Synthesis and antitumor activity of new benzoheterocyclic derivatives of distamycin A

Article abstract of DOI:10.1021/jm9911229

The design, synthesis, and in vivo and in vitro antileukemic activity of a novel series of compounds (13-22 and 34), in which different benzoheterocyclic rings, bearing a nitrogen mustard or a benzoyl nitrogen mustard or an α-bromoacryloyl group as alkylating moieties, are tethered to a distamycin frame, are reported, and structure-activity relationships are discussed. The new derivatives were prepared by coupling nitrogen mustard- substituted, benzoyl nitrogen mustard-substituted, or α- bromoacryloyl-substituted benzoheterocyclic carboxylic acids 23- 32 with desformyldistamycin (33) or in one case with its two- pyrrole analogue 35. With very few exceptions, the activities of compounds bearing the same alkylating moiety are slightly affected by the kind of the heteroatom present on the benzoheterocyclic ring. All novel compounds, with one exception, showed in vitro activity against L1210 murine leukemia cell line comparable to or better than that of tallimustine. The compounds in which the nitrogen mustard and the α-bromoacryloyl moieties are directly linked to benzoheterocyclic ring showed potent cytotoxic activities (IC₅₀ ranging from 2 to 14 nM), while benzoyl nitrogen mustard derivatives of benzoheterocycles showed reduced cytotoxic activities, and one compound (16) of this cluster was the sole derivative devoid of significant activity. Compound 18, a 5-nitrogen mustard N-methylindole derivative of distamycin, showed the best antileukemic activity in vivo, with a very long survival time (%T/C = 457), significantly increased in comparison to tallimustine (%T/C = 133), and was selected for further extensive evaluation. Arrested polymerase chain reaction and direct DNA fragmentation assays were performed for compound 18 and the structurally related compounds 13-17 and 19. The results obtained have shown that both alkylating groups and oligopeptide frames play a crucial role in the sequence selectivity of these compounds.

Full text of DOI:10.1021/jm9911229

J . Med. Chem. 2000, 43, 2675-2684
2675
Syn th esis a n d An titu m or Activity of New Ben zoh eter ocyclic Der iva tives of
Dista m ycin A
Pier Giovanni Baraldi,*,† Romeo Romagnoli,† Italo Beria,§ Paolo Cozzi,§ Cristina Geroni,§ Nicola Mongelli,§
Nicoletta Bianchi,‡ Carlo Mischiati,‡ and Roberto Gambari‡
Dipartimento di Scienze Farmaceutiche and Dipartimento di Biochimica e Biologia Molecolare, Universita` di Ferrara,
Via Fossato di Mortara 17-19, 44100 Ferrara, Italy, and Department of Chemistry, Discovery Research Oncology,
Pharmacia & Upjohn, Nerviano, Milano, Italy
Received J uly 23, 1999
The design, synthesis, and in vivo and in vitro antileukemic activity of a novel series of
compounds (13-22 and 34), in which different benzoheterocyclic rings, bearing a nitrogen
mustard or a benzoyl nitrogen mustard or an R-bromoacryloyl group as alkylating moieties,
are tethered to a distamycin frame, are reported, and structure-activity relationships are
discussed. The new derivatives were prepared by coupling nitrogen mustard-substituted, benzoyl
nitrogen mustard-substituted, or R-bromoacryloyl-substituted benzoheterocyclic carboxylic acids
23-32 with desformyldistamycin (33) or in one case with its two-pyrrole analogue 35. With
very few exceptions, the activities of compounds bearing the same alkylating moiety are slightly
affected by the kind of the heteroatom present on the benzoheterocyclic ring. All novel
compounds, with one exception, showed in vitro activity against L1210 murine leukemia cell
line comparable to or better than that of tallimustine. The compounds in which the nitrogen
mustard and the R-bromoacryloyl moieties are directly linked to benzoheterocyclic ring showed
potent cytotoxic activities (IC50 ranging from 2 to 14 nM), while benzoyl nitrogen mustard
derivatives of benzoheterocycles showed reduced cytotoxic activities, and one compound (16)
of this cluster was the sole derivative devoid of significant activity. Compound 18, a 5-nitrogen
mustard N-methylindole derivative of distamycin, showed the best antileukemic activity in
vivo, with a very long survival time (%T/C ) 457), significantly increased in comparison to
tallimustine (%T/C ) 133), and was selected for further extensive evaluation. Arrested
polymerase chain reaction and direct DNA fragmentation assays were performed for compound
18 and the structurally related compounds 13-17 and 19. The results obtained have shown
that both alkylating groups and oligopeptide frames play a crucial role in the sequence selectivity
of these compounds.
In tr od u ction
group was replaced with different alkylating functions,
such as benzoyl nitrogen mustard, nitrogen mustard,
or R-bromoacryloyl moieties, obtaining the correspond-
ing derivatives 23 (tallimustine or FCE 24517), 3,4 and
4,3 respectively. Tallimustine (2) was selected as an
antineoplastic drug candidate in view of its high activity
against a wide spectrum of experimental tumors. How-
ever, since its clinical evaluation showed severe myelo-
toxicity, its development was discontinued.5
It is important to underline that, as occurs in the case
of distamycin A and its four-pyrrole homologue 5, the
increase in the number of pyrrole units of the oligopep-
tidic frame in 2-4 led to derivatives 6-8, respectively,
which showed increased in vitro cytoxicity and in vivo
antitumor activity.3,4
Recently, we have described6 the synthesis and the
activity of two isosteric derivatives, 9 and 10, of 6 and
8, respectively, in which the N-methylpyrrole directly
linked to the alkylating moiety was replaced by an
N-methylpyrazole. Both these isosteres 9 and 10 showed
antitumor activity against L1210 leukemia, which was
almost equivalent to that exhibited by 6 and 8, respec-
tively. It should be underlined that, on the contrary, the
analogue 11 of compounds 6 and 9, in which a N-
methylimidazole replaced the pyrrole or pyrazole unit,
respectively, appeared about 2 orders of magnitude less
There is currently interest in the study and develop-
ment of low-molecular-weight sequence-selective agents
interacting with double-stranded DNA. These molecules
are oftenly based on natural products and have been
investigated for their ability to interact selectively with
the minor groove of DNA. One of the most studied minor
groove binders is distamycin A.
