Synthesis and preliminary cytotoxicity of nitrogen mustard derivatives of distamycin A

Article abstract of DOI:10.1016/S0960-894X(02)00986-1

Distamycin and nitrogen mustard conjugates, in which the nitrogen mustard unit was coupled to the C-terminus of the pyrrole, were synthesized. The switching of the nitrogen mustard unit from the N-terminus to the C-terminus did not compromise the compound's cytotoxicity. Compound 3, bearing three pyrrole units, was highly toxic to human K562 leukemia cells in vitro with an IC₅₀ value of 0.03 μM. Addition of a trans double bond to the molecule had little effects on cytotoxicity.

Full text of DOI:10.1016/S0960-894X(02)00986-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 459–461
Synthesis and Preliminary Cytotoxicity of Nitrogen
Mustard Derivatives of Distamycin A
Yuqiang Wang,* Susan C. Wright and James W. Larrick
Panorama Research, Inc., 2462 Wyandotte Street, Mountain View, CA 94043, USA
Received 31 January 2002;accepted 24 October 2002
Abstract—Distamycin and nitrogen mustard conjugates, in which the nitrogen mustard unit was coupled to the C-terminus of the
pyrrole, were synthesized. The switching of the nitrogen mustard unit from the N-terminus to the C-terminus did not compromise
the compound’s cytotoxicity. Compound 3, bearing three pyrrole units, was highly toxic to human K562 leukemia cells in vitro with
an IC50 value of 0.03 mM. Addition of a trans double bond to the molecule had little effects on cytotoxicity.
# 2002 Elsevier Science Ltd. All rights reserved.
Toxic side effects associated with chemotherapy are a
major problem in the treatment of neoplastic diseases.
Therefore, a great deal of research has been focused on
the development of more specific therapeutic strategies
to reduce toxicity to normal cells. Nitrogen mustards like
cyclophosphamide, chlorambucil, and melphalan are
widely used antitumor drugs. These drugs mainly alky-
late guanine-N7 in the major groove of DNA and cause
interstrand DNA cross-link, which is believed to be
responsible for the antitumor activity.1 However, these
drugs also alkylate other bases of DNA and cause DNA
single strand breaks and DNA–protein cross-linking.2
It is reasoned that attaching a chemical reactive group
such as a nitrogen mustard to a DNA sequence-specific
binder such as distamycin will result in a molecule that
can alkylate DNA in a sequence-specific manner, and
thus reduce undesired side effects.8
In all distamycin nitrogen mustard derivatives reported,
the nitrogen mustard moiety has been conjugated
through the 4-amino of the pyrrole, leaving the mole-
cule with a positively charged amidino, guanidino or a
ternary amino group at the C-terminus. We now want
to determine if placing a nitrogen mustard moiety at the
C-terminus of the pyrrole will affect the antitumor
activity. Thus, a series of novel distamycin nitrogen
mustard derivatives was designed (Fig. 2).
DNA minor groove binding compounds are a new class
of anticancer agents, which binds to DNA with a high
sequence-specificity.3,4 This high sequence-selective
binding of DNA in the minor groove has been exploited
to develop drugs to increase their therapeutic efficacy.3,4
Distamycin A is a natural antibiotic, which binds to
AT-rich regions of B-DNA with high affinity.5 Con-
jugates of distamycin and nitrogen mustard have been
studied extensively recently, and some compounds of
this class have significant antitumor activity in experi-
mental animals.3,4,6,7 For example, tallimustine, a ben-
zoic acid nitrogen mustard derivative of distamycin, had
been in Phase II clinical trials.8 PUN 166196, a bro-
moacrylic derivative of distamycin, is currently under
clinical investigations (Fig. 1).7
In these new molecules, a nitrogen mustard is placed at
the C-terminus of the pyrrole and an n-butyramido
group was at the N-terminus. Previously, Wang et al.
have found that CC-1065 analogues bearing an
n-butyramido substituent at C4-position of the DNA-
binding pyrrole unit increased antitumor activity in
vitro.9,10 For this reason, all targeted compounds are
incorporated with a C4-butyramido substituted pyrrole.
