Design, synthesis, and preliminary pharmacological evaluation of N-Acyl-3-aminoglutarimides as broad-spectrum chemokine inhibitors in vitro and anti-inflammatory agents in vivo

Article abstract of DOI:10.1021/jm010984i

A series of N-substituted 3-aminoglutarimides have been synthesized and tested for inhibitory activity against a range of chemokines in vitro and for suppression of lipopolysaccharide-induced inflammation in vivo. The results show that they represent the first class of small molecules with broad-spectrum chemokine inhibitory effects. Among the compounds studied, 10 (NR58,4) was the most potent, being active at doses between 5 and 15 nM in vitro and at 0.3 mg kg^-1 in vivo.

Full text of DOI:10.1021/jm010984i

360
J . Med. Chem. 2002, 45, 360-370
Design , Syn th esis, a n d P r elim in a r y P h a r m a cologica l Eva lu a tion of
N-Acyl-3-a m in oglu ta r im id es a s Br oa d -Sp ectr u m Ch em ok in e In h ibitor s in Vitr o
a n d An ti-in fla m m a tor y Agen ts in Vivo
David J . Fox,† J ill Reckless,‡ Stuart G. Warren,† and David J . Grainger*,‡
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K., and Department of Medicine,
University of Cambridge, Box 157, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, U.K.
Received J uly 12, 2001
A series of N-substituted 3-aminoglutarimides have been synthesized and tested for inhibitory
activity against a range of chemokines in vitro and for suppression of lipopolysaccharide-induced
inflammation in vivo. The results show that they represent the first class of small molecules
with broad-spectrum chemokine inhibitory effects. Among the compounds studied, 10 (NR58,4)
was the most potent, being active at doses between 5 and 15 nM in vitro and at 0.3 mg kg-1
in vivo.
In tr od u ction
mains possible that they bind to a range of different
GPCR targets, limiting their application as specific
chemokine inhibitors.6
Chemokines are small protein (8-12 kDa) signaling
molecules1 involved in the regulation of the immune
system both during physiological host defense2 and in
pathological inflammation.3 Neutralizing antibodies and
genetic deletion strategies have been successfully em-
ployed to demonstrate that reduced chemokine signaling
can reduce or abolish pathological inflammation.3 For
example, when mice are treated with the nonspecific
pro-inflammatory stimulus of bacterial lipopolysaccha-
ride (LPS), neutralizing antibodies against a range of
chemokines act synergistically to reduce inflamma-
tion.4,5 These studies clearly demonstrate that the
different chemokines (more than 50 have now been
described) can act in parallel to promote inflammation.
Because inappropriate chemokine activity has been
suggested to participate in a wide range of human
diseases, including asthma, atherosclerosis, cancer, and
autoimmune disorders,3 there has been a large number
of attempts to identify chemokine inhibitors suitable for
use in vivo.6-11 The chemokines signal through interac-
tion with a range of chemokine receptors, all of which
are members of the intensively studied heptahelical
G-protein-coupled receptor (GPCR) superfamily.1 As a
result, most attempts to identify chemokine inhibitors
have relied on screening strategies similar to those that
have been successful for a wide range of other GPCR
ligands. These studies have yielded a number of classes
of compounds that act as receptor antagonists for one
or several of the chemokine receptors.6-11 Of these
compounds, most show structural similarity to antago-
nists of unrelated GPCR families. For example, several
4-hydroxypiperazine derivatives have been shown to act
as chemokine inhibitors and closely resemble the dopam-
ine receptor inhibitor domperinone. Unfortunately, only
a few of the chemokine receptor antagonists described
to date have been shown to have specificity over other
members of the GPCR superfamily. For many, it re-
With few exceptions, the chemokine inhibitors pub-
lished to date inhibit the function of chemokines binding
to one or
a small subset of related chemokine
receptors.9-11 In circumstances where an inflammatory
response is dependent on the parallel action of a range
of chemokines signaling through a number of different
receptors, these selective inhibitors may be largely
ineffective. For example, neutralizing antibodies against
individual chemokines often have little or no effect on
models of inflammation in vivo.12
In marked contrast, we recently described a short
peptide sequence derived from the human chemokine
monocyte chemoattractant protein-1 (MCP-1) that is
able to simultaneously inhibit the activity of a wide
range of chemokines in vitro.13 This peptide (EICADP-
KQKWVQ (single letter amino acid code) termed peptide
3), has no effect on leukocyte migration induced by a
range of other pro-inflammatory signals.13 Thus, peptide
3 is the first example of a broad-spectrum chemokine
inhibitor. Consistent with its effects in vitro, we have
recently demonstrated that a modified peptide of related
structure (NR58-3.14.3) has anti-inflammatory proper-
ties in vivo; NR58-3.14.3 abolished leukocyte recruit-
ment in response to intradermal LPS injection14 and
reduced the pathological inflammation in animal models
of asthma, stroke,15 and atherosclerosis.16
There are generally significant limitations on the use
of synthetic oligopeptides both experimentally and
therapeutically in vivo. For example, most peptides
(including peptide 3 and the related NR58-3.14.3) have
poor oral bioavailability and short plasma half-lives,
often being secreted at the glomerular filtration rate.17
We have therefore performed a structure-activity re-
lationship (SAR) analysis to identify the structural
motifs essential for broad-spectrum chemokine inhibi-
tory activity within the peptide 3 molecule and designed
a series of non-peptide mimetics that possess broad-
spectrum chemokine inhibitory activity in the nanomo-
lar concentration range.
* To whom correspondence should be addressed. Phone: +44 1223
336812. Fax: +44 1223 762770. E-mail: djg15@mole.bio.cam.ac.uk.
† Department of Chemistry.
‡ Department of Medicine.
10.1021/jm010984i CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/19/2001

Novel Class of Chemokine Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 361
Ta ble 1. Effect of Peptide 3 Sequence Variants on Leukocyte
characterized to determine what structural features of
this peptide were likely to be important for broad-
spectrum chemokine inhibitory activity. None of dipep-
tides WV, WQ, and VQ had any detectable activity as
chemokine inhibitors. Similarly the scrambled tripep-
tides VQW and VWQ were inactive (Table 1). The
tripeptides with an N-terminal glutamine (Q) residue
were not synthesized because of potential contamination
with cyclized pyroglutamate-containing peptides. These
experiments confirmed that the chemokine inhibitory
activity of WVQ tripeptide required the presence of each
amino acid constituent in an appropriate spatial ar-
rangement.
Migration Induced by MCP-1a
peptide
ED50 versus MCP-1 (µM)
EICADPKQKWVQ
CADPKQKWVQ
EICADP
KQKWVQ
KQK
8 ( 3
8 ( 5
50 ( 17
6 ( 3
12 ( 4
4 ( 2
inactive
inactive
inactive
>100
25 ( 7
>100
inactive
inactive
WVQ
VWQ
WQV
GVQ
VVQ
YVQ
WVN
WVR
We next synthesized a series of peptides with substi-
tutions of each of the three amino acids in turn.
Substitution of the tryptophan (W) with glycine (G) or
valine (V) completely abolished the activity of the
tripeptide, but tripeptides containing the aromatic
amino acids tyrosine (Y) or phenylalanine (F) had
activity similar to that of the parent tripeptide (Table
1). This suggests that an aromatic group, or at least a
bulky hydrophobic group, at the P1 position is necessary
for broad-spectrum chemokine inhibitory activity.
WVG
a
THP-1 cells were induced to migrate in response to 25ng/mL
MCP-1 (see text) in the presence of various concentrations of the
peptides shown above (single-letter amino acid code). The concen-
tration that reduced migration by 50% (ED50) is shown, and in
each case is the mean ( sem for three separate determinations.
Peptides that did not statistically significantly reduce migration
at 100 µM (the highest concentration tested) are marked “inactive”.
Peptides that reduced migration significantly, but by less than
50%, at 100 µM are marked “>100”.
Replacement of valine (V) at the P2 position with
glycine (G) slightly increased the potency of the tripep-
tide (by approximately 3-fold). It is possible that this
results from increased conformational flexibility of the
tripeptide. We therefore substituted the P2 position with
a range of amino acids with alkyl side chains of varying
length, using leucine, isoleucine, ethylglycine, alanine,
D-alanine, and D-valine in addition to glycine and valine.
A plot of potency against the number of carbon atoms
in the side chain revealed a negative correlation (Figure
1) with the WGQ tripeptide as the most potent.
P ep tid e SAR
We have already performed a limited mutagenesis
study on the full-length peptide 3 sequence in which
variant amino acids from different chemokines were
substituted into the MCP-1-derived peptide 3 sequence.
