.86 (d, J = 16.5 Hz, 2H), 2.73 (s, 5H), 2.58–2.52 (m, 2H), 2.49–2.42 (m, 4H), 2.37 (d, J = 6.6 Hz, 2H), 0.88–0.83 (m, 1H), 0.53–0.49 (m, 2H), 0.15 (s, 2H); ESI-MS m/z: 675.3 [M + H]⁺.

Step 2.

The abovementioned intermediate 100 (0.38 g, 0.56 mmol) was dissolved in 2,2,2-trifluoroacetic acid (8.0 mL, 105 mmol), and the mixture was heated at 70 °C for 2 h. Workup and purification gave 0.102 g (31%) of 51 as a yellow foamy solid. mp 87–88 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.28; ¹H NMR (400 MHz, CD₃OD): δ 8.61 (dd, J = 2.2, 0.8 Hz, 1H), 7.68 (d, J = 2.2 Hz, 1H), 7.51 (d, J = 1.7 Hz, 1H), 7.49 (dd, J = 2.0, 1.0 Hz, 1H), 7.46–7.40 (m, 3H), 7.40–7.37 (m, 1H), 7.27–7.13 (m, 2H), 7.09–7.03 (m, 2H), 7.03–6.95 (m, 2H), 6.93–6.88 (m, 2H), 6.56 (s, 2H), 3.78 (dd, J = 7.8, 4.8 Hz, 2H), 3.39 (dt, J = 8.3, 4.1 Hz, 1H), 3.25 (d, J = 18.6 Hz, 1H), 3.07 (d, J = 16.9 Hz, 1H), 2.74–2.64 (m, 3H), 2.56–2.48 (m, 3H), 2.46 (d, J = 6.2 Hz, 1H), 2.38 (t, J = 7.7 Hz, 3H), 2.34–2.30 (m, 1H), 0.91 (d, J = 4.8 Hz, 2H), 0.56–0.51 (m, 2H), 0.19 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ = 152.3, 146.6, 143.5, 142.7, 139.0, 137.4, 136.2, 135.7, 131.1, 131.0, 129.13, 128.34, 128.27, 128.19, 127.2, 125.9, 125.6, 119.1, 116.9, 91.1, 60.0, 59.9, 55.8, 47.8, 44.6, 35.6, 31.3, 30.5, 29.8, 29.7, 28.3, 23.5, 9.4, 4.1, 3.7; HRMS (ESI) m/z calcd for C₃₉H₄₁N₂O₃ [M + H]⁺: 585.31117; found, 585.3103; HPLC (system 2) t_R = 7.36 min, purity = 100%.

(4bS,8R,8aS,13bR)-7-(Cyclopropylmethyl)-11-phenyl-8a-propoxy-6,7,8,8a,9,13b-hexahydro-5H-4,8-methanobenzofuro[3,2-h]pyrido[3,4-g]quinolin-1-ol (52).

The allyloxy compound obtained in step 1 in the preparation of compound 50 (0.15 g, 0.3 mmol) was treated with 10% palladium(II) carbon (15.0 mg, 10 wt %) in the mixture of CH₂Cl₂ (7 mL) and MeOH (7 mL). The reaction mixture was evacuated under vacuum and flushed with hydrogen three times. The mixture was allowed to stir under a H₂ atmosphere at room temperature for 20 h. The reaction mixture was filtered through a pad of celite and rinsed with EtOH. The solvent was removed under reduced pressure. The residue was purified by chromatography over a column of silica gel using CHCl₃/MeOH (95:5) as the eluent to give 0.1 g (79%) of 52 as a white solid. mp 146–147 °C; TLC (10% MeOH/CH₂Cl₂): R_f = 0.70; ¹H NMR (400 MHz, CDCl₃): δ 8.63 (t, J = 2.1 Hz, 1H), 7.46–7.33 (m, 6H), 6.70 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.60 (s, 1H), 3.66 (dt, J = 7.5, 5.9 Hz, 2H), 3.28–3.13 (m, 2H), 2.84 (d, J = 16.5 Hz, 1H), 2.77–2.62 (m, 2H), 2.55–2.24 (m, 5H), 1.69–1.62 (m, 1H), 1.39 (dq, J = 11.3, 6.9 Hz, 2H), 1.26 (s, 1H), 0.94–0.84 (m, 1H), 0.66 (t, J = 7.4 Hz, 3H), 0.52 (dq, J = 7.6, 3.3 Hz, 2H), 0.21–0.11 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 152.4, 146.7, 143.6, 136.2, 135.8, 131.2, 131.1, 129.2, 128.3, 127.3, 127.2, 119.1, 116.8, 76.6, 62.1, 59.9, 55.8, 47.9, 44.6, 31.4, 30.5, 29.8, 23.6, 23.4, 22.8, 11.1, 9.5, 4.2, 3.7; HRMS (ESI) m/z calcd for C₃₂H₃₅N₂O₃ [M + H]⁺: 495.26422; found, 495.26226; HPLC (system 2) t_R = 5.76 min, purity 100%.

