Synthesis and evaluation of anti-apoptotic activity of L-carnitine cyclic analogues and amino acid derivatives

Article abstract of DOI:10.1016/j.farmac.2004.01.004

Two series of derivatives were synthesised. In one series (R)-4-hydroxy-2-pyrrolidinone was used as a mimic of cyclic L-carnitine analogue and in the second series 3-amino-2-piperidinone was used as a cyclic ornithine analogue. N-Benzyloxycarbonyl derivatives of some amino acids were also prepared. The newly synthesised compounds were tested for their ability to inhibit Fas-activated apoptosis of human Jurkatt T-cell line. The results confirm the previously described anti-apoptotic activity of carnitine and indicate new carnitine and amino acid analogues (1, 3, 6, 7, 20) that inhibit Fas-induced apoptosis.

Full text of DOI:10.1016/j.farmac.2004.01.004

IL FARMACO 59 (2004) 271–277
www.elsevier.com/locate/farmac
Synthesis and evaluation of anti-apoptotic activity of L-carnitine cyclic
analogues and amino acid derivatives
Renata Fringuelli a,*, M. Pilar Utrilla Navarro b,1, Lara Milanese a, Stefano Bruscoli b,
Fausto Schiaffella a, Carlo Riccardi b, Claudio De Simone c
a Department of Drug Chemistry and Technology, University of Perugia, Perugia, Italy
b Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy
c Department of Infectious Disease, University La Sapienza, Rome, Italy
Accepted 8 January 2004
Abstract
Two series of derivatives were synthesised. In one series (R)-4-hydroxy-2-pyrrolidinone was used as a mimic of cyclic L-carnitine analogue
and in the second series 3-amino-2-piperidinone was used as a cyclic ornithine analogue. N-Benzyloxycarbonyl derivatives of some amino
acids were also prepared. The newly synthesised compounds were tested for their ability to inhibit Fas-activated apoptosis of human Jurkatt
T-cell line. The results confirm the previously described anti-apoptotic activity of carnitine and indicate new carnitine and amino acid
analogues (1, 3, 6, 7, 20) that inhibit Fas-induced apoptosis.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Carnitine; Carnitine cyclic analogues; Apoptosis
1. Introduction
effects under these conditions [8-10] which suggests that it
may be acting as an anti-apoptotic agent [11-15]. In particu-
lar, it has been reported that carnitine inhibits caspase activa-
tion and Fas-induced apoptosis thus resulting in the survival
of T lymphocytes [15].
Apoptosis, programmed cell death (PCD), is a physiologi-
cal process that organisms use to eliminate unwanted cells,
including tumour cells, and that is important in animal devel-
opment, as well as in tissue homeostasis [1]. Moreover,
improperly regulated apoptosis can lead to many pathologi-
cal conditions. Inadequate or diminished apoptosis, associ-
ated with uncontrolled cell growth, results in cell accumula-
tion and is a hallmark of cancer [2]. Excessive apoptosis
leads to organ failure, alteration of homeostatic control and
degenerative disorders, including some neuronal diseases
[3]. In fact, apoptosis has been implicated in the aetiology of
several human diseases, such as Alzheimer’s [4], AIDS [5]
and ischemic injury [6,7]. Clearly, the possibility of modu-
lating, inducting or inhibiting apoptosis, by pharmacological
agents necessitates generating and identifying new agents
with potential anti-apoptosis activity. A number of studies
and clinical trials have shown that L-carnitine has therapeutic
As a part of our investigations aimed at discovering and
developing new apoptosis-modulating compounds and
within a project concerning new L-carnitine analogues, two
series of derivatives have been synthesised. In one series,
(R)-4-hydroxy-2-pyrrolidinone was used as a mimic of cy-
clic L-carnitine analogue; in a second series 3-amino-2-
piperidinone was used as a cyclic ornithine analogue. More-
over, due to the structural similarity between carnitine and
some amino acids (AA), such as glutamic acid, glutamine
and asparagine, N-benzyloxycarbonyl derivatives of some
amino acids were also prepared to test the hypothesis that
benzyloxycarbonyl-group (Z) plays a role in the modulation
of apoptosis [16].
All of the newly synthesised compounds were tested for
their ability to inhibit Fas-activated apoptosis of human Jur-
katt T-cell line. The preliminary results confirm the previ-
ously described anti-apoptotic activity of carnitine and also
indicate new carnitine and AA analogues that inhibit Fas-
induced apoptosis.
* Corresponding author.
E-mail address: fringuel@unipg.it (R. Fringuelli).