All of these substitutions had only small effects on the
inhibitory potency of the peptide (ranging from 2 to 10
µM, with Leu4Ile11-peptide 3[3-13] being the most
potent), and none of the substitutions affected the broad
chemokine specificity of the molecule.13 We have there-
fore performed a further SAR with less conservative
changes to the molecule. The 12 amino acid peptide 3
sequence was divided first into two hexapeptides, and
each was tested for the ability to inhibit migration
induced by MCP-1 (Table 1). Both hexapeptides derived
from peptide 3 inhibited MCP-1-induced leukocyte
migration in a ChemoTx migration assay,13 although
only the C-terminal hexapeptide retained the same
potency as the original dodecapeptide. Surprisingly,
when the C-terminal hexapeptide was further divided
into two tripeptides, both inhibited MCP-1-induced
migration with a potency similar to that of the original
dodecapeptide. Thus, the tripeptides KQK and WVQ
appear to be structurally distinct chemokine inhibitors.
An important feature of the peptide 3 dodecapeptide
was the ability to inhibit all chemokines tested with
similar potency. We therefore tested whether the trip-
eptides retained this attribute. WVQ inhibited all the
chemokines tested (MCP-1, MIP-1R, RANTES, IL-8, and
SDF-1R) with similar potency (2-10 µM) and was
therefore indistinguishable from the original peptide 3
sequence in these experiments. In marked contrast, the
KQK tripeptide inhibited MCP-1-induced migration
(ED50 ) 12 µM) but had no effect on migration induced
by MIP-1R, RANTES, IL-8, or SDF-1R. It seems likely,
therefore, that the two tripeptides are inhibiting chemok-
ine-induced signaling by distinct mechanisms.
All the substitutions we tested at the P3 position
resulted in complete loss of chemokine inhibitory activ-
ity (Table 1). The conservative substitution replacing
glutamine (Q) with asparagine (N), as well as with
glycine (G) and arginine (R), abolished the activity. On
the basis of the data in Table 1, we concluded that the
broad-spectrum chemokine inhibitory activity of WVQ-
related tripeptides depended on the presence of a large
hydrophobic or aromatic group at an appropriate dis-
tance from a glutamine-like primary amide.
As an alternative approach for identifying the struc-
tural features in peptide 3 that are essential for broad-
spectrum chemokine inhibition, we synthesized a panel
of 231 peptides in which each amino acid in Leu4Ile11-
Cys13-peptide 3[3-12] was substituted with the other
19 naturally occurring amino acids. This sequence, with
the addition of a C-terminal cysteine residue, was
chosen for the substitution analysis because it is the
most potent linear analogue of peptide 3 described to
date.13 This panel of peptides, all in the linear, fully
reduced state, was then tested for ability to inhibit IL-
8-induced migration of human neutrophils. Most of the
variant peptides retained the activity of the parent
sequence (green squares in Figure 2). However, a
number of substitutions decreased the activity of the
peptide considerably (red squares in Figure 2; darker
red indicating greater loss of activity). Consistent with
the results from the deletion analysis, substitution of
the amino acids in the C-terminus of the molecule
reduced the activity to a much greater extent than
Since our aim was to design a non-peptide chemokine
inhibitor that retained the broad specificity of peptide
3, the KQK tripeptide was not studied further. Further
variations of the WVQ tripeptide were synthesized and

362 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Fox et al.
F igu r e 2. Summary of the structure-activity relationship for
a replacement net analysis of Leu4Ile11Cys13-peptide 3[3-12].
Leu4Ile11Cys13-peptide 3[3-12] was synthesized on a parallel
peptide synthesizer in 231 combinations (including controls)
in which each amino acid in turn was substituted with the
other 19 amino acids or with the cognate D-amino acid (/). Each
of the 231 variants was then tested for the ability to inhibit
IL-8-induced migration of freshly prepared human peripheral
blood polymorphonuclear cells (predominantly neutrophils).
Peptides with activity similar to that of the parent sequence
are shown as green squares (lighter green indicating slight
loss of activity). Peptides with more than 10-fold loss of activity
are shown as red squares (darker red indicating greater loss
of activity such that a dark red square indicates no inhibitory
activity even at 100 µM). The parental sequence was sepa-
rately synthesized 11 times in the panel and was fully active
in each case (indicated by the hatched green squares).
F igu r e 1. Effect of WxQ tripeptides on leukocyte migration.
(a) THP-1 cells were induced to migrate in response to 25 ng/
mL MCP-1 (filled squares), 3.125 ng/mL SDF-1R (filled circles),
or 100 nM fMLP as a non-chemokine chemoattractant (open
squares) in the presence of various concentrations of the
tripeptide WIQ. The number of cells migrating under each
condition is reported as a proportion of the number of cells
that migrated in the absence of WIQ (mean ( sem of triplicate
wells). (b) The concentration of various WxQ tripeptides that
inhibited MCP-1-induced migration of THP-1 cells by 50%
(ED50) was determined for x ) L-isoleucine, L-allo-isoleucine,
L-valine, L-ethylglycine, L-alanine, glycine, D-alanine, and
D-valine. The ED50 was plotted against the number of carbon
atoms in the simple alkyl side chains of the amino acid at the
P2 position (x).
The syntheses of glutamine derivatives 1, 2, and 4-20
were performed using standard peptide coupling re-
agents and conditions. Initially L-glutamine was reacted
with undec-10-enoyl chloride and benzoyl chloride in
THF and aqueous KOH. The N-acylated products 1 and
2, along with commercially available Boc-glutamine 3,
were then each coupled with ammonia and benzylamine
to give the bis primary amides 4-6 and the benzyla-
mides 7-9, respectively (Scheme 1). The acids were also
activated using DCC and N-hydroxysuccinimide without
external nucleophiles to produce the acylaminogluta-
rimides 10-12 by intramolecular nucleophilic attack of
the primary amide nitrogen.
substitutions in the N-terminal region. Every substitu-
tion at position 10 (tryptophan) significantly, and in
many cases completely, reduced the activity of the
peptide, while most (18/19) substitutions at position 12
(glutamine) reduced the activity substantially.
Both the deletion analysis (Table 1) and the substitu-
tion analysis (Figure 2) strongly indicate that the motif
WxQ is responsible for the broad-spectrum chemokine
inhibitory activity in peptide 3, although the substan-
tially increased potency of the retroinverso analogue
NR58-3.14.3 compared with that of peptide 3 suggests
that the context of the WxQ is also important.14
Further acylaminoglutarimides were made by depro-
tection and acylation of the amino group of Boc-
aminoglutarimide 12 (Scheme 2). Coupling of simple
saturated carboxylic acids produced linear nine-, seven-,
and five-carbon-side-chain derivatives 13-15, as well
as a branched eight-carbon-side-chain analogue 16.
Further functionalization of undec-10-enoylaminoglu-
tarimide 10 gave three further analogues (Scheme 3);
the terminal olefin was reduced to give the saturated
eleven-carbon-side-chain compound 17, and the same
functional group was oxidatively cleaved to give 10-
carbon-side-chain acid glutarimide 18. The ring nitrogen
Design a n d Syn th esis of Non -p ep tid e
Br oa d -Sp ectr u m Ch em ok in e In h ibitor s
As a first step toward defining the constituent phar-
macophores needed to yield a broad-spectrum chemok-
ine inhibitor, we selected four candidate glutamine
mimetics (including glutamine itself) shown in Table 2
and three candidate large hydrophobic groups (as tryp-
tophan mimetics) and synthesized the 12 combinations
(Table 2).

Novel Class of Chemokine Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 363
Sch em e 1a
Ta ble 2. Effect of WxQ Mimetics on Leukocyte Migration
Induced by MCP-1a
a
THP-1 cells were induced to migrate in response to 25 ng/mL
a
(a) BOP, Et3N, DMF, NH3. (b) BOP, Et3N, DMF, PhCH2NH2.
(c) DCC, NHS, DMF, ∆.
MCP-1 in the presence of various concentrations of the compounds
shown (from DMSO stock; 1% final concentration). The concentra-
tion that reduced migration by 50% (ED50) is shown. Compounds
that reduced migration statistically significantly, but by less than
50% at 100 µM (the highest concentration tested), are shown as
“>100 µM”.
Sch em e 2a
of the parent glutarimide was also methylated to
produce compound 19.
The structurally related acylaminolactam 20 was
synthesized from L-ornithine (Scheme 4). Acid-catalyzed
formation of the ornithine methyl ester bishydrochloride
was followed by treatment with 2 equiv of sodium
methoxide to give the free bisamino ester, which cyclized
in situ to give the amino lactam. This rather unstable
compound was immediately acylated with the undec-
10-enoyl chloride to give the aminolactam derivative 20.
a
(a) TFA, CH2Cl2. (b) BOP, Et3N, DMF, RCO2H.
inhibitor of MCP-1-induced leukocyte migration (ED50
) 5nM). NR58,4 retains the key properties of peptide
3, inhibiting migration induced by a range of different
chemokines with similar ED50 values (Figure 3) while
having no effect on migration induced by nonchemo-
kines such as C5a (Figure 3). Thus, NR58,4 has proper-
ties and potency similar to those of the retroinverso
peptide analogue NR58-3.14.3 (ED50 ) 3nM) as a broad-
spectrum chemokine inhibitor.
We next performed a more detailed SAR analysis
based on the NR58,4 structure (10) shown in Table 3.
