Bioinspired honokiol analogs and their evaluation for activity on the norepinephrine transporter

Article abstract of DOI:10.3390/molecules23102536

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 -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.

Full text of DOI:10.3390/molecules23102536

molecules
Article
Bioinspired Honokiol Analogs and Their Evaluation
for Activity on the Norepinephrine Transporter
Kristen Stout 1,2, Marketa Bernaskova 3, Gary W. Miller 1,2 , Antje Hufner 3
and Wolfgang Schuehly 4,5,
*
1
Rollins School of Public Health, Emory University, 1518 Clifton Road, NE, Claudia Nance Rollins Bldg,
Atlanta, GA 30322, USA; kristen.stout@gmail.com (K.S.); gwmille@emory.edu (G.W.M.)
Columbia University Mailman School of Public Health, 722 West 168th Street, Room 1411B,
New York, NY 10032, USA
Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, Universitätsplatz 1, University of Graz,
8010 Graz, Austria; mbernaskova@centrum.cz (M.B.); antje.huefner@uni-graz.at (A.H.)
Institute of Pharmaceutical Sciences, Pharmacognosy, Universitätsplatz 4, University of Graz,
8010 Graz, Austria
2
3
4
5
Institute of Biology, Universitätsplatz 2, University of Graz, 8010 Graz, Austria
Correspondence: wolfgang.schuehly@uni-graz.at
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 4 August 2018; Accepted: 26 September 2018; Published: 4 October 2018
Abstract: 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.
Keywords: honokiol derivatives; norepinephrine transporter; HEK cell expressing hNET; biphenyl-
type neolignans; diphenylmethanes; Magnolia officinalis
1. Introduction
For centuries, traditional Chinese and Japanese medicinal preparations from Magnolia bark have
been used to treat neurological diseases, including anxiety and sleep disorders [
preparations led to the identification of the biphenyl neolignans honokiol ( ) and magnolol (
major active constituents [ ]. While these compounds act promiscuously in the periphery [ ], the central
1]. Study of these
H
M) as the
2
3
nervous system activity of H and M has only been linked to interaction with GABAA 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
Molecules 2018, 23, 2536; doi:10.3390/molecules23102536
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2536
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signalling and recycling the neurotransmitter for subsequent release. Given the anxiolytic efficacy of
and , we hypothesized biphenyl neolignans may similarly inhibit NET, a question that has
H
M
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 [
regulation [ ]. 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 [ ]. In our previous investigations, we focused on the diverse biological activities of , and
derivatives thereof, such as COX-1/2, 5-LOX and LTB4-formation [ ], GABAA receptor activity [10 11],
6], and in sleep
7
8
H, M
9
,
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 1–5).
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).
Table 1. Structures of Compounds Based on Honokiol.
R6
5'
OR2
OR1
4'
B
2
R3
1
R5
3'
1'
A
5
R4
Cpd.
R1
R2
R3
R4
R5
R6
H
H1
H2
H3
H4
H5
H6
H7
H
H
H
H
H
H
Me
Me
Me
Me
Et
Me
Me
Me
H
Et
Me
H
H
H
H
NH2
NH-COC11H23
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
allyl
H
H
H
H
H
H
H
H
H
NH-Ala
NH-Phe
H
NH2
NH2
NH-Ac
NH-Ac
H
Et
Et
H
Me
H
H
Me
H
H8
H9
NH-Ac
H
H
NH2
H
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
H21
H22
H23
H24
H25
H26
H27
H28
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1-propenyl
n-propyl
allyl
H
n-propyl
allyl
allyl
allyl
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NO2
H
Me
Me
H
H
H
Me
Et
H
Et
Et
H
Me
Me
H
H
Me
Me
Me
Me
Me
H
Et
H
Et
Me
H
allyl
1-propenyl
2,3-dihydroxypropyl
n-propyl
n-propyl
allyl
2,3-dihydroxypropyl
n-propyl
n-propyl
allyl
allyl
allyl
n-propyl
n-propyl
2-bromopropyl
allyl
n-propyl
n-propyl
allyl
allyl
n-propyl
H
H
NO2
H
Me
n-propyl
allyl
allyl

Molecules 2018, 23, 2536
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Table 1. Cont.
Cpd.
R1
R2
R3
R4
R5
R6
H29
H30
H31
H32
Me
H
Me
H
H
Me
H
H
NH2
H
n-propyl
n-propyl
n-propyl
n-propyl
n-propyl
NH2
H
NH-Ac
H
n-propyl
n-propyl
n-propyl
Me
NH-Ac
0
Table 2. Structures of Compounds Based on Biphenyl-2,2 -diol.
OR1
OR2
R3
R5
R4
R6
Cpd.
R1
R2
R3
R4
R5
R6
M
H
H
H
Me
Me
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
allyl
allyl
H
n-propyl
allyl
allyl
Me
t-butyl
H
H
H
H
H
H
H
H
allyl
allyl
H
n-propyl
allyl
allyl
Me
M1
M2
M3
M4
M5
M6
M7
H
H
H
H
t-butyl
H
0
Table 3. Structures of Compounds Based on Biphenyl-4,4 -diol.
R
R
HO
OH
Cpd.
R
A1
A2
H
allyl
Table 4. Structures of Compounds Based on Bis(2-hydroxyphenyl)methane.
OR2
OR1
2
2'
R5
R3
1'
1
B
A
5'
5
R6
R4
Cpd.
R1
R2
R3
R4
R5
R6
B1
B1a *
B2
B3 *
B4b
B4a
H
allyl
H
H
Me
Me
H
allyl
H
H
H
H
H
allyl
allyl
allyl
allyl
H
H
H
H
H
H
H
H
allyl
H
allyl
allyl
H
H
H
allyl
H
H
Me
* Compounds not tested.

Molecules 2018, 23, 2536
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0
Table 5. Structures of Compounds Based on 2,4 -Dihydroxydiphenylmethane.
OR1
2
2'
R5
R3
1'
1
A
B
4'
OR2
5
R4
Cpd.
R1
R2
R3
R4
R5
C1
C1a *
C2
C3 *
C4c
C4b
C4a
H
allyl
H
H
Me
H
H
allyl
H
H
H
H
H
allyl
H
allyl
allyl
allyl
H
H
H
allyl
H
H
H
H
allyl
allyl
allyl
allyl
allyl
Me
Me
Me
H
* Compounds not tested.
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: (
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. ( ) 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. ( ) Finally, uptake was visualized using high
A) All compounds were screened for fluorescent uptake in human embryonic cidney
B
C
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.

Molecules 2018, 23, 2536
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2. Results and Discussion
Previous studies indicated a significant increase in pharmacological activity when
nitrogen-containing functional groups were introduced into and other compounds [10]. We enhanced
H
our existing compound library with further nitrogen-bearing honokiol derivatives with significantly
changed polarity, either by N-acylation of 3-amino-40-O-methylhonokiol (H1) with a long chain fatty
acid (H2) or an amino acid (H3
group (H5), or di-O-alkylation without or with N-acetylation (H6
,
H4), by formally replacing the methoxy group of H1 with an ethoxy
H7). In contrast to amid H8 of
,
our parent library, in H9 acetamido and O-methyl groups are located at C-50 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).
OMe
OH
n-
C11H23
NH
O
i)
H2
OMe
OH
H2N
ii) or iii)
iv)
OMe
OH
H1
RNH
H3 R = Ala
H4 R = Phe
Scheme 1. Synthesis of honokiol derivatives. Reagents and conditions: i) n-C11H23COCl, pyridine;
ii) Fmoc-L-Ala-Cl, Zn, THF; iii) Fmoc-L-Phe-Cl, Zn, THF; iv) piperidine.
As outlined in Scheme 2, H5 was prepared analogously to H1 from 40-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
0
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.

