Novel N9-arenethenyl purines as potent dual Src/Abl tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.06.042

Novel N⁹-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N⁹ on the purine core which projects hydrophobic substituents into the sele

Full text of DOI:10.1016/j.bmcl.2008.06.042

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4907–4912
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel N9-arenethenyl purines as potent dual Src/Abl tyrosine kinase inhibitors
*
Yihan Wang , William C. Shakespeare, Wei-Sheng Huang, Raji Sundaramoorthi, Scott Lentini, Sasmita Das,
Shuangying Liu, Geeta Banda, David Wen, Xiaotian Zhu, Qihong Xu, Jeffrey Keats, Frank Wang,
Scott Wardwell, Yaoyu Ning, Joseph T. Snodgrass, Mark I. Broudy, Karin Russian, David Dalgarno,
Tim Clackson, Tomi K. Sawyer
ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Novel N9-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described.
The key structural feature is a trans vinyl linkage at N9 on the purine core which projects hydrophobic
substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through
a Horner–Wadsworth–Emmons reaction of N9-phosphorylmethylpurines and substituted benzaldehydes
or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of
both Src and Abl kinase, and several possess good oral bioavailability.
Article history:
Received 1 May 2008
Accepted 13 June 2008
Available online 18 June 2008
Keywords:
N9-arenethenyl purines
Src/Abl tyrosine kinase inhibitors
Ó 2008 Elsevier Ltd. All rights reserved.
Src tyrosine kinase is the prototypical member of a family of ki-
nases (Src Family Kinases, SFKs) that modulate multiple intracellu-
lar signal transduction pathways involved in cell growth,
differentiation, migration and survival.1 There is evidence that
aberrant Src kinase activity is associated with the invasive pheno-
type in both early and advanced solid tumors.2,3 Moreover, recent
data have demonstrated that Src inhibitors can enhance the antitu-
mor efficacy of hormonal and cytotoxic agents as well as regulate
angiogenesis through the control of VEGF expression.4,5 Bcr-Abl,
the constitutively activated fusion protein product of the Philadel-
phia chromosome (Ph) is the principal oncogene underlying the
pathology of chronic myelogenous leukemia (CML). Imatinib is a
potent inhibitor of Bcr-Abl, binding to an inactive confirmation of
the protein.6 Despite its striking success in treating patients in
the chronic phase of the disease, responses are much less durable
in more advanced phases of the disease due to the emergence of
drug resistance. Although the most widely accepted mechanism
for imatinib resistance is the presence of kinase domain muta-
tions,7 cells from patients resistant to imatinib express an activated
form of the SFK Lyn.8 Moreover, Hck (another SFK) and Lyn are
over-expressed and activated in CML blast-crisis patients and their
up-regulation correlates with disease progression and resistance in
cell lines and patients treated with imatinib.9 Therefore, com-
pounds that can inhibit both Src and Abl might be useful in the
treatment of patients who have relapsed on imatinib.
developed as Src inhibitors frequently exhibit potent inhibition of
Abl kinase.10,11 Several second generation Bcr-Abl kinase inhibitors
target both Src and Abl kinases to combat imatinib resistance, and
include: dasatinib, bosutinib, AP23464, PD166326, AZD0530, and
CGP70630.12
Previously, we had described AP23464 as a potent inhibitor of
Src and Abl with IC50s for both kinases <1 nM.13 A crystal structure
of AP23464 bound to the active conformation of Src detailed the
molecular interactions responsible for its high potency including:
(a) two canonical H-bonds to the hinge region, (b) an extensive
network of H-bonds between the hydroxyl group on the phenethyl
substituent and amino acid residues in the selectivity pocket, and
(c) a water-mediated H-bond between the phosphine oxide and
the hydroxyl side chain of Y340.14 To further probe the purine core
as a template for potent Src/Abl inhibitors, alternative 2-atom link-
ers between the template and a pendant hydrophobic substituent
were explored. Herein, we describe a vinyl group as one such link-
age leading to a series of stable, synthetically accessible, purine-
based dual Src-Abl inhibitors (Fig. 1).
