Potent and selective aurora inhibitors identified by the expansion of a novel scaffold for protein kinase inhibition

Article abstract of DOI:10.1021/jm049076m

Potent and selective Aurora kinase inhibitors were identified from the combinatorial expansion of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a novel and versatile scaffold designed to target the ATP pocket of protein kinases. The most potent c

Full text of DOI:10.1021/jm049076m

3080
J. Med. Chem. 2005, 48, 3080-3084
Potent and Selective Aurora Inhibitors Identified by the Expansion of a Novel
Scaffold for Protein Kinase Inhibition
Daniele Fancelli,* Daniela Berta, Simona Bindi, Alexander Cameron, Paolo Cappella, Patrizia Carpinelli,
Cornel Catana,† Barbara Forte, Patrizia Giordano, Maria Laura Giorgini, Sergio Mantegani, Aurelio Marsiglio,
Maurizio Meroni, Juergen Moll, Valeria Pittala`,‡ Fulvia Roletto, Dino Severino, Chiara Soncini, Paola Storici,
Roberto Tonani, Mario Varasi, Anna Vulpetti, and Paola Vianello
Nerviano Medical Sciences - Oncology, via Pasteur 10, 20014 Nerviano, Milan, Italy
Received November 17, 2004
Potent and selective Aurora kinase inhibitors were identified from the combinatorial expansion
of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a novel and versatile scaffold designed
to target the ATP pocket of protein kinases. The most potent compound reported in this study
had an IC50 of 0.027 µM in the enzymatic assay for Aur-A inhibition and IC50s between
0.05 µM and 0.5 µM for the inhibition of proliferation of different tumor cell lines.
One of the emerging targets in oncology drug discov-
ery is represented by Aurora kinases,1-3 a small family
composed of three Ser/Thr protein kinases: Aurora-A,
B, and C. Besides playing a crucial role in mitosis,1,4
where they have been implicated in centrosome matu-
ration, chromosome segregation, and cytokinesis, Au-
rora kinases have been found to be overexpressed in a
number of tumor cell lines and human primary tu-
mors.5,6 Inhibition of the Aurora kinase activity in tumor
cell lines typically leads to the accumulation of polyploid
cells, apoptosis, and block of proliferation.7,8 In vivo, a
“small molecule” inhibitor of Aurora kinases recently
Figure 1. Schematic representation of the 1,4,5,6-tetrahydro-
demonstrated remarkable efficacy in animal tumor
pyrrolo[3,4-c]pyrazole scaffold in the kinase ATP binding
models.8
pocket.
As a part of our program toward the development of
anticancer kinase inhibitors, we have designed new mol-
Scheme 1a
ecules based on the 3-aminopyrazole moiety, a well-
known adenino mimetic pharmacophore present in sev-
eral classes of kinase inhibitors.9-11 The NH2-C-N-
NH pattern of the 3-aminopyrazole moiety, which is
stereochemically well suited to form hydrogen bonding
interactions with the kinase hinge region of the ATP
pocket, was embedded within the 1,4,5,6-tetrahydro-
pyrrolo[3,4-c]pyrazole to give an original scaffold en-
dowed with additional positions for increasing diversity
(Figure 1).12
a Reagents and conditions for R ) R′ ) H: (a) MeOH, H2SO4,
In this paper we report on the synthesis and biological
7 h, reflux; (b) di-tert-butyl dicarbonate, DCM/aq NaHCO3 (1:1),
characterization of potent Aurora kinases inhibitors,
identified by the combinatorial expansion of the 1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazole scaffold.
24 h, 22 °C; (c) MeONa, toluene, 3 h, 80 °C, then 2 N HCl;
(d) hydrazine hydrochloride, EtOH, 3 h, 60 °C, then aq NaHCO3.
3-carbonitriles 4, while cyclization to provide tetra-
hydropyrrolopyrazole 5 was accomplished by treatment
with hydrazine in ethanol. Tetrahydro-pyrrolopyrazoles
substituted at position 6 can be similarly obtained by
carrying out the process on the corresponding substi-
tuted cyanoethylglycines.14
To perform the following combinatorial expansion, a
novel and simple method to load pyrazoles onto solid
support was developed, by treating tetrahydropyrrolopy-
razole 5 with polystyrene isocyanate resin (Scheme 2).
