Synthesis and hybridization properties of the conjugates of oligonucleotides and stabilization agents. Part 3

Article abstract of DOI:10.1016/j.bmc.2004.12.030

New compounds having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently attached through the 5′-phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleotide pd(TGTTTGGC) a

Full text of DOI:10.1016/j.bmc.2004.12.030

Bioorganic & Medicinal Chemistry 13 (2005) 1515–1522
Synthesis and hybridization properties of the conjugates of
oligonucleotides and stabilization agents. Part 3q
Enzo Sottofattori,a,ꢀ Maria Anzaldi,a Mauro Mazzei,a Mariangela Miele,a
Alessandro Balbi,a,* Dmitri S. Pyshnyi,b Olga D. Zakharovab and
Tatyana V. Abramovab
aDipartimento di Scienze Farmaceutiche, Viale Benedetto XV, 3-16132 Genova, Italy
bInstitute of Bioorganic Chemistry, Novosibirsk 63090, Russia
Received 5 November 2004; revised 9 December 2004; accepted 17 December 2004
Abstract—New compounds having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently
attached through the 50-phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleo-
tide pd(TGTTTGGC) and above oligonucleotide conjugates were tested for their thermodynamic response. The Tm data and
thermodynamic parameters for complex formation confirmed the ability of chromone (c-pyrone) derivatives to stabilize strongly
the 7-mer/8-mer complementary complex. Moreover, benzochromone (naphthopyrane) and, surprisingly, tetrahydropyrimidineth-
anone derivatives showed the capacity of stabilizing this 7-mer/8-mer complementary complex. The effect of all these compounds on
the stability of the oligonucleotide complexes (DDG at 37 ꢀC ranged from ꢀ1.2 to ꢀ2.0 kcal/mol) was shown to be comparable to the
effect of one nucleotide base pair and similar to the effect (DDG at 37 ꢀC ranged from ꢀ1.5 to ꢀ2.0 kcal/mol) found for acridine–
oligonucleotide conjugates, which served as a reference in this study.
ꢁ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
One of the perspective approaches to improve the
hybridization ability (as well as nuclease resistance) of
the oligonucleotides is a simple covalent attachment
of different stabilizing agents (SA), usually aromatic
organic molecules. The nature of the SA and the length
of the linker are fundamental in determining the stability
of complementary complexes.
Since antisense oligodeoxynucleotide strategy was pro-
posed for therapeutic use in 1978, major advances have
been made in developing modified oligonucleotides
(ODNs) whose potential use as therapeutic agents has
been claimed in all kinds of infectious diseases, genetic
disorders and cancer.1–7 Whether they are used in the
so-called ꢁantisenseꢂ, ꢁantigeneꢂ or ꢁanticodeꢂ strategies,
the hybridization properties of the ODNs with the com-
plementary sequences of the target nucleic acids are of
fundamental importance. Moreover, an increase of
hydrophobicity and an efficient synthesis could be a
valid strategy to increase cellular uptake, both favouring
therapeutic use and lowering the end price of the poten-
tial drug.
We previously demonstrated that several compounds
from the pyranone family are considered to be promis-
ing SA.8,9 In particular, compounds 4a,b showed major
stabilization abilities compared to other chromones and
coumarins tested. They were comparable to the stabiliz-
ing effects of the well known acridines. Moreover, some
showed interesting capabilities in inhibiting HIV-1
reverse transcriptase.10
In order to form conjugates with both enhanced stabil-
ization ability and biological significance, we planned
to synthesize new pyran derivatives having nitro, carbal-
dehyde and phenyl substituents. In particular, the benzo
moiety could be a substituent in the chromone ring or
fused to form a naphthopyrane ring.
Keywords: Oligonucleotides; Stabilization agents; Pyranone deri-
vatives.
q For Part 1 and 2 see Refs. 8 and 9.
*
Corresponding author. Tel.: +39 0103538368; fax: +39 0103538358;
e-mail: balbi@unige.it
ꢀ In memory of Enzo Sottofattori, who inspired this work.
0968-0896/$ - see front matter ꢁ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.030

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E. Sottofattori et al. / Bioorg. Med. Chem. 13 (2005) 1515–1522
Benzo moiety was chosen to improve lipophilicity, while
the nitro substituent was considered as a potential
photoreactive group and the carbaldehyde moiety was
introduced for its potential capability of inhibiting
HIV-RT, as shown in previous works.10,11
side products. However, this procedure failed to synthe-
size 4b and 7b. A winning alternative strategy was that
of using the protected reagent N-(triphenylmethyl)pen-
tane-1,5-diamine. The amine exchange reaction pro-
ceeded efficiently yielding the protected derivatives,
which gave the desired 4b and 7b by detritylation with
acetic acid (4b) or by spontaneous loss of the trityl
group during crystallization (7b).
This paper describes the synthesis of different
(benzo)chromones (4–7, 11), together with the new
tetrahydropyrimidinethanones (12, 13), the formation
of the complexes with complementary octanucleotide
and the evaluation of thermodynamic parameters from
the analysis of the melting temperature experiments.
Vilsmeier formylation of 1a gave the 2-formylchromone
2, which furnished 5a and 5b in accordance with the
amine exchange method previously described.14 The
reaction is very sensitive to temperature, molar rate
and solvents. Changing these parameters dramatically
modifies the course of the reaction.
