Synthesis and evaluation of 2′-hydroxyethyl trans-apovincaminate derivatives as antioxidant and cognitive enhancer agents

Article abstract of DOI:10.1021/jm070618k

A series of trans-2′-hydroxyethyl and 2′-acyloxyethyl apovincaminates 4b-f and 7b-f has been synthesized and evaluated for their antioxidant and antiamnesic effects. The new esters were prepared from 4a and 7a ethyl esters or from the corresponding carbox

Full text of DOI:10.1021/jm070618k

J. Med. Chem. 2008, 51, 479–486
479
Synthesis and Evaluation of 2′-Hydroxyethyl trans-Apovincaminate Derivatives as Antioxidant
and Cognitive Enhancer Agents
András Nemes,* László Czibula, Csaba Szántay, Jr., Anikó Gere, Béla Kiss, Judit Laszy, István Gyertyán,
Zsolt Szombathelyi, and Csaba Szántay†
Gedeon Richter Ltd., Budapest 10, POB 27, H-1475, Hungary, and Budapest UniVersity of Technology and Economics, Gellért tér 4,H-1521,
Budapest, Hungary
ReceiVed May 30, 2007
A series of trans-2′-hydroxyethyl and 2′-acyloxyethyl apovincaminates 4b-f and 7b-f has been synthesized
and evaluated for their antioxidant and antiamnesic effects. The new esters were prepared from 4a and 7a
ethyl esters or from the corresponding carboxylic acid sodium salt. For starting materials 11a,b, a new
stereoselective trans-reduction was elaborated. From the combined results of the data obtained from in
vitro and in vivo tests and examination of the metabolism, (3R,16S)-2′-hydroxyethyl apovincaminate (7b,
RGH-10885) was identified as the most promising compound, owing to its potent neuroprotective and
antiamnesic activities. The in vivo effectiveness of selected compounds on the cognitive functions was
studied in a one-trial passive avoidance task and a water-labyrinth test.
Introduction
Vinpocetine (1, cis-(3S,16S)-ethyl apovincaminate), a deriva-
tive of vincamine (2), a main alkaloid of Vinca minor L., has
successfully been used in the treatment of CNS disorders of
cerebrovascular origin for decades.1 Cerebrovascular, antihy-
poxic, antiamnesic, and antiischemic effects have been identified
as the main components of the action of 1.2 Compound 1 is
also known to have cytoprotective activity in vitro.2,3 The
experimentally proved components of the neuroprotective effect
of vinpocetine are the inhibition of voltage-dependent sodium
channels, the inhibition of PDE1 enzyme, and the stabilization
of intracellular Ca2+ homeostasis.4,5 However, the mechanism
by which vinpocetine exerts its protective and cognitive effects
Figure 1. Structure of 7b, Vinpocetine (1), Vincamine (2), and
Vinburnine (3).
is complex and it has not been fully understood.
Clinical and experimental data suggest that free radicals,
reactive oxygen species (ROSa), and lipid peroxidation (LPO)
are implicated in the pathogenesis of a wide variety of human
diseases such as brain ischemia, trauma, stroke, subarachnoid
hemorrhage, mild cognitive impairment, and chronic neurode-
generative disorders, such as Alzheimer’s disease (AD) and
Parkinson’s disease.6,7 The central nervous system (CNS) is
particularly sensitive to oxygen free radicals induced damage
because of the high levels of polyunsaturated lipids, high rate
of oxygen consumption, and relatively low concentration of
antioxidant enzymes and natural antioxidants. The free radicals
induced oxidative stress leads to LPO, which is a pathological
factor in mild cognitive impairment (MCI) and in the progres-
sion to Alzheimer’s disease from MCI.8,9
ischemia attack (TIA), stroke], where learning and memory
defects may occur in addition to neurological symptoms of
various severity. Compound 1 is successfully used in the therapy
of memory disturbances following cerebral ischemic states, and
several experimental data have confirmed that 1 prevents the
impairment of cognitive functions caused by various impairing
agents in animal models, too.2,10 It has been shown that 1 exerts
antioxidant/LPO inhibitory activity.11 In our own experiments,
1 possessed a potent cognitive enhancing effect with moderate
LPO inhibitory activity that may be beneficial in the treatment
of cognitive decline.12
Earlier structure–activity relationship (SAR) studies showed
the effects of different changes in the structure of the eburnane
skeleton. The cerebrovascular activity is closely related to a
specific configuration, namely, the natural cis-(3S,16S)-eburnane
skeleton, which can be identified in 1 and 2, as well as in (-)-
eburnamonine (vinburnine, 3),13 the latter two compounds are
also marketed as cerebrovasculars. (Figure 1). In contrast to cis-
(3S,16S)-derivatives, trans-(3S,16R) ethyl ester (4a) and deriva-
tives as (3S,16R)-dihydroeburnamenine-14-methanol, and its
dinor-derivative (5) exhibit significant peripheral vasodilator
effect.14,15 Substitution of the carboxylic ester function with a
dialkylamino-alkylaminomethyl chain on 14-C (6) increases the
neuroprotective, LPO inhibiting activity, coupling with total loss
of vascular and antihypoxic properties. The neuroprotective
Compounds possessing antioxidant activity exert significant
protective action in events of cerebral ischemia [transient
* To whom correspondence should be addressed. Tel.: +36 1 431 4747.
Fax: +36 1 432 6018. E-mail: a.nemes@richter.hu.
†
Budapest University of Technology and Economics and Chemical
Research Center, Hungarian Academy of Sciences, POB17,
H-1525 Budapest.
aAbbreviations: AD, Alzheimer disease; BHT, 2,6-di-tert-butyl-4-me-
thylphenol; LPO, lipid peroxidation; MCI, mild cognitive impairment; MDA,
malondialdehyde; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen
species; SAR, structure-activity relationship; TBA, thiobarbituric acid;
TBAR, thiobarbituric acid reactive substances; TIA, transient ischemia
attack.
10.1021/jm070618k CCC: $40.75
2008 American Chemical Society
Published on Web 01/10/2008

480 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Nemes et al.
Scheme 1. Synthesis of Hydroxyethyl trans-Apovincaminate
Derivativesa
Figure 2. Structure–activity relationships.
effect is not configuration-dependent; cis and trans-compounds
6 showed comparable activities16 (Figure 2).
To continue these investigations, we describe herein the
synthesis of a new series of substituted-alkyl esters of (3S,16R)-
and (3R,16S)-trans-apovincaminic acids characterized with the
general formulas 4 and 7.17 We hypothesized that the com-
pounds with considerable LPO inhibitory efficacy may exhibit
augmented cognition enhancing characteristics. The purpose of
our work was to develop novel apovincaminate derivatives with
antioxidant feature which can be used as drug candidates for
the treatment and/or prevention of the neurodegenerative
diseases. Preliminary in vitro assays of these molecules were
performed in order to explore the structural requirements for
efficient antioxidative activity. The antiamnesic effect of new
compounds with trans configuration of eburnane skeleton was
assessed using a one-trial passive avoidance test. Furthermore
some selected cis- and trans-apovincaminates were tested in a
complex, spatial learning paradigm: the water-labyrinth test.
