Rapid synthesis of Abelson tyrosine kinase inhibitors using click chemistry

Article abstract of DOI:10.1039/b913333j

Protein kinases catalyze the phosphorylation of serine, threonine, tyrosine and histidine residues in proteins. Aberrant regulation of kinase activity has been implicated in many diseases including cancer. Thus development of new strategies for kinase inhibitor design remains an active area of research with direct relevance to drug development. Abelson (Abl) tyrosine kinase is one of the Src-family of tyrosine kinases and is directly implicated in Chronic Myelogenous Leukemia (CML). In this article, we have, for the first time, developed an efficient method for the construction of small molecule-based bisubstrate inhibitors of Abl kinase using click chemistry. Subsequent biochemical screenings revealed a set of moderately potent inhibitors, a few of which have comparable potency to Imatinib (an FDA-approved drug for treatment of chronic myeloid leukemia) against Abl.

Full text of DOI:10.1039/b913333j

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Rapid synthesis of Abelson tyrosine kinase inhibitors using click chemistry†
Karunakaran A. Kalesh,a Kai Liub and Shao Q. Yao*a,b,c
Received 6th July 2009, Accepted 15th September 2009
First published as an Advance Article on the web 14th October 2009
DOI: 10.1039/b913333j
Protein kinases catalyze the phosphorylation of serine, threonine, tyrosine and histidine residues in
proteins. Aberrant regulation of kinase activity has been implicated in many diseases including cancer.
Thus development of new strategies for kinase inhibitor design remains an active area of research with
direct relevance to drug development. Abelson (Abl) tyrosine kinase is one of the Src-family of tyrosine
kinases and is directly implicated in Chronic Myelogenous Leukemia (CML). In this article, we have,
for the first time, developed an efficient method for the construction of small molecule-based
bisubstrate inhibitors of Abl kinase using click chemistry. Subsequent biochemical screenings revealed a
set of moderately potent inhibitors, a few of which have comparable potency to Imatinib (an
FDA-approved drug for treatment of chronic myeloid leukemia) against Abl.
potency. Successful implementation of this method, however,
Introduction
largely depends on the available knowledge of target kinase
Protein kinases catalyze the phosphorylation of serine, threonine,
secondary site-binding scaffolds, which is so far quite limited. Thus
tyrosine and histidine residues in proteins. Reversible phosphory-
combinatorial approaches are necessary to identify such scaffolds
and to generate new bisubstrate inhibitors.6
lation is one of the major modes of cellular signal transduction
and plays regulatory roles in most metabolic pathways. Hence,
virtually every aspect of cellular physiology is inherently linked
to the proper functioning of these enzymes.1 Aberrant regulation
of kinase activity has been implicated in many diseases including
cancer.2 Thus development of new strategies for kinase inhibitor
design remains an active area of research with direct relevance
to drug development. The majority of kinase inhibitors target
the ATP-binding site of the enzymes and, due to the highly
conserved nature of the ATP-binding pocket amongst the various
kinases, such inhibitors usually lack selectivity. An inhibitor that
exploits binding pockets other than the ATP-binding site could
potentially override this problem, as such secondary pockets
(e.g. substrate-binding or allosteric pockets) are less likely to
be conserved. But there exist relatively few strategies for the
rational design of such inhibitors.3-5 The most commonly used
approach makes use of large libraries of synthetic compounds
and natural products coupled with cell-based assays but has
so far, to the best of our knowledge, yielded only a limited
number of hits.4,5 The recent development of kinase inhibitors
with bisubstrate mode of inhibition exploiting both the ATP-
binding site and an allosteric site or a substrate-binding site opens
up exciting opportunities for the rational design of bisubstrate
inhibitors.3,6 The strategy works by simply appending an inhibitor
scaffold that targets the ATP-binding site of a kinase to another
inhibitor scaffold that targets the allosteric/substrate-binding site,
resulting in a bisubstrate inhibitor with improved selectivity and
In this paper we report, for the first time, the use of the highly
modular and efficient click chemistry to assemble bisubstrate
inhibitors of the Abelson (Abl) tyrosine kinase. The approach
is generally applicable for the development of inhibitors against
other protein kinases.
Results and discussion
Abl kinase
The cellular form of Abl is a close relative of the Src family
of tyrosine kinases. It consists of approximately 1150 residues
with two regulatory domains (SH3 and SH2) and a catalytic
domain (SH1) with 47% sequence homology with Src.7 But unlike
Src, the c-Abl kinase adopts a unique autoinhibitory mechanism
involving the interaction of an N-terminal myristoyl group with
a C-terminal myristoyl binding cleft.7,8 This autoinhibition is of
paramount importance as the aberrant activity of this kinase in
a fusion protein form (Bcr-Abl) is directly implicated in Chronic
Myelogenous Leukemia (CML).9 The remarkable clinical success
of the drug Imatinib (or STI571, GleevecTM) and the recent cases
of drug resistance due to mutations in the BCR-Abl gene have
accelerated the search for new Abl inhibitors.10 New development
of Imatinib analogues and molecules possessing unique inhibitory
mechanisms against Abl may offer promising approaches to
tackling the drug resistance problem.11
aDepartment of Chemistry, National University of Singapore, 3 Science Drive
3, Singapore 117543. E-mail: chmyaosq@nus.edu.sg; Fax: (+65) 67791691;
Tel: (+65) 65162925
First-generation “click” inhibitors
We decided to develop a click chemistry-based strategy to generate
potential bisubstrate inhibitors against Abl as the strategy had
previously been shown to be highly modular and efficient for rapid
assembly of bidentate inhibitors against many other enzymes.12,13
We first investigated the possibility of assembling bisubstrate
inhibitors of kinases by using ADP-alkyne, as a promiscuous and
bDepartment of Biological Sciences, National University of Singapore,
Singapore 117543
cNUS MedChem Program of the Office of Life Sciences, National University
of Singapore, Singapore 117543
† Electronic supplementary information (ESI) available: Detailed experi-
mental procedures and results. See DOI: 10.1039/b913333j
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 5129–5136 | 5129
©

View Article Online
easy-to-synthesize ATP surrogate, and previously reported generic
azides to generate a 344-member library (Fig. 1).14
library of azides was synthesized via a highly efficient solid-phase
traceless protocol we recently developed.14 The click assembly of
the two fragments was then carried out in a 384-well plate in
a mixed solvent system containing t-BuOH, water and DMSO
(1 : 1 : 0.1) with CuI (0.1 eq.) as catalyst and tributylamine (0.2 eq.)
as Cu(I) stabilizing base. The triazole formation was found to
complete in 24 h. The crude reactions were evaporated to dryness
and redissolved in DMSO to prepare standard solutions for kinase
inhibition assays and LC-MS characterizations.
