Highly potent activity of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3- ene-1,2-diol in animal models of parkinson's disease

Article abstract of DOI:10.1021/jm2001579

(1R,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 possesses potent antiparkinsonian activity in both MPTP and haloperidol animal models. The use of compound 1 resulted in nearly full recovery of the locomotor and exploratory activities and was as effective as the comparator agent (levodopa). All eight stereoisomers of compound 1 have been synthesized and the influence of the absolute configuration on the antiparkinsonian activity of compound 1 was shown.

Full text of DOI:10.1021/jm2001579

ARTICLE
pubs.acs.org/jmc
Highly Potent Activity of (1R,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-
3-ene-1,2-diol in Animal Models of Parkinson’s Disease
Oleg V. Ardashov, Alla V. Pavlova, Irina V. Il’ina, Ekaterina A. Morozova, Dina V. Korchagina,
Elena V. Karpova, Konstantin P. Volcho,* Tat’yana G. Tolstikova, and Nariman F. Salakhutdinov
N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences, Lavrentiev Avenue, 9,
630090 Novosibirsk, Russian Federation
S Supporting Information
b
ABSTRACT:
(1R,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 possesses potent antiparkinsonian activity in both MPTP and
haloperidol animal models. The use of compound 1 resulted in nearly full recovery of the locomotor and exploratory activities and
was as effective as the comparator agent (levodopa). All eight stereoisomers of compound 1 have been synthesized and the influence
of the absolute configuration on the antiparkinsonian activity of compound 1 was shown.
Scheme 1a
’ INTRODUCTION
Parkinson’s disease (PD) is one of the most common neuro-
logical diseases characterized mostly by motor disturbances and
usually caused by the loss of the nigrostriatal path’s dopamine-
containing cells. The percent of people affected by PD at the age
of 60ꢀ80 is around 1%.1 While symptoms generally appear
around the age of 60, they can be found in much younger people,
which makes it a condition that does not recognize the boundaries
of age, gender, or race.2 Some of the symptoms associated with
PD involve rigidity, bradykinesia, resting tremor, and postural
instability along with cognitive and psychiatric complications.3,4
A number of medications are available to treat the movement-
related (motor) symptoms of PD, including levodopa, dopami-
nergic agonists, anticholinergics, amantadine, and monoamine
oxidase B inhibitors. Levodopa remains the most effective
medicine, in particular for treating rigidity and slowness of
movement. It is usually combined with a dopa decarboxylase
inhibitor that prevents levodopa from being utilized on the
periphery. Most patients initially do well on levodopa. However,
the treatment is associated with several side effects that people
with PD may experience, such as nausea and appetite loss,
involuntary movements, twisting motions and abnormal pos-
tures (dystonia), hallucinations, and paranoia. Prolonged use of
levodopa also gives rise to “on/off” episodes, resulting in addi-
tional complications.2 Therefore, the search for new effective
medications for the medical correction of PD remains important.
a Reagents and conditions: (a) H2O2/H2O, NaOH, MeOH 10 °C, 2 h;
(b) LiAlH4, Et2O, 0 °C, 3.5 h; (c) K10 clay, CH2Cl2, 1 h.
We have recently discovered5 that compound (1R,2R,6S)-1
(Scheme 1) possesses a high antiparkinsonian activity expressed in
the elimination of oligokinesia in C57Bl/6 mice caused by single or
systematic injections of the neurotoxin MPTP. The parkins-
onian syndrome was caused by intraperitoneal administrations of
0.17 mmol/kg (30 mg/kg) doses of MPTP daily for 3 days. A
0.12 mmol/kg (20 mg/kg) dose of (1R,2R,6S)-1 (70% ee) was
administered per os 24 h after the final injection of MPTP.
Hypokinesia was evaluated 1.5 h after the injection of MPTP on
the basis of the locomotor-orientational activity. The studied agent,
(1R,2R,6S)-1 (70% ee), distinctly improved the markers of the
locomotor and exploratory activities of the animals (demonstrated
Received: February 11, 2011
Published: May 02, 2011
r
2011 American Chemical Society
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Table 1. Study of the Antiparkinsonian Activity of (1R,2R,6S)-1 (70% ee) in C57Bl/6 Micea
group, dose
A
B
C
D
E
F
saline
MPTPb
73.3 ( 2.3**
89.3 ( 1.8
80.5 ( 3.9
90.0 ( 1.4**
64.1 ( 4.1
74.8 ( 6.4*
556.7 ( 19.6**
286.6 ( 36.5
439.6 ( 32.9**
4.6 ( 0.2**
2.5 ( 0.2
4.1 ( 1*
1.6 ( 0.6
6.5 ( 1.6*
24.9 ( 3.3**
8.5 ( 2.2
MPTPb and (1R,2R,6S)-1, 0.12 mmol/kg (20 mg/kg)
3.6 ( 0.3**
14.8 ( 3.4*
a A, general locomotor activity (number of acts); B, time of locomotor activity (s); C, movement distance (cm); D, movement speed (cm/s); E, number
of explored holes; F, number of upright postures. (/) P < 0.05, (//) P < 0.01 reliability in comparison with MPTP group. b MPTP (4 ꢁ 20 mg/kg).
Table 2. Study of the Antiparkinsonian Activity of (1R,2R,6S)-1 (70% ee) in Rats during a 30-Day Administration of the Agents
and MPTP (0.23 mmol/kg (40 mg/kg))a
group, dose
A
B
C
D
E
F
saline
80.3 ( 2.5**
50.4 ( 7.9
69.7 ( 2.1**
27.8 ( 4.8
367.5 ( 21.2**
110.4 ( 19.5
3.0 ( 0.2**
0.9 ( 0.2
4.6 ( 0.9
2.7 ( 1.0
4.3 ( 0.7
4.6 ( 0.6
14.6 ( 1.8**
2.6 ( 0.8
MPTP
MPTP and (1R,2R,6S)-1, 0.12 mmol/kg (20 mg/kg)
81.9 ( 5.5**
86.0 ( 3.9**
62.6 ( 5.2**
65.1 ( 2.9**
305.6 ( 30.4**
316.8 ( 16.2**
2.5 ( 0.3**
2.6 ( 0.13**
10.7 ( 1.2**
9.6 ( 1.2**
MPTP and levodopa, 0.05 mmol/kg (10 mg/kg)
a A, general locomotor activity (number of acts); B, time of locomotor activity (s); C, movement distance (cm); D, movement speed (cm/s); E, number
of explored holes; F, number of upright postures. (/) P < 0.05, (//) P < 0.01 reliability in comparison with MPTP group.
by the number of the explored holes and the time of exploratory
reactions); in other words, it removed the symptoms of the PD
induced by the thrice-repeated administration of the neurotoxin.
