Pyrazolo[3,4-c]pyridazines as novel and selective inhibitors of cyclin-dependent kinases

Article abstract of DOI:10.1021/jm058013g

Pyrazolopyridazine 1a was identified in a high-throughput screening carried out by BASF Bioresearch Corp. (Worcester, MA) as a potent inhibitor of CDK1/cyclin B and shown to have selectivity for the CDK family. Analogues of the lead compound have been syn

Full text of DOI:10.1021/jm058013g

J. Med. Chem. 2005, 48, 6843-6854
6843
Pyrazolo[3,4-c]pyridazines as Novel and Selective Inhibitors of
Cyclin-Dependent Kinases
Miguel F. Bran˜a,*,† Mo´nica Cacho,† M. Luisa Garc´ıa,† Elena P. Mayoral,† Berta Lo´pez,†
Beatriz de Pascual-Teresa,† Ana Ramos,† Nuria Acero,# Francisco Llinares,§ Dolores Mun˜oz-Mingarro,†
Olivier Lozach,‡ and Laurent Meijer‡
Facultad de Farmacia, Universidad San Pablo CEU, Urbanizacio´n Monteprı´ncipe, 28668-Boadilla del Monte, Madrid, Spain,
and CNRS, Cell Cycle Group, UPS 2682 & UMR 2775, Station Biologique, B.P. 74, 29682 Roscoff Cedex, Bretagne, France
Received March 1, 2005
Pyrazolopyridazine 1a was identified in a high-throughput screening carried out by BASF
Bioresearch Corp. (Worcester, MA) as a potent inhibitor of CDK1/cyclin B and shown to have
selectivity for the CDK family. Analogues of the lead compound have been synthesized and
their antitumor activities have been tested. A molecular model of the complex between the
lead compound and the CDK2 ATP binding site has been built using a combination of
conformational search and automated docking techniques. The stability of the resulting complex
has been assessed by molecular dynamics simulations and the experimental results obtained
for the synthesized analogues have been rationalized on the basis of the proposed binding mode
for compound 1a. As a result of the SAR study, monofuryl 1o has been synthesized and is one
of the most active compounds against CDK1 of this series.
Introduction
Cyclin-dependent kinases (CDKs) are a family of
serine/threonine kinases that regulate the cell division
cycle phases, apoptosis, transcription, and differentia-
tion in addition to other functions in the nervous
system.1 Each CDK associates with a specific cyclin
regulatory partner to generate the active catalytic
complex. Owing to both their central role in the cell cycle
and their misregulation in a number of cancers2 and
neurodegenerative disorders, CDK inhibitors have gar-
nered attention recently for their potential as anticancer
us to synthesize a new series of pyrazolo[3,4-c]pyr-
idazine analogues of 1a in an attempt to improve
potency against the CDK family while maintaining a
favorable selectivity profile and to carry out a structure-
activity relationship study. The emphasis has been put
on the molecular modulation. Specifically, (a) different
substituents in the aromatic system at C-4 and C-5 have
been introduced, (b) phenyl rings have been substituted
by other groups, (c) the amino group at the C-3 position
has been substituted by other functions, and (d) differ-
ent substituents have been introduced at position 1
(Figure 1).
While this work was in progress, Witherington et al.8
have also identified pyrazolopyridazine 1a as a potent
inhibitor of glycogen synthase kinase-3 (GSK-3) and
CDK2/cyclin A. In this work, this compound also showed
excellent selectivity against the majority of the tested
kinases. This result is not unexpected given the high
homology between these two kinases and CDK1. GSK-3
has a major role in the regulation of the cell cycle,
transcription, and insulin action. As in the case of
CDKs, this type of kinase is involved in cancer and
neurodegenerative diseases and it has also been used
as a target to identify small molecular weight pharma-
cological inhibitors of potential therapeutic interest.
Therefore, inhibitors that show dual specificity for these
kinases could be useful in the treatment of cancer.1,9
therapeutics.1,3,4
The search for small-molecule inhibitors of CDKs has
already led to the discovery of several classes of com-
pounds with high structural diversity.1,3,4 Only reports
on the clinical trial of flavopiridol and UCN-01 as cancer
therapeutics are available,5-7 but several other CDK
inhibitors are currently making their way in the clinic.
Despite striking chemical diversity, most CDK inhibi-
tors share common properties. Thus, they are essentially
low molecular weight, flat, hydrophobic heterocycles and
act by competing with ATP for binding in the kinase
ATP binding site.
Pyrazolopyridazine 1a was identified in a high-
throughput screening carried out by BASF Bioresearch
Corp. (Worcester, MA) as a potent inhibitor of CDK1/
cyclin B. Subsequent screening of this hit against other
kinases revealed the compound to have selectivity for
the CDK family (IC50 (µM) for different kinases: CDK1/
cyclin B ) 6.1, kdr > 50, and lck > 50). This prompted
* To whom the correspondence should be addressed.
Phone: 34913724700. Fax: 34913510475. E-mail: mfbrana@ceu.es.
† Departamento de Qu´ımica, Universidad San Pablo CEU.
# Departamento de Ciencias Ambientales y Recursos Naturales,
Universidad San Pablo CEU.
§ Departamento de Biolog´ıa Celular, Bioqu´ımica y Biolog´ıa Molec-
ular, Universidad San Pablo CEU.
‡ CNRS, Cell Cycle Group.
10.1021/jm058013g CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/08/2005

6844 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Bran˜a et al.
Scheme 2a
a Reagents: (a) diethyl malonate, K2CO3, KI; (b) NH2NH2,
EtOH; (c) Br2, AcOH; (d) NH3; (e) POCl3, dioxane, ∆; (f) NH2NH2,
EtOH.
yl)acetate, by reaction of trifluoromethylphenyl bromide
with diethyl oxalate in the presence of BuLi.13 We have
found that with the use of equivalent amounts of
reactants and a high excess of base, the yield of the
desired diketone can be increased to 25%.
The desired pyridazinones have been obtained fol-
lowing two alternative methods. Compounds 5d-f were
obtained by reaction of diketones 3d-f with an excess
of hydrazine in EtOH, followed by reaction of the
corresponding monohydrazones 4d-f with ethyl cy-
anoacetate and NaOEt. On the other hand, pyridazi-
nones 5a-c and 5g-l were obtained with better yields
by reacting the corresponding 1,2-dicarbonyl compounds
with cyanoacetohydrazide. The reaction mixture was
refluxed in EtOH or DMF except for pyridazinone 5l,
where the condensation between 3l and cyanoacetohy-
drazide was performed at room temperature to isolate
intermediate 7, which was then transformed into 5l by
Figure 1. Structures of pyrazolopyridazine-based CDK1
inhibitors.
Scheme 1a
treatment with sodium in EtOH. When diketone 3i was
refluxed with cyanoacetohydrazide in EtOH, two com-
pounds were isolated. One of them was the expected
reaction product 5i, and the other was characterized as
pyrazolopyridazine 1i. This result is difficult to explain,
unless some amount of hydrazine was present in the
reaction medium from partial hydrolysis of cyanoaceto-
hydrazide.
Compounds 5a-h and 5j-l were transformed into the
corresponding pyridazines 6a-h and 6j-l by treatment
with phosphorus oxychloride. Reaction of these pyri-
dazines with hydrazine yielded the desired pyrazolopy-
ridazines 1a-h and 1j-l.
Compounds 1n and 1o were obtained following the
method outlined in Scheme 2. Thus, condensation of the
corresponding phenacyl halide with diethyl malonate,
gave diester 8, which by reaction with hydrazine fol-
lowed by oxidation with bromine in AcOH gave com-
pound 10. Treatment of ester 10 with aqueous ammonia
a Reagents: (a) NH2NH2, EtOH, ∆; (b) (i) Na, EtOH, ethyl
cyanoacetate; (ii) 1 N HCl; (c) cyanoacetohydrazide, EtOH, or
DMF, ∆; (d) POCl3, dioxane, ∆; (e) NH2NH2, EtOH, ∆.
Results and Discussion
Chemistry. The method utilized for the synthesis of
pyrazolo[3,4-c]pyridazines 1a-l is outlined in Scheme
1. The necessary 1,2-dicarbonyl compounds were com-
mercially available (3a and 3f-l) or easily prepared
(3b-d) following previously described methods.10-12
Diketone 3e was described as a secondary product (10%)
in the synthesis of ethyl 2-oxo-2-(p-trifluoromethylphen-

Pyridazines as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6845
Scheme 3a
inhibitor. The IC50 is determined from dose response
curves. The IC50 value for each of the compounds
determined under these assay conditions is shown in
Tables 1 and 2.
We also tested the synthesized compounds for their
cytotoxic effects against several cancer cell lines. These
include human colon carcinoma (HT-29), human cervical
carcinoma (HeLa), and human prostate carcinoma (PC-
3), and the results are presented in Tables 1 and 2.
To investigate the effect of substitution on the aro-
matic rings, a small number of compounds (1b-f and
1m) were synthesized and their potency in the purified
enzyme assay was measured. Compounds 1c and 1e,
bearing electron-withdrawing groups in the para posi-
tion of both aromatic rings, were found to be inactive
against CDK1/cyclin B and all the cell lines tested. In
contrast, compound 1f, where a donor group is present
in the same position, was able to inhibit the enzyme,
although a 5-fold drop in potency was found, compared
to the lead compound 1a.
The proposed binding mode for compound 1a (see
later) suggests that the aromatic ring in position 5 may
display van der Waals interactions with vicinal amino
acid residues in the enzyme. On the basis of this model,
it was anticipated that derivatives 1b and 1d, where
the aromatic rings bear a bulky substituent (phenyl for
1b and tert-butyl for 1d), would be significantly weaker
inhibitors of CDK1. Indeed, both of them failed to inhibit
CDK1 at all concentrations up to 50 µM.
