Biomimetic synthesis of galantamine: Via laccase/TEMPO mediated oxidative coupling

Article abstract of DOI:10.1039/d0ra00935k

Laccase-mediated intramolecular oxidative radical coupling of N-formyl-2-bromo-O-methylnorbelladine afforded a novel and isolable spirocyclohexadienonic intermediate of galantamine. High yield and conversion of substrate were obtained in the presence of the redox mediator 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). This laccase procedure, with an overall yield of 34%, represents a scalable and environmentally friendly alternative to previously reported syntheses of galantamine based on the use of potassium ferricyanide as an unspecific radical coupling reagent.

Full text of DOI:10.1039/d0ra00935k

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Biomimetic synthesis of galantamine via laccase/
TEMPO mediated oxidative coupling†
Cite this: RSC Adv., 2020, 10, 10897
a
a
a
b
Claudio Zippilli, Lorenzo Botta, Bruno Mattia Bizzarri, Maria Camilla Baratto,
b
a
Rebecca Pogni and Raffaele Saladino
*
Laccase-mediated intramolecular oxidative radical coupling of N-formyl-2-bromo-O-methylnorbelladine
afforded a novel and isolable spirocyclohexadienonic intermediate of galantamine. High yield and
conversion of substrate were obtained in the presence of the redox mediator 2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPO). This laccase procedure, with an overall yield of 34%, represents
a scalable and environmentally friendly alternative to previously reported syntheses of galantamine based
on the use of potassium ferricyanide as an unspecific radical coupling reagent.
Received 31st January 2020
Accepted 5th March 2020
DOI: 10.1039/d0ra00935k
rsc.li/rsc-advances
the cyclization of nitrogen protected N-formyl-2-bromo-O-
methylnorbelladine 4 to yield N-formyl-1-bromo-narwedine 6
Introduction
The oxidative radical coupling is a reaction of pivotal relevance (54% yield) under similar experimental conditions (pathway A,
5
,6,9
in the biosynthesis of polycyclic natural substances, including Fig. 1).
Laccase (EC 1.10.3.2) is the simplest multi-copper
0
the synthesis of bioactive aromatic alkaloids narwedine 2 and oxidoreductase in nature (E ¼ 0.5 to 0.8 V vs. normal
1
,2
galantamine 3 from O-methylnorbelladine 1 (Fig. 1). Gal- hydrogen electrode NHE) able to catalyze the oxidation of
antamine, isolated from the Caucasian snow-drop and from phenols and other organic substrates with concomitant
1
0–12
the bulbs of different species of Amarillidaceae family, is the reduction of molecular dioxygen (O
2 2
) to water (H O).
active principle contained in Remynil® and Razadyne®, two Several examples of the use of laccase in the oxidative radical
1
3
drugs that act as allosteric ligands (APL) of human nicotinic coupling are reported. The industrial interest in this enzyme
3
–5
acetylcholine receptors. In particular, compound 3 controls is related to the high value of its catalytic constant, large
the neuronal release of different neurotransmitters such as substrate promiscuity, and high thermal resistance, associ-
1
4–16
glutamate, g-aminobutyric acid (GABA) and bioactive mono- ated to the use of air as primary oxidant.
In order to
3
amines in the therapy of the Alzheimer's disease. The develop a novel and environmentally friendly synthesis of
biosynthetic pathway of galantamine proceeds by regiose- galantamine 3 as alternative to the K
3
Fe(CN)
6
process, we
0
lective para–ortho (p–o ) intramolecular radical coupling and report here that laccase (Trametes versicolor) efficiently cata-
0
phenolic addition in compound 1 to yield 2, followed by lyzes the one-pot para–ortho (p–o ) oxidative radical coupling
4
reduction and alkylation. At the industrial scale, the intra- of 4 to a novel and isolable spirocyclohexadienonic interme-
molecular radical coupling is realized in very low yield (1.4%) diate of narwedine 6 and galantamine 3, namely 9-bromo-7-
0
by use of potassium ferricyanide K
3
Fe(CN)
6
, due to the methoxy-4 ,6-dioxo-3,4,5a,6-tetrahydrospiro-[benzo[c]azepine-
0
0
0
occurrence of undesired side-reactions involving less hindered 5,1 -cyclohexane]-2 ,5 -diene-2(1H)-carbaldehyde 5 (pathway B,
6
aromatic positions of the substrate (pathway A, Fig. 1).
Fig. 1). The effect of low molecular weight redox-mediators
K
3
Fe(CN)
6
is characterized by acute toxicity, skin corrosion, (laccase mediator system, LMS), including 2,2,6,6-
0
and serious eye and respiratory tract irritation, as well as by tetramethylpiperidine-1-oxyl
(TEMPO),
2,2 -azino-bis(3-
7
unfavorable environmental impact. The role of K Fe(CN) in ethylbenzothiazoline-6-sulfonic
acid)diammonium
salt
3
6
the cyclization of compound 1 has not been reported in detail, (ABTS) and 1-hydroxybenzotriazole (HOBt), in the selectivity
even if the formation of phenoxyl radicals by iron-mediated and yield of the cyclization was evaluated. These compounds
one-electron abstraction process is expected as operative can enhance the efficacy of the radical process by transferring
8
process. Synthetic alternative to the use of K
3
Fe(CN)
6
involves the oxidative redox potential from the active site of the enzyme
1
7–22
to the bulk of the solution.
