2-Acyl-3-carboxyl-tetrahydroisoquinoline Derivatives: Mixed-Type PTP1B Inhibitors without PPARγ Activation

Abstract

A novel series of 2-acyl-3-carboxyl-tetrahydroisoquinoline derivatives were synthesized and biologically evaluated. Among them, (S)-2-{(E)-3-furan-2-ylacryloyl}-7-[(2E,4E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (compound 17u) was identified as a potent protein tyrosine phosphatase 1B (PTP1B) inhibitor without peroxisome proliferator-activated receptor (PPAR) γ activation: PTP1B inhibition IC₅₀=0.19 µM and PPARγ ΕC₅₀>10 µM. Compound 17u exhibited mixed-type inhibition for PTP1B, and this mode of inhibition was rationalized by computational ligand docking into the catalytic and allosteric sites of PTP1B. Compound 17u also showed high oral absorption at 10 mg/kg (per os (p.o.), C_max=4.67 µM) in rats, significantly reduced non-fasting plasma glucose and triglyceride levels with no side effects at 30 mg/kg/d (p.o.) for 4 weeks, and attenuated elevations in plasma glucose levels in the oral glucose tolerance test performed 24 h after its final administration in db/db mice. In conclusion, the substituted 2-acyl-3-carboxyl-tetrahydroisoquinoline is a novel scaffold of mixed-type PTP1B inhibitors without PPARγ activation, and compound 17u has potential as an efficacious and safe anti-diabetic drug as well as a useful tool for investigations on the physiological and pathophysiological effects of mixed-type PTP1B inhibition.

The increased incidence of patients with type 2 diabetes represents one of the most serious healthcare issues worldwide. In type 2 diabetes, hyperglycemia is caused by reduced insulin secretion and/or insulin resistance, which is frequently accompanied by hyperinsulinemia and is a risk factor for macrovasculopathies, including atherosclerosis. Peroxisome proliferator-activated receptor (PPAR) γ agonists, such as pioglitazone and rosiglitazone, improve insulin resistance, and, thus, have been used in the treatment of type 2 diabetes.^1–3) Pioglitazone has been shown to prevent macrovasculopathies in type 2 diabetic patients⁴⁾; however, PPARγ agonists are associated with a risk of edema, heart failure, bone loss, cancer, and obesity.^5–9) To reduce PPARγ-mediated adverse effects, PPARγ and PPARα dual agonists, partial PPARγ agonists, and PPARγ agonists with protein tyrosine phosphatase 1B (PTP1B) inhibition have been synthesized and studied^10–13) (Fig. 1); however, they have yet to be successfully developed. We previously reported a novel series of 2-acyl-3-carboxyl-tetrahydroisoquinoline derivatives with potent PPARγ activation and weak PTP1B inhibition.^14–17) However, even weak PPARγ activation may cause side effects in PPARγ agonist-sensitive patients.

Fig. 1. Chemical Structures of PPARγ Agonists

Pioglitazone, Rosiglitazone, Farglitazar: PPARγ full agonists, KRP-297, Muraglitazar, Imiglitazar: PPARγ and PPARα dual agonists, KY-699: a PPARγ full agonist with PTP1B inhibition, KY-601: a PPARγ and PPARα dual agonist with PTP1B inhibition, KY-755: a PPARγ partial agonist with PTP1B inhibition.

The development of PPARγ-independent insulin sensitizers has long been desired. PTP1B, a non-receptor-type protein tyrosine phosphatase, negatively regulates insulin signals by dephosphorylating insulin receptors and insulin receptor substrate as well as leptin signals through the dephosphorylation of Janus kinase 2 and signal transducer and activator of transcription 3.^18,19) Thus, PTP1B inhibitors exert anti-diabetic and anti-obesity effects. The genetic deletion of PTP1B has been shown to cause resistance to obesity and insulin hypersensitivity.²⁰⁾ A PTP1B antisense oligonucleotide was found to normalize glucose levels and improve insulin resistance in diabetic mice.²¹⁾ A large number of PTP1B inhibitors have been identified as candidates for anti-diabetic and anti-obesity drugs.^22–25)

Among them, orally active ertiprotafib and JTT-551 and parenterally active trodusquemine entered the clinical trial stages; however, their development was discontinued.^26–30) Ertiprotafib²⁶⁾ with a carboxyl moiety exhibits PPARγ and PPARα agonist activities in addition to PTP1B inhibition, and JTT-551^27,28) with carboxyl and amine moieties and trodusquemine^29,30) with a spermine residue are selective PTP1B inhibitors (Fig. 2). A number of structurally and mechanistically different inhibitors have recently been reported: carboxyl-type and non-carboxyl-type inhibitors as well as competitive-type, non-competitive-type, and mixed-type inhibitors.^22–25,31) Mixed-type inhibition is a mixture of competitive and non-competitive inhibition. We also discovered the non-carboxyl type non-competitive PTP1B inhibitor KY-226 without PPARγ activation, which binds to the allosteric site of PTP1B³²⁾ (Fig. 2). The type of structure, mode of action, and formulation (oral or parenteral) that are beneficial for clinical use have not yet been established and may depend on indications because PTP1B inhibitors are expected to become drugs for the treatment of not only diabetes and obesity, but also neuroinflammation and renal failure.^18,22) Furthermore, PTP1B has been implicated in tumorigenesis, podocyte injury, endothelial dysfunction, and retinal light damage.^{18,22,33–35)}

Fig. 2. Chemical Structures of PTP1B Inhibitors

CX08005: competitive-type PTP1B inhibitor, JTT-551: mixed-type PTP1B inhibitor, KY-226, Ertiprotafib, trodusquemine: non-competitive-type PTP1B inhibitors.

In the present study, to identify a carboxyl-type PTP1B inhibitor without PPARγ activation that is suitable for oral and parenteral use, a novel series of 2-acyl-3-carboxyl-tetrahydroisoquinoline derivatives were synthesized and evaluated. Structure–activity relationships were discussed and (S)-2-{(E)-3-furan-2-ylacryloyl}-7-[(2E,4E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (compound 17u) was selected for further evaluations. Compound 17u exhibited potent and mixed-type PTP1B inhibition without PPARγ activation, high bioavailability in rats, and anti-diabetic activity in db/db mice.

Chemistry

The synthetic routes for tetrahydroisoquinoline derivatives are shown in Charts 1 and 2.

Chart 1. Synthesis of 2-Acyl-3-carboxyl-1,2,3,4-tetrahydroisoquinoline Derivatives

(i) Various carboxylic acids, EDC·HCl, CH₂Cl₂; (ii) various carboxylic acids, EDC·HCl, HOBt, CH₂Cl₂; (iii) various acyl chlorides, Et₃N, CH₂Cl₂; (iv) 2,2-dimethyldodecanoic acid, oxalyl chloride, DMF, i-Pr₂NEt, CH₂Cl₂; (v) various alkylbromides, K₂CO₃, DMF; (vi) 3-(3-trifluoromethylphenyl)propan-1-ol, PPh₃, DEAD in toluene, THF; (vii) HCl in i-PrOH, HCO₂H; (viii) sorbic acid, EDC·HCl, CH₂Cl₂; (ix) 3-furan-2-ylacrylic acid, EDC·HCl, CH₂Cl₂; (x) various carboxylic acids, EDC·HCl, CH₂Cl₂; (xi) (E)-3-thiazol-2-ylacrylic acid, EDC·HCl, Et₃N, CH₂Cl₂ (xii) various alcohols, PPh₃, Mitsunobu reagents, THF; (xiii) various alcohols, n-Bu₃P, Mitsunobu reagents, THF; (xiv) aqueous LiOH, MeOH, THF; (xv) aqueous LiOH, MeOH, THF, then t-butylamine, EtOH; (xvi) methanesulfonamide, CDI, DBU, DMF.

Chart 2. Synthesis of Compound 25

(i) HCl in i-PrOH, HCO₂H; (ii) 3-furan-2-ylacrylic acid, EDC·HCl, CH₂Cl₂; (iii) Raney Ni, HCO₂H; (iv) O-(5-methylhexyl)hydroxylamine, EtOH; (v) aqueous LiOH, MeOH, THF.

Compound 1, which was synthesized as described previously,³⁶⁾ was acylated with various carboxylic acids and acyl chlorides to give compounds 3a–i. Compound 2³⁷⁾ was separately alkylated by alkyl bromide with K₂CO₃ or the Mitsunobu reaction to afford 3j, 3k, 3n, and 3t. Compounds 3a–k, 3n, and 3t were converted to 11a–k, 12k, 12n, and 12t by the deprotection of the t-butoxycarbonyl (Boc) group, followed by condensation with sorbic acid or 3-furan-2-ylacrylic acid. Compounds 11l, 12l, 12m, 12o–s, 12u–x, 13l, 13u, 14l, and 15u were prepared from compound 4³⁸⁾ or 5.³⁹⁾ Thus, the 2-position of the tetrahydroisoquinoline of compound 4 or 5 was acylated with the corresponding carboxylic acid to give compounds 6–10. The Mitsunobu reaction of compounds 6–10 with various alcohols afforded 11l, 12l, 12m, 12o–s, 12u–x, 13l, 13u, 14l, and 15u, respectively. The methyl esters of compounds 11a–l, 12k–x, 13l, 13u, 14l, and 15u were hydrolyzed with aqueous LiOH to give 16a–l, 17k–x, 18l, 18u, 19l, and 20u as a free form or t-butylamine salt. The carboxyl groups of 17u, 18l, and 18u were condensed with methanesulfonamide to afford 21u, 22l, and 22u.

The preparation of compound 25 was outlined in Chart 2. The starting material 23 was prepared as described previously.³⁶⁾ The Boc group of compound 23 was deprotected with HCl/HCO₂H, which was acylated with 3-furan-2-ylacrylic acid to afford 24. The cyano group of compound 24 was reduced using Raney Ni to an aldehyde, which was reacted with the corresponding hydroxylamine, followed by hydrolyzation with aqueous LiOH to afford 25.

Results and Discussion

We previously reported various 3-carboxyl-tetrahydroisoquinoline derivatives with PPARγ agonist activities (Fig. 1): a 2-alkyl-3-carboxyl-7-phenyloxazolyl-tetrahydroisoquinoline derivative with PPARγ full agonist activity,³⁷⁾ a 2-acyl-3-carboxyl-7-alkenyloxazolyl-tetrahydroisoquinoline derivative with PPARγ full agonist activity and PTP1B inhibition (KY-699),¹⁶⁾ a 2-acyl-3-carboxyl-7-cycloalkenyloxazol-tetrahydroisoquinoline derivative with PPARγ and PPARα dual agonist activity and PTP1B inhibition (KY-601),¹⁵⁾ and a 2-acyl-3-carboxyl-7-(bulky substituent)oxazolyl-tetrahydroisoquinoline derivative with partial PPARγ agonist activity and PTP1B inhibition (KY-755).¹⁷⁾ In these studies, we found that substituents on the oxazole influenced PPARγ agonist activity, and an aliphatic substituent was preferable for PTP1B inhibitory activity. Therefore, to identify new compounds with PTP1B inhibitory, but not PPARγ agonist activity, a novel series of 2-acyl-3-carboxyl-tetrahydroisoquinoline derivatives with various substituents at the 7-position were synthesized and evaluated. The chemical structures, molecular weights, calculated log D_7.4 (c log D_7.4), and PTP1B inhibitory and PPARγ agonist activities of the compounds synthesized were shown in Tables 1–3.

Table 1. Chemical Structures, Molecular Weights, c log D_7.4, PTP1B Inhibitory and PPARγ Agonist Activities of 7-Substituted 3-Carboxyl-2-hexadienoyl-tetrahydroisoquinoline Derivatives

a) Molecular weight as the free form. b) n=2. c) t-Butylamine salt. N.D.: not determined.

Table 2. Chemical Structures, Molecular Weights, c log D_7.4, PTP1B Inhibitory and PPARγ Agonist Activities of 7-Substituted 3-Carboxyl-2-furanylacryloyl-tetrahydroisoquinoline Derivatives

a) Molecular weight as the free form. b) n=2. N.D.: not determined.

Table 3. Chemical Structures, Molecular Weights, c log D_7.4, PTP1B Inhibitory and PPARγ Agonist Activities of 2,3,7-Substituted Tetrahydroisoquinoline Derivatives

a) Molecular weight as the free form. b) n=2. N.D.: not determined.

Based on the structure of KY-699, the 7-substituent was subjected to various changes to increase PTP1B inhibitory activity and reduce PPARγ agonist activity. The majority of PPARγ agonists have a methoxy or ethoxy chain with aromatic rings, such as pyridine and phenyloxazole, on the phenyl ring at the para-position of thiazolidinedione or the carboxyl group¹⁰⁾ (Fig. 1). KRP-297, a PPARα and PPARγ dual agonist, has an amide group at the meta-position of the thiazolidinedione group⁴⁰⁾ (Fig. 1). In rosiglitazone, the pyridine ring occupies the lipophilic pocket between helix 3 and the β-sheet, and its nitrogen atom makes a hydrogen bond with a water molecule within the ligand pocket.⁴¹⁾ In farglitazar, a carboxyl group interacts with Ser289, His323, Tyr327, Tyr473, and His449, and phenyloxazole with NH of Ser34.⁴²⁾ To reduce PPARγ agonist activity, the alkoxy chain of KY-699 was changed to an amide chain and to a different alkoxy chain from those in PPARγ agonists. Derivatives with amide substituents, a biphenyl methoxy substituent, and longer alkoxy substituents did not exhibit PPAR γ agonist activities (Table 1).

Among amide derivatives, 16a and 16b with an oxazole ring did not exhibit PTP1B inhibitory activities and this may be attributed to their low lipophilicities. Compound 16c exhibited weak PTP1B inhibitory activity despite its very low lipophilicity: its hydroxyl group may interact with the enzyme. Long alkylamide chains (16d–h) enhanced PTP1B inhibitory activity. This activity increased in a manner that was dependent on chain length (lipophilicity). The hydroxyl group (16f) maintained activity despite its low lipophilicity, whereas the branched structure (16g, 16h) resulted in weaker activity than those of normal alkyl structures with similar lipophilicities (16d, 16e). The biphenyl amide structure (16i) showed decreased lipophilicity and weaker activity, while the biphenyl methoxy structure (16j) exhibited higher lipophilicity and stronger activity than 16i. The long alkoxy side chain (16k, 16l) also exhibited strong PTP1B inhibitory activity. Amide derivatives required higher lipophilicity than the alkoxy derivatives with the same activity (16g vs. 16j, 16h vs. 16k, 16e vs. 16l), suggesting that alkoxy chains are more suitable for PTP1B inhibition than amide chains.

The 2-hexadienoyl structures of 16k and 16l were changed to the 2-furylacryloyl structure (17k, 17l), which has been shown to be chemically more stable and enhances oral absorption in KY-755 drivatives.¹⁷⁾ Compounds 17k and 17l exhibited stronger activities than 16k and 16l, and, thus, various 2-furylacryloyl-7-alkoxy derivatives were synthesized (Table 2). The activity of compound 17m was similar to that of 17l, whereas its lipophilicity was higher. Compounds 17n and 17p with low lipophilicities did not exhibit PTP1B inhibitory activity: c log D_7.4 needs to be at least higher than 0.6 for PTP1B inhibition. Although compounds 17k, 17l, and 17o showed similar lipophilicities (c log D_7.4), 17l exhibited the strongest activity followed by 17k and 17o. The alkenyl structure may be preferable to the alkyl structure, and the branched alkenyl structure may not be preferable for PTP1B inhibition. Among the compounds with a side chain having a phenyl ring, 17q, 17r, and 17s exhibited potent activity that was dependent on lipophilicity. While compounds 17t and 17u had similar c log D_7.4 values of approximately 1.0, 17t exhibited weak activity possibly due to a shorter and saturated chain, while 17u showed potent activity, which was similar to that of 17s with a higher c log D_7.4 (2.0). The introduction of a heteroaromatic ring decreased activities (17v, 17w), while the introduction of a 2,3-dihydrobenzodioxine structure (17x) maintained moderate activity in spite of its low c log D_7.4 (0.6). The oxime structure (25) decreased lipophilicity and activity.

The 2-acyl groups of compounds 16l (Table 1) and 17u (Table 2) with the strongest activities were changed (Table 3). In 7-(9-methyldecadienyloxy) derivatives, activity increased in a manner that depended on lipophilicity (16l–19l, 22l). The 3-methanesulfonylamide (22l) derivative exhibited similar activity to that of 18l; however, its molecular weight and c log D_7.4 were markedly higher than those of 18l. In 7-[5-(2,4,6-trifluorophenyl)pentadienyloxy] derivatives, the activities of 18u, 20u, and 21u were all lower than that of 17u, and activity was not necessarily dependent on lipophilicity. Among compounds with strong activities (IC₅₀<0.2), 17u had lower lipophilicity than compounds 17l, 18l, 19l, and 22l, and showed greater oral absorption than 17l at 10 mg/kg in male Sprague–Dawley (SD) rats (C_max=4.67 and 1.96 µM, respectively): its fluorophenyl ring may be more metabolically stable than the alkyl chain. Thus, 17u was selected for further evaluations.

In order to evaluate the inhibitory mode of compound 17u, a kinetic study using the synthetic substrate p-nitrophenylphosphate (pNPP) was performed. Lineweaver–Burk plots demonstrated that 17u inhibited PTP1B activity in a mixed-type manner with a K_i value of 0.34 µM (Fig. 3). In this inhibitory model, the inhibitor bound to the free enzyme (E) and enzyme–substrate complex (ES), and the substrate had lower affinity for the enzyme–inhibitor complex (EI) than for E, with the enzyme–substrate–inhibitor ternary complex (ESI) being non-productive.

Fig. 3. Lineweaver–Burk Plots for Inhibition of the PTP1B Enzyme Reaction by Compound 17u

To gain insights into the binding modes of compound 17u and its mixed-type inhibition, we performed computational ligand docking into the catalytic and allosteric sites of PTP1B. The crystal structures of PTP1B with an “open” tryptophan–proline–aspartate (WPD)-loop (residues 177–185) were selected as the docking templates for the catalytic site. It is important to note that compound 17u was larger and the catalytic site with a “closed” WPD-loop had insufficient space for its binding (unpublished data). The crystal structures of PTP1B complexed with an allosteric inhibitor were also selected for docking to the allosteric site. Details are shown in the Experimental.

