Phthalazinone pyrazoles as potent, selective, and orally bioavailable inhibitors of Aurora-A kinase

Article abstract of DOI:10.1021/jm101346r

The inhibition of Aurora kinases in order to arrest mitosis and subsequently inhibit tumor growth via apoptosis of proliferating cells has generated significant discussion within the literature. We report a novel class of Aurora kinase inhibitors based upon a phthalazinone pyrazole scaffold. The development of the phthalazinone template resulted in a potent Aurora-A selective series of compounds (typically >1000-fold selectivity over Aurora-B) that display good pharmacological profiles with significantly improved oral bioavailability compared to the well studied Aurora inhibitor VX-680.

Full text of DOI:10.1021/jm101346r

312 J. Med. Chem. 2011, 54, 312–319
DOI: 10.1021/jm101346r
Phthalazinone Pyrazoles as Potent, Selective, and Orally Bioavailable Inhibitors of Aurora-A Kinase†
Michael E. Prime,*,‡ Stephen M. Courtney,‡ Frederick A. Brookfield,‡ Richard W. Marston,‡ Victoria Walker,‡ Justin Warne,‡
,^
Andrew E. Boyd,‡ Norman A. Kairies, Wolfgang von der Saal,§ Anja Limberg,§ Guy Georges,§ Richard A. Engh,
Bernhard Goller,§ Petra Rueger,§ and Matthias Rueth§
‡Evotec (UK) Ltd., 114 Milton Park, Abingdon, OX14 4SA, U.K., §Roche Diagnostics GmbH, Pharma Research Penzberg, D-82372 Penzberg,
Germany, and Max-Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Planegg, Germany. ^ Current address: Department of
Chemistry, University of Tromsø, Norway.
Received October 18, 2010
The inhibition of Aurora kinases in order to arrest mitosis and subsequently inhibit tumor growth via
apoptosis of proliferating cells has generated significant discussion within the literature. We report a
novel class of Aurora kinase inhibitors based upon a phthalazinone pyrazole scaffold. The development of the
phthalazinone template resulted in a potent Aurora-A selective series of compounds (typically >1000-fold
selectivity over Aurora-B) that display good pharmacological profiles with significantly improved oral
bioavailability compared to the well studied Aurora inhibitor VX-680.
Introduction
TheAurorakinases(consistingofAuroraA, B, and C) area
family of serine/threonine kinases that are involved in phos-
phorylation events believed to be key for the completion of
critical mitotic events. Although both Aurora-A and Aurora-
B are essential for the correct progression through and com-
pletion of mitosis, they are involved in very different roles
within the cell division cycle.1-3
Aurora-A localizes mainly to the centrosomes and is key for
correct centrosome maturation and separation.4-7 While
Aurora-A is not a classical oncogene, it is highly expressed
in many tumors; its elevation may play an important role in
the support of tumor progression and as such has attracted
significant attention for small molecule inhibition. Further-
more, studies to inhibit the expression of Aurora-A in cells
cause arrest in the cell cycle and subsequent cell death via
apoptosis.8,9
Aurora-B localizes predominantly to the spindle midzones
and has two key roles within the cell-division cascade of
events. Initially Aurora-B is required for the phosphorylation
of histone H3 at serine 10. It then maintains a wait-anaphase
signal until all chromosomes are in the correct orientation for
separation.10-12
The role of Aurora-C within tumorgenesis is less well-defined
(although it has recently been suggested that it may have over-
lapping functions with Aurora-B), and additionally the expres-
sion level of Aurora-C in most somatic tissues is low except in
the testis where expression is high.13,14
Figure 1. Advanced Aurora kinase inhibitors (data obtained from
literature sources).15-20
for phosphohistone H3). Examples described include VX-680
(Vertex, pan Aurora-A/B/C inhibitor), AZD-1152 (Astra-
Zeneca, Aurora-B/C selective inhibitor), and GSK1070916
(GlaxoSmithKline, Aurora-B selective inhibitor) shown in
Figure 1.15-18 There are very few examples of selective
Aurora-A inhibiting molecules, the best characterized being
MLN8054 (Millenium, 43-fold selectivity over Aurora-B,
although alternative data suggest that this selectivity may be
as low as 7-fold).19,20
We set out to prepare a class of compounds that displays
selectivity for Aurora-A over Aurora-B. In this report we
describe the discovery of a potent, Aurora-A selective class of
small molecule inhibitors with >1000-fold selectivity over
Aurora-B (5, Figure 2) which display orally dosed pharma-
cokinetic profiles suitable for in vivo pharmacodynamic studies.
There are a large number of Aurora inhibitors currently
undergoing clinical development, the vast majority of which
display the classical phenotype associated with Aurora-B
inhibition (cells treated with Aurora-B inhibitors fail to stain
†Atomic coordinates and structure factors for the crystal structure of
Aurora A with compound 7 can be accessed using PDB code 3P9J.
*To whom correspondence should be addressed. Phone: (44) 01235
441416. Fax: (44) 01235 441351. E-mail: Michael.Prime@evotec.com.
r
pubs.acs.org/jmc
Published on Web 12/03/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 313
Scheme 2. Improved Synthesis of Phthalazinonesa
Figure 2. General structure of phthalazinone Aurora-A inhibitors.
Chemistry
The general synthesis of the initial phthalazinone analogues
starts from phthalic anhydride and is described in Scheme 1.
Reaction of electron-rich phenylhydrazines with phthalic
anhydride affords the corresponding phthalazine-1,4-diones
in moderate yields (30-50%) in one step, whereas electron-
poor phenylhydrazines (such as 12 and 13 where Y = Cl or F)
require a ring-expansion step using forcing conditions after
formation of the initial isoindole-1,3-diones to give the target
phthalazine-1,4-diones in good yields (70-80%) over two
steps. Bromination using phosphorus oxybromide affords
the bromophthalazinones in moderate yields (50-65%) which
can be converted via palladium-catalyzed Buchwald-Hartwig
coupling into the target phthalazinones in low to moderate
yield (30-40%).21,22
In order to carry out further elaboration of the phthalazi-
none scaffold and fully explore structure-activity relation-
ships, analternativesynthetic routewas required thatdoesnot
rely on the use of limited commercially available substituted
hydrazines (Scheme 2). Hydrazine insertion into phthalic
anhydride affords phthalazine-1,4-dione in quantitative yield.