Modifications were made to the undec-10-enoyl group
as the hydrophobic pharmacophore, in each case retain-
ing the 3-aminoglutarimide group. Loss of the terminal
double bond had very little effect on the activity of the
molecule, as did shortening the alkyl chain (such that
the molecule with a 5-carbon chain 15, the shortest
tested, was approximately 4-fold less active than NR58,4).
In marked contrast, replacement of the undec-10-enoyl
group with a branched alkyl chain (compound 16)
In Vitr o Activity of Non -p ep tid e An a logu es
The ability of each of these molecules to inhibit MCP-
1-induced migration of THP-1 cells was then tested. Two
of the series (using Boc and benzoyl groups as the
hydrophobic pharmacophore) were poorly active ir-
respective of the glutamine mimetic employed (Table
2). In contrast, all four members of the series employing
an undec-10-enoyl group as the hydrophobic pharma-
cophore were active as chemokine inhibitors (Table 2).
Of this series, compound 1, using glutamine itself, had
potency similar to that of the WVQ tripeptide (13 µM).
The C-terminal primary amide of the same molecule (4)
had similar potency, but the C-terminal N-benzylamide
(7) was approximately 3-fold less active. In contrast,
replacement of the glutamine with 3-aminoglutarimide
(10) resulted in a very significant increase in potency.
Compound 10 (also termed NR58,4) was a potent

364 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Fox et al.
Sch em e 3a
Ta ble 3. Effect of NR58,4 Analogues on Leukocyte Migration
Induced by MCP-1a
a
(a) H2, Pd/C, EtOAc. (b) RuCl3, NaIO4, CCl4, CH3CN, H2O. (c)
CsOH, CH3I, DMF.
Sch em e 4a
a
(a) CH3OH, HCl. (b) CH3ONa, CH3OH. (c) CH2dCH(CH2)2COCl,
Et3N, CHCl3.
a
THP-1 cells were induced to migrate in response to 25 ng/mL
MCP-1 in the presence of various concentrations of the compounds
shown (from DMSO stock; 1% final concentration). The concentra-
tion that reduced migration by 50% (ED50) is shown. All the
compounds shown reduced migration by >90% at 100 µM.
(compound 18) reduced activity by only 10-fold com-
pared with NR58,4 (10).
Several compounds were also synthesized in which
the undec-10-enoyl group was retained, but changes
were made to the 3-aminoglutarimide moiety. N-Me-
thylation of the glutarimide ring (19) resulted in some
loss of activity compared with NR58,4 (10), although
this compound was still an active broad-spectrum
chemokine inhibitor (Table 2). However, the 6-deoxo
derivative of NR58,4 (N-undec-10-enoyl-3-aminotet-
rahydropyridin-2-one; 20) was approximately 20-fold
less potent than NR58,4 (10) (Table 2). Thus, of all the
compounds studied here, N-undec-10-enoyl-3-amino-
gluarimide (NR58,4; 10) is the most potent broad-
spectrum chemokine inhibitor in vitro.
F igu r e 3. Effect of NR58,4 (compound 10) on leukocyte
migration. THP-1 cells (for MCP-1 and SDF-1R experiments)
or freshly prepared human polymorphonuclear cells (for IL-8
and C5a experiments) were induced to migrate in response to
the chemokines MCP-1 (filled square), SDF-1R (filled triangle),
IL-8 (open circle), or the non-chemokine control C5a (open
square) in the presence of various concentrations of NR58,4
(10). The vehicle (DMSO) was present at 1% final concentra-
tion in all wells (including the controls). The number of
leukocytes that migrated is shown as the mean ( sem of
triplicate wells, expressed as a percentage of migration occur-
ring in the absence of compound.
P h a r m a cology
Intraperitoneal injection of bacterial lipopolysaccha-
ride (LPS) leads to local recruitment of leukocytes and
increased systemic levels of pro-inflammatory cytokines
such as tumor necrosis factor R (TNF-R). This approach
has been widely used as a crude model of systemic
inflammation in vivo. Although it is unlikely to have
dramatically reduced the potency of the molecule (by
more than 100-fold), suggesting that there may be a
steric requirement for a linear alkyl group at this site
in the molecule. Interestingly, however, introduction of
a charged carboxyl group at the end of a long alkyl chain

Novel Class of Chemokine Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 365
inhibitory activity resided in the C-terminal tripeptide
WxQ. On the basis of this analysis, we designed a series
of simple compounds coupling various hydrophobic
groups to glutamine mimetics and showed that N-acyl-
3-aminoglutarimides were the most active broad-
spectrum chemokine inhibitors in this series. One of
these compounds (NR58,4; 10) was the most potent,
inhibiting migration induced by a range of chemokines
in the concentration range 5-15 nM in vitro and
suppressing LPS-induced TNF-R at doses down to 0.3
mg/kg in vivo.
Importantly, the N-acyl-3-aminoglutarimides retained
the characteristic property of peptide 3: the ability to
inhibit all chemokines tested with very similar potency.
This observation strongly suggests that neither peptide
3 nor the N-acyl-3-aminoglutarimides are functioning
as simple receptor anatagonists. It is very unlikely that
a range of structurally distinct molecules would bind
to and inhibit a range of different (albeit related)
chemokine receptors with similar affinity. Consistent
with this hypothesis, we have recently demonstrated
that peptide 3 does not bind to CCR1, CCR2, CCR4,
CCR5, CXCR1, CXCR2, or CXCR4 overexpressing CHO
cells to any greater extent than to the parental CHO
cells (unpublished data). It is likely, therefore, that both
peptide 3 and N-acyl-3-aminoglutarimides mediate
chemokine inhibition by a mechanism, as yet unidenti-
fied, that is distinct from that of simple receptor
antagonists. These compounds are currently being
tested against a range of signaling pathways in an
attempt to determine the precise molecular target for
broad-spectrum chemokine inhibitors.
F igu r e 4. Effect of NR58,4 (compound 10) on LPS-induced
TNF-R production in vivo. Mice (n ) 6 per group) were
pretreated with NR58,4 (at various doses), thalidomide (TMD),
or NR58-3.14.3 by subcutaneous injection. After 45 min, the
mice were injected with 750 µg of bacterial lipopolysaccharide
(LPS) via the intraperitoneal route. After a further 3 h, the
mice were sacrificed and the level of TNF-R in serum was
measured by ELISA. The mean ((sem) serum TNF-R for each
group of mice is shown (///, p < 0.005; //, p < 0.01; /, p <
0.05 student’s unpaired t test versus mice receiving LPS but
no pretreatment).
any relevance as a model of human sepsis, nevertheless
it represents a rapid screen for anti-inflammatory
activity. As a result, we examined the effects of NR58,4
(compound 10) on LPS-stimulated TNF-R production in
this model. Adult female CD-1 mice were given a single
dose of either the free acid of NR58,4 dissolved in DMSO
vehicle or the potassium salt in saline solution. After
45 min, the animals were given an intraperitoneal
injection containing 750 µg of LPS (from E. coli O111:
B4) in saline. Three hours after injection of LPS, the
mice were sacrificed and the level of TNF-R, a serum
marker of systemic inflammation, was measured by
enzyme-linked immunosorbent assay (ELISA). In mice
that received saline alone with no LPS treatment,
TNF-R levels were very low (<25 pg/mL). However,
injection of LPS resulted in a substantial increase in
TNF-R within 3 h (1525 ( 225 pg/mL). Pretreatment
with NR58,4 (either as a free acid or as the potassium
salt) resulted in a marked reduction in serum TNF-R
levels compared to animals pretreated with vehicle
alone (Figure 4). In contrast, the substantially less
active branched chain analogue 16 had no effect on
serum TNF-R even at the highest dose (data not shown).
In a further experiment, various doses of NR58,4 as
the potassium salt were given to mice 45 min prior to
injection with LPS. Doses as low as 0.3 mg/kg (∼1 µmol/
kg) significantly reduced serum TNF-R levels (65%
reduction; p < 0.05 student’s unpaired t test). Taken
together, these studies demonstrate that NR58,4 has
potent anti-inflammatory effects in vivo.
An important, but unresolved, consideration is the
stereochemistry of the active N-acyl-3-aminoglutarim-
ides we have studied. The chiral starting material for
the synthesis of NR58,4 10 is L-glutamine, and it is
likely that the synthesis of the 3-aminoglutarimide
proceeds with retention of stereochemistry to ultimately
yield the N-acyl-L-3-aminoglutarimides. However, on the
basis of experience with other derivatized aminogluta-
rimides (such as thalidomide), it is likely that the proton
at the chiral center is sufficiently acidic to allow
exchange to occur in a protic solvent at an appreciable
rate leading to racemization. Since the biological activity
of these molecules, both in vitro and in vivo, is assessed
in aqueous medium, we have deliberately avoided
assigning the steroeochemistry of the active forms
because we cannot exclude the possibility that formation
of a small amount of N-acyl-D-3-aminoglutarimide
through racemization is required for biological activity.
