HETEROCYCLES

Paper | Special issue | Vol. 90, No. 1, 2015, pp. 372-404
Received, 15th May, 2014, Accepted, 30th May, 2014, Published online, 12th June, 2014.
DOI: 10.3987/COM-14-S(K)29

■ Synthesis and Evaluations of GLP-1 Secretion and Anti-Diabetic Effect in KKAy Mice of New Tricyclic Compounds

Daisuke Minehira, Daisuke Takeda, Shota Miyawaki, Atsushi Kato, Isao Adachi,* Akira Miyazaki, Ryuta Miyatake, Masahito Umezaki, Kyoko Miura, Yoshiro Kitahara, Kenji Sugimoto, Yuji Matsuya, and Naoki Toyooka*

Graduate School of Science and Technology, Toyama University, Gofuku 3190, Toyama 930-8555, Japan

Abstract

Glucagon-like peptide-1 (GLP-1), which belongs to the family of incretins, plays important role for the regulation of plasma glucose. Accordingly, GLP-1-based therapies for type 2 diabetes have recognized as one of the most interesting target. In this study, we have found the new tricyclic compounds having strong GLP-1 secretion from human intestinal L cells, and anti-diabetic properties in spontaneously obese and diabetic KKAy mice. The most potent compound 5ka was obtained as the unexpected product, and we would like to report the details of the synthesis, structure elucidations, pharmacological activities on secretion of GLP-1, and anti-diabetic effects using diabetic KKAy mice.

INTRODUCTION
Glucagon-like peptide-1 (GLP-1) belongs to the family of incretins, and it is secreted from enteroendocrine L cells following the stimulation by nutrients.1,2 It promotes glucose-stimulated insulin secretion and suppresses postprandial hyperglycemia.3 Furthermore, GLP-1 has also been reported to have various other beneficial effects such as suppressing glucagon release,4 promoting β-cells proliferation,5 suppressing food intake,6 slowing gastric emptying,7 improving fatty liver,8 and cardiovascular protection.9 However, the incretin effect is reduced in patients with type 2 diabetes, mainly due to defective GLP-1 secretion in response to stimulation by various nutrients.10,11 For this reason, although clinically available GLP-1-based therapies, especially DPP-IV inhibitors are currently widely used for treatment of patients with type 2 diabetes, their monotherapy might be insufficient for achievement of treatment goals for blood glucose control. Therefore, restoration of GLP-1 secretion by intestinal L cells could be an important new therapeutic option for the management of metabolic syndrome. Various mechanisms, such as KATP channel,12 Na+/glucose co-transporter 1,13 glucose transporter 2,14 sweet taste receptor,15 TGR-5,16 G protein-coupled receptors,17 and 5-hydroxytryptamine (5-HT)4 receptor,18 have been reported to be involved in inducing GLP-1 secretion. But none of the compounds interacting with these targets are currently clinically available. In this study, we tried to discover a novel small molecule inducing GLP-1 secretion from intestinal L cells. As part of a program directed at studying the discovery of new drug candidate based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa,19,20 we found the new tricyclic compounds having strong GLP-1 secretion from human intestinal L cells, and anti-diabetic properties in spontaneously obese and diabetic KKAy mice. We report herein the synthesis and evaluations of GLP-1 secretion and anti-diabetic effect of these tricyclic compounds.

RESULTS AND DISCUSSION
CHEMISTRY

We planned the synthesis of new tricyclic carboxylic acid 1 as the first target compound. The synthesis began with known lactone 2a,21 which was converted to thiolactone 3a with Lawesson’s reagent. The thiolactone 3a was converted to the ester 4a, which was treated with TMSI to afford the S-lactone 5a as the major product along with 1a. The separation of 5a and 1a was difficult, and repeated column chromatography of the above mixture provided pure 5a in 66% yield. In the case of the ester 4b, derived from 3b via 2b, we got the same result, and the S-lactone 5b was isolated in 64% yield as the major product again (Scheme 1).

At this time, the first target compound 1a or 1b was obtained as a minor product, we tried to synthesize 5 as new target compound. To avoid the tedious separation of 1 and 5 in the final step, we explored another synthetic route as shown in Scheme 2. The lactones 2c-k and 2m were prepared using the same procedure as in Scheme 1. The lactone 2m was transformed into the lactone 2l by the Suzuki-Miyaura coupling reaction.22 The lactones 2c-l were treated with Lawesson’s reagent to afford the thiolactones 3c-l. Treatment of the thiolactones 3c-l with TMSI gave rise to S-lactone 6c-l, which was transformed into the target acids 5c-l via the corresponding esters 7c-l.

PHARMACOLOGICAL ACTIVITY: EFFECTS ON SECRETION OF GLP-1
We investigated the direct effect of the compounds on GLP-1 secretion by intestinal L cells using a human intestinal L cell line (NCI-H716). In this cell line, glucose has been reported to stimulate GLP-1 release at concentrations above 50 mM.15, 23 In this assay system, we found that 5k had a strong activity to induce GLP-1 secretion under normal glucose concentration (5.6 mM) without any leakage of LDH in the culture medium (Figure 1). Their EC50 values for GLP-1 secretion is 100 μM for 5k. In addition to the lowest EC50 value for 5k, Emax value of this compound is the highest (ca. 1300 pM) among all tested compounds. Therefore, we investigated the in vivo effect of 5k on blood glucose both in normal and diabetic mice.

HYPOGLYCEMIC EFFECT OF 5K AFTER AN ORAL GLUCOSE LOAD AND THE INFLUENCE OF A GLP-1 ANTAGONIST (EXENDIN 9-39) ON IT IN C57BL/6J MICE
Acute effect of 5k after an oral glucose load on blood glucose was investigated in normal mice. An oral administration of 5k (100 mg/kg) just before glucose loading suppressed the increase of blood glucose

(Figure 2). Since exendin (9-39) is often used to confirm the involvement of GLP-1 in the hypoglycemic effect of the compounds in vivo,23,24 we investigated the influence of exendin (9-39) on hypoglycemic effect of 5k. The acute hypoglycemic effect of 5k after an oral glucose load is cancelled by subcutaneous injection of exendin (9-39) (24 nmol/kg) prior to administration of 5k. These data indicate that 5k suppresses the increase of blood glucose via stimulation of GLP-1 secretion in vivo. Then, we investigated the anti-diabetic effect of 5k in diabetic mice.

PHARMACOLOGICAL ACTIVITY: ANTI-DIABETIC EFFECTS OF 5K
Effect of long-term treatment with 5k on blood glucose control was investigated using obese and diabetic KKAy mice. Data are summarized in Table 1. Treatment with 5k for 3 weeks had no impact on food intake and body weight, but it lowered both fasting and non-fasting blood glucose. As a result, HbA1c in 5k-treated mice was decreased. In addition, fasting plasma insulin level and fasting plasma glucagon level tended to be lowered after 3 weeks treatment with 5k. The anti-diabetic effect of 5k was almost comparable to that of pioglitazone. On the other hand, although a DPP-IV inhibitor sitagliptin is known to increase plasma GLP-1 level by inhibiting degradation of GLP-1 by DPP-IV, in this study, 3 weeks treatment with sitagliptin in KKAy mice showed no effect on blood glucose and HbA1c. Taken together, stimulation of GLP-1 secretion by 5k may be a new therapeutic option for patients with type 2 diabetes.

STRUCTURE OF THE MOST POTENT COMPOUND 5K
As shown in 2.2, the most potent compound for the secretion of GLP-1 was 5k, which was a 5:1 mixture of 2 compounds. We thought these two compounds would be the stereoisomers 5kb and 5kc produced in the Friedel-Crafts reaction of the commercially available alcohol 8 (Scheme 3).

So we planned more selective synthesis of the major compound 5kc. The silane reduction of the tertiary alcohol 10, derived from the alcohol 8 via the ketone 9, afforded the two products 11b and 11c in the ratio of 3:1 (Scheme 4.). Although the product 11b was identical with the minor product of the Friedel-Crafts reaction the other product 11c was not identical with the major product of the Friedel-Crafts reaction product (Figure 3).

These results suggested that the major product in the Friedel-Crafts reaction was not the stereoisomer 11c but the rearranged product. To determine the structure of the major and rearranged product in the Friedel-Crafts reaction, first we prepared the p-toluenesulfonic acid salt 14 of the major aniline derivative 13b in the silane reduction of 10 (Scheme 4). The X-ray analysis of 14 revealed that this product was the 1,4-trans-substituted compound (Figure 4).

Next we examined the synthesis of p-nitrobenzenesulfonamide derivative 15 of the major product 11a in the Friedel-Crafts reaction. Nitration of the mixture of 11a and 11b followed by hydrogenation of the resulting nitro derivatives 12a and 12b afforded the corresponding anilines 13a and 13b, which were separated in this stage. The major aniline 13a was converted to 15 by the usual manner (Scheme 5). The structure of 15 was determined by the X-ray analysis shown in Figure 5.

The PM6 calculations for the intermediary cations in the Friedel-Crafts reaction revealed that the rearranged cation A is more stable than the cation B, initially produced by the action of the alcohol 8 with AlCl3 (Figure 6). According to these results, we concluded the major product of the Friedel-Crafts reaction was 1,3-cis-substituted derivative 11a.

The pure 1,3-cis-substituted aniline 13a and 1,4-trans-substituted aniline 13b in hand, we tried to synthesize the pure carboxylic acids 5ka and 5kb for the evaluations of the effect of these stereoisomers in the secretion of GLP-1. The aniline 13a was converted to the lactone 2ka, which was transformed into 5ka via thiolactone 3ka, S-lactone 6ka, and ester 7ka using the same procedure as shown in Scheme 2 (Scheme 6).

The regioisomer 5kb was also synthesized as shown in Scheme 7.

PHARMACOLOGICAL EFFECTS OF 5Ka AND 5Kb ON SECRETION OF GLP-1

Then, we evaluated the effect of these regioisomers (5ka and 5kb) on GLP-1 secretion by NCI-H716. Intriguingly, as shown in Figure 7, 5ka showed strong activity of GLP-1 secretion, while 5kb had no effect. Therefore, the active compound is 1,3-cis form (5ka).

CONCLUSION
We prepared 14 tricyclic compounds 5a-5kb having the different substituent on the 6-position. Evaluations of these compounds for the direct effect on GLP-1 secretion by intestinal L cells using a human intestinal L cell line (NCI-H716) revealed that the compound 5k exhibited strong activity to induce GLP-1 secretion. In addition, 5k showed the anti-diabetic effect in diabetic KKAy mice. We also identified the active compound for the above induction of GLP-1 and anti-diabetic effect was 5ka, which has the 1,3-cis-substituted cyclohexylcyclohexyl substituent on the 6-position of tricyclic nuclei. This compound was unexpected product, and was produced in the Friedel-Crafts reaction by the interesting stereoselective rearrangement on this reaction process.

