Optimisation of a novel series of potent and orally bioavailable azanaphthyridine SYK inhibitors

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx

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Optimisation of a novel series of potent and orally bioavailable azanaphthyridine SYK inhibitors q Neil S. Garton a,⇑, Michael D. Barker a, Rob P. Davis a, Clement Douault a, Edward Hooper-Greenhill a, Emma Jones a, Huw D. Lewis a, John Liddle a, Dave Lugo a, Scott McCleary a, Alex G. S. Preston a, Cesar Ramirez-Molina a, Margarete Neu a, Tracy J. Shipley a, Don O. Somers a, Robert J. Watson a, David M. Wilson b a b

GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK AstraZeneca, Hodgkin Building, Chesterford Research Campus, Little Chesterford, Saffron Walden, Cambs. CB10 1XL, UK

a r t i c l e

i n f o

Article history: Received 28 June 2016 Revised 17 August 2016 Accepted 20 August 2016 Available online xxxx Keywords: SYK Spleen Tyrosine Kinase Selective Potent Orally bioavailable Azanaphthyridine Lead optimisation Medicinal chemistry

a b s t r a c t The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay. Ó 2016 Elsevier Ltd. All rights reserved.

Spleen Tyrosine Kinase (SYK) is a 72 kDa cytosolic non-receptor tyrosine kinase that is involved in signal transduction in a variety of cell types, including B lymphocytes, mast cells and macrophages.1 SYK and Zeta-chain-associated protein kinase 70 (ZAP-70) are the only members of the SYK family of protein tyrosine kinases and share a similar domain organisation with two N-terminal SH2 domains and a C-terminal kinase domain. ZAP70 has much lower intrinsic enzyme activity and its expression is mainly restricted to T-cells and NK cells.2 SYK plays a key role in coupling activated immunoreceptors to downstream events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis. Inhibition of SYK mediated immunoreceptor (Ig Fce, Ig Fcc and B-cell receptors) signalling leads to the inhibition of mast cell, macrophage and B-cell activation and subsequent release of inflammatory modulators.3

q All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. ⇑ Corresponding author. E-mail address: [email protected] (N.S. Garton).

Therefore, the discovery of safe small molecule SYK inhibitors has attracted much attention in a number of therapeutic areas, including the treatment of rheumatoid arthritis, B-cell lymphoma and asthma/rhinitis.4–11 Further to the disclosure by Boehringer-Ingleheim12,13 of the Naphthyridine template as a series of potent SYK inhibitors (SYK IC50 up to 20 nM) we were keen to evaluate the scope of this chemotype to deliver potent, selective and bioavailable SYK inhibitors to treat a range of autoimmune diseases. Azanaphthyridine compound 1 was identified as exhibiting moderate SYK potency and modest selectivity over Aurora B kinase, a kinase essential for cell proliferation and hERG, a voltage gated potassium ion channel that mediates the repolarisation current lkr in cardiac action potential (Table 1). Compound 1 also showed cellular activity in both the SYK mechanistic assay (inhibition of anti-IgM induced Erk1/2 phosphorylation in Ramos cells, pERK, pIC50 5.9, n = 4)14 and the human whole blood assay (inhibition of anti-IgM induced CD69 surface expression in primary B cells)14 within 10-fold of the SYK enzyme potency. A minimum 100-fold margin was sought between the human whole blood potency and hERG binding affinity. The compound exhibited

http://dx.doi.org/10.1016/j.bmcl.2016.08.070 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

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Table 1 In vitro profile of 1 and 2 pIC50

NH2

HN N

N

a,b

SYK hWBa,c Aurora Ba,b hERGd

6.6 5.6 5.9 4.8

N

a

c d

NH2

HN N

N

SYK hWBa,c Aurora Ba,b hERGd

7.3 6.3 7.3 4.4

N

1

b

pIC50 a,b

2

N

N

pIC50 values are reported as a mean of P2 experiments. Enzyme inhibition assay using recombinant human protein.14 Human whole blood assay: inhibition of anti-IgM induced CD69 surface expression in primary B cells.14 hERG binding determined using a fluorescence polarisation assay.14