The docking pose of compound 17u within the catalytic site was shown in Fig. 4. In and around the active site (A-site), the furylacryloyl group of compound 17u formed hydrogen bonds with Ser216, Ala217, and Arg221 of the phosphate-binding loop (P-loop, residues 214–221). Carboxylic acid formed electrostatic and/or hydrogen-bonding interactions with Lys120 and water-mediated hydrogen bonds with Asn111 and Glu115. Furthermore, the tetrahydroisoquinoline ring interacted by π–π stacking with Tyr46. The alkenyloxy chain and trifluorophenyl ring formed van der Waals interactions around the secondary non-catalytic aryl phosphate-binding site (B-site). These interactions are commonly observed in the catalytic sites of inhibitor-PTP1B complex structures.^43–45) The docking pose of compound 17u within the allosteric site was shown in Fig. 5. In this structure, compound 17u exhibited a U-shaped conformation and formed aromatic and van der Waals interactions around Phe196 and Phe280 of PTP1B. The observed U-shaped conformation and interaction pattern of compound 17u are characteristic of known allosteric PTP1B inhibitors.^32,46)

Fig. 4. Docking Pose of Compound 17u to the Catalytic Site of PTP1B

Compound 17u (magenta) and PTP1B residues within 4 Å from 17u (gray) are shown as sticks, with the Connolly surface of PTP1B being transparently depicted. Hydrogen bonds and aromatic interactions are shown as dotted lines, and colored cyan and brown, respectively.

Fig. 5. Docking Pose of Compound 17u to the Allosteric Site of PTP1B

Compound 17u (magenta) and PTP1B residues within 4 Å from 17u (gray) are shown as sticks, with the Connolly surface of PTP1B transparently depicted. Aromatic interactions are shown as dotted lines, and colored brown.

In order to clarify the selectivity of compound 17u to the PTP family, we measured inhibitory activities against T-cell protein tyrosine phosphatase (TCPTP), CD45, and leukocyte common antigen-related protein tyrosine phosphatase (LAR). The results obtained showed that compound 17u was the strongest inhibitor of PTP1B (IC₅₀=0.19 µM) followed by TCPTP (IC₅₀=0.55 µM), and more weakly inhibited CD45 (IC₅₀=6.46 µM) and LAR (IC₅₀>10 µM). Thus, the selectivity of compound 17u for PTP1B over TCPTP was only approximately 2-fold, which was smaller than that of KY-226 (approximately 5-fold), an allosteric inhibitor.³²⁾ In the allosteric site, Phe280 in PTP1B is replaced by Cys278 in TCPTP, and, thus, KY-226 is considered to interact more strongly with PTP1B than with TCPTP. In the catalytic site, Ser28 of PTP1B was replaced by His30 of TCPTP, whereas the other residues labeled in Fig. 4 were fully conserved. The lower selectivity for PTP1B over TCPTP of compound 17u than that of KY-226 suggested that compound 17u mainly binds to the catalytic sites of both enzymes. TCPTP has also been reported to negatively regulate insulin and leptin signals.⁴⁷⁾ Weak TCPTP inhibition by compound 17u may potentiate its anti-diabetic and anti-obesity effects through PTP1B inhibition.

To assess the pharmacokinetic properties of 17u, its plasma concentrations were measured after its oral administration at 10 mg/kg to male SD rats. C_max, T_max, T_1/2, area under curve (AUC), V_d and CL values were 4.67 µM, 1 h, 2.47 h, 29.5 µmol·h/L, 3.72 L, and 0.66 L/h·kg, respectively. C_max exceeded its IC₅₀ value for PTP1B inhibition, suggesting that it exhibited in vivo efficacy via the inhibition of PTP1B. Compound 17u was orally administered at 10 and 30 mg/kg/d for 4 weeks to db/db mice (Table 4). It moderately reduced plasma glucose and triglyceride (TG) levels by 17.9 and 37.4%, respectively, at 10 mg/kg/d, and significantly reduced them by 37.4 and 62.9%, respectively, at 30 mg/kg/d. It also moderately attenuated elevations in plasma glucose levels by 14.9% at 10 mg/kg/d and significantly by 41.3% at 30 mg/kg/d in the oral glucose tolerance test. The mode of inhibition (competitive, mixed, or non-competitive) that is the most suitable for therapeutic use has not yet been established: CX08005 (competitive),³¹⁾ JTT-551 (mixed),²⁷⁾ and KY-226 (non-competitive)³²⁾ have all been reported to exert potent anti-diabetic effects experimentally. Compound 17u has high water solubility (797 µg/mL) and, thus, may be widely used as oral, injection and eye drop formulations for the treatment of diseases mediated by PTP1B.

Table 4. Effects of Compound 17u Administered Orally for 4 Weeks to Male db/db Mice

Compound	Glucose (mg/dL)	TG (mg/dL)	Glucose AUC in OGTT (mg·h/dL)
Control	535.6±34.2	336.2±44.7	978.8±94.3
Compound 17u 10 mg/kg/d	439.7±37.1	232.1±65.1	833.3±110.9
Compound 17u 30 mg/kg/d	335.2±57.4*	124.7±25.5**	574.8±91.7*

Mean±S.E. (n=6). * p<0.05, ** p<0.01 vs. Control, the Student’s t-test.

In conclusion, a series of 2-acyl-3-carboxyl-tetrahydroisoquinoline derivatives were synthesized and the 2-acyl-3-carboxyl-tetrahydroisoquinoline structure was shown to be a novel scaffold for a mixed-type PTP1B inhibitor without PPARγ activation. Among the derivatives synthesized, compound 17u has potential as both an oral and parenteral therapeutic agent for the treatment of diseases in which PTP1B is implicated.

Experimental

General

Melting points (mp) 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 nuclear magnetic resonance spectrometer at 400 MHz (JNM-AL400; JEOL Ltd., Tokyo, Japan) using tetramethylsilane (TMS) as an internal standard. IR spectra were recorded with an infrared spectrometer (FT-IR8200PC; Shimadzu Corporation, Kyoto, Japan). Mass spectra were obtained on a QTRAP LC/MS/MS system (API2000; Thermo Fisher Scientific Inc., Foster, CA, 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).

Methyl (2-Isopropyl-5-methyloxazol-4-yl)acetate (26)

A mixture of isobutylamide (13.0 g, 149 mmol) and methyl 4-bromo-3-oxopentanoate (28.0 g, 134 mmol) in toluene (200 mL) was refluxed for 16 h. After cooling to room temperature, water was added to the reaction mixture, which was extracted with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 26 (4.02 g, 15% yield) as a pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.31 (6H, d, J=7.1 Hz), 2.24 (3H, s), 3.01 (1H, septet, J=7.1 Hz), 3.47 (2H, s), 3.70 (3H, s).

(2-Isopropyl-5-methyloxazol-4-yl)acetic Acid (27)

One molar aqueous LiOH solution (40.8 mL, 40.8 mmol) was added to a solution of 26 (4.02 g, 20.4 mmol) in MeOH (40 mL) and tetrahydrofuran (THF) (40 mL), which was stirred at room temperature for 0.5 h. After the addition of 10% aqueous citric acid solution, the reaction mixture was extracted with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 27 (3.39 g, 91%) as a pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.31 (6H, d, J=6.8 Hz), 2.24 (3H, s), 3.04 (1H, septet, J=6.8 Hz), 3.51 (2H, s), 6.80–8.80 (1H, br).

Methyl (S)-2-tert-Butoxycarbonyl-7-[2-(2-isopropyl-5-methyloxazol-4-yl)acetylamino]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3b)

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (3.55 g, 18.5 mmol) was added to a solution of compounds 1³⁶⁾ (4.72 g, 15.4 mmol) and 27 (3.39 g, 18.5 mmol) in CH₂Cl₂ (100 mL), and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 3b (6.43 g, 89% yield) as a pale yellow oil.¹H-NMR (CDCl₃) δ: 1.37 (6H, d, J=7.0 Hz), 1.42–1.55 (9H, m), 2.26 (3H, s), 3.00–3.23 (3H, m), 3.48 (2H, s), 3.57–3.64 (3H, m), 4.39–4.53 (1H, m), 4.60–4.72 (1H, m), 4.73–4.79 (0.5H, m), 5.08–5.15 (0.5H, m), 6.99–7.12 (1.5H, m), 7.22–7.30 (0.5H, m), 7.37–7.46 (0.5H, m), 7.49–7.54 (0.5H, m), 9.20–9.34 (1H, m).

Benzyl (2-Acetoxymethyl-5-methyloxazol-4-yl)acetate (28)

Chloromethyl acetate (13.7 mL, 127 mmol) was added to a suspension of (S)-2-amino-4-(benzyloxy)-4-oxobutanoic acid hydrochloride⁴⁸⁾ (30.0 g, 116 mmol) and Et₃N (41.0 mL, 294 mmol) in CH₂Cl₂ (600 mL) at −10°C, which was stirred at the same temperature for 1 h. The reaction mixture was washed with 10% aqueous citric acid solution and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

Acetic anhydride (50.0 mL, 529 mmol), N-methylmorpholine (46.0 mL, 418 mmol), and N,N-dimethyl-4-aminopyridine (DMAP) (2.60 g, 12.3 mmol) were added to a solution of the residue obtained in toluene (350 mL), which was stirred at 80°C for 45 min. The reaction mixture was poured into cold water and washed with 10% aqueous citric acid solution, saturated aqueous NaHCO₃ solution, and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

Phosphoryl chloride (12.0 mL, 129 mmol) was added to a solution of the residue obtained in toluene (500 mL), and the mixture was refluxed for 1 h. The reaction mixture was poured into cold water and neutralized with saturated aqueous NaHCO₃ solution, which was extracted with toluene. The organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 28 (6.34 g, 18% yield for 3 steps) as a yellow oil. ¹H-NMR (CDCl₃) δ: 2.12 (3H, s), 2.27 (3H, s), 3.54 (2H, s), 5.08 (2H, s), 5.13 (2H, s), 7.28–7.45 (5H, m).

(2-Acetoxymethyl-5-methyloxazol-4-yl)acetic Acid (29)

A solution of 28 (6.34 g, 20.9 mmol) in MeOH (200 mL) was hydrogenated at 0.4 MPa in the presence of Pd-C (640 mg) at room temperature for 1 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. After the addition of i-Pr₂O, the precipitate was collected by filtration to give 29 (2.91 g, 65% yield) as a pale yellow solid. ¹H-NMR (CDCl₃) δ: 2.13 (3H, s), 2.30 (3H, s), 3.49 (2H, s), 3.54 (2H, s), 7.70–8.60 (1H, br).

Methyl (S)-2-tert-Butoxycarbonyl-7-[2-(2-acetoxymethyl-5-methyloxazol-4-yl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3c)

Compound 3c was synthesized from 1³⁶⁾ and 29 according to the procedure for the synthesis of 3b. Yield quant. ¹H-NMR (CDCl₃) δ: 1.42–1.56 (9H, m), 2.16 (3H, s), 2.32 (3H, s), 3.05–3.26 (2H, m), 3.52 (2H, s), 3.57–3.64 (3H, m), 4.40–4.54 (1H, m), 4.68–4.80 (2H, m), 5.13 (2H, s), 7.00–7.19 (1H, m), 7.20–7.50 (2H, m), 8.67–8.76 (1H, br).

Methyl (S)-2-tert-Butoxycarbonyl-7-(dodecanoylamino)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3d)

n-Dodecanoyl chloride (0.26 mL 1.1 mmol) and Et₃N (0.18 mL, 1.3 mmol) were added to a solution of 1³⁶⁾ (300 mg, 0.979 mmol) in CH₂Cl₂ (3 mL) under ice-cooling, and the mixture was stirred at the same temperature for 20 min. After the addition of 10% aqueous citric acid solution, the reaction mixture was extracted with CH₂Cl₂. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 3d (480 mg, quant.) as a colorless oil. ¹H-NMR (CDCl₃) δ: 0.88 (3H, t, J=6.6 Hz), 1.22–1.41 (16H, m), 1.43–1.55 (9H, m), 1.65–1.78 (2H, m), 2.33 (2H, t, J=7.6 Hz), 3.04–3.28 (2H, m), 3.58–3.64 (3H, m), 4.39–4.53 (1H, m), 4.60–4.73 (1H, m), 4.74–4.82 (0.4H, m), 5.08–5.17 (0.6H, m), 7.02–7.12 (2.5H, m), 7.20–7.58 (1.5H, m).

Methyl (S)-2-tert-Butoxycarbonyl-7-(tetradecanoylamino)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3e)

Compound 3e was synthesized from 1³⁶⁾ and tetradecanoyl chloride according to the procedure for the synthesis of 3d. Yield 96%. A colorless oil. ¹H-NMR (CDCl₃) δ: 0.80–0.90 (3H, m), 1.20–1.58 (29H, m), 1.63–1.76 (2H, m), 2.27–2.37 (2H, m), 3.04–3.26 (2H, m), 3.57–3.64 (3H, m), 4.37–4.82 (3H, m), 7.01–7.57 (4H, m).

Methyl (S)-2-(tert-Butoxycarbonyl)-7-(10-methoxymethoxydecanoylamino)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3f)

Compound 3f was synthesized from 1³⁶⁾ and 10-methoxymethoxydecanoic acid⁴⁹⁾ according to the procedure for the synthesis of 3b. Yield 22% yield. A pale brown oil. ¹H-NMR (CDCl₃) δ: 1.22–1.41 (10H, m), 1.42–1.79 (13H, m), 2.33 (2H, t, J=7.3 Hz), 3.07–3.25 (2H, m), 3.36 (3H, s), 3.51 (2H, t, J=6.8 Hz), 3.60–3.65 (3H, m), 4.40–5.18 (5H, m), 7.03–7.53 (4H, m).

Methyl (S)-2-tert-Butoxycarbonyl-7-(2,2-dimethyldecanoylamino)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3g)

Compound 3g was synthesized from 1³⁶⁾ and 2,2-dimethyldecanoyl chloride⁵⁰⁾ according to the procedure for the synthesis of 3d. Yield quant. A brown oil. ¹H-NMR (CDCl₃) δ: 0.83–0.90 (3H, m), 1.18–1.70 (29H, m), 3.06–3.26 (2H, m), 3.59–3.65 (3H, m), 4.41–4.82 (3H, m), 7.03–7.60 (4H, m).

Methyl (S)-2-tert-Butoxycarbonyl-7-(2,2-dimethyldodecanoylamino)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3h)

Oxalyl chloride (0.13 mL 1.6 mmol) and 1 drop of N,N-dimethylformamide (DMF) were added to a solution of 2,2-dimethyldodecanoic acid⁵⁰⁾ (359 mg, 1.57 mmol) in CH₂Cl₂ (2 mL), and the mixture was stirred at room temperature for 20 min. After the addition of compound 1³⁶⁾ (400 mg, 1.31 mmol) and i-Pr₂NEt (0.81 mL, 4.7 mmol), the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was acidified with 10% aqueous citric acid solution, which was extracted with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 3h (740 mg, 91% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 0.81–0.92 (3H, m), 1.18–1.33 (22H, m), 1.42–1.67 (11H, m), 3.05–3.28 (2H, m), 3.57–3.66 (3H, m), 4.40–4.81 (2.4H, m), 5.08–5.17 (0.6H, m), 7.00–7.12 (1.5H, m), 7.20–7.60 (1.5H, m).

Methyl (S)-7-[(Biphenyl-4-carbonyl)amino]-2-tert-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3i)

Biphenyl-4-carboxylic acid (181 mg, 1.61 mmol), 1-hydroxybenzotriazole (HOBt), and EDC·HCl (371 mg, 1.94 mmol) were added to a solution of compound 1³⁶⁾ in CH₂Cl₂ (10 mL), and the reaction mixture was stirred at room temperature for 3 h and refluxed for 14 h. After cooling to room temperature, the reaction mixture was washed with water and saturated aqueous NaHCO₃ solution, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 3i (680 mg, 65% yield for 2 steps) as a white solid. ¹H-NMR (CDCl₃) δ: 1.47 (4.5H, s), 1.53 (4.5H, s), 3.10–3.28 (2H, m), 3.62 (1.5H, s), 3.65 (1.5H, s), 4.45–4.58 (1H, m), 4.66–4.84 (1.5H, m), 5.14–5.19 (0.5H, m), 7.12–7.18 (1H, m), 7.25–7.29 (0.5H, m), 7.38–7.43 (1.5H, m), 7.45–7.51 (2H, m), 7.53–7.58 (0.5H, m), 7.61–7.65 (2H, m), 7.67–7.73 (2.5H, m), 7.79–7.82 (1H, br), 7.91–7.95 (2H, m).

Methyl (S)-7-(Biphenyl-4-ylmethoxy)-2-tert-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3j)

4-Bromomethylbiphenyl (402 mg, 1.63 mmol) and K₂CO₃ (247 mg, 1.79 mmol) were added to a stirred solution of 2³⁷⁾ (500 mg, 1.63 mmol) in DMF (5 mL), followed by stirring at room temperature for 15 h. After the addition of water, the mixture was extracted with AcOEt, washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 3j (790 mg, quant.) as a white solid. ¹H-NMR (CDCl₃) δ: 1.45 (4.5H), 1.52 (4.5H, s), 3.06–3.13 (1.5H, m), 3.18 (0.5H, dd, J=15.8, 2.9 Hz), 3.61 (1.5H, s), 3.64 (1.5H, s), 4.45 (0.5H, d, J=16.1 Hz), 4.50 (0.5H, d, J=16.4 Hz), 4.65–4.78 (1.5H, m), 5.07 (2H, s), 5.11–5.15 (0.5H, m), 6.73–6.85 (2H, m), 7.06 (1H, d, J=8.3 Hz), 7.32–7.37 (1H, m), 7.41–7.50 (4H, m), 7.57–7.63 (4H, m).

Methyl (S)-2-tert-Butoxycarbonyl-7-(7-methyloctyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3k)

Compound 3k was synthesized from 2³⁷⁾ and 7-methyloctanyl-1-bromide⁵¹⁾ according to the procedure for the synthesis of 3j. Yield 44%. A colorless oil. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.8 Hz), 1.13–1.21 (2H, m), 1.25–1.37 (4H, m), 1.40–1.49 (6.5H, m), 1.49–1.55 (5.5H, m), 1.71–1.80 (2H, m), 3.04–3.21 (2H, m), 3.61 (1.5H, s), 3.63 (1.5H, s), 3.91 (2H, t, J=6.6 Hz), 4.43 (0.5H, d, J=16.1 Hz), 4.48 (0.5H, d, J=16.1 Hz), 4.66 (0.5H, d, J=16.1 Hz), 4.69 (0.5H, d, J=16.1 Hz), 4.74 (0.5H, t, J=5.4 Hz), 5.11 (0.5H, dd, J=5.9, 3.2 Hz), 6.61–6.63 (0.5H, m), 6.67–6.74 (1.5H, m), 7.00–7.04 (1H, m).

