N-Benzyl-3-sulfonamidopyrrolidines as novel inhibitors of cell division in E. coli

Article abstract of DOI:10.1016/j.bmcl.2007.09.010

A new small molecule inhibitor of bacterial cell division has been discovered using a high-throughput screen in Escherichia coli. Although the lead screening hit (534F6) exhibited modest inhibition of the GTPase activity of FtsZ (20 ± 5% at 100 μM of compound), a primary target for bacterial cell division inhibitors, several analogs caused potent bacterial growth inhibition with negligible antagonism of FtsZ GTPase activity. A library of analogs has been prepared and several alkyne-tagged photoaffinity probes have been synthesized for use in experiments to elucidate the primary target of this compound.

Full text of DOI:10.1016/j.bmcl.2007.09.010

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6651–6655
N-Benzyl-3-sulfonamidopyrrolidines as novel inhibitors
of cell division in E. coli
Shubhasish Mukherjee,a, Carolyn A. Robinson,b Andrew G. Howe,a,à Tali Mazor,a,à
Peter A. Wood,a,à Sameer Urgaonkar,a Alan M. Hebert,c,§
Debabrata RayChaudhurid,* and Jared T. Shawa,*,–
aBroad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA
bDepartment of Systems Biology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA
cInstitute of Chemistry and Cell Biology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA
dDepartment of Molecular Biology and Microbiology, Tufts University School of Medicine,
136 Harrison Avenue, Boston, MA 0211, USA
Received 11 August 2007; revised 29 August 2007; accepted 4 September 2007
Available online 7 September 2007
Abstract—A new small molecule inhibitor of bacterial cell division has been discovered using a high-throughput screen in Esche-
richia coli. Although the lead screening hit (534F6) exhibited modest inhibition of the GTPase activity of FtsZ (20 5% at
100 lM of compound), a primary target for bacterial cell division inhibitors, several analogs caused potent bacterial growth inhi-
bition with negligible antagonism of FtsZ GTPase activity. A library of analogs has been prepared and several alkyne-tagged photo-
affinity probes have been synthesized for use in experiments to elucidate the primary target of this compound.
Ó 2007 Elsevier Ltd. All rights reserved.
The emergence of antibiotic-resistant strains of bacteria
has prompted a worldwide effort to seek new avenues
for fighting infectious disease.1 Most antibiotics discov-
ered to date target a narrow range of biochemical pro-
cesses in bacteria.2 FtsZ, the prokaryotic analog of
tubulin,3 has been examined as a potential new target
for antimicrobial chemotherapy. Although FtsZ has
been the primary target for small molecules that inhibit
bacterial cell division,4 it is likely that other proteins
essential for bacterial cytokinesis can also be targeted.5
A high-throughput screen has recently been developed
to identify compounds that cause lethal cell filamenta-
tion in E. coli.6,7 This screen revealed new inhibitors of
FtsZ and at the same time yielded several compounds
that caused cell filamentation without inducing the
SOS response or without significantly inhibiting the
GTPase activity of FtsZ. Herein we describe our preli-
minary SAR studies of 534F6, an N-benzyl-3-sulfonam-
idopyrrolidine (Fig. 1), and our initial preparation of
photoaffinity reagents for the identification of this com-
pound’s protein target(s).
534F6 displayed weak inhibition of FtsZ GTPase
(20 5% at 100 lM compound, 4 lM protein), did not
affect steady-state FtsZ polymerization as assayed by
high-speed sedimentation, and induced SOS-indepen-
Keywords: Bacterial cell division; Antimicrobial compounds; Library
synthesis.
*
Corresponding authors. Tel.: +1 5307529979; fax: +1 5307528895
(J.T.S.); tel.: +1 617 636 383; fax: +1 617 636 3007 (D.R.); e-mail
addresses: debu.raychaudhuri@tufts.edu; shaw@chem.ucdavis.edu
Present address: Hudson Alpha Institute for Biotechnology, 127
Holmes Avenue Huntsville, AL 35801, USA.
O
S
H3C
CH3
- MIC = 10 μM
O
H3C
NH
- IC50 (FtsZ) > 100 μM
- extensive filamentation
observed in primary screen
O
à
Undergraduate research associate.
§
N
Present address: Center for Cancer Research, Massachusetts General
Hospital and Department of Pathology, Harvard Medical School,
Charlestown, MA 02129, USA.
F
534F6
–
Figure 1. Lead compound (534F6) from phenotypic HTS for
compounds that induce lethal filamentation in E. coli.
Present address: Department of Chemistry, University of California,
One Shields Avenue, Davis, CA 95616, USA.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.010

