SCH-527123

Discovery of 3,4-Diaminocyclobut-3-ene-1,2-dione-Based CXCR2 Receptor Antagonists for the Treatment of Inflammatory Disorders

Michael P. Dwyer* and Purakattle Biju

Merck Research Laboratories, 2015 Galloping Hill Road Kenilworth, NJ, 07033

Abstract: The CXC chemokine receptor 2 (CXCR2) has attracted a considerable amount of attention as a target for thera- peutic intervention due the key role this receptor plays in a number of inflammatory disorders. Over the past decade, sev- eral classes of potent, selective CXCR2 receptor antagonists have been developed as potential anti-inflammatory agents. These small-molecule chemokine receptor antagonists have demonstrated the ability to inhibit CXCR2-mediated recruit- ment of inflammatory cells in vitro as well as shown efficacy in vivo in various animal models of inflammation. In addi- tion, several of the most advanced CXCR2 receptor antagonists have recently demonstrated promising proof-of-activity results in early human clinical trials. This review details the discovery and development of the 3,4-diaminocyclobut-3- ene-1,2-dione-based CXCR2 receptor antagonist class including SCH 527123 which is currently in mid-stage clinical eva- luation. The medicinal chemistry efforts leading to the discovery of SCH 527123, the in vitro and in vivo pharmacology for this compound, and an overview of the clinical evaluation of SCH 527123 will also be discussed.
Keywords: CXCR2, 3, 4-diaminocyclobut-3-ene-1, 2-dione, SCH 527123.

INTRODUCTION
The recruitment of leukocytes to the sites of tissue dam- age is a normal response to fight infection and remove dam- aged cells. However, the uncontrolled recruitment of leuko- cytes to the site of inflammation can have serious physio- logical consequences to include tissue damage, delayed wound healing, and potential host death [1]. Leukocyte re- cruitment is controlled by the actions of both exogenous and endogenous chemotactic factors. Among some of the princi- ple endogenous chemotactic factors are chemokines or chemoattractant cytokines [2]. These chemokines are a fam- ily of small molecular weight (7-15 kDa) proteins that are subdivided by a distinctive array of four cysteine residues into three main classes, the CXC-chemokines, CC- chemokines, and CX3C-chemokines [3]. Interleukin-8 or IL- 8 (CXCL8) is a 72-amino acid protein that was the first member of the CXC chemokine family that was shown to play a critical role in the migration of neutrophils to sites of inflammation and tissue injury [4]. CXCL8 is known to bind to two G-protein coupled, seven transmembrane receptors which were cloned and identified as CXCR1 and CXCR2 [5,6]. CXCR2 binds with high affinity to CXCL8 as well as other ELR+ (Glu4-Leu5-Arg6)-containing chemokines such as GCP-2 (CXCL6), ENA-78 (CXCL5), and Gro-
(CXCL1) while CXCR1 is less promiscuous and binds to only CXCL8 and CXCL6 with high affinity [7]. Both CXCR1 and CXCR2 are expressed in high levels on human neutrophils but low levels of expression have also been found on fibroblasts, monocytes, endothelial, and melanoma cells [8]. While binding to CXCR1 is believed to be relevant for neutrophil activation and degranulation, chemokine bind- ing to the CXCR2 receptor is believed to be important for neutrophil chemotaxis [9]. When CXCL8 interacts with the

*Address correspondence to this author at the Merck Research Laboratories, 2015 Galloping Hill Road Kenilworth, NJ, 07033; Tel: (908)740-4478;
Fax: (908)740-7152; E-mail: [email protected]

