Identification of potent pyrazolo[4,3-h]quinazoline-3-carboxamides as multi-cyclin-dependent kinase inhibitor

Article abstract of DOI:10.1021/jm901710h

Abnormal proliferation mediated by disruption of the mechanisms that keep the cell cycle under control is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDKs) and cyclins (Cy) and inhibiting their activity are regarded as promising antitumor agents to complement the existing therapies. An expansion of pyrazolo[4,3-h]quinazoline chemical class oriented to the development of three points of variability was undertaken leading to a series of compounds able to inhibit CDKs both in vitro and in vivo. Starting from the CDK selective but poorly soluble hit compound 1, we succeeded in obtaining several compounds showing enhanced inhibitory activity both on CDKs and on tumor cells and displaying improved physical properties and pharmacokinetic behavior. Our study led to the identification of compound 59 as a highly potent, orally bioavailable CDK inhibitor that exhibited significant in vivo efficacy on the A2780 ovarian carcinoma xenograft model. The demonstrated mechanisms of action of compound 59 on cancer cell lines and its ability to inhibit tumor growth in vivo render this compound very interesting as potential antineoplastic agent.

Full text of DOI:10.1021/jm901710h

J. Med. Chem. 2010, 53, 2171–2187 2171
DOI: 10.1021/jm901710h
Identification of Potent Pyrazolo[4,3-h]quinazoline-3-carboxamides as Multi-Cyclin-Dependent
Kinase Inhibitors†
Gabriella Traquandi,* Marina Ciomei, Dario Ballinari, Elena Casale, Nicoletta Colombo, Valter Croci, Francesco Fiorentini,‡
Antonella Isacchi, Antonio Longo, Ciro Mercurio,§ Achille Panzeri, Wilma Pastori, Paolo Pevarello, Daniele Volpi,
Patrick Roussel,^ Anna Vulpetti,# and Maria Gabriella Brasca
Nerviano Medical Sciences Srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy, and ‡Accelera Srl, Viale Pasteur 10, 20014
Nerviano (MI), Italy
Received November 18, 2009
Abnormal proliferation mediated by disruption of the mechanisms that keep the cell cycle under control
is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent
kinases (CDKs) and cyclins (Cy) and inhibiting their activity are regarded as promising antitumor agents
to complement the existing therapies. An expansionofpyrazolo[4,3-h]quinazoline chemicalclass oriented
to the development of three points of variability was undertaken leading to a series of compounds able to
inhibit CDKs both in vitro and in vivo. Starting from the CDK selective but poorly soluble hit compound
1, we succeededin obtaining several compounds showing enhanced inhibitoryactivity both on CDKs and
on tumor cells and displaying improved physical properties and pharmacokinetic behavior. Our study led
to the identification of compound 59 as a highly potent, orally bioavailable CDK inhibitor that exhibited
significant in vivo efficacy on the A2780 ovarian carcinoma xenograft model. The demonstrated
mechanisms of action of compound 59 on cancer cell lines and its ability to inhibit tumor growth in
vivo render this compound very interesting as potential antineoplastic agent.
Introduction
Progression through the different phases of the eukaryotic
cell cycle has beenshown tobecritically dependent on a family
of proteins known as cyclin-dependent kinases (CDKsa),
which specifically form heterodimeric complexes with regula-
tory subunits named cyclins (Cy).1 Different CDK/Cy com-
plexes finely regulate each cell cycle phase transition: CDK2/
Figure 1. Structure of pyrazolo[4,3-h]quinazoline scaffold and hit
compound 1.
CyE, CDK4/CyD, and CDK6/CyD primarily regulate pas-
sage through the G1 phase and the transition from G1 to S
phase, while CDK2/CyA and CDK1/CyA primarily execute
their critical function during the S phase and control progres-
sion through the G2 phase. CDK1/CyB complex intervenes
then as a key initiator of the M phase or mitosis. These CDKs
phosphorylate a wide variety of proteins whose function is
required over the cell cycle, thereby modulating their activity
and consequently affecting cell growth and survival by several
mechanisms.2 A second group of CDKs, which include
CDK7, CDK8, and CDK9, is involved in transcriptional
regulation so that while these CDKs do not directly impinge
on the cell cycle, their inhibition can however affect the
expression of several cell cycle regulators and mitotic regula-
tory kinases, as well as of apoptosis mediators. Another
member of the family, CDK5, plays a critical role in neuronal
and secretory cell function.3 Interphase CDK/Cy complexes
cooperate in phosphorylating the retinoblastoma protein
(pRb), thereby converting it from a repressor to an activator
of transcription factors that mediate the transcription of genes
required for DNA synthesis.4 pRb function is impaired in
most human neoplasms and loss in pRb repressive activity is a
consequence of CDK hyperactivation. Aberrant cell prolif-
eration that occurs in the majority of human malignancies can
often be attributed to a loss of the correct cellcycle control and
in fact a variety of genetic and epigenetic events can cause
†Coordinates of the CDK2 complex with compound 59 have been
deposited in the Protein Data Bank under accession code 2WXV,
together with the corresponding structure factor files.
§Present address: Genextra Group, Milano, Italy.
Present address: Newron, Milano, Italy.
^Present address: Basilea Pharmaceutica LTD, Switzerland.
#Present address: Novartis Pharma AG, Basel, Switzerland.
*To whom correspondence should be addressed. Phone: þ39-0331-
581330. Fax: þ39-0331-581347. E-mail: gabriella.traquandi@
nervianoms.com.
a Abbreviations: CDK, cyclin-dependent kinase; Cy, cyclin; CDK2/
CyA, (cyclin-dependent kinase 2)/(cyclin A); Aur-A, Aurora A; pRb,
retinoblastoma protein; CKI, cyclin-dependent kinase inhibitor; GSK-
3β, glycogen synthase kinase 3β; ERK2, extracellular signal-regulated
kinase 2; ADME, adsorption-distribution-metabolism-excretion; PK,
pharmacokinetics; CL, clearance; Vss, distribution volume; AUC, area
under curve; F, oral bioavailability; BrdU, bromodeoxyuridine; EDC,
N-(3-dimethylaminopropyl)-N0-ethyl-carbodiimide hydrochloride; HOBT,
1-hydroxybenzotriazole; HOBT NH3, 1-hydroxybenzotriazole ammo-
3
nium salt; DIPEA, N,N-diisopropyl-N-ethylamine; TGI, tumor growth
inhibition; PAMPA, parallel artificial membrane permeability assay;
ECACC, european collection of cell cultures; FBS, fetal calf serum; PBS,
phosphate buffered saline; RNase A, ribonuclease A.
r
2010 American Chemical Society
Published on Web 02/08/2010
pubs.acs.org/jmc

2172 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Traquandi et al.
Scheme 1. Synthesis of Final Compounds 1, 14, 19-26, and 28a
a Reagents and conditions: (a) S-methylisothiourea, AcOK, DMF, 90 ꢀC, 74%; (b) NH4OH, MeOH-DMF, 80 ꢀC, 52%; (c) oxone, DMF, rt, 80%;
(d) R3NH2, DMSO, 50-120 ꢀC, 40-72%.
Scheme 2. Synthesis of Final Compounds 18, 27, 29, 30, and 47-49a
a Reagents and conditions: (a) O-methylisourea, K2CO3, acetonitrile, reflux, 16 h, 80%; (b) trimethylsilyl chloride, NaI, rt, 8 h, 78%;
(c) trifluoromethansulfonic anhydride, Et3N, CH2Cl2, from -78 ꢀC to rt, 5 h, 67%; (d) R3NH2, dioxane, rt, 70-85%; (e) EtOH, KOH, reflux,
75-80%; HOBT NH3, EDC, DIPEA, DMF-THF, rt, 61-73%; (f) NH4OH, MeOH-DMF, 70 ꢀC, 60%; (g) CH3NH2, EtOH, 60 ꢀC, 50-78%.
3
Scheme 3. Synthesis of Final Compounds 15-17, 31-36, and 50-61a
a Reagents and conditions: (a) guanidine hydrochloride, EtONa, EtOH, reflux, 12 h, 85%; (b) NH4OH, MeOH-DMF, 70 ꢀC, 50-65%; (c) CH3NH2,
EtOH, 60 ꢀC, 78%; (d) ArCHO, NaBH3CN, AcOH-MeOH-H2O 1/1/1, rt, 58-62%; (e) cycloalkylketone, NaBH(OAc)3, TFA, DMF, rt, 50-73%;
(f) KOH, EtOH, reflux; R1NH2, EDC, HOBT, DIPEA, DMF-THF, rt, 31-51%; (g) KOH, EtOH, reflux; HOBT NH3, EDC, DIPEA, DMF-THF, rt,
3
43-49%; (h) KOH, EtOH, reflux; (COCl)2, R1NHOH, rt, 36-62%.
globaloveractivationofthe cellcycle.5 Therefore, the develop-
ment of agents able to modulate CDK activity may have a
strong impact on the prevention and therapy of cancer.
The cell produces also specific inhibitors of CDKs known
as cyclin-dependent kinase inhibitors (CKIs), which directly
compete with ATP for binding to the CDK active site.6 In
some cancer types, under-expression of these endogenous
CKIs has been demonstrated and these findings have solicited
the effort of medicinal chemists aimed at replacing CKIs with
synthetic small molecule inhibitors. The first CDK inhibitors
evaluated in clinical trials were Flavopiridol (L868275)7 and
7-hydroxystaurosporine (UCN-01).8 The first one has been

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2173
Table 1. R3 Preliminary Explorationa
Scheme 4. Synthesis of Final Compounds 37-46a
a Reagents and conditions: (a) R2NHNH2, AcOH, rt, 63-80%;
(b) dimethylformamide di-tert-butylacetale, DMF, 60 ꢀC; guanidine
hydrochloride, EtONa, EtOH, reflux, 45-72%; (c) cyclopentylketone,
NaBH(OAc)3, TFA, DMF, rt, 65-80%; (d) NH4OH, MeOH-DMF,
70 ꢀC, 75-80%; (e) CH3NH2, EtOH, 60 ꢀC, 78%.
granted orphan drug status for the treatment of chronic
lymphocytic leukemia. A second generation of CDK inhibi-
tors endowed with higher selectivity have also entered clinical
trials, such as Roscovitine (CYC-202)9 and BMS-387032,10
targeting primarily CDK2, but also CDK7 and 9 and
PD0332991,11 targeting CDK4 and 6. Recently, several stu-
dies have suggested that CDKs and their cyclin partners can
substitute and compensate for one another, leading to the
notion that the inhibition of more than one CDK might be
necessary to sustain the suppression of tumor growth.12 For
example, CDK1 is able to replace CDK2 in the case of its
absence or inhibition. Indeed, more recent products under
clinical study are endowed with a multi-CDK inhibitor activ-
ity, such as AT751913 (CDK1, 2, 4, and 5 inhibitor), R54714
(CDK1, 2, and 4 inhibitor), SCH-72796515 (CDK1, 2, 5, and 9
inhibitor), and AZD559716 (CDK1, 2, and 9 inhibitor).
We recently reported on a series of pyrazolo[4,3-h]quinazo-
line-3-carboxamides as CDK inhibitors.17,18 Previous expan-
sion of this class led to the identification of PHA-848125 as a
potent, orally available compound, mainly targeting CDK2
and TRKA, currently undergoing phase II clinical trials.
Further exploring this class (Figure 1) with the objective
to obtain a multi-CDK inhibitor, we identified compound
1, endowed with an interesting activity profile: CDK2/CyA
IC50=0.051 μM, CDK2/CyE IC50=0.074 μM, CDK1/CyB
IC50 = 0.120 μM, CDK5 IC50 = 0.049 μM. Compound 1
presented a significant selectivity when evaluated on a panel
of 35 serine-threonine and tyrosine kinases (data not shown)
but only a moderate antiproliferative activity in A2780 hu-
man ovarian carcinoma cells (IC50=0.933 μM) and very low
solubility (<1.0 μM). An expansion program based on the
development of three diversity points of the scaffold, R1, R2,
and R3, was then undertaken aimed at obtaining more potent
compounds both on CDKs and on tumor cells, with im-
proved physical properties and pharmacokinetic profile.
a Values are the means of two or more experiments.
derivative 5, suitable for introducing diversity point R3 by
nucleophilic substitution, thus, affording final compounds 1,
14, 19-26, and 28.
The low solubility of intermediate 5, together with the
moderate reactivity of the methansulfonyl group toward
nucleophilic substitution, prompted us to carry out step d in
dimethylsulfoxide at high temperatures. In some cases, partial
aromatization of the scaffold central ring occurred in these
conditions, lowering yields and complicating the product
purification process.
An alternative route was also designed to overcome these
issues exploiting the trifluoromethanesulfonate moiety, which
being a better leaving group than the methansulfonyl group,
would have allowed to carry out the reaction more efficiently
already at room temperature, thus, avoiding the central ring
oxidation side reaction. The synthetic pathway to obtain
Chemistry
The synthetic route to prepare the new pyrazolo[4,3-h]-
quinazoline-3-carboxamide series started from compound 2
(Scheme 1), a key intermediate for the development of
this chemical class, whose synthesis we have previously de-
scribed.17,18 In a first approach, the pyrimidine moiety was
built by condensation of compound 2with S-methylisothiourea,
affording 2-thiomethylpyrimidino derivative 3, that was sub-
mitted to ammonolysis to give carboxamide 4. The latter was
then oxidized with oxone to the corresponding sulfonyl

