Microwave synthesis and evaluation of phenacylhomoserine lactones as anticancer compounds that minimally activate quorum sensing pathways in Pseudomonas Aeruginosa

Article abstract of DOI:10.1021/jm8015377

The bacterial quorum sensing (QS) signal molecule 3-oxo-dodecanoyl-L- homoserine lactone (OdDHL) is produced by the opportunistic pathogen Pseudomonas aeruginosa and controls expression of virulence factors associated with life threatening infections in i

Full text of DOI:10.1021/jm8015377

J. Med. Chem. 2009, 52, 1569–1575
1569
Microwave Synthesis and Evaluation of Phenacylhomoserine Lactones as Anticancer
Compounds that Minimally Activate Quorum Sensing Pathways in Pseudomonas aeruginosa
Colin M. Oliver,†,‡ Amy L. Schaefer,§ E. Peter Greenberg,§ and Janice R. Sufrin*,†,‡
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, Program of Molecular
Pharmacology and Cancer Therapeutics, Roswell Park Graduate DiVision, State UniVersity of New York at Buffalo, Buffalo, New York 14263,
Department of Microbiology, UniVersity of Washington, Seattle, Washington 98195
ReceiVed December 5, 2008
The bacterial quorum sensing (QS) signal molecule 3-oxo-dodecanoyl-L-homoserine lactone (OdDHL) is
produced by the opportunistic pathogen Pseudomonas aeruginosa and controls expression of virulence factors
associated with life threatening infections in immune compromised individuals. OdDHL has also demonstrated
anticancer activity, yet its ability to enhance pathogenicity of P. aeruginosa compromises further consideration
as a potential anticancer agent. In search of acylhomoserine lactones that selectively inhibit cancer cell
growth, a library of phenacylhomoserine lactone analogues has been prepared by microwave synthesis and
evaluated for cancer growth inhibition and quorum sensing activation. Comparative SAR analysis demonstrates
that both anticancer and QS signaling systems require long acyl side chains with a 3-oxo substitution for
maximum activity. Compound 12b, 3-oxo-12-phenyldodecanoyl-L-homoserine lactone, was identified as a
lead compound with strong cancer growth inhibitory activity that minimizes activation of QS signaling
pathways in a P. aeruginosa reporter assay.
Introduction
The acylhomoserine lactone (AHLa), 3-oxo-dodecanoyl-L-
homoserine lactone (OdDHL, Figure 1), has been shown to
inhibit the growth of tumorigenic but not nontumorigenic cells.1
In colorectal and prostate cancer cells, OdDHL down-regulates
thymidylate synthase (TS) and enhances the efficacy of the
Figure 1. Structure of 3-oxo-dodecanoyl-L-homoserine lactone
widely used cancer chemotherapeutic agents, 5-fluorouracil (5-
(OdDHL). Structural motifs subject to modification are listed. Figure
FU) and Taxol.2 The effects of OdDHL are highly structure-
adapted from Chhabra et al.10
specific as elimination of the 3-oxo moiety attenuates activity.
A racemic mixture of enantiomers is 50% as active as the
generation compounds with diminished QS signaling activity
L-isomer, indicating specificity.2
is essential to identifying the most promising candidates for drug
In nature, Gram-negative bacteria that enzymatically produce
development. In this respect, the aliphatic hydrocarbon side
AHLs are capable of coordinating expression of population-
chain of OdDHL lacks a structural platform that can be easily
sensitive genes through signaling networks known as quorum
modified. To address these limitations, we have prepared a
library of phenacylhomoserine lactones (PHLs). Phenyl substitu-
tion retains the lipophilic properties of traditional AHLs while
introducing a structural platform amenable to modification. We
sensing (QS).3 OdDHL is a QS signal produced by Pseudomo-
nas aeruginosa.4 Activation of QS pathways is frequently
associated with characteristics that compromise the host organ-
ism (e.g., increased virulence, resistance).5 Five percent of
have evaluated our library of phenacylhomoserine lactone
immune-compromised patients (e.g., cancer) die from nosoco-
analogues (PHLA) for cancer growth inhibitory and QS signal-
mial P. aeruginosa infection.6 Moreover, OdDHL has been
ing activities. By analyzing comparative structure-activity
relationships (SAR), we have identified a lead structure that
potently inhibits cancer cell growth and minimally activates QS.
detected in clinical isolates of P. aeruginosa.7
OdDHL has promise as a cancer treatment modality. How-
ever, its ability to promote P. aeruginosa infection could be
life-threatening in a clinical setting. Development of second-
Chemistry
To dissociate the inhibitory effects of AHLs on cancer cell
growth from activation of quorum sensing signaling, we have
synthesized a library of phenyl-substituted AHL analogues that
vary in acyl chain length and oxidation state at carbon 3.
Methods we have developed for synthesis of our library of
PHLAs, through the combined use of a solid-phase coupling
reagent and microwave chemistry, improve upon those previ-
ously described.8-11 Other syntheses of AHL analogues that
use solid-phase support and/or microwave irradiation require
multiple, time-consuming steps that can include several rounds
of purification.8,9,12,13 Our method provides PHLAs as final
products in one or two rapid steps from commercially available
starting materials (Schemes 1 and 2). Use of a resin-linked
* To whom correspondence should be addressed. Phone: 716.845.4582.
Fax: 716.845.8857. E-mail: Janice.sufrin@roswellpark.org. Address: Roswell
Park Cancer Institute Grace Cancer Drug Center 109 Elm and Carlton
Streets, Buffalo, NY 14263.
†
Department of Pharmacology and Therapeutics, Roswell Park Cancer
Institute.
