A Novel Small Molecule Supports the Survival of Cultured Dopamine Neurons and May Restore the Dopaminergic Innervation of the Brain in the MPTP Mouse Model of Parkinson's Disease

Article abstract of DOI:10.1021/acschemneuro.9b00396

We previously showed that monoterpenoid (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 alleviates motor manifestations of Parkinson's disease in animal models. In the present study, we designed and synthesized monoepoxides of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 and evaluated their biological activity in the MPTP mouse model of Parkinson's disease. We also assessed the ability of these compounds to penetrate the blood-brain barrier (BBB). According to these data, we chose epoxide 4, which potently restored the locomotor activity in MPTP-treated mice and efficiently penetrated the BBB, to further explore its potential mechanism of action. Epoxide 4 was found to robustly promote the survival of cultured dopamine neurons, protect dopamine neurons against toxin-induced degeneration, and trigger the mitogen-activated protein kinase (MAPK) signaling cascade in cells of neuronal origin. Meanwhile, neither the survival-promoting effect nor MAPK activation was observed in non-neuronal cells treated with epoxide 4. In the MPTP mouse model of Parkinson's disease, compound 4 increased the density of dopamine neuron fibers in the striatum, which can highlight its potential to stimulate striatal reinnervation and thus halt disease progression. Taken together, these data indicate that epoxide 4 can be a promising compound for further development, not only as a symptomatic but also as a neuroprotective and neurorestorative drug for Parkinson's disease.

Full text of DOI:10.1021/acschemneuro.9b00396

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Article
A novel small molecule supports the survival of cultured dopamine
neurons and may restore the dopaminergic innervation of
the brain in the MPTP mouse model of Parkinson's disease
Oleg V. Ardashov, Alla V. Pavlova, Arun Kumar Mahato, Yulia Sidorova, Ekaterina A. Morozova,
Dina V. Korchagina, George E. Salnikov, Alexander M. Genaev, Oksana S. Patrusheva,
Nikolay Li-Zhulanov, Tat’yana G. Tolstikova, Konstantin P. Volcho, and Nariman Salakhutdinov
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00396 • Publication Date (Web): 29 Aug 2019
Downloaded from pubs.acs.org on September 1, 2019
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A novel small molecule supports the survival of cultured dopamine neurons and may restore
the dopaminergic innervation of the brain in the MPTP mouse model of Parkinson's disease
a,b
a
c
c
Oleg V. Ardashov, Alla V. Pavlova, Arun Kumar Mahato, Yulia Sidorova, Ekaterina A.
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a,
a
a,b
a
Morozova, Dina V. Korchagina, Georgi E. Salnikov, Alexander M. Genaev, Oksana S.
a
a,b
a
a,b
Patrusheva, Nikolay S. Li-Zhulanov, Tat’yana G. Tolstikova, Konstantin P. Volcho*, Nariman
Salakhutdinova,b
a
N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy
of Sciences, Lavrentiev ave., 9, 630090 Novosibirsk, Russian Federation
b
Novosibirsk State University, Pirogova, 2, 630090 Novosibirsk, Russian Federation
c
Laboratory of Molecular Neuroscience, Institute of Biotechnology, HiLIFe, Viikinkaari 5D,
University of Helsinki 00014, Helsinki, Finland
Abstract
We previously showed that monoterpenoid (1R,2R,6S)-3-methyl-6-(prop-1-en-2-
yl)cyclohex-3-ene-1,2-diol 1 alleviates motor manifestations of Parkinson's disease in animal
models. In the present study, we designed and synthesized monoepoxides of (1R,2R,6S)-3-methyl-6-
(
prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 and evaluated their biological activity in the MPTP
mouse model of Parkinson's disease. We also assessed the ability of these compounds to penetrate
the blood–brain barrier (BBB). According to these data, we chose epoxide 4, which potently
restored the locomotor activity in MPTP-treated mice and efficiently penetrated the BBB, to further
explore its potential mechanism of action. Epoxide 4 was found to robustly promote the survival of
cultured dopamine neurons, protect dopamine neurons against toxin-induced degeneration, and
1
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trigger the mitogen-activated protein kinase (MAPK) signaling cascade in cells of neuronal origin.
Meanwhile, neither survival-promoting effect nor MAPK activation was observed in non-neuronal
cells treated with epoxide 4. In the MPTP mouse model of Parkinson's disease, compound 4
increased the density of dopamine neuron fibers in the striatum, which can highlight its potential to
stimulate striatal reinnervation and thus to halt disease progression. Taken together, these data
indicate that epoxide 4 can be a promising compound for further development, not only as a
symptomatic, but also as a neuroprotective and neurorestorative drug for Parkinson's disease.
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Keywords: Parkinson's disease, neurorestoration, MAPK signaling, ERK pathway, dopamine
neurons, tyrosine hydroxylase.
1
. INTRODUCTION
Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide.
1
PD affects about 1–1.5% of the population over 60 years of age ; the lifetime risk of developing the
2
3
disease is 1.5%. The economic burden of PD in the United States exceeded $14.4 billion in 2010.
With the global phenomenon of population ageing, the number of PD cases is likely to grow
significantly in the future. The major clinical diagnostic criteria for PD include tremor, rigidity,
akinesia, postural instability, and bradyphrenia; depression is the most frequent psychopathological
4
disorder. The diagnostic motor symptoms of PD are caused by progressive degeneration and death
5
of dopamine neurons in the brain of affected people. There is no cure for PD. In other words, none
of the existing medications can prevent, stop or slow down the underlying neuronal degeneration.
Current management of PD is limited to supportive care that partially alleviates disease signs and
symptoms. The dopamine precursor levodopa in combination with peripheral decarboxylase
inhibitor is still the most available effective medication. The main adverse effects of levodopa
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therapy include nausea, motor complications, dyskinesias and the on–off phenomenon, confusion,
hallucinations, orthostatic hypotension, and sleep disturbances.6-8 Importantly, levodopa only
supplements endogenous dopamine but does not prevent progressive degeneration of dopamine
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neurons and may actually even promote it. Therefore, studying the alternative strategies for PD
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treatment, especially the ones targeting neurodegeneration, is rather relevant today.
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We have earlier discovered a small low-toxicity molecule (1R,2R,6S)-3-methyl-6-(prop-1-
en-2-yl)cyclohex-3-ene-1,2-diol 1 (Fig. 1) that can potently alleviate motor symptoms in rodent
models of PD.10-12 We synthesized and tested in vivo a number of derivatives, including all eight
1
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stereoisomers of compound 1. It was found that all of the four functional groups of 1 are needed
1
3
for the compound to exhibit anti-PD activity. Among N-, O-, S- and C-derivatives at the C9
position of diol 1, compounds 2 and 3 with butyl or propylthio substituents exhibited a high
activity.14
When exploring the chemical space around diol, we have synthesized all four isomeric
monoepoxides 4-7 in the present study (Fig. 1) and investigated their ability to influence the
locomotor activity in the MPTP-induced model of PD. The ability of selected epoxides to penetrate
the blood–brain barrier was also evaluated. Since the mode of action and the molecular target(s) of
diol and its derivatives were still to be elucidated, we attempted to address this question. We also
evaluated the ability of the most promising diol epoxide to promote the survival of dopamine
neurons in vitro and in vivo in the MPTP-induced model of PD.
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Figure 1. (1R,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 and its derivatives 2-7
2
. RESULTS AND DISCUSSION
2.1. Synthesis of epoxides 4 and 5
First, we focused on epoxidation of the 3,4 double bond in compound 1. The attempts to
1
5
epoxidize compound 1 with t-BuOOH in the presence of Ti(i-PrO) (the Sharpless system ) or
4
1
6
VO(acac) in accordance with the method described by Brown S. et al have failed: the starting
2
compound 1 was obtained again or a complex mixture of products was formed. The probable reason
for that phenomenon involved secondary transformations of the epoxide group.