Upon “click” assembly, the 344-member library was screened
for inhibition against Abl and Src kinases using a non-radioactive
luminescence-based kinase assay (Fig. 1b); unique and distinctive
inhibitor fingerprints were obtained for the library against the two
kinases. Detailed analysis of the inhibition profiles revealed a clear
preference for short-chain linkers by the Abl inhibitors whereas no
such linker length preference was observed for Src inhibitors. The
preference of the Abl kinase for short-chain azides may point to
the presence of secondary binding sites not too far from its ATP-
binding site. We noted that, in future, this generic 344-member
ADP library might be used as a platform for high-throughput
identifications of preliminary bisubstrate inhibitors against other
protein kinases.
Second-generation “click” inhibitors
Having successfully established the platform for “click” assembly
of bisubstrate kinase inhibitors, we next focused on Abl kinase. As
shown in Scheme 1, we replaced the promiscuous, weak binding,
ADP-alkyne warhead with the head portion of Imatinib, as this
was reasoned to improve the selectivity as well as potency of the
resulting inhibitors. Compared to the ADP-alkyne click library,
the new click products should also be more ‘drug-like’. Short-chain
(2C) azides were chosen based on the results obtained in Fig. 1.
Two clickable warheads (W1 and W2) containing the Imatinib
core structure were designed by replacing the piperazine-linked
benzene ring (Scheme 1; shaded in orange) in the drug with an
alkyne group. Previously results showed that the piperazine moiety
of Imatinib does not contribute significantly to the inhibition of
Abl.10 We conceived that, upon click chemistry, the warhead will
guide the binding of the assembled inhibitor to the ATP-binding
site and, at the same time, project the diversity element (from
the azide) to the adjacent secondary binding site in the kinase
(i.e. substrate-binding or allosteric site). The triazole ring formed
should closely mimic the benzene ring in Imatinib, minimizing
potential structural disruption of the inhibitor. Briefly, W1 was
synthesized in 5 steps starting from acetylpyridine. The enami-
nation reaction of acetylpyridine with N,N-dimethylformamide
dimethylacetal in refluxing ethanol provided the enaminone 5,
which was converted to the pyrimidinyl amine 6 by reacting with
guanidine hydrochloride in refluxing isopropanol in basic medium.
Compound 6 was converted to 7 via a Cu(I)-catalyzed “Ullmann-
Type” reaction as previously reported.16 Subsequent reduction
of 7 via catalytic hydrogenation gave 8. Standard coupling of 8
with propiolic acid using DCC in DMF yielded 9 (W1) in 25.6%
overall yield (in 5 steps). W2 was synthesized similarly from the
key intermediate 11. Subsequent deprotection of 11 gave 12 which
was then coupled with propargyl amine using HATU in DMF to
afford W2 (13.6% yield over 6 steps).
Fig. 1 (a) Synthesis of the ADP-alkyne warhead. (b) The 344-member
“click” library generated using the ADP-alkyne and azides. The heat map
shows the preference of Abl for short-chain linkers/azides.
The ADP-alkyne was synthesized using procedures similar to
the synthesis of GDP-alkyne by Wong et al. (Fig. 1a).15 Briefly,
propargyl alcohol was phosphorylated with crystalline phospho-
rous acid in the presence of iodine as an oxidizing agent and the
resultant propargyl phosphate 1 was isolated as a cyclohexylamine
salt via precipitation from acetone containing cyclohexylamine.
The propargylphosphate-cylohexylamine salt was exchanged for
pyridinium ion form 2 using Dowex 50W-X2 resin in pyridinium
form and subsequently coupled with AMP-morpholidate 3 using
1H-tetrazole as coupling reagent in anhydrous pyridine, affording
the ADP-alkyne 4 in 21.5% yield over 4 steps. The 344-member
5130 | Org. Biomol. Chem., 2009, 7, 5129–5136
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Scheme 1 Synthesis of the two warheads for Imatinib-based click library.
Next, a 90-member inhibitor library based on Imatinib was
assembled, by click chemistry, using the two warheads and 45
azides (2C) (Scheme 1). Briefly, in each reaction a warhead was
mixed with an azide in a MeOH/H2O/DMSO mixed solvent with
catalytic amounts of CuSO4 and sodium ascorbate (see ESI†). The
click chemistry proceeded in quantitative yields in ~12 h at room
temperature. The solvents were evaporated off and the products
were redissolved in DMSO, and characterized by LCMS.
Inhibitor screening
Subsequently, stock solutions of the inhibitors were prepared in
DMSO and used directly for enzymatic screening against Src and
Abl kinases. The results are shown in Fig. 2. Further analysis
revealed that many inhibitors showed better inhibition against
Abl over Src kinase. Eleven of these inhibitors were further
identified (Fig. 2). They were scaled up, purified to homogeneity
(by preparative HPLC) and characterized (by LCMS) before
quantitative inhibition assays were carried out to obtain their IC50
values against Abl kinase (Table 1 and ESI†). The IC50 results
showed that most of the inhibitors exhibited 10–20 fold selectivity
for Abl over Src kinase. None of the azide components were
found to exhibit any significant inhibition against either kinase (at
50 mM concentration; data not shown). The warhead W1 alone
was more potent (IC50 = 1.36 mM) than W2 (IC50 = 27.3 mM)
against Abl kinase; this was presumably at least partly due to the
more appropriate positioning of the amide bond in W1, which
resulted in more favorable hydrogen bonding interactions with
residues located in the ATP-binding pocket of the kinase. The
same phenomenon had previously been observed in the Imatinib-
Fig. 2 Heat-map showing the relative inhibition of the 90-member
Imatinib-based bisubstrate inhibitor library against Src and Abl kinases.
Structures of representative examples are shown on the right.
Abl complex as well.8 W1 was nearly as potent as Imatinib (IC50
=
0.8 mM)17 under identical assay conditions, indicating indeed that
the piperazine moiety in Imatinib was not essential for binding
to Abl. Although the five bisubstrate inhibitors derived from W1
showed only a marginal improvement in potency over W1 against
Abl, with the best inhibitor, W1-SA20, having an IC50 of 0.70 mM
(2-fold improvement over W1), the six W2-derived bisubstrate
inhibitors were >10-fold more potent than W2 alone. This indi-
cates the simultaneous occupancy by these inhibitors of both the
ATP-binding pocket and the adjacent secondary pocket in the ac-
tive site of Abl kinase. The cellular activities of selected compounds
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 5129–5136 | 5131
©

View Article Online
Table 1 IC50 values of the initial hits against Abl and Src
IC50 (in mM)
under argon or nitrogen atmosphere using oven-dried glassware.
