Development and characterization of improved β-lactone-based anti-virulence drugs targeting ClpP

Article abstract of DOI:10.1016/j.bmc.2011.07.047

Here, we report the synthesis and in depth characterization of a second generation β-lactone derived virulence inhibitors. Based on initial results that emphasized the intriguing possibility to disarm bacteria in their virulence the present study develops

Full text of DOI:10.1016/j.bmc.2011.07.047

Bioorganic & Medicinal Chemistry 20 (2012) 583–591
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development and characterization of improved b-lactone-based
anti-virulence drugs targeting ClpP
Evelyn Zeiler a, Vadim S. Korotkov b, Katrin Lorenz-Baath b, Thomas Böttcher b, , Stephan A. Sieber a,
⇑
⇑
a Department Chemie, Center for Integrated Protein Science CIPSM, Technische Universität München, Institute of Advanced Studies IAS, Lichtenbergstrasse 4, 85747 Garching, Germany
b AVIRU, EXIST Transfer of Research, OC II, Lichtenbergstrasse 4, 85747 Garching, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 29 July 2011
Here, we report the synthesis and in depth characterization of a second generation b-lactone derived vir-
ulence inhibitors. Based on initial results that emphasized the intriguing possibility to disarm bacteria in
their virulence the present study develops this concept further and analyses the potential of this strategy
for drug development. We were able to expand the collection of bioactive compounds via an efficient syn-
thetic route. Testing of all compounds revealed several hits with anti-virulence activity. Moreover, we
demonstrated that these molecules act solely by reducing virulence but not killing bacteria which is
an important prerequisite for preserving the useful microbiome. Finally, incubation of lactones with
eukaryotic cell lines indicated a tolerable cytotoxicity which is essential for entering animal studies.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Beta-lactones
MRSA
Antibiotics
Virulence
Disarming pathogens
1. Introduction
disarm pathogens instead of killing them. This is expected to de-
crease the risk of rapid resistance development and preserve the
Bacterial infections pose again a serious threat for human
health. Reasons for this critical development are the rapid emer-
gence and spread of multiresistant strains and the steadily
decreasing number of available drugs. As bacteria are killed by
antibiotics, their excessive use exerts a paramount selective pres-
sure favoring the development of any bypassing mechanism which
increases bacterial survival. This has led to the evolution of resis-
tances against almost any available antibiotic. However, antibiotic
drug discovery declines as result of rapid resistance development
which puts the commercial success of any novel antibiotic project
into jeopardy. The lack of novel cellular targets and corresponding
lead structures for drug development challenges the treatment of
bacterial infections now and in the near future. New concepts are
thus urgently needed. Recently, we discovered compounds of the
b-lactone family as potent inhibitors of the protease ClpP, a central
regulator of virulence in Staphylococcus aureus.1,2 ClpP has been
implicated in regulating the expressional levels of major virulence
factors of S. aureus in quorum sensing dependent manner3,4 and
was thus proposed as novel drug target.5 We demonstrated that
a chemical knockout of ClpP by our b-lactones leads to a global de-
crease in virulence factor production. The novel concept aims to
host’s endogenous microbiome. Based on our initial inhibitor U1
(Fig. 2A) we demonstrated its anti-virulence capacity even against
aggressive and multiresistant S. aureus strains (MRSA).1,2 Here, we
report our efforts to expand the structural space of b-lactones, de-
fine leads for drug development and establish a GMP (good manu-
facturing practice) conform route for their synthesis. Active
compounds were identified by a combination of enzyme inhibition
and virulence factor bioassays and mechanistically supported by
competitive proteomic profiling. We could confirm the hypothesis
that growth of commensal bacteria of the human microbiome is
not adversely affected and that the tolerable toxicity profile with
eukaryotic cell lines makes our lead structures ideal candidates
for a novel generation of anti-infective drugs.
2. Results and discussion
2.1. Expanding the structural space of b-lactones
One aim of the present study is to exploit the structural space
for ClpP inhibition in more detail.6,7 We used a previously estab-
lished strategy to synthesize racemic trans-3,4-substituted b-lac-
tones from thioesters and aldehydes in
(strategy A, Fig. 1).
a one-pot reaction
⇑
Corresponding authors. Present address: Harvard Medical School, Department
The five new derivatives were obtained by this strategy in low
yields (5–15% for lactonization) and comprised different side
groups which aimed to vary the motive of U1 (1) by the hydropho-
bicity (2), the chain length (3), or both (4, 5 and 6) (Fig. 2A). To as-
sess the potency of the b-lactones to inhibit ClpP protease activity
of Biological Chemistry and Molecular Pharmacology, 240 Longwood Ave., Boston,
MA 02115, USA (T. Böttcher). Tel.: +1 617 432 3801 (T. Böttcher), +49 89 289 13302;
fax: +49 89 289 13210 (S.A. Sieber).
E-mail addresses: boettcher@aviru.de (T. Böttcher), stephan.sieber@tum.de
(S.A. Sieber).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.047

584
E. Zeiler et al. / Bioorg. Med. Chem. 20 (2012) 583–591
zone around colonies on sheep blood agar.10 These hemolysins are
known to be under control of ClpP4 and have been reported to play
important roles in pathogenesis.11–14
It is known for S. aureus that the expression of virulence factors
in suspension culture differs significantly from that of bacteria
grown on solid medium.15 This may also reflect the different
modes of pathogenesis during systemic and tissue infections. In
our assay with liquid medium we cultured the bacteria under dif-
ferent lactone concentrations and measured hemolysin production
by the ability of sterile filtered supernatants to lyse SRBCs. Here,
Figure 1. Synthesis of b-lactones according to strategy A.
we performed an in vitro peptidase assay with purified recombi-
nant S. aureus ClpP and a fluorogenic model substrate.
we obtained EC50 values of 11
lM for 1 and 43
lM for 2 whereas
Compounds 1 and 4 lead to peptidase inhibition between 30%
4 had only reached approx. 20% inhibition at 100
lM (Fig. 4A).
and 70% with EC50 values of 3.4 and 5.4
(Fig. 2B). The lactone 2 was found to fully inhibit ClpP peptidase
activity with an EC50 of 4.5 M while all other compounds 3, 5
lM, respectively
The potency of 2 (ED50 = 15 nmol) was also closest to 1 (ED50 = 7 n-
mol) on blood agar plates while 4 showed only incomplete inhibi-
tion of hemolysis (Fig. 4B). All other compounds were either much
less active or inactive which is consistent with our ClpP peptidase
assay. Differences between activities of 1, 2 and 4 in competitive
and hemolysis inhibition experiments could be attributed to differ-
ent kinetics of uptake and stability in the bacterial cell. We thus
l
and 6 had no effect (Fig. 2B and C). Although this assay is useful
to demonstrate the principal inhibition of ClpP activity, it has to
be considered that the natural function of ClpP is to degrade pro-
teins rather than peptides and that ClpP requires for its full prote-
olytic activity interactions with many additional proteins in the
cellular environment, most notably AAA+ ATPases that recognize
and unfold substrate proteins and translocate them for degradation
to ClpP. To investigate if ClpP is also targeted by our compounds in
living cells, we performed a competitive activity based proteome
profiling (ABPP) experiment (Fig. 3A).8
identified
1 and 2 as two potent leads for further drug
development.
To increase yields, avoid the diastereomeric by-product and
establish an up-scalable GMP conform production route, we devel-
oped a modular synthesis strategy (strategy B) applicable for both
compounds (Fig. 5).