CC-1065 analogues bearing a CPI (1,2,8,8a-tetrahydro-
7-methyl-cyclopropa[c]pyrrolo[3,2-e]indol-4-(5H)-one)
subunit with a trans double bond have been found to
have greatly increased antitumor activity in vitro com-
pared to those without a trans double bond.9,10 Dis-
tamycin derivatives with a cinnamic nitrogen mustard
appear to be more potent than those with a benzoic
*Corresponding author.
yuqiangwang@worldnet.att.net
Fax:
+1-650-694-7717;e-mail:
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00986-1

460
Y. Wang et al. / Bioorg. Med. Chem. Lett. 13(2003) 459–461
Scheme 1.
Figure 1. Structures of distamycin A, tallimustine, and PNU 166196.
Figure 2. Structures of new distamycin nitrogen mustards.
nitrogen mustard.7 For these reasons, we choose to
incorporate a trans double bond in some of the new
compounds (4 and 5) to increase their potency. It has
been shown that an increase in the number of pyrrole
units results in an increase of DNA binding, which in
turn leads to an increase in antitumor activity.3,4,7
Therefore, the antitumor activity of the new compounds
is expected to be 3 >2 >1. We herein report the synth-
esis and cytotoxicity of the new distamycin nitrogen
derivatives.
Scheme 2. (a) EDCI;(b) CH 3SO2Cl, (C2H5)3N;(c) LiCl, 31% yield
from 9 to 13;(d) H /Pd/C, 100% yield;(e) 7, EDCI, 55% yield.
2
Acid 99 was coupled to amino 1013 in the presence of
EDCI to produce 11. The latter was treated with
methanesulfonyl chloride to generate the mesyl deriva-
tive 12, which was converted to 13 by treatment with
lithium chloride. The nitro group of 13 was reduced by
hydrogenation affording 14. The latter was then coupled
to 7 in the presence of EDCI to generate target 3.
Compounds 4 and 5 were synthesized by coupling acid
15 with amines 8 and 14, respectively, in the presence of
EDCI (Scheme 3).
Chemistry11
Compounds 6 and 7 were synthesized as described
previously,9 and were coupled to 4-amino-N, N-bis(2-
chloroethyl)aniline (8)12 in the presence of 1-(3-dime-
thylaminopropyl)-3-ethylcarbodiimide (EDCI) to afford
1 and 2, respectively (Scheme 1). When the same strat-
egy was used to synthesize 3, the yield was very low
(<20%). Thus, alternatively, 3 was also synthesized as
shown in Scheme 2.
Scheme 3. (a) 8 or 14, EDCI, 48% yield for 4, and 37% for 5.

Y. Wang et al. / Bioorg. Med. Chem. Lett. 13(2003) 459–461
Table 1. Cytotoxicity against K562 leukemia cells in vitroa
461
References and Notes
Compd
IC50 (mM)b
1. Rajski, S. R.;Williams, R. M. Chem. Rev. 1998, 98, 2723.
2. Farmer, P. B. Pharmacol. Ther. 1987, 35, 301.
3. Reddy, B. S.;Sharma, S. K.;Lown, J. W.
Chem. 2001, 8, 475 and references therein.
4. Denny, W. A. Curr. Med. Chem. 2001, 8, 533 and refer-
ences therein.
5. Dervan, P. B. Science 1986, 232, 464.
1
2
3
4
0.95
0.25
0.03
1.2
0.21
35
Curr. Med.
5
Chlorambucil
6. D’Incalci, M.;Sessa, C. Exp. Opin. Invest. Drugs 1997, 6,
875 and references therein.
aThe assay was set up in triplicate in 96-well flat-bottom microtiter
plates. All cells were seeded at 5000 cells/well in RPMI-1640 plus 10%
FCS. Drugs were added, and the total volume was adjusted to 0.2 mL/
well. Total incubation time was 48 h with the addition of 3H-thymidine
for the last 24 h of incubation. The assay was harvested and radio-
activity was counted.
