Switching between agonists and antagonists at CRTh2 in a series of highly potent and selective biaryl phenoxyacetic acids

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3616–3621

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Switching between agonists and antagonists at CRTh2 in a series of highly potent and selective biaryl phenoxyacetic acids Tim Luker a,⇑, Roger Bonnert a, Jerzy Schmidt b, Carol Sargent b, Stuart W. Paine c, Stephen Thom a, Gary Pairaudeau a, Anil Patel a, Rukhsana Mohammed a, Elizabeth Akam b, Iain Dougall b, Andrew M. Davis a, Phil Abbott a, Steve Brough a, Ian Millichip a, Thomas McInally a a b c

Medicinal Chemistry, AstraZeneca R&D Charnwood, Loughborough, Leicestershire LE11 5RH, UK Bioscience, AstraZeneca R&D Charnwood, Loughborough, Leicestershire LE11 5RH, UK Drug Metabolism and Pharmacokinetics, AstraZeneca R&D Charnwood, Loughborough, Leicestershire LE11 5RH, UK

a r t i c l e

i n f o

Article history: Received 31 March 2011 Revised 18 April 2011 Accepted 21 April 2011 Available online 28 April 2011 Keywords: CRTh2 DP2 Prostaglandin Inflammation Asthma Phenoxyacetic acid

a b s t r a c t A novel series of biaryl phenoxyacetic acids was discovered as potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). A hit compound 4 was discovered from high throughput screening. Modulation of multiple aryl substituents afforded both agonists and antagonists, with small changes often reversing the mode of action. Understanding the complex SAR allowed design of potent antagonists such as potential candidate 34. Ó 2011 Elsevier Ltd. All rights reserved.

The discovery of chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2, also known as DP2) in 2001 as a novel receptor for PGD2 rekindled interest in this prostanoid as an inflammatory mediator.1 In humans, CRTh2 is highly expressed on key cells implicated in the pathology of asthma and other allergic diseases including eosinophils, basophils and a subset of Th2 lymphocytes1,2 Activation of CRTh2 on these cells elicits a range of responses including chemotaxis2 and mediator release.3 These findings have stimulated considerable interest in the development of antagonists of this receptor as novel treatments for asthma and allergic rhinitis.4 A variety of molecular frameworks have emerged as potent CRTh2 ligands and these have been recently reviewed.5 For example, several series of indole acids have emerged, perhaps inspired by early reports of surprising CRTh2 activity from compounds such as Ramatroban6 (1, Fig. 1), originally developed as a thromboxane A2 receptor (TP) antagonist, and Indomethacin (2)2 a non-selective cyclooxygenase inhibitor. Within the indoles, a number of reports have highlighted structural changes required to eliminate agonism and provide potent antagonists.7 A single report has shown that CRTh2 agonism levels can also be modulated by a small structural ⇑ Corresponding author. E-mail address: [email protected] (T. Luker). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.04.101

change within a small set of tetrahydroquinolines.8 The phenoxyacetic acids (such as 3 from Novartis9) are an emerging class of CRTh2 ligands from several groups including our own,5 with a very

CO2H N

F

CO2H

MeO N

1

N S H O O

2 CO2H

CO2H O

O

Cl

O

R

NO2

Cl 3

4 R = NO2 5R=H

Figure 1. Known CRTh2 scaffolds 1–3 and AstraZeneca HTS hits 4–5.

