Design and synthesis of C-2 substituted thiazolo and dihydrothiazolo ring-fused 2-pyridones: Pilicides with increased antivirulence activity

Article abstract of DOI:10.1021/jm100470t

Pilicides block pili formation by binding to pilus chaperones and blocking their function in the chaperone/usher pathway in E. coli. Various C-2 substituents were introduced on the pilicide scaffold by design and synthetic method developments. Experimenta

Full text of DOI:10.1021/jm100470t

5690 J. Med. Chem. 2010, 53, 5690–5695
DOI: 10.1021/jm100470t
Design and Synthesis of C-2 Substituted Thiazolo and Dihydrothiazolo Ring-Fused
2-Pyridones: Pilicides with Increased Antivirulence Activity†
Erik Chorell,‡ Jerome S. Pinkner,§ Gilles Phan, Sofie Edvinsson,‡ Floris Buelens, Han Remaut, ,^ Gabriel Waksman,
Scott J. Hultgren,*,§ and Fredrik Almqvist*,‡
‡
§
Department of Chemistry, Umea University, SE-90187 Umea, Sweden, Department of Molecular Microbiology, Washington University School
˚
˚
of Medicine, St. Louis, Missouri 63110, Institute of Structural and Molecular Biology, University College London/Birkbeck, Malet Street,
London WC1E 7HX, U.K., and ^Department of Cellular and Molecular Interactions, Laboratory for Structural and Molecular Microbiology,
Structural Biology Brussels, Vrije Universiteit Brussel/VIB, 1050 Brussels, Belgium
Received April 17, 2010
Pilicides block pili formation by binding to pilus chaperones and blocking their function in the
chaperone/usher pathway in E. coli. Various C-2 substituents were introduced on the pilicide scaffold
by design and synthetic method developments. Experimental evaluation showed that proper substitu-
tion of this position affected the biological activity of the compound. Aryl substituents resulted in
pilicides with significantly increased potencies as measured in pili-dependent biofilm and hemagglutina-
tion assays. The structural basis of the PapD chaperone-pilicide interactions was determined by X-ray
crystallography.
Introduction
The increasing bacterial resistance to many antibiotics has
resulted in a growing interest in new antibacterial agents with
new modes of action directed toward Gram-negative
bacteria.1 Contrary to broad-range bactericidal or bacterio-
static antibiotics, drugs that specifically target bacterial viru-
lence factors would exert less selective pressure on bacteria
and minimize the risk for horizontal spread of drug-resistant
genes.2-4 It isestimated thatapproximately11 millioncases of
urinary tract infection (UTIa) occur in the U.S. each year,
primarily in women. Treatment of UTI, like other microbial
infections, is exacerbated by increasing antimicrobial resis-
tance. This has been described as an impending “public health
crisis”. In addition, over 900 000 women and men in the U.S.
experience three or more UTI episodes per year. Thus, anti-
biotics do not stop recurrence in this population, independent
of antibiotic resistance, highlighting the need for alternative
therapeutics.
Many Gram-negative bacteria, such as uropathogenic
E. coli (UPEC), produce long filamentous structures called
pili/fimbriae assembled by the chaperone/usher pathway
Figure 1. (i) Structure of pilicide 1. (ii) The pilicide 1 (green) binds
to the N-terminal domain of the chaperone PapD (blue). (iii) The
(CUP pili)5,6 that mediate colonization and invasion of host
tissues.4,7,8 Compounds that target CUP pili have the poten-
tial toblockvirulence in a broad range ofbacterialstrains. The
chaperones that are required for pilus assembly bind to pilus
pilicide (green) occupies a hydrophobic patch formed by I93, L32,
and V56 (purple). (iv) Through this interaction the pilicide occupies
the usher N-terminal binding site on the chaperone-subunit com-
plex and thus pauses pilus assembly (chaperone in blue, subunit in
orange, usher N-terminal domain in yellow).
†Protein coordinates for the PapD-PapH-pilicide 1 and the
PapD-PapH-pilicide 5d crystal complexes have been deposited with
the Protein Data Bank with PDB codes 2xg4 and 2xg5, respectively.
*To whom correspondence should be addressed. For S.J.H.: phone,
314-362-6772; fax, (314) 362-1998; e-mail, hultgren@borcim.wustl.edu.