Molecules 2018, 23, 2536
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X2
H10 R1 = H, R2 = Et, X1, X2 = H
H5 R1 = H, R2 = Et, X1 = NH2, X2 = H
i), ii)
OR2
OR1
H11 R1 = H, R2 = Me, X1 = H, X2 = H
H6 R1 = Et, R2 = Me, X1 = NH2, X2 = H
X1
i), iii), ii)
iv)
H7 R1 = Et, R2 = Me, X1 = NHAc, X2 = H
H12 R1 = Me, R2 = H, X1 = H , X2 = NH2
H9 R1 = Me, R2 = H, X1 = H , X2 = NHAc
iv)
Scheme 2. Synthesis of honokiol derivatives. Reagents and conditions: i) HNO3 (65%), AcOEt;
ii) SnCl2×2 H2O, EtOH; iii) MW irradiation 1. KOH, 2. Me2SO4 or Et2SO4; iv) Ac2O, H2O.
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 2,40-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.
OR2
OR1
OH
OH
iii)
OR
OR
B2
B4a R1, R2 = Me
B4b R1 = H; R2 = Me
ii)
+
OH
OH
B1 R = H
B1a R = allyl
i)
B3
OR1
OH
iii)
OR
OR2
OH
ii)
C2
C4a R1, R2 = Me
C4b R1 = H; R2 = Me
C4c R1 = Me; R2 = H
+
OR
OH
C1 R = H
i)
C1a R = allyl
OH
C3
Scheme 3. Synthesis of diphenylmethane derivatives. Reagents and conditions: i) K2CO3,
CH2=CH–CH2–Br, acetone; ii) ∆, Ph-NMe2; iii) 1. KOH, 2. Me2SO4.

Molecules 2018, 23, 2536
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3. Experimental
3.1. 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). 1H and 13C-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 CDCl3 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
µ
L/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◦
µ
m) 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
F254 (Merck, Darmstadt, Germany). Preparative thin layer chromatography (PTLC) was performed
using glass plates coated with silica 60 F254 (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
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 m, flow rate
µm, Merck) using cyclohexane/ethyl
×
µ
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-40-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. Et2O (4 mL).
The reaction mixture was stirred at room temperature overnight and filtered. The precipitate was
extracted with Et2O (4 mL). NaHCO3 (1 M, 10 mL) was added to the combined filtrate and washings.
The organic phase was separated, and the aqueous phase was extracted with Et2O (3
×
10 mL).
The combined organic phases were washed with brine (10 mL), dried over Na2SO4, concentrated
under reduced pressure, and purified using CC (silica, cyclohexane/AcOEt 5:1) resulting in 107 mg
(59%) of a light brown solid (H2).
Compound H2: IR (ATR, νmax, cm−1): 3304, 2956, 2919, 2847, 2485, 1639, 1606, 1539, 1499, 1467,
1
1411, 1243, 906, 725, 577; H-NMR (CDCl3)
δ 7.57 (bs, 1H, NH), 7.40 (d, J = 1.5 Hz, 1H, H-4), 7.34
(dd, J = 8.4, 2.3 Hz, 1H, H-60), 7.27 (d, J = 2.1 Hz, 1H, H-20), 7.17 (s, 1H, OH), 6.94 (d, J = 8.4 Hz, 1H,
H-50), 6.89 (d, J = 1.9 Hz, 1H, H-6), 6.01 (ddt, J = 16.8, 10.2, 6.6 Hz, 1H, H-2000), 5.96 (ddt, J = 16.8, 10.0,

Molecules 2018, 23, 2536
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6.7 Hz, 1H, H-200), 5.02–5.13 (m, 4H, H-3000, H-300), 3.87 (s, 3H, OCH3), 3.43 (d, J = 6.6 Hz, 2H, H-1000),
3.33 (d, J = 6.7 Hz, 2H, H-100), 2.42 (t, J = 7.5 Hz, 2H, Acyl-Hα), 1.74 (quint, J = 7.3 Hz, 2H, Acyl-Hβ),
1.21–1.42 (m, 16H, Acyl-Hγ to Hω-1), 0.88 (t, J = 6.8 Hz, 3H, Acyl-Hω), 13C-NMR (CDCl3)
δ 172.7 (CO),
156.8 (C-40), 142.3 (C-2), 137.5 (C-200), 136.7 (C-2000), 132.2 (C-5), 130.7 (C-20), 130.7 (C-1), 129.6 (C-10),
129.0 (C-30), 128.1 (C-60), 126.7 (C-6), 126.2 (C-3), 120.3 (C-4), 115.8 (C-300), 115.6 (C-3000), 110.5 (C-50),
55.5 (OCH3), 39.5 (C-100), 37.5 (Acyl-Cα), 34.3 (C-1000), 31.9 (Acyl-Cω-2), {2
×
29.6, 29.4, 2
×
29.3, 29.2
(Acyl-Cγ-to Cω-3)}, 25.7 (Acyl-Cβ), 22.7 (Acyl-Cω-1), 14.1 (Acyl-Cω), ESI+ calcd for C31H43NO3: [M]+
477.324; found ESI-MS m/z (rel. int.): 478.63 [M + H]+ (100). From the same batch, the compound
has been tested in parallel towards CB1/CB2 receptor agonistic activity by Bertini et al. [22]; however,
spectroscopic data and synthesis are only given here.
3-(N-L-Alanyl)-40-O-methylhonokiol (H3)
L-N-(9-Fluorenylmethoxycarbonyl)alanylchloride, Fmoc-L-Ala-Cl: Fmoc-L-Ala-OH
·
H2O was
◦
first dehydrated at 50 C over P2O5 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.
L-3-(N-(9-Fluorenylmethoxycarbonyl)alanyl)-40-O-methylhonokiol (H3a): Zn (dust) was treated
with HCl (2 M, aqueous solution), washed subsequently with water, EtOH and diethylether, dried
in vacuo, and stored under argon. Under argon, H1 (259 mg, 0.841 mmol), Fmoc-L-Ala-Cl (279 mg,
0.846 mmol), and Zn dust (55 mg, 0.841 mmol) were suspended in abs. THF (50 mL) and stirred at RT
overnight. Over a period of two hours, the reaction color turned from brownish to yellowish. Reaction
completion was proven using TLC analysis (cyclohexane/AcOEt = 5:3). Zn dust was filtered off and
the filtrate was evaporated in vacuo. The residue was dissolved in dichloromethane (15 mL), washed
with brine (2
×
5 mL), and dried over Na2SO4. Evaporation of the solvent in vacuo and CC (silica,
cyclohexane/AcOEt = 5:1) yielded in H3a (428 mg; 87%).
3-(N-L-Alanyl)-40-O-methylhonokiol (H3): H3a (401 mg, 0.682 mmol) was dissolved in diethyl
ether (3 mL) and piperidine (1 mL, 1 mmol) was added. The mixture was stirred at RT for 1 h.
The reaction mixture was evaporated under reduced pressure and dichloromethane (5 mL) was added
to the residue. The resulting precipitate was filtered off and the filtrate was concentrated under reduced
pressure to dryness. The residue (377 mg) was purified using CC (silica, dichloromethane/MeOH,
gradient: 0–10% v/v methanol) to yield 220 mg (88%) of H3 as an orange oil.
Compound H3: IR (ATR, νmax, cm−1): 3272, 3075, 2974, 2929, 2834, 1638, 1606, 1590, 1530, 1500,
1
1460, 1440, 1244, 1029, 993, 910, 813, 594; H-NMR (CDCl3 + D2O)
δ 7.35 (dd, J = 8.1, 1.8 Hz, 1H,
H-60), 7.29 (d, J = 2.2 Hz, 1H, H-20), 7.18 (s, 1H, H-4), 6.92 (s, 1H, H-6), 6.91 (d, J = 8.4 Hz, 1H, H-50),
6.01 (ddt, J = 16.8, 9.8, 6.6 Hz, 1H, H-2000), 5.95 (ddt, J = 16.9, 9.9, 6.6 Hz, 1H, H-200), 5.01–5.13 (m, 4H,
H-300, H-3000), 3.85 (s, 3H, O–CH3), 3.73 (q, J = 6.2 Hz, 1H, CH3CH(NH2)CO), 3.42 (d, J = 6.6 Hz, 2H,
H-1000), 3.32 (d, J = 6.2 Hz, 2H, H-100), 2.03 (s, 2H, NH2), 1.45 (d, J = 6.6 Hz, 3H, CH3CH(NH2)CO);
13C-NMR (CDCl3 + D2O)
δ
174.6 (CO), 156.7 (C-40), 143.2 (C-2), 137.5 (C-200), 136.8 (C-2000), 131.9 (C-5),
0
0
0
0
131.0 (C-1), 130.8 (C-2 ), 130.1 (C-1 ), 128.6 (C-3 ), 128.2 (C-6 ), 127.5 (C-6), 125.6 (C-3), 120.6 (C-4), 115.7
(C-300), 115.5 (C-3000), 110.3 (C-50), 55.5 (O–CH3), 50.5 (CH3CH(NH2)CO), 39.4 (C-100), 34.3 (C-1000), 21.1
(CH3CH(NH2)CO); ESI+ calcd for C22H26N2O3: [M]+ 366.194; found ESI-MS m/z (rel. int.): 367.18
[M + H]+ (100).
3-(N-L-Phenylalanyl)-40-O-methylhonokiol (H4)
L-N-(9-Fluorenylmethoxycarbonyl)phenylalanylchloride, Fmoc-L-Phe-Cl: Under dry conditions
300 mg (0.774 mmol) L-N-(9-Fluorenylmethoxycarbonyl)phenylalanin was suspended in dry
dichloromethane (4 mL), thionyl chloride (freshly distilled, 0.776 mL, 10.7 mmol) was added, and the

