Synthesis and growth inhibition activity of α-bromoacrylic heterocyclic and benzoheterocyclic derivatives of distamycin A modified on the amidino moiety

Article abstract of DOI:10.1016/S0968-0896(02)00533-3

The design, synthesis and in vitro activities of novel α-bromoacryloyl pyrazole, imidazole and benzoheterocyclic derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the distamycin frame, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity of α-bromoacrylic derivatives of distamycin.

Full text of DOI:10.1016/S0968-0896(02)00533-3

Bioorganic & Medicinal Chemistry 11 (2003) 965–975
Synthesis and Growth Inhibition Activity of ꢀ-Bromoacrylic
Heterocyclic and Benzoheterocyclic Derivatives of Distamycin A
Modified on the Amidino Moiety
Pier Giovanni Baraldi,a,* Italo Beria,b Paolo Cozzi,b Nicoletta Bianchi,c
Roberto Gambaric and Romeo Romagnolic
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, 44100 Ferrara, Italy
bPharmacia, Global Chemistry, Discovery Research Oncology, Nerviano, Milan, Italy
`
Dipartimento di Biochimica e Biologia Molecolare, Universita di Ferrara, 44100 Ferrara, Italy
c
Received 3 July 2002;accepted 18 October 2002
Abstract—The design, synthesis and in vitro activities of novel a-bromoacryloyl pyrazole, imidazole and benzoheterocyclic deriva-
tives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features are
described, and the structure–activity relationships are discussed. In spite of the relevance of these modifications on the distamycin
frame, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. Therefore, the presence
of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity of a-bromoacrylic
derivatives of distamycin.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
frame, the a-bromoacryloyl derivative of distamycin A
showed good antiproliferative activity
2
a
Distamycin A 1 is a natural antibiotic belonging to the
class of DNA minor groove binding drugs and char-
acterized by an oligopeptidic pyrrolecarbamoyl frame
ending with an amidino moiety.1 This compound binds
reversibly to double helical of B-DNA with high selec-
tivity for AT-rich sequences containing at least four
adenine-thymine (AT) base pairs. Van der Waals forces
and hydrogen bonding play the key role for the DNA
binding, while hydrophobic interactions and electro-
static binding component from the cationic end stabilise
the complex.2 Distamycin A was used as DNAminor
groove sequence-selective vector of alkylating functions,
leading to compounds endowed with relevant cytotoxic
and antitumor activity in comparison to that, very
weak, of distamycin itself.3
(IC50=79.6ꢀ14 nM against L1210 murine leukaemia
cell line), more than two order of magnitude greater
than that of 1,4 further improved in the case of its four
rings homologue 3.4 The increase from three to four of
the number of pyrrolic rings is associated with a 10-fold
increase in growth inhibition potency. This possibly
arises from a tighter DNA binding, depending on the
increased multiplicity of interaction between the pyrro-
lecarbamoyl units and DNA AT-rich sequences.
Recently, the a-bromoacrylamido derivative of four-
pyrrole distamycin homologue 3 was found to bind to
DNA minor groove AT-rich regions,being however
unable to alkylate AT sequences. This finding may sug-
gest that 3 represents the lead of a new class of minor
groove binders.5,6
Among the cytotoxic derivatives in which a poten-
tial alkylating moiety of low chemical reactivity was
tethered to the N-terminal position of the distamycin
The relevant activity and the atypical mechanism of
action of 3 stimulated the synthesis of new halogen-
oacrylic compounds with the aim to optimising their
profile of activity and possibly to help in defining their
mechanism of action. In previous papers we have
described the synthesis and growth inhibition activitie-
sagainst L1210 murine leukaemia cell line of a series of
*Corresponding author. Tel.:+39-0532-291293;fax:+39-0532-
291296;e-mail: pgb@dns.unife.it
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00533-3

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P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
isosteric analogues of the a-bromoacrylamido derivative
of four pyrrole homologue of distamycin A 3.7,8 The
replacing of N-methylpyrrole directly linked to the
a-halogenoacrylic with isosteric five-membered hetero-
cyclic rings such as N-methyl-pyrazole and -imidazole
gave the compounds 4 and 5, respectively,7 or with dif-
ferent benzoheterocyclic rings, such as indole,
N-methylindole, benzofuran and benzothiophen, furn-
ished the derivatives 6, 7, 8 and 9 respectively.8 The
isosters 4 and 5 were 2- and 7-fold less actives than 3
(IC50=6.3ꢀ1.3 nM for 3 vs IC50=13.3ꢀ0.5 nM and
46.9ꢀ13 nM for 4 and 5, respectively). For the derivatives
6–9, the antileukemic activity was only slightly affected by
the kind of heteroatom present in the benzoheterocyclic
ring (IC50=4.1ꢀ1.3 nM, 2.4ꢀ0.4 nM, 6.1ꢀ0.4 nM and
14.3ꢀ5.9 nM for 6, 7, 8 and 9, respectively).
compounds in which the potent cytotoxicity of the par-
ent amidino derivative was fully maintained and in
some cases even increased. This occured not only in the
case of basic amidino-like compounds of different lipo-
philicity and bulk, such as N-methylamidine (10,
IC50=2.7ꢀ0.78 nM) and N,N0-dimethylamidine (11,
IC50=1.9ꢀ0.18 nM), but also by non basic amidino-
derived such as cyanamidine (12, IC50=4.1ꢀ0.3 nM).
The finding that the amidino moiety of the a-bromo-
acryloyl derivative 3 could be replaced by other ami-
dino-like or non basic-amidino-like moieties of different
nature confirms therefore that the presence of a basic
moiety is not an absolute requirement for in vitro
activity against L1210 cell line.9
A feature of 3 is the presence of an amidino moiety that,
due to its strong basic nature (its pKa should be about
12.5), exhibits a complete protonation in any biological
conditions, and may play a key role both for DNA
binding and cell or tissue bioavailability. In compound
3 the replacement of the amidino group with basic or
non-basic amidino moieties of different nature led to
In this article we reported the synthesis and growth
inhibitory activity of a new series of a-bromoacrylic
derivatives, 13–26, structurally related to compounds 4–
9, in which (a) the N-terminal pyrrole was replaced with
Scheme 1. CH3NH2, DMF, rt (for the synthesis of 27), CH3NH2, DMF 80 ꢁC (for the synthesis of 28) NH2CN, NaH, DMF, rt (for the synthesis of
29);(b) HCl in EtOH, rt; c: 33–38, EDC, DIPEA, DMF.

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
967
both pentatomic heteroaromatic units, such as pyrazole
or imidazole rings, and benzoheterocyclic units, such as
indole, N-methylindole, benzofurane and benzothio-
phene and (b) the amidino moiety was replaced by basic
and non-basic groups of different electronic nature,
lipophilicity and bulk. While N-methylamidine deriva-
tives 13, 16, 19, 21, 23 and 25 and N, N-dimethylami-
dine derivatives 14, 17, 20, 22, 24 and 26 are strongly
basic amidine compounds (pKa values for the N-methyl-
amidine and N, N-dimethylamidine residues are 12.37
and 12.68, respectively), cyanoamidines 15 and 18
are not basic at all (pKa=0.256 for the cyanoamidine
fragment).10
Table 1. In vitro activity and resistance index (R.I.) of distamycin
derivatives against L1210 and L1210 murine leukemia cells resistant to
the doxorubicin (DX)
Compound
IC50 (nMꢀS.E.)
R.Ia
L1210
L1210/DX
Distamycin A4
10,069ꢀ1647
79.6ꢀ14
459,000ꢀ5216
700.0ꢀ109
24.0ꢀ2
45.6
8.8
3.8
7
15.5
8.7
18
6.4
3.4
21.7
13
18.3
3.8
4.5
5.3
6.3
3.4
4.9
5.2
3.5
7.9
5.6
3.6
6.1
5.1
3.5
27.6
24
34
6.3ꢀ1.34
13.3ꢀ0.54
46.9ꢀ13
47
93.1ꢀ17
726.9ꢀ6.9
35.8ꢀ5.6
43.6ꢀ1.5
39.0ꢀ2
57
68
4.1ꢀ1.3
78
2.4ꢀ0.39
6.1ꢀ0.93
14.3ꢀ5.89
2.7ꢀ0.78
2.0ꢀ0.18
4.1ꢀ0.27
10.3ꢀ0.26
25.6ꢀ3.75
7.7ꢀ0.34
155.1ꢀ52.9
59.7ꢀ15.6
186ꢀ11.48
4.5ꢀ0.25
7.6ꢀ2.84
4.8ꢀ0.49
2.8ꢀ0.36
9.5ꢀ1.76
6.3ꢀ0.62
10.5ꢀ1.48
17.9ꢀ6.66
21.8ꢀ1.33
88
98
48.3ꢀ2
109
58.6ꢀ6.4
26ꢀ7.2
119
129
13
75.6ꢀ5.71
38.6ꢀ3
Chemistry
The novel derivatives 13–26 were obtained following the
procedure reported in the Scheme 1. The starting mate-
rial utilized for the synthesis of the cited compounds
was Distamycin A 1.1 The N-methylamidine 27 and
N,N-dimethylamidine 28 were prepared by reacting 1,
dissolved in DMF, with 3 equiv of aq CH3NH2, at 25 ꢁC
or 6 equiv of aq CH3NH2 at 80 ꢁC, respectively, whereas
the cyanamidine 29 was prepared from Distamycin A
with 3 equiv of NH2CN sodium salt, obtained in situ
with NaH in DMF. These amidino modified Distamycin
A derivatives 27–29 were then transformed in the corre-
sponding desformyl derivatives 30–32.9 The condensa-
tion of 30–32 with the appropriate a-bromoacryloyl
heterocyclic/benzoheterocyclic-2-carboxylic acid deriva-
tive 33–387,8 was performed using an excess (2 equiv) of
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydro-
chloride (EDCI) as the coupling agent, in DMF as a
solvent, in the presence of Hunig’s base, at room tem-
perature and with identical reaction times (18 h), to
obtain the final compounds 13–26 in good yields after
purification by silica gel flash-chromatography.
