3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 2. Lead optimization

Article abstract of DOI:10.1021/jm0408870

Inhibitors of cyclin-dependent kinases (CDK) such as CDK2/cyclin A-E are currently undergoing clinical trials to verify their potential as new anticancer agents. In a previous article we described the lead discovery process of a 3-aminopyrazole class of CDK2/cyclin A-E inhibitors. The endpoint of this process was PNU-292137, a compound endowed with in vivo antitumor activity in a mouse tumor xenograft model. We optimized this lead compound to improve some physicochemical properties, notably solubility and plasma protein binding. This lead optimization process brought us to the discovery of (2S)-N-(5-cyclopropyl- 1H-pyrazol-3-yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (PHA-533533, 13), a compound with a balanced activity vs druglike profile. Compound 13 inhibited CDK2/cyclin A with a K_i of 31 nM, counteracting tumor cell proliferation of different cell lines with an IC₅₀ in the submicromolar range. Solubility was improved more than 10 times over the starting lead, while plasma protein binding was decreased from 99% to 74%. With exploitation of this globally enhanced in vitro profile, 13 was more active than PNU-292137 in vivo in the A2780 xenograft model showing a tumor growth inhibition of 70%. Proof of mechanism of action was obtained in vivo by immunohistochemical analysis of tumor slices of 13-treated vs untreated animals.

Full text of DOI:10.1021/jm0408870

2944
J. Med. Chem. 2005, 48, 2944-2956
3-Aminopyrazole Inhibitors of CDK2/Cyclin A as Antitumor Agents. 2. Lead
Optimization
Paolo Pevarello,*,† Maria Gabriella Brasca,† Paolo Orsini,† Gabriella Traquandi,† Antonio Longo,† Marcella Nesi,†
Fabrizio Orzi,† Claudia Piutti,† Pietro Sansonna,† Mario Varasi,† Alexander Cameron,† Anna Vulpetti,†
Fulvia Roletto,‡ Rachele Alzani,‡ Marina Ciomei,‡ Clara Albanese,‡ Wilma Pastori,‡ Aurelio Marsiglio,‡
Enrico Pesenti,‡ Francesco Fiorentini, Jim R. Bischoff,‡,§ and Ciro Mercurio‡
Departments Chemistry and Biology, Nerviano Medical Sciences, BUsOncology and BUsPreclinical Science,
Viale Pasteur 10, 20014 Nerviano (MI), Italy
Received October 19, 2004
Inhibitors of cyclin-dependent kinases (CDK) such as CDK2/cyclin A-E are currently
undergoing clinical trials to verify their potential as new anticancer agents. In a previous article
we described the lead discovery process of a 3-aminopyrazole class of CDK2/cyclin A-E
inhibitors. The endpoint of this process was PNU-292137, a compound endowed with in vivo
antitumor activity in a mouse tumor xenograft model. We optimized this lead compound to
improve some physicochemical properties, notably solubility and plasma protein binding. This
lead optimization process brought us to the discovery of (2S)-N-(5-cyclopropyl-1H-pyrazol-3-
yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (PHA-533533, 13), a compound with a bal-
anced activity vs druglike profile. Compound 13 inhibited CDK2/cyclin A with a Ki of 31 nM,
counteracting tumor cell proliferation of different cell lines with an IC50 in the submicromolar
range. Solubility was improved more than 10 times over the starting lead, while plasma protein
binding was decreased from 99% to 74%. With exploitation of this globally enhanced in vitro
profile, 13 was more active than PNU-292137 in vivo in the A2780 xenograft model showing
a tumor growth inhibition of 70%. Proof of mechanism of action was obtained in vivo by
immunohistochemical analysis of tumor slices of 13-treated vs untreated animals.
Chart 1
Introduction
Abnormal proliferation mediated by disruption of the
normal cell cycle mechanisms is a hallmark of virtually
all cancer cells.1 Compounds targeting complexes be-
tween cyclin-dependent kinases (CDK) and cyclins, such
as CDK2/cyclin A, and inhibiting their kinase activity
are regarded as promising antitumor agents to comple-
ment the existing therapies, and there is a continuing
interest in this field.2 In a previous paper we described
the discovery and the lead finding process of a new class
of 3-aminopyrazole CDK2/cyclin A inhibitors.1 PNU-
292137 (Chart 1) was identified as a compound suitable
for in vivo evaluation but also endowed with suboptimal
physicochemical properties in light of its possible pro-
gression into further preclinical development studies.3
In particular, PNU-292137 displayed a low solubility
(in the range 20-50 µM, depending on its crystalline
state) in aqueous buffer (pH 7.0) solubility experiments.
While this solubility did not hamper its evaluation in
animal models as an oral compound, we deemed it could
limit its use in an intravenous continuous infusion
administration mode that is still widely practiced in the
clinical setting. Furthermore, PNU-292137 was shown
to be a strong binder to human serum albumin (HSA)
in a preliminary assay. We wanted to avoid potential
problems (i.e., abnormal pharmacokinetics (PK) param-
eters in humans) further down the road to a clinical
candidate. We thus embarked on a lead optimization
program with the aim of finding new derivatives of
PNU-292137 with comparable inhibitory potency but
with an improved physicochemical profile consistent
with a potential clinical use of a CDK2/cyclin A inhibi-
tor.
We describe here the optimization of PNU-292137 to
produce a compound ((2S)-N-(5-cyclopropyl-1H-pyrazol-
3-yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (13),
PHA-533533) retaining potent CDK2 inhibition in bio-
chemical and cellular assays but endowed with a better
druglike preclinical profile. Furthermore, an in-depth
pharmacological preclinical profile of 13 is also reported.
* To whom correspondence should be addressed. Phone: +39-
0331581927. Fax: +39-0331581347. E-mail: paolo.pevarello@
nervianoms.com.
† Department of Chemistry, BUsOncology.
Chemistry
‡ Department of Biology, BUsOncology.
BUsPreclinical Science.
All target compounds were obtained by using as a key
step the condensation between tert-butyl 3-amino-5-
§ Current address: Aventis Pharma S. A., Centre de Recherche de
Paris, 13, Quai Jules Guesde, F-94400 Vitry-sur-Seine, France.
10.1021/jm0408870 CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/29/2005

3-Aminopyrazole Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2945
Scheme 1a
a Conditions: (a) EDCI, CH2Cl2, room temp; (b) TFA/CH2Cl2 (1/9; v/v), room temp.
Scheme 2a
a Conditions: (a) HCI, 180 °C; (b) TEA, xylene, reflux; (c) ethyl isocyanateacetate, THF, 0 °C; (d) acetone, HCl, reflux.
Scheme 3a
Racemic methyl 4-aminophenylpropionate (42) was
the starting material to afford carboxylic acids 15a, 25a,
27a, 28a, and 36a as outlined in Schemes 4-8.6
Carboxylic acid 29a was finally prepared by alkyla-
tion of commercially available 52 followed by basic
hydrolysis of intermediate 53 (Scheme 9).7
a Conditions: (a) 1,4-dibromobutane, DIPEA, DMF.
SAR and Structural Studies
For the sake of clarity only a limited yet representa-
tive set of compounds is used here to describe the SAR.
More compounds were synthesized to support the SAR
described below.8
Scheme 4a
In a previous paper we described our first efforts on
the 3-aminopyrazole class to the lead compound PNU-
292137 (Chart 1), and we established that a cyclopropyl
ring was an optimal substituent for the 5-position
because of the efficient packing with the aromatic ring
of Phe 80 and other lipophilic residues in the so-called
buried region of the ATP-binding pocket of the kinase.3
On the basis of the SAR learned in this first part of the
class expansion, we knew that we could leverage on the
variety of substitutions accepted on the 3-amino part
of the pyrazole scaffold for achieving a better balance
between activity and “druglike” properties. We then
turned our attention to the R-position of the arylacet-
amido group in position 3 (Chart 1, Table 1). Structural
inspection of this region based on the X-ray structure3
of the previous lead compound PNU-292137 suggested
that a small group may be tolerated. Figure 1 shows
the R-methylphenylacetamido pyrazole compound docked
into the CDK2/cyclin A ATP pocket. The accessible
surface mesh of the binding site indicates the presence
of a small hydrophobic cavity, which can be filled only
by a small lipophilic group (e.g., a methyl group with S
configuration as in Figure 1). Moreover, the docking
binding mode shows that there is not much space to
accommodate substituents in an R configuration. This
hypothesis was experimentally verified with the syn-
thesis of a number of R-substituted 3-phenylacetami-
dopyrazoles (1-6, 8-10). As shown in Table 1, only a
small group, such as a methyl (8-10) or a fluorine (4)
a Conditions: (a) toluene, 60 °C; (b) N-hydroxybenzotriazole
ammonium salt, EDCI, THF/DMF (1:1 v/v), room temp; (c) MeOH,
aqueous Na2CO3, room temp.
cyclopropyl-1H-pyrazole-1-carboxylate and the suitable
carboxylic acid (Scheme 1) as previously reported by us.3
Carboxylic acids employed to obtain final compounds
1-13, 18-20, 22-24, 30-35, and 37 were either
commercially available or known from the literature and
are reported under the section Registry Numbers, while
the synthesis of the remaining ones is described in the
Experimental Section (Schemes 2-9).
Carboxylic acids 14a, 16a, 17a, 21a, and 26a were
prepared starting from racemic 4-aminophenylpropionic
acid (39) according to Scheme 2,4 while by using its (S)-
enantiomer (41), we obtained compound 38a (Scheme
3).5

2946 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Pevarello et al.
Scheme 5a
a Conditions: (a) chloroacetyl isocyanate, dioxane, room temp; (b) 2 N NaOH, MeOH, reflux.
Scheme 6a
a Conditions: (a) triphosgene, TEA, DCM, 0 °C; (b) 2,2-dimethoxyethylamine, DCM; (c) TFA/water, MeCN, room temp; (d) MeOH/
NaOH, room temp.
Scheme 7a
a Conditions: (a) methyl hydrazinocarboxylate, trimethylorthoformate; (b) MeONa; (c) MeOH/NaOH, room temp.
Scheme 8a
a Conditions: (a) 3-chloropropane-1-sulfonyl chloride, TEA, DCM; (b) DBU, DMF; (c) aqueous Na2CO3/MeOH.
Scheme 9a
a Conditions: (a) benzyl chloride, NaH, DMF; (b) LiOH, THF/water.
could fit into the enzyme cavity; more hydrophilic (1-
3, 5) or bulkier groups (6) were less active CDK2/cyclin
A inhibitors. Second, as indicated by the model (Figure
1), there was a clear stereoselectivity, with the S-
enantiomers (2, 9) more active than the R-counterparts
(3, 10).
By comparison of similar compounds with or without
a R-methyl substitution, a trend was observed toward
a better solubility for the R-methyl-substituted com-
pounds, particularly in vehicles containing cosolvents
that are commonly used for in vivo administration. A
possible explanation for this observation may be that
adding an R-substituent to the benzyl carbon atom
causes a disruption of the tight package of the molecule
in the crystal, bringing about improved solubility.
Further expansion of this class retaining an R-methyl
substituent in the arylacetamido moiety was then
planned, and whenever an interesting racemic product
emerged, the S-enantiomer (obtained either by stereo-
specific synthesis or by chiral separation) was evaluated
and verified to be the eutomer.
One of the major concerns regarding PNU-292137,
besides its rather low solubility in water, was the high
plasma protein binding (PPB).3 We had indications that
the naphthyl moiety was likely to play an important role
in imparting this property. We reasoned that by de-
creasing the lipophilic nature of this moiety while
retaining the inhibitory activity on CDK2/cyclin A, we
could achieve better compounds in terms of balanced
activity vs druglike properties. A first set of compounds
was devised by replacing the â-naphthyl moiety of PNU-
292137 with different 4-lactam-1-ylphenyl moieties. The

