Synthesis of lipoamino acids and their activity against cerebral ischemic injury

Article abstract of DOI:10.3390/molecules14104051

A series of lipoamino acids were synthesized and their neuroprotective effect against brain ischemia induced by oxygen-glucose deprivation (OGD) on rat cerebral slices was evaluated. Among these compounds, N-stearoyl-L-tyrosine (4), N-stearoyl-L-serine (5) and N-stearoyl-L-threonine (6) exhibited good neuroprotective activity. We found that the neuroprotective activity of lipoamino acids depended on the acyl group, the presence of a free carboxylic function and a free hydroxyl group at the branched chain of the amino acids. The results also showed that 5 was the most active compound, protecting rat brain slices against OGD as well as hydrogen peroxide (H₂O₂) insult at the range of 1-10 M.

Full text of DOI:10.3390/molecules14104051

Molecules 2009, 14, 4051-4064; doi:10.3390/molecules14104051
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Lipoamino Acids and Their Activity against
Cerebral Ischemic Injury
Li-Yun Yao, Qi Lin, Yin-Yao Niu, Ke-Min Deng, Jian-Hua Zhang and Yang Lu *
Institute of Pharmaceutical Research, Shanghai Jiao Tong University School of Medicine, Shanghai
200025, China
* Author to whom correspondence should be addressed; E-Mail: huaxue@shsmu.edu.cn.
Received: 14 August 2009; in revised form: 22 September 2009 / Accepted: 29 September 2009 /
Published: 12 October 2009
Abstract: A series of lipoamino acids were synthesized and their neuroprotective effect
against brain ischemia induced by oxygen-glucose deprivation (OGD) on rat cerebral slices
was evaluated. Among these compounds, N-stearoyl-L-tyrosine (4), N-stearoyl-L-serine (5)
and N-stearoyl-L-threonine (6) exhibited good neuroprotective activity. We found that the
neuroprotective activity of lipoamino acids depended on the acyl group, the presence of a
free carboxylic function and a free hydroxyl group at the branched chain of the amino
acids. The results also showed that 5 was the most active compound, protecting rat brain
slices against OGD as well as hydrogen peroxide (H2O2) insult at the range of 1–10 M.
Keywords: lipoamino acids; neuroprotection; brain ischemia; oxygen-glucose deprivation
1. Introduction
Cerebral ischemia would lead to brain damage in pathogenetic mechanisms including
excitotoxicity, over production of free radicals, inflammation and apoptosis [1-3]. One of the
challenging brain damage therapies is to inhibit the toxic pathways by activating endogenous
neuroprotective system to stimulate cellular responses against the damage induced by severe ischemic
events [4-6]. A variety of chemical agents, such as glumatate receptor antagonists, antioxidants, anti-
inflammatory and anti-apoptotic agents, have been used to initiate neuroprotection by mediating the
processes [7-9]. Despite aggressive investigations aimed at development of innovative neuroprotective
drugs, few are in fact clinically effective [10].

Molecules 2009, 14
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Recent evidences suggest that N-arachidonoylethanolamine (anandamide, AEA, Figure 1), an
endocannabinoid, plays a crucial neuroprotective role in certain neurodegenerative diseases [11-13].
Therefore, the design, synthesis and study of analogs of AEA with the aim of obtaining compounds
with neuroprotective activity are ongoing research in our group.
Figure 1. The structures of AEA and its endogenous analog (NAGly).
NH
CH2CH2OH
O
AEA
NH
CH2COOH
O
NAGly
As chemical entities, lipoamino acids (LAs, Figure 2), the fatty acid-amino acid conjugates, exist as
endogenous substances with multiple biological activities [14]. They appear to have close relationships
with AEA, not only structurally, but also in terms of biological activities such as analgesia, anti-
inflammatory effects, inhibition of cell proliferation, calcium ion mobilization and neural protection
[15]. For example, N-arachidonoylglycine (NAGly, Figure 1) is an endogenous constituent in many
tissues and might have analgesic properties similar to those reported for AEA [16]. Recently, we found
that stearic acid protected rat brain slices or neurons in vitro against apoptosis induced by OGD,
NMDA or H2O2 [17]. In terms of the structure and activity of both LAs and stearic acid, we deduced
that LAs as amino acid conjugates with stearic acid might show neuroprotective activity. It is
interesting to synthesize a series of N-stearoylamino acids and its analogues and evaluate their
potential neuroprotective activity. By changing the structure and stereochemistry of the aminodonors,
we would be able to find novel neuroprotective agents and fine-tune their SAR toward obtaining potent
protectants. The present paper reports the synthesis of this library of LAs (Figure 2) and evaluation of
their activity against cerebral ischemic injury in vitro.
Figure 2. The general structure of LAs.
R
O
CH
H3C(H2C)n
N
COOH
H
2. Results and Discussion
2.1. Chemical synthesis
A series of N-stearoylamino acids and their derivatives (Table 1) were synthesized using the
procedures below, shown in Scheme 1 [18]. Briefly, commercially available stearic acid was reacted
with N-hydroxysuccinimide to provide octadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester, which was
combined with appropriate amino acids (or amino acid methyl esters) to give N-stearoylamino acids 1-
11 and N-stearoylamino acid methyl esters 12-14. The acylation of amine of amino acids with higher
yield is especially suitable for the synthesis of LAs for its effective chemoselectivity towards the