The laccase/TEMPO mediated
a
intramolecular radical coupling of 4 to 5, followed by simple
Department of Ecological and Biological Sciences, University of Tuscia, Via S. C. De
alkaline catalyzed intramolecular enolic cycloaddition and
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo canonical reduction procedure afforded galantamine 3 in
Lellis 44, Viterbo, 01100, Italy. E-mail: saladino@unitus.it
b
Moro 2, 53100 Siena, Italy
a total yield higher than that previously reported for K
under optimal experimental conditions.
3 6
Fe(CN)
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0ra00935k
1
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 10897–10903 | 10897

View Article Online
RSC Advances
Paper
compounds I and IIa were probably obtained by the undesired
laccase mediated oxidation of the benzylic position in 4 fol-
lowed by elimination of water molecule and hydrolysis of the
23
corresponding enamonium ion intermediate.
Compound 5 was characterized by the presence of a tricyclic
condensed system, containing the azepine ring (B) and two
hexadienonic rings (A and C, respectively), as a mixture (2 : 1
ratio) of two rotamers do to the steric hindrance of the N-formyl
9
protecting group. The structure of 5 was unambiguously
1
13
assigned by different 1D-NMR ( H-NMR, C-NMR and dis-
tortionless enhancement by polarization transfer analysis
1
1
DEPT-135), and 2D-NMR (homonuclear H– H correlated
1
1
spectroscopy COSY, homonuclear H– H overhauser spectros-
1
copy NOESY and H–DEPT-135 heteronuclear single-quantum
coherence HSQC) analyses (for detailed 1D and 2D NMR
1
13
Fig. 1 Synthesis of galantamine 3. Pathway A: synthesis of galantamine spectra see ESI, S2–S25;† representative H-NMR, C-NMR
1
1
1
1
3
from O-methylnorbelladine 1 or N-formyl-2-bromo-O-methyl- HSQC, DEPT-135, H– H COSY and H– H NOESY data for
norbelladine 4 by use of K
synthesis of galantamine
3
Fe(CN)
6
. Pathway B: laccase mediated
compound 5 are reported in ESI, Table S1†). The prevalence of
the keto-tautomer in ring A (with respect to the possible
phenolic alternative) was conrmed by the couple of signals at
.41 ppm and 6.44 ppm (double singlet) in the H-NMR analysis
3 from N-formyl-2-bromo-O-methyl-
norbelladine 4. LMS: laccase mediator system. DBU: 1,8-diazabicyclo
5.4.0]undec-7-ene.
[
1
6
(unchangeable aer D
2
O treatment), corresponding to H-23
proton (Fig. 2), and by the signals at 186 ppm and 181 ppm in
1
3
the C-NMR analysis, corresponding to C-6 and C-10 quater-
nary carbons, respectively (in accordance with the proposed
structure these signals disappeared in the DEPT-135 experi-
ment). The COSY analysis clearly showed the interaction
between H-23 and H-35, while the para–ortho regioselectivity in
the azepine ring B was further conrmed by the NOESY corre-
lation between H-32 and both H-23 and H-35, as well as by the
short-range heteronuclear correlation (HSQC) involving these
hydrogen atoms and the respective connected carbons. In order
Scheme
1 Simplified synthesis of N-formyl-2-bromo-O-methyl-
norbelladine 4 from 2-bromo-5-hydroxy-4-methoxybenzaldehyde I to increase the yield of compound 5, the oxidative radical
and tyramine II; (a) NaBH
4
, MeOH, 3 h, 95%; (b) HCOOC
2
H
5
, DMF, coupling was repeated in the presence of three laccase redox
mediators characterized by different reaction mechanisms,
ꢀ
8
0 C, 24 h, 92%.
0
0
0
namely ABTS (E ¼ 0.5 V), TEMPO (E ¼ 0.75 V), and HOBt (E ¼
1.13 V). The electron-transfer (ET) process is operative in the
case of ABTS, while oxoammonium ion mediated one-electron
transfer mechanism, and radical H-abstraction (HAT), are
Results and discussion
N-Formyl-2-bromo-O-methylnorbelladine 4 was prepared as effective processes in the case of TEMPO and HOBt,
2
4–28
described in Scheme 1. This synthetic pathway simplies the respectively.