HPLC-grade solvents were used for all reactions. Reaction
progress was monitored by TCL on precoated silica plates (Merck
60 F254, 0.25 mm) and spots were visualized by UV or iodine
stain. Flash column chromatography was carried out using Merck
60 F254, 0.040–0.063 mm silica gel. Ion exchange chromatography
was carried out using Dowex 50WX2-400 cation exchange resin
from Sigma-Aldrich. All NMR spectra (1H-NMR, 13C-NMR
and 31P-NMR) were recorded on a Bruker 300 MHz NMR
spectrometer. Chemical shifts are reported in parts per million
referenced with respect to appropriate internal standards or
residual solvent peaks (CDCl3 = 7.26 ppm, CD3OD = 3.31 ppm,
d6-DMSO = 2.50 ppm). The following abbreviations were used
in reporting spectra, s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, dd = doublet of doublets. All analytical
HPLC were carried out on Shimadzu LCMS (IT-TOF) system or
Shimadzu LCMS-2010EV system equipped with an autosampler
Inhibitor
Abl
Src
Cell-based assay (% inh)a
Imatinib
W1
0.8b
c
1.36
2.55
1.67
2.83
0.70
1.00
27.3
2.49
2.16
3.06
1.12
1.61
1.62
>100
-
-
-
-
W1-SA9
W1-SA10
W1-SA17
W1-SA20
W1-SA22
W2
W2-SA3
W2-SA4
W2-SA7
W2-SA13
W2-SA17
W2-SA18
13.5
42.9
9.37
26.6
55.7
35
19
-
>100
38.8
43.1
55.2
17.9
10.4
24.8
-
-
-
32
60
-
a The cell-based assay was done at 50 mM concentration of inhibitors.
See ESI† for details. b Data obtained from reference 15. c “-” = Not
determined.
˚
using reverse-phase Phenomenex Luna 5 mm C18(2) 100 A 50 ¥
3 mm columns. Water with 0.1% TFA and acetonitrile with 0.1%
TFA were used as eluents and the flow rate was 0.6 ml/min. For
enzyme inhibition and IC50 measurements, a Tecan microplate
were further tested in an anti-proliferation assay using the human
K-562 cell line (a well-known p-210 Bcr-Abl expressing chronic
myelogenous leukemia line); preliminary results showed some of
these compounds indeed had some cellular activity in preventing
the growth of K-562 cells. Further biological experiments are
currently being carried out and will be reported in due course.
R
reader (Multimode Reader, Infiniteꢀ200) in luminescence mode
with i-controlTM software was used.
Synthetic procedures and characterizations
Prop-2-ynyl phosphate cyclohexylamine salt (1). Compound 1
was synthesized as previously reported.15 Briefly, to a solution of
propargyl alcohol (30 mL, 516 mmol) and triethylamine (7.5 mL,
54 mmol) was added phosphorous acid (1.23 g, 15 mmol). To
this was added 5.7 g of iodine (22.47 mmol) in small portions
over 5 min with stirring. The stirring was continued for 10
more minutes and then the reaction mixture was poured into
400 mL of acetone containing 15 mL cyclohexylamine. A white
precipitate was formed immediately and the solution was kept
for 2 h, then the precipitate was filtered and recrystallized from
95% ethanol containing a few drops of cyclohexylamine. The
precipitate obtained was dried to give 3.11 g (Yield = 68%) of
1
the propargyl cyclohexylamine salt 1 as a white solid. H-NMR
(300 MHz, D2O) d ppm = 1.1 (2H, m), 1.25 (8H, m), 1.61 (2H, m),
1.75 (4H, m), 1.93 (4H, m), 2.75 (1H, t, J = 2.46 Hz), 3.09 (2H,
m), 4.34 (2H, dd, J = 7.07 Hz, 2.46 Hz). 31P-NMR (121.5 MHz,
D2O) d ppm = 4.17.
Conclusions
In conclusion, we have developed the first click-chemistry ap-
proach for rapid assembly of bisubstrate inhibitors of Abelson
tyrosine kinase. Though selected compounds identified from this
“click-based” library showed only marginal improvement over
Imatinib in their inhibition of Abl kinase, they nevertheless offer
the first glimpse of the generality and utility of our strategy in
future development of bisubstrate inhibitors against a variety of
other protein kinases. Therefore, the strategy should provide a
useful tool in the fields of kinase research and drug discovery, as
well as in the emerging field of Catalomics.18
Prop-2-ynyl phosphate pyridinium salt (2). The propargyl cy-
clohexylamine salt (0.83 g, 2.71 mmol) was dissolved in water
(1.5 ml) and converted to the pyridinium ion form via ion exchange
on a pyridinium form of Dowex 50W-X2 resin. The pyridinium
form of Dowex resin was prepared from the commercially available
H+ form of the resin by repeated elution of a pre-washed (with
water) column of the resin with 10% pyridine. The product 2
was eluted with 30 mL of water in nearly quantitative yield and
concentrated on a rotary evaporator. 1H-NMR (300 MHz, D2O)
d ppm = 2.78 (1H, t, J = 2.46 Hz), 4.41 (2H, dd, J = 7.07 Hz,
2.46 Hz), 7.95 (t, J = 7.23 Hz), 8.50 (m), 8.65 (d, J = 5.28 Hz).
13C-NMR (75 MHz, D2O) d ppm = 53.50, 75.72, 78.96, 127.29,
140.90, 147.07. 31P-NMR (121.5 MHz, D2O) d ppm = 0.48.
Experimental
Materials and methods
All chemicals were purchased from commercial vendors and
used without further purification, unless indicated otherwise.
All reaction requiring anhydrous conditions were carried out
AMP-morpholidate (3). The AMP-morpholidate was synthe-
sized as previously reported with minor modifications.19 Briefly,
5132 | Org. Biomol. Chem., 2009, 7, 5129–5136
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
adenosine monophosphate monohydrate (0.84 g, 2.3 mmol) was
dissolved in a mixture of water (23 ml), t-butanol (23 ml) and
distilled morpholine (0.78 mL, 11.4 mmol, 5 eq.). The mixture
was heated under reflux and DCC (1.9 g, 9.2 mmol, 4 eq.)
in t-butanol (35 ml) was added dropwise over 60 min and the
reaction mixture was maintained under reflux for 2 h. The
progress of the reaction was monitored by TLC using a 6 : 3 :
2 isopropanol/ammonia/water solvent system. Upon completion
of reaction after 2 h, the reaction mixture was cooled to room
temperature; dicyclohexyl urea was filtered off and washed with
water. The filtrate was concentrated and extracted with diethyl
ether to remove the excess of DCC. The aqueous solution was
concentrated and purified by column chromatography on silica gel
using an isopropanol/ammonia/water (6 : 3 : 2) solvent system.