Replacement of the aromatic rings in positions 4 and
5 of the pyrazolopyridazine system in 1a by a π-rich
furan ring (1h) led to a potent CDK1/cyclin B inhibitor
(IC50 ) 1.5 µM), while an inactive compound (1i) was
obtained when a π-deficient ring was introduced. From
an electrostatic point of view, this result is in accordance
with the previous observation that electron-rich aro-
matic rings in positions 4 and 5 have a favorable effect
on the inhibitory activity. The anti-CDK1 activity of 1h
was not accompanied by antitumor activity in the cell
lines tested.
The next modifications of 1a to be examined were the
elimination of one of the aromatic rings in positions 4
and 5 (1l and 1n) and the substitution of one or both of
the aromatic rings by methyl groups (1j and 1k).
Interestingly, only compound 1n maintained the activity
of the lead compound against the enzyme. This finding
suggested that the presence of an aromatic ring in
position 5 of compound 1a is essential for its inhibitory
activity. However, the aromatic ring in position 4 can
be eliminated without resulting in a loss of activity.
Again, the molecular modeling study gave us a possible
explanation for this behavior. According to the proposed
binding mode for compound 1a, which will be discussed
later, the aromatic ring in position 5 is located in a
hydrophobic pocket formed by residues Val18, Ala31,
Val64, Phe80, and Ala144, while the aromatic ring in
position 4 does not establish any relevant interaction.
a Reagents: (a) X(CH2)nO(CH2)2OCOCH3, X ) Br for 2i and X
) I for 2j, Cs2CO3, DMF; (b) 1 N NH4OH.
at room temperature afforded amide 11. Subsequent
treatment with phosphorus oxychloride in dioxane at
reflux temperature afforded chloronitrile 6. Finally,
treatment of 6 with hydrazine gave the desired pyra-
zolopyridazines 1n and 1o. Pyrazolopyridazine 1m was
obtained by reduction of 1c with Raney Ni and hydra-
zine.
Pyrazolopyridazine 2a was obtained by a method
similar to that outlined in Scheme 1 but using ethyl
3-hydrazino-3-oxopropionate instead of cyanoacetohy-
drazide, as described in the literature.14
The reaction of 1a with ethyl isocyanate or phenyl
isocyanate yielded compounds 2b and 2c, respectively.
Similarly, compounds 13 and 14 were obtained from 1o.
Hydrazine 2d was synthesized by reaction of 1a with
NaNO2/HCl followed by reduction of the formed diazo-
nium salt with Na2SO3.
Derivative 2e was obtained by refluxing 1a with
acetyl chloride and Et3N in dioxane. Under these
conditions, acetylation took place selectively at the
amino group in the 3 position. However, when the
reaction was carried out by heating the reactants in the
presence of pyridine, compound 2f was isolated. Di-
acetylation was observed when 1a was treated with
acetic anhydride in AcOH, yielding compound 2g.
Pyrazolopyridazine 2h was synthesized by reaction
of 6a with phenylhydrazine. Finally, compounds 2i and
2j were obtained by reaction of 1a with the correspond-
ing halogeno derivative in the presence of Cs2CO3,
followed by hydrolysis of the ester group present in 12
with 1 N NH4OH, as depicted in Scheme 3.
Biological Activity. Compounds 1 were tested for
their ability to inhibit in vitro the CDK1/cyclin B
complex from starfish (Marthasterias glacialis) oocytes
as described Meijer et al.15 In this assay the transfer of
γ-33P-labeled phosphate from [γ-33P]ATP to histone H1
in the absence and presence of inhibitor is measured
and expressed as a percent of kinase activity without
Pyrazolopyridazine 1g, where the connection of both
phenyl groups by means of a cyclohexane ring rigidified
the system, did not present inhibitory activity. This
result is also in accordance with the proposed binding
mode (Figure 2), where the phenyl groups in 1a are
oriented in a parallel disposition. Obviously, the rigidi-
fication carried out in compound 1g does not allow the

6846 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Table 1. Enzymatic and Cellular Activity for Compounds 1
Bran˜a et al.
compd
R1
R2
HT-29a IC50, µM
HeLab IC50, µM
PC-3c IC50, µM
CDK1/cyclin B IC50, µM
1a
1b
1c
1d
1e
1f
1g
1h
1i
C6H5
C6H5
>100
>100
>100
8.50
>100
>100
>100
8.78
>100
NT
3.00
>50
>50
>50
>50
15.00
>50
1.50
>50
>50
>50
>50
>50
4.00
5.30
0.4d
p-C6H5C6H4
p-NO2C6H4
p-tBuC6H4
p-CF3C6H4
p-CH3OC6H4
2,2′-biphenyl
2-furyl
p-C6H5C6H4
p-NO2C6H4
p-tBuC6H4
p-CF3C6H4
p-CH3OC6H4
2,2′-biphenyl
2-furyl
NT
NT
NT
>100
>100
NT
NT
NT
72.40
NT
>100
>100
>100
>100
73.00
>100
>100
80.80
10.52
>100
>100
>100
>100
30.70
>100
>100
>100
88.30
>100
>100
>100
>100
2-pyridyl
CH3
C6H5
C6H5
p-NH2C6H4
H
H
2-pyridyl
CH3
CH3
H
p-NH2C6H4
C6H5
2-furyl
1j
1k
1l
1m
1n
1o
flavopiridol
olomoucine
purvalanol B
NT
NT
>100
7d
0.006d
a Human colon carcinoma cell line. b Human cervical carcinoma cell line. c Human prostate carcinoma cell line. d From literature.4
Table 2. Enzymatic and Cellular Activity for Compounds 2
compd
R3
R4
HT-29 IC50, µM
HeLa IC50, µM
PC-3 IC50, µM
CDK1/cyclin B IC50, µM
2a
2b
2c
2d
2e
2f
H
OH
>100
>100
>100
NT
>100
>100
>100
>100
>100
>100
>100
60.44
2.93
>100
>100
>100
NT
>100
>100
>100
>100
>100
>100
>50
7.50
9.00
8.50
>50
>50
>50
>50
>50
>50
H
NHCONHEt
NHCONHC6H5
NHNH2
NHCOCH3
NH2
H
H
NT
H
>100
>100
>100
>100
>100
>100
COCH3
COCH3
2g
2h
2i
NHCOCH3
NH2
NH2
NH2
CH2C6H5
CH2O(CH2)2OH
(CH2)2O(CH2)2OH
2j
maintained the inhibitory activity. Surprisingly, com-
pound 2a, with a hydroxyl group in the same position,
proved to be inactive, while the theoretical work de-
scribed later predicts a very similar orientation of
compounds 1a and 2a inside the binding pocket of
CDK2.
Finally, compounds 2f-j were designed to evaluate
the effect of substitution in position 1 of the lead
compound. Hydroxyethoxymethyl and hydroxyethoxy-
ethyl groups present in 2i and 2j, respectively, were
chosen because these fragments are present in some
antiviral agents, such as aciclovir,16 a nucleoside ana-
logue inhibitor of the herpex simplex virus. In aciclovir,
these groups mimic the carbohydrate fragment present
in nucleosides. Our CDKs inhibitors were designed to
occupy the ATP binding site in CDK1. Therefore, the
presence of hydroxylated chains mimicking the carbo-
hydrate fragment of ATP could increase the affinity and
selectivity of the inhibitor. Unfortunately, none of
derivatives (2f-j) exhibited CDK1 inhibitory or antitu-
mor activity, demonstrating that position 1 in the
Figure 2. Binding model of 1a and ATP in the CDK2 active
site. Residues relevant to the discussion are displayed as thick
sticks in yellow.
molecule to adopt that orientation of the phenyl groups
in the binding site.
Pyrazolopyridazines having more lipophilic substit-
uents at C-3 such as acetamide 2e did lead to a loss of
activity. By contrast, the use of other groups capable of
establishing additional hydrogen bonds with the en-
zyme, such as ureas 2b and 2c and hydrazine 2d,

Pyridazines as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6847
pyrazolopyridazine system must be unsubstituted for
CDK1 inhibition. This result is also in agreement with
the modeling work carried out for these compounds and
is described below, since the introduction of substituents
in position 1 would prevent the electrostatic interaction
with Phe82.
Taking into account that the most active compound
tested so far is pyrazolopyridazine 1h and that substitu-
tion at position 4 is not essential for activity, the next
step in our SAR study was the synthesis and biological
evaluation of the monofuryl 1o and their urea and
thiourea derivatives 13 and 14, respectively. Com-
Figure 3. Schematic representation of the binding mode of
compound 1a to CDK2, showing residues with which the
ligand interacts through hydrogen bonds (blue) or through van
der Waals interactions (magenta).
pounds 1o and 13 were active against CDK1 as we had
expected. More interestingly, these two compounds were
also active against GSK-3 with IC50 values of 0.80 and
0.66 µM, respectively, which enhances their potential
utility in the treatment of tumoral affections.
Disappointingly, the synthesized compounds did not
show cytotoxic effects against the cell lines tested. This
fact could be attributed to their difficulty in crossing
cell membranes, even though these compounds fit the
broad Lipinski guidelines, and validation of this argu-
ment will have to await the results of additional studies.
Molecular Modeling. We have carried out a molec-
ular modeling study on the mode of interaction of
compound 1a with CDK2, making use of automated
docking methods and molecular dynamics simulations.
Although the inhibitory activity has been tested against
CDK1, these studies have been carried out with CDK2
because only X-ray structures of CDK2 in complex with
small-molecule ligands have been solved to date, which
limits the structure-based studies to this cyclin-depend-
ent kinase. However, the two protein kinases have
nearly 66% sequence identity overall, and their ATP
binding pockets are quite similar.17 This issue is exten-
sively explored in a recent work carried out by McGraw-
th et al., where a homology model of CDK1/cyclinB is
generated from a high-resolution CDK2/cyclin A crystal
structure.18 It is therefore possible to assume that these
compounds will not show selectivity between these two
kinases. We and other authors have previously made
use of this approximation in similar studies.
