Synthesis and biological activity of N-substitutedtetrahydro-γ- Carbolines containing peptide residues

Article abstract of DOI:10.3762/bjoc.10.13

The synthesis of novel peptide conjugates of N-substituted-tetrahydro- γ-carbolines has been performed using the sequence of the Ugi multicomponent reaction and Cu(I)-catalyzed click chemistry. The effect of obtained γ-carboline-peptide conjugates on the

Full text of DOI:10.3762/bjoc.10.13

Synthesis and biological activity of N-substituted-
tetrahydro-γ-carbolines containing peptide residues
Nadezhda V. Sokolova1,2, Valentine G. Nenajdenko*1,3,§,
Vladimir B. Sokolov2, Daria V. Vinogradova2, Elena F. Shevtsova2,
Ludmila G. Dubova2 and Sergey O. Bachurin*2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 155–162.
1Department of Chemistry, Moscow State University, Leninskie Gory
1, Moscow, 119992, Russia, 2Institute of Physiologically Active
Compounds, Russian Academy of Sciences, Severny proezd 1,
Chernogolovka, 142432, Russia and 3A. N. Nesmeyanov Institute of
Organoelement Compounds, Russian Academy of Sciences, Vavilov
str. 28, 119991 Moscow, Russian Federation
doi:10.3762/bjoc.10.13
Received: 14 October 2013
Accepted: 06 December 2013
Published: 15 January 2014
This article is part of the Thematic Series "Multicomponent reactions II".
Guest Editor: T. J. J. Müller
Email:
Valentine G. Nenajdenko* - nen@acylium.chem.msu.ru;
Sergey O. Bachurin* - bachurin@ipac.ac.ru
© 2014 Sokolova et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
§ Phone: +7 495 9392276; Fax: +7 495 9328846
Keywords:
mitochondrial membrane potential; mitochondrial permeability
transition; multicomponent; peptides; tetrahydro-γ-carbolines; Ugi
multicomponent reaction
Abstract
The synthesis of novel peptide conjugates of N-substituted-tetrahydro-γ-carbolines has been performed using the sequence of the
Ugi multicomponent reaction and Cu(I)-catalyzed click chemistry. The effect of obtained γ-carboline–peptide conjugates on the rat
liver mitochondria was evaluated. It was found that all compounds in the concentration of 30 µM did onot induce depolarization of
mitochondria but possessed some inhibitory effect on the mitochondria permeability transition. The original N-substituted-
tetrahydro-γ-carbolines containing an terminal alkyne group demonstrated a high prooxidant activity, whereas their conjugates with
peptide fragments slightly inhibited both autooxidation and the t-BHP-induced lipid peroxidation.
Introduction
The design and synthesis of new efficient pharmaceutical drugs found to play a crucial role in the pathogenesis of these diseases
for the treatment and prevention of a wide range of neurodegen- [1-3]. Thus, one of the specific symptoms of such pathologies is
erative diseases, such as Alzheimer’s dementia, Parkinsonism a decrease in the ability of mitochondria to regulate the calcium
and the amyotrophic lateral sclerosis, is of considerable interest homeostasis in cells and malfunction of mitochondrial perme-
in modern medicinal chemistry. Mitochondrial dysfunction was ability transition (MPT) that represents a key step in the
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cascades of cell death. From this point of view, mitochondria We expected that the conjugation of tetrahydro-γ-carbolines
and the MPT process are very attractive targets for the search of with synthetic peptides could lead to a new class of promising
new neuroprotective agents [4,5].
neuroprotectors affecting brain mitochondria. Thus, we synthe-
sized N-substituted tetrahydro-γ-carbolines and their peptide
Several promising mitochondria-targeting neuroprotectors have conjugates and investigated the action of the obtained com-
been reported in the literature. Thus, the antihistaminic drug pounds on the mitochondria membrane potential, mitochondrial
dimebon [6,7], which relates to tetrahydro-γ-carboline deriva- permeability transition and lipid peroxidation.
tives, has been found to stabilize and improve mitochondrial
functions in different in vivo and in vitro models [8,9] Results and Discussion
(Figure 1). Another promising class of neuroprotectors are cell- The starting N-substituted tetrahydro-γ-carbolines 3a–d
permeable mitochondria-targeting synthetic small peptides, for containing a terminal alkyne group were prepared in good
example, the SS (Szeto–Schiller) peptide antioxidants [10] yields by heating compounds 1a–d [12,13] with propargyl acry-
(Figure 1). These peptides were found to scavenge hydrogen late (2) in the presence of catalytic amounts of CsF (Scheme 1).
