Identification of N,1,4,4-tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl] amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (PHA-848125), a potent, orally available cyclin dependent kinase inhibitor

Article abstract of DOI:10.1021/jm9006559

The discovery of a novel class of inhibitors of cyclin dependent kinases (CDKs) is described. Starting from compound 1, showing good potency as inhibitor of CDKs but being poorly selective against a panel of serine-threonine and tyrosine kinases, new analogues were synthesized. Enhancement in selectivity, antiproliferative activity against A2780 human ovarian carcinoma cells, and optimization of the physical properties and pharmacokinetic profile led to the identification of highly potent and orally available compounds. Compound 28 (PHA-848125), which in the preclinical xenograft A2780 human ovarian carcinoma model showed good efficacy and was well tolerated upon repeated daily treatments, was identified as a drug candidate for further development. Compound 28 is currently undergoing phase I and phase II clinical trials.

Full text of DOI:10.1021/jm9006559

5152 J. Med. Chem. 2009, 52, 5152–5163
DOI: 10.1021/jm9006559
Identification of N,1,4,4-Tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-dihydro-
1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (PHA-848125), a Potent, Orally Available
Cyclin Dependent Kinase Inhibitor#,†
Maria Gabriella Brasca,* Nadia Amboldi, Dario Ballinari, Alexander Cameron, Elena Casale, Giovanni Cervi,
Maristella Colombo, Francesco Colotta, Valter Croci, Roberto D’Alessio, Francesco Fiorentini,‡ Antonella Isacchi,
Ciro Mercurio,§ Walter Moretti, Achille Panzeri, Wilma Pastori, Paolo Pevarello,^ Francesca Quartieri, Fulvia Roletto,
Gabriella Traquandi, Paola Vianello, Anna Vulpetti,¥ and Marina Ciomei
Business Unit Oncology, Nerviano Medical Sciences Srl, Viale Pasteur 10, 20014 Nerviano (MI), Italy. Present address: Diamond Light Source,
Chilton, Didcot, Oxfordshire, U.K. ‡ Accelera Srl, Viale Pasteur 10, 20014 Nerviano (MI), Italy. § Present address: Genextra Group, Milano,
Italy. ^ Present address: Newron, Milano, Italy. ¥ Present address: Novartis Pharma AG, Basel, Switzerland
Received May 18, 2009
The discovery of a novel class of inhibitors of cyclin dependent kinases (CDKs) is described. Starting
from compound 1, showing good potency as inhibitor of CDKs but being poorly selective against a
panel of serine-threonine and tyrosine kinases, new analogues were synthesized. Enhancement in
selectivity, antiproliferative activity against A2780 human ovarian carcinoma cells, and optimization of
the physical properties and pharmacokinetic profile led to the identification of highly potent and orally
available compounds. Compound 28 (PHA-848125), which in the preclinical xenograft A2780 human
ovarian carcinoma model showed good efficacy and was well tolerated upon repeated daily treatments,
was identified as a drug candidate for further development. Compound 28 is currently undergoing phase
I and phase II clinical trials.
Introduction
recent genetic studies in mice indicating that normal cells are
not dependent on interphase CDKs (CDK4 and CDK2) for
their growth, certain tumor cells, depending on their origin
and their pathogenic spectrum of mutation, may be sensitive
to the inhibition of CDKs.1 Several small molecules inhibitors
of CDKs are in clinical development. Among the first gene-
ration of CDK inhibitors, flavopiridol has been granted
orphan drug status for the treatment of chronic lympho-
cytic leukemia.3 A second generation of inhibitors with
greater selectivity for CDKs such as roscovitine/CYC-202,4
BMS-387032/SNS-0325 (both mainly CDK2 but also
CDK7 and CDK9 inhibitors), PD03329916 (selective CDK4
and CDK6 inhibitor), and recently R5477 (CDK1, -2, -4
inhibitor), SCH-7279658 (CDK1, -2, -5, -9 inhibitor),
AT75199 (CDK1, -2, -4, -5 inhibitor), and AZD559710
(CDK1, -2, and -9 inhibitor) suggest that agents that inhibit
the function of multiple CDKs may be clinically more success-
ful than very selective inhibitors of CDKs.
Cyclin dependent kinases (CDKsa) are a family of serine/
threonine kinases that, in concert with cyclins (positive regu-
lators) and natural inhibitors (CDKI), play a crucial role in
the cell cycle progression.1 Deregulation of the activity of
CDKs, due to alterations of expression and/or genetic muta-
tions of cyclins, CDKs, CDKIs, and other components of the
retinoblastoma protein (pRB) pathway, has been reported in
more than 90% of human neoplasms. For example, cyclins E
and A have been found overexpressed in 50% of breast and
lung cancer whereas decreased levels of the inhibitor p27
indicate a poor prognosis in breast, prostate, colon, gastric,
lung, and esophageal cancer.2 The high frequency of altera-
tions found in the core members of this pathway in human
tumors led to the suggestion that its deregulation, leading to
increased activity of CDK/cyclin complexes, is an obligatory
event for the development of all human cancers. Despite
We recently reported on a series of benzodipyrazoles11 as
CDK2 inhibitors (Figure 1). As a further development of the
fused pyrazole ring systems, we identified a novel series of
pyrazolo[4,3-h]quinazoline-3-carboxamides as CDK inhibi-
tors. Preliminary expansion of this class led to compound 1
that, notwithstanding being a potent inhibitor of CDKs, was
poorly selective against a panel of serine-threonine and tyro-
sine kinases (see Experimental Session, Kinase Assays). The
remarkable antiproliferative activity of this compound against
A2780 human ovarian carcinoma cells and the potency on
CDKs prompted us to start a medicinal chemistry program
with the aim of optimizing its profile with respect to selectivity,
physicochemical properties, and pharmacokinetic profile.
# Coordinates of the CDK2 complexes with compounds 2 and 28 have
been deposited in the Protein Data Base under accession codes 2WIP
and 2WIH, together with the corresponding structure factor files.
† This paper is dedicated to the memory of our colleague Valter Croci.
*To whom correspondence should be addressed. Phone: þ39-0331-
581533. Fax: þ39-0331-581347. E-mail: gabriella.brasca@nervianoms.
com.
a Abbreviations: CDK, cyclin dependent kinase; CDK2/A, (cyclin
dependent kinase 2)/(cyclin A); Aur-A, Aurora A; TRKA, thropomyo-
sin receptor kinase A, catalytic receptor for the neurothrophin nerve
growth factor; NGF, nerve growth factor; pRB, retinoblastoma protein;
PPB, plasma protein binding; EDC, N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole;
HOBt NH3, 1-hydroxybenzotriazole ammonium salt; (()-BINAP,
30
(()-2,2 -bis(diphenylphosphino)-1,10-binaphthalene.
r
pubs.acs.org/jmc
Published on Web 07/15/2009
2009 American Chemical Society

Article
Structural information was used to support the develop-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5153
Chemistry
ment of this class. Specifically, the crystal structure of CDK2/
cyclinA complexed with carboxylic acid 2 was solved
(Figure 2). This compound, which shows a similar potency
but ismoresoluble thancompound1, binds in the ATP pocket
oftheactiveconformationof the kinase:the pyrazoloquinazo-
line ring system occupies the adenine region of the ATP
pocket, while the phenyl moiety points toward the solvent
accessible region. Compound 2 makes two hydrogen bonds
with the protein backbone of the hinge region: the N atom at
position 7 of the pyrazoloquinazoline core interacts with the
backboneNHofLeu83, whilethe adjacent amino group binds
to the carbonyl oxygen of Leu83. In addition, the carboxylate
group of the ligand is within hydrogen bonding distance of the
conserved lysine (Lys33).
The synthetic approach to this class of compounds12
(Scheme 1) started from commercially available 1,2-cyclohex-
anedione that, after conversion into enol ether 3, was con-
densed with diethyl oxalate to provide diketoester 4 that
further reacted with methylhydrazine to form regioselectively
the pyrazole 5. Condensation with N,N-dimethylformamide
di-tert-butyl acetal gave rise to enaminone 6a. The prepara-
tion of intermediate 6b was accomplished by using dime-
done as starting material. The formation of enol ether 7 was
followed by a reaction sequence encompassing reduction of
the conjugated double bond, elimination, and epoxidation to
give intermediate 8. Oxirane ring-opening by methanol led to
enol ether 9, regioisomer of 7. Subsequent condensation with
diethyl oxalate and reaction of resulting 1,3-diketone inter-
mediate with methylhydrazine regioselectively gave rise to the
pyrazole derivative 10. Condensation with N,N-dimethylform-
amide di-tert-butyl acetal afforded the enaminone 6b.
The two enaminones 6a and 6b were condensed with
phenylguanidine carbonate in DMF to afford the arylamino
derivatives 11a and 11b (Scheme 2). Hydrolysis of carboxylic
esters gave the corresponding acids, which were submitted to
the coupling with amines to provide amides 1, 16-21, and 26.
Figure 1. From benzodipyrazole series to hit compound 1.
Figure 2. Crystal structure of pyrazolo[4,3-h]quinazoline-3-carboxylic acid derivative 2 in complex with CDK2/cyclin A (PDB code 2WIP).
Scheme 1. Synthesis of Adducts 6a and 6ba
a Reagents and conditions: (a) p-TSA cat., toluene, EtOH, reflux, 48 h, 66%; (b) 1 M LiN(TMS)2 in THF, diethyl oxalate, Et2O, -50 ꢀC to room temp,
76%; (c) methylhydrazine, CH3COOH, room temp, 6 h, 63%; (d) N,N-dimethylformamide di-tert-butyl acetal, DMF, 60 ꢀC, 8 h, 90%; (e) TiCl4, MeOH,
room temp, 1 h, 92%; (f) 1 M LiAlH4 in THF, 0 ꢀC to room temp, 4 h; 2 M H2SO4; H2O2, 2% NaOH, MeOH, 0-4 ꢀC, 20 h, 78%; (g) KOH, MeOH, room
temp, 20 h, 68%; (h) NaH, diethyl oxalate, THF, reflux, 1 h; methylhydrazine, CH3COOH, room temp, 12 h, 48%; (i) N,N-dimethylformamide di-tert-
butyl acetal, DMF, 60 ꢀC, 2 h, 87%.

5154 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Scheme 2. Synthesis of Final Compounds 1 and 16-41a
Brasca et al.
a Reagents and conditions: (a) phenylguanidine carbonate, DMF, 110 ꢀC, 6 h, 72-90%; (b) 1.5 N KOH in 95% EtOH, room temp; HOBt NH3,
3
EDC, DIPEA, DMF, room temp or R1NH2, EDC, HOBt, DIPEA, DMF, room temp, 46-83%; (c) guanidine hydrochloride, K2CO3, DMF, 110 ꢀC,
92-93%; (d) isopentyl nitrite, CsI, I2, CuI, DME, 65 ꢀC, 40-46%; (e) K2CO3, H2O, dioxane, reflux; HOBt NH3, EDC, DIPEA, DMF, room temp
3
or R1NH2, EDC, HOBt, DIPEA, DMF, room temp, 56-65%; (f) substituted aniline, Pd(OAc)2, (()-BINAP, K2CO3, DMF, 80 ꢀC, 30-71%.