3-Aminopyrazole Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2947
Table 1. SAR of R-Substituted 3-Phenyl-
acetamido-5-cyclopropyl-1H-pyrazoles (1-10)
Table 2. SAR of 3-(4-Lactam-1-yl)phenyl-
acetamido-5-cyclopropyl-1H-pyrazoles (11-20)
a At least two independent experiments were performed for each
compound in order to determine IC50 values. Potency is expressed
as the mean of IC50 values obtained by nonlinear least-squares
regression fitting of the data. The coefficient of variation of the
mean ranges from 10% to 24%.
a At least two independent experiments were performed for each
compound in order to determine IC50 values. Potency is expressed
as the mean of IC50 values obtained by nonlinear least-squares
regression fitting of the data. The coefficient of variation of the
mean ranges from 10% to 24%.
pound 24, an eutomer of 22, bearing an imidazolidinone
heterocyclic moiety. This compound also has a better
solubility and improved PPB, although the increase in
inhibitory activity is not mirrored in the cellular assay.
The structure of this compound in complex with CDK2/
cyclin A was elucidated. In the X-ray structure the
compound is very well defined; the five-membered
imidazolidinone makes favorable hydrophobic interac-
tions with Ile10, as observed for the naphthyl moiety of
PNU-292137 (Figure 2). The carbonyl of the imidazoli-
dinone ring interacts with a water molecule that in turn
interacts with Lys20. The NH group of this moiety is
within hydrogen-bonding distance of Glu8, although
from the electron density this latter residue is obviously
somewhat mobile. This interaction may explain the
further increase in activity over compound 13 and also
the reduced activity when an N-methyl substitution is
made (30-32 vs 22-24). The complex of 13 with CDK2/
cyclin A was also solved at a resolution of 2.6 Å (data
not shown). In this it was difficult to unambiguously
define the orientation of the pyrrolidinone, and hence,
it seems that the NH in 23 helps define the orientation
of the ring. Bulkier groups such as benzyl (29) were
detrimental for activity, solubility, and PPB. Introduc-
ing an unsaturation into the five-membered ring of
imidazolinone did not hamper CDK2/cyclin A inhibitory
activity but considerably worsened cellular activity. This
finding may be correlated at least in part to the lower
cell permeability of 27 and 28 in the Caco-2 cell
penetration assay. Adding a second carbonyl moiety on
the lactamyl ring of 13 to give 21 gave a moderately
Figure 1. Docking of R-methylphenylacetoamidopyrazole
(magenta carbon atoms) in CDK2/cyclin A (white carbon
atoms). The mesh is the accessible surface of the binding site
which is useful for showing steric and chemical complemen-
tarity (yellow, hydrophobic; blue, hydrogen bond acceptor; red,
hydrogen bond donor).12
para position was chosen for the heterocyclic substitu-
tion because it allowed the substituent to directly point
toward the solvent accessible region. A simple unsub-
stituted five-membered lactam (13) was as active as the
parent lead against CDK2/cyclin A (Table 2), while
activity was even improved upon benzo condensation
of 13 (e.g., 20) or substitution of the five-membered ring
with a suitable group (e.g., 15), though 15 and 20 turned
out to be less active in the cellular assay. Increasing
the lactam ring size to a six-membered pyridinone (14)
was detrimental to activity. Compound 13 was also
active in cells in the submicromolar range and scored a
>10-fold increase in buffer solubility over PNU-292137.
Plasma protein binding (PPB) was consistently reduced
from 99% to 74%.
A second set of compounds featuring other five-
membered heterocycles is reported in Table 3. The best
inhibition in the biochemical assay was seen for com-

2948 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Pevarello et al.
Figure 2. Superposition of the CDK2/cyclin A complex with compound 24 (yellow/brown carbon atoms) on the complex with
PHA-292137 (green/cyan carbon atoms).12 It can be seen that the two inhibitors have very similar binding modes. Ile 10 appears
to adopt a different conformation in the two complexes. The electron density for this residue is better defined in the complex with
PHA-292137 than for that with compound 24.
Table 3. SAR of 3-(4-Heterocycl-1-yl)phenyl-
acetamido-5-cyclopropyl-1H-pyrazoles (21-32)
Table 4. SAR of 3-(4-Heterocycl-1-yl)phenyl-
acetamido-5-cyclopropyl-1H-pyrazoles (33-38)
a At least two independent experiments were performed for each
compound in order to determine IC50 values. Potency is expressed
as the mean of IC50 values obtained by nonlinear least-squares
regression fitting of the data. The coefficient of variation of the
mean ranges from 10% to 24%.
of an R-methyl was confirmed and in the series 22-24
and 30-32, the S-enantiomer was the eutomer. How-
ever, none of the compounds of this set reached suf-
ficient activity in cells to warrant more extensive
preclinical studies.
A final set of heterocycles, the oxazolidines 33-35,
the sultam 36, and the pyrrolidines 37 and 38, was then
evaluated (Table 4). The oxazolidine 35 was a potent
CDK2/cyclin A inhibitor, and again, solubility and PPB
were ameliorated over the parent lead PNU-292137.
Also, the pyrrolidine 38, despite a lower activity in the
biochemical assay, behaved very well in the cellular
assay but the overall druglike profile was not enhanced
over the parent lead.
In general the SAR around the heterocyclic part
linked to the phenylacetic moiety shows that (1) it is
possible to get compounds with potency up to the single
digit nanomolar range using several five-membered
heterocycles. Fine-tuned considerations over the relative
a At least two independent experiments were performed for each
compound in order to determine IC50 values. Potency is expressed
as the mean of IC50 values obtained by nonlinear least-squares
regression fitting of the data. The coefficient of variation of the
mean ranges from 10% to 24%.
active compound but without cellular activity. The
position of the carbonyl group into the imidazolidindione
ring was important for determining CDK2/cyclin A
inhibition, since an imidazolidin-2,4-dione (25) was a
potent inhibitor (IC50 ) 17 nM) while the regioisomer
imidazolidin-2,5-dione (26) was about 10 times less
active although there is no apparent structural reason
to explain this finding. Again, the stereoselective effect

3-Aminopyrazole Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2949
Table 5. Kinase Selectivity and Tumor Cell Antiproliferative Activity of Compounds 13 and 35
IC50, nM
CDK2/Aa
CDK2/Ea
CDK1/Ba
CDK5/p25a
CDK4/D1a
>10000 (n ) 4) GSK-3â: 732 (n ) 13) 744 ( 218 639 ( 176 1354 ( 326
>10000 (n ) 2) GSK-3â: 406 (n ) 6) 716 ( 134 751 ( 230 1129 ( 675
other kinases,a,b
A2780
HT-29
HCT116
13 37 (n ) 90) 55 (n ) 6) 208 (n ) 6) 65 (n ) 5)
35 20 (n ) 11) 37 (n ) 4) 243 (n ) 4) 57 (n ) 4)
a At least two independent experiments were performed for each compound in order to determine IC50 values. Potency is expressed as
the mean of IC50 values obtained by nonlinear least-squares regression fitting of the data. The coefficient of variation of the mean ranges
from 10% to 24%. b From a kinase panel of 30 serine-threonine and tyrosine kinases as previously described.3
Figure 3. HT-29 cells were synchronized in G2\M by a nocodazole treatment of 12 h at 75 ng/mL. G2\M blocked HT-29 cells
were recovered by shake-off and reseeded in the presence or absence of compounds at 1 and 3 µM. Flavopiridol at 0.6 µM was
chosen as a positive reference. HT-29 cells were collected 24 h after the release from the nocodazole block and analyzed by FACS
for the cell cycle analysis. Cell lysates obtained from the same cells were used to follow the status of pRb phosphorylation. The
data reported in the picture indicate the ability of both 13 and 24 (as well as flavopiridol) to induce an accumulation of the
hyposphosphorylated forms of pRb compared to the control and nocodazole blocked cells.
Table 6. Percentage of BrdU Incorporation in Treated and
potency of the compounds still remain difficult to make
Untreated HT-29 Cells
because groups are pointing toward the solvent acces-
35
35
13
13
sible region and a high degree of conformational motion
is possible both for the protein and the inhibitor. (2)
Physicochemical properties such as solubility and PPB
can be definitely improved on converting a lipophilic
naphthyl into a suitable heteroaryl moiety.
control flavopiridol (1 µM) (3 µM) (1 µM) (1 µM)
% Brdu 40
positive
cells
1
65
0.7
25
4
At the end of this optimization process two viable
compounds (13 and 35) were further evaluated in order
to choose a suitable compound to be tested in vivo.
Both compounds were tested on a panel of tumor cell
lines including an ovarian carcinoma cell line (A2780)
and two colon carcinoma cell lines (HT-29 and HCT116).
Their activities (EC50) ranged between 0.6 and 1.3 µM
(Table 5).
Biological and Pharmacological Evaluation
By comparison of 13 and 35 across a panel of kinases,
a slightly different kinase profile for the two compounds
was observed (Table 5). Both products exhibited a
double-digit nanomolar inhibition of CDK2/cyclin A,
CDK2/cyclin E, and CDK5/p25. CDK1/cyclin B was
inhibited at low-micromolar concentrations, while either
13 or 35 were inactive on CDK4/cyclin D1 below 10 µM
concentrations. Of a panel of 30 kinases only GSK-3â
was inhibited by either of the two lead compounds;
compound 13 showed an IC50 of 0.73 µM and 35 of 0.41
µM. On the basis of this selectivity profile, we would
not expect a dramatically different outcome for the two
leads in vivo.
Cell Cycle Analysis and CDK Substrate
Phosphorylation
Confirmation that these compounds were inhibiting
CDK2 enzyme activity in cells was obtained through
examination of the phosphorylation status of a CDK
substrate (pRb) by Western blotting (WB), cell cycle
profile, and DNA replication (analyzing the incorpora-
tion of BrdU by immunofluorescence).
More specifically, the cell cycle profile and phospho-
rylation status of pRb were analyzed on HT-29 colon
carcinoma cell line released from a nocodazole block in

