Structure-activity studies of divin: An inhibitor of bacterial cell division

Article abstract of DOI:10.1021/ml400234x

We describe the synthesis and structure-activity relationship (SAR) studies of divin, a small molecule that blocks bacterial division by perturbing the assembly of proteins at the site of cell septation. The bacteriostatic mechanism of action of divin is distinct from other reported inhibitors of bacterial cell division and provides an opportunity for assessing the therapeutic value of a new class of antimicrobial agents. We demonstrate a convenient synthetic route to divin and its analogues, and describe compounds with a 10-fold increase in solubility and a 4-fold improvement in potency. Divin analogues produce a phenotype that is identical to divin, suggesting that their biological activity comes from a similar mechanism of action. Our studies indicate that the 2-hydroxynaphthalenyl hydrazide portion of divin is essential for its activity and that alterations and substitution to the benzimidazole ring can increase its potency. The SAR study provides a critical opportunity to isolate drug resistant mutants and synthesize photoaffinity probes to determine the cellular target and biomolecular mechanism of divin.

Full text of DOI:10.1021/ml400234x

Letter
pubs.acs.org/acsmedchemlett
Structure−Activity Studies of Divin: An Inhibitor of Bacterial Cell
Division
Maoquan Zhou,† Ye-Jin Eun,† Ilia A. Guzei,‡ and Douglas B. Weibel*,†,‡
†Department of Biochemistry, University of WisconsinMadison, Madison, Wisconsin 53706, United States
‡Department of Chemistry, University of WisconsinMadison, Madison, Wisconsin 53706, United States
S
* Supporting Information
ABSTRACT: We describe the synthesis and structure−
activity relationship (SAR) studies of divin, a small molecule
that blocks bacterial division by perturbing the assembly of
proteins at the site of cell septation. The bacteriostatic
mechanism of action of divin is distinct from other reported
inhibitors of bacterial cell division and provides an opportunity
for assessing the therapeutic value of a new class of
antimicrobial agents. We demonstrate a convenient synthetic
route to divin and its analogues, and describe compounds with
a 10-fold increase in solubility and a 4-fold improvement in
potency. Divin analogues produce a phenotype that is identical to divin, suggesting that their biological activity comes from a
similar mechanism of action. Our studies indicate that the 2-hydroxynaphthalenyl hydrazide portion of divin is essential for its
activity and that alterations and substitution to the benzimidazole ring can increase its potency. The SAR study provides a critical
opportunity to isolate drug resistant mutants and synthesize photoaffinity probes to determine the cellular target and
biomolecular mechanism of divin.
KEYWORDS: Divin, antimicrobial, synthesis, SAR
he development of new antimicrobial agents to combat
assembly of the functional divisome without directly affecting
Tthe emergence of multidrug resistance among clinical FtsZ.9 The unique interaction between 1 and the bacterial
pathogens continues to be a challenge.1 One approach to
divisome makes it a useful chemical probe for studying the
dynamics of bacterial division;11 however, its low solubility and
antimicrobial agent discovery centers upon developing
modest activity limits its application and prevents the
identification of its target.
compounds that bind specifically to a cellular target (e.g.,
DNA gyrase).2,3 Another approach that has been gaining
traction recently is the identification of compounds that have
potent bactericidal or bacteriostatic activity through phenotypic
assays/screens, without considering specific cellular targets per
se.4,5 Membrane-targeting antibiotics represent one class of
compounds that have emerged from this approach.6−8
We recently introduced a new compound, divin (1), that was
discovered using the second track of antibiotic development
mentioned above.9 Divin inhibits late stages of bacterial cell
division and produces mother and daughter cells that remain
physically fused together and share a common cytoplasm.9
Peptidoglycan insertion at the septum is blocked in divin-
treated cells, and the result is a bacteriostatic effect in Gram-
negative and Gram-positive bacteria. In our initial studies, the
low solubility of 1 in culture media prevented us from isolating,
characterizing, and deep sequencing spontaneous mutants that
displayed reduced susceptibility to 1. Rather than focusing on
the identification of the cellular target, we studied how divin
altered the spatial and temporal localization of division proteins
at the divisome and found that four late-stage division proteins
(i.e., FtsQ, FtsL, FtsW, and FtsB) were mislocalized from the
division plane. Unlike the classes of molecules that have been
reported to bind FtsZ and inhibit division,10,11 divin inhibits
In this letter, we lay the groundwork for identifying the target
of 1 by performing a structure−activity relationship (SAR)
study. We demonstrate a convenient synthetic route to divin
analogues and describe compounds with improved solubility
and potency. Divin analogues with enhanced solubility will
enable us to isolate drug resistant mutants and use genomics to
identify the drug target. Alternatively, synthesizing a focused
library of analogues has guided us in the design of a
photoaffinity probe to identify the molecular target of divin
using a pull-down approach.9 These studies lay the groundwork
for identifying the molecular target of divin and the
development of therapeutic agents for treating clinical
infections.