First, the ligand was submitted to a Monte Carlo
conformational search, and then the lowest energy
conformation was taken as the starting conformation
for the automated docking into CDK2. The lowest
energy complex predicted by AutoDock is shown in
Figure 2. As shown in this figure, compound 1a binds
to the active site of CDK2, with the pyrazolopyridazine
system in a position similar to that of the purine ring
of the ATP molecule, which is the natural substrate of
the enzyme but in a different orientation. Compound
1a establishes hydrogen bonds with Asp86 and Leu83,
residues that are also involved in interactions with the
ATP molecule, as shown schematically in Figure 3. In
Figure 4. Residue-based van der Waals energy term (kcal/
mol) of the interaction between compound 1a and the CDK2
active site.
the proposed binding mode predicted by AutoDock,
residues Val18, Ala31, Val64, Phe80, and Ala144 form
a hydrophobic pocket where the phenyl ring in position
5 remains allocated, while the phenyl ring in position 4
does not establish any relevant interaction.
The evidence that proteins that constitute therapeutic
targets are mobile is substantial.19 To take into account
protein flexibility, the behavior of the predicted complex
was studied in a dynamic context and the progression
of the root-mean-square (rms) deviation of the coordi-
nates of the solute with respect to the initial structure
was monitored, leading to an average value of 3.186 (
0.623 Å. The progression of this parameter indicates
that the complex does not experience large conforma-
tional changes during the sampling time and, thus, can
be considered to be in a state near equilibrium. To
evaluate the relative contributions of the different
residues to complex stabilization, the 125 structures
collected from the last 500 ps of the simulation were
averaged and energy-minimized, and the interaction
energy between compound 1a and the binding site was
decomposed on a residue basis using the ANAL module
of AMBER (Figures 4 and 5). A superimposition of the
energy-minimized average structure and the initial
structure is shown in Figure 6. As expected from the
calculated rms deviations mentioned above, the complex
remains stable along the simulation time; thus, the
conformational changes are not very relevant. The

6848 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Bran˜a et al.
result does not match the lack of biological activity
demonstrated for this compound. A possible explanation
for this unexpected result may be that compound 2a
could be interacting with the enzyme through the 4,5-
diphenyl-1,2-dihydropyrazolo[3,4-c]pyridazin-3-one tau-
tomeric form (2a1). A RHF/6-31G* geometry optimiza-
tion of the two tautomers led to a 3.8 kcal/mol energy
difference in favor of the 4,5-diphenyl-1H-pyrazolo[3,4-
c]pyridazin-3-ol form (2a2). However, taking into ac-
count that the solvent effect was not considered, we
cannot discard the possibility that compound 2a may
be interacting with the enzyme’s binding site through
the 2a1 tautomeric form, leading to a completely differ-
ent binding mode if compared to 1a and accounting for
the lack of activity of the synthesized derivative 2a.
Figure 5. Residue-based electrostatic energy term (kcal/mol)
of the interaction between compound 1a and the CDK2 active
site.
Conclusions
We have presented a novel series of pyrazolopyr-
idazine-based CDK1 inhibitors. Our SAR study with
these compounds suggest that (i) the phenyl rings can
be substituted by π-rich rings, such as furan, resulting
in a 2-fold increase of inhibition, (ii) substitution at
position 4 is not relevant, while the presence of a
substituent at position 5 is essential for the potency of
CDK1 inhibition, (iii) the introduction of substituents
at N-1 led to a loss of activity, (iv) ethylurea, phenylurea,
and hydrazine moieties in N-3 maintained the potency
of inhibition.
Molecular modeling studies indicate that these mol-
ecules create an interaction pattern analogous to that
found in many other known small-molecule CDK inhibi-
tors for which structural information is available. Ac-
cording to the proposed binding mode, 1a binds to the
active site of CDK2, with the pyrazolopyridazine system
in a position similar to that of the purine ring of the
ATP molecule, which is the natural substrate of the
enzyme but adopting a different orientation. The pro-
posed binding model for compound 1a has allowed us
to rationalize the CDK1 inhibitory activity observed for
the synthesized analogous derivatives.
Figure 6. View of superimposed C(R) traces of the energy-
minimized average structure of the last 500 ps of the MD
simulation (green) and the initial structure (magenta) for
CDK2/1a.
model we propose for the 1a/CDK2 complex gives rise
to rather strong electrostatic interactions with Phe82,
Leu83, Asp86, Gln131, and Asp145.
Leu83 and Asp86 are involved in the formation of two
stable hydrogen bonds with positions 7 and 3 from the
ligand, respectively. These distances were monitored
along the simulation time, demonstrating the stability
of the above-mentioned interactions. These interactions
seem to be especially relevant for the binding affinity
and selectivity of the ligand, since they have also been
found to interact with other known CDK inhibitors such
as purvalanol B and staurosporine.3
On the other hand, Phe82 establishes a strong elec-
trostatic interaction with the NH in position 1 of the
ligand. It can also be seen that the major contributors
to the van der Waals energy term are Ile10, Val18,
Ala31, Phe80, Glu81, Phe82, Leu83, Asp86, Gln131,
Leu134, Ala144, and Asp145.
A similar docking procedure was used to explore the
possible binding modes of the synthesized analogues
(1b-o and 2a-c,e,f). First, the ligands were submitted
to a Monte Carlo conformational search, and then the
lowest energy conformations were taken as the starting
structures for the automated docking into CDK2. This
study reveals that all the synthesized compounds in-
teract with the ATP binding pocket of the kinase.
However, the binding modes predicted by AutoDock
differ significantly from that described above for the lead
compound, with the exception of compound 2a, which
adopts a similar orientation. As mentioned before, this
Experimental Section
General Methods. Melting points (uncorrected) were
determined on a Stuart Scientific SMP3 apparatus. Infrared
(IR) spectra were recorded with a Perkin-Elmer 1330 infrared
1
spectrophotometer. H and 13C NMR δ values were recorded
on a Bruker 300-AC instrument. Chemical shifts (δ) are
expressed in parts per million relative to internal tetrameth-
ylsilane; coupling constants (J) are in hertz. Mass spectra were
run on a HP 5989A spectrometer. Elemental analyses (C, H,
N) were performed on a Perkin-Elmer 2400 CHN apparatus
at the Microanalyses Service of the University Complutense
of Madrid. Unless otherwise stated, all reported values are
within (0.4% of the theoretical compositions. Thin-layer
chromatography (TLC) was run on Merck silica gel 60 F-254
plates. Unless stated otherwise, starting materials used were
high-grade commercial products. The following products were
prepared according to the literature: 3-amino-4,5-diphenyl-
1H-pyrazolo[3,4-c]pyridazine 1a,20 3-amino-4,5-dimethyl-1H-
pyrazolo[3,4-c]pyridazine 1j,21 4,5-diphenyl-1H-pyrazolo[3,4-
c]pyridazin-3-ol 2a.14 Complete 1H and 13C NMR, IR, MS, and
elemental analysis data are given in the Supporting Informa-
tion.
4,4′-Bis(trifluoromethyl)benzil (3e). A solution of 13.00
mmol of 1.60 M n-butyllithium in hexane (8.1 mL) was cooled
to -60 °C, and a solution of 2.86 g (13.00 mmol) of 1-bromo-
4-trifluoromethylbenzene in 25 mL of anhydride Et2O was
added dropwise. The reaction mixture was warmed to room

Pyridazines as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6849
temperature and then added dropwise to a solution of 2.85 g
(19.55 mmol) of diethyl oxalate in 20 mL of anhydride Et2O
cooled to -60 °C. The reaction mixture was warmed to room
temperature, and then the organic layer was washed with 20
mL of water and 10 mL of 2% HCl successively. The combined
extracts were dried over MgSO4 and concentrated to give a
solid, which was purified by flash chromatography (hexane/
Et2O 98:2) to give 3e13 (550 mg, 25%) as a yellow solid. IR
separated by filtration and purified by recrystallization from
the appropriate solvent or by column chromatography using
the appropriate eluents.
5,6-Bis(p-tert-butylphenyl)-4-cyanopyridazin-3(2H)-
one (5d). From Na (108 mg, 4.70 mmol) in EtOH (25 mL),
ethyl cyanoacetate (530 mg, 4.70 mmol) in EtOH (10 mL), and
4d (1.43 g, 4.26 mmol) was obtained 5d (1.60 g, 96%) as a white
solid, mp >300 °C (AcOEt/cyclohexane). IR (KBr): 1655, 2220
1
(KBr): 1670 cm-1. H NMR (CDCl3): δ 8.01 (4H, d, J ) 7.8,
cm-1 1H NMR (CDCl3): δ 1.27 (9H, s, 3CH3), 1.31 (9H, s,
.
ArH), 8.22 (4H, d, J ) 7.8, ArH). Further elution afforded 768
3CH3), 7.01 (2H, d, J ) 8.2, ArH), 7.15 (2H, d, J ) 8.2, ArH),
7.23 (2H, d, J ) 8.2, ArH), 7.37 (2H, d, J ) 8.8, ArH), 11.63
(1H, sa, NH). 13C NMR (CDCl3): δ 31.0, 31.1, 34.6, 34.9, 113.1,
113.8, 125.1, 125.6, 128.8, 129.5, 131.1, 147.5, 152.5, 152.7,
154.2, 158.3. MS (EI): m/z (%) 385 (M+, 36), 370 (100), 355
(1), 340 (1), 328 (3), 314 (11), 272 (4), 91 (2), 57 (24).
mg (24%) of ethyl 2-oxo-2-(p-trifluoromethylphenyl)acetate.13
2-Cyano-N′-(2-oxo-2-phenylethylidene)acetohy-
drazide (7). To a solution of phenylglyoxal (3.0 g, 22.39 mmol)
in 35 mL of absolute EtOH was added 2.22 g (22.39 mmol) of
cyanoacetohydrazide at 0 °C. The mixture was stirred for 24
h at room temperature. The precipitated solid was filtered and
washed with AcOEt. The combined filtrates were concentrated
under reduced pressure to give 7 as a brown oil, which was
used without further purification. 1H NMR (DMSO-d6): δ 4.19
(2H, s, CH2), 7.55 (2H, dd, J ) 7.3, 7.6, ArH), 7.67 (1H, t, J )
7.3, ArH), 7.79 (1H, s, CH), 8.04 (2H, d, J ) 7.3, ArH), 12.30
(1H, sa, NH).
