Synthesis and biological evaluation of new creatine fatty esters revealed Dodecyl creatine ester as a promising drug candidate for the treatment of the creatine transporter deficiency

Article abstract of DOI:10.1021/jm400545n

The creatine transporter deficiency is a neurological disease caused by impairment of the creatine transporter SLC6A8, resulting in mental retardation associated with a complete absence of creatine within the brain and cellular energy perturbation of neuronal cells. One of the therapeutic hypotheses was to administer lipophilic creatine derivatives which are (1) thought to have better permeability through the cell membrane and (2) would not rely on the activity of SLC6A8 to penetrate the brain. Here, we synthesized creatine fatty esters through original organic chemistry process. A screening on an in vitro rat primary cell-based blood-brain barrier model and on a rat primary neuronal cells model demonstrated interesting properties of these prodrugs to incorporate into endothelial, astroglial, and neuronal cells according to a structure-activity relationship. Dodecyl creatine ester showed then a 20-fold increase in creatine content in pathological human fibroblasts compared with the endogenous creatine content, stating that it could be a promising drug candidate.

Full text of DOI:10.1021/jm400545n

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of New Creatine Fatty Esters
Revealed Dodecyl Creatine Ester as a Promising Drug Candidate for
the Treatment of the Creatine Transporter Deficiency
Alexandra Trotier-Faurion,† Sophie Dezard,‡ Frederic Taran,‡ Vassili Valayannopoulos,§
́
́
́
Pascale de Lonlay,§ and Aloïse Mabondzo*,†
†CEA, DSV, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, 91191 Gif sur Yvette, France
‡CEA, DSV, iBiTec-S, Service de Chimie Bio Organique et de Marquage, Gif sur Yvette, France
§Centre de Ref
́ ́ ́ ́ ́ ̂
ence des Maladies Hereditaires du Metabolisme de l′Enfant et de l′Adulte, Hopital Necker-Enfants Malades,
er
́
Paris Descartes, Paris, France
Universite
S
* Supporting Information
ABSTRACT: The creatine transporter deficiency is a neurological disease caused by
impairment of the creatine transporter SLC6A8, resulting in mental retardation associated with
a complete absence of creatine within the brain and cellular energy perturbation of neuronal
cells. One of the therapeutic hypotheses was to administer lipophilic creatine derivatives which
are (1) thought to have better permeability through the cell membrane and (2) would not rely
on the activity of SLC6A8 to penetrate the brain. Here, we synthesized creatine fatty esters
through original organic chemistry process. A screening on an in vitro rat primary cell-based blood−brain barrier model and on a
rat primary neuronal cells model demonstrated interesting properties of these prodrugs to incorporate into endothelial, astroglial,
and neuronal cells according to a structure−activity relationship. Dodecyl creatine ester showed then a 20-fold increase in
creatine content in pathological human fibroblasts compared with the endogenous creatine content, stating that it could be a
promising drug candidate.
Creatine esters are believed to play an important role in the
restoration of cerebral creatine content. First, they are highly
lipophilic and so probably cross biological membranes such as
the BBB by passive transport.15 Second, cellular esterases are
able to biotransform these prodrugs and deliver creatine into
the cells. Two creatine esters have been considered so far
(creatine benzyl ester and creatine ethyl ester). Creatine benzyl
ester showed interesting properties and increased the creatine
pool in mouse hippocampal slices.15 The same was observed
with creatine ethyl ester.16 However, a 12-month clinical study
in patients did not reveal any neuropsychologic improvement,17
likely because of a lack of chemical stability: creatine esters are
highly susceptible to hydrolysis in the gastric environment and
to the effect of plasmatic esterases.18−20 The esters are thus
biotransformed to creatine which has no therapeutic properties.
Herein we present the synthesis of creatine derivatives by
means of an original method for preparing creatine fatty esters
by carrying out a ring-opening step on diprotected creatinine
with a molecule bearing an alcohol functional group. The whole
chemical library of creatine fatty esters was screened for their
ability to cross the BBB and to be internalized in primary
neuronal cells. Moreover, we shed light on the molecular
mechanisms underlying translocation processes using human
fibroblasts from patients affected by the creatine transporter
deficiency.
INTRODUCTION
■
Cerebral creatine deficiency syndromes (CCDS) are a group of
inborn errors of creatine biosynthesis and transport through the
cellular membranes.1 These diseases are associated with severe
neurologic features: mental retardation, expressive speech and
language delay, autistic-like behavior, and epilepsy. They are
characterized by a lack of creatine in the brain1,2 and metabolic
disturbances in the nervous system because the creatine is
involved in the cellular phosphocreatine energy system.3 The
only way to treat patients is to restore the cerebral creatine pool
by bringing creatine into the brain. Nowadays, the treatment of
creatine biosynthesis defects has yielded significant clinical
improvement.4 However, successful therapeutic strategies still
need to be discovered in order to treat the creatine transporter
defect.2,5
Some clinical studies based on the use of creatine
supplemented by amino acids such as L-arginine and L-glycine
showed no improvement of clinical features in long follow-up
of patients.6 The absence of functional creatine transporters at
the blood−brain barrier (BBB) may prevent the entry of
creatine into the brain, thus affecting the cognitive functions.7−9
For instance, creatine amino acids and phosphocreatine−Mg
complex show neuroprotective activity in in vivo animal models
of cerebral stroke, ischemia, or hypoxia.10−12 In addition, a 9-
week treatment with cyclocreatine as treatment in SLC6A8
knockout mice13 resulted in an increase in phosphocreatine and
phosphocyclocreatine 31P-MRS signals as well as normalization
of behavioral test findings.14
Received: April 16, 2013
© XXXX American Chemical Society
A
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Figure 1. Synthesis of creatine fatty ester by ring-opening of protected creatinine by fatty alcohols.
2). These results represent a significant improvement because
such hydrophobic creatine esters were never successfully
obtained by other methods.
RESULTS
■
Chemistry. Although several procedures have already been
described for the preparation of anhydride (WO 2008/101309)
and amide (US 2011/0269986, US 2008/0200705) derivatives
of creatine, efficient methods leading to creatine fatty esters are
still lacking. To the best of our knowledge classical acid-
catalyzed esterification of creatine by alcohols is the only
method described so far (US 2005/0049428). Because the
alcohol partner is used as solvent in this process, the method is
limited to the preparation of small esters like ethyl, propyl and
butyl esters of creatine.
We therefore were interested in developing a more general
method leading to real fatty esters of creatine. Figure 1
describes our general strategy for a brand new synthesis of
creatine fatty esters. The strategy is based on activation of the
electrophilicity of the carbonyl moiety of creatinine 1 by double
protection of this cyclic guanidine. Indeed, the carbamate
derivative of the guanidine moiety should be a good leaving
group and therefore should promote the opening of the
creatinine ring in presence of nucleophiles such as alcohols.