CXCR2 and CXCR1 receptors on neutrophils, an intracellu- lar response occurs which includes calcium flux, degranula- tion, and subsequent chemotaxis [10]. It should be men- tioned that there are conflicting accounts in the literature about whether neutrophil chemotaxis is mediated by one or both of the CXCL8 receptors, CXCR1 and/or CXCR2 [11]. While the exact role of CXC chemokines such as CXLC8 and CXCL1 in human disease is largely undefined, these chemokines have been observed in association with both chronic and acute inflammatory conditions. In humans, ele- vated levels of CXCL8 and CXCL1 have been observed in individuals with arthritis, asthma, and COPD suggestive of the critical role that these chemokines may play in such processes [12]. Due to the central role that several CXC chemokines play in a variety of inflammatory disorders, the development of small-molecule chemokine receptor antago- nists may be of potential therapeutic value [13]. Further- more, the importance of small molecule antagonists of the CXCL8 receptors has been supported by the normal physiol- ogy of mouse gene CXCR2 knockouts [14].
In 1998, Widdowson and coworkers at SmithKline Beecham reported the first small molecule diaryl urea CXCR2 antagonists [15]. Since this initial report, a number of additional series of small molecule CXCR2 receptor an- tagonists have been reported over the past decade and several comprehensive review articles have appeared [16]. Further- more, the potential therapeutic value of a CXCR2 receptor antagonist for the treatment of a variety of inflammatory diseases has been described in several recent reviews [17]. In this review article, an overview of the Schering-Plough/ Pharmacopeia CXCR2 receptor antagonist medicinal chem- istry program from early medicinal chemistry efforts to the identification of the current clinical compound, SCH 527123, is provided. In addition, supporting preclinical data as well as early clinical evaluation of SCH 527123 is de- scribed.

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MEDICINAL CHEMISTRY EFFORTS TOWARDS CXCR2 RECEPTOR ANTAGONISTS
Due to the interest in the development of a CXCR2 re- ceptor antagonist for the treatment of inflammatory disor-

Table 1. Diaryl Cyclobutenedione Derivatives 3-11

O O

ders, initial screening of the Schering-Plough internal com- R N
pound collection with a CXCR2 biochemical assay was con- H
ducted which yielded very few quality hits. Owing to the description of diaryl urea-based CXCR2 receptor antagonists by workers at SmithKline Beecham [15], it was rationalized that replacement of the urea core might offer an opportunity to access a novel series of CXCR2 receptor antagonists in an expedited fashion. The initial focus of this approach centered around the utilization of the 3,4-diaminocyclobut-3-ene-1,2- dione motif to replace the urea functionality since it had been shown to be an effective replacement of a N-cyanoguanidine by workers at Wyeth working on potassium channel openers [18]. While this moiety had previously been reported to be an effective replacement of a -amino acid [19], there were no known examples of utilizing the 3,4-diaminocyclobut-3- ene-1,2-dione moiety as a replacement for a urea.
O2N
O

N N
H H

O2N

OH 1
O O

N N
H H
OH 2

Fig. (1). Urea and cyclobutenedione-based CXCR2 receptor an- tagonists.
Using the SmithKline Beecham CXCR2 urea derivative 1 as a starting point (Fig. 1), replacement of the central urea moiety with the 3,4-diaminocyclobut-3-ene-1,2-dione motif yielded compound 2 which demonstrated good affinity in the CXCR2 receptor binding assay (IC50 = 36 nM) [20]. Owing to the promising CXCR2 activity of this derivative, further SAR work was conducted around the diaryl cyclobutenedi- one series to determine what key structural features were required for potency (Table 1). In parallel to the CXCR2 urea series from SmithKline Beecham [15], the phenolic OH as well as two NH’s of compound 2 were required for CXCR2 potency as demonstrated by compounds 3 and 5 (Table 1) [20]. Additionally, an appropriately placed electron withdrawing group on the left-hand ring was required to mitigate the pKa of the phenolic group to maintain affinity for the CXCR2 receptor similar to observations made in the urea class of CXCR2 receptor antagonists [15]. Owing to concerns about the metabolic fate of the 4-NO2 group found in compound 2, the 3-dimethylcarboxamide derivative 11 (Table 1) was identified which possessed excellent in vitro potency (CXCR2 IC50 = 2 nM) as well as good functional activity in a CXCR2 chemotaxis assay (IC50 = 26 nM) [20].

na = not active up to > 10,000 nM

However, the 3,4-dianilinocyclobut-3-ene-1,2-dione se- ries (typified by 2) exhibited low chemical solubility and poor predicted absorption based upon Caco-2 calculations [20]. Furthermore, oral administration of 11 in rat led to poor compound exposure in the plasma which precluded further

preclinical evaluation of this CXCR2 receptor antagonist series. Owing to the initial optimization work described on the phenolic amide portion of compound 11 described in Table 1, further SAR exploration of the 3,4-diaminocyclo- but-3-ene-1,2-dione lead series focused upon modifications of the right-hand portion of 11 to identify non-anilinic motifs that might address some of the liabilities of the early ana- logs.

