Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators

Article abstract of DOI:10.1021/jm020909n

Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone (30, C₁₂-HSL), is described. Twenty-four single or combination systematic alteration

Full text of DOI:10.1021/jm020909n

J . Med. Chem. 2003, 46, 97-104
97
Syn th etic An a logu es of th e Ba cter ia l Sign a l (Qu or u m Sen sin g) Molecu le
N-(3-Oxod od eca n oyl)-L-h om oser in e La cton e a s Im m u n e Mod u la tor s
Siri Ram Chhabra, Chris Harty, Doreen S. W. Hooi, Mavis Daykin, Paul Williams, Gary Telford,
David I. Pritchard,* and Barrie W. Bycroft*
School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, U.K.
Received April 15, 2002
Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas
aeruginosa signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone (3O, C12-HSL), is
described. Twenty-four single or combination systematic alterations of the structural compo-
nents of 3O, C12-HSL were introduced as described. Given the already defined immunological
profile of the parent compound, 3O, C12-HSL, these compounds were assayed for their ability
to inhibit murine and human leucocyte proliferation and TNF-R secretion by lipopolysaccharide
(LPS) stimulated human leucocytes in order to provide an initial structure-activity profile.
From IC50 values obtained with a murine splenocyte proliferation assay, it is apparent that
acylated L-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a
3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of
3O, C12-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain
were also potent immune suppressive agents. However, structures lacking the homoserine
lactone ring, structures lacking the L-configuration at the chiral center, and those with polar
substituents were essentially devoid of activity. The ability of compounds selected from the
optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell
proliferation and LPS-induced TNF-R secretion indicates the suitability of these compounds
for further investigation in relation to their molecular mechanisms of action in TNF-R driven
immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid
arthritis, and type 1 (autoimmune) diabetes.
In tr od u ction
serine lactone (3O, C12-HSL),4 one of the cell-density-
dependent (quorum sensing) signal molecules derived
from Pseudomonas aeruginosa, also possesses immune
modulatory activity in animals suggested that a more
detailed evaluation of synthetic analogues of this mol-
ecule might ultimately provide a better understanding
of their mechanism of action and lead to the develop-
ment of novel therapeutic agents in due course.
Immunological diseases, including common disorders
such as rheumatoid arthritis, psoriasis, type 1 diabetes
mellitus, and autoimmune thyroid disease, as well as
less common conditions such as multiple sclerosis and
myasthenia gravis, cause chronic morbidity and dis-
ability. In a recent study of 24 well-characterized
autoimmune diseases in the U.S., it was estimated that
approximately 8 500 000, or 1 in 31 individuals in the
U.S., suffer from one of these disorders,1 which are not
successfully treated with existing therapies. The cur-
rently employed immunosuppressive drugs and their
mechanism of action have recently been reviewed.2
Steroid treatment is in the main nonselective, cy-
closporin A (CsA) and FK506 can be nephrotoxic, and
antibody therapy to Th1 cytokines is expensive and can
cause immune-mediated toxicity. Nevertheless, use of
the potent immunosuppressive agents such as CsA,
FK506, and rapamycin, which have a degree of selective
action on cells of the immune system and are derived
from fungal or bacterial sources, has led to a dramatic
increase in the success rate of transplant surgery,
indicating not only the value of immunosuppressive
natural products to clinical medicine but also an un-
derstanding of molecular mechanisms involved.2 The
recent observation3 that N-(3-oxododecanoyl)-L-homo-
Although N-acylhomoserine lactones (AHLs) have so
far largely been considered as effectors of prokaryotic
gene expression, they are also capable of influencing
eukaryotic cell behavior and potentially mediating
disease processes.3,5 In particular, 3O, C12-HSL (but not
the shorter-chain 3O, C6-HSL homologue) suppresses
human and murine leucocyte proliferation and inhibits
LPS-induced secretion of TNF-R and IL-12, an im-
munological response modification associated with many
autoimmune diseases. AHLs also promote other im-
munological responses associated with recovery from
some autoimmune diseases, indicating immunological
selectivity of action,3 although recent papers have
presented contradictory data at high dosage.6,7 What is
not in doubt is the confirmed biological activity of this
class of molecule in immunological assay.
The present study compares immune-mediated re-
sponses in a number of standard cellular assays,
produced by a range of synthetic analogues of 3O, C12-
HSL, where single or combination systematic alter-
ations have been made to the structural components of
* To whom correspondence should be addressed. For B.W.B.: phone,
44 (0) 115 8466250; fax, 44 (0) 115 8466249; e-mail, Barrie.Bycroft@
nottingham.ac.uk. For D.I.P.: phone, 44 (0) 115 9516165; fax, 44 (0)
115 8466296; e-mail, pazdp@gwmail.nottingham.ac.uk.
10.1021/jm020909n CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/28/2002

98 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Chhabra et al.
Ta ble 1. Chemical Structures, Abbreviations, and Mouse
Splenocyte Proliferation Data for All Compounds Synthesized
for SAR Studies
F igu r e 1. Structure of N-(3-oxododecanoyl)-L-homoserine
lactone. A, B, and C are the structural segments that were
modified for SAR studies.
3O, C12-HSL in order to establish the basic inherent
structural requirements for activity.
Ch em istr y
To evaluate the molecular basis for the immune
modulatory activity of the native autoinducer 3O, C12-
HSL,3 single or combination systematic structural varia-
tions were introduced within the structural components
A, B, and C of 3O, C12-HSL (Figure 1).
Chemical syntheses of some of these analogues have
been previously reported by us8-11 and other groups.12-14
N-(3-Oxoalkanoyl) derivatives were prepared by the
acylation of L-homoserine lactone (L-HSL) with activated
3-oxoalkanoic acids. The acids were either prepared
from the commercially available 3-oxo esters or chemi-
cally synthesized from appropriate acid chlorides and
malonates.10 These procedures involved the protection
and deprotection of the 3-oxo substituent, which could
be shortened by employing 5-acyl Meldrum’s acid (2,2-
dimethyl-1,3-dioxane-4,6-dione) intermediates I (Scheme
1) as a combined means of 3-oxo protection and alkanoic
acid activation.15
The 5-acyl Meldrum’s acid derivatives I were pre-
pared in nearly quantitative yields by the DCCI/DMAP
catalyzed acylation of Meldrum’s acid with an appropri-
ate carboxylic acid in CH2Cl2.16 The isolated products
were used without further purification. Their charac-
terization by 1H NMR established not only their identity
but also the presence of H-bonded enolic structures II
with the enol proton resonating at δ 14-15. Amidation
of II with L-HSL HCl in the presence of triethylamine
in CH3CN afforded the desired N-(3-oxoalkanoyl) ana-
logues III in varying yields. Often, small quantities of
side products IV, arising from the amidation of the
exocyclic carbonyl, were present but could be easily
removed by chromatographic separation on a silica
column. Final purification was achieved by RP-HPLC.