The product was concentrated to give 0.86 g (Yield = 90%) of 3
steps without further purification. 1H-NMR (300 MHz, CDCl3) d
2.92 (3H, s), 3.14 (3H, s), 5.65 (1H, d, J = 12.33 Hz), 7.32 (1H,
m), 7.81 (1H, d, J = 12.33 Hz), 8.15 (1H, d, J = 9.18 Hz), 8.62
(1H, d, J = 7.38 Hz), 9.04 (1H, s); 13C-NMR (75 MHz, CDCl3)
d 38.01, 45.85, 92.50, 123.93, 135.73, 136.30, 149.51, 152.05,
155.36, 187.02. ESI-MS (m/z) calculated = 177.102 [M+H+],
(m/z) observed = 177.099 [M+H+]
4-(Pyridin-3-yl) pyrimidin-2-amine (6)21. 3-(Dimethylamino)-
1-(pyridin-3-yl) prop-2-en-1-one (2.64 g, 15 mmol) and guani-
dinium hydrochloride (1.5 g, 15.75 mmol, 1.05 eq.) were mixed in
25 mL of 2-propanol. To the suspension was added 0.7 g of NaOH
(17.5 mmol, 1.17 eq.) and the mixture was refluxed for 18 h. The
◦
reaction was then cooled to 0 C and the precipitate was filtered
off, suspended in water, filtered off once more and washed with
2-propanol and diethyl ether. The residue was dried in oven at 60
to 70 ◦C and the desired product was obtained as a white powder
(1.68 g, Yield = 65%). 1H-NMR (300 MHz, DMSO-d6) d 6.77 (2H,
s), 7.19 (1H, d, J = 5.1 Hz), 7.52 (1H, m), 8.34 (1H, d, J = 5.1 Hz),
8.39 (1H, d, J = 7.89 Hz), 8.68 (1H, d, J = 6.57 Hz), 9.22 (1H,
s); 13C-NMR (75 MHz, CDCl3) d 106.01, 123.71, 132.46, 134.11,
147.95, 151.10, 159.35, 161.56, 163.79. ESI-MS (m/z) calculated =
173.082 [M+H+], (m/z) observed = 173.079 [M+H+]
1
as an off-white solid. H-NMR (300 MHz, D2O) d ppm = 2.84
(4H, m), 3.46 (4H, t, J = 4.35 Hz), 3.96 (2H, m), 4.24 (1H, m),
4.41 (1H, t, J = 4.92 Hz), 4.63 (1H, t, J = 4.92 Hz), 5.90 (1H,
d, J = 4.77 Hz), 7.89 (1H, s), 8.21 (1H, s). 13C-NMR (75 MHz,
D2O) d ppm = 44.51, 63.65, 66.67, 70.01, 74.06, 83.44, 87.21,
117.93, 139.22, 148.21, 152.33, 154.81. ESI-MS (m/z) calculated =
417.128 [M+H+], (m/z) observed = 417.120 [M+H+].
The warhead, ADP-alkyne (4). To the pyridinium salt form of
the propargyl phosphate 2 (~2.5 mmol) was added trioctylamine
(1.1 mL, 2.5 mmol) and anhydrous pyridine (30 ml) and the
mixture was concentrated to an oil. It was subsequently subjected
to repeated co-evaporations with anhydrous pyridine (3 ¥ 4 ml)
followed by the addition of AMP-morpholidate (1.04 g, 2.5 mmol).
The mixture was again subjected to co-evaporations with anhy-
drous pyridine (3 ¥ 4 ml) and finally concentrated to a minimum
volume. The coupling reagent 1H-tetrazole (0.56 g, 7.96 mmol)
and 4 mL of anhydrous pyridine were added and the reaction was
stirred under argon for 48 h. The solvent was then removed under
reduced pressure and the reaction was quenched with saturated
NaHCO3 until no CO2 was liberated. The mixture was washed
with ethyl ether (3 ¥ 20 ml), concentrated and purified by column
chromatography on silica gel using an isopropanol : ammonia :
water (6 : 3 : 2) solvent system. The sample was further purified by
Semi-Prep HPLC on C-8 column using acetonitrile and water as
eluents with tributylamine (0.01%) as ion-pairing agent affording
0.41 g (Yield = 35%) of the ADP-alkyne as an off-white solid.
1H-NMR (300 MHz, D2O) d ppm = 2.76 (1H, t, J = 2.46 Hz),
4.17 (3H, unresolved m), 4.29 (1H, m), 4.42 (1H, m), 4.47 (2H,
unresolved m), 6.03 (1H, d, J = 5.43 Hz), 8.31 (1H, s), 8.49 (1H,
s). 13C-NMR (75 MHz, D2O) d ppm = 54.03, 65.15, 70.07, 74.49,
75.69, 78.88, 83.94, 87.90, 118.25, 142.31, 144.60, 148.09, 149.63.
ESI-MS (m/z) calculated = 466.052 [M+H+], (m/z) observed =
466.052 [M+H+]
N-(2-Methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine
(7)16. 4-(Pyridin-3-yl) pyrimidin-2-amine 6 (0.456 g, 2.65 mmol,
1.1 eq.), CuI (0.12 g, 0.6 mmol, 0.25 eq.) and anhydrous K2CO3
(0.67 g, 4.82 mmol, 2 eq.) were added to a sealed tube (50 ml)
equipped with a rubber septum and a magnetic stirring bar. The
tube was filled with N2 gas and subsequently a mixture of 2-bromo-
1-methyl-4-nitrobenzene (0.518 g, 2.41 mmol, 1 eq.) and DMEDA
(0.064 mL, 0.6 mmol, 0.25 eq.) in anhydrous dioxane (20 ml)
was added by syringe at room temperature. The rubber septum
was quickly replaced with ◦a Teflon screw cap and the reaction
mixture was stirred at 120 C for 24 h. The reaction was cooled
to room temperature and concentrated ammonia (10 ml) and
saturated solution of NaCl (40 ml) were added then the mixture
was extracted with ethyl acetate (5 ¥ 40 ml). The organic layers
were dried over Na2SO4, concentrated under reduced pressure and
the residue was purified by column chromatography on silica gel
to give 0.62 g (Yield = 83.7%) of 7 as a yellow powder. ESI-MS
(m/z) calculated = 308.114 [M+H+], (m/z) observed = 308.120
[M+H+]
6-Methyl-N1-(4-(pyridin-3-yl) pyrimidin-2-yl) benzene-1,3-
diamine (8). To a degassed solution of 0.4 g (1.302 mmol) of
N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine
7 in 50 mL ethyl acetate was added 10% Pd/C (0.04 g) and the
reaction was stirred under a hydrogen atmosphere (balloon) for
3 h until complete disappearance of the starting material. The
reaction was then filtered through celite, concentrated and the
product was obtained as a yellow solid (0.36 g, quantitative yield)
and used in the subsequent steps without further purification.