For F.A.: phone, þ46 90 7866925; fax, þ46 90 7867655; e-mail, fredrik.
almqvist@chem.umu.se.
a Abbreviations: UTI, urinary tract infection; UPEC, uropathogenic
E. coli; CUP, chaperone usher pathway; LDA, lithium diisopropyla-
mide; MWI, microwave irradiation; HA, hemagglutination.
subunits and facilitate their folding by a mechanism termed
donor strand complementation.9-11 Chaperone-subunit com-
plexes are targeted to the outer membrane usher, which
catalyzes pilus assembly by a process known as donor strand
exchange.5,12,13 The chaperone comprises two immunoglobu-
lin-like domains arranged in a boomerang shape. The usher
has a 24-stranded β barrel that forms a channel across the
r
pubs.acs.org/jmc
Published on Web 06/30/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5691
Figure 2. Synthesis of the dihydrothiazolo ring-fused 2-pyridone scaffold (4) from Δ2-thiazolines (2) and Meldrum’s acid derivatives (3). Prior
to biological evaluation all compounds are hydrolyzed (e.g., 4a to 5a). From the oxidized scaffold (6), methods to introduce substituents in
position C-2 on the scaffold have been developed.
outer membrane. The channel is gated by a plug domain.
Ushers also have two periplasmic domains. The N terminal
periplasmic domain interacts in part with a hydrophobic
patch on the N terminal domain of the chaperone exposed
on incoming chaperone/subunit complexes. One class of
compounds termed pilicides, consisting of a dihydrothiazolo
ring-fused 2-pyridone scaffold, has been shown to prevent the
formation of pili by blocking the interaction of chaperone/
subunit complexes with the N terminal domain of the usher,
thus preventing the donor strand exchange reaction and the
formation of chaperone-usher assembled pili, exemplified in
UPEC.14,15 The X-ray crystal structure of pilicide 1 bound to
the P pilus chaperone PapD revealed that 1 binds to the
hydrophobic patch on the N terminal domain of the chaper-
one (formed by residues I93, L32, and V56) which forms the
well-conserved usher binding site (Figure 1).14,16 Competition
binding studies confirmed that the pilicide 1 prevents binding
of chaperone-subunit complexes to the usher N-terminal
domain. Pilicide 1 therefore functions by blocking delivery
of subunits to the usher, thus preventing donor strand ex-
change and inhibiting pilus assembly (Figure 1). Besides being
useful as potential antibacterial agents, pilicides have been
used as chemical tools to study details of pilus assembly and
their role in disease processes.14,17
In the crystal structure showing pilicide 1 bound to the
chaperone-subunit complex there is unoccupied space near
the thiazolo part of the pilicide scaffold (Figure 1iii). We
hypothesized that introducing substituents in this part of the
scaffold could thus lead to favorable interactions between the
chaperone and the pilicide and thereby result in increased
biological activity. We decided to decorate the C-2 position on
thescaffoldtocreateinteractions withthe unoccupiedspace in
the crystal structure to improve pilicide activity. The dihy-
drothiazolo ring-fused 2-pyridone scaffold (4), which consti-
tutes the platform for design of new pilicides, can be
synthesized via an acyl ketene imine cyclocondensation.18,19
This method introduces substituents directly in the C-7 and
C-8 positions on the scaffold, leaving positions C-6 and C-2
open for further improvements of the pilicides (Figure 2).
Previous studies have also described a number of methods for
the introduction of various substituents in position C-6 on the
core structure.20-22 Although this did not lead to any sub-
stantially increased biological activity, substitutions at C-6
allow for the introduction of solubility enhancing substitu-
ents. The C-2 position on the scaffold had not been varied
until recently, when we reported on the decoration of this
position starting from the R,β-unsaturated methyl ester (6)
(Figure 2).23 From this R,β-unsaturated methyl ester substit-
uents were introduced by conjugate additions resulting in an
addition with complete trans selectivity. In addition, substit-
uents were introduced with a retained double bond by the
use of Heck couplings or deprotonation using Lithium diiso-
propylamide (LDA) and subsequently react the lithiated
scaffold with different electrophilic reagents. Together, these
different methods opened up the possibility of introducing a
widerangeof substituents withvarying size, electronicproper-
ties, and spatial arrangement (Figure 2).