Compounds 1-16 (Table 1) were obtained by this
procedure.
hydrolyzed to the alcohol and then oxidized to the
desired acid using J ones’ reagent.
The corresponding fatty acids required for the prepa-
ration of II for the analogues 1-6, 8, 9, 11, 12, 14, and
16 were commercially available. Preparation of 11,
required prior protection of the terminal hydroxyl group
as an acetate (Ac2O and pyridine)17,18 to avoid self-
condensation. Acid hydrolysis of the final protected
product with 1 M HCl afforded the free alcohol. Unde-
candioic acid monomethyl ester required for the prepa-
ration of 10 was obtained by first converting undecan-
dioic acid to its dimethyl ester followed by saponification
with 1 M equiv of NaOH. For compounds 7 and 13, the
corresponding carboxylic acids were produced by J ones’
oxidation19 of the commercially available 10-bromo-1-
decanol and cis-8-dodecen-1-ol, respectively, and to
prepare 15, 11-dodecen-1-yl acetate was purchased and
The amidation of the 5-decanoyl Meldrum’s acid with
tert-butyl carbazate and L-homocysteine thiolactone
respectively furnished analogues 23 and 24 (Scheme 2).
The open-ring analogue 22 was prepared by methanoly-
sis of compound 4 with NaOMe at room temperature.
3-Hydroxy derivatives 16 and 17 were obtained as
mixtures of diastereoisomers in good yields by NaC-
NBH3 reduction of the corresponding 3-oxo derivatives
4 and 610 at pH 3-4 in MeOH. Finally analogues 19-
21 were synthesized by the acylation of L-HSL either
with the corresponding carboxylic acid activated with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride in a water/1,4-dioxane (1:1) solvent system (ref
10) or with the corresponding acid chlorides in DCM in
the presence of triethylamine.

Lactone Analogues as Modulators
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 99
Sch em e 1
from the type of data plot illustrated in Figure 2A,
where the 12 C and 6 C analogues are compared against
the vehicle DMSO. Splenocytes stimulated with con-
canavalin A are stimulated to incorporate [3H]-thymi-
dine into newly synthesized DNA, indicated as counts
per minute. The IC50 for 3O, C12-HSL in this figure
would be calculated to be in the region of 4-6 µM.
Unsaturation in the 12-14 C alkyl chain had little
effect on activity. Interestingly, the terminally unsatur-
ated compounds 14 and 15 were more active than
compounds 12 and 13 with unsaturation within the
chain. Activity increased, albeit slightly, as the double
bond is moved away from the 3-oxoacyl moiety, sug-
gesting that flexibility of the chain is of importance for
eliciting the decrease in mouse splenocyte response.
These studies were conducted only with the (Z)-alkenes
because of the availability of appropriate precursors
from commercial sources.
Sch em e 2
The same range of activity was observed when the
alkyl chain was terminally substituted with bromine (7
and 8), hydroxyl (11), and CO2Me (10) groups, although
the last, more polar compound was much less active.
However, the analogue with a free CO2H functionality
(9) had markedly less activity. It thus appears that
lipophilicity is important for activity.
The presence of either a 3-oxo or a 3-hydroxyl sub-
stituent (compare compounds 6 and 18 and 3 and 17)
appears to retain activity, since their absence in 19, 20,
and 21 caused a distinct drop in activity compared to
2, 3, and 4. The 3-hydroxy derivatives were assayed as
mixtures of the diastereoisomers because they could not
be resolved easily by standard HPLC protocols.
Resu lts a n d Discu ssion
The present study was undertaken to determine the
broad structural requirements for the immune modula-
tory activity of 3O, C12-HSL, following our initial
observation that this molecule possessed immunological
activity whereas the shorter-chain analogue 3O, C6-HSL
lacked such activity.3
Opening of the lactone ring structure demonstrates
that the homoserine lactone is critical for the immuno-
suppressive activity based on the observed IC50 values
for 4 and 22. The ring-opened analogue methyl ester
22 also had poor activity. Furthermore, the thio ana-
logue 24 was only weakly active compared to 4. Com-
plete removal of the HSL as represented by 23 rendered
the molecule totally inactive. Finally the L-configuration
of the homoserine lactone moiety also appears to be
critical, since the D-isomer had considerably reduced
activity (IC50 ) 100 µM).
To confirm and extend our earlier findings3 that the
parent molecule also inhibited mitogen-driven prolifera-
tion of human lymphocytes, the 3-oxo compounds with
chain lengths ranging from 8 to 14 C (1-6 listed in
Table 1) were selected for assay against Con A stimu-
lated peripheral blood monocyte (PBMC), using three
separate donors. These data are presented in Table 2,
and a representative data plot is given in Figure 2B.
Analysis of this table and figure illustrates once again
that bioactivity in immunological assay appears to
increase with increasing chain length (8-12 C); the 3O,
C6-HSL compound remains inactive (Figure 2B).
A total of 24 compounds were synthesized and char-
acterized. Their purity was established by elemental
analyses. Their homogeneity was confirmed by reverse-
phase HPLC using a linear gradient of acetonitrile in
water (70-100%, v/v) over a 20 min period. The parent
compound N-(3-oxododecanoyl)-L-homoserine lactone
(3O, C12-HSL) (Table 1, compound 4) isolated from the
spent culture of the Gram-negative bacterium Pseudomo-
nas aeruginosa suppresses murine and human leucocyte
proliferation. To determine the molecular basis of this
activity, structure alterations have been performed
initially on three different components of this molecule,
namely, the alkyl chain, the 3-oxo substituent, and the
heterocyclic ring. These compounds were assayed for
their ability to modulate murine and human leucocyte
proliferation stimulated with concanavalin A (ConA)
and TNF-R secretion by LPS stimulated human leuco-
cytes.
Compound activity in the murine splenocyte prolif-
eration assay demonstrated that analogues with an
alkyl chain length shorter than 8 C (data not shown)
had no activity (Table 1, Figure 2A). The upper limit of
the alkyl chain was dictated by analogue solubility.
Thus, our subsequent studies were confined to ana-
logues with a chain length of 8-14 C. Table 1 illustrates
that activity in a murine splenocyte assay gradually
increased when the alkyl chain was lengthened and
peaked at 12 C. However, higher homologues with 13
and 14 C, compounds 5 and 6, had similar activities.
Branched-chain analogue (compound 16) was slightly
less potent than the parent structure (4). Data are
presented as the IC50 of the compound in µM, generated
Two analogues were then taken forward for testing
in a human PBMC assay in which TNF-R secretion is
driven by bacterial lipopolysaccharide. From Table 2 it
can be seen that each compound was bioactive in this
assay; a representative data plot is given in Figure 3.
Our previous publication indicated that 3O, C6-HSL was
totally inactive in preventing LPS-driven TNF-R secre-
tion, supporting our assumption that bioactivity in all
proliferative assays translates to bioactivity in TNF-R

100 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Chhabra et al.
