Cdc7 kinase inhibitors: Pyrrolopyridinones as potential antitumor agents. 1. Synthesis and structure-activity relationships

Article abstract of DOI:10.1021/jm700956r

Cdc7 kinase is an essential protein that promotes DNA replication in eukaryotic organisms. Genetic evidence indicates that Cdc7 inhibition can cause selective tumor-cell death in a p53-independent manner, supporting the rationale for developing Cdc7 small-molecule inhibitors for the treatment of cancers. In this paper, the synthesis and structure-activity relationships of 2-heteroaryl-pyrrolopyridinones, the first potent Cdc7 kinase inhibitors, are described. Starting from 2-pyridin-4-yl-1,5,6,7-tetrahydro-pyrrolo[3,2-c] pyridin-4-one, progress toward a simple scaffold, tailored for Cdc7 inhibition, is reported.

Full text of DOI:10.1021/jm700956r

J. Med. Chem. 2008, 51, 487–501
487
Cdc7 Kinase Inhibitors: Pyrrolopyridinones as Potential Antitumor Agents. 1. Synthesis and
Structure–Activity Relationships
Ermes Vanotti,* Raffaella Amici, Alberto Bargiotti, Jens Berthelsen, Roberta Bosotti, Antonella Ciavolella, Alessandra Cirla,
Cinzia Cristiani, Roberto D’Alessio, Barbara Forte, Antonella Isacchi, Katia Martina,† Maria Menichincheri, Antonio Molinari,
Alessia Montagnoli, Paolo Orsini, Antonio Pillan, Fulvia Roletto, Alessandra Scolaro, Marcellino Tibolla, Barbara Valsasina,
Mario Varasi,‡ Daniele Volpi, and Corrado Santocanale§
NerViano Medical Sciences Srl, Viale Pasteur 10, 20014 NerViano, Milano, Italy
ReceiVed August 2, 2007
Cdc7 kinase is an essential protein that promotes DNA replication in eukaryotic organisms. Genetic evidence
indicates that Cdc7 inhibition can cause selective tumor-cell death in a p53-independent manner, supporting
the rationale for developing Cdc7 small-molecule inhibitors for the treatment of cancers. In this paper, the
synthesis and structure–activity relationships of 2-heteroaryl-pyrrolopyridinones, the first potent Cdc7 kinase
inhibitors, are described. Starting from 2-pyridin-4-yl-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one, progress
toward a simple scaffold, tailored for Cdc7 inhibition, is reported.
Introduction
The inhibition of DNA synthesis has proven a useful strategy
in the treatment of hyper-proliferative disorders.1 However,
because of the toxicity of current DNA replication inhibitors
and because tumor cells can develop resistance to these drugs,2
there is a great need for novel agents that tackle this process
Figure 1. Structure of pyrrolopyridinone 1.
possibly by a different mechanism. In eukaryotic cells, DNA
synthesis is initiated from a number of replication origins in
Upjohn collection) led to the identification of 2-pyridin-4-yl-
the genome.3 Cdc7a kinase is responsible for origin activation
1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one 1 (Figure 1), a
and the establishment of active replication forks. Indeed, Cdc7-
scaffold for MK2 inhibitors, originally developed at Pharmacia
dependent phosphorylation of one or more subunits of the
Corp.,9 as a potent ATP mimetic inhibitor of Cdc7, with an
minichromosome maintenance complex (MCM 2–7)4,5 is thought
IC50 of 10 nM in a Cdc7 kinase assay (using a complex Cdc7FL/
to activate the helicase activity of the complex. Inhibition of
Dbf4FL),10 a Ki value of 3 nM, and greater than 20-fold
Cdc7 kinase by both small interfering RNAs6 and small-
selectivity versus a panel of other kinases. The biological
molecule inhibitors (Montagnoli et al., manuscript in prepara-
properties of this compound will be described in a different work
tion) causes a blockade of DNA synthesis in human cell lines.
(Montagnoli et al., manuscript in preparation).
As a consequence, tumor cells are funnelled into the apoptotic
Starting from scaffold 1, structure–activity relationship (SAR)
pathway in a p53-independent manner, whereas normal cells
studies, designed to identify modifications and substituents that
are arrested in their progression through the cell cycle and are
might increase potency against Cdc7, were carried out.
capable of surviving Cdc7 inhibition for long periods of time.6
The inhibitory activity of putative Cdc7 inhibitors was
These findings support the notion that pharmacological inhibition
determined by a method of assay based on the use of a Dowex
of Cdc7 kinase can be an effective novel strategy for the
resin (see the Experimental Section). For the purposes of
development of oncologic therapeutics and that a small-molecule
determining selectivity, the compounds were evaluated in an
inhibitor of Cdc7 kinase could be a useful drug for the treatment
in-house panel of about 35 Tyr and Ser-Thr kinases. A few of
of cancers.
these, namely, the Cdk2 and GSK3ꢀ kinases, consistent with
This paper reports the discovery of the very first class of Cdc7
their similarity in their ATP binding pockets,11,12 were the most
kinase inhibitors,7,8 namely, the 2-heteroaryl-pyrrolopyridinones.
The screening of our compound library (former Pharmacia-
inhibited by the compounds presented here. The synthesis of
frequently affected. Other kinases were only sporadically
different analogues of 1, sequentially modified at rings A, B
and C, has allowed us to highlight the major features contribut-
ing to the inhibition of the Cdc7 enzyme.
* To whom correspondence should be addressed. Tel. +39-0331-581521.
Fax: +39-0331-581347. E-mail: ermes.vanotti@nervianoms.com.
†
Present address: Dipartimento di Chimica IFM, Università di Torino,
Turin, Italy.
Chemistry
‡
Present address: DAC, Milan, Italy.
§
Present address: National University of Ireland, Galway, Ireland.
aAbbreviations: Cdc7, cell division cycle 7; MCM, minichromosome
maintenance complex; MK2 or MAPKAPK-2, mitogen-activated protein
kinase-activated protein kinase-2; Cdk, cyclin-dependent kinase; GSK,
glycogen synthase kinase; CK, casein kinase; CMGC kinases, cyclin-
dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs),
glycogen synthase kinases (GSKs), and CDK-like kinases (CLKs); CDI,
1,1′-carbonyl diimidazole; EDC, N-(3-dimethylaminopropyl)-N′-ethylcar-
bodiimide; HOBt, 1-hydroxybenzotriazole; PAMPA, parallel artificial
membrane permeation assay.
The preparation of the compounds appearing in this paper is
based on the following different protocols:
1. The Hantzsch reaction between piperidinediones 56 or 57
and bromoketones 64 (Scheme 1) was utilized to prepare
compounds 1, 3-8, 15, 16, 31, 34, 37, 38, 40, 43, 49, and 50.
According to the reaction, heteroarylbromoketones 64a-l
were allowed to react with piperidinedione 56 or 57 in the
presence of either ammonium acetate or a suitable amine
10.1021/jm700956r CCC: $40.75
2008 American Chemical Society
Published on Web 01/18/2008

488 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Scheme 1. Synthesis of Pyrrolopyridinonesa
Vanotti et al.
In this procedure, Meldrum’s acid was allowed to react with
N-Boc-3-aminopropionic acid 54 to provide compound 55,
which was cyclized to protected piperidinedione 56 by refluxing
in ethyl acetate. Piperidinedione 56 was optionally deprotected
to 57 under acidic conditions, with either trifluoroacetic acid in
dichloromethane or hydrochloric acid in dioxane.
Bromoketones 64a-l are commercially available or were
accessed through the halogenation of ketones or ethylenolethers
63, as shown in Scheme 6. This scheme also outlines the
preparation of 63, which is not commercially available.
Results and Discussion
Having established Cdc7 kinase as a potentially viable target
for oncologic studies, a synthetic chemistry program was
initiated, based on testing of our internal collection of Cdk
inhibitors. This was driven by the close sequence similarity of
the two kinases and facilitated by a well-established in-house
research project on Cdk inhibitors. This strategy, supported by
a 65% successful hit rate achieved in a preliminary screening
on 100 Cdk inhibitors, led to the prompt discovery of a few
promising chemical classes, among which the pyrrolopyridino-
nes, represented by compound 1, gained a leading position due
to their favorable characteristics. The latter include low-
molecular-weight, good solubility, low plasma protein binding,
and the possibility of facile synthetic extension of the series.
a Conditions: (a) NH4OAc or R1-NH2, EtOH, rt; (b) TFA, CH2Cl2, rt or
4N HCl in dioxane, MeOH, rt.
Scheme 2. N-Alkylation of Pyrrolea
In the absence of the crystallographic structure of the target
protein, we aimed to build a three-dimensional model of Cdc7
based upon homology with other kinases. In order to identify
the more suitable kinase for modeling, the sequence of the Cdc7
kinase domain was aligned with the sequences of all known
human kinases.17 This alignment highlighted a peculiarity of
Cdc7, namely, an insertion domain spanning from amino acid
203 to 370 that is partially conserved only in the mouse and
Xenopus Cdc7 orthologues and not present in any other known
kinase (Figure 2 in red box).18
When a similarity search was performed with this motif
against a nonredundant protein database, no statistically sig-
nificant hits were obtained. Thus, the lack of conservation of
this Cdc7-specific motif within kinases and other known proteins
might suggest that it has no function in the enzymatic activity
of the kinase. Indeed, removal of most of this sequence resulted
in an active enzyme, and recent work indicates that it contains
a protein–protein interaction domain important for nuclear
import.19
By repeating the similarity search with Cdc7∆203–370 as a
basic sequence, casein kinase 2 (CK2) and several members of
the CMGC kinases, including cyclin-dependent kinase 2 (Cdk2),
were the most important hits.
Because at the time of the analysis the CK2 structure was
not available, a Cdc7 three-dimensional model was built starting
from a Cdc7∆203–370/Cdk2 alignment and Cdk2 structural
information. The model was validated with early Cdc7 inhibitors
and then used for a SAR rationalization, structure-based ligand
design, and activity prediction. In this model, methionine 134
appears to be the gatekeeper residue.
a Conditions: (a) 1. 60% NaH, DMF, rt. 2. R1Br, rt. (b) R1Br, K2CO3,
DMF, 65 °C. (c) TFA, CH2Cl2, rt or 4N HCl in dioxane, MeOH, rt.
R1-NH2, depending on the need to obtain free or N-alkylated
pyrroles, respectively.
2. N-alkylation of the pyrrole ring (Scheme 2) afforded
compounds 22–30, 32, 33, 35, 36, and 39.
The reaction occurs between the protected pyrrolopyridinones
and the appropriate alkyl bromides, under basic conditions, in
the presence of sodium hydride or potassium carbonate.
3. Modifications of rings A and C of already formed
pyrrolopyridinones (Scheme 3) led to compounds 12, 20, 21,
41, 42, and 44–48.
In particular, the brominated aminopyrimidinyl derivative 12
was prepared by direct regioselective bromination of 49 with
N-bromosuccinimide NBS at room temperature. Hydrogenolytic
dehalogenation of lactam 5113 afforded compound 20, whereas
N-formylation of 1, with POCl3 in DMF at room temperature,
led to the N-formyllactam 21. N-alkylated (41, 42, 44, and 45)
and N-acylated (46–48) aminopyrimidinyl derivatives were
obtained from pyrrolopyridinone 5 and its protected analogue
49, the former by a reductive amination reaction, the latter by
exhaustive acylation, followed by selective basic hydrolysis.
4. Finally, in Scheme 4, the synthesis of compounds 14, 18,
and 19 is shown. Compound 14 and pyrrolopyrrolone 19 were
synthesized once more through the Hantzsch reaction, and the
furan analog 18 was obtained by oxidative cycloaddition14 of
the protected piperidinedione to 4-ethynylpyridine.
Compounds 1,7 2,13 10,13 11,13 13,15 17,16 and 51 were
available from the former Pharmacia-Upjohn collection, but their
preparation also is described in the literature. Compound 9 is
commercially available.
Using this Cdc7 model, we explored how compound 1 would
bind in the active site of the kinase and found that two different
binding modes were possible: with either the pyridine nitrogen or
the lactam group interacting with the hinge region (Figure 3).
The first mode (A) presented a more favorable docking
arrangement and better accounted for the subsequent SAR of
this class (Figure 4). In the docking studies, molecules of water
were included in the binding site and appeared to play an
For the synthesis of piperidinediones, the procedure given in
Scheme 5 was used.

