USOO9353116B2
(12) United States Patent (10) Patent No.: US 9,353,116 B2 Garske et al. (45) Date of Patent: May 31, 2016
(54) METHODS AND COMPOSITIONS FOR 2009, 0221614 A1 9, 2009 Taunton et al. KNASE INHIBITION 2011/O144134 A1 6, 2011 Shokat et al. 2011/0224223 A1 9, 2011 Shokat et al. 2011/0275611 A1* 11/2011 AXten et al...... 514,210.21 (71) Applicant: THE REGENTS OF THE 2011/0275651 A1 11, 2011 Dar et al. UNIVERSITY OF CALIFORNLA, 2012,0065154 A1 3/2012 Tanaka et al. Oakland, CA (US) 2014/0243.357 A1 8, 2014 Dar et al. 2015,0031881 A1 1/2015 Tanaka et al. (72) Inventors: Adam L. Garske, San Francisco, CA 2016, OOOO789 A1 1/2016 Shokat et al. (US); Kevan M. Shokat, San Francisco, OTHER PUBLICATIONS CA (US) Chapman et al. (Bioorganic & Medicinal Chemistry Letters, Dec. 7. (73) Assignee: The Regents of the University of 2008, 19,811-813).* California, Oakland, CA (US) Chapman et al. Supplemental content. Ito et al. (Cancer Science, 2003, 94, 3-8).* Aspel et al. (Nature Chemical Biology, 6, 2008, 691-699).* (*) Notice: Subject to any disclaimer, the term of this Aspel et al. (Nature Chemical Biology, 6, 2008, Supplemental con patent is extended or adjusted under 35 tent).* U.S.C. 154(b) by 0 days. J. G. Cannon Chapter Nineteen in Burger's Medicinal Chemistry and Drug Discovery, Fifth Edition, vol. I: Principles and Practice, Wiley (21) Appl. No.: 13/690,785 Interscience 1995, pp. 783-802, 784.* AZam et al., “Activation of tyrosine kinases by mutation of the Filed: Nov.30, 2012 gatekeeper threonine.” Nat Struct Mol Biol, 15(10): 1109-18 (2008). (22) Cameron et al., “PKC Maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity.” Nat Struct Mol (65) Prior Publication Data Biol. 16(6): 624-31 (2009). US 2013/O1377O8A1 May 30, 2013 Chapman et al., “A Small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL.” Bioorganic & Medicinal Chemistry Letters, 19: 811-813 (2009). Related U.S. Application Data Elphicket al. “Using chemical genetics and ATP analogues to dissect protein kinase function.” ACS Chemical Biology, 2: 299-314 (2007). (63) Continuation of application No. Hatzivassililou et al., “RAF inhibitors prime wild-type RAF to acti PCT/US2011/039347, filed on Jun. 6, 2011. vate the MAPK pathway and enhance growth.” Nature, 464(7287): Provisional application No. 61/351,663, filed on Jun. 431-5 (2010). (60) Hindie et al., “Structure and allosteric effects of low-molecular 4, 2010. weight activators on the protein kinase PDK1.” Nat Chem Biol. 5(10): 758-64 (2009). (51) Int. C. Liu et al., “Structural basis for selective inhibition of Src family CI2N 9/12 (2006.01) kinases by PP1.” Chemistry & Biology, 6:671-678 (1999). CI2N 9/99 (2006.01) No Author, Upstate KinaseProfiler Assay Protocols, Jun. 2003 pub CO7D 487/04 (2006.01) lication. C07D 239/94 (2006.01) Okuzumi et al., “Inhibitor Hijacking of Akt Activation.” Nat Chem CO7D 403/2 (2006.01) Biol, 5(7): 484-93 (2009). CO7D 409/2 (2006.01) Zunder et al., “Discovery of drug-resistant and drug-sensitizing AOIN 43/90 (2006.01) mutations in the oncogenic PI3K isoform pl10 alpha.” Cancer Cell, 14(2): 180-92 (2008). A61 K3 1/519 (2006.01) International Search Report and Written Opinion mailed Feb. 3, 2012 (52) U.S. C. in related International Patent Application No. PCT/US2011/ CPC ...... C07D 487/04 (2013.01); C07D 239/94 039347, filed Jun. 6, 2011, 11 pages. (2013.01); C07D 403/12 (2013.01); C07D 409/12 (2013.01); C12N 9/12 (2013.01) * cited by examiner (58) Field of Classification Search None Primary Examiner — Dennis Heyer See application file for complete search history. Assistant Examiner — Daniel M Podgorski (74) Attorney, Agent, or Firm — Mintz Levin Cohn Ferris (56) References Cited Glovsky and Popeo, P.C. U.S. PATENT DOCUMENTS (57) ABSTRACT 5,643,734 A 7, 1997 Henderson The present invention sets forth a new chemical genetic 6,589,950 B1 7/2003 Hayler et al. approach for engineering kinase enzymes with a cysteine 7,271,262 B2 9, 2007 La Greca et al. 7,332,497 B2 2/2008 Hirst et al. gatekeeper residue as well as for developing electrophilic 7,585,868 B2 9/2009 Knight et al. inhibitors thereto. The present invention also provides a Src 8,642,604 B2 2/2014 Knight et al. proto-oncogenic tyrosine kinase with a cysteine gatekeeper 2003/0187001 A1 10, 2003 Calderwood et al. that recapitulates wild type activity and can be irreversibly 2005/0O85472 A1 4/2005 Tanaka et al. 2006.0035912 A1 2/2006 Marx et al. inhibited both in vitro and in cells. The present invention also 2007/0293489 A1 12/2007 Adams et al. provides methods and compositions for modulating kinases 2009, OO29989 A1 1/2009 Adams et al. and for treating kinase-associate diseases. 2009, O124638 A1 5, 2009 Shokat et al. 2009, O181988 A1 7/2009 Tanaka et al. 12 Claims, 22 Drawing Sheets U.S. Patent May 31, 2016 Sheet 1 of 22 US 9,353,116 B2
... 1
Gatekeeper U.S. Patent May 31, 2016 Sheet 2 of 22 US 9,353,116 B2
FG 2
WT types U.S. Patent Sheet 3 of 22 US 9,353,116 B2
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U.S. Patent May 31, 2016 Sheet 4 of 22 US 9,353,116 B2
FG4 y & Co. S 338 . Sir y-Six-i-S
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U.S. Patent May 31, 2016 Sheet 5 of 22 US 9,353,116 B2
FGS
Sir T338C C-SC
SFS
si. 5.
. U.S. Patent May 31, 2016 Sheet 6 of 22 US 9,353,116 B2
F.G. 6
8. SS3 U.S. Patent May 31, 2016 Sheet 7 of 22 US 9,353,116 B2
MW, kDa 7O - 3O -
S - piyr V -
43 -
34 -
SC
3-Actin
U.S. Patent May 31, 2016 Sheet 9 of 22 US 9,353,116 B2
FG. 9
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FG 1) indirubin indu bin-3
-monooxime 8
SS9 y 4 sse X . U.S. Patent May 31, 2016 Sheet 11 of 22 US 9,353,116 B2
FIG 11 3-VS-O BTK 59 CHEK2 (CHK2) 53 EGFR (ErbB1) EGFR (ErbB1) L858R 69 EGFR (ErbB1) L861Q 76 EGFR (ErbB1) T790ML858R 41 ERBB2 (HER2) 66 ERBB4 (HER4) FLT3 D835Y GRK5 LRRK2 LRRK2 G2019S PDGFRA V561D 45 PIK3C2B (PI3K-C2 beta) 65 RPS6KA6 (RSK4) 49 SRMS (Srm) 71 TXK 78 U.S. Patent May 31, 2016 Sheet 12 of 22 US 9,353,116 B2
FIG. 12 WT LRRK2 G2019S LRRK2
156 nM
>1000 nM
>1000 nM
>1000 nM >1000 M U.S. Patent May 31, 2016 Sheet 13 of 22 US 9,353,116 B2
F.G. 13 LRRK2 WT
25OOOO
2OOOOO
15OOOO
1 OOOOO
5OOOO U.S. Patent May 31, 2016 Sheet 14 of 22 US 9,353,116 B2
F.G. 14 LRRK2 GS
45OOOO 4OOOOO 35OOOO 3OOOOO 25OOOO 2OOOOO 150000 1 OOOOO 5OOOO U.S. Patent May 31, 2016 Sheet 15 of 22 US 9,353,116 B2
FG 15
C HN1 c NH 2 N MeO JCl MeO 9 MeO H NN HN NH NN c1r TEA NN MeO al IPA, reflux, 5h MeO n? DCM. O'C MeO r U.S. Patent May 31, 2016 Sheet 16 of 22 US 9,353,116 B2
FG 16 U.S. Patent May 31, 2016 Sheet 17 of 22 US 9,353,116 B2
F.G. 17 3VSO 12 3VSO 5.5h
s f on 7 D 50 s 2 O -8 -7 -6 -5 -4 logCompound (M) U.S. Patent May 31, 2016 Sheet 18 of 22 US 9,353,116 B2
FG 18 R=Thiophene 12 R=Thiophene 5.5h O f 5 10 D 75 SS 50 -8 -7 -6 -5 -4 logCompound (M) U.S. Patent May 31, 2016 Sheet 19 of 22 US 9,353,116 B2
F.G. 19 R=Phenyl 12 R=Ph 5.5h O a 100 D 7 SS
50 -8 -7 -6 -5 -4 logCompound (M) U.S. Patent May 31, 2016 Sheet 20 of 22 US 9,353,116 B2
FG 2
U.S. Patent May 31, 2016 Sheet 21 of 22 US 9,353,116 B2
FG. 21 LRRK2 G2019S in-vitro Kinase Assay
8 Phenyl O A CUN-18 2 A CJN-25 $ CJN-26 c C CJN-27 s o CJN-28 s & CJN-29 A CJN-30
log Cmpd(M) U.S. Patent May 31, 2016 Sheet 22 of 22 US 9,353,116 B2
S. C HN IOl NH HN JOl3:1-R MeO JCl MeO Q9 MeO nN HM NH NN CI-R, TEA NN MeO NIPA,-- reflux sh MeO 2 - DCM, OC - 2 US 9,353,116 B2 1. 2 METHODS AND COMPOSITIONS FOR It is known in the field that mutations in Leucine-Rich KNASE INHIBITION Repeat Kinase 2 (Lrrk-2) can lead to Parkinsons Disease. Also, it is thought that Parkinson's Disease (PD) is caused by CROSS-REFERENCES TO RELATED uncontrolled apoptosis of dopaminergic neurons. Because APPLICATIONS inhibition of Lrrk-2 kinase activity can inhibit the apoptotic effects, there is a need to develop inhibitors for Lrrk-2 to This application claims the benefit of U.S. Provisional provide treatments for Parkinson's Disease. Patent Application 61/351,663, filed Jun. 4, 2010, and Inter As such, there is a need in the field to develop kinase national Patent Application PCT/US2011/039347, filed Jun. gatekeeper residue mutations which do not diminish kinase 6, 2011, which are hereby incorporated by reference in their 10 activity or ATP affinity as well as small molecules which entirety for all purposes. inhibit these kinases. There is also a need to develop effective Lrrk-2 inhig. Surprisingly, the present invention solves these STATEMENT AS TO RIGHTS TO INVENTIONS as well as other problems in the field. MADE UNDER FEDERALLY SPONSORED 15 RESEARCH ORDEVELOPMENT REFERENCE BRIEF SUMMARY OF THE INVENTION TO A “SEQUENCE LISTING ATABLE, ORA COMPUTER PROGRAM LISTINGAPPENDIX In one aspect, the present invention provides a compound SUBMITTED AS ANASCII TEXT FILE having the formula: The Sequence Listing written in file -122-1.TXT, created on Jan. 23, 2013, 229,376 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes. This invention was made with Government support under 25 Grant Nos. 5F32CA138103-2 and 1R01EB001987-16, N1 N\ awarded by the National Institutes of Health. The Govern (R -- it 2NN X ment has certain rights in this invention. N V L2 V BACKGROUND OF THE INVENTION 30 R2 (II) Kinases, which constitute a large family of enzymes (>500 in humans), catalyze the transfer of the Y-phosphate of ATP to protein substrates. Reversible phosphorylation plays a para L4 mount role in cell signaling processes and is regulated by 35 kinases and phosphatases. Accordingly, kinases are critical mediators of a myriad of signal transduction processes. Aber N21 N rant kinase activity is linked to cancer as well as metabolic, 1sN | 21H-L-R); or immunological, and nervous system disorders. As a result, RI -L N kinases have emerged as an important class of drug targets for 40 human disease. However, due to the conserved nature of the (III) active sites of the protein kinase family, it is difficult to obtain selective inhibitors for any one kinase. There are at least 518 kinases, such as those which catalyze L4 the transfer of the gamma phosphate of ATP to protein and 45 Small molecule Substrates and are involved in cell signaling processes. Small molecules provide a means for delineating 4N 1s kinase signaling because they are fast acting and dosable. e-'ts j-i-tN4 ---L-R2). However, because all kinase active sites recognize ATP, it is difficult to develop selective ATP-competitive inhibitors. 50 Several years ago, a chemical genetic strategy for selective X is =N- or =C(L-R)-. Ring A is, in each instance, kinase inhibition was developed with reversible inhibitors independently selected from cycloalkyl, heterocycloalkyl, (U.S. Patent Publication No. 2009/0221614). The chemical aryl, or heteroaryl; L', L. L. L. L. and L are, in each genetic strategy involves the engineered mutation of a con instance, independently selected from a bond, —C(O)—, served bulky residue in the kinase active site known as “the 55 C(O)N(R) - C(O)O S(O), S(O)N(R') , gatekeeper” to a small residue Such as glycine or alanine (See -O-, - N(R)-, - N(R)C(O)N(R) , substituted or Bishop AC, et al. (1998) Design of allele-specific inhibitors unsubstituted alkylene, substituted or unsubstituted het to probe protein kinase signaling. Curr Biol 8(5):257-266: eroalkylene, substituted or unsubstituted cycloalkylene, sub and Bishop AC, et al. (2000) A chemical switch for inhibitor stituted or unsubstituted heterocycloalkylene, substituted or sensitive alleles of any protein kinase. Nature 407(6802):395 60 unsubstituted arylene, or substituted or unsubstituted het 401). The engineered active site can then accommodate an eroarylene, whereing is an integer from 0 to 2: R. R. R. R. inhibitor capable of occupying the newly formed binding R. R. R', and R are, in each instance, independently pocket. While this strategy has utility, mutation of the gate selected from hydrogen, halogen, —CN. —OH, -NH2, keeper residue to a Small amino acid may impair the activity -COOH, -CONH, NO, -SH, -SOCl, -SOH, of the kinase and the selective inhibition can only be applied 65 —SOH, -SONH, substituted or unsubstituted alkyl, sub to one kinase at a time. In addition, it is sometimes not stituted or unsubstituted heteroalkyl, substituted or unsubsti possible to achieve the desired potency. tuted cycloalkyl, substituted or unsubstituted heterocy US 9,353,116 B2 3 4 cloalkyl, substituted or unsubstituted aryl, or substituted or tively. An analog-sensitive (AS) protein kinase has an engi unsubstituted heteroaryl; a is an integer from 0 to 2: b is an neered glycine or alanine gatekeeper and may be selectively integer from 0 to 5; and c is an integer from 0 to 4. inhibited by a bulky inhibitor. An electrophile-sensitive (ES) In a second aspect, the present invention provides a recom protein kinase contains an engineered cysteine gatekeeper binant kinase comprising a cysteine Substitution at a gate and may be selectively inhibited by an electrophilic inhibitor. keeper amino acid position. FIG. 2 shows the crystal structure of compound 9 bound In a third aspect, the present invention provides a co-crystal covalently to c-Src-ES1. The experimental electron density of comprising a recombinant kinase and a compound of pro c-Src-ES1 at 2.20 A resolution is shown (2Fo-F map at lo). vided herein (e.g. formula I, II, or III). (A) The pyrazolopyrimidine portion of compound 9 (green) In a fourth aspect, the present invention provides an iso 10 interacts with the hinge region of c-Src (Met-341 and Glu lated nucleic acid comprising a polynucleotide sequence 339), while the sulfonamide group makes a hydrogen bonds encoding a recombinant kinase provided herein. with Glu-310 of the OC helix (B) Electron density reveals a In a fifth aspect, the present invention provides a method of covalent linkage between Cys-338 and compound 9. The inhibiting a recombinant kinase provided herein, comprising oxygen atoms of the Sulfonamide interact with the backbone contacting the recombinant kinase with an effective amount 15 of Asp-404 and via a water molecule with Phe-405, both of of an inhibitor provided herein, thereby inhibiting the recom which are part of the DFG-motif of the kinase (C) Compari binant kinase. son of structural features of compound 9 bound to c-Src-ES1 In a sixth aspect, the present invention provides a com and a known pyrazolopyrimidine compound bound to WT pound having the formula: c-Src. Both compounds engage the hinge region in a similar fashion and bind the OC helix in the “in” conformation. Furthermore, both compounds participate in hydrogen bond (XV) ing interactions with Glu-310 and backbone amides of the DFG-motif. However while the known pyrazolopyrimidine compound binds in the “DFG-out” conformation, compound L4 25 9 engages the “DFG-in' orientation. The sulfhydryl of the Cys-338 points in the opposite direction relative to the hydroxyl group of Thr-338 in order to facilitate a covalent XI N N bond with compound 9. l 2 ---L-R). FIG. 3 shows an assay for MOK inhibition by cysteine -L x 21 30 gatekeeper-targeting compounds. (top) FLAG-MOK R expressed in COST cells was immunoprecipitated and assayed in vitro with a myelin basic protein (MBP) substrate X' and X’ are, in each instance, independently —N— or and inhibitors at a concentration of 1 uM. Autoradiography is =C(-L-R)-. Ring A is as defined above. R', R, and Rare shown. (center) Quantification of the percent MBP phospho as defined above. L', L, and L are as defined above. The 35 rylated from three independent experiments with associated variables band c are as defined above. standard errors. All values are normalized relative to the In a seventh aspect, the present invention provides a MOK+DMSO lane. (bottom) Western blot of loading con method of inhibiting a Lrrk-2 kinase, the method comprising trols for FLAG-MOK are shown. contacting the Lrrk-2 kinase with an effective amount of a FIG. 4 shows a cellular dose response analysis for inhibi Lrrk-2 inhibitor, thereby inhibiting the Lrrk-2 kinase. 40 tion of v-Src-ES1 (I338C) with electrophilic inhibitors. Cells In an eighth aspect, the present invention provides a transfected NIH-3T3 with either v-Src-ES1 or I338T v-Src method of forming a recombinant kinase, comprising trans were treated with electrophilic inhibitors or non-reactive ana forming a cell with a nucleic acid as set forth herein, thereby logs for one hour (see the far right column of each run, e.g. 10 forming a recombinant kinase as set forth herein. uM 11; 10 uM 11; 10 uM 14: 10 uM 14). Kinase activity was In a ninth aspect, the present invention provides a method 45 monitored by blotting for global phosphotyrosine levels. of treating a kinase-associated disease or condition, in a Actin blots were included to control for protein content. patient in need thereof, said method comprising administer FIG. 5 shows relative rates of wild type c-Src and T338C ing to said patient a therapeutically effective amount of a c-Src following treatment with PP1, 13 or 9 and purification compound of the present invention, thereby treating a kinase by gel filtration. Assay was done in triplicate, and average associated disease or condition. 50 values with standard errors are given. In a tenth aspect, the present invention provides a method FIG. 6 shows ESI-oa-TOF mass spectral analysis of cova of treating a Lrrk-2-associated disease or condition, in a lent labeling of T338C c-Src and WT c-Src with compound 9. patient in need thereof, said method comprising administer T338C c-Src (a) or WT c-Src (b)(15uM) was incubated with ing to said patient a therapeutically effective amount of a two equivalents of compound 9 and analyzed by full-protein compound of the present invention, thereby treating a Lrrk 55 mass spectrometry after 5 minutes of reaction. A 372 Damass 2-associated disease or condition. change occurs upon covalent labeling. Deconvoluted mass In an eleventh aspect, the present invention provides a kit spectra are shown. comprising, a recombinant kinase or a nucleic acid provided FIG. 7 shows analysis of the activity of v-Src gatekeeper herein; and instructions for using the kit. variants in cells by Western blot. NIH-3T3 cells lines were 60 infected with several v-Src gatekeeper variants. The kinase BRIEF DESCRIPTION OF THE DRAWINGS activity of the variants was analyzed by blotting for global phosphotyrosine levels (pTyr). The Src and actin blots FIG. 1 shows a schematic of the chemical genetic strategies account for Src expression levels and total protein content, for inhibiting protein kinases. Kinases are depicted on top, respectively. e.g. WT, AS, and ES, and inhibitors types are represented on 65 FIG.8 shows the amino acid sequence (SEQID NO: 2) of the bottom. Wild type (WT) kinases generally harbor hydro Src and also the nucleic acid sequence (SEQ ID NO: 1) phobic gatekeeper residues and may not be inhibited selec encoding therefor. US 9,353,116 B2 5 6 FIG. 9 shows inhibition of Lrrk-2 kinase activity. in the present invention. A “lower alkyl or “lower alkylene’ FIG. 10 shows inhibition of Lrrk-2 kinase activity. is a shorter chain alkyl or alkylene group, generally having FIG. 11 shows selectivity of compound 19 (3-vs-Q) in the eight or fewer carbon atoms. Invitrogen SelectScreen Kinase Assay. The term "heteroalkyl by itself or in combination with FIG. 12 shows SAR analysis and inhibition as dependent 5 another term, means, unless otherwise stated, a stable straight on a vinylsulfonamide in the 3 position. or branched chain, or combinations thereof, consisting of at FIG. 13 shows SAR analysis and inhibition as dependent least one carbon atoms and at least one heteroatom selected on a vinylsulfonamide in the 3 position. from the group consisting of O. N. P. Si and S, and wherein the FIG. 14 shows SAR analysis and inhibition as dependent nitrogen, phosphorus, and Sulfur atoms may optionally be on a vinylsulfonamide in the 3 position. 10 oxidized and the nitrogen heteroatom may optionally be quat ernized. The heteroatom(s) O, N, P and S and Si may be FIG. 15 shows the synthesis of compounds suitable for use placed at any interior position of the heteroalkyl group or at with the present invention. the position at which alkyl group is attached to the remainder FIG. 16 shows in vitro kinase assay data for wild type and G2019S Lrrk-2. of the molecule. Examples include, but are not limited to, 15 —CH2—CH2—O—CH, —CH2—CH NH CH, FIG. 17 shows toxicity profiles. LD50 of compound 19 CH, CH, N(CH)—CH, —CH, S CH, CH, (3-vs-Q). LD50–3.92 uM; All other compounds -50 uM. CH2—CH2—, —S(O)—CH, —CH2—CH2—S(O) FIG. 18 shows toxicity profiles. LD50 of compound 19 CH, —CH=CH-O CH, Si(CH), —CH2— (3-vs-Q). LD50–3.92 uM; All other compounds -50 uM. CH=N OCH, -CH=CH-N(CH) CH, O CH, FIG. 19 shows toxicity profiles. LD50 of compound 19 —O—CH2—CH, and —CN. Up to two or three heteroat (3-vs-Q). LD50–3.92 uM; All other compounds -50 uM. oms may be consecutive, such as, for example, —CH2— FIG. 20 shows immunocytochemistry. Top left: Staruaspo NH OCH and —CH2—O Si (CH). Similarly, the term rine, TUNEL stain; Top Right: G2019S mutant, -drug; Bot "heteroalkylene' by itself or as part of another substituent tom: G2019S mutant, +Th. means a divalent radical derived from heteroalkyl, as exem FIG. 21 shows assay data. 25 plified, but not limited by, —CH2—CH2—S CH-CH FIG. 22 shows a synthesis of compounds suitable for use and CH, S CH-CH NH-CH For het with the present invention. eroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, DETAILED DESCRIPTION OF THE INVENTION alkyleneamino, alkylenediamino, and the like). Still further, 30 for alkylene and heteroalkylene linking groups, no orienta I. General tion of the linking group is implied by the direction in which Provided herein, interalia, are methods and compositions the formula of the linking group is written. For example, the for imparting to a kinase the capability of being inhibited by formula —C(O)OR'— represents both —C(O)OR and a heterocyclic compound e.g., a cysteine Substituted kinase —R'OC(O)—. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the having a gatekeeper amino acid residue within an ATP bind remainder of the molecule through a heteroatom, such as ing site of a kinase replaced with a cysteine residue. Also C(O)R', C(O)NR', NR'R", OR', SR', and/or provided are methods and compositions for inhibiting a - SOR". Where "heteroalkyl is recited, followed by recita kinase with a heterocyclic compound. Furthermore, methods tions of specific heteroalkyl groups, such as —NR'R' or the and compositions are provided for determining a biological 40 like, it will be understood that the terms heteroalkyl and activity of a kinase and treating kinase-associate diseases. In —NR'R" are not redundant or mutually exclusive. Rather, the addition, methods and compositions are provided for inhib specific heteroalkyl groups are recited to add clarity. Thus, the iting a Lrrk-2 kinase. term "heteroalkyl should not be interpreted hereinas exclud II. Definitions ing specific heteroalkyl groups, such as —NR'R' or the like. The term “alkyl by itself or as part of another substituent, 45 The terms “cycloalkyl and "heterocycloalkyl, by them means, unless otherwise stated, a straight (i.e. unbranched) or selves or in combination with other terms, represent, unless branched chain, or combination thereof, which may be fully otherwise stated, cyclic versions of “alkyland "heteroalkyl, saturated, mono- or polyunsaturated and can include di- and respectively. Additionally, for heterocycloalkyl, a heteroatom multivalent radicals, having the number of carbonatoms des can occupy the position at which the heterocycle is attached to ignated (i.e. C-Co means one to ten carbons). Examples of 50 the remainder of the molecule. Examples of cycloalkyl saturated hydrocarbon radicals include, but are not limited to, include, but are not limited to, cyclopentyl, cyclohexyl, 1-cy groups such as methyl, ethyl, n-propyl, isopropyl. n-butyl, clohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. t-butyl, isobutyl, sec-butyl, homologs and isomers of for Examples of heterocycloalkyl include, but are not limited to, example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, unsaturated alkyl group is one having one or more double 55 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofu bonds or triple bonds. Examples of unsaturated alkyl groups ran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahy include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-iso drothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The pentenyl, 2-(butadienyl), 2.4-pentadienyl, 3-(1,4-pentadi terms “cycloalkylene' and "heterocycloalkylene' refer to the enyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher divalent derivatives of cycloalkyl and heterocycloalkyl, homologs and isomers. 60 respectively. The term “alkylene' by itself or as part of another substitu The term “aryl' means, unless otherwise stated, a polyun ent means a divalent radical derived from an alkyl, as exem saturated, aromatic, hydrocarbon Substituent which can be a plified, but not limited, by —CHCHCHCH , single ring or multiple rings (preferably from 1 to 3 rings) —CH-CH=CH=CH , —CH2C=CCH , which are fused together (e.g. naphthyl) or linked covalently. —CH2CH2CH(CH2CHCH)CH2—. Typically, an alkyl (or 65 The term "heteroaryl refers to aryl groups (or rings) that alkylene) group will have from 1 to 24 carbon atoms, with contain heteroatoms (in at least one ring in the case of mul those groups having 10 or fewer carbonatoms being preferred tiple rings) selected from N, O, and S, wherein the nitrogen US 9,353,116 B2 7 8 and Sulfur atoms are optionally oxidized, and the nitrogen same nitrogen atom, they can be combined with the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be atom to form a 4-, 5-, 6-, or 7-membered ring. For example, attached to the remainder of the molecule through a carbon or —NR'R'" is meant to include, but not be limited to, 1-pyrro heteroatom. Non-limiting examples of aryl and heteroaryl lidinyl and 4-morpholinyl. From the above discussion of sub groups include phenyl, 1-naphthyl 2-naphthyl, 4-biphenyl, stituents, one of skill in the art will understand that the term 1-pyrrolyl 2-pyrrolyl, 3-pyrrolyl 3-pyrazolyl, 2-imidazolyl, “alkyl is meant to include groups including carbon atoms 4-imidazolyl pyrazinyl, 2-oxazolyl, 4-oxazolyl 2-phenyl-4- bound to groups other than hydrogen groups. Such as oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isox haloalkyl (e.g., —CF and —CH2CF) and acyl (e.g., —C(O) azolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, CH, —C(O)CF, —C(O)CHOCH, and the like). 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrim 10 Similar to the substituents described for alkyl radicals idyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimida above, exemplary Substituents for aryland heteroaryl groups Zolyl, 5-indolyl, 1-isoquinolyl, 6-isoquinolyl 2-quinoxali (as well as their divalent derivatives) are varied and are nyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Thus, the selected from, for example: halogen, —OR', —NR'R'", term "heteroaryl' include fused ring structures in which at - SR", -halogen, -SiR'R"R", OC(O)R', C(O)R', least one ring includes at least two double bonds. Substituents 15 COR', C(O)NR'R", OC(O)NR'R", NR"C(O)R', for each of above noted aryland heteroaryl ring systems are NR' C(O)NR"R", NR"C(O)OR', NR C selected from the group of acceptable substituents described (NR'R"R") NR", NR C(NR'R")—NR", S(O)R', below. The terms “arylene' and "heteroarylene' refer to the —S(O).R', -S(O)NR'R", NRSOR', CN and - NO, divalent radicals of aryland heteroaryl, respectively. —R', —CH(Ph), fluoro(C-C)alkoxo, and fluoro(C-C) For brevity, the term “aryl when used in combination with alkyl, in a number ranging from Zero to the total number of other terms (e.g., aryloxo, arylthioxo, arylalkyl) includes both open valences on aromatic ring system; and where R', R", R" aryl and heteroaryl rings as defined above. Thus, the term and R" are preferably independently selected from hydro “arylalkyl is meant to include those radicals in which an aryl gen, Substituted or unsubstituted alkyl, Substituted or unsub group is attached to an alkyl group (e.g., benzyl, phenethyl, stituted heteroalkyl, substituted or unsubstituted cycloalkyl, pyridylmethyl and the like) including those alkyl groups in 25 substituted or unsubstituted heterocycloalkyl, substituted or which a carbon atom (e.g., a methylene group) has been unsubstituted aryl and substituted or unsubstituted het replaced by, for example, an oxygen atom (e.g., phenoxym eroaryl. When a compound of the invention includes more ethyl 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the than one R group, for example, each of the R groups is like). However, the term “haloarylas used herein is meant to independently selected as are each R', R", R" and R" groups cover only aryls substituted with one or more halogens. 30 when more than one of these groups is present. Where a heteroalkyl, heterocycloalkyl, or heteroaryl Two of the substituents on adjacent atoms of the aryl or includes a specific number of members (e.g. "3 to 7 mem heteroaryl ring may optionally form a ring of the formula bered”), the term “member referrers to a carbon or heteroa -T-C(O) (CRR), U , wherein T and U are indepen tOm. dently —NR— —O—, —CRR - or a single bond, and q is The term “oxo' as used herein means an oxygen that is 35 an integer of from 0 to 3. Alternatively, two of the substituents double bonded to a carbon atom. on adjacent atoms of the aryl or heteroaryl ring may Each of above terms (e.g., “alkyl, "heteroalkyl.” optionally be replaced with a substituent of the formula -A- “cycloalkyl, and "heterocycloalkyl, "heteroaryl as well as (CH), B–, wherein A and B are independently their divalent radical derivatives) are meant to include both CRR' , —O— —NR— —S , —S(O)— —S(O) , substituted and unsubstituted forms of the indicated radical. 40 —S(O)NR' or a single bond, and r is an integer of from 1 Preferred substituents for each type of radical are provided to 4. One of the single bonds of the new ring so formed may below. optionally be replaced with a double bond. Alternatively, two Substituents for alkyl, heteroalkyl, cycloalkyl, heterocy of the substituents on adjacent atoms of the aryl or heteroaryl cloalkyl monovalent and divalent derivative radicals (includ ring may optionally be replaced with a Substituent of the ing those groups often referred to as alkylene, alkenyl, het 45 formula—(CRR"). X (C"R") , where sand dare inde eroalkylene, heteroalkenyl, alkynyl, cycloalkyl, pendently integers of from 0 to 3, and X is —O— —NR' . heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can S , S(O)— —S(O) , or S(O)NR' . The sub be one or more of a variety of groups selected from, but not stituents R. R', R" and R" are preferably independently limited to: OR', -O, -NR', -N OR', NR'R'', SR', selected from hydrogen, substituted or unsubstituted alkyl, -halogen, -SiR'R"R", OC(O)R', -C(O)R', -COR', 50 substituted or unsubstituted cycloalkyl, substituted or unsub C(O)NR'R", OC(O)NR'R", NR"C(O)R', NR C stituted heterocycloalkyl, substituted or unsubstituted aryl, (O)NR"R", NR"C(O)OR, NR C(NR'R")—NR", and substituted or unsubstituted heteroaryl. S(O)R', S(O).R', S(O)NR'R", NRSOR', CN As used herein, the term "heteroatom' or “ring heteroa and —NO in a number ranging from Zero to (2 m'+1), where tom' is meant to include oxygen (O), nitrogen (N), Sulfur (S), m" is the total number of carbonatoms in such radical. R', R", 55 phosphorus (P), and silicon (Si). R" and R" each preferably independently refer to hydrogen, The terms “halo' or “halogen by themselves or as part of substituted or unsubstituted heteroalkyl, substituted or another Substituent, mean, unless otherwise stated, a fluorine, unsubstituted cycloalkyl, substituted or unsubstituted hetero chlorine, bromine, or iodine atom. Additionally, terms such as cycloalkyl, Substituted or unsubstituted aryl (e.g., aryl Substi “haloalkyl.” are meant to include monohaloalkyl and polyha tuted with 1-3 halogens), substituted or unsubstituted alkyl, 60 loalkyl. For example, the term “halo (C-C)alkyl is mean to alkoxy or thioalkoxy groups, or arylalkyl groups. As used include, but not be limited to, trifluoromethyl, 2.2.2-trifluo herein, an “alkoxy' group is an alkyl attached to the remain roethyl, 4-chlorobutyl, 3-bromopropyl, and the like. der of the molecule through a divalent oxygen radical. When A "size-limited substituent’ or "size-limited substituent a compound of the invention includes more than one R group, group, as used herein means a group selected from all of the for example, each of the R groups is independently selected as 65 substituents described above for a “substituent group.” are each R', R", R" and R" groups when more than one of wherein each substituted or unsubstituted alkyl is a substi these groups is present. When R' and R" are attached to the tuted or unsubstituted C-Co each Substituted or unsubsti US 9,353,116 B2 10 tuted heteroalkyl is a substituted or unsubstituted 2 to 20 larger than cysteine. A bulky residue may be methionine, membered heteroalkyl, each substituted or unsubstituted leucine, phenylalanine and threonine In some embodiments, cycloalkyl is a Substituted or unsubstituted C-C cycloalkyl, the bulky reside may be larger than leucine, isoleucine and and each substituted or unsubstituted heterocycloalkyl is a threonine. In some embodiments, the bulky reside includes a substituted or unsubstituted 4 to 8 membered heterocy cyclic moiety. cloalkyl. “Electrophilic' is used herein in accordance with its plain A“lower substituent’ or “lower substituent group, as used ordinary meaning and refers to a chemical group having a herein means a group selected from all of the Substituents tendency to attract, acquire or accept electrons or react at described above for a “substituent group, wherein each sub electron-rich sites. stituted or unsubstituted alkyl is a substituted or unsubstituted 10 “Nucleophilic' is used herein in accordance with its plain C-C alkyl, each substituted or unsubstituted heteroalkyl is a ordinary meaning and refers to a chemical group having a substituted or unsubstituted 2 to 8 membered heteroalkyl, tendency to donate electrons (e.g. lower electron density) or each substituted or unsubstituted cycloalkyl is a substituted or react at electron poor sites. unsubstituted C-C cycloalkyl, and each substituted or “Electrophilic moiety” as used herein refers to a functional unsubstituted heterocycloalkyl is a substituted or unsubsti 15 group or chemical Substituent that is electrophilic. Example tuted 5 to 7 membered heterocycloalkyl. electrophilic moieties include, but are not limited to vinylsul In Some embodiments, each Substituted group described in fonamides, acrylamides, epoxides, and fluoromethylketones. the compounds herein is substituted with at least one substitu As defined herein, the term “electrophilic substituent is a ent group. More specifically, in some embodiments, each substituent that is electrophilic. An electrophilic substituent, substituted alkyl, substituted heteroalkyl, substituted electrophilic moieties and electrophilic chemical groups are cycloalkyl, substituted heterocycloalkyl, substituted aryl, typically electron-poor functional groups and can react with substituted heteroaryl, substituted alkylene, substituted het an electron-donating group, such as a nucleophile, by accept eroalkylene, substituted cycloalkylene, substituted heterocy ing an electron pair. In some embodiments, the electrophilic cloalkylene, substituted arylene, and/or substituted het Substituent, moiety or chemical group of a compound is eroarylene described in the compounds herein are substituted 25 capable of reacting with a cysteine residue. In some embodi with at least one Substituent group. In other embodiments, at ments, the electrophilic Substituent, moiety or chemical least one orall of these groups are Substituted with at least one group is capable of forming a covalent bond with a cysteine size-limited Substituent group. Alternatively, at least one or residue within the ATP binding site of the kinase. The cova all of these groups are substituted with at least one lower lent bond is usually formed between the electrophilic sub Substituent group. 