Synthesis of new P3CS derivatives and their mitogenic activity on in vitro mice splenocytes

Article abstract of DOI:10.1016/S0014-827X(02)00012-5

Vaccination against tumors represents a relevant issue in current human cancer therapy. The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-Cys-Ser (P₃CS) and analogs with longer aminoacidic sequence are polyclonal activators for B-lymphocytes. Previous study reported that their N-2,2,2-trichloroethoxycarbonyl (Troc) derivatives increase immunocyte mitogenic activity. Therefore, in order to obtain compounds of greater activity and to investigate relationships between molecular structure of S-glyceryl skeleton and biological activity, we synthesized new Troc derivatives of P₃CS. The mitogenicity of compounds was determined in vitro, by measuring in vitro [³H]-thymidine incorporation into splenocytes from Balb/c mice. Concentrations of compounds ranged from 0 to 64 μg/ml. In particular, S-[2,3-bis(trichloroethoxycarbonyloxy)]-N-trichloroethoxycarbonyl dipeptide derivative exhibited significant mitogenic activity endowed with high pharmacological potency. These new series of compounds could be used as potent immunoadjuvants for the development of novel synthetic vaccines for tumor immunotherapy.

Full text of DOI:10.1016/S0014-827X(02)00012-5

Il Farmaco 58 (2003) 329ꢀ336
/
www.elsevier.com/locate/farmac
Synthesis of new P3CS derivatives and their mitogenic activity on in
vitro mice splenocytes
Maria Pappalardo a,*, Ennio Bousquet a, Agata Annino a, Gabriella Lombardo b,
Renato Bernardini b, Giuseppe Ronsisvalle a
a Department of Pharmaceutical Science, School of Pharmacy, School of Medicine, University of Catania, Viale Andrea 6, 95125 Catania, Italy
b Department of Pharmacology, School of Medicine, University of Catania, Viale Andrea 6, 95125 Catania, Italy
Received 7 February 2002; accepted 18 October 2002
Abstract
Vaccination against tumors represents a relevant issue in current human cancer therapy. The N-terminal part of the lipoprotein
from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-Cys-Ser (P3CS) and analogs with longer aminoacidic sequence
are polyclonal activators for B-lymphocytes. Previous study reported that their N-2,2,2-trichloroethoxycarbonyl (Troc) derivatives
increase immunocyte mitogenic activity. Therefore, in order to obtain compounds of greater activity and to investigate relationships
between molecular structure of S-glyceryl skeleton and biological activity, we synthesized new Troc derivatives of P3CS. The mito-
genicity of compounds was determined in vitro, by measuring in vitro [3H]-thymidine incorporation into splenocytes from Balb/c
mice. Concentrations of compounds ranged from 0 to 64 mg/ml. In particular, S-[2,3-bis(trichloroethoxycarbonyloxy)]-N-
trichloroethoxycarbonyl dipeptide derivative exhibited significant mitogenic activity endowed with high pharmacological potency.
These new series of compounds could be used as potent immunoadjuvants for the development of novel synthetic vaccines for tumor
immunotherapy.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Tripalmitoyl-S-glyceryl-Cys-Ser derivative; Tumor immunotherapy; Lipopeptides; Immunoadjuvant; Synthetic vaccine; Mitogenicity
1. Introduction
aminoacids [4], bearing three N-terminal fatty acids
bound to glycerocysteine as part of a biologically active
region [5]. The natural lipoprotein contains a mixture of
different fatty acids, palmitic acid being the main
component, whereas the synthetic analog S-[2,3-bis(pal-
mitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-
(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine (P3Cys-
Ser-Ser-Asn-Ala) contains palmitoyl residues only.
The synthetic lipopentapeptide P3Cys-Ser-Ser-Asn-
Ala exhibits the same mitogenic properties as the native
lipoprotein and being chemically synthesized, does not
contain endotoxin contaminations as found in various
other bacterial products [6,7]. Variations in the native
lipopeptide structure related to chain length and amino
acid sequence of the peptide moiety, as well as mod-
ifications of the lipoaminoacid P3Cys-OH, lead to
reduction or even complete loss of the adjuvant activity
[8].
The lipopeptides derived from the N-terminal portion
of Braun’s lipoprotein, the major component of the
outer membrane of Escherichia coli and other Enter-
obacteriacee, are potent B-lymphocyte activators [1ꢀ3].
/
They constitute novel and potent immunoadjuvants in
mice, rabbit and other species, markedly enhancing the
immune response when covalently coupled to antigens
or given in mixture with conventional antigens, inducing
antigen-specific antibodies in vivo and in vitro.
