Creatinyl amino acids-new hybrid compounds with neuroprotective activity

Article abstract of DOI:10.1002/psc.1379

Prolonged oral creatine administration resulted in remarkable neuroprotection in experimental models of brain stroke. However, because of its polar nature creatine has poor ability to penetrate the blood-brain barrier (BBB) without specific creatine transporter (CRT). Thus, synthesis of hydrophobic derivatives capable of crossing the BBB by alternative pathway is of great importance for the treatment of acute and chronic neurological diseases including stroke, traumatic brain injury and hereditary CRT deficiency. Here we describe synthesis of new hybrid compounds-creatinyl amino acids, their neuroprotective activity in vivo and stability to degradation in different media. The title compounds were synthesized by guanidinylation of corresponding sarcosyl peptides or direct creatine attachment using isobutyl chloroformate method. Addition of lipophilic counterion (p-toluenesulfonate) ensures efficient creatine dissolution in DMF with simultaneous protection of guanidino group towards intramolecular cyclization. It excludes the application of expensive guanidinylating reagents, permits to simplify synthetic procedure and adapt it to large-scale production. The biological activity of creatinyl amino acids was tested in vivo on ischemic stroke and NaNO₂-induced hypoxia models. One of the most effective compounds-creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. These data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. Copyright

Full text of DOI:10.1002/psc.1379

Research Article
Received: 2 March 2011
Revised: 5 April 2011
Accepted: 7 April 2011
Published online in Wiley Online Library: 6 June 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1379
Creatinyl amino acids – new hybrid compounds
with neuroprotective activity
Sergey Burov,a∗ Maria Leko,a Marina Dorosh,a Anatoliy Dobrodumova
and Olga Veselkinab
Prolonged oral creatine administration resulted in remarkable neuroprotection in experimental models of brain stroke.
However, because of its polar nature creatine has poor ability to penetrate the blood–brain barrier (BBB) without specific
creatine transporter (CRT). Thus, synthesis of hydrophobic derivatives capable of crossing the BBB by alternative pathway is
of great importance for the treatment of acute and chronic neurological diseases including stroke, traumatic brain injury and
hereditary CRT deficiency. Here we describe synthesis of new hybrid compounds – creatinyl amino acids, their neuroprotective
activity in vivo and stability to degradation in different media. The title compounds were synthesized by guanidinylation of
corresponding sarcosyl peptides or direct creatine attachment using isobutyl chloroformate method. Addition of lipophilic
counterion (p-toluenesulfonate) ensures efficient creatine dissolution in DMF with simultaneous protection of guanidino group
towards intramolecular cyclization. It excludes the application of expensive guanidinylating reagents, permits to simplify
synthetic procedure and adapt it to large-scale production. The biological activity of creatinyl amino acids was tested in vivo
on ischemic stroke and NaNO2-induced hypoxia models. One of the most effective compounds – creatinyl-glycine ethyl ester
increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain
stroke model when applied both before and after ischemia. These data evidenced that creatinyl amino acids can represent
c
promising candidates for the development of new drugs useful in stroke treatment. Copyright ꢀ 2011 European Peptide
Society and John Wiley & Sons, Ltd.
Keywords: creatinyl amino acids; sarcosine guanidinylation; creatine p-toluenesulfonate; brain stroke; neuroprotective activity
Introduction
ofintramolecularcyclizationandformationofcreatinine(2-amino-
3-methyl-4H-imidazol-5-one). Authors postulated better stability
of Cr ethyl ester and related derivatives in acidic media and their
cleavage by esterases resulted in Cr release; however they did not
mention any experimental data confirming these statements. Al-
though Cr esters are less polar as compared with natural molecule
and can penetrate the BBB irrespective to CRT, recently it was
shown that their enzymatic or non-enzymatic cleavage is accom-
panied by creatinine formation instead of expected free Cr [8–10].
In this article we describe synthesis of new hybrid com-
pounds – creatinyl amino acids, their neuroprotective activity
in vivo and stability to degradation in different media.
