Synthesis and antitumour activity of new dendritic polyamines-(imide-DNA-intercalator) conjugates: Potent Lck inhibitors

Article abstract of DOI:10.1016/S0223-5234(02)01362-4

A series of dendritic polyamines-(imide-DNA-intercalators) conjugates with different connectivity in their basic chain were synthesised and evaluated as antitumour compounds. Although their antiproliferative activity against HT-29 was not significant, con

Full text of DOI:10.1016/S0223-5234(02)01362-4

Eur. J. Med. Chem. 37 (2002) 541–551
www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumour activity of new dendritic
polyamines–(imide–DNA-intercalator) conjugates: potent Lck
inhibitors
Miguel F. Bran˜a a,*, Gema Dom´ınguez a, Beatriz Sa´ez a, Cynthia Romerdahl b,
Simmon Robinson b, Teresa Barlozzari b
a Departamento de Qu´ımica Orga´nica y Farmace´utica, Facultad de Ciencias Experimentales y Te´cnicas, Uni6ersidad San Pablo-CEU,
Boadilla del Monte 28668 Madrid, Spain
b BASF Bioresearch Corporation, 100 Research Dri6e, 01605 Worcester, MA, USA
Received 7 September 2001; accepted 28 February 2002
Abstract
A series of dendritic polyamines–(imide–DNA-intercalators) conjugates with different connectivity in their basic chain were
synthesised and evaluated as antitumour compounds. Although their antiproliferative activity against HT-29 was not significant,
´
conjugates 13 and 16 showed a promising profile as inhibitors of Lck. © 2002 Editions scientifiques et me´dicales Elsevier SAS.
All rights reserved.
Keywords: Antitumour; Dendritic polyamines; Intercalators; Imides; Lck
1. Introduction
and Elinafide, have been selected for phase II clinical
trials [5,6].
Two potential targets for drug design in cancer are
DNA and tyrosine-kinases. Therefore, the design of
new intercalators and the discovery of specific inhi-
bitors of protein tyrosine-kinases are two important
approaches in the search of new chemotherapeutic
agents.
DNA intercalating agents are among the most com-
mon anticancer drugs used in the clinical therapy of
human tumours. These agents are characterised by the
presence of flat chromophores bearing electron deficient
polyconjugated areas bounded to polar groups [1].
Their antitumour activities are derived from DNA dis-
tortion and altered nuclear protein interaction as a
consequence of reversible complex formation [2].
In our laboratory we have discovered the antitumour
potential of a series of mono and bisintercalating agents
bearing naphthalimide chromophores [3,4].
Nowadays, there is an increasing interest in tethering
polyamines to known cancer drugs and biological ac-
tive agents in order to improve their activity and spe-
cificity [7,8]. Some authors have reported the
combination of polyamines and DNA intercalators but
they have employed linear [9,10] or macrocyclic
polyamines [11], and their results suggest that the ap-
pended polyamine is a value-added fragment.
On the other hand, dendrimers are hyperbranched
polymers that emanate from a central core, have a
defined number of generations and functional end
groups, and are synthesised stepwise by a repetitive
reaction sequence. Branching is dependent on building
Two compounds from this class (Fig. 1), Amonafide
* Correspondence and reprints
E-mail address: mfbrana@ceu.es (M.F. Bran˜a).
Fig. 1. Mono and bisintercalating agents.
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(02)01362-4

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M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
way to increase the binding ability to DNA and, there-
fore the potency of the intercalator. Thus, the increase
of potential hydrogen bonds and favourable electro-
static interactions between the protonated amino
groups of the dendrimeric chains and the negatively
charged sugar phosphate backbone should largely in-
crease the affinity of these polymeric structures for
DNA.
In this regard, Cohen and coworkers found that the
conjugation of a drug to a polyammonium cation in-
creases the DNA binding affinity and that the
polyamine conjugate was more potent inhibiting tu-
mour growth than the unconjugated drug [14,15].
The design of our conjugates is predicated upon the
well-known affinity of polyamines for DNA and the
established binding modes of naphthalimides [16].
DNA intercalation models predict that a multitude of
anionic site charges are available for binding additional
basic functions beyond those present in the intercalator
framework [17]. Since polyamines exist as polycations
in vivo, the conjugates which have an appended polyca-
tionic tail bind to these available sites, thereby increa-
sing the binding affinity and potency of the intercalator.
Moreover, studies have indicated that polyammonium
cations (PACs) have a very high DNA affinity but are
loosely bound and can ‘read’ DNA rapidly. These
properties make PACs and related polycations ideal for
drug delivery when the drug needs to reach specific sites
in DNA [18]. Therefore, the combination of these
agents (dendritic polyamines and DNA intercalators) is
expected to provide new DNA targetering vectors.
This approach was initially supported by molecular
modeling studies, in which it has been determined that
these compounds are likely to bind a DNA octamer of
a model d[GCACGTCG]2 sequence through the central
5%-CpG-3% step, via either the major or the minor
groove [19].
Fig. 2. PPI dendritic–(imide–DNA-intercalator) conjugates.
Fig. 3. PAMAM dendritic–(imide–DNA-intercalator) conjugates.
block valence (this includes the core). Thus, a core
possessing one reactive moiety, such as a primary
amine, is divalent and will accommodate two
monomers, assuming a neutral trisubstituted amine
product. Branching therefore proceeds in a 1–2 man-
ner. PAMAM dendrimers and PPI dendrimers branch
this way and both have amino terminal groups. PPI
(polypropylenimine dendrimers) have N-branching cen-
ters and N-connectivity centers, whereas PAMAM
(polyamidoamine dendrimers) have N-branching cen-
ters as well as amide connectivity. Although dendrimers
have been employed in genic therapy [12,13] and they
have been described as able to form a complex with
DNA, their modification to join an intercalating chro-
mophore as antitumour compounds has never been
described. Thus, the synthesis and the pharmacological
profile of such compounds could be of great interest.
In this work we first tried to improve the therapeutic
properties of known imides by conjugating the DNA-
intercalator chromophore to a dendritic polyamine as a
Our first approach was to synthesise conjugates of
dendritic polyamines with chains of amine ramification
and amine connectivity polypropylenimine dendrimers
(PPI-dendrimers) [20] (Fig. 2). In a second approach,
we have modified the connectivity of the dendritic chain
with an amide group, which possesses the ability to
form additional hydrogen bonds to generate PAMAM
(polyamidoamine)
dendritic–(imide–DNA-intercala-
tor) conjugates (Fig. 3). In this case the distance be-
tween nitrogen atoms in the PAMAM chain was as in
Elinafide, optimum according to our experience.
