HETEROCYCLES

Paper | Regular issue | Vol. 83, No. 6, 2011, pp. 1315-1328
Received, 23rd February, 2011, Accepted, 7th April, 2011, Published online, 12th April, 2011.
DOI: 10.3987/COM-11-12187

■ Polycyclic N-Heterocyclic Compounds. Part 70: Synthesis of 5-Amino-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridines and Related Compounds. Evaluation of Effects on Lipoprotein Lipase mRNA Expression

Kensuke Okuda,* Hideyasu Takechi, Takashi Hirota, and Kenji Sasaki

Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan

Abstract

Reaction of 3-(3-cyanopropoxy)thieno[2,3-b]pyridine-2-carbonitriles with potassium tert-butoxide gave 5-amino-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridines via a Truce-Smiles rearrangement. The 5-amino group was transformed to the chloro derivatives which were allowed to react with various nucleophiles. Effects of the newly synthesized compounds on lipoprotein lipase mRNA expression were also evaluated. The previously unreported parent compound, furo[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine, was also synthesized.

INTRODUCTION
Formation of carbon–carbon (C–C) bonds is a central issue in synthetic organic chemistry. In this regard, the Truce-Smiles rearrangement is among those useful rearrangement reactions that provide access to complex structures from simple precursors through formation of new C-C bonds.1-5
We have previously reported the application of the Truce-Smiles rearrangement for the synthesis of many aromatic fused dihydrofuro[2,3-b]pyridines (1) in one step from cyano aromatic ring having a 3-cyanopopoxy group adjacent to cyano group (2). Thus, base-mediated Truce-Smiles rearrangement of 2 followed by intramolecular cyclization (Scheme 1) produces 1 in moderate to good yields.6-8 5-Substituted derivatives of 1 were also accessible by usual SNAr reactions. An in vitro screening evaluation of these compounds and derivatives to measures their effects on lipoprotein lipase (LPL) mRNA expression in 3T3-L1 preadipocytes was performed as part of our continuing program to develop agents for hyperlipemia.9

With a goal to develop more potent compounds, we have pursued the preparation of other fused dihydrofuropyridines derivatives using our rearrangement methodology. Thus, we sought to expand this rearrangement reaction system to 3-(3-cyanopropoxy)thieno[2,3-b]pyridine-2-carbonitriles (3) as a route to 5-amino-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridines (4). This is a fairly challenging substrate because the electron rich thiophene is a less reactive candidate for the Truce-Smiles rearrangement. Herein we report the details of the Truce-Smiles type rearrangement reaction for a series of 3. We also report preparation of 5-substituted derivatives as well as a dihydrofuran ring cleaved derivative.9 The effect of these fused dihydrofuropyridines on LPL mRNA expression, one of key targets for development of diabetes drug, was evaluated. Finally, the parent skeleton, furo[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (5) was also synthesized.

RESULTS AND DISCUSSION
The starting compounds were readily obtained as follows. Reaction of 3-hydroxythieno[2,3-b]pyridine-2-carbonitrile10 (6a) with 4-chlorobutyronitrile in the presence of potassium carbonate in DMF gave the desired 3a (73%) (Scheme 2). In the IR spectrum of 3a, the hydroxy band present in 6a had disappeared, strongly indicating that 3a is an O-alkyl derivative, not a C-alkyl derivative. Compound 6b was prepared from ethyl 6-methyl-2-sulfanylnicotinate11 (7b) by two steps and 6c was prepared from 3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carbonitrile12 (9c) directly. A similar reaction of 6b and 6c with 4-chlorobutyronitrile afforded the desired 3b (82%) and 3c (78%), respectively.

The initial reaction of 3a with potassium tert-butoxide in 1,4-dioxane at room temperature produced 4a in 59% yield (Table 1, Run 1). In the IR spectrum of 4a, the cyano band was absent and amino bands were observed at 3400, 3325, and 3200 cm-1. In the 1H NMR spectrum of 4a, two dihydrofuran methylene resonances appeared at 3.54 and 4.65 ppm, respectively. The two deuterium oxide exchangeable protons of the amino group appeared at 6.47 ppm. These data are consistent with the structure of 4a and assignment was further supported by FAB-MS and elemental analysis. If the solvent were changed from 1,4-dioxane to THF, the reaction did not proceed at room temperature, but reflux conditions gave 4a in 53% yield (Run 2). The best yield was obtained when DMF was used as solvent at room temperature (72%, Run 3). Sodium hydride did not improve the product yield (Run 4). Compounds 3b and 3c were also treated with potassium tert-butoxide in DMF to give 4b (68%) and 4c (61%), respectively.

With the goal of synthesizing additional derivatives for biological evaluation, the 5-amino groups of 4a–c were transformed to 5-chloro derivatives (10a–c) as potential intermediates for coupling with nucleophiles. Thus the 5-amino derivatives 4a–c were treated with sodium nitrite and concd hydrochloric acid to give 10a–c in 66–70% yield (Scheme 3). Next, 10a was subjected to reactions with various nucleophiles (2-aminoethanol, 3-amino-1-propanol, ethylene glycol, 1,3-propanediol, 2-sulfanylethanol, 3-sulfanyl-1-propanol, pyrrolidine, piperidine, and morpholine) to give derivatives 11–19 in 41–79% yield (Scheme 4 and 5).

