Conformationally restrained ceramide analogues: effects of lipophilic modifications on the antiproliferative activity.

Article abstract of DOI:10.1016/S0014-827X(02)00002-2

Conformationally restrained analogues of ceramide containing thiouracil or uracil moieties in their polar head, substituted with an ethyl group in their 6-positions, proved to inhibit cell proliferation and induce apoptosis. A series of new thiouracil and

Full text of DOI:10.1016/S0014-827X(02)00002-2

Il Farmaco 58 (2003) 85Á89
/
www.elsevier.com/locate/farmac
Short Communication
Conformationally restrained ceramide analogues: effects of lipophilic
modifications on the antiproliferative activity
Marco Macchia a,*, Michela Antonello a, Simone Bertini a, Valeria Di Bussolo b,
Stefano Fogli c, Elisa Giovannetti c, Filippo Minutolo a, Simona Rapposelli a,
Romano Danesi c
a Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Chimica Bioorganica, Universita` di Pisa, via Bonanno 33, 56126 Pisa, Italy
c Divisione di Farmacologia e Chemioterapia, Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Universita` di Pisa, Via
Roma 55, 56126 Pisa, Italy
Received 13 June 2002; accepted 30 July 2002
Abstract
Conformationally restrained analogues of ceramide containing thiouracil or uracil moieties in their polar head, substituted with
an ethyl group in their 6-positions, proved to inhibit cell proliferation and induce apoptosis. A series of new thiouracil and uracil
analogues of ceramide possessing several 6-alkyl- or 6-arylalkyl-substituents, were synthesized and tested as inhibitors of cell
proliferation. The lipophilic substituents introduced in the 6-position were pure alkyls (n-propyl, n-butyl, i-butyl, neo-pentyl), or
aryl-alkyls (2-phenylethyl). Although a significant antiproliferative activity was maintained in most compounds synthesized, none of
them showed any improvement with respect to their 6-ethyl-substituted counterparts.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Apoptosis; Ceramide analogues; Thiouracil; Uracil; Lipophilicity
1. Introduction
production of intracellular ceramide, both by sphingo-
myelin hydrolysis and by de novo synthesis, is stimu-
lated by stress factors (including ionizing radiation,
cytokines, etc.) occurring outside the cell membrane
[3,4], which eventually cause the apoptotic death of the
cell. As a consequence, the suppression of the produc-
tion of growth inhibitory lipids, such as ceramide, may
lead to uncontrolled cell proliferation, which eventually
develops cancer.
It has actually been verified that there is a close
correlation between cancer cell proliferation and low
levels of intracellular ceramide [5]. Therefore, either
inhibition of the ceramide metabolism (inhibition of
ceramidase) or the use of ceramide mimics would open
promising perspectives in cancer therapy, by restoring
the growth inhibitory effect of endogenous ceramide and
regulating the abnormal growth in tumor cells. Cera-
mide itself cannot be used since it is too lipophilic to
cross the cell membrane. Appropriate ceramide mimics
should be metabolically stable, able to permeate through
the membrane and to reach reasonable intracellular
Ceramide, a lipidic second messenger involved in the
sphingomyelin cycle, plays a crucial role in the processes
of induction of apoptosis and inhibition of cell prolif-
eration [1]. Its metabolism involves a hydrolysis step
operated by the enzyme ceramidase to produce sphin-
gosine, which is then phosphorylated by a specific kinase
to sphingosine-1-phosphate, a metabolite which, unlike
ceramide, stimulates cell growth and proliferation (Fig.
1). The dynamic balance between levels of sphingolipid
metabolites, ceramide and sphingosine-1-phosphate, is
an important factor that determines whether a cell
survives and proliferates or undergoes an apoptotic
process. Another metabolic pathway that produces
ceramide is a de novo synthesis operated by a ceramide
synthase, in response to stress signals [2]. An increased
* Correspondence and reprints.