Distamycin A (1) is a naturally occurring antibiotic,1
characterized by the presence of an oligopeptidic pyr-
rolecarbamoyl frame ending with an amidino moiety,
which reversibly binds to the minor groove of DNA with
a strong preference for adenine-thymine (AT)-rich se-
quences containing at least four AT base pairs.2 Dista-
mycin A has been used as a DNA sequence-selective
carrier of alkylating functions, leading to compounds
which are substantially more cytotoxic than distamycin
itself.
Mongelli and co-workers synthesized several semi-
synthetic analogues of distamycin A wherein the formyl
* To whom correspondence should be addressed. Tel: +39-(0)532-
291293. Fax: +39-(0)532-291296. E-mail: pgb@ifeuniv.unife.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
‡ Dipartimento di Biochimica e Biologia Molecolare, Universita` di
Ferrara.
§ Pharmacia & Upjohn.
10.1021/jm9911229 CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/27/2000

2676 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Baraldi et al.
Sch em e 1a
cytotoxic than its pyrrole or pyrazole counterparts.
Moreover, the analogue 12 of compounds 8 and 10, in
which a N-methylimidazole replaced the pyrrole or
pyrazole unit, respectively, showed in vitro cytoxicity
against L1210 cells which was found to be equivalent
to or slightly lower than that of 8 and 10, the antileu-
kemic activity in vivo being higher than that exhibited
by 10.
a
Reagents: (a) EDC, Hunig’s base, DMF, 18 h, rt.
performed using a slight excess (1.5 equiv) of 1-ethyl-
3-[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDC) as coupling agent, in dry DMF as solvent, in the
presence of Hunig’s base, at room temperature and with
identical reaction times (18 h). Compounds 13-22 and
34 were prepared in acceptable yields, after purification
by silica gel flash chromatography.
The synthesis of heterocyclic acids 23-26, bearing the
benzoic acid mustard (BAM), was performed by coupling
ethyl 5-aminoindole-2-carboxylate (36),9 ethyl 1-methyl-
4-aminoindole-2-carboxylate (37),10 ethyl 5-aminoben-
zofuran-2-carboxylate (38), and ethyl 5-amino-1(3H)-
benzoimidazole-2-carboxylate (39), respectively, to
p-[bis(2-chloroethyl)amino]benzoyl chloride (40)11 to
give the corresponding amido esters 41-44, which after
purification by flash chromatography were submitted
to mild alkaline hydrolysis to yield the corresponding
acids 23-26, respectively (Scheme 2).
For the synthesis of acid intermediates 27-29, the
amino esters 36-38 were converted in good yields to
the corresponding N,N-bis(2-hydroxyethyl) derivatives
45-47, respectively, by reaction with a large excess (5
equiv) of ethylene oxide in methanol. Subsequent treat-
ment with phosphorus oxytrichloride (POCl3) afforded
the corresponding dichloro nitrogen mustards 48-50,
which were transformed into the desired acids 27-29
by acid hydrolysis (Scheme 3).
The synthetic approach employed for the preparation
of the R-bromoacryloyl derivatives 30-32 shown in
Scheme 4 was carried out by conversion of the nitro
carboxylic acids 51,12 52,10 and 5313 into the correspond-
ing tert-butyl esters 54-56 in good yield by treating 52
Following these results, we report in this paper the
synthesis and the biological evaluation of the new
distamycin A derivatives 13-22, in which the pyrrole
ring bearing a nitrogen mustard, benzoyl nitrogen
mustard, or R-bromoacryloyl alkylating moiety of com-
pounds 6-8 has been replaced by different benzohet-
erocycles such as indole, N-methylindole, benzoimida-
zole, and benzofuran.7a Since pyrrole is susceptible to
oxidative breakdown, these new derivatives have been
prepared as potentially more stable minor groove bind-
ers aimed to improved the relative instability of the
polypyrrolic skeleton. The choice of these benzohetero-
cycles can be also justified due to their importance in
increasing the binding to DNA and the selectivity of
alkylation of CC-1065 analogues such as U-71,184,
adozelesin, and bizelesin.7b
Ch em istr y
The synthetic route followed for the synthesis of
derivatives 13-22 is outlined in Scheme 1. The key step
was the coupling between benzoheterocyclic carboxylic
acids 23-32 bearing the alkylating moieties and N-
deformyldistamycin (33), obtained from distamycin A
according to a reported procedure.1a For the preparation
of compound 34, the acid 31 was coupled with the
known amino amidine 35.8
The condensations of the acylating agents 23-32 with
deformyldistamycin (33) and of the acid 31 with 35 were

Benzoheterocyclic Derivatives of Distamycin A
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2677
Sch em e 2a
incorporating both the phenyl and the vinylic double
bond of the cinnamic moiety confers rigidity to the
alkylating region of the molecule.
a
Reagents: (a) 40, Et3N, CH2Cl2, 18 h, rt; (b) KOH, water/
dioxane, 3 h, rt.
The compounds 13-22 and 34 were also tested in vivo
against L1210 murine leukemia cells, and some repre-
sentative molecules (18 and 21) were also tested on
M5076 murine reticulosarcoma. The results obtained
are summarized in Table 1.
Sch em e 3a
Derivatives 13-15 containing the BAM as alkylating
moiety showed significant cytotoxicity against L1210
cell line with IC50 values comparable to that reported
for tallimustine (2) but lower than those reported for
both its pyrrole 63 and pyrazole 96 homologues. Deriva-
tives 13-15 were at least 30-fold more cytotoxic than
the benzoimidazole derivative 16, which showed anti-
proliferative activity similar to that reported for the
imidazole distamycin analogue 11.6 The poor activity
of 16 may be related to a different DNA recognition
sequence, due to the possible role of the N(3) lone pair
of the benzoimidazole ring17 which may be capable to
accommodate hydrogen bonding with the guanine 2-ami-
no group and thus provide for GC selectivity. This could
alter the strict preference for AT sequence allowing GC
recognition and then mixed AT/GC sequences.
