Functionalization of bicyclic 2-pyridones targeting pilus biogenesis in uropathogenic Escherichia coli

Article abstract of DOI:10.1016/j.tetlet.2007.04.142

Substituted bicyclic 2-pyridones, termed pilicides, prevent pilus assembly in uropathogenic Escherichia coli. Based on the bioactive methyl ester protected 2-pyridone 4, further functionalization at C-6 has yielded a set of new compounds, which have been

Full text of DOI:10.1016/j.tetlet.2007.04.142

Tetrahedron Letters 48 (2007) 4543–4546
Functionalization of bicyclic 2-pyridones targeting pilus
biogenesis in uropathogenic Escherichia coli
Nils Pemberton,a Jerome S. Pinkner,b Jennifer M. Jones,b Lotta Jakobsson,a
Scott J. Hultgrenb and Fredrik Almqvista,*
a
˚
˚
Department of Chemistry, Umea University, SE-90187 Umea, Sweden
bDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, United States
Received 5 February 2007; revised 18 April 2007; accepted 27 April 2007
Available online 3 May 2007
Abstract—Substituted bicyclic 2-pyridones, termed pilicides, prevent pilus assembly in uropathogenic Escherichia coli. Based on the
bioactive methyl ester protected 2-pyridone 4, further functionalization at C-6 has yielded a set of new compounds, which have been
evaluated for their ability to inhibit pilus formation in uropathogenic E. coli. The key intermediate in the synthesis was formylated 2-
pyridone 5, which could be obtained via a Vilsmeier reaction. This versatile intermediate was converted into secondary and tertiary
amines via reductive amination and could also be converted to other interesting functionalities using simple chemical
transformations.
Ó 2007 Elsevier Ltd. All rights reserved.
Several Gram negative pathogens use adhesive organ-
elles called pili/fimbriae to initiate infection of host cells.
The adhesive pili fibers are assembled via a highly con-
served mechanism called the chaperone/usher pathway
which is used by a multitude of bacterial pathogens.
We have recently shown that the bicyclic 2-pyridone 1
and its aminomethylated derivative 2a (Fig. 1) block
the formation of both type 1 and P pili in uropathogenic
Escherichia coli,1 thus paving the way for these com-
pounds to serve as possible leads towards broad-spec-
trum antibacterial agents.
An efficient synthesis of ring-fused di-substituted 2-pyri-
dones has previously been developed and the key step
was a cyclocondensation between a D2-thiazoline and
an acyl-ketene generated from acyl Meldrum’s acids.2–4
However, the large hydrophobic substituents present
in the active di-substituted 2-pyridones results in poor
water solubility that limited their utility. This issue
was addressed by introducing more hydrophilic substit-
uents at C-6 of the 2-pyridone scaffold (see Fig. 1). By
using a modified Mannich reaction, symmetrical tertiary
aminomethylated 2-pyridones 2a could be prepared and
primary amine 2b was synthesized via a three-step pro-
cedure based on a cyano-group as the key intermediate.5
The cyano functionality was introduced via a micro-
wave-assisted Rosenmund von Braun reaction, and
could then be reduced to yield the primary aminomethyl-
ated 2-pyridone 2b in good yield. Unfortunately, the
somewhat harsh reaction conditions did result in low
optical purity of the final product (8% ee). In vivo eval-
uations of these compounds showed that several of the
tertiary aminomethylated 2-pyridones 2 (e.g. 2a) had
maintained or in some cases even improved their ability
to attenuate pilus assembly in E. coli. In addition, these
more water soluble compounds were much easier to
evaluate in biological assays.1
S
S
S
N
N
N
6
CO H
2
CO Li
2
CO Li
2
O
N
O
O
NH
O
2
2b
1
2a
Figure 1. 2-Pyridone 1 prevents pili formation in uropathogenic
E. coli. Further functionalization at C-6 has yielded the more water
soluble aminomethylated compounds 2a and 2b.
Keywords: 2-Pyridone; Formylation; Antibacterial agent; Pilicide;
Biofilm inhibitor.