The coordinates from the AP23464:Src crystal structure were
used to build a model of Src for docking studies. Docking revealed
that a trans double bond15 on N9 of purine template could properly
orient a 2,6-disubstituted phenyl ring in the selectivity pocket in a
manner similar to dasatinib16 and PD166326 (Fig. 2A). Alterna-
tively, a vinyl-linked 4-substituted indazole was predicted to pre-
serve the intricate series of H-bonds observed in the AP23464
crystal structure (Fig. 2B).
Abl shares significant sequence homology with Src and, in its
active conformation, bears remarkable structural resemblance
with most SFKs. As a result, ATP-competitive compounds originally
Initially,
a Horner–Wadsworth–Emmons (HWE) approach
between N9 phosphine oxides and substituted benzaldehydes was
pursued to obtain the target molecules.17 The synthesis (Scheme
1)18 was initiated from readily accessible 6-chloro-2-iodo-9-THP-
* Corresponding author.
E-mail address: Yihan.wang@ariad.com (Y. Wang).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.042

4908
Y. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4907–4912
Hydrophobic pocket
OH
Hydrophobic pocket
Hydrophobic pocket
NH
N
N
N
N
N
N
N
N
N
N
N
N
HN
N
HN
HN
A
B
O
P
AP23464
O
P
O
P
Figure 1. Evolution of novel 9-arenethenyl purines as dual Src/Abl inhibitors. Dotted lines indicate H-bonds.
Figure 2. Models of AP23464, compound A, compound B docked in Src. Green, carbon; blue, nitrogen; red, phosphorus. Dotted lines indicate H-bonds.
purine 1.19 SNAr reaction of 1 at C6 with 4-(dialkylphosphoryl)-
benzeneamine20 using standard conditions yielded intermediate
2. A subsequent Negishi coupling reaction at C2 with isopropylzinc
chloride furnished 3. Deprotection of the THP group yielded inter-
mediate 4 which was alkylated with dipropyl tosylmethyl phos-
phine oxide to give the HWE reagent 5. Coupling of 5 with
various aldehydes produced the desired trans isomers together with
small amount of the cis isomers. Although successful, the limited
availability of structurally diverse benzaldehydes, tedious prepara-
tionofHMEreagents5, togetherwithlowyieldsoftheHWEreaction,
prompted us to pursue an alternative synthetic strategy.
Ultimately, a Heck coupling reaction between substituted 9-
vinylpurines and a series of diverse aryl halides was developed to
achieve the synthesis of 9-arenethenyl purines.21 Briefly, 9-vinyl-
purines 818 were reacted with anilines in the presence of i-Pr2NEt
in DMF, to yield intermediate vinylpurines 9. Subsequent Heck
coupling of 9 (X = H, Cl) with aryl halides under standard condi-
tions furnished exclusively the desired trans isomer. This reaction
is high yielding and quite robust, tolerating a variety of R1 and R2
groups, and without the need for protection and deprotection
strategies. Compound 10 could be further elaborated by substitu-
tion at C2 ( X = Cl) with amines or alcohols, thus generating com-
pounds 12a–j with either an N or O linkage. Where X = I,
intermediate 9 was first reacted with alkylzinc reagents then sub-
sequently underwent a Heck reaction to generate 6c–e. Com-
pounds 6c–e have C-linked R3 substitutions at C2. Direct Heck
coupling of iodo intermediates 9 with aryl halides was avoided
due to the concern that 2-iodopurines might compete with aryl ha-
lide substrates (Scheme 2).
All aryl halides employed in the Heck coupling step were
commercially available except for target compound 10g which
incorporated a substituted indazole. In this case, the required 4-bro-
mo-5-methylindazole intermediate was synthesized from 2,5-dim-
ethylbromobenzene according to Scheme 3.22 The synthesis ofdialkyl
anilinophenylphosphine oxides has been previously reported.20
Compounds were then evaluated for their Src23 and Abl24 kinase
inhibitory activity as previously described. Comparison of com-
pounds 6a, b versus 7a, b confirmed that the desired trans isomers
were more potent than the corresponding cis compounds against
Src and Abl enzymes by at least 10-fold (Table 1).