Next, we attempted to use non-catalytic methods to oxidize compound 1 with
CH CO H/NaHCO or m-CPBA in CH Cl (Scheme 1). Treatment with CH CO H/NaHCO in
3
3
3
2
2
3
3
3
CH Cl for 3 h at room temperature gave rise to epoxides 4 and 5 with the yields of 16% and 4%,
2
2
respectively; the degree of conversion of diol 1 was 94%. Oxidation of compound 1 with m-CPBA
(1.45 eq.) in CH Cl at 0 °C for one week led to formation of epoxides 4 and 5 with acceptable
2
2
yields. The stereoisomeric epoxides 4 and 5 were isolated in their pure forms by column
chromatography in silica gel; the yields of the products were 26% and 11%, respectively; the degree
of conversion of the starting compound 1 was 91%. In this case, we also detected 2% of epoxide 7 in
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the reaction mixture but failed to isolate it. Finally, when compound 1 was treated with m-CPBA
(
1.66 eq.) in Et O for 3 weeks, the yields of products 4 and 5 were 32% and 10%, respectively; full
2
conversion of diol 1 was achieved. Moreover, a mixture of four stereoisomeric diepoxides 8 was
isolated with a 42% yield.
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O
O
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
[O]
+
+
+
H
1
4
5
O 7
O 8
a) CH CO H, NaHCO , CH Cl , 3 h
16%
4%
-
-
3
3
3
2
2
b) m-CPBA (1.45 eq), CH Cl , 1 week 26%
11%
(2 %)
-
-
2
2
c) m-CPBA (1.66 eq), Et O, 3 week
32%
10%
42%
2
Scheme 1. Epoxidation of compound 1.
2.2 Synthesis of epoxides 6 and 7
Since direct epoxidation of diol 1 proceeds predominantly at the endocyclic double bond, the
iodohydrin approach was used to obtain stereoisomeric compounds 6 and 7. The interaction between
diol 1 and iodine in the presence of water and AcOH in EtOAc led to 62% conversion of 1 within 5
days. 7S- and 7R-epimeric iodohydrins 9 and 10 were obtained as products; the preparative yields
calculated with respect to the consumed diol 1 were 29% and 11%, respectively. Esterification
proceeded as a side process and gave rise to monoacetates 11 and 12 with the yields of 12% and 4%,
respectively. In order to improve this yield, we conducted a similar reaction with diacetate 13, which
17
was obtained from diol 1 with a quantitative yield as described earlier by Ardashov O. et al. The
interaction between diacetate 13 and iodine was much more selective than it was for diol 1 and
predominantly gave rise to 7S-epimer 14 (43% yield), whereas the content of minor 7R-stereoisomer
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15 was only 6%. Moreover, the products of partial hydrolysis 11 and 12 were isolated with the
yields of 2% and 11%, respectively (Scheme 2).
1
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OH
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2 OH
OH
OH
OAc
OH
I , H O
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5
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2
+
I
+
+
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AcOH, EtOAc
days
2% conversion
OH
OH
OAc
6
S
5
H
H
7
R
I
9
8
6
OH
9
OH
10
1
11
12
29%
11%
12%
4%
Ac O
2
NEt , DMAP
3
Et O
2
O
13
14
OAc
O12
OAc
OAc
OH
I , H O
2
2
+
+
11
+
AcOH, EtOAc
5 days
69% conversion
OAc
O
O
OAc
OH
OAc
H
H
S
R
I
I
1
3
OH
4
3%
OH
15
6%
11
2%
12
11%
1
4
100%
1
) i-Pr NEt,
2
OH
OH
OH
OH
OAc
OAc
OH
OH
i-Pr NEt,
2
O
O / H O, 24 h
2
O
O / H2O
4 h
H
H
H
2) NaOH,
O / H O, 16 h
H
R
R
S
S
I
I
2
O
OH
O
OH
2
O
1
0
6
14
7
8
8%
84%
Scheme 2. Synthesis of epoxides 6 and 7.
Iodohydrin 10 was converted to the respective epoxide 6 (with a 88% yield) using i-Pr NEt
2
as a base in aqueous dioxane. A similar process for compound 14 included epoxide formation in the
presence of i-Pr NEt and further saponification by NaOH. The epimeric epoxide 7 was formed with
2
an 84% yield (Scheme 2).
2
.3. Determination of the configuration of epoxide groups in compounds 4-7
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The configuration of epoxide groups in compounds 4 and 5 was determined using the H
1
1
4
5
6
4
NMR data by analyzing the vicinal H- H constants of the C H-C H -C H chain. Proton H in
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5
e
5a
compound 4 takes a position that allows overlapping with both H and H protons with the vicinal
4
constants of 2.2 and 1.9 Hz, respectively (Fig. 2). On the contrary, proton H in compound 5
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5e
interacts only with proton H with the vicinal constant of 5.0 Hz.
Determining the configuration of epoxide groups in compounds 6 and 7 was a challenging
task. A conformational analysis of epoxides 6 and 7 predicts that the oxygen atom in the epoxide
ring in the most energetically favorable forms of these epoxides is oriented towards the hydroxyl
1
proton OH (Fig. 2). This specific conformation is fixed by a strong intramolecular hydrogen bond.
1
1
The presence of this intramolecular H-bond is confirmed by H NMR: the OH signal lies downfield
2
of the OH signal and has a much narrower lineshape. Due to the H-bond fixation of the epoxide
7
conformation, the R/S-configuration of the C atom in both epoxides 6 and 7 can be reliably
determined by NOESY. Indeed, strong cross-peaks are observed in the 2D NOESY spectrum of
1
e
8en
5e
9
5a
epoxide 6. These peaks correspond to NOE between protons H and H , H and CH , H and
3
9
7
CH , being indicative of the R-configuration of C . On the contrary, NOE is observed between
3
5
e
8en
5a
8en
1e
9
protons H and H , H and H , H and CH in the 2D NOESY spectrum of epoxide 7,
3
7
corresponding to the C S-configuration in 7 (Fig. 2).
CH3
O
CH
3
4
O
OH
O
OH
H
2
.2 Hz
OH
OH
OH
OH
4
5
.0 Hz
H5e
H
H
O
H
H
H
H
5e
H
5a
H
5a
1.9 Hz
H
OH
OH
4
5
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1e
H5a
H
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8
en
H
H
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R
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H C
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8ex
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Figure 2. Configuration of epoxide groups in compounds 4-7.
Thus, all four isomeric epoxides 4-7 were successfully synthesized in their pure forms.
Interestingly, when we compared the mass spectra and retention times in GC-MS for epoxides 4-7
with the data on metabolic profiling of diol 1 in vivo, it turned out that epoxide 4 is a metabolite of
diol 1 present in the blood of experimental animals.
It is important that, unlike epoxides 5-7 and other epoxides of similar structure,18,19
compound 4 is unusually stable in acidic conditions such as 10% H SO or montmorillonite clay
2
4
K10.
2
.4. Studying the antiparkinsonian activity of epoxides 4-7
We studied the biological activity of epoxide 4 (identified as a metabolite of compound 1)
and isomeric compounds 5-7 in the MPTP mouse model of PD.
