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Open AccessArticle

Design, Synthesis and Evaluation of Indene Derivatives as Retinoic Acid Receptor α Agonists

ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
*
Author to whom correspondence should be addressed.
Academic Editor: Santosh K. Katiyar
Molecules 2017, 22(1), 32; https://doi.org/10.3390/molecules22010032
Received: 4 November 2016 / Revised: 22 December 2016 / Accepted: 24 December 2016 / Published: 27 December 2016
(This article belongs to the Special Issue Cancer Chemoprevention)

Abstract

A series of novel indene-derived retinoic acid receptor α (RARα) agonists have been designed and synthesized. The use of receptor binding, cell proliferation and cell differentiation assays demonstrated that most of these compounds exhibited moderate RARα binding activity and potent antiproliferative activity. In particular, 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)-carbamoyl)benzoic acid (36d), which showed a moderate binding affinity, exhibited a great potential to induce the differentiation of NB4 cells (68.88% at 5 μM). Importantly, our work established indene as a promising skeleton for the development of novel RARα agonists.
Keywords: retinoic acid receptor; agonist; all-trans-retinoic acid derivative; indene; structure and activity relationship retinoic acid receptor; agonist; all-trans-retinoic acid derivative; indene; structure and activity relationship

1. Introduction

The retinoid signal is mediated in target cells through retinoic acid receptors (RAR) and retinoid X receptors (RXR), both of which are members of the nuclear receptor superfamily. RARs are ligand-dependent transcription factors that act as RAR-RXR heterodimers to modulate gene transcription and thereby regulate a range of metabolic, endocrine and immunologic disorders [1,2]. There are three distinct isoforms RAR (α, -β and -γ), among which RARα is known to play a pivotal role in the control of cellular differentiation and apoptosis, and is therefore an important drug target for cancer therapy and prevention [3]. The natural ligand of RARα, all-trans-retinoic acid (ATRA), has been used to effectively treat acute promyelocytic leukaemia (APL) for nearly thirty years [4]. However, this therapy has its limitations which mainly lie in the structure of ATRA. Due to the presence of conjugated double bonds, ATRA easily undergoes oxidation and/or isomerization in the presence of oxidants, light or excessive heat [5]. To improve the stability, a large number of derivatives have been developed by fusing an aromatic ring in both its hydrophobic and hydrophilic regions to constrain the polyene side chain. The study of the relationships between structure and activity (SAR) has established the structure template of ATRA derivatives as a hydrophobic region and a polar region connected via a linker (Figure 1) [6,7,8]. Further SAR has revealed that the nature of the linker is crucial for the compounds to attain RAR-isotype selectivity and that the amide linker group is a key structural feature for RARα-specificity, presumably due to a favorable hydrogen-bonding interaction between the amide group of the ligand and the hydroxyl group of serine 232 residue present in the ligand binding pocket of RARα [9,10].
Among all the ATRA derivatives, AM80 (Figure 1) is a typical representative approved for therapy in 2005. AM80 can successfully induce complete remission in APL patients for whom ATRA therapy has failed [11,12,13]. This implies the great potential of synthetic RARα agonists in the treatment of APL and has fostered the search for new classes of compounds with improved pharmacologic activities.
To date, most of the developed ATRA derivatives contain a tri-/tetra-methylated six-membered rigid ring in their hydrophobic region. Since it has been reported that the size of the hydrophobic part of the ligands can significantly affect the activity [10], we were interested in studying the impact of a smaller ring system on the activity of derivatives by replacing the hydrophobic part of AM80 with mono-/di- substituted indene derivatives. Specifically, compound 5 was designed by incorporating small alkoxyl or alkyl groups into the indane structure. Keeping the planar configuration of indene by retaining the double bond or the incorporation of a ketone group yielded compounds 6 and 7. Di-substitution of indane with both alkoxyl and alkyl groups gave compounds 8 (Figure 2).

2. Chemistry

Synthesis of indene derivatives 36ap was conducted via procedures reported for the preparation of AM80 with some modifications. Detailed syntheses are shown in Scheme 1. Nitration of commercial available 9 with KNO3 and H2SO4 gave 10, whose carbonyl group was then reduced by NaBH4 to yield 11. Elimination of H2O from 11 gave 12, which could either be reduced to 13 or transformed into 16 [14,15]. Etherification of 16 with alkyl halides in the presence of KOH produced 17a and 17b. Compound 11 could also be alkylated with appropriate halogenoalkane (MeI, EtI, 2-bromo-propane or 1-bromobutane) to afford 14ad. In another synthetic route, 9 was reacted with paraformaldehyde/acetone to give 19a and 19b, which were reduced to yield 20a and 20b. Subsequent nitration of 20a and 20b and reduction of the carbonyl group gave 22a and 22b. Compounds 23a and 23b could be obtained by elimination reaction of 22a and 22b, while coupling of 22a, 22b with trimethyl orthoformate or triethyl orthoformate in the presence of BiCl3 yielded 27a, 28a and 27b, 28b, respectively. Reduction of the nitro group in 14ad, 17a, 17b, 21a, 21b, 23a, 23b, 27a, 27b and 28a, 28b then yielded the hydrophobic moieties 15ad, 18a, 18b, 26a, 26b, 24, 25a, 25b, 29a, 29b and 30a, 30b [16].
The hydrophilic segment 34 was prepared from terephthalic acid (31) after esterification and hydrolysis, followed by chlorination. Coupling of 34 with the hydrophobic part yielded 35ap which were then hydrolyzed to give the target ligands 36ap (Scheme 2).

3. Results and Discussion

3.1. RARα Binding Affinity

The obtained target compounds were tested for their binding affinities to RARα using a time resolved fluorescence resonance energy transfer (TR-FRET) assay with AM80 as the positive control. As shown in Table 1, compound 36a which bears no substituents exhibits modest RARα binding affinity, implying the feasibility of the indene skeleton as a promising platform for novel RARα agonists. With 36b36g being less potent than 36a, it seems that an alkoxyl group is not welcome, especially at the 2-position.
Furthermore, the extension of the π system by the retention of the indene double bond or the incorporation of a ketone group at the 1-position seems to be favorable, with 36j and 36l being more potent than their more saturated counterpart 36h. Interestingly, although an alkoxy-substituent alone is not well tolerated, it contributes to the binding affinity when coexisting with an isopropyl group, which is illustrated by comparison of the results of 36o, 36p with those of 36b and 36c.

3.2. Cell Proliferation Inhibitory Assay

Human promyelocytic leukemia cell lines HL60 and NB4 were employed to determine the effects of the derivatives on cell proliferation [17]. As shown in Table 2, these two cell lines responded quite differently to the tested compounds. Compounds 36bc with small aliphatic ether chains at the 1-position are more potent than 36de with larger ether side chains in HL60 cells, while the results in NB4 cells are the contrary. Alkoxy-substitution at the 2-position (i.e., compounds 36f, 36g) harmed the cell proliferation inhibitory activity of the compounds in both cell lines while the isopropyl group at the same position (compound 36h) is favourable. The extension of the π system by an alkenyl bond or a ketone group, as in 36jl, didn’t affect the compounds’ inhibitory activity in HL60 cells but nulified their activity in NB4 cells. The indene ring tolerates the dual-introduction of a small alkoxyl and an alkyl group at 1- and 2-position, respectively, to retain the antiproliferative activity in HL60 cells. The results of the dual-substituted compounds in the NB4 cells line are a little complicated, with 36n and 36p bearing an ethoxyl group at the 1-position showing moderate activity and 36m and 36o with a methoxyl group at the same position being almost inactive.

3.3. Cell Differentiation Assay Using HL60 and NB4

The effects of 36ag on the differentiation of HL60 and NB4 cells were then assessed. FACS analysis of the granulocyte differentiation marker CD11b revealed that 36d and 36e, which show high binding affinity to RARα (14.88~24.25 nM) and potent proliferation inhibitory activity in NB4 cells (1.86~4.09 μM), have the greatest potential to induce NB4 cell maturation (Table 3), which is in correspondence with the molecular basis of APL [18].

