2-Arylpropionic CXC chemokine receptor 1 (CXCR1) ligands as novel noncompetitive CXCL8 inhibitors

4312

J. Med. Chem. 2005, 48, 4312-4331

2-Arylpropionic CXC Chemokine Receptor 1 (CXCR1) Ligands as Novel

Noncompetitive CXCL8 Inhibitors

Marcello Allegretti,*^,†Riccardo Bertini,^†Maria Candida Cesta,^†Cinzia Bizzarri,^†Rosa Di Bitondo,^†

Vito Di Cioccio,^†Emanuela Galliera,^‡Valerio Berdini,^†Alessandra Topai,^†Giuseppe Zampella,^§Vincenzo Russo,^|

Nicoletta Di Bello,^†Giuseppe Nano,^†Luca Nicolini,^†Massimo Locati,^‡Piercarlo Fantucci,^§Saverio Florio,^|and

Francesco Colotta^†

Dompe´ Research and Development, Dompe´ S.p.A., via Campo di Pile, 67100, L’Aquila, Italy, Centro IDET, Institute of General

Pathology, University of Milan, 20133 Milan, Italy, Department of Biotechnologies and Biosciences, University of Milans

Bicocca, 20122, Milan, Italy, and C.I.N.M.P.I.S. PharmacysChemistry Department, University of Bari, 70125 Bari, Italy

Received November 15, 2004

The CXC chemokine CXCL8/IL-8 plays a major role in the activation and recruitment of

polymorphonuclear (PMN) cells at inflammatory sites. CXCL8 activates PMNs by binding the

seven-transmembrane (7-TM) G-protein-coupled receptors CXC chemokine receptor 1 (CXCR1)

and CXC chemokine receptor 2 (CXCR2). (R)-Ketoprofen (1) was previously reported to be a

potent and specific noncompetitive inhibitor of CXCL8-induced human PMNs chemotaxis. We

report here molecular modeling studies showing a putative interaction site of 1 in the TM

region of CXCR1. The binding model was confirmed by alanine scanning mutagenesis and

photoaffinity labeling experiments. The molecular model driven medicinal chemistry optimiza-

tion of 1 led to a new class of potent and specific inhibitors of CXCL8 biological activity. Among

these, repertaxin (13) was selected as a clinical candidate drug for prevention of post-ischemia

reperfusion injury.

Introduction

bisarylurea derivatives.^18,19N-(2-Bromophenyl)-N′-(2-

hydroxy-4-nitrophenyl)urea (SB225002), the first lead

compound in this class, is >150-fold selective for CXCR2

compared to CXCR1 in a binding assay. In vitro,

SB225002 potently inhibits human and rabbit neutro-

phil chemotaxis induced by CXCL8 and CXCL1 and in

vivo proved to be efficacious in inhibiting CXCL8-

induced PMN margination in rabbits.¹⁸Although

SB225002 is the most thoroughly described CXCL8

inhibitor in the literature, additional candidates with

an improved pharmacokinetic and pharmacodynamic

profile are currently under preclinical investigation.^19,20

We previously reported that both enantiomers of

ketoprofen, a widely used nonsteroidal antiinflamma-

tory drug (NSAID), are potent and selective inhibitors

of CXCL8-induced human PMN chemotaxis not affect-

ing chemokine receptor binding. Interestingly, this

unprecedented biological activity of ketoprofen was

clearly not related to the inhibition of the cyclooxy-

genase (COX) pathway.²¹In this paper, we describe the

identification of an allosteric interaction site between

(R)-ketoprofen (1) and CXCR1. Moreover, medicinal

chemistry driven by the molecular model of interaction

between (R)-ketoprofen (1) and CXCR1 led to a novel

class of potent and specific inhibitors of CXCL8 biologi-

cal activity. Results are consistent with a mechanism

of action whereby inhibitors act as noncompetitive

allosteric blockers interacting with the TM region of

CXCR1, locking this receptor in an inactive conforma-

tion unable to activate intracellular signal transduction.

Chemokines or “chemotactic cytokines” are a class of

8-14 kDa proteins that regulate the trafficking of

leukocytes¹and other biological processes including cell

growth, angiogenesis, and hematopoiesis.^2-4Chemo-

kine receptors belong to the superfamily of seven-

transmembrane G-protein-coupled receptors (7-TM

GPCRs) and, specifically, to the class A (rhodopsin-like)

GPCR family.⁵Disregulation of the chemokines network

has been implicated in a variety of diseases including

rheumatoid arthritis (RA), chronic obstructive pulmo-

nary disease (COPD), asthma, Alzheimer’s disease,

melanoma, and psoriasis.^6-10

CXCL8 belongs to the CXC chemokines family and

plays a major role in the activation and recruitment of

neutrophils.¹¹CXCL8 activates the receptors CXCR1

and CXCR2 expressed on human neutrophil surface,

whereas the closely related chemokine CXCL1 (GRO-

R) is a selective agonist for CXCR2.¹²The role of CXCL8

in acute and chronic human diseases has been recently

reviewed.¹³In particular, CXCL8 has been implicated

in polymorphonuclear (PMN) recruitment and activa-

tion in post-ischaemia reperfusion in which neutralizing

anti-CXCL8 antibodies and high molecular weight

antagonists prevented PMN infiltration and the associ-

ated tissue injury.^14,15To date, only a limited number

of small molecular weight (SMW) CXCL8 inhibitors

have been described.^16,17The main class of SMW CXCL8

inhibitors so far reported in the literature is a class of

* To whom correspondence should be addressed. Phone: +39-0862-

338422. Fax: +39-0862-338219. E-mail: marcello.allegretti@dompe.it.

^†Dompe´ Research and Development.

Results

(R)-Ketoprofen (1) Inhibits CXCL8-Induced G-

Protein Activation. We previously reported that (R)-

and (S)-ketoprofen (1 and 2) are potent and specific

^‡University of Milan.

^§University of MilansBicocca.

^|University of Bari.

Published on Web 05/27/2005

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4313

Table 1. Effect of 2-Arylpropionic Acids on CXCL8-Induced

Human PMN Chemotaxis^a

Figure 1. (R)-Ketoprofen 1 inhibits G-protein activation

induced by CXCL8. The binding of [³⁵S]GTPγS to PMN

membranes stimulated with CXCL8 (300 nM) in the presence

or absence of 1 (1 µM) was measured. Results are shown as

the percentage of induction of [³⁵S]GTPγS binding relative to

unstimulated PMN membranes. Values are the mean ( SD

of two independent experiments: (//) P < 0.01 vs GTP binding

in the absence of 1 (Student’s t test).

inhibitors of CXCL8-induced chemotaxis of human

PMNs in a COX-independent fashion. We sought to

elucidate the mechanism of action of ketoprofen enan-

tiomers. Since 1 and 2 did not affect CXCL8 receptor

binding,²¹we postulated that these molecules might

affect intracellular signal trasduction activated by

CXCL8 receptors. As shown in Figure 1, 1 significantly

inhibited CXCL8-induced activation of the G-protein.

Consistently, signal transduction events downstream to

G-protein activation were also inhibited in cells acti-

vated with CXCL8 in the presence of 1 and 2, including

inhibition of intracellular calcium increase and extra-

cellular signal regulated kinase (ERK)-1/2 activation.²¹

Molecular Modeling Studies. Having found that

1 significantly inhibits CXCL8-induced G-protein acti-

vation, we reasoned that 1 might interact with the

CXCL8 receptor CXCR1, preventing signal transduction

while leaving CXCL8 binding unaffected. The finding

that, in addition to ketoprofen, additional 2-arylpropi-

onic acid NSAIDs inhibit CXCL8 activity (Table 1)

suggested that these molecules might interact with a

homologous binding site in both COX and CXCR1. A

binding model for 1 in COX-1 was built using as a

template the known crystallized structure of the enzyme

in complex with S-flurbiprofen (Figure 2a). S-Flurbi-

profen, as other phenylpropionic NSAIDs, coordinate a

network of polar interactions involving Arg120, Glu254,

Tyr355, and Hys90 at the bottom of a hydrophobic

channel of COX, thus blocking the active site of the

enzyme.^22,23PASS analysis of the TM region of CXCR1,

followed by GRID mapping of the solvent accessible

sites, allowed us to select potential ketoprofen binding

sites and to compare their physicochemical properties

with the COX channel.^24-26In the description of the

binding hypothesis, the Weinstein-Ballesteros number-

ing will be used to emphasize the relative positions of

the CXCR1 TM residues in the helix bundle. A putative

ketoprofen binding site was identified in a cavity

between helices 1, 2, 3, 6, and 7 (Figure 2b). The overall

pattern of interactions is well conserved in the binding

models of 1 with both COX and CXCR1. In both cases,

the inhibitor is well-anchored at the bottom of a

^a(/) % of inhibition at 10^-8M. (//) % of inhibition at 10^-5M.

Figure 2. Comparison between the (R)-ketoprofen 1 binding

mode in COX-1 and the putative CXCR1 allosteric site: key

polar interactions engaged by the carboxylic acid. (a) Binding

model of 1 (green) in the COX-1 (yellow) binding site is shown.

The carboxylic group of the inhibitor is involved in a strong

electrostatic interaction (dashed lines) with the positively

charged guanidinium group of Arg120. The inhibitor bridges

the side chains of Tyr355 and Glu524 in a well-organized polar

network. (b) Orientation of 1 (green) in the putative CXCR1

(light-blue) binding site is shown. In the proposed model an

electrostatic interaction occurs between the hydroxyl group of

Tyr46_1.39and the aromatic ring of the inhibitor. Two additional

polar interactions (dashed lines) orientate the side chains of

two polar residues from TM2 (Lys99_2.64) and TM7 (Glu291_7.35).

The overall interactions pattern of 1 with CXCR1 shows high

similarity with the COX-1 binding mode.

hydrophobic channel where the carboxylic moiety en-

gages a strong ionic bond with a basic amino acid

residue (Arg120 of COX-1 and Lys99_2.64of CXCR1). This

ionic bond is additionally strengthened by an additional

4314 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

polar interaction involving the carbonyl group and a

hydrogen bond donor residue (Tyr355 of COX-1 and

Glu291_7.35of CXCR1). Moreover, the benzoyl group is

accommodated in a well-organized hydrophobic pocket

in COX-1 and CXCR1. This hydrophobic pocket in

CXCR1 involves at least the lipophilic side chains of

Ile43TM1_1.36, Val113TM3_3.28, and Ile283TM7_7.31and the

aliphatic chain of Lys99TM2_2.64. According to the model,

Lys99TM2_2.64is proposed to establish both polar and

hydrophobic interactions with the inhibitor. Finally, in

the CXCR1 binding model, a specific electrostatic in-

teraction apparently directs the phenolic group of

Tyr46_1.39of CXCR1 toward the phenyl ring of 1. This

kind of interaction, involving the π ring system as

hydrogen bond acceptor and the hydroxyl group of

Tyr46_1.39as the hydrogen bond donor, belongs to the

family of weak noncovalent interactions.²⁷OH- π

interactions have been observed in high-resolution

protein structures, and they are important for the

stability of protein structures.²⁸Molecular dynamics

calculations led to the identification of this interaction

as a key feature of the binding mode; nevertheless, the

arrangement of the groups is also compatible with a

CH-π interaction that could be underestimated by the

force field. Specific mutagenesis studies on the nature

of this interaction are still running, but the alanine

scanning mutagenesis studies confirm the relevance of

the interaction between Tyr46_1.39and the inhibitor.

Figure 3. Effect of (R)-ketoprofen 1 on CXCL8-induced

migration of L1.2 transfectants expressing wild-type CXCR1,

mutated Lys99Ala CXCR1, and Glu291Ala. Cell migration was

induced on L1.2 transfectants expressing wild-type CXCR1 (O),

mutated Lys99Ala (0), and Glu291Ala (4) by CXCL8 (10 nM)

in the presence or absence of increasing concentrations of 1.

Effect of 1 on L1.2 untransfected cell migration in response to

CXCL12/SDF1 is also shown. Results are expressed as the

percentage of migration in the absence of 1. Values are the

mean ( SD of three independent experiments: (/) P < 0.05

vs cell migration in the absence of 1; (//) P < 0.01 vs cell

migration in the absence of 1 (Mann-Whitney U test).

Alanine Scanning Mutagenesis of the Interac-

tion Site between (R)-Ketoprofen (1) and CXCR1.

To test the above binding model of 1 with CXCR1, amino

acids of CXCR1 supposed to be directly involved in the

binding of 1 were selected for alanine-replacement

mutagenesis experiments. The alanine-replacement

mutants Lys99Ala and Glu291Ala were expressed in

L1.2 cells. 1 was tested in a wide range of concentrations

(0.1-100 nM) known to affect CXCL8-induced PMN

chemotaxis.²¹Wild-type CXCR1/L1.2 transfectants mi-

gration induced by CXCL8 (10 nM) was significantly

inhibited by 1 (Figure 3), as expected. The inhibition of

wild-type CXCR1/L1.2 transfectant migration was con-

centration-dependent, reaching a maximal inhibition

(74%) at 0.1 µM 1. In contrast, cells expressing the

Lys99Ala CXCR1 mutant completely resisted the action

of 1 while the Glu291Ala CXCR1 mutant had partial

resistance to 1. Receptor expression levels, CXCL8

binding affinity, and chemotactic migration to CXCL8

of the Lys99Ala CXCR1 transfectant were similar to

those of the wild-type CXCR1/L1.2 transfectant (data

not shown). The finding that CXCL8 binding to mutated

CXCR1 is similar to wild-type CXCR1 is in keeping with

the concept that the interaction site between 1 and

CXCR1 is not involved in CXCL8 binding.

Binding of (R)-Ketoprofen (1) to CXCR1 Is Abol-

ished in Lys99Ala CXCR1 Mutant. To provide ad-

ditional evidence that the interaction between 1 and the

amino acid Lys99_2.64is crucial for CXCR1 binding, we

took advantage of the ability of 1 to be photoactivated²⁹

in order to generate a covalent complex with the target

protein. To this aim, wild-type CXCR1/L1.2 and mu-

tated Lys99Ala CXCR1 cell membranes were incubated

with [¹⁴C]-1 and irradiated. The immunoprecipitated

CXCR1 was examined by SDS-PAGE. Radiolabeled

[¹⁴C]-1 binds to wild-type CXCR1/L1.2 membranes. In

Figure 4. Binding of (R)-ketoprofen 1 on CXCR1 is abolished

in the Lys99Ala CXCR1 mutant. Membrane preparations from

L1.2 transfectants expressing wild-type CXCR1 or Lys99Ala

CXCR1 mutant were incubated with [¹⁴C]-1. After photochemi-

cal cross-linking, CXCR1 was immunoprecipitated and ana-

lyzed by SDS-PAGE and Western blot, as indicated. The

arrow marks CXCR1. Data are from one experiment of two

performed.

contrast, binding of 1 to Lys99Ala CXCR1 mutant

membranes was completely abolished (Figure 4). These

data further support the above-described model of

interaction between 1 and CXCR1.

Chemistry. The compounds described in this study

are shown in Tables 1-7, and their synthetic methods

are outlined in Schemes 1-6.

The synthesis of acylsulfonamides (11-27) was car-

ried out according to a disclosed procedure³⁰starting

from chiral or racemic 2-phenylpropionic acids 1-10.

Following this method the carboxylic acid was coupled

to the selected alkyl or arylsulfonamide using 1,1′-

carbonyldiimidazole in the presence of a base at room

temperature. The trifluoromethanesulfonamide 28 was

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4315

Scheme 1^a

compounds 45-47, whereas treatment of 110 and 111

with BBr₃afforded compounds 48 and 49.

