Cytotoxic α-bromoacrylic derivatives of distamycin analogues modified at the amidino moiety

Article abstract of DOI:10.1016/S0960-894X(00)00205-5

The design, synthesis, in vitro and in vivo activities of novel α-bromoacrylic derivatives of distamycin A, modified at the amidino moiety by the replacement with basic or non-basic groups are reported. In spite of the relevance of these modifications of distamycin frame, the new derivatives are potent cytotoxics. The presence of the amidino moiety, is, therefore, not an absolute requirement for the activity. In particular due to a favorable myelotoxicity/cytotoxicity ratio, guanidino derivative PNU 166196 was selected for clinical development. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00205-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1273±1276
Cytotoxic ꢀ-Bromoacrylic Derivatives of Distamycin Analogues
Modi®ed at the Amidino Moiety
Paolo Cozzi, a,* Italo Beria, a Marina Caldarelli, a Cristina Geroni, b
Nicola Mongelli a and Giulia Pennella b
aDepartment of Chemistry, Pharmacia & Upjohn, Discovery Research Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy
bDepartment of Pharmacology, Pharmacia & Upjohn, Discovery Research Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy
Received 4 February 2000; accepted 31 March 2000
AbstractÐThe design, synthesis, in vitro and in vivo activities of novel a-bromoacrylic derivatives of distamycin A, modi®ed at the
amidino moiety by the replacement with basic or non-basic groups are reported. In spite of the relevance of these modi®cations of
distamycin frame, the new derivatives are potent cytotoxics. The presence of the amidino moiety, is, therefore, not an absolute
requirement for the activity. In particular due to a favorable myelotoxicity/cytotoxicity ratio, guanidino derivative PNU 166196
was selected for clinical development. # 2000 Elsevier Science Ltd. All rights reserved.
In recent years several papers dealing with distamycin A
and distamycin-like derivatives as DNA minor groove
binders have been published, however, although many of
these papers have investigated the DNA binding-role of the
oligopyrrolic frame,1 little attention has been paid to the
possible role of the strong basic amidino moiety. This
moiety is typical not only of distamycin A, but also of
other DNA minor groove binders, such as netropsin
and synthetic diarylamidine derivatives as e.g., DAPI,
berenil and pentamidine, which share with distamycin A
a strong selectivity for TA-rich sequences.2 The strong
basic nature of the amidino group, which leads to its
total protonation at any physiological pH, may a€ect
both DNA binding and bioavailability.
reported and showed signi®cant cytotoxicity and in vivo
antileukemic activity.6 PNU 151807, at variance with
tallimustine, was found to bind but not to alkylate DNA
minor groove AT-rich sequences, and it was hypothe-
sized as the lead of a class of minor groove binders
mechanistically di€erent from tallimustine and congener
mustards.7
This hypothesis was strengthened by the results descri-
bed in a previous paper8 in which we reported a series of
halogenoacrylic derivatives of distamycin A and con-
geners prepared with the aim of de®ning the role of the
halogenoacrylic moiety.
We now report the synthesis, in vitro and in vivo activities
of novel a-bromoacrylic derivatives of four pyrrole units
homologues of distamycin A modi®ed at the amidino
moiety (Formula I).
Although distamycin or lexitropsin nitrogen mustard
derivatives, in which the propionamidino moiety was
replaced by aminoalkyl and anilino groups were descri-
bed,3,4 no systematic modi®cation of the amidino moi-
ety was undertaken until we recently reported benzoyl
and cinnamoyl nitrogen mustard derivatives of dis-
tamycin A, in which the amidino moiety was modi®ed
or replaced by groups, basic and non basic, of di€erent
nature.5
The modi®cations include the replacement of the amidino
moiety of the parent PNU 151807 with basic amidino-
derived analogues of di€erent lipophilicity and bulk and
with non-basic groups of di€erent nature.
Recently the a-bromoacrylamido derivative of four
pyrrole homologue of distamycin A, PNU 151807, was
Chemistry
The novel derivatives reported in Table 19 were
obtained by coupling a-bromoacrylamido-pyrrole-
carboxylic acid, via corresponding acid chloride with
desformyldistamycin derivatives appropriately modi®ed
*Corresponding author. Tel.: +39-02-4838-5350; fax: +39-02-4838-
3833; e-mail: paolo.cozzi@eu.pnu.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00205-5