Y. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4907–4912
4909
R2
R2
Cl
HN
HN
a
N
N
N
N
b
N
N
N
N
N
I
N
1
I
N
2
R3
N
3
O
O
O
R2
R2
HN
HN
c
d
N
N
N
N
N
N
H
R3
N
R3
N
Pr
P Pr
O
4
5
R2
R2
HN
HN
e
N
N
N
N
N
+
R3
N
N
R3
N
R1
R1
6 (a, b)
7 (a,b)
Scheme 1. Reagents and conditions: (a) R2–NH2,22 i-Pr2NEt, DMF; (b) R3-ZnX, PdCl2(PPh3)2, Cs2CO3, dioxane; (c) TFA, rt, 15 min; (d) TsOCH2P(O)Pr2, NaH, DMF, rt, o/n;
(e) R1-CHO, NaH, DMF, rt, o/n.
R2
Cl
N
R2
HN
HN
N
N
a
N
N
N
b
N
N
N
N
(X=H,Cl)
X
X
N
X
N
X=H (8a)
X=Cl(8b)
X= I (8c)
10(a-g)
(X=H)
9
R1
d (X=I)
c (X=Cl)
R2
R2
HN
R2
N
HN
HN
b
N
N
N
N
N
N
N
R3
R3
N
Y
N
N
R3
N
6 (c-e)
R1
12(a-j)
(Y=N,O)
11
R1
Scheme 2. Reagents and conditions: (a) R2–NH2, i-Pr2NEt, DMF, 100 °C, 2 h; (b) halobenzene (R1X), Pd(OAc)2, P(o-tol)3, i-Pr2NEt, DMF,
i-Pr2NEt, DMF; (ii) R3-YH (Y = O), NaOEt, DMF; (d) R3ZnX, PdCl2(PPh3)2, DMF, M 60 °C/5 min.
lM 120 °C/10 min. (c) i—R3-YH (Y = N),
l
and 3.58 nM, respectively. Utilizing 10a, various modifications at
R1 were then evaluated including a simple chlorine atom substitu-
tion (10a vs 10b) which was equipotent. Deletion of the 6-methyl
group from 10b confirmed the requirement for 2,6-disubstitution
as a key driver of potency (10b vs 10c) as 10c was >10-fold less po-
tent against both enzymes. We then evaluated the possibility of
mimicking the H-bonding network observed in the AP23464:Src
co-crystal structure by systematically adding in the observed
hydrogen bonds (10d ? 10e ? 10f). While compounds 10d
showed marked decreases in potency against both enzymes rela-
tive to 10b, the indazole derivative 10e, was essentially equipotent
(see Table 1). Moreover, when we incorporated a 5-methyl substi-
tuent, the resulting compound (10f) was at least 10-fold more po-
tent than 10e. Extensive docking studies with 10f revealed that it
could mimic the hydrogen bonding network observed in the
AP23464 co-crystal structure and that the ortho methyl substituent
Br
Br
Br
a
b
NO2
d
NH2
Br
Br
c
N
NH
N
N
Boc
Scheme 3. Reagents and conditions: (a) HNO3, AcOH, 90 °C, 1 h; (b) Fe/AcOH, EtOH,
105 °C, 2 h; (c) NaNO2, HBF4 (40% aqueous solution), KOAc, 18-Crown-6, water,
CHCl3; (d) Boc2O, DMAP, THF.
