Preparation of spermine conjugates with acidic retinoids with potent ribonuclease P inhibitory activity

Article abstract of DOI:10.1016/j.ejmech.2009.01.001

Novel mono- and diacylated spermines, readily obtained using isolable succinimidyl active esters of acidic retinoids for the selective acylation of free spermine or in situ activated acidic retinoids for acylating selectively protected spermine followed b

Full text of DOI:10.1016/j.ejmech.2009.01.001

European Journal of Medicinal Chemistry 44 (2009) 2689–2695
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Preparation of spermine conjugates with acidic retinoids with potent
ribonuclease P inhibitory activity
,
b
,
,
George Magoulas a, Dionysios Papaioannou a , Evangelia Papadimou 1, Denis Drainas b
*
**
a Department of Chemistry, School of Natural Sciences, University of Patras, University Campus, GR-26504 Patras, Greece
b Department of Biochemistry, School of Medicine, University of Patras, University Campus, GR-26504 Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel mono- and diacylated spermines, readily obtained using isolable succinimidyl active esters of
acidic retinoids for the selective acylation of free spermine or in situ activated acidic retinoids for
acylating selectively protected spermine followed by deprotection, were shown to inhibit the ribozyme
ribonuclease P more strongly than the parent retinoids.
Received 15 July 2008
Received in revised form
10 November 2008
Accepted 8 January 2009
Available online 19 January 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Retinoids
All-trans-retinoic acid
Acitretin
Polyamines
Spermine conjugates
Ribonuclease P inhibition
1. Introduction
dermatology, oncology, rheumatology and immunology [1–5]. The
clinical application of synthetic retinoids in the management of
Retinoids constitute a large family of organic compounds
structurally related to the naturally occurring Vitamin A and
analogs, such as all-trans-retinoic acid (ATRA, 1), and a variety of
other synthetic analogs, such as acitretin (ACI, 2) (Fig. 1). Retinoids
can cause specific biological responses upon binding to and
activating special receptors or groups of receptors. Natural and
synthetic retinoids play an important role in vision, cell growth,
reproduction, proliferation and differentiation of various epithelial
or non-epithelial tissues. Although they are already widely used in
systemic and topical treatment of various disorders, retinoids reveal
a considerable number of side effects evenwhen used in therapeutic
doses. Thus, numerous new retinoid analogs have been synthesized
in an attempt to improve the therapeutic index, biological profile
and selectivity of these compounds for clinical application in
recalcitrant and previously incurable neoplastic, inflammatory and
keratinization disorders has introduced
dermatology and other medical fields [6,7].
a real revolution in
It has been recently reported that natural retinoids, like retinoic
acid and retinol, as well as synthetic analogs of retinoic acid, e.g.
isotretinoin (13-cis-retinoic acid), ACI and the arotinoids Ro 13-7410,
Ro 15-0778, Ro 15-1570 and Ro 13-6298 but also other compounds,
e.g. calcipotriol, anthralin and their combination, known for their
antipsoriatic activity, inhibit the ribozyme ribonuclease P (RNase P)
[8–12], which has been isolated and characterized from the slime
mold Dictyostelium discoideum [13] and from normal human
epidermal keratinocytes [14]. These findings advocate the hypoth-
esis that retinoids, in addition to regulating DNA transcription, can
also regulate the activity of enzymes playing key-roles in macro-
molecular biosynthesis, by their implication in post-transcriptional
processes, in which binding to the retinoic acid receptors is not
involved. RNase P is responsible for the ripening of the 50 terminus
of precursor tRNA molecules. RNase P activity has been found in
almost every pro- and eukaryotic organisms studied so far [15]. An
exception, concerning the archaeon Nanoarchaeum equitans has
been published recently [16]. RNase P enzymes are complexes of
RNA with proteins and their activity is mainly attributed to their
RNA subunit. Several findings indicate that the structure of the RNA
Abbreviations: ATRA, all-trans-retinoic acid; ACI, acitretin; Ch, cyclohexyl; DCM,
dichloromethane; SPM, spermine; RAR, retinoic acid receptor; RXR, retinoid X
receptor; RNase P, ribonuclease P; RT, room temperature; TFA, trifluoroacetic acid.
* Corresponding author. Tel./fax: þ30 2610 997156.
** Corresponding author. Tel./fax: þ30 2610 997746.
E-mail addresses: dapapaio@chemistry.upatras.gr (D. Papaioannou), drainas@
med.upatras.gr (D. Drainas).
1
Present address: Department of Pharmacological Sciences and Center for Stem
Cell Research, University of Milano, Via Balzaretti 9, 20133 Milano, Italy.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.001

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Fig. 1. Structures of compounds encountered in the present work.
subunit is of similar size in pro- and eukaryotic organisms and that
the structures of RNase P from different eukaryotic organisms are
similar. For these reasons, it appears that RNase P from D. discoideum
and human epidermal keratinocytes are good models for the
identification and development of new inhibitors.
are essential for normal growth and are involved in the differen-
tiation processes of mammalian cells.
