Highly potent, orally available anti-inflammatory broad-spectrum chemokine inhibitors

Article abstract of DOI:10.1021/jm900133w

A series of 3-acylaminocaprolactams are inhibitors of chemokine-induced chemotaxis. Branching of the side chain α-carbon provides highly potent inhibitors of a range of CC and CXC chemokines. The most potent compound has an ED₅₀ of 40 pM. Selec

Full text of DOI:10.1021/jm900133w

J. Med. Chem. 2009, 52, 3591–3595
3591
Highly Potent, Orally Available Anti-inflammatory Broad-Spectrum Chemokine Inhibitors
David J. Fox,*,§,† Jill Reckless,‡ Hannah Lingard,† Stuart Warren,† and David J. Grainger‡
Department of Chemistry, UniVersity of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K., Department of Medicine,
UniVersity of Cambridge, Box 157, Addenbrooke’s Hospital, Cambridge CB2 2QQ, U.K.
ReceiVed February 3, 2009
A series of 3-acylaminocaprolactams are inhibitors of chemokine-induced chemotaxis. Branching of the
side chain R-carbon provides highly potent inhibitors of a range of CC and CXC chemokines. The most
potent compound has an ED50 of 40 pM. Selected compounds were tested in an in vivo inflammatory assay,
and the best compound reduces TNF-R levels with an ED50 of 0.1 µg/kg when administered by either
subcutaneous injection or oral delivery.
Introduction
Inappropriate inflammation is a key pathogenic component
of many serious diseases. New, potent small-molecule drug
candidates are required for clinical development. Here we
describe the discovery of simple, but extremely potent (40 pM)
Figure 1. Previous broad spectrum chemokine inhibitors.
and orally available anti-inflammatory compounds.
The chemokines1 are a large family of peptides involved in
Scheme 1a
regulating leukocyte trafficking and inflammation in both
physiological and pathological conditions. With approximately
50 ligands and 20 receptors involved in chemokine signaling,2
the system has sufficient complexity to guide leukocytes through
the diverse pathways of a functional immune system. Inhibition
of chemokines and chemokine receptors3-5 is therefore an
important target for medical intervention, with many small
molecule receptor ligands developed as antagonists blocking
chemokine binding at specific members of both the CCR4 and
CXCR5 receptor subclasses.
Peptide6 and small molecule7 broad-spectrum chemokine
inhibitors (BSCIs) such as 1 and 2 (Figure 1) inhibit leukocyte
chemotaxis in vitro due to a range of chemokines and act as
anti-inflammatory agents in vivo.8 So far, the inhibition of
multiple chemokines with BSCIs has had no obvious detrimental
toxicological effects in vivo, possibly as the mechanism of action
of BSCIs is not functionally equivalent to multiple gene
deletions in a range of chemokine receptors.9 However, these
compounds have limited stability, potency, and oral availability.
We have therefore sought an ideal small molecule drug
candidate with BSCI activity to act as a broad anti-inflammatory
agent with similar efficacy to corticosteroids but with a more
acceptable side-effect profile.
Results and Discussion
A library of drug candidates (Scheme 1) was synthesized via
the acylation of either enantiomer of lactam 3 with a range of
acyl chlorides (see Supporting Information). Although simple,
this chemistry yielded a number of very potent chemokine
a Conditions: (i) RCOCl, Na2CO3, rt.
inhibitors (Table 1). In vitro screening was performed by
measuring the ED50 for the inhibition of the migration of human
myelomonocytic THP-1 cells toward the chemokine CCL2.6,7,10
Initially, we found that 2,2-dimethyldodecanoyl substituted
amino-caprolactam (S)-5 is roughly 150 times more potent than
its nonmethylated counterpart (S)-4, with an ED50 of 90 pM
(Scheme 1, Table 1). Its enantiomer (R)-5 is, however, roughly
50 times less potent in this assay. Lactam (S)-5 also inhibited
the chemotaxis of THP-1 cells and human polymorphonuclear
cells induced by other CC and CXC chemokines (Table 2) with
* To whom correspondence should be addressed. Phone: +44 (0)24 7652
4331. Fax: +44 (0)24 7652 4112. E-mail: d.j.fox@warwick.ac.uk.
†
Department of Chemistry, University of Cambridge.
