A new synthesis of alkene-containing minor-groove binders and essential hydrogen bonding in binding to DNA and in antibacterial activity

Article abstract of DOI:10.1039/b901898k

A practical synthesis of alkene-containing minor-groove binders for DNA, related to distamycin, with potential for wide structural diversity is described, based upon the Wittig chemistry of N-alkylpyrrole aldehydes. The compounds prepared have been evalua

Full text of DOI:10.1039/b901898k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A new synthesis of alkene-containing minor-groove binders and essential
hydrogen bonding in binding to DNA and in antibacterial activity†
Nahoum G. Anthony,b David Breen,a Gavin Donoghue,a Abedawn I. Khalaf,a Simon P. Mackay,b
John A. Parkinsona and Colin J. Suckling*a
Received 29th January 2009, Accepted 16th February 2009
First published as an Advance Article on the web 12th March 2009
DOI: 10.1039/b901898k
A practical synthesis of alkene-containing minor-groove binders for DNA, related to distamycin, with
potential for wide structural diversity is described, based upon the Wittig chemistry of N-alkylpyrrole
aldehydes. The compounds prepared have been evaluated for binding to DNA by physical methods
(melting temperature and NMR) and for their antibacterial activity. Significantly, it was found that
alkenes linking the aryl head group of the minor-groove binder promote strong binding to DNA and
high antibacterial activity against Gram-positive bacteria. Conversely, a minor-groove binder
containing an alkene located towards the alkylamino tail group has a low affinity for DNA and does
not show antibacterial activity. These observations suggest an important role for specific hydrogen
bonds in the binding of compounds of this type to DNA, and in their antibacterial activity.
transformations (Scheme 1).5 Also using established methods,3
the morpholinoethyl side chain was added (Scheme 2).
Introduction
Minor-groove binders related to the natural product distamycin
(1) have attracted much attention as antibacterial compounds.1
Structural variation has typically involved increasing the hy-
drophobicity of the analogues by introducing larger N-alkyl
groups than methyl, and larger and more hydrophobic head groups
than formyl. The importance of a low pKa basic tail group has also
been emphasised.2 Recently, we reported that the replacement of
an amide by the isosteric, but non hydrogen bonding, alkene at
the head-group link led to a series of highly active antibacterial
minor-groove binders of which 2 and 3 are examples.3 These
compounds are approximately 100-fold more active than their
corresponding amide isosteres, 4 and 5. The question was therefore
raised whether isosteric substitutions of other amide links in the
distamycin template would be acceptable. To answer this, it was
necessary to develop new synthetic methods for such compounds,
which we refer to as ‘internal alkenes’ to distinguish them from
the head-group alkenes of 2 and 3.
Scheme 1 Synthesis of a key intermediate for the preparation of internal
alkene minor-groove binders.
Results and discussion
The incorporation of 6 into the required minor-groove binders
necessitated the development of reaction conditions for the selec-
tive reduction of the nitro group in the presence of the alkene. After
much experimentation, it was found that reduction with sodium
borohydride in the presence of palladium-on-charcoal6 afforded
clean reduction of the nitro group with no reduction of the alkene,
provided the reaction time was controlled; extended reaction times
led to reduction of the alkene also. The aminopyrrole alkene dimer
7 was then coupled with a range of head groups to afford the
required internal alkene minor-groove binders which were purified
by HPLC (Schemes 2 and 3: see also ESI†); in this way, the
internal alkene analogue 9 of compounds 3 and 5 was prepared.
The over-reduced dimer 8 was also coupled, affording compound
10 with a flexible link between two pyrrole rings; 10 provides
an additional point of comparison for determining the structural
features required for antibacterial activity. In addition to the amide
Although minor-groove binders with internal alkenes have been
prepared before,4 the method did not lend itself to the flexible
synthesis of our target compounds in adequate yields. A practical
route to the key intermediate, 6, was developed from 2-formyl-
N-methylpyrrole in six steps using standard functional group
aWestCHEM, Department of Pure & Applied Chemistry, University of
Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow, G1
1XL, Scotland. E-mail: c.j.suckling@strath.ac.uk; Fax: +44 141 548 5743;
Tel: +44 141 548 2271
bStrathclyde Institute of Pharmacy and Biomedical Sciences, The John
Arbuthnott Building, 27 Taylor Street, Glasgow, G4 0NR, Scotland. E-mail:
simon.mackay@strath.ac.uk; Tel: +44 141 548 2866
† Electronic supplementary information (ESI) available: Scanned1H NMR
spectra of new compounds. See DOI: 10.1039/b901898k
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Scheme 2 Elaboration of intermediate 6 into an internal alkene minor-groove binder, 9, and its reduced alkyl analogue, 10.
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Scheme 3 Synthesis of the first bis-alkenyl minor-groove binder.
head group, that provides the direct analogue of 3, an alkenyl
head group containing a thiazole was also attached to 7 via the
thiazole–quinoline dimer 11 (Scheme 3).7 This provides, to our
knowledge, the first bis-alkenyl minor-groove binder, 12, based
upon the distamycin template. The head-group alkenyl minor-
groove binder, 13, was also prepared.
