Minor groove DNA binders as antimicrobial agents. 1. Pyrrole tetraamides are potent antibacterials against vancomycin resistant Enteroccoci and methicillin resistant Staphylococcus aureus

Article abstract of DOI:10.1021/jm010375a

A new series of short pyrrole tetraamides are described whose submicromolar DNA binding affinity is an essential component for their strong antibacterial activity. This class of compounds is related to the linked bis-netropsins and bis-distamycins, but here, only one amino-pyrrole-carboxamide unit and an amidine tail is connected to either side of a central dicarboxylic acid linker. The highest degree of DNA binding, measured by compound-induced changes in UV melting temperatures of an AT-rich DNA oligomer, was observed for flat, aromatic linkers with no inherent bent, i.e., terephthalic acid or 1,4-pyridine-dicarboxylic acid. However, the antibacterial activity is critically linked to the size of the N-alkyl substiutent of the pyrrole unit. None of the tetraamides with the commonly used methyl-pyrrole showed antibacterial activity. Isoamyl- or cyclopropylmethylene-substituted dipyrrole derivatives have the minimum inhibitory concentrations in the submicromolar range. In vitro toxicity against human T-cells was studied for all compounds. The degree to which compounds inhibited cell growth was neither directly correlated to DNA binding affinity nor directly correlated to antibacterial activity but seemed to depend strongly on the nature of the N-alkyl pyrrole substituents.

Full text of DOI:10.1021/jm010375a

J . Med. Chem. 2002, 45, 805-817
805
Min or Gr oove DNA Bin d er s a s An tim icr obia l Agen ts. 1. P yr r ole Tetr a a m id es
Ar e P oten t An tiba cter ia ls a ga in st Va n com ycin Resista n t En ter occoci a n d
Meth icillin Resista n t Sta ph ylococcu s a u r eu s
Natalia B. Dyatkina,* Christopher D. Roberts, J esse D. Keicher, Yuqin Dai, J oshua P. Nadherny,
Wentao Zhang, Uli Schmitz, Ana Kongpachith, Kevin Fung, Alexander A. Novikov, Lillian Lou, Mark Velligan,
Alexander A. Khorlin,† and Ming S. Chen
Genelabs Technologies, 505 Penobscot Drive, Redwood City, California 94063
Received August 7, 2001
A new series of short pyrrole tetraamides are described whose submicromolar DNA binding
affinity is an essential component for their strong antibacterial activity. This class of compounds
is related to the linked bis-netropsins and bis-distamycins, but here, only one amino-pyrrole-
carboxamide unit and an amidine tail is connected to either side of a central dicarboxylic acid
linker. The highest degree of DNA binding, measured by compound-induced changes in UV
melting temperatures of an AT-rich DNA oligomer, was observed for flat, aromatic linkers
with no inherent bent, i.e., terephthalic acid or 1,4-pyridine-dicarboxylic acid. However, the
antibacterial activity is critically linked to the size of the N-alkyl substiutent of the pyrrole
unit. None of the tetraamides with the commonly used methyl-pyrrole showed antibacterial
activity. Isoamyl- or cyclopropylmethylene-substituted dipyrrole derivatives have the minimum
inhibitory concentrations in the submicromolar range. In vitro toxicity against human T-cells
was studied for all compounds. The degree to which compounds inhibited cell growth was neither
directly correlated to DNA binding affinity nor directly correlated to antibacterial activity but
seemed to depend strongly on the nature of the N-alkyl pyrrole substituents.
In tr od u ction
specificity of peptidic groove binders,7 considerable work
also went into increasing the potency of these com-
pounds by linking together netropsin and distamycin
type molecules. Over 20 years of work has been under-
taken on the improvement of the synthesis of linked
pyrrole polyamides, their DNA binding properties,13,14
and antitumor15 or antiviral activity.16 Lown and co-
workers synthesized netropsin and distamycin dimers
by joining the N terminal ends with a wide range of
dicarboxylic acid linkers (head-to-head linkage). Among
them, distamycin dimers with flexible, aliphatic linkers
showed increased inhibition of proliferation of several
cancer cell lines15 as compared to distamycin and
netropsin alone. These same compounds along with
distamycin dimers containing a terephthalic linkage
(Figure 1) also showed promising anti-HIV activity,
which is related to the inhibition of HIV-1 integrase
catalytic activity.16 This anti-HIV activity has also been
connected with the inhibition of viral reverse tran-
scriptase activity. For the latter, dimers with short rigid
linkers were clearly more potent. Although most of the
linked oligopyrroles exhibit good affinity for AT-rich
DNA sequences, a clear correlation between DNA bind-
ing and biological activity was not observed.15 Beyond
differential cellular uptake and metabolism obscuring
the relationship between DNA binding and antiviral or
anticancer activity, one must also consider that these
rigidly or flexibly linked dimers encounter different
binding modes with a DNA target alone or the ternary
complex (binding to DNA-protein complex) in which
inhibition is accomplished.
The rapid emergence of bacterial infections resistant
to treatment with standard antibiotics constitutes a
serious public health threat1,2 and a new, yet all too
familiar, challenge to medicinal chemistry. Novel anti-
bacterials are needed that are either less prone to give
rise to bacterial resistance or are directed toward novel
biological targets such that combination therapy be-
comes more efficient. While many approved antibacter-
ial drugs inhibit parts of the replication machinery, i.e.,
bacterial polymerases by rifampicin or ribosomes by
aminoglycosides,3 no examples exist that target DNA
directly. Targeting bacterial DNA is promising as it
would involve early steps in cell proliferation and be less
likely to lead to bacterial resistance than targeting
enzymes. This approach, however, would also require
that bacterial DNA could be targeted preferentially over
human DNA to minimize toxicity. The naturally occur-
ring polyamide antibiotics netropsin and distamycin
(Figure 1) are well-characterized DNA binding agents.4-7
These antibiotics bind within the minor groove of DNA
at regions with four or five AT base pairs, respectively.
Not surprisingly, the cytotoxicity of these compounds
precluded clinical use.8,9 Nevertheless, the pyrrole polya-
mides netropsin and distamycin were the inspiration
for a large body of work aimed at the exploration and
improvement of the pharmaceutical potential of DNA
minor groove binders (reviewed in refs 6 and 10-12).
Besides the many efforts to extend the DNA sequence
* To whom correspondence should be addressed. Tel: 650-562-1474.
Fax: 650-368-0709. E-mail: ndyatkina@genelabs.com.
† Current address: Operon Technologies, Inc., 1000 Atlantic Avenue,
Suite 108, Alameda, CA 94501.
From the lack of biological activity of a short ethylene-
linked compound with only one pyrrole unit on each side
10.1021/jm010375a CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/11/2002

806 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Dyatkina et al.
F igu r e 1. Chemical structures of natural antibiotics netropsin and distamycin as compared to a rigid bis-distamycin (compound
8 from ref 15).
Ta ble 1. In Vitro Antimicrobial Activity, Cell Toxicity, and
DNA Binding of Terephthalamides
F igu r e 2. General structure of short pyrrole tetraamides.
MIC (µM)
of the linker, it was concluded that activity is tied to at
∆Tm
BM
toxicity
least two pyrrole units.15 However, considering that the
molecular geometry of such a short oligopyrrole with
only one pyrrole unit and a short rigid linker should be
very similar to that of distamycin itself, one would
expect strong DNA binding affinity and possibly biologi-
cal activity. Recent reports on the antimicrobial activity
of short, rigid minor groove binders carrying amidine
groups create additional support to those expecta-
tions.17,18 Assuming that smaller compounds are more
likely to have suitable cellular uptake and pharmaco-
kinetic properties than bis-netropsins and bis-distamy-
cins, the present study explores the biological activity
of a library of short dipyrrole derivatives with the
general structure as shown in Figure 2. It is well-known
that changes in the overall lipophilicity of a compound
can have dramatic effects on its biological activity.19
This study was driven by the idea that modifications of
the pyrrole units could produce significant changes in
cellular uptake without detrimental effects on DNA
binding and thereby provide antibacterial activity.
compd
R
(°C)a MRSA 4147b UCD-3b (% NCC)c
distamycin methyl
26 >45.5 >45.5 >45.5
79
56
61
4
1
2
3
propyl
30
22.7
11.1
1.4
22.2 >45.5
5.6
0.7
3-methyl-butyl 29
28
5.6
0.7
a
∆Tm, changes in the DNA melting temperature (∆Tm) of an
AT-rich dodecamer, (CGATTATTAAGC)‚(GCTTAATAATCG),
b
upon binding of an equimolar amount of compound. MIC,
minimal inhibitory concentration. c MRSA, methicillin resistant
d
Staphylococcus aureus. BM4147 and UCD3, two strains of VRE.
e Toxicity is measured as a fraction of growth as compared to no
compound control (percent).
22-28 with variations in the positively charged termi-
nal groups.
Synthesis of N-alkylated pyrrole units, the crucial
intermediates for assembly of target tetraamides, was
accomplished starting with 4-nitro-pyrrole 2-carboxylate
2920 (Scheme 1). After the sodium derivative of 29 was
alkylated with alkyl bromides, the corresponding N-
alkyl derivatives 30-33 were hydrolyzed and the liber-
ated acids 34-37 were converted into the chlorides by
refluxing in thionyl chloride, as described earlier for the
N-methyl derivative.21 The reaction with cyclopropyl-
methyl bromide required a longer reaction time and a
large excess of the alkylating agent, as compared with
nonbranched alkyl halides. The condensation of acid
chlorides 34-37 with aminopropionitrile afforded de-
rivatives 38-41 as white crystalline products. Cyano-
ethylamides 38-41 were treated under Pinner reaction
conditions to generate amidine hydrochloride terminal
groups by reaction with hydrochloric acid in ethanol,
followed by treatment with ammonia in ethanol.
Amidines 42-45 were obtained with excellent yield and
were used without further purification. When the etha-
nolic solution of cyanoethylamide 41 was saturated with
HCl, followed by alkylamine treatment at 0-5 °C,
monoalkyl amidine derivatives 46-48 were formed. The
same reaction at an elevated temperature led to the
formation of dialkylated amidine terminal groups. Thus,
the reaction of imino ester derivative of 40 with methyl-
In this paper, we report the synthesis of a select group
of novel, pyrrole tetraamides, their DNA binding prop-
erties, and antibacterial activities. Substantial potency
against vancomycin resistant Enterococci (VRE) and
methicillin resistant Staphylococcus aureus (MRSA) has
been observed. We found that strong DNA binding
properties are necessary for antibacterial acitivity and
that lipophilicity is a strong modulator of activity. We
also optimized our library to reduce cytotoxicity toward
human T-cells.
Ch em istr y
Four groups of linked tetraamides shown in Tables
1-4 have been synthesized. Compounds 1-3 (Table 1),
which have linked pyrroles alkylated with methyl,
propyl, and isoamyl groups, respectively, represent the
first group. The second group (Table 2), compounds
4-14, presents N-isoamylpyrrole units linked with
different dicarboxylic acids. Compounds in Table 3, 15-
21, are N-methyl-cyclopropyl pyrroles linked with aro-
matic dicarboxylic acids. Table 4 presents compounds

Minor Groove DNA Binders as Antimicrobial Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 807
Ta ble 2. In Vitro Antimicrobial Activity, Cell Toxicity, and DNA Binding for N-(3-Methyl-butyl)-1H-pyrrole Dimers with Different
Linkers