‡
Program of Molecular Pharmacology and Cancer Therapeutics, Roswell
Park Graduate Division, State University of New York at Buffalo.
§
Department of Microbiology, University of Washington.
a Abbreviations: 5-FU, 5-fluorouracil; AHL, acylhomoserine lactone;
dFBS, dialyzed fetal bovine serum; DMAP, dimethylaminopyridine; L-HSL,
L-homoserine lactone; PHL, phenacylhomoserine lactone; PHLA, phena-
cylhomoserine lactone analogue; PRF, phenol red free; OdDHL, 3-oxo-
dodecanoyl-L-homoserine lactone; QS, quorum sensing; SRB, sulfor-
hodamine B; W, microwave irradiation; GFP, green fluorescence protein.
10.1021/jm8015377 CCC: $40.75
2009 American Chemical Society
Published on Web 03/04/2009

1570 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
OliVer et al.
Scheme 1a
synthesized and characterized. The phenyl substituent, which
introduces a structural platform amenable to second- and third-
generation modifications, retains the intrinsic lipophilicity of
natural AHLs, a property that enables these small molecules to
cross membranes through unregulated diffusion and act on
intercellular targets.3,14 Moreover, the previously reported
PHLA, 4b, has been shown to possess QS inhibitory activity,
providing additional rationale for a comparative SAR analysis
of a library of PHLAs.12
a (a) PS-carbodiimide (4 equiv); W (1000 W, 1.5 min).
Scheme 2a
PHLAs were evaluated for their ability to inhibit the growth
of three human cancer cell lines: H630 (colorectal-parental),
H630-1 (colorectal-5-FU resistant), and PC3 (prostate). Results
from growth inhibition assays demonstrate that PHLAs contain-
ing an acyl chain length shorter than eight carbons have no
activity (Table 1) regardless of carbon 3 oxidation state. PHLAs
with IC50 values greater than 100 µM were deemed inactive
because growth inhibitory activity at higher concentrations may
be a consequence of nonspecific detergent effects. Compounds
with long acyl chain length (10-14 carbons) maintained the
greatest growth inhibitory activity. Presence of a carbonyl at
carbon 3 drastically enhances activity, confirming previous
reports that 3-oxo substitution is required for anticancer effects
of AHLs.2
a (b) PS-carbodiimide (5 equiv), DMAP (1.05 equiv); 22°C overnight
(c) W (100 W, 2 × 10 min).
carbodiimide coupling reagent provides a platform for quick
and simple purification via filtration, as the major reaction
byproduct, urea, remains bound to the resin. Microwave
irradiation affords dramatic reduction in reaction time.
N-phenacyl-L-homoserine lactones (Table 1, compounds 2 and
3a-12a) were synthesized by resin-bound carbodiimide-medi-
ated direct coupling of commercially available phenacyl car-
boxylic acids with L-homoserine lactone HBr (L-HSL) under
conditions of microwave irradiation (Scheme 1). High energy
microwave reaction (1000 W) allowed for very short reaction
times (30 s to 1.5 min) to completion. Removal of resin-bound
byproduct via filtration yields the final product. Short chain
analogues (<8 carbons) were recrystallized from EtOAc with
hexanes. Products not recrystallized were readily purified by
preparative TLC. Final purification was achieved by RP-HPLC
and structures confirmed by 1H NMR and ESI-MS. Compounds
2, 3a and 4a (Table 1) have been previously reported.13
N-(3-oxo-Phenacyl)-L-homoserine lactones (Table 1, com-
pounds 3b-12b, 13, and 14) were synthesized by preparation
of the corresponding active 3-oxo-phenacyl esters followed by
coupling to L-HSL (Scheme 2). The 3-oxo-phenacyl active esters
were prepared by overnight phenacylation of Meldrum’s acid
(2,2-dimethyl-1,3-dioxane-4,6-dione) in the presence of DMAP
and resin-bound carbodiimide. The isolated phenacyl-Meldrum’s
acid derivatives 1 were used without further purification.
Coupling to L-HSL was achieved in the presence of triethyl-
amine in CH3CN using microwave irradiation. Extended low
energy microwave reactions (100 W, 2 × 10 min) were required
to prevent formation of extensive side products. Regardless,
microwave irradiation decreases reaction time to 20 min from
5 h.10 Final products were readily isolated by gradient flash
chromatography or preparative TLC. Final purification was
QS signaling activity was assessed using a LasR-dependent,
green fluorescence protein (GFP) reporter assay that mimics
natural OdDHL signaling in P. aeruginosa.15 Activation of QS
in response to PHLA is reported as percent activation relative
to OdDHL control (Figure 2). OdDHL was examined at 0.3
µM, which achieves half-maximal induction of the rsaL::gfp
fusion reporter. PHLAs were evaluated at 33 µM or 100× the
concentration of OdDHL. A dose-dependent decrease in activity
was observed for analogues that activated QS to greater than
100% of control (data not shown). SAR analysis of QS signaling
activity demonstrates that compounds with acyl chains between
8-11 carbons retain greatest capacity for QS signaling. A drop
in activity is seen for compounds 12b-14. As with cancer cell
growth inhibition, short chain PHLAs lack functionality while
presence of a 3-oxo substitution enhances QS signaling.
