(S)-1,2,3,4-Tetrahydroisoquinoline Derivatives Substituted with an Acidic Group at the 6-Position as a Selective Peroxisome Proliferator-Activated Receptor γ Partial Agonist

Abstract

A novel series of 2,6,7-substituted 3-unsubstituted 1,2,3,4-tetrahydroisoquinoline derivatives were synthesized to find a peroxisome proliferator-activated receptor γ (PPARγ) partial agonist. Among the derivatives, (E)-7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethoxy]-2-[3-(2-furyl)acryloyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (20g) exhibited potent partial agonist activity (EC₅₀ = 13 nM, maximal response 30%) and very weak protein tyrosine phosphatase 1B (PTP1B) inhibition (IC₅₀ = 1100 nM), indicating a selective PPARγ partial agonist. A computational docking calculation revealed that 20g bound to PPARγ in a similar manner to that of known partial agonists. In male and female KK-A^y mice with insulin resistance and hyperglycemia, 20g at 30 mg/kg for 7 d significantly reduced plasma glucose levels, but not triglyceride levels. The effects of 20g were similar to those of pioglitazone at 10 mg/kg. In conclusion, the 2,6,7-substituted 1,2,3,4-tetrahydroisoquinoline with an acidic group at the 6-position provides a novel scaffold for selective PPARγ partial agonists and 20g exerted anti-diabetic effects via the partial activation of PPARγ.

Introduction

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor belonging to the nuclear receptor family. PPARγ agonists, such as pioglitazone (Fig. 1) and rosiglitazone, have long been used as anti-diabetic drugs to reduce blood glucose levels by improving insulin resistance in type 2 diabetic patients.^1–5) PPARγ agonists increase insulin sensitivity by promoting adipocyte differentiation, resulting in increases in adiponectin, an anti-insulin-resistance adipokine, and decreases in tumor necrosis factor α (TNFα), an insulin resistance-evoking adipokine.²⁾ PPARγ agonists are also expected for the treatment of various diseases, including Alzheimer’s disease, non-alcoholic steatohepatitis, cardiovascular diseases, and cancer.^6–8) However, pioglitazone and rosiglitazone induce edema and increase the risks of weight gain, congestive heart failure, and bone fracture.^8–11) Thus, extensive efforts have been made to develop safer PPARγ agonists, and various PPARα/γ dual agonists and PPAR pan-agonists have been reported. However, their development has mostly been suspended due to the potential risks of cardiovascular events, carcinogenicity, liver injury, and/or renal dysfunction.^12–14) The overactivation of PPAR with PPARα/γ dual agonists and PPAR pan-agonists may lead to carcinogenesis and adverse effects in the liver, heart, and kidneys.^15–19)

Fig. 1. Chemical Structures of PPARγ Full Agonists

PPARγ partial agonists, such as INT-131 (Fig. 2), have been investigated as PPARγ modulators and examined clinically.^20–22) A PPARγ partial agonist is defined to partially activate PPARγ and antagonize the activation of PPARγ by a full agonist, such as farglitazar (Fig. 1). In the concept of PPARγ modulators, the partial activation of PPARγ fully induces insulin sensitization without adverse effects. Although many structurally different PPARγ partial agonists showed higher efficacy with lower toxicity in experimental diabetic animals, none have been successfully developed to date.^10,20–23) Therefore, new partial agonists that are structurally different from known agonists are desired and their efficacy and adverse effects need to be compared with those of full agonists.

Fig. 2. Chemical Structures of PPARγ Partial Agonists

The majority of PPARγ partial agonists have different structures from those of known full agonists^20–28) (Fig. 2). We previously reported various types of PPARγ agonists using the same scaffold, 1,2,3,4-tetrahydroisoquinoline 3-calboxylic acid: a selective PPARγ full agonist, PPARγ full agonist with protein tyrosine phosphatase 1B (PTP1B) inhibition, PPARα/γ dual agonist with PTP1B inhibition, and PPARγ partial agonist with PTP1B inhibition.^{25,26,29–31)} PTP1B negatively regulates the insulin signal and its overexpression has been implicated in insulin resistance; thus, the inhibition of PTP1B is expected to exert synergistic effects with PPARγ activation on insulin sensitization.^32–34) One of the 1,2,3,4-tetrahydroisoquinoline 3-calboxylic acids with PPARα/γ agonist and PTP1B inhibitory activities has been reported to exhibit effective anti-diabetic activities with high safety,³⁵⁾ and this may be due to its weak PPARγ activation and PTP1B inhibition. However, it was not a partial PPARγ agonist because it did not antagonize a PPARγ full agonist (unpublished data). A partial agonist based on 1,2,3,4-tetrahydroisoquinoline-3-calboxylic acid was also reported; however, its maximal activation level was still high (65%).³¹⁾ As described above, our previous studies demonstrated that 3-carboxyl 1,2,3,4-tetrahydroisoquinoline derivatives exerted PPARγ full agonist activity or PPARγ partial agonist activity with relatively high maximal activation. In the present study, the 2,7-substituted 3-unsubstituted 1,2,3,4-tetrahydroisoquinoline structure with an acidic group at the 6-position was revealed to be a novel scaffold for a selective PPARγ partial agonist, and compound 20g was found to possess a partial agonist property with high affinity, low maximal activation, and antagonistic activity, and to show anti-diabetic effects in db/db mice.

Chemistry

(S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid derivative 10 was synthesized as shown in Chart 1. Compound 3 was amidated with methyl 2-amino-3-oxobutanoate hydrochloride via acyl chloride, which was followed by cyclization with I₂, triphenylphosphine (PPh₃) and Et₃N to give oxazole 5. Compound 5 was reduced with lithium aluminium hydride (LiAlH₄) and then chlorinated with SOCl₂ to give 6. The hydroxyl group of compound 7, which was prepared as described previously,²⁹⁾ was alkylated with compound 6 to afford 8. Deprotection of the tert-butoxycarbonyl (Boc) group with HCl in i-PrOH/HCO₂H, followed by acylation with (E)-3-(2-furyl)acrylic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) gave 9. Compound 9 was hydrolyzed with aqueous LiOH, and desired compound 10 was then isolated as a tert-butyl amine salt.

Chart 1. Synthesis of 7-(2-Cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-yl-acryloyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid t-Butylamine Salt (10)

Reagents and conditions: (i) SOCl₂; (ii) methyl 2-amino-3-oxobutanoate hydrochloride, Et₃N, CH₂Cl₂; (iii) I₂, PPh₃, Et₃N, CH₂Cl₂; (iv) LiAlH₄, THF; (v) SOCl₂; (vi) K₂CO₃, DMF; (vii) HCl in i-PrOH, HCO₂H; (viii) (E)-3-(2-furyl)acrylic acid, EDC·HCl, CH₂Cl₂; (ix) LiOH aq., MeOH, THF; (x) t-BuNH₂, AcOEt.

The general approach to the synthesis of 6-substituted-7-(2-cyclopentenyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-yl-propenonyl)tetrahydroisoquinoline derivatives (20a–g) is outlined in Chart 2.

Chart 2. Synthesis of 6-Substituted-7-(2-cyclopentenyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-yl-propenonyl)tetrahydroisoquinoline Derivatives

Reagents and conditions: (i) HMTA, TFA; (ii) BnBr, K₂CO₃, DMF; (iii) 2-methyl-2-butene, NaClO₂, NaH₂PO₄, t-BuOH, H₂O, THF; (iv) LiOH aq., 1,4-dioxane then Boc₂O; (v) MeI, K₂CO₃, DMF; (vi) ClCO₂i-Bu, N-methylmorpholine, NH₃ aq.; (vii) BH₃·THF, THF; (viii) H₂, Pd–C, MeOH; (ix) Zn, AcOH; (x) 37% formalin, NaBH(OAc) ₃, THF; (xi) 6, K₂CO₃, DMF; (xii) POCl₃, Et₃N, CH₂Cl₂, (xiii) NH₂OH·HCl, Et₃N, DMSO; (xiv) isobutyl chloroformate, pyridine, DMF then xylene reflux; (xv) n-Bu₃SnN₃, toluene; (xvi) SO₃·Py, DMSO, i-Pr₂NEt, CH₂Cl₂; (xvii) NH₃ aq., glyoxal aq.; (xviii) HCl in i-PrOH, HCO₂H; (xix) (E)-3-(2-furyl)acrylic acid, EDC·HCl, CH₂Cl₂; (xx) 3-(2-furyl)acrylic acid, EDC·HCl, CH₂Cl₂ then HCl in i-PrOH, i-Pr₂O (xxi) (E)-3-(2-furyl)acrylic acid, (COCl)₂, DMF, Et₃N, CH₂Cl₂; (xxii) LiOH aq., MeOH, THF then t-BuNH₂, AcOEt.