5
The MPTP-induced model is one of the most commonly used animal models of PD. In the
+
brain, MPTP is converted to MPP , which is selectively uptaken by the dopamine transporter into
dopaminergic neurons.20 In dopaminergic neurons, it blocks the electron transport chain in
mitochondria and reduces the cellular ATP levels, which leads to formation of toxic reactive oxygen
species and causes neuronal death. Systemic administration of MPTP causes reproducible lesions in
the nigrostriatal dopaminergic pathway. Although this model has some limitations, MPTP is the
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only known dopaminergic neurotoxin capable of causing clinical presentation indistinguishable
from idiopathic PD in humans.21
Parkinson's disease-like motor symptoms were induced in C57Bl/6 mice by four injections of
MPTP (0.12 mmol/kg (20 mg/kg) per dose, every 2 h) in accordance with the procedure described
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earlier. The studied agents were administered per os 24 h after the last injection of MPTP at a dose
of 20 mg/kg. The main markers of locomotor activity were measured 2 h after administration of the
agent using 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.
Administration of MPTP to mice reduced their locomotor activity, in particular, in
movement distance and duration of locomotor activity parameters (Fig. 3).
Figure 3. The influence of compounds 1, 4-7 (0.12 mmol/kg (20 mg/kg)) on locomotor activity of
MPTP-treated (4×0.12 mmol/kg (4×20 mg/kg)) male C57Bl/6 mice: movement distance (A) and
duration of locomotor activity (B). Mean ± SEM. N = 8 – 10 mice per group. * P < 0.05, ** P <
0.01 compared to the MPTP group, ANOVA with Dunnett's post-hoc test.
Administration of trans-epoxide 4 after MPTP neurotoxin injections led to a virtually
complete recovery in movement distance and a trend towards increasing duration of locomotor
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activity in animals (Fig. 3, A and B). It should be noted that compound 4 given to naïve mice at the
same dose had no effect on animals' locomotor activity (Fig. 4).
Unlike trans-epoxide 4, cis-isomer 5 did not affect the locomotor activity in mice treated
with MPTP (Fig. 3). Epoxide 6 also failed to influence the locomotor activity. Meanwhile, isomeric
epoxide 7 restored the locomotor activity of mice, which was previously reduced by MPTP.
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male C57Bl/6 mice. Mean ± SEM. N = 10 mice per group.
Thus, we found that two out of the four studied epoxides improved motor performance in the
MPTP mouse model of PD. One of these compounds, epoxide 4, is a product of epoxidation of 3,4-
double bound in compound 1, while another one, compound 7, is 7,8-epoxide of 1. It was
demonstrated that the configuration of epoxides played a crucial role in their ability to improve
motor performance in the MPTP mouse model of PD.
Dose reduction of epoxide 4 to 5 mg/kg led to a less pronounced but statistically significant
improvement of locomotor activity in MPTP-treated mice (Fig. 5). Unlike for the results obtained
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for compound 4 at a dose of 20 mg/kg (Fig. 3), restoration of locomotor activity (movement
distance) was incomplete in this case. However, a statistically significant increase in duration of
locomotor activity was observed.
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Figure 5. The influence of compound 4 (0.03 mmol/kg (5 mg/kg)) on locomotor activity of MPTP-
treated (4×0.12 mmol/kg (4×20 mg/kg)) male C57Bl/6 mice. A – movement distance, B – duration
of locomotor activity. Mean ± SEM. N = 10 mice per group.* P < 0.05, ** P < 0.01, **** P <
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.0001 compared to the MPTP group, ANOVA with Dunnett's post-hoc test.
We studied the safety of active epoxides 4 and 7. Neither toxic nor adverse effects were
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observed in response to a single oral 1000 mg/kg dose of these compounds. After 10 days, all
animals were still alive and no abnormalities in general condition were revealed. Hence, these
compounds exhibited low acute toxicity.
The ability of epoxide 4 to penetrate the blood–brain barrier was demonstrated by analyzing
the brain and plasma extracts 1.5 h after oral administration of the epoxide at a 10 mg/kg dose to
rats. Compound 4 was found both in plasma (5.3 μg/mL) and in the brain (3.5 μg/g). These data
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indicate that compound 4 readily penetrates the blood–brain barrier and remains in the bloodstream
for a relatively long time.
It is interesting to note that we also detected a small amount of epoxide 4 in blood of
experimental animals after administration of diol 1. These data indicate that epoxide 4 is a
metabolite of compound 1. In the present study, we showed that it exhibits a biological activity in
the MPTP mouse model of PD.
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According to the results of biological experiments and taking into account the good chemical
stability of epoxide 4, this epoxide was chosen for further studies.
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.5. Insights into the molecular mechanism of action of compound 4
Since the diagnostic symptoms of PD are caused by progressive degeneration of dopamine
neurons, drugs protecting and/or restoring these neurons can significantly improve the management
of PD.23-25 Therefore, we tested the ability of 0.01–10 μM compound 4 to promote the survival of
cultured embryonic dopamine neurons. Midbrain neurons were isolated from mouse embryos
(E13.5) and cultured for five days in the presence of compound 4 or a vehicle. Dopamine neurons
were labelled with antibodies specific to the key enzyme of dopamine synthesis, tyrosine
hydroxylase (TH), imaged using the CellInsight system, and counted using the Cell Profiler
software. Glial cell line-derived neurotrophic factor (GDNF), the protein capable of supporting the
survival of dopamine neurons,26,27 was used as a positive control. Compound 4 promoted the
survival of naïve TH-positive neurons in a dose-dependent manner. In the wells treated with 0.1 μM
and 1 μM compound 4, the number of TH-positive neurons was 2.1- (P < 0.05, ANOVA with
Dunnett's post-hoc test) and 2.3-fold (P < 0.01, ANOVA with Dunnett's post-hoc test) , respectively,
greater than that in vehicle-treated wells (P = 0.028, one-way ANOVA) (Fig. 6A).
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We also studied the effect of compound 4 on the survival of TH-positive neurons in midbrain
cultures treated with 6-hydroxydopamine (6-OHDA), a toxin causing selective death of dopamine
neurons.23 Compound 4 protected cultured dopamine neurons against 6-OHDA-induced
degeneration (Fig. 6B).
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Midbrain cell cultures contain other neuronal types in addition to dopamine neurons (in
particular, a significant proportion of neurons producing neurotransmitter gamma-aminobutyric acid
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(GABA)). Therefore, the effect of compound 4 on the survival of these neurons was investigated.
GABA-positive neurons were detected immuocytochemically using anti-GABA antibodies against
GABA. We found that compound 4 failed to influence the survival of GABA-positive neurons (Fig.
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C). Compound 4 was also unable to promote the proliferation of primary mesencephalic embryonic
fibroblasts (Fig. 6D). These data indicate that the survival-promoting properties of epoxide 4 are
restricted to the cells of neuronal origin and especially to dopamine neurons.
We further attempted to address the possible mechanisms of neuroprotective effect of
epoxide 4 observed in dopamine neurons. It is known that the survival of cultured dopamine neurons
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can be positively affected by neurotrophic factors GDNF and brain-derived neurotrophic factor
(
BDNF).29 Hence, we accessed the ability of epoxide 4 to activate the receptors of these
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neurotrophic factors: receptor tyrosine kinases RET and TrkB, respectively. However, epoxide 4
activated neither RET (Fig. 6E), nor TrkB (Fig. 6F) in cultured murine fibroblasts MG87
overexpressing these receptors. In line with these data, epoxide 4 failed to activate Akt and
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mitogene-activated protein kinase (MAPK/ERK) downstream signaling cascades that are known to
be important for the survival and functioning of mammalian cells in MG87RET fibroblasts (Fig.
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G). Based on these results, we hypothesized that the neuroprotective effect of epoxide 4 can be
mediated by a receptor or receptors other than RET or TrkB expressed in neuronal cells. Therefore,
we assessed the ability of compound 4 to activate the MAPK and AKT signaling cascades in
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immortalized cells of neuronal origin. Indeed, the level of phosphorylated ERK in Neuro2A
neuroblastoma cells was by 32% higher increased in the cells treated with compound 4 compared to
the vehicle-treated cells (P < 0.05, paired t-test), whereas no changes were observed in the level of
pAKT (Fig. 6 H-J). The levels of pERK and pAKT normalized to GAPDH were calculated
separately for each experimental time point and averaged afterwards, because the peaks of pERK
and pAKT activation varied in time between the experiments, making direct comparison
complicated.