1H-NMR (300 MHz, CDCl3) d 2.25 (3H, s), 6.40 (1H, m), 6.98
(1H, s), 7.01 (1H, m), 7.15 (1H, d, J = 5.1 Hz), 7.41 (1H, m), 7.60
(1H, s), 8.33 (1H, m), 8.49 (1H, d, J = 5.1 Hz), 8.71 (1H, m),
9.27 (1H, s). ESI-MS (m/z) calculated = 278.140 [M+H+], (m/z)
observed = 278.101 [M+H+]
3-(Dimethylamino)-1-(pyridin-3-yl) prop-2-en-1-one (5)20.
A
250 mL round-bottomed flask was charged with 6.4 mL
(58.3 mmol) of 3-acetylpyridine, 9.4 mL (70 mmol, 1.2 eq.) of
N,N-dimethylformamide dimethylacetal and 50 mL of ethanol.
The reaction mixture was refluxed overnight, cooled to room tem-
perature and the solvent was removed under reduced pressure. To
the crude residue approximately 50 mL of diethyl ether was added
and the mixture cooled to 0 ◦C. The product 3-(dimethylamino)-
1-(pyridin-3-yl) prop-2-en-1-one was subsequently filtered off as
yellow crystals (7.18 g, Yield = 70%) and used in the subsequent
N-(4-Methyl-3-(4-(pyridin-3-yl) pyrimidin-2-ylamino) phenyl)
propiolamide (9). To a solution of propiolic acid (0.0665 mL,
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 5129–5136 | 5133
©

View Article Online
1.08 mmol, 1.2 eq.) in 8 mL DMF was added 0.223 g (1.08 mmol,
1.2 eq.) of DCC followed by 0.25 g (0.902 mmol) of 6-methyl-
N1-(4-(pyridin-3-yl) pyrimidin-2-yl) benzene-1,3-diamine and the
reaction mixture was stirred at room temperature for 3 h until
the starting material was completely used up. The reaction was
concentrated on a rotary evaporator and the crude product was
purified by column chromatography on silica gel to give 0.2 g
(Yield = 67.3%) of 9 as a pale yellow solid. 13C-NMR (75 MHz,
DMSO-d6) d 17.62, 76.90, 78.52, 107.75, 116.06, 116.45, 128.19,
130.28, 135.97, 136.18, 137.83, 146.87, 149.46, 149.94, 159.56,
160.96, 161.03. ESI-MS (m/z) calculated = 330.135 [M+H+],
(m/z) observed = 330.135 [M+H+]
Methyl 3-iodo-4-methylbenzoate (10)22. A solution of 3-iodo-
4-methyl benzoic acid (0.75 g, 2.86 mmol) and a catalytic amount
of H2SO4 (~ 0.05 ml) in 20 mL methanol was refluxed for 19 h.
The reaction mixture was cooled and the solvent was removed
under reduced pressure. The residue obtained was dissolved in
diethyl ether, washed with saturated NaHCO3 (3¥), water, brine,
dried over Na2SO4 and the solvent removed to give 10 as an orange
viscous liquid (0.73 g, Yield = 92.3%). ESI-MS (m/z) calculated =
276.972 [M+H+], (m/z) observed = 276.964 [M+H+]
5.25 Hz), 8.68 (1H, m), 9.01 (1H, s), 9.26 (1H, s). ESI-MS (m/z)
calculated = 307.118 [M+H+], (m/z) observed = 307.075 [M+H+]
4-Methyl-N-(prop-2-ynyl)-3-(4-(pyridin-3-yl)
pyrimidin-2-yl-
amino) benzamide (13). The acid 12 (0.2 g, 0.653 mmol) was
added to 10 mL of DMF followed by HATU (0.3 g, 0.784 mmol,
1.2 eq.) and DIEA (0.133 mL, 0.784 mmol, 1.2 eq.). After stirring
for 5 min propargylamine (0.054 mL, 0.784 mmol, 1.2 eq.) was
added and the reaction was stirred for 2 h. The solvent was
removed under reduced pressure and the crude product obtained
was purified by column chromatography on silica gel to give
0.15 g (Yield = 67%) of the Warhead-2 as an off-white solid.
1H-NMR (300 MHz, DMSO-d6) d 2.30 (3H, s), 3.10 (1H,s), 4.05
(2H, m), 7.35 (1H, d, J = 8.07 Hz), 7.54 (1H, d, J = 5.07 Hz), 7.61
(1H, m), 7.87 (1H, m), 8.13 (1H, s), 8.60 (1H, d, J = 5.25 Hz),
8.78 (1H, d, J = 8.07 Hz), 8.86 (2H, m), 9.21 (1H, s), 9.35 (1H,
s); 13C-NMR (75 MHz, DMSO-d6) d 18.03, 28.45, 72.67, 81.41,
107.99, 123.26, 124.29, 125.50, 130.23, 131.76, 136.11, 137.68,
138.44, 144.64, 147.66, 158.57, 159.76, 160.10, 160.95, 165.60.
ESI-MS (m/z) calculated = 344.151 [M+H+], (m/z) observed =
344.140 [M+H+]
N-(4-Methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-1-
(2-(2-methylphenylsulfonamido)ethyl)-1H-1,2,3-triazole-4-carbox-
amide [W1-SA20]. 100 mL of a stock solution of W1 (100 mM)
in DMSO was mixed with a 50 mM stock solution of the azide
SA20 (200 mL in DMSO). To this was added 200 mL water, 100 mL
methanol, 50 mL copper sulfate solution (10 mM stock solution in
water) and 50 ml sodium ascorbate solution (50 mM stock solution
in water). The reaction was left overnight on a shaking incubator
at room temperature. Upon completion of the reaction (<12 h),
the crude reaction mixture was purified by reverse phase Semi-
Prep HPLC on a C-18 column. 1H-NMR (300 MHz, DMSO-d6)
d 2.23 (3H, s), 2.51 (3H, s), 3.34 (2H, m), 4.48 (2H, t, J = 5.59 Hz),
7.20 (1H, d, J = 8.37 Hz), 7.35 (2H, m), 7.47 (2H, d, J = 5.25 Hz),
7.53 (1H, m), 7.67 (1H, m), 7.76 (1H, d, J = 7.71 Hz), 7.97 (1H,
t, J = 5.91 Hz), 8.15 (1H, s), 8.55 (1H, d, J = 5.07 Hz), 8.56 (1H,
s), 8.63 (1H, d, J = 7.86 Hz), 8.76 (1H, bs), 9.03 (1H, s), 9.32
(1H, bs), 10.32 (1H, s). ESI-HRMS (m/z) calculated = 570.2036
[C28H28N9O3S], (m/z) observed = 570.2031
Methyl 4-methyl-3-(4-(pyridin-3-yl) pyrimidin-2-ylamino) ben-
zoate (11). 4-(Pyridin-3-yl) pyrimidin-2-amine
6 (0.415 g,
2.41 mmol), CuI (0.12 g, 0.6 mmol, 0.25 eq.) and anhydrous
K2CO3 (0.67 g, 4.82 mmol, 2 eq.) were added to a sealed tube
(50 ml) equipped with a rubber septum and a magnetic stirring
bar. The tube was filled with N2 gas and subsequently a mixture of
methyl 3-iodo-4-methylbenzoate 7 (0.67 g, 2.41 mmol, 1 eq.) and
DMEDA (0.064 mL, 0.6 mmol, 0.25 eq.) in anhydrous dioxane
(20 ml) was added by syringe at room temperature. The rubber
septum was quickly replaced with a Teflon screw cap and the
reaction mixture was stirred at 120 ◦C for 24 h. The reaction was
cooled to room temperature and concentrated ammonia (10 ml)
and saturated solution of NaCl (40 ml) were added and then
extracted with ethyl acetate (5 ¥ 40 ml). The organic layers were
dried over Na2SO4, concentrated under reduced pressure and the
residue was purified by column chromatography on silica gel
to give 0.63 g (Yield = 81%) of 11 as a pale yellow powder.
