Potent and Selective Tetrahydroisoquinoline Kappa Opioid Receptor Antagonists of Lead Compound (3R)‑7-Hydroxy‑N‑[(1S)‑2-methyl-1-(piperidin-1-ylmethyl)propyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (PDTic)

Abstract

Past studies have shown that it has been difficult to discover and develop potent and selective κ opioid receptor antagonists, particularly compounds having potential for clinical development. In this study, we present a structure—activity relationship (SAR) study of a recently discovered new class of tetrahydroisoquinoline κ opioid receptor antagonists which led to (3R)-7-hydroxy-N-{(1S)-2-methyl-1-[(−4-methylpiperidine-1-yl)methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide (12) (4-Me-PDTic). Compound 12 had a K_e = 0.37 nM in a [³⁵S]GTPγS binding assay and was 645- and >8100-fold selective for the κ relative to the μ and δ opioid receptors, respectively. Calculated log BB and CNS (central nervous system) multiparameter optimization (MPO) and low molecular weight values all predict that 12 will penetrate the brain, and pharmacokinetic studies in rats show that 12 does indeed penetrate the brain.

Graphical Abstract

INTRODUCTION

Over the years, it has proven to be very difficult to develop potent and highly selective pure κ opioid receptor antagonists. The first such compound developed was nor-BNI (nor-binaltorphimine) (Figure 1).¹ In 2000, GNTI (5′-guanidino-naltrindole) (Figure 1), a slightly more potent κ opioid receptor antagonist, was developed.² The naltrexone-derived norBNI and GNTI depend on the N-cyclopropylmethyl group for their antagonistic properties. In 2001, we reported the discovery of JDTic (Figure 1), a potent and selective κ opioid receptor antagonist with a novel chemical structure.^3–5 JDTic displayed robust effectiveness in rodent models of depression,⁵ anxiety,⁶ stress induced cocaine relapse,⁵ nicotine withdrawal,⁷ and alcohol seeking, relapse, and withdrawal.^8,9 Besides JDTic, PF-4455242 and LY2456302 have been identified as new classes of κ opioid receptor antagonists. However, both LY2456302 and PF-4455242 have lower potency and selectivity at the κ relative to the μ and δ opioid receptors compared to JDTic, nor-BNI, and GNTI. Nevertheless, JDTic, LY2456302, and PF-4455242 all proceeded through preclinical development and were evaluated in phase 1 clinical studies.^10–13 The compound LY2456302, now known as CERC-501, is the only one still in clinical evaluation to the best of our knowledge. More recently, CYM51317 (structure not available) was reported as a new κ opioid receptor antagonist for migraine prevention.¹⁴

Figure 1.

Structures of JDTic, PF-4455242, LY2456302, nor-BNI, GNTI, 1, and 2.

We recently reported 1 (PDTic), a pure opioid receptor antagonist with a substantially simple tetrahydroisoquinoline moiety, as the lead compound for the design and development of a novel class of potent and selective antagonists for the κ opioid receptor.¹⁵ In this study, we report the design, synthesis, and in vitro opioid receptor binding properties of compounds 3–39 and 41, 43, 45, and 47, which are structural analogues of 1 (structures for these compounds along with JDTic analogues 40, 42, 44, and 46 are given in Tables 1–7), using [³⁵S]GTPγS binding assays. The physiochemical properties of the more potent compounds were calculated to determine if the compound(s) would be predicted to enter the brain and further pharmacokinetic studies in rats were conducted on the most potent and selective κ opioid receptor antagonist identified to determine if the compound did indeed penetrate the brain.

Table 1.

Inhibition of Agonist-Stimulated [³⁵S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors, Importance of the Piperidine Ring

compd	R1, R2	Ke (nM)a			μ/κ	δ/κ
		μ, DAMGO	δ, DPDPE	κ, U69,593
1	−(CH2)5−	144 ± 37	>3000	6.80 ± 2.1	21	>441
3	−(CH2)6−	701 ± 37	>3000	17.0 ± 4.7	41	>177
4	−(CH2)4−	702 ± 120	>3000	48.5 ± 12	14	>62
5		749 ± 59	>3000	2.53 ± 0.6	296	>1186
6		>3000	>3000	396 ± 140	>8	>8
7		915 ± 303	>3000	98.8 ± 28	9	>30
8	C2H5, C2H5	690 ± 110	>3000	41.8 ± 12	16.5	>72
9	C3H7, C3H7	300 ± 27	>3000	14.5 ± 0.5	20.7	>207
10	i-Bu, i-Bu	>3000	>3000	137 ± 24	>22	>22
11		916 ± 130	>3000	61.5 ± 9.3	>15	>49

^aData are mean ± SEM of at least three independent experiments conducted in duplicate. None of the compounds had agonist activity at 10 μM.

Table 7.

Inhibition of Agonist-Stimulated [³⁵S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors, Comparison of Structural Changes in JDTic to Similar Changes in 12

compd	structure	R1, R2	Ke (nM)a			μ/κ	δ/κ
			μ, DAMGO	δ, DPDPE	κ, U69,593
JDTic	A	OH, H	25 ± 0.01	74 ± 2	0.02 ± 0.01	1255	3800
12	B	OH, H	239 ± 22	>3000	0.37 ± 0.09	645	>8100
40b	A	CONH2, H	7.09 ± 2.58	131 ± 23	0.02 ± 0.01	355	6550
41	B	CONH2, H	41.3 ± 11	>3000	1.37 ± 0.32	30	>2190
42b	A	OCH3, H	51.4 ± 15	118 ± 45	0.06 ± 0.	857	1970
43	B	OCH3, H	1200 ± 140	>3000	25.6 ± 6.3	47	>117
44b	A	F, H	7.7 ± 0.9	c	2.20 ± 0.47	3.5
45	B	F, H	>3000	>3000	182 ± 19	>17	>17
46d	A	OH, CH3	210 ± 60	491 ± 120	0.16 ± 0.06	1313	3070
47	B	OH, CH3	752 ± 140	>3000	36.7 ± 5.6	20	>82

^aData are mean ± SEM of at least three independent experiments conducted in duplicate.

^bTaken from ref 21.

^cThis compound was an inverse agonist at the δ opioid receptor with an IC₅₀ of 97 ± 7 nM and percent basal binding of 78 ± 3% (data are mean ± SEM of three independent experiments conducted in duplicate).

^dTaken from ref 25.

RESULTS AND DISCUSSION

Chemistry.

The syntheses of the various analogues substituted at four positions R¹, R², R³, and R⁴ in structure 2 (Figure 1) are outlined in Schemes 1–12. Synthesis of the azepanyl and morpholinyl compounds 3 and 6, respectively, is shown in Scheme 1. l-Valinol (48), prepared according to a literature method,¹⁶ was coupled with Boc-7-hydroxy-d-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Boc-7-hydroxy-d-Tic-OH) using coupling agent N,N′-dicyclohexylcarbodiimide (DCC) to afford 49. The phenol in 49 was protected as the methyl ether using trimethylsilyldiazomethane and the alcohol was oxidized with Dess—Martin periodinane to afford the aldehyde 50. Reductive amination with either azepane or morpholine using sodium triacetoxyborohydride followed by concomitant methyl ether and Boc deprotection with boron tribromide in dichloromethane and subsequent treatment with ammonium hydroxide afforded compounds 3 and 6.

Scheme 1. Synthesis of Analogues 3 and 6^a.

^aReagents and conditions: (a) Boc-7-hydroxy-d-Tic-OH, DCC, HOBt, NEt₃, THF, rt, 72 h; (b) (i) TMSCHN₂, N,N-diisopropylethlamine, CH₃CN, CH₃OH, rt, overnight, (ii) Dess—Martin periodinane, CH₂Cl₂, rt, 1 h; (c) (i) azepane or morpholine, NaBH(OAc)₃, (ii) BBr₃, CH₂Cl₂, −78 °C to rt overnight, (iii) NH₄OH aq, reflux.

Scheme 12. Synthesis of Analogues 39 and 47^a.

^aReagents and conditions: (a) BH₃·SMe₂, THF, reflux, overnight; (b) 37% aq CH₂O, NaBH(OAc)₃, DCE, rt, 24 h.

As shown in Scheme 2, a variety of commercially available amines (pyrrolidine, azabicyclo[2.2.1]heptane, 4-methylpiperazine, diethylamine, dipropylamine, and diisobutylamine) were each subjected to coupling with N-Boc-l-valine (51), using O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (HBTU) and triethylamine in acetonitrile, stirred at room temperature overnight, to provide the tert-butyloxycar-bonyl-protected intermediate, which upon treatment with hydrogen chloride in a solvent mixture of dioxane and acetonitrile or trifluoroacetic acid in dichloromethane provided compounds 52a–f. Amides 52a–f were reduced with borane dimethylsulfide in tetrahydrofuran to furnish diamines 53a–f. Subsequent coupling of the diamines 53a–f with Boc-7-hydroxy-d-Tic-OH using 1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide (EDC) and catalytic amounts of 1-hydroxybenzotriazole (HOBt) and triethylamine in dichloromethane followed by Boc-deprotection (via treatment with methanolic hydrochloric acid or hydrogen chloride in dioxane and acetonitrile) furnished final compounds 4, 5, and 7–10.

Scheme 2. Synthesis of Analogues 4, 5, and 7−10^a.

^aReagents and conditions: (a) appropriate amine [pyrrolidine (for 52a), azabicyclo[2.2.1]heptane (for 52b), 1-methylpiperazine (for 52c), diethylamine (for 52d), dipropylamine (for 52e), diisobutylamine (for 52f)], HBTU, CH₃CN, rt, overnight; (b) TFA in CH₂Cl₂ (for 52a,b) or HCl (4 N in dioxane) in CH₃CN (for 52c−52f); (c) BH₃·SMe₂, THF, reflux, 3 h; (d) (i) Boc-7-hydroxy-d-Tic-OH, EDC·HCl, HOBt, NEt₃, CH₂Cl₂, rt, overnight, (ii) HCl (aq) in CH₃OH (for 4–7) or HCl (4 N in dioxane) in CH₃CN (for 8–10).

The synthesis of a 2-oxopiperidine derived compound 11, as outlined in Scheme 3, commenced from l-valinol (48). The amine in 48 was protected as a benzylcarbamate (54) and the alcohol converted to mesylate using methanesulfonyl chloride to yield 55. Compound 55 was heated at reflux with 2-hydroxypyridine in the presence of tetrabutylammonium bromide (TBAB), potassium carbonate, toluene, and a catalytic amount of water to provide 56.¹⁷ Catalytic hydrogenation of 56 using palladium on carbon catalyst yielded 57, which was subsequently coupled with Boc-7-hydroxy-d-Tic-OH, followed by the tert-butyloxycarbonyl group deprotection using hydrogen chloride in dioxane to provide compound 11.

Scheme 3. Synthesis of Analogue 11^a.

^aReagents and conditions: (a) benzyl chloroformate, N,N-diisopropylethylamine, CH₂Cl₂, 0 °C to rt, overnight; (b) MsCl, pyridine, CH₂Cl₂, 0 °C to rt, overnight; (c) 2-hydroxypyridine, K₂CO₃, TBAB, toluene, H₂O, reflux, overnight; (d) H₂, Pd/C, CH₃OH, 40 psi, rt, 24 h; (e) Boc-7-hydroxy-d-Tic-OH, EDC·HCl, HOBt, NEt₃, CH₂Cl₂, rt, overnight; (f) HCl (4 N in dioxane) in CH₃CN.

A plethora of substituted commercially available piperidines (58a–c, 58f–g, 58i, 58k–n), as well as some lab-prepared known piperidines (58d,e, 58h, 58j), were employed for the synthesis of substituted piperidine analogues 12–26 (Scheme 4). (Difluoromethyl)piperidine (58j) was prepared in three steps from 4-hydroxymethylpiperidine via an aldehyde that was subsequently converted to 1-N-Boc-difluoromethylpiperidine using diethylaminosulfur trifluoride (DAST) as previously reported.¹⁸ For the preparation of trans-3,4-dimethylpiperidine ((±)-58e), the glutaric acid (±)-61 was prepared in three steps following a literature precedence,¹⁹ then was subjected to ring closure by heating in a melt with urea at 180 °C for 3 h to provide 3,4-dimethylpiperidine-2,6-dione as a mixture of the trans and cis isomers in a 2:1 ratio (Scheme 5). Recrystallization of the mixture from ethyl acetate with hexanes provided the pure trans product (±)-62 that was subsequently reduced using sodium borohydride and boron trifluoride etherate in tetrahydrofuran to furnish (±)-58e (Scheme 5). The cis-3,4-dimethylpiperidine ((±)-58d) and 4-ethylpiperidine (58h) were prepared from 3,4-lutidine (63c) and 4-ethylpyridine (63d), respectively, via a three-step protocol reported in the literature,²⁰ followed by subsequent catalytic hydrogenation to provide (±)-58d and 58h (Scheme 6). Each of the piperidines (58a–n) was coupled with Boc-l-valine (51) using HBTU in acetonitrile, followed by removal of the Boc-protection using either hydrogen chloride or trifluoro-acetic acid to provide compounds 59a–n (Scheme 4). Reduction of these amides using borane dimethylsulfide gave the diamines 60a–n. Some of the 4-cyano intermediate (60n) was subjected to an acid catalyzed conversion of the nitrile using aqueous sulfuric acid, stirred at room temperature for 24 h, to furnish the 4-carboxamide (60o). The diamine intermediates 60a–o were next coupled with Boc-7-hydroxy-d-Tic-OH using EDC/HOBt followed by the tert-butyloxycarbonyl group deprotection using methanolic hydrogen chloride or hydrogen chloride in a dioxane acetonitrile mixture to yield the desired final compounds 12–26, respectively (Scheme 4).

Scheme 4. Synthesis of Analogues 12−26^a.

^aReagents and conditions: (a) N-Boc-l-valine (51), HBTU, CH₃CN, rt, overnight; (b) HCl (4 N in dioxane) in CH₃CN, (for 59a–c, 59g, 59i, 59k, 59n)/HCl (aq) in CH₃OH (for 59e–f, 59h, 59j))/TFA in CH₂Cl₂ (for 59l−59m); (c) BH₃·SMe₂ (or BH₃·THF), THF, reflux, 3 h; (d) (i) H₂SO₄, H₂O, at 0 °C then rt for 24 h, (ii) NaOH; (e) (i) Boc-7-hydroxy-d-Tic-OH, EDC·HCl, HOBt, NEt₃, CH₂Cl₂, rt, overnight, (ii) HCl (4 N in dioxane) in CH₃CN (for 12–15, 17–18, 20–22, 26)/HCl (aq) in CH₃OH (for 16, 19, 23–24)/TFA in CH₂Cl₂ (for 25).

Scheme 5. Synthesis of (±)-58e^a.

^aReagents and conditions: (a) urea, 180 °C, 3 h; (b) (i) BF₃·OEt₂, NaBH₄, THF, 2 h, at rt then at reflux, 2 h, (ii) piperazine, H₂O, reflux, overnight.

Scheme 6. Synthesis of Compounds (±)-58d, 58h, and 66a–d^a.

^aReagents and conditions: (a) benzyl bromide, acetone, reflux, 1 h or in a microwave, 100 °C for 5−10 min; (b) NaBH₄, CH₃OH, H₂O, 0 °C then rt, overnight; (c) 1-chloroethyl chloroformate, dichloroethane, reflux 12 h, then reflux with CH₃OH for 1 h (for 66b–d); (d) 10% Pd/C, CH₃OH, CH₂Cl₂, H₂, rt, 50 psi, 48 h (for (±)-58d and 58h).

The pyridines used to prepare compounds 66b–d, (±)-58d, and 58h are shown in Scheme 6. The tetrahydropyridines 66b–d were prepared following a protocol similar to what has been previously reported²⁰ from 4-picoline (63a), 3-picoline (63b), and 3,4-lutidine (63c), respectively, via the N-benzylpyridinium salt, followed by reduction and subsequent cleavage of the benzyl group to give 66b–d. Intermediate 66a, as shown in Scheme 6, was commercially available. Compounds 66a–d were subjected to chemistry analogous to that described for the piperidines: couplings with Boc-l-valine (51) and removal of the Boc-protection to yield 67a–d, reduction with borane dimethylsulfide to give 68a–d, subsequent coupling of those products with Boc-7-hydroxy-d-Tic-OH, and a final Boc-deprotection provided the final compounds 27–30 (Scheme 7).

Scheme 7. Synthesis of Analogues 27−30^a.

^aReagents and conditions: (a) (i) N-Boc-l-valine (51), HBTU, CH₃CN, rt, overnight, (ii) TFA, CH₂Cl₂ (for 67a, 67d)/aq HCl, CH₃OH (for 67b,c); (b) BH₃·SMe₂, THF, reflux, 3 h or LAH, THF, 1 h; (c) (i) Boc-7-hydroxy-d-Tic-OH, EDC·HCl, HOBt, NEt₃, CH₂Cl₂, rt, overnight, (ii) aq HCl, CH₃OH.

As shown in Scheme 8, the diastereomers 31–33 of 12 were synthesized using the different enantiomers of the coupling intermediates. Coupling of 60a with Boc-7-hydroxy-l-Tic-OH yielded the diastereomer 31. On the other hand, the reactions starting with 4-methypiperidine (58a) and N-Boc-d-valine provided the intermediate 69, which was subsequently coupled with either Boc-7-hydroxy-d-Tic-OH or Boc-7-hydroxy-l-Tic-OH to yield 32 and 33, in that order.

Scheme 8. Synthesis of Diastereomers 12 and 31−33^a.

^aReagents and conditions: (a) (i) N-Boc-l-valine (for 60a) or N-Boc-d-valine (for 69), HBTU, CH₃CN, rt, overnight, (ii) HCl (4 N in dioxane) in CH₃CN; (b) BH₃·SMe₂, THF, reflux, 3 h; (c) (i) Boc-7-hydroxy-d-Tic-OH (for 12 and 32) or Boc-7-hydroxy-l-Tic-OH (for 31 and 33), EDC·HCl, HOBt, NEt₃, CH₂Cl₂, rt, overnight, (ii) HCl (4 N in dioxane) in CH₃CN.

Other analogues were synthesized where the isopropyl group, the phenol group, or the tetrahydroisoquinoline ring in 12 were changed or substituted by other groups (Schemes 9–12). Commercially available N-(2-aminoethyl)piperidine (70) was coupled with Boc-7-hydroxy-d-Tic-OH using dicyclohexylcarbodiimide (DCC) as the coupling agent in tetrahydrofuran, followed by removal of the Boc-protection group using methanolic hydrogen chloride to provide analogue 34 (Scheme 9). As shown in Scheme 10, N-Boc-l-cyclo-propylglycine was substituted for Boc-l-valine for coupling with 58a to provide 71. Reduction of 71 using borane dimethylsulfide in THF to give 72 followed by coupling with Boc-7-hydroxy-d-Tic-OH provided 73a, which upon removal of the Boc-protection using hydrogen chloride in methanol furnished the cyclopropyl analogue 35. Coupling 60a with Boc-d-tyrosine in place of Boc-7-hydroxy-d-Tic-OH using EDC/HOBt and triethylamine in dichloromethane, followed by a Boc-deprotection using hydrogen chloride in methanol, provided compound 36. Coupling of 6-hydroxynapthalene-2-carboxylic acid with 60a in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in dimethylformamide as solvent, heated at 100 °C for 3 h, furnished analogue 37. Boc-7-carbamoyl-d-Tic-OH, prepared in three steps as previously reported,²¹ was coupled with diamine 60a to yield 73b, which yielded 41 after the removal of the Boc-protecting group. Similarly, Boc-7-fluoro-d-Tic-OH, also previously reported,²¹ was employed for the synthesis of 73d which upon Boc-deprotection, furnished compound 45. The phenolic hydroxy group in 73c was converted to the methyl ether upon treatment with trimethylsilyl diazomethane followed by the removal of the Boc-protecting group to provide compound 43 (Scheme 10). For the synthesis of analogue 38 (Scheme 11), 6-methoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid (74a)²² was demethylated using hydrogen bromide in acetic acid heated at reflux for 5 h to provide 74b, which was subsequently coupled with 60a using (benzotriazole-1-yloxy) tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and triethylamine in tetrahydrofuran stirred at room temperature overnight to provide 38 as a mixture of diastereomers. Finally, as shown in Scheme 12, the reduced and N-methyl analogues of 12, compounds 39 and 47, were both synthesized in one step from 12. Treatment of 12 in tetrahydrofuran with borane dimethylsulfide heated at reflux overnight furnished the reduced analogue 39. On the other hand, treatment of 12 in dichloroethane with formalin followed by sodium triacetoxyborohydride stirred at room temperature for 24 h yielded the N-methyl analogue 47.

