Synthesis, physicochemical characterization, and biological activities of new carnosine derivatives stable in human serum as potential neuroprotective agents

Article abstract of DOI:10.1021/jm101394n

The synthesis and the physicochemical and biological characterization of a series of carnosine amides bearing on the amido group alkyl substituents endowed with different lipophilicity are described. All synthesized products display carnosine-like properties differentiating from the lead for their high serum stability. They are able to complex Cu²⁺ ions at physiological pH with the same stoichiometry as carnosine. The newly synthesized compounds display highly significant copper ion sequestering ability and are capable of protecting LDL from oxidation catalyzed by Cu²⁺ ions, the most active compounds being the most hydrophilic ones. All the synthesized amides show quite potent carnosine-like HNE quenching activity; in particular, 7d, the member of the series selected for this kind of study, is able to cross the blood-brain barrier (BBB) and to protect primary mouse hippocampal neurons against HNE-induced death. These products can be considered metabolically stable analogues of carnosine and are worthy of additional investigation as potential neuroprotective agents.

Full text of DOI:10.1021/jm101394n

J. Med. Chem. 2011, 54, 611–621 611
DOI: 10.1021/jm101394n
Synthesis, Physicochemical Characterization, and Biological Activities of New Carnosine Derivatives
Stable in Human Serum As Potential Neuroprotective Agents
Massimo Bertinaria,† Barbara Rolando,† Marta Giorgis,† Gabriele Montanaro,† Stefano Guglielmo,†
M. Federica Buonsanti,†, Valentina Carabelli,‡ Daniela Gavello,‡ Pier Giuseppe Daniele,§ Roberta Fruttero,† and
Alberto Gasco*,†
†
ꢀ
Dipartimento di Scienza e Tecnologia del Farmaco, Universita degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy,
Dipartimento di Neuroscienze, Universita degli Studi di Torino, Corso Raffaello 30, 10125 Torino, Italy, and
‡
§
ꢀ
ꢀ
Dipartimento di Chimica Analitica, Universita degli Studi di Torino, Via Pietro Giuria 5, 10125 Torino, Italy.
Present address: CRB Bracco Imaging SpA, c/o Biondustry Park del Canavese, via Ribes 5, Colleretto Giacosa (TO) Italy.
Received October 27, 2010
The synthesis and the physicochemical and biological characterization of a series of carnosine amides
bearing on the amido group alkyl substituents endowed with different lipophilicity are described. All
synthesized products display carnosine-like properties differentiating from the lead for their high serum
stability. They are able to complex Cu2þ ions at physiological pH with the same stoichiometry as
carnosine. The newly synthesized compounds display highly significant copper ion sequestering ability
and are capable of protecting LDL from oxidation catalyzed by Cu2þ ions, the most active compounds
being the most hydrophilic ones. All the synthesized amides show quite potent carnosine-like HNE
quenching activity; in particular, 7d, the member of the series selected for this kind of study, is able to
cross the blood-brain barrier (BBB) and to protect primary mouse hippocampal neurons against HNE-
induced death. These products can be considered metabolically stable analogues of carnosine and are
worthy of additional investigation as potential neuroprotective agents.
Introduction
membranes. Another source of cellular damage is the glyca-
tion of proteins, namely the process whereby reducing sugars
react with protein amino groups generating Schiff’s bases.
These latter products, in turn, can afford advanced glycation
end products (AGEs) able to incorporate additional proteins
through stable cross-links.9 AGEs are toxic for those cells that
are able to recognize them. Carnosine is capable of protecting
proteins against glycation and of reacting with protein carbon-
yl groups to form carnosylated proteins.10-12 Because ROS,
RNS, lipidic oxidation, and glycation products may contri-
bute to the development of many neurodegenerative and
cardiovascular diseases, today there is a great interest in
carnosine and related structures as potential therapeutic tools.
It is known that carnosine is rapidly cleaved to its constit-
uents, histidine and β-alanine, by the carnosinases, which are
a group of ubiquitous dipeptidases belonging to the family of
metalloproteases. So far, two principal isoforms of carnosi-
nase are known, one is a cytosolic form (EC 3.4.13.3) and the
other (EC 3.4.13.20) is known as serum carnosinase because it
is mostly present in the plasma and brain.13,14 The facility of
enzymatic hydrolysis of carnosine and of many related com-
pounds limits their therapeutic potential. The nature of the
metal ion in serum carnosinase is not known with precision,
but it is likely that two Zn2þ ions are present in the catalytic
site. A recent computational study showsthat oneof these two
ions is important in recognizing the carboxylic group of
carnosine.15 If this interaction is lost, it is reasonable to think
that the affinity for the enzyme is strongly reduced. By
contrast, the carboxylic group of carnosine should be less
involved in recognition of the binding site of hPepT1, which is
Carnosine, β-alanyl-L-histidine (1) (Chart 1), is a naturally
occurring dipeptide that in humans is preferentially localized
in skeletal muscles and brain.1 Carnosine can display a variety
of physiological roles, including that of a cytosolic buffering
agent, that of a regulator of content of transition metal ions in
biological fluids and tissues, owing to its ability to form
complexes with these ions, and that of a regulator of macro-
phage function.2-4 It is a potent scavenger of both reactive
oxygen species (ROSa) and reactive nitrogen species (RNS),
which induce peroxidation of unsaturated lipids present in
membranes as well as of toxic reactive R,β-unsaturated alde-
hydes deriving from this oxidation.1,5-8 Acrolein, 4-hydroxy-
trans-2,3-nonenal (HNE), and malondialdehyde are examples
of such products. They are potent bifunctional electrophiles
able to bind nucleophilic centers present in both DNA and
proteins. In addition, the second electrophilic center may
undergo additional attack, affording protein-protein and
DNA-protein cross-linking. The final result is an amplifica-
tion of the cellular damage induced by oxidative attack on
*To whom correspondence should be addressed. Phone: þ39 011
6707670. Fax: þ39 011 6707286. E-mail: alberto.gasco@unito.it.
a Abbreviations: AGEs, advanced glycation end products; HNE,
4-hydroxy-trans-2,3-nonenal; RCS, reactive carbonyl species; ROS,
reactive oxygen species; RNS, reactive nitrogen species; LDL, low
density lipoprotein; HBTU, O-Benzotriazol-1-yl-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole;
DIPEA, di-isopropylethylamine; DMF, dimethylformamide; DCC,
dicyclohexylcarbodiimide; TBARS, thiobarbituric acid reactive sub-
stances; hPepT1, human PepT1 transporter.
r
2010 American Chemical Society
Published on Web 12/23/2010
pubs.acs.org/jmc

612 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Chart 1. β-Alanyl-L-histidine (Carnosine)
Bertinaria et al.
Scheme 2. Preparation of Carnosine Ethylether 12a
Scheme 1. Preparation of Carnosine Carbonamides 7a-na
a Reagents and conditions: (a) NaH, dry DMF, 1 h, rt, then EtBr 18 h, rt;
(b) CF3COOH 1% in CH2Cl2, 0 °C, 2 min; (c) Boc-β-Ala(OH), DCC,
dry CH2Cl2, 0 °C to rt, 18 h; (d) CF3COOH 10% in CH2Cl2, 20 h, rt.
Table 1. Dissociation Constants and Lipophilicity Parameters for
Carnosine, 7a-n, and 12
a
a
a
compd
carnosined
pKa1
pKa2
pKa3
Clog D7.4b
Log D7.4c
2.60
6.79
6.17
6.18
6.17
6.18
6.18
6.21
6.21
9.42
9.16
9.19
9.16
9.19
9.19
9.18
9.18
ND
ND
ND
7a
7b
7c
7d
7e
7f
-4.68
-4.35
-3.81
-3.30
-2.78
-1.72
-0.66
0.39
ND
ND
-2.72
-2.01
-0.88
-0.23
0.85
7g
7h
7i
6.17e
6.16e
6.19
6.22
6.19
6.60
9.21e
9.20e
9.20
9.19
9.20
9.18
1.46
0.93
7l
-1.73
-2.49
-0.99
-2.88
-0.94
-1.76
-0.13
-2.17
7m
7n
12
a Determined by potentiometric titration with GLpKa apparatus;
n g 4, SD < 0.03. b Calculated according to the equation Clog D7.4
=
a Reagents and conditions: (a) DIPEA, HBTU, HOBtcat, 10 min rt,
then 3b-n 1-18 h, rt; (b) DIPEA, HBTU, HOBtcat, 10 min rt then NH3 1
h, rt; (c) piperidine, dry DMF, 1 h, rt; (d) Boc-β-Ala(OH), DCC, dry
CH2Cl2, 0 °C to rt 2-18 h; (e) CF3COOH 10% in CH2Cl2, 20 h, rt.