Compounds 13-15 not only exhibited good activity
in in vitro experiments but also showed a significant
increase in survival time (%T/C) of mice bearing the
lymphocytic leukemia model (L1210). Derivatives 14
and 15 were 8- and 2-fold less potent than tallimustine
(optimal nontoxic dose (OD): OD ) 25 mg/kg for 14,
6.25 mg/kg for 15 vs OD ) 3.13 mg/kg for tallimustine),
while 13 showed a potency similar to that of tallimus-
tine, with a median survival time slightly higher (%T/C
) 163 for 13 vs %T/C ) 133 for tallimustine). In this
series of BAM tetrapeptides 13-15, the compound 14,
which was the less potent in vivo, possessed a %T/C
value comparable to that of tallimustine (%T/C ) 183
for 13 vs %T/C ) 133), while 15 showed a lower value
(%T/C ) 150). Moreover, compounds 13-15 were 2-4-
fold less cytotoxic than the pyrrole 6 and pyrazole 9
counterparts.
a
Reagents: (a) ethylene oxide; (b) POCl3, toluene, rt, 1 h; (c)
37% HCl in water, rt, 3 h.
and 53 with carbonyldiimidazole (CDI) and then with
tert-butyl alcohol14 and 51 with tert-butyl bromide15
(Scheme 4). The amino compounds 57-59, obtained by
catalytic hydrogenation (10% Pd-C) of the correspond-
ing nitro tert-butyl esters 54-56, after treatment with
the R-bromoacrylic acid in the presence of 2 equiv of
EDC afforded 60-62, respectively. Subsequent treat-
ment with trifluoroacetic acid (TFA) at room tempera-
ture afforded the carboxylic acids 30-32.
Resu lts a n d Discu ssion
In Vitr o a n d in Vivo An titu m or Activity. The in
vitro cytotoxic activity of the synthesized compounds
13-22 and 34 was evaluated against the L1210 murine
leukemia cell line and in L1210 sublines resistant to
tallimustine (L1210/tallimustine) and to doxorubicin
(L1210/DX). The cytotoxic activity of the synthesized
compounds has been compared with that of distamycin
A (1), tallimustine (2), and the distamycin derivatives
3-12 and 64. This latter compound is a previously
reported cynnamoyl nitrogen mustard derivative16 of
distamycin A, a vinylogue of tallimustine. Compounds
17-19 should be considered as “conformationally con-
strained” forms of the cinnamic mustard derivative 64
of distamycin A, where the benzoheterocyclic unit
The nature of the alkylating group had a great effect
on the cytoxicity of compounds having the same oli-
gopeptide skeleton. In fact, the presence of a nitrogen
mustard moiety directly linked to the benzoheterocyclic
ring, as in the case of compounds 17-19, instead of a
nitrogen benzoyl mustard moiety, as in the case of

2678 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Baraldi et al.
Sch em e 4a
a
Reagents: (a) (CH3)3C-Br, K2CO3, BTEAC, DMF; (b) CDI, tert-butyl alcohol, DBU, DMF; (c) H2, Pd-C; (d) R-bromoacrylic acid,
EDCI, DMF; (e) CF3CO2H, rt, 1 h.
tives,18 compounds 20-22, bearing the R-bromoacryloyl
Ta ble 1. In Vivo and in Vitro Activity of Distamycin
Derivatives against L1210 Murine Leukemiaa
moiety, gave better results in terms of activity both in
in vivo L1210
vitro and in vivo, in comparison to the corresponding
derivatives 13-15, which possess the BAM function as
in vitro L1210
ip
iv
the alkylating moiety. In fact compounds 20-22 were
at least 10-fold more cytotoxic than 13-15 and, with
the exception of 22, showed %T/C values which were
higher than those reported for the benzoyl mustard
counterparts.
IC50
OD
OD
compd
(nM ( SE)
(mg/kg) %T/C (mg/kg) %T/C
distamycin A3
10069 ( 1647 200
68.5 ( 6.6
3.9 ( 0.6
79.6 ( 14
445 ( 75
19.0 ( 0.6
0.081 ( 0.01
6.3 ( 1.34
34.5 ( 4.6
13.3 ( 0.54
2372 ( 157
46.9 ( 13
95.3 ( 28
61.7 ( 2.49
43.5 ( 7
1422 ( 235
8.0 ( 2.6
14.7 ( 2.4
3.7 ( 1.5
4.1 ( 1.3
2.4 ( 0.39
6.1 ( 0.93
15.3 ( 4.31
9.5 ( 2.71
113
175
138
175
nd
nd
3.1
0.78
nd
nd
nd
0.39
1.6
nd
nd
nd
nd
3.1
25
6.2
nd
0.39
1.6
0.39
12.5
6.2
0.78
3.1
6.2
nd
133
117
nd
tallimustine (2)3
3.1
34
1.6
12.5
nd
0.4
0.39
3.1
3.1
6.2
6.2
6.2
43
53
nd
In the same series 20-22, although the indole deriva-
tive 20 showed the same cytotoxicity as the benzofuran
counterpart 22, the latter compound was 15-fold less
potent in vivo and produced an increased survival time
2-fold higher than that for 20 (OD ) 0.78 mg/kg, %T/C
) 117 for 22 vs OD ) 12.5 mg/kg, %T/C ) 213 for 20).
From the comparison of the compounds bearing the
nitrogen mustard or R-bromoacryloyl unit as the alky-
lating moiety, it was found that derivatives 20 and 21
were more cytotoxic, but less potent in vivo, than the
N-nitrogen mustard counterpart 17 and 18, since 22
was both less cytotoxic and potent than 19. The same
compounds 20-22 maintained cytotoxicity substantially
equivalent to that of the pyrrole 8 and pyrazole 10
analogues, but much higher with respect to the imida-
zole derivative 12.