Compound 10a, an early prototype and devoid of any R3 substi-
tution, was found to potently inhibit Src and Abl with IC50s of 8.31

4910
Y. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4907–4912
Table 1
IC50 values for 9-arenethenyl purines
Compound
R1
R2
R3 or R3-Y
IC50 (nM)
K562
Src
Abl
Ba/F3(WT)
122
O
P
(B)
(A)
Me
Pr
Pr
6a
i-Pr
1.78
5.93
115
Me
HN
7a
6b
7b
A
Ph
Ph
B
B
B
i-Pr
i-Pr
i-Pr
6.58
31.8
376
54.3
73.3
626
964
>1000
>1000
O
P
(C)
Me
Me
10a
10b
A
H
H
8.31
5.56
3.58
2.77
109
109
168
200
HN
Cl
C
Me
Cl
10c
10d
C
C
H
H
126
198
69.4
16.7
1394
954
2006
1541
NH
N
NH
10e
10f
C
C
H
H
17
2.36
355
850
N
NH
<0.46
<0.46
10.5
37.3
Me
10g
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
6c
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
C
C
C
C
C
C
C
C
C
C
C
C
C
H
Me2N
4.0
3.74
1.43
17.3
17.6
19.5
3.02
6.8
368
217
89.1
286
269
123
20.8
73.7
1-Pyrrolidine
1-Morpholine
4-Methyl-1-piperazine
1-Imidazole
MeO
<0.23
<0.23
<0.46
<0.46
<0.46
<0.46
<0.46
<0.46
0.89
23.5
55.7
52.4
40.6
26.7
28.0
8.5
22.7
224
15.8
1.63
1.8
32.7
34.8
51.7
285
35.1
27.1
i-PrO
7.32
1.57
<0.46
0.50
15.8
32.3
<0.46
MeOCH2CH2O
4-(N-Me-piperidine)
3-PyO
i-Pr
Cyclopentyl
NCCH2CH2
6d
6e
1.29
<0.46
38.6
Table 2
PK properties of selected compounds
Compound
Cmax (iv)a (ng/mL)
t1/2 (iv) (h)
Vdss (L/kg)
Cmax (po)b (ng/mL)
t1/2 (po) (h)
F%
10a
10b
10f
10g
6c
4325
4589
7082
5142
1224
3.93
2.74
1.2
1.2
7.5
0.98
2.74
1.9
2.7
5.4
1277
1683
22
NA
478
5
20
17
0
0
25
3.3
NA
NA
5.1
a
Formulation: 50% DMA, 50% 9:1 Peg400/Tween 80; Conc: 2.5 mg/mL; Dose: 5 mg/kg.
fits in a small hydrophobic pocket formed by residues A293, K295
and T338. The presence of the methyl group should also introduce
bias (orthogonality) toward the preferred binding conformation.
Next, we constructed a small focused library at C2 to establish
the SAR of various linkages including N, O, and C-linked deriva-
tives. In general, a wide variety of cyclic and acyclic derivatives
yielded potent, low nanomolar inhibitors against both Src and
Abl, although substitution tended to increase potency primarily
against Src. For example, comparison of 10a, where C2 is unsubsti-
tuted (Src and Abl IC50s of 8.31 and 3.58 nM, respectively) to com-
pounds 12c–12j revealed that Src potencies increased by ꢀ10-fold
where Abl IC50s remained the same or were improved only slightly.
Analysis of the Src/AP23464 crystal structure14 revealed that R3
substituents can make good hydrophobic contact with several res-
idues on the P-loop lining the ribose pocket. By contrast, the P-loop
in Abl is folded differently, such that the side chain of Tyr272 par-
tially fills the ribose pocket, potentially clashing with larger R3
substituents.25

Y. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4907–4912
4911
Table 3
6. Schindler, T.; Bornmann, W.; Pellicena, P.; Miller, W. T.; Clarkson, B.; Kuriyan, J.
Science 2000, 289, 1938.
Kinase selectivity profile of compound 10a
7. Cowan-Jacob, S. W.; Guez, V.; Fendrich, G.; Griffin, J. D.; Fabbro, D.; Furet, P.;
Liebetanz, J.; Mestan, J.; Manley, P. W. Mini-Rev. Med. Chem. 2004, 4, 285.
8. Donato, N. J.; Wu, J.-Y.; Stapley, J.; Gallick, G.; Lin, H.; Arlinghaus, R.; Talpaz, M.
Blood 2003, 101, 690.
9. (a) Dai, Y.; Rahmani, M.; Corey, S. J.; Dent, P.; Grant, S. J. Biol. Chem. 2004, 279,
34227; (b) Donato, N. J.; Wu, J. Y.; Stapley, J.; Lin, H.; Arlinghaus, R.; Aggarwal,
B. B.; Shishodin, S.; Albitar, M.; Hayes, K.; Kantarjian, H.; Talpaz, M. Cancer Res.
2004, 64, 672; (c) Hofmann, W. K.; de Vos, S.; Elashoff, D.; Gschaidmeier, H.;
Hoelzer, D.; Koeffler, H. P.; Ottmann, O. G. Lancet 2002, 359, 481.