Here, we present biological and crystallographic data on
second generation compounds that were synthesized via C-2
substitution of the thiazolo ring-fused 2-pyridone 6, based on
the pilicide 1-PapD complex crystal structure. The C-6
morpholinomethyl substituent in pilicide 1, earlier introduced
for solubility reasons, was not included in this study, as it is
not vital for pilicide activity. The activities of the compounds
were evaluated in pilus dependent biofilm and hemagglutina-
tion assays. Compared to the parent pilicide 5a, C-2 substi-
tuted compounds have significantly improved IC50 in
inhibiting pilus formation. The molecular basis of the ob-
served improved biological activity of the compounds was
determined by elucidating the X-ray crystal structures of
pilicides bound to the PapD-PapH chaperone-subunit com-
plex (PapH is the P pilus termination subunit24). The pilicides
bound to the previously identified binding site on the chaper-
one N-terminal domain near strands F1, C1, and D1. The
introduced substituents were accommodated in a shallow
hydrophobic pocket on the chaperone surface, formed by
residues P30, L32, I93, and P95.
Results and Discussion
The developed methods for C-2 substitution were imple-
mented to introduce a diverse set of substituents on a known
pilicide (4)23 to increase the potency of the compounds in
inhibiting pilus assembly. The generated set of compounds
(4a-d and 6a-f) were then subjected to hydrolysis by three
different methods depending on the nature of the substituents
to givethe correspondingcarboxylic acids(5a-d and 7a-f ) in
80-95% yield (Table 1). The synthesized C-2 substituted
thiazolo and dihydrothiazolo ring-fused 2-pyridones were
biologically evaluated for their ability to block pilus forma-
tion as measured in a pili-dependent biofilm assay on poly-
vinylchloride plastic.25 In this assay, deletion of the type 1
pilus gene cluster in E. coli strain UTI89 completely destroys
the ability of the bacteria to form biofilm. Thus, the amount of
biofilm that is formed in UTI89 grown in the presence of
pilicide is related to the potency of the compound in blocking
piliation. This whole bacteria assay is suitable for screening of

5692 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Chorell et al.
Table 1. Synthesized Compounds Were Hydrolyzed and Evaluated for
Their Ability To Prevent Pili Dependent Biofilm Formation
Table 2. Isolated Yields of C-2 Substituted Unsaturated Thiazolo Ring-
Fused 2-Pyridones
c
bond
C-2/C-3
IC50
(μM)
R
Ma
compd
yieldb
single
single
H-
Me-
1
1
e
5a
5b
5c
5d
7a
7b
7c
7d
7e
7f
quant
92
>800d
54
single
single
MeO-
Ph-
75e
95
167
22
1
2
1
2
2
3
2
double
double
double
double
double
double
Me-
HO(CH3)2C-
Ph-
85
93
90
304
11
NAf
NAf
159
85
90
4-MeO-Ph-
PhCO-
PhNHCO-
80
88
a Method 1: (i) LiOH in THF/MeOH, room temp. (ii) Hþ Method 2:
(i) KOH in THF/MeOH, MWI, 90 ꢀC 25 min. (ii) Hþ Method 3: (i) Et3N,
LiBr in MeCN/H2O, room temp; (ii) Hþ. b Isolated yield. c Calculated
from 16-32 data points on every concentration (Figure S1 in Support-
ing Information). d Figure S1 in Supporting Information. e Hydrolyzed
directly in the methoxy addition.23 f Not active.
a Isolated yield.
Scheme 1
previously been accomplished by a two-step process starting
with oxidationof4a followed by lithiation and bromination.23
To avoid unnecessary purification and isolation of the oxi-
dized ring-fused 2-pyridone 6, a one-pot procedure directly
from 4a to 8 by modifying the developed oxidation method
was envisioned. Consequently, by adjustment of the reaction
conditions and treatment of 4a with 3 equiv of NaH followed
by 3 equiv of BrCCl3 and finally 2 equiv of MeOH, the desired
bromo derivative 8 was synthesized in 91% yield (Scheme 1).
After we successfully established a robust one-pot method
for the key compound 8, our focus was directed to the subse-
quent Suzuki-Miyaura cross-coupling. For this reaction we
adapted a method previously used within our lab for Suzuki-
Miyaura cross-couplings.27 Interestingly, we discovered that
this reaction could be performed with no use of ligands and
still give the same or increased yields without influencing
reaction time and temperature. Hence, seven different aryl-
or heteroarylboronic acids could be coupled to give C-2
substituted thiazolo ring-fused 2-pyridones 6g-m by run-
ning the reaction at 100 ꢀC for 10 min by microwave
irradiation (MWI) with 10 mol % Pd(OAc)2, 2 equiv of
boronic acid, and 1.9 equiv of KF in dry MeOH (Table 2).