2950 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Pevarello et al.
Table 7. In Vitro and in Vivo ADME Parameters for Compounds 13 and 35
in vitro ADME parameters3
in vivo ADME parameters (CD-1 mice)a
solubility
(µM)
Caco-2 cell
permeability
CYP4503A4
(% remaining)
PPB
(%)
clearance
t1/2
(min)
Vss
Fos
(%)
(mL min-1 kg-1
)
(mL kg-1
)
13
35
239
high
96
74
5.8
0.8 (iv)
3.7 (os)
2.8 (iv)
3.1 (os)
280
100
225
moderate
92
73
4.7
573
63
a Pharmacokinetic analysis was performed as previously reported.3
Figure 4. 13 shows 73% tumor growth inhibition (TGI) against a human ovarian cancer model (A2780) transplanted into nude
mice when administered at 7.5 mg/kg twice a day for 20 consecutive days.
the presence or absence of compounds at the indicated
concentrations (Figure 3). It was evident that under
these conditions, at a dose of 3 µM, both compounds
blocked the cells in G0\G1, hampering the progression
of cells in the S phase as highlighted by both the cell
cycle profile and BrdU incorporation data (Table 6).
Regarding the phosphorylation status of pRb, it was
possible to note a clear reduction of the hyperphospho-
rylated form of pRb in the samples obtained by cells
treated with the compounds versus the samples of cells
treated with the vehicle, indicative of an effect on the
activity of CDK2. All together, these data confirmed that
13 and 35 were hitting the target in the cells where they
displayed an antiproliferative effect.
13 a Cmax of 21 µM and an AUC of 96 µM/h were
attained.
In Vivo Antitumor Activity
On the basis of the data presented above, 13 was
prioritized for further in vivo characterization in a
mouse xenograft model to assess its preclinical antitu-
mor activity.
The human ovarian A2780 xenograft mouse model
was used (see Experimental Section), and on the basis
of the plasma levels reached in the preliminary in vivo
PK study, we selected a dose of 7.5 mg/kg, oral, twice a
day. Figure 4 reports the results from this study. At this
dose and scheduling, a 73% tumor growth inhibition
(TGI) was obtained with a maximal body weight loss of
7% and without any signs of toxicity.
Pharmacokinetics and Metabolism in Vitro and
in Vivo
Study of the Molecular Mechanism of Action of
13 in the Tumors
On the right-hand side of Table 7 some in vitro
parameters that we used to prioritize compounds for in
vivo testing are reported. The increase in solubility for
both compounds was about 10-fold over that of the
previous lead PNU-292137. Caco-2 permeability assay
was more favorable for 13 (high permeability) than for
35 (moderate permeability). The plasma protein binding
(PPB) in a high-throughput assay was nearly the same
for the two compounds. In the left part of Table 7 some
in vivo pharmacokinetic parameters are shown (CD1
mice). Both 13 and 35 were characterized by a low
clearance together with a moderate volume of distribu-
tion (Vss). The terminal half-life after oral administra-
tion was comparable for 35 and 13. Although the
terminal half-life in mice was shorter than optimal, it
turned out to be sufficiently long after oral administra-
tion of 13 to elicit an in vivo response (vide infra). Oral
bioavailability (Fos%) was complete for 13 and lower but
still satisfactory for 35. After an oral dose (10 mg/kg) of
The in vivo efficacy result in the A2780 model boosted
an interest in 13 as a candidate for further preclinical
study, but first, we tried to establish a relationship
between the antitumor activity observed in vivo in the
xenograft model and markers of CDK2 inhibition in
tumor sections taken from treated animals.
BrdU incorporation and phosphorylation of pRb on
Ser 795 (this site being phospho-specific for CDK2 and
CDK4) were chosen as markers of CDK2 inhibition.
Sections of A2780 xenograft tumors were collected after
6 days of treatment at 7.5 mg/kg, oral, twice a day, and
processed for the immunohistochemistry (IHC) analysis.
The IHC analysis of BrdU staining indicates a signifi-
cant reduction in the percentage of positive cells in the
tumor sections obtained from the treated group com-
pared to the section obtained from the vehicle group
(Figure 5A), thus demonstrating a clear effect on DNA
replication.

3-Aminopyrazole Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2951
Figure 5. Analysis by IHC of the BrdU incorporation (A) and pRb staining (B) of the sections of A2780 xenograft tumors at the
end of treatment. Tumor sections were fixed in formalin, paraffin-embedded, and stained for BrdU incorporation and phospho-
pRb expression. The analysis shows a significant reduction of both BrdU and phospo-pRb positive cells in the treated vs control
tumors. Representative examples of the stainings are also reported.
directly connected with micro HPLC 1100 Agilent. Organic
solutions, where applicable, were dried over anhydrous Na2-
SO4 and evaporated using a Heidolph WB 2001 rotary evapo-
rator at 15-20 mmHg. Flash column chromatographic sepa-
rations were carried out on 40/60 µm silica gel (Carlo Erba).
Thin-layer chromatography was performed on Whatman silica
gel 60 plates coated with 250 µm layer with fluorescent
indicator. Components were visualized by UV light (λ ) 254
nm) and by iodine vapor. Dichloromethane was distilled from
P2O5 and stored over 4 Å molecular sieves. All experiments
dealing with moisture-sensitive compounds were conducted
under dry nitrogen. Starting materials, unless otherwise
specified, were commercially available (Aldrich, Fluka) and of
the best grade and were used without further purification.
2-[4-(2-Oxopiperidin-1-yl)phenyl]propanoic Acid 14a.
To a solution of 5 g (30.3 mmol) of 39 in 2 mL of 37% HCl, 4.1
mL (45.5 mmol) of δ-valerolactone were added under stirring.
The resulting solution was heated at 150 °C overnight. The
reaction mixture was diluted with water and extracted several
times with dichloromethane. The organic layer was then dried
over Na2SO4 and evaporated to dryness. The residue was
finally purified by chromatography on a silica gel column by
eluting with a mixture CH2Cl2/EtOH 93/7, affording 2.6 g (35%
A comparative analysis of the phospho-Rb positive
cells between the tumor sections obtained from the
vehicle and the treated groups indicates a significant
reduction of 50% (Figure 5B), confirming that the tumor
growth inhibition obtained was related to the inhibition
of the target CDK2.
Conclusion
A pharmacological potent lead had been identified at
a previous stage of lead finding.3 The lead compound
PNU-292137 was systematically modified in the part
of molecule pointing toward the solvent-accessible re-
gion of the CDK2 ATP pocket in order to improve
druglike properties while maintaining good activity in
models predictive of antitumor efficacy. By introducing
a methyl group in the S-configuration on the benzylic
position of PNU-292137 and by changing the naphthyl
moiety into a 4-lactam-1-ylphenyl group, we obtained
13, a candidate for further preclinical investigation with
an improved solubility and lower plasma protein bind-
ing. At a dose of 7.5 mg/kg twice a day for 20 days, 13
was endowed with a tumor growth inhibition (TGI) of
more than 70% without significant toxicity in a human
ovarian A2780 xenograft model in nude mice. A rela-
tionship between the antitumor activity observed in vivo
in the xenograft model and markers of CDK2 inhibition
in tumor sections taken from treated animals was found.
1
yield) of 14a. H NMR (DMSO-d6) δ 1.4 (d, J ) 7.0 Hz, 3 H),
1.8 (m, 4 H), 2.4 (t, J ) 6.4 Hz, 2 H), 3.4 (t, J ) 5.7 Hz, 2 H),
3.9 (m, 1 H), 7.2 (d, J ) 8.5 Hz, 2 H), 7.3 (d, J ) 8.4 Hz, 2 H);
MS (ESI+) m/z 248 (MH+).
The following intermediates were analogously obtained.
2-[4-(2-Methyl-5-oxopyrrolidin-1-yl)phenyl]propa-
1
noic Acid 16a. Yield 41%; H NMR (DMSO-d6) δ 1.0 (d, J )
6.1 Hz, 3 H), 1.3 (d, J ) 7.1 Hz, 3 H), 1.7 (m, 1 H), 2.4 (m, 3
H), 3.8 (m, 1 H), 4.3 (m, 1 H), 7.2 (s, 4 H); MS (ESI+) m/z 265
(MNH4+), 248 (MH+).
Experimental Section
2-[4-(2-Oxo-5-phenylpyrrolidin-1-yl)phenyl]propa-
1
noic Acid 17a. Yield 33%; H NMR (DMSO-d6) δ 1.3 (d, J )
Chemistry. Melting points were determined in open glass
capillaries with a Buchi 535 melting point apparatus and are
uncorrected. Elemental analyses were performed on a Carlo
Erba 1110 instrument, and C, H, and N results were within
7.2 Hz, 3 H), 1.8 (m, 1 H), 2.6 (m, 3 H), 3.5 (q, J ) 7.1 Hz, 1
H), 5.4 (dd, J ) 6.8, 5.2 Hz, 1 H), 7.1 (m, 2 H), 7.2 (m, 1 H),
7.2 (s, 2 H), 7.3 (m, 2 H), 7.4 (d, J ) 8.7 Hz, 2 H), 12.2 (s, 1 H);
MS (ESI+) m/z 327 (MNH4+), 310 (MH+).
1
(0.4% of theoretical values. H NMR spectra were recorded
on a Varian Mercury 400 spectrometer, using the solvent as
internal standard. Chemical shifts are expressed in ppm (δ).
Electron impact (EI) mass spectra (MS) were obtained on a
Finnigan-MAT TSQ 700 triple quadrupole instrument. (ESI)
mass spectra were obtained on an LCQ ion trap thermo
Finnigan.9 HPLC-MS spectra were obtained combining the
MS instrument with HPLC system SSP4000 (Thermo Separa-
tion) with an autosampler Lc Pal (CTC Analytics) and
UV6000LP PDA detector (UV detection 215-400 nm). Exact
mass data ESI(+) were obtained on Micromass Q-Tof Ultima
2-[4-(2,5-Dioxopyrrolidin-1-yl)phenyl]propanoic Acid
21a. A 825 mg sample of 39 (5 mmol) and 600 mg (6 mmol) of
succinic anhydride were dissolved in 30 mL of xylene and 1
mL of triethylamine. The mixture was heated at 150 °C for 3
h under vigorous stirring. After evaporation of the solvent, the
crude was chromatographed on a silica gel column by eluting
with EtOAc to give 956 mg (77% yield) of 21a. 1H NMR
(DMSO-d6) δ 1.4 (d, J ) 7.2 Hz, 3 H), 2.8 (s, 4 H), 3.7 (q, J )
7.2 Hz, 1 H), 7.2 (d, J ) 8.5 Hz, 2 H), 7.4 (d, J ) 8.3 Hz, 2 H),
12.3 (s, 1 H); MS (ESI+) m/z 265 (MNH4+), 248 (MH+).

