Substituted lactam and cyclic azahemiacetals modulate Pseudomonas aeruginosa quorum sensing

Article abstract of DOI:10.1016/j.bmc.2011.07.044

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomose

Full text of DOI:10.1016/j.bmc.2011.07.044

Bioorganic & Medicinal Chemistry 19 (2011) 5500–5506
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Substituted lactam and cyclic azahemiacetals modulate Pseudomonas
aeruginosa quorum sensing
Venkata L.A. Malladi a, Adam J. Sobczak a,à, Natalie Maricic b, Senthil Kumar Murugapiran b,
Lisa Schneper b, John Makemson c, Kalai Mathee b, , Stanislaw F. Wnuk a,
⇑
a Department of Chemistry & Biochemistry, Florida International University, Miami, FL 33199, USA
b Department of Molecular Microbiology and Infectious Diseases, Florida International University, Miami, FL 33199, USA
c Department of Biological Sciences, Florida International University, Miami, FL 33199, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 28 July 2011
Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple
processes including virulence that is critical for establishing infection. The most common QS signaling
molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native
acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-
Keywords:
Azahemiacetals
Azasugars
Pseudomonas aeruginosa
Quorum sensing
negative bacteria. In this work, we have synthesized a set of optically pure c-lactams and their reduced
cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their
effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these
ligands and the simple structural modification such as the length of the alkylthio substituent has notable
effect on activity. The c-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las
signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio
substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the
propylthio analogue strongly stimulated the las QS system at lower concentrations.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Pseudomonas aeruginosa is an important human opportunistic
pathogen affecting immunocompromised individuals, cancer
Quorum sensing (QS) is a type of bacterial cell-to-cell signaling
pathway mediated through the production, release and detection
of the small signaling molecules called autoinducers (AIs).1 Such
communication allows bacterial control of crucial functions in
united communities for enhancement of symbiosis, virulence,
antibiotic production, biofilm formation, and many other processes.
The recent increase in prevalence of bacterial strains resistant to
antibiotics emphasizes the need for the development of a new
generation of antibacterial agents. As QS is utilized by number of
pathogenic bacteria to direct virulence and biofilm formation,
inhibitors/modulators of QS may serve as tools to study or intercept
such community behaviors and might be beneficial as antibacterial
agents.2 The most common QS signaling molecule used by
Gram-negative bacteria are acylhomoserine lactones (AHLs), which
are detected by their cognate regulator (R) proteins.1
patients, burn victims, cystic fibrosis patients and patients with
impaired lung function. It uses two AHL systems called, las and
rhl to mediate QS. LasI/R synthesizes and detects N-(3-oxo-dodec-
anoyl)-L-homoserine lactone (3-oxo-C12-AHL) while RhlI/R synthe-
sizes and detects N-butanoyl-
L-homoserine lactone (C4-AHL)
(Fig. 1a). In addition, P. aeruginosa has a third QS-dependent
pathway, Pseudomonas quinolone signal (PQS) that uses 2-heptyl-
3-hydroxy-4-quinolone as an autoinducer.3 Although certain genes
appear to be regulated by one pathway, for example regulation of
genes involved in rhamnolipid synthesis by the rhl pathway,4 there
is much overlap and crosstalk between the pathways and what
was once thought to be hierarchical regulation, with las activating
rhl, is now known to be much more complex.5 Accumulated evi-
dences clearly indicate the importance of P. aeruginosa QS in
disease.6
Over two decades, several small molecules have been identified
by many research groups as inhibitors of the AHL:R protein com-
plex.7–9 These are mostly AHL-based structures with moderate
changes on the acyl side chain and amide linkage. Some of the most
potent inhibitors prepared by Geske and Blackwell (1a and 2,
Fig. 1b).10 Recently, Meijler and co-workers designed a ligand, 3,
which covalently modified LasR.11 Since AHL is the pharmacophore
⇑
Corresponding author. Tel.: +1 305 348 6195; fax: +1 305 348 3772.
E-mail address: wnuk@fiu.edu (S.F. Wnuk).
Correspondence concerning biological evaluations please direct to Kalai Mathee
at Kalai.Mathee@fiu.edu.
à
On a faculty leave from University of Life Sciences, Department of Chemistry,
Poznan, Poland.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.044

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5500–5506
5501
O
O
O
O
O
a.
b.
N
N
H
H
H
H
O
O
N-butanoyl-L-homoserine
lactone (C4-AHL)
N-(3-oxo-dodecanoyl)-L-homoserine
lactone (3-oxo-C12-AHL)
O
O
O
8
O
O
9
O
O
X
Cl
N
N
N
H
O2N
N
H
H
H
H
H
H
O
O
O
1a: X = O
IC50 = 0.04 µM against LasR
3: IC50 = 1.1µM against LasR
2: IC50 = 0.61 µM against LasR
1b: X = S
N
N
O
O
O
c.
N
OH
5: IC50 = 30 nM against LasR
N
N
7
H
O
4: IC50 =10-15 µM against LasR
Figure 1. (a) AHL-based signal molecules in P. aeruginosa. (b) Examples of potent synthetic QS inhibitors in various Gram-negative bacteria along with their reported IC50
values (tested in the same reporter strain used in the current studies).10,11 (c) Examples of QS inhibitors with a modified AHL scaffold tested in different reporter assays.12,13
present in the natural substrates, AHL-based inhibitors are likely to
modulate R protein activation.
attempted reduction of lactams 7 with LiBEt3H was unsuccessful,
reduction25 of the N-Boc protected lactams 8a–d proceeded
smoothly to afford azahemiacetals 9a–d. Subsequent deprotection
with trifluoroacetic acid afforded 5(S)-(alkylthiomethyl)pyrrolidin-
2-ols 10a–d as a mixture of isomers in equilibrium25–28 with the
corresponding imines 11a–d in addition to the open form alde-
hydes 12a–d. Structure of the hemiaminal 10a was additionally
confirmed by conversion to the correponding O-benzyloxime
derivative 13a with benzylhydroxylamine hydrochloride in anhy-
drous pyridine.