14
15
16
17
18
19
20
21
22
23
24
25
115.7ꢀ3.4
41.0ꢀ4
970.0ꢀ11
200.2ꢀ4.1
914.8ꢀ15.2
23.5ꢀ7
26.7ꢀ7
38.3ꢀ1.11
15.4ꢀ2
33.3ꢀ9
38.6ꢀ1
53.2ꢀ4
26
Doxorubicin
62.3ꢀ5
601ꢀ37.6
IC50=50% inhibitory concentration as the mean ꢀSE from dose–
response curves of at least three experiments.
aR.I (resistance index)=ratio between IC50 values on resistant cells
ities on L1210 murine leukemia cell line, with IC50 ran-
ged between 2 and 80 nM. For the compounds which
possess the same modified amidine terminus, the repla-
cement of the N-terminal pyrrole ring with indole and
N-methyl indole keep the activity substantially unchan-
ged (compounds 10, 19, 21 and 11, 20, 22). By compar-
ing the activities of compounds in which the N-terminal
pyrrole (compounds 10–12) has been substituted with a
pyrazole (derivatives 13–15), it appears that the pre-
sence of a basic moiety is not an absolute requirement
for in vitro activity. This confirms the finding already
reported9 which contrasts with the common opinion
that electrostatic interactions between the cationic moi-
ety and the negatively charged DNA phosphate residues
may represent one of the main contributions to mole-
cular recognition of distamycin-like derivatives. The-
neutral or positive role played by the modifications of
the amidino moiety is confirmed also in the case of
pyrazole and benzoheterocyclic derivatives 13–15 and
19–26, respectively. These compounds maintain or even
improve the cytotoxicity of the amidine parent com-
pounds 4 and 6–9, respectively. Moreover, in the series
of pyrazolic amidino modified-derivatives 13–15 on the
L1210 cell line, the non-basic cyanamidine derivative 15
is not only more active than the N-methylamidine and
N,N-dimethylamidine derivatives 13 and 14 respectively,
but also more potent than the parent amidine com-
pound 4. On the other hand, the imidazole N-methyl-
amidine 16 and cyanamidine 18 derivatives showed
a decreased activity in comparison to the parent
Results and Discussion
All the newly synthesized compounds have been firstly
assayed in vitro on L1210 murine leukemia cells, in
order to obtain preliminar informations on the effects
on cell growth. We employed the L1210 screening sys-
tem since this cell line has been claimed to be predictors
of clinically useful anticancer drugs.11ꢂ15 L1210 cells
were treated continously for 48 h with increasing
amounts of the tested compounds and the number of
viable cells were compared with that of control
untreated cells. The results obtained demonstrate that
all of the tested compounds displayed growth inhibition
effects, despite with different activities. The growth
inhibitory activities of the synthesized compounds were
expressed as nanomolar concentrations of the com-
pound that inhibited 50% of cell proliferation (IC50)
and are compared with the IC50 values in the same cell
lines of distamycin A 1, Doxorubicin (DX) and the dis-
tamycin derivatives 2–12 (Table 1, left column).
With the only exception of distamycin A, compounds 16
and 18 (IC50=10,4 133 and 186 nM, respectively), all
tested molecules exhibited strong growth inhition activ-

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P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
compound 5, while, on the contrary, the activity of the
imidazolic N,N0-dimethyamidine derivative 17 appears
good and roughly comparable to that of the parent
amidino derivative 5. The low antiproliferative potency
of imidazole derivatives 16 and 18 contrast with the
significant activity of pyrazole counterparts 13 and 15
which possess the basic N-methylamidine and the non-
basic cyanamidine moieties, respectively. These data
indicate a lack of correlation between the basicity of the
amidine-like structure and growth inhibition activity,
where this latter depends only from the isosteric five-
membered heterocyclic ring (N-methyl-pyrazole and
-imidazole) joined to the a-bromoacrylic moiety.
investigated the in vitro antiproliferative activity
measured after 4 days (K562 cells) and 5 days (Raji
cells), when control untreated cells are in the log
phase of cell growth. This is required in order to com-
pare the activities of the same compounds on cell lines
exhibiting different proliferation rates. The growth
inhibitory activities of the synthesized compounds are
shown in Table 2 were expressed as mmolar concentra-
tions of the compound that inhibited 50% of cell pro-
liferation (IC50). Usually, the IC50 values obtained using
K562 cells were lower with respect to those obtained by
using Raji cells. These differences are not unexpected,
since it is well known that cells from different origins
and histotype or exhibiting different cell growth kinetics
could display sharply different IC50 values when
exposed to antiproliferative compounds.18 In any case,
when compounds are compared within the same cell
line, the results obtained are convergent and in agree-
ment from those obtained using the L1210 screening
system.
Table 1 (central and right columns) shows also the growth
inhibitory activities and the resistance index (R.I.) values
of 1–26 on L1210 leukemic cell line resistant to doxo-
rubicin (DX) and compared with that of DX. The results
obtained show that the newly synthesized compounds
13–26 display low R.I. values, ranging from four to eight,
showing that neither the modification on the amidino
moiety nor the heterocycle joined to the a-bromoacryloyl
moiety were involved in the resistance mechanism.
The data reported in the Tables 1 and 2 reveal that
L1210 cells are the most sensitive to these com-
pounds in contrast to K562 and Raji cells , while the
B-lymphoid Raji cell line is generally more resistant
to tested derivatives. The huge differential in the activ-
ity of these compounds toward murine and human cell
lines may be due, in part at least, to the fact that L1210
cells grow far more quickly than both K562 and Raji
cells.
In addition, the second set of experiments was per-
formed using as target cells two human cell lines, the
K562 cell line, from a patient with chronic myelogenous
leukemia in blast crisis16 and the B-lymphoid Raji17 cell
line, from a B-lymphoma. This evaluation was under-
taken in order to ascertain whether the compounds were
cytotoxic to human neoplastic cells. In this case we
In order to determine the efficiency of interaction with
DNA, arrested polymerase-chain reaction (PCR) was
performed on selected compounds 3, 9, 11, 13, 24 and
25. In a single control experiment, the antiproliferative
activity of the same compounds used in PCR assays,
was assayed both on K562 and Raji cells after 5 days
cell culture. We have elsewhere reported that arrested
PCR is a valuable tool to determine difference in the
efficiency of interactions between DNA-binding drugs
and target DNA molecules.19ꢂ22 The results obtained
are shown in Table 3 and suggest that the compounds
tested show the ability to interact with DNA, but only
at high concentrations (5–80 mM). In addition, a good
relationship between efficiency in the arrested PCR
Table 2. Effects of distamycin derivatives on in vitro cell growth of
human erythroleukemic K562 and B-lymphoid Raij cell lines
Compd
IC50 (mMꢀSE)
K562
Raji
Distamycin A4
20ꢀ2.2
1.11ꢀ0.2
35ꢀ2.3
1.7ꢀ0.13
24
34
0.045ꢀ0.013
0.51ꢀ0.032
1.4ꢀ0.07
0.046ꢀ0.01
1.2ꢀ0.11
47
57
3.7ꢀ0.13
68
0.58ꢀ0.035
0.29ꢀ0.05
1.2ꢀ0.13
2.2ꢀ0.14
78
1.2ꢀ0.011
1.4ꢀ0.15
88
98
1.26ꢀ0.09
0.038ꢀ0.0072
0.031ꢀ0.0027
0.083ꢀ0.0021
0.682ꢀ0.13
0.70ꢀ0.11
2.5ꢀ0.16
1.41ꢀ0.12
0.510ꢀ0.023
109
119
0.0462ꢀ33
0.160ꢀ0.023
6.25ꢀ0.23
3.32ꢀ0.28
8.01ꢀ0.36
5.11ꢀ0.35
5.51ꢀ0.48
12.12ꢀ1.7
1.82ꢀ0.23
2.11ꢀ0.22
2.40ꢀ0.29
0.67ꢀ0.0082
2.16ꢀ0.22
1.65ꢀ0.21
1.93ꢀ0.21
7.51ꢀ0.45
0.068ꢀ0.0023
129
13
Table 3. In vitro effects of distamycin derivatives on arrested PCR
and on cell growth both of K562 and Raji cell lines
14
15
16
17
18
19
20
21
22
23
24
25
1.53ꢀ0.23
1.75ꢀ0.23
2.85ꢀ0.28
0.530ꢀ0.09
0.85ꢀ0.11
0.72ꢀ0.11
0.035ꢀ0.007
0.99ꢀ0.19
1.18ꢀ0.14
1.51ꢀ0.10
1.43ꢀ0.34
0.053ꢀ0.0052
Compd
Polymerase-chain
reaction (PCR)
K562
Raji
a
IC50 (mM)
IC50b (mM)
IC50b (mM)
Distamycin A
200
5
30
8
80
50
50
25
0.025
1.5
0.05
1
39
0.045
1.5
0.05
8
3
9
11
13
24
25
1.7
2.5
2.2
2
26
Doxorubicin
aInhibitory concentration (mM) necessary to obtain 50% inhibition of
generation of PCR product.
bConcentration causing 50% inhibition of growth both of human
erythroleukemic K562 and B-lymphoid Raji cells.
