Synthesis of distamycin a polyamides targeting G-quadruplex DNA

Article abstract of DOI:10.1039/b607707b

A number of amide-linked oligopyrroles based on distamycin molecules have been synthesized by solid-state methods, and their interactions with a human intramolecular G-quadruplex have been measured by a melting procedure. Several of these molecules show an enhanced ratio of quadruplex vs. duplex DNA binding compared to distamycin itself, including one with a 2,5-disubstituted pyrrole group. Quadruplex affinity increases with the number of pyrrole groups, and it is suggested that this is consistent with a mixed groove/G-quartet stacking binding mode. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b607707b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of distamycin A polyamides targeting G-quadruplex DNA
Michael J. B. Moore,a Francisco Cuenca,a Mark Searceyb and Stephen Neidle*a
Received 31st May 2006, Accepted 11th July 2006
First published as an Advance Article on the web 14th August 2006
DOI: 10.1039/b607707b
A number of amide-linked oligopyrroles based on distamycin molecules have been synthesized by
solid-state methods, and their interactions with a human intramolecular G-quadruplex have been
measured by a melting procedure. Several of these molecules show an enhanced ratio of quadruplex vs.
duplex DNA binding compared to distamycin itself, including one with a 2,5-disubstituted pyrrole
group. Quadruplex affinity increases with the number of pyrrole groups, and it is suggested that this is
consistent with a mixed groove/G-quartet stacking binding mode.
the tri-N-methyl-pyrrole duplex-DNA binding ligand distamycin
Introduction
A21 (Fig. 1) as a lead molecule. Derivatives of distamycin A have
been previously reported to be inhibitors of the human telomerase
enzyme22 although distamycin A itself lacks activity. Recent NMR
studies23,24 have suggested that distamycin A is also able to interact
with G-quadruplex DNA. Two contrasting alternative models
have been proposed in which (i) distamycin molecules bind as
dimers in two of the four grooves of a quadruplex,23 and (ii) in
which two molecules of distamycin A extend over each of the two
G-tetrad planes in a 4 : 1 binding mode.24
The telomerase enzyme complex, which catalyses the synthesis
of telomeric DNA repeats, is responsible for the maintenance
of telomere integrity in cancer cells, and plays a major role in
their immortalisation.1–3 Telomerase, which is expressed in 80–
90% of cancer cells, and not significantly up-regulated in normal
somatic cells, is therefore a key target for selective therapeutic
intervention.4 One particular strategy involves the targeting not
of the enzyme per se, but its substrate, the single-stranded 3ꢀ
overhang of telomeric DNA.5 In this approach, small-molecule
telomere targeting agents induce this guanine-rich DNA to fold
into an intramolecular quadruplex structure, which cannot be
recognized by the RNA template domain of telomerase and so
cannot act as a substrate for the enzyme itself.6 In addition
quadruplex formation may dissociate telomere ends from physical
association with telomerase and other telomere-binding proteins,7
which in cells then results in the triggering of a DNA damage
response and eventual cell death.8 A large number of such small
quadruplex-binding molecules have been studied,9–16 the majority
of which have high affinity for quadruplex DNAs by virtue of their
possessing a planar aromatic chromophore such as an acridine,
anthraquinone or porphyrin, which can interact with the planar
G-quartet surface of a quadruplex by p–p interactions. A lead
compound, BRACO-19, from one such series, of trisubstituted
acridines, has selectivity for quadruplex vs. duplex DNA,14 and
shows cellular effects consistent with G-quadruplex formation and
telomere targeting17 as well as demonstrating in vivo antitumour
activity in a xenograft model.18
Fig. 1 Distamycin A.
We describe here the solid phase synthesis of a number of
distamycin A polyamides and assess their ability to selectively
bind to telomeric G-quadruplex DNA, in comparison with their
binding to duplex DNA. Distamycin A is a classic DNA minor
groove binder,21 with selectivity for the narrow, deep minor
groove of B-DNA A/T sequences, derived from its planar, curved
isohelical structure.25 The observation of the side-by-side binding
of distamycin A in longer A/T sequences,26 ultimately led to
the design of the sequence-reading oligopyrrole carboxamide
(polyamide) hairpin structures.27 These molecules can be used to
target particular DNA sequences and act as inhibitors of DNA–
protein interactions.28
Key to the successful development of distamycin A polyamide
molecules as quadruplex binding and stabilizing agents is the
ability to selectively target G-quadruplex over duplex DNA. Using
qualitative molecular modeling with the human intramolecular
G-quadruplex structure,29 we reasoned that if a 2,5-disubstituted
pyrrole-carboxamide were also to adopt the isohelicity required
to complement the duplex structure, it would then position the
N-methyl group into rather than out of the groove, causing a
steric clash that would inhibit binding to duplex DNA but would
An extended heteroaromatic chromophore is not an essential
feature of quadruplex-binding ligands, as shown by molecules
such as the cyclic oxazole natural product telomestatin,13 and
by pyridine derivatives19 and triazines20 bearing x-aminoalkyl
substituents. We report here on another category of ligand, based
on the polyamide architecture that has been extensively explored
for sequence-selective binding to duplex DNA. We have taken
aCancer Research UK Biomolecular Structure Group, The School of
Pharmacy, University of London, 29-39 Brunswick Square, London, UK
WC1N 1AX. E-mail: stephen.neidle@pharmacy.ac.uk; Fax: +44 (0)20
7753 5970; Tel: +44 (0)20 7753 5971
bDepartment of Pharmaceutical and Biological Chemistry, The School of
Pharmacy, University of London, 29-39 Brunswick Square, London, UK
WC1N 1AX
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Table 1
n
2,4-Substituted oligomers
2,5-Substituted oligomers
1
2
3
4
5
AcPy2bAlaDp (1)
AcPy3bAlaDp (2)
AcPy4bAlaDp (3)
AcPy5bAlaDp (4)
AcPy6bAlaDp (5)
Ac(2,5-Py)PybAlaDp (6)
Ac(2,5-Py)Py2bAlaDp (7)
Ac(2,5-Py)Py3bAlaDp (8)
Ac(2,5-Py)Py4bAlaDp (9)
Ac(2,5-Py)Py5bAlaDp (10)
have little effect on G-quadruplex affinity. We initially assumed a
structural model with distamycin bound on a terminal G-quartet
of a quadruplex, analogous to the planar chromophore binding
mode observed in quadruplex–ligand crystal structures,30 and thus
distamycin would not be intercalated between quartets.
Scheme 1 Monomer synthesis. i) HNO3 dropwise, Ac2O, −40 ◦C–rt,
(CH3)2CHOH, −40 ◦C ii) 0.05 equiv. DMAP, MeOH, rt, N2 iii) H2 (30 psi
for 20), 10% Pd–C, THF (EtOAc for 20) iv) 1.2 equiv. Boc2O, 1.1 equiv.
TEA, 1,4-dioxane, D v) 2 M aqueous NaOH, MeOH, 60 ◦C vi) HOBt,
EDCI or DCC, DMF, rt, N2.
Although our modeling studies suggested that steric crowding
around a centrally-positioned 2,5-analogue may cause synthetic
problems, we reasoned that the design of end-substituted (either
amino or acid terminus) derivatives would be feasible and would
allow the testing of the design. Loss of isohelicity also accompanies
the synthesis of longer duplex-binding distamycin analogues,31 but
would not be expected to have an effect on a stacking binding mode
to a G-quadruplex structure. Therefore, a series of distamycin A
polyamides (2–11, Table 1) containing 2,5-disubstituted pyrroles
and of increasing length were prepared by solid phase synthesis
and their relative binding affinities for duplex DNA and G-
quadruplex DNA determined by a FRET (fluorescence resonance
energy transfer) assay modified by us32 for this purpose.
mixture contained the 5-nitropyrrole or the 4,5-dinitropyrrole as
described for the related nitration of 1-methylpyrrole-2-carboxylic
acid. Investigation of this reaction indicated that the 5-nitro isomer
13 was present in the nitration mixture. As this route is amenable
to very large scale synthesis33 from cheap starting materials, we
reasoned that the isolation of 13 from the nitration mixture
could give significant amounts of material, albeit with a low but
acceptable yield (18%) at this early stage. Hence this route was
adopted towards a synthesis of both 12 and 13 as steps towards
21 and 19 respectively.
