MED
`
`DOI: 10.1002/cmdc.200600221
`Discovery of Selective Irreversible
`Inhibitors for Bruton’s Tyrosine
`Kinase
`
`Zhengying Pan,*[a] Heleen Scheerens,[b] Shyr-
`Jiann Li,[b] Brian E. Schultz,[b] Paul A. Sprengeler,[a]
`L. Chuck Burrill,[a] Rohan V. Mendonca,[a]
`Michael D. Sweeney,[b] Keana C. K. Scott,[c]
`Paul G. Grothaus,[a] Douglas A. Jeffery,[b]
`Jill M. Spoerke,[b] Lee A. Honigberg,[b]
`Peter R. Young,[b] Stacie A. Dalrymple,[b] and
`James T. Palmer[a]
`
`The importance of B cells in rheumatoid arthritis (RA) patho-
`genesis has been recently demonstrated in several clinical
`studies using the anti-CD20 antibody rituximab, which selec-
`tively depletes B cells. A recent phase III clinical trial led to the
`FDA approval of rituximab for a subset of RA patients. Bruton’s
`tyrosine kinase (Btk), a member of Tec family kinases, is a key
`component in the B-cell receptor signal pathway (BCR).[1] Upon
`activation by upstream kinases (for example, Lyn and Syk), Btk
`phosphorylates and thereby activates phospholipase-Cg (PLC-
`g), leading to several important downstream events including
`calcium ion transportation, NF-kB activation, and (auto)anti-
`body generation. Previous biological studies (genetic loss of
`function[2] and siRNA knockdown[3]) strongly suggest that Btk is
`also a mediator of proinflammatory signals. Taken together,
`these studies indicate Btk may be a potential target for the
`treatment of RA. However, despite the previous discovery of
`LFM-A13 as a selective Btk inhibitor,[4] there is no published
`study that has demonstrated that inhibition of Btk activity
`leads to in vivo efficacy in an animal model of rheumatoid ar-
`thritis.
`As ATP binding sites in kinases are highly conserved, it is a
`formidable task to develop selective ATP competitive kinase in-
`hibitors. Among several approaches, the use of electrophilic in-
`hibitors has been shown as a viable method to achieve selec-
`
`[a] Dr. Z. Pan, Dr. P. A. Sprengeler, L. C. Burrill, R. V. Mendonca,
`Dr. P. G. Grothaus, Dr. J. T. Palmer
`Department of Medicinal Chemistry, Celera Genomics
`180 Kimball Way, South San Francisco, CA 94080 (USA)
`Fax: (+ 1) 650-578-9680
`E-mail: zypan@yahoo.com
`[b] Dr. H. Scheerens, Dr. S.-J. Li, Dr. B. E. Schultz, M. D. Sweeney,
`Dr. D. A. Jeffery, Dr. J. M. Spoerke, Dr. L. A. Honigberg,+ Dr. P. R. Young,
`Dr. S. A. Dalrymple
`Department of Biology, Celera Genomics
`180 Kimball Way, South San Francisco, CA 94080 (USA)
`[c] K. C. K. Scott
`Informatics, Computational Science Group, Celera Genomics
`45 West Gude Drive, Rockville, MD 20850 (USA)
`[+] Current Address:
`Pharmacyclics Inc.
`995 E. Arques Ave., Sunnyvale, CA 94085 (USA)
`Supporting information for this article is available on the WWW under
`http://www.chemmedchem.org or from the author.
`
`tivity.[5] Considering the relative scarcity of knowledge on
`“chemical knockdown” of Btk activity, it is crucial to discover a
`potent and selective tool compound for this kinase. Herein, we
`describe the discovery of a selective, irreversible Btk inhibitor
`and its efficacy in a mouse RA model.