7. Cozzi, P. Il Farmaco 2000, 55, 168 and references therein.
8. Arcamone, F. M.;Animati, F.;Barbieri, B.;Configliacchi, E.;
D’Alessio, R.;Giuliani, F. C.;Lazzari, E.;Menozzi, M.;Mon-
gelli, N.;Penco, S.;Verini, M. A. J. Med. Chem. 1989, 32, 774.
9. Wang, Y.;Gupta, R.;Huang, L.;Luo, W.;Lown, J. W.
Anti-Cancer Drug Des. 1996, 11, 15.
bIC50 values are defined as the minimal drug concentration necessary
to inhibit incorporation of [3H] thymidine by 50%, and are the avera-
ges of three experiments.
10. Fregeau, N. L.;Wang, Y.;Pon, R. T.;Wylie, W. A.;
Lown, J. W. J. Am. Chem. Soc. 1995, 117, 8917.
11. The tested compounds were purified by silica gel column
chromatography, eluting with a mixture of ethyl acetate and
hexane. The compounds were characterized by elemental ana-
Results and Discussion
1
lysis with satisfactory results. Their H NMR and MS spectra
The antitumor activity of the new compounds was
determined against human chronic leukemia K562
cells in vitro, and the results were shown in Table 1.
As expected, compound 3 (IC50: 0.03 mM), bearing
were in agreement with the assigned structures. 1NMR
(DMSO-d6) data of 1–5 and MS spectra are given. (1) 9.69 (s,
1H, NH), 9.54 (s, 1H, NH), 7.51 (d, 2H, C6H4, J=9.2 Hz),
7.15 (d, 1H, Py–H, J=1.8 Hz), 6.88 (d, 1H, Py–H, J=1.8 Hz),
6.72 (d, 2H, C6H4, J=9.2 Hz), 3.80 (s, 3H, NCH3), 3.70 (brs,
8H, CH2CH2Cl). 2.20 (t, 2H, CH2CH2CH3, J=7.4 Hz), 1.62–
1.56 (m, 2H, CH2CH2CH3), 0.95 (t, 3H, CH2CH2CH3,
J=7.3 Hz). EIHRMS calcd for C20H26Cl2N4O2 424.1433,
found 424.1432. (2) 9.86 (s, 1H, NH), 9.72 (s, 1H, NH), 9.61
(s, 1H, NH), 7.53 (d, 2H, C6H4, J=9.2 Hz), 7.25 (d, 1H, Py–
H, J=1.4 Hz), 7.15 (d, 1H, Py–H, J=1.9 Hz), 7.05 (d, 1H, Py-
H, J=1.8 Hz), 6.88 (d, 1H, Py–H, J=1.8 Hz), 6.72 (d, 2H,
C6H4, J=9.1 Hz), 3.83 (s, 3H, NCH3), 3.71 (brs, 8H,
CH2CH2Cl). 2.21 (t, 2H, CH2CH2CH3, J=7.4 Hz), 1.62–1.56
(m, 2H, CH2CH2CH3), 0.90 (t, 3H, CH2CH2CH3, J=7.3 Hz).
FABHRMS calcd for C26H32Cl2N6O3 546.1913, found
546.1903. (3) 9.86 (s, 1H, NH), 9.82 (s, 1H, NH), 9.67 (s, 1H,
NH), 9.58 (s, 1H, NH), 7.53 (d, 2H, C6H4, J=9.2 Hz), 7.24 (s,
1H, Py–H), 7.22 (s, 1H, Py–H), 7.14 (s, 1H, Py–H), 7.05 (m,
2H, Py–H), 6.88 (s, 1H, Py–H), 6.73 (d, 2H, C6H4, J=9.2 Hz),
3.86–3.83 (m, 9H, NCH3), 3.71 (brs, 8H, CH2CH2Cl). 2.20 (t,
2H, CH2CH2CH3, J=7.8 Hz), 1.60–1.58 (m, 2H,
CH2CH2CH3), 0.90 (t, 3H, CH2CH2CH3, J=7.3 Hz).
