Amide isosteres in structure-activity studies of antibacterial minor groove binders

Article abstract of DOI:10.1016/j.ejmech.2011.08.035

Antibacterial minor groove binders related to the natural product, distamycin, are development candidates for novel antibiotics. Alkenes have been found to be effective substitutes for the isosteric amide links in some positions and alkyl groups larger th

Full text of DOI:10.1016/j.ejmech.2011.08.035

European Journal of Medicinal Chemistry 46 (2011) 5343e5355
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Amide isosteres in structure-activity studies of antibacterial minor groove binders
,
b
a
a
b
Abedawn I. Khalafa , Nahoum Anthony , David Breen , Gavin Donoghue , Simon P. Mackay ,
*
Fraser J. Scotta, Colin J. Sucklinga
a WestCHEM Research School, Department of Pure & Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, Scotland,
,
*
United Kingdom
b Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, Scotland,
United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Antibacterial minor groove binders related to the natural product, distamycin, are development candi-
dates for novel antibiotics. Alkenes have been found to be effective substitutes for the isosteric amide
links in some positions and alkyl groups larger than methyl have been found to increase binding to DNA
in both selectivity and affinity. However the impact of other isosteres such as diazenes and the position of
an alkyl group with respect to DNA binding and antibacterial activity are not known. The effects of some
systematic variations in the structure of polyamide minor groove binders are investigated. Isosteres of
the amide link (alkenes and diazenes) are compared: it is shown that all three are competent for binding
to DNA but that alkene links give the tightest binding and highest antibacterial activity; no significant
antibacterial activity was found for compounds with a diazene link. Within a series of alkene linked
compounds, the effect of branched N-alkyl substituents on binding to DNA and antibacterial activity is
investigated: it was found that C3 and C4 branched chains are acceptable at the central pyrrole residue
but that at the pyrrole ring adjacent to the basic tail group, a C4 branched chain was too large both for
DNA binding and for antibacterial activity. The active branched alkyl chain compounds were found to be
especially active against Mycobacterium aurum, a bacterium related to the causative agent of tuberculosis.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 15 March 2011
Accepted 23 August 2011
Available online 30 August 2011
Keywords:
DNA
Minor groove binders
Diazene isosteres
Antibacterial activity
1. Introduction
of a novel class of minor groove binder, diazenyl isosteres of the
amides related to the highly active alkenes. We also describe the
The importance of new antibacterial agents has been widely
recognized as resistance develops to existing drugs [1]. In several
studies, we have shown that minor groove binders for DNA based
loosely upon the structure of the natural product, distamycin 1, are
effective antibacterial agents [2e4]. Key structural features of these
compounds are the presence of an alkene isostere replacement for
the N-terminal amide [3] and branched N-alkyl chains on pyrrole
components [2]. These features are seen in the C-alkyl thiazole, 2,
known as thiazotropsin A, and in the quinolyl alkene, 3a. Because of
the large number of potential structural variations, there is no
systematic understanding of the structureeactivity relationships
associated with antibacterial activity. Moreover other isosteric
replacements for amides than alkenes exist, such as diazenes.
Within the highly active alkene series, the potential significance of
different N-substituents in compounds such as 3a has not been
investigated. In this paper we describe the synthesis and properties
synthesis and properties of N-isopropyl and N-2-methylpropyl
analogues of the N-methylpyrroles in the quinolyl alkene 3a to
shed some light on the positions that are amenable to larger
substituents in such antibacterial minor groove binders.
H
NH2
NH
N
O
+
NH2
O
N
H
H
N
HN
O
N
1
O
N
H
NH
N
O
NMe2
O
N
N
H
H
N
HN
O
2
S
O
N
* Corresponding authors. Tel.: þ44 141 548 2520; fax: þ44 141 548 5743.
E-mail address: abedawn.khalaf@strath.ac.uk (A.I. Khalaf).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.035

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A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
2. Results and discussion
2.1. Design of diazenyl and branched alkyl substituted minor groove
binders
To a first approximation it would be expected that a diazenyl
analogue of 3a such as 3b would have a very similar shape to 3a but
would present different opportunities for binding to DNA. Potential
steric clashes with DNA through the alkenyl hydrogens would be
removed and the
N
O
N
HN
3a
H
N
O
HN
O
O
N
N
N
N
O
O
N
N
N
N
3b
HN
H
N
O
O
HN
O
O
O
N
N
N
H
N
3c
Fig. 1. Left: minimised conformation of 3c dimer (stick model) in the minor groove of
a 50-GCGATATATCGC-30 DNA dodecamer (in grey, surfaced rendering). Right: two views
(the bottom view is rotated by 90ꢁ around the Z axis of DNA when compared with the
left-hand-side and top pictures) of minimised conformations of 3a (alkene, red), 3b
(diazene, blue) and 3c (amide, green) dimers in the minor groove of a 50-GCGATA-
TATCGC-30 DNA dodecamer (not shown for clarity). The 3b dimer does not appear as it
overlaps the 3a dimer almost perfectly. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
HN
O
H
N
HN
O
N
bind to DNA in a similar manner to the alkene but that there might
be quantitative differences with respect to the strength of binding.
Greater differences might be expected between alkene and diazene
on the one hand and amide on the other.
The comparison of amide, alkene, and diazene as isosteric links
in minor groove binders has been investigated in three series
lone pairs of the nitrogen atoms introduced might be able to
take part in hydrogen bonding with DNA and accommodate GC
base pairs. Other effects such as the overall lipophilicity of 3a and
related compounds might also play a role in the biological prop-
erties of diazenyl linked minor groove binders [4]. However in the
first instance, the binding of such compounds to DNA is of interest.
The relationship of the three links in compounds with quinolyl
head groups 3aec has been investigated by molecular modelling to
examine whether isohelical geometry is affected. Calculations were
performed using the cff91 force field [5] docking 3aec into the
minor groove co-ordinates of a 50-GCGATATATGCG-30 DNA olig-
omer duplex using parameters to widen the minor groove [6] so as
to accommodate two ligands binding side-by-side. The DNA olig-
omer was minimized in vacuo using 100 steps of the conjugate
gradient (CG) algorithm and
a distant-dependent dielectric
constant of 4rij, [7], with the terminal base pairs fixed. The ligands
were manually docked to span the central bases of the oligomer
and minimized with the DNA co-ordinates fixed until a derivative of
0.1 kcal molꢀ1 Åꢀ2 was achieved. All three ligand dimers appeared
to adopt the same conformation upon minimisation (Fig. 1), with
minimal heavy atom root mean square deviations (RMSDs)
between them. The RMSDs of the bound 3a dimer to the 3b and 3c
dimers were 0.25 and 0.50 respectively and the RMSD of the 3b
dimer to the 3c dimer was 0.34.
Minimum energy conformations of 3aec were calculated fol-
lowed by superimposition of the ligands as monomers with each
other. This showed that they had to adapt their conformations to
match the requirements of the DNA dodecamer, with 3c (amide
linked ligand) requiring a slightly greater adaptation energy than
3b, and again greater than 3a (Fig. 2 and Table 1). This evaluation
suggests that a diazenyl minor groove binder would be expected to
Fig. 2. Superimposition of 3a (red), 3b (blue) and 3c (green) on their quinoline rings
(on the left-hand-side) showing that the amide group of 3c favours a tighter curvature
when compared with the alkene (3a) and diazene (3b) groups. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)

A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
5345
Table 1
groove binders [3]. A panel of eight compounds (6aed, and 7aed,
see Scheme 2) was assembled in which isopropyl, 2-methylpropyl,
and methyl groups were chosen.
Calculated energies (values are in kcal.molꢀ1) of monomers of 3aec.
Ligand
Minimised energy
Minimised energy in DNA
(for a single ligand) (E2)
Adaptation
in vacuo (E1)
energy (E2-E1)
3a
3b
3c
101.5
102.4
103.8
104.4
105.9
107.4
2.9
3.5
3.7
2.2. Synthesis of diazenyl minor groove binders
To our knowledge, diazenyl substituents have been introduced
to minor groove binders as pendant chromophores but not as an
intrinsic link within the compound [10]; new synthetic routes
were therefore required. The strategy employed was to prepare
the diazenyl dimers with each of the three head groups and to
couple them to the pyrrolyl-pyrrolyl-morpholinoethyl component
that has been used previously (Scheme 1) [3]. For the quinolyl and
3-methoxyphenyl compounds, 3b and 4b, that contain a head
group arene or heteroarene that was unreactive for diazo
coupling, condensation with methyl 4-nitrosobenzoate afforded
the required precursors 10a, b [11,12]. For the 4-dimethylamino
compound, 10c, diazo coupling was used [13]. Compounds 3a,
4a, and 14 were available from previous studies [2,3] and the
remaining alkenyl and amide linked minor groove binders to
complete the panel of compounds were prepared by methods
used routinely using the same coupling strategy. The N-alkyl
pyrroles required for the synthesis of compounds 6e7 were
prepared by alkylation of the potassium salt of ethyl 5-
nitropyrrole-2-carboxylate as described previously [2]; assembly
of the minor groove binders also followed standard methods as
illustrated in Schemes 1 and 2.
including the quinolyl series evaluated above. Additionally, the 3-
methoxyphenyl compounds (4aec) and 4-dimethylaminophenyl
compounds (5aec) have been prepared and studied.
Turning to the effects of alkyl group substitution on the pyrrole
nitrogen atoms, structural studies of thiazotropsin A 2 and other
compounds containing isopropyl substituents have shown the
importance of the isopropyl group in the side-by-side interaction of
two molecules of these minor groove binders in the DNA groove
[8,9]; the intermolecular contacts between the two molecules of 2
show close interactions between the isopropyl group of 2 and the
dimethylamino tail group of its partner in the minor groove of its
preferred double stranded oligodeoxynucleotide [8]. It has further
been shown that if the branched N-alkyl chains in a minor groove
binder are too close together, for example on adjacent pyrrole rings,
the formation of strong 2:1 ligand/DNA complexes does not occur
[2]. In order to understand the extent to which increasing the size
of such N-alkyl groups might be compatible with binding to DNA
and antibacterial activity, we have prepared a series of N-alkyl
analogues of our most active antibacterial alkene-containing minor
Scheme 1. Synthesis of diazenyl minor groove binders.