Molecules 2009, 14
4053
amino rather than hydroxyl group in the side-chains of amino acids. Compounds 15 and 16 were
synthesized following the synthetic route of 4 from palmitic and lauric acids instead of stearic acid,
respectively. Compound 17 was obtained by reducing 4 with NaBH4-I2 by the method reported by
Mckennon et al. [19]. The NaBH4-I2 reagent system, by generating the reactive (acyloxy)borohydride
(RCOOBH2), is more selective towards the carboxylic group rather than the amide group, because of
the presence of the free carboxylic group in LAs [20]. The structures of all synthesized compounds
1
13
were confirmed by H- and C-NMR, and the spectroscopic properties and analytical data were in
accord with the proposed structures.
Scheme 1. The synthetic rout to N-stearoylamino acids and their methyl esters.
O
O
O
O
O
DCC
N
O
N OH
HO
(CH2)16CH3
+
(CH2)16CH3
H3CO
THF
R
O
R
R
HO
NH2
NaHCO3
NH2
O
O
O
Pyridine
H3CO
O
R
HO
(CH2)16CH3
N
H
N
(CH2)16CH3
O
H
O
1-11
12-14
H
N
(CH2)nCH3
HO
R
O
15-17
2.2. Biological activity
2.2.1. Effect of the synthesized compounds on OGD-insulted rat brain slices
The compounds shown in Table 1 were screened for neuroprotective activity against brain ischemia
induced by OGD insult on rat cerebral slices [21]. The screening concentrations of compounds were
determined by our preliminary dose-response study (data not shown). The tissue activity of the brain
slices was expressed as percentage of that of the control before brain injury. In Figure 3, the tissue
activities of brain slices pretreated with 1-7 (66.5%–82.2%) were obviously higher than that of the
brain slices pretreated with no drug (51.8%) (P < 0.05), indicating these compounds could reduce the
OGD-induced neurotoxicity. Among these compounds, 4, 5 and 6, the three LAs with free hydroxyl
groups, showed good tissue activity (77.5%, 82.2% and 76.5% respectively), which was similar to that
of the standard ketamine (76.7%) and they attenuated OGD injury effectively (P < 0.01). It’s worthy to
note that the pretreatment of the brain slices with target compounds did not influence the availability of
brain ischemia model within the effective concentration (data not shown). Compound 4 showed a
significant protective effect on insulted brain slices, but its homologues 15 and 16 had little effect

Molecules 2009, 14
4054
(53.4%, 56.6% respectively), indicating that the length of the alkyl moiety of the LAs plays a critical
role in the neuroprotective effect. N-stearoylamino acid methyl esters 12-14 and N-stearoyl-L-tyrosinol
(17) presented little neuroprotective effect, implying that the presence of a free carboxylic group in the
stearic amides may be necessary for their activity. It is interesting that an identical neuroprotective
effect has been observed for 1 (L-phenylalanine) and 2 (DL-phenylalanine). A detailed SAR study
concerning the stereochemistry of LAs and their neuroprotecive activity is currently under way in our
laboratory.
Table 1. Library of LAs.
Chemical Name
R
CH2
CH2
1. N-stearoyl-L-phenylalanine
2. N-stearoyl-DL-phenylalanine
3. N-stearoyl-L-proline
CH2
CH2
CH
H2C
CH2
OH
4. N-stearoyl-L-tyrosine
5. N-stearoyl-L-serine
6. N-stearoyl-L-threonine
CH2OH
CHOH
CH3
CH2
7. N-stearoyl-L-tryptophan
N
H
CH(CH3)2
CH2SH
8. N-stearoyl-L-leucine
9. N-stearoyl-L-cysteine
10. N-stearoyl-L-histidine
H2C
N
NH
(CH2)4NH2
11. N-stearoyl-L-lysine
12. N-stearoylglycine methyl ester
13. N-stearoyl-L-glutamic acid dimethyl ester
14. N-stearoyl-L-phenylalanine methyl ester
H
(CH2)2COOCH3
CH2
15. N--palmitoyl-L-tyrosine
16. N- lauroyl-L-tyrosine
17. N-stearoyl-L-tyrosinol
—COOH, n = 14
—COOH, n = 10
—CH2OH, n = 16
Brain slices not only maintain anatomic relations and natural synaptic connectivity in vitro, but also
eliminate such in vivo variables as blood flow, temperature, ionic environment, and closely match in
vivo conditions [22]. Therefore, increasing numbers of investigation used brain slice models have been
reported to study brain function and brain protection. On the other hand, TTC method using 2,3,5-
triphenyltetrazolium chloride (TTC) as an histopathologic stain for identification of infarcted rat brain
tissue is simple, objective and sensitive, and has been widely used in the assessment of brain ischemia
in vitro [23,24]. In the present study, we evaluated for the first time the effects of LAs on brain slice
injury, using the TTC method.

