New organometallic imines of rhenium(i) as potential ligands of GSK-3β: Synthesis, characterization and biological studies

Article abstract of DOI:10.1039/c7dt04344a

Substituted amino-piperazine derivatives were synthesized and used as precursors for the preparation of a series of new organometallic Re(i) imine complexes with the general formula [(η⁵-C₅H₄CHN-(CH₂)₅-Pz-R)Re(CO)₃] (Pz-R: -alkyl or aryl piperazine). The piperazine-based ligands were designed to be potential inhibitors of GSK-3β kinase. All the ligands and complexes were fully characterized and evaluated against the HT-29 and PT-45 cancer cell lines, in which GSK-3β plays a crucial role. In this context, we carried out biological evaluation using the MTT colorimetric assay. In terms of structure activity relationship, our findings indicated improved biological activity when aromaticity increased in the organic ligands (3d). In addition, the presence of the rhenium fragment in the imines (5a-d) leads to better activity with IC₅₀ values in the range of 25-100 μM. In addition, our experimental studies were complemented by computational studies, where the volume and electrostatic surface of the organic ligands and organometallic compounds as well as their binding to the kinase protein are calculated.

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Efficient extraction of sulfate from water using
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New organometallics imines of Rhenium(I) as potential ligands of
GSK-3β: Synthesis, characterization and biological studies
Received 00th January 20xx,
Accepted 00th January 20xx
Michelle Muñoz-Ossesa, c, Fernando Godoya,*, Angélica Fierrob, Alejandra Gómeza and Nils Metzler-
Noltec,*
DOI: 10.1039/x0xx00000x
Substituted amino-piperazine derivatives have been synthesized and used as precursors in the preparation of a series of
new organometallic Re(I) imine complexes of the general formula [(η5-C5H4CH=N-(CH2)5-Pz-R)Re(CO)3] (Pz-R: -alkyl or aryl
piperazine). The piperazine-based ligands were designed to be potential inhibitors of the GSK-3β kinase. All ligands and
complexes were full characterized and evaluated in HT-29 and PT-45 cancer cell lines, where GSK-3β plays a crucial role. In
this context, we carried out biological evaluation using the MTT colorimetric assay. In terms of structure activity
relationship, our findings indicate improved biological activity when aromaticity is increased in the organic ligands (3d). In
addition, the presence of the rhenium fragment in the imines (5a-d) leads to better activity with IC50 values in the range of
25-100 μM. Additionally, our experimental studies were complemented by computational studies, where the volume and
the electrostatic surface of the organic ligands and the organometallic compounds as well as binding to the kinase protein
were calculated.
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biological candidates for binding to enzymes, proteins or
receptors.18-20 One of the best known examples is the "Re-
Tamoxifen" compound from Jaouen’s group, where a phenyl
Introduction
Bioorganometallic chemistry is the study of biologically active
molecules that contain carbon directly bonded to metals or
metalloids. The functionalization of metal fragment with
organic ligands and/or conjugated biomolecules (amino acids,
peptides, proteins or carbohydrates) has become a viable
strategy to obtain new compounds that have high interest as
targeted therapeutics.1-14
group was replaced by the cyclopentadienyl ring coordinated
to the organometallic fragment, seeking to improve the
activity of this new compound on the estrogen receptor (ER)
and breast cancer.8 Likewise, cyrhetrenyl sulfonamides have
been shown to be excellent inhibitors of human
carboanhydrase (hCa).21,22
Although most of the work in this area has been focused on
medium and late transition metal complexes, rhenium
derivatives have been studied as potential anticancer agents.15
With respect to cytotoxicity, some rhenium carbonyl
complexes equal or even exceed that of the well-established
anti-cancer drug cisplatin. There are a number of examples
that include the Re(I) tricarbonyl core with ancillary ligands
(N,N; alcoxido/hydroxido; N,O; P,P bidentate).16,17 However,
only a few half-sandwich compounds are known. Cyrhetrenyl
complexes [(Cp–R)Re(CO)3] have been evaluated as potential
One of the proteins that has received a lot of interest in recent
years is glycogen synthase kinase
3 (GSK-3), a serine/
threonine kinase that is encoded by two known genes α and β.
It has been described as a protein involved in glycogen
metabolism.23 However, it has also been shown to be involved
in multiple biological processes by regulating different cellular
functions in metabolic and signaling pathways, relating to the
development of neurodegenerative and cancerous diseases.
GSK-3β, a cytosolic protein, is formed by 433 amino acids and
has a mass of 47 kDa. It is distributed mainly in brain tissue,
especially in neurons, 24 explaining and corroborating its role in
neuronal signalling. Thus, the relationship with various central
25
nervous system diseases such as Alzheimer's disease,
schizophrenia,26 bipolar disorder,27 as well as tissue
inflammation28 can be understood.
In addition, studies indicate that GSK-3
β could act as a
potential tumour suppressor due to its ability to phosphorylate
pro-oncogenic molecules such as cyclin D1729 and β-catenin.30
On the other hand, recent studies have shown that GSK-3
β
plays a fundamental role as a positive regulator of the
proliferation of cancer cells through different signalling
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pathways, 24,31 directly related to the development of various For the construction of new organometallic compounds, the organic
types of cancer such as renal,32 pancreatic,33 prostate,34 and cyrhetrenyl fragments were first synthesized independently. It
ovarian,35 colon,36 leukemia,37-38 lung,39 thyroid,40 mammary,41 is important to mention that almost all of the compounds that are
neuroblastoma,42 and glioblastoma.43 For this reason, GSK-3
has been proposed as a viable therapeutic target for the Generally, the synthesis of these new ligands gave pure compounds
β
described below have not been reported in the literature until now.
treatment of a broad spectrum of different types of cancer.
Several studies indicate that this protein is overexpressed in
the cytoplasm and nucleus of colon and pancreas cancer
cells36, 44, suggesting a direct relationship between the activity
in acceptable yields.