Creatine (α-methylguanidino acetic acid; Cr) plays an essential role
in the cellular energy metabolism. It is phosphorylated by creatine
kinase, producing phosphocreatine, a high-energy compound
that takes part in synthesis of ATP, replenishing the energy stores
even in the absence of oxygen and glucose (Figure 1). Cr is
synthesized presumably in the kidney, liver and pancreas with
subsequent delivery to the energy consuming tissues like muscles
and brain. Under normal circumstances its penetration through
the blood–brain barrier (BBB) is mediated by specific protein,
creatine transporter (CRT), containing 635 amino acids [1].
It was shown that oral Cr administration resulted in remarkable
neuroprotection in experimental models of brain stroke, amy-
otrophiclateralsclerosis,HuntingtonandParkinsondiseases[2–5].
However because of the limited capacity of CRT and poor ability of
polar Cr molecule to penetrate the BBB, neuroprotective effect can
be achieved only after prolonged treatment lasting at least sev-
eral weeks. Thus, synthesis of hydrophobic derivatives capable of
crossing the BBB by alternative pathway is of great importance for
the therapy of acute and chronic neurological diseases including
stroke, traumatic brain injury and hereditary CRT deficiency.
Numerous Cr analogs synthesized so far comprise different
homologs, cyclic structures and compounds with modified func-
tional groups (Figure 2). One of the most active and well-known
derivatives – cyclocreatine (2-amino-4,5-dihydro-1H-imidazole-1-
acetic acid) can serve as a substrate for creatine kinase, suppress
cancer cell growth and exhibit neuroprotective activity in case of
prolonged administration [3,6]. Vennerstrom and Miller [7] sug-
gested Cr esterification as a convenient method for the prevention
Materials and Methods
General
All the amino acids and reagents were obtained from Acros Or-
ganics (Morris Plains, NJ, USA), Aldrich (Steinheim, Germany), Merk
Chemicals (Darmstadt, Germany) and IRIS Biotech (Marktredwitz,
Germany). Amberlite IRA-67 was purchased from Sigma (St. Louis,
MO, USA). SP Sephadex C-25 was purchased from GE Health-
care Bio-Science AB (Uppsala, Sweden). TLC was performed on
∗
Correspondenceto:Sergey Burov,InstituteofmacromolecularcompoundsRAS,
V.O., Bolshoi pr. 31, 199004 St. Petersburg, Russia. E-mail: burov@hq.macro.ru
a
Institute of Macromolecular Compounds RAS, 199004 St. Petersburg, Russia
Closed Joint-Stock Company ‘‘Vertex’’, 199026 St. Petersburg, Russia
b
c
J. Pept. Sci. 2011; 17: 620–626
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

CREATINYL AMINO ACIDS WITH NEUROPROTECTIVE ACTIVITY
wasstirredovernightatroomtemperaturethenfilteredandfiltrate
was diluted with EtOAc (300 ml) and washed successively with 1N
H2SO4, 5% NaHCO3 and brine. The organic layer was dried over
anhydrous MgSO4 and solvent was evaporated in vacuum.
Protected peptide amides
Nα-BOC protected peptide ester (20 mM) was dissolved in 6M
methanolic ammonia (200 ml), closed tightly and allowed to stay
at room temperature till the disappearance of the starting material
controlled by TLC (about 48h). Then the solvent was evaporated
in vacuum.
Trifluoroacetates of sarcosyl peptides
Nα-BOC protected peptide (20 mM) was dissolved in TFA (40 ml) at
roomtemperatureandstirredfor15 min.ThenTFAwasevaporated
in vacuum at 25 ◦C and residue was crystallized from diethyl ether
(200 ml).
Figure 1. Structures of creatine (A), phosphocreatine (B) and creatinine (C).
Creatinyl amino acids and peptides
DIEA (3.48 ml, 20 mM) was added to stirred suspension of
sarcosyl peptide trifluoroacetate (10 mM) and benzotriazole-
1-carboxamidinium tosylate (10 mM) in DMF (5 ml) at room
temperature. The reaction mixture was stirred for 48 or 72 h and
then DMF was evaporated in vacuum. The residue was dissolved
in 0.001 M pyridine–acetate buffer (50 ml) and loaded on SP
Sephadex C-25 column (25 mm × 150 mm) equilibrated with
0.001 M pyridine–acetate buffer. The product was eluted with a
linear gradient of 0.001–0.5 M pyridine–acetate buffer containing
20% of isopropyl alcohol. The solvent was evaporated in vacuum
and residue was crystallized from MeCN.