Molecules 2009, 14
Figure 3. The tissue activity of rat brain slices pretreated with compounds 1-17 (10 μM) in
4055
the OGD-induced brain injury model. Brain slices were incubated with different
compounds 30 min prior to and during OGD applications. The viability of brain slices was
evaluated by using TTC staining method. Data are means ± SD from six independent
experiments. ## P <0.01 vs control group. *P <0.05, ** P <0.01 vs OGD group.
120
100
**
**
**
**
*
80
60
40
20
0
*
*
*
##
OGD
2.2.2. Effect of 5 on three types of insulted brain slices
OGD, glutamate (Glu) or H2O2 damaged models can reflect the pathological characteristics of
different phases of ischemia injury (metabolism disorder, toxic amino acid and oxidative stress) [25].
In order to evaluate the possible mechanism of neuroprotective action of these new molecules, we
observed the neuroprotective effect of N-stearoyl-L-serine (5), the most effective one, against three
types of damage brain cortical and hippocampal slices. As shown in Figure 4, compound 5 can
concentration-dependently protect brain slices against OGD as well as H2O2 insult at the range of 1~10
μM (Figures 4A, 4C), and has no protective effect on 1 mM Glu injured brain slices (Figure 4B). The
protective effect became less at 20 μM, implying that 5 at concentration higher than 20 μM might
show the cytotoxicity.
Since the earlier consequences of applying OGD model in brain ischemia are both increasing
release of the excitatory amino acid and excessive activation of oxidative free radicals [26], the
observation that 5 might defend against oxidative stress and couldn’t inhibit the excitotoxicity of
glutamate suggested that neuroprotective effect of 5 might be mediated, at least in part, by an anti-
oxidative stress mechanism. These results are in agreement with our recent report showing that
neuroprotective effect of N-stearoyl-L-tyrosine (4) may be attributed to reduction in lipid peroxidation
and DNA fragmentation [27]. The mechanism studies of 4 and 5 could provide experimental
foundation for investigating of their analogues. The detailed mechanism study of these compounds is
currently under way in our laboratory.

Molecules 2009, 14
Figure 4. The tissue activity of rat brain slices pretreated with N-stearoyl-L-serine (5) on
4056
rat brain slices induced by OGD, Glu, or H2O2 insult. Brain slices were incubated with 5 30
min prior to and during OGD (A), 1 mM glutamate (B), or 2 mM H2O2 (C) applications.
The viability of cortical and hippocampal slices was evaluated by using TTC staining
##
*
method. (n = 6). Data are mean ± SD. P < 0.01 vs control group. P < 0.05, ** P < 0.01
vs injury group.
A
120
100
80
60
40
20
0
cortical slices
hippocampal slices
**
**
**
**
**
**
*
*
*
*
##
##
control
0
1
5
10
20
ketamine(10)
Concentration of agents (uM)
OGD
B
cortical slices
120
hippocampal slices
100
80
60
40
20
0
*
*
##
##
control
0
1
5
10
20
MK-801
Concentration of agents (uM)
glutamate (1 mM)
C
cortical slices
120
100
80
60
40
20
0
hippocampal slices
**
**
*
**
*
*
*
##
##
control
0
1
5
10
20
MK-801
Concentration of agents (uM)
H2O2 (2 mM)