The antibacterial activity of the internal alkene minor-groove
binders 9 and 12 and the alkyl-linked compound 10 was compared
with that of the corresponding head-group alkene minor-groove
binders (MGBs) 3 and 13 against a group of Gram-positive
bacteria, the type of bacterium that is most susceptible to this
class of MGB. The lack of activity of the flexible alkyl-linked
compound 10 was to be expected but emphasised the importance
of an intrinsically and stably curved ligand for binding to DNA.
Surprisingly, however, the internal alkene MGBs 9 and 12 were
essentially inactive (Table 1). It is notable that in 12, the beneficial
presence of the head-group alkene does not compensate for the
deleterious effect of the internal alkene, suggesting the importance
of a specific interaction between DNA and the MGB at the
position of the internal alkene.
If DNA is the crucial target for antibacterial activity of these
compounds, it would be expected that the binding to DNA might
be affected by such structural changes. Studies of the melting
temperatures (Tm) suggest that this is a contributor to the lack
of antibacterial activity of the internal alkenes.8 Using a DNA
target of GCGATATATGCG/CGCTATATACGC (target 1), the
results shown in Table 2 were obtained. The target was chosen
to have a Tm sufficiently above room temperature to give reliable
data and to have a binding site appropriate for predominantly
pyrrole-containing MGBs, which prefer AT-rich regions. These
data show that the increase in Tm caused by binding the internal
alkene MGB 9 is very much lower than that of the corresponding
compound 3 with the internal amide. Such a result could be
explained either by a molecular interaction such as a specific
hydrogen bond or by a significant change of shape of the internal
alkene MGB. With respect to the possibility of hydrogen bonding,
a similar phenomenon was evident in the antibacterial activity of
the quinolyl MGB 2 and its naphthyl analogue 14;9 in this case, the
Table 1 Antibacterial activity (MIC, mg mL-1) of alkenyl MGBs against
two strains of Staphylococcus aureus and one of Streptococcus faecalis
Table 2 Tm measurements to target 1
Compound
S. aureus 1
S. aureus 2
S. faecalis
Compound
Tm Observed/◦C
DTm/◦C
9
>100
>100
>100
6.25
>100
>100
>100
12.5
100
>100
100
50
No ligand
47.0
59.0
47.0
67.5
47.0
—
10.0
0
20.5
0
10
12
3
5
9
2
13
3.12
6.25
25
14
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non-hydrogen-bonding naphthyl compound 14 was also inactive.
In parallel with the antibacterial activity, a significant stabilisation
of target 1 oligomer was only found with 2, which contains the
hydrogen-bonding quinolyl group. A similar comparison of 12
and 13 by Tm measurement has not yet been possible because the
DNA target of these MGBs is not known.
To evaluate the possibility that the shape of the internal alkene
MGB might differ significantly from its amide prototype, molec-
ular modeling was carried out. Structures for compounds 3 and 9
were calculated using Gaussian 03 using a b3lyp Hamiltonian and
a 6-31G* basis set10 It is known that the morpholinoethyl side-
chain of these MGBs does not make ordered contacts with DNA
when bound3 and consequently, superimposition of the amide and
alkene isosteres concentrated upon the regions containing the head
group and the first two pyrroles (stopping at the alkene/amide
isostere). Fig. 1 shows the result and indicates that the head
group and first two pyrroles are essentially superimposable, with
conformational change starting at the alkene/amide bond towards
the morpholino tail. This confirms the expectation that there is
no substantial change in conformation between internal alkene
compounds compared with their isosteres and conformational
change is therefore unlikely to account for the reduced DNA
binding of internal alkene MGBs. Attention then focused on the
possibility of specific hydrogen bonds being involved. This has
been investigated by NMR.
Fig. 2 NOEs to H25 and H17 recorded for the 2 : 1 complex formed
between 3 and the decamer self-complementary oligonucleotide sequence
shown. Assignments are colour-coded as follows: ligand-to-DNA contact
(red); intra-ligand contact (black); inter-ligand contact (green). NMR data
were acquired at 500 MHz using a mixing time of 50 ms and recorded at
a sample temperature of 7 ◦C. Atom numbering is shown only for those
atoms associated with NOEs in this figure.
this interpretation, as noted above in previous work3,9 we found
that a quinolyl head group (1) gave high antibacterial activity, but
the corresponding compound with a naphthyl head group (14) was
inactive. This again suggests the importance of a hydrogen-bond
acceptor in this region of the molecule as we have argued before.2
Moreover, there is evidence for such a hydrogen bond in the NMR
study of 3, which also shows a significant NOE between the aryl
methoxy group and A3NH.
