Biarylpyrimidines: a new class of ligand for high-order DNA recognition.

Article abstract of DOI:10.1039/b301554h

Biarylpyrimidines bearing omega-aminoalkyl substituents have been designed as ligands for high-order DNA structures: spectrophotometric, thermal and competition equilibrium dialysis assays showed that changing the functional group for substituent attachme

Full text of DOI:10.1039/b301554h

Biarylpyrimidines: a new class of ligand for high-order DNA
recognition†
Peter M. Murphy,a Victoria A. Phillips,a Sharon A. Jennings,ab Nichola C. Garbett,c Jonathan B.
Chaires,c Terence C. Jenkinsb and Richard T. Wheelhouse*a
a School of Pharmacy, University of Bradford, Bradford, West Yorkshire, UK BD7 1DP.
E-mail: r.t.wheelhouse@bradford.ac.uk; Fax: 44 1274 235600; Tel: 44 1274 234710
b Yorkshire Cancer Research Laboratory of Drug Design, Tom Connors Cancer Research Centre, University
of Bradford, Bradford, West Yorkshire, UK BD7 1DP
c
Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson,
Mississippi, 39216-4505, USA
Received (in Cambridge, UK) 12th February 2003, Accepted 5th March 2003
First published as an Advance Article on the web 9th April 2003
Biarylpyrimidines bearing w-aminoalkyl substituents have
been designed as ligands for high-order DNA structures:
spectrophotometric, thermal and competition equilibrium
dialysis assays showed that changing the functional group
for substituent attachment from thioether to amide switches
the structural binding preference from triplex to tetraplex
DNA; the novel ligands are non-toxic and moderate
inhibitors of human telomerase.
additions to pyrimidine;3 however, the authors’ experience with
Suzuki arylation of porphyrins7 suggested that arylpyrimidines
could be similarly prepared. Thus a concise biarylpyrimidine
synthesis was developed that allowed the ready introduction of
structural diversity (Scheme 1). 4,6-Diiodopyrimidine8 was
reacted with arylboronic acids in the presence of Pd(Ph3P)4 to
afford substituted biaryl pyrimidines 3a–e directly in up to 90%
isolated yields. The amides 2 were prepared by hydrolysis of
ester 3a to acid 3f and subsequent PyBOP-mediated coupling
with the side-chains. Access to the thioethers, 1, was via
thiophenol 3g obtained from nucleophilic displacement of
bromoarene 3b with NaSEt.9 Compounds bearing basic side-
chains were isolated as hydrobromide salts.‡
High-order (three- and four-stranded) nucleic acid secondary
structures are important targets in drug design1 and ligands with
precisely-defined DNA structure selectivity have a range of
potential therapeutic applications.2 In contrast to biphenyl ring
systems, 4,6-diphenylpyrimidine 1a is planar in its crystal form
and binds modestly to duplex DNA by intercalation.3 Moreover,
the width of the putative intercalating portion of the molecule
(13.2 Å) is a closer match to the cross-sections of tetraplex
(10.8–13.0 Å)4 or triplex (7 or 9–12.4 Å)5 nucleic acid
structures, rather than to DNA duplexes which offer threading
distances of 5.2–7.3 Å. It was also envisaged that the planar
chromophore could be extended by substitution of amides for
the thioether groups (i.e. 1?2) and thereby further direct
nucleic acid-binding preferences towards higher-order struc-
tures.
The effects of the thioethers 1a–f and amides 2a–g on the
thermal denaturation10 of duplex and triplex DNA were
determined; DTm values are presented in Table 1. The melting
curve for poly(dA)·[poly(dT)]2 triplex DNA in the absence of
1
ligand showed two sequential thermal transitions: Tm = 54.2
2
°C (triplex ? duplex + single strand) and Tm = 76.8 °C (melt
to single strands). The thioethers 1 exhibited selective stabilisa-
tion of triplex DNA, increasing the temperature of the triplex
1
melting transition (DTm 5 20 °C) whilst little affecting
2
poly(dA)·poly(dT) duplex melting (DTm 5 3 °C). The
presence of the charged ammonium groups in the side-chains
was essential for triplex stabilisation: the hydroxyethyl (1c) and
methoxyethyl (1d) derivatives showed no evidence of inter-
action with either triplex or duplex DNA, despite the possibil-
ities for DNA–ligand hydrogen bonding. The corresponding
amides 2c,d showed similarly negligible effects. The extent of
Biarylpyrimidines have hitherto been prepared by condensa-
tion of 1,3-diketones with formamide6 or sequential aryl lithium
Table 1 DTm values (°C)a determined for calf thymus duplex (CT) or triplex
DNA with ligands 1a–f and 2a–g.
c
CT DNAb
poly(dA)·[poly(dT)]2
1
2
DTm
Duplex melt
DTm
DTm
Compound
Triplex melt
Duplex melt
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
12.2
10.1
0.8
1.3
1.6
10.5
20.2
4.0
0.3
1.2
0.6
0.2
1.1
2.8
7.7
8.1
1.2
0.8
5.1
14.2
13.7
1.7
13.9
17.4
0.7
5.7
0.8
9.7
11.2
12.5
1.4
0.1
2.3
0.8
Scheme 1 Reagents and conditions: (i) Pd(Ph3P)4/K2CO3/PhMe+MeOH
9+1/D; (ii) NaOH/H2O/D, HCl, 100%; (iii) NaSEt/DMF/D; (iv)
AcOH+THF+H2O 4+2+1, 45 °C, 4 h; (v) Z(CH2)2Cl/NaOH/EtOH, 587%,
HBr; (vi) Z(CH2)2NH2/PyBOP/Et3N/CH2Cl2/RT, HBr, 570 %.
21.3d
25.0d
25.3d
13.9
13.8
2g
a
DTm = Tm(DNA + ligand) 2 Tm(DNA). b 50 µM DNA (in base pairs), 20
µM ligand; Tm for CT DNA = 68.3 °C. c 50 µM triplex (in base triads), 25
µM ligand. d Ligand destabilises the DNA triplex; see text for Tm1 and Tm
† Electronic supplementary information (ESI) available: experimental
details of UV melting studies and example spectroscopic and analytical
data. See http://www.rsc.org/suppdata/cc/b3/b301554h/
2
values in the absence of ligand. All data are mean values (±0.1 °C).
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CHEM. COMMUN., 2003, 1160–1161
This journal is © The Royal Society of Chemistry 2003

