Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding

Article abstract of DOI:10.1021/jm100504d

Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7 inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells while reversibly arresting cell cycle progression in prima

Full text of DOI:10.1021/jm100504d

7296 J. Med. Chem. 2010, 53, 7296–7315
DOI: 10.1021/jm100504d
Cdc7 Kinase Inhibitors: 5-Heteroaryl-3-Carboxamido-2-Aryl Pyrroles as Potential
Antitumor Agents. 1. Lead Finding
Maria Menichincheri,*,† Clara Albanese,† Cristina Alli,† Dario Ballinari,† Alberto Bargiotti,† Marina Caldarelli,†
Antonella Ciavolella,† Alessandra Cirla,† Maristella Colombo,† Francesco Colotta,† Valter Croci,† Roberto D’Alessio,†
Matteo D’Anello,† Antonella Ermoli,† Francesco Fiorentini,‡ Barbara Forte,† Arturo Galvani,† Patrizia Giordano,†
‡
†
†
†
†
†
Antonella Isacchi,† Katia Martina,†,§ Antonio Molinari, Jurgen K. Moll, Alessia Montagnoli, Paolo Orsini, Fabrizio Orzi,
€
†
†
†
†
†
ꢀ †
Enrico Pesenti, Antonio Pillan, Fulvia Roletto, Alessandra Scolaro, Marco Tato, Marcellino Tibolla, Barbara Valsasina,
Mario Varasi,†, Paola Vianello,†, Daniele Volpi,† Corrado Santocanale,†,^ and Ermes Vanotti†
†Nerviano Medical Sciences Srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy, and ‡Accelera Srl, Viale Pasteur 10,
20014 Nerviano (MI), Italy. §Present address: Universitaꢀ degli Studi di Torino, Italy. Present address: IEO European Institute of Oncology,
Milan, Italy. ^Present address: NUI Galway, Ireland.
Received April 26, 2010
Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7
inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells
while reversibly arresting cell cycle progression in primary fibroblasts. This implies that Cdc7 kinase
could be considered a potential target for anticancer therapy. We previously reported that pyrrolopyr-
idinones (e.g., 1) are potent and selective inhibitors of Cdc7 kinase, with good cellular potency and
in vitro ADME properties but with suboptimal pharmacokinetic profiles. Here we report on a new
chemical class of 5-heteroaryl-3-carboxamido-2-substituted pyrroles (1A) that offers advantages of
chemistry diversification and synthetic simplification. This work led to the identification of compound
18, with biochemical data and ADME profile similar to those of compound 1 but characterized by
superior efficacy in an in vivo model. Derivative 18 represents a new lead compound worthy of further
investigation toward the ultimate goal of identifying a clinical candidate.
Introduction
replication.4 In cells, phosphorylation of MCM2 at Ser40
and Ser53 is completely dependent on Cdc7 activity5 and is
essential for the initiation of DNA replication and cell growth.6
Cdc7 inhibition via RNA interference or prototype small mole-
cules causes tumor cells to enter apoptosis in a p53 independent
manner while simply arresting cell cycle progression in normal
cells.7,8 Furthermore, Cdc7 levels are increased in many cancer
cell lines and primary tumors, such as breast and lung cancers,
compared to matched normal tissues, and this overexpression
often correlates with a poor prognosis.9,10 These findings
suggest that alterations in Cdc7 protein abundance or activity
may occur during tumor genesis and have important conse-
quences for cell survival, thereby implying that Cdc7 kinase
could be considered a target for anticancer therapy.11
The inhibition of DNA synthesis is a well-established
strategy in the treatment of hyper-proliferative disorders.1
However, the toxicity and tumor cell resistance associated
with current DNA replication inhibitors2 make the discovery
ofnovel agentstargetingthisprocess by adifferentmechanism
an urgent need. In eukaryotic cells, DNA synthesis is initiated
by multiple origins of replication in the genome.3 Cdc7a is a
serine/threonine kinase that promotes the firing of the DNA
replication origin by phosphorylating one or more subunits of
the MCM DNA helicase complex (MCM2-7), thus leading
to the unwinding of double-stranded DNA at the origins of
Our group12-14 and other investigators15 have initiated a
synthetic chemical effort to identify and characterize Cdc7
inhibitors. In our previous publications,12,13 we reported on
the structure-activity relationship studies and lead optimi-
zation effort around the pyrrolopyridinone chemical class,
which proved to be a valuable core source of potent and
selective Cdc7 kinase inhibitors. This chemotype, exemplified
by compound 1 (Figure 1), is generally characterized by
favorable physicochemical and in vitro ADME properties,
moderate to good potency in cell proliferation assays, and has
a known cellular mechanism of action. However, despite this
promising profile, our best analogues selected for in-depth
in vivo studies generally displayed a suboptimal pharmaco-
kinetic behavior in animal species.
*To whom correspondence should be addressed. Phone: þ39-
0331-581928. Fax: þ39-0331-581347. E-mail: maria.menichincheri@
nervianoms.com.
aAbbreviations: Cdc7, cell division cycle 7; Cdk, cyclin-dependent kinase;
TAO (or SULU), thousand and one amino acid protein; CK, casein kinase;
GSK, glycogen synthase kinase; ATP, adenosine triphosphate MCM,
minichromosome maintenance complex; ADME, adsorption-distribution-
metabolism-excretion; PK, pharmacokinetics; CL, clearance; Vss, distribu-
tion volume; AUC, area under curve; F, oral bioavailability; PAMPA,
parallel artificial membrane permeability assay; TGI, tumor growth
inhibition; BrdU, bromodeoxyuridine; 5-FU, fluorouracil; siRNA,
small interfering RNA; CDI, 1,10-carbonyldiimidazole; EDC, N-(3-
dimethylaminopropyl)-N0-ethyl-carbodiimide hydrochloride; HOBt,
1-hydroxybenzotriazole; HOBt NH3, 1-hydroxybenzotriazole ammo-
nium salt; DIEA, N,N-diisopropyl-N-ethylamine; MBHA, 4-methyl-
benzhydrylamine); TBTU, O-(benzotriazol 1-yl)-N,N,N0,N0-tetra-
methyluronium tetrafluoroborate; Xantphos, 4,5-bis(diphenylphosphino)-
9,9-dimethyl-xanthene.
r
pubs.acs.org/jmc
Published on Web 09/27/2010
2010 American Chemical Society

Article
In light of these results, we decided to initiate a medicinal
chemistry program with the goal of identifying a related
chemical series with improved in vivo PK properties, which
also maintains the attractive features typical of the pyrrolo-
pyridinone chemical class.
With compound 1 as a starting point, we extensively
explored substitution patterns of rings A, B, and C as well as
different lactam ring sizes. The opening of the lactam ring was
envisioned as an attractive strategy which would offer a new
point of diversity for medicinal chemistry intervention.
In fact, the evolution of compound 1A from 1 implies the
possibility of introducing a varietyof different R-groups atthe
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7297
2-positionofthepyrrole (Figure1), providing ease ofchemical
diversification as well as elimination of stereogenic centers
associated with substitutions at the lactam ring.
In this paper, we describe the expansion and development
of this new series of 5-heteroaryl-3-carboxamido-2-substituted
pyrrole derivatives, which identified new lead compounds. In
particular, compound 18 (Figure 2) represents a novel proto-
type Cdc7 kinase inhibitor, with biochemical and ADME
properties profile similar to those of compound 1 but char-
acterized by far superior biological efficacy in in vivo models.
In addition, this simple scaffold is amenable to broad optimi-
zation, considering the large number of derivatives easily
accessible by either phenyl ring substitution or replacement
through straightforward synthetic approaches.
Chemistry
Our strategy for the formation of the pyrrole ring and the
subsequent preparation of our target pyrrolecarboxamides is
based on two protocols, namely the Hantzsch cyclization16
and the pyrrole ring closure of R-cyano-γ-ketoesters to yield
2-halopyrroles, versatile starting materials for various syn-
thetic transformations.
Figure 1. Evolution of the pyrrolopyridinone scaffold.
In particular, the Hantzsch synthesis of pyrroles was used
to prepare compounds 4-29 (Scheme 1A) and compound 32
(Scheme 1B).
This procedure (Scheme 1A) generally implies the reaction
of a series of heteroaromatic bromoketones (2a, 2c-e) with
Figure 2. Compound 18 (see Table 3).
Scheme 1. Synthesis of 2-Substituted Pyrroles via Hantzsch Cyclizationa
a Conditions: (a) NaH, THF or DMF, 0 ꢀC to rt, β-keto ester; (b) NH4OAc, EtOH, rt; (c) 4 M NaOH, EtOH, reflux or 1.5 M KOH/EtOH, reflux for Het =
pyridin-4-yl and R = Me; (d) HOBt NH3, DIEA, EDC HCl, THF, 0 ꢀC to rt, or HOBt NH3, DIEA, TBTU, DMF, rt; (e) from 3, CDI, DMF, 45 ꢀC, then
3
3
3
amine in EtOH, rt; (f) from 10, 2NHCl, MeOH, 50ꢀC; (g) methyl 3-aminocrotonate, DMF, rt for Het = pyridin-4-yl and R = Me; (h) 4 M HCl, dioxane, rt for
obtaining 7 derived from I = 4-tert-butoxycarbonylamino-3-oxo-butyric acid ethyl ester; (i) NH2COOtBu, Xantphos, Pd(OAc)2, Cs2CO3, dioxane, 140 ꢀC.