10. Nagar, B.; Bormann, W. G.; Pellicena, P.; Schindler, T.; Veach, D. R.; Miller, W.
T.; Clarkson, B.; Kuriyan, J. Cancer Res. 2002, 62, 4236.
Kinase
Enzyme IC50 (nM)
Kinase
Enzyme IC50 (nM)
Src (SFK)
Abl
Fms
EphB1
c-Raf
EphB4
Flt4
<0.3
1
3
Kit
Flt1
414
591
Abl(T315I)
Aurora
CDK2/CyclinA
EphA7
Flt3
>1000
>1000
>1000
>1000
>1000
>1000
6
21
30
151
221
PDGFR
a
InsR
11. Klejman, A.; Schreiner, S. J.; Nieborowska-Skorska, M.; Slupianek, A.; Wilson,
M.; Smithgall, T. E.; Skorski, T. EMBO J. 2002, 21, 5766.
12. (a) Martinelli, G.; Soverini, S.; Rosti, G.; Baccarani, M. Leukemia 2005, 19, 1872;
(b) Weisberg, E.; Manley, P. W.; Cowan-Jacob, S. W.; Hochhaus, A.; Griffin, J. D.
Nat. Rev. Cancer 2007, 7, 345; (c) Boschelli, D. H.; Boschelli, F. Drugs Future
2007, 32, 481.
13. (a) Azam, M.; Nardi, V.; Shakespeare, W. C.; Metcalf, C. A.; Bohacek, R. S.; Wang,
Y.; Sundaramoorthi, R.; Sliz, P.; Veach, D. R.; Bornmann, W. G.; Clarkson, B.;
Dalgarno, D. C.; Sawyer, T. K.; Daley, G. D. Proc. Natl. Acad. Sci. 2006, 103, 9244;
(b) Brunton, V. G.; Avizienyte, E.; Fincham, V. J.; Serrels, B.; Metcalf, C. A., III;
Sawyer, T. K.; Frame, M. C. Cancer Res. 2005, 65, 1335; (c) O’Hare, T.; Pollock, R.;
Stoffregen, E. P.; Keats, J. A.; Abdullah, O. M.; Moseson, E. M.; Rivera, V. M.;
Tang, H.; Metcalf, C. A., III; Bohacek, R. S.; Wang, Y.; Sundaramoorthi, R.;
Shakespeare, W. C.; Dalgarno, D.; Clackson, T.; Sawyer, T. K.; Deininger, M. W.;
Druker, B. J. Blood 2004, 104, 2532.
14. Dalgarno, D.; Stehle, T.; Narula, S.; Schelling, P.; Van Schravendijk, M. R.;
Adams, S.; Andrade, L.; Keats, J.; Ram, M.; Jin, L.; Grossman, T.; MacNeil, I.;
Metcalf, C.; Shakespeare, W.; Wang, Y.; Keenan, T.; Sundaramoorthi, R.;
Bohacek, R.; Weigele, M.; Sawyer, T. Chem. Biol. Drug Des. 2006, 67, 46.
15. Noronha, G.; Cao, J.; Zneg, B.; Mak, C.; McPherson, A.; Renick, J.; Pathak, V. P.;
Chow, C.; Palanki, M.; Soll, R. M.; Lohse, D. L.; Hood, J. D.; Dneprovskaia, E. US
Patent 2007/0161645 (some of the compounds disclosed in this patent have a
phenethenyl structure in the hydrophobic pocket).
16. Lombardo, L. J.; Lee, F. Y.; Chen, P.; Norris, D.; Barrish, J. C.; Behnia, K.;
Castaneda, S.; Cornelius, L. A. M.; Das, J.; Doweyko, A. M.; Fairchild, C.; Hunt, J.
T.; Inigo, I.; Johnston, K.; Kamath, A.; Kan, D.; Klei, H.; Marathe, P.; Pang, S.;
Peterson, R.; Pitt, S.; Schieven, G. L.; Schmidt, R. J.; Tokarski, J.; Wen, M.-L.;
Wityak, J.; Borzilleri, R. M. J. Med. Chem. 2004, 47, 6658.