We used an ABPP probe analogue of U1 (U1P) equipped with a
terminal alkyne tag for click chemistry (CC) that has been previ-
ously demonstrated as selective probe for ClpP9 and applied it to
living cells of S. aureus. When ClpP is targeted in live cells, pre-
incubation of the cells with a 20-fold excess of the novel b-lactones
should decrease or abolish labeling by U1P (Fig. 3A). Indeed, only
inhibitors 1, 2 and 4 significantly reduced labeling by the ABPP
probe (Fig. 3B). This demonstrates that ClpP is the preferred target
of these lactones in living cells and underlines the results of the
ClpP inhibition assay.
10-Undecenoyl chloride was coupled with 2-mercaptopyridine
in dichloromethane in good yields (92%). The resulting thioester
7 was deprotonated with LHMDS in THF and the enolate formed
was trapped as silyl ketene acetal 8 with TBSCl. A diastereoselec-
tive tandem Mukaiyama aldol-lactonization (TMAL)16 of 8 with
the aldehydes 3-phenylpropanal or 3-(3-pyridyl)propanal 9 cata-
lyzed by ZnCl2 in dichloromethane gave 1 and 2, respectively.
The overall yield of the new synthesis was 45% for 1 and 33% for
2, compared to 20%2 and 15% (present study) with strategy A,
respectively. The novel strategy completely avoids the use of haz-
ardous class 1 solvents17 and uses only Zn2+ as class 3 metal cata-
lyst with minimal safety concerns as specified by the European
Medicines Agency.18 This allows GMP conform synthesis for future
GLP (good laboratory practice) studies.
To test the efficacy of the compounds against virulence of living
cells of the pathogen S. aureus we used agar plate and liquid med-
ium based hemolysis assays. Production of
a-toxin and b-toxin
leads to rupture of sheep red blood cells (SRBCs) resulting in a clear
Figure 2. Second generation b-lactones as ClpP inhibitors. (A) U1 (1) and a set of novel b-lactones (2–6). (B and C) peptidase activity of recombinant S. aureus ClpP with
different b-lactones.

E. Zeiler et al. / Bioorg. Med. Chem. 20 (2012) 583–591
585
Figure 3. ClpP is targeted by certain b-lactones in living cells. (A) Principle of competitive ABPP using probe U1P. (B) Competitive labeling of ClpP in the proteome of live S.
aureus cells by ABPP.
Figure 4. Hemolysis assays with sheep red blood cells. (A) Using spent culture supernatants of S. aureus cultures in liquid medium incubated with b-lactones and (B) agar
plate based assay.
Figure 5. Improved synthesis of b-lactones (strategy B) as applied for compounds 1
and 2.
Figure 6. Growth of commensal bacteria remains unaffected upon treatment with
1 even at a high concentration of 1 mM.

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E. Zeiler et al. / Bioorg. Med. Chem. 20 (2012) 583–591
Figure 7. Cell toxicity measured by the MTT assay with (A) NIH3T3 fibroblasts and (B) HaCat keratinocytes.
We hypothesized that b-lactone based virulence inhibitors do
4. Experimental part
4.1. Syntheses
not affect the integrity of the endogenous human microbiome. To
this aim, we performed growth assays with various commensal
species of the human body flora. Gram positive and negative spe-
cies colonizing the skin (S. epidermidis and P. aeruginosa) and the
intestinal tract (E. coli and E. faecalis) were not severely inhibited
even at extremely high concentrations of 1 mM 1, implying
unprecedented advantages over existing antibiotics (Fig. 6).
Although slightly increased growth of Escherichia coli was observed
under these conditions, this effect only occurred in experiments
conducted in well-plates but not in larger cultures.
Growth of S. aureus was not significantly reduced which is
essential for our concept to avoid selective pressure on the bacte-
rial pathogens. We may thus conclude that although compound 1
inhibits ClpP and suppresses virulence of S. aureus, the growth of
this and other bacterial species remains unaffected.
Next we were interested to examine the cytotoxicity of our lead
structures against eukaryotic cells. As primary indications of our
compounds could be topical treatment of infections we used two
skin cell lines. The MTT tetrazolium salt assay for metabolic activ-
ity as a measure for cytotoxicity was performed on NIH3T3 mouse
fibroblasts and human HaCat keratinocytes.
The results of these assays indicate especially low cytotoxicity
for 1 in NIH3T3 cells (Fig. 7A). Metabolic activity of NIH3T3 fibro-
blasts dropped for 1 only to approx. 80% and remained unaffected
4.1.1. General
All chemicals were of reagent grade or better and used without
further purification. Chemicals and solvents were purchased from
Sigma Aldrich, Acros Organics, ABCR GmbH & Co. KG and SAFC Sup-
ply solutions. For all reactions, only commercially available solvents
of purissimum grade, dried over molecular sieve and stored under
argon atmosphere were used. Solvents for chromatography and
workup purposes were generally of technical grade and purified be-
fore use by distillation. Temperatures were measured externally. All
experiments were performed under nitrogen or argon atmosphere.
Column chromatography was performed on silica gel (Merck,
0.035–0.070 mm, mesh 60 Å). For TLC analysis, Merck TLC Silica
Gel 60 F254 aluminium sheets were used. 1H NMR and 13C NMR
spectra were recorded on a Varian NMR-System 600 or 300
(600 MHz, 300 MHz), a Bruker AV 500 or AV 360 (500 MHz,
360 MHz) and referenced to the residual proton and carbon signal
of the deuterated solvent, respectively. Mass spectra in ESI mode
were obtained by a Thermo Finnigan LTQ FT or a Thermo Finnigan
LCQ classic in combination with the HPLC-System Hewlett Packard
Agilent 1100 FT. EI-MS was obtained by a Thermo Finnigan MAT
8200 (70 eV). IR spectra were measured by a FT/IR-4100 instrument
from JASCO.
up to 1 mM while 2 exhibited its EC50 at 50
cytes were more sensitive for 1 and gave EC50 values of 91 and
75 M for 1 and 2, respectively (Fig. 7B). These data indicate mod-
lM. HaCat keratino-
l
4.1.2. Synthesis of S-aryl thioates
erate cytotoxicity that needs to be further evaluated in living
organisms.
4.1.2.1. S-Phenyl-10-undecene thioate (10).
S-Phenyl-10-
undecene thioate (10) was prepared as described before.7 To a stir-
ring solution of thiophenol (1.02 mL, 10.0 mmol) and NEt3
(1.40 mL, 10.0 mmol) in toluene (20 mL) was added drop wise over
30 min a solution of 10-undecenoyl chloride (2.20 mL, 10.2 mmol)
in toluene (30 mL). The reaction was monitored by TLC and was
completed after 20 min. The mixture was then washed with satu-
rated NaHCO3 solution (30 mL) and brine (10 mL), dried over
MgSO4 and the solvent was removed under reduced pressure. Flash
column chromatography on silica (iso-hexane/ethyl acetate 25:1)
yielded 2.49 g (88%) of S-phenyl-10-undecene thioate (10) as col-
orless oil, Rf = 0.43. The spectral data are identical to those pub-
lished previously.7
3. Conclusion
We here report novel b-lactones as efficient inhibitors of bacterial
virulence. These were identified by ClpP target enzyme inhibition, pro-
teomic in situ experiments and inhibition of hemolysin production in
living S. aureus cells. We defined lead structures for future drug devel-
opment and established an improved up-scaled, diastereoselective and
GMP conform synthesis. While the compounds abolish virulence via
inhibition of the protease ClpP, there is no adverse effect on the bacte-
rial growth of S. aureus and members of the natural human body flora.
