1,4,5,6-Tetrahydropyrrolo[3,4-c]pyrazoles: Identification of a potent aurora kinase inhibitor with a favorable antitumor kinase inhibition profile

Article abstract of DOI:10.1021/jm060897w

The optimization of a series of 5-phenylacetyl 1,4,5,6-tetrahydropyrrolo[3, 4-c]pyrazole derivatives toward the inhibition of Aurora kinases led to the identification of compound 9d. This is a potent inhibitor of Aurora kinases that also shows low nanomol

Full text of DOI:10.1021/jm060897w

J. Med. Chem. 2006, 49, 7247-7251
7247
1,4,5,6-Tetrahydropyrrolo[3,4-c]pyrazoles: Identification of a Potent Aurora Kinase Inhibitor
with a Favorable Antitumor Kinase Inhibition Profile
Daniele Fancelli,† Ju¨rgen Moll, Mario Varasi,‡ Rodrigo Bravo, Roberta Artico,§ Daniela Berta, Simona Bindi,
Alexander Cameron, Ilaria Candiani, Paolo Cappella, Patrizia Carpinelli, Walter Croci, Barbara Forte, Maria Laura Giorgini,
Jan Klapwijk,| Aurelio Marsiglio, Enrico Pesenti, Maurizio Rocchetti, Fulvia Roletto, Dino Severino, Chiara Soncini,
Paola Storici, Roberto Tonani, Paola Zugnoni, and Paola Vianello*
NerViano Medical Sciences S.r.l. Viale Pasteur 10, 20014 NerViano, Milan, Italy
ReceiVed July 28, 2006
The optimization of a series of 5-phenylacetyl 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivatives toward
the inhibition of Aurora kinases led to the identification of compound 9d. This is a potent inhibitor of
Aurora kinases that also shows low nanomolar potency against additional anticancer kinase targets. Based
on its high antiproliferative activity on different cancer cell lines, favorable chemico-physical and
pharmacokinetic properties, and high efficacy in in vivo tumor models, compound 9d was ultimately selected
for further development.
Introduction
Several 5-amido derivatives endowed with good Aurora-A
inhibitory activity were initially identified through a preliminary
combinatorial expansion of the pyrrolopyrazole scaffold. All
these early hits were characterized by an arylacetic substituent
at position 5 such as compounds 2 and 3 (Table 1).11
To highlight areas for possible optimization of the inhibitory
activity, the crystal structure of compound 3 was solved as a
complex with Aurora-A kinase and refined at a resolution of
2.0 Å (Figure 1).
As would be expected, the binding mode of 3 is very similar
to that reported for PHA-680632 (1). However, the 5-amido-
thiophene substituent adopts a different spatial position to the
urea-containing compound. Rather than being situated under the
glycine-rich loop, as is the case for the diethyl-phenyl urea group
of PHA-680632, the amido-thiophene is directed away from
the loop and packs around Leu 263. It was envisaged, therefore,
that a substituent at the pro-R position of the benzylic methylene
could adopt a position similar to the diethyl benzene of PHA-
680632 under the glycine-rich loop and near to the amino
nitrogen of Lys 162. In the structure reported here, such a
substituent would be blocked by the presence of the activation
loop that adopts a position with His 280 packing onto the aryl
ring of the inhibitor. However, because this loop is very flexible
and in other Aurora-A structures is not seen, its position in this
structure was not thought to be of importance with regard to
what substituents would fit in the binding site. Substitutions at
the pro-S position were thought to be less favorable due to the
proximity of Val 147.