Subsequent dibromination followed by monohydrolysis gives
the desired bromophthalazinone in good yield (60% over two
steps). Substitution of the amide is then achieved using sodium
hydride and an alkylating agent in moderate to high yield
(>55%). The final desired phthalazinones are prepared
a Reagents and conditions: (a) hydrazine, EtOH, 80 ꢀC; (b) POBr3,
DCE, 120 ꢀC, 24 h; (c) AcOH, 120 ꢀC, 2 h; (d) NaH, R2X, DMF, 2 h (3)
Pd2dba3, NaOtBu, 2-(di-tert-butylphosphino)biphenyl, toluene/EtOH
(10:1), 100 ꢀC, 20 h.
using the previously described Buchwald-Hartwig palladium
coupling.
Results and Discussion
Extensive analogue synthesis and design based around a
number of phthalazine and pyridazine scaffolds was carried
Scheme 1. General Synthesis of Phthalazinonesa
a Reagents and conditions: (a) EtOH, 80 ꢀC; (b) glycerol, 180 ꢀC, 24 h; (c) POBr3, DCE, 120 ꢀC, 20 h; (d) Pd2dba3, NaOtBu, 2-(di-tert-butyl-
phosphino)biphenyl, toluene/EtOH (10:1), 100 ꢀC, 20 h.

314 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Prime et al.
out which showed only weak (1-10 μM) activity against
Aurora-A (Figure 3). All of these inhibitors were relatively
linear in structure, closer inspection of the published X-ray
structure of VX-680 with Aurora-A suggested that it may be
possible to deviate from linearity.23 Hence, we moved the aryl
group from the para-position to the meta-position (position
relative to the aminoimidazole group) and designed the
phthalazinone 5 (Figure 2).
While utilization of simple hydrazine insertion led to the
discovery of compound 6 which showed improved activity
toward Aurora-A, we were pleased to discover that substitution
with a phenyl group in initial analogue 7 not only led to
improved potency against Aurora-A but also displayed ex-
cellent selectivity over Aurora-B. Subsequent substituted analo-
gues 8 and 9 displayed similar activities but also improved
cellular activity in the HCT116 assay (Table 1).
Inspection of the binding mode for compound 7 after
cocrystallization with Aurora-A (Figure 4) indicates that the
donor-acceptor-donor motif of the aminopyrazole provides
key (and expected) interactions between compound 7 and the
hinge region of Aurora-A (Glu-211, Ala-213, Pro-214).
The proximity of the R2 group (substituted phenyl or a
methylene linked substituted phenyl in most inhibitors de-
scribed here) to Aurora-A Thr-217 is the most likely con-
tributor to selectivity. The corresponding residue in Aurora-B
is Glu leading to a steric clash with the phenyl R2 group
(Figure 5). The extension of the substitution at the R2 position
by a methylene spacer (compounds 19-26) indicates a slight
removal of this clash, with a slight increase in Aurora-B activity
observed. Additionally, the strength of the inhibition appears to
depend on the chemical nature of the 4-subsitution; i.e., NO2
finds an especially favorable binding site. In this description,
the lack of inhibition of Aurora-B by compound 16 seems
anomalous.
The strong effect of the loss of the methyl group at the R1
position comparing compounds 9 and 14 in Aurora-A also
may seem surprising. The methyl group binds in a largely
hydrophobicpocket. Itdoes not completelyfillthe pocket, but
Figure 3. Selected templates investigated as Aurora kinase inhibitors.
Table 1. Screening Data for Phthalazinone Analoguesa
IC50, μM
compd
R1
R2
Aur-A
Aur-B
HCT116
Colo205
MCF7
6
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Ph
0.65
22
N/T
7.8
N/T
2.9
N/T
1.6
7
0.031
0.029
0.023
0.024
0.24
>100
>100
>100
>100
>50
>100
>100
N/T
N/T
>100
>100
>100
69
8
(4-OMe)Ph
(4-Me)Ph
(3-Me)Ph
(4-tBu)Ph
(4-Cl)Ph
0.7
3.7
3.6
3.6
9
0.24
2.3
>30
2.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
3.2
0.06
0.82
0.8
N/T
2.2
0.78
11.4
1.6
0.093
0.037
1
(4-F)Ph
(4-Me)Ph
1.6
N/T
N/T
1.5
N/T
N/T
4.4
N/T
N/T
12.6
2.6
N/A
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Ph
CH2Ph
14
0.065
0.049
0.025
0.014
0.056
0.032
0.036
0.093
0.27
CH2(3,5-di-F)Ph
(4-OMe)Ph
0.5
2.3
3.8
0.57
1.4
>30
>30
4.5
CH2(4-F)Ph
CH2(4-SO2Me)Ph
CH2(4-OMe)Ph
CH2(3-OMe)Ph
CH2(4-NHAc)Ph
CH2(4-pyridine)
CH2(3-pyridine)
CH2(4-NO2)Ph
CH2C(O)(4-OMe)Ph
CH2C(O)(3-OMe)Ph
CH2C(O)(4-CF3)Ph
CH2C(O)Ph
CH2(3-methylthiazole)
Me
17
4.5
34
46
1.7
1.2
4.2
3.6
>30
>30
>30
>30
13.4
>30
>30
>30
>30
>30
>30
>30
1.6
28
15
0.66
4.2
>30
13
27
20.2
2.9
0.017
0.109
0.067
0.1
12.7
0.93
>100
67
4.1
1.4
N/T
>30
>30
>30
>30
>30
>30
1.7
>30
2.7
0.72
>100
>100
24
>30
5.44
4.18
5.2
0.071
0.12
0.27
17
30
isopropyl
isobutyl
0.035
0.115
0.066
0.55
0.4
1.1
34
15
N/T
N/T
N/T
4.2
4.5
CH2CF3
CH2(4-NHAc)Ph
1.8
>30
34
>30
a N/T = not tested. Results are the average of triplicate data; SD = 5-10%.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 315
Figure 4. Crystal structure of phthalazinone 7 and Aurora-A, with
hydrogen bonds to the hinge region shown in yellow.