Studies of structural features other than the AHL scaffold as
tools to understand the R type protein interaction with AHLs are
limited,12 although the recent X-ray crystal structure of LasR with
its native ligand and triphenyl mimics ought to facilitate the
rational design of QS inhibitors.14,15 Only a few examples of
inhibitors with the altered lactone ring structure of AHL have been
reported.12,13,16,17 For example, Smith et al. reported 3-oxo-C12
-
(2-aminocyclohexanone) (4, Fig. 1c) as a strong antagonist of LasR
system,12 while Muh et al. identified two LasR inhibitors having a
phenyl and tetrazole ring (e.g., 5), with IC50 in nM range.13 It is
To increase the polarity/solubility of the lactam and azahemiac-
etal analogues in the testing media, we also prepared aza ana-
logues with hydroxyl groups at C3 and C4. The N-Boc protected
noteworthy that in the LuxR system
c-thiolactone analogue 1b
showed inhibition while the corresponding
e
-lactam (caprolactam)
1,4-dideoxy-1,4-imino-
from -gulonic- -lactone, served as a suitable starting material
for the synthesis of dihydroxy -lactams 17 and 18. Thus, mesyla-
D
-ribitol (14),29–31 conveniently prepared
analogue was reported to lack LuxR binding.18 To explore further
effects of non-native AHL scaffold on QS, we have designed novel
D
c
c
lactam ligands. Here, we report optically pure
c
-lactams and cyclic
tion of ribitol 14 followed by the selective oxidation32 of the result-
ing 15 afforded 16. Displacement of the mesylate 16 with sodium
hexane- and nonanethiolate produced 5-alkylthiomethyl lactams
17 and 18 in high yields which were deprotected with TFA to give
(5S)-(hexyl- or nonylthiomethyl)-3,4-dihydroxypyrrolidin-2-ones
19 and 20 (Scheme 2). Reduction of 5-alkylthiomethyl lactams
17 and 18 with LiBEt3H afforded cyclic azahemiacetals 21 and
22. Subsequent deprotection with TFA provided (5S)-(alkylthiom-
ethyl)-3,4-dihydroxypyrrolidin-2-ols 23 and 24 as a complex mix-
ture of azahemiacetals existing in equilibrium with dehydrated
form (imine) as well as with open aldehyde and dimeric forms,
as reported for such class of 4-azaribofuranoses.25,28,33 These aza-
azahemiacetals, bearing alkylthiomethyl substituents with differ-
ent carbon chain lengths (C3–C12), which are capable of either
inhibiting or, in some cases, inducing P. aeruginosa QS pathways.
The lactam ring was chosen because it is a more stable isoster of
lactone ring present in AHL inhibitors. Moreover, the
and cyclic azahemiacetal ligands were further modified in a such
c-lactam
way that they resemble S-ribosyl-L-homocysteine, which is known
to regulate QS through the LuxS-mediated biosynthesis of AI-2,1,19–
21 in which the ribose oxygen is replaced with a nitrogen atom and
the homocysteine unit is substituted with a simple alkylthiol
chain. Although, P. aeruginosa does neither harbor LuxS nor
produce AI-2, AI-2 does alter P. aeruginosa gene expression.22
hemiacetals can be considered as aza analogues of S-ribosyl-L-
homocysteine28 (SRH) in which (a) ribose oxygen is replaced by
nitrogen atom and (b) the homocysteine moiety is substituted with
n-alkylthiols with different length of carbon chain.
2. Results and discussion
2.1. Design and synthesis
2.2. Screening against las signaling
The (S)-5-(bromomethyl)pyrrolidin-2-one (6), a key substrate
To determine the effect of the lactams (7, 19, and 20) and the
cyclic azahemiacetal derivatives (10, 23 and 24) on the P. aeruginosa
las AHL-mediated pathway, a las-dependent b-galactosidase repor-
ter (PlasI-lacZ) was expressed with lasR inEscherichia coli.34,35 Almost
no activity was observed in absence of exogenous AHL demonstrat-
ing the AHL-dependence of the system (0.21 0.31 Miller units). As
expected, and in agreement with published data,34,35 addition of
exogenous 3-oxo-C12-AHL activated the PlasI-lacZ (55.4 1.5 Miller
units). The lactams and their azahemiacetal counterparts were
screened at a concentration of 0.2–1.2 mM for their activity against
for the synthesis of
c-lactam analogue 7 was conveniently pre-
pared from
L
-pyroglutamic acid.23 Displacement of bromide in 6
with sodium propanethiolate produced 5(S)-(propylthiometh-
yl)pyrrolidin-2-one (7a, 96%; Scheme 1). Since it was previously
demonstrated that the length of the side chain is crucial for deter-
mining the agonistic and antagonistic activity,24 lactams contain-
ing C6, C9 and C12 alkylthio chain lengths (7b–d) were also
analogously prepared. A set of the cyclic azahemiacetals (N,O-ace-
tals or hemiaminals) 10 with a hydroxyl group instead of a car-
bonyl oxygen at C2 was synthesized as well. Thus, although,

5502
V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5500–5506
R
R
H
N
N
Br
R'
N
c
R'
S
OH
a
S
O
O
9 R = Boc
10 R = H
7 R = H
8 R = Boc
b
d
6
a R' = C3H7, b R' = C6H13
c R' = C9H19, d R' = C12H25
(
H2O)
OBn
H
N
O
NH2
NH2
N
e
C3H7S
RS
RS
H
13a
12
11
Scheme 1. Reagents and conditions: (a) R0SH/NaH/DMF; (b) (Boc)2O/DMAP/CH2Cl2; (c) LiEt3BH/THF/CH2Cl2/ꢀ78 °C; (d)TFA/rt; (e) BnONH2/pyr.