moderate selectivity with five kinases showing activity within tenfold of SYK (Aurora A pIC50 5.9, Aurora B pIC50 5.9, LRRK2 pIC50 5.9, KDR pIC50 5.8, JAK1 pIC50 5.7) in a panel of over 40 kinases tested. X-ray crystallography of 115 (Fig. 1) showed the azanaphthyridine core nitrogen forming a mono-dentate acceptor hinge interaction with ALA451 and indicates that the protonated terminal amine forms important hydrogen bond interactions with ARG498 and ASP512 in the sugar pocket whilst the linker amine interacts with a glycerol molecule derived from the crystal cryoprotection buffer. The tolyl ring is co-planar with the azanaphthyridine core and points out towards solvent and could be exploited as a potential handle to tune the physical–chemical properties of the molecule. Initial work focussed on reducing the lipophilicity of 1 (c Log P 3.3, Chrom Log D 2.8) in order to reduce hERG potency. We quickly identified dimethylaminopyridine 2 as a less lipophilic starting point (c Log P 2.1, Chrom Log D16 2.2) which showed greater potency in the SYK biochemical assay and importantly demonstrated sub-micromolar activity in the human whole blood assay with reduced activity at hERG albeit with a loss of selectivity over Aurora B Kinase. Next we turned our attention to optimisation of the butylamine sidechain (Table 2). The truncated fluorinated analogue 3 surprisingly resulted in only a moderate drop in SYK potency yet a complete loss of activity in the human whole blood assay was observed. Both 4 and 5 were equipotent with 3 at SYK with 4 being inactive and 5 exhibiting low but measurable activity in the hWB assay.

Figure 1. X-ray crystallography of 1 indicating hydrogen bonds to SYK15 and showing Fo-Fc (compound omitted) 3r electron density at 1.43 Å resolution.

Constraint of the amine chain to homochiral (S) piperidine 6 locks the protonated amine in a favourable position for interaction with ARG498 and ASP512 (Fig. 2)15 and resulted in a threefold increase in SYK potency combined with a decrease in Aurora B activity. The increase in lipophilicity was accompanied by an increase in hERG activity and we looked to abrogate hERG altogether by incorporation of an oxygen atom into our molecule to reduce both lipophilicity and pKa. (S)-Morpholine analogue 7 retained potency at SYK and was equipotent to the piperidine in whole blood. Selectivity was improved over hERG but reduced over Aurora B. The (R)-morpholine enantiomer, 8 was tenfold less potent at SYK with the expected reduced potency in whole blood. Interestingly Aurora B potency appeared to be independent of the stereochemistry of the molecule. This was also true of the difluoropiperidine enantiomers 9 and 10 both of which exhibited a near tenfold increase in activity at SYK without this translating into increased potency in hWB. The increased level of hERG binding precluded further study of these molecules. The SYK potency of racemic difluoropiperidine regioisomer 11 was lower than that observed with compounds 9 and 10 whilst the (S)-methylpiperazine enantiomer 12 also showed lower potency than both piperidine 6 and morpholine 7 analogues bearing the same stereochemistry. Interestingly enantiomers 12 and 13 exhibit similar SYK and human whole blood potency to each other unlike that observed for other analogues despite different orientations of the methyl piperazine being observed in the protein by Xray crystallography (not shown). However these compounds have different hERG potencies suggesting SAR at hERG in this region of the molecule that could be exploited to achieve selectivity. Pharmacokinetic profiling of exemplars 6 and 7 in rat (3 mg/kg po (n = 3), 1 mg/kg iv (n = 1)) showed both to have high liver blood flow clearance (CLb 98 & 96 mL/min/Kg), a high volume (Vss 17 & 10 L/Kg) and a bioavailability of 10% and 11% respectively, possibly as a result of the dimethylaminophenyl moiety. Hence we turned our attention to investigate other areas of the molecule that could deliver a similar in vitro profile with improved PK, beginning with modifications to the bicyclic core (Table 3). Naphthyridine (NAP) analogues have been described in the literature as SYK inhibitors and in our hands 15 showed similar potency at SYK and pERK (pIC50 6.1, n = 2) without an increased potency in human whole blood cells relative to Aza-NAP 14. Pyridimidine regioisomer 16 and pyrimidinone 17 were significantly less active than 14 and discouraged further investigation of these cores. Molecular modelling predictions based on X-ray crystallography suggested that 3-N-methylation of 17 would be tolerated and indeed N-methylpyrimidinone 18 resulted in a modest increase in potency at SYK and in whole blood demonstrating that substitution at the 3 position is tolerated, however the overall profile was not sufficiently improved over Aza-NAP 14 to encourage further exploration. Interestingly a moderate improvement in the selectivity over hERG was also observed as a result of this

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N. S. Garton et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx Table 2 In vitro profiles of Aza-NAP’s 2 to 13

HN N

R1 N

N N

Compound No.