Trichloroacetylation proceeded as previously reported.33 Nitra-
tion under the usual conditions was followed by fractional crystal-
lization to isolate pure 4-nitro-regioisomer 12. Chromatography of
the crystallization solvent gave pure 13 in 18% yield, along with
further amounts of 12. Esterification of 13 (step ii, Scheme 1)
was rapidly effected with catalytic DMAP in methanol at room
temperature in 80% yield. Conditions utilizing sodium methoxide,
as previously described,33 were not required and, in our hands,
this reaction also proceeds efficiently with the 2,4-regioisomer
12. The difference in reactivity and solubility between the 2,5-
disubstituted intermediates and their 2,4-counterparts manifested
itself in several of the subsequent synthetic steps. Hydrogenation
of 14 to 15 was achieved under milder conditions (4.5 h at ambient
pressure and temperature) than those typically observed with the
2,4-disubstituted counterpart, where reaction required 30 psi of
hydrogen and remained, at times, irreproducibly stubborn to even
these conditions. The pyrrole amine 15, obtained as a red residue,
was immediately protected to give a mixture of mono- and di-Boc
products 16 (isolated yield 57%) and 17 (isolated yield 2%). In the
case of 2,4-disubstituted substrate 20 under identical conditions
only the mono-protected product was observed. Hydrolysis of
16 by heating with a 1 : 1 methanol–water solution of sodium
Results
Monomer synthesis
Solid phase synthesis provides a rapid and effective route to
extended polyamides.33,34 For the synthesis to proceed, large
amounts of the HOBt active ester of both the 2,4 and 2,5-
regioisomers were required (Scheme 1). Synthesis of 19 and 21
followed modifications of the literature procedure reported for
21.33 A first point of consideration was a synthesis of 14 as a
step towards 19. The preparation of 14 has been achieved by
nitration of commercially available 1-methylpyrrole-2-carboxylic
acid followed by esterification to give a mixture which on
separation affords 14 (11%) and the regioisomer methyl 4-nitro-
1-methylpyrrole-2-carboxylate (42%).35 A small amount of 2,4-
dinitro-1-methylpyrrole may also be isolated.36 The nitration of
11, prepared from 1-methylpyrrole, has also been reported to
proceed regioselectively with isolation of the 4-nitro-isomer in
reasonable yield through fractional crystallization from isopropyl
alcohol at −20 ◦C.33 It had not been reported whether the nitrating
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hydroxide (7 equiv.) afforded 18 (79%). DCC or EDCI mediated
esterification of the acid 18 gave 19 in 94% yield (6% overall yield
from 11).
Solid phase synthesis
Manual solid phase synthesis of polyamides from 19 and 21 was
undertaken using standard coupling conditions.33,34 Chloranil has
been described as an effective colorimetric test for the presence
of aromatic amines37 and in this synthesis we utilized a chloranil
solution to assess coupling of the pyrrole amines. The syntheses
of 2,4-pyrrole oligomers 1–5 proceeded as planned with two
notable exceptions. Coupling between 21 and resin bound Py5
amine required an extended overnight reaction time period beyond
the standard 45 minutes. Acetylation of the resin bound Py6 amine
also required treatment beyond the standard 30 minute period.
Repeated treatments with a mixture of Ac2O, DIPEA in DMF of
1 h, 1.5 h and 2 h proceed with little conversion as indicated by the
chloranil test. However, utilization of a freshly prepared mixture
containing acetyl chloride (12 equiv.) and DIPEA (12 equiv.)
in DMF proved successful in reducing the reaction times to
30 minutes in subsequent experiments.
The synthesis of the 2,5-pyrrole oligomer series 6–10 proved
more demanding. To date, solid phase synthesis of polyamides
possessing a centrally positioned 2,5-pyrrole has not been dis-
closed. The synthesis of trimer 7 was attempted first and achieved
directly on the solid phase. Coupling of 19 to the resin bound
Py2 amine required repeated couplings and longer reaction times
consisting of three cycles of 45, 90 and 90 minutes duration.
However resin-bound Boc(2,5-Py)Py2 deprotection and acetyla-
tion proceeded smoothly. Resin cleavage by treatment with 3-
dimethylaminopropylamine gave 7 in a reasonable 55% yield
following purification by amine scavenge.
Attempts at the introduction of the 2,5-substituted pyrrole at the
N-terminus of longer polyamide chains (n > 2) proved fruitless.
LC/MS analysis of cleavage products indicated not only failed
acetylation but also failed couplings between the resin bound
pyrrole amines and 19 raising concerns over both the efficiencies of
this synthetic step and the sensitivity of chloranil as an analytical
tool. An alternative approach to step-wise solid phase synthesis
of polyamides has involved the coupling of a ‘preformed’ solution
phase prepared dimer into the growing polyamide chain.33,38 In this
way the troublesome coupling of 19 to a resin pyrrole amine could
be by-passed. We also sought to utilize the observed increased
coupling efficiency of HOAt active esters and hence 26 was
prepared.34 Synthesis of 25 (Scheme 2) was initially achieved
by the DMAP mediated coupling of 19 to 20 following Boger’s
related procedure39 to give 24 (53%) followed by hydrolysis and
esterification. However the expense incurred by the low yielding
synthesis of 13 towards 19 and the expense of the latter’s use in
solid phase syntheses prompted the use of haloform chemistry40
to direct a cheaper preparation of 24 and then 26.
Scheme 2 Dimer active ester synthesis. i) 20, DMAP (0.2 equiv.), THF,
N2, rt, 2 h, 81% ii) 30 psi H2, 10% Pd–C, EtOH, rt, 3 h iii) Boc2O, TEA,
1,4-dioxane, 85 ◦C, 16 h, 44% for 2 steps iv) NaOH, H2O–MeOH (1 : 1),
39% (27) v) EDCI, HOAt, DMF, rt, N2 21% (25 over 2 steps), 66% (28)
vi) Ac2O, DMAP, CH2Cl2, N2, 38% vii) 19, EDCI, DMAP, DMF, rt, N2,
53%.
the required extended acetylation limited the use of chloranil
but definitive LC/MS analysis of cleavage products indicated a
lack of success. Rather than carrying out the final acetylation
on bead we sought to undertake this in solution and hence the
dimer Ac(2,5-Py)PyOAt (28) was adopted as a synthetic target.
This was prepared by acetylation of 23 followed by hydrolysis
and re-esterification. The subsequent success in coupling 28 to
the appropriate pyrrole amine obviated the need to carry out the
troubling solid phase acetylation and brought with it ultimately
the synthesis of the remaining oligomers 6 and 8–10.
Quadruplex-binding assay
Polyamides were examined in a FRET (fluorescence resonance
energy transfer) assay to assess both their G-quadruplex DNA and
duplex DNA stabilising abilities. The assay was conducted using
the previously reported protocol.32 In brief, changes in the FRET
signal upon melting of telomeric G-quadruplex DNA and a duplex
DNA probe were observed under conditions approximating those
used in the crystallisation of the human intramolecular G-
quadruplex DNA structure29 (50 mM potassium cacodylate buffer,
pH 7.4). Analysis of this data provides changes in melting point
(DTm), a measure of DNA stabilization and ligand binding affinity.
