Synthesis and biological evaluation of analogues of the kinase inhibitor nilotinib as Abl and Kit inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.11.111

The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced b

Full text of DOI:10.1016/j.bmcl.2012.11.111

Bioorganic & Medicinal Chemistry Letters 23 (2013) 682–686
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of analogues of the kinase
inhibitor nilotinib as Abl and Kit inhibitors
Damien Y. Duveau a, Xin Hu a, Martin J. Walsh a, Suneet Shukla b, Amanda P. Skoumbourdis a,
Matthew B. Boxer a, Suresh V. Ambudkar b, Min Shen a, Craig J. Thomas a,
⇑
a National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, United States
b Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investi-
gated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues
where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these ana-
logues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the
monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a–c to the bind-
ing pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the
stabilizing effect from its juxtamembrane region.
Received 25 October 2012
Revised 20 November 2012
Accepted 26 November 2012
Available online 10 December 2012
Keywords:
CML
Kit
Published by Elsevier Ltd.
Abl
Kinase
Molecular editing
The receptor tyrosine kinase Kit (also known as c-Kit, or CD117)
is involved in a host of cellular processes, including cell differenti-
ation, proliferation and survival.1 Kit is found on the surface of
hematopoietic stem cells (HSC), common myeloid progenitors,
common lymphoid progenitors, myeloblasts, megakaryocytes,
and mast cells. Upon binding of its ligand, the stem cell factor
(SCF), the receptor dimerizes, leading to the activation of the cyto-
plasmic kinase domain. The phosphorylated tyrosine residues of
Kit are then involved in a series of signal transduction events
including the PI3K pathway, the Jak–Stat pathway, Src-family ki-
nases, and the Ras–Erk pathway.2 Gain-of-function mutations of
Kit are associated with a number of cancers such as acute myeloid
leukemia (AML), chronic myeloid leukemia (CML), mast cell leuke-
mia, and gastrointestinal stromal tumors (GIST). Another cause of
CML is the oncogenic breakpoint cluster region protein-abelson ki-
nase (Bcr–Abl) fusion protein, caused by the Philadelphia chromo-
some.3 Bcr–Abl becomes constitutively active, and unregulated
phosphorylation of proteins found in HSC leads to uncontrolled
growth and survival.
was observed in patients because of mutations of the Abl protein.
The second generation kinase inhibitor nilotinib (Tasigna,
Novartis) (Fig. 1) also targets Abl and Kit and was approved in
2007 for the treatment of CML. The drug was then advanced to
phase III clinical trials for the treatment of GIST resistant to imati-
nib, but was discontinued in 2011 because of a lack of superior effi-
cacy compared to the current standard of care. Understanding the
polypharmacology of nilotinib (and other kinase inhibitors) is par-
amount to understanding their ultimate utility, and it is likely that
the capacity for nilotinib to target both Abl and Kit, along with
other kinases such as PDFRa, accounts for the clinical utility of
the drug. Pathway redundancy via Kit signaling, in particular,
may abrogate Bcr–Abl inhibition requiring that Kit must be inhib-
ited in addition to Bcr–Abl for efficacy to be realized.4
Molecular design of nilotinib (1) was based upon the crystal
structure of the imatinib–Abl complex. Binding to the inactive
form of Abl was maintained through the [4-(3-pyridinyl)-2-
pyrimidinyl]aniline portion of imatinib and of nilotinib. However,
nilotinib relies on fewer hydrogen-bonding interactions with
amino acid residues of the hinge region of Abl, and on a larger
number of hydrophobic interactions. Such van der Waals interac-
tions are found by the 4-methyl imidazole moiety and especially
by the trifluoromethyl group. In fact, structural studies have shown
that one of the CF3 fluorines interacts with the carbonyl of the
Asp381 backbone in Abl.5 Olsen showed that protein backbone
Imatinib (Gleevec, Novartis) (Fig. 1) is an inhibitor of the
Bcr–Abl fusion protein, Kit and of the receptor tyrosine kinases
platelet-derived growth factor receptor subtype alpha (PDGFRa).