Mammalian Aurora kinases constitute a small family of three
closely related Ser/Thr protein kinases, namely, Aurora-A, -B,
and -C. They have different functions, subcellular localization,
or timing of expression and are essential to secure the correct
progress of cell cycle during mitosis or meiosis.1,2 In fact, they
are key regulators of major mitotic events such as centrosome
maturation and separation, mitotic spindle assembly, chromo-
some separation, and cytokinesis.2 Several lines of evidence
indicate Aurora-A and -B as attractive targets for pharmacologi-
cal intervention in oncology because, for example, both are
found to be overexpressed in cancer.3,4 The gene locus of
Aurora-A maps to 20q13, a chromosomal region frequently
amplified in a variety of human tumors.5 Aurora-A can act as
an oncogene because overexpression of Aurora-A leads to cell
transformation in vitro,6 and transgenic mice overexpressing
Aurora-A in the mammary gland develop mammary tumors at
a high incidence.7 Finally, “small molecule” inhibitors of Aurora
kinases demonstrate efficacy in animal tumor models, and the
first compounds have entered clinical studies.8-10
We have previously described a novel series of Aurora
kinases inhibitors identified from the combinatorial expansion
of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a ver-
satile scaffold designed to target the ATP pocket of protein
kinases.11 In particular, the development of the pyrrolopyrazole
subclasses characterized by a urea substituent at position 5 led
to the identification of PHA-680632 (1), which demonstrated
high in vitro antiproliferative activity on a wide range of cancer
cell lines and significant tumor growth inhibition in different
animal tumor models at well-tolerated doses.12
Following this hypothesis and assuming that the phenyl group
of compound 2 would adopt the same position as the thiophene
of compound 3, we undertook the synthesis of a small series of
pyrrolopyrazoles branched to the CH2 of the phenylacetyl moiety
in position 5, mainly with H-bond acceptor groups targeting a
possible interaction with the Lys 162 hydrogens. In this series,
we kept fixed at position 3 the already optimized 4-(1-methyl-
piperazin-4-yl)benzamide, which in the analogue 5-urea sub-
classes provided inhibitors endowed with adequate aqueous
solubility and high potency in cellular assays.
In this paper, we describe the optimization of the analogue
5-amido-pyrrolopyrazole subclasses, up to the identification and
preliminary characterization of a potent Aurora inhibitor selected
for evaluation as a potential anticancer agent.
* To whom correspondence should be addressed. Phone: +39.0331.-
581333. Fax: +39.0331.581347. E-mail: paola.vianello@nervianoms.com.
† Present address: Congenia, Italy.
The general synthetic route outlined in Scheme 1 was used
to prepare the target compounds 9a-f.
The synthesis of the common intermediate 7 from the 5-Boc-
protected 3-amino-tetrahydropyrrolo[3,4-c]pyrazole scaffold (4)
‡ Present address: DAC, Italy.
§ Present address: Italfarmaco, Italy.
| Present address: GlaxoSmithKline, U.K.
Present address: Universita` degli Studi di Milano, Italy.
10.1021/jm060897w CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/01/2006

7248 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Brief Articles
Table 1. Structure and Aurora-A Inhibition of PHA-680632 and Initial
5-Amido-pyrrolopyrazoles
Scheme 1
compd
X
Ar
Aur-A, IC50,a nM
1
2
3
NH
CH2
CH2
2,6-diethylphenyl
phenyl
2-thienyl
27
140
65
a IC50 values are reported as the mean of at least three individual
determinations. Variability around the mean was <25%.
Reactants and conditions: (a) EtCOOCl, THF, 20 h, 22 °C; (b)
p-piperazinylbenzoic acyl chloride, DIEA, THF, 16 h, 22 °C; (c) HCl (12
equiv, 4 N in dioxane), DCM, 24 h, 22 °C; (d) acylchloride, DIEA, DCM
6-8 h, 22 °C, or carboxylic acid, TBTU, DIEA, DMF, 24 h, 22 °C; (e)
Et3N 10% in MeOH, 3-6 h, 22 °C.
Table 2. Structure and Aurora-A Inhibition of
5-R-Substituted-(phenylacetyl)pyrrolopyrazoles
Figure 1. Structure of Aurora-A in complex with compound 3 (yellow,
tan carbon atoms) superposed on the structure with PHA-680632 (1;
light blue carbon atoms).
was performed as described in our previous report through
protection of the pyrazole moiety with ethyl carbamate, N-
acylation with p-piperazinylbenzoic acyl chloride, and Boc
removal with HCl in dioxane.11 The available pyrrolidinic
nitrogen of 7 easily reacted with activated acids to give
compounds 8a-f, which by treatment with triethylamine in
methanol furnished the final compounds 9a-f.
Compounds 9a-f were initially evaluated for their Aurora-A
inhibitory activity in a biochemical assay and their ability to
block cell proliferation (Table 2). Accumulation of cells with
g4 N DNA content, the phenotype typically induced by most
known Aurora inhibitors is an indication for inhibiting also
Aurora-B.13,14
Aur-A
Aur-B
HCT-116
cell cycle
block
anti-
inhibition inhibition proliferation EC50,a,b
cmpd
R
R′
IC50,a nM IC50,a nM
IC50,a nM
nM
2
H
F
OH
Me
OMe
H
H
H
H
H
H
Me
OMe
130
9
6
24
13
452
354
n.t.
