Identification of N-(4-piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H- pyrazole-3-carboxamide (AT7519), a novel cyclin dependent kinase inhibitor using fragment-based X-ray crystallography and structure based drug design

Article abstract of DOI:10.1021/jm800382h

The application of fragment-based screening techniques to cyclin dependent kinase 2 (CDK2) identified multiple (>30) efficient, synthetically tractable small molecule hits for further optimization. Structure-based design approaches led to the identification of multiple lead series, which retained the key interactions of the initial binding fragments and additionally explored other areas of the ATP binding site. The majority of this paper details the structure-guided optimization of indazole (6) using information gained from multiple ligand-CDK2 cocrystal structures. Identification of key binding features for this class of compounds resulted in a series of molecules with low nM affinity for CDK2. Optimisation of cellular activity and characterization of pharmacokinetic properties led to the identification of 33 (AT7519), which is currently being evaluated in clinical trials for the treatment of human cancers.

Full text of DOI:10.1021/jm800382h

4986
J. Med. Chem. 2008, 51, 4986–4999
Identification of N-(4-Piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxamide
(AT7519), a Novel Cyclin Dependent Kinase Inhibitor Using Fragment-Based X-Ray
Crystallography and Structure Based Drug Design†
Paul G. Wyatt,‡, Andrew J. Woodhead,‡,* Valerio Berdini, John A. Boulstridge,‡ Maria G. Carr,‡ David M. Cross,#
Deborah J. Davis,| Lindsay A. Devine,§ Theresa R. Early,‡ Ruth E. Feltell,| E. Jonathan Lewis,# Rachel L. McMenamin,|
Eva F. Navarro,‡ Michael A. O’Brien,‡ Marc O’Reilly,§ Matthias Reule,# Gordon Saxty,‡ Lisa C. A. Seavers,|
Donna-Michelle Smith,# Matt S. Squires,| Gary Trewartha,‡ Margaret T. Walker,‡ and Alison J.-A. Woolford‡
Astex Therapeutics Ltd, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, United Kingdom
ReceiVed April 3, 2008
The application of fragment-based screening techniques to cyclin dependent kinase 2 (CDK2) identified
multiple (>30) efficient, synthetically tractable small molecule hits for further optimization. Structure-based
design approaches led to the identification of multiple lead series, which retained the key interactions of the
initial binding fragments and additionally explored other areas of the ATP binding site. The majority of this
paper details the structure-guided optimization of indazole (6) using information gained from multiple
ligand-CDK2 cocrystal structures. Identification of key binding features for this class of compounds resulted
in a series of molecules with low nM affinity for CDK2. Optimisation of cellular activity and characterization
of pharmacokinetic properties led to the identification of 33 (AT7519), which is currently being evaluated
in clinical trials for the treatment of human cancers.
Introduction
efficiency (LEa)12 and as such form a small number of very
high quality interactions. It is possible to optimize fragments
to relatively low molecular weight leads with good drug-like
properties, and this can be achieved with a limited number of
molecules, particularly if good structural data is available. LE
is the ratio of free binding affinity to molecular size, depicted
mathematically as LE ) -∆G /HAC ≈ -RT ln(IC50)/HAC,
where the HAC (heavy atom count) includes all non-hydrogen
atoms. This concept can be used to compare hits of widely
differing structures and activities and is also a simple way of
determining if the optimization of a hit into a lead has been
carried out efficiently.
Background to Fragment-Based Drug Discovery. Astex
has previously described the use of fragment-based X-ray
crystallographic screening to identify low-affinity fragment hits
for a range of targets,1,2 and the area in general has been
reviewed extensively over recent years.3–7 Fragment-based
screening approaches have become widely used throughout the
pharmaceutical industry and can now be regarded as a compli-
mentary approach to high-throughput screening. Fragments are
low-molecular-weight compounds3 (typically 100-250 Da) with
generally low binding affinities (>100 µM) and, as a result,
very sensitive biophysical screening methods are frequently used
to detect them, such as X-ray crystallography,1,8 nuclear
magnetic resonance spectroscopy (NMR),9 and surface plasmon
resonance (SPR).10 Fragment screening has a number of
advantages over conventional screening methodologies. First,
only small libraries of compounds are needed for screening
purposes (∼200-2000), due to the much greater probability of
complimentarity between each fragment and the target than is
expected for larger, drug-like compounds.11 Second, despite their
often very low affinity, fragments generally possess good ligand
Inhibition of Cyclin Dependent Kinases. The cyclin-
dependent kinases (CDKs) are a family of serine-threonine
protein kinases, which are key regulatory elements in cell cycle
progression. The activity of CDKs is critically dependent on
the presence of their regulatory partners (cyclins), whose levels
of expression are tightly controlled throughout the different
phases of the cell cycle.13–15 Loss of cell cycle control resulting
in aberrant cellular proliferation is one of the key characteristics
of cancer,16 and it is anticipated that inhibition of CDKs may
provide an effective method for controlling tumor growth and
hence an effective weapon in cancer chemotherapy.17,18
CDK2/cyclin E, CDK4/cyclin D and CDK6/cyclin D prima-
rily regulate progression from the G1 (Gap1) phase to the S
phase (DNA synthesis) of the cell cycle through phosphorylation
of the retinoblastoma protein (Rb).19,20 Subsequent progression
through S phase and entry into G2 (Gap2) is thought to require
the CDK2/cyclin A complex. Complexes of CDK1 and the A
or B type cyclins regulate both the G2 to M phase transition
†
Coordinates of the CDK2 complexes with compounds 6, 7, 8, 10, 11,
12, 14, 15, 18, 22, 23, 28, 29, and 33 have been deposited in the Protein
Data Bank under accession codes 2VTA, 2VTH, 2VTM, 2VTJ, 2VTR,
2VTS, 2VTI, 2VTL, 2VTN, 2VTO, 2VTP, 2VTQ, 2VTT, and 2VU3,
together with the corresponding structure factor files.
* To whom correspondence should be addressed. Phone: +44 (0)1223
226287. Fax: +44 (0)1223 226201. E-mail: a.woodhead@astex-therapeutics.
com.
‡
Medicinal Chemistry.
Structural Biology.
Biology.
Computational Chemistry and Informatics.
DMPK.
§
|
a Abbreviations: CDK, cyclin dependent kinase; DCM, dichloromethane;
DFG, region of conserved amino acids, aspartic acid, phenyl alanine, glycine;
EDC, 1-(3-dimethyaminopropyl)-3-ethylcarbodiimide; HPꢀCD, hydrox-
ypropyl-ꢀ-cyclodextrin; HOBT, 1-hydroxybenzotriazole hydrate; HOAt,
1-hydroxy-7-azabenzotriazole; LE, ligand efficiency; NMP, 1-methyl-2-
pyrrolidinone; NPM, nucleophosmin; Rb, retinoblastoma protein; T/C, mean
tumor volume of treated animals divided by the mean control tumor volume.
#
Current address: Drug Discovery Unit, Division of Biological Chem-
istry & Drug Discovery College of Life Sciences, James Black Centre,
University of Dundee, Dow Street, Dundee, DD1 5EH, United Kingdom.
Phone: +44 (0)1382 386231. E-mail: p.g.wyatt@dundee.ac.uk.
10.1021/jm800382h CCC: $40.75
2008 American Chemical Society
Published on Web 07/26/2008