IC50=concentration of the compound (mM) causing 50% inhibition of
cell proliferation as the meanꢀSE from dose–response curves of at
least three experiments.

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
969
Figure 1. DNase I footprinting assay. In this experiment a 32P-labelled g-globin gene PCR product was produced using 32P-labelled forward (50-
GGA ATG ACT GAA TCG GAA CAA GGC-30) and unlabelled reverse (50-CTG CTG AAG GGT GCT TCC TTT TAT-30) PCR primer. An
aliquot of the footprinting probe was incubated in 50 mL in the absence (ꢂ) or in the presence of 1 (a), 5 (b) and 10 (c) mM compound 24. After 30
min incubation at room temperature the samples were treated with 1U/reaction of DNAse I (2 min), ethanol precipitated and analysed by electro-
phoresis on a sequencing gel. The nucleotide sequences corresponding to the DNase I footprints are indicated. ATA/TATA motifs are boxed.
Arrows indicate DNase I hypersensitive sites. Asterick indicates full sized PCR products.
assay and inhibitory effects of cell growth of K562 and
Raji cells was obtained (Table 3).
regions (arrowed). Interestingly, all the footprints obtained
contain TAT or TATA elements, sustaining the concept
that these drugs belongs to AT-binders. Accordingly, lack
of footprints is related to a lack of TAT/TATA elements
(Fig. 1, left side of the panel). Similar results were
obtained with the other compounds, when used at con-
centrations allowing efficient binding to DNA.
With respect to selectivity of the interaction with
the DNA, an example of the results obtained is
shown in Figure 1, depicting the DNase I foot-
printing23 obtained using compound 24 at different
concentrations (1, 5 and 10 mM, respectively). As
clearly appreciable, when a DNA mimicking the
g-globin gene promoter is incubated with DNase I after
treatment with increasing amounts of compound 24,
clear footprints are detectable (Fig. 1, right side of the
panel) only at 10 mM, as well as DNase I hypersensitive
In conclusion, the positive role played by some
modifications of the amidino moiety is confirmed also
in the case of pyrazole and benzoheterocyclic deriva-
tives, and some of these compounds maintain or even
improve the growth inhibitory activity of the parent

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P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
compounds 4 and 6–9, respectively. On the other hand,
the imidazolic amidine derivatives 16 and 18 showed
a decreased activity in comparison to the parent
compound 5, and this may be due to the drug’s
ability to enter the cell. The new synthesized com-
pounds 13–26 showed the capability to interact with
DNA at high concentrations with selectivity for AT-rich
sequences. Although the mechanism of action of the
a-bromoacryloyl derivatives 13–26 is still unknown, it
may be hypothised that they are a new class of minor
groove binders acting through a mechanism different
from a direct DNA alkylation.
and DBU (0.9 mL) were added. The mixture was stirred
for 2 h, poured in cooled water (50 mL) and the result-
ing crystalline solid collected and washed with water,
affording the desired t-butyl-5-nitro benzothiophen-2-
carboxylate as a white solid (963 mg, 69% yield);
mp=175–177 ꢁC;IR (KBr): 1717, 1533, 1370, 1370,
1
1343, 1253, 1156 and 831 cmꢂ; H NMR (CDCl3) d:
1.63 (s, 9H), 7.96 (d, J=8.8 Hz, 1H), 8.09 (s, 1H), 8.27
(dd, J=8.8 and 2.2 Hz, 1H), 8.75 (d, J=2.2 Hz, 1H).
The t-butyl-5-nitro benzothiophen-2-carboxylate (5
mmol) was reduced over 10% Pd/C (100 mg) in a mix-
ture of dioxane (10 mL) and methanol (10 mL) at room
temperature under 55 psi pressure. After reduction, the
catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure. The residue,after
purification by flash-chromatography (EtOAc–petroleum
ether, 1:1, v/v) was dissolved in EtOAc and precipitated
with an excess of petroleum ether. The t-butyl-5-amino-
benzothiophen-2-carboxylate was obtained as a yellow
Experimental
Chemical materials and methods. General procedure
All reactions were carried out under Argon atmosphere,
unless otherwise described. Standard syringe techniques
were applied for transferring anhydrous solvents. Reac-
tion courses and product mixtures were routinely mon-
itored by thin-layer chromatography on silica gel
(precoated F254 Merk plates), the spots were examined
with UV light and visualized with aq KMnO4. Infrared
spectra were recorded on a Perkin-Elmer 1710 spectro-
photometer. 1H NMR spectra were recorded in the
given solvent with a Bruker AC 200 spectrometer. Che-
mical shifts are reported as (d) values in parts per mil-
lion. The splitting pattern abbreviations are as follows: s
(singlet), d (doublet), dd (double doublet), t (triplet), br
(broad), and m (multiplet). Matrix-assisted laser deso-
rbion ionization time-of-flight (MALDI-TOF) mass
spectrometry of all synthesized compounds was con-
ducted using a Hewlett Packard G 2025 A LD-TOF
instrument. The samples were analyzed in the linear
mode with 28 kV accelerating voltage, mixing them with
a saturated solution of a-cyano-4-idroxycinnamic acid
matrix. Melting points (mp) were determined using a
Buchi-Tottoli apparatus and are uncorrected. All pro-
1
oil (1.25 g, >95%). H NMR (CDCl3)d: 1.59 (s, 9H),
3.44 (bs, 2H), 7.44 (d, J=8.4 Hz, 1H), 7.62 (s, 1H), 8.22
(dd, J=8.4 and 2.2 Hz, 1H), 8.55 (d, J=2.2 Hz, 1H).
To a solution of a-bromoacrylic acid (600 mg, 4 mmol)
in dry DMF (5 mL), a solution of EDC (383 mg, 2
mmol) in dry DMF (5 mL) was slowly added. After 1 h,
the solution obtained was added to a solution of t-butyl-
5-aminobenzothiophen-2-carboxylate (500 mg, 2 mmol)
in dry DMF (5 mL). The reaction was stirred at room
temperature for 18 h, and then concentrated under
reducedpressure. The residue was dissolved with a mix-
ture of EtOAc (10 mL) and water (5 mL), the organic
phase dried over Na2SO4, evaporated to dryness in
vacuo and the resulting crude residue purified by flash
chromatography (EtOAc–petroleum ether, 2:8, v/v)
furnished t-butyl-5-bromoacrylamidobenzothiophen-2-
1
carboxylate as a orange oil (587 mg, 77% yield). H
NMR (CDCl3) d: 1.61 (s, 9H), 6.17 (d, J=1.6 Hz, 1H),
6.38 (d, J=1.6 Hz, 1H) 7.50 (dd, J=8.6 and 2.2 Hz,
1H), 7.81 (d, J=8.6 Hz, 1H), 7.94 (s, 1H), 8.27 (d,
J=2.2 Hz, 1H), 8.49 (bs, 1H).
1
ducts reported showed H NMR spectra in agreement
with the assigned structures. Mass spectra were recor-
ded on a Nermag R10, 10C spectrometer. Elemental
analyses, conducted by the Mycroanalytical Laboratory
of the Chemistry Department of the University of Fer-
rara, were within 0.4% of the theorical values calculated
for C, H, Br, Cl and N. Column chromatography was
carried out with Merck silica gel (230–240 mash). All
compounds obtained commercially were used without
further purification. Organic solutions were dried over
anhydrous MgSO4. Methanol was distilled from mag-
nesium turnings, dioxane was distilled from calcium
hydride and anhydrous DMF was distilled from calcium
chloride and stored over molecular sieves (3 A). In high-
pressure hydrogenation experiments, a Parr shaker on a
high-pressure autoclave was used.
A solution of t-butyl-5-bromoacrylamidobenzothio-
phen-2-carboxylate (381 mg, 1 mmol) in CF3COOH (1
mL), was stirred at room temperature for 3 h. The sol-
vent was removed under reduced pressure and the
resulting residue was suspended in CH2Cl2 (3 mL) ,
collected by filtration and triturated with ethyl ether (5
mL) to obtain 38 as a white powder (300 mg, 88%
yield);mp=190–195 ꢁC;IR (KBr): 3292, 1658, 1535,
1
1302 and 892 cmꢂ1; H NMR (DMSO-d6) d: 6.33 (d,
J=3.2 Hz, 1H), 6.78 (d, J=3.2 Hz, 1H) 7.70 (dd, J=9
and 1.4 Hz, 1H), 7.99 (d, J=9 Hz, 1H), 8.11 (s, 1H),
8.36 (d, J=1.4 Hz, 1H), 10.5 (bs, 1H).