This ring-opening reaction followed by carbamate deprotection
would thus generate the desired creatine esters.
Carbamate Deprotection of the Creatine Fatty Esters 3.
The deprotection of the CBz groups of creatine esters 3 was
carried out under hydrogen using palladium-supported catalysis
(Figure 1). This step was found to be quite delicate because of
the polarity of the guanidine part of the molecules which do not
facilitate product recovery. After screening of a panel of
reaction conditions, palladium over alumina was finally selected
and products were successfully recovered after washing the
catalyst with methanol. Using these conditions, creatine esters
were obtained in quantitative yields except for products 4c and
4e, which required purification on reversed phase column.
These products were isolated in poor and moderate yields,
respectively, because of their relative instability on silica gel
(Table 3).
Pharmacology. Stability of Creatine Fatty Esters. The
main pitfall for experimentations with creatine fatty esters is
their chemical stability.18−20 Thus, particular attention has been
paid to finding a way to maintain satisfactory stability of our
products during the experiments. The degradation of the esters
occurs by intramolecular cyclization, leading to biologically
inactive creatinine and alcohols. We showed that the rate of
degradation is higher with the longer carbon chains. Thus, 60
min of incubation at 37 °C degraded creatine octadecyl ester by
87%, dodecyl ester by 72%, and octyl ester by 21.25% (Table
4). This degradation can be sustained when the creatine esters
are in acidic solution.18−20 In our experimental conditions, the
dilution of samples in acetonitrile supplemented by 5% formic
acid stopped the undergoing degradation.
Creatine Fatty Esters Are Not Toxic As Regards of Cell
Viability and Do Not Alter Blood−Brain Barrier Integrity.
None of the experimental conditions showed a decrease in the
cell viability of brain endothelial cells, glial cells, or neuronal
cells compared with a 100% vehicle, suggesting that none of the
tested conditions are toxic in terms of cell viability (Table 7).
The permeability of Lucifer Yellow (LY Papp), a paracellular
route marker compound, was then evaluated in in vitro cell-
based rat BBB model in the presence of the creatine fatty ester.
Whatever the creatine fatty ester used in the experiments, the
LY Papp value was below the limit range of 5 × 10−6 cm·s−1,21
suggesting that the creatine fatty esters did not compromise the
integrity of the in vitro cell-based rat BBB model and that the
cell monolayer is intact.
Protection of Creatinine. First attempts to protect the
guanidine moiety of creatinine with Boc2O gave very poor
yields under either aqueous or anhydrous conditions (Figure
1).
Fmoc double protection was also found to be difficult but,
fortunately, the reaction with the benzoyl chloroformate yielded
87% of diprotected creatinine 2c after crystallization in hexane
(entry 4, Table 1).
a
Table 1. Protection of Creatinine
yield
entry
1
reagent
solvent
base
product
(%)
Boc2O (5 equiv) dioxane/
water
NaOH (1
equiv)
2a
traces
2
3
4
Boc2O (6 equiv) DCM
DIEPA (6
equiv)
2a
2b
2c
traces
21
FmocCl (3
equiv)
DCM
DIEPA (3
equiv)
CBzCl (3 equiv) DCM
DIEPA (3
equiv)
87
a
Reactions were carried out with 0.1 M reactants at room temperature
overnight.
Translocation of Creatine Fatty Esters through the in Vitro
Cell-Based Rat BBB Model. Having demonstrated the integrity
of the BBB cell monolayer in the presence of different creatine
fatty esters (Table 7), we investigated the ability of the creatine
fatty esters to reach the brain. This was determined by an in
vitro cell-based model of rat BBB.21 We demonstrated that the
creatine esters were able to penetrate into the brain endothelial
cells according to a structure−activity relationship (Table 6).
After a 60-min incubation of 10 μg·mL−1 of creatine fatty esters,
Nucleophilic Addition of Alcohols to Diprotected Crea-
tinine. Double CBz-protected creatinine 2c undergoes
spontaneous ring-opening at room temperature once dissolved
in methanol to afford creatine methyl ester 3a (entry 1, Table
2). Heating up to 80 °C is however needed for alcohols bearing
longer chain. We were particularly pleased to observe that the
reaction is still effective with long, hydrophobic alcohols. The
octadecyl creatine ester 3h was for example successfully
obtained using only 2 equiv of stearyl alcohol (entry 9, Table
B
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a
Table 2. Nucleophilic Addition of Alcohols to Compound 2c
entry
alcohol
CH3OH
stoichiometry (equiv)
conditions
yield (%)
product
1
2
3
4
5
6
7
8
9
as solvent
25 °C, 2 h
80 °C, 4 h
60 °C, 4 h
80 °C, 4 h
80 °C, 5 h
80 °C, 7 h
80 °C, 7 h
80 °C, 16 h
80 °C, 5 h
92
72
60
40
25
47
55
25
50
3a
3b
3c
3d
3e
3f
CH3CH2OH
as solvent
CH3(CH2)3OH
CH3(CH2)7OH
CH3(CH2)8OH
CH3(CH2)11OH
CH3(CH2)15OH
CH3(CH2)17OH
CH3(CH2)17OH
10
4
8
8
6
3g
3h
3h
1,5
2
a
Reactions were carried out with 0.1 M reactants.
Table 3. Deprotection of the CBz Groups
Table 5. Multiple Reaction Monitoring Parameters for the
Detection of Creatine, Creatinine, and Creatine Ethyl (4b/
C2), Butyl (4c/C4), Octyl (4d/C8), Nonyl (4e/C9),
Dodecyl (4f/C12), and Octadecyl (4h/C18) Fatty Esters by
HPLC-MS/MS
reactant
solvent
product
yield (%)
3c
3d
3e
3f
DCM
4c
4d
4e
4f
7
>95
50
DCM/MeOH (1/1)
DCE
compd/
chain length
transition
(ES+)
parent
ion m/z
product
ion m/z
collision
energy (eV)
tube
lens
DCM/MeOH (1/4)
DCM/MeOH (1/2)
DCM/MeOH (1/1)
DCM/MeOH (1/2)
>95
>95
>95
>95
3f
4f
creatine
(CR)
T1
132.156
90.185
10
100
3g
3h
4g
4h
creatinine
(CRN)
T1
114.124
86.196
10
75
a
Table 4. Degradation of Creatine Esters at 37 °C
T2
T1
T2
T3
T1
T2
T3
T1
T2
T3
T1
T2
T3
T1
T2
T3
T1
T2
T3
114.124
160.153
160.153
160.153
188.197
188.197
188.197
244.236
244.236
244.236
258.225
258.225
258.225
300.285
300.285
300.285
384.364
384.364
384.364
44.254
90.195
10
15
12
12
15
12
12
20
15
15
13
20
15
25
20
15
35
25
20
75
20
4b/C2
4c/C4
4d/C8
4e/C9
4f/C12
4h/C18
creatine ester concentration (μg·mL−1
)
132.126
118.175
90.175
20
octadecyl
ester
20
octyl ester
dodecyl ester
70
t: 0 min
2.07 0.18
1.63 0.04
21.25
6.47 0.60
1.81 0.69
72
5.74 0.10
0.76 0.07
87
132.144
146.166
90.165
70
t: 60 min at 37 °C
70
loss by spontaneous
degradation (% initial
dosage)
80
132.137
202.185
90.175
80
a
80
Creatine ester concentration was quantified by HPLC-MS/MS at
100
100
100
110
110
110
115
115
115
initial and after a 60 min incubation at 37 °C. The loss of product by
spontaneous degradation has been expressed as % of initial dosage.