Some of the new compounds showed considerable antioxidant
and antiamnesic activities. The most active compounds, 7b and
7c, were found among the (3R,16S)-trans series. The first tests
concerning the metabolism demonstrated a quick metabolism
of 7c to 7b. Hence, we selected compound 7b (RGH-10885)17
to investigate its in vivo efficacy in different cognitive tests.
a Reagents and conditions: (a) Pd/C, Et3N, DMF; (b) (i) NaBH4, AcOH,
EtOH; (ii) NH4OH, 73%; (c) (i) KOH/H2O, EtOH, 35 °C; (ii) NaNO2/
H2O, AcOH, 15 °C; (iii) HCl/H2O, 69%; (d) p-toluenesulfonic acid, toluene,
reflux, 75%; (e) HO(CH2)2OH, KOt-Bu, 80 °C (R ) H), 75%; (f) NaOH/
EtOH, reflux, 96%; (g) Cl(CH2)2OR, DMF, 100 °C, 80–85%.
enantiomer 8b. Finally, compounds 4b-f and 7b-f were
isolated as mineral or organic acid salts (Table 1).
Results and Discussion
Biochemistry. Two in vitro methods, enzyme-catalyzed19
lipid peroxidation (NADPH-induced LPO) and nonenzymic lipid
peroxidation (Fe2+-stimulated LPO)20 in rat whole brain was
used to study the antioxidant effect of the compounds. Different
components of neuronal tissue were used as lipid substrates:
plasma membrane (rat brain homogenate) and membrane from
rat brain endoplasmic reticulum (microsomes). LPO, which is
a chain reaction, was induced by a different mechanism, such
Chemistry
as an enzymic (adding of NADPH in the presence of Fe3+
/
The synthetic route followed to obtain the title compounds
is summarized in Scheme 1. (15aS)-Diethyl ethyl-hexahydroin-
dolo[2,3-a]pyrano[3,2-i]quinolizine-14,14-dicarboxylate (8a),
previously reported as intermediate for the synthesis of 1 and
ADP complex) and a nonenzymic way (by addition of Fe2+).
This method is based on the oxidation of polyunsaturated fatty
acids (PUFAs) in biologic membranes, giving rise to a variety
of lipid breakdown products such as malondialdehyde (MDA).
By reacting MDA with thiobarbituric acid (TBA), a pink
chromogene is formed, which can be detected by UV–vis
spectrophotometry. The absorption of thiobarbituric acid reactive
substances (TBARs) was measured spectrophotometrically at
532 nm.6 On the basis of the concentration-effect correlations
of the tested compounds, the IC50 values were determined. The
results from both experiments are listed in Table 2. The
cerebroprotective 1, idebenone, DL-R-tocopherol, quercetin, and
butylated hydroxytoluene or 2,6-ditert-butyl-4-methylphenol
(BHT) were used as reference compounds. Idebenone, a
benzoquinone derivative (coenzyme Q10 analog) with a potent
free radical scavenger and antioxidant properties, has been used
for the oral treatment of Alzheimer’s type dementia.21,22 DL-
R-tocopherol is the best known isomer of natural antioxidant
vitamin E.23 Treatment with idebenone and R-tocopherol was
also protective on Aꢀ-induced learning and memory deficits in
rats.24 The flavonol quercetin is an efficient LPO inhibitor and
free radical scavenger.24 Dialkyl phenol BHT has been used as
2
18 was the starting material for compounds 7a-f. Compound
8a in the presence of a base as triethylamine exists as
hexahydroindolopyranoquinolizine, catalytic reduction of which
led stereoselectively to cis-octahydroindoloquinolizine 9. In
acidic medium, the pyrano-ring transforms to the open-chained
form 10a with addition of a proton.18
A modified stereoselectivity was achieved by the reduction
of compound (15aS)-8a under mild acidic conditions with
sodium tetrahydroborate. After treatment with ammonium
hydroxide, trans-diethoxycarbonylethyl-octahydroindoloquino-
lizine 11a was isolated. The transformation of indoloquinolizinyl
diester (11a) into ethyl indoloquinolizinyl-pyruvate oxime (12a)
was carried out without isolation of the intermediate monoester.
Heating oxime 12a in boiling toluene with p-toluenesulfonic
acid furnished ethyl (3R,16S)-apovincaminate (7a).14,18 Trans-
esterification of compound 7a to 7b,14 then acylation, or reaction
of sodium trans-apovincaminate (13a Na salt) with a halo-alkyl
reagent are the alternative methods to obtain compounds 7b-f.
Similarly, compounds 4b-f were prepared from (15aR)-

2′-Hydroxyethyl trans-ApoVincaminate DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 481
Table 1. Physical Properties of Apovincaminic Acids and Esters 4b-f, 7b-f, 13a,b, and 14
cmpd
R
salt
procedure
yield (%)
mp (°Csolv.
)
[R]D (°solv., c)
4b
4c
4d
4e
4f
7b
7c
7d
7e
7f
13a
13b
14
HO(CH2)2
HCl
HCl
HCl
HCl
CH3SO3H
(+)-tartaric acid
HCl
HCl
HCl
CH3SO3H
A
B
C
C
C
A
B
C
C
C
85
74
75
67
77
82
72
69
71
74
94
92
67
235–237b
222–224c
226–228e
218–220d
200–202e
157–159b
223–224c
219–220e
216–217d
197–199e
201–202g
200–201g
170–171b
+123.0 (1)a
+107.4 (1)a
+107.3 (0.2)a
+84.8 (0.2)a
+73.3 (0.2)a
–72.1 (1)f
CH3CO2(CH2)2
C2H5CO2(CH2)2
C6H5CO2(CH2)2
4-ClC6H4CO2(CH2)2
HO(CH2)2
CH3CO2(CH2)2
C2H5CO2(CH2)2
C6H5CO2(CH2)2
4-ClC6H4CO2(CH2)2
H
–118.9 (0.2)a
–111.9 (1)a
–86.6 (1)a
–73.3 (1)a
–133.0 (1)f
+130.8 (1)f
+172.5 (1)f
H
HO(CH2)2
A
a Solvent: methanol. b Solvent: ethanol. c Solvent: 2-propanol. d Solvent: chlorobenzene. e Solvent: acetone. f Solvent: dimethylformamide. g Solvent:
water.
Table 2. Inhibition of Enzymic Lipid Peroxidation (400 µM NADHP-Induced LPO in Rat Brain Microsomes) and Nonenzymic Lipid Peroxidation (200
µM Fe2+-Induced LPO in Rat Brain Homogenate) by (3S,16R)-trans-Apovincaminic Esters 4a-f and (3R,16S)-trans-Apovincaminic Esters 7a-f and
the Corresponding Acids 13a and 13b and Reference Drugs, such as Vinpocetine, Idebenone, DL-R-Tocopherol, Quercetine, and BHTa
NADH-induced LPO
Fe2+-induced LPO
cmpd No.
