Prodrugs of a CXC chemokine-12 (CXCL12) neutraligand prevent inflammatory reactions in an asthma model in vivo

Article abstract of DOI:10.1021/ml200017d

Chalcone 4 (compound 1) is a small molecule that neutralizes the CXC chemokine CXCL12 and prevents it from acting on the CXCR4 and CXCR7 receptors. To overcome its poor solubility in aqueous buffers, we designed highly soluble analogues of compound 1, phosphate, l-seryl, and sulfate, all inactive by themselves on CXCL12 but when cleaved in vivo into 1, highly active locally at a low dose in a mouse airway hypereosinophilia model.

Full text of DOI:10.1021/ml200017d

Letter
pubs.acs.org/acsmedchemlett
Prodrugs of a CXC Chemokine-12 (CXCL12) Neutraligand Prevent
Inflammatory Reactions in an Asthma Model in Vivo
Vincent Gasparik,† Francois Daubeuf,† Muriel Hachet-Haas,‡ Francois Rohmer,† Patrick Gizzi,‡
̧ ̧
Jacques Haiech,† Jean-Luc Galzi,‡ Marcel Hibert,*,† Dominique Bonnet,† and Nelly Frossard†
†Laboratoire d'Innovation Ther
67401 Illkirch, France
‡Biotechnologie et signalisation cellulaire, UMR 7242 CNRS/Universite
́
apeutique, UMR 7200 CNRS/Universite
́
de Strasbourg, Faculte
́
de Pharmacie, 74 route du Rhin,
́
de Strasbourg, ESBS, Bld Seb
́
astien Brant, 67412 Illkirch, France
S
* Supporting Information
ABSTRACT: Chalcone 4 (compound 1) is a small molecule
that neutralizes the CXC chemokine CXCL12 and prevents it
from acting on the CXCR4 and CXCR7 receptors. To over-
come its poor solubility in aqueous buffers, we designed highly
soluble analogues of compound 1, phosphate, L-seryl, and sul-
fate, all inactive by themselves on CXCL12 but when cleaved
in vivo into 1, highly active locally at a low dose in a mouse
airway hypereosinophilia model.
KEYWORDS: prodrug, solubility, CXCL12 chemokine, CXCR4 receptor, GPCR, asthma
he chemokine CXCL12 plays a pivotal role in normal and
pathological situations, including brain development,
hydroxyl moiety of compound 1: a phosphate, an L-seryl, and
a sulfate group. Prodrugs 2, 4, and 5 were prepared according
to Scheme 1 starting from compound 1, itself obtained
following a Claisen−Schmidt reaction with a 67% overall
yield.12
T
hematopoiesis, and chronic inflammation.1 In screening a
proprietary chemical library,2 we recently identified compounds
belonging to the chalcone family and preventing CXCL12 from
binding to its CXCR4 or CXCR7 receptors.3 These compounds
have an original mechanism of action: they bind to the che-
mokine rather than to the receptors. The highest affinity molecule,
chalcone 43 (compound 1 in Scheme 1, now commercialized by
Sigma-Aldrich, C7870), inhibits both chemotaxis of human
peripheral blood lymphocytes and eosinophil infiltration in a
mouse model of airway hypereosinophilia.3 The observation of
these effects highlights the enormous potential of such a ligand
neutralization strategy.3−5 Because of its mechanism of action,
compound 1 does not affect the resting/basal level of the re-
ceptors for either calcium or chemotactic responses.3 By analogy
to the activity of neutralizing antibodies, we named compound
1 a “neutraligand”.4 It is a tool that complements traditional
antagonists of the CXCR4 receptor, such as AMD3100, AMD3465,
ALX40-4C, and T22/T140,6−8 but has the disadvantage of
poor solubility in aqueous buffer (9 μM). The aim of the work
reported here was to increase its solubility to enable in vivo
access to its pharmacological targets. We report the design and
synthesis of highly soluble derivatives of compound 1 that
behave as prodrugs9,10 converted into the active compound 1
and have in vivo anti-inflammatory activity at low doses upon
intranasal administration in a mouse model of asthma.
