Synthesis and Evaluation of a Novel Series of 2,7-Substituted-6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline Derivatives as Selective Peroxisome Proliferator-Activated Receptor γ Partial Agonists

Abstract

A novel series of 7-substituted-2-[3-(2-furyl)acryloyl]-6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline derivatives were synthesized to clarify structure–activity relationships for peroxisome proliferator-activated receptor γ (PPARγ) partial agonist activity and identify more efficacious PPARγ partial agonists with minor adverse effects. Among the derivatives synthesized, compound 26v with a 2-(2,5-dihydropyrrol-1-yl)-5-methyloxazol-4-ylmethoxy group at the 7-position of the tetrahydroisoquinoline structure exhibited stronger PPARγ agonist and antagonist activities (EC₅₀ = 6 nM and IC₅₀ = 101 nM) than previously reported values for compound 1 (EC₅₀ = 13 nM and IC₅₀ = 512 nM). Compound 26v had very weak protein tyrosine phosphatase 1B (PTP1B) inhibitory activity and showed higher oral absorption (C_max = 11.4 µg/mL and area under the curve (AUC) = 134.7 µg·h/mL) than compound 1 (C_max = 7.0 µg/mL and AUC = 63.9 µg·h/mL) in male Sprague-Dawley (SD) rats. A computational docking calculation revealed that 26v bound to PPARγ in a similar manner to that of compound 1. In male Zucker fatty rats, 26v and pioglitazone at 10 and 30 mg/kg for 4 weeks similarly reduced plasma triglyceride levels, increased plasma adiponectin levels, and attenuated increases in plasma glucose levels in the oral glucose tolerance test, while only pioglitazone decreased hematocrit values. In conclusion, 6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline derivatives provide a novel scaffold for selective PPARγ partial agonists and 26v attenuates insulin resistance possibly by adiponectin enhancements with minor adverse effects.

Introduction

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor belonging to the nuclear receptor family and a master gene regulator of adipocyte differentiation.¹⁾ PPARγ agonists, such as pioglitazone and rosiglitazone (Fig. 1), have been clinically used as anti-diabetic drugs because they attenuate insulin resistance in type 2 diabetic patients.^2–5) PPARγ agonists promote adipocyte differentiation, resulting in increases in adiponectin, an anti-insulin resistance adipokine, and decreases in tumor necrosis factor α (TNFα), an insulin resistance-inducing cytokine, leading to enhanced insulin sensitivity.^6,7) However, pioglitazone and rosiglitazone induce hemodilution and increase the risks of weight gain, congestive heart failure, and bone fracture by the full activation of PPARγ.^2,3,8) In contrast to PPARγ full agonists, PPARγ partial agonists have been expected to be safer PPARγ agonists with potent efficacy. PPARγ partial agonists partially activate PPARγ and antagonize the activation of PPARγ by PPARγ full agonists, such as farglitazar and rosiglitazone⁹⁾ (Fig. 1), and are considered to act as PPARγ modulators.¹⁰⁾ PPARγ modulators partially activate PPARγ and fully induce insulin sensitization without adverse effects, while PPARγ full agonists maximally activate PPARγ and induce insulin sensitization as well as adverse effects. Many structurally different PPARγ partial agonists have been reported to exhibit higher efficacies with lower toxicities in experimental diabetes; however, none have been successfully developed to date possibly due to low efficacy and/or safety.¹⁰⁾ Therefore, new partial agonists with different structures are desired and their efficacies and adverse effects need to be investigated.

Fig. 1. Chemical Structures of PPARγ Agonists

Reported PPARγ partial agonists are structurally classified into three chemotypes: thiazolidinedione, carboxylic acid, and sulfonamide types^9,11–15) (Fig. 1). We previously reported various types of PPARγ agonists using the same scaffold, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid: a selective PPARγ full agonist, PPARγ full agonist with protein tyrosine phosphatase 1B (PTP1B) inhibition, PPARα/γ dual agonist with PTP1B inhibition, and PPARγ partial agonist with PTP1B inhibition.^9,16–19) PTP1B negatively regulates the insulin signal, and, thus, the inhibition of PTP1B is expected to exert synergistic effects with PPARγ activation on insulin sensitization. Among 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids, a compound with PPARγ partial agonist activity and PTP1B inhibitory activity has been reported to exhibit effective anti-diabetic activity without PPARγ-related side effects.^9,20) They are considered to exert effective anti-diabetic effects via the synergy of PPARγ partial activation and PTP1B inhibition. However, PTP1B inhibition may promote carcinogenesis and exacerbate inflammatory responses.^21,22) Therefore, it is important to identify and develop structurally new PPARγ partial agonists without PTP1B inhibitory activity. We recently synthesized a series of 2-substituted-3-unsubstituted-1,2,3,4-tetrahydroisoquinoline derivatives with various acidic groups at the 6-position, and identified 7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethoxy]-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline as a selective and potent PPARγ partial agonist¹⁵⁾ (1, Fig. 1). Compound 1 exerted potent anti-diabetic effects in KK-Ay mice, type 2 diabetic mice, however, its safety has not yet been proven. In the present study, a series of 7-substituted-2-[(E)-3-(2-furyl)acryloyl]-6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline derivatives were synthesized and structure–activity relationships for PPARγ partial agonist activity were discussed. A compound with a (2,5-dihydropyrrol-1-yl)-5-methyloxazol-4-ylmethoxy group at the 7-position (26v) was identified as the most potent PPARγ partial agonist and exerted similar insulin-sensitizing effects to pioglitazone, but with weaker adverse effects.

Chemistry

The synthesis of 7-substituted-2-[(E)-3-(2-furyl)acryloyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline derivatives is outlined in Charts 1 and 2. In Chart 1, the ester group of compound 2a, which was prepared as described previously,²³⁾ was reduced with sodium borohydride (NaBH₄) and then brominated with tetrabromomethane (CBr₄) and triphenylphosphine (PPh₃) to give 3a. Compounds 8b–p were separately synthesized from various acyl chlorides (4b–e, 4j) or carboxylic acids (5f–i, 5k–p) via compounds 6b–p and 7b–p. Acyl chlorides (4b–e, 4j) or carboxylic acids (5f–i, 5k–p) were amidated with methyl 2-amino-3-oxobutanoate hydrochloride to give compounds 6b–p. Compounds 6b–p were cyclized with I₂, PPh₃, and triethylamine (Et₃N) to afford methyl oxazole-5-carboxylates (7b–p). The ester group of compounds 7b–p was transformed to a chloromethyl group by reduction with lithium aluminium hydride (LiAlH₄) and then chlorinated with SOCl₂ to give 8b–p. The halides obtained (3a, 8b–p) were reacted with compound 9¹⁵⁾ to afford 10a–p. The carbamoyl moiety of compounds 10a–p was converted to nitriles 11a–p by POCl₃ and Et₃N, and these compounds were then treated with n-Bu₃SnN₃ to give tetrazole derivatives (12a–p). The tert-butoxycarbonyl (Boc) group of 12a–p was removed with HCl in i-PrOH, followed by acylation with (E)-3-(2-furyl)acrylic acid to afford 2-acyl-tetrahydroisoquinoline derivatives (13a–p).

Chart 1. Synthesis of 7-Substituted-2-[(E)-3-(2-furyl)acryloyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline Derivatives 13a–p

Reagents and conditions: (i) NaBH₄, MeOH, THF; (ii) CBr₄, PPh₃, CH₂Cl₂; (iii) Methyl 2-amino-3-oxobutanoate hydrochloride, Et₃N, CH₂Cl₂; (iv) (COCl)₂, DMF, CH₂Cl₂ then methyl 2-amino-3-oxobutanoate hydrochloride, Et₃N; (v) Methyl 2-amino-3-oxobutanoate hydrochloride, EDC·HCl, i-Pr₂NEt, CH₂Cl₂; (vi) I₂, PPh₃, Et₃N, CH₂Cl₂; (vii) LiAlH₄, THF; (viii) SOCl₂, CH₂Cl₂; (ix) K₂CO₃, DMF; (x) Cs₂CO₃, DMF; (xi) POCl₃, Et₃N, CH₂Cl₂; (xii) n-Bu₃SnN₃, toluene; (xiii) HCl in i-PrOH, HCO₂H; (xiv) (E)-3-(2-furyl)acrylic acid, (COCl)₂, DMF, i-Pr₂NEt, CH₂Cl₂; (xv) (E)-3-(2-furyl)acrylic acid, EDC·HCl, Et₃N, CH₂Cl₂.

The seven other derivatives were synthesized by the different routes shown in Chart 2. The oxazole moieties 18q–w were obtained more conveniently by substitution at the 2-position of 14 or 15²⁴⁾ than by the route of cyclization to oxazoles in Chart 1. Namely, compound 14 or 15 was reacted with various amines to obtain the corresponding 2-tertiaryamino-5-methyloxazole-4-carboxylates 16q, 16u, 17r–t, 17v, and 17w, and these esters were then reduced by LiAlH₄ to afford 18q–w. tert-Butyl 7-hydroxy-6-[1(2)-methoxymethyltetrazol-5-yl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylates (22 and 23) were separately synthesized. The carbamoyl group of compound 19¹⁵⁾ was converted to tetrazole 21 via nitrile 20 in the same procedure to the conversion of 10 to 12. The tetrazole moiety of compound 21 was protected by the methoxymethyl (MOM) group, and benzyl protection was then removed by hydrogenation to obtain a mixture of 22 and 23. Each regioisomer (22 and 23) was isolated by silica gel column chromatography. The hydroxyl group of compounds 22 and 23 at the 7-position was then alkylated with 18q–w by the Mitsunobu reaction to give 24s, 24u, 25q, 25r, 25t, 25v, and 25w. The MOM and Boc groups of 24s, 24u, 25q, 25r, 25t, 25v, and 25w were removed and the amino moiety generated at the 2-position was acylated with (E)-3-(2-furyl)acrylic acid to obtain 26q–w.

Chart 2. Synthesis of 7-Substituted-2-[(E)-3-(2-furyl)acryloyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline Derivatives 26q–w

Reagents and conditions: (i) imidazole, NaH, DMF; (ii) amine, (i-Pr₂NEt); (iii) 3-pyrroline, K₂CO₃, DMF; (iv) LiAlH₄, THF; (v) POCl₃, Et₃N, CH₂Cl₂; (vi) n-Bu₃SnN₃, toluene; (vii) MOMCl, K₂CO₃, DMF; (viii) H₂, Pd-C, MeOH; (ix) 18q–w, ADDP, n-Bu₃P, toluene; (x) HCl in i-PrOH, HCO₂H; (xi) (E)-3-(2-furyl)acrylic acid, (COCl)₂, Et₃N, CH₂Cl₂.

Results and Discussion

We previously reported that 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is a scaffold for PPARγ agonists and we synthesized various types of derivatives as PPARγ full agonists, PPARα/γ agonists, and PPARγ partial agonists.^9,16–19) Among them, the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-based PPARγ partial agonist (KY-755, Fig. 1) exhibited potent activity with a high maximal activation level (EC₅₀ = 85 nM, max 65%) and exerted stronger anti-diabetic effects than rosiglitazone, a PPARγ full agonist.⁹⁾ Rosiglitazone, but not KY-755, exerted adverse effects, such as hemodilution and hepatomegalysis, potentially via the activation of PPARγ. KY-755 exhibits PPARγ partial agonist activity and PTP1B inhibitory activity, which may contribute to its anti-diabetic effects. We then demonstrated that 3-unsubstituted-2,7-substituted-1,2,3,4-tetrahydroisoquinoline with an acidic group at the 6-position is a scaffold for a PPARγ partial agonist, and also that the 6-tetrazolyl derivative (1, Fig. 1) exhibited stronger partial agonist activity (EC₅₀ = 13 nM, Max 30%) than KY-755 with very weak PTP1B inhibitory activity.¹⁶⁾ We revealed that compound 1 showed a similar binding mode to previously reported partial agonists (amorfrutin 2 and nTZDpa, Fig. 1)^13–15) in computational docking calculations: 6-tetrazole forms a salt bridge with Arg288 (Helix 3) and a hydrogen bond with Ser342 (β-strand 3), and oxazole at 7-substituent interacts with Cys285 (Helix 3). Cyclopentenyl on the oxazole ring is speculated to have a lipophilic interaction with the PPARγ protein; however, its influence on activity remains unclear. Thus, in the present study, various substituents were introduced into the 2-position of the oxazole ring and structure–activity relationships were investigated.

The PPARγ agonist activities of the synthesized compounds were assessed as transactivation activity in COS-1 cells containing the full-length human PPARγ1 plasmid co-transfected with the human retinoid X receptor α (RXRα) plasmid and reporter plasmid pGL3-PPREx4-tk-luc. Activation responses were expressed as relative values to maximal activation by farglitazar as a standard PPARγ full agonist as previously reported.^{9,15,17–19)} Antagonist activity against farglitazar (10⁻⁷ M) was assessed to identify the PPARγ partial agonist because partial agonists with high affinity and low efficacy antagonize the full agonist. The effects of the compounds on PTP1B activities were examined using a human PTP1B enzyme. Plasma concentrations after the oral administration of the selected compounds at 10 mg/kg were evaluated, and insulin-sensitizing effects and adverse effects were investigated in male Zucker fatty rats, which are leptin receptor-deficient obese rats. All animal experiments in the present study were conducted according to the guidelines for animal experiments of our institute and the guidelines for animal experimentation approved by the Japanese Association of Laboratory Animal Science.

