Bioorganic & Medicinal Chemistry Letters 24 (2014) 2206–2211

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Purine derivatives as potent Bruton’s tyrosine kinase (BTK) inhibitorsfor autoimmune diseases

http://dx.doi.org/10.1016/j.bmcl.2014.02.0750960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +1 6092523226 (Q.S.), +1 6092523121 (G.V.D).E-mail addresses: qing.shi@bms.com (Q. Shi), george.delucca@bms.com

(G.V. De Lucca).

NN

NNH

HN

O

NN

NNH

HN

O

NNN

BTK IC500.150 µM

NN N

N

BTK IC5012.7 µM

NNN

BTK IC50>50 µM

NN

BTK IC50>50 µM

NN N

N

BTK IC500.038 µM

NNN

BTK IC500.213 µM

NN

BTK IC500.574 µM

NN N

N

BTK IC500.067 µM

1

3 4 5

6 7

2

8 9 10

NO

O

NO

O

Figure 1. Heterocyclic replacement of the imidazo[1,2-a]pyrazine co

Qing Shi ⇑, Andrew Tebben, Alaric J. Dyckman, Hedy Li, Chunjian Liu, James Lin, Steve Spergel,James R. Burke, Kim W. McIntyre, Gilbert C. Olini, Joann Strnad, Neha Surti, Jodi K. Muckelbauer,Chiehying Chang, Yongmi An, Lin Cheng, Qian Ruan, Katerina Leftheris, Percy H. Carter, Joseph Tino,George V. De Lucca ⇑Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, NJ 08543-4000, United States

a r t i c l e i n f o

Article history:Received 1 January 2014Revised 26 February 2014Accepted 28 February 2014Available online 13 March 2014

Keywords:BTKAutoimmune diseasesPurine

a b s t r a c t

Investigation of various heterocyclic core isosteres of imidazopyrazines 1 & 2 yielded purine derivatives 3& 8 as potent and selective BTK inhibitors. Subsequent SAR studies of the purine series led to the discov-ery of 20 as a leading compound. Compound 20 is very selective when screened against a panel of 400kinases and is a potent inhibitor in cellular assays of human B cell function including B-Cell proliferationand CD86 cell surface expression and exhibited in vivo efficacy in a mouse PCA model. Its X-ray co-crystalstructure with BTK shows that the high selectivity is gained from filling a BTK specific lipophilic pocket.However, physical and ADME properties leading to low oral exposure hindered further development.

� 2014 Elsevier Ltd. All rights reserved.

Bruton’s tyrosine kinase (BTK) is a member of the Tec family ofnon-receptor tyrosine kinases. BTK is expressed in most hemato-poietic cells such as B cells, mast cells, and macrophages but notin T cells and plays a critical role in the signaling pathways down-stream of the B cell receptor as well as the FccRIIa, FccRIIIa andFceRI receptors.1 Mutations in the BTK gene lead to defects in B celldevelopment at the pre-B cell stage causing X-linked agammaglob-ulinemia in humans (XLA) and less severe X-linked immunodefi-ciency (xid) in mice.1g,h BTK deficient mice are resistant to mouseanimal models of lupus1i as well as collagen-induced arthritis(CIA).1j A small molecule inhibitor of BTK activity represents anintriguing approach to treat autoimmune diseases such as rheuma-toid arthritis, multiple sclerosis and systemic lupus erythemato-sus.2 In addition to autoimmune diseases, many pharmaceuticalcompanies have investigated small molecules irreversible covalentinhibitors of BTK as an approach for B-Cell malignancies3 whichhas recently culminated with the approval of Ibrutinib for MantleCell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL).3d

At the beginning of our program we were interested in revers-ible BTK inhibitors to target autoimmune diseases and were at-tracted to reports of the imidazo[1,2-a]pyrazines represented bycompounds 1 and 2 in Figure 1.4 Using compounds 1 and 2 as a

starting point, we first examined a variety of heterocyclic isosteresof the imidazo[1,2-a]pyrazine core. As shown in Figure 1, purines 3and 8, imidazo[4,5-c]pyridine 5, imidazo[1,2-b]pyridazine 9, andimidazo[1,2-a]pyridine 10 are all good replacements of the originalcore. Among all the cores that were examined, purine derivativesdisplayed the most potent BTK inhibition.

re.