Scheme 9. Synthesis of Analogue 34^a.

^aReagents and conditions: (a) (i) Boc-7-hydroxy-d-Tic-OH, DCC, HOBt, THF, rt, overnight, (ii) aq HCl, CH₃OH.

Scheme 10. Synthesis of Analogues 35−37, 41, 43, and 45^a.

^aReagents and conditions: (a) (i) N-Boc-l-cyclopropylglycine (for 71) or N-Boc-l-valine (for 59a), HBTU, CH₃CN, rt, overnight, (ii) HCl (4 N in dioxane) in CH₃CN; (b) BH₃·SMe₂, THF, reflux, 3 h; (c) appropriate acid [Boc-d-tyrosine (for 36) or Boc-7-hydroxy-d-Tic-OH (for 73a and 73c) or Boc-7-carbamoyl-d-Tic-OH (for 73b) or Boc-7-fluoro-d-Tic-OH (for 73d)], DCC (for 36) or EDC·HCl (for 73a, 73c,d) or HBTU (for 73b), HOBt, NEt₃, DCM, rt, overnight; (d) aq HCl, CH₃OH; (e) 6-hydroxynaphathalene-2-carboxylic acid, EEDQ, DMF, 100 °C, 3 h; (f) (i) TMSCHN₂ (2 M in Et₂O), DIPEA, CH₃CN:CH₃OH (4:1), rt; (ii) HCl (4 N in dioxane) in CH₃CN, rt, overnight.

Scheme 11. Synthesis of Analogue 38^a.

^aReagents and conditions: (a) HBr (48% aq), AcOH, 105 °C, 5 h; (b) 60a, BOP, NEt₃, THF, rt, overnight.

Pharmacology Studies.

As we recently reported, the structurally simple tetrahydroisoquinoline analogue 1 had a K_e = 6.80 nM at κ, with K_e values of 144 and >3000 nM at the μ and δ opioid receptors, respectively.¹⁵ Thus, the compound was 21- and >441-fold selective for the κ opioid receptor relative to the μ and δ opioid receptors, respectively.¹⁵ Given these encouraging results, we set out to examine the structure—activity relationships of a variety of 1 analogues. All synthesized compounds were evaluated for antagonist activity at the μ, δ, and κ opioid receptors using our established assays¹⁵ that measure the ability of antagonists to inhibit agonist-stimulated [³⁵S]GTPγS binding in membranes prepared from CHO cells expressing human opioid receptors. Concentration response curves of U69,593 (κ), DAMGO (μ), or DPDPE (δ) were run in the absence and presence of a single concentration of test compound. K_e values were calculated with the equation K_e = [L]/(ER — 1) where [L] is the concentration of test compound and ER is the ratio of EC₅₀ values in the presence and absence of test compound. K_e values were considered valid when the ER was at least 4. Compounds were also evaluated for agonist activity in the assays and none of our synthesized analogues displayed any activity at 10 μM final concentration.

To determine the effect of changes in the piperidine ring size and type of ring on the κ potency and selectivity of this new class of κ opioid receptor antagonists, we synthesized and tested the larger ring size eight membered azepane, the smaller ring size, five-membered pyrrolidine analogues 3 and 4, respectively, as well as the bicyclic analogue 5 (Table 1). Similar to 1, compounds 3, 4, and 5 were pure opioid antagonists. With a K_e = 48.5 nM at the κ opioid receptor, the pyrrolidine analogue 4 was seven times less potent at the κ opioid receptor than 1. With a K_e = 17 nM at the κ opioid receptor, the azepane analogue 3 was only 2.5 times less potent at the κ opioid receptor than 1, and with a K_e = 2.53 nM, the bicyclic analogue 5 was 2.7 times more potent than 1. Compounds 4 and 3 had μ/κ values of 14 and 41, respectively, and like 1, were not very selective for the κ opioid receptor relative to the μ opioid receptor. In contrast, 5 had a μ/κ value of 296 and was highly selective for the κ relative to the μ receptor. With a δ/κ value of >1186, 5 was also highly selective for the κ receptor relative to the δ receptor. With δ/κ values of >62 and >177, 4 and 3, respectively, were a little less selective for the κ opioid receptor relative to the δ opioid receptor than 1. Changing the methylene group at the 4-position of the piperidine ring of 1 with an oxygen or N-methyl group to give the morpholino and N-methylpiperazine ring analogues, 6 and 7, respectively, resulted in large losses in potency for all three receptors. Overall results from these changes to the piperidine ring of 1 suggest that the piperidine analogue 1 is a better lead compound than the pyrrolidine and azepane analogues 4 and 3, respectively, as well as the morpholino and N-methylpiperazino analogues 6 and 7, respectively.

To determine if the intact piperidine ring in 1 is needed for high potency at the κ opioid receptor, the simple N,N-diethyl (8), N,N-dipropyl (9), and N,N-diisobutyl (10) analogues were synthesized and evaluated for their potency and selectivity as κ opioid receptor antagonists (Table 1). Even though all three compounds were pure opioid antagonists, they were no better than 1. The N,N-dipropyl analogue 9, with a K_e = 14.5 nM at κ and μ/κ and δ/κ values of 20.7 and >207, respectively, has the best κ potency and selectivity of the three analogues. However, the results did not suggest that 9 would be a better lead compound than 1.

To gain information concerning the need for the basic nature of the amino group in the piperidine ring of 1, the 2-oxopiperidine analogue 11 was synthesized and evaluated (Table 1). The observation that 11 had a K_e of 61.5 nM at the κ opioid receptor suggests that the amino nature of 1 is required. However, low κ potency could be due to steric or other factors.

Because the importance that methyl groups can have in drug discovery is well-documented,^23,24 we investigated the opioid receptor efficacy of the monomethyl and dimethyl analogues 12–17 (Table 2). Compounds 13–17 were tested as isomeric mixtures, where the incorporated piperidine was either a racemate or, in the case of 17, purchased and used as a cis/trans mixture. These methylated-1 analogues had K_e values at the κ opioid receptors ranging from 0.37 to 3.46 nM (Table 2). Compound 12, with a K_e = 0.37 nM at the κ receptor and μ/κ and δ/κ values of 646 and >8100, respectively, was the most potent and selective κ opioid receptor antagonist. However, 15, which has a K_e = 1.26 nM at κ with μ/κ and δ/κ values of 86 and 2381, respectively, has good κ opioid receptor potency and selectivity. Compounds 16 and 17 with K_e values at the κ opioid receptor of 2.3 and 3.46 nM, respectively, were also a little more potent than 1. It is possible that one of the isomers of compounds 13–17 may be more potent than that observed for the mixture of isomers.

Table 2.

Inhibition of Agonist-Stimulated [³⁵S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors, Importance of Methyl Substituent of 1

Compd	Ke (nM)a			μ/κ	δ/κ
	μ,DAMGO	δ, DPDPE	κ, U69,593
1	144 ± 37	>3000	6.80 ± 2.1	21	>441
12	239 ± 22	>3000	0.37 ± 0.09	646	>8100
13b	139 ± 1.0	>3000	15.6 ± 2.3	9	>192
14b	44.9 ± 11	>3000	15.1 ± 4.2	3	>199
15b	108 ± 26	>3000	1.26 ± 0.08	86	>2381
16b	50.9 ± 7.7	2350 ± 390	2.3 ± 0.64	22	1022
17b	30.3 ± 1.0	>3000	3.46 ± 0.54	9	>867

^aData are mean ± SEM of at least three independent experiments conducted in duplicate. None of the compounds had agonist activity at 10 μM.

^bThese compounds are mixtures of isomers.

The high κ antagonist potency and selectivity of 12 led us to investigate the opioid receptor properties of compounds where the 4-methyl group was replaced by other substituents such as the 4,4-dimethyl 18, 4-ethyl 19, 4-trifluoromethyl 20, 4-(difluoromethyl) 21, 4-methoxy 22, 4-(dimethylamino) 23, 4,4-difluoro 24, 4-cyano 25, and the 4-carboxamide 26. These analogues of 12 were synthesized and tested in the [³⁵S]GTPγS binding assay (Table 3) and found to have κ opioid receptor K_e values ranging between 2.48 and 12.7 nM and thus were 7–34 times less potent κ opioid receptor antagonists than the 4-methyl analogue 12. With μ/κ values ranging from 14 to 51, none of the compounds were very selective for the κ relative to the μ opioid receptor. Because the δ/κ values for the compounds in Table 3 ranged from >236 to 1004, all of the compounds are highly selective for the κ relative to the δ opioid receptor.

Table 3.

Inhibition of Agonist-Stimulated [³⁵S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors, Importance of the Piperidine 4-Methyl Group

compd	R1	R2	Ke (nM)a			μ/κ	δ/κ
			μ, DAMGO	δ, DPDPE	κ, U69,593
12	CH3	H	239 ± 22	>3000	0.37 ± 0.09	645	>8100
18	CH3	CH3	142 ± 25	847 ± 180	3.57 ± 1.5	40	>237
19	CH2CH3	H	74.8 ± 23	2490 ± 620	2.48 ± 0.70	30	1004
20	CF3	H	67.9 ± 12	>3000	4.69 ± 0.29	14	>640
21	CHF2	H	70.9 ± 9.4	>3000	3.45 ± 1.2	21	>870
22	OCH3	H	198 ± 48	>3000	6.53 ± 1.8	30	>459
23	N(CH3)2	H	318 ± 8.4	>3000	8.08 ± 1.2	39	>371
24	F	F	604 ± 83	>3000	11.8 ± 3.0	51	>254
25	CN	H	647 ± 150	>3000	12.7 ± 3.1	51	>236
26	CONH2	H	129 ± 26	>3000	3.22 ± 0.78	40	>932

^aData are mean ± SEM of at least three independent experiments conducted in duplicate. None of the compounds had agonist activity at 10 μM.

To determine the effect on opioid antagonist potency by adding a 3,4-double bond to 1, 12, and 15 or 16, we synthesized and evaluated corresponding analogues 27–30 (Table 4). The addition of the 3,4-double bond to 1 to give 27 results in a 6.4-fold increase in κ antagonist potency: K_e = 6.8 nM for 1 (Table 1) compared to a K_e = 1.07 nM for 27 (Table 4). The change also resulted in a small increase in κ relative to μ selectivity. In contrast, the addition of a 3,4-double bond to 12 (Table 2) to give 28 caused a 2.4-fold loss in κ antagonist potency (K_e = 0.37 nM for 12 compared to K_e = 0.88 nM for 28) (Table 4). The change also caused a 20-fold increase in μ antagonist potency (K_e = 239 nM for 12 compared to K_e = 11.8 nM for 28), thus reducing its selectivity for the κ receptor relative to the μ opioid receptor. Compound 29, which is the 3,4-double bond analogue of 13 (Table 2) resulted in very little change in the κ opioid receptor potency, K_e = 15.6 nM for 13 and 9.43 nM for 29. Both compounds have very low selectivity at the κ relative to the μ opioid receptor; however, both compounds were highly selective for the κ relative to the δ opioid receptor. The most striking change resulted from adding a 3,4-double bond to 15 or 16, which both afford analogue 30. The K_e for the κ opioid receptor only changed from 1.26 nM for 15 to K = 1.81 nM for 30. However, the K_e value at the μ opioid receptor changed from 108 nM for 15 to K_e = 1.09 nM for 30. Thus, 15 (Table 2) and 30 (Table 4) have about equal κ opioid receptor potency at both the μ and κ opioid receptors. This results in 30 not being selective for the κ relative to the μ receptor. With a δ/κ value of 1066, 30 is highly selective for both the μ and the κ relative to the δ opioid receptor.

Table 4.

Inhibition of Agonist-Stimulated [³⁵S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors, Effects of Adding a 3,4-Double Bond

compd	R1	R2	Ke (nM)a			μ/κ	δ/κ
			μ, DAMGO	δ, DPDPE	κ, U69,593
27	H	H	37.7 ± 7.9	>3000	1.07 ± 0.11	35	>2804
28	CH3	H	11.8 ± 1.2	>3000	0.88 ± 0.18	13.4	>3410
29	H	CH3	28.9 ± 3.7	>3000	9.43 ± 2.9	3	>318
30	CH3	CH3	1.09 ± 0.11	1930 ± 330	1.81 ± 0.27	0.6	1066

^aData are mean ± SEM of at least three independent experiments conducted in duplicate. None of the compounds had agonist activity at 10 μM.

Compound 12 has two asymmetric centers and thus three possible diastereomers (31–33). To confirm 12 had the best overall properties, the three isomers 31–33 were synthesized and tested. The three compounds had K_e values for the κ opioid receptor ranging between 9.29 and 42.8 nM and thus were much less potent κ opioid receptor antagonists than 12. Compound 31, with a K_e = 9.29 nM and μ/κ and δ/κ values of 103 and >323, respectively, was the most κ potent and selective of the three diastereomers (Table 5), but 12 retained the preferred chirality.

Table 5.

Inhibition of Agonist-Stimulated [³⁵S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors for 12 and Three Isomers

compd	Ke (nM)a			μ/κ	δ/κ
	μ, DAMGO	δ, DPDPE	κ, U69,593
12	239 ± 22	>3000	0.37 ± 0.09	645	>8100
31	955 ± 280	>3000	9.29 ± 3.2	103	>323
32	479 ± 150	>3000	11.5 ± 1.9	42	>261
33	>3000	>3000	42.8 ± 4.0	>70	>70

^aData are mean ± SEM of at least three independent experiments conducted in duplicate. None of the compounds had agonist activity at 10 μM.

To determine the importance of the isopropyl group on the efficacy of 1 and 12, the analogues 34 and 35 were synthesized and evaluated. Replacement of the isopropyl groups in 1 with a hydrogen to give 34 changes its κ opioid receptor K_e value from 6.80 nM to a K_e = 113 nM, a 16.6-fold loss in κ opioid receptor antagonist potency (Table 6). Replacement of the isopropyl group in 12 with a similar sized cyclopropyl group results in a change at the κ opioid receptor from a K_e = 0.37 nM for 12 (Table 2) to a K_e = 5.58 nM for 35, a 15-fold loss in antagonist potency (Table 6). This unexpected finding suggests that the isopropyl group may be essential for the high κ potency and selectivity of 12. Replacing the 7-hydoxyl-d-Tic group in 12 with the d-tyrosine in 36 results in an over 1000-fold loss in κ antagonist potency, showing the importance of the intact 7-hydroxy-d-Tic group to the κ opioid potency and selectivity of 12.

Table 6.

Inhibition of Agonist-Stimulated [³⁵S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors, Importance of the Isopropyl and 7-Hydroxy-d-Tic Groups

compd	Ke (nM)a			μ/κ	δ/κ
	μ, DAMGO	δ, DPDPE	κ, U69,593
34	>3000	>3000	113 ± 21	>27	>27
35	586 ± 130	>3000	5.58 ± 1.6	105	>538
36	>3000	>3000	387 ± 89	>8	>8
37	431 ± 110	>3000	20.5 ± 4.6	21	>146
38	153 ± 37	>3000	11.1 ± 2.9	14	>270
39	569 ± 110	>3000	17.2 ± 5.8	33	>174

^aData are mean ± SEM of at least three independent experiments conducted in duplicate. None of the compounds had agonist activity at 10 μM.

The need for the 7-hydroxytetrahydroisoquinoline group in 12 was further substantiated by the finding that analogues 37 and 38, which have a hydroxynaphthalene and 7-hydroxy-1,2,3,4-tetrahydronaphthalene group, respectively, replacing the 7-hydroxytetrahydroisoquinoline group in 12, had K_e values of 20.5 and 11.1 nM, respectively, at the κ opioid receptor (Table 6). Analogue 39, which has the amide group reduced to a methyleneamino group, has a K_e = 17.2 nM, thus showing that the amide carbonyl group is also needed for the high κ potency of 12.

In Table 7, the effect of replacing the phenolic hydroxyl group in 12 with other substituents was determined and the results are compared to similar analogues of JDTic.²¹ Replacing the 7-hydroxyl substituent in 12 with a carboxamido (41, K_e = 1.37 nM), methoxy (43, K_e = 25.6 nM), and fluoro (45, K_e = 182 nM) substituent resulted in a 3.7-, 69-, and 492-fold loss in κ opioid receptor antagonist potency, respectively. In addition, the compounds were much less selective for the κ relative to the μ opioid receptor. A comparison of the results for 12 to the results for similar substituted JDTic analogues are given in Table 7. While not as potent as JDTic, compound 12, which like JDTic has a hydroxyl group in position 7, is a potent and selective κ opioid receptor antagonist. The JDTic analogue 40, which has a carboxamido substituent in position 7, is as potent as JDTic.²¹ However, compound 41, which has a carboxamido in place of the hydroxyl in 12, is 3.7 times less potent than 12. Additionally, compound 42, which is the 7-methoxy analogue of JDTic, is only 3-fold less potent than JDTic and, like JDTic, is highly selective for the κ opioid receptor relative to the μ and δ opioid receptors.²⁵ In contrast, compound 43 (the methoxy analogue of 12) with a K_e = 25.6 nM at the κ opioid receptor, is 69 times less potent than 12 and is not very selective for κ relative to μ. The 7-fluoro analogue 44, with a K_e = 2.20 nM, is 110-fold less potent than JDTic.²¹ In comparison, the 7-fluoro analogue of 12, compound 45 with a K_e = 182 nM at the κ opioid receptor, is 492-fold less potent than 12. Thus, changes in the 7-hydroxyl group of 12 are more sensitive to similar changes in JDTic and thus do not follow the same SAR found in JDTic.²¹

The difference between the SAR of the new tetrahydroisoquinoline analogues in this study and the SAR of JDTic analogues is also shown by comparing the results obtained for 46 and 47, which are the N-methyl analogues of JDTic and 12, respectively. The JDTic analogue 46, with a K_e = 0.16 nM at the κ opioid receptor, is a potent κ opioid receptor antagonist.²⁶ In contrast, 47 has a K_e = 36.7 nM at the κ opioid receptor and is thus a much weaker κ opioid receptor antagonist.

Table 8 shows a comparison of the calculated physiochemical properties of 12 and 15 to those of JDTic. Both 12 and 15 have TPSA (topological polar surface area) values of 64.60 Ų compared to 84.83 for JDTic. The cLogP values for 12 and 15 are 2.32 and 2.49, respectively, compared to 3.60 for JDTic. The log BB values for 12 and 15 are −0.46 and −0.44, respectively, compared to −0.57 for JDTic. The CNS MPO values for 12 and 15 are 4.5 and 4.1, respectively, compared to a 3.1 value for JDTic. Compounds that have TPSA values less than 76 Å2,^3,27 cLogP values in the range of 2−4,²⁸ calculated logBB values less than −1,^28,29 and CNS MPO values greater than 4,³⁰ are predicted to penetrate the brain; therefore, both compounds 12 and 15 are predicted to penetrate the brain. In addition, CNS penetration has been correlated with lower molecular weights.³¹ The fact that molecular weights of 359.5 and 373.5 Da for 12 and 15 are 106 and 92 Da, respectively, less than JDTic, which is known to penetrate the brain, further suggests that both 12 and 15 will penetrate the brain.