The formylation of the starting material 11³⁶⁾ afforded a mixture of regioisomers 12, which was used without the isolation of each regioisomer. The mixture was treated with benzyl bromide (BnBr) and K₂CO₃, followed by the oxidation and isolation of a regioisomer to obtain 13. The trifluoroacetyl group of 13 was removed by hydrolysis with aqueous LiOH, and then treated with di-tert-butyl dicarbonate (Boc₂O) to afford 14. Compounds 15a, 15d, and 15h are obtained by esterification, amidation, and reduction, respectively, from compound 14. Deprotection of the benzyl group of compounds 15a, 15d, and 15h gave compounds 17a, 17d, and 17h. Separately, the nitro group of 16³⁷⁾ was reduced by zinc and methylated by reductive alkylation to afford 17b. The conversion of 17a, 17b, 17d, and 17h to 18a, 18b, 18d, and 18h was accomplished by alkylation with 6. The carbamoyl moiety of 18d was converted to a cyano group (compound 18c), which was treated with n-Bu₃SnN₃ to give tetrazole 18g. Compound 18c was treated with hydroxylamine hydrochloride, which was cyclized to give the 4H-[1,2,4]oxadiazol-5-one-3-yl 18f. The imidazolyl derivative 18e was prepared from 18h by oxidation and cyclization. The Boc groups of 18a–g were removed and acylated with (E)-3-(2-furyl)acrylic acid to give 19a, 20b–g. Compound 19a was hydrolyzed by aqueous LiOH to afford 20a. Compound 20a was isolated as a t-butylamine salt, and 20b and 20e were isolated as hydrochloride salts.

The synthesis of (E)-2-[3-(2-furyl)acryloyl]-7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethylsulfanyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26) is outlined in Chart 3.

Chart 3. Synthesis of (E)-2-[3-(2-Furyl)acryloyl]-7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethylsulfanyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26)

Reagents and conditions: (i) MeI, K₂CO₃, DMF; (ii) H₂, Pd–C, MeOH; (iii) N,N-dimethylthiocarbamoyl chloride, Et₃N, DMAP, 1,4-dioxane; (iv) n-tetradecane; (v) K₂CO₃ aq., MeOH then Boc₂O; (vi) MeONa in MeOH, MeOH; (vii) 6, Cs₂CO₃, CH₃CN; (viii) LiOH aq., MeOH, THF; (ix) ClCO₂i-Bu, N-methylmorpholine, NH₃ aq., THF; (x) POCl₃, Et₃N, CH₂Cl₂, (xi) n-Bu₃SnN₃, toluene; (xii) HCl in i-PrOH, HCO₂H; (xiii) (E)-3-(2-furyl)acrylic acid, (COCl)₂, DMF, Et₃N, CH₂Cl₂; (xiv) LiOH aq., MeOH, THF.

Intermediate 13 was esterified with methyl iodide (MeI) and K₂CO₃, followed by deprotection of the benzyl group to give compound 21. Compound 22 was synthesized via the Newman–Kwart rearrangement of the corresponding O-aryl dimethylthiocarbamate, which was prepared from compound 21. The protection group of 22 was changed from trifluoroacetyl to the Boc group, which was hydrolyzed with MeONa solution to give 23. Compound 23 was alkylated with compound 6, followed by hydrolysis and amidation to give compound 24. Compound 24 was converted to tetrazole 25 by the same procedure from 16d to 16g, and the Boc group of 25 were then deprotected with HCl/HCO₂H and acylated with (E)-3-(2-furyl)acrylic acid to give 26.

Results and Discussion

We previously reported various series of 1,2,3,4-tetrahydroisoquinoline 3-calboxylic acid derivatives.^{25,26,29–31)} Among them, a series of 1,2,3,4-tetrahydroisoquinoline 3-calboxylic acid with a 7-bulky substituent exhibited PPARγ partial agonist activity (EC₅₀ = 48–193 nM); however, their maximal activation levels were relatively high (50–90%),³¹⁾ which were higher than those of reported partial agonists. On the other hand, the derivatives with 3-acetyl and 3-difluoroethyl exhibited potent activities (EC₅₀ = 30 and 62 nM) and lower maximal activation (21 and 24%); however, their oral absorption was reduced due to low water solubility.³¹⁾ To find a PPARγ partial agonist with low maximal activation and high water solubility, novel 3-unsubstituted 7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethoxy]-2-[3-(2-furyl)acryloyl]-1,2,3,4-tetrahydroisoquinoline derivatives with an acidic, basic or neutral substituent at the 6-position were synthesized and evaluated for PPARγ partial agonist activity. The PPARγ agonist activities of synthesized compounds were assessed as transactivation activity in COS-1 cells containing the full-length human PPARγ1 plasmid co-transfected with the human retinoid X receptor α (RXRα) plasmid and reporter plasmid pGL3-PPREx4-tk-luc. Activation responses were expressed as relative values to the maximal activation by farglitazar, a PPARγ full agonist. Antagonist activity against farglitazar (10⁻⁷ M) was also assessed. The effects of the compounds on PTP1B activities were also examined using a human PTP1B enzyme because reported 1,2,3,4-tetrahydroisoquinoline 3-calboxylic acid derivatives exhibit PTP1B inhibitory activities to some extent. Plasma concentrations after the oral administration of a selected compound at 10 mg/kg were evaluated and anti-diabetic effects were investigated in KK-A^y mice, a type 2 diabetic animal. All animal experiments in the present study were conducted according to the guidelines for animal experiments of our institute and the guidelines for animal experimentation approved by the Japanese Association of Laboratory Animal Science.

The PPARγ agonist and antagonist activities and PTP1B inhibitory activity of the compounds synthesized were shown in Table 1. The 1,2,3,4-tetrahydroisoquinoline 3-calboxylic acid derivative (10) was a PPARγ full agonist (EC₅₀ = 528 nM, maximal activation 98.7%). Compound 20a with 6-carboxyl exhibited PPARγ partial agonist activity (EC₅₀ = 179 nM, maximal activation 28.5%), indicating that 6-carboxyl is more suitable than 3-carboxyl for PPARγ partial agonism. Compound 20c with the 6-cyano group had no agonist activities, and compounds with 6-dimethylamino (20b), 6-amide (20d), and 6-imidazole (20e) all showed low activation levels at 1000 nM (21.4–26.9%). However, they caused cytotoxicity at higher concentrations, and thus maximal activation levels and antagonistic activities were unable to be measured. It remains to be determined whether 20b, 20d and 20e are PPARγ partial agonists.

Table 1. Chemical Structures, Molecular Weights, clog D_7.4, and PTP1B Inhibitory and PPARγ Agonist Activities of 6-Substituted-7-(2-cyclopentenyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-yl-propenonyl)tetrahydroisoquinoline Derivatives

a) Molecular weight as the free form. b) n = 2. c) IC₅₀ value calculated from another test (n = 3). d) t-Butylamine salt. e) HCl salt. f) The activation level at 1000 nM. The maximal activation level was not observed due to cytotoxicity at higher concentrations and the EC₅₀ value was not obtained. g) Not determined.

The compound with 6-oxadiazolone (20f) showed approximately 4-fold higher activity (EC₅₀ = 44 nM, maximal activation 27.6%), while that with 6-tetrazole (20g) showed approximately 14-fold higher activity (EC₅₀ = 13 nM, maximal activation 30.0%) than that of 6-carboxyl (20a). The order of acidity was 20a > 20g > 20f, and the order of lipophilicity was 20f > 20a > 20g. These compounds are considered to bind to the PPARγ protein via ionic and/or hydrophobic interactions. Thioether derivative 26 showed lower activity for PPARγ than its corresponding ether derivative 20g (discussed below). Three derivatives substituted with an acidic group at the 6-position (20a, 20f, and 20g) showed very weak PTP1B inhibitory activities (IC₅₀ = 8200, 3500, and 1100 nM, respectively), which were approximately 50–90-fold lower than their PPARγ partial agonist activities, suggesting selective PPARγ agonists.

To gain structural insights into the mechanisms underlying partial agonism by our compounds, we performed computational docking calculations on 20a, 20f, 20g, and 26 into the human PPARγ proteins. Full agonists (compounds 1, 2, and 10) were also docked in the same manner for comparisons. The human PPARγ protein structures were derived from the Protein Data Bank (PDB; www.rcsb.org),³⁸⁾ and 5Y2O (complexed with a full agonist) and 4A4V chain A (complexed with a partial agonist) were selected as the docking templates. Computational docking calculations were performed for both structures, followed by energy minimization to refine the docking pose. As a result, full agonists 1, 2, and 10 were more stably docked into the template 5Y2O³⁹⁾ than 4A4V,²⁷⁾ and 20a, 20f, and 20g into the template 4A4V than 5Y2O. The molecular orientation of the compounds was similar in each series (Figs. 3, 4). The docking scores (kcal/mol) of the compounds 1, 2, 10, 20a, 20f, and 20g were −8.6, −8.4, −8.0, −9.1, −10.1, and −10.4 for 4A4V and −10.3, −10.6, −8.7, −7.8, −8.6, and −8.6 for 5Y2O, respectively. In our study, the docking score and experimental negative logarithm of the half maximal effective concentration (pEC₅₀) value were not correlated well, but an order of magnitude difference in the agonist activity could be verified by the docking score. By contrast, the orientation of 26 in its top-ranked pose was different from that of other partial agonists, and the pose with an orientation similar to them showed lower docking score (−7.6 kcal/mol). The bulky substitution from ether to thioether might disturb the correct positioning of the compound deep inside the binding pocket, leading the lower agonist activity.

Fig. 3. Docking Poses of Full Agonists to PPARγ

(a) Crystal structure of pioglitazone (PDB code 5Y2O). (b–d) Docking poses of compounds 1 (b), 2 (c), and 10 (d). Compounds and PPARγ residues described in the manuscript are shown as sticks and labeled. Hydrogen bonds and an aromatic interaction are depicted as cyan and green dotted lines, respectively. Secondary structures (Helix (H) and β-strand (S)) of PPARγ were predicted and depicted by Maestro (Schrödinger Suite 2018-1) and H3 is partially hidden for visibility (corresponding to residues 286–288).