2. Chemistry
2.1. Synthesis of stabilizing agents
The reaction pathways depicted in Scheme 2 account for
the formation of the formylbenzochromone derivative
11 and may give an explanation of the critical reaction.
In fact, this compound was only obtainable via the pro-
tected derivative 10, which was, in turn, easily obtained
from 9 by amine exchange with N-(triphenylmethyl)pro-
pane-1,3-diamine at room temperature. Any other
attempt to directly obtain 11 failed. The tetrahydro-
pyrimidine derivative 12 was the main product, the yield
of which depending only on temperature and molar rate.
Following the experimental conditions that gave 12 in
its highest yield, we were also able to obtain the tetra-
hydropyrimidine derivative 13 from chromone 2.
The synthesis of the intermediate and target compounds
was performed by the reaction pattern shown in
Schemes 1 and 2.
The starting molecules 1a,b and 8 were obtained by fol-
lowing our well-established method from substituted
phenols or 2-naphthol and N,N0-(dimethyl)malonamide
in the presence of phosphorus oxychloride.12,13 The
reaction of 1a,b with mixed nitric and sulfuric acid gave
compounds 3 and 6 in excellent yields, which were, in
turn, treated with propane(pentane)-1,3-(1,5)-diamine.
The course of the amine exchange reaction from dimeth-
ylamino to a,x-diaminoalkyl groups depends on the
number of methylenes (3 or 5) and the substrate. Com-
pounds 3 and 6 react smoothly with 1,3-diaminopro-
pane to afford 4a and 7a in good yield and without
2.2. Synthesis of oligonucleotides conjugates
All conjugars were attached to the 50-end of the 7-mer
oligonucleotide according to previously described
R
O
O
CON(CH3)2
CH2
O2N
NO2
O2N
NO2
+
COOC2H5
OH
O
N(CH3)2
O
N(CH3)2
R1
i
3
6
ii
iv
O
ii
R
O
NO2
O2N
NO2
Z
O
N(CH3)2
viii
vi
R1
O
1a, b
3a
Z=NH(CH2)5NHCPh3
O
iii
O2N
NO2
v
O
Y
O
O
NH(CH2)nNH2
X
vii
CHO
O
NH(CH2)nNH2
4, 5
O
N(CH3)2
7a: n=3
7b: n=5
NO2
2
1a: R=CH3, R1=CH(CH3)2
1b: R=H, R1=C6H5
4a: X=Y=NO2, n=3
4b: X=Y=NO2, n=5
5a: X=H, Y=CHO, n=3
5b: X=H, Y=CHO, n=5
Scheme 1. Reagents and conditions: (i) POCl3; (ii) HNO3, H2SO4; (iii) N,N-dimethylformamide, POCl3; (iv) N-(triphenylmethyl)pentane-
1,5-diamine; (v) CH3COOH–H2O (3:1); (vi) propane-1,3-diamine; (vii) propane-1,3-diamine or pentane-1,5-diamine; (viii) propane-1,3-diamine or
N-(triphenylmethyl)pentane-1,5-diamine.

E. Sottofattori et al. / Bioorg. Med. Chem. 13 (2005) 1515–1522
1517
O
O
O
O
CON(CH3)2
CH2
i
CHO
+
COOC2H5
OH
N(CH3)2
iv
NH(CH2)3NHCPh3
10
8
ii
v
HN
O
O
N
H
iii
CHO
CHO
O
O
N(CH3)2
O
NH(CH2)3NH2
HCl
OH
12
9
11
OH
O
HN
vi
N
2
H
13
Scheme 2. Reagents and conditions: (i) POCl3; (ii) N,N-dimethylformamide, POCl3; (iii) propane-1,3-diamine; (iv) N-(triphenylmethyl)propane-1,3-
diamine; (v) HCl; (vi) propane-1,3-diamine.
methods15,16 with some modifications (see Experi-
mental). The major modification included the use of
N,N-dimethylaminopyridine (DMAP) as a catalyst
instead of N-methylimidazole, which is recommended
in the ꢁclassical approachꢂ.17 In our case DMAP gener-
ally led to an increased yield of the oligonucleotide
conjugates and facilitated their recovery from the reac-
tion mixture. However, in some cases the conjugate
yields seem to be sensitive to the position of the linker
group in the (benzo)chromone moiety.
with the complementary 8-mer. The parameters were
calculated from optical melting curves in the assumption
of an ꢁall-or-nothingꢂ model.22
The results show that all (benzo)chromone conjugates
exhibit a higher affinity to the complementary 8-mer as
compared to the parent 7-mer/8-mer complex. The sta-
bilization effect (DDG, at 37 ꢀC) ranged from ꢀ1.2 to
ꢀ2.0 kcal/mol, which is comparable to an effect of one
A–T base pair and is almost superimposable to that ob-
served for acridine conjugates.9 Similar results on the
same 7-mer/8-mer and relative (7-mer/12-mer) model
systems were obtained earlier for phenazinium and
ethidium conjugates.19,23
All oligonucleotide conjugates were isolated and puri-
fied by reverse phase HPLC, the homogeneity of the
chromatographic fractions was monitored by the diode
array detector.