Table 1. R1 Modificationa
IC50, μM
compd
R1
CDK2/A
Aur-A
A2780
solubility at pH 7, μM
1
H
CH3
0.002
0.019
0.081
0.074
3.90
0.050
0.312
0.213
0.364
>10
0.50
2.63
3
1
1
7
1
1
1
16
17
18
19
20
21
cyclopropyl
CH2CH2OH
cyclopentyl
phenyl
3.82
2.38
>10
>10
0.58
0.574
1.053
>10
benzyl
a Values are the mean of two or more experiments.
0.374
Alternatively, the two enaminones 6a and 6b were condensed
with guanidine hydrochloride in DMF to afford the fused
pyrimidine derivatives 12a and 12b, which were converted into
the corresponding iodo derivatives 13a and 13b. Hydrolysis
of carboxylic esters gave the corresponding acids, which were
submitted to the coupling with amines to provide amides
14a,15a and 14b,15b. Palladium catalyzed coupling with
substituted anilines furnished the arylamino derivatives
22-25 and 27-41.
bulkier residues such as cyclopentyl, phenyl, and benzyl
(19-21) caused a marked decrease in activity on CDK2/
cyclinA. However, no particular improvement in terms
of selectivity vs the serine-threonine kinase Aurora A
(used as indicator of compound selectivity) and of cellular
antiproliferative activity was achieved moving from the pri-
mary amide 1 to the most interesting secondary amides
16-18.
Exploration of the solvent accessible region of the ATP
binding site of CDK2/cyclin A was realized by introducing
solubilizing moieties on the phenyl ring to give arylamines
22-25 (Table 2).
Early comparison of analogues 22 and 23 with parent
compounds 1 and 16, respectively, indicated that introduction
of the 4-methylpiperazinyl moiety on the phenyl ring para
position afforded potent enzyme inhibitors with an improve-
ment of the antiproliferative activity in A2780 human ovarian
carcinoma cells as well as solubility in neutral buffer. Intro-
duction of the 4-methylpiperazinyl group in the meta position
Results and Discussion
An initial set of amides 1 and 16-21 (Table 1) was
synthesized in order to explore the phosphate region of the
ATP binding site of the CDK2/cyclinA, which accom-
modates the carboxamide groups. The primary carboxamide
1 proved to be the most active compound. Analogously,
secondary amides bearing small substituents such as methyl,
cyclopropyl, and hydroxyethyl (16-18) displayed interes-
ting enzymatic activity as CDK2/cyclin A inhibitors, whereas

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5155
Table 2. R1, R2, R3 Modificationsa
IC50, μM
compd
R1
R2
R3
CDK2/A
Aur-A
A2780
solubility at pH 7, μM
22
23
24
25
H
4-methylpiperazin-1-yl
4-methylpiperazin-1-yl
H
H
H
0.002
0.045
0.001
0.014
0.053
0.175
0.030
0.051
0.03
0.19
0.03
0.12
156
211
47
CH3
H
4-methylpiperazin-1-yl
H
H
morpholin-4-yl
10
a Values are the mean of two or more experiments.
Table 3. R1, R2, R3 Modificationsa
IC50, μM
compd
R1
R2
R3
CDK2/A
Aur-A
A2780
solubility at pH 7, μM
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
H
H
4-methylpiperazin-1-yl
4-methylpiperazin-1-yl
H
0.024
0.017
0.045
0.031
0.137
0.248
0.029
0.047
0.055
0.059
0.036
0.160
0.059
0.220
0.035
0.083
>10
0.582
1.051
1.00
0.17
0.05
0.20
0.53
0.37
0.70
0.17
0.39
0.24
0.57
0.35
0.87
0.50
3.01
0.11
0.5
<1
98
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
4-methylpiperazin-1-yl
191
182
46
H
morpholin-4-yl
H
morpholin-4-yl
0.649
0.625
1.343
1.584
1.615
1.184
0.535
0.638
0.855
1.437
>5
H
1-methylpiperidin-4-yloxy
51
H
1-methylpiperidin-4-yloxy
183
160
170
162
133
121
56
H
4-methylpiperazin-1-ylmethyl
H
4-methylpiperazin-1-ylmethyl
H
morpholin-4-ylmethyl
H
morpholin-4-ylmethyl
H
dimethylamino
H
H
dimethylamino
H
dimethylaminomethyl
41
dimethylaminomethyl
H
206
193
2.940
a Values are the mean of two or more experiments.
(24) or of the morpholin-4-yl group in the para position of the
phenyl ring (25) was compatible with the maintenance of the
CDK2 inhibitory activity and potency in the cellular anti-
proliferative assay, albeit solubility in neutral buffer was
diminished. Unfortunately, improvement in the selectivity vs
Aurora A, as well as vs many of the kinases in our panel, was
not achieved with this series, and the exploration of the small
CDK2 cavity at the back of the pocket (buried region) was
considered. Size and shape of this cavity vary among the
different kinases and are often exploited for improving selec-
tivity. The buried region of CDK2, defined by Ala31, Val64,
Phe80, and Ala144,13 is small mainly because of the presence
of a hindered and rigid gatekeeper residue such as Phe80.
Structure-activity relationship (SAR) data generated by
us11,13,14 and others15 onmultiplechemicalclasseshave shown
that occupation of this buried area by small hydrophobic
moieties is beneficial to CDK2 binding. By visual inspection
and modeling experiments, position 4 of the pyrazoloquinazo-
line skeleton was identified as suitable for placing a moiety
such as a dimethyl group that could increase the hydrophobic
contact in the CDK2 buried region. Compound 26 (Table 3)
showed interesting CDK2 inhibitory activity and potency
in the A2780 cells assay. Moreover, incorporation of the
dimethyl moiety at the 4-position resulted in a significant
improvement of selectivity against Aurora A for compound
26 when compared to compound 1, supporting the working
hypothesis.
With the aim of improving solubility in neutral buffer, an
expansion on the 4,4-dimethylsubstituted pyrazoloquinazo-
line skeleton was performed by keeping the 3-substituent fixed
as primary or N-methylamide and varying the arylamines at
position 8. Introduction of the 4-methylpiperazinyl moiety in
the para position of the phenyl ring (27 and 28) provided
compounds with good activity against CDK2/cyclinA and in
A2780 cell proliferation assay as well as good solubility in
neutral buffer. Selectivity toward Aurora A was reasonably
improved by the presence of the 4,4-dimethyl moiety, in
particular when comparing 28 with 23. As far as buffer
solubility is concerned, compound 28, albeit less potent than
compound 27, displayed higher solubility. The binding mode

5156 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Brasca et al.
Table 4. In Vitro Physicochemical ADME Properties of Selected
Compoundsa
in vitro ADME properties
solubility in 5%
dextrose, mg/mL
CYP4503A4,
% remaining
compd
PPB, %
27
28
32
34
40
3.4
10
82
85
63
50
77
99
98
10
10
100
99
10
95
a Dosed in 5% dextrose as hydrochloride salt.
Table 5. In Vivo Pharmacokinetic Parameters of Selected Compoundsa
in vivo
PK (mouse),
10 mg/kg iv
in vivo
PK (mouse),
10 mg/kg os
t1/2
h
,
CL, Vss,
(mL/h)/kg mL/kg
t1/2
h
,
Cmax
μM
,
AUC,
μM h F, %
Figure 3. Crystal structure of compound 28 (PHA-848125) in
complex with CDK2/cyclin A (PDB code 2WIH).
compd
3
28
32
34
40
2.6
1.9
1.8
1.4
1873
2621
1898
3200
4822
5273
3227
4040
2.9
3.9
4.2
3.4
2.35
0.60
0.77
0.57
8.29
2.51
3.37
1.97
85
38
42
28
of the 4,4-dimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazo-
line skeleton was confirmed by the crystal structure of
compound 28 in complex with CDK2/cyclinA (Figure 3).
Compound 28 is anchored, in a similar manner as
compound 2, to the ATP pocket of CDK2/cyclinA by three
hydrogen bonds to the protein backbone of the hinge
region (Leu83) and to the conserved Lys33. Selectivity en-
hancement of compound 28 with respect to 23 could be
explained by the more favorable hydrophobic interactions
of the 4,4-dimethyl group with the CDK2 gatekeeper residue
Phe80 (compared with the Aurora A gatekeeper Leu210) and
by the presence of the smaller side chain of CDK2-Val64
(Aurora A-Leu194).
a Dosed in 5% dextrose as hydrochloride salt.
Table 6. Selectivity Profile of Compound 28a
IC50, μM
CDK2/ CDK4/ CDK5/ CDK2/ CDK1/ CDK7/
B
A
D1
p35
E
H
TRKA
0.053
0.045
0.160
0.265
0.363
0.398
0.150
a Values are the mean of two or more experiments.
plasma protein binding (98% bound), and good solubility
(10 mg/mL in 5% dextrose as hydrochloride salt).
CDK2/cyclinA inhibitory activity was retained by intro-
duction of the 4-methylpiperazinyl groupinthe metaposition,
as in compound 29, but a lower potency in the cellular
antiproliferative assay was observed with respect to com-
pound 28. Introduction of the morpholin-4-yl group in the
para and meta positions of the phenyl ring led to compounds
30 and 31, respectively, that were less potent on the enzyme
and in the cellular antiproliferative assays and less soluble in
neutral buffer than compound 28. The role of the 4-methyl-
piperazinyl moiety, as far as potency and solubility are
concerned, was explored by replacement with different solu-
bilizing groups attached to the phenyl ring in the para
and meta positions (32-41). All these compounds, with
the exception of 37 and 39, displayed good activity on
CDK2/cyclinA and from moderate to good selectivity vs
Aurora A. In particular, the para substituted compounds
were endowed with a higher potency in the antiproliferative
assay than the corresponding meta derivatives (compare 32,
34, 36, 38, 40 to 33, 35, 37, 39, 41). All compounds of this
series showed good solubility except for the dimethylamino
derivatives 38 and 39.
Compounds 27, 28, 32, 34, 40 were selected for further
assessment on the basis of their potency on the enzyme,
antiproliferative activity, and preliminary solubility in neutral
buffer. Further selection criteria were applied using preli-
minary in vitro physicochemical ADME properties (solubi-
lity in 5% dextrose, stability to human CYP4503A4,
human plasma protein binding) and in vivo pharmacoki-
netic data as key parameters. As reported in Table 4,
compound 28 emerged among these because of its high
stability to human CYP4503A4 (85% remaining), acceptable
In addition, 28 possessed better pharmacokinetic para-
meters than the other compounds tested so far (32, 34, and
40 in Table 5). In vivo results in healthy nudemouse(10 mg/kg
by iv administration) suggested a half-life of 2.6 h, with a
moderate clearance (about 36% of the hepatic blood flow).