300
48
99
79
220
50
500
110
>200c
100
80
9a
9b
9c
9d
9e
9f
97
21
31
1210
630
n.t.
n.t.
n.t.
n.t.
H
a IC50 values are reported as the mean of at least three individual
determinations. Variability around the mean was <25%. b Concentration
to induce 50% accumulation of cells with g4 N DNA (G2/M and polyploidy
cell populations). c EC50 comprised between 200 and 1000 nM.
Data shown in Table 2 indicate that the benzylic substitution
at the pro-R position significantly increases inhibitory potency
in this series and resulted in compounds endowed with potent
antiproliferative and cell cycle block activity.
associated electron density indicated that the methyl group
occupied the position of the phenyl ring in 9d and the ring
position of the methoxy (unpublished). It should be noted,
however, that even compound 9c, which is unable to form a
hydrogen bond with Lys 126, is much more active than the
unsubstituted compound 2.
Here we report the preliminary in vitro and in vivo profile
of 9d, which emerged from a deeper characterization of this
series as the compound with the best balance in terms of tumor
target kinase inhibition profile, pharmaceutical properties for
development, and efficacy in animal models.
To confirm the binding hypothesis, the structure of Aurora-A
was solved in complex with compound 9d. Although the crystals
only diffracted to 3 Å, the electron density for the inhibitor
clearly showed that the compound bound as expected, with the
benzyl group positioned as the thiophene of the previous
structure and the amino nitrogen of Lys 162 within hydrogen-
bonding distance of the oxygen of the pro-R methoxy substituent
(2.6 Å; see Supporting Information). In contrast, when data were
collected from crystals of Aurora-A in complex with a com-
pound with a methyl group at the pro-S position (9e), the

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7249
Table 3. Selectivity Profile of Compound 9d
kinase
IC50,a µM
kinase
IC50,a,b µM
Aurora-A
Aurora-B
Aurora-C
ABL
TRKA
RET
0.013
0.079
0.061
0.025
0.030
0.031
0.047
0.155
VEGFR3
C-KIT
VEGFR2
CDK2/cyA
STLK2
FLT3
0.161
0.407
0.432
0.462
0.621
0.669
3.5
FGFR1
LCK
PLK1
othersc
>10
a Values are the mean from two independent dose-response curves;
variation was generally (25%. b Kinase assays were carried out as described
in ref 19. c LYN, IR, GSK3â, NIM-1, PAK4, p38R, PDK1, CK2, CHK1,
Cdc7/Dbf4, ZAP70, PKAR, IKK2, MET, EGFR, ERK2, AKT1, IKKi,
PKCâ, and SULU.
Figure 2. Flow cytometric analysis of DNA content in human colon
carcinoma cells (HCT-116) treated for 24 h with increasing concentra-
tions of 9d.
Table 4. Antiproliferative Activity of Compound 9d
cell line
IC50,a µM
HeLa
MCF7
HCT-116
A-2780
0.140
0.080
0.031
0.028
a Values are the mean from three or more independent dose-response
curves; variation was generally 25%.
Figure 3. Western blot analysis of phospho-histone H3 in HCT-116
cells. An antibody recognizing R-tubulin was used as loading control.
Compound 9d was seen to be a potent, ATP-competitive
inhibitor of Aurora-A kinase, with an apparent Ki value of 2.5
( 0.3 nM. Its selectivity was evaluated in an in-house panel
representing diverse families of Tyr and Ser-Thr kinases. Data
reported in Table 3 show that, besides inhibiting all three Aurora
kinases, 9d also inhibits an additional set of protein kinases,
namely, ABL, RET, TRKA, and FGFR1 in the nanomolar range.
Notably, all of them have been independently suggested as
viable targets for oncology drug discovery. In particular, the
therapeutic value of Imatinib clearly validated the approach of
ABL inhibition for the treatment of chronic myelogenous
leukemia15 (CML), while oncogenic activation of RET tyrosine
kinase has been associated with medullary thyroid carcinomas
and, together with TRKA activation, to papillary thyroid
carcinomas.16,17 In addition, the activated TRKA oncogene has
been associated with other human malignancies, in particular,
prostate cancer.18
We expect that the particular kinase inhibition profile of 9d,
in which an antimitotic activity is complemented by the
inhibition of targets relevant for specific cancer types, could
offer promising opportunities for clinical development and
therapeutic use, although we may face the risk of a decreased
tolerance in patients.