Ta ble 2. Mouse Cell Proliferation, Human PBMC Proliferation, and Human TNF-R Releases Data for Selected Compounds
human PBMC proliferation, IC50 (µM)
mouse cell proliferation,
human TNF-R release,
compound
IC50 (µM)
donor A
donor B
donor C
IC50 (µM)
1, 3O, C8-HSL
2, 3O, C10-HSL
3, 3O, C11-HSL
4, 3O, C12-HSL
5, 3O, C13-HSL
6, 3O, C14-HSL
∼400
250
130
60
60
35
a
a
30
10
30
15
230
65
45
35
25
30
a
a
a
15
a
35
21
8.9
4
4.3
6.3
30
a
Not determined.
F igu r e 4. The comparative potency of 3O, C12-HSL compared
with two standard immune suppressive agents dexmethasone
and cyclosporin A was assessed in a concanavalin A driven
murine splenocyte proliferation assay. It is clear from this
experiment that cell proliferation was completely inhibited by
cyclosporin along the dose range used, with dexmethasone
exhibiting an IC50 in the region of 10-3-10-4 µM. 3O, C12-HSL
was the least potent compound in this assay.
response range illustrated in this publication. Figure 4
clearly illustrates the superior potency of cyclosporin A
and dexamethasone in cell proliferative assays. How-
ever, potency does not always equate with efficacy in
vivo, and the effective dose range for 3O, C12-HSL
illustrated in the present study may in fact be sufficient
for application to some immunological diseases.
F igu r e 2. Dose-response curves of 3O, C12-HSL and 3O, C6-
HSL in the proliferation of (A) murine and (B) human
leukocytes, peripheral blood monocytes, in ConA stimulated
assays. DMSO was used as a diluent control. The results are
presented to illustrate stimulations to ConA compared with
medium (Med) and background (Bkg).
Con clu sion s
From these initial structure-activity-relationship
studies, it is apparent that the L-homoserine lactone
ring acylated with a 3-oxo or 3-hydroxy 12-14 carbon
acyl side chain is optimal for immune suppressive
activity. The overall lipophilic character of the com-
pound also appears to be important for activity. Termi-
nal substitution of the acyl chain with a bromine or
hydroxyl functionality retains activity, while polar car-
boxylic acid or methyl ester substitution decreases
activity. Unsaturation within the side chain also retains
activity. 3-Hydroxy derivatives, even as a mixture of
diastereoisomers, were equipotent with the correspond-
ing 3-oxo derivatives. These preliminary observations
using cellular assays have established a general picture
of structure-activity-relationships and allowed the
selection of a number of active compounds for more
detailed evaluation in well-established animal models
of autoimmune disease.
F igu r e 3. Dose-response curves of 3O, C12-HSL in the
secretion of TNF-R by E. coli LPS-stimulated human leuko-
cytes. DMSO was used as a diluent control. The results are
presented to illustrate stimulations to ConA compared with
medium (Med) and background (Bkg).
On the basis of the studies described here, methods
are now available for the synthesis of radioisotope and
fluorescence labeled compounds for further molecular
mechanistic studies to identify molecular targets for
these compounds in eukaryotic cells. To support this
secretion assays. Nevertheless, after a basic structure-
activity profile for 3O, C12-HSL was secured in immune
assay, it is now possible to explore further chemical
modifications to improve on the rather narrow dose-

Lactone Analogues as Modulators
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 101
layer chromatography on silica plates. Further purification was
achieved by RP-HPLC.
work, sites for attachment to solid supports within the
acyl side chain of active molecules have been identified
and currently affinity matrixes are being constructed
for the isolation and identification of the target pro-
teins.20
N-(3-Oxoocta n oyl)-L-h om oser in e La cton e (1). Mp: 78-
80 °C. RP-HPLC: tR ) 5.23 min. IR (KBr): 3290 (NH), 1790
(ring CdO), 1715 (ketone CdO), 1650 (amide CdO), 1545, 1175
1
cm-1. H NMR (CDCl3): δ 0.89 (3H, t, J ) 7.0 Hz, CH3), 1.29
(4H, m, CH3CH2CH2), 1.59 (2H, m, CH2CH2CO), 2.22 (1H, m,
4R-H), 2.52 (2H, t, J ) 7.3 Hz, CH2CO), 2.76 (1H, m, 4â-H),
3.47 (2H, s, COCH2CO), 4.27 (1H, m, 5R-H), 4.48 (1H, td, J )
1.3 and 9 Hz, 5â-H), 4.58 (1H, m, 3-H), 7.64 (1H, br s, NH).
EI-MS m/z (%): 241.1312 (11, M+, C12H19NO4 requires m/z
241.1314), 185 (65), 143 (39), 113 (28), 102 (68), 99 (49), 71
(36), 57 (86). Anal. (C12 H19NO4) C, H, N.
N-(3-Oxod eca n oyl)-L-h om oser in e La cton e (2). Mp: 79-
81 °C. RP-HPLC: tR ) 6.77 min. IR (KBr): 3294 (NH), 1785
(ring CdO), 1716 (ketone CdO), 1648 (amide CdO), 1545, 1176
cm-1. 1H NMR (CDCl3): δ 0.88 (3H, t, CH3), 1.21 (8H, m, CH3-
(CH2)4), 1.58 (2H, m, CH2CH2CO), 2.25 (1H, m, 4R-H), 2.55
(2H, t, CH2CO), 2.74 (1H, m, 4â-H), 3.48 (2H, s, COCH2CO),
4.29 (1H, m, 5R-H), 4.48 (1H, td, 5â-H), 4.58 (1H, m, 3-H), 7.73
(1H, d, NH). ES-MS m/z: 270.1 (MH+, C14H24NO4 requires m/z
270.17). Anal. (C14H23NO4) C, H, N.
Exp er im en ta l Section
Ch em istr y. L-Homoserine was obtained from NovaBiochem
(Nottingham, U.K.). Carboxylic acids and other starting
materials were purchased from Aldrich Chemical Co. The
solvents used were HPLC grade. Dichloromethane was dried
by storing it over anhydrous calcium chloride and distillation.
Melting points were determined with a Kofler hot stage and
are uncorrected. Infrared spectra through the range 4000-
600 cm-1 were obtained with KBr pellets or as thin films using
an Avatar 360 Nicolet FT-IR spectrophotometer. 1H NMR
spectra were recorded on Varian EM 390 and Bruker AMX-
250 spectrophotometers operating at 90 and 250 MHz, respec-
tively, and were recorded with tetramethylsilane as an internal
standard. 13C NMR data were obtained in deuteriochloroform
with a Bruker AMX-250 instrument operating at 62.9 MHz.
Chemical shifts are reported relative to an internal CDCl3
standard on a broad band decoupled mode, and assignments
were done using a DEPT pulse sequence. Electrospray mass
spectra (ES-MS) were recorded using Micromass VG platform
and Micromass LCT spectrometers for low- and high-resolution
spectra, respectively. Elemental analyses were obtained with
a Perkin-Elmer 240 D elemental analyzer.