R1

N c Log P/Chrom Log D10

pIC50 SYK

NH2

2

CF3

3

OH

4

a,b

a,c

hWB

Aurora B

7.3

6.3

6.9

<5.0

6.7

a,b

d

e

hERG

HSA (%)

7.3

4.4

89

7.3

<4.3

96

3.2/5.7

<5.0

7.0

4.5

92

2.0/3.6

6.8

5.4

7.1

<4.3

96

1.4/2.6

7.8

6.5

6.6

4.6

92

2.7/2.3

7.6

6.5

7.0

<4.3

88

2.0/2.2

6.7

5.8

7.1

<4.3

89

2.0/2.2

8.6

6.8

7.1

5.1

95

2.1/3.7

7.5

5.8

7.0

4.7

95

2.1/3.7

7.0*

5.8

6.3

<4.3

93

3.0/4.2

7.3

6.4

5.7

<4.3

91

2.2/2.0

7.1

6.6

6.4

4.8

93

2.2/1.9

2.1/2.2

O 5

NH2

NH

6

NH

7

O NH

8

O

NH 9

F

F NH

10

F

F

NH 11

F F 12

13 * a b c d e

NH N

NH N

Racemic compound. pIC50 values are reported as a mean P2 experiments. Enzyme inhibition assay using recombinant human protein.14 Human whole blood assay: inhibition of anti-IgM induced CD69 surface expression in primary B cells.14 hERG binding determined using a fluorescence polarisation assay.14 HSA as measured by a chromatographic method.17

Figure 2. X-ray crystallography of 6 indicating hydrogen bonds from piperidine to SYK15 and showing Fo-Fc (compound omitted) 3r electron density at 1.95 Å resolution.

methylation despite the increase in lipophilicity again demonstrating SAR against hERG for this template. The previously described dimethylaminopyridine ring system, combined with NAP analogue 19 and the quinoline equivalent 20 delivered highly potent SYK inhibitors but showed approximately a 30-fold drop in potency in human whole blood. PK profiling of 19 in the rat (3 mg/kg po (n = 3), 1 mg/kg iv (n = 1)) showed an improved clearance and volume of distribution (CLb 34 mL/min/Kg, Vss 4 L/Kg) relative to 6 but bioavailability remained poor (4%). Given the preferred profile (lower lipophilicity and higher free fraction) of the Aza-NAP core over the NAP core, we next investigated the role of the heteroatom linker in the azanaphthyridine core series (Table 4). Replacement of the nitrogen atom by carbon (albeit with racemic piperidine) 21 resulted in reduced potency in both enzyme and cell assays. Incorporation of oxygen was well tolerated with piperidine enantiomers showing either excellent 22 or good 23 potency at SYK and in whole blood along with an moderately increased hERG liability. The (S)-enantiomer 22 had an improved pharmacokinetic profile in rat (3 mg/kg po (n = 3), 1 mg/kg iv (n = 1)) relative to the nitrogen-linked analogue 6 (CLb 39 mL/ min/Kg, Vss 8 L/Kg) but similar poor bioavailability (11%).

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Table 3 In vitro profiles of alternative bicyclic cores 14 to 20

HN

R1

N

N

N

19

Compound No.