Drug discovery is based upon identification of a lead
compound that can be accelerated through the developmental
pipeline. For compounds from this study to qualify as leads,
we established several criteria: (1) ready accessibility to
starting materials, (2) inexpensive and straightforward syn-
thesis and purification, (3) significant cancer growth inhibi-
tory activity, (4) structurally specific cancer growth inhibitory
activity (i.e., inactive non-3-oxo- or phenacyl-D-homoserine
lactone analogue), and (5) selectivity for anticancer versus
QS signaling activity. All compounds in the PHLA library
meet criterion 1 as starting materials were commercially
available. The microwave-assisted syntheses described herein
qualify all compounds for criterion 2. As described, com-
pounds with acyl chain length 10-14 carbons and 3-oxo
substitution (10b-14) maintain highest cancer cell growth
inhibitory activity. Compounds 10a and 11a, which lack a
3-oxo substitution, inhibit growth of PC3 cancer cells. Yet
compound 12a does not inhibit growth of cancer cells,
indicating that the activity of 12b is highly structure-specific.
Comparative SAR analysis of biological activities points to
12b as the compound that retains the greatest selectivity
between cancer growth inhibitory and QS signaling activities
(Figure 3).
1
achieved by RP-HPLC and structures confirmed by H NMR
and ESI-MS. Compound 4b has been previously reported.13
Results and Discussion
The current study was undertaken to dissect the broad
structural requirements for inhibition of cancer cell growth
versus QS activation. The ultimate goal of this study was to
identify potential lead compounds that retain anticancer proper-
ties while minimizing QS signaling activity. OdDHL, the
naturally produced P. aeruginosa AHL, has demonstrated
growth inhibitory activity at µM concentrations in a variety of
cancer cell lines.1,2 OdDHL-mediated growth inhibition is
selective for tumorigenic human cells.1
To confirm 12b as the lead compound from these studies,
control structures were synthesized and evaluated for compari-
son. OdDHL was used for reference. Table 2 demonstrates that
To determine the structural basis for the distinct biological
activities of OdDHL, a series of phenyl-substituted AHLs was

Phenacylhomoserine Lactones as Anticancer Compounds
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1571
Table 1. Structures, Yields, and IC50 Values of PHLA Library in Three Human Cancer Cell Lines
a Compounds previously reported.13 Recrystallized yield.
lead compound 12b preserves cancer growth inhibitory activity
when compared to OdDHL in three cancer cell lines, while the
enantiomer, D-12b, is inactive.
minimize activation of QS signaling in P. aeruginosa.
Comparative SAR analysis of a library of PHLAs for cancer
growth inhibitory and QS signaling activity identified 12b
as a lead structure for further biological evaluation and
structural modification. Compound 12b inhibits the growth
of cancer cells at low µM concentrations with little activation
of QS signaling pathways. The activity of 12b is structure-
Conclusions
The overall goal of this study was to identify lead
compounds with cancer growth inhibitory activity that

1572 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
OliVer et al.
Figure 2. Activation of QS. Expression of GFP in response to OdDHL or PHLA. Data presented as percent QS activation relative to OdDHL and
represents the average of three independent experiments. Open bars represent series a, which lack a 3-oxo substitution. Closed bars represent series
b, which possess a 3-oxo substitution.
Figure 3. Comparative SAR analysis. Comparison of PC3 cancer cell growth inhibitory and QS signaling activities for series b PHLAs. IC50 data
presented on inverse axis. Selectivity for anticancer activity is determined by distance of QS activation line from IC50 line. Greater positive distance
(QS activation line below IC50 line) indicates greater selectivity for anticancer activity.
Table 2. Comparison of Anticancer Activity of Lead and Control
Structures
of this series could yield an agent that enhances current
chemotherapeutic regimens while suppressing nosocomial P.
aeruginosa infections that plague immune-compromised
patients. This study presents the first comparative analysis
of the distinct biological effects of AHLs on cancer and
bacterial QS.
IC50 (µM)
compd
H630
H630-1
PC3
OdDHL
12b
D-12b
34 ( 10
16 ( 2
>100
12 ( 4
13 ( 4
>100
5 ( 3
4 ( 2
>100
Experimental Methods
Chemistry. Synthetic procedures were adapted from methods
previously described.10,11 All starting materials were purchased from
commercial sources (Aldrich, Milwaukee, WI; Karl Industries,
Aurora, OH; Lancaster Synthesis, Pelham, NH; Oakwood Products,
West Columbia, SC). Analytical thin layer chromatography (TLC)
was conducted on silica plates (Analtech). Developed plates were
examined under UV light and stained with ninhydrin. Preparative
TLC was conducted on 1000 µm silica plates (Analtech). Flash
specific as elimination of the 3-oxo substitution or use of
the D-HSL enantiomer attenuates activity. The phenyl sub-
stituent of 12b offers a platform amenable to modification.
Such modifications could address any residual QS activation
and/or enhance solubility to yield third-generation analogues
with improved drug characteristics. OdDHL increases the
activity of 5-FU and Taxol.2 Thus, continued development

Phenacylhomoserine Lactones as Anticancer Compounds
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1573
chromatography was performed with a Flashmaster Personal
(Argonaut Technologies). Analytical reverse-phase high-perfor-
mance liquid chromatography (RP-HPLC) was conducted on an
Agilent LC system using a Luna C18(2) 4.6 mm × 250 mm, 5 µm
column (Phenomenex, Torrance, CA). NMR spectra were recorded
on a Bruker AMX 400 MHz, Varian VNMRS 400 MHz, or Bruker
AMX 600 MHz instrument at room temperature. Electrospray mass
spectra (ESI-MS) were recorded on a Bruker Esquire-LC ion trap
mass spectrometer.