It was approved in 2001 for the treatment of CML, and in 2003
for the treatment of GIST. Despite its success, resistance to the drug
fragments H–C
a–C@O provide a favorable ‘fluorophilic’ environ-
⇑
Corresponding author. Tel.: +1 301 217 4079; fax: +1 301 217 5736.
E-mail address: craigt@mail.nih.gov (C.J. Thomas).
ment for the C–F bond, including multipolar interactions between
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.11.111

D. Y. Duveau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 682–686
683
N
N
CF3
N
N
N
N
N
O
H
N
HN
N
HN
N
H
N
O
N
Imatinib
Nilotinib (1)
Figure 1. Structure of imatinib and of nilotinib.
N
N
CF3
CF3
N
N
N
N
N
O
N
H
N
HN
HN
N
H
N
O
N
NS-187
Nilotinib (1)
CF3
CF3
O
O
Cl
N
O
N
N
H
O
HN
N
N
S
N
N
H
N
N
H
H
O
HG-7-85-01
Sorafenib
Figure 2. Structures of kinase inhibitors bearing a trifluoromethyl group.
the intrinsically polar C–F and C@O bonds.6 A significant number of
molecular probes and drug candidates targeting kinases feature a
trifluoromethyl group (Fig. 2). Examples include NS1877 (a Bcr–
Abl and Lyn kinase inhibitor), HG-7-85-018 (a Bcr–Abl, Kit, PDGFR,
and Src family kinase inhibitor), and sorafenib9 (Nevaxar, Bayer/
Onyx)(a c-Raf, Kit, isoform specific VEGFR inhibitor). Since protein
kinases display a high degree of homology at their active sites,
ATP-competitive small molecule kinase inhibitors often possess
activity against several targets. In the particular case of nilotinib,
the activity profile is well established.10 In this study, we endeav-
ored to define the influence of the trifluoromethyl group present in
nilotinib with respect to its polypharmacology.
A modular synthetic sequence was developed to access nilotinib
analogues (Scheme 1). The syntheses of analogues 2a–c com-
menced with the Ullman-type coupling between aryl iodide 3a–c
and 4-methyl-1H-imidazole, using a procedure developed by
Buchwald and co-workers.11 Three challenges associated with this
reaction were (1) the poor solubility of the reagents in the reaction
mixture, (2) the lack of reactivity of the starting aryl iodide
(especially 3b), and (3) the possible isomerization of 4-methyl-
1H-imidazole during the reaction. Using the more soluble cesium
carbonate as base and polyethylene glycol as a co-solvent to aceto-
nitrile improved the solubility of the reagents, affording a clear
reaction solution when stirred at reflux. Activation of the copper
catalyst with 4,7-dimethoxy-1,10-phenanthroline12 allowed not
only the reaction to proceed in higher yields than when using
copper(I) iodide and ethylene diamine were used, but also led to
the formation of N-aryl imidazoles 4a–c as single isomers. The
nitro group was reduced using tin(II) chloride-dihydrate in metha-
nol. Formation of the amide bond between benzoic acid 6 and ani-
lines 5a–c was more difficult than expected. Common procedures
such as EDCI or DCC coupling in DCM, DCE, or acetonitrile failed;
HATU coupling in DMF was not successful either; deprotonation
of the aniline using potassium tert-butoxide in THF, followed by
treatment of the resulting anilide13 with the methyl ester of 6 also
failed. Eventually, conversion of 6 to the corresponding acyl chlo-
ride, using thionyl chloride in the polar aprotic solvent NMP, fol-
lowed by addition of anilines 5a–c provided nilotinib analogues
2a–c.14 The low solubility of 5a–c and of 6 in solvents such as
DCM, DCE or THF, along with the poor reactivity of anilines 5a–c
may account for the difficulty to form the amide bond. The nature
of the substitution on the aromatic ring of aryl iodide 3 had a great
influence on its reactivity, as well as the reactivity of its subsequent
synthetic intermediates. Thus, fluorinated aryl imidazole 4b was
consistently obtained in lower yield, as compared to 4a and 4c. Sim-
ilarly, aniline 5b was less reactive toward the acyl chloride of 6 in
the final coupling step leading to analogue 2b. Dilution of the reac-
tion mixture with ice-cold water led to the precipitation of the
crude products which could then be collected by filtration. The sol-
ids thus obtained were finally purified by reverse-phase HPLC.