In this regard, specific studies are currently ongoing to address
two crucial questions: first, whether the inhibition of these
additional targets contributes to the antitumoral effects or is
rather obscured by the dominant phenotype induced by aurora
inhibition; and second, whether the PK/PD requirements for the
inhibition in vivo of these different targets are compatible with
administration schedules, which might be limited by the
antimitotic nature of the compound.
Table 5. Individual and Average Pharmacokinetic Parameters Estimated
in Male CD-1 Mice Following a Single IV Bolus at the Nominal Dose
of 10 mg/kg with 9d in Mice
parameter
M1
M2
M3
mean
SD
C
max (µM)
8.0
0.97
5.4
4.1
3.2
11.1
1.8
8.3
2.7
4.1
9.6
1.5
5.8
4.0
3.8
9.6
1.4
6.5
3.6
3.7
1.5
0.4
1.5
0.8
0.4
T1/2,z (h)
AUC (µM‚h)
CL (L/h/kg)
V
ss (L/kg)
tested cell line (data not shown). As an example, the cell cycle
profile of HCT-116 cells treated for 24 h with different
concentrations of the compound is reported in Figure 2.
Finally, 9d induces a complete suppression of phosphorylation
of the substrate of Aurora kinases, histone H3 at serine 10, which
is widely regarded as a marker of Aurora B inhibition (Figure
3).20
Major pharmacokinetic parameters were obtained in vivo after
iv administration of 10 mg/kg of 9d to male CD-1 mice
cannulated into the superior vena cava (Table 5).
Plasma levels were detectable up to 6 h post-dosing, giving
an apparent average terminal half-life of about 1.4 h. The
clearance was about 70% of the hepatic blood flow and the
volume of distribution higher than the total body water,
indicating extensive tissue distribution.
These pharmacokinetic properties, coupled with a high
aqueous solubility (>15 mg/mL as the hydrochloride salt at pH
5.5) and a high stability of the resulting solution (shelf life: 36
months at 5 °C) makes compound 9d well-suited for develop-
ment as an injectable therapeutic agent.
In vivo, 9d was seen to be efficacious in a range of tumor
models. As an example, Figure 4 illustrates the dose-dependent
growth inhibition induced in the HL-60 xenograft model. Tested
at doses between 7.5 mg/kg and 30 mg/kg given iv bid for 5
days, the compound showed good dose dependency and a tumor
growth inhibition of up to 98%. At the highest dose (30 mg/
Kg) there was evidence of tumor regression and, in two cases
out of eight, cures. The maximal and reversible body weight
loss at this dose was 16%.
Compound 9d demonstrated potent antiproliferative activity
(IC50 values ranging from 28 to 140 nM, Table 4) in cell lines
originating from various human tumor types such as HCT-116
(colon carcinoma), A2780 (ovarian carcinoma), MCF7 (breast
carcinoma), and HeLa (cervical adenocarcinoma).
Similarly to what has been described for other Aurora-A and
-B inhibitors, the incubation of different tumor cell lines with
9d results in a substantial increase in 4 N DNA and polyploid
(g8 N DNA) populations. We observed that the extent of
polyploidy induction depends on the genetic background of the

7250 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Brief Articles
DMF (50 µL). After stirring the mixture at room temperature
overnight, volatiles were carefully removed under reduced pressure.
The resulting acyl chlorides were dissolved in dichloromethane (5
mL) and added to a solution of ethyl 3-{[4-(4-methylpiperazin-1-
yl)benzoyl]amino}-5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-car-
boxylate trihydrochloride 7 (300 mg, 0.59 mmol) in DCM (10 mL)
and DIEA (610 µL, 3.6 mmol) under stirring at room temperature.
The resulting suspension was stirred 16 h at room temperature. After
solvent removal, the residue was taken up with AcOEt (100 mL)
and the organic layer washed with aqueous NaHCO3 and brine and
dried over Na2SO4. Solvent was evaporated, and the residue was
purified on silica gel flash chromatography (DCM/MeOH, 95:5-
93:7).
Figure 4. Antitumor activity of 9d against HL-60 human tumor
xenograft implanted in SCID mice. Arrows indicate dosing (bid × 5).
Synthesis of 8b. Mandelic acid (304 mg, 2 mmol) was dissolved
in DMF (10 mL), DIPEA (650 mg, 5 mmol), and TBTU (740 mg,
2.4 mmol) were added, and the mixture was stirred for 15 min at
room temperature.