The resulting urea linker between one of the ring
nitrogens and the resin is stable to the acidic conditions
employed in the synthesis and sufficiently labile to allow
a clean cleavage of the final compounds by alkaline
hydrolisis. After acylation of the amino group at posi-
Chemistry The synthesis of the 3-amino-tetrahydro-
pyrrolo[3,4-c]pyrazole scaffold, protected as the N-tert-
butyloxycarbonyl derivative at position 5, is described
in Scheme 113,14 (R, R′ ) H). Treatment of commercial
N-(2-cyanoethyl)glycine with sulfuric acid and methanol
afforded methyl ester 2, which was protected at the
nitrogen with tert-butoxycarbonyl to give 3. Reaction of
3 with sodium methoxide furnished 4-oxo-pyrrolidine-
* To whom correspondence should be addressed. Phone:
+39-0331-58-1546. Fax: +39-0331-58-1757. E-mail: daniele.fancelli@
nervianoms.com.
† Current address: Pfizer Global Research and Development, Ann
Arbor Laboratories, 2800 Plymouth Rd., Ann Arbor, MI 48105.
‡ Current address: Universita` degli Studi di Catania, Dipartimento
di Scienze Farmaceutiche, Viale Andrea Doria 6, 95125 Catania, Italy.
10.1021/jm049076m CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/10/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3081
Scheme 2a,b
the aim of rapidly advancing toward promising cellular
activity, we initially focused our attention on entries
11-14 (Table 1).
This subset of 4-tert-butylbenzamide derivatives
emerged on the basis of the antiproliferative effect on
the human colon carcinoma cell line HCT-116, in com-
bination with high inhibitory activities on Aurora-A.
Despite their promising activity, entries 11-14 are poor-
ly soluble compounds (<15 µM in buffer pH 7), thus
hampering their further development. The subsequent
lead optimization was primarily directed to the prepara-
tion of soluble analogues. Based on the hypothesis that
the tert-butyl is directed toward the outside of the en-
zyme (Figure 1), and having in mind that the substitu-
ents placed in that region have often been used for the
optimization of the physicochemical properties in other
kinase inhibitor series, we planned the parallel synthe-
sis of several arrays of analogues of compounds 11-14,
bearing different hydrophilic moieties at position 4′.
a Solid-phase conditions, PG ) CONH-Polystyrenic resin:
(a) PS-NCO, DCM, 20 h, 22 °C; (b) RCOCl, DIEA, DCM, 16 h, 22
°C; (c) TFA/DCM (1:1), 3 h, 22 °C; (d) R′NCO or R′COOH, TBTU,
NMM, DCM or DMF, 24 h, 22 °C; (e) NaOH aq, MeOH, 72 h, 40
°C, then HCl 35%. bSolution-phase conditions, PG ) COOEt:
(a) EtCOOCl, THF, 20 h, 22 °C; (b) RCOCl, DIEA, THF, 16 h, 22
°C; (c) HCl (12 equiv, 4 N in dioxane), DCM, 24 h, 22 °C; (d) R′NCO
or R′COOH, TBTU, NMM, DCM or DMF, 24 h, 22 °C; (e) Et3N
10% in MeOH, 3-6 h, 22 °C
Table 1 shows the results from a series of 4-methyl-
piperazin-1-yl analogues (entries 15-18). Compound 18
showed improved potency and greatly increased aqueous
solubility (>200 µM in buffer pH 7), and so was selected
for more extensive characterization.
tion 3 and treatment with TFA to unmask the dihydro-
pyrrole nitrogen, reaction of the intermediate 8a with
isocyanates led to ureas while coupling with carboxylic
acids in the presence of TBTU and NMM furnished
amides. Using aqueous NaOH in MeOH, final products
were cleaved from the resin, and after neutralization
with HCl, crude mixtures were filtered through
Si-cartridges.