5346
A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
Scheme 2. Synthesis of branched alkyl substituted compounds.
2.3. Assessment of binding to DNA
oligoDNA duplexes [14]. The oligonucleotide chosen as target for
the current study was GCGATATATGCG and its complement; this
was selected because of the AT run and because its melting
temperature in the absence of minor groove binding was expected
to be in the range 45e50 ꢁC. If binding occurs, the melting
temperature will usually be increased, in cases of strong binding up
to and above 20 ꢁC. However, binding may still be occurring if no
increase in melting temperature is observed although such a result
indicates that the oligonucleotide duplex is not strongly stabilized
by the minor groove binder. It is important, therefore, to use further
techniques for studying binding to DNA and in our work, capillary
electrophoresis has been especially useful [15]. It has been found
that DNA complexes with minor groove binders usually show
different retention times in capillary electrophoresis from the
The accumulated evidence with our alkene and amide con-
taining minor groove binders strongly suggests that they bind most
readily to AT rich regions of DNA; this is consistent with the pres-
ence of the two pyrroles and the phenyl group, all of which are
incompatible with binding to GC base pairs. There are many tech-
niques available to investigate the binding of ligands to DNA and in
this study, we have focused on two, melting temperature
measurements and capillary electrophoresis. Melting temperature
measurements give an indication of the ability of a minor groove
binder to stabilize the DNA duplex of an oligonucleotide and have
been widely used in studies of this class of compound and the
melting temperature can be correlated with specific binding to

A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
5347
Table 3
uncomplexed oligonucleotide. In principle, this makes it possible
both to compare the affinity of a DNA binder to a given sequence
and to investigate the stoichiometry of binding [16]. However since
a DNA-oligo complex peak may overlap with the parent oligo peak
in some cases, the absence of a new peak in a CE experiment does
not of itself show that binding is not occurring. In both of these
methods, therefore, positive results can be taken as conclusive but
negative results are inconclusive. The data we have obtained for the
isosteres panel are shown in Table 2 and those for the N-alkyl panel
in Table 3.
Evaluation of binding of branched alkyl chain minor groove binders. 2 equivalents of
minor groove binder were used.
Compound
R1
R2
Tm (ꢁC)
Tm increase
3a
6a
6b
6c
6d
14
7a
7b
7c
7d
Me
iPr
Me
Me
Me
iPr
sBu
Me
Me
Me
iPr
66
58.9
47.1
47.0 66.0
47.1 63.1
64.0
63.9
61.1
19
10
0
18
15
16
16
13
19
16
sBu
Me
Me
Me
iPr
sBu
Me
Me
67.1
63.9
2.3.1. Assessment of amide isosteres
sBu
The observed Tm depends upon the detailed experimental
conditions and the affinity of the ligand for the particular oligo-
nucleotide target selected. In these experiments, it was necessary in
some cases to use four equivalents of ligand to observe a significant
change in Tm and in some, two Tms were observed, one corre-
sponding to the free oligonucleotide and the other to the ligand/
DNA complex. Although far from a quantitative measurement
equivalent to a dissociation constant, the observations reported
here give an indication of the affinity of the three series of ligands
and for the DNA target selected and emphasise that the results are
obtained under equilibrium conditions.
methoxy groups. In terms of binding to this oligonucleotide, the
diazenyl isostere 4b behaves very similarly in that there is little
duplex stabilization but evidence for binding from CE. This is the
first clear evidence for the similarity in molecular recognition terms
between alkene and diazene. In contrast, the amide linked minor
groove binder 4c had a significant but smaller increase in Tm
although unlike its alkene isostere 4a, an additional peak was not
observed in the CE trace.
All the compounds containing a quinolyl head group (3aec)
bound strongly to the target oligonucleotide according to Tm
measurements. In the case of the amide linked minor groove binder
3c, there was no additional peak observed in the CE experiment
which, as noted above, could be due to overlapping peaks. It is
probable that the quinoline group itself plays a major role in binding
to DNA which would make it difficult to perceive differences in
affinity according to the link present. Footprinting studies have
shown that the quinolyl alkenyl ligand 3a binds exceptionally
strongly to larger DNA oligonucleotides at sub-micromolar concen-
trations and at several AT containing sites (Prof. Keith Fox, unpub-
lished observations), in support of the dominant effect of the quinolyl
group in these cases. With respect to our modelling study, it is clear
that the binding affinity of the dimers compensates for any adapta-
tion energy penalties incurred through geometrical rearrangements
to have compatible isohelicity with the groove floors.
The 4-dimethylamino head group series (5aec) showed
different pattern. In this series, the amide 4c had both
a
a substantial Tm increase and showed a new peak in the CE trace.
The diazene isostere 4b showed similar evidence of binding from
both measurements. However the alkene isostere 4a showed very
little evidence of binding to this particular target by either tech-
nique; at present, we have no explanation for this observation.
Taken together these results emphasise that multiple interac-
tions govern the association of a given minor groove binder with
a particular DNA target and it is probable that some of the
compounds investigated here have different oligonucleotide
targets. On the basis of the available evidence, however, it appears
that the diazenyl compounds are less effective ligands for DNA than
their amide or alkene isosteres for this particular representative
type of AT rich sequence. Bearing in mind the similarity in geom-
etry observed in the molecular modelling, it appears that both the
electronic and hydrogen bonding properties of the diazene link
may be relevant in reducing binding to this short oligonucleotide
compared with the alkene [24, 26].
The 3-methoxyphenyl head group provided
a series of
compounds (4aec) with apparently ambiguous behaviour. The
alkene linked compound 4a has strong antibacterial properties but
apparently did not stabilize this particular DNA duplex according to
Tm measurements. Nevertheless, the observation of a new peak in
the CE is good evidence for complexation and this has been
confirmed by a detailed NMR study of this ligand [17]; the inter-
action of 4a with its DNA host was resolved at atomic detail and
shows evidence for specific hydrogen bonding of amide and
2.3.2. Branched N-alkyl minor groove binders panel
The introduction of the isopropyl group into thiazatropsin A 2
was an important structural innovation together with the thiazole,
both being hydrophobic groups [8]. The extent to which branched
alkyl groups can modulate DNA binding and biological activity is
therefore of general interest in this field, as explained above. Taking
two of our lead antibacterial compounds as prototypes (3a and 14),
we therefore prepared a series of eight new compounds to evaluate
the effect of isopropyl and 2-methylpropyl (s-butyl) groups as
pyrrole nitrogen substituents with respect to DNA binding and
biological activity. The results for the DNA binding study are shown
in Table 3.
It is clear from these data that the N-alkyl group at the C-
terminal pyrrole (R1) is more strongly sensitive to substitution by
the larger alkyl groups than the internal pyrrole (R2); with the
quinolyl head group (6a and 6b) stabilisation of this duplex
appears to be very sensitive to the size of the N-alkyl group, the
isopropyl substituted compound 6a binding less strongly than the
methyl and the 2-methylbutyl 6b. It is possible that with the
quinolyl head group the ligandeligand association is more inti-
mate close to the quinolyl group than with other smaller head
groups and that the larger alkyl groups adjacent to the quinolyl
Table 2
Evaluation of ligand binding to GCGATATATGCG and its complement.
Sample
Link
Equiv
1st Tm
2nd Tm
D
Tm
CE results
Oligo target
3a (quinoline)
e
e
4
2
4
4
48
66
46
48
46
e
e
e
alkene
e
18
20
17
17
New peak
e
68
65
65
3b
3c
diazo
amide
New peak
No new peak
4a (3-MeO)
4b
4c
alkene
diazo
amide
4
4
4
2
46
49
60
58
e
e
e
e
e
New peak
New peak
No new peak
1
12
10
5a (4-NMe2)
5b
5c
alkene
diazo
amide
4
4
4
50
56
48
e
2
8
18
No New peak
New peak
New peak
e
66

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A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
group in the dimer prevent the association necessary for 2:1
ligand/DNA binding. Our previous work has shown that these
minor groove binders preform dimers before binding with DNA,
rather than associate sequentially [18].
drawing conclusions about the biological mechanism of action and
have drawn attention to the importance of the bacterial cell wall
and the depolarisation of the membrane by minor groove binders
[19]. Consistent with these observations, in preliminary studies
with selected compounds from our library, we have found that our
compounds do not inhibit DNA biosynthesis but show some inhi-
bition of bacterial DNA gyrase. Other studies have shown conclu-
sively that minor groove binders interfere with DNA processing
enzymes (gyrase and helicase) [20,21].
2.4. Biological activity
The antibacterial activity of all compounds described here has
been evaluated against representative strains of Gram positive and
Gram negative bacteria as described previously [2,3]. No significant
activity with respect to Gram negative bacteria was found but some
compounds were strongly antibacterial against Staphylococcus
aureus (two strains), Streptococcus faecalis, and Mycobacterium
aurum (Table 4, compounds with quantified activity from this study
and reference compounds 3a and 4a only shown). In addition,
measurable but not significant activity was found for the diazenyl
compound 3b against the fungi Aspergillus niger and Candida albi-
cans. From the point of view of the configuration, structure, and
binding to DNA of the quinolyl diazenyl linked compound 3b, the
lack of antibacterial activity is notable, a fact that emphasizes the
large number of factors involved in obtaining antibacterial activity,
including access to the target as well as intrinsic affinity. The same
is true of the diazenyl analogue 4b with the 3-methoxyphenyl head
group, although this compound did not stabilize the DNA duplex
studied. None of the 4-dimethylaminophenyl compounds 5 were
antibacterial which may in part be due to the lack of a crucial
hydrogen bond to the head group that we have described else-
where [4]. On the other hand, the antibacterial activity of the
branched alkyl compounds 6 and 7 is in line with results from
previous studies [3] with the quinolyl head group showing espe-
cially strong activity against both S. aureus and M. aurum; the latter
activity is the strongest that we have found in this series of
compounds.
In addition to the potential importance of specific hydrogen
bonds, the lipophilicity of our compounds has also been connected
with antibacterial activity [4]. There remain two major questions
about the biological activity of our minor groove binders namely
the origin of the antibacterial activity and the reason for the lack of
toxicity to several mammalian cell lines and the answers to these
questions require more detailed microbiological studies which we
are seeking to address.
3. Experimental
3.1. General
1H and 13C NMR spectra were measured on a Bruker DPX-
400 MHz spectrometer with chemical shifts given in ppm (
d
values), relative to proton and carbon traces in solvent. 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. 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. Chroma-
tography was carried out using 200e400 mesh silica gels, or using
reverse-phase HPLC on a water system using a C18 Luna column
with the gradient given in Table 5.
In our studies of antibacterial minor groove binders there has
been an association between antibacterial activity and binding to
DNA as measured by several methods [2e4,16]. Thus where no or
weak DNA binding was observed, little antibacterial activity was
found. It has even been possible to show that specific hydrogen
bonding sites in the minor groove binder at the head group or at
one of the internal amides is important for both DNA binding and
antibacterial activity [17]. However this connection is far from
proving that DNA binding is the sole or primary mechanism of
antibacterial activity. Recent studies have cautioned against
3.2. Monomers and tail groups
The following were prepared as previously described:
3.2.1. Monomers [22]
1-Methyl-4-nitro-1H-pyrrole-2-carboxylic acid 2,2,2-Trichloro-
1-(1-methyl-1H-pyrrol-2-yl)ethanone
2,2,2-Trichloro-1-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethanone
N-[3-(Dimethylamino)propyl]-1-methyl-4-nitro-1H-pyrrole-2-
carboxamide
Table 4
Antibacterial and antifungal activity of branched N-alkyl substituted minor groove
binders and diazenyl minor groove binders. Compounds were tested at 500
Escherichia coli) and 200
M (for S. aureus). nd ¼ not determined.
mM (for
m
Ethyl 1-methyl-4-nitro-1H-pyrrole-2-carboxylate
Compound
E. coli
S. aureus
S. aureus
M. aurum
% of control
% of control
MIC (
m
M)
MIC (mM)
3.2.2. Dimers [2,3,22]
6a
6b
6c
6d
7a
7b
7c
7d
94.7
74.2
80.9
0.8
0.3
2.3
2.9
8.5
2.8
6.7
3.2
<1
nd
<1
<1
4
16
16
8
2
nd
2
2
8
16
8
31
N-[3-(Dimethylamino)propyl]-1-methyl-4-{[(1-methyl-4-nitro-
1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide
69.0
103.2
115.6
98.6
Table 5
100.9
Programme for HPLC puritication of minor groove binders.
Compound
S. aureus 1
MIC ( M)
3.1
>100
12.5
>100
S. aureus 2
MIC ( M)
12.5
>100
25
S. faecalis
A. niger
C. albicans
Time/Pump
A
B
Flow rate (ml/min)
m
m
MIC (
m
M)
MIC (
m
M)
MIC (mM)
0
90
30
10
90
90
10
70
90
10
10
4
4
4
4
0
3a
3b
4a
5a
25
>100
6.25
>100
50
50
50
25
25
100
100
28
33
38
40
>100
>100