4. Materials and Methods

4.1. General Information

Unless otherwise noted, all reagents were purchased from commercial suppliers and used without further purification. Anhydrous THF was distilled from Na prior to use. Reactions were monitored by thin layer chromatography using TLC Silica gel 60 F254 supplied by Qingdao Puke Separation Material Corporation (Qingdao, China). Silica gel for column chromatography was 200–300 mesh and was supplied by Qingdao Marine Chemical Factory (Qingdao, China). Characterization of intermediates and final compounds was done using NMR spectroscopy and mass spectrometry. 1H-NMR spectra (500 MHz) were determined in CDCl3 on an Advance III MHz spectrometer (Bruker, Bremen, Germany) with TMS as internal standard. Chemical shifts are expressed in parts per million (ppm) and coupling constants in Hz. Mass spectra (ESI-MS) were recorded on an Esquire-LC-00075 spectrometer (Bruker, Bremen, Germany). HRMS were recorded on a 6224 TOF LC/MS spectrometer (Agilent, Santa Clara, CA, USA). Purity was confirmed on a Agilent 1100 series HPLC system equipped with a C18 column (Eclipse XDB-C18, 5 μm, 4.6 × 250 mm) eluted in gradient mode with CH3CN in H2O (from 10% to 95%). Melting points were measured with a B-540 melting-point apparatus (Büchi, Flawil, St. Gallen, Switzerland) and are uncorrected.