63
138
188
725
144
200
125
163
nd
nd
74
133
200
nd
nd
nd
83
96
106
116
126
13
14
15
16
17
18
19
20
21
22
34
6416
nd
nd
163
183
150
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
174
457
200
213
192
117
133
267
a
IC50 ) 50% inhibitory concentration as the mean ( SE from
dose-response curves of at least three experiments; ip ) treatment
was performed intraperitoneally on day 1 after tumor ip trans-
plant; iv ) treatment was performed intravenously on day 1 after
tumor iv transplant; OD ) optimal dose, optimal nontoxic dose <
LD10 (weight loss < 20% with respect to the starting weight); %T/C
) median survival time of treated vs untreated mice × 100; nd )
not determined.
A fairly marked correlation between antitumor activ-
ity and length of the polypyrrolic skeleton was observed
for compounds 21 and 34, where the derivative 34 with
two N-methylpyrrolic units was 5-fold less cytotoxic
than the corresponding pyrrole homologue 21. This is
in agreement with the hypothesis that DNA binding,
which depends on the multiplicity of interactions be-
tween the pyrrolecarbamoyl units and AT-rich se-
quences of DNA, is crucial for cytotoxicity.19 Derivative
34, despite a potency 2-fold higher than that of 21 (OD
) 6.25 vs 3.13 mg/kg), was slightly less effective than
the latter in vivo (T/C% ) 192 vs 133 for 21 and 34,
respectively).
compounds 13-16, proved to be effective in increasing
the cytotoxic activity by a factor of up to 10.
Especially noteworthy was the compound 18, which
although 2- or 4-fold less active in vitro (IC50 ) 14.7
nM for 18 vs IC50 ) 8.04 nM for 17, 3.75 nM for 19)
and 4-fold less potent in vivo with respect to 17 and 19
(OD ) 1.56 mg/kg for 18 vs OD ) 0.39 mg/kg both for
17 and 19), exhibited a %T/C of 457, which was almost
3 times higher than that of 17 (%T/C ) 174) and 19
(%T/C ) 200). Compounds 17-19, although 2 orders of
magnitude less cytotoxic than the nitrogen mustard
derivative 7 with four pyrrole units, showed superior
activity in vivo (%T/C ) 174, 457, and 200 for 17-19,
respectively, vs %T/C ) 133 for 7).
In a preliminary in vivo evaluation against the M5076
solid tumor, compound 21 was almost 4-fold less toxic
than 18 (OD ) 0.93 mg/kg for 18 vs OD ) 3.13 mg/kg
for 21), with an increased %T/C slightly lower (%T/C )
124 for 18 vs %T/C ) 154 for 21), while both compounds
showed the same trend of inhibition of tumor growth
(%TI ) 92 for 18 vs %TI ) 96 for 21).
In Table 2 the resistance index (RI) values of 1-22,
34, and 64 on leukemia cell lines resistant to doxoru-
As reported for other series of distamycin deriva-

Benzoheterocyclic Derivatives of Distamycin A
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2679
Ta ble 2. Cytotoxicity and Resistance Index (RI) of Distamycin
Derivatives against L1210 Leukemia Cells Resistant to
Doxorubicin (DX) and Tallimustinea
L1210/DX
L1210/tallimustine
IC50
(µM ( SE)
IC50
(µM ( SE)
compd
RI
RI
distamycin A3
459 ( 50.1
2.6 ( 0.2
45.6
38.5
nd
68.0
175.4
nd
nd
3.8
326.2
7
2
15.5
5.7
6.8
7.8
2.7
142 ( 13.4
0.55 ( 0.06
nd
12.4 ( 2.5
75.6 ( 42.3
1.24 ( 0.3
nd
0.17 ( 0.013
10.4 ( 1.2
0.55 ( 0.13
5 ( 0.082
0.65 ( 0.14
0.88 ( 0.039
1.2 ( 0.13
12.9 ( 0.58
nd
14.1
8
nd
tallimustine (2)3
34
nd
43
5.4 ( 1
78.1 ( 6.3
nd
156
53
169.9
65.4
nd
27.0
302.3
41.4
2.1
13.9
9.2
19
295.8
nd
11.6
5.3
22.8
34.9
64.8
33.8
10.1
63
74
nd
83
0.024 ( 0.0002
11.3 ( 1.2
0.093 ( 0.017
4.7 ( 1.2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
34
6416
0.73 ( 0.069
0.54 ( 0.039
0.42 ( 0.041
0.34 ( 0.01
3.8 ( 0.59
0.059 ( 0.0007
0.11 ( 0.01
0.07 ( 0.01
0.036 ( 0.0056
0.043 ( 0.015
0.039 ( 0.002
0.041 ( 0.0009
0.13 ( 0.10
7.3 0.093 ( 0.00021
7.4 0.078 ( 0.002
18.7 0.085 ( 0.008
8.7
18
6.4
2.7
17.6
0.14 ( 0.03
0.15 ( 0.036
0.21 ( 0.019
0.15 ( 0.076
0.11 ( 0.023
8.00
a
IC50 ) 50% inhibitory concentration as the mean ( SE from
dose-response curves of at least three experiments; RI (resistance
index) ) ratio between IC50 values on resistant cells and sensitive
cells.
bicin (Dx) and tallimustine are reported. The results
obtained show that, for the newly synthesized com-
pounds 13-22 and 34, only 13, 17, 18, and 34 display
low RI values for leukemia cell lines resistant to
doxorubicin and tallimustine. For each new derivative,
with the exception of 18, which was more active in the
tallimustine-resistant L1210 leukemia line, it was
interesting to note that the RI values in L1210/talli-
mustine cells were at least 2-fold higher than that
reported in L1210/DX cells, showing that neither the
alkylating moiety nor the benzoheterocycle were in-
volved in the resistance mechanism.