Molecules 2018, 23, 2536
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mixture was sonicated at RT for 45 min. The sonication bath was heated up to 40 ◦C over that period
of time. Dichloromethane and the excess of thionyl chloride were removed in vacuo to yield 294 mg
(94%) of Fmoc-L-Phe-Cl as a grey solid, which was used without further purification in the next step.
3-(N-L-Phenylalanyl)-40-O-methylhonokiol (H4): Zn dust was treated with aqueous HCl (2 M),
washed subsequently with water, ethanol, and diethylether, dried in vacuo, and stored under argon.
Under argon, H1 (75 mg, 0.25 mmol), Fmoc-L-Phe-Cl (103 mg, 0.254 mmol), and Zn dust (17 mg,
0.25 mmol) were suspended in dry THF (10 mL) and stirred for 4 h. Reaction completion was proven
using TLC analysis (cyclohexane/AcOEt = 5:3). Piperidine (0.5 mL, 5 mmol) was added and the
reaction mixture was stirred for 20 min at RT, the solvent was evaporated under reduced pressure
and the residue was diluted with dichloromethane (5 mL), filtered, and evaporated under reduced
pressure. The residue (170 mg) was purified using CC (silica, cyclohexane/AcOEt, gradient: 0–100%
v/v AcOEt) to yield 50 mg (45%) of H4 as an orange oil.
Compound H4: IR (ATR, νmax, cm−1): 3270, 3073, 2974, 2834, 1638, 1605, 1530, 1500, 1453, 1439 s,
1244, 1028, 910; 1H-NMR (CDCl3)
δ
9.69 (bs, 1H, CONH), 7.39 (dd, J = 8.4,
≈
2 Hz, 1H, H-60), 7.30–7.36
0
(m, 3H, Ph-Hmeta Ph-Hpara), 7.22–7.30 (m, 3H, H-2 , Ph-Hortho), 7.10 (s, 1H, H-4), 6.95 (s, 1H, H-6), 6.92
(d, J = 8.4 Hz, 1H, H-50), 6.01 (ddt, J = 16.9, 9.9, 6.6 Hz, 1H, H-2000), 5.97 (ddt, J = 16.9, 10.2, 6.6 Hz,
1H, H-200), 5.02–5.14 (m, 4H, H-300, H-3000), 3.86 (s, 3H, OCH3), 3.82 (m, 1H, PhCH2CH(NH2)CO), 3.43
(d, J = 6.6 Hz, 2H, H-1000), 3.34 (m, 1H, PhCH2CH(NH2)CO), 3.33 (d, J = 6.9 Hz, 2H, H-100), 2.84 (dd,
0
J = 13.6, 9.2 Hz, 1H, PhCH2CH(NH2)CO); 13C-NMR (CDCl3)
δ 173.7 (CO), 156.6 (C-4 ), 143.5 (C-2), 139.1
00
000
0
0
(Ph-Cipso), 137.5 (C-2 ), 136.8 (C-2 ), 131.8 (C-5), 131.2 (C-1), 130.8 (C-2 ), 130.2 (C-1 ), 129.3 (Ph-Cortho),
128.8 (Ph-Cmeta), 128.6 (C-30), 128.2 (C-60), 127.6 (C-6), 127.0 (Ph-Cpara), 125.7 (C-3), 120.5 (C-4), 115.7
(C-300), 115.5 (C-3000),010010.3 (C-50), 56.3 (PhCH2CH(NH2)CO), 55.5 (OCH3), 40.5 (PhCH2CH(NH2)CO),
00
39.3 (C-1 ), 34.3 (C-1 ); ESI+ calcd for C28H30N2O3: [M]+ 442.226; found ESI-MS m/z (rel. int.): 442.20
[M + H]+ (100).
3-Amino-40-O-ethylhonokiol (H5)
40-O-Ethyl-3-nitro-honokiol (H5a): Aqueous nitric acid (65%, 0.474 mL, 6.79 mmol) was added
under intense stirring within
Schuehly et al. [12] supplemental; cpd 16b) in AcOEt (5 mL) at RT. The reaction mixture was stirred for
≈
5 s to a solution of H10 (200 mg, 0.679 mmol; synthesis see
60 s and quenched with NaHCO3 (1 M, 10 mL). The organic phase was separated, and the aqueous
phase was extracted with AcOEt (3
(10 mL), dried over Na2SO4, concentrated under reduced pressure, and purified using CC (silica,
×
10 mL). The combined organic layers were washed with brine
0
cyclohexane/AcOEt 99:1) to yield 120 mg (50%) of 4 -O-ethyl-3-nitro-honokiol (H5a) as a dark yellow oil.
Compound H5a: IR (ATR, νmax, cm−1): 3177, 3078, 2978, 2903, 1638, 1607, 1537, 1502, 1314, 1244,
1045, 912, 672, 555 cm−1; 1H-NMR (CDCl3)
δ
110.03 (s, 1H, OH), 7.90 (d, J = 2.2 Hz, 1H, H-4), 7.45 (d,
0
J = 2.2 Hz, 1H, H-6), 7.38 (dd, J = 8.4, 2.2, 1H, H-6 ), 7.33 (d, J = 2.2 Hz, 1H, H-2 ), 6.92 (d, J = 8.4 Hz, 1H,
H-50), 6.03 (ddt, J = 16.9, 10.3, 6.6 Hz, 1H, H-2000), 5.96 (ddt, J = 16.5, 10.3, 6.6 Hz, 1H, H-200), 5.03–5.17
(m, 4H, H-300 and H-3000), 4.10 (q, J = 7.2 Hz, 2H, OCH2CH3), 3.45 (d, J = 6.4 Hz, 2H, H-1000), 3.40 (d,
J = 6.6 Hz, 2H, H-100), 1.46 (t, J = 6.9 Hz, 3H, OCH2CH3); 13C-NMR (CDCl3)
δ
156.6 (C-40), 151.3 (C-2),
138.9 (C-6), 136.8 (C-2000), 136.0 (C-200), 133.8 (C-3), 132.9 (C-1), 131.7 (C-5), 130.7 (C-20), 128.5 (C-30),
128.2 (C-60), 127.4 (C-10), 122.8 (C-4), 117.1 (C-3000), 115.6 (C-300), 11−1.0 (C-50), 63.7 (OCH2CH3), 38.9
00
000
(C-1 ), 34.5 (C-1 ), 14.9 (OCH2CH3); ESI– calcd for C20H21NO4: [M] 339.147; found ESI-MS m/z (rel.
int.): 338.21 ([M − H]− (100).
3-Amino-40-O-ethylhonokiol (H5): SnCl2
2H2O (1.62 g, 7.16 mmol) was added to a solution of
×
H5a (270 mg, 0.796 mmol) in abs. MeOH (10 mL). The reaction mixture was stirred for 48 h at RT,
concentrated under reduced pressure and diluted with AcOEt (15 mL). The spumy precipitate resulting
from the addition of aqueous NaHCO3 (1 M, 20 mL) was filtered off with Celite® (Sigma-Aldrich, Buchs,
Switzerland) and rinsed with AcOEt (30 mL). The organic layer was separated from the combined filtrate
and washings, dried over Na2SO4, and concentrated under reduced pressure. The residue (127 mg) was
purified using PTLC (silica, cyclohexane/AcOEt = 5:1) to yield 65 mg (26%) of H5 as a brown oil.