4.2. Chemistry

6-Nitro-2,3-dihydro-1H-inden-1-one (10). To a solution of 2,3-dihydro-1H-inden-1-one (9, 1.32 g, 10.0 mmol) in concentrated sulfuric acid (10 mL), KNO3 (1.21 g, 12.0 mmol) in concentrated sulfuric acid (10 mL) was added dropwise at −5 °C in 30 min. The mixture was stirred at −5 °C for 4 h. After adding ice water slowly, the mixture was partitioned between water and CH2Cl2. The organic layer was washed with a saturated aqueous solution of NaHCO3 and brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 7/1) to give 10 (1.10 g, 62%) as a beige solid. m.p. 71~74 °C; 1H-NMR: δ 8.59 (d, J = 1.9 Hz, 1H), 8.47 (dd, J = 8.4, 2.2 Hz, 1H), 7.68 (t, J = 8.4 Hz, 1H), 3.33–3.25 (m, 2H), 2.89–2.78 (m, 2H); ESI-MS: m/z [M + H]+ 178.
6-Nitro-2,3-dihydro-1H-inden-1-ol (11). To a solution of 10 (1.77 g, 10.0 mmol) in a mixed solution of MeOH/THF (2:1, 20 mL), NaBH4 (1.52 g, 40.0 mmol) was added in portions. The mixture was stirred at room temperature for one hour. After the addition of water (40 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 2/1) to give 11 (1.64 g, 92%) as a white solid. m.p. 74~77 °C; 1H-NMR: δ 8.26 (d, J = 1.8 Hz, 1H), 8.15 (dd, J = 8.3, 2.1 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 5.32 (t, J = 6.2 Hz, 1H), 3.14 (m, 1H), 2.98–2.85 (m, 1H), 2.64–2.58 (m, 1H), 2.09–1.97 (m, 1H); ESI-MS: m/z [M + H]+ 180.
5-Nitro-1H-indene (12). To a solution of 11 (1.79 g, 10.0 mmol) in anhydrous toluene (20 mL), TsOH (1.72 g, 10.0 mmol) was added at room temperature and the mixture was refluxed for 2 h. The solvent was removed by distillation. After adding water (40 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of NaHCO3 and brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 10/1) to give 12 (1.38 g, 86%) as a white solid, m.p. 78~81 °C; 1H-NMR: δ 8.23 (d, J = 2.1 Hz, 1H), 8.11 (dd, J = 8.2, 2.1 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 6.96 (d, J = 5.5 Hz, 1H), 6.76 (dt, J = 5.4, 1.9 Hz, 1H), 3.52 (s, 2H);ESI-MS: m/z [M + H]+ 162.
1-Methoxy-6-nitro-2,3-dihydro-1H-indene (14a). To a solution of 11 (90 mg, 0.5 mmol) and CH3I (0.31 mL, 5.0 mmol) in anhydrous THF (2 mL), CH3ONa (108 mg, 2.0 mmol) was added at 0 °C. The mixture was stirred at room temperature for 12 h. After adding water (20 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 30/1) to give 14a (39 mg, 41%) as a pale yellow liquid. 1H-NMR: δ 8.26 (d, J = 2.0 Hz, 1H), 8.17 (dd, J = 8.3, 2.2 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 4.87 (dd, J = 6.5, 4.5 Hz, 1H), 3.46 (s, 3H), 3.20–3.13 (m, 1H), 2.95–2.89 (m, 1H), 2.52–2.40 (m, 1H), 2.25–2.13 (m, 1H); ESI-MS: m/z [M + H]+ 194.
1-Ethoxy-6-nitro-2,3-dihydro-1H-indene (14b). The title compound was prepared (34 mg, 33%) as a pale yellow liquid from 11 and CH3CH2I in a similar method with that described for 14a. 1H-NMR: δ 8.24 (d, J = 2.1 Hz, 1H), 8.14 (dd, J = 8.3, 2.2 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 4.95 (q, J = 6.5 Hz, 1H), 3.70–3.60 (m, 2H), 3.18–3.12 (m, 1H), 2.93–2.84 (m, 1H), 2.50–2.43 (m, 1H), 2.19–2.12 (m, 1H), 1.27 (t, J = 7.0 Hz, 3H). ESI-MS: m/z [M + H]+ 208.
1-Isopropoxy-6-nitro-2,3-dihydro-1H-indene (14c). To a stirred solution of compound 11 (180 mg, 1.0 mmol) and isopropyl bromide (183 mg, 1.5 mmol) in anhydrous CH2Cl2 (2 mL), dry mercury oxide/tetrafluoroboric acid (190 mg, 0.5 mmol) was added. The mixture was stirred at room temperature for 2 h and then treated successively with NaHCO3 and 3 M potassium hydroxide until basic. The precipitated mercury oxide was filtered off and the filtrate was extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 25/1) to give 14c (42 mg, 19%) as a pale yellow liquid. 1H-NMR: δ 8.19 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.3, 2.2 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 5.03 (t, J = 6.3 Hz, 1H), 3.93–3.84 (m, 1H), 3.15–3.09 (m, 1H), 2.94–2.82 (m, 1H), 2.54–2.48 (m, 1H), 2.12–2.05 (m, 1H), 1.29 (d, J = 6.1 Hz, 3H), 1.26 (d, J = 6.1 Hz, 3H). ESI-MS: m/z [M + H]+ 222.
1-Butoxy-6-nitro-2,3-dihydro-1H-indene (14d). The title compound was prepared as a pale yellow liquid (69 mg, 29%) from 11 and 1-bromobutane in a manner similar to that described for 14c. 1H-NMR: δ 8.22 (s, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 4.93 (t, J = 5.5 Hz, 1H), 3.69–3.50 (m, 2H), 3.20–3.07 (m, 1H), 2.97–2.83 (m, 1H), 2.55–2.42 (m, 1H), 2.22–2.02 (m, 1H), 1.70–1.56 (m, 2H), 1.41 (m, 2H), 0.94 (t, J = 6.4 Hz, 3H);ESI-MS: m/z [M + H]+ 236.
5-Nitro-2,3-dihydro-1H-inden-2-ol (16). To a stirred solution of compound 12 (805 mg, 5.0 mmol) in anhydrous THF, diborane (10 mmol) in diethyl sulfide (5 mL) was added dropwise at 0 °C. The mixture was stirred at room temperature for 2 h. A small amount of water was added until no bubbles were generated, then 30% hydrogen peroxide (2.8 mL) was added followed by the addition of 1 N NaOH (0.6 mL). The mixture was stirred at room temperature for another 1 h. After adding water (50 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 3/1) to give 16 (295 mg, 33%) as a white solid. m.p. 90~92 °C; 1H-NMR: δ 8.10 (s, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 4.85–4.77 (m, 1H), 3.34–3.24 (m, 2H), 3.01 (m, 2H); ESI-MS: m/z [M + H]+ 180.
2-Methoxy-5-nitro-2,3-dihydro-1H-indene (17a). The title compound was prepared from 16 and iodomethane in a manner similar to that described for 14a as a pale yellow liquid (41%). 1H-NMR: δ 8.08 (s, 1H), 8.04(d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 4.33–4.29 (m, 1H), 3.38 (s, 3H), 3.24–3.19 (m, 2H), 3.11–3.06 (m, 2H);ESI-MS: m/z [M + H]+ 194.
2-Ethoxy-5-nitro-2,3-dihydro-1H-indene (17b). The title compound was prepared from 16 and iodoethane in a manner similar to that described for 14a as a pale yellow liquid (26%). 1H-NMR: δ 7.97 (d, J = 11.5 Hz, 2H), 7.27 (s, 1H), 4.37–4.30 (m, 1H), 3.48 (q, J = 7.0 Hz, 2H), 3.15 (dd, J = 17.0, 6.3 Hz, 2H), 2.99 (dt, J = 9.1, 4.3 Hz, 2H), 1.14 (t, J = 7.0 Hz, 3H);ESI-MS: m/z [M + H]+ 208.
2-Methylene-2,3-dihydro-1H-inden-1-one (19a). To a solution of 9 (1.32 g, 10.0 mmol) and paraformaldehyde (1.50 g, 5.0 eq) in glacial acetic acid (20 mL), morpholine (0.5 mL) was added. The mixture was refluxed under nitrogen for 2 h. The glacial acetic acid was removed by distillation. After adding water (50 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 7/1) to give 19a (0.45 g, 31%) to give a yellow liquid. 1H-NMR: δ 7.89 (d, J = 7.6 Hz, 1H), 7.62 (t, J = 7.4 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.4 Hz, 1H), 6.39 (s, 1H), 5.65 (s, 1H), 3.78 (s, 2H); ESI-MS: m/z [M + H]+ 145.
2-(Propan-2-ylidene)-2,3-dihydro-1H-inden-1-one (19b). To a solution of 9 (1.32 g, 10.0 mmol) in anhydrous acetone (20 mL), NaOH (132 mg, 3.3 mmol) was added at room temperature. The mixture was stirred at room temperature for 4 h and then neutralized with 1 N HCl. Acetone was removed by distillation. After adding water (50 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 7/1) to give 19b (0.77 g, 45%) as a yellow solid. m.p. 98~100 °C; 1H-NMR: δ 7.81 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 7.1 Hz, 1H), 7.47 (s, 1H), 7.37 (s, 1H), 3.65 (s, 2H), 2.45 (s, 3H), 2.01 (s, 3H); ESI-MS: m/z [M + H]+ 173.
2-Methyl-2,3-dihydro-1H-inden-1-one (20a). To a solution of 19a (1.44 g, 10 mmol) in EtOAc (20 mL), 10% Pd/C (20% weight of compound 19a) was added. The mixture was stirred overnight under a hydrogen atmosphere at room temperature. Insoluble materials were removed by filtration and washed with EtOAc. The filtrate was evaporated to dryness under reduced pressure to give 20a (1.43 g, 98%) as a colorless transparent liquid. 1H-NMR: δ 7.76 (d, J = 7.7 Hz, 1H), 7.59 (d, J = 7.6, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H), 3.45–3.36 (m, 1H), 2.79–2.68 (m, 2H), 1.35–1.30 (m, 3H); ESI-MS: m/z [M + H]+ 147.
2-Isopropyl-2,3-dihydro-1H-inden-1-one (20b). The title compound was prepared from 19b in a manner similar to that described for 20a as a colorless transparent liquid (97%). 1H-NMR: δ 7.75 (d, J = 7.7 Hz, 1H), 7.59 (td, J = 7.6, 1.1 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.39–7.34 (m, 1H), 3.16 (dd, J = 17.4, 8.1 Hz, 1H), 2.95 (dd, J = 17.4, 4.0 Hz, 1H), 2.82–2.79 (m, 1H), 2.45–2.42 (m, 1H), 1.07 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H); ESI-MS: m/z [M + H]+ 175.