Molecules 2009, 14
3. Experimental
3.1. General
4057
Chemicals from Sinopharm Chemical Reagent Co. were used without further purification. Melting
points were determined on a WRS-1A melting point apparatus and are uncorrected. Optical rotations
were measured at 20±2°C with a Perkin-Elmer 241MC polarimeter. The progress of reactions was
monitored by silica gel GF254 TLC plates. Column chromatography was performed on silica-gel 60
1
13
(100-200 mesh ASTM). H- and C-NMR spectra were recorded at 400 and 100 MHz, respectively,
on a Bruker AM-400 spectrometer in chloroform-d solutions (unless stated otherwise), with TMS as
internal standard. Chemical shifts were reported in ppm (δ) downfield from TMS. All the coupling
constants (J) are in hertz. Mass spectra were recorded on an Agilent 1100 LC/MSD mass spectrometer.
All reported yields correspond to yields of purified products.
3.2. Preparation of N-stearoylamino acids and their derivatives
3.2.1. Synthesis and spectroscopic data of N-stearoylamino acids 1-11
Dicyclohexylcarbodiimide (DCC, 9 g, 43.6 mmol) was added to a solution of stearic acid (10 g,
35.2 mmol) and N-hydroxysuccinimide (6 g, 52.1 mmol) in THF (80 mL) and the mixture was cooled
in an ice-bath under stirring for 16 h [28]. After filtration, evaporation and recrystallization in
isopropanol, octadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester was obtained with a yield of 90%, m.p.
76~78 °C. To a solution of the appropriate amino acid (5.5 mmol) and NaHCO3 (15 mmol) in water
(20 mL) was added dropwise octadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (5 mmol) in THF
(20 mL). The mixture was stirring for 6 h at room temperature. The solvent was removed under
reduced pressure. The residue was dissolved in water and adjusted with HCl (1 M) to pH 3, and the
solution was extracted with ethyl acetate (2 times × 50 mL). The combined organic extracts were
successively washed with water and saturated NaCl. Then the organic phase was dried over anhydr.
Na2SO4 and evaporated to dryness. Compounds 1-6 and 8 were obtained through recrystallization from
n-hexane, Compounds 10 and 11 from chloroform and 7 and 9 were purified by column
chromatography over silica gel (eleuent: 40:1 EtOAc-CH3OH).
N-Stearoyl-L-phenylalanine (1). Yield 80%; white solid; m.p. 66-68 °C; [α] 21 : +20.9° (c 0.92, CHCl3);
D
1H-NMR: δ (ppm) 7.27 (m, 3H of Ph), 7.15 (d, J = 6.6 Hz, 2H of Ph), 6.07 (d, 1H, J = 7.3 Hz, NH),
4.90 (t, 1H, J = 6.0 Hz, CHN), 3.13-3.23 (m, 2H, CH2Ph), 2.17 (t, 2H, J = 4 Hz, CH2CON), 1.55 (m,
13
2H, CH2), 1.25 (m, 28H, 14 × CH2), 0.88 (t, 3H, J =6.9 Hz, CH3); C-NMR: δ (ppm) 174.6, 174.1
(2C, acid and amide C=O), 135.7 (i-C of Ph), 129.3 (2 × o-CH of Ph), 128.6 (2 × m-CH of Ph), 127.2
(p-CH of Ph), 53.1 (CHN), 37.3 (CH2Ph), 36.4 (CH2-amide), 33.3(CH2), 31.9 (CH2), 29.7-29.2 (12 ×
CH2), 25.5 (CH2), 22.6 (CH2), 14.1 (CH3); ESI-MS: [M-1]+ m/z 430.1.
N-Stearoyl-DL-phenylalanine (2). Yield 67%; white solid; m.p. 93-95 °C; [α] 21 : 0° (c 0.92, CHCl3);
D
1H-NMR and 13C-NMR spectra were identical to those of 1; ESI-MS: [M-1]+ m/z 430.1.