808 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Dyatkina et al.
Ta ble 4. In Vitro Antimicrobial Activity, Cell Toxicity, and
DNA Binding for N,N′-Bis-[1-(3-methyl-butyl)-1H-pyrrol-3-yl]-
terephthalamide with Different Positively Charged Tails
Ta ble 3. In Vitro Antibacterial Activity, Cell Toxicity, and
DNA Binding of Linked N-(Cyclopropylmethyl)-1H-pyrrole
Derivatives
activated ester with pentafluorophenol.24 The product
of the reaction of the corresponding dicarboxylic acid
and pentafluorophenyl trifluoroacetate in dimethylfor-
mamide (DMF) was purified by flash chromatography
on silica gel in toluene and stored at room temperature.
Amino components 42a -50a and 52a were obtained by
the catalytic reduction (Pd/C) of the corresponding nitro
compounds 42-50 and 52.16 Coupling was carried out
at 60 °C for 10 hours and resulted in the desired
tetraamides 1-26. Reaction between activated tere-
phthalic acid and Boc-protected 51a yielded in Boc-
protected derivative, which was isolated by chromatog-
raphy on silica gel and then deprotected with HCl in
ethanol to get the target compound 27. The amino
functions of 27 were converted to guanidines with
pyrazole-1-carboxamidine, as described earlier,25 to get
pyrrole dimer 28 with guanidinium tails. The final
products were purified by preparative reverse phase
HPLC and transformed into hydrochlorides by precipi-
tation with ether from HCl/ethanol. All final products
were homogeneous by HPLC; structures were confirmed
amine in ethanol at 60 °C yielded dimethyl amidine 49,
while the reaction with 1,3-diaminopropane under the
same conditions afforded cyclic amidine 50.22 Most of
the amidine analogues were isolated by precipitation
from methanol with ether and were used without
additional purification; only cyclic product 50 had to be
isolated by preparative high-performance liquid chro-
matography (HPLC). Aminolysis of the N-isoamyl pyr-
role derivative 32 with ethylenediamine at 60 °C for 50
h provided (2-amino-ethyl)amide 51 with 62% yield after
chromatographic purification. To make this unit com-
patible with the coupling reaction, we had to protect the
amino moieties with Boc-protecting groups.
The novel linked pyrroles designed and evaluated in
this paper required the coupling of pyrrole units with
dicarboxylic acids as outlined in Scheme 2. We have
used commercially available dicarboxylic acids as cen-
tral linkers; only 2-methylterephthalic and 2,5-dimeth-
ylterephalic acids were synthesized from corresponding
bromobenzenes as described in the literature.23 Treat-
ment of 1,4-dibromo-2-methylbenzene or 1,4-dibromo-
2, 5-dimethylbenzene with tert-butyllithium, followed by
saturation with CO2, yielded mono- and dimethyl-
terephthalic acids, respectively, in moderate yields.
Dicarboxylic acids were activated via formation of an
1
with H nuclear magnetic resonance (NMR) and high-
resolution mass spectroscopy (HRMS) spectra.
Resu lts a n d Discu ssion s
DNA Bin d in g. DNA binding affinities were evalu-
ated using changes in the DNA melting temperature
(∆Tm) of an AT-rich dodecamer, (CGATTATTAAGC)‚

Minor Groove DNA Binders as Antimicrobial Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 809
Sch em e 1. Synthesis of Pyrrole Flanking Unitsa
a
Reaction conditions: (a) 2 M NaOH, EtOH, 55 °C. (b) SOCl2, 80 °C. (c) Aminopropionitrile (2.1 equiv), toluene. (d) HCl (gas) at 5 °C
in ethanol. (e) NH3 (gas) at 5 °C in ethanol for 42-45, monoalkylamine at 5 °C in ethanol for 46-48, methylamine at 60 °C in ethanol
for 49. (f) 1,3-Propanediamine at 60 °C in ethanol. (g) Ethylenediamine, 50 h, 60 °C. (h) DiBoc-carbonate, DMF.
Sch em e 2. Coupling Scheme for Synthesis of Linked Pyrrolesa
a
Reaction conditions: (a) Pentafluorophenyltrifluoroacetate (2.2 equiv), diisopropylethylamine (2.2 equiv), DMF. (b) H2, 5% Pd/C (Degussa
type), MeOH, 25 psi, 30 min. (c) DMF, 20 h, 55 °C.
(GCTTAATAATCG), upon binding of an equimolar
amount of compound. The values of ∆Tm are listed in
Tables 1-4. The Tm for free DNA at 5 µM was 35 °C.
The range of ∆Tm for these compounds is from 10 to 30
°C with compound 1 having the highest ∆Tm and
compound 6 having the lowest value. The ∆Tm value is
related to binding constants via base pair dissociation
enthalpy and the temperature dependence of the bind-
ing enthalpy.26,27 For several compounds, we also de-
termined the apparent binding constant (KD) by a
fluorescent ethidium displacement assay (data not
shown)28,29 such that values of ∆Tm can also be inter-
preted in a coarse but absolute manner. For example,
compounds 1-3 in Table 1 showed apparent dissociation
constants (KD) of 30-200 nM (data not shown). Com-
pounds with some of the lower ∆Tm values, e.g., 5 and
8 (11 °C), gave values of 1 and 3 µM, respectively.
Biologica l Assa ys. Antibacterial activity was tested
against a wide panel of species, and values for minimal
inhibitory concentrations (MIC) for two strains of VRE,
BM4147 and UCD3, and a strain of MRSA are reported
in Tables 1-4. The compound concentrations tested for
the pyrrole dimers ranged from 0.7 to 45.5 µM (see
Experimental Section).

810 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Dyatkina et al.
VRE strains. This trend parallels the lipophilicity of the
pyrrole side chains with the isoamyl substituent being
the most active. Compound 3 represents the first linked
monopyrrole with good activity against Gram-positive
microorganisms.
Lipophilicity of potential drugs is strongly related to
their capacity to cross cellular membranes, and it is
conveniently expressed as the partioning coefficient for
19
a water-octanol system, log Po/w
.
We determined log
Po/w for compounds 1-3 experimentally because the
prediction of this parameter for highly charged com-
pounds is notoriously difficult. Whereas compounds 1
and 2 are not lipophilic, with log Po/w values of -2 and
-1.1, respectively, compound 3 is clearly lipophilic, with
a log Po/w value of +1.4, and is in a range where the
majority of bioavailable drugs are found.32 It is likely
that the stronger antibacterial activity of compound 3
is related to improved cellular uptake, given that the
DNA binding capacity is essentially unchanged. How-
ever, the possibility that the pyrrole side chains play a
direct role in the inhibition of a ternary complex cannot
be ruled out but such discussion is beyond the scope of
this paper.
F igu r e 3. Molecular model of compound 1 docked into the
distamycin binding site in DNA dodecamer with sequence
GCGAAATTTCGC. Compound 1 is shown in stick format with
ICM atom type colors. Color coding: A:T pairs are blue:green,
G:C pairs are magenta:purple.
Next, we focused on optimization of the motif pre-
sented by compound 3 beginning with the central linker.
Results for compounds 4-14 are presented in Table 2.
Prompted by the known cytotoxicity of similar com-
pounds,15 we assessed compound toxicity in human
T-cells using a colorimetric cell proliferation assay.30
These data are reported in Tables 1-4 as the fraction
of cellular activity to cleave a tetrazolium salt (WST-1)
as compared to untreated cell cultures. Although the
T-cell assay is only a simple, cursory predictor of the
potential toxicity in humans, the results revealed
interesting chemical trends and enabled further com-
pound optimization.
It is evident that good DNA binding properties (∆Tm
>
20 °C) are a prerequisite for antibacterial activity. Data
for compounds 3-6 show that abandoning the flat
geometry for bulkier, aliphatic linkers destroys DNA
binding and activity. While the cyclohexa-1,3-diene-1,4-
linked compound 4 appears very similar to parent
compound 3, cyclohexane-1,4-linked compound 5 dem-
onstrated only moderate activity against MRSA and was
not active against VRE. Compound 6 with the flexible
adipic acid linker has lost significant antibacterial
activity. Our modeling studies suggest that the more
bulky aliphatic linkers would produce van der Waals
clashes with the narrow minor groove of AT-rich re-
gions. The observed SAR for compounds 3-6 demon-
strates that DNA binding is necessary for antibacterial
activity.
Str u ctu r e-Activity Rela tion sh ip (SAR) of P yr -
r ole Ter tr a a m id es. Prompted by the antiviral activity
and DNA binding properties of netropsin and distamy-
cin dimers with a terephthalic linkage,15 we synthesized
compound 1 (Table 1), which, not surprisingly, turned
out to exhibit excellent DNA binding properties. Mo-
lecular modeling suggests that this particular scaffold
fits well into the narrow minor groove of a DNA AT
tract. Figure 3 shows a model of compound 1 docked
into the distamycin receptor site (AAATTT) generated
with the flexible docking program.31 Our approach of
docking into the minor groove of the distamycin DNA
cocrystal structure allowed a semiquantitative compari-
son with distamycin itself. Compound 1 not only has
improved van der Waals interactions with the groove
but head-to-head geometry of this compound is more
suitable for hydrogen bonding to minor groove acceptor
atoms. Consistent with molecular modeling, ∆Tm for
compound 1 was 30 °C, which is 4 °C higher than the
value for distamycin. From the model in Figure 3, it was
also clear that the pyrrole nitrogens, projecting away
from the groove, lend themselves for modification
toward increasing compound lipophilicity. Pyrrole dimers
2 and 3, N-alkylated with propyl and isoamyl groups,
respectively, (Table 1) indeed retain excellent DNA
binding properties, but most importantly, they become
potent antibacterials. Whereas compound 1 is only
mildly active against MRSA and one of the VRE strains,
compound 3 exhibits MIC of 1.4 µM against MRSA and
is active at submicromolar concentrations against both
The result for compound 6 is surprising in light of
earlier results for similarly linked distamycin dimers
for which good binding to calf thymus DNA and anti-
leukemic activity has been reported.15 We must consider
that binding mode and the preferred sequence of bis-
distamycins are unclear and elusive. These dimers
might bind with only one-half of the molecule, or they
could be binding to partially mixed sequences, which
better accommodate the aliphatic linker.
Table 2 shows other effects resulting from structural
variation of the linker. Substitution in the aromatic ring
of the linker affects DNA binding and antibacterial
activity to a much smaller degree than the geometry of
the linker. Compound 7, linked with 2,5-pyridine dicar-
boxylic acid, showed mildly reduced DNA binding and
MIC values similar to parent compound 3. Using
pyrazine dicarboxylic acid as a linker, DNA binding
again slightly drops, but antibacterial activity is re-
tained. The change of linker geometry, as in compound
8, caused a sharp drop in DNA binding and anti-VRE