4-Cyano-5,6-bis(p-trifluoromethylphenyl)pyridazin-
3(2H)-one (5e). From Na (233 mg, 1.01 mmol) in EtOH (15
mL), ethyl cyanoacetate (114 mg, 1.01 mmol) in EtOH (10 mL),
and 4e (330 mg, 0.92 mmol) was obtained 5e (339 mg, 90%)
as a white solid, mp 231-233 °C (toluene/hexane).
4-Cyano-5,6-bis(p-methoxyphenyl)pyridazin-3(2H)-
one (5f).24 From Na (180 mg, 7.70 mmol) in EtOH (75 mL),
ethyl cyanoacetate (0.87 g, 7.70 mmol) in EtOH (10 mL), and
monohydrazone of 4,4′-dimethoxybenzil23 (2.0 g, 7.00 mmol)
was obtained 5f (2.0 g, 86%) as a yellow solid, mp 235-273 °C
(absolute EtOH).
Diethyl 2-[2-(2-Furyl)-2-oxoethyl]malonate (8o). A solu-
tion of 320 mg (2.0 mmol) of diethyl malonate, 387 mg (2.0
mmol) of 2-bromo-1-(2-furyl)ethanone,22 71 mg (0.43 mmol) of
potassium iodide, and 650 mg (4.7 mmol) of potassium carbon-
ate in 30 mL of anhydride acetone was stirred for 24 h at room
temperature. After this time, the solvent was eliminated under
reduced pressure and the crude of reaction was neutralizated
(1 N HCl). The residue was extracted with CHCl3, and the
collected organic layers were dried over MgSO4 and concen-
trated to give a liquid, which was purified by flash chroma-
tography (hexane/AcOEt 7:3) to give 8o (339 mg, 63%) as a
Method B. Cyanoacetohydrazide (2 equiv) was added in
portions to a solution of the corresponding diketone (1 equiv)
in absolute EtOH. The mixture was heated at reflux temper-
ature until the reaction was completed (TLC). After the
solution was cooled, the formed solid was isolated by filtration
and purified by recrystallization from the appropriate solvent
or by column chromatography using the appropriate eluents.
4-Cyano-5,6-di(2-furyl)pyridazin-3(2H)-one (5h). From
2,2′-furyl (1.5 g, 7.90 mmol) in absolute EtOH (30 mL) and
cyanoacetohydrazide (1.56 g, 15.8 mmol) was obtained 5h (1.1
g, 54%) as a brown solid purified by flash chromatography
(hexane/AcOEt 9:1), mp 250-253 °C (absolute EtOH).
4-Cyano-5,6-di(2-pyridyl)pyridazin-3(2H)-one (5i). From
1,2-di(2-pyridyl)ethane-1,2-dione (2 g, 9.5 mmol) in absolute
EtOH (75 mL) and cyanoacetohydrazide (1.68 g, 0.017 mmol)
was obtained 5i (538 mg, 21%) as a white solid purified by
flash chromatography (CHCl3/MeOH 98:2), mp 198 °C (dec).
The mother liquor was concentrated and the crude product
was purified by flash chromatography (CHCl3/MeOH 95:5) to
give 3-amino-4,5-di(2-pyridyl)-1H-pyrazolo[3,4-c]pyridazine 1i
(429 mg, 16%) as a yellow solid, mp 264 °C (dec) (absolute
EtOH). IR (KBr): 3110, 3200, 3280, 3380, 3440 cm-1. 1H NMR
(DMSO-d6): δ 6.18 (2H, sa, NH2), 6.72 (1H, dd, J ) 6.7, 9.1,
pyridylH), 6.92 (1H, m, pyridylH), 7.54 (1H, m, pyridylH), 7.87
(2H, m, pyridylH), 7.99 (1H, m, pyridylH), 8.14 (1H, d, J )
6.7, pyridylH), 8.78 (1H, d, J ) 4.9, pyridylH), 11.90 (1H, sa,
NH). 13C NMR (DMSO-d6): δ 98.6, 103.4, 113.8, 116.0, 119.9,
120.8, 122.1, 123.2, 124.0, 131.3, 137.3, 143.0, 148.4, 155.5,
160.3. MS (EI): m/z (%) 277 (100). Anal. (C15H17N7) C, H, N.
Method C. To a solution of the corresponding diketone (1
equiv) in DMF was added, at 80 °C, a solution of cyanoaceto-
hydrazide (2 equiv) in DMF. The mixture was heated at 100
°C until the reaction was completed (TLC). Then the solution
was concentrated under vacuum. The residue was purified by
recrystallization from the appropriate solvent or by column
chromatography using the appropriate eluents.
colorless liquid. IR (film): 1670, 1720, 1740 cm-1 1H NMR
.
(CDCl3): δ 1.28 (6H, t, J ) 7.1, CH3), 3.49 (2H, d, J ) 7.1,
CH2), 4.03 (1H, t, J ) 7.1, CH), 4.24 (4H, q, J ) 7.1, CH2),
6.56 (1H, m, furylH), 7.25 (1H, m, furylH), 7.61 (1H, s, furylH).
13C NMR (CDCl3): δ 13.7, 37.0, 46.4, 61.5, 112.1, 117.3, 146.5,
151.7, 168.5, 185.2.
General Procedure for the Preparation of Monohy-
drazones. A suspension of the corresponding diketone in
absolute EtOH containing an excess of NH2NH2‚H2O was
heated at reflux temperature until the reaction was completed
(TLC). After the solution was cooled, the formed solid was
isolated by filtration and purified by recrystallization from the
appropriate solvent or by column chromatography using the
appropriate eluents.
Monohydrazone of 4,4′-Di-tert-butylbenzil (4d). From
4,4′-di-tert-butylbenzil23 (1.84 g, 5.70 mmol) and NH2NH2‚H2O
(285 mg, 5.70 mmol) in absolute EtOH (30 mL) was obtained
4d (1.48 g, 77%) as a white solid, mp >300 °C (absolute EtOH).
IR (KBr): 1620, 3260, 3390 cm-1 1H NMR (CDCl3): δ 1.35
.
(18H, s, 6CH3), 6.25 (2H, sa, NH2), 7.29 (2H, d, J ) 8.8, ArH),
7.47 (2H, d, J ) 8.8, ArH), 7.54 (2H, d, J ) 8.3, ArH), 7.92
(2H, d, J ) 8.3, ArH). 13C NMR (CDCl3): δ 31.1, 31.2, 34.8,
34.9, 124.8, 126.1, 126.7, 128.6, 130.1, 135.4, 145.8, 152.1,
155.0, 191.7. MS (EI): m/z (%) 292 (10), 279 (5), 161 (100),
145 (7), 116 (7), 91 (8), 77 (1).
Monohydrazone of 4,4′-Bis(trifluoromethyl)benzil (4e).
From 3e (383 mg, 1.11 mmol) and NH2NH2‚H2O (111 mg, 2.22
mmol) in absolute EtOH (25 mL) was obtained 4e (336 mg,
84%) as a white solid purified by flash chromatography
(hexane/AcOEt 95:5).
General Procedure for the Preparation of Pyridazi-
nones. Method A. EtOH, dried by distillation from Mg/I2, was
added to a flask containing Na (1.1 equiv) under argon
atmosphere. After the Na reacted, a solution of ethyl cyanoac-
etate (1.1 equiv) in EtOH, dried by distillation from Mg/I2, was
added dropwise to the cold (0-5 °C) alkoxide solution. The
solution was stirred at room temperature for 30 min. The
corresponding monohydrazone (1 equiv) was then added as a
solid. After the reaction mixture was heated at reflux tem-
perature until the reaction was completed (TLC), it was cooled
and poured into 1 N HCl. The formed precipitated was
5,6-Bis(4-biphenyl)-4-cyanopyridazin-3(2H)-one (5g).
From 4,4′-diphenylbenzil10 (1.34 g, 3.70 mmol) in DMF (15 mL)
and cyanoacetohydrazide (0.73 mg, 7.37 mmol) in DMF (6 mL)
was obtained 5g (786 mg, 50%) as a yellow solid, mp >300 °C
(dioxane).
4-Cyano-5,6-bis(p-nitrophenyl)pyridazin-3(2H)-one (5c).
From 4,4′-dinitrobenzil11 (2 g, 6.67 mmol) in DMF (15 mL) and
cyanoacetohydrazide (1.32 g, 13.33 mmol) in DMF (10 mL) was
obtained 5c (1.3 g, 54%) as a yellow solid purified by flash
chromatography (CH2Cl2/MeOH 99:1), mp >300 °C.
4-Cyano-5-phenylpyridazin-3(2H)-one (5l).25 EtOH (44
mL), dried by distillation from Mg/I2, was added to a flask

6850 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Bran˜a et al.
containing Na (270 mg, 11.74 mmol) under argon atmosphere.
After the Na reacted, a solution of 7 (1.26 g, 5.86 mmol) in
EtOH (25 mL) was added dropwise to the cold (0-5 °C)
alkoxide solution. The solution was heated at reflux temper-
ature for 3 h. After the solution was cooled, the solvent was
removed under reduced pressure, the residue was dissolved
in water (10 mL), and the pH was adjusted to 5 with 6 N HCl.
The precipitate was isolated by filtration and purified by
recrystallization from AcOEt/hexane to give 5l (782 mg, 68%)
as a brown solid, mp 218-220 °C.
Ethyl 6-(2-Furyl)-3-oxo-2,3,4,5-tetrahydropyridazine-
4-carboxylate (9o). To a solution of 8.11 g (30.23 mmol) of
8o in 60 mL of absolute EtOH cooled to 0 °C was added
dropwise 15.13 g (30.23 mmol) of NH2NH2‚H2O with stirring.