2952 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Pevarello et al.
2-(4-{[(2-Ethoxy-2-oxoethylamino)carbonyl]amino}-
phenyl)propanoic Acid 40. A 4 g (0.02 mol) sample of 39
was dissolved in 100 mL of THF. After the mixture was cooled
to 0 °C, 2.96 mL of ethyl isocyanatoacetate (0.026 mol) were
added. The mixture was stirred at room temperature for 24
h. The solvent was then evaporated, and the crude was
redissolved in water. The pH was then adjusted to 4 by adding
aqueous KHSO4. The crude product was extracted with ethyl
acetate and finally crystallized from a mixture AcOEt/acetone,
Methyl 2-[4-(2,4-Dioxoimidazolidin-1-yl)phenyl]pro-
panoate 45. A 1 g (5.6 mmol) sample of 42 and 0.5 mL (5.6
mmol) of 2-chloroacetylisocyanate were dissolved in 60 mL of
dioxane. After 3 h at room temperature 1.8 mL (11.2 mmol) of
DBU were added together with an additional 40 mL of dioxane.
The resulting solution was stirred overnight at room temper-
ature. The solvent was then removed under vacuum, and the
residue was redissolved in CH2Cl2 and washed with diluted
HCl. The organic phase was dried over Na2SO4, concentrated,
and chromatographed on a silica gel column by eluting with a
mixture CH2Cl2/MeOH 97/3, affording 367 mg (25% yield) of
45. 1H NMR (DMSO-d6) δ 1.4 (d, J ) 7.1 Hz, 3 H), 3.6 (s, 3 H),
3.8 (q, J ) 7.1 Hz, 1 H), 4.4 (s, 2 H), 7.2 (d, J ) 8.5 Hz, 2 H),
7.5 (d, J ) 8.8 Hz, 2 H), 11.1 (s, 1 H); MS (ESI-) m/z 261 (M
- H).
1
giving 3.5 g (60% yield) of 40. H NMR (DMSO-d6) δ 1.2 (t, J
) 7.1 Hz, 3 H), 1.3 (d, J ) 7.2 Hz, 3 H), 3.5 (q, J ) 7.1 Hz, 1
H), 3.8 (d, J ) 5.9 Hz, 2 H), 4.1 (q, J ) 7.1 Hz, 2 H), 6.4 (t, J
) 6.0 Hz, 1 H), 7.1 (d, J ) 8.7 Hz, 2 H), 7.3 (d, J ) 8.5 Hz, 2
H), 8.7 (s, 1 H), 12.1 (s, 1 H); MS (ESI+) m/z 312 (MNH4+),
295 (MH+).
2-[4-(2,5-Dioxoimidazolidin-1-yl)phenyl]propanoic Acid
26a. To a solution of 500 mg (1.7 mmol) of 40 in 20 mL of
acetone, 20 mL of 6 N HCl were added. The resulting solution
was refluxed for 2 h, and the solvent was removed under
reduced pressure. The residue was taken up with CH2Cl2 and
washed with water. The organic phase was dried over Na2-
SO4 and evaporated, and 350 mg (84% yield) of 26a were
obtained by crystallization from diethyl ether. 1H NMR
(DMSO-d6) δ 1.4 (d, J ) 7.1 Hz, 3 H), 3.7 (q, J ) 7.1 Hz, 1 H),
4.0 (s, 2 H), 7.3 (d, J ) 8.5 Hz, 2 H), 7.4 (m, 2 H), 8.2 (s, 1 H),
12.3 (s, 1 H); MS (ESI+) m/z 266 (MNH4+), 249 (MH+).
2-[4-(2,4-Dioxoimidazolidin-1-yl)phenyl]propanoic Acid
25a. A 330 mg (1.26 mmol) sample of 45 was dissolved in 15
mL of MeOH and 2 mL (4 mmol) of 2 N NaOH. The resulting
solution was refluxed for 2 h and cooled to room temperature,
and 2 mL of 2 N HCl were added. The solvent was removed
under vacuum, and the residue was redissolved in AcOEt and
washed with water. The organic layer was dried with Na2SO4
and evaporated to dryness, affording 300 mg (96% yield) of
1
25a. H NMR (DMSO-d6) δ 1.35 (d, J ) 7.11 Hz, 3 H), 3.79
(m, 1 H), 4.42 (s, 2 H), 7.33 (d, J ) 8.80 Hz, 2 H), 7.49 (d, J )
8.65, 2 H), 11.15 (s, 1 H); MS (ESI+) m/z 249 (MH+).
(2S)-2-[4-(1-Pyrrolidinyl)phenyl]propanoic Acid 38a.
A 16.5 g (0.1 mol) sample of 41 was dissolved in 450 mL of
N,N-dimethylformamide, and 15.3 mL (0.13 mol) of 1,4-
dibromobutane and 69.6 mL (0.4 mol) of N,N-diisopropylethyl-
amine were added. The mixture was heated under stirring at
90 °C for 2 h. The solvent was then evaporated under vacuum,
the residue was redissolved in water, and the resulting solution
was extracted several times with AcOEt. The organic phase
was then dried over anhydrous sodium sulfate and concen-
trated. The crude was finally purified on a Florisil (200-300
mesh) column by using n-hexane/AcOEt 7/3 as eluant, leading
to 13.2 g (60% yield) of 38a. 1H NMR (DMSO-d6) δ 1.3 (d, J )
7.1 Hz, 3H), 1.9 (m, 4 H), 3.2 (m, 4 H), 3.5 (q, J ) 7.1 Hz, 1 H),
6.5 (d, J ) 8.8 Hz, 2 H), 7.0 (d, J ) 8.4 Hz, 2 H), 12.0 (s, 1 H);
MS (ESI+) m/z 220 (MH+).
Methyl 2-[4-(2-Oxo-2,3-dihydro-1H-imidazol-1-yl)phe-
nyl]propanoate 48. A 1 g (5.58 mmol) sample of 42 was
dissolved in 10 mL of CH2Cl2, and 0.9 mL of triethylamine
was added in a nitrogen atmosphere. The solution was cooled
to 0 °C, and 1.78 g (5.58 mmol) of triphosgene were cautiously
added portionwise. The reaction mixture was then stirred at
room temperature for 6 h. After this time the solution was
rapidly washed with ice/water, dried over Na2SO4, and evapo-
rated to dryness, giving 46, which was employed for the next
step without any purification. The crude product was dissolved
in 10 mL of CH2Cl2, and 0.69 mL (5.58 mmol) of aminoacetal-
dehyde dimethylacetal was added. The reaction mixture was
stirred at room temperature for 5 h, washed with aqueous
NaHCO3, dried over Na2SO4, and evaporated in vacuo, afford-
ing 47 as a crude intermediate. Compound 47 was then
dissolved in 5 mL of MeCN, 2 mL of water, and 2 mL of
trifluoroacetic acid. The mixture was stirred at room temper-
ature for 6 h. The solvent was finally removed under reduced
pressure, and the residue was diluted with CH2Cl2 and washed
with aqueous NaHCO3. The organic phase was dried over Na2-
SO4 and concentrated. The crude was purified by chromatog-
raphy on a silica gel column (cyclohexane/AcOEt 4/1), affording
1-[4-(2-Methoxy-1-methyl-2-oxoethyl)phenyl]-5-oxo-
proline 43. A 3 g (16.74 mmol) sample of 42 was dissolved in
60 mL of toluene, and 2.85 g (16.74 mmol) of 6,6-dimethyl-
5,7-dioxaspiro[2.5]octane-4,8-dione were added. The mixture
was heated at 60 °C for 12 h under stirring. After cooling to
room temperature, the precipitated product was collected by
filtration and washed with a mixture of toluene and cyclohex-
1
1
411 mg (30% overall yield) of 48. H NMR (DMSO-d6) δ 1.3
ane (3.9 g, 80% yield). H NMR (DMSO-d6) δ 1.33 (d, J ) 7.0
(d, J ) 7.2 Hz, 3 H), 3.6 (s, 3H), 3.8 (q, J ) 7.2 Hz, 1 H), 6.6
(dd, J ) 3.0, 2.5 Hz, 1 H), 6.9 (dd, J ) 3.1, 2.2 Hz, 1 H), 7.3 (d,
J ) 8.5 Hz, 2 H), 7.6 (d, J ) 8.7 Hz, 2 H), 10.2 (s, 1 H); MS
(ESI+) m/z 247 (MH+).
Hz, 3 H), 2.25 (m, 2 H), 3.46 (dd, J ) 9.1, 7.9 Hz, 1 H), 3.60 (s,
3H), 3.78 (m, 3H), 7.33 (d, J ) 8.6 Hz, 2 H), 7.53 (d, J ) 8.3
Hz, 2 H); MS (ESI+) m/z 292 (MH+).
1-[4-(2-Methoxy-1-methyl-2-oxoethyl)phenyl]-5-oxo-
prolinamide 15a. A 3 g (10.3 mmol) sample of 43 was
dissolved in 100 mL of 1/1 DMF/THF mixture. The resulting
solution was cooled to 0 °C, and 3.94 g (20.6 mmol) of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide and 3.59 mL (20.6
mmol) of N-ethyl-N′,N′-diisopropylamine were added. After 30
min under stirring at the same temperature 3.13 g (20.6 mmol)
of N-hydroxybenzotriazole ammonium salt were added. The
mixture was then maintained at room temperature overnight,
the solvent was removed under reduced pressure, and the
residue was redissolved with CH2Cl2. After being washed with
water, the organic layer was dried over Na2SO4 and evaporated
to dryness, affording intermediate 44, which was submitted
to the next step without any further purification. The crude
was dissolved in 200 mL of methanol, and an aqueous solution
of 2.18 g (20.6 mmol) of Na2CO3 was added. After being stirred
overnight, the solution was made acidic with diluted HCl and
extracted with ethyl acetate, affording 1.42 g (50% yield
2-[4-(2-Oxo-2,3-dihydro-1H-imidazol-1-yl)phenyl]pro-
panoic Acid 27a. A 246 mg (1.0 mmol) sample of 48 was
dissolved in 10 mL of MeOH, and 1 mL of 2 N NaOH (2 mmol)
was added. After 6 h at room temperature MeOH was removed
and water was added to the residue. The solution was acidified
with 2 N HCl and extacted with a mixture CH2Cl2/MeOH 95/
5. The organic layer was dried over Na2SO4 and evaporated
1
in vacuo to give 200 mg (86% yield) of 27a. H NMR (DMSO-
d6) δ 1.4 (d, J ) 7.2 Hz, 3 H), 3.7 (q, J ) 7.2 Hz, 1 H), 6.6 (dd,
J ) 3.0, 2.5 Hz, 1 H), 6.9 (dd, J ) 3.1, 2.2 Hz, 1 H), 7.3 (d, J
) 8.5 Hz, 2 H), 7.6 (d, J ) 8.7 Hz, 2 H), 10.2 (s, 1 H), 12.3 (s,
1 H); MS (ESI+) m/z 250 (MNH4+), 233 (MH+).
Methyl 2-[4-(5-Oxo-1,5-dihydro-4H-1,2,4-triazol-4-yl)-
phenyl]propanoate 49. A 1 g (5.58 mmol) sample of 42 was
dissolved in 10 mL of methanol, and 0.7 mL (5.3 mmol) of
trimethylorthoformate together with 531 mg (5.3 mmol) of
methyl hydrazinocarboxylate and 20 mg (0.1 mmol) of p-
toluenesulfonic acid were added. The resulting solution was
stirred at 65 °C for 2 h, then 858 mg of sodium methoxide
dissolved in methanol were added. After the mixture was
stirred for 24 h at room temperature, the solvent was removed
1
overall) of 15a. H NMR (DMSO-d6) δ 1.34 (d, J ) 7.1 Hz, 3
H), 2.25 (m, 2 H), 3.41 (dd, J ) 9.2, 8.1 Hz, 1 H), 3.78 (m, 3H),
7.13 (s, 1 H), 7.30 (d, J ) 8.7 Hz, 2 H), 7.51 (m, 3 H); MS (ESI+)
m/z 277 (MH+).