conventional procedure. Briey, commercially available 2-
9
As a general procedure, compound 4 (380 mg, 1.0 mmol)
bromo-5-hydroxy-4-methoxybenzaldehyde and 4-hydroxy- dissolved in 1,4-dioxane (5 mL) and sodium acetate buffer (20
I
phenylethylamine (tyramine) II were coupled with NaBH
MeOH at 25 C to yield III in 95% yield. Compound III was mmol or 1000 U mmol ) in the presence of the appropriate
4
in mL, 0.5 M, pH 4.5) mixture was treated with laccase (100 U
ꢀ
ꢁ1
ꢁ1
successively reacted with ethyl formate (HCOOC
2
H
5
) in DMF at redox mediator (from 0.3 to 0.6 equivalents with respect to
ꢀ
ꢀ
80 C to afford desired 4 in 92% yield. The oxidative radical substrate) under gentle stirring at 25 C for 3.0 h in air. Irre-
coupling was performed by addition of laccase from Trametes spective from the experimental conditions, laccase (100 U
ꢁ
1
ꢁ1
versicolor (1000 U mmol ) to compound 4 (380 mg, 1.0 mmol) mmol ) in the presence of ABTS or TEMPO (0.3 equivalents)
in 1,4-dioxane (5 mL) and sodium acetate buffer (20 mL; 0.5 M; afforded 5 in a yield similar to that previously obtained with
ꢀ
ꢁ1
pH 4.5) mixture under gentle stirring at 25 C for 3.0 h in air. 1000 U mmol of laccase alone (Table 1, entries 2 and 3,
,4-Dioxane was used to circumvent the problem of the low respectively, versus entry 1). Conversely, degradative and over-
1
solubility of 4 in the buffer. Under these experimental condi- oxidation side-processes prevailed when the reaction was per-
tions, the spirocyclohexadienonic derivative 5 was isolated in formed in the presence of HOBt (Table 1, entry 4). Since the
low yield (18%), besides higher amounts of the compound I, N- work-up of the laccase/ABTS cyclization produced an emulsion
formyl-tyramine IIa, and unreacted substrate (Scheme 2; Table of difficult treatment, the oxidation was further optimized with
1, entry 1). In accordance with data previously reported, TEMPO in a larger amount (0.6 equivalents) as selected redox
10898 | RSC Adv., 2020, 10, 10897–10903
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
EPR signal of TEMPO decreased rapidly in the absence of
compound 4, as a consequence of the formation of the corre-
sponding oxoammonium ion (Fig. 3, Panel A). On the other
hand, the EPR signal decreased slowly in the presence of
compound 4 due to the expected regeneration of the native
radical species by laccase (Fig. 3, Panel B). In Table 2 are re-
ported the quantitative EPR data recorded for the cyclization at
different reaction times and at the two indicated concentration
values of TEMPO, both in the absence (Table 2 entries 1 and 3)
and in the presence of compound 4 (Table 2, entries 2 and 4).
The role of the nitrogen-protecting group in the efficacy of
the reaction was then evaluated by analyzing the oxidation of
unprotected 2-bromo-O-methylnorbelladine III under optimal
experimental conditions (Scheme 2). In this latter case, the
corresponding tricyclic intermediate 5a was isolated in very low
yield besides the compound I and tyramine II (Table 1, entry 9),
conrming the benecial role of the formyl protecting group in
Scheme 2 Biomimetic laccase-mediated synthesis of galantamine 3
23
starting
Compounds I, II and IIa are probably due to the undesired laccase same procedure applied to N-methyl-2-bromo-O-methyl-
catalyzed benzylic oxidation of 4; (a) laccase Tv, mediator, O , 1,4-
, 91%; (c) L-selectride,
from
N-formyl-2-bromo-O-methylnorbelladine
4. avoiding undesired oxidative side-processes. Moreover, the
2
norbelladine 7, bearing an alkyl electron donating group
instead of the electron withdrawing formyl moiety, afforded the
isoindoline derivative 10 as the only recovered product in 76%
yield besides the unreacted substrate (Scheme 3). In line with
data previously reported for the oxidation of aromatic benzyl-
amines with inorganic copper salts, compound 10 was probably
obtained by formation of the corresponding quaternary amine
N-oxide 8 (due to the activation of the nitrogen atom by the
methyl substituent), followed by elimination of water to imi-
nium ion 9 and successive phenolic intramolecular addition
dioxane/sodium acetate buffer; (b) DBU, CH
2
Cl
2
ꢀ
4
THF, ꢁ78 C; (d) LiAlH , THF, r.t., 61%.
mediator. In this latter case, compound 5 was obtained in
appreciable yield (38%) (Table 1, entry 5). The increase of the
ꢀ
reaction temperature (50 C), as well as the use of acetonitrile
(
CH CN) as an alternative solvent did not improve the yield of 5
3
(
Table 1, entries 6 and 7, respectively). Better results were nally
ꢁ1
obtained with 1000 U mmol of laccase and 0.6 equivalents of
29
(
Scheme 3). The activity of isoindoline derivatives as acetyl-
TEMPO, in which case compound 5 was isolated in 70% yield
choline esterase (AChE) inhibitors against Alzheimer's disease
(Table 1, entry 8). The EPR spectra conrmed the occurrence of
30
has beenreported. Galantamine 3 was nally obtained from 5
a catalytic cycle during the oxidation of 4 with laccase and
TEMPO (Fig. 3) (for detailed EPR spectra see ESI, S26 and S27†).
Measurements were performed in situ by treatment of
compound 4 (0.3 mmol) with laccase (0.14 mM) using two
concentrations of TEMPO (0.6 mM and 6 mM) in 1,4-dioxane/
buffer medium (1.2 mL, 1,4-dioxane : buffer 1 : 4 ratio) The
by a two steps procedure (Scheme 2), including the synthesis of
N-formyl-1-bromo-narwedine
6
by treatment with 1,8-
diazabicyclo[5.4.0]undec-7-ene DBU (91%), followed by
conventional reduction with both L-selectride and lithium
7,31,32
aluminum hydride (61%).
Table 1 Laccase-mediated oxidative radical coupling of N-formyl-2-bromo-O-methylnorbelladine 4 and 2-bromo-O-methylnorbelladine III to
a
spirocylohexadienonic derivatives 5 and 5a
Laccase (U
Conversion
(%)
ꢁ
1
Entry
Substrate
mmol
)
Mediator (eq.)
