Concise synthesis of spergualin-inspired molecules with broad-spectrum antibiotic activity

Article abstract of DOI:10.1039/c4md00572d

There is a growing need to identify new, broad-spectrum antibiotics. The natural product spergualin was previously shown to have promising anti-bacterial activity and a privileged structure, but its challenging synthesis had limited further exploration. For example, syntheses of spergualin and its analogs have been reported in approximately 10 linear steps, with overall yields between 0.3 and 18%. Using the Ugi multi-component reaction, we assembled spergualin-inspired molecules in a single step, dramatically improving the overall yield (20% to 96%). Using this strategy, we generated 43 new analogs and tested them for anti-bacterial activity against two Gram-negative and four Gram-positive strains. We found that the most potent analogue, compound 6, had MIC values between 4 and 32 μg mL^-1 against the six strains, which is a significant improvement on spergualin (MIC ~ 6.25 to 50 μg mL^-1). These studies provide a concise route to a broad-spectrum antibiotic with a novel chemical scaffold. This journal is

Full text of DOI:10.1039/c4md00572d

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CONCISE ARTICLE
Concise synthesis of spergualin-inspired
molecules with broad-spectrum antibiotic
Cite this: DOI: 10.1039/c4md00572d
activity†
Victoria A. Assimon,a Hao Shao,b Sylvie Garneau-Tsodikovac
ab
*
and Jason E. Gestwicki
There is a growing need to identify new, broad-spectrum antibiotics. The natural product spergualin was
previously shown to have promising anti-bacterial activity and a privileged structure, but its challenging
synthesis had limited further exploration. For example, syntheses of spergualin and its analogs have been
reported in approximately 10 linear steps, with overall yields between 0.3 and 18%. Using the Ugi multi-
component reaction, we assembled spergualin-inspired molecules in a single step, dramatically improving
the overall yield (20% to 96%). Using this strategy, we generated 43 new analogs and tested them for anti-
bacterial activity against two Gram-negative and four Gram-positive strains. We found that the most potent
analogue, compound 6, had MIC values between 4 and 32 μg mL−1 against the six strains, which is a signifi-
cant improvement on spergualin (MIC ~ 6.25 to 50 μg mL−1). These studies provide a concise route to a
broad-spectrum antibiotic with a novel chemical scaffold.
Received 20th December 2014,
Accepted 26th March 2015
DOI: 10.1039/c4md00572d
www.rsc.org/medchemcomm
hydroxyl at carbon 15 and installing an aromatic group at the
11 position (Fig. 1) greatly increased chemical stability.7b
Introduction
Spergualin was first isolated from culture broths of Bacillus
laterosprus in 1981 and shown to have broad-spectrum anti-
bacterial activity.1 This compound has a modular structure
consisting of a guanidino group and a spermidine-like poly-
amine linked through a peptide (Fig. 1).2 Spergualin is struc-
turally distinct from other antibiotics used in the clinic,3
building interest in re-visiting this privileged scaffold. How-
ever, there has been limited exploration of this molecule since
the 1990s.4 One of the major reasons is that the synthesis of
spergualin is protracted. Typical routes produce spergualin
and its analogs in low yield (0.3 to 18%) over at least 10
steps.5 Another issue is the poor chemical stability of
spergualin, which rapidly hydrolyzes in aqueous buffers and
consequently has a short lifetime in vivo.6 Towards these
goals, we recently reported an improved synthetic approach
that features the Ugi multi-component reaction.7 This route
improved the overall yield of spergualin derivatives to between
31 and 47%, while also reducing the number of synthetic
transformations (by 4 or 5 steps) and expanding the scope of
accessible analogs. Further, it was found that removing the
While these initial efforts were informative, we hoped to sup-
port the creation of a greater number of analogues by further
reducing the number of synthetic transformations.
Here, we report a concise route to spergualin-inspired
molecules, most created in a single step in yields between 20
and 96%. Using this approach, we created a library of ~40
analogues and tested each compound for the ability to
inhibit growth of Bacillus anthracis, Bacillus cereus, Bacillus
subtilis, Escherichia coli, Haemophilus influenzae, and Staphy-
lococcus aureus. We found that the most potent molecule,
compound 6, had broad-spectrum, anti-bacterial activity, with
minimum inhibitory concentration (MIC) values between 4
and 32 μg mL−1. This activity is significantly better than the
natural product (MIC values between 6.25 and 50 μg mL−1).
