Synthesis of a novel dimeric bis-benzimidazole with site-selective DNA-binding properties

Article abstract of DOI:10.1016/S0040-4039(02)01595-2

We describe the synthesis of bis(3-{4-[2′-(4-methoxyphenyl)-3H,3′H-[5,5′] bibenzimidazolyl-2-yl]phenoxy}propyl)methylamine 8, which has high affinity for the oligonucleotide sequence [A.T]₄-[G.C]-[A.T]₄.

Full text of DOI:10.1016/S0040-4039(02)01595-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 7239–7241
Synthesis of a novel dimeric bis-benzimidazole with site-selective
DNA-binding properties
Xiao-Wen Sun,a Stephen Neidleb,† and John Manna,*
aSchool of Chemistry, Queen’s University Belfast, Stranmillis Road, Belfast BT9 5AG, UK
bBiomolecular Structure Unit, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
Received 20 June 2002; revised 23 July 2002; accepted 31 July 2002
Abstract—We describe the synthesis of bis(3-{4-[2%-(4-methoxyphenyl)-3H,3%H-[5,5%]bibenzimidazolyl-2-yl]phenoxy}propyl)-
methylamine 8, which has high affinity for the oligonucleotide sequence [A.T]4-[G.C]-[A.T]4. © 2002 Published by Elsevier Science
Ltd.
In cancer chemotherapy there is a move away from the
traditional drugs, which damage DNA in a non-selec-
tive fashion, to new agents that interact non-covalently
and modify the process of DNA replication. The possi-
ble parent of this new class of compounds is Hoechst
33258 1, which recognises A/T sequences in human
DNA and was shown to inhibit binding of the so-called
TATA-box binding protein to DNA, and also to be an
effective inhibitor of mammalian DNA topoisomerase
I.1 It was shown to have activity against the mouse
leukaemia L1210, and became the subject of several
phase I clinical trials, but toxicity precluded further
advanced trials.2
lung, colon and breast tumours (Table 1).4 There was
also highly sequence-selective binding across four con-
secutive A/T base pairs of the dodecanucleotide
sequence d(CGCGAATTCGCG), which is a model for
longer oligonucleotide sequences in the A/T-rich region
of the minor groove.
N
N
N
H
N
H
RO
OR
2a R = H, 2b R = Me, 2c R = (CH2)3NMe2
OH
With the aim of improving both the degree of binding
and the biological potency, we embarked upon a syn-
thesis of the dimer of compound 2c and our synthetic
route is shown in Scheme 1. In our original synthesis of
compounds 2, the condensation between an aryl alde-
hyde and 3,3%,4,4%-tetraaminobiphenyl was effected in
N
N
NH
N
H
N
N
1 Hoechst 33258
Me
Table 1. IC50 values (mM) following a 96 h exposure to
test compounds
Our redesign of this molecule began with a computer
modelling study and the subsequent synthesis of a
range of head-to-head bis-benzimidazoles3 of general
structure 2. More recent biological evaluation has
revealed that all of these compounds possess modest to
good activities against a range of cultured human can-
cer cell lines with compound 2c possessing the best
overall potency—IC50 values down to 50 nM against
Cell line
Compound 2b
Compound 2c
A2780 (ovarian)
A2780-PtR
13.5
5.1
3.8
16.5
0.174
0.645
0.232
0.175
0.275
0.235
0.115
0.240
0.375
0.051
0.158
0.167
0.140
0.077
CH1 (ovarian)
SKOV-3 (ovarian)
MCF-7 (breast)
RKO (colon)
H630 (colon)
OAW42 (ovary)
H838 (lung)
* Corresponding author.
† New address: School of Pharmacy, University of London, London
WC1N 1AX.
0040-4039/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0040-4039(02)01595-2