2174 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Table 2. R2 and R1 Modificationa
Traquandi et al.
a Values are the means of two or more experiments.
the proper intermediate entailed the condensation of2 with O-
methylisourea, providing derivative 6 (Scheme 2), which
afforded, through methoxy group hydrolysis, intermediate
7. Thetriflate8 was finallyobtained by reactionwithtrifluoro-
methansulfonic anhydride. Substituted amino groups were
introduced in intermediate 8 by nucleophilic substitution with
the proper amine, followed by transformation of the ester
group into amide. This step was performed by hydrolysis of
the carbetoxy group under basic conditions to the correspon-
ding potassium carboxylate, followed by condensation with
N-hydroxy-benzotriazole ammonium salt, as a synthetic
equivalent of ammonia, to give the final amides 18 and 29.
Alternatively, final compounds were obtained by ammono-
lysis with ammonium hydroxide (27, 48) or by aminolysis with
methylamine (30, 47, 49).
starting from 10b by reductive amination with the proper
cycloalkylketone, thus, affording final compounds 51, 53,
55, 57, 59, and 61.
In some cases, for reactivity reasons, it was profitable to
invert the order of the sequence steps so that R3 was first
introduced into the scaffold from intermediate 9 via reductive
amination with the suitable cycloalkylketone, then the R1
diversity point was constructed by several methodologies.
Thus, ammonolysis with ammonium hydroxide afforded
compounds 54 and 58, while basic hydrolysis to the corre-
sponding carboxylic acids followed either by condensation
with the suitable amine or with HOBT NH3 provided com-
3
pounds 31-33 and 50, 52, 56, and 60, respectively. Alterna-
tively, a sequence of basic hydrolysis, acyl chloride formation
and reaction with the proper substituted hydroxylamine
afforded compounds 34-36.
Diversity point R3 could also be introduced in the scaf-
fold by condensing the versatile intermediate 2 with guani-
dine to give compound 9 (Scheme 3) that allowed the
preparation of 10a and 10b, by ammonolysis or by amino-
lysis with methylamine, respectively. R3 was introduced
either starting from 10a, by reductive amination with the
suitable arylaldehyde giving final compounds 15-17, or
The diversity point R2 was regioselectively introduced into
the pyrazole moiety of the scaffold by condensation of
compound 11, prepared as previously described,17,18 with
the suitable substituted hydrazine, providing intermediates
12a-i (Scheme 4); reaction of 12a-i with N,N-dimethylfor-
mamide di-tert-butyl acetale, followed by condensation with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2175
Table 3. R3 and R1 Optimizationa
a Values are the means of two or more experiments.
guanidine hydrochloride afforded compounds 13a-i. R3 was
then introduced in the scaffold from 13a-i by reductive
amination with cyclopentyl ketone and R1 diversity point
was finally built by ammonolysis with ammonium hydroxide
(37-44 and 46) or by aminolysis with methylamine (45).
relative to Aur-A being >20ꢀ. Indeed, Aur-A, consistently
with its similarity to CDK2 in its ATP binding pocket, was the
most frequently cross-affected kinase in the selectivity panel
used to characterize our compounds and, therefore, in the
SAR tables, Aur-A IC50 will be reported as an index of
selectivity. Unfortunately, good activity on CDK2/CyA and
good selectivity were generally accompanied by poor solubi-
lity (14-17) and low antiproliferative activity on cells, with
the exception of compounds 16, 17, and 19, which showed an
IC50 < 1 μM. To improve the solubility, some alkyl deriva-
tives bearing hydrophilic or basic groups were also prepared
(24-26), which still displayed, with the exception of 26, good
inhibitory potency on the enzyme and selectivity versus Aur-
A, but theiractivityoncells was poor. Conversely, exploration
of the subclass of cycloalkyl derivatives (27-29) was quite
promising. Good solubility was achieved with compound 28,
but its potency against CDK2/CyA was the lowest in this
subclass; instead, derivatives 27 and 29, even though still
scarcely soluble, were potent enzyme inhibitors and, in addi-
tion, showed a significant improvement in the antiprolifera-
tive activity in A2780 human ovarian carcinoma cells and an
Results and Discussion
We initially focused our attention on R3 modifications by
pursuing a synthetic strategy that allowed the introduction of
this group in a late step, thus, enabling a convenient parallel
approach (Table 1). While the unsubstituted compound 10a
did not show any biological activity, benzyl derivative 1
showed an interesting activity profile, suggesting that the
involved diversity point deserved a more in-depth investiga-
tion. In this perspective a series of substituted arylmethyl and
heteroarylmethyl derivatives (14-23) were synthesized. With-
in this series several compounds (14-18 and 20-23) showed
good activity on CDK2/CyA that was used as a sensor of
compound activity toward the CDK family and, in several
cases, also good selectivity, based on their potency ratio

2176 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Traquandi et al.
excellent selectivity. Based on its potency on the enzyme and
on cells, we selected compound 29, bearing a cyclopentyl
group as R3, as an interesting hit from which to start further
expansion. An investigation of the R1 and R2 role on a series
of cyclopentyl derivatives was then accomplished (Table 2).
We could observe that activity on CDK2/CyA was negatively
affected upon increasing R1 steric hindrance. In fact, second-
ary amides bearing small substituents such as methyl (30)
displayed interesting activity as CDK2/CyA inhibitors,
whereas bulkier residues (31-33) caused a marked decrease
in activity on the enzyme complex, even though an improve-
ment in the solubility in neutral buffer was observed. Hydro-
xamatederivatives (34-36) conserved a certain affinityfor the
enzyme, but poor activity on cells and low solubility were
observed for these conpounds.
R2 modulation provided compounds endowed with sig-
nificant activity on the enzyme (37-46) and also on cells
(37-38, 40, 42-44, and 46) and good selectivity versus Aur-A.
Some derivatives showed a very poor solubility (37-40,
42, and 43), while the solubility parameter was improved
for compounds bearing hydrophilic or basic substituents
(41 and 44-46). Nevertheless, we observed that R2 modi-
fication caused a general loss of selectivity within the kinase
panel (data not shown). We could conclude that a bulky R2
was tolerated and was suitable to modulate the solubility
but could be critical for the selectivity and moreover did
not represent a significant advantage with respect to methyl,
in terms of atom economy and synthetic complexity. A more
focused study of the R3 substituent role was then under-
taken by a further expansion of the cycloalkyl derivative
subclass. In this perspective, compounds with R1 as either
hydrogen or methyl and R2 as methyl (Table 3) were
synthesized. We could observe that secondary methyl-
amides (49, 51, 53, 55, and 57), although having good
solubility, were less potent than the corresponding primary
amides (48, 50, 52, 54, and 56) both on enzyme and on cells.
Interestingly, compounds 58-61 showed good activity
against CDK2/CyA and in A2780 cell proliferation assay.
As far as buffer solubility is concerned, compound 59, albeit
less potent than compound 58 on cells, displayed higher
solubility. Compounds 50, 52, and 59 were then selected for
further assessment on the basis of their potency on the
enzyme, antiproliferative activity, and preliminary solubi-
lity in neutral buffer.
Table 4. Selectivity Profile of Compounds 50, 52, and 59a
kinase
50
52
59
CDK2/CyA
CDK2/CyE
CDK1/CyB
CDK5/p25
CDK4/CyD1
GSK-3β
ERK2
Aur-A
other kinasesb
0.002
0.007
0.005
0.009
nd
0.003
0.016
0.006
0.010
0.350
0.048
0.226
0.67
0.002
0.008
0.006
0.006
0.176
0.358
0.622
3.565
>10
0.028
0.212
>10
>10
>10
a Values are the means of two or more experiments. b c-ABL, ACK1,
ALK, AKT1, Aur-B, BRK, BUB1, CDC7/DBF4, CHK1, CK2,
EEF2K, EGFR, FAK, FGFR1, FLT3, IGF1R, IKK1, IKK2, IR,
JAK1, JAK3, C-KIT, LCK, LYN, MAPKAPK2, MET, MNK2,
MST4, NEK6, NIM, PAK4, PDGFR, PDK1, PERK, PIM1, PIM2,
PKAR, PKCβ, PLK1, RET, SYK, SULU1, TYK, TRKA, VEGFR2,
VEGFR3, ZAP70.
The three selected hits were subsequently screened against a
panel of 55 serine-threonine and tyrosine kinases and results
are reported in Table 4. Concerning the members of the
CDK family that were part of the panel, compounds 50, 52,
and 59 turned out to be potent inhibitors of CDK2/CyA,
CDK2/CyE, CDK1/CyB, and CDK5/p25, and to inhibit also
CDK4/CyD1, although with lower potency. Among all the
other enzymes in the panel, only GSK-3β (ratio vs CDK2/
CyA of 14ꢀ, 16ꢀ, and 179ꢀ, respectively, for 50, 52, and 59),
Figure 2. Crystal structure of selected compound 59 in complex
with CDK2/CyA.
Figure 4. Decrease of pRb phosphorylation induced by compound
59 in cells after 24 h treatment.
Figure 3. Effects of compound 59 on cell cycle progression and DNA synthesis after 24 h treatment.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2177
Table 5. In Vitro ADME for Selected Compound 59a
preliminary in vitro ADME
CLintr hepatocytes
mL/min/kg
PAMPA permeability
10-6 cm/sec
Caco2 cells
permeability
solubility 5%
dextrose (mg/mL)
CYP4503A4
(% remaining)
PPB (%)
69
5
11.9
high
4.0
88
a Dosed in 5% dextrose as hydrochloride salt.
Table 6. In Vivo Pharmacokinetic Parameters for Selected Compound 59a
in vivo PK (mouse) 10 mg/kg iv
in vivo PK (mouse) 10 mg/kg os
t
1/2 (h)
1.7
CL (mL/h/kg)
Vss (mL/kg)
t1/2 (h)
Cmax (μM)
AUC (os; μM h)
F (%)
3
814.2
1390.2
3.4
2.87
15.31
55.4
a Dosed in 5% dextrose as hydrochloride salt.
Figure 5. Compound 59 shows 38, 46, and 75% tumor growth inhibition (TGI) against a human ovarian cancer model (A2780) transplanted
into nude mice when orally administered respectively at doses of 5 (square), 10 (triangle), and 20 (cross) mg/kg bid for 10 consecutive days.
Vehicle-treated (control) curve is reported with open circles.
and ERK2 (106ꢀ, 75ꢀ, and 311ꢀ, respectively) were inhib-
ited in the high nanomolar range, while Aur-A was inhibited
in a submicromolar range (ratio vs CDK2/CyA of 223ꢀ) only
by 52. All the other tested kinases were substantially not
inhibited (IC50>10 μM).
inserted glycine (Gly216) in Aur-A that is absent in CDK2.19
This additional residue in Aur-A induces a different protein
conformation between the hinge region and the beginning of
the C-terminal domain, which in combination with the pre-
sence of Leu139 (CDK2 Ile10) and Thr217 (CDK2 Asp86),
tends to render the accommodation of nonplanar groups less
favorable. In contrast, the CDK2 pocket, probably due to a
better shape complementarity, can well tolerate a cycloalkyl
ring.
We focused our attention on compound 59 as the most
potent and selective multi-CDK inhibitor in the series. Its
distinctive selectivity profile was explained by obtaining the
crystal structure of the compound in complex with CDK2/
CyA (Figure 2). The amino-pyrimidine group is anchored at
the hinge region by two hydrogen bonds to the nitrogen and
oxygen backbone atoms of Leu83, while the carbonyl oxygen
of the amide forms a hydrogen bond with the conserved
Lys33. Interaction between the amide nitrogen and the car-
boxyl group of Asp145 that generally is present for com-
pounds bearing a primary amide (data not shown) is lost
for this compound because the secondary amide adopts a
CIS conformation. This conformation can explain the experi-
mental data indicating that amide steric hindrance tends to
negatively affect activity on CDK2. The methansulfonyl
group of the piperidine ring makes a hydrogen bond with
backbone nitrogen of Asp86 and an additional weak interac-
tion with Lys89, which is solvent exposed and very flexible.
Interestingly, the piperidine ring or more generally a cycloalkyl
ring is detrimental for activity against Aur-A and therefore
crucial for compound selectivity. This moiety occupies the
solvent accessible region, a part of the ATP pocket where Aur-
A and CDK2 are structurally different for the presence of an
The pharmacological profile of compound 59 was then
characterized in depth.
The effects of compound 59 on the cell cycle progression
and DNA synthesis were analyzed using flow cytometry
analysis and BrdU incorporation, respectively, on A2780
ovarian carcinoma cells in exponential growth in the presence
or absence of compound, for 24 h, at 1 and 3 μM. Compound
59 modulates cell cycle with an increase in G2/M phase and a
reduction in S phase as expected for an inhibitor able to block
both CDK2 and CDK1 activity. The reduction of S phase
population was linked to a strong reduction of the percentage
of BrdU incorporating cells, meaning that DNA synthesis in
these cells was impaired (Figure 3). In addition, the effect on
the phosphorylation status of a known CDK substrate such
as pRb was analyzed in cells treated with compound 59 at
1 and 3 μM. A clear reduction of the hyperphosphorylated form
of pRb was observed in the extracts of treated cells in compari-
son with the untreated cells, confirming the inhibitory effect
exerted by the compound on the activity of CDK2 (Figure 4).