These studies introduce an improved synthetic route and ini-
tial structure–activity relationships, guiding the design of
more potent, broad-spectrum antibiotics.
Results and discussion
Studies of spergualin as an antibiotic are hindered by the
poor overall yield and limited reaction scope in previous syn-
theses, as well as the rapid hydrolysis of the products in basic
buffers. The stability issue has previously been overcome
through removing the hydroxyl at position 15, to produce
15-deoxyspergualin (15-DSG),5 and installing a bulky group
at position 11 (Fig. 1).7b However, the overall yields are still
a Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109, USA
b Department of Pharmaceutical Chemistry, University of California at San
Francisco San Francisco, CA 94158, USA. E-mail: jason.gestwicki@ucsf.edu
c Department of Pharmaceutical Sciences, University of Kentucky, Lexington,
KY 40506, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4md00572d
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purified yields ranged from 20 to 96% (see the ESI† for syn-
thesis and characterization), representing a dramatic increase
in overall yield compared to previous reports.
To explore the antibacterial activity of these compounds,
we tested them for the ability to suppress bacterial growth
using a 96-well, OD600 turbidity platform. Each library member
was initially screened at a single concentration (200 μM) in
triplicate against six bacterial strains, including two Gram-
negatives (E. coli and H. influenzae) and four Gram-positives
(B. anthracis, B. cereus, B. subtilis, and S. aureus). We defined
the negative control as the growth of each bacterial strain in
the presence of 5% DMSO and the positive control was 200
μM ampicillin (Fig. 2a and S1†). Compounds that decreased
turbidity relative to DMSO were considered “active”. We found
that 21 molecules were active against at least one of the six
bacterial strains. B. anthracis was the most susceptible, while
only two compounds were active against E. coli (Fig. 2b).
The ten molecules with activity against both Gram nega-
tives and positives (Fig. 2c) were then subject to confirmatory
re-testing to determine their minimum inhibitory concentra-
tion (MIC) values. Only two compounds (6 and 12) had MIC
values < 256 μg mL−1 against all six bacterial strains
(Fig. 3a, b). The best of these, compound 6, had MIC values
between 4 and 32 μg mL−1. These MIC values are even better
than those of spergualin (MIC values 6.25 to 50 μg mL−1), so
it was chosen for further study (Fig. 3c). This compound was
first re-synthesized and its activity confirmed in the MIC
assay (see ESI†). Next, we tested compound 6 in liquid broth
cultures (Fig. 3d) and found that it also dose-dependently
inhibited bacterial growth of all six strains in that platform
(Fig. S2 and S3†). In control experiments, we confirmed that
none of the synthetic precursors to compound 6 (i.e. benzyl
amine, 3-mercaptopropionic acid, 4-bromobenzaldehyde or
spermidine, an alkyl polyamine) were active (Fig. S4†),
suggesting that the functionalized peptide is the relevant
pharmacophore.
To explore the potential structure–activity relationships
(SAR) around compound 6, we purchased structurally similar
molecules (compounds 6a–d) (Fig. S5a†) and tested their
activity against the six strains. None of these analogues were
significantly active (MIC > 128 μg mL−1) (Fig. S5b†), but this
information, combined with the previous results, helped
refine our knowledge of the pharmacophore. Specifically, the
minimal pharmacophore appears to be the peptide region
with pendant aromatic groups at the C11 and N12 positions.
Short, flexible alkyl chains terminated with electron-rich
groups, such as thiols and amines, were preferred at either
end (Fig. S5c†). Future work will explore the SAR in more
detail. One goal of those studies will be to explore the effects
of stereochemistry at position 11 on potency, as all the mole-
cules reported here are racemic mixtures. Previous studies
suggested that the stereochemistry at this position has mod-
est effects on anti-tumor activity,9 but this issue needs to be
resolved for antibacterial activity.