Molecules 2009, 14
4058
1
N-Stearoyl-L-proline (3). Yield 50%; white solid; m.p. 87-89 °C; [α] 22 : +32.1° (c 1.0, CHCl3); H-
D
NMR: δ (ppm) 4.81-4.66 (m, 5H of pyrrolidine), 3.22 (m, 2H of pyrrolidine), 2.13 (t, 2H, J = 3.5 Hz,
13
CH2CON), 1.55 (m, 2H, CH2), 1.23 (m, 28H, 14 × CH2), 0.89 (t, 3H, J = 7 Hz, CH3); C-NMR: δ
(ppm) 174.7, 174.2 (2 × C=O, acid and amide), 61.7 (N-CH of pyrrolidine), 53.4 (N-CH2 of
pyrrolidine), 37.3(CH2), 36.4 (CH2-amide), 31.9 (CH2), 29.7-29.1(13 × CH2), 25.5 (CH2-pyrrolidine),
22.6 (CH2-pyrrolidine), 14.1 (CH3); ESI-MS: [M+Na]+ m/z 404.2.
N-Stearoyl-L-tyrosine (4). Yield 50%; white solid; m.p. 134-136 °C; [α]18 : +41.7° (c 1.0, CH3OH); 1H-
D
NMR: δ (ppm) 6.99 (d, J = 10.4 Hz, 2H of Ph), 6.73 (d, J = 10.2 Hz, 2H of Ph), 5.94 (s, 1H, NH), 4.82
(t, 1H, J = 6.0 Hz, CHN), 3.1 (m, 2H, CH2Ph), 2.17 (t, 2H, J = 7.8 Hz, CH2CON), 1.56 (m, 2H, CH2),
13
1.26 (m, 28H, 14 × CH2), 0.88 (t, 3H, J = 6.8 Hz, CH3); C-NMR: δ (ppm) 174.1,173.9 (2 × C=O,
acid and amide), 155.3 (C-OH of Ph), 133.5(i-C of Ph),130.4 (2 × o-CH of Ph), 115.8 (2 × m-CH of
Ph), 53.4 (CHN), 36.8 (CH2Ph), 36.5 (CH2-amide), 31.9 (CH2), 31.5 (CH2), 29.7-29.2 (12 × CH2), 25.6
(CH2), 22.6 (CH2), 14.0 (CH3); ESI-MS: [M+1]+ m/z 448.2.
1
N-stearoyl-L-serine (5). Yield 60%; white solid; m.p. 100-102 °C; [α]19 : +21.2° (c 1.0, CH3OH); H-
D
NMR (CD3OD): δ (ppm) 4.48 (t, 1H, J = 4.4 Hz, N-CH), 3.86 (m, 2H, CH2O), 2.26 (t, 2H, J = 7.4 Hz,
13
CH2CON), 1.62 (m, 2H, CH2), 1.28 (m, 28H, 14 × CH2), 0.89 (t, 3H, J = 6.8 Hz, CH3); C-NMR
(CD3OD): δ (ppm) 176.7, 173.8 (2 × C=O, acid and amide), 63.3 (CH2O), 56.3 (CHN), 37.2 (CH2-
amide), 33.3 (CH2), 31.1-30.6 (12 × CH2), 27.1 (CH2), 24.0 (CH2), 14.7 (CH3); ESI-MS: [M+1]+ m/z
372.1.
N-Stearoyl-L-threonine (6). Yield 56%; white solid; m.p. 62-64 °C; [α] 22 : +12.5° (c 0.92, CH3OH);
D
1H-NMR (CD3OD): δ (ppm) 6.8 (s, 1H, NH), 4.51 (d, 1H, J = 7.8 Hz, CHN), 4.43 (s, 1H, CHO), 2.3 (t,
2H, J = 7.4 Hz, CH2CON), 1.63 (d, 2H, J = 6.7 Hz, CH2), 1.25 (m, 28H, 14 × CH2), 1.21 (d, 3H, J =
6.0 Hz, CH3), 0.88 (dd, 3H, J1 = 6.7, J2 = 7.0 Hz, CH3); 13C-NMR (CD3OD): δ (ppm) 177.2, 174.1 (2 ×
C=O , acid and amide), 66.5 (CHO), 59.6 (CHN), 36.9 (CH2-amide), 34.2 (CH2), 30.3-29.6 (12 ×
CH2), 26.1 (CH2), 23.3 (CH2), 17.5 (CH3), 14.1 (CH3); ESI-MS: [M+1]+ m/z 389.2.
N-Stearoyl-L-tryptophan (7). Yield 30%; brown solid; m.p. 88-90 °C; [α] 20 : +28° (c 1.0, CHCl3);
D
1HNMR: δ (ppm) 8.37 (s, 1H, NH of indole), 7.54 (d, 1H, J = 7.8 Hz, N-CH of indole), 7.3-6.96 (m,
4H, Ph-H of indole), 6.14 (d, 1H, J = 7.6 Hz, NH), 4.93 (m, 1H, CHN), 3.32 (m, 2H, CH2-indole), 2.07
(t, 2H, J = 7.4 Hz, CH2CON), 1.48 (m, 2H, CH2), 1.25 (m, 28H, 14 × CH2), 0.88 (t, 3H, J = 7 Hz,
13
CH3); CNMR: δ (ppm) 175.1, 174.3 (2 × C=O , acid and amide), 136.1 (N-C of indole)，127.8 (C,
indole)，123.2 (N-CH of indole)，122.2，119.7 (2 × CH, Ph of indole)，118.4 (C of indole)，111.4,
109.5 (2 × CH, Ph of indole), 53.3 (CHN), 36.4(CH2-amide), 31.9 (CH2-indole), 29.7-27.1 (13 × CH2),
25.4 (CH2), 22.6 (CH2), 14.1 (CH3)；ESI-MS: [M+1]+ m/z 471.2.
1
N-Stearoyl-L-leucine (8). Yield 49%; white solid; m.p. 95-97 °C; [α] 20 : −10.3° (c 1.0, CHCl3); H-
D
NMR: δ (ppm) 5.9 (m, 1H, NH), 4.62 (m, 1H, CHN), 2.23 (t, 2H, J = 7.5 Hz, CH2CON), 1.72 (m, 2H,
CH2), 1.62 (m, 2H, CH2), 1.60 (m, 1H, CH), 1.27 (m, 28H, 14 × CH2), 0.95 (d, 6H, J = 4.3 Hz,
2×CH3), 0.88 (t, 3H, J = 7 Hz, CH3); 13C-NMR: δ (ppm) 176.1, 174.1 (2 × C=O, acid and amide), 50.9