The 3-aminoglutarimide group is not a common
constituent of biologically active compounds, with one
notable exception: thalidomide. Although originally
used as a sedative,18 it was later discovered to be a
selective inhibitor of TNF-R biosynthesis19 and hence
to have anti-inflammatory properties.20 It is these anti-
inflammatory properties that led to the continued
clinical use of thalidomide, most notably to reduce the
impact of leprosy infection.21 At present, however, the
molecular basis for suppression of TNF-R production by
thalidomide remains unknown. Our studies of simpler
N-acyl derivatives of 3-aminoglutarimide suggest that
inhibition of chemokine activity is a plausible mecha-
nism for the reduction of TNF-R biosynthesis following
Discu ssion
Using the ability to inhibit migration of the human
myelomonocytic cell line THP-1 in response to various
chemokines, we have begun to identify the essential
structural motifs present in peptide 3 which are neces-
sary for its broad-spectrum chemokine inhibitory prop-
erties. Both deletion analysis and substitution analysis
of the peptide demonstrated that the majority of the

366 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Fox et al.
recorded with broad-band proton decoupling, and assignments
were aided by distortionless enhancement by polarization
transfer (DEPT) analysis. Melting points were measured on a
Stuart Scientific melting point apparatus (SMP 1) and are
uncorrected. Infrared spectra were recorded on a Perkin-Elmer
1600 (FT-IR) spectrophotometer. Mass spectra were recorded
on a Kratos (MS890) single-beam spectrometer or a micromass
platform with a HP1050 LC system. Microanalyses were
carried out by the staff of the University Chemical Laboratory
using Carlo Erba 1106 or Perkin-Elmer 240 automatic analyz-
ers.
thalidomide treatment, since other chemokine inhibitors
are known to reduce TNF-R biosynthesis.14 Consistent
with this suggestion, we find that thalidomide does
inhibit MCP-1-induced THP-1 migration (ED50 ) 60
µM), although it is at least 10 000 times less potent than
NR58,4 10. Because the well-publicized teratogenic side
effects of thalidomide are likely to result from oxidation
of the aryl ring22,23 (substitution of all 4 aryl protons
with fluorine24 abolishes the teratogenic properties of
the molecule), it is unlikely that NR58,4 10 will have
teratogenic properties similar to those of thalidomide.
However, it will be important to rigorously exclude any
possible teratogenic effects of N-acyl-3-aminoglutarim-
ides prior to consideration of their use as anti-inflam-
matory agents in humans.
The injection of NR58,4 10 into mice reduced TNF-R
production in response to endotoxin injection to an
extent similar to that of the modified peptide NR58-
3.14.3,14 suggesting that both molecules have similar
anti-inflammatory properties in vivo. At high doses of
NR58,4 (above 50 mg/kg), we observed anaesthetic-like
effects with the mice becoming comatose for 4-6 h
before recovering with apparently no chronic toxic
effects. However, at lower doses (which still have
maximal anti-inflammatory properties) we did not
observe any acute toxicity, consistent with our previous
observation using NR58-3.14.3 that, at least in the short
term, broad-spectrum chemokine inhibition may be well
tolerated.14-16 However, NR58,4 is at least 5-fold more
potent than NR58-3.14.3 in vivo (despite having similar
potencies in vitro), retaining activity at 100 nmol/kg
(whereas NR58-3.14.3 is inactive below 5 µmol/kg in the
LPS endotoxemia model). This difference is likely to
result from the pharmacodynamic profiles of the two
molecules; NR58-3.14.3 is cleared from plasma at the
glomerular filtration rate (plasma t1/2 ) 20 min; ref 17),
whereas the hydrophobic NR58,4 may have a much
longer plasma residence time. The cheap, simple syn-
theses of N-acyl-3-aminoglutarimides such as NR58,4
10, together with any improvements in pharmacody-
namics, are likely to render these small molecules the
broad-spectrum chemokine inhibitors of choice over
NR58-3.14.3 for further investigation of the pharmacol-
ogy of this class of drugs in vivo.
Optical rotations were recorded on a Perkin-Elmer 241
polarimeter (using the sodium D line; 589 nm). Specific
rotations are given in units of 10-1 deg dm2 g-1
.
(S)-N-r-Un d ec-10-en oylglu t a m in e 1. L-(S)-Glutamine
(14.62 g, 0.1 mol) and KOH (11.2 g, 0.2 mol) were dissolved in
water (200 mL) at 20 °C. A solution of undec-10-enoyl chloride
(20.3 g, 0.1 mol) in THF (200 mL) was then added over 10
min. The reaction mixture was stirred for 16 h at 20 °C and
was then reduced in vacuo to a volume of approximately 250
mL. Concentrated HCl (ca. 12 M) was added dropwise until
the solution was pH 1. The white precipitate was collected by
filtration, then washed with water (100 mL) and diethyl ether
(100 mL). The solid was then dissolved in refluxing toluene
(500 mL), and the excess water present was removed by
distillation at ambient pressure using a Dean-Stark trap. The
solution was then allowed to cool to 20 °C. After 16 h, the white
solid precipitate was collected via filtration and recrystallized
from EtOAc. This solid was dried in vacuo to give (S)-N-R-
undec-10-enoylglutamine as a white powder (9.36 g, 30%): mp
112-113 °C; [R]20 (c 1.13, MeOH) -4.5; νmax/cm-1 1732 (Cd
D
O, acid), 1685, 1670, 1642, 1621 (CdO, amide), 1545, 1523 (N-
H, amide). Anal. (C16H28N2O4) C, H, N: calcd C 61.5, H 9.0, N
1
9.0; found C 61.3, H 9.0, N 8.6. H NMR: δH 12.40 (1H, br s,
OH), 8.00 (1H, d, J 7.5, C10H19CONH), 7.25 (1H, br s, CONH2),
6.72 (1H, br s, CONH2), 5.77 (1H, ddt, J 17.0, 10.5 and 6.5,
CH)CH2), 4.97 (1H, dq, J 17.0 and 1.5, CHdCH2), 4.91 (1H,
ddt, J 10.0, 2.5 and 1.2, CHdCH2), 4.11 (1H, ddd, J 9.0, 8.0
and 5.0, NCH), 2.12-2.05 (4H, m, CH2CONH and CH2-
CONH2), 1.98 (1H, br q, J 6.5, CH2CHdCH2), 1.90 (1H, dtd, J
14.0, 8.0, and 5.0, CH2CH2CONH2), 1.71 (1H, dtd, J 14.0, 9.0,
and 6.5, CH2CH2CONH2), 1.46 (2H, br qn, J 7.0, CH2), 1.32
(2H, br qn, J 7.0, CH2), 1.28-1.18 (8H, m, CH2 × 4). 13C
NMR: δC 174.0, 173.9, 172.8 (CdO), 115.0 (CHdCH2), 129.3
(CH)CH2), 51.9 (NCH), 35.5, 33.6, 31.8, 29.2 (×2), 29.0, 28.9,
28.7, 27.3, 25.6 (CH2). HRMS (+ESI) C16H28N2O4Na: calcd
335.1947; found 335.1935.
(S)-N-r-Ben zoylglu ta m in e 2. Benzoyl chloride (7.03 g, 50
mmol) dissolved in THF (200 mL) was added slowly to a stirred
solution containing water (200 mL), L-(S)-glutamine (7.30 g,
50 mmol), KOH (2.80 g, 50 mmol), and K2CO3 (6.90 g, 50
mmol). The reaction was then stirred at ambient temperature
for 15 h. Concentrated HCl (ca. 12 M) was added dropwise
until the solution was pH 1, and the solvents were then
removed to dryness under reduced pressure with the aid of
the addition of toluene. The resulting solid was recrystallized
from toluene/MeOH to give (S)-N-R-benzoylglutamine as a
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. All solvents were
distilled before use. THF was freshly distilled from lithium
aluminum hydride, while CH2Cl2 was freshly distilled from
calcium hydride. Triphenylmethane was used as an indicator
for THF. Methanol was freshly distilled from sodium meth-
oxide. DMF and DMSO were dried by stirring over and
distilling from calcium hydride (at reduced pressure when
necessary) and were stored over activated 4 Å molecular sieves.
All reactions were carried out with oven-dried glassware, and
all reactions in nonaqueous solutions were carried out under
an atmosphere of argon.
Flash column chromatography was carried out using Merck
Kieselgel 60 (230-400 mesh). Thin-layer chromatography was
carried out on commercially available precoated plates (Merck
Kieselgel 60F254). Proton and carbon NMR spectra were
recorded on Bruker DRX400 or DPX400 Fourier transform
spectrometers using an internal deuterium lock with DMSO-
d6 as solvent. Chemical shifts are quoted in parts per million
downfield of tetramethylsilane, and values of coupling con-
stants (J ) are given in hertz and are quoted to the nearest 0.5
Hz (or nearest 0.1 Hz for J < 2 Hz). Carbon NMR spectra were
white solid (5.25 g, 42%): mp 146-147 °C; [R]20 (c 0.95,
D
MeOH) -9.1; νmax/cm-1 1710 (CdO, acid), 1655, 1634, 1608
(CdO, amide), 1538 (N-H, amide). Anal. (C12H14N2O4) C, H,
N: calcd C 57.5, H 5.6, N 11.2; found C 57.1, H 5.5, N 11.1.