Methyl (S)-2-tert-Butoxycarbonyl-7-(4-methylpentyloxy)-3,4-dihydro-1H-isoquinoline-3-carboxylate (3n)

Compound 3n was synthesized from 2³⁷⁾ and 4-methylpentylbromide according to the procedure for the synthesis of 3j. Yield 87%. A pale brown oil. ¹H-NMR (CDCl₃) δ: 0.91 (6H, t, J=6.6 Hz), 1.28–1.35 (2H, m), 1.45–1.52 (9H, m), 1.57–1.63 (1H, m), 1.73–1.80 (2H, m), 3.02–3.22 (2H, m), 3.61–3.63 (3H, m), 3.90 (2H, t, J=6.6 Hz), 4.41–4.50 (1H, m), 4.64–4.74 (1.5H, m), 5.08–5.15 (0.5H, m), 6.62–6.72 (2H, m), 7.02 (1H, d, J=8.0 Hz).

Methyl (S)-2-tert-Butoxycarbonyl-7-[3-(3-trifluoromethylphenyl)propoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (3t)

PPh₃ (1.40 g, 5.34 mmol) and 2.2 M diethyl azodicarboxylate (DEAD) in toluene (2.4 mL, 5.3 mmol) were added to a solution of 3-(3-trifluoromethylphenyl)propan-1-ol⁵²⁾ (911 mg, 4.46 mmol) and 2³⁷⁾ (1.37 g, 4.46 mmol) in THF (10 mL), and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 12t (873 mg, 40% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 1.35–1.61 (9H, m), 2.05–2.16 (2H, quintet, J=7.8 Hz), 2.86 (2H, t, J=7.8 Hz), 3.05–3.27 (2H, m), 3.58–3.68 (3H, m), 3.93 (2H, t, J=7.8 Hz), 4.39–4.54 (1.2H, m), 4.60–4.78 (1.8H, m), 6.58–6.77 (2H, m), 6.90–7.07 (1H, m), 7.30–7.50 (4H, m).

Methyl (S)-[(E,E)-2-Hexa-2,4-dienoyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (6)

Sorbic acid (1.30 g, 11.6 mmol) and EDC·HCl (2.59 g, 13.5 mmol) were added to a solution of compound 4³⁸⁾ (2.00 g, 9.65 mmol) in CH₂Cl₂ (30 mL), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was washed with saturated NaHCO₃ solution and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 6 (2.71 g, 90% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 1.82–1.89 (3H, m), 3.00–3.28 (2H, m), 3.58–3.63 (3H, m), 4.52 (0.3H, d, J=17.3 Hz), 4.66–4.79 (1.3H, m), 4.89–4.97 (0.7H, m), 5.50–5.56 (0.7H, m), 5.84–6.04 (0.7H, br), 6.04–6.38 (3H, m), 6.46–6.60 (0.3H, br), 6.60–6.72 (2H, m), 3.97–7.03 (1H, m), 7.28–7.38 (1H, m).

Methyl (S)-[(E)-3-Furan-2-ylacryloyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (7)

Compound 7 was synthesized from 4³⁸⁾ and (E)-3-furan-2-ylacrylic acid according to the procedure for the synthesis of 6. Yield 85%. A white solid. ¹H-NMR (CDCl₃) δ: 3.03–3.34 (2H, m), 3.60–3.64 (3H, m), 4.54–4.62 (0.3H, m), 4.75–4.90 (1.4H, m), 4.96–5.07 (0.6H, m), 5.36 (0.7H, s), 5.55–5.60 (0.7H, m), 5.86 (0.3H, s), 6.45–6.50 (1H, m), 6.56–6.61 (1H, m), 6.56–6.73 (2.3H, m), 6.90 (0.7H, d, J=15.1 Hz), 6.98–7.05 (1H, m), 7.43–7.56 (2H, m).

Methyl (S)-7-Hydroxy-[(E)-2-(3-phenylacryloyl)]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (8)

Compound 8 was synthesized from 4³⁸⁾ and (E)-3-phenylacrylic acid according to the procedure for the synthesis of 6. Yield 85%. A white solid. ¹H-NMR (CDCl₃) δ: 3.03–3.32 (2H, m), 3.62 (3H, s), 4.57 (0.3H, d, J=17.1 Hz), 4.76–4.91 (1.4H, m), 4.97–5.06 (0.7H, m), 5.54–5.61 (0.6H, m), 5.88 (0.7H, s), 6.31 (0.3H, s), 6.64–6.79 (2.3H, m), 6.95–7.05 (1.7H, m), 7.33–7.41 (3H, m), 7.47–7.58 (2H, m), 7.69–7.78 (1H, m).

Methyl (S)-7-Hydroxy-2-[(E)-3-thiophen-2-ylacryloyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (9)

Compound 9 was synthesized from 4³⁸⁾ and (E)-3-thiophen-2-ylacrylic acid⁵³⁾ according to the procedure for the synthesis of 6. Yield 81%. A white solid. ¹H-NMR (CDCl₃) δ: 3.03–3.34 (2H, m), 3.60–3.65 (3H, m), 4.52–4.60 (0.3H, m), 4.76–4.85 (1.4H, m), 4.95–5.03 (0.6H, m), 5.53–5.59 (0.7H, m), 5.76–6.25 (0.7H, s), 6.25–6.40 (0.3H, s), 6.40 (0.4H, d, J=15.1 Hz), 6.65–6.73 (2H, m), 6.77 (0.6H, d, J=14.9 Hz), 6.98–7.07 (2H, m), 7.22–7.28 (1H, m), 7.32–7.37 (1H, m), 7.83–7.91 (1H, m).

Methyl (S)-7-Hydroxy-2-[(E)-3-thiazol-2-ylacryloyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (10)

(E)-3-Thiazol-2-ylacrylic acid⁵⁴⁾ (519 mg, 3.34 mmol), EDC·HCl (699 mg, 3.65 mmol) and Et₃N (0.55 mL, 4.0 mmol) were added to a solution of compound 5³⁹⁾ (740 mg, 3.04 mmol) in CH₂Cl₂ (10 mL), and the reaction mixture was stirred at room temperature for 45 min. The reaction mixture was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 10 (950 mg, 91% yield) as a white solid. ¹H-NMR (CDCl₃) δ: 3.02–3.35 (2H, m), 3.63 (3H, s), 4.54–4.62 (0.2H, m), 4.77–5.10 (2.1H, m), 5.55–5.60 (0.7H, m), 5.97–6.03 (1H, br), 6.64–6.70 (2H, m), 6.97–7.06 (1H, m), 7.42–7.54 (2H, m), 7.76–7.84 (1H, m), 7.88–7.98 (1H, m).

Methyl (S)-7-[2-(2,5-Dimethyloxazol-4-yl)acetylamino]-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11a)

EDC·HCl (375 mg, 1.80 mmol) were added to a solution of compound 1³⁶⁾ (500 mg, 1.63 mmol) and 2-(2,5-dimethyloxazol-4-yl)acetic acid⁵⁵⁾ (266 mg, 1.71 mmol) in CH₂Cl₂ (5 mL), and the reaction mixture was stirred at room temperature for 40 min. The reaction mixture was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure to give 3a (crude).

Compound 3a was dissolved in HCO₂H (4 mL), to which 8.6 M HCl in i-PrOH (0.31 mL, 2.7 mmol) was added under ice-cooling, and followed by stirring at the same temperature for 30 min. The reaction mixture was basified with saturated aqueous NaHCO₃ solution, which was extracted twice with CHCl₃. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

Sorbic acid (192 mg, 1.71 mmol) and EDC·HCl (344 mg, 1.79 mmol) were added to a solution of the residue obtained in CH₂Cl₂ (5 mL), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was washed with water and saturated aqueous NaHCO₃ solution, and then dried over Na₂SO₄. The solvent was removed under reduced pressure and the residue obtained was purified by column chromatography to give 11a (585 mg, y=82% for 3 steps) as a pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.83–1.90 (3H, m), 2.26 (3H, s), 2.45 (3H, s), 3.04–3.15 (1H, m), 3.20–3.27 (1H, m), 3.47 (2H, s), 3.58–3.61 (3H, m), 4.69–4.86 (2H, m), 4.89–5.00 (0.4H, m), 5.52–5.59 (0.6H, m), 6.07–6.38 (3H, m), 7.04–7.12 (1H, m), 7.14–7.19 (1H, m), 7.28–7.40 (1.6H, m), 7.51–7.56 (0.4H, m), 8.93–9.01 (1H, m).

Methyl (S)-2-[(2E,4E)-Hexa-2,4-dienoyl]-7-[2-(2-isopropyl-5-methyloxazol-4-yl)acetylamino]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11b)

Compound 3b (440 mg, 0.933 mmol) was dissolved in HCO₂H (2 mL), to which 8.6 M HCl in i-PrOH (0.33 mL, 2.8 mmol) was added under ice-cooling, and followed by stirring at the same temperature for 30 min. The reaction mixture was basified with saturated aqueous NaHCO₃ solution, which was extracted twice with CHCl₃. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

Sorbic acid (113 mg, 1.01 mmol) and EDC·HCl (211 mg, 1.10 mmol) were added to a solution of the residue obtained in CH₂Cl₂ (2 mL), and the reaction mixture was stirred at room temperature for 50 min. The reaction mixture was washed with water, and then dried over Na₂SO₄. The solvent was removed under reduced pressure and the residue obtained was purified by column chromatography to give 11b (585 mg, y=78% for 2 steps) as a pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.37 (6H, d, J=7.1 Hz), 1.80–1.90 (3H, m), 2.26 (3H, s), 3.00–3.13 (2H, m), 3.19–3.30 (1H, m), 3.48 (2H, s), 3.57–3.62 (3H, m), 4.51–4.85 (2H, m), 4.89–5.00 (0.4H, m), 5.50–5.60 (0.6H, m), 6.06–6.38 (3H, m), 7.03–7.12 (2H, m), 7.28–7.49 (1.4H, m), 7.55–7.63 (0.6H, m), 9.26–9.37 (1H, m).

Methyl (S)-7-[2-(2-Acetoxymethyl-5-methyloxazol-4-yl)acetylamino]-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11c)

Compound 11c was synthesized from 3c according to the procedure for the synthesis of 11b. Yield 62% for 2 steps. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.78–1.90 (3H, m), 2.16 (3H, s), 2.33 (3H, s), 3.02–3.34 (2H, m), 3.52 (2H, s), 3.59 (3H, s), 4.49–4.60 (0.3H, m), 4.68–4.85 (1.7H, m), 4.89–5.00 (0.4H, m), 5.13 (2H, s), 5.50–5.59 (0.6H, m), 6.05–6.37 (3H, m), 7.03–7.20 (2H, m), 7.22–7.43 (1.3H, m), 7.49–7.59 (0.7H, m), 8.71–8.85 (1H, m).

Methyl (S)-7-Dodecanoylamino-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11d)

Compound 11d was synthesized from 3d according to the procedure for the synthesis of 11b. Yield quant. A colorless oil. ¹H-NMR (CDCl₃) δ: 0.88 (3H, t, J=7.1 Hz), 1.20–1.41 (16H, m), 1.65–1.78 (2H, m), 1.87 (3H, d, J=6.6 Hz), 2.34 (2H, t, J=7.6 Hz), 3.02–3.35 (2H, m), 3.60 (3H, s), 4.48–4.61 (0.3H, m), 4.68–4.85 (1.5H, m), 4.90–5.00 (0.5H, m), 5.52–5.60 (0.7H, m), 6.07–6.37 (3H, m), 7.03–7.12 (1.5H, m), 7.19–7.63 (2.5H, m).

Methyl (S)-7-Tetradecanoylamino-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11e)

Compound 11e was synthesized from 3e according to the procedure for the synthesis of 11b. Yield 83% for 2 steps. A yellow oil. ¹H-NMR (CDCl3) δ: 0.88 (3H, t, J=6.6 Hz), 1.20–1.37 (20H, m), 1.65–1.77 (2H, m), 1.82–1.90 (3H, m), 2.30–2.37 (2H, m), 3.04–3.34 (2H, m), 3.60 (3H, s), 4.50–5.02 (2H, m), 5.53–5.60 (1H, m), 6.07–6.37 (3H, m), 7.04–7.64 (5H, m).

Methyl (S)-7-(10-Formyloxydecanoylamino)-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11f)

Compound 11f was synthesized from 3f according to the procedure for the synthesis of 11b. Yield 35% for 2 steps. A colorless oil. ¹H-NMR (CDCl₃) δ: 1.26–1.40 (10H, m), 1.60–1.77 (4H, m), 1.83–1.89 (3H, m), 2.30–2.40 (2H, m), 3.04–3.33 (2H, m), 3.60 (3H, s), 4.16 (2H, t, J=6.6 Hz), 4.51–5.01 (2.4H, m), 5.53–5.59 (0.6H, m), 6.08–6.37 (3H, m), 7.05–7.64 (5H, m), 8.05 (1H, s).

Methyl (S)-7-(2,2-Dimethyldecanoylamino)-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11g)

Compound 11g was synthesized from 3g according to the procedure for the synthesis of 11b. Yield 78% for 2 steps. ¹H-NMR (CDCl₃) δ: 0.83–0.90 (3H, m), 1.22–1.62 (20H, m), 1.82–1.90 (3H, m), 3.04–3.35 (2H, m), 3.61 (3H, s), 4.50–5.03 (1H, m), 5.53–5.60 (2H, m), 6.07–6.37 (3H, m), 7.05–7.66 (5H, m).

(S)-7-(2,2-Dimethyldodecanoylamino)-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11h)

Compound 11h was synthesized from 3h according to the procedure for the synthesis of 11b. Yield 74% for 2 steps. ¹H-NMR (CDCl₃) δ: 0.87 (3H, t, J=6.8 Hz), 1.18–1.33 (22H, m), 1.53–1.61 (2H, m), 1.80–1.92 (3H, m), 3.00–3.35 (2H, m), 3.61 (3H, s), 4.50–5.03 (2.4H, m), 5.50–5.60 (0.6H, m), 6.06–6.38 (3H, m), 7.04–7.13 (1.5H, m), 7.23–7.45 (3H, m), 7.57–7.69 (0.5H, m).

Methyl (S)-7-[(Biphenyl-4-carbonyl)amino]-2-[(2E,4E)-hexadienoyl]-1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (11i)

Compound 11i was synthesized from 3i according to the procedure for the synthesis of 11b. Yield 85% for 2 steps. A colorless oil. ¹H-NMR (CDCl₃) δ: 1.82–1.89 (3H, m), 3.07–3.22 (1H, m), 3.24–3.37 (1H, m), 3.61 (3H, s), 4.54–4.63 (0.25H, m), 4.74–4.90 (1.5H, m), 4.94–5.04 (0.5H, m), 5.57–5.61 (0.75H, m), 6.08–6.38 (3H, m), 7.11–7.18 (1H, m), 7.31–7.43 (2H, m), 7.45–7.51 (2H, m), 7.55–7.57 (0.25H, br), 7.60–7.64 (2H, m), 7.68–7.73 (2H, m), 7.74–7.76 (0.75H, br), 7.91–7.95 (3H, m).

Methyl (S)-7-(Biphenyl-4-ylmethoxy)-2-[(2E,4E)-hexadienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11j)

Compound 11j was synthesized from 3j according to the procedure for the synthesis of 11b. Yield 65% for 2 steps. A white solid. ¹H-NMR (CDCl₃) δ: 1.83–1.88 (3H, m), 3.03–3.31 (2H, m), 3.61 (3H, s), 4.55 (0.3H, d, J=17.6 Hz), 4.75 (0.7H, d, J=15.4 Hz), 4.78 (0.7H, d, J=15.4 Hz), 4.91–4.51 (0.6H, m), 5.07 (2H, s), 5.54–5.58 (0.7H, m), 6.08–6.37 (3H, m), 6.74–6.78 (0.7H, m), 6.80–6.88 (1.3H, m), 7.04–7.11 (1H, m), 7.30–7.39 (2H, m), 7.42–7.51 (4H, m), 7.57–7.63 (4H, m).

Methyl (S)-2-[(2E,4E)-Hexadienoyl]-7-(7-methyloctyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (11k)

Compound 11k was synthesized from 3k according to the procedure for the synthesis of 11b. Yield 80% for 2 steps. A colorless oil. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.14–1.21 (2H, m), 1.25–1.37 (4H, m), 1.39–1.48 (2H, m), 1.48–1.58 (1H, m), 1.72–1.80 (2H, m), 1.83–1.89 (3H, m), 3.02–3.29 (2H, m), 3.60 (3H, s), 3.91 (2H, t, J=6.6 Hz), 4.54 (0.3H, d, J=18.1 Hz), 4.73 (0.7H, d, J=15.4 Hz), 4.78 (0.7H, d, J=15.4 Hz), 4.90–4.99 (0.6H, m), 5.54 (0.7H, dd, J=5.8, 3.4 Hz), 6.08–6.37 (3H, m), 6.63–6.67 (0.7H, m), 6.69–6.76 (1.3H, m), 7.01–7.07 (1H, m), 7.30–7.38 (1H, m).

(E)-7-Methyloct-2-enal (30)

One molar diisobutylaluminium hydride (DIBAL) solution in toluene (14.7 mL, 14.7 mmol) was added to a solution of ethyl (E)-7-methyloct-2-enoate⁵⁶⁾ (900 mg, 4.88 mmol) in CH₂Cl₂ (30 mL) at −10°C, which was stirred at the same temperature for 30 min. After the addition of saturated NH₄Cl solution, the mixture was stirred at room temperature for 30 min. The precipitate was removed by filtration, and the organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure.

MnO₂ (8.49 g, 97.7 mmol) was added to a solution of the residue obtained in CH₂Cl₂ (30 mL), and the mixture was stirred at room temperature for 20 min and 40°C for 4 h. The precipitate was removed by filtration, and the solvent was removed under reduced pressure to give 30 (520 mg, 76% yield for 2 steps) as a pale yellow oil. ¹H-NMR (CDCl₃) δ: 0.89 (6H, d, J=6.6 Hz), 1.17–1.29 (2H, m), 1.47–1.60 (3H, m), 2.29–2.34 (2H, m), 6.19 (1H, dd, J=15.6, 7.8 Hz), 6.85 (1H, dt, J=15.6, 6.8 Hz), 9.50 (1H, d, J=7.8 Hz).