Ring R2

N

20

R1

N

N c Log P/Chrom Log D10

pIC50 a,b

N

NH

HN

N

N

R2

NH

HN

a,c

SYK

hWB

Aurora B

7.6

6.1

7.7

a,b

d

e

hERG

HSA (%)

6.2

ND

91

3.8/3.0

6.1

5.8

4.8

95

4.5/3.0

6.7

5.4

5.7

5.0

91

3.6/2.8

6.8

5.4

5.3

5.1

91

2.9/2.4

7.1

5.9

6.7

4.6

90

3.1/2.8

8.1 8.2

6.1 6.3

6.5 7.0

<4.3 4.4

94 95

3.3/2.1 3.7/2.2

NH

14

N NH

15

16

N N

NH

N

O 17

NH

HN N O

18 19 20

NH

N N See above See above

ND: not determined. a pIC50 values are reported as a mean P2 experiments. b Enzyme inhibition assay using recombinant human protein.14 c Human whole blood assay: inhibition of anti-IgM induced CD69 surface expression in primary B cells.14 d hERG binding determined using a fluorescence polarisation assay.14 e HSA as measured by a chromatographic method.17

Table 4 In vitro profiles of alternative linkers 21 to 23

R3 N

NH N

N N

Compound No.

R3

(R/S)

NH CH2 O O

S R/S S R

c Log P/Chrom Log D10

pIC50 a,b

6 21 22 23

N

SYK

hWB

7.8 6.6* 8.4 7.6

6.5 6.0 6.9 6.3

a,c

Aurora B

a,b

6.6 6.8 7.2 7.5

d

e

hERG

HSA (%)

4.6 4.8 5.0 4.9

92 91 90 91

2.7/2.3 2.9/2.4 2.8/2.2 2.8/2.2

ND: Not determined. * Racemic compound. a pIC50 values are reported as a mean P 2 experiments. b Enzyme inhibition assay using recombinant human protein.14 c Human whole blood assay: inhibition of anti-IgM induced CD69 surface expression in primary B cells.14 d hERG binding determined using a fluorescence polarisation assay.14 e HSA as measured by a chromatographic method.17

In order to improve the oral absorption of the series whilst maintaining our good in vitro profile we looked to replace the dimethylaminopyridine group (Table 5). Incorporation of saturated ring systems such as 24 and 25 led to over one hundredfold decrease in SYK potency along with a tenfold drop in potency at Aurora B. Thiophene 26 is tolerated as a replacement for the dimethylaminopyridine group with increased hERG binding.

Further investigation of 5-membered rings such as furan 27 and the pyrazole 28 both demonstrated that oxygen and nitrogen atoms adjacent to the bicyclic core was disfavoured at SYK. Less lipophilic pyrazole regioisomer 29 showed acceptable SYK lysate and human whole blood potency whilst the methylated analogue 30 delivered excellent cellular potency for this series with ameliorated hERG activity. However the Aurora B activity remained a

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N. S. Garton et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx Table 5 In vitro profiles aryl ring replacements 24 to 31

O

HN N

NH N

Compound No.

N

N

N N

31

R4

N

NH

N

R4

c Log P/Chrom Log D10

pIC50 SYK

6

N

a,b

a,c

hWB

Aurora B

7.8

6.5

<4.6

a,b

d

e

hERG

HSA (%)

6.6

4.6

92

2.7/2.3

ND

5.4

<4.3

70

1.9/ND

5.2

ND

5.1

4.3

59

1.0/0.6

NMe2

24

N

25

N

26

S

7.7

6.0

6.5

5.3

92

3.7/3.1

27

O

7.2

6.1

6.2

4.7

ND

3.2/2.7

28

N N H

6.6

ND

5.6

<4.3

79

2.2/2.9

29

N H

N

7.3

6.5

6.4

<4.3

76

2.0/0.7

N

7.5

6.9

6.6

<4.3

75

1.5/1.2

7.4

6.7

6.8

<4.3

78

1.7/1.0

30 31

NH O NH

N

see above

ND: not determined. a pIC50 values are reported as a mean P2 experiments. b Enzyme inhibition assay using recombinant human protein.14 c Human whole blood assay: inhibition of anti-IgM induced CD69 surface expression in primary B cells.14 d hERG binding determined using a fluorescence polarisation assay.14 e HSA as measured by a chromatographic method.17