2178 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Traquandi et al.
Analysis of the cell cycle profile, DNA synthesis, and
CDK2 substrate phosphorylation status were consistent with
an antiproliferative effect mediated by multi-CDK inhibition.
A preliminary in vitro ADME study (solubility in 5%
dextrose, stability to human CYP4503A4, plasma protein
binding, or PPB) was accomplished and in vivo pharmacoki-
netic data were determined as key-parameters. As reported
in Table 5, compound 59 revealed high metabolic stability
to human CYP4503A4 (88% remaining), good plasma pro-
tein binding (69% bound), good solubility (4.0 mg/mL in
5% dextrose as hydrochloric salt), and good permeability
(PAMPA and Caco2 cells).
The compound, after intravenous administration, showed
a low clearance (CL), which accounted for approximately
15% of the hepatic blood flow. The volume of distribution
(Vss 2-fold of the total body water) suggested a good tissue
distribution. The terminal half-life was 1.7 h. After oral
administration at a dose of 10 mg/kg, compound 59 showed
Thin-layer chromatography was performed on Merck silica gel
60 F254 precoated plates. Column chromatography was con-
ducted either under medium pressure on silica (Merck silica gel
40-63 μm) or on prepacked silica gel cartridges (Biotage).
Components were visualized by UV light (λ: 254 nm) and by
iodine vapor. 1H NMR spectra were recorded at a constant
temperature of 28 ꢀC on a Varian INOVA 400 spectrometer
operating at 400.45 MHz and equipped with a 5 mm Indirect
Detection PFG Probe (1H{15N-31P}). Chemical shifts were
referenced with respect to the residual solvent signals (DMSO-
d6: 2.50 ppm for 1H). Data are reported as follows: chemical shift
(δ), multiplicity (s = singlet, bs = broad signal, d = doublet,
t = triplet, q = quartet, quin = quintet, sxt = sixtet, dd =
doublet of doublets, ddd = doublet of doublet of doublets, dt =
doublet of triplets, td = triplet of doublets, m = multiplet),
coupling constants (Hz), and number of protons. Electrospray
(ESI) mass spectra were obtained on a Finnigan LCQ ion trap.
Unless otherwise specified, all final compounds were homoge-
neous (purity of not less than 95%), as determined by high-
performance liquid chromatography (HPLC). HPLC-UV-MS
analyses, used to assess compound purity, were carried out
combining the ion trap MS instrument with HPLC system
SSP4000 (Thermo Separation Products) equipped with an
autosampler LC Pal (CTC Analytics) and UV6000LP diode
array detector (UV detection 215-400 nm). Instrument control,
data acquisition and processing were performed by using Xca-
libur 1.2 software (Finnigan). HPLC chromatography was run
at room temperature, and 1 mL/min flow rate, using a Waters X
Terra RP 18 column (4.6 ꢀ 50 mm; 3.5 μm). Mobile phase A was
ammonium acetate 5 mM buffer (pH 5.5 with acetic acid)/
acetonitrile 90:10, and mobile phase B was ammonium acetate
5 mM buffer (pH 5.5 with acetic acid)/acetonitrile 10:90; the
gradient was from 0 to 100% B in 7 min then hold 100% B for
2 min before re-equilibration. ESI(þ) high resolution mass
spectra (HRMS) were obtained on a Waters Q-Tof Ultima
directly connected with micro-HPLC 1100 Agilent as previously
described.20
high oral bioavailability (55%) with an AUC of 15.31 μM h,
3
Cmax of 2.87 μM, and half-life of 3.4 h (Table 6). Overall,
compound 59 showed low clearance, suitable volume of
distribution, and high oral bioavailability in the experimental
conditions used in the study.
On the basis of the data presented above, compound 59
proceeded to further in vivo characterization in the human
A2780 xenograft mouse model. The doses of 5, 10, and
20 mg/kg were selected and administered orally twice a day,
for 10 consecutive days (on days 8-17 from tumor cell
injection). A pharmacokinetic analysis was also performed
in a satellite group of three mice after the last oral adminis-
tration at 20 mg/kg twice a day, for 10 consecutive days,
and confirmed the good and dose-proportional exposure
obtained with the compound 59 (AUC = 56.21 μM h; Cmax
3
of 6.40 μM). Figure 5 illustrates the results from this study.
Compound 59 caused a dose-dependent inhibition of A2780
tumor growth up to 75% at the dose of 20 mg/kg on day 18
(P < 0.0001 vs control group, Mann-Whitney U-test) and a
corresponding tumor growth delay of 6.4 days.
Elemental analyses were performed on a Carlo Erba 1110
instrument, and C, H, and N results were within (0.4% of
theoretical values unless specified. All reactants were commer-
cially available or were prepared as previously described.18
Ethyl 1-Methyl-8-(methylsulfanyl)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxylate (3). To a solution of ethyl (6E)-
6-[(dimethylamino)methylidene]-1-methyl-7-oxo-4,5,6,7-tetra-
hydro-1H-indazole-3-carboxylate 2 (9.0 g, 69 mmol, prepared
according to ref 17) in dry DMF (350 mL), dry potassium
acetate (13.4 g, 138 mmol), and S-methylisothiourea sulfate
(19.18 g, 69 mmol) were added. The mixture was maintained
at 90 ꢀC under stirring for 8 h. The solvent was then evaporated,
the residue dissolved with dichloromethane and washed with
water. The organic layer was dried over Na2SO4 and evapo-
rated. The crude was finally triturated with diethyl ether
Conclusions
Exploration of the pyrazoloquinazoline class focused on
R1, R2, and R3 modifications afforded several compounds
that showed good potency as multi-CDK inhibitors, good
selectivity when tested on a panel of serine-threonine and
tyrosine kinases, and antiproliferative activity in A2780 hu-
man ovarian carcinoma cells. Optimization of the physical
properties and pharmacokinetic profile led to the identifica-
tion of the highly potent, orally available compound 59.
Analysis of the cell cycle profile, DNA synthesis, and CDK2
substrate phosphorylation status were consistent with an
antiproliferative effect mediated by multi-CDK inhibition.
The compound exhibited good efficacy in vivo on A2780
xenograftmodel whenorallyadministeredatdose of 20mg/kg
bid for 10 consecutive days. The complete pharmacological
profile of compound 59 will be reported in a separate paper.
1
and collected by filtration to give 3 (15.5 g, 74%). H NMR
(400 MHz, DMSO-d6) δ ppm 1.31 (t, J = 7.1 Hz, 3H), 2.56
(s, 3H), 2.91 (m, 2H), 3.00 (m, 2H), 4.29 (d, J=7.1 Hz, 2H), 4.33
(s, 3H), 8.55 (s, 1H); LCMS (ESI) m/z 305 (M þ H)þ.
1-Methyl-8-(methylsulfanyl)-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxamide (4). A suspension of 3 (13.0 g,
0.043 mol) in a mixture of methanol (200 mL), DMF (200 mL),
and 33% NH4OH (200 mL) was stirred at 65 ꢀC in a closed bottle
for about 8 h. The solvent was then evaporated to dryness, the
residue dissolved with dichloromethane and washed with water.
The organic layer was dried over Na2SO4 and evaporated. The
crude was purified by flash chromatography on a silica gel
column (cyclohexane/ethyl acetate, 8/2), giving 4 (6.16 g, 52%).
1H NMR(400 MHz, DMSO-d6) δ ppm 2.56 (s, 3H), 2.88(m, 2H),
3.01 (m, 2H), 4.30 (s, 3H), 7.27 (bs, 1H), 7.36-7.69 (m, 1H), 8.53
(s, 1H); LCMS (ESI) m/z 276 (M þ H)þ.
Experimental Section
Chemistry. All solvents and reagents, unless otherwise stated,
were commercially available, of the best grade, and were used
without further purification. All experiments dealing with
moisture-sensitive compounds were conducted under dry nitro-
gen or argon. Organic solutions were evaporated using
a Heidolph WB 2001 rotary evaporator at 15-20 mmHg.
1-Methyl-8-(methylsulfonyl)-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxamide (5). A solution of 4 (6.0 g, 0.022 mol)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2179
in DMF (1000 mL) and potassium monopersulfate triple salt
(oxone, 40.57 g, 0.066 mol) was stirred at room temperature for
16 h. Water and ethyl acetate were then added and the layers
separated. The organic phase was finally dried over Na2SO4 and
evaporated. The residue was triturated with diethyl ether and
5 (5.40 g, 80%) was collected by filtration. 1H NMR (400 MHz,
DMSO-d6) δ ppm 3.08 (s, 4H), 3.45 (s, 3H), 4.31 (s, 3H), 7.33 (bs,
1H), 7.55 (bs, 1H), 8.93 (s, 1H); LCMS (ESI) m/z 308 (M þ H)þ.
8-(Benzylamino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]qu-
inazoline-3-carboxamide (1). To a solution of 5 (400 mg, 1.30
mmol) in dry DMSO (20 mL), benzylamine (0.28 mL, 2.60
mmol) was added. After 6 h at 95 ꢀC under nitrogen atmosphere,
the solvent was evaporated. The residue was then dissolved with
dichloromethane and washed with water. The organic layer was
dried over Na2SO4 and evaporated to dryness. By chromato-
graphy on a silica gel column (dichloromethane/acetone, 9/1)
1 was obtained (304 mg, 70%). 1H NMR (400 MHz, DMSO-d6)
δ ppm 2.68-2.76 (m, 2H), 2.84-2.99 (m, 2H), 4.16 (bs, 3H), 4.53
(d, J = 6.2 Hz, 2H), 7.15-7.23 (m, 2H), 7.25-7.35 (m, 4H), 7.40
(bs, 1H), 7.68 (bs, 1H), 8.21 (s, 1H); LCMS (ESI) m/z 335 (M þ
H)þ; HRMS (ESI) calcd for C18H18N6O þ Hþ, 335.1615; found,
335.1610; Anal. (C18H18N6O) C, H, N.
(d, J = 7.8 Hz, 1H), 7.39 (bs, 1H), 7.71 (td, J = 7.6, 1.83 Hz, 2H),
8.22 (s, 1H), 8.47-8.50 (m, 1H); LCMS (ESI) m/z 336 (M þ H)þ;
Anal. (C17H17N7O) C, H, N.
1-Methyl-8-{[2-(morpholin-4-yl)ethyl]amino}-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (24). Yield, 46%; H
1
NMR (400 MHz, DMSO-d6) δ ppm 2.41 (m, 4H), 2.67-2.77 (m,
2H), 2.88-3.02 (m, 2H), 3.43 (q, J = 6.5 Hz, 2H), 3.57 (m, 4H),
4.30 (s, 3H), 6.92 (t, J = 5.9 Hz, 1H), 7.22 (bs, 1H), 7.41 (bs, 1H),
8.20 (s, 1H); LCMS (ESI) m/z 358 (M þ H)þ; HRMS (ESI) calcd
for C17H23N7O2 þ Hþ, 358.1986; found, 358.1971; Anal.
(C17H23N7O2) C, H, N.
8-[(2-Hydroxyethyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxamide (25). Yield, 48%; 1H NMR(400
MHz, DMSO-d6) δ ppm 2.67-2.98 (m, 4H), 3.34-3.60 (m, 4H),
4.29 (s, 3H), 4.65 (s, 1H), 6.95 (t, J = 5.7 Hz, 1H), 7.22 (s, 1H),
7.41 (s, 1H), 8.19 (s, 1H); LCMS (ESI) m/z 289 (M þ H)þ; HRMS
(ESI) calcd for C13H16N6O2 þ Hþ, 289.1408; found, 289.1410;
Anal. (C13H16N6O2) C, H, N.
1-Methyl-8-{[2-(piperidin-1-yl)ethyl]amino}-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (26). Yield, 40%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.38 (m, 2H), 1.44-1.58 (m,
4H), 2.38 (bs, 4H), 2.46 (bs, 2H), 2.72 (m, 2H), 2.93 (m, 2H), 3.41
(q, J = 6.3 Hz, 2H), 4.30 (s, 3H), 6.86 (bs, 1H), 7.22 (bs, 1H), 7.41
(bs, 1H), 8.20 (s, 1H); LCMS (ESI) m/z 356 (M þ H)þ; Anal.
(C18H25N7O) C, H, N.
Compounds 14, 19-26, and 28. By employment of the above-
described procedure, starting from 5 and using the suitable
amine, compounds 14, 19-26, and 28 were prepared.
8-[(3-Methoxybenzyl)amino]-1-methyl-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (14). Yield, 72%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 2.71 (t, J = 7.6 Hz, 2H),
2.92 (t, J = 7.6 Hz, 2H), 3.71 (s, 3H), 4.17 (s, 3H), 4.49 (d, J = 6.3
Hz, 2H), 6.73-6.80 (m, 1H), 6.89 (d, J = 1.3 Hz, 1H), 6.89-6.92
(m, 1H), 7.15-7.28 (m, 2H), 7.39 (s, 1H), 7.66 (t, J = 6.6 Hz, 1H),
8.21 (s, 1H); LCMS (ESI) m/z 365 (M þ H)þ; HRMS (ESI)
calcd for C19H20N6O2 þ Hþ, 365.1721; found, 365.1725; Anal.
(C19H20N6O2 C, H, N.
8-[(1-Ethylpiperidin-4-yl)amino]-1-methyl-4,5-dihydro-1H-py-
razolo[4,3-h]quinazoline-3-carboxamide (28). Yield, 63%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.00 (t, J = 7.2 Hz, 3H),
1.42-1.57 (m, 2H), 1.86-1.93 (m, 2H), 1.93-2.02 (m, 2H), 2.33
(q, J = 7.2 Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H), 2.87 (m, 2H), 2.93
(t, J = 7.62 Hz, 2H), 3.57-3.76 (m, 1H), 4.29 (s, 3H), 6.98 (d, J =
7.9 Hz, 1H), 7.21 (s, 1H), 7.42 (s, 1H), 8.19 (s, 1H); LCMS (ESI)
m/z 356 (M þ H)þ; HRMS (ESI) calcd for C18H25N7O M þ Hþ,
356.2193; found, 356.2184.
1-Methyl-8-{[3-(4-methylpiperazin-1-yl)benzyl]amino}-4,5-di-
hydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (19). Yield,
66%; 1H NMR (400 MHz, DMSO-d6) δ ppm 2.21 (s, 3H), 2.43
(m, 4H), 2.67-2.75 (m, 2H), 2.86-2.97 (m, 2H), 3.05-3.12 (m,
4H), 4.19 (s, 3H), 4.46 (d, J = 6.2 Hz, 2H), 6.72-6.80 (m, 2H),
6.89-6.96 (m, 1H), 7.08-7.16 (m, 1H), 7.21 (s, 1H), 7.40 (s, 1H),
7.62 (s, 1H), 8.21 (s, 1H); LCMS (ESI) m/z 433 (M þ H)þ;
HRMS (ESI) calcd for C23H28N8O þ Hþ, 433.2459; found,
433.2454; Anal. (C23H28N8O) C, H, N.
1-Methyl-8-{[4-(4-methylpiperazin-1-yl)benzyl]amino}-4,5-di-
hydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (20). Yield,
68%; 1H NMR (400 MHz, DMSO-d6) δ ppm 2.26 (bs, 3H),
2.43-2.56 (m, 4H), 2.71 (m, 2H), 2.92 (m, 2H), 3.09 (s, 4H), 4.21
(s, 3H), 4.42 (d, J = 6.2 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 7.18
(d, J = 8.7 Hz, 2H), 7.21 (s, 1H), 7.40 (t, 1H), 7.56 (t, J = 6.1 Hz,
1H), 8.20 (s, 1H); LCMS (ESI) m/z 433 (M þ H)þ; Anal.
(C23H28N8O) C, H, N.
1-Methyl-8-[(pyridin-4-ylmethyl)amino]-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (21). Yield, 45%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.68-2.78 (m, 2H), 2.86-2.98 (m,
2H), 4.07 (bs, 3H), 4.54 (d, J = 6.2 Hz, 2H), 7.21 (bs, 1H),
7.27-7.34 (m, 2H), 7.40 (bs, 1H), 7.78 (t, J = 6.8 Hz, 1H), 8.22 (s,
1H), 8.38-8.54 (m, 2H); LCMS (ESI) m/z 336 (M þ H)þ; HRMS
(ESI) calcd for C17H17N7O þ Hþ, 336.1568; found, 336.1572;
Anal. (C17H17N7O) C, H, N.
1-Methyl-8-[(pyridin-3-ylmethyl)amino]-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (22). Yield, 40%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.68-2.97 (m, 4H), 4.16 (s, 3H), 4.55
(d, J = 6.2 Hz, 2H), 7.21 (s, 1H), 7.32 (dd, J = 7.8, 4.7 Hz, 1H),
7.40 (s, 1H), 7.63-7.81 (m, 1H), 7.72 (dt, J = 7.7, 1.8 Hz, 1H), 8.23
(s, 1H), 8.42 (dd, J = 4.7, 1.5 Hz, 1H), 8.55 (d, J = 1.8 Hz, 1H);
LCMS (ESI) m/z 342 (M þ H)þ; Anal. (C17H17N7O) C, H, N.
1-Methyl-8-[(pyridin-2-ylmethyl)amino]-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (23). Yield, 42%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.68-2.76 (m, 2H), 2.92 (m, 2H),
4.04 (bs, 3H), 4.60 (d, J = 6.1 Hz, 2H), 7.10-7.28 (m, 2H), 7.31
Ethyl 8-Methoxy-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxylate (6). To a solution of 2 (2.0 g, 7.2 mmol)
in acetonitrile (200 mL), O-methylisourea sulfate (17.4 g, 70.6
mmol) and potassium carbonate (10.0 g, 72.4 mmol) were added.
The reaction mixturewas stirredat reflux for 16 h. The solvent was
then evaporated, and the residue was dissolved with dichloro-
methane and washed with water. The organic layer was dried over
dry Na2SO4 and concentrated. After flash chromatography on a
silica gel column (dichloromethane) 6 was obtained (1.7 g, 80%).
1H NMR (400 MHz, DMSO-d6) δ ppm 1.31 (t, J = 7.1 Hz, 3H),
2.87-2.93 (m, 2H), 2.99 (m, 2H), 3.95 (s, 3H), 4.29 (q, J = 7.1 Hz,
2H), 4.33 (s, 3H), 8.52 (s, 1H); LCMS (ESI) m/z 289 (M þ H)þ.
Ethyl 8-Hydroxy-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxylate (7). Toa solutionof 6 (1.5 g, 5.2 mmol)
in acetonitrile (90 mL), sodium iodide (1.6 g, 10.6 mmol), and
trimethylsilylchloride (1.5 mL, 12 mmol) were added in sequence.
After 24 h under stirring and nitrogen atmosphere at room
temperature, the solvent was evaporated, and the residue was
dissolved with a mixture dichloromethane/methanol 4/1 and
washed with a saturated aqueous solution of sodium thiosulfate.
The organic layer was dried over Na2SO4 and evaporated to
dryness. The residue was crystallized from methanol leading 7
(1.1 g, 78%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.30 (t, J =
7.1 Hz, 3H), 2.71 (m, 1H), 2.93 (m, 1H), 4.29 (q, J = 7.1 Hz, 2H),
4.27 (s, 3H), 7.85 (s, 1H), 11.69 (s, 1H); LCMS (ESI) m/z 275
(M þ H)þ; HPLC purity 80%.
Ethyl 1-Methyl-8-{[(trifluoromethyl)sulfonyl]oxy}-4,5-dihy-
dro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (8). A solution
of 7 (0.60 g, 2.19 mmol) and triethylamine (0.31 mL, 2.19 mmol)
in dichloromethane (60 mL) was stirred at -78 ꢀC for 5 h. After
this time, trifluoromethansulfonic anhydride (0.72 mL, 2.19
mmol) was added. The reactionwas stirredovernight andallowed
to come to room temperature, washed with aqueous NaHCO3,
dried over Na2SO4, and evaporated to dryness. The residue
was triturated with diethyl ether/acetone and 8 was collected by
filtration (0.60 g, 67%). 1H NMR (400 MHz, DMSO-d6) δ ppm