Product(s)
Yield (%)
1
2
3
4
5
6
7
8
9
4
4
4
4
4
4
4
4
1000
100
100
100
100
100
100
1000
1000
—
ABTS
TEMPO
HOBt
80
76
81
73
83
71
68
>98
80
5(I)[IIa]
5(I)[IIa]
5(I)[IIa]
(I)[IIa]
5(I)[IIa]
5(I)[IIa]
5(I)[IIa]
5(I)[IIa]
5a(I)[II]
18(31)[27]
19(27)[27]
21(30)[28]
(27)[27]
38(20)[19]
30(22)[18]
25(23)[16]
70(16)[13]
8(37)[33]
b
b
b
c
TEMPO
d
TEMPOe
TEMPOc
TEMPO
c
III
TEMPO
a
The reaction was performed treating the substrate in 1,4-dioxane and sodium acetate buffer with laccase in the presence or absence of the redox
ꢀ
b
mediator under gentle stirring at 25 C for 3.0 h in an air atmosphere. The experiment was conducted in triplicate. 0.3 equivalents of mediator with
c
d
ꢀ
respect to substrate. 0.6 equivalents of mediator with respect to substrate. The reaction was performed with 0.6 equivalents of mediator at 50 C.
e
ꢀ
The reaction was performed with 0.6 equivalents of mediator in acetonitrile at 25 C.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 10897–10903 | 10899

View Article Online
RSC Advances
Paper
N-formyl-1-bromo-narwedine 6 was obtained in a total yield
(
65%) higher than that previously reported for the K Fe(CN)
3
6
6
procedure (54%). Moreover, the total yield for galantamine 3
was 34%. Further studies are ongoing to evaluate the use of
supported laccase in the synthesis of compound 5, as well as,
the laccase mediated synthesis of novel bioactive isoindoline
derivatives starting from N-alkyl-narwedine derivatives.
Experimental
Materials, methods and experimental section
Reagents and laccase from Trametes versicolor were obtained
from commercial suppliers (Sigma-Aldrich Srl, Milan, Italy).
Chemical reactions were monitored using thin layer chroma-
tography on precoated aluminium silica gel Merck 60 F254
plates and an UV lamp was used for visualization. Merck silica
gel 60 (230–400 mesh) was used for ash chromatography. All
Fig. 2 3D structure of compound 5 representing COSY (black arrows)
and NOESY (red arrows) interactions.
1
13
products were dried in high vacuum (10–3 mbar). H NMR,
C
NMR, DEPT-135, COSY, NOESY and HSQC spectra were recor-
ded on a Bruker Avance DRX400 (400 MHz/100 MHz) spec-
trometer. Chemical shis for protons are reported in parts per
million (d scale) and internally referenced to the CD
3
OD, CDCl3,
DMSO-d or (CD O, signal at d 3.33 ppm, 7.28 ppm, 2.50 ppm
6
3 2
)
and 3.31 ppm respectively. Coupling constants (J) are reported
in Hz. Multiplicities are reported in the conventional form: s ¼
singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, br ¼
broad. Mass spectra (MS) data were obtained using an Agilent
ꢁ1
1
100 LC/MSD VL system (G1946C) with a 0.4 mL min ow rate
using a binary solvent system of 95 : 5/methyl alcohol : water.
Enzyme activity assay
Fig. 3 Redox cycle of the transformation of 4 into 5 performed by
0
The enzyme activity was assayed by using 2,2 -azino-bis(3-
laccase and mediated by TEMPO. EPR signal of TEMPO in absence
(
Panel A) and in the presence (Panel B) of the substrate (entry 2 and 4 in ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS)
Table 2).
procedure. ABTS (5.0 mM), sodium acetate buffer (2.0 mL, pH
5
.0), and the enzyme solution (200 mL) were used as standard
solution. The formation of the cation radical was detected by
measuring the increase of absorbance at 420 nm (3420
¼
Conclusions
ꢁ1
ꢁ1
3
6 000 M cm ). One unit of laccase activity has been dened
In conclusion, a novel and biomimetic synthesis of galantamine as the amount of enzyme that catalyzed the oxidation of 1.0 mmol
ꢀ
was developed under environmentally friendly conditions by of ABTS in 200 mL reaction mixture at 25 C during 1.0 min.
laccase/TEMPO mediated oxidative radical coupling of N-
formyl-2-bromo-O-methylnorbelladine
4
to spirocyclohex- EPR analysis
adienonic derivative 5. In the optimal experimental conditions
CW (Continuous wave) X-band (9 GHz) EPR spectra were
recorded at room temperature with a Bruker E580 Elexsys Series
ꢁ
1
(1000 U mmol of laccase and 0.6 equivalents of TEMPO) the
3
a
Table 2 EPR analysis and spin density quantitation (spins per mm ) of TEMPO during the oxidation of compound 4 with laccase
Entry
t ¼ 0 min
t ¼ 30 min
t ¼ 60 min
t ¼ 120 min
t ¼ 180 min
t ¼ 240 min
b
14
14
14
14
14
14
1
1.5 ꢂ 10
1 ꢂ 10
0.7 ꢂ 10
0.7 ꢂ 10
0.3 ꢂ 10
0.8 ꢂ 10
c
14
14
14
14
14
14
2
3 ꢂ 10
2.3 ꢂ 10
2 ꢂ 10
1.4 ꢂ 10
1.3 ꢂ 10
1 ꢂ 10
d
15
14
14
14
14
14
3
8 ꢂ 10
4 ꢂ 10
2 ꢂ 10
0.9 ꢂ 10
1.3 ꢂ 10
1.2 ꢂ 10
e
15
15
15
15
15
15
4
1.6 ꢂ 10
1.5 ꢂ 10
1.2 ꢂ 10
0.9 ꢂ 10
0.9 ꢂ 10
0.6 ꢂ 10
a
b
c
Error value ꢃ 0.1. Spin quantitation in the presence of TEMPO (0.6 mM) and laccase (0.14 mM). Spin quantitation in the presence of TEMPO
d
e
(
0.6 mM), laccase (0.14 mM) and compound 4 (0.25 mM). Spin quantitation in the presence of TEMPO (6 mM) and laccase (0.14 mM). Spin
quantitation in the presence of TEMPO (6 mM), laccase (0.14 mM) and compound 4 (0.25 mM).