O O

N
N N
H H
O OH
11

ring were well-tolerated leading to derivatives with compa- rable binding affinities for the CXCR2 and CXCR1 receptors as compound 17 while maintaining good oral drug exposure in rat [22]. Replacement of the phenyl ring of 17 with either a 2-thienyl (20) or 2-furyl (21) motif led to comparable CXCR2 receptor affinities while improving the affinity for the CXCR1 receptor as illustrated in Table 3. Unfortunately, both of these heteroaryl derivatives suffered from poor bioavailability in rat which limited further profiling of these derivatives [22]. However, introduction of a 5-methyl sub- stituent onto the heterocyclic ring afforded compound 22 and 23 which demonstrated improved the drug exposure upon oral administration in rat versus the 5-H derivatives with a minimal effect on the binding affinities at both CXCR2 and CXCR1 receptors (Table 3). Compound 23 (also known as SCH 527123) exhibited excellent binding affinity at the

O O CXCR2 (IC50 = 2.6 nM) and the CXCR1 (IC50 = 36 nM)
receptors as well as excellent exposure of drug upon oral
N administration in rat [22] (Table 3). Furthermore, the 5-

N N
H H
O OH
12

Fig. (2). Aniline and branched alkyl cyclobutenedione derivatives.

Towards this end, initial structural modifications of com- pound 11 focused upon replacement of the right-hand aniline ring with either cycloalkyl or branched alkyl functionality and compound 12 emerged from these efforts (Fig. 2). This compound demonstrated good affinity for the CXCR2 recep- tor (IC50 = 15 nM) and good functional activity in a CXCR2 chemotaxis assay (IC50 = 19 nM) [20]. More importantly, compound 12 displayed improved organic solubility and better predicted absorption based upon Caco-2 data than the corresponding dianilino derivative 11 [20]. Oral administra- tion of compound 12 produced reasonable plasma exposure in rat which was a significant improvement from the earlier diaryl cyclobutenedione CXCR2 derivatives [21,22] (Table 2).
With compound 12 in hand, additional SAR work was conducted exploring non-anilinic right-hand side derivatives which quickly focused upon a series of benzyl derivatives (Table 2). While the initial benzyl derivative 13 possessed modest potency in the CXCR2 receptor binding assay, in- corporation of -benzylic substitution revealed a trend in terms of preferred stereochemistry for this series of com- pounds. While the enantiomeric -methyl derivatives 14 and 15 showed modest to comparable CXCR2 binding affinity respectively versus derivative 12, the ethyl-substituted de- rivatives 16 and 17 revealed a significant difference in the overall biological profile between the (R)- and (S)- enantiomers illustrated in Table 2 [22]. The (R)-enantiomer
17 demonstrated superior binding affinities for both the CXCR2 and CXCR1 receptors as well as a higher level of drug exposure upon oral administration in rat than the corre- sponding (S)-enantiomer 16 (Table 2). Based upon these SAR findings, chemistry efforts were focused upon explora- tion of substitution around the aromatic ring of 17 as well as potential replacement of the phenyl ring with various hetero- cycles. As illustrated in Table 3, both electron withdrawing
(18) and electron-donating (19) substituents on the phenyl

methylfuryl derivative 23 demonstrated a dose dependent inhibition of human polymorphoneutrophil (hPMN) chemo- taxis induced either by CXCL1 or CXCL8 that was superior to benzyl analog 17 in the same assay [22].
Using the 5-methylfuryl motif as a constant, the influence of variation of the (R)--substituent of compound 23 was briefly examined (Table 4). Several -branched alkyl or cy- cloalkyl sidechains demonstrated excellent affinity for both the CXCR2 and CXCR1 receptor albeit at the expense of oral exposure in rat as illustrated by compounds 25-27 in Table 4 [22]. Furthermore, compound 24 demonstrated dose dependent inhibition of human polymorphoneutrophil (hPMN) chemotaxis induced either by CXCL1 or CXCL8 but at a reduced level compared to ethyl analog 23 [22].
Owing to the importance of the (R)--ethyl sidechain and the substituted furyl moiety found in compound 23, system- atic SAR studies were conducted to explore the role of sub- stitution around the furyl ring as well as replacement of the furyl moiety with other heterocycles [23,24]. In the substi- tuted furyl series, both 4-alkyl/branched alkyl and 4-halo derivatives (28 and 29) showed excellent affinities toward both the CXCR2 and CXCR1 receptors with good compound plasma levels upon oral administration in rat (Fig. 3). In ad- dition, furyl derivative 28 showed good exposure in both dog and monkey upon oral administration and was efficacious in several in vivo rodent models of inflammation [23]. Re- placement of the furyl motif with other heterocycles yielded derivatives such as 30 which retained affinity for the CXCR2 receptor (IC50 = 3 nM) while exhibiting a more noticeable reduction in affinity for the CXCR1 receptor (IC50 = 150 nM) versus the corresponding furyl derivatives [24]. In addi- tion, heterocyclic derivatives such as 30 generally showed lower exposure of drug upon oral administration in rat as well as weaker functional activity in both CXCR2- and CXCR1-mediated chemotaxis assays using recombinant cells (Ba/F3) than the corresponding furyl derivatives [24]. Outside of the heterocyclic derivatives to replace the furyl moiety, recent efforts focused upon the introduction of highly substituted phenyl derivatives such as compound 31 in place of the furyl motif shown in Fig. (3). [25]. Compound 31 demonstrated single-digit nanomolar binding affinity for