The synthesis of 2-arylpropionic acids was performed

following several methods depending on the different

substitution pattern of the aromatic ring. The choice of

different methods was guided by the commercial avail-

ability of the reagents to derivatize trifluoromethane-

sulfonate intermediates. Simple 4-alkyl derivatives 77

and 78 were prepared by Friedel-Crafts acylation of

the corresponding alkylbenzene. The following Willgerodt

rearrangement³¹of the intermediate ketone and ester

hydrolysis afforded the desired compounds (Scheme 3).

Treatment of the methyl ester of the commercial 2-(4-

hydroxyphenyl)propionic acid (84) with trifluoromethane-

sulfonic anhydride allowed us to obtain intermediate

117, which was transformed into the compounds 80, 81,

85, and 86 by reaction with the appropriate alkylzinc

or arylzinc bromide and following methyl ester basic

hydrolysis. Stille coupling of 117 with the appropriate

organotin reagent afforded compound 79. Alkylation of

the potassium salt of the above methyl ester by alkyl

iodide and hydrolysis afforded 4-alkoxy derivatives 82

and 83 (Scheme 4). Starting from the 3-hydroxyphenyl-

acetic acid, intermediate 119 was prepared (Scheme 5)

as described for 117. Compound 95 was obtained from

119 as described above for 81. When the same inter-

mediate and the appropriate alkyltin reagents were

used, compounds 88 and 89 were obtained after basic

hydrolysis, while the reaction with triisopropyl alumi-

num gave compound 91. The classical Stille procedure

for the coupling of trifluoromethanesulfonates with

alkenyltributyltin reagents³²was followed for the syn-

thesis of unsaturated intermediates 120-124 that, after

hydrogenation on Pd/C and hydrolysis, afforded the final

compounds 90, 92, 93, and 96. By hydration of the

double bond of intermediate 123 and hydrolysis, com-

pound 97 was obtained. Direct hydrolysis of intermedi-

ate 123 afforded compound 94. Finally, Scheme 6

outlines the preparation of compounds 98-100. The

direct acidic hydrolysis of the commercial 2-(3-carboxy-

phenyl)propionic acid afforded compound 100. The

treatment of 2-(3-carboxyphenyl)propionic acid with

Meldrum’s acid sodium salt allowed us to isolate the

intermediate 125, which after acidic hydrolysis yielded

^aConditions: (a) CDI, DBU, CH₂Cl₂, R′SO₂NH₂; (b) SOCl₂,

reflux; (c) CF₃SO₂NH₂, TEA, room temp; (d) NH₂NH₂, EtOH,

reflux.

prepared by direct coupling of the corresponding acid

chloride to commercial trifluoromethanesulfonamide in

the presence of triethylamine (Scheme 1).

The synthesis of (R)-2-(4-isobutylphenyl)propionyl

amides 29-35 and 37-71 was performed as described

in Scheme 2. Most amines were commercially available;

the others were prepared according to known proce-

dures, as described in the Experimental Section. (R)-2-

(4-Isobutylphenyl)propionic acid ((R)-ibuprofen) 3 was

coupled to the corresponding amines by two different

strategies. According to the first route, 3 was treated

with thionyl chloride at reflux to give the acid chloride,

which was reacted with the desired amine in the

presence of triethylamine at room temperature to give

the final compounds 29-35, 43, 44, 54, 55, 58, 59, 62-

64, 68-71 and the intermediates 112 and 114. The

acidic hydrolysis of 112 and 114 by 3 M HCl afforded

compounds 65 and 66 in high yield. Treatment of

compound 62 with iodomethane yielded the correspond-

ing quaternary ammonium salt 67. Treatment of com-

pounds 58 and 59 with diazomethane yielded the

corresponding ethers 60 and 61. Alternatively, the direct

coupling between the acid 3 and amines with 1,3-

dicyclohexylcarbodiimide and 1-hydroxybenzotriazole

was performed. Following this procedure, the final

compounds 37-42, 50-53, 56, 57 and the intermediates

109-111 were obtained in good to high yield. The basic

hydrolysis in nonracemizing conditions (1 equiv of 1 N

NaOH in 1,4-dioxane) of 50, 51, and 109 afforded

Scheme 2^a

^aConditions: (a) SOCl₂, reflux; (b) RNH₂, TEA, room temp; (c) DCC, HOBT, TEA, DMF, room temp; (d) 1 N NaOH, 1,4-dioxane, room

temp; (e) 3 M HCl, room temp; (f) CH₃I, THF, room temp; (g) BBr₃, CH₂Cl₂, -10 °C; (h) CH₂N₂, Et₂O, room temp.

4316 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

Scheme 3^a

CXCL8 inhibitors with a marked selectivity (2 logs)

versus the classical COX inhibition. On the other hand,

(R)-enantiomers of 2-phenylpropionic acids have been

reported to undergo chiral inversion in vivo in several

animal species.³³Although the interconversion of 1 to

2 was not observed in humans, significant interconver-

sion to 2 was detected when 1 was administered to rats

and dogs.³⁴Since in vivo generated (S)-2-phenylpropi-

onic acids do have COX-related antiinflammatory activ-

ity, it was important to design novel selective phenyl-

propionic derived CXCL8 inhibitors.

To this aim, on the basis of the above-discussed

binding model, acylmethanesulfonamides, because of

their fairly high acidity, were evaluated as potential

bioisosteres of the carboxylic acid resistant to in vivo

interconversion. The chiral inversion is an enzymatic

process that mainly occurs in the liver and involves the

formation of a coenzyme A thioester intermediate.³⁵

Thus, we hypothesized that the enzymatic inversion of

metabolically stable carboxylic derivatives should be

disallowed. The CXCL8 inhibitory activity was well-

conserved when phenylpropionic acids of Table 1 were

converted to (R)-2-arylpropionylmethanesulfonamides

(Table 2). By contrast, the same methanesulfonamides

were found to be considerably less potent than the

corresponding carboxylic acids as COX inhibitors. In

^aConditions: (a) AlCl₃, ClCOCH₂CH₃, CH₂Cl₂, reflux; (b)

AgNO₃, I₂, CH(OCH₃)₃, MeOH, room temp; (c) NaOH, MeOH,

reflux.

compound 99. The reduction with NaBH₄to intermedi-

ate 126 and the following hydrolysis afforded compound

98. Starting from the commercial acid 87 and following

the same procedure described above, compound 101 was

also prepared in good yield. Starting from selected

arylpropionic acids, compounds 102-108 were synthe-

sized following the above-described procedures.

Structure-Activity Relationship Studies. (R)-2-

Phenylpropionic acids 1 and 3 were found to be potent

Scheme 4^a

^aConditions: (a) concentrated H₂SO₄, MeOH, room temp; (b) ⁱPr₂EtN, (CF₃SO₂)₂O, CH₂Cl₂, -15 °C; (c) RZnBr, Pd(PPh₃)₄, LiCl, THF;

(d) KOH, MeOH, room temp; (e) RI, K₂CO₃, acetone, room temp; (f) Bu₃SnR′, Pd₂dba₃, AsPh₃, LiCl, CuI, NMP, 90 °C.

Scheme 5^a

^aConditions: (a) concentrated H₂SO₄, MeOH, room temp; (b) ⁱPr₂EtN, (CF₃SO₂)₂O, CH₂Cl₂, -15 °C; (c) 60% NaH, CH₃I, THF, -25 °C;

(d) SnR₄, Pd₂dba₃, AsPh₃, LiCl, CuI, NMP, 90 °C; (e) AlR₃, Pd(PPh₃)₄, THF, reflux; (f) Bu₃SnR′, Pd₂dba₃, AsPh₃, LiCl, CuI, NMP, 90 °C;

(g) H₂, 10% Pd/C, EtOH; (h) KOH, MeOH, room temp; (i) (1) BH₃, THF, 0 °C; (2) H₂O₂, NaOH; (l) RZnBr, LiCl, Pd(PPh₃)₄, THF, reflux.

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4317

Table 3. (R)-2-(4-Isobutylphenyl)propionylsulfonamides^a

Scheme 6^a

^aConditions: (a) SOCl₂, reflux; (b) (1) Meldrum’s acid sodium

salt, pyridine, CH₂Cl₂, 0-5 °C; (2) AcOH/H₂O, reflux; (c) concen-

trated HCl, 1,4-dioxane, reflux; (d) NaBH₄, THF/H₂O, room temp.

^a(/) % inhibition at 10^-8M. n.a.: compound not active at 10^-6

M.

Table 2. Effect of 2-Arylpropionylmethanesulfonamides on

at 10^-5M. On the basis of the bioisosterism hypothesis,

derivatization of inactive phenylpropionic/acetic acids

of Table 1 to acylmethanesulfonamides was expected to

afford inactive compounds. Thus, it was surprising to

find that acylmethanesulfonamide 18 was much more

potent than the parent tiaprofenic acid 8. Compound 8

shows a close structural similarity with ketoprofen but,

interestingly, is significantly more acidic than the other

phenylpropionic acids of Table 1.³⁶The high potency of

the corresponding methanesulfonamide 18 suggests that

the pK_adecrease disfavors the interaction with CXCR1.

Under the assumption that the carboxylic group engages

in an ionic interaction with Lys99_2.64and hydrogen

bonding with Glu291_7.35, it is conceivable that the

protonation of the ꢀ-amino group of Lys99_2.64occurs in

the TM pocket of CXCR1.

CXCL8-Induced Human PMN Chemotaxis^a

To gain further insight on the SAR, we decided to

change the nature of the polar group (Table 3). (R)-

Ibuprofen 3 was chosen as a scaffold because of its

intrinsic low potency in the PGE production assay. First,

we changed the nature of the sulfonamide residue to

yield a set of analogues of 13. The size of the alkyl

substituent of the sulfonamide strongly affects the

potency of the inhibitors (21, 22), and the introduction

of phenyl or alkylamino groups resulted in complete

activity loss (24-27). Replacement of the methyl by a

trifluoromethyl group has only minimal effect on the

steric hindrance while strongly enhancing the acidic

properties of the molecule. The lack of activity of

trifluoromethanesulfonamide 28 supports the proposed

model in which the inhibitor binds CXCR1 in the

protonated form.

On the basis of these SAR data, we decided to move

from acidic to neutral amide compounds designed to

^a(/) % of inhibition at 10^-8M. (//) % of inhibition at 10^-5M.

n.a.: compound not active at 10^-6M. n.t.: not tested.

engage a double hydrogen bond with the Lys99_2.64

/

particular, 13 inhibited CXCL8-induced chemotaxis

with IC₅₀) 1 nM while it did not affect lipopolysaccha-

ride (LPS) induced prostaglandin E (PGE) production

Glu291_7.35couple. This modification was supposed to

increase the CXCL8/COX selectivity. Results are sum-

marized in Table 4. The acidic hydroxamic analogue 29

4318 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

Table 4. Effect of (R)-2-(4-Isobutylphenyl)propionylamides on CXCL8-Induced Human PMN Chemotaxis^a

^a(/) % of inhibition at 10^-8M. (//) % of inhibition at 10^-5M. n.a.: compound not active at 10^-6M. n.t.: not tested.

is approximately as potent as the methanesulfonamide

13 but, as expected, retains significant COX inhibitory

activity. Interestingly, several neutral propionamides

potently inhibited CXCL8, and the primary amide 31

was one of the most potent CXCL8 inhibitors in this

A model for the binding of 31 in CXCR1 was built

using the same procedure as described for 1. The polar

interactions engaged by 31 in the TM pocket are

represented in Figure 5a. The effect of 31 on the CXCL8-

induced chemotaxis of CXCR1/L1.2 transfectant and

Lys99Ala and Glu291Ala mutants confirms that the

neutral amide 31 binds CXCR1 in the same region of

the carboxylic acid 1 (Figure 5b). Interestingly, whereas

the Lys99Ala mutant was found to be completely

resistant to the action of 1, both Lys99Ala and Glu291Ala

mutants similarly showed partial resistance to the

action of 31. This result is in keeping with a shift from

an ionic bond to a double hydrogen bond interaction

between the amide moiety of 31 and the side chains of

Lys99_2.64and Glu291_7.35in the receptor site.

class. Furthermore, the lack of effect of 31 at 10^-5

M

on PGE production confirmed the hypothesis on selec-

tivity. A considerable loss in affinity was generally

observed with N-alkyl-substituted secondary amides

(32, 33).

The lack of activity of N,N-dialkylamides 34 and 35

and of the methyl ester 36 is in agreement with a

binding mode in which both CO and NH groups replace

the carboxylic moiety in the interaction with Lys99_2.64

and Glu291_7.35

.

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4319

N-Phenylamide, like the N-alkyl analogues, showed

only moderate affinity for the receptor. Conversely, the

insertion of a heteroatom in the 2-position (with respect

to the carbon bearing the amino group) of the aromatic

ring dramatically increased the inhibitory potency. A

plausible explanation for SAR in the series of N-

arylamides is that an increase in the π-bond character

of the C-N bond results in a significant potency

enhancement. Indeed, the presence of a heteroatom with

basic or hydrogen bond acceptor characteristics could

determine, in the most active compounds (38, 41-44),

a significant dextral shift of the amido/imido equilibri-

um, thus tuning the binding ability of the amido group.

Unfortunately compounds 38, 41, and 42 retained a

remarkable inhibitory effect on COX activity and thus

were not selected for further in vivo characterization.

An additional objective of our work was the selection of

candidates with high water solubility suitable for

parenteral administration. To this aim, we introduced

several functional groups with hydrophilic characteris-

tics on the nitrogen atom of 31. The results reported in

Table 4 show that, although simple alkylamides are only

modest CXCL8 inhibitors, a variety of hydrophilic side

chains can be accommodated in the receptor binding

pocket to improve the water solubility of the inhibitors.

Amides of 3 with R-amino acids 45-51 are potent and

specific CXCL8 inhibitors. In this series, the stereo-

chemistry of both chiral centers strongly influences the

receptor affinity. The four diastereomeric alanylamides

were synthesized and tested in the chemotaxis assay;

only the diastereoisomer 45 was efficacious in inhibiting

CXCL8 activity. In addition, 45 was not efficacious as

a COX inhibitor. The replacement of the alanine methyl

group with functionalized groups (48 and 49) invariably

resulted in a high activity loss. The esterification of the

potent inhibitors 45 and 47 (50 and 51) dramatically

reduced the potency, suggesting a key role of the acidic

carboxylic moiety. Also, the position of the acidic func-

tion is crucial for biological activity (52 and 53). In fact,

the elongation of the spacer between the amidic and

carboxylic moieties led to a substantial activity loss. A

hydroxyl group on the alkyl chain appears to be poorly

tolerated (54, 58, and 59), whereas its transformation

into methyl ether generated active compounds 55, 60,

and 61. Interestingly, as shown by the direct comparison

between compounds 56 and 57, the introduction of an

ethereal oxygen along the chain restored the activity of

the long-chain amido alcohol. These results suggest that

hydrogen bond acceptor groups along the N-alkyl chain

strongly reinforce the affinity for the receptor. This

hypothesis was further confirmed by the activity data

of a series of aminoalkylamides. N,N-Dimethylamino-

alkylamides 62-64 exhibited a potency comparable to

that of the most potent compounds in the series. It was

initially surprising to find out that basic, positively

charged groups were well-tolerated in the receptor

pocket. Data shown in Table 4 suggest that the tertiary

alkylamino group behaves as a hydrogen bond acceptor

rather than as a basic or positively charged group.