132.135
342.346
90.125
we found 70.6
7.80 nmol·mg−1 of proteins of dodecyl
132.146
258.195
90.185
creatine ester in brain endothelial cell lysate, which was 10
times that with octadecyl (6.46 1.42 nmol·mg−1) and nonyl
(8.14
3.95 nmol·mg−1) creatine ester and more than 200
132.136
342.346
times that with octyl creatine ester (0.33 0.10 nmol·mg−1 of
proteins). None of the ethyl and butyl creatine ester was
detected in BBB endothelial cell lysates. When we looked at the
astroglial lysate, we observed that not only had the creatine
esters crossed the brain endothelial cells but they had also
enter the neurons because we found 257 76.8 nmol·mg−1 and
53.9 10.7 nmol·mg−1 in the neuronal cell lysate, respectively,
entered the astroglial cells. About 31.6
13.2 nmol·mg−1
corresponding to 58.2
administered dose, respectively. 5.88
11.3% and 32.2
1.45% of
proteins and 13.3 4.50 nmol·mg−1 proteins of dodecyl and
nonyl creatine esters were detected in astroglial lysates,
respectively, whereas octyl and octadecyl creatine esters were
0.44 nmol·mg−1
proteins (11.4 0.43% of initial dose) of octyl creatine fatty
ester were retrieved in the cell lysate, whereas the ethyl creatine
poorly detectable (0.24
0.04 nmol·mg−1 and 0.12
0.07
ester was poorly detectable in the cell lysate (1.47
0.34
nmol·mg−1 proteins, respectively). Ethyl and butyl creatine
esters were undetectable in our experimental conditions.
Translocation of Creatine Fatty Ester in an in Vitro
Primary Neuronal Cell Culture Model. We next analyzed the
translocation of these creatine prodrugs into primary cortical
neuronal cells, which are the target cells (Table 6) expressing
neuronal cell markers such as microtubule associated protein-2
(MAP2) and neurofilament proteins (data not shown).
Intracellular creatine ester content was determined and
compared with the residual content in the supernatant after
60 min of incubation. Dodecyl and octadecyl were best able to
nmol·mg−1 proteins equivalent to 0.16
0.01% of the
administered dose).
These data indicated that dodecyl creatine ester compound
was best incorporated in brain endothelial cells and astroglial
cells and was thus able to diffuse through the BBB to the
neurons.
Pharmacological Activity of Creatine Fatty Esters. The
pharmacological activity of creatine fatty esters was evaluated in
terms of their capacity to restore the creatine pool into the cells.
We first attempted to determine the esterase activity (Figure 2)
in the pathological model using pediatric patients’ fibroblasts,
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which lack a functional SLC6A8 transporter. We noticed that
each fibroblast batch was able to convert the 4-nitrophenyl
acetate into p-nitrophenol, with peak activity reached at 30 min
for control K and CTp3 and 80 min for DTp1 and VLp2. This
indicates that fibroblasts expressed functional esterases and
allowed us to perform a 60 min incubation. We then evaluated
the uptake of dodecyl creatine ester (Figure 3) in human
fibroblasts. This compound accumulated in human fibroblast
lysates at between 100 and 150 nmol·mg−1 of proteins in 60
min. The same profile was obtained for the control K (107
8.92 nmol·mg−1 of proteins), the child with a functional
creatine transporter, and the pathological DTp1 (123 11.2
nmol·mg−1 of proteins), VLp2 (149
8.26 nmol·mg−1 of
proteins), and CTp3 (107 20.5 nmol·mg−1 of proteins). This
suggests that the dodecyl creatine ester is able to accumulate
inside the fibroblasts even if the creatine transporter SLC6A8 is
no longer functional.
The therapeutic strategy is based on the conversion of the
creatine ester into creatine, so we measured the impact of the
treatment with dodecyl creatine ester on the creatine cell
content (Figure 4). A 5.8-fold increase in the creatine content
was noted in the control K compared with the same
experimental conditions without the dodecyl creatine ester
treatment. In comparison, the endogenous creatine content
detectable in fibroblasts from control K cells was 33.6 1.24
nmol·mg−1 of proteins. Considering the endogenous value in
these cells, the increase in creatine was 2.6-fold, but the most
interesting observations were made on fibroblasts from patients
suffering from a SLC6A8 creatine transporter deficiency
syndrome. Whereas the creatine loading in the cells in the
absence of dodecyl creatine ester was below our lowest
quantitation limit of 0.05 μg.mL−1, the creatine content
increased after a 60 min incubation with dodecyl ester up to
66.1
4.33, 76.1
2.19, and 55.4
7.78 nmol·mg−1 of
proteins in the DTp1, VLp2, and CTp3 patients, respectively.
When considering the initial endogenous creatine content,
these values were increased 23.9-, 20.0-, and 21.6-fold,
respectively, assuming that the dodecyl creatine ester may
have been taken up by cell esterases and converted into creatine
to replenish the cell’s energy supply.
DISCUSSION AND CONCLUSION
■
Cerebral creatine deficiency is a neurological disorder which
induces severe neurodevelopmental delays in children. One of
the established etiologies is deficiency in creatine transporter
because of mutations of the encoding gene.22 Therapeutic
strategies rely on the use of a creatine prodrug, which enters
neuronal cells by a passive transport and is converted into
creatine. On the basis of the principle that increasing drug
lipophilicity will promote the diffusion through the BBB, we
aimed to develop creatine esters linking long and fatty carbon
chains to the creatine structure. Due to the presence of cellular
esterases, these prodrugs could be converted in creatine within
the appropriate target cells.