The PPARγ agonist and antagonist activities of the compounds synthesized were shown in Tables 1 and 2. The derivatives with no or small side chains on the oxazole ring (13a, 13b) exhibited very weak PPARγ partial agonist activities, while those with ethyl (13c) and isobutyl (13d) had weak PPARγ partial agonist and antagonist activities. The derivative with the tert-butyl group on the oxazol ring (13e) exhibited more potent PPARγ partial agonist activity with lower maximal activation (EC₅₀ = 15 nM, Max 16.7%) than compound 13d with isobutyl, and its activity was similar to that of compound 1, indicating that steric bulkiness on the oxazole ring is important for PPARγ partial agonist activity. Thus, derivatives with various aliphatic cyclosubstituents were synthesized: PPARγ partial agonist activity was enhanced in ring size- and carbon number-dependent manners. The PPARγ partial agonist activity of compound 13f with cyclopropylmethyl was 2.5-fold that of compound 13d with isobutyl, suggesting that a cyclosubstituent is preferable for PPARγ partial agonist activity than a branched chain substituent. Among cycloalkyl derivatives (13i, 13j, 13k, 13l), PPARγ agonist activity increased in a ring size-dependent manner, and among them, 13l exhibited the most potent activity (EC₅₀ = 14 nM), which was similar to that of compound 1.

Table 1. Chemical Structures, Molecular Weights, clog D_7.4, and PPARγ Agonist and Antagonist Activities of 7-Substituted-2-[(E)-3-(2-furyl)acryloyl]-6-tetrahydroisoquinoline Derivatives 13a–l

a) n = 2. b) The maximal activation level relative to the level maximally activated by farglitazar. c) The maximal inhibition level against farglitazar (10⁻⁷ M). d) Cited from a previous study (ref. 15). e) The activation level at 10000 nM. Activation did not reach the maximal level at 10000 nM. f) The inhibition level at 10000 nM. Inhibition did not reach the maximal level at 10000 nM.

Table 2. Chemical Structures, Molecular Weights, clog D_7.4, and PPARγ Agonist and Antagonist Activities of 7-Substituted-2-[(E)-3-(2-furyl)acryloyl]-6-tetrahydroisoquinoline Derivatives 13m–p and 26q–w

a) n = 2. b) The maximal activation level relative to the level maximally activated by farglitazar. c) The maximal inhibition level against farglitazar (10⁻⁷ M). d) The activation level at 10000 nM. Activation did not reach the maximal level at 10000 nM. e) The inhibition level at 10000 nM. Inhibition did not reach the maximal level at 10000 nM.

Derivatives with heteroaromatic and nitrogen-containing substituents were then synthesized (Table 2). The introduction of heteroaromatic substituents (26q, 13n, and 13o) decreased PPARγ partial agonist activity possibly due to basicity and/or low lipophilicity. The activities of compounds with an aliphatic amino group were also dependent on carbon numbers. The activities of compounds with a cycloaliphatic amino group were dependent on their ring size, except for 26v, which was the most potent among the derivatives synthesized. In Tables 1 and 2, all derivatives with PPARγ partial agonist activity also exhibited PPARγ antagonist activity. The order of partial agonist activity in derivatives with 5-membered cyclosubstituents was as follows: dihydropyrrolyl (26v) > cyclopentenyl (1) > cyclopentyl (13k) > pyrrolidinyl (26u) > pyrrolyl (13m) > furyl (13p) > imidazolyl (26q). Unexpectedly, the activity of compound 26u (pyrrolidinyl) was weaker than compound 26s (diethylamino), while compound 13f (cyclopropylmethyl) was stronger than compound 13d (isobutyl), suggesting the effects of different steric structures. Comparisons of compound 1 vs. 13k and 26v vs. 26u indicated that the insertion of a double bond structure may be preferable for activity.

Regarding compound 26v, which had the strongest PPARγ partial agonist activity, we performed computational ligand docking to obtain information on the binding mode to PPARγ. Compound 26v was docked to each of the PPARγ protein structures, 5Y2O chain A (co-crystal structure with full agonist) and 4A4V chain A (co-crystal structure with partial agonist), using the same protocol for compound 1.¹⁵⁾ As a result, compound 26v was more stably docked into 4A4V than 5Y2O (Glide docking score −10.3 versus −8.5), similar to compound 1. The binding mode of compound 26v was also similar to that of compound 1 (Fig. 2). The tetrazolyl group of compound 26v at the 6-position formed a hydrogen bond with Ser342 of β-strand 3 and a salt bridge with Arg288 of Helix 3. Compound 26v appeared to exhibit partial agonism by stabilizing Helix 3 and the β-strand, as previously reported for amorfrutin 2 and compound 1.¹⁵⁾ Furthermore, the cyclopentenyl moiety in compound 1 and dihydropyrrolyl moiety in 26v on the oxazole ring sterically fit into the subpocket composed of residues such as Cys285 and Ser289 of Helix 3, Ile326, Tyr327, and Leu330 of Helix 5, and Phe363 and Met364 of Helix 7 (Fig. 2). Although the activity of compound 26v was approximately 2-fold higher than that of compound 1, calculated binding free energy did not significantly differ between the two compounds (26v: ΔG_bind = −81 kcal/mol, 1: ΔG_bind = −82 kcal/mol). Compound 26v exhibited more potent PPARγ agonist activity than compound 1 in the cell-based luciferase assay; however, no significant differences were observed in calculated ΔG_bind between the two compounds. It currently remains unclear why compound 26v was more potent than compound 1 in the luciferase assay: the intracellular concentration of the former may be higher than that of the latter.

Fig. 2. Docking Poses of Compounds 1 and 26v to PPARγ

Compounds were docked into the PPARγ protein structure 4A4V and then energy-minimized. The PPARγ residues described in the manuscript are shown as sticks and labeled. Hydrogen bonds and salt bridges are depicted as cyan and orange dotted lines, respectively. The secondary structures (Helix (H) and β-strand (S)) of PPARγ and the protein surfaces from the residues Cys285, Ser289, Ile326, Tyr327, Leu330, Phe363, and Met364 were depicted by Maestro (Schrödinger Suite 2018-1). H12 is hidden in the right panels for visibility.

Among the synthesized derivatives, compounds 13e, 13l, 26s, and 26v exhibited similar potent activities to compound 1; therefore, their effects on PTP1B activity and oral absorption were examined (Table 3). These derivatives all exhibited very weak PTP1B inhibitory activity, suggesting selective PPARγ partial agonists. In male Sprague-Dawley (SD) rats, C_max and area under the curve (AUC) values after the oral administration of compounds 13e, 13l, and 26s at 10 mg/kg were markedly smaller than those of compound 26v. Compound 26v showed higher oral absorption than compound 1 (Table 3) and had a longer half-life (T_1/2) (26v: 5.6 h, 1: 4.4 h). The time to reach the maximum concentration (T_max) and the volume of distribution (Vd/F) of both compounds were similar (26v: T_max = 3 h and Vd/F = 0.6 L/kg, 1: T_max = 3 h and Vd/F = 1.0 L/kg), whereas the clearance of compound 26v was approximately 2-fold less than that of compound 1 (26v: CL_0–24h = 74.4 mL/h·kg, 1: CL_0–24h = 158.4 mL/h·kg). Compound 26v may be metabolically more stable than compound 1. Therefore, compound 26v was selected for further evaluations of efficacy and adverse effects. We previously compared the anti-diabetic activity of compound 1 with that of pioglitazone in male and female KK-Ay mice; the oral administration of compound 1 and pioglitazone for 1 week reduced plasma glucose and triglyceride levels, and adverse effects were not observed because of the short period of administration. In the present study, compound 26v and pioglitazone (10 and 30 mg/kg) were orally administered to male Zucker fatty rats, which are leptin receptor-deficient obese rats with insulin resistance, for 4 weeks and their efficacies and adverse effects were examined. Compound 26v and pioglitazone dose-dependently and similarly lowered plasma triglyceride and non-esterified fatty acids (NEFA) levels and increased plasma adiponectin levels (Table 4). Compound 26v inhibited the increase in plasma glucose levels induced by the oral administration of glucose in the oral glucose tolerance test (OGTT) test, indicating that compound 26v mitigated insulin resistance (Fig. 3 and Table 4). These results demonstrated that compound 26v was nearly equipotent to pioglitazone as an insulin sensitizer, and these effects may have been due to enhancements in adiponectin as reported previously.¹⁾ On the other hand, pioglitazone, but not compound 26v, reduced hematocrit values, suggesting hemodilution as an adverse effect (Table 5). Pioglitazone and compound 26v at 10 mg/kg similarly increased body weight gain, whereas pioglitazone induced a significantly larger increase than compound 26v at 30 mg/kg (Table 5). Therefore, compound 26v exerted similar potent insulin-sensitizing effects to pioglitazone, but with weaker adverse effects.

Table 3. Chemical Structures, PPARγ Agonist Activities, PTP1B Inhibitory Activities, and Pharmacokinetic Parameters of 7-Substituted-2-[(E)-3-(2-furyl)acryloyl]-6-tetrahydroisoquinoline Derivatives at 10 mg/kg after Oral Administration to SD Rats

a) n = 2. b) n = 3. c) n = 2. d) Cited from a previous study (ref. 15).

Table 4. Effects of 26v Orally Administered for 4 Weeks on Plasma Triglyceride, Non-esterified Fatty Acid (NEFA), Adiponectin, and Glucose Levels in the Oral Glucose Tolerance Test (OGTT) in Male Zucker Fatty Rats

Compound	Dose (mg/kg)	Triglyceride (mg/dL)	NEFA (mEq/L)	Adiponectin (µg/dL)	Glucose in OGTT (mg/dL)
Control		274 ± 20.1	1.04 ± 0.10	5.0 ± 0.2	356 ± 19.5
26v	10	162 ± 11.0	0.70 ± 0.12	8.3 ± 0.4**	293 ± 9.7**
	30	109 ± 6.9	0.48 ± 0.05**	9.9 ± 0.9**	271 ± 8.7**
Pioglitazone	10	146 ± 10.5	0.56 ± 0.13*	9.0 ± 0.4**	278 ± 6.7**
	30	95 ± 13.1	0.43 ± 0.03**	10.4 ± 0.6**	283 ± 11.2**

Mean ± S.E. (n = 5–6). * p < 0.05, ** p < 0.01 vs. Control, Dunnett’s method.

Fig. 3. Effects of 26v on Plasma Glucose Elevations in a Glucose Tolerance Test in Male Zucker Fatty Rats

Values are the mean ± standard error (S.E.) (n = 6). * p < 0.05, ** p < 0.01 vs. control, Dunnett’s method.

Table 5. Adverse Effects of Oral Administration for 4 Weeks on Body Weight Gain and Hematocrit (HCT) in Male Zucker Fatty Rats

Compound	Dose (mg/kg)	Body weight gain (g)	HCT (%)
Control		130.6 ± 5.3	45.6 ± 0.8
26v	10	165.8 ± 4.3**	43.0 ± 0.9
	30	167.7 ± 6.2**	44.2 ± 0.7
Pioglitazone	10	169.7 ± 6.1**	42.4 ± 0.8*
	30	190.6 ± 5.6**, †	41.4 ± 1.4**

Mean ± S.E. (n = 5–6). * p < 0.05, ** p < 0.01 vs. Control, Dunnett’s method. ^†p < 0.05 vs. 26v (30 mg/kg), the Student’s t-test.

In conclusion, we synthesized 7-substituted 6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline derivatives and found that compounds with a cycloaliphatic amino group on the oxazole at the 7-position of tetrahydroisoquinoline exhibited potent PPARγ partial agonist activity. Among the derivatives examined, compound 26v with a 2-(2,5-dihydropyrrol-1-yl)-5-methyloxazol-4-ylmethoxy group at the 7-position exhibited the most potent PPARγ partial agonist activity and exerted good anti-diabetic and insulin resistance-mitigating effects in male Zucker fatty rats. Compound 26v may be a novel candidate anti-diabetic drug with minor adverse effects.

Experimental

General

Melting points were measured on a melting point apparatus (Yamato MP-21; Yamato Scientific Co., Ltd., Tokyo, Japan) and were uncorrected. ¹H-NMR spectra were obtained on a NMR spectrometer at 400 MHz (JNM-AL400; JEOL Ltd., Tokyo, Japan) using tetramethylsilane (TMS) as an internal standard. IR spectra were recorded with a Fourier transform (FT)-IR spectrometer (HORIBA FT-720, HORIBA, Kyoto, Japan). Mass spectra were obtained on an electrospray ionization (ESI)-MS spectrometer (Expression CMS-L, Advion, Ithaca, NY, U.S.A.). Column chromatography was performed on a silica gel (Daisogel No.1001W; Daiso Co., Ltd., Osaka, Japan). Reactions were monitored by TLC (TLC silica gel 60F254, Merck KGaA, Darmstadt, Germany).

4-Hydroxymethyl-5-methyloxazole

NaBH₄ (30.0 g, 0.973 mol) was added to a solution of 2a²³⁾ (30.6 g, 0.197 mmol) in tetrahydrofuran (THF) (600 mL) at room temperature and the reaction mixture was warmed to 60 °C. MeOH (220 mL) was then added to the mixture and stirred at the same temperature for 0.5 h. The mixture was poured into water (1 L) and extracted once with AcOEt and twice with CHCl₃. The combined organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 4-hydroxymethyl-5-methyloxazole (11.4 g, 51% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.34 (3H, s), 3.52–3.92 (1H, br), 4.52 (2H, d, J = 4.6 Hz), 7.75 (1H, s).

4-Bromomethyl-5-methyloxazole (3a)

CBr₄ (36.8 g, 0.111 mol) was added to a solution of 4-hydroxymethyl-5-methyloxazole (11.4 g, 0.101 mol) and PPh₃ (29.1 g, 0.111 mol) in CH₂Cl₂ (400 mL) under ice-cooling, and the mixture was stirred at the same temperature for 0.5 h. The reaction mixture was washed with saturated aqueous NaHCO₃ solution and dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and Et₂O (300 mL) was added to the residue obtained. The precipitate was removed by filtration, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 3a (12.8 g, 72% yield) as an oil. ¹H-NMR (CDCl₃) δ: 2.34 (3H, s), 4.39 (2H, s), 7.75 (1H, s).

Methyl 2,5-Dimethyloxazole-4-carboxylate (7b)

Acetyl chloride (4b) (3.18 mL, 44.8 mmol) and Et₃N (15.2 mL, 89.6 mmol) were added to a suspension of methyl 2-amino-3-oxobutanoate hydrochloride (5.00 g, 29.8 mmol) in CH₂Cl₂ (50 mL) under ice-cooling, and the mixture was then stirred at the same temperature for 0.5 h. The reaction mixture was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure to obtain compound 6b as a crude product.