Table 8.

Summary of Calculated Physiochemical Properties for Compounds 12, 15, and JDTic

compd	TPSA	cLogP	log BBa	CNS MPO	MW
JDTic	84.83	3.60	−0.57	3.1	465.6
12	64.60	2.32	−0.46	4.5	359.5
15	64.60	2.49	−0.44	4.1	373.5

^aEquation 6 from ref 29. [log BB = −0.0148 × TPSA + 0.152 × log P + 0.139] was used for calculations of log BB values.

Pharmacokinetic Studies.

The pharmacokinetics of 12 was assessed in plasma and brain following a single dose to determine uptake into brain (Figure 2). In plasma, 12 reached a C_max of 333 ng/mL at 1 h after dosing, while in brain, a C_max of 239 ng/mL was achieved at 4 h post dose (Table 9). The half-life was determined to be 30.7 h in plasma and 57.2 h in brain. The ratio of brain:plasma concentration rose between 1 and 72 h and then declined. Compound 12 crossed the blood—brain barrier and persisted in plasma and brain for 168 h post dose. The AUC_last was approximately 10-fold higher in brain compared with plasma and the clearance from the brain was 11.2 times slower than from the plasma. The behavior of 12 was similar to that of JDTic, which at a dose of 5 mg/kg in male rats had a half-life of 28.4 h in plasma and 51.8 h in brain, and the AUC_last was approximately 7-fold higher in brain.²⁶ Of four JDTic analogues investigated previously, only RTI-194 ((3R)-7-hydroxy-N-[(1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylbutyl]-1,2,3,4-tet- rahydro-3-isoquinoline-carboxamide) had a similar longer half-life in brain, and AUC_last was approximately10 fold higher in brain.²⁶

Figure 2.

Concentration—time and brain to plasma ratio (B/P) plots for 12 in Sprague—Dawley rats after a 5 mg/kg sc dose. Values represent mean ± SD, N = 3 for all data. Where no error bars are visible, the range is smaller than the dimension of the data point. The solid lines showing brain and plasma concentrations (left axis) are the best-fit lines to an apparent terminal elimination phase.

Table 9.

Pharmacokinetic Parameters for 12 in Male Sprague—Dawley Rats

parameter	plasma	brain
t1/2 (h)	30.7	57.2
Cmax (ng/mL or ng/g)	333	239
Tmax (h)	1	4
clearance (mL/h/kg)	2625	234
AUClast (h·ng/mL or h·ng/g)	1864	18765
AUCtotal (h·ng/mL or h·ng/g)	1905	21400
ratio of brain/plasma AUClast	10.07	10.07

CONCLUSIONS

A SAR study directed toward the recently discovered new structural class of κ opioid receptor antagonists based on lead structure 1 involving changes to the piperidine ring, absolute stereochemistry, the 7-hydroxy group on the tetrahydroisoquinoline, and the isopropyl group, led to the identification of the potent and selective κ opioid receptor antagonist 12. Log BB, CNS MPO, and molecular size predicted that 12 would penetrate the brain. Follow up pharmacokinetic studies in rats showed that 12 did indeed readily penetrate the brain. These studies strongly suggest that compound 12 should be considered for further development.

EXPERIMENTAL SECTION

Melting points were determined using a MEL-TEMP II capillary melting point apparatus. Nuclear magnetic resonance (¹H NMR and 13C NMR) spectra were obtained on a Bruker Avance DPX-300 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm) with reference to internal solvent. Mass spectra (MS) were run on a PerkinElmer Sciex API 150 EX mass spectrometer equipped with ESI (turbospray) source. Elemental analyses were performed by Atlantic Microlab Inc., Atlanta, GA. The purity of compounds (>95%) was established by elemental analysis. Optical rotations were measured on an AutoPol III polarimeter, purchased from Rudolf Research. Analytical thin-layer chromatography (TLC) was carried out using EMD silica gel 60 F₂₅₄ TLC plates. TLC visualization was achieved with a UV lamp or in an iodine chamber. Flash column chromatography was done on a CombiFlash Rf system using ISCO prepacked silica gel columns or using EM Science silica gel 60 Å (230−400 mesh). Solvent system: CMA80 (or DMA80) = 80:18:2 CHCl₃ (or CH₂Cl₂):CH₃OH:conc NH₄OH. Unless otherwise stated, reagent-grade chemicals were obtained from commercial sources and were used without further purification. All moisture- and air-sensitive reactions and reagent transfers were carried out under dry nitrogen. See Supporting Information for the synthesis of all intermediate compounds.

General Synthetic Procedures.

General Method 1. Coupling of Amines with Boc-7-hydroxy-d-Tic-OH.

To a solution of the amine (1.0 equiv) in CH₂Cl₂ (30−50 mL) was added Boc-7-hydroxy-d-Tic-OH (1.1 equiv), EDC (1.2 equiv), HOBt (0.11 equiv), and NEt₃ (5.0−8.0 equiv). The mixture was stirred at room temperature overnight. Saturated aqueous NaHCO₃ (30 mL) was added to the mixture and the organic product extracted with CH₂Cl₂ (3 × 30 mL). The combined organic layers were dried (Na₂SO₄), filtered through Celite, and concentrated in vacuo. Purification of the residue was performed on silica gel eluted with CMA80 (or DMA80) in CH₂Cl₂ to provide the desired Boc-protected product that was then subjected to general method 2 for the cleavage of the Boc group. Alternatively, acid in THF (0.1 M) was treated with DCC (1.2 equiv) and HOBt (1.1 equiv). After 1 h at room temperature, the amine (1.2 equiv) was added. If the amine was a hydrochloride salt, NEt₃ (3 equiv) was also added. After 12 h, the reaction mixture was filtered, concentrated, and the residue subjected to silica gel chromatography to afford the desired amide.

General Method 2. Removal of Boc-Protection: Method 2a.

The Boc-protected compound in CH₃CN was treated with HCl (4 M in 1,4-dioxane, 4 equiv) and stirred at room temperature overnight. The solvent was then removed in vacuo, and the residue was neutralized with 1 N NaOH until the pH of 8−9 was obtained. Purification of the residue on silica gel and eluted with CMA80 (or DMA80) in CH₂Cl₂ provided the product. Method 2b: Alternatively, the Boc-protected compound was dissolved in CH₃OH (5 mL) then treated with 6 N aq HCl (5 mL) and stirred at room temperature overnight. The solvent was evaporated and the residue purified as above.

Preparation of the Hydrochloride Salts.

Hydrochloride salts were prepared by dissolving the compound freebase in cold methanol, adding a slight excess of 2 N HCl in diethyl ether, then evaporating to dryness under vacuum.

(3R)-N-[(1S)-1-(Azepan-1-ylmethyl)-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (3).

Compound 50 (0.15 g, 0.4 mmol) and homopiperidine (110 mg, 1 mmol) in 1,2-dichloroethane (3 mL) were treated with sodium triacetoxyborohydride (200 mg, 0.9 mmol). After 12 h, the solution was washed with saturated aqueous NaHCO₃, dried (Na₂SO₄), and subjected to chromatography on silica gel eluting with a gradient up to 10% CH₃OH in EtOAc to afford 99 mg (52%) of the Boc protected intermediate. The intermediate was dissolved in CH₂Cl₂ (10 mL) and treated with BBr₃ (5 mL, 1.0 M in CH₂Cl₂, 5 mmol) at −78 °C, then allowed to warm to room temperature overnight. The solution was cooled to −78 °C, quenched with methanol, concentrated, and then dissolved in dilute aqueous NH₄OH (1:1). The aqueous solution was brought to a reflux, then cooled and the solvent removed in vacuo. The residue obtained was purified by chromatography on silica gel eluted with a gradient of 0−50% CMA80 in CH₂Cl₂ to afford 3 freebase. ¹H NMR (300 MHz, CDCl₃) δ 7.23 (d, J = 9.42 Hz, 1H), 6.85 (d, J = 8.29 Hz, 1H), 6.49 (d, J = 8.29 Hz, 1H), 6.43 (s, 1H), 4.14 (td, J = 7.30, 14.41 Hz, 1H), 3.57−3.76 (m, 2H), 3.30 (dd, J = 5.09, 11.49 Hz, 1H), 2.75−3.03 (m, 5H), 2.69 (d, J = 6.78 Hz, 2H), 2.35 (dd, J = 11.96, 15.92 Hz, 1H), 1.77−1.94 (m, 1H), 1.46−1.78 (m, 8H), 0.94 (dd, J = 1.79, 6.69 Hz, 6H). 13C NMR (75 MHz, CDCl₃) δ 173.5, 154.9, 137.2, 130.4, 125.1, 113.8, 112.2, 57.4, 56.8, 55.0, 50.6, 48.1, 31.4, 29.7, 27.4, 25.5, 19.1, 18.0. MS (ESI) m/z 360.4 (M + H)⁺. The freebase 3 was converted to 25.5 mg (14% from 50) of the dihydrochloride salt as a pale-yellow powder: mp 160−164 °C (fusion), [α]d²⁵ +65.5° (c 0.165, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₂·1.5H₂O) C, H, N.

(3R)-7-Hydroxy-N-[(1S)-2-methyl-1-(pyrrolidin-1-ylmethyl)-propyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (4).

Compound 53a (1.03 g, 6.02 mmol) was coupled with Boc-7-hydroxy-d-Tic-OH (1.77 g, 6.02 mmol) following the protocol described in general method 1 to provide the Boc protected product Boc-4 (1.43 g, 55% yield): ¹H NMR (300 MHz, CDCl₃) δ 7.81 (br s, 1H), 6.91 (d, J = 8.10 Hz, 1H), 6.48−6.70 (m, 2H), 5.94−6.31 (m, 1H), 4.58−4.87 (m, 1H), 4.31−4.57 (m, 2H), 3.56−3.81 (m, 1H), 3.07−3.31 (m, 2H), 2.91 (d, J = 10.17 Hz, 1H), 2.08−2.35 (m, 4H), 1.71−1.91 (m, 1H), 1.57 (br s, 4H), 1.38−1.50 (m, 9H), 1.07−1.34 (m, 1H), 0.77 (dd, J = 6.69, 13.28 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.5, 134.0, 129.0, 123.8, 114.7, 113.1, 81.3, 60.4, 57.9, 56.5, 54.0, 53.1, 44.9, 44.5, 30.9, 30.2, 28.3, 23.4, 18.8, 17.5. MS (ESI) m/z 432.3 (M + H)⁺. Boc-4 (1.43 g, 3.32 mmol) was subjected to Boc-cleavage according to general method 2b to provide amine 4 (839 mg, 76% yield): ¹H NMR (300 MHz, CDCl₃) δ 7.14 (d, J = 10.17 Hz, 1H), 6.78 (d, J = 8.10 Hz, 1H), 6.22−6.39 (m, 2H), 5.75−6.17 (m, 1H), 4.16 (t, J = 10.55 Hz, 1H), 3.59−3.73 (m, 1H), 3.47−3.58 (m, 1H), 3.20 (dd, J = 5.27, 11.68 Hz, 1H), 2.95 (t, J = 12.15 Hz, 1H), 2.84 (dd, J = 5.09, 16.58 Hz, 1H), 2.72 (br s, 2H), 2.61 (br s, 2H), 2.27 (dd, J = 2.64, 12.43 Hz, 1H), 2.01−2.15 (m, 1H), 1.70−1.92 (m, 5H), 0.75−1.02 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.4, 155.1, 137.6, 130.8, 125.0, 113.2, 112.0, 57.2, 56.5, 54.0, 51.8, 48.5, 32.0, 28.9, 23.1, 19.0, 18.0. MS (ESI) m/z 332.5 (M + H)⁺. A white solid was obtained of the dihydrochloride salt of 4. Mp 148 °C (dec.); [α]d²⁵ = +67.3° (c 1.1, CH₃OH). Anal. (C₁₉H₃₁Cl₂N₃O₂·H₂O) C, H, N. To make a fumarate salt, 4 (376.6 mg, 1.14 mmol) was dissolved in chloroform (2 mL) in a vial and treated with fumaric acid in MeOH (2.0 equiv. 3.5 mL, 0.65 M in MeOH) and allowed to stand in a refrigerator overnight. The excess solvent was then removed under reduced pressure, and the residue salt was redissolved in minimal amount of MeOH. The fumarate salt was recrystallized from MeOH using diethyl ether to provide 329 mg of 4·2C₄H₄O₄ as a beige solid; mp 152 °C (dec.); [α]d²⁵ = +65.0° (c 1.1, CH₃OH). Anal. (C₂₇H₃₇N₃O₁₀·1.25H₂O) C, H, N.

(3R)-N-[(1S)-1-(7-Azabicyclo[2.2.1]hept-7-ylmethyl)-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (5).

A solution of dicyclohexylcarbodiimide (DCC) (120 mg, 0.58 mmol) in THF (2 mL) was treated with a THF (3 mL) solution of HOBt (72 mg, 0.54 mmol) and 7-hydroxy-Boc-d-Tic-OH (150 mg, 0.51 mmol) and stirred at room temperature for 1 h. Amine 53b (140 mg, 0.77 mmol) and NEt₃ (0.2 mL, 1.4 mmol) were added to the suspension and stirred for an additional 12 h at room temperature. The solids were removed by filtration and the filtrate concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel eluted with a gradient of 0−50% DMA80 in CH₂Cl₂. The fractions containing the product were concentrated and the residue dissolved in CH₃OH (5 mL) and treated with 6 N HCl aq (5 mL). After 12 h, the solvent was removed in vacuo and the residue partitioned between CH₂Cl₂ and 7 M aq NH₄OH. The organic layer was dried (Na₂SO₄) and concentrated. The resulting residue was subjected to chromatography on silica gel eluting with a gradient of 0−75% DMA80 in CH₂Cl₂ to afford 36.4 mg (10%) of the freebase 5. ¹H NMR (300 MHz, CDCl₃) δ 7.11 (d, J = 9.61 Hz, 1H), 6.78 (d, J = 8.10 Hz, 1H), 6.44 (dd, J = 2.35, 8.19 Hz, 1H), 6.32 (d, J = 2.26 Hz, 1H), 3.86−4.00 (m, 1H), 3.36−3.68 (m, 4H), 3.08 (dd, J = 5.09, 11.68 Hz, 1H), 2.81 (dd, J = 4.90, 16.39 Hz, 1H), 2.51−2.71 (m, 1H), 2.40 (dd, J = 2.54, 12.90 Hz, 1H), 2.13−2.30 (m, 1H), 1.63−1.91 (m, 5H), 1.23−1.44 (m, 4H), 0.72−0.94 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.4, 155.2, 137.4, 130.6, 125.0, 113.8, 112.1, 59.8, 56.7, 52.2, 48.4, 48.0, 31.7, 29.7, 28.2, 18.8, 18.3. The freebase was converted into a pale-yellow powder as the dihydrochloride salt: MS (ESI) m/z 358.4 (M + H)⁺; mp 194−198 °C (fusion); [α]d²⁵ = +72° (c 0.10, CH₃OH). Anal. (C₂₁H₃₃Cl₂N₃O₂·1.5H₂O) C, H, N.

(3R)-7-Hydroxy-N-[(1S)-2-methyl-1-(morpholin-4-ylmethyl)-propyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (6).

A solution of 50 (0.15 g, 0.4 mmol) in dichloroethane (3 mL) was treated with morpholine (0.09 mL, 1 mmol) then sodium triacetoxyborohydride (200 mg, 0.9 mmol). After 12 h, the solution was washed with saturated aqueous NaHCO₃, dried (Na₂SO₄), and subjected to chromatography on silica gel eluting with EtOAc to afford 100 mg (54%) of the Boc-protected intermediate. The Boc intermediate was dissolved in CH₂Cl₂ (10 mL), cooled to −78 °C, treated with BBr₃ (5 mL, 1.0 M in CH₂Cl₂, 5 mmol), and allowed to warm to room temperature overnight. The solution was cooled to −78 °C, quenched with methanol, concentrated, and then dissolved in 50% dilute aqueous NH₄OH. The aqueous solution was brought to a reflux, then cooled and concentrated. The residue was subjected to chromatography on silica gel eluting with a gradient of 0−50% CMA80 in CH₂Cl₂ to afford 6. ¹H NMR (300 MHz, CDCl₃) δ 7.10 (d, J = 9.80 Hz, 1H), 6.91 (d, J = 8.29 Hz, 1H), 6.60 (dd, J = 2.45, 8.29 Hz, 1H), 6.45 (d, J = 2.07 Hz, 1H), 4.11−4.18 (m, 1H), 3.63−3.78 (m, 6H), 3.37 (dd, J = 5.18, 10.83 Hz, 1H), 2.98 (dd, J = 5.09, 16.39 Hz, 1H), 2.47−2.68 (m, 4H), 2.29−2.43 (m, 3H), 1.85 (dd, J = 6.78, 11.87 Hz, 1H), 0.89−0.96 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 154.7, 137.1, 130.4, 125.3, 114.0, 112.2, 66.6, 60.5, 56.7, 53.8, 49.7, 47.8, 31.1, 29.8, 19.2, 17.8. MS (ESI) m/z 348.3 (M + H)⁺. The freebase was converted to 42.2 mg (44%) of the dihydrochloride salt as a white powder: mp 186−190 °C (fusion), [α]d²⁵ = +62° (c 0.16, CH₃OH). Anal. (C₁₉H₃₁Cl₂N₃O₃·1.5H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-methylpiperizin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Trihydrochloride (7).

Diamine 53c (937 mg, 5.06 mmol) and Boc-7-hydroxy-d-Tic-OH (1.6 g, 5.31 mmol) in a solvent mixture of THF (4 mL) and CH₃CN were coupled using HBTU (2.32 g, 6.07 mmol) (instead of EDC/HOBt) and triethylamine (2.8 mL, 20.5 mmol) to provide Boc-protected product Boc-7 (1.82 g mg, 78% yield): ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J = 8.1 Hz, 1H), 6.67 (d, J = 8.3 Hz, 1H), 6.65 (s, 1H), 5.97−6.41 (m, 1H), 4.71−4.80 (br s, 1H), 4.40−4.58 (m, 2H), 3.83 (br s, 1H), 3.18−3.24 (dd, J = 2.5, 15.3 Hz, 1H), 2.95−3.02 (m, 1H), 2.74−2.82 (m, 2H), 2.20−2.25 (m, 2H), 1.85−1.92 (m, 2H), 1.51 (s, 9H), 1.40−1.60 (m, 3H), 1.12−1.33 (m, 6H), 0.85−0.95 (m, 3H), 0.45−0.59 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.2, 134.0, 129.1, 123.9, 114.7, 113.2, 81.1, 65.8, 59.5, 57.9, 54.3, 53.8, 51.2, 45.1, 34.1, 31.4, 30.8, 30.5, 29.6, 28.4 (3Cs), 21.8, 18.5, 16.6. MS (ESI) m/z 460.4 (M + H)⁺. Boc-7 (689 mg, 1.5 mmol) was treated according to the general method 2b for removal of the Boc-protection to provide 348 mg (64%) of the freebase amine 7: ¹H NMR (300 MHz, CD₃OD) δ 6.97 (d, J = 8.3 Hz, 1H), 6.66 (dd, J = 8.4, 2.4 Hz, 1H), 6.56 (d, J = 2.5 Hz, 1H), 4.07−4.16 (m, 2H), 3.95−4.01 (m, 1H), 3.78−3.82 (dd, J = 5.0, 4.4 Hz, 1H), 2.87−3.09 (m, 2H), 2.60−2.80 (m, 6H), 2.48 (s, 3H), 2.45−2.57 (m, 3H), 1.78−1.87 (m, 1H), 0.93−0.96 (m, 6H). ¹³C NMR (75 MHz, CD₃OD) δ 174.0, 157.1, 135.4, 131.1, 124.7, 115.6, 113.5, 60.7, 58.4, 57.7, 55.4, 52.8, 52.6, 47.1, 45.1, 32.2, 31.8, 20.2, 18.5, 18.3. MS (ESI) m/z 360.3 (M + H)⁺. A white solid was obtained as the trihydrochloride salt of 7: mp >240 °C; [α] ²⁵ = +71.2° (c 1.1, CH₃OH) Anal. (C₂₀H₃₅Cl₃N₄O₂·1.75H₂O) C, H, N.