It is also interesting to note that in the comparison
between compounds 13 and 14, 17 and 18, 20 and 21
which bear the same alkylating moieties, N-methylation
of the indole nucleus had an important effect only for
the %T/C value of 18. Finally, nitrogen mustard and
R-bromoacryloyl derivatives showed comparable cyto-
toxicity against L1210 murine leukemia cells, superior
to that reported for BAM derivatives with the same
oligopeptidic frame.
Ar r ested P olym er a se Ch a in Rea ction (P CR) a n d
Dir ect DNA F r a gm en ta tion Assa y. To determine the
influence of the alkylating group and the oligopeptide
skeletons on sequence selectivity, two series of deriva-
tives, 13-16 and 17-19, were compared, employing
molecular studies, such as arrested PCR and a direct
DNA fragmentation assay.
First, we determined the effect of compounds 13-16
and 17-19 on PCR-mediated amplification of two
genomic sequences: one rich in A+T, the other rich in
G+C. The human estrogen receptor (ER) gene (A+T/
G+C ) 3.46) and the human Ha-ras oncogene (A+T/
G+C ) 0.37) were chosen as model systems, as they are
suitable for use in determining sequence-selective bind-
F igu r e 1. Effects of distamycin and distamycin analogues
13-19 on PCR-mediated amplification of estrogen receptor
(ER; left side of the panel) and Ha-ras (right side of the panel)
genomic sequences. For PCR-mediated amplification the target
DNA was 20 ng of human genomic DNA or 2 ng of pBLCAT1-
ERCAT8; PCR buffer, Taq DNA polymerase, and the four
dNTPs were added as elsewhere described.21 Before PCR,
target DNA was incubated in the absence (-) or presence (a-
h) of DNA-binding compounds (a ) 1 µM, b ) 2 µM, c ) 5 µM,
d ) 10 µM, e ) 20 µM, f ) 50 µM, g )100 µM, h ) 200 µM) for
10 min. Conditions of Ha-ras PCR were: denaturation, 92 °C,
45 s; annealing, 62 °C, 45 s; elongation, 72 °C, 30 s (25 cycles).
Conditions of ER PCR were: denaturation, 92 °C, 45 s;
annealing, 55 °C, 1 min; elongation, 72 °C, 1 min (25 cycles).
Amplified DNA was analyzed by electrophoresis on 2.5%
agarose, 0.5 mg/mL ethidium bromide. Specific ER and Ha-
ras PCR products are arrowed. Specificity of ER and Ha-ras
PCR products was confirmed by Southern blotting and hy-
bridization to specific 32P-labeled probes as well as isolation
of the arrowed PCR products and direct sequencing (ref 22
and data not shown).
ing of DNA-binding compounds when amplified by
PCR.20,21 We have recently published the nucleotide
sequence of a 3.2-kb genomic region located upstream
of the estrogen receptor sequence22 originally designated
exon-1 and demonstrated that this region contains A+T-
rich sequences recognized by distamycin A and dista-
mycin analogues.23 The human Ha-ras oncogene se-
quence, on the other hand, is G+C-rich and therefore
interacts with low efficiency with distamycin, being, on
the contrary, efficiently recognized by G+C-selective
binders, such as chromomycin and mithramycin.20
Figure 1 shows a representative example of the PCR
experiment performed using distamycin A (1) and
distamycin analogues; Table 3 shows all the results
obtained, expressed as the inhibitory activity (IC50) of

2680 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Baraldi et al.
Ta ble 3. Inhibitory Activity (IC50) of Distamycin Derivatives
on the Generation of ER and Ha-ras PCR Products
sponding to compounds 17-19 shows that a different
pattern of fragmentation is generated after the direct
DNA fragmentation assay (see the fragments corre-
sponding to the site 5′-TATGTACGTGTGC-3′), suggest-
ing differences of binding activity of compounds 17-
19. Further experiments employing other genomic
experimental systems exhibiting similar GT-rich nucle-
otide sequences will be necessary in order to determine
whether this observation could be generalized. In any
case, it should be emphasized that major arrested sites
of PCRs (Figure 2) extensively overlap with the nucle-
otide sequences corresponding to the fragments gener-
ated when the direct DNA fragmentation assay was
performed (Figure 3).
a
a
PCR IC50 (µM)
PCR IC50 (µM)
compd
ER
Ha-ras
compd
ER
Ha-ras
distamycin A
20
7.5
5
200
15
15
16
17
18
19
10
1
1
50
20
20
15
13
14
15
3
20
3
a
Inhibitory concentration (mM) necessary to obtain 50% inhibi-
tion of generation of PCR products of human Ha-ras and ER
sequences.
distamycin derivatives and the generation of ER and
Ha-ras PCR products.
In agreement with already published reports,20,21
distamycin A (1) inhibits ER PCR (Figure 1, left side of
the panel) but exhibits lower capacity to inhibit Ha-ras
PCR (Figure 1, right side of the panel), thus confirming
a selective binding of distamycin to A+T-rich gene
sequences. IC50 of distamycin was found to be 20 µM in
the case of ER PCR and over 200 µM in the case of Ha-
ras PCR. By contrast, compounds 13-15 and 17-19 are
effective inhibitors of both ER and Ha-ras PCR (Figure
1), maintaining however sequence selectivity for ER
A+T-rich gene sequences (see Table 3 for semiquanti-
tative analysis). These data suggest that sequence-
selective binding typical of distamycin is to some extent
maintained by these compounds. As reported in Table
3, when compounds 13-15 are compared to the corre-
sponding 17-19, no major differences were found with
respect to inhibitory activity on the generation of Ha-
ras PCR products. By contrast, compounds 17-19 were
found to be consistently more active than the corre-
sponding 13-15 in inhibiting the generation of ER PCR
products (Figure 1 and Table 3).
To further investigate sequence selectivity of com-
pounds 13-16 and 17-19, arrested PCR fragments
generated using a 32P-labeled ER PCR primer were
analyzed by gel electrophoresis and the sites of arrest
identified. The results obtained are shown in Figure 2
and demonstrate that the sequence selectivity of com-
pounds 13-16 and 17-19 is readily observed. The
nucleotide sequences of the sites of arrest are also shown
in Table 4.