Consequently, we might expect less selective pressure and thus a re-
duced resistance development as well as the preservation of the hu-
man microbiome. Severe side effects through the destruction of the
commensal body flora and the rapid resistance development repre-
sent the most challenging issues with classical antibiotic therapy.
Our results qualify the concept of ClpP inhibition as a promising
strategy for potential therapeutic intervention and demonstrate
the great value of our compounds as leads for a novel generation
of anti-infective drugs.
4.1.2.2. S-Phenyl-N-(tert-butoxycarbonyl)-11-aminoundecane
thioate (11).
noundecanoic acid (16) (301 mg, 1.00 mmol) in CH2Cl2 (5 mL) was
added thiophenol (153 L, 1.50 mmol), DCC (227 mg, 1.10 mmol)
To a solution of N-(tert-butoxycarbonyl)-11-ami-
l
and DMAP (14 mg, 0.10 mmol) and the reaction mixture was stirred
for 6 h. The remaining precipitate was filtered off and the residue
was washed with CH2Cl2 (20 mL). The solvent was removed
under reduced pressure. Flash column chromatography on silica

E. Zeiler et al. / Bioorg. Med. Chem. 20 (2012) 583–591
587
(iso-hexane/ethyl acetate 8:1) afforded 270 mg (69%) of the product
11 as a white solid, Rf = 0.26. Mp 59 °C. 1H NMR (599 MHz, CDCl3) d
7.40–7.38 (m, 5H, S-phenyl), 4.48 (s, 1H, NH), 3.09 (dd, J = 13.0,
6.4 Hz, 2H, CH2N), 2.66–2.61 (m, 2H, COCH2), 1.69 (dt, J = 15.1,
7.4 Hz, 2H, CH2), 1.48–1.40 (m, 2H, CH2), 1.43 (s, 9H, OtBu), 1.37–
1.23 (m, 12H, (CH2)6). 13C NMR (151 MHz, CDCl3) d 197.52, 155.93,
134.43, 129.24, 129.10, 127.93, 78.95, 43.69, 40.60, 30.03, 29.43,
4.1.3.2. 4-(N,N-Dimethylamino)-N’-(6-hydroxyhexyl)benzamide
(14). To a stirred solution of 2,5-dioxopyrrolidin-1-yl 4-(N,N-
dimethylamino)benzoate (13) (220 mg, 0.90 mmol) in acetonitrile/
water 1:1.2 (3 mL) was added NEt3 (750 L, 5.4 mmol) and 6-ami-
l
no-1-hexanol (106 mg, 0.90 mmol). The reaction was monitored by
TLC (ethyl acetate, Rf = 0.24) and was completed after 30 min. Then
the mixture was extracted with ethyl acetate (2 ꢁ 20 mL), the com-
bined organic layers were washed with brine (2 mL) and dried over
MgSO4 to yield 191 mg (80%) 4-(N,N-dimethylamino)-N0-(6-
hydroxyhexyl)benzamide (14) as a pale yellow solid. Mp 91 °C.
1H NMR (360 MHz, CDCl3) d 7.68 (d, J = 8.9 Hz, 2H, Harom), 6.67
(d, J = 8.9 Hz, 2H, Harom), 6.14 (s, 1H, NH), 3.64 (t, J = 6.5 Hz, 2H,
NHCH2), 3.44 (dd, J = 13.1, 6.9 Hz, 2H, CH2OH), 3.01 (s, 6H,
N(CH3)2), 2.20 (br s, 1H, OH), 1.68–1.32 (m, 8H, (CH2)4). 13C NMR
(91 MHz, CDCl3) d 167.55, 152.39, 131.82, 128.29, 121.52, 111.09,
29.28, 29.22, 29.18, 28.90, 28.41. IR (film)
m
= 3366 cmꢀ1, 2919,
2852, 1699, 1684, 1646, 1523, 1446, 1430, 1410, 1390, 1365, 1328,
1281, 1241, 1213, 1173, 1117, 1008, 983, 965, 908, 893, 869. HRMS
(ESI) calcd for C22H35NNaO3S [M+Na]+ 416.2235, found 416.2230.
4.1.2.3. S-Pyridin-2-yl-10-undecene thioate (7).
To a stirred
solution of 2-pyridylmercaptane (6.6 g, 60 mmol) in CH2Cl2
(150 mL) was added 10-undecenoyl chloride (10 g, 50 mmol) at
0 °C followed by Et3N (15 mL, 108 mmol). The solution was stirred
for 2 h at rt, quenched with 1 N HCl, phases were separated and the
organic phase was washed with saturated NaHCO3 solution and
then dried over Na2SO4. Volatiles were removed under reduced
pressure yielding a yellow oil that was purified by flash column
chromatography (hexane/ethyl acetate 2:1, Rf = 0.76). Yield:
12.7 g (92%). 1H NMR (360 MHz, CDCl3) d 8.62–8.65 (m, 1H, pyri-
dine), 7.72–7.78 (m, 1H, pyridine), 7.61–7.65 (m, 1H, pyridine),
7.20 (ddd, J = 5, 7, 1 Hz, 1H, pyridine), 5.76–5.89 (m, 1H, CH@CH2),
4.92–5.05 (m, 2H, CH2@CH), 2.71 (t, J = 7 Hz, 2H, CH2), 2.00 2.09 (m,
2H, CH2), 1.68–1.79 (m, 2H, CH2), 1.25–1.45 (m, 10H, (CH2)5). 13C
NMR (91 MHz, CDCl3): 196.59, 151.73, 150.39, 139.15, 137.04,
130.09, 123.40, 114.17, 44.24, 33.77, 29.21, 29.17, 29.02, 28.91,
62.58, 45.82, 40.12, 39.62, 26.53, 8.60. IR (film)
m ,
= 3318 cmꢀ1
3306, 2926, 2856, 2816, 1747, 1684, 1604, 1549, 1513, 1444,
1412, 1364, 1328, 1300, 1230, 1204, 1173, 1131, 1061, 946, 925,
872, 829, 807, 768. HRMS (ESI) calcd for C15H25N2O2 [M+H]+
265.1916, found 265.1907.
4.1.3.3. N-(tert-Butoxycarbonyl)-12-aminododecan-1-ol (15). To a
mixture of NaOH (43 mg, 1.1 mmol), in water/tert-BuOH 1:1
(1.0 mL) was added di-tert-butyl dicarbonate (225 lL, 1.05 mmol)
and 12-amino-dodecan-1-ol (199 mg, 1.00 mmol). After the viscous
mixture was stirred for 18 h, 0.3 M HCl solution (1.5 mL) was added
and the aqueous layer was extracted with ethyl acetate (3 ꢁ 30 mL).
The combined organic layers were washed with brine (5 mL) and
dried over MgSO4. The solvent was removed under reduced pressure
and 294 mg (98%) of the product was obtained as white solid. Mp
75 °C. 1H NMR (300 MHz, CDCl3) d 4.51 (s, 1H, NH), 3.66 (dd, J = 11.1,
6.5 Hz, 2H, NHCH2), 3.11 (dd, J = 13.2, 6.6 Hz, 2H, CH2OH), 1.73–1.16
(m, 21H, (CH2)10, OH), 1.46 (s, 9H, OtBu). 13C NMR (151 MHz, CDCl3)
d 155.94, 78.96, 63.03, 40.60, 32.77, 30.03, 29.53, 29.48, 29.48, 29.37,
28.89, 25.41. IR (film)
m
= 2925 cmꢀ1, 2855, 1627, 1575, 1560,
1449, 1417, 1253, 1081, 838, 783. MS (ESI) positive ion mode: m/
z = 278 ([M+H]+, 100%). HRMS (ESI) calcd for C16H24NOS [M+H]+
278.1579, found 278.1564.