2-Methyl-6-nitro-2,3-dihydro-1H-inden-1-one (21a). The title compound was prepared from 20a in a manner similar to that described for 10 as a yellow solid (67%). m.p. 64~66 °C; 1H-NMR: δ 8.58 (d, J = 2.1 Hz, 1H), 8.46 (dd, J = 8.4, 2.2 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 3.55–3.50 (m, 1H), 2.91–2.79 (m, 2H), 1.37 (d, J = 7.3 Hz, 3H); ESI-MS: m/z [M + H]+ 192.
2-Isopropyl-6-nitro-2,3-dihydro-1H-inden-1-one (21b). The title compound was prepared from 20b in a manner similar to that described for 10 as a yellow solid (73%). m.p. 72~76 °C; 1H-NMR: δ 8.55 (d, J = 2.0 Hz, 1H), 8.44 (dd, J = 8.4, 2.2 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 3.28 (dd, J = 18.3, 8.2 Hz, 1H), 3.04 (dd, J = 18.3, 4.1 Hz, 1H), 2.82-2.79 (m, 1H), 2.51–2.40 (m, 1H), 1.07 (d, J = 6.9 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H); ESI-MS: m/z [M + H]+ 192.
2-Methyl-6-nitro-2,3-dihydro-1H-inden-1-ol (22a). The title compound was prepared from 21a in a manner similar to that described for 11 as a white solid (86%). m.p. 81~84 °C; 1H-NMR: δ 8.58 (d, J = 5.0 Hz, 1H), 8.06 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 5.72 (d, J = 6.0 Hz, 1H), 4.62–4.59 (s, 1H), 3.10–3.05 (m, 1H), 2.58–2.53 (m, 1H), 2.24–2.14 (m, 1H), 1.20 (d, J = 6.7 Hz, 3H);ESI-MS: m/z [M + H]+ 194.
2-Isopropyl-6-nitro-2,3-dihydro-1H-inden-1-ol (22b). The title compound was prepared from 21b in a manner similar to that described for 11 as a white solid (89%). m.p. 90~93 °C; 1H-NMR: δ 8.15–8.04 (m, 1H), 7.70 (m, 1H), 7.41 (m, 1H), 3.22 (d, J = 3.3 Hz, 1H), 3.54 (dd, J = 17.9, 7.3 Hz, 1H), 3.15 (dd, J = 17.9, 9.8 Hz, 1H), 2.05–1.92 (m, 1H), 1.55 (m, 1H), 1.14 (t, J = 5.5 Hz, 3H), 1.09 (d, J = 6.5 Hz, 3H)); ESI-MS: m/z [M + H]+ 222.
2-Methyl-5-nitro-1H-indene (23a). The title compound was prepared from 22a in a manner similar to that described for 12 as a white solid (81%). m.p.: 68~72 °C; 1H-NMR: δ 7.76 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 5.02 (d, J = 5.9 Hz, 1H), 3.41 (s, 8.7 Hz, 2H), 1.32 (d, J = 7.2 Hz, 3H); ESI-MS: m/z [M + H]+ 176.
2-Isopropyl-5-nitro-1H-indene (23b). The title compound was prepared from 22b in a manner similar to that described for 12 as a white solid (82%). m.p. 78~81 °C; 1H-NMR: δ 8.07 (d, J = 1.8 Hz, 1H), 8.00 (dd, J = 8.1, 1.8 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 6.56 (s, 1H), 3.44 (s, 2H), 2.90–2.73 (m, 1H), 1.25 (s, 3H), 1.24 (s, 3H); ESI-MS: m/z [M + H]+ 204.
1-Methoxy-2-methyl-6-nitro-2,3-dihydro-1H-indene (27a). To a solution of 22a (106 mg, 0.55 mmol) and trimethyl orthoformate (1 mL) in anhydrous CH2Cl2 (2 mL), bismuth trichloride (173 mg, 0.55 mmol) was added at room temperature. The mixture was stirred at room temperature for 7 h and then treated with aqueous 1 N NaHCO3 until basic. After adding water (50 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 30/1) to give 27a (27 mg, 24%) as a yellow oil. 1H-NMR: δ 8.22 (s, 1H), 8.15 (dd, J = 8.3, 2.1 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 4.42 (d, J = 4.4 Hz, 1H), 3.52 (s, 3H), 3.31–3.26 (m, 1H), 2.64–2.56 (m, 1H), 2.56–2.50 (m, 1H), 1.19 (d, J = 7.0 Hz, 3H); ESI-MS: m/z [M + H]+ 208.
1-Ethoxy-2-methyl-6-nitro-2,3-dihydro-1H-indene (27b). The title compound was prepared from 22b and triethyl orthoformate in a manner similar to that described for 27a as yellow oil (33%). 1H-NMR: δ 8.26 (s, 1H), 8.16 (d, J = 8.3 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 4.92 (d, J = 6.4 Hz, 1H), 3.65 (d, J = 7.0 Hz, 2H), 2.80–2.73 (m, 2H), 2.65 (q, J = 6.5, 1H), 1.33 (d, J = 6.6 Hz, 3H), 1.31–1.27 (m, 3H); ESI-MS: m/z [M + H]+ 222.
2-Isopropyl-1-methoxy-6-nitro-2,3-dihydro-1H-indene (28a). The title compound was prepared from 22a in a manner similar to that described for 27a as yellow oil (27%). 1H-NMR: δ 8.20 (d, J = 2.0 Hz, 1H), 8.18 (dd, J = 8.2, 2.2 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 4.51 (d, J = 3.2 Hz, 1H), 3.34 (s, 3H), 3.00–2.86 (m, 2H), 2.09–2.01 (m, 1H), 2.00–1.95 (m, 1H), 1.07 (d, J = 6.3 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H); ESI-MS: m/z [M + H]+ 236.
1-Ethoxy-2-isopropyl-6-nitro-2,3-dihydro-1H-indene (28b). The title compound was prepared from 22b and triethyl orthoformate in a manner similar to that described for 27a as yellow oil (19%). 1H-NMR: δ 8.25 (d, J = 4.4 Hz, 1H), 8.14 (dd, J = 8.3, 2.2 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 4.62 (d, J = 5.3 Hz, 1H), 3.20 (q, J =8.5 Hz, 2H), 2.83–2.75 (m, 2H), 2.66–2.56 (m, 1H), 2.19–2.06 (m, 1H), 1.09 (d, J = 6.5 Hz, 5H), 1.01–0.99 (m, 3H); ESI-MS: m/z [M + H]+ 250.
Dimethyl Terephthalate (32). To a solution of terephthalic acid (31, 6.0 g, 36.0 mmol) in methanol (150 mL), thionyl chloride (7.7 mL, 108 mmol) was added dropwise at 0 °C. The mixture was stirred at room temperature for 17 h and then saturated potassium carbonate solution was added until no bubbles were generated. The methanol was removed by distillation. After adding water (40 mL), the mixture was partitioned between water and ether. The organic layer was washed with a saturated aqueous solution of NaHCO3 and brine, dried over anhydrous Na2SO4, and concentrated under vacuum to give 32 (6.84 g, 98%) as a white solid. m.p. 141~143 °C (ether); ESI-MS: m/z [M + H]+ 195.
4-(Methoxycarbonyl)benzoic acid (33). To a solution of 32 (2.0 g, 10.0 mmol) in methanol and ether (methanol:ether = 1:1, 20 mL), a solution of KOH (0.58 g, 10.0 mmol) in methanol and water (methanol:water = 10:1, 10 mL) was added dropwise at 0 °C. The mixture was stirred at room temperature for 24 h. After adding water (50 mL), the mixture was partitioned between water and ether. Then treating the aqueous layer successively with 1 N HCl until pH = 1. The mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of brine, dried over anhydrous Na2SO4, and concentrated under vacuum to give 33 (1.0 g, 56%) as a white solid. m.p. 188~192 °C.
4-((2,3-Dihydro-1H-inden-5-yl)carbamoyl) benzoate (35a). To a solution of 12 (80.5 mg, 0.5 mmol) in EtOAc (20 mL), 10% Pd/C (20% net weight of compound 19a) was added. The mixture was stirred overnight under a hydrogen atmosphere at room temperature. Insoluble materials were removed by filtration and washed with EtOAc. The filtrate was evaporated to dryness under reduced pressure to give 13 as brown oil (65 mg, 98%). To a solution of 33 (150 mg, 0.8 mmol) in thionyl chloride (4 mL), a drop of pyridine was added and refluxed for 24 h. The thionyl chloride was removed by distillation and get 34 as pale yellow solid. 34 was used directly in the next reaction. To a solution of 13 (65 mg, 0.5 mmol) in anhydrous pyridine (2 mL), 34 was added in CH2Cl2 (2 mL) dropwise at 0 °C. The mixture was stirred at room temperature for 7 h and then the methanol was removed by distillation. The organic layer was treated successively with 1 N HCl until acidic. The mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 3/1) to give 35a (118 mg, 80%) as a pale yellow solid. m.p. 138~142 °C; 1H-NMR: δ 8.15 (d, J = 6.9 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 7.60 (s, 1H), 7.30 (d, J = 8.1 Hz,1H), 7.22 (d, J = 7.9 Hz, 1H), 3.96 (s, 3H), 2.95–2.89 (m, 4H), 2.13–2.07 (m, 2H);ESI-MS: m/z [M + H]+ 296.
Methyl 4-((3-methoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35b). The title compound was prepared from 14a in a manner similar to that described for 35a as a pale yellow solid (87%). m.p. 148~151 °C; 1H-NMR: δ 8.15 (d, J = 8.3 Hz, 2H), 7.92 (d, J = 8.3 Hz, 2H), 7.86 (s, 1H), 7.72 (s, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 4.83 (dd, J = 6.5, 4.2 Hz, 1H), 3.96 (s, 3H), 3.43 (s, 3H), 3.11–3.01 (m, 1H), 2.85–2.77 (m, 1H), 2.42–2.32 (m, 1H), 2.11–2.08 (m, 1H); ESI-MS: m/z [M + H]+ 326.