Compounds 15 and 19 were the most active com-
pounds (inhibition of generation of full-length PCR
product was obtained at 2 µM concentration). The
analysis of the nucleotide sequences corresponding to
the sites of arrest of the PCR demonstrate that the
arrested sites (5′-AGTTAAAA-3′ and 5′-TAAT-3′) were
found when the arrested PCR was performed in the
presence of all the compounds tested. The arrested site
5′-TTTAACTT-3′ was preferentially found in the pres-
ence of compounds 14 and 15, also being clearly detect-
able in the presence of compounds 13 and 17-19. The
arrested site 5′-ATGTGTGTGTGTA-3′ was preferen-
tially found in the presence of compounds 18 and 19.
A first conclusion gathered from the data shown in
Figure 2 is that both alkylating groups and oligopeptide
skeletons play a crucial role in determining the sequence
selectivity of these compounds. Direct DNA fragmenta-
tion assays (Figure 3) confirmed these conclusions. In
particular, it should be noted that compounds 17-19
are more active in this assay than compounds 13-16.
In addition, comparative analysis of the lanes corre-
Furthermore, it should be noted that a good relation-
ship exists between activity in the arrested PCR assay
(Table 3) and in vitro cytotoxicity (Table 1). For instance,
compounds 17-19 were found to be more active than
compounds 13-15 in both the in vitro cytotoxicity tests
and the arrested PCR assays. Accordingly, compound
19 was found to be more active than 17 and 18 in both
the in vitro cytotoxicity tests and the arrested PCR and
direct DNA fragmentation assays. Compound 16 was
consistently found to be less active than compounds 13-
15.
Con clu sion s
All the novel compounds, with the exception of
compound 16, showed in vitro activity against L1210
murine leukemia cell line comparable to or better than
that of tallimustine. Compounds in which the nitrogen
mustard and the R-bromoacryloyl moieties are directly
linked to the benzoheterocyclic ring showed very high
equipotent activities, ranging from 2 to 14 nM, while
benzoyl nitrogen mustard derivatives of benzohetero-
cycles showed lower activities, with one compound (16)
of this cluster being the only derivative devoid of
significant activity. Despite the fact that in vivo anti-
leukemic activity was found to be generally poorly
correlated with cytotoxicity, the exceptional antileuke-
mic activity of 18 is noteworthy. Therefore, compound
18 has been selected for further extensive evaluation
on murine solid tumors and human xenograft. The data
obtained in arrested PCR and direct DNA fragmentation
assays suggest that both the alkylating groups and the
oligopeptide frames play a crucial role in determining
the sequence selectivity of these compounds. In conclu-
sion, the results presented in this paper suggest that
the synthesis of various derivatives of distamycin A may
be a useful approach to cancer chemotherapy, leading
to the production of structurally related compounds
exhibiting significant antitumor activity.
Exp er im en ta l Section
Ch em ica l Ma ter ia ls a n d Meth od s. Gen er a l P r oced u r e.
All reactions were carried out under argon atmosphere, unless
otherwise described. Standard syringe techniques were applied
for transferring anhydrous solvents. Reaction courses and
product mixtures were routinely monitored by thin-layer
chromatography on silica gel (precoated F254 Merk plates), the
spots were examined with UV light and visualized with
1
aqueous KMnO4. H NMR spectra were recorded in the given
solvent with a Bruker AC 200 spectrometer. Chemical shifts
are reported as δ values in ppm. The splitting pattern
abbreviations are as follows: s (singlet), d (doublet), dd (double
doublet), t (triplet), br (broad), and m (multiplet). Melting

Benzoheterocyclic Derivatives of Distamycin A
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2681
F igu r e 2. Arrested PCR: analysis of arrested sites. In this experiment, ER target DNA was prepared by PCR. After incubation
in the absence (-) or presence of (a) 0.5, (b) 2, and (c) 10 µM DNA-binding compounds, a further 10 cycles of PCR were performed
with a 32P-labeled ER reverse PCR primer. After PCR, the samples were heated at 92 °C for 5 min and layered onto a sequencing
gel. The nucleotide sequences of the sites of PCR arrest are indicated. Asterisks indicate full-sized PCR products. At these compound
concentrations distamycin A generates no appreciable sites of arrest of ER PCR (data not shown).
Ta ble 4. Arrested PCR: Sequence Recognition by Distamycin
Derivatives
which was precipitated from EtOAc/petroleum ether and used
without purification for the next step.
Gen er a l P r oced u r e B for th e Syn th esis of BAM Ester s
41-44. A solution of p-[N,N-bis(2-chloroethyl)amino]benzoyl
chloride (40) (310 mg, 1.1 mmol) in anhydrous CH2Cl2 (5 mL)
was added dropwise in small portions to a mixture of amino
ester 36-39 (1 mmol) and triethylamine (0.14 mL, 1 mmol)
in anhydrous CH2Cl2 (5 mL) cooled to 0 °C. The reaction
mixture was stirred at room temperature for 18 h and
concentrated under reduced pressure yielding a brown solid,
which was dissolved in EtOAc (20 mL) and washed with 2 N
hydrochloric acid (2 × 10 mL). The organic layer was dried
(Na2SO4) and concentrated and the resulting residue precipi-
tated from EtOAc/petroleum ether to give 41-44.
Gen er a l P r oced u r e C for t h e Syn t h esis of 23-26. A
well-stirred solution of 41-44 (1 mmol) in dioxane (4 mL) was
treated with 2 N KOH in water (1 mL) and stirred at room
temperature for 3 h. The clear solution was evaporated to
remove dioxane, diluited with water (5 mL), cooled on an ice-
water bath and acidified with 2 N hydrochloric acid to pH 2.