Secondary and primary amines (65 and 66) with dual

hydrogen bond acceptor and donor characteristics showed

negligible inhibitory potency in strict analogy with

amido alcohols 54, 58, and 59. Furthermore, the posi-

tively charged related quaternary ammonium salt de-

Figure 5. Binding of compound 31 in the TM region of

CXCR1. (a) Details of the polar interactions of 31 (green) in

the putative CXCR1 (light blue) binding site are shown. In

the proposed model an electrostatic interaction occurs between

the hydroxyl group of Tyr46_1.39and the aromatic ring of the

inhibitor. The amide group engages two hydrogen bond

interactions (dashed lines) with the side chains of Lys99_2.64

and Glu291_7.35. An additional electrostatic interaction orien-

tates the phenol group of Tyr258_6.51toward Glu291_7.35. (b)

Effect of 31 on CXCL8-induced migration of L1.2 transfectants

expressing wild-type CXCR1, mutated Lys99Ala CXCR1, and

Glu291Ala. Cell migration was induced on L1.2 transfectants

expressing wild-type CXCR1 (O), mutated Lys99Ala (0), and

Glu291Ala (4) by CXCL8 (10 nM) in the presence or absence

of increasing concentrations of 31. Effect of 31 on L1.2

untransfected cell migration in response to CXCL12/SDF1 (/)

is also shown. Results are expressed as the percent of migra-

tion in the absence of 31. Values are the mean ( SD of three

independent experiments: (/) P < 0.05 vs cell migration in

the absence of 31; (//) P < 0.01 vs cell migration in the absence

of 31 (Mann-Whitney U test).

The proposed binding mode anticipates that amide

derivatives with putative trans geometries should be

strongly favored in setting the double polar interaction

with the two residues from TM2 and TM7. Additionally,

the good potency of 13 and 29 supports a model in which

a trans amide bond is in the correct orientation for the

receptor binding. Hence, in principle, the cis/trans amide

isomer ratio could strongly influence the SAR in this

series. Several external and internal parameters influ-

ence the magnitude of the rotational barrier separating

cis and trans isomers, and the most stable isomer in

solution is not easily predictable. Nevertheless, on the

basis of literature data,^37,38it is conceivable to assume

for SAR purposes that trans isomers are the most

abundant at least for N-arylamides 37-44 and R-amino-

acylamides 45-51.

4320 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

Table 6. Effect of Ring Substituents of Racemic

Table 5. IC₅₀Values for (R)- and (S)-Enantiomers of

Representative CXCL8 Inhibitors

2-Arylpropionic Acids on CXCL8-Induced Human PMN

Chemotaxis^a

rivative 67 completely lost the inhibitory activity. In

conclusion, the similar trend observed in the series of

amido alcohols and ethers and aminoalkylamides sup-

ports the hypothesis of a common binding mode for the

different chemical classes herein described in which the

ether oxygen and the amino nitrogen are involved in a

supplementary hydrogen bond interaction. In contrast,

hydrogen bond donors, such as alcoholic and primary/

secondary amino groups, are not tolerated in the CXCR1

receptor pocket. It is also noteworthy that the inclusion

of the hydrogen bond acceptor moiety in five- or six-

membered cycles with reduced structure flexibility led

to inactive compounds (68-71). From the receptor

binding point of view, it is likely that the acceptor

moiety engages favorable interactions with other amino

acids in the TM region. Nevertheless, all the most potent

compounds along the three classes share a heteroatom

in such a position that the formation of an intramolecu-

lar hydrogen bond interaction with the amide group is

allowed. Thus, it is possible to speculate that the

hydrogen bond acceptor group cooperates in reinforcing

the binding of the amide group with Lys99_2.64and

Glu291_7.35rather than establishing additional interac-

tions. The simple methyl ketone 72 is a highly potent

and selective CXCL8 inhibitor (IC₅₀) 7 nM), but a

dramatic loss of activity was observed when a set of

analogues was designed on the basis of the SAR criteria

previously learned. Considering the marked reactivity

of the carbonyl group, it is likely that a covalent

interaction with the Lys99_2.64ꢀ-amino group can replace

the double polar interaction in the binding pocket.

Further information on the inhibitor’s binding mode

with CXCR1 arise from a comparison of the IC₅₀values

of different pairs of enantiomeric phenylpropionic de-

rivatives (Table 5). (R)-Ibuprofen 3 and (S)-ibuprofen 4

exhibited, as previously described for ketoprofen enan-

tiomers,²¹a similar potency as CXCL8 inhibitors. By

contrast, a progressive increase of the eudismic ratio

was observed when the enantiomers of 2-(4-isobutyl-

^a(/) % of inhibition at 10^-8M. (//) 3:1 E/Z mixture. n.a.:

compound not active at 10^-6M.

phenyl)propionylmethanesulfonamide (13 and 14) and

2-(4-isobutylphenyl)propionamide (30 and 73) were

compared in the CXCL8-induced chemotaxis assay.

Noticeably, the primary amide 73 was also found to be

inactive at high concentration (10^-5M), and a similar

trend has also been described above for compounds 45

and 46. Taking into account our binding mode hypoth-

esis, a plausible explanation is that the shift from

partially ionic to polar hydrogen bond interactions could

result in a subtle rearrangement of the amino acids in

the binding pocket.

Since 2-arylpropionic amides and sulfonamides were

potent and selective inhibitors of CXCL8 activity, we

decided to investigate the influence of the substituents

on the phenyl ring. In this paper only a limited number

of examples will be discussed. To this aim, racemic

phenylpropionic acids were synthesized. As expected on

the basis of the preliminary observations reported for

NSAIDs, the size and nature of the substituents are

crucial in determining the affinity for the receptor, and

modifications of the phenyl ring substituents generated

molecules ranging from highly potent inhibitors to

completely inactive ones. In Table 6 inhibition at 10^-8

M of some racemic 2-arylpropionic acids is reported in

order to highlight the most potent compounds; however,

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4321

Table 7. Derivatives of (R)-2-Phenylpropionic Acids Selected

phase, inactive carboxylic acids with a theorical pK_aless

than 4.3 were not excluded from the derivatization

program.

as CXCL8 Inhibitors^a

The compounds 13, 45, and 56 showed the desired

characteristics of potency, selectivity, and water solubil-

ity and were selected as viable candidates for pharma-

cological in vivo studies. The comparison of their

pharmacokinetic and pharmacodynamic profile led

us to select 13, N-[(R)-2-(4-isobutylphenyl)propionyl]-

methanesulfonamide (repertaxin), for preclinical and

clinical development. To date, repertaxin has been

proved to be efficacious in several models of reperfusion

injury (RI) and is currently undergoing phase II clinical

trials.^39,40In vivo studies confirmed the hypothesis that

(R)-2-phenylpropionamides and (R)-2-phenylpropionyl-

sulfonamides do not interconvert to the (S)-enantiomers.

The metabolic profile of repertaxin has been widely

characterized in several animal species, and the forma-

tion of the corresponding (S)-enantiomer is not observed

in rats, mice, dogs, and humans (data not shown).

Discussion

^a(/) % inhibition at 10^-8M.

The results reported here describe SAR studies of a

novel class of noncompetitive allosteric inhibitors of the

CXCL8 receptor CXCR1. Photoactivation and alanine

scanning mutagenesis experiments show that the pro-

totypic inhibitor 1 binds to the TM region of CXCR1 in

a pocket defined by TM helices 1, 2, 3, 6, and 7. The

identification of the molecular interactions that take

place between ketoprofen and CXCR1 guided the site-

directed design of a novel class of potent and specific

blockers of CXCL8 activity. Among the CXCL8 blockers

described here, 13 was selected as a clinical candidate

that is now in phase II clinical studies for the prevention

of reperfusion injury.

We previously described that 1 inhibits CXCL8-

induced chemotaxis of human PMN. We describe here

that 1 interacts with a pocket defined by TM helices 1,

2, 3, 6, and 7 of CXCR1. The carboxylic moiety of 1

establishes with Lys99TM2_2.64of CXCR1 a strong ionic

interaction reinforced by an additional hydrogen bond

with the side chain of and Glu291TM7_7.35. Additional

polar and hydrophobic interactions contribute to deter-

mine the affinity of 1 at the binding site of CXCR1.

In the proposed model an electrostatic interaction

occurs between the hydroxyl group of Tyr46TM1_1.39and

the phenylpropionic ring of the inhibitor; the benzoyl

substituent engages hydrophobic interactions with

the side chains of Ile43TM1_1.36, Val113TM3_3.28, and

Ile283TM7_7.31and with the aliphatic chain of

several inactive compounds recovered inhibitory potency

at higher concentrations (10^-6M). The replacement of

the 4-isobutyl group (74) with smaller alkyl chains (75-

77) in the same position led to a moderate potency

decrease, whereas larger hydrophobic groups (78-81,

85, and 86) dramatically reduced receptor affinity,

leading to completely inactive compounds. 2-(4-Alkoxy-

phenyl)propionic acid analogous 82 and 83 exhibited

weak to moderate activity as CXCL8 inhibitors, whereas

the 4-hydroxyphenyl analogue 84 was inactive at maxi-

mal concentration. Next, we examined the effect of

hydrophobic groups on the 3-position (87-101). Inter-

estingly, the compounds with flexible alkyl chains on

the 3-position were only moderately active, whereas the

introduction of a branch on the benzylic carbon invari-

ably resulted in potent inhibitors. The 3-isopropyl

derivative 90 was the most active compound in the

series, but larger aliphatic (93) and aromatic substitu-

ents (96) were well-tolerated too. The forced perpen-

dicular orientation of a branched substituent in the

3-position seems crucial for a correct arrangement of

the inhibitor in the binding pocket. Replacing the

methyl group by a hydroxyl group (98) on the branch

surprisingly had only minimal effect on the potency,

whereas the oxidized 3-acetyl (99) and 3-carboxy (100)

analogues lack biological activity.

Finally, on the basis of the activity data of all the

tested compounds, we synthesized several 2-aryl-

propionamides (102-104, 106-108) and the 2-aryl-

propionylsulfonamide 105 starting from selected active

phenylpropionic acids of Table 6. The results are

reported in Table 7. The structure-activity trend

observed in the series of ibuprofen derivatives was well-

conserved when different phenylpropionic scaffolds were

examined, and this observation strongly supports the

hypothesis of a common binding mode for the whole

class of CXCL8 inhibitors. Thus, the screening of

racemic phenylpropionic acids is a reliable strategy to

select novel scaffolds for the lead optimization phase,

although the carboxylic acid pK_avalue must be taken

into account. As a consequence, in the screening

Lys99TM2_2.64

.

Agonist activation of GPCR induces conformational

changes that are still poorly understood, but evidence

is emerging that receptor proteins exist in multiple

conformational states. Microdomains have been identi-

fied in several GPCRs that could function as activation

switches, and in particular, the rearrangement of

specific residues of TM2 and TM7 could fine-tune the

active/inactive conformational equilibrium.⁴¹The find-

ing that 1 inhibits the signal transduction induced by

CXCL8 without affecting CXCL8 binding strongly sug-

gests that the interaction between 1 and CXCR1 locks

CXCR1 in an inactive conformation unable to activate

downstream signaling events. All in all, the data in this

4322 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

paper suggest that 1 is not a classical competitive

inhibitor of CXCR1. This concept is further strength-

ened by the finding that mutation of residues in the

binding site of 1 in CXCR1 does not alter CXCL8 bind-

ing in terms of affinity constant and biological response.

of 13 on CXCR1 (1 nM) and CXCR2 (100 nM) trans-

fected cells can be explained by the lack of specific

hydrophobic residues in CXCR2 involved in the recogni-

tion of the isobutyl group.³⁹There is conflicting infor-

mation as to whether CXCL8-induced chemotaxis of

PMN is mediated by one or both of the CXCL8 recep-

tors.^18,44The high potency of 13 as inhibitor of the

human PMN-induced chemotaxis, together with data on

transfected cells, supports a prominent role of CXCR1.

13 has been proven to be efficacious and well-tolerated

in several ischemia/reperfusion in vivo animal mod-

els.^39,40On the basis of the pharmacological results, 13

has been selected as a candidate drug for the prevention

and treatment of delayed graft function (DGF) and is

currently undergoing phase II clinical trials.

The identification of the molecular interactions be-

tween 1 and CXCR1 prompted an extensive medicinal

chemistry study aimed at identifying potent and specific

blockers of CXCL8 activity. Several derivatives belong-

ing to different chemical classes were synthesized and

tested in CXCL8-induced chemotaxis of human PMNs.

The SAR data suggest that specific polar interhelical

interactions engaged by the amide moiety are key

requirements for the general inhibitory property of the

class, whereas hydrophobic interactions established by

the ring substituent play a crucial role in determining

the potency. The replacement of the ionic interaction

between the carboxylic moiety of phenylpropionic acids

with a double hydrogen bond interaction between the

novel inhibitors and the Glu291TM7_7.35/Lys99TM2_2.64

couple resulted in very potent inhibitors with negligible

residual activity on COXs. This observation is consistent

with the binding mode of phenylpropionic NSAIDs in

COXs in which the acidic moiety engages a crucial ionic

interaction with Arg120.

It has been proposed⁴²that allosteric sites in the TM

domain of GPCRs may represent a valuable target for

rational drug design of potent, noncompetitive inhibitors

that block agonist induced G-protein activation. Since

chemokines are relatively large ligands, it is evident

that SMW antagonists can hardly compete for receptor

binding. Onuffer and Horuk⁴³have recently discussed

the theoretical possibility of identifying chemokine

inhibitors able to disrupt receptor function without

displacing chemokine binding, but to date, the general

use of chemokine binding as the primary assay in most

of the screening programs hardly limited the discovery

of noncompetitive inhibitors. The results herein reported

show that the use of a functional assay (e.g., chemotaxis)

in the primary screening phase, even if onerous, gives

the opportunity of selecting novel classes of noncompeti-

tive inhibitors. Moreover, since it is generally assumed

that structural motifs might be well-conserved along

subfamilies of the rhodopsin-like GPCR family, it is

conceivable that the identification of the above-described

allosteric site in CXCR1 might represent a novel strat-

egy of general interest for the rational design of novel

classes of compounds targeting other members of the

peptidergic receptors subfamily of GPCRs. Currently,

homology-based structural analysis of the main phar-

macologically relevant peptidergic and not peptidergic

receptors is being carrying out. Preliminary results

confirm that a subfamily of GPCRs share the allosteric

motif, and on the basis of this structural approach, we

have now successfully generated potent and specific

inhibitors for other chemoattractant receptors.

Experimental Section

A. Biology. Reagents. Chemokines were from Pepro Tech

(London, U.K.). Chemicals and protease inhibitors were from

Sigma (St. Louis, MO). The L1.2 lymphoma cell line was from

ATCC (Rockville, MD). Diff-Quik was from Dade Behring

(Switzerland). G418 was from Life Technologies (Grand

Island, NY). Polycarbonate filters were from Neuroprobe Inc.

(Pleasanton, CA). Transwell filters were from Costar (Costar,

Cambrige, MA). Cellulose nitrate membrane filters were from

Whatman International Ltd, (England). [³⁵S]GTPγS (1250

Ci/mmol) and [¹⁴C]-(R)-ketoprofen (87 Ci/mmol) were from

Amersham (Bucks, U.K.). Antihuman CXCR1 was from Bio-

surce International Inc., Camarillo, CA). pcDNA3 was from

Invitrogen.

Animals. Female CD1 mice were obtained from Charles

River Laboratories (Calco, LC, Italy). Animals were housed

and acclimatized for 1 week under controlled temperature (20

( 2 °C), humidity (55 ( 10%), and lighting (7 a.m. to p.m.).

Standard sterilized food and water were supplied ad libitum

during acclimatization and experiments.

All the procedures were performed in the animal facilities

according to ethical guidelines for the conduct of animal

research (Authorization Italian Ministry of Health No. 47/

2001-B; Italian Legislative Decree 116/92, Gazzetta Ufficiale

della Repubblica Italiana No. 40, February 18, 1992, EEC

Council Directive 86/609 OJ358, December 12, 1987; NIH

Guide for the Care and Use of Laboratory Animals; NIH

Publication No. 85-23, 1985).

