Phenyl sulfur mustard derivatives of distamycin A

Article abstract of DOI:10.1016/S0960-894X(00)00295-X

The design, synthesis, and cytotoxic activity of novel benzoyl and cinnamoyl sulfur mustard derivatives of distamycin A are described and structure-activity relationships are discussed. These sulfur mustards are more potent cytotoxics than corresponding nitrogen mustards in spite of the lower alkylating power, while their sulfoxide analogues are substantially inactive. Cinnamoyl sulfur mustard derivative (7) proved to be one of the most active distamycin-derived cytotoxics, about 1000 times more potent than melphalan. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00295-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1653±1656
Phenyl Sulfur Mustard Derivatives of Distamycin A
Paolo Cozzi,a,* Italo Beria,a Marina Caldarelli,a Laura Capolongo,b
Cristina Geroni,b Stefania Mazzinic and Enzio Raggc
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
cDepartment of Molecular Sciences, Faculty of Agriculture, University of Milan Milan, Italy
Received 2 February 2000; accepted 18 May 2000
AbstractÐThe design, synthesis, and cytotoxic activity of novel benzoyl and cinnamoyl sulfur mustard derivatives of distamycin A
are described and structure±activity relationships are discussed. These sulfur mustards are more potent cytotoxics than corre-
sponding nitrogen mustards in spite of the lower alkylating power, while their sulfoxide analogues are substantially inactive. Cin-
namoyl sulfur mustard derivative (7) proved to be one of the most active distamycin-derived cytotoxics, about 1000 times more
potent than melphalan. # 2000 Elsevier Science Ltd. All rights reserved.
DNA minor groove binders represent a class of antitumor
agents whose DNA sequence speci®city may lead to a
high selectivity of action.1 The main representatives of
this class which reached the clinic are the antitumor
agents derived from CC-1065, i.e., adozelesin, carzelesin
and bizelesin,2 and tallimustine (1).3
which proved to be signi®cantly more cytotoxic than
tallimustine.7 We synthesised also the ethyl-chloroethyl
half-mustard analogue (3) of tallimustine and its half-
mustard cinnamic congener (4), which showed cytotoxi-
cities substantially equivalent to, or better than those of
the corresponding two arm nitrogen mustard derivatives
1 and 2.7 This underlines the mechanistic diversity of
these compounds from classical nitrogen half-mustards
which are substantially non cytotoxic,8 possibly due to
the impossibility of crosslinking the two DNA strands.9
The latter is a benzoyl nitrogen mustard derivative of
non-cytotoxic distamycin A4 which exhibits a high ecacy
against a variety of murine tumors and human xeno-
grafts. Tallimustine was shown to bind to the minor groove
AT-rich sequences, and in contrast with conventional
nitrogen mustards, to alkylate at adenine N(3) sites with
no evidence of guanine N(7) alkylation.5
The activity of distamycin half-mustards derivatives
prompted us to the synthesis of sulfur mustard analogues
of tallimustine and congeners, as one-arm alkylating
derivatives of minor groove binder distamycin A.
The rationale that led to the synthesis of tallimustine was
to tether to the distamycin frame, which plays the role of
minor groove binding ligand, a potentially very mild
alkylating moiety represented by the benzoic acid para-
nitrogen mustard (BAM). The aim was to avoid, as
much as possible, unspeci®c alkylation of both intra and
extracellular biological nucleophiles. The weak alkylating
power of BAM,6 is caused by the poor electronic avail-
ability of the aniline-type nitrogen, further decreased by
aromatic conjugation with the para carbonyl group.
A further motivation for the synthesis of phenyl sulfur
mustard derivatives of distamycin was represented by
the foreseen low reactivity of the thiiranium (sulfonium)
cation, which should be the alkylating intermediate
of sulfur mustards.10 The low reactivities is caused by
the aromatic conjugation with the electron withdrawing
carbonyl or vinylcarbonyl function. These sulfur
mustards should ful®l the rationale already followed in
the case of tallimustine, with the aim to avoid aspeci®c
reactivity as much as possible.