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different molecular framework. To the best of our knowledge, only CRTh2 antagonists have been previously reported from this chemo-type. A high throughput screen of the AstraZeneca compound collection identified 4 as a relatively potent CRTh2 ligand (binding7c IC50 28 nM). Interestingly, compounds such as 4, containing multiple potentially toxic functional groups such as nitro, are often excluded from pharmaceutical screening collections.10 In this case, the supplier had given incorrect structural information, suggesting 4 to be mono-nitro 5 which passed preliminary compound quality filters. Ultimately both were independently prepared and 5 was found to have somewhat reduced CRTh2 activity (binding IC50 251 nM). Successful replacement of both nitro groups was seen as critical for the series. An initial range of R1 substituents were prepared (6–14) and the results are summarized in Table 1. A potency trend with inductive effects became apparent, for example, the rp physicochemical descriptor for R1 could explain some of the variance in CRTh2 binding potency. Clearly the sample size is small and further data and descriptors are needed to strengthen this understanding. Trifluoromethyl was found to be optimal within this initial set, with chloro also a reasonable nitro replacement. Several compounds (5, 9–12) were also functionally active and behaved as antagonists of PGD2 driven Ca2+ flux in HEK cells.7c

Table 1 Variation of R1

CO2H O

R

5 6 7 8 9 10 11 12 13 14

1

R1

CRTh2 binding IC50a (nM)

rpb

Rat Hep/Hum Mic CLintc

CRTh2 HEK Ca2+ IC50d (nM)

NO2 H Cl CF3 Me Br 2-Thiazole CN SO2Me NH2

251 41% 10 lM 251 45 794 316 1778 355 541 8912

0.78 0.00 0.23 0.54 0.17 0.23 0.48 0.66 0.72 0.66

<3/<3 — — 3/<3 <3/12 7/<3 8/9 <3/6 6/<3 64/<3

178 — — — 708 158 501 251 Agonist 17% 10 lM

a

Radioligand binding SPA binding assay (3H-PGD2), mean of n >2 measurements. Hammett para substituent constant. c Rat hepatocyte intrinsic clearance (ll/min/1  106 cells)/human liver microsomes intrinsic clearance (ll/min/mg). d Antagonism of PGD2 mediated Ca2+ flux in HEK cells transfected with human CRTh2. b

Table 2 SAR for modulation of aryl substituents

OH 3 R

O

2

R

O 4

R 1

R R1 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 a

CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 F CF3 CF3 Cl CF3 CF3 CF3 CF3 Cl Cl Et Cl Cl Cl Cl

R2

R3

SO2Me H SEt H SO2NEt2 H Cl H OMe H CN H N(Me)SO2Me H H CN H SO2Et H H H H N(Me)SO2Me H N(Me)SO2Me H N(Me)SO2Me H H CN H CN H CN SO2Me H SO2Et H SO2Et H –SO2–(CH2)2– SO2Et H SO2Me H SO2Me H SO2Et H SO2Et H SO2Et H SO2Et H

R4 H H H H H H H H H Ph OBu Cl Me Cl F Me H Cl Cl Me H H Cl H F Me Cl CF3

CRTh2 binding IC50a (nM) 36 8.9 1.3 50 251 126 0.8 <0.4 56 2.0 10 0.1 0.4 2.2 0.2 0.2 1.7 0.2 0.3 0.9 126 19 0.6 79 7.9 4.1 1.5 293

Agonism Eos CD11b IAb *

Agonist Antagonist Antagonist — — — 0.35 0.37 — 0.48 1.00 0.92 0.62 0.37 0.21 0.43 Antagonist 0.25 Antagonist Antagonist Antagonist Antagonist* Antagonist 0.12 Antagonist — Antagonist —

Rat Hep CLintc

Hum Mic CLintd

Log D7.4

<3 — 18 <3 47 — <3 <3 <3 20 9 — <3 — <3 <3 5 5 <5 4 13 <3 5 13 4 <3 <3 <3

<3 130 9 11 12 — 13 <3 12 <3 6 — <3 — <4 <3 <3 <3 <3 <3 6 7 10 <3 <3 <3 7 4

0.9 1.5 0.7 0.9 0.3 — 0.8 0.1 0.7 1.4 1.0 — 0.6 1.2 0.2 0.2 0.3 0.5 0.3 0.5 — 0.8 0.8 — 0.9 0.7 0.4 0.6