1H-NMR (300 MHz, CDCl3) d 2.39 (3H, s), 3.95 (3H, s), 7.19
(1H, d, J = 5.25 Hz), 7.29 (1H, m), 7.42 (1H, m), 7.72 (1H, m),
8.41 (1H, m), 8.49 (1H, d, J = 5.1 Hz), 8.71 (1H, m), 8.98 (1H, s),
9.29 (1H, s); 13C-NMR (75 MHz, CDCl3) d 18.86, 52.67, 108.90,
122.90, 124.20, 125.04, 129.20, 131.06, 133.01, 133.88, 135.10,
138.12, 149.15, 152.12, 159.75, 161.01, 163.00, 167.66. ESI-MS
(m/z) calculated = 321.135 [M+H+], (m/z) observed = 321.130
[M+H+]
N-(4-Methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-1-
(2-(2-methylphenylsulfonamido)ethyl)-1H-1,2,3-triazole-4-carbox-
amide [W2-SA13]. 100 mL of a stock solution of W2 (100 mM)
in DMSO was mixed with a 50 mM stock solution of the azide
SA13 (200 mL in DMSO). To this was added 200 mL water,
100 ml methanol, 50 mL copper sulfate solution (10 mM stock
solution in water) and 50 mL sodium ascorbate solution (50 mM
stock solution in water). The reaction was left overnight on a
shaking incubator at room temperature. Upon completion of
the reaction (<12 h), the crude reaction mixture was purified
4-Methyl-3-(4-(pyridin-3-yl) pyrimidin-2-ylamino) benzoic acid
(12). LiOH (0.24 g, 10 mmol, 8 eq.) was dissolved in a 3 : 1
mixture of methanol and water (12 ml). To this was added the
methyl ester 11 (0.4 g, 1.25 mmol) and the reaction mixture was
stirred for 8 h at room temperature. The reaction was acidified
to pH = 1 with 1 N HCl. The solvent was removed under
reduced pressure and the residue obtained was purified by column
chromatography on silica gel to obtain 0.23 g (Yield = 60%) of
12 as an off-white solid. 1H-NMR (300 MHz, DMSO-d6) d 2.30
(3H, s), 7.29 (1H, d, J = 8.07 Hz), 7.45 (1H, d, J = 5.28 Hz), 7.53
(1H, m), 7.64 (1H, m), 8.22 (1H, s), 8.45 (1H, m), 8.52 (1H, d, J =
1
by reverse phase Semi-Prep HPLC on a C-18 column. H-NMR
(300 MHz, DMSO-d6) d 2.29 (6H, s), 3.20 (2H, m), 4.37 (2H, t, J =
6.00 Hz), 4.49 (2H, d, J = 5.43 Hz), 7.33 (1H, d, J = 7.89 Hz), 7.49
(4H, m), 7.63 (1H, d, J = 7.89 Hz), 7.79 (1H, m), 7.95 (2H, m),
8.14 (1H, s), 8.57 (1H, d, J = 5.28 Hz), 8.72 (1H, d, J = 8.04 Hz),
8.83 (1H, bs), 8.94 (1H, t, J = 5.76 Hz), 9.17 (1H, s), 9.32 (1H,
bs). ESI-HRMS (m/z) calculated = 624.1912 [C29H28FN9O3SNa],
(m/z) observed = 624.1939
5134 | Org. Biomol. Chem., 2009, 7, 5129–5136
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Click assembly of inhibitors
The inhibition assays with Abl and Src kinases were performed
at nearly equal activities of the two kinases at a concentration
of ~50 nM for Abl and ~90 nM for Src kinases respectively. The
substrate peptides used are shown below.
Abl substrate peptide: KKGEAIYAAPFA-NH2
Src substrate peptide: KVEKIGEGTYGVVYK-NH2
The inhibitors were screened against the closely related kinases
Abl and Src in a 384-well plate or 96-well plate as described above.
The luminescence intensity from each well was measured and the
inhibition potency was calculated using the following relation.
The 344-member library formed from ADP-alkyne and azides.
The click-assembly of the two fragments (ADP-alkyne and azides)
was carried out in a 384-well plate in a mixed solvent system
containing t-BuOH, water and DMSO (1 : 1 : 0.1) with CuI
(0.1 eq.) as catalyst and tributylamine (0.2 eq.) as Cu(I) stabilizing
base. Other commonly employed click-catalytic systems such as
CuSO4-sodium ascorbate and Cu-CuSO4 were not successful. The
triazole formation was found to complete in 24 h. The crude
reactions were evaporated to dryness and redissolved in DMSO
to prepare standard solutions for kinase inhibition assays and
LC-MS characterizations.
Potency = 1 - Remaining activity
The average value of potency across the entire library was
calculated and the heat-maps were generated by assigning the
standard deviation values to each compound.
The 90-member Imatinib analogue library formed from the two
warheads and azides. The following optimized conditions were
used for the click-assembly of the inhibitors:
The alkyne warhead (W1 or W2) = 5 mM (stock solution
concentration = 50 mM in DMSO), 20 mL,
IC50 evaluation
The azide = 5 to 7.5 mM (stock solution concentration = 50 to
75 mM in DMSO), 20 mL,
Concentration-dependent experiments were performed to confirm
the potency of the initial hits identified. All the 11 hits were
purified by Semi-Preparative reverse-phase HPLC using water
with 0.1% TFA and acetonitrile with 0.1% TFA as eluents on
a Luna 5 m C18(2) 100A column (50 ¥ 30 mm, 5 micron) at a
flow rate of 10 ml/min before subjecting them to the detailed
inhibition measurements. The dose-dependent inhibition assays
were performed by varying the concentration of the inhibitors
under fixed enzyme concentration of 50 nM as mentioned before.
