Bioinspired Honokiol Analogs and Their Evaluation for Activity on the Norepinephrine Transporter

In traditional Asian medicinal systems, preparations of the root and stem bark of Magnolia species are widely used to treat anxiety and other nervous disturbances. The biphenyl-type neolignans honokiol and magnolol are the main constituents of Magnolia bark extracts. In the central nervous system, Magnolia bark preparations that contain honokiol are thought to primarily interact with γ-aminobutyric acid A (GABAA) receptors. However, stress responses inherently involve the noradrenergic system, which has not been investigated in the pharmacological mechanism of honokiol. We present here interactions of honokiol and other synthesized biphenyl-type neolignans and diphenylmethane analogs with the norepinephrine transporter (NET), which is responsible for the synaptic clearance of norepinephrine and the target of many anxiolytics. Of the synthesized compounds, 16 are new chemical entities, which are fully characterized. The 52 compounds tested show mild, non-potent interactions with NET (IC50 > 100 µM). It is thus likely that the observed anxiolytic effects of, e.g., Magnolia preparations, are not due to direct interaction with the noradrenergic system.


Introduction
For centuries, traditional Chinese and Japanese medicinal preparations from Magnolia bark have been used to treat neurological diseases, including anxiety and sleep disorders [1]. Study of these preparations led to the identification of the biphenyl neolignans honokiol (H) and magnolol (M) as the major active constituents [2]. While these compounds act promiscuously in the periphery [3], the central nervous system activity of H and M has only been linked to interaction with GABA A receptors [4].
It is possible that these biphenyl-type neolignans exert pharmacological activity on other CNS targets. In Western medicine, anxiolytic and antidepressant therapeutics (e.g., norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, or tricyclic antidepressants) generally increase noradrenaline levels by inhibiting the norepinephrine transporter (NET) [5]. NET localizes to the presynaptic membrane, where it acts to clear synaptic norepinephrine, thereby terminating signalling and recycling the neurotransmitter for subsequent release. Given the anxiolytic efficacy of H and M, we hypothesized biphenyl neolignans may similarly inhibit NET, a question that has not been addressed in the context of active ingredients from Magnolia-containing preparations. In addition to anxiolytic treatment, NET inhibitors are effective in the treatment of depression and attention deficit hyperactive disorder (ADHD). It is also considered a target for therapeutics against neurodegenerative diseases, such as Alzheimer's and Parkinson's disease [6], and in sleep regulation [7]. Thus, these compounds may offer therapeutic efficacy for a myriad of CNS disturbances.
Biphenyls are considered privileged structures due to their over-proportionally frequent biological activity [8]. In our previous investigations, we focused on the diverse biological activities of H, M, and derivatives thereof, such as COX-1/2, 5-LOX and LTB 4 -formation [9], GABA A receptor activity [10,11], CB 2 receptor activity [12], and toxicity on several cancer cell lines [13]. In this paper, we focus on the activity of the compounds at the NET. We tested 32 compounds of our already existing compound library and 16 additional compounds, of which 14 are new chemical substances (see . Altogether 52 compounds have been evaluated in a norepinephrine (NE) uptake assay using a human embryonic kidney cell line stably expressing human NET (hNET) and human vesicular monoamine transporter 2 (VMAT2); human VMAT2 (hVMAT2) was added in addition to hNET to more faithfully recapitulate norepinephrine