On the other hand, other attractive targets for antitu-
moural agents are tyrosine-kinases and the signaling
pathways in which they participate.
Tyrosine-specific protein kinases are composed of
two subfamilies: (1) the receptor tyrosine-kinases, which
are integral membrane proteins; and (2) their nonrecep-
tor cytoplasmatic counterparts. The former, upon coor-
dination of specific extracellular ligands, forms

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
543
aggregates and subsequently suffers phosporylation of
key tyrosine residues. Cytoplasmatic signaling proteins,
including nonreceptor tyrosine-kinases, coordinate to
these phosphotyrosine (pTyr) residues through Src ho-
mology 2 (SH2) domains [21]. This binding event trig-
gers the activation of specific intracellular signaling
pathways, ultimately leading to a cellular response in
reaction to the extracellular stimulus. For example,
antigen presentation to T cells results in the clustering
of T-cell receptors (TcR) and subsequent TcR tyrosine
phosphorilation by Src family member LcK (lymphoid
T-cell tyrosine-kinase) [22]. These TcR pTyr moieties
serve as high-affinity binding sites for the SH2 domains
of cytoplasmatic signaling proteins, which generate
complexes that are required for ensuing downstream
events such as an increase in cytoplasmatic Ca2+ levels
and the ultimate production of interleukin-2. In short,
SH2 domains play a critical role in organizing coherent
signal transducing complexes that are essential for the
appropriate cellular response to extracellular stimuli.
Constituitively, active signal transduction pathways
have been identified in a variety of disease states.
Ligands that are able to disrupt these inappropriately
hyperstimulated pathways, by inhibition of Lck, by
blocking SH2 domain-dependent interactions, may ulti-
mately find utility as therapeutic agents. Lawrence and
coworker [23] have reported that diamines with a hy-
drophobic substituent and substituents that could form
a hydrogen bond with residues in the SH2 domain
might promote Lck SH2 affinity.
bis-cyanoethylations were accomplished in acetic glacial
acid, compound 7 was obtained in acid free methanol.
These conditions required a longer reaction time, but
compound 7 did not need further purification. Cleavage
of the tert-butoxycarbonyl group was carried out at
room temperature with trifluoroacetic acid in
dichloromethane–anisole (1:1), providing compound 8,
with the free amine group (Fig. 4).
Imide derivatives were prepared by nucleophilic addi-
tion of the dendritic amine 8 to the appropriate 1,8-
naphthalic
anhydride-3-substituted
to
provide
compounds 9–11. We also synthesised the imide deriva-
tive of 3,4-diphenyl maleic anhydride 12. It is notewor-
thy that this system exhibits relevant cytostatic activity
[26,27]. Finally, reduction of the end cyano groups of
compounds 9–12 under the described conditions, led us
to the target molecules 13–16 (Fig. 5).
The synthesis of dendritic polyamines–(DNA-inter-
calators) conjugates with PAMAM chains was carried
out employing a divergent procedure already described
by Tomalia [28], but using a core with an intercalating
chromophore. In this way, synthesis started from N-(2-
aminoethyl)-1,8-naphthalimide (17), which was synthe-
sised from 1,8-naphthalic anhydride and ethylene-
diamine. A two-step process was developed: first ex-
haustive Michael addition of methyl acrylate to the
amine core 17, followed by exhaustive amidation of the
resulting esters 18, 20 and 22 with excess of ethylenedia-
mine to give us the conjugates 19, 21 and 23, in good
overall yields (Fig. 6).
Another goal of this work was to know the pharma-
cological profile of these conjugates in other different
and important targets like Lck. In our laboratory we
are interested in testing some of these conjugates (which
have amine groups, hydrophobic substituent and amide
groups) for the inhibition of Lck, because this biologi-
cal system was available in the laboratory and this
enzyme is of great interest in the treatment of autoim-
mune diseases and T-cell based leukemias and
lymphomas.
3. Pharmacology
In vitro cytotoxicity of members of our synthetic
series was established using the HT-29 (human colon
carcinoma cell line) and a standard (MMT) assay [29].
The Lck inhibitory activity of some conjugates was
determinated by an enzyme-linked immunosorbent as-
say (ELISA) using the universal tyrosine-kinase sub-
strate Poly(Glu, Tyr) 4:1 at 5 mM ATP [30].
2. Chemistry
4. Results and discussion
To carry out the synthesis of conjugates with chains
of amine branching and amine connectivity, we first
built the dendritic chain by the divergent procedure
described by Vo¨gtle and coworkers [24] and Wo¨rner et
al. [25], starting from N-tert-butoxycarbonyl derivative,
2, which was synthesised from ethylenediamine (1) with
di-tert-butylcarbonate in dichloromethane. The addi-
tion of acrylonitrile to 2 in acetic glacial acid gave us
dicyano 3. The hydrogenation of 3, with Ni-Raney in a
solution of 1.4 M NaOH provided compound 4. This
sequence of reactions was carried out successively to
obtain compounds 5–7, in good yields. Although first
In vitro cytotoxity against human colon carcinoma
cell line HT-29 and in vitro inhibitory assay Lck was
carried out (Table 1). The values obtained for both
types of (polyamine dendritic–DNA intercalator) con-
jugates against HT-29 were not remarkable, probably
due to poor permeabilities. However, compounds 13, 16
and 21 showed significant inhibitory activity against
Lck and the best values were observed when the den-
dritic chain had amine ramification and amine connec-
tivity (PPI chain), and eight amine primary groups (13
and 16). We observed that compounds with similar or

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M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
no significant values of HT-29 exhibited the best results
for inhibition of Lck. It must be noted that a slight
increase in cytotoxic activity did not produce a better
inhibitory activity. We must also consider that HT-29
and Lck are different biological systems and there is no
correlation between the values obtained. HT-29 is an in
Fig. 4. (a) (Boc)2O, CH2Cl2, 0 °C r.t., 16 h, 50%. (b) Acrylonitrile, AcOH or MeOH; 3: 24 h, reflux, 96%; 5: 24 h, reflux, 90%; 7: 1 h, 10 °C,
4 days, 60 °C, 82%. (c) H2, Ni-Raney, 1.4 M NaOH in 95% EtOH; 4: 16 h, 80%; 6: 36 h, 70%. (d) TFA, CH2Cl2–anisol, 1:1, 20 min, 80%.