Preparation of Creatinyl Amino Acids using Creatine
p-Toluenesulfonate. General Procedure
Figure 2. The main classes of creatine analogs, described in the literature.
p-Toluenesulfonic acid (10 mM, 1.9 g) was added to a suspension
of Cr monohydrate (10 mM, 1.49 g) in DMF (10 ml). The suspension
was stirred for 10 min till complete Cr dissolution and then
hydrochloride of C-terminally protected amino acid derivative
(10 mM) was added. The reaction mixture was cooled to −10 ◦C
and isobutyl chloroformate (1.38 ml, 10 mM) was added followed
byNMM(2.2 ml, 20 mM)dropwiseadditionfor10 min. Afterstirring
for 1 h at −10 ◦C and 3 h at room temperature NMM hydrochloride
was filtered off and filtrate was evaporated in vacuum. The
residue was dissolved in water and treated by Amberlite IRA-
67 (acetate form) to remove p-toluenesulfonate ions. The water
wasevaporatedinvacuumandresiduewascrystallizedfromMeCN
(5 ml). The obtained white solid was dissolved in absolute ethanol
(5 ml) and allowed to stay for 24 h at −20 ◦C. The precipitate
containing creatine and creatinine was filtered off and filtrate was
evaporated in vacuum. The residue was crystallized from diethyl
ether.
Merck F 254 and Silufol plates using specified solvent system.
For visualization of creatine, creatinyl amino acids and creatinine
sodium nitroprusside [11] spray was utilized. Analytical RP-HPLC
was carried out using Beckman System Gold with a Phenomenex
Luna C18 column (4.6 mm × 150 mm, 5 µm) using UV detection at
210 nm, solvent system: A: 0.1% H3PO4 in H2O, B: 0.1% H3PO4 in
50% MeCN/H2O at gradient rate of 1% per min and flow rate of
1 ml/min. Mass spectra were recorded on Finnigan LCQ ion-trap
mass spectrometer with an ESI source in the positive mode. An-
alytical RP-HPLC and electrospray MS confirmed the purity and
molecular mass of the synthesized compounds. 1H NMR spectra of
final products were recorded at 400 MHz on a Bruker Avance 400
spectrometer. Calibration was carried out with solvent residual
signals as internal standards.
Preparation of Creatinyl Amino Acids by Guanidinylation
of Sarcosyl Peptides. General Procedure
Protected peptide esters
RP-HPLCEvaluationofCreatinylAminoAcidsStabilityinWater
Solution and Blood Plasma
Boc-Sar-OH(9.5 g, 50 mM)andHOBt(6.75 g, 50 mM)weredissolved
in DMF (40 ml) at 0 ◦C. DCC (10.3 g, 50 mM) solution in DMF (20 ml)
was added and the reaction mixture was stirred for 10 min in
an ice bath. Then hydrochloride of amino acid ester (45 mM) and
triethylamine (6.3 ml, 45 mM) were added. The reaction mixture
Stock solutions of creatinyl amino acids were prepared by dissolv-
ing test compounds in bidistilled water at a final concentration
2 mg/ml. All solutions were used immediately upon preparation.
An aliquot of fresh stock solution was diluted tenfold, analyzed
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BUROV ET AL.
by RP-HPLC and then incubated for 3 h at room temperature. The
amount of intact creatinyl amino acid was assayed by RP-HPLC.
The stability study in human and rat blood plasma were carried
out in analogous way. The assay was initiated upon the addition
of test compound stock solution (200 µl) to 1 ml of plasma and the
mixture was incubated at 37 ◦C. 200 µl aliquots of the incubations
were taken for the following time points: 0, 60 and 180 min.
The aliquots were mixed with 18 µl of 100% TCA and incubated
at −24 ◦C for at least 15 min to precipitate serum proteins. The
supernatant was collected for each sample after centrifugation
at 6000 g for 5 min and analyzed by RP-HPLC. These assays were
performed in triplicate.
sections. The slices were placed in a petri dish containing 0.1% TTC
for 15 min at 37 ◦C. Lesion areas were calculated from summed,
measured areas (ImageJ software, National Institute of Mental
Health, Bethesda, MD, USA) of unstained tissue in mm2. Data are
expressed as mean SD.
