DNA cleavage by aromatic amines

Article abstract of DOI:10.1016/S0960-894X(01)00688-6

A series of aryl amines was found to induce cleavage of DNA. Subsequent refinement led to an efficient family of dimeric derivatives capable of cleavage at low concentration. Initial investigations suggest this is an unprecedented mode of DNA cleavage, wh

Full text of DOI:10.1016/S0960-894X(01)00688-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1–4
DNA Cleavage by Aromatic Amines
Philip M. Warner,* Jiwei Qi, Bin Meng, Gang Li, Longfei Xie,
Ahmed El-Shafey and Graham B. Jones
Department of Chemistry, Northeastern University, 360 Huntington Ave., Boston, MA 02115, USA
Received 22 January 2001; accepted 11 October 2001
Abstract—A series of aryl amines was found to induce cleavage of DNA. Subsequent refinement led to an efficient family of dimeric
derivatives capable of cleavage at low concentration. Initial investigations suggest this is an unprecedented mode of DNA cleavage,
which may be ultimately applied to the development of sequence-specific agents. # 2001 Elsevier Science Ltd. All rights reserved.
DNA cleavage by natural or designed molecules has
long been recognized asa potentially valuable way to
affect cell proliferation, forming the basis of a number
of effective cancer chemotherapeutics.1 Hydrolysis of
the phosphate linkage of and electrophilic alkylation of
its base pairs are two of the most obvious approaches
for DNA degradation. Another paradigm, popularized
by minor groove bindersincluding the enediyne anti-
biotics, and intercalators such as the anthraquinones, is
to initiate cleavage via attack on the deoxyribose moi-
ety. The former case involves attack by carbon-cen-
tered, non-diffusible radicals,2 whereasthe latter
involvesthe production of oxygen-centered radicals
(e.g., HOꢀ).3 We have shown that bisdiazonium ions
structurally similar to the d(A/T) specific minor groove
binding drug NSC-101327 can cleave supercoiled DNA
at low concentrations.4 These cleavages were initiated
either thermally or photochemically. In the former case,
it was advantageous, although not necessary, to add
metal ions. In light of the discoveries of both Dervan
and Lown that dimeric derivativesof ditsamycin were
effective asminor groove binders[e.g., (2-PyN) 2-C3
5
and the bis-Lexitropsins6] we have also synthesized
dimeric aminesthat can be converted to dimeric dia-
zonium ions; these also proved to be effective cleavers.7
Prompted by this discovery, we wished to undertake a
systematic study of such triarylamide-bridged amines,
since (i) they are readily prepared, in contrast to com-
parable distamycin type templates and (ii) variation of
functionality can be conducted at ease. Our synthetic
route to second generation analogues called for sys-
tematic variation of aryl substituents, to enable us to
study the relative contribution of electron modulating
functionality to the activity of the diazonium ions.
Accordingly, triaryl amides 2a–c were produced, using
sequential coupling methods (Scheme 1).8
The original plan called for diazotization of 2 (isoamyl
nitrite, acetic acid, 0 ꢁC) and subsequent investigation
of DNA cleaving ability. Though thisindeed proved
*Corresponding author. Fax: +1-617-373-8795; e-mail: pwarner@lynx.neu.edu
0960-894X/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00688-6

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P. M. Warner et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1–4
Scheme 1. Synthesis of substituted triarylamides.
successful, to our surprise, during our model studies it
wasin fact revealed that 2a–c themselves were capable
of unwinding ÈXI74 DNA, resulting in substantial
conversion to RfII form within 12 h at 37 ꢁC. Pre-
liminary analysis showed that the nature of the appen-
dage Y influenced the efficiency of the cleavage, with the
apparent trend 2a>2c>2b warranting further investi-
gation (Table 1, entries 2–5). Prolonged exposure resul-
ted in conversion to RfIII DNA, and subsequent
degradation. With these intriguing data in hand, we
elected to follow our initial plan in the form of dimeric
structures related to the (2-PyN)2-C3 and bis-Lexi-
tropsin class, using modifications of the existing route
(Scheme 2). The aryl amines 5 proved an order of mag-
nitude more active at DNA cleavage, and showed some
selectivity within the series, particularly on extended
exposure (Table 1, entries 6–11). As had been observed
by Dervan and Lown, the nature of the tether length
between the two binding entitiescan be expected to play
a role in the binding and therefore cleavage events.
Accordingly, based on 5a, further analogueswere pre-
pared with variable tether lengths, using variations on
the synthetic procedures adopted in Scheme 2. The
analogues, 6a–f all showed appreciable cleavage, but 6a
and 6c were most effective (Table 1, entries 12–17).
These observations are significant, suggesting that
designed amines are clearly capable of inducing efficient
DNA cleavage at certain concentrations.
With the results in hand, a series of mechanistic probes
was designed; firstly, it was determined that inhibition
of DNA cleavage by 6c wasonly effective using >1000-
fold excess of the minor groove binder distamycin A
9
(entries18–20).
Thisisin tsark contrats to earlier
Table 1. DNA cleavage induced by arylaminesa
Entry
Amine concn (mM)
Time (h)
Additive
RfI (%)
RfII (%)
RfIII (%)
1
2
3
4
5
6
7
8
—
—
12
12
12
12
48
24
24
24
48
48
48
24
24
24
24
24
48
24
24
24
12
12
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
90
1
44
11
0
35
36
34
7
10
0
10
99
56
89
75
65
64
66
93
90
99
100
76
63
65
62
99
75
35
5
0
0
0
0
25
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
2a
2b
2c
2a
5a
5b
5c
5a
5b
5c
6a
6c
6d
6e
6f
1.0
1.0
1.0
1.0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
0
24
37
35
38
1
25
65
95
27
26
6c
6c
6c
6c
6c
6c
—
10 mM DAb
100 mM DA
1000 mM DA
10 mMc M2+/3+
Ar/N2
73
74
aAll incubationsconducted with amines(6 mL at indicated concn) and ÈX174 bacteriophage DNA (6 mL of 0.14 mM) in Tris–acetate–EDTA buffer
in the absence of light at pH 8.2/37 ꢁC unless noted. Gels electrophoresed on agarose (0.7%) at 80 V/2.5 h, stained (ethidium bromide), then sub-
jected to scanning densitometry.
bDA, distamycin A.
c10 mM aliquotsof metal chloridesused.