Reaction of 13 and amine 20 catalysed by DMAP gave 22 (81%)
which on successive hydrogenation and Boc protection afforded
24 (44% over two steps). Hydrolysis and esterification gave 25 (8%
overall yield from 13). The use of dimer 25, rather than monomer
19, in coupling with resin bound pyrrole amines, proceeded with
greater ease. However the subsequent deprotection acetylation
cycle proved problematic. A bead colour change to purple on
G-Quadruplex DNA stabilization
As polyamide length increases, an increase in G-quadruplex (G4)
DTm was observed within both series of ligands 1–5 and 6–10.
This increased G4 stabilization is not linear, being small for the
tetramer 3 and even more so for compound 8. Comparing G4
stabilization across the series it is apparent that the 2,5-pyrrole
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dimer and trimer, compounds 6 and 7, possess greater G4 DNA
binding affinity than the corresponding 2,4-pyrrole oligomers 1
and 2. This trend however is reversed when longer polyamides are
considered. The 2,4-polyamides 4 and 5 are more potent than their
2,5-pyrrole counterparts 9 and 10.
G-Quadruplex versus duplex DNA stabilization
All compounds (except 5) have decreased G4–duplex stabilization
compared to distamycin itself. G4 DNA stabilization for the 2,4-
pyrrole oligomers 1–5 follows the trend set for duplex DNA
stabilization, and all display 2–4 fold selectivity for duplex
DNA over G4 DNA. All the 2,5-pyrrole oligomers 6–10 display
selectivity for duplex DNA, with the one notable exception being
the trimer 7 which displays a preference for G4 DNA. This
preference is qualified by the modest DTm vales for both G4
DNA (3.3 ◦C) and duplex DNA (1.3 ◦C) although the ratio of
melting temperatures (duplex DNA DTm–G-Quadruplex DNA
DTm) is 0.4 whereas for the other polyamides reported here this
ratio is always >1. The selectivity shown by 7 is a consequence
more of its low duplex DNA stabilization than significant G-
quadruplex stabilization. Polyamides 8 and 9 have comparable
G4 DNA stabilization to 7 but also have significantly increased
duplex DNA affinity. Here the addition of a pyrrole carboxamide
seems advantageous for duplex binding but not for G-quadruplex
binding. There is an increase in G4 stabilization observed for 9
relative to 7, however this is accompanied by increased duplex
stabilization. Compound 5 is notable in showing a high level of
binding to both duplex and quadruplex DNA, so that the ratio of
melting temperatures favours quadruplex some 3-fold more than
distamycin itself.
Duplex DNA stabilization
A near-perfect linear increase of DTm with polyamide length
was observed within the 2,4-pyrrole series 1–5. The binding of
compounds 6–10 to duplex DNA is not so consistent. Thus
tetramer 8 has a high DTm (16 ◦C) relative to the trimer 7
(1.3 ◦C) and pentamer 9 (8.5 ◦C). Across the two series, 2,5-
pyrrole tetramer 8 has increased duplex DNA binding affinity
relative to the 2,4-pyrrole tetramer 3. This relationship exists for
the dimers 6 and 1 but the magnitude of the difference is small. For
the remaining members, substitution of a 2,4-pyrrole heterocycle
(Py) with a 2,5-pyrrole heterocycle (2,5-Py) results in decreased
duplex DNA binding affinity. Distamycin A, as control, has a
◦
DTm of 20.5 C, much higher than the stabilization produced by
the synthetic trimers 2 (3.8 ◦C) and 7 (1.3 ◦C).
Discussion
The concept that incorporation of 2,5-pyrroles will promote
selective G-quadruplex binding is borne out in principle by these
FRET results. Comparison of melting temperature ratios (G-
quadruplex DNA DTm–duplex DNA DTm) for dimers 1 (0.3) and
6 (0.55) indicates that incorporation of the 2,5-pyrrole into the
polyamide scaffold has resulted in modest selectivity towards G4
DNA by decreasing duplex DNA binding affinity. However this
favourable effect is diluted with considering longer sequences. 2,4-
Pyrrole polyamides 4 and 5 have similar selectivity for G4 DNA
over duplex DNA compared to their 2,5-pyrrole counterparts
9 and 10. The unexpected finding here that the longer length
polyamides show the greatest quadruplex stabilization, suggests
that these may be binding in the grooves of a low-energy form
of the human intramolecular quadruplex structure. However the
short length of groove in these structures appears to be insufficient
We expected that polyamides beyond five contiguous rings would
display reduced duplex DNA affinity,16 as a consequence of the
problem of helical phasing. The results of this study indicate that
a plateau is yet to be reached after which introduction of addi-
tional pyrrole carboxamides for compounds 1–5 is penalized and
duplex DNA stabilization decreases, at least for the experimental
conditions employed here (Table 2). The DTm values show that
duplex DNA stabilization increases markedly after addition of a
◦
pyrrole carboxamide to the pentamers 4 (DTm = 16.3 C) and 9
(8.5 ◦C) to give hexamers 5 (25.3 ◦C) and 10 (22 ◦C) respectively.
In addition the viability of the concept of elongating polyamides
to enhance G-quadruplex binding at the expense of duplex DNA
affinity is demonstrated by the present FRET results. Compounds
4, 5 and 9 induce greater G4 stabilization than distamycin itself,
and thus show enhanced relative quadruplex affinity.
Table 2 G-Quadruplex DNA and duplex DNA stabilization (FRET) for compounds 1–10, distamycin and the established quadruplex-binding molecule,
BRACO-1914
Compound
Duplex DNAc/[DTm]D
G-Quadruplex DNAb/[DTm]Q
Ratio [DTm]D–[DTm]Q
1
1
0.3
1.8
2.7
7.2
14
1.1
3.3
2.4
3.8
9.1
31
3.33
4.75
4.00
2.26
1.81
1.81
0.39
6.67
2.24
2.42
2
3.8
10.8
16.3
25.3
2
1.3
16
8.5
22
11
3
4
5
6
7
8
9
10
BRACO-1914
Distamycin A
20.5
3.5
5.86
a Values reported are the average of two determinations at a compound concentration of 10 lM. b Using the labeled G-quadruplex telomeric sequence (5ꢀ-
FAM-d[GGG(TTAGGG)3]-TAMRA-3ꢀ). c Duplex DNA sequence d[TATATATATA] linked by hexaethyleneglycol and labeled with FAM and TAMRA
at 5ꢀ and 3ꢀ ends respectively.
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to accommodate the five or even six pyrrole-amide units in
compounds 4, 5 or 9. This apparent inconsistency may be resolved
by a binding model in which one end of these conformationally
flexible polyamides stacks onto G-quartet ends, and the other is
bound in a quadruplex groove. Such a model is stereochemically
feasible (Fig. 2) although as yet there is no experimental evidence
to support it.
provided by the School of Pharmacy. TLC analysis was carried out
on silica gel (Merck 60F-254) with visualization at 254 and 366 nm
and flash chromatography carried out with BDH silica gel (BDH
153325P). Treatment of an organic solution in the usual manner
refers to stepwise drying with magnesium sulfate, filtration and
then evaporation of the filtrate in vacuo. Reagents and chemicals
(including anhydrous 1,4-dioxane), excepting HBTU and HOBT
(Novabiochem), were purchased from Sigma-Aldrich. Solvents
were purchased from BDH. Anhydrous THF was distilled from
the ketal formed from sodium and benzophenone. The polyamide
monomer 21 was prepared via the intermediates 11, 12 and 20
according to the literature procedure33 as was acid 29.42
Polyamide synthesis
Solid phase synthesis of polyamides employed manual
methodologies.33,34 Thus this resin (0.75 mmol g−1) was placed
in a peptide reaction vessel and covered in DMF to swell for 1 h.