Cdc7 Kinase Inhibitors. 1.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 489
Scheme 3. Synthesis of Pyrrolopyridinones from Other Pyrrolopyridinonesa
a Conditions: (a) NBS, DMF, rt; (b) 4N HCl in dioxane, MeOH, rt; (c) 10% Pd-C/H2, MeOH, rt; (d) POCl3, DMF, rt; (e) RCHO, NaBH(OAc)3, DMF,
TFA, rt; (f) RCOCl (2 equiv), TEA, THF, rt; (g) 1N NaOH, rt; (h) 4N HCl in dioxane, rt.
Scheme 4. Synthesis of Miscellaneous Compoundsa
a Conditions: (a) EtOH, NH4OAc, rt; (b) 2-bromo-1-(2-aminopyrimidin-4-yl)ethanone (64b), rt; (c) (NH4)2Ce(NO3)6, CH3CN, 0 °C; (d) 4N HCl in dioxane,
MeOH, rt; (e) CDI, KOOCCH2COOEt, MgCl2, THF, rt, then 50 °C; (f) 2-bromo-1-pyridin-4-ylethanone (64a), 60% NaH, 0 °C; (g) NH4OAc, AcOH, THF,
rt; (h) NaOH, EtOH, water, reflux; (i) EDC hydrochloride, HOBt, N-ethyldiisopropylamine, CH2Cl2, DMF, rt.
Scheme 5. Synthesis of Piperidinedionesa
a Conditions: (a) Meldrum’s acid, CH2Cl2, EDC hydrochloride, DMAP, 0 °C to rt; (b) EtOAc, reflux; (c) TFA, CH2Cl2, rt or 4N HCl in dioxane, rt.
important role in the binding of compound 1 and, in general,
all of its close derivatives.
profitably replaced by other rings and if position 2 of the pyrrole
is optimal for the attachment of the heteroaryl moiety. The set
of compounds presented in Table 1 offers the opportunity to
answer these questions. In most analogues, a nitrogen at position
1′ is present, a condition considered essential for activity, as
duly demonstrated by compounds 2, 8, and 9, in which,
respectively, the repositioning or the absence of the nitrogen
Our initial work focused on exploring variants of the three-
ring systems that constitute pyrrolopyridinone 1, involving the
pyridinyl ring A, the pyrrole nucleus B, and the lactam ring C,
and studying the effect these parts of the molecule have on
potency. First, we investigated if the pyridine ring could be

490 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Scheme 6. Synthesis of Bromoketones 64a
Vanotti et al.
a Conditions: (a) 48% HBr, Br2, AcOH, 60 °C; (b) Boc2O, DMAP, CH3CN, rt; (c) 1-ethoxyvinyl tri-n-butyltin, Pd(PPh3)4, DMF, 100 °C; (d) NBS, THF,
rt; (e) phenylguanidine carbonate, NaOEt, EtOH, reflux; (f) 88% HCOOH, rt; (g) tert-butyldimethylsilyl trifluoromethanesulfonate, TEA, CH2Cl2, rt.
atom, or the removal of ring A, led to inactive derivatives,
consistent with the proposed binding mode (Figure 4). Moreover,
moving the heteroaryl ring from position 2 to the adjacent
position 3 resulted in the inactive compound 14, possibly a
consequence of the radical change in the geometry of the
molecule.
Figure 2. Alignment of Cdc7 orthologues showing the characteristic motif of human Cdc7 (hereafter called Cdc7-insertion domain) poorly conserved
in other species and not found in other kinases.

Cdc7 Kinase Inhibitors. 1.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 491
necessary determinant for activity. The influence of N-alkylation
of pyrrole on activity (16) deserves attention and will be
examined in more detail later. An alkyl substituent at position
3 (15) appears to be detrimental, because it causes a decrease
in activity. This is most likely due to its unfavorable interaction
with the large gatekeeper residue methionine (Met 134, Figure
4) in the narrow hydrophobic pocket toward which it points. In
contrast, the presence of a vinylidene group, bridging the pyrrole
with the adjacent pyridine ring (13), demonstrated a considerable
improvement of activity, probably due to an optimized fitting
of this flat moiety within the hydrophobic pocket.
Finally, we examined modifications of the lactam ring of
compound 1 (Table 3).
Six- and five-membered lactam rings (compounds 1 and 19,
respectively) showed good inhibitory activity on the enzyme,
whereas the azepinone analogue 20 did not. The free NH of
the lactam seems important for activity, likely because the
hydrogen is well placed for binding to aspartate. This is
evidenced by the fact that the N-formyl derivative 21 is poorly
active.
In summary, from the investigation of variations in rings A,
B, and C, several compounds have emerged that display good
inhibitory activity against the Cdc7 enzyme. Also, as outlined
in Table 4, they show good activity against the A-2780 ovarian
cancer cell line.
Figure 3. Possible binding modes of pyrrolopyridinone 1 in the
homology model of Cdc7 kinase.
These compounds vary in their ring A substitution pattern,
the region which binds well to the hinge region of the enzyme.
Ring C can adopt only two profitable geometries, and ring B
has to be a pyrrole. When challenged for cellular activity, only
a few compounds (3, 5, 12, and 13, Table 4) were as active,
approximately, as the reference compound 1. Of these, those
carrying a substituent at position 3′ or 5′ (fluoro in 3, bromo in
12, and vinylidene in 13) suffer from a severe loss of selectivity,
and only compound 5 shows a selectivity comparable to that
of 1, at least 20-fold versus other kinases.
In conclusion, on the basis of the potency against the Cdc7
enzyme, the antiproliferative activity on the A-2780 cell line,
and selectivity, only the parent compound 1 and the 2-ami-
nopyrimidin-4-yl analogue 5 were considered suitable for further
structural modifications and were subjected to N-substitutions
of the pyrrole ring (both series) and to functionalization of the
amino group of the pyrimidine.
Figure 4. Predicted binding site of Cdc7 homology model with
insertion of ATP (orange) and pyrrolopyridinone 1 (white). The sulfur
atom of the gatekeeper Met 134 is in yellow.
Some compounds with a substituent at different positions of
the heteroaryl moiety (ring A, Figure 1) were also examined. It
is noteworthy that, while substituents at position 3′ or 5′ (fluoro
in 3, bromo in 12, and vinylidene in 13, retained or increased
activity, a substituent at position 2′ resulted in different
outcomes. In fact, a chloro substituent (10) showed less activity
and represents a disturbance to binding, and the presence of an
amino group also is somewhat detrimental in pyridine (11 vs
1) but slightly favorable in the pyrimidine (5 vs 4). However,
its influence is unclear when it is embedded in a ring (6 vs 7),
the differences presumably depending on the electronic distribu-
tion in the system. In conclusion, as is apparent from Table 1,
derivatives 3, 4, 5, 6, 11, 12, and 13 increase or retain activity
when compared to 1.
As anticipated, N-substitution of the pyrrole hampered activity
in the pyridyl series. On the contrary, the aminopyrimidinyl
homologues tolerated N substitution, providing some derivatives
as potent as the unsubstituted precursor (Table 5).
For this reason, we extended our investigations to several other
compounds of the latter series, and these substantially confirmed
our previous results with only two exceptions (Table 6).
One possible explanation of the difference between the two
series is that the pyrrole binds to the protein backbone by the
intermediacy of one or two molecules of water. This binding
possibility, definitively lost in the pyridinyl series, can be
restored in aminopyrimidines (Figures 3A and 5) by the NH2
of the pyrimidine ring, provided that R1 is not a lipophilic bulky
substituent, as evidenced especially by compounds 37 and 38.
Next, we concentrated our studies on substitutions on the
central core of the pyrrolopyridinone 1 (Table 2).
Most N-substituted aminopyrimidinyl analogues, when pro-
vided with a short lipophilic chain or a small rigid residue
(compounds 26, 28, 29 and 31–35), showed IC50 values in the
low nanomolar range, whereas with larger cyclic groups, activity
declined. The ideal chain length seems to be C2-C3, and the
presence of a hydrophilic terminus is less favorable (compound
27). The more hydrophilic piperazinyl ring behaved better than
In Table 2, pyrrole, pyrazole, and furan derivatives demon-
strate that, when the pyrrole NH is alkylated (16) or when the
whole ring is replaced with a different heteroaromatic system
(17–18), a significant loss in potency is experienced. By
retaining the pyridine as the A ring system (see Figure 1), the
presence of hydrogen on the nitrogen of the pyrrole is a

492 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Vanotti et al.
Table 1. SAR: Substituent Variations in the Pyrrole Ring at Position 2 (IC50, µM)a
a IC50 values are reported as the mean ( standard deviation (n g 2).
Table 2. SAR: Variations of the Central Core B (IC50, µM)a
Table 3. SAR: Variations of the Lactam Ring C (IC50, µM)a
a IC50 values are reported as the mean ( standard deviation (n g 2).
investigated. As shown in Table 7, alkylations (41, 42, 44, and
45), arylation (43), and acylations (46–48) led to a much reduced
activity, the only exception being the phenyl derivative (43),
which is quite active on Cdc7 but poorly selective. In conclusion,
functionalization of the amino group of the aminopyrimidinyl
ring must be avoided in order to preserve the most favorable
profile of biological activity.
This refining process led to the definition of the basic scaffold
bs (Figure 6) characterized by structural requirements that
provide the best balance between activity and selectivity: (1)
an unsubstituted pyridin-4-yl or 2-aminopyrimidin-4-yl rings at
position 2 of the pyrrole ring, (2) a pyrrole ring with a free NH
and no other substitutions, and (3) a six-membered lactam, ideal
for size and rigidity, with free NH. Scaffold bs is characterized
a IC50 values are reported as the mean ( standard deviation (n g 2).
cyclohexyl (see 40 vs 37), possibly a sign of binding to water
or to a polar residue in the enzyme.
On the whole, N-substitution of the pyrrole provided very
active compounds that regrettably proved equipotent across a
broad spectrum of kinases, a finding that caused us to abandon
the N-substituted pyrroles in favor of free pyrroles.
Having examined variations in the three main parts of the
molecule, modifications at the amino group of pyrimidine were