Attaching a polyamine on another bioorganic molecule results in
the formation of a polyamine conjugate. Depending on the structure
of the non-polyamine moiety, these conjugates are designed to
exhibit improved biological activity on particular cellular targets or
combine the activities of the constituent molecules. The conjugates
6–10 (Fig. 1) of the present work were synthesized in an attempt to
combine the biological profiles of polyamines and retinoids and are
suitable for structure–activity relationship studies. Thus, in conju-
gates 6 and 7 one molecule of an acidic retinoid (compound 1 or 2) is
attached at N-1 of SPM whereas in conjugates 8–10 two molecules
of an acidic retinoid are attached to either the positions N-1 and N-
12 (conjugates 8 and 9) or the positions N-4 and N-9 of SPM
(conjugate 10). It was anticipated that the polyamine affinity for
nucleic acids, e.g. the RNA part of RNase P, would enforce the binding
of natural and synthetic retinoids on the same molecule. Recently,
N1,N3-diretinoyl-1,3-diaminopropane was synthesized, from 1,3-
diaminopropane and retinoyl chloride, as potential antitumour
agent but showed low cytostatic activity [21].
On the other hand, linear polyamines, like spermine (SPM, 3)
and their compounds with other natural products, collectively
coined as polyamine conjugates, are widely distributed in living
organisms and exhibit interesting biological properties. In order to
determine structure–biological activity relationships and possibly
identify lead compounds for the development of polyamine-based
pharmaceuticals, a variety of polyamine analogs and conjugates
have been synthesized [17–20]. Due to their polycationic nature,
polyamines interact strongly with nucleic acids and play an
important role in their biosynthesis and metabolism. They stabilize
DNA conformation and can induce conformation changes through
the formation of intra- or intermolecular bridges. Polyamines cause
specific modifications of specialized RNA molecules, stabilize
ribonucleases and stimulate the action of ribonucleases and ribo-
zymes. They exert pleiotropic effects on protein synthesis, which

G. Magoulas et al. / European Journal of Medicinal Chemistry 44 (2009) 2689–2695
2691
Scheme 1. Activation of acidic retinoids using HOSu in the presence of DCC. Reagents and conditions: HOSu/DCC, THF or DMF, 30 min at 0 ꢀC then RT overnight, 81–85%.
2. Results and discussion
work were the commercially available all-trans-retinoic acid and
acitretin. Taking into consideration the fact that the succinimidyl
esters of cinnamic acids are hydrolytically stable, can be readily
purified, if necessary, with flash column chromatography (FCC) and
selectively acylate primary amino functions in the presence of
secondary ones [22,23], we chose to activate the acidic retinoids
also in the form of their corresponding ‘active’ esters with N-
hydroxysuccinimide (HOSu). The required succinimidyl esters 4
2.1. Synthesis
Key-reaction in the synthesis of the SPM conjugates described in
the present work is the coupling of an acidic retinoid or a suitably
activated derivative with either free SPM (direct method) or suitably
protected SPM (indirect method). The acidic retinoids used in this
Scheme 2. Selective mono- and diacylation of SPM by isolated syccinimidyl esters of acidic retinoids. Reagents and conditions: (i) [4/5:SPM ¼ 1:3], 0 ꢀC to RT, DCM, 41–45%;
(ii) [4/5:SPM ¼ 2:1], 0 ꢀC, 1 h, DCM, 78–80%.

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Scheme 3. Indirect N4,N9-diacylation of SPM. Reagents and conditions: (i) CF3CO2Et, MeCN, overnight reflux, 93%; (ii) 1/PyBrOP/iPr2NEt, 1.5 h, 0 ꢀC to RT, 70%; (iii) 5 N aq. NaOH/
MeOH, 2 h, RT, 67%.
and 5 were simply obtained (Scheme 1) by treating the acidic
retinoids 1 and 2, respectively, with HOSu in the presence of the
coupling agent N,N0-dicyclohexylcarbodiimide (DCC) together with
small quantities of a less polar byproduct, which was shown by ESI-
MS to be the rearranged compounds (12 and 13, respectively) of the
initial adduct 11 of the retinoids with DCC. The succinimidyl esters
4 and 5 could be readily obtained pure in 81–85% yields, following
purification by FCC. When crude esters 4 and 5 were directly
reacted with SPM, the aforementioned byproducts of the activation
reaction remained in the reaction solvent, as expected, unchanged.
Formation of these N-acylurea byproducts could be completely
suppressed by using a larger excess of HOSu (e.g. acidic reti-
noid:HOSu ¼ 1:2). However, under these conditions, the activation
rate was significantly lower and additional portions of DCC were
necessary to drive the reaction to completion.
Esters 4 and 5 were then used to acylate SPM. Indeed, direct
reaction of active esters 4 and 5 with SPM (in the molar ratio 2:1)
produced the diacylated SPM derivatives 8 and 9 (Scheme 2) which
precipitated out from the reaction mixture as their corresponding
bishydroxysuccinimidate salts. These compounds were thus
obtained pure in 78–80% yields, by simple filtration. We then
turned our attention to the selective monoacylation of SPM on its
primary amino functions. Although an indirect multi-step prepa-
ration of the desired compounds 6 and 7 in rather low yields has
been already reported [24], we considered of interest to examine
the possibility of more direct methods. Indeed, direct reaction of an
excess of SPM with active esters 4 and 5 (ratio of SPM to active
ester ¼ 3:1), followed by RP-HPLC purification of the reaction
mixture thus obtained, afforded the requested compounds 6 and 7,
respectively, as the corresponding tristrifluoroacetate salts in
41–45% yields (Scheme 2).