*
Corresponding author. Tel.: +46 907866925; fax: +46 90138885;
e-mail: fredrik.almqvist@chem.umu.se
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.04.142

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N. Pemberton et al. / Tetrahedron Letters 48 (2007) 4543–4546
Encouraged by these positive results we were interested
in investigating the possibility to further fine-tune the
aminomethyl-substituent to obtain compounds with
enhanced potency. The limitations of the earlier devel-
oped methodologies did not make them suitable for this
purpose, as the previously developed Mannich reaction
was performed by first preparing the methylene iminum
salts in two steps, and the route via the cyano-group was
performed in a time consuming manner over several
steps and yielded the final product with poor ee. Thus
a new method for the preparation of a small series of
aminomethylated 2-pyridones was needed. In this
Letter, we present synthetic routes to primary, second-
ary and tertiary amines. All the synthetic routes
developed were based on a formyl substituted 2-pyri-
done as the key intermediate. The formyl group could
also be transformed to an alcohol and a carboxylic acid
by using robust and efficient transformations.
The formylated 2-pyridone was then reductively ami-
nated to secondary and tertiary aminomethylated 2-
pyridones 6a–g in good to excellent yields (76–95%)
(Scheme 1).8 The reactions were performed in CH2Cl2
and MeOH, allowing the amine and the aldehyde to
˚
react for 30 min in the presence of 3 A molecular sieves
prior to the addition of NaBH(OAc)3. This was done
to prevent the formation of alcohol 9, which was
obtained as a by-product if all the reagents were added
together at the start. Primary aminomethylated 2-pyri-
done 8 was prepared by first converting aldehyde 5 to
the corresponding oxime 7, which was transformed to
the desired primary amine in moderate yield using zinc
in acetic acid.9 This synthetic pathway had to be devel-
oped as initial efforts to perform reductive amination
with different sources of ammonia (NH4OAc, NH3
saturated MeOH) resulted only in dimerization. Impor-
tantly, this pathway also yielded the primary aminome-
thylated 2-pyridone without any loss of optical purity,
which had been the major drawback of the previously
developed method via Rosenmund von Braun cyanation
followed by reduction.
To introduce the desired formyl functionality the Vils-
meier reaction was employed.6 Starting with classical
conditions using DMF and POCl3, the desired formyl-
ated 2-pyridone was obtained, however, the conversion
was slow and after 15 h at room temperature a large
amount of unreacted starting material still remained.
Therefore, a preformed chloromethylene iminium salt
was used instead, providing a higher concentration of
the reactive electrophilic species. Commercially avail-
able ClÀMe2N+@CHCl (Arnold’s reagent) and 2-pyri-
done 4 were reacted in refluxing acetonitrile. This
resulted in full conversion after 3 h and compound 5
was isolated in excellent yield (93%) after simple filtra-
tion through a silica plug.7
The formylated 2-pyridone 5 could also easily be
reduced to alcohol 9 using BH3ÆSMe2 in THF (Scheme
1).10 Oxidation of 5 to the carboxylic acid 10 also proved
straightforward. Compound 10 was prepared in excel-
lent yield using a chemoselective sodium chlorite oxida-
tion.11,12 DMSO was used as the solvent which prevents
oxidation of the sulfide by functioning as a hypochlo-
rous acid scavenger and being oxidized to dimethyl sul-
fone. All 12 compounds were then hydrolyzed to their
corresponding lithium carboxylates 11a–k (0.1 M aq
S
N
CO Me
2
O
4
a
S
N
e
f
CO Me
2
OH
O
S
S
9 (80%)
c
N
N
R
CO Me
2
CO Me
2
O
O
O
7 R = -CH=NOH
8 R = -CH -NH (61% over two steps)
5 (93%)
d
S
N
2
2
HO
b
CO Me
2
O
O
10 (98%)
6a R=morpholine (85%)
6b R=NHBn (92%)
6c R=NHhexyl (90%)
S
N
6d R=NH(CH ) NMe (87%)
2 3
2
6e R=piperidine (76%)
6f R=NHMe (81%)
6g R=NHcyclohexyl (95%)
CO Me
2
R
O
6a-g
À
+
˚
Scheme 1. Reagents and conditions: (a) Cl Me2N @CHCl, MeCN, reflux; (b) amine, CH2Cl2–MeOH 7:3, 3 A mol sieves, NaBH(OAc)3; (c)
NH2OHÆHCl, pyridine, EtOH, reflux; (d) Zn powder, AcOH, rt; (e) 2 M BH3ÁSMe2, THF, rt; (f) NaClO2, NaH2PO4, H2O, DMSO, rt.