17. Boesen, T.; Madsen, C.; Henriksen, U.; Dahl, O. J. Chem. Soc., Perkin Trans. 1 2000,
2015.
18. Wang, Y.; Huang, W.-S.; Sundaramoorthi, R.; Zhu, X.; Thomas, R. M.;
Shakespeare, W. C.; Dalgarno, D. C.; Sawyer, T. K. PCT Int. Appl., WO 2007/
021937.
19. Brun, V.; Legraverend, M.; Grierson, D. S. Tetrahedron Lett. 2001, 42, 8161.
20. Shakespeare, W. C.; Metcalf, C. A.; Sawyer, T. K.; Wang, Y.; Sundaramoorthi, R.;
Dalgarno, D. C.; Bohacek, R.; Weigele, M. Synthesis of 4-(dialkyl-phosphoryl)-
benzeneamine, PCT Int. Appl., WO 2005009348.
21. Huang, W.-S.; Wang, Y.; Sundaramoorthi, R.; Thomas, R. M.; Wen, D.; Liu, S.;
Lentini, S.; Das, S.; Banda, G.; Sawyer, T. K.; Shakespeare, W. C. Tetrahedron Lett.
2007, 48, 7388.
To assess the cellular activities of these compounds, we used
both K562 (a Bcr-Abl positive human derived CML cell line) and
Ba/F3 cells transfected with wild-type (WT) Bcr-Abl.26 Compound
10a inhibited the proliferation of K562 and Ba/F3 cell lines with
IC50s of 109 and 168 nM, respectively, which is similar to imatinib
(272 and 650 nM, respectively). The cellular activities of the com-
pounds with R1 or R3 modifications generally track well with their
Abl enzyme inhibitory potencies. The only exceptions are com-
pounds 10e and 12b; their cellular data are more in line with their
Src inhibitory activities rather than that of Abl, the reason for
which is currently unknown. One of the most potent kinase inhib-
itors of this series, compound 10g, demonstrated a 10-fold increase
in cellular potencies relative to 10a (Table 1).
To probe the ADME properties of this series we evaluated sev-
eral compounds in a rat pharmacokinetics model. Interestingly, de-
spite our early concerns about the potential metabolic instability of
the vinyl linkage, several compounds possessed good PK proper-
ties. Compounds 10a, 10b, and 6c were all characterized as having
moderate to high volumes of distribution (Vdss) and when dosed
orally, they were rapidly absorbed and demonstrated favorable
half-lives (t1/2). On average, their oral bioavailability (F%) in these
studies was 20%. Compounds 10e and 10f, both of which contain
an indazole ring, were not orally bioavailable, presumably due to
the rapid bioconjugation of the indazole NH (Table 2).
To evaluate its selectivity profile, compound 10a was screened
in an enzymatic assay against a panel of 35 tyrosine and serine–
threonine kinases.27 The data confirmed the potencies against both
Src (SFK) and Abl, and revealed low to double digit nM potencies
against Fms, EphB1, c-Raf and EphB4. The compound was inactive
against those kinases with larger gate-keeper residues such as Aur-
ora (Leu), CDK2 (Phe), InsR (Phe), and Abl containing the T315I
point mutant (Table 3).
In conclusion, a novel series of 9-arenethenyl purines possess-
ing a trans double bond were identified as potent inhibitors of
Src and Abl tyrosine kinases. Several compounds in this class po-
tently inhibited the proliferation of cell lines driven by Bcr-Abl
with at least 10-fold greater potency than imatinib. Interestingly,
the N-linked double bond was not rapidly metabolized, yielding
compounds with good pharmacokinetic properties.
22. Hutchison, A.; Yuan, J.; Lee, K.; Maynard, G.; Chenard, B. L.; Liu, N.; Guo, Q.;
Guo, Z.; Hrnciar, P. PCT Int. Appl., WO 2004043925.