In contrast to the above results, a similar reaction of 10a with the anilide anion (3 eq.), prepared by reaction of aniline with sodium hydride in dry 1,4-dioxane, produced a 47% yield of the furan ring opened vinyl derivative 20 that retains the chloro atom (Scheme 6). Typical vinyl protons were observed at 5.85–7.14 ppm in the 1H NMR spectrum. The strong basicity of the anilide anion (aniline pKa 30.6)13 causes the anilide anion to function as a base rather than a nucleophile in this experiment. When 10a was treated with sodium hydride (6 eq.) alone in dry 1,4-dioxane under reflux, the yield of 20 was slightly increased to 51% yield.

With these derivatives in hand, effects on LPL mRNA expression in 3T3-L1 preadipocytes were examined using an in vitro screening test for hyperlipemia. Troglitazone14 was employed as a reference compound and GAPDH was chosen for the house keeping gene. The LPL/GAPDH mRNA ratio was evaluated as the relative values of LPL/GAPDH ratio from vehicle control group and tests were done in triplicate. None of the compounds showed significant activity (data not shown).
Finally, we pursued routes to the parent ring skeleton, unsubstituted furo[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (5), since the parent ring system itself had not been reported. We first attempted dechlorination of 10a by catalytic hydrogenolysis using H2/10% Pd-C but only unreacted starting material was recovered. Next we investigated a two step dechlorination strategy (Scheme 7). The 5-chlorine atom of compound 10a was substituted by hydrazine and the resulting hydrazine derivative was treated with CuSO4 to give 5-unsubsituted 21 in 52% yield (2 steps).15 Oxidation of 21 with DDQ gave the desired parent compound 5 in 57% yield.

CONCLUSION
In summary, we have developed a method for the synthesis of 5-amino-1,2-dihydrofuro[2,3-b]- pyrido[3’,2’:4,5]thieno[3,2-d]pyridines (4) via a Truce-Smiles rearrangement. The 5-amino group was transformed to the chloro derivative (10a), which was allowed to react with various nucleophiles to give 5-substituted derivatives. None of the new compounds showed significant effects on LPL mRNA expression. In addition, the synthesis of the previously unreported parent compound, furo[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (5), was achieved.