It was shown6 that (1R,2R,6S)-1 (70% ee) enhanced the
inhibitory effect of high doses of levodopa (1.0 mmol/kg (200
mg/kg)) and thereby potentiated the dopaminergic system.
Moreover, in the arecoline tremor test, which makes it possible
to estimate the effect on muscarinic cholinergic receptors, the
compound decreased the duration of arecoline induced tremor,
exhibiting an M-anticholinergic activity. The combination of
these properties is probably responsible for the antiparkinsonian
activity of (1R,2R,6S)-1.
Hypokynesia was evaluated with the “open field” test 1.5 h after
the injection of MPTP.
To confirm the results obtained earlier in this work, we
additionally used the protocol for the MPTP mouse model of
Parkinson’s disease published in Nature Protocols.13 It involves
one injection of MPTP ((0.12 mmol/kg (20 mg/kg) per dose) to
mice of C57Bl/6 line every 2 h for a total of four doses over an 8 h
period in 1 day. The studied agent was administered per os 24 h
after the last injection of MPTP in a dose of 0.12 mmol/kg
(20 mg/kg). Hypokynesia was measured 2 h after the adminis-
tration of the agent with the “open field” test for 2 min. The
effectiveness of the studied medication was evaluated according
to its ability to reduce the symptoms of hypokinesia induced
by MPTP.
As can be seen from Table 1, (1R,2R,6S)-1 in a dose of
0.12 mmol/kg (20 mg/kg) sufficiently improved both the loco-
motor (columns BꢀD) and exploratory (columns E, F) activ-
ities. Thus, (1R,2R,6S)-1 (70% ee) demonstrated a reliable
antiparkinsonian activity in this model, too.
We also note the lowacute toxicity of compound (1R,2R,6S)-1,
the LD50 of which amounts to 4250 mg/kg.6
Because of this combination of traits, further study of the
antiparkinsonian activity of this substance holds much promise.
’ RESULTS
A lengthier experiment, which involved a 30-day administra-
tion of MPTP, (1R,2R,6S)-1, and a reference agent (levodopa),
was performed on rats of the Wistar line. Rats are less sensitive to
the MPTP effect, so it is reasonable to use them in a prolonged
experiment. The parkinsonian syndrome was induced via intra-
peritoneal administrations of MPTP (0.23 mmol/kg (40 mg/kg)
doses daily) for 30 days, while the control group received saline.
Compound (1R,2R,6S)-1 (70% ee) in a dose of 0.12 mmol/kg
(20 mg/kg) or the levodopa reference in a dose of 0.05 mmol/kg
(10 mg/kg) was administered per os every day 4 h after the
MPTP injections. Hypokinesia was evaluated from the locomotor
and exploratory activity on the 30th day after the start of the
experiment with the “open field” test for 2 min; the experimental
data are presented in Table 2.
The experiment showed that the 0.12 mmol/kg (20 mg/kg)
dose of (1R,2R,6S)-1 (70% ee) exhibited a distinct antiparkinso-
nian activity after a 30-day administration of the (1R,2R,6S)-1 and
MPTP, which was demonstrated by a nearly full recovery of the
animal’s locomotor (general locomotor activity, movement dis-
tance, movement speed, time of locomotor activity) and explora-
tory (an increase in the number of explored holes and assumption
Study of the Antiparkinsonian Activity of (1R,2R,6S)-1
with 70% ee. Compound (1R,2R,6S)-1 was previously synthe-
sized from verbenone (ꢀ)-2 by epoxidation with hydrogen
peroxide, reduction of verbenone epoxide (ꢀ)-3 to cis-verbenol
epoxide (ꢀ)-4 using LiAlH4, and subsequent rearrangement of
this epoxide into the desired substance in the presence of
montmorillonite clay (Scheme 1).7,8 Because of the commercial
availability of verbenone with up to 70% ee, we also used
(1R,2R,6S)-1 with 70% ee in our previous work.5 Consequently,
we also studied the possible antiparkinsonian activity of
(1R,2R,6S)-1 with the same 70% ee in this work.
In animal studies, neurotoxins such as 1-methyl-4-phenyl-
1,2,3,6-tetrahydroxypyridine (MPTP) and neuroleptic drugs
(e.g., haloperidol) are commonly used to create experimental models
of PD, which may be used to model certain aspects of the disease, such
as catalepsy, motor imbalance, and slowing of movement.9ꢀ11
In our previous article5 we used the MPTP model of PD in
accordance with the Russian “Guide for Preclinical Trials of New
Pharmacological Substances”.12 The model includes the intraper-
itoneal injection of MPTP (0.17 mmol/kg (30 mg/kg)) to mice
15 min before the administration of the studied substances.
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Scheme 2a
Figure 1. Duration of catalepsy evoked by haloperidol.
a Reagents and conditions: (a) Pb(OAc)4, C6H6, 65 °C, 1 h; (b) KOH,
MeOH, H2O, 24 h; (c) Na2Cr2O7, H2SO4, Et2O, 0 °C (1 h), rt (24 h);
(d) H2O2, NaOH, MeOH, H2O, 10 °C, 2 h; (e) LiAlH4, Et2O, 0 °C, 3.5
h; (f) K10 clay, CH2Cl2, 1 h.
antiparkinsonian activity during in vivo experiments on mice and
rats in models induced by MPTP and haloperidol.
Synthesis of All Stereoisomers of Compound 1 with High
Optical Purity. As is known, the absolute configuration of chiral
compounds tends to be critical for the manifestation of various
biological activities. Compound 1 has three asymmetric centers
and hence eight stereoisomers. Synthesis of all stereoisomers
with high (no less than 90% ee) optical purity is necessary for an
in-depth study of the influence of the absolute configuration of
compound 1 on its antiparkinsonian activity.
Synthesis of the first enantiomers, compounds (1R,2R,6S)-1
and (1S,2S,6R)-1, was based on the commercially available (ꢀ)-
and (þ)-R-pinenes (ꢀ)- and (þ)-5 with high optical purity
(Scheme 2).
Figure 2. Percent of animals in the group with catalepsy evoked by
haloperidol.