The reaction resulted in good to excellent yields (72-97%,
Table 2) with the furane boronic acids as the only exceptions
resulting in thiazolo ring-fused 2-pyridones 6l and 6m in 46%
and 34% isolated yield, respectively (Table 2).
the synthesized compounds and provides more relevant bio-
logical information than elementary in vitro binding assays.
The results are summarized in Table 1.
These experiments revealed that the addition of substitu-
ents in the C-2 position of the pilicide scaffold significantly
enhanced the potency of the pilicides, resulting in increased
inhibition of pili dependent biofilm formation. The phenyl
substituted 5d and 7c proved to be the best pilicides with
significantly improved potency compared to their parent lead
5a. Also, the methyl substituents proved to increase the
potency. However, the influence of the spatial arrangement
on activity is different between the phenyl and methyl sub-
stituents. In the phenyl case, the unsaturated 7c is more potent
than the saturated counterpart 5d, whereas in the methyl case
the situation is the opposite; saturated 5b is more potent than
the unsaturated 7a. The remaining substituents resulted in
15-30 times lower potency compared to the most active 7c
(5c, 7f, and 7b) or were completely inactive (7d and 7e).
On the basis of the results with phenyl substituents and the
unsaturated analogue in particular, an additional investiga-
tion with C-2 aryl and heteroaryl substituents was performed.
We decided to use the Suzuki-Miyaura cross-coupling to
introduce these substituents.26 To implement these cross-
coupling reactions, a bromo-substituted unsaturated ring-
fused 2-pyridone 8 was desired. The synthesis of 8 had
To expand this set of compounds, benzyl substituted
analogue 6n was synthesized in 75% yield via lithiation
followed by addition of benzyl bromide using the previously
described procedure.23 Before being evaluated, the com-
pounds were hydrolyzed (KOH in THF/MeOH and MWI
for 10-20 min at 90 ꢀC, method 2). This rendered the
corresponding carboxylic acids (7g-n) in 50-92% yield
(Scheme 2).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5693
Scheme 2
Table 3. Biofilm IC50 and HA Titer Data for the C-2 Aryl Substituted
7g-n and 9
a
bond
C-2/C-3
C-2
substituent
IC50
( μM)
HA
titerb
compd
5a
5d
single
single
H-
>800c
22
7
Ph-
Ph-
4
7c
7g
double
double
double
double
double
double
double
double
double
double
11
11
4.5
3
3-tolyl-
4-tolyl-
7h
7i
13
5-indole-
6-(1,4-bensodioxane)-
3-thiophene-
3-furane-
2-furane-
Bn-
NAd
NAd
25
7j
7k
3.5
3
7l
7m
78
61
Scheme 3
7n
9
DMSOe
7
39
Ph-
8
8
f
a Calculated from 16-32 data points on every concentration (Figure
S1 in Supporting Information). b Number of wells with agglutination
(2n). E. coli was grown in the presence of pilicide (250 μM), and HA titers
were determined, average of dublicate runs. c Figure S1 in Supporting
Information. d Not active. e All compounds were dissolved in DMSO.
f Solely UTI89.
In addition to this, it has been shown that the carboxylic
acid is important for retaining biological activity28 and that
the use of carboxylic acid isosteres (e.g., tetrazoles or
acylsulfonamides) could lead to increased pilicide activity.29
Hence, 7c was also transformed into its corresponding methy-
lacylsulfonamide 9, which had proven to be one of the most
promising isosteres, in 60% yield (Scheme 3).
Finally, the Suzuki coupled aryl and heteroaryl com-
pounds, the benzyl derivative, and the methylacylsulfo-
namide acid isostere were all biologically evaluated for
their ability to prevent pili dependent biofilm formation
(Table 3).
The X-ray crystal structure of pilicide 1 bound to the P pilus
chaperone PapD previously revealed that the pilicide binds a
conserved hydrophobic patch on the chaperone N-terminal
domain formed by residues L32, V56, and I93, which coin-
cides with the usher binding site. Subsequently, pilicide 1 was
shown to block the binding of chaperone-subunit complexes
to the usher in an in vitro binding assay. Thus, we hypothe-
sized that pilicide 1 should be capable of binding to chaper-
one-subunit complexes to exert its mechanism in blocking
binding of the complex to the usher. This hypothesis was
tested by determining the X-ray crystal structure of pilicide 1
bound to the PapD-PapH chaperone-subunit complex.