7298 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Menichincheri et al.
Scheme 2. Synthesis of 2-Aryl Pyrroles via R-Cyano-γ-ketoesters Cyclization and Suzuki Couplinga
a Conditions: (a) ethyl cyanoacetate, NaOEt, DIEA, THF, rt; (b) 33% HBr/AcOH, Et2O, CH2Cl2, 0 ꢀC to rt; (c) 4 M NaOH, EtOH, reflux;
(d) HOBt NH3, DIEA, EDC HCl, THF, 0 ꢀC to rt; (e) RB(OH)2, (Ph3P)2PdCl2, LiCl, 1 M Na2CO3, toluene/EtOH, 100 ꢀC; (f) Rink amide-MBHA
3
3
resin, DMF, DIEA, EDC HCl, HOBt, rt; (g) RB(OH)2, (PPh3)2PdCl2, LiCl, Cs2CO3, DMF/H2O, 100 ꢀC; (h) CF3COOH, CH2Cl2, rt; (i) 4 N HCl/
3
dioxane, Et2O, 0 ꢀC to rt; (l) HCOONH4, 10% Pd-C, rt.
the sodium enolates of the appropriate β-ketoesters (general
structure I) in the presence of ammonium acetate.
The alternative approach, based on the cyclization of
R-cyano-γ-ketoesters to obtain the final compounds, exploits
the reactivity of halogens at the position 2 of the pyrrole ring
under different reaction conditions, such as the Suzuki coupling,
which allows easy access to a variety of 2-aryl pyrroles.
In fact, the action of hydrohalogenic acids in organic solvents
on R-cyano-γ-ketoesters provided the 2-halo pyrroles21 that
were transformed into the final compounds 36a, 37-40,
42-54 (Scheme 2A), and into compound 57 (Scheme 2B).
As depicted in Scheme 2, the 2-bromo derivatives 34a-c
and the 2-chloro compound 55 were obtained upon treatment
of the corresponding R-cyano-γ-ketoesters 33a-c alterna-
tively with hydrobromic and acetic acid in ethyl ether-
dichloromethane solvent or with hydrochloric acid in dioxane.
Compound 55 was subjected to hydrogenolytic conditions to
finally achieve the 2-unsubstituted amide 57 (Scheme 2B),
while the bromo derivatives 34a-c were hydrolyzed to the
corresponding carboxylic acids 35a-c (Scheme 2A), which
represented key intermediates both for solution and for solid
phase synthesis of the final derivatives.
Once obtained, the esters were hydrolyzed to the corre-
sponding carboxylic acid derivatives (general structures IIa,
IIc-e) and in turn transformed into primary amides by means
of hydroxybenzotriazole ammonium salt in the presence
of a coupling reagent or with ammonium chloride and
TBTU.17 For the synthesis of compound 4, methyl 3-amino
crotonate replaced the β-ketoester in the Hantzsch cycliza-
tion step. Direct amination of the carboxylic esters upon
heating with concentrated aqueous or methanolic ammonia
proved unsuccessful. In a few cases, secondary amides were
obtained by acid activation with 1,10-carbonyldiimidazole
and reaction with the appropriate primary amine in etha-
nol (27-29).
Compound 32 (Scheme 1B) was prepared from the 20-chloro
pyridinyl precursor 30, obtained in turn via Hantzsch reac-
tion with 3-oxo-3-phenylpropionic acid ethyl ester and
R-bromoketone 2f. One-step amination-deprotection reac-
tion was accomplished with palladium acetate, using t-butyl
carbamate as the aminating agent and Xantphos as the ligand,
under controlled microwave heating.18
β-Ketoesters, when not commercially available, were pre-
pared by applying the carboxylic acid homologation proce-
dure. The acid was activated as the imidazolide and allowed to
react with either commercial monoethylmalonate potassium
salt19 in the presence of magnesium chloride or ethyl hydrogen
malonate and magnesium ethoxide.20
Subsequently, 2-bromo pyrroles 35a-c were transformed
into the amides 36a-c and subjected to the Suzuki reaction,
thus providing the corresponding 2-aryl pyrroles (Scheme 2A)
under solution phase conditions (compounds 37-40). In
particular, the Suzuki coupling was carried out by heating
the amides 36a-c with commercially available boronic acids
and bis(triphenylphosphine) palladium(II) dichloride. For
solidphase reactions, Rinkamide-MBHA resin22 was allowed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7299
Scheme 3. Synthesis of 2ea
a Conditions: (a) tributyl(1-ethoxyvinyl)stannane, (PPh3)2PdCl2, DMF, 70 ꢀC; (b) 30% aq NH4OH, EtOH, 100 ꢀC; (c) NBS, THF/water, rt.
Scheme 4. Synthesis of N-Alkyl Pyrrolesa
a Conditions: (a) alkyl halide, Cs2CO3, DMF, 85 ꢀC (for 58 and 60); (b) TFA, CH2Cl2, rt (for 60 deriving from alkyl halide = iodoethanol
tetrahydropyranylether); (c) CF3CH2OTf, K2CO3, CH3CN, 18-crown-6, reflux (for 59); (d) NaH, ethyl benzoylacetate, THF, 0 ꢀC to rt; (e) NH4OAc,
EtOH, rt; (f) LiOH, THF/MeOH/water, reflux; (g) NH4Cl, DMA, DIEA, TBTU, 80 ꢀC; (h) 4 M HCl in dioxane, rt; (i) EtNH2, AcOH, microwaves,
170 ꢀC; (l) 4 M NaOH, EtOH, reflux; (m) HOBt NH3, DIEA, EDC HCl, THF, 0 ꢀC to rt.
3
3
to react with the acids 35a,c to provide intermediates 41a,c,
from whichtheproductswere cleavedwithtrifluoroacetic acid
in dichloromethane at room temperature, directly yielding the
primary amides 42-54.
Scheme 5. Synthesis of 71 and 72 Analoguesa
Bromoketones 2a-f, used for both synthetic approaches,
are either commercially available (2a and 2f) or were accessed
through halogenation of ketones12,23 (2b and 2c) or enolethers12
(2d and 2e). The synthesis of 2e is described in Scheme 3.
Commercially available 2,4-dichloro-5-fluoro-pyrimidine
was allowed to react with tributyl(1-ethoxyvinyl)stannane
in the presence of bis(triphenylphosphine) palladium(II)
dichloride in N,N-dimethylformamide to afford the corre-
sponding 4-vinyl ether. The amino group was introduced at
position 2 upon treatment with aqueous concentrated
ammonia in ethanol under heating with microwaves, and
bromination of the resulting vinyl ether to R-bromo-ketone
was accomplished with N-bromosuccinimide in aqueous
tetrahydrofuran.
In Scheme 4, the preparation of N-alkylated pyrroles
58-60, 64, 65, and 67 is outlined. Direct alkylation of amide
18 as well as of the intermediate ester derivative 61 with alkyl
halides and cesium carbonate in N,N-dimethylformamide
provided the corresponding derivatives 58, 60, 64, and 65.
Compound 18 was treated instead with 2,2,2-trifluoroethyl
trifluoromethanesulphonate, potassium carbonate, and crown-
ether, providing compound 59.
a Conditions: (a) NaH, THF, 0 ꢀC to rt; (b) Lawesson’s reagent,
toluene, reflux; (c) chromatography; (d) 4 M NaOH, EtOH, water,
reflux; (e) HOBt NH3, DIEA, EDC HCl, THF, 0 ꢀC to rt.
3
3
A different approach was exploited for the preparation of
the N-ethyl derivative 67, based on the use of ethylamine in the
Hantzsch pyrrole ring formation reaction.
The furan and thiophene analogues 71 and 72 were
simultaneously prepared through the Paal-Knorr con-
densation synthesis24 (Scheme 5) by exposing 1,4-dike-
tone 68 to the action of the Lawesson’s reagent in toluene
at reflux temperature. The mixture of the two homo-
logues could be separated easily by flash chromatog-
raphy.

7300 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Scheme 6. Synthesis of 74a
Menichincheri et al.
a Conditions: (a) NIS, DMF, rt; (b) tributylvinyl stannane, 2,6-dimethyl-4-tert-butyl phenol, Pd(PPh3)4, dioxane, DMF, 110 ꢀC.
Table 1. SAR: Opening the Lactam Ring (IC50, μM)a
Table 2. SAR: Variations of Aryl Substituent R (IC50, μM)a
compd
R
Cdc7
A2780b compd
R
Cdc7
A2780b
11
13
14
15
16
17
42
43
H
0.009 ( 0.003
0.008 ( 0.004
0.023 ( 0.014
0.014 ( 0.006
2.0
1.5
2.4
2.5
2.0
1.2
4.9
1.8
37
44
38
45
46
47
48
49
o-OMe 0.126 ( 0.011
m-OMe 0.058 ( 0.029
p-OMe 0.023 ( 0.007
o-NO2 0.039 ( 0.019
m-NO2 0.044 ( 0.016
p-NO2 0.117 ( 0.051 >10
o-CN 0.131 ( 0.054 2.8
m-CN 0.216 ( 0.042 >10
6.5
7.3
2.6
2.3
9.2
o-F
m-F
p-F
m-Br 0.056 ( 0.021
p-Br 0.073 ( 0.028
m-Me 0.162 ( 0.155
p-Me 0.060 ( 0.010
a IC50 values are reported as the mean ( standard deviation (n g 2).
b IC50 values are reported as the mean of 2-3 experiments with a
coefficient of variation below 35%.