30 stituent, moiety or chemical group and the Sulfhydryl group In other embodiments of the compounds herein, each Sub of the cysteine and may be a reversible or irreversible bond. In stituted or unsubstituted alkyl is a substituted or unsubstituted some embodiments, the covalent bond is irreversible. C-C alkyl, each substituted or unsubstituted heteroalkyl is As used herein, the terms “protein kinase' or “kinase' are a substituted or unsubstituted 2 to 20 membered heteroalkyl, used in accordance with its plain ordinary meaning and each substituted or unsubstituted cycloalkyl is a substituted or 35 referst to an enzyme that is capable of phosphorylating an unsubstituted C-C cycloalkyl, and/or each Substituted or amino acid residue, e.g. an amino acid residue on a protein. unsubstituted heterocycloalkyl is a substituted or unsubsti Typically specific serine, threonine, or tyrosine residues are tuted 3 to 8 membered heterocycloalkyl. In some embodi phosphorylated. Thus, protein kinase encompasses serine ments of the compounds herein, each Substituted or unsub protein kinases, threonine protein kinases, and tyrosine pro stituted alkylene is a substituted or unsubstituted C-Co 40 tein kinases. An "inhibitor of a protein kinase' is a compound alkylene, each substituted or unsubstituted heteroalkylene is a or agent that reduces the activity of a protein kinase. In some substituted or unsubstituted 2 to 20 membered heteroalky embodiments, a “protein kinase inhibitor is a compound that lene, each Substituted or unsubstituted cycloalkylene is a reduces the activity of the protein kinase by binding to the Substituted or unsubstituted C-C cycloalkylene, and/or each protein kinase. Thus, a “protein kinase inhibitor can inhibit substituted or unsubstituted heterocycloalkylene is a substi 45 activity of the enzyme in a competitive, or a noncompetitive tuted or unsubstituted 3 to 8 membered heterocycloalkylene. a. In some embodiments, each substituted or unsubstituted As defined herein, the term "cysteine substituted kinase' alkyl is a Substituted or unsubstituted C-C alkyl, each Sub refers to a recombinant kinase where a gatekeeperamino acid stituted or unsubstituted heteroalkyl is a substituted or unsub residue (e.g. within an ATP binding site of the kinase) is stituted 2 to 8 membered heteroalkyl, each substituted or 50 replaced with a cysteine residue. Similarly, a 'glycine Substi unsubstituted cycloalkyl is a substituted or unsubstituted tuted kinase' refers to a recombinant kinase where a gate Cs-C, cycloalkyl, and/or each Substituted or unsubstituted keeper amino acid residue (e.g. within an ATP binding site of heterocycloalkyl is a substituted or unsubstituted 5 to 7 mem the kinase) is replaced with a glycine residue, and a "alanine bered heterocycloalkyl. In some embodiments, each substi Substituted kinase' refers to a kinase where a gatekeeper tuted or unsubstituted alkylene is a substituted or unsubsti 55 amino acid residue (e.g. within an ATP binding site of the tuted C-C alkylene, each substituted or unsubstituted kinase) is replaced with a alanine residue. heteroalkylene is a substituted or unsubstituted 2 to 8 mem As defined herein, the term “fused rings' refers to a ring bered heteroalkylene, each substituted or unsubstituted system with two or more rings having at least one bond and cycloalkylene is a Substituted or unsubstituted C-C, two atoms in common. cycloalkylene, and/or each substituted or unsubstituted het 60 The terms “nucleic acid, "oligonucleotide.” “polynucle erocycloalkylene is a substituted or unsubstituted 5 to 7 mem otide.” and like terms typically refer to polymers of deoxyri bered heterocycloalkylene. In some embodiments, the com bonucleotides or ribonucleotides in either single—or double pound is a chemical species set forth in the Examples section stranded form, and complements thereof. The term below. “nucleotide' typically refers to a monomer. The terms A "bulky residue' in an amino acid residue having a side 65 encompass nucleic acids containing known nucleotide ana chain group that is larger (i.e. having more atoms and tending logs or modified backbone residues or linkages, which are to fill more space) than glycine and alanine, and optionally synthetic, naturally occurring, and non-naturally occurring, US 9,353,116 B2 11 12 which have similar binding properties as the reference nucleic sequence, that insertion will not correspond to a numbered acid, and which are metabolized in a manner similar to the amino acid position in the reference sequence. In the case of reference nucleotides. Examples of Such analogs include, truncations or fusions there can be stretches of amino acids in without limitation, phosphorothioatcs, phosphoramidatcs, either the reference or aligned sequence that do not corre methyl phosphonates, chiral-methyl phosphonates, 2-O-me spond to any amino acid in the corresponding sequence. thyl ribonucleotides, and peptide-nucleic acids (PNAs). The terms “numbered with reference to” or “corresponding Unless otherwise indicated, a particular nucleic acid to, when used in the context of the numbering of a given sequence also implicitly encompasses conservatively modi amino acid or polynucleotide sequence, refers to the number fied variants thereof (e.g., degenerate codon Substitutions) ing of the residues of a specified reference sequence when the and complementary sequences, as well as the sequence 10 given amino acid or polynucleotide sequence is compared to explicitly indicated. Specifically, degenerate codon Substitu the reference sequence. tions may be achieved by generating sequences in which the A “conservative substitution” as used with respect to third position of one or more selected (or all) codons is sub amino acids, refers to the Substitution of an amino acid with a stituted with mixed-base and/or deoxyinosine residues chemically similar amino acid. Amino acid Substitutions (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et 15 which often preserve the structural and/or functional proper al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., ties of the polypeptide in which the substitution is made are Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is known in the art and are described, for example, by H. used interchangeably with gene, cDNA, mRNA, oligonucle Neurath and R. L. Hill, 1979, in “The Proteins. Academic otide, and polynucleotide. Press, New York. The most commonly occurring exchanges Nucleic acids “hybridize' when they associate, typically in are isoleucine/valine, tyrosine? phenylalanine, aspartic acid/ solution. Nucleic acids hybridize due to a variety of well glutamic acid, lysinefarginine, methionine/leucine, aspartic characterized physico-chemical forces, such as hydrogen acid/asparagine, glutamic acid/glutamine, leucine/isoleu bonding, solvent exclusion, base stacking and the like. As cine, methioninetisoleucine, threonine?serine, tryptophan/ used herein, the term “stringent hybridization wash condi phenylalanine, tyrosine? histidine, tyrosine?tryptophan, tions’ in the context of nucleic acid hybridization experi 25 glutamine/arginine, histidine/asparagine, histidine/ ments, such as Southern and Northern hybridizations, are glutamine, lysine?.asparagine, lysine? glutamine, lysine? sequence dependent, and are different under different envi glutamic acid, phenylalanine/leucine, phenylalanine/me ronmental parameters. An extensive guide to the hybridiza thionine, serine/alanine, serine/asparagine, Valine/leucine, tion of nucleic acids is found in Tijssen, 1993, “Laboratory and valine/methionine. In some embodiments, there may be Techniques in Biochemistry and Molecular Biology-Hybrid 30 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, ization with Nucleic Acid Probes. Part I, Chapter 2 (Elsevier, at least 7, at least 8, at least 9, at least 10, at least 15, at least N.Y.), which is incorporated herein by reference. 20, at least 25, at least 30, at least 35, or at least 40 conserva The terms “peptide.” “polypeptide and “protein’ are used tive substitutions. interchangeably herein to refer to a polymer of amino acid The term "amino acid substitution set or “substitution set” residues. 35 refers to a group of amino acid Substitutions. A Substitution The term “amino acid refers to naturally occurring and set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or synthetic amino acids, as well as amino acid analogs. Natu more amino acid Substitutions. rally occurring amino acids are those encoded by the genetic The term "isolated refers to a nucleic acid, polynucle code, as well as those amino acids that are later modified, e.g., otide, polypeptide, protein, or other component that is par hydroxyproline, Y-carboxyglutamate, and O-phosphoserine. 40 tially or completely separated from components with which it Amino acid analogs refers to compounds that have the same is normally associated (other proteins, nucleic acids, cells, basic chemical structure as a naturally occurring amino acid, etc.). In some embodiments, an isolated polypeptide or pro i.e., an O-carbon that is bound to a hydrogen, a carboxyl tein is a recombinant polypeptide or protein. group, an amino group, and an R group, e.g., homoserine, A nucleic acid (such as a polynucleotide), a polypeptide, or norleucine, methionine Sulfoxide, methionine methyl Sulfo 45 a cell is “recombinant' when it is artificial or engineered, or nium. Such analogs have modified R groups (e.g., norleucine) derived from or contains an artificial or engineered protein or or modified peptide backbones, but retain the same basic nucleic acid (e.g. non-natural or not wild type). For example, chemical structure as a naturally occurring amino acid. a polynucleotide that is inserted into a vector or any other Amino acids may be referred to herein by either their heterologous location, e.g., in a genome of a recombinant commonly known three letter symbols or by the one-letter 50 organism, such that it is not associated with nucleotide symbols recommended by the IUPAC-IUB Biochemical sequences that normally flank the polynucleotide as it is Nomenclature Commission. Nucleotides, likewise, may be found in nature is a recombinant polynucleotide. A protein referred to by their commonly accepted single-letter codes. expressed in vitro or in vivo from a recombinant polynucle An amino acid or nucleotide base “position' is denoted by otide is an example of a recombinant polypeptide. Likewise, a number that sequentially identifies each amino acid (or 55 a polynucleotide sequence that does not appear in nature, for nucleotide base) in the reference sequence based on its posi example a variant of a naturally occurring gene, is recombi tion relative to the N-terminus (or 5'-end). Due to deletions, nant. insertions, truncations, fusions, and the like that must be “Identity” or “percent identity.” in the context of two or taken into account when determining an optimal alignment, more polypeptide sequences, refers to two or more sequences in general the amino acid residue number in a test sequence 60 or Subsequences that are the same or have a specified percent determined by simply counting from the N-terminus will not age of amino acid residues that are the same (e.g., share at necessarily be the same as the number of its corresponding least about 70%, at least about 75%, at least about 80%, at position in the reference sequence. For example, in a case least about 85%, at least about 88% identity, at least about where a variant has a deletion relative to an aligned reference 89%, at least about 90%, at least about 91%, at least about sequence, there will be no amino acid in the variant that 65 92%, at least about 93%, at least about 94%, at least about corresponds to a position in the reference sequence at the site 95%, at least about 96%, at least about 97%, at least about of deletion. Where there is an insertion in an aligned reference 98%, or at least about 99% identity) over a specified region to US 9,353,116 B2 13 14 a reference sequence, when compared and aligned for maxi that is operably linked to a suitable control sequence capable mum correspondence over a comparison window, or desig of effecting the expression in a suitable host of the polypep nated region as measured using a sequence comparison algo tide encoded in the DNA sequence. An "expression vector rithms or by manual alignment and visual inspection. has a promoter sequence operably linked to the DNA Optimal alignment of sequences for comparison and deter sequence (e.g., transgene) to drive expression in a host cell, mination of sequence identity can be determined by a and in Some embodiments a transcription terminator sequence comparison algorithm or by visual inspection (see, Sequence. generally, Ausubel et al., infra). When optimally aligning The term “expression' includes any step involved in the sequences and determining sequence identity by visual production of the polypeptide including, but not limited to, inspection, percent sequence identity is calculated as the 10 transcription, post-transcriptional modification, translation, number of residues of the test sequence that are identical to post-translational modification, and secretion. the reference sequence divided by the number of non-gap The term “operably linked’ refers to a configuration in positions and multiplied by 100. When using a sequence which a control sequence is appropriately placed at a position comparison algorithm, test and reference sequences are relative to the coding sequence of the DNA sequence Such entered into a computer, Subsequence coordinates and 15 that the control sequence influences the expression of a sequence algorithm program parameters are designated. The polypeptide. sequence comparison algorithm then calculates the percent An amino acid or nucleotide sequence (e.g., a promoter sequence identities for the test sequences relative to the ref- sequence, signal peptide, terminator sequence, etc.) is "het erence sequence, based on the program parameters as known erologous' to another sequence with which it is operably in the art, for example BLAST or BLAST 2.0. For example, 20 linked if the two sequences are not associated in nature. comparison can be conducted, e.g., by the local homology The terms “transform' or “transformation, as used in ref algorithm of Smith & Waterman, 1981, Adv. Appl. Math. erence to a cell, means a cell has a non-native nucleic acid 2:482, by the homology alignment algorithm of Needleman sequence integrated into its genome or as an episome (e.g., & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for plasmid) that is maintained through multiple generations. similarity method of Pearson & Lipman, 1988, Proc. Natl. 25 The term “culturing refers to growing a population of Acad. Sci. USA 85:2444, or by computerized implementa- microbial cells under suitable conditions in a liquid or solid tions of these algorithms (GAP BESTFIT. FASTA, and medium. TFASTA in the Wisconsin Genetics Software Package, The term “introduced, as used in the context of inserting a Genetics Computer Group, 575 Science Dr. Madison, Wis.). nucleic acid sequence into a cell, means conjugated, trans Thus alignment can be carried out for sequences that have 30 fected, transduced or transformed (collectively “trans deletions and/or additions, as well as those that have substi- formed') or otherwise incorporated into the genome of, or tutions, as well as naturally occurring, e.g., polymorphic or maintained as an episome in, the cell. allelic variants, and man-made variants. As defined herein, the term "gatekeeper amino acid resi The phrase “substantial sequence identity” or “substantial due' or “gatekeeper residue” refers to a residue (e.g. within identity.” in the context of two nucleic acid or polypeptide 35 the ATP binding site of a kinase) that is capable of controlling sequences, refers to a sequence that has at least 70% identity or modulating the ability of a kinase substrate to bind to the to a reference sequence. Percent identity can be any integer kinase. For example, in some embodiments, the accessibility from 70% to 100%. Two nucleic acid or polypeptide of a protein kinase substrate to the ATP binding site is con sequences that have 100% sequence identity are said to be trolled by the gatekeeper residue. In certain embodiments, the “identical. A nucleic acid or polypeptide sequence are said to 40 gatekeeper residue controls the ability of the Substrate to have “substantial sequence identity to a reference sequence access or bind a hydrophobic pocket adjacent to the ATP when the sequences have at least about 70%, at least about binding site. (Elphick et al. ACS Chemical Biology, 2:299 75%, at least 80%, at least 85%, at least 90%, at least 91%, at 314, 2007). As defined herein, a natural gatekeeper residue least 92%, at least 93%, at least 94%, at least 95%, at least refers to a gatekeeper residue identified in a wild-type kinase. 96%, at least 97%, at least 98%, or at least 99% or greater 45 Examples of gatekeeper residues include, e.g., Thr338 of sequence identity as determined using the methods described c-Src (v-Src numbering, see Liu et al., Chemistry & Biology, herein, Such as BLAST using standard parameters as 6:671-678, 1999), and Thr 493 of rsk2 (see US Application described above. No. 2009/0221614). Gatekeeper residues in other kinases, The term “pre-protein’ refers to a protein including an e.g., gatekeeper residues corresponding to Thr338 of c-Src amino-terminal signal peptide (or leader sequence) region 50 can be readily identified by structure-based sequence align attached. The signal peptide is cleaved from the pre-protein ment of kinase domain of various Src or non-Src kinases. The by a signal peptidase prior to secretion to result in the following is a structure-based sequence alignment of several “mature' or “secreted protein. kinase domains (see U.S. Patent Publication No. 2009/ A “vector is a DNA construct for introducing a DNA 0221614). The gatekeeper residues referred to herein are sequence into a cell. A vector may be an expression vector highlighted in bold italics:
SEQ ID Name Sequence NO :
SC - - - -PEAFLQEAOVMK--KLRHEKLVOLYAVVSEEP- - -IYIVEYM 52
rsk2 - - - -KRDPTEEIEILLR-YGOHPNIITLKDVYDDGKY--VYWWFELM 53
nek2 -EVEKOMLVSEVNLLR--ELKHPNIVRYYDRIIDRTNTTLYIVMEYC 54
mekk1 QEEVVEALREEIRMMS--HLNHPNIIRMLGATCEKSN--YNLEEWM 55 US 9,353,116 B2
- Continued
SEQ ID Name Sequence NO : msk1 ---MEANTOKEITALK-LCEGHPNIVKLHEVFHDOLH--TFLVMELL 56 plk1 -PHOREKMSMEISIHR--SLAHOHV VGFHGFFEDNDF--VFWWEELC f
Additional gatekeeper residues in various kinases can be in various kinases corresponding to Thr338 of v-Src are identified by sequence alignment (see Liu et al., Chemistry & highlighted in bold underlined in the sequence alignment Biology, 6:671-678, 1999). For example, gatekeeper residues below:
338
Sequence SEQ 338 ID Name Start NO :
W-Src. (318) RHEKLVOLYAMVSE------EP SK- - GSLLDFLKGEMGKY 58
c-Src. (318) RHEKLVOLYAVVSE------EPIYIVEYMSK--GSLLDFLKGETGKY 59
Lick (296) QHQRLVRLYAVVTQ------EPIYIIEEYMEN--GSLVDFLKTPSGIK 60
Fyn (319) KHDKLVOLYAVVSE------EPIYIV:EYMNK--GSLLDFLKDGEGRA 61
c - Yes (325) RHDKLVPLYAVVSE------EPIYIVEFMSK--GSLLDFLKEGDGKY 62
Yrk (318) RHDKLVOLYAVVSE------EPIYIV:EFMSQ--GSLLDFLKDGDGRY 63
c-Fgr (311) RHDKLVOLYAVVSE------EPIYIVEFMCH--GSLLDFLKNPEGOD 64
Lyn (295) QHDKLVRLYAVVTRE------EPIYIIgEYMAK--GSLLDFLKSDEGGK 65
Hck (318) QHDKLVKLHAVVTK------EPIYIISEFMAK--GSLLDFLKSDEGSK 66
Blk (287) QHERLVRLYAVVTR------EPIYIV.EYMAR--GCLLDFLKTDEGSR 67
Abil (313). KHPNLVQLLGVCTRE------PPFYIIgEFMTY--GNLLDYLRECNROE 68
Btk (473) SHEKLVOLYGVCTKQ------RPIFIIEEYMAN--GCLLNYLREMRHR 69
Csik (244) RHSNLVQLLGVIVEEK------GGLYIVEEYMAK--GSLVDYLRSRGRSV 70
PDGFR (660). PHLNVVNLLGACTKG------GPIYIIEEYCRY--GDLVDYLHRNKHTF 71.
p38 (85) GLLDVFTPARSLEEF ------NDVVLVEHLMGA---DLNNIVKCOKLTDD 72
ZAP-70 (394) DNPYIVRLIGVCQA------EALMLVEMAGG--GPLHKFL-VGKREE 73
JAK2 (906) OHDNIVKYKGVCYSAGR------RNLRLIEYLPY--GSLRDYLOKHKER 74
PKA (99) NFPFLVKLEFSFKDN------SNLYMVEYVPG--GEMFSHLRRIGR 7s
CamK II (68) KHPNIVRLHDSISEE------GHHYLIEDLVTG--GELFEDIVAREY 76
Cok2 (59) NHPNIWKLLDWIHTE------NKLYLVEEFLHQ- - -DLKKFMDASALTG 77 US 9,353,116 B2 17 18 “Control’ or “control experiment' is used in accordance treated by administration of a pharmaceutical composition as with its plain ordinary meaning and refers to an experiment in provided herein. Non-limiting examples include humans, which the Subjects or reagents of the experiment are treated as other mammals and other non-mammalian animals. in a parallel experiment except for omission of a procedure, Abbreviations used herein have their conventional mean reagent, or variable of the experiment. In some instances, the ing within the chemical and biological arts. control is used as a standard of comparison in evaluating Where substituent groups are specified by their conven experimental effects. tional chemical formulae, written from left to right, they "Contacting is used in accordance with its plain ordinary equally encompass the chemically identical Substituents that meaning and refers to the process of allowing at least two would result from writing the structure from right to left, e.g., distinct species (e.g. chemical compounds including biomol 10 ecules, or cells) to become sufficiently proximal to react, —CH2O— is equivalent to —OCH2—. interact or physically touch. It should be appreciated, how As used herein, the symbol, ever, the resulting reaction product can be produced directly from a reaction between the added reagents or from an inter mediate from one or more of the added reagents which can be 15 produced in the reaction mixture. The term “contacting includes incubating an inhibitor with the kinase. As defined herein, the term “inhibition”, “inhibit, “inhib iting and the like in reference to a kinase-inhibitor interar indicates the point of attachment of a substituents to the action means negatively affecting (e.g. decreasing) the activ remainder of a molecule. ity of the kinase relative to the activity of the kinase in the A “substituent group, as used herein, means a group absence of the inhibitor. Thus, inhibition includes, at least in selected from the following moieties: part, partially or totally blocking stimulation, decreasing, pre (A) —OH, -NH. —SH, —CN, —CF, NO, oxo, venting, or delaying activation, or inactivating, desensitizing, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, or down-regulating signal transduction. Similarly an “inhibi 25 unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, tor” is a compound that inhibits kinase activity, e.g., by bind unsubstituted aryl, unsubstituted heteroaryl, and ing, partially or totally block stimulation, decrease, prevent, (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or delay activation, or inactivate, desensitize, or down-regu and heteroaryl, substituted with at least one substituent late signal transduction. selected from: “Disease' or “condition” refer a state of being or health 30 (i) oxo, OH, -NH, SH, —CN, —CF, NO, halo status of a patient or subject capable of being treated with the gen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubsti compounds provided herein. Examples of disorders or con tuted cycloalkyl, unsubstituted heterocycloalkyl, unsubsti ditions include, but are not limited to, cancer, cardiovascular tuted aryl, unsubstituted heteroaryl, and disease, hypertension, Syndrome X, depression, anxiety, (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, glaucoma, human immunodeficiency virus(HIV) or acquired 35 and heteroaryl, substituted with at least one substituent immunodeficiency syndrome (AIDS), neurodegeneration, selected from: Alzheimer's disease, Parkinson's disease, cognition (a) oxo. —OH, -NH2, —SH, —CN, —CF, NO, enhancement, Cushing's Syndrome, Addison's Disease, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, osteoporosis, frailty, muscle frailty, inflammatory diseases, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, osteoarthritis, rheumatoid arthritis, asthma and rhinitis, adre 40 unsubstituted aryl, unsubstituted heteroaryl, and nal function-related ailments, viral infection, immunodefi (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or ciency, immunomodulation, autoimmune diseases, allergies, heteroaryl, substituted with at least one substituent selected wound healing, compulsive behavior, multi-drug resistance, from oxo, OH, -NH - SH, —CN, —CF, NO, halo addiction, psychosis, anorexia, cachexia, post-traumatic gen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubsti stress syndrome, post-Surgical bone fracture, medical catabo 45 tuted cycloalkyl, unsubstituted heterocycloalkyl, unsubsti lism, major psychotic depression, mild cognitive impairment, tuted aryl, and unsubstituted heteroaryl. psychosis, dementia, hyperglycemia, stress disorders, antip A "size-limited substituent’ or "size-limited substituent sychotic induced weight gain, delirium, cognitive impair group, as used herein means a group selected from all of the ment in depressed patients, cognitive deterioration in indi substituents described above for a “substituent group.” viduals with Down's syndrome, psychosis associated with 50 wherein each substituted or unsubstituted alkyl is a substi interferon-alpha therapy, chronic pain, pain associated with tuted or unsubstituted C-C alkyl, each Substituted or gastroesophageal reflux disease, postpartum psychosis, post unsubstituted heteroalkyl is a substituted or unsubstituted 2 to partum depression, neurological disorders in premature 20 membered heteroalkyl, each substituted or unsubstituted infants, and migraine headaches. In some instances, “disease' cycloalkyl is a substituted or unsubstituted C-C cycloalkyl, or “condition” refer to cancer. In some further instances, 55 and each substituted or unsubstituted heterocycloalkyl is a "cancer refers to human cancers and carcinomas, sarcomas, substituted or unsubstituted 4 to 8 membered heterocy adenocarcinomas, lymphomas, leukemias, etc., including cloalkyl. Solid and lymphoid cancers, kidney, breast, lung, bladder, A “lower substituent’ or “lower substituent group, as used colon, ovarian, prostate, pancreas, stomach, brain, head and herein means a group selected from all of the Substituents neck, skin, uterine, testicular, glioma, esophagus, and liver 60 described above for a “substituent group, wherein each sub cancer, including hepatocarcinoma, lymphoma, including stituted or unsubstituted alkyl is a substituted or unsubstituted B-acute lymphoblastic lymphoma, non-Hodgkin’s lympho C-C alkyl, each substituted or unsubstituted heteroalkyl is a mas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) substituted or unsubstituted 2 to 8 membered heteroalkyl, and Hodgkin’s lymphoma, leukemia (including AML. ALL, each substituted or unsubstituted cycloalkyl is a substituted or and CML), and multiple myeloma. 65 unsubstituted Cs-C7 cycloalkyl, and each Substituted or “Patient' or “subject in need thereof refers to a living unsubstituted heterocycloalkyl is a substituted or unsubsti organism Suffering from or prone to a condition that can be tuted 5 to 7 membered heterocycloalkyl. US 9,353,116 B2 19 20 The compounds of the present invention may exist as salts. It will be apparent to one skilled in the art that certain The present invention includes such salts. Examples of appli compounds of this invention may exist in tautomeric forms, cable salt forms include hydrochlorides, hydrobromides, sul all such tautomeric forms of the compounds being within the fates, methanesulfonates, nitrates, maleates, acetates, cit Scope of the invention. rates, fumarates, tartrates (eg (+)-tartrates, (-)-tartrates or Unless otherwise stated, structures depicted herein are also mixtures thereof including racemic mixtures, succinates, meant to include all stereochemical forms of the structure; benzoates and salts with amino acids such as glutamic acid. i.e., the R and S configurations for each asymmetric center. These salts may be prepared by methods known to those Therefore, single Stereochemical isomers as well as enantio skilled in art. Also included are base addition salts such as meric and diastereomeric mixtures of the present compounds Sodium, potassium, calcium, ammonium, organic amino, or 10 are within the scope of the invention. magnesium salt, or a similar salt. When compounds of the Unless otherwise stated, structures depicted herein are also present invention contain relatively basic functionalities, acid meant to include compounds which differ only in the pres addition salts can be obtained by contacting the neutral form ence of one or more isotopically enriched atoms. For of such compounds with a sufficient amount of the desired 15 example, compounds having the present structures except for acid, either neat or in a suitable inert solvent. Examples of the replacement of a hydrogen by a deuterium or tritium, or acceptable acid addition salts include those derived from the replacement of a carbon by 'C- or 'C-enriched carbon inorganic acids like hydrochloric, hydrobromic, nitric, car are within the scope of this invention. bonic, monohydrogencarbonic, phosphoric, monohydrogen The compounds of the present invention may also contain phosphoric, dihydrogenphosphoric, Sulfuric, monohydro unnatural proportions of atomic isotopes at one or more of gensulfuric, hydriodic, or phosphorous acids and the like, as atoms that constitute such compounds. For example, the com well as the salts derived organic acids like acetic, propionic, pounds may be radiolabeled with radioactive isotopes. Such isobutyric, maleic, malonic, benzoic, Succinic, Suberic, as for example tritium (H), iodine-125 ('I) or carbon-14 fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolyl (''C). Allisotopic variations of the compounds of the present Sulfonic, citric, tartaric, methanesulfonic, and the like. Also 25 invention, whether radioactive or not, are encompassed included are salts of amino acids such as arginate and the like, within the scope of the present invention. and salts of organic acids like glucuronic or galactunoric The term “pharmaceutically acceptable salts' is meant to acids and the like. Certain specific compounds of the present include salts of active compounds which are prepared with invention contain both basic and acidic functionalities that relatively nontoxic acids or bases, depending on the particular 30 Substituent moieties found on the compounds described allow the compounds to be converted into either base or acid herein. When compounds of the present invention contain addition salts. relatively acidic functionalities, base addition salts can be The neutral forms of the compounds are preferably regen obtained by contacting the neutral form of Such compounds erated by contacting the salt with a base or acid and isolating with a sufficient amount of the desired base, either neat or in the parent compound in the conventional manner. The parent 35 a suitable inert Solvent. Examples of pharmaceutically form of the compound differs from the various salt forms in acceptable base addition salts include Sodium, potassium, certain physical properties, such as solubility in polar Sol calcium, ammonium, organic amino, or magnesium salt, or a VentS. similar salt. When compounds of the present invention con Certain compounds of the present invention can exist in tain relatively basic functionalities, acid addition salts can be unsolvated forms as well as Solvated forms, including 40 obtained by contacting the neutral form of Such compounds hydrated forms. In general, the Solvated forms are equivalent with a sufficient amount of the desired acid, either neat or in to unsolvated forms and are encompassed within the scope of a suitable inert Solvent. Examples of pharmaceutically the present invention. Certain compounds of the present acceptable acid addition salts include those derived from invention may exist in multiple crystalline or amorphous inorganic acids like hydrochloric, hydrobromic, nitric, car forms. In general, all physical forms are equivalent for the 45 bonic, monohydrogencarbonic, phosphoric, monohydrogen uses contemplated by the present invention and are intended phosphoric, dihydrogenphosphoric, Sulfuric, monohydro to be within the scope of the present invention. gensulfuric, hydriodic, or phosphorous acids and the like, as Certain compounds of the present invention possess asym well as the salts derived from relatively nontoxic organic metric carbon atoms (optical or chiral centers) or double acids like acetic, propionic, isobutyric, maleic, malonic, ben bonds; the enantiomers, racemates, diastereomers, tau 50 Zoic, Succinic, Suberic, fumaric, lactic, mandelic, phthalic, tomers, geometric isomers, stereoisometric forms that may be benzenesulfonic, p-tolylsulfonic, citric, tartaric, methane defined, in terms of absolute stereochemistry, as (R)- or (S)- Sulfonic, and the like. Also included are salts of amino acids or, as (D)- or (L)-foramino acids, and individual isomers are Such as arginate and the like, and salts of organic acids like encompassed within the scope of the present invention. The glucuronic or galactunoric acids and the like (see, for compounds of the present invention do not include those 55 example, Berge et al., “Pharmaceutical Salts”, Journal of which are known in art to be too unstable to synthesize and/or Pharmaceutical Science, 1977, 66, 1-19). Certain specific isolate. The present invention is meant to include compounds compounds of the present invention contain both basic and in racemic and optically pure forms. Optically active (R)- and acidic functionalities that allow the compounds to be con (S)-, or (D)- and (L)-isomers may be prepared using chiral verted into either base or acid addition salts. synthons or chiral reagents, or resolved using conventional 60 In addition to Salt forms, the present invention provides techniques. When the compounds described herein contain compounds, which are in a prodrug form. Prodrugs of the olefinic bonds or other centers of geometric asymmetry, and compounds described herein are those compounds that unless specified otherwise, it is intended that the compounds readily undergo chemical changes under physiological con include both E and Z geometric isomers. ditions to provide the compounds of the present invention. The term “tautomeras used herein, refers to one of two or 65 Additionally, prodrugs can be converted to the compounds of more structural isomers which exist in equilibrium and which the present invention by chemical or biochemical methods in are readily converted from one isomeric form to another. an ex vivo environment. For example, prodrugs can be slowly US 9,353,116 B2 21 22 converted to the compounds of the present invention when As used herein, the term “mutated’ refers to a kinase with placed in a transdermal patch reservoir with a suitable a non-natural (e.g. non-wild type) amino acid sequence. A enzyme or chemical reagent. mutated kinase is typically recombinant (e.g. engineered). In The terms “a” “an or “acn)', when used in reference to a some embodiments as described below, the mutated kinase group of Substituents herein, mean at least one. For example, has a cysteine residue substitution at the gatekeeper amino where a compound is substituted with “an alkyl or aryl, the acid position. As used herein, the term “unmutated’ refers to compound is optionally Substituted with at least one alkyl the corresponding kinase wherein the mutation (e.g. a cys and/or at least one aryl. Moreover, where a moiety is substi teine residue is Substituted for a gatekeeper amino acid posi tuted with an R substituent, the group may be referred to as tion) is not present (e.g. the natural or wild-type sequence). “R-substituted.” Where a moiety is R-substituted, the moiety 10 Thus, in some instances, unmutated refers to the wild-type or is substituted with at least one R substituent and each R natural kinase. In some other instances, the corresponding substituent is optionally different. kinase is another recombinant kinase having similar but dis Description of compounds of the present invention are tinct Substitutions. limited by principles of chemical bonding known to those As used herein the term “not substantially lower when skilled in the art. Accordingly, where a group may be substi 15 referring to k means that the k is not less than a thou tuted by one or more of a number of substituents, such sub sandth, i.e. /1000, of the corresponding k, used for compari stitutions are selected so as to comply with principles of son. In some embodiments, the k is not less than a hun chemical bonding and to give compounds which are not dredth, i.e. /100, of the corresponding k, used for inherently unstable and/or would be known to one of ordinary comparison. In some instances, the k is not less than a tenth, skill in the art as likely to be unstable under ambient condi i.e. /10, of the corresponding k, used for comparison. In tions, such as aqueous, neutral, and several known physi some instances, the k is not less than a quarter, i.e. "/4, of the ological conditions. For example, a heterocycloalkyl or het corresponding k, used for comparison. In some instances, eroaryl is attached to the remainder of the molecule via a ring the k is not less than half, i.e. /2, of the corresponding k, heteroatom in compliance with principles of chemical bond used for comparison. For example, an engineered or mutated ing known to those skilled in the art thereby avoiding inher 25 kinase may have ak that is not substantially lower than the ently unstable compounds. corresponding k of the corresponding wild-type or natural The terms “treating or “treatment” refers to any indicia of or unmutated kinase. Success in the treatment or amelioration of an injury, pathol As used herein the terms “not substantially lower” when ogy or condition, including any objective or Subjective referring to K, means that the K is not less than a thou parameter Such as abatement; remission; diminishing of 30 sandth, i.e. /1000, of the corresponding K, used for compari symptoms or making the injury, pathology or condition more son. In some embodiments, the K is not less than a hun tolerable to the patient; slowing in the rate of degeneration or dredth, i.e. /100, of the corresponding K, used for decline; making the final point of degeneration less debilitat comparison. In some instances, the K is not less than a tenth, ing; improving a patient's physical or mental well-being. The i.e. /10, of the corresponding K, used for comparison. In treatment or amelioration of symptoms can be based on 35 some instances, the K is not less than a quarter, i.e. "/4, of the objective or subjective parameters; including the results of a corresponding K, used for comparison. In some instances, physical examination, neuropsychiatric exams, and/or a psy the K is not less than half, i.e. /2, of the corresponding K. chiatric evaluation. For example, the certain methods pre used for comparison. For example, an engineered or mutated sented herein Successfully treat cancer by decreasing the inci kinase may have a K that is not Substantially lower than the dence of cancer and or causing remission of cancer. 