Conclusions
3-{[4-(4-Methylpiperazin-1-yl)benzoyl]amino}-5,6-dihydropyr-
rolo[3,4-c]pyrazole-1(4H)-carboxylate trihydrochloride 7 (508 mg,
1 mmol) was added in one portion, and the mixture was stirred 8
h at room temperature. DCM (50 mL) was added, and the organic
layer was washed with water and saturated aqueous NaHCO3 and
dried over Na2SO4, and the solvent was evaporated. The crude
residue was purified by silica gel flash chromatography (DCM/
MeOH, 80:20) to give 160 mg of the title compound (25%).
Synthesis of 9a-f. A solution of 8a-f (0.19mmol) in MeOH
(5 mL) and Et3N (0.32 mL) was stirred at 30 °C for 3 h. The
resulting mixture was evaporated under reduced pressure, and the
residue was taken up with Et2O and re-evaporated (three times).
The final compounds were purified by crystallization (MeOH/H2O;
9b), silica gel flash chromatography (DCM/MeOH, 9:1; 9a and
9c-e), and preparative HPLC (9f).
The data presented in this report were the starting point for
a deeper evaluation of safety and efficacy of compound 9d,
which has been ultimately selected for clinical studies. The
therapeutic value of compound 9d for the treatment of both solid
tumors and leukemias is currently under investigation in
advanced clinical trials.
Experimental Section
All reagents and solvents were purchased from commercial
suppliers of the best grade and used without further purification.
Flash chromatography was performed on silica gel (Merck grade
9385, 60Å). The following chromatographic methods were used
to assess compound purity. Chromatographic Method A: HPLC/
MS was performed on a Waters X Terra RP 18 (4.6 × 50 mm, 3.5
m) column using a Waters 2790 HPLC system equipped with a
996 Waters PDA detector and a Micromass mod. ZQ single
quadrupole mass spectrometer, equipped with an electrospray (ESI)
ion source. Mobile phase A was ammonium acetate 5 mM buffer
(pH 5.5 with acetic acid/acetonitrile 95:5), and mobile phase B was
H2O/acetonitrile (5:95), with a gradient from 10 to 90% B in 8
min and hold at 90% B for 2 min. UV detection was at 220 and
254 nm. Flow rate was 1 mL/min, with an injection volume of 10
µL. A full scan was done, with a mass range from 100 to 800 amu.
The capillary voltage was 2.5 KV, the source temperature was 120
°C, and the cone was 10 V. Retention times and purity refer to UV
detection at 220 nm. Mass are given as the m/z ratio. Chromato-
graphic Method B: HPLC was performed on a Waters X Terra
RP 18 (4.6 × 50 mm, 3.5 m) column using a Waters 2795 HPLC
system equipped with a 996 Waters PDA detector and a S.E.D-
.E.R.E Sedex 55 evaporative light scattering (ELS) detector. Mobile
phase A was pH 10, 0.05% aqueous ammonia/acetonitrile (95:5),
and mobile phase B was H2O/acetonitrile (5:95), with a gradient
from 10 to 90% B in 8 min and hold at 90% B for 2 min. UV
detection was at 220 and 254 nm. The ELS detector conditions
were as follows: gas was air, temperature was 34 °C, pressure was
2.3 bar, gain was 9, flow rate was 1 mL/min, and injection volume
was 10 µL. Retention times and purity refer to ELS detection. When
necessary, compounds have been purified by preparative HPLC on
a Waters X-Terra RP18 (19 × 100 mm, 5 um) column using a
Waters preparative HPLC2525 equipped with a 996 Waters PDA
detector and a Micromass mod. ZQ single quadrupole mass
spectrometer with electrospray ionization, positive mode. Mobile
phase A was water with 0.01% formic acid, and mobile phase B
was acetonitrile, with a gradient from 10 to 90% B in 8 min and
Acknowledgment. The authors are grateful to the group of
Biochemical Screening of Nerviano Medical Science; in par-
ticular, Dr. Cristina Alli has been precious with her contribution
to the selectivity profile of our best compounds.
Note Added after ASAP Publication. The Conclusions
paragraph was omitted and a typographical change was made
in the Introduction section in the version published ASAP
November 1, 2006; the corrected version was published ASAP
November 6, 2006.
Supporting Information Available: Atomic coordinates and
experimental structure factors have been deposited in the PDB. The
complex with compound 3 has been given code 2j4z and that with
9c has been given code 2j50. Analytical characterization of
compounds 9a-f, crystallographic methods, Aurora assay, cell
proliferation assay, cell cycle analysis by flow cytometry, and
Western blot analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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