Fig. 1 Chemical structure of the natural product, spergualin, as well
as its derivatives. See the text for citations and more information.
un-optimized, with the major losses coming during purifica-
tion and workup. We envisioned a route to spergualin-like
analogs that might improve access (Table 1). Key early stud-
ies showed that benzyl protection of the amine at position 12
dramatically improved the ease of purification without nega-
tively impacting biological activity (data not shown), so we
designed the library to include this feature. Specifically, the
Ugi reaction proceeded through the condensation of
benzylamine with a variety of isocyanides, carboxylic acids
and aldehydes to probe the requirements in the guanidine
(R–), 11 position aromatic (R1–) and polyamine (R2–) regions
(Table 1). Most of the components were commercially avail-
able or accessible in a single step.
The individual components were combinatorially assem-
bled to generate a library of 43 molecules (Table 1). Briefly,
benzylamine (1 equiv.) and an aldehyde (1 equiv.) were mixed
in methanol at room temperature until imine formation was
detected by thin layer chromatography (~30 minutes). The
reaction was then purified by column chromatography on sil-
ica gel using a hexane and ethyl acetate gradient, resulting in
compounds 1 and 13–43. Compounds 2–12, which were
derived from tert-butyl IJ4-isocyanobutyl) carbamate,8 were
first subjected to a boc deprotection prior to purification by
column chromatography on basic alumina oxide using an
ethyl acetate gradient and methanol gradient. The final
Finally, we wanted to explore whether compound 6 was
bactericidal or bacteriostatic. S. aureus cultures were treated
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Table 1 Synthesis of spergualin-inspired compounds 1–43
Table 1 (continued)
27
28
Compound
R
R1
R2
29
30
31
32
33
1
2
3
4
5
6
7
8
9
34
35
36
37
38
39
40
41
42
43
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
for 24 hours at the MIC value (8 μg mL−1) or 2× the MIC value
(16 μg mL−1) and the resulting samples were plated on solid
agar to count colony forming units (CFUs). At both concen-
trations, compound
6 was clearly bactericidal (>3 log10
decrease in CFUs) (Fig. 4). Taken together, these results sug-
gest that compound 6 is a promising scaffold for further
development.
Conclusion
The natural product spergualin has promising antibacterial
activity, but its cumbersome synthesis and limited stability
have hindered its use. Using a route that features the Ugi
multi-component reaction, we generated a small library of
synthetically tractable analogs and tested them for anti-
bacterial activity. The results showed that short and flexible
alkyl chains terminated with electron-rich groups at either
end of the peptide-based pharmacophore were necessary for
26
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Fig. 2 Identification of spergualin-inspired analogs with broad spectrum anti-bacterial activity. (A) Representative screening data of compounds
against S. aureus and E. coli in a bacterial growth assay. Dotted lines represents the average growth and standard deviations in presence of the
controls. Results are the average of triplicates and the error bars represent SEM. See the ESI† for the results from other strains. (B) Distribution of
screening hits among the six different bacteria tested. (C) Venn diagram depicting the distribution of screening hits amongst Gram negative and
Gram positive bacteria.
activity. Most notably, we found that compound 6 had partic-
ularly promising broad-spectrum, anti-bacterial activity
against six strains. The concise and convergent assembly of
compound 6 is expected to accelerate discovery of anti-
bacterial molecules.
dramatic changes to the pharmacophore may not be toler-
ated. Regardless, the improved route and preliminary SAR
provided here warrant further exploration.
Experimental section
The mechanism-of-action (MOA) of spergualin is not yet
known and its targets in bacteria are unclear, so these mole-
cules could additionally serve as useful chemical probes. To
this point, MOA studies with spergualin have not been practi-
cal because of its poor stability and its tendency to rapidly
hydrolyze. Compound 6 represents a chance to possibly iden-
tify the target responsible for broad spectrum, bactericidal
activity. This next step will be particularly powerful because
the structure of compound 6 has significantly diverged from
that of the natural product. After the MOA and target are
identified, it will be essential to re-explore whether com-
pound 6 and its analogs share the same profile as spergualin.
Finally, although the activity of compound 6 is promising,
the potency of this series needs to be further improved.
Because MIC values of 4 μg mL−1 were obtained in a relatively
small library of ~40 analogs, we are optimistic about the pos-
sibility of additional potency after more medicinal chemistry.