N. Pemberton et al. / Tetrahedron Letters 48 (2007) 4543–4546
4545
Table 1. The ability to prevent pili formation was evaluated in a hemagglutination assay
S
S
0.1 M LiOH (aq)
THF
N
N
R
R
CO Me
2
CO Li
2
O
O
5, 6a-g, 7-10
2a, 11a-k
Compound
R
HA-titera 3.6 mM pilicide
HA-titera 1.8 mM pilicide
HA-titera 0.72 mM pilicide
16
2a
–CH2-morpholine
–CHO
16b
128
16c
16
—
—
—
—
16
—
—
—
—
16
—
128
11a
11b
11c
—
–CH2NHBn
–CH2NHhexyl
–CH2NH(CH2)3NMe2
–CH2-piperidine
–CH2NHMe
–CH2NHcyclohexyl
–CH@NOH
–CH2NH2
16c
32c
32c
d
d
11d
11e
—
—
8b
32
d
d
11f
—
—
11g
11h
11i
32c
32c
128
—
—
16
—
c
—
11j
11k
No pilicide
–CH2OH
–CO2Li
—
8b
128
128b
128
E.coli was grown in the presence of pilicide at different concentrations and HA-titers were determined.
a Highest dilution that still provides hemagglutination (see text for details).
b Duplicate runs.
c Precipitation of compound in broth.
d Inhibition of bacterial growth.
LiOH in THF) and evaluated for their ability to prevent
pili formation in uropathogenic E. coli. (Table 1). Bacte-
ria were grown in the presence of these substrates at dif-
ferent concentrations (3.6, 1.8, 0.7 mM) and the level of
piliation was subsequently determined in a hemaggluti-
nation assay (HA).13 A low HA-titer indicates that less
pili are formed, and is thus a verification of an efficient
pilicide. Compound 2a bearing a morpholine moiety has
previously been evaluated in several different assays and
has been recognized as the most promising pilicide so
far.1 Several of the new compounds: 11b, 11c, 11e, 11g
and 11j exhibited improved or comparable in vivo activ-
ity compared to 2a (Table 1). Unfortunately, the low
water solubility of secondary amines 11b, 11c and 11g
resulted in some precipitation in the bacterial broth even
at the lowest concentrations (Table 1, column 5). How-
ever, it should be noted that even though the limited
water solubility complicates the evaluation of these com-
pounds they still seem to have a clear effect.
previously most effective compound 2a, suggesting that
further substitution in this readily accessible position is
a viable approach towards more potent pilicides.
Acknowledgement
We thank the Swedish Natural Science Research Coun-
cil and the JC Kempe foundation for financial support.
References and notes
˚
1. Pinkner, J. S.; Remaut, H.; Buelens, F.; Miller, E.; Aberg,
V.; Pemberton, N.; Hedenstro¨m, M.; Larsson, A.; Seed,
P.; Waksman, G.; Hultgren, S. J.; Almqvist, F. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 17897–17902.
˚
2. Emtena¨s, H.; Ahlin, K.; Pinkner, J. S.; Hultgren, S. J.;
Almqvist, F. J. Comb. Chem. 2002, 4, 630–639.
3. Emtena¨s, H.; Alderin, L.; Almqvist, F. J. Org. Chem.
2001, 66, 6756–6761.