Fig. 4. Docking Poses of Partial Agonists to PPARγ

(a) Crystal structure of amorfrutin 2 (PDB code 4A4V). (b–e) Docking poses of compounds 20a (b), 20f (d), and 20g (e), and the superposition of those of 10 (orange) and 20a (magenta) (c). Compounds and PPARγ residues described in the manuscript are shown as sticks and labeled. Hydrogen bonds and a salt bridge are depicted as cyan and orange dotted lines, respectively. Secondary structures (Helix (H) and β-strand (S)) of PPARγ were predicted and depicted by Maestro (Schrödinger Suite 2018-1) and H3 is partially hidden for visibility (corresponding to residues 286 and 287).

PPARγ full agonists, such as thiazolidinediones (TDZ), are known to interact with Helix 12 in the activation function 2 (AF-2) pocket of PPARγ and stabilize the active conformation via direct hydrogen bonding to Tyr473 (Helix 12), which allowed Helix 12 to dock to Helix 3 and Helix 11.⁴⁰⁾ In 5Y2O, pioglitazone also showed that the nitrogen atom of the TZD ring interacts with Tyr473 (Helix 12) via a direct hydrogen bond (Fig. 3a). Furthermore, two carbonyl groups of the TZD ring formed hydrogen bonds with His323 (Helix 4/5), Ser289 (Helix 3), and His449 (Helix 10/11). The phenyl and ethylpyridyl groups formed hydrophobic interactions with Cys285 (Helix3), Leu330 (Helix 4/5), Ile281 (Helix3), and Ile341 (β-strand 3). These interactions were considered to stabilize Helix 12 into an active conformation.

Among previously reported 2,7-substituted 1,2,3,4-tetrahydroisoquinoline 3-carboxyl acids, compound 1 exhibited PPARγ full agonist activity (EC₅₀ = 22 nM, maximal activation 104%) and weak PPARα agonist activity (EC₅₀ = 142 nM),²⁵⁾ and compound 2 exhibited PPARγ full agonist activity (EC₅₀ = 70 nM, maximal activation 102%)²⁶⁾; both were revealed here to have similar binding modes to that of pioglitazone. In compound 1, the phenyl ring in the 7-substituent was attached to the side of Helix 11 and Helix 12, forming interactions with Tyr473 (Helix 12) and His449 (Helix 10/11) by aromatic and/or hydrophobic interactions (Fig. 3b). The 7-substituent in compound 2 was also close to the side of Helix 11 and Helix 12 and may form van der Waals interactions around Tyr473 (Helix 12) and His449 (Helix 10/11) (Fig. 3c). Oxazole interacted with Ser289 (Helix 3) in compounds 1 and 2, and the carbonyl oxygen of the 2-position acyl group in compound 1 interacted with Ser342 (β-strand 3) via hydrogen bonds, whereas the furan ring in compound 2 formed aromatic and/or hydrophobic interactions along Helix 3 and β-strand 3. In compound 10 (EC₅₀ = 528 nM, maximal activation 98.7%), the carbonyl oxygen of the 2-position acyl group formed hydrogen bonds with Ser342 (β-strand 3) and the 7-position substituent interacted around Tyr473 (Helix 12) and His449 (Helix 10/11), whereas the linker length at the 7-position of compound 10 (methoxy) was shorter than those of compounds 1 and 2 (ethoxy) (Fig. 3d). Therefore, the interaction may be weaker than compounds 1 and 2, resulting in weak agonist activity.

On the other hand, unlike full agonists, partial agonists are known to preferentially stabilize other regions of the ligand-binding domain, particularly the β-sheet, and partially recruit a coactivator, resulting in PPARγ partial agonism.⁴⁰⁾ In 4A4V, the carboxyl group of amorfrutin 2 interacted with Ser342 (β-strand 3) by a direct hydrogen bond (Fig. 4a). In addition, the n-pentyl and isoprenyl groups, which are compact side chains of amorfrutin 2, formed weakly hydrophobic interactions with Met348 (β-strand 4), Ile341 (β-strand 3), Leu330 (Helix 4/5), and Leu353 (Helix 6). Compound 20a substituted with carboxyl at the 6-position showed a different binding mode from that of 10 substituted with carboxyl at the 3-position: changes in the molecular orientation and the disappearance of interactions with Helix 11 and Helix 12, which may be due to the introduction of the 6-substituent, at which the carbonyl oxygen forms a hydrogen bond with Glu343 (loop) and oxazole with Cys285 (Helix 3) (Figs. 4b, c). The present results demonstrated that the 3-carboxyl substituent was preferable for interactions with the PPARγ protein as a full agonist, and the 6-carboxyl substituent as a partial agonist. In addition to the interactions shown in compound 10, a nitrogen atom of 6-oxadiazolone in 20f interacted with Glu291 (Helix 3) via a hydrogen bond, and 6-tetrazole in 20g forms a salt bridge with Arg288 (Helix 3) and a hydrogen bond with Ser342 (β-strand 3) (Figs. 4d, e). Amorfrutin 2 does not form the salt bridge with Arg288 (Helix 3), and, thus, may exhibit weaker activity (EC₅₀ = 1200 nM) than 20g. A similar binding mode to β-sheet and Helix 3 was shown in other reported partial agonists, such as nTZDpa (Fig. 2, PDB: 2Q4S). As described above, the order of the partial agonist activity 20g > 20f > 20a may be explained by the interaction with the PPARγ protein.

Among the derivatives, compound 20g with the lowest lipophilicity (c log D_7.4 = 0.95) showed the most potent PPARγ partial agonist properties in the transactivation assay. 20g showed no PPARα or PPARδ agonist activity (EC₅₀ > 10000 nM). The plasma C_max of 20g at 10 mg/kg after oral administration reached 7.0 ± 2.1 µg/mL in Sprague-Dawley (SD) rats and 19.8 ± 0.6 µg/mL in KK-A^y mice, indicating good oral absorption. Compound 20g and pioglitazone were administered as a food admixture for 7 d and their anti-diabetic effects were compared. Despite very low maximal activation (30%), compound 20g at 30 mg/kg significantly lowered blood glucose, which was similar to pioglitazone, a full agonist at 10 mg/kg (Table 2). In the present experiments, neither 20g nor pioglitazone exerted severe adverse effects. Repeated dosing for a longer period may be required to clarify the less adverse effects of 20g than those of pioglitazone.

Table 2. Effects of 20g Administered Orally for 1 Week to KK-A^y Mice

		Control	20g 30 mg/kg/d	Pioglitazone 10 mg/kg/d
Male	Glucose (mg/dL)	584.5 ± 28.8	474.2 ± 33.4*	450.4 ± 35.2*
	TG (mg/dL)	646.2 ± 72.6	661.6 ± 75.2	611.6 ± 63.6
	Body weight (g)	49.1 ± 0.9	51.7 ± 1.1	51.8 ± 1.3
Female	Glucose (mg/dL)	448.0 ± 19.6	290.5 ± 43.0**	241.2 ± 30.7**
	TG (mg/dL)	583.0 ± 48.6	595.5 ± 82.9	663.9 ± 85.2
	Body weight (g)	48.8 ± 1.0	50.3 ± 0.8	51.9 ± 1.3

Mean ± S.E. (n = 7). * p < 0.05, ** p < 0.01 vs. Control, the Student’s t-test.

In summary, the present study found that 7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethoxy]-2-[3-(2-furyl)acryloyl]-1,2,3,4-tetrahydroisoquinoline derivatives substituted with an acidic group at the 6-position are selective PPARγ partial agonists. Computational docking calculations revealed the interaction mode of these partial agonists as well as 1,2,3,4-tetrahydroisoquinoline-based full agonists. Among the three partial agonists, 20g with 6-tetrazole had the most potent activity and lowest lipophilicity, and showed good oral absorption and experimental anti-diabetic activity.

Experimental

General

Melting points were measured on a melting point apparatus (Yamato MP-21; Yamato Scientific Co., Ltd., Tokyo, Japan) and were uncorrected. ¹H-NMR spectra were obtained on a NMR spectrometer at 400 MHz (JNM-AL400; JEOL Ltd., Tokyo, Japan) using tetramethylsilane (TMS) as an internal standard. IR spectra were recorded with a HORIBA FT-720 spectrometer (HORIBA, Kyoto, Japan). Mass spectra were obtained on an Expression CMS-L (Advion, Ithaca, 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-[(Cyclopent-3-enecarbonyl)amino]-3-oxobutyrate (3)

Cyclopent-3-enecarboxylic acid (2) (5.00 g, 44.6 mmol) was dissolved in SOCl₂ (9.80 mL, 134 mmol) and stirred at room temperature for 1 h. The reaction mixture was concentrated in a vacuum and the residue was dissolved in toluene and concentrated.

Et₃N (18.7 mL, 134 mmol) was added dropwise to a suspention of the obtained residue and methyl 2-amino-3-oxobutanoate hydrochloride (8.20 g, 48.9 mmol) in CH₂Cl₂ (80 mL) under ice-cooling, which was then stirred at the same temperature for 0.5 h. After the addition of a 10% aqueous citric acid solution, the reaction mixture was extracted with AcOEt, washed with brine, and dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography to give 3 (6.93 g, 69% yield for 2 steps) as an oil. ¹H-NMR (CDCl₃) δ: 2.39 (3H, s), 2.60–2.70 (4H, m), 3.08 (1H, quintet, J = 7.8 Hz), 3.82 (3H, s), 5.27 (1H, d, J = 6.6 Hz), 5.68 (2H, s), 6.58–6.73 (1H, br).