(3R)-N-{(1S)-1-[(4,4-Diethylamino)methyl]-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (8).

Diamine 53d (336 mg, 1.45 mmol) in CH₂Cl₂ (30 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (426 mg, 1.45mmol) following the protocol described in general method 1 to provide 200 mg (32%) of the product Boc-8: 1H NMR (300 MHz, CDCl₃) δ 7.78 (br s, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.66 (dd, J = 2.6, 8.3 Hz, 1H), 6.61 (s, 1H), 6.25−6.40 (m, 1H), 4.70−4.85 (m, 1H), 4.43−4.55 (m, 2H), 3.77 (br s, 1H), 3.17−3.24 (dd, J = 3.8, 15.6 Hz, 1H), 2.95−3.02 (dd, J = 6.2, 16.2 Hz, 1H), 2.51−2.78 (m, 2H), 2.25−2.50 (m, 3H), 1.83−2.12 (m, 2H), 2.05 (s, 1H), 1.49 (s, 9H), 1.18−1.35 (m, 4H), 0.78−1.07 (m, 11H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.6, 134.1, 129.1, 124.2, 114.6, 113.2, 81.5, 56.7, 52.9, 51.7, 46.5, 44.9, 30.9, 29.4, 28.4 (3Cs), 21.0, 18.8, 17.4, 14.1. MS (ESI) m/z 434.5 (M + H)⁺. A solution of Boc-8 (517 mg, 1.19 mmol) in CH₃CN (20 mL) was subjected to Boc cleavage following the general method 2a to provide the freebase 8: ¹H NMR (300 MHz, CD₃OD) δ 6.98 (d, J = 8.6 Hz, 1H), 6.68 (dd, J = 8.4, 2.6 Hz, 1H), 6.57 (d, J = 2.6 Hz, 1H), 4.06−4.21 (m, 2H), 4.11 (s, 1H), 3.83−3.89 (dd, J = 5.1, 11.3 Hz, 1H), 3.10−3.33 (m, 5H), 3.36 (s, 1H), 2.84−3.07 (m, 2H), 1.84−1.95 (m, 1H), 1.28−1.34 (m, 5H), 0.91−1.05 (m, 6H). ¹³C NMR (75 MHz, CD₃OD) δ 174.5, 157.2, 134.7, 131.2, 124.7, 115.7, 113.5, 58.1, 55.4, 51.2, 47.3, 32.6, 31.3, 20.0, 18.5, 9.2. MS (ESI) m/z 334.5 (M + H)⁺. The freebase was converted into a white solid as the dihydrochloride salt (332 mg, 84%): mp 152−155 °C; [α] ²⁵ = +70.5° (c 1.1, CH₃OH). Anal. (C₂₁H₃₇Cl₂N₃O₂·1.25H₂O) C, H, N.

(3R)-N-{(1S)-1-[(Dipropylamino)methyl]-2-methylpropyl}−7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (9).

To a solution of 53e (544 mg, 2.1 mmol) in CH₂Cl₂ (45 mL) was added Boc-7-hydroxy-d-Tic-OH (677 mg, 2.31 mmol), EDC (483 mg, 2.52 mmol), HOBt (35.3 mg, 0.23 mmol), and NEt₃ (0.7 mL, 5.04 mmol) and reacted as described in general method 1 to provide Boc-9 (864.8 mg, 89% yield): ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 9.5 Hz, 1H), 6.59 (s, 1H), 5.90−6.00 (m, 1H), 4.38−4.67 (m, 3H), 3.69 (br s, 1H), 3.47 (s, 1H), 3.16 (d, J = 11.4 Hz, 1H), 2.94−3.02 (dd, J = 6.7, 4.7 Hz, 1H), 1.94−2.40 (m, 7H), 1.50 (s, 9H), 1.26−1.34 (m, 4H), 0.76−0.85 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 172.0, 155.6, 134.2, 129.0, 124.1, 123.9, 114.7, 113.1, 81.5, 56.7, 55.9, 54.2, 51.9, 50.3, 44.9, 31.1, 30.0, 28.9, 28.4, 28.3, 19.6, 18.8, 16.9, 11.8. MS (ESI) m/z 462.8 (M + H)⁺. Boc-9 was subjected to Boc cleavage following general method 2a to provide 533 mg (79% yield) of the freebase 9: ¹H NMR (300 MHz, CD₃OD) δ 6.91 (d, J = 8.1 Hz, 1H), 6.61 (dd, J = 8.3, 2.9 Hz, 1H), 6.50 (d, J = 2.9 Hz, 1H), 3.90−4.30 (m, 1H), 3.92 (d, J = 2.2 Hz, 1H), 3.53−3.58 (dd, J = 10.7, 4.5 Hz, 1H), 3.37 (s, 1H), 2.92−2.99 (dd, J = 5.2, 3.4 Hz, 1H), 2.74−2.82 (m, 1H), 2.33−2.64 (m, 6H), 1.91−1.97 (m, 1H), 1.43−1.56 (m, 4H), 0.88−1.04 (m, 12H). ¹³C NMR (75 MHz, CD₃OD) δ 175.4, 156.9, 137.3, 130.9, 125.6, 115.1, 113.3, 58.3, 57.4, 57.2, 56.0, 53.0, 48.1, 32.3, 31.6, 21.0, 20.8, 20.3, 19.3, 17.7, 12.3. MS (ESI) m/z 362.4 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 9: mp 160 °C (sublime); [α]d²⁵ = +55.1° (c 1.1, CH₃OH). Anal. (C₂₁H₃₇Cl₂N₃O₂·2H₂O) C, H, N.

(3R)-N-{(1S)-1-{[Bis(2-methylpropyl)amino]methyl}−2-methyl-propyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (10).

The diamine 53f (865 mg, 2.82 mmol) in CH₂Cl₂ (30 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (803 mg, 2.74 mmol) following the protocol described in general method 1 to provide 537 mg (40%) of Boc-10: ¹H NMR (300 MHz, CDCl₃) δ 7.78 (br s, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.66 (dd, J = 2.6, 8.3 Hz, 1H), 6.61 (s, 1H), 6.25−6.40 (m, 1H), 4.70−4.85 (m, 1H), 4.43−4.55 (m, 2H), 3.77 (br s, 1H), 3.17−3.24 (dd, J = 3.8, 15.6 Hz, 1H), 2.95−3.02 (dd, J = 6.2, 16.2 Hz, 1H), 2.51−2.78 (m, 2H), 2.25−2.50 (m, 3H), 2.05 (s, 1H), 1.90−2.18 (m, 6H), 1.50 (s, 9H), 1.46−1.70 (m, 3H), 0.70−0.96 (m, 18H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.6, 134.3, 129.0, 124.3, 114.8, 113.1, 81.5, 64.1, 56.9, 56.6, 52.0, 44.9, 31.4, 29.4, 28.4 (3Cs), 28.0, 26.4, 26.3, 20.9, 20.8, 19.6, 15.7. MS (ESI) m/z 490.7 (M + H)⁺. Boc-10 (655 mg, 1.34 mmol) in CH₃CN (20 mL) was subjected to Boc cleavage following the general method 2a afford the freebase 10: ¹H NMR (300 MHz, CD₃OD) δ 6.93 (d, J = 8.6 Hz, 1H), 6.61 (dd, J = 2.8, 8.4 Hz, 1H), 6.51 (d, J = 2.1 Hz, 1H), 3.96−4.01 (m, 2H), 3.95 (s, 1H), 3.53−3.59 (dd, J = 4.7, 11.5 Hz, 1H), 2.94−3.01 (dd, J = 4.3, 15.8 Hz, 1H), 2.72−2.81 (dd, J = 10.5, 15.8 Hz, 1H), 2.48−2.55 (dd, J = 7.6, 13.4 Hz, 1H), 2.23−2.30 (dd, J = 7.2, 13.3 Hz, 1H), 2.05−2.20 (m, 5H), 1.67−1.80 (m, 2H), 0.89−1.05 (m, 18H). ¹³C NMR (75 MHz, CD₃OD) δ 175.1, 156.9, 137.1, 130.8, 125.5, 115.1, 113.2, 58.9, 58.2, 53.3, 47.9, 32.4, 30.7, 27.7, 21.5, 20.5, 16.8. MS (ESI) m/z 374.5 (M + H)⁺. The freebase was converted into a white solid as the dihydrochloride salt (332 mg, 72% yield): mp 178−180 °C; [α]d²⁵ = +75.5° (c 1.1, CH₃OH). Anal. (C₂₂H₃₇Cl₂N₃O₂·1.5H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(2-oxopiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride (11).

Compound 57 (400 mg, 2.2 mmol) was coupled with Boc-7-hydroxy-d-Tic-OH (638 mg, 2.2 mmol) following the protocol described in general method 1 to provide Boc-11 (389 mg, 39%): ¹H NMR (300 MHz, CDCl₃) δ 6.86−7.15 (m, 1H), 6.50−6.77 (m, 2H), 6.34 (br s, 1H), 4.73 (br s, 1H), 4.55 (d, J = 16.58 Hz, 2H), 4.25−4.48 (m, 1H), 3.73−4.02 (m, 1H), 3.25 (br s, 1H), 2.87−3.21 (m, 4H), 2.67 (br s, 1H), 2.11−2.33 (m, 2H), 1.62−1.84 (m, 2H), 1.40−1.62 (m, 12H), 0.86 (br s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 172.1, 171.4, 155.9, 134.3, 128.8, 123.8, 114.4, 113.0, 80.9, 56.6, 54.9, 51.8, 47.6, 45.0, 44.3, 42.1, 31.8, 30.6, 30.3, 28.3, 22.7, 20.8, 19.0, 17.7. MS (ESI) m/z 460.3 (M + H)⁺. Boc-11 (370 mg, 0.81 mmol) was subjected to Boc-cleavage according to general method 2a to provide the amine 11 (245 mg, 85%): ¹H NMR (300 MHz, CDCl₃) δ 7.09−7.38 (m, 1H), 6.85 (d, J = 8.29 Hz, 1H), 6.54−6.71 (m, 1H), 6.40−6.53 (m, 1H), 4.21−4.40 (m, 1H), 3.99−4.19 (m, 1H), 3.48−3.79 (m, 3H), 3.34 (dd, J = 4.52, 11.30 Hz, 1H), 2.88−3.19 (m, 2H), 2.65 (dd, J = 2.83, 13.37 Hz, 1H), 2.20−2.53 (m, 3H), 1.38−1.89 (m, 5H), 1.13−1.34 (m, 1H), 0.70−1.01 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.6, 171.4, 155.3, 136.8, 130.0, 124.7, 113.9, 112.1, 56.9, 51.4, 48.7, 47.8, 44.8, 41.8, 32.2, 30.8, 22.9, 20.9, 19.4, 18.3. MS (ESI) m/z 360.4 (M + H)+. A white solid was obtained as the hydrochloride salt of 11: mp 154−157 °C (dec.); [α]d²⁵ = +68.8° (c 1.1, CH₃OH). Anal. (C₂₀H₃₀ClN₃O₃·0.5H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-methylpiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (12).

Compound 60a (908 mg, 3.53 mmol) in CH₂Cl₂ (50 mL) was treated with Boc-7-hydroxy-d-Tic-OH (1.14 g, 3.88 mmol), EDC (812 mg, 4.23 mmol), HOBt (59 mg, 0.39 mmol), and NEt₃ (1.2 mL, 8.48 mmol) and reacted as described in general method 1 to provide 1.1 g (68%) of the Boc-12: ¹H NMR (300 MHz, CDCl₃) δ 6.96 (d, J = 9.2 Hz, 1H), 6.65 (d, J = 9.7 Hz, 1H), 5.90−6.00 (m, 1H), 6.60 (s, 1H), 4.41−4.58 (m, 2H), 3.80 (br s, 1H), 3.45 (s, 1H), 3.17−3.29 (m, 1H), 2.95−3.02 (d, J = 11.4 Hz, 1H), 2.40−2.63 (m, 2H), 2.03−2.25 (m, 2H) 1.70−1.87 (m, 3H), 1.50 (s, 9H), 1.40−1.60 (m, 3H), 1.02−1.30 (m, 3H), 0.78−0.88 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 171.6, 155.6, 134.1, 129.1, 123.9, 114.7, 113.1, 81.4, 59.3, 56.9, 56.5, 54.3, 53.5, 51.3, 45.1, 44.8, 34.2, 34.0, 30.5, 30.2, 28.4 (3Cs), 21.8, 18.9, 17.4. MS (ESI) m/z 460.2 (M + H)⁺. Boc-12 (1.1 g, 2.3 mmol) in CH₃CN (20 mL) was subjected to Boc-cleavage using general method 2a to provide compound 12 (594 mg, 72%) as the freebase: ¹H NMR (300 MHz, CD₃OD) δ 6.96 (d, J = 8.7 Hz, 1H), 6.68 (dd, J = 8.4, 2.9 Hz, 4.16−4.21 (m, 1H), 1H), 6.58 (d, J = 2.5 Hz, 1H), 4.09 (d, J = 3.3 Hz, 1H), 3.96−4.06 (m, 1H), 3.83−3.89 (dd, J = 5.6, 5.2 Hz, 1H), 3.58−3.64 (m, 1H), 3.37 (s, 1H) 3.31−3.39 (m, 2H), 3.10−3.17 (m, 2H), 3.00−3.07 (m, 1H), 2.86−2.96 (m, 2H), 2.71−2.79 (m, 1H), 1.96 (s, 1H), 1.78−1.91 (m, 3H), 1.39−1.63 (m, 3H), 0.97−1.00 (m, 9H). ¹³C NMR (75 MHz, CD₃OD) δ 174.5, 157.2, 135.0, 131.2, 124.5, 115.7, 113.5, 60.7, 58.1, 55.5, 53.5, 51.1, 50.0, 47.5, 32.6, 32.5, 31.5, 30.1, 21.6, 20.0, 18.5. MS (ESI) m/z 360.3 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 12: mp 180 °C (dec.); [α]d²⁵ = +65° (c 1.1, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₂·1.25H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[((3RS)-3-methylpiperidin-1-yl)methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (13).

Diamine 60b (839 mg, 4.55 mmol) and Boc-7-hydroxy-d-Tic-OH (1.6 g, 5.46 mmol) were coupled according to the general method 1 to provide desired product Boc-13 (1.6 g, 59%) as a mixture of diastereomers: ¹H NMR (300 MHz, CDCl₃) δ 6.82−7.18 (m, 2H), 6.51−6.82 (m, 3H), 5.85−6.40 (m, 1H), 4.68 (br s, 1H), 4.50−4.64 (m, 1H), 4.29−4.49 (m, 1H), 3.65−3.92 (m, 1H), 3.08−3.34 (m, 2H), 2.81−3.08 (m, 1H), 2.58 (br s, 2H), 1.95−2.30 (m, 2H), 1.85 (d, J = 15.45 Hz, 1H), 1.69 (d, J = 9.98 Hz, 1H), 1.50 (s., 9H), 1.34−1.61 (m, 4H), 0.67−0.97 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 171.6, 155.5, 134.1, 129.1, 124.0, 114.7, 113.0, 81.1, 62.6, 60.4, 59.3, 54.6, 53.2, 51.4, 44.9, 32.8, 30.7, 30.1, 28.4, 28.3 (3 C’s), 25.3, 19.6, 18.8, 17.5. MS (ESI) m/z 460.5 (M + H)⁺. Boc-13 (1.24 g, 2.69 mmol) was treated as described in general method 2a to remove compound 13 was obtained as a mixture of diastereomers: 1H NMR (300 MHz, CD₃OD) δ 6.86−6.95 (m, 1H), 6.61 (dd, J = 2.45, 8.10 Hz, 1H), 6.49 (d, J = 2.26 Hz, 1H), 3.97−4.09 (m, 1H), 3.83−3.97 (m, 2H), 3.44−3.67 (m, 3H), 2.87−3.09 (m, 2H), 2.64−2.87 (m, 2H), 2.32−2.61 (m, 2H), 1.75−2.00 (m, 2H), 1.45−1.75 (m, 4H), 0.77−1.06 (m, 9H). 13C NMR (75 MHz, CD₃OD) δ 175.3, 156.8, 137.4, 130.9, 125.6, 115.0, 113.3, 66.9, 63.8, 62.7, 61.7, 58.2, 55.8, 54.5, 52.3, 34.0, 32.3, 26.4, 20.0, 18.5, 18.0. MS (ESI) m/z 360.4 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 13: mp 178 °C (dec.); [α]d²² = +75.5° (c 1.1, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₂·H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[((2RS)-2-methylpiperidin-1-yl)methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (14).

The diamine 60c (1.37 g, 5.34 mmol) and Boc-7-hydroxy-d-Tic-OH (1.57 g, 5.34 mmol) were coupled according to the general method 1 to provide desired product Boc-14 (1.1 g, 44%) as a mixture of diastereomers: ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J = 9.3 Hz, 1H), 6.67 (d, J = 7.6 Hz, 1H), 6.48 (d, J = 3.2 Hz, 1H), 5.70−6.40 (br m, 3H), 4.70−4.84 (m, 1H), 4.43−4.58 (m, 2H), 3.98 (br s, 1H), 3.63−3.85 (m, 3H), 3.25−3.60 (m, 3H), 3.13−3.23 (m, 1H), 2.82−3.05 (m, 2H), 2.10−2.60 (m, 2H), 1.78−1.98 (m, 1H), 1.65−1.69 (m, 1H), 1.50 (s, 9H), 1.30−1.45 (m, 1H), 1.02−1.29 (m, 2H), 0.74−0.96 (m, 6H). 13C NMR (75 MHz, CDCl₃) δ 173.4, 155.6, 135.1, 134.0, 130.0, 129.1, 124.0, 114.5, 113.1, 81.6, 62.4, 56.0, 52.2, 47.1, 34.2, 33.8, 30.7, 29.9, 28.4 (3Cs), 25.7, 23.2, 18.7, 16.4. MS (ESI) m/z 460.5 (M + H)⁺. Boc-14 (1.1 g, 2.4 mmol) was treated according to the general method 2a for removal of the Boc-protection to provide 853 mg, (99% yield) of the freebase compound 14 as a mixture of diastereomers: ¹H NMR (300 MHz, CD₃OD) δ 6.95 (d, J = 8.3 Hz, 1H), 6.63 (dd, J = 8.2, 2.6 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 4.01−4.10 (m, 1H), 3.93−3.95 (dd, J = 5.6, 5.2 Hz, 1H), 3.53−3.64 (m, 2H), 3.35 (s, 1H), 3.31−3.39 (m, 2H), 3.10−3.17 (m, 2H), 3.00−3.07 (m, 1H), 2.80−2.99 (m, 4H), 2.38−2.56 (m, 1H), 1.80−1.87 (m, 1H), 1.55−1.72 (m, 2H), 1.39−1.47 (m, 1H), 0.85−0.96 (m, 9H). ¹³C NMR (75 MHz, CD₃OD) δ 175.4156.7, 137.2, 131.0, 125.6, 115.1, 113.3, 67.0, 63.5, 61.9, 61.2, 58.3, 52.2, 50.0, 43.1, 32.6, 32.2, 31.8, 20.0, 18.5, 18.1. MS (ESI) m/z 360.3 (M + H)+. A white solid was obtained as the dihydrochloride salt of 14: mp 164−166 °C; [α]d²³ = +76.2° (c 1.1, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₂·1.25H₂O) C, H, N.

(3R)-N-[(1S)-1-{[(3RS,4RS)-3,4-Dimethylpiperidin-1-yl]methyl}−2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (15).