6652
S. Mukherjee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6651–6655
Table 1. GTPase Inhibition and antimicrobial activities of 5a–i
dent E. coli cell filamentation (data not shown). Despite
a certain degree of similarity to sulfonamide antibiotics,
such as sulfamethoxazole, compound 534F6 exhibited
markedly different effects on E. coli. Sulfamethoxazole
showed modest lethality (MIC > 80 lM) and did not
cause E. coli (AcrAB efflux pump knockout strain
DRC 39)4d to filament. Based on these observations,
we set out to develop a library synthesis of 534F6 in
an effort to optimize potency and eventually determine
the protein target of this compound.
R3
O
S
O
S
R2
R1
R3
O
O
H3C
H3C
NH
NH
R2
R1
OHC
(1)
N
NH
NaBH(OAc)3
3
5a-h
F
F
R
i-Bu
NH
O
#
9
CHO
NH
O
10 Ph
11 OPh
12 CF3
13 Br
OHC
OHC
OHC
6
7
8
R
Oi-Pr
We began by investigating the SAR of 534F6. Since the
configuration of this compound was unknown, we pre-
pared each of the two enantiomers as a test for the influ-
ence of stereochemistry on activity. Although no
synthesis of 534F6 had previously been reported, we
were able to convert commercially available (S)-1 and
(R)-1 into (S)- and (R)-534F6, respectively, in three steps
(Scheme 1). We were delighted to find that the (R)-enan-
tiomer caused lethal E. coli filamentation with an MIC
of 10 lM, whereas the (S)-enantiomer neither induced
cell filamentation nor killed E. coli up to 80 lM. This re-
sult suggests that 534F6 is reasonably selective in its
interaction with its target or targets.
O
S
O
S
O
O
H3C
H3C
NH
NH
HO2C
EDCI; 51%
Oi-Pr
(2)
N
O
NH
F
3
5i
F
Entry Aldehyde Product
(% yield) at 100 lMa (%)
FtsZ GTPase inhibition MICb
(lM)
1
2
3
4
5
6
7
8
9
9
10
11
8
5a (87)
5b (90)
5c (93)
5d (86)
5e (83)
5f (72)
5g (83)
5h (94)
5i
13.7
9.4
<10
<10
20
12.3
14.6
24.9
26.9
29.9
31.5
31.9
>80
>80
>80
>80
>80
>80
7
12
6
Using our synthetic route to 534F6, we were able to pre-
pare an initial series of analogs to establish the influence
of the N-benzyl substituent. Intermediate 3 was con-
densed with a series of aromatic aldehydes to produce
a series of analogs featuring different ortho-, meta-,
and para substituents (eq 1). As Table 1 shows, intro-
duction of fused rings or polar para substituents was
deleterious to the activity of these compounds. Replace-
ment of the isopropoxy group with either the isosteric
isobutyl group, a phenyl ring, or a phenoxy group re-
tained activity. Replacement of the benzyl amine with
a benzoyl amide greatly diminished the antimicrobial
activity of this compound (eq 2).
13
—
a Four micromolar of FtsZ was used in the GTPase assays (see Ref.
4d). Values shown are % of the GTPase activity in DMSO-containing
control reactions.
b AcrAB efflux pump-deficient E. coli DRC 39 was used (see Ref. 4d).
was employed in Iroritm kans and the synthesis was
tracked using 2D-barcoding.9 The first step of the syn-
thesis was a reductive amination with a protected amine
that would later be functionalized. We initially explored
phthaloyl (phth) and tetrachlorophthaloyl (tcph) pro-
tecting groups for the primary amine, but found the for-
mer to be preferable once the conditions were adjusted
to account for the precipitated phthalyl hydrazide. In
order to explore the structural elements that might con-
tribute to activity, we prepared protected amine cores
A1–A7. These were condensed onto the aldehyde start-
ing material in six different reaction batches, then pooled
for deprotection, and split for attachment of the sulfonyl
groups. A series of sulfonyl chlorides were employed in
the last step to yield a total of 210 compounds after
cleavage. The compounds were tested for growth inhibi-
tion and cell filamentation in E. coli. None showed im-
proved activity and the majority appeared to be less
potent than 534F6. Although we expected diminished
activity from the hydroxyethoxy substituent, we had
hoped that an optimized core and sulfonyl substituent
would compensate.
Based on these preliminary results, we developed a solid
phase synthesis of 210 analogs of 534F6, mindful of the
importance of the N-benzyl substituent. Since the substi-
tuent at the para position had proven to be crucial for
activity, we elected to keep this structural feature invari-
ant. We replaced the isopropoxy group of 534F6 with a
hydroxyethyl group as a point of attachment to solid
phase synthesis resin.8 Approximately 35 mg of resin
O
S
O
S
O
H3C
O
CH3
1) pyridine
23 ºC, 12 h
Cl
NH
H2N
H2N
Bn
Bn
N
N
2) H2, Pd/C, HOAc
23 ºC, 12 h
NH
(S)-1
2
F
F
3
Oi-Pr
steps 1-3
NaBH(OAc)3
DCE, 23 ºC
12 h
(R)-1
OHC
4
We next focused on a narrower selection of core
structures with a single benzyl substituent. Amine core
structures A2, A4, and A6 were selected and each was
N-benzylated using p-isobutylbenzyaldehyde and
NaBH(OAc)3. These three amines were converted in
parallel to the corresponding sulfonamides using excess
quantities of sulfonyl chlorides B1–B15 and a scavenger
O
S
O
O
H3C
O
H3C
S
NH
NH
Oi-Pr
Oi-Pr
N
N
F
F
(R)-534F6
(S)-534F6
Scheme 1. Synthesis of the enantiomers of 534F6.