According to the methods suggested in the article,17 (ꢀ)- and
(þ)-verbenones 2 were obtained by the interaction of R-pinenes
5 with Pb(OAc)4, subsequent saponification of acetates 6, and
the oxidation of trans- and cis-verbenols 7 and 8. The following
transformations of verbenones 2 into compounds (1R,2R,6S)-1
and (1S,2S,6R)-1 were performed according to the previously
developed methods.7,18 Thus, compound (1R,2R,6S)-1 was
synthesized from R-pinene 5 in six steps with a 12% overall
yield; the yield of compound (1S,2S,6R)-1 amounted to 11%.
The enantiomeric excess of products 1 was defined by GCꢀMS
using a chiral column; it was caused by the optical purity of the
starting pinenes and amounted to 93% for (1R,2R,6S)-1 and 98%
for (1S,2S,6R)-1.
of vertical poses) activities. Compound (1R,2R,6S)-1 (70% ee)
works as effectively as the reference agent (levodopa) in this test.
Cataleptic immobility induced in rodents by typical neuroleptics
(e.g., haloperidol, chlorpromazine, fluphenazine) is a behavioral
method to study the nigrostriatal function and its modulation
by other neurotransmitters.14 Haloperidol blocks dopamine D2
receptors in the striatum and is used to evoke drug-induced
parkinsonism.15 Drugs that attenuate haloperidol-induced motor
disorders might reduce the extrapyramidal signs of PD.16
After the administration of the drugs (10 min) to rats of the
Wistar line, catalepsy was induced via the intraperitoneal admin-
istration of haloperidol in a dose of 4.0 μmol/kg (1.5 mg/kg) of
body weight. The severity of catalepsy was measured 30, 60, 120,
180, and 240 min after the administration of haloperidol using
the parallel bars method. We recorded the time spent by an
animal in a cataleptic state and evaluated the general duration of
catalepsy and the percent of cataleptic animals in the group.
Compound (1R,2R,6S)-1 (70% ee) significantly blocks the
development of catalepsy evoked by haloperidol, which showed
itself as a decrease in catalepsy time, haloperidol’s time course
(Figure 1), and the percent of cataleptic animals (Figure 2).
Thus, compound (1R,2R,6S)-1 (70% ee) demonstrated potent
The key stage of the synthesis of the second enantiomer pair,
(1S,2R,6S)-1 and (1R,2S,6R)-1, was synthesis of trans-verbenol
epoxides (þ)- and (ꢀ)-9 and their further rearrangement into
the corresponding isomers of diol 1 (Scheme 3); trans-verbenols
(þ)- and (ꢀ)-7 were obtained by the literature procedure.19
The main difficulty in the synthesis of epoxides 9 was the low
stability of this compound even in weak acid media, which
made it impossible to use peracids, generally used for similar
oxidations. For the epoxidation of trans-verbenol 7, we used the
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Scheme 3a
a Reagents and conditions: (a) (1) KOH, MeOH, H2O, 24 h; (2) separation on SiO2; (b) t-BuOOH, VO(acac)2, toluene, reflux, 40 min; (c) K10 clay,
CH2Cl2, 1 h.
Scheme 4a
a Reagents and conditions: (a) (1) (COCl)2, DMSO, NEt3, CH2Cl2, ꢀ25 °C, 40 min; (2) separation on SiO2; (b) (1) LiAlH4, Et2O, 0 °C, 5 h; (2)
separation on SiO2.
VO(acac)2/t-BuOOH system, which had previously been suc-
cessfully used for the epoxidation of allylic alcohols.20 The
rearrangement of epoxides 9 in the presence of K10 clay resulted
in the obtaining of the desired diol of (1S,2R,6S)-1. After
the rearrangement of trans-verbenol epoxides 9, the yield of
compound 1 was significantly smaller than that obtained for
cis-verbenol epoxides 4 (12ꢀ17% versus 40ꢀ48%).
but proceeds further and is followed by CꢀC bond cleavage,
ultimately resulting in carbonyl compounds and carboxylic acids.
This behavior is typical not only for the rather stringent oxidants
such as sodium dichromate, chromic anhydride, and potassium
permanganate but also for the well-known mild oxidants, such as
iodoxybenzoic acid (IBX), DessꢀMartin periodinane (DMP),
pyridinium chlorochromate (PCC), and manganese dioxide.25ꢀ28
We tested the oxidation of (1R,2R,6S)-1 using a broad set of
oxidizing systems: Fꢀetizon’s reagent,29 manganese dioxide
(Attenborough method),30 sodium dichromate in the presence
Attempts to obtain the cis-epoxides of verbenone and verbe-
nol, which could serve for the synthesis of the remaining four
isomers of compound 1 through the corresponding bromohy-
drins, failed because of the ring-opening of cyclobutane under the
reaction conditions.21
of sulfuric acid, Collins reagent (CrO3 2Py),31 pyridinium
3
chlorochromate (PCC) applied to alumina,32 NaOCl in the
presence of TEMPO nitroxide,33 and t-BuOOH with VO(acac)2,
IBX,34 DMP,35 and the Swern oxidation.36 In all cases, the yield
of (5S,6R)-10 was low; the best result was obtained with the
Swern system. The preparative yield of (5S,6R)-10 during the
Swern oxidation amounted to 37%. Using the reduction of
(5S,6R)-10 with LiAlH4 followed by column chromatography,
we obtained the desired full cis-isomer of (1R,2R,6S)-1 with a
51% yield. In addition, the initial (1R,2R,6S)-1 isomer was also
isolated (11%). Similarly, we synthesized (1S,2R,6R)-1 with an
overall yield of 21% in two steps (Scheme 4).
One of the most effective methods of the configuration
inversion of the hydroxy group is the Mitsunobu reaction.22,23
It involves the interaction with diethyl azodicarboxylate, p-nitro-
benzoic acid, and triphenylphosphine to form nitrobenzoate, with
the inversion at the hydroxyl-bearing carbon atom and hydrolysis
at the second stage. Unfortunately, using the Mitsunobu reaction
for altering the configuration of the hydroxy group in (1R,2R,6S)-
1 resulted, after all the necessary manipulations, in the obtaining
of the initial compound with no changes in the configuration.
Another approach to the inversion of the allylic hydroxyl
group can be its oxidation into corresponding hydroxy ketone 10
followed by its reduction. Considering the steric hindrance
caused by the isopropenyl group, one can expect a preferential
formation during the reduction of ketone 10 of the desired
isomer. Indeed, it is known that the reduction of carvone to
carveol occurs mostly on the side with lesser steric hindrances,
giving a hydroxyl group in the cis-position in relation to the
isopropenyl group.24
Two more stereoisomers, (1S,2S,6S)- and (1R,2R,6R)-1, were
synthesized from (þ)- and (ꢀ)-carvones 11 according to
Scheme 5. Synthesis of (5S,6S)-10 via the intermediate forma-
tion of (ꢀ)-12 and (ꢀ)-trans-13 is described in literature.37 Note
that ref 38 suggested a modified version of the method in ref 37,
offering simplified handling and processing and higher yields of
the desired products. When we attempted to use it, however,
most of its steps turned out to be irreproducible. As a result, we
synthesized (ꢀ)-trans-13 by the procedure described in ref 37.