Table 2. Branched Alkyl and Benzyl Cyclobutenedione Derivatives 12-17

O O
N
N R
H
O OH

Compd.
R
IC50 for CXCR2 (nM) IC50 for CXCR1 (nM) Rat Pk (10 mg/kg, PO) AUC ( M.hr)

12

N H

15

910

6.4

13

N H

236

na

14

N H

244

na

15

N H

17

3058

16

N H

234

na

1.0

17

N H

6.8

254

17.4
na = not active at >10,000 nM.

Table 3. Aryl and Heteroaryl Cyclobutenedione Derivatives 18-23

O O
N
N R
H
O OH

Compd.
R IC50 for CXCR2 (nM) IC50 for CXCR1 (nM) Rat Pk (10 mg/kg, PO) AUC ( M.hr)

18

F
N H

4.9

197

18.3

(Table 3) Contd….

Compd.
R IC50 for CXCR2 (nM) IC50 for CXCR1 (nM) Rat Pk (10 mg/kg, PO) AUC ( M.hr)

19
N O
H
O

5.0

145

31.7

20
N S
H

6.0

81

2.5

21
N O
H

3.8

26

1.4

22
N S
H

5.3

235

14.1

23
N O
H

2.6

36

49.0

Table 4. 5-Methylfuryl Cyclobutenedione Derivatives 23-27

O O
N
N N
H H
O OH

Compd.
R IC50 for CXCR2 (nM) IC50 for CXCR1 (nM) Rat Pk (10 mg/kg, PO) AUC ( M.hr)
24 Me 5.4 775 34.0
23 Et 2.6 36 49.0
25 Cp 3.6 55 1.4
26 i-Pr 6.2 34 3.2
27 t-Bu 3.8 11 2.6

the CXCR2 receptor with related analogs exhibiting good drug exposure in both rat and monkey upon oral administra- tion [25]. No further preclinical information has been re- ported on these more highly substituted phenyl analogs to date. Lastly, a recent report has illustrated the effective re- placement of the (R)-ethyl -side chain of the 3,4-diamino- cyclobut-3-ene-1,2-dione CXCR2 receptor antagonist series with various fluorinated side chains [26]. The -CF3 deriva- tive 32 (Fig. 3) displayed single-digit nanomolar binding

affinity for both the CXCR2 and CXCR1 receptors while exhibiting excellent exposure levels in rat upon oral admini- stration of the compound [26].
Additional SAR work on the left-hand phenolic ring has been carried out which compliments the early SAR work reported in this region [27]. Slight structural modifications in the amide functionality or appropriate substitution around the aromatic ring containing the phenol group led to slight improvements in affinity for the CXCR1 receptor [27].

O O O O

N
N N
H H
O OH

N
N N
H H
O OH 30

28: R = i-Pr R
29: R = Cl

O O

O O
CF3

N N N
H H
O OH 31 O
CN

N N
H H
OH 32

Fig. (3). Substituted furyl and heteroaryl cyclobutenedione derivatives.
While compound 33 (Fig. 4) demonstrated excellent binding O O
affinities to both the CXCR2 and CXCR1 receptors, this S

compound displayed a reduced level of drug exposure upon oral administration in rat versus the simple dimethylamide 17 [27]. Finally, a recent report described the replacement of the phenolic ring of the 3,4-diaminocyclobut-3-ene-1,2- dione class with a hydroxyl thiophene moiety as shown in (Fig. 4) [28]. Compound 34 was reported to have compara-

N N
N
N H
O OH
35

ble binding affinities to both the CXCR2 and CXCR1 recep- O O
tors as the phenolic derivative however no further evaluation S

of the thiophene series has been reported to date [28].