The phosphate derivative 2 was obtained by phosphorylation
of the phenol group with dibenzylphosphonate in CCl4, followed
by benzyl removal in the presence of trimethylsilylbromide (37%
overall yield).13 An alternative one-pot procedure involved
phosphorylation of the 4-hydroxyphenyl in the presence of
phosphorus oxichloride, followed by basic hydrolysis to obtain the
disodium phosphate analogue with a 45% yield.14 The L-serine
conjugate of compound 1 was synthesized in two steps. First, an
ester was prepared with Boc-L-Ser(tBu)-OH in the presence of 1-
(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDCI) and a
catalytic amount of N,N-dimethyl-4-aminopyridine (4-DMAP).
Subsequent deprotection in acidic media provided the expected
prodrug 4 as the hydrochloride salt, with a 70% overall yield.
The sulfate analogue 5 was obtained by direct sulfation of the
4′-hydroxyphenyl with a chlorosulfonic acid/pyridine mixture and
then treatment with aqueous NaOH to obtain the sodium salt
(34%).15 The structure and purity of all compounds were
1
assessed by H, 13C NMR, high-resolution mass spectrometry
(HRMS), and RP-HPLC analyses.
Thermodynamic solubility was measured in an aqueous 4-
(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer
(pH 7.4, 22 °C). As expected, the solubility of the phosphate 2
was very high, so high that saturation was not achieved at a 30 mM
The requirements for suitable prodrugs of compound 1 were
(1) inclusion of a functional group enzymatically cleavable in
vivo,11 (2) improved aqueous solubility to facilitate in vivo in-
tranasal administration, and (3) rapid and efficient synthesis to
allow large-scale preparation. To fulfill these requirements, we
decided to introduce three types of functional groups on the
Received: April 21, 2011
Accepted: December 9, 2011
Published: December 9, 2011
© 2011 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Prodrugs 2, 4, and 5 Preparation from Compound 1
a
Reagents: (i) (a) POCl3 (3 equiv), Et3N (10 equiv), CH2Cl2, room temperature, overnight; (b) aqueous NaOH. (ii) Boc-Ser(tBu)-OH (1.5 equiv),
DMAP (0.1 equiv), EDCI (1.5 equiv), CH2Cl2, room temperature, 1 h. (iii) 4 N HCl/dioxane, 60 °C, 4 h. (iv) (a) Chlorosulfonic acid (10 equiv),
pyridine, room temperature, overnight; (b) aqueous NaOH.
Table 1. Characterization of Compounds 1, 2, 4, and 5
d
stability half-life (T1/2
)
a
b
c
e
compd
isolated yield (%)
CHI
Log D calcd
solubility (μM)
PBS
murine serum
lung homogenate
binding inhibition (%)
90 10 (Ki = 53 31nM)
1
2
4
5
90
43
76
64
3.4
1.0
2.7
2.1
9
1
>10 h
>10 h
12 min
>10 h
>10 h
2 h
>6 h
40
70
34
>30000
<15 min
<5 min
30 min
5
2
42
3
<5 min
15
5
1434
3
>10 h
4
3
a
Determined by RP-HPLC14 (C18 Luna column, 2 mL/min, 365 nm, solvent A = pH 7.4 ammonium acetate buffer, solvent B = acetonitrile).
Calculated from CHI. Measured at pH 7.4, HEPES buffer, 24 h of incubation, 22 °C. 37 °C. Inhibition of CXCL12-TR binding to EGFP-
b
c
d
e
CXCR4 determined by FRET at 5 μM.
concentration. Its solubility is thus at least 3000 times higher
than the 9 μM solubility of compound 1. The solubility mea-
sured for the sulfate 5 under the same experimental conditions
was 1430 μM, that is, greater than that required for satisfactory
drug development. The yield of the serine 4 was better than
that of the other two compounds (70%), but its solubility was
only moderately better than that of compound 1 (4.6-fold; 42 μM
as compared to 9 μM).