3
3
2
CLOGP - log [1 þ 10(pKa -pH) þ 10(pKa -pKa -2pH)]; CLOGP for
windows, v.1.0 Biobyte Corp., Claremont, CA, USA. c Determined by
shake-flask technique; n g 6, SD <0.2. d Reported values are in
agreement with those reported in literature28 (pKa1 = 2.59, pKa2
=
6.77, pKa3 = 9.37). e Data obtained with 20-34% methanol as cosol-
vent; aqueous pKa values were obtained by extrapolation at 0% metha-
nol using the Yasuda-Shedlovsky procedure.51 ND = not determined.
the main intestinal transporter involved in the absorption of
both dietary peptides and peptidomimetics.16
On these bases, we designed a series of carnosine carbon-
amide derivatives bearing on the amide nitrogen alkyl or aryl
groups characterized by different lipophilicity (compounds
7a-n). This paper reports synthesis, dissociation constants
and lipophilicity determination, the ability to complex Cu2þ
ions, andthe stabilityin humanserum of theseproducts. Their
capacitytoinhibit human LDLautoxidationinduced byCu2þ
and to sequester HNE, chosen as a model of reactive carbonyl
species (RCS), is also considered. Finally, the ability to cross
the BBB in an in vivo ratmodeland the cytoprotectiveefficacy
on primary mouse hippocampal neurons is shown in the case
of 7d, one of the most promising members of the series.
(Boc-β-Ala(OH)) using dicyclohexylcarbodiimide (DCC) as
the coupling agent and then deprotected using 10% (v/v)
CF3COOH in CH2Cl2 at rt to afford the expected ditrifluoro-
acetates 7a-n. In Scheme 2, the synthetic pathway followed
for the preparation of the ether 12 that was used for a
comparison in the Cu2þ complexing studies is reported.
Ditrityl protected alcohol 817 underwent Williamson ether-
ification with ethylbromide in the presence of an excess of
NaH in dry DMF, giving intermediate 9. Selective detrityla-
tion of this product carried out using 1% (v/v) CF3COOH in
CH2Cl2 at 0 °C afforded the expected free amine 10. This
intermediate was coupled with Boc-β-Ala(OH), by the usual
DDC-mediated procedure, obtaining 11. Removal of the
protecting groups was achieved with 10% (v/v) CF3COOH
in CH2Cl2 to afford the target compound 12.
Dissociation Constants (pKas) and Lipophilicity. Dissocia-
tion constants (pKas) of the new carnosine carbonamides and
of ether derivative 12 were determined by potentiometric
titration in aqueous solution using a GLpKa apparatus.
Their values are collected in Table 1, with those of carnosine
measured in the same conditions. For solubility reasons, in
the case of compounds 7h and 7i, the measurements were
carried out using methanol as a cosolvent and the results
were extrapolated to zero cosolvent. As expected, pKa values
of imidazole rings lie in a very narrow range, just a little lower
than the value of the carnosine imidazole ring. The values of
the dissociation constants of the basic center in the lateral
Results and Discussion
Chemistry. The synthetic pathway used to prepare com-
pounds 7a-n is reported in Scheme 1. The commercially
available NR-fluorenylmethoxycarbonyl-N-trityl-L-histidine
(Fmoc-His(Trt)-OH) (2) was coupled with the appropriate
amines 3b-n using the O-benzotriazol-1-yl-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate (HBTU)/1-hydroxy-
benzotriazole (HOBt)/di-isopropylethylamine (DIPEA)
protocol to obtain the corresponding intermediate amides
4b-n. Fmoc deprotection with piperidine in DMF was
achieved at room temperature (rt) to afford the expected
amines 5b-n. The simple amido derivative 5a was obtained
directly by action of ammonia on 2. Free amines so obtained
were coupled with tert-butoxycarbonyl protected β-alanine

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 613
chain are the same, within the experimental errors. At
physiological pH (7.4), all the products exist prevalently as
a mixture of monocharged and dicharged cations, unlike
carnosine, which is a mixture of a zwitterion and a tricharged
ion (þþ-).
Copper Complexes. Carnosine is able to form complexes
with a number of metals. This property has been widely
studied, and the complexes with Cu2þ have received parti-
cular attention in view of their physiological roles.27,28 At
neutral pH values at 35 °C, when the molar concentrations of
carnosine and Cu2þ are equal, the predominant complex is
represented by the neutral dimeric species [Cu2L2H2]0. A
structure in solution related to the structure found in the
solid state has been proposed for this complex, namely two
Cu2þ ions, each bound to an amino group, dissociated amido
function, N3 imidazole nitrogen, and carboxylate oxygen.29
Compounds 7a, 7d, 7g, 7l, and 7m were chosen as represen-
tative members of the structural variety of carnosine amides
here described to investigate the capacity of complexing
Cu2þ of the series. Compound 12 and carnosine were also
studied for comparison. The complex formation between
copper(II) and the different carnosine derivatives has been
investigated by means of the classical pH-metric technique.
The stability constants of complexes have been expressed by
The lipophilicity studies were carried out by shake-flask
technique using as partition pair buffered water (pH 7.4)/n-
octanol. The obtained distribution coefficients (log D7.4) are
reported in Table 1. The very high hydrophilicity of the
compounds 7a-c prevented the direct measurement of their
log D7.4. Consequently, these molecular descriptors were
calculated from the related pKa and CLOGP values
(CLOGP for windows, v.1.0 Biobyte Corp., Claremont,
CA, USA). As shown in Table 1, the lipophilicity of the
series is modulated over a very large range (about 5.5 log
units). As expected, it increases in the aliphatic series with the
length of the chain: the simple amide 7a is the most hydrophilic
member and its dodecyl derivative 7i the most lipophilic.
Stability in Human Serum. As aforementioned, carnosine
is rapidly hydrolyzed by carnosinases. Therefore, one of the
aspects that must be addressed when working with potential
drugs structurally related to carnosine is their metabolic
stability. A number of structural modifications of carnosine
were carried out in order to obtain products with higher
stability in biological media. The most common approach to
obtain carnosinase-stable derivatives consisted in the re-
moval of the carboxylic group from carnosine structure,
thus obtaining β-alanylhistamine (carcinine). Carcinine
and some of its N-substituted derivatives proved stable to
enzymatic hydrolysis and were endowed with free radical
scavenging ability.18,19 Other modifications have regarded
the peptide bond present in the parent molecule which was
transformed into a sulfonamide, affording derivatives stable
to carnosinase activity.20 Acetylation on primary amine
afforded the prodrug derivative N-acetylcarnosine, which
proved more stable to carnosinase activity than parent
L-carnosine,13,19 moreover, substitution of the β-alanyl moiety
with 2,3-diamino propionic acid, with different degrees of
N-acetylation, afforded carnosinase stable compounds which
were able to exert protective action against hydroxyl radical
and peroxynitrite anion.21 The inversion of configuration of
the COOH group switching from L- to D-carnosine proved a
successful approach. D-Carnosine and some synthesized
derivatives were stable to hydrolysis in human serum and
shared some common features with natural L-carnosine; in
particular, D-carnosine was able to inhibit R-crystallin fibril-
lation and of disassembling R-crystallin amyloid fibrils.22
Some D-carnosine analogues showed efficient sequester-
ing of RCS.23 Surprisingly enough, only a few approaches
involving carnosine COOH group manipulation have been
reported so far. In particular, amide formation with
β-cyclodextrin affording carnosinase stable macromolecules
endowed with antioxidant properties in different biological
media was reported.24,25 Other fluorinated amphiphilic al-
kylamides were synthesized and proposed as metal coordi-
nating agents, however, their stability against carnosinase
activity was not tested.26 Carnosine and the carnosine
amides 7a-n were studied by RP-HPLC for their stability
in human serum at 37 °C. Unlike the lead (t1/2 = 5 min), all
the products were completely stable over 3 h. This result is in
keeping with the previously mentioned hypothesis that the
carnosine carboxylic group binds a Zn2þ ion present in the
active cleft of the enzyme, so playing a paramount role in the
hydrolysis of the substrate.