40 corresponding K, of the corresponding wild-type or natural An "effective amount' is an amount sufficient to contribute or unmutated kinase. to the treatment, prevention, or reduction of a symptom or “Disease' or “condition” refer a state of being or health symptoms of a disease. An "effective amount” may also be status of a patient or subject capable of being treated with the referred to as a “therapeutically effective amount.” A “reduc compounds provided herein. Examples of disorders or con tion of a symptom or symptoms (and grammatical equiva 45 ditions include, but are not limited to, cancer, cardiovascular lents of this phrase) means decreasing of the severity or fre disease, hypertension, Syndrome X, depression, anxiety, quency of the symptom(s), or elimination of the symptom(s). glaucoma, human immunodeficiency virus(HIV) or acquired A "prophylactically effective amount of a drug is an amount immunodeficiency syndrome (AIDS), neurodegeneration, of a drug that, when administered to a subject, will have the Alzheimer's disease, Parkinson's disease, cognition intended prophylactic effect, e.g., preventing or delaying the 50 enhancement, Cushing's Syndrome, Addison's Disease, onset (or reoccurrence) a disease, or reducing the likelihood osteoporosis, frailty, muscle frailty, inflammatory diseases, of the onset (or reoccurrence) of a disease or its symptoms. osteoarthritis, rheumatoid arthritis, asthma and rhinitis, adre The full prophylactic effect does not necessarily occur by nal function-related ailments, viral infection, immunodefi administration of one dose, and may occur only after admin ciency, immunomodulation, autoimmune diseases, allergies, istration of a series of doses. Thus, a prophylactically effec 55 wound healing, compulsive behavior, multi-drug resistance, tive amount may be administered in one or more administra addiction, psychosis, anorexia, cachexia, post-traumatic tions. An “activity decreasing amount as used herein, refers stress syndrome, post-Surgical bone fracture, medical catabo to an amount of antagonist required to decrease the activity of lism, major psychotic depression, mild cognitive impairment, an enzyme relative to the absence of the antagonist. A “func psychosis, dementia, hyperglycemia, stress disorders, antip tion disrupting amount as used herein, refers to the amount 60 sychotic induced weight gain, delirium, cognitive impair of antagonist required to disrupt the function of an osteoclast ment in depressed patients, cognitive deterioration in indi or leukocyte relative to the absence of the antagonist. viduals with Down's syndrome, psychosis associated with As used herein, the phrase ATP-binding pocket' refers to interferon-alpha therapy, chronic pain, pain associated with the active site of a kinase that binds ATP. The active site of the gastroesophageal reflux disease, postpartum psychosis, post kinase where ATPbinds is the set of amino acid residues that 65 partum depression, neurological disorders in premature are able to interact with and or bind to an ATP molecule oran infants, and migraine headaches. In some instances, “disease' ATP competitive inhibitor. or “condition” refer to cancer. In some further instances, US 9,353,116 B2 23 24 "cancer refers to human cancers and carcinomas, sarcomas, leukemia, Rieder cell leukemia, Schilling's leukemia, stem adenocarcinomas, lymphomas, leukemias, etc., including cell leukemia, subleukemic leukemia, and undifferentiated Solid and lymphoid cancers, kidney, breast, lung, bladder, cell leukemia. colon, ovarian, prostate, pancreas, stomach, brain, head and The term "sarcoma' generally refers to a tumor which is neck, skin, uterine, testicular, glioma, esophagus, and liver 5 made up of a Substance like the embryonic connective tissue cancer, including hepatocarcinoma, lymphoma, including and is generally composed of closely packed cells embedded B-acute lymphoblastic lymphoma, non-Hodgkin’s lympho in a fibrillar or homogeneous Substance. Sarcomas which can mas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) be treated with a combination of antineoplastic thiol-binding and Hodgkin’s lymphoma, leukemia (including AML. ALL, mitochondrial oxidant and an anticancer agent include a 10 chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosar and CML), and multiple myeloma. coma, myxosarcoma, osteosarcoma, Abemethy’s sarcoma, As used herein, the term "kinase-associated disease' refers adipose sarcoma, liposarcoma, alveolar soft part sarcoma, to a disease or condition that is mediated, at least in part, by a ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, kinase. chorio carcinoma, embryonal sarcoma, Wilms tumor sar As used herein, the term “Lrrk-2-associated disease' refers 15 coma, endometrial sarcoma, stromal sarcoma, Ewing's sar to a disease or condition that is mediated, at least in part, by a coma, fascial sarcoma, fibroblastic sarcoma, giant cell Sar Lrrk-2 kinase. coma, granulocytic sarcoma, Hodgkin’s sarcoma, idiopathic As used herein, the term “cancer refers to all types of multiple pigmented hemorrhagic sarcoma, immunoblastic cancer, neoplasm or malignant tumors found in mammals, sarcoma of B cells, lymphoma, immunoblastic sarcoma of including leukemia, carcinomas and sarcomas. Exemplary T-cells, Jensen’s sarcoma, Kaposi's sarcoma, Kupffer cell cancers include cancer of the brain, breast, cervix, colon, sarcoma, angiosarcoma, leukosarcoma, malignant mesen head & neck, liver, kidney, lung, non-Small cell lung, mela chymoma sarcoma, parosteal sarcoma, reticulocytic sar noma, mesothelioma, ovary, sarcoma, stomach, uterus and coma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, Mcdulloblastoma. Additional examples include, Hodgkin’s and telangiectaltic sarcoma. Disease, Non-Hodgkin’s Lymphoma, multiple myeloma, 25 The term "melanoma’ is taken to mean a tumor arising neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary from the melanocytic system of the skin and other organs. thrombocytosis, primary macroglobulinemia, primary brain Melanomas which can be treated with a combination of anti tumors, cancer, malignant pancreatic insulanoma, malignant neoplastic thiol-binding mitochondrial oxidant and an anti carcinoid, urinary bladder cancer, premalignant skin lesions, cancer agent include, for example, acral-lentiginous mela testicular cancer, lymphomas, thyroid cancer, neuroblastoma, 30 noma, amelanotic melanoma, benign juvenile melanoma, esophageal cancer, genitourinary tract cancer, malignant Cloudman's melanoma, S91 melanoma, Harding-Passey hypercalcemia, endometrial cancer, adrenal cortical cancer, melanoma, juvenile melanoma, lentigo maligna melanoma, neoplasms of the endocrine and exocrine pancreas, and pros malignant melanoma, nodular melanoma, Subungal mela tate Cancer. noma, and Superficial spreading melanoma. The term “leukemia' refers broadly to progressive, malig 35 The term "carcinoma’ refers to a malignant new growth nant diseases of the blood-forming organs and is generally made up of epithelial cells tending to infiltrate the surround characterized by a distorted proliferation and development of ing tissues and give rise to metastases. Exemplary carcinomas leukocytes and their precursors in the blood and bone marrow. which can be treated with a combination of antineoplastic Leukemia is generally clinically classified on the basis of (1) thiol-binding mitochondrial oxidant and an anticancer agent the duration and character of the disease-acute or chronic; (2) 40 include, for example, acinar carcinoma, acinous carcinoma, the type of cell involved; myeloid (myelogenous), lymphoid adenocystic carcinoma, adenoid cystic carcinoma, carcinoma (lymphogenous), or monocytic; and (3) the increase or non adenomatosum, carcinoma of adrenal cortex, alveolar carci increase in the number abnormal cells in the blood-leukemic noma, alveolar cell carcinoma, basal cell carcinoma, carci or aleukemic (Subleukemic). The Pass leukemia model is noma basocellulare, basaloid carcinoma, basosquamous cell widely accepted as being predictive of in vivo anti-leukemic 45 carcinoma, bronchioalveolar carcinoma, bronchiolar carci activity. It is believed that a compound that tests positive in the noma, bronchogenic carcinoma, cerebriform carcinoma, cho Psss assay will generally exhibit some level of anti-leukemic langiocellular carcinoma, chorionic carcinoma, colloid car activity in vivo regardless of the type of leukemia being cinoma, comedo carcinoma, corpus carcinoma, cribriform treated. Accordingly, the present invention includes a method carcinoma, carcinoma en cuirasse, carcinoma cutaneum, of treating leukemia, and, preferably, a method of treating 50 cylindrical carcinoma, cylindrical cell carcinoma, duct carci acute nonlymphocytic leukemia, chronic lymphocytic leuke noma, carcinoma durum, embryonal carcinoma, encephaloid mia, acute granulocytic leukemia, chronic granulocytic leu carcinoma, epiermoid carcinoma, carcinoma epitheliale kemia, acute promyelocytic leukemia, adult T-cell leukemia, adenoides, exophytic carcinoma, carcinoma ex ulcere, carci aleukemic leukemia, a leukocythemic leukemia, basophylic noma fibroSum, gelatiniformi carcinoma, gelatinous carci leukemia, blast cell leukemia, bovine leukemia, chronic 55 noma, giant cell carcinoma, carcinoma gigantocellulare, myelocytic leukemia, leukemia cutis, embryonal leukemia, glandular carcinoma, granulosa cell carcinoma, hair-matrix eosinophilic leukemia, Gross leukemia, hairy-cell leukemia, carcinoma, hematoid carcinoma, hepatocellular carcinoma, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic Hurthle cell carcinoma, hyaline carcinoma, hypemephroid leukemia, stem cell leukemia, acute monocytic leukemia, carcinoma, infantile embryonal carcinoma, carcinoma in situ, leukopenic leukemia, lymphatic leukemia, lymphoblastic 60 intraepidermal carcinoma, intraepithelial carcinoma, leukemia, lymphocytic leukemia, lymphogenous leukemia, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large lymphoid leukemia, lymphosarcoma cell leukemia, mast cell cell carcinoma, lenticular carcinoma, carcinoma lenticulare, leukemia, megakaryocytic leukemia, micromyeloblastic leu lipomatous carcinoma, lymphoepithelial carcinoma, carci kemia, monocytic leukemia, myeloblastic leukemia, myelo noma medullare, medullary carcinoma, melanotic carci cytic leukemia, myeloid granulocytic leukemia, myelomono 65 noma, carcinoma molle, mucinous carcinoma, carcinoma cytic leukemia, Naegeli leukemia, plasma cell leukemia, muciparum, carcinoma mucocellulare, mucoepidermoid car multiple myeloma, plasmacytic leukemia, promyelocytic cinoma, carcinoma mucosum, mucous carcinoma, carcinoma US 9,353,116 B2 25 26 myxomatodes, nasopharyngeal carcinoma, oat cell carci converted to the compounds of the present invention when noma, carcinoma ossificans, ostcoid carcinoma, papillary placed in a transdermal patch reservoir with a suitable carcinoma, periportal carcinoma, preinvasive carcinoma, enzyme or chemical reagent. prickle cell carcinoma, pultaceous carcinoma, renal cell car Certain compounds of the present invention can exist in cinoma of kidney, reserve cell carcinoma, carcinoma sarco- 5 unsolvated forms as well as Solvated forms, including matodes, Schneiderian carcinoma, Scirrhous carcinoma, car hydrated forms. In general, the Solvated forms are equivalent cinoma Scroti, signet-ring cell carcinoma, carcinoma to unsolvated forms and are encompassed within the scope of simplex, Small-cell carcinoma, Solanoid carcinoma, spheroi the present invention. Certain compounds of the present dal cell carcinoma, spindle cell carcinoma, carcinoma spon invention may exist in multiple crystalline or amorphous giosum, Squamous carcinoma, squamous cell carcinoma, 10 forms. In general, all physical forms are equivalent for the String carcinoma, carcinoma telangiectaticum, carcinoma uses contemplated by the present invention and are intended telangiectodes, transitional cell carcinoma, carcinoma to be within the scope of the present invention. tuberosum, tuberous carcinoma, Verrucous carcinoma, and Certain compounds of the present invention possess asym carcinoma villosum. 15 metric carbon atoms (optical centers) or double bonds; the The term “pharmaceutically acceptable salts' is meant to racemates, diastereomers, geometric isomers and individual include salts of the active compounds which are prepared isomers are encompassed within the scope of the present invention. with relatively nontoxic acids or bases, depending on the The compounds of the present invention may also contain particular Substituents found on the compounds described unnatural proportions of atomic isotopes at one or more of the herein. When compounds of the present invention contain atoms that constitute such compounds. For example, the com relatively acidic functionalities, base addition salts can be pounds may be radiolabeled with radioactive isotopes. Such obtained by contacting the neutral form of Such compounds as for example tritium (H), iodine-125 (I) or carbon-14 with a sufficient amount of the desired base, either neat or in (''C). All isotopic variations of the compounds of the present a Suitable inert Solvent. Examples of pharmaceutically 25 invention, whether radioactive or not, are encompassed within the scope of the present invention. acceptable base addition salts include Sodium, potassium, As used herein, the term "salt” refers to acid or base salts of calcium, ammonium, organic amino, or magnesium salt, or a the compounds used in the methods of the present invention. similar salt. When compounds of the present invention con Illustrative examples of acceptable salts are mineral acid (hy tain relatively basic functionalities, acid addition salts can be drochloric acid, hydrobromic acid, phosphoric acid, and the obtained by contacting the neutral form of Such compounds 30 like) salts, organic acid (acetic acid, propionic acid, glutamic with a sufficient amount of the desired acid, either neat or in acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. a Suitable inert Solvent. Examples of pharmaceutically As used herein, the term "isomers' refers to compounds acceptable acid addition salts include those derived from having the same number and kind of atoms, and hence the inorganic acids like hydrochloric, hydrobromic, nitric, car 35 same molecular weight, but differing in respect to the struc bonic, monohydrogencarbonic, phosphoric, monohydrogen tural arrangement or configuration of the atoms. phosphoric, dihydrogenphosphoric, Sulfuric, monohydro As used herein, the term “tautomer, refers to one of two or gensulfuric, hydriodic, or phosphorous acids and the like, as more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another. well as the salts derived from relatively nontoxic organic 40 III. Introduction acids like acetic, propionic, isobutyric, maleic, malonic, ben Provided herein are, interalia, novel methods and compo Zoic, Succinic, Suberic, fumaric, lactic, mandelic, phthalic, sitions for inhibiting a protein kinase, e.g., a cysteine Substi benzenesulfonic, p-tolylsulfonic, citric, tartaric, methane tuted kinase, determining the function of a protein kinase in a Sulfonic, and the like. Also included are salts of amino acids cell, and treating kinase-associated diseases and conditions. Such as arginate and the like, and salts of organic acids like 45 Certain heterocyclic compounds having an electrophilic Sub glucuronic or galactunoric acids and the like (see, for stituent provided herein that specifically, and optionally irre versibly, inhibit cysteine substituted kinases. In some example, Berge et al., “Pharmaceutical Salts”, Journal of embodiments, the heterocyclic compound comprises two or Pharmaceutical Science, 1977. 66, 1-19). Certain specific more fused rings and an electrophilic Substituent. In some compounds of the present invention contain both basic and 50 embodiments, at least one of the two or more fused rings acidic functionalities that allow the compounds to be con comprises a nitrogen atom. In some embodiments, the het verted into either base or acid addition salts. erocyclic compounds inhibit a cysteine Substituted kinase, The neutral forms of the compounds are preferably regen i.e., a kinase having a cysteine residue in the gatekeeper erated by contacting the salt with a base or acid and isolating ss position of the ATP binding site. In some embodiments, the the parent compound in the conventional manner. The parent heterocyclic compounds also inhibit a kinase not having a form of the compound differs from the various salt forms in cysteine residue in the gatekeeper position (e.g. of the ATP certain physical properties, such as solubility in polar Sol binding site). VentS. IV. Compounds In addition to Salt forms, the present invention provides 60 The present invention provides compounds Suitable for use compounds, which are in a prodrug form. Prodrugs of the with the methods and assays described herein. compounds described herein are those compounds that In some other embodiments, the heterocyclic compounds readily undergo chemical changes under physiological con useful for inhibiting a kinase include two or more fused rings ditions to provide the compounds of the present invention. which include at least one heteroatom selected from N, O, or Additionally, prodrugs can be converted to the compounds of 65 S. In some embodiments, the fused rings are substituted with the present invention by chemical or biochemical methods in a ring selected from Substituted or unsubstituted cycloalkyl, an ex vivo environment. For example, prodrugs can be slowly substituted or unsubstituted aryl, or substituted or unsubsti US 9,353,116 B2 27 28 tuted heteroaryl. In some other embodiments, the cycloalkyl, -continued heterocycloalkyl, aryl, or heteroaryl is substituted with a sub (II) stituent selected from substituted or unsubstituted alkyl, sub stituted or unsubstituted heteroalkyl, substituted or unsubsti L4 tuted aryl, substituted or unsubstituted heteroaryl. In other embodiments, the ring which Substitutes the fused rings is an N21 N aryl or heteroaryl. In some embodiments, the ring which Substitutes the fused rings is an aryl. In some other embodi 1sSa | H-L-R); or 10 -L N 2 ments, the ring which Substitutes the fused rings is an aryl R which is substituted with an electrophilic substituent that is (III) capable of accepting electron density from a cysteine gate keeper residue of a protein kinase. In some embodiments, the L4 electrophilic Substituent is capable of forming a covalent 15 bond to the Sulfhydryl group of the cysteine gate keeper residue. 4YN 1s In some embodiments, the compound is a Substituted or e-'t j-i-t ---L-R2). unsubstituted phenyl-derivatized pyrazolopyrimidine, e.g. s N4 3-phenyl-substituted pyrazolopyrimidines, having an elec trophilic substituent. In some other embodiments, the present invention provides 3-phenyl-Substituted pyrazolopyrim In these formula, X is =N or=C(-L-R) ; Ring A is, in idines which are synthesized with an electrophilic groups at each instance, independently cycloalkyl, heterocycloalkyl, positions expected to be in close proximity to the gatekeeper aryl, or heteroaryl; L', L. L. L. L. and L are, in each residue. In some embodiments, compound is a Substituted or 25 instance, independently selected from a bond, —C(O)—, unsubstituted quinazoline having an electrophilic Substituent. - C(O)N(R) , C(O)O , S(O). (i.e. S , In some embodiments, compound is a Substituted or unsub S(O)— or S(O). ), S(O)N(R) , —O , stituted 4-anilinoquinazoline, e.g. Michael acceptor-deriva - N(R)-, - N(R)C(O)N(R)-, substituted or unsubsti tized 4-anilinoquinazolines, having an electrophilic Substitu tuted alkylene, substituted or unsubstituted heteroalkylene, ent. In some embodiments, the compound is a Substituted or 30 substituted or unsubstituted cycloalkylene, substituted or unsubstituted benzyl-derivatized pyrazolopyrimidine having unsubstituted heterocycloalkylene, substituted or unsubsti an electrophilic Substituent. In some embodiments, the com tuted arylene, or substituted or unsubstituted heteroarylene, pound is a Substituted or unsubstituted pyrazolopyrimidine whereing is independently an integer from 0 to 2: R', R. R. having an electrophilic Substituent pyrazolopyrimidine. R. R. R. R', and Rare, in each instance, independently In some other embodiments, the electrophilic substituentis 35 selected from hydrogen, halogen, —CN. —OH, -NH2, an electrophilic ATP-binding pocket moiety (i.e. a chemical -COOH, -CONH, NO, -SH, -SOCl, -SOH, moiety that interacts with amino acids that form part of the —SOH, -SONH, substituted or unsubstituted alkyl, sub ATP-binding pocket). In other embodiments, the electro stituted or unsubstituted heteroalkyl, substituted or unsubsti philic Substituent is a vinylsulfonamide, a vinylsulfone, an tuted cycloalkyl, substituted or unsubstituted heterocy acrylamide, a chloroacetamide, an O-chloroacetamide, an 40 epoxide, or a fluoromethylketones. cloalkyl, substituted or unsubstituted aryl, or substituted or In Some embodiments the compounds described herein are unsubstituted heteroaryl; a is an integer from 0 to 2: b is an inhibitors of kinases (“kinase inhibitors') such as an inhibitor integer from 0 to 5; and c is an integer from 0 to 4. In some of a recombinant cysteine gatekeeper kinase ("cysteine gate embodiments, A is aryl (e.g. phenyl). In some embodiments, keeper kinase inhibitor). In some other embodiments, the 45 R’ is hydrogen. In some embodiments, Rand Rare hydro cysteinegatekeeper kinase inhibitor includes an ATP-binding gen. R', R. R. R. R. R. R. and R may also indepen pocket moiety (e.g. an ATP-binding pocket moiety including dently be hydrogen, halogen (e.g. —Cl or —F), —CN. a heterocyclic moiety) covalently bound to an electrophilic - OH, -NH,-COOH,-CONH, NO, -SH, substi moiety capable of binding the thiol of the gatekeeper cysteine tuted or unsubstituted alkyl, substituted or unsubstituted het residue of the cysteine gatekeeper kinase. In some embodi 50 eroalkyl, substituted or unsubstituted cycloalkyl, substituted ments, the inhibitor is one or more of the compounds set forth or unsubstituted heterocycloalkyl, substituted or unsubsti in Table 1 of Formulas (I) to (XXIX) (e.g. Formula (I) to tuted aryl, or substituted or unsubstituted heteroaryl. (XIV)). In some embodiments, R' is hydrogen, halogen, —CN, In some embodiments, the compounds has the formula: -OH, -NH, -COOH, -CONH, NO, -SH, 55 —SO.C1, SOH, -SOH, -SO.NH R-substituted or unsubstituted alkyl, R-substituted or unsubstituted het eroalkyl, R-substituted or unsubstituted cycloalkyl, R-sub stituted or unsubstituted heterocycloalkyl, R-substituted or unsubstituted aryl, or R-substituted or unsubstituted het 60 eroaryl. R is independently halogen, —CN, -OH, -NH2, . . . \, -COOH, -CONH, NO, -SH, -SOCl, -SOH, a —SOH, -SONHR'-substituted or unsubstituted alkyl, N4 N^V R"-substituted or unsubstituted heteroalkyl, R'-substituted V 65 or unsubstituted cycloalkyl, R'-substituted or unsubstituted R2 heterocycloalkyl, R'-substituted or unsubstituted aryl, or R"-substituted or unsubstituted heteroaryl. US 9,353,116 B2 29 30 R" is independently halogen, —CN, -OH, -NH2, R" is independently halogen, —CN, OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, —SOH, -SONH2, R'-substituted or unsubstituted alkyl, —SOH, -SONH2, R-substituted or unsubstituted alkyl, R'-substituted or unsubstituted heteroalkyl, R'-substituted R-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R'-substituted or unsubstituted or unsubstituted cycloalkyl, R-substituted or unsubstituted heterocycloalkyl, R'-substituted or unsubstituted aryl, or heterocycloalkyl, R-substituted or unsubstituted aryl, or R'-substituted or unsubstituted heteroaryl. R-substituted or unsubstituted heteroaryl. In some embodiments, R is hydrogen, halogen, —CN, In some embodiments, R is hydrogen, halogen, —CN, –OH, -NH, -COOH, -CONH, NO, -SH, - OH, -NH, -COOH, -CONH, NO, -SH, —SOCl, -SOH, -SOH, -SONH, R*-substituted or 10 - SOCl, -SOH, -SOH, -SONH, R-substituted or unsubstituted alkyl, R'°-substituted or unsubstituted het unsubstituted alkyl, R-substituted or unsubstituted het eroalkyl, R'-substituted or unsubstituted cycloalkyl, R'- eroalkyl, R-substituted or unsubstituted cycloalkyl, R substituted or unsubstituted heterocycloalkyl, R'-substi substituted or unsubstituted heterocycloalkyl, R-substi tuted or unsubstituted aryl, or R'-substituted or 15 tuted or unsubstituted aryl, or R-substituted or unsubstituted heteroaryl. unsubstituted heteroaryl. R" is independently halogen, —CN, -OH, -NH2, R’ is independently halogen, —CN, OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, —SOH, -SONH2, R'-substituted or unsubstituted alkyl, - SOH, -SONH2, R’-substituted or unsubstituted alkyl, R'-substituted or unsubstituted heteroalkyl, R'-substituted R’-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R'-substituted or unsubstituted or unsubstituted cycloalkyl, R-substituted or unsubstituted heterocycloalkyl, R'-substituted or unsubstituted aryl, or heterocycloalkyl, R*-substituted or unsubstituted aryl, or R"-substituted or unsubstituted heteroaryl. R’-substituted or unsubstituted heteroaryl. R' independently is halogen, —CN, -OH, -NH2, R’ is independently halogen, —CN, OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, 25 -COOH, -CONH, NO, -SH, -SOCl, -SOH, —SOH, -SONH2, R-substituted or unsubstituted alkyl, —SOH, -SONH2, R-substituted or unsubstituted alkyl, R"-substituted or unsubstituted heteroalkyl, R'-substituted R-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R'-substituted or unsubstituted or unsubstituted cycloalkyl, R-substituted or unsubstituted heterocycloalkyl, R-substituted or unsubstituted aryl, or heterocycloalkyl, R-substituted or unsubstituted aryl, or R-substituted or unsubstituted heteroaryl. 30 R-substituted or unsubstituted heteroaryl. In some embodiments, Rhydrogen, is halogen, —CN, In some embodiments, R is hydrogen, halogen, —CN, OH, -NH COOH, CONH, NO, SH, OH, -NH COOH, CONH, NO, SH, —SOCl, -SOH, -SOH, -SONH, R-substituted or - SOCl, -SOH, -SOH, -SONH, R-substituted or unsubstituted alkyl, R'-substituted or unsubstituted het unsubstituted alkyl, R-substituted or unsubstituted het eroalkyl, R'-substituted or unsubstituted cycloalkyl, R'- 35 eroalkyl, R-substituted or unsubstituted cycloalkyl, R substituted or unsubstituted heterocycloalkyl, R'-substi substituted or unsubstituted heterocycloalkyl, R-substi tuted or unsubstituted aryl, or R'-substituted or tuted or unsubstituted aryl, or R-substituted or unsubstituted heteroaryl. unsubstituted heteroaryl. R" is independently halogen, —CN, -OH, -NH2, R is independently halogen, —CN, OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, 40 -COOH, -CONH, -NO, -SH, -SOCl, -SOH, —SOH, -SONH2, R'-substituted or unsubstituted alkyl, - SOH, -SONH2, R-substituted or unsubstituted alkyl, R-substituted or unsubstituted heteroalkyl, R-substituted R-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R'-substituted or unsubstituted or unsubstituted cycloalkyl, R-substituted or unsubstituted heterocycloalkyl, R'-substituted or unsubstituted aryl, or heterocycloalkyl, R-substituted or unsubstituted aryl, or R-substituted or unsubstituted heteroaryl. 45 R-substituted or unsubstituted heteroaryl. R' is independently halogen, —CN, -OH, -NH2, R’ is independently halogen, —CN, OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, —SOH, -SONH2, R''-substituted or unsubstituted alkyl, - SOH, -SONH2, R-substituted or unsubstituted alkyl, R7-substituted or unsubstituted heteroalkyl, R'7-substituted R-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R'-substituted or unsubstituted 50 or unsubstituted cycloalkyl, R-substituted or unsubstituted heterocycloalkyl, R''-substituted or unsubstituted aryl, or heterocycloalkyl, R-substituted or unsubstituted aryl, or R7-substituted or unsubstituted heteroaryl. R-substituted or unsubstituted heteroaryl. In some embodiments, R is hydrogen, halogen, —CN, In some embodiments, R is hydrogen, halogen, —CN, –OH, -NH, -COOH, -CONH, NO, -SH, -OH, -NH, -COOH, -CONH, NO, -SH, —SOCl, -SOH, -SOH, -SONH, R-substituted or 55 - SOCl, -SOH, -SOH, -SONH, R7-substituted or unsubstituted alkyl, R-substituted or unsubstituted het unsubstituted alkyl, R7-substituted or unsubstituted het eroalkyl, R-substituted or unsubstituted cycloalkyl, R'- eroalkyl, R-substituted or unsubstituted cycloalkyl, R substituted or unsubstituted heterocycloalkyl, R'-substi substituted or unsubstituted heterocycloalkyl, R-substi tuted or unsubstituted aryl, or R-substituted or tuted or unsubstituted aryl, or R7-substituted or unsubstituted heteroaryl. 60 unsubstituted heteroaryl. R" independently is halogen, —CN, OH, -NH. R7 is independently halogen, —CN, OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, -COOH, -CONH, NO, -SH, -SOCl, -SOH, —SOH, -SONH, R-substituted or unsubstituted alkyl, —SOH, -SONH, R-substituted or unsubstituted alkyl, R'-substituted or unsubstituted heteroalkyl, R'-substituted R-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R'-substituted or unsubstituted 65 or unsubstituted cycloalkyl, R-substituted or unsubstituted heterocycloalkyl, R'-substituted or unsubstituted aryl, or heterocycloalkyl, R-substituted or unsubstituted aryl, or R'-substituted or unsubstituted heteroaryl. R-substituted or unsubstituted heteroaryl. US 9,353,116 B2 31 32 R is independently halogen, —CN, -OH, -NH2, heterocycloalkylene, R-substituted or unsubstituted -COOH, -CONH, -NO, -SH, -SOCl, -SOH, arylene, or R-substituted or unsubstituted heteroarylene. —SOH, -SONH2, R-substituted or unsubstituted alkyl, R is independently halogen, —CN, OH, -NH2, R’-substituted or unsubstituted heteroalkyl, R-substituted -COOH, -CONH, NO, -SH, -SOCl, -SOH, or unsubstituted cycloalkyl, R-substituted or unsubstituted —SOH, -SONH, R7-substituted or unsubstituted alkyl, heterocycloalkyl, R-substituted or unsubstituted aryl, or R-substituted or unsubstituted heteroalkyl, R-substituted R’-substituted or unsubstituted heteroaryl. or unsubstituted cycloalkyl, R-substituted or unsubstituted In some embodiments, R is hydrogen, halogen, —CN, heterocycloalkyl, R-substituted or unsubstituted aryl, or –OH, -NH, -COOH, -CONH, NO, -SH, R-substituted or unsubstituted heteroaryl. —SOCl, -SOH, -SOH, -SONH, R-substituted or 10 R7 is independently halogen, —CN, OH, -NH2, unsubstituted alkyl, R-substituted or unsubstituted het -COOH, -CONH, NO, -SH, -SOCl, -SOH, eroalkyl, R-substituted or unsubstituted cycloalkyl, R'- - SOH, -SONH2, R-substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, R-substi R-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R-substituted or unsubstituted tuted or unsubstituted aryl, or R-substituted or 15 heterocycloalkyl, R-substituted or unsubstituted aryl, or unsubstituted heteroaryl. R-substituted or unsubstituted heteroaryl. R" is independently halogen, —CN, -OH, -NH2, In some embodiments, L is in each instance, indepen -COOH, -CONH, -NO, -SH, -SOCl, -SOH, dently selected from a bond, —C(O) , —C(O)N(R') , —SOH, -SONH2, R-substituted or unsubstituted alkyl, C(O)O , S(O) = (i.e. —S , S(O)— or - S(O)2), R-substituted or unsubstituted heteroalkyl, R-substituted —S(O)N(R7)—, O-, -N(R7) , N(R7)C(O)N or unsubstituted cycloalkyl, R-substituted or unsubstituted (R) , R-substituted or unsubstituted alkylene, R-sub heterocycloalkyl, R-substituted or unsubstituted aryl, or stituted or unsubstituted heteroalkylene, R-substituted or R-substituted or unsubstituted heteroaryl. unsubstituted cyclo alkylene, R-substituted or unsubsti R" is independently halogen, —CN, -OH, -NH2, tuted heterocycloalkylene, R-substituted or unsubstituted -COOH, -CONH, -NO, -SH, -SOCl, -SOH, 25 arylene, or R-substituted or unsubstituted heteroarylene. —SOH, -SONH2, R*-substituted or unsubstituted alkyl, R is independently halogen, —CN, OH, -NH2, R-substituted or unsubstituted heteroalkyl, R-substituted -COOH, -CONH, -NO, -SH, -SOCl, -SOH, or unsubstituted cycloalkyl, R*-substituted or unsubstituted - SOH, -SONH2, R'-substituted or unsubstituted alkyl, heterocycloalkyl, R-substituted or unsubstituted aryl, or R'-substituted or unsubstituted heteroalkyl, R'-substituted R*-substituted or unsubstituted heteroaryl. 30 or unsubstituted cycloalkyl, R'-substituted or unsubstituted In some embodiments, R'', R'', R'7, R. R', R. R. heterocycloalkyl, R'-substituted or unsubstituted aryl, or and Rare independently hydrogen, halogen, CN, OH, R'-substituted or unsubstituted heteroaryl. NH, -COOH, -CONH, -NO, -SH, -SOCl, R" is independently halogen, —CN, OH, -NH2, —SOH, -SOH, -SONH2, unsubstituted alkyl, unsub -COOH, -CONH, -NO, -SH, -SOCl, -SOH, stituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted 35 - SOH, -SONH2, R'-substituted or unsubstituted alkyl, heterocycloalkyl, unsubstituted aryl, or unsubstituted het R'-substituted or unsubstituted heteroalkyl, R'-substituted eroaryl. or unsubstituted cycloalkyl, R'-substituted or unsubstituted In some embodiments, L' is in each instance, indepen heterocycloalkyl, R'-substituted or unsubstituted aryl, or dently selected from a bond, —C(O) , —C(O)N(R) , R'-substituted or unsubstituted heteroaryl. C(O)O , S(O) , S(O)N(R)-, -O-, 40 In some embodiments, L' is in each instance, indepen -N(R)-, - N(R)C(O)N(R)-, R-substituted or dently selected from a bond, —C(O) , —C(O)N(R') , unsubstituted alkylene, R-substituted or unsubstituted het C(O)O , S(O) = (i.e. —S , S(O)— or - S(O)2), eroalkylene, R-substituted or unsubstituted cycloalkylene, —S(O)N(R7)—, O-, -N(R7) , N(R7)C(O)N R-substituted or unsubstituted heterocycloalkylene, R (R) , R-substituted or unsubstituted alkylene, R-sub substituted or unsubstituted arylene, or R-substituted or 45 stituted or unsubstituted heteroalkylene, R-substituted or unsubstituted heteroarylene. unsubstituted cycloalkylene, R-substituted or unsubstituted R is independently halogen, —CN, OH, -NH. heterocycloalkylene, R-substituted or unsubstituted -COOH, -CONH, -NO, -SH, -SOCl, -SOH, arylene, or R-substituted or unsubstituted heteroarylene. —SOH, -SONH, R-substituted or unsubstituted alkyl, R" is independently halogen, —CN, OH, -NH2, R-substituted or unsubstituted heteroalkyl, R-substituted 50 -COOH, -CONH, -NO, -SH, -SOCl, -SOH, or unsubstituted cycloalkyl, R-substituted or unsubstituted - SOH, -SONH2, R-substituted or unsubstituted alkyl, heterocycloalkyl, R-substituted or unsubstituted aryl, or R-substituted or unsubstituted heteroalkyl, R-substituted R-substituted or unsubstituted heteroaryl. or unsubstituted cycloalkyl, R-substituted or unsubstituted R is independently halogen, —CN, OH, -NH. heterocycloalkyl, R-substituted or unsubstituted aryl, or -COOH, -CONH, -NO, -SH, -SOCl, -SOH, 55 R-substituted or unsubstituted heteroaryl. —SOH, -SONH, R-substituted or unsubstituted alkyl, R" is independently halogen, —CN, OH, -NH2, R-substituted or unsubstituted heteroalkyl, R-substituted or unsubstituted cycloalkyl, R-substituted or unsubstituted -COOH, -CONH, NO, -SH, -SOCl, -SOH, heterocycloalkyl, R-substituted or unsubstituted aryl, or —SOH, -SONH, R-substituted or unsubstituted alkyl, R-substituted or unsubstituted heteroaryl. 60 R-substituted or unsubstituted heteroalkyl, R-substituted In some embodiments, L is in each instance, indepen or unsubstituted cycloalkyl, R-substituted or unsubstituted dently selected from a bond, —C(O) , —C(O)N(R) , heterocycloalkyl, R-substituted or unsubstituted aryl, or C(O)O , S(O) = (i.e. —S , , S(O)— or S(O)). R-substituted or unsubstituted heteroaryl. —S(O)N(R)-, O-, -N(R)-, - N(R)C(O)N In some embodiments, L is in each instance, indepen (R) , R-substituted or unsubstituted alkylene, R-sub 65 dently selected from a bond, —C(O) , —C(O)N(R') , stituted or unsubstituted heteroalkylene, R-substituted or C(O)O , S(O) = (i.e. —S , S(O)— or - S(O)2), unsubstituted cycloalkylene, R-substituted or unsubstituted —S(O)N(R)-, -O-, -N(R)-, - N(R)C(O)N US 9,353,116 B2 33 34 (R) , R-substituted or unsubstituted alkylene, R-sub -continued stituted or unsubstituted heteroalkylene, R-substituted or (V) unsubstituted cyclo alkylene, R-substituted or unsubsti N tuted heterocycloalkylene, R-substituted or unsubstituted H-L-R), arylene, or R-substituted or unsubstituted heteroarylene. L4 21 R" is independently halogen, —CN, -OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, —SOH, -SONH2, R-substituted or unsubstituted alkyl, N21 N R-substituted or unsubstituted heteroalkyl, R-substituted 1s | H-L-R): or or unsubstituted cycloalkyl, R-substituted or unsubstituted 10 -L' s 2 heterocycloalkyl, R'-substituted or unsubstituted aryl, or R R-substituted or unsubstituted heteroaryl. (VI) R' is independently halogen, —CN, -OH, -NH2, N -COOH, -CONH, -NO, -SH, -SOCl, -SOH, 15 H-L-R), —SOH, -SONH2, R'-substituted or unsubstituted alkyl, L4 2 R'-substituted or unsubstituted heteroalkyl, R'-substituted or unsubstituted cycloalkyl, R'-substituted or unsubstituted heterocycloalkyl, R'-substituted or unsubstituted aryl, or ri?). R'-substituted or unsubstituted heteroaryl. (R t" s l L 21!" Ré). In some embodiments, L is in each instance, indepen dently selected from a bond, —C(O) , —C(O)N(R') , C(O)O , S(O) = (i.e. —S , S(O)— or - S(O)2), In certain embodiments, L', L. L. L. L. and Lare, in —S(O)N(R)-, O-, -N(R)-, - N(R)C(O)N 25 each instance, independently a bond, —NH-, or Substituted (R) , R-substituted or unsubstituted alkylene, R-sub or unsubstituted C-C alkylene. In certain other embodi stituted or unsubstituted heteroalkylene, R-substituted or ments, L is a bond, -NH-, or unsubstituted C1-C5 alky unsubstituted cycloalkylene, R-substituted or unsubstituted lene. heterocycloalkylene, R-substituted or unsubstituted In some other embodiments, the compounds have the for arylene, or R-substituted or unsubstituted heteroarylene. 30 mula (where the variables are as described above): R" is independently halogen, —CN, -OH, -NH2, -COOH, -CONH, -NO, -SH, -SOCl, -SOH, —SOH, -SONH, R-substituted or unsubstituted alkyl, (VII) R-substituted or unsubstituted heteroalkyl, R-substituted 35 or unsubstituted cycloalkyl, R-substituted or unsubstituted | --L-R): heterocycloalkyl, R-substituted or unsubstituted aryl, or le R-substituted or unsubstituted heteroaryl. R" is independently halogen, —CN, -OH, -NH2, 40 (R-L-H 1S-\ N -COOH, -CONH, -NO, -SH, -SOCl, -SOH, a M —SOH, -SONH, R-substituted or unsubstituted alkyl, NaNsV R-substituted or unsubstituted heteroalkyl, R-substituted L2 or unsubstituted cycloalkyl, R-substituted or unsubstituted V heterocycloalkyl, R-substituted or unsubstituted aryl, or R2 R-substituted or unsubstituted heteroaryl. 45 (VIII) In some embodiments, R. R. R. R. R7 and Rare independently hydrogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted hetero cycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. 50 In some other embodiments, the compounds have the for mula (where the variables are as described above):
55 (IV) (IX)
--L-R): 60 (R-l'. N \ M NaNsV L2 65 V US 9,353,116 B2 35 36 -continued —S(O) , NHC(O)—, or NHS(O), . R may be a (X) substituted or unsubstituted alkyl (e.g. substituted or unsub stituted C to Calkyl). For example, R may be an unsubsti NH2 --L-R): tuted alkyl or alkyl substituted with chloro, fluoro, methyl, difluoromethyl, or trifluoromethyl. In other embodiments, R is ethenyl, ethyl, 2.2.2-trichloroethyl, 2,2-dichloroethyl, (R-L'--N1 N1\ N 2-chloroethyl, 2.2.2-trifluoroethyl, 2,2-difluoroethyl, O NaN/N V 2-fluoroethyl, propyl, isopropyl, 1-propenyl, or 2-propenyl. L2 10 V In some embodiments, the compounds Suitable for use with R2 the present invention have the structure of one of the formula cited herein wherein-L-R is:
(XI)
15 A 1
(XII)
25
30
(XIII)
35
40
(XIV) N H-L-R), 45 HN 2
es N 2 2 50 (R-L'--Sls, 2---L--R4). H In some other embodiments, the compounds suitable for use with the present invention include those wherein L' is a In certain embodiments, at least one-L-R is an electrophilic 55 bond. R' may be hydrogen or NH2. In certain embodiments, moiety (e.g. -L-R is or includes an electrophilic moiety). L' is a bond; and R' is hydrogen. In certain other embodi For example, in some embodiments, -L-R forms an electro ments, L' is a bond; and R' is NH2. In some embodiments, the philic moiety. In other embodiments, one of L or R is an present invention provides a compound where L is a bond; electrophilic moiety (e.g. one of L or R is or includes an and R is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, electrophilic moiety). In some embodiments, L forms an 60 pentyl, cyclopentyl, hexyl, cyclohexyl, methoxy, ethoxy, pro electrophilic moiety. In other embodiments, R forms an elec poxy, or butoxy. In certain embodiments, R is isopropyl or trophilic moiety. In certain other embodiments, L is a bond, cyclopentyl. In other embodiments, R is isopropyl. In other —NH substituted or unsubstituted alkylene, or substituted embodiments, R is cyclopentyl. In some otherembodiments, or unsubstituted heteroalkylene; and R is a substituted or 65 R’ is methoxy. In certain embodiments, c is 2: L is a bond, unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and R is methoxy, ethoxy, propoxy, or butoxy. In other or halogen. In some of the embodiments, L is —C(O)—, embodiments, R is methoxy. US 9,353,116 B2 37 38 In some embodiments, the present invention provides com -continued pounds that have:
10 N NH2 % N N1 N O 4. C.N 15
25 N NH2 O N1N 30 4. 5 2 NDO N
35 NH2 N --
40
NH2 4. 5 N1 N 45 NH2 4. C.N N-\
50
55
NH2 NH2 60 N N1 N \ N 2 2N/ N C.N 65 US 9,353,116 B2 39 40 -continued -continued
NH O 2 \4 N N N2; N M 4. N 10
15
1. NN No e H O 25 N
HN O C 30 19 NN No e
35 O O HN Ol N1\/ Na
40 -O NN No e Q 45 o, t
50 HN 19 NN
55 No e
60
65 US 9,353,116 B2 41 42 -continued -continued (XVII) N (R-R). HN 2 N21 ON 10 -L' 1sN o1 R In some other embodiments, the present invention provides O. O (XVIII) a compound having the below formula (which are useful inter 15 alia, as inhibitors of Lrrk-2 kinases): V/ -N1Ns. 2 H s (XV) HN N21 N L4 1s | H-L-R).2- R2 -L' s 2 x1 N1 N R1 l ---L-R): 25 2 2 -L' X2 O. O (XIX) \/ X' and X’ are, in each instance, independently —N— or 30 --N1 yrs. =C(-L-R)-. Ring A is, in each instance, independently HN 2 selected from cycloalkyl, heterocycloalkyl, aryl, or het eroaryl. L. L. L., and L'are as defined above (e.g., in each instance, independently selected from a bond, —C(O)—, N21 OS —C(O)N(R), C(O)C , S(O) , S(O)N(R) , 35 -O-, - N(R)-, - N(R)C(O)N(R)- ), substituted or -L' 1sN o1 unsubstituted alkylene, substituted or unsubstituted het R eroalkylene, substituted or unsubstituted cycloalkylene, sub stituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted het 40 O (XX) eroarylene, whereing is an integer from 0 to 2). R', R. R. R. R. and Rare as defined above (e.g., in each instance, s ul independently selected from hydrogen, halogen, —CN. N R; or –OH, -NH, -COOH, -CONH, NO, -SH, HN 2 SOCl. —SOH, -SOH, -SONH, substituted or 45 unsubstituted alkyl, substituted or unsubstituted heteroalkyl, N21 N substituted or unsubstituted cycloalkyl, substituted or unsub 2- R2 stituted heterocycloalkyl, substituted or unsubstituted aryl, or 1s | H-L-R). substituted or unsubstituted heteroaryl). The variable b is as -L' s 2 defined above (e.g. an integer from 0 to 5; and c is as defined 50 RI above (e.g. an integer from 0 to 4). In some embodiments, the present invention provides a compound having the formula: O (XXI) 55 s ul (XVI) N R3. HN 21
HN 2 60 Na On
N21 N -L' 1sN o1 R 1sN | 2H-L-R): Ll N 65 In some other embodiments, the compound provided herein has the formula (with the variables as defined above):