N-(3-Oxou n d eca n oyl)-L-h om oser in e La cton e (3). Mp:
80-82 °C. RP-HPLC: tR ) 7.62 min. IR (KBr): 3295 (NH),
1782 (ring CdO), 1717 (ketone CdO), 1646 (amide CdO), 1545,
1177 cm-1 1H NMR (CDCl3): δ 0.9 (3H, t, CH3), 1.27 (10H,
.
m, CH3(CH2)5), 1.59 (2H, m, CH2CH2CO), 2.22 (1H, m, 4R-H),
2.52 (2H, t, CH2CO), 2.76 (1H, m, 4â-H), 3.47 (2H, s, COCH2-
CO), 4.27 (1H, m, 5R-H), 4.48 (1H, td, 5â-H), 4.58 (1H, m, 3-H),
7.64 (1H, d, NH). ES-MS m/z: 284.4 (MH+, C15H26NO4 requires
m/z 284.18). Anal. (C15H25NO4) C, H, N.
Thin-layer chromatography (TLC) was performed using
Merck silica gel 60 GF254 precoated (0.2 mm) alumina plates.
Preparative thin-layer chromatography (PLC) was performed
using Merck silica gel 60 GF254 coated (1.0 mm) glass plates
(20 cm × 20 cm). Flash chromatography was performed using
Merck Kieselgel 60 (230-400 mesh) silica.
N-(3-Oxod od eca n oyl)-L-h om oser in e La cton e (4). Mp:
84-85 °C. RP-HPLC: tR ) 8.81 min. IR (KBr): 3297 (NH),
1778 (ring CdO), 1718 (ketone CdO), 1643 (amide CdO), 1547,
1178, 1015 cm-1 1H NMR (CDCl3): δ 0.9 (3H, t, CH3), 1.27
.
(12H, m, CH3(CH2)6), 1.59 (2H, m, CH2CH2CO), 2.22 (1H, m,
4R-H), 2.52 (2H, t, CH2CO), 2.76 (1H, m, 4â-H), 3.47 (2H, s,
COCH2CO), 4.27 (1H, m, 5R-H), 4.48 (1H, td, 5â-H), 4.58 (1H,
m, 3-H), 7.64 (1H, d, NH). 13C NMR (CDCl3): δ 14.1, 22.7, 23.4,
26.4, 29.0, 29.3, 29.4, 29.9, 31.9, 43.7, 49.0, 66.0, 166.8, 175.3
and 206.3. ES-MS m/z: 298.0 (MH+, C16H28NO4 requires m/z
298.19). Anal. (C16H27NO4) C, H, N.
Analytical reverse-phase high-performance liquid chroma-
tography (RP-HPLC) was performed using an analytical
column (HiChrom Kromasil KR100-5 C 8; 250 mm × 4.6 mm)
with a Waters 625 LC system attached to a Waters 996
photodiode array system operating with a Millenium 2010
Chromatograph Manager. Fractions were eluted with a linear
gradient of acetonitrile in water (70-100%, v/ v) over a 20 min
period at a flow rate of 0.7 mL min-1 and monitored at 210
nm. Semipreparative HPLC was performed with a C8 reverse-
phase preparative column (HiChrom Kromasil KR100-5 C 8;
250 mm × 8.0 mm) using a Gilson system. Fractions were
eluted with an isocratic system, and determined from the
N-(3-Oxotr id eca n oyl)-L-h om oser in e La cton e (5). Mp:
84-86 °C. RP-HPLC: tR ) 10.28 min. IR (KBr): 3292 (NH),
1787 (ring CdO), 1724 (ketone CdO), 1643 (amide CdO), 1541,
1187, 1015 cm-1 1H NMR (CDCl3): δ 0.9 (3H, t, CH3), 1.27
.
(14H, m, CH3(CH2)7), 1.59 (2H, m, CH2CH2CO), 2.22 (1H, m,
4R-H), 2.52 (2H, t, CH2CO), 2.76 (1H, m, 4â-H), 3.47 (2H, s,
COCH2CO), 4.27 (1H, m, 5R-H), 4.48 (1H, td, 5â-H), 4.58 (1H,
analytical HPLC data, at a flow rate of 2.0 mL min-1
.
m, 3-H), 7.64 (1H, d, NH). ES-MS m/z: 312.2 (MH+, C17H30
NO4 requires m/z 312.21). Anal. (C17H29NO4) C, H, N.
-
Syn t h esis of N-(3-Oxoa lk a n oyl)-L-h om oser in e La c-
ton es (Ta ble 1, Com p ou n d s 1-16). Gen er a l Meth od A.
To a dry dichloromethane solution containing 2 mmol of the
appropriate carboxylic acid (20 mL) was added 4-(dimethyl-
amino)pyridine (2.1 mmol), N,N′-dicyclohexylcarbodiimide (2.2
mmol), and Meldrum’s acid (2 mmol). This solution was stirred
at room temperature overnight and then filtered to remove
the precipitated N,N′-dicyclohexylurea. The filtrate was evapo-
rated to dryness, and the residue was redissolved in ethyl
acetate. The ethyl acetate solution was washed with 2 M
hydrochloric acid (20 mL), dried over anhydrous magnesium
sulfate, and concentrated to afford the acylated Meldrum’s
acids, which were used without further purification in the next
step.
To a stirred solution of an acylated Meldrum’s acid (1 mmol)
in acetonitrile (30 mL) was added L-homoserine lactone hydro-
chloride (1 mmol) and triethylamine (1.2 mmol). The mixture
was stirred at room temperature for 2 h and then heated under
reflux for 3 h. The solvent was removed by rotary evaporation
to give a residue that was redissolved in ethyl acetate. The
organic solution was sequentially washed with saturated
sodium hydrogen carbonate solution, 1 M potassium hydrogen
sulfate solution, and saturated sodium chloride solution. After
drying over anhydrous magnesium sulfate, the organic solution
was evaporated and the residue was purified by preparative
N-(3-Oxot et r a d eca n oyl)-L-h om oser in e La ct on e (6).
Mp: 90-91 °C. RP-HPLC: tR ) 11.79 min. IR (KBr): 3296
(NH), 1777 (ring CdO), 1718 (ketone CdO), 1643 (amide Cd
O), 1547, 1178, 1015 cm-1. 1H NMR (CDCl3): δ 0.9 (3H, t, CH3),
1.27 (16H, m, CH3(CH2)8), 1.59 (2H, m, CH2CH2CO), 2.22 (1H,
m, 4R-H), 2.52 (2H, t, CH2CO), 2.76 (1H, m, 4â-H), 3.47 (2H,
s, COCH2CO), 4.27 (1H, m, 5R-H), 4.48 (1H, td, 5â-H), 4.58
(1H, m, 3-H), 7.64 (1H, d, NH). ES-MS m/z: 326.3 (MH+,
C
18H32NO4 requires m/z 326.23). Anal. (C18H31NO4) C, H, N.