Pilicide 1 was found to bind to the same global binding site
as seen in the PapD-pilicide 1 structure; however, substantial
reorientation was observed, possibly as a result of the con-
formational changes in the loop between strands F1 and G1 in
the subunit-bound and free chaperone state (Figure 3i). In the
pilicide 1-PapD structure, the pilicide carboxylic acid and
carbonyl group make an electrostatic interaction with R96
and R58, respectively (Figure 1iii). In thePapD-PapHbound
structure, these interactions are broken. We note that in the
PapD-pilicide 1 complex, the morpholine ring of 1 is in
contact with a neighboring molecule such that the effect of
crystal contacts in the observed reorientation in the PapD-
and PapD-PapH-bound compound cannot be excluded.
Finally, to investigate the structural basis for the observed
improved IC50 after introduction of C-2 substituents, PapD-
PapH crystals were soaked with the best compounds. Soaks
with 5d generated a crystal, showing the pilicide bound
to the chaperone-subunit (PapD-PapH) complex near
the same position as the C-2 unsubstituted 1 (Figure 3ii).
Compared to the pilicide 1-bound form, L32 undergoes
a conformational change when bound to 5d, which creates
a shallow pocket that accommodates the phenyl sub-
stituent at the C2 position (Figure 3, ii and iii). The crystal
structure of the N terminal domain of the FimD usher bound
We found that many of the aryl- and heteroaryl-substituted
compounds efficiently inhibited formation of biofilm. The
benzyl substituted compound 7n had an even lower IC50 than
the most active compound from the first biofilm evaluation
(7c) (Table 3). The tolyl substituted compounds 7g and 7h
were both highly active; 3-tolyl (7g) resulted in a similar
activity as the phenyl substituted analogue 7c (Table 3). Of
the smaller heteroaryls, the thiophene substituted 7k proved
to be more potent than the furan substituted 7l and 7m
(Table 3). Sterically more demanding substituents as in 7i
and 7j were not tolerated, and both of them turned out to be
inactive (Table 3). Interestingly, the acid isosteric acylsulfo-
namide in 9 did not give any additive effect but instead gave a
3-fold decreased potency compared to the carboxylic acid 7c
(Table 3). Next, the best compounds from the biofilm evalua-
tions were further tested using a hemagglutination (HA) assay
(Table 3).14 In this assay, the degree of piliation of the culture
is related to the HA titer. Thus, after growth in the presence of
pilicide, the HA titers of the cultures were determined to
evaluate the relative potencies of the compounds in blocking
pilus formation. Growth of UTI89 in the presence of all five
tested compounds resulted in significantly lower HA titers.
The benzyl substituted 7n and the 3-tolyl substituted 7g
resulted in 16 times increased activity compared to the hydro-
gen substituted compound 5a (Table 3). Interestingly, 7n, 7g,
and the thiophene substituted compound 7k were all more active
than the phenyl substituted 5d and 7c (Table 3). Importantly,
the compounds did not affect the bacterial growth (Figure S2
in Supporting Information).
16
to the FimC-FimH158-279 chaperone-subunit complex
is known. Modeling, based on this information, clearly
shows the clash between pilicide and the usher binding site
(Figure 3iv).

5694 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Chorell et al.
Figure 3. (i) Pilicide 1 bound to the PapD-PapH chaperone-subunit complex (pink) undergoes a reorientation compared to pilicide 1 bound
to the PapD chaperone (cyan). (ii) Pilicide 5d (cyan) binds near the same position as C-2 unsubstitued pilicide 1 (pink) in the PapD-PapH
chaperone-subunit complex. (iii) L32 reorients and thus creates a shallow pocket for the C-2 substituent in 5d (cyan). (iv) Clash between the
usher FimD N-terminal (yellow) and the pilicides (cyan and pink) binding site (chaperone FimC in green and subunit FimH in blue).
Conclusion
General Procedure for the Preparation of 6g-m. The proce-
dure for 8-cyclopropyl-7-naphthalen-1-ylmethyl-5-oxo-2-m-to-
lyl-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid methyl ester
(6g) is representative. Dry MeOH (0.9 mL) was added to
pyridone 8 (20 mg, 0.043 mmol), Pd(OAc)2 (1 mg, 0.004 mmol),
KF (5 mg, 0.81 mmol), and boronic acid (12 mg, 0.085 mmol).