The DDG values found in this study differ significantly
from an estimated value (ꢁ6 kcal/mol at 37 ꢀC) reported
in a past paper8 for 10-mer/20-mer and 14-mer/20-mer
oligonucleotide complexes, while they are comparable
to the values reported in our previous work.9 This incon-
sistency may be attributed to the presence of an internal
hairpin formed by the 20-mer oligonucleotide used in the
cited study.8 In fact, the value of the stabilization effect of
the conjugar is dependent on the specific ternary struc-
ture of the duplex (e.g., formation of internal hairpin
3. Results and discussion
All experiments were performed on the model 7-mer/8-
mer duplex, successfully tested in several studies18–21
for its thermodynamic response.
The Table 1 summarizes the thermodynamic parameters
evaluated for complexes of different 7-mer conjugates
Table 1. Melting temperature data and thermodynamic parameters for the complex formation of d(CCAAACA) and its conjugatesa with
pd(TGTTTGGC)
ODN-SA
UMb
4a*
4b*
7a*
7b*
5a*
5b*
11*
12*
13*
Linker, methylene groups (n)
Tm ( 0.2) (ꢀC)
DTm
––
23.0
––
3
32.1
5
34.9
3
32.0
5
34.2
3
35.8
5
36.1
3
32.4
––
––
32.0
33.2
c
9.1
6.4
1.3
11.9
6.8
1.7
58
9.0
6.3
1.2
11.2
6.7
1.6
57
12.8
6.9
1.8
51
13.1
7.1
2.0
56
9.4
6.5
1.4
9.0
6.3
1.2
9.3
6.7
1.6
ꢀDG ( 0.05); 37 ꢀC (kcal/mol)
5.1
––
44
ꢀDDG c
ꢀDH ( 1) (kcal/mol)
ꢀDDH c
51
51
48
51
51
––
7
143
18
14
7
142
17
13
7
12
4
155
30
7
143
18
7
140
15
ꢀDS ( 3) (kcal/mol K)
125
––
160
35
158
33
162
37
164
39
ꢀDDS c
Buffer used: 0.16 M NaCl, 0.01 M Na2HPO4, 0.1 mM EDTA, pH 7.0; concentration of each oligomer: 1.6 · 10ꢀ5 M.
a To easy recognize the conjugates, number 4a* means 30ACAAACCp-SA (4a) and so on.
b Unmodified duplex.
c The difference between DTm (or DG, or DH, or DS) values for modified and unmodified complexes.

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E. Sottofattori et al. / Bioorg. Med. Chem. 13 (2005) 1515–1522
structures and intercomplex associates). Moreover, the
value of the effect may also depend on the nucleotide
sequence of the duplex in the area of SA location.
the aromatic system of (benzo)chromone occurred, also
maintain a good stabilization effect. These two com-
pounds deserve further experimental investigations to
better understand their role as stabilization agents.
The analysis of the data (Table 1) shows that changes of
the substituents at positions 3, 5, 6, 8 of chromone resi-
due cause only small variations in the stability of respec-
tive oligonucleotide complexes. Furthermore, variations
in the chemical structure from benzo to naphtho pyrone
conjugars do not influence the stability of oligonucleo-
tide complexes, which remain positive for all derivatives.
4. Conclusions
The experimental data quantitatively support the previ-
ous observations8,9 that the conjugars of the pyranone
family are powerful stabilizing agents for oligonucleo-
tide duplexes. These conjugars may also be considered
as potential photoreactive groups for modification of
the oligo and polynucleotides. While the introduction
of different substituents in a c-pyrone aromatic system
does not significantly alter the hybridization abilities
of the oligonucleotide conjugate, it provides a conve-
nient way to control a photosensitivity and photochem-
ical reactivity of the conjugar residue. For this purpose,
7a,b seem to be the most perspective photoreactive com-
pounds to use in future experiments on a sequence spe-
cific photomodification in the oligonucleotide duplexes.
On the other hand, compound 11, due to its similarity
to 5a, is selected for its potential biological activity on
interacting with HIV1-RT. Finally, the tetrahydro-
pyrimidinethanone derivatives 12–13, which have un-
expectedly shown good stabilization capacity of the
conjugates, could pave the way to the synthesis of new
SAs with potential biological activities.
In general, in all cases, the effect of the stabilization of
complexes (as compared to parent 7-mer/8-mer com-
plex) is reflected in the substantial increase of DH of
the complex formation (Table 1). Comparison of the
enthalpy changes for the parent 7-mer/8-mer and the
respective complexes for conjugars 4a*, 4b*, 7a*, 7b*,
5b*, 11* and 13* leads to DDH values, which ranged
from ꢀ4 to ꢀ14 kcal/mol. These values are comparable
to DDH values (ꢀ9 to ꢀ23 kcal/mol) found in the com-
plexes of different phenazine conjugates of the 7-mer
with complementary 12-mer oligonucleotide.23 Such
similarities of the DDH values is a good indication of
the common mechanism of stabilization for both types
of complexes (with phenazine and benzo(naphtho)chro-
mone conjugates).
A number of conclusions can be reached regarding the
influence of the length of a linker group on the complex
stability. While the length of the linker group (n = 3 or
n = 5) has a noticeable effect on the complex stability
(see Table 1), no simple correlation between the linker
length and stability of the complex is observed. In gen-
eral, for complexes with a longer linker group (n = 5)
the changes of enthalpy (see DDH and DDG values in
Table 1) are higher than those for complexes with a
shorter one (n = 3). It may be concluded that one posi-
tion of the conjugar aromatic system over the base pair
is more favorable for stacking interaction in the com-
plexes with longer linker group. The respective changes
of entropy (compare DS, DDS values) for complexes
with linkers n = 3 and 5 exhibit the opposite behaviour,
this result could be interpreted as a higher cost for con-
formational restrictions of the conjugar in complexes
with pentamethylene linker chain versus the ones with
trimethylene linker chain.