10900 | RSC Adv., 2020, 10, 10897–10903
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
7
4
.06–7.02 (m, 4H, 4 ꢂ ArH), 6.86, 6.85 (s, 1H, ArH), 6.79–6.75 (m,
H, 4 ꢂ ArH), 4.56, 4.41 (s, 2H; ArCH N), 3.88, 3.87 (s, 3H,
2
OCH ), 3.44 (t, 2H, J ¼ 14.4, 7.2 Hz, NCH CH Ar), 3.36–3.32 (m,
3
2
2
2
2
H, NCH CH Ar), 2.78 (t, 2H, J ¼ 14.4, 7.2 Hz, NCH CH Ar),
2
2
2
2
13
2 2 3 2
.68–2.64 (m, 2H, NCH CH Ar) ppm. C-NMR ((CD ) CO, 100
MHz): showed the presence of two rotamers. d ¼ 32.4, 34.0, 43.7,
4
1
1
1
4
4.1, 48.4, 50.5, 55.7, 55.8, 111.4, 111.8, 115.2, 115.3, 115.5,
15.9, 116.1, 116.7, 128.1, 128.7, 129.1, 129.6, 129.7, 129.8,
46.5, 146.6, 147.8, 148.1, 155.8, 156.0, 162.6, 162.7 ppm. DEPT-
35 ((CD ) CO, 100 MHz) d ¼ 32.3 (CH ), 34.0 (CH ), 43.7 (CH ),
Scheme 3 Proposed mechanism for the laccase-mediated synthesis
of isoindoline 10 starting from N-methyl-2-bromo-O-methyl-
norbelladine 7.
3
2
2
2
2
4.0 (CH ), 48.4 (CH ), 50.7 (CH ), 55.7 (CH ), 115.2 (CH), 115.3
2
2
2
3
(CH), 115.4 (CH), 115.9 (CH), 116.1 (CH), 116.7 (CH), 129.6 (CH),
1
29.8 (CH), 162.6 (CH), 162.7 (CH) ppm. MS (ESI): m/z for
ꢁ
[C
17
H
18BrNO
4
] : 378.02.
using the Bruker ER4122SHQE cavity. The 1 mm diameter
quartz capillaries were lled in with the samples and analyzed
with the EPR spectrometer. The spin quantitation was carried Procedure for the synthesis of compound 5
out against an internal reference (Bruker) of irradiated solid
Laccase (1000 U) was added to a stirred solution of compound 4
1 mmol, 1 eq.) in 1,4-dioxane (5 mL) and acetate buffer, pH 4.5,
.5 M (20 mL). TEMPO (0.6 mmol, 0.6 eq.) was added imme-
diately. The solution was stirred for 3 h under O atmosphere.
Aer this period the aqueous solution was extracted with ethyl
acetate (3 ꢂ 50 mL). The organic layer was washed with brine,
dried over sodium sulfate and evaporated under reduced pres-
sure. The residue was puried by silica gel gradient column
2
alanine (3 mm length, 5 mm diameter) sealed under N atmo-
(
0
ꢁ
7
sphere, and containing a total of 2.05 ꢂ 10 ꢃ 10% spins,
using the SpinCounting program provided in the Xepr soware
2
(Bruker). Experimental conditions for spectra acquisition were:
n ¼ 9.86 GHz, modulation 0.1 mT, microwave power 2 mW.
Procedure for the synthesis of compound III
Tyramine (II) (0.45 mmol, 1.05 eq.) was added to a solution of 2- chromatography (CHCl
/MeOH 20 : 1) to afford 5 (yield ¼ 70%).
, 400 MHz): showed the presence of two
in MeOH (2 mL) and the mixture was stirred for 2 h at room rotamers. d ¼ 8.18, 7.90 (s, 1H, CHO), 7.05, 6.98 (d, 4H, J ¼
temperature. Aer this period, NaBH
3
1
bromo-5-hydroxy-4-methoxybenzaldehyde (I) (0.43 mmol, 1 eq.)