Methyl 2-Cyclopent-3-enyl-5-methyloxazole-4-carboxylate (4)

PPh₃ (30.7 g, 117 mmol) and Et₃N (33.1 mL, 237 mmol) were added to a solution of I₂ (29.7 g, 117 mmol) in CH₂Cl₂ (460 mL) under ice-cooling, which was then stirred at the same temperature for 10 min. Compound 3 (6.93 g, 30.8 mmol) in CH₂Cl₂ (60 mL) was added dropwise to the reaction mixture under ice-cooling and stirred at the same condition for 0.5 h. The reaction mixture was washed with water and saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 4 (4.76 g, 75% yield) as an oil. ¹H-NMR (CDCl₃) δ: 2.60 (3H, s), 2.70–2.87 (4H, m), 3.58 (1H, quintet, J = 7.6 Hz), 3.89 (3H, s), 5.72 (2H, s).

4-Chloromethyl-2-cyclopent-3-enyl-5-methyloxazole (6)

Lithium aluminum hydride (0.61 g, 16 mmol) was added portionwise to a solution of 5 (4.76 g, 23.0 mmol) in tetrahydrofuran (THF) (95 mL) under ice-cooling and stirred at the same condition for 10 min. Water was added to the reaction mixture. The precipitate was removed by filtration and washed with AcOEt. The filtrate was separated into two layers and the organic layer was washed with water and brine, dried over Na₂SO₄, and then evaporated under reduced pressure.

The residue was dissolved in SOCl₂ (9.10 mL, 120 mmol) and stirred at room temperature for 30 min. The reaction mixture was concentrated in a vacuum and the residue was dissolved in toluene and concentrated to give 6 (4.12 g, 91% yield) as an oil. ¹H-NMR (CDCl₃) δ: 2.31 (3H, s), 2.68–2.84 (4H, m), 3.50–3.61 (1H, m), 4.46 (2H, s), 5.73 (2H, s).

Methyl (S)-2-tert-Butoxycarbonyl-7-(2-cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy)-3,4-dihydro-1H-isoquinoline-3-carboxylate (8)

K₂CO₃ (1.35 g, 9.75 mmol) was added to a solution of 7²⁹⁾ (1.00 g, 3.25 mmol) and 6 (722 mg, 3.90 mmol) in N,N-dimethylformamide (DMF) (10 mL), and the mixture was stirred at 60°C for 2.5 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine, and dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 8 (1.38 g, 91% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.45 (4.5H, s), 1.52 (4.5H, s), 2.31 (3H, s), 2.70–2.85 (4H, m), 3.05–3.22 (2H, m), 3.53–3.65 (4H, m), 4.40–4.52 (1H, m), 4.62–4.72 (1H, m), 4.73–4.79 (0.5H, m), 4.84 (2H, s), 5.08–5.16 (0.5H, m), 5.73 (2H, s), 6.70–6.85 (2H, m), 7.00–7.09 (1H, m).

Methyl 7-(2-Cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-ylacryloyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (9)

A total of 9.1 M HCl in i-PrOH (0.34 mL, 3.1 mmol) was added to a solution of 8 (490 mg, 1.05 mmol) in HCO₂H (1.5 mL) under ice-cooling, and the mixture was stirred at room temperature 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 evaporated under reduced pressure.

The obtained residue was dissolved in CH₂Cl₂ (3.0 mL), (E)-3-(2-furyl)acrylic acid (160 mg, 1.16 mmol) and EDC·HCl (262 mg, 1.37 mmol) were added, and the mixture was stirred at room temperature for 2 h. After the addition of a 10% aqueous citric acid solution, the reaction mixture was extracted with CHCl₃ and washed with saturated aqueous NaHCO₃ solution and brine and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography to give 9 (450 mg, 88% yield, 2 steps) as an oil. ¹H-NMR (CDCl₃) δ: 2.32 (3H, s), 2.69–2.86 (4H, m), 3.05–3.37 (2H, m), 3.55–3.64 (4H, m), 4.55–4.64 (0.3H, m), 4.78–5.03 (3.7H, m), 5.04–5.08 (0.3H, m), 5.57–5.64 (0.7H, m), 5.74 (2H, s), 6.44–6.52 (1H, s), 6.56–6.63 (1H, m), 6.68 (0.3H, d, J = 15.1 Hz), 6.77–6.87 (2H, m), 6.91 (0.7H, d, J = 15.1 Hz), 7.04–7.12 (1H, m), 7.43–7.57 (2H, m).

7-(2-Cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-ylacryloyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid t-Butylamine Salt (10)

A 1.0 M aqueous LiOH solution (2.8 mL, 2.8 mmol) was added to a solution of 9 (450 mg, 0.921 mmol) in MeOH (8.3 mL) and THF (2.8 mL), and the mixture was stirred at room temperature for 40 min. The reaction mixture was washed with Et₂O, and the aqueous layer was acidified with a 1.0 M aqueous HCl solution, which was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was evaporated under reduced pressure to give carboxylic acid as a foam (420 mg). tert-Butylamine (49 µM, 0.46 mmol) was added to the solution of carboxylic acid in AcOEt (2.0 mL) and the reaction mixture was stirred at room temperature for 10 min. After the addition of t-BuOMe, the precipitate was filtered to give 10 (178 mg, 71% yield) as a solid, mp 111–114°C. ¹H-NMR (dimethyl sulfoxide (DMSO)-d₆) δ: 1.08–1.17 (9H, m), 2.30 (3H, s), 2.57–2.83 (4H, m), 2.88–2.97 (1H, m), 3.18–3.61 (2H, m), 4.36–4.45 (0.7H, m), 4.53–4.60 (0.7H, m), 4.76–4.95 (3.3H, m), 5.08–5.14 (0.3H, m), 5.73 (2H, s), 6.54–6.65 (1H, m), 6.70–6.90 (4H, m), 6.95–7.06 (1H, m), 7.24–7.37 (1H, m), 7.70–8.40 (3H, m). IR (ATR) cm⁻¹: 1646, 1604. MS m/z: 473 [M − H]⁻, 475 [M + H]⁺.

7-Hydroxy-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-6-carbaldehyde (12)

Hexamethylenetetramine (HMTA) (86.7 g, 0.618 mol) was added to a solution of 11³⁶⁾ (102 g, 0.416 mol) in trifluoroacetic acid (TFA) (1 L), and the mixture was refluxed for 3 h. After cooling to room temperature, TFA was evaporated under reduced pressure. The obtained residue was poured into cold water (1 L), neutralized with NaHCO₃, and extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 12 (74.0 g, 65% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.83–3.02 (2H, m), 3.80–3.95 (2H, m), 4.70–4.85 (0.9H, m), 5.10–5.25 (1.1H, m), 6.75–6.90 (1H, m), 7.20–7.45 (1H, m), 9.82–9.86 (0.45H, m), 10.23–10.30 (0.55H, m), 10.82–10.86 (0.45H, m), 11.93–11.98 (0.55H, m).

7-Benzyloxy-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-6-carboxylic Acid (13)

A solution of 12 (210 g, 0.769 mol) in DMF (1000 mL) was added to benzyl bromide (92.0 mL, 0.775 mol) and K₂CO₃ (160 g, 1.16 mol), and the mixture was stirred at room temperature for 2 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine, and dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was added to n-hexane (500 mL), which was stirred at room temperature for 40 min, and the precipitate that formed was filtered (281 g).

NaClO₂ (124 g, 1.37 mol) was added to a solution of NaH₂PO₄ (131 g, 1.09 mol) in water (1200 mL), and the mixture was stirred at room temperature for 0.5 h. The reaction mixture was added to a solution of the obtained precipitate (133 g, 0.366 mol) in t-BuOH–THF (2 : 1) (1800 mL) and 2-methyl-2-butane (275 mL, 2.59 mol) at the same temperature, and was then stirred at room temperature for 1.5 h. The reaction mixture was cooled with an ice bath and stirred for 15 min. The precipitate was filtered to give 13 (49.0 g, 35% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.80–2.90 (2H, m), 3.79 (2H, t, J = 5.9 Hz), 4.75 (0.8H, s), 4.78 (1.2H, s), 5.15 (2H, s), 7.15 (0.6H, s), 7.20 (0.4H,s), 7.28–7.50 (6H, m), 12.62 (1H, s).