What is first striking when considering the creatine
derivatives is the huge number of patents: several synthesis
pathways have been described to date, but very few compounds
have been reported in the literature. The only one which has
been extensively described is the creatine ethyl ester.12,16 When
we first tried to reproduce the published methods, it turned out
that they were not convenient for the synthesis of long-chain
creatine esters. An original synthesis process was therefore
implemented, and a library of nine creatine prodrugs was
D
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Table 7. In Vitro Evaluation of Toxicity of Creatine Ethyl (4b/C2), Butyl (4c/C4), Octyl (4d/C8), Nonyl (4e/C9), Dodecyl
a
(4f/C12), and Octadecyl (4h/C18) Fatty Esters
concentration
LY Papp (× 10−6
MTT endothelial cells (% of
control)
MTT astroglial cells (% of
control)
LDH neurons (ratio compared to
ctrl)
(μg·mL−1
)
cm/s)
4b/C2
4c/C4
4d/C8
4e/C9
4f/C12
4h/C18
1
10
1
1.54 0.28
1.13 0.47
1.23 0.55
1.81 0.69
0.93 0.33
2.42 1.26
1.49 0.33
1.18 0.25
0.70 0.05
1.70 0.49
−
−
−
−
−
−
0.53
0.53
0.54
0.58
0.65
0.64
0.52
0.45
0.67
0.69
0.65
0.69
76.9 21.5
−
90.6 9.48
−
10
1
−
10
1
84.6 13.0
122 20.4
−
−
−
−
10
1
143 33.2
−
145 19.9
5/10
1
87.8 13.0
−
−
10
−
95.3 6.21
88.3 8.81
a
Three tests assessing the cellular toxicity were performed. The Lucifer Yellow permeability (LY Papp) showed values less than 5 × 106 cm·s−1 for all
conditions, indicating that none of them induced BBB disruption. The mitochondrial function was not altered in any conditions as showed by the
MTT test, in which the cell viability did not differ of more than 20% from the control, and by the LDH test, in which the ratio compared with the
control was below 1, meaning that no conditions induced neurotoxicity. −, test not performed.
Figure 2. Activity of cellular esterases in fibroblasts. The activity of
cellular esterases in human fibroblasts is represented by the optical
density of p-nitrophenol as a function of time for control subject K
(black circle) and three patients (DTp1, VLp2, CTp3) with creatine
transporter deficiency.
prepared. Only six of these were considered during the
pharmacological studies. None of the six compounds decreased
cell viability or increased BBB permeability, and so they were
usable for further investigations on their brain penetrability and
diffusion, using the in vitro BBB model. This revealed a
structure−activity relationship: the longer the carbon chain is
the greater the entry in both brain endothelial and astroglial
cells. The compound showing the most interesting properties
was the dodecyl creatine ester, whereas the shortest carbon
chains (ethyl to octyl) displayed limited diffusion through cell
membranes. The octadecyl creatine ester was detected in brain
endothelial cells but not in astroglial cells, indicating that it was
probably stuck into the endothelial phospholipid bilayer. In the
BBB model, the detection of the creatine ester did not correlate
with an increase in intracellular creatine content but with an
increase in the creatine signal in the blood compartment. This
observation suggested two hypotheses. First, in the BBB model,
the creatine transporter is functional and it seems to mediate
the efflux of creatine from blood to brain and from brain to
Figure 3. Quantitative determination of dodecyl creatine ester.
blood. Second, the creatine content of healthy cells is very high:
Dodecyl creatine ester is expressed as nmol of ester per mg of
55.5
26.9 and 173.4
38.8 μM in endothelial cells and
proteins in the cellular extract in the fibroblasts of three patients
compared with the control subject. Student’s t test was used to
determine significant differences between control and patient, **
stands for p < 0.005.
astroglial cells, respectively. This compared with 16.2 1.42
μM of exogenous creatine provided by the 5% serum in cell
culture media and indicates that these cells may rely on a
creatine biosynthesis in addition to the uptake from the
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EXPERIMENTAL SECTION
■
Chemistry. All the reagents were from Aldrich (Steinheim,
Germany). TLC was performed on Merck F 254 plates using the
specified solvent system. Analytical and preparative LC-MS were
performed on a Waters Autopurify system. The elution was done on a
reversed phase column (Waters XBridge C18 100 mm × 4.6 mm,
particles of 3.5 μm particles) with three methods depending on the
polarity of the different compounds: the time T is in min.
Generic:
T0: 95/5 H2O/ACN (+1/1000 HCO2H)
T8: 100% ACN + 1/1000 HCO2H
T13 min: 100% ACN + 1/1000 HCO2H
Figure 4. Quantitative determination of creatine. The amount of
creatine is expressed as nmol of ester per mg of proteins in the cellular
extract in the fibroblasts of the control subject and three patients in the
presence (+) or absence (−) of dodecyl creatine ester.
Eluent 1:
T0: 95/5 H2O/ACN (+1/1000 HCO2H)
T5: 100% ACN + 1/1000 HCO2H
T13 min: 100% ACN + 1/1000 HCO2H
Eluent 2:
T0: 95/5 H2O/ACN (+1/1000 HCO2H)
T2: 100% ACN + 1/1000 HCO2H
T13 min: 100% ACN + 1/1000 HCO2H
supernatant. Creatine esters taken up in this way may be turned
into the desired creatine, but it remains unclear whether
creatine metabolism can be saturated. In this hypothesis, excess
creatine could be released in the blood compartment.
Interestingly, the same profile was obtained in a primary
neuronal model. The dodecyl and octadecyl creatine esters
penetrated cells well, unlike the ethyl creatine ester, which was
scarcely detectable in the cell lysate. Penetration of the octyl
creatine ester was intermediate.
All the compounds were identified by LC-MS and NMR spectra
(Bruker UltraShield 400 MHz for proton and 100 MHz for carbon
13). Most purifications were performed on Combiflash Teledyne Isco
on silica columns with UV detection. Additional analyses were done
when possible (IR on Perkin-Elmer 2000 FT-IR, melting points on
Buchi melting point B-545). Proton NMR assignments were based on
̈
the integration on the singulet of the N-methyl group (3H). The α
methylene of the carboxy group also gave a singlet, which was used to
calculate the NMR purity of the compounds. The analytical method
LC-MS was used to determine purity confirming ≥95% purity for all
compounds.