β
pqr = [CupLqHr]/[Cu]p[L]q[H]r (for reaction pCu þ qL þ rH =
CupLqHr, where L is the carnosine derivative, p, q, and r
are the stoichiometric coefficients and charges are omitted
for sake of simplicity). It must be pointed out that for ligands
here studied, the dissociation of both peptide and amide
hydrogens takes place at pH values too high to allow a
reliable evaluation of the relative pKa in aqueous solution. As
a consequence, the concentration [L] in the expression of βpqr
takes into account only the hydrogen ion dissociation from
protonated imidazole and amino nitrogens. If the presence of
a metal ion promotes further dissociation of hydrogen ion
from peptide and amide group, in the above reaction, the
species Hþ is subtracted and the index r becomes negative.
Thus, for all the complexes for which dissociation takes place
from peptides and/or amide groups, the r index is negative.
The measurements were carried out at t = 25 °C and ionic
strength I = 0.15 M. The elaboration of experimental pH-
metric data, in order to calculate the values of formation
constants, has been performed by BSTAC program.30 The
results are listed in Table 2. From an inspection of these data,
it can be observed that for all derivatives in which the
carboxylate group has been transformed into amido or an
N-substituted amido group, the stoichiometry of complexes
is the same as for carnosine, but the stability is always
significantly lower. This indicates that the contribution of
carboxylate to the formation of the different species is more
relevant if compared to that of amido group; moreover, the
different N-substituents on amido group does not affect to a
great extent the stability of copper(II) complexes. The stabil-
ity of 11-1 complex (logβ11-1 = 0.16) for the ligand 12,
which does not contain an amido group, is further lowered
with respect to all the other ligands studied and, in addition,
12 does not form dimeric species, suggesting a participation,
however, less significant with respect to carboxylate, of an
amido group in the complex formation for all the above
ligands (a weak coordination by carbonyl in 22-2 species
and a more significant coordination by deprotonated amido
group in 22-3 or 22-4 species). It is very likely that the
structures of 22-2, 22-3, and 22-4 are similar to that
reported for carnosine.28
The stability constants of Table 2 allow us to calculate the
species distribution for the different complexes and the ratio
between the free and total copper(II) concentration. If
assuming [Cu]total = 2.5 ꢀ 10-6 M and [carnosine]total
=
1 ꢀ 10-4 M, the values of log([Cu]total/[Cu]free) at pH 7.4,

614 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Bertinaria et al.
Table 2. Formation Constants (log βpqr)a of Copper(II) Complexes for Carnosine and Derivatives 7a, 7d, 7g, 7l, 7m, 12, and Logarithm of the Ratio
between [Cu2þ
compd
]
total and [Cu2þ
]
free
b
log β111
log β110
log β11-1
log β22-2
log β22-3
log β22-4
log β120
log ([Cu2þ
]
tot /[Cu2þ
]
)
free
7a
7d
12.62(5)
12.22(4)
12.47(5)
12.71(4)
12.69(5)
13.58(4)
13.30c
6.82(3)
6.72(2)
6.59(3)
7.05(3)
7.01(3)
7.30(2)
8.47c
1.15(8)
1.25(7)
1.55(6)
1.53(7)
1.50(7)
0.16(5)
2.44c
5.09(3)
5.40(2)
5.52(2)
6.35(2)
6.20(2)
-2.82(4)
-3.09(5)
-2.46(4)
-2.41(4)
-2.84(4)
-12.23(5)
-12.67(6)
-11.68(5)
-12.32(5)
-12.74(6)
13.86(4)
13.47(4)
13.81(4)
13.27(4)
13.26(4)
13.30(4)
14.05d
2.89
2.93
3.19
3.00
3.09
1.95
3.80
7g
7l
7m
12
carnosine
8.35c
a In parentheses errors ((3σ) in the last significant figures. b Calculated for [Cu2þ
]
total = 2.5 ꢀ 10-6 M and [ligand]total = 1 ꢀ 10-4 M at pH 7.4.
c According to lit.28 d According to lit.53
Figure 1. Effect of carnosine and compounds 7a and 7h on kinetics of conjugated diens formation during copper-induced LDL oxidation. The
figure shows typical experimental kinetic profiles obtained by incubating the compounds (100 μM) at 37 °C with 50 μg mL-1 of LDL in PBS in
the presence of 2.5 μM CuSO4. Conjugated diene formation was assessed monitoring over 6 h the changes in absorbance at 234 nm.
range from 2.89 to 3.19 for all carnosine derivatives (Table 2).
These values, if compared with the value calculated for
carnosine (3.80), clearly indicate that, although it is weaker
than that in lead, the sequestering ability of all the above
autoxidation, initiated by the addition of 2.5 μM CuSO4, was
followed spectrophotometrically by detecting the formation of
conjugated dienes at 234 nm. Typical examples of such experi-
ments are reported in Figure 1 for carnosine, the simple amide
derivative 7a, and its n-decyl analogue 7h. According to
literature,38 three parameters were used to characterize the
kinetics of LDL oxidation: the maximal accumulation of
oxidation products (ODmax), the Δlag time (Δtlag), i.e. the
duration of the period prior to onset of rapid lipidic peroxida-
tion (propagation phase) compared to the control, and the
propagation rate of the oxidation (R). ODmax of all the amides
was the same as the control. R and Δtlag derived for each
compound from the corresponding curves are collected in
Table 3, together with those measured for carnosine and the
control. Analysis of the data indicates that the more hydro-
philic products 7a-f, l-n, bearing shorter alkyl substituent
groups or the cyclohexylmethyl and benzyl substituents, are
able to reduce R with respect to the control in a manner similar
to carnosine. By contrast, all the remaining more lipophilic
members of the series behave similarly to the control, with the
only partial exception of 7h, which induced a slight increase of
the propagation rate of the oxidation. Analysis of Δtlag values
again indicates an influence of lipophilicity. Hydrophilic com-
pounds are the most active members of the series, showing a
Δtlag near that of carnosine, while the less hydrophilic ones
display lower activity. When Δtlag is plotted against log D7.4, the
diagram of Figure 2 is obtained. It shows that the ability of
ligands is highly significant. The value of log([Cu]total/[Cu]free
)
is significantly lower for 12 (1.95) in which a N-substituted
amido group is not present.