7-Benzyloxy-2-tert-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-6-carboxylic Acid (14)

A 1.0 M aqueous LiOH solution (640 mL, 640 mmol) was added to a solution of 13 (92.6 g, 0.244 mol) in 1,4-dioxane (640 mL), and the mixture was stirred at room temperature for 0.5 h. Boc₂O (69.2 g, 0.317 mol) was added to the obtained mixture, which was then stirred at the same temperature for 1 h. AcOEt was added to the reaction mixture, which was washed with 10% aqueous citric acid solution, water, and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was added to n-hexane (100 mL), which was stirred at room temperature for 1 h. The precipitate was filtered to give 14 (88.0 g, 94% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.78–2.88 (2H, m), 3.58–3.68 (2H, m), 4.59 (2H, s), 5.26 (2H, s), 6.85 (1H, s), 7.37–7.48 (5H, m), 7.98 (1H, s), 10.76 (1H, s).

tert-Butyl 7-Benzyloxy-6-carbamoyl-3,4-dihydro-1H-isoquinoline-2-carboxylate (15d)

N-Methylmorpholine (27.1 g, 0.268 mol) and isobutyl chloroformate (34.8 mL, 0.268 mol) were added dropwise to a suspension of 14 (97.4 g, 0.254 mol) in THF (2 L) under ice-cooling, which was stirred at the same temperature for 20 min. A 28% aqueous NH₃ solution (97 mL, 1.4 mol) was added dropwise to the obtained mixture, and the mixture was stirred further at the same temperature for 2 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure to give 15d (117 g, quant.) as a solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.82 (2H, t, J = 5.4 Hz), 3.64 (2H, t, J = 5.4 Hz), 4.59 (2H, s), 5.26 (2H, s), 6.85 (1H, s), 7.39–7.46 (5H, m), 7.98 (1H, s), 9.80–11.20 (2H, br).

tert-Butyl 7-Benzyloxy-6-hydroxymethyl-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (15h)

A total of 1.0 M BH₃·THF (25.1 mL, 25.1 mmol) was added dropwise to a solution of 14 (3.20 g, 8.35 mmol) in THF (25 mL) under ice-cooling, and the mixture was stirred at room temperature for 2 h. The reaction mixture was added to a 1.0 M aqueous NaOH solution (30 mL, 30 mmol), which was stirred at room temperature for 1 h. CHCl₃ was added to the mixture, separated into two layers, and the organic layer was washed with water and dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 15h (3.20 g, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.25 (1H, t, J = 6.6 Hz), 2.71–2.80 (2H, m), 3.57–3.69 (2H, m), 4.53 (2H, s), 4.69 (2H, d, J = 6.6 Hz), 6.68 (1H, s), 7.07 (1H, s), 7.30–7.44 (5H, m).

Methyl 2-tert-Butoxycarbonyl-7-hydroxy-1,2,3,4-tetrahydroisoquinolin-6-carboxylate (17a)

MeI (0.325 mL, 5.22 mmol) and K₂CO₃ (721 mg, 5.22 mmol) were added to a solution of 14 (1.00 g, 2.61 mmol) in DMF (10 mL), which was then stirred at room temperature for 16 h. AcOEt was added to the reaction mixture, washed with water and saturated brine, and then dried over Na₂SO_4. The solvent was evaporated under reduced pressure to give 14a as a colorless oil (crude).

A solution of crude 14a in MeOH (50 mL) was hydrogenated at 0.4 MPa in the presence of 10% Pd–C (100 mg) at room temperature for 86 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 17a (730 mg, 91% yield for 2 steps) as a white solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.72–2.82 (2H, m), 3.57–3.67 (2H, m), 3.94 (3H, s), 4.55 (2H, s), 6.73 (1H, s), 7.60 (1H, s), 10.50–10.55 (1H, br).

tert-Butyl 6-Carbamoyl-7-hydroxy-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (17d)

A solution of 15d (28.7 g, 75.0 mmol) in MeOH (560 mL) was hydrogenated at 0.3 MPa in the presence of 10% Pd–C (2.9 g) at room temperature for 75 min. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 17d (8.8 g, 40% yield) as an oil. NMR (CDCl₃) δ: 1.49 (9H, s), 2.76 (2H, t, J = 5.8 Hz), 3.63 (2H, t, J = 5.8 Hz), 4.54 (2H, s), 5.56–6.22 (1H, br), 6.74 (1H, s), 7.13 (1H, s), 11.83–11.99 (1H, br).

tert-Butyl 7-Hydroxy-6-hydroxymethyl-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (17h)

Compound 17h was synthesized from 15h according to the procedure for the synthesis of 17d. Yield 66%. A pale yellow oil. ¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.16–2.33 (1H, br), 2.67–2.75 (2H, m), 3.56–3.65 (2H, m), 4.49 (2H, s), 4.81 (2H, s), 6.64 (1H, s), 6.81 (1H, s), 7.14–7.19 (1H, br).

tert-Butyl 6-Amino-7-hydroxy-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (27)

An 85% Zn powder (1.15 g, 15 mmol) was added to a solution of 16³⁷⁾ (0.88 g, 3.0 mmol) in acetic acid (5 mL), and the mixture was stirred at same temperature for 0.5 h. The reaction mixture was added to water and the Zn powder was removed by filtration. The filtrate was basified with saturated aqueous NaHCO₃ solution, which was extracted with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 27 (597 mg, 76% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.61–2.73 (2H, m), 3.52–3.65 (2H, m), 4.41 (2H, s), 6.52 (1H, s), 6.57 (1H, s).

tert-Butyl 6-Dimethylamino-7-hydroxy-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (17b)

A total of 37% formalin (8.49 mL, 113 mmol) and NaBH(OAc)₃ (2.87 g, 13.6 mmol) were added to a solution of 27 (597 mg, 2.26 mmol) in THF (12 mL), and the mixture was stirred at room temperature for 1 h. The reaction mixture was basified with saturated aqueous NaHCO₃ solution, which was extracted with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 17b (661 mg, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.63 (6H, s), 2.69–2.77 (2H, m), 3.54–3.69 (2H, m), 4.48 (2H, s), 6.67 (1H, s), 6.90 (1H, s).

Methyl 2-tert-Butoxycarbonyl-7-[2-(cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (18a)

Compound 18a was synthesized from 17a and 6 according to the procedure for the synthesis of 8. Yield 84%. A white solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.34 (3H, s), 2.69–2.85 (6H, m), 3.50–3.67 (3H, m), 3.85 (3H, s), 4.55 (2H, s), 4.97 (2H, s), 5.74 (2H, s), 6.86 (1H, s), 7.58 (1H, s).

tert-Butyl 7-[2-(Cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy]-6-dimethylamino-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (18b)

Compound 18b was synthesized from 17b and 6 according to the procedure for the synthesis of 8. Yield 35%. A brown oil. ¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.30 (3H, s), 2.68–2.84 (12H, m), 3.50–3.70 (3H, m), 4.48 (2H, s), 4.90 (2H, s), 5.72 (2H, s), 6.64 (1H, s), 6.73 (1H, s).

tert-Butyl 6-Cyano-7-[2-cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (18c)

Et₃N (8.27 mL, 59.3 mmol) and POCl₃ (3.32 mL, 35.6 mmol) were added to a solution of 18d (4.60 g, 11.9 mmol) in CH₂Cl₂ (90 mL), and the mixture was stirred at room temperature for 2 h. The reaction mixture was poured into water, neutralized with K₂CO₃, and separated into two layers. The organic layer was dried over Na₂SO₄, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 18c (3.16 g, 72% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.36 (3H, s), 2.67–2.86 (6H, m), 3.51–3.67 (3H, m), 4.57 (2H, s), 5.00 (2H, s), 5.74 (2H, s), 6.91 (1H, s), 7.32 (1H, s).

tert-Butyl 6-Carbamoyl-7-[2-cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (18d)

Compound 18d was synthesized from 17d and 6 according to the procedure for the synthesis of 8. Yield 64%. A white solid. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.32 (3H, s), 2.67–2.86 (6H, m), 3.52–3.68 (3H, m), 4.59 (2H, s), 4.97 (2H, s), 5.58–5.67 (1H, br), 5.74 (2H, s), 6.81 (1H, s), 7.93–8.02 (2H, m).

tert-Butyl 7-[2-(Cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy]-6-hydroxymethyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (18h)

Compound 18h was synthesized from 17h and 6 according to the procedure for the synthesis of 8. Yield 91%. A white solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.31 (3H, s), 2.68–2.85 (7H, m), 3.48–3.67 (3H, m), 4.53 (2H, s), 4.60 (2H, s), 4.91 (2H, s), 5.72 (2H, s), 6.74 (1H, s), 7.02 (1H, s).

tert-Butyl 7-(2-Cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy)-6-formyl-1,2,3,4-tetrahydroiso Quinoline-2-carboxylate (28)

DMSO (0.93 mL, 13 mmol), i-Pr₂NEt (2.2 mL, 13 mmol), and SO₃·Py (1.25 g, 7.83 mmol) were added to a solution of 18h (1.15 g, 2.61 mmol) in CH₂Cl₂ (20 mL), which was then stirred at room temperature for 30 min. After the addition of a 1.0 M aqueous HCl solution, the mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 28 (1.10 g, 96% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.32 (3H, s), 2.69–2.86 (6H, m), 3.53–3.68 (3H, m), 4.59 (2H, s), 4.97 (2H, s), 5.74 (2H, s), 6.87 (1H, s), 7.60 (1H, s), 10.40 (1H, s).

tert-Butyl 7-[2-(Cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy]-6-(1H-imidazol-2-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (18e)

A 40% aqueous glyoxal solution (63 µL, 0.55 mmol) and 28% aqueous NH₃ solution (0.15 mL, 2.3 mmol) were added to a solution of 28 (200 mg, 0.465 mmol) in MeOH (1 mL), which was then stirred at room temperature for 14 h. After the addition of saturated aqueous NaHCO₃ solution, the mixture was extracted twice with CHCl₃. The organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 18e (167 mg, 77% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.31 (3H, s), 2.75–2.93 (6H, m), 3.53–3.69 (3H, m), 4.59 (2H, s), 5.00 (2H, s), 5.79 (2H, s), 6.81 (1H, s), 7.00–7.20 (2H, m), 8.10 (1H, s), 11.50–11.80 (1H, br).

tert-Butyl 7-[2-(Cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy]-6-(5-oxo-[1,2,4]oxadiazolidin-3-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (18f)

Et₃N (1.19 mL, 8.54 mmol) was added to a suspension of NH₂OH·HCl (594 mg, 8.54 mmol) in DMSO (1 mL) and stirred at room temperature for 10 min. The precipitate was removed by filtration and washed with THF. After the removal of THF under a vacuum, the obtained residue was added to 18c and stirred at 85°C for 15 h. After the addition of water, the mixture was extracted twice with AcOEt. The organic layer was washed with water and brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure.