Tertiary amine 11e and alcohol 11j also maintained their
effect at lower concentrations (Table 1) and further eval-
uation of 2a, 11e and 11j was performed in a biofilm
assay.1 (data not shown) confirming their ability to
inhibit pili formation. In agreement with the data from
the HA-titer, 11j was confirmed as the most efficient
pilicide with 50% inhibition of biofilm growth at 90 lM.
4. Emtena¨s, H.; Taflin, C.; Almqvist, F. Mol. Div. 2003, 7,
165–169.
5. Pemberton, N.; Aberg, V.; Almstedt, T.; Westermark, A.;
Almqvist, F. J. Org. Chem. 2004, 69, 7830–7835.
6. Vilsmeier, A.; Haack, A. Ber. Dtsch. Chem. Ges. 1927, 60,
119.
˚
7. Experimental procedure for formylation: 2-Pyridone 4 was
added to
a stirred solution of Arnold’s reagent
(ClÀMe2N+@CHCl) (4 equiv) in acetonitrile (0.12 mmol/
ml) at rt. The solution was refluxed for 3 h, then
concentrated and dissolved in CH2Cl2, washed with
saturated NaHCO3 (aq), extracted with CH2Cl2 and the
combined organic phases were dried over Na2SO4(s),
filtered and concentrated. Further purification by filtration
through a short silica plug (EtOAc) yielded compound 5
In conclusion, by developing a method to introduce a
formyl functionality at C-6 of the bioactive 2-pyridone
4, we have been able to prepare a set of new derivatives
which have been evaluated as inhibitors for pilus assem-
bly in E. coli. Several of the new compounds show main-
tained or improved in vivo activity compared to the

4546
N. Pemberton et al. / Tetrahedron Letters 48 (2007) 4543–4546
(93%): [a]D À297 (c 1.0, CHCl3); IR k 1748, 1633, 1470,
dissolved in acetic acid (0.020 mmol/mL) and stirred at
room temperature for 20 h. The zinc dust was removed by
filtration and the organic phase carefully neutralized with
saturated NaHCO3 (aq). The aqueous phase was extracted
with CH2Cl2 and the combined organic phases dried over
Na2SO4(s), filtered and concentrated. Purification by
column chromatography yielded primary amine 8 (61%):
Data was in agreement with published data.5 The enan-
tiomeric excess was 86% as determined by chiral HPLC
(compared to 86% ee for the starting material 4).
1406, 1211, 1154, 1012, 792; 1H NMR (400 MHz, CDCl3)
d 10.39 (s, 1H), 8.18 (d, J = 8.8, 1H), 7.87 (d, J = 8.4, 1H),
7.70 (d, J = 8.3, 1H), 7.50–7.61 (m, 2H), 7.24–7.29 (m,
1H), 6.76 (d, J = 6.6, 1H), 5.76 (dd, J = 8.9, 2.5, 1H), 5.23
(d, J = 14.6, 1H), 5.09 (d, J = 14.6, 1H), 3.89 (s, 3H), 3.77
(dd, J = 11.9, 9.0, 1H), 3.59 (dd, J = 11.9, 2.5, 1H), 1.30–
1.39 (m, 1H), 0.63–0.76 (m, 2H), 0.51–0.58 (m, 2H); 13C
NMR (100 MHz, CDCl3) d 190.6, 167.9, 162.0, 160.6,
156.9, 134.4, 133.4, 131.8, 128.5, 126.5, 125.9, 125.5, 125.2,
123.0, 122.9, 118.6, 115.3, 63.2, 53.2, 31.4, 31.1, 11.0, 7.6,
7.1; HRMS (FAB) calcd for [M+H]+ C24H22NO4S
420.1270, obsd 420.1267.