Cell Lines and Membrane Preparations.

All in vitro molecular pharmacology evaluations were carried out in CHO cells stably expressing 3X-HA N-terminal-tagged human MOR, DOR, or KOR constructs. The creation and evaluation of the cells, along with their established K_D values in response to ³H-diprenorphine, are described in our previous work.^{32,33,57–59} The cells were grown in 1:1 DMEM/F12 medium with 10% heat-inactivated fetal bovine serum and a 1× penicillin/streptomycin additive, all Gibco brand (Thermo Fisher). Propagation cultures were further maintained with 500 μg/mL G418 (Gibco/Thermo Fisher). Cells were cultured for no more than 20 passages before bringing up a fresh stock. For experiments, cells were plated and grown to confluency in 3 × 15 cm dishes, collected using 5 mM ethylenediaminetetraacetic acid in dPBS (no calcium or magnesium), and the resulting cell pellets were stored at −80 °C until use. All cells were monitored for mycoplasma contamination by DAPI stain and imaging, and all cells used in this project were mycoplasma negative.

Radioligand Competition Binding Assay.

Competition binding experiments were carried out versus a fixed concentration of ³H-diprenorphine using a protocol previously reported in our published work.^{32,33,58–60} The reactions were carried out using a fixed amount of membrane protein in each experiment (~25 μg/well), a fixed concentration of ³H-diprenorphine (~1–2 nM), and concentration curves of the experimental compound or positive control (naloxone for MOR and DOR, U50,488 for KOR) in a 200 μL reaction volume. Vehicle (100%, no competitor) and nonspecific binding (NSB; 10 μM naloxone or U50,488) controls were included on each plate. Reactions were incubated at room temperature for 1 h. The resulting data were normalized to vehicle (100%) and NSB (0%) controls and fitted with a 1 site nonlinear regression competition binding curve using GraphPad Prism 8.0. The affinity (K_i) was calculated for each drug using the previously measured K_D for ³H-diprenorphine in each line (MOR = 5.4 nM; DOR = 1.7 nM; KOR = 1.2 nM)⁶¹ and reported as the mean ± SEM of N ≥ 3 independent experiments.

In Vitro [³⁵S]-GTPγS Coupling Functional Assay.

³⁵S-GTPγS coupling experiments were performed as reported in our published work.^32,33 Membrane protein (15 μg/well) was combined with 0.1 nM ³⁵S-GTPγS (PerkinElmer), and concentration curves of the experimental compound or positive control agonist (MOR–DAMGO; DOR–SNC80; KOR–U50,488) in a 200 μL reaction volume were plotted. For DOR antagonist experiments, membrane protein was preincubated with concentration curves of the experimental compound or naloxone positive control for 5 min prior to adding 100 nM SNC80. Vehicle-treated controls were included on each plate. The reactions were incubated at 30 °C for 1 h. The resulting data were normalized to the stimulation caused by the positive control agonist (100%) or vehicle (0%) and fitted with three variable nonlinear regression curves using GraphPad Prism 8.0. These curves were used to calculate potency (EC₅₀/IC₅₀) and efficacy (E_max/I_max) of each compound. The efficacy values were calculated in relation to the positive control agonist or antagonist (100%). Each value was reported as the mean ± SEM of N ≥ 3 independent experiments.

Log D Determination.

The distribution coefficient (log D) was determined by the method of Wilson et al.⁶² A volume of 5 μL of DMSO solutions of the test compounds was added to a mixture of equal volumes of 50 mM phosphate buffer and 1-octanol and vortex-mixed at 800 rpm for a period of 24 h. Subsequent to centrifugation at 14,000 rpm for 30 min, 1 μL of each layer was analyzed by liquid chromatography–mass spectrometry (LC–MS)/MS. Log D was determined using the peak areas obtained from each layer. The compounds were added at 50 nM concentration in order to limit the precipitation of the compound and have the mixture within the dynamic range of LC–MS/MS instrumentation.

Aqueous Solubility.

Aqueous solubility was determined by the method of Zhou et al.⁶³ using a miniaturized shake-flask approach, under conditions of pH 6.8 and an analyte concentration of 1.0 mM. Aqueous solutions of the analyte were incubated at room temperature in the chamber of a Whatman (Piscataway, NJ) Mini-UniPrep syringeless filter for 24 h while shaking gently (600 rpm). Subsequent to incubation, filter plungers were pushed down to the bottom of the syringeless filter chamber assemblies, allowing the filtrate to enter the plunger compartment. Following an additional 30 min of incubation at room temperature, filtrates were diluted with 50:50 acetonitrile/water + 0.1% formic acid and analyzed by LC–MS/MS. Analyte concentrations were determined by the interpolation of the peak area ratio from a calibration curve formed by the matrix spiked with the authentic reference material, over a calibration range of 0.05–12.5 μM (pH 6.8). The results are presented in Table S1.