PPh₃ (14.5 g, 57.3 mol) and Et₃N (15.2 mL, 109 mmol) were added to a solution of I₂ (15.0 g, 57.3 mmol) in CH₂Cl₂ (150 mL) under ice-cooling, and the mixture was then stirred at the same temperature for 10 min. Compound 6b in CH₂Cl₂ (20 mL) was added to the reaction mixture and stirred at the same temperature for 0.5 h. The reaction mixture was washed with water and saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 7b (2.00 g, 19% yield, 2 steps) as a solid. ¹H-NMR (CDCl₃) δ: 2.44 (3H, s), 2.59 (3H, s), 3.89 (3H, s).

Methyl 2-Ethyl-5-methyloxazole-4-carboxylate (7c)

Compound 7c was prepared according to the procedure for the synthesis of 7b. Yield 39% for 2 steps. ¹H-NMR (CDCl₃) δ: 1.33 (3H, t, J = 7.8 Hz), 2.59 (3H, s), 2.77 (2H, t, J = 7.8 Hz), 3.89 (3H, s).

Methyl 2-Isobutyl-5-methyloxazole-4-carboxylate (7d)

Compound 7d was prepared according to the procedure for the synthesis of 7b. Yield 60% for 2 steps. ¹H-NMR (CDCl₃) δ: 0.97 (6H, d, J = 6.6 Hz), 2.09–2.21 (1H, m), 2.57–2.63 (5H, m), 3.89 (3H, s).

Methyl 2-tert-Butyl-5-methyloxazole-4-carboxylate (7e)

Compound 7e was prepared according to the procedure for the synthesis of 7b. Yield 63% for 2 steps. ¹H-NMR (CDCl₃) δ: 1.39 (9H, s), 2.60 (3H, s), 3.89 (3H, s).

Methyl 2-Cyclopropylmethyl-5-methyloxazole-4-carboxylate (7f)

(COCl)₂ (4.72 mL, 55.0 mmol) and 2 drops of dimethylformamide (DMF) were added to a solution of cyclopropanecarboxylic acid (5f) (5.50 g, 57.3 mmol) in CH₂Cl₂ (100 mL), and the mixture was then stirred at room temperature for 1 h. Methyl 2-amino-3-oxobutanoate hydrochloride (11.1 g, 66.2 mmol) and Et₃N (23.0 mL, 165 mmol) were added under ice-cooling, and the mixture was stirred at the same temperature for 1 h. The reaction mixture was washed with 10% aqueous citric acid solution, water, and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure to obtain compound 6f as a crude product.

PPh₃ (25.5 g, 63.4 mol) and Et₃N (27.1 mL, 194 mmol) were added to a solution of I₂ (24.7 g, 97.3 mmol) in CH₂Cl₂ (280 mL) under ice-cooling, and the mixture was then stirred at the same temperature for 10 min. Compound 6f in CH₂Cl₂ (40 mL) was added to the reaction mixture and stirred at the same temperature for 0.5 h. The reaction mixture was washed with water and saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 7f (3.58 g, 24% yield, 2 steps) as an oil. ¹H-NMR (CDCl₃) δ: 0.24–0.29 (2H, m), 0.54–0.60 (2H, m), 1.07–1.18 (1H, m), 2.60 (3H, s), 2.65 (2H, d, J = 7.1 Hz), 3.89 (3H, s).

Methyl 2-Cyclobutylmethyl-5-methyloxazole-4-carboxylate (7g)

Compound 7g was prepared according to the procedure for the synthesis of 7f. Yield 26% for 2 steps. ¹H-NMR (CDCl₃) δ: 1.69–1.95 (4H, m), 2.06–2.18 (2H, m), 2.58 (3H, s), 2.68–2.81 (1H, m), 2.83 (2H, d, J = 7.6 Hz), 3.88 (3H, s).

Methyl 2-Cyclopentylmethyl-5-methyloxazole-4-carboxylate (7h)

Compound 7h was prepared according to the procedure for the synthesis of 7f. Yield 66% for 2 steps. ¹H-NMR (CDCl₃) δ: 1.18–1.30 (2H, m), 1.50–1.70 (4H, m), 1.73–1.84 (2H, m), 2.26–2.34 (1H, m), 2.59 (3H, s), 2.73 (2H, d, J = 7.6 Hz), 3.89 (3H, s).

Methyl 2-Cyclopropyl-5-methyloxazole-4-carboxylate (7i)

Compound 7i was prepared according to the procedure for the synthesis of 7f. Yield 60% for 2 steps. ¹H-NMR (CDCl₃) δ: 0.98–1.14 (4H, m), 1.99–2.09 (1H, m), 2.56 (3H, s), 3.88 (3H, s).

Methyl 2-Cyclobutyl-5-methyloxazole-4-carboxylate (7j)

Compound 7j was prepared according to the procedure for the synthesis of 7b. Yield 58% for 2 steps. ¹H-NMR (CDCl₃) δ: 1.90–2.00 (2H, m), 2.29–2.50 (4H, m), 2.60 (3H, s), 3.58–3.63 (1H, m), 3.89 (3H, s).

Methyl 2-Cyclopentyl-5-methyloxazole-4-carboxylate (7k)

Compound 7k was prepared according to the procedure for the synthesis of 7f. Yield 29% for 2 steps. ¹H-NMR (CDCl₃) δ: 1.59–1.73 (2H, m), 1.79–1.96 (4H, m), 2.01–2.11 (2H, m), 2.59 (3H, s), 3.13–3.23 (1H, m), 3.89 (3H, s).

Methyl 2-Cyclohexyl-5-methyloxazole-4-carboxylate (7l)

Compound 7l was prepared according to the procedure for the synthesis of 7f. Yield 58% for 2 steps. ¹H-NMR (CDCl₃) δ: 1.20–1.41 (2H, m), 1.53–1.65 (2H, m), 1.66–1.86 (4H, m), 2.01–2.11 (2H, m), 2.59 (3H, s), 3.13–3.23 (1H, m), 3.89 (3H, s).

Methyl 5-Methyl-2-pyrrol-1-yloxazole-4-carboxylate (7m)

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (20.5 g, 107 mmol) was added to a solution of pyrrole-1-carboxylic acid²⁵⁾ (5m) (11.9 g, 107 mmol) in CH₂Cl₂ (200 mL), and the mixture was stirred at room temperature for 0.5 h. Methyl 2-amino-3-oxobutanoate hydrochloride (10.0 g, 59.7 mmol) and i-Pr₂NEt (27.7 mL, 161 mmol) were then added under ice-cooling, and the mixture was stirred at the same temperature for 1 h. After the addition of a 10% aqueous citric acid solution, the mixture was extracted with CH₂Cl₂. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue obtained was purified by column chromatography to obtain compound 6m.

PPh₃ (27.6 g, 105 mol) and Et₃N (29.4 mL, 211 mmol) were added to a solution of I₂ (26.7 g, 105 mmol) in CH₂Cl₂ (500 mL) under ice-cooling, and the mixture was stirred at the same temperature for 15 min. Compound 6m in CH₂Cl₂ (30 mL) was added to the reaction mixture and stirred at the same temperature for 0.5 h. The reaction mixture was washed with water and saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 7m (5.32 g, 43% yield, 2 steps) as a solid. ¹H-NMR (CDCl₃) δ: 2.65 (3H, s), 3.93 (3H, s), 6.33 (2H, t, J = 2.2 Hz), 7.38 (2H, t, J = 2.2 Hz).

Methyl 5-Methyl-2-pyridin-3-yloxazole-4-carboxylate (7n)

Compound 7n was prepared according to the procedure for the synthesis of 7f. Yield 35% for 2 steps. ¹H-NMR (CDCl₃) δ: 2.74 (3H, s), 3.96 (3H, s), 7.40 (1H, ddd, J = 8.0, 4.9, 0.7 Hz), 8.32–8.37 (1H, m), 8.70 (1H, dd, J = 4.9, 1.5 Hz), 9.26–9.30 (1H, m).

Methyl 5-Methyl-2-pyridin-4-yloxazole-4-carboxylate (7o)

Compound 7o was prepared according to the procedure for the synthesis of 7f. Yield 27% for 2 steps. ¹H-NMR (CDCl₃) δ: 2.75 (3H, s), 3.97 (3H, s), 7.89–7.94 (2H, m), 8.72–8.78 (2H, m).

Methyl (2-Fur-2-yl)-5-methyloxazole-4-carboxylate (7p)

Compound 7p was prepared according to the procedure for the synthesis of 7f. Yield 55% for 2 steps. ¹H-NMR (CDCl₃) δ: 2.70 (3H, s), 3.93 (3H, s), 6.52–6.55 (1H, m), 7.08–7.10 (1H, m), 7.54–7.57 (1H, m).

tert-Butyl 6-Carbamoyl-7-(5-methyloxazol-4-yl)methoxy-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10a)

K₂CO₃ (4.80 g, 34.7 mmol) was added to a solution of 3a (2.93 g, 16.6 mmol) and 9¹⁵⁾ (4.06 g, 13.9 mmol) in DMF (80 mL), and the mixture was stirred at 60 °C for 0.5 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography. The solid residue was rinsed with i-Pr₂O-n-hexane (1 : 1) to give 10a (4.60 g, 85% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.38 (3H, s), 2.77–2.86 (2H, m), 3.58–3.68 (2H, m), 4.59 (2H, s), 5.04 (2H, s), 5.81–5.94 (1H, br), 6.83 (1H, s), 7.79 (1H, s), 7.82–7.91 (1H, br), 7.98 (1H, s).

tert-Butyl 6-Carbamoyl-7-(2,5-dimethyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10b)

LiAlH₄ (245 mg, 6.45 mol) was added portionwise to a solution of 7b (1.00 g, 6.45 mmol) in THF (10 mL) under ice-cooling, and the mixture was stirred at the same temperature for 0.5 h. After the addition of water and AcOEt, the precipitate was removed by filtration, and the filtrate was separated into two layers. The organic layer was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure.

SOCl₂ (0.57 mL, 7.8 mmol) was added to a solution of the residue obtained in CH₂Cl₂ (5 mL), and the mixture was stirred at room temperature for 0.5 h. The reaction mixture was evaporated under reduced pressure to afford compound 8b as a crude product.

K₂CO₃ (570 mg, 4.13 mmol) was added to a solution of crude 8b and compound 9¹⁵⁾ (800 mg, 2.75 mmol) in DMF (10 mL), and the mixture was stirred at 50 °C for 14 h and at 80 °C for 24 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 10b (683 mg, 63% yield, 3 steps) as an oil. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.42 (3H, s), 2.47 (3H, s), 2.77–2.86 (2H, m), 3.58–3.70 (2H, m), 4.59 (2H, s), 4.96 (2H, s), 5.65–5.78 (1H, br), 6.81 (1H, s), 7.82–7.94 (1H, br), 7.98 (1H, s).

tert-Butyl 6-Carbamoyl-7-(2-ethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10c)

LiAlH₄ (650 mg, 17.1 mol) was added portionwise to a solution of 7c (2.87 g, 17.0 mmol) in THF (60 mL) under ice-cooling, and the mixture was stirred at the same temperature for 0.5 h. After the addition of water and AcOEt, the precipitate was removed by filtration, and the filtrate was separated into two layers. The organic layer was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure.

SOCl₂ (3.16 mL, 43.3 mmol) was added to a solution of the residue obtained in CH₂Cl₂ (40 mL), and the mixture was stirred at room temperature for 15 min. The reaction mixture was added to water and extracted with CH₂Cl₂. The organic layer was washed with saturated brine and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure to afford compound 8c as a crude product.

Cs₂CO₃ (1.67 g, 5.13 mmol) was added to a solution of crude 8c and 9¹⁵⁾ (330 mg, 2.07 mmol) in DMF (10 mL), and the mixture was stirred at 80 °C for 1 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 10c (633 mg, 45% yield, 3 steps) as a solid. ¹H-NMR (CDCl₃) δ: 1.32 (3H, t, J = 7.6 Hz), 1.50 (9H, s), 2.31 (3H, s), 2.74 (2H, t, J = 7.6 Hz), 2.77–2.85 (2H, m), 3.58–3.68 (2H, m), 4.59 (2H, s), 4.97 (2H, s,), 5.63–5.74 (1H, br), 6.82 (1H, s), 7.89–7.98 (1H, br), 7.97 (1H, s).

tert-Butyl 6-Carbamoyl-7-(2-isobutyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10d)

Compound 10d was prepared according to the procedure for the synthesis of 10b. Yield 49% for 3 steps. ¹H-NMR (CDCl₃) δ: 0.97 (6H, d, J = 6.6 Hz), 1.50 (9H, s), 2.06–2.18 (1H, m), 2.31 (3H, s), 2.59 (2H, d, J = 7.1 Hz), 2.79–2.85 (2H, m), 3.59–3.68 (2H, m), 4.59 (2H, s), 4.97 (2H, s), 5.57–5.66 (1H, br), 6.81 (1H, s), 7.89–7.96 (1H, br), 7.97 (1H, s).

tert-Butyl 7-(2-tert-Butyl-5-methyloxazol-4-ylmethoxy)-6-carbamoyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10e)

Compound 10e was prepared according to the procedure for the synthesis of 10b. Yield 59% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.36 (9H, s), 1.50 (9H, s), 2.31 (3H, s), 2.79–2.84 (2H, m), 3.60–3.68 (2H, m), 4.59 (2H, s), 4.97 (2H, s), 5.60–5.68 (1H, br), 6.82 (1H, s), 7.97 (1H, s), 8.06–8.11 (1H, br).

tert-Butyl 6-Carbamoyl-7-(2-cyclopropylmethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10f)

Compound 10f was prepared according to the procedure for the synthesis of 10c. Yield 70% for 3 steps. ¹H-NMR (CDCl₃) δ: 0.21–0.29 (2H, m), 0.55–0.62 (2H, m), 1.06–1.16 (1H, m), 1.50 (9H, s), 2.33 (3H, s), 2.63 (2H, d, J = 7.1 Hz), 2.76–2.86 (2H, m), 3.57–3.71 (2H, m), 4.59 (2H, s), 4.98 (2H, s), 5.62–5.75 (1H, br), 6.81 (1H, s), 7.91–8.03 (2H, m).