EXPERIMENTAL
All melting points were determined on a Yanagimoto micro-melting point apparatus, and are uncorrected. Elemental analyses were performed on a Yanagimoto MT-5 CHN corder elemental analyzer. The FAB-mass spectra were obtained on a VG 70 mass spectrometer and m-nitrobenzyl alcohol was used as the matrix. The IR spectra were recorded on a JASCO FT/IR-200 spectrophotometer and frequencies are expressed in cm-1. The 1H NMR spectra were recorded on a Varian VXR-200 instrument operating at 200 MHz or Hitachi R-1500 instrument operating at 60 MHz with TMS as an internal standard. Column chromatography was performed on silica gel (IR-60-63-210-W, Daiso). TLC was carried out on Kieselgel 60F254 (Merck) or silica gel 70FM (Wako).
3-(3-Cyanopropoxy)thieno[2,3-b]pyridine-2-carbonitrile (3a). To a solution of 3-hydroxythieno- [2,3-b]pyridine-2-carbonitrile10 (6a, 10.0 g, 56.8 mmol) in DMF (150 mL) were added K2CO3 (15.7 g, 114 mmol) and 4-chlorobutyronitrile (11.8 g, 114 mmol) and the mixture was stirred at 80 °C for 3.5 h. Solid was removed by filtration and the mother liquid was evaporated in vacuo. Ice water (500 mL) was poured into the residue and the resulting precipitate was collected by filtration. Recrystallization from cyclohexane-benzene gave 3a (10.1 g, 73%) as colorless needles, mp 99–101 °C; IR (Nujol) 2240, 2210 (CN) cm-1; 1H NMR (200 MHz, CDCl3) δ 2.31 (quin, J = 6.5 Hz, 2H, 2’-H), 2.66 (t, J = 6.5 Hz, 2H, 3’-H), 4.84 (t, J = 6.5 Hz, 2H, 1’-H), 7.41 (dd, J = 8.1, 4.6 Hz, 1H, 5-H), 8.20 (dd, J = 8.1, 1.7 Hz, 1H, 4-H), 8.76 (dd, J = 4.6, 1.7 Hz, 1H, 6-H); MS m/z 244 (MH+). Anal. Calcd for C12H9N3OS: C, 59.24; H, 3.73; N, 17.27. Found: C, 59.16; H, 3.88; N, 17.11.
Ethyl 2-(cyanomethylsulfanyl)-6-methylnicotinate (8b). To a solution of ethyl 6-methyl-2- sulfanylnicotinate11 (7b, 4.50 g, 22.8 mmol) in DMF (50 mL) were added K2CO3 (6.30 g, 45.6 mmol) and chloroacetonitrile (3.40 g, 45.0 mmol) and the mixture was stirred at 80 °C for 2 h. Solid was removed by filtration and the mother liquid was evaporated in vacuo. Ice water (300 mL) was poured into the residue and the resulting precipitate was collected by filtration. Recrystallization from cyclohexane gave 8b (3.90 g, 72%) as colorless needles, mp 76–78 °C; IR (Nujol) 2245 (CN), 1705 (CO) cm-1; 1H NMR (60 MHz, CDCl3) δ 1.41 (t, J = 7.0 Hz, 3H, CH2CH3), 2.62 (s, 3H, CH3), 3.93 (s, 2H, -CH2CN), 4.40 (q, J = 7.0 Hz, 2H, -CH2CH3), 7.02 (d, J = 8.0 Hz, 1H, 5-H), 8.19 (d, J = 8.0 Hz, 1H, 4-H); MS m/z 237 (MH+). Anal. Calcd for C11H12N2O2S: C, 55.91; H, 5.12; N, 11.86. Found: C, 56.08; H, 5.22; N, 11.58.
3-Hydroxy-6-methylthieno[2,3-b]pyridine-2-carbonitrile (6b). To a solution of 8b (5.00 g, 21.2 mmol) in DMF (100 mL) was added CaO (3.50 g, 62.4 mmol) and the reaction was then stirred at 100 °C for 3 h. Solid was removed by filtration and the mother liquid was evaporated in vacuo. Ice water (300 mL) was poured into the residue and the mixture was neutralized with 1N HCl aq. The resulting precipitate was collected by filtration. The mother liquid was extracted with EtOAc (200 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue and the precipitate described above were combined and recrystallized from EtOAc to give 6b (3.30 g, 82%) as colorless needles, mp 188–192 °C (dec.); IR (Nujol) 3400 (OH), 2210 (CN) cm-1; 1H NMR (60 MHz, DMSO-d6) δ 2.61 (s, 3H, CH3), 7.41 (d, J = 8.4 Hz, 1H, 5-H), 8.30 (d, J = 8.4 Hz, 1H, 4-H), 12.44 (br, D2O exchangeable, 1H, OH); MS m/z 191 (MH+); Anal. Calcd for C9H6N2OS·H2O: C, 51.91; H, 3.87; N, 13.45. Found: C, 51.96; H, 3.95; N, 13.36.