Performing SAR studies required that we develop a flexible
synthetic strategy to manipulate elements of 1 while enabling
maximum diversification of the structure. We developed a
synthetic approach centered upon a sequential, three
component coupling of the benzimidazole, the linker, and the
Received: June 19, 2013
Accepted: July 29, 2013
© XXXX American Chemical Society
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2-hydroxy-1-naphthaldehyde components of divin (Figure
1).12,13 Each of the three components could be modified
independently before they were coupled to produce a range of
analogues for characterization and biological testing.
and as conformational isomers (cis/trans) around the amide
N−C(O) bond.14,15 In our study, we obtained a crystal
structure of 1·EtOH (Scheme 2), which demonstrated that in
Scheme 2. Alternative Synthesis of 1 and Conformational
a
Isomerization
Figure 1. Retrosynthetic strategy for the synthesis of divin and its
analogues.
a
Reagents and conditions: (a) Pd(PPh3)4/PhSiH3, 98%.
To prepare analogues with variations in the bottom region of
the compound, we reacted 2-methyl-1H-benzimidazole 2 with
methyl 3-bromopropionate in the presence of anhydrous
K2CO3 to prepare ester 3 in 96% yield (Scheme 1). We
the solid state the imine of 1 exists in the E configuration and
the N−C(O) existed as trans rotamers (CO anti to the NH).
In solution, the N−C(O) group of 1 interconverts between cis
1
and trans rotamers (Scheme 2). In the H NMR spectra of 1,
Scheme 1. Synthesis of Divin Analogues by Varying the
a
we observed two sets of nuclei resonances for NCH, −CH2−
C(O), and some of the protons in the benzylidene moiety. The
signals belonging to NCH and −CH2−C(O) protons were
observed at 8.89 (trans), 8.80 (cis) ppm and 2.72 (trans), 3.15
(cis) ppm, respectively. The 13C NMR spectra of 1 indicated
two sets of resonances for most of the carbon nuclei. We
Structure of the Naphthalenyl Moiety
1
assigned the two rotamers based on the H downfield shift
(0.43 ppm) of −CH2C(O)− in the cis-isomer (CO syn to
the NH) as compared with trans-isomer and 13C downfield
shift of carbonyl carbon of the cis-isomer (170.71 ppm) relative
to the trans-isomer (CO anti to the NH, 165.95 ppm), which
arises due to the reduced contribution of the conjugative form
N+C−O− present in the cis-isomer.
The two rotamers equilibrated thermally in solution and the
relative percentage varied depending on the polarity of the
1
solvent. We determined the ratio of the two rotamers by H
NMR, as they interconvert too fast to be differentiated by
reverse phase HPLC analysis (Figure S3, Supporting
Information). As the polarity of the solvent increased, we
observed that the percentage of the transC(O)‑N isomer
increased. On the basis of the integrated ratio of the relevant
1
peaks of 1 in the H NMR spectrum, 60% of the compound
was in the trans-isomer at 25 °C in CDCl3/CD3OD (95/5%),
74% in DMSO-d6 (neat), and 83% in D2O/DMSO-d6 (60/
40%).