Utilizing two commercial organizations (Reaction Biology Corp.
www.reactionbiology.com and DiscoveRx http://www.kinome-
scan.com/), we generated IC50 and Kd values for nilotinib (1) and
analogues 2a–c for a panel of kinases (Table 1). The activity profile
of nilotinib versus a broad panel of kinases had been reported by
Davis et al.15 and those results were used as a starting point in
our study to focus on the kinases involved in the signaling path-
ways primarily targeted by nilotinib.
The results obtained in both enzymatic assays were in good
agreement with those previously reported for nilotinib.16 When
comparing the four compounds activities within the functional as-
say (Table 1), nilotinib (1) was the most active toward Abl by a
hundred fold (IC50 <2.5 nM). The second most active analogue
was methyl analogue 2c (IC50 = 135.8 nM), followed by 2b
(IC50 = 750 nM) and 2a (IC50 = 2953 nM). These results underscore
the importance of a methyl or of a trifluoromethyl group for the

684
D. Y. Duveau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 682–686
Cs2CO3, Cu2O
R
SnCl2-2H2O
MeOH, 70 °C
4,7-dimethoxy-1,10-phenanthroline
R
R
PEG-CH3CN, 80 °C
HN
+
O2N
I
N
O2N
N
H2N
N
N
N
R= H
R= H
R= F
R= CH3
R= H
4a
4b
4c
5a
3a
R= F
R= F
3b
3c
5b
5c
R= CH3
R= CH3
1-acid, SOCl2, NMP, 80 °C
2-5a-c, NMP, 60 °C
3-H2O, 5 °C
N
N
R
N
N
N
N
O
HN
CO2H
HN
N
H
N
N
6
R = H
R = F
R = CH3
2a
2b
2c
Scheme 1. Syntheses of analogues 2a–c.
Table 1
Table 2
Inhibitory activities of compounds 1 and 2a–c
Dissociation constants of compounds 1 and 2a–c
a
a
Kinase
Compound IC50 (nM)
Kinase
Compound Kd (nM)
1
2a
2b
2c
1
2a
2b
2c
Abl
Kit
DDR2
FLT3
LCK
LYN
PDGFR
PDGFRb
<2.54
14.70
<2.54
9548
108.2
1281
<2.54
60.11
2953
749.8
9.96
135.8
54.94
17.22
882.0
1448
3534
8.20
Abl
Kit
DDR2
FLT3
PDGFR
3.6
17
6.2
40,000
74
29
9.4
3.5
14
58
24
3.4
14
5.6
1200
65
32.50
653.3
973.9
44,620
45,420
83.87
35,400
4.4
320
1400
14
178.6
117.8
11,050
15,150
25.96
>5000
a
PDGFRb
16
7.2
8.5
12
a
a
Assay details are provided by Reaction Biology Corp. via the internet at http://
www.kinomescan.com/.