N-(12-Br om o-3-oxod od eca n oyl)-L-h om oser in e La cton e
(7). Mp: 92-94 °C. RP-HPLC: tR ) 8.01 min. IR (KBr): 3288
(NH), 1786 (ring CdO), 1716 (ketone CdO), 1641 (amide Cd
O), 1539, 1187 cm-1. 1H NMR (CDCl3): δ 1.28 (10H, m, BrCH2-
CH2(CH2)5), 1.42 (2H, m, BrCH2CH2), 1.58 (2H, m, CH2CH2-
CO), 2.27 (1H, m, 4R-H), 2.54 (2H, t, CH2CO), 2.75 (1H, m,
4â-H), 3.48 (2H, s, COCH2CO), 3.53 (2H, t, CH2Br), 4.27 (1H,
m, 5R-H), 4.44 (1H, td, 5â-H), 4.61 (1H, m, 3-H), 7.74 (1H, d,
NH). ES-MS m/z: 376.3 and 378.3 (MH+, C16H27NO4Br
requires m/z 376.1 and 378.1). Anal. (C16H26NO4Br) C, H, N.
N-(13-Br om o-3-oxotr id eca n oyl)-L-h om oser in e La cton e
(8). Mp: 100-102 °C. RP-HPLC: tR ) 7.96 min. IR (KBr):
3288 (NH), 1786 (ring CdO), 1716 (ketone CdO), 1641 (amide
CdO), 1539, 1187 cm-1 1H NMR (CDCl3): δ 1.28 (12H, m,
.

102 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Chhabra et al.
BrCH2CH2(CH2)6), 1.42 (2H, m, BrCH2CH2), 1.58 (2H, m, CH2-
CH2CO), 2.27 (1H, m, 4R-H), 2.54 (2H, t, CH2CO), 2.75 (1H,
m, 4â-H), 3.48 (2H, s, COCH2CO), 3.53 (2H, t, CH2Br), 4.27
(1H, m, 5R-H), 4.44 (1H, td, 5â-H), 4.61 (1H, m, 3-H), 7.74 (1H,
d, NH). ES-MS m/z: 390.6 and 392.6 (MH+, C17H29NO4Br
requires m/z 390.3 and 392.3). Anal. (C17H28NO4Br) C, H, N.
N-(13-Ca r boxy-3-oxotr id eca n oyl)-L-h om oser in e La c-
ton e (9). Mp: 91-94 °C. RP-HPLC: tR ) 3.44 min. IR (KBr):
3300 (NH), 2930, 2854, 1777 (ring CdO), 1717 (ketone CdO),
1647 (amide CdO), 1544, 1178 cm-1. 1H NMR (CDCl3): δ 1.31
(12H, m, HO2C(CH2)2(CH2)6), 1.59 (4H, m, HO2CCH2CH2 and
CH2CH2CO), 2.33 (2H, t, HO2CCH2), 2.40 (1H, m, 4R-H), 2.55
(2H, t, CH2CO), 2.70 (1H, m, 4â-H), 3.51 (2H, s, COCH2CO),
4.28 (1H, m, 5R-H), 4.47 (1H, td, 5â-H), 4.66 (1H, m, 3-H), 7.88
(1H, d, NH), 8.90 (1H, br s, CO2H). ES-MS m/z: 356.2 (MH+,
CHCH2), 2.27 (1H, m, 4R-H), 2.54 (2H, t, CH2CO), 2.69 (1H,
m, 4â-H), 3.47 (2H, s, COCH2CO), 4.29 (1H, m, 5R-H), 4.47
(1H, m, 5â-H), 4.61 (1H, m, 3-H), 4.94 (2H, m, CH2CHCH2),
5.79 (1H, m, CH2CHCH2), 7.77 (1H, br d, NH). ES-MS m/z:
323.6 (MH+, C18H30NO4 requires m/z 324.2). ES-HRMS m/z:
346.1991 (M + Na, C18H29NO4Na requires m/z 346.1994).
N-(6-Meth yl-3-oxou n d eca n oyl)-L-h om oser in e La cton e
(16). Mp: 72-74 °C. RP-HPLC: tR ) 8.75 min. IR (KBr): 3289
(NH), 1786 (ring CdO), 1716 (ketone CdO), 1649 (amide Cd
O), 1543, 1176 cm-1
.
1H NMR (CDCl3): δ 0.87 (6H, 2 × t,
CHCH3 and CH2CH3), 1.26 (8H, m, CH3(CH2)4), 1.38 (2H, m,
COCH2CH2), 1.63 (1H, m, CH3CH), 2.28 (1H, m, 4R-H), 2.53
(2H, t, COCH2), 2.57 (1H, m, 4â-H), 3.50 (2H, s, COCH2CO),
4.27 (1H, m, 5R-H), 4.44 (1H, dt, 5â-H), 4.63 (1H, m, 3-H), 7.77
(1H, d, NH). ES-MS m/z: 298.2 (MH+, C16H28NO4 requires m/z
298.2). Anal. (C16H27NO4) C, H, N.
C
18H30NO6 requires m/z 356.2). Anal. (C18H29NO6) C, H, N.
N -(12-Me t h oxyca r b on yl-3-oxod od e ca n oyl)-L-h om o-
.Syn th esis of N-(3-Hyd r oxya cyl)-L-h om oser in e La c-
ton es (Ta ble 1, Com p ou n d s 17-18). Gen er a l Meth od B.
To a solution of the N-(3-oxoacyl)-L-homoserine lactone (1
mmol) in methanol (5 mL) was added sodium cyanoborohy-
dride (1.5 mmol). The reaction mixture was maintained at pH
3-4 by addition of 3% HCl in methanol. Three further
additions of sodium cyanoborohydride (3 × 1.5 mmol) were
made at 4 h intervals while the pH was kept at 3-4. The
solvent was removed by rotary evaporation, and the residue
was extracted with hot ethyl acetate (4 × 20 mL). The ethyl
acetate extracts were combined and concentrated to yield the
crude product, which was purified by silica column chroma-
tography (10% MeOH/DCM). Further purification was achieved
by RP-HPLC.
N-(3-Hyd r oxyd od eca n oyl)-L-h om oser in e La cton e (17).
Mp: 94-96 °C. RP-HPLC: tR ) 7.75 min. IR (KBr): 3297
(NH), 1778 (ring CdO), 1643 (amide CdO), 1547, 1178, 1015
cm-1. 1H NMR (CDCl3): δ 0.85 (3H, t, CH3), 1.26 (12H, m, CH3-
(CH2)6), 1.42-1.52 (2H, m, CH2CH2CHOH), 1.67 (2H, m, CH2-
CHOH), 2.19 (1H, m, 4R-H), 2.40 (2H, m, CH2CO), 2.81 (1H,
m, 4â-H), 3.18 (1H, br d, OH), 4.01 (1H, m, CHOH), 4.31 (1H,
m, 5R-H), 4.49 (1H, td, 5â-Η), 4.57 (1Η, m, 3-H), 6.62 (1H, d,
NH). ES-MS m/z: 300.4 (MH+,C16H30NO4 requires m/z 300.2).
