Synthesis and biological evaluation of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4- d]pyrimidin-4-one inhibitors of cyclin-dependent kinases

Article abstract of DOI:10.1021/jm020455u

Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]-pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC₅₀

Full text of DOI:10.1021/jm020455u

5894
J. Med. Chem. 2004, 47, 5894-5911
Synthesis and Biological Evaluation of
1-Aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one Inhibitors of
Cyclin-Dependent Kinases
Jay A. Markwalder,* Marc R. Arnone, Pamela A. Benfield, Michael Boisclair,† Catherine R. Burton,
Chong-Hwan Chang, Sarah S. Cox, Philip M. Czerniak, Charity L. Dean, Deborah Doleniak, Robert Grafstrom,
Barbara A. Harrison, Robert F. Kaltenbach, III, David A. Nugiel, Karen A. Rossi, Susan R. Sherk, Lisa M. Sisk,
Pieter Stouten, George L. Trainor, Peter Worland,† and Steven P. Seitz
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, and GPC Biotech,
One Kendall Square, Cambridge, Massachusetts
Received October 16, 2002
Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]-
pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1
complex-mediated phosphorylation of a protein substrate with IC50s in the low micromolar
range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed
in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen
bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray
diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative
cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)-
pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the
6-arylmethyl group resulted in ∼100-fold increases in CDK4 affinity, giving ligands that were
equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects
in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is
enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse
xenograft model.
Introduction
or their endogenous peptide inhibitors (CKIs) are mu-
tated in a large proportion of human tumors.7 One
family of CKIs, the CIP/KIP class, is relatively promis-
cuous, having affinity for CDK2, CDK3, CDK4, and
CDK6.8 CKIs in the INK4 class, however, are highly
selective for the closely related CDKs 4 and 6, suggest-
ing the possibility of selective inhibition by small
molecule mimics. For these reasons we sought to
discover small-molecule protein kinase inhibitors that
were selective for CDK/cyclin complexes, specifically
CDK4/cyclin D1.
How applicable this approach is to cancer chemo-
therapy is still an unresolved question. While a number
of small-molecule CDK inhibitors have been disclosed,
clinical experience is limited to the ATP-competitive
agent flavopiridol.9 Flavopiridol is relatively nonselec-
tive in its inhibition of the various CDKs; it also appears
to inhibit other protein kinases. More recently, great
strides have been made in the search for CDK-selective
kinase inhibitors,1 including the discovery of a highly
selective CDK4/cyclin D inhibitor by optimization of a
nonselective lead.10
High-throughput screening of a subset (∼160 000
compounds) of DuPont’s compound library against
CDK4/cyclin D1 was performed with a readout of
percent inhibition of pRb phosphorylation at 20 µM.
Discrete IC50 values were obtained for compounds that
were active upon retest and appeared chemically at-
tractive. This screening strategy identified several series
of inhibitors with modest potency (IC50 < 50 µM).
One intriguing lead uncovered via this strategy was
the 4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one 1 (Fig-
Controlling the cell cycle by inhibition of the proteins
that regulate its progression is an attractive strategy
for addressing cancer and other diseases associated with
abnormal cellular proliferation.1 One family of such
proteins, the cyclin-dependent kinases (CDKs), is made
up of at least nine highly homologous enzymes that in
association with specific regulatory subunits (cyclins)
control progression of the cell cycle.2 CDK/cyclin activity
oscillates with cyclin expression/degradation and is
further regulated by the action of several families of
protein kinases and phosphatases.3,4 A series of check-
points serves to ensure the viability of progeny cells by
preventing progression of cells with damaged DNA, an
inappropriate chromosome count, or for which necessary
structural features or conditions of nourishment do not
exist. Passage through an initial G1 restriction point
occurs upon release by the retinoblastoma protein (pRb)
of the transcription factor E2F.5 This is triggered by
phosphorylation of several S/T residues on pRb, the
primary substrate of CDK4/cyclin D. The fate of cells
that enter G1 but which do not progress through the
G1 or subsequent checkpoints is to undergo apoptosis.6
Since tumor cells have misregulated cell cycles, it has
been postulated that they may be especially sensitive
to agents that restore checkpoint control. The impor-
tance of these kinase pathways is highlighted by the
fact that the genes encoding CDKs, their cyclin partners,
* To whom correspondence should be addressed. Phone: (609) 252-
4349. Fax: (609) 252-6804. E-mail: jay.markwalder@bms.com.
† GPC Biotech.
10.1021/jm020455u CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/27/2004

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5895
Aminopyrazole-4-carboxamides bearing other groups
at the 3-position were prepared by the well-precedented
routes shown in Schemes 2-4. Scheme 2 is analogous
to Scheme 1, except that the methylthio leaving group
is replaced by ethoxy in 5 and 6 and that the carboxa-
mide in 2 may be replaced by a nitrile group. These react
with 2,4,6-trichlorophenylhydrazine to give pyrazoles 7
and 8, the former being hydrolyzed to the desired
amides 8 by treatment with concentrated sulfuric acid.
Hydrazones, available from condensation of hydrazines
and aldehydes or aldehyde equivalents (Scheme 3), can
be brominated to give bromohydrazones 9. In what is
probably the most versatile route, these react readily
with salts of malononitrile and cyanoacetamide to afford
7 and 8, respectively. Incorporation of an acetate
equivalent to give 6-methyl-1-(2,4,6-trichlorophenyl)-
pyrazolopyrimidin-4-ones 10 was accomplished as in
Scheme 1. Scheme 4 illustrates the conversion of tet-
racyanoethylene to 3-cyano- and 3-carboxamidopyra-
zolopyrimidines.16
Figure 1. Lead structure from high-throughput screening
against CDK4/cyclin D1.
Scheme 1a
a (a) R1NHNH2, MeOH, reflux; (b) CH3CONH2, ∆; (c) CH3CO2Et,
NaOEt, EtOH, reflux.
ure 1). Although this ring system occurs in a number
of biologically active agents, including coronary dilatat-
ing agents11 and corticotropin-releasing factor (CRF)
antagonists,12 we were hopeful that this was coinciden-
tal and that a divergent SAR for CDK inhibition would
emerge. To explore this possibility, compounds were
prepared that had diverse groups at the substitutable
positions of the pyrazolopyrimidine core.
Standard alkylation conditions (Scheme 5) were used
to convert 10i to its N-methyl derivative 11. The
chloropyrimidine 12a, prepared by heating 10i in POCl3
at reflux, underwent smooth addition-elimination reac-
tion to give methoxypyrimidine 12b. Aminopyrimidine
12c was prepared from 7i via the intermediacy of
imidate 13.
Chemistry
Condensation of trichlorophenylhydrazine with 3-oxo-
valeronitrile (Scheme 6) furnished the pyrazole 14,
which readily underwent electrophilic aromatic substi-
tution with chlorosulfonic acid. Heating the resulting
sulfonamide 15 with base and ethyl acetate in ethanol
furnished cyclic sulfonamide 16, albeit in low yield.
Scheme 7 illustrates the incorporation of diverse
groups at the 6-position, a process accomplished by
refluxing the R6 carboxylic ester and sodium ethoxide
with 3w or 8i in ethanol. This process affords direct
The preparation of a series of 6-methyl-3-(methylthio)-
pyrazolopyrimidin-4-ones was accomplished by a route
analogous to those in the literature (Scheme 1).13
Accordingly, hydrazines were allowed to react with
dithioketeneacetal 2 in refluxing methanol to afford
3-aminopyrazole-4-carboxamides 3.14 These were con-
verted to the target compounds 4 by heating at reflux
with ethyl acetate and an alkali metal alkoxide in
ethanol.15
Scheme 2a
a (a) R3C(OEt)3, Ac2O, ∆; (b) 2,4,6-trichlorophenylhydrazine, MeOH, reflux; (c) H2SO4; (d) CH3CO2Et, NaOEt, EtOH, reflux.
Scheme 3a
a (a) R3CHO, EtOH; (b) N-bromosuccinimide, DMF, 0 °C; (c) malononitrile, NaOEt, EtOH, relux; (d) cyanoacetamide, NaOEt, EtOH,
reflux.

5896 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
Scheme 4a
a Reagents: (a) tetracyanoethylene, EtOH, 69%; (b) (i) Ac2O, (ii) AcOH, H2O, 18%; (c) H2SO4, 52%.
Scheme 5a
a (a) TsOme, K2CO3, DMAC, 44%; (b) POCl3, reflux; NaOMe, MeOH; (d) CH3C(OEt)3, Ac2O, ∆; (e) NH3, EtOH.
Scheme 6a
a Reagents: (a) (i) EtOH, reflux, (ii) HCl, EtOH, 48%. (b) (i) ClSO3H, (ii) NH3, THF, 75%, (c) EtOAc, NaOEt, EtOH, reflux, 7%.
access to products of high purity and tolerates a wide
variety of functional groups. The pyrazolopyrimidines
are isolated by quenching the reactions with aqueous
acid, filtering, and rinsing the insoluble products to
remove any excess reagents. Unsaturated esters did not
react cleanly under these conditions, but they could be
prepared by conventional elimination methods (18c to
21). Scheme 8 illustrates this and other reactions to
further derivatize 6-substituted pyrazolopyrimidines.
Aliphatic amino esters were felt to be too nucleophilic
to be used directly in the cyclization step, so the
corresponding tert-butyl carbamates were instead em-
ployed. Removal of the tert-butoxycarbonyl protecting
group with HCl in dioxane afforded 20e and 20f,
respectively. Phenols could be used in the cyclization
step without protection, or they could be prepared from
the corresponding anisoles by treatment with BBr3 in
CH2Cl2 (19 to 22). Aminophenylacetic esters reacted
well under these cyclization conditions, and the result-
ing anilines could be converted to anilide derivatives

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5897
Scheme 7
Scheme 8a
a (a) m-CPBA, CH2Cl2, DMF; (b) PhCH3, ∆; (c) BocN(Me)(CH2)nCO2Et, EtOH, reflux; (d) HCI, dioxane; (e) BBr3; (f) coupling agent (see
Experimental Section).
such as 23 or 24 by treatment with appropriate elec-
the o-halogens with a small nonhalogen substituent was
allowed. The best compound prepared in this series is
trichlorophenyl derivative 4w. The weak inhibition
exhibited by 4u and 4x further highlights the delicate
balance of steric and electronic factors that define
acceptable R1 substituents.
trophiles in the presence of a base.
Results and Discussion
SAR trends for the pyrazolopyrimidine 1-position are
summarized in Table 1.17 Replacement of the substi-
tuted phenyl moiety with alkyl or unsubstituted aryl
groups gave completely inactive compounds, as shown
by entries 4a-f in the table. Similarly, meta- or para-
substituted phenyl R1 groups did not prove to be
satisfactory replacements. Of the monosubstitued R1
phenyl groups surveyed, only the o-chloro appeared to
be satisfactory (entry 4k). Early analogues having
disubstituted aryl groups (Entries 4l-r) likewise ex-
hibited only minimal potency. Only with the 2,6-
dihalophenyl derivatives 4s and 4t were inhibitors
comparable in potency to the 8 µM IC50 observed for 1
realized. The trisubstituted phenyl R1 derivatives proved
more satisfactory. While rearrangement of the phenyl
substituents in 1 to give the 2,4-dichloro-6-(trifluoro-
methyl)phenyl derivative 4u resulted in a loss of activ-
ity, entry 4v demonstrated that replacement of one of
Our enthusiasm over having modestly potent, low
molecular weight CDK4 inhibitors was tempered some-
what by our desire to have inhibitors that did not bind
in the ATP pocket. We were concerned that ATP
antagonists would exhibit poor selectivity toward other
kinases and potentially toward other classes of enzymes
having nucleoside binding pockets.18 We were thus
gratified to find that while 4w exhibited little selectivity
for any particular CDK (Table 2), it did not appear to
inhibit the serine/threonine kinases protein kinase A
(PKA) or C (PKC). Further, 4w showed little affinity
for the tyrosine kinase c-Abl. A modest level of cellular
potency (IC50 ) 2.5 µM) was observed for 4w when
assayed for growth inhibition of the HCT116 line (vide
infra). This growth inhibition compares favorably with
an IC50 value of 9.2 µM against the Ag1523 line of

5898 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
this is found upon comparison of the 1H NMR spectrum
of 10m with those of other pyrazolopyrimidinones
prepared thus far. The occurrence of the hydroxyl proton
in 10m as a sharp triplet at 4.81 ppm is indicative of
intramolecular hydrogen bonding. Further, the pyrimi-
dinone NH, which normally resonates at 12-13 ppm
(DMSO-d6) is shifted to 10.5 ppm. This implies a
profound distortion of the electronics of the pyrimidi-
none ring.
Table 1. CDK4/Cyclin D1 Inhibition of 6-Methyl-3-
(methylthio)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones17b
step 1
yield,
%
step 2
K4/D1
methoda
(yield, %)
IC50,
no.
R1
µM
The data presented in Table 4 provide a persuasive
argument for the pyrimidinone motif as the preferred
tautomer in these compounds. The lack of activity
observed for the N- and O-methyl derivatives 11 and
12b suggests that the presence of a hydrogen-bond
donor is critical. That it must be donated from the ring
nitrogen is established by the lack of activity of 12c,
which, like ATP, is in all probability present in solution
as the aminopyrimidine tautomer. The sulfonamide 16
was also shown to be inactive, presumably because the
tetrahedral sulfonyl group cannot attain the appropriate
geometry to act as a hydrogen-bond acceptor. Taken
together these observations suggest that the pyrimi-
dinone forms a bidentate hydrogen bond.
We now turned to the preparation of a series of R6
analogues. The availability of a broader range of kinase
assays for routine compound screening now allowed us
to better evaluate issues of selectivity. As the previous
iteration of screening (Table 3) had identified 10i, a
compound with a 2-fold improvement in potency (over
4w) toward CDK4/cyclin D1 but 4-fold improvement
toward CDK2/cyclinE, we prepared to address the
possibility that this series was intrinsically CDK2-
selective.
1
2,6-dichloro-4-(CF3)phenyl
benzyl
8
>150
>200
>100
>210
>180
>180
>160
>160
>160
>83
19
4a
4b
4c
4d
4e
4f
15
33
34
50
b
B (9)
n-butyl
B (32)
B (29)
B (43)
B (90)
B (77)
B (9)
tert-butyl
2-hydroxyethyl
phenyl
2-pyridyl
92
94
74
83
88
66
87
94
84
69
76
64
59
65
73
44
64
88
76
4g
4h
4i
4j
4k
4l
3-chlorophenyl
4-chlorophenyl
4-isopropylphenyl
2-methoxyphenyl
2-chlorophenyl
4-chloro-2-methylphenyl
B (62)
B (16)
B (35)
B (16)
B (51)
B (21)
B (72)
B (28)
B (34)
B (53)
B (56)
B (67)
B (42)
B (57)
B (76)
A (90)
B (66)
74
4m 2,3-dichlorophenyl
50
4n
4o
4p
4q
4r
4s
4t
2,4-dimethylphenyl
48
2,4-dichlorophenyl
37
2,5-dimethylphenyl
25
2,5-dichlorophenyl
23
2-chloro-5-(CF3)phenyl
2-chloro-6-fluorophenyl
2,6-dichlorophenyl
15
6.8
5.3
4u
4v
2,4-dichloro-6-(CF3)phenyl
2,4-dichloro-6-methylphenyl
29
11
4w 2,4,6-trichlorophenyl
4x 2,4,6-trimethylphenyl
a Method A: Scheme 1, conditions b. Method B: Scheme 1,
conditions c. b Prepared in 59% yield by treatment of 5-amino-4-
cyano-3-(methylthio)-4-phenylpyrazole (Maybridge) with concen-
trated sulfuric acid.
2.1
37
Just as at R3, it initially appeared that only a very
narrow range of substituents would be tolerated at R6
(Table 5). In our steric series (first 8 entries), only the
ethyl analogue 18b showed comparable potency to 10i
toward CDK 4/cyclin D1. R-Branching or placement of
polar functional groups close to the pyrazolopyrimidine
core proved detrimental to activity. While compounds
bearing aryl groups at this position (entries 17n, 18e)
bound poorly to CDK4/cyclin D1, those having an
intervening methylene or ethylene tether had reason-
able IC50s. The improvement in CDK2/cyclin E inhibi-
tion realized by tethering an aryl group at this position
is even more profound, as demonstrated by 18i. At 24
nM, this compound is 10-fold selective for CDK2/cyclin
E vs CDK1/cyclin B and >100-fold selective for CDK2/
cyclin E over CDK4/cyclin D. The benzyl-substituted
pyrimidinone 18i also showed improved cellular potency
(Table 6) over 4w. Placing a benzyl group at the R6
position in the R3 ) SMe series also improves CDK2/
cyclin E inhibition (17p). Comparison of the enzyme
inhibitory data for 18i and 17p supports the conclusion
drawn from Table 4 that the R3 ) ethyl series has
slightly greater potency against CDKs than the SMe
series.
human fibroblasts. The NCI HCT116 line was chosen
for routine cellular assays, because it expresses high
levels of CDK/cyclin proteins,19 therefore posing a
challenge to this class of kinase inhibitors. In addition,
the HCT 116 line can be grown as a xenograft in nude
mice. For comparison, the IC50s of staurosporine and
racemic flavopiridol in the above assays are listed in
the table.
With these data in hand we sought improved CDK
inhibitors through modification of the 3-(methylthio)
substituent. Table 3 details the SAR trends realized
through these modifications. Of the electron-withdraw-
ing groups sampled at this position, only the trifluoro-
methyl analogue 10d inhibits CDK4/cyclin D1 to a
significant degree. Compounds 10e-k comprise a brief
steric series that appears to define a very narrow range
of allowable substitution for this position. While ethyl
appears to be slightly better than methylthio, smaller
or larger linear alkyl groups are significantly worse.
Finally, while placement of a hydroxymethyl group at
R3 results in a modest loss of potency versus the same-
sized alkyl analogue 10i, the hydroxyethyl homologue
10m was devoid of activity. A plausible explanation for
Table 2. Spectrum of Activity of Pyrazolopyrimidin-4-one 4w17b
enzyme and cellular IC50s (mM)
no.
PKA
PKC
>500
110
0.033
c-Abl
K4/D1
K2/E
K1/B
HCT116
Ag1523
4w
>500
>500
0.08
420
160
1.1
2.1
0.090
-
0.36
0.95
0.029
6.2
0.52
0.032
2.5
0.49
0.006
9.2
6.7
(()-flavopiridol
staurosporine