H-NMR (CDCl
3
(0.47 mmol, 1.1 eq.) was 7.6 Hz, 4 ꢂ CH]CHC]O), 6.80, 6.74 (d, 4H, J ¼ 8 Hz, 4 ꢂ CH]
4
added to the reaction mixture and it was stirred for 10 min at CHC]O), 6.44, 6.41 (s, 1H, CHC]O), 5.96, 5.93 (s, 1H, CH]
ꢀ
0
C and 3 h at room temperature. The reaction mixture was COCH
3
), 4.35, 4.10 (s, 2H, CH
2
N), 3.85 (s, 6H, 2 ꢂ OCH
3
), 3.49 (t,
4
2
H, J ¼ 13.6, 6.8 Hz, 2 ꢂ NCH CH ), 2.79 (t, 4H, J ¼ 12.8, 6.4 Hz,
then ltered over Celite® and the ltrate was evaporated under
reduced pressure. The residue was puried by silica gel gradient
2
2
1
3
ꢂ NCH CH ) ppm. C-NMR (CDCl , 100 MHz): showed the
2
2
3
column chromatography (CHCl /MeOH/NH OH 10 : 1 : 0.1) to presence of two rotamers. d ¼ 32.6, 34.3, 40.7, 45.1, 46.0, 49.8,
3
4
1
afford III (yield ¼ 95%). H-NMR ((CD ) CO, 400 MHz): d ¼ 7.07 56.4, 56.5, 107.5, 107.7, 115.7, 115.9, 128.8, 129.9, 130.5, 131.2,
3
2
(
s, 1H, ArH), 7.05 (d, 2H, J ¼ 2 Hz, 2 ꢂ ArH), 7.02 (s, 1H, ArH), 143.3, 155.0, 158.9, 163.3, 181.6, 186.9 ppm. DEPT-135 (CDCl
3
,
6
.76, 6.75 (dd, 2H, J ¼ 8.4, 2 Hz, 2 ꢂ ArH), 3.85 (s, 3H, OCH
.75 (s, 2H, ArCH N), 2.83–2.79 (m, 2H, NCH CH
Ar), 2.74, 2.71 (CH
CH Ar) ppm.
3
), 100 MHz) d ¼ 32.6 (CH
2
), 34.3 (CH
2
), 40.7 (CH
2 2
), 45.1 (CH ), 46.0
3
2
2
2
2
), 49.8 (CH ), 56.4 (CH
2
3
), 56.5 (CH
3
), 107.5 (CH), 107.7 (CH),
1
3
(
(
dd, 2H, J ¼ 14.8, 6.8 Hz, NCH
(CD
2
2
C-NMR 115.6 (CH), 115.9 (CH), 129.9 (CH), 130.4 (CH), 131.1 (CH), 163.3
ꢁ
(CH) ppm. LR-MS (ESI): m/z for [C H BrNO ] : 376.03.
3
)
2
CO, 100 MHz) d ¼ 35.2, 50.6, 52.4, 55.7, 111.4, 115.0,
17 16
4
1
1
5
15.4, 116.7, 129.5, 130.9, 132.0, 146.0, 147.1155.5 ppm. DEPT-
35 ((CD ) CO, 100 MHz) d ¼ 35.2 (CH ), 50.6 (CH ), 52.4 (CH ), Procedure for the synthesis of compound 6
3
2
2
2
2
5.7 (CH ), 115.0 (CH), 115.4 (CH), 116.7 (CH), 129.5 (CH) ppm.
3
A mixture of 5 (1 mmol, 1.0 eq.) and DBU (1.8 mmol, 1.8 eq.) in
dichloromethane (20 mL) was stirred at 0 C until no started
ꢁ
MS (ESI): m/z for [C16
H
18BrNO
3
] : 350.07.
ꢀ
material was detected by TLC. The mixture was then poured
into HCl (0.5 M, 40 mL) and extracted with dichloromethane (3
Procedure for the synthesis of compound 4
Ethyl formate (2.8 mL) was added to a solution of III ꢂ 40 mL). The organic layer was then dried over sodium sulfate
(
0.35 mmol, 1.0 eq.) in DMF (1 mL) and the mixture was stirred and evaporated under reduced pressure. The residue was puri-
ꢀ
for 24 h at 80 C. Aer the reaction, ethyl formate was removed ed by silica gel gradient column chromatography (CHCl
3
/
1
under reduced pressure and ethyl acetate was added to the MeOH 40 : 1) to afford 6 (yield ¼ 91%). H-NMR (DMSO-d
6
, 400
reaction mixture. The organic layer was washed with an MHz): showed the presence of two rotamers. d ¼ 8.13, 8.11 (s,
aqueous solution of LiCl (3% aq.) and then with brine and dried 1H, CHO), 7.26–7.23, 7.21–7.18 (dd, 1H, J ¼ 10.4, 2 Hz, COCH]
over sodium sulfate. The organic layer was evaporated under CH), 7.13, 7.09 (s, 1H, ArH), 5.99–5.96 (s, 1H, COCH]CH), 5.44–
reduced pressure. The residue was puried by silica gel gradient 5.40, 5.03–4.99, 4.76–4.72 (d, 2H, J ¼ 16.2, ArCH N), 4.30–4.26
2
column chromatography (CHCl /MeOH 15 : 1) to afford 4 (yield (d, 1H, J ¼ 15.6 Hz, CHOAr), 4.03–4.00, 3.72–3.65, 3.14–3.08,
3
1
¼
92%). H-NMR ((CD
3
)
2
CO, 400 MHz): showed the presence of 2.83–2.79, 1.99–1.92 (m, 4H, NCH
2
CH
2
), 3.83 (s, 3H, OCH
3
)
1
3
two rotamers. d ¼ 8.32, 8.01 (s, 1H, CHO), 7.16, 7.13 (s, 1H, ArH), 2.31–2.28, 2.39–2.20 (d, 2H, J ¼ 14.0 Hz, CHCH
2
CO). C-NMR
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 10897–10903 | 10901

View Article Online
RSC Advances
Paper
(
DMSO-d
6
, 100 MHz): showed the presence of two rotamers. d ¼ ¼ 8.4 Hz, ArH), 6.99 (s, 1H, ArH), 6.77 (d, 2H, J ¼ 8.4 Hz, ArH),
3
1
1
4.3, 37.6, 45.6, 45.7, 49.8, 50.0, 51.1, 56.6, 87.8, 112.1, 113.2, 3.87 (s, 3H, OCH ), 3.75 (s, 2H, ArCH N), 2.83 (br s, 4H, NCH -
16.6, 116.7, 127.1, 128.3, 128.4, 131.7, 131.9, 144.5, 144.6, CH Ar), 2.42 (s, 2H, ArCH N) ppm. C-NMR (CDCl , 100 MHz):
44.8, 145.0, 147.3, 147.5, 162.9, 195.1 ppm. MS (ESI): m/z for 154.3, 146.2, 134.6, 131.0, 128.2, 125.8, 122.6, 116.7, 115.3,
3
2
2
1
3
2
2
3
+
[C
17
H16BrNO
4
] : 378.03.