Boc
Boc
H
N
MsO
N
N
RS
O
c
O
d
RS
O
O
O
O
O
OH OH
17 R = C6H13
18 R = C9H19
16
19 R = C6H13
20 R = C9H19
b
e
Boc
N
Boc
N
H
N
RO
RS
OH
d
RS
OH
OH OH
O
O
O
O
23 R = C6H13
24 R = C9H19
14 R = H
15 R = Ms
21 R = C6H13
22 R = C9H19
a
Scheme 2. Reagents and conditions: (a) MsCl/NEt3/CH2Cl2/rt; (b) NaIO4/hydrated RuO2/EtOAc/H2O/rt; (c) RSH/NaH/DMF; (d)TFA/H2O; (e) LiEt3BH/THF/CH2Cl2/ꢀ78 °C.
the las system (Table 1). At 0.29 mM concentration, the propylthio
lactam 7a slightly but insignificantly inhibited LasR activity and its
corresponding azahemiacetal 10a significantly stimulated (approx-
imately by 2.3-fold) las reporter activity (p-value <0.01). This was
dodecyl chain 10d showed moderate but significant inhibitory
activity (Table 1).
The ribolactam analogues 19 and 20 and their cyclic azahemiac-
etal counterparts 23 and 24 were found to significantly inhibit las
activity at all concentrations tested but only with moderate po-
tency. The azahemiacetals (23 and 24) appears slightly more active
(Table 1).
dependent upon the addition of 2 lM of 3-oxo-C12-AHL as 10a
did not stimulate b-galactosidase activity in the absence of exoge-
nous AHL (data not shown). At the concentration of 0.57 mM, the
azahemiacetal 10a was found to enhance las reporter activity by
15% (p-value <0.01). However, las reporter activity was inhibited
by 69% (p-value <0.05) and 86% (p-value <0.01) at 0.87 and
1.14 mM, respectively. In contrast, the lactam analogue 7a inhibited
las activity at all concentrations tested. Cell growth was not inhib-
ited by the addition of lactam and azahemiacetal compounds at the
tested concentrations (data not shown).
Among other lactam analogues tested, the percent inhibition of
las promoter activity increased in a concentration dependent man-
ner (Table 1). Inhibition potency also increased as the alkylthio
chain length increased. Specifically, nonylthio lactam 7c and dode-
cylthio lactam 7d were found to possess greatest inhibition at all
concentrations tested. At the lowest concentration tested (0.19
and 0.17 mM, respectively), nonylthio lactam 7c modestly inhib-
ited while dodecylthio lactam 7d significantly inhibited las activity
(48%; p-value <0.01). On the contrary, among the cyclic azahemi-
acetals analogues, no general trend was observed between chain
length and percent inhibition. Unlike the propylthio azahemiacetal
10a, hexylthio azahemiacetal 10b did not significantly stimulate
QS at the lowest concentration tested (0.23 mM) but inhibited las
activity 100% at all higher concentrations used (p-value <0.01). In
comparison, azahemiacetals containing nonyl side chain 10c and
2.3. Screening against the rhl pathway
To determine the effect of the lactams (7, 19, and 20) and the
cyclic azahemiacetal derivatives (10, 23 and 24) on the P. aerugin-
osa rhl AHL-mediated pathways, a rhl-dependent b-galactosidase
reporter (PrhlA-lacZ) was expressed with rhlR in E. coli.4 As expected,
and in agreement with published data,4 exogenous C4-AHL acti-
vated the rhl b-galactosidase reporter (266 20 Miller units). There
was minimal activity in the absence of C4-AHL (0.12 2.72 Miller
units). In general, except for the cyclic azahemiacetals with shorter
alkylthio chain 10a and 10b, the rest either had no effect or stim-
ulated rhl expression (Table 1). The lactam analogues 7a, 7c and 7d
did not modulate rhl activity, while hexylthio lactam 7b signifi-
cantly stimulated (p-value <0.01) rhl QS activities at higher concen-
trations. In contrast to lactams, cyclic azahemiacetals with shorter
alkylthio chain 10a (at higher concentration) and 10b (at all con-
centration) significantly inhibited rhl activity, while analogues
10c and 10d with longer alkyl chain were inactive. The hexylthio
azahemiacetal 10b completely inhibited rhl signaling at concentra-
tions of 0.46 mM and higher (p-value <0.02). The strong inhibition
observed with propylthio 10a and hexylthio 10b azahemiacetal

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5500–5506
5503
Table 1
Effect of lactam (7a-d, 19, 20) and cyclic azahemiacetal (10a-d, 23, 24) derivatives on las and rhl systems
Compound
Concentration (mM)
Activitya (%) standard deviationb
Compound
Concentration (mM)
Activitya (%) standard deviationb
las
Rhl
100 0c
104 24
118 0.8
104 13
108 17
las
rhl
AHLc
7a
0.002
0.29
0.58
0.87
1.16
0.23
0.47
0.70
0.93
0.19
0.39
0.58
0.78
0.17
0.33
0.50
0.67
0.20
0.40
0.61
0.81
0.17
0.35
0.52
0.69
100
90 19
72 17
51
43
106 17
87 12
0
10a
0.29
0.57
0.86
1.14
0.23
0.46
0.69
0.92
0.19
0.39
0.58
0.77
0.17
0.33
0.50
0.66
0.20
0.40
0.60
0.80
0.17
0.34
0.52
0.69
228à 39
115 50
31⁄ 2 0
121⁄
138⁄
41
6
3
1
1
7
9
11⁄
109
0à
7
4
0
0
0
9
9à
26 0.2
7b
7c
7d
19
20
126
8
10b
126 29
130 31
145à 19
0.2 0.3
74⁄
61⁄
72⁄
26⁄
18à
6à
5
9
7
8
1
3
0à
0à
0⁄
0
0
0à
95
3
10c
74
93 12
80 13
100 11
99
98 13
93 27
91
96
94
105
109 12
116 18
115 30
108 12
137 46
65 17
57à
4
47à 21
3
52à 11
103
106
109
106
7
6
8
5
10d
72à
56à
45à
34à
3
4
4
7
32
3
2
2
18à
11à
8
5
8
3
96 0.14
78à 0.08
79à 0.5
68à 0.6
93 0.5
132⁄ 36
23
84à 0.7
129 10
68à 0.44
51à 0.37
42à 0.36
78à 0.32
148
161à
106
9
7
3
8
24
98
127⁄ 14
116
121 14
5
87à
80
6
7
7
117à
78à
1
115⁄ 11
71à 0.4
53à 0.5
9
63à
135à
5
a
The P. aeruginosa PlasI-lacZ and PrhlA-lacZ expressions in E. coli tested at the indicated concentrations (0.2–1.2 mM) against 2
respectively. The units are presented as percent activity relative to the DMSO treated control.