Piperazine derivatives
of this kinase and the development of these types of cancer. Piperazines 1a and 1b were synthesized using a combination of
Additionally, recent studies report that GSK-3
plays a crucial methods described in literature63-65 (Scheme 1). The 1H NMR
β
role in cell growth, regulating the activity of the nuclear spectrum of 1a shows the presence of two broad singlets at 2.88
growth factor through the transcription of genes. Thus, the and 2.53 ppm corresponding to the protons of the piperazine
inhibition of the protein attenuates the proliferation of colon fragment, and at 2.19 ppm a multiplet that integrates for one
and pancreas cancer cells and inducing cellular apoptosis24, proton indicates the presence of the mono-substituted ligand.
29,31,33,36,45. This information suggests GSK-3
β as a potential
However, it was impossible to obtain 1b using the same
methodology as for 1a, not even employing Cs2CO3 and CuI as a
catalyst. In both cases, the 1H NMR spectrum only shows the signals
corresponding to the starting reagents. This can be attributed to the
higher energy required to activate the C (sp2)-Br bond present in
the bromoarene; so a new synthesis methodology was applied
using phenylboronic acid56 instead of bromobenzene as the initial
reagent (Scheme 1). In this case, 1b was obtained as a pale yellow
solid in 30% yield. The 1H NMR spectrum shows two multiplets at
3.14 and 3.03 ppm corresponding to the protons of the piperazine
fragment and a singlet centered at 1.78 ppm, which integrates for
one proton corroborating the mono-substitution. Elemental
analysis, 13C NMR and ESI-MS are concordant with what was
expected for 1a-b. Besides, an HPLC of 1b confirms that the ligand
was obtained in analytically pure form.
new therapeutic target in human cancer.
The three-dimensional structure of GSK-3β was obtained by X-
ray diffraction.46-47 The amino acids that make up the binding
site correspond to Ile62, Val70, Ala83, Lys85, Glu97, Met101,
Val110, Asp133, Tyr134, Val135, Pro136, Glu137, Thr138,
Arg141, Lys183, Gln185, Asn186, Leu188, Cys199, Asp200,
which generate a cavity that is mainly polar. In general, the
pharmaceutical industry has focused on the design of organic
compounds as potential activators or inhibitors of kinases. For
example, activity on GSK-3β and in cancer cell lines such as HT-29
was studied using piperazine derivatives, by a number of research
groups.48-52 These results indicate an important role for the
piperazine fragment.
Scheme 1. Synthesis of piperazine ligands: 1-cyclohexyl piperazine
Incorporating a metal or organometallic fragment has been an
alternative strategy to design new compounds with biological
activity. The metal centre may play a structural and electronic role
associated with the distribution and spatial orientation of the
organic ligands in the binding cavities of biological targets. With the
right choice of metals, highly stable complexes are generated,
usually with octahedral geometry. Half-sandwich Ruthenium(II)
complexes have been evaluated as potential inhibitors of kinase
proteins, some of those exhibiting good affinity for the GSK
protein.53-56 In the same way, cyrhetrene complexes functionalized
with aminoquinoline, nitrofurane and glucosamine fragments, have
been used as anticancer57-58 and antimalarial59-60 agents. The
proven biological activity of rhenium complexes, together with the
high stability of the Re(I) fragment and coupled with the high
lipophilicity of cyrhetrene fragments, are some of the advantages of
this metal fragment in the design of new compounds which exhibit
a beneficial response to various biological targets.
(1a) and 1-phenyl piperazine (1b).
Cyano derivatives
The cyano ligands 2a-d (Scheme 2) were obtained in high yields.53-55
The IR spectra of 2 in CH2Cl2 show a frequency at 2230 cm-1, which
confirms the presence of the C≡N group. This frequency is
consistent with related organic species (ν(CN): 2210-2360 cm-1).
Also, the 1H NMR spectra ratify the substitution reaction for the
formation of ligands 2a-d. In all cases, the chemical shift,
multiplicity and coupling constants confirm the presence of the
compounds. The presence of a cyano group is also confirmed by a
signal at 119 ppm in the 13C NMR spectra.
Here, based on the structural GSK-3β characteristics46-47 and the
relevance in designing new ligands for kinase enzymes, a series of
new cyrhetrene imine and amino piperazine derivatives were
synthesized and then evaluated on cell lines where GSK-3β plays a
crucial role.24, 29-31, 61, 62
Results and discussion
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appearance of a single new resonance at a lower frequency around
7.8 ppm, which is assigned to the imine proton. Likewise, only one
set of triplets is observed which is related to the presence of the
five-membered ring in the structures. In addition, the signal of the
methylene protons adjacent to the imine moiety in 5a-d is shifted
to higher frequency with respect to that observed for the
precursors 3a-d. However, as expected, the proton resonances of
the substituted piperazines in 5 are invariant to those observed for
the amine ligands 3.
Amine derivatives
Subsequently, compounds 2 were reduced in the presence of LiAlH4
using anhydrous ether or THF (Scheme 2). After work up the amine
ligands 3a-d were isolated as white, air-stable oily materials, which
are soluble in polar solvents. In the case of 3c-d, these were purified
by column chromatography using neutral alumina as the stationary
phase, while 3a and 3b were obtained in pure form without further
purification.
These new organometallic Schiff bases were observed in a single
isomeric form, which could be assigned to the E-isomer to avoid
steric hindrance. This is also in line with related cyrhetrene imine
complexes having a C=N bond, which have been fully characterized
by conventional spectroscopic techniques and X-ray diffraction69. In
all cases, a typical fragmentation pattern was observed for the
rhenium complexes in high-resolution mass spectra of imines 5a-d.
Elemental analyses (see Experimental Section) also are in
Solution IR (CH2Cl2) spectra of 3 show the disappearance of the
cyano band observed in the starting materials 2, giving a tentative
account of the reduction of this group. Furthermore, 1H NMR
(CDCl3) spectra of 3 show the presence of a new triplet centered
around 2.7 ppm, which integrates for two protons and can be
attributed to the methylene group adjacent to the amino moiety.
This confirms successful reduction of 2 to the ligands 3.