tert-Butyl 6-Carbamoyl-7-(2-cyclobutylmethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10g)

Compound 10g was prepared according to the procedure for the synthesis of 10c. Yield 78% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 1.71–1.81 (2H, m), 1.82–1.97 (2H, m), 2.06–2.18 (2H, m), 2.30 (3H, s), 2.65–2.89 (5H, m), 3.59–3.69 (2H, m), 4.59 (2H, s), 4.96 (2H, s), 5.61–5.74 (1H, br), 6.80 (1H, s), 7.88–8.02 (2H, m).

tert-Butyl 6-Carbamoyl-7-(2-cyclopentylmethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10h)

Compound 10h was prepared according to the procedure for the synthesis of 10b. Yield 43% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.18–1.30 (2H, m), 1.46–1.70 (13H, m), 1.76–1.86 (2H, m), 2.25–2.35 (4H, m), 2.70 (2H, d, J = 7.3 Hz), 2.79–2.86 (2H, br), 3.59–3.68 (2H, br), 4.59 (2H, s), 4.97 (2H, s), 5.62–5.69 (1H, br), 6.81 (1H, s), 7.82–8.01 (2H, m).

tert-Butyl 6-Carbamoyl-7-(2-cyclopropyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10i)

Compound 10i was prepared according to the procedure for the synthesis of 10b. Yield 37% for 3 steps. ¹H-NMR (CDCl₃) δ: 0.98–1.07 (4H, m), 1.49 (9H, s), 1.94–2.07 (1H, m), 2.28 (3H, s), 2.76–2.86 (2H, m), 3.57–3.70 (2H, m), 4.58 (2H, s), 4.93 (2H, s), 5.67–5.71 (1H, br), 6.79 (1H, s), 7.92 (1H, s), 7.95–8.00 (1H, br).

tert-Butyl 6-Carbamoyl-7-(2-cyclobutyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10j)

Compound 10j was prepared according to the procedure for the synthesis of 10b. Yield 31% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 1.91–2.13 (2H, m), 2.32 (3H, s), 2.34–2.44 (4H, m), 2.78–2.86 (2H, m), 3.52–3.70 (3H, m), 4.59 (2H, s), 4.97 (2H, s), 5.63–5.72 (1H, br), 6.82 (1H, s), 7.92–7.82 (2H, m).

tert-Butyl 6-Carbamoyl-7-(2-cyclopentyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10k)

Compound 10k was prepared according to the procedure for the synthesis of 10b. Yield 24% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 1.61–1.92 (6H, m), 1.99–2.10 (2H, m), 2.31 (3H, s), 2.78–2.85 (2H, m), 3.10–3.20 (1H, m), 3.60–3.67 (2H, m), 4.59 (2H, s), 4.97 (2H, s), 5.64–5.70 (1H, br), 6.81 (1H, s), 7.96–8.02 (2H, m).

tert-Butyl 6-Carbamoyl-7-(2-cyclohexyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10l)

Compound 10l was prepared according to the procedure for the synthesis of 10b. Yield 69% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.30–1.85 (10H, m), 1.49 (9H, s), 2.30 (3H, s), 2.68–2.78 (1H, m), 2.78–2.87 (2H, m), 3.58–3.70 (2H, m), 4.58 (2H, s), 4.97 (2H, s), 5.61–5.70 (1H, br), 6.81 (1H, s), 7.93–8.08 (2H, m).

tert-Butyl 6-Carbamoyl-7-[5-methyl-2-(1H-pyrrol-1-yl)oxazol-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10m)

Compound 10m was prepared according to the procedure for the synthesis of 10c. Yield 24% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.38 (3H, s), 2.82 (2H, t, J = 5.6 Hz), 3.64 (2H, t, J = 5.6 Hz), 4.60 (2H, s), 4.99 (2H, s), 5.63–5.75 (1H, br), 6.33 (2H, t, J = 2.2 Hz), 6.82 (1H, s), 7.31 (2H, t, J = 2.2 Hz), 7.84–7.94 (1H, br), 7.99 (1H, s).

tert-Butyl 6-Carbamoyl-7-(5-methyl-2-pyridin-3-yloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10n)

Compound 10n was prepared according to the procedure for the synthesis of 10c. Yield 54% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.47 (3H, s), 2.82 (2H, t, J = 5.6 Hz), 3.59–3.70 (2H, br), 4.61 (2H, s), 5.09 (2H, s), 5.65–5.75 (1H, br), 6.86 (1H, s), 7.41 (1H, ddd, J = 8.0, 4.9, 0.7 Hz), 7.85–7.96 (1H, br), 8.00 (1H, s), 8.24–8.29 (1H, m), 8.69 (1H, dd, J = 4.9, 1.5 Hz), 9.20–9.25 (1H, m).

tert-Butyl 6-Carbamoyl-7-(5-methyl-2-pyridin-4-yloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10o)

Compound 10o was prepared according to the procedure for the synthesis of 10c. Yield 54% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.48 (3H, s), 2.77–2.86 (2H, m), 3.59–3.68 (2H, m), 4.61 (2H, s), 5.10 (2H, s), 5.62–5.73 (1H, br), 6.86 (1H, s), 7.81–7.89 (3H, m), 8.00 (1H, s), 8.71–8.77 (2H, m).

tert-Butyl 6-Carbamoyl-7-(2-fur-2-yl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (10p)

Compound 10p was prepared according to the procedure for the synthesis of 10b. Yield 12% for 3 steps. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.42 (3H, s), 2.81 (2H, t, J = 5.1 Hz), 3.64 (2H, t, J = 5.1 Hz), 4.60 (2H, s), 5.06 (2H, s), 5.66–5.74 (1H, br), 6.54 (1H, dd, J = 3.4, 2.0 Hz), 6.82 (1H, s), 6.97–7.00 (1H, dd, J = 3.4, 0.7 Hz), 7.54–7.57 (1H, m), 7.82–7.91 (1H, br), 7.98 (1H, s).

tert-Butyl 6-Cyano-7-(5-methyloxazol-4-yl)methoxy-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11a)

Et₃N (8.27 mL, 59.3 mmol) and POCl₃ (3.32 mL, 35.6 mmol) were added to a solution of 10a (4.60 g, 11.9 mmol) in CH₂Cl₂ (90 mL), and the mixture was stirred at room temperature for 2 h. The reaction mixture was poured into water, neutralized with K₂CO₃, and separated into two layers. The organic layer was dried over Na₂SO₄, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 11a (3.16 g, 72% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.43 (3H, s), 2.79 (2H, t, J = 5.6 Hz), 3.62 (2H, t, J = 5.6 Hz), 4.58 (2H, s), 5.08 (2H, s), 6.91 (1H, s), 7.32 (1H, s), 7.74 (1H, s).

tert-Butyl 6-Cyano-7-(2,5-dimethyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11b)

Compound 11b was prepared according to the procedure for the synthesis of 11a. Yield 82%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.35 (3H, s), 2.40 (3H, s), 2.72–2.81 (2H, m), 3.58–3.67 (2H, m), 4.58 (2H, s), 4.99 (2H, s), 6.88 (1H, s), 7.26 (1H, s).

tert-Butyl 6-Cyano-7-(2-ethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11c)

Compound 11c was prepared according to the procedure for the synthesis of 11a. Yield 95%. ¹H-NMR (CDCl₃) δ: 1.32 (3H, t, J = 7.6 Hz), 1.49 (9H, s), 2.36 (3H, s), 2.72 (2H, t, J = 7.6 Hz), 2.76 (2H, t, J = 5.6 Hz), 3.62 (2H, t, J = 5.6 Hz), 4.58 (2H, s), 5.00 (2H, s), 6.91 (1H, s), 7.31 (1H, s).

tert-Butyl 6-Cyano-7-(2-isobutyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11d)

Compound 11d was prepared according to the procedure for the synthesis of 11a. Yield 83%. ¹H-NMR (CDCl₃) δ: 0.97 (6H, d, J = 6.6 Hz), 1.49 (9H, s), 2.07–2.17 (1H, m), 2.36 (3H, s), 2.57 (2H, d, J = 7.1 Hz), 2.72–2.79 (2H, m), 3.58–3.67 (2H, m), 4.56 (2H, s), 5.01 (2H, s), 6.90 (1H, s), 7.31 (1H, s).

tert-Butyl 7-(2-tert-Butyl-5-methyloxazol-4-ylmethoxy)-6-cyano-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11e)

Compound 11e was prepared according to the procedure for the synthesis of 11a. Yield 73%. ¹H-NMR (CDCl₃) δ: 1.36 (9H, s), 1.49 (9H, s), 2.36 (3H, s), 2.70–2.79 (2H, m), 3.58–3.68 (2H, m), 4.57 (2H, s), 5.01 (2H, s), 6.88–7.00 (1H, br), 7.32 (1H, s).

tert-Butyl 6-Cyano-7-(2-cyclopropylmethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11f)

Compound 11f was prepared according to the procedure for the synthesis of 11a. Yield 81%. ¹H-NMR (CDCl₃) δ: 0.22–0.28 (2H, m), 0.54–0.62 (2H, m), 1.06–1.17 (1H, m), 1.49 (9H, s), 2.37 (3H, s), 2.62 (2H, d, J = 7.1 Hz), 2.76 (2H, t, J = 5.6 Hz), 3.62 (2H, t, J = 5.6 Hz), 4.58 (2H, s), 5.02 (2H, s), 6.90 (1H, s), 7.32 (1H, s).

tert-Butyl 6-Cyano-7-(2-cyclobutylmethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11g)

Compound 11g was prepared according to the procedure for the synthesis of 11a. Yield 73%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 1.69–1.95 (4H, m), 2.08–2.17 (2H, m), 2.35 (3H, s), 2.68–2.81 (5H, m), 3.62 (2H, t, J = 5.9 Hz), 4.57 (2H, s), 5.00 (2H, s), 6.90 (1H, s), 7.31 (1H, s).

tert-Butyl 6-Cyano-7-(2-cyclopentylmethyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11h)

Compound 11h was prepared according to the procedure for the synthesis of 11a. Yield 64%. ¹H-NMR (CDCl₃) δ: 1.18–1.30 (2H, m), 1.46–1.69 (13H, m), 1.76–1.86 (2H, m), 2.28 (1H, septet, J = 7.8 Hz), 2.36 (3H, s), 2.69 (2H, d, J = 7.3 Hz), 2.72–2.80 (2H, m), 3.57–3.66 (2H, m), 4.57 (2H, s), 5.01 (2H, s), 6.90 (1H, s), 7.31 (1H, s).

tert-Butyl 6-Cyano-7-(2-cyclopropyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11i)

Compound 11i was prepared according to the procedure for the synthesis of 11a. Yield 94%. ¹H-NMR (CDCl₃) δ: 0.96–1.07 (4H, m), 1.49 (9H, s), 1.95–2.03 (1H, m), 2.32 (3H, s), 2.75 (2H, t, J = 5.6 Hz), 3.62 (2H, t, J = 5.6 Hz), 4.57 (2H, s), 4.96 (2H, s), 6.89 (1H, s), 7.31 (1H, s).

tert-Butyl 6-Cyano-7-(2-cyclobuyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11j)

Compound 11j was prepared according to the procedure for the synthesis of 11a. Yield 85%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 1.88–2.12 (2H, m), 2.29–2.45 (7H, m), 2.68–2.78 (2H, m), 3.49–3.65 (3H, m), 4.58 (2H, s), 5.00 (2H, s), 6.91 (1H, s), 7.32 (1H, s).

tert-Butyl 6-Cyano-7-(2-cyclopentyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11k)

Compound 11k was prepared according to the procedure for the synthesis of 11a. Yield 77%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 1.62–1.71 (2H, m), 1.73–1.93 (4H, m), 2.00–2.10 (2H, m), 2.35 (3H, s), 2.73–2.78 (2H, m), 3.14 (1H, quintet, J = 7.9 Hz), 3.59–3.66 (2H, m), 4.57 (2H, s), 5.00 (2H, s), 6.91 (1H, s), 7.31 (1H, s).

tert-Butyl 6-Cyano-7-(2-cyclohexyl-5-methyloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11l)

Compound 11l was prepared according to the procedure for the synthesis of 11a. Yield 77%. ¹H-NMR (CDCl₃) δ: 1.20–2.10 (10H, m), 1.49 (9H, s), 2.35 (3H, s), 2.67–2.74 (1H, m), 2.74–2.80 (2H, m), 3.58–3.69 (2H, m), 4.56 (2H, s), 5.00 (2H, s), 6.90 (1H, s), 7.31 (1H, s).

tert-Butyl 6-Cyano-7-[5-methyl-2-(1H-pyrrol-1-yl)oxazol-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11m)

Compound 11m was prepared according to the procedure for the synthesis of 11a. Yield 89%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.43 (3H, s), 2.76 (2H, t, J = 5.9 Hz), 3.63 (2H, t, J = 5.9 Hz), 4.59 (2H, s), 5.04 (2H, s), 6.31 (2H, t, J = 2.2 Hz), 6.93 (1H, s), 7.31 (2H, t, J = 2.2 Hz), 7.33 (1H, s).

tert-Butyl 6-Cyano-7-(5-methyl-2-pyridin-3-yloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11n)

Compound 11n was prepared according to the procedure for the synthesis of 11a. Yield 77%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.52 (3H, s), 2.77 (2H, t, J = 5.6 Hz), 3.63 (2H, t, J = 5.6 Hz), 4.60 (2H, s), 5.13 (2H, s), 6.95 (1H, s), 7.34 (1H, s), 7.39 (1H, dd, J = 8.1, 4.9 Hz), 8.23–8.30 (1H, m), 8.67 (1H, dd, J = 4.9, 1.4 Hz), 9.19–9.25 (1H, m).

tert-Butyl 6-Cyano-7-(5-methyl-2-pyridin-4-yloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11o)