l
M of 3-oxo-C12-AHL or 2 lM of C4-AHL,
b
⁄p-value <0.05, p-value <0.02, àp-value <0.01 according to a paired, two-tailed Student t-test. Statistically significant values are in bold.
c
The exogenous AHLs added to the las and rhl systems are 3-oxo-C12-AHL and C4-AHL, respectively. The 100% las and rhl activities refer to 55.4 1.5 and 266 20 Miller
units, respectively.
analogues having side chain lengths similar to C4-AHL is in agree-
ment with the structure activity relationship reported for various
synthetic AHL mimetics targeting RhlR.36
The hexyl-ribolactam analogue 19 significantly stimulated
(p-value <0.01) rhl activity, while its cyclic azahmiacetal counter-
part 23 had no effect (Table 1). The nonyl-azahemiacetal analogue
24 had significant stimulatory activity at 0.34 mM (p-value <0.05;
Table 1).
with the natural ligand for binding to the regulator, LasR or RhlR,
or alter binding of the QS regulator to the promoter. Alternatively,
it is possible that the compounds with longer side chains affect the
membrane and that the las pathway is more sensitive to these
changes. Future experiments can address these possibilities. For
example, quantification of extracellular AHL would reveal if the
compounds affect AHL import. If this were the mechanism of ac-
tion, it is expected that there would be less extracellular AHL in
the absence of compound than in the presence. If the compound
competes for binding to the regulator, inhibition should be over-
come by increased AHL concentration. Given the central role the
las and rhl QS pathways play in P. aeruginosa virulence, inhibitors
such as the ones described here, have significant potential as
therapeutics.
3. Conclusions
We have designed and synthesized a set of optically pure
c-lac-
tams with alkylthiomethyl substitution at carbon and their N,O-
c
acetal counterparts. These ligands were evaluated for their effect
on P. aeruginosa AHL-dependent las and rhl QS pathways isolated
in E. coli. Lactam analogues 7 showed selectivity between two QS
systems, acting as inhibitors against las signaling and weak activa-
tors against rhl signaling, possibly due to differences in the active
sites of their cognate R proteins or transport of the native signaling
molecule. Antagonism of las activity increased with the length of
the alkylthio chain. Interestingly, the cyclic azahemiacetal deriva-
tive with shorter propylthio chain (10a) significantly stimulated
las signaling at lower concentrations while strongly inhibiting both
QS systems at higher concentrations. At least with 10a, the stimu-
lation of the las system only occurs in the presence of exogenous
AHL. It is possible that heterodimeric LasR is more active as com-
pared to homodimers. The ribolactam (19 and 20) and cyclic aza-
hemiacetal (24) analogs inhibited las and stimulated rhl
moderately. Of all the compounds tested, the 5-(hexylthiometh-
yl)pyrrolidin-2-ol (10b) appears most potent inhibitor against both
las and rhl systems. The mechanism of inhibition is still unknown.
Since the effect tested utilized a whole cell assay, the QS inhibition
could occur at multiple steps in the pathway. For example, the
compound could affect the import of the natural ligand, compete
4. Methods
General experimental methods are described in Supplementary
data. The 1H and 13C NMR spectra were determined with solutions
in CDCl3 unless otherwise noted.