Preparation of N-[5-({[5-({[3-amino-3-(methylimino)-
propyl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]amino}-
carbonyl)-1-methyl-1H-pyrrol-3-yl]-4-(formylamino)-1-
methyl-1H-pyrrole-2-carboxamide dihydrochloride (27).
To a stirred solution of distamycin A (2 g, 3.86 mmol)
in DMF (50 mL) was added methylamine (40% in
water) (0.32 mL, 3.86 mmol) at room temperature. The
Synthesis of the 5-ꢀ-bromoacrylamido 2-benzothiophene-
2-carboxylic acid (38). A solution of 5-nitro-benzothio-
phen-2-carboxylic acid24 (1.15 g., 5 mmol) in dry DMF
(10 mL) cooled at 0 ꢁC was treated with CDI (1.1 g, 6.5
mmol). After 1 h at room temperature, t-butanol (1 mL)

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
971
solution was stirred for 24 h, and after this time methyl-
amine (0.08 mL, 0.097 mL) was added. The mixture was
stirred for another 5 h, then evaporated. The crude
product purified by chromatography on a silica gel col-
umn, using DCM–MeOH (8:2, v/v) as eluent, furnished
the compound 27 (1.5 g, 73% yield) as a brown solid;
mp=156–159 ꢁC;IR (KBr): 3417, 1637, 1465, 1432, 1257
and 845 cmꢂ1; 1H NMR(DMSO-d6) d 2.61 (m, 2H), 2.78
(s, 3H), 3.50 (m, 2H), 3.81 (s, 3H), 3.86 (s, 3H), 3.87 (s,
3H), 6.93 (m, 2H), 7.06 (d, J=1.8 Hz, 1H), 7.18 (m, 2H),
7.22 (d, J=1.8 Hz, 1H), 8.21 (d, J=1.6 Hz, 1H), 8.22 (m,
reaction was filtered, the residue washed with dry ethyl
ether (5 mL) and then dried on P2O5. The crude product
has been used without purification for the next reaction.
Preparation of 4-amino-N-[5-({[5-({[3-amino-3-(methyli-
mino)propyl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]
amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]-1-methyl-1H-
pyrrole-2-carboxamide dihydrochloride(30). Brown solid,
mp=168–170 ꢁC;IR (KBr): 3417, 1637, 1466, 1431 and
1
1258 cmꢂ1; H NMR(DMSO-d6) d 2.61 (m, 2H), 2.80
(d, J=6.0 Hz, 3H), 3.40-3.60 (m, 2H), 3.79 (s, 3H), 3.85
(s, 3H), 3.90 (s, 3H), 6.91 (d, J=1.7 Hz, 1H), 7.05 (d,
J=1.7 Hz, 1H), 7.08 (d, J=1.7 Hz, 1H), 7.11 (d, J=1.7
Hz, 1H), 7.19 (d, J=1.7 Hz, 1H), 7.25 (d, J=1.7 Hz,
1H), 8.25 (t, J=5.8 Hz, 1H), 8.63 (s, 1H), 9.20 (s, 1H),
9.65 (m, 1H), 10.18 (s, 1H), 10.20 (s, 3H);FAB-MS
(MALDI–TOF): 470.5 [M+1]+.
1H), 8.50–9.5 (bs, 3H), 9.91 (s, 1H), 9.93 (s, 1H), 10.12
+
(s, 1H);FAB-MS (MALDI–TOF): 497.5 [M+1]
.
Preparation of 4-(formylamino) -1-methyl -N-[1-methyl-
5-({[1-methyl-5-({[3-(methylamino) -3-(methylimino) pro-
pyl]amino}
carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-
1H-pyrrol-3-yl]-1H-pyrrole-2-carboxamide dihydrochlor-
ide (28). To a stirred solution of distamycin A (1.5 g,
2.89 mmol) in DMF (20 mL) heated at 80 ꢁC were
added three portions of methylamine (40% in water)
(2.5 mL), waiting 1 h after the last addition. After 1 h
from the addition of the last portion of methylamine the
mixture was evaporated. The crude product purified by
chromatography on a silica gel column, using DCM–
MeOH (9:1, 8:2 and then 7:3) as eluent, furnished the
compound 28 (500g, 32% yield) as a yellow solid;
mp=168–170 ꢁC;IR (KBr): 3424, 1652, 1456, 1422,
Preparation of 4-amino-1-methyl-N-[1-methyl-5-({[1-
methyl-5-({[3-(methylamino)-3-(methylimino)propyl]amino}
carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1H-pyrrol-3-yl]-
1H-pyrrole-2-carboxamide dihydrochloride (31). Brown
solid, mp=187–189ꢁC;IR (KBr): 3434, 1648, 1512, 1445
and 1287 cmꢂ1; 1H NMR(DMSO-d6) d 2.72 (m, 2H), 2.80
(d, J=6.0 Hz, 3H), 3.02 (d, J=6.0 Hz, 3H), 3.50 (m, 2H),
3.82 (s, 3H), 3.86 (s, 3H), 3.92 (s, 3H), 6.92 (d, J=1.8 Hz,
1H), 7.02 (d, J=1.8 Hz, 1H), 7.06 (d, J=1.8 Hz, 1H), 7.12
(d, J=1.8 Hz, 1H), 7.19 (d, J=1.8 Hz, 1H), 7.24 (d,
J=1.8 Hz, 1H), 7.90 (bs, 1H), 8.38 (t, J=5.9 Hz, 1H),
8.35(m, 1H), 9.62 (m, 1H), 9.96 (s, 1H), 10.18 (s, 1H), 10.22
(bs, 3H);FAB-MS (MALDI–TOF): 484.6 [M+1]+.
1
1274, 1237 and 872 cmꢂ1; H NMR (DMSO-d6) d 2.71
(m, 2H), 2.78 (s, 3H), 3.02 (s, 3H), 3.49 (m, 2H), 3.80 (s,
3H), 3.84 (s, 3H), 3.85 (s, 3H), 6.92 (m, 2H), 7.05 (d,
J=1.8 Hz, 1H), 7.20 (m, 2H), 7.22 (d, J=1.8 Hz, 1H),
8.12 (d, J=1.6 Hz, 1H), 8.31 (m, 1H), 9.93 (bs, 2H), 10.12
Preparation of 4-amino -N-[5-({[5-({[(3Z)-3-amino-3-(cy-
anoimino)propyl]amino}carbonyl)-1-methyl-1H-pyrrol-3-
yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]-1-methyl-
(s, 1H);FAB-MS (MALDI–TOF): 511.1 [M+1] +
.
Preparation of N-[5-({[5-({[(3-amino-3-(cyanoimino)pro-
pyl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]amino}car-
bonyl)-1-methyl-1H-pyrrol-3-yl]-4-(formylamino)-1-
methyl-1H-pyrrole-2-carboxamide Hydrochloride (29).
To a stirred solution of NH2CN(650 mg, 15.5 mmol) in
dry DMF (50 mL) was slowly added NaH (50% sus-
pension in mineral oil) (375 mg of suspension corre-
sponding to 15.5 mmol of pure NaH). After 300,
Distamycin A (2 g, 3.86 mmol) was dissolved in the
solution and the mixture stirred for another 5 h. After
this time, the solution was acidified (pH=4) with acetic
acid and then evaporated. The residue purified by col-
umn chromatography using DCM and MeOH (9:1, v/v)
as eluent afforded 1.8 g. (92% yield) of compound 29 as
a cream solid, mp=123–126 ꢁC;IR (KBr): 2256, 1648,
1H-pyrrole-2-carboxamide hydrochloride (32). Green
solid, mp=112–114 C;IR (KBr): 3251, 2251, 1713,
!
1667, 1651, 1557, 1440, 1260, 1102 and 778 cmꢂ1; H
1
NMR (DMSO-d6) d 2.70 (m, 2H), 3.55 (m, 2H), 3.82 (s,
3H), 3.85 (s, 3H), 3.91 (s, 3H), 6.88 (m, 1H), 7.02 (d,
J=1.8 Hz, 1H), 7.10 (d, J=1.8 Hz, 1H), 7.12 (d, J=1.8
Hz, 1H), 7.20 (d, J=1.8 Hz, 1H), 7.28 (d, J=1.8 Hz, 1H),
8.28 (bs, 1H), 8.35(bs, 1H), 9.91(s, 1H), 10.12 (s, 1H),
10.15 (bs, 3H);FAB-MS (MALDI–TOF): 479.5 (M+1)+.
General procedure for the synthesis of compounds (13–
26). A solution of amidino-modified N-deformyldista-
mycin A dihydrochloride (compounds 30–31, 0.5 mmol)
in dry DMF (5 mL) was cooled to 5 ꢁC and N, N’diiso-
propylethylamine (86 mL, 0.5 mmol) was added. After
10 min, the acids 33–38 (0.6 mmol, 1.2 equiv) and then
EDC (192 mg, 1 mmol) were added. The reaction mix-
ture was stirred at room temperature for 18 h, then 2 N
hydrochloric acid was added until pH=4. The solvent
was evaporated in vacuo and the crude residue purified
by flash chromatography to yield an oil, which was
precipitated from MeOH/diethyl ether.