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5899
Table 3. CDK4/Cyclin D1 Inhibition of 6-Methyl-1-
(2,4,6-trichlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]-
pyrimidin-4-ones17b
Table 4. CDK/Cyclin Inhibition of 3-Ethyl-6-methyl-1-
(2,4,6-trichlorophenyl)pyrazolo[3,4-d]pyrimidin-4-one (10i) and
Isosteres 17b
step 1
step 2
yield,
%
K4/D1
methoda
(% yield)
IC50,
no.
R3
µM
4w
methylthio
cyano
2.1
10a
10b
10c
10d
10e
10f
E (69)
(18)b
14
12
43
64
81
75
13
78
81
47
58
66
>28
sulfonylmethyl
carboxamido
trifluoromethyl
phenyl
(60)c
(52)b
D (31)
A (49)
D (15)
A (68)
B (34)
A (70)
A (67)
F (43)
D (40)d
C (44)d
3.1
>120
>48
>6.5
5.7
benzyl
10g
10h
10i
n-butyl
n-propyl
ethyl
1.1
10j
methyl
3.5
10k
10l
H
12
hydroxymethyl
2-hydroxyethyl
7.0
10m
>140
a Method A: Scheme 2, conditions a-c. Method B: Scheme 2,
conditions a, b. Method C: Scheme 3, conditions a-c, e. Method
D: Scheme 3, conditions a, b, d. Method E: Scheme 4. Method F:
Scheme 2, conditions b, c. b See Scheme 4. c Prepared from 4w by
oxidation with excess m-CPBA. d In this sequence, the alcohol
group was protected as a tert-butyldiphenylsilyl ether.
These observations prompted us to prepare the series
of 6-(arylmethyl)pyrazolopyrimidin-4-ones shown in
Table 6. Substitution of the ortho-position to give the
anisole derivative 19a proved detrimental to activity.
Interestingly, the hydroxymethyl isostere 19b showed
little change from the unsubstituted parent compound.
While π-donating substituents were not favorable in the
ortho-position, the 2-pyridyl analog 19c proved to be
even worse. Placement of a single substituent at the
meta-position generally resulted in moderately potent
and CDK2/cyclin E-selective compounds with generally
unimpressive cellular potency. Para-substituted com-
pounds, on the other hand, were generally better
enzyme inhibitors than 18i and were usually potent in
cells. No real gains accrued from making 3,4-disubsti-
tuted analogues until 3,4-dimethoxybenzyl derivative
19s was deprotected to give the key catechol 22s. While
the CDK2/cyclin E inhibition displayed by this com-
pound is unremarkable, against CDK4/cyclin D1 22s
has an IC50 of 44 nM and is 102 and 68 times more
potent than 19s and the unsubstituted analogue 18i,
respectively. Examination of the ratio of CDK4/cyclin
D1 inhibition for several other anisole/phenol pairs
shows that this observation is not anomalous. The
m-phenols 22q, 22g, and 19u are 24, 74, and 190 times
more potent against CDK4/cyclin D1 than the corre-
sponding anisoles. The data for compound 23 imply that
this phenomenon is associated with the acidity of the
meta-substituent. While aniline 19h shows modest
CDK4/cyclin D1 potency, the more acidic sulfonamide
23 is fairly potent and balanced in its inhibition of
CDK4/cyclin D1 and CDK2/cyclin E. The improvements
in CDK inhibition detailed above do not appear to have
come at the expense of selectivity over the other protein
kinases in our panel. The IC50s for 22s against PKA,
PKC, and c-Abl are >100 µm.
The p-aniline derivative 19k offered an attractive
handle for pursuing the effects of larger groups on CDK
SAR. Accordingly, the anilide derivatives shown in
Table 7 were prepared. While only subtle changes in
kinase inhibition were realized with amides 24, they are
profoundly different from the parent aniline in their
effects upon transformed cell lines. Relatively large
substituents, including the protected glycinamide 24b
are accommodated at the para-position. Charged groups
are well-tolerated at this position, suggesting that they
may be solvent-exposed. We felt that placement of a
basic amine in this position would result in compounds
having higher aqueous solubility while maintaining
enzyme affinity and cellular potency. Glycinamide 24c
(log P ) 3.3), while an excellent enzyme inhibitor,
showed only modest activity in the HCT116 growth
inhibition assay. Homologation of 24c to give the N,N-
dimethylglycinamide 24d resulted in increased lipophi-
licity and a reduced number of hydrogen-bond donors.
While 24d had comparable in vitro properties to the

5900 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
Table 5. CDK4/Cyclin D1 and CDK2/Cyclin E Inhibition of
1-(2,4,6-Trichlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]-
pyrimidin-4-ones17b
Table 6. CDK/Cyclin Inhibition of 6-(Arylmethyl)-1-(2,4,6-
trichlorophenyl)-3-ethyl-4,5-dihydro-1H-pyrazolo[3,4-d]-
pyrimidin-4-ones17b
IC50 (µM)
IC50 (µM)
%
no.
R3
R6
% yield K4/D1 K2/E
no.
R
yield K4/D1 K2/E K1/B HCT116
4w
SMe methyl
90
78
2.1
1.1
95
0.36
0.14
2.7
18i
H
3.0 0.024 0.26
0.60
3.4
10i
17a
17b
17c
18a
17d
18b
21
Et
methyl
H
19a 2-methoxy
22a 2-hydroxy
91
91a
88
87
89
95
>22
>11
0.10
0.12
5.5
SMe
27
19b 2-(hydroxy methyl)
19c 2-aza
2.9 0.027 >0.27
9.7 0.23
0.49
3.7
0.19
2.1
SMe isopropyl
59
10
SMe n-propyl
52
9.1
>6.3
4.3
0.94
2.9
>50
>57
>48
28
27
15
6.5
240
13
10
2.4
44
>116
36
>21
20
7.1
3.6
3.0
11
19d 3-amino-2-methyl
19e 3-methyl
>2.2 0.019 >0.27
>2.2 0.039 >0.56
Et
isobutyl
36
0.11
0.40
0.096
0.31
SMe cyclopropyl
35
19f 3-ethoxycarbonylmethyl 42
>1.9
0.15
Et
Et
ethyl
vinyl
84
19g 3-methoxy
76
69
97
92
89
81
58
96
80
89
5.0 0.032 0.41
2.9 0.040 0.27
1.3 0.017 0.22
3.0 0.038 0.49
2.7 0.015 0.15
2.2 0.016 0.15
1.7
0.47
0.30
0.55
0.42
0.60
(67)a
100
100
69
19h 3-amino
17e
20e
17f
20f
17g
17h
18c
17i
17j
SMe BocN(Me)CH2
SMe MeNHCH2HCl
SMe BocN(Me)CH2CH2
SMe MeNHCH2CH2‚HCl
SMe CH3CH(OH)
19i 3-aza
19j 4-aza
19
6.6
19k 4-amino
100
66
19l 4-methoxy
19m 4-hydroxy
2.0 0.016 0.10 0.099
>1.2 0.058
SMe CH3SCH2
52
0.31
19n 4-bromo
2.3
0.34
2.1
0.50
9.5
0.32
0.61
0.72
0.30
0.13
0.67
0.40
0.47
Et
CH3SCH2CH2
80
19o 4-dimethylamino
19p 4-methoxy-3-methyl
22p 4-hydroxy-3-methyl
19q 3-methoxy-4-methyl
19r 4-hydroxy-3-methoxy
19s 3,4-dimethoxy
22s 3,4-dihydroxy
19t 3,5-dihydroxy
19u 3-hydroxy-4-methoxy
22q 3-hydroxy-4-methyl
22g 3-hydroxy
>2.1 0.023 >0.26
>2.1 0.067 0.87
SMe MeO2C
20
SMe HO(CH2)3
SMe HO(CH2)5
81
0.76
0.42
0.25
1.6
91a >2.2 0.027 >0.27
17k
18d
17l
80
97
88
57
2.1
0.11
Et
HO(CH2)4
87
6.6 0.076 0.86
SMe CH2F
94
4.5
0.11 >0.51
93a 0.044 0.020 0.24
50 0.059 0.022
17m SMe CF3
100
55
17n
18e
17o
18g
18h
18i
SMe 2-furyl
Et
3,4-dimethoxyphenyl
14
0.58
65 0.024 0.019 0.20
90a 0.088 0.023 0.52
90a 0.068 0.014 0.19
SMe CH2-2-thienyl
83
Et
Et
Et
CH2CH2-phenyl
CH2CH2-imidazol-4-yl
benzyl
89
1.8
0.62
0.024
0.044
23
3-methylsulfonamido
83b
0.16
0.26
55
88
a Prepared by demethylation of the corresponding anisole with
17p
SMe benzyl
84
BBr3. b Prepared by treatment of 19h with methanesulfonyl
a Prepared by oxidation and thermal elimination of 18c.
chloride and pyridine in CH2Cl2.
Table 7. CDK/Cyclin Inhibition of 6-(4-Acylamino)benzyl)-3-
ethyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]-
pyrimidin-4-ones17b
parent glycinamide, further refinement of its physical
parameters led to a significant increase in cellular
potency. To assess its selectivity against other protein
kinases, dimethylglycinamide 24d was assayed against
PKA and c-Abl, and no significant inhibition (IC50s >
150 µM) was observed. Compound 24d has an IC50 of
0.19 µM against the Ag1523 line of human fibroblasts.
Expressed as a multiple of the HCT116 IC50, this yields
a ratio of 5.6:1, a number little different from the ratio
obtained for racemic flavopiridol and 4w. The data in
Table 8 demonstrate that 24d inhibits growth in Rb+,
Rb-, p53+, and p53- cell lines. The latter point is of
importance for a series of CDK inhibitors, since the p53
pathway is misregulated in a large number of cancers.20
While an inhibitor selective for the cyclin D-associated
CDKs would be expected to inhibit growth preferentially
in the Rb+ lines,10b the broad spectrum of antiprolifera-
tive activity observed for 24d is unsurprising in light
of its modest selectivity for CDK2.
IC50 (µM)
no.
R
K4/D K2/E K1/B HCT log P
19k
H
2.7 0.015 0.15 0.42 3.8
1.3 0.009 0.038 0.029 3.9
1.7 0.016 0.18 0.16 4.9
0.46 0.013 0.07 0.12 3.3
0.59 0.018 0.10 0.034 4.2
24a CH3CO
24b (CH3)3COCONHCH2CO
24c H2NCH3CO‚HCl
24d (CH3)2NCH2CO‚HCl
best model, and compounds were hand-docked into the
active site using InsightII v97.0 (MSI, San Diego, CA).
The preferred binding mode for 10i (blue) is presented
in Figure 2 with ATP shown in magenta and the
proposed bidentate hydrogen bond to Val96 indicated
by dashed lines.
Our modeling experiments started from the assump-
tion that the pyrimidinone is forming a bidentate
hydrogen bond in the region where the aminopyrimidine
moiety of ATP binds.21 Using the Modeller program22
a
series of cyclin-bound CDK4 homology models was
generated based on crystallographic data from CDK2/
cyclin A.23 The Whatif24 program was used to select the
To obtain the binding mode of these inhibitors, the
crystal structure of 22s complexed to CDK2 was deter-
mined (1.85 Å resolution). The entire molecule is well

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5901
Table 8. Antiproliferative Effects of N,N-Dimethylglycinamide
24d against Selected Cell Lines
IC50
PRb
p53
cell line
HCT116
(nM)
function
function
34
12
16
57
16
14
15
13
+
-
+
?
+
-
+
+
-
+
-
-
MDA MB468
NCI H460
A498
T47D
+
+
-
MCF7
DU145
COLO205
+
Figure 4. Caliper-estimated tumor mass plots for 24d (b),
racemic flavopiridol [(()-fp, 2], and vehicle (9). Compounds
were dosed ip, q2d × 7 in female nude mice bearing NCI H460
tumors (see Experimental Section).
dine N5 to Leu83 carbonyl oxygen (2.67 Å), pyrimi-
dinone carbonyl oxygen to backbone nitrogen of the
same residue (3.04 Å), and pyrazole N2 to Nꢀ of Lys33
(2.90 Å). The observation of these first two interactions
validates our homology model, which predicts a biden-
tate hydrogen bond to the corresponding residue in
CDK4 (Val96). The C3 ethyl group from the pyrazole
ring is pointing deeper into the pocket and has hydro-
phobic interactions with the Val 64 side chain. Near the
ethyl group (3.40 Å) there is a water molecule that forms
hydrogen bonds with the side chain and backbone
nitrogen of Asp145 (2.70, 3.02 Å, respectively). The tri-
chlorophenyl group provides a hydrophobic interaction
with either the glycine-rich loop or the C-terminal
domainsone of the o-chlorines with Ile10 and Gly11 and
the other with Gln131 and Ala144swhile the p-chlorine
chlorine is directed toward Thr14 and Lys129. These
interactions allow the trichlorophenyl group to bridge
the N-terminal and C-terminal domains. The R6 aryl
group is projected out of the enzyme, with the phenol
moiety on the opposite side of the phenyl ring from
Phe82 (His95 in CDK4). If the same binding mode
applies to CDK4, rotating the aryl ring 180° would place
the m-hydroxyl group within hydrogen-bonding distance
of His95, possibly explaining the improvement in CDK4/
cyclinD potency of m-phenols over compound not able
to donate a hydrogen bond from this position. Other
residues within 3.5 Å of ST550 are Gly13, Val18, Ala
31, Phe82, His84, Lys129, and Asn132.
Figure 2. Homology model of CDK4 with 10i (magenta) and
ATP (light blue) bound. Hydrogen bonds between the pyrimi-
dinone N5 and Val96 carbonyl and the pyrimidinone carbonyl
and Val96 N are shown by dashed lines.
To assess the viability of this series of compounds in
vivo we dosed 24d in nude mice harboring xenografts
from the NCI H460 cell line. Like HCT116, this line
expresses high levels of CDK/cyclin proteins. The re-
sponse curves for an ip, q2d × 7 dosing schedule are
shown in Figure 4. Tumor masses were estimated by
caliper measurement. The percentage tumor growth
inhibition (TGI) values were obtained by comparing the
tumor volumes from animals receiving compound with
those receiving vehicle (4% aqueous mannitol) only.
TGIs of 41% and 49% were observed for 24d (10 mg/
kg) and racemic flavopiridol (7.5 mg/kg), respectively.
In a study with HCT116 xenografts,26 mice dosed ip
(q1d × 19) with 10 mg/kg of 24d and 7.5 mg/kg of
racemic flavopiridol exhibited TGIs of 46 and 44%,
Figure 3. Crystal structure of 22s bound to CDK2. Hydrogen
bonds between the pyrimidinone N5 and Leu83 carbonyl and
the pyrimidinone carbonyl and Leu 83 N are shown by dashed
lines.
defined as shown in Figure 3 with the final electron
density. The inhibitor molecule binds as ATP and other
reported inhibitors,25 in the deep cleft between the
N-terminal and C-terminal domains. There are three
hydrogen bonds between the protein and 22s: pyrimi-