Compound 11o was prepared according to the procedure for the synthesis of 11a. Yield 59%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.53 (3H, s), 2.77 (2H, t, J = 5.6 Hz), 3.63 (2H, t, J = 5.6 Hz), 4.60 (2H, s), 5.13 (2H, s), 6.94 (1H, s), 7.34 (1H, s), 7.81–7.86 (2H, m), 8.69–8.75 (2H, m).

tert-Butyl 6-Cyano-7-(2-fur-2-yl-5-methyl-oxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (11p)

Compound 11p was prepared according to the procedure for the synthesis of 11a. Yield 85%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.48 (3H, s), 2.76 (2H, t, J = 5.6 Hz), 3.62 (2H, t, J = 5.6 Hz), 4.58 (2H, s), 5.12 (2H, s), 6.51–6.55 (1H, m), 6.91 (1H, s), 6.97 (1H, d, J = 3.6 Hz), 7.33 (1H, s), 7.52–7.55 (1H, m).

tert-Butyl 7-(5-Methyloxazol-4-yl)methoxy-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12a)

n-Bu₃SnN₃ (3.25 mL, 11.9 mmol) was added to a solution of 11a (1.15 g, 3.11 mmol) in toluene (12 mL), and the mixture was refluxed for 18 h. After cooling to room temperature, the reaction mixture was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 12a (1.15 g, 90% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.42 (3H, s), 2.83–2.91 (2H, m), 3.67 (2H, t, J = 5.8 Hz), 4.64 (2H, s), 5.14 (2H, s), 6.88 (1H, s), 8.03–8.06 (1H, m), 8.18–8.21 (1H, m).

tert-Butyl 7-(2,5-Dimethyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12b)

Compound 12b was prepared according to the procedure for the synthesis of 12a. Yield 76%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.35 (3H, s), 2.59 (3H, s), 2.84–2.92 (2H, m), 3.60–3.72 (2H, m), 4.63 (2H, s), 5.08 (2H, s), 6.88 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-Ethyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12c)

Compound 12c was prepared according to the procedure for the synthesis of 12a. Yield 67%. ¹H-NMR (CDCl₃) δ: 1.50 (3H, t, J = 7.6 Hz), 1.51 (9H, s), 2.35 (3H, s), 2.84–2.92 (2H, m), 2.89 (2H, q, J = 7.6 Hz), 3.67 (2H, t, J = 5.6 Hz), 4.63 (2H, s), 5.08 (2H, s), 6.88 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-Isobutyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12d)

Compound 12d was prepared according to the procedure for the synthesis of 12a. Yield 78%. ¹H-NMR (CDCl₃) δ: 1.03 (6H, d, J = 6.6 Hz), 1.51 (9H, s), 2.26–2.37 (4H, m), 2.76 (2H, d, J = 7.1 Hz), 2.85–2.91 (2H, m), 3.64–3.70 (2H, m), 4.63 (2H, s), 5.09 (2H, s), 6.88 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-tert-Butyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12e)

Compound 12e was prepared according to the procedure for the synthesis of 12a. Yield 89%. ¹H-NMR (CDCl₃) δ: 1.50–1.53 (18H, m), 2.36 (3H, s), 2.85–2.93 (2H, m), 3.63–3.72 (2H, m), 4.64 (2H, s), 5.08 (2H, s), 6.89 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-Cyclopropylmethyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12f)

Compound 12f was prepared according to the procedure for the synthesis of 12a. Yield 49%. ¹H-NMR (CDCl₃) δ: 0.28–0.35 (2H, m), 0.63–0.70 (2H, m), 0.89–0.98 (1H, m), 1.49 (9H, s), 2.36 (3H, s), 2.77 (2H, d, J = 7.1 Hz), 2.88 (2H, t, J = 5.6 Hz), 3.67 (2H, t, J = 5.6 Hz), 4.64 (2H, s), 5.10 (2H, s), 6.89 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-Cyclobutylmethyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12g)

Compound 12g was prepared according to the procedure for the synthesis of 12a. Yield 62%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 1.75–2.02 (4H, m), 2.13–2.24 (2H, m), 2.34 (3H, s), 2.84–2.91 (2H, m), 2.92–2.99 (3H, m), 3.62–3.72 (2H, m), 4.63 (2H, s), 5.08 (2H, s), 6.88 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-Cyclopentylmethyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12h)

Compound 12h was prepared according to the procedure for the synthesis of 12a. Yield 43%. ¹H-NMR (CDCl₃) δ: 1.25–1.35 (2H, m), 1.51 (9H, s), 1.57–1.73 (4H, m), 1.80–1.92 (2H, m), 2.35 (3H, s), 2.49 (1H, septet, J = 8.0 Hz), 2.85–2.92 (4H, m), 3.63–3.73 (2H, m), 4.63 (2H, s), 5.09 (2H, s), 6.88 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-Cyclopropyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12i)

Compound 12i was prepared according to the procedure for the synthesis of 12a. Yield 89%. ¹H-NMR (CDCl₃) δ: 0.87–0.97 (4H, m), 1.50 (9H, s), 2.09–2.21 (1H, m), 2.32 (3H, s), 2.80–2.94 (2H, m), 3.60–3.73 (2H, m), 4.62 (2H, s), 5.05 (2H, s), 6.87 (1H, s), 8.17 (1H, s).

tert-Butyl 7-(2-Cyclobutyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12j)

Compound 12j was prepared according to the procedure for the synthesis of 12a. Yield 74%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.03–2.21 (2H, m), 2.35 (3H, s), 2.41–2.51 (2H, m), 2.52–2.63 (2H, m), 2.85–2.91 (2H, m), 3.65–3.77 (3H, m), 4.64 (2H, s), 5.09 (2H, s), 6.89 (1H, s), 8.19 (1H, s).

tert-Butyl 7-(2-Cyclopentyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12k)

Compound 12k was prepared according to the procedure for the synthesis of 12a. Yield 42%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 1.64–1.80 (2H, m), 1.83–1.94 (2H, m), 1.96–2.08 (2H, m), 2.14–2.24 (2H, m), 2.35 (3H, s), 2.85–2.91 (2H, m), 3.18–3.31 (1H, m), 3.64–3.70 (2H, m), 4.63 (2H, s), 5.08 (2H, s), 6.88 (1H, s), 8.18 (1H, s).

tert-Butyl 7-(2-Cyclohexyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12l)

Compound 12l was prepared according to the procedure for the synthesis of 12a. Yield 94%. ¹H-NMR (CDCl₃) δ: 1.32–2.24 (10H, m), 1.50 (9H, s), 2.34 (3H, s), 2.80–2.94 (3H, m), 3.60–3.74 (2H, m), 4.63 (2H, s), 5.07 (2H, s), 6.88 (1H, s), 8.17 (1H, s).

tert-Butyl 7-[5-Methyl-2-(1H-pyrrol-1-yl)oxazol-4-ylmethoxy]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12m)

Compound 12m was prepared according to the procedure for the synthesis of 12a. Yield 61%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.41 (3H, s), 2.88 (2H, t, J = 5.9 Hz), 3.68 (2H, t, J = 5.9 Hz), 4.63 (2H, s), 5.09 (2H, s), 6.41 (2H, t, J = 2.2 Hz), 6.88 (1H, s), 7.58 (2H, t, J = 2.2 Hz), 8.20 (1H, s).

tert-Butyl 7-(5-Methyl-2-pyridin-3-yloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12n)

Compound 12n was prepared according to the procedure for the synthesis of 12a. Yield 75%. ¹H-NMR (CDCl₃) δ: 1.52 (9H, s), 2.50 (3H, s), 2.88 (2H, t, J = 5.8 Hz), 3.68 (2H, t, J = 5.8 Hz), 4.63 (2H, s), 5.17 (2H, s), 6.89 (1H, s), 7.53 (1H, ddd, J = 8.0, 4.9, 0.7 Hz), 8.21 (1H, s), 8.67–8.72 (1H, m), 8.72–8.77 (1H, m), 9.28–9.34 (1H, m).

tert-Butyl 7-(5-Methyl-2-pyridin-4-yloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (12o)

Compound 12o was prepared according to the procedure for the synthesis of 12a. Yield 83%. ¹H-NMR (CDCl₃) δ: 1.52 (9H, s), 2.50 (3H, s), 2.85 (2H, t, J = 5.9 Hz), 3.67 (2H, t, J = 5.9 Hz), 4.57–4.68 (2H, br), 5.13 (2H, s), 6.78–6.92 (1H, br), 8.03–8.10 (2H, m), 8.18 (1H, s), 8.78–8.84 (2H, m).

2-[(E)-3-(2-Furyl)acryloyl]-7-(5-methyloxazol-4-yl)methoxy-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13a)

To a solution of 12a (1.15 g, 2.79 mmol) in HCO₂H (1 mL) was added 8.6 M HCl in i-PrOH (1.00 mL, 8.60 mmol), and the mixture was stirred at room temperature for 15 min. After the addition of Et₂O, the precipitate that formed was collected by filtration to give a deprotected amine.

(COCl)₂ (0.11 mL, 1.3 mmol) and 3 drops of DMF were added to a solution of (E)-3-(2-furyl)acrylic acid (175 mg, 1.27 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at room temperature for 15 min. The above amine was then added at room temperature followed by i-Pr₂NEt (0.60 mL, 3.5 mmol) portionwise under ice-cooling, and the mixture was stirred at the same temperature for 0.5 h. The reaction mixture was washed with 10% aqueous citric acid solution, water, and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue was rinsed with AcOEt to give 13a (306 mg, 51% yield for 2 steps) as a solid, mp 91–94 °C. ¹H-NMR (CDCl₃) δ: 2.43 (3H, s), 2.91–3.08 (2H, m), 3.87–4.01 (2H, m), 4.91 (2H, s), 5.16 (2H, s), 6.48 (1H, dd, J = 3.4, 1.7 Hz), 6.58 (1H, d, J = 3.4 Hz), 6.88 (1H, d, J = 14.9 Hz), 6.96 (1H, s), 7.46 (1H, d, J = 1.7 Hz), 7.52 (1H, d, J = 14.9 Hz), 8.05 (1H, m), 8.23 (1H, s). IR (attenuated total reflectance (ATR)) cm⁻¹: 1649, 1601. MS m/z: 431 [M − H]⁻, 455 [M + Na]⁺.

7-(2,5-Dimethyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13b)

Compound 13b was prepared according to the procedure for the synthesis of 13a. Yield 20% for 2 steps. A white solid. mp 251–253 °C. ¹H-NMR (CDCl₃) δ: 2.35 (3H. s), 2.59 (3H, s), 2.90–3.10 (2H, m), 3.85–4.05 (2H, m), 4.85–4.98 (2H, m), 5.09 (2H, s), 6.45–6.52 (1H, m), 6.58 (1H, d, J = 3.4 Hz), 6.87 (1H, d, J = 15.1 Hz), 6.94 (1H, s), 7.45–7.50 (1H, br), 7.52 (1H, d, J = 15.1 Hz), 8.20 (1H, s). IR (ATR) cm⁻¹: 1653, 1614. MS m/z: 445 [M − H]⁻, 447 [M + H]⁺.

7-(2-Ethyl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13c)

Compound 13c was prepared according to the procedure for the synthesis of 13a. Yield 68% for 2 steps. A pale yellow solid. mp 249–251 °C. ¹H-NMR (CDCl₃) δ: 1.50 (3H, t, J = 7.6 Hz), 2.36 (3H, s), 2.90 (2H, q, J = 7.6 Hz), 2.94–3.06 (2H, m), 3.86–4.03 (2H, m), 4.90 (2H, s), 5.10 (2H, s), 6.44–6.50 (1H, m), 6.58 (1H, d, J = 3.4 Hz), 6.87 (1H, d, J = 15.1 Hz), 6.95 (1H, s), 7.44–7.48 (1H, m), 7.51 (1H, d, J = 15.1 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹: 1645. MS m/z: 459 [M − H]⁻, 461 [M + H]⁺.

2-[(E)-3-(2-Furyl)acryloyl]-7-(2-isobutyl-5-methyloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13d)

Compound 13d was prepared according to the procedure for the synthesis of 13a. Yield 33% for 2 steps. A pale brown solid. mp 148–152 °C. ¹H-NMR (CDCl₃) δ: 1.03 (6H, d, J = 6.6 Hz), 2.26–2.37 (4H, m), 2.76 (2H, d, J = 7.1 Hz), 2.92–3.05 (2H, m), 3.86–4.00 (2H, m), 4.90 (2H, s), 5.10 (2H, s), 6.45–6.50 (1H, m), 6.57 (1H, d, J = 3.2 Hz), 6.87 (1H, d, J = 14.9 Hz), 6.95 (1H, s), 7.45–7.49 (1H, m), 7.52 (1H, d, J = 14.9 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹;1648, 1608. MS m/z: 487 [M − H]⁻, 489 [M + H]⁺, 511 [M + Na]⁺.

7-(2-tert-Butyl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13e)

To a solution of 13e (560 mg, 1.20 mmol) in HCO₂H (1.5 mL) was added 8.6 M HCl in i-PrOH (0.42 mL, 3.6 mmol) under ice-cooling, and the mixture was stirred at room temperature for 15 min. Et₂O was added to the reaction mixture and the precipitate was filtered to give deprotected amine.

(E)-3-(2-Furyl)acrylic acid (160 mg, 2.39 mmol) and EDC·HCl (460 mg, 2.40 mmol) were added to a solution of the solid obtained and Et₃N (0.80 mL, 5.7 mmol) in CH₂Cl₂ (10 mL), and the mixture was stirred at room temperature for 1 h. After the addition of a 10% aqueous citric acid solution, the reaction mixture was extracted with CH₂Cl₂, washed with saturated aqueous NaHCO₃ solution and brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue obtained was purified by column chromatography to give 13e (287 mg, 49% yield, 2 steps) as a white solid. mp 214–216 °C. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.37 (3H, s), 2.90–3.10 (2H, m), 3.85–4.03 (2H, m), 4.86–4.96 (2H, m), 5.09 (2H, s), 6.45–6.52 (1H, m), 6.58 (1H, d, J = 3.4 Hz), 6.88 (1H, d, J = 15.1 Hz), 6.96 (1H, s), 7.45–7.49 (1H, m), 7.52 (1H, d, J = 15.1 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹: 1647, 1603. MS m/z: 487 [M − H]⁻, 489 [M + H]⁺.