7240
X.-W. Sun et al. / Tetrahedron Letters 43 (2002) 7239–7241
OH
OH
Cl
i
ii
EtO2C
O
N
O
CO2Et
Me
N
Me
N
Me
5
Cl
3
4
OMe
H2N
NH2
NH2
H
N
H2N
H2N
iii
H2N
4HCl
N
6
7
N
N
N
H
HN
O
OMe
OMe
iv
8
5
+
7
Me
N
O
H
N
HN
N
N
Scheme 1. Reagents and conditions: (i) SOCl2, benzene, reflux, 4 h; (ii) EtO2CC6H4OH, NaH, DMF, 80°C, 24 h; (iii)
MeOC6H4COOH, 10% PPMA, 100°C, 2 h; (iv) 10% PPMA, 120°C, 6 h.
nitrobenzene at 150°C, with the nitrobenzene pre-
sumably acting as both solvent and oxidant. The yields
of these reactions were never greater than about 30%
and the non-availability of nitrobenzene in Northern
Ireland necessitated a change of strategy. We now
benzimidazole 2c.4 However, DNA footprinting studies
demonstrated that the compound had a high affinity
(drug concentration for half-maximal footprinting was
0.15 mM) and selectivity for the oligonucleotide
sequence [A.T]4-[G.C]-[A.T]4, which is not a common
motif in human DNA. Further biological evaluation is
underway and these results and all of the DNA binding
studies will be described in due course.
envisaged
a
condensation between diethyl 4,4%-
[(methylimino)bis(propoxy)]dibenzoate 5 and 4-[2-(4-
methoxyphenyl)-1H-benzimidazol-6-yl]benzene-1,2-di-
amine 7 to provide the dimeric bis-benzimidazole 8.
Overall, we have devised an effective method of synthe-
sising a new class of molecules with a high degree of
sequence selectivity for A/T-rich regions of DNA (bind-
ing over a ten base pair sequence), and which do not
exhibit generalised cytotoxicity. Additional studies will
be required in order to fine-tune this selectivity and
hopefully provide compounds with selective anti-
tumour activities.
The amine 5 was prepared (95% crude yield)5 from the
ethyl ester of 4-hydroxybenzoic acid (2 equiv.) and
N,N-bis(3-chloropropyl)-N-methylamine 4, which had
been prepared by treatment of 3,3%-(methylimino)-
dipropan-1-ol6 3 with thionyl chloride. The required
benzimidazole 7 was prepared by a condensation7
between 4-methoxybenzoic acid and 3,3%-diaminobenz-
idine tetrahydrochloride 6 using a solution of phos-
phorus pentoxide in methanesulphonic acid (PPMA)8
(1:10 w/w) as the condensing agent. The yield of 7 was
only modest (32%) but the reaction was more conve-
nient (and certainly less hazardous) than our previous
condensation methodology with nitrobenzene. Finally,
compounds 5 and 7 were combined in the ratio of 1:2
and reacted with the same condensing agent (PPMA) at
120°C for 6 h to provide the desired dimeric bis-benz-
imidazole 8 (31% yield after recrystallisation from
DMF–MeOH–H2O).9
Acknowledgements
We thank the McClay Trust for a Research Fellowship
for X. W. Sun.
References
1. (a) Reddy, B. S. P.; Sondhi, S. M.; Lown, J. W. Pharma-
col. Ther. 1999, 84, 1–111; (b) Neidle, S. Nat. Prod. Rep.
2001, 18, 291–309.
Preliminary biological evaluation of this compound
against a range of cancer cells showed that it was about
ten times less cytotoxic on average than the simpler
2. Kraut, E. H.; Masspeis, L.; Bakerzak, S.; Grever, M. Proc.
Am. Soc. Clin. Oncol. 1988, 7, 62.