1
1442, 1427, 1256 and 845 cmꢂ1; H NMR(DMSO-d6) d
2.70 (m, 2H), 3.45 (m, 2H), 3.82 (s, 3H), 3.84 (s, 3H),
3.85 (s, 3H), 6.85 (m, 2H), 7.91 (d, J=1.8 Hz, 1H), 7.02
(d, J=1.8 Hz, 1H), 7.21 (d, J=1.8 Hz, 1H), 7.22 (d,
J=1.8 Hz, 1H), 8.12 (d, J=1.6 Hz, 1H), 8.05–8.40 (bs,
2H), 9.91 (s, 1H), 9.93 (bs, 2H), 10.08 (s, 1H);FAB-MS
(MALDI–TOF): 507.6 [M+1]+.
General procedure for the synthesis of (30–32). To 5 mL
of a solution obtained by the addition of 24 mL of eth-
anol (95%) to 1 mL of 36% HCl in water was added 1
equiv of compounds 27–29, and the mixture so obtained
stirred at room temperature for 24 h. After this time, the
N-Methyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [3(ꢀ-
bromoacrylamido) pyrazole-5-carboxamido] pyrrole-2-
carboxamido]pyrrole-2-carboxamido] pyrrole-2-carboxa-
mido]propionamidine hydrochloride (13). Following the
general procedure, starting from 30 (200 mg, 0.365

972
P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
mmol), Hunig’s base (63 mL, 0.367 mmol), EDC (150
mg, 0.75 mmol) and the acid 33 (100 mg, 0.365 mmol),
after work-up, the residue was purified by flash chro-
matography using methanol–methylene chloride 3:7
(v:v) as eluent. The resulting oil was precipitated and
yielding the compound 13 as a white solid (175 mg, 63%
yield);mp >300 ꢁC;IR (KBr): 3401, 1637, 1578, 1438,
MS (MALDI-TOF): 735.6 (M+1)+. Anal. calcd for
C30H32BrN13O5: C, 49.05;H, 4.39;Br, 10.88;N, 24.79.
Found: C, 48.88;H, 4.23;Br, 10.71;N, 24.58.
N-Methyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [4(ꢀ-
bromoacrylamido) imidazole-2-carboxamido] pyrrole-2-
carboxamido]pyrrole-2-carboxamido] pyrrole-2-carbox-
amido]propionamidine hydrochloride (16). Following the
general procedure, starting from 30 (270 mg, 0.5 mmol),
Hunig’s base (86 mL, 0.5 mmol), EDC (193 mg, 1 mmol)
and the acid 34 (160 mg, 0.6 mmol), after work-up, the
residue was purified by flash chromatography using
methanol–methylene chloride 3:7 (v:v) as eluent. The
resulting oil was precipitated and yielding the com-
pound 16 as a yellow solid (251 mg, 66% yield);
mp>300 ꢁC;IR (KBr): 3412, 1637, 1542, 1431, 1405,
1259, 1209 and 1107 cmꢂ1; 1H NMR (DMSO-d6) d 2.61
(m, 2H);2.80 (d, J=4.6 Hz, 3H), 3.48 (m, 2H), 3.82 (s,
3H), 3.86 (s, 3H), 3.87 (s, 3H), 3.99 (s, 3H), 6.32 (d, J=3
Hz, 1H), 6.82 (d, J=2.8 Hz, 1H), 6.94 (s, 1H), 7.08 (s,
1H), 7.19 (s, 2H), 7.25 (s, 1H), 7.29 (s, 1H), 7.55 (s, 1H),
8.24 (m, 1H), 8.59 (s, 1H), 9.14 (s, 1H), 9.56 (bs, 1H),
9.95 (s, 1H), 10.01 (s, 1H), 10.15 (s, 1H), 10.59 (s,
1H);FAB-MS (MALDI–TOF): 725.6 (M+1)+. Anal.
calcd for C30H36BrClN12O5: C, 47.41;H, 4.77;Br,
10.51;Cl, 4.66;N, 22.11. Found: C, 47.22;H, 4.56;Br,
10.26;Cl, 4.51;N, 21.98.
1
1405 and 1261 cmꢂ1; H NMR (DMSO-d6) d: 2.59 (m,
2H), 2.83 (d, J=4.8 Hz, 3H);3.52 (m, 2H), 3.82 (s, 3H),
3.86 (s, 3H), 3.89 (s, 3H), 4.07 (s, 3H), 6.34 (d, J=3.4
Hz, 1H), 6.82 (d, J=3.4 Hz, 1H), 6.94 (d, J=1.6 Hz,
1H), 7.07 (d, J=1.6 Hz, 1H), 7.11 (d, J=1.6 Hz, 1H),
7.20 (d, J=1.6 Hz, 1H), 7.26 (d, J=1.6 Hz, 1H), 7.34
(d, J=1.6 Hz, 1H), 7.38 (s, 1H), 8.26 (m, 1H), 8.64 (s,
1H), 9.16 (s, 1H), 9.58 (bs, 1H), 9.96 (s, 1H), 10.04 (s,
1H), 10.53 (s, 1H), 11.07 (s, 1H);FAB-MS (MALDI–
TOF): 725.6 (M+1)+. Anal. calcd for C30H36BrClN12O5:
C, 47.41;H, 4.77;Br, 10.51;Cl, 4.66;N, 22.11. Found: C,
47.25;H, 4.61;Br, 10.23;Cl, 4.46;N, 22.02.
N, N0-Dimethyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4
[3-(ꢀ-bromoacrylamido) pyrazole-5-carboxamido] pyr-
role-2-carboxamido]pyrrole-2-carboxamido]
pyrrole-2-
carboxamido]propionamidine hydrochloride (14). Fol-
lowing the general procedure, starting from 31 (166 mg,
0.3 mmol), Hunig’s base (51 mL, 0.3 mmol), EDC (120
mg, 0.6 mmol) and the acid 33 (85 mg, 0.32 mmol), after
work-up, the residue was purified by flash chromato-
graphy using methanol–methylene chloride 2:8 (v:v) as
eluent. The resulting oil was precipitated and yielding
the compound 14 as a white solid (165 mg, 71% yield);
mp>300 ꢁC;IR (KBr): 3392, 1647, 1560, 1432, 1403,
N, N0-Dimethyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4
[4-(ꢀ-bromoacrylamido) imidazole-2-carboxamido] pyr-
role-2-carboxamido]pyrrole-2-carboxamido]
pyrrole-2-
carboxamido]propionamidine hydrochloride (17). Fol-
lowing the general procedure, starting from 31 (166 mg,
0.3 mmol), Hunig’s base (51 mL, 0.3 mmol), EDC (120
mg, 0.6 mmol) and the acid 34 (85 mg, 0.32 mmol), after
work-up, the residue was purified by flash chromato-
graphy using methanol–methylene chloride 3:7 (v:v) as
eluent. The resulting oil was precipitated and yielding
the compound 17 as a white solid (174 mg, 75% yield);
mp>300 ꢁC;IR (KBr): 3400, 1647, 1550, 1432, 1426,
1
1257, 1109 and 888 cmꢂ1; H NMR (DMSO-d6) d: 2.72
(m, 2H), 2.77 (d, J=4.6 Hz, 3H), 3.00 (d, J=4.4 Hz,
3H), 3.34 (m, 2H), 3.84 (s, 3H), 3.86 (s, 3H), 3.98 (s,
3H), 4.05 (s, 3H), 6.33 (d, J=3.2 Hz, 1H), 6.80 (d,
J=3.2 Hz,1H), 6.93 (s, 1H), 7.06 (s, 1H), 7.09 (s, 1H),
7.19 (s, 1H), 7.24 (s, 1H), 7.31 (s, 1H), 7.36 (s, 1H), 8.31
(m, 1H), 8.70 (q, J=4.8 Hz, 1H), 9.44 (q, J=4.8 Hz,
1H), 9.96 (s, 1H), 10.02 (s, 1H), 10.50 (s, 1H), 11.05 (s,
1H);FAB-MS (MALDI–TOF): 739.6 (M+1) +. Anal.
calcd for C31H38BrClN12O5: C, 48.10;H, 4.95;Br,
10.32;Cl, 4.58;N, 21.71. Found: C, 47.89;H, 4.73;Br,
10.13;Cl, 4.37;N, 21.37.
1
1260 and 775 cmꢂ1; H NMR (DMSO-d6) d: 2.73 (m,
2H), 2.78 (d, J=4.8 Hz, 3H), 3.01 (d, J=4.6 Hz, 3H),
3.46 (m, 2H), 3.80 (s, 3H), 3.84 (s, 3H), 3.92 (s, 3H), 3.98
(s, 3H), 6.31 (d, J=3 Hz, 1H), 6.81 (d, J=3 Hz, 1H),
6.93 (s, 1H), 7.08 (s, 1H), 7.18 (s, 1H), 7.20 (s, 1H), 7.25
(s, 1H), 7.28 (s, 1H), 7.54 (s, 1H), 8.33 (m, 1H), 8.73 (q,
J=4.8 Hz, 1H), 9.50 (q, J=4.8 Hz, 1H), 9.95 (s, 1H),
10.00 (s, 1H), 10.15 (s, 1H), 10.59 (s, 1H);FAB-MS
(MALDI–TOF): 739.6 (M+1)+. Anal. calcd for
C31H38BrClN12O5: C, 48.10;H, 4.95;Br, 10.32;Cl, 4.58;
N, 21.52. Found: C, 47.89;H, 4.78;Br, 10.12;Cl, 4.46;
N, 21.55.