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P. Cozzi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1273±1276
Formula 1.
Table 1.
Intermediate nitrile was prepared in 75% yield, reacting
distamycin A with succinic anhydride and K2CO3 in
DMF, following an original procedure recently published
by us (Scheme 4).10
Compound
B
In vitroa
IC50
In Vivo
OD
T/C%
1b
2
3
4
5
6
7
8
6.31Æ1.34
2.67Æ0.78
1.86Æ0.18
1.48Æ0.84
2.46Æ0.52
1.85Æ0.17
8.58Æ1.55
4.13Æ0.27
9.00Æ2.43
18.1Æ2.27
1.56
3.13
1.56
n.d.
n.d.
1.56
6.25
3.13
6.25
12.5
200
150
258
n.d.
n.d.
196
186
157
169
157
.
C(NH)NH2 HCl
.
C(NCH3)NH2 HCl
.
C(NCH3)NHCH3 HCl
.
C(NH)N(CH3)2 HCl
.
C-Imidazolin-2-yl HCl
.
NHC(NH)NH2 HCl
Intermediate guanidino derivative was prepared, di€er-
ently from other intermediates, by a step-by-step total
synthesis from guanidinoethylamine, by iterative acylation
with 4-nitro-pyrrolecarboxylic acid chloride and catalytic
reduction of the nitro group, as previously reported5
(Scheme 5).
C(NOH)NH2
C(NCN)NH2
CONH2
9
10
CN
aIC50=50% inhibitory concentration as the meanÆS.E. from dose±
response curves of at least two experiments, determined after 48 h of con-
tinuous exposure against L1210 cells. For in vivo studies cells were injected
iv at day 0 and mice were treated iv the day after tumor injection;
O.D.=optimal (non-toxic) dose <LD10. %T/C=median survival time of
treated versus untreated miceÂ100. L1210 murine leukemia cell lines were
obtained from NCI, Bethesda, USA.
Results and Discussion
Tested compounds were assayed in vitro and in vivo on
L1210 murine leukemia cells, evaluating cytotoxicity
and antileukemic activity as previously described.11
bCompound 1=PNU151807.
Table 1 data clearly show that the potent cytotoxicity of
the parent amidino derivative PNU 151807 (1) is fully
maintained not only by basic amidino-derived compounds
of di€erent lipophilicity and bulk, such as N-methylami-
dine (2), N,N0-dimethylamidine (3), N,N-dimethylamidine
(4), 2-imidazoline (5), and by guanidine derivative (6), but
also by non-basic amidino-derived compounds such as
amidoxime (7) and cyanoamidine (8), and even by non-
basic, non-amidino-derived amide 9. Only nitrile (10)
shows a modest decrease of activity. Alkylamidino and
guanidino derivatives appear even more potent cyto-
toxics than the parent amidino compound. These data
not only indicate that the amidino moiety is not an
at the amidino moiety, with yields ranging from 30 to
70% (Scheme 1).
Latter intermediates in the case of amidino-derived
compounds were easily obtained from distamycin A and
a suitable amine derivative, in DMF at 70 ꢀC, with
yields ranging from 30 to 80% (Scheme 2).
Intermediate amide was prepared in 90% yield by alka-
line hydrolysis in CH3CN/H2O at 70 ꢀC, of the amidino
moiety of distamycin A (Scheme 3).
Scheme 1.
Scheme 2.