On leave from Departamento de Farmacologia, Facultad de Farmacia,
1
Universidad de Granada (Spain)
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.01.004

272
R. Fringuelli et al. / IL FARMACO 59 (2004) 271–277
HO
O
O
Me2N
O
2. Chemistry
O
a
b
OH
OH
OH
N
Z
HN
Z
HN
Z
O
O
O
12
Starting from (R)-4-hydroxy-2-pyrrolidinone (P), (R)-4-
acetoxy-2-pyrrolidinone (1) [17] was obtained by acylation,
(R)-4-[(4-chlorobenzyl)oxy]-2-pyrrolidinone (2) was pre-
pared by alkylation with 4-chlorobenzyl chloride, and (R)-4-
lauroyloxy-2-pyrrolidinone (3) was synthesised by conden-
sation with lauric acid, dicyclohexylcarbodiimide (DCC),
4-dimethylaminopyridine (DMAP) in Py/CH2Cl2. The acy-
lation of (R)-4-hydroxy-1-methyl-2-pyrrolidinone (4) [18]
led to (R)-4-acetoxy-1-methyl-2-pyrrolidinone (5), while the
condensation with lauric acid afforded (R)-4-lauroyloxy-1-
methyl-2-pyrrolidinone (6). (Scheme 1).
Starting from 3-amino-2-piperidinone [19], N-(2-
oxopiperidin-3-yl)dodecanamide (7) was obtained by con-
densation with lauric acid which was then transformed by
acylation into N-acetyl-N-(1-acetyl-2-oxopiperidin-3-yl)
dodecanamide (8). The reaction of 3-amino-1-methyl-2-
piperidinone (9) [20] with lauric acid, under the usual condi-
tions, led to N-(1-methyl-2-oxopiperidin-3-yl)dodecanamide
(10). (Scheme 2).
11
c
Me2N
O
OMe
HN
Z
O
13
Scheme 3. Glutamic acid derivatives
a: paraformaldehyde, p-toluenesulfonic acid, C6H6; b: Me2NH·HCl,
NaOH/H2O; c: CH2N2, CHCl3.
nPrO
O
HO
O
a
NH2
NH2
HN
Z
HN
Z
O
O
15
14
Scheme 4. Glutamine derivatives
a: SOCl2, n-PrOH.
4-{[(Benzyloxy)carbonyl]amino}-5-(dimethylamino)-5-
oxopentanoic acid (12) was prepared starting from
N-[(benzyloxy)carbonyl]glutamic acid [21,22] through the
cyclic intermediate 11, and then converted into 13 by reaction
with diazomethane. (Scheme 3).
Me
H
HO
O
N
O
O
N
b
c
O
O
HN
Z
NHMe
HN
HN
Z
a
Z
17
18
19
HO
O
O
HN
Z
NH2
d
nPrO
O
O
16
HN
Z
NH2
OAc
OCO(CH2)10CH3
20
O
O
O
O
N
H
N
Me
Scheme 5. Asparagine derivatives
a: K2CO3, DMF; b: t-BuOK, MeI, DMF; c: NaBH4, i-PrOH/toluene/H2O;
d: SOCl2, n-PrOH.
6
1
a
c
Starting from N2-[(benzyloxy)carbonyl]glutamine (14),
n-propyl N2-[(benzyloxy)carbonyl]glutaminate (15) was
synthesised by reaction with thionyl chloride and dry
n-propanol. (Scheme 4).
OCO(CH2)10CH3
OH
OH
OAc
d,e,f
a
c
O
O
O
N
H
N
H
N
N
Me
Me
P
4
3
5
b
3-[(Benzyloxy)carbonylamino]-4-hydroxy-N-methylbuta-
was obtained starting from N2-
Cl
namide (19
)
O
[(benzyloxy)carbonyl]asparagine (16), which was at first
transformed into 3-[(benzyloxy)carbonylamino]pyrrolidine-
2,5-dione (17) [23], then N-methylated [24] and successively
reducted with NaBH4 in i-propanol/toluene/water (6:1:1).
Starting from 16, n-propyl N2-[(benzyloxy)carbonyl]-a-
asparaginate (20) was obtained by reaction with thionyl chlo-
ride and dry n-propanol. (Scheme 5).
N
H
2
Scheme 1. (R)-4-Hydroxy-2-pyrrolidinone derivatives
a: Ac2O, Py; b: NaH, 4-chlorobenzyl chloride, DMF; c: DCC, Py, lauric
acid, DMAP, CH2Cl2; d: TBSCl, 1H-imidazole, DMF; e: NaH, Mel, DMF; f:
HCl, MeOH.