N-Cyano-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [3-(ꢀ-
bromoacrylamido) pyrazole-5-carboxamido] pyrrole-2-
carboxamido]pyrrole-2-carboxamido] pyrrole-2-carbox-
amido]propionamidine (15). Following the general pro-
cedure, starting from 32 (206 mg, 0.4 mmol), Hunig’s
base (68 mL, 0.4 mmol), EDC (192 mg, 1 mmol) and the
acid 33 (110 mg, 0.42 mmol), after work-up, the residue
was purified by flash chromatography using methanol–
methylene chloride 2:8 (v:v) as eluent. The resulting oil
was precipitated and yielding the compound 15 as a
white solid (165 mg, 71% yield);mp >300 ꢁC;IR (KBr):
N-Cyano-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [4-(ꢀ-
bromoacrylamido) imidazole-2-carboxamido] pyrrole-2-
carboxamido]pyrrole-2-carboxamido] pyrrole-2-carbox-
amido]propionamidine (18). Following the general pro-
cedure, starting from 32 (206 mg, 0.4 mmol), Hunig’s
base (68 mL, 0.4 mmol), EDC (192 mg, 1 mmol) and the
acid 34 (110 mg, 0.42 mmol), after work-up, the residue
was purified by flash chromatography using methanol–
methylene chloride 1:9 (v:v) as eluent. The resulting oil
was precipitated and yielding the compound 18 as a
2256, 1637, 1576, 1467, 1405, 1260, 1113 and 889 cmꢂ1
;
1H NMR(DMSO-d6) d: 2.48 (m, 2H), 3.34 (m, 2H), 3.80
(s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 4.05 (s, 3H), 6.30 (d,
J=3.4 Hz, 1H), 6.79 (d, J=3.4 Hz, 1H), 6.88 (s, 1H),
7.04 (s, 1H), 7.09 (s, 1H), 7.20 (s, 1H), 7.25 (s, 1H), 7.31
(s, 1H), 7.36 (s, 1H), 8.34 (bs, 2H), 9.96 (s, 1H), 9.98 (s,
1H), 10.02 (s, 1H), 10.51 (s, 1H), 11.03 (s, 1H);FAB-

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
973
white solid (170 mg, 58% yield);mp >300 ꢁC;IR (KBr):
3412, 2252, 1674, 1643, 1558, 1496, 1433, 1223 and 798
C35H39BrClN11O5: C, 51.96;H, 4.86;Br, 9.88;Cl, 4.38;
N, 19.04. Found: C, 50.72;H, 4.47;Br, 10.16;Cl, 4.47;
N, 19.66.
1
cmꢂ1; H NMR (DMSO-d6) d: 2.64 (m, 2H), 3.35 (m,
2H), 3.81 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 3.99 (s, 3H),
6.31 (d, J=3 Hz, 1H), 6.81 (d, J=3.2 Hz, 1H), 6.88 (s,
1H), 7.06 (s, 1H), 7.16 (s, 1H), 7.21 (s, 1H), 7.26 (s, 1H),
7.28 (s, 1H), 7.55 (s, 1H), 8.22 (bs, 3H), 9.94 (s, 1H),
9.99 (s, 1H), 10.19 (s, 1H), 10.58 (s, 1H);FAB-MS
(MALDI–TOF): 735.6 (M+1)+. Anal. calcd for
C30H32BrN13O5: C, 49.05;H, 4.39;Br, 10.88;N,
24.79. Found: C, 48.88;H, 4.24;Br, 10.67;N, 24.58.
N-Methyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [1-
methyl-5(ꢀ-bromoacrylamido)
indole-2-carboxamido]
pyrrole-2-carboxamido]pyrrole-2-carboxamido] pyrrole-
2-carboxamido]propionamidine hydrochloride(21). Fol-
lowing the general procedure, starting from 30 (275 mg,
0.5 mmol), Hunig’s base (86 mL, 0.5 mmol), EDC (192
mg, 1 mmol) and the acid 36 (194 mg, 0.6 mmol), after
work-up, the residue was purified by flash chromato-
graphy using methanol–methylene chloride 2:8 (v:v) as
eluent. The resulting oil was precipitated and yielding
the compound 21 as a red solid (271 mg, 69% yield);
mp>300 ꢁC;IR (KBr): 3400, 1638, 1559, 1433, 1404,
N-Methyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [5(ꢀ-
bromoacrylamido) indole-2-carboxamido] pyrrole-2-car-
boxamido]pyrrole-2-carboxamido] pyrrole-2-carboxami-
do]propionamidine hydrochloride(19). Following the
general procedure, starting from 30 (270 mg, 0.5 mmol),
Hunig’s base (86 mL, 0.5 mmol), EDC (192 mg, 1 mmol)
and the acid 35 (183 mg, 0.6 mmol), after work-up, the
residue was purified by flash chromatography using
methanol–methylene chloride 2:8 (v:v) as eluent. The
resulting oil was precipitated and yielding the com-
pound 19 as a white solid (320 mg, 83% yield);
mp>300 ꢁC;IR (KBr): 3394, 1646, 1559, 1442, 1405,
1261, 1108 and 1062 cmꢂ1; 1H NMR (DMSO–d6) d 2.60
(m, 2H), 2.79 (d, J=4.8 Hz, 3H), 3.51 (m, 2H), 3.81 (s,
3H), 3.86 (s, 3H), 3.89 (s, 3H), 6.28 (d, J=3.0 Hz, 1H),
6.76 (d, J=3.0 Hz, 1H), 6.95 (s, 1H), 7.09 (s, 1H), 7.21
(s, 1H), 7.27 (s, 1H), 7.30 (s, 1H), 7.36 (s, 1H), 7.40 (s,
1H), 7.40 (m, 2H), 8.01 (s, 1H), 8.26 (t, J=5.7 Hz, 1H),
8.60 (s, 1H), 9.14 (s, 1H), 9.53 (m, 1H), 9.96 (s, 1H),
10.06 (s, 1H), 10.21 (s, 1H), 10.44 (s, 1H), 11.73 (s, 1H);
FAB-MS (MALDI–TOF): 774.7 (M+1)+. Anal. calcd
for C34H37BrClN11O5: C, 51.36;H, 4.69;Br, 10.05;Cl,
4.46;N, 19.38. Found: C, 51.13;H, 4.48;Br, 9.79;Cl,
4.33;N, 19.15.
1
1257 and 1110 cmꢂ1; H NMR (DMSO-d6) d 2.61 (m,
2H), 2.79 (d, J=4.6 Hz, 3H), 3.49 (m, 2H), 3.81 (s, 3H),
3.85 (s, 3H), 3.88 (s, 3H), 4.02 (s, 3H), 6.29 (d, J=2.6
Hz, 1H), 6.78 (d, J=2.6 Hz, 1H), 6.93 (s, 1H), 7.03 (d,
1H), 7.13 (d, 1H), 7.21 (m, 2H), 7.26 (s, 1H), 7.34 (s,
1H), 7.51 (d, J=6.6 Hz, 2H), 8.05 (s, 1H), 8.25 (bs, 1H),
8.60 (s, 1H), 9.16 (s, 1H), 9.56 (bs, 1H), 9.96 (s, 1H),
10.02 (s, 1H), 10.27 (s, 1H), 10.47 (s, 1H);FAB-MS
(MALDI–TOF): 774.7 (M+1)+. Anal. calcd for
C35H39BrClN11O5: C, 51.96;H, 4.89;Br, 9.88;Cl, 4.38;
N, 19.04. Found: C, 51.77;H, 4.67;Br, 9.76;Cl, 4.22;N,
18.89.
N,N 0-Dimethyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [1-
methyl - 5(ꢀ - bromoacrylamido)indole - 2 - carboxamido]-
pyrrole-2-carboxamido]pyrrole-2-carboxamido] pyrrole-
2-carboxamido] propionamidine hydrochloride (22). Fol-
lowing the general procedure, starting from 31 (166 mg,
0.3 mmol), Hunig’s base (51 mL, 0.3 mmol), EDC (112
mg, 0.6 mmol) and the acid 36 (97 mg, 0.6 mmol), after
work-up, the residue was purified by flash chromato-
graphy using methanol–methylene chloride 3:7 (v:v) as
eluent. The resulting oil was precipitated and yielding
the compound 22 as a white solid (213 mg, 86.6% yield);
mp>300 ꢁC;IR (KBr): 3398, 1643, 1560, 1412, 1400,
N,N0-Dimethyl-3-[1-methyl-4[1-methyl-4[1H-5(ꢀ-bromo-
acrylamido)indole-2-carboxamido]pyrrole-2-carboxamido]-
pyrrole-2-carboxamido]propionamidine hydrochloride (20).