The volume of distribution (Vss about 7-fold of the total body
water) pointed toward a high tissue distribution. Compound
28, after oral administration ata dose of 10 mg/kg, showed the
highestoral bioavailability(85%) withanAUC of 8.29μM h,
3
a Cmax of 2.35 μM, and a half-life of 2.9 h.
Compound 28 displayed a combination of cellular potency
and physicochemical properties that made it suitable for
further profiling as an orally active CDK inhibitor. It was
subsequently screened against a panel of 43 serine-threonine
and tyrosine kinases. As shown in Table 6, among the
members of the CDK family that were assessed, 28 turned
out to be a potent inhibitor of CDK2/cyclin A but also, albeit
less potent, an inhibitor of CDK4/cyclinD1 (ratio vs CDK2/
cyclinA of 3.5ꢀ), CDK5/p35 (6ꢀ), CDK2/cyclinE (8ꢀ),
CDK1/cyclin B (8.5ꢀ), and CDK7/cyclin H (3ꢀ). Among
all the other enzymes in the panel, only TRKA was inhibited
in the same nanomolar range by compound 28. It is worth
noting that overexpression of TRKA correlates with an
aggressive phenotype and poor clinical outcome in malignant
solid tumors such as prostate,16 pancreatic,17 and breast
cancer.18
The effects of compound 28 on the cell cycle progression
and DNA synthesis (Table 7) were analyzed using flow
cytometry analysis and BrdU incorporation, respectively, on
A2780 ovarian carcinoma cells in exponential growth in the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5157
Conclusions
presence or absence of compound, for 24 h at 1 μM. At
this concentration, the compound was able to show a clear
reduction of S phase population, which was associated with
an increase of G1 population as expected for a CDK2/
cyclinA inhibitor. The reduction of S phase population was
linked to a strong reduction of the percentage of BrdU incor-
porating cell, meaning that DNA synthesis in these cells was
impaired.
In addition, the effect on the phosphorylation status of a
known CDK substrate such as retinoblastoma protein (pRb)
was analyzed in cells treated with compound28 at 1 and 3 μM.
A clear reduction of the hyperphosphorylated form of pRb
and an accumulation of the hypophosphorylated form of pRb
were observed in the extracts of treated cells in comparison
with the untreated cells, indicative of an effect on the activity
of CDK2 (Figure 4).
On the basis of the data presented above, compound 28 was
prioritized for further in vivo characterization in the human
ovarian A2780 xenograft mouse model. The doses of 20, 30,
and 40 mg/kg were selected and administered orally twice a
day for 10 consecutive days on the basis of the plasma levels
reached in the preliminary in vivo PK study. Figure 5 illus-
trates the resultsfrom thisstudy. Compound28caused a dose-
dependent inhibition of A2780 tumor growth up to 91% at
a dose of 40 mg/kg.
Starting from compound 1, showing good potency as CDK
inhibitor but being poorly selective against a panel of serine-
threonine and tyrosine kinases, a series of new analogues was
synthesized. A remarkable enhancement in selectivity toward
Aurora A was achieved through the incorporation of the
dimethyl group at the 4-position of the skeleton. Several
of the prepared compounds showed antiproliferative activity
against A2780 human ovarian carcinoma cells mediated by
CDK2 inhibition. Optimization of the physical properties and
pharmacokinetic profile led to the identification of the highly
potent, orally available compound 28 (PHA-848125). The
inhibition of TRKA highlighted the value of 28 due to the
involvement of this kinase in the development of solid tumors
such as prostate, pancreatic, and breast cancer. The evalua-
tion of this aspect will be reported elsewhere. Compound 28 is
currently undergoing phase I and phase II clinical trials.
Experimental Section
Chemistry. All solvents and reagents, unless otherwise stated,
were commercially available, were of the best grade, and were
used without further purification. All experiments dealing with
moisture-sensitive compounds were conducted under dry
nitrogen or argon. Organic solutions were evaporated using
a Heidolph WB 2001 rotary evaporator at 15-20 mmHg. Thin-
layer chromatography was performed on Merck silica gel 60
F
254 precoated plates. Column chromatography was conducted
Table 7. Effects on Cell Cycle Progression and DNA Synthesis of
Compound 28 at 1 μMa
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 28 ꢀC on a Varian Inova 400
spectrometer in DMSO-d6 or CDCl3 using the residual solvent
signal as reference. Chemical shifts (δ) are reported in parts per
million (ppm) and coupling constants (J) in Hz. The following
abbreviations are used for multiplicities: s=singlet; bs=broad
signal; d=doublet; t=triplet; m=multiplet; dd=doublet of
doublets. Electrospray (ESI) mass spectra were obtained on a
Finnigan LCQ ion trap. Unless otherwise specified, all final
compounds were homogeneous (purity of not less than 95%), as
G0/G1, %
S, %
G2/M, %
BrdU incorporation, %
þ49
-68
-40
-75
a Versus control cells.
Figure 4. Compound 28 decreases the amount of pRB phos-
phorylation in treated cells in comparison with untreated cells.
Figure 5. Compound 28 shows 53%, 76%, and 91% tumor growth inhibition (TGI) against a human ovarian cancer model (A2780)
transplanted into nude mice when orally administered respectively at doses of 20 (9), 30 (2), and 40 (ꢀ) mg/kg b.i.d. for 10 consecutive days.

5158 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Brasca et al.
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 at 215-400 nm).
Instrument control, data acquisition, and processing were per-
formed by using Xcalibur 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 (4.6mmꢀ50 mm,
3.5 μm). Mobile phase A was 5 mM ammonium acetate buffer
(pH 5.5 with acetic acid)/acetonitrile, 90:10, and mobile phase B
was 5 mM ammonium acetate buffer (pH 5.5 with acetic acid)/
acetonitrile, 10:90. The gradient was from 0 to 100% B in 7 min
and then held at 100% B for 2 min before requilibration. ESI(þ)
high resolution mass spectra (HRMS) were obtained on a
Waters Q-Tof Ultima directly connected to a micro-HPLC
1100 Agilent as previously described.19 Elemental analyses were
performed on a Carlo Erba 1110 instrument, and C, H, and N
results were within (0.4% of theoretical values unless otherwise
specified. Melting points are uncorrected.
δ 7.48 (s, 1H), 4.26 (q, J=6.9 Hz, 2H), 4.12 (s, 3H), 3.12 (s, 6H),
2.88-2.92 (m, 2H), 2.79-2.83 (m, 2H), 1.29 (t, J=6.9 Hz, 3H);
LCMS (ESI) m/z 278 (M þ H)þ.
3-Methoxy-5,5-dimethyl-cyclohex-2-en-1-one (7). A solution
of 5,5-dimethyl-1,3-cyclohexanedione (80.0 g, 0.57 mol) in
anhydrous methanol (600 mL) was treated with 1 M solution
of titanium chloride (TiCl4) in dichloromethane (17.2 mL).
After being stirred for 1 h at room temperature, the mixture
was slowly poured into a cold 5% NaHCO3 solution and
extracted with diethyl ether (450 mL ꢀ 6). The organic layers
were collected, washed with brine, dried over Na2SO4, and
evaporated to dryness, affording 7 (80.7 g, 92%) as a pale-
1
yellow oil. H NMR (400 MHz, CDCl3) δ 5.35 (s, 1H), 3.68
(s, 3H), 2.26 (s, 2H), 2.19 (s, 2H), 1.05 (s, 6H).
4,4-Dimethyl-7-oxa-bicyclo[4.1.0]heptan-2-one (8). A solution
of 7 (80.0 g, 0.519 mol) in anhydrous THF (270 mL) was treated
dropwise with a 1 M solution of LiAlH4 in THF (182 mL) under
argon atmosphere and keeping the temperature of the reaction
between 0 and 5 ꢀC. The temperature was allowed to rise to
25 ꢀC, and the mixture was stirred for 4 h. The resulting slurry
was cooled in an ice bath, quenched with ethyl acetate (30 mL),
and poured with caution into a cooled 2 M H2SO4 solution.
The aqueous solution was then extracted with diethyl ether
(300 mLꢀ3), dried over Na2SO4, and evaporated under reduced
pressure to remove most of the solvent. The crude material was
dissolved in methanol (500 mL), cooled to 0 ꢀC, and treated with
30% hydrogen peroxide (265 mL, 2.6 mol). The resulting
solution was treated dropwise with a 2% NaOH solution
(142 mL, 0.067 mol), keeping the reaction temperature around
0 ꢀC. The mixture was allowed to stay at 4 ꢀC for 20 h and was
then diluted with water (900 mL) and extracted with ethyl
ether (450 mL ꢀ 4). The extracts were collected, washed with
5% sodium metabisulfite solution and with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue
was purified by distillation under vacuum to obtain 8 (56.7 g,
78%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ 3.59
(t, J=4.6 Hz, 1H), 3.20 (d, J=3.8 Hz, 1H), 2.54 (d, J=13.1 Hz,
1H), 1.91 (part of AB system, J=14.6 Hz, 1H), 1.87 (part of AB
system, J=14.6, 4.3, 2.1, 1H), 1.74 (d, J=13.1, 2.1, 1.1 Hz, 1H),
0.97 (s, 3H), 0.85 (s, 3H).
2-Ethoxycyclohex-2-en-1-one (3). 1,2-Cyclohexandione (50.0 g,
0.445 mol) was dissolved in a mixture of toluene (1.0 L) and
ethanol (0.5 L). p-Toluenesulfonic acid (10.0 g, 52 mmol) was
added and the solution heated under reflux for 2 days. The
solvent was then evaporated and the residue dissolved with
dichloromethane and washed with a saturated solution of
NaHCO3. The organic layer was dried over Na2SO4 and con-
centrated. The crude was purified by chromatography on a silica
gel column (cyclohexane/ethyl acetate, 98/2) to give 3 as an oil
(41.41 g, 66%). 1H NMR (400 MHz, DMSO-d6) δ 5.99 (t, J=4.5
Hz, 1H), 3.67 (q, J=6.8 Hz, 2H), 2.33-2.39 (m, 4H), 1.82-1.88
(m, 2H) 1.20 (t, J=6.8 Hz, 3H); LCMS (ESI) m/z 141 (M þ H)þ.
Ethyl (3-Ethoxy-2-oxocyclohex-3-en-1-yl)(oxo)acetate (4).
LiN(TMS)2 (1 M in THF, 325 mL) was added dropwise
at -50 ꢀC to a solution of 3 (41.40 g, 0.29 mol) in diethyl ether
(310 mL). After 30 min at the same temperature, diethyl oxalate
(44.2 mL, 0.325 mol) was added. The solution was kept at room
temperature overnight. Water (300 mL) was added, the pH
adjusted to 4-5 by adding 1 N HCl, and the resulting solution
extracted with ethyl acetate. The organic layer was dried over
Na2SO4 and evaporated to dryness. The crude was purified by
chromatography on a silica gel column (dichloromethane) to
yield 4 as an oil (52.9 g, 76%). 1H NMR (400 MHz, DMSO-d6)
δ 5.93 (m, 1H), 4.23 (q, J=7.1 Hz, 2H), 3.70 (q, J=6.9 Hz, 2H),
3.27 (m, 1H), 2.06-2.58 (m, 4H), 1.25 (t, J=7.1 Hz, 3H), 1.20
(t, J=6.9 Hz, 3H); LCMS (ESI) m/z 241 (M þ H)þ; HRMS
(ESI) calcd for C12H16O5 þ Hþ 241.1070, found 241.1072.