Anal. (C16H29NO4) C, H, N.
ser in e La cton e (10). Mp: 66-68 °C. RP-HPLC: tR ) 5.60
min. IR (KBr): 3295 (NH), 1778 (ring CdO), 1731 (ester Cd
O), 1717 (ketone CdO), 1643 (amide CdO), 1547, 1177 cm-1
.
1H NMR (CDCl3): δ 1.27 (10H, m, CH3OCOCH2CH2(CH2)5),
1.59 (4H, m, CH2CH2CO and CH3OCOCH2CH2), 2.22 (1H, m,
4R-H), 2.25 (2H, t, CH3OCOCH2), 2.52 (2H, t, CH2CO), 2.76
(1H, m, 4â-H), 3.47 (2H, s, COCH2CO), 3.58 (3H, s, CH3O),
4.27 (1H, m, 5R-H), 4.48 (1H, td, 5â-H), 4.58 (1H, m, 3-H), 7.64
(1H, d, NH). ES-MS m/z: 356.0 (MH+, C18H30NO6 requires m/z
356.2). Anal. (C18H29NO6) C, H, N.
N-(12-Hyd r oxy-3-oxod od eca n oyl)-L-h om oser in e La c-
ton e (11). Mp: 64-66 °C. RP-HPLC: tR ) 4.30 min. IR
(KBr): 3293 (NH), 1781 (ring CdO), 1715 (ketone CdO), 1649
(amide CdO), 1547, 1174 cm-1. 1H NMR (CDCl3): δ 1.29 (10H,
m, HOCH2CH2(CH2)5), 1.62 (4H, m, HOCH2CH2 and CH2CH2-
CO), 1.88 (1H, m, OH), 2.27 (1H, m, 4R-H), 2.54 (2H, t, CH2-
CO), 2.75 (1H, m, 4â-H), 3.48 (2H, s, COCH2CO), 3.63 (2H, t,
CH2OH), 4.27 (1H, m, 5R-H), 4.44 (1H, td, 5â-H), 4.61 (1H, m,
3-H), 7.74 (1H, d, NH). ES-MS m/z: 314.1 (MH+, C16H28NO5
requires m/z 314.2). ES-HRMS m/z: 336.1808 (M + Na, C16H27
NO5Na requires m/z 336.1787).
-
N-(3-Oxo-7Z-tetr adecen oyl)-L-h om oser in e Lacton e (12).
Mp: 59-60 °C. RP-HPLC: tR ) 10.14 min. IR (KBr): 3293
(NH), 1780 (ring CdO), 1717 (ketone CdO), 1645 (amide Cd
N-(3-H yd r oxyt et r a d eca n oyl)-L-h om oser in e La ct on e
(18). Mp: 110-112 °C. RP-HPLC: 10.41 min. IR (KBr): 3360
(OH), 3297 (NH), 1775 (ring CdO), 1645 (amide CdO), 1547,
O), 1547, 1175 cm-1 1H NMR (CDCl3): δ 0.84 (3H, t, CH3),
.
1.27 (8H, m, CH3(CH2)4), 1.57 (2H, m, CH2CH2CO), 1.96 (4H,
m, CH2CHCHCH2), 2.26 (1H, m, 4R-H), 2.50 (2H, t, CH2CO),
2.68 (1H, m, 4â-H), 3.43 (2H, s, COCH2CO), 4.24 (1H, m, 5R-
H), 4.43 (1H, m, 5â-H), 4.58 (1H, m, 3-H), 5.30 (2H, m, CHCH),
7.72 (1H, br d, NH). ES-MS m/z: 323.8 (MH+, C18H30NO4
requires m/z 324.2). Anal. (C18H29NO4) C, H, N.
1
1190, 1176 cm-1. H NMR (CDCl3): δ 0.85 (3H, t, CH3), 1.26
(16H, m, CH3(CH2)8), 1.42-1.52 (2H, m, CH2CH2CHOH), 1.67
(2H, m, CH2CHOH), 2.19 (1H, m, 4R-H), 2.40 (2H, m, CH2-
CO), 2.81 (1H, m, 4â-H), 3.18 (1H, br d, OH), 4.01 (1H, m,
CHOH), 4.31 (1H, m, 5R-H), 4.49 (1H, td, 5â-Η), 4.57 (1Η, m,
3-H), 6.62 (1H, d, NH). 13C NMR: δ 14.1, 22.7, 25.5, 29.4, 29.5,
29.6, 29.6, 29.7, 30.4, 31.9, 37.0, 37.0, 42.6, 49.2, 66.1, 68.7,
173.0, 175.4. ES-MS m/z: 328.3 (MH+, C18H34NO4 requires m/z
328.2). Anal. (C18H33NO4) C, H, N.
N-(3-Oxo-10Z-t et r a d ecen oyl)-L-h om oser in e La ct on e
(13). Mp: 55-57 °C. RP-HPLC: tR ) 10.09 min. IR (KBr):
3293 (NH), 1777 (ring CdO), 1718 (ketone CdO), 1648 (amide
CdO), 1548, 1175 cm-1. 1H NMR (CDCl3): δ 0.90 (3H, t, CH3),
1.30 (8H, m, CH3CH2 and (CH2)3(CH2)2CO), 1.58 (2H, m, CH2-
CH2CO), 1.99 (4H, m, CH2CHCHCH2), 2.30 (1H, m, 4R-H), 2.54
(2H, t, CH2CO), 2.71 (1H, m, 4â-H), 3.48 (2H, s, COCH2CO),
4.26 (1H, m, 5R-H), 4.45 (1H, m, 5â-H), 4.60 (1H, m, 3-H), 5.35
(2H, m, CHCH), 7.76 (1H, br d, NH). ES-MS m/z: 323.8 (MH+,
Syn th esis of N-Acyl-L-h om oser in e La cton es (Ta ble 1,
Com p ou n d s 19-21). Gen er a l Meth od C. To a suspension
of L-homoserine lactone hydrochloride (1 mmol) in dry DCM
(20 mL) at 0 °C was added triethylamine (2.4 mmol). The
solution was stirred at 0 °C for 30 min when the alkanoyl
chloride (1 mmol) was added dropwise over 5 min. The reaction
mixture was allowed to come to room temperature and was
stirred for 4 h. The solvent was removed by rotary evaporation.
The residue was redissolved in ethyl acetate and washed
sequentially with saturated sodium hydrogen carbonate solu-
tion (2 × 20 mL), 1 M potassium hydrogen sulfate solution (2
× 20 mL), and brine (1 × 10 mL). After the mixture was dried
over MgSO4, the ethyl acetate was removed by rotary evapora-
tion to leave the crude product that was purified by column
chromatography on silica in ethyl acetate. Further purification
was achieved by RP-HPLC.