US 9,353,116 B2 45 46 -continued -continued O!O -
No e 10 1. NN
H O N 15 No 4. O S N HN O O V S. o= -O SN NH No e HN
25 O O 1. NN o 1 No e NH O 30 V F; OES F NH F 19 NN 35 HN
No e O
40 No % V -n- O NH orn NH 45
HN O 1. NN 50 - NN No e No %
O O 55 V V
o=NH oNH
60 HN
1. O NN - NN N 2 65 No % US 9,353,116 B2 47 48 -continued -continued Osu ; or o=NH
10 HN 19 NN No e 15 C.
O S N \ 1S
o=NH
O V OES HN 25 NH -O NN No e 30 In certain embodiments, L is selected from a bond, —NH , —C(O) , S(O)—, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. R may be selected from substituted or unsubstituted alkyl, sub 35 stituted or unsubstituted heteroalkyl, or halogen. In other embodiments, R is methyl; difluoromethyl; trifluoromethyl; ethenyl; ethyl; 2.2.2-trichloroethyl; 2.2-dichloroethyl: 2-chloroethyl; 2.2.2-trifluoroethyl: 2,2-difluoroethyl; or V 2-fluoroethyl; propyl; isopropyl: 1-propenyl: 2-propenyl: OES 40 butyl; tert-butyl; napthyl; thiophene: 2-chloro-thiophene: t s phenyl: 2-methyl-phenyl: 3-methyl-phenyl, 4-methyl-phe nyl: 2-phenyl-phenyl: 3-phenyl-phenyl: 4-phenyl-phenyl 2-chloro-thiophene; or 3-chloro-thiophene. As described above, the term “inhibitor” may refer to an 45 inhibitor of a recombinant kinase comprising a cysteine Sub stitution at a gatekeeper amino acid position (i.e. a cysteine gatekeeper kinase inhibitor) and includes a compound described herein such as the compound of Formulae (I) to (XIV). In some embodiments, the inhibitors are able to 50 covalently bind to cysteine. In some other embodiments, the inhibitors inhibit the kinase by bonding to the sulfylhydryl group of the cysteine residue at the gatekeeper amino acid position. In some embodiments, a compound provided herein may be a Lrrk-2 kinase inhibitor. In some embodiments, the 55 Lrrk-2 kinase inhibitor is one or more of the compounds set forth in Table Z and/or a compound of Formula (XV) to (XXIX). A person having ordinary skill in the art would immedi ately take into account the widely known principles of chemi 60 cal when considering the description of compounds provided herein. Accordingly, where a group may be substituted by one or more of a number of Substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable 65 and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aque ous, or neutral conditions. US 9,353,116 B2 49 50 It will be apparent to one skilled in the art that certain be converted to the compounds of the present invention by compounds of this invention may exist in tautomeric forms, chemical or biochemical methods in an ex vivo environment. all such tautomeric forms of the compounds being within the For example, prodrugs can be slowly converted to the com Scope of the invention. pounds of the present invention when placed in a transdermal Unless otherwise stated, structures depicted herein are also patch reservoir with a Suitable enzyme or chemical reagent. meant to include compounds which differ only in the pres In some embodiments, each Substituted group described ence of one or more isotopically enriched atoms. For example, compounds having the present structures except for above for the compounds of the present invention is substi the replacement of a hydrogen by a deuterium or tritium, the tuted with at least one Substituent group. More specifically, in replacement of a carbon by 'C- or 'C-enriched carbon, or 10 some embodiments, each substituted alkyl, substituted het the replacement of an iodine by I, are within the scope of eroalkyl, substituted cycloalkyl, substituted heterocy this invention. All isotopic variations of the compounds of the cloalkyl, substituted aryl, substituted heteroaryl, substituted present invention, whether radioactive or not, are encom or unsubstituted alkylene, substituted or unsubstituted het passed within the scope of the present invention. eroalkylene, substituted or unsubstituted cycloalkylene, sub The compounds of the present invention also include the 15 salts, hydrates, Solvates and prodrug forms. The compounds stituted or unsubstituted heterocycloalkylene, substituted or of the present invention also include the isomers and metabo unsubstituted arylene or substituted or unsubstituted het lites of those described in Formula (I)-(XXIX). eroarylene described above is substituted with at least one Salts include, but are not limited, to Sulfate, citrate, acetate, Substituent group. In other embodiments, at least one or all of oxalate, chloride, bromide, iodide, nitrate, bisulfate, phos these groups are Substituted with at least one size-limited phate, acid phosphate, phosphonic acid, isonicotinate, lac Substituent group. Alternatively, at least one or all of these tate, Salicylate, citrate, tartrate, oleate, tannate, pantothenate, groups are substituted with at least one lower substituent bitartrate, ascorbate. Succinate, maleate, gentisinate, fuma group. rate, gluconate, glucaronate, Saccharate, formate, benzoate, In other embodiments of the compounds described above, glutamate, methanesulfonate, ethanesulfonate, benzene 25 Sulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methyl each substituted or unsubstituted alkyl is a substituted or ene-bis-(2-hydroxy-3-naphthoate)) salts. Other salts include, unsubstituted C-C alkyl, each Substituted or unsubstituted but are not limited to, salts with inorganic bases including alkylene is a Substituted or unsubstituted C-C alkylene, alkali metal salts such as Sodium salts, and potassium salts; each substituted or unsubstituted heteroalkyl is a substituted alkaline earth metal salts such as calcium salts, and magne 30 or unsubstituted 2 to 20 membered heteroalkyl, each substi sium salts; aluminum salts; and ammonium salts. Other salts tuted or unsubstituted heteroalkylene is a substituted or with organic bases include salts with diethylamine, diethano unsubstituted 2 to 20 membered heteroalkylene, each substi lamine, meglumine, and N,N'-dibenzylethylenediamine. tuted or unsubstituted cycloalkyl is a substituted or unsubsti The neutral forms of the compounds can be regenerated by tuted C-C cycloalkyl, each Substituted or unsubstituted contacting the salt with a base or acid and isolating the parent 35 cycloalkylene is a Substituted or unsubstituted C-Cs compound in the conventional manner. The parent form of the cycloalkylene, each substituted or unsubstituted heterocy compound differs from the various Saltforms in certain physi cal properties, such as Solubility in polar solvents, but other cloalkyl is a substituted or unsubstituted 4 to 8 membered wise the salts are equivalent to the parent form of the com heterocycloalkyl, each substituted or unsubstituted heterocy pound for the purposes of the present invention. 40 cloalkylene is a substituted or unsubstituted 4 to 8 membered Certain compounds of the present invention can exist in heterocycloalkylene, each substituted or unsubstituted aryl is unsolvated forms as well as Solvated forms, including a substituted or unsubstituted C or Caryl, each substituted hydrated forms. In general, the Solvated forms are equivalent or unsubstituted arylene is a substituted or unsubstituted C or to unsolvated forms and are encompassed within the scope of Cs arylene, each Substituted or unsubstituted heteroaryl is a the present invention. Certain compounds of the present 45 Substituted or unsubstituted Cs or C. heteroaryl, and each invention may exist in multiple crystalline or amorphous substituted or unsubstituted heteroarylene is a substituted or forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended unsubstituted Cs or Cheteroarylene. to be within the scope of the present invention. Alternatively, each substituted or unsubstituted alkyl is a Certain compounds of the present invention possess asym 50 substituted or unsubstituted C-C alkyl, each substituted or metric carbon atoms (optical centers) or double bonds; the unsubstituted alkylene is a substituted or unsubstituted C-Cs enantiomers, racemates, diastereomers, tautomers, geometric alkylene, each substituted or unsubstituted heteroalkyl is a isomers, stereoisometric forms that may be defined, in terms substituted or unsubstituted 2 to 8 membered heteroalkyl, of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- each substituted or unsubstituted heteroalkylene is a substi for amino acids, and individual isomers are encompassed 55 tuted or unsubstituted 2 to 8 membered heteroalkylene, each within the scope of the present invention. The compounds of substituted or unsubstituted cycloalkyl is a substituted or the present invention do not include those which are known in unsubstituted C-C cycloalkyl, each substituted or unsubsti art to be too unstable to synthesize and/or isolate. The present tuted cycloalkyl is a substituted or unsubstituted C-C, invention is meant to include compounds in racemic and cycloalkylene, each substituted or unsubstituted heterocy optically pure forms. Optically active (R)- and (S)-, or (D)- 60 and (L)-isomers may be prepared using chiral synthons or cloalkyl is a substituted or unsubstituted 3 to 6 membered chiral reagents, or resolved using conventional techniques. heterocycloalkyl, and each substituted or unsubstituted het The present invention also provides compounds which are erocycloalkylene is a substituted or unsubstituted 3 to 6 mem in a prodrug form. Prodrugs of the compounds described bered heterocycloalkylene. herein are those compounds that readily undergo chemical 65 V. Kinases changes under physiological conditions to provide the com In some embodiments, the present invention provides a pounds of the present invention. Additionally, prodrugs can recombinant kinase comprising a cysteine Substitution at a US 9,353,116 B2 51 52 gatekeeperamino acid position (also referred to as a "cysteine ing wild type kinase. The activity is considered “not substan gatekeeper kinase', a "recombinant kinase of the present tially lower when the activity is not less than 5-fold less, e.g., invention' or a “recombinant kinase set forth herein'). For 4-fold, 3-fold, or 2-fold less than the reference kinase. In example, the recombinant kinase can comprise a sequence having a cysteine Substitution at the position corresponding to some cases, the term “not substantially lower is determined Thr338 of c-Src, such as the positions shown for SEQ ID in terms of percentage, and a not substantially lower activity NOs:58-77, and sequences having substantial identity is at least 50% of the reference kinase, e.g. higher than 50% of thereto. That is, the recombinant kinase can comprise a the activity of a wild type kinase. In some embodiments, the sequence having at least about 85,90, 92,93, 94, 95, 96.97, activity is 60, 70, 75, 80, 85,90, 95% or higher of the activity 98, or 99% identity to a sequence of any one of SEQ ID 10 of the reference kinase. NOs:58-77, with a cysteine substitution at the position cor The present invention provides methods for evaluating the responding to Thr338 of c-Src. use of a cysteine gatekeeper kinase. In some embodiments, the recombinant kinase can have a In some embodiments, the recombinant kinase includes a sequence of SEQ ID NO:2 (T338C c-Src), or a sequence recombinant kinase is selected from Src (e.g., c-Src (SEQID having Substantial identity thereto. In some embodiments the 15 recombinant kinase can comprise a sequence having at least NOs:2-23 or 59) or v-Src (SEQID NO:46-50 or 58); MOK; about 85,90, 92,93, 94, 95, 96, 97,98, or 99% identity to the Sgk494; Yak/Yrk; SRPK1; CDK; DICTY-I; PAK/STE20; or sequence of SEQ ID NO:2 with a cysteine at the position Ctrl/DPYK1 with a cysteine at the gatekeeper position (at the corresponding to 338 (with reference to the full length amino acid position corresponding to 338 of the c-Src protein sequence of SEQ ID NO:3). In some embodiments, the of SEQ ID NO:3. In some embodiments, the recombinant recombinant kinase comprises less than the full length of kinase is a recombinant Src and the gatekeeper amino acid SEQ ID NO:2 or 3 (and substantially identical variants position is T338. thereof), but retains the cysteine substitution at the position In some embodiments, the recombinant kinase has a 25 greater catalytic efficiency than the corresponding wild type corresponding to amino acid 338 of SEQID NO:3. In some kinase. For example, the kinase activity is greater than 100% embodiments, the recombinant kinase comprises at least 8, of the reference kinase (e.g., wild type c-Src of SEQ ID 10, 12, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, NO:3). In some embodiments, the activity is 1.2-fold, 1.5- 175, 200 or more contiguous amino acids of SEQID NO:2 or fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold a Substantially identical sequence over that span retaining a 30 or higher than the reference. In some embodiments, the cata cysteine at the amino acid position corresponding to 338 of lytic efficiency is measured as the ratio of ki/K. SEQID NO:3. In some embodiments, the recombinant kinase further In some embodiments, the recombinant kinase can have a comprises an additional amino acid substitution correspond sequence of any one of SEQ ID NOS:24-45 or a sequence ing to position V323 of c-Src (see, e.g., SEQID NOs: 10-23). having Substantial identity thereto. These kinase sequences 35 That is, the recombinant kinase can be a cysteine gatekeeper have a naturally occurring gatekeeper cysteine, i.e. a cysteine kinase, i.e., comprising a sequence having Substantial identity at the position corresponding to amino acid 338 in c-Src (SEQ to any one of SEQID NOS:2-77 with a cysteine at the position ID NO:2 shows the T338C c-Src, while SEQID NO:4 shows corresponding to amino acid 338 of SEQID NO:3, and addi the wild type T338 c-Src sequence). In some embodiments tionally include a Substitution at the position corresponding to the recombinant kinase can comprise a sequence having at 40 amino acid323 of SEQID NO:3. One of skill will understand least about 85,90, 92,93, 94, 95, 96, 97,98, or 99% identity that the positions corresponding to those of SEQID NO:3 can to the sequence of any one of SEQ ID NOS:24-45 with a be ascertained for other kinase sequences. cysteine at the position corresponding to amino acid 338 of In Some embodiments, the recombinant kinase further c-Src. In some embodiments, the recombinant kinase com 45 comprises an additional amino acid substitution correspond prises less than the full length of any one of SEQID NOS:24 ing to the position M314 of c-Src, e.g. to gly (G) or ala (A). 45 (and substantially identical variants thereof), but retains For example, the recombinant kinase can be a cysteine gate the cysteine at the position corresponding to amino acid 338 keeper kinase, i.e., comprising a sequence having Substantial of c-Src. In some embodiments, the recombinant kinase com identity to any one of SEQID NOS:2-77 with a cysteine at the prises at least 8, 10, 12, 20, 25, 30,35, 40, 50, 60, 70, 80,90, 50 position corresponding to amino acid 338 of SEQID NO:3, 100, 120, 150, 175, 200 or more contiguous amino acids of and additionally include a substitution at the position corre any one of SEQ ID NOS:24-45 or a substantially identical sponding to amino acid 314 of SEQID NO:3. One of skill will sequence over that span retaining a cysteine at the position understand that the positions corresponding to those of SEQ corresponding to 338 of SEQID NO:2. 55 ID NO:3 can be ascertained for other kinase sequences. In some other embodiments, the recombinant kinase has a In some embodiments, the recombinant kinase includes kactivity that is not substantially lower than the kactivity Substitutions at two or all three positions corresponding to of the corresponding wild-type kinase. In some embodi positions 338,314 and 323 of c-Src (SEQID NO:3). In some ments, the k activity that is not substantially lower than the embodiments, the recombinant kinase comprises a sequence k activity of the corresponding wild-type kinase. In some 60 having substantial identity to SEQ ID NO:2 with a C at the embodiments, the recombinant kinase has a K binding affin position corresponding to amino acid 338 of c-Src (the full ity for ATP of the recombinant kinase is not substantially length sequence of SEQID NO:3), and also has a substitution lower than the K binding affinity for ATP of the correspond at the position corresponding to amino acid 314 of c-Src. In ing wild type kinase. In some embodiments, the K binding 65 Some embodiments, the recombinant kinase comprises a affinity for ATP of the recombinant kinase is not substantially sequence having substantial identity to SEQID NO:2 with a lower than the K binding affinity for ATP of the correspond C at the position corresponding to amino acid 338 (of the full US 9,353,116 B2 53 54 length sequence of SEQID NO:3), and also has a substitution TABLE 1 at the position corresponding to amino acid 323 of c-Src. In Some embodiments, the recombinant kinase comprises a sequence having substantial identity to SEQID NO:2 with a C at the position corresponding to amino acid 338 of c-Src (the full length sequence of SEQ ID NO:3), and also has a Substitution at the positions corresponding to amino acids 314 and 323 of c-Src. Again, one of skill will be able to determine the corresponding amino acid positions for kinases with 10 sequences that are not perfectly aligned with c-Src. Rd In some embodiments, the cysteine gatekeeper kinase has an additional amino acid Substitution of alanine (A) or serine / N Re" (S) at the position corresponding to V323 of c-Src (V323A 15 N \ NH (c-Src-ES2) or V323S (c-Src-ES3). In some other embodi ments, the recombinant kinase having an additional amino acid substitution at VAL323 has a greater catalytic efficiency of the corresponding recombinant kinase that does not have an additional amino acid substitution at VAL323. In some embodiments, the catalytic efficiency is measured as the ratio of ki/K. V y” In some embodiments, the corresponding Substitutions can WT T338C be performed in other kinases. A person having ordinary skill c-Src c-Src. 25 Com- ICso ICso in the art would understand which amino acids correspond to pound in R. R. Re Ra (nM) (nM) VAL 323 in other kinases. 1 0 iPr NH, NHCOCHCH, 2319 419 In some embodiments, the present invention provides 2 0 iPr H NHCOCHCH, >SOOO >SOOO methods and compositions for modifying the microenviron 3 0 iPr NH, NHSOCHCH, 1004 111 ment around the cysteine gatekeeper by alteration of one 30 4 () iPr H NHSOCHCH >SOOO >SOOO nearby residue (e.g. Val323) in order to impact inhibitor 5 0 iPr NH, H NHSOCHCH 899 145 6 0 iPr NH, NHCOCHCI >5000 817 potency. For example, liberating additional space with a 7 1 iPr NH, NHCOCHCH, >SOOO 2762 V323A mutation resulted in a 5-fold increase in potency for 8 1 iPr NH, H NHCOCHCH >5000 >5000 13, while the V3232S mutation had a 12-fold effect. In some 9 1 iPr NH, NHSOCHCH, >SOOO 150 35 10 1 Pr NH, H NHSOCHCH, 3083 1759 embodiments, the present invention provides methods of 11 1 iPr NH, NHSOCHCH >SOOO 3497 boosting potency which may allow dosing levels sufficient to 12 1 iPr NH, NHCOCHCI >SOOO >SOOO substantially minimize off-target effects with MOK kinase 13 1 iPr NH, COCHF >SOOO 338 14 1 Pr NH, COCH, >SOOO 4520 (the effects due to MOK inhibition can be taken into account 15 1 Me NH NHSOCHCH- >SOOO 3161 by comparing effects in WT vs. ES expressing cells). 40 16 1 tBu NH NHSOCHCH- >SOOO 618 A. Forming a Kinase 17 1 Cp NH NHSOCHCH- >SOOO 196 In some other embodiments, the present invention provides Com a method of forming a recombinant kinase described herein, pound Re Rd. wherein the method includes transforming a cell with a 45 18 NHCOCHCH, H >SOOO 1661 nucleic acid encoding a recombinant kinase described herein, 19 NHSOCHCH H >SOOO 1004 thereby forming a recombinant kinase described herein. In 2O H NHSOCHCH, 2170 S60 Some embodiments, the recombinant kinase is selected from IC50 values for electrophile derivatized pyrazolopyrimidines and 4-anilinoquinazolines against WT c-Src and T338C c-Src. Scaffolds are depicted such that the hinge-binding Src.: MOK; Sgk494; Lrrk-2; Yak/Yrk; SRPK1; CDK; element is located on the left. Note that for covalent inhibitors IC50 values are time dependent. In these assays, the inhibitors were preincubated with the Src for ten minutes DICTY-I; PAK/STE20; or Ctrl/DPYK1 as described herein. 50 prior to assay initialization by addition of ATP B. Structure Activity Relationship Studies—Inhibition of Src An array of 3-benzyl-substituted pyrazolopyrimidines modified with electrophiles or isosteric and unreactive nega In some embodiment, the present invention provides a tive control groups at the meta and para positions were syn series of 3-phenyl-substituted pyrazolopyrimidines with 55 thesized and screened against WT and T338C c-Src (com electrophilic groups at positions expected to be in close proX pounds 7-17. Table 1). The benzyl functionalized compounds imity to the gatekeeper residue and as set forth in Table 1. In inhibitored wild type c-Src (ICso values >5uM). Compound, Some other embodiments, the electrophiles include meta and 9, which is functionalized with a vinylsulfonamide, exhibited para Substituents of the 3-phenyl ring and vinylsulfonamides an ICs value of 150 nM. An unreactive control compound 11 as well as acrylamides and chloroacetamides. A meta-substi 60 resulted in a 23-fold drop in potency. A fluoromethylketone tuted vinylsulfonamide, 3 inhibited T338C relative to WT bearing compound, 13, yielded an ICs value of 338 nM. c-Src (>9-fold increase), while a para-substituted version, 5. which was >13-fold more potent than the corresponding elicited a ~6-fold improvement (Table 1). Acrylamides (1) ketone, 14. and chloroacetamides (6) were also shown to be inhibitors. The present invention also provides methods of determin Under the assay conditions used (10 min preincubation with 65 ing the activity effects of modifying the N1 position of pyra inhibitor prior to addition of ATP) ICso values under 5uM for Zolopyrimidines by a structure activity relationship (SAR). In either 2 or 4 for WT or T338C c-Src were not shown. Some embodiments, this includes using the pyrazolopyrimi US 9,353,116 B2 55 56 dine scaffold with a benzyl-linked m-vinylsulfonamide, see fonamide makes a direct hydrogen bond to the side chain compounds 9, 15-17; Table 1 This analysis revealed that Glu310 (FIGS. 2A, C). In crystal structures of wild type Src, secondary alkyl groups such as isopropyl (9) and cyclopentyl the hydroxyl of the gatekeeper threonine is often directed (17) moieties inhibit T338C c-Src. These results indicate that towards the C4-exocyclic amine of the adenine portion of Substitution at N1 can be used to modulate potency against ATP mimetics. T338C c-Src. Accordingly, the present invention provides In the 9 c-Src-ES1 co-crystal structure, the sulfhydryl of methods of modulating the potency against kinases, such as Cys338 adopts a distinct rotamer to accommodate the bulky c-Src. C-3 benzyl group and facilitate a covalent bond (FIG. 2C). The present invention provides methods and compositions The flexible ethylsulfonamide moiety is situated to allow that are suitable for use with a variety of kinases, e.g. recom 10 the covalent linkage with Cys338 (FIG.2B). The side chain of binant, wild type, natural, mutant, and unmutated. In some Met314, a critical component of the hydrophobic spine, is embodiments, these kinases include c-Src, Src.: Src.: MOK; dramatically shifted relative to its position in other c-Src Sgk494; Yak/Yrk; SRPK1; CDK; DICTY-I; PAK/STE20; or structures (FIGS. 2B, C). Movement of Met314 may prevent Ctrlf DPYK1. a steric clash with the ethylsulfonamide moiety of 9. 15 VII. Pharmaceutical Compositions In some embodiments, the recombinant kinases described In some embodiments, the present invention provides a herein include an approximate 15 residue His tag in addition pharmaceutical composition comprising a compound as set to the sequence for the actual protein, e.g. linker and heptamer forth herein (e.g. a compound of Formula (I)-CXXIX)) and a for specific TEV protein cutting. In some instances TEV may pharmaceutically acceptable excipient. be cut at residue 248,249, or 250. It is understood by those in "Pharmaceutically acceptable excipient' and “pharmaceu the art that the DNA sequence can be optimized with respect tically acceptable carrier refer to a substance that aids the to the code or sequence without affecting the primary protein administration of an active agent to and absorption by a Sub encoded thereby. ject and can be included in the compositions of the present The following sets forth gatekeeper residues. In some invention without causing a significant adverse toxicological embodiments, the gatekeeper residue is cysteine. In some 25 effect on the patient. Non-limiting examples of pharmaceu embodiments the kinase is natural, wildtype, or recombinant. tically acceptable excipients include water, NaCl, normal As described herein, a Cysgatekeeper is an attractive target saline solutions, lactated Ringers, normal Sucrose, normal for the inhibitory compounds described herein. Representa glucose, binders, fillers, disintegrants, lubricants, coatings, tive kinases having a naturally occurring Cys at the gate sweeteners, flavors and colors, and the like. One of skill in the keeper position include the entries of Table 2 following. As 30 art will recognize that other pharmaceutical excipients are customary in the art, the terms “GI: number,” “GI: No.” and useful in the present invention. the like refer to a unique sequence identifier (i.e., “GenBank The compounds and compositions of the present invention Identifier”) for a sequence. can be prepared and administered in a wide variety of oral, parenteral and topical dosage forms. Oral preparations TABLE 2 35 include tablets, pills, powder, dragees, capsules, liquids, loZ enges, cachets, gels, syrups, slurries, Suspensions, etc., Suit SEQ ID NO: GI: No. Species able for ingestion by the patient. The compounds of the 24 4587987 Arabidopsis thaliana present invention can also be administered by injection, that 25 19424095 Arabidopsis thaliana 26 1785621 Arabidopsis thaliana is, intravenously, intramuscularly, intracutaneously, Subcuta 27 467827O Arabidopsis thaliana 40 neously, intraduodenally, or intraperitoneally. Also, the com 28 4678272 Arabidopsis thaliana pounds described herein can be administered by inhalation, 29 4678273 Arabidopsis thaliana for example, intranasally. Additionally, the compounds and 30 4678277 Arabidopsis thaliana 31 4886274 Arabidopsis thaliana compositions of the present invention can be administered 32 3O4709S Arabidopsis thaliana transdermally. The GR modulators of this invention can also 33 334.188021 Arabidopsis thaliana 45 be administered by intraocular, intravaginal, and intrarectal 34 929.4588 Arabidopsis thaliana routes including Suppositories, insufflation, powders and 35 1112O792 Arabidopsis thaliana 36 1112O796 Arabidopsis thaliana aerosol formulations (for examples of steroid inhalants, see 37 8777331 Arabidopsis thaliana Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995: Tiwa, 38 7106391 Mits musculus Ann. Allergy Asthma Immunol. 75:107-111, 1995). Accord 39 170572O Carassius at traits 50 ingly, the present invention also provides pharmaceutical 40 6648996 Capsicum annium 41 763O151 Leishmania major compositions including a pharmaceutically acceptable car 42 S139689 Homo sapiens rier or excipient and either a compound of Formula I, or a 43 48.6948 Trichomonas vaginalis pharmaceutically acceptable salt of a compound of Formula I. 44 2546884.46 Plasmodium falciparum For preparing pharmaceutical compositions from the com 45 13509297 Dictyostelium discoideum 55 pounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations VI. Co-Crystals of Kinase and a Compound include powders, tablets, pills, capsules, cachets, supposito The present invention provides co-crystals of a kinase and ries, and dispersible granules. A Solid carrier can be one or a compound, e.g. co-crystal structure of T338C c-Src with a more substances, which may also act as diluents, flavoring vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor 60 agents, binders, preservatives, tablet disintegrating agents, or is provided, see Example 34. an encapsulating material. Details on techniques for formu In the 9-c-Src-ES1 co-crystal structure, the pyrazolopyri lation and administration are well described in the scientific midine pharmacophore interacts with the backbone amides of and patent literature, see, e.g., the latest edition of Reming Glu339 and Met341 of the hinge region (FIG. 2A). The oxy ton's Pharmaceutical Sciences, Maack Publishing Co., Easton gen atoms of the Sulfonamide hydrogen bond directly to the 65 Pa. (“Remington's'). backbone amide of Asp404 and to that of Phe-A05 via a water In powders, the carrier is a finely divided solid, which is in molecule (FIG. 2B, C). Additionally, the nitrogen of the sul a mixture with the finely divided active component. In tablets, US 9,353,116 B2 57 58 the active component is mixed with the carrier having the more Sweetening agents, such as Sucrose, aspartame or sac necessary binding properties in Suitable proportions and com charin. Formulations can be adjusted for osmolarity. pacted in the shape and size desired. The powders and tablets Also included are solid form preparations, which are preferably contain from 5% or 10% to 70% of the active intended to be converted, shortly before use, to liquid form compound. preparations for oral administration. Such liquid forms Suitable solid excipients include, but are not limited to, include solutions, Suspensions, and emulsions. These prepa magnesium carbonate; magnesium Stearate; talc; pectin; dex rations may contain, in addition to the active component, trin; starch; tragacanth; a low melting wax, cocoa butter, colorants, flavors, stabilizers, buffers, artificial and natural carbohydrates; Sugars including, but not limited to, lactose, Sweeteners, dispersants, thickeners, solubilizing agents, and Sucrose, mannitol, or Sorbitol, starch from corn, wheat, rice, 10 the like. potato, or other plants; cellulose Such as methyl cellulose, Oil Suspensions can be formulated by Suspending a GR hydroxypropylmethyl-cellulose, or sodium carboxymethyl modulator in a vegetable oil. Such as arachis oil, olive oil, cellulose; and gums including arabic and tragacanth; as well sesame oil or coconut oil, or in a mineral oil Such as liquid as proteins including, but not limited to, gelatin and collagen. paraffin, or a mixture of these. The oil Suspensions can con If desired, disintegrating or solubilizing agents may be added, 15 tain a thickening agent, such as beeswax, hard paraffin or Such as the cross-linked polyvinyl pyrrolidone, agar, alginic cetyl alcohol. Sweetening agents can be added to provide a acid, or a salt thereof. Such as sodium alginate. palatable oral preparation, such as glycerol, Sorbitol or Dragee cores are provided with Suitable coatings Such as sucrose. These formulations can be preserved by the addition concentrated Sugar Solutions, which may also contain gum of an antioxidant such as ascorbic acid. As an example of an arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther. glycol, and/or titanium dioxide, lacquer Solutions, and Suit 281:93-102, 1997. The pharmaceutical formulations of the able organic solvents or solvent mixtures. Dyestuffs or pig invention can also be in the form of oil-in-water emulsions. ments may be added to the tablets or dragee coatings for The oily phase can be a vegetable oil or a mineral oil, product identification or to characterize the quantity of active described above, or a mixture of these. Suitable emulsifying compound (i.e., dosage). Pharmaceutical preparations of the 25 agents include naturally-occurring gums, such as gum acacia invention can also be used orally using, for example, push-fit and gum tragacanth, naturally occurring phosphatides. Such capsules made of gelatin, as well as soft, sealed capsules as soybean lecithin, esters or partial esters derived from fatty made of gelatin and a coating Such as glycerol or Sorbitol. acids and hexitol anhydrides, such as Sorbitan mono-oleate, Push-fit capsules can contain GR modulator mixed with a and condensation products of these partial esters with ethyl filler or binders such as lactose or starches, lubricants such as 30 ene oxide. Such as polyoxyethylene Sorbitan mono-oleate. talc or magnesium Stearate, and, optionally, stabilizers. In soft The emulsion can also contain Sweetening agents and flavor capsules, the GR modulator compounds may be dissolved or ing agents, as in the formulation of syrups and elixirs. Such Suspended in Suitable liquids, such as fatty oils, liquid paraf formulations can also contain a demulcent, a preservative, or fin, or liquid polyethylene glycol with or without stabilizers. a coloring agent. For preparing Suppositories, a low melting wax, Such as a 35 VIII. Administration mixture offatty acid glycerides or cocoa butter, is first melted The compositions of the present invention can be delivered and the active component is dispersed homogeneously by transdermally, by a topical route, formulated as applicator therein, as by stirring. The molten homogeneous mixture is Sticks, Solutions, Suspensions, emulsions, gels, creams, oint then poured into convenient sized molds, allowed to cool, and ments, pastes, jellies, paints, powders, and aerosols. thereby to solidify. 40 The compositions of the present invention can also be Liquid form preparations include Solutions, Suspensions, delivered as microspheres for slow release in the body. For and emulsions, for example, water or water/propylene glycol example, microspheres can be administered via intradermal Solutions. For parenteral injection, liquid preparations can be injection of drug-containing microspheres, which slowly formulated in Solution in aqueous polyethylene glycol solu release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. tion. 45 7:623-645, 1995; as biodegradable and injectable gel formu Aqueous solutions suitable for oral use can be prepared by lations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as dissolving the active component in water and adding Suitable microspheres for oral administration (see, e.g., Eyles, J. colorants, flavors, stabilizers, and thickening agents as Pharm. Pharmacol. 