Molecules 2018, 23, 2536
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Compound H5: IR (ATR, νmax, cm−1): 3544, 3370, 3075, 2977, 2900, 1637, 1607, 1503, 1486,1474,
0 0
9 Hz, 1H, H-6 ), 7.25 (s, 1H, H-2 ), 6.93 (d,
1242, 1215, 1122, 1044, 908, 808; 1H-NMR (CDCl3)
δ
7.26 (d
≈
J = 8.1 Hz, 1H, H-50), 6.61 (s, 1H, H-4), 6.51 (s, 1H, H-6), 6.00 (ddt, J = 16.9, 9.9, 6.6 Hz, 1H, H-2000), 5.96
(ddt, J = 17.0, 9.9, 6.9 Hz, 1H, H-200), 5.02–5.14 (m, 4H, H-300, H-3000), 4.19 (bs, 2H, NH2), 4.09 (q, J = 7.0
000
00
Hz, 2H, OCH2CH3), 3.43 (d, J = 6.6 Hz, 2H, H-1 ), 3.28 (d, J = 6.9 Hz, 2H, H-1 ), 1.46 (t, J = 7.0 Hz, 3H,
OCH2CH3); 13C-NMR (CDCl3)
δ
156.4 (C-40), 139.0 (C-2), 137.9 (C-200), 136.6 (C-2000), 133.6 (C-3), 132.4
(C-5), 130.3 (C-20), 129.9 (C-30), 129.0 (C-10), 127.9 (C-1), 127.7 (C-60), 010020.5 (C-6), 115.8 (C-3000), 115.7
(C-4), 115.4 (C-3 ), 111.9 (C-5 ), 63.7 (OCH2CH3), 39.6 (C-1 ), 34.5 (C-1 ), 14.9 (OCH2CH3); ESI+ calcd
00
0
00
C20H23NO2: [M]+ 309.173; found ESI-MS m/z (rel. int.): 310.11 [M + H]+ (100).
3-Amino-2-O-ethyl-40-O-methylhonokiol (H6)
In a microwave process vial, KOH (132 mg, 2.36 mmol) was added to a solution of
40-O-methyl-3-nitro-honokiol (synthesis see [10]) (192 mg, 0.590 mmol) in abs. MeOH (2 mL) and
the mixture was stirred at RT for 10 min. Diethyl sulfate (155
the reaction mixture was irradiated in a microwave oven at 90 C for 60 min. After cooling to
µ
◦ L, 2.36 mmol) was added and
RT, the reaction mixture was neutralized with aqueous HCl (2 M). MeOH was evaporated under
◦
reduced pressure (40 C, 100 mbar). The residue was extracted with dichloromethane (3
×
2 mL),
the organic phase washed with water (3
pressure, resulting in 198 mg of crude 2-O-ethyl-4 -O-methyl-3-nitro-honokiol, which was used without
×
5 mL), dried over Na2SO4, and concentrated under reduced
0
further purification.
0
SnCl2
×
2H2O (1.09 g, 4.84 mmol) was added to a solution of crude 2-O-ethyl-4 -O-methyl-3-nitro-
honokiol (190 mg, 0.54 mmol) in abs. EtOH (10 mL). The reaction mixture was stirred at RT for 48 h.
The spumy precipitate resulting from the addition of aqueous NaHCO3 (1 M, 20 mL) was filtered using
Celite®, the residue was extracted with EtOH (50 mL). Combined organic layers were evaporated
under reduced pressure (100 mbar). The residue was extracted with dichloromethane (3 × 10 mL),
which was washed with brine (3
×
5 mL), dried over Na2SO4, and concentrated under reduced
pressure to yield 107 mg of a crude product, which after purification using PTLC (silica, cyclohexane/
AcOEt = 5:1), yielded 26 mg (14%) of H6.
Compound H6: IR (ATR, νmax, cm−1): 3417, 3310, 3076, 2975, 2928, 2836, 1674, 1638, 1607, 1522,
1502, 1435, 1245, 1026,0910, 812, 600, 523; 1H-NMR (CDCl3)
δ
7.47 (d, J = 2.2 Hz, 1H, H-20), 7.46 (dd,
0
J = 8.1, 2.2 Hz, 1H, H-6 ), 6.92 (d, J = 8.4 Hz, 1H, H-5 ), 6.60 (s, 2H, H-4, H-6), 6.07 (ddt, J = 16.6, 10.2, 6.2
Hz, 1H, H-2000), 6.00 (ddt, J = 16.9, 9.9, 6.9 Hz, 1H, H-200), 5.04–5.17 (m, 4H, H-300, H-3000), 3.96 (bs, 2H,
NH2), 3.89 (s, 3H, OCH3), 3.57 (q, J = 6.9 Hz, 2H, OCH2CH3), 3.46 (d, J = 6.6 Hz, 2H, H-1000), 3.33 (d,
J = 6.9 Hz, 2H, H-100), 1.16 (t, J = 7.1 Hz, 3H, OCH2CH3); 13C-NMR (CDCl3)
δ
156.4 (C-40), 142.1 (C-2),
140.0 (C-3), 137.6 (C-200),137.0 (C-2000), 136.0 (C-5), 134.4 (C-1), 131.0 (C-10), 130.5 (C-20), 128.0 (C-30),
127.6 (C-60), 120.5 (C-6), 115.5 (C-300), 115.2 (C-3000), 114.7 (C-4), 109.9 (C-50), 67.6 (C–OCH2CH3), 55.4
(OCH3), 39.9 (C-100), 34.3 (C-1000), 15.7 (OCH2CH3); ESI+ calcd for C21H25NO2: [M]+ 323.189; found
ESI-MS m/z (rel. int.): 324.23 [M + H]+ (100).
3-Acetylamino-2-O-ethyl-40-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
bath. After cooling to RT, the reaction mixture was quenched with aqueous NaHCO3 (1 M, 2 mL)
and extracted with dichloromethane (3 2 mL). The combined extracts were washed with aqueous
µ
L) was added. The flask was rotated at 80 ◦C for about 10 min in a water
×
NaHCO3 (1 M, 2 mL), water (2 mL), dried over Na2SO4, 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.
Compound H7: IR (ATR, νmax, cm−1): 3469, 3368, 3075, 2974, 2928, 2903, 2834, 1638, 1606, 1504,
1438, 1243, 1209, 1030, 909, 810, 596; 1H-NMR (CDCl3)
δ
8.17 (s, 1H, H-4), 7.96 (bs, 1H, NH), 7.38 (s,
1H, H-20), 7.37 (dd, J
≈
8, 2.2 Hz, 1H, H-60), 6.90 (d, J = 8.1 Hz, 1H, H-50), 6.86 (d, J = 1.1 Hz, 1H, H-6),