Fig. 1 Superimposition of 3 (green) on 9 (blue) maximising the overlap
of the head and central part of the MGBs. The key alkene and amide
bonds are coloured red. The major differences are to be seen at the tail
(right-hand side) which is known to be disordered from NMR studies.3
Taken together, these results indicate that potent antibacterial
activity of alkene-containing MGBs requires a contribution not
only from hydrophobic interactions, as shown by the effect of head
groups, but also specific hydrogen bonds, as shown by the amide
link between the two pyrrolyl amino acids next to the alkylamino
tail group.
The head-group alkene MGB 3 was studied bound 2 : 1 to a sim-
ilar recognition sequence as target 1, but a shorter sequence more
suited to NMR experiments (target 2, the self-complementary
oligonucleotide, CGA3TAT6A7T8CG) showed significant NOEs
between ligand NH of the inter-pyrrole peptide bond (replaced in
9 by an alkene) and DNA hydrogens T6H1¢, A7H4¢, A7H1¢, A7H2
and T8H1¢ (Fig. 2). Importantly, these data establish the peptide
bond as oriented with NH on the concave edge of the ligand, thus
making it available to hydrogen-bond acceptors on the floor of the
DNA minor groove. Whilst NOEs do not prove the presence of
hydrogen bonds, our NMR-based solution structure calculated for
the DNA complex of 3 implies that the most likely H-bond of this
Experimental
General
1H-, 13C-, and 31P-NMR were carried out on a Bruker DPX-
400 MHz spectrometer with chemical shifts given in ppm (d
values), relative to proton and carbon traces in solvent or, in the
case of 31P, phosphoric acid was used as an external standard
(d = 0). Coupling constants are reported in Hz. IR spectra were
recorded on a Perkin Elmer, 1 FT-IR spectrometer. Elemental
analysis was carried out on a Perkin Elmer 2400, analyser series 2.
8
˚
NH is to the H-bond acceptor T O2 (model distance = 2.16 A).
In addition, the proton chemical shift of 9.8 ppm is consistent
with expectations for a hydrogen-bonded peptide NH, and is in
agreement with chemical shift values for H-bonded NH in ligands
of a similar nature bound to DNA.9 Further, and consistent with
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Table 3 Programme for HPLC purification of MGBs
Ice-water (5 mL) was then added, at which point the product
precipitated and was collected by filtration, before being dried
under reduced pressure (0.44 g, 81%) m.p. = 158–161 ◦C. nmax
(NaCl): 3129, 2971, 2620, 1680, 1554, 1532, 1488, 1512, 1318,
1258, 1209, 1441 cm-1. dH (CDCl3): 1.36 (3H, t, CH2CH3, J = 7.1),
3.69 (3H, s, N-Me), 3.93 (3H, s, N-Me), 4.31 (2H, q, CH2CH3, J =
Time/min
A
B
Flow rate/mL min-1
0
28
33
38
40
90
30
10
90
90
10
70
90
10
10
4
4
4
4
0
=
=
7.1), 6.52 (1H, d, CH CH, J = 16.0), 6.77 (1H, d, CH CH, J =
16.0), 6.83 (1H, d, Ar-H, J = 1.6), 6.88 (1H, d, Ar-H, J = 1.6), 7.10
(1H, d, Ar-H, J = 1.6), 7.45 (1H, d, Ar-H, J = 1.6). HRFABMS:
Found 303.1221; C15H17N3O4 requires 303.1219.
Mobile phase, A = Water + 0.1% TFA, B = Acetonitrile + 0.1% TFA.
1-Methyl-4-[(E)-2-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethenyl]-1H-
pyrrole-2-carboxylic acid 6
Mass spectra were obtained on a Jeol JMS AX505. Anhydrous
solvents were obtained from a Puresolv purification system, from
Innovative Technologies, or purchased as such from Aldrich.
Melting points were recorded on a Reichert hot-stage microscope,
and are uncorrected. Chromatography was carried out using 200–
400 mesh silica gels, or using reverse-phase HPLC on a water
system using a C18 Luna column with the gradient given in Table 3.