The reaction vessel was drained and the resin washed (DCM (2 ×
30 s) and DMF (1 × 30 s)). The Boc group was removed with
92.5 : 5 : 2.5 TFA–phenol–water (TPW) (1 × 30 s, 1 × 20 min)
and the resin washed. Coupling consisted of the addition of the
appropriate active ester (4 equiv.) in DMF, neat DIPEA (12 equiv.)
and then shaking for the stated time. Colorimetric analysis of
deprotection and coupling cycles was undertaken. Ninhydrin was
used in the first coupling cycle and then subsequently a 2% DMF
solution of chloranil (Avocado) was used as an indicator.22 With a
pyrrole amine on bead a resin sample was observed to turn brown
to black otherwise a resin sample was colourless. Splitting was
achieved by additional washing of the resin (2 × DCM) after a
completed coupling cycle and drying under suction. The resin was
weighed and divided by mass according to the desired splitting
factor. Solid phase acetylation was carried out by shaking with
freshly prepared mixture A for thirty minutes. The preparation of
larger, especially mixed, polyamides required repeated treatments
with mixture A hence in these cases this was substituted for
mixture B. Mixture A contained acetic anhydride (12 equiv.)
and DIPEA (12 equiv.) in DMF whilst mixture B contained
DMAP (2.5 equiv.) as an extra additive. Cleavage was undertaken
by ◦heating resin with N,N-dimethylaminopropylamine (DAP) at
60 C overnight. Product polyamides were isolated as oily solids
according to Chamberlin and Krutzik.34 Excess cleaving reagent
DAP was removed from polyamide products by use of a nucle-
ophile scavenging anhydride resin (Novabiochem MP anhydride),
followed by purification by ion exchange chromatography (Isolute
SCX2, Biotage). In this manner, polyamides 1–4 were constructed
using a split synthesis from PAMbAlaBoc resin. Polyamide 2 has
been reported previously.43 The polyamide AcPy6bAlaDp (5) was
prepared independently. Polyamide 7 was prepared by the coupling
of 19 to the resin Py2 amine followed by acetylation. Following
inefficiencies with this protocol, polyamides 6, 8–10 were prepared
by coupling 28 to the respective resin pyrrole amine. The columns
used were 500 mg SCX-2 columns. Samples were loaded into the
column, dissolved in 2 ml of methanol. Non-basic compounds
were washed off using methanol (2 × 2 ml) and 0.1 M ammonia in
methanol (1 × 2 ml). The basic product was collected using 1 M
ammonia in methanol (1 × 2 ml). The methanolic ammonia was
evaporated off to yield the free base.
Fig. 2 A qualitative molecular model of a six-repeat pyrrole polyamide
molecule (shown in space-filling mode), analogous to compound 5, docked
onto the crystal structure of the human 22-mer quadruplex structure29
(with the bases and backbone shown in cartoon form). Two repeats of the
polyamide are located in a quadruplex groove, and the others are stacked
on a terminal G-quartet surface.
Inhibition of telomerase by distamycin A and compound 7
was determined by the modified PCR based TRAP assay which
was performed as previously described.32,41 The two compounds
were examined in this way to a concentration of 100 lM and
concomitant controls were carried out to verify that there was
no interference with the correct functioning of Taq polymerase
at these concentrations. Distamycin exhibited a telEC50 of 25 lM
whereas compound 7 showed no telomerase inhibitory activity
at 100 lM. However, both compounds showed significant PCR
inhibition, suggesting that the positive results in the TRAP assay
were not necessarily indicative of telomerase inhibition per se.
Experimental
General
Microwave irradiation was performed with an Emrys Optimizer
from Personal Chemistry. Melting points (mp) were measured
with a Stuart Scientific SMP1 melting point apparatus and are
uncorrected. IRspectrawererecordedusingaPerkinElmer SPEC-
TRUM 1000 FT-IR Spectrometer. Proton NMR spectra were
acquired in CDCl3, (Sigma-Aldrich) CD3OD (GOSS), DMSO-
d6 (GOSS) solutions with chemical shift (d) reported in ppm
relative to the internal standard TMS or for CD3OD solutions
relative to the residual solvent resonance (d = 3.31). Carbon
NMR spectra were recorded in CDCl3 solutions referenced to
the solvent peak (d = 77.0 ppm). LC/MS was performed using a
Waters system combining a 2695 separation module, a Micromass
ZQ spectrometer and a 2996 photodiode array detector. Mass
spectrometry and elemental analysis services (ESI, HRMS) were
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Biophysical studies
d ppm 4.28 (s, 3H), 7.15 (d, 2H, J = 4.7 Hz), 7.44 (d, 2H, J =
4.9 Hz); 13C NMR (CDCl3) d 36.3, 95.2, 111.3, 120.4, 125.2, 142.9,
174.1.
All oligonucleotides and their fluorescent conjugates were pur-
chased from Eurogentec (Southampton, UK). DNA was initially
dissolved as a stock 50 lM solution in purified water; further
dilutions were carried out in the relevant buffer.
Methyl 5-nitro-1-methylpyrrole-2-carboxylic acid ester (14)35.
DMAP (38 mg, 0.3 mmol, 0.05 equiv.) was added portionwise
to a stirred suspension of 13 (1.567 g, 5.8 mmol) in anhydrous
CH3OH (20 ml) under N2. The suspension was warmed slightly
to aid dissolution. After 15 min, a pale yellow solid precipitated
and the resulting suspension was stirred for a further 1.25 h. The
solid (14) was collected by filtration and a◦second crop isolated.
Tota◦l yield 14 (852 mg, 80%): mp 104–106 C (MeOH) (lit.20 mp
114 C); IR (cm−1) 3121, 2363, 1710, 1532, 1495, 1476, 1303,
The ability of the compounds to stabilise G-quadruplex DNA
was investigated using a fluorescence resonance energy transfer
(FRET) assay modified to be used as a high-throughput screen
in a 96-well format. The labelled oligonucleotide F21T (5ꢀ-
FAM-dGGG(TTAGGG)3-TAMRA-3ꢀ; donor fluorophore FAM:
6-carboxyfluorescein; acceptor fluorophore TAMRA: 6-carboxy-
tetramethylrhodamine) used as the FRET probe was diluted from
stock to the correct concentration (400 nM) in a 50 mM potassium
cacodylate buffer (pH 7.4) and then annealed by heating to 85 ◦C
for 10 min, followed by cooling to room temperature in the heating
block.
Compounds were stored as 10 mM stock solutions in DMSO;
final solutions (at 2 × concentration) were prepared using 10 mM
HCl in the initial 1 : 10 dilution, after which 50 mM potassium
cacodylate buffer (pH 7.4) was used in all subsequent steps. The
maximum HCl concentration in the reaction volume (at a ligand
concentration of 20 lM) is thus 200 lM, well within the range
of the buffer used. Relevant controls were also performed to
ascertain a lack of interference with the assay. 96-Well plates (MJ
Research, Waltham, MA) were prepared by aliquoting 50 ll of the
annealed DNA into each well, followed by 50 ll of the compound
solutions. Measurements were made on a DNA Engine Opticon
(MJ Research) with excitation at 450–495 nm and detection at
515–545 nm. Fluorescence readings were taken at intervals of
0.5 ◦C over the range 30–100 ◦C, with a constant temperature being
maintained for 30 s prior to each reading to ensure a stable value.
Final analysis of the data was carried out using a script written
in the program Origin 7.0 (OriginLab Corp., Northampton, MA).