Migration Assay. Cell migration of human PMN was

evaluated using a 48-well microchemotaxis chamber.⁴⁰CXCL8

(1 nM) was seeded in the lower compartment. Cell suspensions

(1.5 × 10⁶/mL), preincubated at 37 °C for 10 min in the

presence or absence of compounds, were seeded in the upper

compartment. The two compartments of the chemotactic

chamber were separated by a 5 µm polycarbonate filter (PVP-

free for PMN chemotaxis). The chamber was incubated at 37

°C in air with 5% CO₂for 45 min. At the end of incubation,

filters were removed, fixed, and stained with Diff-Quik, and

five oil immersion fields at high magnification (100×) were

counted after sample coding. Cell migration of L1.2 was

evaluated in 5 µm pore size Transwell filters.⁴⁶The Transwell

filter was incubated at 37 °C in air with 5% CO₂for 4 h, and

the cells were counted in a Bu¨rker chamber.

[

³⁵S]GTPγS Assay. [³⁵S]GTPγS assays were performed as

previously described⁴⁷with the following modifications: a total

of 20 µg of PMN plasma membranes was incubated in buffer

(143 mM NaCl, 50 mM triethanolamine, pH 7.3, 1 mM EDTA,

5 mM MgCl₂, and 20 µM GDP) at 30 °C for 20 min in the

presence or absence of 1. CXCL8 (300 nM) was added and

followed 10 min later by [³⁵S]GTPγS (0.8 nM). The total volume

was 100 µL. The reaction was allowed to proceed at 30 °C for

15 min and terminated by filtration through 0.45 µM cellu-

lose nitrate membrane filters. The filters were washed two

times with 2 mL of washing buffer (50 mM Tris-HCl, pH 7.3,

5 mM MgCl₂, 1 mM EDTA) and transferred to scintillation

vials containing 10 mL of scintillation fluid, and the bound

Among the diverse CXCL8 inhibitors shown in this

work, the clinical candidate repertaxin (13) was selected

for further characterization. The binding site of 13 is

highly homologous in the two CXCL8 receptor subtypes

CXCR1 and CXCR2.³⁹Accordingly, 13 is a noncompeti-

tive allosteric blocker of both receptor subtypes, as

assessed by experiments with CXCR1/L1.2 and CXCR2/

L1.2 transfected cells.³⁹The difference in the IC₅₀values

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4323

[³⁵S]GTPγS was determined by an L96500 multipurpose

calculated for C, H, and N and reported only with symbols.

High-performance liquid chromatography (HPLC) was per-

formed on a Waters 600E apparatus with a Waters 486 UV

detector (monitoring at 254 nm) equipped with a Chiralcel OJ

column and using a mixture consisting of 75:15:10 n-hexane/

EtOH/ⁱPrOH and trifluoroacetic acid (TFA) (0.5%)

All reagents and solvents were purchased from Sigma-

Aldrich or Lancaster and used without further purification.

Reaction courses and product mixtures were monitored by

thin-layer chromatography on silica gel (precoated F₂₅₄

Macherey-Nagel plates); the spots were examined with UV

light and visualized with I₂.

scintillation counter.

Mutagenesis of CXCR1 and Cell Transfection. The

human CXCR1 open reading frame was PCR amplified from

a CXCR1/pCEP4 plasmid (kindly provided by Dr. P. M.

Murphy, NIH, Bethesda, MD). Receptor mutant was obtained

by standard two-step PCR. Wild-type and mutated receptors

were cloned in the mammalian expression vector pcDNA3. The

nucleotide sequence of each construct was confirmed by double

strand DNA sequencing. The mouse L1.2 lymphoma cell line

was transfected by electroporation and selected with G418 (800

µg/mL).

The following 2-arylpropionic acids are commercially avail-

able as racemic mixtures: ketoprofen (87), ibuprofen (74),

flurbiprofen, naproxen (7), thiaprofenic acid (8), indoprofen (9),

carprofen (10), 2-phenylpropionic acid (75), 2-(4-methyl)-

phenylpropionic acid (76), and 2-(4-hydroxy)phenylpropionic

acid (84).

General Procedure for the Optical Resolution of

Racemic 2-Arylpropionic Acids. Compounds 1-6, 90, and

93 were obtained by optical resolution of the related racemic

2-arylpropionic acids as described⁴⁸using the R-(+) or S-(-)-

R-phenylethylamine as resolving agent.

(R)-2-(3-Benzoylphenyl)propionic Acid (1). Yield 38%.

Anal. (C₁₆H₁₄O₃) C, H.

(S)-2-(3-Benzoylphenyl)propionic Acid (2). Yield 37%.

Anal. (C₁₆H₁₄O₃) C, H.

(R)-2-(4-Isobutylphenyl)propionic Acid (3). Yield 35%.

Anal. (C₁₃H₁₈O₂) C, H.

(S)-2-(4-Isobutylphenyl)propionic Acid (4). Yield 33%.

Anal. (C₁₃H₁₈O₂) C, H.

Photochemical Cross-Linking of CXCR1. Membranes

prepared from both wild-type L1.2 CXCR1 and mutated

Lys99Ala CXCR1 cells were incubated with [¹⁴C]-1 (0.1 mM)

at room temperature for 10 min. CXCL8 (80 nM) was added

and followed 10 min later by irradiation (10 cm distance) with

an hand-held UV illuminator at 0 °C for 30 min. After

centrifugation (30000g) at 4 °C for 15 min, membrane suspen-

sions were solubilized and homogenized in a glass-glass

homogenizer. After centrifugation (10000g) at 4 °C for 10 min,

the supernatants were subjected to immunoprecipitation with

10 µg of antihuman CXCR1. The immune complexes were

resolved by SDS-PAGE (15% gel). After electrophoresis, the

gel was stained with Comassie Blue, dried, and exposed to

autoradiography at -80 °C for 1 month.

Macrophage Preparation and LPS-Induced PGE₂Pro-

duction Assay. Peritoneal exudate cells were collected from

peritoneal washing 5 days after ip inoculum of 3% thioglycolate

(Difco, Detroit, MI) in saline (1.5 mL for mouse). Cells were

plated at a density of 6 × 10⁵/well in 96-well plates in RPMI

1640 (Sigma, St. Louis, MO) medium, and nonadherent cells

were removed by repeated washing 2 h later. Compounds were

then added to the macrophages at 10 µM, and 30 min later

LPS (from E. coli 0.55: B5, Difco; 1 µg/mL) was added. Control

wells received saline or DMSO (Sigma) at the appropriate

dilution. Culture supernatants were harvested 4 h after LPS

stimulation for PGE₂production measured with an EIA kit

(Amersham; sensitivity of 2.5 pg/well).

B. Molecular Modeling. The starting rhodopsin-based

CXCR1 molecular model has been extracted from the GPCRDB

database, whereas (S)-flurbiprofen bound COX-1 from the

Protein Data Bank (designation 1EQH). Conformational analy-

sis of the ligands has been performed with molecular mechan-

ics (MM) methods. Steepest descent (SD) and conjugate

gradient (CG) algorithms are as implemented in the DIS-

COVER (Insight II package, release 2000, Accelrys Inc., San

Diego, CA) package using the CVFF force field. After reiterated

minimizations (SD + CG) of the ligand in the putative receptor

binding cavity, the energy-refined model was used as input of

NVT (305 K) molecular dynamics (MD) for 1000 ps starting

from 5 to 305 K, by incrementing 50 K per 2 ps of simulation.

A supplementary elastic potential term (50 kcal/Å²) has been

added on the R carbon of CXCR1 to preserve the secondary

structure of the TM bundle during the simulation. Following

gradual annealing (from 305 to 5 K, 50 K per 2 ps), the

obtained structure has been minimized by MM (CG). To

confirm the reproducibility of the model, starting from different

conformations, the overall calculation has been repeated three

times, and the three final structures were identical. The

stability of the final structure was assessed through 50 ps of

high-temperature MD calculations (from 5 to 1000 K, 50 K

per 2 ps) with an increasing tethering potential (50 kcal/Å²

up to 200 for higher temperature values) associated with the

R carbon of CXCR1. After the annealing step (from 1000 to 5

K), a final MM (CG) calculation followed. All simulations have

been performed on an SGI O2 R12000 (IRIX6.5) instrument.

(R)-2-(2-Fluorobiphenyl-4-yl)propionic Acid (5). Yield

30%. Anal. (C₁₅H₁₃FO₂) C, H.

(S)-2-(2-Fluorobiphenyl-4-yl)propionic Acid (6). Yield

35%. Anal. (C₁₅H₁₃FO₂) C, H.

Synthesis of Acylsulfonamides (Scheme 1). N-[(R)-2-

(3-Benzoylphenyl)propionyl]methanesulfonamide (11).

To a cooled mixture (0-5 °C) of 1 (2.0 g, 7.88 mmol) in CH₂Cl₂

(30 mL), 1,1′-carbonyldiimidazole (CDI) (1.72 g, 7.88 mmol)

was added. After the mixture was stirred for 2 h at 0-5 °C,

methanesulfonamide (0.79 g, 7.88 mmol) and diazobicyclo-

[5.4.0]undec-7-ene (DBU) (1.22 mL, 7.88 mmol) were added.

The mixture was left stirring for 4 h. Glacial AcOH (0.94 mL)

was added, the reaction mixture was diluted with CH₂Cl₂(10

mL), and the organic layer was washed with 10% buffer

NaH₂PO₄(pH 4) (4 × 15 mL) and water (3 × 10 mL), dried

over Na₂SO₄, filtered, and concentrated under reduced pres-

sure to give a crude residue that was purified by flash

chromatography (CH₂Cl₂/CH₃OH 99:1) to afford 11 as a

colorless oil (1.92 g, 74% yield). Anal. (C₁₇H₁₇NSO₃) C, H, S,

N.

N-[(S)-2-(3-Benzoylphenyl)propionyl]methanesulfon-

amide (12). Following the same procedure described for 11

and starting from 2 (2.0 g, 7.88 mmol), after workup and

purification by flash chromatography (CH₂Cl₂/CH₃OH 99:1),

12 was obtained as a transparent oil (1.84 g, 70.5% yield).

Anal. (C₁₇H₁₇NO₃S) C, H, S, N.

N-[(R)-2-(4-Isobutylphenyl)propionyl]methanesulfon-

amide (13). Following the same procedure described for 11

and starting from 3 (2.0 g, 9.7 mmol), after workup and

purification by flash chromatography (CH₂Cl₂/CH₃OH 95:5),

13 was obtained as a white powder (2.28 g, 83% yield). Anal.

(C₁₄H₂₁NO₃S) C, H, S, N.

N-[(S)-2-(4-Isobutylphenyl)propionyl]methanesulfon-

amide (14). Following the same procedure described for 11

and starting from 4 (2.0 g, 9.7 mmol), after workup and

purification by flash chromatography (CH₂Cl₂/CH₃OH 95:5),

14 was obtained as a white powder (2.20 g, 80% yield). Anal.

(C₁₄H₂₁NO₃S) C, H, S, N.

N-[(R)-2-(2-Fluorobiphenyl-4-yl)propionyl]methane-

sulfonamide (15). Following the same procedure described

for 11 and starting from 5 (2.0 g, 8.18 mmol), after workup

and purification by flash chromatography (CH₂Cl₂/CH₃OH 95:

C. Chemistry. General Experimental Procedures. Op-

tical rotations were measured on a Perkin-Elmer 241 pola-

rimeter, and the [R]²⁵values are given in 10^-1deg cm²g^-1

.

D

¹H NMR spectra were recorded on a Bruker ARX 300 spec-

trometer. Melting points were determined using a Bu¨chi

capillary melting point apparatus and are uncorrected. Ele-

mental analyses were within (0.4% of the theoretical values

4324 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

5), 15 was obtained as a white powder (1.79 g, 68% yield). Anal.

(C₁₆H₁₆FNO₃S) C, H, S, N.

(1.52 g, 9.7 mmol), after workup and purification by flash

chromatography (CH₂Cl₂/CH₃OH 98:2), 25 was obtained as a

white powder (2.85 g, 85% yield). Anal. (C₁₉H₂₃NO₃S) C, H, S,

N.

N-[(R)-2-(4-Isobutylphenyl)propionyl]pyrid-3-ylsul-

fonamide Hydrochloride (26). Following the same proce-

dure described for 11 and starting from 3 (2.0 g, 9.7 mmol)

and pyrid-3-ylsulfonamide (1.53 g, 9.7 mmol), after workup

gaseous HCl was bubbled into an ethereal solution of the

crude. 26 was isolated by filtration in the form of a hydro-

chloride as a white powder (1.71 g, 46% yield). Anal. (C₁₈H₂₃-

ClN₂O₃S) C, H, S, N.

4-Chloro-N-[(R)-2-(4-isobutylphenyl)propionyl]ben-

zenesulfonamide (27). Following the same procedure de-

scribed for 11 and starting from 3 (2.0 g, 9.7 mmol) and

4-chlorobenzenesulfonamide (1.86 g, 9.7 mmol), after workup

and purification by flash chromatography (CH₂Cl₂/CH₃OH 99:

1), 27 was obtained as a white powder (2.14 g, 58% yield). Anal.

(C₁₉H₂₂ClNO₃S) C, H, S, N.

N-[(S)-2-(2-Fluorobiphenyl-4-yl)propionyl]methane-

sulfonamide (16). Following the same procedure described

for 11 and starting from 6 (2.0 g, 8.18 mmol), after workup

and purification by flash chromatography (CH₂Cl₂/CH₃OH 95:

5), 16 was obtained as a slightly white powder (1.84 g, 70%

yield). Anal. (C₁₆H₁₆FNO₃S) C, H, S, N.

N-[2-(6-Methoxynaphthalen-2-yl)propionyl]methane-

sulfonamide (17). Following the same procedure described

for 11 and starting from 7 (2.0 g, 8.68 mmol), after workup

and purification by flash chromatography (CH₂Cl₂/CH₃OH 99:

1), 17 was obtained as a pale-yellow powder (1.56 g, 78% yield).

Anal. (C₁₅H₁₇NO₄S) C, H, S, N.

N-[2-(5-Benzoylthiophen-2-yl)propionyl]methane-

sulfonamide (18). Following the same procedure described

for 11 and starting from 9 (2.0 g, 7.68 mmol), after workup

and purification by flash chromatography (CH₂Cl₂/CH₃OH 98:

2), 18 was obtained as a white powder (1.68 g, 65% yield). Anal.

(C₁₅H₁₅NO₄S₂) C, H, S, N.

N-[(R)-2-(4-Isobutylphenyl)propionyl]trifluoromethane-

sulfonamide (28). 3 (0.56 g, 2.71 mmol) was dissolved in

SOCl₂(5 mL), and the resulting solution was left stirring at

reflux for 3 h. After cooling at room temperature, the mixture

was evaporated under reduced pressure. The crude acyl

chloride was diluted with dry CH₂Cl₂(10 mL), and triethyl-

amine (0.83 mL, 5.96 mmol) was added. A solution of trifluoro-

methanesulfonamide (0.4 g, 2.71 mmol) in dry CH₂Cl₂(5 mL)

was added dropwise, and the resulting mixture was left under

stirring at room temperature for 4 h. The mixture was diluted

with CH₂Cl₂(10 mL), washed with water (2 × 10 mL), and

extracted with a saturated solution of NaHCO₃(2 × 10 mL).

The basic aqueous phase was acidified with 37% HCl to pH 2

until the complete precipitation of the acid. The pure com-

pound 28 was obtained by filtration as a slightly yellow powder

(0.65 g, 70% yield). Anal. (C₁₄H₁₈F₃NO₃S) C, H, S, N.