According to the literature, the oxidation of vicinal diols often
does not stop at the stage of hydroxy ketone or diketone formation
We used NH4F HF instead of the previously suggested HF to
3
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Scheme 5a
a Reagents and conditions: (a) LDA, TMSCl, THF, ꢀ80 °C (20 min), 0 °C (1.5 h); (b) 1) m-CPBA, CH2Cl2, 0 °C, 1.5 h, (2) separation on SiO2; (c)
NH4F HF, EtOH, H2O, 6 h; (d) (1) LiAlH4, Et2O, 0 °C, 4 h; (2) separation on SiO2.
3
Figure 3
Table 3. Study of the Antiparkinsonian Activity of the Stereoisomers of Compound 1 (0.12 mmol/kg (20 mg/kg)) in C57Bl/6
Mice after a Single Administration of MPTP (0.17 mmol/kg (30 mg/kg))a
no.
group, dose
A
B
C
D
E
F
1
saline
84.1 ( 1.9**
50.6 ( 8.1
78.6 ( 5.0**
57.9 ( 6.0
84.1 ( 1.9**
71.8 ( 6.4
64.9 ( 8.7
80.2 ( 6.2*
83.0 ( 2.4**
69.9 ( 4.7
28.5 ( 10.1*
82.4 ( 5.8
77.4 ( 8.2
65.5 ( 8.8
58.3 ( 3.9**
27.8 ( 4.8
64.0 ( 5.5**
34.9 ( 4.3
58.3 ( 3.9**
47.9 ( 4.5
45.5 ( 8.2
55.6 ( 6.9
75.9 ( 1.2**
46.8 ( 4.4
15.0 ( 5.4**
56.6 ( 6.2
55.7 ( 7.4
46.9 ( 6.0
276.8 ( 27.8**
107.8 ( 20.6
332.6 ( 39.6**
147.8 ( 20.3
276.8 ( 27.8**
218.1 ( 26.2
212.4 ( 48.7
245.9 ( 34.6*
417.1 ( 21.2**
211.8 ( 29.1
55.9 ( 20.9**
257.7 ( 36.6
253.3 ( 38.1
158.8 ( 32.7
2.3 ( 0.2**
0.9 ( 0.2
2.7 ( 0.3**
1.1 ( 0.2
2.3 ( 0.2**
1.8 ( 0.2
1.7 ( 0.4
1.2 ( 0.2
3.4 ( 0.2**
1.7 ( 0.3
0.4 ( 0.2**
2.1 ( 0.3
2.1 ( 0.3
1.3 ( 0.3
15.6 ( 3.3
2.6 ( 0.8
6.5 ( 1.3**
2.8 ( 0.9
3.2 ( 0.9
2.5 ( 0.9
6.5 ( 1.3**
1.9 ( 0.5
4.0 ( 1.3
3.1 ( 0.6*
5.4 ( 1.0
6.3 ( 1.8
1.7 ( 0.6*
7.9 ( 1.6
10.3 ( 1.0
4.7 ( 2.1
2
MPTP
3
MPTP and (1R,2R,6S)-1
MPTP and (1S,2S,6R)-1
saline
13.1 ( 2.5**
4.4 ( 1.4
4
5
15.6 ( 3.3**
8.9 ( 1.5
6
MPTP
7
MPTP and (1S,2R,6S)-1
MPTP and (1R,2S,6R)-1
Saline
3.5 ( 1.0
8
14.4 ( 3.6**
17.6 ( 5.4
11.3 ( 3.8
0.9 ( 0.6*
8.1 ( 2.0
9
10
11
12
13
14
MPTP
MPTP and (1R,2S,6S)-1
MPTP and (1S,2R,6R)-1
MPTP and (1S,2S,6S)-1
MPTP and (1R,2R,6R)-1
8.1 ( 1.6
3.9 ( 1.4
a A, general locomotor activity (number of acts); B, time of locomotor activity (s); C, movement distance (cm); D, movement speed (cm/s); E, number
of explored holes; F, number of upright postures. (/) P < 0.05, (//) P < 0.01 reliability in comparison with MPTP group.
remove the trimethylsilyl protection in (ꢀ)-trans-13, which
allowed us to increase the yield from 40% to 80% at this stage.
Compound (1S,2S,6S)-1 (99.5% ee) was obtained by the
reduction of the ketone group into the alcohol group in
(5S,6S)-10 with LiAlH4 followed by the separation of the
diastereomers.
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Similarly, from (þ)-carvone 11 (97% ee) we synthesized
(1R,2R,6R)-1 (97% ee) with an overall yield of 17% over the
sequence (Scheme 5).
Thus we managed to successfully synthesize all eight stereo-
isomers of compound 1 with no less than 93% ee.
We synthesized all eight stereoisomers of compound 1 from the
available monoterpenoids (þ)- and (ꢀ)-R-pinenes and (þ)- and
(ꢀ)-carvones for the first time. According to our observations, the
absolute configuration of compound 1 greatly influences its
antiparkinsonian activity in the test with MPTP, from nearly full
removal to a sharp increase in the symptoms of the parkinsonian
syndrome.
Antiparkinsonian Activity of the Stereoisomers of Com-
pound 1. We studied the antiparkinsonian activity of the
stereoisomers of compound 1 (Figure 3) on a model with a
single administration of MPTP neurotoxin to mice of C57Bl/6
line, which was injected intraperitoneally in a 0.17 mmol/kg
(30 mg/kg) dose 15 min before the administration of the studied
isomer (0.12 mmol/kg (20 mg/kg)) by the method described in
the literature.12 Hypokinesia was evaluated 1.5 h after the
injection of MPTP on the basis of the locomotor and exploratory
activities with the “open field” test for 2 min.
According to our study of the antiparkinsonian activity of
(1R,2R,6S)-1 (93% ee), which amounts to 85% of the previously
studied (1R,2R,6S)-1 (70% ee), the compound is characterized
by high activity and restores the locomotor and exploratory
activities almost completely to the rates of the saline group except
the number of the explored holes (Table 3, no. 3). The minor
(1S,2S,6R)-1 isomer demonstrated an unreliable, although visi-
ble, antiparkinsonian activity (Table 3, no. 4).