C
18H30NO4 requires m/z 324.2). Anal. (C18H29NO4) C, H, N.
N-(3-Oxo-12-tr id ecen oyl)-L-h om oser in e La cton e (14).
Mp: 83-85 °C. RP-HPLC: tR ) 8.74 min. IR (KBr): 3297
(NH), 1778 (ring CdO), 1718 (ketone CdO), 1643 (amide Cd
1
O), 1547, 1177, 1015 cm-1. H NMR (CDCl3): δ 1.25 (12H, m,
(CH2)5(CH2)2CO), 1.57 (2H, m, CH2CH2CO), 2.04 (2H, m, CH2-
CHCH2), 2.27 (1H, m, 4R-H), 2.54 (2H, t, CH2CO), 2.69 (1H,
m, 4â-H), 3.47 (2H, s, COCH2CO), 4.29 (1H, m, 5R-H), 4.47
(1H, m, 5â-H), 4.61 (1H, m, 3-H), 4.94 (2H, m, CH2CHCH2),
5.79 (1H, m, CH2CHCH2), 7.77 (1H, br d, NH). ES-MS m/z:
310.2 (MH+, C17H28NO4 requires m/z 310.2). Anal. (C17H27NO4)
C, H, N.
N-Deca n oyl-L-h om oser in e La cton e (19). Mp: 132-134
°C. RP-HPLC: tR ) 8.48 min. IR (KBr): 3318 (NH), 1777 (ring
N-(3-Oxo-13-tetr adecen oyl)-L-h om oser in e Lacton e (15).
Mp: 76-78 °C. RP-HPLC: tR ) 10.14 min. IR (KBr): 3297
(NH), 1778 (ring CdO), 1718 (ketone CdO), 1643 (amide Cd
CdO), 1646 (amide CdO), 1549, 1172, 1013 cm-1 1H NMR
.
(CDCl3): δ 0.88 (3H, t, CH3), 1.26 (12H, m, CH3(CH2)6), 1.67
(2H, m, COCH2CH2), 2.13 (1H, m, 4R-H), 2.25 (2H, t, COCH2),
2.86 (1H, m, 4â-H), 4.28 (1H, m, 5R-H), 4.44 (1H, td, 5â-H),
1
O), 1547, 1177, 1015 cm-1. H NMR (CDCl3): δ 1.25 (12H, m,
(CH2)6(CH2)2CO), 1.57 (2H, m, CH2CH2CO), 2.04 (2H, m, CH2-

Lactone Analogues as Modulators
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 103
4.56 (1H, m, 3-H), 6.12 (1H, d, NH). ES-MS m/z: 256.2 (MH+,
suspensions were prepared by removing the spleen and placing
them into RPMI 1640 medium (Sigma, Poole, U.K.). Spleens
were forced through 70 µm pore size wire gauzes using the
plunger from a 5 mL syringe to produce a single cell suspen-
sion. Cells were pelleted by centrifugation, and erythrocytes
were lysed with 0.017 M Tris, 0.144 M ammonium chloride
buffer, pH 7.2. Leukocyte pellets were washed twice with
RPMI 1640 medium with 2% (v/v) fetal calf serum (FCS) and
resuspended in complete T-cell culture medium (CTCM)
consisting of RPMI 1640 medium with 5% FCS, 2 mM
L-glutamine, and 5 × 10-5 M 2-mercaptoethanol (Sigma, Poole,
U.K.). Test compounds were studied using doubling-down
dilutions ranging from 1 mM to 0.1 µM in a final volume of
200 µL of CTCM containing ConA (Sigma, Poole, U.K.) at 1
µg/mL and 100 000 spleen cells. Following incubation for 48
h at 37 °C in 5% CO2/air, 0.25 µCi [3H]-thymidine (Amersham
Pharmacia Biotech, U.K.) in 10 µL of RPMI 1640 medium was
added and cells were incubated for a further 24 h. Cells were
collected onto fiberglass filters with a filtermate harvester
(Packard Bioscience, Pangbourne, U.K.). After the addition of
25 µL of MicroScint-O (Packard Bioscience, Pangbourne, U.K.)
to each well, these filters were counted with a Packard
TopCount scintillation counter.
C
14H26NO3 requires m/z 256.2). Anal. (C14H25NO3) C, H, N.
N-Un d eca n oyl-L-h om oser in e La cton e (20). Mp: 129-
130 °C. RP-HPLC: tR ) 9.85 min. IR (KBr): 3319 (NH), 1778
(ring CdO), 1646 (amide CdO), 1549, 1173, 1013 cm-1 1H
.
NMR (CDCl3): δ 0.88 (3H, t, CH3), 1.28 (14H, m, CH3(CH2)7),
1.64 (2H, m, CH2CH2CO), 2.12 (1H, m, 4R-H), 2.25 (2H, t, CH2-
CO), 2.84 (1H, m, 4â-H), 4.28 (1H, m, 5R-H), 4.43 (1H, td, 5â-
H), 4.52 (1H, m, 3-H), 6.04 (1H, d, NH). ES-MS m/z: 270.4
(MH+, C15H28NO3 requires m/z 270.2). Anal. (C15H27NO3) C,
H, N.
N-Dod eca n oyl-L-h om oser in e La cton e (21). Mp: 128-
129 °C. RP-HPLC: tR ) 11.36 min. IR (KBr): 3318 (NH), 1778
(ring CdO), 1647 (amide CdO), 1549, 1173, 1013 cm-1 1H
.
NMR (CDCl3): δ 0.88 (3H, t, CH3), 1.26 (16H, m, CH3(CH2)8),
1.62 (2H, m, CH2CH2CO), 2.12 (1H, m, 4R-H), 2.25 (2H, t, CH2-
CO), 2.88 (1H, m, 4â-H), 4.28 (1H, m, 5R-H), 4.48 (1H, td, 5â-
H), 4.55 (1H, m, 3-H), 6.02 (1H, d, NH). 13C NMR: δ 14.11,
22.67, 25.42, 29.20, 29.31, 29.31, 29.44, 29.44, 29.59, 30.75,
36.21, 49.30, 66.11, 173.75, 175.49. ES-MS m/z: 284.4 (MH+,
C
16H30NO3 requires m/z 284.2). Anal. (C16H29NO3) C, H, N.
N-(3-Oxod od eca n oyl)-L-h om oser in e Meth yl Ester (22).
A solution of compound 4 (1 mmol) in MeOH (5 mL) was
stirred with a solution of sodium methoxide (1 mmol) in
methanol (5 mL) for 6 h at room temperature. The methanol
was removed, and the residue was redissolved in ethyl acetate.
The solution was washed with water and brine, dried (MgSO4),
and rotary-evaporated to give N-(3-oxododecanoyl)-L-homo-
Con A Mit ogen -St im u la t ed P r olifer a t ion of H u m a n
P BMC. With ethical clearance and full donor consent, blood
specimens were obtained from healthy human volunteers.