Ethyl 1-Methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-car-
boxylate (5). Compound 4 (30.0 g, 0.125 mol) was dissolved
in glacial acetic acid (150 mL), and methylhydrazine (6.6 mL,
0.125 mol) was added. The mixture was stirred at room tem-
perature for 6 h. The solvent was evaporated, the crude dis-
solved with water, the solution made basic with 30% NH4OH
and extracted with dichloromethane. The organic layer was
dried over Na2SO4 and concentrated. The residue was purified
by chromatography on a silica gel column (dichloromethane)
and crystallized from a mixture n-hexane/diethyl ether to give 5
as a white solid (17.5 g, 63%). 1H NMR (400 MHz, DMSO-d6)
δ 4.33 (q, J=6.9 Hz, 2H), 4.15 (s, 3H), 2.90 (t, J=6.2 Hz, 2H),
2.51-2.53 (m, 2H), 2.03-2.07 (m, 2H), 1.33 (t, J=6.9 Hz, 3H);
LCMS (ESI) m/z 223 (M þ H)þ; HRMS (ESI) calcd for
C11H14N2O3 þ Hþ 223.1077, found 223.1077.
2-Methoxy-5,5-dimethyl-cyclohex-2-en-1-one (9). A solution
of 8 (44.0 g, 0.31 mol) in methanol (150 mL) was added to a
solution of 85% potassium hydroxide (20.7 g, 0.31 mol) in
methanol (450 mL) at room temperature. The mixture was kept
at this temperature for 20 h. After cooling, the solution was
diluted with water (1.2 L) and extracted with diethyl ether
(350 mLꢀ5). The organic extracts were collected, washed with
brine, dried over Na2SO4, and evaporated under vacuum to
remove most of the solvent. The crude material was purified
1
by distillation to obtain 9 (32.8 g, 68%) as an oil. H NMR
(400 MHz, DMSO-d6) δ 5.83 (t, J=4.4 Hz, 1H), 3.49 (s, 3H),
2.29 (s, 2H), 2.28 (d, J=4.4 Hz, 2H), 0.98 (s, 6H).
Ethyl 1,4,4-Trimethyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-
3-carboxylate (10). A sample of 60% sodium hydride in
mineral oil (2.41 g, 60.3 mmol) was suspended in anhydrous
THF (60 mL) under argon atmosphere and treated with a
solution of 9 (6.2 g, 40.2 mmol) in anhydrous THF (50 mL).
After 15 min, a solution of diethyl oxalate (8.17 mL, 60.3 mmol)
in anhydrous THF (50 mL) was added and the mixture was
refluxed for 1 h. The slurry was diluted with water (800 mL),
acidified with 1 N HCl (50 mL), and extracted with ethyl acetate
(500 mLꢀ2). The organic layers were collected, washed with
brine, dried over Na2SO4, and evaporated to dryness to obtain
an orange oil, which was dissolved in acetic acid (65 mL) and
treated dropwise with a solution of methylhydrazine (2.14 mL,
40.2 mmol) in acetic acid (20 mL). The solution was stirred at
room temperature overnight. The mixture was then diluted with
water (800 mL) and extracted with ethyl acetate (500 mLꢀ2).
The organic extracts were washed with brine, dried over
Na2SO4, and evaporated to dryness. The crude material was
Ethyl (6E)-6-[(Dimethylamino)methylidene]-1-methyl-7-oxo-
4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (6a). Compound
5 (16.0 g, 0.072 mol) was dissolved in DMF (100 mL), and N,N-
dimethylformamide di-tert-butyl acetal (32 mL, 0.13 mol) was
added. The mixture was stirred at 60 ꢀC for 8 h. The solvent was
evaporated and the solid crystallized from ethanol to give 6a as
1
a white solid (17.9 g, 90%). H NMR (400 MHz), DMSO-d6)

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5159
chromatographed on silica gel (dichloromethane/ethyl acetate,
100/5) to obtain 10 (4.82 g, 48%). 1H NMR (400 MHz, CDCl3)
δ 4.43 (q, J=7.0 Hz, 2H), 4.19 (s, 3H), 2.61 (t, J=6.4 Hz, 2H),
1.98 (t, J=6.4 Hz, 2H), 1.49 (s, 6H), 1.42 (t, J=7.0 Hz, 3H).
Ethyl (6E)-6-[(Dimethylamino)methylidene]-1,4,4-trimethyl-
7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (6b). A so-
lution of 10 (4.8 g, 19.2 mmol) in anhydrous DMF (30 mL) was
treated with N,N-dimethylformamide di-tert-butyl acetal (9.19
mL, 38.35 mmol) at 60 ꢀC for 2 h. The mixture was evaporated to
dryness, and the crude material was crystallized from n-hexane
to give 6b (5.1 g, 87%). 1H NMR (400 MHz, DMSO-d6) δ 7.49
(s, 1H), 4.27 (q, J=7.1 Hz, 1H), 4.09 (s, 3H), 3.11 (s, 6H), 2.76
(s, 2H), 1.32 (s, 6H), 1.29 (t, J=7.1 Hz, 3H).
temperature overnight. The reaction mixture was poured into
water (25 mL) and extracted with dichloromethane (4 ꢀ
25 mL). The combined organic extracts were washed with water
and dried over Na2SO4, and the solvent was removed under
reduced pressure. The crude was purified by flash chromatogra-
phy on silica gel (dichloromethane/methanol, 98/2) to afford 16
(141 mg, 92%): mp >250 ꢀC; 1H NMR (400 MHz, DMSO-d6) δ
9.50 (s, 1H), 8.40 (s, 1H), 8.07 (q, J=4.9 Hz, 1H), 7.69-7.71 (m,
2H), 7.30 (m, 2H), 6.96 (m, 1H), 4.33 (s, 3H), 2.99 (t, J=7.9 Hz,
2H), 2.81 (t, J=7.9 Hz, 2H), 2.73 (d, J=4.9 Hz, 3H); LCMS (ESI)
m/z 335 (M þ H)þ; HRMS (ESI) calcd for C18H18N6O þ Hþ
335.1615, found 335.1627.
Compounds 17-21. By employment of the above-described
procedure, starting from 2 and using suitable amines, com-
pounds 17-21 were prepared.
Ethyl 1-Methyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxylate (11a). Phenylguanidine carbo-
nate (2.13 g, 13.31 mmol) was added to a suspension of
compound 6a (3.0 g, 10.81 mmol) in DMF (30 mL), and the
mixture was stirred at 110 ꢀC for 6 h. After the mixture was
cooled, the solvent was evaporated and the residue was dis-
solved in dichloromethane and washed with water. The organic
layer was then dried over Na2SO4 and concentrated. The residue
was triturated with diethyl ether and filtered to give 11a as a
N-Cyclopropyl-1-methyl-8-(phenylamino)-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (17). Yield, 55%; mp 186
ꢀC; 1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.40 (s, 1H),
8.13 (d, J=4.5 Hz, 1H), 7.68-7.70 (m, 2H), 7.27-7.31 (m, 2H),
6.93-6.98 (m, 1H), 4.32 (s, 3H), 2.98 (t, J=7.8 Hz, 2H), 2.81 (t,
J=7.8 Hz, 2H), 2.78-2.84 (m, 1H), 0.57-0.71 (m, 4H); LCMS
(ESI) m/z 361 (M þ H)þ; HRMS (ESI) calcd for C20H20N6O þ
Hþ 361.1772, found 361.1779. Anal. (C20H20N6O) C, H, N.
N-(2-Hydroxyethyl)-1-methyl-8-(phenylamino)-4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline-3-carboxamide (18). Yield, 68%; mp
>250 ꢀC; 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.40 (s,
1H), 7.93 (t, J=5.7 Hz, 1H), 7.69-7.71 (m, 2H), 7.28-7.32 (m,
2H), 6.94-6.98 (m, 1H), 4.73 (t, J=5.4 Hz, 1H), 4.34 (s, 3H),
3.49 (m, 2H), 3.30 (m obscured by water signal, 2H), 2.99 (t, J=
7.9 Hz, 2H), 2.81 (t, J=7.9 Hz, 2H); LCMS (ESI) m/z 365 (M þ
H)þ; HRMS (ESI) calcd for C19H20N6O2 þ Hþ 365.1721, found
365.1727; HPLC purity 90%.
1
white solid (2.72 g, 72%). H NMR (400 MHz, DMSO-d6) δ
9.52 (s, 1H), 8.42 (s, 1H), 7.69-7.71 (m, 2H), 7.28-7.32 (m, 2H),
6.94-6.98 (m, 1H), 4.36 (s, 3H), 4.30 (q, J=7.2 Hz, 2H), 2.98 (t,
J=7.7 Hz, 2H), 2.84 (t, J=7.7 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H);
LCMS (ESI) m/z 350 (M þ H)þ; HRMS (ESI) calcd for
C19H19N5O2 þ Hþ 350.1611, found 350.1614.
Ethyl 1,4,4-Trimethyl-8-(phenylamino)-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxylate (11b). By employment of
the above-described procedure, starting from 6b, compound 11b
1
was prepared. Yield, 90%; H NMR (400 MHz, DMSO-d6) δ
N-Cyclopentyl-1-methyl-8-(phenylamino)-4,5-dihydro-1H-pyra-
zolo[4,3-h]quinazoline-3-carboxamide (19). Yield, 72%; mp
>250 ꢀC; 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.40
(s, 1H), 7.88 (d, J=7.8 Hz, 1H), 7.69-7.71 (m, 2H), 7.28-7.32
(m, 2H), 6.94-6.98 (m, 1H), 4.33 (s, 3H), 4.17-4.25 (m, 1H),
2.98 (t, J=7.8 Hz, 2H), 2.81 (t, J=7.8 Hz, 2H), 1.50-1.89 (m,
8H); LCMS (ESI) m/z 389 (M þ H)þ.
9.53 (s, 1H), 8.41 (s, 1H), 7.68-7.70 (m, 2H), 7.28-7.32 (m, 2H),
6.94-6.98 (m, 1H), 4.34 (s, 3 H), 4.29 (q, J=7.1 Hz, 2H), 2.73 (s,
2H), 1.32 (s, 6H), 1.31 (t, J=7.15 Hz, 3H); LCMS (ESI) m/z 378
(M þ H)þ; HRMS (ESI) calcd for C21H23N5O2 þ Hþ 378.1925,
found 378.1919.