The mixture was heated in a sealed tube by MWI at 100 ꢀC for
10 min. The resulting mixture was diluted with CH2Cl2, washed
with saturated aqueous NaHCO3, and the aqueous layer was
extracted with CH2Cl2. The combined organic layers were
concentrated under reduced pressure and purification by par-
allel flash chromatography [heptane/EtOAc 100:0 to 0:100] to
give pyridone 6g (20 mg, 96% yield) as a yellow foam. 1H NMR
(400 MHz, CDCl3) δ 0.77-0.84 (m, 2H), 1.02-1.10 (m, 2H),
1.76-1.86 (m, 1H), 2.41 (s, 3H), 3.88 (s, 3H), 4.55 (s, 2H), 5.94 (s,
1H), 7.21-7.26 (m, 2H), 7.30-7.36 (m, 1H), 7.37-7.44 (m, 3H),
A new route for fast and convenient synthesis of C-2 aryl
substituted pilicides was developed and applied. Biofilm and
HAassays revealedthatproperdecorationof this positioncan
significantly improve the antivirulence activity of the com-
pounds. The most rewarding substituents proved to be phe-
nyls in general and the benzyl and tolyl substituents in
particular. Smaller heteroaryls (thiophene and furanes) also
improved activity, whereas sterically more demanding groups
as indoles and 1,4-benzodioxanes abolished all activity. Com-
bining a C-2 phenyl substituent with an acid isoster did
not give any additive effect; instead a drop in activity was
observed.
X-ray crystallography of the pilicides bound to the PapD-
PapH chaperone-subunit complex revealed that introduc-
tion of a C-2 phenyl substituent induced a reorientation of one
of the chaperone (PapD) residues (L32), generating a shallow
pocket that accommodates this substituent.
7.45-7.52 (m, 2H), 7.75-7.81 (m, 1H), 7.81-7.91 (m, 2H). 13
C
NMR (100 MHz, CDCl3) δ 7.9 (2C), 10.9, 21.4, 36.2, 53.3, 111.7,
111.9, 123.7, 125.4, 125.5 (2C), 125.7, 126.2, 127.3, 127.6, 128.6,
128.8, 128.9, 129.0, 129.1, 130.8, 131.9, 133.9, 134.2, 139.0,
146.3, 153.4, 158.9, 161.8.
General Procedure for the Preparation of 7a,c,d,f-n. The
procedure for 8-cyclopropyl-7-naphthalen-1-ylmethyl-5-oxo-
2-m-tolyl-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid (7g) is
representative. KOH (s) (20 equiv) was added to 6g (17 mg,
0.0355 mmol) dissolved in THF (1 mL) and MeOH (0.5 mL).
The suspension was heated by microwave irradiation for 25 min
at 90 ꢀC before being cooled to room temperature. The mixture
was diluted in CH2Cl2 and washed twice with 1 M HCl. The
combined organic layers were concentrated and purified by
column chromatography [CH2Cl2/MeOH 97:3 f CH2Cl2/
MeOH 95:5 and 1% AcOH] to give 7g (15.2 mg, 92% yield)
which was lyophilized from MeCN/H2O to give a light-yellow
solid. 1H NMR (400 MHz, CDCl3) δ 0.68-0.75 (m, 2H),
0.94-1.03 (m, 2H), 1.66-1.76 (m, 1H), 2.32 (s, 3H), 4.45 (s,
2H), 6.12 (s, 1H), 7.02-7.09 (m, 1H), 7.14-7.18 (m, 1H),
7.20-7.25 (m, 1H), 7.27-7.38 (m, 1H), 7.37-7.41 (m, 1H),
Experimental Section
General. Flash column chromatography (eluents given in
˚
brackets) was performed on silica gel (Matrex, 60 A, 35-
70 μm, Grace Amicon). Parallel flash chromatography was
performed on a Gradmaster parallel Jones chromatograph. 1H
and 13C NMR spectra were recorded on a Bruker DRX-400 in
CDCl3 [residual CHCl3 (δH 7.26 ppm) or CDCl3 (δC 77.0 ppm)
as internal standard] at 298 K. IR spectra were recorded on an
ATI Mattson Genesis series FTIR spectrometer. Microwave
reactions were carried out using a monomode reactor (Smith
Creator, Biotage AB) in Teflon septa capped 0.5-2 or 2-5 mL
Smith process vials with stirring. Reaction times refer to irradia-
tion time at target temperature, as measured by IR sensor.
1
Purities of key compounds were >95% as determined by H
NMR and HPLC.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5695
7.41-7.50 (m, 3H), 7.70-7.80 (m, 2H), 7.82-7.88 (m, 1H). 13
C
(11) Vetsch, M.; Puorger, C.; Spirig, T.; Grauschopf, U.; Weber-Ban,
E. U.; Glockshuber, R. Pilus chaperones represent a new type of
protein-folding catalyst. Nature 2004, 431, 329–332.