Molecules 2018, 23, 2536
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6.03 (ddt,0J00= 16.8, 9.9, 6.9 Hz, 1H, H-2000), 5.98 (ddt, J = 16.9, 9.9, 6.6 Hz, 1H, H-200), 5.02–5.15 0(0m0 , 4H,
00
H-3 , H-3 ), 3.87 (s, 3H, OCH3), 3.52 (q, J = 7.1 Hz, 2H, OCH2CH3), 3.42 (d, J = 6.6 Hz, 2H, H-1 ), 3.38
(d, J = 7.0 Hz, 2H, H-1000), 2.21 (s, 3H, CH3CO),001.14 (t, J = 7.0 Hz, 3H, OCH2CH3); 13C-NMR (CDCl3)
δ
000
168.09 (CO), 156.7 (C-4 ), 143.1 (C-2), 137.3 (C-2 ), 136.9 (C-2 ), 136.2 (C-5), 133.5 (C-1), 131.9 (C-3), 130.4
(C-10), 130.3 (C-20), 128.4 (C-30), 127.6 (C-60), 125.3 (C-6), 110800.7 (C-4), 115.8 (C-300), 115.4 (C-3000), 110.2
0
00
(C-5 ), 68.7 (O–CH2–CH3), 55.4 (OCH3), 40.0 (C-1 ), 34.3 (C-1 ), 24.9 (CH3–C=O), 15.6 (OCH2CH3); ESI+
calcd for C23H27NO3: [M]+ 365.199; found ESI-MS m/z (rel. int.): 366.29 [M + H]+ (100).
50-(N-Acetylamino)-2-O-methyl-honokiol (H9)
In a 10 mL round-bottom flask, H12 (for synthesis, see Reference [11]) (37 mg, 0.125 mmol) was
suspended with water (1 mL), and acetic anhydride (0.21 mmol, 20
allowed to rotate for 10 min at 80 ◦C in a water bath. After cooling to RT, the reaction mixture was
quenched with aqueous NaHCO3 (1 M, 1 mL) and extracted with dichloromethane (3 1.5 mL).
µL) was added. The flask was
×
The combined extracts were washed with aqueous NaHCO3 (1 M, 1.5 mL) and water (1.5 mL), dried
over Na2SO4, and concentrated under reduced pressure. The crude product (29 mg) was purified
using PTLC (silica, cyclohexane/AcOEt 5:3), yielding 16 mg (39%) of H9, a light orange solid.
Compound H9: IR (ATR, νmax, cm−1): 3276, 3075, 3001, 2917, 2848, 1750, 1637, 1595, 1540, 1480,
1239, 1239, 1139, 1025, 910, 873, 809; 1H-NMR (CDCl3)
δ
7.62 (s, br 1H, NH), 7.18 (s, 1H, H-20), 7.10
(
d ≈ 8 Hz, 1H, H-4), 7.07 (s, 2H, H-6, H-60), 6.89 (d, J = 8 Hz, 1H, H-3), 6.06 (ddt, J = 16.9, 10.3, 6.6 Hz,
1H, H-2000), 5.97 (ddt, J
≈
17, 9.9, 6.9 Hz, 1H, H-200), 5.03–5.16 (m, 4H, H-300, H-3000), 3.77 (s, 3H, OCH3),
3.50 (d, J = 5.9 Hz, 2H, H-1000), 3.35 (d, J = 6.6 Hz, 2H, H-100), 2.22 (s, 3H, CO-CH3); 13C-NMR (CDCl3)
δ
170.5 (CO), 154.7 (C-2), 146.0 (C-40), 137.7 (C-200), 136.8 (C-2000), 132.3 (C-5), 130.9 (C-6), 130.4 (C-50),
130.2 (C-30), 129.6 (C-1), 128.8 (C-20), 128.2 (C-4), 125.2 (C-10), 121.4 (C-60), 115.7 (C-3000), 115.6 (C-300),
111.4 (C-3), 55.8 (OCH3), 39.4 (C-100), 35.9 (C-1000), 23.7 (CO–CH3); ESI+ calcd for C21H23NO3: [M]+
337.168; found ESI-MS m/z (rel. int.): 338.08 [M + H]+ (100).
Bis(2-Allyloxyphenyl)-methane (B1a)
B1 (400 mg, 2.0 mmol) was O-allylated with allylbromide (968 mg, 0.70 mL, 8.0 mmol) and K2CO3
(2.21 g, 16 mmol) in acetone (10 mL). The reaction mixture was stirred for 5 h with reflux, and a further
12 h at RT and filtered. The residue was extracted with acetone (50 mL) and the combined organic
solutions were evaporated under reduced pressure yielding B1a (559 mg, 73%).
Compound B1a: IR (ATR, νmax, cm−1): 3025, 2920, 2905, 1595, 1584, 1491, 1450, 1421, 1248, 1116,
1103, 1022, 924, 751 cm−1; 1H-NMR (CDCl3)
δ
7.19 (td, J = 7.6, 1.5 Hz, 2H, H-4), 7.14 (dd, J = 7.5, 1.2 Hz,
8 Hz, 2H, H-5), 6.89 (d, 2H, J = 7.5 Hz, H-3), 6.02–6.13 (m, 2H, CH2–CH=CH2),
5.42 (dquint, J = 17.2, 4.3 Hz, 2H, CH2–CH=CH2), 5.27 (dquint, J = 10.5, 1.4 Hz, 2H, CH2–CH=CH2),
4.58 (dq, J = 5.0, 1.5 Hz, 2H, CH2–CH=CH2), 4.10 (s, 2H, Ar–CH2–Ar); 13C-NMR (CDCl3)
156.5
2H, H-6), 6.90 (t, J
≈
δ
(C-2), 133.6 (CH=CH2), 130.5 (C-6), 129.6 (C-1), 126.9 (C-4), 120.5 (C-5), 116.8 (CH=CH2), 111.6 (C-3),
30.8 (O–CH2), 29.8 (Ar–CH2–Ar); EI+ calcd for C19H20O2: [M]+ 280.1463; found EI-MS m/z (rel. int.):
280.1465 [M]+ (100). Compound B1a has been mentioned in several patents; however, chemical data
were not given therein.
2-Allyl-6-(3-allyl-2-hydroxybenzyl)-phenol (B2) and 4-Allyl-2-(3-allyl-2-hydroxybenzyl)-phenol (B3
)
Claisen rearrangement of B1a (561 mg, 2 mmol) in N,N-diethylaniline (4 mL) following the
procedure of Chattopadhyay et al. [21] resulted in B2 (410 mg; 73%) and B3 (89 mg; 15%) as white wax
like solids.
Compound B2: IR (KBr, νmax, cm−1): 3407 (br, OH), 3073, 3035, 2975, 2937, 1639, 1590, 1461, 1448,
1372, 1294, 1234, 1210, 1192, 1086, 999, 922, 912, 754 cm−1; 1H-NMR (CDCl3)
δ 7.20 (dd, J = 7.7, 1.5 Hz,
2H, H-6), 7.02 (d, J = 7.7 Hz,2H, H-4), 6.88 (t, J = 7.7 Hz, 2H, H-5), 6.43 (s, br, 2H, OH), 6.06 (ddt,
J = 16.8, 10.3, 6.2 Hz, 2H, CH2–CH=CH2), 5.18–5.24 (m, 4H, CH2–CH=CH2), 3.97 (s, 2H, Ar–CH2–Ar),
3.45 (d, J = 6.6 Hz, 4H, CH2–CH=CH2); 13C-NMR (CDCl3)
δ
151.4 (C-2), 136.6 (CH2–CH=CH2), 129.1
(C-6), 128.7 (C-4), 127.1 (C-1), 125.8 (C-3), 121.1 (C-5), 116.6 (CH2–CH=CH2), 35.4 (CH2–CH=CH2), 30.8