5902 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
5-Amino-1-n-butyl-4-carboxamido-3-(methylthio)pyra-
respectively (based on measured tumor masses upon
termination of the study at day 19).
1
zole (3b): mp 114-116 °C; H NMR (DMSO-d6) δ 6.77 (br s,
2H), 6.38 (s, 2H), 3.85 (t, 2H, J ) 7.3 Hz), 2.42 (s, 3H), 1.57-
1.67 (m, 2H), 1.19-1.31 (m, 2H), 0.89 (t, 3H, J ) 7.3 Hz). MS
(m/z) 229 (M + H)+.
Conclusion
Optimization of the CDK4/cyclin D1 screening hit 1
has led to a series of potent 6-arylmethylpyrazolopy-
rimidin-4-one inhibitors that show modest selectivity for
CDK2/cyclin E over other CDKs. This series appears to
be selective for CDKs over several other protein kinases.
The best CDK4/cyclin D1 inhibitors in this series are
50-330 times more potent than the screening hit and
are characterized by having a relatively acidic hydrogen-
bond donor in the meta-position of the pyrazolopyrimi-
dine R6 benzyl. The best antiproliferative activity is seen
in those compounds having anilide groups at the para-
position of the pyrazolopyrimidine R6 benzyl. Modeling
and X-ray crystallography experiments indicate that
groups at this position are projected out of the enzyme
and into solvent, offering opportunities to modulate
physical properties (solubility, partition coefficient, etc.)
without altering enzyme inhibition. The N,N-dimeth-
ylglycinamide 24d is highly potent in a range of anti-
proliferative assays and shows activity in mouse xeno-
graft models.
5-Amino-1-tert-butyl-4-carboxamido-3-(methylthio)-
1
pyrazole (3c): mp 137-139 °C; H NMR (DMSO-d6) δ 6.81
(br s, 2H), 6.33 (d, 2H, J ) 8.0 Hz), 2.39 (s, 3H), 1.48 (s, 9H);
MS (m/z) 227 (M - H)-.
5-Amino-4-carboxamido-1-(2-hydroxyethyl)-3-(meth-
ylthio)pyrazole (3d): 1H NMR (DMSO-d6) δ 6.74 (br s, 2H),
6.24 (s, 2H), 4.91 (t, 1H, J ) 5.1 Hz), 3.89 (t, 2H, J ) 5.7 Hz),
3.59-3.71 (m, 2H), 2.38 (s, 3H).
5-Amino-4-carboxamido-3-(methylthio)-1-phenylpyra-
zole (3e): mp 147-148 °C; 1H NMR (CDCl3) δ 7.45-7.99 (m,
4H), 7.37-7.42 (m, 1H), 5.73 (s, 2H), 2.59 (s, 3H); MS (m/z)
249 (M + H)+.
5-Amino-4-carboxamido-3-(methylthio)-1-(pyrid-2-yl)-
pyrazole (3f): mp 192-193 °C; 1H NMR (DMSO-d6) δ 8.39-
8.42 (m, 1H), 7.92-7.99 (m, 1H), 7.81 (dd, 1H, J ) 8.4, 0.9
Hz), 7.70 (s, 2H), 7.24-7.28 (m, 1H), 6.74 (br s, 2H), 2.54 (s,
3H); MS (m/z) 250 (M + H)+.
5-Amino-4-carboxamido-1-(3-chlorophenyl)-3-(meth-
1
ylthio)pyrazole (3g): mp 138-140 °C; H NMR (CDCl3) δ
7.45 (t, 1H, J ) 1.8 Hz), 7.25-7.37 (m, 2H), 7.18 (dd, 1H, J )
6.2, 1.7 Hz), 5.93 (s, 2H), 2.41 (s, 3H); MS (m/z) 283 (M + H)+.
5-Amino-4-carboxamido-1-(4-chlorophenyl)-3-(meth-
1
ylthio)pyrazole (3h): mp 168-169 °C; H NMR (CDCl3) δ
7.46-7.53 (m, 4H), 5.70 (s, 2H), 2.58 (s, 3H); MS (m/z) 283
(M + H)+.
Experimental Section
5-Amino-4-carboxamido-1-(4-isopropylphenyl)-3-(meth-
ylthio)pyrazole (3i): mp 86-87 °C; 1H NMR (CDCl3) δ
7.44 (d, 2H, J ) 8.1), 7.35 (d, 2H, J ) 7.8 Hz), 5.66 (s, 2H),
2.96 (m, 1H), 2.58 (s, 3H), 1.27 (d, 6H, J ) 6.9 Hz); MS (m/z)
291 (M + H)+.
5-Amino-4-carboxamido-1-(2-methoxyphenyl)-3-(meth-
ylthio)pyrazole (3j): mp 220-221 °C; 1H NMR (CDCl3) δ
7.38-7.45 (m, 2H), 7.05-7.12 (m, 2H), 5.63 (s, 2H), 3.88 (s,
3H), 2.58 (s, 3H); MS (m/z) 279 (M + H)+.
5-Amino-4-carboxamido-1-(2-chlorophenyl)-3-(meth-
ylthio)pyrazole (3k): mp 164-165 °C; 1H NMR (DMSO-d6)
δ 7.65 (d, 1H, J ) 7.9 Hz), 7.47-7.56 (m, 3H), 6.76 (br s, 2H),
6.34 (d, 2H, J ) 5.5 Hz), 2.41 (s, 3H); MS (m/z) 283 (M + H)+.
5-Amino-4-carboxamido-1-(4-chloro-2-methylphenyl)-
3-(methylthio)pyrazole (3l): mp 148-149 °C; 1H NMR
(DMSO-d6) δ 7.36 (s, 1H), 7.24-7.33 (m, 2H), 5.39 (s, 2H), 2.57
(s, 3H), 2.16 (s, 3H); MS (m/z) 298 (M + H)+.
5-Amino-4-carboxamido-1-(2,3-dichlorophenyl)-3-(meth-
ylthio)pyrazole (3m): 1H NMR (CDCl3) δ 7.77-7.83 (m, 1H),
7.45-7.53 (m, 2H), 6.77 (br s, 2H), 6.45 (s, 2H), 2.40 (s, 3H);
MS (m/z) 315 (M - H)-.
General Procedures. Melting points were determined on
a Thomas-Hoover Uni-melt apparatus and are uncorrected.
1H NMR spectra were recorded on dilute solutions in CDCl3
or DMSO-d6 at 300 MHz on Varian Unity instruments.
Chemical shifts are reported in parts per million (δ) downfield
from tetramethylsilane. Low-resolution mass spectral analyses
were performed on HP5988A (NH3-CI) and Micromass Plat-
form II (ESI) instruments. High-resolution mass spectra were
obtained on VG70-VSE (NH3-DCI) and Finnigan MAT95S
(ESI) instruments. Combustion analyses were performed by
Quantitative Technologies Inc., Whitehouse, NJ, and are
within (0.4% of the theoretical values. Reactions were per-
formed under an atmosphere of dry nitrogen or argon in flame-
dried glassware and were monitored for completeness by thin-
layer chromatography (TLC) using silica gel 60 F-254 (0.25
mm) plates. Visualization of TLC plates was accomplished by
I2 vapor, phosphomolybdic acid in ethanol, ceric ammonium
molybdate in aqueous methanol, or UV light absorption at 254
nm. Flash column chromatography was performed by the
method of Still27 using 230-400 mesh silica gel (E Merck).
Tetrahydrofuran was distilled from potassium/benzophenone
ketyl immediately prior to use. Other solvents and reagents
were purchased from commercial sources and were used
without further purification.
5-Amino-4-carboxamido-1-(2,4-dichlorophenyl)-3-(meth-
1
ylthio)pyrazole (3o): mp 168-169 °C; H NMR (CDCl3) δ
7.59 (s, 1H), 7.42 (s, 2H), 5.51 (br s, 2H), 2.58 (s, 3H); MS
(m/z) 315 (M - H)-.
Compounds 1, 2, and 4w were prepared according to the
procedures disclosed by Chen.12
5-Amino-4-carboxamido-1-(2,5-dimethylphenyl)-3-(meth-
ylthio)pyrazole (3p): 1H NMR (DMSO-d6) δ 7.25 (d, 1H,
J ) 7.5 Hz), 7.19 (dd, 1H, J ) 8.1, 0.9 Hz), 7.07 (s, 1H), 6.77
(br s, 2H), 6.15 (d, 2H, J ) 6.0 Hz), 2.41 (s, 3H), 2.29 (s, 3H),
1.98 (s, 3H); MS (m/z) 275 (M - H)-.
5-Amino-4-carboxamido-1-(2,5-dichlorophenyl)-3-(meth-
ylthio)pyrazole (3q): mp 196-197 °C; 1H NMR (DMSO-d6)
δ 7.68 (d, 1H, J ) 8.7 Hz), 7.67 (d, 1H, J ) 1.9 Hz), 7.61 (dd,
1H, J ) 8.4, 2.6 Hz), 6.77 (br s, 2H), 6.48 (s, 1H), 6.46 (s, 1H),
2.41 (s, 3H); MS (m/z) 317 (M + H)+.
5-Amino-4-carboxamido-1-(2-chloro-5-(trifluorometh-
yl)phenyl)-3-(methylthio)pyrazole (3r): mp 196-197 °C;
1H NMR (CDCl3) δ 7.78 (s, 1H), 7.71 (s, 2H), 7.61 (dd, 1H,
J ) 8.4, 2.6 Hz), 5.58 (br s, 2H), 2.59 (s, 3H); MS (m/z) 351
(M + H)+.
Representative Procedure for the Preparation of
5-Amino-3-(methylthio)pyrazole-4-carboxamides (3). 5-
Amino-4-carboxamido-1-(2,4-dimethylphenyl)-3-(methyl-
thio)pyrazole (3n). To a stirred solution of 621 mg (2.94
mmol) of 2 in 10 mL of MeOH was added 609 mg (3.53 mmol)
of 2,4-dimethylphenylhydrazine hydrochloride followed by 610
µL (4.41 mmol) of Et3N. The solution was stirred overnight at
reflux, cooled to ambient temperature, and treated with 30
mL of water. The resulting solid was filtered and rinsed with
water. Recrystallization from EtOAc-hexanes afforded 681 mg
1
(84%) of 3n as an off-white solid: mp 148-149 °C; H NMR
(DMSO-d6) δ 7.18 (s, 1H), 7.12 (s, 2H), 6.78 (br s, 2H), 6.13 (d,
2H, J ) 6.6 Hz), 2.41 (s, 3H), 2.31 (s, 3H), 1.99 (s, 3H); MS
(m/z) 277 (M + H)+.
5-Amino-1-benzyl-4-carboxamido-3-(methylthio)pyra-
zole (3a): mp 118-120 °C; 1H NMR (CDCl3) δ 7.18-7.36 (m,
5H), 5.21 (s, 2H), 5.16 (s, 2H), 2.54 (s, 3H); MS (m/z) 245 (M +
H - H2O)+.
5-Amino-4-carboxamido-1-(2-chloro-6-fluorophenyl)-3-
(methylthio)pyrazole (3s): mp 169-170 °C; 1H NMR (CDCl3)
δ 7.37-7.50 (m, 2H), 7.21(td, 1H, J ) 8.2, 1.6 Hz), 5.48 (br s,
2H), 2.58 (s, 3H); MS (m/z) 301 (M + H)+.