1-Methyl-8-phenylamino-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxylic Acid (2). To a suspension of 11a (0.50 g,
1.43 mmol) in anhydrous ethanol (25 mL), 1.5 M KOH in 95%
ethanol (3.7 mL, 5.55 mmol) was added under good stirring, and
the mixture was left at room temperature overnight. After the
mixture was cooled in an ice bath, 1 N HCl was added until
neutral pH was attained. Water was added and the resulting
precipitate collected by filtration to give 2 (390 mg, 85%) as a
white solid: mp >250 ꢀC; 1H NMR (400 MHz, DMSO-d6)
δ 12.68 (bs, 1H), 9.51 (s, 1H), 8.41 (s, 1H), 7.79-7.71 (m, 2H),
7.28-7.32 (m, 2H), 6.94-6.98 (m, 1H), 4.34 (s, 3H), 2.96 (t, J=
7.6 Hz, 2H), 2.83 (t, J=7.6 Hz, 2H); LCMS (ESI) m/z 322 (M þ
H)þ. Anal. (C17H15N5O2) C, H, N.
1-Methyl-N-phenyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxamide (20). Yield, 60%; mp >250 ꢀC;
1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 9.53 (s, 1H), 8.43
(s, 1H), 7.80-7.83 (m, 2H), 7.70-7.73 (m, 2H), 7.30-7.35 (m,
4H), 7.06-7.10 (m, 1H), 6.95-6.99 (m, 1H), 4.41 (s, 3H), 3.05 (t,
J=7.9 Hz, 2H), 2.86 (t, J=7.9 Hz, 2H); LCMS (ESI) m/z 397
(M þ H)þ.
N-Benzyl-1-methyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo-
[4,3-h]quinazoline-3-carboxamide (21). Yield, 68%; mp 202 ꢀC;
1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.69 (t, J=6.3
Hz, 1H), 8.40 (s, 1H), 7.69-7.71 (m, 2H), 7.22-7.32 (m, 5H),
7.28-7.32 (m, 2H), 6.94-6.98 (m, 1H), 4.42 (d, J=6.3 Hz, 2H),
4.35 (s, 3H), 3.00 (t, J=7.9 Hz, 2H), 2.82 (t, J=7.9 Hz, 2H);
LCMS (ESI) m/z 411 (M þ H)þ.
1-Methyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxamide (1). A solution of 2 (150 mg, 0.46 mmol) in
anhydrous DMF (5 mL) was treated with N,N-diisopropylethy-
lamine (0.12 mL, 0.69 mmol) and EDC (131 mg, 0.69 mmol).
The mixture was treated with 1-hydroxybenzotriazole ammo-
1,4,4-Trimethyl-8-phenylamino-4,5-dihydro-1H-pyrazolo[4,3-
h]quinazoline-3-carboxamide (26). To a suspension of 11b (0.50
g, 1.32 mmol) in anhydrous ethanol (25 mL), 1.5 M KOH in
95% ethanol (2.64 mL, 3.96 mmol) was added under good
stirring, and the mixture was left at room temperature over-
night. After the mixture was cooled in an ice bath, 1 N HCl was
added until neutral pH was attained. Water was added, and the
resulting precipitate was collected by filtration (420 mg, 91%).
1H NMR (400 MHz, DMSO-d6) δ 12.82 (s, 1H), 9.51 (s, 1H),
8.40 (s, 1H), 7.68-7.71 (m, 2H), 7.28-7.32 (m, 2H), 6.94-6.98
(m, 1H), 4.34 (s, 3H), 2.71 (s, 2H), 1.33 (s, 6H). A solution of
the above carboxylic acid (349 mg, 1.0 mmol) in anhydrous
DMF (10 mL) was treated with N,N-diisopropylethylamine
(0.349 mL, 2.0 mmol) and EDC (269 mg, 1.4 mmol). The
nium salt20 (HOBt NH3) (105 mg, 0.69 mmol). The mixture was
3
kept at room temperature overnight and then was diluted with
water, and the resulting precipitate was collected by filtration to
1
afford 1 (132 mg, 90%): mp >250 ꢀC; H NMR (400 MHz,
DMSO-d6) δ 9.48 (s, 1H), 8.40 (s, 1H), 7.68-7.71 (m, 2H), 7.45
(bs, 1H), 7.29 (m, 2H), 7.24 (bs, 1H), 6.93-6.97 (m, 1H), 4.32 (s,
3H), 2.98 (t, J=7.8 Hz, 2H), 2.81 (t, J=7.8 Hz, 2H); LCMS (ESI)
m/z 321 (M þ H)þ; HRMS (ESI) calcd for C17H16N6O þ Hþ
321.1458, found 321.1458. Anal. (C17H16N6O) C, H, N.
N,1-Dimethyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxamide (16). To a solution of 2 (150 mg, 0.46
mmol) in anhydrous DMF (5 mL) were added N,N-diisopropy-
lethylamine (0.12 mL, 0.69 mmol), HOBT (93 mg, 0.69 mmol),
EDC (131 mg, 0.69 mmol), 2 M methylamine in THF (0.345 mL,
0.69 mmol), and the reaction mixture was stirred at room
mixture was treated with HOBt NH3 (213 mg, 1.4 mmol).
3
The mixture was kept at room temperature for 5 h. The mix-
ture was diluted with water and the resulting precipitate was

5160 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Brasca et al.
collected by filtration to afford 26 (320 mg, 92%) as a yellow
solid: mp 158 ꢀC; 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H),
8.39 (s, 1H), 7.68-7.71 (m, 2H), 7.55 (bs, 1H), 7.28-7.31
(m, 2H), 7.29 (bs, 1H), 6.94-6.98 (m, 1H), 4.31 (s, 3H), 2.69
(s, 2H), 1.33 (s, 6H); LCMS (ESI) m/z 349 (M þ H)þ. Anal.
(C19H20N6O) C, H, N.
Ethyl 8-Amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxylate (12a). Potassium carbonate (7.87 g, 57 mmol)
and guanidine hydrochloride (5.44 g, 57 mmol) were added to a
solution of compound 6a (16.0 g, 57 mmol) in DMF (60 mL), and
the mixture was stirredat 110ꢀCfor 6 h. After cooling, the mixture
was poured into water (300 mL) and the resulting precipitate was
collected by filtration and dried to give 12a (14.33 g, 92% yield)
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H),
6.55 (bs, 2H), 4.32 (s, 3H), 4.30 (q, J=7.1 Hz, 2H), 2.91 (t, J=7.7
Hz, 2H), 2.74 (t, J=7.7 Hz, 2H), 1.30 (t, J=7.1 Hz, 3H); LCMS
(ESI) m/z 274 (M þ H)þ; HRMS (ESI) calcd for C13H15N5O2 þ
Hþ 274.1298, found 274.1299.
Ethyl 8-Amino-1,4,4-trimethyl-4,5-dihydro-1H-pyrazolo[4,3-
h]quinazoline-3-carboxylate (12b). By employment of the
above-described procedure, starting from 6b, the compound
12b was prepared. Yield, 93%; 1H NMR (400 MHz, DMSO-d6)
δ 8.16 (s, 1H), 6.56 (bs, 2H), 4.31 (s, 3H), 4.29 (q, J=7.1 Hz, 2H),
2.62 (s, 2H), 1.30 (t, J=7.1 Hz, 3H), 1.29 (s, 6 H); LCMS (ESI)
m/z 302 (M þ H)þ; HRMS (ESI) calcd for C15H19N5O2 þ Hþ
302.1611, found 302.1615.
Ethyl 8-Iodo-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quina-
zoline-3-carboxylate (13a). To a well stirred suspension of 12a
(8.8 g, 32 mmol) in dimethoxyethane (1.2 L) maintained in an
inert atmosphere of argon, cesium iodide (9.13 g, 35 mmol),
iodine (4.45 g, 18 mmol), copper(I) iodide (2.01 g, 10 mmol), and
isopentyl nitrite (7.00 mL, 52 mmol) were added in sequence.
The reaction mixture was stirred vigorously at 65 ꢀC for 18 h.
After the mixture was cooled in an ice-water bath, the solid was
filtered off and the filtrate was diluted with dichloromethane
(2.0 L), washed with 30% ammonium hydroxide (150 mL),
sodium thiosulphate (300 mL), brine, and dried over anhy-
drous Na2SO4. Concentrating to a volume of about 100 mL of
dimethoxyethane, the crude 13a precipitated. It was then filtered
and washed with dimethoxyethane. Flash chromatography on
silica gel (eluant, dichloromethane/methanol, 98/2) yielded the
title compound (5.65 g, 46%). 1H NMR (400 MHz), DMSO-d6),
δ 8.48 (s, 1H), 4.29 (q, J=7.1 Hz, 2H), 4.26 (s, 3H), 3.01 (m, 2H),
2.93 (m, 2H), 1.31 (t, J= 7.1 Hz, 3H); LCMS (ESI) m/z 385
(M þ H)þ; HRMS (ESI) calcd for C13H13IN4O2 þ Hþ 385.0156,
found 385.0154.
8-Iodo-N,1-dimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazo-
line-3-carboxamide (15a). By employment of the above-de-
scribed procedure, starting from 13a and using methylamine,
compound 15a was prepared. Yield, 75%; 1H NMR (400 MHz,
DMSO-d6) δ 8.46 (s, 1H), 8.12 (q, J=4.8 Hz, 1H), 4.23 (s, 3H),
3.02 (dd, J=7.6, 6.6 Hz, 2H), 2.89 (dd J=7.6, 6.6 Hz, 2H), 2.73
(d, J=4.8 Hz, 3H); LCMS (ESI) m/z 370 (M þ H)þ; HRMS
(ESI) calcd for C21H12IN5O þ Hþ 370.0160, found 370.0170.
8-Iodo-1,4,4-trimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazo-
line-3-carboxamide (14b). Potassium carbonate (1.38 g, 10
mmol) in water (10 mL) was added to a solution of compound
13b (1.03 g, 2.5 mmol) in dioxane (20 mL), and the mixture was
stirred under reflux for 30 h. After cooling, the mixture was
poured into water (100 mL) and extracted with ethyl acetate
(2ꢀ100 mL). The aqueous phase was acidified with 2 N HCl
(12 mL), and the resulting precipitate was collected by filtration,
washed with water, dried, and used without further purification.
To a solution of the above carboxylic acid in anhydrous DMF
(10 mL) were added N,N-diisopropylethylamine (0.51 mL,
3.0 mmol), EDC (572 mg, 3.0 mmol), and HOBt NH3 (456
3
mg, 3.0 mmol). The mixture was kept at room temperature
overnight and then diluted with water. The resulting precipitate
was collected by filtration and washed with water to afford 14b
(536 mg, 56%). 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H),
7.51 (bs, 1H), 7.30 (bs, 1H), 4.25 (s, 3H), 2.78 (s, 2H), 1.33
(s, 6H); LCMS (ESI) m/z 384 (M þ H)þ.
8-Iodo-N,1,4,4-tetramethyl-4,5-dihydro-1H-pyrazolo[4,3-h]qui-
nazoline-3-carboxamide (15b). By employment of the above-
described procedure, starting from 13b and using methylamine,
compound 15b was prepared. Yield, 65%; 1H NMR (400 MHz,
DMSO-d6) δ 8.46 (s, 1H), 8.20 (q, J=4.6 Hz, 1H), 4.22 (s, 3H),
2.78 (s, 2H), 2.75 (d, J=4.6 Hz, 3H), 1.32 (s, 6H); LCMS (ESI)
m/z 398 (M þ H)þ; HRMS (ESI) calcd for C14H16IN5O þ Hþ
398.0473, found 398.0476.