The solution was heated at reflux temperature for 3 h. After
the solution was cooled, the solvent was removed under
reduced pressure. The crude of the reaction was purified by
flash chromatography on silica gel (hexane/AcOEt 1:1) to give
9o (3.3 g, 46%) as a white solid, mp 131-132 °C. IR (KBr):
1670, 1730, 3100, 3220 cm-1. 1H NMR (CDCl3): δ 1.29 (3H, t,
J ) 7.1, CH3), 3.06 (1H, dd, J ) 17.0 and 6.6, CHAHB), 3.34
(1H, dd, J ) 17.0 and 8.8, CHAHB), 3.60 (1H, dd, J ) 8.8 and
6.6, CH), 4.26 (2H, q, J ) 7.1, OCH2CH3), 6.51 (1H, dd, J )
1.6 and 3.3, furylH), 6.80 (1H, d, J ) 3.30, furylH), 7.54 (1H,
d, J ) 1.65, furylH), 8.86 (1H, s, NH). 13C NMR (CDCl3): δ
14.0, 25.2, 43.1, 62.3, 112.0, 142.6, 144.7, 149.2, 162.8, 167.7.
MS (ESI): m/z 259 [M + Na]+. Anal. (C11H12N2O2) C, H, N.
143.7, 153.0, 159.2. MS (EI): m/z (%) 445 (M+ + 2, 44), 443
(M+, 100), 408 (34), 382 (2), 366 (41).
5,6-Bis(p-tert-butylphenyl)-3-chloropyridazine-4-car-
bonitrile (6d). From 5d (1.40 g, 3.64 mmol) and POCl3 (11
mL, 120.12 mmol) in dioxane (35 mL) was obtained 6d (1.15
g, 82%) as a yellow solid purified by flash chromatography
(hexane/AcOEt 9:1), mp 182-184 °C.
3-Chloro-5,6-bis(p-nitrophenyl)pyridazine-4-carboni-
trile (6c). From 5c (461 mg, 1.27 mmol) and POCl3 (9 mL,
101.60 mmol) in dioxane (10 mL) was obtained 6c (254 mg,
52%) as a yellow solid purified by flash chromatography
(hexane/AcOEt 8:2), mp 215-217 °C.
3-Chloro-5,6-bis(p-trifluoromethylphenyl)pyridazine-
4-carbonitrile (6e). From 5e (130 mg, 0.32 mmol) and POCl3
(1 mL, 11.84 mmol) in dioxane (4 mL) was obtained 6e (93
mg, 68%) as a white solid purified by flash chromatography
(hexane/AcOEt 8:2).
3-Chloro-5,6-bis(p-methoxyphenyl)pyridazine-4-car-
bonitrile (6f). From 5f (750 mg, 2.25 mmol) and POCl3 (7
mL, 81.84 mmol) in dioxane (15 mL) was obtained 6f (759 mg,
96%) as a yellow solid, mp 144-146 °C (absolute EtOH).
3-Chlorodibenzo[f,h]cinnoline-4-carbonitrile (6g). From
4-cyanodibenzo[f,h]cinnolin-3(2H)-one26 (325 mg, 1.20 mmol)
and POCl3 (4 mL, 39.60 mmol) in dioxane (20 mL) was
obtained 6g (294 mg, 85%) as a yellow solid, mp 239-241 °C
(dioxane).
3-Chloro-5,6-di(2-furyl)pyridazine-4-carbonitrile (6h).
To a solution of 5h (841 mg, 3.30 mmol) in dioxane (27 mL)
was added dropwise, under argon atmosphere and at 0 °C, 10
mL (109.7 mmol) of POCl3. The mixture was heated to reflux
for 7 h. Then, the solution was cooled and poured into ice/
water. The precipitate was isolated by filtration and purified
by flash chromatography (hexane/AcOEt 9:1) to give 6h (680
mg, 76%) as a yellow solid, mp 90-92 °C.
Ethyl 6-(2-Furyl)-3-oxo-2,3-dihydropyridazine-4-car-
boxylate (10o). To a solution of 3.07 g (13.00 mmol) of 9o in
50 mL of acetic acid was added dropwise 0.65 mL (13.00 mmol)
of bromine in 50 mL of acetic acid. The solution was stirred at
room temperature for 2 h. Then, the solvent was removed
under reduced pressure and the residue was dissolved in
water. The crude of reaction was extracted with CHCl3, and
the collected organic layers were dried over MgSO4 and
concentrated to give a solid, which was purified by recrystal-
lization from absolute ethanol to give 10o (2.5 g, 83%) as a
yellow solid, mp 177-179 °C. IR (KBr): 1600, 1660, 1710,
3-Chloro-6-methyl-5-phenylpyridazine-4-carboni-
trile (6k). From 4-cyano-6-methyl-5-phenylpyridazin-3(2H)-
one26 (300 mg, 1.42 mmol) and POCl3 (4 mL, 46.86 mmol) in
dioxane (7 mL) was obtained 6k (244 mg, 75%) as a brown
solid, mp 125-127 °C (MeOH/H2O).
1
2020, 3120, 3140 cm-1. H NMR (CDCl3): δ 1.43 (3H, t, J )
3-Chloro-5-phenylpyridazine-4-carbonitrile (6l).25 To a
solution of 5l (1.68 g, 8.52 mmol) in dioxane (25 mL) was added
26 mL (281 mmol) of POCl3. The mixture was heated to reflux
for 3 h, and then the solution was cooled and poured into ice/
water. The precipitate was collected by filtration, and the
combined filtrates were concentrated under reduced pressure
and purified by flash chromatography on silica gel (hexane/
AcOEt 99:1) to give 6l (108 mg, 6%) as a brown solid, mp 105-
108 °C.
7.1, CH3), 4.46 (2H, q, J ) 7.1, CH2), 6.55 (1H, dd, J ) 3.3 and
1.7, furylH), 6.39 (1H, d, J ) 3.3, furylH), 7.56 (1H, d, J )
1.6, furylH), 8.25 (1H, s, pyridazineH), 11.97 (1H, br s, NH).
13C NMR (CDCl3): δ 14.2, 62.4, 109.3, 112.4, 130.7, 132.2,
138.5, 144.1, 148.3, 158.1, 162.8. MS (ESI): m/z 257 [M + Na]+.
Anal. (C11H10N2O4‚0.25H2O) C, H, N.
6-(2-Furyl)-3-oxo-2,3-dihydropyridazine-4-carboxam-
ide (11o). A solution of 1.60 g (6.83 mmol) of 10o in 60 mL of
concentrated ammonia was stirred at room temperature for
18 h. Then, the solvent was removed under reduced pressure
to give 110 (1.4 g, 99%) as a yellow solid, mp 313 °C (dec). IR
3-Chloro-6-(2-furyl)pyridazine-4-carbonitrile (6o). From
11o (1.58 g, 7.70 mmol) and POCl3 (17 mL, 183.73 mmol) in
dioxane (30 mL) was obtained 6o (1.26 g, 80%) as a yellow
solid purified by flash chromatography (hexane/AcOEt 7:3),
mp 168-169 °C.
1
(KBr): 1600, 1705, 3140, 3320 cm-1. H NMR (DMSO-d6): δ
6.68 (1H, dd, J ) 1.8 and 3.0, furylH), 7.19 (1H, d, J ) 3.0,
furylH), 7.88 (1H, d, J ) 1.8, furylH), 8.12 (1H, s, NH2), 8.40
(1H, s, pyridazineH), 8.84 (1H, s, NH2). 13C NMR (DMSO-d6):
δ 109.7, 112.4, 130.3, 130.7, 138.2, 144.8, 144.8, 148.4, 160.3,
162.4. MS (ESI): m/z 228 [M + Na]+.
General Procedure for the Preparation of Pyrazolo-
[3,4-c]pyridazines. To a solution of the corresponding cloro-
pyridazine in absolute EtOH was added an excess of hydrazine
hydrate in absolute EtOH. The mixture was heated at reflux
temperature until the reaction was completed (TLC). After the
solution was cooled, the formed solid was isolated by filtration
and purified by recrystallization from the appropriate solvent
or by column chromatography using the appropriate eluents.
3-Amino-4,5-bis(4-biphenyl)-1H-pyrazolo[3,4-c]pyr-
idazine (1b). From 6b (250 mg, 0.56 mmol) and NH2NH2‚
H2O (42 mg, 0.84 mmol) in absolute EtOH (5 mL) was obtained
1b (194 mg, 79%) as a yellow solid, mp >300 °C (DMF/AcOEt).
IR (KBr): 3160, 3300, 3430 cm-1. 1H NMR (DMSO-d6): δ 4.75
(2H, sa, NH2), 7.42 (10H, m, ArH), 7.64 (4H, m, ArH), 7.78
(4H, m, ArH), 13.10 (1H, sa, NH). 13C NMR (DMSO-d6): δ
105.9, 126.0, 126.6, 126.7, 127.6, 128.0, 129.0, 129.1, 130.1,
130.2, 130.9, 131.9, 132.5, 136.8, 138.90, 138.91, 139.3, 140.1,
147.7, 149.6, 154.5. MS (EI): m/z (%) 439 (M+, 100), 362 (32).
Anal. (C29H21N5) C, H, N.
General Procedure for the Preparation of Cloropyr-
idazines. A mixture of the corresponding pyridazinone, an
excess of POCl3, and dioxane was heated under reflux until
the reaction was completed (TLC). The solution was cooled and
poured into ice/water. The precipitated was isolated by filtra-
tion and purified by recrystallization from the appropriate
solvent or by column chromatography using the appropriate
eluents.
5,6-Bis(4-biphenyl)-3-chloropyridazine-4-carboni-
trile (6b). From 5b (560 mg, 1.32 mmol) and POCl3 (4 mL,
43.56 mmol) in dioxane (17 mL) was obtained 6b (288 mg, 50%)
as a yellow solid, mp 226-229 °C (dioxane/absolute EtOH).
IR (KBr): 2220 cm-1 1H NMR (DMSO-d6): δ 7.42 (8H, m,
.
ArH), 7.55 (2H, d, J ) 8.5, ArH), 7.68 (4H, m, ArH), 7.75 (2H,
d, J ) 7.0, ArH), 7.84 (2H, d, J ) 7.9, ArH). 13C NMR (DMSO-
d6): δ 113.2, 115.1, 126.3, 126.6, 126.7, 127.9, 128.1, 128.86,
128.92, 130.0, 130.3, 131.5, 133.7, 138.5, 138.7, 140.9, 141.5,
3-Amino-4,5-bis(p-tert-butylphenyl)-1H-pyrazolo[3,4-c]-
pyridazine (1d). From 6d (1.15 g, 2.85 mmol) and NH2NH2‚

Pyridazines as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6851
H2O (360 mg, 7.20 mmol) in absolute EtOH (45 mL) was
obtained 1d (800 mg, 70%) as a yellow solid, mp >300 °C
(absolute EtOH).