Molecules 2009, 14
4059
(CHN), 36.5 (CH2-amide), 31.9 (CH2), 29.7-29.2 (13 × CH2), 25.6 (CH2), 24.9 (CH2), 22.8 (CH), 22.6
(CH3), 21.9 (CH3), 14.1 (CH3); ESI-MS: [M-1]+ m/z 396.1.
N-Stearoyl-L-cysteine (9). Yield 30%; white solid; m.p. 146-148 °C; [α] 20 : +35.4° (c 1.0, CH3OH);
D
1HNMR (CD3OD): δ (ppm) 4.67 (m, 1H, CHN), 3.63 (m, 2H, CH2S), 2.25 (t, 2H, J = 7.2 Hz,
13
CH2CON), 1.62 (m, 2H, CH2), 1.28 (m, 28H, 14 × CH2), 0.89 (t, 3H, J = 6.8 Hz, CH3); CNMR
(CD3OD): δ (ppm) 176.5, 173.8 (2 × C=O, acid and amide), 50.1 (CHN), 37.3 (CH2-amide), 33.3
(CH2S), 31.1-30.6 (13 × CH2), 27.2 (CH2), 24.0 (CH2), 14.7 (CH3); ESI-MS: [M+1]+ m/z 388.0.
N-Stearoyl-L-histidine (10). Yield 56%; white solid; m.p. 127-129 °C; [α] 22 : +5.9° (c 1.0, CH3OH);
D
1H-NMR (CD3OD): δ (ppm) 8.46, 7.12 (m, 2H of imidazole)，4.5 (m, 1H, CHN), 3.04-3.22 (m, 2H,
CH2-imidazole), 2.19 (t, 2H, J = 6 Hz, CH2CON), 1.51 (m, 2H, CH2), 1.28 (m, 28H, 14 × CH2), 0.89
(t, 3H, J = 6.9 Hz, CH3). 13C-NMR (CD3OD): δ (ppm) 176.1, 174.9 (2 × C=O, acid and amide), 135.1
(N-CH of imidazole), 133.1 (N-C of imidazole), 118.5 (NH-CH of imidazole), 54.6 (CHN), 37.4 (CH2-
amide), 33.4 (CH2-imidazole), 31.1-29.7 (13 × CH2), 27.2 (CH2), 24.0 (CH2), 14.7 (CH3); ESI-MS:
[M+1]+ m/z 422.1.
1
N-Stearoyl-L-lysine (11). Yield 50%; white solid; m.p. 85-87 °C; [α] 20 : −28.3° (c 1.0, CH3OH); H-
D
NMR (CD3OD): δ (ppm) 4.19 (m, 1H, CHN), 3.17 (m, 2H, CH2N), 2.51 (m, 2H, CH2CON), 1.71 (m,
2H, CH2), 1.56 (2 × CH2), 1.28 (m, 30H, 15 × CH2), 0.89 (t, 3H, J = 6.8 Hz, CH3); 13C-NMR
(CD3OD): δ (ppm) 170.3, 168.8 (2 × C=O, acid and amide), 48.3 (CHN), 31.6 (CH2NH2), 30.1 (CH2-
amide), 29.2-28.7 (15 × CH2), 28.3 (CH2), 24.5 (CH2), 22.2 (CH2), 14.0 (CH3); ESI-MS: [M-1]+ m/z
411.2.
3.2.2. Synthesis and spectroscopic data of N-stearoylamino acid methyl esters 12-14
A mixture of the appropriate amino acid methyl ester (6.3 mmol) and octadecanoic acid 2,5-dioxo-
pyrrolidin-1-yl ester (7 mmol) in dry pyridine (50 mL) was stirred at room temperature for 24 h. After
evaporation of solvent, the residue was dissolved in dichloromethane (100 mL), and washed with HCl
(1 M, 100 mL × 2), water (100 mL), 5% NaHCO3 (100 mL) and saturated NaCl (100 mL × 2). The
organic layer was dried over anhydr. Na2SO4. After filtration and evaporation of the solvent in vacuo,
the crude product was purified by recrystallization from methanol-chloroform.
N-Stearoylglycine methyl ester (12). Yield 24%; white solid; m.p. 82-84 °C; [α] 20 : 0° (c 1.0, CHCl3);
D
1H-NMR: δ (ppm) 5.97 (m, 1H, NH), 4.04 (d, 2H, J = 5.1 Hz, CH2N), 3.75 (s, 3H, CH3O), 2.23 (t, 2H,
J = 7.5 Hz, CH2CON), 1.62 (m, 2H, CH2), 1.26 (m, 28H, 14 × CH2), 0.87 (t, 3H, J = 7 Hz, CH3); 13C-
NMR: δ (ppm) 173.4, 170.7 (2 × C=O , acid methyl ester and amide), 52.4 (CH3O), 41.3 (CH2N), 36.5
(CH2-amide), 31.9 (CH2), 29.7-29.3 (12 × CH2), 25.6 (CH2), 22.8 (CH2), 14.1 (CH3); ESI-MS: [M+1]+
m/z 356.1
N-Stearoyl-L-glutamic acid dimethyl ester (13). Yield 23%; white solid; m.p. 85-87 °C; [α]1D9 : +18° (c
1.2, CHCl3); 1H-NMR: δ (ppm) 6.15 (d, 1H, J = 7.4 Hz, NH), 4.64 (m, 1H, CHN), 3.77, 3.68 (s, 6H, 2
× CH3O), 2.39 (m, 2H, CH2), 2.21 (t, 2H, J = 7.4 Hz, CH2COO), 2.11 (m, 2H, CH2CON), 1.62 (t, 2H,