Besides, for cyano and amino derivatives, the resonances assigned agreement with the proposed composition. Solutions of imines 5a-d
to the piperazine group are shifted to higher frequencies according (DMSO), were stable for two months. Also, Imines were studied in
to the acceptor character of the substituents on this fragment (R: solution pH 7.0 (0.5% DMSO in an aqueous solution) and were
Ph > Ph-OMe > CH(Ph)2 > Cy). 2D NMR spectra (COSY, HMQC) were stable to hydrolysis. After this time, no resonances were detected
performed in order to correctly assign the protons present in the that could be attributed to either amino-piperazine and cyrhetrene
structures of cyano (2) and amino derivatives (3). Additionally, the carboxaldehyde in the 1 H NMR spectra.
mass spectra of the ligands 2 and 3 (ESI and EI) exhibit the presence
of the molecular ion. It is important to mention that all
chromatograms (HPLC) show the presence of a single peak, which
confirms that all of them are analytically pure, just as shown by
results from elemental analysis.
Scheme 3. Synthesis of new organometallics imines (5a-d).
Biological evaluation on HT-29 and PT45
The cytotoxicity of our new derivatives was studied on different cell
lines by the MTT assay.60 As mentioned above, the protein kinase
GSK-3β (glycogen synthase kinase 3) is involved in the Wnt signaling
pathways. Thus, this isoform of GSK has been related to the cancer
development. The results are summarized in Table 1, and IC50
curves are shown in the Supporting Information.
Scheme 2. Synthesis of new arylpiperazine cyano (2a-d) and amine
(3a-d) ligands.
Organometallics compounds
The [(η5-C5H4CHO)Re(CO)3] (4) complex was synthesized as
reported in the literature65 from the precursor [(η5-C5H5)Re(CO)3]68.
The chromatogram (HPLC) showed the presence of a single peak at
22.68 minutes, indicating 98 % purity.
Table 1: IC50 values of cyano (2a-d), amine (3a-d) and imine
derivatives (5a-d) in HT-29 and PT-45 cancer cell lines as
determined by the MTT assay with 72
(triplicates).
h
incubation time
Compounds
IC50 (ꢀM) HT-29
n. a.
IC50 (ꢀM) PT-45
n. a.
New organometallics cyrhetrenyl imines
2a-d
Through a condensation reaction between the precursor complex 4
[(η5-C5H4CHO)Re(CO)3] and the amine arylpiperazine ligands 3a-d, it
was possible to obtain the organometallic imines 5a-d respectively
as air-stable oily solids, soluble in polar solvents (Scheme 3). The
reaction was monitored by 1H NMR through observing the
disappearance of the aldehyde proton at 9.6 ppm and the
3a-c
n. a.
n. a.
3d
18.11 ± 0.05
22.23 ± 0.04
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4
n. a.
n. a.
n. a.
In-silico evaluation
The proposal of new ligands for GSK-3β raised in this study
contemplates arylpiperazine derivatives substituted with aromatic
and aliphatic substituents and their modification with
organometallic fragments, which could involve different types of
molecular interactions with amino acids in the active site.
Considering the description of the GSK-3β binding cavity, putative
ligands should possess functional groups that generate van der
Waals and coulombic interactions, and have a volume of less than
600 Å3.
5a
5b
5c
5d
97.27 ± 0.06
72.78 ± 0.04
76.38 ± 0.05
30.48 ± 0.03
32.6 ± 0.7
n. a.
90.16 ± 0.05
25.82 ± 0.03
2.2 ± 0.3
Cisplatin
Both, electronic and stereochemical characteristics were evaluated
using computational tools. Area, volume and electrostatic surface
(Figure 1) were determined for the organic ligands arylpiperazine
(3a-d) and the organometallic imines (5a-d).
n. a.: Not active up to a concentration of 250 ꢀM.
Based on the values in Table 1, a difference in the activity related to
different organic functions was observed after the change of C≡N to
NH2 group, and also with increasing the number of the aromatic
ring. Importantly, when the metal was introduced in the molecule
an increase in cytotoxic activity was observed, in addition to an
interesting selectivity toward HT-29. Is important to mention that in
comparison with the standard cisplatin, our compounds 3d and 5a-
b displayed higher activity and improved selectivity (respectively)
on colon cancer cells. This result suggests that rhenium derivatives
offer a strong and attractive alternative to be explored in the design
of new anticancer compound.
This study was performed considering the biological conditions of
the system, which is reflected in the protonation of the amines (3a'-
d'). The volume of the organic ligands varies between 280 and 390
Å3, while for organometallic complexes this ranges from ~ 470 to
560 Å3. Thus, it is possible that all proposed systems could interact
with the binding site of the protein.
On the other hand, a description of the electrostatic potential of
the amino derivatives 3’ shows that in all cases there is a segment
of high electron density determined by the presence of aromatic
rings present in the structures (3b´-d´) and a localized electron
deficiency at the other end, where the terminal ammonium group is
appreciated. On the other hand, the presence of the organometallic
fragment in tricarbonyl imine complexes (5a-d) increases the
polarization of the electrostatic potential in the molecule, i.e. the
concentration of electrons is located mainly on the arylpiperazine
moiety (in red, Figure 1B) and as consequence a deficit of electron
density results in the cyrhetrenyl fragment.
Considering the potential participation of GSK-3β on tumor
suppression and as
a
positive regulator of proliferation, we
rationalize our
experimental results considering the
physicochemical characteristic of the binding cavity of GSK-3β as a
biological target of our new derivatives.
A)
B)
3a’
3b’
5a
5b
-34
350
740
-4134
-21
4092
5c
5d
3c’
3d’
Figure 1. The electrostatic potential from ESP calculations obtained for 3a-d, and 5a-d in A) and B) respectively.
4 | J. Name., 2012, 00, 1-3
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However, unfortunately, the number of compounds with
significant biological activity was not enough in this study to derive
a quantitative relationship between electrostatic potential and
cytotoxicity. Qualitatively, the compounds with higher dipolar
moment (ꢀ, negative charge density concentrated on the
kcal/mol). In Figure 3, a general representation of the complex 3d-
GSK-3β is shown.
arylpiperazine segment) are more active on HT-29 and PT-45.
A
direct relation was observed for the organometallic compounds (5a-
5d), which are displayed in Figure 2. Thus, is possible to assume that
the electron-rich substituent in the nitrogen of piperazine is
advantageous for the interactions with
responsible of the cytotoxic effect.
a biological target
Compound logIC50 HT-29 Dipolar Moment (D)
3a
3b
3c
3d
5a
5b
5c
5d
n. a.
n. a.
n. a.