Statistical analysis of the data was performed using Statistica
software (StatSoft Inc., Tucsa, USA). All comparisons between
treatment groups and the corresponding vehicle groups were
performed using the nonparametric Mann–Whitney U test.
Differences were considered statistically significant at P < 0.05.
Results and Discussion
Biological Activity Studies
Conjugation of Cr with amino acid derivatives permits to
modify physico-chemical and biological properties of synthesized
compounds within wide range and can improve their hydrolytic
stability as compared with Cr esters. For the synthesis of new
drugs potentially useful in the treatment of acute and chronic
neurological diseases such as stroke and traumatic brain injury it is
necessary to attach Cr to hydrophobic moiety ensuring its delivery
through the BBB irrespective to the presence of CRT. Ideally,
preferable amino acid derivatives should be not only safe for the
brain cells but also provide additional advantage of synergistic
neuroprotective action.
The antihypoxic activity of creatinyl amino acids was determined
on white outbred male mice (19–25 g body weight; Pavlov
Institute of physiology RAS) using hemic hypoxia model [12].
The animals were randomized into treatment groups consisting of
10 mice each. Each group received intraperitoneal (i.p.) injection
contained tested compounds (100, 200 and 500 mg/kg) or vehicle
(control, 10 ml/kg of physiological solution), 30 min before the
experiment. The acute hemic hypoxia was produced by the
subcutaneous administration of NaNO2 in a dose of 300 mg/kg.
Then the survival time was recorded.
The neuroprotective activity of synthesized creatine derivatives
was tested on male Wistar rats (220–240 g body weight; Pavlov
Institute of physiology RAS) using ischemic stroke model [13].
Reversible middle cerebral artery occlusion was performed as
described by Longa et al. [14] and Belayev et al. [15]. Tested
compounds were dissolved in 1 ml of distilled water and injected
i.p. to deliver a dose of 55, 137 or 275 mg/kg 1 h before the
initiation of ischemia. Vehicle-treated controls received an equal
volume of physiological solution. After 48 h, the animals were
killed and the brains examined. Infarct volume was assessed using
2,3,5-triphenyltetrazolium chloride (TTC) (MP Biomedicals, Solon,
OH, USA) staining [16]. The brain was removed and cut into 2-mm
Guanidinylation of Sarcosyl Peptides
Preparation of Cr derivatives with neuroprotective properties
encounters several common problems including low Cr solubility
inwaterandorganicsolvents,itspropensityforcyclizationresulted
in creatinine formation and strong influence of any structural
modifications on biological activity. To avoid solubility limitations
and exclude the possibility of intramolecular cyclization, in
our initial experiments synthesis of creatinyl amino acids was
performed by the guanidinylation of sarcosyl peptides [17].
The typical procedure of Cr preparation described in the
Figure 3. Synthesis of creatinyl amino acids by guanidinylation of sarcosyl peptides (A) or acylation by creatine p-toluenesulfonate (B).
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CREATINYL AMINO ACIDS WITH NEUROPROTECTIVE ACTIVITY
Figure 4. 1H and 13C NMR spectra of Cr-Gly-OEt.
literature comprises sarcosine treatment by cyanamide or S-
methylisothiourea in water solution at pH 8.5–9.5 [18,19].
However, our attempts of H-Sar-Gly-OBzl guanidinylation at the
same conditions failed because of the instability of final product
in water–alkaline solution.
Pd black resulted in Cr-Phe-NH2 formation in a yield of 50%.
Nonetheless, this approach to the synthesis of creatinyl amino
acids is apparently impractical because of relatively high price
of protected guanidinylating reagents and possibility of side
reactions at the stage of final deprotection.
Application of protected guanidinylating reagents such as
N,Nꢁ-di-tert-butoxycarbonyl-1H-benzotriazole-1-carboxamidine
[20] and N,Nꢁ-di-benzyloxycarbonyl-1H-benzotriazole-1-carbox-
amidine in DMF with 1 equiv. of TEA permits to obtain Cr(BOC)2-
Gly-OBzl, Cr(BOC)2-Phe-OBzl and Cr(Z)2-Phe-NH2 in a yield of
60–70%. However, BOC group removal using TFA, HCl/dioxane,
HCOOH or HCl/Et2O is accompanied with partial decomposition
of product. Deprotection by HCl/Et2O seems to be the method of
choice, but the yield of desired compounds was only 10–25%. In
the case of benzyloxycarbonyl protection, hydrogenolysis over
Creatinyl amino acids and related derivatives were obtained by
guanidinylation of peptide esters, amides or substituted amides
using benzotriazole-1-carboxamidinium tosylate [21] (Figure 3A).