Recently we synthesised the cinnamic nitrogen mustard
derivative of distamycin (2), i.e., a vinylogue of tallimustine,
In spite of the fact that the traditional de®nition of mus-
tard, applied to cytotoxic N,N-bis-haloethyl derivatives,
arises from the nickname of mustard gas given to bis-
dichloroethyl-sul®de S-(CH2CH2Cl)2, a chemical weapon
used in the course of ®rst world war, the sulfur mustards
*Corresponding author. Fax: +39-02-4838-5351; e-mail: paolo.cozzi@
eu.pnu.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00295-X

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P. C. Cozzi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1653±1656
have found only a limited attention as therapeutic
cytotoxics.11 This was due to the fact that the tethering
of a haloethyl sulfur moiety to an organic template gives
rise to a one-arm alkylating compound with the same
limits cited for nitrogen half-mustards, i.e., the impossi-
bility of crosslinking the two DNA strands. Bifunctionality
of sulfur mustards is apparently required for cytotoxicity,
and sulfur mustards in which the two 2-chloroethyl sub-
stituents are on di€erent sulfur atoms were investigated to
meet this requirement.12
Chemistry
The sulfur mustard derivatives of distamycin A and
reference nitrogen mustard derivatives are reported in
the Table 1. Nitrogen mustard derivatives 1±5 were
synthesised as previously reported7 by coupling the
corresponding benzoic or cinnamic acid mustards with
desformyldistamycin. Novel sulfur mustards distamycin
derivatives and their sulfoxides13 were obtained analo-
gously from corresponding benzoic or cinnamic acid
sulfur mustards and their sulfoxides and desformyldis-
tamycin, by coupling with DCC and HBT in DMF
(yield=45±70%). Benzoic and cinnamic sulfur mustard
intermediates for 6 and 7, were prepared by the reaction of
corresponding 4-mercaptobenzoic and mercaptocinnamic
In this paper we report the synthesis, in vitro activities of
benzoyl and cinnamoyl sulfur mustard derivatives of dis-
tamycin A, 6 and 7, their sulfoxides 8 and 9, and we discuss
their structure±activity relationships also in comparison
with their nitrogen mustard counterparts.
Table 1.
Compounda
A
n
In vitro
IC50 (nM)
Kalkyl
M
Compounda
A
n
In vitro
IC50 (nM)
Kalkyl
M
1
1
1
1
s
s
4
4
1b
0
68.5Æ8.0
9.5Æ2.8
6.2Â10
1.3Â10
6
0
20.6Æ0.1
0.9Æ0.1
1.4Â10
5.6Â10
3
4
2
1
7
1
4
3
4
5
5
3
3
4
5
0
1
0
60.0Æ1.3
2.9Æ0.9
>600
5.5Â10
1.9Â10
1.0Â10
8
9
0
1
>600
>600
5.0Â10
6.2Â10
2.0Â10
10c
980.5Æ642.4
aAll reported compounds are hydrochloride salts.
bTallimustine.
cMelphalan. IC50=50% inhibitory concentration as the mean Æ SE from dose±response curves of at least two experiments, drug sensitivity was
determined after 48 h of continuous exposure against L1210 cells. L1210 murine leukaemia cell lines were obtained from NCI, Bethesda, USA.
k
alkyl=alkylation of 4-(4-nitrobenzyl)pyridine T=66 ꢀC. Nitrogen mustard derivatives 1±5 were described in ref 7.

P. C. Cozzi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1653±1656
1655
acid respectively, with 2-chloroethanol and NaOH at
25ꢀC (yield=90%), followed by chlorination with thionyl
chloride in toluene (yield=70%) in the case of the benzoic
derivative, or with mesyl chloride in pyridine (yield=60%)
in the case of cinnamic compound. Benzoic and cinnamic
sulfoxide mustard intermediates for 8 and 9 were obtained
by oxidation of corresponding 2-chloroethyl sulfur mus-
tards of benzoic and cinnamic acid with NaIO4 in
MeOH/H2O (yield=70%).
Moreover these novel sulfur mustards, compared with
their nitrogen counterparts, show a greater cytotoxicity
and a very low chemical reactivity. While their low
reactivity was hypothesised as a part of the design
rationale, their cytotoxic activity is beyond what could
be foreseen. Cinnamoyl sulfur mustard PNU 193821
proved to be the most potent distamycin-derived cytotoxic
found in our laboratories (IC50=0.9nM against L1210
leukaemia cells, to be compared with IC50=68.5nM for
tallimustine and IC50=980.5nM for melphalan).
Studies aimed to investigate the possible interaction of
these derivatives with DNA oligonucleotides containing
the T4GA consensus sequence identi®ed for tallimustine
have been planned, in order to verify possible mechanistic
analogies or di€erences with the latter, and will be the
subject of a future paper.