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5903
1
5-Amino-4-carboxamido-1-(2,6-dichlorophenyl)-3-(meth-
ylthio)pyrazole (3t): mp 156-159 °C; 1H NMR (CDCl3) δ
7.65 (d, 1H, J ) 8.8 Hz), 7.65 (d, 1H, J ) 7.3 Hz), 7.71 (dd,
1H, J ) 9.3, 6.8 Hz), 6.77 (br s, 2H), 6.50 (s, 2H), 2.39 (s, 3H);
MS (m/z) 317 (M + H)+.
>300 °C; H NMR (DMSO-d6) δ 12.40 (br s, 1H), 8.16 (t, 1H,
J ) 1.0 Hz), 8.05-8.09 (m, 1H), 7.53 (t, 1H, J ) 7.7 Hz), 7.36-
7.41 (m, 1H), 2.59 (s, 3H), 2.40 (s, 3H); MS m/z calcd for
C13H11N4SOCl (M)+ 306.0342, found 306.0340.
1-(4-Chlorophenyl)-6-methyl-3-(methylthio)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4h): yield 62%;
mp >300 °C; 1H NMR (DMSO-d6) δ 12.40 (br s, not integrated),
8.16 (d, 2H, J ) 7.6 Hz), 7.61 (d, 2H, J ) 7.6 Hz), 2.81 (s, 3H),
2.41 (s, 3H); MS m/z calcd for C13H12N4SOCl (M + H)+
307.0418, found 307.0420. Anal. (C13H11N4OSCl‚1/4H2O) C, H,
N, S, Cl.
1-(4-Isopropylphenyl)-6-methyl-3-(methylthio)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4i): yield 16%;
mp 271-273 °C; 1H NMR (CDCl3) δ 11.10 (br s, not integrated),
7.91 (d, 2H, J ) 7.6 Hz), 7.33 (d, 2H, J ) 7.6 Hz), 2.91-3.01
(m, 1H), 2.70 (s, 3H), 2.57 (s, 3H), 1.30 (d, 6H, J ) 7.7 Hz);
MS m/z calcd for C16H18N4SO (M+) 314.1201, found 314.1200.
5-Amino-4-carboxamido-1-(2,4-dichloro-6-(trifluoro-
methyl)phenyl)-3-(methylthio)pyrazole (3u): mp 180-
1
181°C; H NMR (DMSO-d6) δ 8.28 (d, 1H, J ) 2.6 Hz), 8.03
(d, 1H, J ) 2.3 Hz), 6.75 (br s, 2H), 6.57 (d, 2H, J ) 3.3 Hz),
2.37 (s, 3H); MS (m/z) 385 (M + H)+.
5-Amino-4-carboxamido-1-(2,4-dichloro-6-methylphen-
yl)-3-(methylthio)pyrazole (3v): mp 180-182 °C; 1H NMR
(CDCl3) δ 7.41 (d, 1H, J ) 2.2 Hz), 7.28 (d, 1H, J ) 2.2 Hz),
5.37 (br s, 2H), 2.57 (s, 3H), 2.13 (s, 3H); MS (m/z) 331 (M +
H)+.
5-Amino-4-carboxamido-3-(methylthio)-1-(2,4,6-tri-
methylphenyl)pyrazole (3x): mp 163-164 °C; 1H NMR
(DMSO-d6) δ 8.28 (d, 1H, J ) 2.6 Hz), 8.03 (d, 1H, J ) 2.3
Hz), 6.75 (br s, 2H), 6.57 (d, 2H, J ) 3.3 Hz), 2.37 (s, 3H); MS
(m/z) 289 (M - H)-.
1-(2-Methoxyphenyl)-6-methyl-3-(methylthio)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4j): yield 35%;
1
mp 274-275 °C; H NMR (CDCl3) δ 10.58 (br s, 1H), 7.39-
7.48 (m, 2H), 7.08-7.12 (m, 2H), 3.80 (s, 3H), 2.69 (s, 3H), 2.49
(s, 3H); MS m/z calcd for C14H15N4SO2 (M + H)+ 303.0916,
found 303.0895. Anal. (C14H14N4O2S‚1/4MeOH) C, H, N, S.
Representative Procedure for the Preparation of
6-Methyl-3-(methylthio)-4,5-dihydro-1H-pyrazolo[3,4-d]-
pyrimidin-4-ones (4) from 5-Amino-4-carboxamido-3-
(methylthio)pyrazoles (3): 1-(2,4-Dimethylphenyl)-6-
methyl-3-(methylthio)-4,5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-4-one (4n). To a stirred solution of 160 mg (0.58
mmol) of 3n in 5 mL of EtOH was added 0.46 mL (4.6 mmol)
of EtOAc followed by 1.7 mL (4.6 mmol) of a 2.66 M solution
of NaOEt in EtOH. The solution was stirred for 6 h at reflux,
cooled to ambient temperature, and treated with 30 mL of 3%
aqueous HOAc. The resulting solid was filtered and rinsed
with water. Recrystallization from EtOAc-hexanes afforded
126 mg (72%) of 4n as an off-white solid: mp 246-249 °C; 1H
NMR (DMSO-d6) δ 12.15 (br s, 1H), 7.17-7.21 (m, 2H), 7.15
(d, 1H, J ) 8.1), 2.49 (s, 3H), 2.32 (s, 3H), 2.24 (s, 3H), 1.99 (s,
3H); MS (m/z) 299 (M - H)-. Anal. (C15H16N4OS) C, H, N, S.
1-(2-Chlorophenyl)-6-methyl-3-(methylthio)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4k): yield 16%;
1
mp 278-279 °C; H NMR (CDCl3) δ 10.58 (br s, 1H), 7.41-
7.60 (m, 4H), 2.66 (s, 3H), 2.49 (s, 3H); MS m/z calcd for
C13H11N4SOCl (M+) 306.0342, found 306.0320. Anal. (C13H11N4-
OSCl‚1/4H2O) C, H, N, S, Cl.
1-(4-Chloro-2-methylphenyl)-6-methyl-3-(methylthio)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4l): yield
1
51%; mp 268-269 °C; H NMR (DMSO-d6) δ 12.24 (br s, not
integrated), 7.43 (s, 1H), 7.34 (s, 2H), 2.57 (s, 3H), 2.31 (s, 3H),
2.17 (s, 3H); MS m/z calcd for C14H14N4SOCl (M + H)+
321.0577, found 321.0575. Anal. (C14H13N4OSCl‚1/4H2O) C, H,
N, S, Cl.
1-Benzyl-6-methyl-3-(methylthio)-4,5-dihydro-1H-pyra-
zolo[3,4-d]pyrimidin-4-one (4a): yield 9%; mp 262-264 °C;
1H NMR (CDCl3) δ 10.50 (br s, 1H), 7.27-7.35 (m, 5H), 5.43
(s, 2H), 2.62 (s, 3H, CH3), 2.51 (s, 3H, CH3); MS m/z calcd for
C14H14N4SO (M+) 286.0888, found 286.0947.
1-n-Butyl-6-methyl-3-(methylthio)-4,5-dihydro-1H-pyra-
zolo[3,4-d]pyrimidin-4-one (4b): yield 32%; mp 211-213
°C; 1H NMR (DMSO-d6) δ 12.02 (br s, 1H), 4.17 (t, 2H, J ) 7.1
Hz), 2.53 (s, 3H), 2.35 (s, 3H), 1.69-1.80 (m, 2H), 1.20-1.32
(m, 2H), 0.89 (t, 3H, J ) 7.4 Hz); MS (m/z) 251 (M - H)-. Anal.
(C11H16N4OS) C, H, N, S.
1-(2,3-Dichlorophenyl)-6-methyl-3-(methylthio)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4m): yield 21%;
mp >300 °C; 1H NMR (DMSO-d6) δ 12.29 (br s, 1H), 7.86 (dd,
1H, J ) 8.4, 2.2 Hz), 7.50-7.65 (m, 2H), 2.51 (s, 3H), 2.26 (s,
3H); MS m/z calcd for C13H11N4SOCl2 (M + H)+ 341.0031,
found 341.0029.
1-(2,4-Dichlorophenyl)-6-methyl-3-(methylthio)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4o): yield 28%;
1
mp 298-299 °C; H NMR (CDCl3) δ 11.83 (br s, 1H), 7.24 (d,
1H, J ) 2.1 Hz), 7.13 (d, 1H, J ) 8.3 Hz), 7.07 (dd, 1H, J )
8.1, 2.1 Hz), 2.26 (s, 3H), 2.02 (s, 3H); MS m/z calcd for
C13H10N4SOCl2 (M+) 339.9952, found 339.9968. Anal. (C13H10N4-
OSCl2) C, H, N, S, Cl.
1-tert-Butyl-6-methyl-3-(methylthio)-4,5-dihydro-1H-
1
pyrazolo[3,4-d]pyrimidin-4-one (4c): yield 29%; H NMR
(DMSO-d6) δ 11.99 (br s, not integrated), 2.48 (s, 3H), 2.30 (s,
1-(2,5-Dimethylphenyl)-6-methyl-3-(methylthio)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4p): yield 34%;
mp 257-258 °C; 1H NMR (DMSO-d6) δ 12.16 (br s, 1H), 7.12-
7.28 (m, 3H), 2.49 (s, 3H), 2.29 (s, 3H), 2.25 (s, 3H), 1.97 (s,
3H); MS (m/z) 299 (M - H)-. Anal. (C15H16N4OS) C, H, N, S.
3H), 1.62 (t, 9H); MS (m/z) 251 (M - H)-.
1-(2-Hydroxyethyl)-6-methyl-3-(methylthio)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4d): yield 43%;
1
mp 251-253 °C; H NMR (DMSO-d6) δ 11.97 (br s, 1H), 4.81
(t, 1H, J ) 5.3 Hz), 4.17 (t, 2H, J ) 5.9 Hz), 3.68-3.75 (m,
2H), 2.49 (s, 3H), 2.30 (s, 3H); MS (m/z) 239 (M - H)-. Anal.
(C9H12N4O2S‚0.1H2O) C, H, N, S.
1-(2,5-Dichlorophenyl)-6-methyl-3-(methylthio)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4q): yield 53%;
mp 263-264 °C; 1H NMR (DMSO-d6) δ 12.03 (br s, not
integrated), 7.36-7.32 (m, 2H), 7.30 (dd, 1H, J ) 8.4, 2.4 Hz),
2.50 (s, 3H), 2.27 (s, 3H); MS m/z calcd for C13H10N4SOCl2
(M+) 339.9952, found 339.9947. Anal. (C13H10N4OSCl2) C, H,
N, S, Cl.
1-(2-Chloro-5-trifluoromethylphenyl)-6-methyl-3-(meth-
ylthio)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
(4r): yield 56%; mp 259-260 °C; 1H NMR (CDCl3) δ 11.47
(br s, 1H), 7.81 (s, 1H), 7.67-7.75 (m, 2H), 2.66 (s, 3H), 2.53
(s, 3H); MS m/z calcd for C14H11N4SF3OCl (M+) 375.0294,
found 375.0298.
1-(2-Chloro-6-fluorophenyl)-6-methyl-3-(methylthio)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4s): yield
67%; mp 276-277 °C; 1H NMR (CDCl3) δ 11.39 (br s, 1H),
7.39-7.47 (m, 2H), 7.18-7.23 (m, 1H), 2.65 (s, 3H), 2.51 (s,
3H); MS m/z calcd for C13H11N4SFOCl (M+) 325.0326, found
325.0302.
6-Methyl-3-(methylthio)-1-phenyl-4,5-dihydro-1H-pyra-
zolo[3,4-d]pyrimidin-4-one (4e) was prepared as example
4n, except that precipitation from the reaction with water gave
the sodium salt: yield 90%; mp >300 °C; 1H NMR (DMSO-d6)
δ 8.08 (d, 2H, J ) 8.1 Hz), 7.48 (t, 2H, J ) 8.1 Hz), 7.27 (t, 1H,
J ) 8.0 Hz), 2.56 (s, 3H), 2.32 (s, 3H); MS m/z calcd for
C13H13N4SO (M + H)+ 273.0810, found 273.0805. Anal.
(C13H11N4OSNa‚0.25EtOAc) C, H, N, S.
6-Methyl-3-(methylthio)-1-(pyrid-2-yl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (4f): yield 77%; mp 297-
1
299 °C; H NMR (DMSO-d6) δ 8.54 (dd, 1H, J ) 4.7, 0.8 Hz),
7.91-8.02 (m, 2H), 7.37-7.42 (m, 1H), 2.55 (s, 3H), 2.32 (s,
3H); MS m/z calcd for C12H12N5SO (M + H)+ 274.0762, found
274.0759.
1-(3-Chlorophenyl)-6-methyl-3-(methylthio)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4g): yield 9%; mp

5904 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
1-(2,6-Dichlorophenyl)-6-methyl-3-(methylthio)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4t): yield 42%;
mp 306-307 °C; 1H NMR (CDCl3) δ 11.42 (br s, not integrated),
7.50 (d, 2H, J ) 8.1 Hz), 7.41 (dd, 1H, J ) 9.0, 6.0 Hz), 2.66
(s, 3H), 2.51 (s, 3H); MS m/z calcd for C13H10N4SOCl2 (M+)
339.9952, found 339.9954. Anal. (C13H10N4OSCl2) C, H, N, S,
Cl.
NMR (DMSO-d6) δ 7.89 (s, 2H), 6.80 (s, 2H), 2.44-2.49 (m,
2H), 1.50-1.60 (m, 2H), 1.22-1.34 (m, 2H), 0.84 (t, 3H, J )
7.3 Hz); MS (m/z) 343 (M + H)+.
5-Amino-4-cyano-3-phenyl-1-(2,4,6-trichlorophenyl)-
pyrazole (7e): yield 68%; mp 193-194 °C; 1H NMR (DMSO-
d6) δ 8.00 (s, 2H), 7.83 (dd, 2H, J ) 8.3, 1.7 Hz), 7.42-7.53
(m, 3H), 7.09 (s, 2H); MS (m/z) 363 (M + H)+.
1-(2,4-Dichloro-6-trifluoromethenyl)-6-methyl-3-(meth-
ylthio)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
(4u): yield 57%; mp 250-253 °C; 1H NMR (DMSO-d6) δ 12.41
(br s, 1H), 8.38 (d, 1H, J ) 2 Hz), 8.17 (d, 1H, J ) 2 Hz), 2.49
(s, 3H), 2.30 (s, 3H).
5-Amino-4-cyano-3-ethyl-1-(2,4,6-trichlorophenyl)pyra-
zole (7i): yield 83%; mp 152-153 °C (hemietherate); 1H NMR
(DMSO-d6) δ 7.93 (s, 1H), 6.85 (s, 2H), 2.48-2.60 (m, 2H), 1.18
(t, 3H, J ) 7.5 Hz); MS m/z calcd for C12H9N4Cl3 (M+)
313.9893, found 313.9878.
1-(2,4-Dichloro-6-methylphenyl)-6-methyl-3-(methyl-
thio)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
(4v): yield 76%; mp 287-289 °C; 1H NMR (DMSO-d6) δ 12.29
(br s, 1H), 7.71 (d, 1H), 7.55 (s, 1H), 2.48 (s, 3H), 2.25 (s, 3H),
1.97 (s, 3H); MS m/z calcd for C14H12N4SOCl2 (M+) 355.0187,
found 355.0174. Anal. (C14H12N4OSCl2) C, H, N, S, Cl.
6-Methyl-3-(methylthio)-1-(2,4,6-trimethylphenyl-4,5-
dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (4x): yield
5-Amino-4-cyano-3-methyl-1-(2,4,6-trichlorophenyl)-
1
pyrazole (7j): yield 67%; mp 229-230 °C; H NMR (CDCl3)
δ 7.52 (s, 2H), 4.32 (s, 2H), 2.34 (s, 3H); MS (m/z) 299 (M -
H)-.
5-Amino-4-cyano-1-(2,4,6-trichlorophenyl)pyrazole (7k):
yield 49%; MS (m/z) 287 (M + H)+.
5-Amino-4-cyano-3-(2-hydroxyethyl)-1-(2,4,6-trichlo-
rophenyl)pyrazole (7m). To a stirred solution of 1.87 g (6.0
mmol) of 2-(tert-butyldiphenylsilyloxy)propionaldehyde in 30
mL of ethanol was added 1.40 g (6.6 mmol) of 2,4,6-trichlo-
rophenylhydrazine. The solution was stirred for 3 h at reflux,
and about half the solvent was evaporated. The solution was
cooled, poured into ether, and washed twice with 1 N aqueous
HCl. The solution was then washed with aqueous NaHCO3
and then brine, dried (MgSO4), and concentrated under
reduced pressure to afford 5.04 g (quant.) of an oil. A 1.52 g
(3.0 mmol) portion of the crude hydrazone was dissolved in
10 mL of dry DMF, treated with 0.59 g (3.3 mmol) of
N-bromosuccinimide, and stirred for 1 h at ambient temper-
ature. The dark solution was poured into half-saturated brine,
and this mixture was extracted with ether. The organic extract
was washed with water and then brine, dried (MgSO4), and
concentrated under reduced pressure to afford a dark oil.
Malononitrile anion was generated in ethanol by treatment
of 330 mg (5.0 mmol) of malononitrile in 10 mL of ethanol with
2.0 mL of 2.7 M sodium ethoxide in ethanol. This mixture was
treated at ambient temperature with the crude bromohydra-
zone in 10 mL of absolute ethanol, and the reaction was then
refluxed for 30 min. The reaction was cooled to room temper-
ature, stirred another 16 h, and quenched with 10% aqueous
HOAc. The mixture was extracted with ether, and the organic
extract was washed with brine, dried (MgSO4), and concen-
trated under reduced pressure. Chromatography on silica gel
(elution with hexanes, then ether-hexanes) afforded, after
removal of solvents, 1.11 g (65%) of 5-amino-4-cyano-3-(2-(tert-
butyldiphenylsilyloxy)ethyl)-1-(2,4,6-trichlorophenyl)pyra-
zole as a light brown foam: 1H NMR (DMSO-d6) δ 7.89 (s, 2H),
7.58 (dd, 4H, J ) 7.5, 1.5 Hz), 7.33-7.43 (m, 6H), 6.83 (s, 2H),
3.87 (t, 2H, J ) 6.5 Hz), 2.76 (t, 2H, J ) 6.2 Hz), 0.92 (s, 9H).
To a stirred solution of 0.36 g (0.63 mmol) of 5-amino-4-
cyano-3-(2-(tert-butyldiphenylsilyloxy)ethyl)-1-(2,4,6-trichlo-
rophenyl)pyrazole in 10 mL of THF was added 0.73 mL (2.0
mmol) of aqueous 75 wt % solution of TBAF. The solution was
stirred for 2 h and poured into water, and the resulting
mixture was extracted with EtOAc. The organic extract was
washed with brine, dried (MgSO4), and concentrated under
reduced pressure. Chromatography on silica gel (elution with
3:1 hexanes-EtOAc, then 1:1 hexanes-EtOAc) afforded, after
removal of solvent, 187 mg (89%) of 7m as an off-white solid:
1
66%. H NMR (DMSO-d6) δ 12.15 (br s, 1H, NH), 7.17-7.21
(m, 2H, Ph), 7.00 (s, 2H, Ph), 2.48 (s, 3H, SCH3), 2.28 (s, 3H,
CH3), 2.23 (s, 3H, CH3), 1.84 (s, 6H, CH3); MS (m/z) 313 (M -
H)-.
Representative Procedure for the Preparation Alkoxy-
alkylidenemalononitriles (5) from Ortho Esters and
Malononitrile: 2-Cyano-3-methoxyhept-2-enenitrile (5g).
To a stirred solution of 2.64 g (40 mmol) of malononitrile in 9
mL of acetic anhydride was added 6.49 g (40 mmol) of
trimethyl orthovalerate. The solution was stirred for 15 h at
reflux, cooled, and poured into water. The mixture was
extracted with ether, and the organic extract was washed with
aqueous NaHCO3 and then brine. The solution was dried
(MgSO4) and concentrated under reduced pressure to afford
5.9 g (90%) of 5g as a pale amber oil: 1H NMR (CDCl3) δ 4.15
(s, 3H), 2.65 (t, 2H, J ) 7.3 Hz), 1.40-1.69 (m, 4H), 0.98 (t,
3H, J ) 7.2 Hz).
1
2-Cyano-3-methoxycinnamonitrile (5e): yield 81%; H
NMR (CDCl3) δ 7.40-7.68 (m, 5H), 3.94 (s, 3H); MS (m/z) 202
(M + NH4)+.
2-Carboxamido-3-methoxyhex-2-enenitrile (6h). To a
stirred solution of 3.36 g (40 mmol) of cyanoacetamide in 9
mL of acetic anhydride was added 6.72 mL (42 mmol) of
trimethyl orthobutyrate. The solution was stirred for 16 h at
reflux, cooled, and diluted with ether. The resulting solid was
filtered and rinsed with ether to afford 5.9 g (42%) of 5h as
an off-white solid: mp 102-103 °C; 1H NMR (DMSO-d6) δ 7.47
(br s, 1H), 7.24 (br s, 1H), 3.99 (s, 3H), 2.63 (t, 2H, J ) 6.8
Hz), 1.54-1.66 (m, 2H), 0.98 (t, 3H, J ) 7.5 Hz).
5-Amino-3,4-dicyano-1-(2,4,6-trichlorophenyl)pyra-
zole (7a). To a stirred solution of 25.0 g (118 mmol) of 2,4,6-
trichlorophenyl hydrazine in 250 mL of absolute ethanol at 0
°C was added 15.1 g (118 mmol) of tetracyanoethylene (CAU-
TION: Evolves HCN!). The solution was stirred for 10 min
and was warmed to ambient temperature. After stirring for 1
h, the solid was filtered and washed with ethanol to afford
25.4 g (69%) of 7a as a white powder: 1H NMR (DMSO-d6) δ
8.00 (s, 2H), 7.65 (s, 2H); MS (m/z) 310 (M - H)-; IR (KBr)
ν 3390, 2252, 2236, 1652 cm-1
.
Representative Procedure for the Preparation of
5-Amino-4-cyano-1-(2,4,6-trichlorophenyl)pyrazoles (7)
from Alkoxyalkylidenemalononitriles (5): 5-Amino-3-n-
butyl-4-cyano-1-(2,4,6-trichlorophenyl)pyrazole (7g). To
a stirred solution of 4.10 g (25 mmol) of 2-cyano-3-methoxy-
hept-2-enenitrile in 60 mL of MeOH was added 5.92 g (28
mmol) of 2,4,6-trichlorophenylhydrazine. The solution was
stirred for 2 h at reflux, after which time it was cooled to
ambient temperature and poured into 1 N aqueous HCl. The
mixture was extracted with EtOAc, and the organic extract
was washed with aqueous NaHCO3 and then brine. The
solution was dried (MgSO4) and concentrated under reduced
pressure. Chromatography on silica gel (elution with 1:1 THF-
hexanes, then THF) afforded, after evaporation of solvents,
1
mp 158-160 °C; H NMR (DMSO-d6) δ 7.89 (s, 2H), 6.81 (s,
2H), 4.71 (t, 1H, J ) 5.3 Hz), 3.58-3.66 (m, 2H), 2.62 (t, 2H,
J ) 7.4 Hz).
Representative Procedure for the Preparation of
5-Amino-4-carboxamido-1-(2,4,6-trichlorophenyl)pyra-
zoles (8) from 5-Amino-4-cyano-1-(2,4,6-trichlorophenyl)-
pyrazoles (7): 5-Amino-3-n-butyl-4-carboxamido-1-(2,4,6-
trichlorophenyl)pyrazole (8g). A 3.43 g (10 mmol) portion
of 7g was added to 20 mL of concentrated sulfuric acid with
rapid stirring. After 2.5 h the solution was added dropwise by
pipet to a rapidly stirred solution of saturated aqueous
NaHCO3. This mixture was stirred overnight, and the larger
chunks were broken up with a spatula. The solid was then
1
6.6 g (77%) of 7g as an off-white solid: mp 123-124 °C; H