1H NMR: δH 12.50 (1H, br s, OH), 8.70 (1H, d, J 7.5,
PhCONH), 7.85 (2H, br d, J 7.0, ortho-Ph), 7.55-7.48 (1H, m,
para-Ph), 7.47-7.42 (2H, m, meta-Ph), 7.33 (1H, br s, CONH2),
6.80 (1H, br s, CONH2), 4.32 (1H, ddd, J 12.0, 7.5, and 4.0,
NCH), 2.06 (2H, br t, J 7.0, CH2CONH2), 2.12-2.01 (1H, m,
CH2CH2CONH2), 1.98-1.86 (1H, m, CH2CH2CONH2). 13C
NMR: δC 174.0, 173.6, 166.5 (CdO), 134.1 (ipso-Ph), 131.5,
128.4, 127.5 (ortho-, meta-, and para-Ph), 52.6 (NCH), 31.7,
26.3 (CH2). HRMS (+ESI) C12H14N2O4Na: calcd 273.0851;
found 273.0850.
Gen er a l P r oced u r e for Syn t h esis of (S)-N-R-Acyl-
glu ta m in ea m id es 4-6 (Meth od A). The appropriate (S)-N-
R-acylglutamine (1 mmol) was dissolved in DMF (5 mL).

Novel Class of Chemokine Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 367
Triethylamine (0.28 cm3, 2 mmol) and BOP (442 mg, 1 mmol)
were added, and the reaction mixture was stirred for 1 h. The
reaction mixture was then added to liquid ammonia (20 mL)
at -40 °C, and the resulting mixture was allowed to warm to
ambient temperature over 15 h.
173-174 °C; [R]20 (c 0.50, DMSO) -117.2; νmax/cm-1 1662,
D
1627 (CdO, amide), 1538 (N-H, amide). Anal. (C23H35N3O3)
C, H, N: calcd C 68.8, H 8.8, N 10.5; found C 68.6, H 8.7, N,
1
10.4. H NMR: δH 8.31 (1H, t, J 6.0, NHCH2Ph), 7.91 (1H, d,
J 8.0, C10H19CONH), 7.30-7.16 (6H, m, Ph and NH2), 6.71 (1H,
br s, CONH2), 5.76 (1H, ddt, J 17.0, 10.0, and 6.5, CH)CH2),
4.96 (1H, dq, J 17.0 and 1.5, CHdCH2), 4.91 (1H, ddt, J 10.0,
2.0, and 1.0, CHdCH2), 4.30-4.17 (3H, m, NCH and NCH2-
Ph), 2.13-2.03 (4H, m, CH2CO), 1.97 (2H, br q, J 7.0, CH2-
CHdCH2), 1.91-1.81 (1H, m, CH2CH2CONH2), 1.75-1.64 (1H,
m, CH2CH2CONH2), 1.45 (2H, br qn, J 7.0, CH2), 1.31 (2H, br
qn, J 7.0, CH2), 1.26-1.16 (8H, m, CH2 × 4). 13C NMR: δC
174.1, 172.8, 172.0 (CdO), 139.8 (ipso-Ph), 139.3 (CHdCH2),
128.6, 127.4, 127.1 (ortho-, meta-, and para-Ph), 115.1 (CHd
CH2), 52.8 (NCH), 42.4, 35.7, 33.6, 32.0, 29.2 (×2), 29.1, 28.9,
28.7, 28.3, 25.6 (CH2). HRMS (+ESI) C23H35N3O3Na: calcd
424.2576; found 424.2580.
(S)-N-r-Un d ec-10-en oylglu ta m in ea m id e 4. Method A
was used, and the crude precipitated product was removed
from the reaction mixture via filtration, washed with water
(5 mL) and EtOAc (5 mL), and dried in vacuo. The crude
product was recrystallized from MeOH to give (S)-N-R-undec-
10-enoylglutamineamide as a white solid (136 mg, 41%): mp
170-171 °C; [R]20D (c 0.91, DMSO) -50.5; νmax/cm-1 1655, 1637
(CdO, amide), 1540 (N-H, amide). Anal. (C16H29N3O3) C, H,
N: calcd C 61.7, H 9.4, N 13.5; found C 61.7, H 9.4, N 13.5.
1H NMR: δH 7.80 (1H, d, J 8.0, C10H19CONH), 7.26 (2H, br s,
CONH2), 6.96 (1H, br s, CONH2), 6.72 (1H, br s, CONH2), 5.79
(1H, ddt, J 17.0, 10.0, and 6.5, CH)CH2), 4.99 (1H, dq, J 17.0
and 1.5, CHdCH2), 4.93 (1H, ddt, J 10.0, 3.0, and 1.0, CHd
CH2), 4.14 (1H, td, J 8.5 and 5.5, NCH), 2.11 (2H, t, J 7.5,
CH2CO), 2.06 (2H, t, J 8.0, CH2CO), 2.00 (2H, br q, J 7.0, CH2-
CHdCH2), 1.91-1.79 (1H, m, CH2CH2CONH2), 1.74-1.62 (1H,
m, CH2CH2CONH2), 1.47 (2H, br qn, J 6.5, CH2), 1.34 (2H, br
qn, J 6.5, CH2), 1.29-1.20 (8H, m, CH2 × 4). 13C NMR: δC
173.7, 173.6, 172.1 (CdO), 138.8 (CHdCH2), 114.7 (CHdCH2),
52.0 (NCH), 35.3, 33.3, 31.7, 28.8 (×2), 28.7, 28.6, 28.3, 28.0,
25.3 (CH2). HRMS (+ESI) C16H29N3O3Na: calcd 334.2107;
found 334.2099.
(S)-N-r-Ben zoylglu ta m in eben zyla m id e 8. Method B was
used, and the crude precipitated product was removed from
the reaction via filtration, washed with water (5 mL) and
EtOAc (5 mL), and dried in vacuo. The crude product was
recrystallized from H2O/MeOH to give (S)-N-R-benzoylglut-
aminebenzylamide as a white solid (88 mg, 26%): mp 212-
213 °C; [R]20D (c 1.00, DMSO) -25.8; νmax/cm-1 1655, 1622 (Cd
O, amide), 1534 (N-H, amide). Anal. (C19H21N3O3 + _H2O) C,
H, N: calcd C 65.5, H 6.4, N 12.1; found C 65.4, H 6.1, N 12.0.
1H NMR: δH 8.61 (1H, d, J 7.5, PhCONH), 8.45 (1H, t, J 6.0,
NHCH2Ph), 7.88 (2H, br d, J 8.0, ortho-PhCO), 7.51 (1H, t, J
8.0, Ph), 7.45 (2H, t, J 7.5, Ph), 7.33 (1H, br s, CONH2), 7.31-
7.16 (5H, m, Ph), 6.83 (1H, br s, CONH2), 4.39 (1H, ddd, J
9.5, 7.5, and 5.0, NCH), 4.27 (2H, d, J 6.0, CH2Ph), 2.25-2.10
(S)-N-r-Ben zoylglu ta m in ea m id e 5. Method A was used,
and the crude precipitated product was removed from the
reaction via filtration, washed with water (5 mL) and EtOAc
(5 mL), and dried in vacuo. The crude product was recrystal-
lized from MeOH to give (S)-N-R-benzoylglutamineamide as
(2H, m, CH2CONH2), 2.07-1.85 (2H, m, CH2CH2CONH2). 13
C
a white solid (132 mg, 53%): mp 210-211 °C; [R]20 (c 0.63,
NMR: δC 174.2, 171.7, 166.5 (CdO), 139.6, 134.2 (ipso-Ph),
129-126 (m, ortho-, meta-, and para-Ph), 53.8 (NCH), 42.1,
32.1, 27.7 (CH2). HRMS (+ESI) C19H21N3O3Na: calcd 362.1481;
found 362.1476.
D
DMSO) -90.6; νmax/cm-1 1659, 1631 (CdO, amide), 1521 (N-
H, amide). Anal. (C12H15N3O3) C, H, N: calcd C 57.8, H 6.1, N
1
16.9; found C 57.4, H 6.0, N 16.6. H NMR: δH 8.47 (1H, d, J
7.5, PhCONH), 7.86 (2H, br d, J 8.0, ortho-Ph), 7.53-7.48 (1H,
m, para-Ph), 7.47-7.41 (2H, m, meta-Ph), 7.40 (1H, br s,
CONH2), 7.34 (1H, br s, CONH2), 7.00 (1H, br s, CONH2), 6.78
(1H, br s, CONH2), 4.30 (1H, ddd, J 9.0, 8.0, and 5.0, NCH),
2.23-2.10 (2H, m, CH2CONH2), 2.03-1.82 (2H, m, CH2CH2-
CONH2). 13C NMR: δC 174.7, 174.1, 166.8 (CdO), 134.5 (ipso-
Ph), 131.8, 128.6, 127.9 (ortho-, meta-, and para-Ph), 53.7
(NCH), 32.1, 27.7 (CH2). HRMS (+ESI) C12H15N3O3Na: calcd
272.1011; found 272.1008.