neurons in vitro (see characterization in Figure 1). localizes to the presynaptic membrane, where it acts to clear synaptic norepinephrine, thereby terminating signalling and recycling the neurotransmitter for subsequent release. Given the anxiolytic efficacy of H and M, we hypothesized biphenyl neolignans may similarly inhibit NET, a question that has not been addressed in the context of active ingredients from Magnolia-containing preparations. In addition to anxiolytic treatment, NET inhibitors are effective in the treatment of depression and attention deficit hyperactive disorder (ADHD). It is also considered a target for therapeutics against neurodegenerative diseases, such as Alzheimer's and Parkinson's disease [6], and in sleep regulation [7]. Thus, these compounds may offer therapeutic efficacy for a myriad of CNS disturbances. Biphenyls are considered privileged structures due to their over-proportionally frequent biological activity [8]. In our previous investigations, we focused on the diverse biological activities of H, M, and derivatives thereof, such as COX-1/2, 5-LOX and LTB4-formation [9], GABAA receptor activity [10,11], CB2 receptor activity [12], and toxicity on several cancer cell lines [13]. In this paper, we focus on the activity of the compounds at the NET. We tested 32 compounds of our already existing compound library and 16 additional compounds, of which 14 are new chemical substances (see Tables 1 to 5). Altogether 52 compounds have been evaluated in a norepinephrine (NE) uptake assay using a human embryonic kidney cell line stably expressing human NET (hNET) and human vesicular monoamine transporter 2 (VMAT2); human VMAT2 (hVMAT2) was added in addition to hNET to more faithfully recapitulate norepinephrine neurons in vitro (see characterization in Figure 1).         Plate reader-based fluorescent assay using human NET in a validation assay of the screening strategy using the known NET inhibitor desipramine as a positive control. Compound screening workflow: (A) All compounds were screened for fluorescent uptake in human embryonic cidney HEK-hNET-hVMAT2 cells (blue hNET, red VMAT2) using a dye that is a substrate for NET, as indicated by inhibition by addition of a selective NET inhibitor, desipramine. (B) Next, fluorescent counts were confirmed with a radiolabelled uptake of dopamine, which is also a substrate of NET, as demonstrated by the lack of uptake in the presence of desipramine. (C) Finally, uptake was visualized using high content imaging with the Array Scan VTI HCS (Cellomics, Thermo Fisher Scientific, Waltham, MA, USA) to visualize nuclei (stained with DAPI), VMAT2-mCherry, and dye. All experiments were conducted in triplicate and analyzed with nonlinear regression analysis in GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Scale: 10 nm.  Plate reader-based fluorescent assay using human NET in a validation assay of the screening strategy using the known NET inhibitor desipramine as a positive control. Compound screening workflow: (A) All compounds were screened for fluorescent uptake in human embryonic cidney HEK-hNET-hVMAT2 cells (blue hNET, red VMAT2) using a dye that is a substrate for NET, as indicated by inhibition by addition of a selective NET inhibitor, desipramine. (B) Next, fluorescent counts were confirmed with a radiolabelled uptake of dopamine, which is also a substrate of NET, as demonstrated by the lack of uptake in the presence of desipramine. (C) Finally, uptake was visualized using high content imaging with the Array Scan VTI HCS (Cellomics, Thermo Fisher Scientific, Waltham, MA, USA) to visualize nuclei (stained with DAPI), VMAT2-mCherry, and dye. All experiments were conducted in triplicate and analyzed with nonlinear regression analysis in GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Scale: 10 nm.