General Procedure for Synthesis of (E)-tert-Butyl 2-((tert-
butoxycarbonyl)imino)-3-methyl-5-oxoimidazolidine-1-carboxylate
(2c, Supporting Information Figure 1). Benzoylchloroformate (4.2
mL, 3 equiv) was added to a solution of di-isopropyl ethylamine (5.2
mL, 3 equiv) with creatinine (1.124 g, 1 equiv) in 100 mL of
anhydrous dichloromethane under nitrogen. Benzoylchloroformate
was added dropwise in an ice bath. The mixture was stirred for 30 min
in the ice bath and overnight at room temperature.
The reaction is checked by TLC (silica, heptane/ethyl acetate) and
LC-MS. The reaction medium was extracted by addition of
dichloromethane and water. The dichloromethane phase was washed
three times with water and dried with magnesium sulfate.
The dichloromethane solution was concentrated by evaporation
under vacuum and allowed to crystallize. Crude (Z2)-creatinine was
obtained (3.25 g, 87%). The (Z2)-creatinine was purified on silica gel
(heptane/ethyl acetate gradient) for structure determination. The
crude product was used for the esterification step.
(E)-tert-Butyl 2-((tert-Butoxycarbonyl)imino)-3-methyl-5-oxoimi-
dazolidine-1carboxylate (2c). Chemical formula: C20H19N3O5. MW:
381 g·mol−1. Appearance: amber colored solid. 1H NMR (CDCl3, 400
MHz) δ: 3.07 (s, 3H), 4.01 (s, 2H), 5.02 (s, 2H), 5.09 (s, 2H) and
7.34 (m, 10H). 13C NMR (CDCl3, 100 MHz) δ: 31.5, 51.4, 67.8, 70.2,
127.0, 133.7, 136.1, 147.2, 151.7, 159.5, 155.9. FTIR (KBr) ν max:
3361, 2976, 2667, 1800, 1744, 1669, 1623, 1453, 1384, 1318, 1222,
1135, 1072, 981, 902, 871, 799, 728, 695 cm−1. MS (ES+): m/z 382.3.
mp: 124 °C. Rf: 0.25 (5:5, heptane/ethyl acetate, revealed by UV 254
nm).
General Procedure for Synthesis of (CBz)2-Creatine Fatty Esters.
Compound 2c was mixed with excess fatty alcohol without solvent in a
pill box according to Table 2 and heated with stirring at 80 °C for 3−5
h until the mixture became a solution (depending on the melting point
of the fatty alcohol). The reaction mixture was analyzed by LC-MS
analysis and purified on the CombiFlash apparatus.
These experiments show that the incorporation of creatine
fatty esters in endothelial, astroglial, and neuronal cells depends
on the length of the carbonyl chain. We sought a new
therapeutic strategy for creatine transporter deficiency, using
experiments with the pathological model. Human fibroblasts
from patients with cerebral creatine deficiency caused by a
transporter deficiency were used to investigate whether the
dodecyl creatine ester could enter the cells even if it could not
be taken up by the SLC6A8 transporter. Creatine content in
the fibroblasts was increased, especially in the pathological
fibroblasts when treated by dodecyl creatine ester. This
demonstrated that the dodecyl can be acted up by cellular
esterases, which we demonstrated are active in these cells, and
replenishes the creatine content in these cells. Interestingly, the
ethyl creatine ester was not able to penetrate the cells and so
did not increase creatine content. This suggests that on the
basis of published observations,16,17 ethyl creatine ester is not
an effective drugable compound because of its poor cell
membrane permeability. In contrast, dodecyl creatine ester is a
good candidate for development as a treatment option in
patients with creatine deficiency transporter.
However, further investigations are needed for the protection
of dodecyl creatine ester chemical structure from the
degradation by plasma esterases. A vehicle approach such as
the use of Lipid NanoCapsules (LNC) would be a suitable way
to cross the BBB and deliver dodecyl creatine ester into the
parenchyma target cells. Besides a nanovector approach, further
strategy such as the improvement of the chemical template, e.g.,
exploiting the chemical properties of the cationic guanidine to
induce the self-assembly of dodecyl creatine ester could be also
considered. To treat the creatine transporter deficiency, the
challenge will be to protect the dodecyl creatine ester from the
degradation in biological fluids, deliver it through the BBB, and
improve neuronal creatine and phosphocreatine to restore the
neuronal functionality.
(CBz)2-Butyl 2-(2,3-Bis((benzyloxy)carbonyl)-1methylguanidino)-
acetate (3c, Supporting Information Figure 2). Chemical formula:
C24H29N3O6. MW: 455 g·mol−1. Appearance: colorless oil. H NMR
1
(CDCl3, 400 MHz) δ: 0.95 (t, 3H), 1.36 (q, 2H), 1.6 (q, 2H), 3.12 (s,
F
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3H), 4.14 (m, 4H), 5.14 (m, 4H), 7.36 (m, 10H). MS (ES+): m/z
456.19. Rf: 0.75 (5:5, heptane/ethyl acetate, revealed by UV 254 nm).
Purification: Combiflash on silica; solvent, heptane/ethyl acetate;
gradient, 100/0 → 80/20.
(t, 3H), 1.49 (m, 14H), 1.86 (m, 2 H), 3.25 (s, 3H), 4.38 (t, 2H), 4.42
(s, 2H). MS (ES+): m/z: 258.80.
Dodecyl 2-(1-Methylguanidino)acetate (4f, Supporting Informa-
tion Figure 11). Chemical formula: C16H32N3O2. MW: 300 g·mol−1.
1
Appearance: thick white solid. H NMR (MeOD, 400 MHz) δ: 0.90
(CBz)2-Octyl 2-(2,3-Bis((benzyloxy)carbonyl)-1methylguanidino)-
(t, J = 6.8 Hz, 3H), 1.29 (m, 16H), 1.67 (m, 4H), 3.06 (s, 3H), 4.18 (t,
J = 6.8 Hz, 2H), 4.22 (s, 2H). 13C NMR (MeOD, 100 MHz) δ: 12.9,
22.2, 25.4, 28.1, 28.8, 28.9, 29.1, 29.1, 29.2, 29.2, 29.5, 31.5, 32.1, 36.3,
51.0, 61.5, 65.5, 77.4, 77.7, 78.1, 167.8. MS (ES+): m/z 300.8. FTIR
(KBr) ν max: 2921, 2852, 1743, 1669, 1592, 1498, 1466, 1418, 1328,
1220, 1116, 681, 608 cm−1. mp: 101.4 °C.
acetate (3d, Supporting Information Figure 3). Chemical formula:
1
C28H37N3O6. MW: 511 g·mol−1. Appearance: yellow oil. H NMR
(CDCl3, 400 MHz) δ: 0.87 (t, 3H), 1.26 (m, 10H), 1.62 (q, 2H), 3.13
(s, 3H), 4.14 (m, 2H), 4.19 (s, 2H), 5.14 (s, 2H), 5.19 (s, 2H) 7.33
(m, 10H). 13C NMR (CDCl3, 100 MHz) δ: 14.0, 14.1, 20.9, 22.5, 25.7,
28.4, 29.0, 31.6, 38.8, 60.3, 65.5, 67.7, 128.1, 128.2, 128.3, 128.4, 128.5,
156.6, 168.2, 171.0. MS (ES+): m/z 512.2. Rf: 0.7 (5:5, heptane/ethyl
acetate, revealed by UV 254 nm). Purification: Combiflash on silica;
solvent, heptane/ethyl acetate; gradient, 100/0 → 80/20.