EXPERIMENTAL
General information
Melting points are uncorrected. Flash chromatography was performed on Kanto Kagaku silica gel 60N. NMR spectra were recorded on a JEOL a-GX 400 and JEOL JNX-ECX500 spectrometer using CDCl3 as the solvent. Chemical shifts (δ) are given in ppm downfield from TMS and referenced to CHCl3 (7.26 ppm) as an internal standard. Peak multiplicities are designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad and coupling constants in (J) Hz. High-resolution mass spectral data were obtained on a JEOL MStation JMS-700. All commercial reagents were used as received unless otherwise noted.
Synthesis of lactone 2b
According to the literature procedure,25 lactone 2b was synthesized.
6-Cyclohexyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2b). Yield: 28%; mp 175-176 °C; IR (KBr): 3269, 2920, 2849, 1735 cm-1; 1H NMR (300 MHz, CDCl3): δ 9.03 (1H, br), 7.43-7.38 (2H, m), 7.30-7.26 (1H, m), 4.70 (2H, t, J = 6.2 Hz), 3.15 (2H, t, J = 6.2 Hz), 2.60-2.57 (1H, m), 1.94-1.26 (10H, m); 13C NMR (125 MHz, CDCl3): δ 161.74, 140.82, 137.15, 126.94, 124.43, 122.91, 122.14, 117.43, 112.72, 69.48, 44.50, 34.87, 26.93, 26.13, 21.42; MS (EI) m/z 269 (M+); HRMS (EI) calcd for C17H19NO2: 269.1416 (M+), found: 269.1414.

Synthesis of thiolactones 3a and 3b
To a stirred solution of lactone (1 mmol) in toluene (5 mL) was added Lawesson’s reagent (0.55 eq), and the resulting mixture was refluxed for 20–24 h. After cooling, the solvent was removed, and the residue was chromatographed on SiO2 (hexane–acetone = 20:1) to give the corresponding thiolactone.
6-tert-Butyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3a). Yield: 95%; mp 40-42 °C; IR (KBr): 2957, 1541, 1231 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.80 (1H, br), 7.56 (1H, d, J = 1.7 Hz), 7.51 (1H, dd, J = 1.7, 9.0 Hz), 7.35 (1H, d, J = 9.0 Hz), 4.74 (2H, t, J = 6.4 Hz), 3.18 (2H, t, J = 6.4 Hz), 1.38 (9H, s); 13C NMR (75 MHz, DMSO-d6): δ 197.90, 142.94, 137.94, 132.31, 125.85, 123.49, 116.72, 116.39, 112.53, 71.26, 34.42, 31.39, 20.60; MS (EI) m/z 259 (M+); HRMS (EI) calcd for C15H17NOS: 259.1031 (M+), found: 259.1022.
6-Cyclohexyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3b). Yield: 95%; mp 40-42 °C; IR (KBr): 3348, 2922, 2848, 1541, 1226 cm-1; 1H NMR (400 MHz, CDCl3): δ 8.75 (1H, br), 7.40 (1H, s), 7.31 (1H, d, J = 8.7 Hz), 7.27 (1H, dd, J = 1.4, 8.7 Hz), 4.72 (2H, t, J = 6.4 Hz), 3.15 (2H, t, J = 6.4 Hz), 2.57 (1H, tt, J = 3.2, 11.4 Hz), 1.92-1.84 (4H, m), 1.78-1.74 (1H, m), 1.50-1.23 (5H, m); 13C NMR (75 MHz, DMSO-d6): δ 197.93, 139.96, 138.34, 132.26, 127.18, 123.81, 118.34, 116.06, 112.68, 71.26, 43.82, 34.36, 26.49, 25.70, 20.57; MS (EI) m/z 285 (M+); HRMS (EI) calcd for C17H19NOS: 285.1187 (M+), found: 285.1191.

Synthesis of esters 4a and 4b
To a stirred solution of thiolactone (1 mmol) in DMF (5 mL) was added NaH (60%, 1.20 equiv) at 0 °C, and the reaction mixture was stirred at 0 °C for 30 min. To the mixture was added BrCH2CO2t-Bu at 0 °C, and the resulting mixture was stirred at room temperature for 20–24 h. The reaction was quenched with H2O (10 mL), and the aqueous mixture was extracted with Et2O (10 mL × 3). The organic extracts were combined, dried over MgSO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 20:1) to give the corresponding ester.
(6-tert-Butyl-1-thioxo-3,4-dihydro-1H-pyrano[3,4-b]indol-9-yl)acetic acid tert-butyl ester (4a). Yield: quant.; mp 144-145 °C; IR (KBr): 2967, 1748, 1527, 1228 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.56 (1H, d, J = 1.8 Hz), 7.53 (1H, dd, J = 1.8, 8.7 Hz), 7.19 (1H, d, J = 8.7 Hz), 5.47 (2H, s), 4.66 (2H, t, J = 6.4 Hz), 3.17 (2H, t, J = 6.4 Hz), 1.45 (9H, s), 1.36 (9H, s); 13C NMR (75 MHz, CDCl3): δ 196.28, 167.28, 144.19, 139.29, 131.38, 126.46, 122.04, 118.60, 116.69, 109.65, 81.82, 69.93, 46.55, 34.46, 31.36, 27.90, 21.26; MS (EI) m/z 373 (M+); HRMS (EI) calcd for C21H27NO3S: 373.1712 (M+), found: 373.1732.
(6-Cyclohexyl-1-thioxo-3,4-dihydro-1H-pyrano[3,4-b]indol-9-yl)acetic acid tert-butyl ester (4b). Yield: quant.; mp 123-125 °C; IR (KBr): 2925, 2851, 1740, 1525, 1228 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.44 (1H, s), 7.34 (1H, dd, J = 1.7, 9.0 Hz), 7.19 (1H, d, J = 9.0 Hz), 5.49 (2H, s), 4.67 (2H, t, J = 6.4 Hz), 3.17 (2H, t, J = 6.4 Hz), 2.61-2.57 (1H, m), 1.92-1.85 (4H, m), 1.78-1.76 (1H, m), 1.51-1.24 (5H, m), 1.46 (9H, s); 13C NMR (75 MHz, CDCl3): δ 196.63, 167.62, 141.62, 140.05, 131.72, 128.19, 122.71, 118.58, 118.50, 110.04, 82.28, 70.19, 46.92, 44.51, 34.87, 28.17, 27.01, 26.21, 21.59; MS (EI) m/z 399 (M+); HRMS (EI) calcd for C23H29NO3S: 399.1868 (M+), found: 399.1858.

Synthesis of carboxylic acids 5a and 5b
To a stirred suspension of NaI (4.00 equiv) in CHCl3 (3 mL) was added TMSCl (4.00 equiv), and the resulting mixture was stirred at room temperature for 15 min. To a solution of t-butyl ester (1 mmol) in CHCl3 (5 mL) was transferred a solution of TMSI, prepared above, via a cannula, and then the resulting mixture was refluxed for 1 days. After cooling, the reaction was quenched with 10% HCl aq, and the aqueous mixture was extracted with CHCl3 (10 mL × 3). The organic extracts were combined, dried over MgSO4, and evaporated. The residue was chromatographed repeatedly on SiO2 (hexane–acetone = 2:1) to give the carboxylic acid.
(6-tert-Butyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5a). Yield: 66%; mp 237-239 °C; IR (KBr): 2964, 1727, 1623 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 7.67 (1H, s), 7.52 (2H, s), 5.19 (2H, s), 3.47 (2H, t, J = 6.1 Hz), 3.31 (2H, t, J = 6.1 Hz), 1.35 (9H, s); 13C NMR (75 MHz, DMSO-d6): δ 182.80, 169.96, 143.03, 136.61, 127.11, 126.46, 125.75, 123.68, 116.14, 110.41, 46.31, 34.42, 31.44, 30.84, 21.70; MS (EI) m/z 317 (M+); HRMS (EI) calcd for C17H19NO3S: 317.1086 (M+), found: 317.1091.
(6-Cyclohexyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5b). Yield: 64%; mp 245-247 °C; IR (KBr): 2924, 2850, 1717, 1619, 1251 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.45 (1H, s), 7.33 (1H, d, J = 8.5 Hz), 7.21 (1H, d, J = 8.5 Hz), 5.26 (2H, s), 3.43 (2H, t, J = 6.2 Hz), 3.32 (2H, t, J = 6.2 Hz), 2.62-2.58 (1H, m), 1.92-1.85 (4H, m), 1.78-1.76 (1H, m), 1.50-1.38 (4H, m), 1.32-1.26 (1H, m); 13C NMR (75 MHz, DMSO-d6): δ 182.82, 169.96, 140.06, 137.00, 127.08, 127.02, 126.14, 124.04, 117.81, 110.58, 46.31, 43.77, 34.39, 30.83, 26.49, 25.67, 21.69; MS (EI) m/z 343 (M+); HRMS (EI) calcd for C19H21NO3S: 343.1242 (M+), found: 343.1280.

Synthesis of lactones 2c-m
According to the literature procedure,25 known lactones (2c, 2d, 2i, 2j, 2m) and the following lactones were prepared.
6-Ethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2e). Yield: 37%; mp 158-160 °C; IR (KBr): 3297, 1675 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.80 (1H, br), 7.42 (1H, s), 7.33 (1H, d, J = 8.7 Hz), 7.27 (1H, d, J = 8.7 Hz), 4.74 (2H, t, J = 6.5 Hz), 3.16 (2H, t, J = 6.5 Hz), 2.74 (2H, q, J = 7.6 Hz), 1.29 (3H, t, J = 7.6 Hz); 13C NMR (75 MHz, CDCl3): δ 161.70, 136.95, 136.31, 127.38, 124.13, 122.53, 121.75, 118.24, 112.66, 69.36, 28.72, 21.23, 16.02; MS (EI) m/z 215 (M+); HRMS (EI) calcd for C13H13NO2: 215.0946 (M+), found: 215.0945.
6-sec-Butyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2f). Yield: 43%; mp 155-157 °C; IR (KBr): 3280, 2958, 2919, 2871, 1733 cm-1; 1H NMR (300 MHz, CDCl3): δ 9.44 (1H, br), 7.45-7.40 (2H, m), 7.26-7.23 (1H, m), 4.71 (2H, t, J = 6.3 Hz), 3.16 (2H, t, J = 6.3 Hz), 2.70 (1H, sext, J = 6.9 Hz), 1.65 (2H, quint, J = 7.4 Hz), 1.29 (3H, d, J = 7.1 Hz), 0.84 (3H, t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): δ 161.64, 140.12, 137.11, 126.64, 124.34, 122.77, 122.06, 117.95, 112.72, 69.51, 41.73, 31.46, 22.36, 21.57, 12.40; MS (EI) m/z 243 (M+); HRMS (EI) calcd for C15H17NO2: 243.1259 (M+), found: 243.1255.
6-Cyclopentyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2g). Yield: 34%; mp 144-146 °C; IR (KBr): 3296, 2950, 2865, 1707 cm-1; 1H NMR (300 MHz, CDCl3): δ 9.31 (1H, br), 7.46 (1H, s), 7.42 (1H, d, J = 8.7 Hz), 7.31 (1H, d, J = 8.7 Hz), 4.70 (2H, t, J = 6.3 Hz), 3.18-3.04 (3H, m), 2.18-1.57 (8H, m); 13C NMR (75 MHz, CDCl3): δ 161.47, 138.94, 136.91, 126.98, 124.36, 122.76, 122.13, 117.79, 112.61, 69.49, 46.02, 34.97, 25.58, 21.57; MS (EI) m/z 255 (M+); HRMS (EI) calcd for C16H17NO2: 255.1259 (M+), found: 255.1259.
6-Cycloheptyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2h). Yield: 53%; mp 168-170 °C; IR (KBr): 3277, 2921, 2851, 1735 cm-1; 1H NMR (300 MHz, CDCl3): δ 9.36 (1H, br), 7.41 (1H, s), 7.41 (1H, d, J = 8.1 Hz), 7.26 (1H, d, J = 8.1 Hz), 4.70 (2H, t, J = 6.2 Hz), 3.15 (2H, t, J = 6.2 Hz), 2.77 (1H, quint, J = 5.1 Hz), 1.98-1.92 (2H, m), 1.84-1.48 (10H, m); 13C NMR (75 MHz, CDCl3): δ 161.57, 142.55, 136.85, 126.67, 124.23, 122.76, 121.98, 117.11, 112.71, 69.45, 47.04, 37.23, 27.90, 27.23, 21.49; MS (EI) m/z 283 (M+); HRMS (EI) calcd for C18H21NO2: 283.1572 (M+), found: 283.1574.
6-Bicyclohex-4-yl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2k). About 5:1 mixture of 2ka and 2kb; Yield: 26%; IR (KBr): 3289, 2923, 2849, 1699 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.72 (1H, br), 7.43 (1H, s), 7.37 (1H, d, J = 8.5 Hz), 7.30 (1H, s), 4.69 (2H, t, J = 6.2 Hz), 3.15 (2H, t, J = 6.2 Hz), 2.61-2.44 (1H, m), 2.06-0.86 (20H, m).
6-Phenyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2l). To a stirred solution of 2m (266 mg, 1.00 mmol) in 1,4-dioxane (15 mL) were added PhB(OH)2 (134 mg, 1.10 mmol), KBr (131 mg, 1.10 mmol), K3PO4・3H2O (399 mg, 1.50 mmol) and Pd(PPh3)4 (116 mg, 0.10 mmol), and the resulting mixture was refluxed for 13 h. The reaction mixture was diluted with CH2Cl2, and the organic layer was washed with sat. NaHCO3 aq. The organic layer was dried over MgSO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 12:1) to give 2l (77.4 mg, 29%). mp 247-250 °C; IR (KBr): 3278, 1692 cm-1; 1H NMR (400 MHz, CDCl3): δ 8.86 (1H, br), 7.80 (1H, s), 7.64 (1H, d, J = 8.7 Hz), 7.61 (2H, d, J = 7.3 Hz), 7.51 (1H, d, J = 8.7 Hz), 7.44 (2H, t, J = 7.3 Hz), 7.33 (1H, t, J = 7.3 Hz), 4.71 (2H, t, J = 6.2 Hz), 3.19 (2H, t, J = 6.2 Hz); 13C NMR (125 MHz, CDCl3): δ 161.18, 141.50, 137.56, 134.63, 128.80, 127.28, 126.90, 125.07, 123.38, 122.94, 120.97, 119.07, 112.99, 69.47, 21.47; MS (EI) m/z 263 (M+); HRMS (EI) calcd for C17H13NO2: 263.0946 (M+), found: 263.0949.