768.4
a
Assay details are provided by Reaction Biology Corp. via the internet at
www.reactionbiology.com.
a
stronger binding affinity than unsubstituted analogue 2a
binding of the compound to Abl, as reported by Manley et al.5 The
influence of the trifluoromethyl group, and, to a lesser extent, of
the methyl group was also observed for the inhibition of the dis-
coin domain receptor subtype 2 (DDR2), the lymphocyte-specific
tyrosine kinase (LCK) and for the inhibition of LYN: once again, nil-
otinib was the most active compound, followed by methyl ana-
logue 2c. Regarding the platelet-derived growth factor receptor
(PDGFR), nilotinib (1) and methyl analogue 2c were the most active
(IC50 <2.54 and 8.20 nM respectively), compared to 2a and 2b (IC50
<83.87 and 25.96 nM, respectively). The comparative activity be-
tween methylated and non-methylated analogues was particularly
noticeable with PDGFRb (IC50 <60.11 nM for 1 and 768.4 nM for 2c
vs 35400 nM for 2a and >50,000 nM for 2b). On the other hand, Nil-
otinib and analogues 2a–c displayed good inhibitory activity to-
ward Kit, suggesting that unlike Abl, the presence of a methyl or
of a trifluoromethyl group was not required. The same structure–
activity relationship was observed for the four compounds versus
the Fms-like tyrosine kinase 3 (FLT3), with fluorinated analogue
2c being the most active compound (IC50 = 117.8 nM). While 2a
and 2c displayed comparable activites (IC50 = 973.9 and 882.0 nM
respectively), no correlation was observed between 2c and 1
(IC50 = 9548 nM).
(Kd = 9.4 nM for 2b and Kd = 29 nM for 2a). This result may imply
the presence of van der Waals interactions between the fluorine
and a carbonyl of the backbone in the hydrophobic pocket. In addi-
tion, compounds 2a and 2b showed the strongest affinity for Kit
(4.4 and 3.5 nM respectively). A good correlation between the mea-
surement of the inhibition of kinase activity (IC50) and that of the
thermodynamic binding affinity (Kd) was observed for Abl, Kit,
DDR2, and FLT3. While the level of selectivity between compounds
1 and 2a–c was not high in the panel of kinases tested (the Kd val-
ues were in the same order of magnitude), a remarkable difference
in affinity of the fluorinated analogue toward FLT3, whereby fluo-
rinated analogue 2b showed a tighter binding by a factor twenty
compared to the other compounds tested. This result supports
the selective inhibitory activity of 2b as compared to the other ana-
logues (Table 1).
Taken together, those results show that the four compounds
tested fell into two categories: ‘methylated’ analogues (1 and 2c)
versus ‘non methylated’ analogues (2a and 2b), each category pos-
sessing its own selectivity profile. In addition the influence of a
fluorine further tuned the activity profile of the four analogues.
For instance, nilotinib (1) is selective toward Abl and PGDFR
while 2b is more selective toward Kit and FLT3.
a,
The binding modes of these nilotinib analogues to Abl and Kit
were generated by docking them into the binding site followed by
energy minimization refinement. As shown in Figure 3, the small
molecule inhibitor occupied in the ATP binding pocket of Kit17,18
The binding affinity data (Table 2) showed that nilotinib (1) and
methylated analogue 2c displayed stronger binding affinities to-
ward Abl (Kd = 3.6 nM for 1 and Kd = 3.4 nM for 2c), although the
Kd values for 2a and 2b were close. Fluorinated analogue 2b had

D. Y. Duveau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 682–686
685
Figure 3. Predicted binding mode from AutoDock: (A) compound 2c bound in the active site of Abl, (B) nilotinib bound in Kit and (C) structural overlay of the hydrophobic
pocket between Kit and Abl. Compound 2a is illustrated in the binding complex. Residues of Abl are labeled in green and residues of Kit are labeled in black. Surface of the
binding pocket of Abl is colored in cyan and the surface of Kit is colored in yellow.
in a similar manner as observed in the complex of Abl/nilotinib. Key
interactions such as binding at the hinge region, H-bonding with
the gatekeeper Thr-670 (Kit), and aromatic stacking interaction
with Phe-811 of the DFG motif are typically retained in these
kinases. Another remarkable binding feature associated with these
compounds is that the edited group was orientated into a hydro-
phobic pocket at the bottom of the cleft, which is formed by a num-
ber of hydrophobic residues and highly conserved in Abl and Kit
(Fig. 3). The trifluoromethyl group of nilotinib appears to fit more
favorably into the pocket of Abl as compared to the proton and
the fluoride substituent of compounds 2a and 2b that had a signif-
icantly decreased activity. Compared to Abl, the hydrophobic pock-
et in Kit is relatively small (estimated cavity volume is 755.4 and
822.5 Å3 for Kit and Abl, respectively), suggesting that it may have
contributions to the differential activities found herein.