Cdc7 Kinase Inhibitors. 1.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 493
Table 4. SAR: Comparison of the Most Interesting Scaffolds
(IC50, µM)a
(intrinsic clearance lower than 20 mL/min/kg in rat hepato-
cytes)20 but low cellular permeability (Papp lower than 2.10-6
cm/s) in the PAMPA assay.21 The latter property may account
for the moderate cellular activity displayed against the A-2780
cell line. A further SAR study, related to the functionalization
of the unexplored positions 6 and 7, will be presented in due
course.
Conclusions
In this paper, the first known inhibitors of Cdc7 kinase are
revealed. Starting from the basic framework of 1, the SARs
associated with the molecule were investigated. These show that
most functionalizations are detrimental to biological properties.
Strong Cdc7 inhibitory activity is associated with a central
unsubstituted pyrrole nucleus fused with a five- or six-membered
lactam and carrying at position 2 an appropriate, optionally
substituted, heteroaromatic ring. Regarding selectivity, more
stringent conditions are required of ring A, in that it can only
be achieved with a pyridin-4-yl ring, unsubstituted at position
3′, or with a pyrimidin-4-yl residue, unsubstituted at position
5′, and optionally bearing a primary amino function at position
2′ as the substituent.
This study led to the definition of the simple pharmacoph-
ore bs, as a potent inhibitor of Cdc7 yet selective versus
other tested kinases (Montagnoli et al., manuscript in
preparation). This scaffold seems to possess the best geometry
and a favorable entropy in its interaction with the Cdc7
binding site. The decreased potency of most substituted
analogues might be due to distortion of this ideal geometry
or to the positioning of the ligands away from the best binding
mode presented by the naked scaffolds.
Further work, consisting of functionalizing the unexplored
positions 6 and 7, will answer the question as to whether there
are substituents that might impart better characteristics, in
particular, increased intracellular activity, and, in general, a more
appropriate druglike profile that seems to be obligatory for this
demanding kinase.
a IC50 values are reported as the mean ( standard deviation (n g 2).
b IC50values are reported as the mean of 2–3 experiments with a coefficient
of variation below 35%.
Experimental Section
Table 5. SAR: Variations of the Pyrrole Alkylating Group R1 (IC50,
µM)a
I. Chemistry. Unless otherwise noted, solvents and reagents
were obtained from commercial suppliers and used without further
purification. All reactions involving air- or moisture-sensitive
reagents were performed under an argon atmosphere. All final
compounds were purified to >95% purity as determined by high-
performance liquid chromatography (HPLC). Purity was measured
by HPLC 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 Micromass mod. A ZQ single quadrupole
mass spectrometer, equipped with an electrospray ion source (ESI).
Mobile phase A was an ammonium acetate 5 mM buffer (pH 5.5
with acetic acid/acetonitrile 95:5), and mobile phase B was H2O/
acetonitrile (5:95). The following conditions were used: a gradient
from 10 to 90% B in 8 min and held at 90% B for 2 min; UV
detection at 220 and 254 nm; a flow rate of 1 mL/min; an injection
volume of 10 µL; full scan, mass range from 100 to 800 amu. The
capillary voltage was 2.5 kV; the source temperature was 120 °C;
the cone was 10 V. Masses are given as an m/z ratio.
When necessary, compounds were purified by preparative HPLC
on a Waters Symmetry C18 (19 × 50 mm, 5 µm) column using a
Waters preparative HPLC 600 equipped with a 996 Waters PDA
detector and a Micromass mod. A ZMD single quadrupole mass
spectrometer, with electro-spray ionization, in the positive mode,
was used. Mobile phase A was water and 0.01% trifluoroacetic
acid, and mobile phase B was acetonitrile. The following conditions
were used: a gradient from 10 to 90% B in 8 min and held at 90%
B for 2 min; a flow rate of 20 mL/min. Column chromatography
a IC50 values are reported as the mean ( standard deviation (n g 2).
by a low-molecular-weight (around 200), good solubility in
water (>225 µM at pH 7),20 and good metabolic stability

494 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Table 6. Other N-Substituted Aminopyrimidinylpyrrole Derivativesa
Vanotti et al.
a IC50 values are reported as the mean ( standard deviation (n g 2).
million (ppm) downfield from tetramethylsilane or other internal
reference in the appropriate solvent indicated. Data are reported as
follows: chemical shift, multiplicity (s ) singlet, d ) doublet, t )
triplet, q ) quartet, bs ) broad singlet, bd ) broad doublet, bt )
broad triplet, td ) triplet of doublet, ddd ) doublet of doublet of
doublet, m ) multiplet), coupling constants, and number of protons.
Low-resolution mass spectral (MS) data were determined on a
Finnigan MAT LCQ ion trap instrument, equipped with ESI.
High-resolution mass spectra (HRMS) were obtained on a Waters
Q-TOF Ultima instrument, equipped with ESI, and using reserpine
(MW 609.28065) for lock mass correction.
Elemental analyses were performed on a Carlo Erba EA1110
instrument, and C, H, and N values were within ( 0.4% of
theoretical values, unless otherwise noted. Thin-layer chromatog-
raphy was performed on Merck silica gel 60 plates coated with a
250 µM layer with a fluorescent indicator. Components were
visualized by UV light (λ ) 254 and 366 nm) and iodine vapors.
2-(Pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-
4-one hydrochloride (1) and 4-oxo-(2-pyridin-4-yl)-1,4,6,7-
tetrahydropyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl
ester (50). A suspension of 2,4-dioxopiperidine-1-carboxylic acid
tert-butyl ester (56; 2.6 g, 12.24 mmol) and 2-bromo-1-pyridin-4-
ylethanone hydrobromide (64a; 2.6 g, 9.42 mmol) in ethanol (120
mL) was treated with ammonium acetate (2.9 g, 37.7 mmol) at
room temperature. The resulting solution was stirred for 16 h. The
mixture was concentrated under reduced pressure, diluted with ethyl
acetate, and washed with 0.5 N NaOH (pH ) 9). The aqueous layer
was extracted with ethyl acetate (50 mL × 5). The collected organic
layers were combined, dried over anhydrous sodium sulfate, and
evaporated to dryness. The crude material was flash-chromato-
graphed (eluant: dichloromethane/ethanol 10:1), to provide 50 as
a white solid (1.74 g, 59%).
1H NMR (400 MHz DMSO-d6): δ 1.47 (s, 9 H), 2.95 (t, J )
6.40 Hz, 2 H), 3.97 (t, J ) 6.34 Hz, 2 H), 7.11 (d, J ) 2.07 Hz, 1
H), 7.63 (d, J ) 6.22 Hz, 2 H), 8.51 (d, J ) 6.22 Hz, 2 H), 12.14
(bs, 1 H). HRMS (M+H)+ calcd: 314.1499. Found: 314.1505.
The compound was dissolved in anhydrous methanol (30 mL);
4 N HCl in dioxane (7 mL) was added, and the mixture was stirred
at room temperature for 2 h. After concentrating to dryness, the
material was neutralized with 7 N ammonia in methanol, affording
1 as an off-white solid (1.2 g, 88%).
Figure 5. Supposed binding modes of N-alkylated pyridinyl pyrroles
and aminopyrimidinyl pyrroles.
was conducted either under medium pressure on silica (Merck silica
gel 40–63 µm) or on prepacked silica gel cartridges (Biotage) or
on a Horizon system.
1H NMR spectra were routinely recorded on a Varian Inova 400
spectrometer operating at 400 MHz and equipped with a 5 mm
indirect detection PFG probe (1H{15N-31P}). Where reported, a
Varian Inova 500 spectrometer, operating at 500 MHz and equipped
with a 5 mm triple resonance PFG probe (1H{15N-31P}), or a
Varian Mercury 300 spectrometer, operating at 300 MHz and
equipped with a 5 mm switchable probe (15N-31P{1H,19F}) was
used. All observed proton absorptions are reported as parts per

Cdc7 Kinase Inhibitors. 1.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 495
Table 7. SAR: Substitutions R at the Amino Group of Pyrimidine (IC50, µM)a
a IC50 values are reported as the mean ( standard deviation (n g 2).
3.38–3.44 (m, 2 H), 6.68 (d, J ) 2.44 Hz, 1 H), 6.93 (t, J ) 2.74
Hz, 1 H), 7.15–7.23 (m, 1 H), 7.37 (t, J ) 7.80 Hz, 2 H), 7.64 (dd,
J ) 8.35, 1.16 Hz, 2 H), 11.59 (s, 1 H). HRMS (M+H)+ calcd:
213.1022. Found: 213.1020. Anal. calcd (C13H12N2O): C, H, N.
3-Methyl-2-(pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-
c]pyridin-4-one (15). 1H NMR (400 MHz, DMSO-d6): δ 2.66 (s,
4 H), 2.90 (t, J ) 6.83 Hz, 3 H), 3.29–3.47 (m, 2 H), 7.24 (s, 1 H),
8.00 (d, J ) 6.95 Hz, 2 H), 8.71 (d, J ) 7.19 Hz, 2 H), 12.39 (s,
1 H). HRMS (M+H)+ calcd: 228.1131. Found: 228.1134. Anal.
calcd (C13H13N3O): C, H, N.
2-(2-Phenylaminopyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyr-
rolo[3,2-c]pyridin-4-one (43). 1H NMR (400 MHz, DMSO-d6): δ
2.7 (m, J ) 17.1, 6.2, 5.1, 1.5, 0.9 Hz, 1 H), 2.8 (m, J ) 17.1, 6.3,
5.1, 1.5, 0.9 Hz, 1 H), 3.5 (m, J ) 12.2, 6.2, 5.1, 2.5 Hz, 1 H), 3.5
(m, J ) 12.2, 6.3, 5.1, 2.5 Hz, 1 H), 6.9 (m, J ) 7.5, 1.2 Hz, 1 H),
7.3 (m, J ) 8.1, 7.5, 1.6, 0.4 Hz, 1 H), 7.3 (m, J ) 8.1, 7.5, 1.6,
0.4 Hz, 1 H), 7.6 (m, J ) 0.9 Hz, 1 H), 7.6 (m, J ) 8.1, 2.5, 1.2,
0.4 Hz, 1 H), 7.6 (m, J ) 8.1, 2.5, 1.2, 0.4 Hz, 1 H), 7.7 (m, J )
4.8 Hz, 1 H), 8.4 (m, J ) 4.8 Hz, 1 H). HRMS (M+H)+ calcd:
306.1349. Found: 306.1346. Anal. calcd (C17H15N5O): C, 66.87;
H, 4.95; N, 22.94. Found: C, 65.71; H, 4.64; N, 21.44.
Figure 6. The basic scaffold bs.
1H NMR (400 MHz, DMSO-d6): δ 2.94 (t, J ) 6.83 Hz, 2 H),
3.45 (t, J ) 6.83 Hz, 2 H), 7.30 (bs, 1 H), 7.59 (s, 1 H), 8.23 (d,
J ) 7.08 Hz, 2 H), 8.71 (d, J ) 7.08 Hz, 2 H), 12.89 (bs, 1 H).
HRMS (M+H)+ calcd: 214.0975. Found: 214.0975. Anal. calcd
(C12H11N3O): C, H, N.
The following compounds were synthesized starting from the
appropriate haloketone and according to the general procedure
detailed above:
2-(3-Fluoro-pyridin-4-yl)-1,5,6,7-tetrahydropyrrolo[3,2-c]py-
ridin-4-one (3). 1H NMR (400 MHz, DMSO-d6): δ 2.91 (m, 2 H),
3.41 (m, 2 H), 7.08 (bs, 1 H), 7.19 (bs, 1 H), 7.90 (dd, J ) 5.60
Hz, 7.19 Hz, 1 H), 8.48 (d, J ) 5.00 Hz, 1 H), 8.70 (d, J ) 4.15
Hz, 1 H), 12.15 (bs, 1 H). HRMS (M+H)+ calcd: 232.0881. Found:
232.0876. Anal. calcd (C12H10FN3O): C, H, N.
2-(Pyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyri-
din-4-one hydrochloride (4). 1H NMR (400 MHz, DMSO-d6): δ
2.87 (t, J ) 6.83 Hz, 2 H), 3.44 (t, J ) 6.83 Hz, 2 H), 7.19 (bs, 1
H), 7.37 (s, 1 H), 7.89 (d, J ) 5.85 Hz, 1 H), 8.68 (d, J ) 5.85 Hz,
1 H), 9.10 (s, 1 H). HRMS (M+H)+ calcd: 215.0927. Found:
215.0932. Anal. calcd (C11H10N4O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-4-oxo-1,4,6,7-tetrahydro-pyrrolo[3,2-
c]pyridine-5-carboxylic Acid tert-Butyl Ester (49). 1H NMR (400
MHz, DMSO-d6): δ 1.48 (s, 9 H), 2.94 (m, 2 H), 3.96 (m, 2 H),
6.38 (bs, 2 H), 6.95 (d, J ) 5.12 Hz, 1 H), 7.14 (s, 1 H), 8.19 (d,
J ) 5.12 Hz, 1 H), 12.00 (bs,1 H). HRMS (M+H)+ calcd:
330.1561. Found: 330.1566. Anal. calcd (C16H19N5O3): C, H, N.
2-(2-Amino-5-bromo-pyrimidin-4-yl)-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (12). To a solution of 2-(2-aminopyri-
midin-4-yl)-4-oxo-1,4,6,7-tetrahydropyrrolo[3,2-c]pyridine-5-car-
boxylic acid tert-butyl ester (49; 0.2 g, 0.6 mmol) in N,N-
dimethylformamide (2 mL) at room temperature was added
N-bromosuccinimide (0.11 g, 0.6 mmol) in N,N-dimethylformamide
(1 mL) dropwise with stirring. After 3 h, water was added, and the
aqueous solution was extracted with ethyl acetate. The organic layer
was dried over anhydrous sodium sulfate and concentrated to a
crude product that, after flash chromatography (eluant: 96:4
dichloromethane/ethanol), yielded 2-(2-amino-5-bromopyrimidin-
4-yl)-4-oxo-1,4,6,7-tetrahydropyrrolo[3,2-c]pyridine-5-carboxylic
acid tert-butyl ester (0.09 g, 37%). To a solution of this compound
in methanol (4 mL) was added 4 N HCl in dioxane (0.55 mL), and
the mixture was stirred at room temperature for 1 h. After
concentrating to dryness, the material was neutralized with 7 N
ammonia in methanol, and the title compound was isolated as a
pale yellow solid (0.05 g, 65%).
2-(2-Aminopyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-
c]pyridin-4-one (5). 1H NMR (400 MHz, DMSO-d6): δ 2.91 (t, J
) 6.71 Hz, 2 H), 3.36 (t, J ) 6.71 Hz, 2 H), 7.27 (d, J ) 6.70 Hz,
1 H), 7.29 (bs, 1 H), 7.46 (s, 1 H), 7.86 (bs, 2 H), 8.21 (d, J )
6.70 Hz, 2 H). HRMS (M+H)+ calcd: 230.1036. Found: 230.1032.
Anal. calcd (C11H11N5O.0.25 H2O): C, H, N.
2-(1H-Pyrrolo[2,3-b]pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyr-
1
rolo[3,2-c]pyridin-4-one (6). H NMR (400 MHz, DMSO-d6): δ
2.95 (t, J ) 6.71 Hz, 2 H), 3.47 (t, J ) 6.71 Hz, 2 H), 7.10 (s, 1
H), 7.22 (bs, 1 H), 7.29 (s, 1 H), 7.61 (bd, 1 H), 7.71 (bt, 1 H),
8.38 (d, J ) 6.10 Hz, 2 H), 12.26 (bs, 1 H). HRMS (M+H)+ calcd:
253.1084. Found: 253.1076. Anal. calcd (C14H12N4O): C, H, N.
2-(9H-Purin-6-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyri-
din-4-one (7). 1H NMR (400 MHz, DMSO-d6): δ 2.91 (t, J ) 6.83
Hz, 2 H), 3.42 (t, J ) 6.83 Hz, 2 H), 7.16 (bs, 1 H), 7.77 (s, 1 H),
8.59 (s, 1 H), 8.80 (s, 1 H), 12.26 (bs, 1 H). HRMS (M+H)+ calcd:
255.0989. Found: 255.0988. Anal. calcd (C12H10N6O): C, H, N.
1H NMR (400 MHz, DMSO-d6): δ 2.86 (m, 2 H), 3.3–3.5 (m,
2 H), 6.4–6.8 (bs, 2 H), 7.16 (bs, 1 H), 7.51 (s, 1 H), 8.35 (s, 1 H),
11.75 (s, 1 H). HRMS (M+H)+ calcd: 308.0141. Found: 308.0135.
Anal. calcd (C11H10BrN5O): C, H, N.
2-Phenyl-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one (8).
1H NMR (400 MHz, DMSO-d6): δ 2.83 (t, J ) 6.89 Hz, 2 H),