The lipopeptides are non-toxic, non-pyrogenic immu-
noadjuvants, unable to induce tissue damage when
injected; thus, they are ideal candidate molecules to
replace Freund’s adjuvant avoiding side effects of the
latter. The Braun’s lipoprotein is composed of 58
Moreover, the minimal structure for biological activ-
ity is showed by the lipodipeptide N-palmitoyl-S-[2,3-
* Corresponding author.
E-mail address: mrspld@mbox.unict.it (M. Pappalardo).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 0 0 1 2 - 5

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M. Pappalardo et al. / Il Farmaco 58 (2003) 329ꢀ336
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on lipopentapeptide derivatives with altered glycerol
skeleton, suggests that glycerol moiety could play a
more relevant role with respect to cysteinyl part in
recognition process of synthetic lipopeptides [16,17]. It
has also been reported that introduction of 2,2,2-
trichloroethoxycarbonyl (Troc) group on cysteine resi-
due of lipotetra- and pentapeptide greatly enhances the
mitogenic activity [17ꢀ20], likely as a consequence of
/
drastic alterations of physical properties, such as
solubility and micellar state. Therefore, in order to
acquire more information about the relationship be-
tween molecular requirements of S-glycerocysteine
moiety and interactions with the lymphocyte plasma
membrane, we have synthesized new derivatives of
lipodipeptide P3CS (2ꢀ3 Chart 1) characterized by
/
structural modifications obtained with insertion of
Troc groups into the glycerol moiety.
We have also synthesized the compound with Troc
group on cysteine residue (1 Chart 1) for a comparison
of the biological activity with the other lipodipeptides
synthesized.
In addition, to determine whether the compounds
synthesized could exert immunostimulating effects, we
have tested their mitogenic activity on dispersed mouse
splenocytes in culture in vitro.
2. Chemistry
Target compounds were synthesized according to the
reaction sequence shown in Chart 2.
P3CS was obtained as described by Prass et al. [13].
Compound 4 was obtained according to the method
reported by Kurimura et al. [20].
Chart 1. Chemical structure of P3CS derivatives synthesized in this
study.
Alkylation of thiol 4 with racemic 3-bromo-1,2-
propandiol and triethylamine (TEA) gave 6 as mixture
of diastereoisomers. The esterification of diol 6 with
palmitic acid (Pal-OH), N,N?-dicyclohexylcarbodiimide
(DCC) in presence of a catalytic amount of 4-dimethy-
laminopyridine (DMAP) gave 8 which was deprotected
of the tert-butyl group with trifluoroacetic acid (TFA)
affording 11.
Compound 11 was condensed with O-tert-butyl-(S)-
serine tert-butyl ester [H-Ser(But)-(OBut)] by the DCC/
1-hydroxybenzotriazole (HOBT) method to give 14
which was deprotected to afford desired final compound
1.
bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-ser-
ine, P3CS (Chart 1), whereas the lipoaminoacid P3Cys-
OH lacking a polar peptide moiety is almost inactive
[9,10].
Covalent association of lipopeptide immunoadjuvant
P3CS to a glycopeptide has been proposed as a model of
synthetic vaccine and elicited an immune response to
tumor-associated carbohydrate antigens [11,12].
However, little is known on the different contribution
offered to specificity of B-lymphocyte signaling activa-
tion by the plasma membrane interaction of lipid anchor
group and polar headgroup of the mitogenic P3Cys-Ser-
lipopeptide [13,14].
The synthesis of the thiol 5 and its alkylation to give
the compound 7 were carried out according to the
method reported by Wiesmuller et al. [6].
¨
Several studies have reported that activation process
of B-lymphocytes elicited by lipopentapeptide and its
analogs is strongly depending on chirality of asymmetric
carbon atom C-2 of the (S)-glyceryl moiety [6,8,15].
Such evidence, along with the results reported by studies
Esterification of compounds 6 and 7 with 2,2,2-
trichloroethylchloroformate (Troc-Cl) in pyridine af-
forded 9 and 10, respectively.
The deprotection of the tert-butyl groups with TFA
yielded compounds 12 and 13; subsequent coupling with

M. Pappalardo et al. / Il Farmaco 58 (2003) 329ꢀ
/336
331
Chart 2. Preparation of lipopeptides 1ꢀ3.
/
H-Ser(But)-(OBut) was carried out by the DCC/HOBT
method to give 15 and 16.
The final deprotection of the tert-butyl groups by
treatment with TFA yielded desired compounds 2 and 3.