The inversion of the configuration of the hydroxy group in
position 1 during the transfer from (1R,2R,6S)-1 to (1S,2R,6S)-1
resulted in a complete disappearance of the antiparkinsonian
activity (Table 3, no. 7). Its optical antipode, (1R,2S,6R)-1,
however, exhibited a significant activity (Table 3, no. 8), although
it was slightly less than that of the (1R,2R,6S)-1 isomer.
An unexpected result was obtained after the configuration of
the hydroxy group in position 2 was changed. The transfer to a
complete cis-isomer of (1R,2S,6S)-1 led to a sudden and nearly
full inversion of activity, namely, to a strong increase in hypoki-
nesia and a decrease in the animals’ exploratory activity (Table 3,
no. 11). Compound (1S,2R,6R)-1, the enantiomer of (1R,2S,6S)-
1, did not display any reliable activity (Table 3, no. 12), yet it
significantly increased the general locomotor activity.
’ EXPERIMENTAL SECTION
Reagents and solvents were purchased from commercial suppliers and
used as received. Dry solvents were obtained according to the standard
procedures. The K10 clay (Merck) was calcinated at 110 °C for 3 h
immediately before use. GC parameters were as follows: 7820A gas
chromatograph (Agilent Technologies, U.S.); flame-ionization detector;
HP-5 capillary column (0.25 mm L ꢁ 30 m ꢁ 0.25 μm), He as carrier
gas (flow rate 2 mL/min, flow division 99:1). Chirospecific GCꢀMS
parameters were as follows: 6890N gas chromatograph (Agilent Tech-
nologies, U.S.); 5973 INERT mass-selective detector (Agilent Technol-
ogies, U.S.); Cyclosil-B capillary column (0.32 mm L ꢁ 30 m ꢁ
0.25 μm, Agilent Technolgies, U.S.); temperature of the column
thermostat was 50 °C/2 min; temperature gradient from 2 °C/min to
220 °C/5 min; evaporator and interface temperature 250 °C; He as
carrier gas (flow rate 2 mL/min, flow division 99: 1); sweep from m/z 29
to m/z 500; 1 μL sample. Optical rotation parameters were as follows:
1
polAAr 3005 spectrometer; CHCl3 solution. H and 13C NMR para-
meters were as follows: Bruker DRX-500 apparatus at 500.13 MHz (1H)
and 125.76 MHz (13C) in CCl4/CDCl3 1:1 (v/v); chemical shifts δ in
ppm relative to residual CHCl3 [δ(H) 7.24, δ(C) 76.90 ppm], J in Hz.
The structure of the products was determined by analyzing the 1H and
1
13C NMR spectra, H,1H double-resonance spectra, and 13C,1H-type
2D-COSY (J(C,H) = 135 Hz). HR-MS parameters were as follows: DFS
Thermo Scientific spectrometer in a full scan mode (0ꢀ500 m/z, 70 eV
electron impact ionization, direct sample administration). Column
chromatography (CC) was performed on silica gel (60ꢀ200 μm,
Macherey-Nagel). The purity of the target compounds was determined
by gas chromatography methods. All of the target compounds reported
in this paper have a purity of g95%.
(1R,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol
(1R,2R,6S)-1 (70% ee) was obtained from (ꢀ)-verbenone (ꢀ)-2
(Aldrich) (70% ee) according to previously described methods.6,7
(ꢀ)-Verbenone (ꢀ)-2 and (þ)-verbenone (þ)-2 were synthesized
from (ꢀ)-R-pinene (ꢀ)-5 (93% ee) and (þ)-R-pinene (þ)-5 (98% ee)
in accordance with procedure.17
(ꢀ)-Verbenone Epoxide (ꢀ)-3. (ꢀ)-Verbenone (ꢀ)-2 (42.0 g,
280 mmol) was dissolved in methanol (420 mL) and cooled to 10 °C;
33% H2O2 (89 mL) and 6 N NaOH (21 mL) were added. The mixture
was stirred for 2 h at 12ꢀ15 °C. EtOAc (4 ꢁ 300 mL) was used
for extraction, H2O (2 ꢁ 300 mL) for washing, and Na2SO4 for drying.
The solvent was distilled off, and 33.0 g of (ꢀ)-verbenone epoxide
(199 mmol, 71%) (ꢀ)-3 ([R]2D5 ꢀ106.0 (c 1.74, CHCl3)) was obtained.
The spectral characteristics of compound (ꢀ)-3 corresponded to those
described in ref 7.
The (1S,2S,6S)-1 and (1R,2R,6R)-1 enantiomer pair pro-
duced no reliable effect on the locomotor-orientational activity
in these tests (Table 3, nos. 13 and 14), but (1R,2R,6R)-1 led to a
visible decrease in the animals’ exploratory activity.
Thus, it can be said that the absolute configuration of
compound 1 greatly influences the antiparkinsonian activity of
the compound in the test with MPTP, from nearly full removal to
a sharp increase in the symptoms of the parkinsonian syndrome.
’ CONCLUSION
Our study demonstrates that (1R,2R,6S)-1 displays a potent
antiparkinsonian activity in vivo on models with MPTP in mice
and rats and is not inferior to the reference agent (levodopa).
Compound (1R,2R,6S)-1 clearly improves the markers of the
locomotor and exploratory activities, thus removing the signs of
the parkinsonian syndrome induced by neurotoxin injection. The
described activity is proved by a number of tests with varying
duration of toxin and agent administration (1ꢀ30 days).
Compound (1R,2R,6S)-1 almost completely prevents the
development of catalepsy caused by haloperidol, which is de-
monstrated by a notable decrease in the catalepsy duration in
animals, the duration of haloperidol time course, and the percent
of cataleptic animals.
(þ)-Verbenone Epoxide (þ)-3. Similar to the synthesis of (ꢀ)-3,
2.31 g of (þ)-verbenone epoxide (þ)-3 (13.9 mmol, 70%) was obtained
from 3.00 g (20.0 mmol) of (þ)-verbenone (þ)-2.
(ꢀ)-cis-Verbenol Epoxide (ꢀ)-4. A solution of (ꢀ)-verbenone
epoxide (ꢀ)-3 (32.6 g, 196 mmol) in ether (420 mL) was added to a
suspension of LiAlH4 (8.08 g, 213 mmol) in ether (300 mL) at 0 °C. The
mixture was stirred for 3.5 h at 0 °C, after which H2O (20 mL) was added.