1.25
1.99
1.86
1.88
1.48
31.79
32.16
32.95
37.71
1282.77
1397.28
1554.24
1708.03
Figure 3. Compound 3d complexed in GSK-3β and the electrostatic
potential of the binding cavity is shown in surface (Blue: positive
charged Green: non-charged surface Red: negative charged).
Our results for 3d show that the protonated amino groups interact
with a negatively charged segment of the cavity corresponding to
the location of Asp200. Furthermore, the aromatic ring stacks with
Tyr134, Arg141 and Asp133 (in the blue surface in Figure 2). Finally,
our computational description supports the hypothesis that
compounds with a high polarization of electrostatic potential could
be ligands of GSK-3β.
5a
5b
5c
5d
Figure 2. Dipolar moment of organometallic compounds (5a to 5d).
Inset shows specific values of dipolar moment and its relationship
with experimental data.
Experimental
Considering this electronic distribution, and that the union cavity
has amino acids such as tyrosine, aspartate, glutamate, and arginine
(among others), it is possible to indicate that the proposed
compounds could generate coulombic type interactions with the
amino acids present at the binding site of GSK-3β.
General Methods
All reactions were carried out using standard Schlenk technique
under nitrogen atmosphere. All solvents were purified and dried by
conventional methods and were distilled under nitrogen prior to
use, except those used for thin layer and column chromatography,
which were used without purification. [(η5-C5H5)Re(CO)3]57, [(η5-
C5H4CHO)Re(CO)3]58 (4) and Tollens reagent were synthesized
following literature procedures. Piperazine 1-(diphenylmethyl), 1-
(2-methoxyphenyl), and 5-bromovaleronitrile were purchased from
Aldrich and used without purification or treatment.
In order to describe specific interactions in the protein-ligand
complexes, docking studies into the binding cavity were done. It is
important to mention that specific parameters to describe the
coordination of metal as rhenium-cyclopentadienyl (η5) are not
available into the force field. For that reason, the docking was
carried out just with the organic compounds (3a-d). In all cases, the
ligands were located in the same cavity and main interactions were
conserved. The binding energies for the organic compounds were
negative (from -4.41 to -6.98 kcal/mol; more detail in Electronic
Supporting Information). The favorable interactions were observed
to 3d, which displayed the more negative binding energy (-6.98
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to remove excess potassium salts, and the resulting solution is
evaporated to dryness.
Synthetic procedures and characterization
Synthesis of cyclohexyl piperazine HNC4H8N(C6H11) (1a)
1.0 g of piperazine (11.6 mmol) and 1.9 g of bromocyclohexane
(11.6 mmol; d= 1.324 g/ml) in 30 mL of anhydrous CH3CN were
dissolved. 829.3 mg of K2CO3 (6.0 mmol) and 30.0 mg KI (0.2 mmol)
were added and refluxed for 24 h. After this time, the reaction
mixture was filtered and the resulting solution was evaporated to
dryness. The reaction crude was purified by column
chromatography, where from the fraction eluted with chloroform:
methanol (1:1) it was possible to obtain a white solid attributed to
2a: crude was purified by column chromatography on neutral
alumina, where from the fraction eluted with acetone it was
possible to obtain a white oily solid corresponding to 2a (yield 80%;
1.2 g, 4.7 mmol). 1H-NMR (400 MHz, CDCl3) δ: 2.53 (bs, 4H,
NC4H8N); 2.40 (br s, 4H, NC4H8N); 2.31 (m, 4H, CH2); 2.16 (m, 1H,
C6H11); 1.82 (psd, 2H, CH2); 1.73 (psd, 2H, CH2); 1.60 (m, 5H, C6H11);
1.14 (m, 5H, C6H11). 13C-NMR (400 MHz, CDCl3) δ: 119.61 (s, CN);
63.44 (s, C6H11); 57.60 (s, CH2); 53.63 (s, NC4H8N); 48.88 (s, NC4H8N);
29.03 (s, C6H11); 26.33 (s, CH2); 25.89 (s, C6H11); 25.79 (s, C6H11);
23.59 (s, CH2); 17.10 (s, CH2). ESI-MS (m/z) (CH3 OH): 249.98 [M+];
168.12 [M+ - NC4H8N-1- (C6H11)]. Anal. Calc: C, 72.24%; H, 10.91%;
N, 16.85%. Found: C, 70.05%; H, 10.47%; N, 17.13%.
1
compound 1a (yield 48%; 944.0 mg; 5.6 mmol). H-NMR (400 MHz,
CDCl3) δ: 2.88 (t, 4H, JH-H= 5.0 Hz, NC4H8N); 2.53 (t, 4H, JH-H= 5.0 Hz,
NC4H8N); 2.19 (m, 1H, C6H11); 1.91 (bs, 1H, C6H11); 1.85 (m, 2H,
C6H11); 1.77 (m, 2H, C6H11); 1.61 (psd, 1H, NH); 1.20 (m, 4H, C6H11);
1.07 (m, 1H, C6H11) .13C-NMR (400 MHz, CDCl3) δ: 64.22 (s, CH);
50.44 (s, NC4H8N); 46.75 (s, NC4H8N); 28.96 (s, C6H11); 26.51 (s,
C6H11); 26.10 (s, C6H11). ESI-MS (m/z) (CH3OH): 168.92 [M+]. Anal.
Calc: C, 71.37%; H, 11.98%; N, 16.65%. Found: C, 70.71%; H,
11.95%; N, 16.69%.