Compound 60d (864 mg, 4.40 mmol) in CH₂Cl₂ (30 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (1.36 g, 4.62 mmol) following the protocol described in general method 1 to provide Boc-15 (1.38 g, 66%) as a mixture of diastereomers: ¹H NMR (300 MHz, CDCl₃) δ 6.98 (d, J = 8.29 Hz, 1H), 6.67 (d, J = 8.29 Hz, 1H), 6.59 (br s, 1H), 5.76−6.35 (m, 1H), 4.64−4.93 (m, 1H), 4.48−4.63 (m, 1H), 4.33−4.48 (m, 1H), 3.80 (br s, 1H), 3.08−3.32 (m, 2H), 2.81−3.08 (m, 1H), 2.44−2.70 (m, 1H), 1.80−2.39 (m, 6H), 1.69 (d, J = 11.30 Hz, 1H), 1.45−1.58 (m, 11H), 1.29−1.42 (m, 1H), 0.71−1.04 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 171.6, 155.6, 134.0, 129.1, 124.1, 114.7, 113.0, 81.4, 62.4, 59.0, 58.2, 56.5, 54.9, 53.6, 51.6, 44.7, 37.3, 34.3, 33.7, 32.0, 30.6, 29.8, 28.4 (3 C’s), 19.2, 17.1. MS (ESI) m/z 474.7 (M + H)⁺. Boc-15 (1.38 g, 2.92 mmol) in CH₃CN (20 mL) was subjected to Boc cleavage following the general method 2a to provide 15 (1.05 g, 96%) as a mixture of diastereomers: ¹H NMR (300 MHz, CD₃OD) δ 6.93 (d, J = 8.29 Hz, 1H), 6.61 (dd, J = 2.54, 8.19 Hz, 1H), 6.44−6.54 (m, 1H), 3.85−4.06 (m, 2H), 3.44−3.64 (m, 1H), 2.67−3.01 (m, 3H), 2.09−2.63 (m, 4H), 1.74−1.98 (m, 2H), 1.39−1.72 (m, 3H), 1.05−1.37 (m, 2H), 0.81−1.02 (m, 12H). ¹³C NMR (75 MHz, CD₃OD) δ 175.3, 156.8, 137.4, 130.9, 125.6, 115.0, 113.2, 63.7, 61.5, 58.2, 56.1, 54.8, 54.1, 53.2, 52.5, 38.7, 35.4, 35.1, 33.6, 32.3, 20.0, 17.9. MS (ESI) m/z 374.3 (M + H)⁺. A beige solid was obtained as the dihydrochloride salt of 15: mp 158−162 °C; [α]d²³ = +69.4° (c 1.1, CH₃OH). Anal. (C₂₂H₃₇Cl₂N₃O₂·0.75H₂O) C, H, N.

(3R)-N-[(1S)-1-{[(3RS,4SR)-3,4-Dimethylpiperidin-1-yl]methyl}−2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (16).

Compound 60e (184 mg, 0.927 mmol) in CH₂Cl₂ (30 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (327 mg, 1.11 mmol) following the protocol described in general method 1 to provide Boc-16 (276 mg, 63%) as a mixture of diastereomers: ¹H NMR (300 MHz, CDCl₃) δ 6.99 (d, J = 8.48 Hz, 1H), 6.53−6.81 (m, 2H), 4.69 (br s, 1H), 4.49−4.63 (m, 1H), .28−4.49 (m, 1H), 3.75−3.91 (m, 0H), 3.50−3.61 (m, 1H), 3.20 (dd, J = 3.01, 14.88 Hz, 1H), 2.98 (d, J = 14.32 Hz, 1H), 2.07−2.30 (m, 1H), 1.50 (br s, 9H), 1.40 (d, J = 7.16 Hz, 1H), 1.02−1.29 (m, 6H), 0.75−0.97 (m, 7H), 0.64 (br s, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 171.5, 155.6, 134.6, 129.2, 128.9, 124.2, 114.6, 113.0, 81.4, 65.8, 58.3, 56.9, 54.9, 53.5, 50.9, 45.0, 37.5, 37.3, 34.3, 34.4, 30.1, 28.7, 28.4, 19.2, 17.0. MS (ESI) m/z 474.7 (M + H)⁺. Boc-16 (276 mg, 0.584 mmol) in CH₃OH (20 mL) was subjected to Boc cleavage following the general method 2b to provide 16 (218 mg, 91%) as a mixture of diastereomers: ¹H NMR (300 MHz, CD₃OD) δ 6.93 (d, J = 8.29 Hz, 1H), 6.61 (dd, J = 2.54, 8.19 Hz, 1H), 6.44−6.54 (m, 1H), 3.85−4.06 (m, 2H), 3.44−3.64 (m, 1H), 2.67−3.01 (m, 3H), 2.09−2.63 (m, 4H), 1.74−1.98 (m, 2H), 1.39−1.72 (m, 3H), 1.05−1.37 (m, 2H), 0.81−1.02 (m, 12H). ¹³C NMR (75 MHz, CD₃OD) δ 175.3, 156.8, 137.4, 130.9, 125.6, 115.0, 113.2, 63.7, 61.5, 58.2, 56.1, 54.8, 54.1, 53.2, 52.5, 38.7, 35.4, 35.1, 33.6, 32.3, 20.0, 17.9. MS (ESI) m/z 374.3 (M + H)⁺. A beige solid was obtained as the dihydrochloride salt of 16: mp 178−183 °C; [α]d²³= +82.1° (c 0.2, CH₃OH). Anal. (C₂₂H₃₇Cl₂N₃O₂·1.5H₂O) C, H, N.

(3R)-N-{(1S)-1-[(3,5-Dimethylpiperidin-1-yl)methyl]-2-methylpropyl}−7-hydroxy-1,2,3,4 tetrahydroisoquinoline-3-carboxamide Dihydrochloride (17) (Mixture of Isomers).

The diamine 60f (766 mg, 3.57 mmol) and Boc-7-hydroxy-d-Tic-OH (1.26 g, 4.29 mmol) were treated according to the general method 1 to provide Boc-17 (1.6 g, 93%) as a mixture of isomers: ¹H NMR (300 MHz, CDCl₃) δ 6.97 (d, J = 9.3 Hz, 1H), 6.67 (d, J = 7.6 Hz, 1H), 6.63 (d, J = 3.2 Hz, 1H), 5.94 (br s, 1H), 4.70−4.84 (m, 1H), 4.41−4.58 (m, 2H), 3.79 (br s, 1H), 3.30 (s, 3H), 3.10−3.22 (m, 2H), 2.95−3.02 (m, 1H), 2.42−2.60 (m, 2H), 1.75−2.28 (m, 6H), 1.50 (s, 9H), 1.21−1.30 (m, 2H), 0.78−0.90 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.6, 155.7, 134.0, 134.0, 129.1, 124.2, 114.9, 113.3, 81.3, 60.4, 58.8, 55.3, 51.3, 50.8, 44.8, 30.6, 30.4, 30.2, 28.4 (3Cs), 18.9, 17.4. MS (ESI) m/z 476.6 (M + H)⁺. Boc-17 (1.5 g, 2.43 mmol) was treated as described in general method 2a to remove the Boc-protection and provide 845 mg (93%) of freebase 17 as a mixture of isomers: ¹H NMR (300 MHz, CD₃OD) δ 6.94 (d, J = 8.4 Hz, 1H), 6.62 (dd, J = 2.0, 8.4 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 4.00−4.05 (m, 1H), 3.91−3.95 (m, 2H), 3.52−3.57 (dd, J = 4.8, 10.2 Hz, 1H), 3.37 (s, 1H), 2.74−2.98 (m, 4H), 2.41−2.46 (dd, J = 3.7, 12.6 Hz, 1H), 2.24−2.38 (m, 2H), 2.12−2.20 (td, J = 2.4, 11.4 Hz, 1H), 1.80−1.95 (m, 1H), 1.44−1.65 (m, 4H), 1.25−1.35(m, 2H), 1.13−1.18 (t, J = 7.0 Hz, 1H), 1.07−1.09 (m, 3H), 1.45−1.62 (m, 2H), 0.91−0.96 (m, 6H). 13C NMR (75 MHz, CD₃OD) δ 175.3, 156.8, 137.5, 130.9, 125.6, 115.0, 113.3, 58.2, 57.8, 56.8, 53.2, 52.8, 51.9, 50.0, 48.0, 35.2, 32.8, 32.0, 26.8, 20.1, 18.1, 17.3. MS (ESI) m/z 374.2 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 17: mp 158 °C (dec.); [α]d²² = +74.5° (c 1.1, CH₃OH). Anal. (CH₃₇Cl₂N₃O₃·1.5H₂O) C, H, N.

(3R)-N-{(1S)-1-[(4,4-Dimethylpiperidin-1-yl)methyl]-2-methylpropyl}−7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (18).

The diamine 60g (764 mg, 2.82 mmol) in dichloromethane (50 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (826 mg, 2.82 mmol) following the protocol described in general method 1 to provide 673 mg (50%) of Boc-18: ¹H NMR (300 MHz, CDCl₃) δ 6.97 (d, J = 8.2 Hz, 1H), 6.66 (dd, J = 2.6, 8.4 Hz, 1H), 6.54 (s, 1H), 5.92−6.22 (m, 1H), 4.71−4.88 (m, 1H), 4.39−4.57 (m, 2H), 3.79 (br s, 1H), 3.21 (d, J = 14.2 Hz, 1H), 2.99 (dd, J = 6.0, 14.6 Hz, 1H), 2.83 (s, 1H), 2.78−2.88 (m, 1H), 2.05−2.27 (m, 4H), 1.00−1.93 (m, 1H), 1.50 (s, 9H), 1.18−1.35 (m, 3H), 0.78−0.88 (m, 12H). ¹³C NMR (CDCl₃) δ 171.4, 155.6, 134.0, 129.2, 124.2, 114.8, 113.0, 81.5, 59.3, 56.5, 54.5, 51.3, 50.0, 44.8, 38.6, 38.5, 30.7, 30.2, 29.6, 28.4 (3Cs), 28.2, 28.1, 19.0, 17.4. MS (ESI) m/z 474.7 (M + H)⁺. Boc-18 (673 mg, 1.42 mmol) in CH₃CN (20 mL) was subjected to Boc cleavage following the general method 2a to provide 18 as the freebase: ¹H NMR (300 MHz, CD₃OD) δ 6.96 (d, J = 8.6 Hz, 1H), 6.65 (dd, J = 8.4, 2.6 Hz, 1H), 6.52 (d, J = 2.6 Hz, 1H), 4.16−4.21 (m, 1H), 4.09 (d, J = 3.3 Hz, 1H), 3.93−4.16 (m, 3H), 3.61−3.76 (m, 2H), 3.36 (s, 1H), 2.74−2.99 (m, 6H), 1.82−1.91 (m, 1H), 1.46−1.63 (m, 4H), 1.14−1.24 (m, 1H), 0.91−1.05 (m, 12H). ¹³C NMR (75 MHz, CD₃OD) δ 175.2, 157.0, 136.4, 131.0, 125.1, 115.3, 113.4, 61.0, 58.1, 57.1, 51.6, 51.1, 50.0, 47.9, 38.0, 32.6, 31.9, 29.0, 28.3, 20.0, 18.5, 18.3. MS (ESI) m/z 374.5 (M + H)⁺. A white solid was obtained as the dihydrochloride salt (390 mg, 73%): mp 192−195 °C; [α]d25 = +73.7° (c 1.1, CH₃OH). Anal. (C₂₂H₃₇Cl₂N₃O₂·H₂O) C, H, N.

(3R)-N-{(1S)-1-[(4-Ethylpiperidin-1-yl)methyl]-2-methylpropyl}−7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (19).

The amine 60h (133 mg, 0.67 mmol) was dissolved in CH₂Cl₂ (5 mL) and added to a solution of 7-hydroxy-Boc-d-Tic-OH (223 mg, 0.75 mmol), EDC·HCl (306 mg, 1.5 mmol), and catalytic HOBt (14 mg, 0.1 mmol) in CH₂Cl₂ (10 mL). The reaction mixture was stirred overnight, then was concentrated and purified by chromatography on silica gel eluted with a gradient of 0−35% DMA80 in CH₂Cl₂. The fractions containing the product were concentrated to afford Boc-19, which was then treated with and concentrated from CH₃OH (5 mL) and HCl (6 M, 5 mL). The concentrated residue was subjected to chromatography on silica gel eluting with 1:2 DMA80:CH₂Cl₂ to afford 77.5 mg (31%) 19 freebase. ¹H NMR (300 MHz, CDCl₃) δ 7.16−7.37 (m, 1H), 6.86 (d, J = 8.29 Hz, 1H), 6.56 (dd, J = 2.35, 8.19 Hz, 1H), 6.44 (d, J = 2.07 Hz, 1H), 4.22 (t, J = 9.89 Hz, 1H), 3.61 (s, 2H), 3.39 (d, J = 10.93 Hz, 1H), 3.25 (dd, J = 4.99, 11.59 Hz, 1H), 3.06 (d, J = 11.11 Hz, 1H), 2.75−2.94 (m, 2H), 2.30−2.52 (m, 2H), 2.11−2.29 (m, 1H), 1.93−2.10 (m, 1H), 1.62−1.92 (m, 3H), 1.10−1.49 (m, 5H), 0.93 (d, J = 6.78 Hz, 6H), 0.77−0.89 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 155.0, 137.0, 130.5, 124.9, 113.8, 112.2, 59.7, 56.8, 55.8, 52.3, 49.5, 48.0, 36.9, 31.7, 30.7, 30.5, 29.6, 28.8, 19.0, 18.1, 11.2. MS (ESI) m/z 374.2 (M + H)⁺. The freebase was converted into a white powder as the dihydrochloride salt: mp 176−180 °C (fusion); [α]d²⁵ = +75.0° (c 0.20, CH₃OH). Anal. (C₂₂H₃₇Cl₂N₃O₂·H₂O) C, H, N.

(3R)-7-Hydroxy-N-[(1S)-2-methyl-1-{[4-(trifluoromethyl)-piperidin-1-yl]methyl}propyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (20).

The diamine 60i (766 mg, 3.57 mmol) and Boc-7-hydroxy-d-Tic-OH (1.26 g, 4.29 mmol) were treated as described in the general method 1 to provide Boc-20 (1.6 g, 93% yield): ¹H NMR (300 MHz, CDCl₃) δ 6.98 (d, J = 9.3 Hz, 1H), 6.68 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 3.2 Hz, 1H), 5.82−6.17 (m, 1H), 4.70−4.84 (m, 1H), 4.38−4.55 (m, 2H), 3.81 (br s, 1H), 3.21 (d, J = 16.8 Hz, 1H), 2.99 (dd, J = 13.8, 6.6 Hz, 1H), 2.62−2.78 (m, 2H), 2.10−2.20 (m, 2H), 1.63−2.02 (m, 6H), 1.51 (s, 9H), 1.21−1.30 (m, 2H), 0.79−0.87 (m, 6H). ¹³C NMR (300 MHz, CDCl₃) δ 171.3, 155.6, 134.0, 129.1, 127.5 (q, J_CF = 272 Hz), 124.1, 114.8, 113.0, 81.1, 60.4, 59.4, 52.6, 52.1, 51.3, 44.9, 40.2 (q, J_CF = 23.2 Hz), 39.5 (q, J_CF = 27.6 Hz), 29.9, 28.3 (3Cs), 24.5, 19.1, 17.1. MS (ESI) m/z 514.6 (M + H)⁺. Boc-20 (1.6 g, 3.03 mmol) was treated as described in the general method 2a to remove the Boc protection to provide 1.1 g (88%) of 20 as the freebase: 1H NMR (300 MHz, CD₃OD) δ 6.95 (d, J = 8.4 Hz, 1H), 6.63 (dd, J = 8.4, 2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 3.89−4.03 (m, 3H), 3.59 (dd, J = 7.7, 2.9 Hz, 1H), 2.78−3.09 (m, 4H), 2.40−2.47 (m, 2H), 2.04−2.12 (m, 2H), 1.81−1.98 (m, 4H), 1.50−1.65 (m, 2H), 0.89−1.04 (m, 6H). 13C NMR (75 MHz, CD₃OD) δ 175.4, 156.8, 137.4, 130.9, 120.9 (J_CF = 272 Hz), 125.5, 115.1, 113.3, 61.2, 58.2, 54.2, 53.0, 52.5, 47.8, 41.4 (q, J_CF = 26.9 Hz), 32.4, 32.3, 25.7, 20.0, 18.0. MS (ESI) m/z 376.5 (M + H)⁺. A beige solid was obtained as the dihydrochloride salt of 20: mp 200−203 °C; [α]d²² = +67.1° (c 2.01, CH₃OH). Anal. (C₂₁H₃₂Cl₂F₃N₃O₃·H₂O) C, H, N.

(3R)-N-[(1S)-1-{[4-(Difluoromethyl)piperidin-1-yl]methyl}−2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carbox-amide Dihydrochloride (21).

Compound 60j (994 mg, 4.66 mmol) in CH₂Cl₂ (40 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (1.0 g, 3.41 mmol) following the protocol described in general method 1 to provide 1.22 g (73%) of the Boc-protected 21: ¹H NMR (300 MHz, CDCl₃) δ 6.98 (d, J = 8.29 Hz, 1H), 6.67 (dd, J = 2.07, 8.10 Hz, 1H), 6.54 (br s, 1H), 5.52 (d, J = 4.71 Hz, 1H), 4.76 (br s, 1H), 4.37−4.60 (m, 2H), 3.74−3.87 (m, 1H), 3.24 (dd, J = 3.01, 15.45 Hz, 1H), 2.96 (dd, J = 5.84, 15.26 Hz, 1H), 2.67 (br s, 2H), 1.98−2.27 (m, 2H), 1.82 (dd, J = 6.12, 11.40 Hz, 2H), 1.55−1.73 (m, 3H), 1.50 (s, 9H), 1.18−1.43 (m, 2H), 0.95−1.16 (m, 1H), 0.83 (dd, J = 6.69, 16.67 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.5, 155.6, 133.8, 129.2, 124.1, 118.8, 114.7, 113.0, 81.6, 59.4, 56.3, 53.2, 52.0, 51.1, 44.8, 39.7, 30.3, 28.4, 24.7, 19.1, 17.4. MS (ESI) m/z 496.6 (M + H)⁺. Boc-deprotection was accomplished following the general method 2b to provide compound 21 (873 mg, 90%) as the freebase: ¹H NMR (300 MHz, CDCl₃) δ 7.12 (d, J = 9.61 Hz, 1H), 6.85 (d, J = 8.29 Hz, 1H), 6.57 (d, J = 7.35 Hz, 1H), 6.42 (s, 1H), 5.23−5.73 (m, 1H), 4.04 (td, J = 4.52, 9.04 Hz, 1H), 3.74 (s, 2H), 3.06 (d, J = 10.55 Hz, H), 2.81−3.01 (m, 2H), 2.38−2.65 (m, 2H), 2.24−2.37 (m, 1H), 2.02 (t, J = 11.11 Hz, 1H), 1.54−1.90 (m, 4H), 1.17−1.53 (m, 2H), 0.62−0.99 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.6, 155.1, 136.6, 130.1, 124.7, 118.6, 114.1, 112.3, 59.8, 56.7, 53.8, 51.6, 50.5, 50.0, 47.4, 39.6, 30.9, 30.2, 24.5, 19.2, 17.7. MS (ESI) m/z 396.4 (M + H)⁺. A beige solid was obtained as dihydrochloride salt, 21·2HCl: mp 186−188 °C; [α]d²⁰ = +154° (c 1.1, CH₃OH). Anal. (C₂₁H₃₃Cl₂F₂N₃O₂·H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-methoxypiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (22).

