Chemical Platform for the Preparation of Synthetic Orally Active Peptidomimetics with Hemoregulating Activity

Article abstract of DOI:10.1002/cmdc.201600157

A novel chemical platform based on branched piperazine-2,5-dione derivatives (2,5-diketopiperazines) for creating orally available biologically active peptidomimetics has been developed. The platform includes a diketopiperazine scaffold with “built-in” fu

Full text of DOI:10.1002/cmdc.201600157

DOI: 10.1002/cmdc.201600157
Communications
Chemical Platform for the Preparation of Synthetic Orally
Active Peptidomimetics with Hemoregulating Activity
Vladislav Deigin,*[a] Olga Ksenofontova,[a] Alexey Khrushchev,[a] Oleg Yatskin,[a]
Alexandra Goryacheva,[b] and Vadim Ivanov[a]
A novel chemical platform based on branched piperazine-2,5-
dione derivatives (2,5-diketopiperazines) for creating orally
available biologically active peptidomimetics has been devel-
oped. The platform includes a diketopiperazine scaffold with
“built-in” functionally active peptide fragments covalently at-
tached via linkers. The concept was applied to two hemosti-
mulatory drugs, the dipeptide thymogen (GluTrp) and the tri-
peptide stemokin (IleGluTrp). Preparation of a series of respec-
tive derivatives is described. Of the five synthesized analogues,
three demonstrated high hemostimulatory activity in vivo on
intact mice and on ex vivo irradiated bone marrow cells. Pros-
pects of further development of the concept are discussed.
The major disadvantage of this compound class—low stabili-
ty under non-invasive methods of administration—limits the
use of peptide preparations in medical practice.[5] Among the
common ways of increasing enzymatic stability of peptide mol-
ecules is the introduction of elements with a cyclic structure,
which as a rule are resistant to proteolytic degradation.[6–8] In
the scientific[7] and patent literature,[9] derivatives of cyclic di-
peptides (piperazine-2,5-diones or 2,5-diketopiperazines, DKP)
are reported to have been introduced as elements attached to
the N or C termini of an active peptide, or incorporate one or
two residues of an active peptide in their structure as a new
group of protease-resistant peptidomimetics.
This approach was applied in the present work to the dipep-
tide Glu-Trp (thymogen) 1, isolated in 1985 from the extract of
bovine thymus,[10] and its tripeptide analogue Ile-Glu-Trp (ste-
mokin) 2. Both preparations are registered in Russia as drugs
under the trade names Thymogenꢀ and Stemokinꢀ. Prepara-
tions are used in clinical practice in the form of injections.[11]
Thymogenꢀ stimulates the differentiation and proliferation of
T-lymphocytes and promotes the activity of neutrophils, mono-
cytes, and NK cells, providing a regulatory effect on cellular
and humoral immunity.[12] It was also shown that Thymogenꢀ
promotes proliferation of damaged bone marrow cells.[13] Ste-
mokinꢀ exhibits an increased affinity for bone marrow cells
and is known as a hemostimulatory and immunoadjuvant
agent.[14]
Peptides regulate a broad range of biochemical reactions in
living organisms, and serve as potential prototypes for numer-
ous drug preparations.[1,2] Despite various problems related to
the development of peptide drugs, the number of pharma-
ceuticals based on peptides and peptidomimetics is constantly
growing; at present more than 70 peptide preparations have
been registered worldwide, including 14 original peptide drug
products developed and registered in Russia.[3,4]
Several hundred peptides as potential drug candidates are
currently at various stages of preclinical and clinical investiga-
tions in many countries. However, the total share of peptides
in the global pharmaceutical market (less than 1%) corre-
sponds neither to the major role that peptides play in vital ac-
tivity of higher organisms, nor to the range of opportunities
that multifunctional peptide molecules provide for the creation
of new derivatives applicable for regulating virtually any meta-
bolic process.