The advanced curve-fitting function in Origin 7.0 was used for
calculation of DTm values.
1
1247; H NMR (CDCl3) d ppm 3.90 (s, 3H), 4.32 (s, 3H), 6.90
(d, 2H, J = 4.6 Hz), 7.13 (d, 2H, J = 4.5 Hz); 13C NMR
(CDCl3) d ppm 35.2, 52.1, 112.0, 115.7, 126.9, 141.3, 160.6.
Found C, 45.89; H, 4.30; N, 15.10. Calcd C, 45.66; H, 4.38; N,
15.21%.
Methyl 5-amino-1-methylpyrrole-2-carboxylate (15). Methyl
ester 14 (146 mg, 0.8 mmol) in anhydrous THF was stirred under
H2 in the presence of 10% Pd–C (30 mg) for 4.5 h. The reaction
mixture was filtered through Celite, the Celite was washed with
EtOAc and the combined filtrate and washings were concentrated
to give 15 as a red residue which was submitted directly to the next
synthetic step. Yield (123 mg, quantitative).
Methyl 5-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-carb-
oxylate (16) and methyl 5-[(di-tert-butoxycarbonyl)amino]-1-
methylpyrrole-2-carboxylate (17). A solution of the amine 15
(618 mg, 4 mmol) and Et3N (0.6 mL, 446 mg, 4.4 mmol,
1.1 equiv.) in 1,4-dioxane was treated portionwise with Boc2O
(1.050 g, 4.8 mmol, 1.2 equiv.). The solution was then heated under
reflux and reaction progress monitored by TLC. On completion,
the solution was concentrated to give a mixture of 16 and 17.
Purification by flash chromatography (1 : 4 EtOAc–hexane) gave
17 (30 mg, 2%) and 16 (577 mg, 57%): 16 mp 87–88 ◦C; IR (cm−1)
1
3300, 2978, 1697, 1561, 1486, 1239, 1154, 1105, 747; H NMR
Synthetic chemistry
(CDCl3) d 1.49 (s, 9H), 3.76 (s, 3H), 3.79 (s, 3H), 6.02 (d, 1H, J =
3.6 Hz), 6.50 (bs, 1H), 6.89 (d, 1H, J = 4.2 Hz); 13C NMR (CDCl3)
d ppm 161.6, 153.5, 132.2, 119.2, 116.7, 103.0, 81.3, 50.9, 31.4,
28.1; HRMS (+ESI) m/z 255.1351 (MH+, C12H19N2O4 requires
255.1339). 17 Mp 110–112 ◦C; IR (cm−1): 2978, 1759, 1732, 1702,
5-Nitro-2-(trichloroacetyl)-1-methylpyrrole (13). A solution of
11 (10.10 g, 0.045 mol) in Ac2O (60 ml) was treated dropwise
with nitric acid (69%, 8 ml) over a period of 0.5 h at −40 C in
◦
a CCl4–dry ice bath. The reaction solution was allowed to warm
to rt and then stirred for 3 h. The solution was then cooled to
−40 ◦C, isopropyl alcohol (53 ml) added and the temperature
maintained at −40 ◦C for 0.5 h and −22 ◦C overnight. The
resultant brown solution was concentrated, poured into H2O and
the resulting mixture was extracted with CHCl3. The organic
extract was worked up in the usual manner to give a brown
residue which solidified on standing. The solid was triturated
with ice-cold EtOAc upon which a white solid (12), deposited.
The solid was collected by filtration and washed with ice-cold
EtOAc. The combined filtrate and washings were concentrated
and stored at −22 ◦C overnight to provide a second crop of
12. The brown residue resulting from evaporation of the filtrate
was submitted to flash column chromatography (1 : 4 EtOAc–
hexane) to afford, first, 13 as a pale green solid and ◦then a further
amount of 12. Yield 13 (1.093 g, 18%): mp 67–69 C; IR (cm−1)
1692, 1540, 1503, 1353, 1290, 1210, 1183, 1130;1H NMR (CDCl3)
1
1367, 1249, 1105; H NMR (CDCl3) d ppm 1.35 (s, 18H), 3.63
(s, 3H), 3.74 (s, 3H), 5.91 (d, 1H, J = 4.2 Hz), 6.83 (d, 1H, J =
4.2 Hz); 13C NMR (CDCl3) d 27.7, 31.1, 50.9, 83.3, 105.6, 116.2,
120.2, 132.3, 150.6, 161.5; HRMS (+ESI) m/z 355.1862 (MH+,
C17H27N2O6 requires 355.1864).
5-[(tert-Butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylic
acid (18). A slurry of 6 (175 mg, 0.69 mmol) in a 1 : 1 solution
of CH3OH–2 M aq. NaOH (2.56 ml) was heated at 60 ◦C for
2 h. The resulting pink solution was allowed to cool to rt, washed
with diethyl ether, acidified to pH = 1 (universal indicator paper)
with 10% aq. HCl and extracted with EtOAc. The combined
organic extracts were treated in the usual manner to give a tan
residue which was dissolved in CH2Cl2 and a volume of hexane
(4 × volume of CH2Cl2 used) was added. The resulting slurry was
concentrated. This was repeated three times to give 19 as an orange
3484 | Org. Biomol. Chem., 2006, 4, 3479–3488
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solid (130 mg, 79%): mp 110–113 ◦C; IR (cm−1) 3317, 2981, 1697,
1651, 1530, 1461, 1365, 1246; H NMR (DMSO-d6) d ppm 1.44
(s, 9H), 3.63 (s, 3H), 5.90 (d, 1H, J = 4.1 Hz), 6.74 (d, 1H, J =
4.1 Hz), 8.99 (s, 1H), 11.97 (s, 1H); 13C NMR (DMSO-d6) d ppm
27.9, 31.2, 79.4, 102.2, 116.0, 118.8, 133.2, 153.5, 161.8.
3H), 3.89 (s, 3H), 5.99 (m, 1H), 6.32 (bs, 1H), 6.56 (d, 1H, J =
4.1 Hz), 6.75 (d, 1H, J = 2.0 Hz), 7.40 (d, 1H, J = 1.9 Hz), 7.77
(bs, 1H); 13C NMR (CDCl3) d ppm 28.2, 31.4, 36.7, 51.1, 81.3,
103.2, 108.3, 110.7, 119.7, 121.0, 121.9, 122.8, 130.9, 153.9, 159.3,
161.6; HRMS (+ESI) m/z 377.1819 (MH+, C18H25N4O5 requires
377.1826).
1
1,2,3-Benzotriazolyl-5-[(tert-butoxycarbonyl)amino]-1-methyl-
pyrrole-2-carboxylate (19). The acid 18 (130 mg, 0.5 mmol) was
dissolved in DMF (2 ml) and HOBt (80 mg, 0.5 mmol, 1 equiv.) and
DCC (156 mg, 0.76 mmol, 1.4 equiv.) were added. The reaction
mixture was microwaved at 80 ◦C for 20 minutes in fixed time
mode at which point TLC analysis indicated reaction completion.
The reaction mixture was filtered, and then evaporated to give an
oil which was dissolved in EtOAc and passed through a silica plug
to give 19 as a brown solid (177 mg, 94%): mp 109–113 ◦C, IR
Compound 24 was also prepared from the reaction between 19
and 20 in 53% yield after purification by flash chromatography.