Table 2Optimization of the purine N-9 substituents

NN N

N

R

NH

HN

O

ON

O

CompoundR Selectivity (X)

BTK IC50 (lM) JAK2/BTK

FLT3/BTK

ITK/BTK

20 H 0.008 383 51 6.48 Me 0.038 4.9 3.3 0.13

21 0.17 281 0.34 0.16

22 0.14 324 20 345

23 >50 1.0 0.10 1.0

24 OO

29 1.7 1.7

25 OHO

0.97 42 36 2.4

Figure 2. Compound 8 docked into a BTK homology model.

Table 1Evaluation of the para-substituent of the C-6 aniline

NN N

NHN

O

1

342

56 78

9

NH

R

Compound R BTK IC50 (lM)

11 OHO

0.064

8 ONO

0.038

12 NHNO

0.025

13 NNOHO

0.028

14 NNO

0.017

15 O NO

0.143

16 NO

0.043

17 NH

NO

0.087

18 NH

OHO

0.126

19 NN 0.019

Procedure for the BTK enzyme assay has been described in Ref. 6.

Figure 3. The ‘gatekeeper pocket’ in the BTK homology model.

Q. Shi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2206–2211 2207

In order to understand the dramatic potency differences amongthe heterocyclic cores, we employed molecular modeling to studythe interactions of these compounds with BTK protein. Compound8, as an example, was docked into a BTK homology model con-structed from the crystal structure of the homologous kinase ITK(Fig. 2). The model predicts that N-7 (see structure in Table 1 fornumbering) of the purine core and the NH of the C-6 aniline sub-stituent form hydrogen bonds with M477 in the hinge region.These two hydrogen bond interactions are critical to BTK enzyme

potency. Compounds lacking N-7, such as molecules 4, 6 and 7,are inactive in BTK enzyme assay. Replacing the NH of the C-6 ani-line of 8 with an oxygen also eliminates BTK enzyme potency (BTKIC50 > 25 lM).

The model also shows that the benzene ring of the C-6 anilinefalls into a narrow groove formed by L408 and G480 and makeshydrophobic interactions with these residues. To fit within the ste-ric constraints imposed by L408 and G480, the aniline ring must re-main nearly coplanar with respect to the bicyclic core. Wehypothesized that the aniline ring of compounds 3, 5 and 8 couldadopt a coplanar conformation due to their N-1 on the bicyclic corethat reduces steric interaction.5 On the other hand, the aniline ringof 9 and 10 will be forced to adopt an out-of-plane conformationdue to a clash with the C–H on the bicyclic core adjacent to theaniline substituent, consistent with their weaker potency. Our

Table 3Methyl substituent on the C-2 phenyl ring

NN N

H

NNH

HN

O

21 3

46 5

ON

O

CompoundR Selectivity (X)

BTK IC50 (lM) JAK2/BTK SRC/BTK FLT3/BTK ITK/BTK

20 0.008 383 304 51 6.4

26 0.033 37 14 13 0.7

27 0.065 229 187 37 1.0

28 0.026 24 5 28 1.7

Table 4Evaluation of the side chain

N

N NH

NR

NHN

O

O

CompoundR Selectivity (X)