Department of Medicine, University of Cambridge.
Current Address: Department of Chemistry, University of Warwick,
‡
§
Gibbet Hill Road, Coventry, CV4 7AL, U.K.
10.1021/jm900133w CCC: $40.75
2009 American Chemical Society
Published on Web 05/08/2009

3592 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Brief Articles
Table 1. In Vitro Inhibition of CCL2 Induced Chemotaxis of THP-1
Leukocytesa
lactam
ED50 (nM)
lactam
ED50 (nM)
(S)-4
(S)-5
(R)-5
(S)-6
(S)-7
15
0.09
4.5
0.08
0.2
0.5
1
3
5
10
4
(S)-14
(S)-15
(R)-15
(S)-16
(S)-17
(S)-18
(S)-19
(S)-20
(S)-21
(S)-22
(S,S)-23
7
0.09
0.44
0.8
0.09
0.5
(S)-8
(S)-9
0.8
(S)-10b
(S)-11
(S)-12
(S)-13
0.07
0.04
9
200
a , human myelomonocytic THP-1 cells. b A 50:50 mixture of diastere-
oisomers at the 2′-stereocenter.
Table 2. In Vitro Inhibition of Chemotaxis by (S)-5a
chemattractant
cell typea
ED50 (nM)
CCL2
CCL3
CCL5
CXCL8
CXCL12
C5a
THP-1
THP-1
THP-1
PMNC
THP-1
PMNC
0.09
0.15
0.9
0.3
1
Figure 2. X-ray crystal structures of compounds (S)-15, (S)-17, and
(S)-21 (one of three in the unit cell). Ellipsoids are shown at 50%
probability.
>1000
a Human myelomonocytic THP-1 cells, or human peripheral blood
polymorphonuclear cells (PMNC).
(S)-21 are shown in Figure 2. In the crystal of compound
(S)-17, the methyl group is in the equatorial position while
the amide substituent is in the axial position. The energy
difference between this conformation and one with an axial
methyl and equatorial amide is probably small, and either
conformation may be the active one. The side chain of the
“axial-methyl” conformation of (S)-17 can however be
overlaid with the majority of the carbons of the adamantane
in compound (S)-15 (Scheme 1).
Representative potent compounds (S)-5, (S)-15, (S)-17, and
(S)-21 were then tested for anti-inflammatory potency in vivo.
As previously, we adopted the murine sublethal lipopolysac-
charide (LPSa ) induced endotoxemia as a rapid in vivo screen
of anti-inflammatory activity, using serum tumour necrosis
factor-R (TNF-R) measured 2 h after an intraperitoneal LPS
injection as the end point. The lactams when administered
via either the subcutaneous or oral routes inhibited LPS-
stimulated TNF-R production with an ED50 of as little as 0.1
µg/kg and maximal effect of ∼90%. (Table 3, Figure 3, (S)-
21) This extraordinary anti-inflammatory potency is 1000
times greater than the previous unbranched lactams7 and
roughly 30 times more potent than the widely used “gold-
standard” anti-inflammatory steroid dexamethasone (see
Supporting Information for details). Interestingly, shortening
of the long lipophilic side-chain (5-21) increases oral
potency by over 1000 times.
The pharmacokinetics of compounds (S)-5, (S)-15, (S)-17,
and (S)-21 following single dose administration in rat were also
determined (Table 4). Consistent with the observations in the
endotoxemia model, the shortened side chain of (S)-21 compared
with (S)-5 resulted in a marked increase in exposure by both
intravenous and oral administration as well as a substantial
increase in fractional bioavailability by the oral route. The
volume of distribution was also lower (close to total body water),
suggesting that unwanted tissue accumulation resulting from
the lipophilicity of long-chain-containing lactam (S)-5 was
essentially absent with trimethylacetylated lactam (S)-21.