However, the relatively narrow scope of the SAR suggests that
Synthesis of tert-butyl IJ4-isocyanobutyl) carbamate
The preparation of this isocyanide was based on literature
precedent.8 Specifically, to a 500 mL 3-necked RBF equipped
with an addition funnel and purged with N2IJg) was added
1,4-diaminobutane (15 g, 170.2 mmol) dissolved in 60 mL of
dioxane. Using the addition funnel, boc anhydride (3.7 g,
17.02 mmol) dissolved in 60 mL of dioxane was added
dropwise over 1.5 h. The addition of boc anhydride resulted
in the formation of a white precipitate. The reaction was
allowed to stir overnight at RT. The next morning, the solvent
was removed the under vacuum resulting in a white solid to
which 100 mL of water was added. The resulting insoluble
material was removed by gravity filtration. The filtrate was
then extracted with DCM (3 × 100 mL). The organic layers
were dried with anhydrous sodium sulfate and concentrated
to give N-boc-1,4-diaminobutane, a light yellow oil, in 80–
89% yield. Next, to a 100 mL RBF purged with N2IJg) was
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Fig. 3 Compound 6 has broad-spectrum antibacterial activity. (A) Summary of the screening campaign. From the screens, we focused on com-
pounds 6 and 12 as being active against all six strains. (B) Chemical structures of lead compounds, 6 and 12. (C) Minimum inhibitory concentrations
(MICs) of compounds 6 and 12 against six bacterial strains, compared to the natural product, spergualin. MIC assay were performed at least twice
in triplicate. (D) Compound 6 shows a dose-dependent inhibition of bacterial growth. Growth curve experiments were performed twice in tripli-
cate. Representative graphs are shown.
Fig. 4 Compound 6 has a bactericidal mode of action. (A) Representative time-kill spot titer plates of S. aureus in the presence of compound 6 (0,
8, and 16 μg mL−1) over time (0 and 4 hours). (B) Representative time-kill curves of S. aureus in the presence of 0, 8, and 16 μg mL−1 of compound 6.
added N-boc-1,4-diaminobutane (2.8 g, 14.9 mmol) diluted in
25 mL of DCM. The reaction was then cooled using an ice
bath. Once cooled, DIC (2.3 mL, 14.9 mmol) was to added
dropwise and the resulting white mixture was to allowed stir
overnight at RT. The next morning, the reaction was
subjected to gravity filtration. The resulting filtrate was
washed 2 × 50 mL with saturated sodium bicarbonate. The
organic layers were pooled, dried with anhydrous sodium sul-
fate, and concentrated to give the intermediate tert-butyl
IJ4-formamidobutyl)carbamate. Next, in a 100 mL RBF purged
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with N2IJg) was added tert-butyl IJ4-formamidobutyl)carbamate
(1 g, 4.6 mmol) dissolved in 10 mL of DCM and TEA (1.9 mL,
13.8 mmol). This reaction was cooled using an ice bath. Once
cooled, phosphoryl chloride (0.44 mL, 4.6 mmol) was added
dropwise, causing the reaction mixture to turn orange. This
mixture was allowed to stir at RT for 30 minutes. Afterwards,
potassium carbonate (6.4 g, 4.6 mmol) dissolved in water was
added dropwise and the reaction was allowed to stir for an
additional 30 minutes. The reaction was then transferred to
separatory funnel, the organic layer was removed and saved.
The remaining aqueous layer was extracted with 5 × 20 mL of
DCM. The combined organic layers were dried with anhy-
drous sodium sulfate and then concentrated under vacuum.
This crude product was purified by column chromatography
on silica gel using a hexane : ethyl acetate gradient. The puri-
fied product eluted at 50 : 50 hexane : ethyl acetate. Solvent
removal resulted in a yellow oil in 20–43% yield. 1H NMR
(500 MHz, chloroform-d) δ 4.65 (s, 1H), 3.42 (t, J = 6.5 Hz,
1H), 3.16–3.13 (m, 2H), 1.74–1.68 (m, 2H), 1.65–1.59 (m, 2H),
1.43 (s, 9H).