Minor Groove DNA Binders as Antimicrobial Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 811
activity. Similar effects of this meta-linker have been
reported for the bis-netropsin and bis-dystamicin
dimers.16
groove binding, as evidenced by the high ∆Tm values of
compounds 20 and 21. Among the compounds in Table
3, thiophene-linked compound 20 is among those with
the best activity profile. A comparison of the group of
cyclopropylmethyl compounds 15-19 (Table 3) with the
isoamyl compounds in Table 2 shows that ∆Tm values
are slightly but systematically increased, whereas an-
tibacterial activity is only mildly reduced. Most notice-
able, however, is the dramatically reduced toxicity
against T-cells with most compounds in Table 3 exhibit-
ing percent NCC values above 80%.
The second approach to optimization of the parent
compound 3 was modification of the tail groups bearing
positive charges (Table 4). In addition to the alkylated
amidine derivatives, compounds 22-25, tails with a
primary amine and a guanidinium moiety were evalu-
ated as well (compounds 27 and 28, respectively).
Alkylation of the amidine moiety had very little effect
on DNA binding and antibacterial activity. Interest-
ingly, compounds 22 and 23 with small alkyl groups,
methyl and ethyl, respectively, show a large reduction
in toxicity. In contrast, analogue 24, containing the
longer propyl groups, showed a high toxicity more
similar to that of the parent compound 3. Cyclic, bis-
alkylated compound 26 is the most active in the entire
pyrrole tetraamide series and showed reasonable toxic-
ity. The other charged tails tested here did not lead to
improved compound properties. The primary amine tail
for compound 27 caused a significant loss in DNA
binding and antibacterial activity. Transformation of the
amino terminus to guanidine restored activity, but
toxicity against T-cells became very high indicated by
a percent NCC value of 5%. For compounds 26 and 27,
the correlation between activity and DNA binding is
obvious.
On the other hand, the potency of dimers 10 and 13
indicates that one exocyclic substituent on the aromatic
unit of the linker, i.e., nitro or methyl groups, respec-
tively, is well-tolerated. Modeling studies suggest that
the small reduction in DNA binding activity is related
to a minor van der Waals clash between the exocylic
substiutent and the adjacent carbonyl oxygen, since the
preferred, docked conformation requires near coplanar-
ity. This steric effect would be even more aggravated
for the linker of compound 11. The two ortho-substitu-
ents of 1,4-naphthalene dicarboxylic acid indeed cause
slightly reduced activity and DNA binding. The hypoth-
esis that only linker geometries compatible with good
minor groove binding confers activity is again confirmed
by compound 14, which exhibits two exocyclic methyl
groups at positions 2 and 5. Because they appear on
either side of the ring, one would always prohibit the
molecule from penetrating the groove deeply enough to
engage in sufficient hydrophobic interactions. The activ-
ity and DNA binding pattern are essentially that of
compound 5, which has the bulky cyclohexane linker.
The most surprising results came from compound 12,
which has a 2,6-naphthalene dicarboxylic acid linker
that should be compatible with good groove binding, as
seen by modeling and verified by its high ∆Tm value.
The fact that compound 12 does not exhibit any activity
against VRE and MRSA suggests that the length of the
linking unit, which shapes the overall geometry, is also
related to antibacterial activity.
The results presented in Table 2 show that the
general framework of two N-isoamylpyrroles linked with
terephthalic or substituted terephthalic acids provides
antibacterial activity in the 0.7-2.8 µM ranges. How-
ever, the toxicity against human T-cells remained
significantly high. For the most active compounds, e.g.,
3, 4, and 7, cells grew less than 25% in the presence of
compound as compared to the no compound control.
Adding a nitro or methyl group to the linker, compounds
10 and 13, somewhat improves toxicity. It is not
surprising that most of the poor minor groove binders
barely inhibit T-cell growth. However, toxicity of com-
pound 8 (6% NCC) is an exception and suggests that
the meta-geometry of the linker leads to a different, yet
unknown, mode of action in human T-cells. Unexpect-
edly, the highest toxicity is seen for inactive compound
12, which also suggests that multiple targets might be
involved.
Con clu sion s
The present study demonstrates that short pyrrole
tetraamides with head-to-head geometry strongly bind
to the minor groove of AT tracts of DNA. In comparison
with distamycin, molecular models of our head-to-head
scaffold showed improved hydrophobic interactions as
well as improved hydrogen bonding to the floor of the
groove. Good DNA binding properties were found to be
a requirement for good antibacterial activity. The most
revealing conclusion to be drawn from the present data
is that lipophilic, alkyl substituents on the nitrogen
atom of the pyrroles produce strong antibacterial activ-
ity indicated by submicromolar MIC values against both
MRSA and VRE. Among the various N-substituents
evaluated, isoamyl and cyclopropylmethyl groups led to
the most active compounds. From data reported in this
study, it appears that it is the lipophilicity of the
compounds, probably related to improved cellular up-
take, that modulates antibacterial activity for those
compounds that exhibit sufficiently strong DNA binding.
As uptake would also increase for other cells than the
bacterial targets, significant toxicity would be expected.
Strong toxicity against human T-cells was indeed
observed for some compounds, but this toxicity did not
correlate with antibacterial activity or DNA binding.
However, the degree of toxicity was found to be very
sensitive to the alkyl substituents on the pyrrole units.
The cyclopropylmethyl group dramatically reduced tox-
To reduce cell toxicity but retain antibacterial activity
along two different paths, we further optimized the
structure of the most active compounds. The first
utilized additional modifications of the N-alkyl moiety
of pyrrole units, which yielded the interesting group of
compounds 15-21 (Table 3) that all carry a cyclopro-
pylmethyl group. This group was chosen as a more
restrained, smaller mimic of the isoamyl moiety. Com-
pounds 15-19 all exhibit the successful linking units
introduced in Table 2. In addition to the 1,4-linked
aromatic geometries, we also introduced two five-
membered ring aromatic dicarboxylic acid linker,
thiophene and 3,5-pyrazole dicarboxylic acid. This new
linker geometry was very compatible with good minor

812 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Dyatkina et al.
(78%); mp 124-125 °C. ES MS: 263.97 (M + H+). H1 NMR:
δ 0.02-0.04 and 0.09-0.12 (m, 2H, CH2), 0.89-1.00 (m, 1H,
CH), 2.37-2.41 (t, 2H, CH2-CN), 3.06-3.11 (dd, 2H, CH2-
NH), 3.86-3.88 (d, 2H, CH2-N), 7.09 and 7.87 (d, 1H, pyrrole),
8.45 (t, 1H, NH).
1-(Meth yl)-4-n itr o-1H-p yr r ole-2-ca r boxylic Acid (2-Cy-
a n o-eth yl)a m id e, 38. A literature procedure20 gave 38 in 74%
yield based on pyrrole 29; mp 134-136 °C (literature20 mp
135-136 °C).
1-(P r op yl)-4-n itr o-1H-p yr r ole-2-ca r boxylic Acid (2-Cy-
a n o-eth yl)a m id e, 39. A literature procedure20 gave 39 in 71%
yield based on pyrrole 29; mp 120-122 °C (literature20 mp
120.5-121 °C).
icity against T-cells without a concurrent drop in
antibacterial activity. This observation leaves room for
the speculation that activity is not a simple matter of
lipophilicity and DNA binding but that a more complex
mechanism is at work, which might involve a specific
interaction of the pyrrole substituents in a ternary
DNA-protein complex.
This study has produced a series of compounds with
promising activity against antibiotic resistant strains
of Staphylococcus aureus and Enterococci. It is note-
worthy that the putative mode of action of these
compounds involves DNA binding and has not been
previously exploited for antibacterial pharmaceutical
design. The responsiveness of compound design to alter
toxicity against human T-cells is particularly promising.
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
(2-Cya n o-eth yl)a m id e, 40. The title compound was synthe-
sized from 1-(3-methyl-butyl)-4-nitro-1H-pyrrole-2-carboxylic
acid 3621 (4.5 g, 20 mmol) as described above for compound
41. The yield of 40 was 3.7 g (68%); mp 112 °C. ES MS: 279.32
(M + H+). H1 NMR: δ 0.83-0.86 (m, 6H, CH3), 1.40-1.51 (m,
1H, CH), 1.51-1.61 (m, 2H, CH2-CH), 2.68-2.72 (m, 2H,
CH2-CN), 3.35-3.42 (m, 2H, CH2-NH), 4.33-4.37 (m, 2H,
CH2-N), 7.38 and 8.15 (d, 1H, pyrrole), 8.56 (t, 1H, NH).
Gen er a l P r oced u r e for t h e Syn t h esis of Am id in e
Der iva tives. A solution of (2-cyano-ethyl)amidine 38-41 (5
mmol) in 50 mL of dry ethanol was cooled to 0-5 °C and
saturated with HCl gas. The mixture was sealed and refriger-
ated for 20 h. The mixture was allowed to warm to room
temperature, and ethanol was evaporated. The resulting imino
ester was dissolved in 50 mL of anhydrous ethanol and
saturated with ammonia gas to get amidine 42-45. The
treatment of the imino ester with 30 mmol of alkylamine or
alkyldiamine resulted in the formation of mono- (46-48) or
bis-substituted (49, 50) amidines. The sealed mixture was kept
overnight at 0-5 °C to get amidines or monoalkylated amidines
42-48. Bis-substituted derivatives 49 and 50 were formed
under heat overnight at 60 °C. The solvent was evaporated.
The solid was dissolved in 5 mL of methanol, and ether (35
mL) was added to precipitate the target product. This proce-
dure was used for the preparation the following compounds.
1-Meth yl-4-n itr o-1H-p yr r ole-2-ca r boxylic Acid (2-Ca r -
ba m im id oyl-eth yl)a m id in e, 42. This compound was pre-
pared from N-methylpyrrole derivative 38. The corresponding
imino ester was treated with ammonia gas to afford 1.1 g (92%)
of amidine 42; mp 269-274 °C (literature20 mp 268-272 °C).
1-P r op yl-4-n itr o-1H-p yr r ole-2-ca r boxylic Acid (2-Ca r -
ba m im id oyl-eth yl)a m id in e, 43. This compound was pre-
pared from N-propylpyrrole derivative 39. The corresponding
imino ester was treated with ammonia gas in ethanol to afford
1.25 g (95%) of amidine 43; mp 215 °C (literature20 mp 215-
216 °C).
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
(2-Ca r ba m im id oyl-eth yl)a m id in e, 44. This compound was
prepared from N-(3-methyl-butyl)pyrrole derivative 40. The
corresponding iminoester was treated with ammonia gas in
ethanol to afford 1.38 g (96%) of amidine 44; mp 182-184 °C.
ES MS: 296.38 (M + H+). H1 NMR: δ 0.88-0.86 (d, 6H, CH3),
1.43-1.61 (m, 3H, CH and CH2-CH), 2.61-2.65 (m, 2H, CH2-
CN), 3.49-3.55 (m, 2H, CH2-NH), 4.33-4.38 (m, 2H, CH2-
N), 7.51 and 8.18 (d, 1H, pyrrole), 8.73 (t, 1H, NH).
1-(Cyclop r op ylm eth yl)-4-n itr o-1H-p yr r ole-2-ca r boxyl-
ic Acid (2-Ca r ba m im id oyl-eth yl)a m id in e, 45. This com-
pound was prepared from N-(cyclopropylmethyl)pyrrole de-
rivative 41. The corresponding imino ester was treated with
ammonia gas in ethanol to afford 1.25 g (90%) of amidine 45;
mp 242 °C. ES MS: 280.31 (M + H+). H1 NMR: δ 0.36-0.47
(m, 4H, CH2), 1.21-1.38 (m, 1H, CH), 2.51-2.60 (t, 2H, CH2-
CN), 3.48-3.52 (m, 2H, CH2-NH), 4.19 (d, 2H, CH2-N), 7.46
and 8.20 (d, 1H, pyrrole), 8.56, 8.67 and 8.98 (bs, 1H, NH).
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
[2-(N-Meth ylca r ba m im id oyl)eth yl]a m id en e, 46. This com-
pound was prepared from N-(3-methyl-butyl)pyrrole derivative
40. The corresponding imino ester was treated with 1 M
solution of aminomethane in methanol at 5 °C to afford 1.25
g (82%) of amidine 46 as colorless foam. ES MS: 310.94 (M +
Exp er im en ta l Section
Ch em istr y. All chemicals used were of reagent grade.
Melting points were determined on a Mel-temp apparatus
(Laboratory Devices, Inc.) and are uncorrected. 1H NMR
spectra were recorded on a Varian Mercury VX-300 MHz.
Chemical shifts are reported in parts per million relative to
the solvent residual signal. The samples were prepared in
methylsulfoxide-d6 unless otherwise specified. The peaks were
assigned based on gCOSY experiments. Electrospray mass
spectra were recorded on a spectrometer Mariner-EMS (PE-
Biosystems). HRMS (fast atom bombardment (FAB)) were
recorded on
a VG-ZAB by the UCR Mass Spectrometry
department. HPLC purification was carried out on a Vydac
12 µm C18 (2.2 cm × 25 cm) column using a solvent gradient
with two solvents: 0.1% trifluoroacetic acid (TFA) in water
(A) and 0.1% TFA in acetonitrile (B). Unless otherwise stated,
the applied conditions for purification were 10-70% eluent B
gradient over 40 min with a flow rate of 10 mL/min. The
monitoring was at 254 nm.
1-Cyclop r op ylm et h yl-4-n it r o-1H -p yr r ole-2-ca r b oxyl-
ic Acid Eth yl Ester , 33. 4-Nitro-1H-pyrrole-2-carboxylic acid
ethyl ester 29, prepared as in ref 20 (5 g), was dissolved in 50
mL of anhydrous EtOH, and 50 mL of 1 M sodium ethylate
was added and then followed with the addition of 10 mL of
cyclopropylmethyl bromide. The reaction mixture was heated
at 80 °C. Sodium methylate (25 mL) and cyclopropylmethyl
bromide (5 mL) were added twice, at 5 h and at 10 h. The
reaction mixture was heated for 5 more hours, cooled to room
temperature, and partitioned between water and chloroform.
The organic phase was washed with water, dried with sodium
sulfate, and evaporated. The residue was purified on a silica
gel column using toluene/ethyl acetate (9:1 v/v) as the eluant
to afford 4.8 g (74%) as white crystals; mp 55-56 °C. H1
NMR: δ 0.37-0.42 and 0.65-0.72 (m, 2H, CH2), 1.22-1.28
(m, 1H, CH), 1.37 (t, 3H, CH3), 4.23 (d, 2H, CH2-N), 4.32 (q,
2H, CH2-ethyl), 7.44 and 7.81 (d, 1H, pyrrole).
1-Cyclop r op ylm et h yl-4-n it r o-1H -p yr r ole-2-ca r b oxyl-
ic Acid , 37. Ethyl ester 33 (4.5 g, 19 mmol) was suspended in
20 mL of methanol, 2 M NaOH (10 mL) was added, and the
mixture was stirred at 50 °C for 2 h. The clear solution was
diluted with water (50 mL), and 1 N HCl was added dropwise
to get pH 2.5. The white residue was filtered, washed with
water, and dried to get 3.8 g (96%) of 37 as white crystals; mp
216 °C. H1 NMR: δ 0.37-0.41 and 0.45-0.52 (m, 2H, CH2),
1.26-1.32 (m, 1H, CH), 4.18 (d, 2H, CH2-N), 7.27 and 8.28
(d, 1H, pyrrole).
1-(Cyclop r op ylm eth yl)-4-n itr o-1H-p yr r ole-2-ca r boxyl-
ic Acid (2-Cya n o-eth yl)a m id e, 41. The acid 37 was sus-
pended in SOCl2 (20 mL), and the mixture was refluxed for 4
h until a clear solution was obtained, cooled, evaporated, and
dried by coevaporation with toluene (10 mL × 3). The obtained
chloroanhydride, without purification, was dissolved in toluene
(10 mL), and 3-aminopropionitrille (2.7 mL, 38 mmol) was
added. The mixture was kept for 1 h at ambient temperature
and evaporated. The white precipitate was suspended in 0.1
N HCl, filtered, washed with water, and dried to yield 3.7 g