(S)-N-r-ter t-Bu toxyca r bon ylglu ta m in eben zyla m id e 9.
Method B was used, and the crude reaction was partitioned
between water (30 mL) and EtOAc (3 × 50 mL). The combined
organic extracts were washed with 0.1 M HCl (2 × 50 mL),
dried (Na2SO4), and reduced in vacuo. The residue was
recrystallized from EtOAc to give (S)-N-R-tert-butoxycarbon-
ylglutaminebenzylamide as a white solid (74 mg, 22%): mp
151-152 °C; [R]20 (c 0.66, MeOH) -15.3; νmax/cm-1 1694
D
(CdO, carbamate), 1657 (CdO, amide), 1534 (N-H, amide).
Anal. (C17H25N3O4) C, H, N: calcd C 60.9, H 7.5, N 12.5; found
C 60.5, H 7.4, N 12.5. 1H NMR: δH 8.30 (1H, t, J 6.0, NHCH2-
Ph), 7.30-7.16 (6H, m, Ph and NH2), 6.91 (1H, d J 8.0, NHCH),
6.67 (1H, s, NH2), 4.28 (1H, dd, J 15.5 and 6.0, CH2Ph), 4.23
(1H, dd, J 15.5 and 6.0, CH2Ph), 3.89 (1H, td, J 8.5 and 5.5,
NCH), 2.15-2.00 (2H, m, CH2CONH2), 1.90-1.77 (1H, m, CH2-
CH2CONH2), 1.75-1.61 (1H, m, CH2CH2CONH2), 1.36 (9H,
s, CH3 × 3). 13C NMR: δC 175.0, 173.2, 156.5 (CdO), 140.6
(ipso-Ph), 129.4, 128.1, 127.9 (ortho-, meta-, and para-Ph), 79.3
(S)-N-r-ter t-Bu toxyca r bon ylglu ta m in ea m id e 6. Method
A was used, and the crude reaction was partitioned between
water (30 mL) and EtOAc (3 × 50 mL). The combined organic
extracts were washed with water (2 × 50 mL), dried (Na2SO4),
and reduced in vacuo. The residue was recrystallized from
EtOAc to give (S)-N-R-tert-butoxycarbonylglutamineamide as
a white solid (91 mg, 37%): mp 116-117 °C; [R]20 (c 1.0,
D
MeOH) -1.4; νmax/cm-1 1685 (CdO, carbamate), 1645 (CdO,
amide), 1521 (N-H, amide). Anal. (C10H19N3O4) C, H, N: calcd
1
(OCMe3), 55.6 (NCH), 43.1 (CH2Ph), 32.8 (CH2), 29.4 (CH3
3), 28.8 (CH2). HRMS (+ESI) C17H25N3O4Na: calcd 358.1743;
found 358.1730.
×
C 49.0, H 7.8, N 17.1; found C 48.7, H 7.7, N 17.0. H NMR:
δH 7.26 (1H, s, NH), 7.22 (1H, s, NH), 6.96 (1H, s, NH), 6.78-
6.69 (2H, m, NH × 2), 3.78 (1H, td, J 8.5 and 5.5, NCH), 2.12-
1.98 (2H, m, CH2CONH2), 1.85-1.72 (1H, m, CH2CH2CONH2),
Gen er a l P r oced u r e for th e Syn th esis of 3-Acyla m in o-
glu ta r im id es (Meth od C). The appropriate N-R-acylated
glutamine (10 mmol) was dissolved in DMF (20 mL) along with
N-hydroxysuccinimide (1.15 g, 10 mmol) and 1,3-dicyclohexy-
lcarbodiimide (2.06 g, 10 mmol). The reaction mixture was
heated to 80 °C for 6 h and then allowed to cool. The DMF
was removed under reduced pressure, and EtOAc (100 mL)
was added to the residue. The resulting suspension was
filtered, retaining the filtrate. Then the solid was extracted
with additional EtOAc (100 mL), and the combined filtrate
solution was reduced in vacuo.
1.69-1.58 (1H, m, CH2CH2CONH2), 1.34 (9H, s, CH3 × 3). 13
C
NMR: δC 175.0, 174.9, 156.3 (CdO), 79.0 (OCMe3), 54.9 (NCH),
32.6 (CH2), 29.2 (CH3 × 3), 28.8 (CH2). HRMS (+ESI)
C
10H19N3O4Na: calcd 268.1273; found 268.1270.
Gen er a l P r oced u r e for Syn t h esis of (S)-N-R-Acyl-
glu ta m in eben zyla m id es 7-9 (Meth od B). The appropriate
(S)-N-R-acylglutamine (1 mmol) was dissolved in DMF (5 mL).
Triethylamine (0.28 cm3, 2 mmol), BOP (442 mg, 1 mmol), and
PhCH2NH2 (214 mg, 2 mmol) were added, and the reaction
mixture was stirred for 15 h.
(S)-N-r-Un dec-10-en oylglu tam in eben zylam ide 7. Method
B was used, and the crude precipitated product was removed
from the reaction via filtration, washed with water (5 mL) and
EtOAc (5 mL), and dried in vacuo. The crude product was
recrystallized from EtOAc/MeOH to give (S)-N-R-undec-10-
enoylglutamineamide as a white solid (204 mg, 51%): mp
(S)-3-(Un d ec-10-en oyla m in o)glu ta r im id e 10. Method C
was used, and the residue was subjected to silica column
chromatography (diethyl ether) and then recrystallized from
EtOAc to give (S)-3-(undec-10-enoylamino)glutarimide as a
white solid (1.79 g, 61%): mp 124-125 °C; [R]20 (c 1.07,
D
MeOH) -13.9; νmax/cm-1 1720 (CdO, imide), 1653 (CdO,

368 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
amide), 1538 (N-H, amide). Anal. (C16H26N2O3) C, H, N: calcd
Fox et al.
49.4 (NCH), 35.7, 31.7, 31.3, 29.2, 29.0 (×2), 25.6, 24.8, 22.5
(CH2), 14.4 (CH3). HRMS (+ESI) C14H24N2O3Na: calcd 291.1685;
found 291.1674.
1
C 65.3, H 8.9, N 9.5; found C 65.3, H 8.9, N 9.5. H NMR: δH
10.74 (1H, br s, CONHCO), 8.08 (1H, d, J 8.5, C10H19CONH),
5.75 (1H, ddt, J 17.0, 10.0, and 6.5, CHdCH2), 4.96 (1H, dq, J
17.0 and 1.5, CHdCH2), 4.90 (1H, ddt, J 10.0, 2.0, and 1.0,
CHdCH2), 4.49 (1H, q, J 8.5, NCH), 2.74-2.62 (1H, m, CH2-
CONHCO), 2.46 (1H, dt J 17.0 and 4.0, CH2CONHCO), 2.09
(2H, t, J 7.5, C9H17CH2CONH), 1.98 (2H, br q, J 7.5, CH2CHd
CH2), 1.92-1.82 (2H, m, NCHCH2CH2CONH), 1.47 (2H, br qn,
J 7.0, CH2), 1.29 (2H, br qn, J 6.0, CH2), 1.28-1.18 (8H, m,
CH2 × 4). 13C NMR: δC 173.0, 172.3, 172.3 (CdO), 138.9 (CHd
CH2), 114.7 (CHdCH2), 49.0 (NCH), 35.3, 33.3, 31.0, 28.8 (×2),
28.6, 28.5, 28.3, 25.3, 24.5 (CH2). HRMS (+ESI) C16H26N2O3-
Na: calcd 317.1841; found 317.1827.
(S)-3-(Hep ta n oyla m in o)glu ta r im id e 14. Method D was
used (101 mg, 42%): mp 119-120 °C; [R]20 (c 0.63, MeOH)
D
-37.1; νmax/cm-1 1716 (CdO, imide), 1646 (CdO, amide), 1548
(N-H, amide). Anal. (C12H20N2O3) C, H, N: calcd C 60.0, H
1
8.4, N 11.7; found C 59.9, H 8.5, N 11.6. H NMR: δH 10.73
(1H, br s, CONHCO), 8.09 (1H, d, J 8.5, C6H13CONH), 4.50
(1H, q, J 8.5, NCH), 2.69 (1H, ddd, J 17.5, 7.5, and 3.5, CH2-
CONHCO), 2.46 (1H, dt, J 17.5 and 3.5, CH2CONHCO), 2.10
(2H, t, J 7.5, C5H11CH2CONH), 1.95-1.83 (2H, m, NCHCH2-
CH2CONH), 1.48 (2H, br qn, J 7.0, CH2), 1.31-1.18 (6H, m,
CH2), 0.84 (3H, t, J 7.0, CH3). 13C NMR: δC 173.1, 172.3 (×2)
(CdO), 49.0 (NCH), 35.4, 31.0 (×2), 28.3, 25.3, 24.5, 22.1 (CH2),
14.0 (CH3). HRMS (+ESI) C12H20N2O3Na: calcd 263.1372;
found 263.1368.