Ethyl 1-methyl-4-[(E)-2-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethe-
nyl]-1H-pyrrole-2-carboxylate (0.200 g, 0.63 mmol) was dissolved
in ethanol (3 mL). To this solution, sodium hydroxide (0.072 g,
1.81 mmol) in water (5 mL) was added and the solution was
heated under reflux for 1 h. The solution was then cooled to
◦
0 C at which point a precipitate formed. This was collected by
(1-Methyl-4-nitro-1H-pyrrol-2-yl)methanol11
filtration and dissolved in water (10 mL) the solution was then
cooled to 0 ◦C and dilute hydrochloric acid added until a pH
of 2 was reached, at which point the product 6 precipitated as a
yellow solid (0.110 g, 67%) m.p. = no distinct m.p., decomposes
at >230 ◦C. nmax (NaCl): 3200–2800, 3134, 2923, 2613, 1670,
1557, 1536, 1493, 1517, 1312, 1259 cm-1. dH (CDCl3): 3.71 (3H, s,
1-Methyl-4-nitro-1H-pyrrole-2-carbaldehyde
6
(0.40
g,
2.08 mmol) was placed in anhydrous ethanol (50 mL) under
nitrogen. Sodium borohydride (0.04 g, 1.04 mmol) was added in
small portions over 5 min and the solution allowed to stir for
20 min. Water (10 mL) was then added slowly to quench the
reaction and the solution was then extracted with ethyl acetate
(2 ¥ 20 mL). The organic layer was then dried (MgSO4), filtered,
and the solvent removed under reduced pressure to yield the
required product as a light-brown solid (0.32 g, 98%), m.p. =
=
N-Me), 3.83 (3H, s, N-Me), 6.74 (1H, d, CH CH, J = 16.0), 6.89
=
(1H, d, Ar-H, J = 1.6), 6.90 (1H, d, CH CH, J = 16.0), 7.13 (1H,
d, Ar-H, J = 1.6), 7.23 (1H, d, Ar-H, J = 1.6), 7.92 (1H, d, Ar-H,
J = 1.6), 12.30 (1H, s, COOH). HRFABMS: Found 275.0905;
C13H13N3O4 requires 275.0906. Found: C, 56.28; H, 4.51.; N,
15.64; C13H13N3O4 requires C, 56.73; H, 4.76; N, 15.27%.
◦
89–90 ◦C, (Lit. 90.5–91.5 C).11 nmax (KBr): 3521, 3131, 2934,
2888, 1490, 1412, 1520, 1337 cm-1. dH (CDCl3): 3.67 (3H, s,
N-Me), 4.40 (2H, d, CH2, J = 5.4), 5.18 (1H, t, OH, J = 3.0),
6.57 (1H, d, Ar-H, J = 1.6), 7.92 (1H, d, Ar-H, J = 1.6).
1-Methyl-4-[(E)-2-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethenyl]-N-
[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide
Diethyl (1-methyl-4-nitro-1H-pyrrol-2-yl)methylphosphonate
1-Methyl-4-[(E)-2-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethenyl]-1H-
pyrrole-2-carboxylic acid 6 (0.400 g, 1.51 mmol) and ethy-
laminomorpholine (196 mL, 1.52 mmol) were dissolved in DMF
(2.0 mL), HBTU (1.137 g, 3.00 mmol) was added and the solution
was allowed to stir overnight. Water (5.0 mL) was then added
and the solution extracted with ethyl acetate (2 ¥ 10 mL); the
organic layer was dried over (MgSO4), and the solvent removed
under reduced pressure. The residue was dissolved in ethyl acetate,
and hexane was added until the product precipitated as an orange
solid (0.360 g, 62%) m.p. >230 ◦C. nmax (KBr): 3429, 3143, 2925,
1630, 1518, 1300, 845 cm-1. dH (DMSO): 3.15 (2H, d, CH2, J =
11.5), 3.28 (2H, m, CH2), 3.32 (4H, m, 2(CH2), 3.65 (2H, t,
CH2, J = 11.5), 3.68 (3H, s, N-Me), 3.84 (3H, s, N-Me), 4.02
(1-Methyl-4-nitro-1H-pyrrol-2-yl)methanol (0.10, 0.64 mmol) was
dissolved in dichloromethane (5 mL), and thionyl chloride (5 mL)
was added slowly. The solution was then heated under reflux
for 15 min, and the excess thionyl chloride was removed under
reduced pressure. The residue was then heated under reflux in
triethylphosphite (3 mL) for 1 h at 160 ◦C, and the excess
triethylphosphite was removed under vacuum (1.5 mmHg at 70 ◦C)
to give the desired product initially as a brown oil, which solidified
after 48 h at 0–4 ◦C to a brown semi-solid (0.17 g, 98%). nmax
(NaCl): 3137, 2985, 1556, 1438, 1519, 1346, 1308, 1163 cm-1. dH
(CDCl3): 1.20 (6H, t, CH3, J = 6.8), 3.08 (2H, d, (CH2)P, J = 20.4),
3.65 (3H, s, N-Me), 4.01 (4H, q, (CH2)CH3, J = 6.8), 6.54 (1H, d,
Ar-H, J = 1.6), 7.39 (1H, d, Ar-H, J = 1.6), dP (CDCl3), 23.44.
HRFABMS: Found 276.0875; C10H17N2O5P requires 276.0873.
=
(2H, d, CH2, J = 11.5), 6.60 (1H, d, (C CH), J = 16.2), 6.93
=
(3H, m, 2Ar-H and C CH), 7.20 (1H, d, Ar-H, J = 1.8), 7.96
(1H, d, Ar-H, J = 1.8), 8.33 (1H, broad s, NH). HRFABMS:
Found 388.1983; C19H26N5O4+ requires 388.1985. Found: C, 58.19;
H, 6.07; N, 18.42; C19H25N5O4 requires C, 58.90; H, 6.50; N,
18.08%.