Figure 1.
Plate reader-based fluorescent assay using human NET in a validation assay of the scree-ning strategy using the known NET inhibitor desipramine as a positive control. Compound screening workflow: (A) All compounds were screened for fluorescent uptake in human embryonic cidney HEK-hNET-hVMAT2 cells (blue hNET, red VMAT2) using a dye that is a substrate for NET, as indicated by inhibition by addition of a selective NET inhibitor, desipramine. (B) Next, fluorescent counts were confirmed with a radiolabelled uptake of dopamine, which is also a substrate of NET, as demonstrated by the lack of uptake in the presence of desipramine. (C) Finally, uptake was visualized using high content imaging with the Array Scan VTI HCS (Cellomics, Thermo Fisher Scientific, Waltham, MA, USA) to visualize nuclei (stained with DAPI), VMAT2-mCherry, and dye. All experiments were conducted in triplicate and analyzed with nonlinear regression analysis in GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Scale: 10 nm.

Results and Discussion
Previous studies indicated a significant increase in pharmacological activity when nitrogen-containing functional groups were introduced into H and other compounds [10]. We enhanced our existing compound library with further nitrogen-bearing honokiol derivatives with significantly changed polarity, either by N-acylation of 3-amino-4 -O-methylhonokiol (H1) with a long chain fatty acid (H2) or an amino acid (H3, H4), by formally replacing the methoxy group of H1 with an ethoxy group (H5), or di-O-alkylation without or with N-acetylation (H6, H7). In contrast to amid H8 of our parent library, in H9 acetamido and O-methyl groups are located at C-5 and C-2, respectively. In the course of the syntheses, intermediates were fully characterized by their chemical and physical properties, but not tested in the biological assay. Compound data is provided for all synthesized chemicals, including compounds mentioned in the literature for which chemical and physical data were not fully provided.
All synthesized compounds were tested for pharmacological activity at the NET and compared with selective NET inhibitor, desipramine ( Figure 1). Compounds with any apparent pharmacological activity were further characterized by radioactive uptake and high content imaging. No compound showed robust NET activity ( Table 1).
As outlined in Scheme 1, N-acylations of H1 were performed using the corresponding carboxylic acid chlorides [14,15]. An Fmoc-protection group was used for the preparation of the peptide-type amides H3 and H4 [16,17] (see Scheme 1). N-acylation of H6 resulted in H7 (see Scheme 2).

Results and Discussion
Previous studies indicated a significant increase in pharmacological activity when nitrogen-containing functional groups were introduced into H and other compounds [10]. We enhanced our existing compound library with further nitrogen-bearing honokiol derivatives with significantly changed polarity, either by N-acylation of 3-amino-4′-O-methylhonokiol (H1) with a long chain fatty acid (H2) or an amino acid (H3, H4), by formally replacing the methoxy group of H1 with an ethoxy group (H5), or di-O-alkylation without or with N-acetylation (H6, H7). In contrast to amid H8 of our parent library, in H9 acetamido and O-methyl groups are located at C-5′ and C-2, respectively. In the course of the syntheses, intermediates were fully characterized by their chemical and physical properties, but not tested in the biological assay. Compound data is provided for all synthesized chemicals, including compounds mentioned in the literature for which chemical and physical data were not fully provided.
All synthesized compounds were tested for pharmacological activity at the NET and compared with selective NET inhibitor, desipramine ( Figure 1). Compounds with any apparent pharmacological activity were further characterized by radioactive uptake and high content imaging. No compound showed robust NET activity ( Table 1).
As outlined in scheme 1, N-acylations of H1 were performed using the corresponding carboxylic acid chlorides [14,15]. An Fmoc-protection group was used for the preparation of the peptide-type amides H3 and H4 [16,17] (see Scheme 1). N-acylation of H6 resulted in H7 (see Scheme 2). As outlined in Scheme 2, H5 was prepared analogously to H1 from 4′-O-ethylhonokiol (H10) via nitration with nitric acid followed by reduction with SnCl2 × 2H2O [10,18,19]. To avoid N-alkylation in the synthesis of di-O-alkylated amine H6, the O-alkylation of the hydroxy group of 4′-O-methylhonokiol honokiol (H11) had to be performed after nitration and prior to reduction with SnCl2 × 2 H2O. N-acetylation of H6 with acetic anhydride resulted in H7 [10]. H9 was prepared by a similar N-acetylation of the corresponding amine H12.  To go one step further, we tested several dioxygenated diphenylmethanes to see if insertion of a methylene group between the two aromatic moieties affects biological activity. As outlined in Scheme 3, the synthesis started from bis(2-hydroxyphenyl)methane (B1) and 2,4′-dihydroxydiphenylmethane (B1), which are commercially available but were also prepared from salicylic alcohol and phenol [20]. Double O-allylation and Claisen rearrangement according to Chattopadhyay et al. [21] yielded mainly the ortho rearrangement products (B2 and C2 resp.) together with little of the para rearrangement products (B3 and C3 resp.). B2 and C2 were submitted to incomplete O-methylation to yield the monoethers B4a, C4b, and C4c together with the respective diether B4a or C4a. To go one step further, we tested several dioxygenated diphenylmethanes to see if insertion of a methylene group between the two aromatic moieties affects biological activity. As outlined in Scheme 3, the synthesis started from bis(2-hydroxyphenyl)methane (B1) and 2,4 -dihydroxydiphenylmethane (B1), which are commercially available but were also prepared from salicylic alcohol and phenol [20]. Double O-allylation and Claisen rearrangement according to Chattopadhyay et al. [21] yielded mainly the ortho rearrangement products (B2 and C2 resp.) together with little of the para rearrangement products (B3 and C3 resp.). B2 and C2 were submitted to incomplete O-methylation to yield the monoethers B4a, C4b, and C4c together with the respective diether B4a or C4a. To go one step further, we tested several dioxygenated diphenylmethanes to see if insertion of a methylene group between the two aromatic moieties affects biological activity. As outlined in Scheme 3, the synthesis started from bis(2-hydroxyphenyl)methane (B1) and 2,4′-dihydroxydiphenylmethane (B1), which are commercially available but were also prepared from salicylic alcohol and phenol [20]. Double O-allylation and Claisen rearrangement according to Chattopadhyay et al. [21] yielded mainly the ortho rearrangement products (B2 and C2 resp.) together with little of the para rearrangement products (B3 and C3 resp.). B2 and C2 were submitted to incomplete O-methylation to yield the monoethers B4a, C4b, and C4c together with the respective diether B4a or C4a.