A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
5349
1-Methyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-pyrrole-2-
carboxamide
1-Methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide
water (10 mL) whilst stirring. The reaction mixture was heated at
reflux for 2 h, then cooled to 0 ꢁC (ice bath) and acidified with
concentrated hydrochloric acid whilst stirring. The precipitate was
filtered off, washed with water (2 ꢂ 15 mL) and dried at 50 ꢁC under
reduced pressure for 8 h to give the product as a white solid
(445 mg, 78%), m.p. 194e195 ꢁC. nmax (KBr): 3155, 2983, 1682, 1535,
1507, 1287, 1246, 1180, 1137, 1115. dH (DMSO-d6): 13.16 (1H, bs,
CO2H), 8.35 (1H, d, NH, J ¼ 2.0), 7.27 (1H, d, NH, J ¼ 2.0), 5.42 (1H,
septet, CH3CHCH3, J ¼ 6.7), 1.42 (6H, d, CH3CHCH3, J ¼ 6.7)
3.2.3. Nitroso compounds [11]
1-Methoxy-3-nitrosobenzene
Ethyl 4-nitrosobenzoate
3.3.5. 1-sec-Butyl-4-nitro-1H-pyrrole-2-carboxylic acid 16b
3.3. New monomers
Ethyl
1-sec-butyl-4-nitro-1H-pyrrole-2-carboxylate
15b
(614 mg, 2.56 mmol) was dissolved in ethanol (4 mL) and to this
was added a solution of sodium hydroxide (420 mg, 10.50 mmol) in
water (10 mL) whilst stirring. The reaction mixture was heated at
reflux for 2 h, cooled to 0 ꢁC (ice bath) and acidified with concen-
trated hydrochloric acid whilst stirring. The precipitate was filtered
off, washed with water (2 ꢂ 15 mL) and dried at 50 ꢁC under
reduced pressure for 4 h to give the product as white solid at a yield
of (565 mg, 96%), m.p. 153e156 ꢁC. nmax (KBr): 2923, 1682, 1511,
1292,1242,1183 cmꢀ1 dH NMR (CDCl3): 7.81 (1H, d, NH, J ¼ 2.0), 7.58
(1H, d, NH, J ¼ 2.0), 5.34 (1H, sextet, CH2CHCH3, J ¼ 6.9), 1.78e1.88
(2H, m, CH3CH2CH), 1.50 (3H, d, CH2CH3, J ¼ 7.1), 0.91 (3H, t,
CH3CH2, J ¼ 7.4) dC: (CDCl3) 164.57; 136.16; 124.18; 121.70; 114.91;
55.79; 30.80; 21.38; 10.29. HRMS FAB: found 212.0796; C9H13N2O4
requires 212.0797 [M þ 1]þ.
3.3.1. General alkylation procedure
Ethyl 4-nitro-1H-pyrrole-2-carboxylate was dissolved in DMF
(40 ml, anhydrous) under nitrogen gas and to this was added
potassium metal (2 eq.) whilst maintaining the nitrogen atmo-
sphere within the flask. The reaction mixture was heated to 100 ꢁC
and left at that temperature for 1 h, with stirring. The reaction
mixture was allowed to cool to 50e60 ꢁC then the required bro-
moalkane (2 eq.) and potassium iodide (2 eq.) were added, still
maintaining the inert atmosphere, and then the reaction mixture
was heated, with stirring, to 100 ꢁC and left for 5 h at that
temperature. The reaction mixture was left stirring for a further
12 h at room temperature. The DMF was removed at 50 ꢁC under
reduced pressure and the crude product was dissolved in DCM
(50 mL) and the salt was extracted with water (2 ꢂ 20 mL). The
organic layer was dried over magnesium sulphate, filtered and the
solvent removed at 30 ꢁC under reduced pressure. The crude
product was purified by flash column chromatography using an
appropriate mobile phase. The material with the required Rf value
was collected and the solvent was removed at 30 ꢁC under reduced
pressure to furnish the desired product.
3.3.6. General procedure for coupling the pyrrole carboxylic acid
with aminoethylmorpholine
The pyrrole carboxylic acid was dissolved in thionyl chloride
(4 mL) then the reaction mixture was heated to reflux and left for 3 h.
Excess thionyl chloride was removed at 30 ꢁC under reduced pressure
andtheacidchloridesoformedwasusedwithoutfurtherpurification.
N-aminoethyl morpholine (1.2 eq.) was dissolved in DCM (20 mL,
anhydrous) to which NMM (2 eq.) was added. The acid chloride was
dissolved in DCM (5 mL, anhydrous) then added dropwise to the
aminoethylmorpholine solution with stirring. The reaction mixture
was left stirring for 15 h. The crude product was extracted with
a solution of sodium carbonate (540 mg, 5.09 mmol) inwater (25 mL)
and the organic layer was dried over magnesium sulphate, filtered
and the solvent was removed at 30 ꢁC under reduced pressure. The
crude product obtained was purified by flash column chromatog-
raphy using ethyl actetate as the mobile phase. Material with the
required Rf value was collected and the solvent removed at 30 ꢁC
under reduced pressure to afford the desired product.
3.3.2. Ethyl 1-isopropyl-4-nitro-1H-pyrrole-2-carboxylate 15a
From ethyl 4-nitro-1H-pyrrole-2-carboxylate (2.370 g,
12.87 mmol) and potassium metal (977 mg, 25 mmol) and 2-
bromopropane (2.345 mL, 25 mmol) with potassium iodide
(4.150 g, 25 mmol). The purification used 1:6 ethyl actetate:hexane
as the mobile phase affording an off-white solid (850 mg, 29%), m.p.
60e62 ꢁC. nmax (KBr): 3155, 2984,1713,1536,1502,1285,1223,1175,
1135, 1110, 1084 cmꢀ1 dH (DMSO-d6): 7.80 (1H, d, NH, J ¼ 2.0), 7.45
(1H, d, NH, J ¼ 2.0), 5.50 (1H, septet, CH3CHCH3, J ¼ 6.6), 4.33 (2H, q,
OCH2CH3, J ¼ 7.1), 1.50 (6H, d, CH3CHCH3, J ¼ 6.7), 1.38 (3H, t,
CH2CH3, J ¼ 7.6).
3.3.3. Ethyl 1-sec-butyl-4-nitro-1H-pyrrole-2-carboxylate 15b
From ethyl 4-nitro-1H-pyrrole-2-carboxylate (1.536 g,
8.34 mmol) and potassium metal (585 mg, 14.96 mmol) with 2-
bromobutane (1.640 mL, 15.01 mmol) and potassium iodide
(2.132 g, 12.84 mmol) as described. The purification used 1:3 ethyl
acetate:hexane as the mobile phase affording a pale yellow oil
(614 mg 31%). nmax (NaCl): 3143, 2971, 2935, 2879, 1716, 1537, 1505,
1295,1220, 1176,1104 cmꢀ1 dH (CDCl3): 7.75 (1H, d, NH, J ¼ 2.0), 7.74
(1H, d, NH, J ¼ 2.0), 5.38 (1H, sextet, CH2CHCH3, J ¼ 6.9), 4.32 (2H, q,
OCH2CH3, J ¼ 7.1), 1.76e1.85 (2H, m, CH3CH2CH), 1.48 (3H, d, CHCH3,
3.3.7. 1-Isopropyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-pyrrole-
2-carboxamide 17a
From 1-isopropyl-4-nitro-1H-pyrrole-2-carboxylic acid 16a
(350 mg, 1.77 mmol) and aminoethylmorpholine (250
ml,
1.91 mmol) using NMM (250 l, 2.27 mmol). The product was a pale
m
yellow solid (394 mg, 72%), m.p. 107e110 ꢁC. nmax (KBr): 3312, 3121,
2928, 1638, 1560, 1529, 1503, 1420, 1172, 1110 cmꢀ1 dH (CDCl3): 7.75
(1H, d, NH, J ¼ 2.2), 7.06 (1H, d, NH, J ¼ 1.9), 6.93 (1H, bs, CONH), 5.51
(1H, septet, CH3CHCH3, J ¼ 6.7), 3.79 (4H, t, CH2CH2OCH2CH2,
J ¼ 4.6), 3.54 (2H, q, NHCH2CH2N, J ¼ 5.4), 2.68 (2H, t, NHCH2CH2N,
J ¼ 6.0), 2.61 (4H, bt, CH2CH2NCH2CH2, J ¼ 4.4), 1.48 (6H, d,
CH3CHCH3, J ¼ 6.6) dC: (CDCl3) 160.66; 135.47; 126.18; 121.75;
107.30; 66.32; 57.01; 53.26; 50.06; 35.92; 23.70. HRMS FAB: found
311.1721; C14H33N4O4 requires 311.1719 [M þ 1]þ.
J ¼ 6.8), 1.38 (3H, t, CH2CH3, J ¼ 7.1), 0.89 (3H, t, CH3CH2, J ¼ 7.4) dC
:
(CDCl3) 160.18; 135.89; 123.02; 111.91; 60.80; 51.80; 30.81; 21.38;
14.29; 10.41. HRMS FAB: found 240.1109; C11H17N2O4 requires
240.110 [M þ 1]þ.
3.3.4. 1-Isopropyl-4-nitro-1H-pyrrole-2-carboxylic acid 16a
3.3.8. 1-sec-Butyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-pyrrole-
2-carboxamide 17b
Ethyl
1-isopropyl-4-nitro-1H-pyrrole-2-carboxylate
15a
(650 mg, 2.87 mmol) was dissolved in ethanol (4 mL) and to this
was added a solution of sodium hydroxide (420 mg, 10.50 mmol) in
From 1-sec-butyl-4-nitro-1H-pyrrole-2-carboxylic acid 16b
(99 mg, 0.47 mmol) and aminoethylmorpholine (125 ml, 0.95 mmol)