E-mail address: mmacchia@farm.unipi.it (M. Macchia).
´
0014-827X/02/$ - 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 0 2 - 2

86
M. Macchia et al. / Il Farmaco 58 (2003) 85Á89
/
Fig. 1. Schematic representation of the sphingomyelin cycle and effect of extracellular factors on ceramide generation cell proliferation and death.
concentrations. Some early examples of ceramide mi-
mics, which showed promising results were C2- and C6-
ceramide [5], in which the long side chain at the amidic
portion is replaced by an acetyl or a hexanoyl group,
respectively. Other attempts to produce ceramide ana-
logues concerned the replacement of the alkenyl chain
with a styryl [6] or an allylic fluoride [7], or the
introduction of a sugar moiety, to form a-galactosylcer-
amides [8].
We have recently synthesized new conformationally
restricted analogues of C2-ceramides, compounds 1a
and 2a [9], which, respectively, possess a thiouracil (Xꢀ
S) or a uracil (XꢀO) ring in the place of the polar head
of C2-ceramide. Moreover, they are substituted in the 6-
position of the polar ring with an ethyl side chain (Rꢀ
/
/
/
CH3CH2), closely resembling the structural motif of C2-
ceramide. Both compounds (1a and 2a) showed excellent
antiproliferative properties when tested on the human T-
cell acute leukemia cells CCRF-CEM and they proved
to be practically devoid of any toxicity when adminis-
tered (up to 200 mg/kg) to animal models [9].
On the basis of these results, we have developed a
series of new analogues of compounds 1a and 2a,
bearing bulkier and more lipophilic substituents in the
place of the simple 6-ethyl group. We, therefore,
synthesized thiouracil and uracil derivatives containing
an n-propyl (1b, 2b), an n-butyl (1c, 2c), an i-butyl (1d,
2d), a neo-pentyl (1e, 2e), or a 2-phenylethyl (1f, 2f)
group in their 6- positions.
reacted with the appropriate acid chloride (4bÁ
produce the related b-ketoester 5bÁf, which was not
purified, but was used as a crude intermediate for the
next step. Subsequent condensation of 5bÁf with
thiourea in the presence of sodium ethoxide in refluxing
absolute ethanol afforded thiouracil derivatives 1bÁ
/
f) to
/
/
/f
2. Chemistry
[10]. The thioracil ring was then converted into a uracil
ring by exchanging the sulfur with an oxygen atom
Compounds 1bÁ
/
f and 2bÁf were synthesized as shown
/
in Scheme 1. Ethyl palmitate (3) was treated with
lithium diisopropylamide (LDA) in THF to generate
the corresponding lithium enolate, which was then
provided by reaction of compounds 1bÁ
ing 10% aqueous solution of chloroacetic acid, affording
uracil derivatives 2bÁf [11,12].
/
f with a reflux-
/

M. Macchia et al. / Il Farmaco 58 (2003) 85Á
/
89
87
Scheme 1.
3. Results and discussion
duced in the 6-position of reference compounds 1a and
2a. Furthermore, also in this series, there are no
significant differences between the activities of thiouracil
The antiproliferative activities of compounds 1bÁ
/f
(1bÁ
The decrease in the activity observed with these new
derivatives (1bÁf and 2bÁf) with respect to the pre-
viously reported ones (1a and 2a) [9] might be due to
their increased lipophilicity, which could be responsible
for a lower efficiency in the delivery process of these
molecules inside the tumor cells, where they produce
their antiproliferative effect.
These results tend to indicate that in general sub-
stituents of a simple aliphatic and aromatic nature in the
6-position of thiouracil (type 1) and uracil (type 2)
derivatives do not significantly disturb their antiproli-
ferative activities, although small ethyl groups (1a and
2a) seem to be preferred. As no improvements were
obtained by increasing the lipophilic character of the 6-
substituents, it would be wise in the near future to
synthesize new compounds of both types (1 and 2)
containing in this same position polar moieties, in order
to evaluate the effects of a lowered lipophilicity in this
region on the cell-proliferation inhibitory properties of
these kinds of molecules.