Kinase Inhibition and Binding Mode Compound
18 is a potent inhibitor of Aurora kinases (IC50
)
27 nM, 135 nM, and 120 nM versus Aurora-A, -B, and
-C, respectively). Screened against a panel of enzymes
representing diverse families of Tyr and Ser-Thr
kinases, it exhibited >25-fold selectivity for Aurora-A
enzyme over 19 out of the 20 kinases evaluated (data
available in the Supporting Information). The minor
cross-reactivity with FGFR1 (IC50 ) 0.4 µM) is not
expected to be involved in the antiproliferative and cell
cycle block effects on tumor cells exhibited by the
compound.
According to Scheme 2a shown above, 22 acyl chlo-
rides were used in step b and 46 acylating agents in
step d to produce an initial set of about 1000 compounds,
to be routinely used in the screens against different
kinase targets. Building block selection was carried out
by first filtering the combined sets of commercial and
proprietary reagents according to synthetic constraints
and medicinal chemistry criteria and then by clustering
the resulting lists by structural similarity. One repre-
sentative reagent per cluster was finally selected.
Synthetic steps a-c were carried out on a multigram
scale, while intermediates 8a were portioned in smaller
aliquots. Typically, by working on 200 mg of resin 8a,
about 10-40 mg of final compound 10 were obtained
as dry powder in adequate purity for screening purposes
(identity confirmed by 1H NMR and MS. HPLC raw area
% >90 at 220 and 254 nm; experimental details are
available on line in the Supporting Information). A close
analogue solution-phase process, based on the protection
of the pyrazole ring nitrogen as ethyl carbamate (Scheme
2b), was also developed to allow large scale productions.
In particular, by using 4-(4-methyl-piperazin-1-yl)-
benzoyl chloride in step b and 2,6-diethyl-phenylisocy-
anate in step d, the process outlined in Scheme 2b
allowed multigram productions of the compound 18 in
high yield and purity.
The crystal structure of 18 was solved as a complex
with the kinase domain of Aurora-A and refined at a
resolution of 2.5 Å. As can be seen in Figure 2 the
compound makes the expected hydrogen bonding inter-
actions with the hinge region that are shown in Figure
1, while the 2,6-disubstitution of the phenylurea at
position 5 forces the phenyl ring approximately perpen-
dicular to the pyrrolopyrazole. The Nꢀ atom of Lys 162
is situated directly above the phenyl ring at a distance
of 3.5 Å and is within hydrogen bonding distance of the
carboxyl oxygen of the ligand (∼3.1 Å).
Cell Assays Incubation of HCT-116 cells with com-
pound 18 for 24 h led to an accumulation of cells with
g4 N DNA content (Figure 3). This phenotype is con-
sistent with the expected molecular mechanism of action
and the crucial role of Aurora kinases for cell cycle
progression. A similar phenotype has been seen with
two previously described Aurora kinase inhibitors.7,8
To further demonstrate that in cells compound 18 acts
as an Aurora kinase inhibitor, we analyzed its effect on
histone H3 phosphorylation. In particular the Aurora-B
kinase family member has been shown to be critical for
phosphorylation of histone H3 at Ser10 and this has
been correlated with mitosis and chromosome condensa-
tion. As shown in Figure 4 compound 18 is able to
inhibit histone H3 phosphorylation on Ser10 in HCT-
116 cells.
Discussion In a screen against Aurora kinase A, the
library gave an inhibition hit rate (IC50 < 10 µM)
exceeding 10%, with 7% exhibiting sub-micromolar
activity. At this stage, without the need to analyze the
SAR in depth, it became apparent that all the most
potent Aurora inhibitors in this set were characterized
by 4′-substituted benzamido groups at position 3. With

3082 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Brief Articles
Table 1. Aurora-A Inhibition of Representative 1,4,5,6-Tetrahydropyrrolo[3,4-c]pyrazoles
a Enzyme inhibition IC50 µM; b Antiproliferation IC50 µM. c Values are mean from two or more independent dose-response curves;
variation was generally (25%. d SD ) 0.011, n ) 15. e SD ) 0.017, n )10.
Figure 4. Phosphorylation of histone H3 after treatment of
HCT-116 cells for 24 h with 1 µM 18. Normalization of the
total protein was done using an antibody which recognizes
histone H1.