The isomerization of the other analogues in solution
depended on their structures. In general, analogues containing
a C-2-naphthalenyl or phenyl hydroxyl group (i.e., 5a and 5i)
had an isomerization pattern that was similar to 1 and consisted
of a mixture of transC(O)‑N and cisC(O)‑N isomers in which the
trans-isomer was the major product. Analogues lacking a C-2
naphthalenyl or a free hydroxyl group (i.e., 5b-5i) primarily
existed as the cisC(O)‑N isomer in solution. Analogue 5c
(Scheme 2) contained an allyl-protected hydroxyl group. In
CDCl3/CD3OD (95/5%), 5c primarily existed in the cis-isomer
a
Reagents and conditions: (a) methyl 3-bromopropionate, K2CO3,
DMF, 85 °C, 96%; (b) hydrazine, MeOH, 65 °C, 85%; (c) aldehyde,
AcOH, EtOH, reflux, 79%.
synthesized the key intermediate 3-(2-methyl-1H-benzimida-
zol-1-yl) propanohydrazide 4 by hydrazinolysis of 3. Com-
pounds 1 and 5a−j were prepared by the condensation of 4 and
corresponding aldehydes with yields ranging from 32% to 93%
at a scale of 0.1−0.5 mmol.
1
(84%) according to H NMR measurements. We removed the
allyl protecting group in 5c using Pd(PPh3)4/PhSiH3 and
produced 1 in which the trans-isomer was the major product.
It is well-known that N-acylhydrazones may exist as
geometrical isomers (E/Z) with respect to the imine bonds
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We selected two linkers, one lacking a methylene group and
the other containing an additional methylene group, to explore
the relationship between the length of the linker and the
activity of analogues (Scheme 3). Specifically, we reacted 2-
Scheme 4. Synthesis of Divin Analogues by Varying the
Benzimidazole Moiety
a
Scheme 3. Synthesis of Divin Analogues Containing
a
Different Linker Lengths
a
Reagents and conditions: (a) methyl bromoacetate or ethyl 4-
bromobutyrate, K2CO3, DMF, 85 °C; (b) hydrazine, MeOH, 65 °C;
(c) aldehyde, AcOH, EtOH, reflux.
methyl-1H-benzimidazole (2) with methyl 3-bromoacetate or
ethyl 4-bromobutyrate in the presence of K2CO3 to yield the
corresponding ester 6a and 6b in 43% and 56% yield,
respectively. After hydrazinolysis of 6a and 6b using hydrazine
hydrate, we reacted the resulting hydrazides 7a and 7b with 2-
hydroxy-1-naphthaldehyde to form the analogues 8a and 8b in
88% and 92% yields, respectively.
To efficiently prepare divin analogues containing variations
in the top region of the molecule, the benzimidazole moiety, we
initially attempted to couple the linker to 2-methoxy-1-
naphthaldehyde, followed by alkylation of the product with 2-
methyl-1H-benzimidazole. This strategy was unsuccessful, and
we used the above 3-step sequence to prepare analogues
containing variations to the benzimidazole region of 1. We
alkylated imidazoles (A-I) with methyl 3-bromopropionate to
afford the corresponding esters 9a−h in 40−91% yields
(Scheme 4). Hydrazinolysis of 9a−h using hydrazine hydrate
provided 10a−h in 59−96% yield. We prepared analogues
11a−j by condensing 10a−h with 2-hydroxy-1-naphthaldehyde.
Compounds 9b, 9c, 10b, and 10c were inseparable mixtures
of C-5 and C-6 substituted regioisomers arising from N1/N3
competitive alkylation.16 From these intermediates, we
synthesized analogue 11c as a mixture of regioisomers. Four
a
Reagents and conditions: (a) benzimidazoles, K2CO3, DMF, 85 °C;
(b) hydrazine, MeOH, 65 °C; (c) aldehyde, AcOH, EtOH, reflux.
presence of pyridine at 25 °C, which yielded diester 12 in 45%
yield (Scheme 5). We also reduced 1 to the corresponding
amine 13 in 43% yield.
a
Scheme 5. Other Modifications to the Structure of Divin
1
sets of signals in the H and 13C NMR spectra indicated that
11c existed as both C-5/C-6 regio- and C(O)−N trans/cis
isomers. As expected, the C-5/C-6 dichloro-substituted
analogue 11j produced a single regioisomer. The two
regiosiomers 11d and 11e that we produced from 10c were
separable by flash chromatography. All of the other analogues
existed as a mixture of transC(O)‑N and cisC(O)‑N rotamers in
solution that were similar to 1.