Results and Discussion
All tested compounds were assayed in vitro on L1210
murine leukaemia cells, evaluating cytotoxicity as pre-
viously described.14 The chemical reactivity of the mustard
moieties were evaluated by determining the rate of
alkylation of 4-(4-nitrobenzyl) pyridine (NBP) following
classical procedures.15
References and Notes
These sulfur mustard derivatives show a cytotoxicity/
reactivity ratio more favourable than the corresponding
two and one-arm nitrogen mustards (6 versus 1, 3, 5 and
7 versus 2, 4). The NBP alkylation rate of benzoyl sulfur
mustard 6 is about four times lower than that of corre-
sponding nitrogen mustards 1 and 3 and only little
greater than that of nitrogen half-mustard 5, however
its cytotoxicity is about three times greater than that of
1 and 3 and about 30 times greater than that of 5. Cin-
namoyl sulfur mustard 7, PNU 193821, which shows a
chemical reactivity equivalent to that of tallimustine and
its half-mustard 3, is more than 60 times more potent
than the latter. Compound 7 is about three times less
reactive, but about 1000 times more cytotoxic, against
L1210 leukaemia cells, than the classical nitrogen mustard
melphalan (10).
1. Zunino, F.; Animati, F.; Capranico, G. Curr. Pharmaceut.
Des. 1995, 1, 83.
2. See e.g. Aristo€, P. A. In Advances in Medicinal Chemistry,
JAI Press Inc. 1993; Vol. 2, pp 67±110 and references therein.
3. Pezzoni, G.; Grandi, M.; Biasoli, G.; Capolongo, L.; Balli-
nari, D.; Giuliani, F. C.; Barbieri, B.; Pastori, A.; Pesenti, E.;
Mongelli, N.; Sprea®co, F. Br. J. Cancer 1991, 64, 1047.
4. Arcamone, F.; Penco, S.; Orezzi, P. G.; Nicolella, V.; Pir-
elli, A. Nature 1964, 203, 1064.
5. Broggini, M.; Coley, H. M.; Mongelli, N.; Pesenti, E.;
Wyatt, M. D.; Hartley, J. A.; D' Incalci, M. Nucleic Acid
Research 1995, 23, 81.
6. Bardos, T. J.; Datta-Gupta, N.; Hebborn, P.; Triggle, D. J.
J. Med. Chem. 1965, 8, 167.
7. Cozzi, P.; Beria, I.; Biasoli, G.; Caldarelli, M.; Capolongo,
L.; D'Alessio, R.; Geroni, C.; Mazzini, S.; Ragg, E.; Rossi, C.;
Mongelli, N. Bioorg. Med. Chem. Lett. 1997, 7, 2985.
8. Brendel, M.; Ruhland, A. Mutation Res. 1984, 133, 51.
9. Sunters, A.; Springer, C. J.; Bagshave, K. D.; Souhami, R.
L.; Hartley, J. A. Biochem. Pharmacol. 1992, 44, 59.
10. See e.g. Ross, W. C. The Chemistry of Cytotoxic Alkylat-
ing Agents, In Advances Cancer Res. 1953, 1, 397.
11. See e.g., Chemistry of Antitumor Drugs, Remers W.A. In
Antineoplastic Agents, Remers W. A. Ed.; John Wiley, 1984.
12. See e.g. Davis, W.; Ross, W. C. J. Med. Chem. 1965, 8
757.
Noteworthy, the corresponding sulfoxide derivatives 8
and 9 of sulfur mustards 6 and 7 show a poor alkylating
power versus NBP, about one order of magnitude lower
than that shown by sulfur mustards and tallimustine.
This suggests that the formation of a thiiranium cation,
which is no more possible in the case of sulfoxides, may
play a key role in the alkylation mechanism of sulfur
mustards towards NBP. More relevant is the substantial
lack of cytotoxicity of sulfoxide derivatives 8 and 9,
which suggests that the formation of a thiiranium cation
is possibly also the basis of the mechanism leading to
cytotoxicity.