BTK IC50 (lM) JAK2/BTK SRC/BTK FLT3/BTK BMX/BTK ITK/BTK

20HN

O0.008 383 304 60 4.5 6.4

29HN

O0.032 993 222 155 2.3 6.9

30HN

O

F3C0.048 36 35 14 9.0 6.5

31HN

O0.083 10 16 11 2.0 10

32HN

O0.169 5.4 5.8 3.1 1.3 3.9

33HN

O0.109 4.5 20 5.4 1.2 9.2

34 H2N 0.077 5.4 7.1 3.8 0.7 2.1

35 H3C 0.035 5.0 11 6.0 0.8 2.1

36HN

HNO

0.018 13 26 9.1 1.0 5.2

37HN

HNO

0.049 5.6 9.2 1.3 0.8 2.4

38 SHNOO

0.027 74 92 16 1.8 45

39 SHNOO 0.041 18 11 3.8 2.4 11

2208 Q. Shi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2206–2211

hypothesis was confirmed with ab initio minimizations of modelsystems consisting of the bicyclic core and unsubstituted anilinering at the B3LYP/6-31G⁄⁄ level of theory. As anticipated, the anglebetween the plane of the bicyclic core and aniline ring was zero de-grees for cores 3, 5 and 8 and 33 degrees for cores 9 and 10 at theirminimum energy conformations.

The para-substituent of the C-6 aniline extends towards a sol-vent exposed region in the model. This implies that the para-sub-stituent would not affect the BTK enzyme potency. To verify, weselected a wide range of hydrophilic groups as para-substituents

(Table 1). Indeed, very small effect on the BTK potency was ob-served. The BTK IC50 fluctuated only within a small range from17 nM to 143 nM as the substituent changed from acid to a varietyof amides to amine. This part of the molecule could therefore beused as a handle to balance the physical properties of the overallmolecule.

The ‘gatekeeper’ region then attracted our attention. Due to theresidue differences at this region among BTK (T474), Janus kinase 2(JAK2) (M929), Fms-like tyrosine kinase 3 (Flt3) (F691), and Inter-leukin-2-inducible T-cell kinase (ITK) (F434),7 we hypothesized

Figure 4. X-ray co-crystal structure of compound 20 in human BTK (PDB code4NWM). Hydrogen bond interactions between BTK and the bicyclic purine core ofcompound 20 from the BTK/compound 20 co-crystal structure are highlighted.Hydrogen bonds to compound 20 and within the water network are shown asyellow and red dashed lines, respectively.

Figure 5. The tert-butyl pocket from BTK/compound 20 co-crystal structure.

Q. Shi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2206–2211 2209

that growing the molecule into this ‘gatekeeper’ region (Fig. 3)from the purine N-9 substituents would gain better selectivityagainst these kinases. Different sized N-9 substituents from hydro-gen to a large 3-phenylpropyl group were selected to probe thisarea (Table 2). We observed that the off target kinase activitiesdid decrease with the size of the N-9 substituent. However, thisalso decreased BTK potency. Of all the N-9 substituents examined,2-phenylethyl 23 was optimal in balancing the kinase selectivityover JAK2 (324 fold), Flt3 (20 fold), and ITK (345 fold) while main-taining potency against BTK (140 nM). Compound 20, though bear-ing the smallest N-9 substituent, gave the best BTK potency (8 nM)along with excellent JAK2 selectivity (383 fold) (vide infra).

We then examined the substitution pattern of the central ben-zene ring (Table 3). Lacking the 2-methyl group, compound 26loses BTK potency and especially kinase selectivity. Walking themethyl group to the 4- or 6-position, compounds 27 and 28 loseBTK potency and kinase selectivity as well. The 2-methyl grouphelps position the 1-benzamide and 3-purine groups, due to thesteric interactions, to get the best potency and selectivity. A methylgroup is also the ideal size to interact with L528 at the base of theATP binding site (Fig. 2).

Next, we studied the SAR of the side chain off the central ben-zene ring (Table 4). We evaluated a variety of R groups includingalkyl, amines, amides, ureas and sulfonamides. Amides turnedout to be superior to ureas or sulfonamides in both BTK potencyand kinase selectivity. Among amides, the BTK potency and selec-tivity increased with the lipophilicity of the side chain. The optimalresult was obtained with a tert-butylphenyl amide moiety in com-pound 20.