13b
4a
4b
4c
R
configuration
IC50 (µM)
IC50 (µM)
H
C2H5
3S,16R
3S,16R
3S,16R
3S,16R
3S,16R
3S,16R
3S,16R
3R,16S
3R,16S
3R,16S
3R,16S
3R,16S
3R,16S
3R,16S
3S,16S
3S,16S
>>300
258 (237–278)
13.4 (11.8–15.0)
13.6 (12.0–15.2)
15.2 (11.9–18.6)
15.8 (14.3–17.2)
14.8 (10.3–19.3)
15.7
238 (237–239)
14.4 (14.0–14.8)
13.7 (11.6–15.8)
9.8 (7.8–11.9)
21 (7.5–34.5)
18.2 (15.3–21.0)
14.6 (9.2–19.9)
209 (157–261)
N.D.
3.0 (1.6–4.4)
9.1 (6.1–12.1)
3.8 (2.3–5.3)
4.4
HO(CH2)2
CH3CO2(CH2)2
C2H5CO2(CH2)2
C6H5CO2(CH2)2
4-ClC6H4CO2(CH2)2
H
4d
4e
3.4
2.7
>>300
3.5 (2.8–4.1)
7.0 (5.0–9.1)
3.6 (2.3–4.9)
5.0
3.8
2.7
137 (75.2–199)
>>30
1.3 (0.9–1.6)
273 (148–399)
8.5 (6.9–10.1)
3.2 (2.1–4.4)
4f
13a
7a
7b
7c
7d
7e
7f
1
14
C2H5
HO(CH2)2
CH3CO2(CH2)2
C2H5CO2(CH2)2
C6H5CO2(CH2)2
4-ClC6H4CO2(CH2)2
C2H5
HO(CH2)2
idebenone
DL-R-tocopherol
quercetin
BHT
31.5 (19–44)
35.7 (10.7–60.7)
10.7 (8.9–12.6)
6.2 (5.3–7.1)
a Values are taken from 1-8 experiments performed in triplicate, expressed as means of IC50 in µM, with the confidence intervals in parenthesis. Values
without confidence intervals are for a single determination. N.D. ) not determined.
a radical scavenging antioxidant in food industry for many years
and as an inhibitor of LPO in experimental pharmacology.25,26
Figures 3 and 4 indicate the effect of (3S,16R)-trans-esters 4b,c
and (3R,16S)-trans-esters 7b,c and reference drugs such as
vinpocetine and idebenone on NADPH (400 µM) evoked LPO
in rat brain microsomes and Fe2+ (200 µM) evoked LPO in rat
brain homogenates, respectively.
tion, propagation, and termination steps of LPO evoked by Fe2+
in rat brain homogenate.
Trans-apovincaminic acid esters (4a-f, 7a-f) were more
active in inhibition of LPO than esters with cis-configuration
(1, 14), as shown by their much lower IC50 values. It is therefore
assumed that interaction of trans-ring-fused apovincaminates
with membrane lipids is stronger than that of cis-ring-fused
esters, thus, they more potently protect the brain membrane
against the free radical-evoked oxidative damages.
It was found that all new compounds with trans-apovincami-
nate moiety (4b-f, 7b-f) were active inhibitors of LPO both
in rat cerebral microsomes and in rat brain homogenate induced
by NADPH or Fe2+, respectively. Compounds decreased the
formation of TBARs in a concentration-dependent manner. IC50
values of the compounds were in micromolar range (Table 2).
The inhibition of microsomal LPO could be explained by the
withdrawal of electron from NADPH cytochrome c reductase,
which is the enzyme involved in the generation of free radical
and in the initiation of LPO in the presence of NADPH and
Fe3+/ADP complex. The compounds can react with the initia-
There was no difference between the inhibitory action of
alkyl-esters and substituted alkyl-esters of (3R,16S)-apovin-
caminate (7a-f) and (3S,16R)-apovincaminate (4a-f) on LPO.
The configuration of the ethyl substituent at the D/E ring
junction of eburnane skeleton did not change the inhibitory
effects of these compounds on LPO.
On the one hand, esterification of the 14-carboxylic group
has a marked effect on LPO. On the other hand, the acylation,
for example, esterification of the 2-hydroxyethyl group of trans-

482 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Nemes et al.
Table 3. Percent Value of Inhibition of Diazepam-Induced Amnesia by
(3S,16R)-trans-Apovincaminic Esters (4b-e) and (3R,16S)-trans-
Apovincaminic Esters (7b-f) in the One-Trial Passive Avoidance Test
in Mice
inhibition of diazepam-induced amnesia (%)
cmpd No.
4b
4c
4d
4e
7b
7c
7d
7e
0.1 mg/kg p.o.
10.0 mg/kg p.o.
50
72
64
23
74
79
16
0
56
44
24
16
65
100
4
0
7f
3
0
15
100
1 vinpocetine
Figure 3. Effect of (3S,16R)-trans-apovincaminic esters 4b,c and
(3R,16S)-trans-apovincaminic esters 7b,c and reference drugs vinpo-
cetine and idebenone on NADPH (400 µM) evoked LPO in rat brain
microsomes. Each point shown is the mean ( SEM of 3-8 separate
experiments performed in triplicate, determined from 7-9 concentrations.
modifications in the ester side-chain (R substituents), whereas
the change the cis-ring fusion to trans in the apovincaminic
skeleton increased the antioxidant activity of the compounds.
Behavioral Tests for Screening In Vivo Drug Effect. The
in vivo effectiveness of the various chemical structures on the
learning memory functions was studied in experimental amnesia
models using a passive avoidance test and a water-labyrinth
learning task in mice and rats, respectively.
In the passive avoidance paradigm, as the first step in testing
of antiamnesic activity of the compounds, vinpocetine at an oral
dose of 10 mg/kg exerted a marked protective effect against
anterograde amnesia induced by diazepam. However, some of
trans-derivatives also proved to be active (Table 3). Two
compounds (7b and 7c) with trans-(3R,16S)-apovincaminate
moiety showed the most considerable antiamnesic activity
against diazepam-induced cognitive impairment at both doses
tested (Table 3).
Their (3S,16R)-enantiomers, 4b and 4c (Table 1), seemed to
be also effective. The memory improving effect of 7b and 7c
compounds appeared already at the lower 0.1 mg/kg oral dose
and was comparable to that of vinpocetine (cis-configuration)
exerted at 10 mg/kg dose. Whereas compound 7c showed a
quick metabolism to 7b, this latter structure has been chosen
for further in vivo examination.
Figure 4. Effect of (3S,16R)-trans-apovincaminic esters 4b,c and
(3R,16S)-trans-apovincaminic esters 7b,c and reference drugs vinpo-
cetine and idebenone on Fe2+ (200 µM) evoked LPO in rat brain
homogenates. Each point shown is the mean ( SEM of 3-8
independent experiments performed in triplicate, determined from 6-9
concentrations.