Octadecyl 2-(1-Methylguanidino)acetate (4h, Supporting In-
formation Figure 12). Chemical formula: C22H45N3O2. MW: 383
1
g·mol−1. Appearance: white solid. H NMR (MeOD, 400 MHz) δ:
0.86 (t, J = 8 Hz, 3H), 1.26 (m, 30H), (t, J = 7.2 Hz, 2H), 3.03 (s,
3H), 4.12 (t, J = 8 Hz, 2H), 4.31 (s, 2H). 13C NMR (MeOD, 100
MHz) δ: 14.0, 22.6, 25.7, 29.1, 29.3, 29.4, 29.4, 29.5, 29.6, 29.6, 31.8,
32.7, 63.0, 114.9, 127.9, 128.2. MS (ES+): m/z 384.4. FTIR (KBr) ν
max: 3415, 2958, 2918, 2850, 2397, 2303, 1742, 1588, 1468, 1263,
1209, 1163, 1071, 967 cm−1. mp: 55.5 °C
General Procedure for Synthesis of Creatine Ethyl Ester. Pure
thionyl chloride (2.5 mL) was added through a condenser under
nitrogen to a solution of creatine (1.23g −9.38 mmol) in dried ethanol
(80 mL) according to US 2005/0049428.The solution was refluxed for
1 h and then allowed to cool to room temperature. Creatine ethyl ester
hydrochloride was recovered by crystallization in a 50% yield.
Ethyl 2-(1-Methylguanidino)acetate HCl (4b, Supporting In-
formation Figure 7). Chemical formula: C6H13N3O2. MW: 159
g·mol−1. Appearance: crystal white needles. 1H NMR (D2O, 400
MHz) δ: 1.26 (t, J = 7 Hz, 3H), 3.05 (s, 3H), 4.25 (q, J = 7 Hz, 2H),
4.24 (s, 2H). MS (ES+): m/z: 160.
Pharmacology. In Vitro Primary Cell-Based Rat Blood−Brain
Barrier Model. We first tested the capacity of the creatine fatty esters
to cross the in vitro BBB model, which consisted of a coculture of rat
primary endothelial and astroglial cells. Primary rat astroglial cells were
seeded at a density of 2 × 104 cells/well in 1500 μL on a 12-well plate.
The astroglial culture medium was a mixture of α-MEM/F-12 (Life
Technologies) supplemented with 5% FBS (Lonza), 1% human serum
(Sigma Aldrich), 1% penicillin/streptomycin/neomycin (Life Tech-
nologies), and 0.4% FGF (Millipore). Then 24 h later, Transwell
inserts (Costar; pore size, 0.4 μm; diameter, 12 mm; surface, 1.12 cm2)
were placed inside the wells and primary rat endothelial cells were
plated out on the upper layer at a density of 8 × 104 cells/insert in 500
μL of EBM-2 basal medium (Lonza) supplemented with the EGM-
2MV kit (Lonza). The chambers containing endothelial cells and
astroglial cells were considered as the apical and basolateral
compartments, respectively. The plates were incubated at 37 °C in
an atmosphere containing 5% CO2, and the BBB model formed
confluent monolayers within 12 days.21,23
After 12 days, the integrity of this BBB model was assessed (see
below), and the creatine fatty esters were screened. The apical and
basolateral media were replaced by specific transport buffer (150 mM
NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2, 6 mM NaHCO3,
2.8 mM glucose, and 5 mM Hepes) in which the creatine fatty esters
were dissolved at 1 and 10 μg·mL−1. After 60 min of incubation,
supernatants were diluted 5-fold in acetonitrile/5% formic acid and
cells were scrapped in a mixture 20% water/76% acetonitrile/4%
formic acid. An HPLC-MS/MS was then performed to detect the
creatine fatty esters in each compartment and in endothelial and
astroglial cell lysates. The creatine fatty esters which were concerned in
this experiment were 4b, 4c, 4d, 4e, 4f, and 4h.
( C B z ) 2 - N o n y l 2 - ( 2 , 3 - B i s ( ( b e n z y l o x y ) c a r b o n y l ) -
1methylguanidino)acetate (3e, Supporting Information Figure 4).
Chemical formula: C29H39N3O6. MW: 525 g·mol−1. Appearance:
1
yellow oil. H NMR (CDCl3, 400 MHz) δ: 0.87 (t, 3H), 1.25 (m,
14H), 1.61 (q, 2H), 3.12 (s, 3H), 3.92 (s, 2H), 4.16 (m, 2H), 5.14 (s,
2H), 5.16 (s, 2H) 7.36 (m, 10H). MS (ES+): m/z 526.11. Rf: 0.7 (5:5,
heptane/ethyl acetate, revealed by UV 254 nm) Purification:
Combiflash on silica; solvent, heptane/ethyl acetate; gradient, 100/0
→ 80/20.
(CBz)2 -Dodecyl 2-(2, 3-Bis((benzyloxy)carbonyl)-
1methylguanidino)acetate (3f, Supporting Information Figure 5).
Chemical formula: C32H45N3O6. MW: 567 g·mol−1. Appearance:
1
yellow oil. H NMR (CDCl3, 400 MHz) δ: 0.88 (t, 3H), 1.25 (m,
18H), 1.62 (q, 2H), 3.07 (s, 3H), 4.16 (m, 4H), 5.14 (s, 2H), 5.19 (s,
2H) 7.35 (m, 10H). 13C NMR (CDCl3, 100 MHz) δ: 8.0, 8.5, 14.0,
22.6, 25.7, 28.4, 29.1, 38.9, 45.7, 52.9, 53.3, 63.3, 65.5, 67.7, 126.9,
128.0, 128.4, 128.5, 156.5, 168.1. MS (ES+): m/z 568.3. Rf: 0.45 (5:5,
heptane/ethyl acetate, revealed by UV 254 nm). Purification:
Combiflash on silica; solvent, heptane/ethyl acetate; gradient, 100/0
→ 80/20.
(CBz)2 -Octadecyl 2-(2,3-Bis((benzyloxy)carbonyl)-1-
methylguanidino)acetate (3h, Supporting Information Figure 6).