Table 2. Inhibition of Cell Proliferation by Compound 18
cell line
HL-60
0.13
A-2780
0.11
HT-29
0.08
HeLa
0.41
IC50, µMa
Figure 2. Superposition of the structure of Aurora-A in
complex with 18 (turquoise carbon atoms) on that of the active
form of Aurora-A in complex with ADP and TPX215 (brown
carbon atoms).
a Antiproliferation IC50. Values are the mean from three or more
independent dose-response curves; variation was generally (25%.
of a number of parameters (enzyme inhibition, cell
activity, physicochemical aspects) across a diverse series
of inhibitors. The optimization of tetrahydropyrrolo-
[3,4-c]pyrazoles toward additional kinase targets, as
well as the ongoing work aimed to explore the efficacy
of compound 18 in a range of in vivo models, will be the
subjects of future reports.
Experimental Section
Figure 3. Cell cycle profile of HCT-116 cells treated for 24 h
with 1 µM compound 18.
All reagents and solvents were purchased from commercial
suppliers of the best grade and used without further purifica-
tion. Flash chromatography was performed on silica gel (Merck
grade 9385, 60 Å). The following chromatographic methods
were used to assess compound purity.
Chromatographic method A: HPLC/MS performed on a
Waters X Terra RP 18 (4.6 × 50 mm, 3.5 µm) 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). Gradient from 10 to 90% B in 8 min,
hold 90% B 2 min. UV detection at 220 and 254 nm. Flow rate
1 mL/min. Injection volume 10 µL. Full scan, mass range from
100 to 800 amu. Capillary voltage was 2.5 kV; source temper-
Finally, the potent antiproliferative effect exhibited
by 18 on the HCT-116 cells was confirmed on a panel
of different tumor cell lines (Table 2).
Conclusions The tetrahydropyrrolo[3,4-c]pyrazoles
represent a novel class of compounds designed to target
the ATP pocket of protein kinases. The high potential
of tetrahydropyrrolo[3,4-c]pyrazoles for the development
of protein kinase inhibitors is exemplified by the rapid
identification of compound 18, a potent and selective
Aurora kinase inhibitor, able to block cell cycle and
tumor cell proliferation in vitro in the nanomolar range.
The selection of 18 was guided by early consideration

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3083
ature was 120 °C; cone was 10 V. Retention times and purity
refer to UV detection at 220 nm. Mass values are given as
m/z ratio.
slowly added to a mixture of tert-butyl 3-amino-4,6-dihydro-
1H-pyrrolo[3,4-c]pyrazole-5-carboxylate (20 g, 89 mmol) and
DIEA (92 mL, 528 mmol) in THF (500 mL) at 0-5 °C. The
reaction was kept at the same temperature for 2 h, allowed to
reach r.t., and stirred overnight. The obtained mixture was
evaporated to dryness and the resulting residue extracted with
AcOEt and water. The organic layer was separated, dried over
sodium sulfate, and evaporated to dryness. The residue was
purified by flash chromatography (ethyl acetate/cyclohexane
4:6 to 7:3) to give 19 g (72%) of the title compound as a white
solid. 1H NMR (500 MHz, DMSO-d6) δ ppm 1.25, 1.26 (2 t,
J ) 7.1 Hz, 3 H, rotamers), 1.42 (s, 9 H), 4.11-4.19 (m, 2 H),
4.25 (q, J ) 7.1 Hz, 2 H), 4.41-4.47 (m, 2 H), 5.65 (s, 2 H).
LRMS (pos. ESI, M + H+) m/z 297.
Chromatographic method B: HPLC performed on a Waters
X Terra RP 18 (4.6 × 50 mm, 3.5 µm) 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). Gradient from 10 to 90% B in 8 min, hold 90% B 2 min.
UV detection at 220 and 254 nm. ELS detector: gas was air,
temperature was 34 °C, pressure was 2.3 bar, gain was 9. Flow
rate 1 mL/min. Injection volume 10 µL. Retention times and
purity refer to ELS detection.