N(Ac)CO(CH2)10CH3
O
NHCO(CH2)10CH3
b
N
N
H
O
a
Ac
NH2
O
3. Results and Discussion
7
8
N
H
NHCO(CH2)10CH3
NH2
c,d,e
We here present data showing the anti-apoptotic activity
of newly synthesised compounds.
a
N
O
N
O
Me
Me
9
The previously reported anti-apoptotic activity of car-
nitine was demonstrated in experimental systems where cell
death was induced by triggering Fas. Therefore, experiments
were performed to evaluate the influence that the synthesised
10
Scheme 2. 3-Amino-2-piperidinone derivatives
a: DDC, Py, lauric acid, DMAP, CH2Cl2; b: Ac2O, reflux; c: Et3N, (Boc)2O,
CH2Cl2; d:NaH, MeI, DMF; e: CF3CO2H.

R. Fringuelli et al. / IL FARMACO 59 (2004) 271–277
273
80
70
60
50
40
30
20
10
0
A
*
60
*
*
*
*
*
50
*
*
*
*
*
*
*
40
30
20
10
0
Compound (10 µg/mL)
-
-
C
P
1
2
3
4
5
6
7
8
10
Compound (10
µ
M)
-
-
C
1
3
6
7
11
13 15
20
α
Fas (200 ng/mL)
-
+
+
+
+
+
+
+
+
+
+
+
+
α
Fas (200 ng/mL)
-
+
+
+
+
+
+
+
+
+
+
Fig. 3. Jurkatt cells (106 cell/mL) were incubated in the absence (-) or
presence (+) of 200 ng/mL anti-Fas plus different compound (10 µM) as
shown in the figure. The percentage of cells undergoing apoptosis was
determined by PI staining. Representative data are shown with mean percen-
tage apoptosis SD from three experiments. C = carnitine. * P<0.05
80
70
60
50
40
30
20
10
0
B
*
*
*
*
comparable to that of carnitine. In particular, some of the
five-member (P, 1, 3, 4, 6) and one of the six-member (7)
cyclic analogues, showed a significant inhibitory activity,
while other five-member (2, 5) and six-member (8, 10) cyclic
analogues did not significantly inhibit Fas-induced apoptosis
(Figure 1-A). Results of the experiment, that tested the AA
derivatives (Figure 1-B), indicate that the activities of some
of the compounds (A, 11, 16, 17, 18, 20) were comparable to
that of carnitine, while other compounds (G, Gn, 13, 14, 15,
19) did not significantly inhibit apoptosis.
The compounds were also tested at different concentra-
tions ranging between 1 and 100 µg/mL. The results of a
representative experiment, reported in Figure 2, confirm that
1 and 6 had an inhibitory activity comparable, or slightly
superior, to that of carnitine, while 13 and 15 did not signifi-
cantly inhibit apoptosis.
*
*
*
Compound (10 µg/mL)
Fas (200 ng/mL)
-
-
C
G
11 13 Gn 14 15
A
16 17 18 19 20
α
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Fig. 1. Jurkatt cells (106 cell/mL) were incubated in the absence (-) or
presence (+) of 200 ng/mL anti-Fas plus different compounds (10 µg/mL) as
shown in the figure. The percentage of cells undergoing apoptosis was
determined by PI staining. Representative data are shown with mean percen-
tage apoptosis SD from three experiments. C = carnitine; G = glutamic
acid; Gn = glutamine; A = asparagine. * P<0.05
compounds had on Fas-induced apoptosis. Human Jurkatt
cell line was treated with anti-Fas mAb alone or in combina-
tion with different compounds and apoptosis was evaluated
by propidium iodide (PI) staining and flow cytometry analy-
sis as previously described [25].