5. Experimental
5.1. General
Melting points of synthesized compounds were deter-
mined with a Fisher–Johns apparatus and are uncor-
rected. The IR spectra were recorded in potassium
bromide disks on a Perkin–Elmer 398 IR spectrophoto-
meter. The 1H NMR spectra were obtained on a Hitachi
Perkin–Elmer R 600 (60 MHz) and on a Varian Gemini
200 (200 MHz) spectrometers with TMS as the internal
standard (d = 0). NMR signals are quoted as singlet (s),
doublet (d), doublet of doublets (dd), triplet (t), quartet
(q), multiplet (m), broad (br). The purity of the
compounds was checked by TLC on silica gel 60-F254
precoated plates and the spots were located under UV
light or by iodine vapour or with ninidrine. Elemental
analyses were performed in the Microanalysis Labora-
tory of the Department of Pharmaceutical Sciences
(Genoa) on a Carlo Erba 1106 Elemental Analyzer.
UV absortion spectra of ODNs and ODN-SAs were re-
The resulting stabilization effect of the linker length
seems to be dependent on the chemical structure of the
conjugar. All c-pyrones tested showed higher stability
with the longer linker function (n = 5) than the shorter
linker group (n = 3). On the other hand, the shorter lin-
ker group (n = 3) is advantageous for a-pyrones (couma-
rins).8 However, as has already been demonstrated for
other conjugars (phenazine, acridine, ethidium), the
optimal length of the linker may be specific for the par-
ticular chemical structure of the pyranone and also on
the nucleotide sequence of oligonucleotide complex
(see review).6
corded with
spectrophotometer.
a
Shimadzu UV-2100 UV–vis
5.2. HPLC
The liquid chromatograph was a Perkin–Elmer Series
4 (Norwalk, CT, USA) equipped with a Rheodine
7125 (Berkeley, CA, USA) injector valve with 20 lL
or 1 mL loop (respectively, for analytical or
preparative purpose). The diode array detector was a
Perkin–Elmer LC-235. Retention times, peak areas
Finally, it is worth noting that the tetrahydropyrimidin-
ethanone derivatives 11 and 13, in which a disruption of

E. Sottofattori et al. / Bioorg. Med. Chem. 13 (2005) 1515–1522
1519
1
and UV spectra were recorded on Perkin–Elmer LCI-
100 integrator.
1640, 1600, 1580 (NO2) cmꢀ1; H NMR (DMSO-d6): d
3.03 (s, 6H, CH3), 8.20 (s, 1H, arom H), 8.41 (s, 1H,
arom H), 8.65 (d, 2H, arom H), 8.79 (d, 2H, arom H).
Anal. Calcd for C17H12N4O8: C, 51.01; H, 3.02; N,
14.00. Found: C, 51.20; H, 3.00; N, 13.88.
The anionic exchange chromatography was performed
on a stainless steel column (250 · 10 mm) filled by
us with Partisil-10 SAX (Whatman, USA). The
reverse-phase column was a stainless steel column
(250 · 10 mm) filled by us with 10 mm LiChrosorb
RP18 (Merck, Germany).
5.3.3. Compounds 2 and 9. In a flask, cooled in an ice
bath and protected from moisture by a calcium chloride
tube, POCl3 (0.92 g, 6.0 mmol) was added dropwise to
2 mL of N,N-(dimethyl)formamide. The resulting solu-
tion was stirred for 30 min at room temperature. A sus-
pension of 4 mmol of a suitable pyranone (1a, 8) in
DMF (10 mL) was then added and heated at the temper-
ature and time indicated for each compound. The mix-
ture was then cooled and poured onto crushed ice.
The solution was alkalinized with sodium carbonate
giving a colourless solid which was collected, washed
with H2O, dried and crystallized with an appropriate
solvent giving the following compounds.
5.3. Synthesis of key intermediates
5.3.1. Compounds 1a,b and 8. Phosphorous oxychloride
(0.15 mol) was added dropwise by stirring to 0.11 mol
of N,N0-(dimethyl)malonamide which was contained in
an ice bath-cooled flask protected from moisture with
a calcium chloride drying tube. After the addition, the
ice-bath was removed and the mixture was kept at room
temperature for 30 min. A solution (a suspension for 2-
naphthol) of a suitable phenol (0.10 mol) in 60 mL of
chlorobenzene was added to the yellow solution, and
the resulting mixture was heated at a temperature and
for the time indicated for each compound. After cooling,
a solution of 136 g of trihydrate sodium acetate in
350 mL of water was added and the mixture was heated
by stirring at 80 ꢀC for 90 min. The layers were sepa-
rated and the aqueous phase was extracted twice with
chloroform. The combined organic portions were
washed with brine and evaporated under reduced pres-
sure to yield a dark red oil. The oil was stirred at room
temperature with 250 mL of 2 N NaOH and 80 mL of
petroleum ether. The obtained solid was filtered, washed
with water and crystallized, giving the following
compounds.
5.3.3.1.