In the absence of the crystallographic structure of the target
protein, we used a homology model of Cdc7 kinase based on
Cdk2 kinase, as reported in our previous publications.12,13
The docking studies and the SAR of the pyrrolopyridinone
series correlates well with a binding mode where the nitrogen
of the pyridyl ring accepts a hydrogen bond from the hinge
region, while the lactamic moiety forms hydrogen bonds
between the carbonyl and the conserved lysine residue and
between the NH and the conserved aspartate via electrostatic
interaction. In the present work, we therefore assumed a
similar binding mode and orientation in the ATP pocket also
within this novel series of compounds.
The first set of compounds prepared maintained the pri-
mary amide and were varied only at position 2 of the pyrrole,
with substituents spanning from hydrogen to larger alkyl and
aromatic groups (Table 1).
The data in Table 1 clearly prove that the tolerance toward
the different substitutions is very limited, as only the phenyl
derivative 11 retained good activity on Cdc7 kinase, compar-
able to that of our reference compound 1. Moving from
phenyl tocyclohexyl and 4-pyridyl analogues 9 and 12, respec-
tively, the potency dropped nearly 20- to 30-fold, as for the
unsubstituted compound 57 and the bromo derivative 36a.
The alkyl derivatives 4-6 suffered a 50-fold decrease in
potency, whereasthe aminoderivatives7, 10, and 26displayed
an even greater loss of activity, showing IC50 values in the
micromolar range. Finally, the benzyl analogue 8 also proved
to be much less active, nearly 90-fold with respect to com-
pound 11.
Given the excellent potency as well as good selectivity over
Cdk2 demonstrated by compound 11, we decided to investi-
gatea possiblestructural interpretation. Asjudged from a pre-
liminary modeling analysis, the phenyl derivative 11 looked
particularly interesting compared to the other derivatives in
Table 1 because it showed a potentially favorable binding
mechanism to Cdc7 kinase. A T-shaped interaction between
a IC50 values are reported as the mean ( standard deviation (n g 2).
Compound 18 was variously halogenated as described
elsewhere25 to obtain derivatives 73 and 75-77. The 4-iodo
pyrrole derivative 73 was in turn transformed into the 4-allyl
derivative 74 by vinylation via the Stille cross-coupling reac-
tion protocol.26 The reaction occurred by heating 73 with
tributylvinylstannane in the presence of tetrakis(triphenyl-
phosphine)palladium(0) in dioxane/DMF (Scheme 6).
Results and Discussion
As we mentioned earlier, the opening of the lactam ring was
the first step toward the design of a novel chemical series of
Cdc7 kinase inhibitors based on the 5-heteroaryl-3-carboxa-
mido-2-substituted pyrrole template.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7301
Figure 3. Predicted binding mode of compound 11 in the Cdc7 kinase homology model.
Table 3. SAR: Variations of Ring A (IC50, μM)a
a IC50 values are reported as the mean ( standard deviation (n g 2). b IC50 values are reported as the mean of 2-3 experiments with a coefficient of
variation below 35%.
the phenyl R group of the ligand and the phenylalanine of the
glycine-rich loop appeared feasible (Figure 3).27 Cdk2 kinase
would be unable to perform such interaction, having a tyro-
sine residue at that position. In fact, the two front-runners 1
This binding hypothesis is confirmed by the weaker activity
of derivatives 4-7, 9, 10, 26, 36a, and 57 due to the lack of the
aromatic moiety at position 2 of the pyrrole nucleus. Further-
more, this model explains the poor activity of compound 8,
where the conformational freedom of the benzyl substituent
disfavors an efficient interaction between the phenyl ring of
the ligand and the phenyl ring of the phenylalanine residue.
The reduced activity of compound 12 can be explained by the
desolvation penalty of burying the pyridine in a clearly
hydrophobic pocket. In the docking studies, molecules of
and 11 are equipotent against the Cdc7 kinase (Cdc7 IC50
=
0.010 μM and Cdc7 IC50 = 0.009 μM, respectively), but 11 is
5 times more selective over Cdk2/CycA kinase (Cdk2/CycA
IC50=0.240 μM for 1 and Cdk2/CycA IC50=1.1 μM for 11).
The same ratio holds true in the aminopyrimidinyl series,
(e.g., 18) when compared to its lactamic homologue.

7302 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Table 4. SAR: Variations of the Central Core B (IC50, μM)
Menichincheri et al.
Table 6. SAR: Variations of the Pyrrole N-Substituent (IC50, μM)a
a IC50 values are reported as the mean ( standard deviation (n g 2).
b IC50 values are reported as the mean of 2-3 experiments with a
coefficient of variation below 35%.
Another important site for exploration is ring A, which
models suggest interacts with the hinge region of the ATP
binding site. For this reason, we replaced ring A of compound
11 with other nitrogen containing heterocycles able to form or
even improve the hydrogen bondinginteraction withthe hinge
region.
Table 3 summarizes the biochemical data on Cdc7 kinase,
antiproliferativeactivityagainst A2780 cell lines, andthe main
cross-reactivities against a panel of 51 tyrosine and serine-
threonine kinases.
a IC50 values are reported as the mean ( standard deviation (n g 2).
b IC50 values are reported as the mean of 2-3 experiments with a
coefficient of variation below 35%.
Table 5. SAR: Variations of Side Chain R (IC50, μM)a
The addition of an amino group onto the pyridyl ring
(compound 32) able to form additional H-bond interactions,
has a detrimental effect on the activity both in biochemical
and cellular assay. On the contrary, the replacement of the
pyridyl ring with an amino-pyrimidinyl heterocycle, as in
compound 18, has a minor impact on potency but provides
submicromolar cellular potency in the proliferation assay.
The introduction of a phenyl ring on the amino group
(compound 19) has insteada moderately negative effect on the
biochemical and cellular potency and also affects selectivity,
as GSK3β represents the main target affected in the double-
digit nanomolar range.
compd
R
Cdc7
A2780b
3
OH
NH2
0.090 ( 0.007
0.022 ( 0.013
>10
>10
0.8
7.2
18
27
28
29
NHMe
NHiPr
NHBn
>10
>10
>10
>10
aIC50 values are reported as the mean ( standard deviation (n g 2).
bIC50 values are reported as the mean of 2-3 experiments with a
coefficient of variation below 35%.
Additionofhalogenson the heterocyclicringin compounds
20, 39, 75, and 76 generally leads to an improvement of
potency and cellular activity, however, all these derivatives,
except 20, display a less satisfactory selectivity profile. The
outstanding impact of the 2-aminopyrimidinyl residue on the
cell based assay prompted us to maintain this residue in all
subsequent derivatives.
Modifications of the central core B were also explored
(Table 4). Whereas the halogen substituted derivatives 73
and 77 proved to still be active and very potent in A2780 cells,
a vinyl group at that position is deleterious (compound 74).
When the central nucleus was replaced with either a furan or a
thiophene ring (compounds 71 and 72, respectively) a con-
siderable loss of activity was observed both in the biochemical
and in the cellular assay.
water were included in the binding site as they might play an
important role in the binding of compound 11 and its close
analogues.
Having established the importance of a 2-phenyl substitu-
ent for the activity against Cdc7 kinase, we decided to inves-
tigate the role of the substitution pattern on the aromatic ring.
As illustrated in Table 2, a clear trend in terms of electronic
influence does not emerge, and only the small fluorine atom
seems to give an activity comparable to that of the unsub-
stituted derivative 11, in particular for the ortho regioisomer 13.
This might suggest that the steric hindrance of the sub-
stituent might affect unfavorably the potency of the com-
pound despitea potentiallygood contribution tothe T-shaped
interaction.
On the other hand, the good potency of most of these
compounds in the biochemical assay was counterbalanced by
a disappointing micromolar activity in the A2780 cellular
assay, making them not appealing for additional studies.
Next we focused our attention on the carboxamido group
(Table 5). As already mentioned, we knew from our previous
studies on the pyrrolopyridinone series that the lactamic
moiety should participate in relevant interactions with the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7303
Table 7. SAR: Variations of Aromatic Ring C (IC50, μM)a
a IC50 values are reported as the mean ( standard deviation (n g 2). b IC50 values are reported as the mean of 2-3 experiments with a coefficient of
variation below 35%.
Figure 4. (A) In vitro mechanism of action (MoA) of compound 18. A2780 cells were incubated for up to 24 h with 9 and 12 μM concentrations
of 18. Proteins were prepared and analyzed by Western blot. Phosphorylation status of MCM2 protein at Cdc7-dependent (Ser-40) phospho-
site was assessed using specific antiphosphopeptide antibody. Detection of cleaved active caspase 3 indicates that cells are undergoing apoptotic
cell death. (B) DNA synthesis and BrdU incorporation analysis in A2780 cells treated with compound 18. Logarithmically growing A2780 cells
were treated for the indicated times with 9 μM 18 and labeled with BrdU before fixation. DNA content and BrdU positive cells (%) were
measured by FACS.
conserved lysine and aspartate residues. As expected, the lac-
tamic ring-opening did not affect the activity on Cdc7 kinase
in the case of the primary amide (compound 18). However
when secondary amides were tested (compounds 27-29), a
complete loss of activity has been observed likely as a result of
the syn conformation adopted by the substituted amide bond
with respect to the anti conformation of the lactam, thus
hampering efficient H-bond interactions. The acid derivative
3 is still active against Cdc7 kinase in the nanomolar range,
but it is devoid of cellular activity possibly due to poor cell
permeability.