Synthesized compounds were converted to acetate salts by cation
exchange chromatography on SP Sephadex C-25 and crystallized
from acetonitrile. To elucidate the influence of attached amino
acid on physico-chemical properties and stability of Cr derivatives,
we prepared a set of analogs, shown in Table 1. The purity of
synthesizedcompoundswasdeterminedby1HNMRspectroscopy,
analytical RP-HPLC and ESI MS (Figure 4; Table 1).
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BUROV ET AL.
Table 1. List of creatinyl amino acids and peptides
Table 2. Influence of reagents ratio on the yield of Cr-Gly-OEt
ESI MS
Cr
p-TSA
DCC
DIEA
HCl∗H-Gly-OEt
Yield (%)a
Cr-containing compounds
Yielda (%)
MW
[M + H]+
1
1
2
2
2
1
1
2
2
2
1
1
2
1
1
1
2
1
1
2
1
2
1
1
2
11
13
45
44
26
1a Cr-Gly-OEt
22(1), 25(2)
12(1)
29(1), 18(2)b
216.12
188.09
215.14
278.14
230.14
306.17
277.15
278.14
293.15
294.13
244.15
287.20
273.14
444.24
519.29
217.15
189.11
216.16
279.14
231.15
307.18
278.17
279.15
294.16
295.15
245.18
288.16
274.16
445.26
520.33
1b Cr-Gly-OH
1c Cr-Gly-NHEt
1d Cr-Gly-OBzl
1e Cr-Gly-OiPr
2a Cr-Phe-OEt
2b Cr-Phe-NH2
2c Cr-Phe-OH
21(1)
a Quantified by RP-HPLC at 220 nm.
20(2)
22(1)
46(1), 10(2)b
20(1)
RP-HPLC. The influence of reagents ratio on the yield of Cr-Gly-
OEt is summarized in Table 2. Although an excess amount of
Cr p-toluenesulfonate increases the yield of final product, the
purification procedure becomes difficult because of side products
formation and the presence of starting material in the reaction
mixture. The mixed anhydride method was most advantageous
in terms of reaction completeness and simplicity of subsequent
purification. Better results were obtained by using 1 equiv. of Cr
hydrate instead of anhydrous Cr and dropwise addition of NMM
to the reaction mixture.
Purification of synthesized creatinyl amino acids primarily
comprises removal of p-toluenesulfonic acid, Cr and creatinine.
Our preliminary experiments revealed that application of cation
exchange chromatography on Rexyn 101(H), SP Sephadex C-
25 or Toyopearl SP-650M in pyridine–acetate buffer permitted
to obtain Cr-Gly-OEt in acetate form, but the purity was low.
Moreover, the procedure requires the use of large buffer volumes.
To simplify the treatment of crude product we investigated utility
of anion-exchange resin (IRA-67) at the stage of p-toluenesulfonic
acid removal. Application of IRA-67 in free-base form resulted
in significant decomposition of Cr-Gly-OEt accompanied by
creatinineformation,whileresininacetateformdoesnotinfluence
the product stability.
3a Cr-Tyr-NH2
3b Cr-Tyr-OH
38(1)
16(1)
4 Cr-GABA-OEt
5 H-Lys(Cr)-OEt
6 Cr-Gly-Gly-OEt
7 Cr-Glu-Arg-OEt
8 Cr-Phe-Arg-Gly-OEt
29(1)
26(1), 6(2)b
15(2)
24(1)c
37(1)c
a Total yield of products, obtained using different synthetic ap-
proaches: (1) guanidinylation of sarcosyl peptides; (2) acylation by Cr
p-toluensulfonate.
b Significant loss of product at the stage of crystallization.
c Yields were calculated starting from sarcosyl peptides.