Fig. 5. (a) Aromatic anhydride, absolute EtOH; 9: r.t., 18 h, 92%; 10: reflux; 18 h, 83%; 11: reflux; 48 h, 60%; 12: reflux, 2 h, 80%. (b) H2,
Ni-Raney, 1.4 M NaOH in 95% EtOH; 13: 48 h, 52%; 14: 24 h, 60%; 15: 24 h; 49%, 16: 16 h, 77%.

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
545
Fig. 6. (a) Methylacrilate, MeOH, reflux; 18: 6 h, 81%; 20: 16 h, 62%; 22: 48 h, 67%. (b) Ethylenediamine excess, MeOH, r.t.; 19: 58%; 21: 60%;
23: 40%.

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M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
Table 1
to conjugate an intercalating chromophore as an antitu-
mour compound has never been described. Biological
assays showed no relevance for the activity of these
compounds against HT-29 cell lines and values range
around 44– \100 mM. However, results obtained for
the inhibition of Lck seem promising and an excellent
starting point for further development of this type of
approximation in the search for new antitumour com-
pounds. It appears that Lck expression is largely
confined to T cells, and its over-expression may con-
tribute to certain T-cell tumours [31,32]. Thus, selective
inhibition of the Lck enzyme may be of interest in the
treatment of several types of lymphomas, such as non-
Hodgkin’s [33] which express the Lck enzyme. These
results open up a new area of future research.
Biological activities against HT-29 and Lck
Compounds
HT-29 IC50 mM
Lck IC50 mM
8
9
10
11
12
13
14
15
16
19
21
23
\100
\100
\100
\100
57
3.8
\50
33.8
5.02
\50
0.39
44
a
a
–
–
–
a
a
–
100
\100
\100
0.10
6.1
1.78
a
a
–
–
–
–
a
Amonafide
Elinafide
38
0.014
a
a Not determined.
6. Experimental
vitro assay, whereas the determination of inhibition of
Lck is an ex vivo assay.
6.1. Chemistry
The values obtained for conjugates with PPI chains
with different chromophores (13 vs. 16) were insignifi-
cant against HT-29 and it should be noted that a slight
improvement was shown with naphthalimide chro-
mophore. Nevertheless, these compounds showed a sig-
nificant effect in the inhibition of Lck. Thus, the change
of the naphthalimide chromophore for the 3,4-diphenyl
maleic chromophore practically did not alter the acti-
vity against Lck and it would always be around optimal
values.
The results for conjugates with PAMAM chains, in
which the ramification grade increased (19 vs. 21)
against HT-29, was not relevant. However, the values
obtained against Lck were relevant in both cases, and it
can be deduced that the increasing in the ramification
grade is a favourable factor in the inhibition of Lck,
but the absolute values are less than conjugates with
PPI chains.
In conjugates with end cyano groups, the introduc-
tion of the chromophore in the PPI chain did not
improve the results against HT-29. However, the acti-
vity against Lck was better without the chromophore.
We can deduce that the presence of end cyano groups is
an unfavourable factor for the inhibition of Lck and
HT-29 (9, 12 vs. 13, 16), whereas one favourable factor
for the inhibition of Lck is the presence of amine
primary groups (8 vs. 9–12).
1
The H- and 13C-NMR spectra were recorded in a
Bru¨cker AC 300 spectrometer with Me4Si as internal
standard. IR spectra were taken on a Perkin–Elmer
1330 spectrometer. For purification of crude reaction
mixtures by flash chromatography, Merck silica gel
(230–400 mesh) or neutral aluminum oxide (70–230
mesh) was used as the stationary phase. Identification
of products was made by analytical TLC (Merck Kie-
segel 60F-254), UV light (u=254 nm), and 5% phos-
phomolydic acid solution in 95% EtOH were used to
develop the plates. Aldrich celite 545 was employed as
a filtrating agent. Reagents and solvents were handled
by using standard syringe techniques. DMF and
CH2Cl2 were distilled over calcium hydride. All other
solvents were reagent grade. Ni-Raney (slurry in water)
was purchased from Aldrich. Analyses indicated by the
symbols of the element were within 90.4% of theoreti-
cal values.
6.1.1. Synthesis of
N-tert-butoxycarbonylaminoethylamine (2)
Di-tert-butyldicarbonate (9.07 g, 41.5 mmol) dis-
solved in CHCl3 (200 mL) was added dropwise to a
solution of ethylenediamine (5 g, 83.1 mmol) in 80 mL
of CHCl3 at 0 °C. The reaction mixture was stirred 16
h at room temperature (r.t.) and washed with water.
The organic phase was dried over Na2SO4, evaporated
to dryness in vacuo to obtain a colourless oil which was
purified by silica gel chromatography (CHCl3–MeOH
5:0.1). Yield: 3.3 g (50%) (colourless oil). 1H-NMR
(CDCl3): l 1.39 (9H, 3CH3), 1.90 (s, 2H, NH2), 2.74 (t,
2H, CH2, CH2NH2, J=6.0 Hz), 3.12 (q, 2H, CH2,
CH2NH, J=5.5 Hz), 5.23 (bs, 1H, NH). 13C-NMR
(CDCl3): l=156.0, 78.9, 43.0, 41.6, 28.2. IR (neat):
3400, 2980, 1720 cm−1. Anal. (C7H16N2O2) C, H, N.
5. Conclusions
In conclusion, in this work we have synthesised the
first examples of dendritic polyamines–(imide–DNA-
intercalator) conjugates. Although dendritic polyamines
have been employed in gene therapy, their modification

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
547
6.1.2. General procedure for synthesis of oligonitriles
6ia cyanoethylation (3, 5, 7)
l 155.9, 118.8, 78.8, 52.9, 52.0, 51.9, 51.4, 51.3, 49.4,
38.2, 29.6, 28.4, 24.8, 16.8. IR (neat): 2980, 2240, 1725
cm−1. Anal. (C49H82N16O2) C, H, N.
6.1.2.1. Method A. To a solution of the amine (10
mmol) in a large excess of acrylonitrile (50 mL), glacial
AcOH (20 mmol per primary amine fuction) was added
and the mixture was refluxed for 24 h. The excess of
acrylonitrile was evaporated in vacuo to obtain a
residue, which was dissolved in CHCl3 (30 mL) and
added to 10 mL of concentrated aq. NH4OH. The
organic layer was separated, washed with water and
dried over Na2SO4. Evaporation of the solvent and
neutral alumine chromatography of the obtained oil
yielded the pure products.
