Synthesis of 6β-D-glucosyl and 6-nitroxy (-)-galanthamine derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1691/ph.2007.6.6661

Galanthamine is an alkaloid approved for the treatment of Alzheimer's disease. In this paper the syntheses and the anticholinesterase activities of new glucosyl and nitroxy derivatives substituted on position 6 are reported. Compounds 2, 3 and 5 presented a percentage of inhibition of 35.22%, 47.48% and 67.89 % respectively.

Full text of DOI:10.1691/ph.2007.6.6661

ORIGINAL ARTICLES
1
Dipartimento di Chimica Farmaceutica e Tossicologica , Universit a` di Napoli “Federico II”, Napoli, Dipartimento di Farmacolo-
2
gia “Giorgio Segre” , Universit a` degli Studi di Siena, Siena, Italy
Synthesis of 6b-d-glucosyl and 6-nitroxy (–)-galanthamine derivatives
as acetylcholinesterase inhibitors
1
1
1
1
2
2
1
1
E. Perissutti , F. Fiorino , B. Severino , F. Frecentese , P. Massarelli , C. Nencini , V. Santagada , G. Caliendo
Received July 27, 2006, accepted October 14, 2006
Prof. Guiseppe Caliendo, Dipartimento di Chimica Farmaceutica e Tossicologica, Universit a` di Napoli
Federico II”, Via D. Montesano 46, 80131 Napoli, Italy
“
caliendo@unina.it
Pharmazie 62: 403–405 (2007)
doi: 10.1691/ph.2007.6.6661
Galanthamine is an alkaloid approved for the treatment of Alzheimer’s disease. In this paper the synth-
eses and the anticholinesterase activities of new glucosyl and nitroxy derivatives substituted on posi-
tion 6 are reported. Compounds 2, 3 and 5 presented a percentage of inhibition of 35.22%, 47.48%
and 67.89 % respectively.
1
. Introduction
in CNS (Emson et al. 1995). On the other hand, their dif-
ferent sterical hindrance could allow us to better define
the impact of the substituent’s encumbrance in position 6
on the galanthamine binding mode to the AchE active site
(Greenblatt et al. 1999).
Alzheimer’s disease (AD) is a progressive neurodegenera-
tive disorder of the central nervous system (CNS), asso-
ciated with cholinergic insufficiency, resulting in dementia
that is characterized by profound memory impairments,
emotional disturbance and personality changes (Bartus
et al. 1982; Dunnet et al. 1993; Weinstock 1997). Cur-
rently, the most effective way of improving the reduced
acetylcholine levels is treatment with reversible competi-
tive inhibitors of acetylcholinesterase. Galanthamine is an
alkaloid found in the bulbs and flowers of the common
snowdrop (Galanthus nivalis L.) and of several other
members of the Amaryllidaceae family. It is approved for
the treatment of AD as hydrobromide salt under the name
2
. Investigations, results and discussion
(–)-Galanthamine was been synthesized according to a
procedure reported in the literature (Czollner et al. 1998).
The syntheses of tetra-acetyl derivative 2 and desacetyl
derivative 3 were carried out following the synthetic pro-
cedure reported in Scheme 1. (–)-Galanthamine (1) was
treated with 2,3,4,6-tetra-O-acetyl-a-d-glucopyranosyl bro-
mide affording the 2,3,4,6-tetra-O-acetyl-b-d-glucopyrano-
syl derivative 2. Acetyl groups were removed in dry
methanol with catalytic amounts of 0.1 M sodium metha-
nolate affording the desired 6b-d-glucosyl(–)-galantha-
mine (3), without methyl ether as by-product. The nitroxy
derivative 5 was prepared as reported in Scheme 2, start-
ing from (–)-Galanthamine (1) which was treated with
SOCl2 to give the corresponding 6a-chloride derivative 4.
Treatment with AgNO3 in CH3CN converted compound 4
into 6b-nitroxy-(–)-galanthamine 5.
1
1
Nivalin or Reminyl . Limited supplies and high costs of
Galanthamine isolation from natural sources (Shieh et al.