The structures of synthesized compounds were con-
firmed by analysis of the infrared (IR), 13C NMR and
FAB-MS spectra.
standard. FAB-MS was recorded on a Finnigan MHT
90 mass spectrometer. Analytical thin-layer chromato-
graphy was performed on Silica gel plates 60 F254
(Merck) with detection by quenching of UV fluores-
cence and by spraying with 50% NaClO and 1% KI in
soluble starch. Column chromatography was carried out
on Merck Silica gel 60 (mesh size 0.040ꢀ0.063 mm).
/
3.2. S-[2,3-Dihydroxy-(2RS)-propyl]-N-2,2,2-
trichloroethoxycarbonylcysteine tert-butyl ester (6)
3. Experimental
3.1. Chemistry
3-Bromo-1,2-propanediol (5.6 g, 36.1 mmol) and
TEA (3.6 g, 36.1 mmol) were added to N-2,2,2-
trichloroethoxycarbonylcysteine tert-butyl ester (4)
(1.27 g, 3.6 mmol) in dry dimethylformamide (DMF)
13C NMR spectra were recorded on a Varian Inova-
200 spectrometer, using tetramethylsilane as an internal

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M. Pappalardo et al. / Il Farmaco 58 (2003) 329ꢀ336
/
(30 ml). After stirring for 15 min at 80 8C and 15 h at
r.t., CH2Cl2 (300 ml) was added to the reaction mixture.
The CH2Cl2 solution was washed with 5% citric acid
(Troc-CH2); 95.2 (Troc-CCl3); 153.9 (Troc-CO); 173.3
(Pal-CO); 174.6 (Cys-COOH).
(3ꢁ
/
150 ml) and water (saturated with N2). After drying
3.5. S-[2,3-Bis-(palmitoyloxy)-(2RS)-propyl]-N-
2,2,2-trichloroethoxycarbonyl-(R)-cysteinyl-O-tert-
butyl-(S)-serine tert-butyl ester (14)
over sodium sulfate, the solvent was removed in vacuo.
The oily residue was purified by silica gel column
chromatography using CHCl3/MeOH/n-Hexane (15:1:1
v/v/v) as eluent to give 6 (1.15 g, 75%) as an yellow oil.
IR (neat)/cm: 3330, 1739. 13C NMR (CDCl3) d: 27.9
(COOtBu-CH3); 35.7 (S-glyceryl-CH2); 36.6 (Cys-CH2);
52.6 (Cys-CH); 64.9, 65.2 (S-glyceryl-OCH2); 70.2 (S-
glyceryl-CH); 74.6 (Troc-CH2); 83.2 (tBu-Cq); 95.2
(Troc-CCl3); 153.9 (Troc-CO); 168.9 (Cys-CO).
11 (0.25 g, 0.3 mmol) was activated in CH2Cl2 (6 ml)
with DCC (70 mg, 0.34 mmol) and HOBT (45 mg, 0.34
mmol) in DMF (3 ml) at 0 8C. After 30 min H-Ser(But)-
OBut (75 mg, 0.34 mmol) was added. After being stirred
for 15 h at r.t., the precipitated dicyclohexylurea was
filtered off and the filtrate was concentrated in vacuo.
The residue was dissolved in AcOEt (5 ml) and
dicyclohexylurea was filtered off again. After evapora-
tion of the solvent, the residue was dissolved in CH2Cl2
3.3. S-[2,3-Bis-(palmitoyloxy)-(2RS)-propyl]-N-
2,2,2-trichloroethoxycarbonyl-(R)-cysteine tert-butyl
ester (8)
(50 ml) and washed with 5% NaHCO3 (3ꢁ
/
25 ml) and
water (3ꢁ25 ml). After drying over Na2SO4, the solvent
/
was evaporated in vacuo. The residue was chromato-
graphed on silica gel with CH2Cl2/MeOH/C6H6 (7:0.5:3
v/v/v) as an eluent to give 14 as a colourless oil (0.18 g,
60%). Rf: 0.6 (ethyl acetate saturated with water).
13C NMR (CDCl3) d: 14 (Pal-CH3); 22.6, 24.9 (Pal-
CH2); 27.3 (OtBu-CH3); 27.9 (COOtBu-CH3); 29.1,
29.4, 29.6, 31.9 (Pal-CH2); 34 (S-glyceryl-CH2); 34.2
(Pal-CH2); 34.3 (Cys-CH2); 53.1 (Cys-CH); 54.5 (Ser-
CH); 61.2 (Ser-CH2); 63.5 (S-glyceryl-OCH2); 70.2 (S-
glyceryl-CH); 74.3 (OtBu-Cq); 74.7 (Troc-CH2); 83.2
(COOtBu-Cq); 95.3 (Troc-CCl3); 154.4 (Troc-CO); 169.8
(Ser-CO); 170 (Cys-CO); 173.3 (Pal-CO).