Identification of AT7519
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4987
Figure 2. X-ray crystal structure of ATP (adenosine triphosphate)
bound into the active site of CDK2 (1HCL). The ligand is anchored in
place with two hydrogen bonds, one between the backbone carbonyl
of Glu81 and the 6-amino group and one between the backbone NH of
Leu83 and the N1 position of the adenine ring. An additional favorable
electrostatic interaction is made between the hydrogen atom at the C2
position and the carbonyl of Leu83. The ribose and phosphate groups
form multiple polar interactions, one of which involves coordination
to the catalytic magnesium (gray/silver sphere) via the phosphate groups
along with Asp145 and Asn132. Other key features to notice are that
ATP does not interact with the solvent accessible region or the
hydrophobic pocket between the gatekeeper residue and the DFG region.
Figure 1. CDK inhibitors currently under investigation in clinical trials.
and mitosis.15,17 However, not all members of the CDK family
are involved exclusively in cell cycle control; CDK2/cyclin E
plays a role in the p53 mediated DNA damage response pathway
and also in gene regulation.21–24 CDKs 7, 8, and 9 are implicated
in the regulation of transcription, and CDK5 plays a role in
neuronal and secretory cell function.25–27
Thus inhibiting CDK enzyme activity may affect cell growth
and survival via several different mechanisms and therefore
represents an attractive target for therapeutics designed to arrest,
or recover control of, the cell cycle in aberrantly dividing cells.
Accumulating evidence from genetic knockouts of the CDKs
and/or their cyclin partners and from siRNA studies suggests
significant redundancy in their regulation of key cell cycle
events.28–32 In addition, the effects of CDK inhibitors on cell
proliferation and the induction of apoptosis are not fully
reconciled with the current understanding of the biological
functions of individual CDKs and the CDK family as a whole.
Therefore, an inhibitor active against more than one of the key
CDKs may have additional benefits in terms of antitumor
activity.
Not surprisingly, with the wealth of underlying biological
rationale, the development of chemical modulators of CDKs as
new anticancer agents has engendered significant interest, with
several compounds in clinical and preclinical development.33,34
First generation CDK inhibitors such as 1 (Flavopiridol/
L868275)35 and 7-hydroxystaurosporine36 (UCN-01) have been
evaluated in the clinic for some time, and recently 1 has been
granted orphan drug status for the treatment of chronic lym-
phocytic leukemia.37 Inhibitors with greater selectivity for the
CDKs such as 2 (roscovitine/CYC-202),38 3 (BMS-387032/
SNS-032)39 (both primarily target CDK2, but also possess
significant CDK7 and 9 activity), and 4 (PD0332991)40 (a
selective CDK4/6 inhibitor) (Figure 1) are currently being
evaluated in phase I and II clinical trials, but limited results
have been published to date.
Hit Identification. Apo crystals of CDK2 were soaked with
cocktails of targeted fragments (4 fragments per cocktail). The
screening set of about 500 compounds was made up from a
focused kinase set, a drug fragment set, and compounds
identified by virtual screening against the crystal structure of
CDK2.1 Multiple (>30) low-affinity fragment hits were identi-
fied that bind in the adenosine 5′-triphosphate (ATP) binding
site. A conserved structural feature of all the bound fragments
was one or more hydrogen bonding interactions to key backbone
residues at the hinge region of CDK2 (Glu81 and Leu83). ATP
itself adopts a similar binding mode, as illustrated in Figure
2.41 The compounds shown in Figure 3 are a representative
selection of the hits identified during fragment screening
(compounds 5-8). The hits had only low potency (40 µM to 1
mM) but were highly efficient binders given their low molecular
weight (<225) and limited functionality. An important consid-
eration during our fragments-to-leads phase is pursuing multiple
series in parallel in order to have two or more series for
optimization in the later stages of the project. A key feature of
this process was the collection of multiple protein-ligand crystal
structures to guide iterative cycles of optimization.1,2
To enable the design process, a detailed analysis of the ATP
binding site of CDK2 and binding mode of known CDK
inhibitors was carried out. The analysis identified a number of
key interactions and regions of the protein to target in order to
optimize activity and physicochemical properties (Figure 2).
Hydrogen bonds to the backbone carbonyl and NH of Leu83
and the backbone carbonyl of Glu81 were commonly observed
with bound fragments and more potent ligands. Making all three
of these interactions with the hinge appeared to be a potential
way to design a very potent and ligand efficient inhibitor. Other
key areas to explore included the relatively small region between
the gatekeeper residue (Phe80) and the catalytic aspartic acid
(Asp145) of the DFG motif and a hydrophobic pocket leading
to the solvent exposed region (defined by Phe82, Ile10, Leu134,
and side chain methylene of Asp86). A number of inhibitors in
the literature appeared to interact with Asp86, and this was
targeted with some early compounds.42,43 The solvent accessible
region toward Lys89 was identified as potentially suitable for
modulating physicochemical properties, particularly for increas-

4988 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Wyatt et al.
Figure 3. Fragment-protein cocomplexes of four low-molecular-weight hits identified by fragment-based X-ray crystallographic screening (5-8).
On the left is shown the fragment structure and available IC50 data, in the center a pictorial representation of the protein-ligand complex, and the
right-hand column provides a description of the experimentally determined binding mode. Key: red spheres, water molecules; purple dashed lines,
protein-ligand and water-ligand hydrogen bonds; blue dashed lines, other electrostatic interactions. The PDB code for compound 5 is 1WCC.

Identification of AT7519
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4989
Figure 4. Fragment to lead optimization of pyrazine-based inhibitors. Compound 5 possesses reasonable growth points toward the gatekeeper
residue (Phe80) and from the amino group out toward the solvent exposed region. Hydrophobic space filling by substitution at the 2-amino position
with an aryl group gave compound 9 (7 µM; LE ) 0.50), displaying a 150-fold jump in activity over the starting fragment 5. Perhaps surprisingly,
the structural data quality for this compound was poor, with no electron density observed for the aryl group, possibly due to the aryl group being
able to bind in a number of conformations. Introduction of a sulfonamide at the 4-position of the aryl group forces an intramolecular salt bridge
between Asp86 and Lys89 to break and allows for the formation of a further H-bonding interaction between the sulfonamide and the backbone NH
of Asp86. In spite of this additional interaction, only a modest increase in activity is observed for 10 (1.9 µM; LE ) 0.43).51 Further modification
of this group or replacement of the 6-chloro substituent suggested that optimization beyond low micromolar activity was not straightforward, so this
series was not pursued further. Key: red spheres, water molecules; purple dashed lines, protein-ligand and water-ligand hydrogen bonds; blue
dashed lines, other electrostatic interactions.
Figure 5. Fragment to lead optimization of pyrazolopyrimidine-based inhibitors. Fragment 8 binds to CDK2 as described in Figure 3. The binding
mode is very similar to that of Roscovitine (2) and other bicyclic templates described in the literature. Substitution of compound 8 at the 7-position
with a hydrogen bond donor allowed a third interaction to be formed with the protein backbone at the hinge region (carbonyl of Leu83). The amine
could be substituted with a range of functionalities and the isopropyl group being particularly effective. Compound 11 gave a 700-fold jump in
binding affinity (IC50 ) 1.5 µM, LE ) 0.50) and an improvement in ligand efficiency over the starting fragment. Growing out from the 5-position
allowed the opportunity to access the ribose and phosphate binding regions of the active site. Introduction of basic functionality in the phosphate
binding pocket was well tolerated, with this strategy producing the high affinity lead 12 (IC50 ) 0.03 µM, LE ) 0.45). The crystal structure shows
the 4-amino group to be forming hydrogen bonds with the carboxylate of Asp145 and the side chain carbonyl of Asn132, mimicking the Mg2+
observed in many ATP bound kinase complexes. Compound 12 also displayed good cellular activity (HCT116 cell IC50 ) 0.29 µM), however, this
did not translate into in vivo activity and work on this series was abandoned in favor of more promising compounds. Key: red spheres, water
molecules; purple dashed lines, protein-ligand and water-ligand hydrogen bonds; blue dashed lines, other electrostatic interactions.