(S)-3-(Ben zoyla m in o)glu ta r im id e 11. Method C was
used, and the residue was subjected to silica column chroma-
tography (diethyl ether) and then recrystallized from EtOAc
to give (S)-3-(benzoylamino)glutarimide as a white solid (626
(S)-3-(P en ta n oyla m in o)glu ta r im id e 15. Method D was
mg, 27%): mp 180 °C (dec); [R]20 (c 0.60, MeOH) -55.1; νmax
/
used (62 mg, 29%): mp 144-145 °C; [R]20 (c 0.35, MeOH)
D
D
cm-1 1702 (CdO, imide), 1642 (CdO, amide), 1528 (N-H,
-40.3; νmax/cm-1 1699 (CdO, imide), 1618 (CdO, amide), 1550
amide). Anal. (C12H12N2O3) C, H, N: calcd C 62.1, H 5.2, N
(N-H, amide). Anal. (C10H16N2O3) C, H, N: calcd C 56.6, H
1
1
12.1; found C 61.6, H 5.2, N 11.8. H NMR: δH 10.85 (1H, s,
7.6, N 13.2; found C 56.2, H 7.5, N 12.9. H NMR: δH 10.74
CONHCO), 8.75 (1H, d, J 8.5, PhCONH), 7.89-7.84 (2H, m,
ortho-Ph), 7.57-7.53 (1H, m, para-Ph), 7.52-7.46 (2H, m,
meta-Ph), 4.78 (1H, ddd, J 12.5, 8.5, and 5.5, NCH), 2.80 (1H,
ddd, J 17.5, 13.5, and 5.5, CH2CONH), 2.55 (1H, dt, J 17.5
and 3.5, CH2CONH), 2.13 (1H, qd, J 13.0 and 4.5, CH2CH2-
CONH), 1.98 (1H, dtd, J 13.0, 5.5, and 3.0, CH2CH2CONH2).
13C NMR: δC 173.1, 172.3, 166.2 (CdO), 134.0 (ipso-Ph), 131.6,
128.5, 127.4 (ortho-, meta-, and para-Ph), 49.6 (NCH), 31.1,
24.3 (CH2). HRMS (+ESI) C12H12N2O3Na: calcd 255.0746;
found 255.0755.
(1H, br s, CONHCO), 8.10 (1H, d, J 8.5, C4H9CONH), 4.50 (1H,
q, J 8.5, NCH), 2.74-2.63 (1H, m, CH2CONHCO), 2.46 (1H,
dt, J 17.5 and 3.5, CH2CONHCO), 2.10 (2H, t, J 7.5, C3H7CH2-
CONH), 1.96-1.83 (2H, m, NCHCH2CH2CONH), 1.47 (2H, br
qn, J 7.5, CH2), 1.27 (2H, sext, J 7.5, CH2CH3), 0.85 (3H, t, J
7.5, CH3). 13C NMR: δC 173.1, 172.3 (×2) (CdO), 49.0 (NCH),
35.1, 31.0, 27.5, 25.3, 24.5 (CH2), 13.8 (CH3). HRMS (+ESI)
C
10H16N2O3Na: calcd 235.1059; found 235.1047.
(S)-3-(2-P r opylp en ta n oyla m in o)glu ta r im ide 16. Method
D was used (201 mg, 79%): mp 146-147 °C; [R]20 (c 0.50,
D
MeOH) -47.2; νmax/cm-1 1703 (CdO, imide), 1651 (CdO,
(S)-3-(ter t-Bu toxycar bon ylam in o)glu tar im ide 12.Method
C was used, and the residue was subjected to silica column
chromatography (diethyl ether) and then recrystallized from
EtOAc to give (S)-3-(tert-butoxycarbonylamino)glutarimide as
amide), 1517 (N-H, amide). Anal. (C13H22N2O3) C, H, N: calcd
1
C 61.4, H 8.7, N 11.0; found C 61.3, H 8.8, N 11.0. H NMR:
δH 10.71 (1H, br s, CONHCO), 8.14 (1H, d, J 8.0, Pr2-
CHCONH), 4.50 (1H, ddd, J 13.5, 8.5, and 5.5, NCH), 2.69
(1H, ddd, J 17.5, 13.0 and 6.0, CH2CONHCO), 2.46 (1H, dt, J
17.0 and 4.0, CH2CONHCO), 2.17 (1H, m, Pr2CHCONH),
1.99-1.80 (2H, m, NCHCH2CH2CONH), 1.50-1.37 (2H, m,
CH2), 1.34-1.14 (6H, m, CH2), 0.83 (3H, t, J 7.0, CH3), 0.82
(3H, t, J 7.0, CH3). 13C NMR: δC 176.8, 174.9, 174.0 (CdO),
49.1 (NCH), 45.6 (CHCO), 35.3, 35.2, 31.2, 24.7, 20.4 (×2)
(CH2), 14.4 (×2) (CH3). HRMS (+ESI) C13H22N2O3Na: calcd
277.1528; found 277.1531.
a white solid (1.30 g, 57%): mp 152-153 °C; [R]20 (c 0.63,
D
MeOH) -55.2; νmax/cm-1 1723 (CdO, imide), 1685 (CdO,
carbamate), 1538 (N-H, carbamate). Anal. (C10H16N2O4) C, H,
N: calcd C 52.6, H 7.1, N 12.3; found C 53.1, H 7.1, N, 12.1.
1H NMR: δH 10.72 (1H, s, CONHCO), 7.11 (1H, d, J 8.5,
NHCH), 4.25-4.13 (1H, m, NCH), 2.68 (1H, ddd, J 18.0, 8.5,
and 6.5, CH2CONH), 2.45 (1H, dt, J 17.5 and 3.5, CH2CONH),
2.01-1.82 (2H, m, CH2CH2CONH), 1.36 (9H, s, CH3 × 3). 13C
NMR: δC 175.0, 174.5, 157.4 (CdO), 80.1 (OCMe3), 52.3 (NCH),
32.9 (CH2), 30.1 (CH3 × 3), 25.7 (CH2). HRMS (+ESI)
C10H16N2O4Na: calcd 251.1008; found 251.1012.
(S)-3-(Un d eca n oyla m in o)glu ta r im id e 17. (S)-3-(Undec-
10-enoylamino)glutarimide (294 mg, 1 mmol) was dissolved
in EtOAc (20 mL), and 10% Pd on carbon (50 mg) was added.
The reaction mixture was stirred under hydrogen (1 atm) at
ambient temperature for 15 h and then was filtered. The
solution was reduced in vacuo, and the residue was recrystal-
lized from EtOAc to give (S)-3-(undecanoylamino)glutarimide
Gen er a l P r oced u r e for th e Syn th esis of 3-Acyla m in o-
glu ta r im id es (Meth od D). (S)-3-(tert-Butoxycarbonylamino)-
glutarimide 12 (228 mg, 1 mmol) was dissolved in CH2Cl2 (3
mL), and trifluoroacetic acid (1 mL) was added. The reaction
was stirred for 1 h and then reduced in vacuo to give a thick
oil. The residue was redissolved in DMF (2 mL), and to this
solution was added BOP (442 mg, 1 mmol), triethylamine (1
mL), and the appropriate carboxylic acid to be incorporated
as a side chain (1 mmol). The reaction mixture was stirred at
ambient temperature for 15 h and was then partitioned
between water (20 mL) and EtOAc (3 × 50 mL). The combined
organic layers were washed with 0.1 M HCl, dried (Na2SO4),
and reduced in vacuo to give the crude product. This residue
was subjected to silica column chromatography (EtOAc) and
then recrystallized (EtOAc) to give the 3-acylaminoglutarim-
ide.
as a white solid (278 mg, 94%): mp 131-132 °C; [R]20 (c 0.6,
D
MeOH) -18.3; νmax/cm-1 1716 (CdO, imide), 1650 (CdO,
amide), 1547 (N-H, amide). Anal. (C16H28N2O3) C, H, N: calcd
1
C 64.8, H 9.5, N 9.5; found C 64.8, H 9.6, N 9.5. H NMR: δH
10.76 (1H, br s, CONHCO), 8.11 (1H, d, J 8.5, C10H21CONH),
4.49 (1H, dt, J 10.0 and 8.0, NCH), 2.69 (1H, ddd, J 18.0, 11.0,
and 7.0, CH2CONHCO), 2.45 (1H, dt, J 17.5 and 4.0, CH2-
CONHCO), 2.08 (2H, t, J 7.5, C9H19CH2CONH), 1.95-1.80
(2H, m, NCHCH2CH2CONH), 1.47 (2H, br qn, J 6.5, CH2),
1.25-1.18 (14H, m, CH2), 0.82 (3H, br t, J 7.0, CH3). 13C
NMR: δC 173.7, 173.0 (×2) (CdO), 49.7 (NCH), 36.0, 32.1, 31.7,
29.7 (×2), 29.6, 29.5, 29.3, 26.0, 25.1, 22.9 (CH2), 14.7 (CH3).
HRMS (+ESI) C16H28N2O3Na: calcd 319.1998; found 319.1983.