To better understand the SAR in a structural context, a co-crys-tal structure of compound 20 and BTK was obtained (PDB code4NWM).8 In agreement with the homology model, the co-crystalstructure shows that the N-7 of the purine core and the NH ofthe C-6 aniline form hydrogen bonds with the backbone amideNH and carbonyl of M477 (Fig. 4). The crystal structure revealsadditional interactions between purine N-3, N-9 and a water net-work that bridges to a conserved lysine K430 and the gatekeeperresidue T474. This network would be disrupted by any N-9 substi-tuent other than hydrogen, which explains the SAR in Table 2. Incontrast, Jak2, Flt3 and ITK have hydrophobic gatekeeper residuesthat pack well against the methyl group at N-9 of compound 8,thereby enhancing their potency and reducing BTK selectivity.

The structure also made clear the specific interactions with thetert-butyl benzamide moiety (Fig. 5). The tert-butyl group is in awell-defined hydrophobic pocket, that is, the specificity pocket.3b,9

This pocket is comprised of residues from the G-loop, Q412 andF413, and the activation loop, L542, S543, V546, and Y551. Thebenzene ring forms hydrophobic interactions with Q412 andY413. The amide carbonyl forms hydrogen bonds to the backboneNH of F413 and the backbone carbonyl of G414 via a bridgingwater molecule. Collectively, these interactions contribute to thehigher potency of compound 20 versus the other analogs lackingthese contacts.

The side chains of residues S543, V546, and Y551 are presum-ably responsible for the high selectivity gained from filling theBTK specificity pocket. In the literature, sequence analysis of 491human kinases shows that the V546/Y551 pair occurs in only threeother kinases, BMX, ITK, and TXK while the triple S543/V546/Y551is completely unique to BTK.9 The kinase selectivity profile of com-pound 20 shown in Tables 4 and 5 is in accordance to this analysis.

Compound 20 was screened against the Ambit kinase panel ofover 400 kinases. Only 3.1% of the kinases showed less than 33%control binding at 1 lm. Consistent with this measure of selectiv-ity, biochemical kinase assays against 40 kinases showed com-pound 20 to be more potent against BTK than others, includingthe other Tec family kinases (BMX, ITK, TEC and TXK) (Table 5).

Compound 20 was also found to be very potent in cellular as-says and to have a good correlation between enzyme potencyand cell based activities. In B cells stimulated through the B cellreceptor (BCR), compound 20 selectively inhibited signaling andfunctional endpoints, including calcium flux and phosphorylationof BTK in human Ramos B cells,6,10 production of IL-6 and prolifer-ation of human tonsilar B cells,11,12 and surface expression of CD86on human or mouse peripheral blood B cells13,14 (Table 6).

Further profiling of compound 20 is listed in Table 7. Unfortu-nately, the metabolic stability of 20 was low in human and rat liver

O.D. values are the MEAN +/- SEM of 14 tissue samples per treatment group

Vehicle 3 10 300.00

0.05

0.10

0.15

0.20

Compound 20, mg/kg, IP, -1hr

* *Dye

Con

tent

(O.D

.)

Figure 6. Compound 20 in mouse Passive Cutaneous Anaphylaxis (PCA) model.

NN N

H

N

Cl

ClNN N

N

Cl

Cl

THP

H2N

NN N

NHN

ClTHP

ONO

NO

O

97% 92%

BHN O

O95%

NN N

NHN

HN

ON

THPO

NN N

H

NHN

HN

ON

O

O

90%

O

a b

c

d

40 41 42

4320

O

Scheme 1. Reagents and conditions: (a) DHP, TsOH, EtOAc, rt; (b) KOtBu, THF, 0 �C-rt; (c) Pd(Ph3P)4, 2M aq K3PO4, toluene/EtOH, 100 �C; (d) 1N aq HCl, MeOH, rt.

Table 7Partial in vivo and in vitro profile of compound 20

Liver microsomes stability (%remaining):

Human 14%; Rat 36%; Mouse 68%

CYP inhibition (IC50): 3A4 BFC 9.8lM, 3A4 BZR>40 lM;2C8 1.4 lM; 2C9 0.4 lM; 2C193.4 lM; 2D6>40 lM

Mouse Cytotox IC50: 7.7 lMHerg Flux IC50: >80 lMPAMPA: 403 nm/sCaco2: <15 nm/s (both directions)aq. soluability: <0.001 mg/mL at pH 1 and 6Mouse PK (30 mg/kg dose, Methocel

suspension through IPadministration):