The IC50 values for each inhibitor were calculated from the
percentage activity vs. log [concentration of inhibitor] curves
generated using the GraphPad Prism software.
CuSO4 = 0.1 mM,
Sodium ascorbate = 1 mM,
Reaction volume = 200 mL (100 mL water + 40 mL DMSO
+ 60 mL ,methanol)
Briefly, the alkyne warhead and the azide were mixed in the
above mentioned solvent system in the presence of the indicated
amounts of CuSO4 and sodium ascorbate. The reactions were
shaken for 12 h at room temperature upon which the entire
warhead was converted to the triazole (monitored by LC-MS).
The reactions were concentrated and redissolved in DMSO to
prepare standard solutions for enzyme inhibition assays.
Cell culture and proliferation assay
Human leukemic cell lines K-562 (p-210 Bcr-Abl expressing
chronic myelogenous leukemia) were maintained in RPMI-1640
medium supplemented with L-glutamine and 10% FBS. The cells
(0.2–0.4 ¥ 106 per ml) were plated in duplicate in 96-well plates
containing two different concentrations (50 and 10 mM) of the 11
inhibitor hits against recombinant Abl identified from the in-vitro
assay. After incubation at 37 ◦C in 5% CO2 for 48 h, the effect of
the compounds on the cell proliferation was determined by the
XTT (sodium 3¢-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis
(4-methoxy-6-nitro) benzene sulfonic acid hydrate) colorimetric
dye reduction method. Briefly, the procedure for the assay was as
follows. The XTT was dissolved in hot RPMI media (37 ◦C) at a
concentration of 1 mg/mL. Immediately before use, the electron
coupling reagent PMS (N-methyl dibenzopyrazine methyl sulfate)
was added to the XTT solution giving a PMS concentration of
125 mM (The PMS was stored as 100 mM stock solution in saline
at 4 ◦C). 25 mL of this XTT/PMS solution was added to each well
in the 96-well plate containing ~100 mL per well culture giving a
final concentration of 0.2 mg/mL XTT and 25 mM PMS. After
incubation at 37 ◦C in 5% CO2 for 2–6 h the absorbance of each well
was measured at a wavelength of 450 nm using Tecan microplate
reader and effect of the compounds on the cell proliferation was
determined by comparing with the DMSO (no inhibitor) controls.
Kinase inhibition assays
R
The kinase inhibition assay was performed with Kinase-Gloꢀ
Plus Luminescent Kinase assay kit from Promega following
the manufacturer’s instructions. Briefly, the kinase, the peptide
substrate, ATP and the inhibitor were mixed in the kinase reaction
buffer (100 mM Tris, pH = 7.5, 10 mM MgCl2) at a volume of
25 mL in a flat-bottom solid white 384-well plate (or 55 mL in a
flat-bottom solid white 96-well plate). The incubation was allowed
to continue for 30 min at 37 ◦C and the kinase reaction was then
quenched by the addition of an equal volume of the Kinase-Glo
reagent. After 5 min of incubation the luminescence readouts from
the wells were measured using a Tecan microplate reader with i-
control software. The ATP and substrate peptide concentrations
used in the assay were 5 mM and 50 mM, respectively. The following
control reactions were also performed simultaneously.
1. Enzyme control (No kinase, Buffer+Substrate+ATP).
2. Inhibitor control.
(No inhibitor+Buffer+Kinase+Substrate+ATP).
3. Substrate control (No substrate, Buffer+Kinase+ATP).
4. Buffer alone.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 5129–5136 | 5135
©

View Article Online
10 (a) P. W. Manley, S. W. Cowan-Jacob and J. Mestan, Biochim. Biophys.
Acta, 2005, 1754, 3; (b) M. E. Gorre, M. Mohammed, K. Ellwood, N.
Hsu, R. Paquette, P. N. Rao and C. L. Sawyers, Science, 2001, 293, 876;
(c) N. von Bubnoff, F. Schneller, C. Peschel and J. Duyster, Lancet,
2002, 359, 487.
11 (a) T. O’Hare, D. K. Walters, M. W. N. Deininger and B. J. Druker,
Cancer Cell, 2005, 7, 117; (b) P. La Rosee, A. S. Corbin, E. P. Stoffregen,
M. W. Deininger and B. J. Druker, Cancer Res., 2002, 62, 7149; (c) J. S.
Tokarski, J. A. Newitt, C. Y. J. Chang, J. D. Cheng, M. Wittekind, S. E.
Kiefer, K. Kish, F. Y. F. Lee, R. Borzillerri, L. J. Lombardo, D. Xie, Y.
Zhang and H. E. Klei, Cancer Res., 2006, 66, 5790; (d) P. W. Manley, W.
Breitenstein, J. Bru¨ggen, S. W. Cowan-Jacob, P. Furet, J. Mestan and
T. Meyer, Bioorg. Med. Chem. Lett., 2004, 14, 5793.
Acknowledgements
Funding support was provided by Ministry of Education (R143-
000-390-315), the Agency for Science, Technology and Research
(A*Star) of Singapore (R154-000-274-305), and the National
Research Foundation (Competitive Research Programme R143-
000-218-281).
References
1 (a) J. A. Adams, Chem. Rev., 2001, 101, 2271; (b) T. Hunter, Cell, 2000,
100, 113; (c) J. A. Ubersax and J. E. Ferrell, Jr, Nat. Rev. Mol. Cell
Biol., 2007, 8, 530.
2 (a) P. Cohen, Nat. Rev. Drug Discovery, 2002, 1, 309; (b) C. L. Sawyers,
Cancer Cell, 2002, 1, 13; (c) D. S. Krause and R. A. Van Etten,
N. Engl. J. Med., 2005, 353, 172.
12 For recent reviews, see: (a) K. A. Kalesh, P.-Y. Yang, R. Srinivasan and
S. Q. Yao, QSAR Comb. Sci., 2007, 26, 1135; (b) A. Brik, C. Y. Wu and
C.-H. Wong, Org. Biomol. Chem., 2006, 4, 1446.
13 (a) V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chem., Int. Ed., 2002, 41, 2596; (b) C. W. Tornoe, C. Christensen
and M. Meldal, J. Org. Chem., 2002, 67, 3057; (c) H. C. Kolb and K. B.