(12) Remaut, H.; Tang, C.; Henderson, N. S.; Pinkner, J. S.; Wang, T.;
Hultgren, S. J.; Thanassi, D. G.; Waksman, G.; Li, H. L. Fiber
formation across the bacterial outer membrane by the chaperone/
usher pathway. Cell 2008, 133, 640–652.
NMR (100 MHz, CDCl3) δ 8.3 (2C), 11.4, 21.5, 36.7, 111.3,
113.7, 124.2, 126.0, 126.1, 126.2, 126.7, 127.9, 128.1, 128.3,
128.7, 129.3, 129.38, 129.43, 129.6, 131.1, 132.4, 134.5, 134.8,
139.4, 148.0, 154.4, 160.0, 164.5.
Structure Determination of Pilicides 1 and 5d Bound to
PapD-PapH. The PapD-PapH complex (where for PapH,
residues 1-22 of the mature protein are removed for stability)
was purified and crystallized as previously described.30 Com-
plexes with pilicide 1 and 5d were formed by a 24 h incubation of
PapD-PapH crystals in the crystallization solution (10 mM
cobalt chloride, 100 mM MES, pH 6.5, and 1.8 M ammonium
sulfate) containing 2 mM compound.
(13) Dodson, K. W.; Jacobdubuisson, F.; Striker, R. T.; Hultgren, S. J.
Outer-membrane PapC molecular usher discriminately recognizes
periplasmic chaperone pilus subunit complexes. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 3670–3674.
˚
(14) Pinkner, J. S.; Remaut, H.; Buelens, F.; Miller, E.; Aberg, V.;
€
Pemberton, N.; Hedenstrom, M.; Larsson, A.; Seed, P.; Waksman,
G.; Hultgren, S. J.; Almqvist, F. Rationally designed small com-
pounds inhibit pilus biogenesis in uropathogenic bacteria. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 17897–17902.
(15) Aberg, V.; Almqvist, F. Pilicides-small molecules targeting bacter-
Acknowledgment. F.A. was supported by the Swedish
Natural Research Council, the Knut and Alice Wallenberg
Foundation, and JC Kempe Foundation (SJCKMS). S.J.H.
was supported by NIH Grants AI048689, AI049950, AI029549,
and DK086378. G.W. was supported by MRC Grant 85602.
H.R. was supported by a VIB Young PI project grant and the
Odysseus program of the FWO-Vlaanderen.
ial virulence. Org. Biomol. Chem. 2007, 5, 1827–1834.
(16) Nishiyama, M.; Horst, R.; Eidam, O.; Herrmann, T.; Ignatov, O.;
Vetsch, M.; Bettendorff, P.; Jelesarov, I.; Grutter, M. G.;
Wuthrich, K.; Glockshuber, R.; Capitani, G. Structural basis of
chaperone-subunit complex recognition by the type 1 pilus
assembly platform FimD. EMBO J. 2005, 24, 2075–2086.
(17) Aberg, V.; Fallman, E.; Axner, O.; Uhlin, B. E.; Hultgren, S. J.;
Almqvist, F. Pilicides regulate pili expression in E. coli without
affecting the functional properties of the pilus rod. Mol. BioSyst.
2007, 3, 214–218.
€
(18) Emtenas, H.; Alderin, L.; Almqvist, F. An enantioselective ketene-
Supporting Information Available: Synthetic procedures and
characterization data of all compounds, growth curves for 5d,
7c, 7f, and 9, details of data quality and structure refinement for
structure determination of 1 and 5d bound to PapD-PapH.
This material is available free of charge via the Internet at http://
pubs.acs.org.
imine cycloaddition method for synthesis of substituted ring-fused
2-pyridinones. J. Org. Chem. 2001, 66, 6756–6761.
(19) Chorell, E.; Edvinsson, S.; Almqvist, F. Improved procedure for
the enantioselective synthesis of dihydrooxazolo and dihydrothia-
zolo ring-fused 2-pyridones. Terahedron Lett. 2010, 51, 2461-2463.
(20) Aberg, V.; Sellstedt, M.; Hedenstrom, M.; Pinkner, J. S.; Hultgren,
S. J.; Almqvist, F. Design, synthesis and evaluation of peptidomi-
metics based on substituted bicyclic 2-pyridones. Targeting virulence
of uropathogenic E. coli. Bioorg. Med. Chem. 2006, 14, 7563–7581.