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Taken together, our results suggest that compound 4 may be promising both for treatment of
disease progression in PD and alleviation of PD symptoms. The receptor of compound 4 is yet to be
determined. Furthermore, we cannot rule out the possibility that epoxide 4 has no neuroprotective
properties; instead, it might influence the secretion of various neurotrophic factors supporting the
survival of dopamine neurons as it has been shown for selegiline.33,34
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Figure 6. The biological effects of compound 4 in cultured cells. The influence of compound 4 on
the survival of cultured naïve dopamine neurons (A), on the cultured dopamine neurons treated with
6
-OHDA (B), on the survival of GABA neurons (C) and murine mesencephalic embryonic
fibroblasts (D). The influence of compound 4 on phosphorylation of receptors of neurotrophic
factors GDNF (E) and BDNF (F). The influence of compound 4 on MAPK/ERK and AKT signaling
cascades in murine fibroblasts MG87RET transfected with GDNF family receptor alpha 1 (GFRα1)
(G) or in neuroblastoma Neuro 2A cells (H) shown by Western blotting. The concentration of
compound 4 applied to Neuro 2A cells (H) was 100 M. Quantitative analysis of pAKT (I) and
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pERK (J) levels in Neuro2A cells treated with vehicle or compound 4 (100 M) for 1–60 min. The
quantitative data are presented as Mean ± SEM. IP – immunoprecipitation; WB – Western blotting;
pY – phosphotyrosine; GAPDH – glyceraldehyde 3-phosphate dehydrogenase, loading control;
VEH – vehicle. A – * P < 0.05 ANOVA with Dunnett's post-hoc test compared to vehicle, N = 4; H
– * P < 0.05, paired t-test, compared to vehicle, N = 5.
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.6. Evaluation of the neuroprotective/neurorestorative properties of epoxide 4 in the
nigrostriatal dopamine system
According to the in vitro data showing the positive influence of compound 4 on the survival
of cultured dopamine neurons, we decided to evaluate the neurorestorative properties of diol
epoxide in the nigrostriatal dopamine system in vivo in the MPTP-induced mouse model of PD. The
bodies of nigrostriatal dopamine neurons are located in the brain region known as substantia nigra
pars compacta (SNpc), while their axons project to the brain region called the striatum. Therefore,
we quantified the density of TH-positive fibers in the striatum and the number of TH-positive cells
in SNpc. The mice were treated with 4×20 mg of MPTP to induce degeneration of dopamine
neurons. Two different doses (5 and 20 mg/kg) were used to study the effect of epoxide 4 in this
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model. Epoxide 4 was administered orally to mice 24 h after the last MPTP injection and then for
additional 14 days (5 daily administrations per week for 3 weeks). The murine brains were collected
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0 days after the lesion. Administration of MPTP reduced the density of TH-positive fibers in the
striatum twofold as compared to that in vehicle-treated mice (Fig. 7A). Treatment of MPTP mice
with both 5 and 20 mg/kg of compound 4 significantly increased the density of TH-positive fibers in
the striatum as compared to that in MPTP-treated mice (P = 0.0053 and P = 0.0329, respectively,
ANOVA with Dunnett's post-hoc test) (Fig. 7A).
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We also studied the effect of epoxide 4 on the number of TH-positive neurons in the SNpc.
Significant changes in the number of TH-positive neurons were observed in none of treatment
groups.
The data presented in Fig. 6 suggest that compound 4 can either stimulate the outgrowth and
arborization of neurites of dopamine neurons in vivo, which is expected to increase the number of
dopaminergic synapses in the striatum and halt the disease progression, or at least stimulate the
expression of TH, which will likely elevate dopamine content in the striatum and alleviate
symptoms of PD. It is unclear whether compound 4 can also stimulate regeneration of dopamine cell
bodies in SNpc, since their loss induced by MPTP in this particular experiment was mild, if any at
all. However, we noticed in the in vitro experiments that epoxide 4 can activate ERK (Fig 6H, 6J),
but not AKT (Fig 6I). In neurons, AKT is the major regulator of neuronal survival,35,36 while
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ERK1/2 is important for neurite growth rather than for survival. Thus, the in vitro data showing
pERK activation in cultured cells taken together with the results of analyzing the TH-positive fiber
density may indicate that the mechanism of neurorestoration induced by epoxide 4 is mediated by
ERK and includes stimulation of striatal reinnervation by dopamine neurons.
It is important to mention that neurodegeneration in Parkinson's disease starts from axons.
The motor symptoms of PD appear when dopamine concentration in the putamen drops by 70%–
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80%. Meanwhile, the loss of dopamine neuron bodies in the SNpc at disease onset is only 30%3.
Moreover, some bodies of dopamine neurons remain in the brain of PD patients even at the very late
stage (> 20 years after the diagnosis), while dopamine axons in the putamen disappear almost
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Our results do not give grounds for drawing a conclusion whether epoxide 4 is able to protect
the bodies of dopamine neurons in SNpc. However, it might protect or stimulate axonal regrowth
into the striatum. Therefore, our data and the features of PD progression in patients support that
epoxide 4 should be further developed not only as symptomatic treatment but also as a potential
neuroprotective/neurorestorative therapeutic agent against PD. It will also be interesting to test
epoxide 4 in other models of neurodegenerative diseases (e.g., Alzheimer’s and Huntington’s
diseases), as well as in other nervous system disorders associated with neuronal loss (e.g.,
neuropathic pain). The cellular receptor of epoxide 4 seems to be expressed in the cells of neuronal
origin, but its molecular identity remains to be determined. It is possible that other types of neurons
than dopamine neurons can also respond positively to compound 4, thus providing grounds for
developing disease-modifying treatment for multiple conditions caused by neuronal cell death.
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Figure 7. The influence of compound 4 (5 and 20 mg/kg) on the density of TH-positive fibers in the
striatum (A, B) and the number of TH-positive cells (C) in SNpc of MPTP-treated (4×0.12 mmol/kg
(4×20 mg/kg)) male C57Bl/6 mice. The data are presented as Mean ± SEM, N = 3–6 per group. * P
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.7. Conclusions
All four isomeric epoxides of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-
diol 1 have been synthesized. Epoxidation of double bonds at positions 3,4 (epoxide 4) and 7,8
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(epoxide 7) was found to give rise to biologically active compounds capable of restoring the
locomotor activity in the MPTP-induced mouse model of PD. Epoxide 4 was identified to be an
active metabolite of 1. Both compounds were able to penetrate the blood–brain barrier, but epoxide
4 did so with higher efficacy. Moreover, epoxide 4 remained in the bloodstream longer than epoxide
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
did. Interestingly, epoxide 4 is also a metabolite of diol 1 found in vivo. Based on all these data,
we selected epoxide 4 to study the possible mechanism of its biological activity in the PD model.
We found that epoxide 4 protects cultured dopamine neurons against spontaneous and toxin-induced
death and selectively activates ERK in the cells of neuronal origin. In animals treated with MPTP,
epoxide 4 increases the density of TH-positive fibers in the striatum. Our data indicate that epoxide
4
may produce positive immediate and long-term effects in the PD model; the latter ones might be
related to neurorestoration. Hence, epoxide 4 may encourage the development of disease-modifying
treatment against PD.
3. Methods
3
.1. General methods and materials: Reagents and solvents were purchased from commercial
suppliers and used as received. Dry solvents were obtained according to the standard procedures.