Figure 6. Cellular effect of 16 on MCF7 cells: (A) normal cycling
cell, immunostained for Aurora-A (green); (B) cells incubated with
16 displaying improper centrosome formation. Cells were incubated
with 10 μM 16 for 24 h. DNA is in blue, and Aurora-A is in green.
Compounds 7, 16, and 33 (which all display good biochem-
ical potency with moderate activity against all cell lines) were
selected for further in vitro and in vivo profiling (Table 2).
There were no significant cytochrome P450 or hERG related
issues. The compounds showed moderate to high stability in
microsomes and hepatocytes and were taken on for pharmaco-
kinetic analysis.
All three compounds displayed plasma concentrations
above the cellular IC50 at the half-life and also show substan-
tially improved oral bioavailability compared with VX-680 as
illustrated in Figure 7.
Conclusion
The phthalazinone class of general structure 5 represents a
novel class of Aurora-A inhibitors displaying over 1000-fold
selectivity against Aurora-B. The compounds also display
good oral bioavailability and good plasma concentrations,
which is encouraging for pharmacodynamic testing. Further
development of this class of compound has been carried out to
address improvements in cellular activity, and the results of
this development along with initial pharmacodynamic studies
will be reported separately.
Figure 5. Pictorial representation of clash generated between R2
phenyl and Glu residue (shown in yellow) in Aurora-B.
the adjoining surfaces are partially polar and can accommo-
date water molecules. In the absence of inhibitor, water can be
found bound to the hinge and the ATPa pocket will be filled
with largely structured water. Compound 14, however, allows
neither the hydrophobic interactions made by the methyl
group nor the binding of an appropriately structured water
molecule, leaving instead an energetically unfavorable hole. A
similar comparison between compounds 23 and 36 shows a
smaller effect. However, the lessened effect arises primarily
because compound 23 binds to Aurora-A more weakly than
compound 9 and the energetic disadvantage of a putative
disruptive water remains.
Experimental Section
Crystallography. A triple mutant (K124A, T287A, T288A)
kinase domain construct (residues 124-391) of Aurora-A ex-
pressed in E. coli was used for crystallization.24 The threonine to
alanine mutations were located in the activation loop at the
phosphorylation sites and eliminated heterogeneous autopho-
sphorylation that prevented purification to homogeneity. The
protein was concentrated to between 10 and 20 mg/mL and
mixed in a ratio of 24:1 with a 50 mM DMSO solution of the
inhibitor. Crystals were produced by mixing the protein-inhibi-
tor solution with a PEG grid crystallization screen in volumes of
100 nL þ 200 nL, 200 nL þ 200 nL, and 300 nL þ 200 nL using a
MicroSys8 nanoliter pipetter into Greiner and/or Corning low
birefringence 96-well sitting drop crystallization plates at 4 ꢀC.
The crystallization optimization screen consisted of a set of
Several compounds display phenotypic Aurora-A inhibi-
tion when tested against the MCF7 cell line, with poorly
formed centrosomes observed (such as compound 16, Figure 6),
and cells subsequently arrest at the G2M stage.
a Abbreviations: ATP, adenosine triphosphate; DCE, 1,2-dichlor-
oethane; hERG, the human ether-a-go-go-related gene; HPLC, high
pressure liquid chromatography; PEG, polyethylene glycol.

316 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Prime et al.
Table 2. In Vitro/in Vivo Profiling of Selected Phthalazinone Inhibitors
7
16
33
Aurora-A (μM)
HCT 116 (μM)
0.03
7.8
0.065
1.5
1.9
0.035
0.4
aqueous solution (μg/mL)
Cyp 3A4, 2C9, 2D6 (μM)
hERG
CLhep ((μL/min)/106 cells)
CLmic ((μL/min)/mg Prot)
Cmax (μM), 30 mg/kg po
1.2
7
>50, >50, >50
>50, >50, >50
20% at 10 μM
r 2.3
>50, 42, >50
N/T
r 2.4
N/T
N/T
N/T
25
h 0, m 15.7
1.4
2.3
h 7.4, m 28
13
4.2
T
1/2 (h)
2.4
Aurora-A kinase or Aurora-B kinase with test compound and
30 μM ATP. The reaction buffer was 10ꢀ kinase buffer (Cell
Signaling catalog no. 9802) supplemented with 1 μg/mL I-block.
Reactions were stopped after 40 min by addition of 25 mM
EDTA. After washing, substrate phosphorylation was detected
by addition of anti-phospho-histone H3 (Ser 10) 6G3 mAb (Cell
Signaling catalog no. 9706) and sheep anti-mouse pArb-HRP
(Amersham catalog no. NA931V), followed by colorimetric
development with TMB (3,30,5,50-tetramethylbenzidine from
Kirkegaard & Perry Laboratories). After readout of the adsor-
bance, IC50 values were calculated using a nonlinear curve fit
(XLfit software (ID Business Solution Ltd., Guilford, Surrey,
U.K.)). All screening data are the average of triplicate results
with a standard deviation of between 5% and 10%.
CellTiter-Glo Assay in HCT 116 Cells. HCT 116 cells (human
colon carcinoma, ATCC no. CCl-247) were cultivated in RPMI
1640 medium with GlutaMAX I (Invitrogen, catalog no. 61870-
010), 2.5% fetal calf serum (FCS, Sigma catalog no. F4135), and
100 units of penicillin/mL and 100 μg of streptomycin/mL
(=Pen/Strep from Invitrogen, catalog no.15140). Cells were
seeded in 384-well plates, 1000 cells per well, in the same
medium. The following day the test compounds were added
in various concentrations ranging from 30 to 0.0015 μM
(10 concentrations, 1:3 diluted). After 5 days the CellTiter-Glo
assay was carried out according to the instructions of the manu-
facturer (CellTiter-Glo luminescent cell viability assay, from
Promega). The cell plate was equilibrated to room temperature
for approximately 30 min, and then the CellTiter-Glo reagent
was added. The contents were carefully mixed for 15 min to induce
cell lysis. After 45 min the luminescent signal was measured in
Victor 2 (scanning multiwall spectrophotometer, Wallac). All
screening data are the average of triplicate results with a standard
deviation of between 5% and 10%.
Figure 7. Plasma concentrations of phthalazinones 16 and 33.