108.8, 64.5, 58.4, 56.0, 53.2, 35.6 ppm. 35.6, 53.2, 56.0, 58.4,
6
4.5, 108.8, 115.3, 116.7, 122.6, 125.8, 128.2, 131.0, 134.6, 146.2,
ꢁ
Procedure for the synthesis of compound 3
157.3 MS (ESI): m/z for [C17
H
18BrNO
3
] : 362.08.
A solution of L-selectride (2.35 mmol, 2.35 eq.) in THF (2.35 mL,
1
.0 M solution) was added to a solution of 6 (1 mmol, 1.0 eq.) in
ꢀ
Conflicts of interest
THF (20 mL) and the mixture was stirred for 6 h at ꢁ78 C. Aer
this period, a suspension of LiAlH
4
(2.35 mmol, 2.35 eq.) in THF
There are no conicts to declare.
(6 mL) was added to the reaction mixture and it was stirred for
12 h at room temperature. Aer this time, an aqueous solution
of sodium sulfate was added to the reaction mixture that was
then extracted with chloroform. The organic layer was dried
Acknowledgements
over sodium sulfate and evaporated under reduced pressure. This work was supported by funds from: MIUR Ministero del-
The residue was dissolved in methanol and 47% aq. HBr in l'Istruzione, dell'Universit `a della Ricerca Italiano, project PRIN
methanol was added and the solution was stirred for 1 h at less 2017, ORIGINALE CHEMIAE in Antiviral Strategy – Origin and
ꢀ
than 30 C. Galantamine 3 was then isolated by ltration, Modernization of Multi-Component Chemistry as a Source of
washed with MeOH, and dried under reduced pressure (yield ¼ Innovative Broad Spectrum Antiviral Strategy, cod. 2017BMK8JR
1
6
1
5
1
1
1%). H-NMR (DMSO-d , 400 MHz): d ¼ 9.80 (br, 1H), 6.85 (d, (L. B. and R. S.).
6
H, J ¼ 8.0 Hz), 6.81 (d, 1H, J ¼ 8.4 Hz), 6.17 (d, 1H, J ¼ 10 Hz),
.91–5.89 (dd, 1H, J ¼ 4, 14 Hz), 4.80 (d, 1H, J ¼ 13.2 Hz), 4.60 (s,
H), 4.50 (s, 1H), 4.35 (bs, 1H), 4.10 (s, 1H), 3.76 (m, 4H), 3.51 (d, Notes and references
1
3
H), 2,85 (bs, 1H) 2.50–2.10 (m, 3H), 1.90 (bs, 1H) ppm. C-
1
2
A. I. Scott, Q. Rev., 1965, 19, 1.
D. H. Barton, R. Kirby, J. B. Taylor and G. M. Thomas, J.
Chem. Soc., 1963, 4545–4558.
M. Villarroya, A. G. Garc ´ı a, J. Marco-Contelles and
M. G. L ´o pez, Expert Opin. Invest. Drugs, 2007, 16, 1987–1988.
B. Janssen and B. Sch ¨a fer, ChemTexts, 2017, 3, 7.
J. Marco-Contelles, M. d. C. Carreiras, C. Rodr ´ı guez,
M. Villarroya and A. G. Garc ´ı a, Chem. Rev., 2006, 106, 116–
NMR (DMSO-d
6
, 100 MHz): 31.4, 46.8, 55.3, 56.1, 59.9, 86.8,
1
12.4, 122.6, 125.7, 130.4, 133.5, 145.4, 146.9 ppm. MS (ESI): m/z
for [C17
+
H
3
20BrNO ] : 366.08.
3
Procedure for the synthesis of compound 7
4
5
Formaldehyde (0.22 mmol, 1.05 eq.) was added to a solution of
III (0.21 mmol, 1 eq.) in MeOH (2 mL) and the mixture was
stirred for 3 h. Aer this period, NaBH (0.23 mmol, 1.1 eq.) was
added to the reaction mixture and stirred for 10 min at 0 C and
4
133.
ꢀ
6
7
8
9
B. K u¨ enburg, L. Czollner, J. Fr ¨o hlich and U. Jordis, Org.
Process Res. Dev., 1999, 3, 425–431.
M. Node, S. Kodama, Y. Hamashima, T. Katoh, K. Nishide
and T. Kajimoto, Chem. Pharm. Bull., 2006, 54, 1662–1679.