Synthesis of thiolactones 3c-l
To a stirred solution of lactone (1 mmol) in toluene (5 mL) was added Lawesson’s reagent (0.55 eq), and the resulting mixture was refluxed for 20–24 h. After cooling, the solvent was removed, and the residue was chromatographed on SiO2 (hexane–acetone = 20:1) to give the corresponding thiolactone.
4,9-Dihydro-3H-pyrano[3,4-b]indole-1-thione (3c). Yield: 93%; mp 115-116 °C; IR (KBr): 3347, 1537, 1208 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 11.67 (1H, br), 7.70 (1H, d, J = 8.2 Hz), 7.47 (1H, d, J = 8.3 Hz), 7.36 (1H, ddd, J = 1.4, 6.9, 8.2 Hz), 7.11 (1H, ddd, J = 1.1, 6.9, 8.3 Hz), 4.71 (2H, t, J = 6.5 Hz), 3.16 (2H, t, J = 6.5 Hz); 13C NMR (75 MHz, DMSO-d6): δ 198.04, 139.42, 132.08, 126.87, 123.73, 121.85, 120.49, 116.21, 112.84, 71.29, 20.52; MS (EI) m/z 203 (M+); HRMS (EI) calcd for C11H9NOS: 203.0405 (M+), found: 203.0439.
6-Methyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3d). Yield: 78%; mp 148-149 °C; IR (KBr): 3349, 1538, 1232 cm-1; 1H NMR (400 MHz, CDCl3): δ 8.76 (1H, br), 7.40 (1H, s), 7.30 (1H, d, J = 8.5 Hz), 7.24 (1H, dd, J =1.2, 8.8 Hz), 4.74 (2H, t, J = 6.5 Hz), 3.15 (2H, t, J = 6.5 Hz), 2.44 (3H, s); 13C NMR (75 MHz, DMSO-d6): δ 197.95, 138.02, 132.18, 129.30, 128.97, 123.91, 120.75, 115.64, 112.60, 71.24, 21.09, 20.52; MS (EI) m/z 217 (M+); HRMS (EI) calcd for C12H11NOS: 217.0561 (M+), found: 217.0542.
6-Ethyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3e). Yield: 86%; mp 122-123 °C; IR (KBr): 3349, 2949, 1531, 1231 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.80 (1H, br), 7.39 (1H, s), 7.30 (1H, d, J = 8.5 Hz), 7.26-7.23 (1H, m), 4.71 (2H, t, J = 6.6 Hz), 3.13 (2H, t, J = 6.6 Hz), 2.72 (2H, q, J = 7.5 Hz), 1.27 (3H, t, J = 7.5 Hz); 13C NMR (75 MHz, CDCl3): δ 198.06, 137.59, 137.35, 132.19, 128.73, 124.83, 119.60, 115.52, 112.11, 71.18, 28.91, 21.29, 16.12; MS (EI) m/z 231 (M+); HRMS (EI) calcd for C13H13NOS: 231.0718 (M+), found: 231.0716.
6-sec-Butyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3f). Yield: 94%; orange oil; IR (neat): 3357, 2957, 2925, 1541, 1229 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.82 (1H, br), 7.39-7.24 (3H, m), 4.73 (2H, t, J = 6.5 Hz), 3.16 (2H, t, J = 6.5 Hz), 2.69 (1H, sext, J = 7.0 Hz), 1.64 (2H, quint, J = 7.3 Hz), 1.29 (3H, d, J = 7.1 Hz), 0.84 (3H, t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): δ 197.94, 140.60, 137.72, 132.09, 127.59, 124.60, 118.92, 115.60, 112.11, 71.09, 41.58, 31.26, 22.18, 21.21, 12.30; MS (EI) m/z 259 (M+); HRMS (EI) calcd for C15H17NOS: 259.1031 (M+), found: 259.1034.
6-Cyclopentyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3g).
Yield: 94%; yellow oil; IR (neat): 3348, 2955, 1533, 1214 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.73 (1H, br), 7.38 (1H, s), 7.26 (2H, s), 4.67 (2H, t, J = 6.4 Hz), 3.11-2.99 (3H, m), 2.05-1.49 (8H, m); 13C NMR (75 MHz, CDCl3): δ 198.07, 139.58, 137.69, 132.25, 128.06, 124.75, 118.81, 115.60, 112.09, 71.19, 45.98, 34.90, 25.57, 21.38; MS (EI) m/z 271 (M+); HRMS (EI) calcd for C16H17NOS: 271.1031 (M+), found: 271.1028.
6-Cycloheptyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3h). Yield: 92%; yellow oil; IR (neat): 3338, 2919, 2851, 1541, 1229 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.78 (1H, br), 7.40 (1H, s), 7.32 (1H, d, J = 8.0 Hz), 7.27 (1H, d, J = 8.0 Hz), 4.73 (2H, t, J = 6.5 Hz), 3.16 (2H, t, J = 6.5 Hz), 2.75 (1H, quint, J = 5.1 Hz), 1.97-1.91 (2H, m), 1.83-1.57 (10H, m); 13C NMR (75 MHz, CDCl3): δ 197.99, 143.22, 137.56, 132.19, 127.82, 124.68, 118.24, 115.69, 112.14, 71.16, 47.02, 37.17, 27.91, 27.75, 21.31; MS (EI) m/z 299 (M+); HRMS (EI) calcd for C18H21NOS: 299.1344 (M+), found: 299.1344.
6-Chloro-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3i). Yield: quant.; mp: 155-157 °C; IR (KBr): 3219, 1537, 1228 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.88 (1H, br), 7.61 (1H, s), 7.35 (2H, s), 4.76 (2H, t, J = 6.4 Hz), 3.15 (2H, t, J = 6.4 Hz); 13C NMR (75 MHz, DMSO-d6): δ 198.06, 137.61, 132.99, 126.84, 124.86, 124.70, 120.95, 115.50, 114.51, 71.42, 20.38; MS (EI) m/z 237 (M+); HRMS (EI) calcd for C11H8NOSCl: 237.0015 (M+), found: 237.0001.
6-Methoxy-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3j). Yield: 93%; mp 153-155 °C; IR (KBr): 3326, 1534, 1219 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.79 (1H, br), 7.31 (1H, d, J = 9.1 Hz), 7.08 (1H, dd, J = 2.1, 9.1 Hz), 6.95 (1H, d, J = 2.1 Hz), 4.74 (2H, t, J = 6.5 Hz), 3.86 (3H, s), 3.15 (2H, t, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3): δ 197.97, 154.94, 134.50, 132.58, 124.97, 119.60, 115.27, 113.32, 101.21, 71.21, 55.74, 21.39; MS (EI) m/z 233 (M+); HRMS (EI) calcd for C12H11NO2S: 233.0510 (M+), found: 233.0512.
6-Bicyclohex-4-yl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3k). About 5:1 mixture of 3ka and 3kb; Yield: 82%; IR (KBr): 2919, 2849, 1542, 1230 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.78 (1H, br), 7.42 (1H, s), 7.34-7.27 (2H, m), 4.73 (2H, t, J = 6.5 Hz), 3.16 (2H, dt, J = 2.7, 6.5 Hz), 2.63-2.50 (1H, m), 1.98-0.86 (20H, m).
6-Phenyl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3l). Yield: 76%; yellow oil; IR (neat): 3364, 1542, 1232 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.90 (1H, br), 7.81 (1H, s), 7.67 (1H, d, J = 9.9 Hz), 7.63 (2H, d, J = 7.5 Hz), 7.48 (1H, d, J = 9.9 Hz), 7.46 (2H, t, J = 7.5 Hz), 7.35 (1H, t, J = 7.5 Hz), 4.79 (2H, t, J = 6.5 Hz), 3.23 (2H, t, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3): δ 198.01, 141.17, 138.34, 134.97, 132.66, 128.73, 127.67, 127.11, 126.93, 125.31, 119.88, 112.58, 71.24, 31.02, 21.39; MS (EI) m/z 279 (M+); HRMS (EI) calcd for C17H13NOS: 279.0718 (M+), found: 279.0718.