Further substitutions on the pyrrole nitrogen were consid-
ered (Table 6), in particular the alkylation of the pyrrole
nitrogen. At first, the insertion of the small methyl group
(compound 58) was checked and found to be detrimental to
Cdc7 kinase inhibitory activity. Not surprisingly, the ethyl
derivative 67 performed much better both in the biochemical
and in the cellular assay compared to 58, enforcing the trend
already observed with the pyrrolopyridinones class.12 The
trifluoroethyl derivative 59 still maintained a good activity
on Cdc7 but was poorly active in cells, and variations of the
ethyl group such as hydroxyethyl, aminoethyl, and amino-
propyl substitutions in compounds 60, 64, and 65 were all
unsuccessful in terms of biochemical and cellular activity.
Because the aminopyrimidinyl ring A, the 4-unsubstituted-
pyrrolyl ring B, and the primary carboxamido group (Figure 1)
emerged as promising structural moieties to achieve satisfac-
tory biological activity, we maintained this core structure in
undertaking further study of substituent R. In this work, we
mainly focused on heteroaromatic groups. It is evident from
the data in Table 7 that compounds 22-25 and 40 show a
good combination of Cdc7 kinase inhibition and cellular

7304 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Table 8. In Vitro ADME Parameters of Selected Derivatives
Menichincheri et al.
activity. All other compounds displayed an unsatisfactory inhi-
bitory activity on Cdc7 kinase and/or a poor cellular potency.
The biochemical data confirmed the importance of an aro-
matic substituent for good activity and a strong preference
for phenyl bioisosters, in particular the thiophenyl derivatives
22 and 40. The disappointing biochemical activity of the
20-pyridinyl compound 21 is also in line with the previous
observation on 40-pyridinyl analogue 12.
The study of the mechanism of action in A2780 cells of
those compounds, which best performed in terms of biochemical
potency, selectivity profile, and cellular activity, in partic-
ular compounds 18, 20, 22-25, 40, 67, 73, and 77, permitted
further prioritization. Therefore the capacity of these inhibi-
tors to halt the cell cycle and DNA synthesis by bromodeoxy-
uridine (BrdU) incorporation block, to down-modulate the
Ser40 phosphorylation of MCM2 substrate, and to induce
cleaved caspase 3, was evaluated.13
Despite the good cellular activity of derivatives 24, 25, and
77, their mechanism of action could not be clearly elucidated
in vitro and thus they have not been investigated further.
However, the antiproliferative activity of the other analogues
was demonstrated to be correlated with the inhibition of
cellular Cdc7 kinase, as exemplified by the data for compound
18 (Figure 4).
InFigure4A, theinhibitionofSer40phosphorylationof the
MCM2 substrate was evaluated in a time-course experiment
at different compound concentrations. Both at 9 μM and at
12 μM, the inhibition was complete at 24 h. In panel B, the cell
cycle profile was studied on A2780 cells, using fluorescence-
activated cell sorting. Cell population peaks showed a G1/S
block and a strong decrease of bromodeoxyuridine incorpora-
tion (from 40% to 6%) indicated an impairment of DNA
synthesis in the cells. Both caspase 3 activation and sub-G1
accumulation demonstrated that cells treated with compound
18 were undergoing apoptosis.
Solubility, permeability, plasma protein binding, and in-
trinsic clearance in rat hepatocytes data (in vitro ADME
properties) of the most interesting compounds 18, 20, 22, 23,
40, 67, and 73 are summarized in Table 8.
Most compounds showed good to excellent solubility at
neutral pH, moderate permeability in the PAMPA assay, and
modest plasma protein binding. The intrinsic clearance, eval-
uated in rat hepatocytes, ranges in most cases from low to
moderate but is clearly higher for compounds 40 and 73,
and these data were confirmed by their high in vivo
clearance value. For all these compounds, in vitro profiles
were considered acceptable and PK was evaluated in mice
(Table 9).
Table 9 reports the main in vivo PK parameters of the
previously described derivatives, measured after iv and per os
administration. Among them, compounds 20, 40, and 73 were
judged unsatisfactory. In particular, compound 20 shows very
short per os half-life, low exposure, and modest bioavailabil-
ity. Compound 73 is characterized by the worst clearance and
exposure values, making it unsuitable for oral administration.
Compound 40 also displays modest per os exposure and a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7305
Table 9. In Vivo ADME Parameters of Selected Derivatives (Harlan nu/nu Mice)a
PK data (iv), doseb: 10 mg/kg
PK data (per os), doseb: 10 mg/kg
compd
C
max (μM) AUC¥ (μM h) CL (mL/min/kg)
Vss (mL/kg)
t1/2 (h) Cmax (μM) Tmax (h) AUC¥ (μM h) t1/2 (h) Fc (%)
3
3
18
20
22
23
40
67
73
30.0 ( 0.2
11.6 ( 0.7
11.3 ( 3.3
20.9 ( 1.9
13.7 ( 2.2
10.5 ( 1.3
119.2 ( 22
0.5 ( 0.1
50.0 ( 2.7
33.5 ( 11.8
27.3 ( 2.0
31.5 ( 5.4
58.3 ( 8.2
4.8 ( 0.9
560.0 ( 33.0
485.0 ( 143.0
264.0 ( 22.4
723.0 ( 43.0
480.0 ( 40.0
213.6 ( 23.5
1035.0 ( 96.0
0.16
0.18
0.15
0.31
0.14
0.79
0.16
5.2 ( 1.9
4.5 ( 0.8
8.9 ( 0.2
4.7 ( 1.6
4.6 ( 2.2
37.4 ( 16
0.1 ( 0.0
0.25
0.25
0.25
0.4
3.8 ( 1.1
2.1 ( 0.6
11.2 ( 1.4
11.2 ( 2.7
2.4 ( 1.0
86.8 ( 31.7
0.1 ( 0.0
0.8
0.2
0.7
0.9
0.5
1.2
31
18
51
65
22
73
3
24.7 ( 6.1
58.4 ( 5.3
25.6 ( 1.0
31.0 ( 3.2
96.4 ( 6.7
1.4 ( 0.1
0.25
0.25
0.25
122.3 ( 15.7
a n=6 animals per study. b Dosed in 5% dextrose; iv=intravenous administration; per os=oral administration. c Bioavailability.
Figure 5. Compound 18 has antitumor activity in rodents. CDI nu/nu mice carrying subcutaneous A2780 human ovarian carcinoma were
treated with either vehicle or 18 by oral administration. Curves indicate tumor growth in vehicle-treated (black line) or 18-treated mice for 5
days (dark bar) at 90 mg/kg (triangles) three times a day. Data are represented as mean ( SEM.
Table 10. Selectivity Profile of Compound 18a
kinase
IC50 (μM)
kinase
IC50 (μM)
Cdc7
0.022 ( 0.013
0.394 ( 0.114
0.42 ( 0.15
0.89 ( 0.02
1.39
Cdk2/A
Cdk1/B
JAK2
2.8
3.1
Cdk9/T
TAO1
CK2
3.3
3.7
PKCβ
IKK2
othersb
GSK3β
Cdk2/E
3.8
2.7
>10
a Where possible, IC50 values are reported as the mean ( standard
deviation (n g 2). bABL, AKT1, ALK, AUR1, AUR2, BRK, Cdk4/D1,
Cdk5/P25, CHK1, EEF2K, EGFR1, ERK2, FAK, FGFR1, FLT3,
IGFR1, IKKi, IR, JAK3, KIT, LCK, MET, MK2, MPS1, MST4, NEK6,
NIM1, P38R, P38β, PAK4, PDGFR, PDK1, PERK, PIM1, PKAR,
PLK1, RET, SYK, TRKA, VEGFR3 and ZAP70.
Figure 6. In vivo target modulation by compound 18. A2780
xenografted mice were treated either with vehicle (lanes 1-6) or
with 3ꢀ90 mg/kg of 18 (lanes 7-12) and sacrificed after 6 h. Tumors
were explanted and analyzed by Western blot using antibodies
against phosphorylated MCM2 at Ser40. Levels of total MCM2
and lamin B were also measured as loading control.
The correlation of the in vivo antitumor activity with Cdc7
inhibition was evaluated by Western blot on A2780 tumors
explanted from controls and animals treated with 3 ꢀ 90 mg/kg
of 18 and sacrificed after 6 h (Figure 6).
The inhibition of Ser40 phosphorylation of MCM2 sub-
strate was clearly observed in the tumors of treated animals.
A good antitumor efficacy (TGI = 68%) was also obtained
in the xenograft colorectal cancer model HCT-116 with the
compound18administeredorally followingthe sameschedule
reported above. A lower but still significant antitumor activity
(max TGI = 41%) was obtained in COLO-205 colorectal
cancer xenograft model.
borderline bioavailability. The remaining four compounds,
18, 22, 23, and 67, are reasonably bioavailable, with very good
F values for derivatives 23 and 67. These data convinced us to
pursue these four compounds further.
These four compounds were challenged in efficacy experi-
ments to evaluate their potential for in vivo treatment. Thus
derivatives 18, 22, 23, and 67 were administered orally as
a methocel suspension to nude mice carrying subcutaneous
A2780 human ovarian carcinoma. From these preliminary
results, compound 67 showed signs of toxicity at low dosages,
while compounds 22 and 23 proved to be poorly efficacious in
vivo. However, compound 18 displayed an excellent in vivo
activity, exceeding 90% TGI (tumor growth inhibition) after
three times per day administration (90 mg/kg) for five con-
secutive days (Figure 5).