The binding and functional activities of nilotinib (1) and 2a–c
versus Kit suggest that this kinase is much more tolerant of struc-
tural deviance at this specific molecular site. The protonated com-
pound 2a, while retained the same activity as nilotinib to Kit,
exhibited ꢀ100 fold higher selectivity over Abl. This raised an
interesting issue, as the binding interactions of 2a in Kit and Abl
are highly conserved. Further analysis indicated that Kit possesses
a unique juxtamembrane region at the N-terminal end followed by
the kinase domain, which extends to the back door of the solvent
exposed region of the substrate binding pocket. It is likely that
the juxtamembrane loop participates in the binding interaction
by stabilizing the inhibitor in the pocket. To test this hypothesis,
MD simulations were performed to investigate the dynamics of
inhibitor binding. As shown in Figure 4, the complex of 2a bound
to Abl exhibited high flexibility, but was significantly stabilized
in the Kit binding complex. In contrast, the complexes of nilotinib
in Abl and Kit were comparable and remained stable during the
simulations. Therefore, a plausible explanation for the higher
inhibitory activity and selectivity of these analogues to Kit is that,
Figure 4. RMSD plot of the inhibitor binding complexes with (A) Abl and (B) Kit
from MD simulations.
while the trifluromethyl group plays a dominant role in nilotinib
binding to Abl, lack of such van der Waals interaction and shape
complementarity in Kit is largely compensated by the stabilizing
effect from the juxtamembrane region.
To gain further insight into this possible explanation we exam-
ined nilotinib (1) and 2a–c within a series of functional assays at
selected, well characterized Kit mutants (D816V, D816H, T670I,
V560G, V654A) (Reaction Biology Corp. www.reactionbiology.
com). Residue Asp-816 lies in front of the DFG motif in the activa-
tion loop. Though it is not directly involved in the binding interac-
tions with inhibitors, mutation of this residue with a His or Val is
expected to affect the conformation of the activation loop and, con-
sequently the binding affinity of small molecules. This was, in fact,
>200 nM versus D816H. The gatekeeper residue Thr-670 forms a
hydrogen bond and plays a vital role in inhibitor binding and each
agent in this study lost activity (>1 lM) versus the T670I mutant. It
is worth noting that mutant T670I, while disrupting the key H-
bonding interaction, also likely poses a steric hindrance to inhibitor
in the pocket. Val-654 is found at the bottom of the binding pocket
which contributes to the hydrophobic interaction. The decreased
activity of these compounds (1, 2a and 2c >1 lM; 2b >400 nM)
against the mutant V654A corroborated the importance of the
found with each agent possessing >1 lM activity versus D816V and
hydrophobic interaction in the pocket. More interestingly, these

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D. Y. Duveau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 682–686
2. Roskoski, R. Biochem. Biophys. Res. Commun. 2005, 337, 1.
3. Kurzrock, R.; Kantarjian, H. M.; Druker, B. J.; Talpaz, M. Ann. Intern. Med. 2003,
138, 819.
4. Belloc, F.; Airiau, K.; Jeanneteau, M.; Garcia, M.; Guerin, E.; Lippert, E.; Moreau-
Gaudry, F.; Mahon, F. X. Leukemia 2009, 23, 679.
analogues showed increased inhibitory activities to mutant V560G
(nilotinib (1) and 2a–c <10 nM). Val560 lies in the juxtamembrane
region and a replacement of Val to Gly may possibly make the juxt-
amembrane loop more flexible allowing more facile coverage of
the solvent-exposed region and further stabilize inhibitor binding.