The partition coefficients of compounds 1, 2, 4, and 5 at pH
7.4, expressed as Log D, are derived directly from measure-
ments of their chromatographic hydrophobicity index (CHI),
as compared with reference compounds.16 Log D decreased
from 3.4 for the original compound 1 to 2.7 for the serine 4, 2.1
for the sulfate 5, and 1.0 for the phosphate 2.
The stability of the four compounds was evaluated by LC-MS
in three media: phosphate buffer saline (PBS), mouse serum, and
mouse lung homogenates (Table 1). All experiments were
conducted with extemporaneously prepared solution. Com-
pound 1 at 10 μM is stable after incubation in all three media
(T1/2 > 6−10 h). The phosphate 2 showed good stability in
PBS, with 50% of it converted into 1 after 2 h of incubation in
mouse serum (Table 1 and Figure 1) and within less than 15
min in lung homogenates (Table 1). The serine ester 4 was
rapidly hydrolyzed into 1 with a T1/2 = 12 min in PBS; it dis-
appeared in less than 5 min in mouse serum and lung homo-
genate. This instability is the combined effect of the strong
electron withdrawing effect of the protonated aminogroup acti-
vating the ester linkage toward hydrolysis and intramolecular
assistance of this aminogroup, as already reported for α-aminoester
prodrugs.17 The sulfate 5 was stable in PBS and murine serum
(T1/2 > 10 h) but rapidly converted into 1 in lung homogenates
(T1/2 = 30 min). We therefore confirmed the high rate of
conversion of each prodrug candidate into 1 in lung homo-
genates (Table 1).
Figure 1. RP-HPLC chromatogram (C18 Luna column, 2 mL/min,
365 nm, solvent A = pH 7.4 ammonium acetate buffer, solvent B =
acetonitrile). Gradient: 0−0.2 min, 0% B; 0.2−2.7 min, 0−100% B;
2.7−3.2 min, 100% B; 3.2−3.4 min, 100−0% B; and 3.4−6.2 min, 0%
B. Positive ESI-MS spectrum of compound 2 at t0 and t180 at 37 °C in
mouse serum.
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In vitro inhibition of binding of Texas Red (TR)-labeled
CXCL12 (CXCL12-TR, 100 nM) to enhanced green fluorescent
protein (EGFP)-tagged CXCR4 by compounds 1, 2, 4, and 5 at 5
μM was determined by fluorescence resonance energy
transfer (FRET) (Table 1 and Figure 2) as previously described.3
and lymphocyte recruitment, although those were more moderate
(Figure 3). Because topical administration is a well-accepted and
preferred route for treatment of inflamed airways, especially in
asthma, we developed our model with intranasal administration
of the compounds in mice to mimic the inhalation route used
in humans.
We first investigated the activity of compound 1 and com-
pared it with that of phosphate 2, the most soluble compound,
after intranasal administration to the airways (2 h before each
challenge with OVA or saline on 3 consecutive days) (Figure 3A).
We evaluated the activity of 1 and 2 administered at doses of
22 nmol/kg, that is, the dose corresponding to the maximal
solubility of 1 in PBS (9 μM, 25 μL/mouse). The compounds
per se had no effect in the control unsensitized groups. In the
OVA-challenged mice, 1 had no effect on inflammatory cell
recruitment (Figure 3A). By contrast, the phosphate 2 signi-
ficantly inhibited OVA-induced eosinophil (56
9%) and
neutrophil recruitment, with a trend toward significance for ly-
mphocytes (Figure 3A). The dose−response activity of phos-
phate 2 on eosinophil recruitment showed an IC50 of 10 nmol/kg
(Figure 3B), thereby suggesting that the prodrug provides more
efficient distribution of compound 1 in the target tissue. Con-
trastingly, no effect of 2 was noticed on macrophage recruitment,
corroborating results on the activity of anti-CXCR4 or -CXCL12
neutralizing antibodies18 or CXCR4 antagonist.19 LC-MS analysis
shows that 1 appears in lung tissue as early as 15 min after
intranasal administration of 2, thus revealing its hydrolysis into
the active 1, thereby delivered at high concentrations to the lung.