Cmax: 3.802 lM; AUC (tot):32.468 lM⁄h; t1/2: 4.3 h

Table 5Kinases selectivity of compound 20

BTK IC50: 0.008 lM SRC IC50: 2.434 lM IKK-1 IC50: >50 lMBMX IC50: 0.037 lM JAK2 IC50: 3.068 lM IKK-2 IC50: >50 lMITK IC50: 0.052 lM JAK3 IC50: 33.740 lM IRAK4 IC50: >50 lMTEC IC50: 0.014 lM HER1 IC50:

>16.670 lMGSK-3B IC50: >50 lM

TXK IC50: 0.055 lM InsR IC50: 9.142 lM IGF-1R IC50:17.910 lM

FLT3 IC50: 0.479 lM MK2 IC50: 13.330 lMMAPKAPK3 IC50:

0.2130 lM

Table 6Cell potency of compound 20

Cell assays IC50 (lM)

Calcium flux in Ramos B cell 0.009pBTK in Ramos B cells (western blot) �0.050IL-6 in Human Tonsilar B cells 0.030Human B cell proliferation 0.008Human CD86-BCR surface expression 0.142Mouse CD86-BCR surface expression 0.112

Procedures of the above cell-based assays are described in Refs. 6,10–14.

2210 Q. Shi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2206–2211

microsomes while moderate in mouse. Biotransformation studiesshowed the major metabolic pathway was the oxidation of thetert-butylphenyl moiety. Due to its poor Caco2 permeability, pooraqueous solubility and moderate mouse liver microsomal stability,acceptable exposure in mouse was only obtained through IPadministration.

In vivo efficacy of compound 20 was then evaluated in an FceRI-driven mouse Passive Cutaneous Anaphylaxis (PCA) model. Theprocedure15 was modified from the technique described by Wer-shil et al.16 At 10 mg/kg dose, compound 20 reduced the dye con-tent by 74% compared to vehicle (Fig. 6) demonstrating goodin vivo efficacy.

A representative synthesis of purine analogs is exemplified bythe synthesis of 20 as shown in Scheme 1. Commercially available2,6-dichloro-9H-purine 40 was selectively protected with a THPgroup at the N-9 position to give 41. Reaction of 41 and the corre-sponding aniline with potassium tert-butoxide afforded 42 regio-selectively. Suzuki coupling between 42 and the correspondingboronic ester yielded 43, which was then deprotected under acidiccondition to give 20.

Herein we reported purine derivatives as a class of potent BTKinhibitors. Structure-activity relationship studies led to the discov-ery of 20 as one of the most potent and selective compounds. Com-pound 20 was also efficacious in a mouse PCA model. However, thelow oral exposure hindered its further development. This issue hasto be addressed to advance this class of BTK inhibitors.

References and notes

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5. Shewchuk, L.; Hassel, A.; Wisely, B.; Rocque, W.; Holmes, W.; Veal, J.; Kuyper, L.F. J. Med. Chem. 2000, 43, 133.

6. Liu, Q.; Batt, G.D.; De Lucca, V.G.; Shi, Q.; Tebben, A. US8084620.7. van Linden, O. P. J.; Kooistra, A. J.; Leurs, R.; de Esch, I. J. P.; de Graaf, C. J. Med.

Chem. 2014, 57, 249.8. A baculovirus construct of His-TEV-hBTK(E396–S659) was expressed in Sf9