4990 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Wyatt et al.
Figure 6. CDK2 cocrystal structures of compounds 6, 14, and 15. Key: red spheres, water molecules; purple dashed lines, protein-ligand hydrogen
bonds; arrows indicate potential vectors for substitution.
Table 1. CDK2 Inhibition of Selected Indazole and Pyrazole Amidesa
ing water solubility. Many of these interactions are discussed
in an interesting review by Liao on the molecular recognition
of protein kinase binding pockets.44
Results and Discussion
Summary of Fragment to Lead Optimization. Figure 3
shows four representative fragment hits identified by structural
screening of CDK2. A number of considerations were taken
into account when deciding which of the hits should be worked
on further, and these included LE, vectors suitable to access
the key regions highlighted in Figure 2, novelty, and synthetic
tractability. Of the fragments described, compound 7 was
assessed not to have suitable vectors for optimization and, in
addition, the chemistry did not appear to be very tractable. As
a result, this compound did not enter hit-to-lead chemistry.
Compounds 5, 6, and 8 were deemed to have suitable vectors
and the chemistry sufficiently tractable to warrant further work.
The optimization of compounds 5 and 8 is outlined in brief in
Figures 4 and 5, respectively; the optimization of compound 6
will be discussed in detail in the following sections.
Fragment to Lead Optimization of Compound 6. The “hit
to lead” chemistry of 6 focused primarily on two vectors (from
the 3 and 5 positions of the indazole ring, see Figure 6) suitable
to access pockets identified by the kinase structural analysis
(Figure 2). Structural data from Astex fragments and known
CDK inhibitors suggested that formation of an additional
hydrogen bonding interaction to the carbonyl of Leu83 was a
possibility.45,46 This was achieved by linking an aryl amide to
the 3-position of indazole, resulting in 13 (IC50 ) 3 µM; LE )
0.42) (Table 1). The phenyl ring was twisted out of plane and
occupies the hydrophobic pocket formed by the backbone of
the linker region and side chains of Ile10 and Leu134. Addition
of a sulfonamide group at the 4-position of the phenyl ring
afforded 14 with submicromolar activity (IC50 ) 0.66 µM; LE
) 0.38). The sulfonamide picks up two further interactions, both
to Asp86, a direct hydrogen bond to the backbone NH and a
water-mediated interaction to the carboxylate side chain (Figure
6).42,43
Substitution of the indazole ring at the 4 or 5 positions
resulted in relatively small increases in CDK2 activity, while
as expected, substitutions at the 6 or 7 positions were poorly
tolerated (data not shown) due to the close proximity of Phe80.
An alternative strategy was pursued in parallel and rather than
seeking to increase the potency of 14 by continuing to add
molecular weight, the system was simplified by removal of the
fused benzene ring to afford the pyrazole 15 (IC50 ) 97 µM;
a Compound were assayed 2 or more times. b IC50 data for 2 was
generated in house and compares well with values quoted in the literature
(within 2 fold).47
LE ) 0.39). Despite a drop in activity, the LE was similar to
the more elaborated compounds and the binding mode as

Identification of AT7519
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4991
Figure 7. CDK2 cocrystal structures of compounds 18, 22, and 23, demonstrating the use of 2,6-disubstitution on the phenyl ring (23) to stabilize
the induced twist observed for the benzamide of 22 on binding to CDK2. Key: red spheres, water molecules; purple dashed lines, protein-ligand
and water-ligand hydrogen bonds.
Table 2. Pyrazole Diamide Structure-Activity Relationships (SAR)
confirmed by the X-ray crystal structure remained identical to
the starting fragment, which encouraged us to pursue this
strategy further. The change of hinge binder provided a
significantly different vector and improved access to the DFG
region between the gatekeeper residue (Phe80) and the catalytic
aspartate (Asp145) (Figure 2). It appeared that derivatizing the
pyrazole at the 4-position presented an opportunity to grow into
this pocket (Figure 6). Accordingly, introduction of a 4-amino
group as a synthetic handle gave 17 which resulted in a modest
increase in activity (IC50 ) 85 µM; LE ) 0.35). Introduction
of a hydrogen bond acceptor gave compounds such as the amide
18, leading to a 100-fold increase in activity and improved LE
(IC50 ) 0.85 µM; LE ) 0.44). An X-ray structure of 18 bound
into CDK2 showed that the increase in activity was at least in
part due to a water mediated hydrogen bond from the acetamide
carbonyl oxygen to the backbone NH of Asp145 (Figure 7).
Another important observation is that the planarity of 18 is
achieved due to an intramolecular hydrogen bond between the
acetamide NH and the benzamide carbonyl, allowing the
compound to fit into the narrow binding pocket.
Ongoing with this work were attempts to replace the amide
at the 3-position of the pyrazole with alternative groups that
could maintain the hydrogen bonding interaction to the backbone
carbonyl of Leu83 while maintaining the planarity of the system.
For example, a 2-benzimidazole group proved to be an effective
replacement for the aryl amide. 16 (IC50 ) 25 µM; LE ) 0.45)
is more active than the corresponding amide 15. Further
optimization of 16 led to the identification of an alternative series
a Compounds were assayed 2 or more times unless indicated * where n
) 1.
with excellent kinase and cell activity. Details of the develop-
ment of this alternative series will follow in a subsequent
publication.
µM, LE ) 0.39); however, the protein-ligand crystal structure
(Figure 7) provided a number of important insights into the
binding mode of this compound. First, a small amount of protein
movement had occurred, allowing the aromatic ring to be
accommodated. Second the phenyl ring was significantly twisted
out of plane of the amide, with a torsion angle of 51°, an
energetically unfavorable conformation. It was postulated that
stabilization of this twist by diortho substitution of the phenyl
ring might be beneficial. The X-ray structure confirmed that 23
bound to CDK2 as predicted (Figure 7), resulting in a 45-fold
increase in kinase activity for the addition of only two heavy
atoms and with a ligand efficiency very similar to the starting
fragment (IC50 ) 0.003 µM, LE ) 0.45).
The protein-ligand structure of 18 indicated that the methyl
of the acetamide group is in very close proximity to the side
chain carboxylate of Asp145 (approximately 3.5 Å), making
the pocket relatively small. Some protein flexibility is observed
in this region of the binding site, so in order to probe this area
further, a limited number of amides with diverse properties were
synthesized (Table 2). A range of simple functionalities such
as 19 and 20 did not afford a significant increase in activity
over 18; however, interesting levels of kinase activity were
obtained with directly attached monocycles such as the cyclo-
hexyl amide 21 and benzamide 22, and these compounds also
showed some indication of cellular activity. The benzamide 22
was particularly interesting, showing only a small increase in
binding affinity and a decrease in ligand efficiency (IC50 ) 0.14
Although 23 exhibited good kinase activity and its pharma-
cokinetic (PK) properties indicated it to be a good lead molecule,