2180 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Traquandi et al.
1.31 (t, J = 7.1 Hz, 3H), 3.07 (s, 4H), 4.27 (s, 3H), 4.31 (q, J = 7.1
Hz, 2H), 8.84 (s, 1H); LCMS (ESI) m/z 407 (M þ H)þ.
(t, J = 7.6 Hz, 2H), 2.92 (t, J = 7.6 Hz, 2H), 3.60-3.75 (m, 1H),
4.28 (q, J = 7.1 Hz, 2H), 4.32 (s, 3H), 6.96 (d, J = 5.4 Hz, 1H),
8.19 (s, 1H); LCMS (ESI) m/z 356 (M þ H)þ; HRMS (ESI) calcd
for C19H25N5O2 þ Hþ, 356.2081; found, 356.2081. A solution of
the above-described ethyl carboxylate (250 mg, 0.70 mmol) in a
mixture of methanol (10 mL), DMF (10 mL), and 33% NH4OH
(5 mL) was stirred at 70 ꢀC in a closed bottle for 8 h. The solvent
was removed under reduced pressure and the residue dissolved
with dichloromethane and washed with water. The organic layer
was dried over Na2SO4 and evaporated. The crude was purified by
flash chromatography on a silica gel column (dichloromethane/
1-Methyl-8-{[4-(methylsulfonyl)benzyl]amino}-4,5-dihydro-1H-py-
razolo[4,3-h]quinazoline-3-carboxamide (18). A solution of 8 (1.5 g,
3.69 mmol) and 4-methylsulfonyl benzylamine hydrochloride
(1.6 g, 7.38 mmol) in dry 1,4-dioxane (100 mL) was stirred at
room temperature for 6 h, then the solvent was removed in vacuo.
The residue was taken up with dichloromethane and washed with
aqueousNaHCO3. The organiclayerwas thendried over Na2SO4
and evaporated. The crude was triturated with diethyl ether
and filtered, giving ethyl 1-methyl-8-{[4-(methylsulfonyl)benzyl]-
amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxy-
late (2.3 g, 70%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.29
(t, J = 7.1 Hz, 3H), 2.75 (t, J = 7.6 Hz, 2H), 2.91 (t, J = 7.6 Hz,
2H), 3.16 (s, 3H), 4.08 (s, 3H), 4.27 (q, J = 7.1 Hz, 2H), 4.62 (d,
J = 6.2 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.86 (m, 3H), 8.24
(s, 1H); LCMS (ESI) m/z 442 (M þ H)þ. HRMS (ESI) calcd for
C21H23N5O4S þ Hþ, 442.1543; found, 442.1543. A suspension of
the above-described ethyl carboxylate (2.0 g, 4.54 mmol) in dry
ethanol (40 mL) was treated with a 1.5 M solution of KOH in
ethanol (9.10 mL, 13.62 mmol) at reflux temperature for 2 h.
After cooling in an ice bath, the resulting precipitate was collected
by filtration to give potassium 1-methyl-8-{[4-(methylsulfonyl)-
benzyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-car-
boxylate (1.54 g, 75%). 1H NMR (400 MHz, DMSO-d6) δ ppm
2.63 (t, J = 7.6 Hz, 2H), 2.87 (t, J = 7.6 Hz, 2H), 3.16 (s, 3H), 3.97
(s, 3H), 4.60 (d, J = 6.1 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.67
(t, J = 6.3 Hz, 1H), 7.85 (d, J = 8.4 Hz, 2H), 8.13 (s, 1H); LCMS
(ESI) m/z 414 (M þ H)þ. HRMS (ESI): calcd for C19H19N5O4S þ
Hþ, 414.1231; found, 414.1234. A solution of the above-described
carboxylic acid potassium salt (298 mg, 0.66 mmol) was dissolved
in a mixture of dry DMF (5 mL) and dry THF (5 mL). After
cooling to 0 ꢀC, N,N-diisopropyl-N0-ethylamine (DIPEA; 0.46
mL, 2.64 mmol) and N-(3-dimethylaminopropyl)-N0-ethylcarbo-
diimide hydrochloride (EDC; 380 mg, 1.98 mmol) were added in
sequence to the solution. The reaction mixture was maintained
at the same temperature for 30 min and then N-hydroxybenzo-
1
methanol) affording 27 (137 mg, 60%). H NMR (400 MHz,
DMSO-d6) δ ppm 1.05-1.42 (m, 5H), 1.54-1.99 (m, 5H), 2.71
(t, J = 7.6 Hz, 2H), 2.93 (t, J = 7.6 Hz, 2H), 3.61-3.78 (m, 1H),
4.29 (s, 3H), 6.93 (d, J = 6.9 Hz, 1H), 7.21 (s, 1H), 7.42 (s, 1H),
8.18 (s, 1H); LCMS (ESI) m/z 327 (M þ H)þ; HRMS (ESI) calcd
for C17H22N6O þ Hþ, 327.1928; found, 327.1928; Anal. (C17H22-
N6O) C, H, N.
1-Methyl-8-[(1-methylpiperidin-4-yl)amino]-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (48). By employment
of the above-described procedure, starting from 8, and using
1-methylpiperidin-4-yl-amine, compound 48 was prepared. Yield,
50%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.44-1.64 (m, 2H),
1.89 (m, 2H), 1.96-2.08 (m, 2H), 2.20 (s, 3H), 2.71 (t, J = 7.6 Hz,
2H), 2.79 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H), 3.58-3.73 (m, 1H),
4.29 (s, 3H), 6.99 (d, J = 6.3 Hz, 1H), 7.21 (s, 1H), 7.42 (s, 1H),
8.19 (s, 1H); LCMS (ESI) m/z 342 (M þ H)þ; HRMS (ESI) calcd
for C17H23N7O4 þ Hþ, 342.2037; found, 342.2024.
8-(Cyclohexylamino)-N-1-dimethyl-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxamide (47). A solution of ethyl 8-(cy-
clohexylamino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxylate (200 mg, 0.56 mmol), prepared as described
for the synthesis of compound 27 in a mixture of ethanol (30 mL)
and 33 wt % methylamine (10 mL) in absolute ethanol, was
stirred at 60 ꢀC in a closed bottle for 8 h. The solvent was then
removed in vacuo and the crude purified by flash chromatogra-
phy on a silica gel column (dichloromethane/methanol/triethy-
lamine 95/5/5) affording 47 (149 mg, 78%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.24 (m, 4H), 1.61 (m, 2H), 1.75 (m, 2H), 1.94
(m, 2H), 2.70-2.74 (m, 2H), 2.75 (d, J = 4.7 Hz, 3H), 2.95
(m, 2H), 3.69 (m, 1H), 4.31 (s, 3H), 6.94 (d, J = 5.6 Hz, 1H), 8.06
(q, J = 4.7 Hz, 1H), 8.20 (s, 1H); LCMS (ESI) m/z 341 (M þ H)þ;
HRMS (ESI) calcd for C18H24N6O þ Hþ, 341.2084; found,
341.2089.
triazole ammonium salt (HOBT NH3; 300 mg, 1.98 mmol) was
3
added under stirring. The mixture was warmed to room tempera-
ture and kept at this temperature overnight. The solvent was then
evaporated and the residue taken up with dichloromethane and
washed with aqueous NaHCO3. The organic layer was dried over
Na2SO4 and the solvent removed in vacuo. After trituration with
1
diethyl ether, 18 (167 mg, 61%) was collected by filtration. H
NMR (400 MHz, DMSO-d6) δ ppm 2.72 (m, 2H), 2.92 (m, 2H),
3.16 (s, 3H), 4.07 (bs, 3H), 4.62 (d, J = 6.2 Hz, 2H), 7.21 (bs, 1H),
7.39 (bs, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.79-7.84 (m, 1H), 7.86
(d, J = 8.5 Hz, 2H), 8.22 (s, 1H); LCMS (ESI) m/z 413 (M þ H)þ;
HPLC purity 90%.
By employment of the above-described procedure, starting
from 8, and using the suitable amine, compounds 30 and 49 were
prepared.
8-(Cyclopentylamino)-N-1-dimethyl-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxamide (30). Yield, 51%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.43-1.79 (m, 6H), 1.87-2.03
(m, 2H), 2.74 (d, J =4.7Hz, 3H), 2.77(t, J = 7.6Hz, 2H), 2.98(t,
J = 7.6 Hz, 2H), 4.19 (s, 1H), 4.30 (s, 3H), 7.67-7.90 (m, 1H),
8.10 (q, J = 4.7 Hz, 1H), 8.22 (s, 1H); LCMS (ESI) m/z 327 (M þ
H)þ; HRMS (ESI) calcd for C17H22N6O þ Hþ, 327.1928; found,
327.1933; HPLC purity 95%.
N-1-Dimethyl-8-[(1-methylpiperidin-4-yl)amino]-4,5-dihydro-
1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (49). Yield, 50%;
1H NMR (400 MHz, DMSO-d6) δ ppm 1.46-1.60 (m, 2H), 1.88
(m, 2H), 1.98-2.07 (m, 2H), 2.20 (s, 3H), 2.68-2.75 (m, 2H), 2.73
(d, J = 4.7 Hz, 3H), 2.76-2.82 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H),
3.61-3.73 (m, 1H), 4.29 (s, 3H), 6.99 (d, J = 7.6 Hz, 1H), 8.04
(q, J = 4.7 Hz, 1H), 8.19 (s, 1H); LCMS (ESI) m/z 356 (M þ H)þ;
HRMS (ESI) calcd for C18H25N7O þ Hþ, 356.2193; found,
356.2176; HPLC purity 95%.
8-(Cyclopentylamino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-
h]quinazoline-3-carboxamide (29). By employment of the above-
described procedure, starting from 8, and using cyclopentyla-
mine, compound 29 was prepared. Yield, 55%; 1H NMR (400
MHz, DMSO-d6) δ ppm 1.47-1.78 (m, 6H), 1.86-1.99 (m, 2H),
2.71 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H), 4.08-4.20 (m, 1H), 4.31
(s, 3H), 7.06 (d, J = 6.8 Hz, 1H), 7.22 (s, 1H), 7.41 (s, 1H), 8.19
(s, 1H); LCMS (ESI) m/z 313 (M þ H)þ; HRMS (ESI) calcd for
C16H20N6O þ Hþ, 313.1772; found, 313.1774.
8-(Cyclohexylamino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxamide (27). A solution of 8 (1.5 g, 3.69 mmol)
and cyclohexylamine (0.84 mL, 7.33 mmol) in dry 1,4-dioxane
(100 mL) was stirred at room temperature for 6 h, and then the
solvent was removed in vacuo. The residue was taken up with
dichloromethane and washed with aqueous NaHCO3. The or-
ganic layer was then dried over Na2SO4 and evaporated. The
crude was triturated with diethyl ether and filtered, giving ethyl
8-(cyclohexylamino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]-
Ethyl 8-Amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxylate (9). To a solution of 2 (16.0 g, 0.06 mol) in
ethanol (600 mL), sodium ethylate (3.90 g, 0.06 mol) and
guanidine hydrochloride (5.44 g, 0.06 mol) were added in se-
quence. The mixture was stirred under reflux for 12 h. The solvent
was then evaporated, the residue taken up with dichloromethane
1
quinazoline-3-carboxylate (1.07 g, 82%). H NMR (400 MHz,
DMSO-d6) δ ppm 1.06-1.35 (m, 5H), 1.30 (t, J = 7.1 Hz, 3H),
1.53-1.64 (m, 1H), 1.68-1.78 (m, 2H), 1.85-2.01 (m, 2H), 2.74