1H NMR spectra were recorded on a Varian Inova 400
operating at 400.45 MHz or on a Varian Inova 500 operating
at 499.76 MHz; both instruments are equipped with a Indirect
detection probe (1H {15N-31P}. The residual signal of the
deuterated solvent was used as internal reference, the chemi-
cal shifts are expressed in ppm (δ).
[(2-Cyanoethyl)amino]acetic Acid Methyl Ester (2). Six
milliliters of H2SO4 96% (0.112 mol) was added dropwise to a
suspension of [(2-cyanoethyl)amino]acetic acid (9.3 g,
0.078 mol) in methanol (100 mL). The reaction mixture was
refluxed for 7 h, the solvent evaporated under vacuum, and
the residue diluted with NaOH 20% until pH 8. The aqueous
layer was extracted with DCM (3 × 50 mL), and the separated
organic phase was dried over anhydrous Na2SO4. The solvent
was evaporated to dryness to give 9.32 g of a yellow oil (84%),
which was used in the next step without further purification.
LRMS (pos. ESI, M + H+) m/z 143.
[tert-Butoxycarbonyl-(2-cyanoethyl)amino]acetic Acid
Methyl Ester (3). Di-tert-butyl dicarbonate (27.63 g,
0.127 mol) and NaHCO3 sat. solution (90 mL) were added to
a mixture of methyl [(2-cyanoethyl)amino]acetate in DCM
(90 mL). The resulting solution was stirred for 24 h at 22 °C,
the organic layer was separated, and the aqueous layer was
washed with DCM (3 × 50 mL). The recollected organic
fractions were dried over anhydrous Na2SO4, and the solvent
was evaporated to dryness. The obtained residue was purified
on a silica gel column by using a mixture hexane/EtOAc (4:1)
to give 13.4 g of the title compound as a colorless oil (88%).
1H NMR (500 MHz, DMSO-d6) δ 1.45 (s, 9 H), 2.67 (m, 2 H),
3.46 (t, J ) 6.7 Hz, 2 H), 3.63 (d, J ) 6.8 Hz, 3 H), 3.98
(d, J ) 2.6 Hz, 2 H). LRMS (pos. ESI, M + H+) m/z 243.
4-Cyano-1-N-(tert-butoxycarbonyl)pyrrolidin-3-one (4).
Sodium methylate (3.02 g, 0.0543 mol) was added to a solution
of methyl [tert-butoxycarbonyl-(2-cyanoethyl)amino]acetic acid
methyl ester (13.2 g, 0.0543 mol) in anhydrous toluene
(120 mL). The reaction mixture was stirred for 3 h at 80 °C,
cooled to 0 °C, and filtered. The obtained white solid was
suspended in 1 N HCl (55 mL), stirred for 2 h at 22 °C, and
then cooled to 0 °C and filtered again. The white solid was
washed with water and dried under vacuum to give 9.5 g of
the title compound (83%). 1H NMR (500 MHz, DMSO-d6)
δ 1.39 (s, 9 H), 4.02 (m, 4 H), 12.12 (br s, 1 H). LRMS (pos.
ESI, M + H+) m/z 211.
5-tert-Butyl 1-Ethyl 3-{[4-(4-Methylpiperazin-1-yl)ben-
zoyl]amino}-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicar-
boxylate (7b). Oxalyl chloride (23.2 mL, 265 mmol) was added
to a suspension of 4-(4-methyl-1-piperazinyl)benzoic acid
(11.7 g, 53 mmol) in DCM (320 mL) and DMF (0.52 mL). After
refluxing the mixture for 6.5 h, volatiles were carefully
removed under reduced pressure. The resulting 4-methyl-
piperazinobenzoyl chloride dihydrochloride was portion-wise
added to a solution of 5-tert-butyl 1-ethyl 3-amino-4,6-
dihydropyrrolo[3,4-c]pyrazole-1,5-dicarboxylate
(13.1
g,
44.3 mmol) in dry THF (620 mL) and DIEA (54.4 mL,
0.32 mol) under stirring at room temperature. The resulting
suspension was stirred 16 h at room temperature, and 1 h at
40 °C. After solvent removal, the residue was taken up with
AcOEt (600 mL) and the organic layer washed with aqueous
Na2CO3 and brine and dried over Na2SO4. Solvent was
evaporated, and the residue was triturated with a mixture of
Et20 (135 mL) and AcOEt (15 mL), filtered, and dried under
vacuum to give 20 g (90%) of the title compound as a white
solid. 1H NMR (500 MHz, DMSO-d6) δ ppm 1.36 and 1.37
(2 t, J ) 7.1 Hz, 3 H, rotamers), 1.50 (s, 9 H), 2.27 (s, 3H),
2.48 (m, 4 H), 3.32-3.38 (m, 4 H), 4.42 (q, J ) 7.1 Hz, 2 H),
4.50-4.65 (m, 4 H), 7.0 (d, J ) 9.0 Hz, 2 H), 7.97 and 7.8 (2 d,
J ) 9.0, 2 H, rotamers), 11.15 and 11.16 (2 s, 1 H, rotamers).