Antioxidant Activity. Oxidative stress occurs in cells, tissues,
and organs when the balance between the concentration of the
prooxidant reactive species and the antioxidant capacity is
broken in favor of the prooxidant forces. Oxidative stress is
involved in many pathological processes including aging, cancer,
chronic inflammation, diabetic complications, cardiovascular
diseases, including atherosclerosis and stroke, as well as a
number of neurodegenerative disorders.31,32 In particular, there
is strong evidence that LDL-oxidation is increased in neurode-
generative and cognitive impairing diseases such Alzheimer’s
disease (AD) and vascular dementia.33-36 As aforementioned,
carnosine displays antioxidant properties; in particular, it is
reported that carnosine was able to increase lag time for
TBARS appearance and reduce the maximal rate of LDL
oxidation catalyzed by Cu2þ. In the same work, conflicting
results were obtained when hydroperoxides formation was
monitored.37 In our work, we tested carnosine and all the
newly synthesized amides 7a-n (100 μM) for their ability to
suppress conjugated dienes formation during copper-mediated
LDL (50 μg protein mL-1) oxidation. The time course of

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 615
Table 3. Antioxidant Activity and HNE Scavenging Ability of Derivatives 7a-n, 12, and Carnosine
antioxidant activity
HNE scavenging ability
HNE
HNE
scavenged
(%) ( SEM b
6 h
HNE
scavenged
(%) ( SEM b
24 h
R (nmol min-1
mg-1 LDL prot)
( SEM a
scavenged
(%) ( SEM b
1 h
Δtlag (min)
( SEM a
compd
carnosine
67 ( 2
64 ( 3
75 ( 2
68 ( 3
68 ( 2
82 ( 6
79 ( 3
43 ( 2
9 ( 5
-3 ( 3
85 ( 5
85 ( 4
49 ( 5
-4 ( 1
5.8 ( 0.3
7.7 ( 0.4
6.8 ( 0.6
7.0 ( 0.3
7.7 ( 0.3
5.7 ( 0.3
6.7 ( 0.4
11.8 ( 0.4
21.0 ( 1.0
14.5 ( 0.2
5.6 ( 0.1
5.2 ( 0.2
10.0 ( 0.3
12.2 ( 0.4
20.4 ( 1.7
10.2 ( 1.7
8.0 ( 1.6
8.4 ( 1.0
12.5 ( 1.9
12.5 ( 1.7
12.0 ( 1.0
12.8 ( 2.3
ND
59.6 ( 1.6
31.6 ( 1.4
35.2 ( 0.8
32.9 ( 1.1
42.7 ( 2.0
36.2 ( 1.2
35.4 ( 1.9
49.7 ( 1.9
ND
88.8 ( 1.6
77.7 ( 1.7
75.4 ( 2.4
70.9 ( 2.2
81.0 ( 1.6
75.2 ( 1.3
75.9 ( 2.3
86.7 ( 1.8
ND
7a
7b
7c
7d
7e
7f
7g
7h
7i
ND
ND
ND
7l
12.8 ( 3.2
7.2 ( 1.4
8.8 ( 1.6
17.9 ( 1.6
28.2 ( 2.8
29.3 ( 1.6
32.6 ( 1.6
60.7 ( 1.2
62.5 ( 2.1
72.8 ( 0.6
80.7 ( 0.8
86.2 ( 0.3
7m
7n
12
a Obtained by CuSO4-induced human LDL oxidation assay in the presence of compounds at 100 μM. R values were calculated from ΔA234 as a
function of time, using ε234 = 29500 M-1 cm-1 for conjugated lipid peroxides. For control LDL samples, R = 12.1 ( 0.5 nmol min-1 mg-1 LDL prot.
b Determined in phosphate buffer (pH 7.4, 1 mM) at 37 °C; HNE (50 μM), test compound (1 mM). Scavenging % was calculated according to the
following formula: scavenging (%) = 100 - {[ (amount of HNE left after t h in the presence of the scavenger)/(amount of HNE left after t h in the
control)] ꢀ 100}. ND = not determined.
the presence of the plateau. The different ability to sca-
venge Cu2þ of the products as consequence of their dif-
ferent lipophilicity could also partly justify their different
ability in influencing the propagation rate of the oxida-
tion. Compound 12, which does not contain amido group,
was also tested for its ability to reduce LDL oxidation. It
proved unable to modify the three parameters which
characterize the kinetics of LDL oxidation. This result is
in keeping with the very low Cu2þ sequestering ability
shown by 12.
HNE-Quenching Activity. The products formed by lipid
peroxidation are degraded to reactive aldheydes such as
Figure 2. Relationship between antioxidant activity (expressed as
HNE, malondialdehyde, and alkenals. HNE has been de-
Δtlag) and log D7.4 for carnosine amide derivatives 7a-n; compound
monstrated to cause neuronal death.41-44 Moreover HNE-
12 is also showed for comparison.
protein adducts have been detected in the brain of patients
carnosine amides to increase the lag time of the copper-
catalyzed LDL peroxidation linearly increases with hydrophi-
licity and then reaches a plateau. It is known that Cu2þ ions can
form LDL-copper complexes with binding sites of apolipo-
protein, and the amount of copper bound to LDL increases
with increasing concentrations of copper ions until the copper
binding sites become saturated.39 These complexes are able to
produce free radicals at the LDL surface by interacting with
preformed LDL-associated hydroperoxides (LOOH), thus in-
ducing peroxidation.40 As aforementioned, the ability of the
amide analogues of carnosine to sequester Cu2þ ions at phy-
siological pH is roughly similar. The lipophilicity of com-
pounds, expressed by log D7.4, should reasonably mimic their
capacity of distribution between the LDL and the aqueous
phase. Consequently, the linear tract of the plot in Figure 2
could be justified by the fact that, under the adopted experi-
mental conditions, the higher the hydrophilicity is, the higher the
amount of ligand in aqueous phase free to complex Cu2þ ions
and thus able to decrease the onset of rapid lipidic peroxidation.
Under the threshold log D7.4 value of about -1.5, the amount
of ligand sequestered by the LDL phase is so small that any
further reduction of lipophilicity does not produce any appreci-
able variation in ligand aqueous concentrations and, conse-
quently, in the amount of chelated Cu2þ ions; this could justify
with AD, therefore HNE is considered to play a crucial role
in oxidative injury of biomolecules related to AD.45-47 In the
Introduction, we have already briefly discussed the biologi-
cal role of carnosine as a quencher of R,β-unsaturated
aldehydes. This reaction has been object of detailed
studies.6,7,48 Two adducts have been isolated as principal
reaction products working in phosphate buffer, pH 7.4,
using HNE as a model of reactive aldehyde. The former is
an imidazole-based Michael adduct, stabilized as a five-
membered cyclic hemiacetal and the latter a 13-membered
ring Schiff base Michael adduct.6,7,23,49 To evaluate whether
carnosine amides display carnosine-like properties as
quenchers of HNE, all the products were incubated in
phosphate buffer, pH 7.4, with HNE at 37 °C. Samples
relative to different incubation times were directly analyzed
by RP-HPLC to measure HNE consumption. The % HNE
quenched increased with the time and after 24 h reached a
plateau. The results after 1, 6, and 24 h are summarized in
Table 3. Carnosine, taken as reference, after 24 h was able to
block about 89% of HNE. The most active product among
the carnosine amides was the n-octyl substituted compound
7g, which was as active as carnosine within the experimental
error. For all the other compounds, the quenching effect on
HNE fell in the range 62-81%. These results indicate that all

616 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Bertinaria et al.
Figure 3. In vitro cytotoxicity on hippocampal neurons. (A) Percentage of cell death induced by different compounds on hippocampal
neurons after 24 h exposure. (B) Percentage of cytoprotection after 24 h exposure of hippocampal cells to HNE (10 μM) and carnosine 100 μM
(gray bar) or 7d 100 μM (black bar). (C) Images at the optical microscope at 20ꢀ magnification. On the left, a group of control cells after 24 h. In
the middle, a group of cell exposed for 24 h to HNE 10 μM. Cell death is evident. In the last panel, on the right, a group of cells after 24 h
incubation with HNE 10 μM plus 7d 100 μM. Reported results are given as mean ( SEM for n (n = 4-7) experiments. Statistical significance
was calculated by using Student’s paired t test. Values of p < 0.05 were considered significant.
the canosine amides here described display quite potent
carnosine-like HNE quenching activity.
rats. Blood and brain tissues samples were collected from rats
assigned to 1.5 or 3 h post-treatment time points. Samples were
analyzed by RP-HPLC (see Experimental Section), and
concentration of 7d was determined. At 3 h, the level of 7d in
brain was 1.1 ( 0.4 μg/g tissue, and at 1.5 h, the level of 7d
was about 0.6 μg/g tissue, approaching the detection limit of
this technique. In blood, 7d was only detectable at 1.5 h at a
concentration of 0.26 μg/mL. The obtained data indicates
how levels of 7d in brain increased between 1.5 and 3 h of
administration, whereas levels of 7d in the serum decreased
during this time interval.
Protection from HNE-Induced Cell Death. On the bases of
the results we obtained from the experiments of HNE-
quenching activity, we decided to test the ability of com-
pound 7d, chosen as an example of this new series of carnosine
derivatives, to protect primary mouse hippocampal neurons
against HNE-induced death. Carnosine was taken as refer-
ence. At first, the adverse effects of 7d (100 μM), HNE (10
and 20 μM), and carnosine (100 μM) on hippocampal
neurons were evaluated (Figure 3A). Then, mouse hippo-
campal neurons were pretreated either with the selected
compound or with carnosine and then exposed to HNE
10 μM concentration. After 24 h exposure, the percentage of cell
death was evaluated (Figure 3B) by Trypan Blue exclusion in
order to stain just dead cells due to loss of membrane integrity.