1
994; Kuenburg et al. 1999) led to the development of
several synthetic routes for the preparation of (ꢀ)-ga-
lanthamine, epigalanthamine and narwedine (Kametani
et al. 1971a, b; Barton et al. 1962; Kametani et al. 1969;
Shimizu et al. 1977; Vlakow et al. 1989; Marco-Contelles,
J. et al. 2006). Moreover a variety of synthetic galantha-
mine derivatives have been previously described including
C-ring derivatives, quaternary ammonium derivatives (Han
et al. 1992) or esters and carbamates of 3-O-dimethyl-ga-
lanthamine (Bores et al. 1996) as well as some galantha-
mine analogues (Lewin et al. 2005). Most of these com-
pounds are characterized by the unsubstituted hydroxyl
group in position 6.
In order to better evaluate the role of the hydroxyl group
in position 6, we synthesized new glucosyl and nitroxy
galanthamine analogues. These substituents were selected
starting from two different considerations: firstly, these
groups represents important biological scaffolds both as
transporting carriers substrates (glucosyl derivatives) (Bell
et al. 1993; Barret et al. 1999) than as useful NO donors
These new products were tested in vitro on rat brain
homogenate and the results are reported in the Table as
percentage of inhibition at highest tested concentration
ꢁ4
(10 M). Even if lower in comparison with galanthamine
(inhibition 89.88 %; IC50 5.86 mM, the results supported
the hypothesis of Greenblatt et al. (1999, 2004). In agree-
ment with their reports, our derivatives showed that the
increasing of the steric hindrance in position 6 provides a
lower interaction of galanthamine in the enzyme active
site; in fact, the percentage of inhibition of our com-
pounds increased when 2,3,4,6-tetra-O-acetyl-b-d-gluco-
pyranosyl derivative (2) was converted into 6b-d-gluco-
Pharmazie 62 (2007) 6
403

ORIGINAL ARTICLES
ꢂ
Scheme 1 Reagents and conditions: (i) 2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl bromide, AgOTfl, anhydrous CH2Cl2, ꢁ10 C. (ii) 0.1 M sodium
methanolate, rt, 3 h
OAc
OH
O
O
O
OAc
OAc
O
OH
OH
OH
H
O
H
O
H
O
AcO
HO
i
H CO
ii
H CO
3
H CO
3
3
N
N
N
CH3
CH3
CH
3
(
-)-Galanthamine
1
2
3
ꢂ
Scheme 2 Reagents and conditions: (i) SOCl2, toluene, 55 C. (ii) AgNO3, CH3CN, rt, 48 h
OH
Cl
ONO2
H
H
H
O
O
O
H CO
3
i
H CO
3
ii
H CO
3
N
N
N
CH
CH3
CH3
3
(
-)-Galanthamine
1
4
5
Table: In vitro AChE inhibition of compounds 1, 2, 3 and 5
were performed on a LCQ Thermoquest-Ion trap mass spectrometer. Re-
sults obtained were within ꢀ 0.4% of the theoretical values. All reactions
were followed by thin-layer chromatography (TLC), carried out on Merck
silica gel 60 F254 plates with fluorescent indicator and the plates were
visualized with UV light (254 nm). Preparative chromatographic purifica-
tions were performed using silica gel column (Kieselgel 60). Organic solu-
tions were dried over anhydrous Na2SO4 and concentrated with B u¨ chi ro-
tary evaporator at low pressure while, in case of aqueous solution, the final
product was recovered by lyophilization.
For the pharmacological assays all the used solvents and powders were for
analysis and were purchased from Sigma Chemical Co., St. Louis, MO,
U.S.A. The measurements were carried out with a Beckman DU-650 spec-
trophotometer (Beckman Instruments Inc., Fullerton, CA, USA).
Compd.
% Inhibitiona
Mꢀ ꢀ s.d.)