7-(2-Cyclopropylmethyl-5-methyloxazol-4-yl)methoxy-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13f)

Compound 13f was prepared according to the procedure for the synthesis of 13e. Yield 20% for 2 steps. A pale brown solid. mp 168–171 °C. ¹H-NMR (CDCl₃) δ: 0.29–0.36 (2H, m), 0.63–0.70 (2H, m), 1.29–1.40 (1H, m), 2.37 (3H, s), 2.77 (2H, d, J = 7.1 Hz), 2.92–3.08 (2H, m), 3.85–4.01 (2H, m), 4.90 (2H, s), 5.11 (2H, s), 6.45–6.50 (1H, m), 6.58 (1H, d, J = 3.2 Hz), 6.88 (1H, d, J = 14.9 Hz), 6.96 (1H, s), 7.47 (1H, s), 7.52 (1H, d, J = 14.9 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹: 1649, 1608. MS m/z: 485 [M − H]⁻, 487 [M + H]⁺, 509 [M + Na]⁺.

7-(2-Cyclobutylmethyl-5-methyloxazol-4-yl)methoxy-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13g)

Compound 13g was prepared according to the procedure for the synthesis of 13e. Yield 28% for 2 steps. A white solid. mp 163–166 °C. ¹H-NMR (CDCl₃) δ: 1.74–2.02 (4H, m), 2.13–2.24 (2H, m), 2.35 (3H, s), 2.89–3.05 (5H, m), 3.59–3.68 (2H, m), 4.90 (2H, s), 5.09 (2H, s), 6.44–6.51 (1H, m), 6.58 (1H, d, J = 3.4 Hz), 6.88 (1H, d, J = 14.9 Hz), 6.95 (1H, s), 7.46–7.50 (1H, m), 7.51 (1H, d, J = 14.9 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹: 1649, 1608. MS m/z: 499 [M − H]⁻, 501 [M + H]⁺, 523 [M + Na]⁺.

1-[7-(2-Cyclopentylmethyl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13h)

Compound 13h was prepared according to the procedure for the synthesis of 13e. Yield 48% for 2 steps. A white solid. mp 156–159 °C. ¹H-NMR (CDCl₃) δ: 1.21–1.38 (2H, m), 1.53–1.78 (4H, m), 1.80–1.97 (2H, m), 2.36 (3H, s), 2.49 (1H, heptet, J = 7.6 Hz), 2.87 (2H, d, J = 7.6 Hz), 2.90–3.07 (2H, m), 3.85–4.05 (2H, m), 4.84–4.97 (2H, m), 5.10 (2H, s), 6.47 (1H, dd, J = 3.4, 1.7 Hz), 6.58 (1H, d, J = 3.4 Hz), 6.88 (1H, d, J = 14.9 Hz), 6.95 (1H, s), 7.45–7.49 (1H, m), 7.51 (1H, d, J = 14.9 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹: 1649, 1608. MS m/z: 513 [M − H]⁻, 537 [M + Na]⁺.

7-(2-Cyclopropyl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13i)

Compound 13i was prepared according to the procedure for the synthesis of 13e. Yield 16% for 2 steps. A pale yellow solid. mp 184–186 °C (decomp.). ¹H-NMR (CDCl₃) δ: 1.11–1.20 (2H, m), 1.23–1.33 (2H, m), 2.12–2.21 (1H, m), 2.33 (3H, s), 2.90–3.07 (2H, m), 3.86–4.00 (2H, m), 4.89 (2H, s), 5.06 (2H, s), 6.44–6.51 (1H, m), 6.57 (1H, d, J = 3.4 Hz), 6.87 (1H, d, J = 15.1 Hz), 6.94 (1H, s), 7.44–7.48 (1H, m), 7.51 (1H, d, J = 15.1 Hz), 8.20 (1H, s). IR (ATR) cm⁻¹: 1648, 1606. MS m/z: 471 [M − H]⁻, 473 [M + H]⁺.

1-[7-(2-Cyclobutyl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13j)

Compound 13j was prepared according to the procedure for the synthesis of 13e. Yield 48% for 2 steps. A white solid. mp 161–164 °C (decomp.). ¹H-NMR (CDCl₃) δ: 2.02–2.22 (2H, m), 2.36 (3H, s), 2.38–2.65 (4H, m), 2.89–3.11 (2H, m), 3.71 (1H, quintet, J = 8.5 Hz), 3.86–4.03 (2H, m), 4.83–4.95 (2H, m), 5.10 (2H, s), 6.45–6.52 (1H, m), 6.58 (1H, d, J = 3.4 Hz), 6.87 (1H, d, J = 15.1 Hz), 6.95 (1H, s), 7.45–7.50 (1H, m), 7.51 (1H, d, J = 15.1 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹: 1649, 1608. MS m/z: 485 [M − H]⁻, 487 [M + Na]⁺.

1-[7-(2-Cyclopentyl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13k)

Compound 13k was prepared according to the procedure for the synthesis of 13e. Yield 40% for 2 steps. A pale yellow solid. mp 170–172 °C (decomp.). ¹H-NMR (CDCl₃) δ: 1.60–1.80 (2H, m), 1.80–1.95 (2H, m), 1.95–2.08 (2H, m), 2.13–2.27 (2H, m), 2.36 (3H, s), 2.90–3.05 (2H, m), 3.29 (1H, quintet, J = 8.3 Hz), 3.86–4.00 (2H, m), 4.85–4.93 (2H, m), 5.09 (2H, s), 6.47 (1H, dd, J = 3.4, 1.9 Hz), 6.58 (1H, d, J = 3.4 Hz), 6.87 (1H, d, J = 15.1 Hz), 6.95 (1H, s), 7.45–7.49 (1H, m), 7.51 (1H, d, J = 15.1 Hz), 8.20 (1H, s). IR (ATR) cm⁻¹: 1649, 1608. MS m/z: 499 [M − H]⁻, 501 [M + H]⁺.

7-(2-Cyclohexyl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13l)

Compound 13l was prepared according to the procedure for the synthesis of 13e. Yield 33% for 2 steps. A white solid. mp 163–167 °C (decomp.). ¹H-NMR (CDCl₃) δ: 1.30–1.50 (2H, m), 1.62–1.80 (4H, m), 1.84–1.95 (2H, m), 2.13–2.25 (2H, m), 2.35 (3H, s), 2.81–2.92 (1H, m), 2.93–3.10 (2H, m), 3.86–4.02 (2H, m), 4.90 (2H, s), 5.09 (2H, s), 6.45–6.53 (1H, m), 6.54–6.64 (1H, m), 6.87 (1H, d, J = 14.9 Hz), 6.95 (1H, s), 7.44–7.48 (1H, m), 7.51 (1H, d, J = 14.9 Hz), 8.20 (1H, s). IR (ATR) cm⁻¹: 1610. MS m/z: 513 [M − H]⁻, 515 [M + H]⁺.

2-[(E)-3-(2-Furyl)acryloyl]-7-(5-methyl-2-pyrrol-1-yloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13m)

Compound 13m was prepared according to the procedure for the synthesis of 13a. Yield 38% for 2 steps. A yellow solid. mp 228–232 °C. ¹H-NMR (CDCl₃) δ: 2.41 (3H, s), 2.84–3.09 (2H, m), 3.86–4.00 (2H, m), 4.78–4.96 (2H, m), 4.98–5.14 (2H, m), 6.36–6.43 (2H, m), 6.45–6.51 (1H, m), 6.58 (1H, d, J = 2.7 Hz), 6.87 (1H, d, J = 14.9 Hz), 6.88–6.99 (1H, m), 7.44–7.50 (1H, m), 7.52 (1H, d, J = 14.9 Hz), 7.53–7.62 (2H, m), 8.21 (1H, s). IR (ATR) cm⁻¹: 1651, 1610. MS m/z: 496 [M − H]⁻.

2-[(E)-3-(2-Furyl)acryloyl]-7-[5-methyl-2-(pyridin-3-yl)oxazol-4-ylmethoxy]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13n)

Compound 13n was prepared according to the procedure for the synthesis of 13a. Yield 31% for 2 steps. A white solid. mp 220–222 °C (decomp.). ¹H-NMR (CDCl₃) δ: 2.50 (3H, s), 2.87–3.07 (2H, m), 3.86–3.98 (2H, m), 4.89 (2H, s), 5.16 (2H, s), 6.45–6.51 (1H, m), 6.59 (1H, d, J = 3.2 Hz), 6.88 (1H, d, J = 15.2 Hz), 6.91–6.99 (1H, m), 7.47 (1H, d, J = 1.5 Hz), 7.52 (1H, dd, J = 8.0, 5.2 Hz), 7.52 (1H, d, J = 15.2 Hz), 8.22 (1H, s), 8.67 (1H, d, J = 8.0 Hz), 8.73 (1H, d, J = 5.2 Hz), 9.30 (1H, s). IR (ATR) cm⁻¹: 1649, 1604. MS m/z: 508 [M − H]⁻.

2-[(E)-3-(2-Furyl)acryloyl]-7-[5-methyl-2-(pyridin-4-yl)oxazol-4-ylmethoxy]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13o)

Compound 13o was prepared according to the procedure for the synthesis of 13a. Yield 54% for 2 steps. A pale brown solid. mp 228–231 °C (decomp.). ¹H-NMR (CDCl₃) δ: 2.51 (3H, s), 2.86–3.08 (2H, m), 3.86–3.98 (2H, m), 4.82–4.96 (2H, m), 5.17 (2H, s), 6.45–6.52 (1H, m), 6.59 (1H, d, J = 2.9 Hz), 6.88 (1H, d, J = 15.1 Hz), 6.92–6.99 (1H, br), 7.47 (1H, d, J = 1.7 Hz), 7.52 (1H, d, J = 15.1 Hz), 8.04–8.13 (2H, m), 8.23 (1H, s), 8.79–8.86 (2H, m). IR (ATR) cm⁻¹: 1649, 1604. MS m/z: 508 [M − H]⁻.

1-{7-[2-(Fur-2-yl)-5-methyloxazol-4-ylmethoxy]}-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (13p)

n-Bu₃SnN₃ (2.12 mL, 7.69 mmol) was added to a solution of 11p (670 mg, 1.54 mmol) in toluene (7 mL), and the mixture was refluxed for 24 h. After cooling to room temperature, the reaction mixture was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 12p as a crude product.

To a solution of crude 12p in HCO₂H (2 mL) was added 8.6 M HCl in i-PrOH (0.45 mL, 3.9 mmol), and the mixture was stirred at room temperature for 15 min. After the addition of Et₂O, the precipitate formed was collected by filtration to give a deprotected amine.

(E)-3-(2-Furyl)acrylic acid (175 mg, 2.60 mmol) and EDC·HCl (500 mg, 2.60 mmol) were added to a solution of the above obtained amine and Et₃N (0.27 mL, 1.94 mmol) in CH₂Cl₂ (10 mL), and the mixture was stirred at room temperature for 1.5 h. The reaction mixture was washed with 10% citric acid solution, water, and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue obtained was purified by column chromatography to give 13p (367 mg, 48% yield for 3 steps) as a white solid. mp 250–252 °C. ¹H-NMR (CDCl₃) δ: 2.47 (3H, s), 2.89–3.08 (2H, m), 3.86–4.02 (2H, m), 4.85–4.95 (2H, m), 5.17 (2H, s), 6.46–6.50 (1H, m), 6.58 (1H, d, J = 3.2 Hz), 6.60–6.65 (1H, m), 6.88 (1H, d, J = 15.1 Hz), 6.95 (1H, s), 7.44–7.49 (2H, m), 7.52 (1H, d, J = 15.1 Hz), 7.60–7.64 (1H, m), 8.22 (1H, s). IR (ATR) cm⁻¹: 1651, 1620. MS m/z: 497 [M − H]⁻, 499 [M + H]⁺.

Methyl 3-Bromo-2-oxobutyrate

To a solution of 2-oxobutyric acid (10 g, 99 mmol) in MeOH (200 mL) was added 8.6 M HCl in i-PrOH (5.0 mL, 43 mmol), and the mixture was refluxed for 21 h. After cooling to room temperature, the reaction mixture was evaporated under reduced pressure. AcOEt was added to the residue, neutralized with saturated aqueous NaHCO₃ solution, and separated into two layers. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure.

CuBr₂ (83.0 g, 0.372 mol) in CHCl₃ (620 mL) was added to a solution of the residue obtained in AcOEt, and the mixture was refluxed for 5 h. After cooling to room temperature, the reaction mixture was filtrated, and the filtrate was then evaporated under reduced pressure. AcOEt was added to the residue and washed with water, saturated aqueous NaHCO₃ solution, and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give methyl 3-bromo-2-oxobutyrate (13.4 g, 70% yield for 2 steps) as an oil. ¹H-NMR (CDCl₃) δ: 1.14 (3H, t, J = 7.3 Hz), 2.88 (2H, q, J = 7.3 Hz), 3.87 (3H, s).

Methyl 2-Amino-5-methyloxazole-4-carboxylate

Urea (6.18 g, 0.103 mol) was added to a solution of methyl 3-bromo-2-oxobutyrate (13.4 g, 68.7 mmol) in EtOH (67 mL), and the mixture was refluxed for 12 h. After cooling to room temperature, the reaction mixture was evaporated under reduced pressure. AcOEt was added to the residue, neutralized with saturated aqueous NaHCO₃ solution, and separated into two layers. The organic layer was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The solid obtained was rinsed with Et₂O to give methyl 2-amino-5-methyloxazole-4-carboxylate (3.70 g, 34% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.50 (3H, s), 3.85 (3H, s), 4.85–5.04 (2H, br).