Minor Groove DNA Binders as Antimicrobial Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 813
H+). H1 NMR: δ 0.86-0.88 (d, 6H, CH3-isopentyl), 1.43-1.61
(m, 3H, CH and CH2-CH), 2.60-2.65 (t, 2H, CH2-amidine),
3,37 (s, 3H, CH3-NH), 3.49-3.55 (m, 2H, CH2-NHCO), 4.33-
4.38 (t, 2H, CH2-N), 7.51 and 8.18 (d, 1H, pyrrole), 8.73 (t,
1H, NHCO).
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
[2-(N-Eth ylca r ba m im id oyl)eth yl]a m id en e, 47. This com-
pound was prepared from N-(3-methyl-butyl)pyrrole derivative
40. The corresponding imino ester was treated with a 1 M
solution of aminoethane in methanol at 5 °C to afford 1.3 g
(80%) of amidine 47 as a colorless foam. ES MS: 324.79 (M +
H+). H1 NMR: δ 0.88-0.86 (d, 6H, CH3-isopentyl), 1.07-1.12
(t, 3H, CH3-ethyl), 1.43-1.61 (m, 3H, CH and CH2-CH),
2.52-2.56 (t, 2H, CH2 CH2-amidine), 3.12-3.21 (m, 2H, CH2-
ethyl), 3.49-3.55 (m, 2H, CH2-NHCO), 4.33-4.38 (t, 2H,
CH2-N), 7.39 and 8.20 (d, 1H, pyrrole), 8.54-8.60 (NH-
amidine), 9.38 (t, 1H, NHCO).
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
[2-(N-P r op ylca r ba m im id oyl)eth yl]a m id en e, 48. This com-
pound was prepared from N-(3-methyl-butyl)pyrrole derivative
40. The corresponding imino ester was treated with 1-amino-
propane at 5 °C to afford 1.25 g (77%) of amidine 48 as a
colorless foam. ES MS: 338.44 (M + H+). H1 NMR: δ 0.88-
0.86 (d, 9H, CH3-isopentyl, CH3-propyl), 1.07-1.12 (t, 3H,
CH3-ethyl), 1.43-1.61 (m, 3H, CH2-CH), 2.50-2.53 (m, 2H,
CH2-amidine), 3.08-3.14 (m, 2H, CH2-propyl), 3.29-3.36 (m,
2H, CH2-propyl), 3.43-3.50 (m, 2H, CH2-NHCO), 4.33-4.38
(t, 2H, CH2-N), 7.43 and 8.20 (d, 1H, pyrrole), 8.62-8.90 (m,
2H, NH-amidine), 9.39 (t, 1H, NHCO).
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
[2-(N,N′-Dim et h ylca r b a m im id oyl)et h yl]a m id e, 49. This
compound was prepared from N-(3-methyl-butyl)pyrrole de-
rivative 40. The corresponding imino ester was treated with
1 M solution of methylamine in methanol at 50 °C to afford
1.25 g (77%) of amidine 49 as a colorless foam. ES MS: 324.74
(M + H+). H1 NMR: δ 0.85-0.87 (d, 6H, CH3-isopentyl), 1.43-
1.61 (m, 3H, CH and CH2-CH), 2.76-2.79 (m, 5H, CH2-
amidine and CH3-NH), 2.96-2.98 (d, 3H, CH3-NH), 3.46-
3.55 (m, 2H, CH2-NHCO), 4.34-4.39 (t, 2H, CH2-N), 7.51
and 8.18 (d, 1H, pyrrole), 9.76 (t, 1H, NHCO).
2-Meth yl-ter ep h th a lic Acid . A stirred solution of 1,4-
dibromo-2-methyl-benzene (1.0 g, 4.0 mmol) in anhydrous
tetrahydrofuran (40.0 mL) and tetramethylethylenediamine
(4.8 mL, 31.8 mmol) was cooled to -70 °C (CO2/acetone bath)
under an argon atmosphere. tert-Butyllithium (18.8 mL, 32.0
mmol) as 1.7 M pentane solution was added, and the reaction
mixture was stirred for 2.5 h. The yellow reaction solution was
transferred to a 1000 mL round-bottom flask containing excess
solid CO2. After the CO2 sublimated, the reaction was quenched
with 5% NH4Cl (50 mL) and acidified with 1 M HCl to reach
pH 3. The resulting mixture was extracted with ethyl acetate,
dried with Na2SO4, and evaporated. The mixture of mono- and
dicarboxylic acids was crystallized from MeOH to give the
2-methyl-terephthalic acid (240 mg, 33% yield). MS: 179.01
(M - H). 1H NMR: δ 13.2 (bs, 2H, COOH), 7.85 (m, 3H, C6H4),
2.58 (s, 3H, CH3).
2,5-Dim eth yl-ter ep h th a lic Acid . The synthesis was per-
formed using 1,4-dibromo-2,5-dimethyl-benzene as described
above for 2-methyl-terephthalic acid. The reaction mixture was
extracted with ethyl acetate (20 mL × 4). The ethyl acetate
solution was washed with 1 M NaOH (3 × 20 mL). The
combined aqueous fractions were acidified to pH 3 with 1 M
HCl. The white precipitate was filtered and crystallized from
aqueous methanol to afford 444 mg (40%) of 2,5-dimethyl-
terephthalic acid. MS: 193.03 (M - H). 1H NMR: δ 13.09 (bs,
2H, COOH), 7.69 (s, 2H, C6H4), 2.48 (m, 6H, C6H4).
Gen er a l Meth od for th e P r ep a r a tion of Activa ted
Dica r boxylic Acid s. Dicarboxylic acid (2 mmol) was sus-
pended in 4 mL of dry DMF, and diisopropylethylamine (4.8
equiv) was added followed with pantafluorophenyltrifluorac-
etate (4.8 mmol). The reaction was kept at room temperature
for 1 h and evaporated. The flash chromatography on silica
gel (toluene/ethyl acetate 9:1 v/v) provided the corresponding
dipentafluorophenyl ester as white crystals or a clear oil. All
of the compounds were homogeneous on thin-layer chroma-
tography (toluene/ethyl acetate 7:3 v/v) and had similar 19F
NMR spectra. For example, the dipentafluorophenul ester of
1,4-naphthaline dicarboxylic acid 19F NMR: -45 692.62 (t),
-44 406.44 (t), -42 983.05 (d).
Gen er a l Meth od for th e Syn th esis of Lin k ed P yr r ole.
To a stirred solution of nitropyrrole derivatives 42-50 (0.15
mmol) in methanol (20 mL) was added 10% Pd/C (Degussa
type, Aldrich) (0.1 g). The flask was evacuated, flushed three
times with hydrogen, and finally filled with hydrogen at 25-
30 psi. The resultant suspension was stirred vigorously at
ambient temperature for 30 min. The suspended material was
filtered; the filtrate was evaporated to dryness. The resulting
aminopyrrole derivatives 42a -50a were used for the next step
without purification. The solution of freshly prepared ami-
nopyrrole derivatives 42a -50a in 2 mL of dry DMF was added
to the dipentafluorophenyl ester of dicarboxylic acid (0.07
mmol). The reaction mixture was stirred for 15 h at 55 °C,
cooled, and purified by HPLC (Vydac 12 µm C18 2.2 cm × 25
cm column, 10-70% acetonitrile gradient over 40 min, flow
rate 10 mL/min) to give corresponding linked dipyrrole as a
bis-trifluoroacetate salt. This salt was dissolved in 2 mL of
methanol saturated with HCl, 35 mL of diethyl ether was
added, and the precipitate of linked dipyrrole as bis-HCl salt
was separated and dried. The average yield was 45-55%.
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
[2-(1,4,5,6-Tetr ah ydr opyr im idin -2-yl)eth yl]am ide, 50. This
compound was prepared from N-(3-methyl-butyl)pyrrole de-
rivative 40. The corresponding imino ester was treated with
1,3-diaminopropane at 50 °C. The crude reaction mixture was
evaporated and purified by preparative HPLC to get 1.36 g
(61%) of amidine 50 as TFA salt. ES MS: 364. 37 (M + H+).
H1 NMR: δ 0.85-0.87 (d, 6H, CH3), 1.42-1.61 (m, 3H, CH2-
CH of pyrrole), 1.79-1.84 (m, 2H, CH2CH2CH2), 2.55-2.60 (t,
2H, CH2-amidine), 3.20-3.30 (m, 4H, CH2CH2CH2), 3.40-
3.52 (m, 2H, CH2-NHCO), 4.33-4.38 (t, 2H, CH2-N), 7.48
and 8.18 (d, 1H, pyrrole), 8.15 and 9.78 (bs, 1H, NH-amidine),
8.89 (t, 1H, NHCO).
1-(3-Meth yl-bu tyl)-4-n itr o-1H-pyr r ole-2-car boxylic Acid
21
(2-Am in o-eth yl)a m id e, 51. Compound 32 (1.3 g, 5 mmol)
was dissolved in diethylamine (20 mL). This solution was kept
for 50 h at 60 °C and evaporated. The chromatography on silica
gel (CHCl3/CH3OH/NH4OH 9:1:0.05 v/v/v) provided the title
product 51 (830 mg, 62%) as a clear oil. ES MS: 269.33 (M +
H+).
N,N′-Bis-[5-(2-car bam im idoyl-eth ylcar bam oyl)-1-m eth -
yl-1H-p yr r ol-3-yl]ter ep h th a la m id e, 1. The reaction of re-
duced nitropyrrole 42 with the dipentafluorophenyl ester of
terephthalic acid gave 1. H1 NMR: δ 2.63 (t, 4H, CH2-
amidine), 3.43-3.54 (m, 4H, CH2NH), 3.83 (s, 6H, CH3), 6.99
and 7.31 (d, 2H, pyrrole), 8.05 (s, 4H, C6H4), 8.03 (t, 1.5H,
CONH), 8.69 and 9.02 (s, 3H, amidine), 10.52 (s, 1.3 H,
CONH). HRMS (FAB): m/z 549.2709 [(M + H)+ calcd for
2-({1-[1-(3-Meth yl-bu tyl)-4-n itr o-1H-p yr r ol-2-yl]m eth -
a n oyl}a m in o)eth yl]ca r ba m ic Acid ter t-Bu tyl Ester , 52.
Amine 51 was dissolved in DMF (20 mL), and diBoc-carbonate
(2.18 g, 10 mmol) was added. The reaction was kept for 1 h at
ambient temperature and evaporated. The residue was dis-
solved in chloroform (30 mL), washed with 0.1 M HCl (10 × 2
mL), 5% NaHCO3 (10 × 2 mL), and water, dried over sodium
sulfate, and evaporated. The crude compound 52 was crystal-
lized from toluene/hexane (4:1 v/v) to afford 1.27 g (69%) of
white crystals; mp 170-172 °C. H1 NMR: δ 0.85-0.87 (d, 6H,
CH3), 1.34 (s, 9H, Boc), 1.42-1.61 (m, 3H, CH2-CH of pyrrole),
3.03-3.08 and 3.18-3.22 (each m, 2H, NHCH2CH2NH), 4.33-
4.38 (t, 2H, CH2-N), 6.85 (t, 1H, NHBoc), 7.48 and 8.18 (d,
1H, pyrrole), 8.36 (t, 1H, NHCO).
C
26H33N10O4, 549.2686].
N,N′-Bis-[5-(2-ca r ba m im id oyl-eth ylca r ba m oyl)-1-p r o-
p yl-1H-p yr r ol-3-yl]ter ep h th a la m id e, 2. The reaction of
reduced nitropyrrole 43 with the dipentafluorophenyl ester of
terephthalic acid gave 2. H1 NMR: δ 0.79 (t, 6H, CH3), 1.62-
1.69 (m, 4H, CH2CH3), 2.61-2.65 (t, 4H, CH2-amidine), 3.47-
3.51 (m, 4H, CH2-NH), 4.20-4.25 (t, 4H, CH2N), 6.99 and 7.35