PEG, pH, and salt additive screens, using PEG3350, PEG2000,
and PEG4000. Plates were stored and imaged at 4 and 20 ꢀC in a
Rhombix (DCA/Kendro/Thermo) based temperature controlled
hotel and microscope imager system. Hexagonal crystals typically
appeared within 1 or 2 days, but the largest crystals could take
up to a month to grow and reach lengths of ∼300 μm.
Crystals were harvested using paratone N as cryoprotectant
and frozen in liquid nitrogen. Data were collected at the Swiss
Light Source in Villigen, Switzerland. The measured crystal
was hexagonal, with P6122 symmetry, with cell constants a =
˚
˚
˚
b = 83.1 A and c =169.2 A, and diffracted to 2.8 A. The data
were 99.7% complete for the resolution range used in refinement
˚
(2.8-24.7 A). The data were integrated and scaled using XDS.
Generic Experimental for the Synthesis of Phthalazinone
Derivatives. Commercially available reagents and solvents
(HPLC grade) were used without further purification. H NMR
Model building was done with MOLOC (www.moloc.ch) and
refinement with the CCP4 program suite. The final R-factor was
21.5% without modeling explicit solvent positions.
1
spectra were recorded on a Bruker 400 MHz spectrometer in deu-
terated solvents. Chemical shifts (δ) are in parts per million. Thin-
layer chromatography (TLC) analysis was performed with
Kieselgel 60 F254 (Merck) plates and visualized using UV light.
Analytical HPLC-MS was performed on Shimadzu LCMS-
2010EV systems using reverse phase Atlantis dC18 columns
(3 μm, 2.1 mm ꢀ 50 mm), gradient 5-100% B (A = water/0.1%
formic acid, B = acetonitrile/0.1% formic acid) over 3 min,
injection volume 3 μL, flow rate 1.0 mL/min. UV spectra were
recorded at 215 nm using a Waters 2788 dual wavelength UV
detector. Mass spectra were obtained over the range m/z 150-
850 at a sampling rate of 2 scans per second using Waters LCT or
analytical HPLC-MS on Shimadzu LCMS-2010EV systems
using reverse phase Water Atlantis dC18 columns (3 μm, 2.1 mm ꢀ
100 mm), gradient 5-100% B (A = water/0.1% formic acid,
B = acetonitrile/0.1% formic acid) over 7 min, injection volume
3 μL, flow rate 0.6 mL/min. UV spectra were recorded at 215 nm
using a Waters 2996 photodiode array. Data were integrated
and reported using Shimadzu PsiPort software. All compounds
prepared for biochemical screening displayed purities determined by
HPLC as g95%.
Disorder apparent from the electron density map prevented
modeling of the N-terminus (residues 124 and 125), much of the
activation loop (residues 280-290, 304-306), and the C-termi-
nus (residues 389-391). The glycine loop showed disorder but
was modeled into the apparent predominant conformation.
Difference density for the inhibitor confirmed the details of the
hinge binding to the aminopyrazole and the relative placement
of the phthalazinone and phenyl rings, in particular the proxi-
mity of the phenyl ring to the sole amino acid exchange position
that distinguishes Aurora-A from Aurora-B at the ATP binding
site (Thr-317).
Inhibition Assays. Both Aurora-A and Aurora-B kinase ac-
tivities were measured by enzyme-linked immunosorbent assay
(ELISA). Maxisorp 394-well plates (Nunc) were coated with
recombinant fusion protein comprising residues 1-15 of histone
H3 fused to the N-terminus of glutathione S-transferase. Plates
were then blocked with a solution of 1 mg/mL I-block (Tropix
catalog no. T2015, highly purified form of casein) in phosphate-
buffered saline. Kinase reactions were carried out in the wells of
the ELISA plate by combining an appropriate amount of mutant

Article
2-Phenyl-2,3-dihydrophthalazine-1,4-dione. Phenylhydrazine
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 317
8.48 (d, J = 8.1 Hz, 1H), 8.35 (d, J = 6.8 Hz, 1H), 7.98-7.86
(2H, m), 7.54-7.47 (2H m), 7.37 (t, J = 7.7 Hz, 1H), 7.16 (d, J =
7.1 Hz, 1H), 6.23 (1H, s), 2.37 (3H, s), 2.17 (3H, s). tR = 1.09 min.
m/z (ESþ) (M þ H)þ 332.34.
(59.4 g, 0.55 mol) was added in one portion to a stirred mixture
of phthalic anhydride (74.0 g, 0.5 mol) in acetic acid (500 mL) at
room temperature. The mixture was heated to 125 ꢀC for 2 h and
then allowed to cool to room temperature. The suspension was
poured into water (500 mL), and the precipitate was filtered. The
precipitate was stirred in 1 M Na2CO3 (400 mL) and the remaining
undissolved solid removed by filtration. This solid was washed
with two further 400 mL portions of 1 M Na2CO3. The basic
solutions were combined and acidified by dropwise addition of
concentrated HCl until gas evolution ceased. A white precipitate
formed and was filtered and dried for 18 h in a vacuum oven
(50 ꢀC) to give the phthalazinone (46.3 g, 39% yield) as a white
solid. δH (400 MHz, DMSO-d6) 11.7 (1H, br s), 8.3 (1H, d), 8.03
(1H, d), 7.93 (2H, m), 7.67 (1H, d), 7.51 (1H, t), 7.4 (1H, t). tR =
1.04 min. m/z (ESþ) (M þ H)þ 239.
2-(4-tert-Butylphenyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-
2H-phthalazin-1-one (11). 11 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.36 (1H, s), 8.46 (d, J = 7.8 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H),
8.00-7.89 (2H, m), 7.62 (d, J = 8.8 Hz, 2H), 7.50 (d, J =
8.8 Hz, 2H), 6.24 (1H, s), 2.21 (3H, s), 1.34 (9H, s). tR = 1.30 min.
m/z (ESþ) (M þ H)þ 374.40.