Inspection of the Figure 3A indicates that, when compared
to the control, HNE was able to kill 61-84% of treated cells
in a concentration dependent manner while no adverse effect
was seen either with carnosine or 7d. Analysis of Figure 3B
shows that carnosine has, if any, a very low cytoprotective
efficacy (6 ( 4%), this is probably due to its instability to
carnosinase, which is known to be present in mouse brain,50
while 7d triggers highly significant protective effect (40 (
17%). Figure 3C shows the damage induced by HNE on the
neural network (middle image) compared to the control (left
image). The destruction is visible at the axonal level and also
in the matrix surrounding the processes; moreover, there is a
reduction in the total number of neurons and visible signs of
necrosis. The presence of 7d clearly reduces these effects
(right image).
Conclusion
This paper describes synthesis, physicochemical, and bio-
logicalcharacterizationof a seriesofcarnosine amides bearing
on the amido group alkyl substituents endowed with different
lipophilicity. All the products were able to display carnosine-
like properties, morover, they were stable over 3 h of incuba-
tion in human serum at 37 °C, unlike the lead that was rapidly
cleaved into its constituents. All the synthesized compounds
were capable of affording copper complexes at physiological
pH with the same stoichiometry as carnosine and of display-
ing highly significant copper ion sequestering ability. The
products were capable of protecting LDL from oxidation
catalyzed by Cu2þ ions, the most active compounds being the
most hydrophilic ones. All the amides triggered quite potent
carnosine-like HNE quenching activity. 7d, the compound
chosen as the representative example of the series, was able to
protect primary mouse hippocampal neurons against HNE-
induced death. 7d was also capable of penetrating rat brain
after in vivo administration. These products can be con-
sidered metabolically stable analogues of carnosine, worthy
of additional investigation as potential neuroprotective
agents.
HPLC-Detection of 7d in Rat Serum and Brain. To further
explore the neuroprotective potential of this class of com-
pounds, we determined whether 7d enters the brain by
administering 7d (20 mg/kg) intravenously to a group of

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 617
Experimental Section
the water phase was washed with CH2Cl2 (2ꢀ 20 mL) and then with
EtOAc (2 ꢀ 20 mL) and evaporated to afford the final product.
β-Alanyl-L-histidinamide Ditrifluoroacetate (7a). The product
was recrystallized from dry MeOH/Et2O and freeze-dried to
afford 7a as an amorphous semisolid material; overall yield:
28%. 1H NMR (DMSO): δ 8.81 (s, 1H, ImH2), 8.46 (d, 1H, J =
8.4 Hz, NHCH), 7.86 (s, br, 3H, NH3þ), 7.52 (s, br, 1H, CONHH),
7.28 (s, 1H, ImH5), 7.26 (s, br, 1H, CONHH), 4.55-4.48 (m, 1H,
CH), 3.17-2.85 (m, 4H, 2 CH2), 2.61-2.43 (m, 2H, COCH2).
13C NMR (DMSO): δ 172.1, 169.7, 134, 130.3, 116.9, 51.8, 35.3,
32.2, 27.3.
Chemistry. Melting points were measured with a capillary
€
apparatus (Buchi 540). Compounds 7a-h and 12 were highly
hygroscopic amorphous semisolids or foams. The determina-
tion of their melting point was affected by the complex thermal
behavior of these compounds; consequently, the melting point
was not reported. All the compounds were routinely checked by
1H and 13C NMR (Bruker Avance 300) at 300 and 75 MHz,
respectively, and mass spectrometry (Finnigan-Mat TSQ-700).
The following abbreviations are used to indicate the peak
multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, m=
multiplet. Flash column chromatography was performed on
silica gel (Merck Kieselgel 60, 230-400 mesh ASTM) using the
reported eluents. Thin layer chromatography (TLC) was carried
out on 5 cmꢀ20 cm plates (Fluka) with a 0.2 mm layer thickness.
Unless otherwise stated, anhydrous magnesium sulfate was used
as the drying agent for the organic phases. Analysis (C, H, N) of
the target compounds was performed by Service de Microana-
β-Alanyl-N-methyl-L-histidinamide Ditrifluoroacetate (7b). The
product was recrystallized from dry MeOH/Et2O and freeze-dried
to afford 7b as a white amorphous foam; overall yield 29%. H
1
NMR (DMSO): δ 8.89 (s, 1H, ImH2), 8.52 (d, 1H, J=8.1 Hz,
NHCH), 8.03-8.01 (m, 1H, NHCH3), 7.89 (s, br, 3H, NH3þ), 7.30
(s, 1H, ImH5), 4.56-4.49 (m, 1H, CH), 3.17-2.83 (m, 4H, 2 CH2),
2.57 (d, 3H, J=4.5 Hz, CH3), 2.51-2.41 (m, 2H, COCH2). 13
C
NMR (DMSO): δ 170.1, 169.5, 134, 133.7, 116.6, 51.7, 35, 32, 27,
25.6.
ꢁ
ꢀ
ꢀ
lyse, Universite de Geneve, Geneve (CH), and REDOX (Monza),
and the results were within (0.4% of the theoretical. HNE (4-
hydroxy-trans-2,3-nonenal) was prepared by acid treatment (1 mM
HCl) of HNE-DMA (4-hydroxy-trans-2,3-nonenal-dimethylacetal;
Sigma). Compound 817 was synthesized according to the litera-
ture. Preparative HPLC was performed on a Lichrospher C18
column (250 mmꢀ25 mm, 10 μm) (Merck Darmstadt, Germany)
with a Varian ProStar mod-210 with Varian UV detector mod-
325 with a flow rate of 39 mL/min; the detection was performed
at 224 nm.
β-Alanyl-N-ethyl-L-histidinamide Ditrifluoroacetate (7c). The
product was recrystallized from dry MeOH/Et2O and freeze-
dried to afford 7c as a white amorphous foam; overall yield 78%.
1H NMR (CD3OD): δ 8.68 (s, 1H, ImH2), 7.29 (s, 1H, ImH5),
4.70-4.60 (m, 1H, CH), 3.21-3.04 (m, 6H, 3 CH2), 2.66-2.64
(m, 2H, COCH2), 1.10-1.06 (t, 3H, J=7.2 Hz, CH3). 13C NMR
(CD3OD): δ 172.3, 171.9, 135.1, 131.5, 118.3, 53.8, 36.8, 35.4,
32.7, 28.5, 14.7.
β-Alanyl-N-propyl-L-histidinamide Ditrifluoroacetate (7d). The
product was recrystallized from dry MeOH/Et2O and freeze-dried
to afford 7d as a white amorphous foam; overall yield: 41%. 1H
NMR (DMSO): δ 8.84 (s, 1H, ImH2), 8.50 (d, 1H, J=8.4 Hz,
NHCH), 8.06 (t, 1H, J=10.8 Hz, CONHCH2), 7.90 (s, br, 3H,
NH3þ), 7.28 (s, 1H, ImH5), 4.57-4.50 (m, 1H, CH), 3.16-2.85 (m,
6H, 3 CH2), 2.58-2.42 (m, 2H, COCH2), 1.39 (q, 2H, J=7.2 Hz,
CH2CH3), 0.91 (t, 3H, J=7.2 Hz, CH3). 13C NMR (DMSO): δ
170.5, 170.4, 134.6, 130.7, 117.6, 52.8, 41.3, 36, 32.9, 28.2, 23, 12.1.
β-Alanyl-N-butyl-L-histidinamide Ditrifluoroacetate (7e). The
product was recrystallized from dry MeOH/Et2O and freeze-
dried to afford 7e as a white amorphous foam; overall yield 31%.