814 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Dyatkina et al.
(s, 2H, pyrrole), 8.04 (s, 4H, C6H4), 8.28 (t, 2H, NHCO), 8.69
and 9.01 (s, 6H, amidine). HRMS (FAB): m/z 605.3298 [(M +
H)+ calcd for C30H41N10O4, 605.3312].
a m id e}, 9. The reaction of reduced nitropyrrole 44 with the
dipentafluorophenyl ester of pyrazine-2,5-dicarboxylic acid
gave 9. H1 NMR: δ 0.88 (d, 12H, CH3), 1.42-1.55 (m, 6H, CH2-
CH of pyrrole), 2.37-2.43 (t, 4H, CH2-amidine), 3.17-3.25
(m, 4H, CH2NH), 4.27-4.32 (t, 4H, CH2N), 7.05 and 7.42 (d,
2H, pyrrole), 8.21-8.25 (t, 1.5H, CONH), 8.63 and 9.04 (s, 3H,
amidine), 9.26 (s, 2H, pyrazine), 11.00 (s, 1.3H, CONH-
pyrrole). HRMS (FAB): m/z 663.3821 [(M + H)+ calcd for
N,N′-Bis-[5-(2-ca r b a m im id oyl-et h ylca r b a m oyl)-1-(3-
m eth yl-bu tyl)-1H-p yr r ol-3-yl]ter ep h th a la m id e, 3. The re-
action of reduced nitropyrrole 44 with the dipentafluorophenyl
ester of terephthalic acid gave 3. H1 NMR: δ 0.88 (d, 12H,
CH3), 1.42-1.55 (m, 6H, CH2CH of pyrrole), 2.61-2.65 (t, 4H,
CH2-amidine), 3.48-3.51 (m, 4H, CH2NH), 4.30-4.32 (t, 4H,
CH2N), 6.97 and 7.36 (d, 2H, pyrrole), 8.05 (c, 4H, C6H4), 8.29
(t, 1.5H, CONH), 8.70 and 9.02 (s, 3H, amidine), 10.52 (s, 1.3H,
CONH-pyrrole). HRMS (FAB): m/z 661.3920 [(M + H)+ calcd
for C34H49N10O4, 661.3938].
C
33H48N11O4, 663.3843].
N1,N4-Bis-[5-(2-car bam im idoyl-eth ylcar bam oyl)-1-m eth -
yl-1H-p yr r ol-3-yl]-2-n itr o-ter ep h th a la m id e, 10. The reac-
tion of reduced nitropyrrole 44 with the dipentafluorophenyl
ester of nitroterephthalic acid gave 10. H1 NMR: δ 0.88 (d,
12H, CH3), 1.48-1.59 (m, 6H, CH2CH of pyrrole), 2.61-2.66
(m, 4H, CH2-amidine), 3.48-3.52 (m, 4H, CH2NH), 4.28-4.35
(t, 4H, CH2-N), 6.88, 6.99, 7.24, and 7.37 (d, 1H, pyrrole),
7.83-7.86 (d, 1H, C6H4), 8.25-8.32 (m, 1.5H, CONH), 8.32-
8.40 (d, 1H, C6H4), 8.60-8.71 (m, 5H, amidine and C6H4), 9.04
(s, 3H, amidine), 10.77 and 10.81 (s, 0.5H, CONH-pyrrole).
HRMS (FAB): m/z 706.3777 [(M + H)+ calcd for C34H48N11O4,
706.3789].
Na p h th a len e-1,4-d ica r bocylic Acid Bis-{[5-(2-ca r ba m -
im id oyl-et h ylca r b a m oyl)-1-(3-m et h yl-bu t yl)-1H-p yr r ol-
3-yl]a m id e}, 11. The reaction of reduced nitropyrrole 44 with
the dipentafluorophenyl ester of naphthalene-1,4-dicarboxylic
acid gave 11. H1 NMR: δ 0.90 (d, 12H, CH3), 1.50-1.58 (m,
6H, CH2CH of pyrrole), 2.59-2.64 (t, 4H, CH2-amidine), 3.43-
3.56 (m, 4H, CH2NH), 4.29-4.33 (t, 4H, CH2N), 6.92 and 7.38
(d, 2H, pyrrole), 7.60-7.63 (q, 2H, naphthalene), 7.69 (s, 2H,
naphthalene), 8.18-8.21 (q, 2H, naphthalene), 8.23-8.26 (m,
1.5H, CONH), 8.60 and 8.97 (s, 3H, amidine), 10.60 (s, 2H,
CONH-pyrrole). HRMS (FAB): m/z 711.4067 [(M + H)+ calcd
for C38H51N10O4, 711.4095].
Na p h th a len e-2,6-d ica r bocylic Acid Bis-{[5-(2-ca r ba m -
im id oyl-et h ylca r b a m oyl)-1-(3-m et h yl-bu t yl)-1H-p yr r ol-
3-yl]a m id e}, 12. The reaction of reduced nitropyrrole 44 with
the dipentafluorophenyl ester of naphthalene-2,6-dicarboxylic
acid gave 12. H1 NMR: δ 0.90 (d, 12H, CH3), 1.50-1.58 (m,
6H, CH2CH of pyrrole), 2.59-2.64 (t, 4H, CH2-amidine), 3.43-
3.56 (m, 4H, CH2NH), 4.29-4.33 (t, 4H, CH2N), 7.00 and 7.38
(d, 2H, pyrrole), 8.06-8.15 (m, 4H, naphthalene), 8.26-8.32
(m, 1.5H, CONH), 8.59 (s, 2H, naphthalene), 8.66 and 9.00 (s,
3H, amidine), 10.61 (s, 2H, CONH-pyrrole). HRMS (FAB):
m/z 711.4084 [(M + H)+ calcd for C38H51N10O4, 711.4095].
Cycloh exa -1,3-d ien e-1,4-d ica r boxylic Acid Bis-{[5-(2-
ca r ba m im id oyl-eth ylca r ba m oyl)-1-(3-m eth yl-bu tyl)-1H-
p yr r ol-3-yl]a m id e}, 4. The reaction of reduced nitropyrrole
44 with the dipentafluorophenyl ester of cyclohexa-1,3-diene
dicarboxylic acid gave 4. H1 NMR: δ 0.86 (d, 12H, CH3), 1.40-
1.53 (m, 6H, CH2CH of pyrrole), 2.50 (s, 4H, CH2 of cyclohexa-
diene), 2.61-2.65 (t, 4H, CH2-amidine), 3.48-3.51 (m, 4H,
CH2NH), 4.23-4.28 (t, 4H, CH2N), 6.87 and 7.26 (d, 2H,
pyrrole), 6.90 (s, 2H, cyclohexadiene), 8.20-8.22 (m, 1.5H,
CONH), 8.64 and 9.02 (s, 3H, amidine), 9.94 (s, 2H, CONH-
pyrrole). HRMS (FAB): m/z 663.4105 [(M + H)+ calcd for
C
34H51N10O4, 663.4105].
Cycloh exa n e-1,4-d ica r boxylic Acid Bis-{[5-(2-ca r ba m -
im id oyl-et h ylca r ba m oyl)-1-(3-m et h yl-b u t yl)-1H-p yr r ol-
3-yl]a m id e}, 5. The reaction of reduced nitropyrrole 44 with
the dipentafluorophenyl ester of cyclohexane-1,4-dicarboxylic
acid gave 5. H1 NMR: δ 0.86 (d, 12H, CH3), 1.40-1.60 (m,
10H, CH2-CH of pyrrole and CH2 of cyclohexane), 1.80-2.00
(s, 4H, CH2 of cyclohexane), 2.52-2.67 (t, 4H, CH2-amidine),
3.48-3.51 (m, 4H, CH2NH), 4.23-4.28 (t, 4H, CH2N), 6.69-
79 and 7.12-7.20 (m, 2H, pyrrole), 8.16-8.22 (m, 1.5H,
CONH), 8.62 and 8.98 (s, 3H, amidine), 9.66 and 9.79 (s, 2H,
CONH-pyrrole). HRMS (FAB): m/z 667.4429 [(M + H)+ calcd
for C34H55N10O4, 667.4408].
H exa d ioic Acid Bis-{[5-(2-ca r b a m im id oyl-et h ylca r -
ba m oyl)-1-(3-m eth yl-bu tyl)-1H-p yr r ol-3-yl]a m id e}, 6. The
reaction of reduced nitropyrrole 44 with the dipentafluorophen-
yl ester of adipic acid gave 6. H1 NMR: δ 0.86 (d, 12H, CH3),
1.40-1.54 (m, 10H, CH2CH of pyrrole and 2CH2 of adipic acid),
2.19-2.23 (m, 4H, CH2 of adipic acid), 2.61-2.65 (t, 4H, CH2-
amidine), 3.48-3.51 (m, 4H, CH2NH), 4.19-4.24 (t, 4H, CH2N),
6.70 and 7.14 (d, 2H, pyrrole), 8.18-8.21 (m, 1.5H, CONH),
8.62 and 8.98 (s, 3H, amidine), 9.82 (s, 2H, CONH-pyrrole).
HRMS (FAB): m/z 605.3298 [(M + H)+ calcd for C30H41N10O4,
605.3312].
P yr id in e-2,5-d ica r boxylic Acid Bis-{[5-(2-ca r ba m im -
id oyl-eth ylca r ba m oyl)-1-(3-m eth yl-bu tyl)-1H-p yr r ol-3-yl]-
a m id e}, 7. The reaction of reduced nitropyrrole 44 with the
dipentafluorophenyl ester of pyridine-2.5-dicarboxylic acid
gave 7. H1 NMR: δ 0.88 (d, 12H, CH3), 1.40-1.51 (m, 6H, CH2-
CH of pyrrole), 2.61-2.65 (t, 4H, CH2-amidine), 3.48-3.51
(m, 4H, CH2NH), 4.28-4.35 (t, 4H, CH2N), 6.98, 7.12, 7.39,
and 7.41 (d, 1H, pyrrole), 8.19 (d, 1H, pyridyl-H4), 8.20-8.45
(m, 1.5H, CONH), 8.51 (dd, 1H, pyridyl-H3), 8.64 and 9.02
(d, 3H, amidine), 9.16 (d, 1H, pyridyl-H6), 10.29 and 10.31
(s, 0.5 H, CONH-pyrrole). HRMS (FAB): m/z 662.3901 [(M
+ H)+ calcd for C33H48N10O4, 662.3891].
P yr id in e-2,4-d ica r boxylic Acid Bis-{[5-(2-ca r ba m im -
id oyl-eth ylca r ba m oyl)-1-(3-m eth yl-bu tyl)-1H-p yr r ol-3-yl]-
a m id e}, 8. The reaction of reduced nitropyrrole 44 with the
dipentafluorophenyl ester of pyridine-2,5-dicarboxylic acid
gave 8. H1 NMR: δ 0.88 (d, 12H, CH3), 1.48-1.55 (m, 6H, CH2-
CH of pyrrole), 2.61-2.66 (t, 4H, CH2-amidine), 3.48-3.52
(m, 4H, CH2NH), 4.28-4.35 (t, 4H, CH2N), 7.00, 7.11, 7.39,
and 7.42 (d, 1H, pyrrole), 8.10-8.12 (d, 1H, pyridyl), 8.25-
8.40 (m, 1.5H, CONH), 8.55 (s, 1H, pyridyl), 8.84-6.86 (d, 1H,
pyridyl), 8.71 and 8.04 (s, 3H, amidine), 10.75 and 10.93 (s,
0.5 H, CONH-pyrrole). HRMS (FAB): m/z 662.3870 [(M +
H)+ calcd for C33H48N11O4, 662.3891].
N1,N4-Bis-[5-(2-car bam im idoyl-eth ylcar bam oyl)-1-m eth -
yl-1H-p yr r ol-3-yl]-2-m eth yl-ter ep h th a la m id e, 13. The re-
action of reduced nitropyrrole 44 with the dipentafluorophenyl
ester of 2-methylterephthalic acid gave 13. H1 NMR: δ 0.88
(d, 12H, CH3), 1.48-1.55 (m, 6H, CH2CH of pyrrole), 2.40 (s,
3H, methyl), 2.60-2.66 (t, 4H, CH2 amidine), 3.48-3.52 (m,
4H, CH2NH), 4.24-4.32 (t, 4H, CH2N), 6.86, 6.95, 7.27, and
7.33 (d, 1H, pyrrole), 7.48 (d, 1H, terephthalyl), 7.80-7.84 (m,
2H, terephthalyl), 8.20-8.38 (m, 1.5H, CONH), 8.64 and 8.99
(s, 3H, amidine), 10.34 and 10.40 (s, 0.5 H, CONH-pyrrole).
HRMS (FAB): m/z 675.4113 [(M + H)+ calcd for C35H51N10O4,
675.4095].
N1,N4-Bis-[5-(2-car bam im idoyl-eth ylcar bam oyl)-1-m eth -
yl-1H-p yr r ol-3-yl]-2,5-d im eth yl-ter ep h th a la m id e, 14. The
reaction of reduced nitropyrrole 44 with the dipentafluorophen-
yl ester of 2,5-dimethylterephthalic acid gave 14. H1 NMR: δ
0.87 (d, 12H, CH3), 1.41-160 (m, 6H, CH2CH of pyrrole), 2.40
(s, 3H, CH3-C6H4), 2.53-2.60 (m, 4H, CH2-amidine), 3.43-
3.56 (m, 4H, CH2NH), 4.22-4.32 (t, 4H, CH2N), 6.86, 6.95, 7.27
and 7.33 (d, 1H, pyrrole), 7.47-7.49 (d, 1H, C6H3), 7.80-7.84
(m, 2H, C6H3), 8.20-8.30 (m, 1.5H, CONH), 8.64 and 8.99 (s,
3H, amidine), 10.34 and 10.40 (s, 0.5H, CONH-pyrrole).
HRMS (FAB): m/z 689.4241 [(M + H)+ calcd for C36H53N10O4,
689.4251].
N,N′-Bis-[5-(2-ca r b a m im id oyl-et h ylca r b a m oyl)-1-(cy-
clop r op yl-m et h yl)-1H -p yr r ol-3-yl]t er ep h t h a la m id e, 15.
The reaction of reduced nitropyrrole 45 with the dipentafluo-
rophenyl ester of terephthalic acid gave 15. H1 NMR: δ 0.28-
0.47 (m, 8H, CH2-cyclopropyl), 1.13-1.28 (m, 2H, CH-
cyclopropyl), 2.61-2.65 (t, 4H, CH2-amidine), 3.49-3.53 (m,
P yr a zin e-2,5-d ica r boxylic Acid Bis-{[5-(2-ca r ba m im -
id oyl-eth ylca r ba m oyl)-1-(3-m eth yl-bu tyl)-1H-p yr r ol-3-yl]-