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5905
filtered and rinsed with water. Vacuum drying at 70 °C for 3
h afforded 3.56 g (98%) of 8g as an off-white solid: mp 103-
105 °C; 1H NMR (DMSO-d6) δ 7.89 (s, 2H), 6.58 (br s, 2H),
6.32 (d, 2H, J ) 4.8 Hz), 2.67 (t, 2H, J ) 7.5 Hz), 1.48-1.58
(m, 2H), 1.22-1.34 (m, 2H), 0.83 (t, 3H, J ) 7.4 Hz).
5-Amino-4-carboxamido-3-phenyl-1-(2,4,6-trichloro-
phenyl)pyrazole (8e): yield 89%; 1H NMR (CDCl3) δ 7.59-
7.65 (m, 2H), 7.53 (s, 2H), 7.42-7.57 (m, 3H); MS m/z calcd
for C16H11N4OCl3 (M+) 379.9998, found 379.9999.
5-Amino-4-carboxamido-3-ethyl-1-(2,4,6-trichlorophen-
yl)pyrazole (8i): yield 99%; mp 186-188 °C; 1H NMR (CDCl3)
δ 7.89 (s, 1H), 6.58 (br s, 2H), 6.35 (s, 2H), 2.70 (m, 2H), 1.10
(t, 3H, J ) 7.5 Hz); MS m/z calcd for C12H11N4OCl3 (M+)
331.9998, found 331.9989.
solid: 1H NMR (CDCl3) δ 7.54 (s, 2H), 5.78-6.03 (m, 2H),
5.62-5.76 (m, 2H); 19F NMR (CDCl3) δ -61.5; MS (m/z) 373
(M + H)+.
5-Amino-3-benzyl-4-carboxamido-1-(2,4,6-trichloro-
phenyl)pyrazole (8f): yield 15%; 1H NMR (CDCl3) δ 7.54
(s, 2H), 7.26-7.38 (m, 5H), 5.42 (s, 2H), 5.31 (s, 2H), 4.17 (s,
2H); MS (m/z) 373 (M + H)+.
5-Amino-4-carboxamido-3-(hydroxymethyl)-1-(2,4,6-
trichlorophenyl)pyrazole (8l): yield 40%. 1H NMR (DMSO-
d6) δ 7.89 (s, 2H), 6.48 (s, 2H), 6.15 (t, 1H, J ) 7.3 Hz), 4.01
(d, 2H, J ) 7.3 Hz); MS (m/z) 335 (M + H)+.
3-Cyano-6-methyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-
1H-pyrazolo[3,4-d]pyrimidin-4-one (10a). A solution of 10
g (32 mmol) of 7a in 100 mL of acetic anhydride was refluxed
2 days. The mixture was cooled and concentrated under
reduced pressure. The residue was taken up in 200 mL of
acetic acid and 10 mL of water and was heated to reflux. After
refluxing overnight, the solvent was removed under reduced
pressure and the residue was chromatographed (silica gel, 5%
methanol/CH2Cl2) and then triturated in EtOAc to give 2.1 g
(18%) of 10a as a white powder: 1H NMR (DMSO-d6) δ 12.92
(br s, 1H), 8.06 (s, 2H), 2.33 (s, 3H); MS (m/z) 371 (M + NH4)+;
IR (KBr) ν 2242, 1688 cm-1. Anal. [C13H6N5OCl3‚0.05CH2Cl2,
(CH2Cl2 detected in NMR)] C, H, N, Cl.
6-Methyl-3-methanesulfonyl-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (10b): A
suspension of 150 mg (0.4 mmol) of 4w in 5 mL of 1:1 MeOH-
CH2Cl2 was treated with 140 mg (0.8 mmol) of m-CPBA. The
suspension was stirred for 20 min, becoming clear as the solids
dissolve. The reaction was quenched with 0.5 mL of 10%
aqueous NaHSO3 and then poured into 10% aqueous Na2CO3,
and the mixture was extracted with EtOAc. The organic
extract was dried (MgSO4), concentrated under reduced pres-
sure, and recrystallized from EtOH to give 95 mg (60%) of 10b
as an amorphous solid: 1HNMR (DMSO-d6) δ 12.9 (br s, 1 H),
8.1 (s, 2 H), 3.6 (s, 3 H), 2.4 (s, 3 H), HRMS calcd for
C13H10N4O3SCl3 (M + H)+ 406.9539, found 406.9558.
3-Carboxamido-6-methyl-1-(2,4,6-trichlorophenyl)-4,5-
dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (10c). A solu-
tion of 250 mg (0.71 mmol) of 10a in 6 mL of concentrated
sulfuric acid was stirred overnight. The mixture was poured
into ice water and the resulting solid was filtered and washed
with water. The solid was triturated in boiling ethanol to give
137 mg (52%) of 10c as a white powder: 1H NMR (DMSO-d6)
δ 12.95 (br s, 1H), 9.46 (s, 1H), 8.04 (s, 2H), 8.02 (s, 1H), 2.34
(s, 3H); MS (m/z) 372 (M + H)+. Anal. (C13H8N5O2Cl3) C, H,
N, Cl.
5-Amino-4-carboxamido-3-methyl-1-(2,4,6-trichloro-
1
phenyl)pyrazole (8j): yield 99%; mp 223-224 °C; H NMR
(DMSO-d6) δ 7.88 (s, 2H), 6.59 (br s, 2H), 6.36 (d, 2H, J ) 4.4
Hz), 2.25 (s, 3H); MS (m/z) 317(M - H)-.
5-Amino-4-carboxamido-1-(2,4,6-trichlorophenyl)pyrazole
(8k): yield 88%; MS (m/z) 305 (M + H)+.
5-Amino-4-carboxamido-3-n-propyl-1-(2,4,6-trichloro-
phenyl)pyrazole (8h): To a stirred solution of 480 mg (2.85
mmol) of 6h in 11 mL of MeOH was added 698 mg (3.30 mmol)
of 2,4,6-trichlorophenylhydrazine. The solution was stirred for
3 h at reflux, cooled, and treated with 4 mL of water. The
mixture was filtered, and the crude solid was chromatographed
on silica gel (elution with 3:1 then 1:1 ether-hexanes).
Concentration of the appropriate fractions afforded 800 mg
1
(81%) of 8h as an off-white solid: mp 177-178 °C. H NMR
(DMSO-d6) δ 11.82 (s, 1H), 7.97 (s, 1H), 7.63 (s, 2H), 2.60-
2.72 (m, 2H), 1.53-1.69 (m, 2H), 0.97 (t, 3H, J ) 7.5 Hz); MS
m/z calcd for C13H13N4OCl3 (M)+ 346.0155, found 346.0163.
5-Amino-4-carboxamido-3-(2-hydroxyethyl)-1-(2,4,6-
trichlorophenyl)pyrazole (8m): A solution of 0.090 g (0.27
mmol) of 7m in 4 mL of concentrated sulfuric acid was stirred
for 2 h at ambient temperature. The solution was added
dropwise to a rapidly stirred saturated aqueous solution of
sodium carbonate. The mixture was extracted twice with ethyl
acetate, and the combined organic extracts were washed
(brine), dried (MgSO4), and concentrated under reduced pres-
sure to afford 72 mg (76%) of 8m as a foam. 1H NMR (DMSO-
d6) δ 7.96 (s, 2H), 6.36 (br s, 2H), 4.23 (t, 2H, J ) 5.9 Hz), 2.73
(t, 2H, J ) 4.7 Hz); MS (m/z) 331 (M + H - H2O)+.
Representative Procedure for the Preparation of
5-Amino-4-carboxamido-1-(2,4,6-trichlorophenyl)pyra-
zoles (8) from Aldehydes or Aldehyde Equivalents and
2,4,6-Trichlorophenylhydrazine: 5-Amino-4-carboxamido-
1-(2,4,6-trichlorophenyl)-3-trifluoromethylpyrazole (8d).
To a stirred solution of 2.88 g (20 mmol) of 1-ethoxy-2,2,2-
trifluoroethanol in 15 mL of ethanol was added 3.81 g (18
mmol) of 2,4,6-trichlorophenylhydrazine. The solution was
stirred for 16 h at reflux, cooled, and poured into water. A little
brine was added, and the mixture was then extracted with
ether. The organic extract was washed with brine, dried over
MgSO4, and concentrated to give the hydrazone as a chalky,
off-white solid. A stirred solution of 583 mg (2.00 mmol) of this
hydrazone in 10 mL of dry DMF was treated with 374 mg (2.10
mmol) of N-bromosuccinimide, and the solution was stirred
for 1 h at ambient temperature. The reaction was diluted with
ether and washed three times with water and once with brine,
dried over MgSO4, and concentrated under reduced pressure.
Cyanoacetamide anion was generated in ethanol by treatment
of 127 mg (1.5 mmol) of cyanoacetamide in 5 mL of ethanol
with 1.11 mL of 2.7 M sodium ethoxide in ethanol. This
mixture was treated at ambient temperature with the crude
bromohydrazone in 5 mL of absolute ethanol, and the reaction
was then stirred for 16 h at ambient temperature. The reaction
was quenched with 10% aqueous HOAc, and the mixture was
extracted with ether. The organic extract was washed with
brine, dried over MgSO4, and concentrated under reduced
pressure. Chromatography on silica gel (elution with 3:1
hexanes-EtOAc, then 1:1 hexanes-EtOAc) afforded, after
removal of solvent, 230 mg (31%) of 8d as an amorphous
Representative Procedure for the Preparation of
6-Methyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-pyra-
zolo[3,4-d]pyrimidin-4-ones (10) from 5-Amino-4-car-
boxamido-1-(2,4,6-trichlorophenyl)pyrazoles (8): 3-n-
Butyl-6-methyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (10g). To a stirred solution
of 180 mg (0.50 mmol) of 8g in 6 mL of EtOH was added 0.46
mL (4.6 mmol) of EtOAc followed by 1.13 mL (3.0 mmol) of a
2.66 M solution of NaOEt in EtOH. The solution was stirred
for 16 h at reflux, cooled to ambient temperature, and treated
with 10 mL of 10% aqueous HOAc. The resulting solid was
filtered and rinsed with 6 mL of 1:1 methanol-water and then
6 mL of 1:1 ether-hexanes. The solid was then briefly air-
dried to afford 145 mg (75%) of 10g as an off-white solid: mp
1
219-222 °C; H NMR (DMSO-d6) δ 12.25 (br s, 1H), 7.95 (s,
2H), 2.82 (t, 2H, J ) 7.3 Hz), 2.26 (s, 3H), 1.81-1.97 (m, 2H),
1.21-1.35 (m, 2H), 0.85 (t, 3H, J ) 7.4 Hz); MS m/z calcd for
C16H16N4OCl3(M + H)+ 385.0390, found 385.0382. Anal.
(C16H15N4OCl3) C, H, N, Cl.
6-Methyl-1-(2,4,6-trichlorophenyl)-3-trifluoromethyl-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (10d): yield
43%; mp >300 °C; 1H NMR (CDCl3) δ 7.55 (s, 2H), 2.55 (s,
3H); MS m/z calcd for C13H6N4OF3Cl3 (M+) 395.9559, found
395.9559.
6-Methyl-3-phenyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-
1H-pyrazolo[3,4-d]pyrimidin-4-one (10e) was prepared as
example 10g except that precipitation from the reaction with