To further evaluate the inhibitory activity of some of the
different compounds, experiments were performed using
equimolar concentrations. The results reported in Figure 3
show that anti-Fas-induced apoptosis was inhibited when
carnitine, carnitine cyclic analogues andAA derivatives were
tested at the equimolar concentration of 10 µM. Carnitine
was active at this concentration which was 5 times less than
that used in the experiments reported in Figure 1, in which
10 µg/mL was equal to 50.5 µM. These results thus confirm-
ing that carnitine activity also occurred at lower concentra-
tions (Figure 2). The results in Figure 3 indicate that the
cyclic analogues 1, 3, 6, 7 and the AA derivative 20 showed
anti-apoptotic activity, while 11, 13 and 15 did not inhibit
Fas-induced apoptosis. In contrast to the results reported in
Figure 1, 11 showed a slight but not significant inhibition and
this was due to the lower concentration used with respect to
that reported in Figure 1, in that 10 µM was equal to
2.9 µg/mL. As expected from the results reported in Figure 1,
13 and 15 were not active at the 10 µM concentration. All the
other compounds listed in Figure 3 were significantly active
as in the experiments reported in Figure 1. In comparison
with the 10 µg/mL, 10 µM corresponds to a concentration
reduction of 5 times for carnitine (10 µM is equal to
2 µg/mL), 7 times for 1 (10 µM is equal to 1.42 µg/mL), and
3 times for 3, 6 and 7 (10 µM is about 3.3 µg/mL), thus
Results of representative experiments are shown in Fig-
ures 1, 2 and 3. The results obtained when carnitine cyclic
analogues and AA derivatives were tested at the concentra-
tion of 10 µg/mL are reported in Figure 1. The results from
two different experiments (A and B), confirm previous obser-
vations that carnitine inhibits Fas-induced apoptosis. The
results also indicate that the activities of some of the carnitine
cyclic analogues (A) and some of the AA derivatives (B) are
70
60
50
C
1
40
6
13
30
15
20
10
0
100
10
1
µ
( g/mL)
Fig. 2. Percentage of inhibition of anti-Fas-induced apoptosis in Jurkatt cells
by different compounds in a dose-dependent manner. Percentage of cells
undergoing apoptosis was determined by PI staining. C = carnitine.

274
R. Fringuelli et al. / IL FARMACO 59 (2004) 271–277
indicating that these synthesised compounds (1, 3, 6, 7) have
an activity comparable to that of carnitine.
4-Chlorobenzyl chloride (0.28 g, 1.78 mmol) in dry DMF
(5 mL) was then added dropwise and the mixture was main-
tained at room temperature overnight. The solution was
poured into water, extracted with EtOAc, evaporated to dry-
ness and the residue was purified on Al2O3 eluting with
CHCl3 to give 2 as an amorphous solid (0.10 g, 30%).
1H-NMR (CDCl3) d: 2.45 (1H, dd, J = 18.0 and 2.0 Hz,
CHCHHCO), 2.75 (1H, dd, J = 18.0 and 5.1 Hz, CHCH-
HCO), 3.20 (1H, dd, J = 10.2 and 2.0 Hz, CHCHHNH), 3.50
(1H, dd, J = 10.2 and 5.1 Hz, CHCHHNH), 4.30-4.55 (3H, m,
CHOCH2), 7.10-7.35 (5H, m, aromatic H and NH).
Taking into consideration the small number of new com-
pounds in every series, we could only advance a tentative
hypothesis based on structure-activity relationships. In the
series of (R)-4-hydroxy-2-pyrrolidinone derivatives we can
note that by transforming the hydroxyl group into the corre-
sponding lauroyl ester (compounds 3 and 6) the activity was
maintained or improved slightly depending on the simulta-
neous nitrogen alkylation. Applying this result to the
3-amino-2-piperidinone series, the transformation of the
amino group into lauroyl amide maintained the anti-
apoptotic activity (compound 7) that decreased with the
simultaneous alkylation of the endocyclic nitrogen (com-
pounds 8 and 10).
In the series of amino acids, N-benzyloxycarbonyl deriva-
tives were synthesised to test the hypothesis that the Z-group
plays a role in the modulation of apoptosis but it leads to a
positive effect only in the case of five-member cyclic inter-
mediates (compounds 11, 17 and 18).
In conclusion, the preliminary results here described con-
firm that carnitine antagonises Fas-induced apoptosis and
point out that some new carnitine analogues at least have
similar activity. Future studies will be devoted to improve the
activity by nitrogen quaternarization and to analyse the
mechanism of anti-apoptotic activity.
4.3. (R)-4-Lauroyloxy-2-pyrrolidinone (3)
DCC (0.41 g, 1.98 mmol) and dry Py (0.22 mL) were
added to a solution of lauric acid (0.40 g, 1.98 mmol) in dry
CH2Cl2 (10 mL). A suspension of P (0.20 g, 1.98 mmol) and
DMAP (0.24 g, 1.98 mmol) in dry CH2Cl2 (10 mL) was then
added to the resulting mixture and maintained at room tem-
perature for 48 h. After filtration under vacuum, the resulting
solution was evaporated to dryness and chromatographed on
flash SiO2 eluting with cyclohexane/EtOAc 3:7. 3 was ob-
tained as a white solid which crystallized from MeOH,
0.34 g, 60%, mp 89-90°C. 1H-NMR (CDCl3) d: 0.90 (3H, t,
J = 6.6 Hz, CH3), 1.25-1.50 (16 H, m, aliphatic H), 1.55-1.70
(2H, m, OCOCH2CH2), 2.35 (2H, t, J = 7.6 Hz,
OCOCH2CH2), 2.40 (1H, dd, J = 17.0 and 2.4 Hz, CHCH-
HCO), 2.75 (1H, dd, J = 17.0 and 7.0 Hz, CHCHHCO), 3.40
(1H, dd, J = 11.5 and 1.5 Hz, CHCHHNH), 3.80 (1H, dd,
J = 11.5 and 5.9 Hz, CHCHHNH), 5.40-5.50 (1H, m, CH2CH
CH2), 6.80 (1H, bs, NH).