S. Mukherjee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6651–6655
6653
Solid phase, split-pool synthesis of Library 1:
o- or m- linkage
210 compounds
HO
O
1) H2NNH2 or
O
N
A1-A7
EtSH/DBU (for NNos)
R
i-Pr
i-Pr
ZO
N
S
X
n
N
m
O
Si
O
O
NaBH(OAc)3
2) B1-B15, Et3N
3) HF-Pyr
H
O
X
NH
O
*
n
m
O
= Z
n, m = 0, 1; X = CH or NNos [NSO2(o-NO2C6H4)]
Solution pase, scavenger-based synthesis of Library 2:
H
N
O
O
45 compounds
O
1) p-(i-Bu)C6H4CHO, NaBH(OAc)3
2) H2NNH2
O
S
R
O
S
H
S
R
O
NH
i-Bu
i-Bu
i-Bu
A2, A4, A6
R
N
3) B1-15, PS-N(i-Pr)2;
PS(NHCH2CH2)2-NH2
N
N
N
Building Blocks A1-A7
Building Blocks B1-B15
R1
O
O
#
R1 R2 R3
R4
O
O
R2
R3
S
Cl
S
NPhth
N
N
Cl
B5
H
H
H
H
H
H
H
H
H
NPhth
PhthN
PhthN
NH
NH
H3C
B6
CH3
H
H
H
H
H
H
H
H
H
H
H
Cl
HN
HN
O
O
B7
CH3
OCH3
Cl
B5-B15
R4
S
A1
A2
A3
A4
B8
H
H3C
i-Pr
Cl
B9
H
B1
B2
O
S
NO2
B10
B11
H
Ph
O
O
O
N
Cl
H
Cl Cl
PhthN
O
O
S
NH
B12 CH3
B13 Cl
H
H
F
F
S
N
CF3
H
HN
NPhth
Cl
HN
B14
B15
H
H
Cl
H
A5
A6
A7
B3
B4
Scheme 2. Solid- and solution-phase synthesis of analogs of 534F6.
resin for removal of the excess reagent.10 These 45
compounds were tested and compound 14 was found
to be the most potent, with an MIC of 10 lM. Examina-
tion of the E. coli culture treated with 5 lM 14 showed
extensive filamentation (Fig. 2A). Compound 15,
featuring the same sulfonamide and benzyl groups on
a different core, exhibited an MIC of 20 lM, but little
filamentation was observed at 10 lM (Fig. 2B)
(Scheme 2).
In a parallel effort, we have prepared several derivatives
of 534F6 for use in target identification. We designed
several compounds that would serve as photoaffinity re-
H
H
OHC
R
BocN
BocN
R
21a: R =
21b: R = N3, 79%
1) HCl, dioxane, 55 ºC, 1 h
i-Bu, 97%
NaBH(OAc)3
DCE, 23 ºC, 12 h
NH
N
20
2)
p-IC6H4SO2Cl, Et3N, CH2Cl2
23 ºC, 12 h
O
S
O
S
O
O
NH
NH
N3
CH3
CH3
N
N
I
I
22b, 50%
22a, 75%
Figure 2. Escherichia coli DRC39 treated for 15 h with (A) 5 lM 14,
(B) 10 lM 15.
1) Pd(OAc)2, Bu4NOAc
DMF, 55 ºC, 3 h
2) TBAF, rt, 2 h
H
Si(
i-Pr)3
O
S
O
S
NH
H3C
CH3
O
O
NH
N3
N
N
23a: R =
i
-Bu, 35%; MIC <12 μM
23b, 37%; MIC >40 μM
H
H
OH
1) NaN3, CuI, EtOH/H2O (7:3)
sodium ascorbate, (CH3NHCH2)2
MW, 100 ºC, 40 min; 81%
CHO
2) PCC, CH2Cl2, 23 ºC, 3 h; 90%
20b
I
N3
Scheme 3. Synthesis of 19, a benzophenone derivative of 534F6.
Scheme 4. Synthesis of 23a and 23b, alkyne-tagged derivatives of 5a.