496 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Vanotti et al.
3-(2-Aminopyrimidin-4-yl)-1,5,6,7-tetrahydropyrrolo[3,2-c]-
pyridin-4-one (14). A solution of piperidine-2,4-dione (57; 2.86 g,
25.3 mmol) and ammonium acetate (5.2 g, 67.5 mmol) in absolute
ethanol (75 mL) was stirred at room temperature for 45 min; then,
1-(2-amino-pyrimidin-4-yl)-2-bromo-ethanone hydrobromide (64b;
5 g, 16.8 mmol) was added, and the reaction mixture stirred at
room temperature for 18 h. After solvent removal, the residue was
adsorbed onto a Horizon column and eluted with dichloromethane/
methanol/30% aqueous ammonia in a ratio of 93:7:0.7 (then 90:
10:1). In this way, the title compound was obtained (1.27 g, 33%
yield). The main impurity is the 2-isomer 5, which was isolated in
13% yield (0.49 g) from the less polar fractions.
1H NMR (400 MHz, DMSO-d6): δ 2.97 (t, J ) 7.70 Hz, 2 H),
3.64 (t, J ) 7.70 Hz, 2 H), 7.05 (d, J ) 5.24 Hz, 1 H), 7.23 (s, 2
H), 8.31 (d, J ) 5.24 Hz, 1 H), 8.42 (s, 1 H), 9.71 (s, 1 H), 11.67
(s, 1 H). HRMS (M+H)+ calcd: 230.1036. Found: 230.1037. Anal.
calcd (C11H11N5O): C, H, N.
1H NMR (400 MHz, DMSO-d6): δ 1.30 (t, J ) 7.07 Hz, 3 H),
1.41 (s, 9 H), 4.22 (d, J ) 7.07 Hz, 2 H), 4.50 (d, J ) 5.49 Hz, 2
H), 6.91 (s, 1 H), 7.15 (d, J ) 2.68 Hz, 1 H), 7.70 (dd, J ) 4.63,
1.59 Hz, 2 H), 8.52 (dd, J ) 4.63, 1.59 Hz, 2 H), 11.94 (s, 1 H).
To a solution of 2-(tert-butoxycarbonylaminomethyl)-5-(pyridin-
4-yl)-1H-pyrrole-3-carboxylic acid ethyl ester (0.2 g, 0.6 mmol)
in ethanol/water 3:1 (30 mL) was added sodium hydroxide (0.6 g).
The solution was heated to reflux overnight, partially concentrated,
and brought to pH 6–7 with 2 N HCl. The precipitate was filtered
and dried, yielding 2-(tert-butoxycarbonylamino-methyl)-5-pyridin-
4-yl-1H-pyrrole-3-carboxylic acid (0.19 g, quantitative yield).
1H NMR (400 MHz, DMSO-d6): δ 1.36 (s, 9 H), 4.47 (s, 2 H),
7.04 (s, 1 H), 7.19 (s, 1 H), 7.67 (d, J ) 5.86 Hz, 2 H), 8.45 (d, J
) 6.15 Hz, 2 H), 12.06 (bs, 1 H).
To a solution of 2-(tert-butoxycarbonylamino-methyl)-5-(pyridin-
4-yl)-1H-pyrrole-3-carboxylic (22 mg, 0.07 mmol) in dioxane (2
mL) was added 4 N HCl in dioxane (1 mL), and the solution was
shaken for 18 h at room temperature. The precipitate was filtered
and dried, thus affording 2-aminomethyl-5-(pyridin-4-yl)-1H-pyr-
role-3-carboxylic acid hydrochloride (53; 17 mg, 98% yield).
1H NMR (400 MHz, DMSO-d6): δ 4.41 (s, 2 H), 7.58 (s, 1 H),
8.24 (d, J ) 5.86 Hz, 2 H), 8.46 (bs, 2 H), 8.73 (d, J ) 6.74 Hz,
2 H), 13.53 (bs, 1 H).
2-Pyridin-4-yl-6,7-dihydro-5H-furo[3,2-c]pyridin-4-one (18).
To a stirred, cooled (0 °C) suspension of 2,4-dioxopiperidine-1-
carboxylic acid tert-butyl ester (56; 75 mg, 0.35 mmol) and
4-ethynylpyridine hydrochloride (490 mg, 3.5 mmol) in acetonitrile
(15 mL) was added ceric ammonium nitrate (390 mg, 0.7 mmol)
in three portions. After being stirred for 6 h, the solvent was
evaporated, 5% K2CO3 (aq) was added, and the mixture was
extracted with ethyl acetate and the extract dried over anhydrous
sodium sulfate and concentrated. The crude residue was purified
by flash chromatography (eluant: ethyl acetate). The N-Boc
protected product (10 mg, 0.03 mmol) was dissolved in methanol
(1 mL) containing 4 N HCl in dioxane (0.5 mL) and stirred for
24 h. The solvent was evaporated; 5% K2CO3 (aq) was added, and
the mixture was extracted with ethyl acetate, dried over anhydrous
sodium sulfate, and concentrated. The crude residue was purified
by flash chromatography (eluant: 6:1 ethyl acetate/methanol) to yield
the title compound (7 mg, 11% yield).
To a solution of 53 (17 mg, 0.066 mmol) in N,N-dimethylfor-
mamide/dichloromethane (1:1, 5 mL) were added 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (13 mg, 0.066
mmol), hydroxybenzotriazole (9 mg, 0.066 mmol), and diisopro-
pylethylamine (34 mg, 0.26 mmol). The solution was left shaking
overnight at room temperature and was concentrated. The crude
product was purified by chromatography (eluant: 98:2 dichloro-
methane/methanol, then 90:10), thus affording the title compound
(13 mg, 45% yield).
1H NMR (400 MHz, DMSO-d6): δ 4.31 (s, 2 H), 6.95 (d, J )
1.95 Hz, 1 H), 7.64 (d, J ) 6.22 Hz, 3 H), 8.52 (d, J ) 6.22 Hz,
2 H), 12.19 (s, 1 H). HRMS (M+H)+ calcd: 200.0818. Found:
200.0820. Anal. calcd (C11H9N3O): C, H, N.
2-(Pyridin-4-yl)-5,6,7,8-tetrahydro-1H-pyrrolo[3,2-c]azepin-
4-one (20). A mixture of 2-(2-chloropyridin-4-yl)-5,6,7,8-tetrahy-
dro-1H-pyrrolo[3,2-c]azepin-4-one (51; 10 mg, 0.04 mmol) and
10% Pd on carbon (20 mg) in methanol (5 mL) was hydrogenated
at room temperature and 40 psi of hydrogen for 6 h. After filtration
through celite and solvent evaporation, the title compound was
obtained (6 mg, 65% yield).
1H NMR (400 MHz, DMSO-d6): δ 2.39 (t, J ) 7.44 Hz, 2 H),
3.55 (dt, J ) 7.44, 2.07 Hz, 2 H), 7.40 (s, 1 H), 4.47 (d, J ) 4.63,
2 H), 8.50 (d, J ) 4.76, 2 H), 8.57 (bs, 1H). HRMS (M+H)+ calcd:
215.0815. Found: 215.0812.
2-(Pyridin-4-yl)-5,6-dihydro-1H-pyrrolo[3,4-b]pyrrol-4-one
(19). A solution of N-Boc glycine (2 g, 11.4 mmol) and 1,1′-
carbonyldiimidazole (2.8 g, 17 mmol) in 18 mL of anhydrous
tetrahydrofuran was shaken at room temperature for 3 h. Magnesium
chloride (2.18 g, 22.8 mmol) and potassium ethylmalonate (3.9 g,
22.8 mmol) in anhydrous tetrahydrofuran (20 mL) were added. The
temperature was brought to 50 °C and the suspension shaken for
18 h, then filtered. The solvent was removed, and the raw product
was dissolved in ethyl acetate and washed with 5% sodium bisulfate
(aq, × 3), sodium bicarbonate (aq), and then brine. The organic
solution was dried over anhydrous sodium sulfate, and the solvent
was removed. The crude product was purified by chromatography
(eluant: 15:85 ethyl acetate/n-hexane, then 80:20), yielding 4-tert-
butoxycarbonylamino-3-oxobutyric acid ethyl ester (52; 2 g, 71%).
1H NMR (400 MHz, DMSO-d6): δ 1.81-1.63 (m, 2 H),
2.92-2.79 (m, 2 H), 3.14-2.96 (m, 2 H), 6.91 (s, 1 H), 7.60 (d, J
) 5.51 Hz, 2 H), 8.54 (d, J ) 5.51 Hz, 2 H), 8.64 (t, J ) 5.70 Hz,
1 H), 11.89 (s, 1 H). HRMS (M+H)+ calcd: C13H13N3O + H1,
228.1131. Found: 228.1129. Anal. calcd (C13H13N3O): C, H, N.
4-Oxo-2-(pyridin-4-yl)-1,4,6,7-tetrahydropyrrolo[3,2-c]pyri-
dine-5-carbaldehyde (21). To a fresh solution of POCl3 (0.06 mL,
0.6 mmol) in N,N-dimethylformamide (3 mL) was added 2-(pyridin-
4-yl)-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one hydrochloride
(1; 50 mg, 0.2 mmol), and the mixture was stirred at room
temperature for 18 h. After the addition of water, the precipitate
was filtered, washed with water, saturated with NaHCO3 (aq), and
dried. The title compound was obtained as a white solid (40 mg,
83%).
1H NMR (400 MHz, DMSO-d6): δ 1.15 (t, J ) 7.03 Hz, 3 H),
1.37 (s, 9 H), 3.55 (s, 2 H), 3.82 (d, J ) 5.86 Hz, 2 H), 4.06 (q, J
) 7.33 Hz, 2 H), 7.07 (t, 1 H).
A mixture of 52 (1 g, 4.1 mmol) and 60% sodium hydride (0.41
g, 10.2 mmol) in anhydrous tetrahydrofuran (20 mL) was stirred
for 1 h at room temperature and then cooled to 0 °C. A suspension
of 2-bromo-1-(pyridin-4-yl)ethanone hydrobromide (64a; 1.4 g, 5
mmol) in anhydrous tetrahydrofuran (10 mL) was added dropwise
and the mixture stirred at 0 °C for 4 h. After solvent removal, the
residue was dissolved in ethanol/acetic acid (1:1, 30 mL), and
ammonium acetate (1.27 g, 16.4 mmol) was added. The solution
was stirred at room temperature for 5 h. After concentration, the
crude product was dissolved in ethyl acetate, washed with brine
(× 3), and dried over anhydrous sodium sulfate. The solvent was
removed, and the impure product was purified by chromatography
(eluant: 98:2 dichloromethane/methanol), thus providing 2-(tert-
butoxycarbonylaminomethyl)-5-(pyridin-4-yl)-1H-pyrrole-3-car-
boxylic acid ethyl ester (0.5 g, 35% yield).
1H NMR (400 MHz, DMSO-d6): δ 3.07 (t, J ) 6.52 Hz, 2 H),
4.01 (t, J ) 6.58 Hz, 2 H), 7.56 (s, 1 H), 7.98 (d, J ) 4.76 Hz, 2
H), 8.69 (d, J ) 4.63 Hz, 2 H), 9.37 (s, 1 H), 12.66 (s, 1 H). HRMS
(M+H)+ calcd: 242.0924. Found: 242.0931. Anal. calcd
(C13H11N3O2): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-cyclopropylmethyl-1,5,6,7-tet-
rahydropyrrolo[3,2-c]pyridin-4-one (34). A mixture of 1-(2-
aminopyrimidin-4-yl)-2-bromoethanone hydrobromide (64b; 0.9 g,
3 mmol), cyclopropylmethylamine (1.03 mL, 12 mmol), and
piperidin-2,4-dione (0.51 g, 4.5 mmol), dissolved in absolute ethanol
(10 mL), was stirred under heating at 70 °C for 3 h in a glass
pressure tube. Ethanol was evaporated off, and the crude reaction
mixture was purified by flash chromatography (eluant: 4:1 dichloro-