The diamine 60k (766 mg, 3.57 mmol) and Boc-7-hydroxy-d-Tic-OH (1.26 g, 4.29 mmol) were treated according to the general method 1 to provide Boc-22 (1.6 g, 93%): ¹H NMR (300 MHz, CDCl₃) δ 6.97 (d, J = 9.3 Hz, 1H), 6.67 (d, J = 7.6 Hz, 1H), 6.63 (d, J = 3.2 Hz, 1H), 5.94 (br s, 1H), 4.70−4.84 (m, 1H), 4.41−4.58 (m, 2H), 3.79 (br s, 1H), 3.30 (s, 3H), 3.10−3.22 (m, 2H), 2.95−3.02 (m, 1H), 2.42−2.60 (m, 2H), 1.75−2.28 (m, 6H), 1.50 (s, 9H), 1.21−1.30 (m, 2H), 0.78−0.90 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.6, 155.7, 134.0, 134.0, 129.1, 124.2, 114.9, 113.3, 81.3, 60.4, 58.8, 55.3, 51.3, 50.8, 44.8, 30.6, 30.4, 30.2, 28.4 (3Cs), 18.9, 17.4. MS (ESI) m/z 476.6 (M + H)+. Boc-22 (1.2 g, 2.4 mmol) was treated as described in general method 2a to remove the Boc-protection to provide 731 mg (80%) of 22 as the freebase: ¹H NMR (300 MHz, CD₃OD) δ 6.95 (d, J = 8.4 Hz, 1H), 6.63 (dd, J = 8.4, 2.5 Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H), 3.92−4.0 (m, 3H), 3.56−3.58 (m, 1H), 3.33 (s, 3H), 3.21−3.26 (m, 1H), 2.70−2.98 (m, 4H), 2.41−2.44 (m, 2H), 2.08−2.27 (m, 2H), 1.81−1.90 (m, 3H), 1.45−1.62 (m, 2H), 0.91−0.96 (m, 6H). ¹³C NMR (75 MHz, CD₃OD) δ 175.4 156.8, 137.4, 130.9, 125.6, 115.0, 113.3, 77.7, 61.1, 58.2, 55.8, 52.5, 52.1, 50.0, 48.0, 32.6, 32.4, 31.8, 20.0, 18.0. MS (ESI) m/z 376.5 (M + H)+. A white solid was obtained as the dihydrochloride salt of 22: mp 184−186 °C; [α]d²⁰ = +75.1° (c 1.1, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₃·H₂O) C, H, N.

(3R)-N-[(1S)-1-{[4-(Dimethylamino)piperidin-1-yl]methyl}−2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Trihydrochloride (23).

Compound 60l (994 mg, 4.66 mmol) in CH₂Cl₂ (40 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (1.4 g, 1.0 equiv) following the protocol described in general method 1 to provide 1.94 g, (85%) of the Boc-protected 23: ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J = 8.10 Hz, 1H), 6.53−6.71 (m, 2H), 4.72 (br s, 1H), 4.33−4.56 (m, 2H), 3.78 (br s, 1H), 3.11−3.28 (m, 1H), 2.95 (dd, J = 6.03, 15.26 Hz, 1H), 2.25 (br s, 5H), 1.61−1.84 (m, 5H), 1.47 (s, 9H), 1.22 (t, J = 7.16 Hz, 1H), 0.80 (dd, J = 6.69, 17.99 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.6, 133.9, 129.1, 124.0, 121.8, 114.7, 112.9, 81.6, 60.4, 58.6, 56.6, 54.9, 51.6, 50.1, 44.9, 33.7, 30.7, 30.2, 28.3, 21.0, 19.2, 17.3. MS (ESI) m/z 489.7 (M + H)⁺. Removal of the Boc protection following the general method 2b furnished freebase 23 (1.38 g, 72%): ¹H NMR (300 MHz, CD₃OD) δ 6.76 (d, J = 8.29 Hz, 1H), 6.44 (d, J = 8.10 Hz, 1H), 6.34 (s, 1H), 3.73−3.87 (m, 3H), 3.42 (dd, J = 4.80, 9.89 Hz, 1H), 2.83−3.05 (m, 3H), 2.58−2.83 (m, 3H), 2.20−2.40 (m, 2H), 2.00−2.15 (m, 2H), 1.82−1.98 (m, 2H), 1.60−1.81 (m, 4H), 1.44−1.59 (m, 1H), 1.22−1.42 (m, 2H), 1.08 (t, J = 7.16 Hz, 1H), 0.93 (t, J = 7.16 Hz, 1H), 0.77 (t, J = 6.12 Hz, 6H). 13C NMR (75 MHz, CD₃OD) δ 175.3, 156.9, 137.3, 130.9, 125.6, 115.1, 113.4, 63.8, 61.0, 58.2, 54.7, 53.5, 52.6, 47.9, 45.3, 41.8, 39.1, 35.9, 32.3, 28.9, 20.1, 18.2. MS (ESI) m/z 389.5 (M + H)⁺. A beige solid was obtained as the trihydrochloride salt of 23: mp 184 °C (dec); [α]d²⁰ = +91.6° (c 0.14, CH₃OH). Anal. (C₂₂H₃₉Cl₃N₄O₂·1.75H₂O) C, H, N.

(3R)-N-{[(1S)-1-[4,4-Difluoropiperidin-1-yl]methyl]-2-methyl-propyl}−7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (24).

Compound 60m (992 mg, 4.807 mmol) in CH₂Cl₂ (40 mL) was coupled with Boc-7-hydroxy-d-Tic-OH (1.41 g, equiv) following the protocol described in general method 1 to provide 1.73 g, (75%) of the Boc-protected 24: ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J = 8.10 Hz, 1H), 6.53−6.71 (m, 2H), 4.72 (br s, 1H), 4.33−4.56 (m, 2H), 3.78 (br s, 1H), 3.11−3.28 (m, 1H), 2.95 (dd, J = 6.03, 15.26 Hz, 1H), 2.25 (br s, 5H), 1.61−1.84 (m, 5H), 1.47 (s, 9H), 1.22 (t, J = 7.16 Hz, 1H), 0.80 (dd, J = 6.69, 17.99 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.6, 133.9, 129.1, 124.0, 121.8, 114.7, 112.9, 81.6, 60.4, 58.6, 56.6, 54.9, 51.6, 50.1, 44.9, 33.7, 30.7, 30.2, 28.3, 21.0, 19.2, 17.3. MS (ESI) m/z 482.4 (M + H)⁺. A solution of Boc-24 (1.733 g, 4.77 mmol) in CH₃OH (20 mL) was subjected to Boc cleavage following the general method 2b to provide freebase 24 (1.25 g, 69%): ¹H NMR (300 MHz, CDCl₃) δ 7.15 (d, J = 9.61 Hz, 1H), 6.90 (d, J = 8.29 Hz, 1H), 6.54−6.72 (m, 1H), 6.46−6.53 (m, 1H), 3.95−4.16 (m, 1H), 3.72−3.88 (m, 2H), 3.47 (dd, J = 5.18, 10.27 Hz, 1H), 3.02 (dd, J = 4.99, 16.29 Hz, 1H), 2.60−2.75 (m, 3H), 2.39−2.53 (m, 3H), 1.78−1.99 (m, 5H), 0.70−0.97 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.7, 155.1, 136.6, 130.1, 124.8, 121.8, 114.1, 112.3, 58.9, 56.7, 51.0, 50.2, 47.3, 33.7, 30.6, 30.3, 19.3, 17.7. MS (ESI) m/z 382.8 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 24: mp 195−197 °C; [α]d²⁰ = +67.9° (c 0.5, CH₃OH). Anal. (C₂₀H₃₁Cl₂F₂N₃O₂·H₂O) C, H, N.

(3R)-N-{(1S)-1-[(4-Cyanopiperidin-1-yl)methyl]-2-methylprolyl}−7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (25).

Diamine 60n (470 mg, 2.41 mmol) and Boc-7-hydroxy-d-Tic-OH (705 mg, 2.41 mmol) were treated according to the general method 1 to provide Boc-25 (558 mg, 49%): ¹H NMR (300 MHz, CDCl₃) δ 7.97 (br s, 1H), 6.93−7.06 (m, 1H), 6.60−6.78 (m, 2H), 4.76 (br s, 1H), 4.37−4.59 (m, 2H), 3.82 (br s, 1H), 3.23 (d, J = 17.71 Hz, 2H), 2.88−3.03 (m, 1H), 2.05−2.60 (m, 6H), 1.57−1.82 (m, 4H), 1.38−1.56 (m, 9H), 0.66−0.91 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.6, 155.5, 142.9, 134.0, 129.2, 124.2, 121.6, 114.8, 113.2, 81.0, 59.5, 51.1, 50.9, 44.8, 33.8, 30.6, 28.4, 28.4, 28.3, 28.1, 25.6, 24.9, 21.0, 19.2, 17.2. (ESI) m/z 471.4 (M + H)⁺. A solution of Boc-25 (300 mg, 0.64 mmol) in methylene chloride was treated with trifluoroacetic acid (1 mL) and stirred at room temperature overnight to remove the Boc-protection. The reaction mixture was concentrated, neutralized by addition of DMA80, and concentrated down again, and the remaining residue was subjected to silica gel chromatography eluting with a gradient of DMA80 in CH₂Cl₂ to afford 213 mg (90%) of the freebase 25: ¹H NMR (300 MHz, CDCl₃) δ 7.24 (d, J = 8.85 Hz, 1H), 6.82−6.97 (m, 1H), 6.54−6.70 (m, 1H), 6.38−6.52 (m, 1H), 5.47 (br s, 2H), 3.94−4.08 (m, 1H), 3.77−3.91 (m, 2H), 3.54 (dd, J = 5.09, 9.80 Hz, 1H), 2.88−3.02 (m, 1H), 2.43−2.85 (m, 5H), 2.30−2.42 (m, 1H), 1.72−1.94 (m, 4H), 0.82−1.06 (m, 6H). 13C NMR (75 MHz, CDCl₃) δ 172.9, 155.0, 135.7, 130.1, 124.7, 121.6, 114.3, 112.4, 59.9, 56.5, 51.4, 50.5, 46.9, 31.0, 30.8, 30.0, 28.3, 25.8, 19.3, 17.7. (ESI) m/z 371.3 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 25: mp 182 °C (dec); [α]d²⁰ = +82.2° (c 1.1, CH₃OH). Anal. (C₂₁H₃₂Cl₂N₄O₂·0.75H₂O) C, H, N.

(3R)-N-{(1S)-1-[(4-Carbamoylpiperidin-1-yl)methyl]-2-methylprolyl}−7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (26).

Diamine 60o (526 mg, 2.47 mmol) and Boc-7-hydroxy-d-Tic-OH (796 mg, 2.71 mmol) were reacted according to the general method 1 to provide Boc-26 (882 mg, 73% yield): ¹H NMR (300 MHz, CDCl₃) δ 7.57−8.47 (m, 1H), 6.85−7.12 (m, 1H), 6.56−6.79 (m, 2H), 5.78−6.46 (m, 1H), 4.26−5.34 (m, 4H), 3.50−3.95 (m, 1H), 3.39 (d, J = 15.82 Hz, 1H), 3.10−3.31 (m, 2H), 2.74−3.07 (m, 2H), 2.64 (d, J = 17.14 Hz, 1H), 2.16−2.45 (m, 3H), 1.85 (dd, J = 26.56, 44.27 Hz, 1H), 1.38−1.56 (m, 11H), 1.12−1.31 (m, 3H), 0.69−1.05 (m, 4H). 13C NMR (75 MHz, CD₃OD) δ 180.9, 161.2, 157.5, 141.8, 136.2, 129.9, 125.1, 115.4, 114.0, 82.1, 61.5, 57.9, 54.3, 52.9, 49.9, 47.5, 45.2, 43.5, 39.0, 35.9, 32.2, 29.9, 28.8 (3C’s), 19.9. MS (ESI) m/z 489.5 (M + H)⁺. Boc-26 (730 mg, 1.5 mmol) was treated according to the general method 2a to provide 462 mg (80%) of the freebase 26: ¹H NMR (300 MHz, CD₃OD) δ 6.94 (d, J = 8.29 Hz, 1H), 6.62 (d, J = 8.10 Hz, 1H), 6.52 (d, J = 2.07 Hz, 1H), 3.95 (d, J = 6.22 Hz, 2H), 3.62 (q, J = 6.97 Hz, 2H), 2.74−3.23 (m, 5H), 2.34−2.56 (m, 2H), 1.56−2.12 (m, 4H), 1.19 (t, J = 7.06 Hz, 3H), 1.10 (t, J = 7.25 Hz, 3H), 0.80−1.04 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 175.1, 156.9, 136.5, 131.0, 125.3, 115.3, 113.4, 65.0, 61.3, 58.0, 55.2, 53.8, 52.0, 50.0, 47.8, 47.7, 45.4, 36.7, 32.1, 29.9, 19.9. MS (ESI) m/z 388.5 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 26: mp 184−186 °C; [α]d²⁵ = +62.2° (c 1.1, CH₃OH). Anal. (C₂₁H₃₄C_l2N₄O₃·1.5H₂O) C, H, N.

(3R)-N-[(1S)-1-(3,6-Dihydropyridin-1(2H)-ylmethyl)-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (27).

7-Hydroxy-Boc-d-Tic-OH (285 mg, 0.97 mmol), EDC·HCl (305 mg, 1.6 mmol), catalytic HOBt (53 mg, 0.4 mmol), and the amine 68a (156 mg, 0.93 mmol) in CH₂Cl₂ (15 mL) were treated with NEt₃ (0.42 mL, 3.0 mmol) and stirred for 4 h at room temperature. The reaction mixture was then concentrated in vacuo and purified by chromatography on silica gel eluted with a gradient of 0−35% DMA80 in CH₂Cl₂. The Boc-intermediate containing fractions were concentrated, and the residue was dissolved in CH₃OH (5 mL) and HCl (6 M, 5 mL). After 1 h, the reaction mixture was concentrated. The resulting residue was purified by chromatography on silica gel, eluting with a gradient of 0−25% DMA80 in CH₂Cl₂ to afford freebase 27. ¹H NMR (300 MHz, CDCl₃) δ 7.06 (d, J = 10.36 Hz, 1H), 6.67 (d, J = 8.29 Hz, 1H), 6.34 (dd, J = 2.45, 8.10 Hz, 1H), 6.27 (d, J = 2.26 Hz, 1H), 5.62−5.79 (m, 2H), 4.13−4.31 (m, 1H), 3.54−3.68 (m, 1H), 3.36−3.54 (m, 2H), 3.12 (dd, J = 5.09, 11.87 Hz, 1H), 2.63−2.87 (m, 4H), 2.24−2.50 (m, 3H), 1.84−2.06 (m, 2H), 1.62−1.79 (m, 1H), 0.78−0.95 (m, 6H). 13C NMR (75 MHz, CDCl₃) δ 173.4, 154.9, 137.4, 130.7, 125.7, 125.1, 124.1, 113.8, 111.9, 59.5, 56.6, 51.3, 50.8, 49.3, 48.6, 32.2, 29.3, 24.7, 19.3, 18.0. MS (ESI) m/z 344.4 (M + H)⁺. The freebase was converted into 75.6 mg (20% from 68a) of a white powder as the dihydrochloride salt: mp ∼175 °C (dec.); [α]d²⁵ = +75° (c 0.10, CH₃OH). Anal. (C₂₀H₃₁Cl₂N₃O₂·H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-methyl-3,6-dihydropyridin-1(2H)-yl)methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (28).

A solution of Boc-7-hydroxy-d-Tic-OH (78 mg, 0.25 mmol), EDC·HCl (95 mg,0.5 mmol), catalytic HOBt, and the amine 68b (33 mg, 0.18 mmol) in CH₂Cl₂ (10 mL) was treated with NEt₃ (0.21 mL, 1.5 mmol). After 12 h, the reaction mixture was concentrated and purified by chromatography on silica gel eluting with a gradient of 0−40% DMA80 in CH₂Cl₂. The Boc-intermediate containing fractions were dissolved in CH₃OH (5 mL) and HCl (6 M, 5 mL). After 1 h, the reaction mixture was concentrated. The residue was purified by chromatography on silica gel, eluting with a gradient of 0−50% DMA80 in CH₂Cl₂ to afford the freebase 28. ¹H NMR (300 MHz, CDCl₃) δ 7.06−7.19 (m, 1H), 6.72 (d, J = 8.29 Hz, 1H), 6.39 (dd, J = 2.54, 8.19 Hz, 1H), 6.33 (d, J = 2.26 Hz, 1H), 5.47 (br s, 1H), 4.19−4.36 (m, 1H), 3.65 (s, 1H), 3.57 (s, 1H), 3.39−3.51 (m, 1H), 3.19 (d, J = 6.97 Hz, 1H), 2.70−2.94 (m, 4H), 2.44−2.57 (m, 1H), 2.37 (dd, J = 3.20, 12.43 Hz, 2H), 1.85−2.04 (m, 2H), 1.69−1.84 (m, 1H), 1.66 (s, 3H), 0.83−1.02 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 154.9, 137.4, 133.4, 130.8, 125.0, 118.0, 113.4, 111.8, 59.1, 56.6, 51.6, 50.8, 50.7, 49.4, 48.7, 32.2, 29.4, 22.6, 19.3, 18.0. MS (ESI) m/z 358.2 (M + H)⁺. The freebase was converted into 32.5 mg (40%) of a white powder as the dihydrochloride salt: mp 184−188 °C (fusion); [α]d²⁵ = +80° (c 0.10, CH₃OH). Anal. (C₂₁H₃₃Cl₂N₃O₂·1.25H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(5-methyl-3,6-dihydropyridin-1(2H)-yl)methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (29).

A solution of Boc-7-hydroxy-d-Tic-OH (290 mg, 1 mmol), EDC·HCl (380 mg, 2 mmol), catalytic HOBt (14 mg, 0.1 mmol), and the amine 68c (167 mg, 0.91 mmol) in CH₂Cl₂ (10 mL) was treated with NEt₃ (0.2 mL, 1.4 mmol). After 12 h, the reaction mixture was concentrated and purified by chromatography on silica gel eluting with a gradient of 0−50% DMA80 in CH₂Cl₂. The Boc-intermediate containing fractions were concentrated and the residue dissolved in CH₃OH (5 mL) then treated with HCl (6 M, 5 mL). After 1 h, the reaction mixture was concentrated. The residue was purified by reverse-phase chromatography on C-18 silica gel, eluting with 25% CH₃CN in water (0.1% TFA). The product containing fraction was evaporated then applied to silica gel and eluted with 25% DMA80 in CH₂Cl₂ to afford 17.7 mg (5% from 67c) of the freebase 29. ¹H NMR (300 MHz, CDCl₃) δ 7.10 (d, J = 10.17 Hz, 1H), 6.75 (d, J = 7.91 Hz, 1H), 6.26−6.38 (m, 2H), 5.49 (br s, 1H), 4.29 (t, J = 11.49 Hz, 1H), 3.61−3.75 (m, 1H), 3.51−3.61 (m, 1H), 3.12−3.30 (m, 2H), 2.65−2.95 (m, 4H), 2.26−2.54 (m, 3H), 1.92−2.18 (m, 2H), 1.66−1.88 (m, 4H), 0.84−1.04, (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 154.9, 137.4, 131.0, 130.6, 125.1, 119.9, 113.6, 111.9, 59.0, 56.6, 55.1, 49.4, 48.6, 32.1, 29.3, 24.4, 21.0, 19.2, 18.1. MS (ESI) m/z 358.3 (M + H)⁺. The freebase was converted to a white powder as the dihydrochloride salt: mp 86−90 °C (fusion), [α]d²⁵ +77° (c 0.10, CH₃OH). Anal. CH₃OH). Anal (C₂₁H₃₃Cl₂N₃O₂·H₂O) C, H, N.

(3R)-N-{(1S)-1-[(4,5-Dimethyl-3,6-dihydropyridin-1(2H)-yl)-methyl]-2-methylpropyl}−7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (30).