X.-W. Sun et al. / Tetrahedron Letters 43 (2002) 7239–7241
7241
3. (a) Neidle, S.; Mann, J.; Rayner, E. L.; Baron, A.; Opoku-
Boahen, Y.; Simpson, I. J.; Smith, N. J.; Fox, K. R.;
Hartley, J. A.; Kelland, L. R. Chem. Commun. 1999,
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acid (1.82 g, 12 mmol) was added to this solution and the
mixture was stirred at 100°C for 2 h. The solution was
cooled to room temperature, poured into ice water and
neutralised with saturated Na2CO3 solution. The resulting
solid was filtered under reduced pressure, washed with
water and dried. It was then extracted with hot MeOH
and the extract was evaporated to give the crude product,
which was purified by flash column chromatography on
silica gel eluting with DCM/MeOH (10:1) to give com-
pound 7 (1.28 g, 32%) as a yellow solid; mp 219–220°C; IR
(KBr)/cm−1 3367, 2926, 1611, 1522, 1471, 1386, 1255,
1178, 1025, 809 and 732; lH (500 MHz; DMSO-d6) 8.11
(2H, d, J=8.9, ArH), 7.57 (1H, s, ArH), 7.53 (1H, d,
J=8.2, ArH), 7.32 (1H, dd, J=8.2 and 1.5, ArH), 7.11
(2H, d, J=8.9, ArH), 6.90 (1H, d, J=2.0, ArH), 6.75 (1H,
dd, J=7.9 and 2.0, ArH), 6.60 (1H, d, J=7.9, ArH), 4.54
(4H, br s, 2×NH2) and 3.85 (3H, s, OCH3); m/z (EI) 330
(M+, 8%), 195 (8), 184 (33), 168 (100), 153 (27), 141 (17)
and 115 (23); HRMS (EI) calcd for C20H18N4O (M+)
330.1481, found 330.1468.
4. Van den Berg, H. W., School of Oncology, QUB, personal
communication.
5. Synthesis of diethyl 4,4%-[(methylimino)bis(propoxy)]-
dibenzoate 5: To a suspension of sodium hydride (0.80 g,
60% dispersion in mineral oil, 20 mmol) in dry DMF (10
cm3) was added a solution of ethyl 4-hydroxybenzoate
(3.32 g, 20 mmol) in dry DMF (8 cm3) dropwise. After the
evolution of hydrogen gas ceased, a solution of N,N-bis(3-
chloropropyl)-N-methylamine 4 (1.84 g, 10 mmol) in dry
DMF (2 cm3) was added dropwise and the reaction mix-
ture was stirred at 80°C for 24 h. It was then cooled in an
ice bath, water was added slowly and the mixture was
extracted with EtOAc. The combined organic extracts
were washed with saturated Na2CO3 solution and brine
successively, dried over MgSO4, filtered and evaporated to
yield compound 5 (4.20 g, 95%) as a viscous yellow oil
used directly for the next step without further purification.
IR (film)/cm−1 2954, 1712, 1607, 1512, 1465, 1367, 1276,
1255, 1168, 1104, 1024 and 770; lH (300 MHz; CDCl3;
Me4Si) 7.96 (4H, d, J=8.9, ArH), 6.86 (4H, d, J=8.9,
ArH), 4.34 (4H, q, J=7.1, 2×CO2CH2CH3), 4.03 (4H, t,
J=6.3, 2×OCH2), 2.55 (4H, t, J=7.0, 2×NCH2), 2.26 (s,
3H, NCH3), 1.96 (4H, quintet, J=6.6, 2×CH2) and 1.38
(6H, t, J=7.1, 2×CO2CH2CH3); lC (75 MHz; CDCl3;
Me4Si) 166.4, 162.7, 131.5, 122.8, 114.0, 66.1, 60.6, 53.9,
42.3, 27.0 and 14.4; m/z (EI) 443 (M+, 1%), 250 (15), 121
(49), 97 (41), 85 (60), 69 (52), 57 (100) and 43 (98); HRMS
(EI) calcd for C25H33NO6 (M+) 443.2308, found 443.2314.
6. (a) Bencini, A.; Bianchi, A.; Borselli, A.; Ciampolini, M.;
Garcia-Espana, E.; Dapporto, P.; Micheloni, M.; Paoli, P.;
Ramirez, J. A.; Valtancoli, B. Inorg. Chem. 1989, 28,
4279–4284; (b) Granier, C.; Guilard, R. Tetrahedron 1995,
51, 1197–1208.
8. Eaton, P. E.; Carlson, G. R.; Lee, J. T. J. Org. Chem.
1973, 38, 4071–4073.
9. Synthesis
of
bis(3-{4-[2%-(4-methoxyphenyl)-3H,3%H-
[5,5%]bibenzimidazolyl - 2 - yl]phenoxy}propyl)methylamine
8: 4-[2-(4-Methoxyphenyl)-1H-benzimidazol-6-yl]benzene-
1,2-diamine 7 (0.60 g, 1.8 mmol) was dissolved in PPMA
(4.55 cm3) and diethyl 4,4%-[(methylimino)bis(pro-
poxy)]dibenzoate 5 (0.40 g, 0.9 mmol) was added to this
solution. The reaction mixture was stirred at 120°C for 6
h, then the solution was cooled to room temperature,
poured into ice water and neutralised with saturated
Na2CO3 solution. The resulting solid was filtered under
reduced pressure, washed with water and dried. It was
recrystallised from DMF–MeOH–H2O to give pure com-
pound 8 (0.27 g, 31%) as a yellow solid; mp >300°C; IR
(KBr)/cm−1 3420, 2925, 1652, 1611, 1541, 1489, 1446,
1385, 1251, 1174, 1026, 834 and 803; lH (500 MHz;
DMSO-d6) 8.14 (8H, t, J=8.6, ArH), 7.95–7.40 (12H, m,
ArH), 7.12 (4H, d, J=8.9, ArH), 7.09 (4H, d, J=8.8,
ArH), 4.10 (4H, t, J=6.0, 2×OCH2), 3.85 (6H, s, 2×
OCH3), 2.56 (4H, m, 2×NCH2), 2.26 (3H, s, NCH3) and
1.92 (4H, m, 2×CH2); m/z (LSI) 976 (M+H+, 100%), 544
(27) and 433 (56); HRMS (LSI) calcd for C61H54N9O4
(M+H+) 976.4299, found 976.4333.
7. Synthesis of 4-[2-(4-methoxyphenyl)-1H-benzimidazol-6-
yl]benzene-1,2-diamine 7: 3,3%-Diaminobenzidine tetra-
hydrochloride dihydrate 6 (4.76 g, 12 mmol) was dissolved
in PPMA (30 cm3, 1:10 solution by weight of phosphorus
pentoxide in methanesulphonic acid). When the bubbles of
hydrogen chloride gas were eliminated, 4-methoxybenzoic