3-Amino-4,5-bis(p-nitrophenyl)-1H-pyrazolo[3,4-c]pyr-
idazine (1c). From 6c (271 mg, 0.71 mmol) and NH2NH2‚H2O
(53 mg, 1.07 mmol) in absolute EtOH (6 mL) was obtained 1c
(166 mg, 62%) as a yellow solid purified by flash chromatog-
raphy (CH2Cl2/hexane 8:2), mp >300 °C (AcOEt).
mixture of CH2Cl2/THF 5:3 (8 mL) was obtained 2b (174 mg,
72%) as a red solid isolated by concentrating the solvent under
vacuum, mp 132-134 °C. IR (KBr): 1715, 3310, 3350, 3460
cm-1. 1H NMR (CDCl3): δ 1.30 (3H, t, J ) 7.1, CH3), 3.51 (2H,
m, CH2), 6.23 (2H, sa, 2NHCO), 7.23-7.47 (10H, m, ArH), 7.94
(1H, sa, NH). 13C NMR (CDCl3): δ 14.5, 35.3, 127.4, 127.8,
128.8, 129.2, 129.3, 130.2, 133.3, 136.7, 144.8, 147.3, 152.8.
MS (EI): m/z (%) 316 (M+ - 42, 26), 288 (100), 72 (32).
1-(4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)-3-
phenylurea (2c). From phenyl isocyanate (0.076 mL, 0.70
mmol) in anhydrous CH2Cl2 (1 mL) and 1a20 (200 mg, 0.70
mmol) in a mixture of CH2Cl2/THF 5:3 (5 mL) was obtained
2c (94 mg, 33%) as a yellow solid isolated by filtration, mp
239-241 °C.
1-Ethyl-3-[5-(2-furyl)1-H-pyrazolo[3,4-c]pyridazin-3-yl]-
urea (13). To a solution of ethyl isocyanate (71 mg, 1.00 mmol)
in anhydrous THF (5 mL) was added at 0 °C a solution of 1o
(201 mg, 1.00 mmol) in anhydrous THF (10 mL). The mixture
was stirred at room temperature for 24 h. Then, the solvent
was removed under reduced pressure and the crude of reaction
was purified by flash chromatography on silica gel (CHCl3/
EtOH 9.9:0.1) to give 13 (150 mg, 55%) as a red solid, mp 172-
174 °C.
1-Ethyl-3-[5-(2-furyl)-1H-pyrazolo[3,4-c]pyridazin-3-yl]-
thiourea (14). The starting materials were ethyl isothiocy-
anate (87 mg, 1.00 mmol) in anhydrous CH2Cl2 (5 mL) and 1o
(201 mg, 1.00 mmol) in a mixture of CH2Cl2/THF 5:3 (10 mL).
After 20 h, the solvent was removed under reduced pressure
and the crude of reaction was purified by flash chromatography
on silica gel (CHCl3/EtOH 9:1) to give 14 (80 mg, 28%) as a
yellow solid, mp 240-242 °C.
3-Amino-4,5-bis(p-trifluoromethylphenyl)-1H-pyrazolo-
[3,4-c]pyridazine (1e). From 6e (180 mg, 0.42 mmol) and
NH2NH2‚H2O (42 mg, 0.84 mmol) in absolute EtOH (10 mL)
was obtained 1e (120 mg, 67%) as a yellow solid purified by
flash chromatography (CHCl3/MeOH 99:1), mp 162-163 °C
(absolute EtOH).
3-Amino-4,5-bis(p-methoxyphenyl)-1H-pyrazolo[3,4-c]-
pyridazine (1f). From 6f (300 mg, 0.85 mmol) and NH2NH2‚
H2O (214 mg, 4.27 mmol) in absolute EtOH (25 mL) was
obtained 1f (286 mg, 97%) as a yellow solid purified by flash
chromatography (CHCl3/AcOEt 8:2), mp 129-130 °C (absolute
EtOH).
3-Amino-4,5-di(2-furyl)-1H-pyrazolo[3,4-c]pyridazine
(1h). From 6h (200 mg, 0.74 mmol) and NH2NH2‚H2O (55 mg,
1.11 mmol) in absolute EtOH (6 mL) was obtained 1h (136
mg, 69%) as a brown solid purified by flash chromatography
(CHCl3/MeOH 95:5), mp 250-252 °C (absolute EtOH).
3-Amino-5-methyl-4-phenyl-1H-pyrazolo[3,4-c]pyr-
idazine (1k). From 6k (300 mg, 1.31 mmol) and NH2NH2‚
H2O (98 mg, 1.96 mmol) in absolute EtOH (11 mL) was
obtained 1k (161 mg, 55%) as a yellow solid purified by flash
chromatography (AcOEt/hexane 9:1), mp 122-127 °C (AcOEt).
3-Amino-4-phenyl-1H-pyrazolo[3,4-c]pyiridazine (1l).25
From 6l (35 mg, 0.16 mmol) and NH2NH2‚H2O (12 mg, 0.24
mmol) in absolute EtOH (2 mL) was obtained 1l (30 mg, 88%)
as a yellow solid purified by flash chromatography (HCl3/
MeOH 99:1), mp 219-221 °C (AcOEt).
(4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)hydra-
zine Trihydrochloride (2d). A suspension of 1a20 (0.50 g,
1.74 mmol) in H2O (2 mL) was added dropwise to a solution
of NaNO2 (120 mg, 1.74 mmol) and concentrated HCl in H2O
(2 mL) at 0 °C for 48 h. After cooling, the formed solid was
filtered under vacuum and purified by recrystallization from
absolute EtOH to give 2d (760 mg, 75%), mp 134-136 °C. IR
3-Amino-5-phenyl-1H-pyrazolo[3,4-c]pyiridazine (1n).27
From 3-chloro-6-phenylpyridazine-4-carbonitrile 2028 (300 mg,
1.40 mmol) and NH2NH2‚H2O (105 mg, 2.10 mmol) in absolute
EtOH (12 mL) was obtained 1n (193 mg, 96%) as a yellow solid
purified by flash chromatography (CHCl3/EtOH 9:1), mp >300
°C (H2O).
3-Amino-5-(2-furyl)-1H-pyrazolo[3,4-c]pyiridazine (1o).
From 6o (206 mg, 1.00 mmol) and NH2NH2‚H2O (101 mg, 2.00
mmol) in absolute EtOH (12 mL) was obtained 1o (193 mg,
96%) as a yellow solid purified by flash chromatography
(CHCl3/EtOH 9:1), mp 206-208 °C (H2O).
3-Amino-4,5-bis(p-aminophenyl)-1H-pyrazolo[3,4-c]-
pyridazine (1m). A solution of 1c (85 mg, 0.23 mmol) in a
mixture of THF (3.5 mL) and MeOH (1.5 mL) was added
dropwise to a solution of NH2NH2‚H2O (310 mg, 6.20 mmol)
and Raney nickel (57 mg) in absolute MeOH (2 mL) under
argon atmosphere. The mixture was heated at reflux temper-
ature for 1 h, and Raney nickel was separated by filtration
and washed with MeOH. The combined filtrates were concen-
trated under reduced pressure, and the residual solid was
crystallized from MeOH to give 1m (38 mg, 52%), mp 227 °C
(dec).
1-Amino-3H-dibenzo[f,h]pyrazolo[3,4-c]cinnoline (1g).
To a solution of 6g (313 mg, 1.08 mmol) in DMF (15 mL) was
added 81 mg (1.62 mmol) of NH2NH2‚H2O. The mixture was
heated at 110 °C for 8 h. Then, the solution was cooled and
poured into ice/water. The precipitate was isolated by filtration
and purified by recrystallization from DMF/H2O to give 1g (150
mg, 50%), mp 235-239 °C.
1
(KBr): 3190, 3290, 3440 cm-1. H NMR (CDCl3): δ 3.46 (1H,
sa, OH), 3.70 (4H, sa, 2CH2), 3.91 (2H, sa, NH2), 4.04 (2H, t,
J ) 5.0, CH2), 4.76 (2H, t, J ) 5.0, CH2), 7.24-7.42 (10H, m,
ArH). 13C NMR (CDCl3): δ 46.7, 61.6, 69.5, 72.6, 107.1, 127.6,
127.8, 128.7, 129.1, 129.4, 130.4, 132.5, 133.4, 136.9, 146.3,
151.0, 152.8. MS (ESI): m/z 398 [M + Na]+. Anal. (C21H21N5O2)
C, H, N.
3-Acetamide-4,5-diphenyl-1H-pyrazolo[3,4-c]pyr-
idazine (2e). A mixture of 1a20 (200 mg, 0.70 mmol), acetyl
chloride (157 mg, 2.00 mmol), and two drops of Et3N in dioxane
(4 mL) was heated under reflux for 30 min. Then, the solvent
was removed under reduced pressure and the crude of reaction
was purified by flash chromatography on silica gel (CHCl3/
MeOH 99:1) to give 2e (180 mg, 78%) as a white solid, mp
262-264 °C (lit.29 mp 228-229 °C, absolute EtOH/toluene).
IR (KBr): 1670, 3185, 3210, 3440 cm-1. 1H NMR (DMSO-d6):
δ 1.48 (3H, s, CH3), 7.15 (2H, m, ArH), 7.30 (8H, m, ArH), 9.81
(1H, s, NHCO), 12.92 (1H, s, NH). 13C NMR (DMSO-d6): δ
21.9, 110.6, 127.69, 127.74, 127.9, 128.3, 129.9, 130.5, 131.8,
132.7, 137.5, 138.6, 152.0, 154.9, 170.0. MS (EI): m/z (%) 329
(M+, 43), 286 (100), 77 (9), 51 (6). Anal. (C19H15N5O) C, H, N.