In Vitro Mouse Liver Microsomal Stability.

Metabolic stability of lead compounds was assessed in vitro by the method of Ackley et al.⁶⁴ Briefly, mouse liver microsome preparations (Corning Life Sciences, Woburn, MA) were isolated from CD-1 mice (male mice, 8–10 weeks of age). Assays were conducted using 0.123 mg/mL protein concentration (total protein concentration in the microsomal solution) and 1.0 μM drug concentration under incubation conditions of 37 °C. Metabolic stability was determined following 0, 5, 15, 30, and 60 min of incubation time. The samples were analyzed by reversed phase LC using a triple quadrupole mass spectrometer. Compound specific transitions of the parent ion to product ion were monitored, and the percent remaining was calculated based on the peak area of 5–60 min time points (relative to time zero). Half-life calculations were carried out using the formula t_1/2 = −ln(2)/k, where k (min⁻¹) is the turnover rate constant (the slope) estimated from a log-linear regression of the percentage compound remaining versus time.

In Vitro Human Liver Microsomal Stability.

Metabolic stability of the compounds in human liver microsomes was determined by the method described above using pooled human liver microsome preparations from 20 male donors (Corning Life Sciences, Woburn, MA). Assays were conducted as described above, and the calculated in vitro half-life of the compounds is presented in Table S1.

LC–MS/MS Analysis. LC–MS/MS analysis was conducted using an Agilent (Santa Clara, CA) 6460 triple quadrupole mass spectrometer coupled with an Agilent LC system. The LC system consists of a binary pump, degasser, column heater, and autosampler. Chromatographic separation was performed on a Waters Atlantis T3 3 μm 3.0 × 50 mm analytical column using a ballistic gradient of the mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.75 mL/min. The mobile phase was heated to a temperature of 45 °C.

Animals.

Male CD-1 mice (25–35 g), purchased from Charles Rivers, were housed in a climate-controlled room on a regular 12 h light/dark cycle with lights on at 7:00 am with food and water available ad libitum. Initially, four to five animals were housed per cage. All procedures were performed during the 12 h light cycle according to the policies/recommendations of the International Association for the Study of Pain and the NIH guidelines for laboratory animals and with IACUC approval from the University of Arizona (17–223). All behaviors were evaluated in n = 5–15 male mice by an observer blinded to the injection content with results compared to those of mice treated with vehicle (10% DMSO, 10% Tween 80, 80% saline) or saline where appropriate.

Antinociception.

Antinociceptive effects were evaluated using the tail-flick assay. Naïve mice were baselined in the 55 °C warm water tail-withdrawal test, and the time to reflexively withdraw the distal third of the tail was recorded. Doses of morphine or the test compound were injected icv, sc, iv, or po, and antinociception was assessed at 10, 15, 20, 30, 45, 60, 80, 120, and 180 min post injection. Percent antinociception was calculated using eq 1

$$\begin{gathered} \%\text{MPE}\left(\text{maximal}\text{possible}\text{effect}\right) \\ =100\times \left(\text{test}\text{−}\text{control}\right)/\left(\text{cutoff}\text{−}\text{control}\right) \end{gathered}$$ (1)

where control is the predrug latency, test is the postdrug latency, and cutoff is the maximal length of the stimulus, allowing 10 s for 55 °C tail-withdrawal. Antinociceptive A₅₀ values and 95% confidence intervals were determined using linear regression software (GraphPad by Prism 7.0). Opioid activity of the test compounds was assessed by pretreating animals with naloxone (10 mg/kg intraperitoneal, −10 min) followed by an sc injection of the calculated A₉₀ dose of the test compound. If a compound did not produce a full agonist effect, then the dose that produced the greatest antinociceptive effect was used. Antinociception was assessed in the 55 °C warm water tail-withdrawal test at the time of maximal percent effect. A positive response to a fixed dose of naloxone was indicated when greater than 80% reduction in the antinociceptive effect of the agonist was observed.

Tolerance Regimen.

Mice were injected twice daily (8 a.m. and 5 p.m.) with an approximate A₉₀ dose of morphine or A₉₀ dose of compound 20 for 3 days. Antinociceptive dose–response curves were generated on the morning of the fourth day using the procedures described above.

Physical Dependence/Precipitated Withdrawal.