4.1.2.4. S-Pyridin-2-yl tetradecane thioate (12).
S-Pyridin-2-
yl tetradecane thioate (12) was synthesized analogous to 7 from 2-
pyridylmercaptane (3.3 g, 30 mmol), tetradecanoyl chloride (8 g,
25 mmol) and Et3N (10 mL, 80 mmol) in CH2Cl2 (75 mL). Yield:
6.4 g (80%). 1H NMR (360 MHz, CDCl3) d 8.62–8.65 (m, 1H, pyri-
dine), 7.72–7.78 (m, 1H, pyridine), 7.62–7.65 (m, 1H, pyridine)
7.26–7.32 (m, 1H, pyridine), 2.71 (t, J = 7 Hz, 2H, CH2), 1.22–1.32
(22H, m, (CH2)11), 0.89 (t, J = 7 Hz, 3H, CH3). 13C NMR (91 MHz,
CDCl3) d 196.6, 151.8, 150.4, 139.15, 137.0, 130.1, 123.4, 44.3,
31.9, 29.66, 29.63, 29.37, 29.34, 29.23, 25.4, 22.7, 14.1. HRMS
(ESI) calcd for C19H31NOS [M+H]+ 321.2126, found 321.2195.
29.23, 28.40, 26.77, 25.69. IR (film) m
= 3368 cmꢀ1, 2919, 2851, 1685,
1653, 1558, 1523, 1481, 1469, 1457, 1443, 1389, 1364, 1337, 1288,
1266, 1243, 1225, 1173, 1142, 1059, 1027, 994, 978, 867, 781, 741,
720, 679, 661. HRMS (ESI) calcd for C17H35NNaO3 [M+Na]+ 324.2515,
found 324.2509.
4.1.3.4. N-(tert-Butoxycarbonyl)-11-aminoundecanoic acid
(16). The product was prepared as described for 15. To a mixture
of NaOH (213 mg, 5.30 mmol), in water/tert-BuOH 1:1 (1.2 mL)
was added di-tert-butyl dicarbonate (964 lL, 1.05 mmol) and 11-
aminoundecanoic acid (863 mg, 4.30 mmol). After stirring for 18 h
and work up 518 mg (40%) of the product (16) was obtained as
white solid. Mp 64 °C. 1H NMR (300 MHz, CDCl3) d 4.56 (s, 1H, NH),
3.11 (dd, J = 12.9, 6.5 Hz, 2H, NHCH2), 2.34 (t, J = 7.2 Hz, 2H,
CH2COOH), 1.70–1.57 (m, 2H, CH2), 1.54–1.40 (m, 2H, CH2), 1.46 (s,
9H, OtBu), 1.44–1.22 (m, 12H, (CH2)6). 13C NMR (151 MHz, CDCl3) d
179.30, 155.98, 79.02, 41.67, 40.60, 34.39, 34.21, 29.98, 29.39,
4.1.3. Synthesis of further starting compounds
4.1.3.1. 2,5-Dioxopyrrolidin-1-yl 4-(N,N-dimethylamino)benzo-
ate (13).
4-(N,N-Dimethylamino)benzoic acid (332 mg,
2.00 mmol), N-hydroxysuccinimide (244 mg, 2.10 mmol) and 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide, (EDAC) (402 mg,
2.1 mmol) were suspended in CH2Cl2 (10 mL). After 20 min the
suspension became a clear violet solution. The reaction mixture
was monitored by TLC (iso-hexane/ethyl acetate 1:1, 1% AcOH,
Rf = 0.30) and the reaction was completed after 2.5 h. Then water
(10 mL) was added and the aqueous layer was extracted with
dichloromethane (2 ꢁ 10 mL). The combined organic layers were
washed with brine (10 mL), dried over MgSO4 and the solvent
was removed under reduced pressure to yield 451 mg (86%) of
13 as a white solid. Mp 181 °C. 1H NMR (300 MHz, CDCl3) d 7.99
(d, J = 9.1 Hz, 2H, Harom), 6.67 (d, J = 9.2 Hz, 2H, Harom), 3.09 (s,
6H, N(CH3)2), 2.89 (s, 4H, (CH2)2). 13C NMR (75 MHz, CDCl3) d
169.83, 161.92, 154.41, 132.55, 110.81, 110.65, 39.98, 25.70. IR
29.28, 29.18, 29.02, 28.40. IR (film)
m
= 3365 cmꢀ1, 2976, 2917,
2852, 1698, 1682, 1523, 1471, 1443, 1428, 1410, 1388, 1364, 1328,
1280, 1239, 1213, 1186, 1171, 1116, 1072, 1058, 1044, 1025, 1008,
980, 963, 921, 902, 870, 781, 738, 719. ꢀ HRMS (ESI) calcd for
C
16H31NNaO4 [M+Na]+ 324.2151, found 324.2145. The spectral
data are identical to those published in literature.20
4.1.4. General procedure for the synthesis of aldehydes from
alcohols
Aldehydes were prepared from the respective alcohols by
Swern oxidation.
(film)
1182, 1070, 1012, 979, 944, 825, 755. HRMS (ESI) calcd for
13H15N2O4 [M+H]+ 263.1032, found 263.1028. The spectral data
are identical to those published in literature.19
m
= 1735 cmꢀ1, 1604, 1535, 1436, 1375, 1269, 1248, 1207,
4.1.4.1. 3-(3-Pyridyl)propanal (9).
oxalyl chloride (532 L, 6.20 mmol) in CH2Cl2 (15 mL) was added
at ꢀ78 °C successively DMSO (881 L, 12.4 mmol), 3-(3-pyridyl)-
To a stirred solution of
C
l
l

588
E. Zeiler et al. / Bioorg. Med. Chem. 20 (2012) 583–591
1-propanol (712 mg, 5.17 mmol) in CH2Cl2 (1.5 mL) and NEt3
(3.96 mL, 28.4 mmol). The reaction mixture was stirred at
ꢀ78 °C and monitored by TLC (iso-hexane/ethyl acetate 1:1,
Rf = 0.16). After 4 h it was warmed up to room temperature.