2-(4-Chlorophenyl)-2,3-dihydrophthalazine-1,4-dione. 4-Chloro-
phenyl hydrazine hydrochloride (5.00 g, 28.0 mmol) was added
in one portion to a stirred mixture of phthalic anhydride (3.70 g,
25.0 mmol) in acetic acid (50 mL) at room temperature. The
mixture was heated to 125 ꢀC for 2 h and then allowed to cool
to room temperature. The suspension was poured into water
(100 mL), and the precipitate was filtered. The precipitate was
stirred in 1 M Na2CO3 (100 mL) and the remaining undissolved
solid removed by filtration. This solid was washed with a further
100 mL portion of 1 M Na2CO3. The basic aqueous solutions
were combined and acidified by dropwise addition of concen-
trated HCl until gas evolution ceased. A white precipitate formed
and was filtered and dried for 18 h in a vacuum oven (50 ꢀC) to
give the phthalazinone (270 mg, 4% yield). The solid insoluble in
1 M Na2CO3 was stirred in glycerol (50 mL) and heated to 150 ꢀC
for 10 h. The reaction mixture was then diluted with water (50 mL),
and 4 M HCl was added dropwise until a precipitate formed. The
precipitate was filtered, resuspended in MeOH (30 mL), and
collected by filtration. The product was dried under vacuum
to give the desired phthalazinone (3.6 g, 72% yield) as a white
solid. δH (400 MHz, DMSO-d6) 12.10 (1H, br s), 8.3 (1H, d),
7.9-8.0 (3H, m), 7.7 (2H, d), 7.6 (2H, d). tR = 1.27 min. m/z (ESþ)
(M þ H)þ 273, 275.
4-Bromo-2-phenyl-2H-phthalazin-1-one. Phosphorus oxybro-
mide (3.13 g, 10.8 mmol) was added to a stirred suspension of
2-phenyl-2,3-dihydrophthalazine-1,4-dione (1.30 g, 5.4 mmol)
in 1,2 dichloroethane (15 mL). The mixture was heated to 100 ꢀC
for 18 h before being cooled and poured into water (100 mL).
The aqueous layer was made basic with 1 M Na2CO3 and then
extracted into DCM (3 ꢀ 100 mL). The organic layers were
combined, dried (MgSO4), and concentrated in vacuo. The residue
was purified by silica column chromatography (20% ethyl acetate/
hexane) to give the bromophthalazinone (0.770 g, 48% yield) as
a white solid. δH (400 MHz, DMSO-d6) 8.2 (1H, dd), 7.91 (1H, td),
7.89 (1H, td), 7.35-7.55 (5H, m). tR = 1.51 min. m/z (ESþ)
(M þ H)þ 301, 303.
4-(5-Methyl-2H-pyrazole-3-ylamino)phenyl-2H-phthalazin-1-
one (7). Degassed toluene (6 mL) and ethanol (3 mL) were added
in one portion to a mixture of 4-bromo-2-phenyl-2H-phthala-
zin-1-one (0.75 g, 2.5 mmol), sodium tert-butoxide (0.34 g,
3.5 mmol), 3-amino-5-methylpyrazole (0.29 g, 3 mmol), tris-
(dibenzylidineacetone)dipalladium (0.11 g, 0.125 mmol), and
2-(di-tert-butylphosphino)biphenyl (0.07 g, 0.25 mmol) under
nitrogen. The reaction mixture was heated to 100 ꢀC for 20 h
with stirring and then cooled to room temperature. Diethyl ether
(10 mL) was added and the resulting precipitate was filtered
to give the crude product as a gray solid. The crude product
was triturated with acetonitrile (2 mL) to give the target com-
pound (0.045 g, 40% overall yield) as an off-white solid. δH
(400 MHz, DMSO-d6) 11.93 (1H, s), 9.31 (1H, s), 8.53 (d, J =
8.0 Hz, 1H), 8.38 (d, J = 8.0 Hz, 1H), 7.92-7.99 (2H, m), 7.88
(d, J = 8.6 Hz, 1H), 7.77 (t, J = 8.1 Hz, 1H), 7.34 (t, J = 7.4 Hz,
1H), 6.24 (1H, s), 2.18 (3H, s). tR = 1.11 min. m/z (ESþ) (M þ H)þ
318.29.
This material was then brominated and used in the Buchwald
reaction as described previously.
2-(4-Chlorophenyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-
phthalazin-1-one (12). 12 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
11.97 (1H, s), 9.34 (1H, s), 8.51 (d, J = 7.8 Hz, 1H), 8.38 (d,
J = 7.8 Hz, 1H), 7.99-7.89 (2H, m), 7.80 (d, J = 8.9 Hz, 2H),
7.58 (d, J = 8.9 Hz, 2H), 6.24 (1H, s), 2.20 (3H, s). tR = 1.16 min.
m/z (ESþ) (M þ H)þ 352.30.
2-(4-Fluorophenyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-
phthalazin-1-one (13). 13 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
12.17 (1H, s), 9.51 (1H, s), 8.73 (d, J = 7.8 Hz, 1H), 8.59 (d, J =
7.8 Hz, 1H), 8.20-8.10 (1H m), 7.99-7.95 (2H, m), 7.55 (t, J =
9.0 Hz, 2H), 6.45 (1H, s), 2.40 (3H, s). tR = 1.07 min. m/z (ESþ)
(M þ H)þ 336.31.
4-(5-Methyl-1H-pyrazol-3-ylamino)-2H-phthalazin-1-one (6).
6 was synthesized according to the general procedure as de-
scribed above. δH (400 MHz, DMSO-d6) 8.98 (1H, s), 8.37 (d,
J = 8.2 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 7.96-7.82 (2H, m),
6.18 (1H, s), 2.22 (3H, s). tR = 1.27 min. m/z (ESþ) (M þ H)þ
242.15.
4-(1H-Pyrazol-3-ylamino)-2-p-tolyl-2H-phthalazin-1-one (14). 14
was synthesized according to the general procedure as described
above. δH (400 MHz, DMSO-d6) 12.05 (1H, s), 9.22 (1H, s), 8.31
(d, J = 7.8 Hz, 1H), 8.17 (d, J = 7.8 Hz, 1H), 7.82-7.64 (2H, m),
7.38 (d, J = 8.3 Hz, 2H), 7.02 (d, J = 8.3 Hz, 2H), 6.28 (1H, s),
2.09 (3H, s). tR = 1.11 min. m/z (ESþ) (M þ H)þ 318.32.
