Warning: include_once(../article_type.php): failed to open stream: No such file or directory in /home/suxhorbncfos/public_html/omcij/OMCIJ.MS.ID.555792.php on line 120
Warning: include_once(): Failed opening '../article_type.php' for inclusion (include_path='.:/opt/alt/php56/usr/share/pear:/opt/alt/php56/usr/share/php') in /home/suxhorbncfos/public_html/omcij/OMCIJ.MS.ID.555792.php on line 120
Anthraquinone derivatives have antineoplasmatic effects. Our goal was to design and synthesize, for the first time, an anthraquinone derivative conjugated with a couple of immunomodulatory peptide analogues of Myelin Basic Protein MBP [1-11] [4Y] and a peptide analogue of IL-2Rb receptor with inhibitory effects. MBP [1-11] [4Y] was the analogue of our choice because it has previously shown that is a very strong binder to T cell receptor and could limit cellular infiltration into the Central Nervous System, protecting male mice from EAE. Furthermore, IL-2Rβ (107-118) epitope, has shown inhibitory effect on proliferation assays in PBMCs.
Multiple Sclerosis (MS) is an inflammatory autoimmune disease of the Central Nervous System (CNS), characterized by activation of T-cells subset, CD4+ and other cells of the immune system. After the activation and proliferation of the above cells, they infiltrate into the CNS through the Blood Brain Barrier (BBB). Then, they are secondary activated against the Myelin sheath. The destruction of the Myelin sheath leads to neurological dysfunction and the emerge of neurological symptoms. MS is a devastated disease resulting in neurological disability if not treated from early stages [1-4].
Myelin Protein consists of major and minor lipoproteins which form the Myelin sheath. The major proteins are MBP, PLP and MOG. T-cells are activated against specific epitopes which are called immunodominant epitopes such as MBP83-99, MBP1-11, PLP139-145 and MOG35-55 [4-6]. Furthermore, IL-2, is a cytokine, playing a critical role on the activation of the immune system against foreign attacks by viruses and bacteria. It is known that the IL-2 receptor is ex-pressed on the surface of activated T-cells and not on the “rest” T cells [7-9]. The design was based on the binding information of the high affinity IL-2 receptor with
specific epitopes, linear and cyclic, of the extracellular segment of the β chain. Monoclonal antibodies are bound to these epitopes and prevent the connection of the cytokine with the IL-2R.
1,4-bis(substituted alky-1-amino)-anthraquinones are considered as molecules with antineoplasmatic properties [10-12]. These compounds intercalate with the DNA helix and inhibit the DNA transcription. They have also shown a strong inhibitory effect on Topoisomerase II enzyme and demonstrate a down regulation effect on Pgp activity, resulting in inhibition of mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) [13-15]. Moreover, such derivatives act against Leukemia P-388 in mice . 9,10-Dihydroxy-2,3-dihydro-1,4- anthraquinone (leucoquinizarin), is the derivative of our choice in the present synthesis.
Numerous of organic compounds have been synthesized so far to modulate the MS patients’ immune system. In this study, we describe the synthesis of mono substituted amino, ethyl-amino anthraquinone and bis substituted 2-amino, ethyl-amino anthraquinone derivatives con-jugated with mutated peptide analogues (APLs) of the immunodominant epitope MBP(1-11) with mutation at position 4 (4Y) and a peptide inhibitor of the
IL-2Rb receptor. The design of the peptide analogues was based
on the primary structure of the MBP1-11 epitope and β subunit
receptor extracellular area, IL-2Rβ (107-118). The peptide
RELFRQSLSQRETALV does not share any amino acid sequence
with the extracellular area of the IL-2 receptor β-subunit and in
the present study was used as control.
In addition, the epitope AcMBP1-11 when injected in PL/J
mice induced EAE. Replacement of Lys at position 4 with Ala and
Tyr results in peptide analogues with antagonistic effects [17-
20]. Moreover, these peptide analogues demonstrate stronger
binding affinity characteristics to Major Histocompatibility
Complex (MHC). Based on the above, we synthesized 4 peptidylanthraquinone
compounds (mono and bis substituted) to discover
new molecules towards the potential immunomodulation of MS. It
is worth to note that the described conjugation was carried out in
liquid phase for the first time.
Synthesis of the mono and 1,4 bis-ethylene-diamine substituted anthraquinone.