1-Acetyl-3-amino-4,5-diphenyl-1H-pyrazolo[3,4-c]pyr-
idazine (2f). A mixture of 1a20 (400 mg, 1.39 mmol) and acetyl
chloride (0.14 mL, 1.96 mmol) in pyridine (3 mL) was heated
under reflux for 1 h. Then, the pH was adjusted to 5 with
aqueous hydrochloric acid. The precipitate formed was col-
lected by filtration to give 2f (305 mg, 66%) as a brown solid,
mp 246-248 °C (toluene). IR (KBr): 1695, 3195, 3250, 3485
General Procedure for the Preparation of Ureas. To
a solution of the corresponding isocyanate in anhydrous CH2-
Cl2 was added, at 0 °C, a solution of the corresponding
aminopyrazolo[3,4-c]pyridazine in a mixture of CH2Cl2/THF
5:3. The mixture was stirred at room temperature for 20 h.
Then, the desired compound was isolated by filtration or
concentrated the solvent under vacuum.
1
cm-1. H NMR (DMSO-d6): δ 2.77 (3H, s, CH3), 5.31 (2H, s,
NH2), 7.35 (7H, m, ArH), 7.48 (3H, m, ArH). 13C NMR (DMSO-
d6): δ 24.4, 112.5, 127.9, 128.2, 128.8, 129.3, 129.4, 130.1,
131.8, 133.1, 136.5, 149.6, 153.3, 154.6, 167.0. Anal. (C19H15N5O)
C, H, N.
1-(4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)-3-
ethylurea (2b). From ethyl isocyanate (48 mg, 0.68 mmol) in
anhydrous CH2Cl2 (1 mL) and 1a20 (180 mg, 0.68 mmol) in a
3-Acetamide-1-acetyl-4,5-diphenyl-1H-pyrazolo[3,4-c]-
pyridazine (2g). A mixture of 1a20 (200 mg, 0.70 mmol) in
acetic anhydride (1 mL) and acetic acid (1 mL) was heated

6852 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Bran˜a et al.
under reflux for 90 min. Then, the solution was cooled and
poured into ice/water. The precipitate was isolated by filtration
and purified by recrystallization to give 2g (150 mg, 58%) as
a white solid, mp 174-175 °C (AcOEt/hexane). IR (KBr): 1785,
vacuum and purified by recrystallization from absolute EtOH
to give 2j (760 mg, 75%), mp 134-136 °C. IR (KBr): 3190,
3290, 3440 cm-1. 1H NMR (CDCl3): δ 3.46 (1H, sa, OH), 3.70
(4H, sa, 2CH2), 3.91 (2H, sa, NH2), 4.04 (2H, t, J ) 5.0, CH2),
4.76 (2H, t, J ) 5.0, CH2), 7.24-7.42 (10H, m, ArH). 13C NMR
(CDCl3): δ 46.7, 61.6, 69.5, 72.6, 107.1, 127.6, 127.8, 128.7,
129.1, 129.4, 130.4, 132.5, 133.4, 136.9, 146.3, 151.0, 152.8.
MS (ESI): m/z 398 [M + Na]+. Anal. (C21H21N5O2) C, H, N.
CDK1/Cyclin B Enzyme Inhibition. Biochemical Re-
agents. Sodium orthovanadate, EGTA, EDTA, Mops, â-glyc-
erophosphate, phenyl phosphate, sodium fluoride, dithiothre-
itol (DTT), glutathione agarose, glutathione, bovine serum
albumin (BSA), nitrophenyl phosphate, leupeptin, aprotinin,
pepstatin, soybean trypsin inhibitor, benzamidine, and histone
H1 (type III-S) were obtained from Sigma Chemicals. [γ-32P]-
ATP (PB 168) was obtained from Amersham.
The GS-1 peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE)
was synthesized by the Peptide Synthesis Unit, Institute of
Biomolecular Sciences, University of Southampton, Southamp-
ton SO16 7PX, U.K.
Buffers. Homogenization buffer consisted of 60 mM â-glyc-
erophosphate, 15 mM p-nitrophenyl phosphate, 25 mM Mops
(pH 7.2), 15 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM
sodium vanadate, 1 mM NaF, 1 mM phenyl phosphate, 10 µg
of leupeptin/mL, 10 µg of aprotinin/mL, 10 µg of soybean
trypsin inhibitor/mL, and 100 µM benzamidine. Buffer A
consisted of 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM
Tris-HCl, pH 7.5, 50 µg of heparin/mL. Buffer C was the same
as the homogenization buffer but with 5 mM EGTA, no NaF,
and no protease inhibitors.
Kinase Preparations and Assays. Kinase activities were
assayed in buffer A or C (unless otherwise stated), at 30 °C,
at a final ATP concentration of 15 µM. Blank values were
subtracted and activities calculated as pmoles of phosphate
incorporated for a 10 min incubation. The activities are usually
expressed in percent of the maximal activity, i.e., in the
absence of inhibitors. Controls were performed with appropri-
ate dilutions of dimethyl sulfoxide.
CDK1/cyclin B was extracted in homogenization buffer from
M. phase starfish (Marthasterias glacialis) oocytes and purified
by affinity chromatography on p9CKShs1-sepharose beads, from
which it was eluted by free p9CKShs1 as previously described.15
The kinase activity was assayed in buffer C, with 1 mg of
histone H1/mL, in the presence of 15 µM [γ-33P]ATP (3000 Ci/
mmol, 1 mCi/mL) in a final volume of 30 µL. After 10 min of
incubation at 30 °C, 25 µL aliquots of supernatant were spotted
onto P81 phosphocellulose papers and treated as described
above.
In Vitro Cytotoxicity Assays. The cell lines used were
human colon carcinoma (HT-29) (ATCC, HTB 38), human
cervical carcinoma (HeLa) (ATCC, CCL 2), and human pros-
tate carcinoma (PC-3) (ECACC, 90112714). For each experi-
ment, cultures were seeded from frozen stocks. Each cell line
was maintained in its appropriate medium and was incubated
at 37 °C in a 5% CO2 atmosphere.
All cell lines were in the logarithmic phase of growth when
the assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) was carried out. Cells were harvested and
seeded into 96-well tissue culture plates at a density of 2.5 ×
103 cells/well in 150 µL aliquots of medium. The concentrations
tested were serial dilutions of a stock solution (1 × 10-5 M in
DMSO) with phosphate-buffered saline (PBS) and were added
24 h later. The assay was ended after 72 h of drug exposure,
and PBS was used as a negative control and doxorubicine as
a positive control.
3160, 3550 cm-1 1H NMR (DMSO-d6): δ 1.46 (3H, s, CH3-
.
CONH), 2.90 (3H, s, CH3CON), 7.17 (2H, m, ArH), 7.35 (8H,
m, ArH), 10.32 (1H, sa, NH). 13C NMR (DMSO-d6): δ 21.7,
24.3, 114.9, 127.7, 127.9, 128.2, 128.6, 129.9, 130.2, 131.6,
133.1, 136.6, 142.8, 153.7, 155.4, 167.3, 169.8.
3-Amino-1-benzyl-4,5-diphenyl-1H-pyrazolo[3,4-c]pyr-
idazine (2h). To a solution of 6a20 (500 mg, 1.72 mmol) in
absolute EtOH (20 mL) was added 667 mg (3.42 mmol) of
benzylhydrazine and 0.71 mL (5.13 mmol) of Et3N. The
mixture was stirred at reflux temperature for 24 h. Then, the
solvent was removed under reduced pressure and the crude
of reaction was purified by flash chromatography on silica gel
(CHCl3/EtOH 20:1) to give 2h (225 mg, 35%) as a yellow solid,
mp 167-169 °C (AcOEt/hexane). IR (KBr): 3180, 3280, 3450
1
cm-1. H NMR (DMSO-d6): δ 3.87 (2H, sa, NH2), 5.73 (2H, s,
CH2), 7.26-7.52 (15H, m, ArH). 13C NMR (DMSO-d6): δ 50.7,
106.9, 127.6, 127.8, 128.4, 128.6, 128.7, 129.0, 129.4, 130.4,
130.8, 132.3, 133.5, 136.8, 137.0, 146.4, 151.0, 152.6. Anal.
(C24H19N5) C, H, N.
2-[(3-Amino-4,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-
1-yl)methoxy]ethyl Acetate (12i). Cs2CO3 (284 mg, 0.87
20
mmol) was added in portions to a solution of 1a (250 mg,
0.87 mmol) in DMF (6 mL). The mixture was stirred at room
temperature for 30 min. Then, 2-bromomethoxyethyl acetate14
(172 mg, 0.87 mmol) was added and the solution was stirred
for 24 h. Then, the solvent was eliminated under vacuum and
the formed solid was extracted with AcOEt. The combined
extracts were dried over MgSO4 and concentrated to give a
solid, which was purified by flash chromatography (CH2Cl2/
MeOH 98:2) to give 12i (122 mg, 35%), mp 143-144 °C (AcOEt/
1
hexane). IR (KBr): 1740, 3340, 3470 cm-1. H NMR (CDCl3):
δ 2.07 (3H, s, CH3), 3.90 (2H, dd, J ) 4.4, 4.9, CH2), 3.97 (2H,
sa, NH2), 4.23 (2H, dd, J ) 4.4, 4.9, CH2), 5.97 (2H, s, CH2N),
7.25-7.44 (10H, m, ArH). 13C NMR (CDCl3): δ 20.8, 63.1, 67.4,
75.5, 107.9, 127.7, 127.8, 128.7, 129.1, 129.3, 130.2, 132.5,
133.0, 136.6, 147.2, 152.0, 153.4, 170.8. Anal. (C22H21N5O3) C,
H, N. Further elution afforded 127 mg (51%) of 1a.20
2-[2-(3-Amino-4,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-
1-yl)ethoxy]ethyl Acetate (12j). Cs2CO3 (1.14 g, 3.49 mmol)
was added in portions to a solution of 1a20 (1.00 g, 3.49 mmol)
in DMF (24 mL). The mixture was stirred at room temperature
for 30 min. Then, 2-(2-iodoethoxy)ethyl acetate30 (900 mg, 3.49
mmol) was added and the solution was stirred for 24 h. Then,
the solvent was eliminated under vacuum and the formed solid
was extracted with AcOEt. The combined extracts were dried
over MgSO4 and concentrated to give a solid, which was
purified by flash chromatography (CHCl3/EtOH 9:1) to give
12j (1.14 g, 78%), mp 157-159 °C (AcOEt). IR (KBr): 1730,
1
3340, 3485 cm-1. H NMR (CDCl3): δ 2.02 (3H, s, CH3), 3.75
(2H, t, J ) 4.9, CH2), 3.88 (2H, sa, NH2), 4.05 (2H, t, J ) 5.5,
CH2), 4.17 (2H, m, CH2), 4.75 (2H, t, J ) 5.5, CH2), 7.25-7.42
(10H, m, ArH). 13C NMR (CDCl3): δ 20.8, 46.2, 63.4, 68.5, 68.9,
106.9, 127.5, 127.8, 128.7, 129.0, 129.4, 130.3, 132.2, 133.5,
137.0, 146.1, 150.9, 152.9, 170.9. Anal. (C23H23N5O3) C, H, N.