FABHRMS calcd for C32H38Cl2N8O4 668.2393, found
668.2360. (4) 9.69 (s, 1H, NH), 9.66 (s, 1H, NH), 7.54 (d, 2H,
C6H4, J=8.4 Hz), 7.38 (d, 1H, CH¼CH, J=15.5 Hz), 6.72 (d,
2H, C6H4, J=8.9 Hz), 6.46 (s, 1H, Py–H), 6.38 (d, 1H, Py–H,
J=15.5 Hz), 3.70 (brs, 8H, CH2CH2Cl). 3.64 (s, 3H, NCH3),
2.19 (t, 2H, CH2CH2CH3, J=7.4 Hz), 1.62–1.55 (m, 2H,
CH2CH2CH3), 0.87 (t, 3H, CH2CH2CH3, J=7.3Hz).
FABHRMS calcd for C22H29Cl2N4O2 451.1668, found
451.1675. (5) 9.88 (s, 1H, NH), 9.67 (s, 1H, NH), 9.58 (s, 1H,
NH), 7.54 (d, 2H, C6H4, J=8.8Hz), 7.36 (d, 1H, CH¼CH,
J=15.5Hz), 7.28 (s, 1H, Py–H), 7.17 (d, 1H, Py–H, J=1.5Hz),
6.91 (d, 1H, Py–H, J=1.8 Hz), 6.73 (d, 2H, C6H4, J=9.2Hz),
6.47 (s, 1H, Py–H), 6.37 (d, 1H, Py–H, J=15.5Hz), 3.83 (s, 3H,
NCH3), 3.71 (brs, 8H, CH2CH2Cl). 3.64 (s, 3H, NCH3), 2.19 (t,
2H, CH2CH2CH3, J=7.4Hz), 1.59–1.55 (m, 2H, CH2CH2CH3),
0.89 (t, 3H, CH2CH2CH3, J=7.3 Hz). FABHRMS calcd for
C28H34Cl2N6O3 572.2069, found 572.2069.
three pyrroles, is more potent than
2
(IC50:
0.25 mM), bearing two pyrroles, which is more potent
than 1 (IC50: 0.95 mM), bearing one pyrrole. Gen-
erally, for distamycin nitrogen mustards, the potency
of the compound will increase approximately 10-fold
with the addition of one pyrrole unit up to a total
of four pyrroles. Interestingly, it appears that
switching the nitrogen mustard unit from the N-ter-
minus to the C-terminus of the pyrrole did not
compromise the compound’s cytotoxicity. For exam-
ple, the cytotoxicity of most distamycin nitrogen
mustard conjugates is in the range of high mM to
middle nM.7
In contrast to the finding with distamycin cinnamic
nitrogen mustard derivatives, in which, a trans double
bond appears to increase potency,7 the addition of a
trans double bond in the new molecules, 4 and 5,
had little effect on cytotoxicity. For example, the
cytotoxicity of 1 and 4 is almost the same (IC50 value
of 0.95 and 1.2 mM). There is little difference between
the potency of 2 and 5 (IC50 value of 0.25 and
0.21 mM).
Conclusion
For the first time, distamycin and nitrogen mustard
conjugates, in which the nitrogen mustard unit was
coupled to the C-terminus of the pyrrole, were syn-
thesized. The switching of the nitrogen mustard unit
from the N-terminus to the C-terminus did not com-
promise the compound’s cytotoxicity. Compound 3,
bearing three pyrrole units, was highly toxic to
human K562 leukemia cells in vitro. Addition of a
trans double bond to the molecule had little effect on
cytotoxicity.
12. Palmer, B. D.;Wilson, W. R.;Pullen, S. M.;Denny, W. A.
J. Med. Chem. 1990, 33, 112.
13. Gourdie, T. A.;Valu, K. K.;Gravatt, G. L.;Boritzki,
T. J.;Baguley, B. C.;Wakelin, L. P. G.;Wilson, W. R.;
Woodgate, P. D.;Denny, W. A. J. Med. Chem. 1990, 33, 1177.