) 8.85 Hz), 4.232-4.156 (m, 1H), 2.559 (t, 2H, J ) 7.6 Hz),
2.417-2.312 (m, 1H), 2.185-2.063 (m, 1H), 2.092 (t, 2H, J )
7.4 Hz), 1.601-1.440 (m, 4H), 1.321-1.248 (m, 4H). ESI-MS m/z:
[M + Na]+ calcd for C17H23NNaO3, 312.2; found, 312.2; [2M +
Na]+ calcd for C34H46N2NaO6, 601.3; found, 601.0.
N-8-Phenyloctanoyl-L-homoserine Lactone (8a). Melting point
1
99.3-100.4 °C. RP-HPLC: tr ) 21.98 min. H NMR (600 MHz,
DMSO-d6) δ 8.266 (d, 1H, NH, J ) 8.0 Hz), 7.288-7.128 (m,
5H, Ar-H), 4.554-4.471 (m, 1H), 4.332 and 4.328 (dt, 1H, J )
8.9 Hz), 4.231-4.153 (m, 1H), 2.559 (t, 2H, J ) 7.9 Hz),
4.414-4.312 (m, 1H), 2.182-2.060 (m, 1H), 2.091 (t, 2H, J )
7.5 Hz), 1.603-1.441 (m, 4H), 1.318-1.213 (br s, 6H). ESI-MS
m/z: [M + Na]+ calcd for C18H25NNaO3, 326.2; found, 326.3; [M
+ K]+ calcd for C18H25KNO3, 342.2; found, 342.2; [2M + Na]+
calcd for C36H50N2NaO6, 629.4; found, 629.0.
Synthesis of N-Phenacyl-L-homoserine Lactones: General
Method A. To a mixture of PS-carbodiimide resin (0.620 mmol,
1.24 mmol/g, Argonaut Technologies), phenacyl carboxylic acid
(0.230 mmol), and DCM (5.0 mL) in a 10 mL reactivial (Pierce)
was added a solution of (S)-(-)-R-amino-γ-butyrolactone HBr
(0.155 mmol, Aldrich) in MeOH (0.5 mL). After microwave
irradiation (1000 W, 1.5 min), PS-carbodiimide resin was removed
by vacuum filtration and solvents were removed in vacuo. The
reaction product was: (1) recrystallized from EtOAc with Hex or
(2) purified by preparative TLC (EtOAc:Hex, 80:20 v/v). Purity
was confirmed by HPLC (linear gradient, 30 min, 10-100%
CH3CN in H2O + 0.1% TFA).
N-9-Phenylnonanoyl-L-homoserine Lactone (9a). RP-HPLC:
1
tr ) 23.69 min. H NMR (400 MHz, DMSO-d6) δ 8.257 (d, 1H,
NH, J ) 8.1 Hz), 7.290-7.126 (m, 5H, Ar-H), 4.552-4.467 (m,
1H), 4.333 and 4.328 (dt, 1H, J ) 8.8 Hz), 4.230-4.155 (m, 1H),
2.557 (t, 2H, J ) 7.7 Hz), 2.412-2.313 (m, 1H), 2.181-2.055 (m,
1H), 2.089 (t, 2H, J ) 7.5 Hz), 1.600-1.435 (m, 4H), 1.334-1.209
(br m, 8H). ESI-MS m/z: [M + Na]+ calcd for C19H27NNaO3, 340.2;
found, 340.3.
N-2-Phenylethanoyl-L-homoserine Lactone (2).13 Melting point
130.7-132.0 °C. RP-HPLC: tr ) 11.40 min. 1H NMR (600 MHz,
DMSO-d6) δ 8.563 (d, 1H, NH, J ) 7.9 Hz), 7.329-7.200 (m,
5H, Ar-H), 4.594-4.510 (m, 1H), 4.339 and 4.334 (dt, 1H, J )
8.85 Hz), 4.239-4.163 (m, 1H), 3.466 (s, 2H), 2.439-2.352 (m,
1H), 2.190-2.075 (m, 1H). ESI-MS m/z: [M + Na]+ calcd for
C12H13NNaO3, 242.1; found, 242.1; [2M + Na]+ calcd for
C24H26N2NaO6, 461.2; found, 461.1.
N-10-Phenyldecanoyl-L-homoserine Lactone (10a). RP-HPLC:
1
tr ) 25.39 min. H NMR (600 MHz, DMSO-d6) δ 8.267 (d, 1H,
NH, J ) 8.0 Hz), 7.292-7.125 (m, 5H, Ar-H), 4.556-4.470 (m,
1H), 4.333 and 4.328 (dt, 1H, J ) 8.8 Hz), 4.233-4.152 (m, 1H),
2.556 (t, 2H, J ) 7.8 Hz), 2.422-2.315 (m, 1H), 2.185-2.056 (m,
1H), 2.089 (t, 2H, J ) 7.4 Hz), 1.602-1.436 (m, 4H), 1.256 (m,
10H). ESI-MS m/z: [M + Na]+ calcd for C20H29NNaO3, 354.2;
found, 354.3.
N-3-Phenylpropanoyl-L-homoserine Lactone (3a).13 Melting
point 150.0-150.8 °C. RP-HPLC: tr ) 13.25 min. 1H NMR (600
MHz, DMSO-d6) δ 8.357 (d, 1H, NH, J ) 7.7 Hz), 7.295-7.148
(m, 5H, Ar-H), 4.573-4.491 (m, 1H), 4.338 and 4.333 (dt, 1H,
J ) 8.85 Hz), 4.236-4.160 (m, 1H), 2.820 (t, 2H, J ) 7.7 Hz),
2.419 and 2.415 (dt, 2H, J ) 7.6 Hz and 8.15 Hz), 2.388-2.323
(m, 1H), 2.154-2.035 (m, 1H). ESI-MS m/z: [M + Na]+ calcd
for C13H15NNaO3, 256.1; found, 256.3; [M + K]+ calcd for
C13H15KNO3, 272.1; found, 272.2 1; [2M + Na]+ calcd for
C26H30N2NaO6, 489.2; found, 489.1.