49:669-674, 1997). Both transdermal desired. Aqueous Suspensions Suitable for oral use can be and intradermal routes afford constant delivery for weeks or made by dispersing the finely divided active component in 50 months. water with viscous material. Such as natural or synthetic The pharmaceutical compositions of the present invention gums, resins, methylcellulose, sodium carboxymethylcellu can be provided as a salt and can be formed with many acids, lose, hydroxypropylmethylcellulose, Sodium alginate, poly including but not limited to hydrochloric, Sulfuric, acetic, vinylpyrrolidone, gum tragacanth and gum acacia, and dis lactic, tartaric, malic, Succinic, etc. Salts tend to be more persing or wetting agents such as a naturally occurring 55 soluble in aqueous or other protonic solvents that are the phosphatide (e.g., lecithin), a condensation product of an corresponding free base forms. In other cases, the preparation alkylene oxide with a fatty acid (e.g., polyoxyethylene Stear may be a lyophilized powder in 1 mM-50 mM histidine, ate), a condensation product of ethylene oxide with a long 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), 5.5, that is combined with buffer prior to use. a condensation product of ethylene oxide with a partial ester 60 In another embodiment, the compositions of the present derived from a fatty acid and a hexitol (e.g., polyoxyethylene invention are useful for parenteral administration, Such as Sorbitol mono-oleate), or a condensation product of ethylene intravenous (IV) administration or administration into a body oxide with a partial ester derived from fatty acid and a hexitol cavity or lumen of an organ. The formulations for adminis anhydride (e.g., polyoxyethylene Sorbitan mono-oleate). The tration will commonly comprise a solution of the composi aqueous Suspension can also contain one or more preserva 65 tions of the present invention dissolved in a pharmaceutically tives such as ethyl or n-propyl p-hydroxybenzoate, one or acceptable carrier. Among the acceptable vehicles and Sol more coloring agents, one or more flavoring agents and one or vents that can be employed are water and Ringer's solution, US 9,353,116 B2 59 60 an isotonic sodium chloride. In addition, sterile fixed oils can plished by co-formulation, i.e., preparing a single pharma conventionally be employed as a solvent or Suspending ceutical composition including both active agents. In other medium. For this purpose any bland fixed oil can be employed embodiments, the active agents can be formulated separately. including synthetic mono- or diglycerides. In addition, fatty In another embodiment, the active and/or adjunctive agents acids Such as oleic acid can likewise be used in the preparation 5 may be linked or conjugated to one another. of injectables. These solutions are sterile and generally free of IX. Nucleic Acids undesirable matter. These formulations may be sterilized by In some embodiments, the present invention provides an conventional, well known sterilization techniques. The for isolated nucleic acid comprising a nucleic acid sequence mulations may contain pharmaceutically acceptable auxil encoding a recombinant kinase provided herein (i.e. a recom iary Substances as required to approximate physiological con 10 binant kinase comprising a cysteine Substitution at a gate ditions such as pH adjusting and buffering agents, toxicity keeper amino acid position). This is also referred to herein as adjusting agents, e.g., sodium acetate, sodium chloride, a “nucleic acid of the present invention.” Thus, provided potassium chloride, calcium chloride, Sodium lactate and the herein are nucleic acids that encode the cysteine gatekeeper like. The concentration of the compositions of the present kinases described herein, e.g., recombinant kinases having a invention in these formulations can vary widely, and will be 15 cysteine in the position corresponding to amino acid 338 of selected primarily based on fluid volumes, viscosities, body c-Src (SEQID NO:3). weight, and the like, in accordance with the particular mode In Some embodiments, the nucleic acid sequence encodes a of administration selected and the patient’s needs. For IV sequence oran enzymatically functional fragment thereof, set administration, the formulation can be a sterile injectable forth in SEQ ID NOS 2-77. The enzymatically functional preparation, such as a sterile injectable aqueous or oleaginous fragment may be 50, 100, 150, or 200 bases in length. In some Suspension. This Suspension can be formulated according to embodiments, the nucleic acid encodes a polypeptide having the known art using those Suitable dispersing or wetting substantial identity to any one of SEQID NOS:2-77 wherein agents and Suspending agents. The sterile injectable prepara the polypeptide has a cysteine at the position corresponding to tion can also be a sterile injectable solution or Suspension in a amino acid 338 of c-Src. One of skill will understand that a nontoxic parenterally-acceptable diluent or solvent, Such as a 25 number of nucleic acid sequences will encode the some solution of 1,3-butanediol. polypeptide, due to the degeneracy of the nucleic acid code. In another embodiment, the formulations of the composi In some embodiments, the nucleic acid encodes a polypeptide tions of the present invention can be delivered by the use of encoding any one of SEQID NOS:2-77, wherein the polypep liposomes which fuse with the cellular membrane or are tide has a cysteine at the position corresponding to 338 of endocytosed, i.e., by employing ligands attached to the lipo 30 c-Src, or a sequence having at least 75, 80, 85,90,91, 92,93, some, or attached directly to the oligonucleotide, that bind to 94, 95, 96, 97,98, or 99% identity thereto. In some embodi surface membrane protein receptors of the cell resulting in ments, the polypeptide is shorter than the full length of any endocytosis. By using liposomes, particularly where the lipo one of SEQ ID NOS:2-77, but retains enzymatic (kinase) Some Surface carries ligands specific for target cells, or are activity. In some embodiments, the polypeptide is at least 25, otherwise preferentially directed to a specific organ, one can 35 30, 40, 50, 75, 80, 100,120, 150, 200 or more amino acids in focus the delivery of the compositions of the present inven length, and has substantial identity over the corresponding tion into the target cells in vivo. (See, e.g., Al-Muhammed, J. length of the selected sequence (selected from the sequences Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Bio consisting of SEQ ID NOS:2-77, having a C at the position technol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. corresponding to 338 of c-Src). For the non-identical amino 46:1576-1587, 1989). 40 acids, one of skill will understand that conservative amino The pharmaceutical preparation is preferably in unit dos acid Substitutions can be included. age form. In Such form the preparation is Subdivided into unit In some embodiments, the nucleic acid encodes a polypep doses containing appropriate quantities of the active compo tide having substantial identity to any one of SEQID NOS:2- nent. The unit dosage form can be a packaged preparation, the 77 wherein the polypeptide has a cysteine at the position package containing discrete quantities of preparation, such as 45 corresponding to amino acid 338 of c-Src, and an additional packeted tablets, capsules, and powders in vials orampoules. amino acid substitution at the position corresponding to Also, the unit dosage form can be a capsule, tablet, cachet, or amino acid 323 of c-Src and/or the position corresponding to lozenge itself, or it can be the appropriate number of any of amino acid 314 of c-Src. One of skill will understand that a these in packaged form. number of nucleic acid sequences will encode the same The quantity of active componentina unit dose preparation 50 polypeptide, due to the degeneracy of the nucleic acid code. may be varied or adjusted from 0.1 mg to 10000 mg, more In some embodiments, the nucleic acid encodes a polypeptide typically 1.0 mg to 1000 mg, most typically 10 mg to 500 mg. encoding any one of SEQID NOS:2-77, wherein the polypep according to the particular application and the potency of the tide has a cysteine at the position corresponding to 338 of active component. The composition can, if desired, also con c-Src, and optionally one or both of the substitutions at posi tain other compatible therapeutic agents. 55 tions corresponding to amino acids 314 or 323 of c-Src, or a The compounds described herein can be used in combina sequence having at least 75, 80, 85,90,91, 92,93, 94, 95, 96, tion with one another, with other active agents known to be 97, 98, or 99% identity thereto. In some embodiments, the useful in modulating a protein kinase, or with adjunctive polypeptide is shorter than the full length of any one of SEQ agents that may not be effective alone, but may contribute to ID NOS:2-77, but retains enzymatic (kinase) activity. In some the efficacy of the active agent. 60 embodiments, the polypeptide is at least 25, 30, 40, 50, 75,80, In some embodiments, co-administration includes admin 100, 120, 150, 200 or more amino acids in length, and has istering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, Substantial identity over the corresponding length of the or 24 hours of a second active agent. Co-administration selected sequence (selected from the sequences consisting of includes administering two active agents simultaneously, SEQID NOS:2-77, having a C at the position corresponding approximately simultaneously (e.g., within about 1, 5, 10, 15. 65 to 338 of c-Src, and optionally one or both of the substitutions 20, or 30 minutes of each other), or sequentially in any order. at positions corresponding to amino acids 314 or 323 of In some embodiments, co-administration can be accom c-Src). US 9,353,116 B2 61 62 In some other embodiments, the present invention provides the transgenic mouse or rat expresses a recombinant kinase an expression cassette comprising a nucleic acid of the comprising a cysteine Substitution at a gatekeeperamino acid present invention. In yet other embodiments, the expression position as described above. Methods of producing a trans cassette is a recombinant viral vector. In some other embodi genic mouse or rat that expresses recombinant proteins and ments, the expression cassette of is inside of a host cell. In enzymes are well-known in the art. A detailed description for other embodiments, the expression cassette is selected from such procedures may be found elsewhere, for example at U.S. mammalian, non-mammalian, mouse, rat, or human. In some Pat. No. 4.736,866, the contents of which are incorporated by embodiments, the recombinant kinase is inside a cell. In some reference in their entirety for all purposes. other embodiments, the cell is selected from mammalian, A. Descriptions of SEQID NOS (1-51) Follows. non-mammalian, mouse, rat, or human. Thus, in some 10 The following sets forth SEQID NOs: 1-51 that are suitable embodiments, a transgenic mouse or rat is provided, wherein for use with the compositions, methods, and kits herein:
SEQ ID NO: Description 1 DNA construct for T338C c-Src (251-533) 2 Protein encoded by SEQID NO: 1 3 Gaius gaits proto-oncogene (c = -Src.) 4 c-Src (251-533) 5 c-Src (251-533) with GHM at N-terminal 6 T338X)c-Src (251-533) 7 GHM-T338Xc-Src (251-533) (GHM at N-terminal) 8 T338Clc-Src (251-533) (c-Src “ES1) 9 GHM-T338Cc-Src (251-533) (GHM at N-terminal) (c-Src “ES1) 10 T338X, V323Xc-Src (251-533) 11 GHM-T338X, V323X c-Src (251-533) (GHM at N-terminal) 12 T338C, V323Xc-Src (251-533) 13 GHM-T338C, V323X c-Src (251-533) (GHM at N-terminal) 14 T338C, V323Alc-Src (251-533) (c-Src “ES2) 15 GHM-T338C, V323 Alc-Src (251-533) (GHM at N-terminal) (c-Src “ES2) 16 T338C, V323Sc-Src (251-533) (c-Src “ES3) 17 GHM-T338C, V323Sc-Src (251-533) (GHM at N-terminal) (c-Src “ES3) 18 T338C, V323Dc-Src (251-533) (c-Src “ES4) 19 GHM-T338C, V323Dc-Src (251-533) (GHM at N-terminal) (c-Src “ES4) 2O T338C, V323Ec-Src (251-533) (c-Src “ES5') 21 HM-T338C, V323Ec-Src (251-533) (GHM at N-terminal) (c-Src “ES5') 22 T338C, V323H]c-Src (251-533) (c-Src “ES6) 23 GHM-T338C, V323H]c-Src (251-533) (GHM at N-terminal) (c-Src “ES6') 24 Kinases with gatekeeper Cys (Arabidopsis thaliana) 4587987 25 Kinases with gatekeeper Cys (Arabidopsis thaliana) 19424095 26 Kinases with gatekeeper Cys (Arabidopsis thaliana) 1785621 27 Kinases with gatekeeper Cys (Arabidopsis thaliana) 4678270 28 Kinases with gatekeeper Cys (Arabidopsis thaliana) 4678272 29 Kinases with gatekeeper Cys (Arabidopsis thaliana) 4678273 30 Kinases with gatekeeper Cys (Arabidopsis thaliana) 4678277 31 Kinases with gatekeeper Cys (Arabidopsis thaliana) 488.6274 32 Kinases with gatekeeper Cys (Arabidopsis thaliana) 3047095 33 Kinases with gatekeeper Cys (Arabidopsis thaliana) 334188021, 15238494 34 Kinases with gatekeeper Cys (Arabidopsis thaliana) 9294588 35 Kinases with gatekeeper Cys (Arabidopsis thaliana) 11120792 36 Kinases with gatekeeper Cys (Arabidopsis thaliana) 11120796 37 Kinases with gatekeeper Cys (Arabidopsis thaliana) 8777331 38 Kinases with gatekeeper Cys (Mus musculus) 7106391 39 Kinases with gatekeeper Cys (Carassius auratus) 1705720 40 Kinases with gatekeeper Cys (Capsicum annuum) 6648996 41 Kinases with gatekeeper Cys (Leishmania major) 7630151 42 Kinases with gatekeeper Cys (Homo sapiens) 5139689 43 Kinases with gatekeeper Cys (Trichomonas vaginalis) 48.6948 44 Kinases with gatekeeper Cys (Plasmodium falciparum) 254688446/3845 109 45 Kinases with gatekeeper Cys (Dictyostelium discoideum) 13509297 46 v-Src (Roussarcoma virus) 47 I338Xv-Src 48 I338Civ-Src 49 I338Tv-Src 50 I338Giv-Src 51 Artificial sequence (Substrate for methods of testing)
An exemplary DNA construct useful for the methods the art. An additional spacer (e.g., SEQ ID NO:2, residues described herein was synthesized (SEQID NO: 1). This con 21-22) may be present in expressed proteins, which spacers struct encodes the expressed protein set forth in SEQ ID 60 residues may occupy the N-terminal position(s) of the NO:2. The expressed protein includes a His (SEQID NO:78) expressed protein after protease cleavage (e.g., TEV protease tag sequence at the N-terminal, useful for purification of cleavage). Thus, it is understood that reference to “c-Src recombinantly expressed protein as known in the art. The (251-533)' and variants thereof herein contemplates expressed protein further includes a spacer sequence (i.e., expressed proteins having one or more amino acids at the DYDIPTT, (SEQID NO:79), SEQ ID NO:2 residues 7-13) 65 N-terminal which may result from the process of recombinant and a tobacco etch viral (TEV) protease site (i.e., ENLYFQG, protein production. For example, after the action of the TEV (SEQIDNO: 80), SEQID NO:2, residues 14-20) as known in protease on the protein of SEQID NO:2, the expressed c-Src US 9,353,116 B2 63 64 (251-533) protein may include the N-terminal tripeptide gopeptide sequence resulting from processing of the recom "GHM. It is understood that absent indication otherwise, the binant protein is set forth in SEQ ID NO:15. These doubly numbering of c-Src proteins and variants as discussed herein Substituted proteins are also known as the so-called c-Src follows the numbering of the full c-Src protein (SEQ ID “ES2 variant. NO:3). For example, full length c-Src (SEQ ID NO:3) con Further exemplary of this embodiment, there is provided tains 533 residues. Accordingly, residues 23-305 of SEQID TC, VSlc-Src(251-533) (SEQID NO:16), and the cor NO:2 correspond to residues 251-533 of SEQID NO:3. c-Src responding protein having an N-terminal oligopeptide (251-533) is expressly set forth in SEQ ID NO:4. A recom sequence resulting from processing of the recombinant pro binantly expressed and processed protein of c-Src (251-533), tein (SEQID NO:17). These doubly substituted proteins are as described above, having the N-terminal tripeptide "GHM’ 10 also known as the so-called c-Src “ES3 variant. is set forth in SEQID NO:5. In some embodiments, a c-Src variant is provided wherein Further exemplary of this embodiment, there is provided the residue at the position equivalent to Thr of c-Src (SEQ TC, V-Dic-Src(251-533) (SEQID NO:18), and the cor ID NO:3) is substituted with another amino acid. In some responding protein having an N-terminal oligopeptide embodiments, the Substituted amino acid is a naturally occur 15 sequence resulting from processing of the recombinant pro ring amino acid, as known in the art. Exemplary recombi tein (SEQID NO:19). These doubly substituted proteins are nantly expressed proteins having this Substitution are set forth also known as the so-called c-Src "E54” variant. in SEQID NO:6 and SEQID NO:7, wherein SEQ ID NO:7 Further exemplary of this embodiment, there is provided further includes the N-terminal tripeptide "GHM as TC, VEc-Src(251-533) (SEQID NO:20), and the cor described above. Similarly, in some embodiments, a V-Src responding protein having an N-terminal oligopeptide variant is provided wherein the residue at the position equiva sequence resulting from processing of the recombinant pro lent to Thr of v-Src (SEQ ID NO:46) is substituted with tein (SEQID NO:21). These doubly substituted proteins are another amino acid. An exemplary recombinantly expressed also known as the so-called c-Src “ES5' variant. protein having this substitution is set forth in SEQID NO:47. Further exemplary of this embodiment, there is provided Specific exemplary recombinantly expressed proteins having 25 TC, VHlc-Src(251-533) (SEQID NO:22), and the cor a C, T or G substitution at position 338 of v-Src (i.e., I338C responding protein having an N-terminal oligopeptide v-Src. I338Tv-Src. I338Giv-Src), are set forth in SEQ ID sequence resulting from processing of the recombinant pro NO: 48, SEQID NO:49 and SEQID NO:50, respectively. tein (SEQID NO:23). These doubly substituted proteins are In some embodiments, a protein kinase is provided having also known as the so-called c-Src “ES6' variant. a Thr to CyS Substitution at the position corresponding to 30 X. Methods residue 338 of c-Src (i.e., T338C substitution). The protein may be a fragment of full length c-Src. Recombinantly A. General expressed protein variants of c-Src (251-533) having a T338C In some embodiments, the present invention provides substitution (i.e., T338Cc-Src.(251-533)) are set forth in methods of determining the role of a kinase in a cell. In certain SEQID NO:8 and SEQID NO:9. It is understood that within 35 embodiments, the methods include determining the depen the context of protein descriptive names, bracketed (i.e., “I’) dence of transformed cells on aberrant oncogenic signaling entries denote Substitution(s), and that parenthetic entries by the EGFR kinase. In other embodiments, the determining after the protein name denote the corresponding residues of includes assaying inhibitor-induced conformational changes the fragment. For example, “T338Cc-Src(251-533)” refers of kinases. In other embodiments, the methods include elu to the fragment of c-Src from residue 251 to residue 533, 40 additionally having a Thr to Cys substitution at position 338 cidating the mechanisms of inhibitor-induced Akt hyperphos (c-Src numbering). These proteins are also known as “c-Src phorylation. In some embodiments, the methods include ES1’ proteins. It is further understood that, as customary in transactivation of RAF dimmers. the art, the term “XNNNY' refers to substitution of residue In some other embodiments, the present invention provides “X” at position “NNN’ with residue “Y” 45 a chemical genetic approach based on engineered shape In Some embodiments, a plurality of Substitutions of c-Src. complementarity between the kinase active site and a small or fragment thereof, are provided. For example, in some embodiments, a protein having double Substitutions at resi molecule inhibitor, which allows systematic discovery of an dues T and V* of c-Src is provided. In some embodi inhibitor for a particular kinase. In some embodiments, a ments, a protein having double substitutions at residues T. 50 conserved hydrophobic residue in the kinase active site and V of a fragment of c-Src (e.g., c-Src(251-533)) is known as the 'gatekeeper' is mutated to a small residue Such provided. See SEQ ID NO:10. In some embodiments, the as glycine or alanine to generate a uniquely targetable mutant fragment of c-Src includes an N-terminal oligopeptide kinase termed an analog-sensitive (AS) allele. sequence resulting from processing of the recombinant pro In certain other embodiments, the present invention pro tein as described above. See SEQID NO:11. 55 In some embodiments, there is provided a T338C substi vides methods of making engineered kinase which can be tution of c-Src, or fragment thereof (e.g., c-Src.(251-533), in targeted with sterically bulky analogs of natural kinase inhibi combination with a substitution at position 323. See SEQID tors, which are capable of occupying the enlarged engineered NO:12. In some embodiments, such doubly substituted frag kinase pocket (FIG. 1). In some embodiments, the methods ment of c-Src includes an N-terminal oligopeptide sequence 60 include wildtype kinases which may be resistant to inhibition resulting from processing of the recombinant protein as by the bulky analog as the result of a steric clash with natu described above. See SEQID NO:13. rally occurring gatekeeper residues (e.g. Met, Leu, Phe, Thr, In some embodiments, a T338C substitution of c-Src, or fragment thereof (e.g., c-Src.(251-533), in combination with a Gln and others). In yet other instances, the wild type kinases specific substitution at position 323 is provided. For example, 65 may not be resistant to inhibition by the bulky analog as the TC, VAlc-Src(251-533) is set forth in SEQID NO:14, result of a steric clash with naturally occurring gatekeeper and the corresponding protein having an N-terminal oli residues (e.g. Met, Leu, Phe, Thr, Gln and others). US 9,353,116 B2 65 66 B. Structure Activity Relationship (SAR) Analysis Table 3 shows compounds 9 and 13 exhibit time-dependent In order to determine the effects of modifying the group at inhibition against T338C c-Src. Compounds were preincu this position, a structure activity relationship (SAR) analysis bated with the enzyme prior to initializing the reaction with was performed on the pyrazolopyrimidine scaffold with a ATP benzyl-linked m-vinylsulfonamide (compounds 9, 15-17; XI. Methods of Inhibiting a Kinase Table 2). This analysis revealed that secondary alkyl groups In some other embodiments, the present invention provides a method of imparting to a kinase the capability of being such as isopropyl (9) and cyclopentyl (17) moieties elicited inhibited by a heterocyclic compound, comprising replacing optimal activity against T338C c-Src. Relative to isopropyl a gatekeeperamino acid residue within an ATPbinding site of substitution, tert-butyl (16) and methyl (15) derivatization 10 a kinase with a cysteine residue thereby forming a cysteine resulted in 4- and 21-fold drops in potency, respectively. Substituted kinase. Collectively, these results indicate that substitution at N1 In some embodiments, the present invention provides a can be used to modulate potency against T338C c-Src. IN method of inhibiting a recombinant kinase as set forth herein, Some instances, Michael acceptor-derivatized 4-anilino 15 comprising contacting the recombinant kinase with an effec quinazolines were synthesized and evaluated as inhibitors tive amount of an inhibitor, thereby inhibiting the recombi (compounds 18-20; Table 2). nant kinase. In some embodiments, the inhibitor is capable of In some embodiments, the ES kinase alleles should be forming a covalent bond to the cysteine at the gatekeeper useful for a host of other applications. For example, fluores amino acid position of the recombinant kinase. In some cently labeled versions of the inhibitors could be used to embodiments, the inhibitor is a compound as set forth herein, quantitatively probe the occupancy of kinase active sites to e.g. a compound of formulas I-XXIX. In some other embodi determine the percent activity required for signaling events. ments, the method further comprises determining a level of In some other embodiments, the present invention provides a inhibition for the recombinant kinase. In some embodiments, method for determining the properties of pseudokinases, for 25 the determining of said level of inhibition for the recombinant which there is no good readout of active site occupancy. In kinase comprises: determining an amount of enzymatic activ certain embodiments, the present invention sets forth the use ity of the recombinant kinase in the presence of the inhibitor; of irreversible inhibitors and allows for the validation of tar determining an amount of enzymatic activity of the recombi get specificity. 30 nant kinase in the absence of the inhibitor, and comparing the In some embodiments, the present invention provides amount of enzymatic activity of the recombinant kinase in the methods of evaluating the reversibility of inhibition of a presence of the inhibitor with the amount of enzymatic activ kinase as set forth herein. In some embodiments, an electro ity of the recombinant kinase in the absence of the inhibitor, philic inhibitors covalently interact with the cysteine gate thereby determining a level of inhibition for the recombinant keeper. In one instance, two compounds, 9 and 13, were 35 kinase. In some embodiments, the enzymatic activity is assayed accordingly. Both compounds inhibited T338C c-Src selected from phosphorylation of a non-specific protein tar in a time-dependent manner (Table 3). get, phosphorylation of a specific protein target, consumption In addition, when T338C c-Src was treated with either of ATP, or cell growth. inhibitor and purified by gel filtration and the inhibitory activ 40 In other embodiments, the present invention provides a ity against the kinase was retained. See FIG. 5. In contrast, in method as set forth herein wherein the recombinant kinase is the case of WT c-Src, inhibitory activity was lost after gel in a cell. In some embodiments, the methods set forth herein filtration. Importantly, inhibition by PP1, a reversible Src further comprise determining a function of the recombinant kinase in the cell, by: determining an amount of enzymatic inhibitor, was abrogated in the cases of both WT and T338C 45 c-Src following gel filtration (FIG.5). Full protein mass spec activity of the recombinant kinase in the presence of the trometry suggested specific labeling of T338C relative to WT inhibitor in the cell; determining an amount of enzymatic c-Src for 9 (FIG. 2). However, under similar conditions, an activity of the recombinant kinase in the absence of the inhibi adduct formation with 13 was not observed, possibly due to a tor in the cell; and comparing the amount of enzymatic activ reversible covalent interaction. The results suggest that cova 50 ity of the recombinant kinase in the presence of the inhibitor lent binding of the electrophilic inhibitors depend on the with the amount of enzymatic activity of the recombinant presence of a cysteine gatekeeper, i.e. T338C c-Src as elec kinase in the absence of the inhibitor, thereby determining a trophile-sensitive c-Src1 (c-Src-ES1). function of the recombinant kinase in the cell. In some embodiments, the enzymatic activity is selected from phos 55 TABLE 3 phorylation of a specific protein target. In other embodiments, the methods as set forth herein Compounds 9 and 13 exhibit time-dependent inhibition against include a recombinant kinase is selected from Src.: MOK; T338C c-Src. Compounds were preincubated with the enzyme prior to initializing the reaction with ATP. Sgk494; Lrrk-2; Yak/Yrk; SRPK1; CDK; DICTY-I; PAK/ 60 STE20; or Ctrl/DPYK1. In some embodiments, the recombi Preincubation Time (min nant kinase is Src. In some embodiments, the present invention provides a 2 2O 40 Compound T338C c-Src ICso (nM) method of inhibiting a Lrrk-2 kinase, comprising contacting the Lrrk-2 kinase with an effective amount of a Lrrk-2 inhibi 9 981 309 138 13 1281 318 136 65 tor, thereby inhibiting the recombinant Lrrk-2 kinase. A Lrrk-2 inhibitor is compound of Formula (XV) to (XXIX) including embodiments thereof. US 9,353,116 B2 67 68 XII. Methods of Treating ing administering to said patient a therapeutically effective In some embodiments, the present invention provides a amount of a Lrrk-2 inhibitor, thereby treating a Lrrk-2-asso method of treating a kinase-associated disease or condition, ciated disease or condition. In some embodiments, the dis in a patient in need thereof. The method includes administer ease or condition is a neurodegenerative disease selected ing to the patient a therapeutically effective amount of a from Parkinson's Disease. compound provided herein, thereby treating a kinase-associ In some embodiments, the methods further include the step ated disease or condition. In some embodiments, the com of allowing the cell to express the recombinant kinase. pound is a kinase inhibitor capable of forming a covalent XIII. Tables Relevant to the Methods and Assays Herein bond to the cysteine at the gatekeeper amino acid position of 10 the recombinant kinase. In some embodiments, the com TABLE 4 pound is a compound as set forth herein, e.g. a compound of Relative ka/K, for a series of c-Src variants formulas (I)-CXXIX) including embodiments thereof. In cer tain embodiments, the kinase-associated disease or condition c-Src Variant rel. k.ca/Kn is selective from cancer, immunological disorders, neurologi ES1 cal disorders, neurodegenerative disorders, infections, meta ES2 ES3 bolic diseases, Leishmania major, Zoonotic cutaneous leish ES4 maniasis, Plasmodium falciparum, malaria, Trichomonas ES5 vaginalis, and trichomiasis. In certain other embodiments, the ES6 cancer is selected from neoplasm or malignant tumors found in mammals; leukemia; carcinomas and sarcomas; cancer of Table 5a-5c show relative catalytic efficiency for T338C the brain, breast, cervix, colon, head and neck, liver, kidney, c-Src with second-site mutations (ES1=T338C: ES2=T338C/ lung, non-small cell lung, ovary, testicle, stomach, uterus; V323A; ES3=T338C/V323S, ES4=T338C/V323D; melanoma; mesothelioma; Medulloblastoma; Hodgkin’s 25 ES5=T338C/V323E: ES6=T338C/V323H). Data were fitted Disease, Non-Hodgkin’s Lymphoma; multiple myeloma; to the Michaelis-Menten equation and standard errors of the neuroblastoma, rhabdomyosarcoma; primary thrombocyto fits are reported. Data are unitless. sis; primary macroglobulinemia; primary brain tumors; Table 5 shows kinome-wide screening of a panel of inhibi malignant pancreatic insulanoma; malignant carcinoid; uri tors. Compounds were screened using the SelectScreenTM nary bladder cancer; premalignant skin lesions; lymphomas; 30 platform developed by LifeTechnologies. Zlyte (a, measures thyroid cancer; neuroblastoma; esophageal cancer, genitouri kinase activity), Adapta (b, measures kinase activity) and nary tract cancer; malignant hypercalcemia; endometrial can Lantha assays (c., measures ATP binding) were performed. cer; adrenal cortical cancer, neoplasms of the endocrine and Inhibition data are represented in a heat map format. exocrine pancreas; or prostate cancer. In yet other embodi 35 Table 6 shows comparison of selectivity of 13, 1NA-PP1 ments, the disease or condition is a neurodegenerative disease and 1 NM-PP1. All kinases for which >40% inhibition was selective from Parkinson's disease. observed in a kinome wide Zlyte screen (Life Technologies) In some other embodiments, the present invention also are shown. Legend for Tables 5-6: <40% inhibition (gray); provides a method of treating a Lrrk-2-associated disease or 40%-80% inhibition (white); 80% inhibition (diagonal condition, in a patient in need thereof, said method compris stripes). TABLE 5a
Conc Compound
ity ity ity ity Activity Km a Activity Km a
R1B (ALK4) Activity Km a
BK1 (GRK2) Activity Km a BK2 (GRK3) Activity Km a
(PKB alpha) Activity Km a KT2 (PKB beta) Activity Km a KT3 (PKB gamma) Activity Km a Activity Km a MPKA1 B1 G1 Activity Km a MPKA2AB1 G1 Activity Km a
URKA (Aurora A) ity Km app :::::::::::::::::::::::::::::::::::::::::::::
US 9,353,116 B2 71 72 TABLE 5a-continued
DYRK1A Activi DYRK1B Activi DYRK3 Activi DYRK4 Activi
EGFR (ErbB1) Activi EGFR (ErbB1) L858R Activi EGFR (ErbB1) L861Q Activi EGFR (ErbB1) T790M Activi EGFR (ErbB1) T790M L858R | Activi EPHA1 Activi EPHA2 Activi EPHA4 Activi EPHA5 Activi Activi Activi Activi Activi Activi
Activi Activi Activi Activi
(VEGFR1)
FR FYN US 9,353,116 B2 73 TABLE 5a-continued
GSK3A (GSK3 alpha) Activi GSK3B (GSK3 beta) Activi Activi HIPK1 (Myak) Activi HIPK2 Activi HIPK3 (YAK1) Activi HIPK4 Activi GF1R KBKB (IKK beta) KBKE (IKK epsilon) NSR (NSRR (IRR) RAK4
AK2 Activi AK2 JH1 JH2 Activi
w
A K C w KDR (VEGFR2) A CWE. KIT Activity KITT67OI Activity LCK Activity LTK (TYK1) Activity LYNA Activity Kmap LYNB Activity Kmap
MAP2K1 (MEK1) Activity 1OO K2 (MEK2) Activi MAP2K6 (MKK6) Activi MAP3K8 (COT) Activi MAP3K9 (MLK1) Activi MAP4K2 (GCK) Activi MAP4K4 (HGK) Activi MAP4K5 (KHS1) Activi US 9,353,116 B2 75 76 TABLE 5a-continued
MAPK1 (ERK2) Activi MAPK10 (JNK3) Activi MAPK11 (p38 beta) Activi MAPK12 (p38 gamma) Activi MAPK13 (p38 delta) Activi MAPK14 (p38 alpha) Activi MAPK14 (p38 alpha) Direct Activi MAPK3 (ERK1) Activi MAPK8 (JNK1) Activi MAPK9 (JNK2) Activi MAPKAPK2 Activi MAPKAPK3 Activi MAPKAPK5 (PRAK) Activi MARK1 (MARK) Activi
ity K ity
MERTK (cMER) Km app MET (cMet) Km app MET M1250T Km app
y
NEK2 NEK4 NEK6 NEK7 NEK9 NTRK1 (TRKA) NTRK2 (TRKB) NTRK3 (TRKC) ity US 9,353,116 B2 77 78 TABLE 5a-continued
PAK2 (PAK65) Activi PAK3 Activi PAK4 Activi PAK6 Activi PAK7 (KIAA1264) Activi PASK Activi PDGFRA (PDGFRalpha) Activi PDGFRAD842V Activi y PDGFRAT674I PDGFRA V561D PDGFRB (PDGFR beta) PDK1 PDK1 Direct
y
M1 Activi M2 Activi y KN1 (PRK1) Activi y LK1 Activi y LK2 Activi PLK3 Activi Activi
D KCB2 (PKC beta II) Activi KCD (PKC delta) Activi KCE (PKC epsilon) Activi KCG (PKC gamma) Activi D KCH (PKC eta) Activi KCI (PKC iota) Activi PRKCN (PKD3) Activi PRKCQ (PKC theta) Activi PRKCZ (PKCzeta) Activi PRKD1 (PKC mu) Activi
PRKD2 (PKD2) Activity Km app PRKG1 PRKG2 (PKG2) Activity Km app PRKX Activity Km app PTK2 (FAK) Activity Km app PTK2B (FAK2) Activity Km app PTK6 (Brk) Activity Km app US 9,353,116 B2 79 80 TABLE 5a-continued
RAF1 (cRAF) Y34ODY341D | Activi RET Ac W RETV804L Activi RETY791F Activi ROCK1 Activi ROCK2 Activi ROS1 Activi RPS6KA1 (RSK1) Activi RPS6KA2 (RSK3) Activi RPS6KA3 (RSK2) Activi RPS6KA4 (MSK2) Activi RPS6KA5 (MSK1) Activi RPS6KA6 (RSK4) Activi RPS6KB1 (p70S6K) Activi SGK (SGK1) Activi SGK2 Activi SGKL (SGK3) Activi
y
SRCN1 Activi SRMS (Srm) Activi SRPK1 Activi SRPK2 Activi STK22B (TSSK2) Activi STK22D (TSSK1) Activi STK23 (MSSK1) Activi STK24 (MST3) Activi STK25 (YSK1) Activi STK3 (MST2) Activi STK4 (MST1) Activi Activi Activi Activi TEK (Tie2) Activi
TYRO3 (RSE) Activi YES1 Activi ZAP70 Activi
US 9,353,116 B2 85 86 Table 6 Comparison of selectivity of 13, 1NA-PP1 and TABLE 7 1NM-PP1. All kinases for which >40% inhibition was c-Src variant ICsa (nM observed in a kinome wide Zlyte screen (Life Technologies) Compound ES1 ES2 ES3 are shown. 111 63 131 N/S
TABLE 6 10 N1 N-\
Kinase tested 1NM-PP1 l 2 N
BMX 15 N - BTK CSF1R (FMS) 3 CSNK1E (CK1 epsilon) 150 2O7 424 EGFR (ErbB1) L858R
EGFR (ErbB1) T790M EGFR (ErbB1) T790M L858R EPHA1 EPHA2 EPHA4 25 EPHA5 EPHA8 51 EPHB1 EPHB2 30 338 67 29 EPHB3 EPHB4 FGR 35
40 13
FRK (PTK5) IC50 values of a panel of electrophilic inhibitors against c-Src-ES variants with second-site FYN mutations (ES1 = T338C, ES2 = T338CV323A; ES3 = T338CV323S). HCK XIV. Kit LCK 45 In some other embodiments, the present invention provides LYNA a kit comprising, a recombinant kinase described herein (see LYNB section (V) above) or a nucleic acid described herein (See section IX) and instructions for using the kit. The instructions MAP4K4 (HGK) for using the kit describe the steps set forth in a method MAP4K5 (KHS1) 50 provided herein (see section X, XI and XII). MINK In some embodiments, the present invention provides a kit PRKACA (PKA) for testing for inhibition of kinase activity comprising a het PRKCN (PKD3) erocyclic compound, wherein the heterocyclic compound PRKD1 (PKC mu) 55 comprises two or more fused rings and an electrophilic Sub P RKD2 (PKD2) stituent, wherein at least one of the two or more fused rings RKGI comprises a nitrogen atom, and a cysteine Substituted kinase PTK6 (Brk) wherein a gatekeeper amino acid residue within an ATPbind RE ing site of the kinase is replaced with a cysteine residue. 60 RETY791F XV. Examples Chemical Synthesis. Reactions were performed in flame S RC dried flasks under argon with magnetic stirring. All Hand S RMS (Srm) 'C NMR spectra were recorded on a Varian Innova 400 YES1 65 spectrometer and referenced to solvent peaks. "H chemical shifts are reported in 6 (ppm) as S (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or br (broad). Low resolu US 9,353,116 B2 87 88 tion mass spectra (LC/ESI-MS) were recorded on a Waters A solution of tetrahydrofuran (20 mL), compound 22 (219 Micromass ZQ equipped with a Waters 2695 Separations mg, 0.817 mmol) and diisoproylethylamine (156 uL, 0.895 Module and a XTerra MS C18 3.5 mm column (Waters). mmol) was cooled to 0°C. Acryloyl chloride (67 uL, 0.828 RP-HPLC was performed on a Varian ProStar solvent deliv mmol) was added and the reaction was allowed to proceed for ery system equipped with a Zorbax300-SS C18 columnusing 1 hour and afterwards concentrated in vacuo. The residue was CHCN/HO/0.1% TFA (1-100% gradient) and monitoring dissolved in dichloromethane (20 mL) and washed with satu at 260 nm. rated Sodium bicarbonate (20 mL). The aqueous layer was extracted with dichloromethane (2x20 mL). The combined 10 organic layers were dried with MgSO filtered and concen EXAMPLE1 trated in vacuo. The product was purified by preparative RP HPLC and lyophilized (70 mg, 26% yield): 'H NMR (400 Preparation of 3-(3-aminophenyl)-1-isopropyl-1H MHz, DMSO) & 10.40 (s, 1H), 8.38 (s, 1H), 8.04 (s, 1H), 7.73 pyrazolo 3,4-dipyrimidin-4-amine (22) 15 (d. J=8.0, 1H), 7.52 (t, J=7.9, 1H), 7.39 (d. J=7.7, 1H), 6.47 (dd, J=17.0, 10.1, 1H), 6.29 (dd, J=17.0, 2.0, 1H), 5.80 (dd. J=10.1, 2.0, 1H), 5.11 (hept, J=6.6, 1H), 1.51 (d. J=6.7, 6H): This compound was prepared in a similar procedure to that 'C NMR (100 MHz, DMSO) & 163.47, 154.87, 151.90, used for (18}. 150.94, 144.73, 139.53, 132.54, 131.68, 129.88, 127.31, 123.42, 119.96, 119.22, 96.91, 48.80, 21.74; M+H" calcu EXAMPLE 2 lated for CH NO 323.1, found 323.5.
Preparation of 3-(4-aminophenyl)-1-isopropyl-1H EXAMPLE 4 pyrazolo 3,4-dipyrimidin-4-amine (23) 25 Preparation of N-(3-(4-amino-1-isopropyl-1H-pyra This compound was prepared in a similar procedure to that Zolo 3,4-dipyrimidin-3-yl)phenyl)ethenesulfonamide (3) used for (18}. 30
EXAMPLE 3
NH2 O Preparation of N-(3-(4-amino-1-isopropyl-1H-pyra 35 NH Cln/ Zolo 3,4-dipyrimidin-3-yl)phenyl)acrylamide (1) 2 / N-1\c N1 N-\ NEt3 0° C.