5906 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
water gave the sodium salt: yield 64%; mp >300 °C; 1H NMR
(DMSO-d6) δ 8.39 (d, 2H, J ) 7.9 Hz), 8.00 (s, 2H), 7.39-7.49
(m, 3H), 2.30 (s, 3H); MS m/z calcd for C18H12N4OCl3 (M +
H)+ 405.0077, found 405.0081. Anal. (C18H10N4ONaCl3‚1/2H2O)
C, H, N.
3-Benzyl-6-methyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-
1H-pyrazolo[3,4-d]pyrimidin-4-one (10f): yield 81%; mp
266-270 °C; 1H NMR (DMSO-d6) δ 12.26 (s, 1H), 8.00 (s, 2H),
7.21-7.60 (m, 4H), 7.12-7.17 (m, 1H), 4.20 (s, 2H), 2.26 (s,
3H); MS (m/z) 417(M - H)-. Anal. (C19H13N4OCl3‚1/2cyclohex-
ane) C, H, N, Cl.
7.98 (s, 2H), 4.09 (s, 3H), 2.93 (q, 2H, J ) 7.6 Hz), 2.49 (s,
3H), 1.27 (t, 3H, J ) 7.6 Hz); MS m/z calcd for C15H14N4OCl3
(M + H)+ 371.0233, found 371.0257. Anal. (C15H13N4OCl3‚
1/3H2O) C, H, N, Cl.
4-Amino-3-ethyl-6-methyl-1-(2,4,6-trichlorophenyl)-1H-
pyrazolo[3,4-d]pyrimidine (12c). A solution of 548 mg (1.42
mmol) of 13 in 20 mL of saturated ammonia in ethanol was
stirred for 2 days. The solvent was removed under reduced
pressure and the residue was chromatographed (silica gel, 50
to 75% EtOAc/hexanes) to give 122 mg (24%) of 12c as a white
powder: 1H NMR (DMSO-d6) δ 7.93 (s, 2H), 2.97 (q, 2H, J )
7.7 Hz), 2.27 (s, 3H), 1.20 (t, 3H, J ) 7.7 Hz); MS (m/z) 356
(M + H)+. Anal. (C14H12N5Cl3) C, H, N, Cl.
Ethyl N-(3-Ethyl-4-cyano-6-methyl-1-(2,4,6-trichloro-
phenyl)pyrazol-5-yl)acetimidate (13). A stirred solution of
5.0 g (15.8 mmol) of 7i in 5.64 g (34.7 mmol) of triethyl
orthoacetate and 3.54 g (34.7 mmol) of acetic anhydride was
heated to 130 °C. After stirring for 3 h, the solvent was
removed under reduced pressure and the residue was chro-
matographed (silica gel, 25-33% EtOAc/hexanes) to give 5.57
g (91%) of 13 as a white powder: 1H NMR (DMSO-d6) δ 7.92
(s, 2H), 3.97 (q, 2H, J ) 6.9 Hz), 2.67 (q, 2H, J ) 7.5 Hz), 2.09
(s, 3H), 1.21 (t, 3H, J ) 7.5 Hz), 1.03 (t, 3H, J ) 6.9 Hz); MS
(m/z) 385 (M + H)+.
5-Amino-3-ethyl-1-(2,4,6-trichlorophenyl)pyrazole (14).
To a stirred solution of 10.0 g (47 mmol) of 2,4,6-trichlorophen-
ylhydrazine in 200 mL of ethanol was added 4.6 g (47 mmol)
of 3-oxovaleronitrile and the solution was heated to reflux.
After refluxing overnight, the solvent was partially removed
under reduced pressure and the resulting solid was filtered.
The solid was taken up in 250 mL of ethanol and 75 mL of
concentrated HCl was slowly added. The solution was refluxed
for 4 h and the solution was concentrated under reduced
pressure. The residue was diluted with EtOAc and washed
with 1 N aqueous NaOH and brine, and dried (MgSO4). The
solvent was removed under reduced pressure to afford 6.66 g
(48%) of 14 as a white solid: 1H NMR (CDCl3) δ 7.47 (s, 2H),
5.52 (s, 1H), 3.52 (br s, 1H), 2.61 (q, 2H, J ) 7.7 Hz), 1.25
(t, 3H, J ) 7.7 Hz); MS (m/z) 290 (M + H)+.
5-Amino-3-ethyl-4-sulfonamido-1-(2,4,6-trichlorophen-
yl)pyrazole (15). Compound 14 (4.45 g, 15.3 mmol) was added
to 7.0 mL of chlorosulfonic acid at 0 °C in portions. After
stirring for 1 h the solution was heated on a steam bath for
10 min. The mixture was poured into ice/water and the
resulting solid was filtered and washed well with water. The
solid was taken up in 100 mL of THF at 0 °C and the solution
was saturated with ammonia. After stirring for 2 days, the
solvent was removed under reduced pressure and the residue
was chromatographed (silica gel, 5 to 10% methanol/CH2Cl2)
to give 4.25 g (75%) of 15 as a solid: 1H NMR (CDCl3) δ 7.51
(s, 2H), 4.80 (br s, 4H), 2.84 (q, J ) 7.7 Hz, 2H), 1.33 (t, J )
7.7 Hz, 3H); MS (m/z) 369 (M + H)+.
3-Ethyl-6-methyl-1-(2,4,6-trichlorophenyl)-4-thia-1,2,5,7-
tetraazaindene 4,4-dioxide (16). To a solution of 100 mg
(0.27 mmol) of 15 in 4 mL of ethanol was added 166 mg (2.0
mmol) of ethyl acetate and 0.5 mL of 21% sodium ethoxide in
ethanol. The solution was heated to reflux. After refluxing for
2 days, the mixture was diluted with EtOAc, washed with
water and brine, and dried (MgSO4). The solvent was removed
under reduced pressure and the residue was chromatographed
(silica gel, 10% methanol/CH2Cl2) to give 7 mg (7%) of 16 as a
solid: 1H NMR(CD3OD) δ 7.81 (s, 2H), 2.86 (q, 2H, J ) 7.7
Hz), 2.29 (s, 3H), 1.36 (t, 3H, J ) 7.7 Hz); HRMS calcd for
C13H12N4O2Cl3S (M + H)+ 392.9747, found 392.9766.
6-Methyl-3-n-propyl-1-(2,4,6-trichlorophenyl)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-4-one (10h): yield 13%;
1
mp 244-246 °C; H NMR (DMSO-d6) δ 12.3 (br s, 1H), 7.99
(s, 2H), 2.80-2.88 (m, 2H), 2.29 (s, 3H), 1.69-1.83 (m, 2H),
0.89 (t, 3H, J ) 7.4 Hz); MS m/z calcd for C15H13N4OCl3 (M)+
370.0155, found 370.0157.
3-Ethyl-6-methyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-
1H-pyrazolo[3,4-d]pyrimidin-4-one (10i): yield 78%; 1H
NMR (DMSO-d6) δ 12.31 (br s, 1H), 7.99 (s, 2H), 2.88(q, 2H,
J ) 7.3 Hz), 2.50 (s, 3H), 1.27 (t, 3H, J ) 7.3 Hz). MS m/z
calcd for C14H11N4OCl3 (M)+ 355.9998, found 355.9988. Anal.
(C14H11N4OCl3) C, H, N, Cl.
3,6-Dimethyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (10j): yield 81%; mp >300
1
°C; H NMR (DMSO-d6) δ 12.26 (br s, 1H), 7.96 (s, 2H), 2.45
(s, 3H), 2.26 (s, 3H); MS m/z calcd for C13H9N4OCl3 (M)+
341.9842, found 341.9840. Anal. (C13H9N4OCl3) C, H, N, Cl.
6-Methyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-pyra-
zolo[3,4-d]pyrimidin-4-one (10k): yield 47%. 1H NMR
(CDCl3) δ 12.2 (bs, 1H), 8.25 (s, 2H), 7.3 (s, 1H), 2.6 (s, 3H);
MS m/z calcd for C12H8N4OCl3 (M + H)+ 328.9764, found:
328.9778.
3-(Hydroxymethyl)-6-methyl-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (10l): yield
1
58%. H NMR (DMSO-d6) δ 12.40 (s, 1H), 7.97 (s, 2H), 5.26-
5.37 (m, 1H), 4.68 (br s, 2H), 2.79 (t, 2H, J ) 7.5 Hz), 2.28 (s,
3H); MS (m/z) 357 (M - H)-.
3-(2-Hydroxyethyl)-6-methyl-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (10m):
1
yield 66%; H NMR (DMSO-d6) δ 10.54 (s, 1H), 7.97 (s, 2H),
4.81 (t, 1H, J ) 5 Hz), 3.64-3.68 (m, 2H), 2.79 (t, 2H, J ) 7.5
Hz), 1.96 (s, 3H); MS (m/z) 371 (M - H)-.
5,6-Dimethyl-3-ethyl-1-(2,4,6-trichlorophenyl)-4,5-di-
hydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (11). To a stirred
solution of 250 mg (0.70 mmol) of 10i in 10 mL of N,N-
dimethylacetamide were added 130 mg (0.70 mmol) of methyl
p-toluenesulfonate and 113 mg (0.82 mmol) of potassium
carbonate, and the mixture was heated to 100 °C. After stirring
overnight, the solvent was removed under reduced pressure
and the residue was diluted with water and extracted with
EtOAc. The combined organic layers were washed with water
and dried (MgSO4). The solvent was removed under reduced
pressure and the residue was chromatographed (silica gel,
2.5% methanol/CH2Cl2) to give 115 mg (44%) of 11 as a white
powder: 1H NMR(CDCl3) δ 7.50 (s, 2H), 3.58 (s, 3H), 3.05(q,
J ) 7.7 Hz), 2.53 (s, 3H), 1.39 (t, J ) 7.7 Hz, 3H); MS (m/z)
371 (M + H)+. Anal. (C15H13N4OCl3) C, H, N, Cl.
3-Ethyl-4-methoxy-6-methyl-1-(2,4,6-trichlorophenyl)-
1H-pyrazolo[3,4-d]pyrimidine (12b). A solution of 100 mg
(0.28 mmol) of 10i in 1 mL of POCl3 was stirred for 30 min at
reflux. The solution was cooled and poured onto ice, and
resulting white solid was then stirred vigorously for a few
minutes. The mixture was filtered, and the product was rinsed
with water and air-dried. The white solid was suspended in
1:3 ether-hexanes and filtered. The filtrate was concentrated
under reduced pressure and suspended in 2 mL of MeOH. This
suspension was treated with 0.05 mL of a 4.4 M solution of
NaOMe in MeOH, warmed to reflux, and stirred for 30 min.
The mixture was cooled to ambient temperature, and 6 mL of
water was added. The white solid was filtered, rinsed with
water then hexanes, and air-dried to afford 34 mg (35%) of
12b as a white solid: mp 154-158 °C; 1H NMR (DMSO-d6) δ
Representative Procedures for the Preparation of
3-(Methylthio)-1-(2,4,6-tichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-ones (17) and 3-Ethyl-1-(2,4,6-
trichlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrim-
idin-4-ones (18 and 19) from 5-amino-4-carboxamido-1-
(2,4,6-trichlorophenyl)pyrazoles (3w and 8i): 6-Benzyl-
3-ethyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (18i). To a stirred solution
of 167 mg (0.50 mmol) of 8i in 6 mL of absolute ethanol was

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5907
added 0.43 mL (3.0 mmol) of methyl phenylacetate followed
by 1.13 mL (3.0 mmol) of 2.7 M NaOEt in ethanol. The solution
was stirred for 18 h at reflux, cooled slightly, and quenched
with 5 mL of 10% aqueous HOAc. The mixture was cooled,
filtered, rinsed with 1:1 MeOH-water and then 3:1 hexanes-
ether, and air-dried to afford 192 mg (88%) of 18i as an off-
white solid: mp 240-242 °C; 1H NMR (DMSO-d6) δ 12.50
(br s, 1H), 7.99 (s, 2H), 7.15-7.30 (m, 5H), 3.85 (s, 2H), 2.83
(q, 2H, J ) 7.5 Hz), 1.23 (t, 3H, J ) 7.5 Hz); MS (m/z) 431
(M - H)-. Anal. (C20H15N4OCl3) C, H, N, Cl.
6-(5-Hydroxypentyl)-3-(methylthio)-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
1
one (17k): yield 80%; mp 193-197 °C; H NMR (DMSO-d6)
δ 12.35 (br s, 1H), 7.97 (s, 2H), 4.29 (t, 1H, J ) 5.3 Hz), 3.24-
3.34 (m, 2H), 2.43-2.54 (m, 2H), 1.49-1.61 (m, 2H), 1.17-
1.40 (m, 4H); MS m/z calcd for C17H18N4SO2Cl3 (M + H)+
447.0216, found 447.0198.
6-Fluoromethyl-3-(methylthio)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (17l): yield
81%; mp 248-250 °C; 1H NMR (DMSO-d6) δ 12.74 (br s, 1H),
7.99 (s, 2H), 5.25 (d, 2H, J ) 46.2 Hz), 2.50 (s, 3H); MS (m/z)
391 (M - H)-. Anal. (C13H8N4OSFCl3) C, H, N, S, Cl.
3-(Methylthio)-1-(2,4,6-trichlorophenyl)-6-trifluoro-
methyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
(17m): yield 100%; mp 228-230 °C; 1H NMR (DMSO-d6) δ
8.06 (s, 2H), 2.56 (s, 3H); MS m/z calcd for C13H7N4SOF3Cl3
(M + H)+ 428.9358, found 428.9354. Anal. (C13H6N4OSF3Cl3)
C, H, N, S, Cl.
6-Fur-2-yl-3-(methylthio)-1-(2,4,6-trichlorophenyl)-4,5-
dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (17n): yield
55%. 1H NMR (DMSO-d6) δ 12.65 (br s, 1H), 8.05 (s, 2H), 7.98
(d, 1H, J ) 1 Hz), 7.71 (d, 1H, J ) 6 Hz), 6.73-6.77 (m, 1H),
2.57 (s, 3H); MS m/z calcd for C16H10N4SO2Cl3 (M + H)+
426.9604, found 426.9605.
3-(Methylthio)-6-(thien-2-ylmethyl)-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one (17o): yield 83%; mp 218-222 °C; 1H NMR (DMSO-d6) δ
12.69 (br s, 1H), 8.03 (s, 2H), 7.37-7.42 (m, 1H), 6.90-6.99
(m, 2H), 4.10 (s, 2H), 2.52 (s, 3H); MS m/z calcd for C17H11N4-
OS2Cl3 (M + H)+ 456.9490, found 456.9518. Anal. (C16H9N4-
OS2Cl3) C, H, N, S, Cl.
3-Methylthio-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (17a): yield 59%; mp >300
1
°C; H NMR (DMSO-d6) δ 12.53 (br s, 1H), 8.10 (s, 1H), 8.03
(s, 2H), 2.53 (s, 3H); MS m/z calcd for C12H7N4SOCl3
(M+) 359.9406, found 359.9399. Anal. (C12H7N4SOCl3) C, H,
N, Cl, S.
6-Isopropyl-3-(methylthio)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (17b): yield
59%; mp 238-240 °C; 1H NMR (CDCl3) δ 10.90 (br s, 1H), 7.51
(s, 2H), 2.88-3.01 (m, 1H), 2.64 (s, 3H), 1.33 (d, 6H, J ) 7.2
Hz); MS m/z calcd for C15H13N4SOCl3 (M+) 401.9876, found
401.9894. Anal. (C15H13N4OSCl3) C, H, N, S, Cl.
3-(Methylthio)-6-propyl-1-(2,4,6-trichlorophenyl)-4,5-
dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (17c): yield
1
1
52%; mp 236-241°C; H NMR (CDCl3) δ H NMR (CDCl3) d
12.40 (br s, 1H), 8.02 (s, 2H), 2.47-2.59 (m, 5H), 1.56-1.67
(m, 2H), 0.86 (t, 3H, J ) 7.5 Hz); MS m/z calcd for C15H13N4-
SOCl3 (M+) 408.9876, found 401.9873. Anal. (C15H13N4OSCl3)
C, H, N, S, Cl.
6-Cyclopropyl-3-(methylthio)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (17d): yield
1
35%; mp 250-252 °C; H NMR (DMSO-d6) δ 12.60 (br s, not
3-(Methylthio)-6-benzyl-1-(2,4,6-trichlorophenyl)-4,5-
dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (17p): yield
84%; mp 238-240 °C; 1H NMR (DMSO-d6) δ 12.63 (br s, 1H),
8.00 (s, 2H), 7.20-7.29 (m, 5H), 3.87 (s, 2H), 2.51 (s, 3H); MS
(m/z) 451 (M + H)+. Anal. (C19H13N4SOCl3) C, H, N.
integrated), 7.99 (s, 2H), 1.97-2.03 (m, 1H), 2.51 (s, integrates
with solvent), 0.99-1.08 (m, 2H), 0.84-0.91 (m, 2H); MS m/z
calcd for C13H10N4SOCl (M+) 399.9719, found 399.9727. Anal.
(C15H11N4OSCl3) C, H, N, S, Cl.
6-(N-(tert-Butoxycarbonyl)-N-methylaminomethyl)-3-
(methylthio)-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (17e): yield 100%; 1H NMR
(CDCl3) δ 10.05 (br s, not integrated), 7.51 (s, 2H), 4.26 (q,
2H), 2.92 (br s, 3H), 2.63 (s, 3H), 1.45 (br s, 9H); MS m/z calcd
for C19H21N5SO3Cl3 (M + H)+ 504.0431, found 504.0435. Anal.
(C19H20N5SO3Cl3) C, H, N, S, Cl.
6-(2-(N-(tert-Butoxycarbonyl)-N-methylamino)ethyl)-
3-(methylthio)-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (17f): yield 69%; mp 201-
202 °C; 1H NMR (DMSO-d6) δ 12.40-12.52 (br s, 1H), 7.96 (s,
2H), 3.40-3.53 (m, 2H), 3.65-3.74 (m, 2H), 2.64 (s, 3H), 2.49
(s, 3H), 1.16-1.32 (m, 9H). Anal. (C20H22N5O3SCl3) C, H, N,
S, Cl.
3-Ethyl-6-isobutyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-
1H-pyrazolo[3,4-d]pyrimidin-4-one (18a): yield 36%; mp
1
219-222 °C; H NMR (DMSO-d6) δ 12.24 (br s, 1H), 7.96 (s,
2H), 2.84 (q, 2H, J ) 7.6 Hz), 2.38 (d, 2H, J ) 7.3 Hz), 1.89-
2.03 (m, 1H), 1.25 (t, 3H, J ) 7.5 Hz), 0.81 (d, 6H, J ) 6.6
Hz); MS (m/z) 399 (M + H)+. Anal. (C17H17N4OCl3) C, H, N,
Cl.
3, 6-Diethyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one (18b): yield 84%; mp 202-
1
203 °C; H NMR (DMSO-d6) δ 12.25 (br s, 1H), 7.97 (s, 2H),
2.85 (q, 2H, J ) 7.4 Hz), 2.51 (q, 2H, J ) 7.4 Hz), 1.24 (t, 3H,
J ) 7.4 Hz), 1.08 (t, 3H, J ) 7.4 Hz); MS m/z calcd for
C15H13N4SOCl3 (M+) 370.0155, found 370.0141. Anal. (C15H13N4-
OCl3) C, H, N, Cl.
(()-6-(1-Hydroxyethyl)-3-(methylthio)-1-(2,4,6-trichlo-
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one (17g): yield 66%; 1H NMR (DMSO-d6) δ 12.49 (br s, 1H),
8.02 (s, 2H), 5.76 (d, 1H), 4.47-4.59 (m, 1H), 2.57 (s, 3H), 1.30
(d, 3H); MS m/z calcd for C14H11N4SO2Cl3 (M)+ 403.9668,
found 403.9653. Anal. (C14H11N4O2SCl3) C, H, N, S, Cl.
3-(Methylthio)-6-methylthiomethyl-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
3-Ethyl-6-(2-methylthioethyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (18c): yield
80%; mp 196-198 °C; 1H NMR (DMSO-d6) δ 12.36 (br s, 1H),
8.00 (s, 2H), 2.82-2.93 (m, 4H), 2.75 (t, 2H), 2.03 (s, 3H), 1.28
(t, 3H, J ) 7.5 Hz); MS (m/z) 415 (M - H)-. Anal. (C16H15N4-
OSCl3) C, H, N, S, Cl.
3-Ethyl-6-(4-hydroxybutyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (18d): yield
80%. 1H NMR (CDCl3) δ 11.57 (br s, 1H), 7.51 (s, 2H), 3.73 (q,
2H, J ) 6.0 Hz), 3.05 (q, 2H, J ) 7.6 Hz), 2.78 (t, 2H, J ) 7.5
Hz), 1.82-1.97 (m, 2H), 1.58-1.69 (m, 2H), 1.42 (t, 3H, J )
7.7 Hz); MS m/z calcd for C17H18N4O2Cl3 (M+) 415.0495, found
415.0519. Anal. (C17H17N4O2Cl3) C, H, N, Cl.
6-(3,4-Dimethoxyphenyl)-3-ethyl-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one (18e): yield 14%; mp 262-263 °C; 1H NMR (DMSO-d6) δ
12.44 (br s, 1H), 7.99 (s, 2H), 7.63 (br s, 1H), 7.57 (d, 1H), 7.03
(d, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 2.89 (q, 2H, J ) 7.5 Hz),
1.28 (t, 3H, J ) 7.5 Hz); MS (m/z) 477 (M - H)-.
3-Ethyl-6-(2-phenylethyl)-1-(2,4,6-trichlorophenyl)-4,5-
dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (18g): yield
89%; mp 228-231 °C; 1H NMR (DMSO-d6) δ 12.30 (br s, <1H),
7.97 (s, 2H), 7.08-7.22 (m, 5H), 2.78-2.88 (m, 6H), 1.24 (s,
1
one (17h): yield 52%; mp 219-222 °C; H NMR (DMSO-d6)
δ 12.50 (br s, 1H), 8.02 (s, 2H), 3.50 (s, 2H), 2.54 (s, 3H), 2.05
(s, 3H); MS m/z calcd for C14H11N4S2OCl3 (M)+ 419.9440, found
419.9448. Anal. (C14H11N4OS2Cl3) C, H, N, S, Cl.
6-Methoxycarbonyl-3-(methylthio)-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one (17i): yield 20%; mp 214-218 °C; 1H NMR (DMSO-d6) δ
12.98 (br s, 1H), 8.06 (s, 2H), 3.88 (s, 3H), 2.55 (s, 3H); MS
m/z calcd for C143H9N4SO3Cl3 (M + H)+ 420.9539, found
420.9503. Anal. (C14H9N4O3SCl3) C, H, N, S, Cl.
6-(3-Hydroxypropyl)-3-(methylthio)-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one (17j): yield 81%; mp 219-220 °C; 1H NMR (DMSO-d6) δ
7.97 (s, 2H), 3.31-3.37 (m, 2H), 2.56 (t, 2H, J ) 7.6 Hz), 2.48
(s, 3H), 1.63-1.72 (m, 2H); MS (m/z) 417 (M - H)-. Anal.
(C15H13N4O2SCl3) C, H, N, S, Cl.