4.1. 5-(Propylthiomethyl)pyrrolidin-2-one [7a(5S)]
Procedure A: Propanethiol (50 lL, 42 mg, 0.55 mmol) was added
dropwise to a stirred suspension of NaH (35 mg, 0.875 mmol, 60%/
mineral oil) in dry DMF (1 mL) under Ar atmosphere at 0 °C. After
10 min (till gas evolution has ceased), solution of compound 623
[(5S), 82 mg, 0.46 mmol] in dry DMF (1 mL) was added dropwise,
and after 15 min the reaction mixture was allowed to warm to
ambient temperature. After 12 h the resulting mixture was
quenched with water at 0 °C, volatiles were evaporated, and the
residue was column chromatographed (EtOAc?10% MeOH/EtOAc)
to give 7a(5S) (77 mg, 96%) as a colorless oil: 1H NMR d 0.98 (t,
J = 7.3 Hz, 3H), 1.60 (sx, J = 7.3 Hz, 2H), 1.76–1.87 (m, 1H),

5504
V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5500–5506
2.25–2.34 (m, 1H), 2.34–2.45 (m, 2H), 2.52 (t, J = 7.3 Hz, 2H), 2.54
(dd, J = 7.7, 13.2 Hz, 1H), 2.68 (dd, J = 5.5, 13.2 Hz, 1H), 3.80 (‘quint’,
J = 5.5 Hz, 1H), 6.73 (br s, 1H); 13C NMR d 13.4, 23.1, 26.6, 30.2,
34.7, 38.6, 53.9, 178.0; MS (APCI) m/z 174 (MH+). HRMS (AP-ESI)
m/z calcd for C8H15NNaOS [M+Na]+ 196.0772; found 196.0779.
NaHCO3/H2O), washed (brine) and dried (MgSO4). The resulting
oil was chromatographed (30%?40% EtOAc/hexane) to give N-
tert-butoxycarbonyl-5-(propylthiomethyl)pyrrolidin-2-ol [9a(5S);
104 mg, 96%] as a colorless oil of the mixture of anomers/rota-
mers: MS (ESI) m/z 274 (10, [Mꢀ1]+), 258 (100, [Mꢀ17]+). Step
b. Procedure D: Compound 9a (104 mg, 0.37 mmol) in TFA
(4.0 mL) was stirred at room temperature for 2 h. Volatiles were
evaporated to give 10a (62 mg, 96%) as a light yellow oil of a mix-
ture of isomers accompanied by ꢁ25% of the aldehyde 12a [1H
NMR d 8.89 (s, ꢁ0.25H); and 13C NMR d 180.8]; MS (ESI) m/z
158 (100, [Mꢀ17]+).
4.2. 5-(Hexylthiomethyl)pyrrolidin-2-one [7b(5S)]
Treatment of 622 [(5S), 823 mg, 4.62 mmol] in dry DMF (6 mL)
with a thiolate solution in dry DMF (6 mL) generated from hexa-
nethiol (682 lL, 573 mg, 4.86 mmol), and NaH (204 mg,
5.09 mmol, 60%/mineral oil) by Procedure A [column chromatogra-
A solution of crude 10a (5S; 8 mg, 0.046 mmol) and O-ben-
zylhydroxylamine hydrochloride (48 mg, 0.3 mmol) in anhydrous
pyridine (1 mL) was stirred under an atmosphere of nitrogen at
room temperature for 12 h. Pyridine was evaporated to afford 4-
amino-5-(propylthio)pentanal O-benzyloxime [13a(4S)] of suffi-
cient purity (ꢁ90%) for spectroscopic characterization together
with the excess of BnONH2 used: MS (ESI) m/z 281 (60, MH+),
158 (100, [MꢀBnONH]+), (APCI) m/z 281 (100, MH+).
phy (80% EtOAc/hexane?5% MeOH/EtOAc)] gave 7b(5S) (932 mg,
94%) as a colorless oil: [a]
D = +40.7 (c 0.03, CHCl3); 1H NMR d
0.90 (t, J = 7.0 Hz, 3H), 1.24–1.33 (m, 4H), 1.33–1.42 (m, 2H), 1.58
(‘quint’, J = 7.4 Hz, 2H), 1.78–1.87 (m, 1H), 2.27–2.46 (m, 3H) 2.53
(dd, J = 8.0, 13.4 Hz, 1H), 2.54 (t, J = 7.3 Hz, 2H), 2.70 (dd, J = 5.3,
13.2 Hz, 1H), 3.81 (‘quint’, J = 6.6 Hz, 1H), 6.47 (br s, 1H); 13C
NMR d 14.0, 22.5, 26.8, 28.5, 29.7, 30.1, 31.4, 32.7, 38.7, 53.8,
177.7; MS (ESI) m/z 216 (100, MH+); HRMS (TOF MS-ESI) m/z calcd
for C11H21NOSNa [M+Na]+ 238.1236; found 238.1252.
4.6. 5-(Hexylthiomethyl)pyrrolidin-2-ol [10b(5S)]
4.3. N-tert-Butoxycarbonyl-5-(propylthiomethyl)pyrrolidin-2-
one [8a(5S)]
Step a. Treatment of 8b (178 mg, 0.56 mmol) in CH2Cl2 (3 mL)
with LiEt3BH (1 M soln in THF, 1.41 mL, 1.41 mmol), by procedure
C [quenched with MeOH (4 mL) at low temp., column chromatog-
raphy (30%?40% EtOAc/hexane)] gave N-tert-butoxycarbonyl-5-
Procedure B: DMAP (114 mg, 0.93 mmol), and (Boc)2O (398 mg,
1.82 mmol) were added to a stirred solution of compound 7a
(77 mg, 0.445 mmol) in CH2Cl2 (2 mL) at ambient temperature un-
der Ar atmosphere. After 48 h, the reaction mixture was quenched
with H2O (5 mL) and partitioned between CH2Cl2//NaHCO3/H2O.