40 l 2 N DCM NH2 O N - NH2 C ---21 22 DIPEA 0°C. O 1N-\ THF 45 l 2 N Ost-AI NH
50 NH2 22 h N1 N O NH 55 4N NH2 2. N1 N 60 A solution of dichloromethane (2 mL), compound 22 (20 mg, 0.075 mmol) and triethylamine (11 uL, 0.079 mmol) was cooled to 0°C. 2-chloro-1-ethane sulfonyl chloride (7 uL. 0.067 mmol) was added and the reaction was allowed to 65 proceed for 1 hour prior to addition of saturated sodium bicarbonate (10 mL) and extraction with dichloromethane (3x10 mL). The combined organic layers were dried with US 9,353,116 B2 89 90 MgSO filtered and concentrated in vacuo. The product was & 10.57 (s, 1H), 8.41 (s, 1H), 7.95 (s, 1H), 7.67 (d. J=7.6, 1H), purified by preparative RP-HPLC and lyophilized (5.6 mg, 7.53 (t, J=7.9, 1H), 7.41 (d. J=7.7, 1H), 5.11 (hept, J=6.7, 1H), 23% yield): 'H NMR (400 MHz, DMSO) & 10.22 (s, 1H), 4.30 (s. 2H), 1.51 (d. J=6.7, 6H); 'C NMR (100 MHz, 8.31 (s, 1H), 7.49 (t, J–7.9, 1H), 7.45 (s, 1H), 7.38 (d. J=7.8, DMSO) & 165.06, 154.82, 151.89, 150.90, 144.68, 139.03, 1H), 7.27 (d. J=7.3, 1H), 6.86 (dd, J=16.4, 10.0, 1H), 6.17 (d. 132.62, 128.28, 123.81, 120.02, 119.24, 96.92, 48.84, 43.55, J=16.4, 1H), 6.08 (d. J=9.9, 1H), 5.08 (hept, J=6.7, 1H), 1.50 21.77; M+H" calculated for CHCINO 345.1, found (d. J=6.7, 6H). 'C NMR (100 MHz, DMSO) & 155.01, 3454. 151.96, 151.19, 144.29, 138.50, 136.32, 133.09, 130.22, 127.79, 123.73, 12009, 119.20, 96.94, 48.85, 21.73; M+H" 10 calculated for CHNOS 3.59.1, found 359.4. EXAMPLE 6
EXAMPLE 5 Preparation of N-(4-(4-amino-1-isopropyl-1H-pyra 15 Zolo 3,4-dipyrimidin-3-yl)phenyl)ethenesulfonamide (5) Preparation of N-(3-(4-amino-1-isopropyl-1H-pyra Zolo 3,4-dpyrimidin-3-yl)phenyl)-2-chloroaceta mide (6) NH2
NH2 O 25 Cls/ NH2 / N-1N NH2 C --- NEt3 0° C. N1 N N DIPEATHF 0°C. N \ DCM 30 l 2 N N - se 5 23 22 35 C K Y 40 -2' NH \S NH2 NH2 N1 N-\ 45 1N-\
50
A solution of THF (20 mL), compound 22 (200 mg, 0.75 55 mmol) and DIPEA (143 uL, 0.821 mmol) was cooled to 0°C. Chloroacetylchloride (54 uL, 0.67 mmol) was added and the A solution of dichloromethane (5 mL), compound 23 (45 reaction was allowed to proceed for 1 hour and afterwards mg, 0.168 mmol) and triethylamine (71 uL, 0.509 mmol) was concentrated in vacuo. The residue was dissolved in dichlo 60 cooled to 0°C. 2-chloro-1-ethane sulfonyl chloride (16 uL. romethane (20 mL) and washed with saturated sodium bicar 0.148 mmol) was added and the reaction was allowed to bonate (20 mL). The aqueous layer was extracted with dichlo proceed for 1 hour prior to addition of saturated sodium romethane (2x20 mL). The combined organic layers were bicarbonate (10 mL) and extraction with dichloromethane dried with MgSO, filtered and concentrated in vacuo. The 65 (2x10 mL). The combined organic layers were dried with product was purified by preparative RP-HPLC and lyo MgSO filtered and concentrated in vacuo. The product was philized (30.4 mg, 13% yield): 'H NMR (400 MHz, DMSO) purified by preparative RP-HPLC and lyophilized (8.7 mg, US 9,353,116 B2 91 92 16% yield): 'H NMR (400 MHz, DMSO) & 10.30 (s, 1H), EXAMPLE 8 8.36 (s, 1H), 7.60 (d. J=8.5, 2H), 7.31 (d. J=8.6, 2H), 6.86 (dd. J=16.4, 9.9, 1H), 6.21 (d. J=16.4, 1H), 6.09 (d. J=9.9, 1H), 5-amino-1-isopropyl-3-(3-nitrophenyl)-1H-pyrazole 5.08 (hept, J=6.7, 2H), 1.49 (d. J=6.7, 6H); 'C NMR (100 5 4-carbonitrile (26) MHz, DMSO) & 155.44, 152.03, 151.64, 14.4.22, 138.54, 136.17, 129.25, 127.98, 12741, 119.52, 96.95, 48.66, 21.75; NO M+H" calculated for CH NOS 359.1, found 359.5. isopropylhydrazine 10 NEt EXAMPLE 7 EtOH NC 21 o1 Preparation of 2-(methoxy(3-nitrophenyl)methylene)malononitrile 15 CN (25) 25
NO 1) malonoitrile, DIPEA, THF 2) dimethylsulfate -e-
C O 25 24 26
NO Compound 25 (5 g, 21.8 mmol), isopropylhydrazine 30 hydrochloride (2.41 g, 21.8 mmol) (purchased from Ryan Scientific) and triethylamine (6.40 mL, 46.0 mmol) were allowed to react in ethanol (145 mL) at room temperature for 1 hr. After concentrating the reaction mixture, it was purified NC 21N1 by Silica chromatography using a chloroform/methanol sol 35 vent system (methanol gradient increased with time from CN 0-10%). The relevant fractions were concentrated in vacuo to 25 yield a yellow powder (4.65 g, 79% yield): 'H NMR (400 MHz, DMSO) & 8.59 (s, 1H), 8.24 (m, 2H), 7.78 (t, J=8.2, 1H), 6.82 (br, 2H), 4.53 (hept, J =6.5, 1H), 1.36 (d.J=6.5, 6H). 40 'C NMR (100 MHz, DMSO) & 153.04, 148.76, 146.98, A solution of 3-nitrobenzoylchloride (25 g, 134 mmol), 133.97, 132.22, 131.19, 123.80, 120.37, 116.28, 70.74, malononitrile (9.74 g. 147 mmol) and THF (140 mL) was 48.64, 22.02; M+H" calculated for CHNO. 272.1, cooled to 0°C. DIPEA (59 mL, 335 mmol) was added drop found 272.3. wise and the reaction was allowed to warm to room tempera 45 ture and proceed for 2 hours. Afterwards, dimethylsulfate (38 EXAMPLE 9 mL, 399 mmol) was added and the temperature was raised to 70° C. for 4 hours. Next, the reaction mixture was brought to room temperature and allowed to proceed for an additional 12 Preparation of 5-amino-1-isopropyl-3-(3-nitrophe hours. EtOAc (200 mL) was added to the reaction mixture in 50 nyl)-1H-pyrazole-4-carboxamide (27) addition to brine (200 mL). The organic and aqueous layers were separated and the aqueous layer was extracted with EtOAc (4x25 mL). The combined organic layers were dried with MgSO and concentrated in vacuo. The material was 55 purified over a silica column using chloroform:hexane (90: 10) initially and eluted with pure chloroform. After concen trating the fractions containing the product, a yellow oil was triturated with diethyl ether to yield a solid (11.3 g, 37% yield): 'H NMR (400 MHz, DMSO) & 8.63 (s, 1H), 8.51 (d. J=7.7, 1H), 8.16 (d. J=7.7, 1H), 7.94 (t, J=7.8, 1H), 3.93 (s, 3H); 'CNMR (100 MHz, DMSO) & 183.66, 147.82, 135.23, 131.00, 129.55, 127.15, 124.08, 113.31, 111.87, 66.71, 61.87; M+H calculated for CHNO. 228.0, found 65 27 214.10 (product appears to hydrolyze during LC/MS analy sis). US 9,353,116 B2 93 94 Compound 26 (100 mg, 0.369 mmol) was added to con- (d. J–7.8, 1H),8.23 (d. J=8.1, 1H),8.13 (s, 1H), 7.75 (t, J=8.0, centrated sulfuric acid (1 mL) and heated to 65° C. for 3 1H), 5.06 (hept, J=6.6, 1H), 1.52 (d. J=6.7, 6H); C NMR hours. Afterwards, the reaction mixture was poured into ice (100 MHz, DMSO) & 157.77, 152.48, 148.16, 148.06, water and the pH was brought to 14 with 10 MNaOH. The s 143.85, 133.58, 129.95, 123.05, 12207, 103.02, 49.07, aqueous material was extracted several times with dichlo- 21.74; M+H" calculated for CHNO. 300.1, found romethane. The combined organic layers were dried over 3OO.O. MgSO filtered and concentrated to a solid (91 mg. 85% yield): 'H NMR (400 MHz, DMSO) & 8.32 (s, 1H), 8.21 (d, to J=8.2, 1H), 7.98 (d. J–7.7, 1H), 7.69 (t, J=8.0, 1H), 6.20 (br. EXAMPLE 11 2H), 4.51 (hept, 1H), 1.35 (d. J=6.5, 6H); 'C NMR (100 MHz, DMSO) & 166.17, 149.37, 147.65, 145.50, 135.63, 134.92, 129.69, 122.79, 122.43,95.43, 47.11, 21.50; M+H" is Preparation of 3-(1-isopropyl-1H-pyrazolo 3,4-d calculated for CHNO. 290.1, found 290.0. pyrimidin-3-yl)aniline (28)
EXAMPLE 10 2O
Preparation of 1-isopropyl-3-(3-nitrophenyl)-1H pyrazolo 3,4-dipyrimidin-4(5H)-one (28) 1) SOCl, cat. DMF 2) H2, Pd/C, NEt3, MeOHFEtOAC -e-
1N-\ - N N - 29
Compound 28 (2 g. 6.68 mmol) was mixed with thionyl 2- chloride (12.5 mL, 171 mmol) and ten drops of DMF and 55 heated to 80° C. for forty minutes. Afterwards, the reaction mixture was poured onto 300 mL of ice and the pH was adjusted to 8 with saturated sodium carbonate. The solution was extracted with dichloromethane (3x150 mL). The com bined organic layers were dried over MgSO and concen Compound 27 (1 g, 3.46 mmol) was added to formamide 60 trated in vacuo (2.04 g, yield 96%). The resulting solid (1 g, (1.167 mL, 29.3 mmol) and heated to 160° C. for 40 hours. 3.34 mmol) was dissolved in MeOH/EtOAc (30 mL/20 mL) Afterwards, the reaction mixture was allowed to cool to room and reacted with 10% Pd/C (540 mg) and triethylamine (466 temperature and diluted into ice cold water. The mixture was uL, 3.34 mmol) under a H, atmosphere for 24 hours. The filtered and a solid was collected (94.3 mg, 91% yield): 'H reaction mixture was filtered over celite and concentrated. NMR (400 MHz, DMSO) & 12.37 (s, 1H), 9.32 (s, 1H), 8.81 The material was resuspended in dichloromethane (100 mL), US 9,353,116 B2 95 96 which was washed with saturated sodium bicarbonate (2x100 EXAMPLE 13 mL) prior to drying with MgSO and concentrating in vacuo. The resulting Solid was purified over silica using a dichlo Preparation of N-(3-(1-isopropyl-1H-pyrazolo 3,4-d romethane/methanol (0-5%) solvent system (487 mg, 58% 5 pyrimidin-3-yl)phenyl)ethenesulfonamide (4) yield): "H NMR (400 MHz, DMSO) & 9.59 (s, 1H), 9.02 (s, 1H), 7.34 (s, 1H), 7.23 (d. J=7.6, 1H), 7.18 (t, J=7.7, 1H), 6.66 (d. J–7.7, 1H), 5.30 (br, 2H), 5.21 (kept, J=6.7, 1H), 1.56 (d. NH2 J=6.7, 6H); 'C NMR (100 MHz, DMSO) & 154.60, 153.03, 10 151.87, 149.28, 143.17, 132.14, 129.59, 114.65, 114.32, Cls/WN-1 nic 112.06, 111.85, 48.53, 21.72: M+H" calculated for N1 N-\ O CHNs 254.1, found 254.0. l N NEt 0°C. 15 4. 5- DCM EXAMPLE 12
29 O Preparation of N-(3-(1-isopropyl-1H-pyrazolo 3,4-d Ost-A pyrimidin-3-yl)phenyl)acrylamide (2) t
25 N O N 2 M --- l N N 30 N1N N DIPEA, O C. l N THF 2.
4. - 35 Compound 4 was prepared by the same procedure that was used for compound 3 (18% yield): "H NMR (400 MHz, 29 DMSO) & 10.17 (s, 1H), 9.61 (s, 1H), 9.08 (s, 1H), 7.90 (t, J=1.9, 1H), 7.82 (d. J=7.7, 1H), 7.49 (t, J=7.9, 1H), 7.29 (ddd, J=8.2, 2.2, 0.9, 1H), 6.87 (dd, J=16.4, 9.9, 1H), 6.17 (d. 40 J=16.4, 1H), 6.07 (d. J=9.9, 1H), 5.23 (hept, J=6.7, 1H), 1.57 h (d, J=6.7, 6H); 'C NMR (100 MHz, DMSO) & 15456, 152.69, 151.97, 141.98, 138.59, 136.20, 132.46, 130.16, NH 127.98, 122.09, 119.78, 117.64, 111.94, 48.89, 21.71; M+H calculated for CH-NOS 344.11, found 344.2. 45 N1 N EXAMPLE 1.4 DO4. N Preparation of N-(3-((4-amino-1-isopropyl-1H-pyra 50 Zolo 3,4-dipyrimidin-3-yl)methyl)phenyl)acrylamide (7)
55 Compound 2 was prepared by the same procedure that was used for compound 1 (49% yield): "H NMR (400 MHz, NH2 DMSO) & 10.36 (s, 1H), 9.66 (s, 1H), 9.08 (s, 1H), 8.41 (t, NH2 acrolyl chloride N \ DIPEA, O C. J=1.8, 1H), 7.84 (m, 2H), 7.52 (t, J=7.9, 1H), 6.48 (dd, J=17.0, He 10.0, 1H), 6.32 (dd, J=17.0, 2.0, 1H), 5.81 (dd, J=10.0, 2.0, M THF 1H), 5.24 (kept, J=6.6, 1H), 1.58 (d. J=6.7, 6H); C NMR sells (100 MHz, DMSO) & 163.39, 154.53, 152.74, 151.97, 142.36, 139.75, 132.03, 131.77, 129.75, 127.24, 121.84, 65 119.79, 117.31, 111.98, 48.81, 21.73; M+H" calculated for CHNO308.1, found 308.6. US 9,353,116 B2 97 98 -continued A solution of dichloromethane (20 mL), compound 30 (190 mg. 0.673 mmol) and triethylamine (188 uL. 1.35 NH2 O mmol) was cooled to 0°C. 2-chloro-1-ethane sulfonyl chlo 5 ride (70 uL, 0.670 mmol) was added and the reaction was N1 N N NH -- allowed to proceed for 1 hour prior to addition of saturated N sodium bicarbonate (20 mL) and extraction with dichlo 2N/ romethane (3x20 mL). The combined organic layers were dried with MgSO filtered and concentrated in vacuo. The 10 product was purified by preparative RP-HPLC and lyo - philized (14 mg, 6% yield): 'H NMR (400 MHz, DMSO) & 9.95 (s, 1H), 8.31 (s, 1H), 7.21 (t, J=8.1, 1H), 6.97 (m, 3H), 6.69 (dd, J=16.4, 9.9, 1H), 5.97 (d. J=16.5, 1H), 5.93 (d. A solution of THF (20 mL), compound 30 (200 mg. 0.708 mmol) (prepared as in Dar et al. Dar, 2008 #18) and N,N- 15 J=9.9, 1H), 5.00 (hept, J–6.7, 1H), 4.38 (s. 2H), 1.46 (d. diisopropylethylamine (136 uL, 0.781 mmol) was cooled 0° J=6.7, 6H); C NMR (100 MHz, DMSO) & 154.31, 151.46, C., at which point freshly distilled acryloyl chloride (52 uL. 150.23, 144.31, 137.91, 136.26, 136.16, 129.25, 127.42, 0.642 mmol) was added. After one hour, the reaction mixture 123.79, 119.26, 117.52, 97.91, 48.59, 32.81, 21.70; M+H" was concentrated in vacuo. The material was resuspended in calculated for C7HoNOS 373.1, found 373.4. dichloromethane (20 mL), which was washed with saturated Sodium bicarbonate (20 mL). The aqueous layer was extracted with dichloromethane (3x20 mL) and the organic EXAMPLE 16 layers were subsequently combined, dried over MgSO, fil tered and concentrated to a solid. The material was purified by RP-HPLC and lyophilized to a white powder (113 mg, 47% 25 Preparation of N-(4-((4-amino-1-isopropyl-1H-pyra yield): 'H NMR (400 MHz, DMSO) 10.06 (s, 1H), 8.33 (s, Zolo 3,4-dipyrimidin-3-yl)methyl)phenyl)acrylamide 1H), 7.54 (s, 1H), 7.50 (d. J=8.1, 1H), 7.24 (t, J=7.9, 1H), 6.96 (d. J–7.7, 1H), 6.40 (dd, J=17.0, 10.1, 1H), 6.22 (dd, J=17.0, (8) 2.0, 1H), 5.72 (dd, J=10.1, 1.9, 1H), 4.99 (hept, J=6.5, 1H), 4.40 (s. 2H), 1.46 (d. J=6.7, 6 H); C NMR (100 MHz, 30 DMSO) & 163.06, 153.94, 151.30, 149.68, 144.69, 139.16, 139.08, 131.86, 128.82, 126.78, 123.59, 119.34, 117.51, 97.90, 48.74, 33.03, 21.70; M+H calculated for CHNO337.2, found 337.4. 35 NH2 NH2 EXAMPLE 1.5 acrolyl chloride Preparation of N-(3-((4-amino-1-isopropyl-1H-pyra N \ DIPEA, O C. Zolo 3,4-dpyrimidin-3-yl)methyl)phenyl)ethene 40 2 M THF sulfonamide (9) N - 31 O 45 NH2 Cln/ Snu1\ NH / C h N N y 2 NEt 0°C. 2 50 NH N - DCM NH2
30 N1N 55 2NNY. NH2 \ N1 N N H -29 N S. 60 2N/ A solution of THF (20 mL), compound 31 (200 mg, 0.708 65 mmol) (prepared as in Dar et al. Dar, 2008 #18) and N,N- diisopropylethylamine (136 uL, 0.781 mmol) was cooled 0° C., at which point freshly distilled acryloyl chloride (52 uL. US 9,353,116 B2 99 100 0.642 mmol) was added. After one hour, the reaction mixture dried with MgSO, filtered and concentrated in vacuo. The was concentrated in vacuo. The material was resuspended in product was purified by preparative RP-HPLC and lyo dichloromethane (20 mL), which was washed with saturated philized (14 mg, 6% yield): 'H NMR (400 MHz, DMSO) & Sodium bicarbonate (20 mL). The aqueous layer was 9.91 (s, 1H), 8.30 (s, 1H), 7.16 (d. J=8.3, 2H), 7.05 (d. J=8.4, extracted with dichloromethane (3x20 mL) and the organic 2H), 6.74 (dd, J=16.4, 9.9, 1H), 6.07 (d. J=16.4, 1H), 6.00 (d. layers were subsequently combined, dried over MgSO, fil J=10.0, 1H), 5.03-4.94 (m. 1H), 4.34 (s. 2H), 1.44 (d. J=6.6, tered and concentrated to a solid. The material was purified by 6H); 'C NMR (100 MHz, DMSO) & 1540, 1514, 149.8, RP-HPLC and lyophilized to a white powder (78 mg, 30% 144.7, 136.3, 136.0, 133.9, 129.3, 127.5, 120.0, 97.9, 48.6, 10 32.3, 21.7: M+H" calculated for CHNOS 373.1, yield): 'H NMR (400 MHz, DMSO) & 10.09 (s, 1H), 8.31 (s, found 373.4. 1H), 7.57 (d.J=8.5, 2H), 7.18 (d. J=8.5, 2H), 6.41 (dd, J=17.0, 10.1, 1H), 6.22 (dd, J=17.0, 2.1, 1H), 5.73 (dd, J=10.1, 2.1, 1H), 5.00 (hept, J=6.7, 1H), 4.36 (s.3H), 1.44 (d. J=6.7, 6H): EXAMPLE 1.8 'C NMR (100 MHz, DMSO) & 1630, 153.9, 1513, 149.6, 15 145.1, 137.4, 133.4, 1319, 128.7, 126.7, 119.5, 97.8, 48.7, 32.5, 21.6; M+H" calculated for CHNO337.2, found Preparation of N-(3-((4-amino-1-isopropyl-1H-pyra 337.4. Zolo 3,4-dipyrimidin-3-yl)methyl)phenyl)ethane sulfonamide (11) EXAMPLE 17
Preparation of N-(4-((4-amino-1-isopropyl-1H-pyra 25 Zolo 3,4-dpyrimidin-3-yl)methyl)phenyl)ethene O sulfonamide (10) NH2 s/ NH / n-1 30 N N N 2 C N DIPEA 0°C. NH, MO 2 -- NH2 Ns/ N-1\c N - THF 35 N \ NEt30 C. 30 M DCM 4. - 40 31 NH K N- 2O N1 N N H O 45 2 N Ost-AI NH N - NH2 11 50 N1 N 2 Y. A solution of tetrahydrofuran (5 mL), compound 30 (50 N - 55 mg, 0.177 mmol) and diisopropylethylamine (34 uL, 0.195 mmol) was cooled to 0° C. Ethanesulfonylchloride (15 uL. 10 0.159 mmol) was added and the reaction was allowed to proceed for one hour. After one hour, the reaction mixture was concentrated in vacuo. The material was resuspended in A solution of dichloromethane (20 mL), compound 31 60 dichloromethane (10 mL), which was washed with saturated (200 mg. 0.708 mmol) and triethylamine (200 uL. 1.43 Sodium bicarbonate (10 mL). The aqueous layer was mmol) was cooled to 0°C. 2-chloro-1-ethane sulfonyl chlo extracted with dichloromethane (3x10 mL) and the organic ride (70 uL, 0.670 mmol) was added and the reaction was layers were subsequently combined, dried over MgSO, fil allowed to proceed for 1 hour prior to addition of saturated 65 tered and concentrated to a solid. The combined organic sodium bicarbonate (20 mL) and extraction with dichlo layers were dried with MgSO filtered and concentrated in romethane (3x20 mL). The combined organic layers were vacuo. The product was purified by preparative RP-HPLC US 9,353,116 B2 101 102 and lyophilized (25 mg, 38% yield): 'H NMR (400 MHz, 6H); 'C NMR (100 MHz, DMSO) & 164.5, 1540, 151.3, DMSO) 89.72 (s, 1H), 8.34 (s, 1H), 7.23 (t, J=7.8, 1H), 7.09 150.0, 144.6, 139.2, 138.6, 128.9, 124.0, 119.3, 117.5, 97.9, (s, 1H), 7.03 (d, J =7.9, 1H), 6.98 (d. J=7.6, 1H), 5.01 (hept, 48.7, 43.6, 33.0, 21.7: M+H" calculated for CHCINO J=6.7, 1H), 4.40 (s. 2H), 3.01 (q, J=7.3, 2H), 1.46 (d. J=6.7, 359.1, found 359.2. 6H), 1.13 (t, J=7.3, 3H); 'C NMR (100 MHz, DMSO) & 5 154.11, 151.38, 150.04, 144.45, 139.67, 138.54, 129.28, 123.66, 119.19, 117.45, 97.89, 48.57, 44.89, 32.84, 21.68, EXAMPLE 20 7.90; M+H" calculated for CHNOS 375.1, found 375.6. 10 Preparation of EXAMPLE19 2-(methoxy(3-bromobenzyl)methylene)malononitrile (33) 15 Preparation of N-(3-((4-amino-1-isopropyl-1H-pyra Zolo 3,4-dpyrimidin-3-yl)methyl)phenyl)-2-chloro acetamide (12) 1) oxalyl chloride, DCM 2) malononitrile, DIPEA,THF 2) dimethylsulfate Br Hibs
O HO O
NH2 C --- 25 32 NH, DIPEA 0° C. N1 N-\ -e-THF l 2 N Br 30 N J NC 30 33 35
NH2 O A solution of 3-bromoacetyl acetic acid (5 g, 23.3 mmol) 40 was mixed with oxalyl chloride (10 mL, 121 mmol) in dichlo -- romethane (75 mL) at room temperature for 30 minutes and 2 N N N then concentrated in vacuo. To the resulting solid was added malononitrile (1.69 g, 25.6 mmol) and THF (25 mL). After cooling to 0°C. DIPEA (10.1 mL, 58.1 mmol) was added 45 dropwise and the reaction was allowed to warm to room temperature and proceed for 2 hours. Afterwards, dimethyl sulfate (6.60 mL, 69.3 mmol) was added and the temperature A solution of tetrahydrofuran (10 mL), compound 30 (100 was raised to 70° C. for 4 hours. Next, the reaction mixture mg, 0.354 mmol) and diisopropylethylamine (68 uL, 0.390 50 was brought to room temperature and allowed to proceed for mmol) was cooled to 0°C. Chloroacetylchloride (25.4 uL. an additional 12 hours. EtOAc (50 mL) was added to the 0.313 mmol) was added and the reaction was allowed to reaction mixture in addition to brine (50 mL). The organic and proceed for one hour. After one hour, the reaction mixture was aqueous layers were separated and the aqueous layer was concentrated in vacuo. The material was resuspended in 55 extracted with EtOAc (4x25 mL). The combined organic dichloromethane (10 mL), which was washed with saturated layers were dried with MgSO and concentrated in vacuo. Sodium bicarbonate (10 mL). The aqueous layer was The material was purified over a silica column using chloro extracted with dichloromethane (3x10 mL) and the organic form:hexane (90:10) initially and eluted with pure chloro layers were subsequently combined, dried over MgSO, fil form. After concentrating the fractions containing the prod tered and concentrated to a solid. The combined organic 60 uct, an amber oil was triturated with diethyl ether to yield a layers were dried with MgSO filtered and concentrated in solid (2.057 g., 32% yield): 'H NMR (400 MHz, DMSO) & vacuo. The product was purified by preparative RP-HPLC 7.56 (m, 2H), 7.38 (t, J=7.7, 1H), 7.31 (d. J=7.7, 1H), 4.19 (s. and lyophilized (35 mg, 31% yield): 'H NMR (400 MHz, 2H), 4.01 (s, 3H); C NMR (100 MHz, DMSO) & 187.96, DMSO) & 10.22 (s, 1H), 8.32 (s, 1H), 7.46 (s, 1H), 7.41 (d. 65 135.22, 131.33, 131.18, 130.77, 127.18, 122.32, 113.75, J=8.2, 1H), 7.25 (t, J =7.8, 1H), 6.99 (d. J=7.6, 1H), 5.00 112.03, 65.62, 60.00, 35.60; M+H calculated for (hept, J=6.7, 1H), 4.40 (s. 2H), 4.20 (s. 2H), 1.46 (d, J =6.7, CHBrNO 274.9, 276.9 (50:50), found 274.8, 276.5. US 9,353,116 B2 103 104 EXAMPLE 21 -continued
Preparation of 5-amino-3-(3-bromobenzyl)-1-isopro NH2 pyl-1H-pyrazole-4-carbonitrile (34) N1 N Br 2 Y. isopropylhydrazine NEt3 10 -e- N - Br EtOH 35 NC a\ 15 CN Compound 34 (6.09 g, 19.1 mmol) was added to forma 33 mide (26.6 mL, 668 mmol) and heated to 160° C. for 27 hours. Afterwards, the reaction mixture was allowed to cool to room temperature and diluted into ice cold water (50 mL). A vis cous material was filtered and dissolved in EtOAc. This solu tion was washed with brine and concentrated in vacuo (6.23g. 91% yield): 'H NMR (400 MHz, DMSO) & 8.13 (s, 1H), 7.49 (s, 1H), 7.37 (dt, J=7.5, 1.7, 1H), 7.22 (m, 2H), 4.95 (hept, J=6.6, 1H), 4.38 (s. 2H), 1.43 (d. J=6.7, 6H); 'C NMR (100 25 MHz, DMSO) & 157.90, 155.39, 153.22, 142.01, 141.86, 131.16, 130.52, 129.00, 127.40, 121.56, 98.39, 47.76, 32.66, 21.71: M+H" calculated for CHBrNs 346.0, 348.0 found 346.0: 348.0. 30 Compound 33 (3.124 g, 11.3 mmol), isopropylhydrazine hydrochloride (1.27 g, 11.5 mmol) (purchased from Ryan EXAMPLE 23 Scientific) and triethylamine (6.40 mL, 46.0 mmol) were allowed to react in ethanol (75 mL) at room temperature for 1 hr. After concentrating the reaction mixture, it was purified by 35 Preparation of 3-(3-acetylbenzyl)-1-isopropyl-1H silica chromatography using a chloroform/methanol solvent pyrazolo 3,4-dipyrimidin-4-amine (14) system (methanol gradient increased with time from 0-10%). The relevant fractions were concentrated in vacuo to yield a yellow powder (3.15 g, 87% yield): 'H NMR (400 MHz, DMSO) & 7.41 (m, 2H), 7.26 (td, J=7.7, 1.2, 1H), 7.21 (dt, 40 J–7.7, 1.3, 1H), 6.50 (s. 2H), 4.38 (hept, J=6.5, 1H), 3.81 (s, 2H), 1.27 (d. J=6.5, 6H); C NMR (100 MHz, DMSO) & NH l 151.01, 150.15, 141.24, 131.05, 130.53, 129.16, 127.45, Br 1No SnBus 121.55, 115.16, 71.77, 47.24, 32.88, 21.35: M+H" calcu N N Pd(PPh3)4 45 N -e- lated for CHBrN, 319.0, 321.0 found 318.9: 321.0. 2 M Toluene EXAMPLE 22 N - Preparation of 3-(3-bromobenzyl)-1-isopropyl-1H 50 34 pyrazolo 3,4-dipyrimidin-4-amine NH2
N O 55 N 2 Y. N - formamide 60 160° C. 14
Anhydrous toluene (10 mL) was degassed prior to addition of 34 (3.5 g, 10.1 mmol) Tributyl(1-ethoxyvinyl)tin (4.081 65 mL, 12.1 mmol), tetrakis(triphenylphosphine) palladium (1.169 g, 10 mol%) and heating to 120°C. After 16 hours, the reaction mixture was concentrated in vacuo. Next, a THF/1M US 9,353,116 B2 105 106 HCl solution (33 mL/10 mL) was added to the brownish 132.65, 128.85, 127.74, 126.52, 109.01, 83.67, 49.08, 33.51, material and the reaction was allowed to proceed at room 27.19, 26.58, 21.63; M+H" calculated for CHNO temperature for 12 hours. Afterwards, EtOAc (175 mL) was 510.2, found 510.1. added to the mixture, which was washed with saturated 5 sodium bicarbonate (700 mL) and extracted with 1 M HCl EXAMPLE 25 (2x525 mL). The pH was adjusted to a value of 13 and the mixture was extracted with EtOAc (2x525 mL). The organic layers were dried with sodium sulfate and concentrated in Preparation of 1-(3-((4-amino-1-isopropyl-1H-pyra vacuo. The material was purified by silica chromatography Zolo 3,4-dpyrimidin-3-yl)methyl)phenyl)-2-fluoro using a chloroform/methanol solvent system (methanol gra 10 ethanone (13) dient increased with time from 0-8%). The relevant fractions were concentrated in vacuo to yield a solid (2.035 g. 65% yield): "H NMR (400 MHz, DMSO) & 8.13 (s, 1H), 7.91 (s, 1H), 7.79 (d. J–7.5, 1H), 7.48 (d. J=7.7, 1H), 7.43 (t, J=7.6, 15 1) LHMDS, 1H), 4.97 (hept, J=6.7, 1H), 4.46 (s.2H), 2.53 (s.3H), 1.44 (d. N-fluorobenzenesul J=6.7, 6H); 'C NMR (100 MHz, DMSO) & 19780, 157.96, finimide, THF 155.40, 153.24, 142.22, 139.80, 136.84, 133.15, 128.75, O 2) TFA, DCM 128.10, 126.32, 98.40, 47.71, 32.94, 26.70, 21.77; M+H" -e- calculated for C2HNO 310.1, found 310.0.
EXAMPLE 24
Preparation of 3-(3-acetylbenzyl)-1-isopropyl-1H 25 pyrazolo 3,4-dipyrimidin-4-di-t-butoxycarbonyl amine (14)
30
NH 2 (Boc)2O, DMAP O He N1 N-\ THF 35 Compound 35 (253 mg, 0.496 mmol) was mixed with anhydrous THF (2 mL) and cooled to -78°C., whereupon 1.0 M LHMDS in THF (0.645 mL, 0.645 mmol) was added dropwise via Syringe and allowed to react for 15 minutes. N-fluorobenzenesulfinimide (250 mg. 0.794 mmol) in THF (2 mL) was then added dropwise and the reaction mixture was allowed to come to room temperature over 30 minutes. The reaction mixture was cooled to -78°C. and saturated ammo nium chloride (100 mL) was added dropwise. The reaction mixture was extracted with EtOAc (70 mL), and the resulting organic layer was washed with Saturated Sodium bicarbonate (1x70 mL) and brine (1x70 mL). The organic layer was concentrated in vacuo to give a yellow oil. The resulting product was purified over a silica column (hexane/ethyl acetate solvent system) and fractions containing the monof luorinated product and unreacted material (35) were pooled Compound 14, di-tert-butyldicarbonate and dimethylami 55 (they were inseparable). This combined mixture (93 mg) was reacted with TFA (1.5 mL) in DCM for 5 hours at room nopyridine were mixed and allowed to react for 3 hours at temperature and then concentrated in vacuo. The Boc-depro room temperature. Afterwards, the reaction mixture was tected material was then resuspended in EtOAc (30 mL) and diluted with EtOAC and washed with 1 MHC1 and brine. The washed with saturated sodium bicarbonate (1x30 mL) and organic solution was dried with MgSO and concentrated in 60 brine (1x30 mL). After concentrating the organic layer, the vacuo. The material was purified over a silica column using a monofluorinated product was purified by preparative TLC hexane/ethyl acetate solvent system (1.834g, 55% yield): 'H using a 8% MeOH/CHC1 solvent system (22 mg, 14% yield): NMR (400 MHz, DMSO) & 8.92 (s, 1H), 7.81 (d. J=7.9, 1H), H NMR (400 MHz, DMSO) 89.91 (s, 1H), 8.30 (s, 1H), 7.16 7.77 (s, 1H), 7.41 (t, J=7.7, 1H), 7.29 (d. J=7.7, 1H), 5.20 (d. J=8.3, 2H), 7.05 (d. J=8.4, 2H), 6.74 (dd, J=16.4, 9.9, 1H), (hept, J–6.7, 1H), 4.25 (s. 2H), 2.53 (s, 3H), 1.55 (d. J=6.7, 65 6.07 (d. J=16.4, 1H), 6.00 (d. J=10.0, 1H), 5.03-4.94 (m, 1H), 6H), 1.28 (s, 18H). 'C NMR (100 MHz, DMSO) & 197.42, 4.34 (s. 2H), 1.44 (d. J=6.6, 6H); 'C NMR (100 MHz, 154.90, 154.28, 153.19, 149.83, 141.77, 138.25, 136.88, DMSO) & 1540, 1514, 149.8, 144.7, 136.3, 136.0, 133.9, US 9,353,116 B2 107 108 129.3, 127.5, 120.0, 97.9, 48.6, 32.3, 21.7; M+H" calcu -continued lated for CHFNO328.1, found 328.1. NH2 O EXAMPLE 26 Y20 N1 N-\ N Preparation of N-(3-((4-amino-1-methyl-1H-pyra N S. Zolo 3,4-dpyrimidin-3-yl)methyl)phenyl)ethene 2N/
sulfonamide (15) 10
NH s/ 15 Compound 37 was prepared by the method of Dar et al. 2 c/ N-1 no {Dar, 2008 #18. Compound 16 was prepared by the same method used for compound 9 (10% yield): 'H NMR (400 N1 N N NH2 NEt3 MHz, DMSO) & 9.95 (s, 1H), 8.30 (s, 1H), 7.21 (t, J=8.1, 1H), N DCM 6.97 (m,3H), 6.69 (dd, J=16.4.9.9, 1H), 5.94 (m, 2H), 4.36 (s, 2N/ 2H), 1.71 (s, 9H); 'C NMR (100 MHz, DMSO) & 154.5, N 152.3, 149.6, 142.5, 139.6, 137.9, 136.2, 129.1, 127.4,123.8, 36 119.1, 117.4, 99.1, 60.2, 32.8, 28.8: M--H* calculated for C.HNOS 387.2, found 387.5.