Cdc7 Kinase Inhibitors. 1.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 497
methane/methanol) and then by crystallization from methanol (0.42
g, 49% yield).
H), 5.85–5.99 (m, 1 H), 7.27 (d, J ) 6.71 Hz, 1 H), 7.36 (s, 1 H),
7.44 (s, 1 H), 7.99 (s, 2 H), 8.14 (d, J ) 6.70 Hz, 1 H). HRMS
(M+H)+ calcd: 272.1506. Found: 272.1510. Anal. calcd
(C14H17N5O.0.7 H2O): C, H, N.
1H NMR (400 MHz, DMSO-d6): δ 0.30–0.48 (m, 4 H),
1.12–1.26 (m, 1 H), 2.97 (t, J ) 6.77 Hz, 2 H), 3.45 (t, J ) 6.46
Hz, 2 H), 4.61 (d, J ) 7.07 Hz, 2 H), 7.34–7.44 (d, J ) 6.95 Hz,
1 H and one s, 1 H), 7.71 (s, 1 H), 8.15 (d, J ) 6.95 Hz, 1 H), 8.31
(s, 1 H). HRMS (M+H)+ calcd: 284.1506. Found: 284.1500. Anal.
calcd (C15H17N5O): C, 56.34; H, 5.67; N, 21.906. Found: C, 50.06;
H, 5.68; N, 19.64.
2-(2-Aminopyrimidin-4-yl)-1-benzyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (39). To a solution of 2-(2-aminopyri-
midin-4-yl)-4-oxo-1,4,6,7-tetrahydropyrrolo[3,2-c]pyridine-5-car-
boxylic acid tert-butyl ester (49, 300 mg, 0.9 mmol) in anhydrous
N,N-dimethylformamide (5 mL) was added 60% NaH (58 mg),
and the mixture was stirred for 2 h at room temperature. Benzyl
bromide (0.25 mL) was added, and the reaction mixture was stirred
overnight. The mixture was poured in water and extracted with
ethyl acetate. The organic layers were dried over anhydrous sodium
sulfate and concentrated. The residue was purified by flash
chromatography (eluant: 10:1 dichloromethane/methanol) to yield
2-(2-aminopyrimidin-4-yl)-1-benzyl-4-oxo-1,4,6,7-tetrahydro-pyr-
rolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (240 mg,
0.58 mmol) that was subjected to deprotection by dissolution in
dry dioxane (200 mL) and methanol (4 mL) followed by treatment
with 4 N HCl in dioxane (2 mL). After being stirred for 2 h at
room temperature followed by being concentrated to dryness,
the material was neutralized with 7 N ammonia in methanol, and
the title compound was obtained as a white solid (154 mg, 75%
yield).
The following compounds were synthesized according to the
general procedure detailed above:
1-Methyl-2-(pyridin-4-yl)-1,5,6,7-tetrahydropyrrolo[3,2-c]pyri-
1
din-4-one (16). H NMR (400 MHz, DMSO-d6): δ 2.87 (t, J )
6.89 Hz, 1 H), 3.45 (td, J ) 6.95, 2.56 Hz, 2 H), 3.65 (s, 3 H),
6.66 (s, 1 H), 7.06 (m, 1 H), 7.50 (d, J ) 6.10 Hz, 2 H), 8.59 (d,
J ) 6.22 Hz, 2 H). HRMS (M+H)+ calcd: 228.1131. Found:
228.1126. Anal. calcd (C13H13N3O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-cyclobutyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (31). 1H NMR (400 MHz, DMSO-d6): δ
1.68–1.89 (m, 2 H), 2.38–2.59 (m, 3 H), 3.11–3.21 (m, 2 H),
3.24–3.63 (m, 2 H), 5.66–5.82 (m, 1 H), 7.30 (d, J ) 6.71 Hz, 1
H), 7.42 (s, 1 H), 7.46 (s, 1 H), 8.16 (d, J ) 6.83 Hz, 1 H), 8.26
(bs, 2 H). HRMS (M+H)+ calcd: 284.1506. Found: 284.1512. Anal.
calcd (C15H17N5O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-cyclohexyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (37). 1H NMR (400 MHz, DMSO-d6): δ
1.15–2.04 (m, 10 H), 2.98–3.07 (m, 2 H), 3.52–3.61 (m, 2 H),
4.04–4.17 (m, 1 H), 7.40–7.49 (m, 2 H), 8.36–8.51 (m, 5 H),
8.58–8.65 (m, 1 H). HRMS (M+H)+ calcd: 312.1819. Found:
312.1816. Anal. calcd (C17H21N5O): C, H, N.
1H NMR (400 MHz, DMSO-d6): δ 2.87–2.94 (m, 2 H),
3.39–3.51 (m, 2 H), 6.01 (s, 2 H), 7.01–7.07 (m, 2 H), 7.19–7.25
(m, 1 H), 7.26–7.34 (m, 3 H), 7.39 (s, 1 H), 7.71 (s, 1 H), 8.00 (s,
2 H), 8.09 (d, J ) 6.83 Hz, 1 H). HRMS (M+H)+ calcd: 320.1506.
Found: 320.1497. Anal. calcd (C17H22N6O): C, H, N.
The following compounds were synthesized according to the
general procedure detailed above:
2-(2-Aminopyrimidin-4-yl)-1-phenyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (38). 1H NMR (400 MHz, DMSO-d6): δ
2.90–2.99 (m, 2 H), 3.48–3.58 (m, 2 H), 7.54–7.68 (m, 7 H),
8.36–8.75 (m, 4 H). HRMS (M+H)+ calcd: 306.1349. Found:
306.1342. Anal. (C17H15N5O): C, H, N.
1-Ethyl-2-(pyridin-4-yl)-1,5,6,7-tetrahydropyrrolo[3,2-c]-
pyridin-4-one (22). 1H NMR (400 MHz, DMSO-d6): δ 1.28
(t, J ) 7.19 Hz, 3 H), 2.97 (t, J ) 6.83 Hz, 2 H), 3.11–3.30 (m, 2
H), 4.21 (q, J ) 7.19 Hz, 2 H), 7.18 (s, 1 H), 7.29 (s, 1 H), 8.00 (s,
1 H), 8.01 (s, 1 H), 8.74 (s, 1 H), 8.76 (s, 1 H). HRMS (M+H)+
calcd: 242.1288. Found: 242.1291. Anal. calcd (C14H15N3O): C, H,
N.
1-(2-Hydroxyethyl)-2-(pyridin-4-yl)-1,5,6,7-tetrahydropyrrolo-
[3,2-c]pyridin-4-one (23). 1H NMR (400 MHz, DMSO-d6): δ 2.97
(t, J ) 6.89 Hz, 2 H), 3.28–3.53 (m, 2 H), 3.65 (t, J ) 5.43 Hz, 2
H), 4.25 (t, J ) 5.49 Hz, 2 H), 7.13–7.18 (m, 1 H), 7.26–7.31 (m,
1 H), 8.12–8.16 (m, J ) 6.22 Hz, 2 H), 8.74 (s, 1 H), 8.76 (s, 1 H).
HRMS (M+H)+ calcd: 258.1237. Found: 258.1231. Anal. calcd
(C14H15N3O2 0.6 H2O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-(1-methyl-piperidin-4-yl)-1,5,6,7-
tetrahydropyrrolo[3,2-c]pyridin-4-one (40). 1H NMR (400 MHz,
DMSO-d6): δ 2.07–2.18 (m, 4 H), 2.79 (d, J ) 4.63 Hz, 3 H),
3.12–3.20 (m, 2 H), 3.21–3.54 (m, 6 H), 5.66–5.86 (m, 1 H), 7.27
(s, 1 H), 7.41 (s, 1 H), 7.46 (s, 1 H), 8.07 (bs, 2 H), 8.16 (d, J )
6.46 Hz, 1 H), 10.45–10.71 (m, 1H). HRMS (M+H)+ calcd:
327.1928. Found: 327.1937. Anal. calcd (C17H22N6O.0.5 H2O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-(2-fluoroethyl)-1,5,6,7-tetrahy-
dropyrrolo[3,2-c]pyridin-4-one (35). A mixture of 2-(2-ami-
nopyrimidin-4-yl)-4-oxo-1,4,6,7-tetrahydropyrrolo[3,2-c]pyridine-
5-carboxylic acid tert-butyl ester (49; 100 mg, 0.3 mmol),
1-bromo-2-fluoroethane (0.09 mL, 1.2 mmol), and potassium carbonate
(166 mg, 1.2 mmol) in anhydrous N,N-dimethylformamide (2 mL)
was stirred under heating at 65 °C for 4 h. After cooling, the reaction
mixture was treated with water and ethyl acetate; the organic layer
was extracted with brine and then dried over anhydrous sodium
sulfate. The crude product was purified by flash chromatography
(eluant: 95:5 dichloromethane/ethanol). Pure 2-(2-aminopyrimidin-
4-yl)-1-(2-fluoroethyl)-4-oxo-1,4,6,7-tetrahydropyrrolo[3,2-c]py-
ridine-5-carboxylic acid tert-butyl ester, obtained as a solid (90
mg, 80% yield), was deprotected in dry methanol (5 mL) and 4
N HCl in dioxane (0.6 mL), by stirring the solution at room
temperature for 3 h. After concentration to dryness, the material
was neutralized with 7 N ammonia in methanol, and the title
compound was obtained as a white solid (71 mg, 95% yield).
1H NMR (400 MHz, DMSO-d6): δ 2.91–2.98 (m, 2 H),
3.40–3.50 (m, 2 H), 4.67–4.75 (m, 1 H), 4.80–4.85 (m, 1 H),
4.85–4.90 (m, 1 H), 4.90–4.97 (m, 1 H), 7.40 (d, J ) 6.83 Hz, 1
H and 1 singlet, 1H), 7.76 (s, 1 H), 8.14 (d, J ) 6.82 Hz, 1 H),
8.17–8.51 (m, 2 H). HRMS (M+H)+ calcd: 276.1255. Found:
276.1244. Anal. calcd (C13H14FN5O): C, H, N.
2-(Pyridin-4-yl)-1-(2,2,2-trifluoroethyl)-1,5,6,7-tetrahydro-
1
pyrrolo[3,2-c]pyridin-4-one (24). H NMR (400 MHz, DMSO-
d6): δ 2.98 (t, J ) 6.71 Hz, 2 H), 3.41–3.55 (m, 2 H), 5.22 (q, J )
8.86 Hz, 2 H), 7.04 (s, 1 H), 7.39 (s, 1 H), 7.97 (s, 2 H), 8.77 (s,
1 H), 8.79 (s, 1 H). HRMS (M+H)+ calcd: 296.1005. Found:
296.0998. Anal. calcd (C14H12F3N3O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-methyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (25). 1H NMR (400 MHz, DMSO-d6): δ
2.83–2.89 (m, 2 H), 3.40–3.47 (m, 2 H), 3.96 (s, 3 H), 6.54 (s, 2
H), 6.89 (d, J ) 5.37 Hz, 1 H), 7.01 (s, 1 H), 7.09 (s, 1 H), 8.14
(d, J ) 5.37 Hz, 1 H). HRMS (M+H)+ calcd: 244.1193. Found:
244.1194. Anal. calcd (C12H13N5O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-ethyl-1,5,6,7-tetrahydropyr-
1
rolo[3,2-c]pyridin-4-one (26). H NMR (400 MHz, DMSO-d6):
δ 1.29 (t, J ) 7.08 Hz, 3 H), 2.99 (t, J ) 6.78 Hz, 2 H), 3.48 (t,
J ) 6.77 Hz, 2 H), 4.62 (q, J ) 7.00 Hz, 2 H), 7.38 (s, 1 H), 7.40
(d, J ) 7.00 Hz, 1 H), 7.72 (s, 1 H), 8.16 (d, J ) 7.00 Hz, 1 H).
HRMS (M+H)+ calcd: 258.1349. Found: 258.1349. Anal. calcd
(C13H15N5O): C, H, N.
2-(2-Aminopyrimidin-4-yl)-1-(2-hydroxy-ethyl)-1,5,6,7-tet-
1
rahydropyrrolo[3,2-c]pyridin-4-one (27). H NMR (400 MHz,
The following compound was synthesized according to the
general procedure detailed above:
2-(2-Aminopyrimidin-4-yl)-1-isopropyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (30). 1H NMR (400 MHz, DMSO-d6): δ
1.50 (d, J ) 6.95 Hz, 6 H), 3.05–3.14 (m, 2 H), 3.39–3.48 (m, 2
DMSO-d6): δ 2.97 (t, J ) 6.77 Hz, 2 H), 3.36–3.50 (m, 2 H), 3.66
(t, J ) 5.37 Hz, 2 H), 4.58 (t, J ) 5.30 Hz, 2 H), 7.33 (d, J )
10.12 Hz, 1 H), 7.35 (d, J ) 7.19 Hz, 1 H), 7.69 (s, 1 H), 8.09 (d,
J ) 6.95 Hz, 1 H). HRMS (M+H)+ calcd: 274.1298. Found:
274.1291. Anal. calcd (C13H15N5O2): C, H, N.