The water-labyrinth test is a classical maze paradigm making
use of the motivation of the rats to escape from the water.27
The method is appropriate for measuring the potential improving
effect of test compounds on memory disturbance induced by
diazepam, as an impairing agent. Some selected compounds
were investigated in this complex learning task. 3S,16S)-
Apovincaminic acid ethylester, 1, its 3-epimer 3R,16S)-apovin-
caminic acid ethylester 7a, 3R,16S)-apovincaminic acid hy-
droxyethylester, 7b, the 3-epimer (3S,16S)-apovincaminic acid
hydroxyethylester 14 were compared with the primary in vivo
metabolites of ethylesters,28 the epimeric cis-apovincaminic28
(15) and trans-apovincaminic (13a) acids.
The control animals produced a progressive improvement in
the water-labyrinth acquisition from day to day, while diazepam
at a dose of 5 mg/kg ip disrupted the normal learning process,
resulting in a significant increase in the number of directional
turning errors.
Figure 5 presents the percent reversal of diazepam-induced
amnesia exerted by various apovincaminic acid derivatives given
at an oral dose of 5 mg/kg. In the group of the compounds
studied (3R,16S)-apovincaminic acid ethylester 7a produced the
most moderate protective effect (just missed the 5% level of
statistical significance) against learning deficit induced by
diazepam (Figure 5), while (3R,16S)-apovincaminic acid hy-
apovincaminate enantiomers with acetyl-, propionyl-, benzoyl-,
and 4-chlorobenzoyl groups did not change the antioxidant
efficacy of the compounds. Probably the esterification of the
14-carboxylic group enhances the lipophilicity of the com-
pounds, which is involved in the increase of inhibitory potency
of the compounds on LPO.
Compounds (4a-f, 7a-f) were 2–5-fold more potent in
inhibition of LPO evoked by NADPH and Fe3+-ADP complex
in microsomes than in Fe2+-induced LPO in brain homogenate.
Fe2+ stimulates membrane LPO more than Fe3+ does. It can
be explained by the greater solubility of Fe2+ salts in solution,
higher reactivity of alkoxy radicals, and the faster rate of
decomposition of lipid peroxides by Fe2+
.
Inhibitory effect of these compounds on LPO was comparable
to antioxidant effect of well-known neuroprotective idebenone
and flavonoid quercetine. trans-Apovincaminic acid esters
(4a-f, 7a-f) were more active in inhibition of LPO than
synthetic DL-R-tocopherol.
These results indicate that a series of 2′-hydroxyethyl and
2′-acyloxyethyl trans-apovincaminates possess potent antioxi-
dant activity shown by their strong inhibitory action on LPO
induced by different agents. Structure–activity analysis within
a series of analogues indicated a high tolerance toward structural

2′-Hydroxyethyl trans-ApoVincaminate DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 483
for the treatment of oxidative stress-related neurodegenerative
disorders and mild cognitive impairments.
Experimental Section
Chemistry. Melting points were determined with a Büchi 510
apparatus and are uncorrected. The [R]D observed using a Perkin-
Elmer 243B polarimeter. IR spectra were recorded on a Perkin-
1
Elmer Spectrum 1000 FT-IR spectrometer using KBr pellets. H
NMR spectra were recorded on a Varian INOVA 500 spectrometer
(500 MHz for 1H). MS spectrometric measurements were performed
on a Finnigan MAT 95SQ mass spectrometer using EI (70 eV,
220 °C source temperature) ionization method. High-resolution MS
measurements were carried out on a Finnigan MAT 95XP mass
spectrometer; perfluorotributylamine was the reference compound
using EI ionization technique. Where analyses are indicated by
symbols of the elements, analytical results obtained for those
elements were (0.4% of the theoretical values. All reagents and
solvents were of commercial quality. Starting (15aS) and (15aR)
diethyl 15a-ethyl-hexahydroindolo[2,3-a]pyrano[3,2-i]quinolizine-
14,14-dicarboxylates (8a,b) were prepared according to published
method.18
Figure 5. Percent restoring effect of vincaminic acid derivatives in
diazepam-induced memory deficiency in the water-labyrinth test
(calculated as described in Data Analysis). *p < 0.05, ***p < 0.001,
ANOVA followed by posthoc Duncan test was performed on group
means pooled over days.
droxyethylester 7b and (3S,16S)-apovincaminic acid hydroxy-
ethylester 14 proved to be the most effective molecules with
restoring effects of 81.1 and 80.3%, respectively. (3S,16S)-
Apovincaminic acid ethylester 1 (vinpocetine) and the primary
metabolites (15 and 13a) showed less but statistically significant
memory improving activity against diazepam amnesia (their
protective effect was measured to be 56.6, 66.7, and 71.3%,
respectively).
(1S,12bR)-1-(2′,2′-Diethoxycarbonylethyl)-1-ethyl-1,2,3,4,
6,7,12,12b-octahydroindolo[2,3-a] quinolizine 11a. To a mix-
ture of compound 8a (50 g, 0.11 mol), ethanol (500 mL), and acetic
acid (15 mL) sodium tetrahydroborate (9g, 0.197 mol) in ethanol
(300 mL) was added at 20–25 °C for 1 h under nitrogen, then 25%
NH4OH solution (15 mL) was added and the mixture was stirred
for 2 h at 50 °C. After completion of the reaction, water (270 mL)
was added at 20 °C and stirred for 1 h at 5 °C. The crystals obtained
were filtered and washed with water (2 × 200) and ethanol (40
It appears, that the alternation in the cis–trans-configuration
of eburnane skeleton alone did not modify the memory
improving property of the compounds, however, the hydroxy-
ethylesters possess a higher biological activity in the water-
labyrinth memory test. Putatively, the higher in vivo effective-
ness of the hydroxyethylesters is due to their better metabolic
stability.
mL) to give 34.8 g (73%) of 11a, mp 99 °C (ethanol), [R]D
)
1
+25° (c 1, DMF). H NMR (DMSO-d6, δTMS ) 0.00 ppm; data
are given according to the eburnane skeletal numbering): 0.52 (3H,
t, H3-21); 1.16 (1H, td, Hax-17); 1.21 (6H, t, OCH2CH3 × 2); 1.43
(1H, m, Heq-18); 1.46 (1H, m, Heq-17); 1.51 (1H, m, Hx-20); 1.59
(1H, m, Hy-20); 1.67 (1H, m, Hax-18); 2.35 (1H, m, Hax-19); 2.39
(1H, dd, Hx-15); 2.49 (1H, m, Hax-5); 2.53 (1H, dd, Hy-15); 2.54
(1H, m, Heq-6); 2.75 (1H, m, Hax-6); 2.95 (2H, m, Heq-19, Heq-5);
3.31 (1H, s, H-3); 3.67 (1H, t, H-14); ∼4.19 (4H, m, (4H, OCH2CH3
x 2); 6.94 (1H, t, H-10); 7.02 (1H, t, H-11); 7.34 (1H, d, H-9);
7.35 (1H, d, H-12); 9.98 (1H, brs, NH). MS (EI) m/z (%): 426
(M+, 13.7), 425 (10.2), 411 (1.2), 381 (5.5), 353 (1.8), 267 (100),
253 (1.5), 237 (4.7), 197 (3.8), 170 (4.2), 169 (4.5). HRMS calcd
for C25H34N2O4, 426.25131; found, 426.25221 (delta: 2.1 ppm).