Methyl 4-((3-ethoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35c). The title compound was prepared from 14b in a manner similar to that described for 35a as a pale yellow solid (76%). m.p. 153~156 °C; 1H-NMR: δ 8.16 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 7.82 (s, 1H), 7.71 (s, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 4.96–4.89 (m, 1H), 3.97 (s, 3H), 3.64 (q, J = 6.8, 2H), 3.09–3.03 (m, 1H), 2.83–2.77 (m, 1H), 2.44–2.36 (m, J = 6.7 Hz, 1H), 2.12–2.06 (m, 1H), 1.25 (t, J = 7.0 Hz, 3H); ESI-MS: m/z [M + H]+ 340.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35d). The title compound was prepared from 14c in a manner similar to that described for 35a as a pale yellow solid (79%). m.p. 150~153 °C ; 1H-NMR: δ 8.16 (d, J = 7.7 Hz, 2H), 7.93 (d, J = 7.6 Hz, 2H), 7.80 (s, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 5.01 (t, J = 5.9 Hz, 1H), 3.96 (s, 3H), 3.92–3.83 (m, 1H), 3.03 (s, 1H), 2.78 (dt, J = 15.7, 7.8 Hz, 1H), 2.44 (d, J = 6.2 Hz, 1H), 2.03 (dt, J = 13.1, 8.5 Hz, 1H), 1.25 (s, 6H); ESI-MS: m/z [M + H]+ 354.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35e). The title compound was prepared from 14d in a manner similar to that described for 35a as a pale yellow solid (73%). m.p. 159~160 °C; 1H-NMR: δ 8.16 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.81 (s, 1H), 7.66 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.24 (s, 1H), 4.93–4.85 (m, 1H), 3.96 (s, 3H), 3.57 (dd, J = 15.4, 6.7 Hz, 2H), 3.04 (ddd, J = 16.8, 11.4, 7.0 Hz, 1H), 2.79 (s, 1H), 2.40 (dq, J = 8.2, 6.2 Hz, 1H), 2.15–1.99 (m, 1H), 1.67–1.58 (m, 2H), 1.41 (dd, J = 13.2, 7.5 Hz, 2H), 0.96–0.89 (m, 3H); ESI-MS: m/z [M + H]+ 368.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35f). The title compound was prepared from 17a in a manner similar to that described for 35a as a yellow solid (66%). m.p. 134~135 °C; 1H-NMR: δ 8.16 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.75 (s, 1H), 7.63 (s, 1H), 7.31 (d, J = 5.7 Hz, 1H), 4.28 (t, J = 5.3 Hz, 1H), 3.97 (s, 3H), 3.39 (s, 3H), 3.21–3.13 (m, 2H), 3.03–2.95 (m, 2H); ESI-MS: m/z [M + H]+ 326.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35g). The title compound was prepared from 17b in a manner similar to that described for 35a as a yellow solid (71%). m.p. 137~139 °C; 1H-NMR: δ 8.16 (d, J = 8.3 Hz, 2H), 7.92 (d, J = 8.3 Hz, 2H), 7.75 (s, 1H), 7.62 (s, 1H), 7.30 (d, J = 9.3 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 4.37 (t, J = 5.0 Hz, 1H), 3.56 (d, J = 7.0 Hz, 2H), 3.21-3.14 (m, 2H), 3.04–2.93 (m, 2H), 1.23 (t, J = 7.0 Hz, 3H); ESI-MS: m/z [M + H]+ 340.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35h). The title compound was prepared from 23b in a manner similar to that described for 35a as a pale yellow solid (87%). m.p. 144~149 °C; 1H-NMR: δ 8.19 (d, J = 8.3 Hz, 2H), 7.98 (s, 1H), 8.00–7.93 (m, 2H), 7.84 (s, 1H), 7.54 (d, J = 8.2 Hz, 1H), 3.98 (d, J = 4.4 Hz, 3H), 3.21–3.11 (m, 3H), 2.79–2.70 (m, 3H), 1.25 (d, J = 20.0 Hz, 6H); ESI-MS: m/z [M + H]+ 338.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35k). The title compound was prepared from 21a in a manner similar to that described for 35a as a pale yellow solid (77%). m.p. 167~171 °C; 1H-NMR: δ 8.17 (s, 2H), 8.11 (d, J = 6.4 Hz, 1H), 7.96 (s, 2H), 7.82 (s, 1H), 7.50 (d, J = 8.2 Hz, 1H), 3.97 (s, 3H), 3.41 (dd, J = 16.4, 7.3 Hz, 1H), 2.80–2.75 (m, 1H), 2.75 (s, 1H), 1.33 (d, J = 7.3 Hz, 3H); ESI-MS: m/z [M + H]+ 324.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35l). The title compound was prepared from 21b in a manner similar to that described for 35a as a pale yellow solid (89%). m.p. 170~174 °C; 1H-NMR: δ 8.21 (d, J = 8.2 Hz, 1H), 8.18 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.2 Hz, 2H), 7.86 (s, 1H), 7.53 (d, J = 8.3 Hz, 1H), 3.15 (dd, J = 17.4, 8.0 Hz, 1H), 2.94 (dd, J = 17.4, 3.8 Hz, 1H), 2.72 (dt, J = 8.1, 4.1 Hz, 1H), 2.43–2.33 (m, 1H), 1.06 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H; ESI-MS: m/z [M + H]+ 352.
Methyl 4-((3-isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35m). The title compound was prepared from 27a in a manner similar to that described for 35a as a pale yellow solid (91%). m.p. 147~149 °C; 1H-NMR: δ 8.16 (dd, J = 8.2, 4.4 Hz, 2H), 7.93 (d, J = 7.2 Hz, 2H), 7.73 (s, 1H), 7.48 (d, J = 7.1 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 4.41 (d, J = 3.6 Hz, 1H), 3.96 (s, 3H), 3.49 (s, 3H), 3.25–3.17 (m, 1H), 2.57–2.50 (m, 1H), 2.43 (dd, J = 15.8, 3.2 Hz, 1H), 1.17 (d, J = 7.0, 3H); ESI-MS: m/z [M + H]+ 340.
Methyl 4-((3-ethoxy-2-methyl-2, 3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35n). The title compound was prepared from 27b in a manner similar to that described for 35a as a pale yellow solid (86%). m.p. 140~142 °C; 1H-NMR: δ 8.16 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 7.82 (s, 1H), 7.70 (s, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 4.49 (d, J = 4.6 Hz, 1H), 3.97 (s, 3H), 3.71 (q, J = 7.0 Hz, 2H), 3.19 (dd, J = 15.7, 7.6 Hz, 1H), 2.57–2.45 (m, 1H), 2.42 (dd, J = 15.6, 5.8 Hz, 1H), 1.27 (t, J = 6.9 Hz, 3H), 1.18 (d, J = 7.0 Hz, 3H); ESI-MS: m/z [M + H]+ 354.
4-((2-Isopropyl-3-methoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35o). The title compound was prepared from 28a in a manner similar to that described for 35a as a pale yellow solid (87%). m.p. 152~155 °C; 1H-NMR: δ 8.16 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 7.89 (s, 1H), 7.72 (s, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 4.70 (d, J = 5.0 Hz, 1H), 3.97 (s, 3H), 3.48 (s, 3H), 3.08 (dd, J = 16.2, 8.4 Hz, 1H), 2.60 (dd, J = 16.2, 6.3 Hz, 1H), 2.31 (tt, J = 8.4, 6.4 Hz, 1H), 1.84 (dq, J = 13.4, 6.7 Hz, 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H); ESI-MS: m/z [M + H]+ 368.
Methyl 4-((3-ethoxy-2-isopropyl-2,3-dihydro-1H-inden-5-yl)carbamoyl) benzoate (35p). The title compound was prepared from 28b in a manner similar to that described for 35a as a pale yellow solid (97%). m.p. 146~149 °C; 1H-NMR: δ 8.15 (s, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.74 (s, 1H), 7.55 (s, 1H), 7.17 (d, J = 7.9 Hz, 1H), 4.56 (d, J = 5.0 Hz, 1H), 3.96 (s, 3H), 3.00 (q, J =8.0 Hz, 2H), 2.68–2.58 (m, 2H), 2.21–2.16 (m, 1H), 1.72–1.63 (m, 1H), 1.02 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.6 Hz, 6H); ESI-MS: m/z [M + H]+ 368.
4-((2,3-Dihydro-1H-inden-5-yl)carbamoyl) benzoate (35i). To a solution of 23a (175 mg, 1.0 mmol) and Fe (392 mg, 7.0 mmol) in EtOH (20 mL), AcOH (0.8 mL) was added. The mixture was refluxing for 2 h under a nitrogen atmosphere at room temperature. Insoluble materials were removed by filtration and washed with EtOAc. The filtrate was evaporated to dryness under reduced pressure. The mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue 25a was used directly in the next reaction. To a solution of 33 (150 mg, 0.8 mmol) in thionyl chloride (4 mL), a drop of pyridine was added and refluxed for 24 h. The thionyl chloride was removed by distillation and get 34 as pale yellow solid. To a solution of 25a (45 mg, 0.3 mmol) in anhydrous pyridine (2 mL), 34 was added in CH2Cl2 (2 mL) dropwise at 0 °C. The mixture was stirred at room temperature for 7 h and then the methanol was removed by distillation. The organic layer was neutralized with 1 N HCl. The mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc = 3/1) to give 35i (77 mg, 84%) as a pale yellow solid. m.p. 157~159 °C; 1H-NMR: δ 8.15 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.2 Hz, 2H), 7.83 (s, 1H), 7.62 (s, 1H), 7.34 (d, J = 7.9 Hz, 1H), 6.48 (s, 1H), 3.96 (s, 3H), 3.29 (s, 2H), 2.17 (s, 3H); ESI-MS: m/z [M + H]+ 338.
Methyl 4-((2-isopropyl-1H-inden-5-yl)carbamoyl) benzoate (35j). The title compound was prepared from 23b in a manner similar to that described for 35i as a pale yellow solid (67%). m.p. 155~156 °C; 1H-NMR: δ 8.16 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 7.78 (s, 1H), 7.63 (s, 1H), 7.35 (s, 1H), 6.50 (s, 1H), 3.97 (s, 3H), 3.34 (s, 2H), 2.81–2.76 (m, 1H), 1.24 (d, J = 6.8, 3H); ESI-MS: m/z [M + H]+ 336.
4-((2,3-Dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36a). To a solution of 35a (56 mg, 0.2 mmol) in MeOH (2 mL), 0.5 N LiOH (0.4 mL) was added dropwise at 0 °C. The mixture was stirred at room temperature for 48 h and then neutralized with 1 N HCl. The MeOH was removed by distillation. After adding water (10 mL), the mixture was partitioned between water and EtOAc. The organic layer was washed with a saturated aqueous solution of brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (eluent: hexane/EtOAc/AcOH = 30/10/1) to give 36a (52 mg, 92%) as a white solid, purity: 97%. m.p. >250 °C; 1H-NMR: δ 13.26 (s, 1H), 10.30 (s, 1H), 8.14–7.92 (m, 4H), 7.68 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H), 2.93–2.76 (m, 4H), 2.07–1.97 (m, 2H); ESI-MS: m/z [M − H] 280.
4-((3-Methoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36b). The title compound was prepared from 35b in a manner similar to that described for 36a as a white solid (91%), purity: 96%. m.p. >250 °C; 1H-NMR: δ 10.38 (s, 1H), 8.15–8.01 (m, 4H), 7.86 (s, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 8.2 Hz, 1H), 4.86–4.72 (m, 1H), 3.33(s, 3H),3.00–2.87 (m, 1H), 2.81–2.69 (m, 1H), 2.36–2.32 (m, 1H), 1.99–1.93 (m, 1H); ESI-MS: m/z [M − H] 310. HRMS (ESI) calcd [M + H]+ for C18H18NO4 312.1230, found 312.1234.