At first, the 1,4 dihydroxy-anthraquinone (1.5mmol) was
dissolved in 25ml of methanol. Boc protected ethylene-diamine
(1.5mmol) was added dropwise. The procedure was executed
in N2 atmospheric conditions for 30min. The duration of the
reaction was 1h reflux at temperature of 50-55oC. Control of the
temperature is crucial due to the formation of “by-products” which
decrease the final yield . After 1h, the solution was freeze and
was oxygen-exposed for 15h to get mild oxidation at atmospheric
conditions. Two main products were isolated. The mono-ethylenediamine
substituted anthraquinone product (blue light colored)
and the bis-ethylene-diamine anthraquinone (dark blue colored).
Both were Boc protected. The removal of Boc protecting group was
accomplished by treating the solution with TFA/DCM (65/35, v/v)
for ap-proximately 2h. Both products, were monitored by HPLC
and were identified by Thin Layer Chromatography (TLC) and
Mass Spectrometry. In the following Schemes 1 & 2, the synthesis
of the two products is described. The products were identified by
ESI-MS, purified and isolated using HPLC semi-preparative and a
Nucleosil 100-5 C18 (250x4mm) and 5μm particle diameter, using
AcN and H2O gradient eluent (10% -60% AcN). More details are
included in Table 1.
The synthesis of the Bocamino protected peptide analogues
used in this study has been accomplished in solid phase using
Fmoc/tBu methodology. The mutated peptides (APLs) had amino
acid alterations in critical positions for their activity known by
recent literature. This synthetic procedure has been extendedly
described by our group [22,23].
Conjugation of bis-ethylene-diamine anthraquinone
with peptide analogues of MBP[1-11] [4Y] and IL-2Rβ
The conjugation of peptide analogues was carried out in liquid
phase using N,N’-Dicyclo-hexyl-carbodiimide (DCC) as coupling
agent. The excess of the protected intermediate that was used
was 4x fold. Triethylamine was used as base, distilled Dimethyl
formamide (DMF) as solution and the reaction was accomplished
after 24h. The final products were identified by TLC and HPLC.
The final deprotection mixture was consisted of Trifluoracetic
acid (TFA) and specific scavengers [Dithiothreitol (DTT), anisole
and water (25/65/4/6, v/v, 7ml) in Dichloromethane (DCM).The
reaction was terminated after 5h and the final products 1 and 2
were identified and isolated as described above (Scheme 2).
Preliminary assays of the synthesized analogues 3a and 4a
were carried out using T cell line specific for AcMBP [1-9]. The
proliferation assays were carried out in different concentrations,
with a range of 0.00001μg/ml to 10μg/ml. The proliferation value
effect of AcMBP1-9 epitope T-cell lines was used as a reference.
Moreover, the proliferation of the analogue 3a was higher than
the control in the concentration of 0.00001μg/ml. Whereas, in
the concentration of 10μg/ml showed lower proliferation effect
compared to control. The epitope IL-2 Rβ (107-118) has shown
a decrease of 27.95% of PBMC and a decrease of 56.69% of IL-2
secretion compared to the control. The analogue 3b showed
a respective decrease of PBMCs of MS patients but not of IL-2
This synthetic approach describes for the first time
the conjugation of two immunodominant epitopes and an
anthraquinone derivative in liquid phase. The peptide analogues
were synthesized in solid phase according to Fmoc/tBu
methodology. The anthraquinone derivatives were synthesized
according to literature in liquid phase as well as the conjugation
procedure. The isolation of mono and bissubstituted derivatives
was accomplished using cation exchange chromatographic
techniques. The characterization of the final conjugated products
was carried out using IR and Mass Spectra.
The preliminary assays have shown that the IL-2Rβ (107-118)
interfere with the extracellular β subunit of IL-2 receptor and may
prevent the binding of IL-2. In addition, the synthesized analogues
inhibit the activation and proliferation of T cells by an antigen or
mitogen. This could happen because both analogues are found to
have toxic characteristics. The IL-2 is a very important cytokine
(Th1 immunological response), for the immune system activation.
The regulation of IL-2 is a crucial step towards relief of the disease
The use of several different peptide analogues conjugated
to anthraquinone derivatives could be an attractive approach
towards its biological evaluation. This new approach is giving
us a new generation of molecules consist of two or more organic
compounds and could be potential immunotherapeutic. Many
research groups have used different carriers such as nanoparticles
or PEGylated parts to combine organic molecules with biological
activity, aiming in new synthetic compounds with limited side
effects, to a specific target within the body. In conclusion, the
synthesis of conjugated compounds consists of peptide analogues
and anthraquinone derivatives could be a different methodology
towards the modulation of the disease and to design more effective
mimetic analogues against MS.