Compound 18 provided a clean selectivity profile when
tested on a panel of 51 kinases, with just three kinases
inhibited below the micromolar range (Cdk9/T, TAO1, and
CK2) but with at least a 20-fold selectivity ratio (Table 10).

7306 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Menichincheri et al.
Table 11. Antiproliferative Activity of Compound 18
IC50 (μM)a
Column chromatography was conducted either under me-
dium pressure on silica (Merck silica gel 40-63 μm) or on
prepacked silica gel cartridges (Biotage) or on a Horizon system.
When necessary, compounds were purified by preparative
HPLC on a Waters XTerra Prep RP18 (19 mm ꢀ 100 mm,
5 μm) column using a Waters FractionLynx System equipped
with a Waters 2996 PDA detector and a Waters ZQ single
quadrupole mass spectrometer, with electro-spray ionization,
in the positive mode, was used. Mobile phase A was water and
0.05% NH4OH, in H2O pH10/acetonitrile 95/5 and mobile
phase B was acetonitrile. The following conditions were used:
a gradient from 10 to 90% B in 8 min and held at 100% B for
2 min; a flow rate 20 mL/min.
cell line
A2780
HCT116
origin
p53 5-FU gemcitabine 18
ovary
colon
þ
þ
-
-
-
-
-
-
-
þ
-
þ
þ
-
þ
þ
14
12
0.035
0.006
0.5
0.8
1.8
2
HCT116-E6 colon p53-
HeLa
50
cervix
colon
colon
>100
57
90
>10
4
SW480
COLO205
K562
1.7
5.7
2.5
8.9
7.5
6.6
5.9
7.7
3.7
9.2
7
3
0.6
leukaemia
astrocytoma
ovary
>100
>100
>100
48
SF-268
OVCAR-8
HCT-15
Jurkat
0.01
0.003
0.01
0.03
0.08
0.02
0.003
0.02
0.18
1H NMR spectra were acquired at 25 ꢀC in DMSO-d6 on a
Varian Inova 400 spectrometer operating at 400 MHz and
equipped with a 5 mm 1H{15N-31P} Z-axis-PFG indirect detec-
tion probe and on a Varian Mercury 300 spectrometer operating
at 300 MHz and equipped with a 5 mm 15N-31P{1H,19F}
switchable probe. Residual not-deuterated solvent signal was
used as reference with δ = 2.50 ppm for DMSO-d5. Data are
reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, quint = quintet, bs = broad
singlet, dd = doublet of doublet, td = triplet 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.
ESI(þ) high-resolution mass spectra (HRMS) were obtained on
a Waters Q-Tof Ultima directly connected with micro HPLC
1100 Agilent as previously described.28
colon
leukaemia
breast
87
64
MCF7
NHDF
PC-3
human fibroblasts
prostate
30
30
SF-539
U2OS
gliosarcoma
osteosarcoma
43
16
7.9
5.8
a IC50 values are reported as the mean of 2-3 experiments with a
coefficient of variation below 35%.
In addition, compound 18 demonstrated a good antiproli-
ferative activity against a panel of several cell lines indepen-
dently of the p53 status and in cellular models resistant to
5-FU and/or gemcitabine, well-known inhibitors of DNA
replication elongation (Table 11).
Elemental analyses were performed on Carlo Erba EA1110
instrument, and C, H, and N values were within (0.4% of
theoretical values unless otherwise noted.
Thin-layer chromatography was performed on Merck silica
gel 60 plates coated with 0.25 mm layer with fluorescent
indicator. Components were visualized by UV light (λ = 254
and 366 nm) and iodine vapors.
Conclusions
Starting from our previous series of pyrrolopyridinone
compounds, we have developed a new chemical class of
Cdc7 kinase inhibitors, the 5-heteroaryl-3-carboxamido-
2-substituted pyrrole derivatives. This class of compounds is
amenable to straightforward diversification through both
solution and solid phase synthesis, is characterized by good
potency against the target, satisfactory activity in cells, and
good in vitro ADME properties. From this expansion, a
potent, selective, cellularly efficacious, and orally available
derivative, 18, wasidentified. It displays verygood activity ina
A2780 xenograft model with ex vivo target inhibition.
On the basis of these data, derivative 18 has emerged as
a new promising lead compound worthy of further studies
aimed at the selection of a potential candidate for clinical
development.
Compounds 73, 75, 76, and 77 were prepared as described in
the literature.25
1-(2-Amino-5-fluoro-4-pyrimidinyl)-2-bromo-ethanone (2e).
To a solution of 2,4-dichloro-5-fluoro-pyrimidine (1.23 g, 7.24
mmol) in N,N-dimethylformamide (14 mL), tributyl-(1-ethoxy-
vinyl)-stannane (2.7 mL, 7.9 mmol) was added, followed by
bis(triphenylphosphine) palladium(II) dichloride (0.103 g, 0.145
mmol). The mixture was heated at 70 ꢀC for 1 h, cooled, a
saturated aqueous solution of potassium fluoride was added,
and the mixture was stirred at room temperature for 0.5 h. After
dilution with water/diethylether and filtration through a Celite
pad, the organic phases were washed thoroughly with water,
dried over anhydrous sodium sulfate, and concentrated. The crude
material was purified with the Horizon system (25 mm column),
eluting with n-hexane/EtOAc 95:5. 2-Chloro-4-(1-ethoxy-vinyl)-
5-fluoro-pyrimidine was obtained (1.24 g, 84%). 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.32 (t, J = 6.95 Hz, 3H), 3.95 (q,
J = 6.99 Hz, 2H), 4.88 (d, J = 2.80 Hz, 1H), 5.20 (d, J = 2.93 Hz,
1H), 8.90 (d, J = 3.17 Hz, 1H).ESI (þ) MS: m/z 203 (MHþ).
A solution of the above obtained enol ether (15.52 g, 76.73
mmol) in absolute ethanol (25 mL) and 30% aqueous ammonia
(50 mL) was warmed under shaking at 100 ꢀC for 1.5 h in a Parr
apparatus. After cooling, ethanol was removed and the com-
pound was extracted with dichloromethane. The crude material
was purified with the Horizon system, eluting with n-hexane/
EtOAc 1:1. There was obtained 4-(1-ethoxy-vinyl)-5-fluoro-
pyrimidin-2-ylamine (9.01 g, 64%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.29 (t, J = 7.01 Hz, 3H), 3.87 (q, J = 6.95
Hz, 2H), 4.62 (d, J = 2.44 Hz, 1H), 4.91 (dd, J = 2.38, 0.55 Hz,
1H), 6.64 (bs, 2H), 8.28 (d, J = 3.54 Hz, 1H). ESI (þ) MS: m/z
184 (MHþ).
Experimental Section
1. Chemistry. Unless otherwise noted, all solvents and re-
agents 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 routinely measured by HPLC on a Waters X Terra
RP 18 (4.6 mm ꢀ 50 mm, 3.5 μm) column using a Waters 2790
HPLC system equipped with a 996 Waters PDA detector and
Micromass model A ZQ single quadrupole mass spectrometer,
equipped with an electrospray (ESI) ion source. Mobile phase A
was an ammonium acetate 5 mM buffer (pH 5.2 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.
To a solution of the above prepared amino derivative (0.521 g,
2.78 mmol) in tetrahydrofuran (25 mL), water (1.7 mL) was
added followed by N-bromosuccinimide (0.512 mg, 2.87 mmol).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7307
The mixture was stirred at room temperature for 1.5 h. The
solvent was evaporated, and the residue was stirred thoroughly
in methanol and filtered to obtain the title compound 1-(2-
amino-5-fluoro-pyrimidin-4-yl)-2-bromo-ethanone (0.511 g, 77%).
1H NMR (400 MHz, DMSO-d6) δ ppm 4.70 (s, 2H), 6.94
(bs, 2H), 8.50 (d, J = 2.93 Hz, 1H). ESI (þ) MS: m/z 234-
236 (MHþ).
346 (MHþ). HRMS (M þ H)þ calcd for C18H23N3O4 þ Hþ,
346.1761; found, 346.1764.
To a solution of 2-(tert-butoxycarbonylamino-methyl)-5-
pyridin-4-yl-1H-pyrrole-3-carboxylic acid ethyl ester (0.345 g,
1 mmol) in 30 mL of ethanol/water 3:1, 1.05 g of sodium
hydroxide was added. The solution was heated at 70 ꢀC for 18
h, and then cooled. 2 N HCl was added to reach pH 6-7, and the
precipitate was filtered, yielding 0.274 g (85%) of 2-(tert-butoxy-
carbonylamino-methyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxy-
lic acid as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm
1.36 (s, 9H), 4.47 (s, 2H), 7.04 (s, 1H), 7.19 (s, 1H), 7.67 (d, J =
5.86 Hz, 2H), 8.45 (d, J = 6.15 Hz, 2H), 12.06 (bs, 1H).
A solution of 2-(tert-butoxycarbonylamino-methyl)-5-pyri-
din-4-yl-1H-pyrrole-3-carboxylic acid (26 mg, 0.081 mmol),
HOBt (16 mg, 1.5 equiv), ammonium chloride (8 mg, 2 equiv),
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP, 63 mg, 1.5 equiv), and DIEA (0.05 mL) in
N,N-dimethylformamide (3 mL) was stirred at room tempera-
ture for 6 h. The reaction mixture was concentrated and purified
by reverse phase flash chromatography. The obtained protected
amide was treated with 4 M HCl in dioxane to yield the title
2-Methyl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide hydrochlo-
ride (4). A solution of 2-bromo-1-pyridin-4-ylethanone hydro-
bromide (2a) (3.00 g, 10.8 mmol) and methyl 3-aminocroto-
nate (1.84 g, 1.5 equiv) in anhydrous N,N-dimethylformamide
(15 mL) was stirred for 15 h at room temperature. The reaction
mixture was diluted with ethyl acetate and the precipitate
filtered off. The mother liquor was basified with aqueous 10%
potassium carbonate solution and extracted with ethyl acetate.