Minor Groove DNA Binders as Antimicrobial Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 815
4H, CH2NH), 4.13-4.16 (d, 4H, CH2-N), 6.99 and 7.40 (d, 1H,
pyrrole), 8.04 (s, 4H, C6H4), 8.28-8.32 (t, 2H, CONH), 8.67
and 9.00 (s, 3H, amidine), 10.50 (s, 2H, CONH-pyrrole).
HRMS (FAB): m/z 629.3296 [(M + H)+ calcd for C32H41N10O4,
629.3312].
N,N′-Bis-[5-(2-ca r b a m im id oyl-et h ylca r b a m oyl)-1-(cy-
clop r op yl-m et h yl)-1H -p yr r ol-3-yl]-2-n it r ot er ep h t h a la -
m id e, 16. The reaction of reduced nitropyrrole 45 with the
dipentafluorophenyl ester of 2-nitroterephthalic acid gave 16.
H1 NMR: δ 0.19-0.22 and 0.31-0.39 (d, 4H, cyclopropyl),
1.00-1.20 (m, 2H, CH), 2.45-2.60 (m, 4H, CH2-amidine),
3.43-3.56 (m, 4H, CH2NH), 4.18 (d, 2H, CH2N), 6.58, 6.69,
6.97 and 7.10 (d, 1H, pyrrole), 7.55 (d, 1H, C6H3), 8.11-8.28
(m, 2.2H, CONH and C6H3), 8.30-8.35 (m, 4H, amidine and
C6H3), 8.69 (s, 3H, amidine), 10.45 and 10.47 (s, 1H, CONH-
pyrrole). HRMS (FAB): m/z 674.3183 [(M + H)+ calcd for
C32H40N11O4, 674.3163].
N,N′-Bis-{1-(3-m eth yl-bu tyl)-5-[2-(N-m eth ylca r ba m im -
id oyl)eth ylca r ba m oyl]- 1H-p yr r ol-3-yl}ter ep h th a la m id e,
22. The reaction of reduced nitropyrrole 46 with the dipen-
tafluorophenyl ester of terephthalic acid gave 22. H1 NMR: δ
0.88 (d, 12H, CH3), 1.44-1.58 (m, 6H, CH2CH of pyrrole),
2.59-2.63 (t, 4H, CH2-amidine), 2.78 (d, 6H, methyl), 3.48-
3.51 (m, 4H, CH2NH), 4.26-4.30 (t, 4H, CH2-N), 6.96 and 7.35
(d, 2H, pyrrole), 8.04 (s, 4H, C6H4), 8.27 (t, 1.5H, CONH), 8.58
and 9.15 (s, 2H, amidine), 9.57 (d, 2H, amidine), 10.51 (s, 1.3H,
CONH-pyrrole). HRMS (FAB): m/z 689.4253 [(M + H)+ calcd
for C36H53N10O4, 689.4251].
N,N′-Bis-{1-(3-m et h yl-b u t yl)-5-[2-(N-et h ylca r b a m im -
id oyl)eth ylca r ba m oyl]- 1H-p yr r ol-3-yl}ter ep h th a la m id e,
23. The reaction of reduced nitropyrrole 47 with the dipen-
tafluorophenyl ester of terephthalic acid gave 23. H1 NMR: δ
0.88 (d, 12H, CH3), 1.05-1.11 (m, 6H, CH2CH3), 1.44-1.58 (m,
6H, CH2CH of pyrrole), 2.59-2.63 (t, 4H, CH2-amidine), 2.78
(d, 6H, methyl), 3.13-3.20 (m, 4H, CH2CH3), 3.48-3.51 (m,
4H, CH2NH), 4.26-4.30 (t, 4H, CH2N), 7.55 and 8.13 (d, 2H,
pyrrole), 8.04 (s, 4H, C6H4), 8.70-8.80 (t, 1.5H, CONH), 8.67,
9.23 and 9.64 (bs, 1H, amidine), 10.51 (s, 1.3H, CONH-
pyrrole). HRMS (FAB): m/z 717.4548 [(M + H)+ calcd for
P yr id in e-2,5-d ica r boxylic Acid Bis-{[5-(2-ca r ba m im -
idoyl-eth ylcar bam oyl)-1-(cyclopr opyl-m eth yl)-1H-pyr r ol-
3-yl]a m id e}, 17. The reaction of reduced nitropyrrole 45 with
the dipentafluorophenyl ester of pyridine-2,5-dicarboxylic acid
gave 17. H1 NMR: δ 0.20-0.35 (m, 8H, CH2-cyclopropyl),
1.13-1.28 (m, 2H, CH-cyclopropyl), 2.58-2.65 (m, 4H, CH2-
amidine), 3.49-3.53 (m, 4H, CH2NH), 4.13-4.16 (d, 4H,
CH2N), 6.90, 7.02, 7.33 and 7.37 (d, 1H, pyrrole), 8.05 (d, 1H,
C6H3), 8.18-8.32 (m, 2H, CONH), 8.41 (d, 1H, C6H3), 8.59 and
8.92 (s, 3H, amidine), 9.07 (s, 1H, C6H3), 10.50 (s, 2H, CONH-
pyrrole). HRMS (FAB): m/z 630.3261 [(M + H)+ calcd for
C
38H57N10O4, 717.4564].
N,N′-Bis-{1-(3-m eth yl-bu tyl)-5-[2-(N-p r op ylca r ba m im -
id oyl)eth ylca r ba m oyl]- 1H-p yr r ol-3-yl}ter ep h th a la m id e,
24. The reaction of reduced nitropyrrole 48 with the dipen-
tafluorophenyl ester of terephthalic acid gave 24. H1 NMR: δ
0.84-0.95 (m, 18H, CH3), 1.42-1.60 (m, 10H, CH2CH of
pyrrole and CH2 of propyl), 2.61-2.65 (t, 4H, CH2-amidine),
3.09-3.19 (m, 4H, CH2 of propyl), 3.48-3.51 (m, 4H, CH2NH),
4.30-4.32 (t, 4H, CH2N), 6.97 and 7.35 (d, 2H, pyrrole), 8.05
(c, 4H, C6H4), 8.24 (t, 1.5H, CONH), 8.62 and 9.11 (s, 3H,
amidine), 9.50 (m, 2H, amidine), 10.51 (s, 1.3H, CONH-
pyrrole). HRMS (FAB): m/z 745.4884 [(M + H)+ calcd for
C
31H40N11O4, 630.3265].
P yr a zin e-2,5-d ica r boxylic Acid Bis-{[5-(2-ca r ba m im -
idoyl-eth ylcar bam oyl)-1-(cyclopr opyl-m eth yl)-1H-pyr r ol-
3-yl]a m id e}, 18. The reaction of reduced nitropyrrole 46 with
the dipentafluorophenyl ester of pyrazine-2,5-dicarboxylic acid
gave 18. H1 NMR: δ 0.29-0.46 (m, 8H, CH2-cyclopropyl),
1.16-1.28 (m, 2H, CH-cyclopropyl), 2.61-2.65 (t, 4H, CH2-
amidine), 3.50-3.54 (m, 4H, CH2NH), 4.13-4.16 (d, 4H,
CH2N), 7.09 and 7.50 (d, 1H, pyrrole), 8.28-8.32 (t, 2H,
CONH), 8.64 and 8.99 (s, 3H, amidine), 9.27 (s, 2H, pyrazine),
11.02 (s, 2H, CONH-pyrrole). HRMS (FAB): m/z 631.3211
[(M + H)+ calcd for C30H39N12O4, 631.3217].
N,N′-Bis-[5-(2-ca r b a m im id oyl-et h ylca r b a m oyl)-1-cy-
clop r op yl-m eth yl-1H-p yr r ol-3-yl]-2-m eth ylter ep h th a la -
m id e, 19. The reaction of reduced nitropyrrole 45 with the
dipentafluorophenyl ester of 2-nitroterephthalic acid gave 19.
H1 NMR: δ 0.29-0.32 and 0.41-0.49 (m, 4H, cyclopropyl),
1.10-1.30 (m, 2H, CH), 2.42 (s, 3H, CH3), 2.52-2.68 (m, 4H,
CH2-amidine), 3.45-3.56 (m, 4H, CH2NH), 4.13-4.15 (d, 2H,
CH2N), 6.89, 6.99, 7.32 and 7.40 (d, 1H, pyrrole), 7.48-7.50
(d, 1H, C6H3), 7.81-7.85 (m, 2H, C6H3), 8.27-8.28 (t, 1.5H,
CONH), 8.63 and 8.97 (d, 3H, amidine), 10.34 and 10.40 (s,
1H, CONH-pyrrole). HRMS (FAB): m/z 643.3499 [(M + H)+
calcd for C33H43N10O4, 643.3469].
Th iop h en e-2,5-d ica r boxylic Acid Bis-{[5-(2-ca r ba m -
im id oyl-eth ylca r ba m oyl)-1-(cyclop r op yl-m eth yl)-1H-p yr -
r ol-3-yl]a m id e}, 20. The reaction of reduced nitropyrrole 45
with the dipentafluorophenyl ester of thiophene-2,5-dicarboxyl-
ic acid gave 20. H1 NMR: δ 0.28-0.47 (m, 8H, CH2-cyclo-
propyl), 1.13-1.28 (m, 2H, CH-cyclopropyl), 2.61-2.65 (t, 4H,
CH2-amidine), 3.49-3.53 (m, 4H, CH2NH), 4.13-4.16 (d, 4H,
CH2-N), 6.96 and 7.33 (d, 1H, pyrrole), 7.96 (s, 2H, thiophene),
8.28-8.32 (t, 2H, CONH), 8.64 and 8.99 (s, 3H, amidine), 10.50
(s, 2H, CONH-pyrrole). HRMS (FAB): m/z 635.2869 [(M +
H)+ calcd for C30H39N10O4, 635.2876].
1H-P yr a zole-3,5-d ica r boxylic Acid Bis-{[5-(2-ca r ba m -
im id oyl-eth ylca r ba m oyl)-1-(cyclop r op yl-m eth yl)-1H-p yr -
r ol-3-yl]a m id e}, 21. The reaction of reduced nitropyrrole 45
with the dipentafluorophenyl ester of pyrazole-3,5-dicarboxylic
acid gave 21. H1 NMR: δ 0.19-0.22 and 0.31-0.39 (m, 4H,
cyclopropyl), 1.00-1.20 (m, 2H, CH), 2.45-2.60 (m, 4H, CH2-
amidine), 3.43-3.56 (m, 4H, CH2NH), 4.18 (d, 2H, CH2N), 6.62,
6.69, 7.05 and 7.15 (d, 1H, pyrrole), 7.06 (s, 1H, pyrazole),
0.8.21-8.38 (t, 1.2H, CONH), 8.54 and 8.89 (s, 3H, amidine),
10.13 and 10.45 (s, 2H, CONH-pyrrole). HRMS (FAB): m/z
619.3200 [(M + H)+ calcd for C29H39N12O4, 619.3217].
C
40H61N10O4, 745.4877].
N,N′-Bis-{5-[2-(N,N′-d im eth ylca r ba m im id oyl)eth ylca r -
b a m oyl]-1-(3-m et h yl-b u t yl)-1H -p yr r ol-3-yl}t er ep h t h a l-
a m id e, 25. The reaction of reduced nitropyrrole 49 with the
dipentafluorophenyl ester of terephthalic acid gave 25. H1
NMR: δ 0.87 (d, 12H, CH3), 1.41-1.52 (m, 6H, CH2CH of
pyrrole), 2.61-2.65 (t, 4H, CH2 CH2-amidine), 2.77 and 2.99
(d, 3H, CH3-amidine), 3.48-3.51 (m, 4H, CH2NH), 4.27-4.31
(t, 4H, CH2N), 6.96 and 7.35 (d, 2H, pyrrole), 8.04 (s, 4H, C6H4),
8.33-8.38 (t, 1.2H, CONH), 8.74 and 9.52 (d, 1.5H, amidine),
10.52 (s, 2H, CONH-pyrrole). HRMS (FAB): m/z 717.4587
[(M + H)+ calcd for C38H57N10O4, 717.4564].
N,N′-Bis-{1-(3-m et h yl-b u t yl)-5-[2-(1,4,5,6-t et r a h yd r o-
p y r i m i d i n -2-y l )e t h y l c a r b a m o y l ]-1 H -p y r r o l -3 -y l}-
ter ep h th a la m id e, 26. The reaction of reduced nitropyrrole
50 with the dipentafluorophenyl ester of terephthalic acid gave
26. H1 NMR: δ 0.88 (d, 12H, CH3), 1.45-1.55 (m, 6H, CH2CH
of pyrrole), 1.78-1.89 (m, 4H, tetrahydopyrimidinyl), 2.61-
2.65 (t, 4H, CH2 -amidine), 3.20-3.40 (m, tetrahydopyrimidinyl
and water), 3.48-3.51 (m, 4H, CH2NH), 4.27-4.31 (t, 4H,
CH2N), 6.99 and 7.36 (d, 2H, pyrrole), 8.04 (s, 4H, C6H4), 8.33-
8.38 (t, 1.3H, CONH), 9.67 (s, 3H, amidine), 10.52 (s, 1H,
CONH-pyrrole). HRMS (FAB): m/z 741.4569 [(M + H)+ calcd
for C40H57N10O4, 741.4564].
N,N′-Bis-[5-(2-am in oeth ylcar bam oyl)-1-(3-m eth ylbu tyl)-
1H-p yr r ol-3-yl]ter ep h th a la m id e, 27. To a stirred solution
of nitropyrrole derivative 52 (0.15 mmol) in methanol (20 mL)
was added 10% Pd/C (Degussa type, Aldrich) (0.1 g). The flask
was evacuated, flushed three times with hydrogen, and finally
filled with hydrogen at 25-30 psi. The resultant suspension
was stirred vigorously at ambient temperature for 30 min. The
suspended material was filtered; the filtrate was evaporated
to dryness. The resultant aminopyrrole derivative 52a was
used for the next step without purification. The solution of
freshly prepared aminopyrrole derivative 52a in 2 mL of dry
DMF was added to the dipentafluorophenyl ester of dicar-
boxylic acid (35 mg, 0.07 mmol). The reaction mixture was
stirred for 15 h at 55 °C, cooled, and evaporated. The dark
solid was dissolved in 5 mL of methanol, 2 mL of 4 N HCl in
dioxane was added, and the reaction was kept for 40 min at