3-(3-Cyanopropoxy)-6-methylthieno[2,3-b]pyridine-2-carbonitrile (3b). To a solution of 6b (1.40 g, 7.36 mmol) in DMF (20 mL) were added K2CO3 (2.00 g, 14.5 mmol) and 4-chlorobutyronitrile (1.50 g, 14.5 mmol) and the reaction mixture was stirred at 100 °C for 2 h. Solid was removed by filtration and the mother liquid was evaporated in vacuo. Ice water (150 mL) was poured into the residue and the resulting precipitate was collected by filtration. Recrystallization from cyclohexane-benzene gave 3b (1.55 g, 82%) as colorless needles, mp 113–115 °C; IR (Nujol) 2250, 2210 (CN) cm-1; 1H NMR (200 MHz, CDCl3) δ 2.29 (quin, J = 6.0 Hz, 2H, 2’-H), 2.65 (t, J = 6.0 Hz, 2H, 3’-H), 2.70 (s, 3H, CH3), 4.81 (t, J = 6.0 Hz, 2H, 1’-H), 7.25 (d, J = 8.3 Hz, 1H, 5-H), 8.05 (d, J = 8.3 Hz, 1H, 4-H); MS m/z 258 (MH+). Anal. Calcd for C13H11N3OS: C, 60.68; H, 4.31; N, 16.33. Found: C, 60.84; H, 4.46; N, 16.30.
3-Hydroxy-4,6-dimethylthieno[2,3-b]pyridine-2-carbonitrile (6c) and 3-Hydroxy-4,6- dimethylthieno[2,3-b]pyridine-2-carboxamide (6c’). A solution of 3-amino-4,6-dimethylthieno- [2,3-b]pyridine-2-carbonitrile12 (9c, 5.00 g, 24.6 mmol) in 6N HCl aq. (100 mL) was refluxed for 3 h. After cooling, the solution was neutralized with NaHCO3 and the resulting precipitate was collected by filtration. The mother liquid was extracted with EtOAc (200 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue and the precipitate described above were combined and chromatographed on silica gel. The eluate of cyclohexane-EtOAc (2:1) was evaporated in vacuo and the residue was recrystallized from EtOAc to give 6c (2.20 g, 44%) as colorless needles. The further eluate of EtOAc was evaporated in vacuo and the residue was recrystallized from MeOH to give 6c’ (1.90 g, 35%) as colorless needles. 6c: mp 241–245 °C (dec.); IR (Nujol) 3450 (OH), 2210 (CN) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.53 (s, 3H, CH3), 2.65 (s, 3H, CH3), 7.17 (s, 1H, 5-H), 12.53 (br, D2O exchangeable, 1H, OH); MS m/z 205 (MH+). Anal. Calcd for C10H8N2OS·H2O: C, 54.04; H, 4.53; N, 12.60. Found: C, 54.26; H, 4.80; N, 12.56. 6c’: mp 252–254 °C; IR (Nujol) 3340, 3280, 3150 (NH and OH), 1685 (CO) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.53 (s, 3H, CH3), 2.66 (s, 3H, CH3), 7.13 (s, 1H, 5-H), 7.96 (br, D2O exchangeable, 2H, NH2), 13.33 (br, D2O exchangeable, 1H, OH); MS m/z 223 (MH+). Anal. Calcd for C10H10N2O2S: C, 54.04; H, 4.53; N, 12.60. Found: C, 54.16; H, 4.77; N, 12.86.
3-(3-Cyanopropoxy)-4,6-dimethylthieno[2,3-b]pyridine-2-carbonitrile (3c). To a solution of 6c (2.00 g, 9.79 mmol) in DMF (25 mL) were added K2CO3 (2.70 g, 19.5 mmol) and 4-chlorobutyronitrile (2.00 g, 19.3 mmol) and the mixture was stirred at 80 °C for 4 h. Solid was removed by filtration and the mother liquid was evaporated in vacuo. Ice water (150 mL) was poured into the residue and the resulting precipitate was collected by filtration. Recrystallization from cyclohexane-benzene gave 3c (2.08 g, 78%) as colorless needles, mp 148–150 °C; IR (Nujol) 2240, 2200 (CN) cm-1; 1H NMR (200 MHz, CDCl3) δ 2.30 (quin, J = 6.2 Hz, 2H, 2’-H), 2.61 (s, 3H, CH3), 2.64 (t, J = 6.2 Hz, 2H, 3’-H), 2.65 (s, 3H, CH3), 4.80 (t, J = 6.2 Hz, 2H, 1’-H), 7.00 (s, 1H, 5-H); MS m/z 272 (MH+). Anal. Calcd for C14H13N3OS: C, 61.97; H, 4.83; N, 15.49. Found: C, 61.90; H, 4.97; N, 15.41.