`An initial campaign to scan for scaffolds capable of inhibit-
`ing Btk’s kinase activity identified compound 1 as having
`
`8.2 nm potency against Btk in a FRET based biochemical enzy-
`mology assay. In agreement with a previous study,[6] screening
`against more than 100 kinases at Ambit Biosciences[7] showed
`that 1 inhibited activity of certain Tec and Src family kinases
`(Table 1). Interestingly, 1 shows only modest inhibitory activity
`against Itk, another Tec family kinase, probably due to the dif-
`ference at the “gatekeeper” residue.[8]
`
`Table 1. Selectivity screening results of compound 1.[a]
`
`Kinase
`
`Activity remaining [%]
`
`Kinase
`
`Activity remaining [%]
`
`Abl
`Aurora A
`CSK
`EGFR
`INSR
`JAK1
`JNK3
`Kit
`p38a
`Syk
`
`5.1
`> 30
`1.4
`3.5
`> 30
`> 30
`> 30
`0.7
`> 30
`> 30
`
`Btk
`Fgr
`Fyn
`Hck
`Lck
`Lyn
`Src
`Yes
`Bmx
`Itk
`
`0
`0
`0.2
`0.3
`0.1
`0
`0.1
`0
`0
`10
`
`[a] Binding assay at Ambit Biosciences, concentration of 1 is 10 mm.
`
`We realized that these two kinases could have SAR homolo-
`gy[9] because of compound 1’s high potency towards Btk and
`Lck, and constructed a homology model based on the known
`X-ray structures of Btk kinase domain 1K2P[10] and Lck 1QPE[11]
`(Figure 1). The amino acids that were within contact range of
`compound 1 were identified in this homology model (Table 2).
`Alignment of Btk and Lck sequences revealed Cys 481 in Btk as
`a nucleophilic site, which could potentially form a covalent
`complex between Btk and an electrophilic inhibitor. Further se-
`quence analysis indicated that this cysteine was at the same
`position as Cys 773 in EGFR family kinases that were the target
`of several irreversible kinase inhibitors in clinical trials, for ex-
`ample, CI-1033[12] and HKI-272.[13] Based on sequence align-
`ments of 491 kinases,[14] there are only 10 kinases with a cys-
`teine at this position: Blk, Btk, Bmx, EGFR, ErbB2, ErbB4,
`Itk,
`
`58
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`maining recombinant Btk activity after 60 min incubation of
`the kinase and various concentrations of inhibitors in buffer
`(Table 3). We confirmed the irreversibility of these compounds
`using two methods: 1) after recombinant Btk was pretreated
`with compounds,
`its activity was not recovered by multiple
`washings with inhibitor-free medium;[15] and 2) a major peak
`was observed by mass spectrometry corresponding to the mo-
`lecular weight of a 1:1 covalent complex between compound
`4 and Btk.[16]
`All these compounds were highly potent inhibitors with IC50
`values in the subnanomolar to single digit nanomolar range
`except compound 5. Their cellular (Ramos cell) inhibitory po-
`tencies in a Ca2 + flux assay[17] ranged from 3 to 92 nm. Three
`types of Michael acceptors, acrylamide, vinyl sulfonamide, and
`propiolamide, exhibited strong interactions with Btk. Adding a
`trans-oriented methyl group to the vinyl group decreased po-
`tency as shown by compound 5, which was 28-fold less potent
`than 4. This presumably relates to the reduced electrophilicity
`of the more substituted olefin. Consistent with a previous
`report,[18] compound 15 with a tertiary amine group regained
`some potency compared to 5, even though it still suffered a
`potency drop compared to 13. It was interesting to observe
`that compound 10 was about 6-fold more potent than 9, pre-
`sumably because of the difference in the electrophile orienta-
`tion. Finally, R configuration was determined as the slightly
`preferred absolute stereochemistry configuration by two sets
`of enantiomers (11 versus 12 and 13 versus 14).
`Kinases are low abundance proteins, which are tightly regu-
`lated in cells. It is well-known that biochemical assays with re-
`combinant kinases in simplified conditions may not fully reflect
`an inhibitor’s capability of mod-
`ulating kinase activities in live
`cells.[19] Therefore we further
`characterized properties of these
`compounds in cellular systems
`(Table 4).
`Even though com-
`pounds 1 and 4 showed limited
`selectivity in biochemical enzy-
`mology experiments with puri-
`fied kinases, it was very encour-
`aging to observe that 4 exhibited significant selectivity im-
`provements in cellular assays.