General synthesis of analogs
In a 50 mL RBF, benzylamine (1 mmol), an aldehyde
(1 mmol), and 5 mL of methonal were mixed at RT until
imine formation was detected by thin layer chromatography
(~30 minutes). Next, a carboxylic acid (1 mmol) and an iso-
cyanide (1 mmol) were added and allowed to react overnight.
This reaction was then purified by column chromatography
on silica gel using a hexane and ethyl acetate gradient,
resulting in compounds 1 and 13–43. Molecules 2–12, com-
pounds that contained the tert-butyl IJ4-isocyanobutyl) carba-
mate starting material, were then subjected to
a boc
deprotection before purification. Briefly, the boc protected
peptoid was dissolved in DCM (10 mL) and treated with 85%
phosphoric acid (3 equiv.). This mixture was allowed to stir
overnight at RT. Afterwards, 10 mL of water was added and
then the reaction mixture's pH was neutralized using 10%
NaOH. This mixture was quenched with saturated sodium
bicarbonate and then extracted 3 × 10 mL with ethyl acetate.
The combined organic layers were dried, concentrated, and
then subjected to column chromatography on basic alumina
oxide using an ethyl acetate gradient and methanol gradient.
Compound yields ranged from 20–96%. See the ESI† for addi-
tional details and characterization.
General synthesis for guanidylated acids
The preparation of guanidylated acids was based on literature
precedent.10 To a 3-necked RBF equipped with a condenser and
N2IJg) inlet, was added either pentanoic, hexanoic, or octanoic
acid (1 mmol). The flask was purged with N2IJg). Afterwards,
8 mL of anhydrous DCM was added and the flask was heated
to 55–60 °C using an oil bath. Once heated, N-methyl-N-
IJtrimethylsilyl)trifluoroacetamide (0.4 mL, 2.2 mmol) was added
dropwise. The resulting cloudy mixture was allowed to reflux
for 2 h. Afterwards, the reaction was removed from heat and
was allowed to cool to RT. TEA (0.15 mL, 1.1 mmol) was added,
Laboratory growth and maintenance of bacterial strains
The following bacterial strains were used: Bacillus anthracis
34F2 Sterne, Bacillus cereus ATCC 11778, Bacillus subtilis 168,
Escherichia coli K-12 (MG1655), Haemophilus influenza ATCC
51907, and Staphylococcus aureus RN4220. H. influenzae was
grown in Brain Heart Infusion (BHI) media supplemented with
hemin and β-nicotinamide adenine dinucleotide hydrate.11
Broth cultures of H. influenzae were prepared by scraping bacte-
ria from agar plates and suspending into fresh supplemented
BHI medium to the desired OD600. All other bacterial strains
were grown in Luria–Bertani (LB) medium. Inoculum for liquid
culture assays was prepared by diluting an overnight LB broth
culture, grown and 37 °C with shaking (200 rpm), into fresh liq-
followed
by 1,3-di-boc-2-IJtrifluoromethylsulfonyl)guanidine
(0.430 g, 1.1 mmol) and an additional 2 mL of DCM. The
reaction flask was re-purged with N2IJg) and allowed to stir
for 4–5 h at RT. During this time the reaction mixture clari-
fied. Afterwards, the reaction was washed in the following
manner: 2 × 8 mL of brine, 1 × 8 mL water, and 1 × 8 mL 10%
citric acid. The combined organic layers were dried with anhy-
drous sodium sulfate and then concentrated under vacuum.
This crude product was purified by column chromatography
on silica gel using a hexane: ethyl acetate gradient. The puri-
fied product eluted at 50: 50 hexane : ethyl acetate. IJZ)-5-IJ2,3-
bisIJtert-butoxycarbonyl)guanidino)pentanoic acid. White solid
in 48.3% yield. 1H NMR (400 MHz, chloroform-d) δ 8.48 (s,
1H), 3.48 (s, 2H), 2.41 (t, J = 6.9 Hz, 2H), 1.75–1.59 (m, 4H), 1.49
(s, 18H). IJZ)-6-IJ2,3-bisIJtert-butoxycarbonyl)guanidino)hexanoic
acid. White solid in 41% yield. 1H NMR (400 MHz, chloro-
form-d) δ 8.36 (s, 1H), 3.41 (q, J = 8.0 Hz, 2H), 2.34
(t, J = 7.4 Hz, 2H), 1.71–1.61 (m, 2H), 1.61–1.54 (m, 2H),
1.48 (d, J = 2.7 Hz, 18H), 1.44–1.34 (m, 2H). IJZ)-8-IJ2,3-bisIJtert-
butoxycarbonyl)guanidine)octanoic acid. White solid in 63%
yield. 1H NMR (400 MHz, chloroform-d) δ 8.28 (s, 1H), 3.36
(q, J = 7.2 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.65–1.57 (m, 2H),
1.56–1.50 (m, 2H), 1.47 (d, J = 2.4 Hz, 18H), 1.31 (s, 6H).