Compound 20 was administered to male CD-1 mice at a dose of 32 mg/kg, sc, twice a day (9 a.m. and 5 p.m.) for 3 days. On the fourth day, 4 h after the morning administration of compound 20, the mice were given naloxone (30 mg/kg, ip) to precipitate withdrawal. Animals were immediately placed in clear acrylic cylinders, and behaviors were monitored through video capture for 40 min. BL behaviors for each animal were recorded 40 min prior to naloxone administration. Monitored behaviors included urine output, presence/absence of diarrhea, paw tremor, ptosis, and a number of droppings, vertical jumps, backward walking steps, and wet dog shakes to calculate an overall withdrawal score. All videos were observed and scored by an individual blinded to treatment.

Respiratory Depression.

Respiratory depression was measured by determining the average weight in freely moving, conscious male CD1 mice (25–30 g) using whole body plethysmography chambers (Data Sciences International, St Paul, MN). Chambers were maintained at room temperature, and flow and composition of the gas were set by mass flow controllers. Vehicle (10% DMSO, 10% Tween 80, 80%; 10 mL/kg, sc), saline (10 mL/kg IP), morphine (10 mg/kg, ip), or compound 20 (3.2, 10, 32 or 75 mg/kg, sc) was administered following a 30 min BL. Mice remained in the chambers after injection for a 7 min room air reading (0% CO₂), followed by a 7 min 5% concentration of carbon dioxide/oxygen mixture challenge. Minute ventilation, tidal volume, and respiratory rate were recorded for each measure, BL, room air, and 5% CO₂ concentration challenge. It should be noted that compound 20 was in solution for all of the doses except the 75 mg/kg dose; at the time of injection, the compound had precipitated out of solution and was therefore administered as a suspension.

Conditioned Place Preference/Aversion.

The basal time spent in each chamber (Panlab, Barcelona, Spain) was recorded for individual mice (CD-1, male, 25–35 g) over 15 min. Any mouse spending more than 80% of the total time in any one chamber or less than 20% of the total time in the end chambers was excluded from further testing (n = 0). Animals received injections both morning and afternoon and were placed and confined to one chamber or the other 10 min following injection for a 15 min duration such that all pairing combinations were executed (e.g., if an animal received drug in the morning and was placed in the dotted chamber, then in the afternoon, the same animal received vehicle and was placed in the opposite (striped) chamber). Injections took place 4 h apart, as shown in Figure 10. On day 5, injections occurred in the morning and testing was performed in the afternoon. Mice were placed in the corridor with the doors to both chambers in place. Once the software was started, the doors were lifted and the animals were allowed free access to all chambers for a 15 min duration. Compound 20 was given at 20 mg/kg, and morphine sulfate was given at 10 mg/kg; all injections were performed at 10 mL/kg (sc).

Statistical Analyses.

Numbers of animals required for individual behavioral outcomes were determined in GPower3.1 to give 80% power to detect a 20% difference when α = 0.05. Results were statistically significant when p ≤ 0.05. Individual assays were analyzed as follows: tail flick (Figures 4 and 5): time × treatment, two-way RM ANOVA Bonferroni post hoc; tail flick (Figure 6): one-way ANOVA Bonferroni post hoc; DRC (Figure 7) nonlinear regression; PD (Figure 8): one-way ANOVA/behavior; RD (Figure 9): time × treatment two-way RM ANOVA Bonferroni post hoc; and CPP/CPA (Figure 10F,G): one-way ANOVA/behavior Bonferroni post hoc.

Computational Docking Study.

Crystal structures of the agonist-bound mouse MOR (PDB ID 5C1M), antagonist-bound human DOR (PDB ID 4N6H), and antagonist-bound human KOR (PDB ID 4DJH) were used in the docking studies. Protein structures were prepared using Schrödinger Small Molecule Drug Discovery Suite. Conserved water molecules were kept, and modified residues in crystal structures were changed back to the wildtype. The 3D structures of the compounds were generated using LigPrep in Schrödinger. Ligands in the crystals were used to define the binding site for docking. All compounds were docked to the MOR, DOR, and KOR using induced fit docking protocol (flexible ligand and protein within 5 Å of ligand poses) implemented in Schrödinger.⁶⁵

ABBREVIATIONS

ADME

absorption, distribution, metabolism, and excretion

CHO

Chinese hamster ovary

CPA

conditioned place aversion

CPM

cyclopropylmethyl

CPP

conditioned place preference

DAMGO

[d-Ala²,Me-Phe,Gly-ol⁵]enkephalin

DOR

δ opioid receptor

[³⁵S]GTPγS

guanosine-5′-O-(3[³⁵S]thio-triphosphate)

KOR

κ opioid receptor

MOR

μ opioid receptor

MPE

maximum possible effect

TLC

thin-layer chromatography