4-(1H-Indazol-3-ylamino)-2-phenyl-2H-phthalazin-1-one (15). 15
was synthesized according to the general procedure as described
above. δH (400 MHz, DMSO-d6) 12.48 (1H, s), 9.37 (1H, s),
8.46-8.37 (2H, m), 8.06-7.91 (2H, m), 7.71 (d, J = 8.1 Hz, 1H),
7.52 (d, J = 6.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 1H), 7.36-7.18
(4H, m), 7.01 (t, J = 8.0 Hz, 1H). tR = 1.29 min. m/z (ESþ)
(M þ H)þ 354.14.
2-(4-Methoxyphenyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-
phthalazin-1-one (8). 8 was synthesized according to the general
procedure as described above. δH (400 MHz, DMSO-d6) 11.72
(1H, s), 9.05 (1H, s), 8.28 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8
Hz, 1H), 7.76-7.66 (2H m), 7.40 (d, J = 6.8 Hz, 2H), 6.84 (d,
J = 6.8 Hz, 2H), 6.03 (1H, s), 3.61 (3H, s), 1.97 (3H, s). tR = 1.03
min. m/z (ESþ) (M þ H)þ 348.34.
4-(1H-Pyrazol-3-ylamino)-2-p-tolyl-2H-phthalazin-1-one (9).
9 was synthesized according to the general procedure as de-
scribed above. δH (400 MHz, DMSO-d6) 11.70 (1H, s), 9.04 (1H, s),
8.27 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.78-7.68
(2H m), 7.35 (d, J = 8.3 Hz, 2H), 7.06 (d, J = 8.3 Hz, 2H),
6.01 (1H, s), 2.14 (3H, s), 1.94 (3H, s). tR = 1.11 min. m/z (ESþ)
(M þ H)þ 318.32.
4-Bromo-2H-phthalazin-1-one. 2,3-Dihydro-1,4-phthalazine-
dione (12.5 g, 78 mmol) was suspended in dichloroethane (200 mL),
and phosphorus pentabromide (50.0 g, 116 mmol) was added in
one portion. The mixture was heated to reflux for 24 h. A further
portion of phosphorus pentabromide (20.0 g, 70 mol) was added
and the mixture heated for a further 24 h. The mixture was cooled to
room temperature and poured into ice-water (500 mL). The
4-(5-Methyl-1H-pyrazol-3-ylamino)-2-m-tolyl-2H-phthalazin-
1-one (10). 10 was synthesized according to the general proce-
dure as described above. δH (400 MHz, DMSO-d6) 9.28 (1H, s),

318 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Prime et al.
resulting precipitate was filtered and washed with water to give a
crude mixture of monobrominated and dibrominated product
(22.8 g). This crude material was suspended in acetic acid (230 mL)
and heated to 120 ꢀC for 2 h. The mixture was cooled to room
temperature and poured into ice-water and the resulting pre-
cipitate filtered. The solid was washed with water and dried to
give the title compound (10.4 g, 60% yield) as a white solid.
δH (400 MHz, DMSO-d6) 12.95 (1H, s), 8.25 (1H, dd),
8.03 (1H, ddd), 7.96-7.90 (2H, m). tR = 1.02 min. m/z (ESþ)
(M þ H)þ 225, 227.
11.88 (1H, s), 9.23 (1H, s), 8.44 (d, J = 7.8 Hz, 1H), 8.32 (d,
J = 7.8 Hz, 1H), 7.92-7.86 (2H, m), 7.24 (t, J = 7.9 Hz, 1H),
6.96 (1H, s), 6.93 (d, J = 7.6 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H),
6.15 (1H, s), 5.21 (2H, s), 3.72 (3H, s), 2.18 (3H, s). tR
=
1.32 min. m/z (ESþ) (M þ H)þ 362.35.
N-{4-[1-Oxo-4-(1H-pyrazol-3-ylamino)-1H-phthalazin-2-yl-
methyl]phenyl}acetamide (23). 23 was synthesized according to
the general procedure as described above. δH (400MHz, DMSO-d6)
9.91 (1H, s), 9.16 (1H, s), 8.46-8.36 (1H, m), 8.29 (dd, J = 7.6,
1.7 Hz, 1H), 7.86 (qd, J = 6.7, 6.6 Hz, 2H), 7.51 (d, J = 8.5 Hz,
2H), 7.28 (d, J = 8.5 Hz, 2H), 6.10 (1H, s), 5.14 (2H, s), 2.17
(3H, s), 1.99 (3H, s). tR = 1.38 min. m/z (ESþ) (M þ H)þ 375.29.
2-(Methyl-4-pyridine)-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-
phthalazin-1-one (24). 24 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.38 (1H, s), 8.70 (d, J = 6.6 Hz, 2H), 8.44 (d, J = 7.8 Hz, 1H),
8.31 (dd, J = 7.8, 1.3 Hz, 1H), 7.98-7.86 (2H, m), 7.69 (d, J =
6.6 Hz, 2H), 6.06 (1H, s), 5.46 (2H, s), 2.17 (3H, s). tR = 1.08 min.
m/z (ESþ) (M þ H)þ 333.21.
2-Benzyl-4-bromo-2H-phthalazin-1-one. 4-Bromo-2H-phtha-
lazin-1-one (10.38 g, 46 mmol) was dissolved in DMF (60 mL).
To this was added NaH (60%, 1.55 g, 46.2 mmol) as a DMF
suspension (20 mL). The mixture was stirred at room tempera-
ture for 30 min. Then benzyl bromide (13.82 g, 50.8 mmol) was
added in one portion as a solution in DMF (20 mL). The reaction
mixture was stirred for 2 h. Then the DMF was removed under
reduced pressure and the resulting crude material purified by
column chromatography (gradient elution, 100% heptane to 20%
ethyl acetate/heptane) to give the title compound (8.16 g, 56%
yield) as a white solid. δH (400 MHz, DMSO-d6) 8.30 (1H, dd),
8.03 (1H, ddd), 7.97-7.91 (2 H, m), 7.34-7.27 (5H, m), 5.31
(2H, s). tR = 1.11 min. m/z (ESþ) (M þ H)þ 315, 317
This material was then used in the Buchwald reaction as
described previously.