peroxide and peroxynitrite and inhibit lipid peroxidation in
vitro. By reducing mitochondrial reactive oxygen species, they The corresponding protected azidopeptides 5 [14,15] are acces-
inhibit MPT and cytochrome c release, thus protecting cells sible by the Ugi multicomponent reaction [16-19] of chiral
from oxidative cell death [11].
isocyanoazides 4 [20] with carbonyl compounds, amines and
Boc-protected amino acids (Scheme 2). As we showed before,
the racemization of the chiral centre of the isocyanoazide does
not occur under the conditions of the Ugi reaction [20]. This ap-
proach permits to prepare a broad variety of azidopeptides using
multicomponent methodology.
The modification of N-substituted tetrahydro-γ-carbolines 3a–d
by peptide fragments was performed using Cu(I)-catalyzed
click chemistry – one of the most effective conjugation methods
[21,22]. Thus, heating the educts with Cu(II)/sodium ascorbate
in a biphasic mixture of CH2Cl2/H2O during 1 h provided com-
pounds 6a–g (Scheme 3). According to the 13C NMR spectra,
the click reaction proceeds regioselective in all cases affording
the desired conjugates 6a–g in good yields.
Figure 1: Structures of dimebon and SS peptides.
Scheme 1: Synthesis of starting N-substituted tetrahydro-γ-carbolines 3a–d.
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Beilstein J. Org. Chem. 2014, 10, 155–162.
Scheme 2: Synthesis of peptides 5 through the Ugi reaction.
Next, we turned our attention to the deprotection of the amine experiments were repeated using three different preparations of
function in the peptide residues, in order to obtain water-soluble isolated rat liver mitochondria.
conjugates. Thus, N-Boc protecting groups were removed from
compounds 6a–g with 2 M HCl in methanol to give the corres- Mitochondrial membrane potential was monitored using safra-
ponding dihydrochloride salts 7a–g in nearly quantitative yields nine O [23]. The ability of the compounds to affect MPT was
(Scheme 4).
assessed by monitoring mitochondria swelling caused by
calcium chloride addition in a plate reader (Victor3, Perkin
The final dihydrochloride salts 7a–g were tested on rat liver Elmer). Swelling rate (Vmax) was calculated as a slope of the
mitochondria (RLM) using standards tests: lipid peroxidation steepest portion of the plot of swelling (light scattering) versus
(LP), mitochondrial membrane potential (ΔΨm) and Ca2+- time (dA530 × 1000 × min−1).
induced mitochondrial permeability transition (MPT).
N-substituted tetrahydro-γ-carbolines 3a–d possessed a pro-
On the day of the experiment, adult male Wistar rats fasted oxidant activity. These compounds potentiated the t-BHP-
overnight were euthanized in a CO2 chamber followed by induced LP and also induced LP of liver mitochondria. Their
decapitation. The procedure is in compliance with the Guide- peptide conjugates not only lost pro-oxidant activity but,
lines for Animal Experiments at IPAC RAS. Rat liver mito- moreover, some of these compounds could inhibit both mito-
chondria were isolated by conventional differential centrifuga- chondrial lipid auto-oxidation and the t-BHP-induced LP
tion as previously described [23]. All experiments were (Table 1).
provided with mitochondria energized by succinate in the pres-
ence of rotenone.
At the concentration of 100 µM (200 nmol/mg mitochondria)
the studied compounds caused a week decrease of ΔΨm and
The influence of compounds on spontaneous or induced by tert- demonstrated no significant influence on mitochondrial
butylhydroperoxide (t-BHP) LP was studied by the standard swelling (data not shown). But at higher pharmacologically
assay [24]. The extent of LP was measured spectrophotometri- relevant concentration of 30 µM (60 nmol/mg mitochondria) all
cally (λmax = 532 nm) and malondialdehyde–thiobarbituric acid compounds did not affect the ΔΨm and increased the resistance
adduct concentrations (MDA, expressed in nmol/mg protein) of mitochondria to calcium-induced MPT (Table 2). The results
were obtained by interpolation with a MDA standard curve of biological evaluation revealed an antioxidant and mitoprotec-
from commercially available 1,1,3,3-tetramethoxypropane. All tive potential of the new synthesized peptide-modified N-substi-
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Scheme 3: Synthesis of N-substituted tetrahydro-γ-carbolines containing protected peptide residues.
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Scheme 4: Synthesis of dihydrochloride salts 7a–g.
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Table 1: The influence of N-substituted tetrahydro-γ-carbolines and their peptide conjugates on LP of rat liver mitochondria.