The organic layer was dried over anhydrous sodium sulfate,
concentrated, and purified by flash chromatography (ethyl
acetate). The oily residue was dissolved in methanol and treated
with excess 4 M HCl in dioxane. The precipitate was immedi-
ately filtered, washed with ethyl acetate, and dried, yielding
2-methyl-5-pyridin-4-yl-1H-pyrrole-3-carboxylic acid methyl
1
1
compound (12 mg, 52%). H NMR (400 MHz, DMSO-d6) δ
ester hydrochloride as a white solid (0.752 g, 28% yield). H
ppm 4.22-4.29 (m, 2H), 7.52 (d, J = 9.63 Hz, 2H), 7.74 (s, 2H),
7.93 (s, 1H), 8.39 (s, 3H), 8.68 (d, J = 4.27 Hz, 2H), 12.70 (s, 1H).
ESI (þ) MS: m/z 217 (MHþ). HRMS (M þ H)þ calcd for
C11H12N4O þ Hþ, 217.1084; found, 217.1081.
NMR (400 MHz, DMSO-d6) δ ppm 2.57 (s, 3H), 3.77 (s, 3H),
7.64 (d, J = 2.68 Hz, 1H), 8.15 (d, J = 6.95 Hz, 2H), 8.71 (d, J =
6.95 Hz, 2H), 12.56 (bs, 1H). ESI (þ) MS: m/z 217 (MHþ).
The ester (0.175 g, 0.69 mmol), dissolved in 2 mL of ethanol
and 1.5 M KOH in ethanol (2.3 mL), was refluxed for 15 h. The
solution was cooled and neutralized with ammonium chloride.
The yellow precipitate was filtered and washed with water to
afford 0.114 g (80%) of 2-methyl-5-pyridin-4-yl-1H-pyrrole-
3-carboxylic acid as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ ppm 2.51 (s, 3H), 7.09 (d, J = 2.80 Hz, 1H), 7.62 (d, J = 6.22
Hz, 2H), 8.50 (d, J = 6.22 Hz, 2H), 11.84 (bs, 2H). ESI (þ) MS:
m/z 203 (MHþ).
2-Ethyl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide (5). To a
cooled (0 ꢀC) solution of ethyl 3-oxopentanoate (1.21 g, 8.3
mmol) in anhydrous tetrahydrofuran (200 mL), sodium hydride
(60% in mineral oil, 0.923 g, 21 mmol) was added cautiously.
After 5 min, 4-(bromoacetyl)pyridine hydrobromide 2a (3.05 g,
10.8 mmol) was added and the thick solution was stirred at 0 ꢀC
for 5 h. The solvent was replaced with ethanol (120 mL), and
ammonium acetate (1.93 g, 25 mmol) was added. The mixture
was stirred at room temperature for 18 h. After concentration,
ethyl acetate was added and the organic layer was washed first
with saturated aqueous Na2CO3, then with water, dried over
anhydrous sodium sulfate, and concentrated. The residue was
purified by flash chromatography (ethyl acetate), yielding 1.22 g
(59%) of 2-ethyl-5-pyridin-4-yl-1H-pyrrole-3-carboxylic acid
The acid (0.083 g, 0.4 mmol) was dissolved in anhydrous
tetrahydrofuran (1.5 mL) and anhydrous N,N-dimethylform-
amide (1.5 mL) in the presence of DIEA (0.14 mL, 2 equiv). To
the solution, cooled to 0 ꢀC, EDC HCl (0.152 g, 2 equiv) and
3
HOBt NH3 (0.120 g, 2 equiv) were added. The reaction mixture
3
was left overnight at room temperature. Water was added, and
the slurry was extracted with ethyl acetate (ꢀ2). The organic
layer was concentrated, and the residue was dissolved in ethanol
and treated with 2 M aqueous HCl. The precipitate was filtered
and dried to yield 0.041 g (42%) of the title compound as
a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.55
(s, 3H), 6.94 (bs, 1H), 7.35 (bs, 1H), 7.65 (d, J = 2.56 Hz, 1H),
7.95 (d, J = 6.83 Hz, 2H), 8.68 (d, J = 6.95 Hz, 2H), 12.33
(bs, 1H). ESI (þ) MS: m/z 202 (MHþ). HRMS (ESI): calcd for
C11H11N3O þ Hþ, 202.0975; found, 202.0971.
1
ethyl ester as a yellow solid. H NMR (400 MHz, DMSO-d6)
δ ppm 1.22 (t, J = 7.44, 3H), 1.30 (t, J = 7.07, 3H), 2.94 (q, J =
7.56, 2H), 4.21 (q, J = 7.07, 2H), 7.09 (d, J = 2.80 Hz, 1H), 7.64
(m, 2H), 8.50 (m, 2H), 11.83 (bs, 1H). ESI (þ) MS: m/z 245
(MHþ). HRMS (ESI): calcd for C14H16N2O2 þ Hþ, 245.1284;
found, 245.1283.
2-Ethyl-5-pyridin-4-yl-1H-pyrrole-3-carboxylic acid ethyl ester
(1.22 g, 5 mmol), dissolved in 10 mL of 4 M aq NaOH and 10 mL
of ethanol, was refluxed for 2 h. The solution was cooled and
neutralized with acetic acid. The precipitate was filtered and
washed with water and acetone to afford 0.756 g (70%) of
2-ethyl-5-pyridin-4-yl-1H-pyrrole-3-carboxylic acid (general struc-
ture IIa, R = Et) as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.24 (t, J = 7.50 Hz, 3H), 2.99 (q, J = 7.36 Hz, 2H), 7.59 (d,
J = 2.68 Hz, 1H), 8.16 (d, J = 6.95 Hz, 2H), 8.70 (d, J = 6.95 Hz,
2H), 12.39 (bs, 1H). ESI (þ) MS: m/z 217 (MHþ). HRMS (ESI):
calcd for C12H12N2O2 þ Hþ, 217.0971; found, 217.0973
2-Aminomethyl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide di-
hydrochloride (7). 4-tert-Butoxycarbonylamino-3-oxo-butyric
acid ethyl ester (1.06 g, 4.08 mmol) and 0.410 g of sodium
hydride (60% in mineral oil) dissolved in 20 mL of anhydrous
tetrahydrofuran were stirred 1 h at room temperature and then
cooled to 0 ꢀC. A suspension of 1.22 g (4 mmol) of 2-bromo-1-
pyridin-4-ylethanone hydrobromide (2a) in 10 mL of anhydrous
tetrahydrofuran was added dropwise and the mixture stirred at
0 ꢀC for 4 h. The resulting solution was concentrated, the residue
was dissolved in 30 mL of ethanol/acetic acid 1:1, and 1.25 g of
ammonium acetate was added. The solution was stirred for 5 h,
the solvent was removed, and the raw product was dissolved in
ethyl acetate, washed (ꢀ3) with brine, and dried over anhydrous
sodium sulfate. The solvent was removed and the crude was
purified by flash chromatography over silica gel, thus providing
0.463 g of 2-(tert-butoxycarbonylamino-methyl)-5-pyridin-4-yl-
1H-pyrrole-3-carboxylic acid ethyl ester (34%). 1H NMR (400
MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.07 Hz, 3H), 1.41 (s, 9H),
4.22 (d, J = 7.07 Hz, 2H), 4.50 (d, J = 5.49 Hz, 2H), 6.91 (s, 1H),
7.15 (d, J = 2.68 Hz, 1H), 7.70 (dd, J = 4.63, 1.59 Hz, 2H), 8.52
(dd, J = 4.63, 1.59 Hz, 2H), 11.94 (s, 1H). ESI (þ) MS: m/z
Acid (0.136 g, 0.63 mmol) was dissolved in anhydrous N,N-
dimethylformamide (5 mL) in the presence of DIEA (0.6 mL,
6 equiv). To the solution, cooled to 0 ꢀC, O-benzotriazol-1-yl-
N,N,N0,N0-tetramethyluronium tetrafluoroborate (TBTU, 0.412 g,
2 equiv) and HOBt NH3 (0.211 g, 2 equiv) were added. The
3
reaction mixture was left overnight at room temperature and
then poured into water. The aqueous solution was extracted (5 ꢀ
5 mL) with ethyl acetate, the combined organic layers were
washed with 1 M aqueous NaOH, dried, and concentrated. The
crude material was treated with ethyl ether and filtered, yielding
1
0.064 g (46%) of the title compound as an off-white solid. H
NMR (400 MHz, DMSO-d6) δ ppm 1.17 (t, J = 7.44 Hz, 3H),
2.94 (q, J = 7.48 Hz, 2H), 6.68 (bs, 2H), 7.17 (d, J = 2.80 Hz,

7308 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Menichincheri et al.