General
Microwave reactions were carried out on a CEM Corp. Discover laboratory microwave equipped with an Explorer unit (CEM Corp. Matthews, NC, USA). Infrared spectra were recorded on a Bruker Alpha Platinum ATR spectrometer (Bruker, Kennewick, WA, USA) or as KBr pellets on a Perkin-Elmer 281 B spectrometer (PerkinElmer, Waltham, MA, USA). 1 H and 13 C-NMR spectra were recorded on a Varian 400 MHz UnityINOVA spectrometer (400 and 100 MHz, respectively, Varian, Palo Alto, CA, USA) and unless otherwise stated in CDCl 3 with undeuterated solvent as an internal standard (7.26 ppm and 77.0 ppm, respectively). For convenience, atoms were numbered according to structures in Tables 1, 4 and 5 with double-primed numbers for substituents at ring A and triple primed numbers for substituents at ring B, respectively. ESI-MS were recorded in ESI positive and negative mode on an LC Ultimate 3000 (Thermo, San José, CA, USA) with DAD detection in line with a Thermo Scientific LTQ XL mass spectrometer. Column: Knauer (Berlin, Germany) RP-18 (1.8 µm; 125 × 2.1 mm) with a guard cartridge at a flowrate of 150 µL/min. ESI-MS were recorded on an Agilent Technologies HP 7890A instrument fitted with detector HP 5975C VL MSD (70 eV, ion source 250 • C, quadrupole temperature 150 • C, Santa Clara, CA, USA). An Agilent HP-5MS column (30 m, ID 0.25 mm, film 5%pheny l95%, methylpolysiloxane 0.25 µm) was used. The oven temperature was kept at 45 • C for 2 min and programmed to increase to 300 • C at a rate of 3 • C/min, then kept constant at 300 • C for 20 min, with a total run time of 64.5 min. Helium was used as a carrier gas. The injection volume was 1 µL (≈0.5% solution) and a split ratio of 1:50.
For high resolution mass spectrometry, a Waters GCT Premier instrument was used with electron impact ionization (70 eV) at an ion source temperature of 200 • C.