498 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Vanotti et al.
2-(2-Aminopyrimidin-4-yl)-1-(2,2,2-trifluoroethyl)-1,5,6,7-tet-
rahydropyrrolo[3,2-c]pyridin-4-one (28). H NMR (400 MHz,
2-(2-Isobutylaminopyrimidin-4-yl)-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (42). H NMR (400 MHz, DMSO-d6):
1
1
DMSO-d6): δ 3.01 (t, J ) 6.70 Hz, 2 H), 3.43–3.51 (m, 2 H), 5.89
(d, J ) 7.76 Hz, 2 H), 7.40 (d, J ) 6.70 Hz, 1 H), 7.50 (s, 1 H),
7.74 (s, 1 H), 8.23 (d, J ) 6.55 Hz, 1 H). HRMS (M+H)+ calcd:
312.1067. Found: 312.1072. Anal. calcd (C13H12F3N5O): C, H,
N.
2-(2-Aminopyrimidin-4-yl)-1-propyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (29). 1H NMR (400 MHz, DMSO-d6): δ
0.85 (t, J ) 7.32 Hz, 3 H), 1.57–1.70 (m, 2 H), 2.91–3.00 (m, 2
H), 3.42–3.52 (m, 2 H), 4.56 (t, J ) 7.19 Hz, 2 H), 7.31–7.39 (m,
2 H), 7.65 (s, 1 H), 8.12 (d, J ) 6.82 Hz, 1 H). HRMS (M+H)+
calcd: 272.1506. Found: 272.1509. Anal. calcd (C14H17N5O): C,
H, N.
2-(2-Aminopyrimidin-4-yl)-1-butyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (32). 1H NMR (400 MHz, DMSO-d6): δ
0.87 (t, J ) 7.44 Hz, 3 H), 1.22–1.34 (m, 2 H), 1.52–1.63 (m, 2
H), 2.91–2.99 (m, 2 H), 3.42–3.51 (m, 2 H), 4.60 (t, J ) 7.19 Hz,
2 H), 7.32 (d, J ) 6.95 Hz, 1 H), 7.34 (s, 1 H), 7.61 (s, 1 H),
7.97 (s, 2 H), 8.12 (d, J ) 6.70 Hz, 1 H). HRMS (M+H)+
calcd: 286.1662. Found: 286.1669. Anal. calcd (C15H19N5O): C,
H, N.
2-(2-Aminopyrimidin-4-yl)-1-isobutyl-1,5,6,7-tetrahydropyr-
rolo[3,2-c]pyridin-4-one (33). 1H NMR (400 MHz, DMSO-d6): δ
0.82 (d, J ) 6.71 Hz, 6 H), 1.80–1.94 (m, 1 H), 2.88–2.98 (m, 2
H), 3.42–3.54 (m, 2 H), 4.43 (d, J ) 7.20 Hz, 2 H), 7.32 (d, J )
6.83 Hz, 1 H), 7.36 (s, 1 H), 7.62 (s, 1 H), 7.97 (s, 2 H), 8.12 (d,
J ) 6.71 Hz, 1 H). HRMS (M+H)+ calcd: 286.1662. Found:
286.1663. Anal. calcd (C15H19N5O): C, H, N.
δ 0.93 (d, J ) 6.71 Hz, 6 H), 1.81–1.95 (m, 1 H), 2.87 (t, J ) 6.83
Hz, 2 H), 3.24 (s, 2 H), 3.41 (td, J ) 6.98, 2.38 Hz, 2 H), 6.71–6.94
(m, 1 H), 6.87 (d, J ) 5.24 Hz, 1 H), 7.04 (d, J ) 2.32 Hz, 1 H),
7.06 (t, J ) 2.38 Hz, 1 H), 8.16 (d, J ) 5.12 Hz, 1 H), 11.66 (s,
1 H). HRMS (M+H)+ calcd: 286.1662. Found: 286.1664. Anal.
calcd (C15H19N5O): C, H, N.
2-(2-Benzylaminopyrimidin-4-yl)-1,5,6,7-tetrahydropyrrolo[3,2-
1
c]pyridin-4-one (44). H NMR (500 MHz, DMSO-d6): δ 2.87 (t,
J ) 6.85 Hz, 2 H), 3.42 (td, J ) 6.85, 2.44 Hz, 2 H), 4.68 (s, 2 H),
6.92 (d, J ) 5.18 Hz, 1 H), 7.07 (d, J ) 2.28 Hz, 1 H), 7.08 (t, J
) 2.51 Hz, 1 H), 7.21–7.26 (m, 1 H), 7.27–7.48 (m, 1 H), 7.32 (t,
J ) 7.54 Hz, 2 H), 7.39 (d, J ) 7.76 Hz, 2 H), 8.19 (d, J ) 5.18
Hz, 1 H), 11.73 (s, 1 H). HRMS (M+H)+ calcd: 320.1506. Found:
320.1511. Anal. calcd (C18H17N5O): C, H, N.
N-[4-(4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-
yl)pyrimidin-2-yl]benzamide (48). To a mixture of 2-(2-aminopy-
rimidin-4-yl)-4-oxo-1,4,6,7-tetrahydropyrrolo[3,2-c]pyridine-5-car-
boxylic acid tert-butyl ester (49; 0.13 g, 0.39 mmol) and triethylamine
(0.22 mL, 1.58 mmol) in dry tetrahydrofuran (5 mL) was added
benzoyl chloride (0.09 mL, 0.79 mmol), and the mixture was stirred
at room temperature overnight. To this, 1 N NaOH (4 mL) was
added; after 0.5 h, the solvent was evaporated, the residue was
dissolved in water, and the aqueous phase was extracted with ethyl
acetate (20 mL × 2). The organic phase was washed with saturated
NH4Cl (aq) then dried over anhydrous sodium sulfate. After solvent
evaporation, the crude product was purified by flash chromatography
(97:3 dichloromethane/methanol) to give 2-(2-benzoylaminopyri-
midin-4-yl)-4-oxo-1,4,6,7-tetrahydropyrrolo[3,2-c]pyridine-5-car-
boxylic acid tert-butyl ester, as a solid (0.14 g, 83%).
2-(2-Aminopyrimidin-4-yl)-1-(4,4,4-trifluorobutyl)-1,5,6,7-tet-
1
rahydropyrrolo[3,2-c]pyridin-4-one (36). H NMR (400 MHz,
1H NMR (400 MHz, DMSO-d6): δ 1.49 (s, 9 H), 2.98 (t, J )
6.34 z, 2 H), 3.98 (t, J ) 6.34 Hz, 2 H), 7.35 (s, 1 H), 7.51–7.66
(m, 4 H), 8.00 (m, 2 H), 10.82 (s, 1 H), 12.11 (bs, 1 H).
To a solution of this product (0.05 g, 0.115 mmol) in tetrahy-
drofuran (1 mL) was added 4 N HCl in dioxane (0.2 mL), and the
reaction was left under stirring at room temperature for 2 h. After
concentration to dryness, the material was neutralized with 7 N
ammonia in methanol, and the title compound was obtained as a
solid (0.036 g, 94%).
DMSO-d6): δ 1.80–1.90 (m, 2 H), 2.35–2.49 (m, 2 H), 2.96 (t, J )
6.77 Hz, 2 H), 3.47 (ddd, J ) 6.80, 2.26 Hz, 2 H), 4.64 (t, J )
7.32 Hz, 2 H), 7.35 (d, J ) 6.71 Hz 1 H), 7.36 (s, 1 H), 7.68 (s, 1
H), 7.94–8.04 (m, 2 H), 8.13 (d, J ) 6.71 Hz, 1 H). HRMS
(M+H)+ calcd: 340.1380. Found: 340.1373. Anal. calcd
(C15H16F3N5O): C, H, N.
2-[2-(Cyclohexylmethylamino)pyrimidin-4-yl]-1,5,6,7-tetrahy-
dropyrrolo[3,2-c]pyridin-4-one (45). 2-(2-Aminopyrimidin-4-yl)-
1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one (5; 140 mg, 0.61
mmol), trifluoroacetic acid (0.56 mL, 7.33 mmol), and cyclohexa-
necarbaldehyde (0.15 mL, 1.25 mmol) were mixed in N,N-
dimethylformamide (10 mL). Sodium triacetoxyborohydride (390
mg, 1.84 mmol) was added and the reaction mixture stirred at
room temperature for 20 h. Additional cyclohexanecarbaldehyde
(0.15 mL, 1.25 mmol) and sodium triacetoxyborohydride (390
mg, 1.84 mmol) were added, and the reaction mixture was stirred
20 h longer. The reaction was quenched with 0.33 N NaOH (50
mL), and the product was extracted with dichloromethane (50
mL). The dichloromethane extract was dried over anhydrous
sodium sulfate, and the solvent was evaporated. The residue was
purified by flash chromatography (dichloromethane/methanol, 95:
5) to give the product as a beige-colored solid (80 mg, 40%
yield).
1H NMR (DMSO-d6): δ 2.91 (t, J ) 6.77 Hz, 2 H), 3.44 (2 H),
7.22 (bs, 1 H), 7.36 (1 H), 7.57 (m, 2 H), 7.64 (m, 2 H), 8.03 (m,
2 H), 8.59 (d, J ) 5.73 Hz, 1 H), 11.26 (bs, 1 H), 12.13 (bs, 1 H).
HRMS (M+H)+ calcd: 334.1298. Found: 334.1308. Anal. calcd
(C18H15N5O2): C, H, N.
The following compounds were synthesized according to the
general procedure detailed above:
N-[4-(4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-
1
yl)pyrimidin-2-yl]acetamide (46). H NMR (400 MHz, DMSO-
d6): δ 2.31 (s, 3 H), 2.92 (t, J ) 6.71, 2 H), 3.51 (m, 2 H), 7.26
(bs, 1 H), 7.37 (1 H, s), 7.60 (d, J ) 6.10 Hz, 1 H), 8.50 (d, J )
6.10 Hz, 1 H), 11.11 (bs, 1 H), 12.22 (bs, 1 H). HRMS (M+H)+
calcd: 272.1142. Found: 272.1150. Anal. calcd (C13H13N5O2): C, H, N.
N-[4-(4-Oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-
yl)-pyrimidin-2-yl]isobutyramide (47). 1H NMR (400 MHz,
DMSO-d6): δ 1.14 (s, 3 H), 1.16 (s, 3 H), 2.92 (m, 2 H), 3.44 (m,
2 H), 7.25 (bs, 1 H), 7.36 (s, 1 H), 7.58 (d, J ) 5.85 Hz, 1 H), 8.50
(d, J ) 5.85 Hz, 1 H), 11.03 (bs, 1 H), 12.16 (bs, 1 H). HRMS
(M+H)+ calcd: 300.1455. Found: 300.1469. Anal. calcd
(C15H17N5O2): C, H, N.
2,4-Dioxopiperidine-1-carboxylic acid tert-butyl ester (56)
and 2,4-dioxopiperidine (57). To a solution of N-Boc-ꢀ-alanine
(25 g, 132 mmol), 2,2-dimethyl-1,3-dioxane-4,6-dione (20.9 g, 145
mmol), and 4-dimethylaminopyridine (24.2 g, 198 mmol) in
anhydrous dichloromethane (700 mL) at 0 °C was added 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (30.4 g,
158 mmol), and the resulting solution was stirred overnight at room
temperature. The reaction mixture was washed (500 mL × 4) with
5% potassium bisulfate (aq). The organic layer was dried over
anhydrous sodium sulfate, filtered, and concentrated, thereby
affording crude [3-(2,2-dimethyl-4,6-dioxo-[1,3]-dioxan-5-yl)-3-
1H NMR (400 MHz, DMSO-d6): δ 0.87–1.29 (m, 5 H),
1.43–1.85 (m, 6 H), 2.87 (t, J ) 6.77 Hz, 2 H), 3.18–3.31 (m, 2
H), 3.38–3.49 (m, 2 H), 6.67–6.95 (m, 1 H), 6.85 (d, J ) 5.24 Hz,
1 H), 7.03 (d, J ) 2.19 Hz, 1 H), 7.06 (t, J ) 2.07 Hz, 1 H), 8.15
(d, J ) 5.12 Hz, 1 H), 11.65 (s, 1 H). HRMS (M+H)+ calcd:
326.1975. Found: 326.1979. Anal. calcd (C18H23N5O): C, 66.44;
H, 7.12; N, 21.52. Found: C, 65.49; H, 7.22; N, 20.64.
The following compounds were synthesized according to the
general procedure detailed above:
2-(2-Propylaminopyrimidin-4-yl)-1,5,6,7-tetrahydropyrrolo-
[3,2-c]pyridin-4-one (41). 1H NMR (400 MHz, DMSO-d6): δ 0.93
(t, J ) 7.38 Hz, 3 H), 1.51–1.64 (m, 2 H), 2.87 (t, J ) 6.89 Hz,
2 H), 3.34–3.48 (m, 4 H), 6.81 (s, 1 H), 6.86 (d, J ) 5.24 Hz,
1 H), 7.03 (d, J ) 2.32 Hz, 1 H), 7.05 (t, J ) 2.07 Hz, 1 H),
8.16 (d, J ) 5.24 Hz, 1 H), 11.66 (s, 1 H). HRMS (M+H)+
calcd: 272.1506. Found: 272.1509. Anal. calcd (C14H17N5O): C,
H, N.