Chemical formula: C38H57N3O6. MW: 651 g·mol−1. Appearance:
1
yellow oil. H NMR (CDCl3, 400 MHz) δ: 0.88 (t, J = 8.0 Hz, 3H),
1.26 (m, 30H), 1.629 (t, J = 8.0 Hz, 2H), 3.13 (s, 3H), 4.14 (t, J = 8.0
Hz, 2H), 4.19 (s, 2H), 5.15 (s, 2H), 5.20 (s, 2H), 7.33 (m, 10H). MS
(ES+): m/z 652.0. Rf: 0.17 (5:5, heptane/ethyl acetate, revealed by UV
254 nm). Purification: Combiflash on silica; solvent, heptane/ethyl
acetate; gradient, 95/05 → 80/20.
General Procedure for Synthesis of Creatine Fatty Esters. First, 10
mg·mL−1 pure (CBz)2 creatine fatty ester was dissolved in anhydrous
dichloromethane/methanol solution (1/2) or acetonitrile/methanol
(1/1) under nitrogen. Then 5% Pd/Al2O3 was added and the reaction
mixture was degassed under vacuum, frozen, and purged three times
with hydrogen. The medium was allowed to reach room temperature
and react with vigorous stirring.
The reaction was monitored by TLC (silica, heptane/ethyl acetate)
and LC-MS. When the reaction was complete, generally after 3 h, the
creatine fatty ester solution was filtered (0.5 μm filter).
Butyl 2-(1-Methylguanidino)acetate (4c, Supporting Information
Figure 8). Chemical formula: C8H17N3O2. MW: 187 g·mol−1
Appearance: colorless oil. H NMR (D2O, 400 MHz) δ: 0.9 (t, J =
7 Hz, 3H), 1.37 (m, J = 7 Hz, 2H), 1.65 (m, J = 7 Hz, 2H), 3.07 (s,
3H), 4.24 (m, 4H). MS (ES+): m/z: 188.4.
.
1
Octyl 2-(1-Methylguanidino) Acetate (4d, Supporting Informa-
tion Figure 9). Chemical formula: C12H25N3O2. MW: 244 g·mol−1.
1
Appearance: thick white solid. H NMR (MeOD, 400 MHz) δ: 0.90
The integrity of the cell-based BBB models was demonstrated by
measuring the flux of [14C]-sucrose, [3H]-vinblastine, and [3H]-
propranolol (Perkin-Elmer) through the monolayer. Transwells with
rat endothelial cells monolayers were transferred to new 12-well plates.
A specific transport buffer was added: 500 μL to the apical
compartment and 1500 μL to the basolateral compartment. After 60
min of incubation at 37 °C of 0.1 μCi/mL of [14C]-labeled sucrose, 1
μCi/mL of [3H]-propranolol in the apical compartment, and 0.1 μCi/
mL of [3H]-vinblastine in the apical and basolateral compartment,
(t, J = 5.2 Hz, 3H), 1.31 (m, 12H), 3.04 (s, 3H), 3.30 (t, J = 8.0 Hz,
2H), 3.93 (s, 2H). 13C NMR (MeOD, 100 MHz) δ: 12.9, 22.2, 25.5,
29.4, 29.4, 29.5, 31.4, 36.5, 55.8, 66.8, 169.9, 186.1. MS (ES+): m/z:
244.1. FTIR ν max: 3455, 2953, 2849, 2085, 1636, 1493, 1396, 1363,
1249, 1110, 1064, 1028, 898, 823.
Nonyl 2-(1-Methylguanidino) Acetate (4e, Supporting Informa-
tion Figure 10). Chemical formula: C13H27N3O2. MW: 258 g·mol−1.
1
Appearance: thick white solid. H NMR (MeOD, 400 MHz) δ: 1.09
G
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supernatants from both apical and basolateral compartments were
collected. The amount of tracer that passed through the endothelial
monolayer was determined by scintillation counting and the
permeability Papp of each compound was assessed using the formula
(A):
490 and 690 nm. Results are expressed as [DO(490 nm) − DO(690 nm)],
ratio to the control. A ratio equal to or less than 1 indicates an absence
of neuronal cell toxicity.
Human Primary Fibroblasts Cell Culture. Human fibroblasts were
obtained from skin biopsies through a gift of the Centre de Ref
́ ́
erence
des Maladies Hereditaires du Metabolisme at the Necker Hospital in
́
́
́
[Xbasolateral] × VB
T × S × [X0]
Paris. Three patients with cerebral creatine deficiency caused by lack of
the creatine transporter functionality and 1 control were studied. All of
the mutations were previously described in Valayannopoulos et al.,
2013:24 p.Asn336del c.1006_1008delAAC (patient 1, DTp1) and p.
(Gly499del) c.1497_1500delGAG (patient 2, VLp2) as P3 and P4,
respectively, or in Valayannopoulos et al., 2012:25 p.(G414del)
c.1221_1223delTTC (patient 3, CTp3) as P2. The fibroblasts were
plated out at 3 × 103 cells per well in 6-well plates in a DMEM
medium (Life Technologies) supplemented with 10% fetal bovine
serum (Lonza), 1% penicillin/streptomycin/neomycin (Life Tech-
nologies), 1% sodium pyruvate (Sigma Aldrich), and 1% L-glutamine
(Life Technologies). They were cultured for 6 days by replacing the
medium every 2−3 days. The incubation of creatine esters consisted in
replacing the medium by HBSS (Life Technologies) in which the
compound was diluted to 10 μg·mL−1. After 1 h at 37 °C, 5% CO2, the
supernatant was diluted 5-fold in acetonitrile/5% formic acid and cells
were scrapped in 20% water/76% acetonitrile/4% formic acid. An
HPLC-MS/MS was then performed to detect the creatine fatty esters
in the cell lysates.
P XA→B
=
app
(A)
X is the compound for which the permeability is assessed, [Xbasolateral
]
the concentration of the compound X in the basolateral compartment
at the end of the incubation, VB the total volume of the basolateral
compartment (1.5 mL), T the time of the incubation, S the transwell
surface area, and [X0] the concentration of compound X at T0.
Validated BBB models have sucrose permeability below 8 × 10−6
cm·s−1, propranolol permeability above 16 × 10−6 cm·s−1, and
vinblastine permeability ratio above 2.