2-(Dimethylamino)-8-isopropyl-5-methyl-4-
oxo-4H-1-benzopyran-3-carbaldehyde (2). From 2-(di-
methylamino)-8-isopropyl-5-methyl-4H-1-benzopyran-4-
one at 120 ꢀC for 30 min (yield 85%); mp 134–5 ꢀC
(cyclohexane).26
5.3.3.2. 3-(Dimethylamino)-1-oxo-1H-naphtho[2,1-b]-
pyran-2-carbaldehyde (9). From 3-(dimethylamino)-
naphtho[2,1-b]pyran-1(1H)one at 95 ꢀC for 90 min
(yield 84%); mp 218–9 ꢀC (EtOH).27
5.3.4. 2-{5-[(Triphenylmethyl)amino]pentylamino}-8-iso-
propyl-5-methyl-3,6-dinitro-4H-1-benzopyran-4-one (3a).
The mixture of 3 (1.0 g, 2.98 mmol), N-(triphenyl-
methyl)pentane-1,5-diamino (1.50 g, 4.35 mmol) and
20 mL of acetonitrile was refluxed for 1 h. After cooling,
removal of the solvent afforded a yellow solid. After
recrystallization from ethanol, a pure 3a was obtained
(yield 1.2 g, 64%); mp 202–203 ꢀC; IR m 3300, 2900,
5.3.1.1. 2-(Dimethylamino)-8-isopropyl-5-methylchro-
mone (1a). From thymol at 110 ꢀC for 5 h (yield 65%);
mp 151–2 ꢀC (ligroin).12
1
5.3.1.2. 2-(Dimethylamino)-8-phenylchromone (1b).
From biphenyl-2-ol at 100 ꢀC for 4 h (yield 60%); mp
175–6 ꢀC (ligroin).13
1640, 1600, 1200 cmꢀ1; H NMR (CDCl3): d 1.20 (d,
6H, CH3 isopropyl), 1.4 (m, 6H, CH2), 2.2 (t, 2H, tri-
tyl-NHCH2), 2.7 (t, 2H, CH2NH), 2.85 (s, 3H, CH3),
4.50 (m, 1H, CH isopropyl), 4.90 (s, 1H, NH), 7.18
(m, 3H, 4-trityl), 7.27 (m, 6H, 3,5-trityl), 7.48 (m, 6H,
2,6-trityl), 7.90 (s, 1H, H-7), 10.20 (s, 1H, NH). Anal.
Calcd for C37H38N4O6: C, 70.01; H, 6.03; N, 8.83.
Found: C, 69.97; H, 5.99; N, 8.71.
5.3.1.3. 3-(Dimethylamino)-1H-naphtho[2,1-b]pyran-
4(4H)-one (8). From 2-naphthol at 85 ꢀC for 5 h (yield
71%); mp 190–1 ꢀC (ethanol).24
5.3.2. Compounds 3 and 6. A solution of nitric acid
(d = 1.52) in 5 mL of sulfuric acid was slowly added to
a solution of 8.0 mmol of the suitable chromone in
15 mL of sulfuric acid at 0 ꢀC by stirring. The resulting
solution was kept at room temperature for 30 min then
poured onto crushed ice. The resulting yellow solid was
filtered, washed with water and crystallized giving the
following compounds.
5.4. Synthesis of stabilizing agents
5.4.1.
2-[(3-Aminopropyl)amino]-8-isopropyl-5-methyl-
3,6-dinitro-4H-1-benzopyran-4-one (4a). A warm solu-
tion of 2-(dimethylamino)-8-isopropyl-5-methyl-3,6-
dinitro-4H-1-benzopyran-4-one 3 (1.0 g, 2.98 mmol) in
toluene (30 mL) was added dropwise to a solution of
propane-1,3-diamine (0.22 g, 2.98 mmol) in toluene
(15 mL) heated at 110 ꢀC. This reaction mixture was
allowed to stir at the same temperature for 1 h, then
evaporated under reduced pressure. The resulting solid
was dissolved in chloroform and co-evaporated with
silica gel (2 g) under reduced pressure. The silica gel
was applied to the top of a silica gel column, which
was then eluted with ethyl acetate giving 4a as yellow
5.3.2.1. 2-(Dimethylamino)-8-isopropyl-5-methyl-3,6-
dinitrochromone (3). From 24.0 mmol of nitric acid
(yield 78%); mp 199–200 ꢀC (ethyl acetate).25
5.3.2.2.
2-(Dimethylamino)-3,6-dinitro-8-(40-nitro-
phenyl)chromone (6). From 36.0 mmol of nitric acid
(yield 58%); mp 188–190 ꢀC (toluene); IR m 1710 (CO),

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E. Sottofattori et al. / Bioorg. Med. Chem. 13 (2005) 1515–1522
crystals from isopropylic alcohol (yield 0.36 g, 33%); mp
165 ꢀC (dec); IR m 1750 (CO), 1640, 1570–1550 (NO2)
cmꢀ1; 1H NMR (CDCl3): d 1.40 (d, 6H, CH3 isopropyl),
2.20 (t, 2H, CH2), 2.40 (s, 3H, CH3), 3.30 (m, 1H, CH
isopropyl), 3.60 (m, 4H, CH2), 3.65 (m, 1H, NH), 7.80
(s, 1H, H-7), 11.00 (s, 2H, NH2, deuterium oxide
exchangeable). Anal. Calcd for C16H20N4O6: C, 52.74;
H, 5.53; N, 15.38. Found: C, 52.54; H, 5.54; N, 15.08.