Molecules 2018, 23, 2536
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(Ar–CH2–Ar); EI+ calcd for C19H20O2: [M]+ 280.1463; found EI-MS m/z (rel. int.): 280.1469 [M]+ (58).
Compound B2 has been mentioned in several patents; however, chemical data were not given therein.
Compound B3: IR (KBr, νmax, cm−1): 3387 (OH), 3077, 2853, 1638, 1611, 1592, 1506, 1465, 1434,
1
1256, 1232, 1215, 1107, 996, 914, 759 cm−1. H-NMR (CDCl3)
δ
7.17 (dd, J = 7.8, 1.5 Hz, 1H, H-6), 7.06
(d, J = 2.2 Hz,1H, H-60), 6.98 (dd, J = 7.3, 1.5 Hz, 1H, H-4), 6.91 (dd, J = 8.4, 2.2 Hz, 1H, H-40), 6.84 (t,
J = 7.5 Hz, 1H, H-5), 6.74 (d, J = 8.0 Hz, 1H, H-30), 6.31 (s, vb, 2H, OH), 6.02 (ddt, J =010 6.8, 10.3, 6.2 Hz,
00
00
1H, 2 -H), 5.92 (ddt, J = 16.9, 10.3, 6.6 Hz, 1H, H-2 ), 5.19 (dq, J
≈
16, 1.7 Hz, 1H, H-3 ), 5.18 (dq, J ≈ 9
,
1.5 Hz, 1H, H-300), 5.03 (dq, J
≈
17, 1.8 Hz, 1H, H-3000), 5.04 (dq, J
≈
9, 1.3 Hz, 1H, H-3000), 3.89 (s, 2H,
151.3
Ar–CH2–Ar), 3.41 (d, J = 6.2 Hz, 2H, H-100), 3.28 (d, J = 7.0 Hz, 2H, H-1000); 13C-NMR (CDCl3)
δ
(C-2), 151.2 (C-20), 137.8 (C-2000), 136.5 (C-200), 132.8 (C-50), 130.8 (C-60), 129.1 (C-6), 128.8 (C-4), 128.0
(C-40), 127.1 (C-1), 126.6 (C-10), 125.6 (C-3), 121.2 (C-5), 116.9 (C-300), 116.0 (C-30), 115.4 (C-3000), 39.4
(C-1000), 35.6 (C-100), 30.8 (Ar–CH2–Ar); EI+ calcd for C19H20O2: [M]+ 280.1463; found EI-MS m/z (rel.
int.): 280.1464 [M]+ (100).
2,40-Diallyloxy-diphenylmethane (C1a)
C1 (3.77 g, 18.8 mmol) was allylated with allylbromide (8.97 g, 6.5 mL, 74.1 mmol) and K2CO3
(20.8 g, 0.15 mol) in acetone (100 mL) as described above yielding the diallyloxy-diphenylmethane
(4.12 g, 78%).
Compound C1a: IR (ATR, νmax, cm−1): 3077, 3022, 2915, 2860, 1610, 1599, 1584, 1508, 1490, 1451,
1
1237, 1221, 1020, 996, 921, 749 cm−1; H-NMR (CDCl3)
δ
7.22 (td, J
≈
8, 1.3 Hz, 1H, H-4), 7.21 (d,
J ≈ 8 Hz, 2H, H-20 and H-60), 7.14 (d, J = 7.7 Hz, 1H, H-6), 6.94 (t, J = 7.3 Hz, 1H, H-5), 6.90 (d, 1H,
J ≈ 8 Hz, H-3), 6.89 (d, 2H, J = 8.4 Hz, H-30 and H-50), 6.03–6.17 (m, 2H, H-200 and H-2000), 5.45 (dq,
J = 17.2, 1.5 Hz, 1H, H-3000), 5.44 (dq, J = 17.3, 1.5 Hz, 1H, H-300), 5.32 (dq, J = 10.5, 1.5 Hz, 1H, H-3000),
5.31 (dq, J = 10.5, 1.5 Hz, 1H, H-300), 4.58 (dt, J = 5.1, 1.5, 2H, H-100), 4.55 (dt, J = 5.1, 1.5, 2H, H-1000),
4.02 (s, 2H, Ar–CH2–Ar); 13C-NMR (CDCl3)
δ
156.7(C-40), 156.2 (C-2), 2
×
133.5 (2
×
CH=CH2), 133.3
(C-10*), 130.4 (C-1*), 130.2 (C-6), 129.8 (C-20 and C-60), 127.2 (C-C-4), 120.6 (C-5), 117.4 (C-3000), 116.9
(C-300), 114.5 (C-30 and C-50), 111.7 (C-3), 68.8 and 68.7 (2
×
O–CH2), 35.1 (Ar–CH2–Ar); EI+ calcd for
C19H20O2: [M]+ 280.1463; found EI-MS m/z (rel. int.): 280.1 [M]+ (45), 133.1 [C6H4–O–allyl]+ (100).
2-Allyl-4-(3-allyl-2-hydroxybenzyl)-phenol (C2) and 2-Allyl-4-(5-allyl-2-hydroxybenzyl)-phenol (C3
)
Claisen rearrangement of C1a (14.5 g, 51.7 mmol) in N,N-diethylaniline (115 mL) following the
procedure of Chattopadhyay et al. [21] resulted in C2 (10.6 g; 73%) and C3 (1.2 g; 8.3%)
Compound C2: IR (ATR, νmax, cm−1): 3507 (br, OH), 3076, 3009, 2977, 2906, 1637, 1609, 1591, 1501,
1461, 1432, 1328, 1255, 1182, 996, 911, 751 cm−1; 1H-NMR (0CDCl3)
δ
6.95–7.06 (m, 040H, H-4,H-6, H-20,
0
000
H-6 ), 6.86 (t, J = 7.5 Hz, 1H, H-5), 6.74 (d, J = 7.7 Hz, 1H, H-5 ), 5.94–6.09 (m, 2H, H-2 , H-2 ), 5.12–5.20
(m, 4H, H-300, H-3000), 4.99 (s, b, 2H, OH), 3.92 (s, 2H, Ar–CH2–Ar), 3.41 (d, J = 6.2 Hz, 2H, H-100), 3.38
000
0 00 000
δ 152.6 (C-4 ), 152.4 (C-2), 136.5 (C-2 ), 136.4 (C-2 ), 132.1
(d, J = 6.2 Hz, 2H, H-1 ); 13C-NMR (CDCl3)
(C-10), 130.7 (C-20), 129.1 (C-6), 128.6 (C-4), 128.0 (C-60), 127.6 (C-1), 125.6 (C-3, C-30), 120.6 (C-5), 116.4
(C-300, C-3000), 116.0 (C-50), 35.7 (Ar–CH2–Ar), 35.3 (C-100), 35.10 (C-1000); EI+ calcd for C19H20O2: [M]+
280.1463; found EI-MS m/z (rel. int.): 280.1465 [M]+ (100).
Compound C3: IR (ATR, νmax, cm−1): 3277 (br, OH), 3078, 3013, 2977, 2902, 1638, 1608, 1502, 1434,
1341, 1247, 1207, 1094, 992, 793, 634, 617 cm−1; 1H-NMR (CDCl3)
δ
6.90–7.04 (m, 4H, H-4, H-6, H-20,
H-60), 6.0608–6.78 (m 2H, H-3, H-50), 5.88–6.07 (m, 2H, H-200, H-2000), 5.10–5.20 (m, 2H, H-3000), 4.99–5.10 (m,
000
2H, H-3 ), 4.87 (s, 1H, OH), 4.57 (s, 1H, OH), 3.89 (s, 2H, Ar–CH2–Ar), 3.38 (d, J = 6.2 Hz, 2H, 1 -H), 3.30
(d, J = 6.6 Hz, 2H, H-10); 13C-NMR (CDCl3) 152.6 (C-40), 152.1 (C-2), 137.9 (C-200), 136.4 (C-2000), 132.2
δ
0
0
0
0
000
(C-5), 132.0 (C-1 ), 131.0 (C-6), 130.2 (C-2 ), 127.9 (C-6 ), 127.7 (C-4), 127.1 (C-1), 125.6 (C-3 ), 116.3 (C-3 ),
116.0, 115.8 (C-3, C-50), 115.3 (C-300), 39.3 (C-100), 35.7 (Ar–CH2–Ar), 35.1 (C-1000 EI+ calcd for C19H20O2:
[M]+ 280.1463; found EI-MS m/z (rel. int.): 280.1 [M]+ (100), 147.1 [CH2–C6H4–O–allyl]+ (100).
General Procedure for the Methylation of Dihydroxy-Diphenylmethanes