IC50 Values
(
1
2
3
5
89.88 ꢀ 1.20
35.22 ꢀ 0.94
47.48 ꢀ 1.33
67.89 ꢀ 0.57
5.86 ꢀ 0.345
ꢁ
ꢁ
45.09 ꢀ 2.596
a
ꢁ4
%
inhibition at highest tested concentration (10 M)
syl(–)-galanthamine (3), while the best result was ob-
tained with the introduction of the nitroxyl group. Com-
pound 5, characterized by lower hindrance on the enzyme
catalytic site, showed, in fact, a percentage of inhibition of
3
.1.2 (–)-(4a a,6b)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6 H-
benzofuro [3a,3,2-e,f] [2] benzazepin-6-(2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranoside (2)
To a solution of (–)-galanthamine 1 (0.39 g, 1.36 mmol) and triethylamine
(0.185 mL, 1.36 mmol) in anhydrous dichloromethane (10 mL) was added
AgOTfl (0.35 g, 1.36 mmol). The mixture was stirred under nitrogen atmo-
6
7.89% with a IC50 value of 45.09 mM.
In conclusion, the new derivatives, even if characterized
by a lower enzymatic inhibition, could represent a new
valuable strategy in order to improve the pharmacokinetic
and pharmacological properties of the bioactive isomer of
galanthamine. In fact we could hypothesize that, by means
of glucose carriers, the novel glucosyl derivatives could
have a better bioavailability in the CNS, as well as for the
nitroxyl derivative we could hypothesize a release of NO,
an important neuromediator over BBB. These will be in-
vestigated in further in vivo studies.
ꢂ
sphere and cooled to ꢁ10 C. 2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl
bromide (0.56 g, 1.36 mmol) was added and then the mixture was stirred
for 2 h. After 12 h at room temperature the suspension was neutralized
with 2,3,5-collidine and centrifuged. The supernatant was concentrated,
redissolved in acetonitrile and purified by preparative HPLC (eluent: A,
0
.1% trifluoroacetic acid in acetonitrile, B, 0.1% trifluoroacetic acid in
water; linear gradient from 0 to 70% over 25 min, flow rate: 1 ml/min)
and lyophilized affording the pure product 2 (yield: 60%). M. p: 96–
ꢂ
25
ꢂ
1
98 C. [a]D : –132.5 (c ¼ 1, CHCl ). H NMR (CDCl , 500 MHz) d
3
3
(ppm): 6.68 (q, 2 H), 6.02 (m, 2 H), 5.27–5.14 (m, 3 H), 5.08 (d, 1 H),
4
3
.63 (bs, 1 H), 4.45 (bs, 1 H), 4.26 (dd, 1 H), 4.15 (dd, 1 H), 4.02 (d, 1 H),
.83 (s, 3 H), 3.79 (m, 1 H), 3.71 (d, 1 H), 3.28 (t, 1 H), 3.06 (d, 1 H), 2.69
(
dd, 1 H), 2.40 (s, 3 H), 2.10 (m, 2 H), 2.06, 2.04, 2.03 and 2.02 (4s, 12 H,
13
acetyl), 1.47 (dd, 1 H); C NMR (CDCl3, 500 MHz) d (ppm): 169.1,
69.2, 170.1, 170.4, 145.9, 144.2, 133.3, 129.5, 127.8, 127.1, 122.2,
3. Experimental
1
3
.1. Synthesis
111.5, 99.1, 88.8, 72.7, 71.3, 68.2, 62.1, 61.9, 60.6, 56.1, 54.1, 48.4, 42.3,
þ
3
3.9, 30.1, 20.5. ESI-MS: 618.8 [M þ H] .
3
.1.1. General procedures
C31H39NO12
All reagents were commercial products purchased from Aldrich. Melting
points were determined using a Kofler hot-stage apparatus and are uncor-
1
13
3.1.3. (–)-(4a a,6b)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6 H-
benzofuro [3a,3,2-e,f] [2] benzazepin-6-b-D-glucopyranoside (3)
rected. H NMR and C NMR spectra were recorded on Bruker AMX-500
spectrometer in CDCl3 or DMSO-d6. Chemical shifts are reported in ppm
using Me4Si as internal standard. The following abbreviations are used to
describe peak patterns when appropriate: s (singlet), d (doublet), dd (dou-
ble doublet), t (triplet), m (multiplet). Mass spectra of the final products
A solution of 2 (0.50 g, 0.81 mmol) in anhydrous methanol (15 mL) was
treated with catalytic amounts of 0.1 M sodium methanolate (1 mL) and
stirred at room temperature. After 3 h the solution was neutralized by ion-
404
Pharmazie 62 (2007) 6

ORIGINAL ARTICLES
þ
exchange resin (Amberlite IR-120 H , purchased from Aldrich), the resin
was filtered off and the filtrate was concentrated in vacuo. The residue was
directly purified by preparative HPLC (eluent: A, 0.1% trifluoroacetic acid
in acetonitrile, B, 0.1% trifluoroacetic acid in water; linear gradient from 0
to 60% over 25 min, flow rate: 1 mL/min) and lyophilized affording the
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ꢂ
25
ꢂ
pure product 3 (yield: 80%). M. p.: 88–90 C. [a] : –123.6 (c ¼ 1,
D
1
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2
(
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(
1
3
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7
4
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þ
50.3 [M þ H] .