3
D
1
[]
(1R,2R,6S)-3-Methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol (1) (
-49.1 (c 2.6, CHCl ))
3
2
D
5
was synthesized from (-)-verbenone (Aldrich) ([] -210.5 (c 0.77, CHCl )) according to
3
previously described procedure.39,40
Spectral and analytical investigations were carried out at Collective Chemical Service center
1
13
of Siberian Branch of Russian Academy of Sciences. H- and C-NMR Spectra: Bruker DRX-500
1
13
apparatus at 500.13 MHz ( H) and 125.76 MHz ( C) in CDCl , CDCl / CCl 1 : 1 (v/v)or CDCl /
3
3
4
3
CD OD 10 : 1 (v/v); chemical shifts  in ppm rel. to residual CHCl [(H) 7.24, (C) 76.90 ppm], J
3
3
1
13
in Hz. The structure of the products was determined by analyzing the Н- and С-NMR spectra,
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13
1
Н, Н double-resonance spectra and С, Н-type 2D-COSY (J(С,Н) = 135 Hz). NMR spectra for
1
13
compounds 6 and 7: Bruker AV-600 spectrometer (resonance frequency for H 600.30 MHz, for C
1
13
1
50.95 MHz). Chemical shifts for H and C measured relative to the internal standard CDCl (δ
3
1
13
7.24 ppm for Н, 76.90 ppm for С). For structure determination and NMR signal assignment in 6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
13
and 7 2D correlation spectra H- H (COSY, NOESY) and H- C (HSQC, HMBC) were used. GC:
7
820A gas chromatograph (Agilent Tech., USA); flame-ionization detector; HP-5 capillary column
(0.25 mmol Ø × 30 m × 0.25 µm), He as carrier gas (flow rate 2 ml/min, flow division 99 : 1).
GC/MS: Agilent 7890A with a quadrupole mass spectrometer Agilent 5975C as a detector, HP-5MS
quartz column, 30000x0.25 mm, He (1 atm) as carrier gas. Optical rotation: polAAr 3005
spectrometer; CHCl soln. HR-MS: DFS Thermo Scientific spectrometer in a full scan mode (15-
3
500 m/z, 70 eV electron impact ionization, direct sample administration).
Conformational analysis of the epoxides 6 and 7 was performed with the computer program
packages ChemAxon Marvin,41 VeraChem Vconf42 and Tinker.43 The geometries of all the
conformers were optimized by DFT (Priroda program,44,45 PBE functional, L1 basis (Λ01, cc-
pVDZ analog). Quantum chemical calculations were carried out on the computational cluster at the
Information Computation Center, Novosibirsk State University. 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 no less than 95% (GC-MS data). Target compounds successfully passed the virtual
filter for known classes of assay interference compounds (PAINS).48
46
47
3.2. (1S,2S,3R,4S,6R)-1-methyl-4-(prop-1-en-2-yl)-7-oxabicyclo[4.1.0]heptane-2,3-diol (4) and
(
1R,2S,3R,4S,6S)-1-methyl-4-(prop-1-en-2-yl)-7-oxabicyclo[4.1.0]heptane-2,3-diol (5).
.2.1. Epoxidation of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol (1)
with peracetic acid. Method a (Scheme 1). A mixture of AcOH (200 ml), 35% H O (200 ml) and
3
2
2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H SO (5 ml) was stored for 5 days. Then a part of this mixture (100 ml) was extracted with CH Cl
2
2
4
2
(
2 x 50 ml) and combined extract was dried over MgSO . The concentration of AcOOH was 0.0925
4
moles per 100 ml of the solution. Grinded Na CO (2 g) and then the AcOOH solution (7 ml, 0.648
2
3
mmol) were added under stirring to the solution of diol 1 (1.00 g, 5.95 mmol) in CH Cl (4 ml). The
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reaction mixture was stirred for 3 h, diluted with water (40 ml), separated, and the water phase was
extracted with CH Cl (3 x 20 ml) and EtOAc (3 x 30 ml). The combined organic layers were
2
2
desiccated over Na CO . The desiccant was filtered off, the solvent was evaporated. The residue
2
3
(762 mg) was separated by CC on silica gel (17 g) using 0-100% EtOAc gradient in hexane as
eluent. This procedure gave starting diol 1 (64 mg, 0.38 mmol, conv. 94%), (1S,2S,3R,4S,6R)-1-
methyl-4-(prop-1-en-2-yl)-7-oxabicyclo[4.1.0]heptane-2,3-diol (4, 179 mg, 0.973 mmol, 16%
calculated on reacted diol 1) and (1R,2S,3R,4S,6S)-1-methyl-4-(prop-1-en-2-yl)-7-
oxabicyclo[4.1.0]heptane-2,3-diol (5, 40 mg, 0.217 mmol, 4%)
Compound 4. []D24 -19.8 (c 1.06, CHCl ). H NMR (500 MHz, CDCl /CCl 1 : 1 (v/v)): δ
1
3
3
4
1
0
9
5e
1
.44 (s, C H ), 1.77 (s, C H ), 1.88 (ddd, H , J = 14.7, J5e,6a = 4.5, J5e,4 = 2.2 Hz), 2.07 (ddd,
5e,5a
3
3
5
a
6a
= 12.0, J6a,5e = 4.5 Hz), 2.29 (d, C1-
6a,5a
H , J
= 14.7, J5a,6a = 12.0, J5a,4 = 1.9 Hz), 2.21 (dd, H , J
5
a,5e
4
2e
1e
OH, JOH,1e = 10.0 Hz), 3.31 (dd, H , J
= 2.2, J4,5a = 1.9 Hz), 3.58 (s, H ), 3.76 (dd, H , J
=
4,5e
1e,OH
8
8’ 13
10.0, J1e,2e = 2.5 Hz), 4.79 (br. s, H ), 4.94 (br. s, H ). C NMR (125 MHz, CDCl /CCl 1 : 1 (v/v)):
3
4
1
2
3
4
5
6
7
δ 71.32 (d, C ), 69.43 (d, C ), 58.44 (s, C ), 63.32 (d, C ), 23.12 (t, C ), 35.95 (d, C ), 144.98 (s, C ),
8
9
10
+
1
11.78 (t, C ), 22.92 (q, C ), 22.17 (q, C ). HR-MS: 166.1000 (M -H O, C H O ; calc. 166.0988).
2 10 14 2
Compound 5. []D26 -24.1 (c 0.64, CHCl ). H NMR (500 MHz, CDCl /CCl 1 : 1 (v/v)): δ
1
3
3
4
10
9
5e
1
2
.38 (s, C H ), 1.75 (s, C H ), 2.02 (dddd, H , J = 15.8, J5e,6a = 7.5, J5e,4 = 5.0, J5e,1e = 0.6 Hz),
5e,5a
3
3
5
a
6a
= 12.0, J6a,5e = 7.5 Hz), 3.09 (d, C1-
6a,5a
.07 (dd, H , J
= 15.8, J5a,6a = 12.0 Hz), 2.37 (dd, H , J
5
a,5e
4
2
3e
OH, JOH,1e = 11.3 Hz), 3.16 (d, H , J = 5.0 Hz), 3.32 (br. s, C -OH), 3.62 (dm, H , J
= 11.3
3e,OH
4,5e
2e
8
8’
Hz), 3.93 (br. s, H ), 4.78 (dd, H , J = 1.7, J8,6a = 0.7 Hz), 4.84 (dd, H , J = 1.7, J8’,6a = 1.5
8
,8’
8’,8
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3
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2
3
Hz). C NMR (125 MHz, CDCl /CCl 1 : 1 (v/v)): δ 73.39 (d, C ), 70.19 (d, C ), 60.83 (s, C ),
3
4
4
5
6
7
8
9
10
6
0.43 (d, C ), 23.67 (t, C ), 37.42 (d, C ), 145.24 (s, C ), 111.74 (t, C ), 22.01 (q, C ), 19.85 (q, C ).