5350
A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
using NMM (125
m
l, 1.14 mmol) giving the product as a pale orange
1H-pyrrole-2-carboxylic acid (150 mg, 0.88 mmol) used as the acid
chloride in the presence of NMM (250 l, 2.27 mmol).The purifica-
oil, (112 mg, 74%). nmax (NaCl): 3335, 2965, 1650, 1292, 1116 cmꢀ1 dH
(CDCl3): 7.72 (1H, d, NH, J ¼ 1.6), 7.01 (1H, d, NH, J ¼ 1.7), 6.55 (1H, bs,
m
tion used 99:1 ethyl actetate:triethylamine as the mobile phase
affording the product as a pale yellow solid (120 mg, 35%), m.p.
161e164 ꢁC. nmax (KBr): 3400, 3312, 3130, 2961, 2932, 2854, 2814,
1658, 1621, 1569, 1535, 1501, 1456, 1254, 1204, 1186, 1170, 1143,
1116 cmꢀ1 dH (CDCl3): 7.65 (1H, bs, CONH), 7.61 (1H, d, NH, J ¼ 2.2),
7.36 (1H, d, NH, J ¼ 2.2), 7.19 (1H, d, NH, J ¼ 2.2), 6.45e6.55 (2H, m,
NH and CONH), 5.34 (1H, sextet, CH2CHCH3, J ¼ 7.0), 4.05 (3H, s,
CONH), 5.34 (1H, sextet, CH2CHCH3,
J
¼
6.8), 3.74 (4H, t,
CH2CH2OCH2CH2, J ¼ 4.4), 3.48 (2H, q, NHCH2CH2N, J ¼ 5.6), 2.59 (2H,
t, NHCH2CH2N, J ¼ 5.6), 2.51 (4H, t, CH2CH2NCH2CH2, J ¼ 4.4),
1.75e1.85 (2H, m, CH3CH2CH),1.47 (3H, d, CHCH3, J ¼ 6.8), 0.88 (3H, t,
CH3CH2, J ¼ 7.6) dC: (CDCl3) 160.56; 135.65; 126.92; 127.9 21.94;
106.65; 66.94; 56.81; 55.30; 53.36; 35.64; 30.93; 21.52; 10.40. HRMS
FAB: found 325.1872; C15H25N4O4 requires 325.1872 [M þ 1]þ.
NCH3), 3.77 (4H, t, CH2CH2OCH2CH2,
J
¼
5.2), 3.49 (2H, q,
NHCH2CH2N, 5.7), 2.50e2.63 (6H, m, NHCH2CH2N and
J
¼
3.4. New dimers
CH2CH2NCH2CH2),1.70e1.85 (2H, m, CH3CH2CH), 1.44 (3H, d, CHCH3,
J ¼ 7.0), 0.85 (3H, t, CH3CH2, J ¼ 7.4) dC: (CDCl3) 161.75; 157.40;
135.04; 126.97; 126.97; 124.34; 121.21; 113.04; 106.91; 102.80;
67.00; 57.04; 53.88; 53.36; 37.99; 35.52; 31.05; 21.78; 10.71. HRMS
FAB: found 447.2361 C21H31N6O6 requires 447.2356 [M þ 1]þ.
3.4.1. General procedure for the coupling reaction to form the
dimers
The compound containing the pyrrole ring attached to the tail
group was dissolved in ethanol (10 mL), cooled to 0 ꢁC (ice bath)
and then to this was added 10% palladium over charcoal (2 weight
eq.) under nitrogen. The reaction mixture was hydrogenated at
room temperature and atmospheric pressure for 3 h. The catalyst
was removed over Kieselguhr and the solvent removed at 50 ꢁC
under reduced pressure to give the amine as an amorphous solid
which was used in the next step without further purification. The
pyrrole carboxylic acid (1 eq.) was dissolved in thionyl chloride
(3 mL) and this was heated under reflux for 3 h. Excess thionyl
chloride was removed at 50 ꢁC under reduced pressure and the
resulting acid chloride was used without further purification. The
amine was dissolved in DCM (10 mL, anhydrous) to which NMM (2
eq.) was added at room temperature with stirring. The acid chloride
was dissolved in DCM (10 mL, anhydrous) then it was added to the
amine solution, dropwise, with stirring. The reaction was left for
14 h. The solvent was removed at 50 ꢁC under reduced pressure
then the crude product was dissolved in DCM (30 mL) and
extracted with a solution of sodium carbonate (600 mg, 5.66 mmol)
in water (20 mL). The organic layer was collected, dried over
magnesium sulphate and the solvent removed at 50 ꢁC under
reduced pressure. The crude product was purified by flash column
chromatography using an appropriate mobile phase. Material with
the required Rf value was collected and the solvent removed at
50 ꢁC under reduced pressure to afford the desired product.
3.4.4. 4-{[(1-Isopropyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1-
methyl-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 18c
From
1-methyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-
pyrrole-2- carboxamide (116 mg, 0.41 mmol) and 1-isopropyl-4-
nitro-1H-pyrrole-2-carboxylic acid (70 mg, 0.35 mmol) used as
the acid chloride in the presence of NMM (140 ml, 1.27 mmol). The
purification used ethyl actetate as the mobile phase affording the
product as a pale brown solid, (72 mg, 40%), m.p. 105e115 ꢁC. nmax
(KBr): 3294, 3138, 2933, 1659, 1533, 1402, 1282, 1233, 1202, 1174,
1149, 1114, 1061 cmꢀ1 dH (CDCl3): 7.81 (1H, d, NH, J ¼ 2.2), 7.60 (1H,
bs, CONH), 7.20 (1H, d, NH, J ¼ 2.2), 7.16 (1H, d, NH, J ¼ 1.8), 6.54 (1H,
d, NH, J ¼ 1.8), 6.44 (1H, bs, CONH), 5.53 (1H, septet, CH3CHCH3,
J ¼ 6.7), 3.94 (3H, s, NCH3), 3.76 (4H, t, CH2CH2OCH2CH2, J ¼ 4.5),
3.49 (2H, q, NHCH2CH2N, J ¼ 5.7), 2.59 (2H, t, NHCH2CH2N, J ¼ 5.7),
2.52 (4H, t, CH2CH2NCH2CH2, J ¼ 4.5), 1.52 (6H, d, CH3CHCH3,
J ¼ 6.7) dC: (CDCl3) 161.55; 157.66; 135.57; 126.08; 123.83; 122.12;
120.65; 119.01; 107.23; 103.19; 66.89; 57.02; 53.30; 50.27; 36.69;
35.38; 23.74. HRMS FAB: found 433.2201; C20H29N6O5 requires
433.2199 [M þ 1]þ.
3.4.5. 4-{[(1-sec-Butyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1-
methyl-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 18d
From
1-methyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-
pyrrole-2-carboxamide (164 mg, 0.58 mmol) and 1-sec-butyl-4-
nitro-1H-pyrrole-2-carboxylic acid (90 mg, 0.42 mmol) used the
3.4.2. 1-Isopropyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 18a
acid chloride in the presence of NMM (200 ml, 1.82 mmol). The
From
1-isopropyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-
purification used ethyl actetate as the mobile phase affording the
product as a pale yellow solid, (83 mg, 44%), m.p.168e178 ꢁC. nmax
(KBr): 3317, 3136, 2854, 1658, 1566, 1533, 1494, 1292, 1230, 1142,
1112, 1072 cmꢀ1 dH (CDCl3): 7.88 (1H, bs, CONH), 7.75 (1H, d, NH,
J ¼ 1.8), 7.22 (1H, d, NH, J ¼ 1.8), 7.18 (1H, d, NH, J ¼ 1.8), 6.55e6.65
(2H, m, NH and CONH), 5.37 (1H, sextet, CH2CHCH3, J ¼ 6.8), 3.93
(3H, s, NCH3), 3.79 (4H, t, CH2CH2OCH2CH2, J ¼ 4.6), 3.51 (2H, q,
pyrrole-2-carboxamide (350 mg, 1.13 mmol) and 1-methyl-4-nitro-
1H-pyrrole-2-carboxylic acid (192 mg, 1.13 mmol) used as the acid
chloride in the presence of NMM (300 ml, 2.72 mmol). The purifica-
tion used 99:1 ethyl actetate:triethylamine as the mobile phase
affording product as a pale orange solid, (360 mg, 74%), m.p.
97e100 ꢁC. nmax (KBr): 3394, 3311, 3128, 2959, 2928, 2854, 2813,
1657,1573,1535,1501,1253,1202,1189,1177,1141,1114,1069 cmꢀ1 dH
(CDCl3): 7.61 (1H, d, NH, J ¼ 1.8), 7.58 (1H, bs, CONH), 7.39 (1H, d, NH,
J ¼ 1.7), 7.18 (1H, d, NH, J ¼ 1.7), 6.51 (1H, d, NH, J ¼ 1.7), 6.45 (1H, bs,
CONH), 5.54 (1H, septet, CH3CHCH3, J ¼ 6.7), 4.05 (3H, s, NCH3), 3.76
(4H, t, CH2CH2OCH2CH2, J ¼ 4.6), 3.49 (2H, q, NHCH2CH2N, J ¼ 5.6),
2.59 (2H, t, NHCH2CH2N, J ¼ 6.1), 2.52 (4H, t, CH2CH2NCH2CH2,
J ¼ 4.4), 1.45 (6H, d, CH3CHCH3, J ¼ 6.7) dC: (CDCl3) 161.73; 157.47;
135.02; 127.00; 126.34; 123.43; 121.24; 113.06; 107.12; 103.36;
66.83; 57.12; 53.32; 48.48; 38.02; 35.42; 23.82. HRMS FAB: found
433.2202; C20H29N6O5 requires 433.2199 [M þ 1]þ.
NHCH2CH2N,
J
¼
5.6), 2.55e2.77 (6H, m, NHCH2CH2N and
CH2CH2NCH2CH2), 1.75e1.88 (2H, m, CH3CH2CH), 1.50 (3H, d,
CHCH3, J ¼ 6.8), 0.89 (3H, t, CH3CH2, J ¼ 7.4). dC: (CDCl3) 161.56;
157.73; 135.71; 126.69; 123.79; 122.34; 120.72; 119.05; 107.04;
103.22; 66.89; 57.05; 55.50; 53.31; 36.69; 35.37; 30.89; 21.59;
10.42. HRMS FAB: found 447.2365; C21H31N6O5 requires 447.2356
[M þ 1]þ.
3.5. Head groups
3.5.1. Methyl 4-[(E)-3-quinolinyldiazenyl]benzoate 9a
3.4.3. 1-sec-Butyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 18b
To a solution of 3-aminoquinoline (0.05 g; 0.34 mmol) in glacial
acetic acid was added a solution of methyl 4-nitrosobenzoate, 8
(0.06 g; 0.34 mmol) in glacial acetic acid (2 mL) with stirring at
room temperature. The solution was then stirred at 70 ꢁC under
From
1-sec-butyl-N-[2-(4-morpholinyl)ethyl]-4-nitro-1H-
pyrrole-2-carboxamide (250 mg, 0.77 mmol) and 1-methyl-4-nitro-