3-Aminopyrazole Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2953
under vacuum and water was added to the residue. The
resulting solution was acidified with 23% HCl and extracted
with CH2Cl2. The organic layer was dried over Na2SO4 and
evaporated to afford 827 mg (60% overall yield) of 49. 1H NMR
(DMSO-d6) δ 1.4 (d, J ) 7.1 Hz, 3 H), 3.6 (s, 3 H), 3.9 (q, J )
7.1 Hz, 1 H), 7.4 (d, J ) 8.4 Hz, 2 H), 7.6 (d, J ) 8.5 Hz, 2 H),
8.3 (d, J ) 1.4 Hz, 1 H), 11.9 (s, 1 H); MS (ESI+) m/z 265
(MNH4+), 248 (MH+).
2-[4-(5-Oxo-1,5-dihydro-4H-1,2,4-triazol-4-yl)phenyl]-
propanoic Acid 28a. A solution of 800 mg (3.25 mmol) of 49
and 3.25 mL of 1 N NaOH in 20 mL of MeOH was stirred at
room temperature for 5 h. The solvent was then removed, and
the residue was diluted with water, acidified with 1 N HCl,
and extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and evaporated to afford 643 mg (85% yield) of 28a.
1H NMR (DMSO-d6) δ 1.4 (d, J ) 7.3 Hz, 3 H), 3.7 (q, J ) 7.1
Hz, 1 H), 7.4 (d, J ) 8.4 Hz, 2 H), 7.6 (d, J ) 8.4 Hz, 2 H), 8.3
(d, J ) 1.3 Hz, 1 H), 11.9 (s, 1 H), 12.3 (s, 1 H); MS (ESI+)
m/z 251 (MNH4+), 234 (MH+).
temperature overnight. After this time CH2Cl2 was added and
the solution was washed with 5% citric acid, aqueous NaHCO3,
and brine. The organic layer was dried over Na2SO4 and
evaporated to dryness.
The crude was dissolved in 16 mL of CH2Cl2 and 1.6 mL of
trifluoroacetic acid and stirred at room temperature for 3 h.
The solvent was then removed under reduced pressure, and
the residue was redissolved in CH2Cl2 and washed with
aqueous NaHCO3. The organic layer was dried over Na2SO4
and evaporated to dryness. The crude was purified by column
chromatography (CH2Cl2/MeOH 95/5), affording 110 mg (31%
overall yield) of 25. 1H NMR (DMSO-d6) δ 0.60 (m, 2 H), 0.86
(m, 2 H), 1.33 (d, J ) 7.09 Hz, 3 H), 1.80 (m, 1 H), 3.79 (m, 1
H), 4.39 (s, 2 H), 6.10 (s, 1 H), 7.32 (d, J ) 8.79 Hz, 2 H), 7.48
(m, 2 H), 10.30 (s, 1 H), 11.11 (s, 1 H), 11.96 (s, 1 H); MS (ESI+)
m/z 354 (MH+); HRMS (ESI+) calcd for C18H19N5O3 + H
354.1561, found 354.1558. HPLC purity (as area %): 100.
By using the suitable carboxylic acid, the following com-
pounds were analogously prepared.
2-[4-(1,1-Dioxidoisothiazolidin-2-yl)phenyl]propa-
noic Acid 36a. To a solution of 1 g (5.58 mmol) of 42 in 10
mL of CH2Cl2 and 1.5 mL (10 4 mmol) of Et3N, 0.9 mL (7.4
mmol) of 3-chloropropane-1-sulfonyl chloride was added. The
mixture was stirred overnight at room temperature, washed
with 1 N HCl, and evaporated to dryness. The resulting crude
50 was dissolved in 8 mL of DMF, and 1 mL (6.7 mmol) of
DBU was added. After being stirred for 3 h at room temper-
ature, the reaction mixture was poured into 250 mL of hexane/
AcOEt 1/1 and washed with 1 N HCl and brine. The organic
layer was dried over Na2SO4 and evaporated to give 1.2 g of
crude 51, which was employed without any further purifica-
tion. Compound 51 was dissolved in 30 mL of MeOH, and 10
mL of saturated aqueous Na2CO3 were added. After the
mixture was stirred at room temperature for 16 h, MeOH was
removed in vacuo and the residue was diluted with water and
washed with AcOEt. The aqueous layer was then acidified with
1 N HCl and extracted with AcOEt. The organic phase was
dried over Na2SO4 and evaporated to afford 900 mg (60%
overall yield) of 36a. 1H NMR (DMSO-d6) δ 1.3 (d, J ) 7.2 Hz,
3 H), 2.4 (m, 2 H), 3.5 (t, J ) 7.5 Hz, 2 H), 3.6 (q, J ) 7.1 Hz,
1 H), 3.7 (t, J ) 6.5 Hz, 2 H), 7.1 (d, J ) 8.7 Hz, 2 H), 7.3 (d,
J ) 8.7 Hz, 2 H), 12.2 (s, 1 H); MS (ESI+) m/z 287 (MNH4+),
270 (MH+), 178.
2-[4-(3-Benzyl-2-oxoimidazolidin-1-yl)phenyl]propa-
noic Acid 29a. To a solution of 248 mg (1 mmol) of 52 in 5
mL of dry DMF cooled to 0 °C, 48 mg (1.2 mmol) of NaH 60%
in mineral oil were added. After 30 min, the reaction mixture
was treated with 0.13 mL (1.1 mmol) of benzylbromide, and
the temperature was allowed to reach room temperature. After
2 h under stirring the mixture was poured into ice/water,
acidified with 2 N HCl, and extracted with AcOEt/hexane 1/1.
The organic layer was dried over Na2SO4 and evaporated
under vacuum, giving crude 53 that, without any further
purification, was dissolved in 10 mL of THF/water 1/1. The
resulting solution was treated with 40 mg (1.58 mmol) of LiOH
and stirred for 3 h at room temperature. The reaction mixture
was diluted with AcOEt and water. The aqueous phase was
acidified with 2 N HCl and extracted with CH2Cl2. The organic
phase was then dried over Na2SO4 and concentrated under
reduced pressure to afford 180 mg (55% overall yield) of 29a.
1H NMR (DMSO-d6) δ 1.3 (d, J ) 7.2 Hz, 3 H), 3.3 (m, 2 H),
3.6 (q, J ) 7.1 Hz, 1 H), 3.8 (m, 2 H), 4.4 (s, 2 H), 7.2 (d, J )
8.7 Hz, 2 H), 7.3 (m, 1 H), 7.3 (m, 2 H), 7.3 (m, 2 H), 7.5 (d, J
) 8.8 Hz, 2 H), 12.2 (s, 1 H); MS (ESI+) m/z 342 (MNH4+),
325 (MH+).
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-methoxy-2-phenyl-
acetamide 1. Yield 65%; 1H NMR (DMSO-d6) δ 0.60 (m, 2
H), 0.86 (ddd, J ) 8.36, 6.46, 3.96 Hz, 2 H), 1.80 (tt, J ) 8.44,
5.06 Hz, 1 H), 3.28 (s, 3 H), 4.82 (s, 1 H), 6.07 (s, 1 H), 7.35
(m, 5 H), 10.20 (s, 1 H), 12.10 (s, 1 H); MS (ESI+) m/z 272
(MH+), 240, 212; HRMS (ESI+) calcd for C15H17N3O + H
272.1393, found 272.1396. HPLC purity (as area %): 100.
(2R)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-methoxy-2-
1
phenylacetamide 2. Yield 60%; H NMR (DMSO-d6) δ 0.61
(m, 2 H), 0.86 (m, 2 H), 1.81 (m, 1 H), 3.28 (s, 3 H), 4.82 (s, 1
H), 6.08 (s, 1 H), 7.38 (m, 5 H), 10.23 (s, 1 H); MS (ESI+) m/z
272 (MH+), 240; HRMS (ESI+) calcd for C15H17N3O2 + H
272.1393, found 272.1405. HPLC purity (as area %): 95.
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-methoxy-2-
1
phenylacetamide 3. Yield 66%; H NMR (DMSO-d6) δ 0.61
(m, 2 H), 0.86 (m, 2 H), 1.81 (m, 1 H), 3.28 (s, 3 H), 4.83 (s, 1
H), 6.08 (s, 1 H), 7.38 (m, 5 H), 10.23 (s, 1 H); MS (ESI+) m/z
272 (MH+), 240; HRMS (ESI+) calcd for C15H17N3O2 + H
272.1393, found 272.1393. HPLC purity (as area %): 95.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-fluoro-2-phenyl-
acetamide 4. Yield 71%; 1H NMR (DMSO-d6) δ 0.6 (m, 2 H),
0.9 (m, 2 H), 1.8 (m, 1 H), 6.0 (d, J ) 47.4 Hz, 1 H), 6.1 (s, 1
H), 7.4 (m, 3 H), 7.5 (m, 2 H), 10.7 (s, 1 H), 12.1 (s, 1 H); MS
(ESI+) m/z 260 (MH+), 240, 212; HRMS (ESI+) calcd for
C14H14FN3O + H 260.1194, found 260.1190. HPLC purity (as
area %): 95.
(2S)-2-Amino-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-
phenylacetamide 5. Yield 48%; 1H NMR (DMSO-d6) δ
0.63 (m, 2 H), 0.89 (m, 2 H), 1.83 (m, 1 H), 5.00 (m, 1 H), 6.15
(s, 1 H), 7.70 (m, 5 H), 8.68 (s, 2 H), 10.95 (s, 1 H); MS (ESI+)
m/z 257 (MH+), 240; HRMS (ESI+) calcd for C14H16N4O + H
257.1397, found 257.1401. HPLC purity (as area %): 95.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2,2-diphenylacet-
1
amide 6. Yield 70%; H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9
(m, 2 H), 1.8 (ddd, J ) 13.5, 8.5, 5.1 Hz, 1 H), 5.2 (s, 1 H), 6.2
(s, 1 H), 7.2 (m, 2 H), 7.3 (m, 8 H), 10.7 (s, 1 H), 12.0 (s, 1 H);
MS (EI) m/z 317 (M+); HRMS (ESI+) calcd for C20H19N3O +
H 318.1601, found 318.1605. Anal. (C20H19N3O) C, H, N. HPLC
purity (as area %): 95.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-oxo-2-phenylacet-
1
amide 7. Yield 66%; H NMR (DMSO-d6) δ 0.67-0.72 (m, 2
H), 0.90-0.97 (m, 2 H), 1.86-1.94 (m, 1 H), 6.27 (s, 1 H), 7.59
(t, J ) 7.76 Hz, 2 H), 7.74 (t, J ) 7.46 Hz, 1 H), 7.94 (d, J )
7.46 Hz, 2 H), 11.14 (s, 1 H), 12.28 (s, 1 H); MS (ESI) m/z 256
(MH+); HRMS (ESI+) calcd for C14H13N3O2 + H 256.1080,
found 256.1086. HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2,4-dioxoimi-
dazolidin-1-yl)phenyl]propanamide 25. A 300 mg (1.21
mmol) sample of 25a was dissolved in 20 mL of CH2Cl2 and
12 mL of DMF, 460 mg (2.40 mmol) of 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide were added, and the resulting
solution was cooled to 0°C. After the mixture was stirred for
1 h at the same temperature, 225 mg (1 mmol) of tert-butyl
3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylate in 4 mL of
CH2Cl2 were added. The mixture was allowed to reach room
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-phenylpropan-
amide 8. Yield 75%; 1H NMR (DMSO-d6) δ 0.59 (m, 2 H), 0.86
(m, 2 H), 1.34 (d, J ) 7.04 Hz, 3 H), 1.80 (ddd, J ) 13.50, 8.51,
4.99 Hz, 1 H), 3.81 (q, J ) 7.04 Hz, 1 H), 6.11 (s, 1 H), 7.19 (t,
J ) 7.04 Hz, 1 H), 7.28 (t, J ) 7.48 Hz, 2 H), 7.33 (m, 2 H),
10.31 (s, 1 H), 11.98 (s, 1 H); MS (ESI+) m/z 256 (MH+), 124;
HRMS (ESI+) calcd for C15H17N3O + H 256.1444, found
256.1443. HPLC purity (as area %): 100.