10. Experimental procedure for reduction: Formylated 2-pyri-
done 5 was dissolved in THF (0.07 mmol/ml) at 0 °C, then
2 M BH3 Á SMe2 in THF (1.1 equiv) was added dropwise
over 15 min. The mixture was stirred at rt for 1 h,
quenched with methanol and concentrated. The residue
was co-concentrated from methanol and then purified by
column chromatography to yield 9 (80%): [a]D À148 (c 2.0,
CHCl3); IR k 3419, 3001, 2950, 2872, 1739, 1627, 1501,
8. General procedure for reductive amination: 1.0 equiv of
amine was added to a stirred solution of aldehyde 5 in
˚
CH2Cl2–MeOH 7:3 and 3 A mol sieves at 0 °C, and
stirring was continued for 30 min. Sodium triacetoxy-
borohydride (1.8 equiv) was then added and the mixture
was allowed to attain rt and stirred for 3 h. The reaction
was washed with saturated NaHCO3 (aq), extracted with
CH2Cl2 and the combined organic phases were dried over
Na2SO4(s). Purification by column chromatography
yielded the aminomethylated 2-pyridones 6a–g. Data for
compound 6b: [a]D À128 (c 1.2, CHCl3); IR k 3009, 2953,
1748, 1631, 1557, 1500, 1209, 791, 726; 1H NMR
(400 MHz, CDCl3) d 8.08 (d, J = 8.3 Hz, 1H), 7.89 (d,
J = 8.1 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.51–7.62 (m,
2H) 7.10–7.36 (m, 6H) 6.80 (d, J = 7.0 Hz, 1H), 5.68 (dd,
J = 8.6, 2.5 Hz, 1H), 4.66 (d, J = 16.3 Hz, 1H), 4.57 (d,
J = 16.3 Hz, 1H) 3.85 (s, 3H) 3.49–3.74 (m, 6H) 1.35–1.43
(m, 1H) 0.45–0.69 (m, 4H); 13C NMR (100 MHz, CDCl3)
d 168.5, 161.4, 153.0, 145.8, 139.4, 134.3, 133.4, 131.6,
128.6, 128.0 (split), 126.8, 126.6, 126.0, 125.6, 125.3, 124.2,
123.8, 122.9, 114.4, 63.2, 53.1 (split), 45.2, 31.8, 31.3, 11.6,
7.3, 7.0; HRMS (FAB) calcd for [M+H]+ C31H31N2O3S
511.2055, obsd 511.2042. The enantiomeric excess was
76% as determined by chiral HPLC (compared to 76% ee
for the starting material 4).
1
1210, 1008, 792, 773; H NMR (400 MHz, CDCl3) d 8.16
(d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.71 (d,
J = 8.2 Hz, 1H), 7.50–7.62 (m, 2H) 7.28–7.33 (m, 1H) 6.83
(d, J = 6.9 Hz,1H), 5.68 (dd, J = 8.7, 2.4 Hz, 1H), 4.71 (d,
J = 16.3 Hz, 1H), 4.62 (d, J = 16.3 Hz, 1H), 4.52 (d,
J = 12.8 Hz, 1H), 4.43 (d, J = 12.8 Hz, 1H) 3.84 (s, 3H)
3.68 (dd, J = 11.8, 8.7 Hz, 1H) 3.51 (dd, J = 11.8, 2.4 Hz,
1H) 1.34–1.44 (m, 1H) 0.60–0.69 (m, 2H) 0.43–0.56 (m,
2H); 13C NMR (100 MHz, CDCl3) d 168.4, 161.7, 151.9,
146.4, 133.9, 133.6, 131.6, 128.7, 127.0, 126.2, 125.7, 125.5,
125.4, 124.1, 122.8, 114.7, 63.1, 58.6, 53.2, 31.6, 31.4, 11.6,
7.3, 7.0; HRMS (FAB) calcd for [M+H]+ C24H24NO4S
422.1426, obsd 422.1423. The enantiomeric excess was
76% as determined by chiral HPLC (compared to 76% ee
for the starting material 4).
11. Fang, X. Q.; Bandarage, U. K.; Wang, T.; Schroeder, J.
D.; Garvey, D. S. Synlett 2003, 489–492.