1H NMR (CD3OD): δ, 8.74 (d, 1H, J=1.2 Hz, ImH2), 7.30 (d,
1H, J=1.2 Hz, ImH5), 4.68-4.64 (m, 1H, CH), 3.30-3.02 (m,
6H, ImCH2, NHCH2, CH2NH3þ), 2.71-2.58 (m, 2H, COCH2),
General Procedure for Preparation of 7a-n. To a stirred
solution of Fmoc-His-(Trt)-OH (2) (2.5 g; 4 mmol) in dry
DMF (40 mL), DIPEA (1.03 mL; 6 mmol), HBTU (2.29 g;
6 mmol), and HOBt (0.08 g; 0.6 mmol) were added. After 10 min,
the appropriate amine 3a-n (6 mmol) was added; the reaction
mixture was allowed to stir at rt until TLC showed complete
consumption of starting material (1-18 h). The solvent was
removed under reduced pressure (oil pump) and the residue
taken up with CH2Cl2 (40 mL) and washed with water (3ꢀ30 mL)
and brine (30 mL), then dried and evaporated under reduced
pressure. The residual oil was purified by flash chromatography
eluting with CH2Cl2/MeOH 9.9/0.1 to 9.5/0.5 to afford the
desired intermediate 4b-n (56-100%). To a stirred solution
of the obtained intermediate (2.59 mmol) in dry DMF (23 mL),
piperidine (1.15 mL; 11.6 mmol) was added and the reaction
mixture was stirred at rt for 1 h. The solvent was evaporated
under reduced pressure, and the solid residue was taken up with
CH2Cl2 (30 mL) and washed with water (3 ꢀ 30 mL) and brine
(30 mL). The organic phase was dried (Na2SO4), and the crude
product was purified by flash chromatography eluting with
CH2Cl2/MeOH 9.8/0.2 to 8/2 to yield the desired intermediates
5b-n (50-100%). When ammonia was used as the amine
nucleophile in the coupling reaction with activated 2, amidation
and fmoc-deprotection were achieved in one step, leading to
desired 5a, which was isolated as a white solid after filtration
from cold (0 °C) CH2Cl2 in 86% yield. To a stirred solution of
the free amines 5a-n (1.75 mmol) and Boc-β-Ala(OH) (0.35 g;
1.83 mmol) in dry CH2Cl2 (30 mL) kept at 0 °C, DCC (0.36 g;
1.75 mmol) was added, the ice bath was removed, and the
reaction was stirred at rt for 2-18 h. The reaction mixture was
cooled to 0 °C. The precipitate was filtered and washed with cold
(0 °C) CH2Cl2. The liquid phase was washed with water (3 ꢀ
30 mL) and brine (30 mL) and then dried and evaporated under
reduced pressure to leave a white solid. The crude material was
purified by flash chromatography eluting with CH2Cl2/MeOH
9.8/0.2 to 9.5/0.5 to give the desired derivatives 6a-n (41-98%)
as white foams. The obtained products were dissolved in CH2Cl2
(21 mL), treated with CF3COOH (2.1 mL), and stirred at rt for
20 h. The solvent was evaporated under reduced pressure, and
the semisolid residue was treated with water (30 mL). The formed
precipitate was filtered off through a sintered glass funnel, and
1.49-1.35 (m, 4H, CH2CH2), 0.93 (t, 3H, J=7.2 Hz, CH3). 13
C
NMR (CD3OD): δ 172.3, 172, 135.1, 131.4, 118.4, 53.8, 40.3, 36.8,
32.7, 32.4, 28.41, 21, 14.1.
β-Alanyl-N-hexyl-L-histidinamide Ditrifluoroacetate (7f). The
product was purified by RP-HPLC eluting with MeOH/H2O 60/
40 þ 0.1% TFA to give pure 7f as a white amorphous foam;
1
overall yield 49%. H NMR (CD3OD): δ 8.81 (s, 1H, ImH2),
7.33 (s, 1H, ImH5), 4.70-4.65 (m, 1H, CH), 3.31-3.03 (m, 6H,
ImCH2, NHCH2, CH2NH3þ), 2.73-2.56 (m, 2H, COCH2),
1.49-1.44 (m, 2H, NHCH2CH2), 1.33-1.28 (m, 6H, 3 CH2),
0.90 (t, 3H, J=6.9 Hz, CH3). 13C NMR (CD3OD): δ 172.3,
171.9, 135, 131.1, 118.4, 53.7, 40.6, 36.8, 32.69, 32.66, 30.3, 28.3,
27.7, 23.6, 14.4.
β-Alanyl-N-octyl-L-histidinamide Ditrifluoroacetate (7g). The
product was recrystallized from dry MeOH/Et2O and freeze-
dried to afford 7g as white amorphous foam; overall yield 41%.
1H NMR (CD3OD): δ 8.78 (s, 1H, ImH2), 7.33 (s, 1H, ImH5),
4.70-4.65 (m, 1H, CH), 3.30-3.08 (m, 6H, NHCH2, ImCH2,
CH2NH3þ), 2.68-2.62 (m, 2H, COCH2), 1.49-1.44 (m, 2H,
NHCH2CH2), 1.30-1.28 (m, 10H, 5 CH2), 0.90 (t, 3H, J=6.9 Hz,
CH3). 13C NMR (CD3OD): δ 172.3, 171.9, 135, 131.2, 118.4, 53.7,
40.7, 36.8, 33.0, 32.7, 30.42, 30.41, 30.36, 28.3, 28, 23.7, 14.5. 1H
NMR in agreement with those reported for the hydrochloride
derivative.26
β-Alanyl-N-decyl-L-histidinamide Ditrifluoroacetate (7h). The
product was purified by RP-HPLC eluting with MeOH/H2O 70/
30 þ 0.1% TFA to give 7h as a white amorphous solid; overall

618 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Bertinaria et al.
1
yield 29%. H NMR (CD3OD): δ 8.81 (s, 1H, ImH2), 7.33 (s,
2.84 mmol) and Boc-β-Ala(OH) (0.67 g; 3.54 mmol) in dry
CH2Cl2 (20 mL) kept at 0 °C, DCC (0.73 g; 3.54 mmol) was
added, the ice bath was removed, and the reaction was stirred at
rt for 15 h. The reaction mixture was cooled to 0 °C, and the
obtained precipitate was filtered and washed with cold (0 °C)
CH2Cl2. The liquid phase was washed with water (3 ꢀ 30 mL)
and brine (30 mL) and then dried and evaporated under reduced
pressure to leave a white solid. The crude material was purified
by flash chromatography eluting with CH2Cl2 þ 2% MeOH, to
give 1.60 g (97%) of 11 as a white foam. The obtained product was
dissolved in CH2Cl2 (41 mL), treated with CF3COOH (4.1 mL;
55 mmol), and stirred at rt for 20 h. The solvent was evaporated
under reduced pressure, and the dark oily residue was treated
with water (20 mL). The formed precipitate was filtered off
through a sintered glass funnel, and the liquid phase was extracted
with EtOAc (3 ꢀ 20 mL). The aqueous layer was evaporated
under reduced pressure to leave 1.12 g (87%) of 12 as a colorless
semisolid material. The product was recrystallized twice from
dry MeOH/Et2O and freeze-dried. 1H NMR (CD3OD): δ 8.74
(s, 1H, ImH2), 7.3 (s, 1H, ImH5), 4.33-4.29 (m, 1H, CH), 3.56-
3.45 (m, 4H, 2 CH2O), 3.15-3.11 (t, 2H, J=6.6 Hz, CH2NH3þ),
3.07-2.87 (m, 2H, ImCH2), 2.59 (t, 2H, J=6.6 Hz, COCH2),
1.19 (t, 3H, J=6.9 Hz, CH3). 13C NMR (CD3OD): δ 172.1, 134.9,
132.3, 118, 72.2, 67.8, 49.9, 37, 32.8, 27.9, 15.4.