4992 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Table 3. Summary of SAR for Compounds 23-29
Wyatt et al.
a Compounds were assayed 2 or more times unless indicated * where n
) 1. b The plasma clearance data was determined after IV administration
to BALB/c mice at 0.2 mg/kg according to Pharmacokinetic Study Methods.
Figure 8. CDK2 cocrystal structures of compounds 28 and 29. 29
forms an additional hydrogen bond between the ring nitrogen of the
piperidyl group and the carboxylate of Asp86. This may explain the
observed improvement in binding affinity. Key: red spheres, water
molecules; purple dashed lines, protein-ligand and water-ligand
hydrogen bonds.
with moderate plasma clearance (40 mL/min/kg) after intrave-
nous (iv) dosing in mice, its antiproliferative cell activity against
HCT116 colon cancer cells was only moderate (1.4 µM). One
explanation for this moderate cell activity may be due to low
cell permeability. 23 has a ClogP of 2.4, however, the measured
value is approximately 2 log units higher, presumably due to
internal hydrogen bonding reducing the overall polarity of the
molecule. This relatively high lipophilicity may be detrimental
to cell permeability, and in an attempt to address this, further
optimization of the series was sought by replacing the lipophilic
4-fluorophenyl group of 23 (Table 3). In general, other aromatic
groups (data not shown) and simple alkyl groups such as in 24
gave good kinase activity but only moderate cell activity.
However, the directly attached cycloalkyl ring of 25 gave an
improvement in kinase activity and was the first compound in
this series with submicromolar cell activity. Because of its
reasonable cellular activity the PK properties of 25 were
determined in mice and it was found to have high plasma
clearance (65 mL/min/kg). A potential cause of this was
oxidative metabolism of the highly lipophilic cyclohexyl group,
and in an attempt to address this, modifications were made to
the cyclohexyl ring. Although a number of 4-substituted
cyclohexyl derivatives such as 26 and 27 exhibited good kinase
and cell activity, most had relatively high plasma clearances.
Introduction of a nitrogen atom into the ring affording 28 and
29 gave compounds with good CDK2 and cell potency. It
became apparent from making these small polar changes that
modulating physicochemical properties was just as important
as increasing kinase activity when attempting to improve cell
potency. Interestingly, the 3-piperidinyl isomer 29 exhibited an
increase in CDK1 as well as CDK2 activity compared to 28.
This increase in activity can be explained by the presence of a
conserved carboxylate residue in CDK1 and CDK2 (Asp86 in
CDK2) with which the 3-piperidinyl nitrogen of 29 can interact.
The X-ray structure of 29 bound to CDK2 (Figure 8) confirms
this interaction, the protonated nitrogen being 2.65 Å from the
carboxylate of Asp86. Compound 28 was considered to be a
promising lead due to its reasonable cell activity and acceptable
plasma protein binding (PPB) (Table 5); as a consequence of
this, further in vivo characterization of 28 was performed.
Despite showing moderate plasma clearance (43 mL/min/kg),
the compound was dosed to mice bearing HCT116 tumor
xenografts to determine the level of compound in the tumor.
After a single 10 mg/kg dose of 28 was administered via the ip
route, reasonable, albeit somewhat variable levels of compound
appeared to distribute into tumor (AUC ) 3422 ( 2478 h ·ng/
g) (Table 5). The compound was much more rapidly cleared
from plasma, with negligible material remaining after 7 h (data
not shown). In an attempt to determine whether compound levels
in tumor were a potential indicator of in vivo efficacy, 28 was
evaluated for antitumor activity in a mouse xenograft model.
The hydrochloride salt of compound 28 was dosed ip bid (twice
daily) at 18.2, 9.1, and 4.6 mg/kg to SCID mice bearing early
stage HCT116 human colon carcinoma xenografts for 10 days.
Compound 28 showed a clear dose-response for antitumor

Identification of AT7519
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4993
Table 4. Substituted Benzamide SAR for Compounds 30-33
effectively than fluorine. Because previous compounds showed
persistence in tumor in spite of moderate to high plasma
clearance (e.g., 28), further compounds (31 and 33) were dosed
to HCT116 tumor bearing mice at 10 mg/kg to determine tumor
distribution properties. A similar trend to compound 28 was
observed, with significant levels of both 31 and 33 present in
tumor (AUC ) 3325 ( 543 and 6260-6340 h ·ng/g, respec-
tively) (Table 5). Compound 33 in particular showed good tumor
exposure.
The promising in vitro kinase and antiproliferative cell
activity, coupled with low PPB and reasonable tumor distribu-
tion for both 31 and 33, led them to be evaluated in vivo for
potential antitumor efficacy. Compound 31 showed 38% tumor
growth inhibition (%T/C ) 62) in the HCT116 mouse xenograft
model at 10 mg/kg, although the dose and schedule were not
optimized. Compound 33 showed significant efficacy in the
same tumor type producing tumor growth inhibition of 87%
(%T/C ) 13) when dosed at 9.1 mg/kg ip bid for 10 days (Table
5), which warranted further investigation. A similar beneficial
effect was observed for 33 in the A2780 (human ovarian
carcinoma cell line) mouse xenograft model. Details of this and
further characterization of 33 is described in the following
section.
Characterization of Compound 33. Kinase Selectivity
Profile. Compound 33 was profiled more widely against a panel
of kinases (see Supporting Information). In addition to CDKs
1 and 2 (IC50s 190 nM and 47 nM, respectively), 33 potently
inhibited a number of other CDKs (4 and 5 in particular, IC50s
67 nM and 18 nM, respectively), but had lower activity against
other kinases tested (more detailed selectivity data will be
published in a subsequent paper). One explanation for the
observed selectivity over some kinases (Aurora A, IR kinase,
MEK, PDK1, c-abl, IC50 > 10 µM) is shown in Figure 9a. All
these kinases possess an additional glycine residue (in between
the amino acids corresponding to Gln85 and Asp86 of CDK2),
which causes the main chain to bulge into the ATP binding
pocket resulting in a clash with the piperidine of 33.
Cell-Based Activity. Compound 33 is a potent inhibitor of
HCT116 cell proliferation (used as a primary screen during lead
optimization). Following 72 h exposure, 33 potently inhibited
the proliferation of a range of human tumor cell lines (over 100
cell lines have been tested), with compound 33 showing sub 1
µM activity against more than 75 (data not shown). Compound
33 had reduced antiproliferative activity against the nontrans-
formed fibroblast cell line, MRC-5, but more significantly, it
did not affect the viability of noncycling MRC-5 cells at doses
up to 10 µM (Table 6). These data suggest that the antiprolif-
erative activity is cell cycle related and not due to general
cytotoxicity to nondividing cells.
a Compounds were assayed 2 or more times. b The plasma clearance
data was determined after IV administration to BALB/c mice at 0.2 mg/kg
according to Pharmacokinetic Study Methods.
activity although the dose schedule was not optimized. The 18.2
mg/kg dose group showed tumor growth inhibition of 86%
(%T/C ) 14), however this dose was not well tolerated. At a
tolerated dose of 9.1 mg/kg, growth inhibition was 46% (%T/C
) 54) (Table 5) and the 4.6 mg/kg group showed 22% growth
inhibition (%T/C ) 78). These data suggested that distribution
of compounds in tumor and their persistence there might be a
useful indicator of in vivo activity when considered alongside
protein binding and cell potency. The further impact of
compound disposition on biomarker modulation will be dis-
cussed in a subsequent paper.
Further optimization of 28 aimed at increasing activity against
CDK2 and subsequently improve cell activity was explored by
reoptimizing the 2,6-difluorophenyl moiety (Table 4). Attempts
to block potential metabolism of the phenyl ring by substitution
of the 4-position, e.g., in 30 resulted in somewhat reduced
inhibitory activity against the kinase. Replacement of one of
the fluorines of 28 with small substituents such as methoxy and
chloro to give 31 and 32 was well tolerated and resulted in
increased kinase and cell activity (Table 4). The 2,6-dichlo-
rophenyl derivative 33 gave an increase in kinase and cell
activity, as the chlorine atoms filled this lipophilic pocket more
The mechanism of action of 33 in cells was investigated by
monitoring the phosphorylation state of substrates specific for
the various CDKs, following treatment with 33 for 24 h. These
studies indicated that inhibition of phosphorylation of the CDK1
substrate PP1R (Thr320) and the CDK2 substrates Rb (Thr821)
and Nucleophosmin (NPM) (Thr199) (data to be published in
Table 5. Activity, Distribution, and Initial Efficacy Parameters for Compounds 28, 31, and 33
HCT116a
IC50(µM)
Plasma protein binding
(% bound)
Efficacy screening protocol in
HCT116 tumor xenograft model
b
Compound
Tumor AUC(0-t) h·ng/g
% T/Ce
28
31
33
0.31
0.052
0.082
84
75
42
3422 ( 2478 (n ) 4)
3325 ( 543 (n ) 3)
6260-6340
9.1 mg/kg/dose bid QDx10c
10 mg/kg/dose bid QDx9d
9.1 mg/kg/dose bid QDx10c
54
62
13
a Compounds were assayed 2 or more times. b Mean ( SD for n determinations or range for n ) 2 determinations. c Efficacy study conducted in SCID
mice following ip administration. d Efficacy study conducted in BALB/c nude mice following ip administration. e %T/C is the mean tumor volume of treated
animals divided by the mean control tumor volume expressed as a percentage.