The preparation of SPM acylated at its secondary amino func-
tions by acidic retinoids is exemplified in Scheme 3 with the
preparation of the SPM conjugate 10. Thus, selective tri-
fluoroacetylation of the primary amino functions of SPM with
CF3CO2Et gave SPM derivative 14 [25,26]. The latter was then
acylated on the remaining free secondary amino functions with
ATRA in the presence of the powerful coupling agent bromo-
tripyrrolidinophosphonium hexafluorophosphate (PyBrOP) and
iPr2NEt, to afford the fully protected SPM derivative 15. From this
compound, the projected conjugate 10 was readily obtained
through routine deprotection of the primary amino function by
alkaline hydrolysis.
2.2. Effect of spermine conjugates with acidic retinoids
on RNase P activity
We have developed a method through which we can estimate
rapidly the biological activity of the spermine–acidic retinoid
conjugates. This method is based on the effect of the polyamine–
retinoid conjugates on RNase P activity and is described in detail in
Section 4. Due to the high hydrophobicity of the conjugates, all
Fig. 2. Double reciprocal plot (1/v versus 1/[pre-tRNA]) for RNase P reaction in the presence of conjugate 8. The reaction was carried out at the indicated concentrations in the
presence or absence of inhibitor. All reactions were carried out at 37 ꢀC in 20
L buffer D in the presence of 10% DMSO. (A) Without inhibitor, with conjugate 8 at (,) 2 M, (-)
M, (:) 4 M, (C) 5 M. Right panel: Replot of the slopes of the double reciprocal lines versus inhibitor (I) concentrations.
m
m
3
m
m
m

G. Magoulas et al. / European Journal of Medicinal Chemistry 44 (2009) 2689–2695
2693
Table 1
and monoacylation seem to lead to compounds presenting weaker
inhibition. The synthesis and the biological evaluation of additional
conjugates of ATRA and ACI, as well as other acidic retinoids, with
other polyamines are currently in progress to determine the effect
of the nature of the acidic retinoid and of the polyamines on the
inhibitory activity of these conjugates on RNase P.
Equilibrium constants derived from primary and secondary plots.a
Retinoids
Ki (m
M)
Spermine conjugates with acidic retinoids
Ki (mM)
Retinol
ATRA
Isotretinoin
ACI
Ro 13-7410
Ro 15-0788
Ro 15-1570
Ro 13-6298
ATRA þ SPM
1475b
15
SPM–ATRA conjugate 6
SPM–ATRA conjugate 8
SPM–ATRA conjugate 10
SPM–ACI conjugate 7
SPM–ACI conjugate 9
2.4
0.5
1.1
2.45
0.95
20b
8
45c
2800c
3600c
4350c
15
4. Experimental section
4.1. General
a
The Ki values are calculated from the negative intercept of the slope replots (see
Capillary melting points were taken on a Bu¨chi SMP-20 appa-
ratus and are uncorrected. IR spectra were recorded as KBr pellets,
unless otherwise stated, on a Perkin–Elmer 16PC FT-IR spectro-
Fig. 2).
b
Ref. [8].
Ref. [9].
c
photometer. 1H NMR spectra were obtained at 400.13 MHz and 13
C
NMR at 100.62 MHz on a Bruker Avance 400 DPX spectrometer at
ambient temperature with the exception of compounds 8 and 9
which were recorded at 70 ꢀC. Electron-Spray Ionization (ESI) mass
spectra were obtained on a Micromass-Platform LC spectrometer
for solutions of the measured compounds in MeOH. The high
resolution MS (HRMS) experiments were carried out in a hybrid Q-
Star Pulsar-i (MDS Sciex Applied Biosystems, Toronto, Canada) mass
spectrometer equipped with an ion spray ionization source.
Microanalyses were performed on a Carlo Erba EA 1108 Elemental
Analyzer. Preparative HPLC was performed on a Waters system
equipped with a 600E system controller using a Lichrosorb RP-18
assays were carried out in the presence of 10% DMSO. At this
concentration, DMSO marginally affects (Ki ¼ 5 M) the catalytic
parameters of D. discoideum RNase P [8]. All synthesized conjugates
exert distinct inhibitory effects on D. discoideum RNase P activity.
The type of this inhibition was elucidated by a detailed kinetic
analysis. In order to carry out such analysis, the initial velocity in
the presence or absence of the conjugate was determined from the
initial slopes of time plots (not shown). The data were plotted in
double reciprocal plots (1/v versus 1/[S]) with increasing concen-
trations of the conjugates and the Ki values (the dissociation
constant of the inhibitor (I), where I is a spermine–acidic retinoid
conjugate, with the enzyme) were determined from the replots of
the slopes of the double reciprocal plots versus the inhibitor’s
concentration. These replots lead to the graphical determination of
Ki values from the negative intercept of the line with the I-axis.
Fig. 2 shows the double reciprocal plot and the slope replot (right
panel) for conjugate 8. The linearity of the slope replot is indicative
of simple competitive inhibition and leads to the graphical deter-
reversed phase preparative column (30 ꢁ 300 mm) with a 10
mm
Bondapak C18 packing material. Separations were achieved with
a stepped linear gradient of acetonitrile (containing 0.08% TFA) in
water (containing 0.08% TFA) over 60 min at a flow rate of 9 mL/
min. Elution of the compounds was determined from the absor-
bances at 254 and 230 nm (Waters 996 Photodiode Array Detector).