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The corresponding diol (1 eq) was dissolved in aqueous KOH (10%, 2.5 eq) and stirred for 10
min. Me2SO4 (1.2 eq) was added and the resulting mixture was stirred for 20 min at RT and for
◦
1 h at 95 C. After cooling to RT, the mixture was acidified with aqueous HCl (2M) and extracted
with chloroform (3
×
). The organic layers were dried over Na2SO4 and concentrated under reduced
pressure. The residue was chromatographed over silica with cyclohexane/AcOEt = 5:1.
Bis(3-allyl-2-methoxyphenyl)methane (B4a) and 6-allyl-2-(3-allyl-2-methoxybenzyl)-phenol (B4b
)
Methylation of 420 mg (1.5 mmol) B2 resulted in 39 mg of starting material, 40 mg B4a (8.6%) and
249 mg B4b (57%).
Compound B4a: IR (ATR, νmax, cm−1): 3001, 2978, 2941, 2827, 1463, 1427, 1291, 1164, 1080, 1009,
911, 766 cm−1; 1H-NMR (CDCl3)
7.09 (dd, 2H, J = 7.6 and 1.3 Hz, H-4), 7.00 (t, 2H, J = 7.5 Hz, H-5),
δ
6.93 (dd, 2H, J = 7.4 and 1.3 Hz, H-6), 5.97–6.09 (m, 2H, -CH=CH2), 5.06–5.21 (m, 4H, -CH=CH2), 4.10 (s,
2H, Ar–CH2–Ar), 3.72 (s, 6H, OCH3), 3.46 (d, 4H, J 6.6 Hz, CH2-CH=CH2); 13C-NMR (CDCl3) δ 154.4
(C-2), 137.3 (-CH=CH2), 134.0 (C-1), 132.9 (C-3), 128.9 (C-6), 128.5 (C-4), 124.1 (C-5), 115.8 (-CH=CH2),
34.0 (-CH2-CH=CH2), 29.6 (Ar–CH2–Ar). EI+ calcd for C21H24O2: [M]+ 308.1776; found EI-MS m/z
(rel. int.): 308.1776 [M]+ (100).
Compound B4b: IR (ATR, νmax, cm−1): 3320 (br, OH), 3001, 2978, 2975, 2944, 2908, 1641, 1632, 1591,
1
1462, 1446, 1430, 1220, 1086, 998, 910, 780, 604 cm−1; H-NMR (CDCl3)
δ
7.35 (s,01H, OH), 7.18 (dd,
0
J = 7.1, 1.8 Hz, 1H, H-6 ), 7.13 (d, J = 7.1 Hz, 1H, H-6), 7.08 (dd, J
≈
7,
≈
2 Hz, 1H, H-4 ), 7.06 (t, J = 7.5 Hz,
1H, H-50), 7.00 (d, J = 7.7 Hz, 1H, H-4), 6.81 (t, J = 7.5 Hz, 1H, H-5), 5.92–6.08 (m, 2H, H-20 and H-2 ),
000
000
00
000
5.03–5.15 (m, 4H, H-3 and H-3 ), 3.92 (2
×
s, 5H, Ar–CH2–Ar and OCH3), 3.47 (d, J = 6.3 Hz, 2H, H-1 ),
3.40 (d, J = 6.6 Hz, 2H, H-100); 13C-NMR (CDCl3) 154.2 (C-20), 151.2 (C-2), 137.0 (C-200), 136.8 (C-2000),
δ
2 × 133.0, (C-10 and C-30), 129.1 (C-40), 128.9 (C-60), 128.6 (C-4), 128.4 (C-6), 127.2 (C-3), 126.6 (C-1), 125.3
(C-5 ), 120.1 (C-5), 116.2 (C-3 ), 115.4 (C-3 ), 62.3 (OCH3), 34.7 (C-1 ), 33.8 (C-1 ), 31.4 (Ar–CH2–Ar); EI+
0
000
00
00
000
calcd for C21H24O2: [M]+ 294.1620; found EI-MS m/z (rel. int.): 294.1619 [M]+ (100).
3,30-Diallyl-2,40-dimethoxy-diphenyl-methan (C4a), 6-allyl-2-(3-allyl-4-methoxybenzyl)-phenol (C4b
and 2-allyl-4-(3-allyl-2-methoxybenzyl)-phenol (C4c)
)
Methylation of 2.10 g (7.5 mmol) C2 resulted in 143 mg of starting material, 318 mg C4a (14%),
665 mg C4b (30%) and 442 mg C4c (20%).
Compound C4a: IR (ATR, νmax, cm−1): 3001, 2977, 2939, 2833, 1638, 1500, 1463, 1429, 1248, 1125,
1080, 1033, 1010, 910, 768 cm−1; 1H-NMR (CDCl3)
δ 7.07 (dd, J = 7.3, 1.5 Hz, 1H, H-4), 6.98–7.02 (m, 3H,
H-20, H-5 and H-60), 6.96 (dd, J = 7.6, 1.3 Hz, 1H, H-6), 6.78 (d, J = 8.4 Hz, 1H, H-50), 5.92–6.07 (m, 2H,
H-200 and H-2000), 5.06–5.13 (m, 2H, H-300), 4.99–5.06 (m, 2H, H-3000), 3.96 (s, 2H, Ar–CH2–Ar), 3.80 (s,
3H, C40-OCH3), 3.68 (s, 3H, C2-OCH3), 3.46 (d, J = 6.3 Hz, 2H, H-100), 3.36 (d, J = 6.6 Hz, 2H, H-1000);
13C-NMR (CDCl3) 156.3 (C-2), 155.6 (C-20), 137.3 (C-200), 137.1 (C-2000), 134.7 (C-1), 133.0 (C-3), 132.8
δ
(C-10), 130.2 (C-20), 129.0 (C-6), 128.5 (C-4), 128.4 (C-30), 127.5 (C-60), 120.5 (C-5), 115.8 (C-300), 115.3
(C-3000), 110.3 (C-50), 61.2 (C2-OCH3), 55.4 (C40-OCH3), 34.9 (Ar–CH2–Ar), 34.3 (C-1000), 34.0 (C-100); MS
(ESI+) m/z (%): calculated for C21H24O2: [M]+ 308.178; found: EI-MS m/z (rel. int.): 308.4 [M]+ (100).
D4b: IR (ATR, νmax, cm−1): 3526 (br, OH), 3003, 2976, 2905, 2834, 1637, 1608, 1592, 1499, 1461,
1247, 1182, 1032, 996, 910, 752 cm−1; 1H-NMR (CDCl3) 6.97–7.02 (m, 4H, H-4, H-6, H-20 and H-60),
δ
6.82 (t, J = 7.5 Hz, 1H, H-5), 6.76 (d, J = 8.8 Hz, 1H, H-50), 5.90–6.05 (m, 2H, H-200and H-2000), 5.11–5.17
(m, 2H, H-300), 4.98–5.05 (m, 2H, H-3000), 4.94 (s, 1H, OH), 3.91 (s, 2H, Ar–CH2–Ar), 3.78 (s, 3H, OCH3),
3.38 (d, J = 6.2 Hz, 2H, H-100), 3.34 (d, J = 6.5 Hz, 2H, H-1000); 13C-NMR (CDCl3)
δ
152.9 (C-40), 152.4
(C-2), 136.9 (C-2000), 136.5 (C-200), 131.4 (C-10), 130.2 (C-20), 129.1 (C-60), 128.8 (C-30), 128. 6 (C-4), 127.6
(C-1), 127.2 (C-6), 125.5 (C-3), 120.5 (C-5), 116.4 (C-300), 115.3 (C-3000), 55.4 (OCH3), 35.8 (Ar–CH2–Ar),
35.3 (C-100), 34.3 (C-1000); MS (EI) m/z (%): calculated for C20H22O2: [M]+ 294.162; found: EI-MS m/z
(rel. int.): 294.4 [M]+ (80), 253.2 (65).
D4c: IR (ATR, νmax, cm−1): 3393 (br, OH), 3005, 2977, 2940, 2911, 2830, 1638, 1610, 1591, 1505, 1464,
1428, 1251, 1198, 1080, 996, 910, 768 cm−1; 1H-NMR (CDCl3)
δ
7.09 (dd, J = 7.3, 1.7 Hz, 1H, H-4), 7.01 (t,
J = 7.3 Hz, 1H, H-5), 6.93–6.99 (m, 3H, H-20, H-6 and H-60), 6.73 (d, J = 7.7 Hz, 1H, H-50), 5.94–6.08 (m,