5-Amino-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (4a). To a solution of 3a (2.00 g, 8.22 mmol) in dry DMF (20 mL) was added t-BuOK (1.80 g, 16.0 mmol) and the mixture was stirred for 6 min at room temperature. Ice water (100 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from DMF gave 4a (1.44 g, 72%) as yellow micro crystals, mp > 300 °C; IR (Nujol) 3400, 3325, 3200 (NH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 3.54 (t, J = 8.5 Hz, 2H, 1-H), 4.65 (t, J = 8.5 Hz, 2H, 2-H), 6.47 (br, D2O exchangeable, 2H, NH2), 7.54 (dd, J = 7.9, 4.7 Hz, 1H, 9-H), 8.42 (dd, J = 7.9, 1.7 Hz, 1H, 10-H), 8.71 (dd, J = 4.7, 1.7 Hz, 1H, 8-H); MS m/z 244 (MH+). Anal. Calcd for C12H9N3OS: C, 59.24; H, 3.73; N, 17.27. Found: C, 59.34; H, 4.09; N, 16.97.
5-Amino-8-methyl-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (4b). To a solution of 3b (1.00 g, 3.89 mmol) in dry DMF (10 mL) was added t-BuOK (870 mg, 7.75 mmol) and the mixture was stirred for 6 min at room temperature. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from DMF gave 4b (680 mg, 68%) as yellow micro crystals, mp > 300 °C; IR (Nujol) 3450, 3275, 3150 (NH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.63 (s, 3H, CH3), 3.50 (t, J = 8.5 Hz, 2H, 1-H), 4.64 (t, J = 8.5 Hz, 2H, 2-H), 6.41 (br, D2O exchangeable, 2H, NH2), 7.39 (d, J = 8.1 Hz, 1H, 9-H), 8.28 (d, J = 8.1 Hz, 1H, 10-H); MS m/z 258 (MH+). Anal. Calcd for C13H11N3OS: C, 60.68; H, 4.31; N, 16.33. Found: C, 60.55; H, 4.50; N, 16.21.
5-Amino-8,10-dimethyl-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (4c). To a solution of 3c (1.00 g, 3.69 mmol) in dry DMF (10 mL) was added t-BuOK (820 mg, 7.31 mmol) and the mixture was stirred for 6 min at room temperature. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from DMF gave 4c (610 mg, 61%) as yellow micro crystals, mp > 300 °C; IR (Nujol) 3435, 3275, 3160 (NH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.55 (s, 3H, CH3), 2.82 (s, 3H, CH3), 3.68 (t, J = 8.6 Hz, 2H, 1-H), 4.50 (t, J = 8.6 Hz, 2H, 2-H), 6.33 (br, D2O exchangeable, 2H, NH2), 7.16 (s, 1H, 9-H); MS m/z 272 (MH+). Anal. Calcd for C14H13N3OS: C, 61.97; H, 4.83; N, 15.49. Found: C, 61.97; H, 5.20; N, 15.38.
5-Chloro-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (10a). To a stirred suspension of 4a (2.00 g, 8.22 mmol) in concd HCl (50 mL) cooled in an ice water bath (0–5 °C) was added dropwise NaNO2 (1.70 g, 24.6 mmol) in water (4.0 mL) and the mixture was then stirred for 1h. After the end point of the reaction was confirmed with KI-starch paper, the mixture was basified with NaHCO3. The resulting precipitate was collected by filtration. The mother liquid was extracted with EtOAc (100 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue and the precipitate described above were combined and recrystallized from 1,4-dioxane to give 10a (1.52 g, 70%) as colorless needles, mp 278–280 °C; 1H NMR (200 MHz, DMSO-d6) δ 3.73 (t, J = 8.7 Hz, 2H, 1-H), 4.84 (t, J = 8.7 Hz, 2H, 2-H), 7.65 (dd, J = 8.0, 4.7 Hz, 1H, 9-H), 8.54 (dd, J = 8.0, 1.7 Hz, 1H, 10-H), 8.81 (dd, J = 4.7, 1.7 Hz, 1H, 8-H); MS m/z 263 (MH+), 265 (MH+ + 2). Anal. Calcd for C12H7ClN2OS: C, 54.86; H, 2.69; N, 10.66. Found: C, 54.79; H, 2.87; N, 10.49.
5-Chloro-8-methyl-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (10b). To a stirred suspension of 4b (2.00 g, 7.77 mmol) in concd HCl (50 mL) cooled in an ice water bath (0–5 °C) was added dropwise NaNO2 (1.60 g, 23.2 mmol) in water (4.0 mL) and the reaction was then stirred for 1h. After the end point of the reaction was confirmed with KI-starch paper, the mixture was basified with NaHCO3. The resulting precipitate was collected by filtration. The mother liquid was extracted with EtOAc (100 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue and the precipitate described above was combined and recrystallized from 1,4-dioxane to give 10b (1.44 g, 67%) as colorless needles, mp > 300 °C; 1H NMR (200 MHz, DMSO-d6) δ 2.66 (s, 3H, CH3), 3.70 (t, J = 8.7 Hz, 2H, 1-H), 4.83 (t, J = 8.7 Hz, 2H, 2-H), 7.50 (d, J = 8.2 Hz, 1H, 9-H), 8.39 (d, J = 8.2 Hz, 1H, 10-H); MS m/z 277 (MH+), 279 (MH+ + 2). Anal. Calcd for C13H9ClN2OS: C, 56.42; H, 3.28; N, 10.12. Found: C, 56.54; H, 3.26; N, 9.96.
5-Chloro-8,10-dimethyl-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (10c). To a stirred suspension of 4c (1.00 g, 3.69 mmol) in concd HCl (20 mL) cooled in an ice water bath (0–5 °C) was added dropwise NaNO2 (760 mg, 11.0 mmol) in water (2.0 mL) and the reaction was stirred for 1h. After the end point of the reaction was confirmed with KI-starch paper, the mixture was basified with NaHCO3. The resulting precipitate was collected by filtration. The mother liquid was extracted with EtOAc (100 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue and the precipitate described above were combined and recrystallized from 1,4-dioxane to give 10c (710 mg, 66%) as colorless needles, mp 283–285 °C; 1H NMR (200 MHz, DMSO-d6) δ 2.57 (s, 3H, CH3), 2.88 (s, 3H, CH3), 3.93 (t, J = 8.7 Hz, 2H, 1-H), 4.67 (t, J = 8.7 Hz, 2H, 2-H), 7.28 (s, 1H, 9-H); MS m/z 291 (MH+), 293 (MH+ + 2). Anal. Calcd for C14H11ClN2OS: C, 57.83; H, 3.81; N, 9.63. Found: C, 57.84; H, 4.18; N, 9.50.