General Information on Syntheses
Compounds were synthesized as described below. Solvents were of analytical quality, if not stated otherwise. The purity of all synthesized compounds was verified using NMR and analytical HPLC. Analytical thin layer chromatography (TLC) was performed using aluminium foil coated with silica 60 F 254 (Merck, Darmstadt, Germany). Preparative thin layer chromatography (PTLC) was performed using glass plates coated with silica 60 F 254 (Merck). Detection was done using UV/254 nm and spraying with molybdophosphoric acid and subsequent heating. Compound mixtures were separated using column chromatography (CC) on silica gel 60 (63-200 µm, Merck) using cyclohexane/ethyl acetate mixtures. Further purification was performed using preparative HPLC (Varian Prepstar with a Dynamax Rainin detector; column SepServ, Berlin, Germany, 250 × 21 mm, RP-18, 7 µm, flow rate 15 mL). Honokiol (purity > 98%) was purchased from APIChem Technology Co. (Hangzhou, China). Proton NMR spectra of the newly described compounds are given under "Supplementary Materials".

3-Dodecanoylamino-4 -O-methylhonokiol (H2)
Dodecanoyl chloride (0.34 mmol, 80 µL) was added with intense stirring at room temperature (RT) to a solution of 100 mg (0.34 mmol) of H1 and pyridine (0.41 mmol, 33 µL) in abs. Et 2 O (4 mL). The reaction mixture was stirred at room temperature overnight and filtered. The precipitate was extracted with Et 2 O (4 mL). NaHCO 3 (1 M, 10 mL) was added to the combined filtrate and washings. The organic phase was separated, and the aqueous phase was extracted with Et 2 O (3 × 10 mL). The combined organic phases were washed with brine (10 mL), dried over Na 2 SO 4 , concentrated under reduced pressure, and purified using CC (silica, cyclohexane/AcOEt 5:1) resulting in 107 mg (59%) of a light brown solid (H2). . From the same batch, the compound has been tested in parallel towards CB 1 /CB 2 receptor agonistic activity by Bertini et al. [22]; however, spectroscopic data and synthesis are only given here.

3-(N-L-Alanyl)-4 -O-methylhonokiol (H3)
L-N-(9-Fluorenylmethoxycarbonyl)alanylchloride, Fmoc-L-Ala-Cl: Fmoc-L-Ala-OH · H 2 O was first dehydrated at 50 • C over P 2 O 5 in vacuo for 12 h. Under dry conditions 400 mg (1.28 mmol) L-N-(9-Fluorenylmethoxycarbonyl)alanin was suspended in 5 mL dichloromethane, thionyl chloride (freshly distilled, 1.23 mL, 17.7 mmol) was added, and the mixture was sonicated at RT. After 7 min, the reaction mixture was a homogeneous solution and it was sonicated for an additional 15 min. Dichloromethane and the excess of thionyl chloride were removed in vacuo to yield 404 mg (96%) of Fmoc-L-Ala-Cl as a grey solid, which was used without further purification for a second step.

3-Acetylamino-2-O-ethyl-4 -O-methylhonokiol (H7)
In a 10 mL round-bottom flask, H6 (34 mg, 0.105 mmol) was mixed with water (0.3 mL), and acetic anhydride (0.21 mmol, 20 µL) was added. The flask was rotated at 80 • C for about 10 min in a water bath. After cooling to RT, the reaction mixture was quenched with aqueous NaHCO 3 (1 M, 2 mL) and extracted with dichloromethane (3 × 2 mL). The combined extracts were washed with aqueous NaHCO 3 (1 M, 2 mL), water (2 mL), dried over Na 2 SO 4 , and concentrated under reduced pressure. The crude product (27 mg) was purified using PTLC (cyclohexane/AcOEt = 5:3), yielding 21 mg (55%) of H7 as a brownish oil. long-term treatment with the compounds could change tissue expression of receptors or affect the tissue level of norepinephrine, potentially by modulating enzymes that produce and metabolize the neurotransmitter. Future experiments are needed to determine if chronic administration of drugs may change noradrenergic signaling. Within the context of these experi-ments, however, we see no evidence for acute modulation of NET activity by biphenyl neolignans or derivatives. It is also of note that serotonergic signaling also plays a major role in anxiety and depression. Though beyond the scope of this work, investigation of honokiol derivatives at serotonin receptors may provide further information as to how these compounds exert their anxiolytic effect.