Condensing 4 with 2-acetyloxy-1-naphthaldehyde yielded 1,
presumably due to the instability of C-2 phenol ester. To
acylate the phenol group of 1, we treated 1 with Ac2O in the
a
Reagents and conditions: (a) Ac2O/Pyr., 45%; (b) NaCNBH3,
MeOH, 43%.
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Treating Gram-negative infections can be difficult as the
outer membrane reduces the bioavailability of antibiotics. We
hence decided to screen analogues of 1 against Gram-negative
bacterial strains. We determined the minimum inhibitory
concentration (MIC) of each analogue against the model
Gram-negative bacteria Caulobacter crescentus CB15N (wild
type) and Escherichia coli BW25113 ΔtolC using a CLSI
protocol based on serial dilution;17,18 E. coli BW25113 ΔtolC
lacks a central component of one family of efflux drug pumps,
and the genetic modification increases the susceptibility of cells
to antibiotics. The results are listed in Table 1.
change the MIC significantly; the only exception was the 3-fold
reduction in MIC of the CF3 analogue 11a against E. coli
BW25113 ΔtolC. Analogue 11a had an MIC that was nearly
identical to 1 against C. crescentus CB15N. The benzene ring in
the benzimidazole group was not essential, as removing it (i.e.,
11g) did not change the MIC significantly. Introducing new
substituents on the benzene ring improved the MIC of
analogue (i.e., 11c, 11e, and 11j) against E. coli BW25113
ΔtolC; the alterations did not change the activity against C.
crescentus CB15N. The dichloro analogue 11j had a MIC
against E. coli BW25113 ΔtolC that was 2-fold lower than 1.
A key goal of this study was to introduce structural
modifications that improved the solubility of 1 without affecting
its activity. We determined the solubility of a set of divin
analogues in M8 media (with 2% DMSO) using a light
scattering assay.19,20 M8 is the media we used to study the
activity of divin and analogues against E. coli BW25113 ΔtolC
cells and a serial of clinic pathogenic strains of bacteria. The
results indicated that some modifications led to large
improvements in solubility while maintaining the biological
activity of the analogues (Table 2). Compound 8b contained a
Table 1. MIC of Divin Analogues against C. crescentus
CB15N (Wild Type) and E. coli BW25113 ΔtolC Cells; MIC
Values are Reported in μM
MIC
MIC
a
b
a
b
compd
CR
EC
compd
CR
7.5
EC
1
5
12.5
50
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
12
>50
>50
7.5
5a
5b
5c
5d
5e
5f
15
>20
5
>20
>20
>20
>20
>20
>20
>20
5
>50
>50
50
10
5
25
Table 2. Solubility of Active Divin Analogues in M8 Media
(with 2% DMSO); Solubility is Expressed as μM
12.5
12.5
12.5
12.5
12.5
6.3
>50
>50
>50
>50
12.5
>50
25
7.5
7.5
5
compd
solubility
compd
solubility
5g
5h
5i
1
20
11f
11g
11h
11i
11j
30
10
90
20
90
5
8a
20
3.8
>40
40
8b
120
30
5j
>20
5
50
11a
11c
11e
8a
8b
13
50
210
90
5
10
a
b
C. crescentus CB15N cells. E. coli BW25113 ΔtolC cells.
linker that was longer than 1, and the analogue was 6 times
more soluble than 1 in M8 media. Introducing a chloride into
the benzimidazole ring (11c) increased the solubility 10-fold
compared to 1.