12. Experimental procedure for oxidation: To a solution of
formylated 2-pyridone
5 in DMSO (0.1 mmol/mL),
NaH2PO4 (2 equiv), dissolved in water (0.5 mmol/mL),
was added dropwise at room temperature; the mixture was
then kept on ice and NaClO2 (4 equiv), dissolved in water
(2 mmol/mL), was added dropwise over 30 min. After
stirring the reaction for 1 h at room temperature, the
reaction mixture was poured into a separation funnel
containing ice-cooled 1 M HCl. The aqueous phase was
extracted with CH2Cl2 and the combined organic phases
concentrated. The residue was dissolved in H2O: CH3CN,
8:2 and lyophilized to yield carboxylic acid 10 (98%): [a]D
À155 (c 1.1, CHCl3); IR k 1712, 1592, 1450, 1216, 1141,
791, 773; 1H NMR (400 MHz, DMSO-d6) d 13.86 (s, 1H),
8.26 (d, J = 8.3 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.78 (d,
J = 8.3 Hz, 1H) 7.54–7.66 (m, 2H) 7.33–7.40 (m, 1H), 6.79
(d, J = 7.2 Hz, 1H), 5.79 (dd, J = 9.3, 1.8 Hz, 1H) 4.93 (m,
2H) 3.95 (dd, J = 12.0, 9.5 Hz, 1H) 3.78 (s, 3H) 3.70 (dd,
J = 12.0, 1.8 Hz, 1H), 1.22–1.32 (m, 1H) 0.41–0.69 (m,
4H); 13C NMR (100 MHz, CDCl3) d 167.4, 165.1, 165.0,
163.6, 154.2, 134.8, 133.7, 132.0, 128.7, 126.6, 126.0, 125.6,
125.4, 123.3, 123.1, 118.5, 112.6, 64.0, 53.7, 33.2, 31.4,
12.0, 8.2, 7.5; HRMS (FAB) calcd for [M+H]+
C24H22NO5S 436.1219, obsd 436.1199. The enantiomeric
excess was 76% as determined by chiral HPLC after
methylation (TMSCl, MeOH) (compared to 76% ee for
the starting material 4).
Data for compound 6c: IR k 2948, 2927, 2858, 2362, 2337,
1749, 1631, 1498, 1452, 1211, 1170, 790, 771; HRMS
(FAB) calcd for [M+H]+ C30H37N2O3S 505.2525, obsd
505.2508.
Data for compound 6d: IR k 2942, 2816, 2765, 1747, 1629,
1501, 1211, 792, 732; HRMS (FAB) calcd for [M+H]+
C29H36N3O3S 506.2474, obsd 506.2477.
Data for compound 6e: IR k 2930, 2849, 1751, 1633, 1497,
1207, 793, 771; HRMS (FAB) calcd for [M+H]+
C29H33N2O3S 489.2212, obsd 489.2205.
Data for compound 6f: IR k 1747, 1629, 1449, 1209, 791,
772; HRMS (FAB) calcd for [M+H]+ C25H27N2O3S
435.1742, obsd 435.1743.
Data for compound 6g: IR k 2923, 2850, 1750, 1632, 1501,
1209, 791, 771; HRMS (FAB) calcd for [M+H]+ C30H35-
N2O3S 503.2368, obsd 503.2361.
9. Experimental procedure for the synthesis of primary amine
8: Aldehyde 5 was dissolved in ethanol (0.05 mmol/ml)
and hydroxylamine hydrochloride (6 equiv) and pyridine
(0.5 mmol/mL) were added. The mixture was refluxed for
3 h, allowed to cool to rt and concentrated. The residue
was dissolved in CH2Cl2 and washed with water. The
aqueous phase was extracted with CH2Cl2 and the
combined organic phases dried over Na2SO4(s), filtered
and concentrated to yield the oxime as a yellow solid in
quantitative yield. The oxime and Zn dust (6 equiv) were
13. Slonim, L. N.; Pinkner, J. S.; Branden, C. I.; Hultgren, S.
J. EMBO. J. 1992, 11, 4747–4756.