N-11-Phenylundecanoyl-L-homoserine Lactone (11a). RP-
HPLC: tr ) 26.48 min. 1H NMR (400 MHz, DMSO-d6) δ
8.280-8.206 (m, 1H, NH), 7.303-7.125 (m, 5H, Ar-H),
4.549-4.459 (m, 1H), 4.361-4.290 (m, 1H), 4.231-4.147 (m, 1H),
2.411-2.317 (m, 1H), 2.188-1.989 (br m, 3H), 1.676-1.378 (br
m, 6H), 1.245-1.131 (br m, 12H). ESI-MS m/z: [M + Na]+ calcd
for C21H31NNaO3, 368.2; found, 368.3.
N-12-Phenyldodecanoyl-L-homoserine Lactone (12a). RP-
N-4-Phenylbutanoyl-L-homoserine Lactone (4a).13 Melting
1
HPLC: tr ) 28.67 min. H NMR (600 MHz, DMSO-d6) δ 8.267
1
point 76.2-77.3 °C. RP-HPLC: tr ) 15.14 min. H NMR (600
(d, 1H, NH, J ) 8.1 Hz), 7.300-7.125 (m, 5H, Ar-H), 4.560-4.468
(m, 1H), 4.333 and 4.328 (dt, 1H, J ) 8.8 Hz), 4.236-4.154 (m,
1H), 2.554 (t, 2H, J ) 7.6 Hz), 2.418-2.328 (m, 1H), 2.193-2.060
(m, 1H), 2.090 (t, 2H, J ) 7.45 Hz), 1.600-1.440 (m, 4H),
1.300-1.208 (m, 14H). ESI-MS m/z: [M + Na]+ calcd for
C22H33NNaO3, 382.2; found, 382.3.
MHz, DMSO-d6) δ 8.307 (d, 1H, NH, J ) 7.7 Hz), 7.307-7.146
(m, 5H, Ar-H), 4.575-4.490 (m, 1H), 4.341 and 4.336 (dt, 1H, J
) 8.9 Hz and 8.7 Hz), 4.240-4.160 (m, 1H), 2.572 (t, 2H, J ) 7.6
Hz), 4.424-4.339 (m, 1H), 2.211-2.083 (m, 1H), 2.129 (t, 2H, J
) 7.6 Hz), 1.798 (m, 2H). ESI-MS m/z: [M + Na]+ calcd for
C14H17NNaO3, 270.1; found, 270.1; [2M + Na]+ calcd for
C28H34N2NaO6, 517.2; found, 517.0.
Synthesis of N-(3-oxo-Phenacyl)-L-homoserine Lactones:
General Method B. PS-carbodiimide resin (0.504 mmol, 1.26
mmol/g, Argonaut Technologies), phenacyl carboxylic acid (0.252
mmol), 2,2-dimethyl-1,3-dioxane-4,6-dione (0.252 mmol), 4,4-
dimethylaminopyridine (0.265 mmol), and DCM (5.0 mL) were
added to a 10 mL reactivial (Pierce). After stirring at room
temperature overnight, PS-carbodiimide resin was removed by
vacuum filtration and solvents were removed in vacuo. The resulting
residue was dissolved in EtOAc (10 mL) and washed with 0.1 N
HCl (10 mL). The organic layer was washed with H2O (3 × 20
mL) and dried (MgSO4) to yield a yellow oil. Without further
purification, the yellow oil product was dissolved in CH3CN (5
mL) and transferred to a 10 mL reactivial (Pierce) with triethylamine
(0.237 mmol) and: (1) (S)-(-)-R-amino-γ-butyrolactone HBr (0.198
mmol) for L-isomer or (2) (R)-(+)-R-amino-γ-butyrolactone HCl
(0.198 mmol) for D-isomer. After microwave irradiation (100 W,
2 × 10 min), solvents were removed in vacuo. The resulting residue
was redissolved in EtOAc (10 mL + 0.5 mL H2O) and washed
sequentially with NaHCO3 (1 × 10 mL), 1.0 M KHSO4 (1 × 10
mL), and sat. NaCl (1 × 10 mL). The organic layer was dried
(MgSO4), concentrated and purified by gradient flash chromatog-
raphy (20 g Isolute Flash SI II silica column cartridge, 0-80%
EtOAc in Hex, 10 mL/min, 47 min). Purity was confirmed by HPLC
(linear gradient, 30 min, 10-100% CH3CN in H2O + 0.1% TFA).
N-5-Phenylpentanoyl-L-homoserine Lactone (5a). Melting
1
point 132.5-134.0 °C. RP-HPLC: tr ) 16.85 min. H NMR (600
MHz, DMSO-d6) δ 8.299 (d, 1H, NH, J ) 8.0 Hz), 7.304-7.134
(m, 5H, Ar-H), 4.562-4.473 (m, 1H), 4.335 and 4.329 (dt, 1H, J
) 9.0 Hz), 4.235-4.154 (m, 1H), 2.571 (t, 2H, J ) 7.2 Hz),
2.419-2.313 (m, 1H), 2.184-2.064 (m, 1H), 2.137 (t, 2H, J )
7.0 Hz), 1.613-1.471 (m, 4H). ESI-MS m/z: [M + Na]+ calcd for
C15H19NNaO3, 284.1; found, 284.1; [M + K]+ calcd for
C15H19KNO3, 300.1; found, 300.1; [2M + Na]+ calcd for
C30H38N2NaO6, 545.3; found, 545.0.