Compound purity was assessed by analytical HPLC (Nucleosil-120
C18, 250 ꢁ 4.00 mm) with a linear gradient of 20–100% acetonitrile
(containing 0.08% TFA) in water (containing 0.08% TFA) over 30 min
at a flow rate of 1 mL/min. Lyophilization of the pure products was
performed by a Labconco Freezone 4.5 system. Flash Column
Chromatography (FCC) was performed on Merck silica gel 60 (230–
400 mesh) and Thin Layer Chromatography (TLC) on Merck silica
gel F254 films (0.2 mm) precoated on aluminium foil. The solvent
systems used were: (A) PhMe/EtOAc (95:5), (B) PhMe/EtOAc (9:1),
(C) PhMe/EtOAc (1:1), (D) CHCl3/MeOH/conc. NH3 (6:4:0.4), (E)
CHCl3/MeOH/conc. NH3 (5:5:0.5). Spots were visualized with UV
light at 254 nm and ninhydrin (where applicable). All solvents used
were dried according to standard procedures prior to use. Experi-
ments involving retinoids were routinely conducted under an
atmosphere of Ar and with protection from light. Drying of solu-
tions was effected with anhydrous Na2SO4, whereas evaporation of
the solvents was performed under reduced pressure in a rotary
evaporator at a bath temperature not exceeding 40 ꢀC.
mination of Ki ¼ 0.5
mM. Similar plots were constructed for all
conjugates. This analysis showed that the type of inhibition of all
conjugates is simple competitive. According to this finding, we
could assign the potency of conjugates solely on the basis of the Ki
value, which is a very good measure for the accurate potency of the
spermine–acidic retinoid conjugates. The Ki values of the effect of
the conjugates on D. discoideum RNase P activity are presented in
Table 1 in comparison to known natural and synthetic retinoids [8]
and arotinoids [9]. Thus, among the conjugates 6–10, the conjugate
8 appears to be the strongest inhibitor. Notably, conjugate 8 is 90,
40, 30 and 16 times more potent than Ro 13-7410, isotretinoin,
ATRA and ACI, respectively. Also, double acylation of SPM (conju-
gates 8 and 9) seems to result in stronger inhibition than single
acylation (conjugates 6 and 7), whereas double acylation of primary
amino functions (e.g. conjugate 8) is more effective than double
acylation of the secondary ones (conjugate 10). Finally, SPM
conjugate 8 is a more effective inhibitor than a combination of the
unconjugated molecules ATRA and SPM.
4.2. General method for the preparation of succinimidyl
esters of acidic retinoids
3. Conclusion
To an ice-cold solution of acidic retinoid 1 or 2 (10 mmol) in dry
THF or DMF (30 mL) were added sequentially HOSu (1.73 g,
15 mmol) and DCC (2.48 g, 12 mmol) and the resulting mixture was
stirred for an additional 30 min at 0 ꢀC and for overnight at ambient
temperature. The precipitated DCU was filtered off and washed
several times with EtOAc. The combined filtrates were washed
sequentially with an ice-cold 5% aqueous (aq.) NaHCO3 solution,
H2O and twice with brine. Drying, followed by filtration and
evaporation of the solvent left a residue. FCC of the residue fol-
lowed by evaporation of the eluant and trituration with Et2O gave
the succinimidyl esters as yellowish crystalline compounds. The
Spermine conjugates (amides) with acidic retinoids, like ATRA
and ACI, were for the first time prepared through the condensation
of either free or selectively protected SPM with the succinimidyl
active esters of the retinoids or with the free acids in the presence
of the coupling agent PyBrOP, followed by deprotection. These
conjugates were invariably stronger inhibitors of the enzyme RNase
P, isolated from the slime mold D. discoideum, than the parent
compounds. The most potent inhibitor in this series was the
diacylated derivative of SPM at the external (primary) amino
functions. Diacylation of the internal (secondary) amino functions

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G. Magoulas et al. / European Journal of Medicinal Chemistry 44 (2009) 2689–2695
byproducts 12 and 13 of the reactions were also isolated in 6–7%
yields as yellow solids and had Rf (A): 0.36, MS (ESIþ): m/z 555.54
(MNa), 533.58 (MH) (for 12) and Rf (B): 0.44, MS (ESIþ): m/z 529.44
(MNa), 507.53 (MH) (for 13).
(COCH2CH2CO), 22.1 (C-18), 19.4 (C-13), 13.7, 13.2 (C-16, C-17) ppm,
Anal. Calcd. for C58H88N6O8: C, 69.85; H, 8.89; N, 8.43. Found: C,
69.56; H, 8.97; N, 8.62. Found: C, 69.56; H, 8.97; N, 8.62. HRMS
(C50H78N4O2 þ Hþ) calcd.: 767.6198. Found: 767.6248.