In conclusion, the influence of the trifluoromethyl group in the
activity profile of nilotnib was investigated, by synthesizing ana-
logues of nilotinib bearing a fluorine (2b), a methyl group (2c), or
no substituent (2a) in that position. Presence of a trifluoromethyl
group in nilotinib (1), and of a methyl group in 2c, leads to tight fit-
ting in Abl, and probably in DDR2 as well. Furthermore, the trifluo-
romethyl group allows van der Waals interactions between one of
the fluorine and the one of the carbonyl backbone located in the
hydrophobic pocket adjacent to the hinge region in Abl, thus fur-
ther enhancing the selectivity of nilotnib toward this kinase. On
the other hand, the corresponding hydrophobic pocket found in
Kit is larger in size, accommodating each one of the four com-
pounds studied. However, the role juxtamembrane region in Kit
may account for the higher binding affinity of 2a and 2b toward
Kit. Fluorinated analogue 2b possesses a good inhibitory activity
toward Kit and FLT3, and binds tightly to those two kinases. Since
kinases possess a high degree of homology, taking advantage of the
subtle difference between a methyl and a trifluoromethyl group, or
between a proton and fluorine may allow tuning of the pharmaco-
logical profile of kinase inhibitors.
5. Manley, P. W.; Cowan-Jacob, S. W.; Fendrich, G.; Mestan, J. Blood 2005, 106,
940A.
6. Olsen, J. A.; Banner, D. W.; Seiler, P.; Wagner, B.; Tschopp, T.; Obst-Sander, U.;
Kansy, M.; Müller, K.; Diederich, F. ChemBioChem 2004, 5, 666.
7. Kimura, S.; Naito, H.; Segawa, H.; Kuroda, J.; Yuasa, T.; Sato, K.; Yokota, A.;
Kamitsuji, Y.; Kawata, E.; Ashihara, E.; Nakaya, Y.; Naruoka, H.; Wakayama, T.;
Nasu, K.; Asaki, T.; Niwa, T.; Hirabayashi, K.; Maekawa, T. Blood 2005, 106,
3948.
8. Weisberg, E.; Choi, H. G.; Ray, A.; Barrett, R.; Zhang, J.; Sim, T.; Zhou, W.;
Seeliger, M.; Cameron, M.; Azam, M.; Fletcher, J. A.; Debiec-Rychter, M.;
Mayeda, M.; Moreno, D.; Kung, A. L.; Janne, P. A.; Khosravi-Far, R.; Melo, J. V.;
Manley, P. W.; Adamia, S.; Wu, C.; Gray, N.; Griffin, J. D. Blood 2010, 115, 4206.
9. Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.; Chen, C.;
Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.;
Rowley, B.; Liu, L.; Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.;
Voznesensky, A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. Cancer Res. 2004, 64,
7099.
10. Weisberg, E.; Manley, P.; Mestan, J.; Cowan-Jacob, S.; Ray, A.; Griffin, J. D. Br. J.
Cancer 2006, 94, 1765.
11. Altman, R. A.; Koval, E. D.; Buchwald, S. L. J. Org. Chem. 2007, 72, 6190.
12. Altman, R. A.; Buchwald, S. L. Org. Lett. 2006, 8, 2779.
13. Ueda, S.; Su, M. J.; Buchwald, S. L. J. Am. Chem. Soc. 2012, 134, 700.
14. Analogue 2a: 1H NMR (400 MHz, DMSO-d6) d 10.54 (s, 1H), 9.53 (s, 1H), 9.28 (s,
1H), 9.17 (s, 1H), 8.70 (d, J = 4.8 Hz, 1H), 8.55 (d, J = 5.1 Hz, 1H), 8.48 (d,
J = 8.0 Hz, 1H), 8.32 (t, J = 2.1 Hz, 1H), 8.29 (d, J = 1.9 Hz, 1H), 7.95 (s, 1H), 7.81
(d, J = 8.4 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.61 (t, J = 8.1 Hz, 1H), 7.58–7.52 (m,
1H), 7.49 (d, J = 5.1 Hz, 1H), 7.47 (m, 1H), 7.44 (d, J = 8.4 Hz, 1H), 2.36 (s, 3H),
2.36 (s, 3H).