potential issue for this series. Unfortunately the blood clearance and volume of distribution remained high for this molecule in the rat (3 mg/kg po (n = 3), 1 mg/kg iv (n = 1)), CLb 136 mL/min/ Kg, Vss 12 L/Kg) resulting in low bioavailability (12%). Although potent oxygen-linked piperidine 31 retained selectivity over hERG and demonstrated moderate clearance and a good volume in the rat (3 mg/kg po (n = 3), 1 mg/kg iv (n = 1)) 50 mL/min/Kg, Vss 3 L/ Kg) the bioavailability was still poor (5%) indicating the absorption could be an issue for this series. We hypothesised that the high basicity of the piperidine ring (pKa 10.0) was contributing to the poor oral exposure and hence sought to address this with a series of less basic analogues in the Aza-NAP series (Table 6). Combination of a morpholine ring with the preferred methylpyrazole ring (pKa 7.7) resulted in compound 32 which was equipotent to the piperidine 30 at SYK and in whole blood and exhibited a minimal drop off between the two, unlike the previously described morpholine 7. Upon oral dosing to the rat (3 mg/kg, n = 3) a 10-fold improvement in AUC was observed. The difluoropiperidine bearing molecules 33–34 did not show the same level of exposure as 32 despite the similar pKa, presumably due to high clearance. Less basic racemic difluoropiperidine entities 35–36 showed improved exposure but were not potent enough in whole blood for further progression. The methylpiperazine analogues 37–38 were more basic than the morpholine

equivalents and also showed insufficient oral exposure when dosed to rats. The screening PK in the rat (3 mg/kg po (n = 3), 1 mg/kg iv (n = 1)) showed morpholine 32 to have moderate clearance (CLb 54 mL/min/Kg), a good half-life (1.7 h) and a moderate volume of distribution (Vss 7 L/Kg) resulting in a bioavailability of 55%. Profiling morpholine 32 against a kinase selectivity panel of over 40 kinases showed moderate selectivity with only five kinases within tenfold of SYK (Aurora B pIC50 7.0, ZAP-70 pIC50 7.0, BTK pIC50 6.6, LRRK2 pIC50 6.6, JAK1 pIC50 6.3). Since 32 had an otherwise desirable profile, including excellent selectivity in human whole blood over hERG (> 100-fold), we chose to evaluate the Aurora B activity further, in a mechanistic cell assay (phosphorylation of Histone-H3 at Ser10 in calyculin stimulated HT29 colon carcinoma cells). 32 showed minimal activity (pIC50 <5) in the Aurora B mechanistic assay and was progressed to the in-vivo efficacy model. We evaluated the efficacy of 32 in the reverse passive Arthus reaction, an immune complex mediated model of inflammation19,20 (Fig. 3). Prophylactic oral dosing of 32 to rats 1 h before ovalbumin challenge reduced the cutaneous reverse passive Arthus reaction in a dose dependent manner by approximately 45% and 85% at 10 mg/kg (1.4 lM) and 30 mg/kg (6.2 lM), respectively, compared to the vehicle control.21

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Table 6 In vitro profiles aryl ring replacements 32 to 38

R5 N

N

N

Compound No.

R5

pIC50 a,b

30 32

N N

HN

NH NH

HN O

HN

hWB

7.5

pKa18

Rat Oral PK a,b

Aurora B

hERG

6.9

6.6

7.2

6.8

8.4

d

f

f

Cmax (ng/mL)

AUC (h.ng/mL)

<4.3

8

43

10.0

7.0

<4.3

96

469

7.7

7.1

7.2

4.6

54

172 (n = 4)

7.9g

8.3*

7.2

7.3

4.6

28 (n = 2)

99 (n = 2)

7.7

6.5*

5.7

6.3

<4.3

96

216

6.9

6.8*

6.2

7.2

<4.3

148

296

7.0

7.0

6.7

5.7

<4.3

12

90

8.7

7.2

7.0

6.2

<4.3

8

37

8.6

NH F

33

SYK

a,c

F O

NH F

34

F

HN

NH

35

F O

F NH

36

F 37 38

HN

F NH

N O

NH N

ND: not determined. * Racemic compound. a pIC50 values are reported as a mean P2 experiments. b Enzyme inhibition assay using recombinant human protein.14 c Human whole blood assay: inhibition of anti-IgM induced CD69 surface expression in primary B cells.14 d hERG binding determined using a fluorescence polarisation assay.14 f n = 3. g Assigned by analogy, pKa data was generated using the opposite (R) enantiomer.