A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
5351
a nitrogen atmosphere for 24 h during which time a brown
3.5.5. Methyl 4-{(E)-[4-(dimethylamino)phenyl]diazenyl} benzoate
12 [13]
precipitate formed and the solution became almost solid. The solid
was collected by filtration. The filtrate was neutralised with satu-
rated aqueous sodium bicarbonate solution (50 mL). Ethyl acetate
(100 mL) was then added and the resulting organic layer separated,
washed with brine and, dried (MgSO4), and concentrated under
reduced pressure to give a brown solid which was combined with
the solid obtained by filtration. The combined solid products were
then purified by flash column chromatography (20% ethyl acetate in
hexane as eluant) to give the required diazo compound 9a as an
orange solid (0.06 g, 60%), m.p. 150e152 ꢁC, nmax (KBr): 3420, 1727,
1603, 1573, 1497, 1462 cmꢀ1 dH (CDCl3): 9.53 (1H, s, CH quinolyl),
8.68 (1H, s, CH quinolyl), 8.25 (2H, d, J ¼ 8.6, CH Ar), 8.22 (1H, d,
J ¼ 8.6, CH Ar), 8.02e8.06 (3H, m, CH Ar), 7.85 (1H, t, J ¼ 7.0, CH Ar),
7.65 (1H, t, J ¼ 7.4, CH Ar), 3.98 (3H, s, CH3). dC (CDCl3): 52.3; 123.12;
124.0; 127.9; 129.6; 129.67; 130.9; 131.5; 132.4; 142.3; 144.6; 145.1;
155.2; 166.7; 178.3. HRFABMS: found 292.1082; C17H14O2N3
(M þ H)þ requires 292.1086.
Methyl 4-aminobenzoate (0.38 g, 2.5 mmol) was disssolved in
hydrochloric acid solution (20% of 12 M hydrochloric acid in
water; 20 mL) and the solution cooled to 0 ꢁC using an ice/salt
bath with vigorous stirring. A solution of sodium nitrite (0.17 g;
2.5 mmol) in water (1 mL) was then added dropwise and the
solution stirred at 0 ꢁC for 30 min to allow formation of the dia-
zonium salt to occur. During this time, N,N-dimethylaniline (0.3 g;
2.5 mmol) dissolved in methanol (5 mL) and sodium hydroxide
(10% soln. in water; 20 mL) was cooled to 0 ꢁC. This solution was
then added dropwise to the diazonium salt solution. The solution
was then adjusted to pH 6 by the addition of 10% aqueous sodium
hydroxide solution. The solution was extracted with ethyl acetate
(100 mL), the organic layer was separated, washed with brine
(1 ꢂ 50 mL) and saturated aqueous sodium bicarbonate solution
(2 ꢂ 50 mL), dried (MgSO4), then concentrated under reduced
pressure to give a brown solid. The brown solid was purified by
flash column chromatography (10% EtOAc in hexane as eluant).
Evaporation of the solvents gave the required diazene 12 as a red
solid (0.42 g, 60%), m.p. 183e184 ꢁC. nmax (KBr): 3358, 2905, 1710,
1606 cmꢀ1 dH (CDCl3): 8.16 (2H, dd, J ¼ 6.8 and J ¼ 1.7, CH Ar),
7.86e7.92 (4H, m, CH Ar), 6.78 (2H, d, J ¼ 9.2 Hz, CH Ar), 4.14 (3H, s,
CH3), 3.11 (6H, s, (CH3)2). HREIMS: found 283.1320; C16H17O2N3
requries 283.1321.
3.5.2. 4-[(E)-3-quinolinyldiazenyl]benzoic acid 10a
The foregoing methyl ester 9a (0.08 g; 0.275 mmol) was dis-
solved in methanol (2 mL) and to this was added a solution of
lithium hydroxide (0.014 g; 2 equiv) in water (10 mL) causing the
ester to precipitate. The mixture was then heated to 70 ꢁC and
stirred for 24 h during which time a strong red colour was
produced. TLC analysis of the mixture showed 100% conversion to
the lithium salt of the acid. The solution was then acidified to pH 6
using hydrochloric acid (10% solution in water). Ethyl acetate
(100 mL) was added to this mixture, the resulting organic layer was
removed, washed with brine (50 mL) and saturated aqueous
sodium bicarbonate solution (2 ꢂ 50 mL) and dried (MgSO4).
Evaporation of the solvents under reduced pressure gave the
required carboxylic acid as an orange solid (0.075 g, 98%), with no
distinct melting point. nmax (KBr): 3445, 1634, 1462, 1425 cmꢀ1 dH
(CDCl3): 9.45 (1H, s, CH quinolyl), 8.93 (1H, s, CH quuinolyl), 8.36
(1H, d, CH Ar, J ¼ 8.4), 8.27 (1H, d, J ¼ 8.0, CH Ar), 8.05e8.19 (4H, m,
CH Ar), 7.95 (1H, t, CH Ar, J ¼ 7.6), 7.69 (1H, t, CH Ar, J ¼ 7.4). dC
(DMSO-d6): 167.4; 166.8; 154.3; 149.2; 144.8; 131.2; 130.7; 130.5;
130.1; 129.7; 128.3; 125.1; 123.1; 122.9. HRFABMS: found 278.0925;
C16H12O2N3 (M þ H)þ requires 278.0924.
3.5.6. 4-{(E)-[4-(Dimethylamino)phenyl]diazenyl}benzoic acid 13
[23]
From the foregoing ester using the method described above as
a red solid 53% yield, m.p. >230 ꢁC (lit. > 230 ꢁC) [22], nmax (KBr):
3374, 3076, 2653, 1681, 1597, 1521 cmꢀ1 dH (DMSO-d6): 8.08 (2H, d,
J ¼ 8.8, CH Ar), 7.81e7.84 (4H, m, CH Ar), 6.86 (2H, d, J ¼ 9.2, CH Ar),
3.08 (6H, s,(CH3)2). HREIMS: found 269.1162; C15H15O2N3 requires
269.1164.
3.6. Amides
3.6.1. Methyl 4-[(3-quinolinylcarbonyl)amino]benzoate 19a [2]
Quinoline 3-carboxylic acid (0.20 g, 1.15 mmol) was dissolved in
methanol (5 mL) and DMF (1 mL); methyl 4-aminobenzoate (0.17 g,
1.15 mmol) was then added followed by DMT-MM (0.30 g,
1.15 mmol). The mixture was then stirred at room temperature for
24 h. During this time the product formed as a white precipitate
which was collected by filtration and dried under reduced pressure
(0.25 g, 70%), m.p. >230 ꢁC (lit. >230 ꢁC) [2], nmax (KBr): 3249, 2994,
1715, 1676, 1600, 1478 cmꢀ1 dH (DMSO-d6): 10.9 (1H, s, NH), 9.36
(1H, d, J ¼ 2.3, CH Ar), 8.9 (1H, d, J ¼ 1.9, CH Ar), 8.12e8.17 (2H, m,
CH Ar), 7.99e8.02 (4H, m, CH Ar), 7.89e7.91 (1H, m, C12), 7.74 (1H, t,
Similarly prepared were:
3.5.3. Methyl 4-[(E)-(3-methoxyphenyl)diazenyl]benzoate 9b
From m-Anisidine (0.23 g; 1.8 mmol) and methyl 4-
nitrosobenzoate (0.3 g; 1.8 mmol) in acetic acid (glacial, 10 mL) at
reflux for 24 h. Purification by flash column chromatography (30%
ethyl acetate/hexane). Removal afforded the required diazene 139,
as an orange solid (0.19 g, 40%), m.p. 93e95 ꢁC. nmax (KBr): 2958,
1727, 1600, 1435 cmꢀ1dH (CDCl3): 8.18e8.21 (2H, m, CH Ar), 7.98
(2H, d, J ¼ 8.6, CH Ar), 7.61 (1H, d, J ¼ 7.7 CH Ar), 7.43e7.49 (m, 2H,
CH Ar), 7.1 (1H, dd, J ¼ 8.2 and J ¼ 1.8, CH Ar), 3.99 (3H, s, OCH3),
3.91(3H, s, OCH3). dC (CDCl3): 52.5; 55.7; 106.0; 117.8; 118.7; 122.8;
130.1; 130.8; 132.1; 154.1; 155.3; 160.6; 166.6. HREIMS: found
270.1002; C15H14N2O3 requires 270.1004. Found: C, 66.4; 5.24; N,
10.01; C15H14N2O3 requires C, 66.66; H, 5.22; N, 10.36%.
J
¼
7.5, C13), 3.85 (3H, s, CH3). HREIMS: found 306.1007;
C18H14N2O3 requires 306.1004.
3.6.2. 4-[(3-Quinolinylcarbonyl)amino]benzoic acid 20a [2]
To
a solution of methyl 4-[(3-quinolinylcarbonyl)amino]
benzoate, 19a, (0.15 g, 0.51 mmol) in methanol (5 mL) a solution of
lithium hydroxide (0.02 g, 0.92 mmol) in water (4 mL) was added.
The resulting suspension was heated at 80 ꢁC with stirring for 5 h.
The solution was then acidified using dilute hydrochloric acid and
the precipitated solid filtered off and dried under reduced pressure
to give the required carboxylic acid, 20a, as a pale brown solid
(0.14 g, 95%), m.p. >230 ꢁC (lit. >230 ꢁC) [2], nmax (KBr): 3320, 3052,
2928, 2473, 1930, 1695, 1542 cmꢀ1 dH (DMSO-d6): 10.9 (1H, s, NH),
9.38 (1H, s, CH Ar), 9.03 (1H, d, J ¼ 2.0, CH Ar), 8.13e8.19 (2H, m, CH
Ar), 7.91e7.99 (5H, m, CH Ar), 7.9 (1H, t, J ¼ 7.1, CH Ar). HRFABMS:
found 293.0924; C17H13O3N2 (M þ H)þ requires 293.0921.
3.5.4. [(E)-(3-Methoxyphenyl)diazenyl]benzoic acid 10b
From the foregoing ester in 85% yield as an orange solid with no
distinct melting point. nmax (KBr): 2934, 2834, 1680, 1600,
1429 cmꢀ1 dH (DMSO-d6): 8.13e8.16 (2H, m, CH Ar), 7.97 (2H, dd,
J ¼ 6.8 and J ¼ 2.0, CH Ar), 7.54e7.56 (2H, m, CH Ar), 7.45 (1H, t,
J ¼ 2.0, CH Ar), 7.18e7.21 (1H, m, CH Ar), 3.99 (3H, s, OCH3). dC
(DMSO-d6): 56.3; 107.1; 117.5; 119.4; 123.4; 131.2; 131.5; 133.8;
154.0; 155.1; 161.0; 167.5. HRFABMS: found 257.0928; C14H13N2O3
(M þ H)þ requires 257.0926.