+
HR-MS: 184.1100 (M , C H O ; calc. 184.1094).
1
0
16
3
3.2.2. Epoxidation of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol (1)
with m-CPBA.
Method b (Scheme 1). A solution of diol 1 (0.312 g, 1.86 mmol) in CH Cl (40 ml) was
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
o
cooled to 0 C. NaHCO (1.55 g, 18.5 mmol) and m-CPBA (m-Cl-C H -CO H, “Acros Organics”
3
6
4
3
0
.589 g, 70-75% of content, ~ 2.5 mmol) were added and the reaction mixture was stored in
o
refrigerator (2 - 5 C) for 1 week. Then water (30 ml) was added and the resulting mixture was
filtered and separated. The water phase was extracted with EtOAc (3 x 15 ml) Combined organic
layers were washed with saturated Na S O (15 ml) and saturated NaCl (20 ml) and dessicated over
2
2
3
Na SO . The desiccant was filtered off, the solvent was evaporated. The residue (293 mg) was
2
4
separated by CC on silica gel (9 g) using 0-100% EtOAc gradient in hexane as eluent. This
procedure gave starting diol 1 (28 mg, 0.17 mmol, conv. 91%), the epoxide 4 (82 mg, 0.446 mmol,
2
6% calculated on reacted diol 1) the epoxide 5 (34 mg, 0.185 mmol, 11%) and the epoxide 7 (10
mg with impurities, 0.04-0.05 mmol, 2-3%).
Method с (Scheme 1). A solution of diol 1 (458 mg, 2.73 mmol) in Et O (30 ml) was cooled
2
o
to 0 C. Then m-CPBA (“Acros Organics” 982 mg, 70-75% of content, ~ 4.2 mmol) was added and
o
the reaction mixture was stored in refrigerator (2 - 5 C) for 3 week. The solvent was evaporated and
the residue was separated by CC on silica gel (17 g) using 0-100% EtOAc gradient in hexane as
eluent. This procedure gave the epoxide 4 (161 mg, 0.875 mmol, 32%) epoxide 5 (49 mg, 0.266
mmol, 10%) and 230 mg (1.15 mmol, 42%) of the mixture of four stereoisomeric diepoxides 8. The
formation of diepoxides is confirmed by the signals of oxirane cycle at 2.5 – 3.5 ppm and by the
1
absence of signals related to double bounds at 4.6 – 6 ppm in H NMR spectra.
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
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5
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5
5
5
5
5
5
6
3
3
.3. (1R,2R,6R)-3-methyl-6-((R)-2-methyloxiran-2-yl)cyclohex-3-ene-1,2-diol (6) and (1R,2R,6R)-
-methyl-6-((S)-2-methyloxiran-2-yl)cyclohex-3-ene-1,2-diol (7).
3.3.1. Interaction of diol 1 with iodine. Iodine (811.4 mg, 3.197 mmol) was grinded in a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mortar and dissolved in EtOAc (5 ml). Acetic acid (1.0 ml) and water (3 ml) were added. Then a
solution of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol (1, 523.6 mg, 3.113
mmol) in 10 ml EtOAc was added. The reaction mixture was stirred for 5 days at r.t. Then it was
washed with saturated solutions of NaHCO (2x20 ml, add slowly) and Na S O (10 ml).The
3
2
2
3
organic phase was desiccated over Na SO . The desiccant was filtered off, the solvent was
2
4
evaporated. The residue (682.8 mg) was separated by CC on silica gel (20 g) using 0-100% EtOAc
gradient in hexane as eluent. This procedure gave starting diol 1 (326.3 mg, 1.940 mmol, conv.
6
2%), (1R,2R,6R)-6-((S)-2-hydroxy-1-iodopropan-2-yl)-3-methylcyclohex-3-ene-1,2-diol (9, 176.1
mg, 0.564 mmol, 29% calculated on consumed diol 1), (1R,2R,6R)-6-((R)-2-hydroxy-1-iodopropan-
2-yl)-3-methylcyclohex-3-ene-1,2-diol (10, 69.4 mg, 0.208 mmol, 11% calculated on reacted diol
1
), (1R,5S,6R)-6-hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enyl acetate (11, 48.2 mg, 0.229
mmol, 12%) and (1R,2R,6S)-2-hydroxy-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-enyl acetate (12,
17.9 mg, 0.085 mmol, 4%).
1
9
2
Compound 9. H NMR (500 MHz, CDCl ): δ 1.52 (s, C H ), 1.73 (br. d, C -OH, J = 5.0
OH,2e
3
3
1
0
5e
6a
5a
7
Hz), 1.82 (m, C H , all J ≤ 2.5 Hz), 2.00 – 2.09 (m, H , H ), 2.25 – 2.35 (m, H ), 2.90 (br. s, C -
3
1
8
2
8’ 2
OH), 3.01 (br. d, C -OH, J
= 3.4 Hz), 3.24 (d, H , J = 10.1 Hz), 3.42 (d, H , J = 10.1 Hz), 3.81
OH,1e
2
e
1e
(br. dd, H , J
= 5.0, J2e,1e = 3.0 Hz), 4.30 (ddd, H , J
= 3.4, J1e,2e = 3.0, J1e,6a = 1.5 Hz),
2
e,OH
1e,OH
4
13
1
2
3
5
1
.65 – 5.69 (m, H ). C NMR (125 MHz, CDCl ): δ 71.26 (d, C ), 72.08 (d, C ), 131.14 (s, C ),
3
4
5
6
7
8
9
10
25.16 (d, C ), 20.71 (t, C ), 37.37 (d, C ), 73.44 (s, C ), 18.39 (t, C ), 27.15 (q, C ), 20.87 (q, C ).
+
HR-MS: 294.0109 (M -H O, C H O I; calc. 294.0111).
2
10 15
2
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Compound 10. []D26 -36.7 (c 0.463, CHCl ). H NMR (500 MHz, CDCl /CD OD): δ 1.30
1
3
3
3
9
10
5e 2
(
s, C H ), 1.75 (m, C H , all J ≤ 2.5 Hz), 2.01 (dddq, H , J = 17.6, J
5.6, J5e,4 = 5.3, J5e,10 = 1.6
3
3
5e,6a
6
a
5a 2
Hz), 2.12 (ddd, H , J
= 11.3, J6a,5e = 5.6, J6a,1e = 1.8 Hz), 2.26 (dddqd, H , J = 17.6, J
=
5a,6a
6a,5a
8
2
8’ 2
1
1.3, J5a,4 = J5a,10 = 2.5, J5a,2e = 1.3 Hz), 3.35 (dd, H , J = 10.2, J = 0.6 Hz), 3.39 (d, H , J = 10.2
8,9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
e
1e
4
Hz), 3.64 (br. d, H , J
= 2.7 Hz), 4.10 (dd, H , J
= 2.7, J1e,6a = 1.8 Hz), 5.62 (ddq, H , J
=
4,5e
2
e,1e
1e,2e
1
3
1
2
5
.3, J4,5a = 2.5, J4,10 = 1.5 Hz). C NMR (125 MHz, CDCl /CD OD): δ 70.40 (d, C ), 71.99 (d, C ),
3
3
3
4
5
6
7
8
9
130.85 (s, C ), 125.18 (d, C ), 21.66 (t, C ), 36.17 (d, C ), 73.04 (s, C ), 17.79 (t, C ), 25.19 (q, C ),
10
+
2
0.73 (q, C ). HR-MS: 294.0110 (M -H O, C H O I; calc. 294.0111).