Pyridine (0.13 mL, 1.6 mmol) and isobutyl chloroformate (0.18 mL, 1.4 mmol) were added to a solution of the obtained residue in DMF (3.0 mL) under ice-cooling and the mixture was stirred at the same temperature for 1 h. After the addition of water, the mixture was extracted twice with AcOEt. The organic layer was washed with water and brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure.

The residue was dissolved in xylene (5.0 mL) and the mixture was refluxed for 3 h. After cooling to room temperature, the mixture was purified by silica gel column chromatography to give 18f (470 mg, 67% yield for 3 steps) as an oil. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.33 (3H, s), 2.68–2.96 (6H, m), 3.58–3.79 (3H, m), 4.60 (2H, s), 5.01 (2H, s), 5.74 (2H, s), 6.83 (1H, s), 7.72 (1H, s), 11.65–11.83 (1H, br).

tert-Butyl 7-[2-Cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (18g)

n-Bu₃SnN₃ (3.25 mL, 11.9 mmol) was added to a solution of 18c (1.15 g, 3.11 mmol) in toluene (12 mL), and the mixture was refluxed for 18 h. After cooling to room temperature, the reaction mixture was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 18g (1.15 g, 90% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 2.36 (3H, s), 2.80–2.98 (6H, m), 3.62–3.75 (3H, m), 4.63 (2H, s), 5.08 (2H, s), 5.81 (2H, s), 6.89 (1H, s), 8.19 (1H, s).

Methyl (E)-7-(2-Cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-yl-acryloyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (19a)

Compound 19a was synthesized from 18a according to the procedure for the synthesis of 9. Yield 85%, 2 steps. A yellow oil. ¹H-NMR (CDCl₃) δ: 2.34 (3H, s), 2.69–2.95 (6H, m), 3.45–3.55 (1H, m), 3.81–3.94 (5H, m), 4.76–4.86 (2H, m), 4.98 (2H, s), 6.44–6.49 (1H, m), 6.57 (1H, d, J = 3.2 Hz), 6.87 (1H, d, J = 15.2 Hz), 6.93 (1H, s), 7.46 (1H, s), 7.50 (1H, d, J = 15.2 Hz), 7.60 (1H, s).

(E)-7-(2-Cyclopent-3-enyl-5-methyloxazol-4-ylmethoxy)-2-(3-furan-2-yl-acryloyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic Acid t-Butylamine Salt (20a)

A 1.0 M aqueous LiOH solution (2.58 mL, 2.58 mmol) was added to a solution of 19a (420 mg, 0.860 mmol) in MeOH (2.5 mL) and THF (7.5 mL), and the mixture was stirred at room temperature for 1 h. The reaction mixture was acidified with a 1.0 M aqueous HCl solution, which was extracted with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. tert-Butylamine (90 µM, 0.88 mmol) was added to a solution of the obtained residue in AcOEt (2.0 mL), and the precipitate was then filtered to give 20a (125 mg, 27% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.24 (9H, s), 2.28 (3H, s), 2.56–2.96 (2H, m), 3.45–3.55 (1H, m), 3.78–3.90 (2H, m), 4.76–4.86 (2H, br), 4.89 (2H, s), 6.44–6.50 (1H, m), 6.56 (1H, d, J = 3.2 Hz), 6.77 (1H, d, J = 15.2 Hz), 6.86 (1H, s), 7.44–7.47 (1H, m), 7.49 (1H, d, J = 15.2 Hz), 7.60 (1H, s). IR (ATR) cm⁻¹: 1646, 1604. MS m/z: 473 [M−H]⁻.

(E)-7-[2-(Cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy]-6-dimethylamino-2-[3-(2-furyl)acryloyl]-1,2,3,4-tetrahydroisoquinoline Hydrochloride (20b)

A total of 8.6 M HCl in i-PrOH (0.29 mL, 2.5 mmol) was added to a solution of 18b (380 mg, 0.838 mmol) in HCO₂H (2 mL), and the mixture was stirred at room temperature for 15 min. The reaction mixture was basified with saturated aqueous NaHCO₃ solution, which was extracted with AcOEt. The organic layer was washed with water and saturated brine, and dried over Na₂SO₄. The solvent was evaporated under reduced pressure.

The obtained residue was dissolved in CH₂Cl₂ (5.0 mL) to which was added (E)-3-(2-furyl)acrylic acid (120 mg, 0.869 mmol) and EDC·HCl (210 mg, 1.10 mmol), and the mixture was stirred at room temperature for 2 h. After the addition of a 10% aqueous citric acid solution, the reaction mixture was extracted with CH₂Cl₂, washed with saturated aqueous NaHCO₃ solution and brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography. A total of 7.3 M HCl in i-PrOH (0.13 mL, 0.95 mmol) and i-Pr₂O (2.0 mL) was added to the obtained residue in AcOEt (2.0 mL), and the precipitate was then filtered to give 20b (281 mg, 55% yield for 3 steps) as a pale yellow solid, mp 91–94°C. ¹H-NMR (CDCl₃) δ: 2.42 (3H, s), 2.70–3.00 (6H, m), 3.23 (6H, s), 3.60–3.77 (1H, m), 3.80–3.96 (2H, m), 4.83 (2H, s), 5.05 (2H, s), 5.74 (2H, s), 6.47 (1H, dd, J = 3.4, 1.9 Hz), 6.58 (1H, d, J = 3.4 Hz), 6.84 (1H, d, J = 15.1 Hz), 7.01 (1H, s), 7.47 (1H, d, J = 1.9 Hz), 7.49 (1H, d, J = 15.1 Hz), 7.91 (1H, s). IR (ATR) cm⁻¹: 1644. MS m/z: 472 [M − H]⁻, 474 [M + H]⁺.

(E)-6-Cyano-7-[2-(cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy)-2-[3-(2-furyl)acryloyl]-1,2,3,4-tetrahydroisoquinoline (20c)

A total of 8.6 M HCl in i-PrOH (0.24 mL, 2.1 mmol) was added to a solution of 18c (300 mg, 0.689 mmol) in HCO₂H (1 mL), and the mixture was then stirred at room temperature for 15 min. The reaction mixture was basified with saturated aqueous NaHCO₃ solution, which was extracted with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure.

(COCl)₂ (0.089 mL, 1.0 mmol) and 1 drop of DMF were added to a solution of (E)-3-(2-furyl)acrylic acid (140 mg, 1.04 mmol) in CH₂Cl₂ (10 mL), and the mixture was stirred at room temperature for 0.5 h. The obtained mixture and Et₃N (0.48 mL, 3.5 mmol) were added to a solution of the crude intermediate in CH₂Cl₂ (5 mL), which was stirred at the same temperature for 2 h. The reaction mixture was washed with water, saturated aqueous NaHCO₃ solution, and saturated brine, and then dried over Na₂SO₄. The obtained residue was added to i-Pr₂O (2.0 mL), and the precipitate was then filtered to give 20b (244 mg, 78% yield for 2 steps) as a white solid, mp 159–161°C. ¹H-NMR (CDCl₃) δ: 2.36 (3H, s), 2.67–2.94 (6H, m), 3.50–3.63 (1H, m), 3.80–3.98 (2H, m), 4.84 (2H, s), 5.01 (2H, s), 5.73 (2H, s), 6.44–6.51 (1H, m), 6.57 (1H, d, J = 3.2 Hz), 6.83 (1H, d, J = 15.1 Hz), 6.99 (1H, s), 7.34 (1H, s), 7.43–7.47 (1H, m), 7.50 (1H, d, J = 15.1 Hz). IR (ATR) cm⁻¹: 2217, 1600. MS m/z: 456 [M + H]⁺.

(E)-6-Carbamoyl-7-[2-(cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy]-2-[3-(2-furyl)acryloyl]-1,2,3,4-tetrahydroisoquinoline (20d)

Compound 20d was synthesized from 18d according to the procedure for the synthesis of 20c. Yield 36% for 2 steps. A white solid. mp 172–174°C. ¹H-NMR (CDCl₃) δ: 2.32 (3H, s), 2.67–2.86 (4H, m), 2.86–3.00 (2H, m), 3.50–3.63 (1H, m), 3.80–3.97 (2H, m), 4.85 (2H, s), 4.98 (2H, s), 5.65 (1H, s), 5.74 (2H, s), 6.44–6.51 (1H, m), 6.56 (1H, d, J = 3.2 Hz), 6.86 (1H, d, J = 15.1 Hz), 6.88 (1H, s), 7.44–7.47 (1H, m), 7.50 (1H, d, J = 15.1 Hz), 7.90–8.00 (1H, br), 8.00 (1H, s). IR (ATR) cm⁻¹: 1602, 1421. MS m/z: 474 [M − H]⁻.