Methyl 2-Chroro-5-methyloxazole-4-carboxylate (14)

t-Butyl nitrite (3.40 mL, 28.7 mmol) and methyl 2-amino-5-methyloxazole-4-carboxylate (1.25 g, 8.01 mmol) were added to a suspension of CuCl₂ (3.31 g, 24.6 mmol) in MeCN (130 mL) at 75 °C under a N₂ atmosphere, and the reaction mixture was stirred at the same temperature for 0.5 h. After cooling to room temperature, AcOEt was added to the mixture obtained, washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to give 14 (3.06 g, 85% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.63 (3H, s), 3.91 (3H, s).

Methyl 2-(Imidazol-1-yl)-5-methyloxazole-4-carboxylate (16q)

To a solution of imidazole (1.78 g, 26.1 mmol) in THF (60 mL) was added portionwise 60% sodium hydride (NaH) (1.04 g, 26 mmol) under ice-cooling, and the mixture was stirred at the same temperature. A solution of 14 (3.06 g, 17.4 mmol) in THF (30 mL) was added to the reaction mixture, which was stirred at room temperature for 2 h. AcOEt was added to the mixture obtained, washed with saturated brine, and dried over Na₂SO₄. The solvent was evaporated under reduced pressure, n-hexane was added to the residue obtained, and the precipitate was collected by filtration to give 16q (1.97 g, 55% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.69 (3H, s), 3.95 (3H, s), 7.17–7.19 (1H, m), 7.58–7.62 (1H, m), 8.24 (1H, s).

Methyl 5-Methyl-2-pyrrolidin-1-yloxazole-4-carboxylate (16u)

Compound 14 (9.00 g, 47.5 mmol) was added to pyrrolidine (20 mL, 0.24 mol), which was stirred at room temperature for 1 h. AcOEt was added to the reaction mixture, the organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to give 16u (10.6 g, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 1.92–1.99 (4H, m), 2.51 (3H, s), 3.48–3.53 (4H, m), 3.86 (3H, s).

Ethyl 2-(N-Ethylmethylamino)-5-methyloxazole-4-carboxylate (17r)

N-Ethylmethylamine (1.13 mL, 13.2 mmol) was added to a solution of 15²⁴⁾ (500 mg, 2.64 mmol), which was stirred at room temperature for 13.5 h. AcOEt was added to the reaction mixture, washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to give 17r (542 mg, 97% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.17 (3H, t, J = 7.1 Hz), 1.37 (3H, t, J = 7.1 Hz), 2.50 (3H, s), 3.02 (3H, s), 3.45 (2H, q, J = 7.1 Hz), 4.34 (2H, q, J = 7.1 Hz).

Ethyl 2-Diethylamino-5-methyloxazole-4-carboxylate (17s)

Diethylamine (1.64 mL, 15.9 mmol) and i-Pr₂NEt (4.55 mL, 26.4 mmol) were added to a solution of 15²⁴⁾ (500 mg, 2.64 mmol) in DMF (20 mL), which was stirred at room temperature for 1 h and 50 °C for 13 h. AcOEt was added to the reaction mixture, washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to give 17s (800 mg, 67% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.18 (6H, t, J = 7.1 Hz), 1.36 (3H, t, J = 7.1 Hz), 2.50 (3H, s), 3.45 (4H, q, J = 7.1 Hz), 4.34 (2H, q, J = 7.1 Hz).

Ethyl 2-(Azetidin-1-yl)-5-methyloxazole-4-carboxylate (17t)

Compound 17t was prepared according to the procedure for the synthesis of 17s. Yield 62%. ¹H-NMR (CDCl₃) δ: 1.36 (3H, t, J = 7.1 Hz), 2.40 (2H, quintet, J = 7.6 Hz), 2.50 (3H, s), 4.14 (4H, t, J = 7.6 Hz), 4.35 (2H, q, J = 7.1 Hz).

Ethyl 2-(2,5-Dihydropyrrol-1-yl)-5-methyloxazole-4-carboxylate (17v)

K₂CO₃ (8.03 g, 58.1 mmol) and 3-pyrroline (3.00 g, 14.5 mmol) were added to a solution of 15²⁴⁾ (5.51 g, 29.1 mmol) in DMF (30 mL), which was stirred at room temperature for 2 h. AcOEt was added to the reaction mixture, the organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to give 17v (4.25 g, 66% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.37 (3H, t, J = 7.1 Hz), 2.52 (3H, s), 4.36 (2H, q, J = 7.1 Hz), 4.33 (4H, s), 5.86 (2H, s).

Ethyl 5-Methyl-2-piperidin-1-yloxazole-4-carboxylate (17w)

Compound 17w was prepared according to the procedure for the synthesis of 17r. Yield quant. ¹H-NMR (CDCl₃) δ: 1.36 (3H, t, J = 7.1 Hz), 1.57–1.65 (6H, m), 2.49 (3H, s), 3.43–3.49 (4H, m), 4.34 (2H, q, J = 7.1 Hz).

4-Hydroxymethyl-2-(1H-imidazol-1-yl)-5-methyloxazole (18q)

LiAlH₄ (250 mg, 6.59 mmol) was added portionwise to a solution of 16q (1.97 g, 9.51 mmol) in THF (60 mL) under ice-cooling, which was stirred at the same temperature for 1 h and room temperature for 1 h. After the addition of water and AcOEt, the mixture was filtrated. The two layers of filtrate were separated, and the organic layer was dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 18q (447 mg, 26% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.38 (3H, s), 2.75–3.25 (1H, br), 4.55 (2H, s), 7.16 (1H, s), 7.52 (1H, s), 8.31 (1H, s).

2-(N-Ethylmethylamino)-4-hydroxymethyl-5-methyloxazole (18r)

Compound 18r was prepared according to the procedure for the synthesis of 18q. Yield 94%. ¹H-NMR (CDCl₃) δ: 1.16 (3H, t, J = 7.1 Hz), 2.19 (3H, s), 2.98 (3H, s), 3.40 (2H, q, J = 7.1 Hz), 4.39 (2H, s), 4.86–5.07 (1H, br).

2-Diethylamino-4-hydroxymethyl-5-methyloxazole (18s)

Compound 18s was prepared according to the procedure for the synthesis of 18q. Yield 99%. ¹H-NMR (CDCl₃) δ: 1.17 (6H, t, J = 7.1 Hz), 2.19 (3H, s), 3.39 (4H, q, J = 7.1 Hz), 4.24–4.76 (1H, br), 4.39 (2H, s).

2-Azetidin-1-yl-4-hydroxymethyl-5-methyloxazole (18t)

Compound 18t was prepared according to the procedure for the synthesis of 18q. Yield 82%. ¹H-NMR (CDCl₃) δ: 2.19 (3H, s), 2.38 (2H, quintet, J = 7.6 Hz), 3.40–3.80 (1H, br), 4.09 (4H, t, J = 7.6 Hz), 4.37 (2H, s).

4-Hydroxymethyl-5-methyl-2-pyrrolidin-1-yloxazole (18u)

Compound 18u was prepared according to the procedure for the synthesis of 18q. Yield 94%. ¹H-NMR (CDCl₃) δ: 1.91–1.98 (4H, m), 2.20 (3H, s), 3.43–3.49 (4H, m), 3.78–4.32 (1H, br), 4.40 (2H, s).

2-(2,5-Dihydropyrrol-1-yl)-4-hydroxymethyl-5-methyloxazole (18v)

Compound 18v was prepared according to the procedure for the synthesis of 18q. Yield 92%. ¹H-NMR (CDCl₃) δ: 2.22 (3H, s), 4.29 (4H, s), 4.41 (2H, s), 5.85 (2H, s).

4-Hydroxymethyl-5-methyl-2-piperidin-1-yloxazole (18w)

Compound 18w was prepared according to the procedure for the synthesis of 18q. Yield 85%. ¹H-NMR (CDCl₃) δ: 1.57–1.65 (6H, m), 2.19 (3H, s), 3.36–3.45 (4H, m), 3.55–3.77 (1H, br), 4.39 (2H, s).

tert-Butyl 7-Benzyloxy-6-cyano-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (20)

Et₃N (112 mL, 804 mmol) and POCl₃ (55.4 mL, 594 mmol) were added to a suspension of 19¹⁵⁾ (90.0 g, 235 mmol) in CH₂Cl₂ (2 L) under ice-cooling, followed by stirring at room temperature for 1 h. The reaction mixture was poured into water, neutralized with K₂CO₃, and separated into two layers. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 20 (61.0 g, 71% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.72–2.80 (2H, m), 3.57–3.67 (2H, m), 4.54 (2H, s), 5.17 (2H, s), 6.72 (1H, s), 7.30–7.47 (6H, m).

tert-Butyl 7-Benzyloxy-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (21)

n-Bu₃SnN₃ (30.0 g, 90.3 mmol) was added to a solution of 20 (10.0 g, 27.4 mmol) in toluene (100 mL), and the mixture was refluxed for 28 h. After cooling to room temperature, the reaction mixture was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 21 (9.60 g, 86% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.77–2.88 (2H, m), 3.60–3.72 (2H, m), 4.60 (0.8H, s), 4.61 (1.2H, s), 5.06 (1.2H, s), 5.20 (0.8H, s), 6.85 (1H, s), 7.20–7.38 (5H, m), 7.52 (0.8H, s), 7.84 (0.2H, s).

tert-Butyl 7-Benzyloxy-6-[1(2)-methoxymethyl-1(2)H-tetrazol-5-yl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate

K₂CO₃ (2.69 g, 19.5 mmol) and chloromethyl methyl ether (1.19 mL, 15.6 mmol) were added to a solution of 21 (5.30 g, 13.0 mmol) in DMF (53 mL), and the mixture was stirred at room temperature for 3 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give tert-butyl 7-benzyloxy-6-[1(2)-methoxymethyl-1(2)H-tetrazol-5-yl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (5.39 g, 92% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.77–2.88 (2H, m), 3.27 (1.8H, s), 3.47 (1.2H, s), 3.60–3.72 (2H, m), 4.60 (0.8H, s), 4.61 (1.2H, s), 5.06 (1.2H, s), 5.20 (0.8H, s), 5.58 (1.2H, s), 5.89 (0.8H, s), 6.85 (1H, s), 7.20–7.38 (5H, m), 7.52 (0.6H, s), 7.84 (0.4H, s).

tert-Butyl 7-Hydroxy-6-[1-methoxymethyl-1H-tetrazol-5-yl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (22) and tert-Butyl 7-Hydroxy-6-[2-methoxymethyl-2H-tetrazol-5-yl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (23)

A solution of tert-butyl 7-benzyloxy-6-[1(2)-methoxymethyl-1(2)H-tetrazol-5-yl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (5.30 g, 11.7 mmol) in MeOH (400 mL) was hydrogenated at 0.12 MPa in the presence of Pd-C (3.0 g) at room temperature for 12 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 22 (2.61 g, 62% yield) and 23 (1.52 g, 36% yield) as a solid.

Compound 22

¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.80–2.88 (2H, m), 3.55 (3H, s), 3.60–3.70 (2H, m), 4.59 (2H, s), 5.84 (2H, s), 6.92 (1H, s), 7.66 (1H, s), 10.29 (1H, s).

Compound 23

¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.80–2.88 (2H, m), 3.53 (3H, s), 3.61–3.70 (2H, m), 4.58 (2H, s), 5.92 (2H, s), 6.85 (1H, s), 7.87 (1H, s), 9.42 (1H, s).

tert-Butyl 7-(2-Diethylamino-5-methyloxazol-4-ylmethoxy)-6-(2-methoxymethyl-2H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (24s)

n-Bu₃P (0.42 mL, 1.7 mmol) and azodicarbonyldipiperidine (ADDP) (420 mg, 1.66 mmol) were added to a solution of 18s (153 mg, 0.830 mmol) and 22 (300 mg, 0.830 mmol) in toluene (10 mL) under ice-cooling, which was stirred at room temperature for 1 h. The reaction mixture was washed with water and saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 24s (326 mg, 74% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.19 (6H, t, J = 7.1 Hz), 1.51 (9H, s), 2.09 (3H, s), 2.81 (2H, t, J = 5.4 Hz), 3.25 (3H, s), 3.39 (4H, q, J = 7.1 Hz), 3.62–3.71 (2H, m), 4.62 (2H, s), 4.76 (2H, s), 5.80 (2H, s), 6.90 (1H, s), 7.36 (1H, s).

tert-Butyl 6-(2-Methoxymethyl-2H-tetrazol-5-yl)-7-(5-methyl-2-pyrrolidin-1-yloxazol-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (24u)

Compound 24u was prepared according to the procedure for the synthesis of 24s. Yield 64%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 1.94–2.02 (4H, m), 2.08 (3H, s), 2.77–2.86 (2H, m), 3.24 (3H, s), 3.41–3.49 (4H, m), 3.62–3.71 (2H, m), 4.62 (2H, s), 4.77 (2H, s), 5.80 (2H, s), 6.96 (1H, s), 7.36 (1H, s).

tert-Butyl 7-[2-(1H-Imidazol-1-yl)-5-methyloxazol-4-ylmethoxy]-6-(2-methoxymethyl-2H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (25q)

n-Bu₃P (1.05 mL, 4.20 mmol) and azodicarbonyldipiperidine (1.06 g, 4.20 mmol) were added to a solution of 18q (250 mg, 1.40 mmol) and 23 (504 mg, 1.39 mmol) in toluene (20 mL) under ice-cooling, which was stirred at room temperature for 1 h. The reaction mixture was washed with water and saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 25q (578 mg, 79% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.40 (3H, s), 2.84 (2H, t, J = 5.4 Hz), 3.47 (3H, s), 3.62–3.72 (2H, m), 4.62 (2H, s), 5.06 (2H, s), 5.86 (2H, s), 6.95 (1H, s), 7.15–7.19 (1H, br), 7.52 (1H, t, J = 1.4 Hz), 7.80 (1H, s), 8.16–8.21 (1H, m).

tert-Butyl 7-[2-(N-Ethylmethylamino)-5-methyloxazol-4-ylmethoxy]-6-(2-methoxymethyl-2H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (25r)