In this work we synthesized Glu-Trp-containing diketopipera-
zines 3–7 (Figure 1) and evaluated their hemostimulating activ-
ity upon systemic and oral administration. Preliminary commu-
nication is given in reference [15]. The selection of the target
molecules was based on the following considerations. Ana-
logues 3–7 contain a 2,5-diketopiperazine fragment in the N-
terminal part of dipeptide 1. In all cases the Ca-carboxyl group
of the glutamic acid residue participates in the formation of
[a] Prof. V. Deigin, Dr. O. Ksenofontova, Dr. A. Khrushchev, Dr. O. Yatskin,
Prof. V. Ivanov
Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Miklukho-Maklaya 16/10, Moscow 117997 (Russia)
E-mail: vdeigin@gmail.com
[b] Dr. A. Goryacheva
Tsyb Medical Radiological Research Center, Ministry of Health of Russia,
249030, Kaluga region, Obninsk (Russia)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600157.
ꢁ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
Figure 1. Cyclic analogues of thymogen (3–6) and stemokin (7).
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the cyclic fragment, while the amide bond of Glu-Trp, in con-
trast to thymogen, is formed by the Cg-carboxyl group. In our
earlier work[13, 16–19] it was shown that such structural modifica-
tion does not impair the biological activity of thymogen and
its analogues.[13,17] As the second amino acid participating in
the formation of the diketopiperazine cycle, alanine 3, hydro-
phobic valine and leucine (4 and 5), and lysine with its amino
function protonated under standard conditions 6 were used.
Peptide 7 is considered as an analogue of stemokin 2, in
which the isoleucine residue is linked to the thymogen-like
fragment via the side chain of the second glutamic acid, incor-
porated into the diketopiperazine cycle. In all cases, residues
that form the diketopiperazine core are not only carriers for
pharmacophores but also act as active components, built into
the structure of the drug candidate.
Synthesis of the substituted diketopiperazines was per-
formed by classical solution-phase methods of peptide chemis-
try according to synthetic schemes shown in Figures 2 and 3.
Standard abbreviations are used as recommended in refer-
ence [20]. Two approaches were investigated for the cycliza-
tion of linear peptides into DKP derivatives. The first (A) includ-
ed cyclization of activated esters of protected dipeptides fol-
lowed by coupling with the third amino acid;[21] the second, B,
involved direct cyclization of linear tripeptide precursor by
heating in pyridine solution at reflux.[22] Samples of 3–5 were
obtained according to both approaches. The second one af-
forded final products with considerably higher overall yields
(27, 35, and 34%, versus 14, 16, and 17%) and was chosen for
the synthesis of compounds 6 and 7 (Figure 3).
Figure 2. Synthetic schemes for peptides 3, 4, and 5.
Cyclization kinetics (Figure 4) were followed by analytical
HPLC. Experimental details of the syntheses are given in the
Supporting Information. An Acquity UPLC system was used
(Waters, USA). The column was an Acquity UPLC BEH C18
(1.7 mm, 2.1ꢂ50 mm). Buffer A consisted of 0.1% formic acid;
buffer B was 0.1% formic acid in acetonitrile. Elution was per-
formed by a linear gradient from 5 to 80% buffer B in buffer A
for 8 min at a flow rate of 0.5 mLminÀ1, with detection at
l 220 nm.
Figure 3. Synthetic schemes for compounds 6 and 7.
Final purification by preparative HPLC [Spherical C18 silica gel
(Sorbent Technology)] afforded the target products 3–7 in 95–
Figure 4. Cyclization kinetics of compound 3 (peak 2) from linear precursor (peak 1).
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97% purity. Physicochemical characteristics of peptides 3–7
and their linear precursors are provided in the Supporting In-
formation (Table S1). The proteolytic stability of peptide 6
along with a series of its derivatives was demonstrated in one
of our earlier works.[7]
Thymogen, stemokin, and other hemoregulatory factors
such as colony-, granulocyte-, and granulocyte–macrophage-
stimulating factors (CSF, G-CSF, and GM-CSF) affect the prolifer-
ation rate and induce differentiation of a variety of blood
cells.[23] In our study we evaluated the impact of peptides 3–7
on initial studies of hematopoiesis in a test system based on
the measurement of colony-forming ability of undifferentiated
bone marrow stem cells (CFU-S) on the spleen of sub-lethally
irradiated recipients (Figure 5).