6.1.3. Synthesis of 8-cascade ethylenediamine
[2]–(1-azabutylidyne)2–propanitrile (8)
Oligonitrile 7 (3.58 g, 3.86 mmol) was dissolved in 50
mL of 50% CHCl3–anisol, and 25 mL of trifluoracetic
acid was slowly added at 0 °C. After stirring for 25 min
the solvent and the TFA were removed under vacuum.
The resulting residue was dissolved in water and ad-
justed to pH\8 by addition of a NaHCO3 saturated
solution. The resulting solution was extracted with
CHCl3 (3×15 mL) and the combined organic layers
dried over Na2SO4, filtered, and concentrated in vacuo
to obtain an oil which was purified by Ku¨gelrorh
distillation to yield 2.5 g (80%) of 8 as an orange oil.
6.1.2.2. Method B. To a cold solution of the polyamine
(1.0 g, 1.9 mmol) in MeOH (18 mL) were added
dropwise 19 mL (288 mmol) of acrylonitrile. After
stirring 1 h at 10 °C and 4 days at 60 °C, the quantita-
tive bis(cyanoethylation) was obtained. The solvent was
evaporated in vacuo to obtain an oil which was purified
by low pressure distillation.
1H-NMR (CDCl3):
l
1.57 (m, 12H, 6CH2,
CH2CH2CH2), 2.40–2.58 (m, 42H, 21CH2, CH2N), 2.70
(m, 2H, CH2, CH2NH2), 2.81 (t, 16H, 8CH2, CH2CN,
J=7.1 Hz). 13C-NMR (CDCl3): l 118.7, 52.3, 51.9,
51.4, 51.3, 49.4, 39.6, 24.8, 24.3, 16.8. IR (neat): 3500,
2240 cm−1. Anal. (C44H74N16) C, H, N.
6.1.2.3. Synthesis of N,N-bis-(2-cyanoethyl)-N%-tert-bu-
toxycarbonyl ethylenediamine (3). Following method A,
from 10.0 g (64.8 mmol) of 2 was obtained 2.1 g (96%)
of 3 as a colourless oil after chromatography (CHCl3–
6.1.4. General procedure for synthesis of primary
amines 6ia hydrogenation with Ni-Raney (4, 6, 13–16)
A 1.4 M solution NaOH in 95% EtOH was added to
a 500 mL hydrogenation vessel filled with the oligoni-
trile and Raney-Ni catalyst. The mixture was hydro-
genated at 60 psi for several hours at r.t. The catalyst
was filtered through celite and washed with 95% EtOH.
After diluting the filtrate with H2O, EtOH was evapo-
rated and the residue extracted several times with
CH2Cl2. Thereby, the NaOH concentration in the aq.
phase was increased in every extraction step. The or-
ganic layers were dried with Na2SO4 and the solvent
evaporated in vacuo yielding the polyamines as oils.
1
MeOH 5:0.1). H-NMR (CDCl3): l 1.39 (s, 9H, 3CH3),
2.46 (t, 4H, 2CH2, CH2CN, J=6.6 Hz), 2.64 (t, 2H,
CH2, NCH2CH2NH, J=6.0 Hz), 2.86 (t, 4H, 2CH2,
CH2N, J=6.0 Hz), 3.16 (q, 2H, CH2, NCH2CH2NH,
J=6.0 Hz), 5.0 (bs, 1H, NH). 13C-NMR (CDCl3): l
155.9, 118.5, 79.0, 52.8, 49.4, 38.2, 28.1, 16.9. IR (neat):
2980, 2240, 1720 cm−1. Anal. (C13H22N4O2) C, H, N.
6.1.2.4. Synthesis of 4-cascade–N-tert-butoxycarbonyl
ethylenediamine [2]–(1-azabutylidyne)–propanitrile (5).
Following method A, from 3.2 g (11.7 mmol) of 4 was
obtained 5.1 g (90%) of 5 as a colourless oil after
chromatography (CHCl3–MeOH 10:0.1). 1H-NMR
(CDCl3): l 1.43 (s, 9H, 3CH3), 1.59 (m, 4H, 2CH2,
CH2CH2CH2), 2.46–2.57 (m, 18H, 9CH2, CH2N), 2.84
(t, 8H, 4CH2, CH2CN, J=6.6 Hz), 3.14 (q, 2H, CH2,
CH2NH, J=6.0 Hz), 4.99 (bs, 1H, NH). 13C-NMR
(CDCl3): l 155.9, 118.7, 79.0, 53.0, 51.5, 51.2, 49.5,
28.4, 24.9, 16.8. IR (neat): 2980, 2240, 1720, 1600
cm−1. Anal. (C25H42N8O2) C, H, N.
6.1.4.1. Synthesis of N,N-bis-(3-aminopropyl)-N%-tert-
butoxycarbonyl ethylenediamine (4). From 4.0 g (15
mmol) of 3, 1.5 g of Ni-Raney and 12 mL of a 1.4 M
solution NaOH in 95% EtOH were hydrogenated for 16
h. In this case the filtrate was concentrated and dis-
solved in a small amount of water. After addition of
NaOH pellets, the amine 4 begins to separate as an oil
which is extracted with CHCl3. The organic layer was
dried over Na2SO4 and the solvent evaporated to ob-
tain 3.2 g (80%) of 4 as an orange oil. 1H-NMR
(CDCl3): l 1.22 (bs, 4H, 2NH2), 1.38 (s, 9H, 3CH3),
1.52 (m, 4H, 2CH2, CH2CH2CH2), 2.41 (t, 4H, 2CH2,
CH2N, J=7.1 Hz), 2.44 (t, 2H, CH2, NCH2CH2NH,
J=6.0 Hz), 2.67 (t, 4H, 2CH2, CH2NH2, J=7.1 Hz),
3.11 (q, 2H, CH2, NCH2CH2NH, J=6.0 Hz), 5.00 (bs,
1H, NH). 13C-NMR (CDCl3): l 156.0, 78.8, 53.1, 51.5,
6.1.2.5. Synthesis of 8-cascade–N-tert-butoxycar-
bonylethylenediamine [2]–(1-azabutylidyne)2–propani-
trile (7). Following method B, from 1.0 g (1.9 mmol) of
6 was obtained 1.5 g (82%) of 7 as a pale yellow oil.
1H-NMR (CDCl3): l 1.42 (s, 9H, 3CH3), 1.58 (m, 12H,
6CH2, CH2CH2CH2), 2.48 (m, 42H, 21CH2, CH2N),
2.83 (t, 16H, 8CH2, CH2CN, J=6.6 Hz), 3.13 (s, 2H,
CH2NH), 5.04 (bs, 1H, NHBOC). 13C-NMR (CDCl3):
40.3, 38.2, 30.7, 28.3. IR (neat): 3500, 2980, 1720 cm−1
Anal. (C13H30N4O2) C, H, N.