Radioligand binding SPA binding assay (3H-PGD2), n >2 measurements. Agonism in eosinophil CD11b, IA refers to intrinsic activity on scale of 0.00–1.00 where antagonist refers to compounds with a value of 60.1 relative to full agonist DKPGD2 (IA = 1.00). c Rat hepatocyte intrinsic clearance (ll/min/1  106 cells). d Human liver microsomes intrinsic clearance (ll/min/mg). * Refers to qualitative data generated in the CRTh2 mediated Ca2+ flux in HEK cell screen (see Table 1) b

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O

O

OH

Me N SO2Me

O

R R

4

1

size increases agonism strongly

size increases binding & agonism

H / Me

sulfonamide gives large potency increase & promotes agonism

OH

CN

R limited effect

cyano gives large potency increase but promotes agonism

O

R

4

1

limited effect size increases binding & agonism

may reduce agonism O H / Me

OH

R

3

SO2Alkyl

O

R limited effect

R

1

size increases agonism

4

alkyl size reduces agonism

sulfone gives large potency increase & some agonism size can increase binding, slight promotion of agonism

Figure 2. Summary of agonism SAR across sulfonamide, nitrile and sulfone sub-types.

Interestingly 13, containing the larger methylsulfone R1 substituent, switched to become a partial agonist in this functional screen, as evidenced by its stimulation of a calcium flux in the absence of PGD2. This agonism was investigated further and 13 was also found to behave as a CRTh2 partial agonist in human eosinophils, promoting the up-regulation of the adhesion molecule CD11b in the absence of DK-PGD2.11 The intrinsic activity (IA) of compounds in the CD11b assay was expressed as a fraction of the maximum response elicited by CRTh2 selective prostanoid DK-PGD2 (IA = 1.00), with 13 having IA = 0.11. With both nitro groups successfully removed, and high CRTh2 potency retained, compound 8 became an interesting lead compound with low rates of in vitro metabolic turn-over in both rat hepatocyte and human microsome incubations, as well as acceptable aqueous solubility (>80 lM in pH 7.4 buffer). Building on these results, a more extensive investigation of additional substituents on the second aryl ring was undertaken (Table 2). Given that the original hit 4 containing a para-nitro substituent on the pendant aryl ring, was significantly more potent than unsubstituted analogue 5, the initial set of new targets focused on functional groups that were either electron withdrawing or contained hydrogen bond acceptors. It was hoped that these would mimic the beneficial potency contribution of the nitro group without the well known toxicity liabilities. Synthetic routes to these compounds have been revealed elsewhere,12 and were generally based around Suzuki coupling to construct the required biaryl bond. Gratifyingly, a number of these compounds (15–23) started to gain additional CRTh2 potency, with sulfones (15), sulfonamides ( 17, 21) and sulfides (16) being noteworthy. Further work on sulfides was terminated due to their unsurprising lack of metabolic stability in oxidative human microsome incubations. Unfortunately, sulfone 15 was found to be an agonist in the HEK Ca2+ flux assay. Additionally, the most encouraging high potency CRTh2 ligand, sulfonamide 21, was also found to be a partial agonist in the CD11b screen with IA = 0.35. Although such partial