Anal. (C25H34N2O4) C, H, N.
Conclusions
In the present study, we showed that all new compounds with
trans-apovincaminate moiety (4b-f, 7b-f) were active inhibi-
tors of LPO both in rat cerebral microsomes and in rat brain
homogenate induced by enzymic (adding of NADPH in the
presence of Fe3+/ADP complex) and nonenzymic way (by
addition of Fe2+).
(1R,12bS)-1-(2′,2′-Diethoxycarbonylethyl)-1-ethyl-1,2,3,4,
6,7,12,12b-octahydroindolo[2,3-a]quinolizine 11b. From 8b,
mp 98 °C (ethanol), [R]D ) -21° (c 1, DMF). Anal. (C25H34N2O4)
C, H, N.
We have demonstrated by the present SAR study that
probably both the change of the ring fusion to trans in the
apovincaminate skeleton and the substitution of ester chain with
2′-hydroxyethyl and 2′-acyloxyethyl groups are responsible for
the increasing inhibitory potency of the compounds on LPO.
Ethyl (1S,12bR)-1-Ethyl-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-
a]quinolizine-1-(2′-hydroxy-imino)-propionate 12a. To (1S,12bR)-
11a (30.0 g, 70 mmol) in EtOH (140 mL), a solution of potassium
hydroxide (3.84 g, 70 mmol) in water (48 mL) was added and stirred
for 2 h under nitrogen at 30–35 °C. The pH was adjusted to 7 with
AcOH (2 mL) and the solution was evaporated in vacuo. The
residue was dissolved in AcOH (230 mL), and a portion of aqueous
acetic acid (80 mL) was distilled. A solution of sodium nitrite (9.2
g, 133 mmol) in water (25 mL) was added at 10 °C and stirred for
2 h at 10–15 °C, and then a mixture of concd HCl (25 mL) and
water (48 mL) was added to give 25 g (69.6% yield) of 12a HCl,
mp 233–236 °C, [R]D ) +23° (c 1, DMF). Aqueous NH4OH 25%
solution (43 mL) was added to give a solution. After 30 min of
stirring, water (85 mL) was added and the mixture was stirred for
1 h, then filtered, and washed with 20% EtOH/water (2 × 20 mL)
to yield 22 g (65%) of 12a, mp 168–170 °C, [R]D ) +53° (c 1,
DMF). 1H NMR (DMSO-d6, δTMS ) 0.00 ppm; data are given
according to the eburnane skeletal numbering): 0.58 (3H, t, H3-
21); 1.23 (3H, t, OCH2CH3); 1.37 (1H, td, Hax-17); 1.40 (1H, m,
Heq-18); 1.50 (1H, m, Hx-20); 1.51 (1H, m, Heq-17); 1.57 (1H, m,
Hy-20); 1.64 (1H, m, Hax-18); 2.33 (1H, td, Hax-19); 2.48 (1H, td,
Besides that they may protect the brain against the free
radical-induced oxidative membrane damage, results obtained
in behavioral studies suggest that some compounds of the series
of 2′-hydroxyethyl and 2′-acyloxyethyl trans-apovincaminates
have considerable cognitive enhancer activity as well. However,
comparing data of LPO assay with memory results, there is not
a close correlation between antioxidant property and cognitive
improving activity of the drugs tested. Although differences in
pharmacokinetic parameters and metabolism of various mol-
ecules studied might at least partly explain the discrepancy
between in vitro and in vivo data, it appears more probable that
the antioxidant property does not underlie the cognitive enhanc-
ing profile of the compounds. The considerable influence of 7b
on lipid peroxidation may be an adventageous additional effect

484 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Nemes et al.
Hax-5); 2.55 (1H, m, Heq-6); 2.75 (1H, m, Hax-6); 2.94 (1H, m,
Heq-5); 2.95 (1H, m, Heq-19); 3.02 (1H, d, Hx-15); 3.23 (1H, d,
Hy-15); 3.33 (1H, s, H-3); 4.22 (2H, q, OCH2CH3); 6.94 (1H, t,
H-10); 7.02 (1H, t, H-11); 7.35 (1H, d, H-9); 7.40 (1H, d, H-12);
10.20 (1H, s, NH); 12.42 (1H, s, OH). MS (EI) m/z (%): 383 (M+,
53.5), 382 (29.9), 366 (52.8), 354 (11.5), 321 (11.7), 310 (13.5),
307 (10.6), 292 (100), 278 (8.8), 267 (30.8), 253 (34.6), 237 (41.2),
223 (8.3), 197 (23.4), 184 (13.5), 170 (25.4), 169 (26.8). HRMS:
calcd for C22H29N3O3, 383.22034; found, 383.21994 (delta: -1.1
ppm). Anal. (C22H29N3O3) C, H, N.
Ethyl (1R,12bS)-1-Ethyl-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-
a]quinolizine-1-(2′-hydroxy-imino)-propionate 12b. From 11b,
mp 168–169 °C, [R]D ) –53° (c 1, DMF). Anal. (C22H29N3O3) C,
H, N.
m, OCH2CH2OH); 6.39 (1H, s, H-15); 7.08 (1H, m, H-10); 7.09
(1H, m, H-11); 7.27 (1H, d, H-12); 7.42 (1H, d, H-9). MS (EI) m/z
(%): 366 (M+, 65.8), 365 (41.0), 351 (6.7), 337 (100), 293 (28.8),
292 (24.3), 277 (11.3), 262 (6.8), 249 (26.5), 248 (31.0). HRMS
calcd for C22H26N2O3, 366.19379; found, 366.19351 (delta: -0.8
ppm).
2′-Hydroxyethyl (3S,16R)-Apovincaminate·HCl, 4b ·HCl. See
Table 1. 1H NMR (DMSO-d6, δTMS ) 0.00 ppm): 0.71 (3H, t, H3-
21); 0.88 (1H, m, Hx-20); 1.73 (1H, td, Hax-17); 1.91 (1H, d, Heq-
18); 2.02 (1H, m, Hax-18); 2.10 (1H, d, Heq-17); 2.13 (1H, m, Hy-
20); 2.94 (1H, d, Heq-6); 3.23 (1H, m, Hax-19); 3.36 (1H, m, Hax-
6); 3.50 (2H, m, Hax-5 + Heq-19); 3.68 (1H, m, Heq-5); 3.70 (2H,
m, OCH2CH2OH); 4.36 (2H, m, OCH2CH2OH); 4.75 (1H, brs, H-3);
6.50 (1H, s, H-15); 7.16 (1H, m, H-10); 7.20 (1H, m, H-11); 7.36
(1H, d, H-12); 7.55 (1H, d, H-9); 10.65 (1H, brs, NH). MS (EI)
m/z (%): 366 (M+, 64.5), 365 (38.2), 351 (4.9), 337 (100), 293
(29.8), 292 (24.2), 277 (10.5), 262 (6.1), 249 (29.5), 248 (35.0).
HRMS calcd for C22H26N2O3, 366.19379; found, 366.19337 (delta:
-1.2 ppm). Anal. (C22H27ClN2O3) C, H, N.