.

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M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
6.1.4.2. Synthesis of 4-cascade–N-tert-butoxycar-
bonylethylenediamine [2]–(1-azabutylidyne)–propan-
IR (neat): 3400, 1740, 1660 cm−1. Anal. (C56H111
N17O2) C, H, N.
-
amine (6). From 5.2 g (10.6 mol) of 5, 5.7 g of Ni-
Raney and 16.6 mL of a 1.4 M solution NaOH in
EtOH was obtained 3.7 g (70%) of 6 as an orange oil.
6.1.4.6. Synthesis of 8-cascade–N-(2-aminoethyl)-2,3-
diphenylmaleimide [2]–(1-azabutylidyne)2–propanamine
(16). From 0.46 g (0.44 mmol) of 12, 0.70 g of Ni-
Raney and 25 mL of a 1.4 M solution NaOH in 95%
EtOH was obtained 0.37 g (77%) of 16 as a yellow oil.
1
Reaction time: 36 h. H-NMR (CDCl3): l 1.34 (bs, 8H,
4NH2), 1.38 (s, 9H, 3CH3), 1.52 (m, 12H, 6CH2,
CH2CH2CH2), 2.38 (m, 18H, 9CH2, CH2N), 2.66 (t,
8H, 4CH2, CH2NH2, J=6.6 Hz), 3.10 (m, 2H, CH2,
CH2NH), 4.93 (bs, 1H, NH). 13C-NMR (CDCl3): l
156.0, 78.7, 53.0, 51.8, 51.7, 51.5, 40.6, 38.2, 30.7, 28.3,
1
Reaction time 36 h. H-NMR (D2O): l 1.26–1.39 (m,
28H, 14CH2, CH2CH2CH2), 2.08–2.27 (m, 42H,
21CH2, CH2N), 2.33 (t, 16H, 8CH2, CH2NH2, J=7.1
Hz), 3.91 (bs, 2H, CH2, CH2NCO), 6.99–6.87 (m, 10H,
Har). 13C-NMR (D2O): l 170.2, 138.1, 129.2, 128.4,
127.8, 127.5, 57.1, 50.3, 50.1, 48.0, 38.3, 27.6, 27.0, 20.8.
24.4. IR (neat): 3400, 2980, 1720 cm−1
(C25H58N8O2) C, H, N.
. Anal.
6.1.4.3. Synthesis of 8-cascade–N-(2-aminoethyl)-1,8-
naphthalimide [2]–(1-azabutylidyne)2–propanamine (13).
From 1.13 g (1.12 mmol) of 9, 1.8 g of Ni-Raney and
25 mL of a 1.4 M solution NaOH in 95% EtOH was
obtained 0.58 g (50%) of 13 as a light brown oil.
IR (neat): 3400, 1740, 1650 cm−1. Anal. (C60H114
N16O2) C, H, N.
-
6.1.5. General procedure for synthesis of imide
deri6ati6es 9, 10, 11, 12
1
Reaction time 48 h. H-NMR (D2O): l 1.40–1.56 (m,
To a solution of the 8-cascade–ethylenediamine [2]–
(1-azabutylidyne)2–propanitrile (8), the corresponding
anhydride was added. The mixture was stirred and the
solvent removed under reduced pressure to obtain the
imide derivatives after chromatography in neutral alu-
minum oxide using CHCl3–MeOH (5:0.1) as eluent.
28H, 14CH2, CH2CH2CH2), 2.32–2.43 (m, 42H,
21CH2, CH2N), 2.70 (m, 16H, 8CH2, CH2NH2), 3.97
(bs, 2H, CH2, CH2NCO), 7.59 (bs, 2H, Har), 8.19 (bs,
4H, Har). 13C-NMR (D2O): l 163.3, 128.9, 127.6,
127.5, 127.4, 57.1, 50.3, 50.1, 48.0, 38.3, 27.6, 27.0, 24.8,
20.9. IR (neat): 3500, 1700, 1610 cm−1
(C56H110N16O2) C, H, N.
. Anal.
6.1.5.1. Synthesis of 8-cascade–N-(2-aminoethyl-1,8-
naphthalimide [2]–(1-azabutylidyne)2–propanitrile (9).
From 0.89 g (1.08 mmol) of 8 and 0.21 g (1.08 mmol)
of 1,8-naphthalic anhydride in absolute EtOH (50 mL)
were stirred 18 h at r.t. to obtain 0.89 g (92%) of 9 as
6.1.4.4. Synthesis of 8-cascade–N-(2-aminoethyl)-3-ni-
tro-1,8-naphthalimide [2]–(1-azabutylidyne)2–propan-
amine (14). From 1.10 g (1.04 mmol) of 10, 1.4 g of
Ni-Raney and 60 mL of a 1.4 M solution NaOH in
95% EtOH was obtained 0.68 g (60%) of 14 as a red oil.
1
a light brown oil. H-NMR (CDCl3): l 1.64 (m, 12H,
6CH2, CH2CH2CH2), 2.50 (t, 24H, 12CH2, CH2CH2N,
J=6.6 Hz), 2.59 (t, 16H, 8CH2, NCH2CH2CN, J=7.1
Hz), 2.75 (t, 2H, CH2, NCH2CH2N(CO)2, J=7.1 Hz),
2.86 (t, 16H, 8CH2, CH2CN, J=7.1 Hz), 4.26 (t, 2H,
NCH2CH2N(CO)2, J=7.1 Hz), 7.78 (t, 2H, Har, J=
7.7 Hz), 8.24 (d, 2H, Har, J=8.2 Hz), 8.5 (d, 2H, Har,
J=7.1 Hz). 13C-NMR (CDCl3): l 163.8, 133.8, 131.3,
130.8, 127.7, 126.7, 122.1, 118.7, 52.3, 51.5, 51.2, 50.9,
49.1, 44.1, 37.6, 24.3, 23.9, 16.5. IR (neat): 2240, 1700,
1610 cm−1. Anal. (C56H78N16O2) C, H, N.
1
Reaction time 24 h. H-NMR (D2O): l 1.10–1.40 (bs,
28H, 14CH2, CH2CH2CH2), 2.29 (m, 42H, 21CH2,
CH2N), 2.45 (m, 16H, 8CH2, CH2NH2), 3.06 (bs, 2H,
CH2NCO), 7.21 (bs, 1H, Har), 7.47 (bs, 1H, Har), 7.52
(bs, 1H, Har), 8.16 (bs, 1H, Har), 8.81 (bs, 1H, Har).