3,4-DIAMINOCYCLOBUT-3-ENE-1,2-DIONE CORE REPLACEMENTS
Additional efforts by the Schering-Plough/Pharmacopeia collaboration have focused upon the replacement of the cen- tral cyclobutenedione core with 1,2,5-thiadiazole-based structural motifs. Towards this end, three structurally related 3,4-diamino-1,2,5-thiadiazole-based CXCR2 receptor an- tagonist classes were identified, prepared, and evaluated. The first of these classes was the 3,4-diamino-1,2,5-thiadiazole- 1,1-dioxide-based series which is shown in (Fig. 5) [29]. Compound 35 exhibited good binding affinities for both CXCR2 (Ki = 11 nM) and CXCR1 (Ki = 61 nM) receptors while methylenedioxyphenyl analog 36 displayed excellent binding affinities for both the receptors (CXCR2 Ki = 3 nM and CXCR1 Ki = 17 nM). Despite potent in vitro profiles,

CO2H O O

N N
H H
O OH
33

O O O O S
S
N O

N N
N
N H
O OH
36

Fig. (5). 3,4-Diamino-1,2,5-thiadiazole-1,1-dioxide-based CXCR2 antagonists.

the 3,4-diamino-1,2,5-thiadiazole-1,1-dioxide class of CXCR2 receptor antagonists did not exhibit any functional activity in a human neutrophil (hPMN) myeloperoxidase (MPO) release assay mediated by either CXCL8 or CXCL1 [30]. In addition, compound 36 was evaluated in a rat car- ageenan induced paw edema model and failed to show any efficacy despite its excellent inhibitory effects of IL-8 recep- tor binding and excellent plasma exposure in rat upon oral administration. It is believed that poor Caco-2 permeability (< 10 nM/sec) and subtle differences in pKa of the phenolic functionality in this series of CXCR2 receptor antagonists (versus the cyclobutenedione series) may in part explain the lack of in vivo activity [30]. A related series of 3,4-diamino-2,5-thiadiazole-1-oxide- based CXCR2 receptor antagonists (Fig. 6) demonstrated improved chemokine receptor binding affinities and solubil- ity compared to the corresponding dioxide analogs [29]. Compound 37 exhibited excellent binding affinity for both CXCR2 (Ki = 2.3 nM) and CXCR1 (Ki = 21 nM) receptors and demonstrated potent functional activity in the human N N neutrophil (hPMN) MPO release assay mediated by either by HO H H 34 CXCL1 (IC50 = 8 nM) or CXCL8 (IC50 = 20 nM). Based Fig. (4). Left-hand side cyclobutenedione modifications. upon excellent in vitro binding affinities and potent func- tional efficacy in MPO release assays, compounds 38 and 39 were progressed into a rat lipopolysaccharide (LPS) neutro- philia model based upon their suitable plasma exposure in rat upon oral administration [30]. Oral administration of a 3 mg/kg dose of compound 38 or 39 demonstrated potent ac- tivity (88% and 90% inhibition of hPMNs, respectively) in a rat neutrophilia model. A structurally related 3,4-diamino-1,2,5-thiadiazole class was recently described [31] and representative examples are shown in (Fig. 7). Compound 40 exhibited good binding affinity for the CXCR2 receptor (Ki = 14 nM) while possess- ing moderate binding affinity for CXCR1 receptor (Ki = 91 nM). Compound 40 demonstrated good functional activity in hPMN MPO release assay mediated by either CXCL1 (EC50 = 21 nM) or CXCL8 (EC50 = 143 nM) chemoattractants [31]. Thiophene derivative 41 retained good CXCR2 receptor binding (Ki = 14 nM) while possessing moderate binding affinity for CXCR1 receptor (Ki = 1300 nM). OTHER CYCLOBUTENEDIONE-BASED CXCR2 RE- CEPTOR ANTAGONISTS Outside the Schering-Plough/Pharmacopeia efforts, sev- eral other companies have investigated cyclobutenedione- based CXCR2 receptor antagonists. Workers at GlaxoS- mithKline independently reported a series of N, N’- dianilinocyclobutenedione derivatives related to compound 11 [32]. Compound 42 is a prototypical derivative of this series which has excellent affinity for the CXCR2 receptor (IC50 = 8 nM) and good aqueous solubility (44 uM), while possessing low clearance and good oral bioavailability (%F = 43) in rat (Fig. 8). In an apparent lead hopping effort off the Schering-Plough/Pharmacopeia CXCR2 series, WuXi PharmaTech in collaboration with Merck reported a series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-dione-based CXCR2 receptor antagonists [33]. Compound 43 demon- strated reasonable affinity for both CXCR2 (Ki = 130 nM) and CXCR1 (Ki = 5200 nM) receptors coupled with good activity in an IL-8-mediated chemotaxis assay in a Chinese hamster ovary (CHO) cell line (IC50 = 75 nM) (Fig. 8). Incu- bation of compound 43 in human liver microsomes at 37oC showed good stability with >50% of drug remaining even after 2 h [33]. Novartis recently reported several reduced variants of the Schering-Plough/Pharmacopeia cyclobu- tenedione-based CXCR2 series including compound 44 shown in (Fig. 8) [34]. While this patent application contains no biological data for this series of compounds, the focus of the application is the preparation of right-hand side saturated heterocyclic variants of the derivatives in Table 3.