Ethyl 1-methyl-4-[(E)-2-(1-methyl-4-nitro-1H-pyrrol-2-
yl)ethenyl]-1H-pyrrole-2-carboxylate
(1-Methyl-4-nitro-1H-pyrrol-2-yl)methylphosphonate (0.50 g,
1.81 mmol) and 4-formyl-1-methyl-1H-pyrrole-2-carboxylate 8
(0.28 g, 1.84 mmol) were taken up in THF (4 mL) under nitrogen,
sodium hydride (0.26 g, 10.83 mmol) was then added in small
portions over 5 min and the solution heated under reflux for 1 h.
Methyl 4-[(3-methoxybenzoyl)amino]benzoate
m-Anisoylchloride (1.038 g, 5.86 mmol) was dissolved in
dichloromethane (25 mL), methyl 4-aminobenzoate (0.886 g,
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5.86 mmol) and triethylamine (1.3 mL, 10.0 mmol).
Dichloromethane (10 mL) was then added dropwise at 0 ◦C and
the solution allowed to return to room temperature overnight,
during this time the product precipitated as a white solid (1.236 g,
74%) m.p. = 79–81 ◦C. nmax (KBr): 3467, 3356, 3070, 2846, 2831,
1709, 1500, 1450 cm-1. dH (DMSO): 3.83 (6H, s, 2OCH3), 7.17
(1H, d of d, Ar-H, J = 2.5 and 8.1), 7.49 (3H, m, Ar-H), 7.93 (4H,
4-{(E)-2-[4-({4-[(3-Methoxybenzoyl)amino]benzoyl}amino)-1-
methyl-1H-pyrrol-2-yl]ethenyl}-1-methyl-N-[2-(4-morpholinyl)-
ethyl]-1H-pyrrole-2-carboxamide trifluoroacetate 9
◦
Yield: 0.031 g, 15%, m.p.>230 C, purity by HPLC = 97% nmax
(KBr): 3421, 2995, 1675, 1675, 1523 cm-1.. dH (DMSO): 3.20 (2H,
m, CH2), 3.34 (2H, m, CH2), 3.58 (4H, m, 2(CH2), 3.68 (3H, s, N-
Me), 3.75 (2H, t, CH2, J = 12.2), 3.90 (3H, s, N-Me), 3.92 (3H, s,
N-Me), 4.02 (2H, d, CH2, J = 12.9 Hz), 6.50 (1H, d, Ar-H, J =
+
m, Ar-H), 10.52 (1H, s, NH). LRMS: Found 286.0; C16H15NO4
requires 286.1.
=
=
1.7), 6.67 (1H, d, (C CH), J = 16.1 Hz), 6.72 (1H, d, (C CH),
J = 16.1), 7.09 (1H, d, Ar-H, J = 1.7), 7.23 (1H, d, Ar-H, J =
1.7), 7.26 (1H, d, Ar-H, J = 1.7), 7.55 (2H, m, Ar-H), 7.63 (1H, m,
Ar-H), 7.98 (4H, m, Ar-H), 8.33 (1H, t, NH, J = 5.5), 9.83 (1H, s,
N+H), 10.18 (1H, s, NH), 10.52 (1H, s, NH). HRFABMS: Found
4-[(3-Methoxybenzoyl)amino]benzoic acid3
Methyl
4-[(3-methoxybenzoyl)amino]benzoate
(1.03
g,
3.80 mmol) was dissolved in ethanol (5 mL); a solution of
sodium hydroxide (0.152 g, 3.80 mmol) in water (15 mL) was then
added and the solution allowed to stir overnight. The solution
was then cooled to 0 ◦C and acidified using dilute hydrochloric
acid solution, until the product precipitated as a white solid
(1.236 g, 74%) m.p. = 89–91 ◦C. nmax (KBr): 3381, 2961, 1690,
1583, 1311 cm-1. dH (DMSO): 3.85 (3H, s, OCH3), 7.17 (1H, d
of d, Ar-H, J = 2.5 and 8.1), 7.50 (3H, m, Ar-H), 7.93 (4H, m,
+
611.3134; C34H39N5O4 requires 611.3133.
4-{2-[4-({4-[(3-Methoxybenzoyl)amino]benzoyl}amino)-1-methyl-
1H-pyrrol-2-yl]ethyl}-1-methyl-N-[2-(4-morpholinyl)ethyl]-1H-
pyrrole-2-carboxamide trifluoroacetate 10
◦
Yield: 0.024 g, 12%, m.p. >230 C, purity by HPLC = 98% nmax
(KBr): 3315, 3020, 1674, 1523 cm-1. dH (DMSO): 2.73 (2H, m,
CH2), 2.77 (2H, m, CH2), 3.20 (2H, m, CH2), 3.31 (2H, m, CH2),
3.54 (4H, m, 2(CH2), 3.58 (3H, s, N-Me), 3.71 (2H, t, CH2, J =
12.2), 3.86 (3H, s, N-Me), 3.91 (3H, s, N-Me), 4.08 (2H, d, CH2,
J = 12.9), 6.02 (1H, d, Ar-H, J = 1.7), 6.75 (1H, d, Ar-H, J =
1.7), 6.89 (1H, d, Ar-H, J = 1.7), 7.15 (1H, d, Ar-H, J = 1.7),
7.26 (2H, d of d, Ar-H, J = 2.5 and 9.1), 7.63 (1H, m, Ar-H), 7.98
(4H, m, Ar-H), 8.22 (1H, t, NH, J = 5.5), 9.62 (1H, s, N+H), 10.04
(1H, s, NH), 10.49 (1H, s, NH). HRFABMS: Found 613.3156;
+
Ar-H), 10.54 (1H, s, NH). LRMS: Found 271.9; C15H14NO4
requires 272.1. Found: C, 65.98; H, 4.80; N, 5.16; C15H13NO4
requires C, 66.41; H, 4.83; N, 5.16%.