LRMS (pos. ESI, M + H+) m/z 499.
Ethyl 3-{[4-(4-Methylpiperazin-1-yl)benzoyl]amino}-
5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate Di-
hydrochloride (8b). A 4 N solution of HCl in dioxane
(122 mL, 488 mmol) was added dropwise to a stirred solution
of 5-tert-butyl 1-ethyl 3-{[4-(4-methylpiperazin-1-yl)benzoyl]-
amino}-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarboxylate
(19.5 g, 39.1 mmol) in dry DCM (240 mL); precipitation of a
white solid occurred almost immediately. The resulting mix-
ture was stirred at room temperature for 24 h; after dilution
with Et2O (100 mL), the solid was filtered, extensively washed
with Et2O, and dried under vacuum at 50 °C to give 19.8 g
(100%) of the title compound, used in the next step without
further purification. 1H NMR (500 MHz, DMSO-d6) δ ppm 1.35
(t, J ) 7.1 Hz, 3 H), 2.80 (d, J ) 4.6 Hz, 3 H), 3.10-3.20
(m, 2 H), 3.23 (td, J ) 13.3, 2.1 Hz, 2 H), 3.38-3.51 (m, 2 H),
4.05 (d, J ) 13.4 Hz, 2 H), 4.41 (q, J ) 7.1 Hz, 2 H), 4.47 (m,
2 H), 4.54 (m, 2 H), 7.07 (d, J ) 9.1 Hz, 2 H), 8.00 (d, J ) 9.0
Hz, 2 H), 10.39 (m, 2 H), 10.94 (br s, 1 H), 11.36 (s, 1 H). LRMS
(pos. ESI, M + H+) m/z 399.
Ethyl 5-(2,6-Diethyl-phenylcarbamoyl)-3-[4-(4-methyl-
piperazin-1-yl)benzoylamino]-5,6-dihydro-4H-pyrrolo-
[3,4-c]pyrazole-1-carboxylate (9b). A solution of diethylphe-
nylisocyanate (6.53 mL, 37.8 mmol) in DCM (20 mL) was
added dropwise to a suspension of ethyl 3-{[4-(4-methyl-
piperazin-1-yl)benzoyl]amino}-5,6-dihydropyrrolo[3,4-c]pyra-
zole-1(4H)-carboxylate trihydrochloride (16 g, 31.5 mmol) in
DCM (370 mL) and DIEA (21.5 mL, 126 mmol). The resulting
solution was stirred at room temperature for 18 h, washed with
water and brine, dried over Na2SO4, and evaporated. The
residue was triturated with a mixture of Et2O (300 mL) and
AcOEt (30 mL), filtered, extensively washed with Et2O, and
dried under vacuum at 40 °C to give 14.4 g (80%) of the title
compound as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.11 (t, J ) 7.5 Hz, 6 H), 1.33 (t, J ) 7.1 Hz, 3 H), 2.20
(s, 3 H,) 2.41 (m, 4 H), 2.55 (q, J ) 7.5 Hz, 4 H), 3.28 (m, 4 H),
tert-Butyl 3-Amino-4,6-dihydro-1H-pyrrolo[3,4-c]pyra-
zole-5-carboxylate (5). A mixture of 4-cyano-1-N-(tert-
butoxycarbonyl)pyrrolidin-3-one (9.0 g, 0.043 mol) and hydra-
zine dihydrochloride (4.5 g, 0428 mol) in ethanol (250 mL) was
stirred at 60 °C for 3 h. After cooling to 0 °C, a solution of sat-
urated sodium bicarbonate (600 mL) was slowly added drop-
wise. The solvent was evaporated under vacuum, the residual
water extracted with AcOEt, and the organic phase dried over
anhydrous Na2SO4. The solvent was evaporated to dryness to
give a crude solid. Purification by flash chromatography over
silica gel eluting with DCM/methanol (46:4), afforded the title
1
compound as a white solid (3.56 g, 31%). H NMR (500 MHz,
DMSO-d6) δ 1.41 (s, 9 H), 4.12 (m, 4 H), 4.99 (br s, 2H), 11.15
(br s, 1 H), LRMS (pos. ESI, M + H+) m/z 225.
