Mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities

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

Synthetic derivatives of the natural product antibiotic novobiocin were synthesized in order to improve their physiochemical properties. A Mannich reaction was used to introduce new side chains at a solvent-exposed position of the molecule, and a diverse panel of functional groups was evaluated at this position. Novobiocin and the new derivatives were tested for their binding to gyrase B and their antibacterial activities against Staphylococcus aureus, Mycobacterium tuberculosis, Francisella tularensis and Escherichia coli. While the new derivatives still bound the gyrase B protein potently (0.07-1.8 μM, IC₅₀), they had significantly less antibacterial activity. Two compounds were identified with increased antibacterial activity against M. tuberculosis, with a minimum inhibitory concentration of 2.5 μg/ml.

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

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5697–5700
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Mannich reaction derivatives of novobiocin with modulated
physiochemical properties and their antibacterial activities
⇑
Arlyn Tambo-ong, Sidharth Chopra, Bryan T. Glaser, Karen Matsuyama, Tran Tran, Peter B. Madrid
Center for Infectious Disease and Biodefense Research, Biosciences Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthetic derivatives of the natural product antibiotic novobiocin were synthesized in order to improve
their physiochemical properties. A Mannich reaction was used to introduce new side chains at a solvent-
exposed position of the molecule, and a diverse panel of functional groups was evaluated at this position.
Novobiocin and the new derivatives were tested for their binding to gyrase B and their antibacterial
activities against Staphylococcus aureus, Mycobacterium tuberculosis, Francisella tularensis and Escherichia
Received 2 May 2011
Revised 5 August 2011
Accepted 5 August 2011
Available online 12 August 2011
coli. While the new derivatives still bound the gyrase B protein potently (0.07–1.8
significantly less antibacterial activity. Two compounds were identified with increased antibacterial
activity against M. tuberculosis, with a minimum inhibitory concentration of 2.5 g/ml.
lM, IC50), they had
Keywords:
Novobiocin
Gyrase B
Tuberculosis
Antibacterial
Aminocoumarin
l
Ó 2011 Elsevier Ltd. All rights reserved.
The therapeutic treatment of bacterial diseases with antibacte-
rial drugs has had a remarkable and profound impact on human
health around the world. Despite the tremendous success of such
drugs, the increase in diseases attributed to drug-resistant and
Since modifying existing classes of drugs to evade resistance
mechanisms has proven to be an effective strategy for multiple
classes of antibacterial drugs, this approach may also be effective
for improving the activities of the antibacterial drug novobiocin
(1). Novobiocin targets the bacterial topoisomerase II B subunit,
referred to as gyrase B, and is the only approved drug against this
target. A natural product isolated from Streptomyces niveus, novo-
biocin was licensed for clinical use in the 1960s and has proven
to be an effective treatment for methicillin-resistant Staphylococcus
aureus (MRSA) infections. The drug has since been withdrawn from
the market, primarily due to the availability of superior drugs. Two
related compounds, clorobiocin and coumermycin A1, are even
more potent inhibitors than novobiocin, but they have not been
approved for clinical use.
multi-drug resistant (MDR) bacterial pathogens indicates
a
growing need for new antibacterial drugs. New therapeutics are
needed for bacterial pathogens that have become resistant to many
of the currently approved drugs as well as bacterial strains that are
naturally resistant to many antibacterial drugs and have become
increasingly pathogenic. Some of the most difficult types of infec-
tions to treat are those involving Gram-negative bacteria and
mycobacteria, since these classes of bacteria often possess multiple
mechanisms that make them resistant to entire classes of antibac-
terial drugs.
The majority of the most effective new treatments that have
been developed for treating drug-resistant strains of bacteria have
come from modifications to existing classes of antibacterial drugs.
Currently, new generations of cephalosporins, carbapenems and
tetracyclines have proven to be some of the most valuable new
drugs for treating drug-resistant infections. The newer generations
of these drugs have been developed to maintain their potency at
their molecular target while circumventing the drug-resistant
mechanisms that have made the older generations of these drugs
less effective. Additionally, some of the modifications to these drugs
have increased the spectrum of activity to include some of the more
difficult to treat pathogens, particularly the Gram-negative ones.
Although novobiocin is a highly potent inhibitor of gyrase B in
vitro, it has a very high level of serum protein binding and there-
fore must be given at high doses for in vivo efficacy.1,2 Also, the
drug is only effective against Gram-positive bacteria, such as S. aur-
eus.3 Given these shortcomings, modifications to novobiocin that
would reduce the drug’s serum protein binding and perhaps
increase its spectrum of activity to include Gram-negative bacteria
and mycobacteria could produce a useful next-generation amino-
coumarin antibiotic.
Many drugs that target Gram-positive bacteria are unable to
penetrate the outer membrane of Gram-negative bacteria. This
membrane is an orthogonal permeability barrier to the cellular
membrane and keeps out many types of drugs that would
otherwise be considered membrane-permeable. The structure of
novobiocin is amphipathic, with a polar noviose sugar moiety and
⇑
Corresponding author. Tel.: +1 650 859 2253; fax: +1 650 859 3153.
E-mail address: peter.madrid@sri.com (P.B. Madrid).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.035