The absence of toxicity of the creatine fatty esters on the endothelial
cells viability was estimated by an MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide, Sigma Aldrich) cell viability test on
endothelial monolayers. The endothelial culture medium was replaced
by a solution of 10 μg·mL−1 of creatine fatty ester in the transport
buffer. The cells were incubated for 75 min at 37 °C in an atmosphere
containing 5% CO2. The solution was then removed, and 1 mg·mL−1
MTT in PBS was added for an overnight incubation. MTT was
reduced by metabolically active cells to insoluble purple formazan dye
crystals. DMSO (Sigma Aldrich) was then added to the wells,
solubilizing the crystals so the absorbance could be read using a
spectrophotometer at wavelengths of 550 and 650 nm. Results are
expressed as [OD(550 nm) − OD(650 nm)], percentage of control.
The Lucifer Yellow (LY, Sigma Aldrich) permeability test was used
to study the effect of creatine fatty esters on BBB integrity. LY was
diluted in transport buffer to a final concentration of 100 μM and
added to the apical compartment during creatine ester incubation.
Fluorescence leakage was determined for LY with 485 nm excitation
and 530 nm emission using a fluorescence plate reader. The LY
permeability (LY Papp) was then assessed using formula (A). An LY
Papp value below 5.10−6 cm·s−1 indicates that the creatine ester did not
damage BBB integrity.
In Vitro Rat Primary Neuronal Model. Primary cortical neurons
were prepared from fetal (E18) rat cortices according to the 86/609
European directive. The pregnant female was anesthetized with
isoflurane, and the embryos were extracted by laparotomy and placed
in a Petri dish containing HBSS supplemented with 5 mM glucose and
10 mM Hepes, pH 7.4. The brain was removed from the skull, and the
cortices were dissected. Two enzymatic dissociations were performed
with 0.25% trypsine−EDTA (Life Technologies) without and then
with DNase (Sigma Aldrich). Cortical cells were then seeded at a
density of 105 cells/cm2 onto poly-D-lysine (Sigma)-coated 24-well
plates in a medium consisting of DMEM (Life Technologies)
supplemented with 10% horse serum (Life Technologies) first. Then
2 h later, the medium was replaced by Neurobasal containing B27 1X
(Life Technologies), 2 mM L-glutamine (Life Technologies), 1 mM
sodium pyruvate (Sigma Aldrich), and 10 U/mL penicillin/
streptomycin/neomycin (Life Technologies). At culture day 4, 3 μM
of cytosine-β-D-arabinose was added. Neuronal cells express typical
markers such as MAP2 and neurofilament (data not shown).
The tests with creatine esters were performed at 10 DIV in HBSS at
1 and 10 μg·mL−1. After 1 h at 37 °C and under 5% CO2, the
supernatant was diluted 5-fold in acetonitrile/5% formic acid and cells
were scrapped in 20% water/76% acetonitrile/4% formic acid. An
HPLC-MS/MS was then performed to detect the creatine fatty esters
in the cell lysates.
Esterase Activity Determination. Esterase activity in fibroblast cells
was determined by incubating 20 μg·mL−1 of cell proteins with 350
μM of 4-nitrophenyl acetate (Sigma Aldrich) in deionized water.
Conversion of the 4-nitrophenyl acetate by esterases leads to p-
nitrophenol, which can be monitored by absorbance spectroscopy at
405 nm.
HPLC-MS/MS Analysis. Liquid chromatography (Shimadzu HPLC
system LC 20AD) with a 2.0 mm × 150 mm Uptisphere Diol HPLC
column (UP6OH, Interchim) was used for elution of creatine esters.
The mobile phase was isocratic at 40/60 (detection of creatine, ethyl
and butyl creatine esters) or 20/80 (detection of octyl, nonyl, dodecyl,
and octadecyl creatine esters) A/B, where solvent A was H2O
containing 0.1% formic acid and solvent B was acetonitrile containing
0.1% formic acid; the flow rate was 0.4 mL/min. Analyte (10 μL) was
injected onto the column placed in an oven at 40 °C. The total run
time was 6 min.
Detection was done by tandem mass spectrometry (Finnigan TSQ
Quantum Discovery with Xcalibur and LC Quan softwares, Thermo)
in positive electrospray mode. Spray voltage was 3.0 kV, and sheath
and auxiliary gas pressures were 50 and 20 (arbitrary units),
respectively. The in-source CID energy was fixed at 12 V, and
capillary temperature was 350 °C. Tube lens and collision energy
values were optimized for each compound and are summarized in
Table 5. Multiple reaction monitoring was used for the detection of
the ion transitions (Table 5). The standard curves showed linearity for
creatine over a range of 0.05−10 μg·mL−1 and of 0.01−5 μg·mL−1 for
ethyl, butyl, octyl, nonyl, dodecyl, and octadecyl creatine esters.
Creatine fatty esters and creatine concentrations and amounts were
determined in each compartment and in endothelial cells, astroglial
cells, and fibroblasts lysates. The amount of creatine fatty esters and
the amount of creatine were standardized to the amount of protein in
each lysate.
Each experiment was performed in triplicate.
Statistical Analysis. Statistical analysis was performed using the
Prism 5.01 program (GraphPad Software, Inc., San Diego SA).
Statistical comparisons were made using a two-tailed unpaired t test
and a variance analysis one-way ANOVA followed by a Bonferroni
post-test. Changes were considered statistically significant at p < 0.05.
A lactate deshydrogenase (LDH) test was performed to assess the
absence of neuronal death. First, 70 μL of LDH lysis solution was
added to each well after the 60 min experiment and was incubated at
37 °C under 5% CO2 for 45 min. Then 50 μL was removed for testing
and 100 μL of LDH assay mixture was added for 30 min at room
temperature. Then 10 μL of 1N HCl was finally added to the wells and
the absorbance was read using a spectrophotometer at wavelengths of
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra of selected compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
H
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Journal of Medicinal Chemistry
Article
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +33 1 69 08 13 21. Fax: +33 1 69 08 59 07. E-mail:
aloise.mabondzo@cea.fr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mickael Kempf, Anne-Cec
́
ile Guyot, and Dr Agnes
̀
̈
Belly (CEA, iBiTec-S, Service de Pharmacologie et d’Immu-
noanalyse, Gif-sur-Yvette, France), David-Alexandre Buisson,
́
Celine Puente, and Emilie Dubois (CEA, iBiTec-S, Service de
Chimie Bioorganique et de Marquage) for their technical skills
and all the patients included in this study. This work was
financially supported by the Fondation Lejeune.
ABBREVIATIONS USED
■
Boc, tert-butoxy carbonyle; CBz, Z = benzyl carbamate =
benzyloxy carbonyl; Fmoc, fluorenylmethoxycarbonyl; FTIR,
Fourier transform infrared spectroscopy; KBr, potassium
bichromate; mp, melting point; NMR, nuclear magnetic
resonance; Papp, permeability; TLC, Thin layer chromatography
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