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2181
and washed with water. The organic layer was then dried over
Na2SO4 andconcentrated. Theresiduewastriturated withdiethyl
ether and 9 was collected by filtration (13.9 g, 85%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz,
3H), 2.74 (m, 2H), 2.92 (m, 2H), 4.28 (q, J = 7.1 Hz, 2H), 4.32 (s,
3H), 6.56 (s, 2H), 8.18 (s, 1H); LCMS (ESI) m/z 274 (M þ H)þ;
HRMS (ESI) calcd for C13H15N5O2 þ Hþ, 274.1298; found,
274.1299.
8-Amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-
3-carboxamide (10a). A suspension of 9 (300 mg, 1.1 mmol) in a
mixture of 33% NH4OH (20 mL), methanol (20 mL), and DMF
(10mL) was stirred at 65 ꢀC for 16h in a closed bottle. The solvent
was evaporated in vacuo, and the residue was taken up with
dichloromethane and washed with aqueous NaHCO3 The or-
ganic phase was dried over Na2SO4 and evaporated, giving 10a
(135 mg, 50%) after trituration with diethyl ether. 1H NMR (401
MHz, DMSO-d6) δ ppm 2.71 (t, J = 7.6 Hz, 2H), 2.92 (m, 2H),
4.29 (s, 3H), 6.52 (s, 2H), 7.21 (bs, 1H), 7.41 (bs, 1H), 8.16 (s, 1H);
LCMS (ESI) m/z 245 (M þ H)þ; HRMS (ESI) calcd for
C11H12N6O þ Hþ, 245.1146; found, 245.1143.
10b (490 mg, 1.9 mmol) in dry DMF (12 mL), 1-acetyl-4-piper-
idone (470 μL, 3.8 mmol), trifluoroacetic acid (1 mL, 12.8 mmol),
and sodium triacetoxyborohydride (886 mg, 4.2 mmol) were
added. After 18 h, 0.33 N NaOH (80 mL, 26.4 mmol) was added
dropwise to the mixture. The precipitate was filtered, washed with
water, and dried in an oven to dryness to give 51 (509 mg, 70%). 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.84 (m, 4H), 2.03 (s, 3H),
2.76(d, J= 4.8, 3H), 2.84 (m, 2H), 3.17 (m, 2H), 4.21 (m, 1H), 4.24
(s, 3H), 7.28 (s, 1H), 8.19 (s, 1H), 9.10 (q, J = 4.8 Hz, 1H); LCMS
(ESI) m/z 384 (M þ H)þ; HRMS (ESI) calcd for C19H25N7O2 þ
Hþ, 384.2142; found, 384.2145.
By employment of the above-described procedure, starting
from 10b and using the suitable cycloalkylketone, compounds
53, 55, 59, and 61 were prepared.
N-1-Dimethyl-8-{[1-(phenylcarbonyl)piperidin-4-yl]amino}-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (53). Yield,
70%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.50 (s, 2H), 1.97 (s,
2H), 2.73 (d, J = 4.7 Hz, 3H), 2.76 (m, 2H), 2.96 (t, J = 7.7 Hz,
2H), 3.05-3.77 (m, 3H), 4.03 (m, 1H), 4.29 (s, 3H), 4.39 (m, 1H),
7.35-7.41 (m, 2H), 7.43-7.47 (m, 3H), 7.58 (s, 1H), 8.08 (q, J =
4.7 Hz, 1H), 8.24 (s, 1H); LCMS (ESI) m/z 891 (2M þ H)þ;HRMS
(ESI) calcd for 2.C24H27N7O2 þ Hþ, 891.4525; found, 891.4491.
N-1-Dimethyl-8-({1-[(4-methylpiperazin-1-yl)carbonyl]piperi-
din-4-yl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-car-
boxamide (55). Yield, 50%; 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.41-1.50 (m, 2H), 1.90 (m, 2H), 2.19 (s, 3H), 2.31 (s, 4H),
2.69-2.75 (m, 2H), 2.73 (d, J = 4.7 Hz, 3H), 2.80-2.89 (m, 2H),
2.94 (t, J = 7.6 Hz, 2H), 3.10-3.17 (m, 4H), 3.58 (dt, J = 13.3, 3.3
Hz, 2H), 3.78-3.93(m, 1H), 4.29(s,3H), 7.07 (s,1H), 8.04 (q, J =
4.7 Hz, 1H), 8.20 (s, 1H); LCMS (ESI) m/z 468 (M þ H)þ; HRMS
(ESI) calcd for C23H33N9O2 þ Hþ, 468.2830; found, 468.2831.
Ethyl 4-{[1-Methyl-3-(methylcarbamoyl)-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazolin-8-yl]amino}piperidine-1-carboxylate (57).
8-[(4-Methoxybenzyl)amino]-1-methyl-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (15). To a solution of 10a
(244 mg, 1.0 mmol) in a mixture of glacial acetic acid/methanol/
water 1/1/1 (30 mL) in a round-bottom flask, p-methoxybenzalde-
hyde (0.88 mL, 6.0 mmol) and then 85% sodium cyanoborohydride
(420 mg, 4.0 mmol) were added. The solution was stirred at room
temperature overnight. The reaction mixture was then poured into
ice-water (200 mL), the pH was adjusted to 10 by addition of
saturated aqueous Na2CO3, and the solution was extracted with
ethyl acetate. The collected organic extracts were washed with
brine until neutral, washed with water, and dried over Na2SO4.
Evaporation of the solvent under reduced pressure left a yellow
solid residue that was purified by flash chromatography on silica gel
(dichloromethane/methanol, 95/5) to yield a yellow compound.
Crystallization from methanol afforded crystalline 15 (225 mg,
62%). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (m, 2H), 2.92
(m, 2H), 3.71 (s, 3H), 4.20 (bs, 3H), 4.45 (s, 2H), 6.86 (d, J=8.8Hz,
2H), 7.21 (bs, 1H), 7.25 (d, J = 8.8 Hz, 2H), 7.40 (bs, 1H), 7.62 (bs,
1H), 8.20 (s, 1H); LCMS (ESI) m/z 365 (M þ H)þ.
1
Yield, 60%; H NMR (400 MHz, DMSO-d6) δ ppm 1.18 (t,
J = 7.1 Hz, 3H), 1.32-1.49 (m, 2H), 1.90 (m, 2H), 2.70-2.75
(m, 1H), 2.73 (d, J = 4.7 Hz, 1H), 2.94 (t, J = 7.7 Hz, 1H), 2.94
(t, J = 7.7 Hz, 1H), 3.84-3.93 (m, 1H), 3.92-3.98 (m, 1H), 4.04
(q, J = 7.1 Hz, 2H), 4.29 (s, 3H), 7.08 (d, J = 5.9 Hz, 1H), 8.04 (q,
J = 4.7 Hz, 1H), 8.21 (s, 1H); LCMS (ESI) m/z 414 (M þ H)þ;
HRMS (ESI) calcd for C20H27N7O3 þ Hþ, 414.2248; found,
414.2263.
N-1-Dimethyl-8-{[1-(methylsulfonyl)piperidin-4-yl]amino}-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (59). Yield,
72%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.59 (m, 2H), 2.02
(m, 2H), 2.73 (m, 2H), 2.75 (d, J = 4.7 Hz, 3H), 2.86 (m, 2H), 2.88
(s, 3H), 2.96 (t, J = 7.6 Hz, 2H), 3.56 (m, 2H), 3.87 (m, 1H), 4.29
(s, 3H), 7.43 (m, 1H), 8.08 (m, 1H), 8.23 (s, 1H); LCMS (ESI) m/z
420 (M þ H)þ; HRMS (ESI) calcd for C18H25N7O3S þ Hþ,
420.1812; found, 420.1814; Anal. (C18H25N7O3S) C, H, N, S.
N-1-Dimethyl-8-{[1-(phenylsulfonyl)piperidin-4-yl]amino}-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (61). Yield,
68%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.59 (m, 2H), 1.98
(m, 2H), 2.56 (m, 2H), 2.74 (m, 2H), 2.72 (d, J = 4.8 Hz, 3H), 2.94
(t, J = 7.6 Hz, 2H), 3.58 (m, 2H), 3.74 (m, 1H), 4.22 (s, 3H), 7.53
(s, 1H), 7.66 (m, 2H), 7.74 (m, 1H), 7.74-7.76 (m, 2H), 8.06 (q,
J = 4.8 Hz, 1H), 8.20 (s, 1H); LCMS (ESI) m/z 482 (M þ H)þ;
HRMS (ESI) calcd for C23H27N7O3S þ Hþ, 482.1969; found,
482.1959.
1-Methyl-8-({1-[(4-methylpiperazin-1-yl)carbonyl]piperidin-4-
yl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxa-
mide (54). To a suspension of 9 (355 mg, 1.30 mmol) in dry DMF
(10 mL), 1-[(4-methylpiperazin-1-yl)carbonyl]piperidin-4-one di-
hydrochloride (562 mg, 2.50 mmol, prepared as described in
ref 17, trifluoroacetic acid (1.3 mL, 16.64 mmol), and sodium
triacetoxyborohydride (528 mg, 2.50 mmol) were added. After 18
h, the mixture was poured into a solution of potassium carbonate
(500 mg in 100 mL of water) and extracted with dichloromethane.
The organic layer was concentrated and the residue purified by
flash chromatography on a silica gel column (dichloromethane/
ethanol 9/1) affording ethyl 1-methyl-8-({1-[(4-methylpiperazin-
By employment of the above-described procedure, starting
from 10a and using the suitable aldehyde, compounds 16 and 17
were prepared.
8-[(4-Bromobenzyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxamide (16). Yield, 60%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.68-3.00 (m, 4H), 4.15 (s, 3H),
4.49 (d, J = 6.3 Hz, 2H), 7.21 (s, 1H), 7.29 (d, J = 8.5 Hz, 2H),
7.40 (s, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.65-7.77 (m, 1H), 8.21 (s,
1H); LCMS (ESI) m/z 413 (M þ H)þ.
8-{[4-(Acetylamino)benzyl]amino}-1-methyl-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (17). Yield, 58%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 2.00 (s, 3H), 2.71 (t, J = 7.7
Hz, 2H), 2.92 (t, J = 7.7 Hz, 2H), 4.18 (s, 1H), 4.46 (d, J = 6.2 Hz,
1H), 7.21 (s, 1H), 7.24 (d, J = 8.5 Hz, 2H), 7.40 (s, 1H), 7.48 (d,
J = 8.5 Hz, 2H), 7.56-7.68 (m, 1H), 8.21 (s, 1H), 9.80 (none, 1H);
LCMS (ESI) m/z 392 (M þ H)þ; HRMS (ESI) calcd for C20H21-
N7O2 þ Hþ, 392.183; found, 392.1835; Anal. (C20H21N7O2) C,H,N.
8-Amino-N-1-dimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazo-
line-3-carboxamide (10b). A solution of 9 (200 mg, 0.73 mmol) in a
mixture of ethanol (10 mL) and 33 wt % methylamine in absolute
ethanol (30 mL) was stirred at 60 ꢀC in a closed bottle for 8 h. The
solvent was then removed in vacuo and the crude was purified by
flash chromatography on a silica gel column (dichloromethane/
methanol/triethylamine, 95/5/5) affording 10b (147 mg, 78%). 1H
NMR (400 MHz, DMSO-d6) δ ppm 2.68-2.73 (m, 2H), 2.73 (d,
J = 4.8 Hz, 3H), 2.93 (t, J = 7.6 Hz, 2H), 4.29 (s, 3H), 6.52 (s,
2H), 7.97-8.06 (m, 1H), 8.16 (s, 1H); LCMS (ESI) m/z 259 (M þ
H)þ; HRMS (ESI) calcd for C12H14N6O þ Hþ, 259.1302; found,
259.1300.
8-[(1-Acetylpiperidin-4-yl)amino]-N,1-dimethyl-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (51). To a suspension of