5352
A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
3.6.3. Methyl 4-[(3-methoxybenzoyl)amino]benzoate 19b [17]
3-Methoxybenzoyl chloride (0.20 g, 1.17 mmol) was dissolved in
dichloromethane (5 mL, dry) to which a solution of methyl 4-
aminobenzoate (0.18 g, 1.17 mmol) in dichloromethane (5 mL, dry)
was added followed by N-methylmorpholine (0.25 mL, 2.34 mmol).
The solution was then stirred at room temperature for 3 h. Sodium
hydroxide solution (aq. 10% w/v, 20 mL) was then added to the
solution, the dichloromethane layer removed, dried (MgSO4), and
the solvent evaporated under reduced pressure to give a pale brown
solid which was purified using silica gel column chromatography
(eluant: 30% ethyl acetate/n-hexane). Removal of the solvent gave
the required amide, as a brown solid (0.20 g, 60%), m.p. 88e90 ꢁC (lit.
79e81 ꢁC) [17], nmax (KBr): 3356, 2942, 1709, 1666, 1522 cmꢀ1 dH
(CDCl3): 8.07 (2H, dd, J ¼ 6.8 and 1.9, CH Ar), 7.75 (2H, dd, J ¼ 6.9 and
J ¼ 1.9, CH Ar), 7.45 (1H, s, CH Ar), 7.40e7.41 (2H, m, CH Ar), 7.10e7.12
(1H, m, CH Ar), 3.92 (3H, s, OCH3), 3.85(3H, s, OCH3). HRFABMS:
found 286.1075; C16H16NO4 (M þ H)þ requires 286.1079.
(2H, d, J ¼ 7.8, CH Ar), 7.64 (2H, d, J ¼ 7.4, CH Ar), 7.49 (2H, d, J ¼ 7.8,
CH Ar), 7.32 (1H, d, J ¼ 16.8, CH]CH), 7.09 (1H, d, J ¼ 16.8, CH]CH),
6.83 (2H, s (broad), CH Ar), 2.96 (6H, s, (CH3)2). HRFABMS: found
268.1341; C17H18NO2 (M þ H)þ requires 268.1338.
3.8. Minor groove binders
3.8.1. 1-Methyl-N-[1-methyl-5-({[2-(4-morpholinyl)ethyl]amino}
carbonyl)-1H-pyrrol-3-yl]-4-({4-[(E)-3-quinolinyldiazenyl]
benzoyl}amino)-1H-pyrrole-2-carboxamide 3b
A solution of the dipyrrole dimer, 1-methyl-4-{[(1-methyl-4-
nitro-1H-pyrrol-2-yl)carbonyl]amino}-N-[2-(4-morpholinyl)
ethyl]-1H-pyrrole-2-carboxamide 11 (0.06 g; 0.15 mmol) in meth-
anol (25 mL) was cooled in an ice/salt bath to 0 ꢁC with stirring
under a nitrogen atmosphere. To this was added palladium on
carbon (10% w/w, 25 mg) slowly in small portions. The reaction
vessel was evacuated and then filled with hydrogen. The reaction
mixture was then sealed and left to stir vigorously for 3 h. The
solution was then filtered under suction through Kieselguhr and the
filtrate evaporated under reduced pressure taking care that the
resulting solid was completely free of solvent. The aminopyrrole
dimer so obtained was used directly without purification or analysis.
The aminopyrrole dimer was dissolved in dry DMF (1 mL) to
3.6.4. 4-[(3-Methoxybenzoyl)amino]benzoic acid 20b [2]
From the foregoing methyl ester using the method described
above in 90% yield as a white solid. m.p. >230 ꢁC (lit. >230 ꢁC) [2],
nmax (KBr): 3401, 2924, 2854,1681,1632,1598,1513,1462,1424 cmꢀ1
dH (DMSO-d6): 10.48 (1H, s, NH), 7.90e7.95 (4H, m, CH Ar),
7.40e7.62 (3H, m, CH Ar), 7.16e7.18 (1H, m, CH Ar), 3.85 (3H, s, CH3).
HRFABMS: found 272.0922; C15H14O4N (M þ H)þ requires 272.0923.
Similarly prepared were:
which was added HBTU (0.11 g; 0.3 mmol;
2 equiv), 4-
methylmorpholine (0.025 mL; 0.45 mmol; 3 equiv) and the dimer
4-[(E)-3-quinolinyldiazenyl]benzoic acid (10a) (0.04 g; 0.15 mmol).
The solution was stirred until all the solid material had dissolved
and allowed to stand overnight. The crude product was purified by
HPLC giving the required minor groove binder 3b, as an orange
solid with no definite melting point (0.33 g, 35%), purity by HPLC:
>95%. nmax (KBr): 3394, 1678, 1554, 1464 cmꢀ1 dH (DMSO-d6): 10.57
(1H, s, NH); 10.0 (1H, s, NH), 9.80 (1H, bs, TFA), 9.46 (1H, d, J ¼ 2.0,
CH quinolyl), 8.94 (1H, d, J ¼ 2.4, quinolyl), 8.28e8.29 (1H, m, NH),
8.22e8.27 (2H, m, CH quinolyl), 8.10e8.16 (4H, m, CH Ar), 7.92 (1H,
t, J ¼ 7.0, CH Ar), 7.76 (1H, t, J ¼ 7.6, CH Ar), 7.37 (1H, d, J ¼ 2.0, CH
pyrrole), 7.23 (1H, d, J ¼ 2.0, CH pyrrole), 7.16 (1H, d, J ¼ 1.6, CH
pyrrole), 7.01 (1H, d, J ¼ 1.6, CH pyrrole), 3.99e4.02 (2H, m, CH2),
3.92 (3H, s, CH3), 3.89 (3H, s, CH3), 3.55e3.70 (6H, m, 3 ꢂ CH2),
3.26e3.29 (2H, m, CH2), 3.12e3.16 (2H, m, CH2).HRFABMS: found
634.2889; C34H36O4Nþ9 requires 634.2890.
3.6.5. Methyl 4-{[4-(dimethylamino)benzoyl]amino}benzoate 19c
[23]
From 4-dimethylaminobenzoyl chloride and methyl 4-
aminobenzoate as a white solid (0.10 g, 30%), m.p. 208e210 ꢁC,
purity by HPLC 94%. nmax (KBr): 3336,1711,1655,1609,1517 cmꢀ1 dH
(DMSO-d6): 10.26 (1H, s, NH), 7.90e7.97 (6H, m, CH Ar), 6.83e6.86
(2H, m, CH Ar), 3.8 (3H, s, CH3), 3.05 (6H, s, (CH3)2). HRCIMS: found
299.1390; C17H19NO4 (M þ H)þ requires 299.1396.
3.6.6. 4-{[4-(Dimethylamino)benzoyl]amino}benzoic acid 20c
From the foregoing ester in 85% yield as a white solid, m.p.
>230 ꢁC, nmax (KBr): 3341, 2561, 1675, 1606, 1502 cmꢀ1 dH (DMSO-
d6): 10.12 (1H, s, NH), 7.86e7.89 (6H, m, CH Ar), 6.86 (2H, d, J ¼ 8.8,
CH Ar), 3.0 (6H, s, (CH3)2). dC (DMSO-d6): 38.8; 111.3; 119.2; 124.7;
129.4; 130.1; 143.8; 152.1; 165.4; 166.9. HRFABMS: found 285.1242
C16H17N2O3 (M þ H)þ requires 285.1239.
Similarly prepared were:
3.8.2. N-[4-({[1-Methyl-5-({[1-methyl-5-({[2-(4-morpholinyl)
ethyl]amino}carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1H-pyrrol-
3-yl]amino}carbonyl)phenyl]-3-quinoline carboxamide 3c
3.7. Alkenes
From
1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
3.7.1. Methyl 4-{(E)-2-[4-(dimethylamino)phenyl]ethenyl}
benzoate 21 [25]
carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-
carboxamide (11) and 4-[(3-quinolinylcarbonyl)amino] benzoic
acid 20a in 15% yield, purity by HPLC >95%. nmax (KBr): 3424, 1676,
1530, 1436, 1406 cmꢀ1 dH (DMSO-d6): 10.87 (1H, s, NH), 10.28 (1H,
s, NH), 9.98 (1H, s, NH), 9.68 (1H, bs, TFA), 9.39 (1H, d, J ¼ 2.0, CH
quinlolyl), 9.01 (1H, d, J ¼ 1.6, CH quinloly), 8.20e8.22 (1H, m, NH),
8.12e8.19 (2H, m, CH quinloyl), 7.80e7.90 (5H, m, CH Ar), 7.75e7.76
(1H, m, CH Ar), 7.33 (1H, d, J ¼ 1.6, CH pyrrole), 7.22 (1H, d, J ¼ 1.6,
CH pyrrole), 7.12 (1H, d, J ¼ 1.6, CH pyrrole), 7.0 (1H, d, J ¼ 2.0, CH
pyrrole), 3.99e4.0 (2H, m, CH2), 3.88 (3H, s, CH3), 3.83 (3H, s, CH3),
3.69e3.73 (2H, m, CH2), 3.54e3.55 (4H, m, 2 ꢂ CH2), 3.26e3.29 (2H,
m, CH2); 3.11e3.15 (2H, m, CH2). HRFABMS: found 649.2887;
C35H37N7Oþ5 requires 649.2902.
Diethyl
(4-(methoxycarbonyl)phenyl)methylphosphonate
(0.10 g; 0.35 mmol) was dissolved in THF (dry, 5 mL) and the solution
was cooled to 0 ꢁC. Sodium hydride (0.13 g; 8 equiv) was then added
slowly in small portions to this solution which was then stirred for
20 min. 4-(Dimethylamino)benzaldehyde (0.052 g; 0.35 mmol) was
dissolved inTHF (dry, 3 mL) and added dropwise to this solution. The
resulting mixture was stirred at room temperature for 4 h. The
reactionwas then quenched withwater (10 mL) causingawhite solid
to precipitate. The precipitate was collected by filtration and dried
under reduced pressure. The product obtained was shown to be
a mixture of the esterand the corresponding carboxylic acid by LRMS
the mixture was therefore hydrolysed directly to the carboxylic acid.
3.8.3. ({4-[(E)-(3-Methoxyphenyl)diazenyl]benzoyl}amino)-1-
methyl-N-[1-methyl-5-({[2-(4-morpholinyl)ethyl]amino}
carbonyl)-1H-pyrrol-3-yl]-1H-pyrrole-2-carboxamide 4b
3.7.2. 4-{(E)-2-[4-(Dimethylamino)phenyl]ethenyl}benzoic acid 22
[25]
From the above mixture in 85% yield, m.p. >230 ꢁC (lit. >230 ꢁC)
[25], nmax (KBr): 3429, 2925, 1681, 1595 cmꢀ1 dH (DMSO-d6): 7.9
From
1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-