1-Methyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-di-
hydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (22). Pd-
(OAc)2 (20 mg, 0.09 mmol), (()-BINAP (55 mg, 0.09 mmol),
and DMF (5 mL) were charged in a round-bottom flask flushed
with argon. The flask was evacuated and backfilled with argon.
The mixture was stirred under argon for 30 min and added to a
mixture of 14a (319 mg, 0.9 mmol), 4-(4-methylpiperazin-1-
yl)phenylamine (515 mg, 2.7 mmol), and potassium carbonate
(1.24 g, 9.0 mmol) in DMF (10 mL). The resulting mixture was
stirred at 80 ꢀC for 3 h under argon. After cooling to room
temperature, the reaction mixture was filtered on a pad of Celite.
The solvent was concentrated, and the crude solid was purified
by flash chromatography on silica gel (eluant, dichloromethane/
Ethyl 8-Iodo-1,4,4-trimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]-
quinazoline-3-carboxylate (13b). By employment of the above-
described procedure, starting from 12b, compound 13b was
prepared. Yield, 40%; 1H NMR (400 MHz, DMSO-d6) δ 8.48
(s, 1H), 4.30 (q, J=7.2 Hz, 2H), 4.26 (s, 3H), 2.81 (s, 2H), 1.32 (s,
6H), 1.31 (t, J=7.2 Hz, 3H); LCMS (ESI) m/z 413 (M þ H)þ;
HRMS (ESI) calcd for C15H17IN4O2 þ Hþ 413.0469, found
413.0472.
1
methanol, 95/5) to afford 22 (230 mg, 61%): mp 211 ꢀC; H
NMR (400 MHz, DMSO-d6) δ 9.25 (bs, 1H), 8.33 (s, 1H), 7.52
(m, 2H), 7.46 (bs, 1H), 7.25 (bs, 1H), 6.90 (m, 2H), 4.31 (s, 3H),
3.05-3.07 (m, 4H), 2.97 (t, J=7.9 Hz, 2H), 2.78 (t, J=7.9 Hz,
2H), 2.46-2.48 (m, 4H), 2.23 (s, 3H); LCMS (ESI) m/z 419 (M þ
H)þ; HRMS (ESI) calcd for C22H26N8O þ Hþ 419.2303, found
419.2297.
8-Iodo-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-
3-carboxamide (14a). To a suspension of 13a (384 mg, 1.0 mmol)
in anhydrous ethanol (10 mL), 1.5 M KOH in 95% ethanol
(2.0 mL, 3.0 mmol) was added under good stirring, and the mix-
ture was left at room temperature overnight. After the mixture
was cooled in an ice bath, the resulting precipitate was collected
by filtration and used without further purification. A suspension
of the above carboxylic acid potassium salt in anhydrous DMF
N,1-dimethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-
4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (23).
By employment of the above-described procedure, starting from
1
15a, compound 23 was prepared. Yield, 65%; H NMR (400
MHz, DMSO-d6) δ 9.27 (s, 1H), 8.34 (s, 1H), 8.08 (q, J=4.7 Hz,
1H), 7.55 (m, 9.2 Hz, 2H), 6.93 (d, J=9.2 Hz, 2H), 4.33 (s, 3H),
3.04-3.08 (m, 4H), 2.97 (t, J=7.9 Hz, 2H), 2.78 (t, J=7.9 Hz,
2H), 2.75 (d, J=4.7 Hz, 3H), 2.45-2.50 (m, 4H), 2.24 (s, 3H);
LCMS (ESI) m/z 433 (M þ H)þ; HRMS (ESI) calcd for
C23H28N8O þ Hþ 433.2459, found 433.2459.
(10 mL) was treated with EDC (269 mg, 1.4 mmol). HOBt NH3
3
(213 mg, 1.4 mmol) was added, and the mixture was kept at
room temperature overnight and then diluted with water. The
resulting precipitate was collected by filtration to afford 14a
(220 mg, 62%). 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H),
7.51 (bs, 1H), 7.29 (bs, 1H), 4.23 (s, 3H), 3.01 (t, J=8.0 Hz, 2H),
2.89 (t, J=8.0 Hz, 2H); LCMS (ESI) m/z 356 (M þ H)þ; HRMS
(ESI) calcd for C11H10IN5O þ Hþ 356.0003, found 356.0005.
Compounds 24 and 25. By employment of the above-described
procedure, starting from 14a and using the suitable substituted
aniline, compounds 24 and 25 were prepared.
1-Methyl-8-{[3-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (24).
Yield, 52%; 1H NMR (400 MHz, DMSO-d6) δ 9.34

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5161
(bs, 1H), 8.39 (s, 1H), 7.43 (bs, 1H), 7.26-7.28 (m, 2H), 7.26
(bs, 1H), 7.15 (t, J=8.0 Hz, 1H), 6.60 (d, J=8.4 Hz, 1H), 4.33
(s, 3H), 3.0-3.5 (bs obscured by water signal, 4H), 2.98 (t, J=
7.8 Hz, 2H), 2.85-2.95 (bs, 4H), 2.80 (t, J=7.8 Hz, 2H), 2.52-
2.59 (bs, 3H); LCMS (ESI) m/z 419 (M þ H)þ; HRMS (ESI)
calcd for C22H26N8O þ Hþ 419.2303, found 419.2306.
1-Methyl-8-[(4-morpholin-4-ylphenyl)amino]-4,5-dihydro-1H-
(bs, 5H), 1.93-2.02 (bs, 2H), 1.66-1.81 (bs, 2H), 1.32 (s, 6H);
LCMS (ESI) m/z 476 (M þ H)þ; HRMS (ESI) calcd
for C26H33N7O2 þ Hþ 476.2768, found 476.2767; HPLC
purity 95%.
N,1,4,4-Tetramethyl-8-({3-[(1-methylpiperidin-4-yl)oxy]phenyl}-
amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (33). Yield, 30%; 1H NMR (400 MHz, DMSO-d6) δ 9.50
(s, 1H), 8.40 (s, 1H), 8.14 (q, J=4.80 Hz, 1H), 7.40 (t, J=2.1 Hz,
1H), 7.26 (ddd, J=8.2, 2.1, 0.7 Hz, 1 H), 7.17 (t, J=8.2 Hz, 1H),
6.55 (ddd, J=8.2, 2.1, 0.7 Hz, 1 H), 4.33 (s, 3H), 4.28-4.34 (m,
1H), 2.74 (d, J=4.8 Hz, 3H), 2.69 (s, 2H), 2.58-2.66 (m, 2H),
2.18-2.25 (bs, 2H), 2.20 (s, 3H), 1.91-1.99 (m, 2H), 1.60-
1.70 (m, 2H), 1.33 (s, 6H); LCMS (ESI) m/z 476 (M þ H)þ;
HRMS (ESI) calcd for C26H33N7O2 þ Hþ 476.2768, found
476.2776.
1
pyrazolo[4,3-h]quinazoline-3-carboxamide (25). Yield, 60%; H
NMR (400 MHz, DMSO-d6) δ 9.25 (bs, 1H), 8.34 (s, 1H), 7.52-
7.56 (m, 2H), 7.44 (bs, 1H), 7.23 (bs, 1H), 6.89-6.93 (m, 2H),
4.31 (s, 3H), 3.73-3.77 (m, 4H), 3.03-3.05 (m, 4H), 2.97 (t, J=
7.8 Hz, 2H), 2.78 (t, J=7.8 Hz, 2H); LCMS (ESI) m/z 406 (M þ
H)þ; HRMS (ESI) calcd for C21H23N7O2 þ Hþ 406.1986, found
406.1991; HPLC purity 90%.
1,4,4-Trimethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-
4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (27).
By employment of the above-described procedure, starting from
14b and using the suitable substituted aniline, tcompound 27
N,1,4,4-Tetramethyl-8-({4-[(4-methylpiperazin-1-yl)methyl]-
phenyl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-
carboxamide (34). Yield, 55%; 1H NMR (400 MHz, DMSO-d6)
δ 9.48 (s, 1H), 8.38 (s, 1H), 8.15 (q, J = 4.8 Hz, 1H), 7.62-
7.65 (m, 2H), 7.19-7.22 (m, 2H), 4.31 (s, 3H), 3.40 (s, 2H), 2.75
(d, J=4.8 Hz, 3H), 2.69 (s, 2H), 2.26-2.45 (bs, 8H), 2.18 (bs,
3H), 1.33 (s, 6H); LCMS (ESI) m/z 475 (M þ H)þ; HRMS (ESI)
calcd for C26H34N8O þ Hþ 475.2928, found 475.2932.
N,1,4,4-Tetramethyl-8-({3-[(4-methylpiperazin-1-yl)methyl]-
phenyl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-
carboxamide (35). Yield, 62%; 1H NMR (400 MHz, DMSO-d6)
δ 9.51 (s, 1H), 8.39 (s, 1H), 8.14 (q, J=4.8 Hz, 1H), 7.73 (bs, 1H),
7.55 (d, J=7.8, Hz, 1H), 7.22 (t, J=7.8 Hz, 1H), 6.87 (d, J=7.8
Hz, 1H), 4.33 (s, 3H), 3.41 (s, 2H), 2.75 (d, J=4.8 Hz, 3H), 2.69
(s, 2H), 2.26-2.47 (bs, 8H), 2.17 (bs, 3H), 1.33 (s, 6H); LCMS
(ESI) m/z 475 (M þ H)þ; HRMS (ESI) calcd for C26H34N8O þ
1
was prepared. Yield, 54%; H NMR (400 MHz, DMSO-d6) δ
9.23 (bs, 1H), 8.32 (s, 1H), 7.53 (bs, 1H), 7.49-7.53 (m, 2H), 7.27
(bs, 1H), 6.87-6.91 (m, 2H), 4.29 (s, 3H), 3.06-3.08 (m, 4H),
2.66 (s, 2H), 2.48-2.55 (m partially obscured by DMSO, 4H),
2.24 (s, 3H), 1.32 (s, 6H); LCMS (ESI) m/z 447 (M þ H)þ;
HRMS (ESI) calcd for C24H30N8O þ Hþ 447.2616, found
447.2616.
Compounds 28-41. By employment of the above-described
procedure, starting from 15b and using the suitable substituted
aniline, the compounds 28-41 were prepared.
N,1,4,4-Tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]-
amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (28). Yield, 65%; mp 190 ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ 9.24 (bs, 1H), 8.32 (s, 1H), 8.13 (q, J=4.8 Hz, 1H),
7.49-7.54 (m, 2H), 6.88-6.92 (m, 2H), 4.29 (s, 3H), 3.05-3.10
(m, 4H), 2.74 (d, J=4.8 Hz, 3H), 2.66 (bs, 2H), 2.44-2.54 (m
obscured by DMSO, 4H), 2.26 (bs, 3H), 1.32 (s, 6H); LCMS
(ESI) m/z 461 (M þ H)þ; HRMS (ESI) calcd for C25H32N8O
þHþ 461.2772, found 461.2772. Anal. (C25H32N8O) C, H, N.