P. Cozzi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1273±1276
1275
Scheme 3.
Scheme 4.
Scheme 5.
absolute requirement for activity, but also a lack of any
correlation between the basicity of the amidine-like
structure and cytotoxicity. This apparently contrasts
with the common opinion that electrostatic interaction
between the cationic moiety and the negatively charged
DNA phosphate residues represents one of the main
contributions to molecular recognition of distamycin
and distamycin-like derivatives.12
In particular the guanidino derivative 6, PNU 166196,
results 20-fold more active than tallimustine in inducing
apoptosis in A2780 human ovarian carcinoma cells,13
circumvents in vitro and in vivo resistance to alkylating
agents and topo I inhibitors and it shows an out-
standing myelotoxicity/cytotoxicity ratio, being its
mean IC50 against a series of tumor cells about eighty
times lower than its IC50 on human CFU-GM hemato-
poietic progenitors cells.14
A lack of correlation between the basicity of the ami-
dine-replacing moiety and cytotoxicity was already
demonstrated by us in the case of nitrogen mustard
derivatives of distamycin A.5 However, the high cyto-
toxic activity of the compounds of this series appears
particularly relevant and occurs also in the case of
modi®cations such as those represented by amidoxime
(7) and amide (9), which led in the mustard series to a
decrease of activity particularly relevant in the case of
amidoxime.5
This compounds, as the parent PNU 151807, appears
unreactive in DNA alkylation assays.14 All these facts
underline the novelty of PNU 166196 for both the pro-
®le of activity and the mechanism of action. PNU
166196 is presently undergoing Phase I Clinic.
References and Notes
1. See e.g., Pindur, U.; Fischer, G. Curr. Med. Chem. 1996, 3,
379 and references therein.
Also in vivo antileukemic activities appear equivalent
to, or even better than, that of the parent PNU 151807.
Some of these compounds, which appear signi®cantly
more cytotoxic than tallimustine and show a favorable
myelotoxicity/cytotoxicity ratio, have been selected for
further extensive evaluation in vitro and on murine solid
tumors and human xenografts.
2. See e.g., Zunino, F.; Animati, F.; Capranico G. Current
Pharmaceutical Design 1995, 1, 83 and references therein.
3. See e.g., (a) Lee, M.; Rhodes, A.L.; Wyatt, M.; Forrow, S.;
Hartley, J.A. Anticancer Drug Design 1993, 8, 173, (b) Wyatt,
M.D.; Garbiras, B.J.; Haskell, M.K.; Lee, M.; Souhami, R.L.;
Hartley, J.A., Anticancer Drug Design 1994, 9, 511, (c) Wyatt,
M.D.; Lee, M.; Hartley, J. A. Anticancer Drug Design 1997, 12, 49.

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4. Brooks, N.; Hartley, J. A.; Simpson, J. E.; Wright, S. R.;
Woo, S.; Centioni, S.; Fontaine, M. D.; McIntyre, T. E.; Lee,
M. Biorg. Med. Chem. 1997, 5, 1497.
2H); 3.00 (s, 3H); 2.79 (s, 3H); 2.73 (m, 2H). (4): 9.0 (b.s., 1H);
8.31 (t, J=5.8 Hz, 1H); 3.46 (m, 2H) 3,22 (s, 3H); 3.03 (b.s.,
3H), 2.77 (t, J=6.5 Hz, 2H). (6): 8.10 (t, J=5.9 Hz, 1H); 7.56
(t, J=5.9, 1H); 7.2 (b.s., 4H); 3.30 (m, 4H). (7): 9.5 (b.s., 2H);
8.5 (b.s., 1H); 7.98 (t, J=5.9 Hz, 1H); 3.38 (m, 2H); 2,31 (m,
2H).(8): 8.3 (b.s., 2H); 8.1 (b.s., 1H); 3.4 (b.s., 2H); 2,6 (b.s.,
2H). (9): 7.94 (t, J=5.9 Hz, 1H); 6.8 (b.s., 2H); 3.33 (m, 2H);
2.30 (t, J=7.2 Hz, 2H). (10): 8.36 (t, J=5.9 Hz, 1H); 3,42 (m,
2H); 2.75 (t, J=6.5 Hz, 2H).
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9. Tested compounds were puri®ed by silica gel column chro-
matography (eluant CH2Cl2:CH3OH: 80:20) and gave satis-
1
factory analytical values and H NMR spectra in agreement
1
with assigned structures. representative H NMR data of the
12. See e.g. Lown, J. W. Chemtracts-Organic Chemistry 1993,
6, 205.
compounds (DMSO-d6) are given. (Bruker AC200 spectro-
meter, d in ppm, TMS as internal standard). (1): 7.94 (t,
J=5.9 Hz, 1H); 7.3 (b.s., 1H); 6.8 (b.s., 1H); 3.3 (m, 2H); 2.30
(t, J=7.2 Hz, 2H). (2): 5 (b.s., 1H); 9.1 (b.s., 1H); 8.5 (b.s.,
1H); 8.22 (t, J=5.9 Hz, 1H); 3.48 (m, 2H); 2.79 (s, 3H); 2.62
(m, 2H). (3): 9.2 (b.s., 2H); 8.33 (t, J=6.0 Hz, 1H); 3.44 (m,
13. Ciomei, M.; Pastori, W.; Cozzi, P.; Geroni, C. Proceedings
of AACR, Philadelphia 1999, 40 Abst.1490, 224.
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Rossi, R.; Farao, M.; Marchini, S.; Cozzi, P. Proceedings of
AACR, San Francisco, 2000, in press.