cells for 66 h post infection at 27 �C. The frozen cell paste was lysed undernitrogen cavitation at 300–400 psi for 30 min at 4 �C and centrifuged at100,000g for 30 min. The supernatant was purified on the Ni-NTA (Qiagen)resin. His-TEV-hBTK of �80% purity was eluted at 0.3 M imidazole in a gradientof 0.03–0.6 M imidazole. The fractions that contained His-TEV-hBTK werepooled together, concentrated and loaded on a Superdex-200 SEC column (26/60) to remove soluble aggregates. This material was then quantitativelycleaved at an engineered site for TEV recognition, followed by chromatographyon nickel-chelate resin to remove the AcTEV protease (InVitrogen) anduncleaved material with a 60% recovery. Final pure cleaved hBTK (E396–S659) was buffer exchanged into 200 mM NaCl, 20 mM Tris/HCl, pH 8.0, and1 mM TCEP. The final sample was aliquoted and stored at �80 �C at 0.34 mg/ml. For protein/compound complex formation, 4.4 ll of Compound 20 (25 mMDMSO stock) was added to 2.0 ml of hBTK at 0.34 mg/ml and incubated at 4 �C,concentrated to 8.5 mg/ml and run over a 26/60 Superdex-200 SEC column.Crystals were grown at room temperature using hanging drop vapor diffusionmethod. The drop consisted of 2 ll protein solution and 2 ll reservoir solutioncontaining 26% (w/v) methyl ether PEG 5000, 0.2 M Tris–HCl, pH 7.25. Macroseeding was performed to initiate crystal growth. The crystals appeared withina few days and continued to grow for 2–3 lweeks. Crystals were flash-cooledin liquid nitrogen for data collection with 20% glycerol and 80% reservoirsolution as cryoprotectant. Diffraction data were collected by ShamrockStructures, Inc. at IMCA-CAT, beamline 17ID at the Advanced Photon Source.hBTK/Compound 6 co-crystals belonged to the space group p212121:a = 44.9 Å, b = 86.5 Å, c = 135.1 Å, a = b = c = 90.0�. The 2.0 Å resolutionstructure was determined by molecular replacement using a previouslydetermined in house BTK structure (unpublished results). The structure ofhBTK + Compound 20 has been deposited to RCSB with PDB code 4NWM.

9. Di Paolo, A. J.; Huang, T.; Balazs, M.; Barbosa, J.; Barck, H. K.; Bravo, J. B.; Carano,A. D. R.; Darrow, J.; Davies, R. D.; DeForge, E. L.; Diehl, L.; Ferrando, R.; Gallion, L.S.; Giannetti, M. A.; Gribling, P.; Hurez, V.; Hymowitz, G. S.; Jones, R.; Kropf, E.J.; Lee, P. W.; Maciejewski, M. P.; Mitchell, A. S.; Rong, H.; Staker, L. B.;Whiteney, J. A.; Yeh, S.; Young, B. W.; Yu, C.; Zhang, J.; Reif, K.; Currie, S. K. Nat.Chem. Biol. 2011, 7, 41.

10. Measure of pBTK in Ramos B cells: After a 1 h pre-incubation of Ramos B cellsin media containing 10% FCS with varying concentrations of the testingcompound at 37 �C, the cells were stimulated with anti-human IgM (F(ab0)2fragment, Jackson ImmunoResearch, catalog #109-006-129) at 50 lg/ml forexactly 2 min at 37 �C. Cells were then fixed and stained with an Alexa647-conjugated anti-phospho-BTK antibody which specifically recognizes pY551(BD Biosciences, 558134) and the amount of phospho-Y551 quantitated by themean fluorescence intensity (MFI) as measured by FACS analysis.

11. Measure of IL-6 in Human Tonsilar B Cells: Human tonsilar B cells wereisolated according to the general procedure of Gillooly et al. [Gillooly, K.M.;Pattoli, M.A.; Taylor, T.L.; Chen, L.; Cheng, L.; Gregor, K.R.; Whitney, G.S.;Susulic, V.; Watterson. S.H.; Kempson, J.; Pitts, W.J.; Booth-Lute, H.; Yang, G.;Davies, P.; Kukral, D.W.; Strnad, J.; McIntyre, K.W.; Darienzo, C.J.; Salter-Cid, L.;Yang, Z.; Wang-Iverson, D.B.; Burke, J.R. J. Pharmacol. Exp. Ther. 2009, 331, 349].The isolated B cells were suspended in media containing 10% FBS and variousconcentrations of the testing compound. After a 1 h incubation at 37 �C, thecells were stimulated with 40 lg/mL F(ab0)2 goat anti-human IgM (Jackson

ImmunoReasearch, cat# 109–006-129) and 10 ng/mL IL-4 (Peprotech, cat#200–04). After an additional 4.5 h incubation, the supernatants were collectedand the levels of IL-6 measured by EIA.