Ionization Constants and Lipophilicity Descriptors. The ioni-
zation constants of compounds were determined by potentio-
metric titration with the GLpKa apparatus (Sirius Analytical
Instruments Ltd, Forest Row, East Sussex, UK). Ionization
constants of carnosine 1 and compounds 7a-g, 7l-n, and 12
were obtained by aqueous titrations by at least four separate
titrations for each compound: different aqueous solutions (ionic
strength adjusted to 0.15 M with KCl) of the compounds (20 mL,
about 1 mM) were initially acidified to pH 1.8 with 0.5 N HCl;
the solutions were then titrated with standardized 0.5 N KOH to
pH 12.2. Because of the low aqueous solubility, ionization
constants measurement of compounds 7h-i required titrations
in the presence of methanol as a cosolvent: at least five different
hydro-organic solutions (ionic strength adjusted to 0.15 M with
KCl) of the compounds (20 mL, about 1 mM in 20-34 wt %
methanol) were initially acidified to pH 1.8 with 0.5 N HCl; the
solutions were then titrated with standardized 0.5 N KOH to pH
12.2. The initial estimates of the psKa values (the apparent ioniza-
tion constants in the water-methanol mixtures) were obtained
and aqueous pKa values were determined by extrapolation to
zero content of the cosolvent according to the Yasuda-
Shedlovsky procedure.51 All the titrations were performed
under nitrogen at 25.0 ( 0.1 °C. The apparent partition coeffi-
cients log D7.4 were obtained by shake-flask procedure at pH 7.4
(phosphate buffer solution with ionic strength adjusted to 0.15
M with KCl); n-octanol was added to the buffer, and the two
phases were mutually saturated by shaking for 4 h. The com-
pounds were solubilized in the buffered aqueous phase at a
concentration of about 0.1 mM, and an appropriate amount of
n-octanol was added. The two phases were shaken for about 20
min, by which time the partitioning equilibrium of solutes was
reached and then centrifuged (10000 rpm, 10 min). The con-
centration of the solutes in the aqueous phase was measured by
UV spectrophotometer (UV-2501PC, Shimadzu) at 230 nm. For
each compound, at least seven log D values were measured.
Copper Complexes. The complex formation between copper-
(II) and carnosine and derivatives 7a, 7d, 7g, 7l, 7m, and 12 was
investigated by means of the classical pH-metric technique with
the GLpKa apparatus. For each compound, at least three separate
titrations were performed: different aqueous solutions of the
compounds and of CuCl2 equimolar (1 mM) were initially
acidified to pH 1.8 with 0.5 N HCl; the solutions were then
titrated with standardized 0.5 N KOH to pH 12.2. The measure-
ments were carried out under nitrogen at 25.0 ( 0.1 °C and
ionic strength adjusted to 0.15 M (KCl). The elaboration
of experimental pH-metric data, in order to calculate the
1H, ImH5), 4.70-4.65 (m, 1H, CH), 3.34-3.03 (m, 6H, NHCH2,
ImCH2, CH2NH3þ), 2.71-2.56 (m, 2H, COCH2), 1.48-1.44
(m, 2H, NHCH2CH2), 1.31-1.29 (m, 14H, 7 CH2), 0.90 (t, 3H, J =
6.9 Hz, CH3). 13C NMR (CD3OD): δ 172.3, 171.9, 135, 131.2,
118.4, 53.7, 40.8, 36.8, 33.1, 32.7, 30.8, 30.7, 30.49, 30.46, 30.37,
28.3, 28, 23.8, 14.5. 1H NMR in agreement with those reported
for the hydrochloride derivative.26
β-Alanyl-N-dodecyl-L-histidinamide Ditrifluoroacetate (7i). The
product was purified by RP-HPLC eluting with MeOH/H2O 80/
20 þ 0.1% TFA to give the desired 7i as a white amorphous solid;
overall yield 14%; mp 72.2-80.7 °C. 1H NMR (CD3OD): δ 8.79
(s, 1H, ImH2), 7.36 (s, 1H, ImH5), 4.75-4.70 (m, 1H, CH), 3.26-
3.02 (m, 6H, ImCH2, NHCH2, CH2NH3þ), 2.71-2.58 (m, 2H,
COCH2), 1.48-1.44 (m, 2H, NHCH2CH2), 1.30-1.29 (m, 18H, 9
CH2), 0.90 (t, 3H, J=6.9 Hz, CH3). 13C NMR (CD3OD): δ 172.5,
172.1, 134.9, 131.1, 118.4, 53.9, 40.8, 36.8, 33.0, 32.7, 30.77, 30.75,
30.71, 30.6, 30.5, 30.4, 30.3, 28.7, 28, 23.7, 18.4.
β-Alanyl-N-cyclohexyl-L-histidinamide Ditrifluoroacetate (7l).
The product was purified by RP-HPLC eluting with MeOH/
H2O 70/30 þ 0.1% TFA and freeze-dried to give 7l as a white
amorphous solid; overall yield 37%; mp 74.5-84.2 °C. 1H NMR
(CD3OD): δ 8.81 (s, 1H, ImH2), 7.34 (s, 1H, ImH5), 4.71-4.66
(m, 1H, CH), 3.30-2.94 (m, 6H, ImCH2, NHCH2cHex, CH2-
NH3þ), 2.66-2.59 (m, 2H, COCH2), 1.73-0.90 (m, 11H, cHexH).
13C NMR (CD3OD): δ 172.5, 172.2, 135.2, 131.3, 118.6, 54, 47.1,
39.3, 37.1, 32.9, 32.1, 28.5, 27.7, 27.2.
β-Alanyl-N-benzyl-L-histidinamide Ditrifluoroacetate (7m). The
product was recrystallized from MeOH/Et2O and freeze-dried to
give 7m as a white amorphous solid; overall yield 20%; mp
158.7-161.9 °C. 1H NMR (CD3OD): δ, 8.74 (s, 1H, ImH2), 7.32-
7.21 (m, 6H, ImH5, 5 ArH), 4.76-4.71 (m, 1H, CH), 4.45-4.30
(m, 2H, NHCH2), 3.20-2.95 (m, 4H, ImCH2, CH2NH3þ), 2.76-
2.58 (m, 2H, COCH2). 13C NMR (CD3OD): δ 172.4, 172, 139.7,
134.9, 129.6, 128.6, 128.4, 118.42, 112.2, 53.8, 44.2, 36.8, 32.7, 28.1.
β-Alanyl-N-(4-butoxbenzyl)-L-histidinamide Ditrifluoroacetate
(7n). The product was purified by preparative RP-HPLC eluting
with MeOH/H2O 60/40 þ 0.1% TFA and freeze-dried to give
the desired product as a white solid; overall yield 17%; mp
70.8-73.2 °C. 1H NMR (CD3OD): δ 8.73 (s, 1H, ImH2), 7.23 (s,
0
0
1H, ImH5), 7.10 (d, 2H, J=8.7 Hz, ArH2 ,6 ), 6.82-6.79 (d, 2H,
0
0
J = 8.7 Hz, ArH3 ,5 ), 4.67 (m, 1H, CH), 4.26-4.23 (m, 2H,
NHCH2), 3.91 (t, 2H, J=6.3 Hz, OCH2), 3.15-3.10 (m, 4H,
ImCH2, CH2NH3þ), 2.62-2.61 (m, 2H, COCH2), 1.72-1.68
(m, 2H, CH2CH2CH2CH3), 1.47-1.45 (m, 2H, CH2CH2CH2-
CH3), 0.94 (t, 3H, J=7.2 Hz, CH3). 13C NMR (CD3OD): δ 172.3,
171.8, 160, 134.9, 131.5, 131, 130, 118.4, 115.5, 68.7, 53.8, 43.7,
36.8, 32.7, 32.5, 28.2, 20.3, 14.2.