7-Azabenzo-1,2,3-triazolyl 4-[5-[(tert-butoxycarbonyl)amino-1-
methylpyrrole-2-yl]-carbonyl]amino-1-methylpyrrole-2-carboxylate
(25). A suspension of the methyl ester 24 (30 mg, 0.08 mmol)
in aq. NaOH (0.8 M, 1 mL, 0.8 mmol, 10 equiv.) and CH3OH
(1 ml) was heated at 50–60 ◦C overnight. The resulting yellow
solution was washed with diethyl ether and acidified with 10%
HCl. Extraction with EtOAc and the usual work up gave a brown
residue. This was dissolved in CH2Cl2 and hexane was added
and the suspension concentrated to dryness. This procedure was
repeated twice to give the corresponding acid as a pale yellow
solid. A solution of this solid (112 mg, 0.31 mmol) in anhydrous
DMF (1.5 ml) was treated with HOAt (42 mg, 0.31 mmol) and
EDCI (60 mg, 0.31 mmol). The reaction mixture was stirred
under a N2 atmosphere for 4 h, filtered through Celite into
ice–water (15 ml) and the resulting suspension was filtered to give
a pale yellow solid. This solid was dissolved in acetone (3 ml) and
added to cold hexane (15 ml). The isolated solid was redissolved
in CH2Cl2 (3 ml) and this solution was added to cold hexanes
(15 ml). The pale yellow solid was collected by filtration and dried
1
(cm−1) 2977, 2931, 1761, 1731, 1558, 1364, 1227, 1149, 1044; H
NMR (CDCl3) d ppm 1.49 (s, 9H), 3.76 (s, 3H), 6.29 (d, 1H, J =
4 Hz), 7.36–7.53 (m, 4H), 7.98 (s, 1H), 8.03 (d, 1H, J = 9 Hz);
13C NMR (CDCl3) d ppm 156.6, 152.8, 143.4, 137.3, 129.2, 128.5,
124.7, 121.4, 120.3, 112, 108.6, 103.5, 81.9, 31.7, 28.2; HRMS
(+ESI) m/z 358.1515 (MH+, C17H20N5O4 requires 358.1510).
Methyl 4-(5-nitro-1-methylpyrrole-2-carboxamido)-1-methyl-
pyrrole-2-carboxylate (22). A solution of 13 (1.608 g, 5.9 mmol)
in anhydrous THF (5 ml) was added to a stirred solution of 21
(902 mg, 5.9 mmol) in anhydrous THF (10 ml) at rt. DMAP
(142 mg, 1.2 mmol, 0.2 equiv.) was added and stirring continued.
After 2 h a yellow solid (22) had precipitated and analysis of
the reaction mixture by LC/MS showed the reaction to be
complete. Filtration gave a first crop (953 mg) and concentrating
the filtrate and crystallisation of the resulting residue from
chloroform–hexane afforded a second◦crop (507 mg). The total
yield of 22 was 1.46 g (81%): mp 166 C (dec.); IR (cm−1) 3342,
◦
in vacuo. Yield 25 (75 mg, 21% over two steps): mp 139–143 C;
1
IR (cm−1) 2981, 2358, 1687, 1555, 1447, 1390, 1243, 1157; H
NMR (CDCl3) d ppm 1.50 (s, 9H), 3.83 (s, 3H), 3.91 (s, 3H), 6.06
(d, 1H, J = 3.5 Hz), 6.24 (bs, 1H), 6.64 (d, 1H, J = 4.2 Hz), 7.25
(d, 1H, J = 1.9 Hz), 7.45 (dd, 1H, J = 4.5, 8.4 Hz), 7.76 (d, 1H,
J = 1.7 Hz), 8.45 (dd, 1H, J = 1.4, 8.4 Hz), 8.74 (dd, 1H, J = 1.4,
4.5 Hz); 13C NMR (CDCl3) d ppm 28.2, 31.5, 36.8, 81.4, 103.1,
111.0, 111.2, 113.3, 115.9, 120.8, 122.3, 123.4, 125.2, 129.5, 131.4,
135.0, 141.0, 151.7, 156.3, 159.2; HRMS (+ESI) m/z 481.1919
(MH+, C22H25N8O5 requires 481.1942).
1
1691, 1670, 1566, 1439, 1348, 1292, 1239; H NMR (DMSO-d6)
d ppm 3.75 (s, 3H), 3.86 (s, 3H), 4.15 (s, 3H), 6.88 (d, 1H, J =
4.6 Hz), 6.92 (d, 1H, J = 2.0 Hz), 7.28 (d, 1H, J = 4.6 Hz), 7.50 (d,
1H, J = 1.9 Hz), 10.53 (bs, 1H); 13C NMR (CDCl3) d ppm 35.4,
36.9, 51.2, 106.4, 108.2, 110.1, 112.4, 112.8, 120.4, 120.9, 121.2,
131.3, 161.3; HRMS (ESI+) m/z 307.1029 (MH+, C13H15N4O5
requires 307.1037).
Methyl
4-(5-acetamido-1-methylpyrrole-2-carboxamido)-1-
methylpyrrole-2-carboxylate (26). Acetic anhydride (175 lL,
190 mg, 1.9 mmol, 1.2 equiv.) and DMAP (484 mg, 4 mmol,
2.5 equiv.) were added to a solution of amine 23 (0.44 g, 1.6 mmol)
in anhydrous CH2Cl2 (15 ml) at rt under N2. Stirring was
continued for 3 h at which point LC/MS analysis showed the
reaction to be complete. The mixture was concentrated, loaded
onto a silica cartridge and eluted with EtOAc to give 26 as a
yellow oil (193 mg, 38%). IR (cm−1) 2359, 1700, 1663, 1625, 1540,
Methyl
(5-amino-1-methylpyrrole-2-carboxamido)-1-methyl-
pyrrole-2-carboxylate (23). A solution of 22 (20 mg, 0.39 mmol)
in ethanol (20 ml) containing 10% Pd–C (15 mg) was shaken
under 30 psi of H2 for 3 h. At reaction completion the mixture
was filtered through Celite and concentrated to give 23 as a red
residue, which was submitted directly to the next synthetic step.
1
1444, 1245; H NMR (DMSO-d6) d ppm 2.04 (s, 3H), 3.65 (s,
Methyl 4-{5-[(tert-butyloxycarbonyl)amino]-1-methylpyrrole-2-
carboxamido}-1-methylpyrrole-2-carboxylate (24). Triethylamine
(0.059 mL, 43 mg, 0.42 mmol, 1.1 equiv.) and Boc2O (70 mg,
0.27 mmol, 0.7 equiv.), were added sequentially and portionwise to
a solution of the amine 23 (0.39 mmol) in anhydrous 1,4-dioxane
(1.7 ml). The resulting solution was heated at 85 ◦C overnight
under N2. The reaction solution was cooled and concentrated to
give a red-brown residue. Purification by flash chromatography
(3 : 1 EtOAc–hexane) gave 24 as a pale brown solid (65 mg, 44%):
3H), 3.73 (s, 3H), 3.83 (s, 3H), 5.95 (d, 1H, J = 4 Hz), 6.84 (d, 1H,
J = 4.1 Hz), 6.87 (d, 1H, J = 1.9 Hz), 7.44 (d, 1H, J = 1.9 Hz),
9.68 (bs, 1H), 9.81 (bs, 1H); 13C NMR (DMSO-d6) d ppm 22.7,
31.5, 36.2, 50.9, 101.7, 108.2, 111.4, 118.5, 120.6, 122.1, 122.9,
131.7, 158.6, 160.7, 169.2; HRMS (+ESI) m/z 319.1409 (MH+,
C15H19N4O4 requires 319.1401).