6654
S. Mukherjee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6651–6655
Scheme 5. Synthesis of 29, an alkyne-tagged and azide-appended derivative of 534F6.
agents to modify their protein targets.11 In addition, we
incorporated terminal alkyne substituents as chemical
tags that would allow us to separate the modified target
from the cellular lysate (Scheme 3).12
agarose in hopes of pulling down the protein target
from a cell lysate of E. coli.
In summary, we have discovered a new compound
(534F6) that appears to inhibit bacterial cell division
without inhibiting FtsZ as the primary target. Initial at-
tempts to prepare alkyne tagged photoaffinity reagents
have revealed regions of the molecule that are not suit-
able for structural variation. We are currently examining
a variety of alternative approaches for identifying the
target of this compound.
We initially explored the possibility of incorporating a
benzophenone group in the para position of the N-ben-
zyl substituent. The requisite 4-formyl benzophenone
(18) was prepared using the palladium coupling reported
by Goossen.13 Reductive amination of 18 yielded 19.
This compound’s weak activity (MIC > 80 lM)
prompted us to explore other options for installing a
photoreactive group (Scheme 4).
Acknowledgments
Our next compound was designed to use an aryl azide as
the photoreactive group. 21a was prepared by reductive
amination of 20. This compound was converted to sul-
fonamide 22a, which was carried on to alkyne 23a. Sul-
fonamidopyrrolidine 23a exhibited an MIC of <12 lM,
indicating that the presence of the alkyne did not affect
the activity. Encouraged by this result, we proceeded
with the synthesis of 23b by a parallel synthetic route.
This synthesis was enabled by the ligand- and copper-
free Sonagashira reaction reported by Verkade,14 which
avoids reduction and cycloaddition of the aryl azide.
Compound 23b exhibited an MIC of >40 lM, confirm-
ing the necessity of a lipophilic group at this site for
activity. The activity of 23a established the viability of
an alkyne on sulfonamide portion of the molecule
(Scheme 5).
J.T.S. thanks the National Institute for Allergy and
Infectious Disease (NIAID, RO3 AI062905) and the
Broad Institute Scientific Planning and Allocation of
Resources Committee (SPARC) for funding of this
research. D.R.C. acknowledges DARPA, the Charles
A. Dana Foundation, and the National Institute of
General Medical Sciences (NIGMS; RO1 GM068025)
for research support. The Hudson-Alpha Institute for
Biotechnology is acknowledged for support of S.M. as
a visiting scientist. The Harvard College Research
Program (HCRP) is acknowledged for a fellowship to
T.M. We thank Profs. Marc Kirschner and Tim Mitch-
ison (Harvard Medical School) for insightful discus-
sions. A portion of this work was conducted in Prof.
Kirschner’s Laboratory. This project has been funded
in part with Federal funds from the NCI’s Initiative
for Chemical Genetics, NIH, under Contract No.
N01-CO-12400. The content of this publication does
not necessarily reflect the views or policies of the
Department of Health and Human Services, nor does
mention of trade names, commercial products or organi-
zations imply endorsement by the U.S. Government.
The activities of 23a and 23b encouraged us to explore
the possibility of a hybrid of these two compounds
with 534F6. Sulfonamide 25 was prepared in two steps
from N-Boc-(R)-3-aminopyrrolidine (20). The Boc
group was removed and the 3-nitro-4-isopropoxy ben-
zyl group was installed by reductive amination.15 The
nitro group was reduced to the corresponding aniline,
which was then diazotized and displaced with azide.
After deprotection with TBAF, compound 29 was
examined for antimicrobial activity and found to have
an MIC of >64 lM. Although installation of the al-
kyne on the sulfonamide only slightly lowered the
activity of 23a relative to 5a, it is apparent that the
combined effect of the ortho azide and the alkyne
greatly diminishes the activity of 29. We are currently
preparing an affinity matrix with 23a using ‘clickable’
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