3-Amino-N-[(1S)-2-ethoxy-1-(1H-imidazol-4-ylmethyl)ethyl]-
propanamide Ditrifluoroacetate (12). In a flame-dried flask
equipped with a CaCl2 guard tube, NaH 60% in mineral oil
(0.83 g; 20.8 mmol) suspended in dry DMF (12.5 mL) was stirred
for 1 h. A solution of 8 (2.50 g; 4.0 mmol) in dry DMF (15 mL)
was added, and the reaction mixture was stirred at rt for 1 h, then
bromoethane (1.09 g; 10 mmol) was added and the reaction
mixture stirred overnight. The mixture was cooled to 0 °C and
excess NaH was destroyed by a slow addition of water. The
obtained suspension was extracted with EtOAc (3 ꢀ 40 mL), the
organic phase was dried (Na2SO4) and evaporated under reduced
pressure. The residue was purified by flash chromatography
eluting with CH2Cl2/EtOAc 9.5/0.5 to 9/1 to yield 9as a white foam
(2.21 g; 85%). To an ice-cooled solution of 9 (2.16 g, 3.3 mmol)
in dry CH2Cl2 (73 mL), CF3COOH (0.73 mL; 9.82 mmol) was
added and the reaction mixture was stirred for 2 min. The mixture
was diluted with 10% (w/v) Na2CO3 aqueous solution and
extracted with EtOAc (3 ꢀ 40 mL). The combined organic layers
were washed with water (50 mL) and dried (Na2SO4) to afford a
crude product which was purified by flash chromatography
eluting with CH2Cl2/MeOH 9.5/0.5 to 8.5/1.5 to yield 1.17 g
(87%) of 10 as a colorless oil. To a stirred solution of 10 (1.17 g;

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 619
values of formation constants, was performed by the BSTAC
program.30
plus DNAse (0,1 mg/mL). Isolated cells were plated onto Petri
dishes, coated with poly-DL-lysine and laminine, at the final
density of 260 cells/mm2. The cells were incubated with 1%
penicillin/streptomycin, 1% glutamax, 2% B-27 supplemented
neurobasal medium in a humidified 5% CO2 atmosphere at
37 °C for 6 days before performing the experiments.
Stability in Human Serum. A solution of each compound
(10 mM) in water was added to human serum (Sigma) preheated
at 37 °C, and the final concentration of the compound was 0.5 mM.
Resulting solutions were incubated at 37 ( 0.5 °C, and at
appropriate time intervals, 500 μL of reaction mixture was
withdrawn and added to 500 μL of acetonitrile containing 0.1%
trifluoroacetic acid in order to deproteinize the serum. The
sample was sonicated, vortexed, and then centrifuged for 10 min
at 2150g. The clear supernatant was filtered by 0.45 μm PTFE
filters (Alltech) and analyzed by RP-HPLC. HPLC analyses
were performed with a HP 1100 chromatograph system (Agilent
Technologies, Palo Alto, CA, USA) equipped with a quaternary
pump (model G1311A), a membrane degasser (G1379A), and a
diode-array detector (DAD) (model G1315B) integrated in the
HP1100 system. Data analysis was done using a HP ChemSta-
tion system (Agilent Technologies). The analytical column was a
Purospher C18-endcapped (250 mm ꢀ 4.6 mm, 5 μm particle size)
(Merck Darmstadt, Germany). The mobile phase consisting of
methanol/20 mM CH3COONa pH 4.5-5 mM SDS (80/20 to 60/
40 in accordance with the polarity of compounds), and the flow
rate was 0.7 mL/min. The injection volume was 20 μL (Rheodyne,
Cotati, CA). The column effluent was monitored at 210 and 223
nm referenced against a 360 nm wavelength. Quantitation was
done by comparison of peak areas with standards chromato-
graphed under the same conditions.
Cytotoxicity Assays. Carnosine and 7d were dissolved in the
culture medium containing Neurobasal (Invitrogen), 2% B-27,
1% pen-strep (Lonza), and 1% ultraglutamine (Lonza) and
used at the final concentration of 100 μM. HNE (Cayman) was
stored at -80 °C in ethanol and then tested at the final concentra-
tion of 10 and 20 μM. After incubating hippocampal cells with
the substances for 24 h, cell viability was determined by Trypan
Blue exclusion, in order to stain just dead cells, due to loss of
membrane integrity. Cells were counted with knowledge of the
treatment history of the culture by comparing the number of
living cells before incubation and after 24 h of treatment.
Substances were tested alone and combined to HNE to assess
not only the cytoprotective activity of carnosine and the carno-
sine analogue 7d but also to exclude a possible toxicity of the
compound. The percentage of cytoprotection was then evalu-
ated by comparing the percentage of cells death after exposure
to HNE and to HNE þ 7d. Data are given as the mean ( SEM
for 4-7 experiments. Statistical significance was calculated by
using Student’s paired t test. Values of p < 0.05 were considered
significant.
HPLC Detection of 7d in Rat Blood and Brain. Male Wistar
rats weighing 200-250 g were used for this experiment. Each rat
received 20 mg/kg of 7d by iv injection of 200 μL of compound
dissolved in 0.9% normal saline. After 1.5 or 3 h, animals were
sacrificed for decapitation, and three rats were used for any time
point. Blood was collected in polystyrene tubes with 500 UI
eparine, and the brain was rapidly removed, rinsed with cold
distilled water, weighed, and homogenized at 4 °C in an equal
volume of cold distilled water using a Potter-Elvehjem homo-
genizer. An equal volume of acetonitrile containing 0.1%
trifluoroacetic acid was added to blood samples or brain tissue
homogenates to precipitate proteins. The samples were soni-
cated, vortexed, and then centrifuged for 10 min at 2150g. Theclear
supernatant was filtered by 0.45 μm PTFE filters and analyzed
by RP-HPLC. HPLC analyses were performed with the same
chromatograph system and stationary phase used for stability in
human serum analyses. The supernatant (100 μL) was eluted
with methanol/20 mM CH3COONa pH 4.5 and 5 mM SDS
(70/30) at a flow rate of 0.7 mL/min. The column effluent was
monitored at 210 and 223 nm (reference 360 nm). Samples from
untreated rats were used either as control or, after adding a
known amount of 7d, as standard.
Antioxidant Activity. LDL Isolation and Oxidation. Human
plasma from healthy donors was provided by Blood Bank (AO
San Giovanni Battista Turin) and added with 0.1% EDTA. The
LDL fraction was isolated by ultracentrifugation through NaCl
discontinuous gradients and collected as the fraction floating at
a density of 1.019-1.063 g/mL. The determination of the lag
phase (tlag) and of the propagation rate (R) was carried out as
previously described.52 EDTA was removed by rapid filtration
through disposable desalting columns Econo-Pac 10 DG (Bio-
Rad). Filtered LDL were diluted with PBS (10 mM phosphate
buffer, pH 7.4) to give a final concentration of 50 μg of LDL
protein/mL and transferred to a 1 cm cuvette with 50 μL of water
alone or 50 μL of tested compound solution in water at a final
concentration of 100 μM. The formation of conjugated dienes
was measured spectrophotometrically in a Varian Cary 50 Bio
spectrophotometer, equipped with a thermostatic control
(37 °C) and an automatically exchangeable multipositions cuv-
ettes holder, operating at 234 nm. Oxidation was initiated by the
addition to the LDL suspension of CuSO4 at a final concentra-
tion of 2.5 μM.
In Vitro HNE Scavenging Studies. HNE Incubation and LC
Analysis. HNE (final concentration 50 μM in 1 mM phosphate
buffer, pH 7.4) was incubated with solution of 7a-n, 12, or with
carnosine (final concentration 1 mM in 1 mM phosphate buffer,
pH 7.4) for different periods (up to 24 h) at 37 °C. Samples for
each different incubation time were directly analyzed by HPLC
to measure HNE consumption, as previously described.6,49
HNE was determined by reverse-phase HPLC using a HP 1100
chromatograph system (Agilent Technologies, Palo Alto, CA,
USA). Reaction mixture (20 μL) were eluted on a Agilent Zorbax
Eclipse XDB-C18 column (150 mm ꢀ 4.6 mm; particle size 5 μm).
The mobile phase was 60% A (water/acetonitrile/formic acid;
9:1:0.01, v/v/v) and 40% B (water/acetonitrile; 1:9, v/v) delivered
at a flow rate of 1 mL/min. The column effluent was monitored
at 223 nm.
Acknowledgment. This work was supported by Regione
Piemonte, “Progetto di Ricerca Sanitaria Finalizzata”, Bando
Regionale 2009. The help from D. Dallorto and Dr. A. Barge
in the brain-penetration experiments is gratefully acknowl-
edged.
Supporting Information Available:
Elemental analyses,
HPLC and MS detection of 7d in rat brain homogenate. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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sterile conditions, kept in cold HBSS (4 °C) with high glucose,
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