agonists would be expected to block the effects of higher efficacy agonists such as the endogenous ligand PGD2, they could theoretically elicit chemotactic1 and pro-inflammatory effects3 in CRTh2 expressing cells. For this reason, progression of these initially promising compounds was terminated. Substituents were also investigated at the meta (R3) and ortho 4 (R ) positions of the aryl ring. A cyano R3 group provided a substantial increase in CRTh2 binding potency over the R2 cyano isomer (22 vs 20), however a methylsulfone in the R3 position (23 vs 15) did not provide any additional potency. The precise positioning of a hydrogen bond acceptor in the distal region of the aromatic ring was able to influence substantial potency gains. 3D overlays suggested that the tetrahedrally-disposed sulfonyl oxygen from the para position may be able to provide a similar interaction vector to the linear cyano substituent from the meta position. Unfortunately cyano 23 was found to a partial agonist. Interestingly, large ortho (R4) substituents such as Ph (24) or BuO (25) also promoted agonism, with the later compound being of similar IA to the full agonist DK-PGD2. It was fascinating that these small variations in molecular framework were able to markedly affect pharmacological efficacy—for example, reversal of the sulfonamide (17 vs 21) or a change in sulfur oxidation state (16 vs 15) provided antagonists. We wished to investigate whether the agonist-promoting tendencies of the more potent R2 and R3 groups (15, 21–22), could be reversed by additional R1 and R4 substituents. This complex SAR is discussed below across the sulfonamides, nitriles and sulfones, with the key findings also summarized in Figure 2. In the sulfonamide R2 sub-series, 26 and 27 were prepared which added additional R4 groups onto ‘parent’ 21. Unfortunately these changes increased IA turning partial agonist 21 into virtually a full agonist 26 which contained the additional chloro R4 group. More encouragingly, changing R1 from CF3 (26) to the smaller F (28) retained high CRTh2 binding potency, while reducing agonism levels from IA = 0.92 to IA = 0.37. In this sub-series, the magnitude

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T. Luker et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3616–3621 Table 3 SAR for modulation of acid side chain

CN SO2Et

R

SO2Me

R

Me

Cl

CF3

CF3

43-49 R

R

CF3

50

51

CRTh2 binding IC50a (nM)

Agonism Eos CD11b IAb

Rat Hep CLintc

Hum Mic CLintd

LogD7.4

1.6

Antagonist*

23

<3

—

355

Borderline (0.1)

10

—

178

Antagonist*

—

—

0.3

3981

Antagonist*

—

—

–

2.5

Antagonist

25

<4

0.8

5650

—

29

<3

2.8

891

Antagonist*

—

—

0.6

0.6

Borderline (0.1)

<3

<3

0.6

0.29

<5

<3

Me 43

CO2H

O

Me 44

CO2H

O

0.4

Et 45

CO2H

O

Me Me 46

CO2H

O

47

CH2

48

O

49

CO2H CONH2

N

O

N N H

N

Me 50

O

CO2H

0.0

Me 51

O

CO2H

0.2

a

Radioligand binding SPA binding assay (3H-PGD2), mean of n >2 measurements. Agonism in eosinophil CD11b, IA refers to intrinsic activity on scale of 0.00–1.00 where antagonist refers to compounds with a value of 60.1 relative to full agonist DK-PGD2 (IA = 1.00). c Rat hepatocyte intrinsic clearance (ll/min/1  106 cells). d Human liver microsomes intrinsic clearance (ll/min/mg). * Refers to qualitative data generated in the CRTh2 mediated Ca2+ flux in HEK cell screen (see Table 1). b

of reduction was not enough to overcome the intrinsic agonismpromoting properties of the R2 group. A larger R1 (methylsulfone) had also promoted agonism in the early unsubstituted analogues (13, Table 1), and thus it seemed that this SAR might be more broadly applicable and that a reduction in R1 size might reduce agonism levels within other potent R2 and R3 scaffolds. In the nitrile sub-series, comparison of ‘parent’ 22 with analogues 29–30 showed a minimal effect of the additional R4 group on agonism. However as hoped, a reduction in R1 size, in this case from CF3 to Cl, gave 31 with exceptional CRTh2 binding potency, no agonism in the CD11b screen and also high metabolic stability—a profile suitable for further characterisation. In the sulfone sub-series, both ‘parent’ 15 and analogue 32 (containing an additional R4 = Cl) were agonists. In this sub-series, it was found that increasing the alkyl sulfone size from methyl to ethyl within R2 itself removed the unwanted agonism, successfully overriding the agonism-promoting effects of both the larger R1 and R4 groups. This gave 33 and 34 as high potency antagonists with high metabolic stability. The ethyl group on the sulfone could also be joined back onto the R3 position (35) which also prevented agonism, albeit at the expense of some binding potency.