The sediment was filtered, and the filtrate was washed with H2O (2 ꢁ
300 mL) and dried with Na2SO4. The solvent was distilled off to get 25.0 g
(149 mmol, 76%) of (ꢀ)-cis-verbenol epoxide (ꢀ)-4([R]2D5 ꢀ76.3 (c 0.92,
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Journal of Medicinal Chemistry
ARTICLE
CHCl3). The spectral data of compound (ꢀ)-4 corresponded to those
described in ref 7.
(þ)-cis-Verbenol Epoxide (þ)-4. Similar to the obtaining of
compound (ꢀ)-4, 0.873 g of (þ)-cis-verbenol epoxide (þ)-4 (5.20 mmol,
75%) was obtained from 1.16 g (6.98 mmol) of (þ)-verbenone epoxide
(þ)-3.
and CH2Cl2 (8 mL) was added. The reaction mixture was stirred for
25 min at ꢀ25 °C. Then triethylamine (2.51 mL, 18 mmol) was added,
and the mixture was stirred for 15 min at ꢀ25 °C. The reaction mixture
was warmed to rt. H2O (20 mL) was added, and the water phase was
extracted with CH2Cl2 (2 ꢁ 10 mL). The combined organic extracts
were washed with 3% HCl (2 ꢁ 20 mL), 5% NaHCO3 (2 ꢁ 15 mL), and
H2O (2 ꢁ 20 mL). Na2SO4 was used for drying. The solvent was
distilled off. The obtained mixture was separated on a column with SiO2
(17 g), with a gradient of EtOAc in hexane from 0% to 100% as eluent, to
get 0.138 g (0.83 mmol, 37%) of (5S,6R)-10 ([R]2D4 ꢀ56.4 (c 1.50,
CHCl3)). The spectral data of (5S,6R)-10 corresponded to those
described in the literature.38
(5R,6S)-6-Hydroxy-2-methyl-5-(prop-1-en-2-yl)-2-cyclo-
hexene-1-one (5R,6S)-10. Similar to the synthesis of (5S,6R)-10,
0.074 g (0.45 mmol, 40%) of (5R,6S)-10 was obtained from 0.188 g
(1.12 mmol) of (1S,2S,6R)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-
ene-1,2-diol (1S,2S,6R)-1 (98% ee).
(1R,2S,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol (1R,2S,6S)-1. A solution of (5S,6R)-10 (0.065 g, 0.39 mmol) in
diethyl ether (7 mL) was added to a suspension of LiAlH4 (0.015 g,
0.39 mmol) in diethyl ether (3 mL) at 0 °C. The mixture was stirred for
5 h at 0 °C, after which 4 drops of H2O were added and the residue was
filtered. Thefiltrate wasdriedwith Na2SO4. The solvent wasdistilled offto
obtain 0.062 g of the product mixture. It was separated on a column of
SiO2 (9 g), with a gradient ofEtOAc inhexane from 0% to 100% as eluent,
to get 0.033 g (0.20 mmol, 51%) of (1R,2S,6S)-1 ([R]2D1 þ14.3 (c 0.63,
CHCl3), 93% ee) and 0.007 g (0.042 mmol, 11%) of (1R,2R,6S)-1.
Data for (1R,2S,6S)-1. 1H NMR (500 MHz, CCl4/CDCl3): δ 1.73
(m, C10H3), 1.80 (br s, C9H3), 1.87ꢀ1.96 (m, H5e), 2.00 (br s, OH),
2.21ꢀ2.30 (m, H6, H5a), 2.43 (br s, OH), 4.04 (br s, H1, H2), 4.84 (br s)
and 4.90 (m, 2H8), 5.48 (dm, H4, J4,5e = 5.5 Hz). 13C NMR (125 MHz,
CCl4/CDCl3): δ 69.30 (d, C1), 71.90 (d, C2), 133.64 (s, C3), 122.99
(d, C4), 24.76 (t, C5), 44.80 (d, C6), 145.69 (s, C7), 111.46 (t, C8), 22.46
(q, C9), 19.16 (q, C10). HR-MS: 150.1041 ([Mþ ꢀ H2O], C10H14O;
calcd 150.1045).
(1S,2R,6R)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol (1S,2R,6R)-1. Similar to the obtaining of (1R,2S,6S)-1, 0.065 g of
product mixture was obtained from 0.072 g (0.43 mmol) of (5R,6S)-6-
hydroxy-2-methyl-5-(prop-1-en-2-yl)-2-cyclohexene-1-one (5R,6S)-10.
It was separated on a column with SiO2 (9 g), with a gradient of EtOAc
in hexane from 0% to 100% as eluent, to get 0.038 g (0.23 mmol, 53%) of
(1S,2R,6R)-1 ([R]1D8 ꢀ40.8 (c 1.23, CHCl3), 98% ee) and 0.016 g (0.095
mmol, 22%) of (1S,2S,6R)-1.
(1R,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-
1,2-diol (1R,2R,6S)-1. A solution of (ꢀ)-cis-verbenol epoxide (ꢀ)-4
(23.6 g, 140 mmol) in CH2Cl2 (200 mL) was added to a suspension of
K10 clay (51 g) in CH2Cl2 (300 mL). It was stirred for 1 h 10 min at rt.
EtOAc (150 mL) was added, the catalyst was filtered, and the solvent was
distilled off to furnish 23.6 g of mixture. It was separated on a column with
SiO2 (150 g), with a gradient of EtOAc in hexane from 0% to
100% as eluent, to obtain 9.44 g (56.2 mmol, 40%) of (1R,2R,6S)-1
([R]2D9 ꢀ84.0 (c 3.47, CHCl3), 93% ee), 5.54 g (33.0 mmol, 24%) of
2-hydroxy-1-[(1S)-2,2,3-trimethylcyclopent-3-en-1-yl]ethanone ([R]D27
ꢀ
25.0 (c 1.07, CHCl3)), 2.02 g (12.0 mmol, 9%) of (ꢀ)-2-(2,2-dimethylcy-
clopent-3-enyl)-2-hydroxypropanal ([R]2D4 ꢀ5.4 (c 0.48, CHCl3)), and
1.04 g (7.76 mmol, 6%) of p-cymene. The spectral characteristics of
(1R,2R,6S)-1, 2-hydroxy-1-[(1S)-2,2,3-trimethylcyclopent-3-en-1-yl]ethanone
and (ꢀ)-2-(2,2-dimethylcyclopent-3-enyl)-2-hydroxypropanal corre-
sponded to those found in refs 7 and 8.