Ethyl (E,E)-9-Methyldeca-2,4-dienoate (31)

A 60% suspension of NaH in mineral oil (163 mg, 6.8 mmol) was added portionwise to a stirred solution of ethyl (diethoxyphosphoryl)acetate (914 mg, 4.08 mmol) in THF (10 mL) under ice-cooling, which was stirred at the same temperature for 10 min. After the addition of 30 (520 mg, 3.71 mmol) in THF (5 mL) under ice-cooling, the reaction mixture was stirred at the same temperature for 15 min. The mixture was acidified with saturated aqueous NH₄Cl solution, followed by extraction with AcOEt and the organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure and the residue obtained was purified by silica gel column chromatography to give 31 (448 mg, 57% yield) as a pale yellow oil. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.15–1.22 (2H, m), 1.29 (3H, t, J=7.1 Hz), 1.37–1.47 (2H, m), 1.48–1.59 (1H, m), 2.11–2.18 (2H, m), 4.19 (2H, d, J=7.1 Hz), 5.78 (1H, d, J=15.4 Hz), 6.07–6.21 (2H, m), 7.21–7.29 (1H, m).

(E,E)-9-Methyldeca-2,4-dien-1-ol (32)

One molar DIBAL solution in toluene (6.4 mL, 6.4 mmol) was added to a solution of 31 (448 mg, 2.13 mmol) in THF (15 mL) at −10°C, which was stirred at the same temperature for 25 min. After the addition of saturated NH₄Cl solution and water, the precipitate was removed by filtration, and the organic layer was then dried over Na₂SO₄. The solvent was removed under reduced pressure to give 32 (344 mg, 96% yield) as a yellow oil. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.13–1.21 (2H, m), 1.23–1.32 (1H, m), 1.34–1.43 (2H, m), 1.47–1.55 (1H, m), 2.03–2.11 (2H, m), 4.13–4.19 (2H, m), 5.66–5.77 (2H, m), 5.99–6.09 (1H, m), 6.16–6.26 (1H, m).

Methyl (S)-[(E,E)-2-Hexa-2,4-dienoyl]-7-(9-methyldeca-2,4-dienyloxy)-1,2,3,4-tetra-hydroisoquinoline-3-carboxylate (11l)

PPh₃ (551 mg, 2.10 mmol) and 2.2 M DEAD in toluene (0.82 mL, 1.8 mmol) were added to a solution of 6 (452 mg, 1.50 mmol) and 32 (260 mg, 1.55 mmol) in THF (10 mL) under ice-cooling, and the mixture was stirred at the same temperature for 2 h. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 11l (105 mg, 16% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.11–1.20 (2H, m), 1.33–1.44 (2H, m), 1.47–1.55 (1H, m), 1.82–1.88 (3H, m), 2.04–2.11 (2H, m), 3.00–3.29 (2H, m), 3.60 (3H, s), 4.47–4.57 (2.3H, m), 4.68–4.82 (1.5H, m), 4.90–4.99 (0.5H, m), 5.51–5.58 (0.7H, m), 5.69–5.79 (2H, m), 6.01–6.37 (5H, m), 6.63–6.79 (2H, m), 7.00–7.08 (1H, m), 7.29–7.38 (1H, m).

Methyl (S)-[2-(3-Furan-2-ylacryloyl)-7-(7-methyloctyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12k)

Compound 3k (1.22 g, 2.81 mmol) was dissolved in HCO₂H (7 mL), to which 8.3 M HCl in i-PrOH (1.0 mL, 8.3 mmol) was added under ice-cooling, and followed by stirring at the same temperature for 1 h. The reaction mixture was basified with saturated aqueous NaHCO₃ solution, which was extracted twice with CHCl₃. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure to give a crude intermediate (960 mg) as a brown solid.

3-Furan-2-ylacryloylic acid (200 mg, 1.45 mmol) and EDC·HCl (332 mg, 1.73 mmol) were added to a solution of the crude intermediate (480 mg, 1.41 mmol) in CH₂Cl₂ (10 mL), and the reaction mixture was stirred at room temperature for 14 h. The reaction mixture was washed with H₂O solution and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 12k (530 mg, 83% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.14–1.21 (2H, m), 1.25–1.37 (4H, m), 1.39–1.58 (3H, m), 1.72–1.81 (2H, m), 3.05–3.34 (2H, m), 3.62 (3H, s), 3.92 (2H, t, J=6.3 Hz), 4.58 (0.25H, d, J=18.0 Hz), 4.81 (0.75H, d, J=15.4 Hz), 4.89 (0.75H, d, J=15.4 Hz), 4.97–5.06 (0.5H, m), 5.57 (0.75H, dd, J=5.8, 3.6 Hz), 6.44–6.49 (1H, m), 6.55–6.60 (1H, m), 6.66–6.77 (2.25H, m), 7.00 (0.75H, d, J=14.9 Hz), 7.03–7.08 (1H, m), 7.43–7.56 (2H, m).

Methyl (S)-2-[(E)-3-Furan-2-ylacryloyl]-7-[(E,E)-9-methyldeca-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12l)

PPh₃ (375 mg, 1.43 mmol) and 2.2 M DEAD in toluene (0.28 mL, 0.73 mmol) were added to a solution of 7 (452 mg, 1.50 mmol) and 30 (260 mg, 1.55 mmol) in THF (10 mL) under ice-cooling, and the mixture was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 12l (144 mg, 27% yield) as a colorless oil. ¹H-NMR (DMSO-d₆) δ: 0.87 (6H, d, J=6.6 Hz), 1.13–1.22 (2H, m), 1.34–1.45 (2H, m), 1.47–1.56 (1H, m), 2.01–2.12 (2H, m), 3.02–3.37 (2H, m), 3.62 (3H, s), 4.47–4.63 (2H, m), 4.76–5.11 (3H, m), 5.55–5.62 (1H, m), 5.69–5.82 (1H, m), 6.00–6.13 (0.7H, m), 6.23–6.38 (1.3H, m), 6.43–6.51 (1H, m), 6.54–6.63 (1H, m), 6.65–6.82 (2H, m), 6.91 (1H, d, J=15.2 Hz), 7.01–7.11 (1H, m), 7.41–7.59 (2H, m).

Methyl (S)-2-[(E)-3-Furan-2-ylacryloyl]-7-nonyloxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12m)

Compound 12m was synthesized from 7 according to a similar procedure for the synthesis of 12l. Yield 49%. A yellow oil. ¹H-NMR (CDCl₃) δ: 0.88 (3H, t, J=6.6 Hz), 1.23–1.49 (12H, m), 1.72–1.81 (2H, m), 3.04–3.34 (2H, m), 3.62 (3H, s), 3.92 (2H, t, J=6.5 Hz), 4.58 (0.3H, d, J=17.8 Hz), 4.77–4.93 (2H, m), 5.55–5.61 (0.7H, m), 6.43–6.50 (1H, m), 6.55–6.61 (1H, m), 6.65–6.77 (2.2H, m), 6.91 (0.8H, d, J=15.1 Hz), 7.03–7.8 (1H, m), 7.42–7.56 (2H, m).

Methyl (S)-2-(3-Furan-2-ylacryloyl)-7-(4-methylpentyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12n)

Compound 12n was synthesized from 3n according to the procedure for the synthesis of 12k. Yield 88% for 2 steps. ¹H-NMR (CDCl₃) δ: 0.92 (6H, d, J=6.6 Hz), 1.29–1.36 (2H, m), 1.56–1.64 (1H, m), 1.72–1.83 (2H, m), 3.03–3.34 (2H, m), 3.62 (3H, s), 3.91 (2H, t, J=6.6 Hz), 4.55–5.08 (2.3H, m), 5.55–5.60 (0.7H, m), 6.44–6.49 (1H, m), 6.55–6.61 (1H, m), 6.66–6.77 (2.3H, m), 6.91 (0.7H, d, J=15.1 Hz), 7.03–7.09 (1H, m), 7.43–7.59 (2H, m).

Methyl (S)-7-[(E,E)-3,7-Dimethylocta-2,6-dienyloxy)-2-[(E)-3-furan-2-ylacryloyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12o)

Compound 12o was synthesized from 6 and (E,E)-3,7-dimethylocta-2,6-dien-1-ol⁵⁷⁾ according to the procedure for the synthesis of 12l. Yield 80%. ¹H-NMR (CDCl₃) δ: 1.61 (3H, s), 1.69 (3H, s), 1.74 (3H, s), 2.06–2.18 (4H, m), 3.05–3.36 (2H, m), 3.63 (3H, s), 4.48–4.62 (2.3H, m), 4.79–4.94 (1.5H, m), 4.98–5.14 (1.5H, m), 5.45–5.52 (1H, m), 5.56–5.62 (0.7H, m), 6.45–6.50 (1H, m), 6.56–6.62 (1H, m), 6.67–6.81 (2.3H, m), 6.92 (0.7H, d, J=15.1 Hz), 7.03–7.10 (1H, m), 7.43–7.57 (2H, m).

Ethyl (E)-7-Isopropoxyhept-2-enoate (33)

Dimethyl sulfoxide (DMSO) (5.3 mL, 75 mmol), i-Pr₂NEt (6.3 mL, 37 mmol), and SO₃·pyridine (5.93 g, 37.3 mmol) were added to a stirred solution of 5-isopropoxypentan-1-ol⁵⁸⁾ (2.18 g, 14.9 mmol) in CH₂Cl₂ (35 mL) under ice-cooling, followed by stirring at room temperature for 15 min. After the addition of 1.0 M aqueous HCl solution, the mixture was extracted twice with CH₂Cl₂, washed with water, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

A 60% suspension of NaH in mineral oil (163 mg, 6.8 mmol) was added portionwise to a stirred solution of ethyl (diethoxyphosphoryl)acetate (3.34 g, 14.9 mmol) in THF (30 mL) under ice-cooling, which was stirred at the same temperature for 15 min. After the addition of the residue obtained in THF (10 mL) under ice-cooling, the reaction mixture was stirred at the same temperature for 30 min. The mixture was acidified with saturated aqueous NH₄Cl solution, followed by extraction with AcOEt, and the organic layer was washed with saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure and the residue obtained was purified by silica gel column chromatography to give 33 (2.56 g, 80% yield for 2 steps) as a colorless oil. ¹H-NMR (CDCl₃) δ: 1.14 (6H, d, J=6.1 Hz), 1.28 (3H, t, J=7.1 Hz), 1.48–1.63 (4H, m), 2.18–2.27 (2H, m), 3.40 (2H, t, J=6.0 Hz), 3.48–3.57 (1H, m), 4.18 (2H, q, J=7.1 Hz), 5.81 (1H, d, J=15.6 Hz), 6.96 (1H, dt, J=15.6, 7.1 Hz).

(E)-7-Isopropoxyhept-2-en-1-ol (34)

Compound 34 was synthesized from 33 according to the procedure for the synthesis of 32. Yield 38%. ¹H-NMR (CDCl₃) δ: 1.15 (6H, d, J=6.1 Hz), 1.23–1.32 (1H, m), 1.40–1.50 (2H, m), 1.52–1.60 (2H, m), 2.04–2.12 (2H, m), 3.40 (2H, t, J=6.5 Hz), 3.50–3.59 (1H, m), 4.05–4.11 (2H, m), 5.59–5.74 (2H, m).

Methyl (S)-2-(3-Furan-2-ylacryloyl)-7-[(E)-7-isopropoxyhept-2-enyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12p)

Compound 12p was synthesized from 7 and 34 according to the procedure for the synthesis of 12l. Yield 39%. ¹H-NMR (CDCl₃) δ: 1.15 (6H, d, J=6.1 Hz), 1.42–1.52 (2H, m), 1.52–1.62 (2H, m), 2.08–2.15 (2H, m), 3.03–3.35 (2H, m), 3.40 (2H, t, J=6.5 Hz), 3.48–3.59 (1H, m), 3.61 (3H, s), 4.43 (2H, d, J=5.6 Hz), 4.58 (0.3H, d, J=17.5 Hz), 4.77–5.08 (2H, m), 5.55–5.61 (0.7H, m), 5.65–5.74 (1H, m), 5.77–5.88 (1H, m), 6.44–6.50 (1H, m), 6.55–6.61 (1H, m), 6.65–6.78 (2.3H, m), 6.91 (0.7H, d, J=14.9 Hz), 7.03–7.08 (1H, m), 7.42–7.56 (2H, m).

Methyl (S)-2-[(E)-3-Furan-2-ylacryloyl]-7-[(E,E)-5-phenylpenta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12q)

Compound 12q was synthesized from 7 and (E,E)-5-phenylpenta-2,4-dien-1-ol⁵⁹⁾ according to the procedure for the synthesis of 12l. Yield 63%. A colorless oil. ¹H-NMR (CDCl₃) δ: 3.05–3.36 (2H, m), 3.62 (3H, s), 4.21 (1H, q, J=7.1 Hz), 4.55–4.67 (2H, m), 4.77–4.94 (1.5H, m), 4.97–5.10 (0.5H, m), 5.55–5.61 (1H, m), 6.44–6.62 (3H, m), 6.67–6.86 (3H, m), 6.91 (1H, d, J=15.1 Hz), 7.04–7.11 (1H, m), 7.18–7.58 (8H, m).

Ethyl (2E,4E)-5-(3-Trifluoromethylphenyl)penta-2,4-dienoate (35)

Compound 35 was synthesized from (E)-3-(3-trifluoromethylphenyl)propenal⁶⁰⁾ according to the procedure for the synthesis of 31. Yield 49%. A white solid. ¹H-NMR (CDCl₃) δ: 1.32 (3H, t, J=7.1 Hz), 4.23 (2H, t, J=7.1 Hz), 6.04 (1H, d, J=15.1 Hz), 6.88–6.97 (2H, m), 7.40–7.49 (2H, m), 7.54 (1H, d, J=7.6 Hz), 7.62 (1H, d, J=7.6 Hz), 7.69 (1H, s).

(2E,4E)-5-(3-Trifluoromethylphenyl)penta-2,4-dien-1-ol (36)

Compound 36 was synthesized from 35 according to the procedure for the synthesis of 32. Yield 49%. A white solid. ¹H-NMR (CDCl₃) δ: 1.40 (1H, t, J=5.4 Hz), 4.25–4.30 (2H, m), 6.00–6.06 (1H, m), 6.41–6.47 (1H, m), 6.56 (1H, d, J=15.6 Hz), 6.84 (1H, dd, J=15.6, 10.5 Hz), 7.40–7.48 (2H, m), 7.51–7.56 (1H, m), 7.62 (1H, s).

Methyl (S)-2-{(E)-3-Furan-2-yl-acryloyl}-7-[(2E,4E)-5-(3-trifluoromethylphenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12r)

Compound 12r was synthesized from 7 and 36 according to the procedure for the synthesis of 12l. Yield 48%. A colorless oil. ¹H-NMR (CDCl₃) δ: 3.03–3.35 (2H, m), 3.62 (3H, m), 4.59–4.65 (2H, m), 4.76–5.08 (2.3H, m), 5.55–5.63 (0.7H, m), 6.00–6.12 (1H, m), 6.45–6.63 (3H, m), 6.67–6.94 (4H, m), 7.04–7.12 (1H, m), 7.41–7.56 (6H, m), 7.63 (1H, s).

Methyl (S)-2-(3E)-[Furan-2-yl-acryloyl]-7-[5-(4-trifluoromethylphenyl)-(2E,4E)-penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12s)

PPh₃ (382 mg, 1.42 mmol) and diisopropyl azodicarboxylate (0.28 mL, 1.4 mmol) were added to a solution of (2E,4E)-5-(4-trifluoromethylphenyl)penta-2,4-dien-1-ol⁶¹⁾ (270 mg, 1.18 mmol) and 7 (387 mg, 1.18 mmol) in THF (5 mL) under ice-cooling, and the mixture was stirred at the same temperature for 1 h. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 12s (130 mg, 21% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 3.05–3.35 (2H, m), 3.62 (3H, s), 4.57–4.63 (2H, m), 4.80–5.05 (2.2H, m), 5.56–5.62 (0.8H, m), 6.02–6.12 (1H, m), 6.45–6.63 (4H, m), 6.67–6.94 (4H, m), 7.05–7.11 (1H, m), 7.44–7.58 (6H, m).

Methyl (S)-2-(3-Furan-2-ylacryloyl)-7-[3-(3-trifluoromethylphenyl)propoxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12t)

Compound 12t was synthesized from 3t according to the procedure for the synthesis of 12k. Yield 60% for 2 steps. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 2.08–2.17 (2H, m), 2.87 (2H, t, J=7.6 Hz), 3.05–3.37 (2H, m), 3.62 (3H, s), 3.94 (2H, t, J=6.1 Hz), 4.54–4.63 (0.4H, m), 4.54–4.63 (1.4H, m), 4.97–5.10 (0.6H, m), 5.56–5.63 (0.6H, m), 6.45–6.51 (1H, m), 6.56–6.63 (1H, m), 6.65–6.95 (2H, m), 6.91 (1H, d, J=15.1 Hz), 7.04–7.10 (1H, m), 7.36–7.57 (6H, m).

Ethyl (2E,4E)-5-(2,4,6-Trifluorophenyl)penta-2,4-dienoate (37)

Compound 37 was synthesized from (2E,4E)-3-(2,4,6-trifluorophenyl)propenal⁶²⁾ according to the procedure for the synthesis of 31. Yield 80%. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.32 (3H, t, J=7.3 Hz), 4.23 (2H, t, J=7.3 Hz), 5.92–6.04 (1H, m), 6.64–6.72 (2H, m), 6.84 (1H, d, J=16.1 Hz), 7.10 (1H, dd, J=16.1, 11.2 Hz), 7.32–7.43 (1H, m).

(2E,4E)-5-(2,4,6-Trifluorophenyl)penta-2,4-dien-1-ol (38)

Compound 38 was synthesized from 37 according to the procedure for the synthesis of 32. Yield 76%. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.38 (1H, t, J=5.8 Hz), 4.27 (2H, t, J=5.8 Hz), 5.98–6.04 (1H, m), 6.38–6.50 (2H, m), 6.63–6.68 (2H, m), 7.01 (1H, dd, J=15.9, 10.5 Hz).

Methyl (S)-2-{(E)-3-Furan-2-ylacryloyl}-7-[(2E,4E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12u)

Compound 12u was synthesized from 7 and 38 according to the procedure for the synthesis of 12s. Yield 82%. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 3.04–3.38 (2H, m), 3.62 (3H, s), 4.55–4.65 (2H, m), 4.77–4.95 (1.5H, m), 4.97–5.10 (0.5H, m), 5.55–5.64 (1H, m) 5.95–6.12 (1H, m), 6.43–6.83 (3H, m), 6.92 (1H, d, J=15.1 Hz), 6.97–7.13 (2H, m), 7.42–7.59 (2H, m).