`In anti-IgM stimulated Ramos
`
`480
`
`G
`G
`
`481
`
`C
`S
`
`528
`
`L
`L
`
`Figure 1. The proposed binding mode of compound 1 in a homology
`model of Btk: the highlighted residues on the left side are Thr-Glu-Tyr-Met;
`the residue at the bottom right corner is the irreversibility handle Cys 481.
`
`Table 2. Selected residues of Lck and Btk.
`
`Kinase
`
`Btk
`Lck
`
`427
`
`V
`V
`
`428
`
`A
`A
`
`474
`
`T
`T
`
`Sequence Number in Btk
`475
`476
`477
`
`E
`E
`
`Y
`Y
`
`M
`M
`
`Jak3, Tec, and Txk. This result reinforced our initial proposal to
`develop a selective inhibitor for Btk through an electrophilic
`center capable of irreversibly in-
`activating the target.
`A series of irreversible inhibi-
`tors were synthesized through
`steps outlined in Scheme 1 with
`compound 4 shown as the ex-
`ample. Intermediate 2 was cou-
`pled with N-Boc-3-hydroxypiperi-
`dine by Mitsunobu reaction to
`give the Boc protected inter-
`mediate 3. After deprotection
`with acid, coupling with acid
`chlorides completed the synthe-
`sis. Their IC50 values were mea-
`sured by determination of
`re-
`
`Scheme 1. Synthesis of irreversible Btk inhibitor 4: a) polymer-bound TPP, DIAD, THF; b) HCl/dioxane; then
`acryloyl chloride, TEA.
`
`ChemMedChem 2007, 2, 58 – 61
`
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`
`www.chemmedchem.org
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`

`MED
`
`Table 3. Assay data for irreversible inhibitors.
`
`Compd
`
`R
`
`Btk
`IC50 [nm]
`
`Ramos Cell
`Ca Flux IC50 [nm][a]
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`0.72
`
`20
`
`0.52
`
`0.58
`
`0.72
`
`3.6
`
`0.58
`
`1.6
`
`1.9
`
`< 0.5
`
`1.4
`
`2.5
`
`10
`
`89
`
`92
`
`9
`
`9
`
`48
`
`3
`
`24
`
`90
`
`10
`
`7
`
`36
`
`[a] A wider range of deviation ( 50 %) was observed for potent compounds including 4, 13, and 14 due to
`their high potency and relatively low solubility in aqueous cell culture media.
`
`line, com-
`cells, a human B cell
`pound 4 greatly reduced the
`phosphorylation of Btk’s
`sub-
`strate PLC-g1 with an IC50 =
`0.014 mm, while the Lyn and Syk
`dependent phosphorylation of
`tyrosine 551 on Btk was inhibited
`(IC50 > 7.5 mm).
`more weakly
`Thus, compound 4 exhibits a
`> 500-fold selectivity between
`Btk and Lyn or Syk in cells.
`In
`contrast, compound 1 exhibits
`only a 4-fold difference in the
`IC50 values for the same assays,
`suggesting the advantages that
`irreversibility can provide. We
`also measured the effect of com-
`pound 4 on Jurkat
`cells, a
`human T cell
`line,
`in which Lck
`and Itk are required for T cell re-
`ceptor mediated Ca2 + flux. Here,
`compound 4 was 11-fold less
`active in inhibiting Ca2 + flux
`than in Ramos cells, supporting
`the expected selectivity for B
`versus T cells.
`The in vivo efficacy of com-
`pound 4 was evaluated in a
`mouse model of rheumatoid ar-
`thritis. Arthritis was induced in
`Balb/c mice by administration of
`anticollagen
`antibodies
`and
`LPS.[20] Compound 4 was admin-
`istrated orally once a day in an
`aqueous suspension for 10 con-
`secutive days, starting one day
`after
`LPS
`administration. As
`shown in Figure 2, compound 4
`inhibited arthritis development
`in a dose dependent manner,
`with a maximum effect (> 95 %
`inhibition) at dose levels of 10
`and 30 mg kg1
`(p < 0.05). The
`plasma concentrations of com-
`pound 4 that induced this maxi-
`mum effect were in the 0.6–
`1.7 mm range at Tmax
`(2 hours)
`and did not need to be sus-
`tained at high levels for 24 h to
`achieve efficacy, which is not
`surprising for an irreversible in-
`hibitor.[21]
`In summary, we have discov-
`ered a series of irreversible inhib-
`itors for Bruton’s tyrosine kinase.