According to HPLC analysis extent of sarcosyl peptide conver-
sion was about 90–100%, however the yield of purified product
was only 40–60% because of its partial loss in the course of IEC
and subsequent crystallization. It should be mentioned that some
creatinyl amino acids can be precipitated in pure form as free
bases by the addition of n-butyl alcohol to the reaction mixture
but, in contrast to acetate salts, their solubility in water is very low.
In attempts to simplify the procedure and exclude the stage of
ion-exchange chromatography we investigated utility of different
organic and inorganic acids for Cr solubilization and its protection
from intramolecular cyclization. It was found that addition of
1 Equiv. of TFA or HCl, but not CH3COOH and H3PO4 ensures
complete Cr dissolution in DMF. While trifluoroacetate is not
pharmaceutically acceptable salt, it can be easily converted to
hydrochloride using well-known techniques [23]. However our
attempts of Cr-Gly-OEt purification by the crystallization in the
form of TFA or HCl salt failed. In addition, application of Cr
hydrochloride decreased the yield of final product by 30–40%
as compared with Cr p-toluenesulfonate. Thus, in our hands
application of p-toluenesulfonic acid for Cr solubilization was
the method of choice both in terms of yield and purity of crude
product.
Acylation of Amino Acids by Creatine p-Toluenesulfonate
Considering that preparation of creatinyl amino acids by guani-
dinylation of sarcosyl peptides comprises three stages the oppor-
tunity to minimize the number of synthetic steps and reagents
requirements seems to be very attractive. To simplify the pro-
cedure we investigated the possibility of direct amino group
acylation by anhydrous Cr or Cr hydrate (Figure 3B). The problem
of low Cr solubility in organic solvents can be overcome by its
conversion to an appropriate salt form. It is known that solubility
of peptides and proteins, in organic solvents can be improved
using p-toluenesulfonate as hydrophobic counterion [22]. In our
experience, addition of p-TSA represents simple and convenient
procedure for dissolution of Cr and other different guanidino or
amidino compounds in DMF and related solvents.
The second obstacle to be overcome is high propensity of
Cr to cyclization in acidic media or in the course of carboxylic
group activation. To minimize side products formation and
simplify the purification of creatinyl amino acids, we investigated
different coupling methods including DCC, active esters and
isobutyl chloroformate. In all experiments application of p-
toluenesulfonate counterion ensures Cr solubility and significant
suppression of intramolecular cyclization. As a model compound
to optimize the reaction conditions we have chosen Cr-Gly-
OEt. While our attempts to prepare Cr active esters failed,
DCC condensation resulted in the formation of desired product.
The completeness of the coupling reaction was determined by
Efficient Cr and creatinine removal is the most problematic
stageofcreatinylaminoacidspurification. InthecaseofCr-Gly-OEt
simplerecrystallizationfromwaterseemstobeimpossiblebecause
of its high solubility. Commonly, the crude product obtained
after ion-exchange chromatography contains about 5–10% of
creatinine.Itscontentcanbereducedupto3–5%bycrystallization
from MeCN, followed by the dissolution of Cr-Gly-OEt in ethyl
alcohol and removal of precipitated creatinine. This protocol
afforded the desired product in a yield of 20–25%. However, in the
cases of hydrophobic creatinyl amino acid derivatives purification
procedure should be modified in order to ensure the maximum
recovery.
c
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CREATINYL AMINO ACIDS WITH NEUROPROTECTIVE ACTIVITY
Figure 5. Suggested mechanism of creatinyl amino acids degradation.
Stability of Creatinyl Amino Acids in Water Solutions
and Blood Plasma
amino acids in the case of their action as substrates of creatine
kinase or ability to release free creatine as a result of enzymatic
degradation. It should be mentioned that in spite of numerous
biological experiments with free Cr, the exact mechanism of
its protective action is still unclear. Recent studies have shown
that in brain stroke model Cr-mediated protection can occur
independent of changes in the bioenergetic status of neurons and
involve improved cerebrovascular function [24].
The neuroprotective activity of creatinyl amino acids was
tested in vivo using two different models: NaNO2-induced hypoxia
(mice) and ischemic stroke (rats). In the first case, sodium
nitrite injection reduces oxygen-carrying capacity of hemoglobin
because of its conversion to methemoglobin and induces severe
vasodilatation resulted in cerebral hypoxia. All tested compounds
were administered intraperitoneally at doses of 100–500 mg/kg.