HN

Cl N

(a)

N

N

N

N

Cl (c)

(b)

N

N

N

Cl

O

R1

R1 (c)

N Cl

HN / O N N

R1 N R4

Scheme 1. Generic reagents and conditions for synthesis (examples 32 & 22). (a) H2NCH2N(Boc)morpholine (1 equiv), DIPEA (1.5 equiv), NMP, MW, 130 °C, 30 min, 73%; (b) HOCH2N(Boc)morpholine (1.2 equiv), NaH (1.2 equiv), DMF, ambient, 18 h; (c) N-methylpyrazoleboronic acid (1.1 equiv), Cs2CO3 (2 equiv), Pd(PPh3)4 (0.03 equiv), dioxan/water (4:1), reflux, 16 h, 98%.

Figure 3. Dose dependent inhibition of immune complex activation of Fc-gamma receptor function with 32.

The compounds described herein were each prepared in a similar manner using our previously reported short route involving two sequential displacements from the versatile intermediate

dichloropyrido[3,4-b]pyrazine (Scheme 1).22 A nucleophilic displacement reaction on the more labile peri-chlorine followed by palladium mediated cross coupling with boronic acids or esters followed by a Boc deprotection of the secondary amine using TFA in DCM yielded the target compounds. Homochiral molecules were obtained either through use of commercially available homochiral starting materials, through homochiral syntheses23 or from chiral resolution of the Boc protected racemic products.

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The progression of 32 was terminated following a positive result in the Ames bacterial mutagenicity assay24,25 when screened against strain TA1537 with and without metabolic activation. No cell cytotoxicity was reported at doses up to 5000 lg/plate. Several other analogues from this series were also found to show a genotoxic potential in the Ames assay. In summary, we have described the lead optimisation of the Aza-NAP series of SYK inhibitors and the important discovery of 32. The medicinal chemistry strategy was focussed on optimising the human whole blood activity (CD69 assay) whilst achieving a sufficient margin over liability kinases and over a 300-fold margin over hERG activity. 32 is a potent and selective SYK inhibitor showing good oral bioavailability and good efficacy in the rat Arthus model. Our efforts to improve the broad spectrum kinase selectivity and to overcome the mutagenicity discovered in this template and deliver preclinical SYK inhibitors will be disclosed in due course.

16.

17. 18.