N-6-Phenylhexanoyl-L-homoserine Lactone (6a). Melting point
1
91.6-92.6 °C. RP-HPLC: tr ) 18.57 min. H NMR (600 MHz,
DMSO-d6) δ 8.276 (d, 1H, NH, J ) 7.9 Hz), 7.291-7.129 (m,
5H, Ar-H), 4.558-4.470 (m, 1H), 4.334 and 4.329 (dt, 1H, J )
9.0 Hz and 8.85 Hz), 4.235-4.154 (m, 1H), 2.557 (t, 2H, J ) 7.55
Hz), 2.410-2.314 (m, 1H), 2.169-2.048 (m, 1H), 2.100 (t, 2H, J
) 7.25 Hz), 1.609-1.488 (m, 4H), 1.329-1.234 (m, 2H). ESI-
MS m/z: [M + Na]+ calcd for C16H21NNaO3, 298.1; found, 298.2;
[2M + Na]+ calcd for C32H42N2NaO6, 573.3; found, 572.9.
N-7-Phenylheptanoyl-L-homoserine Lactone (7a). Melting
1
point 127.8-129.3 °C. RP-HPLC: tr ) 20.28 min. H NMR (600
MHz, DMSO-d6) δ 8.270 (d, 1H, NH, J ) 8.0 Hz), 7.293-7.130
(m, 5H, Ar-H), 4.558-4.470 (m, 1H), 4.334 and 4.329 (dt, 1H, J

1574 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
N-(3-oxo-3-Phenylpropanoyl)-L-homoserine Lactone (3b). RP-
OliVer et al.
1H), 3.328 (s, 2H), 2.555 (t, 2H, J ) 7.9 Hz), 2.525-2.472 (m,
2H and DMSO), 2.456-2.374 (m, 1H), 2.204-2.075 (m, 1H),
1.600-1.506 (m, 2H), 1.488-1.396 (m, 2H), 1.300-1.200 (br m,
8H). ESI-MS m/z: [M + Na]+ calcd for C21H29NNaO4, 382.2; found,
382.2.
N-(3-oxo-12-Phenyldodecanoyl)-L-homoserine Lactone (12b).
RP-HPLC: tr ) 26.01 min. 1H NMR (400 MHz, DMSO-d6) δ 8.587
(d, 1H, NH, J ) 7.7 Hz), 7.289-7.122 (m, 5H, Ar-H), 4.592-4.506
(m, 1H), 4.375-4.307 (m, 1H), 4.242-4.160 (m, 1H), 3.329 (s,
2H), 2.552 (t, 2H, J ) 7.6 Hz), 2.523-2.467 (m, 2H and DMSO),
2.452-2.362 (m, 1H), 2.205-2.068 (m, 1H), 1.597-1.501 (m, 2H),
1.488-1.383 (m, 2H), 1.322-1.171 (m, 10H). ESI-MS m/z: [M +
Na]+ calcd for C22H31NNaO4, 396.2; found, 396.3.
N-(3-oxo-12-Phenyldodecanoyl)-D-homoserine Lactone (D-12b).
RP-HPLC: tr ) 26.04 min. 1H NMR (400 MHz, DMSO-d6) δ 8.580
(d, 1H, NH, J ) 8.0 Hz), 7.285-7.128 (m, 5H, Ar-H), 4.591-4.510
(m, 1H), 4.345 and 4.340 (dt, 1H, J ) 7.7 Hz), 4.241-4.164 (m,
1H), 3.329 (s, 2H), 2.553 (t, 2H, J ) 7.6 Hz), 2.521-2.475 (m,
2H and DMSO), 2.451-2.371 (m, 1H), 2.200-2.074 (m, 1H),
1.591-1.504 (m, 2H), 1.474-1.393 (m, 2H), 1.310-1.177 (m,
10H). ESI-MS m/z: [M + Na]+ calcd for C22H31NNaO4, 396.2;
found, 396.1.
N-(3-oxo-13-Phenyltridecanoyl)-L-homoserine Lactone (13). RP-
HPLC: tr ) 27.07 min. 1H NMR (400 MHz, DMSO-d6) δ
8.573-8.510 (m, 1H, NH), 7.292-7.111 (m, 5H, Ar-H),
4.576-4.483 (m, 1H), 4.363-4.297 (m, 1H), 4.234-4.153 (m, 1H),
3.313 (s, 2H), 2.507-2.335 (m, 5H), 2.192-2.066 (m, 1H),
1.537-1.326 (m, 4H), 1.268-0.956 (m, 12H). ESI-MS m/z: [M +
Na]+ calcd for C23H33NNaO4, 410.2; found, 410.4.
N-(3-oxo-14-Phenyltetradecanoyl)-L-homoserine Lactone (14).
RP-HPLC: tr ) 29.16 min. 1H NMR (400 MHz, DMSO-d6) δ 8.559
(d, 1H, NH, J ) 7.4 Hz), 7.280-7.136 (m, 5H, Ar-H), 4.576-4.517
(m, 1H), 4.344 (t, 1H, J ) 8.9 Hz), 4.236-4.176 (m, 1H), 3.330
(s, 2H), 2.574-2.535 (m, 4H), 2.444-2.375 (m, 1H), 2.187-2.107
(m, 1H), 1.580-1.520 (m, 2H), 1.498-1.412 (m, 2H), 1.295-1.203
(m, 14H). ESI-MS m/z: [M + Na]+ calcd for C24H35NNaO4, 424.3;
found, 424.3. [2M + H]+ calcd for C48H71N2O8, 803.5; found, 803.5.