5-(2-Hydroxyethylamino)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (11). To a suspension of 10a (300 mg, 1.14 mmol) in 1,4-dioxane (5.0 mL) was added 2-aminoethanol (2.10 g, 34.4 mmol) and the mixture was heated at 100 °C for 22 h with stirring. Ice water (50 mL) was poured into the reaction mixture which was then extracted with EtOAc (100 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and then evaporated in vacuo. The residue was recrystallized from DMF to give 11 (143 mg, 44%) as yellow needles, mp 290–292 °C; IR (Nujol) 3440, 3175 (NH and OH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 3.44–3.68 (m, 6H, 1, 1’, and 2’-H), 4.66 (t, J = 8.7 Hz, 2H, 2-H), 7.54 (dd, J = 7.8, 4.7 Hz, 1H, 9-H), 8.42 (dd, J = 7.8, 1.6 Hz, 1H, 10-H), 8.71 (dd, J = 4.7, 1.6 Hz, 1H, 8-H); MS m/z 288 (MH+). Anal. Calcd for C14H13N3O2S: C, 58.52; H, 4.56; N, 14.62. Found: C, 58.36; H, 4.39; N, 14.46.
5-(3-Hydroxypropylamino)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (12). To a suspension of 10a (300 mg, 1.14 mmol) in 1,4-dioxane (5.0 mL) was added 3-amino-1-propanol (2.50 g, 33.3 mmol) and the reaction was heated at 80 °C for 18 h while stirring. Ice water (50 mL) was poured into the reaction mixture which was then extracted with EtOAc (100 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and then evaporated in vacuo. The residue was recrystallized from MeOH to give 12 (141 mg, 41%) as yellow needles, mp 171–173 °C; IR (Nujol) 3340, 3180 (NH and OH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.75 (quin, J = 6.3 Hz, 2H, 2’-H), 3.41–3.58 (m, 6H, 1, 1’, and 3’-H), 4.66 (t, J = 8.6 Hz, 2H, 2-H), 6.78 (br, D2O exchangeable, 1H, NH or OH), 7.55 (dd, J = 8.0, 4.7 Hz, 1H, 9-H), 8.43 (dd, J = 8.0, 1.7 Hz, 1H, 10-H), 8.71 (dd, J = 4.7, 1.7 Hz, 1H, 8-H); MS m/z 302 (MH+). Anal. Calcd for C15H15N3O2S: C, 59.78; H, 5.02; N, 13.94. Found: C, 59.70; H, 4.72; N, 13.72.
5-(2-Hydroxyethoxy)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (13). To a mixture of 10a (300 mg, 1.14 mmol) and ethylene glycol (5.0 mL) was added K2CO3 (470 mg, 3.40 mmol) and the reaction was heated at 100 °C for 9 h with stirring. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from DMF gave 13 (144 mg, 44%) as colorless needles, mp 269–271 °C; IR (Nujol) 3180 (OH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 3.63 (t, J = 8.6 Hz, 2H, 1-H), 3.77 (t, J = 5.0 Hz, 2H, 2’-H), 4.42 (t, J = 5.0 Hz, 2H, 1’-H), 4.77 (t, J = 8.6 Hz, 2H, 2-H), 7.60 (dd, J = 8.0, 4.6 Hz, 1H, 9-H), 8.50 (dd, J = 8.0, 1.5 Hz, 1H, 10-H), 8.76 (dd, J = 4.6, 1.5 Hz, 1H, 8-H); MS m/z 289 (MH+). Anal. Calcd for C14H12N2O3S: C, 58.32; H, 4.20; N, 9.72. Found: C, 58.42; H, 4.13; N, 9.67.
5-(3-Hydroxypropoxy)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (14). To a mixture of 10a (300 mg, 1.14 mmol) and 1,3-propanediol (5.0 mL) was added K2CO3 (470 mg, 3.40 mmol) and the stirred mixture was heated at 100 °C for 10 h. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from DMF gave 14 (174 mg, 50%) as colorless granules, mp 210–212 °C; IR (Nujol) 3300 (OH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.92 (quin, J = 6.3 Hz, 2H, 2’-H), 3.55–3.67 (m, 4H, 1 and 3’-H), 4.47 (t, J = 6.3 Hz, 2H, 1’-H), 4.77 (t, J = 8.8 Hz, 2H, 2-H), 7.59 (dd, J = 8.1, 4.7 Hz, 1H, 9-H), 8.49 (dd, J = 8.1, 1.7 Hz, 1H, 10-H), 8.76 (dd, J = 4.7, 1.7 Hz, 1H, 8-H); MS m/z 303 (MH+). Anal. Calcd for C15H14N2O3S: C, 59.59; H, 4.67; N, 9.27. Found: C, 59.53; H, 4.47; N, 9.18.
5-(2-Hydroxyethylsulfanyl)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (15). To a solution of 10a (300 mg, 1.14 mmol) in DMF (5.0 mL) were added 2-sulfanylethanol (890 mg, 11.4 mmol) and K2CO3 (630 mg, 4.56 mmol) and the mixture was then heated at 80 °C for 5 h with stirring. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from DMF gave 15 (217 mg, 62%) as yellow needles, mp > 300 °C; IR (Nujol) 3200 (OH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 3.30 (t, J = 6.3 Hz, 2H, 1’-H), 3.63–3.72 (m, 4H, 1 and 2’-H), 4.75 (t, J = 8.6 Hz, 2H, 2-H), 7.58 (dd, J = 8.1, 4.6 Hz, 1H, 9-H), 8.49 (dd, J = 8.1, 1.6 Hz, 1H, 10-H), 8.76 (dd, J = 4.6, 1.6 Hz, 1H, 8-H); MS m/z 305 (MH+). Anal. Calcd for C14H12N2O2S2: C, 55.24; H, 3.97; N, 9.20. Found: C, 55.37; H, 3.90; N, 9.24.