2182 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Traquandi et al.
1-yl)carbonyl]piperidin-4-yl}amino)-4,5-dihydro-1H-pyrazolo[4,
3-h]quinazoline-3-carboxylate (315 mg, 50%). 1H NMR (400
MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 1.45 (m, 2H),
1.89 (m, 2H), 2.18 (s, 3H), 2.37 (m, 4H), 2.75 (t, J = 7.7 Hz, 2H),
2.85 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H), 3.13 (m, 4H), 3.57 (m, 2H),
3.87 (m, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.31 (s, 3H), 7.11 (s, 1H),
8.22 (s, 1H); LCMS (ESI) m/z 483 (M þ H)þ; HRMS (ESI) calcd
for C24H34N8O3 þ Hþ, 483.2827; found, 483.2820. A suspension
of the above-described ethyl carboxylate (300 mg, 0.62 mmol) in a
mixture of 33% NH4OH (10 mL), methanol (5 mL), and DMF
(5 mL) was stirred at 65 ꢀC in a closed bottle for 16 h. After that
time, the solvent was removed and the residue was partitioned
between dichloromethane and aqueous NaHCO3. The organic
layer was then dried over Na2SO4 and evaporated affording 54
(170 mg, 61%), after purification by flash chromatography
(dichloromethane/ethanol/33% NH4OH, 90/9/1). 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.87 (m, 4H), 2.44(s, 3H), 2.49
(m, 4H), 2.86 (m, 2H), 3.13 (m, 2H), 3.39 (m, 2H), 3.50 (m, 2H),
3.80 (m, 2H), 4.05 (m, 1H), 4.24 (s, 3H), 7.28 (s, 1H), 7.27 (bs, 1H),
7.45 (bs, 1H), 8.18 (s, 1H); LCMS (ESI) m/z 454 (M þ H)þ;
HRMS (ESI) calcd for C22H31N9O2 þ Hþ, 454.2673; found,
454.2687.
rated with diethyl ether and 31 was collected by filtration (81 mg,
35%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.10 (d, J = 6.6 Hz,
3H), 1.60 (m, 4H), 1.84 (m, 2H), 2.07 (m, 2H), 2.27 (s, 6H), 2.30 (m,
2H), 2.85 (m, 2H), 3.11 (m, 2H), 4.12 (m, 1H), 4.24 (s, 3H), 4.57
(quin, J= 8.0 Hz, 1H), 7.28 (s, 1H), 8.02 (s, 1H), 8.20 (d, J=7.9Hz,
1H); LCMS (ESI) m/z 398 (M þ H)þ; HRMS (ESI) calcd for
C21H31N7O þ Hþ, 398.2663; found, 398.2671.
By employment of the above-described procedure, starting
from 9 and using the suitable amine, compounds 32 and 33 were
prepared.
8-(Cyclopentylamino)-N-[2-(dimethylamino)ethyl]-N-1-dimeth-
yl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (32).
Yield, 45%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.64 (m, 4H),
1.84. (m, 4H), 2.03 (m, 2H), 2.27 (s, 6H), 2.41 (t, J = 6.5 Hz, 2H),
2.87 (m, 2H), 2.92 (s, 3H), 2.98 (m, 2H), 3.70 (t, J = 6.5 Hz, 2H),
4.23 (s, 3H), 4.57 (quin, J = 8.0 Hz, 1H), 7.28 (s, 1H), 8.18 (s, 1H);
LCMS (ESI) m/z 398 (M þ H)þ; HRMS (ESI) calcd for
C21H31N7O þ Hþ, 398.2663; found, 398.2676.
[8-(Cyclopentylamino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-
h]quinazolin-3-yl](4-methylpiperazin-1-yl)methanone (33). Yield,
51%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.64 (m, 4H), 1.86.
(m, 2H), 2.08 (m, 2H), 2.47 (s, 3H), 2.55 (m, 4H), 2.89 (m, 2H),
3.03 (m, 2H), 3.64 (m, 2H), 3.71 (m, 2H), 4.22 (s, 3H), 4.57 (quin,
J = 8.0 Hz, 1H), 7.28 (s, 1H), 8.20 (s, 1H); LCMS (ESI) m/z 396
(M þ H)þ; HRMS (ESI) calcd for C21H29N7O þ Hþ, 396.2506;
found, 396.2518.
1-Methyl-8-{[1-(methylsulfonyl)piperidin-4-yl]amino}-4,5-dihy-
dro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (58). By em-
ployment of the above-described procedure, compound 58 was
1
prepared. Yield, 52%; H NMR (400 MHz, DMSO-d6) δ ppm
1.50-1.66 (m, 2H), 2.02 (m, 2H), 2.74 (t, J = 7.7 Hz, 2H), 2.84-
2.92 (m, 2H), 2.88 (s, 3H), 2.95 (t, J = 7.6 Hz, 2H), 3.52-3.61
(m, 2H), 3.80-3.92 (m, 1H), 4.29 (s, 3H), 7.24 (s, 1H), 7.34
(s, 1H), 7.44 (s, 1H), 8.23 (s, 1H); LCMS (ESI) m/z 406 (M þ H)þ;
HRMS (ESI) calcd for C17H23N7O3S þ Hþ, 406.1656; found,
406.1653.
8-[(1-Acetylpiperidin-4-yl)amino]-1-methyl-4,5-dihydro-1H-py-
razolo[4,3-h]quinazoline-3-carboxamide (50). To a suspension of 9
(5.187 g, 19 mmol) in dry DMF (120 mL), 1-acetyl-4-piperidone
(4.7 mL, 38 mmol), trifluoroacetic acid (10 mL, 128 mmol), and
sodium triacetoxyborohydride (8.862 g, 42 mmol) were added.
After 18 h, 0.33 N NaOH (800 mL, 264 mmol) was added drop-
wise to the mixture. The precipitate was filtered, washed
with water, and dried in oven to dryness to give of ethyl 8-[(1-
acetylpiperidin-4-yl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo[4,
3-h]quinazoline-3-carboxylate (5.3 g, 70%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 1.43 (m, 2H), 1.94
(m, 2H), 2.01 (s, 3H), 2.76 (m, 3H), 2.93 (t, J = 7.8 Hz, 2H), 3.15
(m, 1H), 3.33 (m, 1H), 3.81 (m, 1H), 3.94 (m, 1H), 4.28 (q, J = 7.1
Hz, 2H), 4.32 (s, 3H), 7.13 (d, J = 7.2 Hz, 1H), 8.23 (s, 1H); LCMS
(ESI) m/z 399 (M þ H)þ; HRMS (ESI) calcd for C20H26N6O3 þ
Hþ, 399.2139; found, 399.2140. A suspension of the above-de-
scribed ethyl carboxylate (1.0 g, 2.50 mmol) in anhydrous ethanol
(20 mL) was treated with a 1.5 M solution of KOH in ethanol
(5 mL, 7.5 mmol) at reflux temperature for 1.5 h. After cooling in
ice bath, the resulting precipitate was collected by filtration to
give potassium 8-[(1-acetylpiperidin-4-yl)amino]-1-methyl-4,5-dihy-
dro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (836 mg, 82%) as
a crystalline solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.38
(m, 2H), 1.79-1.99 (m, 2H), 2.00 (s, 3H), 2.64 (m, 2H), 2.74 (td,
J = 12.78, 3.1 Hz, 1H), 2.89 (m, 2H), 2.96 (m, 1H), 3.14 (m, 2H),
3.81 (m, 1H), 3.86-3.99 (m, 1H), 4.17 (s, 3H), 4.23-4.32 (m, 1H),
6.93 (d, J = 7.3 Hz, 1H), 8.12 (s, 1H); LCMS (ESI) m/z 371 (M þ
H)þ; HRMS (ESI) calcd for C18H22N6O3 þ Hþ, 371.1826; found,
371.1825. A suspension of the above-described carboxylic acid
potassium salt (500 mg, 1.23 mmol) in a mixture of dry DMF
(10 mL) and dry THF (10 mL) was cooled to 0 ꢀC, then DIPEA
(0.86 mL, 4.92 mmol) and EDC (708 mg, 3.69 mmol) were added in
sequence. After stirring at the same temperature for 30 min,
8-(Cyclopentylamino)-N-[1-(dimethylamino)propan-2-yl]-1-meth-
yl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (31).
A suspension of 9 (546 mg, 2 mmol) in dry DMF (10 mL) was
treated with cyclopentanone (212 μL, 2.4 mmol), trifluoroacetic
acid (1 mL, 12.8 mmol), and sodium triacetoxyborohydride (632
mg, 3 mmol). After 16 h, 0.5 N NaOH (60 mL, 30 mmol) was
added dropwise to the mixture. The precipitate was filtered,
washed with water, and dried. The residue was then purified
by flash chromatography on a silica gel column (dichloro-
methane/methanol, 99/1) affording ethyl 8-(cyclopentylamino)-
1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxy-
late (500 mg, 73%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.30
(t, J = 7.1 Hz, 3H), 1.44-1.61 (m, 4H), 1.63-1.73 (m, 2H), 1.92
(m, 2H), 2.75 (m, 2H), 2.92 (m, 2H), 4.15 (m, 1H), 4.28 (q, J = 7.1
Hz, 2H), 4.33 (s, 3H), 7.08 (d, J = 5.6 Hz, 1H), 8.20 (s, 1H);
LCMS (ESI) m/z 342 (M þ H)þ; HRMS (ESI) calcd for
C18H23N5O2 þ Hþ, 342.1924; found, 342.1924. A suspension of
the above-described ethyl carboxylate (230 mg, 0.67 mmol) was
suspendedin dryethanol (5 mL) and treated with a 1.5 M solution
of KOH in ethanol (1.33 mL, 2.01 mmol) at reflux temperature
for 1.5 h. After cooling in an ice bath, the resulting precipitate
was collected by filtration to give potassium 8-(cyclopentyla-
mino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-car-
boxylate (210 mg, 89%) as a crystalline solid. 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.39-1.76 (m, 6H), 1.80-1.99 (m, 2H), 2.63 (t,
J = 7.7 Hz, 2H), 2.88 (t, J = 7.7 Hz, 2H), 4.09-4.17 (m, 1H), 4.18
(s, 3H), 6.87 (d, J = 6.8 Hz, 1H), 8.10 (s, 1H); LCMS (ESI) m/z 314
(M þ H)þ; HRMS (ESI) calcd for C16H19N5O2 þ Hþ, 314.1611;
found, 314.1615. To a suspension of the above-described carboxylic
acid potassium salt (210 mg, 0.58 mmol) in DMF (4 mL), 1-hydro-
xybenzotriazole (HOBT, 101 mg, 0.75 mmol) and EDC (144 mg,
0.75 mmol) were added in sequence. The mixture was stirred at room
temperature for 30 min, then N1,N1-dimethylpropane-1,2-diamine
(130 μL, 1 mmol)) was added. After 20 h the reaction mixture was
poured into water and extracted with dichloromethane. The organic
phase was dried over Na2SO4 and concentrated. The crude was then
purified by flash chromatography on a silica gel column (dichloro-
methane/ethanol/33% NH4OH, 94/5.5/0.5). The residue was tritu-
HOBT NH3 (560 mg, 3.69 mmol) was added. The mixture was
3
then maintained at room temperature overnight and then the sol-
vent was removed under reduced pressure. The residue was taken
up with dichloromethane and washed with aqueous NaHCO3. The
organic layer was finally dried over Na2SO4 and the solvent was
evaporated. After trituration with diethyl ether, 50 (272 mg, 60%)
was collected by filtration. 1H NMR (400 MHz, DMSO-d6) δ ppm
1.16-1.52 (m, 3H), 1.83-1.99 (m, 2H), 2.01 (s, 3H), 2.72 (m, 2H),
2.93 (m, 2H), 3.16 (m, 2H), 3.81 (m, 2H), 3.87-4.02 (m, 2H), 4.27
(bs, 2H), 4.29 (s, 3H), 7.10 (bs, 1H), 7.22 (bs, 1H), 7.42 (bs, 1H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2183
8.21 (s, 1H); LCMS (ESI) m/z 370 (M þ H)þ; HRMS (ESI) calcd
for C18H23N7O2 þ Hþ, 370.1986; found, 370.1982.
was stirred at room temperature for 6 h. The solvent was
evaporated and the crude dissolved with water, the solution
made basic with 33% NH4OH and extracted with dichloro-
methane. The organic layer was dried over Na2SO4 and con-
centrated. The residue was chromatographed on a silica gel
column (n-hexane/ethylacetate, 7/3) and triturated with a mix-
ture n-hexane/diethyl ether to give 12a (2.34 g, 63%). 1H NMR
(401 MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 2.06
(quin, J = 6.3 Hz, 1H), 2.56 (dd, J = 7.3, 5.7 Hz, 2H), 2.95 (t,
J = 6.1 Hz, 2H), 4.29 (q, J = 7.1 Hz, 2H), 5.72 (s, 2H), 7.19
(dd, J = 7.8, 1.6 Hz, 2H); LCMS (ESI) m/z 299 (M þ H)þ;
HRMS (ESI) calcd for C17H18N2O3 þ Hþ, 299.1390; found,
299.1404.
By employment of the above-described procedure, com-
pounds 52, 56, and 60 were prepared.
1-Methyl-8-{[1-(phenylcarbonyl)piperidin-4-yl]amino}-4,5-di-
hydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (52). Yield,
45%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.91 (m, 4H), 2.86
(m, 4H), 3.08 (m, 2H), 3.61 (m, 4H), 4.02 (m, 1H), 4.24 (s, 3H),
7.26 (bs, 1H), 7.28 (bs, 1H), 7.47 (bs, 1H), 7.55 (m, 3H), 7.78 (m,
2H), 8.20 (s, 1H); LCMS (ESI) m/z 432 (M þ H)þ; HRMS (ESI)
calcd for C23H25N7O2 þ Hþ, 432.2142; found, 432.2135.
Ethyl 4-[(3-Carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazolin-8-yl)amino]piperidine-1-carboxylate (56).Yield, 50%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.18 (t, J = 7.1 Hz, 3H), 1.39
(m, 2H), 1.91 (m, 2H), 2.69-2.76 (m, 2H), 2.94 (m, 4H), 3.88 (m,
1H), 3.91-3.98 (m, 2H), 4.04 (q, J = 7.1 Hz, 2H), 4.28 (s, 3H), 7.07
(bs, 1H), 7.22 (bs, 1H), 7.42 (bs, 1H), 8.21 (s, 1H); LCMS (ESI) m/z
400 (M þ H)þ; HRMS (ESI) calcd for C19H25N7O3 þ Hþ,
400.2092; found, 400.2095; Anal. (C19H25N7O3) C, H, N.
1-Methyl-8-{[1-(phenylsulfonyl)piperidin-4-yl]amino}-4,5-di-
hydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (60). Yield,
43%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.60 (m, 2H), 1.99
(m, 2H), 2.55 (m, 2H), 2.71 (t, J = 7.6 Hz, 2H), 2.93 (m, 2H), 3.59
(m, 2H), 3.72 (m, 1H), 4.24 (s, 3H), 7.10 (d, J = 7.3 Hz, 1H), 7.23
(s, 1H), 7.41 (s, 1H), 7.62 (m, 2H), 7.75 (m, 1H), 7.77 (m, 2H), 8.19
(s, 1H); LCMS (ESI) m/z 468 (M þ H)þ; HRMS (ESI) calcd for
C22H25N7O3S þ Hþ, 468.1813; found, 468.1808.
By employment of the above-described procedure, starting
from 11 and using the suitable substituted hydrazine, com-
pounds 12b-i were prepared.
Ethyl 7-Oxo-1-phenyl-4,5,6,7-tetrahydro-1H-indazole-3-car-
1
boxylate (12b). Yield, 80%; H NMR (400 MHz, DMSO-d6)
δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.08-2.17 (m, 2H), 2.56 (dd, J =
7.4, 5.5 Hz, 2H), 3.03 (t, J = 6.1 Hz, 2H), 4.34 (q, J = 7.1 Hz,
2H), 7.51 (s, 5H); LCMS (ESI) m/z 285 (M þ H)þ; HRMS (ESI)
calcd for C16H16N2O3 þ Hþ, 285.1234; found, 285.1236.
Ethyl 1-(4-Methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-in-
1
dazole-3-carboxylate (12c). Yield, 72%; H NMR (400 MHz,
DMSO-d6) δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.03-2.15 (m, 2H),
2.54 (dd, J = 7.3, 5.5 Hz, 2H), 3.01 (t, J = 6.1 Hz, 2H), 3.83 (s,
3H), 4.33 (q, J = 7.1 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 7.42 (d,
J = 9.0 Hz, 2H); LCMS (ESI) m/z 315 (M þ H)þ; HRMS (ESI)
calcd for C17H18N2O4 þ Hþ, 315.1339; found, 315.1345.
Ethyl 7-Oxo-1-(4-sulfamoylphenyl)-4,5,6,7-tetrahydro-1H-in-
8-(Cyclopentylamino)-N-hydroxy-N,1-dimethyl-4,5-dihydro-
1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (34). To a sus-
pension of potassium 8-(cyclopentylamino)-1-methyl-4,5-dihy-
dro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (300 mg, 0.85
mmol, prepared as described for the preparation of compound 31)
in dry dichloromethane (60 mL) and a few drops of dry DMF,
oxalyl chloride (85 μL, 1 mmol) was added at 0 ꢀC. The mixture
was stirred at room temperature for 6 h and then evaporated,
dissolved in dry dichloromethane, and dropped into a solution of
methylhydroxylamine hydrochloride (141 mg, 1.70 mmol) and
triethylamine (490 μL, 3.40 mmol) in the same solvent (20 mL),
cooled to 0 ꢀC. After 4 h the mixture was washed with a saturated
solution of NaHCO3, dried over Na2SO4, and evaporated to
dryness. The residue was triturated with diethyl ether and filtered
to give 34 (175 mg, 60%). 1H NMR (400 MHz, DMSO-d6) δ ppm
1.53 (m, 4H), 1.66 (m, 2H), 1.93 (m, 2H), 2.65-2.87 (m, 4H), 3.33
(s, 3H), 4.15 (m, 1H), 4.29 (s, 3H), 7.04 (d, J = 6.6 Hz, 1H), 8.19
(s, 1H), 9.87 (s, 1H); LCMS (ESI) m/z 343 (M þ H)þ; HRMS
(ESI) calcd for C17H22N6O2 þ Hþ, 343.1877; found, 343.1884;
Anal. (C17H22N6O2) C, H, N.
By employment of the above-mentioned procedure and using
the suitable substituted hydroxylamine, compounds 35 and 36
were prepared.
N-Cyclohexyl-8-(cyclopentylamino)-N-hydroxy-1-methyl-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (35). Yield,
55%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.16-1.36 (m, 2H),
1.46-1.80 (m, 12H), 1.84-1.99 (m, 2H), 2.65-2.79 (m, 4H), 4.16
(m, 1H), 4.29 (s, 3H), 4.33 (m, 1H), 7.04 (d, J = 6.8 Hz, 1H), 8.18
(s, 1H), 9.44 (s, 1H); LCMS (ESI) m/z 411 (M þ H)þ; Anal.
(C22H30N6O2) C, H, N.
1
dazole-3-carboxylate (12d). Yield, 70%; H NMR (400 MHz,
DMSO-d6) δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.08-2.17 (m, 2H),
2.55-2.61 (m, 2H), 3.03 (t, J = 6.1 Hz, 2H), 4.35 (q, J = 7.1 Hz,
2H), 7.53 (s, 2H), 7.75 (d, J = 8.8 Hz, 2H), 7.94 (d, J = 8.8 Hz,
2H); LCMS (ESI) m/z 364 (M þ H)þ; HRMS (ESI) calcd for
C16H17N3O5S þ Hþ, 364.0962; found, 364.0961.
Ethyl 7-Oxo-1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-indazole-
3-carboxylate (12e). Yield, 75%; 1H NMR (400 MHz, DMSO-
d6) δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.08-2.19 (m, 2H), 2.57 (dd,
J = 7.3, 5.6 Hz, 2H), 3.03 (t, J = 6.2 Hz, 2H), 4.34 (q, J = 7.1
Hz, 2H), 7.60 (ddd, J = 7.4, 4.7, 1.0 Hz, 1H), 7.62 (dt, J = 7.9,
0.9 Hz, 1H), 8.06 (td, J = 7.7, 1.8 Hz, 1H), 8.56 (ddd, J = 4.7,
1.8, 0.8 Hz, 1H); LCMS (ESI) m/z 286 (M þ H)þ.
Ethyl 1-(2-Hydroxyethyl)-7-oxo-4,5,6,7-tetrahydro-1H-inda-
zole-3-carboxylate (12f). Yield, 72%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 1.99-2.09 (m,
2H), 2.51-2.56 (m, 2H), 2.93 (t, J = 6.1 Hz, 2H), 3.72 (t, J = 5.8
Hz, 2H), 4.25-4.34 (m, 2H), 4.54 (t, J = 5.8 Hz, 2H), 4.85 (bs,
1H); LCMS (ESI) m/z 253 (M þ H)þ; HRMS (ESI) calcd for
C12H16N2O4 þ Hþ, 253.1183; found, 253.1187.
Ethyl 7-Oxo-1-(2,2,2-trifluoroethyl)-4,5,6,7-tetrahydro-1H-
indazole-3-carboxylate (12g). Yield, 68%; 1H NMR (500 MHz,
DMSO-d6) δ ppm1.31(t, J = 7.2Hz, 3H), 2.07 (dt, J =12.5, 6.26
Hz, 2H), 2.59 (dd, J = 7.2, 5.9 Hz, 2H), 2.96 (t, J = 6.1 Hz, 2H),
4.32 (q, J = 7.2 Hz, 2H), 5.48 (q, J = 8.7 Hz, 2H); LCMS (ESI)
m/z 291 (M þ H)þ.
Ethyl 1-(1-Methylpiperidin-4-yl)-7-oxo-4,5,6,7-tetrahydro-1H-
indazole-3-carboxylate (12h). Yield, 79%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 1.82-2.10 (m, 6H),
2.26 (s, 3H), 2.46-2.51 (m, 2H), 2.50-2.58 (m, 2H), 2.89-2.98
(m, 2H), 2.93 (t, J = 6.2 Hz, 2H), 4.30 (q, J = 7.1 Hz, 2H),
4.92-5.10 (m, 1H); LCMS (ESI) m/z 306 (M þ H)þ; HRMS
(ESI) calcd for C16H23N3O3 þ Hþ, 306.1812; found, 306.1801.
Ethyl 1-(1-Acetylpiperidin-4-yl)-7-oxo-4,5,6,7-tetrahydro-1H-
N-Benzyl-8-(cyclopentylamino)-N-hydroxy-1-methyl-4,5-di-
hydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (36). Yield,
62%; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.52 (m, 4H), 1.69
(m, 2H), 1.89 (m, 2H), 2.76 (m, 4H), 4.16 (m, 1H), 4.29 (s, 3H),
4.99 (s, 2H), 7.05 (d, J = 6.6 Hz, 1H), 7.20-7.41 (m, 5H),
8.19 (s, 1H), 9.87 (s, 1H); LCMS (ESI) m/z 419 (M þ H)þ;
HRMS (ESI) calcd for C23H26N6O2 þ Hþ, 419.2190; found,
419.2201.
1
indazole-3-carboxylate (12i). Yield, 76% yield); H NMR (400
Ethyl 1-Benzyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-car-
boxylate (12a). Ethyl (3-ethoxy-2-oxocyclohex-3-en-1-yl)(oxo)-
acetate 11 (3.0 g, 12.5 mmol, prepared as described in ref 17) was
dissolved in glacial acetic acid (15 mL) and benzylhydrazine
dihydrochloride (2.44 g, 12.5 mmol) was added. The mixture
MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 1.73 (m, 1H),
1.95 (m, 3H), 2.00-2.08 (m, 2H), 2.04 (s, 3H), 2.56 (dd, J = 7.2,
5.6 Hz, 2H), 2.64-2.76 (m, 1H), 2.93 (t, J = 6.1 Hz, 2H), 3.20
(m, 2H), 3.87-3.99 (m, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.48
(m, 1H), 5.27 (m, 1H); LCMS (ESI) m/z 334 (M þ H)þ;