N,1,4,4-Tetramethyl-8-{[3-(4-methylpiperazin-1-yl)phenyl]-
amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (29). Yield, 61%; 1H NMR (400 MHz, DMSO-d6) δ 9.30
(s, 1H), 8.37 (s, 1H), 8.14 (q, J=4.9 Hz, 1H), 7.24 (dd, J=8.0, 2.2
Hz, 1H), 7.21 (t, J=2.2 Hz, 1H), 7.12 (t, J=8.0 Hz, 1H), 6.56 (dd,
J=8.0, 2,2 Hz, 1H), 4.31 (s, 3H) 3.09-3.13 (m, 4H), 2.74 (d, J=
4.9 Hz, 3H), 2.67 (bs, 2H) 2.50-2.54 (m obscured by DMSO,
4H), 2.25 (bs, 3H), 1.32 (s, 6H); LCMS (ESI) m/z 461 (M þ H)þ;
HRMS (ESI) calcd for C25H32N8O þ Hþ 461.2772, found
461.2780.
N,1,4,4-Tetramethyl-8-[(4-morpholin-4-ylphenyl)amino]-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (30). Yield,
61%; 1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.33 (s, 1H),
8.13 (q, J=4.9 Hz, 1H), 7.52-7.56 (m, 2H), 6.89-6.93 (m, 2H),
4.29 (s, 3H), 3.73-3.75 (m, 4H), 3.03-3.05 (m, 4H), 2.74 (d,
J=4.9 Hz, 3H), 2.66 (s, 2H), 1.32 (s, 6H); LCMS (ESI) m/z 448
(M þ H)þ; HRMS (ESI) calcd for C24H29N7O2 þ Hþ 448.2456,
found 448.2455.
N,1,4,4-Tetramethyl-8-[(3-morpholin-4-ylphenyl)amino]-4,5-
dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (31). Yield,
71%; 1H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 1H), 8.38 (s, 1H),
8.14 (q, J=4.6 Hz, 1H), 7.28 (dd, J=8.0, 2.0 Hz, 1H), 7.21 (t, J=
2.0 Hz, 1H), 7.14 (t, J=8.0 Hz, 1H), 6.57 (dd, J=8.0, 2.0 Hz, 1H),
4.31 (s, 3H), 3.73-3.75 (m, 4H), 3.07-3.09 (m, 4H), 2.74 (d, J=
4.6 Hz, 3H), 2.68 (s, 2H), 1.32 (s, 6H); LCMS (ESI) m/z 448 (M þ
H)þ; HRMS (ESI) calcd for C24H29N7O2 þ Hþ 448.2456, found
448.2454.
N,1,4,4-Tetramethyl-8-({4-[(1-methylpiperidin-4-yl)oxy]phenyl}-
amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (32). Yield, 36%; 1H NMR (400 MHz, DMSO-d6) δ 9.32
(s, 1H), 8.34 (s, 1H), 8.13 (q, J=4.8 Hz, 1H), 7.54-7.58 (m, 2H),
6.90-6.94 (m, 2H), 4.34-4.41 (m, 1H), 4.28 (s, 3H), 2.82-
2.93 (bs, 2H), 2.74 (d, J=4.8 Hz, 3H), 2.67 (s, 2H), 2.37-2.47
H
þ 475.2928, found 475.2931.
N,1,4,4-Tetramethyl-8-{[4-(morpholin-4-ylmethyl)phenyl]-
amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (36). Yield, 64%; 1H NMR (400 MHz, DMSO-d6) δ 9.49
(s, 1H), 8.38 (s, 1H), 8.14 (q, J=4.9 Hz, 1H), 7.63-7.68 (m, 2H),
7.19-7.25 (m, 2H), 4.31 (s, 3H), 3.56-3.61 (m, 4H), 3.40 (s, 2H),
2.74 (d, J=4.9 Hz, 3H), 2.69 (s, 2H), 2.30-2.37 (bs, 4H), 1.32
(s, 6H); LCMS (ESI) m/z 462 (M þ H)þ; HRMS (ESI) calcd for
C25H31N7O2 þ Hþ 462.2612, found 462.2621.
N,1,4,4-Tetramethyl-8-{[3-(morpholin-4-ylmethyl)phenyl]-
amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (37). Yield, 60%; 1H NMR (400 MHz, DMSO-d6) δ 9.52
(s, 1H), 8.40 (s, 1H), 8.14 (q, J=4.8 Hz, 1H), 7.74 (bs, 1H), 7.56
(d, J=7.8 Hz, 1H), 7.23 (t, J=7.8 Hz, 1H), 6.89 (d, J=7.8 Hz,
1H), 4.34 (s, 3H), 3.54-3.60 (bs, 4H), 3.42 (bs, 2H), 2.74 (d, J=
4.8 Hz, 3H), 2.69 (s,2H), 2.33-2.40 (bs, 4H), 1.33 (s, 6H); LCMS
(ESI) m/z 462 (M þ H)þ; HRMS (ESI) calcd for C25H31N7O2 þ
H
þ 462.2612, found 462.2618; HPLC purity 95%.
8-{[4-(Dimethylamino)phenyl]amino}-N,1,4,4-tetramethyl-
4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (38).
1
Yield, 44%; H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H),
8.40 (s, 1H), 8.16 (q, J=4.8 Hz, 1H), 7.55 (d, J=9.2 Hz, 2H), 6.93
(d, J=9.2 Hz, 2H), 4.33 (s, 3H), 2.88 (s, 6H), 2.76 (d, J=4.8 Hz,
3H), 2.67 (s, 2H), 1.32 (s, 6H); LCMS (ESI) m/z 406 (M þ H)þ;
HRMS (ESI) calcd for C22H27N7O þ Hþ 406.2350, found
406.2349.
8-{[3-(Dimethylamino)phenyl]amino}-N,1,4,4-tetramethyl-
4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (39).
1
Yield, 52%; H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H),
8.37 (s, 1H), 8.14 (q, J=4.8 Hz, 1H), 7.18 (dd, J=8.2, 2.1 Hz,
1H), 7.09 (t, J=8.2 Hz, 1H), 6.97 (t, J=2.1 Hz, 1H), 6.37 (dd, J=
8.2, 2.1 Hz, 1H), 4.32 (s, 3H), 2.88 (s, 6H), 2.74 (d, J=4.8 Hz,
3H), 2.68 (s, 2H), 1.32 (s, 6H); LCMS (ESI) m/z 406 (M þ H)þ;
HRMS (ESI) calcd for C22H27N7O þ Hþ 406.2350, found
406.2346; HPLC purity 90%.
8-({4-[(Dimethylamino)methyl]phenyl}amino)-N,1,4,4-tetra-
methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (40). Yield, 54%; mp 161 ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ 9.51 (bs, 1H), 8.39 (s, 1H), 8.15 (q, J=4.8 Hz, 1H),

5162 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Brasca et al.
7.64-7.68 (m, 2H), 7.20-7.25 (m, 2H), 4.32 (s, 3H), 3.43
(bs, 2H), 2.74 (d, J=4.8 Hz, 3H), 2.69 (s, 2H), 2.21 (bs, 6H),
1.32 (s, 6H); LCMS (ESI) m/z 420 (M þ H)þ; HRMS (ESI) calcd
for C23H29N7O þ Hþ 420.2506, found 420.2509.
Km concentration. The known inhibitor ketoconazole was
included to check the inhibition response. Analysis of both
substrate and metabolite was done by LC/MS/MS.
Plasma Protein Binding. Plasma protein binding was per-
formed as previously described.21
8-({3-[(Dimethylamino)methyl]phenyl}amino)-N,1,4,4-tetra-
methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox-
amide (41). Yield, 62%; mp 181 ꢀC; 1H NMR (400 MHz,
DMSO-d6) δ 9.51 (s, 1H), 8.39 (s, 1H), 8.14 (q, J = 4.8 Hz,
1H), 7.76 (s, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.23 (t, J=7.8 Hz, 1H),
6.88 (d, J=7.8 Hz, 1H), 4.33 (s, 3H), 3.34-3.38 (s obscured by
water, 2H), 2.75 (d, J=4.8 Hz, 3H), 2.69 (s, 2H), 2.17 (s, 6H),
1.33 (s, 6H); LCMS (ESI) m/z 420 (M þ H)þ; HRMS (ESI) calcd
for C23H29N7O þ Hþ 420.2506, found 420.2498.
In Vivo Pharmacokinetics. The pharmacokinetic profiles of
the compounds were 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). 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 10000g for 3 min at 4 ꢀC, and the plasma was
stored at -80 ꢀC until analysis. Samples were analyzed by LC/
MS/MS technique.
Registry Numbers (RN). 1,2-Cyclohexanedione (RN, 765-87-
7), 5,5-dimethyl-1,3-cyclohexanedione (RN, 126-81-8), phenylgua-
nidine carbonate (RN, 14018-90-7), guanidine hydrochloride (RN,
50-01-1), 4-(4-methylpiperazin-1-yl)aniline (RN, 16153-81-4), 3-(4-
methylpiperazin-1-yl)aniline (RN, 148546-99-0), 4-morpholin-4-
ylaniline (RN, 2524-67-6), 3-morpholin-4-ylaniline (RN, 159724-
40-0), 4-[(1-methylpiperidin-4-yl)oxy]aniline (RN, 358789-72-7),
3-[(1-methylpiperidin-4-yl)oxy]aniline (RN, 790667-66-2), 4-[(4-
methylpiperazin-1-yl)methyl]aniline (RN, 70261-82-4), 3-[(4-me-
thylpiperazin-1-yl)methyl]aniline (RN, 198281-55-9), 4-(morpho-
lin-4-ylmethyl)aniline (RN, 51013-67-3), 3-(morpholin-4-ylmethyl)-
aniline (RN, 123207-48-7), N,N-dimethylbenzene-1,4-diamine
(RN, 536-46-9), N,N-dimethylbenzene-1,3-diamine (RN, 3575-
32-4), 4-[(dimethylamino)methyl]aniline (RN, 6406-74-2), 3-
[(dimethylamino)methyl]aniline (RN, 27958-77-6).
Acknowledgment. We thank Antonio Longo, Fabrizio
Orzi, and Marcellino Tibolla for their skilled synthetic chemi-
stry support. We thank Daniela Borghi for skillful assistance
in NMR spectra recording and interpretation, and Clara
Albanese for her support in flow cytometric analysis. We
are also grateful to Nilla Avanzi and the group of Assay
Development.
Supporting Information Available: X-ray crystallographic
studies of compounds 2 and 28; elemental analyses results of
compounds 1, 2, 17, 26, 28; kinase selective profile of compound
28. This material is available free of charge via the Internet at
http://pubs.acs.org.