13C-NMR (D2O): l 165.5, 162.5, 133.9, 132.8, 132.0,
131.6, 131.4, 129.0, 54.2, 54.0, 53.9, 53.7, 53.6, 53.5,
53.0, 52.1, 36.6, 31.2, 31.0, 30.4, 24.8. IR (neat): 3400,
1700, 1610, 1590 cm−1. Anal. (C56H109N17O4) C, H, N.
6.1.4.5. Synthesis of 8-cascade–N-(2-aminoethyl)-3-
amine-1,8-naphthalimide
6.1.5.2. Synthesis of 8-cascade–N-(2-aminoethyl)-3-ni-
tro-1,8-naphthalimide [2]–(1-azabutylidyne)2–propani-
trile (10). From 0.81 g (0.98 mmol) of 8 and 0.24 g (0.98
mmol) of 3-nitro-1,8-naphthalic anhydride in 50 mL of
50% absolute EtOH–CH2Cl2 were refluxed 18 h to
obtain 0.72 g (83%) of 10 as a red oil. 1H-NMR
(CDCl3): l 1.65 (m, 12H, 6CH2, CH2CH2CH2), 2.48–
2.65 (m, 40H, 20CH2, CH2N), 2.76 (m, 2H, CH2,
NCH2CH2N(CO)2), 2.85 (t, 16H, 8CH2, CH2CN, J=
6.6 Hz), 4.26 (t, 2H, CH2, NCH2CH2N(CO)2, J=7.1
Hz), 7.95 (t, 1H, Har, J=7.1 Hz), 8.43 (d, 1H, Har,
J=8.2 Hz), 8.76 (d, 1H, Har, J=7.1 Hz), 9.14 (s, 1H,
Har), 9.23 (s, 1H, Har). 13C-NMR (CDCl3): l 162.8,
[2]–(1-azabutylidyne)2:pro-
panamine (15). From 1.0 g (0.98 mmol) of 11, 1.44 g of
Ni-Raney and 50 mL of a 1.4 M solution NaOH in
95% EtOH was obtained 0.51 g (49%) of 15 as a yellow
1
oil. Reaction time 24 h. H-NMR (D2O): l 1.25–1.33
(bs, 28H, 14CH2, CH2CH2CH2), 2.15–2.30 (m, 42H,
21CH2, CH2N), 2.45 (m, 16H, 8CH2, CH2NH2), 3.03
(bs, 2H, CH2, CH2NCO), 6.82 (bs, 1H, Har), 6.94 (bs,
1H, Har), 7.15 (bs, 1H, Har), 7.49 (bs, 1H, Har), 7.61
(bs, 1H, Har). 13C-NMR (D2O): l 161.9, 158.7, 131.3,
130.9, 130.7, 130.0, 128.9, 126.6, 124.6, 122.7, 54.2,
53.7, 53.6, 53.5, 52.2, 41.7, 40.3, 36.3, 31.0, 30.4, 24.8.

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
549
162.3, 145.9, 135.6, 134.2, 130.8, 129.9, 129.0, 124.3,
6.1.7. General procedure for synthesis of polyesters 18,
20, 22
122.8, 118.8, 52.3, 51.6, 51.3, 51.1, 49.3, 44.2, 38.1, 24.6,
16.6. IR (neat): 2240, 1700, 1610, 1590 cm−1. Anal.
(C56H77N17O4) C, H, N.
Methyl acrylate (75 equiv. per primary amine func-
tion) was added to a solution of conjugate with amine
terminal groups and the mixture was heated to reflux.
MeOH and excess of methylacrylate were eliminated to
yield the polyester as an oil after chromatography in
silica gel using CHCl3–MeOH 10:0.1.
6.1.5.3. Synthesis of 8-cascade–N-(2-aminoethyl)-3-
amine-1,8-naphthalimide
[2]–(1-azabutylidyne)2–pro-
panitrile (11). From 0.98 g (1.19 mmol) of 8 and 0. 25
g (1.19 mmol) of 3-amine-1,8-naphthalic anhydride in
50 mL of 50% absolute EtOH–DMF were refluxed 48
6.1.7.1. N-[2-(N,N%-bis(2-metoxycarbonylethyl)amine)-
ethyl]-1,8-naphthalimide (18). From 987 mg (4.11
mmol) of 17 and 27 mL (308 mmol) of methyl acrylate
in 40 mL of MeOH was obtained 18 as a colourless oil
with 81%. Reaction time: 6 h. 1H-NMR (CDCl3): l
2.48 (t, 4H, 2CH2, NCH2CH2CO2CH3, J=7.1 Hz),
2.81 (t, 2H, CH2, NCH2CH2NCO, J=7.1 Hz), 2.90 (t,
4H, 2CH2, NCH2CH2CO2CH3, J=7.1 Hz), 3.52, (s,
6H, 2CH3), 4.26 (t, 2H, CH2, NCH2CH2NCO, J=7.1
Hz), 7.74 (t, 2H, Har, J=8.2 Hz), 8.20 (d, 2H, Har,
J=8.2 Hz), 8.56 (d, 2H, Har, J=7.1 Hz). 13C-NMR
(CDCl3): l 172.9, 164.1, 133.8, 131.5, 131.2, 128.1,
126.9, 122.6, 122.6, 51.3, 50.9, 49.6, 38.0, 32.8. IR
(neat): 1730, 1700, 1650 cm−1. Anal. (C22H24N2O6) C,
H, N.
1
h to obtain 1.0 g (60%) of 11 as a yellow oil. H-NMR
(CDCl3):
l
1.75–2.10 (m, 14H, 6CH2+NH2,
CH2CH2CH2), 2.53–2.65 (m, 42H, 21CH2, CH2N), 2.84
(t, 16H, 8CH2, CH2CN, J=6.0 Hz), 4.24 (t, 2H, CH2,
CH2N(CO)2, J=7.1 Hz), 7.31 (s, 1H, Har), 7.61 (t, 1H,
Har, J=7.7 Hz), 7.95 (d, 1H, Har, J=7.1 Hz), 8.02 (s,
1H, Har), 8.25 (d, 1H, Har, J=7.1 Hz). 13C-NMR
(CDCl3): l 162.8, 162.3, 133.5, 131.8, 127.1, 122.1,
119.1, 119.0, 118.8, 113.8, 51.5, 51.3, 50.7, 49.4, 44.5,
36.4, 31.4, 18.9, 17.0. IR (neat): 3500, 2240, 1700, 1650
cm−1. Anal. (C56H79N17O2) C, H, N.