uid medium to the desired OD600
.
Antibacterial screening assay
Bacterial inoculum of each strain was prepared to an OD600
of 0.1 as described above. Next, 100 μL of each dilute culture
was added in triplicate to a sterile non-treated CytoOne 96-
well clear bottom plate. To each well, was added 5 μL of
either compound in DMSO or DMSO alone. The final concen-
tration of compound was 200 μM and the concentration of
DMSO was 5%. The plates were covered and incubated at 37
°C with shaking (200 rpm) for 6 to 7 hours. Afterwards, bacte-
rial growth was recorded by measuring OD600 using a
SpectraMax M5 plate reader.
Minimum inhibitory concentration (MIC) assay
MIC experiments were performed using the double dilution
method. Briefly, inoculum of each strain was prepared to an
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OD600 of 0.1 and 200 μL of each dilute culture was added to a Acknowledgements
sterile non-treated CytoOne 96-well clear bottom plate. To the
V. A. A. was supported by a pre-doctoral fellowship from the
NIH (F31 AG043266-02). J. E. G. acknowledges the financial
support of an NSF CAREER award. We thank Dr. Phil Hanna
(University of Michigan) for providing Bacillus anthracis. We
also thank Dr. Xiaokai Li (University of California at San
Francisco) for assistance in preparing this manuscript.
plated dilute cultures, was added 10 μL of compound from a
2-fold dilution series. The final concentrations of the com-
pounds were in the range of 256 to 2 μg mL−1. Plates were
covered and incubated at 37 °C with shaking (200 rpm) for
24 hours before MIC values were determined. All experiments
were performed at least twice in triplicate.
Bacterial growth assay in liquid culture
References
Bacterial cultures were prepared to an OD600 of 0.1 and 200
μL of each dilute culture was added to the wells of sterile
non-treated CytoOne 96-well clear bottom plates. Compounds
(10 μL) from a 2-fold dilution series were then added to a
final concentration between 32 and 1 μg mL−1. Plates were
covered and incubated at 37 °C with shaking (200 rpm). Bac-
terial growth was recorded every 30 minutes by measuring
OD600 using a SpectraMax M5 plate reader. All experiments
were performed at least twice in triplicate.
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Time-kill studies were performed using 5 mL cultures of S.
aureus (OD600 = 0.1) treated with either 250 μL of compound
6 or DMSO. Final compound concentrations were 8 to 16 μg
mL−1. Samples (100 μL) of each culture were removed after 0,
4, and 24 hours, serially diluted 10-fold in sterile phosphate
buffered saline, and spotted (2 μL) on LB agar plates. Finally,
colonies were counted after incubation for 24 hours at 37 °C.
Compound 6 analogues
Compound 6a IJ4-cyano-N-IJ2-oxo-2-IJpiperazin-1-yl)ethyl-N-IJ3-
phenylpropyl)benzamide)) was purchased from ChemDiv. Com-
pound 6b IJN-IJ2-IJbenzylamino)-2-oxoethyl)-4-isopropylcyclohexane-
1-carboxamide) was purchased from Vitas-M Laboratory. Com-
pound 6c IJN-benzyl-2-IJ2-IJisopropylthio)acetamido)acetamide)
and compound 6d IJN-IJ2-IJ4-benzylpiperazin-1-yl)-2-oxoethyl)-
methylbutanamide) were purchased from Enamine. Mass
spectrometry was used to validate the identity of each pur-
chased compound (see the ESI†).
11 J. W. Johnston, Unit 6D 1, Current Protocols Microbiology,
2010, ch. 6.
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