2184 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Traquandi et al.
HRMS (ESI) calcd for C17H23N3O4 þ Hþ, 334.1762; found,
334.1757.
MHz, DMSO-d6) δ ppm 1.33 (t, J = 7.1 Hz, 3H), 2.77 (t, J = 7.8
Hz, 2H), 2.97 (t, J = 7.8 Hz, 2H), 4.32 (q, J = 7.1 Hz, 2H), 5.82
(q, J= 8.9 Hz, 2H), 6.65 (s, 2H), 8.22 (s, 1H); LCMS (ESI) m/z 342
(M þ H)þ.
Ethyl 8-Amino-1-benzyl-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxylate (13a). To a solution of 12a (500 mg, 1.67
mmol) in dry DMF (12 mL), N,N-dimethylformamide di-tert-
butylacetale (4 mL, 16.3 mmol) was added. The mixture was stirred
at 60 ꢀC for 8 h. The solvent was then evaporated and the residue
triturated with diethyl ether to give ethyl (6E)-1-benzyl-6-[(dime-
thylamino)methylidene]-7-oxo-4,5,6,7-tetrahydro-1H-indazole-
3-carboxylate (501 mg, 85%). 1H NMR (401 MHz, DMSO-d6)
δ ppm 1.29 (t, J = 7.1 Hz, 3H), 2.80-2.87 (m, 2H), 2.87-2.96 (m,
2H), 3.12 (s, 6H), 4.26 (q, J = 7.1 Hz, 2H), 5.80 (s, 2H), 7.18-7.22
(m, 2H), 7.23-7.34 (m, 2H), 7.51 (s, 1H); LCMS (ESI) m/z 354
(M þ H)þ; HRMS (ESI) calcd for C20H23N3O3 þ Hþ, 354.1812;
found, 354.1804. To a solution of the above-described ethyl
carboxylate (450 mg, 1.27 mmol) in ethanol (20 mL), sodium
ethylate (86 mg, 1.27 mmol) and guanidine hydrochloride
(121 mg, 1.27 mmol) were added in sequence. The solution was
stirred at reflux for 12 h. The solvent was then evaporated, the
residue taken up with dichloromethane and washed with water.
The organic layer was dried over anhydrous Na2SO4 and con-
centrated. The residue was triturated with diethyl ether and
collected by filtration, giving 13a (377 mg, 85%). 1H NMR
(401 MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 2.75 (t,
J = 7.7 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H), 4.28 (q, J = 7.1 Hz,
2H), 6.07 (s, 2H), 6.63 (bs, 2H), 7.22-7.38 (m, 5H), 8.17 (s, 1H);
LCMS (ESI) m/z 350 (M þ H)þ; HRMS (ESI) calcd for
C19H19N5O2 þ Hþ, 350.1612; found, 350.1618.
Ethyl 8-Amino-1-(1-methylpiperidin-4-yl)-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxylate (13h). Yield, 68%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.31 (t, J = 7.1 Hz, 3H), 1.93 (m,
2H), 2.04 (m, 2H), 2.14-2.24 (m, 4H), 2.25 (s, 3H), 2.72 (m, 2H),
2.87 (bs, 2H), 2.91 (m, 2H), 4.29 (q, J = 7.1 Hz, 2H), 5.55 (tt, J =
11.3, 4.2 Hz, 1H), 6.53 (s, 2H), 6.73-6.74 (m, 1H), 6.77-6.79 (m,
1H), 8.18 (s, 1H); LCMS (ESI) m/z 357 (M þ H)þ; HRMS (ESI)
calcd for C18H24N6O2 þ Hþ, 357.2033; found, 357.2033.
Ethyl 1-(1-Acetylpiperidin-4-yl)-8-amino-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxylate (13i). Yield, 79%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 1.72-2.05
(m, 4H), 2.07 (s, 3H), 2.71-2.76 (m, 2H), 2.73-2.85 (m, 1H), 2.92
(t, J = 7.6 Hz, 2H), 3.26-3.35 (m, 1H), 3.89-4.03 (m, 1H), 4.28
(q, J = 7.1 Hz, 2H), 4.46-4.57 (m, 1H), 5.77-5.95 (m, 1H), 6.60
(s, 2H), 8.19 (s, 1H); LCMS (ESI) m/z 385 (M þ H)þ; HRMS
(ESI) calcd for C19H24N6O3 þ Hþ, 385.1983; found, 385.1983.
1-Benzyl-8-(cyclopentylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxamide (37). To a suspension of 13a (450 mg,
1.29 mmol) in dry DMF (10 mL), cyclopentanone (0.23 mL, 2.58
mmol), sodium triacetoxyborohydride (600 mg, 2.84 mmol), and
trifluoroacetic acid (0.7 mL, 9 mmol) were added in sequence. The
mixture was stirred at room temperature overnight. After this
time, 0.33 N NaOH (54 mL, 18 mmol) was added dropwise to the
mixture. The precipitate was filtered, washed with water, and
dried in an oven to dryness to give ethyl 1-benzyl-8-(cyclopentyla-
mino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate
(430 mg, 80%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.29 (t,
J = 7.1 Hz, 3H), 1.42 (bs, 4H), 1.53-1.65 (m, 2H), 1.75 (bs, 2H),
2.77 (m, 2H), 2.97 (m, 2H), 4.00 (bs, 1H), 4.28 (q, J = 7.1 Hz, 2H),
6.10 (s, 2H), 7.09 (d, J = 7.2 Hz, 1H), 7.17 (m, 2H), 7.25 (m, 1H),
7.31 (m, 2H), 8.21 (s, 1H); LCMS (ESI) m/z 418 (M þ H)þ;
HRMS (ESI) calcd for C24H27N5O2 þ Hþ, 418.2238; found,
418.2236. A solution of the above-described ethyl carboxylate
(300 mg, 0.72 mmol) in a mixture of methanol (10 mL), DMF
(10 mL), and 33% NH4OH (5 mL) was stirred at 70 ꢀC in a close
bottle for 8 h. The solvent was then removed under reduced
pressure and the residue dissolved with dichloromethane and
washed with water. The organic layer was dried over Na2SO4 and
evaporated. The crude was purified by flash chromatography on
a silica gel column (dicloromethane/methanol, 98/2) affording 37
(223 mg, 80%). 1H NMR (400 MHz, DMSO-d6) δ ppm
1.36-1.52 (m, 4H), 1.58-1.83 (m, 4H), 2.79 (t, J = 7.6 Hz,
2H), 3.02 (t, J = 7.6 Hz, 2H), 3.98 (s, 1H), 6.04 (s, 2H), 7.09-7.34
(m, 7H), 7.49 (s, 1H), 7.70 (s, 1H), 8.22 (s, 1H); LCMS (ESI) m/z
389 (M þ H)þ; HRMS (ESI) calcd for C22H24N6O þ Hþ,
389.2085; found, 389.2090.
By employment of the above-described procedure, starting
from intermediates 12b-i, compounds 13b-i were prepared.
Ethyl 8-Amino-1-phenyl-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxylate (13b). Yield, 56%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.80 (m, 2H), 2.99 (m,
2H), 4.33 (q, J = 7.1Hz, 2H), 6.04 (bs, 2H), 7.50 (m, 3H), 7.56 (m,
2H), 8.20 (s, 1H); LCMS (ESI) m/z 336 (M þ H)þ; HRMS (ESI)
calcd for C18H17N5O2 þ Hþ, 336.1455; found, 336.1449.
Ethyl 8-Amino-1-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxylate (13c). Yield, 52%; 1H NMR (400
MHz, DMSO-d6) δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.79 (t, J = 7.5
Hz, 2H), 2.98 (t, J = 7.4 Hz, 2H), 3.84 (s, 3H), 4.32 (q, J = 7.1 Hz,
2H), 6.08 (s, 2H), 7.0 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 9.0 Hz, 2H),
8.19 (s, 1H); LCMS (ESI) m/z 366 (M þ H)þ; HRMS (ESI) calcd
for C19H19N5O3 þ Hþ, 366.1561; found, 366.1564; HPLC purity
85%.
Ethyl 8-Amino-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxylate (13d). Yield, 60%; 1H NMR (400
MHz, DMSO-d6) δ ppm 1.33 (t, J = 7.1 Hz, 3H), 2.82 (t, J = 7.5
Hz, 2H), 3.00 (t, J = 7.4 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 6.12 (s,
2H), 7.43 (s, 2H), 7.81 (d, J = 8.8 Hz, 2H), 7.90-7.97 (m, 2H),
8.23 (s, 1H); LCMS (ESI) m/z 415 (M þ H)þ; HRMS (ESI) calcd
for C18H18N6O4S þ Hþ, 415.1183; found, 415.1174.
By employment of the above-described procedure, starting
from intermediates 13b-i, compounds 38-44 and 46 were
prepared.
Ethyl 8-Amino-1-(pyridin-2-yl)-4,5-dihydro-1H-pyrazolo[4,3-
1
h]quinazoline-3-carboxylate (13e). Yield, 58%; H NMR (400
MHz, DMSO-d6) δ ppm 1.33 (t, J = 7.1 Hz, 3H), 2.81 (t, J = 7.5
Hz, 2H), 2.99 (t, J = 7.5 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 6.00
(s, 2H), 7.56 (ddd, J = 7.5, 4.8, 1.0 Hz, 1H), 7.67 (dt, J = 7.9, 0.9
Hz, 1H), 8.04 (td, J = 7.7, 1.9 Hz, 1H), 8.20 (s, 1H), 8.51 (ddd,
J = 4.8, 1.9, 0.9 Hz, 1H); LCMS (ESI) m/z 337 (M þ H)þ;
HRMS (ESI) calcd for C17H16N6O2 þ Hþ, 337.1407; found,
337.1394.
8-(Cyclopentylamino)-1-phenyl-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxamide (38). Yield, 55%; 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.01-1.62 (m, 8H), 2.79 (t, J = 7.62 Hz, 2H),
3.02 (t, J = 7.62 Hz, 2H), 3.25-3.31 (m, 1H), 6.75 (s, 1H), 7.32 (s,
1H), 7.43-7.56 (m, 5H), 7.59 (s, 1H), 8.16 (s, 1H); LCMS (ESI) m/
z 375 (M þ H)þ; HRMS (ESI) calcd for C21H22N6O þ Hþ,
375.1928; found, 375.1933.
Ethyl 8-Amino-1-(2-hydroxyethyl)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxylate (13f). Yield, 70%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.31 (t, J = 7.1 Hz, 3H), 2.74 (t,
J = 7.6 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H), 3.77-3.86 (m, 2H),
4.29 (q, J = 7.1 Hz, 2H), 4.78 (t, J = 5.8 Hz, 1H), 4.82 (t, J = 5.9
Hz, 2H), 6.53 (s, 2H), 8.17 (s, 1H); LCMS (ESI) m/z 304 (M þ
H)þ; HRMS (ESI) calcd for C14H17N5O3 þ Hþ, 304.1404;
found, 304.1412; HPLC purity 80%.
8-(Cyclopentylamino)-1-(pyridin-2-yl)-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (39). Yield, 50%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.04-1.63 (m, 8H), 2.80 (t,
J=7.7Hz,2H), 3.02(t,J=7.7Hz,2H),3.11-3.61 (m, 1H), 6.77 (s,
1H), 7.35 (s, 1H), 7.56 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.63 (s, 1H),
7.66 (dt, J = 7.9, 0.9 Hz, 1H), 8.02-8.08 (m, 1H), 8.17 (s, 1H), 8.56
(ddd, J = 4.8, 1.8, 0.9 Hz, 1H); LCMS (ESI) m/z 376 (M þ H)þ.
8-(Cyclopentylamino)-1-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (40). Yield, 52%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 0.98-1.66 (m, 8H), 2.77 (t,
Ethyl 8-Amino-1-(2,2,2-trifluoroethyl)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxylate (13g). Yield, 45%; 1H NMR (400