A solution of Boc-7-hydroxy-d-Tic-OH (106 mg, 0.36 mmol), EDC·HCl (112 mg, 0.59 mmol), catalytic HOBt (12 mg), and the amine 68d (55 mg, 0.28 mmol) in CH₂Cl₂ (6 mL) was treated with NEt₃ (0.3 mL, 2.2 mmol). After 12 h, the reaction mixture was concentrated and purified by chromatography on silica gel eluting with a gradient of 0−40% DMA80 in CH₂Cl₂. The Boc-intermediate containing fractions were concentrated then the residue dissolved in CH₃OH (5 mL) and aq HCl (6 M, 5 mL). After 1 h, the reaction mixture was concentrated. The residue was purified by reverse-phase chromatography on C-18 silica gel, eluting with 20% CH₃OH in water (0.1% TFA). The product containing fraction was evaporated then applied to silica gel and eluted with a gradient of 0−40% DMA80 in CH₂Cl₂ to afford 36.8 mg (35% over two steps) of the freebase 30. ¹H NMR (300 MHz, CDCl₃) δ 7.07 (d, J = 10.17 Hz, 1H), 6.72 (d, J = 8.29 Hz, 1H), 6.32 (d, J = 2.26 Hz, 1H), 6.26 (dd, J = 2.35, 8.19 Hz, 1H), 4.20−4.37 (m, 1H), 3.62−3.75 (m, 1H), 3.50−3.62 (m, 1H), 3.12−3.30 (m, 2H), 2.65−2.93 (m, 4H), 2.26−2.56 (m, 3H), 1.87−2.01 (m, 2H), 1.71−1.87 (m, 1H), 1.63 (d, J = 19.97 Hz, 6H), 0.82−1.05 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 173.2, 155.1, 137.4, 130.6, 125.1, 125.0, 122.8, 113.3, 111.9, 58.9, 56.6, 56.0, 51.6, 49.5, 48.6, 32.1, 30.5, 29.5, 19.2, 18.1, 18.1, 16.7. MS (ESI) m/z 372.1 (M + H)⁺. The freebase was converted into a white powder as the dihydrochloride salt: mp 135−139 °C (fusion); [α]d²⁵ = +74° (c 0.10, CH₃OH). Anal. (C₂₂H₃₅Cl₂N₃O₂·1.5H₂O) C, H, N.

(3S)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-methylpiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Di-hydrochloride (31).

The diamine 60a (401 mg, 2.18 mmol) and Boc-7-hydroxy-L-Tic-OH (491 mg, 1.67 mmol) were treated according to the general method 1 to provide Boc-31 (727 mg, 95%): ¹H NMR (300 MHz, CDCl₃) δ 6.96 (d, J = 8.1 Hz, 1H), 6.67 (d, J = 8.3 Hz, 1H), 6.64 (s, 1H), 5.90−6.30 (m, 1H), 4.69−4.79 (br s, 1H), 4.40−4.58 (m, 2H), 3.83 (br s, 1H), 3.21 (dd, J = 2.5, 15.3 Hz, 1H), 2.95−3.02 (m, 1H), 2.74−2.82 (m, 2H), 2.20−2.25 (m, 2H), 1.85−1.92 (m, 2H), 1.51 (s, 9H), 1.40−1.60 (m, 3H), 1.10−1.36 (m, 6H), 0.82−0.95 (m, 4H), 0.45−0.59 (m, 5H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.2, 134.0, 129.1, 123.9, 114.7, 113.2, 81.1, 65.8, 59.5, 57.9, 54.3, 53.8, 51.2, 45.1, 34.1, 31.4, 30.8, 30.5, 29.6, 28.4 (3Cs), 21.8, 18.5, 16.6. MS (ESI) m/z 460.4 (M + H)⁺. Boc-31 (696 mg, 1.5 mmol) was treated according to the general method 2a for removal of the Boc-protection to provide 479 mg (88%) of the freebase 31: ¹H NMR (300 MHz, CD₃OD) δ 6.90 (d, J = 8.3 Hz, 1H), 6.60 (dd, J = 8.4, 2.4 Hz, 2.91−2.98 (m, 2H), 1H), 6.50 (d, J = 2.5 Hz, 1H), 3.90−4.03 (m, 3H), 3.54 (dd, J = 5.3, 4.4 Hz, 1H), 3.37 (s, 1H), 3.31−3.39 (m, 1H), 2.72−2.82 (m, 2H), 2.37−2.51 (m, 2H), 2.09 (td, J = 13.6, 2.8 Hz, 1H), 1.77−2.13 (m, 3H), 1.49−1.65 (m, 3H), 1.11−1.40 (m, 4H), 0.88−0.95 (m, 9H). ¹³C NMR (75 MHz, CD₃OD) δ 175.6, 156.9, 135.3, 137.2, 125.7, 115.1, 113.4, 61.6, 58.3, 55.9, 54.5, 51.1, 52.4, 48.3, 35.3, 32.5, 31.9, 28.9, 22.4, 19.9, 18.1. MS (ESI) m/z 360.3 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 31: mp >230 °C; [α] ²² = −37.0° (c 1.1, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₂·1.25H₂O) C, H, N.

(3R)-7-Hydroxy-N-{(1R)-2-methyl-1-[(4-methylpiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (32).

The diamine 69 (377 mg, 2.04 mmol) and Boc-7-hydroxy-d-Tic-OH (599 mg, 2.04 mmol) were treated according to the general method 1 to provide Boc-32 (689 mg, 73%): ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J = 8.1 Hz, 1H) 6.67 (d, J = 8.3 Hz, 1H), 6.65 (s, 1H), 5.97−6.41 (m, 1H), 4.71−4.80 (br s, 1H), 4.40−4.58 (m, 2H), 3.83 (br s, 1H), 3.18−3.24 (dd, J = 2.5, 15.3 Hz, 1H), 2.95−3.02 (m, 1H), 2.74−2.82 (m, 2H), 2.20−2.25 (m, 2H), 1.85−1.92 (m, 2H), 1.51 (s, 9H), 1.40−1.60 (m, 3H), 1.12−1.33 (m, 6H), 0.85−0.95 (m, 3H), 0.45−0.59 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 155.2, 134.0, 129.1, 123.9, 114.7, 113.2, 81.1, 65.8, 59.5, 57.9, 54.3, 53.8, 51.2, 45.1, 34.1, 31.4, 30.8, 30.5, 29.6, 28.4 (3Cs), 21.8, 18.5, 16.6. MS (ESI) m/z 460.4 (M + H)⁺. Boc-32 (689 mg, 1.5 mmol) was treated according to the general method 2a for removal of the Boc-protection to provide 348 mg (64%) of the freebase 32: ¹H NMR (300 MHz, CD OD) δ 6.90 (d, J = 8.3 Hz, 1H), 6.60 (dd, J = 8.4, 2.4 Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H), 3.90−4.03 (m, 3H), 3.54 (dd, J = 5.3, 4.4 Hz, 1H), 3.37 (s, 1H), 3.31−3.39 (m, 1H), 2.91−2.98 (m, 2H), 2.72−2.82 (m, 2H), 2.37−2.51 (m, 2H), 2.09 (td, J = 13.6, 2.8 Hz, 1H), 1.77−2.13 (m, 3H), 1.49−1.65 (m, 3H), 1.11−1.40 (m, 4H), 0.88−0.95 (m, 9H). ¹³C NMR (75 MHz, CD₃OD) δ 175.6, 156.9, 135.3, 137.2, 125.7, 115.1, 113.4, 61.6, 58.3, 55.9, 54.5, 51.1, 52.4, 48.3, 35.3, 32.5, 31.9, 28.9, 22.4, 19.9, 18.1. MS (ESI) m/z 360.3 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 32: mp >228 °C; [α]d²² = +36.6° (c 1.1, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₂·H₂O) C, H, N.

(3S)-7-Hydroxy-N-{(1R)-2-methyl-1-[(4-methylpiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (33).

The diamine 69 (353 mg, 1.92 mmol) and Boc-7-hydroxy-L-Tic-OH (511 mg, 1.74 mmol) were treated according to the general method 1 to provide Boc-33 (428 mg, 53.5% yield): ¹H NMR (300 MHz, CDCl₃) δ 6.96 (d, J = 8.1 Hz, 1H), 6.68 (d, J = 8.3 Hz, 1H), 6.60 (s, 1H), 6.00−6.29 (m, 1H), 4.70−4.85 (br s, 1H), 4.40−4.61 (m, 2H), 3.86 (br s, 1H), 3.20 (dd, J = 2.5, 15.3 Hz, 1H), 2.94−3.02 (m, 1H), 2.50−2.61 (m, 1H), 2.20−2.23 (m, 2H), 1.70−1.85 (m, 2H), 1.50 (s, 9H), 1.40−1.60 (m, 3H), 1.01−1.25 (m, 2H), 0.78−0.87 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 171.6, 156.0, 134.0, 129.1, 124.0, 114.7, 113.1, 81.1, 59.3, 58.1, 54.3, 53.4, 51.2, 45.1, 34.1, 31.4, 34.0, 30.5, 30.3, 28.4 (3Cs), 21.8, 19.0, 17.4. MS (ESI) m/z 460.4 (M + H)⁺. Boc-33 (428 mg, 0.935 mmol) was treated as described in general method 2a for removal of the Boc-protection to provide 266 mg (79%) of the freebase 33: ¹H NMR (300 MHz, CDCl₃) δ 7.57 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 11.2, Hz, 1H), 6.49 (s, 1H), 5.20−5.40 (m, 3H), 4.11−4.23 (br s, 1H), 3.65−3.73 (m, 2H), 3.35−3.42 (m, 2H), 3.12−3.16 (d, J = 11.6 Hz, 1H), 2.80−2.87 (dd, J = 3.8, 15.8 Hz, 1H), 2.53−2.68 (m, 2H), 2.39 (t, J = 10.9 Hz, 1H), 2.20 (t, J = 10.9 Hz, 1H), 1.77−1.88 (m, 1H), 1.60−1.72 (m, 2H), 1.31−1.53 (m, 3H), 0.86−0.95 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 173.6, 155.1, 136.0, 130.2, 124.4, 114.1, 112.3, 59.5, 58.0, 57.0, 55.2, 52.3, 49.4, 47.4, 32.1, 31.4, 30.0, 29.8, 21.3, 19.2, 18.1. MS (ESI) m/z 360.3 (M + H)⁺. A pale-yellow solid was obtained as the dihydrochloride salt of 33: mp >230 °C; [α]d²² = −77.0° (c 1.1, CH₃OH). Anal. (C₂₁H₃₅Cl₂N₃O₂·0.5H₂O) C, H, N.

(3R)-7-Hydroxy-N-(2-piperidin-1-ylethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (34).

The amine 2-(1-piperidinyl)ethanamine (70) (0.825 g, 6.5 mmol) was added to a solution of dicyclohexylcarbodiimide (DCC) (1.36 g, 6.6 mmol), HOBt (891 mg, 6.6 mmol), and 7-hydroxy-Boc-d-Tic-OH (1.94 g, 6.6 mmol) in THF (20 mL) at 0 °C. The solution was allowed to warm to room temperature overnight, forming a suspension. The solids were filtered, and the filtrate concentrated to a residue which was partitioned between CH₂Cl₂ and satd aq NaHCO₃. The organic layer was separated and dried (Na₂SO₄), then concentrated and the residue subjected to chromatography on silica gel eluting with a gradient of 0−50% DMA80 in CH₂Cl₂ to afford 1.62 g (62%) of the Boc-protected intermediate. ¹H NMR (300 MHz, CDCl₃) δ 6.99 (d, J = 8.10 Hz, 1H), 6.49−6.79 (m, 1H), 6.42 (br s, 1H), 4.64 (br s, 1H), 4.32−4.58 (m, 1H), 3.06−3.41 (m, 2H), 2.79−3.06 (m, 1H), 2.05−2.53 (m, 4H), 1.28−1.72 (m, 11H). The Boc-protected intermediate (1.00 g, 2.6 mmol) was dissolved in MeOH (10 mL) and treated with aq HCl (6 N, 10 mL) at room temperature. After 2 h, the solution was concentrated. The residue was subjected to chromatography on silica gel eluting with a gradient of 0−50% DMA80 in CH₂Cl₂ to afford 0.35 g (44%) of the 34 freebase. ¹H NMR (300 MHz, DMSO-d₆) δ 9.04 (br s, 1H), 7.78 (t, J = 5.46 Hz, 1H), 6.87 (d, J = 8.29 Hz, 1H), 6.53 (dd, J = 2.54, 8.19 Hz, 1H), 6.42 (d, J = 2.45 Hz, 1H), 3.71−3.90 (m, 2H), 3.33 (dd, J = 4.80, 10.08 Hz, 1H), 3.15−3.25 (m, 3H), 2.79 (dd, J = 4.71, 15.82 Hz, 1H), 2.59 (dd, J = 10.17, 15.82 Hz, 1H), 2.19−2.45 (m, 6H), 1.44−1.58 (m, 4H), 1.39 (d, J = 4.90 Hz, 2H). 13C NMR (75 MHz, DMSO-d₆) δ 172.4, 155.2, 136.8, 129.5, 124.3, 113.3, 111.8, 57.5, 56.2, 53.9, 46.8, 35.8, 30.1, 25.5, 24.0. The free base was converted into a pale-yellow powder as the dihydrochloride salt. MS (ESI) m/z 304.5 (M + H)⁺; mp 129−133 °C (fusion); [α]d = +57° (c 1.2, CH₃OH). Anal. (C₁₇H₂₇Cl₂N₃O₂·0.5H₂O) C, H, N.

(3R)-N-[(1S)-1-Cyclopropyl-2-(4-methylpiperidine-1-yl)ethyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (35).

Compound 73a (1.24 g, 2.67 mmol) in CH₃CN (20 mL) was subjected to Boc cleavage following the general method 2b to provide 35 (626 mg, 65%). ¹H NMR (300 MHz, CD₃OD) δ 6.66 (d, J = 8.29 Hz, 1H), 6.36 (dd, J = 2.07, 8.29 Hz, 1H), 6.24 (d, J = 1.70 Hz, 1H), 3.57−3.75 (m, 1H), 3.38 (q, J = 7.22 Hz, 2H), 3.24 (q, J = 7.03 Hz, 2H), 2.34−2.75 (m, 3H), 2.13−2.32 (m, 1H), 1.76−1.91 (m, 1H), 1.52−1.74 (m, 1H), 1.36 (td, J = 2.80, 5.51 Hz, 2H), 0.86−1.15 (m, 7H), 0.59−0.76 (m, 3H), −0.03−0.32 (m, 2H). ¹³C NMR (75 MHz, CD₃OD) d 175.1, 156.8, 137.4, 130.9, 125.6, 115.1, 113.4, 66.9, 63.8, 62.9, 58.4, 56.0, 54.5, 51.7, 35.2, 31.9, 30.3, 22.5, 18.6, 16.2, 15.7. MS (ESI) m/z 358.3 (M + H)⁺. A white solid was obtained as dihydrochloride salt of 35: mp 162−164 °C; [α]d²⁴ = +62.7° (c 1.1, CH₃OH). Anal. (C₂₁H₃₃Cl₂N₃O₂·1.25H₂O) C, H, N.

N-{(1S)-2-Methyl-1-[(4-methylpiperidin-1-yl)methyl]propyl}-d-ty-rosinamide Dihydrochloride (36).

A solution of Boc-d-tyrosine (928 mg, 3.3 mmol), HOBt (0.45 g, 3.3 mmol), and dicyclohexylcarbo-diimide (0.68 g, 3.3 mmol) in THF (10 mL) was stirred for 1 h, forming a suspension. A solution of (2S)-3-methyl-1-(4-methylpiper-idin-1-yl)butan-2-amine (60a) (735 mg, 3.7 mmol) in THF (1.5 mL) was added, then the suspension was stirred overnight at room temperature. The solids were separated by filtration and the filtrate concentrated. The residue was partitioned between EtOAc and satd NaHCO₃. The organic layer was concentrated to a residue which was dissolved in CH₃OH (25 mL) and 6 N HCl (25 mL). After 12 h, the solution was concentrated by approximately half. Sodium hydroxide (5 M) was used to adjust the solution to pH 9. The aqueous was extracted with CH₂Cl₂. The organic layer was dried (Na₂SO₄) and concentrated to a residue which was subjected to chromatography on silica gel eluting with a gradient of 0−50% DMA80 in CH₂Cl₂ to afford 83 mg (7% over two steps) of the desired 36 freebase. ¹H NMR (300 MHz, CDCl₃) δ 7.24 (d, J = 9.23 Hz, 1H), 7.00 (d, J = 8.29 Hz, 2H), 6.78 (d, J = 8.48 Hz, 2H), 4.02 (td, J = 4.36, 9.18 Hz, 1H), 3.48 (dd, J = 4.33, 9.42 Hz, 1H), 3.14 (dd, J = 4.14, 13.75 Hz, 1H), 2.78−2.99 (m, 2H), 2.49 (dt, J = 9.42, 13.75 Hz, 2H), 2.26−2.39 (m, 1H), 2.00−2.15 (m, 1H), 1.78−1.98 (m, 2H), 1.58 (d, J = 11.49 Hz, 2H), 1.10−1.43 (m, 2H), 0.79−0.94 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 174.8, 155.9, 130.3, 128.8, 115.9, 59.5, 57.0, 54.8, 53.3, 50.8, 40.3, 34.0, 33.9, 30.6, 30.5, 21.8, 19.1, 17.6. The freebase was converted into a white powder as the dihydrochloride salt. MS (ESI) m/z 348.0 (M + H)+; mp 161−165 °C (fusion); [α]d25 = −20° (c 0.10, CH₃OH). Anal. (C₂₀H₃₅Cl₂N₃O₂·0.75H₂O) C, H, N.

6-Hydroxy-N-{(1S)-2-methyl-1-[(4-methylpipieridine-1-yl)-methyl]propyl}naphthalene-2-carboxamide Hydrochloride (37).

A solution of 6-hydroxynapthalene-2-carboxylic acid (233 mg, 1.24 mmol), 60a (220 mg, 1.24 mmol), and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (367.1 mg, 1.48 mmol) in DMF was heated at 100 °C for 3 h then transferred to a rotovap and heated for an additional 1 h under reduced pressure until all the solvent was evaporated. The residue was purified on silica gel eluted with ethyl acetate in hexanes to provide 96 mg (22%) of 37 as a clear oil. ¹H NMR (300 MHz, CDCl₃) δ 7.58 (s, 1H), 7.47 (dd, J = 1.60, 8.57 Hz, 1H), 7.15−7.25 (m, 1H), 7.10 (d, J = 8.67 Hz, 1H), 6.74 (dd, J = 2.26, 8.85 Hz, 1H), 6.46−6.58 (m, 1H), 4.29−4.42 (m, 1H), 3.26 (d, J = 11.49 Hz, 1H), 2.89 (d, J = 11.49 Hz, 1H), 2.77 (t, J = 12.24 Hz, 1H), 2.36 (dd, J = 3.86, 12.72 Hz, 1H), 2.13 (t, J = 10.93 Hz, 1H), 1.85−2.03 (m, 2H), 1.69 (d, J = 12.62 Hz, 1H), 1.44−1.61 (m, 1H), 1.10−1.40 (m, 3H), 0.74−1.00 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 168.2, 155.9, 135.9, 130.5, 128.5, 126.5, 126.4, 123.6, 118.8, 109.0, 58.7, 55.6, 51.9, 50.7, 33.6, 33.4, 31.9, 30.4, 29.7, 21.6, 19.0, 18.3. MS (ESI) m/z 355.4 (M + H)⁺. A beige solid was obtained as the hydrochloride salt of 37: mp 128−132 °C; [α]d²¹ = +33° (c 0.11, CH₃OH). Anal. (C₂₂H₃₁ClN₂O₂·0.75H₂O) C, H, N.