The activity of the other two prodrugs, the serine 4 and
sulfate 5 derivatives, was compared to that of the phosphate 2
in the same airway hypereosinophilia mouse model. The intra-
nasally administered dose was 30 nmol/kg. The activity levels
of 4 and 5 shows similarity to that of 2 with significant
reduction of the recruitment of eosinophils (67, 69, and 64%
inhibition, respectively) and neutrophils by the three prodrugs
and at a lower level for lymphocytes, with no effect on macro-
phage recruitment (Figure 4). This clearly indicates that all
three prodrugs release the same amount of the active species 1
when administered to the airways. This finding is consistent
with the rapid release of 1 from the three different prodrugs in
lung homogenates in vitro (Table 1), its limited distribution in
Figure 2. Real-time monitoring of fluorescent CXCL12-TR to EGFP-
CXCR4 at 510 nm as a function of time. Upon addition of CXCL12-
TR (100 nM), the fluorescence intensity at 510 nm declines as a result
of interactions between CXCL12 and CXCR4. Compound 1 inhibits
binding of CXCL12-TR to EGFP-CXCR4, whereas prodrugs 2, 4, and
5 (5 μM) do not.
Compound 1, the reference compound, at 5 μM reduces this
binding by 90% with a Ki of 53 nM. Compounds 2, 4, and
5 are, as expected, almost inactive in this in vitro binding assay
(Figure 2). When serine was substituted in 4, prolonged
incubation times tended to inhibit CXCL12 binding to CXCR4.
The inadequate stability of 4 in buffer or serum (Table 1), due
to the progressive conversion of the inactive prodrug 4 into the
active 1, may explain this result.
The in vivo activity of the prodrugs was assessed in an 8 day
mouse model of airway hypereosinophilia in ovalbumin
(OVA)-sensitized mice challenged with OVA or control saline.
Eosinophil number in the bronchoalveolar lavage fluid in-
creased significantly after OVA challenge, as compared with saline,
in OVA-sensitized animals, as well as macrophage, neutrophil,
Figure 3. (A) Intranasal treatment with compounds 1 and 2 (22 nmol/kg in PBS) 2 h before each challenge in the 8 day mouse model of
hypereosinophilia. Blocks (means) and bars (SEM) of n = 6 mice/group. *p ≤ 0.05 as compared to saline-treated OVA group. (B) Dose−response
inhibition of eosinophil number of compound 2 (percent). Means (filled squares) and bars (SEM) of n = 6 mice/group.
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anhydrous CH2Cl2 (1.5 mL), and N,N-dimethyl-4-aminopyridine
(4-DMAP) (4 mg, 0.034 mmol) was added. To this solution was added a
solution of Boc-L-Ser(OtBu)-OH (136 mg, 0.52 mmol) and EDCI
hydrochloride (100 mg, 0.52 mmol) in anhydrous CH2Cl2 (1.5 mL), after
it was prepared, and stirred for 15 min at room temperature in the dark.
The combined mixture was stirred at room temperature in the dark for
1 h, the solvent was removed, and the crude product was purified by
chromatography on Si40 silica (3/7 ethyl acetate/heptane). Compound 3
was recovered as a pale yellow oil (170 mg, 92% yield).
For (R,E)-4-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)-2-methoxy-
phenyl 2-amino-3-hydroxypropanoate, hydrochloride salt 4, com-
pound 3 (51 mg, 0.096 mmol) was dissolved in a mixture of dioxane
and 4 N HCl aqueous solution (1/1 v/v, 510 μL). The mixture was
heated at 60 °C for 3 h. After completion, the precipitate was
recovered by filtration and triturated twice with ether. After it was
dried, 4 was obtained as a pale yellow solid (30 mg, 76% yield).