6.1.5.4. Synthesis of 8-cascade–N-(2-aminoethyl)-2,3-
diphenylmaleimide [2]–(1-azabutylidyne)2–propanitrile
(12). From 0.80 g (0.97 mmol) of 8 and 0.24 g (0.97
mmol) of 2,3-diphenylmaleic anhydride in 50 mL of
absolute EtOH were refluxed 2 h to obtain 0.82 g (80%)
6.1.7.2. 4-cascade–N-(2-aminoethyl)-1,8-naphthalimide
[2]–1, 4-diaza-5-oxoheptylidine–methylpropanoate (20).
From 19 (458 mg, 0.98 mmol) in 10 mL of MeOH and
13.2 mL (146.7 mmol) of methyl acrylate 20 was ob-
tained as a yellow oil with 62%. Reaction time: 16 h.
1H-NMR (CDCl3): l 2.45 (t, 12H, 6CH2, J=6.6Hz),
2.54 (t, 4H,2CH2, J=6.0 Hz), 2.77 (t, 8H, 4CH2,
J=6.6 Hz), 2.83 (t, 2H, CH2, NCH2CH2NCO, J=7.7
Hz), 2.96 (t, 4H, 2CH2, J=6.6 Hz), 3.27 (c, 4H, 2CH2,
CH2NHCOCH2, J=6.0 Hz), 3.65 (s, 12H, 4MeO),
4.28 (t, 2H, CH2, NCH2CH2NCO, J=7.14 Hz), 7.12
(t, 2H, 2NH), 7.76 (t, 2H, Har, J=7.1 Hz), 8.23 (d,
2H, Har, J=7.7 Hz), 8.57 (d, 2H, Har, J=6.6 Hz).
13C-NMR (CDCl3): l 172.7, 171.9, 163.7, 133.8, 131.3,
130.8, 127.7, 126.7, 122.2, 52.7, 51.3, 50.1, 50.0, 48.9,
37.7, 36.8, 33.6, 32.4. IR (neat): 3300, 1730, 1650, 1630
cm−1. Anal. (C40H56N6O12) C, H, N.
1
of 12 as a yellow oil. H-NMR (CDCl3): l 1.58 (m,
12H, 6CH2, CH2CH2CH2), 2.42–2.54 (m, 40H, 20CH2,
CH2N), 2.71 (m, 2H, CH2, NCH2CH2N(CO)2), 2.80 (t,
16H, 8CH2, CH2CN, J=6.6 Hz), 3.70 (t, 2H, CH2,
NCH2CH2N(CO)2, J=6.6 Hz), 7.44–7.35 (m, 10H,
Har). 13C-NMR (CDCl3): l 170.7, 136.2, 129.8, 128.7,
128.6, 128.5, 118.8, 52.5, 51.8, 51.5, 51.2, 49.5, 36.4,
24.7, 16.8. IR (neat): 2240, 1700, 1640 cm−1. Anal.
(C60H82N16O2) C, H, N.
6.1.6. Synthesis of N-(2-aminoethyl)-1,8-naphthalimide
(17)
1,8-Naphthalenedicarboxylic anhydride (2 g, 10.1
mmol) was added in portions to a solution of 1.01 mL
(15.1 mmol) of ethylenediamine in 250 mL of EtOH
and the mixture was heated to reflux during 6 h. The
mixture was filtered under vacuum and the filtrate was
evaporated under reduced pressure. The residue was
recrystalised from EtOH to give 17 as a solid with 80%.
1H-NMR (CDCl3): l 1.50 (bs, 2H, NH2), 3.08, (t, 2H,
CH2, CH2NH2, J=6.6 Hz), 4.29 (t, 2H,CH2,
CH2N(CO)2), J=6.6 Hz), 7.75 (t, 2H, Har, J=7.7
Hz), 8.21 (d, 2H, Har, J=8.2 Hz), 8.59 (d, 2H, Har,
J=7.1 Hz). 13C-NMR (CDCl3):l 164.9, 134.4, 131.9,
131.7, 128.6, 127.3, 122.9, 43.5, 40.9. IR (neat): 3345,
1700, 1660 cm−1. Anal. (C14H12N2O2) C, H, N.
6.1.7.3. 8-cascade: N-(2-aminoethyl)-1,8-naphthalimide
[2]–(1,4 - diaza - 5 - oxoheptylidine)2–methylpropanoate
(22). From 21 (255 mg, 0.27 mmol) in 20 mL of MeOH
and 7.2 mL (81 mmol) of methyl acrylate 22 was
obtained as a yellow oil with 67%. Reaction time: 48 h.
1H-NMR (CDCl3): l 2.38–2.44 (m, 32H, 16CH2),
2.49–2.54 (m, 8H, 4CH2), 2.56 (bs, 2H, CH2), 2.74 (t,
16H, 8CH2, J=6.6 Hz), 2.79–2.85 (m, 8H, 4CH2), 2.93
(t, 4H, 2CH2, J=6.6 Hz), 3.29 (c, 12H, 6 CH2, J=5.5
Hz), 3.66 (s, 24H, 3OMe), 4.28 (t, 2H, NCH2CH2NCO,
J=7.1 Hz), 7.07 (m, 4H, NHCO), 7.69 (bs, 2H,
NHCO), 7.77 (t, 2H, Har, J=7.1 Hz), 8.23 (d, 2H,
Har, J=8.2 Hz), 8.58 (d, 2H, Har, J=7.7 Hz). 13C-

550
M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
NMR (CDCl3): l 173.0, 172.3, 172.2, 164.0, 134.0,
131.6, 128.1, 127.0, 122.5, 52.8, 52.5, 51.6, 50.2, 49.9,
49.2, 37.1, 33.7, 32.6, 29.6. IR (neat): 3300, 1700, 1680
cm−1. Anal. (C76H120N14O24) C, H, N.
2H, Har), 8.42 (bs, 2H, Har). 13C-NMR (CD3OH): l
177.6, 176.8, 176.0, 166.4, 131.0, 126.2, 122.7, 118.8,
59.3, 43.8, 41.7, 41.5, 37.2, 31.8, 30.7. IR (neat): 3300,
1700, 1650 cm−1. Anal. (C84H152N30O16) C, H, N.
6.1.8. General procedure for amonolysis of polyesters
19, 21, 23
6.2. Pharmacology
Ethylenediamine (54 equiv. per terminal ester group)
was added dropwise to a solution of the polyester in
MeOH and the mixture was stirred at r.t. The excess of
ethylenediamine and MeOH were eliminated under re-
duced pressure. The crude was purified by chromato-
graphy using CHCl3–MeOH 10:0.5 to obtain the
polyamide as an oil.