4. Experimental Procedures
Melting points determined in capillary tubes (Electrother-
mal, Model 9100, melting point apparatus) were uncor-
rected. Element analysis was performed on a Carlo Erba
element analyser 1106, and the data for C, H, and N are
4.4. (R)-4-Hydroxy-1-methyl-2-pyrrolidinone (4)
1
within 0.4% of the theoretical values. H-NMR spectra
It was prepared starting from P according to the literature
were recorded at 200 MHz (Bruker DPX spectrometer) with
Me4Si as internal standard. Chemical shifts are given in ppm
(d) and the spectral data are consistent with the assigned
structures. Column chromatography separations were carried
out on Merck SiO2 40 (mesh 70-230), SiO2 60 (flash, mesh
230-400) or neutral Al2O3 activity III. Reagents and solvents
were purchased from common commercial suppliers and
used as received. Yields of purified product were not opti-
mised. All starting materials were commercially available
unless otherwise indicated.
[18].
4.5. (R)-4-Acetoxy-1-methyl-2-pyrrolidinone (5)
A mixture of 4 (0.17 g, 1.47 mmol), Ac2O (0.15 g,
10.30 mmol) and dry Py (2.5 mL) was stirred at room tem-
perature for 6 h, then poured into water and extracted with
CHCl3. The combined organic layers were washed with a
saturated solution of NaCl, then with water and evaporated to
dryness. The residue was chromatographed on flash SiO2
eluting with CHCl3 to give 5 as a yellow oil (0.13 g, 56.5%).
1H-NMR (CD3OD) d: 2.10 (3H, s, COCH3), 2.40 (1H, dd,
J = 18.0 and 1.5 Hz, CHCHHCO), 2.75-2.90 (4H, m, NCH3
and CHCHHCO), 3.45 (1H, dd, J = 11.5 and 1.6 Hz,
CHCHHNH), 3.85 (1H, dd, J = 11.5 and 5.5 Hz,
CHCHHNH), 5.25-5.35 (1H, m, CH2CH CH2).
4.1. (R)-4-Acetoxy-2-pyrrolidinone (1)
It was prepared starting from (R)-4-hydroxy-2-
pyrrolidinone (P) according to the literature [17].
4.2. (R)-4-[(4-Chlorobenzyl)oxy]-2-pyrrolidinone (2)
4.6. (R)-4-Lauroyloxy-1-methyl-2-pyrrolidinone (6)
NaH (60% mineral oil dispersion, 0.065 g, 1.63 mmol)
was added portionwise to a solution of (0.15 g, 1.48 mmol) in
dry DMF (5 mL) cooled to 0°C, and stirred for 15 min.
DCC (0.36 g, 1.74 mmol) and dry Py (0.2 mL) were added
to a solution of lauric acid (0.35 g, 1.74 mmol) in dry CH2Cl2

R. Fringuelli et al. / IL FARMACO 59 (2004) 271–277
275
(5 mL). A suspension of 4 (0.20 g, 1.74 mmol) and DMAP
(0.21 g, 1.74 mmol) in dry CH2Cl2 (5 mL) was then added to
the resulting mixture and maintained at room temperature for
48 h.After filtration under vacuum, the resulting solution was
evaporated to dryness and chromatographed on flash SiO2
eluting with CHCl3/MeOH 99:1. 6 (0.42 g, 81%) was ob-
tained as a white solid which crystallized from MeOH, mp
CH2Cl2 (15 mL). A suspension of 3-amino-1-methyl-2-
piperidinone 9 (0.25 g, 1.95 mmol) and DMAP (0.24 g,
1.95 mmol) in dry CH2Cl2 (5 mL) was then added to the
resulting mixture and maintained at room temperature for
48 h. After filtration under vacuum the resulting solution was
evaporated to dryness and chromatographed on SiO2 eluting
with CHCl3/MeOH 99:1. 10 (0.42 g, 70%) was obtained as a
white solid which crystallized from cyclohexane/EtOAc, mp
71-72°C. 1H-NMR (CDCl3) d: 0.90 (3H, t, J = 6.8 Hz, CH3),
1.05-2.05 (21H, m, aliphatic H), 2.20 (2H, t, J = 7.8 Hz,
COCH2), 2.60-2.70 (1H, m, CHHCH), 2.95 (3H, s, NCH3),
3.20-3.40 (2H, m, CH2N), 4.15-4.30 (1H, m, CHCH2), 6.55
(1H, bs, CONH).