/
f) and uracil (2bÁ
/
f) derivatives.
and 2bÁf were assessed in the human T-cell acute
/
leukemia cells CCRF-CEM, and were compared with
the data previously obtained with compounds 1a and 2a
[9], using C2-ceramide as the reference compound. The
results are reported in Table 1.
/
/
All the new thiouracil (1bÁ
/
f) and uracil (2bÁf)
/
derivatives, which differ from 1a and 2a because they
contain higher homologues of their 6-ethyl substituent,
generally proved to possess appreciable antiproliferative
activities, although they are lower than that of 1a and
2a. Among them, 1d and 2d, containing isobutyl
substituents in their 6-positions, proved to be the most
active of this new series, with IC50s of 13.2 and 8.7 mM,
respectively, a value which is still better than the IC50
obtained with C2-ceramide (31.6 mM). In general the
differences in the activities obtained with the new
compounds are not remarkable and, therefore, it is not
possible to highlight rational effects of the different
character of the alkyl- and arylalkyl-side chains intro-
Table 1
Effects on cell growth (IC50, inhibitory concentration at the 50% effect
level) by compounds 1aÁ
/
f, 2aÁ
/
f and C2-ceramide
4. Experimental
a
Comp.
IC50 (mM)
b
1a
2a
1.79
7.99
50.19
35.29
31.69
50.19
13.29
8.79
15.19
29.19
20.79
15.69
31.69
/
0.2
0.9
7.2
5.0
5.8
4.3
2.3
1.2
1.9
3.5
2.6
1.5
4.6
4.1. Chemistry
b
/
1b
/
Melting points were determined on a Kofler hot-stage
1
apparatus and are uncorrected. H NMR spectra of all
2b
/
1c
/
2c
/
compounds were obtained with a Varian Gemini-200
instrument operating at 200 MHz; the data are reported
as follows: chemical shift (in ppm) from the Me4Si line
as the external standard, multiplicity (br, broad; s,
singlet; d, doublet; t, triplet; m, multiplet). Mass spectra
were recorded on a VG 70-250S mass spectrometer.
Analytical TLCs were carried out on 0.25 mm layer
silica gel plates containing a fluorescent indicator; spots
were detected under UV light (254 nm). Column
1d
/
2d
/
1e
/
2e
/
1f
/
2f
/
b
C2-ceramide
/
a
P B
/
0.05. ANOVA followed by the TukeyÁKramer post test,
/
versus C2-ceramide.
b
Ref. [9].
chromatography was performed using 230Á400 mesh
/

88
M. Macchia et al. / Il Farmaco 58 (2003) 85Á89
/
silica gel (MachereyÁ
/
Nagel Silica Gel 60 Art. no.
4H), 10.20 (br, 1H, D2O exchangeable), 10.45 (br, 1H,
D2O exchangeable). MS (EI, 70 eV): m/z 380 (Mꢁ).
Compound 1e: 859 mg, 2.18 mmol, 31% yield (two
815381). Sodium sulfate was always used as the drying
agent. Evaporations were performed in vacuo (rotating
evaporator). Synthesis and characterization data of
compounds 1a and 2a have already been published [9].
C2-ceramide was purchased from Sigma, St. Louis, MO.
1
steps). M.p. 58Á
0.88 (t, 3H, Jꢀ
24H), 2.31Á2.46 (m, 4H), 8.92 (br, 1H, D2O exchange-
/
60 8C. H NMR (CDCl3, 200 MHz) d
/
6.5 Hz), 1.06 (s, 9H), 1.16Á1.36 (m,
/
/
able), 9.37 (br, 1H, D2O exchangeable). MS (EI, 70 eV):
m/z 394 (Mꢁ).