Biological Evaluation: Cell Culture. Human colorectal carci-
noma cell lines, H630 (parental) and H630-1 (5-fluorouracil
resistant), and human prostate carcinoma cell line, PC3, were
cultured in RPMI-1640 phenol-red free (PRF) media supplemented
with 10% dialyzed fetal bovine serum (dFBS), without antibiotics.
H630-1 cells were grown in media containing 1 µM 5-fluorouracil
(5-FU) until 1 week prior to experimentation. All cells were
maintained in a humidified incubator at 37 °C and 5% CO2.
Growth Inhibition of Cancer Cell Lines. Growth inhibition
was measured by the sulforhodamine B (SRB) assay in 96-well
plate format adapted from methods previously described.16 Briefly,
the compounds were added to the wells of 96-well plates at 10×
final concentration in EtOH (20 µL) in duplicate or quadruplicate.
After overnight drying, H630, H630-1 and PC3 cells were seeded
at 2 × 103, 4 × 103, and 2 × 103 cells/well, respectively, in RPMI-
1640PRF supplemented with 10% dFBS. After 5 days, media was
removed and cells were fixed with 10% trichloroacetic acid for
g1 h at 4 °C. Plates were washed with shaking (dH2O, 3 × 300
µL/well) and allowed to dry. After 15 min incubation with 0.4%
SRB solution (100 µL/well), plates were washed with shaking (1%
acetic acid, 4 × 300 µL/well) and allowed to dry. Protein-bound
SRB dye was dissolved in 100 mM TRIS (100 µL/well) with
shaking for 5 min and quantified by measurement of optical density
at 570 nm (OD570). IC50 values represent the average of three
independent experiments and were determined from growth curves
generated using Sigmaplot software.
1
HPLC: tr ) 11.17 min. H NMR (400 MHz, DMSO-d6) δ 8.687
(d, 1H, NH, J ) 8.0 Hz), 7.677-7.473 (m, 5H, Ar-H), 4.625-4.538
(m, 1H), 4.348 and 4.344 (dt, 1H, J ) 8.9 Hz), 4.249-4.171 (m,
1H), 3.958 (s, 2H), 2.480-2.393 (m, 1H), 2.208-2.086 (m, 1H).
ESI-MS m/z: [M + Na]+ calcd for C13H13NNaO4, 270.1; found,
270.1.
N-(3-oxo-4-Phenylbutanoyl)-L-homoserine Lactone (4b).13 RP-
1
HPLC: tr ) 12.60 min. H NMR (600 MHz, DMSO-d6) δ 8.602
(d, 1H, NH, J ) 7.5 Hz), 7.335-7.147 (m, 5H, Ar-H), 4.590-4.533
(m, 1H), 4.346 and 4.343 (dt, 1H, J ) 8.9 Hz), 4.233-4.179 (m,
1H), 3.853 (s, 2H), 3.427 (s, 2H), 2.446-2.386 (m, 1H), 2.190-2.106
(m, 1H). ESI-MS (m/z): [M + Na]+ calcd for C14H15NNaO4, 284.1;
found, 284.2; [2 M + Na]+ calcd for C28H30N2NaO8, 545.2; found,
545.1.
N-(3-oxo-5-Phenylpentanoyl)-L-homoserine Lactone (5b). RP-
1
HPLC: tr ) 14.71 min. H NMR (600 MHz, DMSO-d6) δ 8.584
(d, 1H, NH, J ) 7.8 Hz), 7.273-7.142 (m, 5H, Ar-H), 4.574-4.514
(m, 1H), 4.338 and 4.335 (dt, 1H, J ) 8.8 Hz), 4.227-4.170 (m,
1H), 3.368 (s, 2H), 2.859 (t, 2H, J ) 7.65 Hz), 2.767 (t, 2H, J )
7.65 Hz), 2.439-2.377 (m, 1H), 2.178-2.095 (m, 1H). ESI-MS
m/z: [M + Na]+ calcd for C15H17NNaO4, 298.1; found, 298.1; [2M
+ Na]+ calcd for C30H34N2NaO8, 573.2; found, 573.0.
N-(3-oxo-6-Phenylhexanoyl)-L-homoserine Lactone (6b). RP-
1
HPLC: tr ) 16.17 min. H NMR (600 MHz, DMSO-d6) δ 8.559
(d, 1H, NH, J ) 7.9 Hz) 7.290-7.148 (m, 5H, Ar-H), 4.569-4.513
(m, 1H), 4.337 and 4.335 (dt, 1H, J ) 8.8 Hz), 4.227-4.174 (m,
1H), 3.336 (d, 2H, J ) 1.1 Hz), 2.550-2.506 (m, 4H), 2.431-2.374
(m, 1H), 2.168-2.086 (m, 1H), 1.782-1.720 (m, 2H). ESI-MS m/z:
[M + Na]+ calcd for C16H19NNaO4, 312.1; found, 312.1; [2M +
Na]+ calcd for C32H38N2NaO8, 601.3; found, 601.0.
N-(3-oxo-7-Phenylheptanoyl)-L-homoserine Lactone (7b). RP-
1
HPLC: tr ) 17.82 min. H NMR (600 MHz, DMSO-d6) δ 8.555
(d, 1H, NH, J ) 7.8 Hz), 7.277-7.131 (m, 5H, Ar-H), 4.567-4.511
(m, 1H), 4.334 and 4.331 (dt, 1H, J ) 8.8 Hz), 4.222-4.170 (m,
1H), 3.223 (d, 2H, J ) 1.6 Hz), 2.566-2.526 (m, 4H), 2.429-2.374
(m, 1H), 2.175-2.096 (m, 1H), 1.550-1.434 (m, 4H). ESI-MS m/z:
[M + Na]+ calcd for C17H21NNaO4, 326.1; found, 326.2; [2M +
Na]+ calcd for C34H42N2NaO8, 629.3; found, 629.1.