2b: crude was purified by column chromatography using neutral
alumina as the stationary phase. From the fraction eluted with ethyl
acetate, it was possible to obtain compound 2b as an orange solid
(yield 93%; 1.4 g, 5.8 mmol). 1H-NMR (400 MHz, methanol-d4) δ:
7.23 (t, 2H, JH-H = 8.0 Hz, C6H5); 6.96 (d, 2H, JH-H = 8.3 Hz, C6H5); 6.84
(m, 1H, C6H5); 3.18 (t, 4H, JH-H= 4.9 Hz, NC4H8N); 2.63 (t, 4H, JH-H= 4.9
Hz, NC4H8N); 2.46 (m, 4H, CH2); 1.68 (m, 4H, CH2). 1H-NMR (400
MHz, CDCl3) δ: 7.26 (t, 2H, JH-H= 8.0 Hz, C6H5); 6.93 (d, 2H, JH-H= 8.3
Hz, C6H5); 6.85 (m, 1H, C6H5); 3.20 (t, 4H, JH-H= 4.9 Hz, NC4H8N); 2.60
(t, 4H, JH-H= 4.9 Hz, NC4H8N); 2.42 (m, 4H, CH2); 1.72 (m, 4H, CH2)
13C-NMR (400 MHz, methanol-d4) δ: 152.70 (s, C6H5); 130.09 (s,
C6H5); 121.18 (s, C6H5); 119.61 (s, CN); 58.55 (s, CH2); 54.22 (s,
NC4H8N); 50.22 (s, NC4H8N); 26.51 (s, CH2); 24.59 (s, CH2); 17.23 (s,
CH2). ESI-MS (m/z) (CH3OH): 244.29 [M++ H]. Anal. Calc: C, 74.03%;
H, 8.70%; N: 17.27%. Found: C, 72.50%; H, 8.54%; N, 16.84%. HPLC
(H2O: CH3 CN): retention time: 16.78 minutes, 99% purity.
Synthesis of phenyl piperazine HNC4H8N (C6H5) (1b)
500.0 mg of piperazine (5.8 mmol) and 800.0 mg of phenylboronic
acid (6.6 mmol) were dissolved in 20 mL of anhydrous methanol in
the presence of CuI as catalyst (44.0 mg, 0.2 mmol). The reaction
mixture was bubbled with O2 and refluxed for 4 hours. After this
time, 10 ml of anhydrous methanol was added and the mixture was
allowed to reflux at room temperature with constant stirring for 48
hours. Subsequently, the solution is evaporated to dryness. The
reaction crude is washed with ethyl acetate to give a white solid
which corresponds to the unreacted free piperazine. The solution is
evaporated to dryness to give a pale yellow solid attributed to
ligand 1b (yield 30%; 278.0 mg; 1.7 mmol). 1H-NMR (400 MHz,
CDCl3) δ: 7.27 (m, 2H, C6H5); 6.93 (d, 2H, JH-H= 8.2 Hz, C6H5); 6.86 (t,
1H, JH-H= 7.2 Hz, C6H5); 3.14 (m, 4H, NC4H8N); 3.03 (m, 4H, NC4H8N);
1.78 (s, 1H, NH). 13C-NMR (400 MHz, CDCl3) δ: 152.01 (s, C6H5);
129.18 (s, C6H5); 119.77 (s, C6H5); 116.14 (s, C6H5); 50.51 (s,
NC4H8N); 46.31 (s, NC4H8N ESI-MS (m/z) (CH3OH): 162.94 [M+]. Anal.
Calc: C, 74.03%; H, 8.70%; N: 17.27%. Found: C, 68.89%; H, 7.76%;
N, 15.40%. HPLC (H2O: CH3CN): retention time: 3.78 minutes, 98%
purity.
2c: Ligand 2c was purified by column chromatography and
recovered in the fraction eluted with CH2Cl2 in 86% yield (1.3 g, 4.5
1
mmol). H-NMR (400 MHz, CDCl3) δ: 6.94 (m, 4H, C6H4); 3.87 (s, 3H,
OCH3); 3.09 (bs, 4H, NC4H8N); 2.65 (bs, 4H, NC4H8N); 2.42 (t, 4H, JH-H
= 6.3 Hz, CH2); 1.71 (m, 4H, CH2). 13C-NMR (400 MHz, CDCl3) δ:
152.47 (s, C6H4); 141.30 (s, C6H4); 123.07 (s, C6H4); 121.26 (s, C6H4);
119.88 (s, CN); 118.54 (s, C6H4); 111.42 (s, C6H4); 57.74 (s, CH2);
55.48 (s, OCH3); 53.54 (s, NC4H8N); 50.79 (s, NC4H8N); 25.73 (s, CH2);
23.79 (s, CH2); 17.24 (s, CH2). ESI-MS (m/z) (CH3OH): 295.92 [M++
Na+], 273.96 [M+], 205.99 [M + - (CH2)3-CN], 190.18 [M+ - (CH2)4-CN].
EI (m/z): 273.1 [M+]. Anal. Calc: C, 70.30%; H, 8.48%; N: 15.37%.
Found: C, 69.73%; H, 8.31%; N, 15.46%. HPLC (H2O: CH3CN):
retention time: 16.73 minutes.
Synthesis of cyano derivatives: 2HN(CH2)5-NC4H8N-(C6H11) (2a);
2HN(CH2)5-NC4H8N-(C6H5) (2b); 2HN(CH2)5-NC4H8N-(C6H4)-2-(OCH3)
(2c); 2HN(CH2)5-NC4H8N-(CH(C6H5)2) (2d)
2d: the crude was purified by column chromatography, where from
the fraction eluted with ethyl acetate it was possible to obtain a
white solid corresponding to compound 2d (yield 88%, 1.20 g, 3.52
General experimental procedure
1
mmol). H-NMR (400 MHz, CDCl3) δ: 7.41 (d, 4H, JH-H= 7.5 Hz, C6H5);
1.0 g of corresponding piperazine (1a: cyclohexyl piperazine (5.9
mmol); 1b: phenyl piperazine (6.2 mmol); 1c: 2-methoxyphenyl
piperazine (5.2 mmol); 1d: diphenylmethyl piperazine (4.0 mmol)
and equimolar 5-bromovaleronitrilo (d=1.388 g/ml) were dissolved
in 35 ml of anhydrous acetonitrile. 829.3 mg of K2CO3 (6.0 mmol)
and 30.0 mg KI (0.2 mmol) were added and the solution was
refluxed for 16-20 h. After this time, the reaction mixture is filtered
7.26 (t, 4H, JH-H= 7.5 Hz, C6H5); 7.17 (t, 2H, JH-H= 7.4 Hz, C6H5); 4.22
(s, 1H, CH); 2.45 (bs, 8H, NC4H8N); 2.36 (t, 4H, JH-H= 6.7 Hz, CH2);
1.65 (m, 4H, CH2). 13C-NMR (400 MHz, CDCl3) δ: 142.90 (s, C6H5);
128.61 (s, C6H5); 128.10 (s, C6H5); 127.04 (s, C6H5); 119.77 (s, CN);
76.41 (s, CH); 57.49 (s, CH2); 53.65 (s, NC4H8N); 52.02 (s, NC4H8N);
25.89 (s, CH2); 23.68 (s, CH 2); 17.24 (s, CH2). ESI-MS (m/z) (CH3OH):
6 | J. Name., 2012, 00, 1-3
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337.28 [M+]. EI (m/z): 333.1 [M+]. Anal. Calc: C, 79.24%; H, 8.16%; N:
12.60%. Found: C, 78.33%; H, 8.03%; N, 12.52%. HPLC (H2O: CH3CN):
retention time: 20.55 minutes.