5-(3-Hydroxypropylsulfanyl)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (16). To a solution of 10a (300 mg, 1.14 mmol) in DMF (5.0 mL) were added 3-sulfanyl-1-propanol (1.05 g, 11.4 mmol) and K2CO3 (630 mg, 4.56 mmol) and the reaction was then heated at 80 °C for 3.5 h. Ice water (50 mL) was poured into the mixture and the resulting precipitate was collected by filtration. Recrystallization from 1,4-dioxane gave 16 (257 mg, 71%) as yellow needles, mp 189–191 °C; IR (Nujol) 3280 (OH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.83 (quin, J = 6.5 Hz, 2H, 2’-H), 3.33 (t, J = 6.5 Hz, 2H, 1’-H), 3.52 (t, J = 6.5 Hz, 2H, 3’-H), 3.69 (t, J = 8.7 Hz, 2H, 1-H), 4.78 (t, J = 8.7 Hz, 2H, 2-H), 7.61 (dd, J = 8.0, 4.7 Hz, 1H, 9-H), 8.50 (dd, J = 8.0, 1.5 Hz, 1H, 10-H), 8.60 (dd, J = 4.7, 1.5 Hz, 1H, 8-H); MS m/z 319 (MH+). Anal. Calcd for C15H14N2O2S2: C, 56.58; H, 4.43; N, 8.80. Found: C, 56.41; H, 4.21; N, 8.72.
5-(Pyrrolidin-1-yl)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (17). A mixture of 10a (300 mg, 1.14 mmol) and pyrrolidine (5.0 mL) was heated at 80 °C for 7 h with stirring. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from EtOAc gave 17 (240 mg, 71%) as yellow needles, mp 171–173 °C; 1H NMR (200 MHz, DMSO-d6) δ 1.93–2.03 (m, 4H, 3’ and 4’-H), 3.51 (t, J = 8.6 Hz, 2H, 1-H), 3.66–3.75 (m, 4H, 2’ and 5’-H), 4.64 (t, J = 8.6 Hz, 2H, 2-H), 7.52 (dd, J = 8.0, 4.6 Hz, 1H, 9-H), 8.40 (dd, J = 8.0, 1.5 Hz, 1H, 10-H), 8.69 (dd, J = 4.6, 1.5 Hz, 1H, 8-H); MS m/z 298 (MH+). Anal. Calcd for C16H15N3OS: C, 64.62; H, 5.08; N, 14.13. Found: C, 64.65; H, 4.87; N, 14.05.
5-(Piperidin-1-yl)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (18). A mixture of 10a (300 mg, 1.14 mmol) and piperidine (5.0 mL) was heated at 100 °C for 14 h while being stirred. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from n-hexane-benzene gave 18 (281 mg, 79%) as yellow needles, mp 146–148 °C; 1H NMR (200 MHz, DMSO-d6) δ 1.65 (br, 6H, 3’, 4’, and 5’-H), 3.38–3.45 (m, 4H, 2’ and 6’-H), 3.61 (t, J = 8.6 Hz, 2H, 1-H), 4.71 (t, J = 8.6 Hz, 2H, 2-H), 7.57 (dd, J = 8.2, 4.6 Hz, 1H, 9-H), 8.46 (dd, J = 8.2, 1.5 Hz, 1H, 10-H), 8.74 (dd, J = 4.6, 1.5 Hz, 1H, 8-H); MS m/z 312 (MH+). Anal. Calcd for C17H17N3OS: C, 65.57; H, 5.50; N, 13.49. Found: C, 65.72; H, 5.28; N, 13.28.
5-(Morpholin-4-yl)-1,2-dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (19). A mixture of 10a (300 mg, 1.14 mmol) and morpholine (5.0 mL) was heated at 100 °C for 29 h with stirring. Ice water (50 mL) was poured into the reaction mixture and the resulting precipitate was collected by filtration. Recrystallization from MeOH gave 19 (221 mg, 62%) as yellow needles, mp 220–222 °C; 1H NMR (200 MHz, DMSO-d6) δ 3.39 (t, J = 4.6 Hz, 4H, 4H, 3’ and 5’-H), 3.63 (t, J = 8.8 Hz, 2H, 1-H), 3.79 (t, J = 4.6 Hz, 4H, 2’ and 6’-H), 4.73 (t, J = 8.8 Hz, 2H, 2-H), 7.57 (dd, J = 7.9, 4.7 Hz, 1H, 9-H), 8.46 (dd, J = 7.9, 1.5 Hz, 1H, 10-H), 8.74 (dd, J = 4.7, 1.5 Hz, 1H, 8-H); MS m/z 314 (MH+). Anal. Calcd for C16H15N3O2S: C, 61.32; H, 4.82; N, 13.41. Found: C, 61.16; H, 4.63; N, 13.26.