4-[(3-Methoxybenzoyl)amino]benzoyl chloride
4-[(3-Methoxybenzoyl)amino]benzoic acid (0.410 g, 1.42 mmol)
was dissolved in dichloromethane (3 mL).Thionyl chloride (5 mL)
was then added, and the solution was heated under reflux for 1 h.
The solvent and thionyl chloride were removed under reduced
pressure to yield the product 20 as an off-white solid (1.236 g,
74%) m.p. = 65–67 ◦C nmax (KBr): 3464, 3349, 3063, 2839, 2826,
1752, 1585, 1504, 1452 cm-1. dH (DMSO): 3.84 (3H, s, OCH3), 7.18
(1H, d of d, Ar-H, J = 2.5 and 8.1), 7.53 (3H, m, Ar-H), 7.92 (4H,
+
C34H41N6O5 requires 613.3138.
Ethyl 2-(diethoxymethyl)-1,3-thiazole-4-carboxylate12
2,2-Diethoxyethanethioamide (0.654 g, 6.03 mmol) and ethyl
bromopyruvate (1.28 g, 6.10 mmol) were dissolved in ethanol
+
m, Ar-H), 10.51 (1H, s, NH). LRMS: Found 291.0; C15H13ClNO3
˚
(10 mL) in the presence of a 4 A mol. sieve (1.0 g). The solution was
requires 291.1.
then heated under reflux for 45 min and the solvent removed under
reduced pressure. The residue was then dissolved in ethyl acetate
(20 mL) and extracted with sat. sodium bicarbonate solution (2 ¥
20 mL) and brine (2 ¥ 20 mL). The organic fraction was dried
(MgSO4) and the solvent removed under reduced pressure to yield
the required product as a white solid (1.452 g, 93%) m.p. = 70–
72 ◦C. nmax (KBr): 3108, 2981, 2885, 2856, 1735, 1505, 1042 cm-1.
4-{(E)-2-[4-({4-[(3-Methoxybenzoyl)amino]benzoyl}amino)-1-
methyl-1H-pyrrol-2-yl]ethenyl}-1-methyl-N-[2-(4-morpho-
linyl)ethyl]-1H-pyrrole-2-carboxamide trifluoroacetate 9 and
4-{2-[4-({4-[(3-methoxybenzoyl)amino]benzoyl}amino)-1-methyl-
1H-pyrrol-2-yl]ethyl}-1-methyl-N-[2-(4-morpholinyl)ethyl]-1H-
pyrrole-2-carboxamide trifluoroacetate 10
d
H (CDCl3): 1.29 (9H, m, 2CH3), 3.71 (4H, m, 2CH2), 4.47 (2H, q,
CH2 J = 7.8), 5.70 (1H, s, CH), 8.19 (1H, s, Ar-H). HRFABMS:
1-Methyl-4-[(E)-2-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethenyl]-N-
[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide (0.111 g,
0.27 mmol) was dissolved in dioxane (5 mL). Pd/C (10%, 0.100 g)
was then added, followed by a solution of sodium borohydride
(0.030 g, 0.52 mmol) in water (2 mL). The solution was then
allowed to stir for 20 min, before being filtered directly into
a flask containing 4-[(3-methoxybenzoyl)amino]benzoyl chloride
(0.078 g, 0.27 mmol). Saturated aqueous sodium carbonate (2 mL)
was then added and the solution allowed to stir overnight. Water
(5 mL) was added and the solution was extracted with ethyl
acetate (2 ¥ 20 mL). The organic layer was dried (MgSO4) and
the solvent removed under reduced pressure. The residue was
purified by HPLC which allowed both products to be obtained
as yellow/orange solids.
Found 260.0956 (M + H); C11H17NO4S requires 259.0878.