The organic layer was washed (brine), dried (MgSO4) and evapo-
rated. The residue was column chromatographed (30%?40%
EtOAc/hexane) to give 8a(5S) (107 mg, 88%) as a colorless oil: 1H
NMR d 0.95 (t, J = 7.3 Hz, 3H), 1.50 (s, 9H), 1.58 (sx, J = 7.3 Hz,
2H), 1.96–2.04 (m, 1H), 2.06–2.17 (m, 1H), 2.40 (ddd, J = 2.6, 9.6,
17.9 Hz, 1H), 2.50 (‘dt’, J = 4.9, 7.3 Hz, 2H), 2.58–2.67 (m, 1H),
2.60 (dd, J = 9.2, 13.5 Hz, 1H), 2.86 (ddd, J = 0.5, 2.8, 13.5 Hz, 1H),
4.20–4.27 (m, 1H); 13C NMR d 13.3, 21.9, 23.1, 28.0, 31.2, 34.8,
35.4, 57.5, 83.1, 149.8, 174.2; MS (ESI) m/z 274 (10, MH+), 215
(100, [MHꢀ59]+).
(hexylthiomethyl)pyrrolidin-2-ol [9b(5S); 170 mg, 95%)] as
a
colorless oil of a mixture of isomers: MS (ESI) m/z 316 (100,
[Mꢀ1]+), 300 (20, [Mꢀ17]+); HRMS (TOF MS-ESI) m/z calcd for
C
16H31NO3SNa [M+Na]+ 340.1926; found 340.1955. Step b. Com-
pound 9b (38.5 mg, 0.12 mmol) in TFA (0.8 mL) was stirred at
0 °C (ice-bath) for 3 h. The reaction mixture was diluted with ex-
cess of ice-cold CH2Cl2 and neutralized with solid NaHCO3. Result-
ing mixture was stirred for 20 min at ambient temperature and
was decanted. The residual slurry was extracted with fresh portion
of CH2Cl2 and the combined extracts were dried (Na2SO4) and con-
centrated to give crude 10b (25 mg) as a colorless oil. Crude prod-
uct was column chromatographed to give first (0%?0.25% MeOH/
CHCl3) anomeric mixture of azahemiacetals 10b
(a/b, 9:20;
2.0 mg, 8%) as a colorless oil: 1H NMR d 0.89 (t, J = 7.0 Hz, 4.35H),
1.26–1.45 (m, 8.7H), 1.47–1.72 (m, 3.9H), 1.75–1.85 (m, 0.45H),
1.92–2.07 (m, 3.45H), 2.10–2.23 (m, 0.9H), 2.46 (dd, J = 9.5,
13.0 Hz, 1H), 2.52–2.65 (m, 2.9H), 2.93–3.02 (m, 0.9H), 3.23 (dd,
J = 2.5, 13.0 Hz, 1H), 3.60–3.68 (m, 1H), 3.67–3.75 (m, 0.45H),
4.04–4.10 (m, 1H), 4.20–4.27 (m, 0.45H); MS (ESI) m/z 200 (100,
[Mꢀ17]+). Further elution (0.25%?0.5% MeOH/CHCl3) gave imine
11b (5.8 mg, 22%) as a colorless oil: 1H NMR d 0.91 (t, J = 7.0 Hz,
3H), 1.27–1.46 (m, 7H), 1.55–1.65 (m, 2H), 2.02–2.11 (m, 1H),
2.49–2.63 (m, 5H), 2.95 (dd, J = 5.3, 12.8 Hz, 1H), 4.21–4.29 (m,
1H), 7.63 (br t, J = 1.1 Hz, 1H); 13C NMR d 14.0, 22.5, 26.1, 28.6,
29.8, 31.4, 33.0, 37.0, 38.1, 72.9, 167.0; MS (ESI) m/z 200 (100,
MH+).
4.4. N-tert-Butoxycarbonyl-5-(hexylthiomethyl)pyrrolidin-2-
one [8b(5S)]
Treatment of 7b (311 mg, 1.45 mmol) in CH2Cl2 (6 mL) with
DMAP (185 mg, 1.52 mmol), and (Boc)2O (746 mg, 3.42 mmol) by
procedure B [column chromatography (20%?40% EtOAc/hexane)]
gave 8b(5S) (429 mg, 94%) as a colorless oil: 1H NMR d 0.89 (t,
J = 7.0 Hz, 3H), 1.25–1.33 (m, 4H), 1.34–1.42 (m, 2H), 1.55 (s, 9H),
1.59 (‘quint’, J = 7.4 Hz, 2H), 2.01–2.08 (m, 1H), 2.10–2.21 (m,
1H), 2.45 (ddd, J = 2.5, 9.6, 17.9 Hz, 1H), 2.56 (‘dt’, J = 2.9, 7.3 Hz,
2H), 2.62–2.72 (m, 1H), 2.63 (dd, J = 9.3, 13.5 Hz, 1H), 2.91 (dd,
J = 2.7, 13.5 Hz, 1H), 4.24–4.31 (m, 1H); 13C NMR d 14.0, 22.0,
22.5, 28.1, 28.4, 29.8, 31.2, 31.4, 32.9, 35.5, 57.5, 83.1, 149.8,
174.1; MS (ESI) m/z 315 (15, M+), 256 (100, [Mꢀ59]+); HRMS
(TOF MS-ESI) m/z calcd for C16H29NO3SNa [M+Na]+ 338.1760;
found 338.1752.
Note: The composition of crude products after step b depends
strongly on the work up conditions. For example, the reaction mix-
ture contained also ꢁ22% of the aldehyde 12b [1H NMR d 8.91 (s,
ꢁ0.22H)] at pH lower than 7.
4.7. 4-Amino-N-(tert-butoxycarbonyl)-4-deoxy-2,3-O-
4.5. 5-(Propylthiomethyl)pyrrolidin-2-ol [10a(5S)]
isopropylidene-5-O-methanesulfonyl-D-ribono-1,4-lactam (16)
Step a. Procedure C: LiEt3BH (1 M soln in THF, 0.98 mL,
0.98 mmol) was added to a stirred solution of 8a (107 mg,
0.39 mmol) in CH2Cl2 (3 mL) at ꢀ78 °C under N2 atmosphere.