(21) Pemberton, N.; Aberg, V.; Almstedt, T.; Westermark, A.; Almq-
vist, F. Microwave-assisted synthesis of highly substituted amino-
methylated 2-pyridones. J. Org. Chem. 2004, 69, 7830–7835.
(22) Pemberton, N.; Pinkner, J. S.; Jones, J. M.; Jakobsson, L.; Hultgren,
S. J.; Almqvist, F. Functionalization of bicyclic 2-pyridones targeting
piles biogenesis in uropathogenic Escherichia coli. Tetrahedron Lett.
2007, 48, 4543–4546.
(23) Chorell, E.; Das, P.; Almqvist, F. Diverse functionalization of
thiazolo ring-fused 2-pyridones. J. Org. Chem. 2007, 72, 4917–4924.
(24) Baga, M.; Norgren, M.; Normark, S. Biogenesis of Escherichia coli
Pap pili-PapH, a minor pilin subunit involved in cell anchoring
and length modulation. Cell 1987, 49, 241–251.
(25) O’Toole, G. A.; Kolter, R. Initiation of biofilm formation in
Pseudomonas fluorescens WCS365 proceeds via multiple, conver-
gent signalling pathways: a genetic analysis. Mol. Microbiol. 1998,
28, 449–461.
References
(1) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.;
Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad
bugs, no drugs: no ESKAPE! An update from the Infectious
Diseases Society of America. Clin. Infect. Dis. 2009, 48, 1–12.
(2) Cegelski, L.; Marshall, G. R.; Eldridge, G. R.; Hultgren, S. J. The
biology and future prospects of antivirulence therapies. Nat. Rev.
Microbiol. 2008, 6, 17–27.
(3) Clatworthy, A. E.; Pierson, E.; Hung, D. T. Targeting virulence: a
new paradigm for antimicrobial therapy. Nat. Chem. Biol. 2007, 3,
541–548.
(4) Cusumano, C. K.; Hultgren, S. J. Bacterial adhesion. A source of
alternate antibiotic targets. IDrugs 2009, 12, 699–705.
(5) Hung, D.; Hultgren, S. J. Pilus biogenesis via the chaperone/usher
pathway: an integration of structure and function. J. Struct. Biol.
1998, 124, 201–220.
(6) Waksman, G.; Hultgren, S. J. Structural biology of the chaper-
one-usher pathway of pilus biogenesis. Nat. Rev. Microbiol. 2009,
7, 765–774.
(7) Hung, D. L.; Knight, S. D.; Woods, R. M.; Pinkner, J. S.; Hultgren,
S. J. Molecular basis of two subfamilies of immunoglobulin-like
chaperones. EMBO J. 1996, 15, 3792–3805.
(8) Sauer, F. G.; Remaut, H.; Hultgren, S. J.; Waksman, G. Fiber
assembly by the chaperone-usher pathway. Biochim. Biophys.
Acta 2004, 1694, 259–267.
(9) Bann, J. G.; Pinkner, J. S.; Frieden, C.; Hultgren, S. J. Catalysis of
protein folding by chaperones in pathogenic bacteria. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 17389–17393.
(26) Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling reac-
tions of organoboron compounds. Chem. Rev. 1995, 95, 2457–2483.
(27) Sellstedt, M.; Almqvist, F. Synthesis of a novel tricyclic peptido-
mimetic scaffold. Org. Lett. 2008, 10, 4005–4007.
(28) Aberg, V.; Hedenstrom, M.; Pinkner, J. S.; Hultgren, S. J.; Almqvist,
F. C-Terminal properties are important for ring-fused 2-pyridones
that interfere with the chaperone function in uropathogenic E. coli.
Org. Biomol. Chem. 2005, 3, 3886–92.
(29) Aberg, V.; Das, P.; Chorell, E.; Hedenstrom, M.; Pinkner, J. S.;
Hultgren, S. J.; Almqvist, F. Carboxylic acid isosteres improve the
activity of ring-fused 2-pyridones that inhibit pilus biogenesis in E.
coli. Bioorg. Med. Chem. Lett. 2008, 18, 3536–3540.
(10) Sauer, F. G.; Pinkner, J. S.; Waksman, G.; Hultgren, S. J. Chaper-
one priming of pilus subunits facilitates a topological transition
that drives fiber formation. Cell 2002, 111, 543–551.
(30) Verger, D.; Miller, E.; Remaut, H.; Waksman, G.; Hultgren, S.
Molecular mechanism of P pilus termination in uropathogenic
Escherichia coli. EMBO Rep. 2006, 7, 1228–1232.