5698
A. Tambo-ong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5697–5700
a weakly acidic aminocoumarin core structure attached to a non-
polar hydroxybenzoate group ( Fig. 1).4 A potential strategy for
increasing the permeability of novobiocin is to introduce functional
groups that can modulate its permeability properties, thereby
potentially increasing its spectrum of activity.
In order to test the chemistry, novobiocin sodium was reacted
with pyrrolidine (1.5 equiv) and paraformaldehyde (1.1 equiv) in
ethanol in a sealed glass tube heated to 150 °C with microwave
irradiation. The desired product was formed in low yields, but
there was also a significant amount of hydrolysis of the carbamate
group on the noviose group, and this side product was inseparable
by chromatographic methods. This side product was minimized by
the addition of acetic acid (1.5 equiv) to neutralize the slightly
acidic sodium phenolate on the coumarin ring, and this modifica-
tion eliminated the formation of this side product. Finally, it was
found that switching the solvent from ethanol to tetrahydrofuran
(THF) improved the isolated yield of pure product from 14% to
65% in this model system. Subsequently, these conditions were
used for all of the Mannich derivatives, with THF as the solvent.
The resulting products contained a basic amine group; with the
acidic coumarin ring, the resulting compounds were zwitterionic
and required reversed-phase C18 prep-HPLC purification.
In order to create a small set of derivatives functionalized at this
position, we wanted to add the smallest possible amine group to
the novobiocin structure, since the molecular weight of novobiocin
(612 g/mol) was already quite high for a drug. The smallest amine
that produced the desired Mannich product was methylamine, so
this product (2) was scaled up to Gram scale in order to produce
a key intermediate (Scheme 1). This secondary amine intermediate
could then be coupled to a diverse set of side chains to produce a
set of analogs with modulated physiological properties.
New novobiocin analogs were designed using the X-ray crystal
structure of novobiocin bound to a 35-kD fragment of gyrase B5 as
well as other available structure–activity relationship (SAR) infor-
mation (Fig. 1).6–8 From this information, it was clear that the novi-
ose sugar and aminocoumarin components of novobiocin bind
deep into the ATP-binding pocket of the protein and make most
of the key binding interactions. The hydrozybenzoate group also
makes hydrophobic interactions with the protein, but this group
is partially solvent-exposed and more amenable to substitution
from prior SAR data (Fig. 1).7 This structural information suggests
that the position ortho to the phenol group would be ideal to
append an additional solvent-exposed side chain that could modu-
late the overall physiochemical properties of the molecule. The two
closest amino acids to this position are Asp80 and Glu136 (1KIJ
structure numbering), two negatively charged residues, but
Arg135 is also adjacent to this position so it was reasoned that a
neutral functional group should be used as an attachment point
off of the hydroxybenzoate ring.
Previous work on analogs of novobiocin using either synthetic
or chemoenzymatic methods have established that the noviose
group is essential for binding to gyrase B. Compounds such as
clorobiocin, which have an extended group off of the 3-position
of the noviose ring, more deeply penetrate the binding pocket
and generally have greater potency.7,9 A previous effort to improve
the physiochemical properties of novobiocin was done by remov-
ing the entire hydroxybenzoate group and replacing the coumarin
hydroxyl group with amines.10,11 Modifications of the hydroxyben-
zoate group have been done primarily by chemoenzymatic meth-
ods and have produced analogs with modifications to the
isoprenyl functional group.12,13 Most of the modifications that
shorten or significantly alter the isoprenyl group result in losses
of both binding and antimicrobial activities.
Using intermediate 2,
a set of carboxylic acid chlorides,
symmetric anhydrides and acids were selected to explore the ef-
fects of hydrophobic, polar neutral, acidic, and basic functionalities
at this position (Scheme 1). In all cases we attempted to select
groups that were as small as possible, given the molecular weight
of novobiocin. The coupling reactions of intermediate 2 and the
acid chlorides and anhydrides were carried out by a reaction in
pyridine using standard amide formation conditions to give prod-
ucts 3a, 3b, and 3f in 10–35% isolated yields following prep-HPLC
purifications. The reaction of intermediate 2 with acids was
performed with HBTU/EDCI coupling conditions to give products
3c–e and 3g–j in 10–55% yields following purification. The synthe-
sis of some of the basic side-chain derivatives was performed by
directly coupling novobiocin sodium to the appropriate amine
using our standard Mannich conditions to give products 4k–n in
15–55% yields. This set of 14 new analogs was characterized for
purity and identity by LC/MS, HPLC and 1H NMR analysis and
shown to be of >95% purity by HPLC.
Developing chemistry that could be used to make semi-syn-
thetic analogs of novobiocin is challenging because its structure
is not stable in strongly acidic or strongly basic conditions. Addi-
tionally, since the structure of the drug includes many different
functional groups, it was important that the synthetic transforma-
tions be highly regioselective. Based on these criteria, it was
deduced that a Mannich reaction would be ideal for introducing
substitutions ortho to the phenol group since the reaction can be
carried out under neutral conditions and is compatible with all of
the other functional groups on novobiocin.
The introduction of non-polar, polar neutral, acidic, and basic
groups at the solvent-exposed position enabled us to survey the ef-
fects of such modifications on the physiological properties, binding
Figure 1. The structure of novobiocin is comprised of three primary chemical groups: A noviose group, an aminocoumarin group and a hydroxybenzoate group. Previous SAR
data and the crystal structure of novobiocin bound to a 43-kD fragment of gyrase B (1KIJ) indicates that the position ortho to the phenol on the hydroxybenzoate group (white
arrow) is well positioned for appending an additional solvent-exposed side chain.