To a solution of amino-amidine 31(272 mg, 0.5 mmol)
in anhydrous DMF (5 mL) cooled to 0 ꢁC, Hunig’s base
(86 mL, 0.5 mmol) was added. After 5 min the acid 35
(186 mg, 0.6 mmol) and then EDC (192 mg,1 mmol)
were added. The reaction mixture was slowly warmed to
room temperature and allowed to stir for 18 h. After
this time, the solution was acidified with 20% HCl at
pH 4 and DMF was removed under reduced pressure
The resulting solid purified by flash column chromato-
graphy with methanol–methylene chloride 2:8 (v:v)
afforded a yellow oil which was precipitated from
methanol-ethyl ether to give 20 as a yellow solid (288
mg, 71% yield);mp >300 ꢁC;IR (KBr):3400, 1641,
1
1277 and 1089 cmꢂ1; H NMR (DMSO-d6) d 2.73 (m,
2H), 2.78 (d, J=3.2 Hz, 3H), 3.01 (d, J=3.4 Hz, 3H),
3.43 (m, 2H), 3.81 (s, 3H), 3.85 (s, 3H), 3.89 (s, 3H), 4.02
(s, 3H), 6.29 (d, J=3.0 Hz, 1H), 6.76 (d, J=3.0 Hz,
1H), 6.93 (d, J=1.6 Hz, 1H), 7.08 (d, J=1.6 Hz, 1H),
7.12 (d, J=1.4 Hz, 1H), 7.20 (d, J=1.6 Hz, 1H), 7.26 (s,
1H), 7.34 (d, J=1.4 Hz, 1H), 7.46 (s, 1H), 7.57 (d,
J=4.8 Hz, 2H), 8.06 (s, 1H), 8.31 (t, J=6 Hz, 1H), 8.71
(q, J=4.8 Hz, 1H), 9.46 (q, J=5 Hz, 1H), 9.96 (s, 1H),
10.02 (s, 1H), 10.24 (s, 1H), 10.46 (s, 1H);FAB-MS
(MALDI–TOF): 788.7 (M+1)+. Anal. calcd for
C36H41BrClN11O5: C, 52.23;H, 5.02;Br, 9.71;Cl, 4.31;
N, 18.72. Found: C, 52.36;H, 4.89;Br, 9.55;Cl, 4.12;N,
18.56.
1
1578, 1435, 1252 and 1106 cmꢂ1; H NMR (DMSO-d6)
d 2.72 (t, J=4.8 Hz, 2H), 2.78 (d, J=4.6 Hz, 3H), 3.01
(d, J=4 Hz, 3H), 3.49 (m, 2H), 3.81 (s, 3H), 3.86 (s,
3H), 3.89 (s, 3H), 6.28 (d, J=2.6 Hz, 1H), 6.78 (s, 1H),
6.94 (s, 1H), 7.09 (s, 1H), 7.12 (s, 1H), 7.22 (s, 1H), 7.28
(s, 1H), 7.32 (s, 1H), 7.37 (s, 1H), 7.40 (s, 2H), 8.00 (s,
1H), 8.37 (t, J=4.6 Hz, 1H), 8.84 (q, J=4.8 Hz, 1H),
9.61 (q, J=4.8 Hz, 1H), 9.98 (s, 1H), 10.06 (s, 1H),
10.23 (s, 1H), 10.50 (s, 1H), 11.78 (s, 1H);FAB-MS
(MALDI–TOF): 774.6 (M+1)+. Anal. calcd for
N-Methyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [5(ꢀ-
bromoacrylamido)benzofurane-2-carboxamido]pyrrole-2-
carboxamido] pyrrole-2-carboxamido]pyrrole-2-carboxa-
mido]propionamidine hydrochloride (23). Following the
general procedure starting from 30 (275 mg, 0.5 mmol),
Hunig’s base (86 mL, 0.5 mmol), EDC (192 mg, 1 mmol)
and the acid 37 (190 mg, 0.6 mmol), after work-up, the

974
P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
residue was purified by flash chromatography using
methanol–methylene chloride 2:8 (v:v) as eluent to give
23 (308 mg, 78% yield);mp >300 ꢁC;IR (KBr):3328,
50.28;H, 4.47;Br, 9.84;Cl, 4.37;N, 17.25. Found: C,
50.22;H, 4.25;Br, 9.67;Cl, 4.15;N, 17.06.
1637, 1578, 1433, 1262, 1208, 1109 and 802 cmꢂ1; H
N, N 0-Dimethyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4
[5(ꢀ-bromoacrylamido) benzothiopene-2-carboxamido]
pyrrole-2-carboxamido]pyrrole-2-carboxamido] pyrrole-
2-carboxamido]propionamidine hydrochloride (26). Fol-
lowing the general procedure (K), starting from 31 (111
mg, 0.2 mmol), Hunig’s base (35 mL, 0.5 mmol), EDC
(77 mg, 0.4 mmol) and the acid 38 (73 mg, 0.22 mmol),
after work-up, the residue was purified by flash chro-
matography using methanol–methylene chloride 2:8
(v:v) as eluent. The resulting oil was precipitated and
yielding the compound 26 as a red solid (127 mg, 77%
yield);mp >300 ꢁC;IR (KBr): 3255, 1637, 1578, 1523,
1432, 1260 and 1206 cmꢂ1; 1H NMR (DMSO-d6) d 2.72
(m, 2H), 2.78 (d, J=4.8 Hz, 3H), 3.01 (d, J=4.4 Hz,
3H), 3.38 (m, 2H), 3.81 (s, 3H), 3.86 (s, 3H), 3.89 (s,
3H), 6.35 (d, J=3.2 Hz, 1H), 6.81 (d, J=3 Hz, 1H),
6.94 (s, 1H), 7.08 (s, 1H), 7.13 (s, 1H), 7.20 (s, 1H), 7.26
(s, 1H), 7.33 (s, 1H), 7.65 (d, J=8.8 Hz, 1H), 8.00 (d,
J=8.8 Hz, 1H), 8.24 (s, 1H), 8.27 (bs, J=6 Hz, 1H),
1H), 8.36 (s, 1H), 8.66 (q, J=4.6 Hz, 1H), 9.41 (q,
J=4.6 Hz, 1H), 9.95 (s, 1H), 10.03 (s, 1H), 10.48 (s,
1H), 10.68 (s, 1H);FAB-MS (MALDI–TOF): 791.7
(M+1)+. Anal. calcd for C35H38BrClN10O5S: C, 50.88;
H, 4.64;Br, 9.67;Cl, 4.29;N, 16.95. Found: C, 50.68;H,
4.52;Br, 9.34;Cl, 4.08;N, 16.77.
1
NMR (DMSO-d6 ) d2.57 (t, J=6.6 Hz, 2H), 2.81 (d,
J=4.6 Hz, 3H), 3.49 (m, 2H), 3.79 (s, 3H), 3.85 (s, 3H),
3.88 (s, 3H), 6.33 (d, J=3 Hz, 1H), 6.80 (d, J=3 Hz,
1H), 6.93 (d, J=1.8 Hz, 1H), 7.08 (d, J=1.8 Hz, 1H),
7.16 (d, J=1.8 Hz, 1H), 7.19 (d, J=1.8 Hz, 1H), 7.26
(d, J=1.8 Hz, 1H), 7.34 (d, J=1.8 Hz, 1H), 7.70 (m,
3H), 8.17 (s, 1H), 8.16 (bs, 1H), 8.58 (s., 1H), 9.14 (s.,
1H), 9.54 (q, J=4.8 Hz, 1H), 9.96 (s, 1H), 10.06 (s,
1H), 10.47 (s, 1H), 10.77 (s, 1H);FAB-MS (MALDI–
TOF):
761.7
(M+1)+.
Anal.
calcd
for
C34H36BrClN10O6: C, 51.30;H, 4.56;Br, 10.04;Cl,
4.45;N, 17.59. Found: C, 51.05;H, 4.34;Br, 9.89;Cl,
4.33;N, 17.35.
N,N0-Dimethyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4
[5(ꢀ-bromoacrylamido)benzofurane-2-carboxamido]pyr-
role-2-carboxamido]
pyrrole-2-carboxamido]pyrrole-2-
carboxamido]propionamidine hydrochloride (24). Fol-
lowing the general procedure, starting from 31 (166 mg,
0.3 mmol), Hunig’s base (51 mL, 0.3 mmol), EDC (115 mg,
0.6 mmol) and the acid 37 (93 mg, 0.3 mmol), after work-
up, the residue was purified by flash chromatography
using methanol–methylene chloride 3:7 (v:v) as eluent,
yielding the compound 24 as a white solid (190 mg, 78%
yield);mp >300 ꢁC;IR (KBr): 3328, 1637, 1578, 1405,
1
1262, 1209 and 867 cmꢂ1; H NMR (DMSO-d6) d2.72
Biological assays
(m, 2H), 2.78 (d, J=4.6 Hz, 3H), 3.01 (d, J=4.4 Hz,
3H), 3.37 (m, 2H), 3.81 (s, 3H), 3.85 (s, 3H), 3.89 (s,
3H), 6.33 (d, J=3 Hz, 1H), 6.79 (d, J=3 Hz, 1H), 6.94
(s, 1H), 7.08 (s, 1H), 7.16 (s, 1H), 7.20 (s, 1H), 7.26 (s,
1H), 7.34 (s, 1H), 7.68 (m, 3H), 8.17 (s, 1H), 8.32 (bs.,
1H), 8.70 (q, J=4.8 Hz, 1H), 9.46 (q, J=4.8 Hz, 1H),
9.96 (s, 1H), 10.05 (s, 1H), 10.45 (s, 1H), 10.75 (s, 1H);
FAB-MS (MALDI–TOF): 775.7 (M+1)+. Anal. calcd
for C35H38BrClN10O6: C, 51.89;H, 4.73;Br, 9.86;Cl,
4.38;N, 17.29. Found: C, 51.67;H, 4.55;Br, 9.72;Cl,
4.13;N, 17.11.