Crystallographic Methods. Expression, purification, crystal-
lization, and soaking procedures of the CDK2/cyclin A com-
plexes were carried out as previously described.21
Kinase Assays. Kinase assays were performed as previously
described.21 The panel includes c-ABL, AKT1, ALK, Aur-A,
Aur-B, CDC7, CDK2/A, CDK1/B, CDK2/E, CDK4/D1,
CDK5/p25, CDK7/H, CHK1, CK2, EGFR, ERK2, FGFR1,
GSK3β, IGF1R, IKKi, IKK2, IR, C-KIT, LCK, LYN, MAP-
KAPK2, MET, NEK-6, NIM, PAK4, PDGFR, PDK1, PKAR,
PKCβ, PLK1, P38R, P38β, RET, STLK2, SULU1, TRKA,
VEGFR2, VEGFR3, and ZAP-70.
In Vitro Pharmacology. A2780 Cells Proliferation Assay. Cells
were seeded into 96- or 384-well plates at final concentration
ranging from 10 000 to 30 000 cells per cm2 in appropriate
medium plus 10% FCS. After 24 h cells were treated using serial
dilution of compounds in two replicates. At 72 h after treatment
the amount of cells were evaluated using the Cell Titer_Glo
assay (Promega). IC50 values were calculated using a sigmoidal
fitting (Assay Explorer MDL). Experiments were replicated at
least two times.
Flow Cytometry Analysis and BrdU Incorporation. A2780 cells
(human adenocarcinoma ovary, from ECACC) were seeded in
T-75 tissue culture flasks, 25 000 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 compounds at 1 μM for 24 h.
Cells in the supernatant and adherent cells were collected using
0.25% trypsin and 0.02% EDTA. Cells were washed with PBS
and were divided into three samples for flow cytometry analysis
and for immunoblot and BrdU incorporation, as previously
described.21
References
(1) Malumbres, M.; Barbacid, M. Cell cycle, CDKs and cancer: a
changing paradigm. Nat. Rev. Cancer 2009, 9, 153–166.
(2) Malumbres, M.; Barbacid, M. To cycle or not to cycle: a critical
decision in cancer. Nat. Rev. Cancer 2001, 1, 222–231.
(3) Alvocidib: Orphan Drug Status. Presented at the U.S. Food and
Drug Administration, Committee for Orphan Medicinal Products,
84th Plenary Meeting, 2007.
(4) Meijer, L.; Bettayeb, K.; Galons, H. (R)-Roscovitine (CYC202,
Seliciclib). In Inhibitors of Cyclin-Dependent Kinases as Anti-
Tumor Agents; CRC: Boca Raton, FL, 2007; pp 187-225.
(5) Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.; Barbosa,
S. A.; Hunt, J. T.; Rawlins, D. B.; Shan, W.; Ahmed, S. Z.; Qian,
L.; Chen, B.; Zhao, R.; Bednarz, M. S.; Kellar, K. A.; Mulheron,
J. G.; Batorsky, R.; Roongta, U.; Kamath, A.; Marathe,
P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.;
Lee, F. Y. F.; Webster, K. R.; Kimball, S. D. N-(Cyclo-
alkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent
kinase 2. N-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]methyl]thio]-2-
thiazolyl]-4-piperidinecarboxamide (BMS-387032), a highly effi-
cacious and selective antitumor agent. J. Med. Chem. 2004, 47,
1719–1728.
(6) Toogood, P. L.; Harvey, P. J.; Repine, J. T.; Sheehan, D. J.;
VanderWel, S. N.; Zhou, H.; Keller, P. R.; McNamara, D. J.;
Sherry, D.; Zhu, T.; Brodfuehrer, J.; Choi, C.; Barvian, M. R.; Fry,
D. W. Discovery of a potent and selective inhibitor of cyclin-
dependent kinase 4/6. J. Med. Chem. 2005, 48, 2388–2406.
(7) Chu, X. J.; DePinto, W.; Bartkovitz, D.; So, S. S.; Vu, B. T.;
Packman, K.; Lukacs, C.; Ding, Q.; Jiang, N.; Wang, K.; Goelzer,
P.; Yin, X.; Smith, M. A.; Higgins, B. X.; Chen, Y.; Xiang, Q.;
Moliterni, J.; Kaplan, G.; Graves, B.; Lovey, A.; Fotouhi, N.
Discovery of [4-amino-2-(1-methanesulfonylpiperidin-4-ylami-
In Vivo Pharmacology. Evaluation of antitumor efficacy was
performed as previously described.21
High-Throughput Solubility. Solubility at pH 7 was per-
formed as previously described.21
Metabolic Stability. Compounds were dissolved in DMSO at
10 μM. Human cDNA expressed cytochrome P450 isoforms
(supersomes) were purchased from Gentest (Woburn, MA).
All chemicals used were of analytical grade and commerci-
ally available. The potential inhibitory effect was investi-
gated against cDNA expressed human CYP4503A4 super-
some using typical substrates incubated at their respective
no)pyrimidin-5-yl](2,3-difluoro-6-
methoxyphenyl)methanone
(R547), a potent and selective cyclin-dependent kinase inhibitor
with significant in vivo antitumor activity. J. Med. Chem. 2006, 49,
6549–6560.
(8) Parry, D.; Guzi, T.; Seghezzi, W.; Paruch, K.; Doll, R.; Nomeir, A.;
Wang, Y.; Oft, M.; Kirschmeier, P.; Chen, T.; Lees, E. In Vitro and
in Vivo Characterization of SCH-727965, a Novel Potent Cyclin
Dependent Kinase Inhibitor. Presented at the AACR Conference,
Los Angeles, CA, April 14-18, 2007; Poster 4371.
(9) Wyatt, P. G.; Woodhead, A. J.; Berdini, V.; Boulstridge, J. A.;
Carr, M. G.; Cross, D. M.; Davis, D. J.; Devine, L. A.; Early, T. R.;

Article
Feltell, R. E.; Lewis, E. J.; McMenamin, R. L.; Navarro, E. F.;
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5163
6-Substituted pyrrolo[3,4-c]pyrazoles: an improved class of CDK2
inhibitors. ChemMedChem 2007, 6, 841–852.
(15) Chang, Y.-T.; Gray, N. S.; Rosania, G. R.; Sutherlin, D. P.; Kwon,
S.; Norman, T. C.; Sarohia, R.; Leost, M.; Meijer, L.; Schultz, P. G.
Synthesis and application of functionally diverse 2,6,9-trisub-
stituted purine libraries as CDK inhibitors. Chem. Biol. 1999, 6,
361–375.
(16) Papatsoris, A. G.; Liolitsa, D.; Deliveliotis, C. Manipulation of the
nerve growth factor network in prostate cancer. Expert Opin.
Invest. Drugs 2007, 16, 303–309.
O’Brien, M. A.; O’Reilly, M.; Reule, M.; Saxty, G.; Seavers, L. C.
A.; Smith, D.-M.; Squires, M. S.; Trewartha, G.; Walker, M. T.;
Woolford, A. J.-A. Identification of N-(4-piperidinyl)-4-(2,6-
dichlorobenzoylamino)-1H-pyrazole-3-carboxamide (AT7519), a
novel cyclin dependent kinase inhibitor using fragment-based X-
ray crystallography and structure based drug design. J. Med. Chem.
2008, 51, 4986–4999.
(10) Jones, C. D.; Andrews, D. M.; Barker, A. J.; Blades, K.; Daunt, P.;
East, S.; Geh, C.; Graham, M. A.; Johnson, K. M.; Loddick, S. A.;
McFarland, H. M.; McGregor, A.; Moss, L.; Rudge, D. A.;
Simpson, P. B.; Swain, M. L.; Tam, K. Y.; Tucker, J. A.; Walker,
M. The discovery of AZD5597, a potent imidazole pyrimidine
amide CDK inhibitor suitable for intravenous dosing. Bioorg.
Med. Chem. Lett. 2008, 18, 6369–6373.
(11) D’Alessio, R.; Bargiotti, A.; Metz, S.; Brasca, M. G.; Cameron, A.;
Ermoli, A.; Marsiglio, A.; Polucci, P.; Roletto, F.; Tibolla, M.;
Vazquez, M. L.; Vulpetti, A.; Pevarello, P. Benzodipyrazoles: a
new class of potent CDK2 inhibitors. Bioorg. Med. Chem. Lett.
2005, 15, 1315–1319.
(12) Traquandi, G.; Brasca, M. G.; D’Alessio, R.; Polucci, P.; Roletto,
F.; Vulpetti, A.; Pevarello, P.; Panzeri, A.; Quartieri, F.; Ferguson,
R.; Vianello, P.; Fancelli, D. Pyrazolo-quinazoline Derivatives,
Process for Their Preparation and Their Use as Kinase Inhibitors.
Patent WO2004/104007, 2004.
(17) Zhang, Y.; Dang, C.; Ma, Q.; Shimahara, Y. Expression of nerve
growth factor receptors and their prognostic value in human
pancreatic cancer. Oncol. Rep. 2005, 14, 161–171.
ꢀ
(18) Dolle, L.; Adriaenssens, E.; El Yazidi-Belkoura, I.; Le Bourhis, X.;
Nurcombe, V.; Hondermarck, H. Nerve growth factor receptors
and signaling in breast cancer. Curr. Cancer Drug Targets 2004, 4,
463–470.
(19) Colombo, M.; Riccardi-Sirtori, F.; Rizzo, V. A fully automated
method for accurate mass determination using high-performance
liquid chromatography with a quadrupole/orthogonal acceleration
time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom.
2004, 18, 511–517.
(20) Groll, M.; Goetz, M.; Kaiser, M.; Weyher, E.; Moroder, L. TMC-
95-based inhibitor design provides evidence for the catalytic versa-
tility of the proteasome. Chem. Biol. 2006, 13, 607–614.
(21) Pevarello, P.; Brasca, M. G.; Amici, R.; Orsini, P.; Traquandi, G.;
Corti, L.; Piutti, C.; Sansonna, P.; Villa, M.; Pierce, B. S.; Pulici,
M.; Giordano, P.; Martina, K.; Fritzen, E. L.; Nugent, R. A.;
Casale, E.; Cameron, A.; Ciomei, M.; Roletto, F.; Isacchi, A.;
Fogliatto, G.; Pesenti, E.; Pastori, W.; Marsiglio, A.; Leach, K. L.;
Clare, P. M.; Fiorentini, F.; Varasi, M.; Vulpetti, A.; Warpehoski,
M. A. 3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor
agents. 1. Lead finding. J. Med. Chem. 2004, 47, 3367–3380.
(13) Vulpetti, A.; Pevarello, P. An analysis of the binding modes of
ATP-competitive CDK2 inhibitors as revealed by X-ray structures
of protein-inhibitor complexes. Curr. Med. Chem.: Anti-Cancer
Agents 2005, 5, 561–573.
(14) Brasca, M. G.; Albanese, C.; Amici, R.; Ballinari, D.; Corti, L.;
Croci, V.; Fancelli, D.; Fiorentini, F.; Nesi, M.; Orsini, P.; Orzi, F.;
Pastori, W.; Perrone, E.; Pesenti, E.; Pevarello, P.; Riccardi-Sirtori,
F.; Roletto, F.; Roussel, P.; Varasi, M.; Vulpetti, A.; Mercurio, C.