4-(5-Acetamido-1-methylpyrrole-2-carboxamido)-1-methylpyr-
role-2-carboxylic acid (27). The methyl ester 26 (293 mg,
0.92 mmol) was suspende◦d in 1 : 1 NaOH (aq. 2 M)–CH3OH
(4.6 ml) and heated at 45 C for 2 h. The resulting solution was
◦
mp 138–139 C; IR (cm−1) 1701, 1636, 1552, 1446, 1242, 1155,
1107; 1H NMR (CDCl3) d ppm 1.50 (s, 9H), 3.75 (s, 3H), 3.80 (s,
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1
cooled and then washed with diethyl ether, acidified to pH = 1
(universal indicator paper) with 10% HCl and then extracted with
EtOAc. The organic extracts were treated in the usual manner
to give 27 as a pale brown solid which was dried in vacuo. Yield
(108 mg, 39%): mp 213–215 ◦C; IR (cm−1) 3253, 1666, 1626, 1537,
by ion-exchange chromatography to afford 4 (14 mg, 48%). H
NMR (CD3OD) d ppm 1.83 (m, 2H), 1.92 (s, 3H), 2.05 (s, 6H),
2.50 (t, 2H, J = 6.4 Hz), 2.86–2.90 (m, 2H), 3.27 (m, 2H), 3.58 (t,
2H, J = 5.9 Hz), 3.87–3.91 (m, 15H), 6.84 (m, 2H), 6.96 (m, 2H),
7.13 (d, 1H, J = 1.2 Hz), 7.17 (d, 1H, J = 1.2 Hz), 7.21–7.20 (m,
4H); HRMS (+ESI) m/z 826.4091 (MH+, C40H52N13O7 requires
826.4107).
1
1448, 1250; H NMR (acetone-d6) d ppm 1.95 (s, 3H), 3.64 (s,
3H), 3.75 (s, 3H), 5.86 (d, 1H, J = 4 Hz), 6.72 (s, 1H), 6.77 (s, 1H),
7.31 (s, 1H), 9.20 (bs, 1H), 9.34 (bs, 1H), 11.65 (bs, 1H); 13C NMR
(acetone-d6) d ppm 24.0, 33.0, 37.6, 103.3, 110.1, 112.9, 121.7,
121.8, 121.9, 125.1, 133.9, 160.7, 164.4, 170.6; HRMS (+ESI) m/z
305.1220 (MH+, C14H17N4O4 requires 305.1244).
AcPy6bAlaDp (5). PAMbAlaPy6Boc (0.52 mmol) was pre-
pared from PAMbAlaBoc resin employing stepwise coupling of
29 (twice), activated by stirring with HBTU and HOBt in DMF
prior to addition to resin, and then 21 (twice). Deprotection
and acetylation using mixture B (4 × 1 h treatments) followed.
Polyamide isolation gave an oily solid of which a sample was
purified by silica column chromatography eluting with 3 : 6 :
1 CH3OH–CH2Cl2–Et3N to afford 5 (3 mg, 43%). 1H NMR
(CD3OD) d ppm 1.69 (m, 2H), 1.92 (s, 3H), 2.07 (s, 6H), 2.34
(m, 2H), 2.48 (t, 2H, J = 6.5 Hz), 3.22 (m, 2H), 3.56 (t, 2H, J =
6.7 Hz), 3.88–3.93 (m, 18H), 6.78 (d, 1H, J = 1.8 Hz), 6.84 (d, 1H,
J = 1.8 Hz), 6.94–6.95 (m, 4H), 7.13 (d, 1H, J = 1.7 Hz), 7.21–
7.19 (m, 5H); HRMS (+ESI) m/z 948.4621 (MH+, C46H58N15O8
requires 948.4588).
7-Azabenzo-1,2,3-triazolyl 4-(5-acetamido-1-methylpyrrole-2-
carboxamido)-1-methylpyrrole-2-carboxylate (28).
A solution
of the acid 27 (80 mg, 0.25 mmol) in DMF (3 ml) was treated
successively with HOAt (34 mg, 0.25 mmol, 1 equiv.) and EDCI
(48 mg, 0.25 mmol, 1 equiv.) and the resulting mixture was stirred
at rt overnight under N2. The red suspension was filtered into
a stirred ice–water mixture (15 ml) at which point a pale yellow
solid precipitated. The ice–water mixture was allowed to melt and
was then filtered and the isolated solid partially dissolved in a
small volume of CH2Cl2 and added to ice-cold hexanes (15 ml).
The resulting suspension was filtered to give 28 as a pale yellow
solid which was dried under suction and then in vacuo. Yield 28
(70 mg, 66%): mp 159–162 ◦C; IR (cm−1) 3322, 2361, 1780, 1666,
1555, 1392, 957; 1H NMR (CDCl3) d ppm 8.74 (dd, 1H, J = 1.1,
4.5 Hz), 8.45 (dd, 1H, J = 1.1, 8.4 Hz), 7.83 (s, 1H), 7.76 (d, 1H,
J = 1.3 Hz), 7.45 (dd, 1H, J = 4.5, 8.3 Hz), 7.12 (s, 1H), 6.65 (d,
1H, J = 4.1 Hz), 6.09 (d, 1H, J = 4.1 Hz), 3.91 (s, 3H), 3.80 (s,
3H), 2.22 (s, 3H); 13CNMR (CDCl3) d ppm 23.1, 31.7, 36.7, 103.2,
111.3, 111.7, 112.9, 120.7, 122.6, 123.9, 125.2, 126.0, 129.4, 131.2,
134.9, 140.9, 151.6, 156.3, 170.1; HRMS (+ESI) m/z 423.1543
(MH+, C19H19N8O4 requires 423.1524)
2,5-Pyrrole polyamides (AcPyn = 2–6bAlaDp, 6–10)
Ac(2,5-Py)PybAlaDp (6). Resin PAMbAlaBoc (26 mg,
0.02 mmol) was deprotected and coupled to 28 (24 mg,
0.057 mmol, 3 equiv.) with addition of DIPEA (0.041 mL, 30 mg,
0.24 mmol, 12 equiv.) and shaking overnight. The resin was then
capped by shaking with an acetylation mixture containing Ac2O
(12 equiv.) and DIPEA (12 equiv.) in DMF for 1 h. Polyamide
isolation and then purification by ion-exchange chromatography
gave 6 (3 mg, 33%). 1H NMR (CD3OD) d ppm 1.76 (m, 2H), 1.92
(s, 3H), 2.16 (s, 6H), 2.60 (m, 2H), 3.33 (m, 2H), 3.42 (t, 2H, J =
6.8 Hz), 3.57 (t, 2H, J = 6.7 Hz), 3.73 (s, 3H), 3.87 (s, 3H), 6.01
(d, 1H, J = 4.2 Hz), 6.78 (d, 1H, J = 1.9 Hz), 6.81 (d, 1H, J =
4.1 Hz), 7.15 (d, 1H, J = 1.7 Hz); HRMS (+ESI) m/z 460.2675
(MH+, C22H34N7O4 requires 460.2667).
AcPy2bAlaDp (1)41. Resin PAMbAlaPy2Boc (0.032 mmol)
was deprotected and acetylated. Polyamide isolation followed by
purification by ion-exchange chromatography afforded 1 (6 mg,
43%). 1H NMR (CD3OD) d ppm 1.66–1.72 (m, 2H), 2.07 (s, 3H),
2.20 (s, 6H), 2.36 (m, 2H), 2.44–2.49 (m, 2H), 3.19–3.26 (m, 2H),
3.55 (t, 2H, J = 6.7 Hz), 3.87 (s, 3H), 3.89 (s, 3H), 7.18 (d, 1H, J =
1.6 Hz), 7.13 (d, 1H, J = 1.6 Hz), 6.81 (d, 1H, J = 1.6 Hz), 6.76 (d,
1H, J = 1.7 Hz); HRMS (+ESI) m/z 460.2663 (MH+, C22H34N7O4
requires 460.2667).