Growth inhibitory activity on murine L1210 and human
K562 and Raji tumor cell lines. The murine lymphocytic
L1210 leukemia,12 the human chromic myelogenous
K562,16 and the human B-lymphoid Raji17 cell lines were
obtained from the American Type Culture Collection
(ATCC). All the tested compounds were dissolved in
DMSO at 1 mg/mL immediately before the use and diluted
in medium before addition to the cells. The murine lym-
phocytic leukemia cells L1210 and L1210/DX were grown
in vitro as a stationary suspension culture in RPMI 1640
medium (GIBCO) supplemented with 10% FCS (Flow,
Irvine, UK), 2 mM l-glutamine (GIBCO), 10 mM b-
mercaptoethanol, 100 U/mL penicillin and 100 mg/mL
streptomycin. To determine survival after drug expo-
sure, exponentially growing L1210 cells were continously
exposed to various concentrations of drugs for 48 h and
surviving cells were counted in a model ZBI Coulter
Counter (Coulter Electronics, Hialeah, FL). Results were
expressed as IC50 (dose causing 50% inhibition of cell
growth in treated cultures relative to untreated controls).
Human K562 and Raji cell lines were treated with the tes-
ted compounds for 4 and 5 days respectively and then the
cell number/mL determined as elsewhere reported.18ꢂ20 All
experiments were repeated at least twice. For each drug
concentration, duplicate cultures were used. Vehicle or
solvent controls were run with each experiment.
N-Methyl-3-[1-methyl-4 [1-methyl-4 [1-methyl-4 [5(ꢀ-
bromoacrylamido) benzothiopene-2-carboxamido] pyr-
role-2-carboxamido]pyrrole-2-carboxamido]
pyrrole-2-
carboxamido]propionamidine hydrochloride (25). Fol-
lowing the general procedure, starting from 30 (270 mg,
0.5 mmol), Hunig’s base (86 mL, 0.5 mmol), EDC (192
mg, 1 mmol) and the acid 38 (200 mg, 0.6 mmol), after
work-up, the residue was purified by flash chromato-
graphy using methanol–methylene chloride 2:8 (v:v) as
eluent. The resulting oil was precipitated and yielding
the compound 25 as a white solid (313 mg, 77% yield);
mp>300 ꢁC;IR (KBr): 3296, 1634, 1582, 1434, 1262,
1
1207 and 805 cmꢂ1; H NMR (DMSO-d6) d 2.59 (m,
2H), 2.78 (d, J=3 Hz, 3H), 3.50 (m, 2H), 3.81 (s, 3H),
3.85 (s, 3H), 3.89 (s, 3H), 6.36 (s, 1H), 6.83 (s, 1H), 6.94
(s, 2H), 7.08 (s, 1H), 7.13 (s, 1H), 7.20 (s, 1H), 7.27 (s,
1H), 7.34 (s, 1H), 7.67 (d, J=8.8 Hz, 1H), 8.01 (d,
J=8.6 Hz, 1H), 8.27 (s, 1H), 8.37 (s, 1H), 8.59 (bs, 1H),
9.15 (s, 1H), 9.55 (bs, 1H), 9.97 (s, 1H), 10.06 (s, 1H),
10.53 (s, 1H), 10.74 (s, 1H);FAB-MS (MALDI–TOF):
777.7 (M+1)+. Anal. calcd for C34H36BrClN10O5S: C,
PCR primers and arrested PCR and arrested PCR. The
sequences of g-globin gene primers used for polymerase
chain reaction (PCR) were the following: g-globin-for-
ward, 50-GGA ATG ACT GAA TCG GAA CAA
GGC-30, g-globin-reverse, 50a-CTG CTG AAG GGT

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 965–975
975
GCT TCC TTT TAT-30. Taq DNA polymerase was
purchased from FINNZYMES OY (Espoo, Finland)
and added at 2.5 U/25 mL final concentration. For
PCR-mediated amplification the target DNA was 20 ng
of pre-amplified DNA;PCR buffer, Taq DNA poly-
merase and the four dNTPs were added as elsewhere
described. Conditions of PCRs were: denaturatiꢁon,
1 min (30 cycles). Arrested PCR21,22 was performed
after allowing different concentrations of the tested
DNA-binding drugs to interact with the pre-amplified
target DNA. After PCR, the products were analysed by
agarose gel electrophoresis and ethidium bromide
staining as elsewhere reported. The inhibitory activity
was expressed as IC50, causing a 50% inhibition of the
generation of PCR products.
2. Pelton, J. G.;Wemmer, D. E. J. Am. Chem. Soc. 1990, 112,
1393.
3. Marchini, S.;Broggini, M.;Sessa, C.;D’Incalci, M.
Opin. Invest. Drugs 2001, 10, 1703.
Exp.
4. D’Alessio, R.;Geroni, C.;Biasoli, G.;Pesenti, E.;Grandi,
M.;Mongelli, N. Bioorg. Med. Chem. Lett. 1994, 4, 1467.
5. Marchini, S.;Ciro, M.;Gallinari, F.;Geroni, C.;Cozzi, P.;
D’Incalci, M.;Broggini, M. Br. J. Cancer 1999, 80, 991.
6. Marchini, S.;Ciro, M.;Broggini, M. Apoptosis 1999, 4, 39.
7. Baraldi, P. G.;Beria, I.;Cacciari, B.;Capolongo, L.;Cozzi,
ꢁ
92 ꢁC, 1 min;annealing, 55 C, 1 min;elongation, 72 C,
P.;Mongelli, N.;Romagnoli, R.;Spalluto, G.
Chem. Lett. 1996, 6, 1241.
Biorg. Med.
8. Baraldi, P. G.;Romagnoli, R.;Beria, I.;Cozzi, P.;Geroni,
C.;Geroni, C.;Mongelli, N.;Bianchi, N.;Mischiati, C.;
Gambari, R. J. Med. Chem. 2000, 43, 2675.
9. Cozzi, P.;Beria, I.;Caldarelli, M.;Geroni, C.;Mongelli,
N.;Pannella, G. Bioorg. Med. Chem. Lett. 2000, 10, 1273.
10. The pKa values have been calculated using the ACD labs
software verson 5.12 (web site: http://www.acdlabs.com).
11. Suffness, M.;Douros, J. In: Methods in Cancer Research,
Vol. XVI, Part A;De Vita, V.T., Jr. Brusch, H., Eds;Aca-
demic Press;New York, 1979;p 84.
12. Cory, J. G.;Somerville, L.;He, A. W.;Cory, A. H. Adv.
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Footprinting probes and DNase I footprinting. Foot-
printing probes were generated by PCR using the pri-
mers described in the previous section. For PCR-
mediated amplification the target DNA was 20 ng of
genomic DNA. After a first PCR round (20 cycles),
samples were heated at 90 ꢁC for 5 min and a second
PCR round was performed (20 cycles) using a 32P-
labelled g-globin-forward PCR primer. After PCR, each
reaction, containing 32P-labelled PCR products was
resuspended in water, aliquots were incubated with
increasing amounts of the tested compounds (0, 1, 5, 10
mM) and 1 U/reaction of DNAse I was added. After 2
min, 5 mL of loading dye (0.1% xilene-cyanol, 0.1%
bromophenol blue, 0.1 M NaOH–formamide 1:2) was
added and the reactions electrophoresed through a
sequencing gel as described.
17. Kurita-Ochiai, T.;Ochiai, K.;Fukushima, K.
Immun. 1998, 66, 2587.
Infect.
18. Marminon, C.;Anizon, F.;Moreau, P.;Leonce, S.;Pierre,
J. Med. Chem.
A.;Pfeiffer, B.;Renard, P.;Prudhomme, M.
2002, 45, 1330.
19. Bianchi, N.;Osti, F.;Rutigliano, C.;Corradini, F. G.;
Borsetti, E.;Tomassetti, M.;Mischiati, C.;Feriotto, G.;
Gambari, R. Br. J. Haematol. 1999, 104, 258.
20. Baraldi, P. G.;Cacciari, B.;Guiotto, A.;Leoni, A.;
Romagnoli, R.;Spalluto, G.;Mongelli, N.;Howard, P. W.;
Acknowledgements
Thurston, D. E.;Bianchi, N.;Gambari, R.
Chem. Lett. 1998, 8, 3019.
Bioorg. Med.
We wish to thank Pharmacia & Upjohn and Ministero
Universita e Ricerca Scientifica (MUIR) (60%) for
financial support of this work. R.G. is granted by CNR
PF Biotechnology, CNR ‘Agenzia-200000 and Ministero
della Sanita, Ricerca Finalizzata 2001.
21. Passadore, M.;Bianchi, N.;Feriotto, G.;Mischiati, C.;
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References and Notes
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elli, A. Nature 1964, 203, 1064.
24. Zambias, R. A.;Hammond, M. L. Synth. Comm. 1991,
21, 959.