2-(Methyl-3-pyridine)-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-
phthalazin-1-one (25). 25 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.38 (1H, s), 8.68 (d, J = 1.2 Hz, 2H), 8.54 (d, J = 4.2 Hz, 2H),
8.29 (d, J = 7.8 Hz, 2H), 8.17 (dd, J = 7.7, 1.3 Hz, 2H), 8.05
(d, J = 8.1 Hz, 2H), 7.83-7.72 (4H, m), 7.58 (dd, J = 7.8,
5.4 Hz, 2H), 5.95 (2H, s), 5.27 (3H, s), 2.07 (4H, s). tR = 1.05 min.
m/z (ESþ) (M þ H)þ 333.14.
2-Benzyl-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-phthalazin-
1-one (16). 16 was synthesized according to the general proce-
dure as described above. δH (400 MHz, DMSO-d6) 11.89 (1H, s),
9.24 (1H, s), 8.44 (d, J = 7.7 Hz, 1H), 8.31 (d, J = 7.7 Hz, 1H),
7.93-7.82 (2H, m), 7.37-7.30 (4H m), 7.27 (d, J = 7.0 Hz, 1H),
6.09 (1H, s), 5.24 (2H, s) 2.17 (3H, s). tR = 1.60 min. m/z (ESþ)
(M þ H)þ 332.08.
4-(5-Methyl-1H-pyrazol-3-ylamino)-2-(4-nitrobenzyl)-2H-
phthalazin-1-one (26). 26 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.32 (1H, s), 8.34 (d, J = 8.0 Hz, 2H), 8.21(d, J = 8.0 Hz, 2H),
8.0 (d, J = 6.8 Hz, 2H), 7.49-7.85 (2H, m), 7.49 (d, J =
8.8 Hz, 2H), 5.93 (1H, s), 5.29 (2H, s), 2.04 (3H, s). tR = 1.05 min.
m/z (ESþ) (M þ H)þ 377.24.
2-(3,5-Difluorobenzyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-
2H-phthalazin-1-one (17). 17 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.59 (1H, s), 8.44 (d, J = 8.0 Hz, 1H), 8.33 (dd, J = 7.8, 1.2 Hz,
1H), 7.99-7.88 (2H, m), 7.17 (tt, J = 9.4, 2.4 Hz, 2H), 7.08 (dd,
J = 8.3, 2.1 Hz, 2H), 6.12 (1H, s), 5.32 (2H, s), 2.23 (3H, s). tR =
1.17 min. m/z (ESþ) (M þ H)þ 368.10.
2-[2-(4-Methoxyphenyl)-2-oxoethyl]-4-(5-methyl-1H-pyrazol-
3-ylamino)-2H-phthalazin-1-one (27). 27 was synthesized accord-
ing to the general procedure as described above. δH (400 MHz,
DMSO-d6) 9.43 (1H, s), 8.40 (d, J = 8.3 Hz, 1H), 8.22 (d, J =
8.3 Hz, 1H), 8.00 (d, J = 9.0 Hz, 2H), 7.93-7.81 (2H, m), 7.04
(d, J = 9.0 Hz, 2H), 6.12 (1H, s), 5.54 (2H, s), 3.80 (3H, s), 2.11
(3H, s). tR = 1.25 min. m/z (ESþ) (M þ H)þ 390.14.
2-(2,5-Difluorobenzyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-
2H-phthalazin-1-one (18). 18 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
11.88 (1H, s), 9.25 (1H, s), 8.44 (d, J = 7.9 Hz, 1H), 8.32 (d, J =
7.9 Hz, 1H), 7.93-7.85 (2H, m), 7.32-7.21 (3H, m), 5.93 (1H, s),
5.29 (2H, s), 2.16 (3H, s). tR = 1.14 min. m/z (ESþ) (M þ H)þ
368.14.
2-[2-(3-Methoxyphenyl)-2-oxoethyl]-4-(5-methyl-1H-pyrazol-
3-ylamino)-2H-phthalazin-1-one (28). 28 was synthesized accord-
ing to the general procedure as described above. δH (400 MHz,
DMSO-d6) 9.38 (1H, s), 8.43 (d, J = 8.1 Hz, 1H), 8.21 (d, J =
8.1 Hz, 1H), 7.92-7.80 (2H, m), 7.61 (d, J = 7.6 Hz, 1H),
7.49-7.42 (2H, m), 7.21 (d, J = 8.3 Hz, 1H), 6.11 (1H, s),
5.57 (2H, s), 3.77 (3H, s), 2.09 (3H, s). tR = 1.32 min. m/z (ESþ)
(M þ H)þ 390.15.
2-(4-Fluorobenzyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-
phthalazin-1-one (19). 19 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.53 (1H, s), 8.40 (d, J = 7.8 Hz, 1H), 8.31 (dd, J = 7.9, 1.3 Hz,
1H), 7.96-7.85 (2H, m), 7.41 (dd, J = 8.7, 5.6 Hz, 2H), 7.15
4-(5-Methyl-1H-pyrazol-3-ylamino)-2-[2-oxo-2-(4-trifluoro-
methylphenyl)ethyl]-2H-phthalazin-1-one (29). 29 was synthe-
sized according to the general procedure as described above.
(t, J = 8.9 Hz, 2H), 6.11 (1H, s), 5.26 (2H, s), 2.22 (3H, s). tR
=
1.13 min. m/z (ESþ) (M þ H)þ 350.11.
δ
H (400 MHz, DMSO-d6) 11.89 (1H, s), 9.31 (1H, s), 8.48 (d, J =
8.1 Hz, 1H), 8.27 (d, J = 8.8 Hz, 3H), 7.96 (d, J = 8.1 Hz, 3H),
7.88 (t, J =7.5 Hz, 1H), 6.18 (1H, s), 5.67 (2H, s), 2.14 (3H, s). tR =
1.06 min. m/z (ESþ) (M þ H)þ 428.09.
2-(4-Toluylsulfonate)-4-(5-methyl-1H-pyrazol-3-ylamino)-
2H-phthalazin-1-one (20). 20 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.20 (1H, s), 8.35 (d, J = 7.8 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H),
7.89-7.79 (4H, m), 7.53 (d, J = 8.3 Hz, 2H), 6.00 (1H, s),
5.30 (2H, s), 3.08 (3H, s), 2.12 (3H, s). tR = 1.23 min. m/z (ESþ)
(M þ H)þ 410.12.