(1S,2S,6R)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol (1S,2S,6R)-1. Similar to the obtaining of compound (1R,2R,6S)-
1, 0.333 g (1.98 mmol, 40%) of (1S,2S,6R)-1 1 ([R]2D9 þ82.7 (c 0.73,
CHCl3), 98% ee) and 0.095 g (0.56 mmol, 11%) 2-hydroxy-1-[(1R)-
2,2,3-trimethylcyclopent-3-en-1-yl]ethanone ([R]2D7 þ27.6 (c 1.20,
CHCl3)) were obtained from 0.850 g (5.05 mmol) (þ)-cis-verbenol
epoxide (þ)-4.39
(ꢀ)-trans-Verbenol Epoxide (ꢀ)-9. A solution of 5 mg of
VO(acac)2 and 3.0 mL (16.5 mmol) of 5.5 M t-BuOOH in hexane
was added at rt to a solution of 2.00 g (13.2 mmol) of (ꢀ)-trans-verbenol
(ꢀ)-7 in dry toluene (100 mL). The reaction mixture was boiled for
40 min and washed with a saturated solution of NaHCO3 (100 mL) and
H2O (2 ꢁ 100 mL). Na2SO4 was used for drying. The solvent was
distilled off to get 1.97 g (11.7 mmol, 89%) of (ꢀ)-trans-verbenol
epoxide (ꢀ)-9 ([R]2D9 ꢀ118.6 (c 6.67, CHCl3)). 1H NMR (500 MHz,
CCl4/CDCl3): δ 0.87 (s, C9H3), 1.27 (s, C8H3), 1.31 (s, C10H3), 1.66
(d, H7an, J7an,7sin = 9 Hz), 1.79ꢀ1.90 (m, H5, H7sin), 1.92 (dd, H1,
J1,7sin = 6 Hz, J1,5 = 5 Hz), 2.41 (br.s, OH), 3.20 (dd, H3sin, J3sin,4sin
=
4 Hz, J3sin,5 = 1.2 Hz), 3.91 (m, H4sin). 13C NMR (125 MHz, CCl4/
CDCl3): δ 45.50 (d, C1), 61.48 (s, C2), 58.87 (d, C3), 66.87 (d, C4),
46.11 (d, C5), 42.96 (s, C6), 21.78 (t, C7), 26.66 (q, C8), 19.45 (q, C9),
21.89 (q, C10).
(5S,6S)-6-Hydroxy-2-methyl-5-(prop-1-en-2-yl)-2-cyclohexen-
1-one (5S,6S)-10 and (5R,6R)-6-hydroxy-2-methyl-5-(prop-1-en-2-
yl)-2-cyclohexen-1-one (5R,6R)-10 were synthesized from (ꢀ)-car-
vone (-)-11 (99.5% ee) and (þ)-carvone (þ)-11 (97% ee) by the
procedure described in ref 37.
(1S,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol (1S,2R,6S)-1. A solution of 1.57 g (9.35 mmol) of (ꢀ)-trans-
verbenol epoxide (ꢀ)-9 in CH2Cl2 (15 mL) was added to a suspension
of K10 clay (3.10 g) in CH2Cl2 (15 mL). It was stirred for 1 h at rt.
EtOAc (10 mL) was added, clay was filtered, and the solvent was distilled
off. The residue was separated on a column with 17 g of SiO2, with a
gradient of EtOAc in hexane from 0% to 100% as eluent, to get
0.192 g (1.14 mmol, 12%) of (1S,2R,6S)-1 ([R]2D9 ꢀ90.1 (c 6.07,
CHCl3), 93% ee). The spectral data of (1S,2R,6S)-1 corresponded to
those described in the literature.40
(1S,2S,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol (1S,2S,6S)-1. A solution of (5S,6S)-10 (0.067 g, 0.40 mmol) in
ether (4 mL) was added to a suspension of LiAlH4 (0.015 g, 0.39 mmol)
in ether (3 mL) at 0 °C. The mixture was stirred for 4 h at 0 °C, after
which 15 drops of H2O were carefully added. The residue was filtered
through a filter with a SiO2 layer and washed with 35 mL of EtOAc.
Na2SO4 was used for drying and the solvent was distilled off to get
0.070 g of a product mixture, which was separated on a column with SiO2
(9 g), with a gradient of EtOAc in hexane from 0% to 100% as eluent, to
obtain 0.046 g (0.27 mmol, 68%) of (1S,2S,6S)-1 ([R]2D3 þ35.0 (c 1.52,
CHCl3), 99.5% ee) and 0.022 g (0.13 mmol, 32%) of (1S,2R,6S)-1.
(1R,2S,6R)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol (1R,2S,6R)-1. Similar to the synthesis of (1S,2R,6S)-1, 0.278 g
(1.65 mmol, 17%) of (1R,2S,6R)-1 ([R]2D2 þ87.9 (c 1.70, CHCl3),
98% ee) was obtained from 1.50 g (9.87 mmol) of (þ)-trans-verbenol
(þ)-7.
1
Data for (1S,2S,6S)-1. H NMR (500 MHz, CCl4/CDCl3): δ 1.71
(5S,6R)-6-Hydroxy-2-methyl-5-(prop-1-en-2-yl)-2-cyclohex-
ene-1-one (5S,6R)-10. A mixture of DMSO (0.63 mL, 8.8 mmol) and
CH2Cl2 (4 mL) was added to a solution of oxalyl chloride (0.38 mL,
4.5 mmol) in CH2Cl2 (8 mL) at ꢀ25 °C. Then a solution of 0.377 g
(2.24 mmol) of (1R,2R,6S)-1 (93% ee) in DMSO (0.94 mL, 13 mmol)
2
(m, C10H3), 1.72 (br s, C9H3), 2.02 (dddm, H5e, J = 17.3, J5e,6a 6.0,
J5e,4 = 5.0 Hz), 2.02 (dddm, H5a, 2J = 17.3, J5a,6a = 11.2, J5a,4 = 2.5 Hz),
2.36 (ddd, H6a, J6a,1a = 11.2, J6a,5a = 11.2, J6a,5e = 6.0 Hz), 2.71 (br s, OH),
3.17 (br s, OH), 3.55 (dd, H1a, J1a,6a = 11.2 Hz, J1a,2a = 7.3 Hz), 3.97
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Journal of Medicinal Chemistry
ARTICLE
(br d, H2a, J2a,1a = 7.3 Hz), 4.84 (br s) and 4.87 (m, 2H8), 5.34 (m, H4).