Synthesis of S-lactones 6c-l
To a stirred solution of NaI (4.00 equiv) in ClCH2CH2Cl (3 mL) was added TMSCl (4.00 equiv), and the resulting mixture was stirred at room temperature for 15 min. To a solution of thiolactone (1.00 mmol) in ClCH2CH2Cl (5 mL) was transferred a solution of TMSI, prepared above, via a cannula, and then the resulting mixture was refluxed for 3-6 days. After cooling, the reaction was quenched with 10% HCl aq, and the aqueous mixture was extracted with CHCl3 (10 mL × 3). The organic extracts were combined, dried over MgSO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 20:1) to give the corresponding S-lactone including the known compound (6c).26
6-Methyl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6d). Yield: 81%; mp 198-200 °C; IR (KBr): 3306, 1599 cm-1;1H NMR (400 MHz, CDCl3): δ 8.96 (1H, br), 7.41 (1H, s), 7.32 (1H, d, J = 8.4 Hz), 7.22 (1H, dd, J = 1.5, 8.4 Hz), 3.48 (2H, t, J = 6.3 Hz), 3.27 (2H, t, J = 6.3 Hz), 2.46 (3H, s); 13C NMR (100 MHz, CDCl3): δ 183.67, 135.02, 130.21, 129.26, 128.67, 125.99, 124.84, 120.15, 112.14, 31.52, 21.73, 21.43; MS (EI) m/z 217 (M+); HRMS (EI) calcd for C12H11NOS: 217.0561 (M+), found: 217.0563.
6-Ethyl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6e). Yield: 56%; mp 120-123 °C; IR (KBr): 3314, 2962, 1609 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.96 (1H, br), 7.43 (1H, s), 7.35 (1H, d, J = 8.5 Hz), 7.27-7.24 (1H, m), 3.49 (2H, t, J = 6.3 Hz), 3.28 (2H, t, J = 6.3 Hz), 2.75 (2H, q, J = 7.4 Hz), 1.29 (3H, t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): δ 183.83, 136.59, 135.34, 128.55, 128.16, 125.72, 124.99, 118.61, 112.43, 31.58, 28.96, 21.84, 16.21; MS (EI) m/z 231 (M+); HRMS (EI) calcd for C13H13NOS: 231.0718 (M+), found: 231.0716.
6-sec-Butyl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6f). Yield: 71%; brown oil; IR (neat): 3297, 2958, 1609 cm-1; 1H NMR (300 MHz, CDCl3): δ 9.23(1H, br), 7.40-7.37 (2H, d, J = 9.3 Hz), 7.23 (1H, d, J = 1.4 Hz), 3.49 (2H, t, J = 6.2 Hz), 3.30 (2H, t, J = 6.2 Hz), 2.70 (1H, sext, J = 6.9 Hz), 1.65 (2H, quint, J = 7.4 Hz), 1.30 (3H, d, J = 6.9 Hz), 0.85 (3H, t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): δ 183.74, 140.05, 135.47, 128.58, 127.17, 125.68, 125.10, 118.11, 112.39, 41.76, 31.62, 31.46, 22.36, 21.91, 12.43; MS (EI) m/z 259 (M+); HRMS (EI) calcd for C15H17NOS: 259.1031 (M+), found: 259.1034.
6-Cyclopentyl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6g). Yield: 30%; mp 168-170 °C; IR (KBr): 3311, 2948, 1607 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.74 (1H, br), 7.47 (1H, s), 7.36-7.29 (2H, m), 3.49 (2H, t, J = 6.4 Hz), 3.29 (2H, t, J = 6.4 Hz), 3.10 (1H, quint, J = 8.7 Hz), 2.18-2.10 (2H, m), 1.87-1.64 (6H, m); 13C NMR (75 MHz, CDCl3): δ 183.40, 138.92, 135.18, 128.61, 127.75, 125.50, 125.02, 117.97, 112.19, 46.05, 34.98, 31.63, 25.60, 21.91; MS (EI) m/z 271 (M+); HRMS (EI) calcd for C16H17NOS: 271.1031 (M+), found: 271.1026.
6-Cycloheptyl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6h). Yield: 86%; mp 155-157 °C; IR (KBr): 3282, 2918, 2850, 1606 cm-1; 1H NMR (300 MHz, CDCl3): δ 9.05 (1H, br), 7.41 (1H, s), 7.34 (1H, d, J = 8.8 Hz), 7.25 (1H, d, J = 8.8 Hz), 3.47 (2H, t, J = 6.3 Hz), 3.27 (2H, t, J = 6.3 Hz), 2.82-2.73 (1H, m), 2.02-1.22 (12H, m); 13C NMR (75 MHz, CDCl3): δ 183.70, 142.53, 135.26, 128.56, 127.25, 125.62, 125.13, 117.32, 112.42, 47.10, 37.26, 31.60, 27.96, 27.30, 21.88; MS (EI) m/z 299 (M+); HRMS (EI) calcd for C18H21NOS: 299.1344 (M+), found: 299.1344.
6-Chloro-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6i).Yield: 75% (brsm); mp 245-247 °C; IR (KBr): 3316, 1607 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.85 (1H, br), 7.62 (1H, s), 7.38-7.35 (2H, m), 3.52 (2H, t, J = 6.4 Hz), 3.28 (2H, t, J = 6.4 Hz); 13C NMR (125 MHz, Acetone-d6): δ 183.27, 136.25, 130.69, 127.62, 127.54, 126.35, 125.18, 121.07, 115.12, 31.95, 22.18; MS (EI) m/z 237 (M+); HRMS (EI) calcd for C11H8NOSCl: 237.0015 (M+), found: 237.0017.
6-Methoxy-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6j). Yield: 44%; mp 186-187 °C; IR (KBr): 3294, 1611 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.73 (1H, br), 7.32 (1H, d, J = 8.8 Hz), 7.06 (1H, dd, J = 2.5, 8.8 Hz), 6.98 (1H, d, J = 2.5 Hz), 3.87 (3H, s), 3.50 (2H, t, J = 6.4 Hz), 3.27 (2H, t, J = 6.4 Hz) 13C NMR (125 MHz, CDCl3): δ 183.60, 154.71, 132.04, 129.07, 126.01, 124.67, 119.00, 113.52, 100.71, 55.71, 31.51, 21.82; MS (EI) m/z 233 (M+); HRMS (EI) calcd for C12H11NO2S: 233.0510 (M+), found: 237.0509.
6-Bicyclohex-4-yl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6k). About 5:1 mixture of 6ka and 6kb; Yield: 87%; brown oil; IR (neat): 3306, 2921, 2849, 1616 cm-1; 1H NMR (300 MHz, CDCl3): δ 8.98 (1H, br), 7.44 (1H, s), 7.35 (1H, d, J = 8.1 Hz), 7.27 (1H, d, J = 8.1 Hz), 3.49 (2H, t, J = 6.2 Hz), 3.29 (2H, t, J = 6.2 Hz), 2.65-2.51 (1H, m), 1.99-0.99 (20H, m).
6-Phenyl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6l). Yield: 43% (brsm); mp 228-230 °C; IR (KBr): 3292, 1609 cm-1; 1H NMR (300MHz, CDCl3): δ 8.84 (1H, br), 7.83 (1H, s), 7.65 (1H, d, J = 8.8 Hz), 7.63 (2H, d, J = 7.3 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.46 (2H, t, J = 7.3 Hz), 7.35 (1H, t, J = 7.3 Hz), 3.53 (2H, t, J = 6.4 Hz), 3.35 (2H, t, J = 6.4 Hz); 13C NMR (75 MHz, CDCl3): δ 183.40, 141.41, 135.86, 134.41, 129.11, 128.71, 127.21, 127.17, 126.82, 126.28, 125.46, 119.08, 112.64, 31.60, 21.88; MS (EI) m/z 279 (M+); HRMS (EI) calcd for C17H13NOS: 279.0718 (M+), found: 279.0716.