12. Measure of Human B Cell Proliferation: Human B cells from tonsils, isolated asdescribed above, in media containing 10% FCS and various concentrations ofthe test compound were stimulated with AffinPure F(ab0)2 goat anti-humanIgG+IgM (Jackson, Cat#109-006-127) and incubated at 37 �C for 72 h. The cellswere then labeled with [3H]thymidine and incubated overnight at 37 �C. Cellswere then harvested onto filter plates and the amount of [3H]thymidineincorporation, as a measure of B cell proliferation, was determined by liquidscintillation counting.

13. Measure of Human CD86-BCR surface expression: The E-negative fraction ofhuman peripheral blood mononuclear cells, isolated after removal of Tlymphocytes by rosetting with sheep red blood cells, in media containing10% BCS and various concentrations of the testing compound were stimulatedfor 18 h at 3 7�C with AffinPure F(ab0)2 Fragment Goat anti Human IgG + IgM(Jackson Cat#109-006-127). The cells were then fixed and stained with FITC-conjugated mouse anti-human CD20 antibody (BD Pharmingen 555622) andAPC-conjugated mouse anti-human CD86 monoclonal antibody (BDPharmingen 555660) and the amount of CD86 expression quantitated by themean fluorescence intensity (MFI) after gating on the viable (propidium iodideexclusion) CD20-positive B cell population as measured by FACS analysis. Foranalogous experiments measuring the effect of CD40 or TLR4 stimulation onthese endpoints, either human IZ-CD40L or LPS was used to stimulate the cells.To determine the effect of the testing compound CD86 expression in memory Bcells, an additional marker was employed (PE-conjugated mouse anti-humanCD27, BD Pharmingen 555441) to identify CD27+CD20+memory B cells.

14. Measure of Mouse CD86-BCR surface expression: Mouse splenic B cells wereisolated using a nylon wool column from homogenized spleens andresuspended in media containing 10% FCS with various concentrations of thetesting compound at 37 �C before stimulating for 18 h with 10 lg/mL affinPureF(ab0)2 fragment goat anti-mouse IgG + IgM (Jackson, Cat#115-006-068). Cellswere then fixed and stained with an APC-conjugated rat anti-mouse CD45R/B220 and FITC-conjugated rat anti-mouse CD86 (B7–2) and the amount ofCD86 expression quantitated by the mean fluorescence intensity (MFI) aftergating on the viable (propidium iodide exclusion) B220-positive B cellpopulation as measured by FACS analysis.

15. Experimental procedure of mouse PCA study: Female CD-1 mice, 8–10 week ofage (Harlan), were anesthetized (Isoflurane, 3–5% in O2) and their backs wereshaved. Mice were injected intradermally with 40ng anti-DNP IgE monoclonalantibody in 20 lL saline into each of two sites on the back. 24 h later, the micewere challenged by intravenous injection of 100 lg DNP-coupled humanserum albumin in 200 lL saline containing 1% Evans blue dye. Vehicle (0.5%Methocel with 0.1% Tween 80) or compound 20 was administeredintraperitoneally 1 h prior to antigen challenge. At 1 h after antigenchallenge, mice were euthanized and the skin of the back was removed. Full-thickness skin biopsy punches (8 mm diam.) encompassing each of the two IgEinjections sites were collected along with a ‘background’ biopsy from aseparate uninvolved region of the skin. The biopsy specimens were placed intoseparate tubes with 1 ml formamide and the Evans blue dye was extracted at100�C for 3 h. An aliquot of the formamide supernatant was transferred to a96-well flat-bottomed plate and the OD was read at 620 nm. The OD of the‘background’ biopsy supernatant was subtracted from the OD of the ‘IgE’biopsy supernatants to determine a net extravasation of dye.

16. Wershil, B. K.; Mekori, Y. A.; Murakami, T.; Galli, S. J. J. Immunol. 1987, 139,2605.