After 30 min, the reaction mixture was quenched with MeOH
(4 mL) and was allowed to warm to ambient temperature. Vola-
tiles were evaporated and the residue was partitioned (EtOAc//
Step a. Triethylamine (93
(25
lL, mg, 67 mg, 0.66 mmol) and MsCl
l
L, 38 mg, 0.33 mmol) were added dropwise to stirred solution
of 1430 (60 mg, 0.22 mmol) in anhydrous CH2Cl2 (6 mL) at 0 °C (ice-
bath). After 5 min, ice-bath was removed and the reaction mixture
was allowed to stir at ambient temperature for 30 min. The reaction
mixture was quenched with saturated NaHCO3/H2O and was

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5500–5506
5505
extracted with CH2Cl2. The organic layer was washed (brine), dried
(MgSO4) and evaporated to give 1-amino-1,4-anhydro-N-tert-
butoxycarbonyl-1-deoxy-2,3-O-isopropylidene-5-O-methanesul-
4.10. 4-Amino-4-deoxy-5-S-hexyl-5-thio-
(23)
a/b-D-ribofuranose
fonyl-
D
-ribitol 15 (73 mg, 96%) as a mixture (ꢁ3:2) of two rotamers
Treatment of 17 (40 mg, 0.1 mmol) in THF (1 mL) with LiEt3BH
(1 M/THF, 0.26 mL, 0.26 mmol), by procedure C [column chromatog-
raphy (10%?20% EtOAc/hexane)] gave 4-amino-N-(tert-butoxycar-
of sufficient purity to be directly used for next step: 1H NMR d 1.28 (s,
3, CH3), 1.42 (s, 12H, t-Bu, CH3), 2.96 (s, 1.2, Ms), 2.98 (s, 1.8, Ms), 3.39
(dd, J = 12.5, 4.8 Hz, 0.4H), 3.46 (dd, J = 12.5, 4.8 Hz, 0.6H), 3.69 (d,
J = 12.5 Hz, 0.6H), 3.82 (d, J = 12.5 Hz, 0.4H), 4.10–4.14 (m, 0.4H),
4.22–4.30 (m, 1H), 4.22–4.29 (m, 1.4H), 4.45 (dd, J = 10.1, 4.1 Hz,
0.6H), 4.65 (‘d’, J = 5.9 Hz, 1H); 4.72 (‘t’, J = 5.3 Hz, 1H); 13C NMR (ma-
jor) d 24.9, 26.9, 29.6, 37.1, 52.5, 62.4, 68.9, 79.2, 80.4, 81.7, 112.1,
154.2; 13C NMR (minor) d 24.9, 26.9, 29.6, 37.5, 53.1, 62.6, 68.6,
78.5, 80.6, 82.5, 112.1, 153.6; MS (APCI) m/z 352 (10, MH+), 252
(100, [MH2-Boc]+). Step b. RuO2 ꢂ H2O (8.5 mg, 0.064 mmol) was
added to a stirred solution of NaIO4 (172 mg, 0.96 mmol) in H2O
(1 mL) at ambient temperature. After 5 min, a solution of 15
(80 mg, 0.32 mmol) in EtOAc (1 mL) was added dropwise and the
reaction mixture was continued to stir for 12 h. H2O (20 mL) and
EtOAc (20 mL) were added and the separated aqueous layer was fur-
thermore extracted with EtOAc (2 ꢂ 20 mL). The combined organic
layers were washed (brine), dried (MgSO4) and evaporated. The res-
idue was column chromatographed (EtOAc) to give 16 (78 mg, 95%)
as a colorless oil: 1H NMR d 1.37 (s, 3H), 1.44 (s, 3H), 1.54 (s, 9H), 3.01
(s, 3H), 4.39–4.43 (‘m’, 2H), 4.58 (d, J = 5.45 Hz, 1H), 4.64 (dd, J = 11.2,
3.1 Hz, 1H), 4.70 (d, J = 5.45 Hz, 1H); 13C NMR d 25.6, 27.0, 28.0, 37.7,
59.2, 67.0, 74.5, 77.5, 84.7, 112.8, 149.7, 170.2; MS (APCI) m/z 298
(100, [MH2ꢀBoc+MeOH]+).
bonyl)-4-deoxy-5-S-hexyl-3,4-O-isopropylidene-5-thio-a/b-D-ribo
furanose 21 (39 mg, 97%)] as a colorless oil of the mixture of isomers:
MS (ESI) m/z 389 (100, M+); HRMS (TOF MS-ESI) m/z calcd for
C
19H35NO5SNa [M+Na]+ 412.2128; found 412.2117. Deprotection
of 21 (39 mg, 0.1 mmol) with TFA/H2O (0.9:0.1 mL) by Procedure D
gave light yellow oil that was column chromatographed
a
(5%?10% MeOH/EtOAc) to give 23 (22 mg, 88%) as a light yellow
oil. 1H NMR showed a mixture of isomers accompanied by the open
aldehyde form. MS (APCI) m/z 230 (40, MH+), 232 (100, [Mꢀ17]+).
4.11. Anti-quorum sensing assay (b-galactosidase assay)
An overnight (O/N) culture of E. coli DH5
a harboring the plas-
mids pSC11, which contains a PlasI-lacZ translational fusion,34 and
pJN105L, which contains a PBAD-lasR expression plasmid35 grown
in LB media (10 g tryptone, 5 g yeast extract, 5 g sodium chloride
per liter) supplemented with ampillicin (100
cin (15 g/ ml), was diluted to an OD600 of 0.150. At this time,
arabinose (0.2% w/v), N-3-(oxododecanoyl)homoserine lactone
(3-oxo-C12-AHL; 2 M), and either the compound under analysis
lg/ml) and gentamy-
l
l
or solvent (DMSO), was added to the culture (1.5 mL). A negative
control containing only solvent, antibiotic, and arabinose (0.2%
w/v) without 3-oxo-C12-AHL was also assayed (data not shown).