Molecules 2009, 14
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CH2), 1.28 (m, 28H, 14 × CH2), 0.88 (t, 3H, J =6.8 Hz, CH3); 13C-NMR: δ (ppm) 173.3, 173.1, 172.5
(3 × C=O, acid methyl ester and amide), 52.5 (CHN), 51.8, 51.5 (2 × CH3O), 36.6 (CH2-amide), 31.9
(CH2-acid), 30.1 (CH2), 29.7-29.2 (12 × CH2), 27.4 (CH2), 25.6 (CH2), 22.7 (CH2), 14.1 (CH3); ESI-
MS: [M+H]+ m/z 442.2.
N-Stearoyl-L-phenylalanine methyl ester (14). Yield 30%; grey solid; m.p. 70-72 °C; [α] 2D2 : −16.7° (c
1.0, CHCl3); 1H-NMR: δ (ppm) 7.28 (m, 3H of Ph), 7.12 (dd, J1 = 1.5, J2 = 6.7 Hz, 2H of Ph), 5.89 (d,
1H, J =7.6 Hz, NH), 4.92 (m, 1H, CHN), 3.75 (s, 3H, CH3O), 3.13 (m, 2H, CH2Ph), 2.18 (t, 2H, J =
13
6.8 Hz, CH2CON), 1.63 (m, 2H, CH2), 1.29 (m, 28H, 14 × CH2), 0.90 (t, 3H, J =7 Hz, CH3); C-
NMR: δ (ppm) 173.7, 172.3 (2 × C=O , acid methyl ester and amide), 137.6 (i-C of Ph), 129.2 (2 × o-
CH of Ph), 128.6 (2 × m-CH of Ph), 126.6 (p-CH of Ph), 64.3 (CHN), 52.8 (CH3O), 37.0 (CH2Ph),
36.8 (CH2-amide), 31.9 (CH2), 29.7-29.1 (12× CH2), 25.7 (CH2), 22.7 (CH2), 14.2 (CH3); ESI-MS:
[M+1]+ m/z 446.3.
3.2.3. Synthesis and spectroscopic data of N-palmitoyl-L-tyrosine (15)
Hexadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester was synthesized following the synthetic route of
octadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester from palmitic acid instead of stearic acid. Then 15
was prepared from hexadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester instead of octadecanoic acid 2,5-
dioxo-pyrrolidin-1-yl ester by the same procedure of 1-11. Yield 45%; white solid; recrystallization
from n-hexane; m.p. 115-117 °C; [α] 22 : +24.2° (c 1.0, CH3OH); 1H-NMR (CD3OD): δ (ppm) 7.03 (m,
D
2H of Ph), 6.73 (m, 2H of Ph), 4.62 (m, 1H, CHN), 2.8-3.09 (m, 2H, CH2Ph), 2.15 (t, 2H, J = 7.5 Hz,
13
CH2CON), 1.48 (m, 2H, CH2), 1.28 (m, 24H, 12× CH2), 0.89 (t, 3H, J = 7 Hz, CH3); C-NMR
(CD3OD): δ (ppm) 176.4, 175.3 (2 × C=O , acid and amide), 157.6 (C-OH of Ph), 131.5 (i-C of Ph),
129.4, 122.8 (2 × o-CH of Ph), 116.6 (2 × m-CH of Ph), 55.4 (CHN), 37.9 (CH2Ph), 37.1 (CH2-amide),
33.4 (CH2), 31.1-30.4 (10 × CH2), 27.2 (CH2), 24.0 (CH2), 14.7 (CH3); ESI-MS: [M+1]+ m/z 420.1.
3.2.4. Synthesis and spectroscopic data of N-lauroyl-L-tyrosine (16)
Dodecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester was synthesized following the synthetic route of
octadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester from lauric acid instead of stearic acid. Then 16 was
prepared from dodecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester instead of octadecanoic acid 2,5-dioxo-
pyrrolidin-1-yl ester by the same procedure of 1-11. Yield 50%; white solid; recrystallization from n-
1
hexane; m.p. 95-97 °C; [α] 22 : +17.4° (c 1.0, CH3OH); H-NMR (CD3OD): δ (ppm) 6.99 (m, 2H of
D
Ph), 6.73 (m, 2H of Ph), 4.62 (m, 1H, CHN), 2.8-3.09 (m, 2H, CH2Ph), 2.14 (t, 2H, J = 7.5 Hz,
13
CH2CON), 1.48 (m, 2H, CH2), 1.27 (m, 16H, 8 × CH2), 0.88 (t, 3H, J = 6.8 Hz, CH3); C-NMR
(CD3OD): δ (ppm) 176.6, 175.4 (2C, acid and amide C=O), 157.8 (C-OH of Ph), 131.7 (i-C of Ph),
129.6, 123.1 (2 × o-CH of Ph), 116.6 (2 × m-CH of Ph), 55.6 (CHN), 38.2 (CH2Ph), 37.3 (CH2-amide),
33.6 (CH2), 31.3-30.6 (6 × CH2), 27.3 (CH2), 24.2 (CH2), 14.9 (CH3); ESI-MS: [M+H]+ m/z 364.0.