Compound 25r was prepared according to the procedure for the synthesis of 25q. Yield 93%. ¹H-NMR (CDCl₃) δ: 1.16 (3H, t, J = 7.1 Hz), 1.50 (9H, s), 2.19 (3H, s), 2.82 (2H, t, J = 5.4 Hz), 2.98 (3H, s), 3.41 (2H, q, J = 7.1 Hz), 3.49 (3H, s), 3.60–3.72 (2H, m), 4.60 (2H, s), 4.94 (2H, s), 5.87 (2H, s), 6.97 (1H, s), 7.75 (1H, s).

tert-Butyl 7-(2-Azetidin-1-yl-5-methyloxazol-4-ylmethoxy)-6-(2-methoxymethyl-2H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (25t)

Compound 25t was prepared according to the procedure for the synthesis of 25q. Yield 76%. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.19 (3H, s), 2.39 (2H, quintet, J = 7.6 Hz), 2.83 (2H, t, J = 5.4 Hz), 3.50 (3H, s), 3.62–3.72 (2H, m), 4.08 (4H, t, J = 7.6 Hz), 4.60 (2H, s), 4.94 (2H, s), 5.87 (2H, s), 6.94 (1H, s), 7.76 (1H, s).

tert-Butyl 7-[2-(2,5-Dihydropyrrol-1-yl)-5-methyloxazol-4-ylmethoxy]-6-(2-methoxymethyl-2H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (25v)

Compound 25v was prepared according to the procedure for the synthesis of 25q. Yield 65%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.10 (3H, s), 2.76–2.87 (2H, m), 3.25 (3H, s), 3.59–3.73 (2H, m), 4.27 (4H, s), 4.62 (2H, s), 4.79 (2H, s), 5.81 (2H, s), 5.87 (2H, s), 6.90 (1H, s), 7.36 (1H, s).

tert-Butyl 6-(2-Methoxymethyl-2H-tetrazol-5-yl)-7-(5-methyl-2-piperidin-1-yl-oxazol-4-yl-methoxy)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (25w)

Compound 25w was prepared according to the procedure for the synthesis of 25q. Yield 83%. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 1.56–1.65 (6H, m), 2.19 (3H, s), 2.83 (2H, t, J = 5.4 Hz), 3.36–3.45 (4H, m), 3.49 (3H, s), 3.60–3.70 (2H, m), 4.60 (2H, s), 4.94 (2H, s), 5.87 (2H, s), 6.96 (1H, s), 7.75 (1H, s).

2-[(E)-3-(2-Furyl)acryloyl]-7-[2-(imidazol-1-yl)-5-methyloxazol-4-ylmethoxy]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26q)

Compound 26q was prepared according to the procedure for the synthesis of 13a. Yield 50% for 2 steps. A white solid. mp 231–234 °C. ¹H-NMR (CDCl₃) δ: 2.47 (3H, s), 2.86–3.07 (2H, m), 3.86–4.01 (2H, m), 4.82–4.96 (2H, m), 5.12 (2H, s), 6.45–6.51 (1H, m), 6.59 (1H, d, J = 2.9 Hz), 6.88 (1H, d, J = 15.2 Hz), 6.95 (1H, s), 7.20–7.27 (1H, m), 7.45–7.50 (1H, m), 7.52 (1H, d, J = 15.2 Hz), 7.87 (1H, s), 8.18–8.26 (1H, m), 8.28–8.37 (1H, m). IR (ATR) cm⁻¹: 1651. MS m/z: 497 [M − H]⁻, 521 [M + Na]⁺.

7-[2-(N-Ethylmethylamino)-5-methyloxazol-4-ylmethoxy]-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26r)

Compound 26r was prepared according to the procedure for the synthesis of 13a. Yield 59% for 2 steps. A yellow solid. mp 159–163 °C. ¹H-NMR (CDCl₃) δ: 1.25 (3H, t, J = 7.1 Hz), 2.27 (3H, s), 2.90–3.11 (2H, m), 3.19 (3H, s), 3.55–3.68 (2H, m), 3.86–4.03 (2H, m), 4.84–4.95 (2H, m), 5.00 (2H, s), 6.44–6.52 (1H, m), 6.55–6.66 (1H, m), 6.88 (1H, d, J = 15.1 Hz), 6.96 (1H, s), 7.44–7.48 (1H, m), 7.51 (1H, d, J = 15.1 Hz), 8.17 (1H, s). IR (ATR) cm⁻¹: 1649. MS m/z: 488 [M − H]⁻, 490 [M + H]⁺.

7-(2-Diethylamino-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26s)

Compound 26s was prepared according to the procedure for the synthesis of 13a. Yield 45% for 2 steps. A pale brown solid. mp 111–114 °C. ¹H-NMR (CDCl₃) δ: 1.26 (6H, t, J = 7.1 Hz), 2.27 (3H, s), 2.88–3.07 (2H, m), 3.61 (4H, q, J = 7.1 Hz), 3.86–4.01 (2H, m), 4.84–4.95 (2H, m), 5.00 (2H, s), 6.47 (1H, dd, J = 3.4, 1.7 Hz), 6.58 (1H, d, J = 3.4 Hz), 6.88 (1H, d, J = 14.9 Hz), 6.98 (1H, s), 7.44–7.48 (1H, m), 7.51 (1H, d, J = 14.9 Hz), 8.16 (1H, s). IR (ATR) cm⁻¹: 1647. MS m/z: 502 [M − H]⁻, 504 [M + H]⁺.

7-(2-Azetidin-1-yl-5-methyloxazol-4-ylmethoxy)-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26t)

Compound 26t was prepared according to the procedure for the synthesis of 13a. Yield 65% for 2 steps. A pale yellow solid. mp 212–215 °C. ¹H-NMR (CDCl₃) δ: 2.00–2.12 (2H, m), 2.27 (3H, s), 2.88–3.09 (2H, m), 3.61–3.75 (4H, m), 3.85–4.03 (2H, m), 4.89 (2H, s), 5.01 (2H, s), 6.47 (1H, dd, J = 3.4, 1.9 Hz), 6.58 (1H, d, J = 3.4 Hz), 6.88 (1H, d, J = 15.1 Hz), 6.95 (1H, s), 7.43–7.49 (1H, m), 7.51 (1H, d, J = 15.1 Hz), 8.15 (1H, s). IR (ATR) cm⁻¹: 1649. MS m/z: 486 [M − H]⁻.

2-[(E)-3-(2-Fur-2-yl)acryloyl]-7-(5-methyl-2-pyrrolidin-1-yloxazol-4-ylmethoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26u)

Compound 26u was prepared according to the procedure for the synthesis of 13a. Yield 88% for 2 steps. A pale yellow solid. mp 136–138 °C. ¹H-NMR (CDCl₃) δ: 2.00–2.12 (4H, m), 2.27 (3H, s), 2.88–3.09 (2H, m), 3.61–3.75 (4H, m), 3.85–4.03 (2H, m), 4.89 (2H, s), 5.01 (2H, s), 6.47 (1H, dd, J = 3.4, 1.9 Hz), 6.58 (1H, d, J = 3.4 Hz), 6.88 (1H, d, J = 15.1 Hz), 6.95 (1H, s), 7.43–7.49 (1H, m), 7.51 (1H, d, J = 15.1 Hz), 8.15 (1H, s). IR (ATR) cm⁻¹: 1649. MS m/z: 500 [M − H]⁻.

7-[2-(2,5-Dihydropyrrol-1-yl)-5-methyloxazol-4-ylmethoxy]-2-[(E)-3-(2-furyl)acryloyl]-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26v)

Compound 26v was prepared according to the procedure for the synthesis of 13a. Yield 88% for 2 steps. A white solid. mp 239–241 °C. ¹H-NMR (CDCl₃) δ: 2.28 (3H, s), 2.88–3.09 (2H, m), 3.85–4.02 (2H, m), 4.50 (4H, s), 4.83–4.95 (2H, m), 5.03 (2H, s), 5.94 (2H, s), 6.47 (1H, dd, J = 3.2, 1.7 Hz), 6.58 (1H, d, J = 3.2 Hz), 6.88 (1H, d, J = 15.1 Hz), 6.96 (1H, s), 7.44–7.48 (1H, br), 7.51 (1H, d, J = 15.1 Hz), 8.17 (1H, s). IR (ATR) cm⁻¹: 1641. MS m/z: 498 [M − H]⁻, 500 [M + H]⁺. Anal. Calcd for C₂₆H₂₅N₇O₄: C, 62.52; H, 5.04; N, 19.63. Found: C, 62.33; H, 5.01; N, 19.40.

2-[(E)-3-(2-Furyl)acryloyl]-7-(5-methyl-2-piperidin-1-yloxazol-4-yl-methoxy)-6-(tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26w)

Compound 26w was prepared according to the procedure for the synthesis of 13a. Yield 63% for 2 steps. A pale yellow solid. mp 166–170 °C. ¹H-NMR (CDCl₃) δ: 1.57–1.82 (6H, m), 2.26 (3H, s), 2.88–3.07 (2H, m), 3.56–3.71 (4H, m), 3.85–4.03 (2H, m), 4.83–4.94 (2H, m), 5.00 (2H, s), 6.44–6.51 (1H, m), 6.55–6.62 (1H, m), 6.87 (1H, d, J = 14.9 Hz), 6.96 (1H, s), 7.44–7.48 (1H, m), 7.51 (1H, d, J = 14.9 Hz), 8.17 (1H, s). IR (ATR) cm⁻¹: 1647. MS m/z: 514 [M − H]⁻, 516 [M + H]⁺, 538 [M + Na]⁺.

PPARγ Agonist Activities

COS-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nissui Pharmaceutical, Co., Ltd., Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS, JRH Bioscience, Inc., Lenexa, KS), 0.3% NaHCO₃, and 1% penicillin–streptomycin under 5% CO₂ at 37 °C.

A full-length human PPARγ1 plasmid (Open Biosystems, Huntsville, AL, U.S.A.) was electroporated into COS-1 cells (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) with the human RXRα plasmid (GeneCopoeia Inc., Rockville, MD, U.S.A.) and the reporter plasmid pGL3-PPREx4-tk-luc using Nucleofector II (AAD-1001S, Lonza Group Ltd., Basel, Switzerland). Cells were incubated for 24 h in the presence and absence of test compounds in DMEM containing 10% FBS under 5% CO₂ at 37 °C. The medium was removed and luciferase activities were measured using a commercial kit (PicaGene LT7.5; TOYO B-Net Co., Ltd., Tokyo, Japan) and microplate luminescence reader (Dainippon Sumitomo Pharma Co., Ltd.). EC₅₀ values were assessed from concentration-response curves and maximal activation levels relative to the level maximally activated by farglitazar, a PPARγ full agonist, were calculated. Antagonistic activities against farglitazar (10⁻⁷ M) were measured, and the IC₅₀ value and maximal inhibition were calculated.

PTP1B Inhibitory Activity

Inhibitory activities against recombinant human PTP1B (Enzo Life Sciences, Inc., Farmingdale, NY) were measured using pNPP as a substrate. Briefly, PTP1B inhibitory activities were assessed in the absence and presence of the test compounds in 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 5.5) containing the enzyme, 1 mM dithiothreitol (DTT), and 1 mM ethylenediaminetetraacetic acid (EDTA). The reaction was started by the addition of pNPP and stopped by the addition of 1 M NaOH after a 30-min incubation at 37 °C. The absorbance of p-nitrophenol produced was measured at 405 nm.

Plasma Concentration after Oral Administration in Male SD Rats

Male SD rats (7 weeks old; Japan SLC, Inc., Hamamatsu, Japan) were used. The test compound at 10 mg/kg suspended in 0.5% methylcellulose solution was orally administered and a blood sample was then taken from the external jugular vein after 0.25, 0.5, 1, 3, 5, 8, and 24 h. The plasma concentrations of the compounds were assessed using HPLC equipment consisting of a pump (PU-980; JASCO, Tokyo, Japan), UV detector (UV-970; JASCO), autoinjector (AS-950; JASCO), and STR-ODS-II column (5 µm, 4.6 × 150 mm; Shimadzu GLC Ltd., Kyoto, Japan).

Effects in Zucker Fatty Rats

Male Zucker fatty rats (8 weeks old; Clea Japan, Inc., Tokyo, Japan) were allocated into three groups: a control group, 26v-treated group, and pioglitazone-treated group (n = 5–6). Compound 26v and pioglitazone suspended in 0.5% methylcellulose solution were orally administered at 10 or 30 mg/kg/d to rats for 28 d. Blood samples were taken from the jugular vein of non-fasted mice 24 h after the final administration. Plasma triglyceride, NEFA, and adiponectin levels in rats were assessed using commercial kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan and Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and hematocrit values were evaluated using the pocH-100iV DIFF hematology analyzer (Sysmex Corporation, Kobe, Japan). Rats were then fasted overnight and orally administered glucose (2 g/kg). Blood samples were taken from the jugular vein before and 30, 60, and 120 min after the glucose administration, plasma was isolated, and plasma glucose levels were assessed using a commercial kit (Wako Pure Chemical Industries, Ltd.).

Computational Analyses

An ensemble docking simulation of compounds 1 and 26v was performed according to a previous study.¹⁵⁾ Briefly, protein structures (PDB codes 4A4V (chain A) and 5Y2O (chain A)) were preprocessed using Protein Preparation Wizard in Maestro (Schrödinger Suite 2018-1) and the receptor grids for computational ligand docking were generated for the two protein structures using the grid generation feature of Glide (Schrödinger Suite 2018-1). The possible three dimensional (3D) structures of the compounds with various ionization states at pH 7.0 ± 2.0, tautomers, and ring conformations were defined, and multiple conformations were generated using LigPrep and ConfGen (Schrödinger Suite 2018-1). All docking calculations were performed with Glide in the standard precision (SP) mode. The ligand van der Waals radius for non-polar atoms was scaled by factors of 0.8 and 1.0. The conformers generated by ConfGen were docked into the receptor grids, and the top pose for each compound, assessed by the Glide docking score, was collected. Each docked structure was optimized using the OPLS3e force field and 0.05 kcal/mol/Å of convergence with a distance-dependent dielectric constant (ε = 4), and ligand-binding energy was then calculated using MM-GBSA methodology in Prime (Schrödinger Suite 2018-1).

Conflict of Interest

The authors declare no conflict of interest.

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