5908 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
3H, J ) 7.5 Hz); MS m/z calcd for C21H17N4OCl3 (M+)
Hz), 3.90 (s, 2H), 2.83 (q, 2H, J ) 7.4 Hz), 1.23 (t, 3H, J ) 7.5
446.0468, found 446.0466. Anal. (C21H17N4OCl3) C, H, N, Cl.
Hz); MS (m/z) 432 (M - H)-. Anal. (C19H14N5OCl3) C, H, N,
Cl.
3-Ethyl-6-(2-(imidazol-4-yl)ethyl)-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
3-Ethyl-6-(pyrid-4-ylmethyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19j): yield
92%; mp 277-280 °C; 1H NMR (DMSO-d6) δ 12.58 (br s, 1H),
8.43 (d, 2H, J ) 4.0 Hz), 7.96 (s, 2H), 7.22 (d, 2H, J ) 5.8 Hz),
3.91 (s, 2H), 2.84 (q, 2H, J ) 7.4 Hz), 1.24 (t, 3H, J ) 7.5 Hz);
MS (m/z) 432 (M - H)-.
1
one (18h): yield 55%; mp 271-275 °C; H NMR (DMSO-d6)
δ 7.92 (s, 2H), 7.46 (s, 1H), 6.65 (s, 1H), 2.76-2.88 (m, 6H),
1.25 (t, 3H, J ) 7.5 Hz); MS m/z calcd for C21H17N4OCl3 (M+)
436.0373, found 436.0367. Anal. (C18H15N6OCl3‚1/4H2O) C, H,
N, Cl.
3-Ethyl-6-(2-methoxybenzyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19a): yield
91%; mp 188-191 °C; 1H NMR (DMSO-d6) δ 12.31 (br s, 1H),
7.91 (s, 2H), 7.15-7.22 (m, 1H), 6.89-6.98 (m, 2H), 6.80 (dd,
1H, J ) 7.4, 0.9 Hz), 3.82 (s, 2H), 3.67 (s, 3H), 2.85 (q, 2H,
J ) 7.4 Hz), 1.25 (t, 3H, J ) 7.5 Hz); MS (m/z) 461 (M - H)-.
Anal. (C21H17N4O2Cl3) C, H, N, Cl.
6-(4-Aminobenzyl)-3-ethyl-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19k): yield
89%; mp 256-259 °C; 1H NMR (DMSO-d6) δ 12.34 (br s, 1H),
7.97 (s, 2H), 6.89 (d, 2H, J ) 8.4 Hz), 6.41 (d, 2H, J ) 8.4 Hz),
4.94 (s, 2H), 3.61 (s, 2H), 2.82 (q, 2H, J ) 7.6 Hz), 1.23 (t,
3H, J ) 7.5 Hz); MS (m/z) 446 (M - H)-. Anal. (C20H16N5OCl3‚
1/4THF) C, H, N, Cl.
3-Ethyl-6-(2-(hydroxymethyl)benzyl)-1-(2,4,6-trichlo-
3-Ethyl-6-(4-methoxybenzyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19l): yield
81%; mp 261-262 °C; 1H NMR (DMSO-d6) δ 12.43 (br s, 1H),
7.97 (s, 2H), 7.17 (d, 2H, J ) 8.7 Hz), 6.81 (d, 2H, J ) 8.8 Hz),
3.76 (s, 2H), 3.66 (s, 3H), 2.83 (q, 2H, J ) 7.4 Hz), 1.23 (t, 3H,
J ) 7.5 Hz); MS (m/z) 463 (M + H)+. Anal. (C21H17N4O2Cl3) C,
H, N, Cl.
3-Ethyl-6-(4-hydroxybenzyl)-1-(2,4,6-trichlorophen-
yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
(19m): yield 58%; mp 288-291 °C; 1H NMR (DMSO-d6) δ
12.40 (br s, 1H), 9.29 (s, 1H), 7.97 (s, 2H), 7.04 (d, 2H, J ) 8.4
Hz), 6.62 (d, 2H, J ) 8.4 Hz), 3.70 (s, 2H), 2.83 (q, 2H, J ) 7.5
Hz), 1.23 (t, 3H, J ) 7.5 Hz); MS m/z calcd for C20H15N4O2Cl3
(M)+ 448.0261, found 448.0248. Anal. (C20H15N4O2Cl3) C, H,
N, Cl.
6-(4-Bromobenzyl)-3-ethyl-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19n): yield
96%; mp 296-299 °C; 1H NMR (DMSO-d6) δ 12.52 (br s, 1H),
7.96 (s, 2H), 7.45 (d, 2H, J ) 8.4 Hz), 7.19 (d, 2H, J ) 8.5 Hz),
3.83 (s, 2H), 2.85 (q, 2H, J ) 7.6 Hz), 1.23 (t, 3H, J ) 7.5 Hz).
Anal. (C20H14N4OBr Cl3) C, H, N; MS (m/z) 509 (M - H)-.
6-(4-(N,N-Dimethylamino)benzyl)-3-ethyl-1-(2,4,6-tri-
chlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
4-one (19o): yield 80%; mp 238-241 °C; 1H NMR (DMSO-
d6) δ 12.38 (br s, 1H), 7.98 (s, 2H), 7.07 (d, 2H, J ) 8.8 Hz),
6.59 (d, 2H, J ) 8.8 Hz), 3.68 (s, 2H), 2.82 (q, 2H, J ) 7.6 Hz),
2.79 (s, 6H), 1.23 (t, 3H, J ) 7.5 Hz); MS m/z calcd for
C17H18N4O2Cl3 (M + H)+ 476.0771, found 476.0812. Anal.
(C22H20N5OCl3) C, H, N, Cl.
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
1
one (19b): yield 88%; mp 220-222 °C; H NMR (DMSO-d6)
δ 7.95 (s, 2H), 7.33 (dd, 1H, J ) 6.9, 1.8 Hz), 7.06-7.22 (m,
3H), 5.36 (br s, 1H), 4.53 (s, 2H), 3.91 (s, 2H), 2.83 (q, 2H, J )
7.6 Hz), 1.24 (t, 3H, J ) 7.5 Hz); MS m/z calcd for C21H17N4O2-
Cl3 (M)+ 462.0417, found 462.0404. Anal. (C21H17N4O2Cl3) C,
H, N, Cl.
3-Ethyl-6-(pyrid-2-ylmethyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19c): yield
87%; mp 164-169 °C; 1H NMR (DMSO-d6) δ 12.46 (br s, 1H),
8.40-8.43 (m, 1H), 7.91 (s, 2H), 7.69 (ddd, 1H, J ) 7.7, 7.6,
1.8 Hz), 7.26 (d, 1H, J ) 8.0 Hz), 7.18-7.24 (m, 1H), 4.08 (s,
2H), 2.85 (q, 2H, J ) 7.4 Hz), 1.25 (t, 3H, J ) 7.5 Hz); MS
m/z calcd for C19H14N5OCl3 (M+) 433.0264, found 433.0255.
Anal. (C19H14N5OCl3‚1/4HOAc) C, H, N, Cl.
6-(3-Amino-2-methylbenzyl)-3-ethyl-1-(2,4,6-trichloro-
phenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
1
one (19d): yield 89%; mp 242-243 °C; H NMR (DMSO-d6)
δ 12.26 (br s, 1H), 7.94 (s, 2H), 6.74 (d, 1H, J ) 7.7 Hz), 6.47
(d, 1H, J ) 7.7 Hz), 6.30 (d, 1H, J ) 7.4 Hz), 4.76 (br s, 1-2H),
3.78 (s, 2H), 2.84 (q, 2H, J ) 7.6 Hz), 1.92 (s, 3H), 1.24 (t, 3H,
J ) 7.5 Hz); MS (m/z) 462 (M + H)+. Anal. (C21H18N5OCl3) C,
H, N, Cl.
3-Ethyl-6-(3-methylbenzyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19e): yield
95%; mp 238-240 °C; 1H NMR (DMSO-d6) δ 12.47 (br s, 1H),
7.98 (s, 2H), 7.13 (d, 1H, J ) 7.6 Hz), 7.07 (br s, 1H), 6.97-
7.04 (m, 2H), 3.80 (s, 2H), 2.83 (q, 2H, J ) 7.6 Hz), 2.02 (s,
3H), 1.23 (t, 3H, J ) 7.5 Hz, CH3). Anal. (C21H17N4OCl3) C, H,
N, Cl.
6-(3-(Ethoxycarbonylmethyl)benzyl)-3-ethyl-1-(2,4,6-
trichlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimi-
din-4-one (19f): yield 42%; mp 168-169 °C; 1H NMR (CDCl3)
δ 10.41 (br s, 1H), 7.54 (s, 2H), 7.21-7.34 (m, 4H), 4.13 (q,
2H, J ) 7.1 Hz), 3.98 (s, 2H), 3.58 (s, 2H), 3.05 (q, 2H, J ) 7.4
Hz), 1.42 (t, 3H, J ) 7.5 Hz), 1.23 (t, 3H, J ) 7.1 Hz); MS
(m/z) 517 (M - H)-. Anal. (C24H21N4O3Cl3) C, H, N, Cl.
3-Ethyl-6-(3-methoxybenzyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19g): yield
76%; mp 235-238 °C; 1H NMR (DMSO-d6) δ 12.47 (br s, 1H),
7.97 (s, 2H), 7.16 (t, 1H, J ) 7.9 Hz), 6.73-6.85 (m, 3H), 3.81
(s, 2H), 3.66 (s, 3H), 2.83 (q, 2H, J ) 7.5 Hz), 1.23 (t, 3H, J )
7.5 Hz); MS (m/z) 461 (M - H)-. Anal. (C21H17N4O2Cl3) C, H,
N, Cl.
6-(3-Aminobenzyl)-3-ethyl-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19h): yield
69%; mp 236-240 °C; 1H NMR (DMSO-d6) δ 12.41 (br s, 1H),
7.96 (s, 2H), 6.84-6.89 (m, 1H), 6.33-6.38 (m, 3H), 4.97 (br s,
1H), 3.67 (s, 2H), 2.83 (q, 2H, J ) 7.6 Hz), 1.243 (t, 3H, J )
7.5 Hz); MS m/z calcd for C20H16N5OCl3 (M+) 447.0420, found
447.0418.
3-Ethyl-6-(4-methoxy-3-methylbenzyl)-1-(2,4,6-trichlo-
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
1
one (19p): yield 89%; mp 245-247 °C; H NMR (DMSO-d6)
δ 12.40 (br s, 1H), 7.98 (s, 2H), 6.99-7.08 (m, 2H), 6.80 (d,
2H, J ) 9.2 Hz), 3.72 (s, 2H), 3.69 (s, 3H), 2.82 (q, 2H, J )
7.4 Hz), 2.04 (s, 3H), 1.23 (t, 3H, J ) 7.5 Hz); MS (m/z) 477
(M + H)+. Anal. (C22H19N4O2Cl3) C, H, N, Cl.
3-Ethyl-6-(3-methoxy-4-methylbenzyl)-1-(2,4,6-trichlo-
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
1
one (19q): yield 97%; mp 263-265 °C; H NMR (DMSO-d6)
δ 12.44 (br s, 1H), 7.97 (s, 2H), 6.99 (d, 1H, J ) 7.6 Hz), 6.85
(s, 1H), 6.71 (d, 1H, J ) 7.3 Hz), 3.78 (s, 2H), 3.68 (s, 3H),
2.83 (q, 2H, J ) 7.6 Hz), 2.03 (s, 3H), 1.23 (t, 3H, J ) 7.5 Hz);
MS (m/z) 477 (M + H)+.
3-Ethyl-6-(4-hydroxy-3-methoxybenzyl)-1-(2,4,6-trichlo-
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one (19r): yield 88%; mp 260-280 °C; 1H NMR (DMSO-d6) δ
12.44 (br s, 1H), 8.87 (s, 1H), 7.97 (s, 2H), 6.82 (s, 1H), 6.59-
6.65 (m, 2H), 3.70 (s, 2H), 3.65 (s, 3H), 2.82 (q, 2H, J )
7.4 Hz), 1.23 (t, 3H, J ) 7.5 Hz). Anal. (C21H17N4O3Cl3) C, H,
N, Cl.
6-(3,4-Dimethoxybenzyl)-3-ethyl-1-(2,4,6-trichlorophen-
yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
(19s): yield 57%; mp 220-222 °C; 1H NMR (DMSO-d6) δ 12.43
(br s, 1H), 7.97 (s, 2H), 6.88 (d, 1H, J ) 1.5 Hz), 6.74-6.85
(m, 2H), 3.74 (s, 2H), 3.66 (s, 3H), 3.64 (s, 3H), 2.82 (q, 2H,
J ) 7.4 Hz), 1.23 (t, 3H, J ) 7.5 Hz). Anal. (C22H19N4O3Cl3) C,
H, N, Cl.
3-Ethyl-6-(pyrid-3-ylmethyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (19i): yield
97%; mp 257-260 °C; 1H NMR (DMSO-d6) δ 12.56 (br s, 1H),
8.46 (d, 1H, J ) 1.5 Hz), 8.40 (dd, 1H, J ) 4.8, 1.5 Hz), 7.96
(s, 2H), 7.62(dt, 1H, J ) 8.0, 2.0 Hz), 7.28 (dd, 1H, J ) 7.7, 4.7