Molecules 2009, 14
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3.2.5. Synthesis and spectroscopic data of N-stearoyl-L-tyrosinol (17)
To L-tyyrosine (1.81 g, 10 mmol) and NaBH4 (0.91 g, 24 mmol) dissolved in THF (50 ml) were
added dropwise I2 (2.54 g, 10 mmol) in THF (7.5 mL) during 10 min. Then, the mixture was refluxed
for 5 h. It was then cooled to room temperature, and MeOH (5 mL) was added. The solvent was
evaporated and the residue dissolved in 2 M HCl (10 mL). After removal of the solvent in vacuo, the
residue was then suspended in EtOH (30 mL) at 40 °C and the suspension filtered. The filtrate was
concentrated until crystals appear and was kept at 5 °C overnight. The resulting crystals were isolated
by filtration, washed with EtOH, and dried in vacuo to yield 45% of L-tyrosinol hydriodide as colorless
crystals: m.p. 214-216 °C. Then 17 was prepared from L-tyrosinol hydriodide instead of L-tyrosine by
the same procedure of 1-11. Yield 30%; white solid; recrystallization from n-hexane; m.p. 121-123 °C;
[α] 22 : +27.1° (c 1.0, CH3OH); 1H-NMR: δ (ppm) 7.28 (m, 2H of Ph), 7.22 (m, 2H of Ph), 5.78 (d, 1H,
D
J = 7.6 Hz, NH), 4.17 (m, 1H, CHN), 3.66-3.58 (m, 2H, CH2O), 2.86 (m, 2H, CH2Ph), 2.12 (t, 2H, J =
13
7.5 Hz, CH2CON), 1.53 (m, 2H, CH2), 1.25 (m, 28H, 14 × CH2), 0.88 (t, 3H, J = 7.1 Hz, CH3); C-
NMR: δ (ppm) 173.9 (CONH), 155.6 (C-OH of Ph), 137.7 (i-C of Ph), 129.1, 128.6 (2 × o-CH of Ph),
126.6 (2 × m-CH of Ph), 63.3 (CH2OH), 52.8 (CHN), 37.0 (CH2Ph), 36.8 (CH2-amide), 31.9 (CH2),
29.7-29.1 (12× CH2), 25.7 (CH2), 22.7 (CH2), 14.1 (CH3); ESI-MS: [M+H]+ m/z 434.3.
3.3. Neuroprotective effects of the target compounds on rat brain slices in vitro injury models
The slices were prepared as described by Bickler with modification [26]. Whole brain from male
Sprague-Dawley rats (about 100 g ± 50 g) were obtained after decapitation and quickly removed to
ice-cold oxygenated (95 % O2/5 % CO2) normal artificial cerebrospinal fluid (ACSF), which had the
following composition (in mM): NaCl 119, KCl 2.5, CaCl2 2, MgSO4 1, NaH2PO4 1.25, NaHCO3 26.2,
glucose10 (final pH 7.4). Brain slices (400 μm thick) were prepared using a vibrating tissue slicer
(ZQP-86, Xiangshan, Zhejiang, China), then transferred to a ‘slice saver’ containing continuously-
oxygenated normal ACSF at 32-34 °C for 90 min recover. After that, brain slices were transferred to
experimental chambers and randomly assigned to one of following groups: control group, in which
slices were immersed in oxygenated ACSF at 34 °C; OGD group, in which slices were made anoxic by
switching the ACSF into the glucose-free ACSF equilibrated with 95% N2/5% CO2. After 10 min,
slices were reoxygenated in ACSF for 2 h; OGD + compound group, in which slices were incubated
with different 10 μM target compounds (or 1~20 μΜ compound 5) for 30 min prior to and during
OGD application; glutamate group, in which slices were subjected to 1 mM glutamate in magnesium-
free artificial cerebrospinal fluid for 15 min (which had the following composition [in mM]: NaCl 143,
KCl 5.4, CaCl2 1.8, NaH2PO4 1.0, HEPES 2.4, glucose 5.6 pH = 7.4). Slices were reoxygenated in
ACSF for 2 h; Glu + 5 group, in which slices were incubated with 1–20 μΜ 5 for 30 min prior to and
during Glu application; H2O2 group, in which slices were subjected to 2 mM H2O2 for 30 min;
H2O2 + 5 group, in which slices were incubated with different concentrations of 5 (1–20 μM) 30 min
prior to and during H2O2 application.
After insult, slices were immersed into 2% TTC solution at 37 °C for 30 min and the wet weight
was measured after rinsing twice with saline. A mixture of ethanol and dimethylsulfoxide (50:50) was
added at a rate of 20 mL per 1 g of slices. After 24 h extraction in dark, the extracted liquid was added

Molecules 2009, 14
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to 96-well plates (200 μL per well), the absorbance (A) at 490 nm of each well was measured by the
ELISA reader (Elx800uv, Bio-TEK). The viability of brain slices was evaluated from the following
equation: % tissue activities = 100% × (A injury / A control).
4. Conclusions
In summary, we have synthesized a series of N-stearoylamino acids and their analogues by a facile
route, and evaluated their activity against cerebral ischemia in vitro. Among these compounds, 4, 5 and
6 elicited higher activity in protecting rat brain slices against OGD insult. According to the preliminary
SAR study, the neuroprotective activity of these lipoamino acids might be related with the fatty-acyl
moieties of the tested compounds and the presence of a free carboxylic and hydroxyl groups on the
branched chain of the amino acids. The evaluation of neuroprotective effects of 5 with three models
implies that certain LAs could protect brain slices against OGD as well as H2O2 insult. Therefore, they
might serve as candidate for the amelioration of cerebral ischemia and other cerebral insults.
Acknowledgements
This study was supported by National Natural Science Foundation of China (NO. 30873057), Key
Basic Project of Shanghai Municipal Science and Technology Commission (No. JC1413600), Science
and Technology Development Foundation from Shanghai Municipal Health Bureau (NO: 2007085).
The authors express their appreciation to Li-Ping Shi of Shanghai Institute of Organic Chemistry for
her help in 1HNMR and 13CNMR measuring.
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Sample Availability: Samples of compounds 1–17 are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).