For (E)-4-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)-2-methoxy-
phenyl sulfate, sodium salt 5, under argon atmosphere, 1 (100 mg,
0.35 mmol) was dissolved in anhydrous pyridine (10 mL), and the
mixture was cooled to −10 °C. Chlorosulfonic acid (233 μL, 3.5 mmol)
was added dropwise (warning: violent reaction!). The mixture was
stirred at room temperature overnight. The solvent was then
evaporated under reduced pressure, and the residue was dissolved in
water and acidified to pH 2 by addition of 1 N HCl aqueous solution.
The mixture was extracted three times with CH2Cl2. The combined
organic layers were concentrated under reduced pressure. The residue
was dissolved in EtOH (1 mL) and alkalinized with 50% NaOH
aqueous solution (1 equiv). The solvent was evaporated under
reduced pressure at room temperature, and the crude oily residue was
purified by flash chromatography on Reverse-Phase 18 column (eluent
water-acetonitrile). Compound 5 was recovered as a yellow solid (46 mg,
34% yield).
After mice were exposed for 15 min to intranasally administered
compounds 2, 4, and 5, they were killed, and their lungs were col-
lected, homogenized, and solid−liquid extracted with acetonitrile.
After 3000g centrifugation, the supernatant was collected, evaporated,
and resuspended in 50% water−50% acetonitrile, 0.1% TFA before
injection. Analyses were conducted with RP-HPLC, as shown in
Figure 1, or LC-MS.
The activity of each compound was assessed in vivo in an 8 day
model of hypereosinophilia. Briefly, 9 weeks old male Balb/c mice
were sensitized by intraperitoneal injection of 50 μg of OVA (grade V,
Sigma-Aldrich) adsorbed on 2 mg of aluminum hydroxide (Sigma-
Aldrich) in 0.1 mL of saline on days 0, 1, and 2. Mice were challenged
intranasally [10 μg of OVA in 25 μL of saline (12.5 μL/nostril)] on
days 5, 6, and 7. Control mice received intraperitoneal and intranasal
administrations of saline alone. Intranasal administrations were
performed under anesthesia with intraperitoneal 50 mg/kg ketamine
and xylazine at 3.33 mg/kg. Food and water were supplied ad libitum.
Animal experimentation was conducted with the approval of the
government body that regulates animal research in France. Two hours
before each OVA or saline challenge, compound 1 or 2, 4, or 5 in PBS
(25 μL) was administered intranasally.
Figure 4. Intranasal administration of compounds 2, 4, and 5
(30 nmol/kg) 2 h before each challenge in a hypereosinophilic mouse
model. Absolute numbers of cells in BAL are shown. Means (blocks)
and SEM (bars) of n = 6 mice/group. *p ≤ 0.05 as compared to OVA.
vivo, and efficient trapping of the pro-inflammatory chemokine,
CXCL12, locally in the inflamed lung tissue.
Three analogues of compound 1, a neutraligand of the CXC
chemokine CXCL12, bearing a phosphate, an L-seryl, and a
sulfate moiety, are highly soluble and bioavailable when admini-
stered to the airways. They behave as prodrugs, remaining
inactive until compound 1 is released. Intranasal application of
each of these three analogues inhibits eosinophil recruitment in
the airways by ≥50% at a dose as low as 30 nmol/kg and
without any signs of toxicity. This prodrug strategy, which
results in a low-dose application for topical effect, therefore,
appears to be a powerful strategy for obtaining highly soluble
compounds that act directly in the airways and promote the
desired local action while avoiding systemic adverse effects on
other CXCL12-related functions.