References and notes 1. Mocsai, A.; Ruland, J.; Tybulewicz, V. L. J. Nat. Rev. Immunol. 2010, 10, 387. 2. Au-Yeung, B. B.; Deindl, S.; Has, L.; Palacios, A. H.; Levin, S. E.; Kuriyan, J.; Weiss, A. Immunol. Rev. 2009, 228, 41. 3. Ghosh, D.; Tsokos, G. Autoimmunity 2010, 43, 48. 4. Riccaboni, M.; Bianchi, I.; Petrillo, P. Drug Discovery Today 2010, 15, 517. 5. Ruzza, P.; Biondi, B.; Calderan, A. Expert Opin. Ther. Pat. 2009, 19, 1361. 6. Moore, W. J.; Richard, D.; Thorarensen, A. Expert Opin. Ther. Pat. 2010, 20, 1703. 7. Liddle, J.; Atkinson, F. L.; Barker, M. D.; Carter, P. S.; Curtis, N. R.; Davis, R. P.; Douault, C.; Dickson, M. C.; Elwes, D.; Garton, N. S.; Gray, M.; Hayhow, T. G.; Hobbs, C. I.; Jones, E.; Leach, S.; Leavens, K.; Lewis, H. D.; McCleary, S.; Neu, M.; Patel, V. K.; Preston, A. G. S.; Ramirez-Molina, C.; Shipley, T. J.; Skone, P. A.; Smithers, N.; Somers, D. O.; Walker, A. L.; Watson, R. J.; Weingarten, G. G. Bioorg. Med. Chem. Lett. 2011, 21, 6188. 8. Geahlen, R. L. Trends Pharmacol. Sci. 2014, 8, 414. 9. Norman, P. Expert Opin. Ther. Pat. 2014, 24, 573. 10. Lucas, M. C.; Tan, S. L. Future Med. Chem. 2014, 6, 1811. 11. Ferguson, G. D.; Delgado, M.; Plantevin-Krenitsky, V.; Jensen-Pergakes, K.; Bates, R. J.; Torres, S.; Celeridad, M.; Brown, H.; Burnett, K.; Nadolny, L.; Tehrani, L.; Packard, G.; Pagarigan, B.; Haelewyn, J.; Nguyen, T.; Xu, L.; Tang, Y.; Hickman, M.; Baculi, F.; Pierce, S.; Miyazawa, K.; Jackson, P.; Chamberlain, P.; LeBrun, L.; Xie, W.; Bennett, B.; Blease, K. Plos One 2016. http://dx.doi.org/ 10.1371/journal.pone.0145705. 12. Cywin, Charles L.; Bao-Ping, Zhao; McNeila, Daniel W.; Hrapchaka, Matt; Prokopowicz, Anthony S., IIIa; Goldberga, Daniel R.; Morwicka, Tina M.; Gaoa, Amy; Jakesa, Scott; Kashema, Mohammed; Magoldaa, Ronald L.; Sollb, Richard M.; Playerb, Mark R.; Bobko, Mark A.; Rinkerb, James; Des Jarlais, Renee L.; Wintersb, Michael P. Bioorg. Med. Chem. Lett. 2003, 13, 1415. 13. Bao-Ping, Zhao; Bobko, Mark A.; Cywin, Charles L.; Des Jarlais; Renee L.; Heider, Joachim; Jakes, Scott E.; Rinker, James; Player, Mark; Winters, Michael; Sinka, Steven; Natili, Richard; WO 2003/057695. 14. Details of the SYK biochemical assay, Erk1/2 phosphorylation assay, human whole blood assay, Aurora B biochemical assay, hERG binding assay and synthesis of GSK143 are described in detail in the following reference: Atkinson, F. L.; Patel, V. K. WO 2010/097248. 15. Purified SYK truncate (360–635)7 at 5 mg/ml in buffer comprising 20 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM DTT and 1 mM EDTA was incubated with at least 5-fold excess of compound, on ice for approximately 1 h. Cocrystallisation was achieved by sitting drop vapour diffusion carried out using drop ratios of 1:1 or 2:1 of protein/compound mixture to reservoir solution. The reservoir solution for compound 1 was 20% PEG3350, 0.2 M Sodium formate, whilst it was comprised 23% PEG1500, 0.1 M HEPES pH6.8, 7% Tacsimate, 10% glycerol, for compound 6. A crystal was cryoprotected by brief immersion into reservoir buffer containing 10% glycerol for protein/compound 1, or harvested directly from a drop for protein/compound 6, prior to cryofreezing in liquid nitrogen and obtaining synchrotron X-ray diffraction data, at 100 K. Diffraction data for compound 1 were collected at the Diamond Light Source (station I04) using a Pilatus 6 M pixel array detector and were processed and scaled within AUTOPROC (Vonrhein C. et al., Acta Crystallogr. 2011, D67, 293–302) using XDS (Kabsch, W. Acta Crystallogr. 2010, D66, 125–