As with the other sub-series, reducing the size of R1 was a successful strategy, giving further highly potent sulfone antagonists (36–37, 39). When the R1 size increased to ethyl, partial agonism returned (38). The comparison of 15 with 32 suggested that an R4 group might actually be a source of additional CRTh2 binding potency if its agonism-promoting effects could be controlled. Therefore, a set was prepared in which a smaller R1 was fixed as chloro and the size of R4 was incrementally increased. The results from R4 = H (36)/F (39)/Me (40)/Cl (41) indeed showed increasing CRTh2 potency until CF3 (42) which became too large. Gratifyingly, these compounds were devoid of agonism. Changes to the acidic side chain were also investigated on the more promising scaffolds (Table 3). An (R)-a-methyl group was tolerated next to the acid in both the sulfone and nitrile sub-series, (43, 50–51) with no increase in agonism. The (S)-a-methyl enantiomer (44) was far less active, as were the a-ethyl (45) and a,a-dimethyl substituents (46), suggesting limited space around the acid upon binding to the receptor. Interestingly, the propionc acid (47) maintained CRTh2 potency but this 2-carbon linker was less metabolically stable than the equivalent oxyacetic acid (34). Primary amide (48) was poorly active as a bioisostere, but the acidic tetrazole (49) retained some modest CRTh2 activity, perhaps suggesting an ionic recognition event at the receptor. Novartis

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mucosal damage.16 It is therefore suggested that 34 constitutes an excellent pharmacological probe compound. In summary, a series of biaryl phenoxyacetic acids were optimized for potency and antagonism at the CRTh2 receptor. The initial hit 4 had good CRTh2 binding potency but contained multiple potentially toxic functional groups. An investigation of the SAR within the series was carried out via modulation of aromatic substituents and changes to the acidic side chain functionality. This allowed successful replacement of both nitro groups, but also revealed a complex pattern of functional activity with small structural changes promoting dramatic switching between agonism and antagonism. Once the complex substituent-agonism QSAR was understood, careful molecular design enabled the provision of high quality CRTh2 antagonists with encouraging potency, selectivity, cross-over and metabolic stability. Compounds such as 34 are expected to help further evaluation of the function of CRTh2 in a range of inflammatory diseases. References and notes

Figure 3. Distribution of agonism and antagonism across the CRTh2 binding and lipophilicity space investigated.

have also reported some acid side chain SAR within their 2-cycloalkyl phenoxyacetic acid series.9 Within the phenoxyacetic acids, it appeared that agonists and antagonists were distributed across both binding potency and lipophilicity space (Fig. 3). For example, iso-lipophilic compounds with comparable CRTh2 binding potency (e.g., 22, 26, 34 all c log P 3.3, sub-nM at CRTh2) spanned the complete range of antagonist/partial agonist/full agonist properties. Equally, moderate potency higher log D compounds (24, 16) could also be both agonists and antagonists. Indeed, when the CRTh2 ligands were categorized into antagonists and agonists, no statistically meaningful difference in the mean values for a wide range of lipophilicity descriptors could be observed.13 Our work shows that the precise positioning of lipophilicity across molecular substituents is a more precise tool for dissecting complex functional activity SAR. In the CRTh2 series presented, agonism reduction is probably dependent on lipophilicity and/or substituent size increases at R2 and R3 but decreases at R1 and R4. Through this lead optimization program, a number of quality compounds were identified with high CRTh2 binding potency, low potential for agonism, high metabolic stability and high aqueous solubility and these were characterized in more detail. Nitrile 31 was a potent antagonist, blocking PGD2 mediated Ca2+ flux in CHO cells expressing human CRTh2 (IC50 10 nM in presence of 1% BSA14). It had high (>1000-fold) selectivity over DP1 as well as encouraging rat pharmacokinetics (IV: t½ 2.3 h; PO: F% 96%). Compound 3415 was also a high potency antagonist in the Ca2+ flux CHO cell assay (IC50 29 nM in presence of 1% BSA). It was more than 1000-fold selective over related prostanoid targets such as DP1, TP and COX-1. It also showed promising (>1000-fold) selectivity in a more general panel of 111 receptor and enzyme assays and IC50s >50 lM against a panel of CYP p450 isoforms (1A2, 3A4, 2C9, 2C19, 2D6). The compound had high CRTh2 binding potency at the mouse (IC50 1.0 nM), rat (IC50 1.3 nM) and guinea pig (IC50 63 nM) receptors as well as encouraging pharmacokinetics in rat (IV: t½ 1.1 h; PO: F% 42%) and dog (IV: t½ 9.8 h; PO: F% 46%). In a murine 11-day tobacco smoke model 34 blocked accumulation of inflammatory cells in the lung and exerted a protective effect on