(0.6 g, 1.5 mmol) in acetonitrile (10 mL) and heated at
80 ꢀC for 1 h. After cooling, the red precipitate was fil-
tered off and crystallized from ethanol giving 0.3 g (yield
47%) of 7a as red crystals. Mp 218 ꢀC (ethanol). IR m
1
3250, 1640, 1598, 1500 cmꢀ1; H NMR (DMSO-d6): d
1.80 (m, 2H, CH2), 2.85 (m, 4H, CH2), 3.65 (m, 1H,
NH deuterium oxide exchangeable), 7.80 (d, 1H, arom
H), 8.0 (d, 2H, arom H), 8.15 (d, 1H, arom H), 8.20
(d, 2H, arom H), 11.00 (s, 2H, NH2 deuterium oxide
exchangeable). Anal. Calcd for C18H15N5O8: C, 50.35;
H, 3.52; N, 16.31. Found: C, 49.99; H, 3.71; N, 16.12.
5.4.2.
2-[(5-Aminopentyl)amino]-8-isopropyl-5-methyl-
3,6-dinitro-4H-1-benzopyran-4-one acetate (4b). Com-
pound 3a (1.20 g, 1.90 mmol) was added to a solution
of H2O/CH3COOH (1:3), stirring 2 h at 60 ꢀC. Removal
of the solvent under reduced pressure afforded a white
solid, which was stirred with ether for 6 h and filtered
to give the pure 4bÆCH3COOH (yield 0.50 g, 58%); mp
5.4.6. 2-[(5-Aminopentyl)amino]-3,6-dinitro-8-(40-nitro-
phenyl)-4H-1-benzopyran-4-one (7b). A solution of 6
(0.6 g, 1.5 mmol) and N-(triphenylmethyl)pentane-1,5-
diamine (1.0 g, 2.9 mmol) in acetonitrile (20 mL) was re-
fluxed for 1 h. The red precipitate, which formed after
cooling, was filtered off and crystallized from ethanol
giving the already deprotected 7b as red crystals (yield
0.4 g, 58%). Mp 220–21 ꢀC (ethanol). IR m 3300, 2900,
1
210–12 ꢀC; IR m 3300, 2900, 1640, 1600, 1200 cmꢀ1; H
NMR (DMSO-d6): d 1.20 (d, 6H, CH3 isopropyl), 1.4
(m, 6H, CH2), 2.2 (t, 2H, NHCH2), 2.4 (s, 3H, CH3 ace-
tate), 2.7 (t, 2H, CH2NH3+), 2.85 (s, 3H, CH3), 4.50 (m,
1H, CH isopropyl), 4.90 (s, 1H, NH), 5.70 (m, 3H,
1
1640, 1600, 1200 cmꢀ1; H NMR (DMSO-d6): d 1.40
+
NH3 ), 8.10 (s, 1H, H-7). Anal. Calcd for C20H28N4O8:
C, 53.09; H, 6.24; N, 12.38. Found: C, 52.81; H, 6.19;
N, 12.21.
(s, 6H, CH2), 2.20 (t, 2H, NH–CH2), 2.70 (t, 2H,
CH2), 3.65 (m, 1H, NH deuterium oxide exchangeable),
7.70 (d, 1H, arom H), 7.95 (d, 2H, arom H), 8.10 (d, 2H,
arom H), 8.20 (d, 2H, arom H) 11.00 (s, 2H, NH2 deu-
terium oxide exchangeable). Anal. Calcd C20H19N5O8:
C, 52.52; H, 4.19; N, 15.31. Found: C, 52.88; H, 4.00;
N, 15.09.
5.4.3. 2-[(3-Aminopropyl)amino]-8-isopropyl-5-methyl-4-
oxo-4H-1-benzopyran-3-carbaldehyde (5a). A suspension
of 2 (0.8 g, 2.90 mmol) and propane-1,3-diamine (0.21 g,
2.90 mmol) in toluene (8 mL) was kept at room temper-
ature for 2 days, while stirring. The solvent was evapo-
rated and the residue purified on silica gel and eluted
first with cyclohexane–ethyl acetate (1:1 v/v) and second
with cyclohexane–ethyl acetate (1:4 v/v). The second
eluted gave 0.31 g (yield 35%) of 5a as pale yellow crys-
tals. Mp 110–112 ꢀC (cyclohexane–ethyl acetate, 1:2). IR
m 3400, 2900, 1660, 1560, 1200 cmꢀ1; 1H NMR (CDCl3):
d 1.34 (d, 6H, CH3 isopropyl), 2.30 (m, 2H, CH2), 2.82
(s, 3H, CH3), 3.70 (m, 5H, CH2 and CH isopropyl), 7.20
(dd, 2H, arom H), 10.20 (s, 1H, CHO), 10.60 (s, 1H,
NH, deuterium oxide exchangeable), 11.20 (s, 2H,
NH2, deuterium oxide exchangeable). Anal. Calcd for
C17H22N2O3: C, 67.53; H, 7.33; N, 9.26. Found: C,
67.90; H, 7.35; N, 9.20.