4.2.1. Succinimidyl all-trans-retinoate (4)
4.3.2. N1,N12-Bisacitretinylspermine (9)
Yield: 3.38 g (85%), Rf (A): 0.24, m.p.: 173–76 ꢀC, MS (ESIþ): m/z
833.20 (2MK), 818.24 (2MNa), 436.07 (MK), 420.04 (MNa), 398.09
(MH), 283.08 (M-C4H4NO3), IR (KBr): cmꢂ1 1758, 1732, 1626, 1595,
Yield: 2.21 g (78%), yellow powder, Rf (D): 0.45 (free base) and
0.13 (HOSu), m.p.: 167–69 ꢀC, IR (KBr): cmꢂ1 3486, 3274, 1672, 1648,
MS (ESIþ): m/z 820.41 (MH), 819.39 (M), 589.80 (M-C36H54N4O3),
1574,1558, 1H NMR (CDCl3):
d
7.13 (1H, dd, J 12 and 16 Hz, H-5), 6.33
410.41 (MH/2), 1H NMR (CDCl3):
d 8.00 (2H, unresolved t, NHCO),
(2H, d, J 16 Hz, H-4 and H-8), 6.17 (1H, d, J 12 Hz, H-6), 6.16 (1H, d, J
16 Hz, H-9), 5.95 (1H, s, H-1), 2.85 (4H, br s, COCH2CH2CO), 2.38 (3H,
s, H-16), 2.02 (5H, br s, H-17 and H-12), 1.72 (3H, s, H-18), 1.66–1.57
(2H, m, H-13), 1.50–1.44 (2H, m, H-14), 1.01 (6H, s, H-19 and H-20)
6.93 (2H, dd, J 12 and 16 Hz, H-5), 6.68 (2H, s, H-12), 6.66 (2H, d, J
16 Hz, H-4), 6.31 (2H, d, J 16 Hz, H-8), 6.30 (2H, d, J 16 Hz, H-9), 6.26
(2H, d, J 12 Hz, H-6), 5.82 (2H, s, H-2), 3.75 (6H, s, OCH3), 3.16–3.04
(4H, unresolved q, H-10 and H-120), 2.65–2.52 (8H, m, H-30, H-50, H-
80, H-100), 2.27 (6H, s, H-16), 2.24 (6H, s, H-18), 2.17 (6H, s, H-19),
2.05 (12H, s, H-17 and H-20), 1.61–1.48 (8H, m, H-20 and H-110),
1.47–1.36 (4H, unresolved m, H-60 and H-70) ppm, 13C NMR (CDCl3):
ppm, 13C NMR (CDCl3):
d 169.8 (COCH2CH2CO), 161.7 (C-1), 160.1 (C-
3),142.0 (C-7),137.8 (C-10),137.2 (C-5),134.0 (C-4),133.9 (C-8),130.7
(C-11), 130.1 (C-6), 129.3 (C-9), 110.8 (C-2), 39.8 (C-14), 34.4 (C-15),
33.3 (C-12), 29.1 (C-19, C-20), 25.8 (COCH2CH2CO), 21.9 (C-18), 19.4
(C-13), 14.8 (C-16), 13.2 (C-17) ppm, Anal. Calcd. for C24H31NO4: C,
72.52; H, 7.86; N, 3.52. Found: C, 72.69; H, 7.72; N, 3.28.
d
173.85, 173.83 (COCH2CH2CO, C-1), 156.3 (C-3), 156.2 (C-13), 138.3,
138.2, 137.2, 135.7, 134.1, 131.3, 130.0, 129.5, 128.1 (C-4, C-5, C-6, C-7,
C-8, C-9, C-10, C-11, C-15), 123.6, 122.2 (C-12, C-14), 110.7 (C-2), 55.9
(OCH3), 48.5 (C-30, C-100), 44.4 (C-50, C-80), 36.8 (C-10, C-120), 28.7
(C-20, C-110), 26.4 (C-60, C-70), 25.8 (COCH2CH2CO), 21.8 (C-16), 17.8
(C-18), 15.8 (C-19), 13.2 (C-20), 12.4 (C-17) ppm, Anal. Calcd. for
C60H84N6O10: C, 68.68; H, 8.07; N, 8.01. Found: C, 68.94; H, 7.85; N,
7.76. HRMS (C52H74N4O4 þ Hþ) calcd.: 819.5783. Found: 819.5831.
4.2.2. Succinimidyl acitretinate (5)
Yield: 3.43 g (81%), Rf (B): 0.31, m.p.: 177–79 ꢀC, MS (ESIþ): m/z
869.25 (2MNa), 462.04 (MK), 446.33 (MNa), 424.04 (MH), 309.11
(M-C4H4NO3), IR (KBr): cmꢂ1 1751, 1736, 1638, 1590, 1573, 1561, 1H
NMR(CDCl3): d 7.18 (1H, dd, J 12 and 16 Hz, H-5), 6.76 (1H, d, J 16 Hz,
H-4), 6.62 (1H, s, H-12), 6.38 (1H, d, J 16 Hz, H-8), 6.25 (1H, d, J 16 Hz,
H-9), 6.23 (1H, d, J 12 Hz, H-6), 5.97 (1H, s, H-2), 3.82 (3H, s, OCH3),
2.83 (4H, br s, COCH2CH2CO), 2.40 (3H, s, H-16), 2.31 (3H, s, H-18),
2.25 (3H, s, H-19), 2.16 (3H, s, H-20), 2.14 (3H, s, H-17) ppm, 13C NMR
4.4. General direct method for the preparation of
N1-monoacylated spermines
To an ice-cold solution of SPM (0.21 g, 1.05 mmol) in DCM
(10 mL) a solution of active ester 4 or 5 (0.35 mmol) in DCM (10 mL)
was added dropwise over 1 h. The resulting reaction mixture was
stirred at ambient temperature until the consumption of active
ester (monitored by TLC). The reaction mixture was diluted with
DCM and washed sequentially with an ice-cold 5% aqueous (aq.)