Synthesis of Amides (Scheme 2). (R)-N-Hydroxy-2-(4-

isobutylphenyl)propionamide (29). To a solution of (R)-2-

(4-isobutylphenyl)propionyl chloride (prepared as described for

compound 28) (24.5 mmol) in CH₂Cl₂(25 mL), a suspension of

hydroxylamine hydrochloride (2.04 g, 29.4 mmol) and triethyl-

amine (8.2 mL, 58.8 mmol) in CH₂Cl₂(25 mL) was added by

dripping. A 6 N HCl (15 mL) solution was added after stirring

for 4 h. The two phases were separated, and the organic one

was washed with water (2 × 20 mL) and brine (20 mL), dried

over Na₂SO₄, and evaporated under reduced pressure to give

a crude. When pulped in isopropyl ether (15 mL) overnight at

room temperature, pure 29 was isolated by filtration as a white

powder (4.5 g, 83% yield). Anal. (C₁₃H₁₉NO₂) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-methoxypropionamide (30).

Following the same procedure described for 29 and starting

from 3 (2.47 g, 12 mmol) and methoxylamine hydrochloride

(1.2 g, 14.4 mmol), after workup and purification by flash

chromatography (CH₂Cl₂/CH₃OH 98:2), 30 was obtained as a

yellow oil (2.12 g, 75% yield). Anal. (C₁₄H₂₁NO₂) C, H, N.

(R)-2-(4-Isobutylphenyl)propionamide (31). Following

the same procedure described for 29 and starting from 3 (2.47

g, 12 mmol) and gaseous ammonia, after workup and purifica-

tion by crystallization from EtOAc, 31 was obtained as a white

powder (1.38 g, 56% yield). Anal. (C₁₃H₁₉NO) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-methylpropionamide (32).

Following the same procedure described for 29 and starting

from 3 (2.47 g, 12 mmol) and methylamine (2.0 M in THF, 12

mmol), after workup and crystallization from acetone, 32 was

obtained as a white powder (1.57 g, 60% yield). Anal. (C₁₄H₂₁NO)

C, H, N.

N-{2-[4-(1,3-Dihydroisoindol-2-yl)phenyl]propionyl}-

methanesulfonamide (19). Following the same procedure

described for 11 and starting from 8 (2.0 g, 7.11 mmol), after

workup and purification by flash chromatography (CH₂Cl₂/

CH₃OH 99:1), 19 was obtained as a white powder (1.68 g, 65%

yield). Anal. (C₁₈H₁₈N₂O₄S) C, H, S, N.

N-[2-(6-Chloro-9H-carbazol-2-yl)propionyl]methane-

sulfonamide (20). Following the same procedure described

for 11 and starting from 10 (2.0 g, 7.31 mmol), after workup

and purification by flash chromatography (CH₂Cl₂/CH₃OH 99:

1), 20 was obtained as a white powder (1.84 g, 72% yield). Anal.

(C₁₆H₁₅ClN₂O₃S) C, H, S, N.

General Procedure for the Synthesis of Intermediates

Alkyl- and Arylsulfonamides. The noncommercial alkyl-

and arylsulfonamides were prepared according to the described

procedure. A suitable alkyl- or arylsulfonyl chloride was

dissolved in dry Et₂O (or 1,4-dioxane if insoluble), and gaseous

ammonia was bubbled into the solution until the precipitation

of ammonium chloride was complete. After filtration, the

mother liquors were evaporated under reduced pressure and

hot water was added to the crude. The resulting solution was

left cooling at room temperature, and the pure alkyl- or aryl

sulfonamide was isolated as a white solid by filtration and

1

characterized by mp and H NMR analysis.

N-[(R)-2-(4-Isobutylphenyl)propionyl]ethanesulfon-

amide (21). Following the same procedure described for 11

and starting from 3 (2.0 g, 9.7 mmol) and ethanesulfonamide

(1.06 g, 9.7 mmol), after workup and purification by flash

chromatography (CH₂Cl₂/CH₃OH 98:2), 21 was obtained as a

pale-yellow solid (2.31 g, 80% yield). Anal. (C₁₅H₂₃NO₃S) C,

H, S, N.

N-[(R)-2-(4-Isobutylphenyl)propionyl]isopropylsulfon-

amide (22). Following the same procedure described for 11

and starting from 3 (2.0 g, 9.7 mmol) and propane-2-sulfon-

amide (1.19 g, 9.7 mmol), after workup and purification by

flash chromatography (CH₂Cl₂/CH₃OH 98:2), 22 was obtained

as a waxy solid (1.67 g, 55% yield). Anal. (C₁₆H₂₅NO₃S) C, H,

S, N.

2-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)ethanesulfonic

Acid [(R)-2-(4-Isobutylphenyl)propionyl]amide (23). Fol-

lowing the same procedure described for 11 and starting from

3 (2.0 g, 9.7 mmol) and 2-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-

ethanesulfonic acid amide (2.46 g, 9.7 mmol), after workup and

purification by flash chromatography (CH₂Cl₂/CH₃OH 95:5),

23 was obtained as a white powder (3.13 g, 73% yield). Anal.

(C₂₃H₂₆N₂O₅S) C, H, S, N.

2-Aminoethanesulfonic Acid [(R)-2-(4-Isobutylphenyl)-

propionyl]amide (24). 24 was obtained by treatment of 23

(2.0 g, 4.52 mmol) with hydrazine according a known proce-

dure.⁴⁹Pure 24 was isolated as a white powder (1.27 g, 90%

yield). Anal. (C₁₅H₂₄N₂O₃S) C, H, S, N.

(R)-2-(4-Isobutylphenyl)-N-ethylpropionamide (33). Fol-

lowing the same procedure described for 29 and starting from

3 (2.47 g, 12 mmol) and ethylamine (2.0 M in THF, 12 mmol),

after workup and purification by flash chromatography (CH₂Cl₂/

CH₃OH 98:2), compound 33 was obtained as a colorless oil

(1.79 g, 64% yield). Anal. (C₁₅H₂₃NO) C, H, N.

N-[(R)-2-(4-Isobutylphenyl)propionyl]benzenesulfon-

amide (25). Following the same procedure described for 11

and starting from 3 (2.0 g, 9.7 mmol) and benzenesulfonamide

(R)-2-(4-Isobutylphenyl)-N,N-dimethylpropionamide

(34). Following the same procedure described for 29 and

starting from 3 (2.47 g, 12 mmol) and N,N-dimethylamine (2.0

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4325

M in THF, 12 mmol), after workup and purification by flash

chromatography (CH₂Cl₂/CH₃OH 99:1), compound 34 was

obtained as a colorless oil (1.34 g, 48% yield). Anal. (C₁₅H₂₃NO)

C, H, N.

(R)-2-(4-Isobutylphenyl)-1-piperidin-1-ylpropan-1-

one (35). Following the same procedure described for 29 and

starting from 3 (1 g, 4.85 mmol) and piperidine (0.48 mL, 4.85

mmol), after workup and purification by flash chromatography

(n-hexane/EtOAc 8:2), 35 was obtained as a colorless oil (0.88

g, 66% yield). Anal. (C₁₈H₂₇NO) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(isoxazol-2-yl)propion-

amide (44). Following the same procedure described for 29

and starting from 3 (0.5 g, 2.42 mmol) and 2-aminoisoxazole

(0.18 mL, 2.42 mmol), after workup and purification by pulping

in n-hexane, 44 was obtained as a white powder (0.4 g, 60%

yield). Anal. (C₁₆H₂₀N₂O₂) C, H, N.

(S)-2-[(R)-2-(4-Isobutylphenyl)propionylamino]pro-

pionic Acid (45). 51 (2 g, 6.87 mmol) was dissolved in 1,4-

dioxane (9 mL), and 1 N NaOH (9 mL) was added. The

resulting mixture was left stirring at room temperature

overnight. Water and concentrated H₂SO₄were added until

pH 2 was attained, and the aqueous phase was extracted with

CH₂Cl₂(3 × 15 mL). The collected organic extracts were dried

over Na₂SO₄and evaporated under reduced pressure to give

a solid residue that, after pulping in Et₂O, afforded 45 as a

white powder (1.81 g, 95% yield). Anal. (C₁₆H₂₃NO₃) C, H, N.

(R)-2-[(R)-2-(4-Isobutylphenyl)propionylamino]pro-

pionic Acid (46). Following the same procedure described for

45 and starting from 109 (2 g, 6.86 mmol), after workup and

purification by pulping in Et₂O, pure 46 was obtained as a

colorless oil (1.83 g, 96% yield). Anal. (C₁₆H₂₃NO₃) C, H, N.

[(R)-2-(4-Isobutylphenyl)propionylamino]acetic Acid

(47). Following the same procedure described for 45 and

starting from 50 (1 g, 3.60 mmol), after workup and purifica-

tion by pulping in n-hexane, 47 was obtained as a pale-yellow

powder (0.85 g, 90% yield). Anal. (C₁₅H₂₁NO₃) C, H, N.

(S)-3-Hydroxy-2-[(R)-2-(4-isobutylphenyl)propionyl-

amino]propionic Acid (48). To a cooled (-10 °C) solution

of 110 (0.29 g, 0.94 mmol) in CH₂Cl₂(15 mL), BBr₃(1 M in

CH₂Cl₂) (4.6 mL) was added by dripping. The resulting solution

was left at -10 °C for 1 h and at room temperature for 2.5 h.

The reaction was quenched adding, by dripping, water (23 mL).

The two phases were separated and the organic one was

extracted with a saturated solution of NaHCO₃(2 × 10 mL).

The collected aqueous extracts were acidified with 37% HCl

to pH 2 and extracted back with CH₂Cl₂(2 × 10 mL). The

collected organic extracts were dried over Na₂SO₄and evapo-

rated to give pure 48 as a pale-yellow oil (0.20 g, 74% yield).

Anal. (C₁₆H₂₃NO₄) C, H, N.

(R)-2-[(R)-2-(4-Isobutylphenyl)propionylamino]-3-mer-

captopropionic Acid (49). Following the same procedure

described for 48 and starting from 111 (1 g, 3.09 mmol), after

workup and purification, pure 49 was obtained as a slightly

white powder (0.82 g, 86% yield). Anal. (C₁₆H₂₃NO₃S) C, H, S,

N.

[(R)-2-(4-Isobutylphenyl)propionylamino]acetic Acid

Methyl Ester (50). 50 was prepared as described for 37

starting from 3 (5 g, 24.27 mmol) and from a mixture of glycine

methyl ester hydrochloride (3.05 g, 24.27 mmol) and triethyl-

amine (3.38 mL, 24.27 mmol) in DMF (15 mL). After workup

and purification, 50 was obtained as a colorless oil (5.38 g,

80% yield). Anal. (C₁₆H₂₃NO₃) C, H, N.

(S)-2-[(R)-2-(4-Isobutylphenyl)propionylamino]pro-

pionic Acid Methyl Ester (51). 51 was prepared as described

for 37 starting from 3 (5 g, 24.27 mmol) and from a mixture of

L-alanine methyl ester hydrochloride (3.39 g, 24.27 mmol) and

triethylamine (3.38 mL, 24.27 mmol) in DMF (25 mL). After

workup and purification by pulping in n-hexane, 51 was

obtained as a white powder (4.90 g, 69% yield). Anal. (C₁₇H₂₅-

NO₃) C, H, N.

(R)-2-(4-Isobutylphenyl)propionic Acid Methyl Ester

(36). To a solution of 3 (1.03 g, 5 mmol) in CH₃OH (5 mL), a

few drops of concentrated H₂SO₄were added. The resulting

solution was left stirring at room temperature overnight. The

solvent was evaporated, and the residue, dissolved in CH₂Cl₂

(15 mL), was washed with a saturated solution of NaHCO₃(2

× 10 mL) and with brine (2 × 10 mL), dried over Na₂SO₄, and

evaporated to give pure 36 as a colorless oil (1.1 g, quantitative

yield). Anal. (C₁₄H₂₀O₂) C, H.

(R)-2-(4-Isobutylphenyl)-N-phenylpropionamide (37).

To a cooled (0-5 °C) solution of 3 (0.502 g, 2.4 mmol) in DMF

(10 mL), 1-hydroxybenzotriazole hydrate (HOBT) (0.324 g, 2.4

mmol) and 1,3-dicyclohexylcarbodiimide (DCC) (0.495 g, 2.4

mmol) were added. After 1 h of stirring at the same temper-

ature, aniline (0.22 mL, 2.4 mmol) was added, and the

resulting mixture was left stirring overnight at room temper-

ature. DMF was distilled off, and the crude residue was diluted

with CH₃CN (15 mL). The precipitated 1,3-dicyclohexylurea

(DCU) was filtered off, and this treatment was repeated twice

to completely eliminate the residual DCU. The crude was

diluted with CH₂Cl₂(20 mL), washed with 1 N HCl (2 × 10

mL), with a saturated solution of NaHCO₃(2 × 10 mL), and

with brine, dried over Na₂SO₄, and evaporated to give a crude

that was purified by flash chromatography (CH₂Cl₂/CH₃OH

98:2). The pure 37 was obtained as a colorless oil (0.51 g, 76%

yield). Anal. (C₁₉H₂₃N₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(pyrid-2-yl)propionamide

(38). Following the same procedure described for 37 and

starting from 3 (0.502 g, 2.4 mmol) and 2-aminopyridine (0.23

g, 2.42 mmol), after workup and purification by flash chro-

matography (CH₂Cl₂/CH₃OH 98:2), 38 was obtained as a

colorless oil (0.36 g, 54% yield). Anal. (C₁₈H₂₂N₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(pyrid-4-yl)propionamide

(39). Following the same procedure described for 37 and

starting from 3 (0.502 g, 2.4 mmol) and 4-aminopyridine (0.23

g, 2.42 mmol), after workup and purification by flash chro-

matography (CH₂Cl₂/CH₃OH 95:5), 39 was obtained as a

colorless oil (0.48 g, 71% yield). Anal. (C₁₈H₂₂N₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(pyrid-3-yl)propionamide

(40). Following the same procedure described for 37 and

starting from 3 (0.5 g, 2.4 mmol) and 3-aminopyridine (0.23

g, 2.42 mmol), after workup and purification by flash chro-

matography (CH₂Cl₂/CH₃OH 98:2), 40 was obtained as a waxy

solid (0.34 g, 50% yield). Anal. (C₁₈H₂₂N₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(pyrimidin-4-yl)propion-

amide (41). Following the same procedure described for 37

and starting from 3 (0.88 g, 4.27 mmol) and 4-aminopyrimidine

(0.41 g, 4.27 mmol), after workup and purification by pulping

in n-hexane, 41 was obtained as a white powder (0.72 g, 60%

yield). Anal. (C₁₇H₂₁N₃O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(pyrazin-2-yl)propion-

amide (42). Following the same procedure described for 37

and starting from 3 (1.012 g, 5.10 mmol) and aminopyrazine

(0.485 g, 5.10 mmol), after workup and purification by flash

chromatography (CH₂Cl₂to CH₂Cl₂/CH₃OH 95:5), 42 was

obtained as a pale-yellow oil (0.75 g, 52% yield). Anal.

(C₁₇H₂₁N₃O) C, H, N.

3-[(R)-2-(4-Isobutylphenyl)propionylamino]pro-

pionic Acid (52). 52 was prepared by direct coupling of 3 (1

g, 4.85 mmol) with â-alanine (0.45 g, 4.85 mmol) as described

for the preparation of 37. After workup and purification by

flash chromatography (CH₂Cl₂/CH₃OH 98:2), 52 was obtained

as a white powder (0.40 g, 30% yield). Anal. (C₁₆H₂₃NO₃) C,

H, N.

(R)-2-(4-Isobutylphenyl)-N-(thiazol-2-yl)propion-

amide (43). Following the same procedure described for 29

and starting from 3 (0.5 g, 2.42 mmol) and 2-aminothiazole

(0.243 g, 2.42 mmol), after workup and purification by pulping

in n-hexane, 43 was obtained as a white powder (0.48 g, 69%

yield). Anal. (C₁₆H₂₀N₂OS) C, H, S, N.

3-[(R)-2-(4-Isobutylphenyl)propionylamino]butyric Acid

(53). 53 was prepared by direct coupling of 3 (1 g, 4.85 mmol)

with 4-aminobutyric acid (0.50 g, 4.85 mmol) as described for

the preparation of 37. After workup and purification by flash

chromatography (CH₂Cl₂/CH₃OH 99:1), 53 was obtained as a

white powder (0.39 g, 28% yield). Anal. (C₁₇H₂₅NO₃) C, H, N.