Pal-OH (1.6 g, 6.24 mmol), DMAP (80 mg, 0.65
mmol) and DCC (1.3 g, 6.3 mmol) were added to a
stirred solution of diol 6 (1.13 g, 2.65 mmol) in CHCl3
(25 ml).
After being stirred for 4 h at r.t., the mixture was
added of CHCl3 (50 ml) and the precipitated dicyclo-
hexylurea was filtered off. The organic solution was
washed with 5% citric acid (3ꢁ/80 ml), water (80 ml),
5% NaHCO3 (3ꢁ80 ml) and water (3ꢁ
/
/80 ml). After
drying over Na2SO4 and evaporating to dryness, the
residue crystallized from CHCl3/MeOH (1:3 v/v) to yield
a colourless product (1.67 g, 70%). m.p. 42ꢀ43 8C. Rf:
/
0.9 (CHCl3/MeOH/n-Hexane 7.5:0.5:0.5 v/v/v).
IR (KBr)/cm: 3300, 1739. 13C NMR (CDCl3) d: 14
(Pal-CH3); 22.6, 24.9 (Pal-CH2); 27.9 (COOtBu-CH3);
29.1, 29.4, 29.6, 31.9 (Pal-CH2); 33.2 (S-glyceryl-CH2);
34.2 (Pal-CH2); 35 (Cys-CH2); 54.5 (Cys-CH); 63.4 (S-
glyceryl-OCH2); 70.2 (S-glyceryl-CH); 74.6 (Troc-CH2);
83.2 (tBu-Cq); 95.2 (Troc-CCl3); 153.9 (Troc-CO); 168.9
(Cys-CO); 173.3 (Pal-CO).
3.6. S-[2,3-Bis-(palmitoyloxy)-(2RS)-propyl]-N-
2,2,2-trichloroethoxycarbonyl-(R)-cysteinyl-(S)-serine
(1)
TFA (3 ml) was added to 14 (0.3 g, 0.3 mmol). After
being stirred for 2 h at r.t., the mixture was concentrated
in vacuo and the residue was chromatographed on silica
gel with CH2Cl2/MeOH/AcOH (9:1:0.01 v/v/v) as an
eluent to give 1 (0.17 g, 60%) as a white powder. Rf: 0.25
3.4. S-[2,3-Bis-(palmitoyloxy)-(2RS)-propyl]-N-
2,2,2-trichloroethoxycarbonyl-(R)-cysteine (11)
(CHCl3/MeOH/AcOH 9:1:0.01 v/v/v). [a]Dꢂ
1.04, CHCl3).
/
ꢃ3.88 (c
/
13C NMR (CDCl3) d: 14 (Pal-CH3); 22.6, 24.9, 29.1,
29.4, 29.6, 31.9 (Pal-CH2); 34 (S-glyceryl-CH2); 34.2
(Pal-CH2); 34.3 (Cys-CH2); 53.1 (Cys-CH); 54.5 (Ser-
CH); 62.4 (Ser-CH2); 63.5 (S-glyceryl-OCH2); 70.2 (S-
glyceryl-CH); 74.7 (Troc-CH2); 95.3 (Troc-CCl3); 154.8
(Troc-CO); 170.1 (Cys-CO); 173.3 (Pal-CO); 173.7 (Ser-
TFA (3 ml) was added to 8 (0.27 g, 0.3 mmol) at r.t.
After being stirred for 2 h, the acid was removed in
vacuo, and the residue dissolved in CH2Cl2 (50 ml).
After washing with water (3ꢁ30 ml), the solution was
/
dried over Na2SO4 and evaporated to dryness. The
residue was chromatographed on silica gel with CHCl3/
MeOH/n-Hexane (7.5:0.5:0.5 v/v/v) as an eluent to give
11 as a colourless oil (0.22 g, 90%). Rf: 0.4 (ethyl acetate
saturated with water).
13C NMR (CDCl3) d: 14 (Pal-CH3); 22.6, 24.9 (Pal-
CH2); 29.1, 29.4, 29.6, 31.9 (Pal-CH2); 33 (S-glyceryl-
CH2); 34.2 (Pal-CH2); 34.3 (Cys-CH2); 54 (Cys-CH);
63.4 (S-glyceryl-OCH2); 70.2 (S-glyceryl-CH); 74.6
COOH). FAB-MS m/z: 933 (Mꢃ
H)ꢃ.