278.2 [M+]. Anal. Calc: C, 69.27%; H, 9.81%; N: 15.15%. Found: C,
64.68%; H, 9.15%; N, 13.78%.
3d: 182.0 mg of white solid (yield 90%, 0.54 mmol) was obtained.
Synthesis of amino derivatives: 2HN(CH2)5-NC4H8N-(C6H11) (3a);
2HN(CH2)5-NC4H8N-(C6H5) (3b); 2HN(CH2)5-NC4H8N-(C6H4)-2-(OCH3)
(3c); 2HN(CH2)5-NC4H8N-(CH(C6H5)2) (3d)
1H-NMR (CDCl3) δ: 7.41 (d, 4H, JH-H = 7.5 Hz, C6H5); 7.26 (t, 4H, JH-H
=
7.5 Hz, C6H5); 7.17 (t, 2H, JH-H= 7.3 Hz, C6H5); 4.22 (s, 1H, CH); 2.69 (t,
2H, JH-H = 7.0 Hz, CH2); 2.45 (bs, 8H, NC4H8N); 2.33 (t, 2H, JH-H = 7.7
Hz, CH2); 1.66 (broad s, 2H, NH2); 1.47 (m, 4H, CH2); 1.32 (m, 2H,
CH2). 13C-NMR (400 MHz, CDCl3) δ: 142.98 (s, C6H5); 128.54 (s, C6H5);
128.13 (s, C6H5); 126.99 (s, C6H5); 76.43 (s, CH); 58.81 (s, CH2); 53.69
(s, NC4H8N); 52.06 (s, NC4H8N); 42.20 (s, CH2); 33.65 (s, CH2); 26.86
(s, CH2); 25.03 (s, CH2). ESI-MS (m / z) (CH3OH): 338.12 [M+]. EI
(m/z): 338.2 [M+]. Anal. Calc: C, 78.29%; H, 9.26%; N: 12.45%.
Found: C, 78.21%; H, 9.31%; N, 12.12%.
General experimental procedure
200.0 mg of ligand 2 (2a: 0.80 mmol, 2b: 0.82 mmol, 2c: 0.73 mmol,
2d: 0.60 mmol) were dissolved in a Schlenk in 10 mL of dry ether
(anhydrous THF was used for ligands 2a and 2b) and 1.5 ml (4.4
mmol) of a solution of LiAlH4 (2M in THF) was added dropwise. The
solutions were allowed to react with constant stirring for 1 h at 0 ° C
under a nitrogen atmosphere. At the end of this time, 0.5 ml of
methanol and an aqueous solution of KOH (10 ml, 10%) were added
to the reaction mixture. The aqueous phase was extracted with
CH2Cl2 (3 x 10 mL). The latter were dried over Na2SO4. The organic
phases were concentrated to dryness to give the 3 (a-d) ligands.
Synthesis of [(η5-C5H4CHO)Re(CO)3] (4)
The aldehyde cyrhetrene was synthesized using a method described
in literature57. The 4 compound was obtained as a yellow solid
(CH2Cl2, (ʋCO) cm-1]: 2033 (s), 1940 (s), S), 1694 (w) .1H-NMR (CDCl3)
δ: 9.61 (s, 1H, CHO); 6.03 (t, 2H, JH-H = 2.3 Hz, C5H4); 5.48 (t, 2H, JH-H
= 2.3 Hz, C5H4). HPLC (CH3CN: H2O) retention time: 22.68 minutes.
Purity: 98%.
3a: 192.6 mg of white solid (yield 95%, 0.76 mmol) was obtained.
1H-NMR (400 MHz, CDCl3) δ: 2.66 (t, 2H, JH-H = 7.0 Hz, CH2); 2.58 (bs,
4H, NC4H8N); 2.45 (bs, 4H, NC4H8N); 2.30 (t, 2H, JH-H = 7.8 Hz, CH2);
2.19 (m, 1H, C6H11); 1.86 (psd, 2H, C6H11); 1.77 (psd, 2H, CH2); 1.60
(m, 1H, C6H11); 1.46 (m, 4H, CH2); 1.19 (m, 8H, C6H11). 13C-NMR (400
MHz, CDCl3) δ: 63.61 (s, C6H11); 58.96 (s, CH2); 53.91 (s, NC4H8N);
49.06 (s, NC4H8N); 42.22 (s, CH2); 33.82 (s, CH2); 29.10 (s, C6H11);
26.91 (s, CH2); 26.51 (s, CH2); 26.02 (s, C6H11); 25.06 (s, C6H11). ESI-
MS (m/z) (CH3OH): 254.07 [M+]. EI (m/z): 254.3 [M+]. Anal. Calc: C,
71.09%; H, 12.33%; N, 16.58%. Found: C, 66.10%; H, 11.13%; N,
14.18%.
Synthesis of organometallic imines [(η5-C5H4CHNH-(CH2)5-NC4H8N-
1-(C6H11))Re(CO)3]
1(C6H5))Re(CO)3] (5b); [(η5-C5H4CHNH-(CH2)5-NC4H8N-1-(C6H4)-2-
(OCH3))Re(CO)3] (5c);
[(η5-C5H4CHNH-(CH2)5-NC4H8N-1-
(5a);[(η5-C5H4CHNH(CH2)5-NC4H8N-
(CH(C6H5)2)Re(CO)3] (5d).