Ethyl 2-formyl-1,3-thiazole-4-carboxylate12
Ethyl 2-(diethoxymethyl)-1,3-thiazole-4-carboxylate (1.34 g,
5.17 mmol) was dissolved in acetone (100 mL). Hydrochloric
acid solution (12.8 mL, 1 M) was then added and the solution
heated under reflux for 45 min. The solvents were then removed
under reduced pressure and the residue dissolved in ethyl acetate
(40 mL) and extracted with sat. sodium bicarbonate solution (2 ¥
40 mL), and brine (2 ¥ 40 mL). The organic fraction was then
dried (MgSO4) and the solvent removed under reduced pressure
to yield ethyl 2-formyl-1,3-thiazole-4-carboxylate as a light-brown
solid (0.937 g, 98%) m.p. = 65–67 ◦C (Lit. 67–68 ◦C).12
nmax (KBr):
1848 | Org. Biomol. Chem., 2009, 7, 1843–1850
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3116, 2983, 2910, 2814, 1730, 1513, 1060 cm-1. dH (CDCl3): 1.47
(3H, t, CH3 J = 8.0), 4.50 (2H, q, CH2 J = 8.0), 8.52 (1H, d, Ar-H
J = 1.2), 10.08 (1H, d, Ar–COH J = 1.2). HRFABMS: Found
186.0228 (M + H); C7H7NO3S requires 185.0147.
(2 mL) was then added and the solution allowed to stir overnight.
Water (5 mL) was added and the solution extracted with ethyl
acetate (2 ¥ 20 mL), the organic layer was dried and the solvent
removed under reduced pressure. The residue was purified by
HPLC which allowed the product to be obtained as a dark-green
solid (0.044 g, 22%) m.p. >230 ◦C, purity by HPLC = 98%. nmax
(KBr): 3347, 2925, 1685, 1535, 1204 cm-1. dH (DMSO): 3.13 (2H,
m, CH2), 3.29 (2H, m, CH2), 3.55 (4H, m, 2(CH2), 3.57 (3H, s,
N-Me), 3.67 (2H, t, CH2, J = 12.5), 3.84 (3H, s, N-Me), 4.01 (2H,
d, CH2, J = 11.16), 6.60 (1H, d, Ar-H, J = 1.7), 6.63 (1H, d,
Ethyl 2-[(E)-2-(2-quinolinyl)ethenyl]-1,3-thiazole-4-carboxylate
Diethyl 2-quinolinylmethylphosphonate (0.580 g, 2.08 mmol) was
dissolved in anhydrous THF (2 mL). Sodium hydride (0.273 g,
11 mmol) was then added in small portions and the resulting
solution allowed to stir for 10 min. Ethyl 2-formyl-1,3-thiazole-
4-carboxylate (0.387 g, 2.08 mmol) in anhydrous THF (3 mL)
was added dropwise and the solution allowed to stir for 16 h.
Water (5 mL) was then added (dropwise initially) during which
time the required product precipitated as a light-brown/yellow
=
=
(C CH), J = 16.1), 6.70 (1H, d, (C CH), J = 16.1), 7.01 (1H, d,
Ar-H, J = 1.8), 7.18 (1H, d, Ar-H, J = 1.7), 7.25 (1H, d, Ar-H,
J = 1.7), 7.64 (1H, t of d, Ar-H, J = 1.2 and 8.4), 7.82 (1H, t of
=
d, Ar-H, J = 1.2 and 8.4), 7.92 (1H, d, (C CH), J = 16.0), 7.98
(1H, d, Ar-H, J = 8.6), 8.01 (1H, d, Ar-H, J = 8.4), 8.04 (1H, d,
◦
solid (0.232 g, 36%) m.p. = 183–186 C. nmax (KBr): 3124, 3043,
=
Ar-H, J = 8.5), 8.10 (1H, d, (C CH), J = 16.0), 8.34 (1H, t, NH,
2955, 2925, 2899, 2853, 1730, 1612, 1627, 1592, 1553, 1479 cm-1.
J = 5.5), 8.38 (1H, s, Ar-H), 8.46 (1H, d, Ar-H, J = 8.6), 9.55
d
H (CDCl3): 1.34 (3H, t, CH3 J = 8.0), 4.34 (2H, q, CH2 J = 8.0),
(1H, s, N+H), 10.26 (1H, s, NH). HRFABMS: Found 622.2598;
=
7.62 (1H, t, Ar-H J = 7.2), 7.74 (1H, d, C C alkene J = 16.1),
+
C34H39N5O4 requires 622.2595.
=
7.79 (1H, t, Ar-H J = 6.8), 7.98 (3H, m, Ar-H), 8.05 (1H, d, C C
alkene J = 16.1), 8.42 (1H, d, Ar-H J = 8.5), 8.56 (1H, s, Ar-H).
N-[1-Methyl-5-({[1-methyl-5-({[2-(4-morpholinyl)ethyl]-
amino}carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1H-pyrrol-3-
yl]-2-[(E)-2-(2-quinolinyl)ethenyl]-1,3-thiazole-4-carboxamide
trifluoroacetate 13
HRFABMS: Found 310.0772; C17H14N2O2S requires 310.0776.