P. M. Warner et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1–4
3
Scheme 2. Preparation of dimeric polyamines.
results obtained with simple diamines and diazonium
ions,4,7 and, though speculative, may point to an alter-
native mode of binding and recognition to that expected
for polyamidesof thisgeneral clas.
the process to specific DNA sequences and micro-
environments, together with kinetic analysis promises to
lead to new possibilities in the design of anti-
proliferativesand chemical nucleases.
One possibility might be the involvement of trace metal
cations, and to investigate this, analogous cleavage
experimentswere conducted in the presence of 100-fold
excess of Cd2+, Co2+, Cu2+, Fe3+, Hg2+, Ni2+, or
Zn2+salts. In all cases, using a 24 h incubation period,
no change (relative to control) wasobserved (entry 21).
We were also concerned regarding the possible partici-
pation of free radical chain reactionsin the cleavage
process. To address this issue, cleavage induced by 6c
was evaluated in the presence and absence of a 10-fold
excess of oxygen radical scavengers including KI,
DABCO, Tiron, and catalase. No change relative to
control wasobserved, nor wasa difference found when
cleavage wasconducted under simulated deoxygenated
conditions, implying that a radical species is not
involved (entry 22).
Acknowledgements
Thiswork wassupported by grantsfrom the NSF and
NIH.
References and Notes
1. Bailly, C. In Advances in DNA Sequence-Specific Agents;
Jones, G. B., Ed.; Jai: Greenwich, 1998; Vol. 3, p 97.
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J. M.; Plourde, G. W.; Hynd, G.; Huber, R. S.; Mathews, J. E.
J. Am. Chem. Soc. 2000, 121, 1937.
A remaining possibility is that some form of nucleo-
philic attack mechanism involving the arylamines is
responsible, and to investigate this further, cleavage was
examined over a wide pH range. Significantly, cleavage
induced by 6c wasmuted at pH >8.2, yet it was
enhanced at pH 7.3 (80% RfII/24 h) and pH 6.5 (90%
RfII/24 h), relative to control (entry 13). At still lower
pH, uncatalyzed cleavage competes, preventing accurate
controlsfrom being developed. However, it isevident
that when the aniline group(s) is partially protonated
(pKbꢂ9), cleavage isenhanced, which may imply osme
form of proton-transfer mechanism for the initiation of
cleavage. Thiswould contsitute a new mode of DNA
cleavage, and the structure–activity correlations observed
suggest a degree of control may be attainable. Ongoing
research, involving the use of restriction fragments, in
concert with in-depth binding analyses,10 and NMR stud-
ies are designed to shed further light on the process.6,11
3. (a) Boger, D. L.; Ramsey, T. M.; Stubbe, J. J. Am. Chem.
Soc. 1998, 120, 9139. (b) Hecht, S. M. Pure Appl. Chem. 1989,
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7. Qrya, D. P.; Devlin, T. A.; Jebaratnam, D.; Warner, P. M.
U.S. Patent 5,770,736.
8. Satisfactory spectroscopic and analytical data were
obtained for all new compounds; 6c: mp 129–130 ꢁC (dec); MS
(ESI) 789.3 (M+H), 789.2, 670.4, 416.0, 395.5, 305.3, 261.2,
217.1; 13C (DMSO-d6) 30.7, 38.6, 114.5, 116.3, 116.5, 116.6,
117.0, 118.4, 129.1, 129.9, 130.2, 133.2, 136.1, 137.0, 137.2,
An unprecedented method of DNA cleavage via readily
accessible arylamines has been revealed. Application of

4
P. M. Warner et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1–4
140.6, 140.9, 150.2, 167.1, 167.9, 168.1; C45H40N8O6 requires:
C, 68.52, H, 5.11; found: C, 68.88, H, 4.98.
[dG.C]=0.96Â105 MÀ1. Complete detailswill be published in
a full account of thiswork. (b) Pyle, A. M.; Rehmann, J. P.;
Meshoyrer, R.; Kumar, C. V.; Turro, N. J.; Barton, J. K. J.
Am. Chem. Soc. 1989, 111, 3051. (c) Kumar, C. V.; Asuncion,
E. H. J. Am. Chem. Soc. 1993, 115, 8547.
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U.S.A. 1989, 86, 5723. (b) Wade, W. S.; Mrksich, M.; Dervan,
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10. (a) Preliminary binding data,6 using poly [dA.T] and poly
[dG.C] substrates (fluorescence titration followed by Scatch-
ard analysis)10b confirm the candidate compoundsaffinity for
A/T sequences; for example 6a: K25 [dA.T]=4.9Â107 MÀ1
,