Figure 6. Stimulation of intact bone marrow cell proliferation by peptides 1–
7. The ratio of cell colony number on the recipient spleen versus control (in
%) is shown; *p<0.05 relative to control.
Figure 7. Experimental model for the study of therapeutic action of peptides
on damaged bone marrow cells.
Figure 5. Experimental model for the study of intact bone marrow cell stim-
ulation by peptides.
The method originally introduced in reference [24] was used
in our earlier works.[13,19] Suspension of bone marrow cells
taken from the femoral bone of intact mice or mice treated by
peptide 48 h before the bone marrow extraction was intrave-
nously administrated to sub-lethally irradiated (8 Gy) recipient
animals. The immune and hematopoietic systems of such ani-
mals are fully destroyed and do not interfere with donor stem
cells. The donor bone marrow stem cells form colonies on the
surface of the recipient spleen, the number of colonies are
measured by microscopy and subjected to analysis. An in-
crease in colony number from cells taken from the peptide-
treated animal serves as a measure of the stimulatory action of
peptides (Figures 5, 6 and Table S2 in the Supporting Informa-
tion). As seen from Figure 5 and Table S2 of the Supporting In-
formation, linear prototypes 1 and 2 show practically no effect
on the proliferative bone marrow activity of the intact mice
both at intraperitoneal injection and at oral application. In con-
trast, the cyclic analogues 3 and 7 significantly enhance the
colony-forming activity of intact bone marrow cells upon both
routes of administration. Peptides 4–6 were inactive at the
standard 100 mgkgÀ1 intraperitoneal (i.p.) applications.
Figure 8. Restoration of damaged bone marrow cell proliferation activity by
peptides 1–7. The ratio of cell colony number on the recipient spleen versus
control (in %) is shown; *p<0.05 relative to control group, **p<0.05 rela-
tive to irradiated group.
In another set of experiments the action of peptides 1–7 on
damaged bone marrow was studied (Figures 7, 8 and Table S3
in the Supporting Information). As a damaging factor, ex vivo
irradiation of the cell suspension from the femoral marrow at
a dose of 1 Gy was used. Irradiated and control (intact) suspen-
sions were administered intravenously to 8 Gy irradiated recipi-
ents. One hour after transplantation of the irradiated cells the
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[1] D. J. Craik, D. P. Fairlie, S. Liras, D. Price, Chem. Biol. Drug Des. 2013, 81,
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[2] L. Stryer, Biochemistry, 3rd ed., W. H. Freeman & Co., New York, 1988,
Chs. 2, 21, 24; pp. 15–42, 495–515, 575–600.
[3] V. J. Hruby, Nat. Rev. Drug Discovery 2002, 1, 847–858.
[4] Peptide Therapeutics Market – Global Industry Analysis, Size, Share,
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ketresearch.com.
[5] Z. Antosova, M. Mackova, V. Kral, T. Macek, Trends Biotechnol. 2009, 27,
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[6] A. T. Bockus, C. M. McEwen, R. S. Lokey, Curr. Top. Med. Chem. 2013, 13,
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2006.
test peptide or control solution were administered to recipient
i.p. or orally at doses of 10 or 100 mgkgÀ1. Irradiation of bone
marrow cells leads to depletion of colony numbers, and pep-
tide treatment is intended to counteract the damage. The re-
sults obtained (Figure 8 and Table S3 in the Supporting Infor-
mation) clearly demonstrate that linear peptides thymogen
1 and stemokin 2, being highly active under systemic applica-
tion, show no activity in the post-irradiation cell restoration
test upon oral administration. In contrast, cyclic peptides 3, 6,
and 7 show high activity under both routes of administration,
commensurate with the above-mentioned activity of 1 and 2.
The Leu-containing analogue 5 showed some tendency in
activity at systemic application, and the Val-containing ana-
logue 4 showed none. Generally, the behavior of analogues 4–
6 in the two test systems provides another example of poor
predictability of structure–function relationships in complex
biological systems.