4326 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

(R)-2-(4-Isobutylphenyl)-N-(2-hydroxyethyl)propion-

amide (54). Following the same procedure described for 29

and starting from 3 (2 g, 9.6 mmol) and ethanolamine (0.58

mL, 9.6 mmol), after workup and purification by flash chro-

matography (CH₂Cl₂/CH₃OH 98:2), 54 was obtained as a

colorless oil (2.16 g, 90% yield). Anal. (C₁₅H₂₃NO₂) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(2-methoxyethyl)propion-

amide (55). Following the same procedure described for 29

and starting from 3 (2 g, 9.6 mmol) and 2-methoxyethylamine

(0.83 mL, 9.6 mmol), after workup and purification by flash

chromatography (CH₂Cl₂/CH₃OH 99:1), 55 was obtained as a

colorless oil (2.07 g, 82% yield). Anal. (C₁₆H₂₅NO₂) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-[2-(2-hydroxyethoxy)ethyl]-

propionamide (56). Compound 56 was prepared by direct

coupling of 3 (1 g, 4.85 mmol) with 2-(2-aminoethoxy)ethanol

(0.48 mL, 4.85 mmol) as described for the preparation of 37.

After workup and purification by flash chromatography (CH₂Cl₂/

CH₃OH 95:5), 56 was obtained as a colorless oil (0.91 g, 64%

yield). Anal. (C₁₇H₂₇NO₃) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-[2-(5-hydroxypentyl)]pro-

pionamide (57). 57 was prepared by direct coupling of 3 (1

g, 4.85 mmol) with 5-amino-1-pentanol (0.50 g, 4.85 mmol) as

described for the preparation of 37. After workup and purifica-

tion by flash chromatography (n-hexane/EtOAc 9:1), 57 was

obtained as a colorless oil (0.78 g, 55% yield). Anal. (C₁₈H₂₉NO₂)

C, H, N.

(R)-2-(4-Isobutylphenyl)-N-((R)-2-hydroxy-1-methyl-

ethyl)propionamide (58). Following the same procedure

described for 29 and starting from 3 (0.5 g, 2.42 mmol) and

(R)-1-amino-2-propanol (0.19 mL, 2.42 mmol), after workup

and purification by trituration from n-hexane, 58 was obtained

as a white powder (0.25 g, 40% yield). Anal. (C₁₆H₂₅NO₂) C,

H, N.

(R)-2-(4-Isobutylphenyl)-N-((S)-2-hydroxy-1-methyl-

ethyl)propionamide (59). Following the same procedure

described for 29 and starting from 3 (0.5 g, 2.42 mmol) and

(S)-1-amino-2-propanol (0.19 mL, 2.42 mmol), after workup

and purification by pulping in n-hexane, 59 was obtained as

a white powder (0.46 g, 72% yield). Anal. (C₁₆H₂₅NO₂) C, H,

N.

(R)-2-(4-Isobutylphenyl)-N-((S)-2-methoxy-1-methyl-

ethyl)propionamide (60). To a cooled (0-5 °C) solution of

58 (0.4 g, 1.5 mmol) in Et₂O (15 mL), silica gel (0.2 g) was

added. A solution of diazomethane was carefully added by

dripping until a persistent yellow solution was obtained. The

reaction mixture was left stirring until complete disappearance

of starting material (TLC). The reaction was quenched by

adding excess glacial AcOH. All the solvents were evaporated

under reduced pressure, and the residue was diluted with

CH₂Cl₂. The organic phase was filtered to eliminate silica gel,

washed with water, dried over Na₂SO₄, and evaporated under

reduced pressure to give a solid residue that, after purification

by flash chromatography (n-hexane/EtOAc 1:1), afforded pure

60 as a waxy solid (0.33 g, 80% yield). Anal. (C₁₇H₂₇NO₂) C,

H, N.

obtained as a pale-yellow oil (1.15 g, 44% yield). Anal.

(C₁₇H₂₈N₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(3-dimethylaminobutyl)-

propionamide Hydrochloride (64). 4-(Dimethylamino)-

butylamine was prepared starting from commercial N-(4-

bromobutyl)phthalimide and dimethylamine hydrochloride

according to the classical procedure for the Gabriel synthesis

of amines.³⁹Following the same procedure described for 29

and starting from 3 (0.62 g, 3 mmol) and 4-(dimethylamino)-

butylamine (0.35 g, 3 mmol), after workup gaseous HCl was

bubbled into an ethereal solution of the crude. 64 was isolated

by filtration in the form of a hydrochloride as a pale-pink

powder (0.43 g, 42% yield). Anal. (C₁₉H₃₃ClN₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(3-aminopropyl)propion-

amide Hydrochloride (65). To a solution of 112 (1.4 g, 3.9

mmol) in CH₂Cl₂(30 mL), 3 M HCl (4 mL) was added. The

resulting solution was left stirring for 24 h at room temper-

ature. The organic layer was washed with water (2 × 10 mL)

and brine, dried over Na₂SO₄, and evaporated under reduced

pressure to give a residue that, after pulping in Et₂O, afforded

pure 65 in the form of a hydrochloride as a white powder (0.775

g, 66% yield). Anal. (C₁₆H₂₆N₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(3-methylaminopropyl)pro-

pionamide Hydrochloride (66). Starting from 114 and

following the procedure described above for 65, 66 was

obtained, after pulping in Et₂O, in the form of a hydrochloride

as a white powder (0.775 g, 66% yield). Anal. (C₁₇H₂₈N₂O) C,

H, N.

{3-[(R)-2-(4-Isobutylphenyl)propionylamino]propyl}-

trimethylammonium Iodide (67). To a solution of 62 free

base (0.23 g, 0.67 mmol) in THF (10 mL), iodomethane (0.15

mL, 2.42 mmol) was added, and the resulting solution was left

stirring at room temperature overnight. The solvent was

evaporated, and the residue was suspended in isopropyl alcohol

(5 mL) and left stirring for 4 h. Pure 67 was isolated by

filtration as a white powder (0.203 g, 70% yield). Anal. (C₁₉H₃₃-

IN₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-[(1-methyl)piperidin-4-yl]-

propionamide Hydrochloride (68). Following the same

procedure described for 29 and starting from 3 (1.03 g, 5 mmol)

and 4-(aminomethyl)piperidine (0.57 g, 5 mmol), after workup

gaseous HCl was bubbled into an ethereal solution of the

crude. 68 was isolated after solvent removal in the form of a

hydrochloride as a glassy solid (1.14 g, 67% yield). Anal.

(C₁₉H₃₁ClN₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(tetrahydropyran-4-yl)pro-

pionamide (69). Tetrahydropyran-4-ylamine was prepared

starting from commercial 4-chlorotetrahydropyran by treat-

ment with phthalimide⁵⁰and, in the next step, with hydra-

1

zine⁵¹(80% yield). H NMR (CDCl₃) δ 3.95 (m, 2H), 3.40 (m,

2H), 3.11 (m, 1H), 1.90 (m, 2H), 1.55 (m, 2H), 1.40 (bs, 2H,

NH₂). Following the same procedure described for 29 and

starting from 3 (1.03 g, 5 mmol) and tetrahydropyran-4-

ylamine (0.505 g, 5 mmol), after workup and purification by

pulping in n-hexane, 69 was obtained as a glassy solid (0.79

g, 55% yield). Anal. (C₁₈H₂₇NO₂) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-((R)-2-methoxy-1-methyl-

ethyl)propionamide (61). Following the same procedure

described for 60 and starting from 59 (0.4 g, 1.5 mmol), after

workup and purification by flash chromatography (n-hexane/

EtOAc 1:1), 61 was obtained as a waxy solid (0.31 g, 75% yield).

Anal. (C₁₇H₂₇NO₂) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(3-dimethylaminopropyl)-

propionamide Hydrochloride (62). Following the same

procedure described for 29 and starting from 3 (2 g, 9.6 mmol)

and 3-(dimethylamino)propylamine (1.22 mL, 9.6 mmol), after

workup gaseous HCl was bubbled into an ethereal solution of

the crude. 62 was isolated by filtration in the form of a

hydrochloride as a white powder (2.45 g, 78% yield). Anal.

(C₁₈H₃₁ClN₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(1-methylpirrolidin-3-yl)-

propionamide (70). 1-Methylpyrrolidin-3-ylamine was pre-

pared starting from commercial 1-methyl-3-pyrrolidinol trans-

formed into the corresponding bromide according a described

procedure⁵²and then treated with phthalimide⁵⁰and, in the

next step, with hydrazine⁵¹to afford the pure amine as a

colorless oil (75% yield). ¹H NMR (CDCl₃) δ 3.11 (m, 1H), 2.55

(m, 4H), 2.35 (s, 3H), 1.90 (m, 2H), 1.45 (bs, 2H, NH₂).

Following the same procedure described for 29 and starting

from 3 (1.03 g, 5 mmol) and 1-methylpyrrolidin-3-ylamine (0.50

g, 5 mmol), after workup and purification by pulping in

isopropyl ether, 70 was obtained as a waxy solid (0.72 g, 50%

yield). Anal. (C₁₈H₂₈N₂O) C, H, N.

(R)-2-(4-Isobutylphenyl)-N-(tetrahydrofuran-3-yl)pro-

pionamide (71). Tetrahydrofuran-3-ylamine was prepared

starting from commercial 3-hydroxytetrahydrofuran. After its

transformation into the bromide derivative according a de-

scribed procedure,⁵²the following treatments with phthal-

(R)-2-(4-Isobutylphenyl)-N-(3-dimethylaminoethyl)-

propionamide (63). Following the same procedure described

for 29 and starting from 3 (2 g, 9.6 mmol) and 2-(dimethyl-

amino)ethylamine (1.12 mL, 9.6 mmol), after workup 63 was

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4327

imide⁵⁰and, in the next step, with hydrazine⁵¹afforded the

chromatography (n-hexane/EtOAc 9:1) to give methyl 2-[4-

(cyclohexylmethyl)phenyl]propanoate (6 g, 85% yield) as a

colorless oil. The methyl ester hydrolysis was performed as

described for 79. Pure 80 was isolated as a slightly white

powder (5.1 g, 90% yield). Anal. (C₁₆H₂₂O₂) C, H.

1

pure amine as a colorless oil (70% yield). H NMR (CDCl₃) δ

3.85 (m, 2H), 3.40 (m, 2H), 3.12 (m, 1H), 2.10 (m, 1H), 1.85

(m, 1H), 1.45 (bs, 2H, NH₂). Following the same procedure

described for 29 and starting from 3 (1.03 g, 5 mmol) and

tetrahydrofuran-3-ylamine (0.43 g, 5 mmol), after workup and

purification by pulping in n-hexane, 71 was obtained as a waxy

solid (0.8 g, 58% yield). Anal. (C₁₇H₂₅NO₂) C, H, N.

(R)-3-(4-Isobutylphenyl)butan-2-one (72). 72 was pre-

pared according to a described procedure⁵³starting from 3 (4.8

g, 23.2 mmol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (3.34 g,

23.2 mmol). 72 was isolated as a yellow oil (3.5 g, 74%). Anal.

(C₁₄H₂₀O) C, H.

(S)-2-(4-Isobutylphenyl)propionamide (73). Following

the same procedure described for 29 and starting from 4 (2.47

g, 12 mmol) and gaseous ammonia, after workup and purifica-

tion by crystallization from EtOAc, 73 was obtained as a white

powder (1.23 g, 50% yield). Anal. (C₁₃H₁₉NO) C, H, N.

Synthesis of 2-Arylpropionic Acids (Schemes 3-6).

2-(4-Isopropylphenyl)propanoic Acid (77). 115 (5 g, 28.41

mmol) was transformed into the methyl 2-(4-isopropylphenyl)-

propanoate with I₂and AgNO₃following a described proce-

dure³¹and obtained pure enough for the following step of

hydrolysis. The methyl ester was dissolved in MeOH (20 mL)

and treated with 32% NaOH (1 equiv) at reflux. After 4 h, the

solvent was evaporated and the residue was diluted with

CH₂Cl₂and extracted with 1 N NaOH. A 37% HCl solution

was added to the basic aqueous phase until the complete

precipitation of 77 as a pale-yellow powder (2.18 g, 40% yield).

Anal. (C₁₂H₁₆O₂) C, H.

2-(4-Benzylphenyl)propanoic Acid (81). Following the

same procedure described for the 80 and starting from 117 (5

g, 27.1 mmol) and commercial benzylzinc bromide (0.5 M in

THF) (9.61 g, 40.65 mmol), after workup and following

hydrolysis of the methyl ester intermediate, 81 was isolated

as a white powder (5.21 g, 80% yield). Anal. (C₁₆H₁₆O₂) C, H.

2-(4-Isopropoxyphenyl)propanoic Acid (82). To a solu-

tion of 2-(4-hydroxy)phenylpropionic acid (84) (50 g, 0.29 mol)

in CH₃OH (250 mL), concentrated H₂SO₄(0.4 mL) was added.

The resulting solution was left stirring overnight at room

temperature. After solvent evaporation the oily residue was

diluted with CH₂Cl₂and washed with a saturated solution of

NaHCO₃(2 × 100 mL) and water (3 × 150 mL), dried over

Na₂SO₄, and evaporated to give methyl 2-(4-hydroxy)phenyl-

propanoate (51.6 g, 0.28 mol). To a solution of the methyl ester

(2.0 g, 10.85 mmol) in acetone (10 mL), K₂CO₃(1.5 g, 10.85

mmol) was added. The mixture was left stirring for 1 h at room

temperature. Then 2-iodopropane (1.08 mL, 10.85 mmol) was

added by dripping, and the resulting mixture was left under

stirring overnight at room temperature. The solid was filtered

off, and acetone was evaporated under reduced pressure to give

a crude that was dissolved in CH₂Cl₂(15 mL). The organic

layer was washed with 0.5 N NaOH (2 × 10 mL) and water (2

× 10 mL), dried over Na₂SO₄, and evaporated to give the

intermediate methyl 2-(4-isopropoxyphenyl)propanoate as a

colorless oil (1.97 g, 8.73 mmol). The methyl ester was

dissolved in MeOH (10 mL), a 20% alcoholic solution of KOH

(5 mL) was added, and the resulting mixture was left stirring

for 4 h at room temperature. After solvent evaporation under

reduced pressure, the residue was diluted with CH₂Cl₂(10

mL). The organic layer was extracted with 1 N NaOH (2 × 5

mL), and 37% HCl was added to the collected aqueous extracts

until precipitation occurred. 82 was isolated by filtration as a

white powder (1.67 g, 92% yield). Anal. (C₁₂H₁₆O₃) C, H.

2-(4-Methoxyphenyl)propanoic Acid (83). Following the

same procedure described for the 82 and starting from methyl

2-(4-hydroxy)phenylpropanoate (2.0 g, 10.85 mmol) and iodo-

methane (0.67 mL, 10.85 mmol), after workup and following

hydrolysis of the methyl ester intermediate, 83 was isolated

as a white powder (1.86 g, 95% yield). Anal. (C₁₀H₁₂O₃) C, H.

2-(1,1′-Biphenyl-4-yl)propanoic Acid (85). Following the

same procedure described for 80 and starting from 117 (5 g,

27.1 mmol) and commercial phenylzinc bromide (0.5 M in THF)

(9.04 g, 40.65 mmol), after workup and following hydrolysis

of the methyl ester intermediate, 85 was isolated as a white

powder (3.37 g, 55% yield). Anal. (C₁₅H₁₄O₂) C, H.