23. Src enzymatic assay protocol. Inhibition of Src activity by ARIAD
Pharmaceuticals, Inc. compounds (AP compounds) was measured in
a
homogeneous time-resolved fluorescence resonance energy transfer (TR-
FRET) assay using partially purified full length human Src from a baculovirus
expression system (Upstate Biotechnology Inc.) and a biotinylated phospho-
kinase substrate peptide (amino acid Sequence—Biotin-KVEKIGEGTYGVVYK-
NH2) as the substrate peptide (Pierce). Kinase reactions were carried out in
complete LANCE kinase buffer (LKB); 20 mM Na–Hepes, pH 7.4, 0.1 mg/mL
BSA, 1 mM ATP, 10 mM MgCl2, 0.41 mM DTT, in round-bottomed black 96-well
Microfluor plates (Dynex Technologies Inc.) pre-blocked with 1% BSA in PBS at
4 °C overnight. For the kinase reaction, compound dilutions were incubated
with Src (165 pM) and substrate peptide (50 nM) for 2 h at 37 °C in a total
volume of 0.1 mL LKB. The kinase reaction was terminated by addition of
0.05 mL of a kill/detection solution containing 15 lM of a potent ARIAD kinase
Acknowledgements
inhibitor, 6 nM Europium-labeled anti-phosphotyrosine mAb PT66 and 60 nM
allophycocyanin-labeled streptavidin (LANCETM reagents from Perkin-Elmer
Inc.). Assay plates were incubated at room temperature in the dark for 20 min
prior to reading fluorescence at 615 and 665 nm on a Victor2V plate reader
The authors thank Narayana Narasimhan, Patricia Ryan, Qurish
Mohemmad, and Manfred Weigele from ARIAD for useful
discussions.
(Perkin-Elmer). Positive and negative controls and
a standard curve for
phosphorylated PKS1 peptide were included on each plate. Data values were
transferred to an Excel spreadsheet and IC50s were calculated from the
fluorescence at 665 nm by interpolation between the averaged duplicate well
data on 3-fold serial dilutions of AP Compounds. No compound effects on the
615 nm fluorescence were observed.
References and notes
1. Thomas, S. M.; Brugge, J. S. Annu. Rev. Cell Dev. Biol. 1997, 13, 513.
2. Summy, J. M.; Gallick, G. E. Cancer Metastasis Rev. 2003, 22, 337.
3. Sawyer, T.; Boyce, B.; Dalgarno, D.; Iuliucci, J. Expert Opin. Investig. Drugs 2001,
10, 1327.
4. Shah, Y. M.; Rowan, B. G. Mol. Endocrinol. 2005, 19, 732.
5. Pengetnze, Y.; Steed, M.; Roby, K. F.; Terranova, P. F.; Taylor, C. C. Biochem.
Biophys. Res. Commun. 2003, 309, 377.
24. The Abl enzymatic assay protocol is described in Ref. 15.
25. The Abl/10f structure was resolved and will be published elsewhere.
26. K562 and Ba/F3 cell assay format. For the cell proliferation assay, the K-562
human wild-type Bcr-Abl CML cell line and the murine pro-B Ba/F3 cell line
stably transfected with a construct expressing wild-type Bcr-Abl were used. K-
562 was obtained from the American Type Culture Collection (ATCC) and
maintained in Iscove’s modified Dulbecco’s medium with 10% FBS. The Ba/F3

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Y. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4907–4912
cell line was obtained from Brian J. Druker (Howard Hughes Medical Institute,
and then the plates were returned to the incubator at 37 °C for 2 h. The
absorbance in each well was then measured at 490 nm using a Wallac Victor2V
plate reader. The IC50 was calculated by determining the concentration of
compound required to decrease the MTS signal by 50% in best-fit curves using
Microsoft XLfit software, by comparing with baseline, the DMSO control, as 0%
inhibition.
Oregon Health and Science University, Portland, OR, USA) and was maintained
in RPMI 1640 growth medium with 10% FBS. All cells were incubated at 37 °C
in 5% CO2. After the compounds were incubated with the cells for 3 days, the
number of viable cells in each well in 96-well plate was measured using an
MTS assay (Promega). This assay is a colorimetric method for determining the
number of viable cells through measurement of their metabolic activity, which
can be detected by absorbance at 490 nm. MTS reagent was added to all wells
27. The KinaseProfilerTM assay service from Millipore was used. Assay protocols are
available at http://www.millipore.com/drug discovery/svp3/profilerxl.