2′-Hydroxyethyl (3S,16S)-Apovincaminate 14. See Table 1.
1H NMR (DMSO-d6, δTMS ) 0.00 ppm): 0.81 (1H, td, Hax-17);
0.95 (3H, t, H3-21); 1.33 (1H, m, Heq-18); 1.49 (1H, d, Heq-17);
1.58 (1H, m, Hax-18); 1.84 (2H, q, H2-20); 2.42 (1H, m, Heq-6);
2.43 (1H, m, Hax-19); 2.51 (1H, m, Heq-19); 2.92 (1H, m, Hax-6);
3.13 (1H, td, Hax-5); 3.21 (1H, dd, Heq-5); 3.70 (2H, m,
OCH2CH2OH); 4.07 (1H, s, H-3); 4.32 (1H, m) and 4.38 (1H, m),
[OCH2CH2OH]; 4.77 (1H, t, OH); 6.18 (1H, s, H-15); 7.06 (1H,
m, H-10); 7.09 (1H, m, H-11); 7.21 (1H, d, H-12); 7.43 (1H, d,
H-9). MS (EI) m/z (%): 366 (M+, 41.8), 337 (100), 296 (62.0),
293 (7.7), 277 (2.7), 252 (18.7), 249 (7.5). HRMS calcd for
C22H26N2O3, 366.19379; found, 366.19445 (delta: 1.8 ppm). Anal.
(C22H26N2O3) C, H, N.
2′-Acetyloxyethyl (3R,16S)-Apovincaminate 7c. General Pro-
cedure for Compounds 4c and 7c. Procedure B. To 13a sodium
salt (4 g, 11.6 mmol) in DMF (15 mL), 2-chloroethyl acetate (3g,
24mmol) was added. The mixture was stirred under nitrogen at
100 °C for 4 h. After cooling to rt, water (25 mL) was added, and
the solutions was stirred for 1 h, filtered, and washed with water
(3 × 5 mL) to obtain 3.6g (76%) of amorphous 7c.
Ethyl (3R,16S)-Apovincaminate 7a. From a mixture of p-
toluenesulfonic acid hydrate (25 g, 130 mmol) and toluene (360
mL), toluene (60 mL) was distilled. The solution was cooled to 80
°C and 12a (20 g, 52 mmol) was added. The mixture was boiled
under reflux for 4 h. After cooling to rt, water (165 mL) and 25%
NH4OH solution (16.5 mL) was added. The organic layer was
separated, and the water was extracted with toluene (40 mL). The
combined organic layers were washed with water (60 mL) and dried
(MgSO4), and the filtrate was clarified with Al2O3 (5 g) and Norit
(1 g). The filtrate was evaporated and crystallized from ethanol–
water 70:30 (120 mL). The separated crystals were filtered and
washed with 70% aqueous ethanol (10 mL) to obtain 13.8 g (75%)
of 7a, mp 132–134 °C, [R]D ) –145° (c 1, CHCl3), identical with
an authentic sample.14
Ethyl (3S,16R)-Apovincaminate 4a. Compound 4a14 was
obtained from 12b under the same conditions.
(3R,16S)-Apovincaminic Acid 13a. A mixture of compound 7a
(5 g, 14 mmol), ethanol (30 mL), and sodium hydroxide (0.68 g,
17 mmol) was refluxed under nitrogen for 3 h. After completion
of the hydrolysis, the solution was evaporated in vacuo. Acetone
(20 mL) was added to the residue, and the mixture was refluxed
for 10 min. The suspension was cooled to 10 °C, stirred for 1 h,
and filtered to yield 4.6 g (95%) of 13a sodium salt, mp 270 °C
(dec.), [R]D ) -106° (c 1, CH3OH). The crystals were dissolved
in water (35 mL), and the solution was acidified with acetic acid
25% to pH 4 to afford 13a (4.25 g, 94%), mp 201–202, [R]D
)
7c·HCl: Crude 7c was dissolved in 2-propanol (30 mL) and
acidified with HCl 24% in 2-propanol to pH 4 to yield 7c·HCl
(3.4 g, 65%), mp 188–189 °C, [R]D ) –118.9° (c 0.2, MeOH). 1H
NMR (DMSO-d6, δTMS ) 0.00 ppm): 0.70 (3H, t, H3-21); 0.88
(1H, m, Hx-20); 1.73 (1H, td, Hax-17); 1.93 (1H, d, Heq-18); 2.05
(1H, m, Hax-18); 2.11 (1H, d, Heq-17); 2.12 (1H, m, Hy-20); 2.95
(1H, d, Heq-6); 3.23 (1H, m, Hax-19); 3.32 (1H, m, Hax-6); 3.50
(2H, m, Hax-5 + Heq-19); 3.67 (1H, m, Heq-5); 4.35 (2H, m,
OCH2CH2OCO); 4.55 (2H, m, OCH2CH2OCO); 4.74 (1H, brs,
H-3); 6.45 (1H, s, H-15); 7.17 (1H, m, H-10); 7.20 (1H, m, H-11);
7.36 (1H, d, H-12); 7.55 (1H, d, H-9); 10.62 (1H, brs, NH). MS
(EI) m/z (%): 408 (M+, 69.6), 407 (40.5), 393 (2.6), 380 (55.0),
379 (44.5), 366 (5.5), 349 (5.1), 337 (8.5), 321 (11.0), 307 (7.0),
293 (100), 292 (59.7), 277 (12.1), 248 (28.3). HRMS calcd for
C24H28N2O4, 408.20436; found, 408.20407 (delta: -0.7 ppm). Anal.
(C24H29ClN2O4) C, H, N.
1
–133° (c 1, DMF). 13a, 13b: H NMR (DMSO-d6, δTMS ) 0.00
ppm): 0.58 (1H, m, Hx-20); 0.61 (3H, m, H3-21); 1.44 (1H, td,
Hax-17); 1.57 (1H, d, Heq-18); 1.79 (1H, m, Hax-18); 1.81 (1H, m,
Hy-20); 1.96 (1H, d, Heq-17); 2.28 (1H, td, Hax-19); 2.53 (1H, td,
Hax-5); 2.62 (1H, d, Heq-6); 2.80 (1H, m, Hax-6); 2.98 (1H, dd, Heq-
19); 3.01 (1H, s, H-3); 3.04 (1H, dd, Heq-5); 6.28 (1H, s, H-15);
7.05 (1H, m, H-10); 7.07 (1H, m, H-11); 7.31 (1H, d, H-12); 7.41
(1H, d, H-9). MS (EI) m/z (%): 322 (M+, 58.4), 321 (37.8), 307
(6.8), 294 (52.6), 293 (100), 292 (76.6), 277 (10.2), 262 (6.9), 252
(7.7), 249 (35.1), 248 (29.9), 219 (8.8), 123 (8.6). HRMS calcd
for C20H22N2O2, 322.16758; found, 322.16783 (delta: 0.8 ppm).
(3S,16R)-Apovincaminic Acid 13b. From 4a, mp 200–201, [R]D
) +130.8° (c 1, DMF).