5-tert-Butyl 1-Ethyl 3-Amino-4,6-dihydro-pyrrolo[3,4-
c]pyrazole-1,5-dicarboxylate (6b). A solution of ethyl
chlorocarbonate (8.9 mL, 93 mmol) in THF (250 mL) was

3084 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Brief Articles
4.39 (q, J ) 7.1 Hz, 2 H), 4.70 (m, 4 H), 6.95 (d, J ) 9.1 Hz,
2 H), 7.06 (d, J ) 7.4 Hz, 2 H), 7.13 (dd, J ) 8.4, 6.5 Hz, 1 H),
7.82 (s, 1 H), 7.94 (d, J ) 9.0 Hz, 2 H), 11.13 (s, 1 H). LRMS
(pos. ESI, M + H+) m/z 574.
pH 6.8 and 2% SDS. Samples were sonicated for a few seconds
and heated for 3 min at 95 °C. An amount of 20 µg proteins/
well was loaded and separated through SDS-PAGE (12%
acrylamide) and transferred onto nitrocellulose filters. Filters
were saturated in 5% milk in TBS containing 0.05% Tween
20 (TBS-T) for 1 h at r.t. A rabbit polyclonal anti-phospho-H3
(1:250, Upstate biotechnology # 06-570) was incubated for
1 h at room temperature at 4 °C, followed by washes in TBS-T
and incubation with secondary horseredish peroxidase (HRP)
conjugated anti rabbit Ig antibody. HRP-conjugated antibodies
were detected with ECL (Amersham).
N-(2,6-Diethylphenyl)-3-[4-(4-methyl-piperazin-1-yl)-
benzoylamino]-4,6-dihydro-1H-pyrrolo[3,4-c]pyrazole-5-
carboxamide (18). A solution of ethyl 5-(2,6-diethyl-phenyl-
carbamoyl)-3-[4-(4-methyl-piperazin-1-yl)benzoylamino]-5,6-
dihydro-4H-pyrrolo[3,4-c]pyrazole-1-carboxylate (11 g, 19.2
mmol) in MeOH (320 mL) and Et3N (32 mL) was stirred at
30 °C for 3 h. The resulting mixture was evaporated under
reduced pressure and the residue taken up with Et2O and re-
evaporated (three times). The resulting solid was triturated
with a mixture of AcOEt (300 mL) and Et2O (30 mL), filtered,
washed, and dried under vacuum to give 8.0 g (83% yield) of
the title compound as a white powder (mp 240-242 °C).
1H NMR (400 MHz, DMSO-d6) δ ppm 1.15 (t, J ) 7.3 Hz,
6 H), 2.34 (br s, 3 H), 2.55 (q, J ) 7.6 Hz, 4 H), 2.53-2.74
(m, 4 H), 3.29 (m, 4 H), 4.45-4.68 (m, 4 H), 6.99 (d, J ) 7.9
Hz, 2 H), 7.06 (d, J ) 7.3 Hz, 2 H), 7.13 (dd, J ) 8.5, 6.6 Hz,
1 H), 7.67 (br s, 1 H), 7.90 (d, J ) 7.8 Hz, 2 H), 10.56 (br s,
1 H), 12.08 and 12.32 (2 br s, 1 H, tautomers). LRMS (pos ESI,
M + H+) m/z 502. HPLC method A: Rt 3.36 min. purity 100%;
HPLC method B: tR 4.89 min. Purity 100%.