A. Tambo-ong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5697–5700
5699
Scheme 1. Reagents and conditions: (a) Amine (1.1 equiv), paraformaldehyde (1.1 equiv), CH3COOH (2.0 equiv), THF, 120 °C (2 h); (b) acid chlorides and anhydrides
(1.2 equiv), pyridine, rt (1–4 h); (c) acids (1.2 equiv), HBTU (1.2 equiv), DIPEA (3.0 equiv), MeCN, rt (1–4 h).
Table 1
Gyrase B inhibitory and antimicrobial activities of novobiocin derivatives
Compound
c Log Da pH 7.4
Gyrase B IC50
(lM)
Minimum inhibitory concentration (MIC) (lg/mL)
S. aureus ATCC 29213
M. tuberculosis H37Rv
F. tularensis Schu S4
E. coli ATCC 25922
1
2.65
3.63
3.58
1.97
0.60
0.67
À1.70
À0.35
0.62
0.008
1.8
0.125
>64
16
16
>64
16
64
>64
16
>64
>64
>64
>64
>64
>64
4-8
>10
>10
2.5
>10
2.5
>10
>10
10
>10
>10
>10
>10
>10
10
2
>64
64
4–16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4k
4l
4m
4n
0.12
0.09
0.46
0.07
0.18
0.14
0.07
0.23
0.37
0.81
0.63
0.80
0.68
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
À0.02
1.32
1.53
À0.59
2.48
1.39
a
c Log D values were calculated using Chem Axon software.
affinities, and antibacterial activities of the new analogs (Table 1).
The calculated Log D values (at pH 7.4) demonstrated a wide range
of partition coefficients ranging from À1.70 to 3.63. The partition
coefficients alone proved to have no correlation with the binding
affinities or the antibacterial activities, suggesting that this single
property of the compounds is not sufficient to significantly
increase their permeability to bacterial membranes.
The in vitro binding constants for all of the novobiocin deriva-
tives were determined using a fluorescence polarization ligand
displacement method with the full-length gyrase B protein from
Francisella tularensis Schu S4 strain.14 The IC50 values obtained for
binding to gyrase B revealed that all of the derivatives were still
potent binders to gyrase B but had some loss of binding activity
relative to novobiocin (Table 1). Derivatives with sterically bulky
groups, such as 3a, 3b, 3l, and 3m, had the greatest drop in binding
potencies, suggesting that even though this position is solvent-ex-
posed in the crystal structure, some obstruction from protein
residues on the outside of the binding pocket might be clashing
with these groups. The most potent compounds contained either
small non-polar side chains (3b, 0.17
(3c, 0.09 M; and 3e, 0.07 M), or a weakly acidic sulfonamide side
chain (3h, 0.07 M). All of the derivatives with a basic side chain
lM), polar neutral side chains
l
l
l
had greater losses in binding affinities, which was somewhat
expected given that Arg135 is near these substitutions and could
form unfavorable interactions with the positive charges on these
basic side chains.
The antibacterial activities of these new derivatives were
measured against a diverse panel of bacteria that included a