PHARMACOLOGY AND IN VIVO CHARACTERIZA- TION OF SCH 527123
Based upon the preclinical assessment in the 3,4- diamino-cyclobut-3-ene-1,2-dione-based CXCR2 receptor antagonist series from the Schering-Plough/Pharmacopeia efforts, compound 23 (SCH 527123) was selected as the lead candidate to further profile from both an in vitro and in vivo pharmacology perspective.
In an effort to better understand the pharmacology of the 3,4-diamino-cyclobut-3-ene-1,2-dione-based CXCR2 recep- tor antagonists, competition binding assays were conducted with SCH 527123 (compound 23) at both the hCXCR2 and hCXCR1 receptors. Results from these competition experi- ments suggested a potential allosteric interaction between compound 23 and target receptors [35]. In addition, SCH 527123 was shown to inhibit chemokine-stimulated [35S]

O S
N N
N
N H
O OH
37

O S
N N
N
N H
O OH 38

O S
N N
N
N N
H H
O OH
39

Fig. (6). 1,2,5-Thiadiazole -1-oxide-based CXCR2 antagonists.

S N S N

N N
N O
N H
O OH
40

N
N H
O OH
41

Fig. (7). 3,4-Diamino-1,2,5-thiadiazole-based CXCR2 antagonists.

F O O
O
S N N
N H H
O OH
N 42

O O
N N N

O O

N O H

N N
H H
O OH 43 O

N N
H H
OH 44

Fig. (8). Additional cyclobutenedione-based CXCR2 receptor antagonists.

GTPS exchange in hCXCR2 and hCXCR1 membranes in an insurmountable fashion indicative that the compound is a noncompetitive allosteric antagonist at both receptors. To measure the true affinity of SCH 527123 at both hCXCR2 and hCXCR1, [3H]SCH 527123 was prepared and demon- strated saturable, high-affinity binding in membranes ex- pressing either hCXCR2 (Kd = 49 pM) or hCXCR1 (Kd = 4 nM) [35]. While SCH 527123 displayed good affinity for the CXCR1 receptor, the compound is in fact selective for the CXCR2 receptor (>100-fold based upon Kd) due in part to the exceedingly slow dissociation rate from the hCXCR2 receptor (t1/2 = 24 h at rt). In addition, the CXCR2-selective nature of the compound was also supported by functional assessment in CXCR2- and CXCR1-mediated chemotaxis studies in recombinant cells (Ba/F3) and primary hPMN [35]. Furthermore, SCH 527123 inhibited neutrophil chemo- taxis and myeloperoxidase release in response to CXCL8 and CXCL1 without inhibiting response to chemoattractants such as C5a, fMLP, or LTB4 indicative of the selectivity of this compound for the chemokine receptors of interest [35].
In terms of pharmacokinetic properties, SCH 527123 demonstrated good bioavailability across multiple species, including mouse (%F = 44), rat (%F = 33), and cynomolgus monkey (%F = 39) which supported in vivo characterization [36]. Furthermore, this compound was shown to bind with high affinity to the homologs of rodent CXCR2 receptors as well as cynomolgus monkey CXCR2 receptor with Kd’s in the 0.080-0.20 nM range, while inhibiting CXCR2-mediated chemotaxis in the single digit nanomolar range across sev- eral species. Based upon the in vivo pharmacology of SCH 527123 at rodent CXCR2 and cynomolgus monkey CXCR1 and CXCR2 receptors, this compound was profiled in sev- eral models of pulmonary inflammation [36]. Oral admini- stration of SCH 527123 blocked pulmonary neutrophilia (ED50 = 1.2 mg/kg) as well as inhibiting goblet cell hyper- plasia (ED50 ~ 3 mg/kg) in mice following intranasal lipopolysaccharide (LPS) challenge with comparable results observed in rat [36]. In addition, the compound was able to suppress pulmonary neutrophilia (ED50 = 1.3 mg/kg) as well as goblet cell hyperplasia (ED50 0.7 mg/kg) in rat after inter- trachial (i.t.) vanadium pentoxide (V2O5) challenge. In cy- nomolgus monkeys, the compound reduced pulmonary