(S)-3-(Non a n oyla m in o)glu ta r im id e 13. Method D was
used (164 mg, 61%): mp 121-122 °C; [R]20 (c 1.00, MeOH)
D
-32.9; νmax/cm-1 1716 (CdO, imide), 1647 (CdO, amide), 1551
(S)-3-(9-Ca r boxyn on a n oyla m in o)glu ta r im id e 18. (S)-3-
(undec-10-enoylamino)glutarimide (294 mg, 1 mmol) was
dissolved in CCl4 (2 mL) and CH3CN (2 mL), and a solution of
NaIO4 (875 mg, 4.1 mmol) was added. Ruthenium trichloride
hydrate (13 mg, 0.06 mmol) was added, and the reaction
mixture was stirred for 4 h. The reaction was extracted with
EtOAc (3 × 20 mL). The combined organic layers were dried
(Na2SO4) and reduced in vacuo, and the residue was recrystal-
lized from EtOAc to give (S)-3-(9-carboxynonanoylamino)-
(N-H, amide). Anal. (C14H24N2O3) C, H, N: calcd C 62.7, H
1
9.0, N 10.4; found C 63.0, H 8.9, N 10.5. H NMR: δH 10.71
(1H, br s, CONHCO), 8.09 (1H, d, J 8.0, C8H17CONH), 4.50
(1H, q, J 8.0, NCH), 2.75-2.62 (1H, m, CH2CONHCO), 2.46
(1H, dt, J 17.5 and 3.5, CH2CONHCO), 2.09 (2H, t, J 7.5,
C7H15CH2CONH), 1.95-1.82 (2H, m, NCHCH2CH2CONH),
1.48 (2H, br qn, J 7.0, CH2), 1.30-1.17 (10H, m, CH2), 0.84
(3H, br t, J 7.0, CH3). 13C NMR: δC 173.4, 172.7 (×2) (CdO),

Novel Class of Chemokine Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 369
glutarimide as a white solid (268 mg, 86%): mp 126-127 °C;
[R]20D (c 0.50, MeOH) -30.2; νmax/cm-1 1719 (CdO, imide), 1651
(CdO, amide), 1543 (N-H, amide). Anal. (C14H24N2O3) C, H,
(CHdCH2), 50.2 (NCH), 42.5 (NCH2), 36.7, 34.7, 30.2 (×2),
30.0, 29.9, 29.7, 29.3, 26.7, 22.4 (CH2). HRMS (+ESI)
C16H28N2O2Na: calcd 303.2048; found 303.2055.
1
N: calcd C 57.7, H 7.8, N 9.0; found C 57.5, H 7.9, N 8.6. H
Biologica l Assa ys. Compounds were tested for their ability
to inhibit chemokine-induced migration using the microtiter
format trans-well migration assay, exactly as described previ-
ously.14 As noted in the text, assays were performed either
using the human myelomonocytic cell line THP-1 (which
responds to a range of chemoattractants including MCP-1,
MIP-1R, RANTES, SDF-1R, and fMLP but not C5a or IL-8) or
using freshly prepared human peripheral blood neutrophils
(which respond to C5a or IL-8). All migration assays were
performed in Gey’s balanced salt solution containing 1 mg/
mL endotoxin-free BSA (in the absence of fetal calf serum),
allowing migration to occur for 2 h at 37 °C. All compounds
were solubilized in DMSO and added to both the top and
bottom compartments of the trans-well migration plate at a
constant final DMSO concentration of 1%. The number of cells
undergoing migration was measured using the vital dye MTT,
as previously described,14 and is expressed as a percentage of
the number of cells undergoing migration in the presence of
1% DMSO vehicle only. Values are given as the mean and
standard error from triplicate determinations.
NMR: δH 11.98 (1H, br s, OH), 10.77 (1H, s, CONHCO), 8.12
(1H, d, J 8.5, HO2CC8H16CONH), 4.50 (1H, br q, J 8.5, NCH),
2.69 (1H, ddd, J 18.0, 11.0, and 7.0, CH2CONHCO), 2.45 (1H,
dt, J 17.5 and 3.5, CH2CONHCO), 2.15 (2H, t, J 7.5, C7H14CH2-
CO), 2.08 (2H, t, J 7.5, C7H14CH2CO), 1.94-1.80 (2H, m,
NCHCH2CH2CONH), 1.52-1.40 (4H, m, CH2), 1.28-1.17 (8H,
m, CH2). 13C NMR: δC 173.4, 171.9, 171.2, 171.1 (CdO), 49.2
(NCH), 35.6, 34.0, 31.2, 29.0 (×2), 28.9, 28.8, 25.5, 24.8, 24.7
(CH2). HRMS (+ESI) C15H24N2O5Na: calcd 335.1583; found
335.1571.
(S)-1-Meth yl-3-(u n d ec-10-en oyla m in o)glu ta r im id e 19.
(S)-3-(Undec-10-enoylamino)glutarimide (294 mg, 1 mmol) was
dissolved in DMF (2 mL), and CsOH‚H2O (168 mg, 1 mmol)
was added. Methyl iodide (0.124 mL, 2 mmol) was then added,
and the reaction mixture was stirred at ambient temperature
for 16 h. The reaction mixture was partitioned between water
(20 mL) and EtOAc (3 × 20 mL), and the combined organic
layers were washed with 0.1 M HCl (2 × 50 mL), dried (Na2-
SO4), and reduced in vacuo. The residue was subjected to silica
column chromatography (diethyl ether) and then recrystallized
from EtOAc to give (S)-1-methyl-3-(undec-10-enoylamino)-
glutarimide as a white solid (160 mg, 52%): mp 82-83 °C;
[R]20D (c 1.01, MeOH) -16.0; νmax/cm-1 1675 (CdO, imide), 1640
(CdO, amide), 1532 (N-H, amide). Anal. (C17H28N2O3) C, H,
The effect of NR58,4 10 on TNF-R upregulation in vivo was
determined as follows. Adult male CD-1 mice (n ) 6 per group)
were pretreated with various agents (vehicle, NR58,4, thali-
domide or NR58-3.14.3) by subcutaneous injection 45 min prior
to an acute inflammatory challenge with 750 µg of bacterial
lipopolysachharide (from E. coli 0111:B4; Sigma L-4130) via
the intraperitoneal route. After a further 3 h, the animals were
sacrificed and blood was drawn by cardiac puncture. The level
of TNF-R in serum was determined using the Quantikine
ELISA for murine TNF-R (R&D Systems) and is reported as
the mean ( standard error for each group.
1
N: calcd C 66.2, H 9.2, N 9.1; found C 66.0, H 9.2, N, 8.9. H
NMR: δH 8.15 (1H, d, J 8.5, CONH), 5.78 (1H, ddt, J 17.0,
10.0, and 6.5, CHdCH2), 4.96 (1H, dq, J 17.0 and 2.0, CHd
CH2), 4.90 (1H, ddt, J 10.0, 2.0, and 1.0, CHdCH2), 4.49 (1H,
ddd, J 12.5, 8.5, and 6.5 NCH), 2.95 (3H, s, NCH3) 2.77 (1H,
dt, J 17.0, 12.0, and 6.0, CH2CONHCO), 2.63 (1H, dt, J 17.0
and 3.5, CH2CONHCO), 2.09 (2H, t, J 7.0, CH2CONH), 1.98
(2H, br q, J 7.5, CH2CHdCH2), 1.95-1.80 (2H, m, NCHCH2-
CH2CONCH3), 1.48 (2H, br qn, J 7.0, CH2), 1.37-1.18 (10H,
m, CH2 × 5). 13C NMR: δC 172.5, 172.4, 172.2 (CdO), 139.2
(CHdCH2), 114.9 (CH)CH2), 49.8 (NCH), 35.6, 33.5, 31.4, 29.0
(×2), 28.9, 28.8, 28.6, 26.8, 25.5, 23.8 (CH2 and CH3). HRMS
(+ESI) C17H28N2O3Na: calcd 331.1998; found 331.1982.
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solid (1.79 g, 64%): mp 90-91 °C; [R]20 (c 0.50, MeOH) -4.0;
D
ν
max/cm-1 1667 (CdO, lactam), 1634 (CdO, amide), 1535 (N-
H, amide). Anal. (C16H28N2O2) C, H, N: calcd C 68.5, H 10.1,
1
N 10.0; found C 68.5, H 10.1, N 9.9. H NMR: δH 7.90 (1H, d,
J 8.0, C10H19CONH), 7.51 (1H, br s, NCHCONH), 5.77 (1H,
ddt, J 17.0, 10.0, and 6.5, CHdCH2), 4.97 (1H, br d, J 17.0,
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10.0, 8.0, and 6.5, NCH), 3.13-3.04 (2H, m, CH2NH), 2.05 (2H,
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1.87 (1H, m, CH2CH2NH), 1.78-1.62 (2H, m, CH2CH2CH2N),
1.54 (1H, qd, J 10.5 and 4.5, CH2CH2CH2N), 1.50-1.42 (2H,
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× 4). 13C NMR: δC 173.3, 171.4 (CdO), 140.3 (CHdCH2), 116.1
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