Then water (15 ml) was added and the aqueous layer was ex-
tracted with diethyl ether (3 ꢁ 50 mL). The combined organic
layers were washed with brine (5 mL), dried over MgSO4 and
the solvent was removed under reduced pressure. Purification
by Kugelrohr distillation yielded 293 mg (42%) of 9 as a pale yel-
low oil. Bp 107 °C (0.6 mbar). 1H NMR (300 MHz, CDCl3) d 9.77
(t, J = 1.1 Hz, 1H, CHO), 8.43 (m, 1H, 2-pyridine-CH), 8.40 (dd,
J = 4.8, 1.7 Hz, 1H, 6-pyridine-CH), 7.48 (ddd, J = 7.8, 2.3, 1.7 Hz,
1H, 4-pyridine-CH), 7.17 (ddd, J = 7.8, 4.8, 0.8 Hz, 1H, 5-pyri-
dine-CH), 2.91 (t, J = 6.9 Hz, 2H, CH2), 2.77 (ddt, J = 6.9, 2.6,
1.1 Hz, 2H, CH2CHO). 13C NMR (75 MHz, CDCl3) d 200.46 (CHO),
149.75 (pyridine-CH), 147.74 (pyridine-CH), 135.82 (pyridine-
CH), 135.78 (pyridine-Cquart), 123.37 (pyridine-CH), 44.70 (CH2),
25.11 (CH2CHO). All spectral data are identical to those pub-
lished in literature.21
4.1.5.2. (3R⁄,4R⁄)-3-Non-8-enyl-4-(2-(3-pyridyl)ethyl)-oxetan-2-
one (2). solution of diisopropylethylamine (88.9 L,
0.630 mmol) in THF (6 mL) was cooled in an ice bath to 0 °C and
under stirring n-butyllithium (252 L, 0.630 mmol, 2.45 M in hex-
ane) was gradually added via a syringe. The reaction mixture was
stirred for 20 min at 0 °C. Then the ice bath was replaced with an
acetone/dry ice bath and the mixture was cooled to ꢀ78 °C. A solu-
tion of S-phenyl-10-undecene thioate (10) (167 mg, 0.604 mmol)
A
l
l
in THF (750
the mixture at ꢀ78 °C for 2 h a solution of 3-(3-pyridyl)propanal
(9) (81.6 mg, 0.604 mmol) in THF (900 L) was added drop wise
lL) was injected dropwise over 20 min. After stirring
l
over 30 min via a syringe that was externally cooled by an alumin-
ium funnel filled with dry ice. The reaction mixture was stirred for
1.5 h at ꢀ78 °C and then gradually warmed up to ꢀ5 °C. Then half
saturated NH4Cl solution (2 ml) was added and the mixture was di-
luted with ethyl acetate (20 ml). The aqueous layer was extracted
with ethyl acetate (2 ꢁ 20 ml) and the combined organic layers
were washed with brine (5 ml) and dried over MgSO4. The solvent
was removed under reduced pressure to give 225 mg of crude
product as yellow oil. Purification by flash column chromatography
on silica (iso-hexane/ethyl acetate 2:1) yielded 27.1 mg (15%) of
trans-3-non-8-enyl-4-(2-pyridin-3-yl-ethyl)-oxetan-2-one (2) as a
pale yellow oil, Rf = 0.22 and 12.5 mg (7%) of cis-3-non-8-enyl-4-
(2-(3-pyridyl)ethyl)-oxetan-2-one (2b) as a pale yellow oil con-
taminated with 30% of the trans-product, Rf = 0.18. Compound 2:
1H NMR (599 MHz, CDCl3) d 8.48–8.46 (m, 2H, 2-pyridine-CH, 6-
pyridine-CH), 7.51 (ddd, J = 7.8, 2.3, 1.7 Hz, 1H, 4-pyridine-CH),
7.23 (ddd, J = 7.8, 4.8, 0.8 Hz, 1H, 5-pyridine-CH), 5.78 (ddt,
J = 16.9, 10.2, 6.7 Hz, 1H, CH@CH2), 4.97 (ddt, J = 17.1, 2.0, 1.6 Hz,
1H, CH@CH2), 4.91 (ddt, J = 10.2, 2.3, 1.2 Hz, 1H, CH@CH2), 4.20
(ddd, J = 8.6, 4.8, 4.0 Hz, 1H, 4-CH), 3.20 (ddd, J = 8.5, 6.8, 4.0 Hz,
1H, 3-CH), 2.81 (ddd, J = 14.7, 9.6, 5.4 Hz, 1H, 2-ethyl-CHa2), 2.70
(ddd, J = 14.2, 9.3, 7.1 Hz, 1H, 2-ethyl-CHb2), 2.17–2.04 (m, 2H,
CH2), 2.01 (ddt, J = 14.8, 6.7, 1.4 Hz, 2H, CH2CH@CH2), 1.82–1.75
(m, 1H, CHa2), 1.71–1.64 (m, 1 H, CHb2), 1.45–1.22 (m, 10H,
(CH2)5). 13C NMR (151 MHz, CDCl3) d 170.91, 149.74, 147.94,
139.00, 135.80, 135.48, 123.48, 114.22, 76.67, 56.32, 35.88, 33.68,
29.16, 29.08, 28.90, 28.78, 28.63, 27.70, 26.89. IR (film)
4.1.4.2.
(17).
4-(N,N-Dimethylamino)-N0-(6-oxohexyl)benzamide
To a solution of oxalyl chloride (60 L, 0.72 mmol) in
L, 1.44 mmol),
4-(N,N-dimethylamino)-N0-(6-hydroxyhexyl)benzamide
(14)
(159 mg, 0.60 mmol) in CH2Cl2 (3 mL) and NEt3 (460 L,
l
CH2Cl2 (2 mL) was added at ꢀ78 °C DMSO (102
l
l
3.30 mmol). The reaction mixture was stirred at ꢀ78 °C for 5 h
and was then warmed up to ꢀ5 °C. After work up and flash column
chromatography on silica (iso-hexane/ethyl acetate 1:2) 103 mg
(65%) of the product 17 was obtained as white solid, Rf = 0.27. 1H
NMR (300 MHz, CDCl3) d 9.77 (t, J = 1.7 Hz, 1H, CHO), 8.02–7.43
(m, 2H, Harom), 6.71–6.65 (m, 2H, Harom), 6.15 (br s, 1H, NH),
3.45 (td, J = 7.0, 6.0 Hz, 2H, NHCH2), 3.02 (s, 6H, N(CH3)2), 2.46
(td, J = 7.2, 1.7 Hz, 2H, CH2CHO), 1.75–1.57 (m, 4H, (CH2)2), 1.49–
1.35 (m, 2H, CH2). 13C NMR (75 MHz, CDCl3) d 202.55, 167.46,
152.36, 128.30, 121.53, 111.13, 43.74, 40.16, 39.50, 29.62, 26.45,
21.65. IR (film)
m
= 3335 cmꢀ1, 3309, 3276, 2922, 2859, 2818,
1717, 1685, 1607, 1542, 1513, 1444, 1411, 1363, 1328, 1299,
1229, 1203, 1173, 1134, 1064, 993, 946, 829, 768, 732. ꢀ HRMS
(ESI) calcd for C15H23N2O2 [M+H]+ 263.1760, found 263.1755.
m
= 2925 cmꢀ1, 2854, 1815, 1729, 1639, 1575, 1479, 1456, 1423,
1387, 1321, 1304, 1284, 1242, 1190, 1116, 1082, 1042, 1026,
997, 956, 907, 841, 795, 714. HRMS (ESI) calcd for C19H28NO2
[M+H]+ 302.2120, found 302.2115. Compound 2b: 1H NMR
(599 MHz, CDCl3) d 8.49–8.46 (m, 1H, 2-pyridine-CH, 6-pyridine-
CH), 7.51 (ddd, J = 7.8, 2.2, 1.8 Hz, 1H, 4-pyridine-CH), 7.23 (ddd,
J = 7.7, 4.8, 0.7 Hz, 1H, 5-pyridine-CH), 5.78 (ddt, J = 16.9, 10.1,
6.7 Hz, 1H, CH@CH2), 4.99–4.94 (m, 1H, CH@CH2), 4.93–4.89 (m,
1H, CH@CH2), 4.52 (ddd, J = 10.6, 6.4, 3.1 Hz, 1H, 4-CH), 3.61
(ddd, J = 9.0, 7.3, 6.6 Hz, 1H, 3-CH), 2.89 (ddd, J = 14.4, 9.7, 4.9 Hz,
1H, 2-ethyl-CHa2), 2.71 (ddd, J = 14.1, 9.3, 7.3 Hz, 1H, 2-ethyl-
CHb2), 2.10–1.90 (m, 2H, CH2), 1.82–1.73 (m, 1H, CHa2), 1.63–1.44
(m, 2H, CH2), 1.40–1.22 (m, 9H, (CH2)4, CHb2). 13C NMR
4.1.4.3. N-(tert-Butoxycarbonyl)-12-aminododecanal (18). To
a solution of oxalyl chloride (84
was added at ꢀ78 °C DMSO (142
carbonyl)-12-aminododecan-1-ol (15) (250 mg, 0.83 mmol) in
CH2Cl2 (1.3 mL) and NEt3 (630 L, 4.5 mmol). The reaction
lL, 1.0 mmol) in CH2Cl2 (2.5 mL)
lL, 2.0 mmol), N-(tert-butoxy-
l
mixture was stirred at ꢀ78 °C for 3 h and was then warmed up
to room temperature. After work up and flash column chroma-
tography on silica (iso-hexane/ethyl acetate 9:1) 182 mg (73%) of
18 was obtained as white solid, Rf = 0.19. Mp 51 °C. 1H NMR
(400 MHz, CDCl3) d 9.76 (t, J = 1.9 Hz, 1H, CHO), 4.49 (s, 1H, NH),
3.09 (t, J = 7.0 Hz, 2H, NHCH2), 2.41 (td, J = 7.4, 1.9 Hz, 2H,
CH2CHO), 1.43 (s, 9H, OtBu), 1.67–1.21 (m, 18H, (CH2)9). 13C
(151 MHz, CDCl3)
d 171.65, 149.87, 147.91, 139.01, 135.96,
135.73, 123.46, 114.19, 74.14, 52.73, 33.69, 31.84, 29.25, 29.08,
28.91, 28.86, 28.79, 27.52, 23.94.