4-((3-Methoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36c). The title compound was prepared from 35c in a manner similar to that described for 36a as a white solid (99%), purity: 95%. m.p. >250 °C; 1H-NMR: δ 10.37 (s, 1H), 8.07–8.03 (m, 4H), 7.81 (s, 1H), 7.63 (dd, J = 8.0, 2.0 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 4.89–4.83 (m, 1H), 3.60–3.50 (m, 2H), 2.94–2.88 (m, 1H), 2.76–2.69 (m, 1H), 2.36–2.30 (m, 1H), 1.95–1.89 (m, 1H), 1.15 (t, J = 7.0 Hz, 3H); ESI-MS: m/z [M − H] 324. HRMS (ESI) calcd [M + H]+ for C19H20NO4 326.1387, found 326.1392.
4-((3-Isopropoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36d). The title compound was prepared from 35d in a manner similar to that described for 36a as a white solid (97%), purity: 97%. m.p. >250 °C; 1H-NMR: δ 13.24 (s, 1H), 10.36 (s, 1H), 8.05 (s, 4H), 7.74 (s, 1H), 7.64 (dd, J = 8.1, 1.9 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 4.96 (t, J = 6.1 Hz, 1H), 3.93–3.74 (m, 2H), 3.85–3.79 (m, 1H), 2.94–2.84 (m, 1H), 2.73–2.67 (m, 1H), 2.40–2.34 (m, 1H), 1.87–1.81 (m, 1H), 1.17 (d, J = 6.0, 3H) , 1.16 (d, J = 6.5, 3H); ESI-MS: m/z [M − H] 338. HRMS (ESI) calcd [M + H]+ for C20H22NO4 340.1543, found 340.1550.
4-((3-Butoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36e). The title compound was prepared from 35e in a manner similar to that described for 36a as a white solid (90%). purity: 96%. m.p. >250 °C; 1H-NMR: δ 13.26 (s, 1H), 10.37 (s, 1H), 8.05 (d, J = 2.1 Hz, 4H), 7.79 (s, 1H), 7.63 (dd, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 4.89–4.80 (m, 1H), 3.51 (td, J = 6.5, 2.5 Hz, 2H), 2.98–2.85 (m, 1H), 2.78–2.66 (m, 1H), 2.38–2.30 (m, 1H), 1.94–1.86 (m, 1H), 1.56–1.42 (m, 2H), 1.39–1.31 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H); ESI-MS: m/z [M − H] 352. HRMS (ESI) calcd [M + H]+ for C21H24NO4 354.1700, found 354.1703.
4-((2-Methoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36f). The title compound was prepared from 35f in a manner similar to that described for 36a as a white solid (86%), purity: 98%. m.p. >250 °C; 1H-NMR: δ 8.15 (d, J = 8.3 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.80 (s, 1H), 7.62 (s, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 4.31–4.23 (m, 1H), 3.96 (s, 3H), 3.38 (s, 3H), 3.20–3.12 (m, 2H), 3.03-2.94 (m, 2H); ESI-MS: m/z [M − H] 310. HRMS (ESI) calcd [M + H]+ for C18H18NO4 312.1230, found 312.1240.
4-((2-Ethoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36g). The title compound was prepared from 35g in a manner similar to that described for 36a as a white solid (97%), purity: 97%. m.p. >250 °C; 1H-NMR: δ 10.37 (s, 1H), 8.12–8.01 (m, 4H), 7.82 (s, 1H), 7.65 (dd, J = 8.1, 1.7 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 4.94–4.80 (m, 1H), 3.62–3.54 (m, 2H), 2.97–2.88 (m, 1H), 2.78–2.69 (m, 1H), 2.37–2.31 (m, 1H), 1.98–1.90 (m, 1H), 1.16 (t, J = 7.0 Hz, 3H); ESI-MS: m/z [M − H] 324. HRMS (ESI) calcd [M + H]+ for C19H20NO4 326.1387, found 326.1396.
4-((2-Isopropyl-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36h). The title compound was prepared from 35h in a manner similar to that described for 36a as a white solid (94%), purity: 96%. m.p. >250 °C; 1H-NMR: δ 8.07–8.02 (m, 4H), 7.63 (s, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 2.98–2.90 (m, 2H), 2.66–2.52 (m, 2H), 2.15–2.10 (m, 1H), 1.68–1.10 (m, 1H), 0.95 (d, J = 6.5 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H); ESI-MS: m/z [M − H] 322. HRMS (ESI) calcd [M + H]+ for C20H22NO3 324.1594, found 324.1600.
4-((2-Methyl-1H-inden-5-yl)carbamoyl)benzoic acid (36i). The title compound was prepared from 35i in a manner similar to that described for 36a as a white solid (98%), purity: 95%. m.p. >250 °C; 1H-NMR: 10.31 (s, 1H), 8.07–8.02 (m, 4H), 7.78 (s, 1H), 7.48 (d, J = 6.4 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 6.51 (s, 1H), 3.29 (s, 2H), 2.12 (s, 3H); ESI-MS: m/z [M − H] 292. HRMS (ESI) calcd [M + H]+ for C18H16NO3 294.1125, found 294.1129.
4-((2-Isopropyl-1H-inden-5-yl)carbamoyl)benzoic acid (36j). The title compound was prepared from 35j in a manner similar to that described for 36a as a white solid (83%), purity: 95%. m.p. >250 °C; 1H-NMR: δ 13.25 (s, 1H), 10.34 (s, 1H), 8.09–8.06 (m, 4H), 7.72 (s, 1H), 7.47 (dd, J = 8.0, 1.5 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 6.53 (s, 1H), 3.37 (s, 2H), 2.79–2.72 (m, 1H), 1.20 (d, J = 6.5, 3H), 1.19 (d, J = 6.5, 3H); ESI-MS: m/z [M − H] 320. HRMS (ESI) calcd [M + H]+ for C20H20NO3 322.1438, found 322.1440.
4-((2-Methyl-3-oxo-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36k). The title compound was prepared from 35k in a manner similar to that described for 36a as a white solid (92%), purity: 95%. m.p. >250 °C (AcOH–EtOAc–hexane); 1H-NMR: δ 10.60 (s, 1H), 8.09–8.04 (m, 4H), 8.07 (d, J = 2.8 Hz, 1H), 8.00 (dd, J = 8.3, 2.1 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 3.38–3.36 (m, J = 7.4 Hz, 1H), 2.78–2.72 (m, 1H), 2.70–2.65 (m, 1H), 1.20 (d, J = 7.4 Hz, 3H); ESI-MS: m/z [M − H] 308. HRMS (ESI) calcd. [M + H]+ for C18H16NO4 310.1074, found 310.1069.
4-((2-Isopropyl-3-oxo-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36l). The title compound was prepared from 35l in a manner similar to that described for 36a as a white solid (97%), purity: 98%. m.p. >250 °C; 1H-NMR: δ 13.30 (s, 1H), 10.61 (s, 1H), 8.14 (d, J = 1.8 Hz, 1H), 8.08 (d, J = 3.9 Hz, 4H), 8.00 (dd, J = 8.3, 2.0 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 3.13 (dd, J = 17.4, 8.0 Hz, 1H), 2.88 (dd, J = 17.5, 3.8 Hz, 1H), 2.73 (dt, J = 8.0, 4.1 Hz, 1H), 2.30–2.24 (m, 1H), 1.01 (d, J = 6.9 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H); ESI-MS: m/z [M − H] 336. HRMS (ESI) calcd [M + H]+ for C20H20NO4 338.1387, found 338.1384.
4-((3-Methoxy-2-methyl-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36m). The title compound was prepared from 35m in a manner similar to that described for 36a as a white solid (89%), purity: 99%. m.p. >250 °C (AcOH–EtOAc–hexane); 1H-NMR: δ 13.26 (s, 1H), 10.36 (s, 1H), 8.06–8.04 (m, 4H), 7.84 (s, 1H), 7.63 (dd, J = 8.1, 1.8 Hz, 1H), 7.23 (d, J = 8.0, 1H), 4.37 (d, J = 4.3 Hz, 1H), 3.40 (s, 3H), 3.12–3.07 (m, 1H), 2.45–2.34 (m, 2H), 1.12 (d, J = 6.8 Hz, 3H); ESI-MS: m/z [M − H] 324. HRMS (ESI) calcd [M + H]+ for C19H20NO4 326.1387, found 326.1392.
4-((3-Ethoxy-2-methyl-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36n). The title compound was prepared from 35n in a manner similar to that described for 36a as a white solid (97%), purity: 98%. m.p. >250 °C; 1H-NMR: δ 13.25 (s, 1H), 10.36 (s, 1H), 8.13–8.00 (m, 4H), 7.80 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 4.44 (d, J = 4.5 Hz, 1H), 3.68 (q, J = 7.0 Hz, 2H), 3.10–3.05 (m, 1H), 2.40–3.34 (m, 2H), 1.18 (t, J = 7.0 Hz, 3H), 1.13 (d, J = 6.6 Hz, 3H); ESI-MS: m/z [M − H] 338. HRMS (ESI) calcd [M + H]+ for C20H22NO4 340.1543, found 340.1549.
4-((2-Isopropyl-3-methoxy-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36o). The title compound was prepared from 35o in a manner similar to that described for 36a as a white solid (98%), purity: 97%. m.p. >250 °C; 1H-NMR: δ 13.21 (s, 1H), 10.34 (s, 1H), 8.10–8.02 (m, 4H), 7.84 (s, 1H), 7.64 (dd, J = 8.5, 1.5 Hz , 1H), 7.20 (d, J = 8.2 Hz, 1H), 4.65 (d, J = 3.3 Hz, 1H), 3.39 (s, 3H), 3.01–2.96 (m, 1H), 2.55–2.53 (m, 1H), 2.23–2.15 (m, 1H), 1.83–1.76 (m, 1H), 0.95 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H); ESI-MS: m/z [M − H] 352. HRMS (ESI) calcd [M + H]+ for C21H24NO4 354.1700, found 354.1704.
4-((3-Ethoxy-2-isopropyl-2,3-dihydro-1H-inden-5-yl)carbamoyl)benzoic acid (36p). The title compound was prepared from 35p in a manner similar to that described for 36a as a white solid (98%), purity: 98%. m.p. >250 °C; 1H-NMR: δ 13.28 (s, 1H), 10.36 (s, 1H), 8.11–7.99 (m, 4H), 7.80 (s, 1H), 7.66–7.58 (dd, J = 8.0, 2.0 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 4.69 (d, J = 5.6 Hz, 1H), 3.72–3.57 (m, 2H), 3.33–3.31 (m, 1H), 2.94 (q, J = 8.4 Hz, 1H), 2.18–2.12 (m, 1H), 1.81–1.76 (m, 1H), 1.16 (t, J = 7.0 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H); ESI-MS: m/z [M − H] 366. HRMS (ESI) calcd [M + H]+ for C22H26NO4 368.1856, found 368.1863.