1. Hirai, H.; Tanaka, K.; Yoshie, O.; Ogawa, K.; Kenmotsu, K.; Takamori, Y.; Ichimasa, M.; Sugamura, K.; Nakamura, M.; Takano, S.; Nagata, K. J. Exp. Med. 2001, 193, 255. 2. Nagata, K.; Hirai, H.; Tanaka, K.; Ogawa, K.; Aso, T.; Sugamura, K.; Nakamura, M.; Takano, S. FEBS Lett. 1999, 459, 195. 3. Xue, L.; Gyles, S. L.; Wettey, F. R.; Gazi, L.; Townsend, E.; Hunter, M. G.; Pettipher, R. J. Immunol. 2005, 175, 6531. 4. Pettipher, R.; Hansel, T. T. Drug News Perspect. 2008, 21, 317. 5. Norman, P. Expert Opin. Invest. Drugs 2010, 19, 947. 6. (a) Sugimoto, H.; Shichijo, M.; Okano, M.; Bacon, K. B. Eur. J. Pharmacol. 2005, 524, 30; (b) Rosentreter, U.; Boshagen, H.; Seuter, F.; Perzborn, E.; Fiedler, V. B. Arzneim.-Forsch. 1989, 39, 1519; (c) Shizuka, T.; Matsui, T.; Okamoto, Y.; Ohta, A.; Shichijo, M. Cardiovasc. Drug Rev. 2004, 22, 71; (d) Aizawa, H.; Shigyo, M.; Nogami, H.; Hirose, T.; Hara, N. Chest 1996, 109, 338. 7. (a) Hata, A. N.; Lybrand, T. P.; Marnett, L. J.; Breyer, R. M. Mol. Pharmacol. 2005, 67, 640; (b) Gervais, F. G.; Morello, J. P.; Beaulieu, C.; Sawyer, N.; Denis, D.; Greig, G.; Malebranche, A. D.; O’Neill, G. P. Mol. Pharmacol. 2005, 67, 1834; (c) Birkinshaw, T. N.; Teague, S. J.; Beech, C.; Bonnert, R. V.; Hill, S.; Patel, A.; Reakes, S.; Sanganee, H.; Dougall, I. G.; Phillips, T. T.; Salter, S.; Schimdt, J.; Arrowsmith, E. C.; Carrillo, J. J.; Bell, F.; Paine, S. W.; Weaver, R. Bioorg. Med. Chem. Lett. 2006, 16, 4287; (d) Crosignani, S.; Page, P.; Missotten, M.; Colovray, V.; Cleva, C.; Arrighi, J. F.; Atherall, J.; Macritchie, J.; Martin, T.; Humbert, Y.; Gaudet, M.; Pupowicz, D.; Maio, M.; Pittet, P. A.; Golzio, L.; Giachetti, C.; Rocha, C.; Bernardinelli, G.; Filinchuk, Y.; Scheer, A.; Schwarz, M. K.; Chollet, A. J. Med. Chem. 2008, 51, 2227. 8. Mimura, H.; Ikemura, T.; Kotera, O.; Sawada, M.; Tashiro, S.; Fuse, E.; Ueno, K.; Manabe, H.; Ohshima, E.; Karasawa, A.; Miyaji, H. J. Pharmacol. Exp. Ther. 2005, 314, 244. 9. (a) Sandham, D. A.; Aldcroft, C.; Baettig, U.; Barker, L.; Beer, D.; Bhalay, G.; Brown, Z.; Dubois, G.; Budd, D.; Bidlake, L.; Campbell, E.; Cox, B.; Everatt, B.; Harrison, D.; Leblanc, C. J.; Manini, J.; Profit, R.; Stringer, R.; Thompson, K. S.; Turner, K. L.; Tweed, M. F.; Walker, C.; Watson, S. J.; Whitebread, S.; Willis, J.; Williams, G.; Wilson, C. Bioorg. Med. Chem. Lett. 2007, 17, 4347; (b) Ulven, T.; Receveur, J.-M.; Grimstrup, M.; Rist, Ø.; Frimurer, T. M.; Gerlach, L.-O.; Mathiesen, J. M.; Kostenis, E.; Uller, L.; Högberg, T. J. Med. Chem. 2006, 49, 6638. 