NMR (101 MHz, CDCl3)
29.48, 29.44, 29.35, 29.31, 29.24, 29.13, 28.41, 26.76, 22.05. IR
(film)
= 3371 cmꢀ1, 3008, 2981, 2917, 2848, 2722, 1722, 1680,
d
202.99, 77.19, 43.90, 30.04,
4.1.5.3. (3R⁄,4R⁄)-4-(4-(N,N-Dimethylamino)benzamido-5-pen-
m
1513, 1480, 1466, 1411, 1391, 1367, 1318, 1288, 1268, 1245,
1227, 1167, 1119, 1086, 1049, 1006, 980, 865, 784, 765, 750, 723.
tyl)-3-(non-8-enyl)oxetan-2-one (4).
The reaction, as de-
scribed for 2, was performed with S-phenyl-10-undecene thioate
(10) (87 mg, 0.31 mmol) and 4-(N,N-dimethylamino)-N0-(6-
oxohexyl)benzamide (17) (81 mg, 0.31 mmol). Standard workup
and purification by flash column chromatography on silica (iso-
hexane/ethyl acetate 2:1) yielded 16 mg (12%) of the product as
colorless oil, Rf = 0.21. 1H NMR (500 MHz, CDCl3) d 7.73–7.68 (m,
2H, Harom), 6.74 (d, J = 7.2 Hz, 2H, Harom), 6.10 (br s, 1H, NH),
5.82 (ddt, J = 16.9, 10.1, 6.7 Hz, 1H, CH@CH2), 5.04–4.98 (m, 1H,
CH@CH2), 4.95 (ddt, J = 10.3, 2.3, 1.2 Hz, 1H, CH@CH2), 4.23 (ddd,
HRMS (ESI) calcd for
300.2529.
C
17H34NO3 [M+H]+ 300.2539, found
4.1.5. Synthesis of b-lactones according to strategy A
4.1.5.1. General procedure.
b-Lactones were prepared
according to a method developed by Danheiser and Novick6 and
as described before by Böttcher and Sieber2,7 from various S-phenyl
thioates and aldehydes.

E. Zeiler et al. / Bioorg. Med. Chem. 20 (2012) 583–591
589
J = 7.6, 5.6, 4.2 Hz, 1H, 4-CH), 3.46 (dd, J = 13.4, 6.1 Hz, 2H, CH2),
3.18 (ddd, J = 8.7, 6.7, 4.0 Hz, 1H, 3-CH), 3.04 (s, 6H, N(CH3)2), 2.06
(dd, J = 14.5, 6.7 Hz, 2H, CH2), 1.91–1.69 (m, 4H, (CH2)2), 1.68–1.60
(m, 2H, CH2), 1.55–1.27 (m, 16H, (CH2)8). 13C NMR (91 MHz, CDCl3)
d 171.53, 167.30, 151.91, 139.04, 128.30, 114.19, 111.63, 77.97,
77.20, 56.15, 40.47, 39.60, 34.33, 33.70, 29.67, 29.20, 29.11, 28.93,
2.03–2.26 (m, 2H, CH2), 1.61–1.85 (m, 2H, CH2), 1.35–1.46 (m,
1H, CHH), 1.25–1.35 (m, 19H, (CH2)9+CHH), 0.90 (t, J = 7 Hz, 3H,
CH3). 13C NMR (91 MHz, CDCl3): 171.4, 140.2, 128.6, 128.3, 126.4,
77.1, 56.3, 36.2, 31.9, 31.4, 29.64, 29.63, 29.59, 29.49, 29.3, 29.28,
29.24, 27.8, 26.9, 22.7, 14.1. IR (film)
m
= 2924 cmꢀ1, 2853, 1821,
1455, 1560, 1449, 1417, 1253, 1081, 838, 783. HRMS (APCI) calcd
28.80, 27.81, 26.91, 26.55, 24.84. IR (film)
m
= 2924 cmꢀ1, 2854,
for C23H37O2 [M+H]+ 345.2794, found 345.2800.
1812, 1636, 1606, 1541, 1509, 1361, 1328, 1294, 1230, 1203, 1172,
1123, 1064, 994, 945, 908, 876, 829, 767. EI-MS, m/z (%): 428.3
(16) [M]+, 384.3 (21) [MꢀCO2]+, 178.1 (10), 164.0 (43)
[MꢀC17H28O2]+, 148.0 (100) [MꢀC17H30NO2]+, 43.9 (29). HRMS (EI)
calcd for C26H40N2O3 [M]+ 428.30389, found 428.30349.
4.1.6. Synthesis of b-lactones according to strategy B
4.1.6.1. (Z)-2-((1-((tert-Butyldimethylsilyl)oxy)undeca-1,10-die-
nyl)thio)pyridine (8).
LHMDS (13 mL, 13 mmol, 1 M in THF)
was placed in a flask cooled to -78 °C. DMF (1.7 mL) was added
via syringe followed by Et3N (3 mL, 21 mmol) and a solution of TBSCl
(1.7 g, 11 mmol) in THF (7.5 mL) and thioester 7 (3 g, 10.8 mmol) in
THF (20 mL). The solution was stirred 1 h at ꢀ78 °C and then let
warm up to 0 °C over 1 h and quenched with H2O. Phases were sep-
arated; the aqueous layer was extracted with EtOAc (3 ꢁ 20 mL),
and the combined organic phase was dried over Na2SO4. The solvent
was distilled off under reduced pressure and the residue was sepa-
rated by column chromatography (eluent hexane/ethyl acetate
1:10, Rf = 0.31) to give slightly yellow oil. Yield: 3 g (71%). 1H NMR
(360 MHz, CDCl3): 8.42–8.45 (m, 1H, pyridine), 7.53–7.59 (m, 1H,
pyridine), 7.33–7.37 (m, 1H, pyridine), 7.01 (ddd, J = 5, 7, 1 Hz, 1H,
pyridine), 5.77–5.90 (m, 1H, CH@CH2), 5.42 (t, J = 7 Hz, 1H,
CH@C(OTBS), 4.92–5.05 (m, 2H, CH@CH2), 2.16 2.24 (m, 2H, @CH-
CH2-CH2), 2.01–2.11 (m, 2H, @CH-CH2-CH2), 1.25–1.49 (m, 10H,
(CH2)5), 0.89 (s, 9H, tBu), 0.10 (s, 6H, Si(CH3)2) ppm. 13C NMR
(91 MHz, CDCl3): 160.6, 149.4, 139.2, 136.5, 124.1, 121.4, 119.6,
114.2, 33.8, 29.32, 29.30, 29.15, 29.11, 28.9, 26.8, 25.7, 18.1, -4.4.