4.3. Biology

4.3.1. Receptor Binding Assay

All synthesized compounds were tested for their binding affinity by using time resolved fluorescence resonance energy transfer (TR-FRET) assay, which used a LanthaScreen® TR-FRET RAR alpha Coactivator Assay Kit (Invitrogen, Carlsbad, CA, USA). Briefly, all experiments were performed in black 384-well low-volume plates (Corning Inc., Corning, NY, USA) in dark at room temperature. The final assay volume was 20 μL. All dilutions were made in assay buffer (TR-FRET Coregulator Buffer D). The final DMSO concentration was 1%. A mixture of 5 nM RAR alpha LBD-GST, 5 nM TbAnti-GST antibody, 50 nM Fluorescein-D22 was added to the wells. The only variable is the agonist concentration (6.1 × 10−11~1.0 × 10−6 M final concentrations of each retinoid). The mixture was incubated for one hour in dark followed by fluorescence intensity determination on a SpectraMax M5 microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA) with 340 nm and 520 as excitation and emission wavelengths for terbium and 340 and 495 for fluorescein, respectively. Data were analyzed by using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) and FRET signal was determined for all treatments by dividing 520 nm/495 nm signals. Graphs plotted as fold change of FRET signal for compounds treatment over DMSO only treatment.

4.3.2. Inhibition of Cell Proliferation Assay

Cells were seeded in 96-well plates (Corning Inc.) with a density of 2500 cells (NB4 or HL60) per well for overnight. Then, cells were exposed to each of the test compounds 36a36p in gradient concentration of 10−9, 10−8, 10−7, 10−6, 10−5 mol/L. Control cultures were treated with the same volume of DMSO. After 72 hours of incubation, the cell density in each well were fixed by trichloroacetic acid and then measured using the SRB (sulforhodamine B) method. After rinsing, the SRB was solubilized in TrisHCl, and the optical density of each culture was determined with a Bio-Tek Elx 800 absorbance microplate reader (BioTek, Shoreline, WA, USA). The OD of the treated cultures was divided by that of the control cultures treated with solvent alone.