`These
`inhibitors were highly
`potent
`in both in vitro and
`
`60
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`ChemMedChem 2007, 2, 58 – 61
`
`

`

`Table 4. Enzymology and cellular assay data for compounds 1 and 4.
`
`Compd
`
`Btk[a] [nm]
`
`Lck[a] [nm]
`
`Lyn[a] [nm]
`
`Itk[a] [mm]
`
`Btk p551[b] [mm]
`
`pPLC-g1[b] [mm]
`
`Ramos Ca2 + Flux[b] [mm]
`
`Jurkat Ca2 + Flux[b] [mm]
`
`1
`4
`
`8.2
`0.72[b]
`
`4.6
`97
`
`2.5
`14
`
`> 3.0
`1.0
`
`1.4
`> 7.5
`
`0.33
`0.014
`
`0.53
`0.0405
`
`n/a
`0.466
`
`[a] Ki (app) values. [b] IC50 values.
`
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`Ford, M. Galvin, J. L. Gerlach, R. M. Grotzfeld, S. Herrgard, D. E. Insko,
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`Blethrow, D. O. Morgan, K. M. Shokat, Chem. Biol. 1999, 6, 671.
`[9] S. V. Frye, Chem. Biol. 1999, 6, R3-R7.
`[10] C. Mao, M. Zhou, F. M. Uckun, J. Biol. Chem. 2001, 276, 41 435.
`[11] X. Zhu, K. A. Morgenstern, Structure 1999, 7, 651.
`[12] D. W. Fry, Pharmacol. Ther. 2002, 93, 253.
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`Johnson, R. S. Michalak, R. Nilakantan, C. Discafani, J. Golas, S. K. Rabin-
`dran, R. Shen, X. Shi, Y-F. Wang, J. Upeslacis, A. Wissner, J. Med. Chem.
`2005, 48, 1107.
`[14] Sugen and Salk Institute, http://198.202.68.14/human/kinome/phyloge-
`ny.html.
`[15] J. B. Smaill, B. D. Palmer, G. W. Rewcastle, W. A. Denny, D. J. McNamara,
`E. M. Dobrusin, A. J. Bridges, H. Zhou, H. D. Hollis Showalter, R. Thomas
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`[16] Compound 4: 440 Da; Recombinant Btk kinase domain: 33,487 Da;
`Complex (1:1): expected 33,927 Da, observed 33,927 Da. See Supporting
`Information.
`[17] FlexStation, Molecular Devices.
`[18] H.-R. Tsou, N. Mamuya, B. D. Johnson, M. F. Reich, B. C. Gruber, F. Ye, R.
`Nilakantan, R. Shen, C. Discafani, R. DeBlanc, R. Davis, F. E. Koehn, L. M.
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`[21] See Supporting Information for experimental data of exposure levels of
`compound 4.
`
`[22] J. S. Smolen, G. Steiner, Nat. Rev. Drug Discovery 2003, 2, 473.
`
`Received: September 8, 2006
`Published online on December 12, 2006
`
`Figure 2. Efficacy study of compound 4 in a mouse arthritis model. *: vehi-
`cle; ~: 1 mg kg1; &: 3 mg kg1; ^: 10 mg kg1; *: 30 mg kg1.
`
`in vivo assays. Compound 4 showed preference towards Btk
`over closely related kinases. This compound inhibited B cell
`function, and within the BCR pathway it selectively acted to in-
`hibit Btk-dependent processes. Finally, compound 4 demon-
`strated clear dose-dependent efficacy in a mouse arthritis
`model. This study not only presents a strategy to discover se-
`lective kinase inhibitors but also provides convincing evidence
`that Btk is a very attractive target for the treatment of rheuma-
`toid arthritis.[22]
`
`Keywords: biological activity · drug design · enzymes ·
`medicinal chemistry · structural bioinformatics
`
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`ChemMedChem 2007, 2, 58 – 61
`
` 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`www.chemmedchem.org
`
`61
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