13C NMR (125 MHz, CCl4/CDCl3): δ 74.53 (d, C1), 76.16 (d, C2),
134.55 (s, C3), 122.10 (d, C4), 30.29 (t, C5), 48.14 (d, C6), 145.15 (s,
C7), 113.60 (t, C8), 18.85 (q, C9), 18.66 (q, C10). HR-MS: 168.1149
(Mþ, C10H16O2; calcd 168.1145).
(1R,2R,6R)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol (1R,2R,6R)-1. A solution of (5R,6R)-10 (0.057 g, 0.34 mmol) in
ether (4 mL) was added for 3 min to a suspension of LiAlH4 (0.013 g,
0.34 mmol) in ether (3 mL) stirred at 0 °C. The mixture was stirred for
4 h at 0 °C, after which 15 drops of H2O were added and the mixture was
immediately filtered through a filter with a SiO2 layer, washed with
30 mL of EtOAc, and dried over sodium sulfate. The solvent was distilled
off. The obtained product mixture (0.053 g) was separated on a column
with SiO2 (4.5 g, 60ꢀ200 μm; Macherey-Nagel), with a gradient of
EtOAc in hexane from 0% to 100% as eluent, to get 0.029 g (0.17 mmol,
50%) of (1R,2R,6R)-1 ([R]D23 ꢀ38.1 (c 0.93, CHCl3), 97% ee) and
0.015 g (0.089 mmol, 26%) of (1R,2S,6R)-1.
Animals. The experiments were performed on Wistar rats (male)
weighing 200ꢀ220 g and C57BL/6 mice (male) weighing 25ꢀ30 g
(SPF-vivarium of the Institute of Cytology and Genetics of the Siberian
Branch of the Russian Academy of Sciences). The animals were
maintained at 22ꢀ25 °C on a 12 h lightꢀdark cycle with food and
water available ad libitum. All work with animals was performed in strict
accordance with the legislation of the Russian Federation, the regula-
tions of the European Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific Purposes, and the
requirements and recommendations of the Guide for the Care and Use
of Laboratory Animals.
MPTP Mouse Model of Parkinson’s Disease Induced by Single
Administration of MPTP Neurotoxin12. MPTP (0.17 mmol/kg
(30 mg/kg)) was injected intraperitoneally to mice of the C57Bl/6 line
15 min prior to the administration of the studied substances. The compound
under study was administrated per os in a dose of 0.12 mmol/kg (20 mg/kg).
Normal saline was injected to the control group. The effectiveness of the
studied medications was evaluated according to their ability to reduce the
symptoms of hypokinesia induced by MPTP. Hypokinesia caused by
neurotoxin administration was evaluated with the “open field” test performed
for 2 min using Tru Scan (U.S.) 1.5 h after the injection of MPTP, registering
the main markers of the locomotor and exploratory activities: general
locomotor activity (number of acts), time of locomotor activity (s), move-
ment distance (cm), movement speed (cm/s), number of upright postures,
and number of explored holes.
MPTP Mouse Model of Parkinson’s Disease Induced by
4-Fold Administration of MPTP Neurotoxin13. MPTP was
injected intraperitoneally to mice of C57Bl/6 line every 2 and 8 h
period in 1 day in a dose of 0.12 mmol/kg (20 mg/kg) for a total of four
doses. The studied agent was administrated per os 24 h after the last
injection of MPTP in a dose of 0.12 mmol/kg (20 mg/kg). The
effectiveness of the studied medications was evaluated according to
their ability to reduce the symptoms of hypokinesia induced by MPTP.
Hypokinesia caused by neurotoxin administration was evaluated with
the “open field” test performed for 2 min using Tru Scan (U.S.) 2 h after
the administration of the studied agent, registering the main markers of
the locomotor and exploratory activities: general locomotor activity
(number of acts), time of locomotor activity (s), movement distance
(cm), movement speed (cm/s), number of upright postures, number of
explored holes.
MPTP Rat Model of Parkinson’s Disease Induced by
Administration of MPTP Neurotoxin12. MPTP (0.23 mmol/kg
(40 mg/kg)) was injected intraperitoneally to rats 4 h prior to the
administration of the studied substances. Compound (1R,2R,6S)-1 was
administrated per os in a dose of 0.12 mmol/kg (20 mg/kg). Normal
saline was injected to the control group. The effectiveness of the studied
medications was evaluated on the 30th day after the start of the
experiment according to their ability to reduce the symptoms of
hypokinesia induced by MPTP. Hypokinesia caused by neurotoxin
administration was evaluated with the “open field” test performed for
2 min using Tru Scan (U.S.) 2 h after the administration of the studied
agent, registering the main markers of the locomotor and exploratory
activities: general locomotor activity (number of acts), time of locomo-
tor activity (s), movement distance (cm), movement speed (cm/s),
number of upright postures, and number of explored holes.
Catalepsy Mouse Model Induced by Haloperidol41. Neuro-
leptic-induced catalepsy has long been used as an animal model for
screening drugs for parkinsonism.42 Adult male Wistar rats were divided
into two groups each containing six animals. Group I received haloper-
idol 4.0 μmol/kg (1.5 mg/kg) in normal saline and served as the
cataleptic control without any drug treatment. Group II received
(1R,2R,6S)-1 (0.12 mmol/kg (20 mg/kg) per os); 10 min after the
administration of this drug, catalepsy was induced by the intraperitonial
administration of haloperidol in a dose of 4.0 μmol/kg (1.5 mg/kg)
body weight in normal saline. All the behavioral studies were performed
at rt in a calm room without any external interference. The severity of
catalepsy was measured 30, 60, 120, 180, and 240 min after haloperidol
administration using the method of parallel bars. The fore and hind
limbs of a rat were placed on parallel bars (walls) to ensure that the
animal’s back was straight, and the time spent by the animal in an
immobilized state was recorded. The general duration of catalepsy and
the percent of cataleptic animals in the group were evaluated.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
spectra for compounds 2, 6, and 10. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone þ73833308870. Fax: þ73833309752. E-mail: volcho@
nioch.nsc.ru.
’ ABBREVIATIONS USED
CC, column chromatography; DMP, DessꢀMartin periodinane;
IBX, iodoxybenzoic acid; ee, enantiomeric excess; GCꢀMS, gas
chromatographyꢀmass spectrometry; MPTP, 1-methyl-4-phen-
yl-1,2,3,6-tetrahydroxypyridine; PCC, pyridinium chlorochro-
mate; PD, Parkinson’s disease; rt, room temperature
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