4-Chloro-2-hydroxy-1-vinylpyrido[3’,2’:4,5]thieno[2,3-c]pyridine (20). (method a): To a suspension of 10a (200 mg, 0.761 mmol) in dry 1,4-dioxane (20 mL) was added aniline (210 mg, 2.25 mmol) and NaH (55 mg, 2.29 mmol) and the mixture was refluxed for 1 h. After removal of solvent in vacuo, ice water (30 mL) was poured into the residue and the mixture was neutralized with 1N HCl aq. The resulting precipitate was collected by filtration. Recrystallization from EtOAc gave 20 (94.0 mg, 47%) as colorless needles, mp 265–270 °C (dec.); IR (Nujol) 3100 (OH) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 5.85 (dd, J = 11.1, 1.9 Hz, 1H, 2’-H), 5.97 (dd, J = 17.6, 1.9 Hz, 1H, 2’-H), 7.14 (dd, J = 17.6, 11.1 Hz, 1H, 1’-H), 7.59 (dd, J = 8.3, 5.3 Hz, 1H, 8-H), 8.76–8.84 (m, 2H, 7- and 9-H), 11.90 (br, D2O exchangeable, 1H, OH); MS m/z 263 (MH+), 265 (MH+ + 2). Anal. Calcd for C12H7ClN2OS: C, 54.86; H, 2.69; N, 10.66. Found: C, 54.98; H, 2.90; N, 10.66. (method b): To a suspension of 10a (200 mg, 0.761 mmol) in dry 1,4-dioxane (20 mL) was added NaH (110 mg, 4.58 mmol) and the mixture was refluxed for 48 h. After removal of solvent in vacuo, ice water (30 mL) was poured into the residue and the mixture was neutralized with 1N HCl aq. The resulting precipitate was collected by filtration. Recrystallization from EtOAc gave 20 (103 mg, 51%) as colorless needles. All analytical data were in good agreement with values obtained from the compound synthesized by method a.
1,2-Dihydrofuro[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (21). To a suspension of 10a (1.00 g, 3.81 mmol) in 1,4-dioxane (50 mL) was added anhydrous hydrazine (2.44 g, 76.3 mmol) and the reaction was refluxed for 4 d. The resulting precipitate was collected by filtration to give the hydrazine intermediate (770 mg, mp > 300 °C) as yellow micro needles, which was used without further purification. To a mixture of this intermediate in water (12 mL) and acetic acid (12 mL) under reflux was added dropwise 10% CuSO4 aq. (10 mL) and the reaction mixture was further refluxed for 2 h. After cooling to room temperature, the mixture was basified with 10% NaOH aq. and then extracted with EtOAc (100 mL x 3). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue was recrystallized from 1,4-dioxane to give 21 (452 mg, 52%) as colorless micro prisms, mp 231–233 °C; 1H NMR (200 MHz, DMSO-d6) δ 3.73 (t, J = 8.7 Hz, 2H, 1-H), 4.77 (t, J = 8.7 Hz, 2H, 2-H), 7.60 (dd, J = 8.0, 4.7 Hz, 1H, 9-H), 8.52 (dd, J = 8.0, 1.6 Hz, 1H, 10-H), 8.65 (s, 1H, 5-H), 8.76 (dd, J = 4.7, 1.6 Hz, 1H, 8-H); MS m/z 229 (MH+). HRMS m/z 229.0475 (Calcd for C12H9N2OS: 229.0436).
Furo[2,3-b]pyrido[3’,2’:4,5]thieno[3,2-d]pyridine (5). To a mixture of 21 (400 mg, 1.75 mmol) and 1,4-dioxane (50 mL) was added DDQ (1.60 g, 7.14 mmol) and the reaction was refluxed for 5 d. After removal of solvent in vacuo, Et2O (100 mL) was poured into the residue and the solution was washed with 10% NaOH aq. (50 mL x 3), saturated brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue was chromatographed on silica gel. The eluate of n-hexane-EtOAc (1:1) was evaporated and the residue was recrystallized from cyclohexane to give 5 (227 mg, 57%) as colorless needles, mp 243–245 °C; 1H NMR (200 MHz, DMSO-d6) δ 7.32 (d, J = 2.4 Hz, 1H, 1-H), 7.54 (dd, J = 8.0, 4.7 Hz, 1H, 9-H), 7.95 (d, J = 2.4 Hz, 1H, 2-H), 8.61 (dd, J = 8.0, 1.7 Hz, 1H, 10-H), 8.82 (dd, J = 4.7, 1.7 Hz, 1H, 8-H), 8.87 (s, 1H, 5-H); MS m/z 227 (MH+). Anal. Calcd for C12H6N2OS: C, 63.70; H, 2.67; N, 12.38. Found: C, 63.48; H, 2.97; N, 12.28.
Effects on LPL mRNA expression in 3T3-L1 preadipocytes The effects were assayed according to the literature procedure.9

ACKNOWLEDGEMENTS
We are grateful to the SC-NMR Laboratory of Okayama University for 200 MHz 1H NMR experiments. We also thank Dr. K. L. Kirk (NIDDK, NIH) for helpful suggestions.

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