5700
A. Tambo-ong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5697–5700
Gram-positive pathogen (S. aureus), two Gram-negative pathogens
(F. tularensis and Escherichia coli) and a mycobacteria (Mycobacte-
rium tuberculosis). Given that novobiocin has been used as a drug
primarily for S. aureus, we expected the greatest activities against
these bacteria. Novobiocin was quite potent against S. aureus, with
of antibiotics that target Gram-negative bacteria revealed that in
general Gram-negative antibiotics have lower molecular weights
and lower partition coefficients than antibiotics targeting Gram-
positive bacteria.15 In fact, it is believed that many of the Gram-
negative antibiotics must enter the cytosol through porins rather
than diffusion across the outer membrane.
an MIC of 0.125
significantly less potent, with four compounds having MIC values
of 16 g/ml. These four compounds—3b, 3c, 3e, and 3h—were the
lg/ml. The most potent new derivatives were all
l
Acknowledgments
same four compounds that were the most potent in the gyrase B
binding assay. Their binding affinities were approximately one
order of magnitude weaker than novobiocin, but their antibacterial
activities were closer to two orders of magnitude weaker than
novobiocin. These data suggest that the modifications on these
compounds may have actually decreased their ability to enter into
the cytoplasm of S. aureus, even though these four compounds
have calculated partition coefficients ranging from 0.62 to3.58,
around that for novobiocin (2.65).
The project described was supported by Award Number
U01AI082070 from the National Institute of Allergy and Infectious
Diseases. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National
Institute of Allergy and Infectious Diseases or the National Insti-
tutes of Health.
Supplementary data
The antibacterial activities of the new derivatives against M.
tuberculosis (Mtb) were slightly more encouraging, with two
compounds, 3c and 3e, showing a significant increase in potency
relative to novobiocin (Table 1). Interestingly, all of the compounds
that showed any activity against S. aureus showed higher potencies
against Mtb, which is surprising given that novobiocin itself is
much more potent against S. aureus (the MIC value for novobiocin
Supplementary data (Synthetic and biological methods and
analytical characterization of all compounds) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.08.035.
References and notes
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activity at the concentrations tested. Clearly, the modifications
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permeability to the Gram-negative pathogens. The large size of
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