similar potency, making this compound a true broad spectrum
chemokine inhibitor (BSCI). Notably, lactam (S)-5 does not
inhibit chemotaxis induced by the nonchemokine chemoattrac-
tant C5a. Lactam (S)-5 itself does not possess any chemoat-
tractant activity nor exhibits any cell toxicity or any effect on
cell size, which can result in misinterpretation of data from the
chemotaxis assay (see Supporting Information).10
The lactams inhibit migration in response to a range of
chemokines with broadly similar potencies (within a factor
of 10-fold), and changes in the lactam structure have similar
effects on the potency against all the chemokines tested. This
profile indicates that these compounds are not chemokine
receptors antagonists acting at multiple receptors but that the
BSCI compounds bind to cells at a single site and inhibit
chemokine-induced migration by a yet-to-be understood
mechanism. Consistent with this view, related BSCIs have
been shown to neither bind to nor down-regulate any of a
range of chemokine receptors nor to interefere with chemok-
ine ligand or receptor dimerization.8a BSCIs cannot, therefore,
be considered to be traditional chemokine antagonists.
Other 2′,2′-dimethylated compounds (6-9) are also very
potent in the CCL2/THP-1 chemotaxis assay, but monosub-
stituted side-chains (10-12) are less active. Unsaturation in
the chain reduces potency (6 vs 7, 8 vs 9, 10 vs 12). The
3,3-dimethylated compound, (S)-13, is 40 times less potent
than its isomer (S)-5. While bicyclooctane derivative (S)-14
is a relatively poor inhibitor, adamantanes such as 15 are as
potent as noncyclic lactams 5 and 6, and once again the (R)-
enantiomer is markedly less active. The non-2′,2′-disubsti-
tuted analogue 16 is again less active, but significantly loss
of most of the adamantane structure (17-21) leads to little
loss of activity as long as the quaternary 2-carbon of the
side-chain is left intact. Indeed, lactams (S)-20 and (S)-21
are more potent than their larger analogues (70 and 40 pM,
respectively). Chlorination (22) and dimerization (23) reduces
potency. These studies demonstrate that the presence of a
quaternary carbon at the 2-position of the side chain
dramatically increases the potency of aminolactam BSCIs.
X-ray crystal structures of compounds (S)-15, (S)-17, and
a Abbreviations: LPS, lipopolysaccharide; TNF-R, tumour necrosis factor -R.

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3593
Table 3. In Vivo Anti-inflammatory Activity Measured by Inhibition of LPS Induced TNF-R
a
a
lactam
ED50 µg/kg (subcut)
maximum inhibitory effect %b
ED50 µg/kg (oral)
maximum inhibitory effect %b
(S)-5
0.15
2
0.33
0.1
3
70
90
90
90
90
300
0.5
0.33
0.1
d
70
90
90
80
d
(S)-15
(S)-17
(S)-21
dexc
a See Supporting Information for full data. b % relative to control. c Dexamethasone. d Oral assay for dexamethasone was not performed.
inhibition of LPS induced increases in TNF-R, even at
concentrations when the molecular target is likely to be
saturated. Indeed this substantial, but incomplete, inhibition
may contribute to the promising profile of these lactam BSCIs
as anti-inflammatory agents against a broad range of disease
models. However, this same complexity also hampers a full
understanding of the mode of action in vivo, and further
investigations are clearly warranted.
Conclusion
The inclusion of branching at the R-carbon of the side
chains of 3-acylaminocaprolactams greatly increases their in
vitro and in vivo potency as broad-spectrum chemokine
inhibitors. In addition, these features have also increased the
activity of these compounds by oral administration signifi-
cantly. Overall, the ease of chemical synthesis combined with
the outstanding potencies of these molecules should lead to
the first BSCI entering clinical trials as powerful anti-
inflammatory drugs, potentially useful for the treatment of a
wide range of conditions.
Figure 3. Inhibition of LPS induced TNF-R in vivo by (S)-21
(subcutaneous and oral dose) and dexamethasone (subcutaneous dose).
Table 4. Pharmacokinetic Parameters Following a Single Dose in Rat
(S)-5
(S)-15
(S)-17
(S)-21
Experimental Section
Intravenous Route (1 mg/kg)
Synthesis of Selected Compounds. Full synthetic details and
characterization data, including elemental analyses, are given
is the Supporting Information. All compounds tested are >95%
pure by elemental analysis. Proton NMR spectra were recorded
on a Bruker Avance 500 Fourier transform spectrometer using
an internal deuterium lock. Chemical shifts are quoted in parts
per million downfield of tetramethylsilane, and values of
coupling constants (J) are given in Hz.
