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

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.


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.  (1) and representative aromatic RA derivatives (2)(3)(4).
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).

Chemistry
Synthesis of indene derivatives 36a-p 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 14a-d. 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 14a-d, 17a, 17b, 21a, 21b, 23a, 23b, 27a, 27b and 28a, 28b then yielded the hydrophobic moieties 15a-d, 18a, 18b, 26a, 26b, 24, 25a, 25b, 29a, 29b and 30a, 30b [16]. 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). 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 Figure 2. Chemical structures of indene derived compound series 6-8.

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 36b-36g 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.
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.
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.

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 36b-c with small aliphatic ether chains at the 1-position are more potent than 36d-e 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 36j-l, 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.
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.

Cell Differentiation Assay Using HL60 and NB4
The effects of 36a-g 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].

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. 1 H-NMR spectra (500 MHz) were determined in CDCl3 on an Advance III MHz spectrometer (Bruker, 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, 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%-95%). Melting points were measured with a B-540 melting-point apparatus (Büchi, Flawil, St. Gallen, Switzerland) and are uncorrected. (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. 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

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 F 254 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. 1 H-NMR spectra (500 MHz) were determined in CDCl 3 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 CH 3 CN in H 2 O (from 10% to 95%). Melting points were measured with a B-540 melting-point apparatus (Büchi, Flawil, St. Gallen, Switzerland) and are uncorrected.

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 36a-36p 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.

Cell Differentiation Assay
Cells (1 × 10 6 /mL) were treated with compounds at different concentrations based on IC 50 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.

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.