Analogue 2b: 1H NMR (400 MHz, DMSO-d6) d ppm 10.69 (s, 1H), 9.52 (d,
J = 1.8 Hz, 1H), 9.30 (d, J = 1.6 Hz, 1H), 9.19 (s, 1 H), 8.72 (dd, J = 4.8, 1.5 Hz, 1H),
8.56 (d, J = 5.1 Hz, 1H), 8.51 (ddd, J = 8.2, 2.0, 1.8 Hz, 1H), 8.30 (d, J = 1.8 Hz, 1H),
8.12 (m, 1H), 7.95(m, 1H), 7.79 (m, 1H), 7.75 (dd, J = 8.0, 2.0 Hz, 1H), 7.59 (dd,
J = 8.1, 4.8 Hz, 1H), 7.50 (d, J = 5.3 Hz, 1H), 7.49 (dt, J = 9.4, 2.2 Hz, 1H), 7.45 (d,
J = 8.2 Hz, 1H), 2.36 (s, 3H), 2.35 (s, 3H).
Acknowledgments
We thank Jim Bougie, Thomas Daniel and William Leister for
compound purification, as well as Paul Shinn, Danielle VanLeer
and Christopher LeClair for assistance with compound manage-
ment. This research was supported by the Molecular Libraries Ini-
tiative of the National Institutes of Health Roadmap for Medical
Research Grant U54HG005033 and the Intramural Research Pro-
gram of the National Human Genome Research Institute at the Na-
tional Institutes of Health. S. Shukla and S.V. Ambudkar were
supported by the Intramural Research Program of the NIH, Na-
tional Cancer Institute, Center for Cancer Research.
Analogue 2c: 1H NMR (400 MHz, DMSO-d6) d 10.45 (s, 1H), 9.52 (s, 1H), 9.28 (s,
1H), 9.16 (s, 1H), 8.71 (m, 1H), 8.55 (d, J = 5.1 Hz, 1H), 8.48 (d, J = 7.9 Hz, 1H),
8.28 (d, J = 1.9 Hz, 1H), 8.12 (m, 1H), 7.94 (m, 1H), 7.74 (m, 1H), 7.65 (s, 1H),
7.55 (m, 1H), 7.49 (d, J = 5.2 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.32 (s, 1H), 2.40 (s,
3H), 2.35 (s, 6H).
15. Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.; Pallares, G.;
Hocker, M.; Treiber, D. K.; Zarrinkar, P. P. Nat. Biotechnol. 2011, 1046, 29.
16. Manley, P. W.; Cowan-Jacob, S. W.; Fendrich, G.; Jahnke, W.; Fabbro, D. Blood
2008, 118, 727.
17. Mol, C. D.; Dougan, D. R.; Schneider, T. R.; Skene, R. J.; Kraus, M. L.; Scheibe, D.
N.; Snell, G. P.; Zou, H.; Sang, B.-C.; Wilson, K. P. J. Biol. Chem. 2004, 279,
31655.
18. Mol, C. D.; Lim, K. B.; Sridhar, V.; Zou, H.; Chien, E. Y. T.; Sang, B.-C.;
Nowakowski, J.; Kassel, D. B.; Cronin, C. N.; McRee, D. E. J. Biol. Chem. 2003, 278,
31461.
References and notes
1. Lennartsson, J.; Jelacic, T.; Linnekin, D.; Shivakrupa, R. Stem Cells 2005, 23, 16.