2 10 15 2
Compound 11. []D26 -65.3 (c 1.28, CHCl ). H NMR (500 MHz, CDCl ): δ 1.67 (d, C -OH,
1
1
3
3
1
0
9
5e 2
JOH,1e = 4.0 Hz), 1.69 (m, C H , all J ≤ 2.5 Hz), 1.79 (m, C H , all J < 2.0 Hz), 2.03 (dddq, H , J =
3
3
12
5a 2
1
1
7.3, J5e,4 = 5.3, J5e,6a = 4.6, J5e,10 = 1.5 Hz), 2.08 (s, C H ), 2.26 (dddqd, H , J = 17.3, J
=
3
5a,6a
6
a
1.4, J5a,4 = J5a,10 = 2.5, J5a,2e = 1.5 Hz), 2.33 (br. dd, H , J
= 11.4, J6a,5e = 4.6 Hz), 3.91 (ddd,
6a,5a
1
e
8
8’
H , J
= 4.0, J1e,2e = 2.6, J1e,6a = 1.7 Hz), 4.85 (m, H , all J < 2.0 Hz), 4.96 (m, H , all J < 2.0
1
e,OH
2e
4
13
Hz), 5.10 – 5.12 (m, H , all J < 3.0 Hz), 5.76 – 5.79 (m, H ). C NMR (125 MHz, CDCl ): δ 68.57
3
1
2
3
4
5
6
7
(
d, C ), 73.00 (d, C ), 128.32 (s, C ), 127.57 (d, C ), 24.28 (t, C ), 40.50 (d, C ), 145.17 (s, C ),
8
9
10
11
12
+
111.59 (t, C ), 22.36 (q, C ), 20.52 (q, C ), 170.45 (s, C ), 21.01 (q, C ). HR-MS: 168.1149 (M ,
+
C H O ; calc. 168.1145). HR-MS: 168.1145 (M -C H O, C H O ; calc. 168.1145).
1
0
16
2
2
1
2
10 16
2
Compound 12. []D26 -49.4 (c 1.04, CHCl ). H NMR (500 MHz, CDCl ): δ 1.77 (m, C H ,
9
3
3
3
1
0
12
5e
2
all J < 2.0 Hz), 1.78 (m, C H , all J ≤ 2.5 Hz), 1.98 (s, C H ), 2.03 (dddq, H , J = 17.6, J
=
3
3
5e,6a
5a
2
J5e,4 = 5.3, J5e,10 = 1.5 Hz), 2.27 (dddqd, H , J = 17.6, J
= 11.2, J5a,4 = J5a,10 = 2.5, J5a,2e = 1.4
= 11.2, J6a,5e = 5.3 Hz), 3.80 – 3.84
6a,5a
5a,6a
2
6a
Hz), 2.35 (br. d, C -OH, J
≈ 5.0 Hz), 2.47 (br. dd, H , J
OH,2e
2
e
8
8’
1e
(
m, H ), 4.72 (m, H , all J < 2.0 Hz), 4.83 (m, H , all J < 2.0 Hz), 5.12 (dd, H , J
= 2.8, J1e,6a
=
1e,2e
4
13
1
2
1
.7 Hz), 5.65 – 5.69 (m, H ). C NMR (125 MHz, CDCl ): δ 73.50 (d, C ), 69.79 (d, C ), 131.26 (s,
3
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C ), 125.61 (d, C ), 25.35 (t, C ), 38.08 (d, C ), 144.81 (s, C ), 111.12 (t, C ), 22.19 (q, C ), 20.54 (q,
10
11
12
+
C ), 170.70 (s, C ), 20.88 (q, C ). HR-MS: 193.1223 (M -OH, C H O ; calc. 193.1223).
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2
3
.3.2. Interaction of diacetate 13 with iodine. Iodine (247.8 mg, 0.976 mmol) was grinded in
a mortar and dissolved in EtOAc (3 ml). Acetic acid (0.50 ml) and water (1.5 ml) were added. Then
a solution of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diyl diacetate (13, 248.7
mg, 0.986 mmol) in 5 ml EtOAc was added. The reaction mixture was stirred for 5 days at r.t. Then
it was washed with saturated solutions of NaHCO (2x10 ml, add slowly) and Na S O (3 ml).The
0
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2
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8
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0
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9
0
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8
9
0
1
2
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6
7
8
9
0
3
2
2
3
organic phase was desiccated over Na SO . The desiccant was filtered off, the solvent was
2
4
evaporated. The residue (320.9 mg) was separated by CC on silica gel (9 g) using 0-100% EtOAc
gradient in hexane as eluent. This procedure gave starting diacetate 13 (77.4 mg, 0.307 mmol, conv.
69%), (1R,2R,6R)-6-((S)-2-hydroxy-1-iodopropan-2-yl)-3-methylcyclohex-3-ene-1,2-diyl diacetate
(
14, 108.7 mg, summary formed 116.0 mg, 0.293 mmol, 43% calculated on reacted diacetate 13),
mixture of (1R,2R,6R)-6-((R)-2-hydroxy-1-iodopropan-2-yl)-3-methylcyclohex-3-ene-1,2-diol (14)
and epimeric compound 15 (24.1 mg, 15 : 13 = 2.3 : 1, the content of 15 was 16.8 mg, 0.0424 mmol,
6
% calculated on reacted diol 1), (1R,2R,6S)-2-hydroxy-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-
enyl acetate (12, 16.0 mg, 0.0761 mmol, 11%) and (1R,5S,6R)-6-hydroxy-2-methyl-5-(prop-1-en-2-
yl)cyclohex-2-enyl acetate (11, 3.3 mg, 0.0157 mmol, 2%).
1
9
10
Compound 14. H NMR (500 MHz, CDCl ): δ 1.27 (s, C H ), 1.65 (m, C H , all J ≤ 2.5
3
3
3
7
12
14
5
6a
8 2
Hz), 1.93 (br. s, C -OH), 2.00 (s, C H ), 2.08 (s, C H ), 2.15 – 2.28 (m, 2H , H ), 3.36 (d, H , J =
3
3
8
’
2
2e
1e
1
0.5 Hz), 3.46 (d, H , J = 10.5 Hz), 4.96 (br. d, H , J
= 2.7 Hz), 5.17 (dd, H , J
1e,2e
= 2.7, J1e,6a
2e,1e
4
13
1
2
= 1.0 Hz), 5.79 – 5.82 (m, H ). C NMR (125 MHz, CDCl ): δ 68.61 (d, C ), 70.50 (d, C ), 128.20
3
3
4
5
6
7
8
9
(
s, C ), 127.82 (d, C ), 21.69 (t, C ), 40.48 (d, C ), 70.94 (s, C ), 21.82 (t, C ), 23.53 (q, C ), 20.35
1
0
11
12
13
14
+
(
q, C ), 169.89 (s, C ), 21.01 (q, C ), 169.73 (s, C ), 20.90 (q, C ). HR-MS: 336.2016 (M ,
C H O I ; calc. 336.2017).
1
4
21
5
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Compound 15. The NMR spectra of compound 15 were recorded for the mixture of isomers
1
9
1
5 and 14 in a ratio 2.3 : 1. Data for 15: H NMR (500 MHz, CDCl ): δ 1.30 (s, C H ), 1.65 – 1.69
3 3
1
0
12
14
5e
5a
(
m, C H , all J ≤ 2.5 Hz), 2.02 (s, C H ), 2.09(s, C H ), 2.07 – 2.14 (m, H ), 2.18 – 2.23 (m, H ),
3 3 3
6
a
8
2
8’ 2
2.27 (ddd, H , J
= 11.6, J6a,5e = 4.7, J6a,1e = 1.2 Hz), 3.35 (d, H , J = 10.3 Hz), 3.38 (d, H , J =
a,5a
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2e
1e
1
5
0.3 Hz), 5.05 (br. d, H , J
= 2.7 Hz), 5.30 (ddd, H , J
= 2.7, J1e,6a = 1.2, J1e,5e = 0.7 Hz),
2
e,1e
1e,2e
4
13
1
2
3
.78 – 5.81 (m, H ). C NMR (125 MHz, CDCl ): δ 68.76 (d, C ), 70.22 (d, C ), 128.48 (s, C ),
3
4
5
6
7
8
9
10
127.54 (d, C ), 22.49 (t, C ), 39.27 (d, C ), 71.31 (s, C ), 19.71 (t, C ), 24.85 (q, C ), 20.45 (q, C ),
11
12
13
14
1
70.18 (s, C ), 21.15 (q, C ), 169.76 (s, C ), 20.94 (q, C ).