Synthesis of esters 7c-l
To a stirred solution of S-lactone (1 mmol) in DMF (5 mL) was added NaH (60%, 1.2 equiv) at 0 °C, and the reaction mixture was stirred at 0 °C for 30 min. To the mixture was added BrCH2CO2t-Bu (1.2 equiv) at 0 °C, and the resulting mixture was stirred at room temperature for 20–24 h. The reaction was quenched with H2O (10 mL), and the aqueous mixture was extracted with Et2O (10 mL × 3). The organic extracts were combined, dried over MgSO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 20:1) to give the corresponding ester.
(1-Oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7c). Yield: 75%; mp 122-123 °C; IR (KBr): 1745, 1617 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.66 (1H, d, J = 8.0 Hz), 7.42 (1H, t, J = 8.2 Hz), 7.26 (1H, t, J = 8.8 Hz), 7.19 (1H, t, J = 7.5 Hz), 5.17 (2H, s), 3.44 (2H, t, J = 6.3 Hz), 3.34 (2H, t, J = 6.3 Hz), 1.45 (9H, s); 13C NMR (75 MHz, DMSO-d6): δ 185.83, 169.73, 137.60, 132.27, 125.53, 125.36, 120.54, 120.48, 120.25, 110.67, 49.37, 46.17, 29.66, 29.52, 6.17; MS (EI) m/z 317 (M+); HRMS (EI) calcd for C17H19NO3S: 317.1086 (M+), found: 317.1085.
(6-Methyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7d). Yield: 20%; mp: 174-176 °C; IR (KBr): 1743, 1618 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.42 (1H, s), 7.25 (1H, d, J = 8.4 Hz), 7.15 (1H, d, J = 8.4 Hz), 5.14 (2H, s), 3.43 (2H, t, J = 6.5 Hz), 3.30 (2H, t, J = 6.5 Hz), 2.45 (3H, s), 1.44 (9H, s); 13C NMR (125 MHz, CDCl3): δ 183.95, 167.87, 137.07, 130.35, 129.31, 127.95, 125.93, 125.00, 120.41, 109.61, 82.26, 47.17, 31.28, 28.02, 22.36, 21.36; MS (EI) m/z 331 (M+); HRMS (EI) calcd for C18H21NO3S: 331.1242 (M+), found: 331.1247.
(6-Ethyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7e). Yield: 95%; mp 153-155 °C; IR (KBr): 2975, 2930, 1742, 1620 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.43 (1H, s), 7.28 (1H, d, J = 9.6 Hz), 7.17 (1H, d, J = 9.6 Hz), 5.14 (2H, s), 3.43 (2H, t, J = 6.3 Hz), 3.31 (2H, t, J = 6.3 Hz), 2.75 (2H, q, J = 7.6 Hz), 1.46 (9H, s), 1.28 (3H, t, J = 7.6 Hz); 13C NMR (75 MHz, CDCl3): δ 183.64, 167.64, 137.01, 136.77, 128.19, 127.76, 125.94, 124.78, 119.00, 109.59, 82.13, 47.12, 31.28, 28.87, 28.04, 22.39, 16.20; MS (EI) m/z 345 (M+); HRMS (EI) calcd for C19H23NO3S: 345.1399 (M+), found: 345.1402.
(6-sec-Butyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7f). Yield: quant.; mp 103-105 °C; IR (KBr): 2953, 1743, 1635 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.40 (1H, brs), 7.27 (1H, dd, J = 1.5, 8.8 Hz), 7.18 (1H, d, J = 8.8 Hz), 5.14 (2H, s), 3.43 (2H, t, J = 6.3 Hz), 3.32 (2H, t, J = 6.3 Hz), 2.69 (1H, sext, J = 7.0 Hz), 1.64 (2H, quint, J = 7.2 Hz), 1.46 (9H, s), 1.28 (3H, d, J = 6.9 Hz), 0.83 (3H, t, J = 7.2 Hz); 13C NMR (75 MHz, CDCl3): δ 183.66, 167.72, 140.21, 137.20, 127.77, 127.19, 126.07, 124.71, 118.47, 109.60, 82.18, 47.15, 41.70, 31.44, 31.31, 28.09, 22.46, 22.36, 12.41; MS (EI) m/z 373 (M+); HRMS (EI) calcd for C21H27NO3S: 373.1712 (M+), found: 373.1709.
(6-Cyclopentyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7g). Yield: 63%; mp 103-106 °C; IR (KBr): 2952, 1753, 1618 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.47 (1H, s), 7.33 (1H, d, J = 8.8 Hz), 7.18 (1H, d, J = 8.8 Hz), 5.14 (2H, s), 3.43 (2H, t, J = 6.5 Hz), 3.31 (2H, t, J = 6.5 Hz), 3.09 (1H, quint, J = 8.5 Hz), 2.11-2.09 (2H, m), 1.84-1.21 (15H, m); 13C NMR (75 MHz, CDCl3): δ 183.70, 167.72, 139.02, 137.17, 127.85, 127.56, 126.11, 124.71, 118.26, 109.62, 82.24, 47.21, 46.00, 34.98, 31.37, 28.14, 25.60, 22.52; MS (EI) m/z 385 (M+); HRMS (EI) calcd for C22H27NO3S: 385.1712 (M+), found: 385.1711.
(6-Cycloheptyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7h). Yield: 92% (brsm); mp 158-161 °C; IR (KBr): 2923, 2850, 1739, 1632 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.41 (1H, s), 7.27 (1H, d, J = 8.8 Hz), 7.15 (1H, d, J = 8.8 Hz), 5.12 (2H, s), 3.41 (2H, t, J = 6.0 Hz), 3.30 (2H, t, J = 6.0 Hz), 1.96-1.53 (12H, m), 1.45 (9H, s); 13C NMR (75 MHz, CDCl3): δ 183.61, 167.69, 142.67, 136.98, 127.76, 127.29, 126.11, 124.67, 117.71, 109.59, 82.15, 47.10, 47.04, 37.25, 31.31, 28.07, 27.94, 27.28, 22.43; MS (EI) m/z 413 (M+); HRMS (EI) calcd for C24H31NO3S: 413.2025 (M+), found: 413.2024.
(6-Chloro-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7i). Yield: quant.; mp 218-220 °C; IR (KBr): 1739, 1623 cm-1; 1H NMR (500 MHz, DMSO-d6): δ 7.90 (1H, d, J = 2.1 Hz), 7.64 (1H, d, J = 9.0 Hz), 7.34 (1H, dd, J = 2.1, 9.0 Hz), 5.09 (2H, s), 3.66 (2H, t, J = 7.7 Hz), 3.43 (2H, t, J = 7.7 Hz), 1.56 (9H, s); 13C NMR (125 MHz, CDCl3): δ 180.70, 167.59, 136.83, 128.89, 127.73, 126.76, 125.77, 125.51, 120.52, 111.29, 82.72, 47.37, 31.21, 28.11, 22.30; MS (EI) m/z 351 (M+); HRMS (EI) calcd for C17H18NO3SCl: 351.0696 (M+), found: 351.0693.
(6-Methoxy-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7j). Yield: quant.; mp 134-135 °C; IR (KBr): 1741, 1610 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.16 (1H, d, J = 9.1 Hz), 7.09 (1H, dd, J = 2.4, 9.1 Hz), 6.99 (1H, d, J = 2.4 Hz), 5.13 (2H, s), 3.86 (3H, s), 3.42 (2H, t, J = 6.5 Hz), 3.29 (2H, t, J = 6.5 Hz), 1.45 (9H, s); 13C NMR (125 MHz, CDCl3): δ 183.84, 167.81, 154.83, 134.04, 128.20, 125.59, 124.95, 118.97, 110.95, 101.00, 82.30, 55.77, 47.23, 31.22, 28.00, 22.42; MS (EI) m/z 347 (M+); HRMS (EI) calcd for C18H21NO4S: 347.1191 (M+), found: 347.1191.
(6-Bicyclohex-4-yl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7k). About 5:1 mixture of 7ka and 7kb; Yield: 93%; yellow oil; IR (neat): 2923, 2849, 1748, 1634 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.44 (1H, s), 7.30 (1H, d, J = 8.8 Hz), 7.17 (1H, d, J = 8.8 Hz), 5.14 (2H, s), 3.43 (2H, t, J = 5.3 Hz), 3.32 (2H, t, J = 5.3 Hz), 2.61-2.51 (1H, m), 1.97-0.89 (20H, m), 1.46 (9H, s).
(1-Oxo-6-phenyl-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7l). Yield: 98% (brsm); mp 128-130 °C; IR (KBr): 2979, 1741, 1624 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.83 (1H, d, J = 1.7 Hz), 7.67 (1H, dd, J = 1.7, 8.7 Hz), 7.63 (2H, d, J = 7.4 Hz), 7.46 (2H, t, J = 7.4 Hz), 7.35 (1H, t, J = 7.4 Hz), 7.33 (1H, d, J = 8.7 Hz), 5.20 (2H, s), 3.47 (2H, t, J = 5.7 Hz), 3.38 (2H, t, J = 5.7 Hz), 1.47 (9H, s); 13C NMR (75 MHz, CDCl3): δ 183.77, 167.57, 141.35, 137.84, 134.47, 128.68, 128.34, 127.21, 127.16, 126.77, 126.57, 125.18, 119.26, 110.15, 82.42, 47.29, 31.31, 28.12, 22.46; MS (EI) m/z 393 (M+); HRMS (EI) calcd for C23H23NO3S: 393.1399 (M+), found: 393.1402.

Synthesis of carboxylic acids 5c-l
To a stirred solution of NaI (4 equiv) in ClCH2CH2Cl (3 mL) was added TMSCl (4 equiv), and the resulting mixture was stirred at room temperature for 15 min. To a solution of t-butyl ester (1 mmol) in ClCH2CH2Cl (5 mL) was transferred a solution of TMSI, prepared above, via a cannula, and then the resulting mixture was refluxed for 2 days. After cooling, the reaction was quenched with 10% HCl aq, and the aqueous mixture was extracted with CHCl3 (10 mL × 3). The organic extracts were combined, dried over MgSO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 2:1) to give the corresponding carboxylic acid.
(1-Oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5c). Yield: 73%; mp 207-209 °C; IR (KBr): 1720, 1612 cm-1; 1H NMR (500 MHz, DMSO-d6): δ 7.78 (1H, d, J = 8.1 Hz), 7.61 (1H, d, J = 8.5 Hz), 7.42 (1H, t, J = 8.3 Hz), 7.19 (1H, t, J = 7.5 Hz), 5.22 (2H, s), 3.49 (2H, t, J = 6.4 Hz), 3.34-3.32 (2H, m); 13C NMR (75 MHz, DMSO-d6): δ 182.96, 169.91, 138.10, 127.05, 126.93, 126.32, 124.01, 121.16, 120.51, 110.75, 46.33, 30.81, 21.67; MS (EI) m/z 261 (M+); HRMS (EI) calcd for C13H11NO3S: 261.0460 (M+), found: 261.0454.
(6-Methyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5d). Yield: 52%; mp 215-217 °C; IR (KBr): 1715, 1625 cm-1; 1H NMR (500 MHz, DMSO-d6): δ 7.54 (1H, s), 7.50 (1H, d, J = 8.5 Hz), 7.25 (1H, d, J = 8.5 Hz), 5.18 (2H, s), 3.47 (2H, t, J = 6.2 Hz), 3.27 (2H, t, J = 6.2 Hz), 2.41 (3H, s); 13C NMR (75 MHz, CDCl3): δ 182.82, 169.92, 136.72, 129.39, 128.88, 127.08, 125.72, 124.15, 120.16, 110.51, 46.33, 30.81, 21.67, 20.98; MS (EI) m/z 275 (M+); HRMS (EI) calcd for C14H13NO3S: 275.0616 (M+), found: 275.0600.
(6-Ethyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5e). Yield: 76%; mp 231-233 °C; IR (KBr): 2963, 1717, 1616 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.44 (1H, s), 7.31 (1H, d, J = 8.7 Hz), 7.21 (1H, d, J = 8.7 Hz), 5.27 (2H, s), 3.43 (2H, t, J = 6.3 Hz), 3.31 (2H, t, J = 6.3 Hz), 2.75 (2H, q, J = 7.7 Hz), 1.28 (3H, t, J = 7.7 Hz); 13C NMR (125 MHz, DMSO-d6): δ 183.13, 170.21, 137.10, 136.27, 128.10, 127.28, 126.12, 124.29, 119.13, 110.77, 46.36, 30.83, 28.18, 21.68, 16.23; MS (EI) m/z 289 (M+); HRMS (EI) calcd for C15H15NO3S: 289.0773 (M+), found: 289.0776.
(6-sec-Butyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5f). Yield: 68%; mp 184-185 °C; IR (KBr): 2960, 1718, 1624 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.41 (1H, s), 7.31 (1H, d, J = 8.8 Hz), 7.22 (1H, d, J = 8.8 Hz), 5.26 (2H, s), 3.44 (2H, t, J = 5.8 Hz), 3.32 (2H, t, J = 5.8 Hz), 2.70 (1H, d, J = 7.1 Hz), 1.64 (2H, quint, J = 7.4 Hz), 1.28 (3H, d, J = 6.9 Hz), 0.83 (3H, t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): δ 184.23, 174.29, 140.62, 137.12, 127.64, 127.58, 126.59, 124.73, 118.57, 109.55, 46.28, 41.68, 31.42, 31.21, 22.36, 22.33, 12.41; MS (EI) m/z 317 (M+); HRMS (EI) calcd for C17H19NO3S: 317.1086 (M+), found: 317.1086.
(6-Cyclopentyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5g). Yield: 86%; mp 232-234 °C; IR (KBr): 2952, 1718, 1617 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.48 (1H, d, J = 0.82 Hz), 7.37 (1H, d, J = 8.7 Hz), 7.22 (1H, d, J = 8.7 Hz), 5.26 (2H, s), 3.43 (2H, t, J = 5.7 Hz), 3.32 (2H, t, J = 5.7 Hz), 3.10 (1H, quint, J = 9.0 Hz), 2.11-2.09 (2H, m), 1.84-1.63 (6H, m); 13C NMR (125 MHz, CDCl3): δ 184.59, 173.34, 139.60, 137.26, 128.14, 127.78, 126.80, 124.81, 118.46, 109.67, 46.33, 45.91, 34.89, 31.15, 25.49, 22.32; MS (EI) m/z 329 (M+); HRMS (EI) calcd for C18H19NO3S: 329.1086 (M+), found: 329.1083.
(6-Cycloheptyl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5h). Yield: 89%; mp 213-215 °C; IR (KBr): 2923, 2849, 1725, 1627 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.43 (1H, s), 7.32 (1H, d, J = 9.0 Hz), 7.21 (1H, d, J = 9.0 Hz), 5.24 (2H, s), 3.43 (2H, t, J = 6.0 Hz), 3.31 (2H, t, J = 6.0 Hz), 2.77 (1H, m), 2.09-0.86 (12H, m); 13C NMR (75 MHz, DMSO-d6): δ 183.08, 170.23, 142.13, 137.03, 127.26, 127.03, 126.29, 124.16, 117.73, 110.80, 46.33, 46.29, 36.73, 30.84, 27.38, 26.66, 21.68; MS (EI) m/z 357 (M+); HRMS (EI) calcd for C20H23NO3S: 357.1399 (M+), found: 357.1396.
(6-Chloro-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5i). Yield: 50%; mp 221-222 °C; IR (KBr): 1727, 1624 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 7.90 (1H, d, J = 1.9 Hz), 7.69 (1H, d, J = 9.1 Hz), 7.41 (1H, dd, J = 1.9, 9.1 Hz), 5.22 (2H, s), 3.49 (2H, t, J = 6.4 Hz), 3.30 (2H, t, J = 6.4 Hz); 13C NMR (75 MHz, DMSO-d6): δ 183.19, 169.73, 136.48, 128.07, 126.84, 125.58, 125.09, 124.93, 120.35, 112.74, 46.59, 30.73, 21.49; MS (EI) m/z 295 (M+); HRMS (EI) calcd for C13H10O3NSCl: 295.0070 (M+), found: 295.0042.
(6-Methoxy-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5j). Yield: 34%; mp 205-207 °C; IR (KBr): 1715, 1623 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 7.53 (1H, d, J = 9.1 Hz), 7.21 (1H, d, J = 2.5 Hz), 7.06 (1H, d, J = 2.5, 9.1 Hz), 5.18 (2H, s), 3.81 (3H, s), 3.47 (2H, t, J = 6.2 Hz), 3.28 (2H, t, J = 6.2 Hz); 13C NMR (75 MHz, DMSO-d6): δ 182.75, 169.96, 154.10, 133.66, 127.29, 125.56, 124.22, 118.57, 111.89, 100.97, 55.44, 46.44, 30.84, 21.78; MS (EI) m/z 291 (M+); HRMS (EI) calcd for C14H13NO4S: 291.0565 (M+), found: 291.0570.
(6-Bicyclohex-4-yl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5k). About 5:1 mixture of 5ka and 5kb; Yield: 86%; IR (KBr): 2921, 2849, 1722, 1632 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.44 (1H, s), 7.33 (1H, d, J = 9.5 Hz), 7.20 (1H, d, J = 9.5 Hz), 5.26 (2H, s), 3.42 (2H, t, J = 5.7 Hz), 3.32 (2H, t, J = 5.7 Hz), 2.64-2.55 (1H, m), 1.98-0.89 (20H, m).
(1-Oxo-6-phenyl-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5l). Yield: 55%; mp 220-223 °C; IR (KBr): 2923, 1716, 1619 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.84 (1H, s), 7.71 (1H, d, J = 7.4 Hz), 7.62 (2H, d, J = 6.9 Hz), 7.46 (2H, t, J = 6.9 Hz), 7.39 (1H, d, J = 7.4 Hz), 7.37 (1H, t, J = 6.9 Hz), 5.33 (2H, s), 3.46 (2H, t, J = 6.5 Hz), 3.41 (2H, t, J = 6.5 Hz); 13C NMR (125 MHz, DMSO-d6): δ 183.29, 170.17, 140.59, 137.91, 133.15, 128.90, 127.86, 126.96, 126.88, 126.81, 126.55, 124.75, 119.13, 111.50, 46.58, 30.83, 21.66; MS (EI) m/z 337 (M+); HRMS (EI) calcd for C19H15NO3S: 337.0773 (M+), found: 337.0775.