/
3.7. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteine
tert-butyl ester (9)
2,2,2-Trichloroethoxycarbonyl chloride (0.21 g, 1
mmol) was added dropwise under stirring to the

M. Pappalardo et al. / Il Farmaco 58 (2003) 329ꢀ
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333
solution of diol 6 (0.43 g, 1 mmol) in pyridine (8 ml) at
0 8C. After being stirred for 45 min at 0 8C, H2O (2 ml)
was added. After being stirred for 15 min at r.t., the
solution was washed with 5% citric acid, water and
evaporated in vacuo. The oily residue was subjected to
column chromatographed on silica gel with C6H6/Ethyl
acetate (6:1 v/v) as an eluent to give 9 as a colourless oil
(0.5 g, 65%). Rf: 0.4 (C6H6/Ethyl acetate 6:1 v/v).
13C NMR (CDCl3) d: 27.9 (COOtBu-CH3); 33.4, 33.6
(S-glyceryl-CH2); 35.1, 35.3 (Cys-CH2); 54.5 (Cys-CH);
67.3 (S-glyceryl-OCH2); 74.6 (Troc-CH2); 75.4 (S-
glyceryl-CH); 83.5 (COOtBu-Cq); 95.2 (Troc-CCl3);
153.3, 153.6, 153.9 (Troc-CO), 168.8 (Cys-CO).
95.2 (Troc-CCl3); 153.3, 153.6, 154.2 (Troc-CO), 169.8
(Ser-CO); 170.2 (Cys-CO).
3.10. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteinyl-
(S)-serine (3)
TFA (3 ml) was added to 15 (0.27 g, 0.3 mmol). After
being stirred for 30 min at r.t., the mixture was
concentrated in vacuo and the residue was chromato-
graphed on silica gel with CH2Cl2/MeOH/AcOH
(9:1:0.01 v/v/v) as an eluent to give 3 (0.14 g, 60%) as
a white powder. Rf: 0.35 (CHCl3/MeOH/AcOH 9:1:0.01
v/v/v). [a]Dꢂ
/
ꢃ9.28 (c 1.3, CHCl3)
/
13C NMR (CDCl3) d: 32.6 (S-glyceryl-CH2); 34.6
(Cys-CH2); 54.1 (Cys-CH); 54.5 (Ser-CH); 62.4 (Ser-
CH2); 67.5 (S-glyceryl-OCH2); 74.6 (Troc-CH2); 75.4
(S-glyceryl-CH); 95.2 (Troc-CCl3); 153.3, 153.6, 154.2
(Troc-CO), 170.5 (Cys-CO); 174.8 (Ser-COOH). FAB-
3.8. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteine
(12)
TFA (3 ml) was added to 9 (0.23 g, 0.3 mmol) at r.t.
After being stirred for 30 min, the acid was removed in
vacuo. The residue was purified by precipitation from
MS m/z: 808 (Mꢃ
H)ꢃ.
/
diethyl ether/light petroleum 30ꢀ50 8C (1:4 v/v) and
/
3.11. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-palmitoyl-(R)-cysteine tert-butyl ester (10)
dried in vacuo with diphosphorus pentoxide. Yield 0.15
g, 70%. Rf: 0.2 (CH2Cl2/MeOH/AcOH 9:1:0.01 v/v/v).
13C NMR (CDCl3) d: 32.3, 32.4 (S-glyceryl-CH2);
34.5, 34.7 (Cys-CH2); 53.6, 53.8 (Cys-CH); 67.3 (S-
glyceryl-OCH2); 74.6 (Troc-CH2); 75.4 (S-glyceryl-CH);
95.2 (Troc-CCl3); 153.3, 153.6, 154.2 (Troc-CO), 173.7
(Cys-COOH).
2,2,2-Trichloroethoxycarbonyl chloride (0.21 g, 1
mmol) was added dropwise under stirring to the
solution of diol 7 (0.5 g, 1 mmol) in pyridine (8 ml) at
0 8C.
After being stirred for 45 min at 0 8C, H2O (2 ml) was
added. After being stirred for 15 min at r.t., the solution
was washed with 5% citric acid, water and evaporated in
vacuo.
The oily residue was subjected to column chromato-
graphed on silica gel with CHCl3/MeOH/C6H6
(8.5:0.2:0.2 v/v/v) as an eluent to give 10 as a colourless
oil (0.55 g, 65%). Rf: 0.85 (CHCl3/MeOH/n-Hexane
15:1:1 v/v/v).