2-[(E)-2-(2-Quinolinyl)ethenyl]-1,3-thiazole-4-carboxylic acid
2-[(E)-2-(2-Quinolinyl)ethenyl]-1,3-thiazole-4-carboxylate (0.137 g,
0.44 mmol) was suspended in ethanol (2 mL). Sodium hydroxide
(0.052 g, 1.32 mmol) in water (5 mL) was added and the solution
heated under reflux for 2 h. The reaction was then filtered and then
cooled to 0 ◦C. Dilute hydrochloric acid was added dropwise until
the required product precipitated as a yellow solid (0.103 g, 83%)
m.p. = 218–220 ◦C. nmax (KBr): 3469, 3105, 3068, 2923, 2853, 1715,
1640, 1632, 1599, 1541, 1493, 1320 cm-1. dH (CDCl3): 7.74 (1H, t,
N-[1-Methyl-5-({[1-methyl-5-({[2-(4-morpholinyl)ethyl]amino}-
carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1H-pyrrol-3-yl]-2-
[(E)-2-(2-quinolinyl)ethenyl]-1,3-thiazole-4-carboxamide trifluo-
roacetate 13 was prepared using an analogous procedure to the
coupling reaction for compound 12 above (0.020 g, 21%), no
distinct m.p., purity by HPLC = 97%. nmax (KBr): 3421, 3116,
2928, 1677, 1647, 1638, 1556, 1465, 1241, 1204, 1131, 722 cm-1.
dH (CDCl3): 3.17 (2H, m, CH2), 3.39 (2H, m, CH2), 3.57 (4H, m,
CH2), 3.84 (3H, s, NMe), 3.88 (3H, s, NMe), 4.00 (2H, m, CH2),
7.00 (1H, d, Ar-H J = 1.6), 7.22 (2H, m, Ar-H), 7.35 (1H, d, Ar-H
J = 1.6), 7.63 (1H, t, Ar-H J = 6.9 Hz), 7.80 (1H, t, Ar-H J =
=
Ar-H J = 7.2), 7.76 (1H, d, C C alkene J = 16.1), 7.93 (1H, t,
Ar-H J = 7.2), 8.11 (1H, d, Ar-H J = 8.5), 8.05 (1H, d, Ar-H J =
=
8.5), 8.19 (2H, m, Ar-H), 8.24 (1H, d, C C alkene J = 16.1), 8.58
(1H, s, Ar-H), 8.65 (1H, d, Ar-H J = 8.5). HRFABMS: Found
282.0465; C15H10N2O2S requires 282.0463. Found: C, 63.49; H,
4.06; N, 9.49; S, 11.53; C15H10N2O2S requires C, 63.81; H, 3.57; N,
9.92; S, 11.36%.
=
6.9), 7.90 (1H, d, HC CH J = 16.2), 7.96 (1H, d, Ar-H J = 8.6),
=
8.01 (2H, m, Ar-H), 8.09 (1H, d, HC CH J = 16.2), 8.24 (1H, t,
NH J = 5.6), 8.40 (1H, s, Ar-H), 8.45 (1H, d, Ar-H J = 8.6), 9.66
(1H, s, NH+), 9.98 (1H, s, NH), 10.39 (1H, s, NH). HRFABMS:
Found 639.2505; C33H35N8O4S requires 639.2502.
4-[2-({[1-Methyl-4-((E)-2-{1-methyl-4-[({2-[(E)-2-(2-quinolinyl)-
ethenyl]-1,3-thiazol-4-yl}carbonyl)amino]-1H-pyrrol-2-yl}-
ethenyl)-1H-pyrrol-2-yl]carbonyl}amino)ethyl]morpholin-4-ium
trifluoroacetate 12
Melting temperature measurement
DNA oligomers and their complements were melted at a rate of
0.5 ◦C min-1 in 10 mM PBS buffer solution (pH 7.4) with 50 mM
NaCl on a Cary 300 BIO UV–visible spectrophotometer. Each
oligomer (made to a concentration of 6 ¥ 10-6 M ) was mixed with
sufficient MGB to give the appropriate ratio. Samples were heated
to 80 ◦C and cooled to 10 ◦C. The melting temperatures (Tm) of
the hybrids were determined from the derivative maxima.
2-[(E)-2-(2-Quinolinyl)ethenyl]-1,3-thiazole-4-carboxylic acid 11
(0.078 g, 0.26 mmol) was dissolved in dichloromethane (2 mL),
thionyl chloride (5 mL) added and the solution heated under
reflux for 1 h, after which time the solvent was removed
under reduced pressure to give 2-[(E)-2-(2-quinolinyl)ethenyl]-
1,3-thiazole-4-carbonyl chloride as a dark-green/brown solid.
1-Methyl-4-[(E)-2-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethenyl]-N-
[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 6 (0.105 g,
0.26 mmol) was dissolved in dioxane (5 mL). Pd/C (10%, 0.100 g)
was then added, followed by a solution of sodium borohydride
(0.030 g, 0.52 mmol) in water (2 mL). The solution was then
allowed to stir for 15 min, and filtered directly into the flask con-
taining the previously prepared 2-[(E)-2-(2-quinolinyl)ethenyl]-
1,3-thiazole-4-carbonyl chloride. Saturated sodium carbonate
Antibacterial activity measurement
Carried out as described previously.3,9
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©