2954 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
(2R)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-phenylpro-
Pevarello et al.
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.8 (m, 2 H), 1.2 (d, J ) 7.0
Hz, 3 H), 1.8 (m, 2 H), 2.6 (m, 3 H), 3.7 (m, 1 H), 5.4 (dd, J )
7.0, 5.4 Hz, 1 H), 6.1 (s, 1 H), 7.2 (m, 7 H), 7.3 (d, J ) 8.7 Hz,
2 H), 10.2 (m, 1 H), 11.9 (s, 1 H); MS (ESI+) m/z 415 (MH+);
HRMS (ESI+) calcd for C25H26N4O2 + H 415.2128, found
415.2144. HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(1-oxo-1,3-dihy-
dro-2H-isoindol-2-yl)phenyl]propanamide 18. Yield 50%;
1H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.4 (d, J )
7.1 Hz, 3 H), 1.7 (m, 1 H), 3.8 (m, 1 H), 5.0 (s, 2 H), 6.1 (s, 1
H), 7.4 (d, J ) 8.7 Hz, 2 H), 7.5 (m, 1 H), 7.6 (m, 2 H), 7.8 (d,
J ) 7.5 Hz, 1 H), 7.8 (d, J ) 8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0
(s, 1 H); MS (ESI+) m/z 387 (MH+), 236; HRMS (ESI+) calcd
for C23H22N4O2 + H 387.1815, found 387.1811. HPLC purity
(as area %): 95.
1
panamide 9. Yield 72%; H NMR (DMSO-d6) δ 0.6 (m, 2 H),
0.9 (m, 2 H), 1.4 (d, J ) 7.0 Hz, 3 H), 1.8 (m, J ) 8.5, 8.5, 5.1,
5.0 Hz, 1 H), 3.8 (q, J ) 7.0 Hz, 1 H), 6.1 (s, 1 H), 7.2 (m, 1 H),
7.3 (m, 2 H), 7.4 (m, 2 H), 10.4 (s, 1 H), 12.0 (s, 1 H); MS (ESI+)
m/z 256 (MH+); HRMS (ESI+) calcd for C15H17N3O + H
256.1444, found 256.1438. Anal. (C15H17N3O) C, H, N. HPLC
purity (as area %): 100.
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-phenylpro-
panamide 10. Yield 78%; 1H NMR (DMSO-d6) δ 0.6 (m, 2 H),
0.9 (m, 2 H), 1.4 (d, J ) 7.1 Hz, 3 H), 1.8 (m, 1 H), 3.8 (q, J )
7.0 Hz, 1 H), 6.1 (s, 1 H), 7.2 (tt, 1 H), 7.3 (m, 2 H), 7.4 (m, 2
H), 10.4 (s, 1 H), 12.0 (s, 1 H); MS (ESI+) m/z 256 (MH+);
HRMS (ESI+) calcd for C15H17N3O2 + H 256.1444, found
256.1432. HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxopyrroli-
din-1-yl)phenyl]propanamide 11. Yield 74%; 1H NMR
(DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0 Hz, 3
H), 1.8 (m, 1 H), 2.0 (m, 2 H), 2.4 (t, J ) 8.0 Hz, 2 H), 3.8 (m,
1 H), 3.8 (t, J ) 7.1 Hz, 2 H), 6.1 (s, 1 H), 7.3 (d, J ) 8.7 Hz,
2 H), 7.5 (d, J ) 8.7 Hz, 2 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS
(ESI+) m/z 339 (MH+); HRMS (ESI+) calcd for C19H22N4O2 +
H 339.1815, found 339.1827. Anal. (C19H22N4O2) C (calcd 67.44,
found 66.80), H, N. HPLC purity (as area %): 100.
(2R)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(1-oxo-1,3-
dihydro-2H-isoindol-2-yl)phenyl]propanamide 19. Yield
1
49%; H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.4 (d,
J ) 7.1 Hz, 3 H), 1.7 (m, 1 H), 3.8 (m, 1 H), 5.0 (s, 2 H), 6.1 (s,
1 H), 7.4 (d, J ) 8.7 Hz, 2 H), 7.5 (m, 1 H), 7.7 (m, 2 H), 7.8 (d,
J ) 7.6 Hz, 1 H), 7.8 (d, J ) 8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0
(s, 1 H); MS (ESI+) m/z 387 (MH+), 236; HRMS (ESI+) calcd
for C23H22N4O2 + H 387.1815, found 387.1818. HPLC purity
(as area %): 95.
(2R)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-
pyrrolidin-1-yl)phenyl]propanamide 12. Yield 73%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.1
Hz, 3 H), 1.8 (m, 1 H), 2.0 (m, 2 H), 2.4 (t, J ) 8.0 Hz, 2 H),
3.8 (m, 1 H), 3.8 (t, J ) 7.0 Hz, 2 H), 6.1 (s, 1 H), 7.3 (d, J )
8.7 Hz, 2 H), 7.5 (d, J ) 8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0 (s, 1
H); MS (ESI+) m/z 339 (MH+); HRMS (ESI+) calcd for
C19H22N4O2 + H 339.1815, found 339.1816. HPLC purity (as
area %): 100.
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(1-oxo-1,3-
dihydro-2H-isoindol-2-yl)phenyl]propanamide 20. Yield
1
52%; H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.4 (d,
J ) 7.1 Hz, 3 H), 1.8 (m, 1 H), 3.8 (m, 1 H), 5.0 (s, 2 H), 6.1 (s,
1 H), 7.4 (d, J ) 7.0 Hz, 2 H), 7.5 (m, 1 H), 7.7 (m, 2 H), 7.8 (d,
J ) 7.6 Hz, 1 H), 7.8 (d, J ) 8.7 Hz, 2 H), 10.3 (s, 1 H), 12.0
(s, 1 H); MS (ESI+) m/z 387 (MH+), 236; HRMS (ESI+) calcd
for C23H22N4O2 + H 387.1815, found 387.1819. HPLC purity
(as area %): 100.
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-
pyrrolidin-1-yl)phenyl]propanamide 13. Yield 76%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.1
Hz, 3 H), 1.8 (m, 1 H), 2.0 (m, 2 H), 2.4 (t, J ) 8.1 Hz, 2 H),
3.8 (m, 1 H), 3.8 (t, J ) 7.0 Hz, 2 H), 6.1 (s, 1 H), 7.3 (d, J )
8.7 Hz, 2 H), 7.5 (d, J ) 8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0 (s, 1
H); 13C NMR (DMSO-d6): 7.2 (CH-6), 8.1 (CH2-7, CH2-8), 17.9
(CH2-22), 18.8 (CH3-13), 32.7 (CH2-23), 44.8 (CH-12), 48.5
(CH2-21), 92.8 (CH-4), 119.8 (CH-16, CH-18), 127.9 (CH-15,
CH-19), 138.0 (C-17), 138.6 (C-14), 145.8 (C-3), 147.8 (C-5),
171.6 (C-10), 174.0 (C-24); MS (ESI+) m/z 339 (MH+); HRMS
(ESI+) calcd for C19H22N4O2 + H 339.1815, found 339.1804.
Anal. (C19H22N4O2) C, H, N. [R]D +115.0° (c 1.0, MeOH). HPLC
purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxopiperidin-
1-yl)phenyl]propanamide 14. Yield 67%; 1H NMR (DMSO-
d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0 Hz, 3 H), 1.8 (m,
5 H), 2.3 (t, J ) 6.3 Hz, 2 H), 3.5 (t, J ) 5.8 Hz, 2 H), 3.8 (m,
1 H), 6.1 (s, 1 H), 7.2 (d, J ) 8.5 Hz, 2 H), 7.3 (d, J ) 8.4 Hz,
2 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS (ESI+) m/z 353 (MH+),
202; HRMS (ESI+) calcd for C20H24N4O2 + H 353.1972, found
353.1969. HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2,5-dioxopyr-
rolidin-1-yl)phenyl]propanamide 21. Yield 63%; 1H NMR
(DMSO-d6) δ 0.61 (m, 2 H), 0.86 (ddd, J ) 8.50, 6.40, 3.87 Hz,
2 H), 1.37 (d, J ) 7.09 Hz, 3 H), 1.80 (tt, J ) 8.48, 5.04 Hz, 1
H), 2.75 (s, 4 H), 3.87 (q, J ) 7.00 Hz, 1 H), 6.11 (s, 1 H), 7.16
(d, J ) 8.53 Hz, 2 H), 7.43 (d, J ) 8.53 Hz, 2 H), 10.40 (s, 1
H), 11.98 (s, 1 H); MS (ESI+) m/z 353 (MH+); HRMS (ESI+)
calcd for C19H20N4O3 + H 353.1608, found 353.1614. HPLC
purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxoimidazo-
lidin-1-yl)phenyl]propanamide 22. Yield 65%; 1H NMR
(DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0 Hz, 3
H), 1.8 (m, 1 H), 3.4 (m, 2 H), 3.8 (m, 1 H), 3.8 (m, 2 H), 6.1 (s,
1 H), 6.8 (s, 1 H), 7.3 (d, J ) 8.8 Hz, 2 H), 7.4 (d, J ) 8.8 Hz,
2 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS (ESI+) m/z 340 (MH+);
HRMS (ESI+) calcd for C18H21N5O2 + H 340.1768, found
340.1762. HPLC purity (as area %): 100.
(2R)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxoimi-
dazolidin-1-yl)phenyl]propanamide 23. Yield 68%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0
Hz, 3 H), 1.8 (m, 1 H), 3.4 (m, 2 H), 3.8 (m, 1 H), 3.8 (m, 2 H),
6.1 (s, 1 H), 6.8 (s, 1 H), 7.3 (d, J ) 8.7 Hz, 2 H), 7.4 (d, J ) 8.7
Hz, 2 H), 10.3 (s, 1 H), 11.9 (s, 1 H); MS (ESI+) m/z 340 (MH+);
HRMS (ESI+) calcd for C18H21N5O2 + H 340.1768, found
340.1775. HPLC purity (as area %): 100.
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxoimi-
dazolidin-1-yl)phenyl]propanamide 24. Yield 69%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 6.8
Hz, 3 H), 1.8 (tt, J ) 8.4, 5.0 Hz, 1 H), 3.4 (m, 2 H), 3.8 (q, J
) 7.0 Hz, 1 H), 3.8 (m, 2 H), 6.1 (s, 1 H), 6.9 (s, 1 H), 7.3 (d, J
) 8.7 Hz, 2 H), 7.5 (d, J ) 8.7 Hz, 2 H), 10.3 (s, 1 H), 11.9 (s,
1 H); MS (ESI+) m/z 340 (MH+); HRMS (ESI+) calcd for
C18H21N5O2 + H 340.1768, found 340.1767. HPLC purity (as
area %): 100.
1-(4-{2-[(5-Cyclopropyl-1H-pyrazol-3-yl)amino]-1-meth-
yl-2-oxoethyl}phenyl)-2-oxopyrrolidine-3-carboxamide
1
15. Yield 52%; H NMR (DMSO-d6) δ 0.59 (m, 2 H), 0.86 (m,
2 H), 1.33 (d, J ) 6.96 Hz, 3 H), 1.79 (m, 1 H), 2.25 (m, 2 H),
3.46 (dd, J ) 9.12, 7.94 Hz, 1 H), 3.78 (m, 3 H), 6.10 (s, 1 H),
7.13 (s, 1 H), 7.33 (d, J ) 8.66 Hz, 2 H), 7.53 (m, 3 H), 10.31
(s, 1 H), 11.97 (s, 1 H); MS (ESI+) m/z 382 (MH+); HRMS
(ESI+) calcd for C20H23N5O3 + H 382.1874, found 382.1889.
HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-methyl-5-oxo-
pyrrolidin-1-yl)phenyl]propanamide 16. Yield 58%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.1 (d, J ) 6.2
Hz, 3 H), 1.3 (d, J ) 7.0 Hz, 3 H), 1.6 (m, 1 H), 1.8 (m, 1 H),
2.3 (m, 1 H), 2.4 (m, 1 H), 2.5 (m, 1 H), 3.8 (m, 1 H), 4.3 (m, 1
H), 6.1 (s, 1 H), 7.3 (s, 4 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS
(ESI+) m/z 353 (MH+), 202; HRMS (ESI+) calcd for C20H24N4O2
+ H 353.1972, found 353.1984. HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-phenyl-5-oxo-
pyrrolidin-1-yl)phenyl]propanamide 17. Yield 60%; 1H
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2,5-dioxoimi-
dazolidin-1-yl)phenyl]propanamide 26. Yield 42%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.4 (d, J ) 7.0
Hz, 3 H), 1.8 (m, 1 H), 3.9 (q, J ) 7.3 Hz, 1 H), 4.0 (d, J ) 1.1
Hz, 2 H), 6.1 (s, 1 H), 7.2 (d, J ) 8.5 Hz, 2 H), 7.4 (d, J ) 8.4
Hz, 2 H), 8.2 (s, 1 H), 10.4 (s, 1 H), 12.0 (s, 1 H); MS (ESI+)