Fig. 1 (a) Thermal melting curves for poly(dA)·[poly(dT)]2 triplex DNA in the absence and presence of amide ligands 2b, 2e–g (DNA: 50 µM in base triads;
ligands: 25 µM. Competition equilibrium dialysis data for compounds (b) 1b and (c) 2b; for a full description of the nucleic acid structures used, see reference
11.
triplex stabilisation was dependent upon the nature of the basic
side-chains with the bulkier and more hydrophobic groups
In conclusion, substituted biarylpyrimidines present an
intriguing platform for the design of ligands to recognise high-
order nucleic acid structures. Furthermore, a structural switch
has been devised which alters binding preference for triplex or
tetraplex DNA forms. No compounds examined showed
appreciable cytotoxicity in a range of human tumour cell lines
(IC50 4 100 µM), consistent with only weak affinity for duplex-
form DNA. As expected for tetraplex-binding ligands, moderate
inhibition of human telomerase was found (IC50 4 10 µM).§
1
being more effective (e.g. piperidinyl, 1f: DTm = 17.4 °C;
1
diethylamino, 1b: DTm = 20.2 °C) although no correlation
with the pKa of the basic amino group (1a < 1e < 1b) was
apparent.
Minor structural alteration to the amides 2, resulted in a
significantly different pattern of DTm values. The simple
dialkylamino compounds, e.g. 2a, induced a small stabilisation
2
of poly(dA)·poly(dT) duplex (DTm 5 8.1 °C) with little effect
1
on triplex melting (mostly DTm 5 1 °C). Spectrophotometric
titration of dimethylamino compounds 1a or 2a with poly-
(dA)·poly(dT) duplex or poly(dA)·[poly(dT)]2 triplex con-
firmed the lack of interaction of amide 2a with the three-
stranded DNA structure: hypochromicity and sharp isosbestic
points were observed in all titrations except in the case of amide
2a with triplex-form DNA. Remarkable structure-selective
differences were seen for the amide ligands bearing saturated
heterocyclic side chains (2e–g), see Figure 1 panel (a). Ligands
2f,g showed closely similar effects: a marked stabilisation of
Notes and references
‡ Satisfactory spectroscopic and analytical data were obtained for all
compounds. See ESI.†
§ This work was supported by the EPSRC (grant GR/N 37605 to RTW and
TCJ), Yorkshire Cancer Research (to TCJ), National Cancer Institute (grant
CA35635 to JBC) and an EPSRC CASE studentship with Enact Pharma plc.
Mass spectra were obtained from the EPSRC National Mass Spectrometry
Service Centre, University of Wales, Swansea.
2
poly(dA)·poly(dT) duplex (DTm ≈ 14 °C) accompanied by a
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1
specific destabilisation of the triplex structure (DTm ≈ 25 °C)
that preserved the shape of the biphasic melting curve. An
intermediate effect was observed for the morpholino derivative
2e. The structural basis for these differential behaviours and the
selective destabilisation of triplex DNA is uncertain and the
subject of continuing investigations.
The structure preferences and relative binding affinities were
further investigated by competitive equilibrium dialysis.11
Figure 1 panels (b) and (c) show data obtained for compounds
1b and 2b. These results broadly confirmed that neither ligand
family had a strong affinity for any nucleic acid duplex; as
anticipated, preferential interactions with high-order structures
were evident. In agreement with the thermal and spectrophoto-
metric data, thioether 1b showed a strong preference for triplex
DNA (27-fold greater than for the natural duplex, CT DNA).
Significantly less ligand bound to triplex RNA, indicating that
at least part of the bound ligand (probably the pendant side
chains) must align in the grooves of the DNA triplex—an
accommodation thwarted by the 2A-hydroxyl groups in the all-
RNA structure.5 In contrast, the amide 2b bound more strongly
with tetraplex DNA structures (Figure 1, panel (c)) but was less
discriminating in its recognition profile. Uncharged compounds
1c,d and 2c,d showed greatly reduced affinity for nucleic acids
but the structure preferences generally resembled those of their
dimethylamino counterparts. In both series, variation of the
simple dialkyamino groups only influenced binding affinity
(e.g. Z = NMe2 > NEt2) whilst the overall selectivity was
unaffected. However, compounds bearing saturated hetero-
cyclic side-chains, showed distinctly different patterns of
binding preferences, in agreement with their anomalous thermal
melting behaviour.
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Ren and J. B. Chaires, J. Am. Chem. Soc., 2000, 122, 424–425; J. S. Ren
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