4.1.5.4.
cyl]-3-(non-8-enyl)-oxetan-2-one (5).
(3R⁄,4R⁄)-4-[N-(tert-Butoxycarbonyl)-11-aminounde-
The reaction, as de-
scribed for (2), was performed with S-phenyl-10-undecene
thioate (10) (92.3 mg, 0.334 mmol) and N-(tert-butoxycarbonyl)-
12-aminododecanal (18) (100 mg, 0.334 mmol). Standard workup
and purification by flash column chromatography on silica (iso-
hexane/ethyl acetate 8:1) yielded 8 mg (5%) of the product as
white solid, Rf = 0.33. Mp 45 °C. 1H NMR (500 MHz, CDCl3) d 5.83
(ddt, J = 17.0, 10.2, 6.7 Hz, 1H, CH@CH2), 5.05–4.99 (m, 1H,
CH@CH2), 4.96 (ddt, J = 10.3, 2.2, 1.2 Hz, 1H, CH@CH2), 4.51 (s,
1H, NH), 4.23 (ddd, J = 7.0, 6.1, 4.0 Hz, 1H, 4-CH), 3.18 (ddd,
J = 8.8, 6.6, 4.0 Hz, 1H, 3-CH), 3.12 (s, 2H, NHCH2), 2.06 (dd,
J = 14.2, 6.9 Hz, 2H, CH2), 1.92–1.80 (m, 2H, CH2), 1.80–1.19 (m,
30H, (CH2)15), 1.47 (s, 9H, OtBu). 13C NMR (91 MHz, CDCl3) d
171.65, 155.93, 139.06, 114.24, 78.16, 77.21, 56.13, 34.45, 33.74,
30.09, 29.52, 29.50, 29.45, 29.40, 29.28, 29.24, 29.15, 28.96,
28.84, 28.44, 27.88, 26.97, 26.80, 25.04. IR (film)
m
= 3366 cmꢀ1
,
IR (film) m
= 2925 cmꢀ1, 2855, 1627, 1575, 1560, 1449, 1417, 1253,
2979, 2919, 2851, 2359, 2337, 1793, 1686, 1523, 1468, 1389,
1364, 1275, 1241, 1173, 1145, 1080, 1021, 992, 973, 910, 870,
830, 802, 782, 737, 723. EI-MS, m/z (%): 364.3 (31) [MꢀC5H9O2]+,
321.3 (39) [MꢀC6H8O4]+, 57.0 (100) [MꢀC24H42NO4]+, 41.0 (30).
HRMS (ESI) calcd for C28H52NO4 [M+H]+ 466.3896, found 466.3886.
1081, 838, 783. MS (ESI): m/z = 392 ([M+H]+, 100). HRMS (ESI) calcd
for C22H38NOSSi [M+H]+ 392.2443, found 392.2419.
4.1.6.2. (3R⁄,4R⁄)-3-(8-Nonenyl)-4-(2-phenylethyl)oxetan-2-one
(1).
To the solution of ZnCl2 (0.72 g, 5.4 mmol) in CH2Cl2
solution of 3-phenylpropanal (0.37 g,
(18 mL) was added
a
4.1.5.5. (3R⁄,4R⁄)-3-(N-(tert-Butoxycarbonyl)-9-aminononyl)-4-
2.7 mmol) in CH2Cl2 (5 mL) followed by ketene acetal 19 (1.5 g,
3.8 mmol). The reaction mixture was stirred for 4 days. Then the
reaction was quenched with H2O and the organic phase was sepa-
rated and dried over Na2SO4. The solvent was distilled off under re-
duced pressure and the residue was separated by column
chromatography (eluent hexane/ethyl acetate 20:1), Rf = 0.42. Yield:
0.36 g (45%). 1H, 13C NMR and mass-spectra are identical to the pre-
(dec-9-enyl)oxetan-2-one (6).
The reaction, as described for
(2), was performed with 10-undecenal (226 mg, 1.21 mmol) and S-phe-
nyl-N-(tert-butoxycarbonyl)-11-aminoundecane thioate (11) (476 mg,
1.21 mmol). Standard workup and purification by flash column chroma-
tography on silica (iso-hexane/ethyl acetate 8:1) yielded 38 mg (7%) of
the product as white solid, Rf = 0.18. Mp 39 °C. 1H NMR (500 MHz,
CDCl3) d 5.83 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H, CH@CH2), 5.01 (ddt,
J = 17.1, 2.0, 1.6 Hz, 1H, CH@CH2), 4.95 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H,
CH@CH2), 4.51 (s, 1H, NH), 4.23 (ddd, J = 7.3, 6.0, 4.0 Hz, 1H, 4-CH),
3.18 (ddd, J = 8.7, 6.7, 4.0 Hz, 1H, 3-CH), 3.12 (t, J = 6.9 Hz, 2H, CH2N),
2.11 2.02 (m, 2H, CH2), 1.91 1.79 (m, 2H, CH2), 1.78–1.68 (m, 2H, CH2),
1.46 (s, 9H, OtBu), 1.53–1.25 (m, 26H, (CH2)13). 13C NMR (91 MHz, CDCl3)
d171.64, 156.01, 139.12, 114.18, 78.14, 77.23, 56.13, 40.65, 34.44, 33.77,
30.07, 29.40, 29.35, 29.30, 29.25, 29.22, 29.04, 28.87, 28.43, 27.88, 26.97,
viously published data.2 IR (film)
1115, 907, 843, 747.
m
= 2928 cmꢀ1, 2856, 1823, 1450,
4.1.6.3. (3R⁄,4R⁄)-3-(8-Nonenyl)-4-(2-(3-pyridyl)ethyl)oxetan-2-
one (2).
(3R⁄,4R⁄)-3-(8-Nonenyl)-4-(2-(3-pyridyl)ethyl)oxe-
tan-2-one was synthesized analogous to 1 (strategy B) from ketene
acetal (1.0 g, 2.6 mmol), 3-(3-pyridyl)propanal (9) (0.2 g,
8
1.5 mmol) and ZnCl2 (0.4 g, 3.0 mmol) in CH2Cl2 (15 mL). Standard
workup and column chromatography afforded the product (eluent
iso-hexane/ethyl acetate 2:1, Rf = 0.22). Yield: 149 mg (33%). All
spectral data were identical to the data of strategy A.
26.75, 25.03. IR (film)
m
= 2925 cmꢀ1, 2854, 1819, 1704, 1508, 1478,
1457, 1390, 1364, 1268, 1248, 1171, 1125, 1069, 1040, 994, 908, 870,
828, 808, 781, 745. HRMS (EI), m/z (%): calcd for C23H41NO4 [MꢀC4H8]+
395.30356, found 395.30350 (23), 351.31296 (83) [MꢀC5H8O2]+,
307.32382 (100) [MꢀC6H9O4]+. HRMS (ESI) calcd for C27H49NNaO4
[M+Na]+ 474.3559, found 474.3553.
4.2. Compound preparation for biological assays
For biological and biochemical testing, stocks of the compounds
were prepared in DMSO and stored at ꢀ20 °C. Dilutions for assays
were prepared in DMSO and pure DMSO always was included as
control.
4.1.5.6.
(3).