N-(3-oxo-8-Phenyloctanoyl)-L-homoserine Lactone (8b). RP-
1
HPLC: tr ) 19.46 min. H NMR (600 MHz, DMSO-d6) δ 8.554
(d, 1H, NH, J ) 7.7 Hz), 7.269-7.129 (m, 5H, Ar-H), 4.565-4.510
(m, 1H), 4.334 and 4.332 (dt, 1H, J ) 9.0 Hz), 4.222-4.171 (m,
1H), 3.322 (d, 2H, J ) 1.8 Hz), 2.541 (t, 2H, J ) 7.7 Hz), 2.502
(t, 2H, J ) 7.3 Hz), 2.429-2.373 (m, 1H), 2.174-2.092 (m, 1H),
1.571-1.508 (m, 2H), 1.507-1.448 (m, 2H), 1.272-1.211 (m, 2H).
ESI-MS m/z: [M + Na]+ calcd for C18H23NNaO4, 340.2; found,
340.2.
N-(3-oxo-9-Phenylnonanoyl)-L-homoserine Lactone (9b). RP-
1
HPLC: tr ) 21.09 min. H NMR (600 MHz, DMSO-d6) δ 8.553
(d, 1H, NH, J ) 7.6 Hz), 7.271-7.129 (m, 5H, Ar-H), 4.568-4.513
(m, 1H), 4.336 and 4.333 (dt, 1H, J ) 8.8 Hz), 4.224-4.172 (m,
1H), 3.323 (d, 2H, J ) 1.4 Hz), 2.544 (t, 2H, J ) 7.7 Hz), 2.491
(t, 2H, J ) 7.2 Hz), 2.433-2.377 (m, 1H), 2.177-2.098 (m, 1H),
1.570-1.506 (m, 2H), 1.469-1.411 (m, 2H), 1.283-1.218 (m, 4H).
ESI-MS m/z: [M + Na]+ calcd for C19H25NNaO4, 354.2; found,
354.3; [2M + Na]+ calcd for C38H50N2NaO8, 685.3; found, 685.0.
N-(3-oxo-10-Phenyldecanoyl)-L-homoserine Lactone (10b).
RP-HPLC: tr ) 22.73 min. 1H NMR (600 MHz, DMSO-d6) δ 8.552
(d, 1H, NH, J ) 7.7 Hz), 7.268-7.127 (m, 5H, Ar-H), 4.564-4.510
(m, 1H), 4.334 and 4.331 (dt, 1H, J ) 8.9 Hz), 4.221-4.171 (m,
1H), 3.319 (d, 2H, J ) 2.0 Hz), 2.546 (t, 2H, J ) 7.8 Hz),
2.506-2.475(m,2HandDMSO),2.432-2.375(m,1H),2.117-2.097
(m, 1H), 1.572-1.507 (m, 2H), 1.462-1.400 (m, 2H), 1.292-1.171
(m, 6H). ESI-MS m/z: [M + Na]+ calcd for C20H27NNaO4, 368.2;
found, 368.4; [2M + Na]+ calcd for C40H54N2NaO8, 713.4; found,
713.0.
Activation of Quorum Sensing. To assess the ability of PHLAs
to activate quorum sensing, we used the strain Pseudomonas
aeruginosa MW1 (lasI-, rhlI-) carrying pUM15, which contains
an rsaL::gfp transcriptional fusion. The bacteria were grown in LB
with 150 µg/mL of carbenicillin. Assays were similar to those
previously described.15 The reporter culture was inoculated from
an overnight starter culture to an OD600 ) 0.05 with shaking at 37
°C for 1-2 h. Bacterial cell cultures (0.5 mL) in midlogarithmic
N-(3-oxo-11-Phenylundecanoyl)-L-homoserine Lactone (11b).
RP-HPLC: tr ) 24.37 min. 1H NMR (400 MHz, DMSO-d6) δ 8.554
(d, 1H, NH, J ) 7.9 Hz), 7.287-7.127 (m, 5H, Ar-H), 4.588-4.506
(m, 1H), 4.344 and 4.340 (dt, 1H, J ) 9.0 Hz), 4.243-4.168 (m,

Phenacylhomoserine Lactones as Anticancer Compounds
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to achieve a final concentration of 0.3 µM and 33 µM, respectively.
OdDHL at 0.3 µM achieves half-maximal induction of rsaL::gfp
fusion reporter. Tubes were incubated at 37 °C with shaking. After
5 h, fluorescence from 100 µL of sample was measured with an
excitation wavelength of 485 nm and an emission wavelength of
535 nm using a Tecan Genios Pro Platereader. Results represent
the average of three independent experiments.
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Acknowledgment. These studies were supported in part by
NIH/NCI training grants 5T32CA009072 (C.M.O.), CA091114
(J.R.S.), and USPHS GM-59026 (A.L.S. and E.P.G.). Spectral
characterizations of newly synthesized compounds were carried
out in the NMR and Biopolymer Core Facilities at Roswell Park
Cancer Institute. These core facilities are supported in part by
NCI Core Grant CA16056.
Supporting Information Available: HPLC and NMR spectra
for PHLA library. This material is available free of charge via the
Internet at http://pubs.acs.org.
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