It was shown that efficacy of antihypoxic action depends on
the structure of amino acid moiety. Thus, Cr-Phe-NH2 and Cr-
GABA-OEt possessed moderate activity, while Cr-Tyr-NH2 and
Cr-Gly-OEt increase life span of experimental animals about two
times (Figure 6).
Similar results were obtained using ischemic brain injury model.
The efficiency of neuroprotective action was estimated as the
ratio of damaged tissue area to the total area of brain slice
(Figure 7). I.p. administration of Cr-GABA-OEt (1 mmol/kg) and Cr-
Phe-NH2 reduces the size of necrotic zone by 25% in contrast to
more than 50% in the case of intravenous Cr-Gly-OEt injection
1 h before or after ischemia. These data evidenced that creatinyl
amino acids possess pronounced neuroprotective properties. The
mechanisms of their biological action and potential synergistic
effect of attached amino acid residue are under investigation.
Chemical stability of synthesized compounds in aqueous solutions
and their susceptibility to enzymatic degradation in blood plasma
was investigated by means of RP-HPLC and ESI MS. It was shown
that stability of Cr derivatives is strongly dependent on the
structure of adjacent amino acid residue, the kind of counterion
and pH of solution. Creatinyl-glycine esters seem to be less
stable as compared with other analogs and can undergo partial
decomposition in the course of RP-HPLC purification. These data
can be explained by the assumption that the degradation mech-
anism comprises intramolecular cyclization resulted in cleavage
of amide bond and creatinine formation (Figure 5). Creatinine can
be easily identified in water solution by means of HPLC and by
the intensive signal in the mass spectrum (m/z 113). In contrast
to creatinyl-glycine esters, phenylalanine derivatives containing
bulky benzyl side chain are relatively stable irrespectively to the
presenceofcounterion. ThelessstableofsynthesizedCrderivative
is Cr-Pro-Gly-OEt which easily undergoes decomposition both
in water and organic media resulted in creatinine and Pro-Gly
diketopiperazine formation. However, it should be mentioned
that surprisingly high stability of Cr-GABA-OEt remains unclear.
On the basis of these preliminary results we studied resistance
of glycine and phenylalanine derivatives to hydrolysis in water
solutionandenzymaticcleavageinbloodplasma.Itwasfoundthat
in distilled water both Cr-Gly-OBzl and Cr-Phe-NH2 are completely
stable during 3 h at room temperature, while Cr-OBzl at the same
conditions undergoes degradation (up to 60%). In agreement with
theliteraturedata, themaindecompositionproductwasidentified
as creatinine using RP-HPLC and ESI MS technique. In human and
rat blood plasma Cr-Gly-OBzl was stable during at least 3 h at
37 ◦C, whereas Cr-Phe-NH2 concentration decreased up to 85%.
These data suggest that creatinyl amino acids can reach the target
cells without significant decomposition and function as intact
molecules or undergo enzymatic cleavage near the sites of their
action.
Conclusions
Conjugation of Cr with amino acid derivatives permits to alter
physico-chemical properties of the molecule in wide range.
These conjugates can be synthesized in reasonable yield by the
guanidinylation of sarcosyl peptides or direct acylation of amino
acidsusingCrp-toluenesulfonate. Thestructureofattachedamino
acid derivative determines stability of synthesized compound in
water solution and their susceptibility to enzymatic degradation
in blood plasma. Synthesized creatinyl amino acids possess
neuroprotective action in vivo and can represent promising
Neuroprotective Activity of Creatinyl Amino Acids
ConjugationofCrwithdifferentaminoacidderivativesimprovesits
solubility in water and potentially can facilitate its penetration via
cellmembraneandBBB. Considering neuroprotectiveactivityofCr
molecule one can expect similar biological properties of creatinyl
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BUROV ET AL.
30
25
20
15
10
5
0
C
2b
4
3a
1a
Figure 6. The influence of creatinyl amino acids on the life span of experimental animals in hypoxia model. C, control; 1a, Cr-Gly-OEt; 2b, Cr-Phe-NH2; 3a,
Cr-Tyr-NH2; 4, Cr-GABA-OEt.
20
15
10
5
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