4994 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Wyatt et al.
Figure 9. (a) Overlay of C-R traces of the crystal structure of 33 bound into CDK2 (green) with the crystal structures of Aurora A (PDB code
1OL6, yellow), insulin receptor (IR) kinase (1GAG, brown), MEK1 (1S9J, purple), PDK1 (1UU3, blue), and c-abl (1IEP, red), showing the clash
of the kinases (except CDK2) with the piperidine of 33. Aurora A, IR kinase, MEK1, PDK1, and c-abl all have IC50 values >10 µM. (b) CDK2
cocrystal structures of compound 33. Key: red spheres, water molecules; purple dashed lines, protein-ligand and water-ligand hydrogen bonds.
(c) Molecular surface representation of CDK2 with 33 bound in the ATP binding site. The piperidine moiety is pointing out of the pocket toward
solvent. The twisted 2,6-dichlorophenyl is toward the back of the pocket, with the two chlorine atoms efficiently filling small hydrophobic pockets.
Table 6. In Vitro Antiproliferative Activity of 33 in a Panel of Human
Tumor Cell Lines
Origin
Cell line
IC50 (nM)
colon carcinoma
ovarian carcinoma
fibroblast
HCT116
A2780
MRC 5
82
350
980
MRC 5 (nonproliferative)
>10000
Table 7. Mouse PK Data of Compound 33 after iv Administration to
BALB/c Micea
t1/2 (h)
Cl (ml/min/kg)
Vss (L/kg)
1.6 ( 0.9
0.68 ( 0.28
46 (21
a Mean ( SD for n ) 5-7 replicate studies.
a subsequent paper) in HCT116 cells occurred at doses
consistent with the observed antiproliferative effects.
Figure 10. Efficacy of 33 in the early stage A2780 ovarian carcinoma
xenograft mouse model following repeated administration at 7.5 mg/
kg/dose given twice daily by the intraperitoneal route for 8 days against
a matched vehicle-dosed control. Tumor growth curves ( SE for groups
of n ) 12. Growth curves became significantly different from control
from day 5 onward (* p < 0.05, ** p < 0.001). The CDK2 and cellular
IC50 data quoted is the mean based on a minimum of two separate
determinations.
Pharmacokinetics Study. As summarized in Table 7, the
systemic clearance of 33 in BALB/c mice after iv dosing
averaged 46 mL/min/kg with a mean half-life (t1/2) and volume
of distribution (Vss) of 0.68 h and 1.6 L/kg, respectively. 33
showed low oral bioavailability (<1%), which was a common
feature of many closely related basic compounds.
In Vivo Antitumor Activity. Compound 33 was evaluated
for its in vivo antitumor activity in nude BALB/c mice bearing
early stage A2780 human ovarian carcinoma xenografts with a
mean starting volume of approximately 50 mm3 (Figure 10).
In this study, the hydrochloride salt of 33, dissolved in 0.9%
saline, was administered by the intraperitoneal (ip) route, twice
daily, for 8 consecutive days. Tumor growth inhibition at the
end of the experiment was 86% at the 7.5 mg/kg dose level
(%T/C ) 14). A more extensive biological characterization of
compound 33, including a comprehensive cell cycle analysis
and a detailed in vivo efficacy evaluation will be published
separately.
4-nitropyrazole-3-carboxylic acid 34 with 4-fluoroaniline, fol-
lowed by catalytic hydrogenation of 35 with palladium on
carbon. Diamides 18-23 were synthesized by coupling of 17
with acetic anhydride or the appropriate carboxylic acids using
EDC/HOBt.
Esterification of 34 with ethanol under acidic conditions
followed by catalytic hydrogenation of the nitro group gave the
aminopyrazole 36 (Scheme 2). Acylation of 36 with 2,6-
difluorobenzoic acid, followed by basic hydrolysis afforded
pyrazole acid 37. Coupling of 37 with the required amines using
EDC/HOBt gave compounds 24-26. Compounds 27-29
required an additional deprotection step to remove the N-tert-
butoxycarbonyl group. This was achieved under standard acidic
conditions with saturated HCl in EtOAc. The piperidine amides
30-33 were synthesized via the aminopyrazole 38 (Scheme 3),
itself synthesized from the coupling of 4-amino-piperidine-1-
carboxylic acid tert-butyl ester and 34, followed by reduction
of the nitro group. Coupling of 38 with the appropriate
Chemistry. Compounds 13, 14, and 15 were synthesized by
coupling either 1H-indazole-3-carboxylic acid or 1H-pyrazole-
3-carboxylic acid with aniline or 4-aminobenzenesulfonamide.
Compound 16 was prepared by coupling 1H-pyrazole-3-
carboxylic acid with 1,2-diaminobenzene followed by acid
mediated cyclization to form the benzimidazole. Synthesis of
aminopyrazole 17 was achieved (Scheme 1) by coupling

Identification of AT7519
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4995
Scheme 1a
a Reagents and conditions: (a) 4-fluoroaniline, EDC, HOBt, DMF; (b) 10% Pd/C, EtOH, H2; (c) Ac2O, pyridine, or RCO2H, EDC, HOBt, DMF, or
DMSO.
Scheme 2a
a Reagents and conditions: (a) SOCl2, EtOH; (b) 10% Pd/C, EtOH, H2; (c) 2,6-difluorobenzoic acid, EDC, HOBt, DMF; (d) NaOH, MeOH/H2O (1:1);
(e) RNH2, EDC, HOBt, or HOAt, DMF, or DMSO; (f) saturated solution of HCl in EtOAc (where R contains a Boc protected amine).
Scheme 3a
a Reagents and conditions: (a) 4-amino-piperidine-1-carboxylic acid tert-butyl ester, EDC, HOBt, DMF; (b) 10% Pd/C, EtOH, H2; (c) RCO2H, EDC,
DMF; (d) saturated solution of HCl in EtOAc.
carboxylic acids, then acidic deprotection of the Boc protected
piperidine of 39, afforded compounds 30-33. During preclinical
development, compound 33 was synthesized by a six-step route
(similar to Scheme 2) in multikilogram quantities with an overall
yield of 80% and purity >99%, without the requirement for
purification by column chromatography.
used to ensure optimization was carried out efficiently. It quickly
became apparent for indazole based compounds (e.g., 14) that
molecular weight was being added for only small gains in
potency. In contrast, introduction of the acetamide moiety to
the pyrazole system (18) and stabilizing the twisted benzamide
conformation (23) were very efficient methods of increasing
enzyme activity. During later stage lead optimization, LE
became a less important criterion as multiple parameters were
being changed simultaneously. 28 is a good example of a
compound showing a small decrease in enzyme activity,
however, this is offset by a change in physicochemical properties
leading to an improvement in cellular activity.
Compound 33 is a potent, ligand efficient inhibitor of CDK2
(IC50 ) 0.047 µM; LE ) 0.42), with good activity against a
range of human tumor cell lines. It has a good profile against
the major cytochrome P450 isoforms (<30% inhibition at 10
µM for 1A2, 2D6, 3A4, 2C9, 2C19), has good aqueous
thermodynamic solubility either as the acetate or hydrochloride
salt (>25 mg/ml in water or 0.9% saline), and its synthetic
tractability makes it readily amenable to large scale synthesis.
On the basis of these and further data (to be published in a
later paper), compound 33 was selected as a preclinical
development candidate and subsequently entered clinical
development.
Conclusions
High-throughput X-ray crystallographic screening of fragment
libraries was used to identify multiple hits that bound to CDK2.
A number of these fragment hits were elaborated in parallel
with the aid of detailed structural information enabling the
project to take 3 series into late stage lead optimization.
Optimization of the indazole hit 6 eventually led to the discovery
of AT7519 (33). The key compounds in the discovery of 33
are summarized in Scheme 4. Important structural features
identified throughout the optimization process include (Figure
9b): (i) a donor-acceptor-donor interaction anchoring the
molecule to the hinge region of CDK2 (residues Glu81 and
Leu83), (ii) a water mediated hydrogen bond between the
carbonyl of the 4-benzamide group and the backbone N-H of
Asp145, (iii) stabilization of the twisted benzamide conformation
by introduction of two ortho-substituents, (iv) introduction of
the solubilizing aminopiperidine amide group resulted in
improved hydrophobic filling of the region bounded by the
backbone of the hinge and sidechains of residues Phe82, Ile10,
Leu134, and Asp86 and which led to selectivity over non-CDK
kinases, improved cellular activity, and lower plasma clearance.
During the fragment to lead process, structural data and LE were
Experimental Section
Chemistry. Reagents and solvents were obtained from com-
mercial suppliers and used without further purification. Compounds
5-8 were obtained from commercial suppliers. Thin layer chro-