Human peripheral blood mononuclear cells were isolated from
heparinized whole blood by buoyant density centrifugation
over Histopaque 1077 (Sigma, Poole, U.K.) at 600 g for 20 min.
Cells harvested from “buffy” layers were washed twice with
RPMI 1640 medium and resuspended in CTCM. Test com-
pounds were tested using similar dilutions as used for murine
splenocyte studies in 200 µL of CTCM containing 1 µg/mL
ConA and 100 000 PBMC. Human PBMC were incubated for
48 h at 37 °C in 5% CO2/air, followed by pulsing with 0.25 µCi
[3H]-thymidine (see above). After a further incubation of 24
h, cells were collected onto fiberglass filters and then counted
in MicroScint-O with a Packard TopCount scintillation counter.
serine methyl ester as an amorphous solid. RP-HPLC: tR
)
7.95 min. 1H NMR (CDCl3): δ 0.88 (3H, t, CH3), 1.32 (12H, m,
CH3(CH2)6), 1.59 (2H, m, CH2CH2CO), 1.71 (1H, m, â-H), 2.18
(1H, m, â-H), 2.54 (2H, t, CH2CO), 2.76 (1H, m, OH), 3.47 (2H,
s, COCH2CO), 3.60 (1H, m, γ-H), 3.70 (1H, m, γ-H), 3.78 (3H,
s, CO2CH3), 4.78 (1H, m, R-H), 7.74 (1H, br d, NH). ES-MS
m/z: 330.2 (MH+, C17H32NO5 requires m/z 330.2).
3-Oxod od eca n a m id e (23). A solution of 5-decanoyl Mel-
drum’s acid (1 mmol) (prepared by general method A) and tert-
butyl carbazate (1 mmol) in acetonitrile (20 mL) was stirred
at room temperature overnight and then heated under reflux
for 2 h. The solvent was removed, and the residue was
chromatographed on a silica column in ethyl acetate. The
product, N-tert-butoxycarbonyl-3-oxododecanamide, was taken
up in 3 M HCl/ethyl acetate and left at room temperature for
3 h. Evaporation of the solvent afforded 3-oxododecanamide
as crystalline material after crystallization from ethyl acetate.
Mp: 105-106 °C. RP-HPLC: tR ) 8.00 min. IR (KBr): 3382,
TNF -r Secr etion fr om LP S-Stim u la ted Hu m a n P BMC.
Bacterial lipopolysaccharide (LPS) stimulates the production
of a variety of cytokines, including TNF-R, from human PBMC;
these cytokines in turn influence the development of T cells,
supporting a T helper 1 conducive milieu. Human PBMC
prepared from whole blood by buoyant density centrifugation
was resuspended in CTCM. Test compounds were again
studied using similar dilutions as used for murine splenocytes
in 200 µL of CTCM containing 1 × 10-5 µg/mL LPS Escherichia
coli strain 055:B5 (Sigma, Poole, U.K.) and 100 000 PBMC.
Following incubation for 24 h at 37 °C in 5% CO2/air, cell
culture supernatants were collected and tested for TNF-R
levels by “sandwich” ELISA. Briefly, 96-well Nunc MaxiSorp
(Life Technologies, Paisley, U.K.) plates were coated with 50
µL of a 2 µg/mL solution of mouse antihuman TNF-R mono-
clonal antibody (BD Pharmingen, U.K.) in 0.05 M carbonate/
bicarbonate buffer, pH 9.6, overnight at 4 °C. After these plates
were washed three times with PBS-Tween, which contained
phosphate-buffered saline (PBS) with 0.5% (v/v) Tween 20
(Sigma, Poole, U.K.), plates were blocked with 1% (w/v) bovine
serum albumin (BSA) (Sigma, Poole, U.K.) at room tempera-
ture for 2 h. Following three washes with PBS-Tween, 50 µL
of cell culture supernatant was added and the mixture was
incubated overnight at 4 °C; standard human TNF-R (BD
Pharmingen, U.K.) ranging from 2000 to 31.25 pg/mL was
added to each plate. After four washes with PBS-Tween, 50
µL of a second antibody, biotinylated mouse antihuman TNF-R
monoclonal antibody (BD Pharmingen, U.K.), was added at
0.5 µg/mL dilution in 1% BSA in PBS-Tween and incubated
at room temperature for 1 h. Following four washes, the
bonded biotinylated antibody was measured with 50 µL of a
1:1000 dilution of streptavidin/peroxidase (BD Pharmingen,
U.K.). At the end of an hour of incubation at room temperature,
these plates were thoroughly washed six times with PBS-
Tween and the assay developed by the addition of 100 µL of
0.1 mg/mL of 3,3′,5,5′-tetramethylbenzidine substrate (Sigma,
3186 (NH), 1706 (CdO), 1657 (amide CdO) cm-1 1H NMR
.
(CDCl3): δ 0.88 (3H, t, CH3), 1.26 (12H, m, CH3(CH2)6), 1.59
(2H, m, COCH2CH2), 2.53 (2H, t, COCH2), 3.35 (2H, s, COCH2-
CO), 5.55 (1H, br d, NH), 7.07 (1H, br d, NH). ES-MS m/z:
214.2 (MH+, C12H24NO2 requires m/z 214.2). Anal. (C12H23NO2)
C, H, N.
N-(3-Oxododecan oyl)-L-h om ocystein e Th iolacton e (24).
Compound 24 was synthesized from decanoic acid using
general method A replacing L-homoserine lactone with L-
homocysteine thiolactone. Mp: 83-85 °C. RP-HPLC: tR
)
10.54 min. IR (KBr): 3267 (NH), 1702 (CdO), 1648 (amide
CdO), 1561, 1178 cm-1. 1H NMR (CDCl3): δ 0.81 (3H, t, CH3),
1.22 (12H, m, CH3(CH2)6), 1.55 (2H, m, CH2CH2CO), 1.94 (1H,
m, 4R-H), 2.16 (2H, t, CH2CO), 2.70 (1H, m, 4â-H), 3.18 (1H,
m, 5R-H), 3.24 (1H, m, 5â-H), 3.45 (2H, s, COCH2CO), 4.52
(1H, m, 3-H), 6.63 (1H, d, NH). 13C NMR (CDCl3): δ 14.49,
23.04, 23.75, 27.87, 29.38, 29.63, 29.74, 29.77, 31.82, 32.24,
44.22, 48.89, 59.61, 166.76, 205.10, 206.87. ES-MS m/z: 314.1
(MH+, C16H28NO3S requires m/z 314.2). Anal. (C16H27NO3S)
C, H, N.
Im m u n ologica l Assa ys. Con ca n a va lin A (Con A) Mito-
gen -Stim u la ted P r olifer a tion of Mu r in e Sp len ocytes. A
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incorporation of [3H]-thymidine into DNA. Eight-week-old
female BALB/c mice were obtained from Harlan (Bicester,
Oxon, U.K.) and given food and water ad libitum. Splenocyte

104 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Chhabra et al.
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