25 NH2 K EXAMPLE 28 -'29 N1 N-\ H N Preparation of N-(3-((4-amino-1-cyclopentyl-1H N S. pyrazolo 3,4-dipyrimidin-3-yl)methyl)phenyl)ethe 2N/ 30 N nesulfonamide (17) 15
35 O Compound 36 was prepared by the method of Dar et al. NH2 s/ {Dar, 2008 #18. Compound 15 was prepared by the same NH Cl N-1N method used for compound 9 (22% yield): 'H NMR (400 2
MHz, DMSO) & 9.94 (s, 1H), 8.36 (s, 1H), 7.21 (t, J=7.8, 1H), -- 6.98 (m,3H), 6.71 (dd, J=16.4, 9.9, 1H), 6.03 (d. J=16.4, 1H), 40 C2N/ \ NEt3DCM 5.97 (d. J=9.9, 1H), 4.36 (s. 2H), 3.91 (s.3H); 'CNMR (100 N N MHz, DMSO) & 1542, 152.3, 150.4, 144.6, 139.4, 137.9, 136.2, 129.2, 127.6, 124.0, 119.4, 117.5, 97.8, 33.7, 32.8; M+H calculated for CHNOS 345.1, found 345.4. 45 38 EXAMPLE 27 NH2 K Preparation of N-(3-((4-amino-1-tert-butyl-1H-pyra N1 N N H -'29 Zolo 3,4-dpyrimidin-3-yl)methyl)phenyl)ethene N S. sulfonamide (16) 2 N N
55 O NH2 On4. NH C N-1\e 2 N N N NEt3 60 Compound 38 was prepared by the method of Dar et al. -> {Dar, 2008 #18. Compound 17 was prepared by the same 2N/ DCM method used for compound 9 (18% yield): 'H NMR (400 MHz, DMSO) & 9.95 (s, 1H), 8.37 (d. J=6.8, 1H), 7.21 (t, J=8.0, 1H), 6.98 (m, 3H), 6.69 (dd, J=16.4, 9.9, 1H), 5.98 (d. 65 J=16.4, 1H), 5.93 (d. J=9.9, 1H), 5.18 (t, J=7.2, 1H), 4.38 (s, 2H), 2.08 (m, 2H), 1.98 (m, 2H), 1.88 (m, 2H), 1.68 (m, 2H): 'C NMR (100 MHz, DMSO) & 153.7, 1517, 149.6, 144.7, US 9,353,116 B2 109 110 139.5, 137.9, 136.2, 129.1, 127.4, 123.8, 119.2, 117.5, 97.9, -continued 57.2, 32.8, 31.9, 24.3; M+H" calculated for CHNOS 399.2, found 399.4. HN |Clu--- EXAMPLE 29 H 19 NN Preparation of N1-(6,7-dimethoxyquinazolin-4-yl) benzene-1,3-diamine 10 No e 18
NH2 A solution of THF (7 mL), compound 40 (75 mg, 0.253 C 15 mmol) and 1N,N-diisopropylethylamine (96 uL, 0.551 O mmol) was cooled 0° C., at which point freshly distilled 1. n N acryloyl chloride (19 uI, 0.230 mmol) was added. After one hour, the reaction mixture was concentrated in vacuo. The 2 -e-NH2 No N iPr0H material was resuspended in dichloromethane (10 mL), 39 which was washed with saturated sodium bicarbonate (10 mL). The aqueous layer was extracted with dichloromethane (2x10 mL) and the organic layers were Subsequently com bined, dried over MgSO filtered and concentrated to a solid. HN NH2 25 The material was purified by RP-HPLC and lyophilized to a 1.O NN powder (40 mg, 50% yield): 'H NMR (400 MHz, DMSO) & 10.81 (br. 1H), 10.31 (s, 1H), 8.78 (s, 1H), 8.17 (s, 1H), 8.06 2 (s, 1H), 7.41 (m, 3H), 7.25 (s, 1H), 6.47 (dd, J=17.0, 10.1, No N 1H), 6.28 (dd, J=17.0, 19, 1H), 5.79 (dd, J=10.1, 19, 1H), 40 30 4.00 (s, 6H); 'CNMR (100 MHz, DMSO) & 163.32, 158.05, 156.18, 150.11, 149.32, 139.48, 137.32, 131.73, 129.02, Compound 39 was prepared by a previously described 127.18, 119.88, 117.10, 115.49, 107.30, 103.23, 100.68, method Perera, 2008 #110. Compound 39 (300 mg, 1.34 56.57, 56.45; M+H" calculated for CHNO. 351.1, mmol) and 1,3-phenylenediamine (1.78 g. 16.5 mmol) were 35 found 351.4. heated to 90° C. in isopropanol and allowed to react for 1.5 hours, after which the reaction was brought to room tempera ture. The resulting green, Solid product was collected by EXAMPLE 31 filtration and washed with cold isopropanol (173 mg. 44% 40 yield): 'H NMR (400 MHz, DMSO) & 11.01 (br. 1H), 8.72 (s, Preparation of N-(3-(6,7-dimethoxyquinazolin-4- 1H), 8.24 (s, 1H), 7.36 (s, 1H), 7.15 (t, J=8.0, 1H), 7.00 (s, ylamino)phenyl)ethenesulfonamide (19) 1H), 6.93 (d. J=7.9, 1H), 6.62 (d. J=8.0, 1H), 3.99 (s.3H), 3.97 (s.3H); CNMR (100 MHz, DMSO) & 157.85, 155.85, 149.92, 149.21, 137.73, 129.08, 113.52, 112.92, 110.99, 45 107.35, 103.60, 100.86, 56.73, 56.33; M+H" calculated for CHNO. 297.1, found 297.4. O
EXAMPLE 30 HN NH2 / C 50 O NEt 0°C. 1. NN DCM Preparation of N-(3-(6,7-dimethoxyquinazolin-4- ylamino)phenyl)acrylamide (18) No N2 55 40
O O \e HN N 1 \- 60 HN NH2 --- O O DIPEA 0°C. 1. NN 1. NN THF 2 No N 2 No N 65 19 US 9,353,116 B2 111 112 A solution of dichloromethane (10 mL), compound 40 (97 113.97, 107.62, 103.05, 102.79, 56.49, 56.10; M+H" cal mg, 0.327 mmol) and triethylamine (138 uL, 0.989 mmol) culated for CHNO. 297.1, found 297.4. was cooled to 0°C. 2-chloro-1-ethane sulfonyl chloride (30 uL, 0.287 mmol) was added and the reaction was allowed to EXAMPLE 33 proceed for 1 hour prior to removal of a greenish precipitate Preparation of N-(4-(6,7-dimethoxyquinazolin-4- and addition of saturated sodium bicarbonate (10 mL) and ylamino)phenyl)ethenesulfonamide (20) extraction with dichloromethane (2x10 mL). The combined organic layers were dried with MgSO filtered and concen trated in vacuo. The product (organic layers and green pre 10 cipitate combined) was purified by preparative RP-HPLC and lyophilized (40 mg. 36% yield): 'H NMR (400MHz, DMSO) NH2 Cln/ & 10.71 (br. 1H), 10.20 (s, 1H), 8.76 (s, 1H), 8.02 (s, 1H), 7.52 / N-1\c (s, 1H), 7.39 (m, 2H), 7.25 (s, 1H), 7.06 (m, 1H), 6.82 (dd. 15 HN NEt3 0° C. He J=16.4, 9.9, 1H), 6.18 (d. J=16.4, 1H), 6.10 (d. J=9.9, 1H), O DCM 3.99 (s, 6H); CNMR (100 MHz, DMSO) & 157.94, 156.12, 1 NN 150.06, 149.43, 138.29, 137.85, 136.06, 129.40, 128.11, 119.92, 117.13, 115.32, 107.41, 103.15, 100.96, 56.55, No N2 56.42; M+H" calculated for CHNOS 387.1, found 41 387.5. o- / NH EXAMPLE 32 25
HN Preparation of N1-(6,7-dimethoxyquinazolin-4-yl) O benzene-1,4-diamine (41) 30 1. NN No N 2 2O C HN 35
O A solution of dichloromethane (10 mL), compound 41 1. n N NH2 (100 mg, 0.337 mmol) and triethylamine (141 uL. 1.02 e iPr0H mmol) was cooled to 0°C. 2-chloro-1-ethane sulfonyl chlo No N 40 ride (32 uL, 0.304 mmol) was added and the reaction was allowed to proceed for 1 hour prior to removal of a precipitate 39 and addition of saturated sodium bicarbonate (10 mL) and NH2 extraction with dichloromethane (2x10 mL). The combined organic layers were dried with MgSO, filtered and concen 45 trated in vacuo. The product was purified by preparative RP HN HPLC and lyophilized (24 mg, 20% yield): 'H NMR (400 O MHz, DMSO) & 10.81 (br. 1H), 10.13 (s, 1H), 8.75 (s, 1H), 1. NN 8.02 (s, 1H), 7.58 (d. J=8.9, 2H), 7.25 (m, 3H), 6.83 (dd, J=16.4, 10.0, 1H), 6.15 (d. J=16.5, 1H), 6.07 (d. J=10.0, 1H), No N2 50 3.99 (s.3H), 3.98 (s, 3H); C NMR (100 MHz, DMSO) & 41 157.93, 156.16, 150.10, 149.26, 136.23, 135.75, 132.76, 127.82, 125.54, 120.00, 107.16, 103.16, 100.51, 56.55, 56.45; M+H" calculated for CHNOS 387.1, found 3874. Compound 39 was prepared by a previously described 55 method Perera, 2008 #110. Compound 39 (600 mg, 2.68 EXAMPLE 34 mmol) and 1,3-phenylenediamine (3.57 g., 33 mmol) were Gel Filtration and Kinetic Assays heated to 90° C. in isopropanol and allowed to react for 1.5 hours, after which the reaction was brought to room tempera 60 Inhibitors (final concentration 23 uM) were incubated with ture. The resulting solid product was collected by filtration c-Src variants (final concentration 11.5 M) in kinase reac tion buffer (50 mM Tris pH 8, 100 mMNaCl, 1 mM DTT, 5% and washed with cold isopropanol (775 mg, 98% yield): 'H glycerol, 5% DMSO) for 25 minutes at room temperature. NMR (400 MHz, DMSO) & 10.38 (br. 1H), 8.59 (s.1H), 8.00 The solutions (2.6 mL total) were then passed over PD10 (s, 1H), 7.28 (d. J=8.6, 2H), 7.20 (s, 1H), 6.79 (s.1H), 6.66 (d. 65 desalting columns (GE Healthcare) using the kinase reaction J=8.6, 2H), 3.96 (s, 6H); 'C NMR (100 MHz, DMSO) & buffer for elution. Src concentrations were calculated using 157.31, 155.07, 150.47, 149.39, 145.88, 126.53, 125.38, the extinction coefficient for c-Src (51.140 mM'cm) US 9,353,116 B2 113 114 {Seeliger, 2005 #100. Kinase assays were performed as SelectScreen(R) Kinase Profiling Service (Life Technologies described in the experimental section at a final enzyme con Corporation, Madison, Wis.). Compounds were assayed at 1 centration of 10 nM. uMatan ATP concentration equal to the ATP Km.app for the Protein labeling conditions: Kinase labeling reactions assay following the detailed procedures described in the were performed by incubating 30 uL quantities of c-Src vari ants (in 50 mM Tris pH 8, 100 mM. NaCl, 1 mM DTT, 5% SelectScreen R. Customer Protocol and Assay Conditions glycerol) with two equivalents of inhibitor in DMSO (final documents located at www.invitrogen.com/kinaseprofiling. DMSO concentration=2.4%). The covalent labeling reaction In order to identify potential off-targets, a panel of the was quenched by removing 4 LL and adding it to 31 L of electrophilic inhibitors that showed inhibition of c-Src-ES1 0.1% formic acid. The sample was then analyzed by ES1-oa against 307 kinases was screened (Table 5). Compounds that TOF mass spectrometry. 10 were profiled include, but are not limited to, 3, 4, 9, 13, and 20. Excluding 3, all of the compounds had relatively few EXAMPLE 35 off-target effects. The exocyclic amine mimicsN6 of ATP and plays an important role in a hydrogen bonding interaction Crystallization and Data Collection for c-Src-ES-9 with the hinge region of kinases. Several of the kinases for 15 which >80% inhibition was achieved with vinylsulfonamide The c-Src-ES variant was prepared and purified as based inhibitors was observed are those with exposed cys described above, run over a 0.22 Lum PVDF centrifugal filter teines near the active site (e.g. EGFR, HER4, BTK, BMX, and diluted to 1.5-3 mg/ml in 50 mM Tris (pH 8.0), 100 mM TXK). The fluoromethylketone-type compound, 13, had a NaCl, 5% (v/v) glycerol, 1 mM DTT. Compound 9 was clean profile against kinases in the panel. The present inven freshly dissolved in DMSO and added to the protein solutions (1.5-3 equivalents). After 1.5 hours of incubation at room tion provides one of the most selective chemical genetic temperature, the reaction mixtures were spun at 10,000 rpm kinase inhibitor reported to date. and the Supernatants were collected. Hanging drop crystalli Zation conditions were set up by mixing 1:1 protein and EXAMPLE 37 precipitation solutions (100 mM MES (pH 6.5), 50 mM 25 NaOAc, 4-8% PEG 4000). After 24-48 hours at room tem Site Directed Mutagenesis perature, thin plate-like crystals were observed. Crystals were cryoprotected in the crystallization solution Supplemented The T338C mutation was introduced to a pBT-28 vector with 25% glycerol and stored in liquid nitrogen prior to containing a hexahistidine-tagged Src construct using stan obtaining diffraction data at beamline 8.2.2 (wavelength of dard site directed mutagenesis methods. The protein was 1.0088 nm, nitrogen gas stream at 100 K) at the Berkeley Lab 30 produced in E. coli BL21 DE3 cells containing YopH phos Advanced Light Source. Data was processed with HKL2000 phatase and GroEL. The cells were grown in Terrific Broth (HKL Research, Inc.) and Phenix software Adams, #108). containing (kanamycin, 50 mg/mL/streptomycin, 50 Crystal structure of c-Src-ES1 with 9. In order to eluci mg/mL). Cells were grown to an ODoo of 1.2 at 37°C., and date the binding mode for a kinase with a cysteine gatekeeper cooled for 1 hour with shaking at 18°C. Afterwards, the cells and an irreversible inhibitor, an X-ray crystal structure of the 35 were induced for 16 hat 18°C. with 0.2 mMIPTG. Cells were catalytic domain of c-Src-ES1 (residues 251-533) bound to 9 harvested and resuspended in 50 mM Tris (pH 8.0), 500 mM was solved (FIG.2). Co-crystallization through incubation of NaCl, 5% glycerol, 25 mM imidazole for purification over c-Src-ES1 with 9 was performed using hanging-drop vapor Ni-NTA resin. diffusion. The complex was solved by molecular replacement and, contained two molecules in the crystallographic asym 40 EXAMPLE 38 metric unit of the P1 space group. The structure was refined to 2.2A and exhibited electron density for 9 covalently bound to Expression and Purification of c-Src Variants Cys338. Poor electron density was observed near the N-ter minus (residues 251-256) and in flexible regions of the kinase such as the glycine-rich loop (residues 275-278) and the Hexahistidine-tagged recombinant chicken c-Src (residues activation segment (residues 407-424). However, the DFG 45 251-533) was prepared in a similar manner to that described motif at the beginning of the activation segment (residues in Seeliger MA, et al. Protein Sci. 14 (12):3135-3139 with the 404-406) was clearly resolved and was in the conformation modifications used by Blair JA, et al. (2007) Structure associated with an active kinase (DFG-in). guided development of affinity probes for tyrosine kinases The binding mode of 9 (a vinylsulfonamidefunctionalized using chemical genetics. Nat Chem Biol 3(4): 229-238. The compound) with c-Src-ES1 is related to Type I/2 kinase inhi 50 hexahistidine tag was removed with AcTeV protease (Invitro bition. Like Type I inhibitors, Type I/2 inhibitors bind the gen) and concentrations were determined spectrophotometri active conformation of the kinase (DFG-in) and engage in a cally at 280 nm using an extinction coefficient of 52.370 series of hydrogen bonds in the hinge region. Type I/2 are M'cm'. All mutations were introduced using the site-di similar to Type II inhibitors in that they occupy the pocket rected mutagenesis protocol of Zheng L, Baumann U. Rey situated behind the gatekeeper and hydrogen bond to the 55 mond J L (2004) An efficient one-step site-directed and site carboxylate of the conserved glutamate on the OC-helix and saturation mutagenesis protocol. Nucleic Acids Res 32(14): backbone amide of the DFGaspartate (FIG. 2C). The hydro e115. Protein aliquots were stored at -80° C. in 50 mM Tris gen bonds afforded by the tetrahedral arrangement of the (pH 8), 100 mM NaCl, 1 mM DTT and 5% glycerol. sulfone may contribute to the increased potency of 9 relative to 7, which contains an acrylamide. 60 EXAMPLE 39 EXAMPLE 36 In vitro Kinase Assays Kinome-Wide Profiling of Inhibitors In vitro kinase assays for c-Src variants were performed in 65 50 mM Tris (pH 8.0), 10 mM MgCl, and 1 mg/mL BSA. The percent inhibition results in FIG. were generated with When obtaining kinetic parameters (k, K) kinase and biochemical enzymatic kinase assays using the peptide substrate (IYGEFKKK) (SEQID NO:51) concentra US 9,353,116 B2 115 116 tions were 2 nM and 500 uM, respectively, while ATP con phosphate, 2 mM EGTA, 0.8 mM sodium orthovanadate, 0.4 centrations ranged from 2000-0.655 uM. Addition of nonra mM dithiothreitol, 0.1 mM ATP (supplemented with PATP dioactive ATP supplemented with PATP (3,000 Ci/mmol. (3,000 Ci/mmol. NEN)), and 20 ug of a protein substrate NEN) was used to initiate kinase reactions. Time points were (myelin basic protein) at a pH of 8. Inhibitors were used at a selected such that product formation never exceeded 10%. concentration of 1 uM (final DMSO concentration, 2%). Reactions were quenched by spotting 3 ul quantities onto Myelin basic protein phosphorylation was analyzed by SDS phosphocellulose sheets (P81, Whatman). Afterwards, the PAGE and autoradiography. MOK levels were evaluated by Western blot using HRP conjugated ANTI-FLAG M2 anti sheets were washed 3x5 minutes in 0.5% phosphoric acid and body at a 1000:1 dilution. dried. Radioactivity was measured by phosphorimaging and 10 recorded on a Typhoon fluorescence imager (Molecular EXAMPLE 42 Dynamics). Data were plotted as rate (min) versus ATP concentration and fitted to the Michaelis-Menten equation, Inhibition Assays with v-Src Transformed NIH-3T3 V-(k)SI/(K+SI), using Kaleidagraph software (Syn Cells 15 ergy) to extract kinetic parameters. When obtaining ICs val NIH-3T3 cell lines transformed with v-Src gatekeeper ues for the inhibitors, 2% (v/v) DMSO was included in kinase variants were prepared using a procedure similar to that in reactions. In these cases ATP, peptide, and enzyme concen Bishop AC, et al. (1998) Design of allele-specific inhibitors trations were 15 nM, 100 LM and 5 nM, respectively, while to probe protein kinase signaling. Curr Biol 8(5):257-266. inhibitor concentrations ranged from 10,000-0.610 nM. In all Cells were grown to 60-90% confluence in DMEM supple cases, a ten-minute preincubation step between the kinase and mented with fetal bovine serum (10%), penicillin G (100 the inhibitor preceded addition of ATP and a fifteen-minute units/ml) and streptomycin sulfate (100 g/ml) (PenStrep, reaction. The data was fitted to a sigmoidal dose-response UCSF Cell Culture Facility) prior to treatment with kinase curve using Prism 4.0c (GraphPad Software) to obtain ICso inhibitors dissolved in DMSO (final DMSO concentration, values. 25 0.5%). Following 1 hour of incubation with inhibitors at 37° C., cells were harvested in lysis buffer (50 mM Tris (pH 7.4), EXAMPLE 40 300 mM NaCl, 5 mM EDTA, 1% triton, 0.02% NaN, 1x complete mini protease inhibitor (Roche), 1 mM PMSF, Crystallization and Data Collection for c-Src-ES1-9 1xPHOS-stop (Roche), 0.02 uM microcystin, 2 mM sodium 30 orthovanadate), normalized for concentration and analyzed The c-Src-ES1 variant was prepared and purified as by Western blot for global phosphotyrosine levels (4G10. described above, run over a 0.22 Lum PVDF centrifugal filter Millipore, 1:1000). Levels of B-actin (B-actin Antibody, Cell and diluted to 1.5-3 mg/ml in 50 mM Tris (pH 8.0), 100 mM Signaling, 1:1000) and v-Src (Src 32G6 rabbit mAb, Cell NaCl, 5% (v/v) glycerol, 1 mM DTT. Compound 9 was 35 Signaling, 1:1000) were ascertained by Western blot. freshly dissolved in DMSO and added to the protein solutions EXAMPLE 43 (1.5-3 equivalents). After 1.5 hours of incubation at room temperature, the reaction mixtures were spun at 10,000 rpm Blockade of v-Src-ES1 Activity in Cells with and the Supernatants were collected. Hanging drop crystalli Electrophilic Inhibitors Zation conditions were set up by mixing 1:1 protein and 40 precipitation solutions (100 mM MES (pH 6.5), 50 mM I338C (v-Src-ES1, SEQ ID NO:48), I338T (SEQ ID NaOAc, 4-8% PEG 4000). After 24-48 hours at room tem NO:49), I338G (v-Src-AS1, SEQID NO:50) and WT v-Src perature, thin plate-like crystals were observed. Crystals were transformed NIH-3T3 cell lines were generated. Unlike cryoprotected in the crystallization solution Supplemented c-Src, v-Src is constitutively active and harbors an isoleucine with 25% glycerol and stored in liquid nitrogen prior to 45 gatekeeper. The I338TV-Src variant was generated for con sistency with the in vitro c-Src studies. For each cell line, obtaining diffraction data at beamline 8.2.2 (wavelength of global levels of phosphotyrosine were analyzed (FIG. 7). 1.0088 nm, nitrogen gas stream at 100 K) at the Berkeley Lab Importantly, the V-Src-ES1 variant was an excellent mimic of Advanced Light Source. Data was processed with HKL2000 WTV-Src, while the activity of v-Src-AS1 (the mutant used in (HKL Research, Inc.) and Phenix software. 50 previous chemical genetic studies) was markedly diminished EXAMPLE 41 as judged by whole cellphosphotyrosine levels. To determine whether the electrophilic inhibitors function in cells, the Immunoprecipitation and Assay of MOK v-Src-ES1 and I338T V-Src-transformed cell lines were 55 treated with 9 and 13. Both 9 and 13 inhibited v-Src-ES1 in a A plasmid encoding full-length mouse MOK with a dose-dependent manner, while isosteric control compounds FLAG-tag for expression in mammalian cells was used. (11 and 14) showed no activity (FIG. 4). Furthermore, neither Immunoprecipitation from CosT cells was performed using a 9 nor 13 inhibited I338Tv-Src even at levels as high as 10 uM. procedure similar to Miyata Y. Akashi M. Nishida E (1999). Collectively, these results suggest that a kinase with a cys Molecular cloning and characterization of a novel member of 60 teine gatekeeper can be selectively targeted in cells. the MAP kinase superfamily. Genes Cells 4(5):299-309, with the following modification: MOK was directly immunopre EXAMPLE 44 cipitated on ANTI-FLAG M2 magnetic beads (Sigma-Ald Second-site Mutations to Modulate Inhibitor Potency rich). Kinase assays were performed directly on-bead for 60 65 minutes in 30 uL quantities of 50 mM Tris-HCl, 16 mM The design strategy, in order to determine whether further MOPS, 150 mM. NaCl, 10 mM MgCl2, 20 mM B-glycero kinase engineering could enhance potency, was to either US 9,353,116 B2 117 118 enhance the reactivity of the cysteine by installing nearby assays are further provided in FIG. 11. In the table below, hydrophilic/basic residues or to slightly enlarge the area Cmpd 3-vs-Q refers to compound 19. around the cysteine to allow for additional rotational freedom to facilitate optimized thiol-electrophile attack geometry. TABLE 9a
Accordingly, mutations at Val323—a residue within 4 A of 5 the gatekeeper (FIG. 2C) in c-Src were introduced in com bination with T338C. Of the double mutants, V323A/T338C Kinase tested (c-Src-ES2) and V323S/T338C (c-Src-ES3) had substantial activity, while V323D/T338C (c-Src-ES4), V323E/T338C 10 ACVR1 (ALK2) (c-Src-ES5), and V323H/T338C (c-Src-ES6) were inactive (Table 4). Enhanced inhibitor potency was observed for both BMPR1A (ALK3) c-Src-ES2 and c-Src-ES3 when treated with 13 (Table 3). In CAMKK1 (CAMKKA) the latter case, a 12-fold improvement was noted relative to 15 CAMKK2 (CaMKK beta) c-Src-ES1. Interestingly, the potencies of 3 and 9 were not DK8/cyclin C modulated appreciably upon introduction of the additional DK9/cyclin K mutations. Taken together, these results indicate that the judi cious placement of a secondary mutation can be an effective means for modulating inhibitor potency for an ES allele, but 2O that this strategy needs to be evaluated on a case-by-case basis.
EXAMPLE 45 25
Evaluating the Use of a Cysteine Gatekeeper Kinase 30 K1 (MEK1) S218D A recombinant wild type (WT) and T338C c-Src was gen erated. The recombinant wildtype (WT) and the T338C c-Src K3 (MEK3) were assayed for kinase activity, see Table 8. The k value K6 (MKK6) S207E for T338C c-Src (183 min") closely approximated that of 35 WT (159 min)and was ~3.5-fold greater than that of c-Src- K10 (MLK2) AS1 (51.9 min'). The T338C c-Src variant also recapitulated K11 (MLK3) WT in affinity for ATP as determined by the Michaelis con- K14 (NIK) stant (K) values (21.9 LM vs. 31.9 LM), while c-Src-AS1 K2 (MEKK2) (87.5 M) exhibited ~4-fold loss relative to T338C c-Src. " K3 (MEKK3) These effects translate to a 14-fold improvement in catalytic K5 (ASK1) efficiency (k/K) for T338C c-Src in relation to c-Src-AS1. K7IMAP3K7IP1 K1-TAB1) TABLE 8 45 MKNK2 (MNK2) Kinetic parameters for c-Src variants. Values were determined MLCK (MLCK2) by fitting data to the Michaelis-Menten equation. Standard errors associated with the fits are reported. MYLK (MLCK) c-Src. kca, Kn: ATP kca?Kn 50 Variant (min) (IM) (min' M') WT 1594 31.93.O 4.99 O.40 T338C 1833 21.9 1.7 834 - 0.57 STK16 (PKL12) AS1 51.91.9 87.5 - 12.6 O.S92 O.O72 STK17A (DRAK1) 55 Kinetic measurements reveal that in the case of c-Src, the TAOK3 (JIK) ES1 variant is a mimic of wild type activity. Furthermore, the TEC ES1 variant of v-Src is also a mimic of the wild type, which contains a particularly hydrophobic (isoleucine) gatekeeper. TGFBR1 (ALK5) See Tables 9a-9c following. Results on the selectivity of 60 TNK2 (ACK) compound 19 in the Invitrogen SelectScreen(R) Kinase Assay are provided in Tables 9a-9c following. Table 9a is the Lan thaScreenTM heat map., Table 9b is the AdaptaR) heat map. and Table 9c is the Z-lyteTM heat map. Legend for Tables as 9a-9c: <40% inhibition (gray); 40%–80% inhibition (white); 80% inhibition (diagonal stripes). Selected results in the US 9,353,116 B2 119 120 TABLE 9b TABLE 9c-continued
Activity 5
AXL Activity CAMK1 (CaMK1) Activity 1OO BLK Activity CDK7/cyclin H/MNAT1 Activity Km app CDK9/cyclin T1 Activity Kim app BMX Activity CHUK (IKK alpha) Activity Kim app 10 BRAF Activity DAPK1 Activity Kim app BRAF V599E Activity GSG2 (Haspin) Activity Km app BRSK1 (SAD1) Activity RAK1 Activity Km app BTK Activity
LRRK2 G2019S Activity Km app Activity NUAK1 (ARK5) Activity Km app PI4KA (PI4K alpha) Activity 10 Activity PI4KB (PI4K beta) Activity Kim app Activity PIK3C2A (PI3K-C2 Activity Kim app 2O Activity
alpha) PIK3C2B (PI3K-C2 beta) Activity 1OO Activity PIK3C3 (hVPS34) Activity Kim app CDK1/cyclin B Activity PIK3CA/PIK3R1 (p110 Activity Kim app 25 CDK2/cyclin A Activity alpha p85 alpha) CDK5?p25 ity PIK3CD/PIK3R1 (p110 Activity Kim app delta?p85 alpha) PIK3CG (p110 gamma) Activity Km app SPHK1 Activity Km app 30 SPHK2 Activity 1OO
35
40 TABLE 9c
CLK3 CSF1R (FMS) CSK CSNK1A1 (CK1 alpha 1) CSNK1 D (CK1 delta) CSNK1E (CK1 epsilon) CSNK1 G1 (CK1 gamma 1 )
C SNK1G2 (CK1 gamma Activity R1B (ALK4) 2 ) DRBK1 (GRK2) 55
DRBK2 (GRK3) CSNK1G3SN (C ActiviCW
KT1 (PKB alpha) 3)
KT2 (PKB beta) KT3 (PKB gamma) 60 CSNK2A2 (CK2 alpha2) Activi Kim a DAPK3 (ZIPK) Activi
MPKA2AB1 G1 ty Kim a DNA-PK Activi URKA (Aurora A) ty Kim a 65 DYRK1A Activi US 9,353,116 B2
TABLE 9c-continued TABLE 9c-continued
DYRK1B ity NSRR (IRR) Activity Kim app DYRK3 ity 5 RAK4 Activity Kim app DYRK4 ity TK Activity Km app EEF2K ity 2 AK1 Activity Km app EGFR (ErbB1) Activity % AK2 Activity Km app EGFR (ErbB1) L858R Activity 69 10 AK2 JH1 JH2 Activity Km app EGFR (ErbB1) L861Q Activity AK2 JH1 JH2W617F Activity Kim app EGFR (ErbB1) T790M Activity AK3 Activity Km app EGFR (ErbB1) T790M Activity KDR (VEGFR2) Activity Km app L858R 15 KIT Activity Km app
EPHA2 Activity LCK Activity Km app EPHA4 Activity LTK (TYK1) Activity Km app EPHA5 ivity 2O LYNA Activity Km app ivity ity ity ity
25 y BB2 ((HER2 ) y LYNB Activity Km app BB4 (HER4) ity MAP2K1 (MEK1) Activity 100 ity 30 MAP2K2 (MEK2) Activity 100 W y MAP2K6 (MKK6) Activity 100 FGFR1 Activity MAP3K8 (COT) Activity 100
FGFR3 K65OE MAP4K4 (HGK) Activity Km app
FGR Activity MAPK1 (ERK2) Activity Kim app FLT1 (VEGFR1) Activity Kim app 40 MAPK10 (JNK3) Activity 100 FLT3 Activity Km app MAPK11 (p38 beta) Activity Km app FLT3 D835Y Activity Kim app MAPK12 (p38 gamma) Activity Kim app FLT4 (VEGFR3) Activity Km app MAPK13 (p38 delta) Activity Km app FRAP1 (mTOR) Activity Km app 45 FRK (PTK5) Activity Km app FYN Activity Km app GRK4 Activity Km app GRKS Activity Km app GRK6 Activity Kim app MAPK14 (p38 alpha) Activity 100 MAPK14 (p38 alpha) Activity Km app GRK7 Activity Km app Direc GSK3A (GSK3 alpha) Activity Km app MAPK3 (ERK1) Activity Km app GSK3B (GSK3 beta) Activity Km app 55 MAPK8 (JNK1) Activity 100 HCK Activity Km app MAPK9 (JNK2) Activity 100 HIPK1 (Myak) Activity Km app MAPKAPK2 Activity Km app HIPK2 Activity Km app MAPKAPK3 Activity Km app HIPK3 (YAK1) Activity Km app 60 MAPKAPK5 (PRAK) Activity Km app HIPK4 Activity Km app MARK1 (MARK) Activity Km app IGF1R Activity Km app MARK2 Activity Km app IKBKB (IKK beta) Activity Km app MARK3 Activity Km app IKBKE (IKK epsilon) Activity Km app 65 MARK4 Activity Km app INSR Activity Km app US 9,353,116 B2 123 124 TABLE 9c-continued TABLE 9c-continued
MATK (HYL) Activity Kim app Activity MELK Activity Kim app 5 Activity MERTK(cMER) Activity Kim app Activity MET (cMet) Activity Kim app Activity MET M1250T Activity Kim app Activity MINK1 Activity Km app 10 PRKCD (PKC delta) Activity MKNK1 (MNK1) Activity Km app PRKCE (PKC epsilon) Activity MST1R (RON) Activity Kim app PRKCG (PKC gamma) Activity MST4 Activity Km app PRKCH (PKCeta) Activity MUSK Activity Kim app 15 PRKCI (PKCiota) Activity MYLK2 (skMLCK) Activity Kim app PRKCN (PKD3) Activity NEK1 Activity Kim app PRKCQ (PKC theta) Activity NEK2 Activity Kim app PRKCZ (PKC zeta) Activity NEK4 Activity Km app 2O PRKD1 (PKC mu) ity NEK6 Activity Km app
25
NEK7 PRKD2 (PKD2) Activity
NEK9 PRKG1 Activity
NTRK1 (TRKA) 30 PRKG2 (PKG2) Activity RK2 (TRKB) PRKX Activity RK3 (TRKC) PTK2 (FAK) Activity
PAK1 ity PTK2B (FAK2) Activity
PAK2 (PAK65) ity 35 PTK6 (Brk) Activity
PAK3 ity RAF1 (cRAF) Y340D Activity
PAK4 ity Y341D
PAK6 ity RET
PAK7 (KIAA1264) ity RET V804L 40 PASK ity RETY791F
PDGFRA (PDGFR ity ROCK1 alpha)
45
ROCK2 ity PDGFRAD842V Activi y ROS1 ity PDGFRAT674I ity RPS6KA1 (RSK1) ity DGFRA V561D ity RPS6KA2 (RSK3) ity DGFRB (PDGFR beta) ity RPS6KA3 (RSK2) ity DK1 ity RPS6KA4 (MSK2) ity PDK1 Direct D HKG1 Activi D HKG2 Activi y y D M1 y y D M2 Activi D KN1 (PRK1) Activi y y LK1 y y LK2 y y US 9,353,116 B2 125 126 TABLE 9c-continued TABLE 9c-continued
TEK (Tie2) SRCN1 Activi Activi Activi Activi TYRO3 (RSE) ST
Although the foregoing invention has been described in Some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appre ST K3 (MST2) Activity 15 ciate that certain changes and modifications may be practiced STK4 (MST1) Activity within the scope of the appended claims. In addition, each SYK Activity reference provided herein is incorporated by reference in its TAOK2 (TAO1) Activity entirety to the same extent as if each reference was individu TBK1 Activity ally incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 8O
<21 Os SEQ ID NO 1 &211s LENGTH: 918 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: synthetic DNA construct for T338C c-Src (251-533)
<4 OOs SEQUENCE: 1 catcatcatc at cat cacga ttacgatatic ccaacgaccg aaaacctitta ct tccagggc 60 catatgcaga ccCagggact cgc.caaggac gcgtgggaala to coccggga gtcgctgcgg 12O
Ctggaggtga agctggggca gggctgctitt ggagaggtot ggatggggac Ctggaacggc 18O accaccagag togccataaa gactctgaag cc.cggcacca tgtc.ccc.gga ggcctt CCtg 24 O
Caggaag.ccc aagtgatgaa gaagctc.cgg catgaga agc tggttcagct gtacgcagtg 3 OO gtgtcggaag agccCatcta catcgtctgt gagtacatga gcaa.ggggag cct cctggat 360 titcCtgaagg gagagatggg caagtacctg cggctgccac agct cqtcga tatggctgct 42O
Cagattgcat Coggcatggc Ctatgtggag aggatgaact acgtgcaccg agacctgcgg 48O gcggccaa.ca to ctggtggg ggaga acctg gtgttgcaagg tggctgactt toggctggca 54 O cgc.ct catcg aggacaacga gtacacagca cggcaaggtg ccaagttcc c catcaagtgg 6 OO acagc.ccc.cg aggcagc cct Ct atggc.cgg tt Caccatca agtcggatgt CtggtcCttic 660 ggcatcCtgc tigactgagct gaccaccaag cataccCagg gatggt caac 72O agggaggtgc tggaccaggit ggagagggg.c taccgcatgc Cctgc.ccgcc cagtgcc cc 78O gag togctgc atgacct cat tggcggalagg accctgagga gC9gcc cact 84 O tttgagtacc togcaggc citt Cctggaggac tact to acct cgacagagcc cc agtaccag 9 OO US 9,353,116 B2 127 128 - Continued
Cctggagaga acctatag 918
<210s, SEQ ID NO 2 &211s LENGTH: 305 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic T338C c-Src (251-533) protein <4 OOs, SEQUENCE: 2 His His His His His His Asp Tyr Asp Ile Pro Thr Thr Glu Asn Lieu. 1. 5 1O 15 Tyr Phe Glin Gly His Met Gln Thr Glin Gly Lieu Ala Lys Asp Ala Trp 2O 25 3O Glu Ile Pro Arg Glu Ser Lieu. Arg Lieu. Glu Val Llys Lieu. Gly Glin Gly 35 4 O 45 Cys Phe Gly Glu Val Trp Met Gly. Thir Trp Asn Gly Thr Thr Arg Val SO 55 6 O Ala Ile Llys Thr Lieu Lys Pro Gly Thr Met Ser Pro Glu Ala Phe Leu 65 70 7s 8O Glin Glu Ala Glin Val Met Lys Llys Lieu. Arg His Glu Lys Lieu Val Glin 85 90 95 Lieu. Tyr Ala Val Val Ser Glu Glu Pro Ile Tyr Ile Val Cys Glu Tyr 1OO 105 11 O Met Ser Lys Gly Ser Lieu. Lieu. Asp Phe Lieu Lys Gly Glu Met Gly Lys 115 12 O 125 Tyr Lieu. Arg Lieu Pro Gln Lieu Val Asp Met Ala Ala Glin Ile Ala Ser 13 O 135 14 O Gly Met Ala Tyr Val Glu Arg Met Asn Tyr Val His Arg Asp Lieu. Arg 145 150 155 160 Ala Ala Asn. Ile Lieu Val Gly Glu Asn Lieu Val Cys Llys Val Ala Asp 1.65 17O 17s Phe Gly Lieu Ala Arg Lieu. Ile Glu Asp Asn. Glu Tyr Thr Ala Arg Glin 18O 185 19 O Gly Ala Lys Phe Pro Ile Llys Trp Thir Ala Pro Glu Ala Ala Lieu. Tyr 195 2OO 2O5 Gly Arg Phe Thir Ile Llys Ser Asp Val Trp Ser Phe Gly Ile Lieu. Lieu. 21 O 215 22O Thr Glu Lieu. Thir Thr Lys Gly Arg Val Pro Tyr Pro Gly Met Val Asn 225 23 O 235 24 O Arg Glu Val Lieu. Asp Glin Val Glu Arg Gly Tyr Arg Met Pro Cys Pro 245 250 255 Pro Glu. Cys Pro Glu Ser Lieu. His Asp Leu Met Cys Gln Cys Trp Arg 26 O 265 27 O Lys Asp Pro Glu Glu Arg Pro Thr Phe Glu Tyr Lieu. Glin Ala Phe Lieu. 27s 28O 285
Glu Asp Tyr Phe Thr Ser Thr Glu Pro Glin Tyr Gln Pro Gly Glu Asn 29 O 295 3 OO
Lell 3. OS
<210s, SEQ ID NO 3 &211s LENGTH: 533 212. TYPE: PRT <213> ORGANISM: Gallus gallus 22 Os. FEATURE: <223> OTHER INFORMATION: proto-oncogene c-Src US 9,353,116 B2 129 130 - Continued
<4 OOs, SEQUENCE: 3
Met Gly Ser Ser Lys Ser Pro Asp Pro Ser Glin Arg Arg Arg 1. 15
Ser Luell Glu Pro Pro Asp Ser Thir His His Gly Gly Phe Pro Ala Ser 25
Glin Thir Pro Asn Thir Ala Ala Pro Asp Thir His Arg Thir Pro Ser 35 4 O 45
Arg Ser Phe Gly Thir Wall Ala Thir Glu Pro Lys Lell Phe Gly Gly Phe SO 55 6 O
Asn Thir Ser Asp Thir Wall Thir Ser Pro Glin Arg Ala Gly Ala Luell Ala 65 70
Gly Gly Wall Thir Thir Phe Wall Ala Luell Tyr Asp Glu Ser Arg Thir 85 90 95
Glu Thir Asp Luell Ser Phe Gly Glu Arg Lell Glin Ile Wall Asn 105 11 O
Asn Thir Glu Gly Asp Trp Trp Luell Ala His Ser Lell Thir Thir Gly Glin 115 12 O 125
Thir Gly Tyr Ile Pro Ser Asn Wall Ala Pro Ser Asp Ser Ile Glin 13 O 135 14 O
Ala Glu Glu Trp Tyr Phe Gly Ile Thir Arg Arg Glu Ser Glu Arg 145 150 155 160
Lell Luell Luell Asn Pro Glu Asn Pro Arg Gly Thir Phe Lell Wall Arg Glu 1.65 17O
Ser Glu Thr Thr Lys Gly Ala Cys Lieu Ser Wall Ser Asp Phe Asp 18O 185 19 O
Asn Ala Lys Gly Lell Asn Wall Lys His Lys Ile Arg Luell Asp 195 2OO
Ser Gly Gly Phe Tyr Ile Thir Ser Arg Thir Glin Phe Ser Ser Luell Glin 21 O 215 22O
Glin Luell Wall Ala Tyr Tyr Ser His Ala Asp Gly Lell His Arg 225 23 O 235 24 O
Lell Thir Asn Wall Cys Pro Thir Ser Pro Glin Thir Glin Gly Luell Ala 245 250 255
Asp Ala Trp Glu Ile Pro Arg Glu Ser Luell Arg Lell Glu Wall Lys 26 O 265 27 O
Lell Gly Glin Gly Phe Gly Glu Wall Trp Met Gly Thir Trp Asn Gly 27s 28O 285
Thir Thir Arg Wall Ala Ile Lys Thir Luell Pro Gly Thir Met Ser Pro 29 O 295 3 OO
Glu Ala Phe Luell Glin Glu Ala Glin Wall Met Lys Lell Arg His Glu 3. OS 310 315
Luell Wall Glin Lell Tyr Ala Wall Wall Ser Glu Glu Pro Ile Tyr Ile 3.25 330 335
Wall Thir Glu Tyr Met Ser Gly Ser Luell Luell Asp Phe Luell Gly 34 O 345 35. O
Glu Met Gly Tyr Lell Arg Luell Pro Glin Luell Wall Asp Met Ala Ala 355 360 365
Glin Ile Ala Ser Gly Met Ala Wall Glu Arg Met Asn Tyr Wall His 37 O 375
Arg Asp Luell Arg Ala Ala Asn Ile Luell Wall Gly Glu Asn Luell Wall Cys 385 390 395 4 OO
Wall Ala Asp Phe Gly Lell Ala Arg Luell Ile Glu Asp Asn Glu Tyr US 9,353,116 B2 131 132 - Continued
4 OS 41O 415
Thir Ala Arg Glin Gly Ala Lys Phe Pro Ile Llys Trp Thr Ala Pro Glu 42O 425 43 O
Ala Ala Leu Tyr Gly Arg Phe Thir Ile Llys Ser Asp Val Trp Ser Phe 435 44 O 445 Gly Ile Leu Lieu. Thr Glu Lieu. Thir Thr Lys Gly Arg Val Pro Tyr Pro 450 45.5 460 Gly Met Val Asn Arg Glu Val Lieu. Asp Glin Val Glu Arg Gly Tyr Arg 465 470 47s 48O
Met Pro Cys Pro Pro Glu. Cys Pro Glu Ser Lieu. His Asp Leu Met Cys 485 490 495
Glin Cys Trp Arg Lys Asp Pro Glu Glu Arg Pro Thr Phe Glu Tyr Lieu. SOO 505 51O
Glin Ala Phe Leu Glu Asp Tyr Phe Thr Ser Thr Glu Pro Glin Tyr Glin 515 52O 525
Pro Gly Glu Asn Lieu. 53 O
<210s, SEQ ID NO 4 &211s LENGTH: 283 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic Gallus gallus proto-oncogene c-Src. (251-533)
<4 OOs, SEQUENCE: 4 Glin Thr Glin Gly Lieu Ala Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser 1.