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2185
J = 7.6Hz, 2H), 3.01(t, J = 7.6Hz, 2H), 3.20-3.42(m, 1H), 3.80
(s, 3H), 6.77 (d, J = 5.8 Hz, 1H), 7.02 (d, J = 9.0 Hz, 2H), 7.29 (s,
1H), 7.44 (d, J = 8.9 Hz, 2H), 7.56 (s, 1H), 8.14 (s, 1H); LCMS
(ESI) m/z 405 (M þ H)þ; Anal. (C22H24N6O2) C, H, N.
410 (M þ H)þ; HRMS (ESI) calcd for C22H31N7O þ Hþ,
410.2663; found, 410.2668.
Crystallographic Methods. Expression, purification, crystal-
lization, and soaking procedures of the CDK2/cyclin A com-
plexes were carried out as previously described.21
8-(Cyclopentylamino)-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-
1
pyrazolo[4,3-h]quinazoline-3-carboxamide (41). Yield, 48%; H
Kinase Assays. Kinase assays were performed as previously
described.21 The panel includes the following: c-ABL, ACK1,
ALK, AKT1, Aur-A, Aur-B, BRK, BUB1, CDC7/DBF4,
CDK2/CyA, CDK2/CyE, CDK1/CyB, CDK5/p25, CDK4/
CyD1, GSK-3β, CHK1, CK2, EEF2K, EGFR, ERK2, FAK,
FGFR1, FLT3, IGF1R, IKK1, IKK2, IR, JAK1, JAK3, C-
KIT, LCK, LYN, MAPKAPK2, MET, MNK2, MST4, NEK6,
NIM, PAK4, PDGFR, PDK1, PERK, PIM1, PIM2, PKAR,
PKCβ, PLK1, RET, SYK, SULU1, TYK, TRKA, VEGFR2,
VEGFR3, and ZAP70.
In Vitro Pharmacology. A2780 Cells Proliferation Assay. This
cell line was selected for the screening of compounds active
against CDKs because it has a high proliferation rate (doubling
time of 21 h). Cells were seeded into 96 or 384 wells plates at
final concentration ranging from 10000 to 30000 cells per cm2
in appropriate medium plus 10% FCS. After 24 h, cells were
treated with scalar doses of test compounds in two replicates.
Compound dilutions were prepared in 100% DMSO at 1000-
fold concentration, and then diluted in the culture medium to
have in the wells the desired compound dose with a final
DMSO concentration of 0.1% that does not interfere with cell
proliferation. A total of 72 h after the treatment, the cell
number was evaluated using the Cell Titer_Glo Assay
(Promega). IC50s were calculated using a Sygmoidal fitting
(Symyx Assay Explorer). Experiments were replicated at least
two times.
Flow Cytometry Analysis, Immunoblot, and BrdU Incorpora-
tion. A2780 cells (human ovary adenocarcinoma, from
ECACC), were seeded in T-75 tissue culture flasks, 25000
cells/cm2 in RPMI 1640, pH 7.4, 10% FBS, 2 mM L-glutamine,
1ꢀ penicillin-streptomycin, and maintained in 5% CO2 at
37 ꢀC with 96% relative humidity. After 24 h, cells were treated
with the compound at 1 and 3 μM for 24 h, approximately
3- and 10-fold the IC50 measured in 72 h proliferation study.
Both detached and adherent cells were collected using 0.25%
trypsin, 0.02% EDTA, washed in PBS, and divided into three
samples for flow cytometry analysis, immunoblot, and BrdU
incorporation as previously described.21 In particular, the
sample for cell cycle analysis was fixed in ethanol 70% in
PBS for at least 12 h. DNA staining was performed using a
solution of propidium iodide 25 μg/mL in sodium citrate 1%
containing RNase A (2 μg/mL) and 0.002% NP-40 (Sigma).
After 1 h at room temperature, samples were acquired and
analyzed using a FACS-Calibur flow cytometer (Beckton
Dickinson). The DNA histograms were analyzed by Modfit
3.0 (Verity Software House).
In Vivo Pharmacology. All animal studies were carried out in
compliance with Italian Legislative Decree No. 116, dated
January 27, 1992, and the European Communities Council
Directive No. 86/609/EEC concerning the protection of animals
for experimental purposes and according to institutional policy
regarding the care and use of laboratory animals. Female nu/nu
mice (Harlan), 5-6 weeks age (average weight 20-22 g) were
used. A2780 human ovarian carcinoma cells were transplanted
s.c. in athymic mice. Mice bearing a palpable tumor were
selected and randomized into vehicle and treated groups
(Mean: 180 mg). Treatments started the day after randomiza-
tion. Compound 59 was dissolved in 5% dextrose. Treatments
were administered twice a day for 10 consecutive days. Dimen-
sions of the tumors were measured regularly using Vernier
calipers for all the duration of the experiment and tumor masses
and tumor growth inhibition were calculated as described.21 The
effectiveness of the anticancer treatment was also determined as
the delay in the onset of the exponential growth of the tumor
according to the criteria of Bissery et al.22 Tumor growth delay
NMR (400 MHz, DMSO-d6) δ ppm 1.07-1.66 (m, 8H), 2.85
(t, J = 7.4 Hz, 2H), 3.06 (t, J = 7.4 Hz, 2H), 3.27-3.55 (m, 1H),
7.45 (s, 4H), 7.69 (s, 1H), 7.81 (d, J = 8.6 Hz, 2H), 7.93 (d, J =
8.6 Hz, 2H), 8.23 (s, 1H); LCMS (ESI) m/z 454 (M þ H)þ;
HRMS (ESI) calcd for C21H23N7O3S þ Hþ, 454.1656; found,
454.1651; Anal. (C21H23N7O3S) C, H, N, S.
8-(Cyclopentylamino)-1-(2-hydroxyethyl)-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (42). Yield, 51%; 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.39-1.76 (m, 6H), 1.83-1.99 (m,
2H), 2.71 (t, J = 7.7 Hz, 2H), 2.93 (t, J = 7.6 Hz, 2H), 3.84 (t, J =
6.1 Hz, 2H), 4.07-4.24 (m, 1H), 4.71-4.88 (m, 1H), 4.80 (t, J =
6.2 Hz, 2H), 7.03 (d, J = 6.0 Hz, 1H), 7.22 (s, 1H), 7.41 (s, 1H),
8.19 (s, 1H); LCMS (ESI) m/z 343 (M þ H)þ; HRMS (ESI) calcd
for C17H22N6O2 þ Hþ, 343.1877; found, 343.1876.
8-(Cyclopentylamino)-1-(2,2,2-trifluoroethyl)-4,5-dihydro-1H-
1
pyrazolo[4,3-h]quinazoline-3-carboxamide (43). Yield, 53%; H
NMR (400 MHz, DMSO-d6) δ ppm 1.40-1.76 (m, 6H),
1.82-1.98 (m, 2H), 2.76 (t, J = 7.7 Hz, 2H), 2.97 (t, J = 7.7
Hz, 2H), 4.00-4.18 (m, 1H), 5.79 (q, J = 8.8 Hz, 2H), 7.20 (d, J =
6.9 Hz, 1H), 7.39 (s, 1H), 7.44 (s, 1H), 8.24 (s, 1H); LCMS (ESI)
m/z 381 (M þ H)þ; Anal. (C17H19F3N6O) C, H, N.
8-(Cyclopentylamino)-1-(1-methylpiperidin-4-yl)-4,5-dihydro-
1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (44). Yield, 58%;
1H NMR (400 MHz, DMSO-d6) δ ppm 1.4-1.63 (m, 4H), 1.68-
1.79 (m, 2H), 1.83-2.11 (m, 4H), 2.09-2.32 (m, 6H), 2.67-2.73
(m, 2H), 2.92 (t, J = 7.7 Hz, 2H), 2.98 (bs, 2H), 4.17 (sxt, J = 7.0
Hz, 1H), 5.58 (bs, 1H), 7.08 (bs, 1H), 7.24 (bs, 1H), 7.33 (bs, 1H),
8.19 (s, 1H); LCMS (ESI) m/z 396 (M þ H)þ; HRMS (ESI) calcd
for C21H29N7O þ Hþ, 396.2507; found, 396.2507.
1-(1-Acetylpiperidin-4-yl)-8-(cyclopentylamino)-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (46). Yield, 49%; 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.43-1.60 (m, 4H), 1.71
(m, 2H), 1.89-2.04 (m, 6H), 2.06 (s, 3H), 2.65 (bs, 1H), 2.71 (t, J =
7.7 Hz, 2H), 2.85-2.99 (m, 2H), 3.07-3.21 (m, 1H), 4.02 (m, 1H),
4.16 (m, 1H), 4.55 (m, 1H), 5.81 (m, 1H), 7.07 (bs, 1H), 7.21 (s, 1H),
7.41 (s, 1H), 8.20 (s, 1H); LCMS (ESI) m/z 424 (M þ H)þ; HRMS
(ESI) calcd for C22H29N7O2 þ Hþ, 424.2456; found, 424.2457.
8-(Cyclopentylamino)-N-methyl-1-(1-methylpiperidin-4-yl)-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (45). To a
suspension of 13h (1.0 g, 2.80 mmol) in dry DMF (20 mL),
cyclopentanone (0.5 mL, 5.6 mmol), trifluoroacetic acid (1.53
mL, 19.6 mmol), and sodium triacetoxyborohydride (1.3 g, 6.16
mmol) were added. After 18 h, 0.33 N NaOH (119 mL, 39.2
mmol) was added dropwise to the mixture. The precipitate was
filtered and dried in oven to give ethyl 8-(cyclopentylamino)-1-(1-
methylpiperidin-4-yl)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-
1
3-carboxylate (949 mg, 80%). H NMR (400 MHz, DMSO-d6)
δ ppm 1.31 (t, J = 7.1 Hz, 3H), 1.44-1.64 (m, 4H), 1.73 (m, 2H),
1.90-2.02 (m, 6H), 2.13 (m, 2H), 2.25 (bs, 3H), 2.73 (m, 2H), 2.92
(m, 2H), 2.99 (bs, 2H), 4.17(m, 1H), 4.29 (q, J = 7.1Hz, 2H), 5.63
(m, 1H), 7.12 (bs, 1H), 8.21 (s, 1H); LCMS (ESI) m/z 425 (M þ
H)þ; HRMS (ESI) calcd for C23H32N6O2 þ Hþ, 425.2660; found,
425.2658. To a solution of the above-described ethyl carboxylate
(800 mg, 1.89 mmol) in ethanol (50 mL) was added 33 wt %
methylamine in absolute ethanol (15 mL). The solution was
stirred at 60 ꢀC in a closed bottle for 8 h. The solvent was then
removed under reduced pressure and the crude purified by flash
chromatography on a silica gel column (dichloromethane/metha-
nol/triethylamine, 95/5/5) affording 45 (603 mg, 78%). 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.43-1.62 (m, 4H), 1.74 (m, 2H),
1.87-2.06 (m, 6H), 2.13-2.20 (m, 2H), 2.21 (s, 3H), 2.67-2.73
(m, 2H), 2.75 (d, J = 4.8 Hz, 3H), 2.92 (t, J = 7.7 Hz, 2H), 2.96
(bs, 2H), 4.17 (sxt, J = 7.2 Hz, 1H), 5.57 (tt, J = 11.4, 3.9 Hz,
1H), 7.09 (bs, 1H), 7.93 (m, 1H), 8.19 (s, 1H); LCMS (ESI) m/z

2186 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Traquandi et al.
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Plasma Protein Binding. Plasma protein binding was evalu-
ated as previously described.21
In Vivo Pharmacokinetics. The pharmacokinetic profile of the
compounds was investigated in overnight fasted male Nu/Nu
mice following a single dose given intravenously (iv) or orally
(po). The vehicle used was 5% dextrose solution. A total of six
mice were treated (three for each leg). Pharmacokinetic analysis
was also repeated in three A2780 tumor bearing female Balb nu/
nu mice the last day of treatment with 20 mg/kg twice a day orally
for 10 consecutive days. Blood samples of each mouse were
collected from the saphenous vein at predose, 0.083, 0.5, 1, 6,
and 24 h postdosing following iv dosing, and at predose, 0.25, 0.5,
1, 6, and 24 h following oral dosing. Samples were centrifuged at
10000 g for 3 min at 4 ꢀC and the plasma was stored at -80 ꢀC
until analysis. Samples were analyzed by LC/MS/MS technique.
Acknowledgment. We thank Walter Moretti and Piero
Sansonna for their skilled synthetic chemistry support. We
thank Maristella Colombo and Federico Riccardi Sirtori for
skillful assistance in MS spectra recording and interpretation,
and Clara Albanese for her support in flow cytometry analy-
sis. We are also grateful to Nilla Avanzi and the group of
Assay Development and to Marcella Nesi and Luisa Rusconi
for their enlightening advices.
Supporting Information Available: X-ray crystallographic
studies of compound 59; Elemental analysis results of com-
pounds 1, 14, 17, 19-27, 34, 35, 40, 41, 43, 56, and 59. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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