4996 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Wyatt et al.
Scheme 4. Key Compounds in the Optimization of Fragment 6 to the Clinical Candidate 33a
a IC50 data are for CDK2.
matography (TLC) analytical separations were conducted with E.
Merck silica gel F-254 plates of 0.25 mm thickness and were
visualized with UV light (254 nM) and/or stained with iodine,
potassium permanganate, or phosphomolybdic acid solutions fol-
lowed by heating. Standard silica gel chromatography was employed
as a method of purification using the indicated solvent mixtures.
Compound purification was also carried out using a Biotage SP4
system using prepacked disposable SiO2 cartridges (4, 8, 19, 38,
40, and 90 g sizes) with stepped or gradient elution at 5-40 mL/
min of the indicated solvent mixture. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded in the deuterated
solvents specified on a Bruker Avance 400 spectrometer operating
at 400 MHz. Chemical shifts are reported in parts per million (δ)
from the tetramethylsilane internal standard. Data are reported as
follows: chemical shift, multiplicity (br ) broad, s ) singlet, d )
doublet, t ) triplet, m ) multiplet), coupling constants (Hz),
integration. Compound purity and mass spectra were determined
by a Waters Fractionlynx/Micromass ZQ LC/MS platform using
the positive electrospray ionization technique (+ES), or an Agilent
1200SL-6140 LC/MS system using positive-negative switching,
both using a mobile phase of acetonitrile/water with 0.1% formic
acid (see Supporting Information for experimental details).
organic layer was filtered and evaporated under reduced pressure.
The residue was purified by preparative HPLC (method C) to give
10 (11 mg, 6%). 1H NMR (400 MHz, DMSO-d6): δ 10.23 (s, 1H),
8.26 (s, 1H), 8.11 (s, 1H), 7.79 (s, 4H), 7.23 (s, 2H). LC/MS: Rt )
1.08 min, [M + H]+ 285 (method A).
5-Chloro-7-isopropylaminopyrazolo[1,5-a]pyrimidine-3-carbo-
nitrile (11). Isopropylamine (0.12 mL, 2.83 mmol) and potassium
carbonate (652 mg, 4.72 mmol) were added to a solution of 5,7-
dichloropyrazolo[1,5-a]pyrimidine-3-carbonitrile (500 mg, 2.36
mmol) in acetonitrile (10 mL). The resulting mixture was stirred
at room temperature overnight, filtered, and concentrated in vacuo
1
to give 11 (10 mg, 2%). H NMR (400 MHz, Me-d3-OD): δ 8.39
(s, 1H), 6.50 (s, 1H), 4.09-3.98 (m, 1H), 1.39 (d, J ) 6.4 Hz,
6H). LC/MS: Rt ) 2.81 min, [M + H]+ 236 (method A).
5-(4-trans-Amino-cyclohexylamino)-7-isopropylamino-pyrazo-
lo[1,5-a]pyrimidine-3-carbonitrile formate (12). A solution of
5-chloro-7-isopropylamino-pyrazolo[1,5-a]pyrimidine-3-carboni-
trile (180 mg, 0.61 mmol), trans-1,4-cyclohexanediamine (84 mg
0.73mmol) and N,N-diisopropylethylamine (160 µL) in DMF (3.1
mL) was subjected to microwave heating at 140 °C for 1 h. The
reaction mixture was allowed to cool and then purified by RP-
HPLC (method C) to afford 12 as a colorless solid (35 mg, 18%).
1H NMR (400 MHz, DMSO-d6): δ 8.42 (s, 1H), 8.23 (s, 1H),
7.27-7.10 (m, 2H), 5.41 (s, 1H), 3.80 (s, 1H), 3.55 (s, 2H), 2.89
(t, J ) 12.3 Hz, 1H), 1.96 (t, 4H), 1.46-1.32 (m, 2H), 1.32-1.22
(m, 8H). LC/MS: Rt ) 1.03 min (RR), [M + H]+ 314 (method B).
4-(6-Chloropyrazin-2-ylamino)benzenesulphonamide (10). A
suspension of 2,6-dichloropyrazine (100 mg, 0.67 mmol) and
4-sulfamoyl aniline (115 mg, 0.67 mmol) in NMP in a sealed tube
was heated to 250 °C under microwave irradiation for 8 min. The
mixture was diluted with water and extracted with EtOAc. The