NaHCO3 and brine. Drying over Na2SO4, followed by filtration and
evaporation of the solvent left an oily residue from which pure
products 6 and 7 were obtained by preparative RP-HPLC as the
corresponding tristrifluoroacetate salt.
(CDCl3):
d 169.7 (COCH2CH2CO), 161.6 (C-1), 159.8 (C-3), 156.3 (C-
13), 141.5 (C-7), 137.9, 134.3, 133.7, 129.9, 129.8 (C-4, C-5, C-6, C-8, C-
9),136.0,134.0,129.6,128.2 (C-10, C-11, C-14, C-15),110.9,110.0 (C-2,
C-12), 55.5 (OCH3), 25.7 (COCH2CH2CO), 21.4 (C-16),17.5 (C-18),14.7,
13.1 (C-19, C-20), 11.8 (C-17) ppm, Anal. Calcd for C25H29NO5: C,
70.90; H, 6.90; N, 3.31. Found: C, 70.60; H, 7.05; N, 3.12.
4.3. General method for the preparation of N1,N12-bisacylated
spermines
To an ice-cold solution of 3 (0.55 g, 2.7 mmol) in dry DCM
(25 mL) was added ester 4 or 5 (5 mmol). The resulting solution was
stirred for an additional hour at 0 ꢀC and then allowed to reach
ambient temperature. The precipitated product was filtered off,
washed on the filter with DCM and dried under reduced pressure to
give pure product in the form of the corresponding bishydrox-
ysuccinimidate salt.
4.4.1. N1-(All-trans-retinoyl)spermine (6)
Yield: 0.13 g (45%), yellow foam, tR: 16.93 min, IR (KBr): cmꢂ1
3422, 3046, 2928, 1676, 1646, MS (ESIþ): m/z 485.36 (MH), 1H NMR
(d6-DMSO): d
8.87 (2H, br s, H-40), 8.69 (2H, br s, H-90), 8.18 (1H, t, J
8 Hz, NHCO), 8.06 (3H, br s, NHþ3 ), 6.93 (1H, dd, J 12 and 16 Hz, H-5),
6.31 (1H, d, J 16 Hz, H-4), 6.26 (1H, d, J 12 Hz, H-6), 6.20 (1H, d, J
16 Hz, H-8), 6.16 (1H, d, J 16 Hz, H-9), 5.83 (1H, s, H-2), 3.18 (2H, q, J
8 Hz, H-10), 3.05–2.81 (10H, two unresolved m, H-30, H-50, H-80, H-
100, H-120), 2.28 (3H, s, H-16),1.96 (2H, unresolved t, H-12),1.91 (3H,
s, H-17), 1.89 (2H, quintet, J 7 Hz, H-20), 1.74 (2H, quintet, J 7 Hz, H-
110), 1.69 (3H, s, H-18), 1.64 (4H, unresolved m, H-60 and H-70), 1.60–
1.55 (2H, m, H-13), 1.47–1.41 (2H, m, H-14), 1.01 (6H, s, H-19 and H-
4.3.1. N1,N12-Bis(all-trans-retinoyl)spermine (8)
Yield: 2.15 g (80%), yellow powder, Rf (D): 0.40 (free base) and
0.13 (HOSu), m.p.: 149–52 ꢀC, IR (KBr): cmꢂ1 3430, 3317, 1674, 1647,
MS (ESIþ): m/z 790.25 (MNa) 768.34 (MH), 767.34 (M), 1H NMR (d6-
DMSO):
d
7.75 (2H, unresolved t, NHCO), 6.88 (2H, dd, J 12 and
20) ppm, 13C NMR (d6-DMSO):
d 167.3 (C-1),147.2 (C-3),138.4,137.9,
16 Hz, H-5), 6.27 (2H, d, J 16 Hz, H-4), 6.23 (2H, d, J 16 Hz, H-8), 6.17
(2H, d, J 12 Hz, H-6), 6.13 (2H, d, J 16 Hz, H-9), 5.83 (2H, s, H-2), 5.69
(4H, s, H-40 and H-90), 3.18–3.09 (4H, unresolved q, H-10 and H-120),
2.61–2.44 (16H, m, H-30, H-50, H-80, H-100, COCH2CH2CO), 2.25 (6H,
s, H-16), 2.01 (4H, unresolved t, H-12), 1.95 (6H, s, H-17), 1.68 (6H, s,
H-18), 1.63–1.53 (8H, unresolved quintet, H-13, H-20, H-110), 1.48–
1.39 (8H, m, H-14, H-60, H-70), 1.02 (12H, s, H-19 and H-20) ppm, 13C
137.6, 136.7 (C-5, C-7, C-10, C-11), 130.6, 129.9, 129.8, 127.9 (C-4, C-6,
C-8, C-9), 123.1 (C-2), 46.8 (C-50, C-80), 45.4 (C-100), 44.5 (C-30),
40.8–39.5 (C-14, solvent), 36.9 (C-120), 36.2 (C-10), 34.5 (C-15), 33.2
(C-12), 29.4 (C-19, C-20), 26.7 (C-110), 24.4 (C-20), 23.3, 23.2 (C-60, C-
70), 22.0 (C-18), 19.3 (C-13), 13.8, 13.1 (C-16, C-17) ppm, HRMS
(C30H52N4O þ Hþ) calcd.: 485.4214. Found: 485.4286.