2-(4-tert-Butylphenyl)propanoic Acid (78). Following

the same procedure described for 77, 116 (5 g, 26.28 mmol)

was transformed into 78 and isolated pure as a white powder

(2.06 g, 38% yield). Anal. (C₁₃H₁₈O₂) C, H.

2-{4[(1E/Z)-Prop-1-enyl]phenyl}propanoic Acid (79).

To a solution of 117 (0.26 g, 1.12 mmol) in anhydrous 1-methyl-

2-pyrrolidinone (3 mL) under nitrogen and vigorous stirring,

LiCl (0.142 g, 3.366 mmol), CuI (10 mg, 0.056 mmol), AsPh₃

(27 mg, 0.09 mmol), and Pd₂dba₃(21 mg, 0.02 mmol) were

added. After the mixture was stirred for 10 min, 1-propenyl-

tributyltin (prepared as previously described⁵⁴) (0.45 g, 1.35

mmol) was added and the reaction mixture was left stirring

for 5 h at 90 °C. After the mixture was cooled at room

temperature, a saturated solution of KF (10 mL) was added.

After stirring for 15 min′, water was added (5 mL). The

aqueous solution was extracted with Et₂O (3 × 10 mL), and

the collected organic extracts were washed with water (2 ×

10 mL), dried over Na₂SO₄, and evaporated under reduced

pressure to give a crude residue that, after purification by flash

chromatography (n-hexane/EtOAc 9:1), afforded methyl 2-[(4-

prop-1-enyl)phenyl]propanoate as a yellow oil (0.17 g, 75%

yield). The methyl ester was dissolved in MeOH (2 mL), a 20%

alcoholic solution of KOH (1 mL) was added, and the resulting

mixture was left stirring overnight at room temperature. After

solvent evaporation the residue was diluted with water (5 mL)

and washed with Et₂O (2 × 5 mL). The aqueous phase was

acidified with 1 N HCl (2 × 5 mL) to pH 1 and extracted with

EtOAc (2 × 10 mL). The organic collected extracts were dried

over Na₂SO₄and evaporated under reduced pressure to give

a crude residue that, after purification by flash chromatogra-

phy (n-hexane/EtOAc 1:1), afforded pure 79 as a pale-yellow

oil (0.14 g, 90% yield) as a 3:1 mixture of E/Z isomers. Anal.

(C₁₂H₁₄O₂) C, H.

2-(4-Thien-2-ylphenyl)propanoic Acid (86). Following

the same procedure described for 80 and starting from 117 (5

g, 27.1 mmol) and 2-thienylzinc bromide (0.5 M in THF) (9.28

g, 40.65 mmol), after workup and following hydrolysis of the

methyl ester intermediate, 86 was isolated as a waxy solid

(5.35 g, 85% yield). Anal. (C₁₃H₁₂O₂S) C, H, S.

2-(3-Ethylphenyl)propanoic Acid (88). Following the

same procedure described for 79 and starting from 119 (0.35

g, 1.12 mmol) and tetraethyltin (0.27 mL, 1.35 mmol), after

workup and following hydrolysis of the methyl ester interme-

diate, 88 was isolated as a colorless oil (0.11 g, 85% yield).

Anal. (C₁₁H₁₄O₂) C, H.

2-(3-Butylphenyl)propanoic Acid (89). Following the

same procedure described for 79 and starting from 119 (0.35

g, 1.12 mmol) and commercial tetrabutyltin (0.44 mL, 1.35

mmol), after workup and following hydrolysis of the methyl

ester intermediate, 89 was isolated as a pale-yellow oil (0.15

g, 64% yield from 119). Anal. (C₁₃H₁₈O₂) C, H.

2-(3-Isopropylphenyl)propanoic Acid (90). To palladium

on charcoal (10% Pd, 25 mg) a solution of 120 (0.2 g, 1 mmol)

in EtOH (5 mL) was added, and the mixture was stirred under

hydrogen until complete disappearance (4 h) of the starting

material (TLC). After filtration on a Celite pad, concentration

2-[4-(Cyclohexylmethyl)phenyl]propanoic Acid (80).

To a solution of 117 (5 g, 27.1 mmol) in dry THF (20 mL) under

nitrogen atmosphere, LiCl (3.45 g, 81.4 mmol) and Pd(PPh₃)₄

(1.25 g, 1.084 mmol) were added. After the mixture was stirred

for 10 min′ at room temperature, (cyclohexylmethyl)zinc

bromide (0.5 M in THF) (9.85 g, 40.65 mmol) was added, and

the resulting mixture was refluxed overnight. After the

mixture was cooled at room temperature, the solvent was

evaporated and the residue was diluted with EtOAc (20 mL),

washed with water (2 × 20 mL), dried over Na₂SO₄, and

evaporated to give an oily crude that was purified by flash

4328 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Allegretti et al.

of the mother liquors gave pure methyl 2-(3-isopropylphenyl)-

propanoate (0.19 g, 95% yield). Hydrolysis of the methyl ester

was performed as described for 79. Pure 90 was isolated by

flash chromatography (n-hexane/EtOAc 1:1) as a colorless oil

(0.16 g, 90% yield from 120). Anal. (C₁₂H₁₆O₂) C, H.

2-(3-Isobutylphenyl)propanoic Acid (91). To a solution

of 119 (0.35 g, 1.12 mmol) in dry THF (5 mL) under nitrogen

atmosphere, Pd(PPh₃)₄(52 mg, 0.045 mmol) was added. After

the mixture was stirred for 10 min at room temperature,

triisobutylaluminum (1.0 M in hexanes) (1.68 mL, 1.68 mmol)

was added. The resulting mixture was refluxed for 24 h. After

the mixture was cooled at room temperature, the solvent was

evaporated and the residue was diluted with EtOAc (10 mL),

washed with water (2 × 10 mL), dried over Na₂SO₄, and

evaporated under reduced pressure to give an oily crude that

was purified by flash chromatography (n-hexane/EtOAc 95:5)

to give methyl 2-(3-isobutylphenyl)propanoate (0.185 g, 75%

yield) as a colorless oil. The following hydrolysis of the methyl

ester was performed as described for 79. Pure 91 was isolated

by flash chromatography (n-hexane/EtOAc 1:1) as a colorless

oil (0.16 g, 95% yield from 119). Anal. (C₁₃H₁₈O₂) C, H.

2-(3-sec-Butylphenyl)propanoic Acid (92). Following the

same procedure described for 90 and starting from 121 (0.2 g,

0.92 mmol), after workup and following hydrolysis of the

methyl ester intermediate, 92 was isolated as a colorless oil

(0.16 g, 85% yield from 121). Anal. (C₁₃H₁₈O₂) C, H.

2-[3-(1-Ethylpropyl)phenyl]propanoic Acid (93). Fol-

lowing the same procedure described for the 90 and starting

from 122 (0.13 g, 0.56 mmol), after workup and following

hydrolysis of the methyl ester intermediate, 93 was isolated

as a colorless oil (86 mg, 70% yield from 122). Anal. (C₁₄H₂₀O₂)

C, H.

2-{3-[(1E/Z)-Prop-1-enyl]phenyl}propanoic Acid (94).

Intermediate 123 (0.15 g, 0.73 mmol) was treated with KOH

as described for 79, and after workup, 94 was isolated as a

pale-yellow oil (0.13 g, 70% yield from 123) as a 3:1 mixture

of E/Z isomers. Anal. (C₁₂H₁₄O₂) C, H.

2-(3-Benzylphenyl)propanoic Acid (95). Following the

same procedure described for 80 and starting from 119 (2.5 g,

13.55 mmol) and commercial benzylzinc bromide (0.5 M in

THF) (4.8 g, 20.32 mmol), after workup and following hydroly-

sis of the methyl ester intermediate, compound 95 was isolated

as a colorless oil (2.41 g, 74% yield from 119). Anal. (C₁₆H₁₆O₂)

C, H.

2-[3-(1-Phenylethyl)phenyl]propanoic Acid (96). Fol-

lowing the same procedure described for 90 and starting from

124 (0.15 g, 0.56 mmol), after workup and following hydrolysis

of the methyl ester intermediate, compound 96 was isolated

as a colorless oil (0.12 g, 84% yield from 124). Anal. (C₁₇H₁₈O₂)

C, H.

extracted with CH₂Cl₂(2 × 10 mL). The collected organic

extracts were washed with water (2 × 5 mL), dried over

Na₂SO₄, and evaporated under reduced pressure to give 98 as

a pale-yellow oil (0.41 g, 94% yield). Anal. (C₁₁H₁₄O₃) C, H.

2-(3-Acetylphenyl)propanoic Acid (99). Following the

same procedure described for 98 and starting from 125 (1 g,

5.8 mmol), after workup, 99 was isolated as a pale-yellow solid

(1.05 g, 94% yield). Anal. (C₁₁H₁₂O₃) C, H.

3-(1-Carboxyethyl)benzoic Acid (100). Following the

same procedure described for 98 and starting from commercial

2-(3-carboxyphenyl)propanenitrile (2.5 g, 14 mmol), after

workup, 100 was isolated as a white powder (2.5 g, 92% yield).

Anal. (C₁₀H₁₀O₄) C, H.

2-{3-[Hydroxyl(phenyl)methyl]phenyl}propanoic Acid

(101). Methyl 2-(3-benzoyl)phenylpropanoate (0.99 g, 3.7

mmol), prepared as described for 82 starting from acid 87, was

hydrogenated following the procedure described for 90. For our

purposes triethylamine (1:1 with Pd w/w) was added. After

workup and following basic hydrolysis of the intermediate

ester, 101 was isolated by flash chromatography ((n-hexane/

EtOAc 95:5) as a white powder (0.78 g, 78%). Anal. (C₁₆H₁₆O₂)

C, H.

(R)-2-(3-Isopropylphenyl)propionamide (102). Follow-

ing the same procedure described for 31 and starting from the

(R)-enantiomer of 90 (30% yield, [R]²⁵-56° (c 0.2, absolute

D

EtOH)) (35 mg, 0.18 mmol) and gaseous ammonia, after

workup, 102 was obtained as a colorless oil (20 mg, 58% yield).

Anal. (C₁₂H₁₇NO) C, H, N.

(R)-2-[3-(1-Ethylpropyl)phenyl]-N-(3-dimethylamino-

propyl)propionamide Hydrochloride (103). Following the

same procedure described for 62 and starting from the (R)-

enantiomer of 93 (34% yield, [R]²⁵-28° (c 0.5, EtOH)) (80

D

mg, 0.36 mmol) and 3-(dimethylamino)propylamine (46 µL,

0.36 mmol), after workup and purification, 103 was obtained

in the form of a hydrochloride as a colorless oil (84 mg, 68%

yield). Anal. (C₁₉H₃₃ClN₂O) C, H, N.

(R)-2-[3-Isopropyl)phenyl]-N-(3-dimethylaminopro-

pyl)propionamide (104). Following the same procedure

described for 62 and starting from the (R)-enantiomer of 90

(80 mg, 0.41 mmol) and 3-(dimethylamino)propylamine (50 µL,

0.41 mmol), after workup and purification by flash chroma-

tography (CH₂Cl₂/CH₃OH 7:3), 104 was obtained as a colorless

oil (65 mg, 57% yield). Anal. (C₁₇H₂₈N₂O) C, H, N.

N-[(R)-2-[3-(1-Phenylethyl)phenyl]propionylmethane-

sulfonamide (105). Following the same procedure described

for 11 and starting from 127 (89 mg, 0.35 mmol) and methane-

sulfonamide (33 mg, 0.35 mmol), after workup and purification

by pulping in diisopropyl ether, 105 was obtained as a white

powder (34 mg, 30% yield). Anal. (C₁₈H₂₁NO₃S) C, H, S, N.

(R)-2-[3-(1-Phenylethyl)phenyl]-N-(3-dimethylamino-

propyl)propionamide (106). Following the same procedure

described for 62 and starting from the 127 (56 mg, 0.22 mmol)

and 3-(dimethylamino)propylamine (27 µL, 0.82 mmol), after

workup and purification by flash chromatography (CH₂Cl₂/

CH₃OH 85:15), 106 was obtained as a colorless oil (46 mg, 62%

yield). Anal. (C₂₂H₃₀N₂O) C, H, N.

[(R)-2-(3-Benzoylphenyl)propionylamino]acetic Acid

(107). 107 was prepared according to the procedures described

for the synthesis of 45 starting from 1 (0.55 g, 2.16 mmol).

107 was isolated as a glassy solid (0.31 g, 44% yield from 1).

Anal. (C₁₈H₁₇NO₄) C, H, N.

(R)-2-(3-Benzoylphenyl)-N-(3-dimethylaminopropyl)-

propionamide (108). Following the same procedure described

for 62 and starting from 1 (1 g, 3.93 mmol) and 3-(dimethyl-

amino)propylamine (0.49 mL, 3.93 mmol), after workup and

purification by flash chromatography (CH₂Cl₂/CH₃OH 8:2), 108

was obtained as a colorless oil (0.55 g, 41% yield). Anal.

(C₂₁H₂₆N₂O₂) C, H, N.

2-[3-(1-Hydroxypropyl)phenyl]propanoic Acid (97). To

a cooled (0 °C) solution of 123 (0.1 g, 0.49 mmol) in dry THF

(5 mL) under nitrogen, a borane/methyl sulfide complex (5.0

M in Et₂O) (0.11 mL) was added, and the solution was left

stirring for 3 h at 0 °C. Water (0.5 mL) was added by dripping,

and after the mixture was stirred for 20 min′, 38% H₂O₂(0.53

mL) and 1 N NaOH (1.78 mL) were added. After the mixture

was stirred for 30 min at room temperature, the solution was

diluted with water (2 mL) and 1 N HCl was added to obtain

pH 1. The aqueous phase was extracted with EtOAc (2 × 5

mL), and the collected organic extracts were dried over Na₂SO₄

and evaporated under reduced pressure to give an oily crude

that was purified by flash chromatography (n-hexane/EtOAc

8:2) to give methyl 2-[3-(1-hydroxypropyl)phenyl]propanoate

(0.087 g, 80% yield) as a colorless oil. The following methyl

ester hydrolysis was performed as described for 79. Pure 97

was isolated by flash chromatography (n-hexane/EtOAc 1:1)

as a colorless oil (0.077 g, 95% yield from 123). Anal. (C₁₂H₁₆O₃)

C, H.

Synthesis of Intermediates. Methyl (2R)-2-{[(2R)-2-(4-

Isobutylphenyl)propanoyl]amino]propanoate (109). 109

was prepared as described for 37 starting from 3 (2.5 g, 12.13

mmol) and from a mixture of ^D-alanine methyl ester hydro-

chloride (1.69 g, 12.13 mmol) and triethylamine (1.69 mL,

12.13 mmol) in DMF (15 mL). After workup and purification

2-[3-(1-Hydroxyethyl)phenyl]propanoic Acid (98). To

a solution of 126 (0.4 g, 2.3 mmol) in 1.4-dioxane (3 mL), 37%

HCl (2.1 mL) was added, and the resulting solution was

refluxed for 5 h. After the mixture was cooled at room

temperature, the crude was diluted with water (10 mL) and

CXCL8 Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4329

by pulping in n-hexane, 109 was obtained as a white powder

evaporated under reduced pressure to give 115 as a colorless

oil (6.03 g, 33.64 mmol). ¹H NMR (CDCl₃) δ 7.95 (d, 2H, J ) 7

Hz), 7.48 (d, 2H, J ) 7 Hz), 3.00 (q, 2H, J ) 7 Hz), 2.75 (m,

1H), 1.20 (d, 6H, J ) 3 Hz), 1.15 (t, 3H, J ) 7 Hz).

1-(4-tert-Butylphenyl)propan-1-one (116). Following the

same procedure described for 115 but starting from tert-

butylbenzene (5 g, 37.25 mmol), 116 was obtained as a

colorless oil (5.90 g, 31 mmol). ¹H NMR (CDCl₃) δ 7.95 (d, 2H,

J ) 7 Hz), 7.48 (d, 2H, J ) 7 Hz), 3.00 (q, 2H, J ) 7 Hz), 1.35

(s, 9H), 1.15 (t, 3H, J ) 7 Hz).