K. B. Wiberg, H. Maltz and M. Okano, Inorg. Chem., 1968, 7,
for 3 h at room temperature. The reaction mixture was then

ltered over Celite® and the ltrate was evaporated under
reduced pressure. The residue was puried by silica gel gradient
column chromatography (CHCl : MeOH 10 : 1) to afford 7
, 400 MHz): 7.07 (s, 1H, ArH), 7.05
d, 2H, J ¼ 8 Hz, ArH), 7.00 (s, 1H, ArH), 6.77 (d, 2H, J ¼ 8 Hz),
.88 (s, 3H, OCH ), 3.60 (s, 2H, ArCH N), 2.81–2.78 (m, 2H,
3
1
(
(
3
yield ¼ 96%). H-NMR (CDCl
3
8
30–831.
J. Szewczyk, A. H. Lewin and F. I. Carroll, J. Heterocycl. Chem.,
988, 25, 1809–1811.
3
2
1
ArCH CH N), 2.71–2.68 (m, 2H, ArCH CH N), 2.34 (s, 3H,
NCH ) ppm. C-NMR (CDCl , 100 MHz): 32.5, 41.7, 50.8, 59.3,
0.4, 113.8, 114.9, 115.3, 116.9, 129.8, 145.1, 146.4, 153.9 ppm.
MS (ESI): m/z for [C17
2
2
2
2
1
1
0 A. Messerschmidt, Multi-Copper Oxidases, World Scientic
Publishing Co. Pte. Ltd, Singapore, 1997.
1 L. J ¨o nsson, K. Sj ¨o str ¨o m, I. H ¨a ggstr ¨o m and P. O. Nyman,
Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol., 1995,
1
3
3
3
6
ꢁ
H
20BrNO
3
] : 364.02.
1
251, 210–215.
Procedure for the synthesis of compound 10
1
2 M. Mogharabi and M. A. Faramarzi, Adv. Synth. Catal., 2014,
Laccase (1000 U) was added to a stirred solution of compound 7
356, 897–927.
(
0
1 mmol, 1 eq.) in 1,4-dioxane (5 mL) and acetate buffer, pH 4.5, 13 T. Kudanga, G. S. Nyanhongo, G. M. Guebitz and S. Burton,
.5 M (20 mL), and the mixture was stirred for 6 h under O Enzyme Microb. Technol., 2011, 48, 195–208.
atmosphere. Aer this period the aqueous solution was 14 N. Santhanam, J. M. Vivanco, S. R. Decker and K. F. Reardon,
Trends Biotechnol., 2011, 29, 480–489.
2
extracted with ethyl acetate (3 ꢂ 50 mL). The organic layer was
washed with brine, dried over sodium sulfate and evaporated 15 F. Xu, Ind. Biotechnol., 2005, 1, 38.
under reduced pressure. The residue was puried by silica gel 16 S. Riva, Trends Biotechnol., 2006, 24, 219–266.
gradient column chromatography (CHCl
3
/MeOH 15 : 1) to 17 M. Fabbrini, C. Galli and P. Gentili, J. Mol. Catal. B: Enzym.,
1
afford 10 (yield ¼ 76%). H-NMR (CDCl
3
, 400 MHz): 7.04 (d, 2H, J 2002, 16, 231–240.
10902 | RSC Adv., 2020, 10, 10897–10903
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
1
8 V. A. Golubev, Y. N. Kozlov, A. N. Petrov and A. P. Purmal, 25 A. Marjasvaara, J. J ¨a nis and P. J. Vainiotalo, Mass Spectrom.,
Catalysis of Redox Processes by Nitroxyl Radicals, Springer,
Heidelberg, Berlin, 1992.
9 V. V. Abalyaeva and O. N. Emov, Russ. J. Electrochem., 2002,
2008, 43, 470–477.
26 B. Branchi, C. Galli and P. Gentili, Org. Biomol. Chem., 2005,
3, 2604–2614.
1
2
2
2
2
2
3
8, 1093–1097.
27 P. Baiocco, A. M. Barreca, M. Fabbrini, C. Galli and
P. Gentili, Org. Biomol. Chem., 2003, 1, 191–197.
28 C. Galli and P. Gentili, J. Phys. Org. Chem., 2004, 17, 973–977.
0 R. Bourbonnais, D. Leech and M. G. Paice, Biochim. Biophys.
Acta, Gen. Subj., 1998, 1379, 381–390.
1 K. Li, F. Xu and K. L. Eriksson, Appl. Environ. Microbiol., 29 G. Rousselet, P. Capdevielle and M. Maumy, Tetrahedron
999, 65, 2654. Lett., 1995, 36, 4999–5002.
2 O. V. Morozova, G. P. Shumakovich, S. V. Shleev and 30 E. Andrade-Jorge, L. A. S ´a nchez-Labastida, M. A. Soriano-
1
Y. I. Yaropolov, Appl. Biochem. Microbiol., 2007, 43, 523–535.
3 P. Galletti, F. Funiciello, R. Soldati and D. Giacomini, Adv.
Synth. Catal., 2015, 357, 1840–1848.
4 R. Bourbonnais, M. G. Paice, B. Freiermuth, E. Bodie and 32 J. Szewczyk, J. W. Wilson, A. H. Lewin and F. I. Carroll, J.
S. Borneman, Appl. Environ. Microbiol., 1997, 63, 4627. Heterocycl. Chem., 1995, 32, 195–199.
Urs u´ a, J. A. Guevara-Salazar and J. G. Trujillo-Ferrara, Med.
Chem. Res., 2018, 27, 2187–2198.
31 M. C. Carre n˜ o and A. Urbano, Synlett, 2005, 1, 1–25.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 10897–10903 | 10903