EXPERIMENTAL PROCEDURES
■
For (E)-4-(3-(4-chlorophenyl)-3-oxoprop-1-enyl)-2-methoxyphenyl-
phosphate, sodium salt 2, under argon atmosphere, POCl3 (186 μL,
2 mmol) was dissolved in anhydrous CH2Cl2 (8 mL); the mixture was
then cooled to 0 °C, and anhydrous triethylamine (700 μL, 5 mmol)
was added. After 5 min of stirring, compound 1 (114 mg, 0.39 mmol
dissolved in 1 mL of anhydrous CH2Cl2) was added dropwise to the
reaction mixture. The resulting mixture was stirred for 1 h at 0 °C and
then allowed to warm to room temperature overnight. The crude
mixture was then evaporated to dryness under reduced pressure to
remove the excess NEt3 and POCl3. The residue was dissolved in
CH2Cl2 and evaporated to dryness again. The procedure was repeated
twice. Finally, the residue was dissolved in a tetrahydrofuran−water
solution (2/1 v/v, 1.5 mL), and the mixture was vigorously stirred for
1 h. The solution was diluted with water (500 μL) and cooled down to
0 °C. A 0.1 N, NaOH aqueous solution was added dropwise to reach a
pH of 12. After concentration under reduced pressure, compound 5
was isolated by flash chromatography on an RP18 column, with a
linear gradient of acetonitrile in water. Following a freeze/drying step,
compound 2 was recovered as a yellow solid (74 mg, 45% yield).
For (R,E)-4-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)-2-methoxy-
phenyl-3-(tert-butoxy)-2-(tert-butyloxycarbonylamino)-propanoate 3,
under argon atmosphere, 1 (100 mg, 0.35 mmol) was dissolved in
Bronchoalveolar lavage (BAL) was performed 24 h after the last
OVA challenge. Mice were deeply anesthetized by intraperitoneal
injection of 150 mg/kg ketamine and 10 mg/kg xylazine. A plastic
canula was inserted into the trachea, and airways were lavaged by 10
instillations of 0.5 mL of ice-cold saline supplemented with 2.6 mM
ethylenediaminetetraacetic acid (EDTA) (saline-EDTA). BAL fluids
were centrifuged (300g, 5 min, 4 °C) to pellet cells, and erythrocytes
were lysed by hypotonic shock. Cells were resuspended in 500 μL of
ice-cold saline-EDTA, and total cell counts were determined with a
hemocytometer (Neubauer's chamber). Differential cell counts were
assessed on cytologic preparations (Cytospin, Shandon Ltd.) (250 000
cells/mL in ice-cold saline-EDTA) stained with Hemacolor (Merck)
on counts of at least 400 cells and expressed as absolute numbers or
percentage of total cell number.
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Letter
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Detailed synthesis and full characterization of compounds 1, 2,
4, and 5, the solubility and stability determinations, and the in
vitro binding experiments. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
(10) Huttunen, K. M.; Raunio, H.; Rautio, J. Prodrugs-from
Serendipity to Rational Design. Pharmacol. Rev. 2011, 63, 750−771.
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Corresponding Author
*Tel: +33368854232. Fax: +33368854310. E-mail: mhibert@
unistra.fr.
Funding
This work was supported by the French “Minister
Recherche”, ANR, CNRS, INSERM, Region Alsace, and Strasbourg
University.
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́
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We warmly thank Step
́
hanie Riche
́
for her continuous
involvement in this work. We thank Pascale Buisine and
Patrick Wehrung for MS analyses and Cyril Antheaume for
NMR analyses. We thank Pascal Villa for screening.
ABBREVIATIONS
■
BAL, bronchoalveolar lavage; CHI, chromatography hydro-
phobicity index; 4-DMAP, N,N-dimethyl-4-aminopyridine;
EDCI, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide;
EDTA, ethylenediaminetetraacetic acid; EGFP, enhanced
green fluorescent protein; FRET, fluorescence resonance
energy transfer; HEPES, 4-(2-hydroxyethyl)-1-piperazine etha-
nesulfonic acid; HRMS, high-resolution mass spectrometry;
OVA, ovalbumin; SEM, standard error of the mean; TR, Texas
Red; Tris, 2-amino-2-hydroxymethyl-1,3-propanediol
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