NMR (d6-DMSO):
d
173.8 (COCH2CH2CO), 166.9 (C-1), 146.6 (C-3),
4.4.2. N1-Acitretinylspermine (7)
138.2, 137.9, 137.7, 136.9 (C-5, C-7, C-10, C-11), 130.7, 129.8, 129.5,
127.8 (C-4, C-6, C-8, C-9), 123.5 (C-2), 48.8 (C-30, C-100), 46.6 (C-50,
C-80), 40.8–39.5 (C-14, solvent), 36.9 (C-10, C-120), 34.5 (C-15), 33.2
(C-12), 29.4 (C-19, C-20), 28.9 (C-20, C-110), 26.7 (C-60, C-70), 25.8
Yield: 0.12 g (41%), orange foam, tR: 16.16 min, IR (neat): cmꢂ1
3428, 3046, 2941, 1678, 1636, MS (ESIþ): m/z 549.39 (MK), 511.46
(MH), 201.05 (M-SPM), 186.01 (M-C10H24N3), 171.01 (M-C9H22N3),
1H NMR (d6-DMSO): 8.92 (2H, br s, H-40), 8.73 (2H, br s, H-90), 8.26
d

G. Magoulas et al. / European Journal of Medicinal Chemistry 44 (2009) 2689–2695
2695
(1H, t, J 8 Hz, NHCO), 8.06 (3H, br s, NHþ3 ), 7.03 (1H, dd, J 12 and
16 Hz), 6.76 (1H, s, H-12), 6.75 (1H, d, J 16 Hz, H-4), 6.40 (1H, d J
16 Hz, H-8), 6.37 (1H, d, J 12 Hz, H-6), 6.34 (1H, d, J 16 Hz, H-9), 5.91
(1H, s, H-2), 3.82 (3H, s, OCH3), 3.25 (2H, q, J 8 Hz, H-10), 3.08–2.95
(10H, two unresolved m, H-30, H-50, H-80, H-100, H-120), 2.36 (3H, s,
H-16), 2.32 (3H, s, H-18), 2.25 (3H, s, H-19), 2.13 (6H, s, H-17 and H-
20), 1.95 (2H, quintet, J 7 Hz, H-20), 1.83 (2H, quintet, J 7 Hz, H-110),
1.75–1.66 (4H, unresolved m, H-60 and H-70) ppm, 13C NMR (d6-
137.7, 137.4 (C-7, C-10, C-11), 135.4 and 135.3 (C-5), 129.6, 128.8,
128.5, (C-6, C-8, C-9), 127.9 and 127.8 (C-4), 121.4 and 121.2 (C-2),
44.9 (C-30, C-100), 39.6 (C-14), 39.2 (C-10, C-120), 34.3 (C-15), 33.1 (C-
12), 31.3 (C-50, C-80), 28.9 (C-19, C-20), 26.4 (C-60, C-70), 25.1 (C-20, C-
110), 21.7 (C-18), 19.2 (C-13), 14.3 (C-16), 12.8 (C-17) ppm, HRMS
(C50H78N4O2 þ Hþ) calcd.: 766.6125. Found: 766.6132.
4.5. Biological evaluation of conjugates as RNase P inhibitors
DMSO):
d 166.7 (C-1), 156.1 (C-3), 156.0 (C-13), 138.1, 137.0, 135.6,
133.9, 131.1, 129.8, 129.3, 127.9 (C-4, C-5, C-6, C-7, C-8, C-9, C-10,
C-11), 123.5, 122.1, 122.0 (C-12, C-14, C-15), 55.8 (OCH3), 49.0, 48.2
(C-50, C-80), 46.8 (C-100), 46.1 (C-30), 37.1 (C-120), 36.7 (C-10), 28.4 (C-
110), 25.6 (C-20, C-60, C-70), 21.5 (C-16), 17.6 (C-18), 13.6 (C-19), 13.0
(C-20), 12.2 (C-17) ppm, HRMS (C31H50N4O2 þ Hþ) calcd.: 511.4007.
Found: 511.4076.