Compound 1f: 903 mg, 2.11 mmol, 30% yield (two
4.1.1. Synthesis of thiouracil derivatives 1bÁf
/
A solution obtained by dissolving 2.0 g of ethyl
palmitate (7.0 mmol) in 5 ml of anhydrous THF was
added drop by drop, at a temperature of 0 8C under an
argon gas atmosphere, to 4.2 ml of a 2 M solution of
LDA in anhydrous THF. After 30 min of stirring at
0 8C, the reaction mixture was added to a solution
containing 8.5 mmol of the appropriate acyl chloride
1
steps). M.p. 100Á
d 0.88 (t, 3H, Jꢀ
2.96 (m, 6H), 7.18Á
/
102 8C. H NMR (CDCl3, 200 MHz)
/
6.3 Hz), 1.11Á
/
1.40 (m, 24H), 2.27Á
/
/7.34 (m, 5H), 9.65 (br, 1H, D2O
exchangeable), 9.72 (br, 1H, D2O exchangeable). MS
(EI, 70 eV): m/z 428 (Mꢁ).
(4bÁf) in 10 ml of anhydrous THF. The resulting
/
mixture was stirred at room temperature (r.t.) for 12
h, then added to a saturated solution of NH4Cl. The
organic solvent was removed under a vacuum and the
remaining aqueous phase was extracted with diethyl
ether. The combined organic phase was washed with a
saturated aqueous solution of NaCl, dried over anhy-
drous Na2SO4 and then concentrated under a vacuum to
provide a crude residue composed almost exclusively of
4.1.2. Synthesis of uracil derivatives 2bÁ
The appropriate thiouracil derivative 1bÁ
/
f
/
f (0.45
mmol) was treated with a 10% aqueous solution of
chloroacetic acid (11 ml) and the mixture was refluxed
for 16 h. The resulting precipitate was then collected by
suction filtration, washed with absolute ethanol and
diethylether. The crude product so obtained was pur-
ified by silica gel column chromatography using an n-
b-ketoester (5bÁ/f), which was used in the next step
hexaneÁ
uracils 2bÁ
Compound 2b: 52 mg, 0.15 mmol, 33% yield. M.p.
132Á
134 8C. 1H NMR (CDCl3, 200 MHz) d 0.87 (t, 3H,
Jꢀ 6.4 Hz), 1.01 (t, 3H, Jꢀ 7.3 Hz), 1.25 (br, 26H), 2.32
(t, 2H, Jꢀ 7.5 Hz), 2.42 (t, 2H, Jꢀ 7.7 Hz), 8.81 (br,
/EtOAc mixture as the eluant, to afford pure
without further purification. The crude residue was
dissolved in 40 ml of absolute ethanol and then treated
with 8.02 g of thiourea (52.8 mmol) and 14.4 g of
sodium ethoxide (106 mmol). The resulting mixture was
stirred at 90 8C for 1 h, after which time it was cooled to
r.t. and filtered. The filtrate was evaporated under a
vacuum; the residue thus obtained was dissolved in a
10:1 mixture of water and THF. The resulting solution
was cooled to 0 8C and acidified to pH 2 with
concentrated HCl; the precipitate that developed due
to acidification was collected by suction filtration and
washed with small quantities of acetone, providing a
crude residue which was purified by silica gel column
/f.
/
/
/
/
/
1H, D2O exchangeable), 9.74 (br, 1H, D2O exchange-
able). MS (EI, 70 eV): m/z 350 (Mꢁ).
Compound 2c: 57 mg, 0.16 mmol, 35% yield. M.p.
116Á
Jꢀ 6.6 Hz), 0.96 (t, 3H, Jꢀ
28H), 2.32 (t, 2H, Jꢀ 7.8 Hz), 2.43 (t, 2H, Jꢀ
/
120 8C. 1H NMR (CDCl3, 200 MHz) d 0.87 (t, 3H,
/
/
7.1 Hz), 1.16Á
/
1.46 (m,
/
/
7.6 Hz),
8.72 (br, 1H, D2O exchangeable), 9.53 (br, 1H, D2O
exchangeable). MS (EI, 70 eV): m/z 364 (Mꢁ).