13C NMR (CDCl3) d: 14 (Pal-CH3); 22.6, 25.5 (Pal-
CH2); 27.9 (COOtBu-CH3); 29.3, 29.4, 29.6, 31.9 (Pal-
CH2); 35.3 (S-glyceryl-CH2); 35.4 (Cys-CH2); 36.5 (Pal-
CH2); 52.5 (Cys-CH); 68.3 (S-glyceryl-OCH2); 75.2 (S-
glyceryl-CH); 76.3 (Troc-CH2); 83.4 (COOtBu-Cq); 93.8
(Troc-CCl3); 153.4 (Troc-CO); 169.5 (Cys-CO); 173.2
(Pal-CO).
3.9. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteinyl-
O-tert-butyl-(S)-serine tert-butyl ester (15)
12 (0.21 g, 0.3 mmol) was activated in CH2Cl2 (6 ml)
with DCC (70 mg, 0.34 mmol) and HOBT (45 mg, 0.34
mmol) in DMF (3 ml) at 0 8C. After 30 min H-Ser(But)-
OBut (75 mg, 0.34 mmol) was added. After being stirred
for 15 h at r.t., the precipitated dicyclohexylurea was
filtered off and the filtrate was concentrated in vacuo.
The residue was dissolved in AcOEt (5 ml) and
dicyclohexylurea was filtered off again. After evapora-
tion of the solvent, the residue was dissolved in CH2Cl2
(50 ml) and washed with 5% NaHCO3 (3ꢁ/25 ml) and
water (3ꢁ25 ml).
/
After drying over Na2SO4, the solvent was evaporated
in vacuo. The residue was chromatographed on silica gel
with CHCl3/MeOH (9:1 v/v) as an eluent to give 15 as a
colourless oil (0.17 g, 60%). Rf: 0.5 (CHCl3/MeOH 9:1
v/v).
13C NMR (CDCl3) d: 27.3 (OtBu-CH3); 27.9
(COOtBu-CH3); 34.6 (S-glyceryl-CH2); 35.5 (Cys-
3.12. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-palmitoyl-(R)-cysteine (13)
TFA (3 ml) was added to 10 (0.28 g, 0.3 mmol) at r.t.
After being stirred for 30 min, the acid was removed
in vacuo and the residue dissolved in CH2Cl2 (50 ml).
After washing with water (3ꢁ30 ml), the solution was
/
CH2); 53.1 (Cys-CH); 53.8ꢀ
CH2); 67.3 (S-glyceryl-OCH2); 74.3 (OtBu-Cq); 74.6
(Troc-CH2); 75.4 (S-glyceryl-CH); 83.5 (COOtBu-Cq);
/
54.5 (Ser-CH); 61.2 (Ser-
dried over Na2SO4 and evaporated to dryness to give 13
as a colourless oil (0.22 g, 95%). Rf: 0.2 (CH2Cl2/MeOH/
C6H6 8:1:2 v/v/v).

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M. Pappalardo et al. / Il Farmaco 58 (2003) 329ꢀ336
/
13C NMR (CDCl3) d: 14 (Pal-CH3); 22.6, 25.5 (Pal-
4. Biological activity
CH2); 29.3, 29.4, 29.6, 31.9 (Pal-CH2); 32.3 (Cys-CH2);
34.2 (S-glyceryl-CH2); 36.5 (Pal-CH2); 52.5 (Cys-CH);
68.3 (S-glyceryl-OCH2); 74.6 (Troc-CH2); 75.2 (S-
glyceryl-CH); 93.8 (Troc-CCl3); 153.4 (Troc-CO);
173.2 (Pal-CO); 173.5 (Cys-COOH).
4.1. Preparation of immunoadjuvants in DMF/medium
(DM)
The lipopeptide was dissolved in a minimum quantity
of DMF. Then, the solution was diluted in medium,
constituted by RPMI-1640 (GIBCO, Life Technologies,
Paisley, Scotland) and 10% fetal calf serum (FCS,
GIBCO), to obtain a solution 0.5% in DMF, not toxic
for the cells.
3.13. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-palmitoyl-(R)-cysteinyl-O-tert-butyl-(S)-
serine tert-butyl ester (16)
13 (0.23 g, 0.3 mmol) was activated in CH2Cl2 (6 ml)
with DCC (70 mg, 0.34 mmol) and HOBT (45 mg, 0.34
mmol) in DMF (3 ml) at 0 8C. After 30 min H-Ser(But)-
OBut (75 mg, 0.34 mmol) was added. After being stirred
for 15 h at r.t., the precipitated dicyclohexylurea was
filtered off and the filtrate was concentrated in vacuo.
The residue was dissolved in AcOEt (5 ml) and
dicyclohexylurea was filtered off again. After evapora-
tion of the solvent, the residue was dissolved in CH2Cl2
4.2. Animals
Adult female Balb/c mice, 6ꢀ
River Italia S.p.A, Calco, LC, Italy). Mice were housed,
6 per cage, in standard conditions (2491 8C, 12 h light/
dark cycle, commercial rat chow and tap water available
ad libitum) for at least 5 days prior to sacrifice.