The aqueous suspension was extracted with EtOAc (2 × 10
mL) and the organic layers were combined, dried (Na2SO4) and
concentrated. The resulting residue was precipitated from
EtOAc-hexane to give the product 23-26.
compd
18 and 19
17
17-19
13-15 and 17-19
13-19
13-19
nucleotide sequence
5′-ATGTGTGTGTGTA-3′
5′-TGTGTATGT-3′
5′-CGTGTGC-3′
5′-TTTAACTT-3′
5′-TAATT-3′
5′-CAGTTAAAA-3′
points (mp) were determined using a Buchi-Tottoli apparatus
and are uncorrected. All products reported showed 1H NMR
spectra in agreement with the assigned structures. Mass
spectra were recorded on a Nermag R10,10C spectrometer.
Elemental analyses, conducted by the Microanalytical Labora-
tory, Chemistry Department, University of Ferrara, were
within 0.4% of the theorical values calculated for C, H, Br, Cl,
and N. Column chromatography was carried out 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
magnesium turnings, dioxane was distilled from calcium
hydride, and anhydrous DMF was distilled from calcium
chloride and stored over molecular sieves (3 Å). In high-
pressure hydrogenation experiments, a Parr shaker on a high-
pressure autoclave was used.
Gen er a l P r oced u r e D for th e Syn th esis of In ter m ed i-
a tes 45-47. To a solution of amino ester 36-38 (1 mmol) in
MeOH (10 mL) cooled at -10 °C was added cold ethylene oxide
(2.5 mL). The reaction flask was sealed and allowed to reach
room temperature overnight. Methanol and excess of ethylene
oxide were removed by evaporation and the crude residue
purified by flash chromatography on silica gel.
Gen er a l P r oced u r e A for th e Syn th esis of 38 a n d 39.
A solution of the appropriate nitro ester (2 mmol) in 10 mL of
a mixture of MeOH/dioxane (1:1, v/v) was hydrogenated over
50 mg of 10% Pd/C at 60 psi for 5 h. The catalyst was removed
by filtration; the filtrate was concentrated to give a green oil

2682 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Baraldi et al.
F igu r e 3. DNA cross-linking assay. In this experiment a 32P-labeled ER PCR product was produced using a 32P-labeled ER
reverse PCR primer. After production of the ER PCR probe, an aliquot was incubated in 50 µL in the absence (-) or presence of
(a) 0.5, (b) 2, and (c) 10 µM DNA-binding compounds. After 5 h incubation at room temperature the samples were heated at 90
°C for 30 min, ethanol-precipitated, and analyzed by electrophoresis on a sequencing gel. The nucleotide sequences corresponding
to DNA fragmentation are indicated. Asterisks indicate full-sized PCR products.
Gen er a l P r oced u r e E for th e Syn th esis of th e Nitr o-
gen Mu sta r d Ester s 48-50. The compound 45-47 (1 mmol)
was cooled in an ice bath and 2 mL of phosphorus oxychloride
(20 mmol, 2 mL) was added dropwise. The solution was heated
at 100 °C for 1 h, the solvent evaporated under vacum, and
then the residue dissolved in EtOAc (8 mL) and washed with
water (3 mL). The organic phase was dried (Na2SO4) and
concentrated and the crude product purified by flash chroma-
tography on silica gel.
Gen er a l P r oced u r e F for th e Syn th esis of Nitr ogen
Mu sta r d Ca r boxylic Acid s 27-29. The nitrogen mustard
48-50 (1 mmol) was dissolved in 36% hydrochloric acid (4 mL)
and heated at 100 °C for 3 h. The solution was cooled at room
temperature, diluted with water (10 mL) and extracted with
EtOAc (2 × 20 mL). The recombined organic phases were dried
(Na2SO4) and evaporated in vacuo and the residue was purified
by flash chromatography on silica gel.
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, was dissolved in EtOAc and precipitated with
an excess of petroleum ether.
Gen er a l P r oced u r e I for th e Syn th esis of Br om oa cr yl-
a m id o ter t-Bu tyl Ester s 60-62. To a solution of R-bro-
moacrylic acid (600 mg, 4 mmol) in dry DMF (5 mL) was added
a solution of EDC (383 mg, 2 mmol) in dry DMF (5 mL) slowly.
After 1 h, the solution obtained was added to a solution of
amino tert-butyl ester 57-59 (2 mmol) in dry DMF (5 mL).
The reaction was stirred at room temperature for 18 h and
then concentrated under reduced pressure. The residue was
dissolved with a mixture of EtOAc (10 mL) and water (5 mL),
the organic phase dried over Na2SO4 and evaporated to
dryness in vacuo, and the resulting crude residue purified by
flash chromatography.
Gen er a l P r oced u r e G for th e Syn th esis of Nitr o ter t-
Bu tyl Ester s 55 a n d 56. A solution of nitro acid 52-53 (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, tert-
butyl alcohol (1 mL) and DBU (0.9 mL) were added. The
mixture was stirred for 2 h and poured in cooled water (50
mL) and the resulting crystalline solid collected and washed
with water, affording the desired nitro tert-butyl ester 55-
56.
Gen er a l P r oced u r e H for th e Red u ction of Nitr o ter t-
Bu tyl Ester s 54 a n d 56. The nitro tert-butyl ester 54-56 (5
mmol) was reduced over 10% Pd/C (100 mg) in a mixture of
dioxane (10 mL) and methanol (10 mL) at room temperature
Gen er a l P r oced u r e J for th e Syn th esis of 30-32. A
solution of the ester 60-62 (1 mmol) in CF3COOH (1 mL) was
stirred at room temperature for 3 h. The solvent was removed
under reduced pressure and the resulting residue was sus-
pended in CH2Cl2 (3 mL), collected by filtration and triturated
with ethyl ether (5 mL) to obtain 30-32.
Gen er a l P r oced u r e K for th e Syn th esis of Com p ou n d s
13-22. A solution of N-deformyldistamycin A dihydrochloride
(33) (263 mg, 0.5 mmol) in dry DMF (5 mL) was cooled to 5
°C and N,N′-diisopropylethylamine (86 µL, 0.5 mmol) was
added. After 10 min, the acid 23-32 (0.6 mmol, 1.2 equiv) and
then EDC (192 mg, 1 mmol) were added. The reaction mixture
was stirred at room temperature for 18 h, then 2 N hydro-

Benzoheterocyclic Derivatives of Distamycin A
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2683
chloric 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.