Compound 2d: 53 mg, 0.14 mmol, 32% yield. M.p.
chromatography using an n-hexaneÁ
the eluant, to afford pure thiouracils 1bÁ
Compound 1b: 644 mg, 1.76 mmol, 25% yield (two
/EtOAc mixture as
/f.
105Á
Jꢀ 6.4 Hz), 0.99 (d, 6H, Jꢀ
24H), 1.93Á2.03 (m, 1H), 2.31Á
/
108 8C. 1H NMR (CDCl3, 200 MHz) d 0.88 (t, 3H,
1
/
/
6.6 Hz), 1.14Á
/
1.40 (m,
2.36 (m, 4H), 9.08 (br,
steps). M.p. 128Á
d 0.87 (t, 3H, Jꢀ
(br, 26H), 2.35 (t, 2H, Jꢀ
/
130 8C. H NMR (CDCl3, 200 MHz)
6.4 Hz), 1.03 (t, 3H, Jꢀ 7.2 Hz), 1.25
7.4 Hz), 2.43 (t, 2H, Jꢀ 7.5
/
/
/
/
1H, D2O exchangeable), 9.91 (br, 1H, D2O exchange-
able). MS (EI, 70 eV): m/z 364 (Mꢁ).
Compound 2e: 63 mg, 0.17 mmol, 37% yield. M.p.
/
/
Hz), 9.73 (br, 2H, D2O exchangeable). MS (EI, 70 eV):
m/z 366 (Mꢁ).
Compound 1c: 642 mg, 1.69 mmol, 24% yield (two
1
98Á
/
100 8C. H NMR (CDCl3, 200 MHz) d 0.88 (t, 3H,
1
Jꢀ
/
6.3 Hz), 1.00 (s, 9H), 1.15Á
/
1.40 (m, 24H), 2.33Á2.45
/
steps). M.p. 124Á
d 0.87 (t, 3H, Jꢀ
1.10Á1.48 (m, 28H), 2.34 (t, 2H, Jꢀ
/
126 8C. H NMR (CDCl3, 200 MHz)
(m, 4H), 8.37 (br, 1H, D2O exchangeable), 8.85 (br, 1H,
D2O exchangeable). MS (EI, 70 eV): m/z 378 (Mꢁ).
Compound 2f: 72 mg, 0.18 mmol, 39% yield. M.p.
/
6.8 Hz), 0.96 (t, 3H, Jꢀ
/
7.2 Hz),
7.5 Hz), 2.46 (t, 2H,
/
/
Jꢀ 7.6 Hz), 10.12 (br, 2H, D2O exchangeable). MS (EI,
/
118Á
Jꢀ 6.3 Hz), 1.13Á
7.21Á7.32 (m, 5H), 8.79 (br, 1H, D2O exchangeable),
/
120 8C. 1H NMR (CDCl3, 200 MHz) d 0.88 (t, 3H,
1.37 (m, 24H), 2.23Á2.92 (m, 6H),
70 eV): m/z 380 (Mꢁ).
Compound 1d: 748 mg, 1.97 mmol, 28% yield (two
/
/
/
1
/
steps). M.p. 102Á
d 0.87 (t, 3H, Jꢀ
1.12Á1.39 (m, 24H), 1.93Á
/
104 8C. H NMR (CDCl3, 200 MHz)
/
6.4 Hz), 1.00 (d, 6H, Jꢀ
/
6.6 Hz),
2.38 (m,
9.93 (br, 1H, D2O exchangeable). MS (EI, 70 eV): m/z
412 (Mꢁ).
/
/
2.03 (m, 1H), 2.31Á
/

M. Macchia et al. / Il Farmaco 58 (2003) 85Á
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4.2. Assay of cell proliferation [9]
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