Ethyl (2E,4E)-5-Thiazol-2-ylpenta-2,4-dienoate (39)

Compound 39 was synthesized from (E)-3-thiazol-2-ylpropenal according to the procedure for the synthesis of 31. Yield 96%. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.32 (3H, t, J=7.2 Hz), 4.23 (2H, q, J=7.2 Hz), 6.11 (1H, d, J=15.4 Hz), 7.07 (1H, d, J=15.4 Hz), 7.16 (1H, dd, J=15.4, 10.7 Hz), 7.34 (1H, d, J=3.2 Hz), 7.41 (1H, dd, J=15.4, 10.7 Hz), 7.85 (1H, d, J=3.2 Hz).

(2E,4E)-5-Thiazol-2-ylpenta-2,4-dien-1-ol (40)

Compound 40 was synthesized from 39 according to the procedure for the synthesis of 32. Yield 57%. A pale brown oil. ¹H-NMR (CDCl₃) δ: 1.55–1.61 (1H, m), 4.25–4.31 (2H, m), 6.11 (1H, dt, J=15.1, 5.4 Hz), 6.45 (1H, dd, J=15.1, 10.7 Hz), 6.77 (1H, d, J=15.4 Hz), 7.07 (1H, dd, J=15.4, 10.7 Hz), 7.22 (1H, d, J=3.2 Hz), 7.76 (1H, d, J=3.2 Hz).

Methyl (S)-2-[(E)-3-Furan-2-ylacryloyl]-7-[(2E,4E)-5-thiazol-2-ylpenta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12w)

n-Bu₃P (0.57 mL, 2.3 mmol) and 1,1′-(azodicarbonyl)dipiperidine (ADDP) (587 mg, 2.33 mmol) were added to a solution of 40 (300 mg, 1.79 mmol) and 7 (586 mg, 1.79 mmol) in THF (12 mL) under ice-cooling, and the mixture was stirred for 1.5 h. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 12w (581 mg, 61% yield) as a pale yellow oil. ¹H-NMR (CDCl₃) δ: 3.04–3.36 (2H, m), 3.62 (3H, s), 4.55–4.65 (2.3H, m), 4.79–5.09 (2H, m), 5.55–5.63 (0.7H, m), 6.14 (1H, dt, J=15.3, 5.4 Hz), 6.45–6.61 (3H, m), 6.66–6.82 (3.2H, m), 6.91 (0.8H, d, J=15.1 Hz), 7.04–7.13 (2H, m), 7.23 (1H, d, J=3.2 Hz), 7.44–7.57 (2H, m), 7.77 (1H, d, J=3.2 Hz).

Methyl (S)-7-[(E,E)-9-Methyldeca-2,4-dienyloxy]-2-(3-phenylacryloyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (13l)

Compound 13l was synthesized from 8 and 32 according to a similar procedure for the synthesis of 12w. Yield 67%. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.13–1.22 (2H, m), 1.32–1.44 (2H, m), 1.47–1.57 (1H, m), 2.05–2.12 (2H, m), 3.05–3.34 (2H, m), 3.63 (3H, s), 4.49–4.62 (2.2H, m), 4.79–4.93 (1.5H, m), 4.98–5.08 (0.6H, m), 5.57–5.63 (0.7H, m), 5.70–5.81 (2H, m), 6.07 (1H, dd, J=15.0, 10.6 Hz), 6.32 (1H, dd, J=15.1, 10.6 Hz), 6.67–6.81 (2H, m), 6.96–7.10 (2H, m), 7.34–7.43 (3H, m), 7.48–7.61 (2H, m), 7.67–7.80 (1H, m).

Methyl (S)-2-(3-Phenylacryloyl)-7-[(E,E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (13u)

Compound 13u was synthesized from 8 and 40 according to the procedure for the synthesis of 12w. Yield 68%. ¹H-NMR (CDCl₃) δ: 3.06–3.36 (2H, m), 3.63 (3H, s), 4.56–4.64 (2.2H, m), 4.81–4.95 (1.5H, m), 4.99–5.07 (0.6H, m), 5.56–5.63 (0.7H, m), 6.04 (1H, dt, J=15.4, 5.6 Hz), 6.45–6.55 (2H, m), 6.62–6.82 (4H, m), 6.96–7.12 (2H, m), 7.35–7.43 (3H, m), 7.49–7.62 (2H, m), 7.68–7.80 (1H, m).

Methyl (S)-7-[(E,E)-9-Methyldeca-2,4-dienyloxy]-2-[(E)-3-thiophen-2-ylacryloyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (14l)

Compound 14l was synthesized from 7 and 32 according to the procedure for the synthesis of 12l. Yield 36%. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.12–1.22 (2H, m), 1.33–1.44 (2H, m), 1.47–1.55 (1H, m), 2.06–2.12 (2H, m), 3.03–3.34 (2H, m), 3.62 (3H, s), 4.47–4.62 (2.2H, m), 4.77–4.90 (1.5H, m), 4.95–5.06 (0.5H, m), 5.03–5.62 (0.8H, m), 5.70–5.81 (2H, m), 6.02–6.13 (1H, m), 6.32 (1H, dd, J=15.2, 10.6 Hz), 6.56 (0.2H, d, J=14.9 Hz), 6.68–6.82 (2.8H, m), 7.02–7.08 (2H, m), 7.21–7.27 (1H, m), 7.31–7.37 (1H, m), 7.82–7.91 (1H, m).

Methyl (S)-2-[(E)-3-Thiazol-2-ylacryloyl]-7-[(2E,4E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (15u)

Compound 15u was synthesized from 10 and 40 according to the procedure for the synthesis of 12w. Yield 43%. ¹H-NMR (CDCl₃) δ: 3.06–3.38 (2H, m), 3.63 (3H, s), 4.55–4.64 (2.3H, m), 4.82–5.12 (2H, m), 5.55–5.61 (0.7H, m), 6.04 (1H, dt, J=15.2, 5.6 Hz), 6.45–6.55 (2H, m), 6.62–6.83 (4H, m), 6.98–7.11 (2H, m), 7.41–7.48 (2H, m), 7.76–7.96 (2H, m).

(S)-7-[2-(2,5-Dimethyloxazol-4-yl)acetylamino]-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16a)

One molar aqueous LiOH solution (4.0 mL, 4.0 mmol) was added to a solution of 11a (580 mg, 1.33 mmol) in MeOH (2 mL) and THF (2 mL), which was stirred at room temperature for 15 min. After the addition of aqueous HCl solution, the organic layer was removed under reduced pressure. The residue obtained was extracted with CHCl₃, washed with saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of i-Pr₂O, the precipitate was collected by filtration to give 16a (562 mg, quant.) as a pale yellow solid, mp 168–174°C. ¹H-NMR (DMSO-d₆) δ: 1.78–1.85 (3H, m), 2.23 (3H, s), 2.30 (3H, s), 3.04–3.15 (1H, m), 2.96–3.23 (2H, m), 3.40–3.45 (2H, m), 4.30–4.40 (0.4H, m), 4.57–4.66 (0.6H, m), 4.68–4.83 (0.5H, m), 5.11–5.24 (0.5H, m), 6.08–6.38 (3H, m), 7.0–7.18 (2H, m), 7.28–7.57 (2H, m), 9.98–10.07 (1H, m). IR (ATR) cm⁻¹: 1728, 1651. MS m/z: 422 [M−H]⁻, 446 [M+Na]⁺.

(S)-2-[(2E,4E)-Hexa-2,4-dienoyl]-7-[2-(2-isopropyl-5-methyl-oxazol-4-yl)acetylamino]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid tert-Butylamine Salt (16b)

One molar aqueous LiOH solution (1.2 mL, 1.2 mmol) was added to a solution of 11b (280 mg, 0.583 mmol) in MeOH (3 mL) and THF (3 mL), which was stirred at room temperature for 1 h. After the addition of aqueous HCl solution, the organic layer was removed under reduced pressure. The residue obtained was extracted with AcOEt, washed with saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of tert-butylamine (73 µL, 0.69 mmol) to a solution of the residue obtained in EtOH (3 mL), the mixture was stirred at room temperature for 1 h, and the precipitate was then collected by filtration to give 16b (168 mg, 53% yield for 2 steps) as a pale brown solid, mp 200–217°C. ¹H-NMR (DMSO-d₆) δ: 1.13 (9H, s), 1.21 (6H, d, J=6.8 Hz), 1.71–1.84 (3H, m), 2.22 (3H, s), 2.84–3.01 (3H, m), 3.10–3.30 (2H, m), 4.27–4.37 (1H, m), 4.44–4.54 (0.7H, m), 4.68–4.85 (1H, m), 5.03–5.10 (0.3H, m), 5.98–6.45 (2.7H, m), 6.56–6.64 (0.3H, m), 6.95–7.10 (2.7H, m), 7.19–7.43 (2.3H, m), 9.98–10.06 (1H, m). IR (ATR) cm⁻¹: 1651, 1620, 1549. MS m/z: 452 [M+H]⁺, 474 [M+Na]⁺.

(S)-2-[(2E,4E)-Hexa-2,4-dienoyl]-7-[2-(2-hydroxymethyl-5-methyl-oxazol-4-yl)acetylamino]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16c)

Compound 16c was synthesized from 11c according to the procedure for the synthesis of 16a. Yield 19%. A pale yellow solid, mp 150–152°C. ¹H-NMR (DMSO-d₆) δ: 1.75–1.87 (3H, m), 2.28 (3H, s), 2.94–3.33 (2H, m), 3.47 (2H, s), 4.38 (2H, d, J=5.4 Hz), 4.54–4.94 (2H, m), 5.13–5.27 (1H, m), 5.54 (1H, t, J=5.4 Hz), 6.06–6.72 (3H, m), 7.05–7.20 (2H, m), 7.26–7.59 (2H, m), 10.00–10.12 (1H, m), 11.50–13.60 (1H, br). IR (ATR) cm⁻¹: 1728, 1651. MS m/z: 438 [M−H]⁻, 474 [M+Na]⁺.

(S)-7-Dodecanoylamino-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16d)

Compound 16d was synthesized from 11d according to the procedure for the synthesis of 16a. Yield 76%. A pale yellow amorphous, mp 76–77°C. ¹H-NMR (CDCl₃) δ: 0.88 (3H, t, J=7.1 Hz), 1.18–1.36 (16H, m), 1.56–1.72 (2H, m), 1.85 (3H, d, J=6.6 Hz), 2.29 (2H, t, J=7.1 Hz), 2.80–3.30 (2H, m), 4.30–4.68 (2H, m), 4.84–4.91 (0.3H, m), 5.31–5.42 (0.7H, m), 5.70–6.70 (5H, m), 6.84–7.09 (1.5H, m), 7.20–7.52 (2H, m), 7.90–8.44 (0.5H, m). IR (ATR) cm⁻¹: 1728, 1651. MS m/z: 467 [M−H]⁻, 491 [M+Na]⁺.

(S)-7-Tetradecanoylamino-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16e)

Compound 16e was synthesized from 11e according to the procedure for the synthesis of 16a. Yield 85%. A pale yellow solid, mp 89–91°C. ¹H-NMR (CDCl₃) δ: 0.87 (3H, t, J=6.6 Hz), 1.20–1.32 (20H, m), 1.65–1.76 (2H, m), 1.83–1.91 (3H, m), 2.30–2.39 (2H, m), 2.94–3.35 (2H, m), 4.43–4.85 (2H, m), 5.28–5.35 (1H, m), 6.10–6.35 (3H, m), 6.99–7.67 (5H, m). IR (ATR) cm⁻¹: 2921, 1731, 1648. MS m/z: 495 [M−H]⁻.

(S)-2-[(2E,4E)-Hexa-2,4-dienoyl]-7-(10-hydroxydecanoylamino)-1,2,3,4-tetrahydrosoquinoline-3-carboxylic Acid (16f)

Compound 16f was synthesized from 12f according to the procedure for the synthesis of 16a. Yield 96%. A white solid, mp 78–82°C. ¹H-NMR (DMSO-d₆) δ: 1.22–1.34 (10H, m), 1.35–1.46 (2H, m), 1.52–1.62 (2H, m), 1.78–1.87 (3H, m), 2.23–2.35 (2H, m), 3.04–3.22 (2H, m), 3.32–3.43 (2H, m), 4.26–4.93 (2H, m), 5.15–5.24 (1H, m), 6.08–6.73 (3H, m), 7.08–7.59 (4H, m), 9.77–9.83 (1H, br). IR (ATR) cm⁻¹: 1649. MS m/z: 455 [M−H]⁻.

(S)-7-(2,2-Dimethyldecanoylamino)-2-[(2E,4E)-hexa-2,4-dienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16g)

Compound 16g was synthesized from 11g according to the procedure for the synthesis of 16a. Yield 90%. A yellow solid, mp 90–93°C. ¹H-NMR (CDCl₃) δ: 0.83–0.90 (3H, m), 1.22–1.30 (18H, m), 1.51–1.60 (2H, m), 1.82–1.91 (3H, m), 3.98–3.35 (2H, m), 4.48–4.97 (2H, m), 5.30–5.38 (1H, m), 6.06–6.34 (3H, m), 7.03–7.48 (4H, m), 7.68–7.04 (1H, br). IR (ATR) cm⁻¹: 2923, 1733, 1648. MS m/z: 467 [M−H]⁻, 491 [M+Na]⁺.

(S)-7-(2,2-Dimethyldodecanoylamino)-2-hexa-2,4-dienoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16h)

Compound 16h was synthesized from 11h according to the procedure for the synthesis of 16a. Yield 80%. A white amorphous, mp 69–70°C. ¹H-NMR (CDCl₃) δ: 0.87 (3H, t, J=7.1 Hz), 1.19–1.34 (20H, m), 1.51–1.61 (2H, m), 1.80–1.90 (3H, m), 2.94–3.32 (2H, m), 4.38–4.92 (2.2H, m), 5.36–5.45 (0.8H, m), 6.02–6.32 (3H, m), 6.38–6.94 (2H, br), 6.95–7.18 (2H, m), 7.22–7.43 (2H, m), 7.44–7.55 (1H, m), 7.56–7.65 (1H, m). IR (ATR) cm⁻¹: 1651, 1618. MS m/z: 495 [M−H]⁻, 519 [M+Na]⁺.

(S)-7-[(Biphenyl-4-carbonyl)amino]-2-[(2E,4E)-hexadienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid tert-Butylamine Salt (16i)

One molar aqueous LiOH solution (1.2 mL, 1.2 mmol) was added to a solution of 11i (280 mg, 0.583 mmol) in MeOH (3 mL) and THF (3 mL), which was stirred at room temperature for 1 h. After the addition of aqueous HCl solution, the organic layer was removed under reduced pressure. The residue obtained was extracted with AcOEt, washed with saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of tert-butylamine (73 µL, 0.69 mmol) to a solution of the residue obtained in EtOH (3 mL), the mixture was stirred at room temperature for 1 h, and the precipitate was then collected by filtration to give 16i (168 mg, 53% yield for 2 steps) as a pale brown solid, mp 200–217°C (decomp.). ¹H-NMR (DMSO-d₆) δ: 1.14 (9H, s), 1.76–1.83 (3H, m), 2.81 (0.3H, dd, J=15.8, 6.6 Hz), 2.93 (0.7H, dd, J=15.6, 6.1 Hz), 3.24–3.33 (1H, m), 4.38 (0.7H, d, J=17.8 Hz), 4.52–4.57 (0.7H, m), 4.75–4.89 (1.3H, m), 5.09–5.13 (0.3H, m), 6.01–6.38 (2H, m), 6.45 (0.7H, d, J=14.4 Hz), 6.65 (0.3H, d, J=14.9 Hz), 6.98–7.12 (2H, m), 7.38–7.55 (4.7H, m), 7.63–7.66 (0.3H, br), 7.70–8.35 (3H, br), 7.72–7.76 (2H, m), 7.79–7.83 (2H, m), 8.02–8.06 (2H, m), 10.21 (0.7H, s), 10.24 (0.3H, s). IR (ATR) cm⁻¹: 1722, 1649. MS m/z: 465 [M−H]⁻.

(S)-7-(Biphenyl-4-ylmethoxy)-2-[(2E,4E)-hexadienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16j)

One molar aqueous LiOH solution (1.6 mL, 1.6 mmol) was added to a solution of 11j (490 mg, 1.05 mmol) in MeOH (3 mL) and THF (6 mL), which was stirred at room temperature for 1.5 h. After the addition of aqueous HCl solution, the organic layer was removed under reduced pressure. The residue obtained was extracted with AcOEt, washed with saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue was recrystallized from AcOEt and t-BuOMe to give 16j (383 mg, 80% yield) as a white solid, mp 168–174°C. ¹H-NMR (DMSO-d₆) δ: 1.78–1.85 (3H, m), 2.95–3.22 (2H, m), 4.38 (0.4H, d, J=17.8 Hz), 4.63 (0.6H, d, J=16.1 Hz), 4.77 (0.4H, d, J=17.8 Hz), 4.87 (0.6H, d, J=16.1 Hz), 5.09–5.13 (2H, m), 5.15–5.25 (1H, m), 6.08–6.37 (2H, m), 6.51 (0.4H, d, J=14.6 Hz), 6.60 (0.6H, d, J=14.9 Hz), 6.81–6.88 (1H, m), 6.90–6.95 (1H, m), 7.07–7.18 (2H, m), 7.36 (1H, t, J=7.3 Hz), 7.43–7.48 (2H, m), 7.49–7.54 (2H, m), 7.64–7.69 (4H, m), 12.44–12.98 (1H, br). IR (ATR) cm⁻¹: 1724, 1649. MS m/z: 452 [M−H]⁻.

(S)-2-[(2E,4E)-Hexadienoyl]-7-(7-methyloctyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16k)

Compound 16k was synthesized from 11k according to the procedure for the synthesis of 16a. Yield 34%. A white solid, mp 156–158°C. ¹H-NMR (DMSO-d₆) δ: 0.84 (6H, d, J=6.6 Hz), 1.10–1.18 (2H, m), 1.21–1.33 (4H, m), 1.34–1.43 (2H, m), 1.44–1.55 (1H, m), 1.63–1.72 (2H, m), 1.78–1.85 (3H, m), 2.93–3.20 (2H, m), 3.87–3.93 (2H, m), 4.35 (0.4H, d, J=18.1 Hz), 4.61 (0.6H, d, J=15.9 Hz), 4.75 (0.4H, d, J=18.1 Hz), 4.85 (0.6H, d, J=15.9 Hz), 5.14–5.19 (1H, m), 6.08–6.36 (2H, m), 6.51 (0.4H, d, J=14.6 Hz), 6.59 (0.6H, d, J=14.6 Hz), 6.69–6.75 (1H, m), 6.78 (1H, d, J=2.4 Hz), 7.05–7.18 (2H, m), 12.44–12.94 (1H, br). IR (ATR) cm⁻¹: 1722, 1649. MS m/z: 412 [M−H]⁻.