neutrophilia (ED50 =0.3 mg/kg) induced by repeat broncho- scopy and lavage [36]. In a separate study, SCH 527123 in- hibited smoke-induced lung neutrophilia by 50% with con- comitant reduction of -glucuronidase and BALF neutro- phils in a murine cigarette model [37]. Based upon the pre- clinical in vivo pulmonary evaluation described above, SCH 527123 could be beneficial in the treatment inflammatory disorders where pulmonary neutrophilia and mucus hyper- secretion are key components of the disease pathology.
SCH 527123 was also evaluated in several inflammation models such as the carrageenan-induced rat paw edema model and a mouse knee swelling model [38]. The combina- tion of SCH 527123 with either indomethacin (41% reduc- tion in paw edema) or betamethasone (61% reduction in paw edema) exhibited a greater response versus the compound alone at 1 mg/kg (20% reduction) [38]. In the mouse knee swelling model, SCH 527123 (10 mg/kg; 46% inhibition) administered with indomethacin (2 mg/kg; 42% inhibition) resulted in significantly greater reduction of knee swelling (74% inhibition) compared to either agent alone [38]. In ad- dition, single agent administration of SCH 527123 showed a significant reduction in IL-1 production consistent with the known pharmacological action of this compound.
More recently, the evaluation of SCH 527123 for the treatment of melanoma has been reported [39,40]. Oral ad- ministration of SCH 527123 (100 mg/kg) in athymic nude mice that had been pretreated with human A375SM mela- noma cells resulted in a 4.5-fold reduction in human mela- noma tumor volume compared to control group. Moreover, the antitumor efficacy of SCH 527123 was attributed to a decrease in both cell proliferation and angiogenesis while enhancing apoptosis [40]. Oral administration of the agent resulted in reduced chemotaxis and invasion of human A375SM cells compared to the untreated group. Based upon these results, SCH 527123 represents a potential novel therapeutic for the treatment of human malignant melanoma. Additionally, SCH 527123 has been shown to inhibit both tumor growth and angiogenesis in a colon cancer mouse model demonstrating the potential broad utility for such an agent for the treatment of cancer [40].

CLINICAL EVALUATION OF SCH 527123
Based upon the preclinical work summarized above, SCH 527123 was advanced into human clinical trials as a potential therapy for various inflammatory disorders. In Sep- tember 2007, phase I clinical data was reported which evalu- ated the ability of SCH 527123 to inhibit ozone-induced spu- tum neutrophilia in healthy subjects [41]. The efficacy of SCH 527123 (dosed 50 mg, qd, for 4 days) in reducing ozone-sputum neutrophilia was compared to either predniso- lone (50 mg) or placebo in a randomized cross-over study. In
18 healthy patients with known ozone sensitivity, SCH 527123 inhibited ozone-induced sputum neutrophilia more robustly than prednisolone (Table 5). In addition, sputum myeloperoxidase, total-sputum non-squa-mous cells, and IL- 8 levels were all reduced in the treatment group versus pla- cebo [41]. The efficacy observed for SCH 527123 in a vali- dated ozone challenge model [42] demonstrated the potential benefit of this compound to modify the progression of COPD by reducing the extent of airway inflammation.
SCH 527123 was the first orally bioavailable CXCR2 receptor antagonist to enter Phase II clinical trials for the treatment of various inflammatory disorders. The clinical evaluation of SCH 527123 in inflammatory diseases such as COPD, neutrophilic asthma, allergen-induced asthma, and psoriasis is ongoing at the time of this writing of this review [43].