3b: 195.4 mg of white solid (yield 96%, 0.79 mmol) was obtained.
1H-NMR (400 MHz, CDCl3) δ: 7.26 (m, 2H, C6H5); 6.93 (d, 2H, JH-H
=
General experimental procedure:
8.2 Hz, C6H5); 6.85 (t, 1H, JH-H = 7.3 Hz, C6H5); 3.21 (t, 4H, JH-H = 4.9
Hz, NC4H8N); 2.70 (t, 2H, JH-H = 6.9 Hz, CH2); 2.60 (t, 4H, JH-H = 5.0
Hz, NC4H8N); 2.39 (t, 2H, JH-H = 7.5 Hz, CH2); 1.45 (m, 8H, CH2). 13C-
NMR (400 MHz, CDCl3) δ: 151.59 (s, C6H5); 129.34 (s, C6H5); 119.81
(s, C6H5); 59.08 (s, CH2); 53.58 (s, NC4H8N); 49.08 (s, NC4H8N); 42.32
100.0 mg of aldehyde precursor complex 4 (0.3 mmol) and 0.3
mmol of amine ligand 3a-d (3a: 87.1 mg; 3b: 85.0 mg; 3c: 95,4 mg;
3d: 116,0 mg) were dissolved in 20 ml of anhydrous toluene.
Molecular sieve (4Å) was added to the solution, which was refluxed
for 9 h under nitrogen atmosphere with constant stirring. After this
time, the reaction mixture was filtered and the solvent evaporated
under reduced pressure to give a yellow oily solid corresponding to
the organometallic imines 5a-d.
(s, CH2); 34.05 (s, CH ); 27.09 (s, CH2); 25.05 (s, CH2). ESI-MS (m/z)
2
(CH3OH): 248.00 [M+]. Anal. Calc: C, 72.83%; H, 10.19%; N: 16.99%.
Found: C, 68.33%; H, 9.31%; N, 14.67%.
3c: the reaction crude was purified through column
chromatography on neutral alumina, where from the fraction
eluted with acetonitrile it was possible to recover the precursor
ligand 2c that did not react. Finally of the fraction eluted with
methanol, ligand 3c was obtained as a white solid, stable in air and
5a: 1H-NMR (400 MHz, CDCl3) δ: 7.87 (s, 1H, HC = N); 5.82 (t, 2H, JH-
H= 2.2 Hz, C5H4); 5.35 (t, 2H, JH-H= 2.2 Hz, C5H4); 3.47 (t, 2H, JH-H = 6.4
Hz, CH2); 2.60 (bs, 4H, NC4H8N); 2.47 (bs, 4H, NC4H8N); 2.31 (t, 2H,
JH-H = 7.8 Hz, CH2); 1.82 (m, 1H, C6H11); 1.58 (m, 5H, C6H11); 1.24
1
soluble in polar solvents (yield 78%, 158.3 mg, 0.57 mmol). H-NMR
+
(m, 11H, C6H11 and CH2). ESI-MS (m/z) (CH3OH): 599.73 [M + H ].
(CDCl3) δ: 6.92 (m, 4H, C6H4); 3.85 (s, 3H, OCH3); 3.09 (bs, 4H,
NC4H8N); 2.69 (t, 2H, JH-H = 6.8 Hz, CH2); 2.63 (bs, 4H, NC4H8N); 2.40
(t, 2H, JH-H = 7.4 Hz, CH 2); 1.54 (m, 2H, CH2); 1.46 (m, 2H, CH2); 1.36
(m, 2H, CH2). 13C-NMR (400 MHz, CDCl3) δ: 152.42 (s, C6H4); 141.65
(s, C6H4); 122.95 (s, C6H4); 121.10 (s, C6H4); 118.30 (s, C6H4); 111.38
Anal. Calc: C, 48.14%; H, 5.72%; N, 7.02%. Found: C, 48.14%; H,
5.72%; N, 6.92%.
1
5b: H-NMR (400 MHz, CDCl3) δ: 7.88 (s, 1H, HC = N); 7.27 (m, 1H,
(s, C6H4); 58.96 (s, CH2); 55.47 (s, OCH3); 53.66 (s, NC4H8N); 50.78 (s, C6H5); 6.88 (m, 4H, C6H5); 5.82 (t, 2H, JH-H = 2.2 Hz, C5H4); 5.36 (t, 2H,
NC4H8N); 42.29 (s, CH2); 33.96 (s, CH2); 26.92 (s, CH2); 25.02 (s, CH2).
ESI-MS (m/z) (CH3OH): 278.02 [M++ H], 261.30 [M+ - NH 2], EI (m/z):
J
H-H = 2.2 Hz, C5H4); 3.49 (t, 2H, JH-H = 6.5 Hz, CH2); 3.21 (pst, 4H, JH-H
= 4.8 Hz, NC4H8N); 2.60 (pst, 4H, JH-H = 5.0 Hz, NC4H8N); 2.40 (t, 2H,
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J. Name., 2013, 00, 1-3 | 7
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JH-H = 6.3 Hz, CH2); 1.60 (m, 4H, -CH2); 1.35 (m, 2H, -CH2) ESI-MS In order to obtain information regarding the principal Protein-
(m/z) (CH3OH): 593.98 [M + H+]. Anal. Calc: C, 48.63%; H, 4.76%; N,
Ligand interactions the molecular docking of 3a-d with GSK-3β was
done using AutoDock 4.072 suite. In general, the grid maps were
7.09%. Found: C, 48.31%; H, 4.80%; N, 7.05%.
calculated using the autogrid4 option and were located on the
1
5c: H-NMR (400 MHz, CDCl3) δ: 7.88 (s, 1H, HC = N); 6.91 (m, 4H,
center of the receptor cavity. The volumes for the grid maps were
C6H4); 5.82 (t, 2H, JH-H = 2.2 Hz, C5H4); 5.36 (t, 2H, JH-H = 2.2 Hz,
85x 85 x 85 points (a grid-point spacing of 0.375 Å). The autotors
C5H4); 3.86 (s, 3H, OCH3); 3.50 (t, 2H, JH-H = 6.6 Hz, CH2); 3.11 (t, 4H,
option was used to define the rotating bonds in the ligand. In the
JH-H = 4.9 Hz, NC4H8N); 2.64 (t, 4H, JH-H = 4.27 Hz, NC4H8N); 2.40 (pst,
Lamarckian genetic algorithm (LGA) dockings, an initial population
2H, CH2); 1.60 (m, 6H, CH2). ESI-MS (m/z) (CH3OH): 623.82 [M + H+].