(S)-3-(2′,2′-Dimethyl-dodecanoyl)amino-caprolactam(S)-5.(S,S)-
3-Amino-caprolactam hydro-pyrrolidine-5-carboxylate (2 mmol)
and Na2CO3 (6 mmol) in water (25 mL) were added to a solution
of 2,2-dimethyl-dodecanoyl chloride (2 mmol) in dichlo-
romethane (25 mL) at ambient temperature and the reaction
mixture was stirred for 2 h. The organic layer was then separated,
and the aqueous phase was extracted with additional dichlo-
romethane (2 × 25 mL). The combined organic layers were dried
over Na2CO3 and reduced in vacuo. The residue was purified
by silica column chromatography (eluent:EtOAc to 9:1 EtOAc:
MeOH) to give (S)-3-(2′,2′-dimethyl-dodecanoyl)amino-capro-
lactam (543 mg; 80%); δH, (500 MHz, CDCl3) 7.08 (1H, d, J
5.5, CHNH), 6.67 (1H, br s, CH2NH), 4.44 (1H, dd, J 11, 5.5,
CHNH), 3.28-3.15 (2H, m, CH2NH), 2.01 (1H, br d, J 13, ring
CH), 1.98-1.89 (1H, m, ring CH), 1.84-1.72 (2H, m, ring CH),
1.47-1.30 (3H, br m, ring CH + CH2CMe2CONH), 1.27-1.15
(17H, br m, ring CH +(CH2)8) 1.13 (3H, s, CMeMe), 1.12 (3H,
s, CMeMe), and 0.82 (3H, t, J 7, CH2CH3).
(S)-3-(1′-Adamantanecarbonyl)amino-caprolactam (S)-15. (S)-
3-amino-caprolactam hydrochloride (1 mmol) and Na2CO3 (3
mmol) in water (15 mL) were added to a solution of 1-adaman-
tanecarbonyl chloride (1 mmol) in dichloromethane (15 mL) at
ambient temperature and the reaction was stirred for 2 h. The
organic layer was then separated, and the aqueous phase was
extracted with additional dichloromethane (2 × 25 mL). The
combined organic layers were dried over Na2CO3 and reduced
in vacuo. The residue was recrystallized from CH2Cl2/hexanes
to give (S)-3-(1′-adamantanecarbonyl)amino-caprolactam (171
mg, 59%); δH (500 MHz, CDCl3) 7.08 (1H, d, J 5.5, CHNH),
half-life (mins)
46
40
2.0
23700
16
84
0.8
477
11
55
0.6
2900
121
clearance (mL/min/kg)
6.8
0.7
231500
Vss (L/kg)a
exposure (min·ng/mL)
Oral Route (3 mg/kg)
F (%)b
half-life (min)
Cmax (ng/mL)
exposure (min·ng/mL)c
a Volume of distribution at steady state. b F ) fractional bioavailability
by the oral route. c Exposure ) AUC(0ft). d n.d. ) not determined, because
the drug concentration in plasma was below the lower limit of quantitation
of the assay (approximately 2 ng/mL) at all time points.
16
102
60
<1
5
71
168
989
ndd
ndd
ndd
ndd
3
11000
430
493000
In contrast, although lactams (S)-15 and (S)-17 were also
highly active in the endotoxemia model, both compounds
achieve very poor exposure (Table 4). This apparent paradox
is explained by investigation of the metabolic fate of the
compounds. Adamantane (S)-15 is rapidly converted to the
3′-hydroxy derivative, presumably by a cytochrome P450
enzyme, on first pass through the liver, and this hydroxy-
adamantane analogue is as active as the parent compound
both in vitro and in vivo (data not shown). Similarly,
cyclohexane (S)-17 is also rapidly hydroxylated, although
the structures (and biological activities) of its primary
metabolites are not known. Of the compounds studied here,
therefore, it is trimethylacetylamino-lactam (S)-21 that has
the most promising combination of DMPK properties and
anti-inflammatory potency for further development as a
pharmaceutical agent.