Sharpless, Drug Discovery Today, 2003, 8, 1128; (d) A. Brik, Y.-C. Lin,
J. Elder and C.-H. Wong, Chem. Biol., 2002, 9, 891; (e) M. Whiting, J.
Muldoon, Y.-C. Lin, S. M. Silverman, W. Lindstrom, A. J. Olson, H. C.
Kolb, M. G. Finn, K. B. Sharpless, J. H. Elder and V. V. Fokin, Angew.
Chem., Int. Ed., 2006, 45, 1435; (f) M. D. Best, A. Brik, E. Chapman,
L. V. Lee, W.-C. Cheng and C.-H. Wong, ChemBioChem, 2004, 5, 811;
(g) L. V. Lee, M. L. Mitchell, S.-J. Huan, V. V. Fokin, K. B. Sharpless
and C.-H. Wong, J. Am. Chem. Soc., 2003, 125, 9588; (h) J. Wang, M.
Uttamchandani, J. Li, M. Hu and S. Q. Yao, Org. Lett., 2006, 8, 3821;
(i) R. Manetsch, A. Krasinski, Z. Radic, J. Raushel, P. Taylor, K. B.
Sharpless and H. C. Kolb, J. Am. Chem. Soc., 2004, 126, 12809; (j) R.
Srinivasan, M. Uttamchandani and S. Q. Yao, Org. Lett., 2006, 8, 713;
(k) S. L. Ng, P.-Y. Yang, K. Y.-T. Chen, R. Srinivasan and S. Q. Yao,
Org. Biomol. Chem., 2008, 6, 844; (l) P. C. Chen, V. Patil, W. Guerrant,
P. Green and A. K. Oyelere, Bioorg. Med. Chem., 2008, 16, 4839; (m) M.
Hu, J. Li and S. Q. Yao, Org. Lett., 2008, 10, 5529–5531.
3 (a) Y. Liu and N. S. Gray, Nat. Chem. Biol., 2006, 2, 358; (b) C. Pargellis,
L. Tong, L. Churchill, P. F. Cirillo, T. Gilmore, A. G. Graham, P. M.
Grob, E. R. Hickey, N. Moss, S. Pav and J. Regan, Nat. Struct. Biol.,
2002, 9, 268; (c) J. G. Cumming, C. L. McKenzie, S. G. Bowden, D.
Campbell, D. J. Masters, J. Breed and P. J. Jewsbury, Bioorg. Med.
Chem. Lett., 2004, 14, 5389; (d) P. W. Manley, G. Bold, J. Bru¨ggen,
G. Fendrich, P. Furet, J. Mestan, C. Schnell, B. Stolz, T. Meyer, B.
Meyhack, W. Stark, A. Strauss and J. Wood, Biochim. Biophys. Acta,
2004, 1697, 17; (e) P. T. C. Wan, M. J. Garnett, S. M. Roe, S. Lee,
D. Niculescu-Duvaz, V. M. Good, C. M. Jones, C. J. Marshall, C. J.
Springer, D. Barford and R. Marais, Cell, 2004, 116, 855; (f) A. L.
Gill, M. Frederickson, A. Cleasby, S. J. Woodhead, M. G. Carr, A. J.
Woodhead, M. T. Walker, M. S. Congreve, L. A. Devine, D. Tisi,
M. O’Reilly, L. C. A. Seavers, D. J. Davis, J. Curry, R. Anthony,
A. Padova, C. W. Murray, R. A. E. Carr and H. Jhoti, J. Med.
Chem., 2005, 48, 414; (g) K. Parang, J. H. Till, A. J. Ablooglu, R. A.
Kohanski, S. R. Hubbard and P. A. Cole, Nat. Struct. Biol., 2001, 8,
37.
14 R. Srinivasan, L. P. Tan, H. Wu, P.-Y. Yang, K.A. Kalesh and S. Q.
Yao, Org. Biomol. Chem., 2009, 7, 1821.
4 K. Gumireddy, S. J. Baker, S. C. Cosenza, P. John, A. D. Kang, K. A.
Robell, M. V. R. Reddy and E. P. Reddy, Proc. Natl. Acad. Sci. U. S. A.,
2005, 102, 1992.
5 F. J. Adria`n, Q. Ding, T. Sim, A. Velentza, C. Sloan, Y. Liu, G. Zhang,
W. Hur, S. Ding, P. Manley, J. Mestan, D. Fabbro and N. S. Gray, Nat.
Chem. Biol., 2006, 2, 95.
15 L. V. Lee, M. L. Mitchell, S.-J. Huang, V. V. Fokin, K.B. Sharpless and
C.-H. Wong, J. Am. Chem. Soc., 2003, 125, 9588.
16 Y.-F. Liu, C.-L. Wang, Y.-J. Bai, N. Han, J.-P. Jiao and X.-L. Qi, Org.
Process Res. Dev., 2008, 12, 490.
17 M. A. Seeliger, M. Young, M. N. Henderson, P. Pellicena, D. S. King,
A. M. Falick and J. Kuriyan, Protein Sci., 2005, 14, 3135.
6 (a) J. H. Lee, S. Kumar and D. S. Lawrence, ChemBioChem, 2008, 9,
507; (b) S. C. Meyer, C. D. Shomin, T. Gaj and I. Ghosh, J. Am. Chem.
Soc., 2007, 129, 13812.
7 B. Nagar, O. Hantschel, M. A. Young, K. Scheffzek, D. Veach, W.
Bornmann, B. Clarkson, G. Superti-Furga and J. Kuriyan, Cell, 2003,
112, 859.
18 (a) H. Sun, S. Chattopadhaya, J. Wang and S. Q. Yao, Anal. Bioanal.
Chem., 2006, 386, 416; (b) M. Uttamchandani, C. H. S. Lu and S. Q.
Yao, Acc. Chem. Res., 2009, 42, 1183.
19 J. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc., 1961, 83, 649.
¨
20 Z. S. Szaka´cs, S. Be´ni, Z. Varga, L. Orfi, G. Ke´ri and B. Nosza´l, J. Med.
Chem., 2005, 48, 249.
8 T. Schindler, W. Bornmann, P. Pellicena, W. T. Miller, B. Clarkson and
J. Kuriyan, Science, 2000, 289, 1938.
9 M. W. N. Deininger, J. M. Goldman and J. V. Melo, Blood, 2000, 96,
3343.
21 G. B. Bennett, R. B. Mason, L.J. Alden and J. B. Roach, Jr, J. Med.
Chem., 1978, 21, 623.
22 M. Lautens, J.-F. Paquin, S. Piguel and M. Dahlmann, J. Org. Chem.,
2001, 66, 8127.
5136 | Org. Biomol. Chem., 2009, 7, 5129–5136
This journal is
The Royal Society of Chemistry 2009
©