Inhibitors of Cyclin-Dependent Kinases
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5909
6-(3,5-Dihydroxybenzyl)-3-ethyl-1-(2,4,6-trichlorophen-
yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
(19t): 1H NMR (DMSO-d6) δ 12.41 (br s, 1H), 9.15 (br s), 7.96
(s, 2H), 5.99-6.08 (m, 3H), 3.64 (s, 2H), 2.84 (q, 2H, J )
7.4 Hz), 1.24 (t, 3H, J ) 7.5 Hz). Anal. (C20H15N4O3Cl3) C, H,
N, Cl.
Hz), 3.83 (s, 2H), 2.88 (q, 2H, J ) 7.6 Hz), 1.29 (t, 3H, J ) 7.5
Hz); MS (m/z) 449 (M + H)+. Anal. (C20H15N4O2Cl3‚1/3H2O) C,
H, N, Cl.
3-Ethyl-6-(3-hydroxybenzyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (22g): yield
91%; 1H NMR (DMSO-d6) δ 12.45 (br s, 1H), 9.33 (s, 1H), 7.96
(s, 2H), 7.03 (t, 1H, J ) 7.7 Hz), 6.56-6.65 (m, 3H), 3.75 (s,
2H), 2.83 (q, 2H, J ) 7.5 Hz), 1.24 (t, 3H, J ) 7.5 Hz); MS
(m/z) 447 (M - H)-. Anal. (C20H15N4O2Cl3‚1/2H2O) C, H, N, Cl.
3-Ethyl-6-(4-hydroxy-3-methylbenzyl)-1-(2,4,6-trichlo-
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
3-Ethyl-6-(3-hydroxy-4-methoxybenzyl)-1-(2,4,6-trichlo-
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
1
one (19u): yield 65%; mp 241-244 °C; H NMR (DMSO-d6)
δ 12.40 (br s), 8.89 (s, 1H), 7.97 (s, 2H), 6.78 (d, 1H, J ) 8.0
Hz), 6.66 (br s, 1H), 6.02 (br d, 1H, J ) 8.0 Hz), 3.67 (s, 5H),
2.83 (q, 2H, J ) 7.4 Hz), 1.23 (t, 3H, J ) 7.5 Hz); MS (m/z)
479 (M + H)+. Anal. (C21H17N4O3Cl3) C, H, N, Cl.
1
one (22p): yield 91%; mp 260-263 °C; H NMR (DMSO-d6)
δ 12.37 (br s, 1H), 9.17 (s, 1H), 7.98 (s, 2H), 6.96 (br s, 1H),
6.85 (d, 1H, J ) 8.0 Hz), 6.62 (d, 1H, J ) 8.1 Hz), 3.67 (s, 2H),
2.82 (q, 2H, J ) 7.5 Hz), 2.01 (s, 3H), 1.23 (t, 3H, J ) 7.5 Hz);
MS (m/z) 461 (M - H)-. Anal. (C21H17N4O2Cl3) C, H, N, Cl.
3-Ethyl-6-(3-hydroxy-4-methylbenzyl)-1-(2,4,6-trichlo-
rophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-
one (22q): yield 90%. 1H NMR (DMSO-d6) δ 12.49 (br s, 1H),
9.23 (s, 1H), 8.00 (s, 2H), 6.94 (d, 1H, J ) 7.7 Hz), 6.64 (s,
1H), 6.60 (d, 1H, J ) 7.7 Hz), 3.75 (s, 2H), 2.87 (q, 2H, J ) 7.4
Hz), 2.01 (s, 3H), 1.28 (t, 3H, J ) 7.5 Hz); MS m/z calcd for
C21H17N4O2Cl3 (M)+ 462.0417, found 462.0404.
6-(N-Methylaminomethyl)-3-(methylthio)-1-(2,4,6-tri-
chlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
4-one hydrochloride (20e). To a solution of 5 mL of 4 N HCl
in dioxane was added 100 mg (0.20 mmol) of 17e. The solution
was stirred for 1 h and frozen in a dry ice/acetone bath.
Lyophilization afforded 87 mg (100%) of 20e as a white
powder: 1H NMR (DMSO-d6) δ 12.80-12.88 (br s, 1H), 8.96-
9.06 (br s, 2H), 8.01 (s, 2H), 4.12-4.20 (m, 2H), 2.53-2.60 (m,
3H), 2.51 (s, 3H); MS m/z calcd for C14H12N5SO3Cl3 (M)+
402.9828, found 402.9799.
6-(2-(N-Methylamino)ethyl)-3-(methylthio)-1-(2,4,6-tri-
chlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
4-one hydrochloride (20f) was prepared from 17f by the
same procedure used to prepare 21e: yield 100%; mp 232-
233 °C; 1H NMR (DMSO-d6) δ 12.55 (br s, 1H), 8.60-8.72 (m,
2), 7.99 (s, 2H), 3.37 (s, 3H), 3.08-3.21 (m, 2H), 2.92-3.01 (m,
2H), 2.50 (s, 3H). Anal. (C15H15N5OSCl4) C, H, N, S, Cl.
N-(3-{[3-Ethyl-4-oxo-1-(2,4,6-trichlorophenyl)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-6-yl]methyl}phenyl)-
methanesulfonamide (23). To a stirred susension of 47 mg
(0.10 mmol) of 19h in 2.5 mL of 2:2:1 CH2Cl2-ether-pyridine
was added 0.02 mL (0.26 mmol) of methanesulfonyl chloride.
The mixture was stirred for 16 h at ambient temperature and
diluted with ether. The solution was washed twice with 1 N
aqueous HCl and once with brine, dried (MgSO4), and con-
centrated under reduced pressure to afford an off-white solid.
Crystallization from EtOAc-hexanes afforded 44 mg (83%) of
1-(2,4,6-Trichlorophenyl)-3-ethyl-6-vinyl-4,5-dihydro-
1H-pyrazolo[3,4-d]pyrimidin-4-one (21). To a stirred sus-
pension of 1.1 g (2.6 mmol) of 18d in 25 mL of warm CH2Cl2
was added 4 mL of DMF followed by 582 mg (3.38 mmol) of
m-CPBA in three portions over 1 h. The solution was stirred
for 1 h longer, poured into EtOAc, and washed twice with
aqueous NaHCO3 and once with brine. The solution was dried
(MgSO4) and concentrated to afford the crude sulfoxide as a
chalky solid. This material was suspended in 80 mL of toluene
and heated to reflux for 4 h. Solvent was removed under
reduced pressure, and the crude olefin was chromatographed
on silica gel (elution with 1:1 EtOAc-hexanes) to afford, after
concentration, 620 mg (67%) of 21 as a white solid: mp 202-
1
23 as a white solid: mp 246-248 °C; H NMR (DMSO-d6) δ
7.96 (s, 2H), 7.18 (t, 1H, J ) 7.9 Hz), 7.08 (s, 1H), 7.02 (d, 1H,
J ) 8.1 Hz), 6.96 (d, 1H, J ) 7.7 Hz), 3.83 (s, 2H), 2.90 (s,
3H), 2.83 (q, 2H, J ) 7.4 Hz), 1.24 (t, 3H, J ) 7.5 Hz); MS
m/z calcd for C21H19N5SO3Cl3 (M + H)+ 526.0274, found
526.0256.
Representative Procedure for the Preparation of
Anilide Derivatives 24 from Aniline 19k: 2-(Dimethyl-
amino)-N-(4-{[3-ethyl-4-oxo-1-(2,4,6-trichlorophenyl)-4,5-
dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl]methyl}-
phenyl)acetamide Hydrochloride (24d). To a stirred
susension of 70 mg (0.16 mmol) of 19k in 2 mL of CH2Cl2 was
added 0.07 mL (1.32 mmol) of Et3N followed by 52 mg (0.50
mmol) of N,N-dimethylglycine and 96 mg (0.50 mmol) of EDC.
The mixture was stirred for 24 h at ambient temperature and
filtered through a slug of silica gel (elution with 1:1 THF-
hexanes). The appropriate fractions were concentrated under
reduced pressure, redissolved in 5 mL of benzene and 0.1 mL
of 4 N HCl in dioxane, and frozen. Lyophilization afforded 70
mg (80%) of 24d as an off-white solid: mp 288-290 °C (dec);
1H NMR (DMSO-d6) δ 12.50 (br s, 1H), 10.41 (s, 1H), 10.69
(br s, 1H), 7.97 (s, 2H), 7.48 (d, 2H, J ) 8.8 Hz), 7.23 (d, 2H,
J ) 8.4 Hz), 4.09 (s, 2H), 3.82 (s, 2H), 2.83 (q, 2H, J ) 7.5 Hz),
1.23 (t, 3H, J ) 7.5 Hz); MS (m/z) 533 (M + H)+. Anal.
(C24H24N6O2Cl4) C, H, N, Cl.
1
203 °C; H NMR (DMSO-d6) δ 12.39 (br s, 1H), 7.97 (s, 2H),
6.36-6.56 (m, 2H), 5.79 (dd, 1H, J ) 10, 1 Hz), 2.85 (q, 2H,
J ) 7.4 Hz), 1.24 (t, 3H, J ) 7.4 Hz); MS (m/z) 367 (M - H)-.
Anal. (C15H11N4OCl3) C, H, N, Cl.
Representative Procedure for the Preaparation of
6-(Hydroxybenzyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrim-
idin-4-ones (22) from 6-(Methoxybenzyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-ones (19): 6-(3,4-Dihydroxy-
benzyl)-3-ethyl-1-(2,4,6-trichlorophenyl)-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-one(22s).Toastirredsuspension
of 500 mg (1.01 mmol) of 19s in 20 mL of CH2Cl2 was added
10 mL of a 1 M solution of BBr3 in CH2Cl2. The solution was
stirred for 1 h at room temperature and then cooled to 0 °C.
The reaction was quenched with 1 N aqueous HCl, poured into
water, and extracted with 2:1 EtOAc-THF. (Addition of a little
of brine and/or MeOH may be necessary to produce two clear
phases.) The organic extract was washed with half-saturated
brine and then brine. The solution was dried (MgSO4),
concentrated under reduced pressure, and chromatographed
on silica gel (elution with 1:1 hexanes-THF and then THF).
Concentration of the appropriate fractions afforded 40 mg
(93%) of 22s as an off-white, amorphous solid: 1H NMR
(DMSO-d6) δ 12.38 (br s, 1H), 8.79 (t, 1H, J ) 3.3 Hz) 7.97 (s,
2H), 6.56-6.61 (m, 2H), 6.46-6.50 (m, 1H), 3.64 (s, 2H), 2.83
(q, 2H, J ) 7.6 Hz), 1.23 (t, 3H, J ) 7.5 Hz); MS m/z calcd for
C20H15N4O3Cl3 (M)+ 464.0210, found 464.0218.
N-(4-{[3-Ethyl-4-oxo-1-(2,4,6-trichlorophenyl)-4,5-dihy-
dro-1H-pyrazolo[3,4-d]pyrimidin-6-yl]methyl}phenyl)-
acetamide (24a): yield 86%; 1H NMR (DMSO-d6) δ 12.50 (s,
1H), 9.90 (s, 1H), 8.01 (s, 1H), 7.47 (d, 2H, J ) 8.4 Hz), 7.19
(d, 2H, J ) 8.5 Hz), 3.81 (s, 2H), 2.87 (q, 2H, J ) 7.6 Hz), 2.01
(s, 3H), 1.27 (t, 3H, J ) 7.5 Hz); MS m/z calcd for C22H18N5O2-
Cl3 (M)+ 489.0526, found 489.0498.
2-(tert-Butoxycarbonylamino)-N-(4-{[3-ethyl-4-oxo-1-
(2,4,6-trichlorophenyl)-4,5-dihydro-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl]methyl}phenyl)acetamide (24b): yield 65%;
1
mp 243-244 °C; H NMR (DMSO-d6) δ 12.48 (br s, 1H), 9.87
3-Ethyl-6-(2-hydroxybenzyl)-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one (22a): yield
100%; mp 285-289 °C; 1H NMR (DMSO-d6) δ 12.28 (br s, 1H),
9.62 (s, 1H), 7.96 (s, 2H), 7.02-7.07 (m, 1H), 6.91 (d, 1H, J )
6.3 Hz), 6.78 (dd, 1H, J - 6.7, 0.7 Hz), 6.69 (dd, J ) 7.7, 7.3
(br s, 1H), 7.97 (s, 2H), 7.44 (d, 2H, J ) 8.4 Hz), 7.17 (d, 2H,
J ) 8.4 Hz), 6.99 (t, 1H, J ) 5.9 Hz), 3.78 (s, 2H), 3.64 (d, 2H,
J ) 6.2 Hz), 2.83 (q, 2H, J ) 7.5 Hz), 1.35 (s, 9H), 1.23 (t, 3H,
J ) 7.5 Hz); MS m/z calcd for C27H28N6O4Cl3 (M + H)+
605.1238, found 605.1256.

5910 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Markwalder et al.
Table 9. Crystallographic Statistics for CDK2/22s
2-Amino-N-(4-{[3-ethyl-4-oxo-1-(2,4,6-trichlorophenyl)-
4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl]methyl}-
phenyl)acetamide (24c) was prepared from 24b in the same
manner used to prepare 20e from 17e: yield 100%; mp 294-
296 °C; 1H NMR (DMSO-d6) δ 12.50 (br s, 1H), 10.41 (br s,
1H), 7.99-8.16 (m, 3H), 7.97 (s, 2H), 7.46 (d, 2H, J ) 8.5 Hz),
7.22 (d, 2H, J ) 8.4 Hz), 3.81 (s, 2H), 3.68-3.77 (m, 2H), 2.83
(q, 2H, J ) 7.6 Hz), 1.23 (t, 3H, J ) 7.5 Hz); MS (m/z) 505
(M + H)+. Anal. (C22H20N6O2Cl4) C, H, N, Cl.
Enzyme IC50 Determinations. The in vitro assays employ
cell lysates from insect cells expressing either of the kinases
and subsequently their corresponding regulatory units. The
CDK/cyclin lysate is combined in a microtiter-type plate along
with a kinase compatible buffer, 32P-labeled ATP at a concen-
tration of 50 µM, a GST-Rb fusion protein, and the test
compound at varying concentrations. The kinase reaction is
allowed to proceed with the radiolabeled ATP and then
effectively stopped by the addition of a large excess of EDTA
and unlabeled ATP. The GST-Rb labeled protein is sequestered
on a GSH-Sepharose bead suspension, washed, and resus-
pended in scintillant, and the 32P activity is detected in a
scintillation counter. The compound concentration that inhibits
50% of the kinase activity was calculated for each compound.
space group
cell
P212121
refinement resoln (Å) 10.0-1.85
a ) 72.97 R factorb
b ) 73.16
0.169
dimensions (Å)
c ) 54.26
c
max resolution (Å) 1.85
total
observations
unique
reflections
completeness (%) 78
Rfree
0.279
32.7
36 372
av protein
temp factor (Å)
20 060
av inhibitor
35.2
temp factor (Å)
rms bonds (Å)
rms angles (deg)
0.016
3.5
a
Rmerge
0.081
a Rmerge ) ∑h∑j|Ihj - Ih|∑h∑j|Ihj|, where Ihj is the jth observation
of reflection h. b R factor ) ∑h|Fobs| - |Fcalc|∑h|Fobs|, where Fobs and
Fcalc are the observed and calculated structure factor amplitudes,
c
respectively, for reflection h. Rfree ) R factor for a 9.2% subset of
reflections not used in the refinement.
the electron density maps clearly showed the location of the
inhibitor inside the ATP binding site. Iterative cycles of
refinement and model building were performed, resulting in
a final model, which includes residues 1-298, 22s, and 106
water molecules. Data and refinement statistics are presented
in Table 9.
Cellular Growth Inhibition Assay. Effects of compounds
on the growth of the following transformed cell lines were
evaluated in a colorimetric assay using sulforhodamine B
(SRB):28 HCT-116 (human colon carcinoma cell line, ATCC),
MDA-MB-468 (human mammary gland adenocarcinoma,
ATCC), NCI-H460 (human lung carcinoma, ATCC), A-498
(human kidney carcinoma, ATCC), T-47D (human mammary
duct carcinoma, ATCC), MCF 7 (human mammary gland
adenocarcinoma), DU 145 (human prostate carcinoma, ATCC),
COLO 205 (human colon adenocarcinoma, ATCC). Briefly,
exponentially growing cells were seeded in wells of a 96-well
microtiter plate at a concentration to allow for 3-5 doublings
before obtaining 85% confluence. Eighteen hours later, graded
concentrations of test compounds were added to the cell plates.
Plates were incubated for 5-6 days at 37 °C in 5% CO2 or
atmospheric CO2 (MDA-MB-468). Fifty microliters of cold 50%
TCA was gently added to each well, and plates were placed at
4 °C for 1 h. Plates were decanted, rinsed five times with cold
tap water, and allowed to air-dry. Fifty microliters of 0.4% SRB
in 1% acetic acid was added, and the plates were incubated at
room temperature for 15 min. Plates were washed four times
with 1% acetic acid and again allowed to air-dry. Finally, 150
µL of 10 mM Tris base was added, and plates were agitated
on a plate shaker for 5 min before reading optical densities at
570 nm using a BIORAD 3550 plate reader.
Purification and Crystallization of Human CDK2.
CDK2 protein was prepared and purified as described,29 except
for the addition of 10% (v/v) glycerol during the SP-sepharose
and ATP-agarose column steps. Protein was concentrated to
6 mg/mL using a Collodion concentrator against 10 mM
HEPES (pH 7.4), 15 mM NaCl. Crystals were grown at 18 °C
from sitting drops containing premixed and filtered solutions
of 3.0 mg/mL CDK2, 32.5mM HEPES (pH 7.4), 11.3 mM
sodium chloride, 12.5 mM ammonium acetate, 2 mM DTT,
2-4% PEG 4000 against 100 mM HEPES (pH 7.4), 50 mM
ammonium acetate, 2 mM DTT, 4-14% PEG 4000. Crystals
appeared in 1 day and grew for 1 week to approximately 0.25
mm × 0.25 mm in size.
Mouse Xenograft Procedure. HCT116 human colon
carcinoma cells or NCI-H460 cells were implanted subcutane-
ously at a concentration of 1 × 107 cells (0.1 mL) per animal
into the inguinal region of female nude mice. The test drugs
were administered intraperitoneally once a day, every day, for
14 days beginning 7-10 days following cell injection. On the
fifteenth day after initial drug administration, tumors were
removed and weighed, and tumor growth inhibition index (%)
was determined according to the following formula: tumor
growth inhibition index (%) ) (1 - mean net tumor weight of
experimental group/mean net tumor weight of control group)
× 100.
Acknowledgment. We thank Jim Krywko for gen-
erating models of the cyclin-bound conformation of
CDK4 and J. Gerry Everlof for performing log P
measurements.
Supporting Information Available: Table of combustion
and HPLC purity values for the final compounds. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
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