6.2.1. Materials and methods
6.2.1.1. Cytotoxicity assay in HT-29 cells. The cytotoxi-
city of synthesised conjugates was measured using a
standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay. The human carcinoma
cell line, HT-29, was obtained from the American Type
Culture Collection and cultured in the recommended
media. Exponentially growing cells were plated at 3000-
well into 96-well plates in 150 mL complete DMEM
media containing 10% fetal bovine serum (FBS). Cells
were allowed to attach for 24 h before the addition of
a serial (1:4) dilution of the drug in 50 mL of fresh
medium. After 72 h of incubation at 37 °C (5% CO2),
MTT was added to each well and the plates incubated
for 4 h. Formazan crystals formed by MTT metabolism
were solubilised by the addition of 50 mL of 25% SDS
(pH 2) to each well and incubated overnight. The
cellular metabolism of MTT was then quantified by
reading the absorbance of the solubilised product at
550 nm with a 96-well plate reader, IC50 values were
calculated as the concentration of drug inhibiting cell
growth to 50% of controls.
6.1.8.1. N-[2-(N,N-Bis(2-methoxycarbonylethyl)amine)-
ethyl]-1,8 naphthalimide (19). From 18 (1.38 g, 3.32
mmol) in 30 mL of MeOH and 244 mL (364.8 mmol)
of ethylenediamine was obtained 19 (900 mg) with 58%
1
as a yellow oil. Reaction time: 24 h. H-NMR (CDCl3):
l 1.75 (bs, 4H, NH2), 2.45 (t, 2CH2, J=6.0 Hz),
2.76–2.83 (m, 6H, 3CH2), 2.89 (t, 4H, 2CH2, J=6.0
Hz), 3.25 (c, 4H, 2CH2, CH2NHCOCH2, J=6.06 Hz),
4.26 (t, 2H, CH2, NCH2CH2NCO, J=7.1 Hz), 7.32 (t,
2H, 2NH), 7.77 (t, 2H, Har, J=7.7 Hz), 8.23 ( d, 2H,
Har, J=7.7 Hz), 8.59 (d, 2H, Har, J=7.1 Hz). 13C-
NMR (CDCl3): l 172.7, 164.4, 134.4, 131.6, 131.4,
128.1, 127.0, 122.3, 51.0, 50.4, 42.1, 41.5, 38.5, 34.5. IR
(neat): 3500, 1700, 1650 cm−1. Anal. (C24H32N6O4) C,
H, N.
6.1.8.2. 4-Cascade–N-(2-aminoethyl)-1,8-naphthalimide
[2]–1,4-diaza-5-oxoheptylidine–3-aza-4-oxohexanamine
(21). From 20 (338 mg, 0.42 mmol) in 10 mL of MeOH
and 6.06 mL (90.7 mmol) of ethylenediamine was ob-
tained 21 with 60% as a yellow oil. Reaction time: 3
6.2.1.2. ELISA for determination of Lck acti6ity. Hu-
man recombinant Lck catalytic domain was produced
in a baculovirus expression system. Purified protein was
used. The susbstrate Poly(Glu, Tyr) 4:1 for ELISA
plate coating was diluted to 1 mg mL−1 in phosphate-
buffered saline (PBS) containing 0.02% Na azide; 100
mL per well of this polymer solution was used to coat
96-well microtiter ELISA plates. Plates were covered
and incubated overnight at 4 °C. Immediately prior to
use, plates were washed four times using TBS–Tween
20 (25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.2%
Tween 20). The enzyme mix was prepared immediately
prior to use in a kinase buffer. The final concentrations
of components in the Lck kinase assay were 20 mM
Tris–HCl, pH 7.5, 10 mM MgCl2 5 mM ATP and 1 ng
of Lck enzyme. Fifty microlitres of kinase buffer con-
taining Me2SO was added to each well and inhibitor
dilutions were carried out. Compounds were dissolved
in Me2SO, giving final concentrations of 5%. Positive
and negative controls contained the appropiate Me2SO
concentration. Negative controls contained 10 mL of 0.5
M EDTA, which was added prior to the addition of the
enzyme. All assays were carried out in duplicate. The
reaction was started by the addition of 50 mL of kinase
buffer containing diluted enzyme to each well. Plates
1
days. H-NMR (D2O): l 2.18–2.23 (m, 12H, 2CH2+
4NH2), 2.30 (c, 4H, 2CH2, J=6.0 Hz), 2.50–2.54 (m,
8H, 4CH2), 2.63–2.67 (m, 4H, 2CH2), 2.89–2.82 (m,
20H, 10CH2), 3.11 (t, 2H, CH2, J=6.0 Hz), 3.22 (t,
8H, 4CH2, J=5.5 Hz), 3.74 (m, 2H, CH2), 7.35–7.42
(m, 2H, Har), 7.85–7.93 (m, 4H, Har). 13C-NMR
(D2O): l 178.0, 177.4, 167.4, 137.8, 134.1, 133.4, 129.8,
129.3, 123.0, 51.8, 51.4, 42.9, 42.2, 41.8, 41.4, 40.6, 40.5,
38.9, 35.0. IR (neat): 3300, 1700, 1650 cm−1. Anal.
(C44H72N14O8) C, H, N.
6.1.8.3. 4-Cascade–N-(2-aminoethyl)-1,8-naphthalimide
[2]–(1,4-diaza-5-oxoheptylidine)2–3-aza-4-oxohexan-
amine (23). From 22 (42 mg, 0.026 mmol) in 30 mL of
MeOH and 0.75 mL (11.3 mmol) of ethylenediamine
was obtained 23 (900 mg) 40% as a yellow oil. Reaction
time: 4 days. 1H-NMR (CD3OH): l 2.22 (bs, 28H,
14CH2), 2.42 (bs, 16H, 8CH2, CH2NH2), 2.69 (t, 28H,
14CH2, J=6.0 Hz), 3.25 (t, 42H, 21CH2, J=6.0 Hz),
4.01 (bs, 2H, CH2NCO), 7.68 (bs, 2H, Har), 8.23 (bs,

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 37 (2002) 541–551
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tions were terminated by washing the ELISA plates
four times with TBS–Tween. Washed plates were
blocked with 3% BSA–TBS–Tween (100 mL per well)
for 45 min and then washed four times with TBS–
Tween; 100 mL of anti-phosphotyrosine antibody was
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45 min. ELISA plates were finally washed with TBS–
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