Identification of AT7519
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4997
1H-Indazole-3-carboxylic Acid (4-Sulfamoylphenyl)amide (14).
To 1H-indazole-3-carboxylic acid (100 mg, 0.6 mmol) in DMF (5
mL) was added EDC (142 mg, 0.72 mmol), HOBt (100 mg, 0.72
mmol), and sulfanilimide (127 mg, 0.72 mmol). The reaction was
heated at 100 °C for 48 h then cooled and evaporated. The residue
was partitioned between DCM and saturated NaHCO3 solution, then
the resultant solid was collected by filtration, washed with water
and DCM, and then dried. The crude material was purified by RP-
HPLC (method C), and product containing fractions were combined
and evaporated to give 14 as a white solid (12 mg, 6%). 1H NMR
(400 MHz, DMSO-d6): δ 13.87 (s, 1H), 10.68 (s, 1H), 8.24 (d, J
) 8.1 Hz, 1H), 8.09 (d, J ) 8.3 Hz, 2H), 7.80 (d, J ) 8.3 Hz, 2H),
7.69 (d, J ) 8.4 Hz, 1H), 7.48 (t, J ) 7.6 Hz, 1H), 7.32 (t, J ) 7.7
Hz, 1H), 7.26 (s, 2H). LC/MS: Rt ) 2.72 [M + H]+ 317 (method
A).
1H), 10.37 (s, 1H), 8.38-8.26 (m, 1H), 7.92-7.79 (m, 2H),
7.25-7.12 (m, 2H), 6.09 (t, J ) 5.6 Hz, 1H), 4.01 (d, J ) 5.6 Hz,
2H). LC/MS: Rt ) 2.65 min, [M + H]+ 278 (method A).
The following compounds were synthesized using the same
method as used for 19:
4-Benzoylamino-1H-pyrazole-3-carboxylic Acid (4-Fluoro-phen-
yl)-amide (22). Benzoic acid gave 22 as a pink solid (26 mg, 35%).
1H NMR (400 MHz, DMSO-d6): δ 13.55 (br s, 1H), 10.56 (s, 1H),
10.50 (s, 1H), 8.42 (s, 1H), 7.96-7.81 (m, 4H), 7.70-7.55 (m,
3H), 7.27-7.14 (m, 2H). LC/MS: Rt ) 3.96 min, [M + H]+ 324
(method A).
4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid
(4-Fluoro-phenyl)-amide (23). 2,6-Difluorobenzoic acid in DMSO
1
gave 23 as a cream-colored solid (25 mg, 30%). H NMR (400
MHz, DMSO-d6): δ 13.57 (s, 1H), 10.42 (s, 1H), 10.31 (s, 1H),
8.43 (s, 1H), 7.88-7.77 (m, 2H), 7.69-7.58 (m, 1H), 7.27 (dd, J
) 8.6, 8.5 Hz, 2H), 7.17 (dd, J ) 8.7, 8.6 Hz, 2H). LC/MS: Rt
3.76 min, [M + H]+ 361 (method A).
1H-Pyrazole-3-carboxylic Acid Phenylamide (15). 1H-Pyrazole-
3-carboxylic acid (100 mg, 0.9 mmol), EDC (211 mg, 1.1 mmol),
HOBt (147 mg, 1.1 mmol), and aniline (0.089 mL, 0.98 mmol)
were dissolved in DMF (5 mL) and the resulting solution was stirred
for 2 days. The mixture was concentrated under reduced pressure
and partitioned between water and EtOAc. The organic fraction
was dried (MgSO4), filtered, and evaporated in vacuo and the
residue purified by column chromatography to give 15 (97 mg,
4-Amino-1H-pyrazole-3-carboxylic Acid Ethyl Ester (36). Step
1. Thionyl chloride (2.9 mL, 39.8 mmol) was slowly added to a
mixture of 34 (5.68 g, 36.2 mmol) in EtOH (100 mL) at room
temperature then stirred for 48 h. The mixture was evaporated in
vacuo and re-evaporated with toluene to afford 4-nitro-1H-pyrazole-
1
1
58%) as a white solid. H NMR (400 MHz, DMSO-d6): δ 13.44
3-carboxylic acid ethyl ester as a white solid (6.42 g, 96%). H
(s, 1H), 10.01 (s, 1H), 7.93-7.84 (s, 1H), 7.81 (d, J ) 8.0 Hz,
2H), 7.33 (dd, 8.0, 7.7 Hz, 2H), 7.08 (t, J ) 7.7 Hz, 1H), 6.82 (s,
1H). LC/MS: Rt ) 0.98 (RR) [M - H]- 186 (method B).
NMR (400 MHz, DMSO-d6): δ 14.4 (s, 1H), 9.0 (s, 1H), 4.4 (q,
2H), 1.3 (t, 3H).
Step 2. A mixture of 4-nitro-1H-pyrazole-3-carboxylic acid ethyl
ester (6.40 g, 34.6 mmol) and 10% Pd/C (650 mg) in EtOH (150
mL) was stirred under an atmosphere of hydrogen for 20 h. The
mixture was filtered through a plug of celite, evaporated under
vacuum then re-evaporated with toluene to afford 36 as a pink solid
4-Nitro-1H-pyrazole-3-carboxylic Acid (4-Fluoro-phenyl)-amide
(35). 4-Nitro-1H-pyrazole-3-carboxylic acid 34 (10 g; 63.66 mmol)
was added to a stirred solution of 4-fluoroaniline (6.7 mL; 70
mmol), EDC (14.6 g; 76.4 mmol), and HOBt (10.3 g; 76.4 mmol)
in DMF (25 mL) and then stirred at room temperature for 16 h.
The solvent was removed by evaporation under reduced pressure
and the residue triturated with EtOAc/saturated brine solution. The
resultant yellow solid was collected by filtration, washed with 2 M
hydrochloric acid, and then dried under vacuum to give 35 (15.5
1
(5.28 g, 98%). H NMR (400 MHz, DMSO-d6): δ 12.7 (s, 1H),
7.1 (s, 1H), 4.8 (s, 2H), 4.3 (q, 2H), 1.3 (t, 3H) (method A).
4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid
(37). A mixture of 2,6-difluorobenzoic acid (6.32 g, 40.0 mmol),
36 (5.96 g, 38.4 mmol), EDC (8.83 g, 46.1 mmol), and HOBt
(6.23 g, 46.1 mmol) in DMF (100 mL) was stirred at ambient
temperature for 6 h. The mixture was reduced in vacuo, water
added, and the solid formed collected by filtration and air-dried
to give 4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic
acid ethyl ester as the major component of a mixture (15.3 g).
LC/MS: Rt ) 3.11 min, [M + H]+ 295. Crude 4-(2,6-difluoro-
benzoylamino)-1H-pyrazole-3-carboxylic acid ethyl ester (10.2
g) in 2 M aqueous NaOH/MeOH (1:1, 250 mL) was stirred at
ambient temperature for 14 h. Volatile materials were removed
in vacuo, water (300 mL) added and the mixture adjusted to pH
5 using 1 M aqueous HCl. The resultant precipitate was collected
by filtration and dried through azeotrope with toluene to afford
1
g, 97%). H NMR (400 MHz, DMSO-d6): δ 14.25 (s, 1H), 10.73
(s, 1H), 8.96 (s, 1H), 7.72 (dd, J ) 8.4, 4.9 Hz, 2H), 7.22 (t, J )
8.5 Hz, 2H). LC/MS: Rt ) 2.92 min, [M + H]+ 250 (method A).
4-Amino-1H-pyrazole-3-carboxylic Acid (4-Fluoro-phenyl)-
amide (17). Compound 35 (15 g, 60 mmol) was dissolved in 200
mL of EtOH, treated with 1.5 g of 10% palladium on carbon under
a nitrogen atmosphere, then hydrogenated at room temperature and
pressure for 16 h. The catalyst was removed by filtration through
celite and the filtrate evaporated. The crude product was dissolved
in acetone/water (200 mL, 1:1), and after slow evaporation of the
acetone, 17 was collected by filtration as a brown crystalline solid
1
(8.1 g, 62%). H NMR (400 MHz, DMSO-d6): δ 12.77 (s, 1H),
1
9.87 (s, 1H), 7.88-7.76 (m, 2H), 7.25-7.07 (m, 3H), 4.69 (s, 2H).
37 as a pink solid (5.70 g, 83% from 36). H NMR (400 MHz,
LC/MS: Rt ) 1.58 min, [M + H]+ 221 (method A).
DMSO-d6): δ 13.60-13.30 (br s, 1H), 10.06 (s, 1H), 8.25 (s,
1H), 7.68-7.54 (m, 1H), 7.25 (t, J ) 8.4 Hz, 2H). LC/MS: Rt
) 2.33 min (94% purity), [M + H]+
4-Acetylamino-1H-pyrazole-3-carboxylic Acid (4-Fluoro-phen-
yl)-amide (18). Compound 17 (500 mg; 2.27 mmol) was dissolved
in pyridine (5 mL), treated with acetic anhydride (240 µL, 2.5
mmol), and then stirred at room temperature for 16 h. The solvent
was removed by evaporation, and then dichloromethane (20 mL)
and 2 M hydrochloric acid (20 mL) were added. The undissolved
solid was collected by filtration, washed with dichloromethane and
water, and then dried under vacuum. The product 18 was isolated
as an off-white solid (275 mg, 46%). 1H NMR (400 MHz, DMSO-
d6): δ 13.33 (br s, 1H), 10.29 (s, 1H), 9.54 (s, 1H), 8.24 (s, 1H),
7.90-7.77 (m, 2H), 7.18 (t, J ) 8.7 Hz, 2H), 2.12 (s, 3H). LC/
MS: Rt ) 2.96 min, [M + H]+ 263 (method A).
267 (method A).
4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid
(trans-4-Amino-cyclohexyl)-amide (27). A mixture of 37 (100 mg,
0.37 mmol), (trans-4-amino-cyclohexyl)-carbamic acid tert-butyl
ester (98 mg, 0.46 mmol), EDC (86 mg, 0.45 mmol), and HOBt
(60 mg, 0.45 mmol) in DMSO (5 mL) was stirred at ambient
temperature for 16 h. The mixture was reduced in vacuo, then
the residue taken up in CH2Cl2 and washed successively with
saturated aqueous sodium bicarbonate, water, and brine. The
organic portion was dried (MgSO4), filtered, and the solvent
reduced in vacuo. Trituration of the resulting solid with CH2Cl2
gave 27, protected with an N-tert-butoxycarbonyl (t-Boc) group.
This was removed by treatment with saturated EtOAc/HCl at
room temperature for 1 h. The solid that precipitated out of the
reaction mixture was filtered off, washed with ether, and then
dried to give 27 (14 mg, 10%). 1H NMR (400 MHz, Me-d3-
OD): δ 8.45 (s, 1H), 8.36 (s, 1H), 7.63-7.57 (m, 1H), 7.16 (t,
J ) 8.6 Hz, 2H), 3.91-3.84 (m, 1H), 3.18-3.11 (m, 1H),
2.16-2.05 (m, 4H), 2.03 (s, 1H), 1.61-1.50 (m, 4H). LC/MS:
Rt ) 1.75 min, [M + H]+ 364 (method A).
4-(2-Hydroxyacetylamino)-1H-pyrazole-3-carboxylic Acid (4-
fluorophenyl)amide (19). Hydroxyacetic acid (19 mg, 0.25 mmol)
was added to a solution of 17 (50 mg, 0.23 mmol), EDC (53 mg,
0.27 mmol), and HOBt (37 mg, 0.27 mmol) in DMF (5 mL). The
reaction mixture was stirred at room temperature for 24 h. The
solvent was removed under reduced pressure. The residue was
purified by preparative LC/MS (method C) and, after evaporation
of product-containing fractions, yielded 19 as a white solid (26 mg,
41%). 1H NMR (400 MHz, DMSO-d6): δ 13.45 (s, 1H), 10.42 (s,