5.4.7. 3-[3-(Aminopropyl)amino]-1-oxo-1H-naphtho[2,1-b]-
pyran-2-carbaldehyde hydrochloride (11ÆHCl). A solution
of 9 (0.4 g, 1.5 mmol) and (1.5 mmol) of N-(triphenylm-
ethyl)propane-1,3-diamine in 15 mL of toluene was stir-
red at rt for 3 h. Evaporation of the solvent and
purification of the residue by column chromatography
(SiO2; AcOEt) yielded 0.65 g of crude 1-oxo-3-{3-[(tri-
phenylmethyl)amino]-propylamino}-1H-naphtho[2,1-b]-
pyran-2-carbaldehyde 10, which was hydrolyzed with
diluted HCl at 60 ꢀC for 2 h. The H2O was removed by
co-evaporation with benzene and the residue crystallized
with EtOH to give 11ÆHCl (yield 0.3 g, 60%) as white
crystals. Mp 210–213 ꢀC (ethanol). IR m 3400, 2900,
2000, 1660, 1630, 1610, 1560, 1490 cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6): d 2.05 (2H, m, CH2), 2.90
(2H, m, CH2N), 3.72 (2H, m, CH2N), 7.73 (3H, m, arom
CH), 8.07 (1H, d, arom CH), 8.20 (3H, NH3), 8.35 (1H,
d, arom CH), 9.90 (1H, d, arom CH), 10.13 (1H, s,
CHO), 10.25 (1H, NH); 13C NMR (50 MHz, DMSO-
5.4.4. 2-[(5-Aminopentyl)amino)]-8-isopropyl-5-methyl-4-
oxo-4H-1-benzopyran-3-carbaldehyde (5b). Analogously
to the preparation of 5a, 0.8 g (2.90 mmol) of 2 and
0.30 g (2.90 mmol) of pentane-1,5-diamine gave, after
purification on silica gel, 5b as light brown crystals (yield
0.24 g, 25%). Mp 120–2 ꢀC (cyclohexane–ethyl acetate,
d6):
d
27.15
(CH2),
36.46
(CH2),
37.82
(CH2), 100.67 (C), 114.48 (C), 117.46 (CH), 126.43
(CH), 126.54 (CH), 128.87 (CH), 129.26 (CH),
130.36 (C), 131.05 (C), 135.78 (CH), 154.36 (C),
163.05 (C), 177.94 (C), 188.74 (CHO). Anal. Calcd
for C17H17N2O3Cl: C, 61.36; H, 5.15; N, 8.42; Cl,
10.65. Found: C, 61.00; H, 5.25; N, 8.31; Cl, 10.85.
1:1). IR m 3400, 2900, 1660, 1560, 1200 cmꢀ1 1H
;
NMR (CDCl3): d 1.30 (d, 6H, CH3 isopropyl), 2.20
(m, 2H, CH2), 2.80 (s, 3H, CH3), 3.68 (m, 5H, CH2
and CH isopropyl), 7.25 (dd, 2H, arom H), 10.20 (s,
1H, CHO), 10.58 (s, 1H, NH, deuterium oxide
exchangeable), 11.15 (s, 2H, NH2, deuterium oxide
exchangeable). Anal. Calcd for C19H26N2O3: C, 69.06;
H, 7.93; N, 8.48. Found: C, 69.46; H, 7.80; N, 8.31.
5.4.8. 1-(2-Hydroxynaphthalen-1-yl)-2-(tetrahydropyrim-
idin-2(1H)-ylidene)ethanone (12). A suspension of 9
(0.2 g, 0.75 mmol) and propane-1,3-diamine (0.63 mL,
7.5 mmol) in toluene (5 mL) was stirred at 110 ꢀC for
2 h. The solvent was evaporated and the residue purified
by column chromatography (SiO2; AcOEt/EtOH 8:2) to
give 0.1 g (yield 50%) of 12 as brownish yellow-coloured
5.4.5. 2-[(3-Aminopropyl)amino]-3,6-dinitro-8-(40-nitro-
phenyl)-4H-1-benzopyran-4-one (7a). A warm solution
of propane-1,3-diamine (0.14 g, 1.85 mmol) in aceto-
nitrile was added dropwise to a warm solution of 6

E. Sottofattori et al. / Bioorg. Med. Chem. 13 (2005) 1515–1522
1521
oil, which solidifies with Et2O. Mp 226–7 ꢀC (ethanol/
ethyl acetate 1:4). IR m 3320, 1620, 1580, 1510, 1460,
was precipitated with 1 mL of 2% LiClO4 in acetone.
After centrifugation, the supernatant was eliminated
and the precipitate dissolved in 50 lL of 3 M LiClO4
and treated with 1 mL of 2% LiClO4 in acetone. The res-
idue (lithium salt) was dissolved in 1 mL of water, puri-
fied by reverse-phase HPLC, collected and evaporated
under reduced pressure (yield 65%).23 The final residue
was dissolved in water and a precisely measured aliquot
is taken off to measure the absorbance at 260 nm.16
Then the solution was precipitated with 2% LiClO4 in
acetone to give an ODN conjugate (4a*, 4b*, 7a*, 7b*,
5a*, 5b*, 11*, 12* or 13*).
1380, 1370 cmꢀ1 1H NMR (200 MHz, DMSO-d6): d
;
1.88 (t, 2H, CH2), 3.33 (m, 4H, CH2N), 5.05 (m, 1H,
CH), 7.03 (d, 1H, arom CH), 7.27 (m, 1H, arom CH),
7.45 (m, 1H, arom CH), 7.76 (m, 2H, arom CH), 8.30
(d, 1H, arom CH), 9.25 (2H, NH), 13.12 (1H, OH);
13C NMR (50 MHz, DMSO-d6): d 19.94 (CH2), 37.72
(CH2), 37.83 (CH2), 85.36 (CH), 118.10 (C), 119.15
(CH), 122.48 (CH), 124.70 (CH), 126.40 (CH), 128.31
(C), 128.63 (CH), 130.71 (CH), 131.00 (C), 157.24 (C),
159.36 (C), 179.58 (C). Anal. Calcd for C16H16N2O2:
C, 71.62; H, 6.01; N, 10.44. Found: C, 71.82; H, 6.11;
N, 10.56.