10. Davis, A. M.; Keeling, D. J.; Steele, J.; Tomkinson, N. P.; Tinker, A. C. Curr. Top. Med. Chem. 2005, 5, 421. 11. Human granulocytes were prepared from venous blood taken from healthy volunteers using Polymorphprep and re-suspended 5  106 cells/mL. Antibodies, (10 lL of a mix of FITC labelled murine anti-human CD11b and PE labelled murine anti-human CD16) or their respective isotype controls were mixed with 10 lL of agonist (DK-PGD2 or test compound) and 80 lL of purified granulocytes. After incubation at 37 °C for 15 min, the cells were fixed and incubated in the dark for a further 15 min at room temperature. Remaining red blood cells were lysed using a hypotonic shock. The level of CD11b expression was determined by flow cytometry using lack of CD16 expression to distinguish eosinophils from neutrophils. 12. Bonnert, R. V.; Brough, S.; Davies, A.; Luker, T.; McInally, T.; Millichip, I.; Pairaudeau, G.; Patel, A.; Rasul, R.; Thom, S. PCT Int. Appl. WO2004089885, 2004; Chem. Abstr. 2004, 141, 366033. 13. At other biological targets, partial agonists or antagonists have had consistently higher lipophilicity than would be expected for an agonist of comparable affinity. See for example: Parker, M. A.; Kurrasch, D. M.; Nichols, D. E. Bioorg. Med. Chem. 2008, 16, 4661. 14. Dilute bovine serum albumin was used as part of the buffer medium for cost reasons. This is relatively commonplace (see for example Liu, J.; Fu, Z.; Wang, Y.; Schmitt, M.; Huang, A.; Marshall, D.; Tonn, G.; Seitz, L.; Sullivan, T.; Tang, H. L.; Collins, T.; Medina, J. Bioorg. Med. Chem. Lett. 2009, 19, 6419. ). At these dilute serum albumin concentrations and with relatively free compounds this approximation is expected to have minimal impact. However, significant

T. Luker et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3616–3621 differences between protein shifts across species can occur, and so independent plasma protein binding measurements in 100% plasma were undertaken for 34 across multiple species. Relatively consistent values were obtained: human 4.6, rat 5.6, dog 3.0, cyno 2.6 (% free).. 15. Selected data for 34: 1H NMR (DMSO-d6) d 7.80–2.12 (6H, m), 4.63 (2H, s), 3.39– 2.29 (2H, q), 2.23 (3H, s), 1.18–2.11 (3H, t). m/z ( ve) (APCI) 401;

3621

C18H17F3O5S
0.5 H3N requires C, 52.7; H, 4.5; N, 1.5; S, 7.8%. Found: C, 52.4; H, 4.5; N, 1.2; S, 7.8%. 16. Sargent, C.; Stinson, S.; Schmidt, J.; Dougall, I.; Bonnert, R.; Paine, S.; Saunders, M.; Foster, M. Br. J. Pharmacol. 2009, 98, pA2 online. (See https://bps. conference-services.net/resources/344/1686/pdf/JB2009_0098.pdf).