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2H, H-200 and H-2000), 5.06–5.20 (m, 4H, H-300 and H-3000), 4.95 (s, 1H, OH), 3.96 (s, 2H, Ar–CH2–Ar),
3.68 (s, 3H, OCH3), 3.46 (d, J = 6.6 Hz, 2H, H-100), 3.38 (d, J = 6.5 Hz, 2H, H-1000), 13C-NMR (CDCl3)
δ
156.3 (C-2), 152.3 (C-40), 137.3 (C-200), 136.5 (C-2000), 134.6 (C-1), 133.3 (C-10), 133.0 (C-3), 130.1 (C-20),
129.0 (C-6), 128.6 (C-4), 128.2 (C-60), 125.1 (C-30), 124.1 (C-5), 116.3 (C-300), 115.8 (C-3000), 115.7 (C-50),
61.2 (OCH3), 35.2 (C-1000), 34.9 (Ar–CH2–Ar), 33.9 (C-100); MS (EI) m/z (%): calculated for C20H22O2:
[M]+ 294.162; found: EI-MS m/z (rel. int.): 294.4 [M]+ (75), 253.2 (40).
3.2. Biological Assays
Cell line: All screening was done in a line of human embryonic kidney cells stably expressing
the human norepinephrine transporter and the vesicular monoamine transporter 2 (HEK-hNET-
-hVMAT2-mCherry). Human VMAT2 expression, in addition to hNET, more fully recapitulates a
neuronal phenotype in cell culture. Cells were imaged and fully characterized for functionality
(Figure 1). For imaging, cells were fixed in 4% paraformaldehyde and protein expression of
NET determined by immunolabelling (primary antibody: Novus, anti-norepinephrine transporter,
1:1000; secondary antibody: Thermo Fisher, Alexafluor goat anti-rabbit-488, 1:5000) followed by
confocal imaging (Nikon A1R, Nikon Instruments, Melville, NY, USA). Screening: Synthesized
compounds were tested for interaction with NET via a three-step process. First, all compounds
were tested using a commercially available fluorescent assay (neurotransmitter transport uptake kit,
Molecular Devices, San Jose, CA, USA) for the assessment of plasmalemmal monoamine transport
ability [23,24]. Cells were plated in poly-D-lysine (PDL)-coated 96-well plates (Corning Inc., Corning,
NY, USA) to 100% confluence. The following day, cell media (DMEM, 10% fetal bovine serum,
1% penicillin/streptomycin) was removed, and experimental media (Hank’s balanced salt solution,
Sigma-Aldrich) was added. Following a 30-min incubation at 37 ◦C, dye was added to all wells, with
or without compounds (3 nM–100
(3 nM–10 M final concentration, control). Plates were incubated for 1 h, then read on a fluorescent plate
reader (Thermo Fisher). Experiments were conducted in triplicate. Second, the most potent modulators
of transport activity (C4a H8 H11 H16 H20 and M6) were further assessed for radiolabeled dopamine
uptake in the same cell line, as dopamine is a substrate of NET [25 26]. Cells were plated to confluence
µM final concentration) or selective NET inhibitor desipramine
µ
,
,
,
,
,
in 48-well PDL-coated plates the day prior to the experiment. For experiments, media was removed,
cells were rinsed with phosphate buffered saline (PBS), and incubated with or without compounds or
desipramine (10 nM to 10
ten minutes at 37 C. Dopamine (1
µ
M, non-specific uptake measured by addition of 100
µM desipramine) for
◦
µ
M total concentration with 200 nM 3H-dopamine tracer) was added
and uptake proceeded for 10 min at 37 ◦C. Cells were then rinsed three times with PBS, lysed with
0.1 N NaOH, and measured via liquid scintillation counting (Beckman Coulter, Brea, CA, USA). Finally,
each selected compound was visualized with high-content imaging using the Array Scan VTI HCS
(Cellomics). Cells were seeded to 80% confluence in PDL-coated, glass-bottom 96-well plates. Media
was removed and DMEM without phenol red with or without compounds or desipramine (10 nM
to 10
fields per well were taken and total fluorescence in the green channel was reported. No compound
demonstrated potent activity at NET (IC50 > 100 M), as compared with the selective NET inhibitor,
desipramine, which inhibited fluorescent (plate reader: IC50 = 125.3 nM; imaging: IC50 = 65.7 nM
µM) was added. Following a one-hour incubation, cells were imaged. Forty-nine contiguous
µ
,
Figures 1.1 and 3) and radioactive uptake (IC50 = 52.1 nM, Figure 1.2). Dose-Response curves (measured
as fluorescence) for tested compounds can be found in “Supplementary Materials”.
4. Conclusions
Altogether, we tested a series of 52 biphenyl neolignans and derivatives (including
diphenyl-methane analogs) found in biological preparations that are used to treat anxiety and insomnia
in Eastern medicine, for pharmacological efficacy at the NET. No tested compound potently inhibited
uptake via NET, suggesting acutely that these compounds may work exclusively through GABAA
receptor modulation. It is also possible that these compounds still modulate the noradrenergic system;

Molecules 2018, 23, 2536
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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.
Supplementary Materials: Proton NMR spectra of the new compounds and dose-response curves of the tested
compounds are available in “Supplementary Materials”.
Author Contributions: W.S. concept, funding acquisition, HPLC separation, GC-MS measurements and
purity control of compounds, analyzing data, writing the manuscript. K.S. and G.W.M. design and carryout
of pharmacological investigation, data analysis, writing and correcting the manuscript. M.B. design and
performing of the syntheses. A.H. Design of chemical syntheses, performing synthetic research, performing NMR
spectroscopic experiments and data analysis, writing of the manuscript.
Funding: Parts of this work were supported by the Austrian Science Fund (FWF) under a grant “Synthesis of new
GABAA receptor modulators” (P21241).
Acknowledgments: We are indebted to Andreas Leitner for recording IR spectra. We thank R. Bauer and
F. Bucar (Institute of Pharmaceutical Sciences Graz) for the use of preparative HPLC, ESI-MS and GC-MS
facil-ities, respectively.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from WS and AH. The availability can only be
guaranteed for compounds that remain stable under storage conditions.
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