2′-Hydroxyethyl (3R,16S)-Apovincaminate 7b. General
Procedure for Compounds 4b, 7b, and 14 (R ) Hydro-
xyethyl). Procedure A. Compound 7a (15 g, 42 mmol) and KOt-
Bu (1 g, 9 mmol) in ethylene glycol (300 mL) was heated for 4 h
at 100 °C. The solution was cooled to rt, and water (300 mL) was
added. The precipitated crystals were filtered and washed with water
(3 × 200) to afford 14.1 g (91%) of 7b: mp 87–88 °C, [R]D ) –
95 ° (c 1, DMF).
2′-Acetyloxyethyl (3S,16R)-Apovincaminate HCl, 4c ·HCl. See
Table 1. Anal. (C24H29ClN2 O4) C, H, N.
2′-Benzoyloxyethyl (3R,16S)-Apovincaminate 7e. General
Procedure for Compounds 4d-f and 7d-f. Procedure C. To a
solution of 7b (4.44 g, 12 mmol) in chlorobenzene (60 mL),
triethylamine (1.83 g, 18 mmol) and benzoylchloride (2.5 g, 18
mmol) were added. The mixture was stirred for 30 min at 40 °C,
then NaHCO3 15% solution (15 mL) was added. The organic layer
was washed with water (2 × 20 mL), dried, handled with HCl in
dioxane, then chilled, filtered, and washed with acetone to obtain
7e·HCl (4.8 g, 79%), mp 216–217 °C, [R]D ) –111.9° (c 1,
MeOH). 1H NMR (DMSO-d6, δTMS ) 0.00 ppm): 0.65 (3H, t, H3-
21); 0.85 (1H, m, Hx-20); 1.66 (1H, td, Hax-17); 1.91 (1H, d, Heq-
18); 2.01 (1H, m, Hax-18); 2.03 (1H, d, Heq-17); 2.09 (1H, m, Hy-
20); 2.94 (1H, d, Heq-6); 3.21 (1H, m, Hax-19); 3.33 (1H, m, Hax-
6); 3.48 (1H, m, Heq-19); 3.49 (2H, m, Hax-5); 3.68 (1H, m, Heq-
7b Tartrate: The mixture of (+)-L-tartaric acid (9.65 g, 64
mmol) in water (10 mL) and ethanol (115 mL), and 7b (23 g) in
ethanol (190 mL) was stirred in 55 °C. The resulting crystals were
filtered to obtain 30.7 g of 7b tartrate: mp 157–159 °C, [R]D
)
–72 ° (c ) 1, DMF). 1H NMR (DMSO-d6, δTMS)0.00 ppm):
0.55–0.65 (4H, m, H3-21+Hx-20); 1.45 (1H, td, Hax-17); 1.58 (1H,
d, Heq-18); 1.80 (1H, m, Hax-18); 1.84 (1H, m, Hy-20); 1.98 (1H,
d, Heq-17); 2.28 (1H, td, Hax-19); 2.53 (1H, m, Hax-5); 2.63 (1H, d,
Heq-6); 2.80 (1H, m, Hax-6); 2.98 (1H, m, Heq-19); 3.03 (1H, s,
H-3); 3.05 (1H, m, Heq-5); 3.70 (2H, m, OCH2CH2OH); 4.34 (2H,

2′-Hydroxyethyl trans-ApoVincaminate DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 485
5); 4.62 (2H, m, OCH2CH2OCO); 4.70 (2H, m, OCH2CH2OCO);
4.70 (1H, brs, H-3); 6.41 (1H, s, H-15); 7.06 (1H, m, H-11); 7.13
(1H, m, H-10); 7.34 (1H, d, H-12); 7.53 (1H, d, H-9); 7.54 (2H,
m), 7.68 (1H, m); 7.95 (2H, m) [ArH]. MS (EI) m/z (%): 470 (M+,
69.2), 469 (43.5), 455 (4.5), 442 (64.3), 441 (100), 400 (3.1), 349
(5.1), 321 (3.2), 293 (6.0), 277 (10.0), 261 (9.3), 248 (29.7), 149
(48.4), 105 (29.7), 77 (11.9). HRMS calcd for C29H30N2O4,
470.22001; found, 470.21962 (delta: -0.8 ppm). MS (EI) m/z (%):
366 (M+, 64.5), 365 (38.2), 351 (4.9), 337 (100), 293 (29.8), 292
(24.2), 277 (10.5), 262 (6.1), 249 (29.5), 248 (35.0). Anal.
(C29H31ClN2O4) C, H, N.
percent inhibition of TBARS formation (mean values of replicates
were used for calculation) obtained in the presence of our
compounds or well-known reference drugs. IC50 values (i.e.,
concentration of compound giving 50% inhibition of TBARS
formation) were calculated from concentration-effect curves by
sigmoid fitting using Origin 6.0 software (Microcal).
In Vivo Assays. Experimental animals: male Wistar rats (bred
at Gedeon Richter Ltd.) and male NMRI mice (supplied by LATI
Kft, Hungary) weighing 200–220 g and 25–28 g, respectively, were
used in the behavioral studies. The animals, housed five per cage,
were kept at 22 ( 1 °C, on a 12:12 h light/dark cycle (light on at
6:00 a.m.) in a laboratory animal care unit and commercial pellet
rat-mouse feed and tap water were given ad lib.
2′-Benzoyloxyethyl (3S,16R)-Apovincaminate HCl 4e · HCl.
See Table 1. Anal. (C29H31ClN2O4) C, H, N.
All the procedures carried out on animals had been approved by
the local ethical committee and conformed to the rules and
principles of the 86/609/EEC directive.
2′-Propionyloxyethyl (3S,16R)-Apovincaminate HCl, 4d ·HCl.
See Table 1. MS (EI) m/z (%, same as 7d·HCl): 422 (M+, 71.3),
421 (42.3), 407 (2.7), 394 (55.4), 393 (44.3), 349 (5.3), 321 (12.1),
307 (6.2), 293 (100), 292 (57.4), 277 (10.7), 248 (26.2). HRMS
calcd for C25H30N2O4, 422.22001; found, 422.22004 (delta: 0.1
ppm).
1. Passive Avoidance Test in Mice. The passive avoidance
apparatus consisted of an illuminated and a dark chamber (15 ×
15 × 20 cm) separated by a guillotine door. During the habituation
session, each mouse was placed in the lighted compartment and
the time taken to enter the dark chamber was determined (step-
through latency). The animals with higher latency than 30 s were
excluded from the experiment. On the next day, the acquisition
trial was performed. After entering the dark compartment, the door
was closed and the mouse received a footshock (1 mA for 3 s)
within 30 s. A total of 24 hours after the acquisition trial, a single
retrieval trial was conducted and the step-through latency was
measured (300 s was the upper cutoff time). For inducing
anterograde amnesia, the animals were treated intraperitoneally with
3 mg/kg of diazepam 30 min before the acquisition trial. The test
compounds were administered orally at 0.1 or 10 mg/kg doses,
respectively, 1 h prior to the acquisition trial. All the experimental
groups consisted of 10 animals. The percentage value of the
protective effect (P%) was calculated by the following formula
2′-Propionyloxyethyl (3R,16S)-Apovincaminate HCl, 7d ·HCl.