Growth of D. discoideum cells (strain A ꢁ 2 wild type), cell
breakage, and purification of RNase P were essentially carried out
as previously described [13]. RNase P assays were carried out at
37 ꢀC in 20
mL buffer D (50 mM Tris/HCl pH 7.6, 10 mM NH4Cl, 5 mM
MgCl2 and 5 mM dithiothreitol), containing 2–5 fmol pre-tRNAser
substrate (an in vitro labeled transcript of the Schizosaccharomyces
pombe tRNAser gene supSI) and 1.3
mg protein from the RNase P
4.4.3. N4,N9-Bis(all-trans-retinoyl)spermine (10)
fraction. Stock solutions of conjugates were prepared in 100%
dimethylosulfoxide (DMSO). Enzyme assays were carried out in the
presence of 10% DMSO. The reactions were stopped by the addition
To an ice-cold solution of the bistrifluoroacetate salt of SPM
derivative 14 (0.32 g, 0.5 mmol) in anhydrous CHCl3 (1 mL) was
i
added Pr2NEt (0.7 mL, 4 mmol) and a mixture consisted of ATRA
of 5 mL stop dye (80% formamide, 50 mM EDTA, 0.1% bromophenol
(0.3 g,1 mmol) and PyBrOP (0.6 g,1.28 mmol) in small portions over
a period of 1 h. After 30 min at ambient temperature, the reaction
mixture was diluted with CHCl3 and washed sequentially with an
ice-cold 5% aq. NaHCO3 solution (twice) and water (twice). Drying
and evaporation left a residue from which pure intermediate 15 was
obtained through FCC, using as an eluant the solvent system C.
Intermediate 15 (0.3 g, 0.31 mmol) was then dissolved in 4 mL
MeOH and treated with 0.4 mL of a 5 N aq. NaOH solution for 2 h at
ambient temperature. MeOH was then removed under reduced
pressure and the residue was taken up in 20 mL water and extracted
twice with DCM. The combined organic layers were washed twice
with brine, dried, evaporated and subjected to FCC using as an
eluant the solvent system E. Fractions containing the pure conjugate
10 were pooled, washed with water and evaporated under vacuo to
afford the desired product.
blue, 0.1% xylene cyanol). Reaction products were resolved on
a denaturing 10% polyacrylamide/8M urea gel and visualized with
phosphoimager Fujifilm FLA 3000. Gel bands were quantified using
AIDA software.
Acknowledgments
BIOMEDICA Life Sciences S.A. is gratefully acknowledged for the
financial support of this work. In addition, the authors wish to
thank Prof. Giovanni Sindona and Dr. Anna Napoli from the
Department of Chemistry of the University of Calabria (Italy) for the
kind provision of the HRMS analyses.
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Yield: 0.34 g (70%), yellow foam, Rf (C): 0.13, IR (neat): cmꢂ1 3267,
2924, 1714, 1614, MS (ESIþ): m/z 998.90 (MK), 982.81 (MNa), 959.87
(MH),1H NMR (CDCl3):
d 8.35 (2H, unresolved t, NHCOCF3), 6.83 (2H,
dd, J 12 and 16 Hz, H-5), 6.21 (2H, d, J 16 Hz, H-4), 6.20 (2H, d, J 16 Hz,
H-8), 6.11 (2H, d, J 12 Hz, H-6), 6.09 (2H, d, J 16 Hz, H-9), 5.81 (2H, s,
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m, H-30, H-50, H-80, H-100), 2.09 (6H, s, H-16),1.95 (4H, t, J 6 Hz, H-12),
1.92 (6H, s, H-17),1.64 (6H, s, H-18),1.56–1.48 (12H, m, H-13, H-20, H-
60, H-70, H-110),1.39–1.35 (4H, m, H-14), 0.98 (12H, s, H-19 and H-20)
ppm, 13C NMR (CDCl3):
d 169.3 (C-1), 157.5 (q, J 36 Hz, COCF3), 147.7
(C-3), 139.2, 137.7, 137.2, 134.6, (C-5, C-7, C-10, C-11), 129.9, 129.2,
128.4, 127.3 (C-4, C-6, C-8, C-9), 119.2 (C-2), 116.1 (q, J 287 Hz, CF3),
47.9 (C-10, C-120), 41.7 (C-30, C-100), 39.6 (C-14), 36.0 (C-50, C-80), 34.3
(C-15), 33.1 (C-12), 28.9 (C-19, C-20), 27.1 (C-60, C-70), 26.3 (C-20,
C-110), 21.7 (C-18), 19.2 (C-13), 14.4 (C-16), 13.9 (C-17) ppm.
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4.4.5. N4,N9-Bis(all-trans-retinoyl)spermine (10)
Yield: 0.16 g (67%), yellow foam, Rf (E): 0.26, IR (neat): cmꢂ13434,
1620, MS (ESIþ): m/z 807.74 (MK) 790.68 (MNa), 767.73 (M), 1H NMR
(CDCl3): d 6.78 and 6.74 (2H, two dd, J 12 and 16 Hz, H-5), 6.25 and
5.94 (2H, d, J 12 Hz, H-6), 6.22 (2H, d, J 16 Hz, H-4), 6.16 (2H, d, J 16 Hz,
H-8), 6.10 (2H, s, H-2), 6.05 (2H, d, J 16 Hz, H-9), 3.42–3.24 (8H, m, H-
30, H-50, H-80, H-100), 2.66–2.60 (4H, unresolved m, H-10 and H-120),
2.05 (6H, br s, H-16),1.95 (4H, t, J 6 Hz, H-12),1.91 (6H, s, H-17),1.87–
1.79 (4H, m, H-20 and H-110), 1.64 (6H, s, H-18), 1.53–1.44 (8H, m, H-
13, H-60, H-70),1.40–1.36 (4H, m, H-14), 0.95 (12H, s, H-19 and H-20)
ppm, 13C NMR (CDCl3):
d 168.1 and 168.0 (C-1), 145.5 (C-3), 138.1,