816 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Dyatkina et al.
ambient temperature. The reaction mixture was evaporated
and purified by HPLC (Vydac 12 µm C18 2.2 cm × 25 cm
column, 10-70% acetonitrile gradient over 40 min, flow rate
10 mL/min) to give the corresponding linked dipyrrole 27. This
salt was dissolved in 2 mL of methanol saturated with HCl,
35 mL of diethyl ether was added, and the precipitate of linked
dipyrrole 27 as bis-HCl salt was separated and dried to yield
25 mg (52%) based on the dipentafluorophenyl ester of tere-
phthalic acid. H1 NMR: δ 0.88 (d, 12H, CH3), 1.50-1.61 (m,
6H, CH2CH of pyrrole), 2.84-2.95 (m, 4H, CH2-NH2), 3.39-
3.43 (m, 4H, CH2NH), 4.27-4.32 (t, 4H, CH2-N), 6.99 and 7.36
(d, 2H, pyrrole), 7.90-8.10 (bs, 4H, NH2), 8.04 (s, 4H, C6H4),
8.21-8.25 (t, 1.2H, CONH), 10.51 (s, 2H, CONH-pyrrole).
HRMS (FAB): m/z 607.3719 [(M + H)+ calcd for C32H47N8O4,
607.3720].
N,N′-Bis-[5-(2-gu a n id in o-eth ylca r ba m oyl)- 1-(3-m eth -
yl-bu tyl)-1H-p yr r ol-3-yl]ter ep h th a la m id e, 28. The TFA
salt of amine 27 (42 mg, 0.05 mmol) was dissolved in DMF (3
mL), pyrazole-1-carboxamidine (55 mg, 0.5 mmol) was added,
and the reaction was stirred at ambient temperature over-
night. The title product 28 was isolated by HPLC and
transformed to hydrochloride as described above to afford 25
mg (65%). H1 NMR: δ 0.88 (d, 12H, CH3), 1.48-1.58 (m, 6H,
CH2CH of pyrrole), 3.20-3.40 (m, 8H, 2CH2), 4.27-4.32 (t, 4H,
CH2N), 6.96 and 7.35 (d, 2H, pyrrole), 7.58-7.65 and 8.15-
8.23 (t, 2H, CONH and guanidine), 8.04 (s, 4H, C6H4), 10.51
(s, 2H, CONH-pyrrole). HRMS (FAB): m/z 691.4136 [(M +
H)+ calcd for C34H51N12O4, 691.4156].
Dock in g a n d Mod el Bu ild in g. Molecular models of can-
didate molecules were generated using the ab initio, flexible
docking procedure of the ICM 2.8 molecular modeling program
by Molsoft.31,33 Structural coordinates for the “receptor” were
derived from the cocrystal structure of distamycin34 bound to
the minor groove of the DNA duplex (GCCAAATTTCGC)2
(PDB entry 2dnd) from which the ligand was removed. A grid
potential map for the receptor was created for the inner eight
base pairs involving electrostatic, van der Waals, and hydrogen-
bonding energy terms at a grid size of 0.5 Å. For the ligands,
2D to 3D conversion is followed by energy minimization using
the MMFF option as a force field. Starting from random ligand
positions, the first step of docking involves a restrained energy
minimization, which brings the ligand into the center of the
receptor, which, here, was set at the center of the minor groove
in the middle of the AT tract. The second step is comprised of
100 independent Monte Carlo searches of 10 000 steps each
in torsion angle internal coordinate space to find the best
ligand conformation to interact with the receptor. The top 20
conformations are saved and rescored with hard van der Waals
potentials. When this procedure is used to dock distamycin
with full torsional flexibility into its native site, deviation from
the crystal structure coordinates is only 0.7 Å. Most of that is
related to a 0.5 Å 5′ to 3′ shift of the ligand in the groove and
a flipped formyl group. The van der Waals component of the
intermolecular energy was used as an indicator for the quality
of the shape fit of a new ligand. Typically, good DNA binders
(indicated by ∆Tm > 20 °C) showed van der Waals contribu-
tions to interaction energy better than that of distamycin.
and mixed with compounds at various ratios. The temperature
was typically varied from 15 to 95 °C with a ramp rate of 0.2
°C/min. To determine the melting temperature (Tm) where half
of the double-stranded DNA molecules dissociate into two
separated strands,26,27 the first-order derivatives of the absorp-
tion-temperature curve were calculated using the Varian
software, and the maximum of derivatives corresponds to the
melting temperature. The melting temperatures determined
by the derivative method were verified using a standard
hyperchromicity method provided by the Varian software. The
∆Tm value was reported as the difference between melting
temperatures in the presence and in the absence of compounds.
All experiments were at least in duplicate.
Estim a tion of Dr u g-DNA Bin d in g Con sta n ts. An ethid-
ium bromide (EtBr) displacement assay was used to determine
the dissociation constant for binding of compounds to oli-
go1.28,29 Briefly, increasing amounts of compounds were ti-
trated to preformed fluorescent EtBr-DNA complexes. The
dissociation of EtBr-DNA due to compound competition gives
rise to decreases in the fluorescent signal. The competition was
allowed to equilibrate for at least 2 h. The fluorescence was
monitored at an excitation wavelength of 530 nm and emission
wavelength of 620 nm using a 96 well plate fluorimeter
(CytoFluor Series 4000, PerSeptives Biosystems). Values of KD
were determined using a standard competition analysis.
An tiba cter ia l MIC Assa ys. The MIC assays were per-
formed using the microtiter method according to NCCLS
protocol M7-A4.36 Briefly, test compounds were freshly dis-
solved in 100% dimethyl sulfoxide to a stock concentration of
10 mM and diluted in the BHI for VRE or Mueller-Hinton
media for MRSA. Seven 1:2 serial dilutions of compound in
appropriate media were prepared such that the compound test
concentrations ranged from 45.5 to 0.7 µM. MIC values were
called after visual inspection of the wells after 2 days.
T-Cell Ba sed WST Toxicity Tests. Compound toxicity was
tested using the CCRF-CEM Human Leukemia cell line (ATCC
Manassas, Virginia) and cell proliferation reagent WST-1
(Roche Molecular Biochemicals, Mannheim, Germany).30 Growth
medium was RPMI-1640 (ATCC Manassas, Virginia) supple-
mented with 10% of fetal bovine sera (Gibco BRL, Grand
Island, NY) and Gentamicin 0.1 mg/mL (Gibco BRL, Grand
Island, NY). A total of 4 × 104 cells/well was applied to 96
well plates and grown over several days. Concentrations of
compound added to cells at the beginning of the experiment
were 50, 25, 12.5, and 6.3 µM. After the cells were incubated
with WST-1 reagent, absorbance at 440 nm was measured
with a Spectra Max 340 PC microplate spectrophotometer
(Molecular Devices, Sunnyvale, CA) 24, 48, and 72 h after
adding the compound. The toxicity value reported in the
present study is the lowest fraction (%) of WST-1 cleavage
activity left in treated cells as compared to the culture with
no compound (% NCC; % of no compound control), which
typically reflects the value taken 72 h after treatment with
50 µM compound. Experiments were done at least in triplicate.
Ack n ow led gm en t. This work was partially sup-
ported by Grant N65236-98-1-5400 from the Defense
Advanced Research Projects Agency (DARPA) Uncon-
ventional Pathogens Countermeasures (UPC) program.
Exp er im en ta l Log P o/w. The apparent octanol/water parti-
tion coefficients at pH 7.5 (log Po/w) were determined experi-
mentally for selected compounds using the shake-flask method
and subsequent quantitation of UV-detected reverse phase
HPLC traces.35 Typically, 50-80 µg were subjected to equili-
bration between 0.3 mL of potassium phosphate buffer (1 mM,
pH 7.5) and 0.3 mL of buffer-saturated octanol at 30 °C. To
avoid saturation artifacts, compounds were assayed over a 50-
100-fold concentration range through dilution of the initial
samples. The precision of our experimental log Po/w values is
about 0.5 units.
DNA Bin d in g Meth od s. Ligand-DNA interactions were
monitored in a buffer containing 10 mM HEPES, pH 7.2, 0.1
mM EDTA, and 50 mM NaCl. DNA thermal melting was
monitored by UV absorbance at 260 nm on a Cary 100 Bio
UV/vis spectrophotometer. A 12 base pair AT-rich DNA
oligonucleotide (Oligo1: CGATTATTAAGC) was used at 5 µM
Refer en ces
(1) Nash, J . M. The Antibiotics Crisis. Time Magazine 2001, pp 749-
750.
(2) Walsh, C. Molecular mechanisms that confer antibacterial drug
resistance. Nature 2000, 406, 775-781.
(3) Foye, W. O.; Lemke, T. L.; Williams, D. A. Principles of Medicinal
Chemistry; Williams & Wilkins: Media, PA, 1995.
(4) DiMarco, A.; Gaetani, M.; Orezzi, P.; Scotti, T.; Arcamone, F.
Experimental studies on distamycin A-a new antibiotic with
cytotoxic activity. Cancer Chemother. Rep. 1962, 18, 15-19.
(5) Probst, G. W.; Hoehn, M. M.; Woods, B. L. Anthelvencins, new
antibiotics with Anthelmintic Properties. Antimicrob. Agents
Chemother. 1965, 5, 789.
(6) Geierstanger, B.; Wemmer, D. E. DNA binding ligands. Annu.
Rev. Biophys. Biomol. Struct. 1995, 24, 463-493.