See Table 1) Anal. (C25H31ClN2O4) C, H, N.
2′-(4-Chlorobenzoyl)-oxyethyl (3S,16R)-Apovincaminate Meth-
anesulfonate, 4f ·CH3SO3H. See Table 1. MS (EI) m/z (%, same
as 7f · CH3SO3H): 504 (M+, 61.5), 503 (41.8), 476 (81.8), 475
(100), 434 (3.7), 349 (5.6), 321 (4.1), 293 (8.5), 277 (11.8), 261
(10.6), 249 (38.6), 183 (38.6), 139 (29.7), 111 (8.9). HRMS calcd
for C29H29N2O4Cl, 504.18104; found, 504.18049 (delta: -1.1 ppm).
2′-(4-Chlorobenzoyl)-oxyethyl
(3R,16S)-Apovincaminate
CH3SO3H, 7f·CH3SO3H. See Table 1. Anal. (C30H33ClN2O7S) C,
H, N.
Pharmacology. In Vitro Assays. Experimental animals: male
Hannover-Wistar rats (of our own breeding colony, Gedeon Richter)
weighing 180–220 g were used.
1. Effect on the NADPH-Induced Lipid Peroxidation in
Rat Brain Microsomes.19 The whole rat brain was homogenized
in a 10 vol (w/v) of ice-cold 0.25 M sucrose solution. The
homogenate was centrifuged at 15000 g for 10 min at 4 °C.
The supernatant was centrifuged at 78000 g for 60 min at 4 °C.
The pellet, designated as microsomes, was suspended in 0.15 M
KCI solution. The protein content was determined and then adjusted
to 10 mg/mL concentration. The preparation was stored at -70 °C
until use. The microsomes (0.2 mg protein) were incubated in 1.0
mL of medium (50 mM TRIS-HCI (pH 6.8)), 0.2 mM FeCI3, 1
mM KH2PO4, and 0.5 mM ADP for 20 min at 37 °C with or without
compounds. The LPO was induced by adding 0.4 mM NADPH.
After 20 min, the reaction was stopped by adding 0.375 mL of a
stopping solution containing trichloroacetic acid (TCA) of 40% and
5 M HCI in a 2:1 ratio. Acidified samples were mixed with 1 mL
of 1% TBA solution and were placed in boiling water for 10 min.
The samples were centrifuged at 2000 g for 10 min at 4 °C. Optical
density was measured spectrophotometrically at 535 nm in a Hitachi
150-20 double beam spectrophotometer.
Ttreated+DIA - T
Tplacebo - Tplacebo+DIA
P% )
placebo+DIA × 100
(1)
where T means the latency to enter the dark chamber in control
(Tplacebo), memory-impaired (Tplacebo+DIA), and impaired, drug-treated
(Ttreated+DIA) groups.
2.Water-Labyrinth Test in Rats. In the water-labyrinth task,
the rats had to maneuver through three choice points of a labyrinth
system to reach a platform that allowed them to escape from the
water. The water tank (1 m long, 60 cm wide, and 60 cm deep)
was filled with water of 24 ( 2 °C to a depth of 30 cm and a
removable labyrinth-system consisting of vertical metal plates was
placed into the pool. The procedure for testing water-labyrinth
acquisition process was carried out on 4 consecutive days. On day
1 (pretraining day), the rats were habituated to the test environment
without plates and they were conditioned three times to swim in
the tank from the start point to the platform. During the next days
(days 2–4), the labyrinth system was in place and the animals were
trained to swim through the labyrinth in three daily trials separated
by approximately 25 min intertrial rest periods. The number of
directional turning errors committed at all the three choice points
was measured as a variable reflecting learning performance. If the
rat did not find the platform within 5 min, it was assisted to the
end of the labyrinth by the experimenter and the number of errors
was recorded as 12.
The effect of compounds on the memory impairment elicited
by diazepam was investigated at an oral dose of 5 mg/kg given 1 h
prior to the first daily trial. Vinpocetine, cis-, and trans-apovin-
caminic acid were dissolved in 2% ascorbic acid solution, 7b was
dissolved in distilled water, while 7a and 14 were dispersed in 5%
(v/v) Tween 80. A dose of 5 mg/kg diazepam, suspended in 5%
(v/v) Tween 80 solution, was injected intraperitoneally 30 min
before the first swimming. Each study included a nonimpaired
solvent control, a memory-impaired group, and an impaired group
treated with the test compound. There were 10 animals in each
experimental group. trans-Apovincaminic acid was examined in
two separated experiments and data obtained were pooled.
2. Effect on the Fe2+-Induced Lipid Peroxidation in Rat
Brain Homogenate.20 The whole brain was homogenized in 9 vol
of ice-cold Krebs-Ringer’s buffer containing 15 mM HEPES, pH
7.4, 140 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 0.7 mM MgCl2,
1.4 mM KH2PO4, and 10 mM glucose. The rat brain homogenate
(10 mg protein/ml) was used immediately. Cerebral homogenate
(200 µL) was incubated at 37 °C for 20 min with or without
compounds (added in a volume of 5 µL). The Fe2+-induced LPO
was induced by adding 5 µL of 8 mM Fe2(NH4)2(SO4)2 solution.
After 20 min, the reaction was stopped by addition of the stopping
solution (12.5% TCA in 0.8 M HCl). Acified samples were
centrifuged at 2000 g for 10 min at 4 °C. A total of 1 mL of 1%
TBA solution was added to a 0.5 mL aliquot of the supernatant,
and then the samples were placed in boiling water for 20 min. The
optical density was determined at 535 nm with a Hitachi 150-20
spectrophotometer.
Data Analysis. The inhibitory effect of the compounds on LPO
was determined using a minimum of six concentrations, and
experiments were performed 1-8 times. Results are expressed as

486 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Nemes et al.
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Data Analysis. Results are given as percent reversal by the
compound of the amnesia calculated from the group-means of
pooled errors for all the trials in the training days using the following
formula
(12) Paróczai, M. Not published.
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Nerr of amnestic - Nerr of compound
% )
× 100
(2)
Nerr of amnestic - Nerr of control
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Kárpáti, E.; Sohár, P.; Kessel, M.; Kreidl, J. Syntheses and cardio-
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where Nerr means the number of errors.
Statistical comparisons between parameters of each group were
made by ANOVA (two-way repeated measures analysis of variance)
using “groups” as the independent between groups factor and “days”
as the repeated measures factor. Posthoc comparisons (Duncan-
test) were performed in case of a significant between-groups effect.
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Acknowledgment. The authors thank Mr. János Kóti and
Ms. Erika Sziki for the IR spectra, Dr. Viktor Háda for the MS
data, and Ms. Zsuzsanna Sánta for her contribution in the NMR
measurements and interpretations.
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L.; Farkas, M.; Juhász, I.; Gere, A.; Paróczai, M.; Lapis, E.; Szekeres,
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Supporting Information Available: Table of elemental analy-
ses, IR spectra, and NMR spectra for homologue compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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