(S)-[(E,E)-2-Hexa-2,4-dienoyl]-7-(9-methyldeca-2,4-dienyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (16l)

Compound 16l was synthesized from 11l according to the procedure for the synthesis of 16a. Yield 58%. A white solid, mp 102–105°C. ¹H-NMR (DMSO-d₆) δ: 0.84 (6H, d, J=6.6 Hz), 1.09–1.19 (2H, m), 1.29–1.39 (2H, m), 1.45–1.57 (1H, m), 1.77–1.86 (3H, m), 1.99–2.08 (2H, m), 2.93–3.22 (2H, m), 4.37 (0.4H, d, J=17.9 Hz), 4.48–4.56 (2H, m), 4.62 (0.6H, d, J=15.9 Hz), 4.76 (0.4H, d, J=17.9 Hz), 4.86 (0.6H, d, J=15.9 Hz), 5.13–5.22 (1H, m), 5.68–5.82 (2H, m), 6.03–6.38 (4H, m), 6.48–6.65 (1H, m), 6.71–6.83 (2H, m), 7.05–7.19 (2H, m), 12.36–12.97 (1H, br). IR (ATR) cm⁻¹: 1734. MS m/z: 436 [M−H]⁻. Anal. Calcd for C₂₇H₃₅NO₄·0.25H₂O: C, 73.36; H, 8.09; N, 3.17. Found: C, 73.08; H, 7.92; N, 3.10.

(S)-[2-(3-Furan-2-ylacryloyl)-7-(7-methyloctyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17k)

Compound 17k was synthesized from 12k according to the procedure for the synthesis of 16a. Yield 42%. A white solid, mp 153–157°C. ¹H-NMR (DMSO-d₆) δ: 0.84 (6H, d, J=6.6 Hz), 1.11–1.19 (2H, m), 1.22–1.34 (4H, m), 1.34–1.43 (2H, m), 1.50 (1H, heptet), 1.63–1.72 (2H, m), 2.95–3.22 (2H, m), 3.88–3.94 (2H, m), 4.42 (0.4H, d, J=17.6 Hz), 4.71 (0.6H, d, J=15.6 Hz), 4.77 (0.4H, d, J=17.6 Hz), 4.95 (0.6H, d, J=15.6 Hz), 5.20–5.24 (0.6H, m), 5.26–5.30 (0.4H, m), 6.58–6.61 (0.4H, m), 6.61–6.64 (0.6H, m), 6.70–6.76 (1H, m), 6.79–6.82 (0.4H, m), 6.86–6.93 (2H, m), 7.00 (0.6H, d, J=15.4 Hz), 7.06–7.11 (1H, m), 7.37 (0.4H, d, J=14.9 Hz), 7.40 (0.6H, d, J=15.4 Hz), 7.77–7.80 (0.4H, m), 7.82–7.84 (0.6H, m), 12.44–12.94 (1H, br). IR (ATR) cm⁻¹: 1728, 1639. MS m/z: 438 [M−H]⁻, 440 [M+H]⁺.

(S)-2-[(E)-3-Furan-2-yl-acryloyl]-7-[(E,E)-9-methyldeca-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17l)

Compound 17l was synthesized from 12l according to the procedure for the synthesis of 16a. Yield 15%. A white solid, mp 57–60°C. ¹H-NMR (DMSO-d₆) δ: 0.85 (6H, d, J=6.6 Hz), 1.10–1.19 (2H, m), 1.29–1.42 (2H, m), 1.45–1.58 (1H, m), 1.98–2.09 (2H, m), 4.38–4.56 (2.4H, m), 4.67–4.83 (1H, m), 4.97 (0.6H, d, J=12.0 Hz), 5.20–5.32 (1H, m), 4.13–4.19 (2H, m), 5.69–5.83 (2H, m), 6.03–6.14 (1H, m), 6.27–6.39 (1H, m), 6.59–6.66 (1H, m), 6.74–6.95 (3.3H, m), 7.01 (0.7H, d, J=15.1 Hz), 7.07–7.14 (1H, m), 7.35–7.44 (1H, m), 7.76–7.86 (1H, m), 12.37–13.10 (1H, br). IR (ATR) cm⁻¹: 1728. MS m/z: 462 [M−H]⁻.

(S)-2-(3-Furan-2-ylacryloyl)-7-nonyloxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17m)

Compound 17m was synthesized from 12m according to the procedure for the synthesis of 16a. Yield 26%. A pale yellow solid, mp 147–149°C. ¹H-NMR (DMSO-d₆) δ: 0.86 (3H, t, J=6.4 Hz), 1.16–1.44 (12H, m), 1.63–1.74 (2H, m), 3.50–3.23 (2H, m), 3.86–3.93 (2H, m), 4.43 (0.4H, d, J=17.8 Hz), 4.68–4.84 (1H, m), 4.96 (0.6H, J=15.6 Hz), 5.21–5.33 (1H, m), 6.58–6.66 (1H, m), 6.70–6.77 (1H, m), 6.79–6.95 (2.3H, m), 7.01 (0.7H, d, J=15.1 Hz), 7.06–7.13 (1H, m), 7.35–7.46 (1H, m), 7.76–7.86 (1H, m), 12.38–13.03 (1H, br). IR (ATR) cm⁻¹: 1716. MS m/z: 438 [M−H]⁻, 462 [M+Na]⁺.

(S)-2-(3-Furan-2-ylacryloyl)-7-(4-methylpentyloxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17n)

Compound 17n was synthesized from 12n according to the procedure for the synthesis of 16a. Yield 82%. A white solid, mp 163–165°C. ¹H-NMR (DMSO-d₆) δ: 0.89 (6H, d, J=6.6 Hz), 1.25–1.34 (2H, m), 1.52–1.63 (1H, m), 1.63–1.75 (2H, m), 2.96–3.23 (2H, m), 3.91 (2H, t, J=6.2 Hz), 4.43 (0.4H, d, J=17.8 Hz), 4.68–4.82 (1H, m), 4.96 (0.6H, d, J=15.9 Hz), 5.20–5.32 (1H, m), 6.58–6.65 (1H, m), 6.70–6.68 (1H, m), 6.80–6.95 (2.4H, m), 7.01 (0.6H, d, J=15.4 Hz), 7.07–7.13 (1H, m), 7.35–7.44 (1H, m), 7.77–7.85 (1H, m), 12.47–13.00 (1H, br). IR (ATR) cm⁻¹: 1724. MS m/z: 396 [M−H]⁻, 398 [M+H]⁺.

(S)-7-[(E,E)-3,7-Dimethylocta-2,6-dienyloxy]-2-[(E)-3-furan-2-ylacryloyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17o)

Compound 17o was synthesized from 12o according to the procedure for the synthesis of 16a. Yield 30%. A white solid, mp 137–139°C. ¹H-NMR (DMSO-d₆) δ: 1.57 (3H, s), 1.63 (3H, s), 1.69 (3H, s), 2.00–2.13 (4H, m), 2.96–3.24 (2H, m), 4.39–4.52 (2.4H, m), 4.67–4.82 (1H, m), 4.91–5.00 (0.6H, m), 5.04–5.12 (1H, m), 5.20–5.34 (1H, m), 5.37–5.47 (1H, m), 5.59–6.66 (1H, m), 6.72–6.79 (1H, m), 6.82–6.96 (2.4H, m), 7.01 (0.6H, d, J=15.1 Hz), 7.35–7.45 (1H, m), 7.76–7.86 (2H, m), 12.44–13.04 (1H, br). IR (ATR) cm⁻¹: 1728. MS m/z: 448 [M−H]⁻, 450 [M+H]⁺.

(S)-2-(3-Furan-2-ylacryloyl)-7-[(E)-7-isopropoxyhept-2-enyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxyl Acid (17p)

Compound 17p was synthesized from 12p according to the procedure for the synthesis of 16a. Yield 24%. A white solid, mp 112–114°C. ¹H-NMR (DMSO-d₆) δ: 1.05 (6H, d, J=5.8 Hz), 1.32–1.51 (4H, m), 2.00–2.08 (2H, m), 2.96–3.24 (4H, m), 3.43–3.52 (1H, m), 3.38–4.47 (2.4H, m), 4.68–4.84 (1H, m), 4.96 (0.6H, d, J=15.6 Hz), 5.20–5.33 (1H, m), 5.60–5.72 (1H, m), 5.76–5.88 (1H, m), 6.58–6.67 (1H, m), 6.72–6.95 (3.3H, m), 7.01 (0.7H, d, J=15.3 Hz), 7.06–7.14 (1H, m), 7.34–7.45 (1H, m), 7.76–7.86 (1H, m), 12.36–13.10 (1H, m). IR (ATR) cm⁻¹: 1724. MS m/z: 466 [M−H]⁻, 468 [M+H]⁺.

(S)-2-[(E)-3-Furan-2-ylacryloyl]-7-[(E,E)-5-phenylpenta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17q)

Compound 17q was synthesized from 12q according to the procedure for the synthesis of 16a. Yield 12%. A white solid, mp 183–184°C. ¹H-NMR (DMSO-d₆) δ: 2.98–3.24 (2H, m), 4.44 (0.4H, d, J=17.5 Hz), 4.64 (2H, d, J=6.9 Hz), 4.69–4.84 (1H, m), 4.98 (0.6H, d, J=15.9 Hz), 5.21–5.32 (1H, m), 6.01–6.09 (1H, m), 6.50–6.66 (3H, m), 6.76–6.82 (1H, m), 6.87–6.97 (3.4H, m), 7.02 (0.6H, d, J=15.1 Hz), 7.11–7.15 (1H, m), 7.21–7.26 (1H, m), 7.30–7.35 (2H, m), 7.39 (1H, dd, J=15.1, 11.5 Hz), 7.45–7.49 (2H, m), 7.78–7.85 (1H, m), 12.50–12.94 (1H, br). IR (ATR) cm⁻¹: 1728. MS m/z: 454 [M−H]⁻.

(S)-2-{(E)-3-Furan-2-ylacryloyl}-7-[(2E,4E)-5-(3-trifluoromethylphenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17r)

Compound 17r was synthesized from 12r according to the procedure for the synthesis of 16a. Yield 14%. A pale yellow solid, mp 136–138°C. ¹H-NMR (DMSO-d₆) δ: 2.98–3.23 (2H, m), 4.41–4.46 (0.4H, m), 4.64–4.81 (3H, m), 4.95–4.99 (0.6H, m), 5.22–5.29 (1H, m), 6.10–6.15 (1H, m), 6.52–6.63 (2H, m), 6.71–6.81 (2H, s), 6.87–7.03 (3H, m), 7.04–7.17 (2H, m), 7.36–7.42 (1H, m), 7.53–7.57 (2H, m), 7.77–7.83 (3H, m). IR (ATR) cm⁻¹: 1736. MS m/z: 452 [M−H]⁻.

(S)-2-{(E)-3-Furan-2-ylacryloyl}-7-[(2E,4E)-5-(4-trifluoromethylphenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17s)

Compound 17s was synthesized from 12s according to the procedure for the synthesis of 16a. Yield 23%. A pale yellow solid, mp 181–183°C. ¹H-NMR (DMSO-d₆) δ: 2.97–3.18 (2H, m), 4.41–4.46 (0.35H, m), 4.65–4.81 (3.15H, m), 4.95–4.99 (0.5H, m), 5.22–5.29 (1H, m), 6.10–6.20 (1H, m), 6.54–6.63 (2H, m), 6.71 (1H, d, J=15.6 Hz), 6.77–6.83 (1H, m), 6.85–6.97 (2H, m), 7.01 (1H, d, J=15.1 Hz), 7.07–7.17 (2H, m), 7.35–7.42 (1H, m), 7.65–7.70 (4H, m), 7.77–7.83 (1H, d, J=19.8 Hz), 12.50–12.95 (1H, br). IR (ATR) cm⁻¹: 1716,1643. MS m/z: 522 [M−H]⁻.

(S)-2-(3-Furan-2-ylacryloyl)-7-[3-(3-trifluoromethylphenyl)propoxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17t)

Compound 17t was synthesized from 12t according to the procedure for the synthesis of 16a. Yield 27%. A white solid, mp 161–162°C. ¹H-NMR (DMSO-d₆) δ: 2.02–2.10 (2H, m), 2.84 (2H, t, J=7.8 Hz), 2.96–3.24 (2H, m), 3.95 (2H, t, J=6.3 Hz), 4.40–4.47 (0.4H, m), 4.68–4.84 (1H, m), 4.93–5.00 (0.6H, m), 5.21–5.33 (1H, m), 6.60–6.66 (1H, m), 6.74–6.79 (1H, m), 6.87–6.95 (2H, m), 7.02 (1H, d, J=15.2 Hz), 7.08–7.14 (1H, m), 7.35–7.45 (1H, m), 7.50–7.63 (4H, m), 7.78–7.87 (1H, m), 12.50–13.00 (1H, br). IR (ATR) cm⁻¹: 1728. MS m/z: 498 [M−H]⁻, 500 [M+H]⁺.

(S)-2-{(E)-3-Furan-2-ylacryloyl}-7-[(2E,4E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17u)

Compound 17u was synthesized from 12u according to the procedure for the synthesis of 16a. Yield 7.2%. A pale yellow solid, mp 183–185°C. ¹H-NMR (DMSO-d₆) δ: 2.96–3.20 (2H, m), 4.41–4.45 (0.4H, m), 4.62 (2H, s), 4.71–4.82 (1H, m), 4.95–4.99 (0.6H, m), 5.15–5.25 (1H, br), 6.12–6.17 (1H, m), 6.51–6.62 (3H, m), 6.79–7.02 (5H, m), 7.11 (1H, d, J=8.3 Hz), 7.20–7.25 (2H, m), 7.34–7.42 (1H, m), 7.77 (1H, d, J=21.7 Hz), 12.40–13.05 (1H, br). IR (ATR) cm⁻¹: 1720, 1565. MS m/z: 508 [M−H]⁻. Anal. Calcd for C₂₈H₂₂F₃NO₅·0.25H₂O: C, 65.43; H, 4.41; N, 2.73. Found: C, 65.39; H, 4.51; N, 2.69.

Ethyl (2E,4E)-5-Thiophen-3-yl-penta-2,4-dienoate (41)

Compound 41 was synthesized from (E)-3-thiophen-3-yl-propenal⁶³⁾ according to the procedure for the synthesis of 31. Yield 77%. A brown oil. ¹H-NMR (CDCl₃) δ: 1.31 (3H, t, J=7.1 Hz), 4.21 (2H, q, J=7.1 Hz), 5.95 (1H, d, J=15.1 Hz), 6.69 (1H, dd, J=15.6, 11.2 Hz), 6.90 (1H, d, J=15.6 Hz), 7.28–7.43 (4H, m).

(2E,4E)-5-Thiophen-3-yl-penta-2,4-dien-1-ol (42)

Compound 42 was synthesized from 41 according to the procedure for the synthesis of 32. Yield 83%. A pale yellow solid. ¹H-NMR (CDCl₃) δ: 1.34 (1H, t, J=5.9 Hz), 4.22–4.25 (2H, br), 5.89–5.96 (1H, d, J=15.1 Hz), 6.34–6.40 (1H, m), 6.54–6.66 (2H, m), 7.17 (1H, s), 7.22–7.28 (2H, m).

(S)-2-(3-Furan-2-yl-acryloyl)-7-{(2E,4E)5-thiophen-3-yl-penta-2,4-dienyloxy}-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17v)

n-Bu₃P (0.19 mL, 0.783 mmol) and ADDP (197 mg, 0.783 mmol) were added to a solution of 7 (197 mg, 0.602 mmol) and 42 (100 mg, 0.602 mmol) in THF (3 mL) under ice-cooling, and the mixture was stirred at room temperature for 0.5 h. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give crude 12v.

One molar aqueous LiOH solution (0.63 mL, 0.63 mmol) was added to a solution of crude 12v in MeOH (2 mL) and THF (1 mL), which was stirred at 40°C for 1 h. After cooling, water and Et₂O were added to the reaction mixture, and the aqueous layer was then acidified with 1.0 M aqueous HCl solution, followed by extraction with CHCl₃. The organic layer was dried over Na₂SO₄ and the solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give crude 17v. After the addition of t-BuOMe to crude 17v, the precipitate was collected by filtration to give 17v (6.0 mg, 2.2% yield for 2 steps) as a pale brown solid, mp 160–163°C. ¹H-NMR (DMSO-d₆) δ: 2.97–3.19 (2H, m), 4.40–4.45 (0.45H, m), 4.57–4.63 (2H, m), 4.68–4.84 (1H, m), 4.91–5.02 (0.55H, m), 5.15–5.25 (1H, m), 5.92–6.03 (1H, m), 6.44–6.53 (1H, m), 6.58–6.67 (2H, m), 6.73–7.05 (5H, m), 7.07–7.13 (1H, m), 7.30–7.55 (4H, m), 7.80 (1H, d, J=23.4 Hz), 12.40–13.20 (1H, br). IR (ATR) cm⁻¹: 1731, 1641. MS m/z: 460 [M−H]⁻.

Ethyl (2E,4E)-5-Thiazol-2-ylpenta-2,4-dienoate (43)

Compound 43 was synthesized from (E)-5-thiazol-2-yl-propenal⁶⁴⁾ according to the procedure for the synthesis of 31. Yield 96%. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.32 (3H, t, J=7.2 Hz), 4.23 (2H, q, J=7.2 Hz), 6.11 (1H, d, J=15.4 Hz), 7.07 (1H, d, J=15.4 Hz), 7.16 (1H, dd, J=15.4, 10.7 Hz), 7.34 (1H, d, J=3.2 Hz), 7.41 (1H, dd, J=15.4, 10.7 Hz), 7.85 (1H, d, J=3.2 Hz).

(2E,4E)-5-Thiazol-2-ylpenta-2,4-dien-1-ol (44)

Compound 44 was synthesized from 43 according to the procedure for the synthesis of 32. Yield 57%. A pale orange solid. ¹H-NMR (CDCl₃) δ: 1.55–1.61 (1H, m), 4.25–4.31 (2H, m), 6.11 (1H, dt, J=15.1, 5.4 Hz), 6.45 (1H, dd, J=15.1, 10.7 Hz), 6.77 (1H, d, J=15.4 Hz), 7.07 (1H, dd, J=15.4, 10.7 Hz), 7.22 (1H, d, J=3.2 Hz), 7.76 (1H, d, J=3.2 Hz).