of 1500 random individuals with
a population size of 100
Anal. Calc: C, 48.22%; H, 4.86%; N, 6.75%. Found: C, 48.46%; H,
4.98%; N, 6.57%.
individuals, a maximum number of 2.5 x 106 energy evaluations, a
maximum number of 27,000 generations, a mutation rate of 0.02
and
a cross-over rate of 0.80 were employed. The docked
1
5d: H-NMR (400 MHz, CDCl3) δ: 7.86 (s, 1H, HC = N); 7.41 (m, 4H,
compound complexes were built using the lowest docked-energy
binding positions.
C6H5); 7.26 (m, 4H, C6H5); 7.17 (m, 2H, C6H5); 5.80 (t, 2H, JH-H = 2.2
Hz, C5H4); 5.33 (t, 2H, JH-H = 2.2 Hz, C5H4); 4.22 (s, 1H, CH); 3.47 (t,
2H, JH-H = 6.6 Hz, CH2); 2.46 (t, 8H, NC4H8N); 2.35 (t, 2H, JH-H = 7.4 Hz,
CH2); 1.56 (m, 6H, CH2). ESI-MS (m/z) (CH3OH): 683.05 [M + H+].
Anal. Calc: C, 54.53%; H, 5.02%; N, 6.15%. Found: C, 52.85%; H,
5.09%; N, 5.92%.
Conclusions
Four different asymmetric alkyl and aryl cyano-piperazine
derivatives have been synthesized and full characterized. The
reduction of the cyano group with LiAlH4 afforded amino-
piperazines
3
, in quantitative yield. Subsequently, through a
Cell Culture and Cytotoxicity Assays
condensation reaction between cyrhetrenyl aldehyde (
4) and
the amino derivatives (3a-d), new organometallic piperazine-
derived imines of rhenium (5a-d) were obtained in analytically
pure form. All compounds were evaluated in two cancer cell
lines, namely HT-29 and PT-45. With the exception of the
amino-piperazine 3d, only the organometallic compounds
showed appreciable cytotoxic activity. Again, the 3d-derived
For the growth of the cells was used Dulbecco’s Modified Eagle’s
Medium (DMEM). It contains 10% FCS, 1% penicillin and
streptomycin. HT-29 and PT-45 cells were detached from the wells
with trypsin and EDTA, harvested by centrifugation and
resuspended in cell culture medium.
complex 5d showed best activity in the range of 30 ꢀM against
The assays were carried out on 96 well plates with 6000 cells per
well for both cell lines. After 24 h of incubation at 37 °C and 10%
CO2, the cells were treated with the compounds (2a-d, 3a-d, 4 and
both cell lines. In terms of structure-actvity relationship (SAR),
our results indicate that by increasing the aromaticity in the
organic ligands (3d), and incorporating an organometallic
5a-d) with DMSO concentrations of 0.5%, and a final volumen of fragment into the structure of the compounds
(5a-d),
significant biological activity can be achieved on both cell lines.
In addition, the general physicochemical characteristics of our
compounds (3a-d and 5a-d) and the binding site of the kinase
protein could be related indirectly to the biological function. A
computational study showed that 3d interacts with amino
200 μL per well. For this, we used standar solution of each
compound of 20 mM. For a negative control, one series of cells was
left untreated. The cells were incubated for 48 h followed by adding
50 μL MTT (2.5 mg·mL–1). After an incubation time of 2 h, the
medium was removed and 200 μL DMSO were added. The formazan
crystals were dissolved and the absorption was measured at 550
nm, using a reference wavelength of 620 nm. Each test was
repeated in triplicates in at least three independent experiments for
each cell line.70
acids like as Asp200
of GSK-3 . This may present a hint for the highest IC50 value of
this compound, and may provide a clue towards further
, Tyr134, Arg141, Asp133 in the active site
β
optimization of this compound class as organometallic GSK-3
β
inhibitors.
In silico studies
Conflicts of interest
Optimization Geometry and Electrostatic Potential for 3a-d and
5a-d
There are no conflicts to declare.
The compounds were calculated using Gaussian0371 and the partial
charges were corrected using ESP methodology. For organic ligands
Hartree-Fock 6-31G* was used and for organometallic compounds
Semiempiric PM3 was employed. The electrostatic potential
surface was displayed in GaussView.
Acknowledgements
F.G. and A. F. thank the Fondo de Desarrollo Científico
Tecnológico (FONDECYT), Chile, for financial support under project
nº. 1151156 and 1161375 and VRIDEI-Usach.
y
Docking Studies
8 | J. Name., 2012, 00, 1-3
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M.M.O. acknowledge CONICYT for a doctoral fellowship and Project 17 N.A. Illán-Cabeza, A.R. García-García, M.N. Moreno-Carretero,
no. 21130725. Additionality, we acknowledge Vicerrectoría de J.M. Martínez-Martos, M.J. Ramírez-Expósito, J. Inorg. Biochem.,
Investigación, Desarrollo e Innovación of Universidad de Santiago 2005, 99, 1637-1645.
de Chile, for a fellowship for research.
18 A. Vessieres, M.N. Rager, M. Huche, S. Top, A. Vessières, P.
N.M.N. acknowledges the Ruhr University Research School (RUB RS) Pigeon, M. -N. Rager, M. Huché, E. Salomon, C. Cabestaing, J.
for support of internationally mobile researchers.
Vaissermann, G. Jaouen, ChemBioChem, 2004, 5, 1104-1113.
19 S. S. Agasti, A. Chompoosor, C. -C. Yu, P. Ghosh, C. K. Kim, V.M.
Rotello, J. Am. Chem. Soc., 2009, 131, 5728-5729.
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Amino-piperazines and organometallic imines were synthetized and evaluated in HT-
29 and PT-45 cancer cell lines. Computational studies were developed.