Ac(2,5-Py)Py2bAlaDp (7). Prepared from PAMbAlaPyBoc
(29 mg, 0.019 mmol) by coupling to 20 (two cycles required of
45 minutes’ duration) and then 19 (45 minutes). Acetylation,
cleavage and polyamide isolation and purification gave 7 (6 mg,
55%). 1H NMR (CD3OD) d ppm 1.71 (m, 2H), 1.92 (s, 3H), 2.21
(s, 6H), 2.46 (m, 2H), 2.62 (m, 2H), 3.56 (t, 2H, J = 6.8 Hz), 3.75
(s, 3H), 3.88 (s, 3H), 3.91 (s, 3H), 6.02 (d, 1H, J = 4.1 Hz), 6.77
(d, 1H, J = 1.9 Hz), 6.82 (d, 1H, J = 4.1 Hz), 6.92 (d, 1H, J =
1.9 Hz), 7.19 (m, 2H), 7.99 (bs, 1H); HRMS (+ESI) m/z 582.3148
(MH+, C28H40N9O5 requires 582.3147).
AcPy4bAlaDp (3). Resin PAMbAlaPy3Boc (0.033 mmol) was
deprotected and coupled to 20 (4 equiv.) with addition of
DIPEA (12 equiv.) and shaking for 45 minutes. Deprotection
and acetylation was followed by cleavage, polyamide isolation and
purification by ion-exchange chromatography to give 3 (5 mg,
22%). 1H NMR (CD3OD) d ppm 1.69 (m, 2H), 1.92 (s, 3H), 2.07
(s, 6H), 2.31–2.42 (m, 2H), 2.47 (t, 2H, J = 6.5 Hz), 3.21 (m,
2H), 3.55 (t, 2H, J = 6.7 Hz), 3.87–3.91 (m, 12H), 6.79–6.86
(m, 4H), 7.15–7.22 (m, 4H); HRMS (+ESI) m/z 704.3647 (MH+,
C34H46N11O6 requires 704.3627).
Ac(2,5-Py)Py3bAlaDp (8). Prepared from the resin
PAMbAlaPy2Boc (0.0133 mmol) by coupling to 29 for 90 minutes.
Cleavage and polyamide isolation gave 8 (5 mg, 54%). H NMR
(CD3OD) d ppm 1.73 (m, 2H), 2.14 (s, 3H), 2.35 (s, 6H), 2.46–2.54
(m, 4H), 3.21–3.24 (m, 2H), 3.56 (t, 2H, J = 6.6 Hz), 3.73 (s,
3H), 3.86 (s, 3H), 3.90 (s, 3H), 3.90 (s, 3H), 6.01 (d, 1H, J =
4.1 Hz), 6.78 (d, 1H, J = 1.9 Hz), 6.82 (d, 1H, J = 4.1 Hz),
6.928–6.932 (m, 2H), 7.17–7.19 (m, 3H); HRMS m/z 704.3659
(MH+, C34H46N11O6 requires 704.3627).
1
AcPy5bAlaDp (4). Resin PAMbAlaPy4Boc (0.035 mmol) was
deprotected and coupled to 20 (4 equiv.) with addition of
DIPEA (12 equiv.) and shaking for 45 minutes. Deprotection and
acetylation was followed by polyamide isolation and purification
3486 | Org. Biomol. Chem., 2006, 4, 3479–3488
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Ac(2,5-Py)Py4bAlaDp
(9). Prepared
from
resin
10 R. T. Wheelhouse, D. Sun, H. Han, F. X. Han and L. H. Hurley, J. Am.
Chem. Soc., 1998, 120, 3261–3262; F. X. Han, R. T. Wheelhouse and
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S. M. Gowan, L. R. Kelland and M. F. G. Stevens, J. Med. Chem.,
2002, 45, 590–597; R. A. Heald and M. F. G. Stevens, Org. Biomol.
Chem., 2003, 1, 3377–3389.
12 J.-L. Mergny, L. Lacroix, M.-P. Teulade-Fichou, C. Hounsou, L.
Guittal, M. Hoarau, P. B. Arimondo, J.-P. Vigneron, J.-M. Lehn, J.-
F. Riou, T. Garestier and C. He´le`ne, Proc. Natl. Acad. Sci. U. S. A.,
2001, 98, 3062–3067; J.-F. Riou, L. Guittat, P. Mailliet, A. Laoui, E.
Renou, O. Petitgenet, F. Me´gnin-Chanet, C. He´le`ne and J.-L. Mergny,
Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 2672–2677.
PAMbAlaPy2Boc (0.027 mmol) by coupling to 20, a two-
fold split and then coupling to 29 for 90 minutes. Cleavage and
polyamide isolation gave 9 (4 mg, 18%). 1H NMR (CD3OD)
d ppm 1.77 (m, 2H), 2.16 (s, 3H), 2.47 (s, 6H), 2.49 (t, 2H, J =
6.4 Hz), 2.65 (m, 2H), 3.25 (t, 2H, J = 6.7 Hz), 3.57 (t, 2H, J =
6.6 Hz), 3.72 (s, 3H), 3.84 (s, 3H), 3.88 (s, 3H), 3.89 (s, 6H), 6.02
(d, 1H, J = 4.1 Hz), 6.81 (d, 1H, J = 1.8 Hz), 6.83 (d, 1H, J =
4.1 Hz), 6.95–6.96 (m, 3H), 7.18 (d, 1H, J = 1.8 Hz), 7.20 (d,
1H, J = 1.8 Hz), 7.21 (d, 2H, J = 1.8 Hz); HRMS m/z 826.4110
(MH+, C40H52N13O7 requires 826.4107).
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Furihata, Y. Hayakawa and H. Seto, J. Am. Chem. Soc., 2001, 123,
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Med. Chem. Lett., 1999, 9, 2463–2468; M. A. Read, R. J. Harrison, B.
Romagnoli, F. A. Tanious, S. H. Gowan, A. P. Reszka, W. D. Wilson,
L. R. Kelland and S. Neidle, Proc. Natl. Acad. Sci. U. S. A., 2001,
98, 4844–4849; R. J. Harrison, J. Cuesta, G. Chessari, M. A. Read,
S. K. Basra, A. P. Reszka, J. Morrell, S. M. Gowan, C. M. Incles, F. A.
Tanious, W. D. Wilson, L. R. Kelland and S. Neidle, J. Med. Chem.,
2003, 46, 4463–4476; M. J. B. Moore, C. M. Schultes, J. Cuesta, F.
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Ac(2,5-Py)Py5bAlaDp
(10). Prepared
from
resin
PAMbAlaPy3Boc (0.0133 mmol) by coupling to 20 and
then 29 for 135 minutes. Cleavage and polyamide isolation gave
1
10 (5 mg, 40%). H NMR (CD3OD) d ppm 1.66 (m, 2H), 2.14
(s, 3H), 2.19 (s, 6H), 2.31 (t, 2H, J = 7.9 Hz), 2.45 (t, 2H, J =
6.7 Hz), 2.71 (m, 2H), 3.53 (t, 2H., J = 6.5 Hz), 3.74 (s, 3H),
3.86 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H), 3.91 (s, 6H), 6.00 (d, 1H,
J = 4.1 Hz), 6.75 (d, 1H, J = 1.7 Hz), 6.81 (d, 1H, J = 4.1 Hz),
6.92–6.93 (m, 4H), 7.18 (m, 5H); HRMS (+ESI) m/z 948.4636
(MH+, C46H58N15O8 requires 948.4587).
15 E. S. Baker, J. T. Lee, J. L. Sessler and M. T. Bowers, J. Am. Chem. Soc.,
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Acknowledgements
This work has been supported by project and programme grants
to S. N. from Cancer Research UK.
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The Royal Society of Chemistry 2006
©