1
219-220°C. H-NMR (CDCl3) d: 0.90 (3H, t, J = 6.7 Hz,
CH3), 1.25-1.47 (16H, m, aliphatic H), 1.60-1.75 (2H, m,
OCOCH2CH2), 2.30 (2H, t, J = 7.6 Hz, OCOCH2CH2), 2.50
(1H, dd, J = 17.5 and 1.5 Hz, CHCHHCO), 2.85 (1H, dd,
J = 17.5 and 7.0 Hz, CHCHHCO), 2.90 (3H, s, NCH3), 3.35
(1H, dd, J = 11.5 and 1.5 Hz, CHCHHNH), 3.80 (1H, dd,
J = 11.5 and 5.9 Hz, CHCHHNH), 5.25-5.35 (1H, m, CH2CH
CH2).
4.11. 3-{3-[(Benzyloxy)carbonyl]-5-oxo-1,3-oxazolidin-
4-yl}propanoic acid (11)
4.7. N-(2-Oxopiperidin-3-yl)dodecanamide (7)
It was prepared starting from N-[(benzyloxy)carbonyl]-
glutamic acid according to the literature [21].
DCC (0.54 g, 2.63 mmol) and dry Py (0.30 mL) were
added to a solution of lauric acid (0.53 g, 2.63 mmol) in dry
CH2Cl2 (15 mL). A suspension of 3-amino-2-piperidinone
[19] (0.30 g, 2.63 mmol) and DMAP (0.32 g, 2.63 mmol) in
dry CH2Cl2 (10 mL) was then added to the resulting mixture
and maintained at room temperature for 48 h. After filtration
under vacuum, the resulting solution was evaporated to dry-
ness and chromatographed on SiO2 eluting with
CHCl3/MeOH 99:1. 7 (0.55 g, 70%) was obtained as a white
4.12. 4-{[(Benzyloxy)carbonyl]amino}-5-
(dimethylamino)-5-oxopentanoic acid (12)
It was prepared starting from 11 according to the literature
[22].
1
4.13. Methyl 4-{[(benzyloxy)carbonyl]amino}-5-
(dimethylamino)-5-oxopentanoate (13)
solid which crystallized from MeOH, mp 154-156°C. H-
NMR (CDCl3) d: 0.92 (3H, t, J = 6.0 Hz, CH3), 1.05-2.05
(21 H, m, aliphatic H), 2.25 (2H, t, J = 8.0 Hz, COCH2),
2.60-2.70 (1H, m, CHHCH), 3.35-3.40 (2H, m, NHCH2),
4.20-4.40 (1H, m, CHCH2), 6.00 (1H, bs, piperidinonic
NHCO), 6.45 (1H, bs, CONH).
Diazomethane 0.15M solution in Et2O was added drop-
wise to a solution of 12 (0.15 g, 0.49 mmol) until the solution
turned yellow. The mixture was evaporated to dryness and
the residue was chromatographed on flash SiO2 eluting with
cyclohexane/EtOAc 1:1 to give 13 (0.12 g, 76%) as an oil.
1H-NMR (CDCl3) d: 1.70-1.90 and 2.05-2.25 (each 1H, m,
CHCH2CH2), 2.40-2.60 (2H, m, CHCH2CH2), 3.00 and 3.20
(each 3H, s, N(CH3)2), 3.75 (3H, s, OCH3), 4.75-4.85 (1H,
m, CHCH2CH2), 5.15 (2H, s, CH2Ph), 5.80 (1H, d,
J = 8.1 Hz, NH), 7.30-7.40 (5H, m, aromatic H).