4.3.3. Cell Differentiation Assay

Cells (1 × 106/mL) were treated with compounds at different concentrations based on IC50 in Table 2 for how long, temperature (provide the cell culture condition). After the treatment, cells were washed twice with PBS and then fixed with 75% alcohol overnight at −20 °C. The fixed cells were washed with PBS and blocked with 95 μL 3% BSA in PBS for 45 min at room temperature. The cells were incubated with 5 μL CD11b-PE at 4 °C for 45 min with protection from light. The antigens were then determined by a FACSCalibur flow cytometer (BD Biosciences Pharmingen, San Diego, CA, USA). The percentages of positive cells were quantitated using CellQuest Pro software. Cells stained with mouse IgG-PE served as negative controls. Both CD11b-PE and mouse IgG-PE antibodies were purchased from BD Biosciences. At least 10,000 cells were analyzed for each data point.

5. Conclusions

In summary, a series of mono-/di- substituted indene derivatives were designed and synthesized to explore the impact of the size of the hydrophobic region of ATRA derivatives on the bioactivity of related compounds. Binding, antiproliferative and cell differentiation assays showed that most of these compounds retained moderate RARα agonist activity and promising cell proliferation inhibitory activity. In particular, compound 36d with a high RARα binding affinity exhibited a strong ability to inhibit cell proliferation and to induce differentiation in NB4 cells. Structure and activity relationship study indicates that 2-alkylindene, 2-alkylindanone, or 1-alkoxyl-2-alkylindane are generally promising structural features for potent RARα agonists. All these results taken together demonstrate that indene as a promising start point for the development of novel RARα agonists.

Acknowledgments

We thank Jianyang Pan (Pharmaceutical Informatics Institute, Zhejiang University) for performing the NMR spectrometry. We also appreciate national natural science foundation of China (81502914) for financial support.

Author Contributions

G.X., H.Y. and Y.H. conceived, designed and performed the experiments; L.P. and H.Q. performed the antiproliferative activity assay. Y.H. analyzed the data and drafted the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds are available from the authors.
Figure 1. Chemical structures of ATRA (1) and representative aromatic RA derivatives (24).
Figure 1. Chemical structures of ATRA (1) and representative aromatic RA derivatives (24).
Molecules 22 00032 g001
Figure 2. Chemical structures of indene derived compound series 68.
Figure 2. Chemical structures of indene derived compound series 68.
Molecules 22 00032 g002
Scheme 1. The synthetic route of 13, 18a, 18b, 26a, 26b, 29a, 29b, 30a, 30b. Reagents and conditions: (a) KNO3, H2SO4, 0 °C, 4 h; (b) NaBH4, MeOH/THF = 2/1, 1 h; (c) p-toluenesulfonamide, PhMe, reflux, 2 h; (d) H2, 10% Pd/C, EtOAc, r.t., overnight; (e) MeI or EtI, MeONa, THF, r.t., 12 h; (f) (i) diborane, THF, r.t., 2 h; (ii) 30% H2O2, 30% KOH aq, r.t., 1 h; (g) 2-bromo-propane or 1-bromobutane HgO/HBF4, CH2Cl2, r.t., 2 h; (h) paraformaldehyde, AcOH, morpholine, reflux, 2 h; (i) acetone, NaOH, r.t., 4 h; (j) Fe, AcOH, EtOH, reflux, 2 h; (k) methyl orthoformate or triethoxy orthoformate, BiCl3, CH2Cl2, r.t., 7 h.
Scheme 1. The synthetic route of 13, 18a, 18b, 26a, 26b, 29a, 29b, 30a, 30b. Reagents and conditions: (a) KNO3, H2SO4, 0 °C, 4 h; (b) NaBH4, MeOH/THF = 2/1, 1 h; (c) p-toluenesulfonamide, PhMe, reflux, 2 h; (d) H2, 10% Pd/C, EtOAc, r.t., overnight; (e) MeI or EtI, MeONa, THF, r.t., 12 h; (f) (i) diborane, THF, r.t., 2 h; (ii) 30% H2O2, 30% KOH aq, r.t., 1 h; (g) 2-bromo-propane or 1-bromobutane HgO/HBF4, CH2Cl2, r.t., 2 h; (h) paraformaldehyde, AcOH, morpholine, reflux, 2 h; (i) acetone, NaOH, r.t., 4 h; (j) Fe, AcOH, EtOH, reflux, 2 h; (k) methyl orthoformate or triethoxy orthoformate, BiCl3, CH2Cl2, r.t., 7 h.
Molecules 22 00032 sch001
Scheme 2. The synthetic route of 36a36p. Reagents and conditions: (l) MeOH, SOCl2, r.t., 12 h; (m) KOH, MeOH, ether, H2O, r.t., 24 h; (n) SOCl2, reflux, 24 h; (o) amine fragments, Py, CH2Cl2, r.t., 7 h; (p) 0.5% LiOH aq, MeOH, r.t., 48 h.
Scheme 2. The synthetic route of 36a36p. Reagents and conditions: (l) MeOH, SOCl2, r.t., 12 h; (m) KOH, MeOH, ether, H2O, r.t., 24 h; (n) SOCl2, reflux, 24 h; (o) amine fragments, Py, CH2Cl2, r.t., 7 h; (p) 0.5% LiOH aq, MeOH, r.t., 48 h.
Molecules 22 00032 sch002
Table 1. RARα binding affinity of compounds 36a36p.
Table 1. RARα binding affinity of compounds 36a36p.
Molecules 22 00032 i001
CompoundsR1R1R2R3EC50 (nM)
AM80----0.170
36aHH--12.13
36b-OCH3H--129.6
36c-OCH2CH3H--311.5
36d-OCH(CH3)2H--24.25
36e-O(CH2)3CH3H--14.88
36fH-OCH3-->1000
36gH-OCH2CH3-->1000
36hH-CH(CH3)2--42.96
36i---CH3-14.08
36j---CH(CH3)2-3.529
36k----CH31375
36l----CH(CH3)24.677
36m-OCH3-CH3--291.9
36n-OCH2CH3-CH3--146.1
36o-OCH3-CH(CH3)2--3.933
36p-OCH2CH3-CH(CH3)2--9.031
Table 2. Results of cell proliferation inhibitory assay using HL60 and NB4 cells.
Table 2. Results of cell proliferation inhibitory assay using HL60 and NB4 cells.
Molecules 22 00032 i001
CompoundsR1R1R2R3IC50 (μM)
HL60NB4
AM80----0.17013.28
36aHH-->506.97
36b-OCH3H--0.1335.43
36c-OCH2CH3H--0.25>50
36d-OCH(CH3)2H-->501.86
36e-O(CH2)3CH3H-->504.09
36fH-OCH3-->50>50
36gH-OCH2CH3-->5021.99
36hH-CH(CH3)2--0.912.61
36i---CH3->50>50
36j---CH(CH3)2-2.37>50
36k----CH32.66>50
36l----CH(CH3)22.1136.90
36m-OCH3-CH3--0.43>50
36n-OCH2CH3-CH3--0.808.51
36o-OCH3-CH(CH3)2--2.35>50
36p-OCH2CH3-CH(CH3)2--1.137.56
Table 3. Cell differentiation potential of compounds 36ag.
Table 3. Cell differentiation potential of compounds 36ag.
Molecules 22 00032 i002
CompoundsR1R1CD11b (%) a
HL60NB4
AM80--4.59 (10 μM)94.93 (10 μM)
36aHH1.13 (50 μM)0.97 (5 μM)
36b-OCH3Hndnd
36c-OCH2CH3Hndnd
36d-OCH(CH3)2H3.77 (50 μM)68.88 (5 μM)
36e-O(CH2)3CH3H2.87 (50 μM)15.42 (5 μM)
36fH-OCH3ndnd
36gH-OCH2CH35.82 (50 μM)0.49 (20 μM)
a nd: not detected.
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