A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
5353
carboxamide 11 and [(E)-(3-methoxyphenyl)diazenyl] benzoic acid
3.8.7. 4-[(4-{[4-(Dimethylamino)benzoyl]amino}benzoyl)amino]-
10b (0.026 g; 0.1 mmol) to give the product as an orange solid with
no distinct melting point (0.021 g, 35%), purity by HPLC >95%. nmax
(KBr): 3414, 1678, 1600, 1526, 1436 cmꢀ1 dH (DMSO-d6): 10.56 (1H,
s, NH), 10.03 (1H, s, NH), 9.65 (1H, bs, TFA), 8.20e8.26 (1H, m, NH)
8.2 (2H, d, J ¼ 8.4, CH Ar), 8.03 (2H, d, J ¼ 8.4, CH Ar), 7.53e7.57 (2H,
m, CH Ar), 7.46 (1H, s, CH pyrrole), 7.37 (1H, s, CH pyrrole),
7.21e7.24 (2H, m, CH Ar), 7.15 (1H, s, CH pyrrole), 7.01 (1H, s, CH
pyrrole), 4.00e4.01 (2H, m, CH2), 3.89 (3H, s, CH3), 3.88 (3H, s, CH3),
3.84 (3H, s, CH3), 3.67e3.70 (2H, m, CH2), 3.53e3.57 (4H, m,
2 ꢂ CH2), 3.26e3.28 (2H, m, CH2); 3.13e3.17 (2H, m, CH2).
HRFABMS: found 613.2899; C32H37N8Oþ5 requires 613.2887
[M þ 1]þ.
1-methyl-N-[1-methyl-5-({[2-(4-morpholinyl)ethyl]amino}
carbonyl)-1H-pyrrol-3-yl]-1H-pyrrole-2-carboxamide 5c
From 1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 11
and 4-{[4-(dimethylamino)benzoyl] amino}benzoic acid 20c as
a brown solid with no distinct melting point (12 mg, 20%), purity by
HPLC >95%. nmax (KBr): 3414, 2963, 1650, 1604, 1536, 1437 cmꢀ1 dH
(DMSO-d6): 10.19 (1H, s, NH), 10.06 (1H, s, NH), 9.96 (1H, s, NH),
9.72 (1H, bs, TFA), 8.23 (1H, s, NH), 7.88e7.91 (6H, m, CH Ar), 7.31
(1H, s, CH pyrrole), 7.21 (1H, s, CH pyrrole), 7.10 (1H, s, CH pyrrole),
7.00 (1H, s, CH pyrrole), 6.7 (2H, d, J ¼ 9.2, CH Ar), 3.99e4.0 (2H, m,
CH2), 3.87 (3H, s, CH3), 3.83 (3H, s, CH3), 3.64e3.72 (2H, m, CH2),
3.54e3.58 (4H, m, 2 ꢂ CH2), 3.26e3.29 (2H, m, CH2), 3.11e3.15 (2H,
m, CH2), 3.01 (6H, s, (CH3)2). HRFABMS: Found 641.3197;
C34H41N8Oþ5 requires 641.3200 [M þ 1]þ.
3.8.4. 4-({4-[(3-Methoxybenzoyl)amino]benzoyl}amino)-1-methyl-
N-[1-methyl-5-({[2-(4-morpholinyl)ethyl]amino} carbonyl)-1H-
pyrrol-3-yl]-1H-pyrrole-2-carboxamide 4c
From
1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
3.8.8. 1-Isopropyl-4-({[1-methyl-4-({4-[(E)-2-(3-quinolinyl)
ethenyl]benzoyl}amino)-1H-pyrrol-2-yl]carbonyl}amino)-N-[2-(4-
morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 6a
carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-
carboxamide 11 and 4-[(3-Methoxybenzoyl)amino]benzoic acid
20b to give the product as a white solid with no distinct melting
point (12 mg, 20%), purity by HPLC >95%. nmax (KBr): 3421, 1673,
1592, 1525 cmꢀ1 dH (DMSO-d6): 10.45 (1H, s, NH), 10.24 (1H, s, NH),
9.97 (1H, s, NH), 9.59 (1H, bs, TFA), 8.25 (1H, s, NH), 7.91e7.98 (4H,
m, CH Ar), 7.47e7.56 (3H, m, CH Ar), 7.32 (1H, d, J ¼ 1.6, CH pyrrole),
7.20e7.22 (2H, m, CH Ar), 7.11 (1H, d, J ¼ 1.6, CH pyrrole), 7.00 (1H,
d, J ¼ 1.6, CH pyrrole), 4.00e4.03 (2H, m, CH2), 3.88 (3H, s, CH3),
3.86 (3H, s, CH3), 3.84 (3H, s, CH3), 3.64e3.72 (2H, m, CH2),
3.54e3.56 (4H, m, 2 ꢂ CH2), 3.25e3.29 (2H, m, CH2), 3.12e3.16 (2H,
m, CH2) HRFABMS: found 628.2879 calculated for C34H38N7O6þ
requires 628.2884 [M þ 1]þ.
From
1-isopropyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-
carboxamide 18a (35 mg, 0.08 mmol) and 4-[(E)-2-(3-quinolinyl)
ethenyl]benzoic acid (22 mg, 0.08 mmol) using HBTU (60 mg,
0.16 mmol). Purification by HPLC furnished the desired product as
a pale yellow solid, giving a yield of (14 mg, 25%), purity by HPLC
>95% with no distinct melting point. nmax (KBr): 3414, 1674, 1580,
1529, 1406, 1259, 1201, 1133, 838 cmꢀ1 dH NMR (DMSO-d6): 10.38
(1H, s, ArH), 10.00 (1H, s, ArH), 9.61 (1H, bs, TFA), 9.27 (1H, s, ArH,
J ¼ 2.1), 8.57, (1H, s, ArH, J ¼ 2.1), 8.23 (1H, t, CONH, J ¼ 8.0),
7.99e8.04 (5H, m), 7.74e7.83 (4H, m), 7.60e7.72 (4H, m), 7.38 (1H,
d, J ¼ 1.7), 7.34 (1H, d, J ¼ 1.7), 7.13 (1H, d, J ¼ 1.7), 6.98 (1H, d,
J ¼ 1.7), 5.49 (1H, septet, CH3CHCH3, J ¼ 6.7), 3.83 (3H, s, NCH3),
3.99e4.02 (2H, m), 3.61e3.68 (2H, m), 3.53e3.58 (4H, m),
3.25e3.29 (2H, m), 3.05e3.18 (2H, m), 1.34 (6H, d, CH3CHCH3,
J ¼ 6.7). HRMS FAB: found 660.3270; C38H41N7O4 requires 660.3293
obtained for [M þ 1]þ.
3.8.5. 4-[(4-{(E)-[4-(Dimethylamino)phenyl]diazenyl}benzoyl)
amino]-1-methyl-N-[1-methyl-5-({[2-(4-morpholinyl)ethyl]amino}
carbonyl)-1H-pyrrol-3-yl]-1H-pyrrole-2-carboxamide 5b
From 1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 11
and 4-{(E)-[4-(dimethylamino)phenyl] diazenyl}benzoic acid 13 as
a white solid (35 mg, 40%) with no distinct melting point, purity by
3.8.9. 1-sec-Butyl-4-({[1-methyl-4-({4-[(E)-2-(3-quinolinyl)
ethenyl]benzoyl}amino)-1H-pyrrol-2-yl]carbonyl}amino)-N-[2-(4-
morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 6b
HPLC >95%. nmax (KBr): 3422, 2927, 1681, 1647, 1596 cmꢀ1 dH
:
(DMSO-d6) 10.43 (1H, s, NH); 9.98 (1H, s, NH), 9.65 (1H, bs, TFA),
8.23 (1H, m, NH), 8.1 (2H, d, J ¼ 8.4, CH Ar), 7. 22e7.88 (4H, m, CH
Ar), 7.34 (1H, s, CH pyrrole), 7.21 (1H, s, CH pyrrole), 7.13 (1H, s, CH
pyrrole), 7.0 (1H, s, CH pyrrole), 6.8 (2H, d, J ¼ 9.6, CH Ar), 3.99e4.02
(2H, m, CH2), 3.88 (3H, s, CH3), 3.83 (3H, s, CH3), 3.54e3.71 (6H, m,
3 ꢂ CH2), 3.27e3.29 (2H, m, CH2), 3.12e3.15 (2H, m, CH2), 3.09 (6H,
s, (CH3)2). HRFABMS: found 626.3204; C33H40O4Nþ9 requires
626.3203 [M þ 1]þ.
From
1-sec-butyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-
carboxamide 18b (20 mg, 0.045 mmol) and 4-[(E)-2-(3-quinolinyl)
ethenyl]benzoic acid (12 mg, 0.045 mmol) using HBTU (30 mg,
0.08 mmol) as described. Purification by HPLC furnished the
desired product as a bright orange solid, (6 mg, 11%), purity by HPLC
>95%. nmax (KBr): 3420, 2925, 1683, 1639, 1406, 1206, 1136, 803,
723, 609 cmꢀ1 dH (DMSO-d6): 10.36 (1H, s, ArH), 9.98 (1H, s, ArH),
9.66 (1H, bs, TFA), 9.26 (1H, d, ArH, J ¼ 2.1), 8.55, (1H, d, ArH, J ¼ 2.1),
8.22 (1H, m, CONH), 7.99e8.04 (5H, m), 7.74e7.83 (4H, m),
7.60e7.71 (4H, m), 7.33 (2H, m), 7.13 (1H, d, J ¼ 1.7), 6.96 (1H, s),
5.33 (1H, sextet, CH2CHCH3, J ¼ 6.8), 3.99e4.02 (2H, m), 3.88 (3H, s,
NCH3), 3.62e3.68 (2H, m), 3.53e3.58 (4H, m), 3.25e3.29 (2H, m),
3.05e3.18 (2H, m), 1.70e1.74 (2H, m, CH3CH2CH), 1.34 (3H, d,
CHCH3, J ¼ 6.7), 0.75 (3H, t, CH3CH2, J ¼ 7.3). HRMS FAB: found
674.3452; C39H44N7O4 requires 674.3455 [M þ 1]þ.
3.8.6. 4-[(4-{(E)-2-[4-(Dimethylamino)phenyl]ethenyl}benzoyl)
amino]-1-methyl-N-[1-methyl-5-({[2-(4-morpholinyl)ethyl] amino}
carbonyl)-1H-pyrrol-3-yl]-1H-pyrrole-2-carboxamide 5a
From 1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl) carbonyl]
amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 11
and 4-{(E)-2-[4-(dimethylamino) phenyl]ethenyl}benzoic acid 22
as a brown solid with no distinct melting point (29 mg, 40%), purity
by HPLC >95%. nmax (KBr): 3421, 1678, 1594, 1524, 1434, 1400 cmꢀ1
dH (DMSO-d6): 10.35 (1H, s, NH),10.03 (1H, s, NH), 9.80 (1H, bs, TFA),
8.24e8.27 (1H, m, NH), 7.94 (2H, d, J ¼ 8.0, CH Ar), 7.92 (2H, d, J ¼ 8.4,
CH Ar), 7.47e7.49 (2H, d, J ¼ 8.8, CH Ar), 7.00e7.42 (5H, mAr and
pyrrole), 6.77 (2H, d, J ¼ 8.8, CH Ar), 6.57 (1H, s, CH Ar), 4.02e4.04
(2H, m, CH2), 3.9 (3H, s, CH3), 3.88 (3H, s, CH3), 3.67e3.73 (2H, m,
CH2), 3.53e3.57 (4H, m, 2 ꢂ CH2), 3.26e3.30 (2H, m, CH2), 3.11e3.15
(2H, m, CH2), 2.96 (6H, s, (CH3)2). HRFABMS: found 624.0345;
C36H42N7Oþ4 requires 624.0320 [M þ 1]þ.
3.8.10. 4-({[1-isopropyl-4-({4-[(E)-2-(3-quinolinyl)ethenyl]
benzoyl}amino)-1H-pyrrol-2-yl]carbonyl}amino)-1-methyl-N-[2-
(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 6c
From
4-{[(1-isopropyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
amino}-1-methyl-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-
carboxamide 6c (30 mg, 0.07 mmol) and 4-[(E)-2-(3-quinolinyl)
ethenyl]benzoic acid (19 mg, 0.07 mmol) using HBTU (50 mg,