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132) and AIMLESS (Evans, P. R.; Murshudov, G. N. Acta Crystallogr. 2013, D69, 1204–1214) [of the CCP4 programming suite (Winn, M. D. et al., Acta Crystallogr. 2010, D67, 235–242)]. Diffraction data for compound 6 were collected at the ESRF station ID23.1 using an ADSC Q315R CCD detector. These data were processed using MOSFLM (Leslie, A. G. W.; Powell, H. R. Processing diffraction data with MOSFLM. In Evolving Methods for Macromolecular Crystallography, Read, R. J., Sussman, J. L., Eds.; Springer Press, 2007, pp 41– 51) and SCALA (Evans, P. R. Acta Crystallogr. 2006, D62, 72–82) within the CCP4 Programming Suite. The complexes were solved by Fourier Synthesis using SYK protein coordinates determined in-house. Refinements were carried out using REFMAC (Murshudov, G. N.; Vagin, A. A.; Dodson, E.J. Acta Crystallogr. 1997, D53, 240–255) and model building using COOT (Emsley, P.; Cowtan, K. Acta Crystallogr. 2004, D60, 2126–2132). The final Rfactor (and Rfree) achieved for compounds 1 and 6 structures were 15.6% (18.3%) and 17.5% (22.8%) respectively. The structures were deposited into the PDB as entries 5LMA (compound 1) and 5LMB (compound 6). Getting physical in drug discovery II: the impact of Chromatographic hydrophobicity measurements and aromaticity Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Drug Discovery Today 2011, 16, 822. A human serum albumin coated HPLC column method Valko, K.; Nunhuck, S.; Bevan, C.; Reynolds, D. P.; Abrahams, M. H. J. Pharm. Sci. 2003, 92, 2236. Sirius T3 (Sirius Analytical Inc, UK) instrument has been used for pKa determination of the compounds. The pKa determination is based on acidbase titration and the protonation/deprotonation of the molecule is measured either by UV spectroscopy or potentiometrically. The pKa value is calculated from the pH where the 50–50% of the protonated and unprotonated form of the molecules are present. The UV-metric method provides pKa results for samples with chromophores whose UV absorbance changes as a function of pH. It typically requires 5 ll of a 10 mM solution of the samples and the UV absorbance is monitored over 54 pH values in a buffered solution in about 5 min. When the ionization centre is far from the UV chromophore pH-metric method based on potentiometric acid-base titration is used. The pH of each point in the titration curve is calculated using equations that contain pKa, and the calculated points are fitted to the measured curve by manipulating the pKa. The pKa that provides the best fit is taken to be the measured pKa. Usually 0.5– 1 mg of solid material is required for the measurements. When the compound precipitates at some point during the pH titration co-solvent method using methanol is applied using various concentration of co-solvent. The pKa in water is calculated using the Yasuda-Shedlovsky extrapolation method. The pKa value quoted is for the protonated form of the structure shown. Braselmann, S.; Taylor, V.; Zhao, H.; Wang, S.; Sylvain, C.; Baluom, M.; Qu, K.; Herlaar, E.; Lau, A.; Young, C.; Wong, B. R.; Lovell, S.; Sun, T.; Park, G.; Argade, A.; Jurcevic, S.; Pine, P.; Singh, R.; Grossbard, E. B.; Payan, D. G.; Masuda, E. S. J. Pharmacol. Exp. Ther. 2006, 319, 998. Ravetch, J. V.; Bolland, S. Annu. Rev. Immunol. 2001, 19, 275. 32 was prepared as a suspension in 1% methylcellulose and was administered orally using a dose volume of 3 mL/kg. Each treatment group consisted of 8 animals. Male CD rats (175–200 g) obtained from Charles River (UK), were housed in groups of 4 under a 12:12 h light/dark cycle (lights on 07:00 h) with food and water available ad libitum. All experiments were carried out by an observer blind to drug treatments. All procedures were performed under the Home Office Animals Scientific Procedures Act 1986. Reagents were obtained from Sigma chemicals, UK. 32 was administered 1 h (this is a pre-determined but variable time that is compound specific. It is the time that ensures the Cmax is reached at the point of administering the challenge) prior to challenge. Samples were taken to confirm blood concentrations and systemic exposure directly followed by intravenous challenge with 1% ovalbumin in saline (10 mg/kg) containing 1% Evans blue dye. Ten minutes later animals were anaesthetised with isoflurane, shaved dorsolaterally and rabbit anti-OVA IgG (50 lg/50 lL) was injected intradermally on the left side at three adjacent locations. Rabbit polyclonal IgG (50 lg/50 lL) was injected intradermally on the right side to serve as a control. Four hours after challenge, animals were euthanized and skin tissue was removed and local skin edema at each injection site was measured. A punch biopsy of each of the injection sites (12 mm) were obtained and placed in formamide (0.5 mL) for 16 h at 80 °C. Extravasated Evans blue dye concentration was measured via spectrophotometer (Molecular Devices, USA) at OD610. Atkinson, Francis Louis; Atkinson, Stephen John; Barker, Michael David; Douault, Clement; Garton, Neil Stuart; Liddle, John; Patel, Vipulkumar Kantibhai; Preston, Alexander G.; Shipley, Tracy Jane; Wilson, David Matthew; Watson, Robert J. WO 2012/123312. Morie, Toshiya; Kato, Shiro; Harada, Hiroshi; Yoshida, Naoyuki; Matsumoto, Junichi Chem. Pharm. Bull. 1994, 42, 877. Ames, B. N.; Lee, F. D.; Durston, W. E. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 782. Mortelmans, K.; Zeiger, E. Mutat. Res. 2000, 455, 29.

Please cite this article in press as: Garton, N. S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.070