SUMMARY AND PERSPECTIVE
In the past decade since the first small molecule CXCR2 receptor antagonist was reported, a significant effort within the pharmaceutical industry has produced a number of small molecule CXCR2 receptor antagonists [16] that have prom- ising profiles for therapeutic intervention in various inflam- matory disorders. Due to the wealth of preclinical data pro-

filing these derivatives which support the critical role of the CXCR2 receptor and its ligands in human disease [17], addi- tional classes of chemokine modulators/CXCR2 receptor antagonists continue to be described [44]. Furthermore, early clinical data for SCH 527123 (vide supra) as well as positive pulmonary proof-of-activity studies in human for CXCR2 antagonists from both Astra-Zeneca and GlaxoSmithKline further support additional efforts in this area. In 2007, Astra- Zeneca reported that AZD 8309 (believed to be a CXCR2 receptor antagonist) reduced both total sputum cells and spu- tum neutrophlils when compared to placebo after LPS- challenge in 16 patients [45]. In 2009, GlaxoSmithKline re- ported that SB-656933 reduced sputum neutrophil counts was well as total leukocyte counts compared to placebo in an ozone challenge model in 24 patients [46]. The status of CXCR2 receptor antagonists (Fig. 9) that are currently in ongoing clinical trials is summarized in Table 6.
Figure 9 serves to point out the relatively narrow chemi- cal scope of the CXCR2 receptor antagonists that are cur- rently under clinical evaluation (mainly phenol-containing) while several candidates (SB 332235 and AZ 8309) devel- opment activities are believed to have been halted (Table 6). Despite the considerable resources that have been applied towards the development of CXCR2 receptor antagonists across the industry, a number of challenges still exist in this area such poor species homology (rodent versus human), lack of translation of in vitro results into functional activity, as well as lack of a pure CXCR1 antagonist to profile. One of the fundamental questions to be answered in this field is the exact nature of CXCR2 antagonist interactions with the receptor as well as the underlying molecular mechanism of action of these chemokine receptor antagonists. Towards this end, recent studies have explored the existence of a potential nonpeptide antagonist binding site that lies near the intracel- lular C-terminal domain of the CXCR2 receptor which may

Table 5. SCH 527123 Inhibition of Sputum Neutrophilia in Healthy Patients

Screening Treatment
Pre Ozone Post Ozone
Placebo
SCH 527123
Prednisolone
Mean Neutrophils (x 106/ml) 0.43 3.33 2.98 0.13a 0.84b
95% CI 1.7, 5.2 0.07, 2.2 0.5, 1.5
aP < 0.001 vs placebo and prednisolone, bP = 0.001 vs placebo Table 6. Small Molecule CXCR2 Antagonists Currently Under Clinical Evaluation Drug Company Indication Phase SCH 527123 (23) Schering-Plough/Pharmacopeia COPD, asthma, psoriasis II SB 656933 (45) GlaxoSmithKline COPD, CF, ulcerative colitis I/II reparixin (46) Dompe Delay graft-function-kidney (IV) II SB 332235 (47) GlaxoSmithKline COPD I (discontinued) AZ 8309* AstraZeneca COPD, RA I (discontinued) *Structure unknown at time of writing O O O N N N F N N H H O OH 23 N H H O OH HN 45 H O N S O N Cl O H2N H H O OH Cl 46 47 Fig. (9). Known CXCR2 antagonists in clinical trials. explain the 100-fold difference in potency between CXCR2 and CXCR1 that has been observed in several classes of compounds [47]. Furthermore, recent studies of several CXCR2 receptor antagonists from different chemical series represented in Table 6 suggest that a large portion of these compounds are likely allosteric modulators of CXCR2 like SCH 527123 [48,49]. While there is evidence supporting the notion of distinct binding sites for both chemokines and small-molecule antagonists at the human CXCR2 receptor, different classes of molecules may in fact have unique bind- ing sites on the receptor [48]. The development of a better understanding of the exact role of IL-8 and its relevant re- ceptors would aid greatly in the identification of additional CXCR2 receptor antagonists for future evaluation. Finally, the outcome of ongoing mid-stage clinical trials for the CXCR2 receptor antagonists summarized in Table 6 will be critical in determining the long-term therapeutic potential and utility of CXCR2 receptor antagonists for the treatment of various inflammatory conditions for the future. 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