(E)-7-[2-(Cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy)-2-[3-(2-furyl)acryloyl]-6-(1H-imidazol-2-yl)-1,2,3,4-tetrahydroisoquinoline Hydrochloride (20e)

Compound 20e was synthesized from 18e according to the procedure for the synthesis of 20b. Yield 33% for 3 steps. A white solid, mp not determined. ¹H-NMR (CDCl₃) δ: 2.30 (3H, s), 2.53–2.63 (2H, m), 2.68–2.78 (2H, m), 2.78–2.95 (2H, m), 3.49–3.60 (1H, m), 3.78–3.92 (2H, m), 4.77–4.96 (2H, m), 5.21 (2H, s), 5.72 (2H, s), 6.60–6.65 (1H, m), 6.89 (1H, d, J = 3.0 Hz), 7.01 (1H, d, J = 15.1 Hz), 7.39 (1H, d, J = 15.1 Hz), 7.42–7.50 (1H, m), 7.74 (2H, s), 7.82 (1H, s), 7.90 (1H, s), 14.30–14.45 (1H, br). IR (ATR) cm⁻¹: 1645, 1601. MS m/z: 495 [M − H]⁻, 497 [M + H]⁺.

(E)-7-[2-(Cyclopent-3-enyl)-5-methyloxazol-4-ylmethoxy)-2-[3-(2-furyl)acryloyl]-6-(5-oxo-[1,2,4]oxadiazolidin-3-yl)-1,2,3,4-tetrahydroisoquinoline (20f)

Compound 20f was synthesized from 18f according to the procedure for the synthesis of 9. Yield 47% for 2 steps. A white solid, mp 179–181°C. ¹H-NMR (CDCl₃) δ: 2.33 (3H, s), 2.68–2.78 (2H, m), 2.82–2.98 (4H, m), 3.68–3.76 (1H, m), 3.84–3.95 (2H, m), 4.87 (2H, s), 5.02 (2H, s), 5.74 (2H, s), 6.45–6.49 (1H, m), 6.58 (1H, d, J = 3.4 Hz), 6.85 (1H, d, J = 14.9 Hz), 6.90 (1H, s), 7.45–7.47 (1H, m), 7.51 (1H, d, J = 14.9 Hz), 7.74 (1H, s), 11.70–11.85 (1H, br). IR (ATR) cm⁻¹: 1766, 1635. MS m/z: 513 [M − H]⁻.

(E)-7-[2-(Cyclopent-3-eny)-5-methyloxazol-4-ylmethoxy]-2-[3-(2-furyl)acryloyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (20g)

Compound 20g was synthesized from 18g according to the procedure for the synthesis of 9. Yield 59% for 2 steps. A white solid, mp 178–180°C. ¹H-NMR (CDCl₃) δ: 2.37 (3H, s), 2.85–3.06 (6H, m), 3.70 (1H, quintet, J = 8.0 Hz), 3.84–4.03 (2H, m), 4.90 (2H, m), 5.10 (2H, s), 5.81 (2H, s), 6.44–6.52 (1H, m), 6.58 (1H, d, J = 3.2 Hz), 6.88 (1H, d, J = 14.9 Hz), 6.95 (1H, s), 7.47 (1H, s), 7.51 (1H, d, J = 14.9 Hz), 8.21 (1H, s). IR (ATR) cm⁻¹: 1651, 1608. MS m/z: 497 [M − H]⁻, 499 [M + H]⁺. Anal. Calcd for C₂₇H₃₁NO₃S₂: C, 65.06; H, 5.26; N, 16.86. Found: C, 65.19; H, 5.31; N, 16.71.

Methyl 7-Hydroxy-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (21)

MeI (1.99 mL, 32.0 mmol) and K₂CO₃ (5.90 g, 42.7 mmol) were added to a solution of 13 (8.07 g, 21.3 mmol) in DMF (80 mL) under ice-cooling, which was then stirred at room temperature for 3 h. The reaction mixture was added to water and extracted twice with AcOEt. The organic layer was washed with water and brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure.

A solution of the obtained residue in MeOH (217 mL) was hydrogenated at 0.3 MPa in the presence of 10% Pd-C (2.17 g) at room temperature for 1 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 21 (5.67 g, 88% yield for 2 steps) as a solid. NMR (CDCl3) δ: 1.49 (9H, s), 2.76 (2H, t, J = 5.8 Hz), 3.63 (2H, t, J = 5.8 Hz), 4.54 (2H, s), 5.56–6.22 (1H, br), 6.74 (1H, s), 7.13 (1H, s), 11.83–11.99 (1H, br).

Methyl 7-Dimethylthiocarbamoyloxy-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (29)

Et₃N (5.33 mL, 37.4 mmol), DMAP (0.46 g, 3.7 mmol), and N,N-dimethylthiocarbamoyl chloride (4.62 g, 37.4 mmol) were added to a solution of compound 21 (5.67 g, 18.7 mmol) in 1,4-dioxane (60 mL) and the mixture was stirred at 80°C for 15 h. After the addition of water, the mixture was extracted twice with AcOEt. The organic layer was washed with water and brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 29 (7.30 g, quant.) as a solid. ¹H-NMR (CDCl₃) δ: 2.94–3.05 (2H, m), 3.39 (2H, s), 3.46 (4H, s), 3.83 (3H, s), 3.85–3.96 (2H, m), 4.78 (0.7H, s), 4.83 (1.3H, s), 6.89 (0.3H, s), 6.91 (0.7H, s), 7.81 (0.7H, s), 7.83 (0.3H, s).

Methyl 7-Dimethylcarbamoylsulfanyl-2-(2,2,2-trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (22)

Compound 29 (7.64 g, 19.6 mmol) was dissolved in n-tetradecane (80 mL) and the mixture was stirred at 250°C under a N₂ atmosphere for 15 h. After cooling to room temperature, the mixture was purified by silica gel column chromatography to give 22 (1.63 g, 22% yield) as an oil. ¹H-NMR (CDCl₃) δ: 2.92–3.20 (8H, m), 3.81–3.94 (5H, m), 4.77 (0.7H, s), 4.81 (1.3H, s), 7.38 (0.3H, s), 7.40 (0.7H, s), 7.72 (0.7H, s), 7.74 (0.3H, s).

Methyl tert-Butoxycarbonyl-7-dimethylcarbamoylsulfanyl-1,2,3,4-tetrahydroisoquinolin-6-carboxylate (30)

A 0.50 M aqueous K₂CO₃ solution (30 mL, 15 mmol) was added to a solution of compound 22 (1.53 g, 3.92 mmol) in MeOH (15 mL) and the mixture was stirred at room temperature for 1 h. Boc₂O (1.71 g, 7.84 mmol) was added to the reaction mixture and stirred at 50°C for 18 h. After the addition of water, the mixture was extracted twice with AcOEt. The organic layer was washed with brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 30 (1.22 g, 79% yield for 2 steps) as a solid. ¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.80–2.91 (2H, m), 2.95–3.20 (6H, m), 3.60–3.70 (2H, m), 3.86 (3H, s), 4.59 (2H, s), 7.34 (1H, s), 7.70 (1H, s).

Methyl tert-Butoxycarbonyl-7-mercapto-1,2,3,4-tetrahydroisoquinolin-6-carboxylate (23)

A 28% solution of NaOMe in MeOH (0.71 mL, 3.7 mmol) was added to a solution of compound 30 (1.22 g, 3.09 mmol) in MeOH (12 mL) at room temperature under a N₂ atmosphere, and the mixture was stirred at 60°C for 6 h. After the addition of a 10% aqueous citric acid solution, the mixture was extracted twice with AcOEt. The organic layer was washed with brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 23 (999 mg, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.71–2.85 (2H, m), 3.55–3.68 (2H, m), 3.91 (3H, s), 4.52 (2H, s), 4.69 (1H, s), 7.04 (1H, s), 7.79 (1H, s).

Methyl tert-Butoxycarbonyl-7-[(2-cyclopent-3-enyl)-5-methyloxazol-4-ylmethylsulfanyl]-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (31)

Cs₂CO₃ (1.83 g, 5.61 mmol) was added to a solution of 23 (1.21 g, 3.74 mmol) and 6 (890 mg, 4.49 mmol) in DMF (25 mL), and the mixture was stirred at room temperature for 2 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography to give 31 (1.48 g, 82% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.23 (3H, s), 2.66–2.78 (4H, m), 2.78–2.88 (2H, m), 3.47–3.59 (1H, m), 3.60–3.68 (2H, m), 3.88 (3H, s), 3.96 (2H, s), 4.56 (2H, s), 5.72 (2H, s), 7.19 (1H, s), 7.71 (1H, s).