We found that removing, alkylating, or acylating the phenyl
hydroxyl group of divin reduced the activity of analogues (i.e.,
5d, 5b, 5c, and 12), thereby indicating the importance of this
moiety on the activity of the compound. Changing the position
of the phenyl hydroxyl to the C-4 position (i.e., 5e) or replacing
the C-4 hydroxyl group with a dimethylamino or fluoro group
(i.e., 5f and 5g) dramatically reduced the biological activity.
The second aromatic ring of the naphthalenyl moiety was
essential for the activity of compounds. Removing the second
aromatic ring resulted in inactive analogues (i.e., 5a and 5h).
Adding a hydroxyl group to the second ring (i.e., 5i) did not
affect the activity.
We found that the imine bond was essential for the activity of
1. Reducing the imine group (i.e., 13) increased the MIC. The
hydrogen on the imine group was required for activity, as
replacing it with a methyl group (i.e., 5j) produced inactive
analogues. We found that the length of the alkyl linker is not
important for the activity of 1; analogues with a shorter (8a) or
longer linker (8b) had the same MIC as 1 against C. crescentus
CB15N. Analogue 8b was slightly more active against E. coli
BW25113 ΔtolC than 1, while 8a was ∼2-fold less active than
1.
We previously demonstrated that 1 is a bacteriostatic agent
by measuring the time-dependent viability of bacterial cells
treated with 1.9 Treating C. crescentus CB15N cells with 1 at its
MIC (5 μM) arrested cell division after the initiation of the
midcell constriction (Figure 2). To evaluate if the most active
analogues had the same phenotypic effect as 1 on cells, we
imaged C. crescentus CB15N cells treated with divin analogues
(at a concentration equivalent to the MIC) after 17 h of
incubation (Figure 2). All of the compounds produced the
same phenotype, which suggests that they share the same
mechanism of action as 1. We measured the time-dependent
viability of C. crescentus CB15N cells treated with 11c and 11j
(Figure S1, Supporting Information). The results indicated that
analogues 11c and 11j are bacteriostatic, which is similar in
mechanism to 1.9
We also explored the efficacy of 1 and 11j against a panel of
clinically relevant strains of bacteria (Table 3). For these
experiments, our choice of 11j versus 8b or 11c was primarily
due to its improved activity. Compounds 1 and 11j inhibit the
growth of Shigella boydii, Enterobacter aerogenes, Vibrio cholera,
and other clinical pathogens. Analogue 11j has a particularly
low MIC (3 μM) against V. cholera and a 4-fold increase in
potency against E. aerogenes compared to 1.
The benzamidazole ring was the region of divin that was
most tolerant to modification; a range of alterations to the
benzamidazole group led to small or no changes in the MIC of
the corresponding analogues. The methyl group on the
benzimidazole ring was not critical to the activity of 1. We
found that removing or substituting the methyl group with CF3,
CH2N(Me)2, or CH2N3 (i.e., 11f, 11a, 11h, and 11i) did not
In this letter, we describe the synthesis of a set of divin
analogues and an SAR analysis of these compounds against cells
of C. crescentus CB15N and E. coli BW25113 ΔtolC. These
D
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
All experimental details, H and 13C spectroscopic data, and
other related material. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(D.B.W.) Phone: 608-890-1342. E-mail: weibel@biochem.
wisc.edu..
Funding
We acknowledge financial support from the University of
WisconsinMadison Graduate School (Senator Robert
Caldwell Graduate Fellowship to Y.-J.E), the Alfred P. Sloan
Foundation (fellowship to D.B.W.), NIH (grant
1DP2OD008735-01), the Wisconsin Alumni Research Foun-
dation, and the Human Frontiers Science Program (grant
RGY0069/2008-C103).
Notes
The authors declare no competing financial interest.
Figure 2. Divin analogues produced the same cellular phenotype with
Gram-negative bacteria as did 1. This figure shows panels of C.
crescentus CB15N cells treated with different analogues and DMSO
control: (a) 1; (b) 8b; (c) 11c; (d) 11e; (e) 11f; (f) 11g; (g) 11i; (h)
11j; and (i) DMSO.
ACKNOWLEDGMENTS
■
We thank Regan Thomson for insightful comments.
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