Bicyclohexyl-4-one (9)
To a suspension of 4-cyclohexylcyclohexanol 8 (7.17 g, 39.3 mmol) and Celite (20 g) in CH2Cl2 (80 mL) was added PCC (12.7 g, 59.0 mmol) at 0 °C, and the resulting mixture was stirred at the room temperature for 14 h. The catalyst was removed by filtration and the filtrate was concentrated in vacuo. The residue was chromatographed on SiO2 (hexane : EtOAc = 20 : 1) to give 9 (6.78 g, 96%).27

4-Phenyl-bicyclohexyl-4-ol (10)
To a freshly made solution of PhMgBr (30 mmol) in THF (30 mL) was added 9 (2.16 g, 12.0 mmol) in THF (12.5 mL) at 0 °C, and the resulting mixture was stirred at the same temperature for 3 h. The reaction was quenched by the addition of sat. NH4Cl aq, diluted with EtOAc. And then, the organic phase was separated, the aqueous mixture was extracted with EtOAc. The organic extracts were combined, dried over MgSO4, and evaporated. The residue was chromatographed on SiO2 (hexane : EtOAc = 30 : 1) to give 10 (2.88 g, 93%).
1H NMR (500 MHz, CDCl3): 7.55-7.50 (2H, m), 7.38-7.33 (2H, m), 7.29-7.22 (1H, m), 1.88-0.83 (20H, m).

trans-4-Phenyl-1,1'-bi(cyclohexane) (11b) and cis-4-Phenyl-1,1'-bi-(cyclohexane) (11c)
To a solution of 10 (2.88 g, 11.1 mmol) in CH2Cl2 was added slowly Et3SiH (3.60 mL, 22.2 mmol) , BF3・OEt2 (1.39 mL, 11.1 mmol) at -40 °C, and the resulting mixture was stirred at the same temperature for 4 h. The reaction was diluted with CH2Cl2 and neutralized with sat. NaHCO3 aq. The organic phase was separated, dried over Na2SO4 and concentrated in vacuo. The residue was chromatographed on SiO2 (hexane) to give mixture of 11b and 11c (the ratio of compound 11b : 11c = ca. 3 : 1, 2.47 g, 92%).
1H NMR (500 MHz, CDCl3): 8.16-8.13 (2H, m), 7.40 (1H, d, J = 8.6 Hz), 7.34 (1H, d, J = 8.5 Hz), 2.79-2.74 (11c; 1H, m), 2.56 (11b; 1H, tt, J = 3.1, 12.0 Hz), 1.94-0.99 (20H, m).

trans-4-(4-Nitrophenyl)-1,1'-bi(cyclohexane) (12b) and cis-4-(4-Nitrophenyl)-1,1'-bi(cyclohexane) (12c)
To a solution of mixture of 11b and 11c (2.47 g) in CHCl3 (10 mL) was added Ac2O (2.9 mL) at 0 °C. At the same time HNO3 (6.1 mL) was slowly added to Ac2O (10 mL) at 0 °C, and this mixture was added dropwise to the solution, prepared above, at 0 °C. The resulting mixture was stirred at room temperature for 13 h. The reaction was quenched by the addition of NaOH (10% aqueous solution), and the organic layer was separated, dried over Na2SO4 and concentrated in vacuo. The residue was chromatographed on SiO2 (hexane) to give mixture of 12b and 12c (1.53 g, 52%).
1H NMR (500 MHz, CDCl3): 8.16-8.13 (2H, m), 7.40 (1H, d, J = 8.6 Hz), 7.34 (1H, d, J = 8.5 Hz), 2.79-2.74 (12c; 1H, m), 2.56 (12b; 1H, tt, J = 3.1, 12.0 Hz), 1.94-0.99 (20H, m).

4-(trans-[1,1'-Bi(cyclohexane)]-4-yl)aniline (13b) and 4-(cis-[1,1'-Bi(cyclohexane)]-4-yl)aniline (13c)
To a solution of mixture of 12b and 12c (1.53 g, 5.33 mmol) in EtOAc (23 mL) was added 10% Pd/C (60 mg), and the resulting suspension was stirred under a hydrogen atmosphere at 1 atm for 3 days. The catalyst was removed by filtration and the filtrate was concentrated in vacuo. The residue was chromatographed on SiO2 (hexane : EtOAc = 15 : 1) to give 13b (795 mg, 58%), 13c (273 mg, 20%).
4-(trans-[1,1'-Bi(cyclohexane)]-4-yl)aniline (13b). mp 118-120 °C; IR (KBr): 3382, 3312, 2920, 2849, 1516 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.00 (2H, d, J = 8.3 Hz), 6.64 (2H, d, J = 8.3 Hz), 3.53 (2H, br), 2.34 (1H, tt, J = 3.5, 12.4 Hz), 1.89-0.96 (20H, m); 13C NMR (125 MHz, CDCl3): δ 144.08, 138.12, 127.41, 115.09, 43.68, 43.27, 42.92, 34.80, 30.26, 30.21, 26.81; MS (EI) m/z 257 (M+); HRMS (EI) calcd for C18H27N: 257.2143 (M+), found: 257.2141.
4-(cis-[1,1'-Bi(cyclohexane)]-4-yl)aniline (13c). mp 67-69 °C; IR (KBr): 3464, 3371, 2927, 2846, 1520 cm-1; 1H NMR (500 MHz, CDCl3): 7.04 (2H, dd, J = 1.4, 6.3 Hz), 6.64 (2H, dd, J = 2.0, 6.3 Hz), 3.53 (2H, br), 2.57-2.51 (1H, m), 1.85-0.78 (20H, m); 13C NMR (125 MHz, CDCl3): δ 143.96, 137.68, 127.69, 115.12, 41.99, 39.19, 36.67, 30.95, 29.54, 27.41, 26.65; MS (EI) m/z 257 (M+); HRMS (EI) calcd for C18H27N: 257.2143 (M+), found: 257.2144.

cis-3-(4-Nitrophenyl)-1,1'-bi(cyclohexane) (12a) and trans-4-(4-nitrophenyl)-1,1'-bi(cyclohexane) (12b)
To a solution of 4-cyclohexylcyclohexanol 8 (2.42 g, 13.3 mmol, about 1 : 1.3 mixture of cis- and trans-isomers) in dry benzene (60 mL) was slowly added AlCl3 (1.77 g, 13.3 mmol) at room temperature. The resulting mixture was stirred at the same temperature for 17 h. The reaction was quenched by the addition of ice water, and then the organic phase was separated, washed with Na2CO3 (saturated aqueous solution), dried over Na2SO4 and concentrated in vacuo to yield mixture of cis-3-phenyl-1,1'-bi(cyclohexane) 11a and trans-4-phenyl-1,1'-bi(cyclohexane) 11b (the ratio of compound 11a : 11b = ca. 5 : 1). The product was used for the next reaction without further purification. To a solution of crude (3.22 g) in CHCl3 (13 mL) was added Ac2O (2.7 mL) at 0 °C. At the same time HNO3 (1.3 mL) was slowly added to Ac2O (2.7 mL) at 0 °C, and this mixture was added dropwise to the solution, prepared above, at 0 °C. The resulting mixture was stirred at room temperature for 14 h. The reaction was quenched by the addition of NaOH (10% aqueous solution), and the organic layer was separated, dried over Na2SO4 and concentrated in vacuo. The residue was chromatographed on SiO2 (hexane : EtOAc = 70 : 1) to give mixture of 12a and 12b (1.78 g, 47% in two steps).
1H NMR (500 MHz, CDCl3): δ 8.14 (2H, dd, J = 2.0, 7.9 Hz), 7.35 (2H, dd, J = 2.0 Hz, 7.9 Hz), 2.65-2.53 (1H, m), 1.94-0.96 (20H, m)

4-(cis-[1,1'-Bi(cyclohexane)]-3-yl)aniline (13a) and 4-(trans-[1,1'-Bi(cyclohexane)]-4-yl)aniline (13b)
To a solution of 12a and 12b (3.30 g, 11.5 mmol) in EtOAc (15 mL) was added Pd/C (59 mg), and the resulting suspension was stirred under a hydrogen atmosphere at 1 atm for 5 days. The catalyst was removed by filtration and the filtrate was concentrated in vacuo. The residue was chromatographed on SiO2 (hexane : EtOAc = 12 : 1) to give 13a (1.80g, 61%), 13b (531mg, 18%).
4-(cis-[1,1'-Bi(cyclohexane)]-3-yl)aniline (13a). mp 58-60 °C; IR (KBr): 3423, 3348, 2920, 2846, 1517 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.01 (2H, d, J = 8.3 Hz), 6.64 (2H, d, J = 8.3 Hz), 3.54 (2H, br), 2.39 (1H, tt, J = 3.3, 11.9 Hz), 1.89-0.94 (20H, m); 13C NMR (125 MHz, CDCl3): δ 144.09, 138.37, 127.41, 115.10, 43.76, 43.67, 43.44, 38.35, 34.55, 30.16, 30.04, 29.53, 26.87, 26.80; MS (EI) m/z 257 (M+); HRMS (EI) calcd for C18H27N: 257.2143 (M+), found: 257.2147.