3-Aminopyrazole Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2955
m/z 354 (MH+), 203; HRMS (ESI+) calcd for C18H19N5O3 + H
354.1561, found 354.1570. HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-2,3-dihy-
dro-1H-imidazol-1-yl)phenyl]propanamide 27. Yield 58%;
1H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J )
7.1 Hz, 3 H), 1.8 (tt, J ) 8.4, 5.0 Hz, 1 H), 3.8 (q, J ) 6.9 Hz,
1 H), 6.1 (s, 1 H), 6.5 (dd, J ) 3.0, 2.6 Hz, 1 H), 6.9 (dd, J )
3.0, 2.2 Hz, 1 H), 7.4 (d, J ) 8.5 Hz, 2 H), 7.6 (d, J ) 8.7 Hz,
2 H), 10.2 (s, 1 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS (ESI+) m/z
338 (MH+); HRMS (ESI+) calcd for C18H19N5O2 + H 338.1611,
found 338.1615. HPLC purity (as area %): 95.
7.5 (d, J ) 8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0 (s, 1 H); 13C NMR
(DMSO-d6) δ 7.2 (CH-23), 8.1 (CH2-24, CH2-25), 18.8 (CH3-
16), 44.7 (CH-13), 45.3 (CH2-3), 61.9 (CH2-2), 92.8 (CH-22),
118.5 (CH-7, CH-11), 128.1 (CH-8, CH-10), 137.6 (C-6, C-9),
145.8 (C-21), 147.8 (C-18), 155.4 (C-5), 171.6 (C-14); MS (ESI+)
m/z 341 (MH+); HRMS (ESI+) calcd for C18H20N4O3 + H
341.1608, found 341.1597. Anal. (C18H20N4O3) C, H, N. [R]D
+109.0° (c 1.0, MeOH). HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(1,1-dioxido-
isothiazolidin-2-yl)phenyl]propanamide 36. Yield 55%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.1
Hz, 3 H), 1.8 (m, 1 H), 2.4 (m, 2 H), 3.5 (m, 2 H), 3.7 (t, J ) 6.6
Hz, 2 H), 3.8 (q, J ) 7.0 Hz, 1 H), 6.1 (s, 1 H), 7.1 (d, J ) 8.7
Hz, 2 H), 7.3 (d, J ) 8.7 Hz, 2 H), 10.3 (s, 1 H), 12.0 (s, 1 H);
MS (ESI+) m/z 375 (MH+), 224; HRMS (ESI+) calcd for
C18H22N4O3S + H 375.1485, found 375.1488. HPLC purity (as
area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-(4-pyrrolidin-1-
ylphenyl)propanamide 37. Yield 30%; 1H NMR (DMSO-d6)
δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0 Hz, 3 H), 1.8 (m, 1
H), 1.9 (m, 4 H), 3.2 (m, 4 H), 3.7 (q, J ) 7.0 Hz, 1 H), 6.1 (s,
1 H), 6.4 (d, J ) 8.8 Hz, 2 H), 7.1 (d, J ) 8.7 Hz, 2 H), 10.1 (s,
1 H), 11.9 (s, 1 H); MS (ESI+) m/z 325 (MH+), 174; HRMS
(ESI+) calcd for C19H24N4O + H 325.2023, found 325.2029.
Anal. (C19H24N4O) C, H, N. HPLC purity (as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(5-oxo-1,5-dihy-
dro-4H-1,2,4-triazol-4-yl)phenyl]propanamide 28. Yield
1
34%; H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.4 (d,
J ) 7.0 Hz, 3 H), 1.8 (m, 1 H), 3.9 (q, J ) 7.1 Hz, 1 H), 6.1 (s,
1 H), 7.4 (d, J ) 8.7 Hz, 2 H), 7.6 (d, J ) 8.7 Hz, 2 H), 8.3 (d,
J ) 1.4 Hz, 1 H), 10.4 (s, 1 H), 11.9 (s, 1 H), 12.0 (s, 1 H); MS
(ESI+) m/z 339 (MH+); HRMS (ESI+) calcd for C17H18N6O2 +
H 339.1564, found 339.1571. HPLC purity (as area %): 100.
2-[4-(3-Benzyl-2-oxoimidazolidin-1-yl)phenyl]-N-(5-cy-
clopropyl-1H-pyrazol-3-yl)propanamide 29. Yield 56%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.1
Hz, 3 H), 1.8 (m, 1 H), 3.3 (m, 2 H), 3.8 (m, 3 H), 4.4 (s, 2 H),
6.1 (s, 1 H), 7.3 (m, 5 H), 7.3 (m, 2 H), 7.5 (m, 2 H), 10.3 (s, 1
H), 12.0 (s, 1 H); MS (ESI+) m/z 430 (MH+); HRMS (ESI+)
calcd for C25H27N5O2 + H 430.2237, found 430.2250. HPLC
purity (as area %): 100.
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-(4-pyrrolidin-
1
1-ylphenyl)propanamide 38. Yield 32%; H NMR (DMSO-
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(3-methyl-2-oxo-
imidazolidin-1-yl)phenyl]propanamide 30. Yield 60%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.1
Hz, 3 H), 1.8 (m, 1 H), 2.7 (s, 3 H), 3.4 (m, 2 H), 3.7 (dd, J )
9.3, 6.6 Hz, 2 H), 3.8 (m, 1 H), 6.1 (s, 1 H), 7.3 (d, J ) 8.7 Hz,
2 H), 7.5 (m, 2 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS (ESI+) m/z
354 (MH+); HRMS (ESI+) calcd for C19H23N5O2 + H 354.1924,
found 354.1920. HPLC purity (as area %): 100.
(2R)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(3-methyl-
2-oxoimidazolidin-1-yl)phenyl]propanamide 31. Yield 65%;
1H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J )
7.0 Hz, 3 H), 1.8 (tt, J ) 8.4, 5.0 Hz, 1 H), 2.7 (s, 3 H), 3.4 (m,
2 H), 3.7 (dd, 2 H), 3.8 (q, J ) 7.1 Hz, 1 H), 6.1 (s, 1 H), 7.3 (d,
J ) 8.8 Hz, 2 H), 7.5 (d, J ) 8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0
(s, 1 H); MS (ESI+) m/z 354 (MH+), 203; HRMS (ESI+) calcd
for C19H23N5O2 + H 354.1924, found 354.1921. HPLC purity
(as area %): 100.
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(3-methyl-
2-oxoimidazolidin-1-yl)phenyl]propanamide 32. Yield 58%;
1H NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J )
7.0 Hz, 3 H), 1.8 (tt, J ) 8.3, 5.2 Hz, 1 H), 2.7 (s, 3 H), 3.4 (m,
2 H), 3.7 (m, 2 H), 3.8 (q, J ) 7.0 Hz, 1 H), 6.1 (s, 1 H), 7.3 (d,
J ) 8.7 Hz, 2 H), 7.5 (d, J ) 8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0
(s, 1 H); MS (ESI+) m/z 354 (MH+), 203; HRMS (ESI+) calcd
for C19H23N5O2 + H 354.1924, found 354.1918. HPLC purity
(as area %): 100.
N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-ox-
azolidin-3-yl)phenyl]propanamide 33. Yield 70%; 1H NMR
(DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0 Hz, 3
H), 1.8 (m, 1 H), 3.8 (q, J ) 7.0 Hz, 1 H), 4.0 (dd, J ) 8.9, 7.2
Hz, 2 H), 4.4 (m, 2 H), 6.1 (s, 1 H), 7.3 (d, J ) 8.8 Hz, 2 H), 7.5
(d, J ) 8.0 Hz, 2 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS (ESI+)
m/z 341 (MH+); HRMS (ESI+) calcd for C18H20N4O3 + H
341.1608, found 341.1598. HPLC purity (as area %): 100.
(2R)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-
oxazolidin-3-yl)phenyl]propanamide 34. Yield 72%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0
Hz, 3 H), 1.8 (m, 1 H), 3.8 (q, J ) 6.9 Hz, 1 H), 4.0 (m, 2 H),
4.4 (m, 2 H), 6.1 (s, 1 H), 7.3 (d, J ) 8.8 Hz, 2 H), 7.5 (d, J )
8.8 Hz, 2 H), 10.3 (s, 1 H), 12.0 (s, 1 H); MS (ESI+) m/z 341
(MH+); HRMS (ESI+) calcd for C18H20N4O3 + H 341.1608,
found 341.1597. HPLC purity (as area %): 100.
d6) δ 0.59 (m, 2 H), 0.86 (m, 2 H), 1.28 (d, J ) 6.96 Hz, 3 H),
1.80 (m, 1 H), 1.90 (m, 4 H), 3.15 (m, 4 H), 3.65 (q, J ) 7.48
Hz, 1 H), 6.10 (s, 1 H), 6.44 (d, J ) 8.79 Hz, 2 H), 7.12 (d, J )
8.66 Hz, 2 H), 10.12 (s, 1 H), 11.93 (s, 1 H); MS (ESI+) m/z
325 (MH+), 174; HRMS (ESI+) calcd for C19H24N4O + H
325.2023, found 325.2027. Anal. (C19H24N4O) C, H, N. HPLC
purity (as area %): 100.
Registry Numbers. Methoxy(phenyl)acetic acid (RN, 7021-
09-2), (2R)-methoxy(phenyl)acetic acid (RN, 3966-32-3), (2S)-
methoxy(phenyl)acetic acid (RN, 26164-26-1), fluoro(phenyl)-
acetic acid (RN, 1578-63-8), (2S)-[(tert-butoxycarbonyl)-
amino](phenyl)acetic acid (RN, 2900-27-8), diphenylacetic acid
(RN, 117-34-0), oxo(phenyl)acetic acid (RN, 611-73-4), 2-phe-
nylpropanoic acid (RN, 492-37-5), (2R)-2-phenylpropanoic acid
(RN, 7782-26-5), (2S)-2-phenylpropanoic acid (RN, 7782-24-
3), 2-[4-(2-oxopyrrolidin-1-yl)phenyl]propanoic acid (RN, 437982-
74-6), (2R)-2-[4-(2-oxopyrrolidin-1-yl)phenyl]propanoic acid (RN,
492445-55-3), (2S)-2-[4-(2-oxopyrrolidin-1-yl)phenyl]propanoic
acid (RN, 492445-57-5), 2-[4-(1-oxo-1,3-dihydro-2H-isoindol-2-
yl)phenyl]propanoic acid (RN, 31842-01-0), (2R)-2-[4-(1-oxo-
1,3-dihydro-2H-isoindol-2-yl)phenyl]propanoic acid (RN, 53086-
14-9), (2S)-2-[4-(1-oxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]-
propanoic acid (53086-13-8), 2-[4-(2-oxoimidazolidin-1-yl)phe-
nyl]propanoic acid (RN, 492445-87-1), (2R)-2-[4-(2-oxoimida-
zolidin-1-yl)phenyl]propanoic acid (RN, 492445-88-2), (2S)-2-
[4-(2-oxoimidazolidin-1-yl)phenyl]propanoic acid (RN, 492445-
90-6), 2-[4-(3-methyl-2-oxoimidazolidin-1-yl)phenyl]propanoic
acid (RN, 492445-98-4), (2R)-2-[4-(3-methyl-2-oxoimidazolidin-
1-yl)phenyl]propanoic acid (RN, 492445-94-0), (2S)-2-[4-(3-
methyl-2-oxoimidazolidin-1-yl)phenyl]propanoic acid (RN,
492445-96-2), 2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanoic
acid (RN, 437982-65-5), (2R)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)-
phenyl]propanoic acid (RN, 492445-70-2), (2S)-2-[4-(2-oxo-1,3-
oxazolidin-3-yl)phenyl]propanoic acid (RN, 492445-72-4), 2-(4-
pyrrolidin-1-ylphenyl)propanoic acid (RN, 26586-38-9), 2-(4-
nitrophenyl)propanoic acid (RN, 19910-33-9), 2-(4-amino-
phenyl)propanoic acid (RN, 59430-62-5), (2S)-2-(4-aminophe-
nyl)propanoic acid (RN, 118417-73-5), methyl 2-(4-aminophe-
nyl)propanoate (RN, 39718-97-3), methyl 2-[4-(2-oxoimidazolidin-
1-yl)phenyl]propanoate (RN, 492445-82-6)
Kinase Assays. Kinase assays were performed as previ-
ously described.3
(2S)-N-(5-Cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-
oxazolidin-3-yl)phenyl]propanamide 35. Yield 73%; 1H
NMR (DMSO-d6) δ 0.6 (m, 2 H), 0.9 (m, 2 H), 1.3 (d, J ) 7.0
Hz, 3 H), 1.8 (tt, J ) 8.4, 5.1 Hz, 1 H), 3.8 (q, J ) 7.0 Hz, 1 H),
4.0 (m, 2 H), 4.4 (m, 2 H), 6.1 (s, 1 H), 7.3 (d, J ) 8.7 Hz, 2 H),
Crystal Structure of CDK2/Cyclin A with Compound
24. Expression, purification, and crystallization of the CDK2/
cyclin A complex were carried out as previously described.3 A
crystal of CDK2/cyclin A was then soaked with compound 24
in a manner analogous to that described previously for the