13. Tested compounds were puri®ed by silica gel column
chromatography (eluant CH2Cl2:CH3OH:80:20) and gave
1
satisfactory analytical values and H NMR spectra in agree-
1
ment with assigned structures. H NMR data of compounds
6±9 (DMSO-d6) are given. (Bruker AC200 spectrometer, d in
ppm, TMS as internal standard): (6) 10.34 (s, 1H), 9.98 (s,
1H), 9.92 (s, 1H), 8.9 (b.s., 2H), 8.6 (bs, 2H), 8.21 (t, J=5.6
Hz, 1H), 7.91 (m, 2H), 7.47 (m, 2H), 7.32 (d, J=1.7 Hz, 1H),
7.24 (d, J=1.7 Hz, 1H), 7.18 (d, J=1.7 Hz, 1H), 7.10 (d,
J=1.7 Hz, 1H), 7.06 (d, J=1.7 Hz, 1H), 6.95 (d, J=1.7 Hz,
1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.78 (t, J=7.3 Hz,
2H), 3.48 (m, 4H), 2.60 (t, J=6.5 Hz, 2H); (7): 10.23 (s, 1H),
9.96 (s, 1H), 9.91 (s, 1H), 8.9 (b.s., 2H), 8.6 (bs, 2H), 8.21 (t,
J=5.6 Hz, 1H), 7.55 (m, 2H), 7.46 (d, J=15.8 Hz, 1H), 7.41
(m, 2H), 7.30 (d, J=1.7 Hz, 1H), 7.23 (d, J=1.7 Hz, 1H), 7.17
(d, J=1.7 Hz, 1H), 7.01 (d, J=1.7 Hz, 1H), 6.96 (d, J=1.7
Hz, 1H), 6.95 (d, J=1.7 Hz, 1H), 6.77 (d, J=15.8 Hz, 1H),
3.85 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.76 (t, J=7.3 Hz, 2H),
3.49 (m, 2H), 3.40 (t, J=7.3 Hz, 2H), 2.60 (t, J=6.4 Hz, 2H);
(8): 10.56 (s, 1H), 10.00 (s, 1H), 9.92 (s, 1H), 9.0 (bs, 2H), 8.6
Conclusions
Benzoyl and cinnamoyl sulfur mustard derivatives of dis-
tamycin A described here represent a structural novelty
among reported mustards because bifunctionality of sulfur
mustards is apparently required for cytotoxicity. These
compounds con®rm the possibility, ®rst demonstrated
by nitrogen half-mustard derivatives of distamycin,7 of
achieving potent cytotoxic activity by means of one-arm
alkylating derivatives of the latter, i.e., when the reac-
tive one-arm mustard moiety is tethered to a sequence
selective minor groove binding ligand.

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P. C. Cozzi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1653±1656
(bs, 2H), 8.21 (t, J=5.6 Hz, 1H), 8.14 (m, 2H), 7.83 (m, 2H),
7.35 (d, J=1.7 Hz, 1H), 7.24 (d, J=1.7 Hz, 1H), 7.18 (d,
J=1.7 Hz, 1H), 7.12 (d, J=1.7 Hz, 1H), 7.06 (d, J=1.7 Hz,
1H), 6.94 (d, J=1.7 Hz, 1H), 4.0-3.8 (m, 2H), 3.87 (s, 3H),
3.84 (s, 3H), 3.80 (s, 3H), 3.49 (m, 2H), 3.46 (m, 1H), 3.26 (m,
1H), 2.61 (t, J=6.6 Hz, 2H); (9): 10.38 (s, 1H), 9.98 (s, 1H),
9.92 (s, 1H), 8.80 (bs, 4H), 8.22 (t, J=6 Hz, 1H), 7.80 (m, 2H),
7.74 (m, 2H), 7.55 (d, J=15.6 Hz, 1H), 7.32 (d, J=1.7 Hz,
1H), 7.24 (d, J=1.7 Hz, 1H), 7.18 (d, J=1.7 Hz, 1H), 7.06
(d, J=1.7 Hz, 1H), 6.98 (d, J=1.7 Hz, 1H), 6.94 (d, J=1.7
Hz, 1H), 6.93 (d, J=15.6 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H),
3.80 (s, 3H), 3.96±3.76 (m, 2H), 3.48 (m, 2H), 3.45±3.20 (m,
2H), 2.61 (t, J=6.5 Hz, 2H).
14. Geroni, C.; Pesenti, E.; Tagliabue, G.; Ballinari, D.;
Mongelli, N.; Broggini, M.; Erba, E.; D'Incalci, M. Cancer
Res. 1993, 53, 308.
15. Bardos, T. J.; Datta-Gupta, N.; Hebborn, P.; Triggle, D.
J. J. Med. Chem. 1965, 8, 167.