[10] G. M. Yakovlev, V. G. Morozov, V. Kh. Khavinson, V. I. Deigin, A. M. Korot-
kov, Pat. No. RF1582393, 1987.
[11] V. G. Morozov, V. Kh. Khavinson, V. V. Malinin, Peptide Thymomimetics,
Nauka, St. Petersburg, Russia, 2000.
[12] V. Kh. Khavinson, Neuroendocrinol. Lett. 2002, 23, 11–144.
[13] V. I. Deigin, T. N. Semenets, I. A. Zamulaeva, Ya. V. Maliutina, E. I. Seliva-
nova, A. S. Saenko, O. V. Semina, Int. Immunopharmacol. 2007, 7, 375–
382.
[14] T. N. Semenets, O. V. Semina, J. E. Vinogradova, V. I. Deigin, A. M. Pover-
enny, Immunology 2000, 6, 20–22.
[15] V. I. Deigin, A. S. Saenko, Yu. A. Semin, Radiat. Biol. Radioecol. 2011, 51,
530–535.
[16] O. V. Semina, T. N. Semenets, V. I. Deigin, A. M. Korotkov, A. M. Poveren-
ny, Bull. Exp. Biol. Med. 1993, 9, 298–299.
As a result of the present work, the first orally active hemos-
timulatory peptides have been prepared on the basis of our
original concept. Successful application of the concept paves
the way for further application of the Glu-Trp family of pep-
tides in the area of analogues with hemo- and immunosup-
pressive activity. Another aspect of future work will concern
the design and development of orally active chimeric peptide
drugs in which the 2,5-diketopiperazine moiety chemically con-
nects the peptide portion with another pharmacophore, not
necessarily of peptide nature. These studies are currently un-
derway in our laboratory.
[17] V. I. Deigin, A. M. Poverenny, O. V. Semina, Immunol. Lett. 1999, 67, 41–
46.
[18] V. Deigin, A. Poverenny, O. Semina, T. Semenets, Proceedings of Ameri-
can Peptide Symposia 2002, 6, 626–627.
[19] O. V. Semina, T. N. Semenets, V. I. Deigin, A. M. Korotkov, A. M. Poveren-
ny, Radiat. Biol. Radioecol 1993, 33, 808–811.
Acknowledgements
[20] Synthesis of Peptides and Peptidomimetics, Vol. E22a (Eds.: M Goodman,
A Felix, L Moroder, C Toniolo), Georg Thieme Verlag, Stuttgart, 2002.
[21] K. D. Kopple, H. G. Ghazarian, J. Org. Chem. 1968, 33, 862–864.
[22] G. P. Slater, Chem. Ind. 1969, 32, 1092.
This work was supported by the Russian Science Foundation
(project no. 14-50-00131) for IBCH RAS.
[23] D. Bonnet, J. Pathol. 2002, 197, 430–440.
[24] J. E. Till, E. A. McCulloch, Radiat. Res. 1961, 14, 213–222.
Keywords: piperazine-2,5-diones
peptidomimetics · synthetic peptides
·
hematopoiesis
·
Received: March 18, 2016
Revised: June 2, 2016
Published online on && &&, 0000
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COMMUNICATIONS
Robust and bioavailable! The low sta-
bility of peptide pharmaceuticals in
non-invasive administration limits the
use of these compounds in medical
practice. We developed a platform
based on branched piperazine-2,5-
diones for creating orally stable pepti-
domimetics. These derivatives were at-
tached to the N or C termini of an
active linear peptide, increasing their re-
sistance against proteolytic activity. As
a result, the first orally active hemosti-
mulatory peptides have been prepared.
V. Deigin,* O. Ksenofontova,
A. Khrushchev, O. Yatskin, A. Goryacheva,
V. Ivanov
&& – &&
Chemical Platform for the Preparation
of Synthetic Orally Active
Peptidomimetics with Hemoregulating
Activity
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ꢁ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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