The cultures were incubated with shaking for three hours at 37 °C.
4.8. 4-Amino-N-(tert-butoxycarbonyl)-4-deoxy-5-S-hexyl-2,3-O-
isopropylidene-5-thio-D-ribono-1,4-lactam (17)
The conditions for the rhl biomonitor E. coli DH5
a harboring
Treatment of 16 (60 mg, 0.16 mmol] in dry DMF (0.5 mL) with
sodium hexathiolate [generated from hexanethiol (46.8 L,
0.33 mmol)/NaH (14 mg, 0.35 mmol, 60%/mineral oil) in dry
DMF (0.5 mL)] by Procedure [column chromatography
pECP61.5 plasmid, which contains Ptac-rhlR and PrhlA-lacZ4 were
l
essentially same except that the LB medium was only supple-
mented with ampillicin (100
lg/ ml), the O/N culture was diluted
A
to an OD600 of 0.150, induced with 1 mM IPTG, 2
lM C4-HSL and
(5%?10% MeOH/EtOAc)] gave 17 (25 mg, 40%) as a colorless oil
and N-Boc deprotected 17 (24 mg, 38%) as a white crystalline so-
lid. Compound 17 had: 1H NMR d 0.81 (t, J = 7.0 Hz, 3H), 1.16–
1.27 (m, 6H), 1.30 (s, 3H), 1.39 (s, 3H), 1.44–1.59 (m, 11H),
2.36–2.50 (m, 2H), 2.76 (dd, J = 6.2, 14.4 Hz, 1H), 2.82 (dd,
J = 2.7, 14.4 Hz, 1H), 4.31 (dd, J = 2.7, 6.2 Hz, 1H), 4.38 (d,
J = 5.5 Hz, 1H), 4.78 (d, J = 5.5 Hz, 1H); 13C NMR d 14.0, 22.5,
25.5, 27.0, 28.0, 28.3, 29.6, 31.3, 33.7, 33.9, 60.8, 76.1, 77.6,
83.9, 112.3, 149.8, 171.0; MS (APCI) m/z 288 (100, [MH2ꢀBoc]+).
N-Boc deprotected 14 had: 1H NMR d 0.88 (t, J = 7.0 Hz, 3H),
1.25–1.36 (m, 6H), 1.38 (s, 3H), 1.48 (s, 3H), 1.56.-1.62 (m, 2H),
2.52.-2.75 (m, 3H), 2.73 (dd, J = 5.9, 13.4 Hz, 1H), 3.81 (‘t’,
J = 6.1 Hz, 1H), 4.50 (d, J = 5.9 Hz, 1H), 4.69 (d, J = 5.9 Hz, 1H),
5.94 (s, 1H); 13C NMR d 14.0, 29.7, 22.5, 28.5, 31.4, 25.6, 26.9,
33.2, 33.7, 58.0, 76.6, 79.2, 112.7, 173.2; MS (APCI) m/z 288
(100, MH+).
the compounds or the controls added when the OD600 reached
1.0. A negative control containing only solvent, antibiotic and IPTG
(1 mM) without C4-AHL was also assayed (data not shown). After
incubation at 37 °C for 4 h with shaking, b-galactosidase activity
was assayed as described previously.37 Miller units were calculated
as described.38 Assays were repeated at least twice. For each bio-
logical replicate, experimental triplicates were performed and the
average percent activity calculated by dividing the average Miller
units from the samples containing compound or extract by the
average Miller units from the sample containing solvent and mul-
tiplying by 100. Significance of inhibition was determined using a
paired two-tailed Student t-test.
Acknowledgments
We
thank
NIGMS/NCI
[SC1CA138176
(S.F.W.)
and
5R15AT002626 (K.M.)] and FIU’s Doctoral Evidence Acquisition
Fellowship (V.L.M.) for their support.
4.9. 4-Amino-4-deoxy-5-S-hexyl-5-thio-D-ribono-1,4-lactam
(19)
Supplementary data
TFA/H2O (1 mL, 9:1) was added to 17 or N-Boc deprotected 17
(22 mg, 0.07 mmol) and the resulting solution was stirred at 0 °C
for 3 h. Evaporation of volatiles gave light yellow oil that was col-
umn chromatographed (5%?10% MeOH/EtOAc) to give 19 (12 mg,
63%) as a colorless oil: 1H NMR d 0.87 (t, J = 7.0 Hz, 3H), 1.24–1.39
(m, 6H), 1.52–1.59 (m, 2H), 2.50–2.55 (m, 3H), 2.73 (dd, J = 5.5,
13.6 Hz, 1H), 3.71 (‘t’, J = 6.4 Hz, 1H), 4.21 (d, J = 5.0 Hz, 1H), 4.44
(d, J = 5.0 Hz, 1H), 7.11 (s, 1H); 13C NMR d 14.0, 29.6, 14.1, 22.5,
31.4, 32.7, 35.3, 59.9, 69.8, 71.8, 176.0; MS (APCI) m/z 248 (100,
MH+); HRMS (TOF MS-ESI) m/z calcd for C11H21NO3SNa [M+Na]+
270.1134; found 270.1137.
Supplementary data (general experimental methods, synthetic
procedures and characterization data for compounds 7c,d, 8c,d,
10c,d, 18, 20, 22 and 24) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2011.07.044.
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