1H), 7.50 (d, J = 6.22 Hz, 2H), 8.48 (d, J = 6.10 Hz, 2H), 11.54
(s, 1H). ESI (þ) MS: m/z 216 (MHþ). HRMS (M þ H)þ calcd for
C12H13N3O þ Hþ, 216.1132; found, 216.1133.
2-(4-Fluoro-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Hydrochloride (15). From ethyl (4-fluorobenzoyl)acetate and
2-bromo-1-pyridin-4-yl-ethanone hydrobromide 2a in 23% over-
all yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.12 (bs, 2H),
7.32-7.39 (m, 4H), 7.70 (d, J = 2.43 Hz, 1H), 8.15 (d, J = 6.59
Hz, 2H), 8.72 (d, J = 6.59 Hz, 2H), 12.52 (s, 1H). ESI (þ) MS:
m/z 282 (MHþ). HRMS (M þ H)þ calcd for C16H12FN3O þ Hþ,
282.1037; found, 282.1044.
Analogously the following compounds were obtained:
2-iso-Propyl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide (6).
From 4-methyl-3-oxo-pentanoic acid ethyl ester and 2-bromo-
1-pyridin-4-yl-ethanone hydrobromide 2a in 18% overall yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 1.25 (d, J = 7.07 Hz,
6H), 3.99 (qq, J = 6.99, 6.77 Hz, 1H), 7.14 (d, J = 2.56 Hz, 1H),
7.49 (s, 1H), 7.57 (d, J = 6.10 Hz, 2H), 8.48 (d, J = 5.98 Hz, 2H).
ESI (þ) MS: m/z 230 (MHþ). HRMS (M þ H)þ calcd for
C13H15N3O þ Hþ, 230.1288; found, 230.1286.
2-(3-Bromo-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Hydrochloride (16). From ethyl (3-bromobenzoyl)acetate and
2-bromo-1-pyridin-4-yl-ethanone hydrobromide 2a in 18%
1
overall yield. H NMR (400 MHz, DMSO-d6) δ ppm 7.14 (bs,
1H), 7.44 (t, J = 7.93 Hz, 1H), 7.51 (bs, 1H), 7.60-7.65 (m, 1H),
7.69 (d, J = 2.56 Hz, 1H), 7.71-7.75 (m, 1H), 7.93 (t, J = 1,83
Hz, 1H), 8.18 (d, J= 5.85 Hz, 2H), 8.76 (d, J= 6.83 Hz, 2H), 12.38
(bs, 1H). ESI (þ) MS: m/z 342-344 (MHþ). HRMS (M þ H)þ
calcd for C16H12BrN3O þ Hþ, 342.0236; found, 342.0233.
2-(4-Bromo-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxylic acid
amide (17). From ethyl (4-bromobenzoyl)acetate and 2-bromo-
1-pyridin-4-yl-ethanone hydrobromide 2a in 22% overall yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 6.93 (bs, 1H), 7.28 (d,
J = 2.68 Hz, 1H), 7.37 (bs, 1H), 7.62-7.67 (m, 4H), 7.69 (d, J =
6.22 Hz, 2H), 8.54 (d, J = 6.22 Hz, 2H), 11.86 (bs, 1H). ESI
(þ) MS: m/z 342-344 (MHþ). HRMS (M þ H)þ calcd for
C16H12BrN3O þ Hþ, 342.0236; found, 342.0240.
2-Benzyl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide Hydro-
chloride (8). From 3-oxo-4-phenyl-butyric acid ethyl ester and
2-bromo-1-pyridin-4-yl-ethanone hydrobromide 2a in 13%
1
overall yield. H NMR (400 MHz, DMSO-d6) δ ppm 4.39 (s,
2H), 6.97 (bs, 1H), 7.16 (m, 1H), 7.26 (m, 2H), 7.29-7.33 (m,
2H), 7.44 (bs, 1H), 7.63-7.71 (m, 1H), 7.86-8.03 (m, 2H),
8.68 (d, J=6.58 Hz, 2H), 12.43 (bs, 1H). ESI (þ) MS: m/z 278
(MHþ). HRMS (M þ H)þ calcd for C17H15N3O þ Hþ,
278.1288; found, 278.1287.
2-Cyclohexyl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide (9).
From 3-cyclohexyl-3-oxo-propionic acid ethyl ester and 2-bro-
mo-1-pyridin-4-yl-ethanone hydrobromide 2a in 13% overall
1
yield. H NMR (400 MHz, DMSO-d6) δ ppm 1.62-1.84 (m,
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-carbox-
amide (18). From 3-oxo-3-phenyl-propionic acid ethyl ester
and 1-(2-amino-4-pyrimidinyl)-2-bromo-ethanone hydrobro-
mide (2c), ethyl 5-(2-aminopyrimidin-4-yl)-2-phenyl-1H-pyr-
role-3-carboxylate (61) was obtained (46%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.20 (t, J=7.13 Hz, 3H), 4.14 (q, J = 7.07
Hz, 2H), 6.45 (s, 2H), 7.10 (d, J = 5.24 Hz, 1H), 7.33 (d, J = 2.56
Hz, 1H), 7.40-7.49 (m, 3H), 7.61-7.65 (m, 2H), 8.23 (d, J=5.24
Hz, 1H), 12.01 (bs, 1H). ESI (þ) MS: m/z 309 (MHþ). HRMS
(M þ H)þ calcd for C17H16N4O2þHþ, 309.1346; found, 309.1342.
From ester 61, 5-(2-amino-pyrimidin-4-yl)-2-phenyl-1H-pyr-
role-3-carboxylic acid (3) was isolated as a white solid
(quantitative). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.05
(bs, 2H), 7.22 (d, J = 5.73 Hz, 1H), 7.39-7.47 (m, 4H), 7.62-
7.64 (m, 2H), 8.24 (d, J = 5.85 Hz, 1H), 11.99 (bs, 1H), 12.10 (bs,
1H). MS: m/z 279 [M - H]. HRMS (M þ H)þ calcd for
C15H12N4O2 þ Hþ, 281.1033; found, 281.1043.
10H), 3.59-3.71 (m, 1H), 6.67 (bs, 1H), 7.13 (d, J = 2.68 Hz, 1H),
7.21 (bs, 1H), 7.58 (d, J = 6.22 Hz, 2H), 8.48(d, J= 6.10Hz, 2H),
11.13 (bs, 1H). ESI (þ) MS: m/z 270 (MHþ). HRMS (M þ H)þ
calcd for C16H19N3O þ Hþ, 270.1601; found, 270.1602.
4-(3-Carbamoyl-5-pyridin-4-yl-1H-pyrrol-2-yl)-piperidine-1-
carboxylic Acid tert-Butyl Ester (10). From 4-(2-ethoxycarbo-
nyl-acetyl)-piperidine-1-carboxylic acid tert-butyl ester and
2-bromo-1-pyridin-4-yl-ethanone hydrobromide 2a in 15%
1
overall yield. H NMR (400 MHz, DMSO-d6) δ ppm 1.45 (s,
9H), 1.67 (d, J = 12.32 Hz, 2H), 1.76-1.89 (m, 2H), 2.71 (bs,
2H), 3.81-3.91 (m, 1H), 4.12 (d, J=11.10 Hz, 2H), 6.76 (bs,
1H), 7.18 (d, J = 2.56 Hz, 1H), 7.29 (bs, 1H), 7.59 (d, J = 6.22
Hz, 2H), 8.50 (d, J = 6.10 Hz, 2H), 11.22 (bs, 1H). ESI (þ) MS:
m/z 371 (MHþ). HRMS (M þ H)þ calcd for C20H26N4O3 þ Hþ,
371.2078; found, 371.2075.
2-Phenyl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide (11). From
3-oxo-3-phenyl-propionic acid ethyl ester and 2-bromo-1-pyr-
idin-4-yl-ethanone hydrobromide 2a in 22% overall yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 6.90 (bs, 2H), 7.27 (d,
J = 2.56 Hz, 1H), 7.37 (m, 1H), 7.44 (m, 2H), 7.67-7.71 (m,
4H), 8.53 (m, 2H), 11.82 (s, 1H). ESI (þ) MS: m/z 264 (MHþ).
HRMS (MþH)þ calcd for C16H13N3OþHþ, 264.1131; found,
264.1132.
From acid 3 the title compound (18) was obtained as an
orange powder in 43% yield. 1H NMR (400 MHz, DMSO-d6) δ
ppm 6.36 (s, 2H), 6.82 (bs, 1H), 7.02 (d, J = 5.2 Hz, 1H), 7.27 (d,
J = 2.6 Hz, 1H), 7.32 (br s, 1H), 7.32-7.37 (m, 1H), 7.37-7.44
(m, 2H), 7.64 (dd, J = 8.3, 1.3 Hz, 2H), 8.20 (d, J = 5.2 Hz, 1H),
11.63 (bs, 1H). ESI (þ) MS: m/z 280 (MHþ). HRMS (M þ H)þ
calcd for C15H13N5O þ Hþ, 280.1193; found, 280.1189. Anal.
(C15H13N5O) C, H, N.
2-Phenyl-5-(2-phenylamino-pyrimidin-4-yl)-1H-pyrrole-3-car-
boxamide (19). From 3-oxo-3-phenyl-propionic acid ethyl ester
and 2-bromo-1-[2-(phenylamino)-4-pyrimidinyl]-ethanone (2d),
the title compound was obtained in 12% overall yield. 1H NMR
(400 MHz, DMSO-d6) δ ppm 6.89 (bs, 1H), 6.95 (t, J = 7.33 Hz,
1H), 7.29-7.48 (m, 6H), 7.29 (d, J = 5.24 Hz, 1H), 7.66-7.68
(m, 2H), 7.85 (d, J = 7.68 Hz, 2H), 8.43 (d, J = 5.24 Hz, 1H),
9.40 (s, 1H), 11.75 (s, 1H). ESI (þ) MS: m/z 356 (MHþ). HRMS
(M þ H)þ calcd for C21H17N5O þ Hþ, 356.1506; found,
356.1508.