6-Hydroxy-N-{(1S)-2-methyl-1-[(4-methylpipieridine-1-yl)-methyl]propyl}−1,2,3,4-tetrahydronaphthalene-2-carboxamide Hydrochloride (38).

A solution of 6-methoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid (74a)²² (378 mg, 1.833 mmol) in aqueous HBr (48%) (10 mL) and acetic acid (10 mL) was heated at reflux for 5 h then cooled. Solvent was removed in vacuo to provide 6-hydroxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid (74b) which was carried on to the next step without further purification. A solution of 74b, 60a (220 mg, 1.24 mg), BOP (973 mg, 2.2 mmol, 1.2 equiv), and NEt₃ (0.880 mL, 5.5 mmol, 3 equiv) in THF (20 mL) was stirred at room temperature overnight. A saturated aqueous solution of NaHCO₃ (50 mL) was then added to the mixture, followed by extraction using EtOAc (3 × 50 mL). The combined organic layers were dried (Na₂SO₄), filtered over Celite, and concentrated in vacuo. The residue was purified on silica gel eluted with 10% CH₃OH in CH₂Cl₂ to provide a clear oil 563 mg (86% over two steps) of 38 as a mixture of diastereomers. ¹H NMR (300 MHz, CDCl₃) δ 6.82 (t, J = 8.48 Hz, 1H), 6.48−6.70 (m, 2H), 6.02−6.22 (m, 1H), 4.02 (dt, J = 4.99, 9.28 Hz, 1H), 2.94−3.07 (m, 1H), 2.70−2.93 (m, 2H), 2.44−2.70 (m, 6H), 2.08−2.44 (m, 3H), 1.83−1.95 (m, 2H), 1.59−1.79 (m, 3H), 1.12−1.48 (m, 3H), 0.77−1.01 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 176.1, 154.9, 136.9, 129.8, 126.0, 115.3, 113.5, 58.6, 55.2, 52.6, 50.5, 42.2, 36.8, 33.7, 32.1, 30.7, 30.4, 28.6, 26.0, 23.3, 18.7, 17.9. MS (ESI) m/z 359.5 (M + H)⁺. A beige solid was obtained as the hydrochloride salt of 38: mp 118−122 (fusion) °C; [α]d²⁰ = +40.3° (c 0.3, CH₃OH). Anal. (C₂₂H₃₅ClN₂O₂·1.25H₂O) C, H, N.

(3R)-3-[({(1S)-2-Methyl-1-[(4-methylpiperidin-1-yl)methyl]-propyl}amino)methyl]-1,2,3,4-tetrahydroisoquinolin-7-ol Trihydro-chloride (39).

A solution of 12 (113 mg, 0.314 mmol) in anhydrous THF was treated with borane dimethylsulfide (3 mmol, 0.3 mL) and heated at reflux overnight. After cooling, the mixture was quenched with CH₃OH (10 mL) and stirred at room temperature for 1 h. The mixture was treated with an aqueous 2 M HCl solution (5 mL) and heated at reflux for an additional 2 h. The solvent was removed in vacuo and resultant crude material was purified on silica gel, eluted with CMA80 (or DMA80) in CH₂Cl₂, to provide 81.2 mg (75%) of the reduced desired product (39). ¹H NMR (300 MHz, CD₃OD) δ 6.83 (d, J = 8.29 Hz, 1H), 6.53 (dd, J = 2.45, 8.29 Hz, 1H), 6.43 (d, J = 2.26 Hz, 1H), 3.91 (s, 1H), 3.42−3.54 (m, 1H), 3.24 (s, 1H), 2.82−3.10 (m, 4H), 2.38−2.73 (m, 6H), 2.32 (t, J = 10.93 Hz, 1H), 1.66 (d, J = 13.00 Hz, 2H), 1.32−1.56 (m, 2H), 1.06−1.30 (m, 2H), 131.1, 125.2, 115.5, 113.5, 60.4, 59.3, 55.7, 55.5, 53.2, 51.8, 48.0, 33.6, 33.4, 31.9, 30.8, 30.2, 21.8, 19.6, 17.5. MS (ESI) m/z 346.4 (M + H)⁺. The freebase was converted to the trihydrochloride salt of 39 as a white solid: mp 172 °C (sublimes); [α]d²⁰ = +27.3° (c 0.2, CH₃OH). Anal. (C₂₁H₃₈Cl₃N₃O·0.75H₂O) C, H, N.

(3R)-N3-{(1S)-2-Methyl-1-[(4-methylpiperidin-1-yl)methyl]-propyl}−1,2,3,4-tetrahydroisoquinoline-3,7-dicarboxamide Dihydrochloride (41).

Compound 73b (993.4 mg, 2.04 mmol) was treated according to the general method 2b for removal of the Boc-protection to provide 795 mg (100% yield) of the freebase amine 41. 1H NMR (300 MHz, CD₃OD) δ 7.69 (d, J = 9.2 Hz, 1H), 7.61 (s 1H), 7.24 (d, J = 9.0 Hz, 1H), 4.04−4.21 (m, 2H), 3.76 (br s, 1H), 3.40−3.53 (m, 2H), 3.12−3.29 (m, 2H), 2.92−3.10 (m, 2H), 2.80−2.90 (m, 1H), 1.95−1.98 (m, 2H), 1.85−1.93 (m, 2H), 1.62−1.74 (m, 1H), 1.35−1.55 (m, 3H), 0.98−1.01 (m, 9H). 13C NMR (75 MHz, CD₃OD) δ 176.1, 175.3, 171.6, 138.6, 135.6, 130.1, 126.4, 118.0, 111.1, 66.5, 60.6, 58.1, 57.0, 55.0, 53.2, 50.9, 47.3, 32.0, 31.8, 21.9, 19.3, 18.0. MS (ESI) m/z 387.4 (M + H)⁺. An off-white solid was obtained as the dihydrochloride salt of 41: mp 185−187 °C; [α]d²² = +69.2° (c 1.1, CH₃OH). Anal. (C₂₂H₃₆Cl₂N₄O₂·2H₂O) C, H, N.

(3R)-7-Methoxy-N-{(1S)-2-methyl-1-[(4-methylpiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (43).

A solution of 73c (365 mg, 0.793 mmol) and diisopropylethylamine (1 mL, 5.00 equiv) in 5 mL of CH₃CN:CH₃OH (4:1) was treated with trimethylsilyl diazomethane (1.7 mL, 2 M in Et₂O, 3 equiv) and stirred at room temperature overnight. The excess reagent was quenched with AcOH and solvent was removed in vacuo. The residue was extracted from NaHCO₃ with EtOAc (3 × 50 mL). The combined organic layers were dried (Na₂SO₄), filtered over Celite, and concentrated in vacuo. The residue was purified over silica gel, eluted with EtOAc in hexanes to furnish compound Boc-43 (290 mg, 77%): ¹H NMR (300 MHz, CDCl₃) δ 6.90−7.21 (m, 1H), 6.58−6.84 (m, 2H), 5.89−6.27 (m, 1H), 4.36−4.76 (m, 3H), 3.79 (s, 1H), 3.51−3.67 (m, 1H), 3.14−3.40 (m, 1H), 2.90−3.14 (m, 1H), 2.60 (br s, 2H), 2.00−2.28 (m, 2H), 1.63−1.97 (m, 2H), 1.39−1.60 (m, 13H), 0.91−1.34 (m, 4H), 0.76−0.89 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) 171.6, 155.6, 134.0, 129.2, 123.7, 114.5, 113.2, 81.5, 59.3, 56.9, 56.5, 54.3, 53.5, 51.3, 45.1, 44.5, 34.2, 34.0, 30.5, 30.2, 28.3 (3C’s), 21.8, 18.9, 17.4. MS (ESI) m/z 474.6 (M + H)⁺. Boc-43 (290 mg, 0.62 mmol) was subjected to Boc-cleavage according to general method 2a to provide 43 (155 mg, 63%) as the freebase: ¹H NMR (300 MHz, CDCl₃) δ 6.89−7.15 (m, 2H), 6.72 (dd, J = 2.64, 8.29 Hz, 1H), 6.56 (d, J = 2.45 Hz, 1H), 3.89−4.06 (m, 2H), 3.70−3.81 (m, 3H), 3.43−3.58 (m, 1H), 3.11 (dd, J = 5.09, 16.20 Hz, 1H), 2.69−2.94 (m, 4H), 2.23−2.51 (m, 2H), 1.95−2.08 (m, 1H), 1.72−1.94 (m, 2H), 1.42−1.62 (m, 2H), 1.20−1.37 (m, 2H), 0.95−1.16 (m, 2H), 0.83−0.93 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 173.0, 157.9, 136.8, 130.1, 126.4, 112.5, 110.5, 59.8, 56.9, 55.2, 55.1, 53.2, 50.7, 47.7, 34.4, 34.2, 30.7, 30.6, 30.3, 21.8, 19.2, 17.7. MS (ESI) m/z 374.6 (M + H)⁺. A beige solid was obtained as the dihydrochloride salt of 43: mp 116−120 °C; [α]d²⁵ = +69.8° (c 1.1, CH₃OH). Anal. (C₂₂H₃₇Cl₂N₃O₂·0.75H₂O) C, H, N.

(3R)-7-Fluoro-N-{(1S)-2-methyl-1-[(4-methylpiperidin-1-yl)-methyl]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (45).

Compound 73d (1.15 g, 2.48 mmol) was subjected to Boc-cleavage according to general method 2b to provide 45 (588 mg, 66%) as the freebase. ¹H NMR (300 MHz, CDCl₃) δ 6.97−7.23 (m, 2H), 6.83 (dt, J = 2.26, 8.48 Hz, 1H), 6.67−6.76 (m, 1H), 3.89−4.08 (m, 3H), 3.56 (dd, J = 5.27, 9.42 Hz, 1H), 3.10 (dd, J = 5.18, 16.29 Hz, 1H), 2.62−2.93 (m, 3H), 2.16−2.51 (m, 2H), 1.72−2.08 (m, 4H), 1.53 (t, J = 12.90 Hz, 2H), 1.29 (dt, J = 3.58, 6.97 Hz, 1H), 1.16 (dt, J = 3.20, 11.77 Hz, 1H), 0.97−1.10 (m, 1H), 0.83−0.95 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 172.6, 161.0 (J_CF = 244.7 Hz), 137.6 (J_CF = 6.5 Hz), 130.5 (J_CF = 7.7 Hz), 129.8 (J_CF = 2.4 Hz), 113.4 (J_CF = 21.4 Hz), 112.0 (J_CF = 21.0 Hz), 59.8, 56.3, 55.0, 53.2, 50.8, 47.0, 34.5, 34.2, 30.6, 30.5, 30.2, 21.8, 19.2, 17.7. MS (ESI) m/z 362.4 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 45: mp 172−175 °C; [α] 24 = +68.9° (c 1.1, 0.76−0.99 (m, 9H). ¹³C NMR (75 MHz, CD OD) δ 156.8, 135.6, 3 1.25H₂O) C, H, N.

(3R)-7-Hydroxy-2-methyl-N-{(1S)-2-methyl-1-[(4-methylpiperidin-1yl)methy]propyl}−1,2,3,4-tetrahydroisoquinoline-3-carboxamide Dihydrochloride (47).

A solution of 12 (446 mg, 1.24 mmol) in dichloroethane (5 mL) was treated with formalin (0.11 mL, 1.2 equiv) followed by NaBH(OAc)₃ (1.2 g, 5.6 mmol). The reaction mixture was stirred at room temperature for 24 h, then partitioned between CH₂Cl₂ and saturated aqueous NaHCO₃. The organic portion was extracted three times (3 × 30 mL), and combined organic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified on silica gel eluted with ethyl acetate/hexanes to furnish 232 mg (50% yield) of the desired freebase 47. ¹H NMR (300 MHz, CDCl₃) δ 7.09 (d, J = 9.23 Hz, 1H), 6.91 (d, J = 8.10 Hz, 1H), 6.66 (dd, J = 2.35, 8.19 Hz, 1H), 6.56 (d, J = 2.26 Hz, 1H), 3.93−4.05 (m, 1H), 3.78 (d, J = 15.07 Hz, 1H), 3.56 (d, J = 15.07 Hz, 1H), 3.15−3.26 (m, 1H), 2.86−3.05 (m, 2H), 2.62−2.84 (m, 2H), 2.37−2.50 (m, 3H), 2.23−2.36 (m, 2H), 1.73−2.03 (m, 3H), 1.52 (td, J = 3.18, 6.64 Hz, 2H), 1.00−1.38 (m, 4H), 0.83−0.96 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3, 155.2, 135.4, 128.9, 124.2, 114.3, 113.1, 64.6, 59.8, 55.7, 54.8, 53.2, 51.0, 42.3, 34.4, 34.2, 30.6, 28.5, 21.9, 19.3, 17.7. MS (ESI) m/z 374.3 (M + H)⁺. A white solid was obtained as the dihydrochloride salt of 47: mp 186−188 °C; [α]d²⁵ = +61.7° (c 1.1, CH₃OH). Anal. (C₂₂H₃₇Cl₂N₃O₂·H₂O) C, H, N.

[³⁵S]GTPγS Binding Assay.

The [³⁵S]GTPγS assays were conducted using the methods previously reported.¹⁵ Briefly, the binding of the GTP analogue [³⁵S]GTPγS to membranes was determined in a volume of 500 μL. The assay mixture contained 50 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA·2H₂O, 1 mM dithiothreitol, 0.1% BSA, 10 μM GDP, test compounds, and approximately 132000 cpm of [³⁵S]-GTPγS (0.1 nM). Human κ (20 μg), μ (10 μg), or δ (7 μg) opioid receptor-expressing CHO cell membranes was added to each tube. For the κ receptor assays, eight-point concentration response curves of the agonist U69,593 (31.6 μM, 10 μM, 3.16 μM, 316 nM, 100 nM, 31.6 nM, 3.16 nM, 0.316 nM final) were prepared, and the dilutions were coincubated with a single concentration of each test compound. For the μ and δ receptors, the agonists were DAMGO (31.6 μM, 10 μM, 3.16 μM, 316 nM, 100 nM, 31.6 nM, 3.16 nM, 0.316 nM final) and DPDPE (10 μM, 3.16 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM final), respectively. Following a 60 min shaking incubation at 22 °C, the assay was terminated by filtration under vacuum on a Brandel (Gaithersburg, MD, USA) 96-well harvester using presoaked PerkinElmer GF/B glass fiber filters. The filters were rinsed three times with 1 mL washes of ice-cold wash buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 10 mM MgCl₂). Filter plates were dried for 1 h at 55 °C. Microscint 20 (50 μL) was added to each well, and filter-bound radioactivity was counted on a Packard TopCount NXT microplate scintillation and luminescence counter. Total binding (TB) was determined in the absence of compounds, and nonspecific binding (NSB) was determined in the presence of 10 μM final unlabeled GTPγS. Percent specific bound (SB) was calculated using the equation % SB = (SB/MB) × 100, where maximal binding (MB) is calculated by subtracting NSB from TB. Percent SB was plotted against the log of compound concentration. Data were fit to a three-parameter logistic curve to generate EC₅₀ values (GraphPad Prism, GraphPad Software, Inc., San Diego, CA). K_e values were calculated using the equation K_e = [L]/((EC₅₀⁺/EC₅₀⁻) − 1) where [L] is the concentration of test compound, EC₅₀⁺ is the EC₅₀ of the control agonist with test compound, and EC₅₀⁻ is the EC₅₀ of control agonist alone. K_e values were considered valid when the EC₅₀⁺/EC₅₀⁻ ratio was at least 4.

Pharmacokinetic Studies.

In Vivo Study.

Pharmacokinetic studies were conducted at Mispro Biotech Services (RTP, NC) and were approved by the Institutional Animal Care and Use Committee. Animals were housed in facilities that are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal procedures were in accordance with the “Guide for the Care and Use of Laboratory Animals” (National Research Council 2011). Male Sprague—Dawley CD rats (Crl:CD-(SD)) were obtained from Charles River Laboratories (Raleigh, NC). The animals were acclimated for 1 week prior to use on study and had ad libitum access to Picolab 5053 food and Durham City (NC) tap water. Environmental conditions included: room temperature 72 ± 3 °F (22 ± 2 °C), relative humidity 35−65%, and a 12 h light/dark cycle. Animals were 10 weeks old at dosing.

A single subcutaneous dose of 12 at 5 mg/kg was administered in saline (5 mg/mL) at a dose volume of 1 mL/kg. Three rats per time point were euthanized at 1, 4, 24, 72, and 168 h post dose by asphyxiation with CO₂, and blood was collected via cardiac puncture using K₃EDTA as anticoagulant. Plasma was prepared from blood by centrifugation at 2000g for 10 min at 4 °C. Brains were collected and flash frozen in liquid nitrogen and stored at −20 °C until analysis.

Sample Preparation.

Brains were weighed and homogenized at 1:5 (wt:vol) with 50:50 ethanol:water using a Geno/Grinder 2010 (SPEXSamplePrep, Metuchen, NJ) at 1750 rpm for 30 s × 2. Aliquots (50 μL) of plasma or brain homogenate were mixed with 300 μL of methanol containing buspirone as internal standard (50 ng/mL) in duplicate in a 96-well plate. The plate was shaken at 800 rpm for 5 min and then centrifuged at 4000 rpm for 10 min at 4 °C. Aliquots (50 μL) of supernatant were mixed with 50 μL of mobile phase B in a new plate and analyzed by LC/MS-MS.

Standard Curve Preparation.

Stock solutions of 12 were prepared and diluted serially to generate standard curve and quality control spiking solutions. The concentrations in plasma were 1, 10, 50, 100, 500, and 1000 ng/mL for standard curves and 2, 20, and 200 ng/mL for quality control samples. In brain homogenate, standard curve sample concentrations were 0.1, 0.2, 0.5, 5, 10, and 50 ng/mL, and 2 and 20 ng/mL for quality control samples.

LC/MS-MS Conditions.

Chromatography was conducted using an Agilent 1100 binary pump and autosampler (Santa Clara, CA) with injection of 10 μL of the processed samples/calibration standards onto a Phenomenex Luna C8 (150 mm × 4.6 mm, 5 μM) column (Torrance, CA). Mobile phase A consisted of 0.5% formic acid in water with 5 mM ammonium acetate, and mobile phase B consisted of 0.5% formic acid in 85:15 CH₃CN:H₂O with 5 mM ammonium acetate. Chromatography was conducted using a linear gradient starting at initial conditions of 5% B and holding for 1 min before increasing linearly to 95% B over 4 min before returning to initial conditions over 0.1 min. Total run time was 8 min, and flow rate was 0.750 mL/min. Quantitation was achieved by multiple reaction monitoring in positive ion mode using an Applied Biosystems Sciex API4000 (Foster City, CA) mass spectrometer with an electrospray ionization source. Compound 12 was quantitated by monitoring the transition of m/z 360.231 → 148.2, and the internal standard, buspirone, was monitored by m/z 386.243 → 122.1. MS parameters were as follows: CUR = 16, GS1 = 50, GS2 = 40, TEM = 650, CAD = 12, and IS = 2000. Calibration curves were processed using Analyst 1.6.2 software by plotting the analyte to internal standard peak area ratio against the calibration standard concentrations. The limit of quantitation for plasma was 1.0 ng/mL and for brain was 0.6 ng/g.

ABBREVIATIONS USED

[³⁵S]GTPγS

sulfur-35 guanosine-5′-O-(3-thio)triphosphate

DAMGO

[d-Ala2,MePhe4,Gly-ol5]encephalin

DPDPE

[d-Pen2,d-Pen5]encephalin

U69,593

(5α,7α,8β)-(—)-N-meth-yl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-[4.5]dec-8-yl]-benzeneacetamide

EDC

1-ethyl-3(−3-(dimethylamino)-propyl)carbodiimide

HOBt

hydroxybenzotriazole

AUC_last

area under the curve from the time zero to time of last measurable concentration