4.8. N-Acetyl-N-(1-acetyl-2-oxopiperidin-3-yl)
dodecanamide (8)
A solution of 7 (0.15 g, 0.51 mmol) in Ac2O (5 mL) was
refluxed for 3 h. The solution was poured into water and
extracted with EtOAc. The combined organic layers were
evaporated to dryness and purified on flash SiO2 eluting with
CHCl3/MeOH 9:1 to furnish 8 as a colourless oil (0.04 g,
4.14. n-Propyl N2-[(benzyloxy)carbonyl]glutaminate (15)
1
20%). H-NMR (CDCl3) d: 0.90 (3H, t, J = 6.8 Hz, CH3),
1.05-2.05 (21H, m, aliphatic H), 2.20 (2H, t, J = 7.8 Hz,
COCH2), 2.60-2.70 (1H, m, CHHCH), 2.95 (3H, s, NCH3),
3.20-3.40 (2H, m, CH2N), 4.15-4.30 (1H, m, CHCH2), 6.55
(1H, bs, CONH).
Thionyl chloride (0.25 g, 0.21 mmol) was added dropwise
to
a
cooled suspension of N2-[(benzyloxy)carbonyl]
glutamine 14 (0.20 g, 0.71 mmol) in dry n-propanol (5 mL).
The mixture was allowed to warm to room temperature and
stirred for 4 h. After evaporation to dryness, the residue was
purified by chromatography on SiO2 eluting with
CHCl3/MeOH 9:1 to give 15 (0.15 g, 65%), mp 108-113°C.
1H-NMR (DMSO-d6) d: 0.95 (3H, t, J = 7.5 Hz, CH3),
1.50-1.70 (2H, m, CH2CH3), 1.72-1.90 and 1.92-2.15 (each
1H, m, CHCH2CH2), 2.17-2.25 (2H, m, CHCH2CH2), 3.95-
4.15 (3H, m, CHCH2CH2 and OCH2CH2), 5.10 (2H, s,
CH2Ph), 6.85 and 7.30 (each 1H, bs, CONH2), 7.32-7.45
(5H, m, aromatic H), 7.78 (1H, d, J = 6.1 Hz, NH).
4.9. 3-Amino-1-methyl-2-piperidinone (9)
It was prepared according to the literature with MeI in-
stead of iodomethyl phenylacetate [20].
4.10. N-(1-Methyl-2-oxopiperidin-3-yl)dodecanamide (10)
DCC (0.40 g, 1.95 mmol) and dry Py (0.20 mL) were
added to a solution of lauric acid (0.39 g, 1.95 mmol) in dry

276
R. Fringuelli et al. / IL FARMACO 59 (2004) 271–277
4.15. 3-[(Benzyloxy)carbonylamino]pyrrolidine-2,5-dione
(17) [23]
n-propanol (12 mL). The mixture was allowed to warm to
room temperature and stirred for 4 h. After evaporation to
dryness, the residue was purified by chromatography on SiO2
eluting with CHCl3/MeOH 8:2 to give 20 as a white solid
(0.43 g, 75%), mp 122-125°C. 1H-NMR (DMSO-d6) d: 0.88
(3H, t, J = 7.3 Hz, CH3), 1.45-1.70 (2H, m, CH2CH3),
2.35-2.70 (2H, m, CHCH2), 4.00 (2H, t, J = 6.3 Hz,
OCH2CH2), 4.35-4.55 (1H, m, CHCH2), 5.05 (2H, s,
CH2Ph), 7.00 (1H, bs,), 7.20-7.55 (6H, m, aromatic H and
CONHH), 7.65 (1H, d, J = 8.1 Hz, NH).
K2CO3 (2.85 g, 20.65 mmol) was added to a cooled
solution of N2-[(benzyloxy)carbonyl]asparagine 16 (0.50 g,
1.87 mmol) in dry DMF (10 mL). The mixture was allowed
to warm to room temperature and stirred for 5 h, the solution
was then poured into water, neutralized with 3N HCl and
extracted with EtOAc. The combined organic layers were
evaporated to dryness and the residue was flash chromato-
graphed on SiO2 eluting with CHCl3/MeOH 94:6 to give 17
1
(0.36 g, 77%) as an amorphous solid. H-NMR (CDCl3) d:
4.19. Cell culture
2.75 (1H, dd, J = 18.0 and 5.7 Hz, COCHH), 3.05 (1H, dd,
J = 18.0 and 8.8 Hz, COCHH), 4.35-4.45 (1H, m, CHNH),
5.10 (2H, s, CH2Ph), 6.00 (1H, d, J = 6.8 Hz, CHNH),
7.30-7.40 (5H, m, aromatic H), 9.35 (1H, bs, NH).