2956 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Pevarello et al.
Table 8
References
data collection
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space group
P6222
cell dimensions (Å)
resolution (Å)
a ) b ) 183.6, c ) 214.1
30-2.4 (2.49-2-4)a
673 208
(2) (a) Fischer, P. M.; Endicott, J.; Meijer, L. Cyclin-dependent
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Collins, E.; Jaramillo, C.; De Diego, J. E.; Spencer, C. D.;
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F.; Beckmann, R. P.; Dixon, J. T.; Gurganus, T. M.; Rankl, N.
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E.; Campbell, R. M.; Patel, B. K. R.; Brooks, H. B. The discovery
of a new structural class of cyclin-dependent kinase inhibitors,
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12189, 2001.
(9) Colombo, M.; Riccardi-Sirtori, F.; Rizzo, V. A Fully Automated
Method for Accurate Mass Determination with High Perfor-
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Acceleration Time-of-Flight Mass Spectrometer. Rapid Commun.
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Academic Press: New York, 1997; Vol. 276.
no. of measured reflections
no. of unique reflections
completeness (%)
I/σI
82 891
99.9 (100)
16.9 (2.9)
Rmerge (%)
7.3 (59.7)
refinement
Rfactor (%)
23.0
27.6
8978
456
Rfree (%)
no. of protein atoms
no. of nonprotein atoms
rmsd from ideal values
bonds (Å)
0.007
1.27
2.4
angles (deg)
% of residues in disallowed
regions of Ramachandran
plotb (%)
average B (protein)
average B (inhibitor)
50
45
a Values in parentheses are for the highest-resolution shells.
b As defined by Kleywegt and Jones.12
PHA-292137 complex.3 Data were collected at the ESRF
ID14-1 and processed using the HKL suite of programs.10
Refinement was carried out in CNX11 using the structure of
the complex with PHA-292137 as the starting model. The
results are shown in Table 8.
In Vitro Pharmacokinetics. High-throughput solubility,
cell permeability, plasma protein binding, and metabolic
stability (rat hepatocytes) data were acquired according to
previously reported procedures.3
In Vivo Pharmacokinetics. The pharmacokinetics of 13
and 35 was investigated in nu/nu mice as described earlier.3
For both the iv and oral legs of the preliminary PK experi-
ments, compounds 13 and 35 were administered using PEG
400/glucosate as a vehicle.
In Vitro and in Vivo Pharmacology. Cell culture, inhibi-
tion of cell proliferation, cell treatment after synchronization
with nocodazole, DNA content determination, Western blot
analysis, and microscopic analysis of BrdU incorporation were
performed as previously described.3 Evaluation of the in vivo
efficacy was performed as follows: A2780 human ovary
carcinoma (from American Type Culture Collection) was
maintained by subcutaneous (sc) transplantation in athymic
mice using 20-30 mg of tumor brei. For the experiment,
tumors were excised and fragments were implanted SC into
the left flank, 10 mice/group. The treatment started when the
tumors were measurable; the mean tumor weight for all the
groups was 0.13 g (day 8). Compound 13 was administered
after dissolution in 50% PEG 400/glucosate. Data are from a
single experiment, but efficacy was confirmed in similar
experiments using different doses and administration sched-
ules.
(11) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W. Crystallography and NMR System:
A New Software Suite for Macromolecular Structure Determi-
nation. Acta Crystallog., Sect. D 1998, 54, 905-921.
(12) Kleywegt, G. J.; Jones, T. A. Phi/psi-chology: Ramachandran
revisited. Structure 1996, 4, 1395-1400.
(13) McMartin, C.; Bohacek, R. S. QXP: Powerful, rapid computer
algorithms for structure-based drug design. J. Comput.-Aided
Mol. Des. 1997, 11, 333-344.
Acknowledgment. We thank Dr. Daniela Borghi,
Dr. Roberto Biancardi, and Dr. Federico Riccardi Sirtori
of the Analytical and Predevelopment Department of
Pharmacia Italia for skillful assistance in NMR and MS
spectra recording and interpretation.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
JM0408870