LP in the presence of 0.1 mM compound, Influence of 0.1 mM compound on 1.6 mM t-BHP-induced LP,
Compound
nmol MDA/mg protein
nmol MDA/mg protein
no compounds
0.48 ± 0.01
1.72 ± 0.05
1.96 ± 0.04
1.72 ± 0.08
1.81 ± 0.08
0.39 ± 0.02
0.41 ± 0.02
0.39 ± 0.02
0.38 ± 0.02
0.4 ± 0.03
0.91 ± 0.02
7.63 ± 0.05
6.09 ± 0.08
6.05 ± 0.16
7.64 ± 0.09
0.86 ± 0.04
0.87 ± 0.06
0.79 ± 0.01
0.81 ± 0.02
0.92 ± 0.04
0.75 ± 0.02
0.95 ± 0.05
3a
3b
3c
3d
7a
7b
7c
7d
7e
7f
0.37 ± 0.01
0.62 ± 0.02
7g
tuted tetrahydro-γ-carbolines. Further investigations of this oxidant activity, whereas their peptide conjugates inhibited both
class of compounds may allow finding a promising approach to auto-oxidation and the t-BHP-induced lipid peroxidation.
cytoprotection, in particularly, neuroprotection.
Experimental
General Information
Table 2: The influence of the peptide conjugates of N-substituted
tetrahydro-γ-carbolines on rat liver mitochondria swelling.a
1H and 13C NMR spectra were recorded in deuterated solvents
on a Bruker Avance 400 MHz spectrometer. 19F NMR spectra
were recorded on a Bruker DXP 200 MHz spectrometer. 1H and
13C chemical shifts are reported in parts per million (ppm) or δ
values downfield from TMS as internal standard. Deuterated
solvent peaks were used as internal references: CDCl3 at 7.25
and 77.00 ppm. 19F chemical shifts are reported on δ scale (in
ppm) downfield from CF3COOH. Liquid chromatography was
performed using Fluka silica gel 60 (0.063–0.200 mm). Melting
points were determined with an Electrothermal IA9100 Digital
Melting Point Apparatus and are uncorrected. Optical rotations
were measured on a Perkin-Elmer 341 polarimeter at 589 nm.
High-resolution mass spectra (HRMS) were measured on a
MicrOTOF II (Bruker Daltonics) spectrometer.
Compounds
ΔA530/min (% of control)
7a
7b
7c
7d
7e
7f
70.5 ± 4.6
79.9 ± 8.1
80.9 ± 8.0
76.2 ± 7.5
72.4 ± 2.7
70.2 ± 8.6
79.0 ± 6.7
7g
aQuantification of swelling was measured as the maximal velocity of
A530 change after CaCl2 addition. The values were normalized
against the control values, set to 100%. Data are expressed as means
± SD (n = 3 or 4).
Conclusion
In summary, we described the conjugation of N-substituted Compounds 1a–d were obtained from the respective aryl-
tetrahydro-γ-carbolines containing a terminal alkyne group hydrazine hydrochlorides and N-substituted 4-piperidones using
3a–d with various azidopeptides 5 (prepared by Ugi multicom- Fischer indole synthesis [12,13]. Propargyl acrylate (2) was
ponent reaction) through the Cu(I)-catalyzed Huisgen 1,3- obtained from propargyl alcohol and acryloyl chloride
dipolar cycloaddition. The activity of the obtained compounds according to procedure described in [25].
on rat liver mitochondria functional characteristics, such as
mitochondrial transmembrane potential, calcium-induced mito- General procedure for the synthesis of compounds 3a–d: A
chondrial permeability transition and lipid peroxidation of mito- mixture of correspondingly substituted 2,3,4,5-tetrahydro-1H-
chondrial membrane was evaluated. It was found that all com- pyrido[4,3-b]indole 1a–d (2 mmol), 0.22 g (2 mmol) acrylic
pounds at a concentration of 30 µM did not induce depolariza- acid propyn-2-yl ester (2) and 0.1 g (0.66 mmol) of CsF in
tion of mitochondria but possessed some inhibitory effect on the 1 mL of DMF was stirred at 110 °C during 2 h. The solvent was
mitochondria permeability transition. The starting N-substi- removed in vacuo (~3 mmHg) and the product was extracted
tuted tetrahydro-γ-carbolines 3a–d demonstrated a high pro- from the residue with CH2Cl2. The solvent was removed in
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Supporting Information
Supporting Information File 1
General information and characterization data for all
compounds.
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