1

(4.97 g, 70% yield), mp 100-102 °C. H NMR (CDCl₃) δ 7.20

(d, 2H, J ) 7 Hz), 7.05 (d, 2H, J ) 7 Hz), 5.90 (bs, 1H, CONH),

4.60 (m, 1H), 3.75 (s, 3H), 3.58 (q, 1H, J ) 7 Hz), 2.48 (d, 2H,

J ) 7 Hz), 1.90 (m, 1H), 1.55 (d, 3H, J ) 7 Hz), 1.30 (d, 3H, J

) 7 Hz), 0.95 (d, 6H, J ) 7 Hz).

Methyl 3-Hydroxy-2-{[(2R)-2-(4-isobutylphenyl)propan-

oyl]amino}propanoate (110). 110 was prepared as described

for 37 starting from 3 (1.5 g, 7.30 mmol) and from a mixture

of ^L-serine methyl ester hydrochloride (1.13 g, 7.30 mmol) and

triethylamine (1.02 mL, 7.30 mmol). After workup and puri-

fication by pulping in Et₂O, 110 was obtained as a colorless

Methyl 2-{4-[(Trifluoromethanesulfonyloxy)phenyl}-

propanoate (117). To a cooled solution (-15 °C) of methyl

2-(4-hydroxy)phenylpropanoate, prepared as described for 82

(48.5 g, 0.26 mol) in dry CH₂Cl₂(500 mL), N,N-diisopropyl-

ethylamine (53.5 mL, 0.31 mol) was added by dripping for 10

min′. The resulting solution was left stirring for 30 min′ at

-15 °C, and then trifluoromethanesulfonic anhydride (47.6

mL, 0.28 mol) was dropped and the mixture was left stirring

for 1 h at -15 °C and overnight at room temperature. The

mixture was transferred into a separatory funnel and washed

with 1 N HCl (2 × 200 mL), 1 N NaOH (2 × 150 mL), and

water (3 × 200 mL), dried over Na₂SO₄, and evaporated under

vacuum to give 117 as dark, oily residue (80 g) pure enough

to be used in the following step. An aliquot was purified by

flash chromatography (CH₂Cl₂/CH₃OH 98:2) for analytical

1

oil (1.63 g, 73% yield). H NMR (CDCl₃) δ 7.20 (d, 2H, J ) 7

Hz), 7.10 (d, 2H, J ) 7 Hz), 6.45 (bs, 1H, CONH), 4.55 (m,

1H), 4.05 (m, 1H), 3.75 (s, 3H), 3.60 (m, 2H), 2.48 (d, 2H, J )

7 Hz), 2.30 (bs, 1H, OH), 1.85 (m, 1H), 1.48 (d, 3H, J ) 7 Hz),

0.95 (d, 6H, J ) 7 Hz).

Methyl 2-{[(2R)-2-(4-Isobutylphenyl)propanoyl]amino}-

3-mercaptopropanoate (111). 111 was prepared as de-

scribed for 37 starting from 3 (1.5 g, 7.30 mmol) and from a

mixture of l-cysteine methyl ester hydrochloride (1.36 g, 7.30

mmol) and triethylamine (1.02 mL, 7.30 mmol). After workup

and purification by pulping in n-hexane, 111 was obtained as

1

a colorless oil (1.36 g, 60% yield). H NMR (CDCl₃) δ 7.20 (d,

2H, J ) 7 Hz), 7.05 (d, 2H, J ) 7 Hz), 6.45 (bs, 1H, CONH),

4.90 (m, 1H), 4.25 (s, 3H), 3.75 (q, 1H, J ) 7 Hz), 3.15 (dd, 2H,

J₁) 10 Hz, J₂) 6 Hz), 2.60 (d, 2H, J ) 7 Hz), 2.05 (bs, 1H,

SH), 1.90 (m, 1H), 1.65 (d, 3H, J ) 7 Hz), 1.05 (d, 6H, J ) 7

Hz).

1

characterization. H NMR (CDCl₃) δ 7.45 (d, 2H, J ) 7 Hz),

7.25 (d, 2H, J ) 7 Hz), 3.80 (q, 1H, J ) 7 Hz), 3.65 (s, 3H),

1.50 (d, 3H, J ) 7 Hz).

Methyl 2-{3-[(Trifluoromethanesulfonyloxy)phenyl}-

acetate (118). Following the same procedure described for 117

but starting from commercial (3-hydroxyphenyl)acetic acid (50

g, 0.32 mol), 118 was obtained as a dark-red oil (84 g) pure

enough to be used in the following step. An aliquot was purified

by flash chromatography (CH₂Cl₂/CH₃OH 95:5) for analytical

(R)-2-(4-Isobutylphenyl)-N-(3-tert-butoxycarbonyl-

aminopropyl)propionamide (112). N-Boc-(3-aminopropyl)-

amine was prepared as described⁵¹starting from 1,3-bromo-

propane. Pure N-Boc-(3-aminopropyl)amine was obtained as

a pale-yellow oil (80% yield). ¹H NMR (CDCl₃) δ 4.90 (bs, 1H,

CONH), 3.35 (m, 2H), 2.83 (t, 2H, J ) 7 Hz), 1.68 (d, 2H, J )

7 Hz), 1.50 (s, 9H). Following the same procedure described

for 29 and starting from 3 (3.6 g, 17.5 mmol) and N-Boc-(3-

aminopropyl)amine (3.7 g, 17.5 mmol), after workup and

purification by crystallization from MeOH, 112 was obtained

as a white powder (4.8 g, 13.06 mmol). ¹H NMR (CDCl₃) δ 7.25

(d, 2H, J ) 7 Hz), 7.12 (d, 2H, J ) 7 Hz), 5.95 (bs, 1H, NHCO),

4.95 (bs, 1H, CONH), 3.55 (q, 1H, J ) 7 Hz), 3.25 (t, 2H, J )

5 Hz), 3.08 (t, 2H, J ) 5 Hz), 2.45 (d, 2H, J ) 7 Hz), 1.88 (m,

1H), 1.52-1.47 (m, 5H), 1.42 (s, 9H), 0.90 (d, 6H, J ) 7 Hz).

1

characterization. H NMR (CDCl₃) δ 7.45-7.30 (m, 2H), 7.25

(s, 1H), 7.18 (m, 1H), 3.70 (s, 3H), 3.45 (s, 2H).

Methyl 2-{3-[(Trifluoromethanesulfonyloxy)phenyl}-

propanoate (119). To a cooled (-25 °C) solution of crude 118

(82 g, 0.275 mol) in dry THF (500 mL), 60% NaH (11 g, 0.46

mol) was added portionwise. The resulting mixture was left

stirring for 1 h at room temperature and then cooled again at

-25 °C, and iodomethane (20.54 mL, 0.33 mol) was added by

dripping. The mixture was left to warm to 0 °C in 3 h and

was stirred overnight at 0 °C. Glacial AcOH (26.3 mL, 0.46

mol) was added to the mixture, and THF was evaporated. The

oily crude was dissolved in CH₂Cl₂(600 mL), washed with 1

N NaOH (2 × 250 mL) with a saturated solution of NH₄Cl

(300 mL) and with a saturated solution of NaCl (300 mL), dried

over Na₂SO₄, and evaporated under reduced pressure to give

a red crude oil (71.6 g). The oil was purified by flash

chromatography (n-hexane, n-hexane/EtOAc 8:2). 119 was

(3-Aminopropyl)methylcarbamic Acid tert-Butyl Ester

(113). Starting from N-Boc-(3-aminopropyl)amine, the amino

group was protected as described⁵⁰to afford [3-(1,3-dioxo-1,3-

dihydroisoindol-2-yl)propyl]carbamic acid tert-butyl ester. The

compound was treated with NaH/CH₃I (1 equiv), and the

resulting N-methyl derivative was treated with hydrazine⁵¹

1

to give 113 as a colorless oil (80% yield). H NMR (CDCl₃) δ

3.52 (t, 2H, J ) 5 Hz), 3.25 (s, 3H), 2.72 (m, 2H), 2.25 (bs, 2H,

NH₂), 1.70 (m, 2H), 1.50 (s, 9H).

1

obtained as a yellow oil (42.2 g, 49% yield). H NMR (CDCl₃)

δ 7.45-7.30 (m, 2H), 7.25 (s, 1H), 7.18 (m, 1H), 3.85 (q, 1H, J

) 7 Hz), 3.70 (s, 3H), 1.55 (d, 3H, J ) 7 Hz).

{3-[(R)-2-(4-Isobutylphenyl)propionylamino]propyl}-

methylcarbamic Acid tert-Butyl Ester (114). Following the

same procedure described for 29 and starting from 3 (1.8 g,

8.75 mmol) and 113 (1.64 g, 8.75 mmol), after workup and

purification by flash chromatography (CH₂Cl₂/CH₃OH 98:2),

114 was obtained as a colorless oil (2.25 g, 6 mmol). ¹H NMR

(CDCl₃) δ 7.50 (bs, 1H, CONH), 7.32 (d, 2H, J ) 7 Hz), 6.83

(d, 2H, J ) 7 Hz), 3.64 (q, 1H, J ) 7 Hz), 3.41 (t, 2H, J ) 5

Hz), 3.30 (t, 2H, J ) 5 Hz), 3.25 (s, 3H), 2.43 (d, 2H, J ) 7

Hz), 1.97 (m, 2H), 1.82 (m, 1H), 1.50 (s, 9H), 1.35 (d, 3H, J )

7 Hz), 0.90 (d, 6H, J ) 7 Hz).

1-(4-Isopropylphenyl)propan-1-one (115). To a solution

of cumene (5.18 mL, 37.25 mmol) in dry CH₂Cl₂(50 mL),

propionyl chloride (3.23 mL, 37.25 mmol) was added dropwise.

The mixture was cooled to 0-5 °C, and AlCl₃(4.97 g, 37.25

mmol) was added portionwise. The resulting mixture was

refluxed for 3 h. After cooling at room temperature, the

mixture was poured into a 1 N HCl/ice mixture and left stirring

for 30 min′. The two phases were separated, and the aqueous

one was extracted twice with CH₂Cl₂. The collected organic

extracts were washed with water, dried over Na₂SO₄, and

Methyl 2-(3-Isopropenylphenyl)propanoate (120). 120

was prepared according to the same procedure described for

the preparation of 79 starting from 119 (0.35 g, 1.12 mmol)

and from isopropenyltributyltin (prepared as described⁵⁴). It

was isolated by flash chromatography as a colorless oil (0.2 g,

90% yield). ¹H NMR (CDCl₃) δ 7.40-7.28 (m, 2H), 7.20 (s, 1H),

7.15 (m, 1H), 5.30 (s, 1H), 5.02 (s, 1H), 3.75 (q, 1H, J ) 7 Hz),

3.65 (s, 3H), 1.55 (d, 3H, J ) 7 Hz), 1.45 (s, 3H).

Methyl 2-{3-[(1E/Z)-1-Methylprop-1-enyl]phenyl}pro-

panoate (121). 121 was prepared according to the same

procedure described for the preparation of 79 starting from

119 (0.35 g, 1.12 mmol) and from 1-methylprop-1-enyltri-

butyltin (prepared as previously described⁵⁴). It was isolated

(as a mixture of E/Z isomers) by flash chromatography as a

colorless oil (0.23 g, 95% yield). ¹H NMR (CDCl₃) δ 7.40-7.28

(m, 2H), 7.20 (s, 1H), 7.15 (m, 1H), 5.80 (d, 1H, J ) 7 Hz, Z

isomer), 5.36 (q, 1H, J ) 7 Hz, E isomer), 3.70 (q, 1H, J ) 7

Hz), 3.65 (s, 3H), 1.73 (dd, 3H, J ) 7 Hz), 1.50 (d, 3H, J ) 7

Hz), 1.45 (d, 3H, J ) 7 Hz).

4330 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Methyl 2-[3-(1E/Z)-1-Ethylprop-1-enyl)phenyl]pro-

Allegretti et al.

Supporting Information Available: Spectroscopic data

and results from elemental analysis of all the listed com-

pounds. This material is available free of charge via the

Internet at http://pubs.acs.org.

panoate (122). 122 was prepared according to the same

procedure described for the preparation of 79 starting from

119 (0.35 g, 1.12 mmol) and from pent-2-enyltributyltin. The

organotin reagent was prepared as previously described⁵⁵and

obtained pure (as a mixture of E/Z isomers) by purification

from the internal isomer. 122 was isolated by workup as a

colorless oil (0.14 g, 55% yield). ¹H NMR (CDCl₃) δ 7.40-7.28

(m, 2H), 7.20 (s, 1H), 7.15 (m, 1H), 5.84 (q, 1H, J ) 7 Hz, Z

isomer), 5.36 (q, 1H, J ) 7 Hz, E isomer), 3.70 (q, 1H, J ) 7

Hz), 3.65 (s, 3H), 2.02 (m, 2H), 1.84 (d, 3H, J ) 7 Hz), 1.50 (d,

3H, J ) 7 Hz), 1.05 (t, 3H, J ) 5 Hz).

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1

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2-[3-(1-Hydroxyethyl)phenyl]propanenitrile (126). To

a solution of 125 (0.38 g, 2.19 mmol) in THF (4 mL) and water

(1 mL), NaBH₄(95 mg, 2.51 mmol) was added. The mixture

was left stirring for 4 h at room temperature. AcOH (0.4 mL)

and a saturated solution of NaCl (5 mL) were added to the

mixture. The aqueous layer was extracted with Et₂O (2 × 10

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and evaporated under reduced pressure to give 126 as a

1

colorless oil (0.29 g, 78% yield). H NMR (CDCl₃) δ 7.35 (m,

4H), 4.95 (q, 1H, J ) 7 Hz), 4.10 (q, 1H, J ) 7 Hz), 1.75 (d,

3H, J ) 7 Hz), 1.55 (d, 3H, J ) 7 Hz).

(2R)-2-[3-(1-Phenylethyl)phenyl]propanoic Acid (127).

To a cooled (0-5 °C) solution of methyltriphenylphosphonium

bromide (2.04 g, 5.59 mmol) in dry THF (5 mL), butyllithium

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benzoylphenyl)propanoate (1.5 g, 5.59 mmol), prepared from

1 according to the procedure described for 82 in dry THF (5

mL). After the mixture was stirred overnight at room tem-

perature, the solution was diluted with CH₂Cl₂(10 mL). The

organic layer was washed with 10% K₂HPO₄buffer (3 × 15

mL) and water (2 × 20 mL), dried over Na₂SO₄, and evaporated

to give an oily crude that was purified by flash chromatography

(n-hexane/EtOAc 95:5) to give methyl (2R)-2-[3-(1-phenyl-

vinyl)phenyl]propanoate (1.06 g, 71% yield) as a colorless oil.

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hydrogenated for 18 h as described for 90. Pure methyl (2R)-

2-[3-(1-phenylethyl)phenyl]propanoate was isolated as a color-

less oil (0.36, 98% yield). The following ester hydrolysis was

performed as described for 79, but to avoid any racemization,

the solvent used was 1,4-dioxane. Pure 127 was obtained as a

1

colorless oil (0.29 g, 87% yield). [R]²⁵-15° (c 1, MeOH); H

D

NMR (CDCl3) δ 7.32-7.15 (m, 9H), 4.10 (q, 1H, J ) 7 Hz),

3.65 (q, 1H, J ) 7 Hz), 1.55 (d, 3H, J ) 7 Hz), 1.40 (d, 3H, J

) 7 Hz).

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JM049082I

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2-Arylpropionic CXC chemokine receptor 1 (CXCR1) ligands as novel noncompetitive CXCL8 inhibitors

DOI: 10.1021/jm049082i

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