2-[(3-Amino-4,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-
1-yl)methoxy]ethanol (2i). A solution of 12i (2.24 g, 5.56
mmol) in 1 N NH4OH (24 mL) was heated at 50-60 °C for 24
h. After the mixture was cooled, the formed solid was filtered
under vacuum and purified by flash chromatography (CHCl3/
EtOH 9:1) to give 2i (1.40 g, 70%), mp 178-181 °C (absolute
EtOH). IR (KBr): 3295, 3330, 3430 cm-1. 1H NMR (CDCl3): δ
3.76 (2H, m, CH2), 3.84 (2H, m, CH2), 4.00 (2H, sa, NH2), 5.99
(2H, s, CH2N), 7.27-7.44 (10H, m, ArH). 13C NMR (CDCl3): δ
61.9, 71.2, 75.9, 108.2, 127.9, 128.0, 128.9, 129.3, 129.5, 130.4,
132.8, 133.2, 136.7, 147.4, 152.2, 153.6. MS (ESI): m/z 384
[M + Na]+. Anal. (C20H19N5O2) C, H, N.
After a 72 h exposure period, cells were washed twice with
PBS, and then 50 µL/well of MTT reagent (1 mg/mL in PBS;
Sigma) and 150 µL/well of prewarmed medium were added.
The plates were returned to the incubator for 4 h. Subse-
quently, DMSO was added as solvent. Absorbance was deter-
mined at 570 nm with a microplate reader (Opsys MR).
2-[2-(3-Amino-4,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-
1-yl)ethoxy]ethanol (2j). A solution of 12j (1.12 g, 2.69 mmol)
in 1 N NH4OH (60 mL) was heated at 50-60 °C for 48 h. After
the mixture was cooled, the formed solid was filtered under
All experiments were performed at least three times, and
the average of the percentage absorbance was plotted against

Pyridazines as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6853
concentration. Then, the concentration of drug required to
inhibit 50% of cell growth (IC50) was calculated for each
compound.
progressively minimizing its potential energy. The optimiza-
tions were carried out in a continuum medium of relative
permittivitty ꢀ ) 4rij for imitating the solvent environment.
The resulting complex was considered to be the initial struc-
ture for the molecular dynamics simulation in water.
Molecular Dynamics in Water. The system was placed
into a 20 Å radius spherical cap of 328 TIP3P water molecules
centered on the center of mass of the bound ligand. The initial
complex was refined by progressively minimizing its potential
energy. Unrestrained 1000 ps MD simulations at 298 K and 1
atm were then run for the complex using the SANDER module
in AMBER. SHAKE42 was applied to all bonds involving
hydrogens, and an integration step of 2 fs was used through-
out. The simulation protocol involved a series of progressive
energy minimizations followed by a 10 ps heating phase and
10 ps equilibration period before data collection. System
coordinates were saved every 2 ps for further analysis.
Analysis of the MD Trajectories. Three-dimensional
structures and trajectories were visually inspected using the
computer graphics program SYBYL.31 Root-mean-square (rms)
deviations form the initial structures, and interatomic dis-
tances were monitored using CARNAL.40 An average structure
from the last 500 ps of the MD simulations in water was
refined by means of steepest-descent energy minimizations,
and the resulting complex was used for the energy analysis
using ANAL.
Computational Details. Molecular Modeling and Con-
formational Searches. Compounds 1a and the synthetic
derivatives (1b-n and 2a-c,e,f) were model built in SYBYL
6.731 using standard geometries. Conformational searches and
energy minimizations were performed using Macromodel,
version 5.5.32 The Macromodel implementation of the AM-
BER33 all-atom force field was used (denoted AMBER*). All
calculations were performed using the implicit water GB/SA
solvation model of Still et al.34 Conformational searches were
performed using the Monte Carlo method of Goodman and
Still.35 For each search 1000 starting structures were gener-
ated and minimized to an energy convergence of 0.05 (kJ/
mol)/Å using the Polak-Ribiere conjugate gradient minimi-
zation method implemented in Macromodel. Duplicated
structures and those greater than 50 kJ/mol above the global
minimum were discarded. The lowest energy conformer for
each compound was then fully optimized by means of the ab
initio quantum mechanical program Gaussian 9836 and the
3-21G basis set. RHF/6-31G*//3-21G RESP charges37 were
derived together with appropriate bonded and nonbonded
parameters consistent with the AMBER force field38 (Tables
3 and 4 in Supporting Information). These low-energy mini-
mized conformers were selected to be used as input for the
ligand docking process.
CDK2 Model Building and Energy Refinement. For the
flexible ligand docking the CDK2-ATP complex structure was
directly retrieved from the Protein Data Bank (PDB)39 (code
1hck). The ATP molecule, the magnesium ion, and water
molecules were manually removed. Polar hydrogens and
Kollman united-atom partial atomic charges were added by
means of the SYBYL program.31 For the molecular dynamics
simulations the same starting structure was used; however,
some preliminary model building and energy refinement had
to be undertaken. The 10 missing amino acids from the 1hck
crystal structure (Ala31 through Thr41) were directly taken
from the CDK2/cyclin A/ATP complex (code 1fin) after super-
imposition of common parts in both structures. Hydrogens
were added using standard geometries, and their positions
were optimized using the molecular mechanics program
AMBER.40 A short optimization was then undertaken where
only the residues connecting the added residues were allowed
to move (Thr39 through Val44 and Glu28 through Lys33) in a
continuum medium of relative permittivitty ꢀ ) 4rij for
imitating the solvent environment. Finally, an optimization
run restraining all non-H atoms to their initial coordinates
allowed readjustment of covalent bonds and van der Waals
contact without changing the overall conformation of the
protein, which was considered the starting structure for the
molecular dynamics simulations described below.
Acknowledgment. We thank Carlos Pe´rez (Lilly S.
A., Spain) for his help with AutoDock. This work was
supported by the Direccio´n General de Estudios Supe-
riores (Grant No. PB98-0055), CYTED, and the Uni-
versidad San Pablo-CEU (Grant No. 5-99/01). M.C.
thanks Fundacio´n Ramo´n Areces for financial support.
We thank the CIEMAT (Spain) for computer time and
facilities. The kinase assays were supported by the
Ministe`re de la Recherche/INSERM/CNRS “Mole´cules
et Cibles The´rapeutiques” Program and a grant from
the EEC (FP6-2002-Life Sciences and Health, PRO-
KINASE Research Project) (L.M.).
Supporting Information Available: Experimental de-
tails of compounds reported, AMBER parameters and partial
atomic charges for compound 1a (Tables 3 and 4), docking
parameters (Table 5), and microanalysis data for evaluated
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Knockaert, M.; Greengard, P.; Meijer, L. Pharmacological Inhibi-
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(2) Malumbre, M.; Barbacid, M. To Cycle or Not To Cycle: A Critical
Decision in Cancer. Nat. Rev. Cancer 2001, 1, 222-231.
(3) Huwe, A.; Mazitschek, R.; Giannis, A. Small Molecules as
Inhibitors of Cyclin-Dependent Kinases. Angew. Chem., Int. Ed.
2003, 42, 2122-2138.
(4) Sielecki, T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G. L. Cyclin
Dependent Kinase Inhibitors: Useful Targets in Cell Cycle
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(5) Senderowicz, A. M.; Headlee, D.; Stinson, S. F.; Lush, R. M.;
Kalil, N.; Villalba, L.; Hill, K.; Steinberg, S. M.; Figg, W. D.;
Tompkins, A.; Arbuck, S. G.; Sausville, E. A. Phase I Trial of
Continuous Infusion Flavopiridol, a Novel Cyclin-Dependent
Kinase Inhibitor, in Patients with Refractory Neoplasms. J. Clin.
Oncol. 1998, 16, 2986-2999.
(6) Sedlacek, H. H.; Czech, J.; Naik, R.; Kaur, G.; Worland, P.;
Losiewicz, M.; Parker, B.; Carlson, B.; Smith, A.; Senderowicz,
A.; Sausville, E. Flavopiridol (L86-8275, NSC-649890), a New
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(7) Akinaga, S.; Sugiyama, K.; Akiyama, T. UCN-01 (7-Hydroxys-
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Docking. All docking studies were performed with the
program AutoDock (version 3.0).41 The target in each docking
run was CDK2 obtained as described above. Affinity grid files
were generated using the auxiliary program AUTOGRID
(version 3.0). The center of the removed ATP molecule bound
to the active site was chosen as the center of the grids, and
the dimensions of the grid were 60 × 60 × 60 Å3 with grid
points separated by 0.375 Å. The original Lennard-Jonnes and
hydrogen-bonding potentials provided by the program were
used. The parameters for the docking using the Lamarckian
genetic algorithm (LGA) were identical for all docking jobs and
are summarized in Table 5 (Supporting Information).
After docking, the 100 solutions were clustered in groups
with rms deviations less than 1.0 Å. The clusters were ranked
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