1’,3’-cis-6-Bicyclohex-3-yl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2ka)
To a suspension of 13a (105 mg, 0.41 mmol) and concentrated HCl (0.1 mL) in THF (0.63 mL) and H2O (0.42 mL) was slowly added a solution of NaNO2 (31 mg, 0.44 mmol) in H2O (0.15 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. At the same time, to a solution of enol lactone (98 mg, 0.49 mmol) and sodium acetate (134 mg, 1.63 mmol) in THF (0.21 mL) and H2O (0.11 mL) was slowly added the diazonium salt, prepared above, at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. The reaction was diluted with CH2Cl2 and the organic phase was separated, dried over Na2SO4 and concentrated in vacuo to yield hydrazone, which was used for the next reaction without further purification. A solution of the above hydrazone (138 mg) in AcOH (1.2 mL) and HCl (1.2 mL, 1.0 M in AcOH solution) was refluxed for 4h, and cooled to room temperature. The resulting mixture was diluted with CH2Cl2 and the organic phase was separated, washed with H2O, neutralized with NaHCO3 (saturated aqueous solution), dried over Na2SO4 and concentrated in vacuo. The residue was chromatographed on SiO2 (hexane : EtOAc = 15 : 1) to give 2ka (99 mg, 69% in two steps).
brown oil, IR (neat): 3291, 2922, 2850, 1700 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.76 (1H, br), 7.43 (1H, brs), 7.38 (1H, d, J = 8.6 Hz), 7.28 (1H, dd, J = 1.7, 8.6 Hz), 4.69 (2H, t, J = 6.3 Hz), 3.15 (2H, t, J = 6.3 Hz), 2.63-2.59 (1H, m), 1.92-0.96 (20H, m); 13C NMR (125 MHz, CDCl3): δ 161.54, 140.96, 137.02, 127.02, 124.55, 122.89, 122.23, 117.55, 112.59, 69.46, 44.73, 43.77, 43.47, 38.57, 34.81, 30.21, 30.10, 29.57, 26.90, 26.80, 21.47; MS (EI) m/z 351 (M+); HRMS (EI) calcd for C23H29NO2: 351.2198 (M+), found: 351.2199.

1’,3’-cis-6-Bicyclohex-3-yl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3ka)
To a stirred solution of 2ka (43 mg, 0.12 mmol) in toluene (3 mL) was added Lawesson’s reagent (27 mg, 0.067 mmol), and the resulting mixture was refluxed for 18 h. After cooling, the solvent was removed, and the residue was chromatographed on SiO2 (hexane–acetone = 20:1) to give 3ka (50 mg, quant.).
yellow oil, IR (neat): 3356, 2921, 2850, 1540, 1231 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.76 (1H, br), 7.42 (1H, brs), 7.32 (1H, d, J = 8.6 Hz), 7.29 (1H, dd, J = 1.5, 8.6 Hz), 4.74 (2H, t, J = 6.6 Hz), 3.17 (2H, t, J = 6.6 Hz), 2.59 (1H, tt, J = 3.2, 11.8 Hz), 1.92-0.96 (20H, m); 13C NMR (125 MHz, CDCl3): δ 198.23, 141.48, 137.83, 132.30, 128.07, 124.85, 118.53, 115.74, 112.12, 71.15, 44.67, 43.69, 43.41, 38.43, 34.68, 30.16, 30.07, 29.50, 26.84, 26.77, 21.22; MS (EI) m/z 367 (M+); HRMS (EI) calcd for C23H29NOS: 367.1970 (M+), found: 367.1970.

1’,3’-cis-6-Bicyclohex-3-yl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6ka)
To a stirred solution of NaI (85 mg, 0.57 mmol) in ClCH2CH2Cl (10 mL) was added TMSCl (72 µL, 0.57 mmol), and the resulting mixture was stirred at room temperature for 15 min. To a solution of 3ka (52mg, 0.14 mmol) in ClCH2CH2Cl (5 mL) was transferred a solution of TMSI, prepared above, via a cannula, and then the resulting mixture was refluxed for 15 days. After cooling, the reaction was quenched with 10% HCl aq, and the aqueous mixture was extracted with CHCl3. The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 30 : 1) to give 6ka (48 mg, 92%).
mp 147-149 °C; IR (KBr): 3306, 2909, 2848, 1606 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.74 (1H, br), 7.44 (1H, brs), 7.33 (1H, d, J = 8.6 Hz), 7.27 (1H, dd, J = 1.7 Hz, 8.6 Hz), 3.48 (2H, t, J = 6.2 Hz), 3.29 (2H, t, J = 6.2 Hz), 2.60 (1H, tt, J = 3.1 Hz, 11.8 Hz), 1.93-0.96 (20H, m); 13C NMR (125 MHz, CDCl3): δ 183.75, 140.86, 135.45, 128.69, 127.54, 125.86, 125.21, 117.67, 112.31, 44.75, 43.77, 43.48, 38.56, 34.80, 31.54, 30.22, 30.11, 29.58, 26.91, 26.82, 21.80; MS (EI) m/z 367 (M+); HRMS (EI) calcd for C23H29NOS: 367.1970 (M+), found: 367.1970.

1’,3’-cis-(6-Bicyclohex-3-yl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7ka)
To a stirred solution of 6ka (104 mg, 0.28 mmol) in DMF (3 mL) was added NaH (60%, 17mg, 0.42 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for 30 min. To the mixture was added BrCH2CO2t-Bu (63 µL, 0.42 mmol) at 0 °C, and the resulting mixture was stirred at room temperature for 17 h. The reaction was quenched with H2O, and the aqueous mixture was extracted with Et2O. The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 30:1) to give 7ka (130 mg, 95%).
mp 163-165 °C; IR (KBr): 2922, 2851, 1742, 1627 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.44 (1H, brs), 7.30 (1H, dd, J = 1.4, 8.6 Hz), 7.17 (1H, d, J = 8.6 Hz), 5.13 (2H, s), 3.43 (2H, t, J = 6.2 Hz), 3.32 (2H, t, J = 6.2 Hz), 2.60 (1H, tt, J = 3.1, 11.6 Hz), 1.91-0.98 (20H, m), 1.45 (9H, s); 13C NMR (125 MHz, CDCl3): δ 183.89, 167.88, 140.98, 137.30, 127.91, 127.59, 126.25, 124.85, 118.02, 109.62, 82.21, 47.11, 44.71, 43.78, 43.49, 38.51, 34.83, 31.27, 30.23, 30.13, 29.58, 28.01, 26.92, 26.83, 22.40; MS (EI) m/z 481 (M+); HRMS (EI) calcd for C23H39NO3S: 481.2651 (M+), found: 481.2652.

1’,3’-cis-(6-Bicyclohex-3-yl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5ka)
To a stirred solution of NaI (162 mg, 1.08 mmol) in ClCH2CH2Cl (5 mL) was added TMSCl (0.14 mL, 1.08 mmol), and the resulting mixture was stirred at room temperature for 15 min. To a solution of 7ka (130mg, 0.27 mmol) in ClCH2CH2Cl (5 mL) was transferred a solution of TMSI, prepared above, via a cannula, and then the resulting mixture was refluxed for 19 h. After cooling, the reaction was quenched with 10% HCl aq, and the aqueous mixture was extracted with EtOAc. The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 3 : 1) to give 5ka (102 mg, 89%).
mp 219-221 °C; IR (KBr): 2925, 2850, 1723, 1631 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.45 (1H, brs), 7.33 (1H, d, J = 8.6 Hz), 7.21 (1H, dd, J = 1.5, 8.6 Hz), 5.26 (2H, s), 3.42 (2H, t, J = 6.0 Hz), 3.32 (2H, t, J = 6.0 Hz), 2.61 (1H, tt, J = 3.3, 11.7 Hz), 1.90-0.96 (20H, m), 13C NMR (125 MHz, CDCl3): δ 184.46, 174.08, 141.39, 137.26, 128.03, 127.73, 126.79, 124.87, 118.11, 109.60, 46.28, 44.68, 43.77, 43.48, 38.52, 34.76, 31.15, 30.22, 30.12, 29.57, 26.90, 26.82, 22.31; MS (EI) m/z 425 (M+); HRMS (EI) calcd for C25H31NO3S: 425.2025 (M+), found: 425.2029.

1’,4’-trans-6-Bicyclohex-4-yl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (2kb)
To a suspension of 13b (120 mg, 0.47 mmol) and concentrated HCl (0.12 mL) in THF (0.72 mL) and H2O (0.24 mL) was slowly added a solution of NaNO2 (35 mg, 0.50 mmol) in H2O (0.16 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. At the same time, to a solution of enol lactone (112 mg, 0.56 mmol) and sodium acetate (153 mg, 1.86 mmol) in THF (0.48 mL) and H2O (0.12 mL) was slowly added the diazonium salt, prepared above, at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. The reaction was diluted with CH2Cl2 and the organic phase was separated, dried over Na2SO4 and concentrated in vacuo to yield hydrazone. The product was used for the next reaction without further purification. A solution of crude (153 mg) in AcOH (1.3 mL) and HCl (1.3 mL, 1.0 M in AcOH solution) was refluxed for 4 h, and cooled to room temperature. The resulting mixture was diluted with CH2Cl2 and the organic phase was separated, washed with H2O, neutralized with NaHCO3 (saturated aqueous solution), dried over Na2SO4 and concentrated in vacuo. The residue was chromatographed on SiO2 (hexane : EtOAc = 8 : 1) to give 2kb (110 mg, 67% in two steps).
mp 272-273 °C; IR (KBr): 3265, 2919, 2850, 1734 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.67 (1H, br), 7.43 (1H, brs), 7.37 (1H, d, J = 8.6 Hz), 7.28 (1H, dd, J = 1.4, 8.6 Hz), 4.69 (2H, t, J = 6.2 Hz), 3.14 (2H, t, J = 6.2 Hz), 2.55 (1H, tt, J = 3.3, 12.2 Hz), 1.98-0.97 (20H, m); 13C NMR (125 MHz, CDCl3): δ 161.51, 140.75, 137.00, 127.00, 124.56, 122.90, 122.24, 117.55, 112.56, 69.45, 44.64, 43.29, 43.00, 35.05, 30.32, 30.27, 26.84, 21.46; MS (EI) m/z 351 (M+); HRMS (EI) calcd for C23H29NO2: 351.2198 (M+), found: 351.2201.