/
10 weeks old (Charles
/
(50 ml) and washed with 5% NaHCO3 (3ꢁ
/
25 ml) and
4.3. Mouse splenocyte cultures and [methyl-3H]-
thymidine incorporation test
water (3ꢁ25 ml). After drying over Na2SO4, the solvent
/
was evaporated in vacuo. The residue was chromato-
graphed on silica gel with CH2Cl2/MeOH/C6H6 (8:1:2 v/
v/v) as an eluent to give 16 as a colourless oil (0.19 g,
65%). Rf: 0.25 (CH2Cl2/MeOH/C6H6 8:1:2 v/v/v).
13C NMR (CDCl3) d: 14 (Pal-CH3); 22.6, 25.5 (Pal-
CH2); 27.4 (OtBu-CH3); 27.9 (COOtBu-CH3); 29.3,
29.4, 29.6, 31.9 (Pal-CH2); 34.8 (S-glyceryl-CH2); 35.4
(Cys-CH2); 36.5 (Pal-CH2); 52.4 (Cys-CH); 55.1 (Ser-
CH); 61.1 (Ser-CH2); 67.5 (S-glyceryl-OCH2); 73.8
(OtBu-Cq); 74.6 (Troc-CH2); 75.2 (S-glyceryl-CH); 83.4
(COOtBu-Cq); 94.2 (Troc-CCl3); 153.5 (Troc-CO); 169.5
(Ser-CO); 170 (Cys-CO); 173.2 (Pal-CO).
Mice were sacrificed by decapitation, according to
current GLP guidelines. Spleen was sterilely minced
using the tip of a syringe needle (21 gauge) and the
resulting splenocyte suspension was then stratified onto
Ficoll gradient (1:2 w/v) (Ficoll-Paque, Pharmacia-
Biotech, Uppsala, Sweden) and centrifuged at 3000
rpm/15 min, to separate splenocytes from other cellular
elements. The resulting pellet was washed twice in PBS
and suspended in RPMI-1680 medium supplemented
with 10% FCS. A volume of 100 ml (2ꢁ
suspension and 100 ml of each solution containing
graded concentrations (0ꢀ64 mg/ml) of the compounds
/
105 cells) of cell
/
were placed in a 96-well microplate (Tissue Culture Plate
96-well, U-bottom, Falcon, Becton Dickinson Labware,
Oxnard, CA) and incubated at 37 8C for 72 h in a 5%
CO2 atmosphere. Then, cells were incubated for 6 h with
0.625 mCi/well (in 10 ml) of [methyl-3H]-thymidine
(Amersham Life Science, Little Chalfont, TRA-120,
specific activity 5 Ci/mmol), harvested by means of an
automatic apparatus (Skatron INC, Sterling, USA),
collected onto fiber glass filters (Skatron) and mixed
with 3.5 ml of scintillation liquid (Instagel, Beckman).
Radioactivity was counted for 1 min in a b-counter.
Results were expressed as the mean of count per minute.
All experiments were run in triplicate and at least twice.
EC50 was calculated for all used compounds. Percent
increase of mitogenic activity of splenocytes was calcu-
lated on the basis of the maximally active concentration
of each compound and according to the following
formula:
3.14. S-[2,3-Bis-(trichloroethoxycarbonyloxy)-(2RS)-
propyl]-N-palmitoyl-(R)-cysteinyl-(S)-serine (2)
TFA (3 ml) was added to 16 (0.29 g, 0.3 mmol). After
being stirred for 30 min at r.t., the acid was removed in
vacuo and the residue was chromatographed on silica
gel with CH2Cl2/MeOH/AcOH (9:1:0.01 v/v/v) as an
eluent to give 2 as a colourless oil (0.15 g, 60%). Rf: 0.25
(CH2Cl2/MeOH/AcOH 9:1:0.01 v/v/v). [a]Dꢂ
/
ꢃ108 (c
/
0.4, CHCl3).
13C NMR (CDCl3) d: 14 (Pal-CH3); 22.6, 25.5 (Pal-
CH2); 29.3, 29.4, 29.6, 31.9 (Pal-CH2); 34.7 (S-glyceryl-
CH2); 35.2 (Cys-CH2); 36.5 (Pal-CH2); 52.4 (Cys-CH);
55.2 (Ser-CH); 62.7 (Ser-CH2); 67.5 (S-glyceryl-OCH2);
74.6 (Troc-CH2); 75.2 (S-glyceryl-CH); 94.2 (Tro-CCl3);
153.5 (Troc-CO); 170.4 (Cys-CO); 173.2 (Pal-CO); 174.6
(Ser-COOH). FAB-MS m/z: 871 (Mꢃ
H)ꢃ.