The human Jurkatt T cell line was maintained in RPMI
1640 medium (HyClone, Logan, UT) supplemented with
10% FBS (HyClone), 2 mM L-glutamine (Life Technolo-
gies), and 10 µg/mL gentamicin (Life Technologies). Cells
were grown at 37°C in a humidified atmosphere consisting of
5% CO2. For the experiments, cells in log phase growth were
resuspended at a density of 1x106 cells/mL and incubated in
the absence or presence of 200 ng/mL mouse anti-human Fas
mAb (Oncogene, San Diego, CA) for 30 min and subse-
quently incubated with L-carnitine and its analogues.
All compounds were dissolved in DMSO and diluted at
the concentration indicated in the figures with RPMI
1640 medium supplemented with 10% FBS, 2mM
L-glutamine, and 10 µg/mL gentamicin.
4.16. 1-Methyl-3-[(benzyloxy)carbonylamino]pyrrolidine-
2,5-dione (18) [24]
t-BuOK (0.11 g, 0.97 mmol) was added in one portion to a
solution of 17 (0.20 g, 0.81 mmol) in dry DMF (2 mL). The
resulting mixture was stirred for 15 min followed by the
dropwise addition of MeI (0.13 g, 0.97 mmol) in dry DMF
(1 mL). The solution was stirred for an additional 2 h and
then poured into water and extracted with EtOAc. The com-
bined organic layers were evaporated to dryness and the
residue was chromatographed on SiO2 eluting with
CHCl3/MeOH 99:1. 18 (0.17 g, 80%) was obtained as a
white solid which crystallized from MeOH, mp 104-106°C.
1H-NMR (CDCl3) d: 2.80 (1H, dd, J = 17.6 and 5.2 Hz,
COCHH), 2.85-3.10 (4H, m, NCH3 and COCHH), 4.25-4.40
(1H, m, CHNH), 5.15 (2H, s, CH2Ph), 5.75 (1H, d,
J = 5.8 Hz, CHNH), 7.25-7.50 (5H, m, aromatic H).
In all experiments vehicle alone (DMSO) at same concen-
tration of working solution has been used as control.
4.20. Apoptosis evaluation by PI solution
Apoptosis was measured by flow cytometry as described
elsewhere [25]. After culturing, cells were centrifuged and
the pellets were gently resuspended in 1.5 mL hypotonic PI
solution (PI, 50 µg/mL in 0.1% sodium citrate plus 0.1%
Triton X-100, Sigma). Tubes were kept at 4°C in the dark
overnight. The PI-fluorescence of individual nuclei was mea-
sured by flow cytometry with standard FACScan equipment
(Becton Dickinson). The nuclei traversed the light beam of a
488 nmArgon laser.A 560 nm dichroid mirror (DM 570) and
a 600 nm band pass filter (band width 35 nm) were used to
collect the red fluorescence due to PI DNA staining, and data
were recorded in logarithmic scale in a Hewlett Packard (HP
9000, model 310) computer. The percentage of apoptotic cell
nuclei (sub-diploid DNA peak in the DNA fluorescence his-
togram) was calculated with specific FACScan research soft-
ware (Lysis II).
4.17. 3-[(Benzyloxy)carbonylamino]-4-hydroxy-N-
methylbutanamide (19)
NaBH4 (0.22 g, 5.72 mmol) was added portionwise to a
cooled solution of 18 (0.50 g, 1.91 mmol) in
i-propanol/toluene/water (6:1:1). The mixture was stirred at
room temperature for 3 h, then evaporated to dryness. The
residue was purified by flash chromatography on SiO2 elut-
ing with CH2Cl2/MeOH 9:1 to give 19 (0.10 g, 20%) as a
white solid which crystallized from MeOH, mp 164-166°C.
1H-NMR (DMSO-d6) d: 2.10-2.45 (2H, m, CH2CHNH),
2.55 (3H, d, J = 4.5 Hz, NHCH3), 3.20-3.45 (2H, m,
CH2OH), 3.75-3.90 (1H, m, CH2CHNH), 4.75 (1H, t,
J = 5.5 Hz, OH), 5.00 (2H, s, CH2Ph), 7.05 (1H, d, J = 8.3 Hz,
CONHCH), 7.25-7.45 (5H, m, aromatic H), 7.70-7.80 (1H,
m, NHCH3).
4.18. n-Propyl N2-[(benzyloxy)carbonyl]-asparaginate
Acknowledgements
(20)
This study was supported by Sigma Tau – Industrie Far-
maceutiche Riunite S.p.A. and Mendes S.r.l. (contracts:
01/ALSI/2001 and ALSI/2002/C.R./n.3).
Thionyl chloride (0.66 g, 5.64 mmol) was added dropwise
to a cooled suspension of 16 (0.50 g, 1.88 mmol) in dry

R. Fringuelli et al. / IL FARMACO 59 (2004) 271–277
277
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