(genomic DNA, recombinant plasmid, PCR product). For PCR-
mediated amplification the target DNA was 20 ng of genomic
DNA or 2 ng of pBLCAT1-ERCAT8; PCR buffer, Taq DNA
polymerase and the four dNTPs were added as elsewhere
described.21 Conditions of PCRs were: denaturation, 92 °C, 1
min; annealing, 55 °C (ER) or 62 °C (Ha-ras), 1 min; elonga-
tion, 72 °C, 1 min (25 cycles). The effects of DNA-binding drugs
were analyzed after incubating target DNA at room temper-
ature, for 5 min, with increasing amounts of the compounds
followed by PCR. Amplified DNA was analyzed by electro-
phoresis on 2% agarose gels. For sequence analysis of arrested
PCR we first prepared ER target DNA by PCR. After incuba-
tion for 1 h at 37 °C with the distamycin analogues, samples
were heated at 90 °C for 5 min and PCR was performed using
the 32P-labeled ER reverse PCR primer (5′-GCAGAAT-
CAAATATCCAGATG-3′). After PCR, each reaction was re-
suspended in 5 µL of loading dye (0.1% xylene-cyanol, 0.1%
bromophenol blue, 0.1 M NaOH:formamide 1:2) and electro-
phoresed through a sequencing gel as described.21
Dir ect DNA F r a gm en ta tion Assa y. A 32P-labeled ER PCR
product was produced using a 32P-labeled ER reverse PCR
primer (5′-GCAGAATCAAATATCCAGATG-3′). After produc-
tion of the ER PCR probe, an aliquot was incubated in 50 µL
of 0.1 × SSC in the presence of DNA-binding drugs. After 5-h
incubation at room temperature the samples were heated at
90 °C for 30 min and ethanol was precipitated. Each reaction
was resuspended in 5 µL of loading dye (0.1% xylene-cyanol,
0.1% bromophenol blue, 0.1 M NaOH:formamide 1:2) and
electrophoresed through a sequencing gel as described.21
Biologica l Testin g in Vitr o Usin g L1210 Cells. The
murine lymphocytic L1210 leukemia cell line was obtained
from the American Type Culture Collection (ATCC). All the
tested compounds were dissolved in DMSO at 1 mg/mL
immediately before use and diluted in medium before addition
to the cells. The murine lymphocytic leukemia cells L1210,
L1210/DX and L1210/tallimustine were grown in vitro as a
stationary suspension culture in RPMI 1640 medium (GIBCO)
supplemented with 10% fetal calf serum (Flow, Irvine, U.K.),
2 mM L-glutamine (GIBCO), 10 mM â-mercaptoethanol, 100
U/mL penicillin and 100 µg/mL streptomycin. To determine
survival after compound exposure, exponentially growing
L1210 cells were continuously exposed to various concentra-
tions of compounds for 48 h, after which the cytotoxic activity
of the compounds was evaluated by counting surviving cells
using an electronic cell counter ZBI (Coulter Counter Electron-
ics, Hialeah, FL). The cytotoxic activity of the compounds was
calculated from dose-response curves and expressed as IC50
(concentration of test compound to reduce the cell number to
50% of that obtained with untreated control cells). All experi-
ments were repeated at least twice. For each compound
concentration, duplicate cultures were used. Vehicle or solvent
controls were run with each experiment.
Biologica l Tests in Vivo. DBA/2N female mice were used
for implanting with the murine L1210 leukemia. For experi-
ments with leukemia CD2F1 female mice were used. C57B16
female mice were used for implanting with the murine
reticulosarcoma M5076. Charles River Italia (Calco, Como,
Italy) supplied all mice. Mice were 8-17 weeks and weighed
20-22 g at the time of tumor implantation. Animal health was
monitored by serological testing; the animals were free of
infectious pathogens, including mouse hepatitis virus, Sendai
virus and Mycoplasma pulmonis, during the course of experi-
mentation. All compound solutions were prepared immediately
before use and given intravenously (iv) in a volume of 10 mL/
kg of body weight. The vehicle used in preparation of solutions
consisted of 10% Tween 80 and 90% saline.
L1210 murine leukemia (originally obtained from the Na-
tional Cancer Institute, Frederick, MD) was maintained in vivo
by continuous ip passage (106 cells/mouse), for experiments
105 cells/mouse, 10 mice/group, were injected ip or iv. Com-
pounds were administered iv or ip at day 1 after tumor cell
injections. A dose-response was determined in all experi-
ments. Toxicity was evaluated on the basis of the gross autopsy
findings and the weight loss, mainly in terms of reduction of
spleen and liver size.
M5076 reticulosarcoma was maintained in vivo by im serial
transplantation. For experiments, 5 × 105 cells were injected
im in the flank of C57B16 female mice. Animals were 8-10
weeks old at the beginning of the experiments. Compounds
were administered iv at day 3, 7, and 11 after tumor implanta-
tion. Survival time of mice and tumor growth were calculated
and activity was expressed in terms of %T/C and %TI. %T/C
) median survival time treated group vs median survival time
untreated group × 100. The survival time of control mice
injected iv with L1210 is 6 days, while for ip injected mice it
is 8-10 days. %TI ) % inhibition of tumor growth with respect
to control. Compounds were considered active if the %T/C
value was g125.
Ack n ow led gm en t. We thank Pharmacia & Upjohn
and Ministero Universita` e Ricerca Scientifica (MURST)
(60%) for generous financial support of this work. R.G.
is supported by ISS (AIDS-98), PRIN-98, P.F. Biotec-
nologie, and Progetto per la Ricerca Finalizzata 1998
(Ministero della Sanita`). We thank Dr. Alessia Felloni
for performing the arrested PCR experiments reported
herein.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and 1H NMR spectra for compounds 13-32, 34, and
38-62. This material is available free of charge via the
Internet at http://pubs.acs.org.
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