Cdc7 Kinase Inhibitors. 1.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 499
oxo-propyl]-carbamic acid tert-butyl ester (55) that was dissolved
in 600 mL of ethyl acetate and refluxed for 4 h. The volume was
reduced to 150 mL, and the resulting solution was allowed to
crystallize at 4 °C overnight. The solid was filtered and washed
with cold ethyl acetate, affording 56 (18.4 g, 65% yield).
1H NMR (400 MHz, DMSO-d6): δ 1.44 (s, 9 H), 2.44 (m, 2 H),
3.71 (m, 2 H), 4.95 (s, 1 H), 11.2 (bs, 1 H). ESI (+) MS: m/z 214
(MH+). Optionally, the tert-butoxycarbonyl group can be removed
by acidic treatment (4 N HCl in dioxane or tetrahydrofuran) at room
temperature to yield 57.
extracted with ethyl acetate and dried over anhydrous sodium
sulfate. After concentration, the crude oil was purified by chroma-
tography (eluant: ethyl acetate). 6-(1-Ethoxy-vinyl)-9-(tetrahydro-
pyran-2-yl)-9H-purine was obtained pure (63g; 0.54 g) in 94% yield.
A portion of the compound (0.43 g, 1.57 mmol), dissolved in
tetrahydrofuran (24 mL) and water (1.5 mL), was treated with
N-bromosuccinimide (0.28 g, 1.57 mmol) and stirred at room
temperature for 0.25 h. The solution was evaporated under reduced
pressure, taken up with water, and filtered to obtain the title
compound (0.3 g, 81% yield) as a yellow solid.
1H NMR (300 MHz, DMSO-d6): δ 5.12 (s, 2 H), 8.85 (s, 1 H),
9.14 (s, 1 H). ESI (+) MS: m/z 242 (MH+).
2-Bromo-1-(3-fluoropyridin-4-yl)ethanone hydrobromide
(64d). To a stirred solution of commercially available 3-fluoro-4-
acetylpyridine (63d; 5.3 g, 38.1 mmol) in glacial acetic acid (14
mL) and 48% hydrobromic acid (5.3 mL) was added bromine (2
mL, 38 mmol) in glacial acetic acid (5.3 mL) dropwise. After the
addition was completed, the solution was stirred at 60 °C for 2.5 h
and cooled to room temperature, and ethyl acetate (70 mL) was
added. After 0.5 h of stirring, the mixture was filtered, and the
solid was washed thoroughly with ethyl acetate and dried. After
evaporation of the solvent, the title compound was obtained in 82%
yield (9.3 g).
2-Bromo-1-(2-phenylamino-pyrimidin-4-yl)-ethanone (64i).
To a solution of sodium (614 mg, 26.7 mmol) in absolute ethanol
(70 mL) was added phenylguanidine carbonate (4.43 g, 13.35
mmol), followed by a solution in absolute ethanol (20 mL) of
1-dimethylamino-4,4-dimethoxypent-1-en-3-one (61; 5 g, 26.7
mmol), obtained as described in the literature.22 The suspension
was refluxed for 20 h; then, 2/3 of the solvent was removed, and
water (250 mL) was added. The precipitate was extracted with ethyl
acetate, and the organic phase was washed with saturated sodium
dihydrogenphosphate (aq) and with brine, dried over anhydrous
sodium sulfate, and concentrated to yield [4-(1,1-dimethoxyeth-
yl)pyrimidin-2-yl]phenylamine (62; 4.3 g, 62%). This ketal (4.2 g,
16.19 mmol) was dissolved in 88% formic acid (25 mL) and stirred
at room temperature for 2.5 h. The reaction mixture was diluted
with water (200 mL); the precipitate was filtered and washed
copiously with water, yielding 1-(2-phenylaminopyrimidin-4-yl)-
ethanone (63i) as a yellow solid (3.1 g, 90%). To a solution of this
ketone (1.3 g, 6.1 mmol) in dichloromethane (40 mL) were added
triethylamine (5.1 mL, 36.6 mmol) and tert-butyldimethylsilyl
trifluoromethansulphonate (4.2 mL, 18.3 mmol). The orange
solution was stirred overnight at room temperature; diluted with
dichloromethane (150 mL); washed with 5% sodium bicarbonate
(aq, ×2), with water, and with brine; dried over anhydrous sodium
sulfate; and concentrated to give (tert-butyldimethylsilanyl)-{4-[1-
(tert-butyldimethylsilanyloxy)-vinyl]pyrimidin-2-yl}phenylamine
(63m; 2.64 g, 96%). To derivative 63m (1.32 g, 2.94 mmol),
dissolved in tetrahydrofuran (25 mL) and water (3 mL) at room
temperature, was added solid N-bromosuccinimide (0.549 g, 3.09
mmol), and the reaction mixture was stirred for 20 h at room
temperature. After concentration and aqueous workup with ethyl
acetate, the crude material was purified by flash chromatography
(eluant: 4:1 n-hexane/ethyl acetate) to yield the title compound (0.66
g, 46%).
1H NMR (400 MHz, DMSO-d6): δ 4.88 (s, 2 H), 7.75–7.79 (m,
1 H), 8.62 (dd, J ) 4.94, 1.52 Hz, 1 H), 8.79 (d, J ) 2.32 Hz, 1
H). ESI (+) MS: m/z 219 (MH+).
The following compound was synthesized according to the
general procedure detailed above:
2-Bromo-1-(pyrimidin-4-yl)ethanone hydrobromide (64e). 1H
NMR (300 MHz, DMSO-d6): δ 5.0 (s, 2 H), 7.98 (d, J ) 5 Hz, 1
H), 9.12 (d, J ) 5 Hz, 1 H), 9.42 (s, 1 H). ESI (+) MS: m/z 202
(MH+).
4-(2-Bromo-acetyl)pyrrolo[2,3-b]pyridine-1-carboxylic acid
tert-butyl ester (64f). To a suspension of commercially available
4-chloro-1H-pyrrolo[2,3-b]pyridine (58; 0.5 g, 3.2 mmol) and
dimethylaminopyridine (0.04 g, 0.32 mmol) in acetonitrile (10 mL)
was added a solution of di-tert-butyl-dicarbonate (0.86 g, 3.9 mmol)
in acetonitrile (3 mL) dropwise. After stirring for 1 h at room
temperature, the solution was diluted with ice water and extracted
with ethyl acetate. The organic layer was washed with 5% potassium
bisulfate (aq), then with brine, and dried over anhydrous sodium
sulfate. After solvent removal, 4-chloro-pyrrolo[2,3-b]pyridine-1-
carboxylic acid tert-butyl ester was obtained (59; 0.8 g, 96% yield).
Part of this crude product (0.5 g, 1.98 mmol) was dissolved in
anhydrous N,N-dimethylformamide (5 mL), and to the solution,
thoroughly degassed with argon, were added palladium-tetrakis-
(triphenylphosphine) (0.1 g + 0.04 g after 5 h + 0.04 g after 8 h)
and 1-ethoxyvinyltri-n-butyltin (0.96 mL, 2.83 mmol); the solution
was thoroughly degassed with argon and heated with stirring at
100 °C for 10 h. Water was added, and the reaction mixture was
extracted with ethyl acetate and dried over anhydrous sodium
sulfate. After concentration, the crude oil was purified by chroma-
tography (eluant: 5:1 n-hexane/ethyl acetate), and 4-(1-ethoxyvi-
nyl)pyrrolo[2,3-b]pyridine-1-carboxylic acid tert-butyl ester (63f,
0.38 g, 67% yield) was obtained as a yellow oil. To part of this
product (0.37 g, 1.28 mmol), dissolved in tetrahydrofuran (16 mL)
and water (1 mL), was added N-bromosuccinimide (0.23 g, 1.28
mmol), and the solution was stirred at room temperature for 3 h.
After solvent removal, the crude reaction product was purified by
chromatography (eluant: 3:1 n-hexane/ethyl acetate), yielding the
title product (0.2 g, 46% yield).
1H NMR (300 MHz, DMSO-d6): δ 4.65 (s, 2 H), 6.7 (m, 1 H),
6.9 (d, J ) 5 Hz, 1 H), 7.0 (m, 2 H), 7.4 (d, J ) 7.7 Hz, 2 H), 8.4
(d, J ) 5 Hz, 1 H), 9.6 (s, 1 H). ESI (+) MS: m/z 293 (MH+).
II. Registry Numbers (RN). 2-Bromo-1-(4-pyridinyl)-ethanone
hydrobromide (RN, 5349-17-7), 2-bromo-1-(3-fluoro-4-pyridinyl)-
ethanone hydrobromide (RN, 845538-59-2), 2-bromo-1-(pyridin-
4-yl)propan-1-one (RN, 780692-61-7), 2-bromo-1-(3-pyridinyl)-
ethanone (RN, 6221-12-1), R-bromoacetophenone (RN, 70-11-1),
1-(2-amino-4-pyrimidinyl)-2-bromo-ethanone (RN, 106157-91-9),
4-acetylpyrimidine (RN, 39870-05-8), 3-fluoro-4-acetylpyridine
(RN, 87674-21-3), 4-chloro-1H-pyrrolo[2,3-b]pyridine (RN, 55052-
28-3), 6-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (RN, 7306-
68-5), 1-(dimethylamino)-4,4-dimethoxy-1-penten-3-one (RN, 106157-
94-2).
1H NMR (300 MHz, DMSO-d6): δ 1.64 (s, 9 H), 4.72 (s, 2 H),
7.12 (d, J ) 3.9 Hz, 1 H), 7.59 (d, J ) 4.5 Hz, 1 H), 7.83 (d, J )
3.9 Hz, 1 H), 8.89 (d, J ) 4.5 Hz, 1 H). ESI (+) MS: m/z 340
(MH+).
2-Bromo-1-(9H-purin-6-yl)ethanone (64g). To a solution of
commercially available 6-chloro-9-(tetrahydropyran-2-yl)-9H-purine
(60; 0.5 g, 2.1 mmol) in anhydrous N,N-dimethylformamide (5 mL),
thoroughly degassed with argon, were added palladium-tetrakis-
(triphenylphosphine) (0.12 g, 0.1 mmol + 0.06 g after 5 h) and
1-ethoxyvinyltri-n-butyltin (1 mL, 3 mmol); the solution was
thoroughly degassed with argon and warmed at 100 °C under
stirring for 8 h. Water was added, and the reaction mixture was
III. Molecular Modeling. Molecular modeling activities were
performed with the Insight II and Cerius 2 programs (Accelrys).
Docking was also performed with the QXP program. The homology
model of Cdc7 was built with the “Homology” module of Insight
II (Accelrys).
Sequence similarity searches were performed using BLAST23
and visualized by Blixem.24 The alignment was performed using
Clustal W25 and visualized using two different programs: Belvu
(http://www.cgb.ki.se/cgb/groups/sonnhammer/Belvu.html) and Vec-
tor NTI AlignX (Invitrogen).
IV. In Vitro Kinase Assays. Inhibition of kinase activity was
assessed using a Dowex resin capture technique. In this assay, 3