5354
A.I. Khalaf et al. / European Journal of Medicinal Chemistry 46 (2011) 5343e5355
0.14 mmol). Purification by HPLC furnished the desired product as
a bright orange solid, (15 mg, 33%), purity by HPLC >95% with no
distinct melting point. nmax (KBr): 3421, 1676, 1582, 1532, 1410,
1251, 1201, 1132, 971 cmꢀ1 dH (DMSO-d6): 10.38 (1H, s, ArH), 10.04
(1H, s, ArH), 9.54 (1H, bs, TFA), 9.27 (1H, s, ArH, J ¼ 2.1), 8.56, (1H, s,
ArH), 8.24 (1H, t, CONH, J ¼ 8.0), 7.99e8.04 (5H, m), 7.74e7.83 (4H,
m), 7.60e7.71 (4H, m), 7.51 (1H, d, J ¼ 1.7), 7.21 (1H, d, J ¼ 1.7), 7.04
(1H, d, J ¼ 1.7), 7.01 (1H, d, J ¼ 1.7), 5.46 (1H, septet, CH3CHCH3,
J ¼ 6.7), 3.83 (3H, s, NCH3), 3.99e4.02 (2H, m), 3.62e3.68 (2H, m),
3.53e3.58 (4H, m), 3.25e3.29 (2H, m), 3.05e3.18 (2H, m), 1.39 (6H,
d, CH3CHCH3, J ¼ 6.8). HRMS FAB: found 660.3298; C38H41N7O4
requires 660.3274 [M þ 1]þ.
ArH), 9.65 (1H, bs, TFA), 9.06 (1H, d, ArH, J ¼ 2.1), 8.26 (1H, d, J ¼ 2.0),
8.24 (1H, d, J ¼ 2.2), 8.22 (1H, t, J ¼ 4.1), 7.75 (1H, d, C]CH, J ¼ 16.0),
7.63e7.66 (3H, dd, J ¼ 8.8, 3.3), 7.34 (1H, s), 7.33 (1H, d, J ¼ 1.8), 7.25
(1H, d, C]CH, J ¼ 16.1), 7.11 (1H, d, J ¼ 1.8), 6.96e7.00 (3H, m), 5.33
(1H, sextet, CH2CHCH3, J ¼ 6.8), 3.99e4.02 (2H, m), 3.88 (3H, s, NCH3),
3.80 (3H, s, OCH3), 3.62-3.69 (2H, m), 3.51e3.58 (4H, m), 3.25e3.31
(2H, m), 3.05e3.18 (2H, m),1.70e1.74 (2H, quintet, CH3CH2CH, J ¼ 7.7),
1.34 (3H, d, CHCH3, J ¼ 6.8), 0.75 (3H, t, CH3CH2, J ¼ 7.3) HRMS FAB:
found 654.3377; C36H44N7O5 requires 654.3398 [M þ 1]þ.
3.8.14. N-[1-Isopropyl-5-({[1-methyl-5-({[2-(4-morpholinyl) ethyl]
amino}carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1H-pyrrol-3-yl]-
6-[(E)-2-(4-methoxyphenyl)ethenyl] nicotinamide 7c
3.8.11. 4-({[1-sec-butyl-4-({4-[(E)-2-(3-quinolinyl)ethenyl]
benzoyl}amino)-1H-pyrrol-2-yl]carbonyl}amino)-1-methyl-N-[2-
(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide 6d
From
4-{[(1-isopropyl-4-nitro-1H-pyrrol-2-yl)carbonyl]
-1H-pyrrole-2-
amino}-1-methyl-N-[2-(4-morpholinyl)ethyl]
carboxamide 18c (30 mg, 0.07 mmol) and 6-[(E)-2-(4-
methoxyphenyl)ethenyl]nicotinic acid 32 (18 mg, 0.07 mmol)
using HBTU (52 mg, 0.14 mmol). Purification by HPLC furnished the
desired product as a bright orange solid (4 mg, 9%), purity by HPLC
> 95% with no distinct melting point. nmax (KBr): 3445, 2923, 1670,
1650, 1513, 1412, 1201, 1175, 1133 cmꢀ1 dH (DMSO-d6): 10.47 (1H, s,
ArH), 10.01 (1H, s, ArH), 9.51 (1H, bs, TFA), 9.06 (1H, d, ArH, J ¼ 2.1),
8.26 (1H, d, J ¼ 2.0), 8.24 (1H, d, J ¼ 2.1), 8.22 (1H, t, J ¼ 4.1), 7.75 (1H,
d, C]CH, J ¼ 16.1), 7.63e7.66 (3H, dd, J ¼ 8.8, 3.3), 7.50 (1H, d,
J ¼ 1.9), 7.25 (1H, m, C]CH,), 7.21 (1H, d, J ¼ 1.8), 7.03 (1H, d, J ¼ 1.8),
6.96e7.02 (3H, m), 5.46 (1H, septet, CH3CHCH3, J ¼ 6.7), 3.83 (3H, s,
NCH3), 3.99e4.03 (2H, m), 3.61e3.68 (2H, m), 3.53e3.58 (4H, m),
3.25e3.29 (2H, m), 3.05e3.18 (2H, m), 1.38 (6H, d, CH3CHCH3,
J ¼ 6.6). HRMS FAB: Found 662.3074; C35H42N7O5Na requires
662.3067 [M þ Na]þ.
From 4-{[(1-sec-butyl-4-nitro-1H-pyrrol-2-yl)carbonyl] amino}-
1-methyl-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-carboxamide
6d (35 mg, 0.08 mmol) and 4-[(E)-2-(3-quinolinyl)ethenyl]benzoic
acid 15 (22 mg, 0.08 mmol) using HBTU (50 mg, 0.14 mmol) as
described. Purification by HPLC furnished the desired product as
a bright yellow solid (15 mg, 29%), purity by HPLC >95% with no
distinct melting point. nmax (KBr): 3404, 1675, 1583, 1530, 1408,
1242, 1201, 1134, 721 cmꢀ1 dH (DMSO-d6): 10.37 (1H, s, ArH), 10.03
(1H, s, ArH), 9.55 (1H, bs, TFA), 9.26 (1H, d, ArH, J ¼ 2.1), 8.56, (1H, d,
ArH, J ¼ 2.1), 8.22 (1H, m, CONH), 7.99e8.04 (5H, m), 7.74e7.83 (4H,
m), 7.60e7.71 (4H, m), 7.47 (1H, d, J ¼ 1.7), 7.21 (1H, d, J ¼ 1.7),
7.00e7.02 (2H, m), 5.29 (1H, sextet, CH2CHCH3, J ¼ 6.8), 3.99e4.02
(2H, m), 3.83 (3H, s, NCH3), 3.62e3.68 (2H, m), 3.53e3.58 (4H, m),
3.25e3.29 (2H, m), 3.05e3.18 (2H, m),1.70e1.74 (2H, m, CH3CH2CH),
1.38 (3H, d, CHCH3, J ¼ 6.7), 0.76 (3H, t, CH3CH2, J ¼ 7.3) HRMS FAB:
found 674.3425; C39H42N7O4 requires 674.3460 [M þ 1]þ.
3.8.15. N-[1-sec-butyl-5-({[1-methyl-5-({[2-(4-morpholinyl)ethyl]
amino}carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1H-pyrrol-3-yl]-
6-[(E)-2-(4-methoxyphenyl)ethenyl] nicotinamide 7d
3.8.12. N-[5-({[1-Isopropyl-5-({[2-(4-morpholinyl)ethyl]amino}
carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-
yl]-6-[(E)-2-(4-methoxyphenyl)ethenyl] nicotinamide 7a
From 4-{[(1-sec-butyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-
1-methyl-N-[2-(4-morpholinyl)ethyl]- 1H-pyrrole-2-carboxamide
14 (30 mg, 0.07 mmol) and 6-[(E)-2-(4-methoxyphenyl)ethenyl]
nicotinic acid 32 (17 mg, 0.07 mmol) using HBTU (50 mg, 0.13 mmol).
Purification by HPLC furnished the desired product as a bright orange
solid (7 mg, 16%), purity by HPLC >95% with no distinct melting
point. nmax (KBr): 3435, 1674, 1594, 1513, 1412, 1260, 1204, 1175,
1135 cmꢀ1 dH (DMSO-d6): 10.47 (1H, s, ArH), 10.02 (1H, s, ArH), 9.55
(1H, bs, TFA), 9.06 (1H, d, ArH, J ¼ 2.1), 8.22e8.26 (3H, m), 7.75 (1H, d,
C]CH, J ¼ 16.0), 7.63e7.66 (3H, dd, J ¼ 8.8, 3.3), 7.47 (1H, s), 7.25 (1H,
d, C]CH, J ¼ 16.1), 7.21 (1H, d, J ¼ 1.7), 6.96e7.09 (4H, m), 5.33 (1H,
sextet, CH2CHCH3, J ¼ 6.8), 3.99e4.02 (2H, m), 3.83 (3H, s, NCH3), 3.81
(3H, s, OCH3), 3.62e3.69 (2H, m), 3.51e3.58 (4H, m), 3.25e3.31 (2H,
m), 3.05e3.18 (2H, m), 1.70e1.74 (2H, m, CH3CH2CH), 1.34 (3H, d,
CHCH3, J ¼ 6.7), 0.75 (3H, t, CH3CH2, J ¼ 7.2) HRMS FAB: found
654.3378; C36H44N7O5 requires 654.3404 [M þ 1]þ.
From
1-isopropyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-
carboxamide 18a (35 mg, 0.08 mmol) and 6-[(E)-2-(4-
methoxyphenyl)ethenyl]nicotinic acid (21 mg, 0.08 mmol)
coupled with with HBTU (60 mg, 0.16 mmol). Purification by HPLC
furnished the desired product as a pale orange solid (14 mg, 27%)
with no distinct m.p. Purity by HPLC >95%. nmax (KBr): 3413, 1671,
1594, 1513, 1407, 1259, 1174, 1132, 720 cmꢀ1 dH (DMSO-d6): 10.47
(1H, s, ArH), 9.97 (1H, s, ArH), 9.60 (1H, bs, TFA), 9.06 (1H, d, ArH,
J ¼ 2.1), 8.26 (1H, d, J ¼ 2.0), 8.24 (1H, d, J ¼ 2.1), 8.21 (1H, t, J ¼ 4.1),
7.75 (1H, d, C]CH, J ¼ 16.1), 7.63e7.66 (3H, dd, J ¼ 8.8, 3.3), 7.37
(1H, d, J ¼ 1.9), 7.33 (1H, d, J ¼ 1.8), 7.24 (1H, d, C]CH, J ¼ 16.1), 7.11
(1H, d, J ¼ 1.8), 6.96e7.00 (3H, m), 5.49 (1H, septet, CH3CHCH3,
J ¼ 6.7), 3.83 (3H, s, NCH3), 3.99e4.03 (2H, m), 3.61e3.68 (2H, m),
3.53e3.58 (4H, m), 3.25e3.29 (2H, m), 3.05e3.18 (2H, m), 1.34 (6H,
d, CH3CHCH3, J ¼ 6.7) HRMS FAB: Found 640.3261; 640.3247
C35H42N7O5 requires 640.3247 [M þ 1]þ.
3.9. Melting temperature measurement
DNA oligomers and their complements were melted at a rate of
0.5 ꢁC minꢀ1 in 10 mM PBS buffer solution (pH 7.4) with 50 mM
NaCl on a Cary 300 BIO UVevisible spectrophotometer. Each olig-
omer (made to a concentration of 6 ꢂ 10ꢀ6 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.
3.8.13. N-[5-({[1-sec-Butyl-5-({[2-(4-morpholinyl)ethyl]amino}
carbonyl)-1H-pyrrol-3-yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-
yl]-6-[(E)-2-(4-methoxyphenyl)ethenyl]nicotinamide 7b
From
1-sec-butyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)
carbonyl]amino}-N-[2-(4-morpholinyl)ethyl]-1H-pyrrole-2-
carboxamide 18b (30 mg, 0.07 mmol) and 6-[(E)-2-(4-
methoxyphenyl)ethenyl]nicotinic acid (17 mg, 0.07 mmol) using
HBTU (50 mg, 0.13 mmol). Purification by HPLC furnished the desired
product as a pale orange solid, (12 mg, 28%) with no distinct m.p.
Purity by HPLC >95%. nmax (KBr): 3429, 2925, 1667, 1593, 1513, 1406,
1174, 1132, 799 cmꢀ1 dH (DMSO-d6): 10.47 (1H, s, ArH), 9.97 (1H, s,
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