4998 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Wyatt et al.
The following compounds were synthesized in an analogous
manner to 27:
Acknowledgment. We thank Emma Vickerstaffe and
Charlotte Griffiths-Jones for writing the experimental details,
Miles Congreve, Martyn Frederickson, Emma Vickerstaffe,
Michelle Jones, and Chris Murray for proof reading, and
David Rees, Nicola Wallis, and Neil Thompson for useful
discussions. We are also grateful to Molecular Imaging
Research Inc (Ann Arbor, MI), particularly W. R. Leopold,
III, and Erin Trachet, for conducting the xenograft studies
using SCID mice.
4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid
Piperidin-4-ylamide (28). 4-Amino-piperidine-1-carboxylic acid
tert-butyl ester gave 28 (62 mg, 48%). 1H NMR (400 MHz,
DMSO-d6): δ 13.49 (s, 1H), 10.38 (s, 1H), 9.08 (br d, 1H), 8.69
(br s, 2H), 8.32 (s, 1H), 7.69-7.58 (m, 1H), 7.27 (t, J ) 8.6
Hz, 2H), 4.11-3.96 (m, 1H), 3.28 (d, 2H), 3.01-2.88 (m, 2H),
2.00-1.75 (m, 4H). LC/MS: Rt ) 1.57 min, [M + H]+ 350
(method A).
4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-(S)-carboxylic Acid
Piperidin-3-(S)-ylamide Hydrochloride (29). Using HOAt instead
of HOBt, 3-(S)-amino-piperidine-1-carboxylic acid tert-butyl
Supporting Information Available: In vitro kinase selectivity
data; detailed descriptions of HPLC methods; HPLC purity
analysis of final compounds; NMR spectra and HPLC traces for
compounds 10, 12, 14, 15, 18, 22, 23, 28, 33; further synthetic
chemistry experimental; experimental details for biological
assays and DMPK studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
ester gave 29 (55 mg, 43%). H NMR (400 MHz, Me-d3-OD):
δ 8.37 (s, 1H), 8.35 (s, 1H), 7.65-7.56 (m, 1H), 7.17 (t, J )
8.6 Hz, 2H), 4.32-4.22 (m, 1H), 3.49 (dd, J ) 12.2, 4.1, 1H),
3.36 (s, 1H), 3.08-2.94 (m, 2H), 2.15-2.02 (m, 2H), 1.92-1.73
(m, 2H). LC/MS: Rt ) 1.76min, [M + H]+ 350 (method A).
4-[(4-Amino-1H-pyrazole-3-carbonyl)-amino]-piperidine-1-car-
boxylic Acid tert-Butyl Ester (38).
References
Step 1. A solution of 34 (7.3 g, 45.8 mmol), 4-amino-
piperidine-1-carboxylic acid tert-butyl ester (10.2 mg, 51 mmol),
EDC (10.7 g, 55.8 mmol), and HOAt (7.5 g, 55.8 mmol) in DMF
(100 mL), was stirred at room temperature for 16 h. The solvent
was then removed by evaporation under reduced pressure and
the residue triturated with water (250 mL). The resultant cream
solid was collected by filtration, washed with water, and then
dried under vacuum to give 4-[(4-nitro-1H-pyrazole-3-carbonyl)-
amino]-piperidine-1-carboxylic acid tert-butyl ester (13.05 g,
84%). 1H NMR (400 MHz, DMSO-d6): δ12.53 (s, 1H), 7.67 (d,
J ) 8.4 Hz, 1H), 7.10 (s, 1H), 4.60-4.52 (m, 1H), 3.99-3.83
(b m, 4H), 2.88-2.75 (m, 2H), 1.74-1.68 (m, 2H), 1.52-1.37
(m, 11H). LC/MS: Rt ) 2.50 min, [M + H]+ 340 (method A).
Step 2. 4-[(4-Nitro-1H-pyrazole-3-carbonyl)-amino]-piperi-
dine-1-carboxylic acid tert-butyl ester (13.05 g, 38.4 mmol) was
dissolved in EtOH (300 mL) and DMF (75 mL), treated with
10% palladium on carbon (500 mg) and then hydrogenated at
room temperature and pressure for 16 h. The catalyst was
removed by filtration through celite, the filtrate evaporated and
re-evaporated with toluene. The crude material was purified by
flash column chromatography eluting with EtOAc then 2%
MeOH/EtOAc then 5% MeOH/EtOAc. Product containing
fractions were combined and evaporated to give 38 (8.78 g, 74%)
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1
as a brown foam. H NMR (400 MHz, DMSO-d6): δ 12.53 (s,
1H), 7.67 (d, J ) 8.4 Hz, 1H), 7.10 (s, 1H), 4.60-4.50 (m,
1H), 3.98-3.81 (m, 4H), 2.87-2.81 (m, 2H), 1.76-1.66 (m,
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Compound 33 was Made Using the Following General Method.
To a stirred solution of 38 (0.23 mmol), EDC (52 mg; 0.27
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4-(2,6-Dichloro-benzoylamino)-1H-pyrazole-3-carboxylic Acid
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