2-tert-Butoxycarbonyl-7-[(2-cyclopent-3-enyl)-5-methyloxazol-4-ylmethylsulfanyl]-1,2,3,4-tetrahydroisoquinoline-6-carboxylic Acid (32)

A 1.0 M aqueous LiOH solution (9.0 mL, 9.0 mmol) was added to a solution of 31 (1.48 g, 3.05 mmol) in MeOH (9.0 mL) and THF (27 mL), and the mixture was stirred at 40°C for 2 h. The reaction mixture was acidified with a 1.0 M aqueous HCl solution, which was extracted with AcOEt. The organic layer was washed with saturated brine, and dried over Na₂SO₄. The solvent was evaporated under reduced pressure to give 32 (1.26 g, 88% yield) as a white solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.17 (3H, s), 2.56–2.79 (4H, m), 2.79–2.87 (2H, m), 3.46–3.58 (1H, m), 3.59–3.69 (2H, m), 3.96 (2H, s), 4.55 (2H, s), 5.70 (2H, s), 7.20 (1H, s), 7.72 (1H, s).

tert-Butyl 6-Carbamoyl-7-[(2-cyclopent-3-enyl)-5-methyloxazol-4-ylmethylsulfanyl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (24)

Compound 24 was synthesized from 32 according to the procedure for the synthesis of 15d. Yield 84%. A white solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 1.91 (3H, s)、2.62–2.80 (4H, m), 2.80–2.87 (2H, m), 3.43–3.55 (1H, m), 3.59–3.68 (2H, m), 3.88 (2H, s), 4.51 (2H, s), 5.71 (2H, s), 7.20 (1H, s), 7.65 (1H, s).

tert-Butyl 6-Cyano-7-[2-(cyclopent-3-enyl)-5-methyloxazol-4-ylmethylsulfanyl]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (33)

Compound 33 was synthesized from 24 according to the procedure for the synthesis of 18c. Yield quant. A white solid. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 2.12 (3H, s), 2.62–2.78 (4H, m), 2.79–2.87 (2H, m), 3.46–3.58 (1H, m), 3.59–3.68 (2H, m), 3.98 (2H, s), 4.54 (2H, s), 5.72 (2H, s), 7.30 (1H, s), 7.39 (1H, s).

tert-Butyl 7-(2-Cyclopent-3-enyl-5-methyl-oxazol-4-ylmethylsulfanyl)-6-(1H-tetrazol-5-yl)-3,4-dihydro-1H-isoquinoline-2-carboxylate (34)

Compound 34 was synthesized from 33 according to the procedure for the synthesis of 18g. Yield 75%. A white solid. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.14 (3H, s), 2.65–2.75 (2H, m), 2.77–2.87 (2H, m), 2.89 (2H, t, J = 5.6 Hz), 3.54–3.64 (1H, m), 3.66 (2H, t, J = 5.6 Hz), 3.96 (2H, s), 4.61 (2H, s), 5.71 (2H, s), 7.49 (1H, s), 7.91 (1H, s).

2-[(E)-3-(2-Furyl)acryloyl]-7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethylsulfanyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (26)

A total of 8.6 M HCl in i-PrOH (0.39 mL, 3.4 mmol) was added to a solution of 34 (558 mg, 1.13 mmol) in HCO₂H (2 mL), and the mixture was stirred at room temperature for 15 min. After the addition of Et₂O, the precipitate formed was collected by filtration to give a crude intermediate.

(COCl)₂ (0.145 mL, 1.7 mmol) and 1 drop of DMF were added to a solution of (E)-3-(2-furyl)acrylic acid (230 mg, 1.70 mmol) in CH₂Cl₂ (20 mL), and the mixture was stirred at room temperature for 0.5 h. The obtained mixture and Et₃N (0.79 mL, 5.7 mmol) were added to a solution of the crude intermediate in CH₂Cl₂ (5 mL), which was stirred at the same temperature for 3 h. The reaction mixture was washed with water, saturated aqueous NaHCO₃ solution, and saturated brine, and then dried over Na₂SO₄. The residue obtained was purified by silica gel column chromatography to give 26 (290 mg, 50% yield for 2 steps) as a pale yellow solid, mp 158–161°C. ¹H-NMR (CDCl₃) δ: 2.15 (3H, s), 2.64–2.75 (2H, m), 2.77–2.90 (2H, m), 2.94–3.08 (2H, m), 3.47–3.60 (1H, m), 3.83–3.94 (5H, m), 3.97 (2H, s), 4.88 (2H, s), 5.71 (2H, s), 6.43–6.52 (1H, m), 6.58 (1H, d, J = 3.4 Hz), 6.85 (1H, d, J = 14.8 Hz), 7.46 (1H, s), 7.51 (1H, d, J = 14.8 Hz), 7.55 (1H, s), 7.93 (1H, s). IR (ATR) cm⁻¹: 1637, 1536. MS m/z: 513 [M−H]⁻, 515 [M + H]⁺.

PPARγ, PPARα, and PPARδ Agonist Activities

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

The full-length human PPARγ1 plasmid (Open Biosystems, Huntsville, U.S.A.), human PPARα plasmid (GeneCopoeia Inc., Rockville, U.S.A.), or human PPARδ plasmid (GeneCopoeia Inc.) were electroporated into COS-1 cells with the human RXRα plasmid (GeneCopoeia Inc.) and the reporter plasmid pGL3-PPREx4-tk-luc using Nucleofector II (AAD-1001S, Lonza Group Ltd., Basel, Switzerland). Cells were incubated for 24 h in the presence and absence of test compounds in DMEM containing 10% FBS under 5% CO₂ at 37°C. The medium was removed and luciferase activities were measured using a commercial kit (PicaGene LT7.5; TOYO B-Net Co., Ltd., Tokyo, Japan) and microplate luminescence reader (Dainippon Sumitomo Pharma Co., Ltd.). EC₅₀ values were assessed from dose-response curves. The maximal activation level relative to the level maximally activated by farglitazar, a PPARγ agonist (10⁻⁷ M), Wy-14643, a PPARα agonist (10⁻⁵ M), and GW-501516, a PPARδ agonist (10⁻⁷ M), were evaluated.

PTP1B Inhibitory Activity

Inhibitory activities against recombinant human PTP1B (Enzo Life Sciences, Inc., Farmingdale, NY, U.S.A.) were measured using p-nitrophenyl phosphate (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.

Plasma Concentration after Oral Administration in Male SD Rats and KK-A^y Mice

Male SD rats (7 weeks old; Japan SLC, Inc., Hamamatsu, Japan) and male KK-A^y mice (10 weeks old; Clea Japan, Inc., Tokyo, Japan) were used. The test compound at 10 mg/kg suspended in 0.5% methylcellulose solution was administered orally and then blood was taken from the external jugular vein after 0.5, 1, 3, 5, and 8 h in rats, and from the tail vein after 0.5, 1, 4, and 8 h in KK-A^y mice. The plasma concentrations of the compounds were assessed using HPLC equipment consisting of a pump (PU-980; JASCO, Tokyo, Japan), UV detector (UV-970; JASCO), autoinjector (AS-950; JASCO), and STR-ODS-II column (5 µm, 4.6 × 150 mm; Shimadzu GLC Ltd., Kyoto, Japan).

Hypoglycemic and Hypotriglyceridemic Effects in Male and Female KK-A^y Mice

Male KK-A^y mice (12 weeks old; Clea Japan, Inc.) and female KK-A^y mice (19 weeks old; Clea Japan, Inc., Tokyo, Japan) were allocated into three groups: control group, 20g-treated group, and pioglitazone-treated group (n = 7). Mice were given normal powdered food or food mixed with the test compounds for 7 d. Blood samples were taken from the tail veins of non-fasted mice 24 h after the final administration. Plasma glucose and triglyceride levels in mice were assessed using commercial kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Computational Analyses

High-quality (resolution ≤2.0 Å, no mutated residue around the ligand-binding site and no distortion by crystal contacts^41,42)) X-ray crystal structures of human PPARγ complexed with non-covalent agonists were retrieved from the PDB. The structures obtained were also curated by the crystallization condition (pH 7.5 ± 1.0) for the following computational docking calculations under mild pH conditions. Sixteen complex structures were found (PDB codes 1ZGY, 2HWQ, 2YFE (chain A), 3B1M, 3ET3, 3LMP, 3V9T, 3V9V, 3VSO, 4A4V (chain A), 4A4W (chain A), 4F9M, 5GTN, 5HZC (chain A), 5Y2O and 5Y2T (chain A)). These structures were manually divided into two groups according to whether the ligand interacted with Helix 10/11 and/or Helix 12 in the AF-2 pocket of PPARγ. A structural similarity analysis was then performed for each group by calculating the atomic root-mean-square deviation (RMSD) of amino acid residues around the ligand-binding site using the conformer cluster script of Schrödinger Suite 2018-1 (Schrödinger, LLC, New York, U.S.A.). One representative structure was selected from each of the two groups, as the nearest one to the centroid, respectively. Protein structures were then preprocessed using Protein Preparation Wizard in Maestro (Schrödinger Suite 2018-1), which added hydrogen atoms, repaired side chains of incomplete residues, and optimized the hydrogen bonding network. The restrained minimization of protein structures were performed with an RMSD cut-off value of 0.3 Å using the OPLS3 force field. Receptor grids for computational ligand docking were then generated for both of the two protein structures using the grid generation feature of Glide (Schrödinger Suite 2018-1). LigPrep (Schrödinger Suite 2018-1) was used to define the possible 3D structures of compounds with various ionization states at pH 7.0 ± 2.0, tautomers, and ring conformations. Multiple conformations of compounds were then generated using ConfGen (Schrödinger Suite 2018-1). All docking calculations were performed with Glide and the run in standard precision (SP) mode. The ligand van der Waals radius for non-polar atoms was scaled by factors of 0.8 and 1.0. The conformers generated by ConfGen were docked into the receptor grids, and the top pose for each compound, assessed by the Glide docking score, was collected. Each docked structure was then energy-minimized using the OPLS3 force field and 0.05 kcal/mol/Å of convergence with a distance-dependent dielectric constant (ε = 4).

Conflict of Interest

The authors declare no conflicts of interest.

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