5.7. Thermal denaturation of complexes
5.4.9. 1-(2-Hydroxy-3-isopropyl-6-methylphenyl)-2-(tet-
rahydropyrimidin-2(1H)-ylidene)ethanone (13). Analo-
gously to the preparation of 12, 0.2 g (0.75 mmol)
of 2-(dimethylamino)-8-isopropyl-5-methyl-4-oxo-4H-
1-benzopyran-3-carbaldehyde 2 and propane-1,3-dia-
mine (0.63 mL, 7.5 mmol) in toluene (5 mL) was heated
at 110 ꢀC for 3 h. After cooling, the yellow precipitate
was filtered off and crystallized from AcOEt (yield
0.12 g, 58%). Mp 240 ꢀC (AcOEt). IR m 3320, 1620,
5.7.1. Preparation of samples. Aqueous solutions of
appropriate concentrations of ODNs were prepared by
diluting a concentrated solution of the ODN or the
ODN-SA according to molar extinction coefficients at
260 nm at 20 ꢀC. Extinction coefficients were calculated
according to results of total PDE-hydrolysis28 and were
as follows (260 nm): 8-mer 70200; 7-mer 66800. The
extinction coefficients for the ODN-SA were estimated
as a sum of respective values for each oligomer and sta-
bilizing agent. Aqueous solutions of ODN or ODN-SA
were mixed with concentrated buffer solutions. In all
cases a final composition of buffer solution was:
0.16 M NaCl, 0.01 M Na2HPO4, 0.1 mM EDTA,
pH 7.0. Concentration of each oligonucleotide chain
was 1.6 · 10ꢀ5 M.
1580, 1510, 1460, 1380, 1370 cmꢀ1
;
1H NMR
(200 MHz, CDCl3): d 1.19 (s, 3H, CH3), 1.22 (s, 3H,
CH3), 1.6 (2H, NH), 2.00 (m, 2H, CH2), 2.4 (s, 3H,
CH3), 3.38 (m, 5H, 2CH2, CH), 4.68 (s, 1H, CH), 6.62
(d, 1H, arom CH), 7.02 (d, 1H, arom CH), 11.25 (s,
1H, OH); 13C NMR (50 MHz, DMSO-d6): d 20.79
(CH2), 22.56 (CH3), 23.10 (2CH3), 26.91 (CH), 38.70
(2CH2), 86.01 (CH), 121.77 (CH), 125.91 (C), 126.41
(CH), 132.60 (C), 133.58 (C), 155.97 (C), 159.99 (C),
184.02 (C). Anal. Calcd for C16H22N2O2: C, 70.04; H,
8.08; N, 10.21. Found: C, 69.89; H, 8.19; N, 10.32.
5.7.2. Melting curves. Optical melting curves were ob-
tained with the use of a home-made apparatus devel-
oped on the base of UV detector of liquid
chromatograph Milichrom (Orel, Russia) connected to
PC computer. Volume of the optical cell was 2 mL,
the cell path length was 1.2 mm. Temperature of the
optical cell was monitored using thermostat-connected
water jacketed cell holder (rate of temperature change
was 0.5 ꢀC/min.) and controlled by Cu-Constantane
thermocouple calibrated with an accuracy of 0.1 ꢀC.
Thermocouple was connected to the PC through digital
voltmeter SH-1516 (Russia). All the data (absorbance
and temperature) were collected by the PC. Each exper-
imental value of optical density was the integral of the
signal for 10 s. A total of 500–600 points were collected
for each melting curve. The corrections caused by the
water heat volume change were added to the final
melting curve profiles.
5.5. Synthesis of oligonucleotides
The oligonucleotides were synthesized in solution,
deprotected and purified following our previous
routes.15,16 Each oligonucleotide in this preparation
showed a single peak either with an anion exchange col-
umn or with a reversed phase column (see HPLC).
5.6. Synthesis of conjugates of oligonucleotides with
stabilizing agents
Following the general procedure previously described,8
3 lL of a 8% water solution of cetyltrimethylammonium
bromide was added to a solution of the lithium salt of
the completely deblocked ODN (5AU at 260 nm) dis-
solved in 50 mL of water and this mixture was then cen-
trifuged. The former solution (1 lL) was then added and
the mixture again centrifuged. The procedure was
repeated until no more precipitate was observed. The
supernatant was eliminated and the residue was dried
in vacuo overnight over P2O5. A solution of this com-
pound in 60 lL of dry DMSO, 0.010 g of triphenylphos-
phine, 0.010 g of dipyridyldisulfide, 0.005 g of N,N-
dimethylaminopyridine was stirred for 10 min and then
0.002 g of the aminoderivative (4a,b, 7a,b, 5a,b, 11, 12 or
13) and 2 lL of anhydrous triethylamine were added.
After stirring for 1 h at room temperature, the solution
Acknowledgements
Financial support of this research by the MIUR (pro-
gram 2002) is gratefully acknowledged.
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