Lell Arg Lieu. Glu Val Llys Lieu. Gly Glin Gly Cys Phe Gly Glu Val Trp 2O 25 3O
Met Gly. Thir Trp Asn Gly Thr Thr Arg Val Ala Ile Llys Thr Lieu Lys 35 4 O 45
Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Glin Glu Ala Glin Val Met SO 55 6 O Lys Llys Lieu. Arg His Glu Lys Lieu Val Glin Lieu. Tyr Ala Val Val Ser 65 70 7s 8O
Glu Glu Pro Ile Tyr Ile Val Thr Glu Tyr Met Ser Lys Gly Ser Leu 85 90 95
Lell Asp Phe Lieu Lys Gly Glu Met Gly Lys Tyr Lieu. Arg Lieu Pro Glin 1OO 105 11 O
Lell Val Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala Tyr Val Glu 115 12 O 125 Arg Met Asn Tyr Val His Arg Asp Lieu. Arg Ala Ala Asn. Ile Lieu Val 13 O 135 14 O Gly Glu Asn Lieu Val Cys Llys Val Ala Asp Phe Gly Lieu Ala Arg Lieu. 145 150 155 160
Ile Glu Asp Asn. Glu Tyr Thr Ala Arg Glin Gly Ala Lys Phe Pro Ile 1.65 17O 17s
Trp. Thir Ala Pro Glu Ala Ala Leu Tyr Gly Arg Phe Thr Ile Llys 18O 185 19 O
Ser Asp Val Trp Ser Phe Gly Ile Leu Lieu. Thr Glu Lieu. Thir Thr Lys 195 2OO 2O5
Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val Lieu. Asp Glin 21 O 215 22O US 9,353,116 B2 133 134 - Continued
Wall Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu. Cys Pro Glu Ser 225 23 O 235 24 O
Lell His Asp Lieu Met Cys Glin Cys Trp Arg Lys Asp Pro Glu Glu Arg 245 250 255
Pro Thr Phe Glu Tyr Lieu. Glin Ala Phe Leu Glu Asp Tyr Phe Thr Ser 26 O 265 27 O
Thir Glu Pro Glin Tyr Gln Pro Gly Glu Asn Lieu. 27s 28O
<210s, SEQ ID NO 5 &211s LENGTH: 286 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic Gallus gallus proto-oncogene c-Src. (251-533) with GHM at N-terminal
<4 OOs, SEQUENCE: 5 Gly His Met Glin Thr Glin Gly Lieu Ala Lys Asp Ala Trp Glu Ile Pro 1. 5 1O 15 Arg Glu Ser Lieu. Arg Lieu. Glu Val Lys Lieu. Gly Glin Gly Cys Phe Gly 2O 25 3O
Glu Val Trp Met Gly. Thir Trp Asn Gly Thr Thr Arg Val Ala Ile Llys 35 4 O 45
Thir Leu Lys Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Gln Glu Ala SO 55 6 O
Glin Val Met Lys Llys Lieu. Arg His Glu Lys Lieu Val Glin Lieu. Tyr Ala 65 70 75 8O
Wall Val Ser Glu Glu Pro Ile Tyr Ile Val Thr Glu Tyr Met Ser Lys 85 90 95 Gly Ser Lieu. Lieu. Asp Phe Lieu Lys Gly Glu Met Gly Llys Tyr Lieu. Arg 1OO 105 11 O
Lell Pro Glin Lieu Val Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala 115 12 O 125 Val Glu Arg Met Asn Tyr Val His Arg Asp Lieu. Arg Ala Ala Asn 13 O 135 14 O
Ile Lieu Val Gly Glu Asn Lieu Val Cys Llys Val Ala Asp Phe Gly Lieu. 145 150 155 160
Ala Arg Lieu. Ile Glu Asp Asn. Glu Tyr Thr Ala Arg Glin Gly Ala Lys 1.65 17O 17s
Phe Pro Ile Llys Trp Thr Ala Pro Glu Ala Ala Lieu. Tyr Gly Arg Phe 18O 185 19 O
Thir Ile Llys Ser Asp Val Trp Ser Phe Gly Ile Lieu. Lieu. Thr Glu Lieu. 195 2OO 2O5
Thir Thr Lys Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val 21 O 215 22O
Lell Asp Glin Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu. Cys 225 23 O 235 24 O
Pro Glu Ser Lieu. His Asp Lieu Met Cys Glin Cys Trp Arg Lys Asp Pro 245 250 255
Glu Glu Arg Pro Thr Phe Glu Tyr Lieu. Glin Ala Phe Lieu. Glu Asp Tyr 26 O 265 27 O
Phe Thir Ser Thr Glu Pro Glin Tyr Glin Pro Gly Glu Asn Lieu. 27s 28O 285
<210s, SEQ ID NO 6 US 9,353,116 B2 135 136 - Continued
&211s LENGTH: 283 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic Gallus gallus proto-oncogene T338Xc-Src (251-533) 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (88) . . (88) <223> OTHER INFORMATION: Xaa = any naturally occurring amino acid
<4 OOs, SEQUENCE: 6 Glin Thr Glin Gly Lieu Ala Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser 1. 5 1O 15 Lieu. Arg Lieu. Glu Val Llys Lieu. Gly Glin Gly Cys Phe Gly Glu Val Trp 2O 25 3O Met Gly. Thir Trp Asn Gly. Thir Thr Arg Val Ala Ile Llys Thr Lieu Lys 35 4 O 45 Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Gln Glu Ala Glin Val Met SO 55 6 O Llys Llys Lieu. Arg His Glu Lys Lieu Val Glin Lieu. Tyr Ala Val Val Ser 65 70 7s 8O Glu Glu Pro Ile Tyr Ile Val Xaa Glu Tyr Met Ser Lys Gly Ser Lieu. 85 90 95 Lieu. Asp Phe Lieu Lys Gly Glu Met Gly Lys Tyr Lieu. Arg Lieu Pro Glin 1OO 105 11 O Lieu Val Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala Tyr Val Glu 115 12 O 125 Arg Met Asn Tyr Val His Arg Asp Lieu. Arg Ala Ala Asn. Ile Lieu Val 13 O 135 14 O Gly Glu Asn Lieu Val Cys Llys Val Ala Asp Phe Gly Lieu Ala Arg Lieu. 145 150 155 160 Ile Glu Asp Asn. Glu Tyr Thr Ala Arg Glin Gly Ala Lys Phe Pro Ile 1.65 17O 17s Llys Trp Thir Ala Pro Glu Ala Ala Lieu. Tyr Gly Arg Phe Thir Ile Llys 18O 185 19 O Ser Asp Val Trp Ser Phe Gly Ile Leu Lieu. Thr Glu Lieu. Thir Thr Lys 195 2OO 2O5 Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val Lieu. Asp Glin 21 O 215 22O Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu. Cys Pro Glu Ser 225 23 O 235 24 O Lieu. His Asp Lieu Met Cys Glin Cys Trp Arg Lys Asp Pro Glu Glu Arg 245 250 255 Pro Thr Phe Glu Tyr Lieu. Glin Ala Phe Leu Glu Asp Tyr Phe Thir Ser 26 O 265 27 O Thr Glu Pro Glin Tyr Gln Pro Gly Glu Asn Lieu. 27s 28O
<210s, SEQ ID NO 7 &211s LENGTH: 286 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic Gallus gallus proto-oncogene GHM-T338Xc-Src (251-533) (GHM at N-terminal) 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (91) ... (91) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid US 9,353,116 B2 137 138 - Continued
<4 OO > SEQUENCE: 7 Gly His Met Glin Thr Glin Gly Lieu Ala Lys Asp Ala Trp Glu Ile Pro 1.
Arg Gl u Ser Lieu. Arg Lieu. Glu Val Llys Lieu. Gly Glin Gly Cys Phe Gly 2O 25 3O
Glu Wa. l Trp Met Gly. Thir Trp Asn Gly Thr Thr Arg Val Ala Ile Llys 35 4 O 45
Thir Le u Lys Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Gln Glu Ala SO 55 6 O
Glin Wa. l Met Lys Llys Lieu. Arg His Glu Lys Lieu Val Glin Lieu. Tyr Ala 65 70 7s 8O
Wall Wa. l Ser Glu Glu Pro Ile Tyr Ile Val Xaa Glu Tyr Met Ser Lys 85 90 95
Gly Se r Lieu. Lieu. Asp Phe Lieu Lys Gly Glu Met Gly Llys Tyr Lieu. Arg 1OO 105 11 O
Lell P o Glin Lieu Val Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala 115 12 O 125
Wa. l Glu Arg Met Asn Tyr Val His Arg Asp Lieu. Arg Ala Ala Asn 13 O 135 14 O
Ile Le u Val Gly Glu Asn Lieu Val Cys Llys Val Ala Asp Phe Gly Lieu. 145 150 155 160
Ala Air g Lieu. Ile Glu Asp Asn. Glu Tyr Thir Ala Arg Glin Gly Ala Lys 1.65 17O 17s
Phe P o Ile Llys Trp Thr Ala Pro Glu Ala Ala Lieu. Tyr Gly Arg Phe 18O 185 19 O
Thir Il e Llys Ser Asp Val Trp Ser Phe Gly Ile Lieu. Lieu. Thr Glu Lieu. 195 2OO 2O5
Thir Thr Lys Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val 21 O 215 22O
Lell As p Glin Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu. Cys 225 23 O 235 24 O
Pro Gl u Ser Lieu. His Asp Lieu Met Cys Glin Cys Trp Arg Lys Asp Pro 245 250 255
Glu Gl u Arg Pro Thr Phe Glu Tyr Lieu. Glin Ala Phe Lieu. Glu Asp Tyr 26 O 265 27 O
Phe Thir Ser Thr Glu Pro Glin Tyr Glin Pro Gly Glu Asn Lieu. 27s 28O 285
SEQ ID NO 8 LENGTH: 283 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene T338cc-Src (251-533) (c-Src ES1")
<4 OOs, SEQUENCE: 8
Glin Thr Glin Gly Lieu Ala Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser 1. 5 1O 15
Lell Air g Lieu. Glu Val Llys Lieu. Gly Glin Gly Cys Phe Gly Glu Val Trp 2O 25 3O
Met Gl y Thr Trp Asn Gly Thr Thr Arg Val Ala Ile Llys Thr Lieu Lys 35 4 O 45
Pro Gl y Thr Met Ser Pro Glu Ala Phe Leu Gln Glu Ala Glin Val Met SO 55 6 O US 9,353,116 B2 139 140 - Continued
Lys Luell Arg His Glu Luell Wall Glin Luell Tyr Ala Wall Wall Ser 65 70 7s
Glu Glu Pro Ile Tyr Ile Wall Glu Tyr Met Ser Gly Ser Luell 85 90 95
Lell Asp Phe Luell Gly Glu Met Gly Lys Lell Arg Luell Pro Glin 105 11 O
Lell Wall Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala Wall Glu 115 12 O 125
Arg Met Asn Tyr Wall His Arg Asp Luell Arg Ala Ala Asn Ile Luell Wall 13 O 135 14 O
Gly Glu Asn Luell Wall Cys Wall Ala Asp Phe Gly Lell Ala Arg Luell 145 150 155 160
Ile Glu Asp Asn Glu Tyr Thir Ala Arg Glin Gly Ala Phe Pro Ile 1.65 17O 17s
Trp Thir Ala Pro Glu Ala Ala Luell Tyr Gly Arg Phe Thir Ile 18O 185 19 O
Ser Asp Wall Trp Ser Phe Gly Ile Luell Luell Thir Glu Lell Thir Thir 195 2O5
Gly Arg Wall Pro Tyr Pro Gly Met Wall Asn Arg Glu Wall Luell Asp Glin 21 O 215
Wall Glu Arg Gly Tyr Arg Met Pro Pro Pro Glu Pro Glu Ser 225 23 O 235 24 O
Lell His Asp Luell Met Glin Trp Arg Lys Asp Pro Glu Glu Arg 245 250 255
Pro Thir Phe Glu Tyr Lell Glin Ala Phe Luell Glu Asp Phe Thir Ser 26 O 265 27 O
Thir Glu Pro Glin Tyr Glin Pro Gly Glu Asn Luell 27s 28O
SEO ID NO 9 LENGTH: 286 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene GHM-T338cc-Src (251-533) (GHM at N-terminal (c-Src vES1 ) <4 OOs, SEQUENCE:
Gly His Met Glin Thir Glin Gly Luell Ala Lys Asp Ala Trp Glu Ile Pro 1. 5 1O 15
Arg Glu Ser Luell Arg Lell Glu Wall Lys Luell Gly Glin Gly Cys Phe Gly 2O 25 3O
Glu Wall Trp Met Gly Thir Trp Asn Gly Thir Thir Arg Wall Ala Ile 35 4 O 45
Thir Luell Lys Pro Gly Thir Met Ser Pro Glu Ala Phe Lell Glin Glu Ala SO 55 6 O
Glin Wall Met Lys Lell Arg His Glu Luell Wall Glin Luell Tyr Ala 65 70 7s
Wall Wall Ser Glu Glu Pro Ile Ile Wall Glu Met Ser 85 90 95
Gly Ser Luell Luell Asp Phe Lell Gly Glu Met Gly Tyr Luell Arg 105 11 O
Lell Pro Glin Luell Wall Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala 115 12 O 125
Wall Glu Arg Met Asn Wall His Arg Asp Lell Arg Ala Ala Asn US 9,353,116 B2 141 142 - Continued
13 O 135 14 O
Ile Luell Wall Gly Glu Asn Lell Wall Lys Wall Ala Asp Phe Gly Luell 145 150 155 160
Ala Arg Luell Ile Glu Asp Asn Glu Thir Ala Arg Glin Gly Ala Lys 1.65
Phe Pro Ile Lys Trp Thir Ala Pro Glu Ala Ala Lell Gly Arg Phe 18O 185 19 O
Thir Ile Lys Ser Asp Wall Trp Ser Phe Gly Ile Lell Lell Thir Glu Luell 195 2OO
Thir Thir Gly Arg Wall Pro Pro Gly Met Wall Asn Arg Glu Wall 21 O 215 22O
Lell Asp Glin Wall Glu Arg Gly Arg Met Pro Pro Pro Glu Cys 225 23 O 235 24 O
Pro Glu Ser Luell His Asp Lell Met Glin Cys Trp Arg Asp Pro 245 250 255
Glu Glu Arg Pro Thir Phe Glu Luell Glin Ala Phe Lell Glu Asp Tyr 26 O 265 27 O
Phe Thir Ser Thir Glu Pro Glin Tyr Glin Pro Gly Glu Asn Luell 27s 28O 285
SEQ ID NO 10 LENGTH: 283 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene T338X, V323X c-Src (251-533) FEATURE: NAME/KEY: VARIANT LOCATION: (73) . . (88) OTHER INFORMATION: Xaa any naturally occurring amino acid
SEQUENCE: 10
Gln Thr Glin Gly Lell Ala Asp Ala Trp Glu Ile Pro Arg Glu Ser 1. 5 15
Lell Arg Luell Glu Wall Lell Gly Glin Gly Phe Gly Glu Wall Trp 25
Met Gly Thir Trp Asn Gly Thir Thir Arg Wall Ala Ile Lys Thir Luell Lys 35 4 O 45
Pro Gly Thir Met Ser Pro Glu Ala Phe Luell Glin Glu Ala Glin Wall Met SO 55 6 O
Lys Luell Arg His Glu Luell Xaa Glin Luell Ala Wall Wall Ser 65 70 7s
Glu Glu Pro Ile Tyr Ile Wall Xaa Glu Tyr Met Ser Gly Ser Luell 85 90 95
Lell Asp Phe Luell Lys Gly Glu Met Gly Lys Lell Arg Luell Pro Glin 105 11 O
Lell Wall Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala Wall Glu 115 12 O 125
Arg Met Asn Wall His Arg Asp Luell Arg Ala Ala Asn Ile Luell Wall 13 O 135 14 O
Gly Glu Asn Luell Wall Cys Wall Ala Asp Phe Gly Lell Ala Arg Luell 145 150 155 160
Ile Glu Asp Asn Glu Thir Ala Arg Glin Gly Ala Phe Pro Ile 1.65 17O 17s
Trp Thir Ala Pro Glu Ala Ala Luell Tyr Gly Arg Phe Thir Ile Lys 18O 185 19 O US 9,353,116 B2 143 144 - Continued
Ser Asp Wall Trp Ser Phe Gly Ile Luell Luell Thir Glu Lell Thir Thir 195 2OO 2O5
Gly Arg Wall Pro Tyr Pro Gly Met Wall Asn Arg Glu Wall Luell Asp Glin 21 O 215
Wall Glu Arg Gly Tyr Arg Met Pro Pro Pro Glu Cys Pro Glu Ser 225 23 O 235 24 O
Lell His Asp Luell Met Glin Trp Arg Lys Asp Pro Glu Glu Arg 245 250 255
Pro Thir Phe Glu Tyr Lell Glin Ala Phe Luell Glu Asp Phe Thir Ser 26 O 265 27 O
Thir Glu Pro Glin Tyr Glin Pro Gly Glu Asn Luell 27s 28O
SEQ ID NO 11 LENGTH: 286 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene GHM-T338X, V323X c-Src (251-533) (GHM at N-terminal) FEATURE: NAME/KEY: VARIANT LOCATION: (76) ... (91) OTHER INFORMATION: Xaa = any naturally occurring amino acid
SEQUENCE: 11
Gly His Met Glin Thir Glin Gly Luell Ala Lys Asp Ala Trp Glu Ile Pro 1. 5 1O 15
Arg Glu Ser Luell Arg Lell Glu Wall Lys Luell Gly Glin Gly Cys Phe Gly 2O 25
Glu Wall Trp Met Gly Thir Trp Asn Gly Thir Thir Arg Wall Ala Ile 35 4 O 45
Thir Luell Pro Gly Thir Met Ser Pro Glu Ala Phe Lell Glin Glu Ala SO 55 6 O
Glin Wall Met Lys Lell Arg His Glu Luell Xaa Glin Luell Tyr Ala 65 70
Wall Wall Ser Glu Glu Pro Ile Ile Wall Xaa Glu Tyr Met Ser 85 90 95
Gly Ser Luell Luell Asp Phe Lell Gly Glu Met Gly Tyr Luell Arg 105 11 O
Lell Pro Glin Luell Wall Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala 115 12 O 125
Wall Glu Arg Met Asn Tyr Wall His Arg Asp Lell Arg Ala Ala Asn 13 O 135 14 O
Ile Luell Wall Gly Glu Asn Lell Wall Wall Ala Asp Phe Gly Luell 145 150 155 160
Ala Arg Luell Ile Glu Asp Asn Glu Tyr Thir Ala Arg Glin Gly Ala Lys 1.65 17s
Phe Pro Ile Lys Trp Thir Ala Pro Glu Ala Ala Lell Tyr Gly Arg Phe 18O 185 19 O
Thir Ile Lys Ser Asp Wall Trp Ser Phe Gly Ile Lell Lell Thir Glu Luell 195 2OO
Thir Thir Gly Arg Wall Pro Pro Gly Met Wall Asn Arg Glu Wall 21 O 215 22O
Lell Asp Glin Wall Glu Arg Gly Arg Met Pro Pro Pro Glu Cys 225 23 O 235 24 O US 9,353,116 B2 145 146 - Continued
Pro Glu Ser Luell His Asp Lieu Met Cys Glin Cys Trp Arg Lys Asp Pro 245 250 255
Glu Glu Arg Pro Thir Phe Glu Tyr Lieu. Glin Ala Phe Lieu. Glu Asp Tyr 26 O 265 27 O
Phe Thir Ser Thir Glu Pro Glin Tyr Glin Pro Gly Glu Asn Lieu. 27s 285
SEQ ID NO 12 LENGTH: 283 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene T338C, V323X c-Src (251-533) FEATURE: NAME/KEY: VARIANT LOCATION: (73) . . (73) OTHER INFORMATION: Xaa any naturally occurring amino acid
<4 OOs, SEQUENCE: 12
Glin Thr Glin Gly Lell Ala Asp Ala Trp Glu Ile Pro Arg Glu Ser 1. 5 15
Lell Arg Luell Glu Wall Lell Gly Glin Gly Phe Gly Glu Wall Trp 25
Met Gly Thir Trp Asn Gly Thir Thir Arg Wall Ala Ile Lys Thir Luell Lys 35 4 O 45
Pro Gly Thir Met Ser Pro Glu Ala Phe Luell Glin Glu Ala Glin Wall Met SO 55 6 O
Lys Lys Lieu Arg His Glu Lieu Xaa Gln Lieu. Ala Wall Wall Ser 65 70
Glu Glu Pro Ile Tyr Ile Wall Glu Tyr Met Ser Gly Ser Luell 85 90 95
Lell Asp Phe Luell Lys Gly Glu Met Gly Lys Lell Arg Luell Pro Glin 105 11 O
Lell Wall Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala Wall Glu 115 12 O 125
Arg Met Asn Wall His Arg Asp Luell Arg Ala Ala Asn Ile Luell Wall 13 O 135 14 O
Gly Glu Asn Luell Wall Cys Wall Ala Asp Phe Gly Lell Ala Arg Luell 145 150 155 160
Ile Glu Asp Asn Glu Thir Ala Arg Glin Gly Ala Phe Pro Ile 1.65 17O 17s
Trp Thir Ala Pro Glu Ala Ala Luell Tyr Gly Arg Phe Thir Ile Lys 18O 185 19 O
Ser Asp Wall Trp Ser Phe Gly Ile Luell Luell Thir Glu Lell Thir Thir Lys 195 2O5
Gly Arg Wall Pro Tyr Pro Gly Met Wall Asn Arg Glu Wall Luell Asp Glin 21 O 215 22O
Wall Glu Arg Gly Tyr Arg Met Pro Pro Pro Glu Pro Glu Ser 225 23 O 235 24 O
Lell His Asp Luell Met Glin Trp Arg Asp Pro Glu Glu Arg 245 250 255
Pro Thir Phe Glu Tyr Lell Glin Ala Phe Luell Glu Asp Phe Thir Ser 26 O 265 27 O
Thir Glu Pro Glin Tyr Glin Pro Gly Glu Asn Luell 27s 28O US 9,353,116 B2 147 148 - Continued
<210s, SEQ ID NO 13 &211s LENGTH: 286 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic Gallus gallus proto-oncogene GHM-T338C, V323X c-Src (251-533) (GHM at N-terminal) 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (76) . . (76) <223> OTHER INFORMATION: Xaa = any naturally occurring amino acid
<4 OOs, SEQUENCE: 13 Gly His Met Glin Thr Glin Gly Lieu Ala Lys Asp Ala Trp Glu Ile Pro 1. 5 1O 15 Arg Glu Ser Lieu. Arg Lieu. Glu Val Lys Lieu. Gly Glin Gly Cys Phe Gly 2O 25 3O Glu Val Trp Met Gly. Thir Trp Asn Gly Thr Thr Arg Val Ala Ile Llys 35 4 O 45 Thr Lieu Lys Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Glin Glu Ala SO 55 6 O Glin Val Met Lys Llys Lieu. Arg His Glu Lys Lieu. Xaa Glin Lieu. Tyr Ala 65 70 7s 8O Val Val Ser Glu Glu Pro Ile Tyr Ile Val Cys Glu Tyr Met Ser Lys 85 90 95 Gly Ser Lieu. Lieu. Asp Phe Lieu Lys Gly Glu Met Gly Llys Tyr Lieu. Arg 1OO 105 11 O Lieu Pro Glin Lieu Val Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala 115 120 125 Tyr Val Glu Arg Met Asn Tyr Val His Arg Asp Lieu. Arg Ala Ala Asn 13 O 135 14 O Ile Lieu Val Gly Glu Asn Lieu Val Cys Llys Val Ala Asp Phe Gly Lieu. 145 150 155 160 Ala Arg Lieu. Ile Glu Asp Asn. Glu Tyr Thr Ala Arg Glin Gly Ala Lys 1.65 17O 17s Phe Pro Ile Llys Trp Thr Ala Pro Glu Ala Ala Lieu. Tyr Gly Arg Phe 18O 185 19 O Thir Ile Llys Ser Asp Val Trp Ser Phe Gly Ile Leu Lleu. Thr Glu Lieu. 195 2OO 2O5 Thir Thr Lys Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val 21 O 215 22O Lieu. Asp Glin Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu. Cys 225 23 O 235 24 O Pro Glu Ser Lieu. His Asp Lieu Met Cys Glin Cys Trp Arg Lys Asp Pro 245 250 255 Glu Glu Arg Pro Thr Phe Glu Tyr Lieu. Glin Ala Phe Lieu. Glu Asp Tyr 26 O 265 27 O
Phe Thir Ser Thr Glu Pro Glin Tyr Glin Pro Gly Glu Asn Lieu. 27s 28O 285
<210s, SEQ ID NO 14 &211s LENGTH: 283 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic Gallus gallus proto-oncogene T338C, V323Ac-Src (251-533) (c-Src ES2")
<4 OOs, SEQUENCE: 14 US 9,353,116 B2 149 150 - Continued
Glin Thir Glin Gly Lell Ala Asp Ala Trp Glu Ile Pro Arg Glu Ser 15
Lell Arg Luell Glu Wall Lell Gly Glin Gly Phe Gly Glu Wall Trp 25
Met Gly Thir Trp Asn Gly Thir Thir Arg Wall Ala Ile Lys Thir Luell Lys 35 4 O 45
Pro Gly Thir Met Ser Pro Glu Ala Phe Luell Glin Glu Ala Glin Wall Met SO 55 6 O
Lys Luell Arg His Glu Luell Ala Glin Luell Ala Wall Wall Ser 65 70
Glu Glu Pro Ile Tyr Ile Wall Glu Tyr Met Ser Gly Ser Luell 85 90 95
Lell Asp Phe Luell Lys Gly Glu Met Gly Lys Lell Arg Luell Pro Glin 105 11 O
Lell Wall Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala Wall Glu 115 12 O 125
Arg Met Asn Wall His Arg Asp Luell Arg Ala Ala Asn Ile Luell Wall 13 O 135 14 O
Gly Glu Asn Luell Wall Cys Wall Ala Asp Phe Gly Lell Ala Arg Luell 145 150 155 160
Ile Glu Asp Asn Glu Thir Ala Arg Glin Gly Ala Phe Pro Ile 1.65 17O 17s
Trp Thir Ala Pro Glu Ala Ala Luell Tyr Gly Arg Phe Thir Ile Lys 18O 185 19 O
Ser Asp Wall Trp Ser Phe Gly Ile Lieu Lieu Thr Glu Lieu Thr Thr Lys 195 2O5
Gly Arg Wall Pro Tyr Pro Gly Met Wall Asn Arg Glu Wall Luell Asp Glin 21 O 215
Wall Glu Arg Gly Tyr Arg Met Pro Pro Pro Glu Pro Glu Ser 225 23 O 235 24 O
Lell His Asp Luell Met Glin Trp Arg Asp Pro Glu Glu Arg 245 250 255
Pro Thir Phe Glu Tyr Lell Glin Ala Phe Luell Glu Asp Phe Thir Ser 26 O 265 27 O
Thir Glu Pro Glin Tyr Glin Pro Gly Glu Asn Luell 27s 28O
SEO ID NO 15 LENGTH: 286 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene GHM-T338C, V323Ac-Src (251-533) (GHM at N-terminal) (c-Src. wES2 )
<4 OOs, SEQUENCE: 15
Gly His Met Glin Thir Glin Gly Luell Ala Lys Asp Ala Trp Glu Ile Pro 1. 15
Arg Glu Ser Luell Arg Lell Glu Wall Lys Luell Gly Glin Gly Cys Phe Gly 2O 25
Glu Wall Trp Met Gly Thir Trp Asn Gly Thir Thir Arg Wall Ala Ile Lys 35 4 O 45
Thir Luell Pro Gly Thir Met Ser Pro Glu Ala Phe Lell Glin Glu Ala SO 55 6 O
Glin Wall Met Lys Lell Arg His Glu Lys Luell Ala Glin Luell Ala US 9,353,116 B2 151 152 - Continued
65 Val Val Ser Glu Glu Pro Ile Tyr Ile Val Cys Glu Tyr Met Ser Lys 85 90 95 Gly Ser Lieu. Lieu. Asp Phe Lieu Lys Gly Glu Met Gly Llys Tyr Lieu. Arg 1OO 105 11 O Lieu Pro Glin Lieu Val Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala 115 12 O 125 Tyr Val Glu Arg Met Asn Tyr Val His Arg Asp Lieu. Arg Ala Ala Asn 13 O 135 14 O Ile Lieu Val Gly Glu Asn Lieu Val Cys Llys Val Ala Asp Phe Gly Lieu. 145 150 155 160 Ala Arg Lieu. Ile Glu Asp Asn. Glu Tyr Thr Ala Arg Glin Gly Ala Lys 1.65 17O 17s Phe Pro Ile Llys Trp Thr Ala Pro Glu Ala Ala Lieu. Tyr Gly Arg Phe 18O 185 19 O Thir Ile Llys Ser Asp Val Trp Ser Phe Gly Ile Leu Lleu. Thr Glu Lieu. 195 2OO 2O5 Thir Thr Lys Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val 21 O 215 22O Lieu. Asp Glin Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu. Cys 225 23 O 235 24 O Pro Glu Ser Lieu. His Asp Lieu Met Cys Glin Cys Trp Arg Lys Asp Pro 245 250 255 Glu Glu Arg Pro Thr Phe Glu Tyr Lieu. Glin Ala Phe Lieu. Glu Asp Tyr 26 O 265 27 O Phe Thir Ser Thr Glu Pro Glin Tyr Glin Pro Gly Glu Asn Lieu. 27s 28O 285
SEQ ID NO 16 LENGTH: 283 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene V323Sc-Src (251-533) (c-Src ES3") SEQUENCE: 16 Glin Thr Glin Gly Lieu Ala Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser 1. 5 1O 15 Lieu. Arg Lieu. Glu Val Llys Lieu. Gly Glin Gly Cys Phe Gly Glu Val Trp
Met Gly. Thir Trp Asn Gly. Thir Thr Arg Val Ala Ile Llys Thr Lieu Lys 35 4 O 45 Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Gln Glu Ala Glin Val Met SO 55 6 O Llys Llys Lieu. Arg His Glu Lys Lieu. Ser Glin Lieu. Tyr Ala Val Val Ser 65
Glu Glu Pro Ile Tyr Ile Val Cys Glu Tyr Met Ser Lys Gly Ser Lieu. 85 90 95
Lieu. Asp Phe Lieu Lys Gly Glu Met Gly Lys Tyr Lieu. Arg Lieu Pro Glin 1OO 105 11 O
Lieu Val Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala Tyr Val Glu 115 12 O 125 Arg Met Asn Tyr Val His Arg Asp Lieu. Arg Ala Ala Asn. Ile Lieu Val 13 O 135 14 O US 9,353,116 B2 153 154 - Continued
Gly Glu Asn Luell Wall Cys Wall Ala Asp Phe Gly Lell Ala Arg Luell 145 150 155 160
Ile Glu Asp Asn Glu Thir Ala Arg Glin Gly Ala Lys Phe Pro Ile 1.65 17O 17s
Trp Thir Ala Pro Glu Ala Ala Luell Tyr Gly Arg Phe Thir Ile Lys 18O 185 19 O
Ser Asp Wall Trp Ser Phe Gly Ile Luell Luell Thir Glu Lell Thir Thir Lys 195
Gly Arg Wall Pro Tyr Pro Gly Met Wall Asn Arg Glu Wall Luell Asp Glin 21 O 215
Wall Glu Arg Gly Tyr Arg Met Pro Pro Pro Glu Pro Glu Ser 225 23 O 235 24 O
Lell His Asp Luell Met Glin Trp Arg Asp Pro Glu Glu Arg 245 250 255
Pro Thir Phe Glu Tyr Lell Glin Ala Phe Luell Glu Asp Phe Thir Ser 26 O 265 27 O
Thir Glu Pro Glin Tyr Glin Pro Gly Glu Asn Luell 27s 28O
SEO ID NO 17 LENGTH: 286 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: synthetic Gallus gallus proto-oncogene GHM-T338C, V323Sc-Src (251-533) (GHM at N-terminal) (c-Src. wES3 )
<4 OOs, SEQUENCE: 17
Gly His Met Glin Thir Glin Gly Luell Ala Lys Asp Ala Trp Glu Ile Pro 1. 1O 15
Arg Glu Ser Luell Arg Lell Glu Wall Lys Luell Gly Glin Gly Cys Phe Gly 2O 25
Glu Wall Trp Met Gly Thir Trp Asn Gly Thir Thir Arg Wall Ala Ile Lys 35 4 O 45
Thir Luell Pro Gly Thir Met Ser Pro Glu Ala Phe Lell Glin Glu Ala SO 55 6 O
Glin Wall Met Lys Lell Arg His Glu Luell Ser Glin Luell Ala 65 70
Wall Wall Ser Glu Glu Pro Ile Ile Wall Glu Met Ser Lys 85 90 95
Gly Ser Luell Luell Asp Phe Lell Gly Glu Met Gly Tyr Luell Arg 1OO 105 11 O
Lell Pro Glin Luell Wall Asp Met Ala Ala Glin Ile Ala Ser Gly Met Ala 115 12 O 125
Wall Glu Arg Met Asn Tyr Wall His Arg Asp Lell Arg Ala Ala Asn 13 O 135 14 O
Ile Luell Wall Gly Glu Asn Lell Wall Wall Ala Asp Phe Gly Luell 145 150 155 160
Ala Arg Luell Ile Glu Asp Asn Glu Thir Ala Arg Glin Gly Ala Lys 1.65 17O 17s
Phe Pro Ile Lys Trp Thir Ala Pro Glu Ala Ala Lell Gly Arg Phe 18O 185 19 O
Thir Ile Lys Ser Asp Wall Trp Ser Phe Gly Ile Lell Lell Thir Glu Luell 195 2OO 2O5
Thir Thir Gly Arg Wall Pro Pro Gly Met Wall Asn Arg Glu Wall