(3R⁄,4R⁄)-3-Dodecyl-4-(2-phenylethyl)oxetan-2-one
(3R⁄,4R⁄)-3-Dodecyl-4-(2-phenylethyl)oxetan-2-one (3)
was synthesized analogous to (2, Strategy A) from thioester 12
(1.0 g, 3.1 mmol), 3-phenylpropanal (0.42 g, 3.1 mmol) and LHMDS
(3.8 mL, 3.8 mmol, 1.0 M in THF) in THF (40 mL), yield: 96 mg (9%).
Rf = 0.21 (hexane/ethyl acetate 20:1) 1H NMR (360 MHz, CDCl3):
7.18–7.37 (m, 5H, phenyl), 4.25 (ddd, J = 7.5, 5.4, 4.0, 1H, 4-CH),
3.20 (ddd, J = 8.5, 6.8, 3.9 Hz, 1H, 3-CH), 2.67–2.90 (m, 2H, CH2),
4.3. Bacterial strains and culture
Staphylococcus aureus NCTC 8325 (Institute Pasteur, France) and
the derived Staphylococcus aureus NCTC 8325-4 strain,

590
E. Zeiler et al. / Bioorg. Med. Chem. 20 (2012) 583–591
Staphylococcus epidermidis RP62A (Institute Pasteur, France), and
Enterococcus faecalis OG1RF (ATCC, USA) were maintained in BHB
(Brain Heart Broth) medium and Pseudomonas aeruginosa PAO1
(Institute Pasteur, France) and Escherichia coli K12 (DSMZ, Germany)
inLB(Luria-Bertanibroth)medium. Allbacteriaweregrownat 37 °C.
shaking at 37 °C over night. The supernatants were sterile filtered
and 20 L were incubated with 180 L of freshly washed SRBCs
l
l
(30% v/v in PBS) in a 96U well plate (Nunclon) for 30 min at
37 °C. The plate was centrifuged at 2000 g for 5 min and the
absorption of the supernatant was measured at 410 nm with a
plate reader (TECAN, Infinite M200pro). Experiments were per-
formed at least in triplicates.
4.4. Inhibition of ClpP peptidase activity
Inhibition of ClpP peptidase activity of recombinant S. aureus
ClpP expressed in E. coli BL21 and purified by Strep-tag affinity
chromatography was determined by cleavage of the fluorogenic
model substrate SLY-AMC as described previously.2
4.7. Effect on commensal bacteria
Overnight cultures of commensal species were diluted 1:100
in LB or BHB medium and 100
lL thereof incubated with 1 lL
EC50 values for the inhibition of ClpP with lactones were mea-
sured by enzymatic conversion of the fluorogenic substrate N-suc-
cinyl-Leu-Tyr-7-amidomethylcoumarin (SLY-AMC) that has been
described as standard for ClpP activity assays.22 Experiments were
100 mM and DMSO as control in round bottom 96-well
1
plates for 9 h at 37 °C under shaking. Then, OD600 was recorded
in appropriate dilutions. Experiments were carried out in
triplicates.
conducted in 50
100 mM KCl, pH 7.0). In general 2
ity buffer (35 L) with varying concentrations of lactone or DMSO
as control for 10 min at room temperature. Then, SLY-AMC
(0.2 mM, 15 L, 667 M stock) was added to give a total volume
of 50 L containing 2% DMSO. Fluorescence was recorded in Grein-
er 96 Flat Bottom Black Polystyrol well plates by excitation at
340 nm and emission at 450 nm by a TECAN Infinite M200pro.
Experiments were performed at least in triplicates. EC50 values
were calculated from curve fittings by Microcal™ Origin 6.0.
l
L of the activity optimized buffer (50 mM MES,
lg ClpP were incubated in activ-
4.8. Determination of cytotoxicity/MTT assay
l
As a measure of cytotoxicity we determined metabolic activity
of cell lines in response to treatment with b-lactones. The principle
of the MTT assay consists in the reduction of the tetrazolium com-
pound MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan,
Sigma Aldrich) by metabolically active cells to insoluble purple for-
mazan crystals as a measure of cells viability. Two different cell
lines have been used: NIH3T3 mouse fibroblasts and human HaCat
keratinocytes. NIH3T3 cells were cultured in DMEM (high Glucose,
l
l
l
4.5. Competitive proteome profiling
L-Glutamine 4 mM, PAA) + 10% FCS-Gold (PAA). Calcium-sensitive
HaCat cells were maintained in calcium-free DMEM (high-glucose,
The competitive labeling experiments were performed with lac-
tone 1–6 in 20-fold excess to the ABPP probe U1P. Cells of S. aureus
NCTC 8325 were grown to stationary phase. For each experiment
L-Glutamine 2 mM, PAA) + 10% dialyzed FCS (PAA). Cells from sub-
confluent cultures were used for the assay. Precisely, 4.5 ꢁ 103 Ha-
Cat cells or 1.5 ꢁ 103 NIH3T3 cells were plated in 96-well flat-
500
resuspended in 100
at rt either with 1
L of DMSO as labeling control. Then 1 l
l
L of culture were harvested, washed with 500
L PBS. The samples were then incubated 1 h
L of lactone stock (100 mM in DMSO) or with
L of U1P stock (5 mM
l
L PBS and
bottom plates (Nunclon) in 100 lL medium and cultured for 24 h
l
to obtain 30–40% confluent cultures. b-Lactones were diluted
1:100 from DMSO stocks in 100 L of the appropriate culture med-
ium and added to the cells after careful removal of the blank cul-
ture medium. After 24 h incubation 20 L MTT substrate solution
(5 mg/ml in PBS) was added. Following an incubation of 2 h, the
medium was discarded and cells were lysed in 200 L DMSO.
l
l
1
l
in DMSO) was directly added and the samples were incubated
for 1 h at rt. The cells were washed with PBS (3 x 1 mL), resus-
l
pended in 100
4 ꢁ 15 s, 80%). Lysate (43
(CC). 100 M rhodamine-azide (1
lowed by 1 mM TCEP (1 L) and of 100 mM ligand (3
cycloaddition was initiated by the addition of 1 mM CuSO4
(1 L). The reactions were incubated at room temperature for
l
L PBS and lysed by sonication (Bandelin Sonopuls,
L) were subjected to click chemistry
L) was added as reporter fol-
L). The
l
l
The complete dissolution of the formazan salt was checked and
the optical density measured at 570 nm (background subtraction
at 630 nm). A representative assay performed in triplicates is
shown.
l
l
l
l
l
1 h, subjected to SDS PAGE and fluorescence was recorded using
Acknowledgments
a Fujifilm Las-4000 Luminescent Image Analyzer with a Fujinon
VRF43LMD3 Lens, an epi-green illuminator (520 nm) and
575DF20 filter.
a
We thank Mona Wolff for excellent technical assistance. E. Zeil-
er was supported by SFB749 and is a member of TUM-GS. T. Bött-
cher, V. Korotkov and K. Lorenz-Baath gratefully acknowledge
support by an EXIST technology transfer grant of the Federal Min-
istry of Economics and Technology (BMWi). Stephan A. Sieber was
supported by the Deutsche Forschungsgemeinschaft (Emmy Noe-
ther program), SFB749 and CIPSM.
4.6. Hemolysis assays
4.6.1. Agar plate based assay
Hemolysis was tested as described previously.1,2 In short,
5.5 mm WhatmanÒ card discs on 5% sheep blood agar plates (Hei-
pha Diagnostika, Eppelheim, Germany) were inoculated with S.
aureus NCTC 8325 and serial dilutions of b-lactones in DMSO and
incubated over night at 37 °C. Hemolysis zones were determined
and normalized to the DMSO control.
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