(S)-2-[(E)-3-Furan-2-ylacryloyl]-7-[(2E,4E)-5-thiazol-2-ylpenta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17w)

Compound 17w was synthesized from 12w according to the procedure for the synthesis of 16a. Yield 46%. A pale brown oil, mp 181–182°C. ¹H-NMR (DMSO-d₆) δ: 2.96–3.25 (2H, m), 4.44 (0.4H, d, J=17.8 Hz), 4.62–4.85 (3H, m), 4.98 (0.6H, d, J=15.9 Hz), 5.20–5.35 (1H, m), 6.21–6.32 (1H, m), 6.55–6.65 (2H, m), 6.76–6.84 (1H, m), 6.85–7.06 (3H, m), 7.10–7.20 (2H, m), 7.35–7.45 (1H, m), 7.66 (1H, d, J=3.2 Hz), 7.77–7.86 (2H, m), 7.77 (1H, d, J=3.2 Hz), 12.55–12.95 (1H, m). IR (ATR) cm⁻¹: 1730. MS m/z: 461 [M−H]⁻.

Ethyl (2E,4E)-5-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)penta-2,4-dienoate (45)

Compound 45 was synthesized from (E)-3-(2,3-dihydrobenzo[1,4]dioxin-6-yl)propenal⁶⁵⁾ according to the procedure for the synthesis of 31. Yield quant. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.31 (3H, t, J=7.1 Hz), 4.19–4.27 (6H, m), 5.92 (1H, d, J=15.1 Hz), 6.68–6.76 (2H, m), 6.80–6.84 (1H, m), 6.95–6.98 (2H, m), 7.40 (1H, dd, J=15.1, 10.1 Hz).

(2E,4E)-5-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)penta-2,4-dien-1-ol (46)

Compound 46 was synthesized from 45 according to the procedure for the synthesis of 31. Yield 65%. ¹H-NMR (CDCl₃) δ: 1.35 (1H, t, J=5.9 Hz), 4.22–4.28 (6H, m), 5.87–5.94 (1H, d, J=15.1 Hz), 6.35–6.46 (2H, m), 6.62 (1H, dd, J=15.4, 10.5 Hz), 6.80 (1H, d, J=8.3 Hz), 6.88–6.91 (2H, m).

(S)-7-[(2E,4E)-5-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)penta-2,4-dienyloxy]-2-{(E)-3-furan-2-ylacryloyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (17x)

PPh₃ (368 mL, 1.40 mmol) and 2.2 M DEAD in toluene (0.64 mL, 1.4 mmol) were added to a solution of 7 (382 mg, 1.17 mmol) and 46 (255 mg, 1.17 mmol) in THF (5 mL) under ice-cooling, and the mixture was stirred at the same temperature for 10 min. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give crude 12x.

One molar aqueous LiOH solution (0.91 mL, 0.91 mmol) was added to a solution of crude 12x in MeOH (2 mL) and THF (1 mL), which was stirred at 40°C for 1 h. After the addition of 1.0 M aqueous HCl solution, the mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄, and the solvent was then removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 17x (20 mg, 13% yield for 2 steps) as a pale brown solid, mp 84–89°C. ¹H-NMR (DMSO-d₆) δ: 2.97–3.22 (2H, m), 4.22 (4H, s), 4.41–4.45 (0.45H, m), 4.57–4.63 (2H, m), 4.70–4.81 (1H, m), 4.95–4.99 (0.55H, m), 5.23–5.29 (1H, m), 5.91–6.05 (1H, m), 6.44–6.63 (3H, m), 6.75–6.82 (3H, m), 6.85–7.04 (5H, d, J=15.1 Hz), 7.11 (1H, d, J=8.3 Hz), 7.35–7.42 (1H, m), 7.81 (1H, d, J=20.3 Hz), 12.20–12.95 (1H, br). IR (ATR) cm⁻¹: 1716, 1643. MS m/z: 512 [M−H]⁻, 536 [M+Na]⁺.

(S)-7-[(E,E)-9-Methyldeca-2,4-dienyloxy]-2-(3-phenylacryloyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (18l)

Compound 18l was synthesized from 13l according to the procedure for the synthesis of 16a. Yield 59%. A white solid, mp 165–167°C. ¹H-NMR (DMSO-d₆) δ: 0.84 (6H, d, J=6.6 Hz), 1.10–1.19 (2H, m), 1.28–1.41 (2H, m), 1.45–1.58 (1H, m), 1.98–2.08 (2H, m), 2.98–3.26 (2H, m), 4.41 (0.4H, d, J=18.1 Hz), 4.48–4.56 (2H, m), 4.69–4.90 (1H, m), 5.06 (0.6H, d, J=15.8 Hz), 5.20–5.26 (0.6H, m), 5.46–5.54 (0.4H, m), 5.68–5.84 (2H, m), 6.08 (1H, dd, J=14.9, 10.7 Hz), 6.27–6.38 (1H, m), 6.72–6.88 (2H, m), 7.08–7.15 (1H, m), 7.27–7.48 (4H, m), 7.51–7.61 (1H, m), 7.68–7.82 (2H, m), 12.45–13.04 (1H, m). IR (ATR) cm⁻¹: 1720. MS m/z: 472 [M−H]⁻.

(S)-2-(3-Phenylacryloyl)-7-[(E,E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (18u)

Compound 18u was synthesized from 13 according to the procedure for the synthesis of 16a. Yield 42%. A white solid, mp 205–207°C. ¹H-NMR (DMSO-d₆) δ: 2.99–3.28 (2H, m), 4.42 (0.4H, d, J=17.8 Hz), 4.61–4.67 (2H, m), 4.72–4.91 (1H, m), 5.08 (0.6H, m), 5.21–5.26 (0.6H, m), 5.46–5.53 (0.4H, m), 6.11–6.22 (1H, m), 6.45–6.55 (2H, m), 6.77–6.92 (2H, m), 6.97–7.07 (1H, m), 7.11–7.18 (1H, m), 7.18–7.29 (2H, m), 7.28–7.48 (4H, m), 7.51–7.60 (1H, m), 7.71–7.79 (2H, m), 12.47–12.97 (1H, m). IR (ATR) cm⁻¹: 1718. MS m/z: 518 [M−H]⁻, 542 [M+Na]⁺.

(S)-7-[(E,E)-9-Methyldeca-2,4-dienyloxy]-2-[(E)-3-thiophen-2-ylacryloyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (19l)

Compound 19l was synthesized from 14l according to the procedure for the synthesis of 16a. Yield 34%. A white solid, mp 173–175°C. ¹H-NMR (DMSO-d₆) δ: 0.85 (6H, d, J=6.6 Hz), 1.10–1.18 (2H, m), 1.28–1.41 (2H, m), 1.44–1.56 (1H, m), 1.98–2.08 (2H, m), 2.96–3.25 (2H, m), 4.41 (0.5H, d, J=17.8 Hz), 4.99–4.57 (2H, m), 4.72 (0.5H, d, J=15.8 Hz), 4.81 (0.5H, d, J=17.8 Hz), 4.99 (0.5H, d, J=15.8 Hz), 5.20–5.27 (0.5H, m), 5.32–5.43 (0.5H, m), 5.69–5.84 (2H, m), 6.04–6.14 (1H, m), 6.27–6.40 (1H, m), 6.72–6.81 (1H, m), 6.81–6.92 (1H, m), 6.97–7.04 (1H, m), 7.08–7.18 (2H, m), 7.47–7.55 (1H, m), 7.62–7.76 (2H, m), 12.47–12.91 (1H, br). IR (ATR) cm⁻¹: 1724. MS m/z: 478 [M−H]⁻, 480 [M+H]⁺. Anal. Calcd for C₂₈H₃₃NO₄S·0.25H₂O: C, 69.46; H, 6.97; N, 2.89. Found: C, 69.63; H, 6.83; N, 2.89.

(S)-2-[(E)-3-Thiazol-2-ylacryloyl]-7-[(2E,4E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyl-oxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (20u)

Compound 20u was synthesized from 15u according to the procedure for the synthesis of 16a. Yield 40%. A pale yellow solid, mp 189–191°C. ¹H-NMR (DMSO-d₆) δ: 2.98–3.26 (2H, m), 4.47 (0.4H, d, J=17.8 Hz), 4.58–4.67 (2H, m), 4.73–4.86 (1H, m), 5.03 (0.6H, d, J=15.9 Hz), 5.19–5.27 (0.6H, m), 5.36–.44 (0.4H, m), 6.10–6.22 (1H, m), 6.49–6.65 (2H, m), 6.76–7.06 (3H, m), 7.10–7.18 (1H, m), 7.17–7.28 (2H, m), 7.38–7.56 (1H, m), 7.60–7.72 (1H, m), 7.85–8.02 (2H, m), 12.54–13.10 (1H, m). IR (ATR) cm⁻¹: 1722, 1641. MS m/z: 525 [M−H]⁻, 549 [M+Na]⁺.

(S)-N-{2-[(E)-3-Furan-2-ylacryloyl]-7-[(E,E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyl-oxy}-1,2,3,4-tetrahydroisoquinoline-3-carbonyl}methanesulfonamide (21u)

1,1′-Carbonyldiimidazole (CDI) (57 mg, 0.35 mmol) was added to the solution of compound 17u (123 mg, 241 mmol), and the mixture was stirred at room temperature for 0.5 h. Methanesulfonamide (67 mg, 0.70 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.11 mL, 0.71 mmol) were added to the reaction mixture, which was stirred at room temperature for 1 h. After the addition of 1.0 M aqueous HCl solution, the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give crude 21u. Crude 21u was recrystallized from AcOEt and t-BuOMe to give 21u (70 mg, 50%) as a white solid, mp 117–119°C. ¹H-NMR (DMSO-d₆) δ: 2.98–3.20 (5H, m), 4.07–5.08 (5H, m), 6.09–6.21 (1H, m), 6.48–6.72 (3H, m), 6.77–7.08 (5H, m), 7.11–7.27 (3H, m), 7.33–7.43 (1H, m), 7.78–7.87 (1H, m), 11.78–12.08 (1H, m). IR (ATR) cm⁻¹: 1601. MS m/z: 585 [M−H]⁻, 609 [M+Na]⁺.

(S)-N-{7-[(E,E)-9-Methyldeca-2,4-dienyloxy]-2-(3-phenylacryloyl)-1,2,3,4-tetrahydroisoquinoline-3-carbonyl}methanesulfonamide (22l)

Compound 22l was synthesized from 18l according to the procedure for the synthesis of 21u. Yield 18%. A pale yellow solid, mp 123–124°C. ¹H-NMR (DMSO-d₆) δ: 0.84 (6H, d, J=6.6 Hz), 1.08–1.17 (2H, m), 1.27–1.38 (2H, m), 1.42–1.56 (1H, m), 1.97–2.08 (2H, m), 2.98–3.23 (5H, m), 4.45–4.58 (2.1H, m), 4.74–5.03 (2.7H, m), 5.14–5.26 (0.2H, m), 5.67–5.83 (2H, m), 6.03–6.13 (1H, m), 6.33 (1H, dd, J=15.0, 10.6 Hz), 6.75–7.18 (3H, m), 7.32–7.49 (4H, m), 7.50–7.57 (1H, m), 7.65–7.80 (2H, m), 11.85–12.03 (1H, br). IR (ATR) cm⁻¹: 1736. MS m/z: 549 [M−H]⁻, 573 [M+Na]⁺.

(S)-N-{2-[(E)-3-Phenylacryloyl]-7-[(E,E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carbonyl}methanesulfonamide (22u)

Compound 22u was synthesized from 18u according to the procedure for the synthesis of 21u. Yield 27%. A white solid, mp 96–98°C. ¹H-NMR (DMSO-d₆) δ: 2.83–3.18 (5H, m), 4.38–4.52 (0.2H, m), 4.60–4.68 (2H, m), 4.76–5.05 (2.8H, m), 6.11–6.21 (1H, m), 6.48–6.65 (2H, m), 6.76–6.91 (1H, m), 6.95–7.28 (5H, m), 7.35–7.58 (5H, m), 7.65–7.80 (2H, m), 11.88–12.04 (1H, br). IR (ATR) cm⁻¹: 1720. MS m/z: 595 [M−H]⁻, 619 [M+Na]⁺.

Methyl (S)-7-Cyano-2-{(E)-3-furan-2-ylacryloyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (24)

A 8.3 M solution of HCl in i-PrOH (0.36 mL, 3.0 mmol of HCl) was added to a suspension of compound 23 in HCO₂H (2 mL) under ice-cooling, and the mixture was then stirred for 30 min. The reaction mixture was neutralized with saturated aqueous NaHCO₃ solution and extracted with CHCl₃. The organic layer was dried over Na₂SO₄, and the solvent was removed under reduced pressure.

3-Furan-2-ylacryloylic acid (180 mg, 1.30 mmol) and EDC·HCl (249 mg, 1.30 mmol) were added to a solution of the residue obtained in CH₂Cl₂ (5 mL), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was washed with saturated aqueous NaHCO₃ solution and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 24 (330 mg, 98% yield for 2 steps) as a white solid. ¹H-NMR (CDCl₃) δ: 3.17–3.48 (2H, m), 3.63 (3H, s), 4.56–4.61 (0.25H, m), 4.85–4.99 (1.5H, m), 5.07–5.16 (0.55H, m), 5.67–5.73 (0.7H, m), 6.46–6.52 (1H, m), 6.59–6.64 (1H, m), 6.69 (0.25H, d, J=14.9 Hz), 6.87 (0.75H, d, J=14.9 Hz), 7.28–7.33 (1H, m), 7.45–7.58 (4H, m).

2-(5-Methylhexyloxy)isoindole-1,3-dione (47)

Et₃N (3.60 mL, 25.8 mmol) and methanesulfonyl chloride (1.73 mL, 22.4 mmol) were added to a solution of 5-methylhexan-1-ol (2.00 g, 17.2 mmol) in CH₂Cl₂ (40 mL) under ice-cooling, and the mixture was stirred at the same temperature for 15 min. The reaction mixture was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure to afford crude 5-methylhexyl methanesulfonate.

A 60% suspension of NaH in mineral oil (572 mg, 14 mmol) was added portionwise to a stirred solution of N-hydroxyphthalimide (2.34 g, 14.3 mmol) in DMF (30 mL) under ice-cooling, which was stirred at the same temperature for 20 min. After the addition of crude 5-methylhexyl methansulfonate in DMF (10 mL) under ice-cooling, the reaction mixture was stirred at 80°C for 2 h. The mixture was acidified with saturated aqueous NH₄Cl solution, followed by extraction with AcOEt, and the organic layer was washed with saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure and the residue obtained was purified by silica gel column chromatography to give a mixture of compound 47 and 5-methylhexyl methanesulfonate. The mixture obtained was treated using a similar procedure described above to afford compound 47 (3.00 g, 67% yield for 2 steps) as a pale brown oil. ¹H-NMR (CDCl₃) δ: 0.89 (6H, d, J=6.6 Hz), 1.19–1.29 (2H, m), 1.44–1.62 (3H, m), 1.74–1.84 (2H, m), 4.20 (2H, d, J=6.6 Hz), 7.73–7.75 (2H, m), 7.81–7.85 (2H, m).

O-(5-Methylhexyl)hydroxylamine (48)

Hydrazine monohydrate (1.15 g, 23.0 mmol) was added portionwise to a stirred solution of compound 47 (3.00 g, 11.5 mmol) in MeOH (20 mL), which was stirred at room temperature for 75 min. The precipitate was removed by filtration and washed with t-BuOMe. The filtrate was evaporated under reduced pressure to give compound 48 (1.30 g, 86% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 0.87 (6H, d, J=6.6 Hz), 1.15–1.22 (2H, m), 1.27–1.36 (2H, m), 1.48–1.59 (3H, m), 3.66 (2H, d, J=6.6 Hz), 5.10–5.60 (2H, br).

Methyl (S)-7-Formyl-2-{(E)-3-furan-2-ylacryloyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (49)

Raney Ni (270 mg) was added portionwise to a stirred solution of compound 24 (3.00 g, 11.5 mmol) in HCO₂H (3 mL) under 90°C, which was stirred at the same temperature for 15 min. Raney Ni (270 mg) was added to the reaction mixture, which was stirred for 1 h. After cooling, the reaction mixture was neutralized with saturated aqueous NaHCO₃ solution, and then extracted with AcOEt. The organic layer was dried over Na₂SO₄, and the solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 49 (70 mg, 26% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 3.17–3.52 (2H, m), 3.63 (3H, s), 4.64–4.70 (0.3H, m), 4.92–5.20 (2H, m), 5.66–5.74 (0.7H, m), 6.46–6.53 (1H, br), 6.58–6.64 (1H, m), 6.71 (0.25H, d, J=14.9 Hz), 6.91 (0.75H, d, J=14.9 Hz), 7.33–7.40 (1H, m), 7.45–7.58 (2H, m), 7.69–7.76 (2H, m), 9.97 (1H, s).

(S)-2-{(E)-3-Furan-2-yl-acryloyl}-7-[(5-methylhexyloxyimino)methyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (25)

Compound 49 (70 mg, 0.21 mmol) and compound 48 (27 mg, 0.21 mmol) were dissolved in EtOH (1 mL), which was stirred at room temperature for 25 min. Compound 48 (27 mg, 0.21 mmol) was added to the reaction mixture, which was refluxed for 30 min. After the addition of water, the mixture was extracted twice with AcOEt, washed with 10% aqueous citric acid solution and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

One molar aqueous LiOH solution (0.62 mL, 0.62 mmol) was added to a solution of the residue obtained in MeOH (2 mL) and THF (1 mL), and the mixture was stirred at room temperature for 30 min and 40°C for 30 min. The reaction mixture was neutralized with 1.0 M aqueous HCl solution and extracted twice with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 25 (68 mg, 76% yield for 2 steps) as a white solid. mp 52–55°C. ¹H-NMR (DMSO-d₆) δ: 0.86 (6H, d, J=6.6 Hz), 1.13–1.26 (2H, m), 1.28–1.40 (2H, m), 1.45–1.70 (3H, m), 3.08–3.30 (2H, m), 4.09 (2H, d, J=6.4 Hz), 4.43–4.48 (0.4H, m), 4.77–4.87 (1H, m), 5.03–5.07 (0.6H, m), 5.26–5.34 (1H, m), 6.61–6.64 (1H, br), 6.88–6.93 (1.4H, m), 7.06 (0.6H, d, J=15.1 Hz), 7.26–7.28 (1H, m), 7.39–7.44 (2.4H, m), 7.56 (0.6H, m), 7.80–7.84 (1H, m), 8.18 (1H, s), 12.50–13.10 (1H, br). IR (ATR) cm⁻¹: 2952, 1731, 1643. MS m/z: 437 [M−H]⁻, 461 [M+Na]⁺.