3
.3.3. Epoxide 6. A solution of (1R,2R,6R)-6-((R)-2-hydroxy-1-iodopropan-2-yl)-3-
methylcyclohex-3-ene-1,2-diol (10, 19.3 mg, 0.0618 mmol) in dioxane (5 ml) was added to water (3
ml). Then N,N-diisopropylethylamine (57 μL, 0.327 mmol) was added. The reaction mixture was
stirred for 24 h, then concentrated to 1 ml and diluted with 5 ml saturated NaCl. It was extracted
with ethylacetate (3 x 3 ml). The combined organic phase was desiccated over Na SO . The
2
4
desiccant was filtered off, the solvent was evaporated. The residue (15.5 mg) was purified by CC on
silica gel (2.5 g) using 0-100% EtOAc gradient in hexane as eluent. This procedure gave
(
1R,2R,6R)-3-methyl-6-((R)-2-methyloxiran-2-yl)cyclohex-3-ene-1,2-diol (6, 10.0 mg, 0.0543
mmol, 88%).
Epoxide (R)-6. []D29 -74.4 (c 0.597, CHCl ). H NMR: δ 1.38 (s, C H ), 1.80 (m, C H , all
1
9
10
3
3
3
6
a
= 10.0 Hz, J6a,5e = 6.7 Hz, J6a,1e = 1.6 Hz), 2.16 – 2.21 (m, 2H5),
a,5a
J ≤ 2.5 Hz), 2.11 (ddd, H , J
6
8
ex
8en
2e
2.66 (d, H , J
= 3.9 Hz), 3.16 (br.d, H , J
= 3.9 Hz), 3.79 (br.d, H , J
= 2.8 Hz),
8
ex,8en
8en,8ex
2e,1e
1e
4
13
1
2
3
.90 (br. m, H , all ≤ 2.8 Hz), 5.65 – 5.68 (m, H ). C NMR: δ 70.74 (d, C ), 71.68 (d, C ), 131.76
3
4
5
6
7
8
9
(
s, C ), 124.87 (d, C ), 22.82 (t, C ), 34.98 (d, C ), 60.05 (s, C ), 52.46 (t, C ), 20.87 (q, C ), 20.80
1
0
+
(
q, C ). HR-MS: 166.0982 (M -C H O, C H O ; calc. 166.0988).
2
2
10 14
2
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3.3.4. Epoxide 7. A solution of (1R,2R,6R)-6-((S)-2-hydroxy-1-iodopropan-2-yl)-3-
methylcyclohex-3-ene-1,2-diyl diacetate (14, 108.3 mg, 0.273 mmol) in dioxane (10 ml) was added
to water (6 ml). Then N,N-diisopropylethylamine (0.200 ml, 1.148 mmol) was added. The reaction
mixture was stirred for 24 h, then NaOH (51.3 mg, 1.128 mmol) was added. The mixture was stirred
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8
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1
6 h more, then concentrated to 1 ml and diluted with 5 ml saturated NaCl. It was extracted with
ethylacetate (6 x 3 ml). The combined organic phase was desiccated over Na SO . The desiccant
2
4
was filtered off, the solvent was evaporated. The residue (57.1 mg) was purified by CC on silica gel
4.5 g) using 0-100% EtOAc gradient in hexane as eluent. This procedure gave (1R,2R,6R)-3-
methyl-6-((S)-2-methyloxiran-2-yl)cyclohex-3-ene-1,2-diol (7, 42.4 mg, 0.230 mmol, 84%).
(
Epoxide (S)-7. []D26 -76.6 (c 0.950, CHCl ). H NMR: δ 1.42 (d, C H , J
1
9
= 0.6 Hz), 1.79
3
3
9,8en
10
5e
(
m, C H , all J ≤ 2.5 Hz), 1.87 (dddq, H , J
= 16.8 Hz, J5e,4 = J5e,6a = 5.2 Hz, J5e,10-Me = 1.6 Hz),
5e,5a
3
2
5a
6a
1
.92 (br.s, OH-C ), 1.96 (ddm, H , J
= 16.8 Hz, J5a,6a = 11.1 Hz), 2.01 (ddd, H , J
= 11.1 Hz,
= 4.4 Hz),
5
a,5e
6a,5a
1
8ex
J6a,5e = 5.2 Hz, J6a,1e = 1.6 Hz), 2.47 (br.d, OH-C , J
= 3.4 Hz), 2.51 (d, H , J
8ex,8en
OH,1e
8
en
2e
1e
2
.81 (dq, H , J
= 4.4 Hz, J8en,9-Me = 0.6 Hz), 3.81 (br.s, H ), 4.17 (ddd, H , J = 3.4 Hz,
1e,OH
8
en,8ex
4
13
J1e,2e = 2.8 Hz, J1e,6a = 1.6 Hz), 5.59 (ddq, H , J = 5.2 Hz, J4,5a = 2.2 Hz, J4,10 = 1.5 Hz). C NMR:
4,5e
1
2
3
4
5
6
δ 71.16 (d, C ), 71.85 (d, C ), 131.85 (s, C ), 124.49 (d, C ), 22.24 (t, C ), 35.88 (d, C ), 58.92 (s,
7
8
9
10
+
C ), 51.30 (t, C ), 21.19 (q, C ), 20.82 (q, C ). HR-MS: 166.0986 (M -C H O, C H O ; calc.
2
2
10 14
2
1
66.0988).
3.5. Animals. The experiments were performed on C57BL/6 mice (male) weighing 25-30 g and
Wistar rats (male) weighing 200-220 g purcahsed from SPF-vivarium of the Institute of Cytology
and Genetics of the Siberian Branch of the Russian Academy of Sciences. The animals were
o
maintained at 22-25 C on a 12 h light-dark cycle with food and water available ad libitum. All work
with animals was guided by 3R principles and performed in strict accordance with the legislation of
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the Russian Federation, the regulations 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. The use of
experimental animals for isolation of primary cell cultures was approved by the Committee for
Animal Experiments of the University of Helsinki (License number: KEK15-022) and these animals
were treated according European guidelines and regulations of the State of Finland.
1
1
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3
.6. The MPTP mouse model of Parkinson’s disease induced by the fourfold administration of
the MPTP neurotoxin.
MPTP was injected intraperitoneally to mice of C57Bl/6 line every 2 h in 8 h period in one
day in a dose of 0.12 mmol/kg (20 mg/kg) for a total of four doses. The agents were dissolved in
saline containing 0.5% Tween 80 just before use and were administered per os, 1 hour before
testing. The studied agent or saline 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, 2 h after the
administration of the agents. Hypokinesia caused by neurotoxin administration was evaluated with
the “Open field” test performed for 2 min using Tru Scan (USA) 2 h after the administration of the
studied agent, registering the main markers of the locomotor activity: movement distance (cm) and
duration of locomotor activity (s). The data were analyzed using ANOVA with Dunnett’s posthoc
test in GraphPad Prism 6.0 software
3
.7. Influence of compound 4 on naïve mice
The studied agent in a dose of 0.12 mmol/kg (20 mg/kg) or saline was administrated per os
to 8 mice of C57Bl/6 line. The main markers of the locomotor activity (movement distance (cm) and
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