/

M. Pappalardo et al. / Il Farmaco 58 (2003) 329ꢀ
/336
335
ꢀ
ꢁ
stimulated3H-Thyincorporation
basal3H-Thyincorporation
Table 1
EC50 values and max. increase of P3CS and analogs
ꢁ100 ꢄ100
Materials tested
EC50 mg/ml
Max.% increase
mg/ml
P3CS
8.6
9.9
134
584
57
64
32
32
4
Lipopeptide 1
Lipopeptide 2
Lipopeptide 3
4.4. Statistical analysis of results
18.4
1.9
167
All data were subjected to one-way analysis of
variance, followed by a Duncan’s test. Significance
was accepted for P B0.05.
/
lipodipeptide 3 reached its maximal effect at signifi-
cantly lower concentrations (4 mg/ml).
All compounds showed a concentration-related cyto-
toxicity, reflected by the decline of their mitogenic
activity, as indicated by the decline of [3H]-Thy incor-
poration at high concentrations (higher than 64 mg/ml)
(Fig. 1).
The lipodipeptide P3CS and its analogs reported in
this study, are characterized by an amphiphilic structure
constituted of a polar headgroup (Ser residue) and a
lipidic anchor (acylic chains).
5. Results and discussion
To test mitogenic activity of synthetic lipodipeptides
3, compared to P3CS activity, we measured [3H]thy-
midine incorporation in Balb/C mice dispersed spleno-
cytes incubated with graded concentrations of each
compound (Fig. 1).
1
/
ꢀ
All the compounds tested exhibited significant, con-
centration-dependent mitogenic activity.
Such structural elements could differently cooperate
in mitogenicity activity of these compounds.
We used the mitogen Concanavalin A [21], known to
stimulate splenocyte proliferation through a specific
interaction with their membranes, as a positive control.
Among compounds tested, lipodipeptide 1 and 3
showed the highest potency.
Thus, the molecular mechanism underlying lipopep-
tide mitogenicity probably involves either a specific
interaction of the polar headgroup with membrane
binding proteins, or an a specific interaction by insertion
of the idrophobic region into the lipid membrane layer.
Based on mitogenic activity profile showed by our
compounds, the trichloroethoxycarbonyl group on cy-
steine residue (lipodipeptides 1 and 3) seems to con-
tribute positively to specific membrane interactions. In
fact the lipodipeptide 1, bearing Troc group only on the
In addition, lipopeptide 3 was about fourfold more
potent than the reference compound P3CS, whereas
lipopeptide 1 was about equivalent to the latter (Table
1).
Maximal [3H]-thymidine incorporation with lipopep-
tides 1 2 and P3CS was achieved, respectively at the
following concentrations: 32, 32 and 64 mg/ml, whereas
/
ꢀ
Fig. 1. Dose-response curves for [3H]thymidine incorporation splenocytes of Balb/c mice after stimulation with P3CS ('), lipopeptides 1 (m), 2 (%),
3 (j), untreat (2) and Concanavalin A (ꢀ).

336
M. Pappalardo et al. / Il Farmaco 58 (2003) 329ꢀ336
/
[4] V. Braun, V. Bosch, Eur. J. Biochem. 28 (1972) 51ꢀ
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Forsch. 153 (1977) 11ꢀ22.
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69.
cysteine residue, exhibits a mitogenic activity signifi-
cantly higher than P3CS; such proliferative effect on
splenocyte responsiveness is further potentiated by the
presence of Troc groups also on S-glyceryl skeleton
(lipodipeptide 3).
/
¨
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606.
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It is plausible to hypothesize that Troc groups
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tions more effectively than palmitoyl chains [22].
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impact on the biological activity. Such experimental
results seem to suggest that in this our class of
compounds the NH-functionality of cysteine residue
could play a critical role if compared to the S-glyceril
part for the activation process specificity.
Lieberknecht, U. Schmidt, J. Med. Chem. 34 (1991)
1969ꢀ
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541.
/
Possible use of P3CS analogs, if admixed or covalently
bound to antigens or haptens, could be envisioned as
potent immunoadjuvants for development of novel
synthetic antitumor vaccines.
¨
Biochim. Biophys. Acta 900 (1987) 116ꢀ
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Acknowledgements
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[17] T. Shimizu, Y. Iwamoto, Y. Yanagihara, M. Kurimura, K.
This study was supported by MURST, Rome, Italy.
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