1’,4’-trans-6-Bicyclohex-4-yl-4,9-dihydro-3H-pyrano[3,4-b]indole-1-thione (3kb)
To a stirred solution of 2kb (150 mg, 0.43 mmol) in toluene (6 mL) was added Lawesson’s reagent (96 mg, 0.24 mmol), and the resulting mixture was refluxed for 17 h. After cooling, the solvent was removed, and the residue was chromatographed on SiO2 (hexane–acetone = 40:1) to give 3kb (126 mg, 81%).
IR (neat): 3363, 2922, 2850, 1540, 1231 cm-1; 1H NMR (500MHz, CDCl3): δ 8.76 (1H, br), 7.42 (1H, brs), 7.32 (1H, d, J = 8.6 Hz), 7.29 (1H, dd, J = 1.4 Hz, 8.6 Hz), 4.74 (2H, t, J = 6.5 Hz), 3.16 (2H, t, J = 6.5 Hz), 2.53 (1H, tt, J = 3.1 Hz, 12.0 Hz), 1.97-0.97 (20H, m); 13C NMR (125 MHz, CDCl3): δ 198.30, 141.32, 137.87, 132.36, 128.09, 124.92, 118.56, 115.75, 112.11, 71.18, 44.62, 43.28, 42.98, 34.96, 30.28, 26.84, 21.27; MS (EI) m/z 367 (M+); HRMS (EI) calcd for C23H29NOS: 367.1970 (M+), found: 367.1968.

1’,4’-trans-6-Bicyclohex-4-yl-4,9-dihydro-3H-2-thia-9-azafluoren-1-one (6kb)
To a stirred solution of NaI (206 mg, 1.37 mmol) in ClCH2CH2Cl (10 mL) was added TMSCl (0.18 mL, 1.37 mmol), and the resulting mixture was stirred at room temperature for 15 min. To a solution of 3kb (126mg, 0.34 mmol) in ClCH2CH2Cl (10 mL) was transferred a solution of TMSI, prepared above, via a cannula, and then the resulting mixture was refluxed for 4 days. After cooling, the reaction was quenched with 10% HCl aq, and the aqueous mixture was extracted with CHCl3. The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 35 : 1) to give 6kb (101 mg, 80%).
mp 275-276 °C; IR (KBr): 3288, 2915, 2848, 1628 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.74 (1H, br), 7.44 (1H, brs), 7.33 (1H, d, J = 8.6 Hz), 7.27 (1H, dd, J = 1.4, 8.6 Hz), 3.48 (2H, t, J = 6.5 Hz), 3.28 (2H, t, J = 6.5 Hz), 2.55 (1H, tt, J = 3.4, 12.2 Hz), 1.98-0.98 (20H, m); 13C NMR (125 MHz, CDCl3): δ 183.61, 140.68, 135.35, 128.70, 127.52, 125.90, 125.18, 117.70, 112.18, 44.67, 43.30, 43.01, 35.05, 31.54, 30.33, 30.29, 26.85, 21.79; MS (EI) m/z 367 (M+); HRMS (EI) calcd for C23H29NOS: 367.1970 (M+), found: 367.1971.

1’,4’-trans-(6-Bicyclohex-4-yl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid tert-butyl ester (7kb)
To a stirred solution of 6kb (101 mg, 0.28 mmol) in DMF (3 mL) was added NaH (60%, 17 mg, 0.41 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for 30 min. To the mixture was added BrCH2CO2t-Bu (61 µL, 0.41 mmol) at 0 °C, and the resulting mixture was stirred at room temperature for 15 h. The reaction was quenched with H2O, and the aqueous mixture was extracted with Et2O. The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 30:1) to give 7kb (128 mg, 97%).
mp 178-180 °C; IR (KBr): 2921, 2850, 1740, 1641 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.44 (1H, brs), 7.30 (1H, d, J = 8.7 Hz), 7.17 (1H, d, J = 8.7 Hz), 5.13 (2H, s), 3.42 (2H, t, J = 6.4 Hz), 3.31 (2H, t, J = 6.4 Hz), 2.54 (1H, tt, J = 3.3, 12.2 Hz), 1.97-0.98 (20H, m), 1.45 (9H, s); 13C NMR (125 MHz, CDCl3): δ 183.87, 167.90, 140.73, 137.31, 127.90, 127.54, 126.24, 124.84, 118.00, 109.60, 82.21, 47.11, 44.61, 43.30, 43.00, 35.02, 31.26, 30.31, 30.27, 28.01, 26.84, 22.37; MS (EI) m/z 481 (M+); HRMS (EI) calcd for C29H39NO3S: 481.2651 (M+), found: 481.2655.

1’,4’-trans-(6-Bicyclohex-4-yl-1-oxo-3,4-dihydro-1H-2-thia-9-azafluoren-9-yl)acetic acid (5kb)
To a stirred solution of NaI (138 mg, 0.92 mmol) in ClCH2CH2Cl (5 mL) was added TMSCl (0.12 mL, 0.92 mmol), and the resulting mixture was stirred at room temperature for 15 min. To a solution of 7kb (111 mg, 0.23 mmol) in ClCH2CH2Cl (5 mL) was transferred a solution of TMSI, prepared above, via a cannula, and then the resulting mixture was refluxed for 19 h. After cooling, the reaction was quenched with 10% HCl aq, and the aqueous mixture was extracted with EtOAc. The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on SiO2 (hexane–acetone = 3 : 1) to give 5kb (81 mg, 83%).
mp 234-236 °C; IR (KBr): 2908, 2850, 1723, 1632 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.44 (1H, brs), 7.33 (1H, dd, J = 1.5, 8.6 Hz), 7.21 (1H, d, J = 8.6 Hz), 5.25 (2H, s), 3.42 (2H, t, J = 6.3 Hz), 3.31 (2H, t, J = 6.3 Hz), 2.55 (1H, tt, J = 3.3, 12.0 Hz), 1.96-0.97 (20H, m); 13C NMR (125 MHz, CDCl3): δ 184.59, 172.37, 141.15, 137.36, 128.02, 127.81, 126.82, 124.87, 118.11, 109.67, 46.42, 44.60, 43.30, 43.01, 35.01, 31.16, 30.30, 29.68, 26.86, 22.32; MS (EI) m/z 425 (M+); HRMS (EI) calcd for C25H31NO3S: 425.2025 (M+), found: 425.2024.

4-(trans-[1,1'-Bi(cyclohexane)]-4-yl)aniline p-toluenesulfonate (14)
To a stirred solution of 13b (63 mg, 0.25 mmol) in MeOH (3 mL) was added p-toluenesulfonic acid (46.6 mg, 0.25 mmol), and the resulting mixture was stirred at room temperature for 2.5 h. The solvent was concentrated in vacuo to give 14 (107 mg, quant.)
mp 250-251 °C; IR (KBr): 2920, 2849, 1516 cm-1; 1H NMR (500 MHz, DMSO-d6): δ 9.88 (1H, br), 7.48 (2H, d, J = 8.0 Hz), 7.33 (2H, d, J = 8.0 Hz), 7.25 (2H, d, J = 8.0 Hz), 7.11 (2H, d, J = 8.0 Hz), 2.47 (1H, tt, J = 3.2, 12.0 Hz), 2.28 (3H, s), 1.81-0.94 (20H, m); 13C NMR (125 MHz, DMSO-d6): δ 147.65, 145.35, 137.84, 129.11, 128.13, 127.98, 125.48, 123.08, 43.35, 42.72, 42.21, 33.98, 29.72, 29.60, 26.34, 26.31, 20.79.

N-(cis-4-Bicyclohex-3-yl-phenyl)-4-nitro-benzenesulfonamide (15)
To a stirred solution of 13a (108 mg, 0.37 mmol) in pyridine (2 mL) was added 4-nitrobenzenesulfonyl chloride (90 mg, 0.41 mmol), and the resulting mixture was stirred at room temperature for 23 h. The mixture was diluted with CH2Cl2 and washed with 10% HCl aq. The organic layer was dried over Na2SO4, and evaporated. The residue was chromatographed on SiO2 (hexane–CH2Cl2 = 1 : 2) to give 15 (186 mg, quant.).
mp 174-175 °C; IR (KBr): 3220, 2926, 2920, 1535, 1349 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.28 (2H, d, J = 9.2 Hz), 7.90 (2H, d, J = 9.2 Hz), 7.11 (2H, d, J = 8.3 Hz), 6.96 (2H, d, J = 8.3 Hz), 6.44 (1H, br), 2.45 (1H, tt, J = 3.2, 12.0 Hz) 1.89-0.95 (20H, m); 13C NMR (125 MHz, CDCl3): δ 150.15, 146.78, 144.78, 132.69, 128.53, 127.96, 124.21, 122.87, 44.10, 43.59, 43.40, 37.93, 34.28, 30.16, 30.13, 29.39, 26.79, 26.70; MS (EI) m/z 442 (M+); HRMS (EI) calcd for C24H30N2O4S: 442.1926 (M+), found: 442.1928.

GLP-1 secretion studies in vitro
Human enteroendocrine NCI-H716 cells were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.), and were maintained in suspension culture as instructed by the supplier. Two days before each experiment, the cells were seeded in 96-well plates pre-coated with poly-L-lysine (1x105 cells /well). On the day of the experiment, the culture medium was replaced by assay buffer (146mM NaCl, 5mM KCl, 1.5mM CaCl2, 1mM MgSO4, 20 mM N-(2-hydroxyethyl)- piperazine-N-(2-ethanesulfonic acid) (HEPES), 5.6 mM glucose, 2 mg/mL bovine serum albumin, and 10 mM sitagliptin, pH 7.4) with or without test agents, and the cells were incubated for 1 h at 37 °C. Then the GLP-1 level in the assay buffer was measured by ELISA (LINCO, Billerica, MA, U.S.A.).

Acute and long-term effect of 5k on blood glucose in vivo
This study was approved by the Animal Care and Use Committee of Ajinomoto. Male C57BL/6J and KKAy mice were purchased from Clea Japan (Tokyo, Japan) at 5 weeks of age, and each mouse was housed in a polycarbonate cage with wood chip bedding. Water and commercial chow were provided ad libitum. The animal room was kept on a 12-h light/dark cycle (7:00 AM to 7:00 PM, dark; 7:00 PM to 7:00 AM, light), with a temperature range of 22 °C ± 1 °C and a relative humidity of 55% ± 5% throughout the experimental period. The animals were acclimatized to the laboratory condition for 4 weeks.
For evaluation of acute hypoglycemic effect of 5k, C57BL/6J mice were fasted overnight, and were administered either the vehicle (0.5% methylcellulose), or 100 mg/kg of 5k by oral gavage with or without subcutaneous injection of GLP-1 antagonist exendin (9-39) (24 nmol/kg). Then, 2 g/kg of glucose was given orally immediately after 5k administration. Blood samples were collected from the tail vein to measure the blood glucose levels.
For evaluation of long-term effect of 5k on blood glucose control, KKAy mice were divided into 4 groups, and either the vehicle, 5k (100 mg/kg), sitagliptin (10 mg/kg), or pioglitazone (10 mg/kg) was administered orally twice a day for 3 weeks. At the end of the study, blood samples were collected from the tail vein and the blood glucose, HbA1c, plasma insulin, and plasma glucagon levels were measured. Blood glucose was measured with an autoanalyzer (Fuji Dri-Chem 5500; Fujifilm, Tokyo, Japan). Plasma insulin was measured by ELISA (Morinaga, Tokyo, Japan). Plasma glucagon was also measured by ELISA (Yanaihara Institute Inc., Shizuoka, Japan). HbA1c was measured by HPLC (TOSOH, Tokyo, Japan).

Statistical analysis
Results are expressed the mean±SEM. Statistical analysis was performed with StatView software (version 5.0, SAS institute, Cary, NC, USA). Differences were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s test. Statistical significance was accepted at p<0.05.

ACKNOWLEDGEMENT
We would like to thank Professor Daniel L. Comins, North Carolina State University, for very valuable suggestions and discussions about this paper.

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