Despite being small and simple, the molecules described
here will likely display complex pharmacology. For the most
part, this reflects the complexity of the immune system. For
example, in vivo the compounds do not achieve 100%

3594 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Brief Articles
6.67 (1H, br s, CH2NH), 4.47 (1H, ddd, J 11, 5.5, 1.5, CHNH),
3.32-3.17 (2H, m, CH2NH), 2.06-1.94 (5H, m, 2 × ring CH
+ 3 × adamantane CH), 1.90-1.75 (8H, m, 2 × ring CH + 3
× adamantane CH2), 1.72 (3H, br d, J 14.5, 3 × adamantane
CHH), 1.68 (3H, br d, J 14.5, 3 × adamantane CHH), and
1.47-1.32 (2H, m, 2 × ring CH).
when plasma concentrations are rising and the log trapezoidal
rule when concentrations are falling. Following intravenous
dosing the concentration versus time curve was extrapolated from
the first data point back to time zero by assuming an initial
volume of 0.26 L/kg (plasma volume in a rat).
Acknowledgment. We thank Dr John Davies for crystal-
lography and the EPSRC for financial assistance towards the
purchase of the Nonius CCD diffractometer. D.J.G. is a
director of, and D.J.F. is a consultant for, Funxional
Therapeutics Ltd.
(S)-3-(1′-Methylcyclohexanecarbonyl)amino-caprolactam (S)-
17. (S,S)-3-amino-caprolactam hydro-pyrrolidine-5-carboxylate
(5 mmol) and Na2CO3 (15 mmol) in water (25 mL) were added
to a solution of 1-methylcyclohexanecarbonyl chloride (5 mmol)
in dichloromethane (25 mL) at ambient temperature and the
reaction was stirred for 12 h. The organic layer was then
separated, and the aqueous phase was extracted with additional
dichloromethane (2 × 25 mL). The combined organic layers were
dried over Na2CO3 and reduced in vacuo. The residue was
purified by recrystallization from EtOAc/hexane to give the
lactam (540 mg, 43%); δH (500 MHz, CDCl3) 7.12 (1H, d, J 5,
CHNH), 6.52 (1H, br s, CH2NH), 4.48 (1H, ddd, J 11, 5.5, 1.5
CHNH), 3.30-3.16 (2H, m, CH2NH), 2.01 (1H, br d, J 13,
lactam ring CH), 1.98-1.86 (3H, m, lactam ring CH + cyhex
CH × 2), 1.85-1.73 (2H, m, lactam ring CH × 2), 1.56-1.47
(2H, m, cyhex CH × 2), 1.47-1.33 (5H, br m, lactam ring CH
× 2 + cyhex CH × 3) and 1.33-1.25 (3H, m, cyhex
CH × 3).
Supporting Information Available: Synthesis and characteriza-
tion data for all compounds (CIF, PDF), in vitro and in vivo
biological data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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The effect of the compounds on TNF-R upregulation in vivo
was determined exactly as previously,7b,10 except that the vehicle
used was 0.6% DMSO 1% carboxymethycellulose (final concentra-
tions) in sterile water.
For pharmacokinetic analysis, male Sprague-Dawley rats
(three animals per compound per route) were dosed with
compound via the intravenous route (1 mg/mL) or the oral route
by gavage (3 mg/mL). The vehicle for intravenous administration
was 5% DMSO in 0.9% saline; the vehicle for oral administration
was 1% carboxymethylcellulose in 0.9% saline. Animals received
food and water ad libitum throughout. Blood was drawn at 5,
15, 30, 60, 120, 180, 240, and 480 min after dosing, and plasma
was prepared. Drug concentration was determined by LC/MS/
MS following extraction of 50 µL of plasma with 100 µL of
0.1% formic acid in acetonitrile, followed by centrifugation at
6000g for 5 min. An Atlantis column (20 mm × 2.1 mm; Waters)
was used on a Sciex 4000 (Applied Biosystems) operating in
TurboIonSpray positive mode, with a mobile phase gradient of
0.1% formic acid in aqueous buffer to acetonitrile over 3.5 min.
An internal standard of related structure was used to correct for
extraction efficiency. Pharmacokinetic parameters were estimated
by standard noncompartmental methods, using Excel. Area under
the curve (AUC) calculations used the linear trapezoidal rule

Brief Articles
Broad-Spectrum Chemokine Inhibitors Resistant to Metabolism in
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