Minor Groove DNA Binders as Antimicrobial Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 817
(7) Wemmer, D. E. Designed sequence-specific minor groove ligands.
Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 439-461.
(8) Bassetti, D. Distamycin A: a new antibiotic with antiviral action.
Initial clinical trial in cases of chickenpox. G. Mal. Infett.
Parassit. 1968, 20, 827-829.
(9) Bialer, M.; Yagen, B.; Mechoulam, R.; Becker, Y. Structure-
activity relationships of pyrrole amidine antiviral antibiotics. 1.
Modifications of the alkylamidine side chain. J . Med. Chem.
1979, 22, 1296-1301.
(10) Reddy, B. S.; Sondhi, S. M.; Lown, J . W. Synthetic DNA minor
groove-binding drugs. Pharmacol. Ther. 1999, 84, 1-111.
(11) Dervan, P. B.; Burli, R. W. Sequence-specific DNA recognition
by polyamides. Curr. Opin. Chem. Biol. 1999, 3, 688-693.
(12) Chaires, J . B. Drug- -DNA interactions. Curr. Opin. Struct. Biol.
1998, 8, 314-320.
(13) Gursky, G. V.; et al. Synthetic sequence-specific ligands. Cold
Spring Harbor Symp. Quant. Biol. 1983, 47, 367-378.
(14) Khorlin, A. A.; et al. A new type of AT-specific ligand constructed
of two netropsin-like molecules. FEBS Lett. 1980, 118, 311-314.
(15) Lown, J . W.; Krowicki, K.; Balzarini, J .; Newman, R. A.; De
Clercq, E. Novel linked antiviral and antitumor agents related
to netropsin and distamycin: synthesis and biological evaluation.
J . Med. Chem. 1989, 32, 2368-2375.
(23) Gilman, H.; Langham, W.; Moore, F. W. Some Interconversion
Reactions of Organolithium Compounds. J . Am. Chem. Soc.
1940, 61, 2328-2335.
(24) Gamper, H. B.; et al. Facile preparation of nuclease resistant 3′
modified oligodeoxynucleotides. Nucleic Acids Res. 1993, 21,
145-150.
(25) Bernatowicz, M. S.; Wu, Y. L.; Matsueda, G. R. 1H-pyrazole-1-
carboxamidine hydrochloridesan attractive reagent for guany-
lation of amines and its application to peptide synthesis. J . Org.
Chem. 1992, 57, 2497-2502.
(26) Crothers, D. M. Statistical thermodynamics of nucleic acid
melting transitions with coupled binding equilibria. Biopolymers
1971, 10, 2147-2160.
(27) Marky, L. A.; Breslauer, K. J . Calculating thermodynamic data
for transitions of any molecularity from equilibrium melting
curves. Biopolymers 1987, 26, 1601-1620.
(28) Morgan, A. R.; Lee, J . S.; Pulleyblank, D. E.; Murray, N. L.;
Evans, D. H. Review: ethidium fluorescence assays. Part 1.
Physicochemical studies. Nucleic Acids Res. 1979, 7, 547-569.
(29) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C. A simple,
high-resolution method for establishing DNA binding affinity
and sequence selectivity. J . Am. Chem. Soc. 2001, 123, 5878-
5891.
(16) Neamati, N.; et al. Highly potent synthetic polyamides, bisdis-
tamycins, and lexitropsins as inhibitors of human immunode-
ficiency virus type 1 integrase. Mol. Pharmacol. 1998, 54, 280-
290.
(30) Ishiyama, M.; et al. A combined assay of cell viability and in
vitro cytotoxicity with a highly water-soluble tetrazolium salt,
neutral red and crystal violet. Biol. Pharm. Bull. 1996, 19,
1518-1520.
(31) Abagyan, R.; Totrov, M.; Kutznetzov, D. ICMsA new method
for protein modeling and designsApplications to docking and
structure prediction from the distorted native conformation. J .
Comput. Chem. 1994, 15, 488-506.
(32) Oprea, T. I. Property distribution of drug-related chemical
databases. J . Comput. Aided Mol. Des. 2000, 14, 251-264.
(33) Totrov, M.; Abagyan, R. Protein-Ligand Docking as an Energy
Optimization Problem. In Drug-Receptor Thermodynamics: In-
troduction and Applications; Raffa, R. B., ed.; J ohn Wiley & Sons
Ltd.: New York, 2001.
(34) Coll, M.; Frederick, C. A.; Wang, A. H.; Rich, A. A bifurcated
hydrogen-bonded conformation in the d(A.T) base pairs of the
DNA dodecamer d(CGCAAATTTGCG) and its complex with
distamycin. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8385-8389.
(35) Rubas, W.; Cromwell, M. E. M. The effect of chemical modifica-
tions on octanol/water partition (log D) and permeabilities across
Caco-2 monolayers. Adv. Drug Delivery Rev. 1997, 23, 157-162.
(36) NCCLS Methods for dilution antimicrobial susceptibility tests
for bacteria that grow aerobically, 4th edition; Approved Stan-
dard. M7-A4. NCCLS Protocol M7-A4 1997, 17 (2).
(17) Francesconi, I.; et al. 2,4-Diphenyl furan diamidines as novel
anti-Pneumocystis carinii pneumonia agents. J . Med. Chem.
1999, 42, 2260-2265.
(18) Tanious, F. A.; et al. Effects of compound structure on carbazole
dication-DNA complexes: tests of the minor-groove complex
models. Biochemistry 2000, 39, 12091-12101.
(19) Florence, A. T.; Attwood, D. Physicochemical Principles of
Pharmacy; Macmillan: London, 1998.
(20) Turchin, K. F.; Grokhovskii, S. L.; Zhuze, A. L.; Gottikh, B. P.
Ligand possessing activity for definite pairs of DNA bases. II.
Study of the stereochemistry of the chromophore of Distamycin
A by 1H NMR spectroscopy. Russ. J . Bioorg. Chem. (Engl.
Transl.) 1978, 4, 780-790.
(21) Grokhovskii, S. L.; Zhuze, A. L.; Gottikh, B. P. Ligand possessing
activity for definite pairs of DNA bases. 1. Synthesis of disto-
mycin A and its analogues with different numbers of N-methyl
and N-propylpyrrole residues in the molecule. Russ. J . Bioorg.
Chem. (Engl. Transl.) 1975, 1163-1169.
(22) Del Poeta, M.; et al. Structure-in vitro activity relationships of
pentamidine analogues and dication-substituted bis-benzimida-
zoles as new antifungal agents. Antimicrob. Agents Chemother.
1998, 42, 2495-2502.
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