Supporting Information Available: Combinatorial chem-
istry methodology, analytical characterization of compounds
11-17, selectivity data for compound 18, and crystallographic
methods are available free of charge via the Internet at
http://pubs.acs.org.
References
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Aurora Assays. The Aurora proteins were produced in
insect cells as a GST-fusion protein and purified using GST
affinity chromatography and gel filtration. The biochemical
activity of compounds was determined by incubation with
Aurora kinase and substrate, followed by quantitation of the
phosphorylated product. Compounds were 3-fold serially di-
luted from 10 µM to 0.0005 µM and then incubated for 60 min
at room temperature in the presence of ATP/P33γ-ATP mix
(10 µM), CHOCKtide 4× (8 µM) for Aurora-A (2.5 nM); ATP/
P33γ-ATP mix (20 µM), CHOCKtide 4× (8 µM) for Aurora-B
(32 nM) or ATP/P33γ-ATP mix (37 µM), Auroratide (27 µM)
for Aurora-C (0.5 nM) in a final volume of 30 µL of buffer
(HEPES pH 7.5 50 mM, MgCl2 10 mM, DTT 1 mM; NaVO3
3 µM+ 0.2 mg/mL BSA); using 96 U bottom plates. After
incubation, the reaction was stopped by the addition of 100
µL of PBS + 32 mM EDTA + 0.1% Triton X-100 + 500 µM
ATP, containing 1 mg streptavidin-coated SPA beads (biotin
capacity 130 pmol/mg). After 20 min incubation for substrate
capture, 100 µL of the reaction mixture were transferred into
Optiplate (PerkinElmer) 96-well plates containing 100 µL of
5 M CsCl, left to stand for 4 h to allow stratification of beads
to the top of the plate, and counted using TopCount (Packard)
to measure substrate-incorporated phosphate.
Cell Proliferation Assay The human colon cancer cell line
HCT-116 was seeded at 5000 cells/cm2 in a 24-well plate using
F12 medium supplemented with 10% FCS, 2 mM L-glutamine,
and 1% penicillin/streptomycin and maintained at 37 °C, 5%
CO2, and 96% relative humidity. The following day plates were
treated in duplicates with 5 µL of an appropriate dilution of
compounds starting from a 10 mM stock in DMSO. Two
untreated control wells were included in each plate. After
72 h of treatment, medium was withdrawn and cells detached
from each well using 0.5 mL of 0.05% (w/v) trypsin, 0,02%
(w/v) EDTA (Gibco). Samples were diluted with 9.5 mL of
Isoton and counted using a Multisizer 3 cell counter. Data were
evaluated as percent of the control wells: % of CTR ) (treated
- blank)/(control - blank). IC50 values were calculated by
LSW/Data Analysis using Microsoft Excel sigmoidal curve
fitting.
Cell Cycle Analysis by Flow Cytometry (FACS). One
million cells before being fixed by methanol 70% were centri-
fuged at 300g for 5 min. After a wash in PBS, cells were
resuspended in 1 mL of PBS containing propidium iodide
25 µg/mL, Nonidet P-40 0.002%, and RNAse A 12.5 µg/mL.
The cells were kept in the dark for 60-90 min at 37 °C before
FACS analysis. For cell cycle analysis were collected 10 000
events, discriminating cell aggregates by appropriate gates by
BD FACSCalibur.
(14) Fancelli, D.; Pittala, V.; Varasi, M. Combinatorial preparation
of bicyclo pyrazoles as kinase inhibitors for treatment of cancer
and other proliferative disorders. WO 02/012242, 2000.
(15) Bayliss, R.; Sardon, T.; Vernos, I.; Conti, E.; Structural basis of
Aurora-A activation by TPX2 at the mitotic spindle. Mol. Cell
2003, 12(4), 851-862.
Western Blot. Subconfluent HCT-116 cells were lysed
directly on plates in a solution containing 0.125 M TrisHCl
JM049076M