4-(5-Methyl-1H-pyrazol-3-ylamino)-2-(2-oxo-2-phenylethyl)-
2H-phthalazin-1-one (30). 30 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
11.91 (1H, s), 9.31 (1H, s), 8.48 (d, J = 8.2 Hz, 1H), 8.30
(d, J = 8.2 Hz, 1H), 8.09 (d, J = 7.1 Hz, 2H), 7.98-7.87 (2H, m),
7.74-7.70 (1H, m), 7.62-7.58 (2H, m), 6.21 (1H, s), 5.63 (2H, s),
2.15 (3H, s). tR = 1.09 min. m/z (ESþ) (M þ H)þ 360.10.
4-(5-Methyl-1H-pyrazol-3-ylamino)-2-(2-methylthiazol-4-
ylmethyl)-2H-phthalazin-1-one (31). 31 was synthesized accord-
ing to the general procedure as described above. δH (400 MHz,
DMSO-d6) 9.82 (1H, s), 8.39 (d, J = 8.1 Hz, 1H), 8.33 (d, J =
8.1 Hz, 1H), 7.99-7.88 (2H, m), 7.36 (1H, s), 6.16 (1H, s),
5.34 (2H, s), 2.64 (3H, s), 2.26 (3H, s). tR = 1.18 min. m/z (ESþ)
(M þ H)þ 353.18.
2-(4-Methoxybenzyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-
2H-phthalazin-1-one (21). 21 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
11.91 (1H, s), 9.28 (1H, s), 8.47 (d, J = 8.0 Hz, 1H), 8.36 (d, J =
8.0 Hz, 1H), 7.97-7.89 (2H, m), 7.37 (d, J = 8.6 Hz, 2H), 6.94
(d, J = 8.6 Hz, 2H), 6.18 (1H, s), 5.21 (2H, s), 3.76 (3H, s), 2.24
(3H, s). tR = 1.30 min. m/z (ESþ) (M þ H)þ 362.36.
2-(3-Methoxybenzyl)-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-
phthalazin-1-one (22). 22 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)

Article
2-Methyl-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-phthalazin-
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 319
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1-one (32). 32 was synthesized according to the general proce-
dure as described above. δH (400 MHz, DMSO-d6) 11.70 (1H, s),
9.00 (1H, s), 8.23 (d, J = 7.8 Hz, 2H), 8.10 (d, J = 7.8 Hz, 2H),
7.73-7.64 (2H, m), 6.13 (1H, S), 3.44 (3H S), 2.04 (3H, s).
t
R = 0.97 min. m/z (ESþ) (M þ H)þ 389.27.
2-Isopropyl-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-phthalazin-
1-one (33). 33 was synthesized according to the general proce-
dure as described above. δH (400 MHz, DMSO-d6) 11.92 (1H, s),
9.17 (1H, s), 8.43 (d, J=7.9Hz, 1H), 8.28(dd,J= 7.8, 1.1 Hz, 1H),
7.90-7.80 (2H, m), 6.35 (1H, s), 5.25 (dt, J = 13.2, 6.6 Hz, 1H),
2.24 (3H, s), 1.13 (d, J = 6.6 Hz, 6H). tR = 0.97 min. m/z (ESþ)
(M þ H)þ 284.34.
2-Isobutyl-4-(5-methyl-1H-pyrazol-3-ylamino)-2H-phthalazin-1-
one (34). 34 was synthesized according to the general procedure
as described above. δH (400 MHz, DMSO-d6) 11.76 (1H, s), 9.00
(1H, s), 8.2 (d, J = 6.8 Hz, 1H), 8.15 (d, J = 6.8 Hz, 1H),
7.77-7.67 (2H, m), 6.17 (1H, S), 3.71 (d, J = 6.8 Hz, 2H),
2.10-2.07 (4H, m), 0.75 (d, J = 6.6 Hz, 6H). tR = 1.03 min. m/z
(ESþ) (M þ H)þ 298.33.
4-(5-Methyl-1H-pyrazol-3-ylamino)-2-(2,2,2-trifluoroethyl)-2H-
phthalazin-1-one (35). 35 was synthesized according to the
general procedure as described above. δH (400 MHz, DMSO-d6)
9.58 (1H, s), 8.47 (d, J = 7.9 Hz, 1H), 8.34 (d, J = 7.9 Hz, 1H),
8.01-7.83 (2H, m), 6.34 (1H, S), 4.93 (q, J = 9.1 Hz, 2H), 2.25
(3H, s). tR = 1.03 min. m/z (ESþ) (M þ H)þ 323.10
N-{4-[1-Oxo-4-(1H-pyrazol-3-ylamino)-1H-phthalazin-2-yl-
methyl]phenyl}acetamide (36). 36 was synthesized according
to the general procedure as described above. δH (400 MHz,
DMSO-d6) 12.13 (1H, s), 9.87 (1H, s), 9.26 (1H, s), 8.42 (d, J =
6.5 Hz, 1H), 8.21 (d, J = 6.5 Hz, 1H), 7.93-7.78 (2H, m), 7.53
(1H, s), 7.44 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H),
6.38 (1H, s), 5.12 (2H, s), 1.91 (3H, s). tR = 0.92 min. m/z (ESþ)
(M þ H)þ 375.29.
4-(1H-Indazol-3-ylamino)-2-isopropyl-2H-phthalazin-1-one (37).
37 was synthesized according to the general procedure as
described above. δH (400 MHz, DMSO-d6) 8.41 (t, J = 8.1 Hz,
2H), 8.08-7.91 (2H, m), 7.59 (d, J = 8.1 Hz, 1H), 7.49 (d, J =
8.3 Hz, 1H), 7.36 (t, J = 7.1 Hz, 1H), 7.02 (t, J = 7.1 Hz, 1H),
5.24-5.04 (1H, m), 1.07 (d, J = 6.4 Hz, 6H). tR = 1.22 min. m/z
(ESþ) (M þ H)þ 319.14.
Acknowledgment. The authors thank Andrea Challand for
cell staining and Lothar Kling for generating the pharmaco-
kinetic data.
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