PTP1B Inhibitory Activity and Enzyme Selectivity

Inhibitory activities against recombinant human PTP1B (Enzo Life Sciences, Inc., Farmingdale, NY, U.S.A.), recombinant human TCPTP (Enzo Life Sciences, Inc.), recombinant human CD45 PTP (Enzo Life Sciences, Inc.), and recombinant human LAR PTP (Enzo Life Sciences, Inc.) 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 7.2) 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.

PPARγ Agonist Activity

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 (JRH Bioscience, Inc., Lenexa, KS, U.S.A.), 0.3% NaHCO₃, and 1% penicillinstreptomycin under 5% CO₂ at 37°C.

The full-length human PPARγ1 (Open Biosystems, Inc., Huntsville, AL, U.S.A.) was co-transfected with a human RXRα plasmid (GeneCopoeia, Inc., Rockville, MD, U.S.A.) and reporter plasmid pGL3-PPREx4-tk-luc into COS-1 cells using Nucleofector II (AAD-1001S; Lonza, Basel, Switzerland). Cells were then seeded at a density of 1.5×10³ cells/well in 96-well plates and incubated for 24 h in the presence or absence of the test compounds. Luciferase activities were assessed using PicaGene LT7.5 (TOYO B-Net Co., Ltd., Tokyo, Japan). Responses were expressed as % values when the maximal response to farglitazar (100 nM) was taken as 100%.

PTP1B Inhibitory Mode

The inhibitory mode was examined using recombinant human PTP1B and pNPP as the substrate. In order to clarify the inhibitory mode, phosphatase activities were measured at a fixed PTP1B concentration, while the concentrations of the substrate and inhibitor varied.

Various concentrations of 17u were added to 100 mM HEPES buffer (pH 7.2) containing the enzyme (23.3 ng/mL), 1 mM DTT, and 1 mM EDTA. The reaction was started by the addition of pNPP (0.1, 0.15, 0.25, 1, 3, 10 mM) and stopped by the addition of 1 M NaOH after a 30-min incubation at 37°C, and the absorbance of p-nitrophenol produced was then measured at 405 nm.

The 1/s and 1/v values, which were obtained from the substrate concentrations (s) and enzyme activities (v) at 0, 0.2, 0.4, and 0.8 µM of 17u, were plotted on the x- and y-axes, respectively. The inhibitory mode was evaluated from the intersection characteristics of the approximately straight lines obtained.

Water Solubility

A solution of Compound 17u in DMSO was added to Japanese Pharmacopoeia second fluid (JP2, pH 6.8), vigorously shaken at room temperature for 30 min, and then centrifuged at 18000 rpm for 10 min. The concentration of compound 17u in the supernatant was assessed using the Hitachi Elite LaChrom HPLC system (Hitachi High Technologies, Tokyo, Japan) consisting of a COSMOSIL 5C18-MS-II column (4.6 mm×150 mm; Nacalai Tesque), pump (L-2130), autosampler (L-2200), column oven (L-2300), and UV detector (L-2400).

Plasma Concentrations after Oral Administration to Male SD Rats

Male SD rats (7 weeks old; Japan SLC, Inc., Shizuoka, Japan) were used. 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. Test compounds suspended in 0.5% methylcellulose solution were orally administered at 10 mg/kg to rats. Blood samples were taken from the jugular vein of rats 0.25, 0.5, 1, 3, 5, 8, and 24 h after administration. The plasma concentrations of the test compounds were assessed using the Hitachi Elite LaChrom HPLC system (Hitachi High Technologies) consisting of a COSMOSIL 5C18-MS-II column (4.6 mm×150 mm; Nacalai Tesque, Kyoto, Japan), pump (L-2130), autosampler (L-2200), column oven (L-2300), and UV detector (L-2400) in mice.

Anti-diabetic Effects in Male db/db Mice

Male db/db diabetic mice (9 weeks old; Institute for Animal Reproduction, Ibaraki, Japan) were allocated to vehicle-treated and 17u-treated groups (n=6). Compound 17u suspended in 0.5% methylcellulose solution was orally administered at 10 and 30 mg/kg/d to mice for four weeks. On day 28, blood samples were taken from the tail vein and plasma glucose and triglyceride levels were measured.

Mice were fasted overnight and orally administered glucose (0.5 g/kg). Blood was taken from the tail vein before and 30, 60, and 120 min after glucose administration, plasma was isolated, and plasma glucose levels were assessed.

Protein Structures for in Silico Analyses

The crystal structures of PTP1B with an “open” WPD-loop were collected as described previously,⁶⁶⁾ followed by conformer clustering using the hierarchical agglomerative method based on the atomic root mean square deviation (RMSD) of the catalytic sites (Schrödinger Suite 2017-1). Three representative structures (PDB codes 1G7F, 1PH0, and 1T4J) were selected for ligand docking to the catalytic site with the “open” WPD loop. The crystal structures of PTP1B complexed with an allosteric inhibitor (PDB codes 1T48, 1T49, and 1T4J) were also collected for ligand docking to the allosteric site. These protein structures were prepared using the Protein Preparation Wizard in Maestro (Schrödinger Suite 2017-1), which added hydrogen atoms, repaired the side chains of incomplete residues, and optimized the hydrogen-bonding network. The restrained minimization of protein structures was performed with a RMSD cut-off value of 0.3 Å using the OPLS3 force field.

Computational Ligand Docking

Receptor grids for computational ligand docking were generated for all of the five protein structures using the grid generation feature of Glide (Schrödinger Suite 2017-1). In some structures (PDB codes 1G7F and 1PH0), a water molecule had been observed in the catalytic site; therefore, the receptor grids were generated in both the presence and absence of a water molecule. In the compound 17u preparation, LigPrep (Schrödinger Suite 2017-1) was used to generate possible 3D structures with various ionization states at pH 7.0±2.0, tautomers and ring conformations. Multiple conformations of compound 17u were generated using ConfGen (Schrödinger Suite 2017-1). All docking calculations were performed with Glide and run in the standard precision (SP) mode. Ligand van der Waals (vdW) radii for non-polar atoms were scaled by a factor of 1.0. The conformers obtained were docked into the receptor grids, and the top-ranked poses by the Glide docking score were then selected from catalytic and allosteric sites.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Bloomgarden Z. T., Diabetes Care, 28, 488–493 (2005).
• 2) Lebovitz H. E., Banerji M. A., Recent Prog. Horm. Res., 56, 265–294 (2001).
• 3) Tack C. J., Smits P., Neth. J. Med., 64, 166–174 (2006).
• 4) Erdmann E., Dormandy J., Wilcox R., Massi-Benedetti M., Charbonnel B., Vasc. Health Risk Manag., 3, 355–370 (2007).
• 5) Staels B., Cell Metab., 2, 77–78 (2005).
• 6) Lehrke M., Lazar M. A., Cell, 123, 993–999 (2005).
• 7) Krische D., West. J. Med., 173, 54–57 (2000).
• 8) Hussein Z., Wentworth J. M., Nankervis A. J., Proietto J., Colman P. G., Med. J. Aust., 181, 536–539 (2004).
• 9) Schwartz A. V., Sellmeyer D. E., Vittinghoff E., Palermo L., Lecka-Czernik B., Feingold K. R., Strotmeyer E. S., Resnick H. E., Carbone L., Beamer B. A., Park S. W., Lane N. E., Harris T. B., Cummings S. R., J. Clin. Endocrinol. Metab., 91, 3349–3354 (2006).
• 10) Cho N., Momose Y., Curr. Top. Med. Chem., 8, 1483–1507 (2008).
• 11) Devasthale P. V., Chen S., Jeon Y., Qu F., Shao C., Wang W., Zhang H., Cap M., Farrelly D., Golla R., Grover G., Harrity T., Ma Z., Moore L., Ren J., Seethala R., Cheng L., Sleph P., Sun W., Tieman A., Wetterau J. R., Doweyko A., Chandrasena G., Chang S. Y., Humphreys W. G., Sasseville V. G., Biller S. A., Ryono D. E., Selan F., Hariharan N., Cheng P. T., J. Med. Chem., 48, 2248–2250 (2005).
• 12) Hegarty B. D., Furler S. M., Oakes N. D., Kraegen E. W., Cooney G. J., Endocrinology, 145, 3158–3164 (2004).
• 13) Chigurupati S., Dhanaraj S. A., Balakumar P., Eur. J. Pharmacol., 755, 50–57 (2015).
• 14) Otake K., Azukizawa S., Takahashi K., Fukui M., Shibabayashi M., Kamemoto H., Kasai M., Shirahase H., Chem. Pharm. Bull., 59, 876–879 (2011).
• 15) Otake K., Azukizawa S., Fukui M., Shibabayashi M., Kamemoto H., Miike T., Kunishiro K., Kasai M., Shirahase H., Chem. Pharm. Bull., 59, 1233–1242 (2011).
• 16) Otake K., Azukizawa S., Fukui M., Kunishiro K., Kamemoto H., Kanda M., Miike T., Kasai M., Shirahase H., Bioorg. Med. Chem., 20, 1060–1075 (2012).
• 17) Otake K., Azukizawa S., Takeda S., Fukui M., Kawahara A., Kitao T., Shirahase H., Chem. Pharm. Bull., 63, 998–1014 (2015).
• 18) Feldhammer M., Uetani N., Miranda-Saavedra D., Tremblay M. L., Crit. Rev. Biochem. Mol. Biol., 48, 430–445 (2013).
• 19) Lund I. K., Hansen J. A., Andersen H. S., Møller N. P., Billestrup N., J. Mol. Endocrinol., 34, 339–351 (2005).
• 20) Elchebly M., Payette P., Michaliszyn E., Cromlish W., Collins S., Loy A. L., Normandin D., Cheng A., Himms-Hagen J., Chan C. C., Ramachandran C., Gresser M. J., Tremblay M. L., Kennedy B. P., Science, 283, 1544–1548 (1999).
• 21) Zinker B. A., Rondinone C. M., Trevillyan J. M., Gum R. J., Clampit J. E., Waring J. F., Xie N., Wilcox D., Jacobson P., Frost L., Kroeger P. E., Reilly R. M., Koterski S., Opgenorth T. J., Ulrich R. G., Crosby S., Butler M., Murray S. F., McKay R. A., Bhanot S., Monia B. P., Jirousek M. R., Proc. Natl. Acad. Sci. U.S.A., 99, 11357–11362 (2002).
• 22) He R. J., Yu Z. H., Zhang R. Y., Zhang Z. Y., Acta Pharmacol. Sin., 35, 1227–1246 (2014).
• 23) Thareja S., Aggarwal S., Bhardwaj T. R., Kumar M., Med. Chem. Res., 32, 459–517 (2012).
• 24) Qian S., Zhang M., He Y., Wang W., Liu S., Future Med. Chem., 8, 1239–1258 (2016).
• 25) Barr A. J., Future Med. Chem., 2, 1563–1576 (2010).
• 26) Erbe D. V., Wang S., Zhang Y. L., Harding K., Kung L., Tam M., Stolz L., Xing Y., Furey S., Qadri A., Klaman L. D., Tobin J. F., Mol. Pharmacol., 67, 69–77 (2005).
• 27) Fukuda S., Ohta T., Sakata S., Morinaga H., Ito M., Nakagawa Y., Tanaka M., Matsushita M., Diabetes Obes. Metab., 12, 299–306 (2010).
• 28) Ito M., Fukuda S., Sakata S., Morinaga H., Ohta T., Diabetes Res., 2014, 680348 (2014).
• 29) Zasloff M., Williams J. I., Chen Q., Anderson M., Maeder T., Holroyd K., Jones S., Kinney W., Cheshire K., Mclane M., Int. J. Obes., 25, 689–697 (2001).
• 30) Lantz K. A., Hart S. G., Planey S. L., Roitman M. F., Ruiz-White I. A., Wolfe H. R., McLane M. P., Obesity, 18, 1516–1523 (2010).
• 31) Zhang X., Tian J., Li J., Huang L., Wu S., Liang W., Zhong L., Ye J., Ye F., Br. J. Pharmacol., 173, 1939–1949 (2016).
• 32) Morishita K., Shoji Y., Tanaka S., Fukui M., Ito Y., Kitao T., Ozawa S., Hirono S., Shirahase H., Chem. Pharm. Bull., 65, 1144–1160 (2017).
• 33) Kumagai T., Baldwin C., Aoudjit L., Nezvitsky L., Robins R., Jiang R., Takano T., Am. J. Pathol., 184, 2211–2224 (2014).
• 34) Zhang J., Li L., Li J., Liu Y., Zhang C. Y., Zhang Y., Zen K., Arterioscler. Thromb. Vasc. Biol., 35, 163–174 (2015).
• 35) Rajala A., Dilly A. K., Rajala R. V., Cell Commun. Signal., 11, 20 (2013).
• 36) Ohta M., Takahashi K., Kasai M., Shoji Y., Kunishiro K., Miike T., Kanda M., Mukai C., Shirahase H., Chem. Pharm. Bull., 58, 1066–1076 (2010).
• 37) Azukizawa S., Kasai M., Takahashi K., Miike T., Kunishiro K., Kanda M., Mukai C., Shirahase H., Chem. Pharm. Bull., 56, 335–345 (2008).
• 38) Kyoto Pharmaceutical Industries, Ltd., EP1398313 (2004).
• 39) TransTech Pharma Inc., WO2010/114824 (2010).
• 40) Nomura M., Kinoshita S., Satoh H., Maeda T., Murakami K., Tsunoda M., Miyachi H., Awano K., Bioorg. Med. Chem. Lett., 9, 533–538 (1999).
• 41) Gampe R. T. Jr., Montana V. G., Lambert M. H., Miller A. B., Bledsoe R. K., Milburn M. V., Kliewer S. A., Willson T. M., Xu H. E., Mol. Cell, 5, 545–555 (2000).
• 42) Devasthale P. V., Chen S., Jeon Y., Qu F., Ryono D. E., Wang W., Zhang H., Cheng L., Farrelly D., Golla R., Grover G., Ma Z., Moore L., Seethala R., Sun W., Doweyko A. M., Chandrasena G., Sleph P., Hariharan N., Cheng P. T., Bioorg. Med. Chem. Lett., 17, 2312–2316 (2007).
• 43) Puius Y. A., Zhao Y., Sullivan M., Lawrence D. S., Almo S. C., Zhang Z. Y., Proc. Natl. Acad. Sci. U.S.A., 94, 13420–13425 (1997).
• 44) Douty B., Wayland B., Ala P. J., Bower M. J., Pruitt J., Bostrom L., Wei M., Klabe R., Gonneville L., Wynn R., Burn T. C., Liu P. C., Combs A. P., Yue E. W., Bioorg. Med. Chem. Lett., 18, 66–71 (2008).
• 45) Moretto A. F., Kirincich S. J., Xu W. X., Smith M. J., Wan Z. K., Wilson D. P., Follows B. C., Binnun E., Joseph-McCarthy D., Foreman K., Erbe D. V., Zhang Y. L., Tam S. K., Tam S. Y., Lee J., Bioorg. Med. Chem., 14, 2162–2177 (2006).
• 46) Wiesmann C., Barr K. J., Kung J., Zhu J., Erlanson D. A., Shen W., Fahr B. J., Zhong M., Taylor L., Randal M., McDowell R. S., Hansen S. K., Nat. Struct. Mol. Biol., 11, 730–737 (2004).
• 47) Zhang X., Tian J., Li J., Huang L., Wu S., Liang W., Zhong L., Ye J., Ye F., Br. J. Pharmacol., 173, 1939–1949 (2016).
• 48) Wende R. C., Seitz A., Niedek D., Schuler S. M. M., Hofmann C., Becker J., Schreiner P. R., Angew. Chem. Int. Ed., 55, 2719–2723 (2016).
• 49) Ohba Y., Takatsuji M., Nakahara K., Fujioka H., Kita Y., Chem. Eur. J., 15, 3526–3537 (2009).
• 50) Roth B. D., Blankley C. J., Hoefle M. L., Holmes A., Roark W. H., Trivedi B. K., Essenburg A. D., Kieft K. A., Krause B. R., Stanfield R. L., J. Med. Chem., 35, 1609–1617 (1992).
• 51) Crooks P. A., Dwoshin L., Xu R., Ayers J. T., US2003/225142 (2003).
• 52) Actelion Pharmaceuticals Ltd., EP2013209 (2011).
• 53) Nishiyama T., Hatae N., Hayashi K., Obata M., Taninaka K., Yamane M., Oda S., Abe T., Ishikura M., Hibino S., Choshi T., Heterocycles, 95, 251–267 (2017).
• 54) Brown T. H., Blakemore R. C., Blurton P., Durant G. J., Ganellin C. R., Parsons M. E., Rasmussen A. C., Rawlings D. A., Walker T. F., Eur. J. Med. Chem., 24, 65–72 (1989).
• 55) Israili Z. H., Smissman E. E., J. Chem. Eng. Data, 22, 357–359 (1977).
• 56) Kona J. R., King’ondu C. K., Howell A. R., Suib S. L., ChemCatChem, 6, 749–752 (2014).
• 57) Rinkel J., Rabe P., Zur Horst L., Dickschat J. S., Beilstein J. Org. Chem., 12, 2317–2324 (2016).
• 58) Eliel E. L., Nowak B. E., Daignault R. A., Badding V. G., J. Org. Chem., 30, 2441–2447 (1965).
• 59) Charette A. B., Molinaro C., Brochu C., J. Am. Chem. Soc., 123, 12168–12175 (2001).
• 60) Takeda Pharmaceutical Company Ltd., EP2264017 (2010).
• 61) Oida S., Tajima Y., Konosu T., Nakamura Y., Somada A., Tanaka T., Habuki S., Harasaki T., Kamai Y., Fukuoka T., Ohya S., Yasuda H., Chem. Pharm. Bull., 48, 694–707 (2000).
• 62) Takekuma S., Matsuoka H., Ogawa S., Shibasaki Y., Minematsu T., Bull. Chem. Soc. Jpn., 83, 1248–1263 (2010).
• 63) Schuetz R. D., Houff W. H., J. Am. Chem. Soc., 77, 1839–1841 (1955).
• 64) Zhang X., Wang R., Perez G. R., Chen G., Zhang Q., Zheng S., Wang G., Chen Q.-H., Bioorg. Med. Chem., 24, 4692–4700 (2016).
• 65) Koul S., Koul J. L., Taneja S. C., Dhar K. L., Jamwal D. S., Singh K., Reen R. K., Singh J., Bioorg. Med. Chem., 8, 251–268 (2000).
• 66) Shinde R. N., Sobhia M. E., J. Mol. Struct., 1017, 79–83 (2012).