C23H31NO8
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ꢂ
period of 30 min and the solution was stirred for 2 h at 55 C; the solvent
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–
2
3
2
5
:
D
ꢂ
1
282.8 (c ¼ 0.61, CHCl3). H NMR (CDCl3, 500 MHz) d (ppm): 6.69 (q,
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13
(
m, 2 H), 1.48 (dd, 1 H); C NMR (CDCl3, 500 MHz) d (ppm): 145.9,
1
4
44.3, 133.5, 129.5, 127.8, 127.0, 122.2, 111.5, 88.9, 62.2, 60.8, 56.1, 54.0,
þ
8.4, 42.3, 33.9, 30.2. ESI-MS: 306.1 [M þ H] .
C17H20ClNO2
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3
.1.5. (–)-(4a a,6b)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl-6-ni-
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A mixture of 4 (0.07 g, 0.23 mmol) and AgNO3 (0.0714 g, 0.42 mmol) in
acetonitrile (10 mL) was stirred under light exclusion for 48 h. The mixture
was filtered on Celite and the solvent was evaporated under vacuum; the
residue was then dissolved in ethyl acetate, washed with H2O and brine and
then the organic phase was separated, dried with anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified on silica gel
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2
5
ꢂ
1
[
6
4
a] : –261.4 (c ¼ 0.6, CHCl3) H NMR (CDCl3, 500 MHz) d (ppm):
D
.92 (q, 2 H), 6.4 (m, 2 H), 4.88 (bs, 1 H), 4.47 (bs, 1 H), 4.23 (d, 1 H),
.01 (s, 3 H), 3.91 (d, 1 H), 3.72 (t, 1 H), 3.18 (d, 1 H), 2.80 (dd, 1 H), 2.51
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13
(
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ppm): 146.8, 144.9, 133.9, 131.0, 128.5, 127.9, 122.9, 111.9, 89.6, 62.9,
þ
6
1.5, 56.9, 54.9, 49.0, 42.9, 34.6, 30.8. ESI-MS: 333.0 [M þ H] .
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3
.2. In vitro pharmacological assays
The method of Ellman et al. (1961) was used with some modifications. Rat
brain was homogenized (approximately 20 mg of tissue per mL of 0.1 M
phosphate buffer, pH 8.0) with a Polytron Apparatus – Kinematica
GmbH, Littau, Switzerland. The preparation of brain homogenate was car-
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nate was added to a cuvette containing 0.58 mL of phosphate buffer.
1
0
.1 mL of test compound dissolved in 5% DMSO or 0.1 mL of solvent
alone were added too (final concentrations of tested compounds were:
1
incubated at room temperature for 5 min and then at 37 C for additional
ꢁ4
ꢁ5
ꢁ6
ꢁ7
0
, 10 , 10 , 10 M, each performed in sextuple). The mixture was
ꢂ
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5
min. At the end of this procedure 0.1 mL of 0.0033 M DTNB reagent
and 0.020 mL of 0.075 M acetylthiocholine iodide were added to the
photocell. The absorbance was measured at 412 nm at 37 C for 5 min.
Galanthamine was employed as reference compound. The percent of inhi-
bition was calculated as reported in the literature (Han et al. 1991). The
results are shown in the Table.
ꢂ
Acknowledgments: The NMR spectral data were provided by Centro di
Ricerca Interdipartimentale di Analisi Strumentale, Universit a` degli Studi
di Napoli “Federico II”. The assistance of the staff has been gratefully
appreciated.
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Pharmazie 62 (2007) 6
405