500 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Vanotti et al.
(2) Longley, D. B.; Johnston, P. G. Molecular mechanisms of drug
resistance. J. Pathol. 2005, 205, 275–292.
µM MCM2 (10-294) is phosphorylated by 2 nM Cdc7/Dbf4 in the
presence of ATP (1.4 µM) traced with P33-ATP in a kinase buffer
(50 mM Hepes, pH 7.9, 2 mM ꢀ-glycerylphosphate, 2 mM DTT,
3 µM NaVO3, 15 mM MgCl2, 0.2 mg/ml BSA) for 60 min. By the
addition of an acidic suspension of Dowex resin (SIGMA, custom-
prepared resin Dowex 1 × 8 200–400 mesh equilibrated in 150
mM sodium formate, pH 3.00), the unreacted ATP is captured and
separated from the supernatant which contains the phosphorylated
substrate: this is then transferred onto a new plate for radioactivity
counting. The assay is run in a robotized format on 384-well plates.
The kinase activity of Cdc7/Dbf4 was determined by measuring
the rate of phosphorylation of Mcm2 (aa 10-294) in the presence
of ATP traced with radiolabeled ATP (Revidue [γ33P] ATP,
Amersham Pharmacia Biotech, U.K.). The buffer composition was
determined by measuring the rate of substrate transphosphorylation
in experiments where the buffer pH and concentrations of Mg2+
and Mn2+ were varied from 7 to 8, 1 to 20 mM, and 0 to 5 mM,
respectively. Final concentrations of 2 mM ꢀ-glycerylphosphate,
1 mM DTT, 3 µM M Na3VO4, and 0.2 mg/ml BSA were kept
constant. The choice of the buffer providing the highest velocity
was made by using a statistical approach (DOE) and resulted in
(3) Bell, S. P.; Dutta, A. DNA replication in eukaryotic cells. Annu. ReV.
Biochem. 2002, 71, 333–374.
(4) Montagnoli, A.; Valsasina, B.; Brotherton, D.; Troiani, S.; Rainoldi,
S.; Tenca, P.; Molinari, A.; Santocanale, C. Identification of Mcm2
Phosphorylation Sites by S-phase-regulating Kinases. J. Biol. Chem.
2006, 281, 10281–10290.
(5) Sheu, Y. J.; Stillman, B. Cdc7-Dbf4 phosphorylates MCM proteins
via a docking site-mediated mechanism to promote S phase progres-
sion. Mol. Cell 2006, 24, 101–113.
(6) Montagnoli, A.; Tenca, P.; Sola, F.; Carpani, D.; Brotherton, D.;
Albanese, C.; Santocanale, C. Cdc7 inhibition reveals a p53-dependent
replication checkpoint that is defective in cancer cells. Cancer Res.
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Santocanale, C.; Martina, K.; Menichincheri, M. Pyridylpyrrole
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Santocanale, C.; Orsini, P.; Pillan, A. Pyrimidylpyrrole derivatives
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Kurumbail, R. G.; Caspers, N.; Poda, G. I.; Schindler, J. F.; Reitz,
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the choice of HEPES 50 mM, pH 7.9, containing 15 mM Mg2+
.
The rate of transphosphorylation was measured through the selective
capture of unreacted ATP by a strong anion exchange resin (Dowex
1 × 8, formate form, Supelco, PA), essentially according to the
literature.26 Briefly, to 15 µL of reaction mixture was added 60 µL
of a 1:3 (v/v) suspension of resin equilibrated with 150 mM Na-
formate in water, pH 3.0, which also served to stop the reaction.
After careful mixing and then allowing the resin to settle for 1 h,
20 µL of supernatant was carefully withdrawn using a Biomek FX
pipet (Beckman Coulter inc., CA) and added to a 384-well plate
(Optiplate, Packard) containing 60 µL of Microscint 40 (Perkin-
Elmer). The radioactivity remaining in the supernatant that was
incorporated into the positively charged substrate was then measured
using a Top Count NXT (Packard). The assay was run in a robotized
format on 384-well plates. Inhibitory potency evaluation was
performed at 25 °C using a 60 min end-point assay where the
concentrations of ATP and Mcm2 were kept equal to 2xRKm and
saturated (>5xRKm), respectively. The Cdc7/Dbf4 concentration
was kept sufficiently low to avoid substrate depletion within the
duration of the end-point assay and to reduce the limit for tight-
binding inhibitors.
V. Cell Culture. A2780 (European collection of cell culture)
was grown in an I1640 medium supplemented with heat-inactivated
10% fetal calf serum.
VI. Inhibition of Cell Proliferation. Cells were seeded at
different densities ranging from 2500 to 5000 cells/well in black
96-well plates with the appropriate complete medium. After 24 h,
the plates were treated with the compounds to be tested and
incubated for 72 h at 37 °C under a 5% CO2 atmosphere. At the
end of incubation, cells were lysed, and the ATP content in the
well was used as a measure of viable cells. This was determined
using a thermostable firefly luciferase based assay (CellTiter-Glo)
from Promega. IC50 values were calculated using the rate of percent
of growth versus the untreated control.
Acknowledgment. The authors are grateful to the personnel
of the Biochemical and Cellular Screening Department of
Nerviano Medical Sciences. We thank Daniela Borghi, Nicoletta
Colombo, Maristella Colombo, Vittorio Pinciroli, Federico
Riccardi Sirtori, and Marco Tatò for NMR and mass determina-
tions; David B. Reitz, Michael Vazquez, William F. Vernier,
David R. Anderson, Dennis Phillion, Lu Hwang-Fun, and
Matthew W. Mahoney for helpful advice on synthesis; and
Rodrigo Bravo and Giulio Draetta for discussion and support.
(18) Masai, H.; Arai, K. Cdc7 kinase complex: a key regulator in the
initiation of DNA replication. J. Cell. Physiol. 2002, 190, 287–296.
(19) Kim, B. J.; Lee, H. Importin-beta Mediates Cdc7 Nuclear Import by
Binding to the Kinase Insert II Domain, Which Can Be Antagonized
by Importin-alpha. J. Biol. Chem. 2006, 281, 12041–12049.
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Corti, L.; Piutti, C.; Sansonna, P.; Villa, M.; Pierce, B. S.; Pulici, M.;
Giordano, P.; Martina, K.; Fritzen, E. L.; Nugent, R. A.; Casale, E.;
Cameron, A.; Ciomei, M.; Roletto, F.; Isacchi, A.; Fogliatto, G.;
Pesenti, E.; Pastori, W.; Marsiglio, A.; Leach, K. L.; Clare, P. M.;
Fiorentini, F.; Varasi, M.; Vulpetti, A.; Warpehoski, M. A. 3-Ami-
nopyrazole Inhibitors of CDK2/Cyclin A as Antitumor Agents. 1. Lead
Finding. J. Med. Chem. 2004, 47, 3367–3380.
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put Screening: Parallel Artificial Membrane Permeation Assay in the
Description of Passive Absorption Processes. J. Med. Chem. 1998,
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