2,5-Dipyridin-4-yl-1H-pyrrole-3-carboxamide (12). From ethyl
isonicotinoylacetate and 2-bromo-1-pyridin-4-yl-ethanone
hydrobromide 2a in 21% overall yield. H NMR (400 MHz,
1
DMSO-d6) δ ppm 7.02 (bs, 2H), 7.29 (s, 1H), 7.71 (m, 4H), 8.56 (m,
4H), 12.01 (bs, 1H). ESI (þ) MS: m/z 265(MHþ). HRMS (M þ
H)þ calcd for C15H12N4O þ Hþ, 265.1084; found, 265.1078.
2-(2-Fluoro-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Hydrochloride (13). From ethyl (2-fluorobenzoyl)acetate and
2-bromo-1-pyridin-4-yl-ethanone hydrobromide 2a in 20%
overall yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.00 (bs,
1H), 7.29-7.34 (m, 2H), 7.36 (bs, 1H), 7.49-7.60 (m, 2H), 7.73
(d, J = 2.43 Hz, 1H), 8.11 (d, J = 6.59 Hz, 2H), 8.74 (d, J = 6.59
Hz, 2H), 12.56 (s, 1H). ESI (þ) MS: m/z 282 (MHþ). HRMS (M þ
H)þ calcd for C16H12FN3O þ Hþ, 282.1037; found, 282.1035.
2-(3-Fluoro-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Hydrochloride (14). From ethyl (3-fluorobenzoyl)acetate and
2-bromo-1-pyridin-4-yl-ethanone hydrobromide 2a in 24% overall
yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.16 (bs, 2H), 7.29
(m, 2H), 7.52 (m, 2H), 7.74 (s, 1H), 8.23 (d, J = 5.80 Hz, 2H), 8.78
(d, J = 5.80 Hz, 2H), 12.42 (s, 1H). ESI (þ) MS: m/z 282 (MHþ).
HRMS (M þ H)þ calcd for C16H12FN3O þ Hþ, 282.1037; found,
282.1036.
5-(2-Amino-5-fluoro-pyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-
carboxamide (20). From 3-oxo-3-phenyl-propionic acid ethyl
ester and 1-(2-amino-5-fluoro-4-pyrimidinyl)-2-bromo-ethanone
1
2e, the title compound was obtained in 17% overall yield. H
NMR (400 MHz, DMSO-d6) δ ppm 6.34 (s, 2H), 6.87 (bs, 1H),
7.27 (t, J = 2.80 Hz, 1H), 7.33-7.43 (m, 3H), 7.40 (s, 1H),
7.62-7.66 (m, 2H), 8.27 (d, J = 3.41 Hz, 1H), 11.49 (bs,
1H). ESI (þ) MS: m/z 298 (MHþ). HRMS (M þ H)þ calcd
for C15H12FN5O þ Hþ, 298.1099; found, 298.1096. Anal.
(C15H12FN5O) C, H, N.

Article
5-(2-Amino-pyrimidin-4-yl)-2-pyridin-2-yl-1H-pyrrole-3-carbox-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7309
J = 8.41, 7.01, 1.16 Hz, 1H), 7.58 (d, J = 0.73 Hz, 1H),
7.59-7.62 (m, 1H), 7.75 (ddd, J = 8.02, 0.99 Hz, 1H).
amide (21). From ethyl picolinoylacetate and 1-(2-amino-4-
pyrimidinyl)-2-bromo-ethanone hydrobromide (2c),, the title
compound was obtained in 14% overall yield. H NMR (400
From ethyl 3-(1-methyl-1H-indol-2-yl)-3-oxopropanoate
and 1-(2-amino-4-pyrimidinyl)-2-bromo-ethanone hydrobro-
mide (2c), the title compound was obtained in 14% overall
yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 3.58 (s, 3H), 6.38
(bs, 2H), 6.61 (d, J = 0.61 Hz, 1H), 6.88 (bs, 1H), 7.00 (d, J =
5.24 Hz, 1H), 7.08 (t, J = 7.70 Hz, 1H), 7.20 (t, J = 7.70 Hz, 1H),
7.25 (bs, 1H), 7.41 (d, J = 2.44 Hz, 1H), 7.47 (d, J = 7.70 Hz,
1H), 7.59 (d, J = 7.70 Hz, 1H), 8.22 (d, J = 5.24 Hz, 1H), 11.98
(bs, 1H). ESI (þ) MS: m/z 333 (MHþ). HRMS (M þ H)þ calcd
for C18H16N6O þ Hþ, 333.1459; found, 333.1460.
1
MHz, DMSO-d6) δ ppm 6.62 (bs, 2H), 7.03 (d, J = 5.12 Hz, 1H),
7.16 (bs, 1H), 7.33-7.40 (m, 2H), 7.89 (ddd, J = 8.05, 7.56, 1.83
Hz, 1H), 8.25 (d, J = 5.12 Hz, 1H), 8.28 (bs, 1H), 8.43 (d, J =
8.17 Hz, 1H), 8.64 (ddd, J = 4.08, 1.70, 1.00 Hz, 1H), 11.29 (s,
1H). ESI (þ) MS: m/z 281 (MHþ). HRMS (M þ H)þ calcd for
C14H12N6O þ Hþ, 281.1146; found, 281.1145.
5-(2-Amino-pyrimidin-4-yl)-2-thiophen-2-yl-1H-pyrrole-3-carbox-
amide (22). From 3-oxo-3-thiophen-2-yl-propionic acid ethyl
ester and 1-(2-amino-4-pyrimidinyl)-2-bromo-ethanone hydro-
bromide (2c), the title compound was obtained in 10% overall
yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 6.46 (bs, 2H), 6.91
(bs, 1H), 7.04 (d, J = 5.24 Hz, 1H), 7.11 (dd, J = 5.12, 3.66 Hz,
1H), 7.30 (d, J = 2.56 Hz, 1H), 7.44 (bs, 1H), 7.57 (dd, J = 5.12,
1.22 Hz, 1H), 7.66 (dd, J = 3.66, 1.22 Hz, 1H), 8.23 (d, J = 5.24
Hz, 1H), 11.60 (bs, 1H). ESI (þ) MS: m/z 286 (MHþ). HRMS
(M þ H)þ calcd for C13H11N5OS þ Hþ, 286.0757; found,
286.0758. Anal. (C13H11N5OS) C, H, N, S.
5-(2-Amino-pyrimidin-4-yl)-2-(1-methyl-1H-indol-3-yl)-1H-
pyrrole-3-carboxamide (25). Ethyl 3-(1-methyl-1H-indol-3-yl)-
3-oxopropanoate crude yellow oil (0.412 g, quant.) was obtained
1
as described above for its regioisomer. H NMR (400 MHz,
DMSO-d6) δ ppm 1.21 (t, J = 7.07 Hz, 3H), 3.89 (s, 3H), 3.95 (s,
2H), 4.13 (q, J = 7.15 Hz, 2H), 7.24-7.29 (m, 1H), 7.30-7.35
(m, 1H), 7.56 - 7.60 (m, 1H), 8.18 (ddd, J = 7.77, 1.37, 0.73 Hz,
1H), 8.39 (s, 1H).
From ethyl 3-(1-methyl-1H-indol-3-yl)-3-oxopropanoate
and 1-(2-amino-4-pyrimidinyl)-2-bromo-ethanone hydrobro-
mide (2c), the title compound was obtained in 18% overall yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 3.87 (s, 3H), 6.35 (bs,
2H), 6.73 (bs, 1H), 6.91 (bs, 1H), 6.98 (d, J = 5.24 Hz, 1H), 7.11
(ddd, J = 7.99, 7.07, 0.91 Hz, 1H), 7.22 (ddd, J = 8.17, 7.13,
0.91 Hz, 1H), 7.32 (d, J = 2.68 Hz, 1H), 7.50 (dt, J = 8.05, 0.85
Hz, 1H), 7.51 (dt, J = 8.32, 0.84 Hz, 1H), 7.80 (s, 1H), 8.17 (d,
J = 5.24 Hz, 1H), 11.36 (bs, 1H).. ESI (þ) MS: m/z 333 (MHþ).
HRMS (M þ H)þ calcd for C18H16N6O þ Hþ, 333.1459; found,
333.1463.
5-(2-Amino-pyrimidin-4-yl)-2-benzo[b]thiophen-5-yl-1H-pyr-
role-3-carboxamide (23). A solution of 1-benzothiophene-5-
carboxylic acid (9.71 g, 54.5 mmol) and 1,10-carbonyldiimida-
zole (13.04 g, 80.1 mmol) in tetrahydrofuran (100 mL) was
stirred at room temperature for 40 min. After addition of MgCl2
(10.44 g, 109 mmol) and monoethylmalonate potassium salt
(14.72 g, 87 mmol) in tetrahydrofuran (100 mL), the mixture was
heated for 3 h at 45 ꢀC under stirring. After solvent removal,
the residue was dissolved in ethyl acetate and washed with 5%
KHSO4 aqueous solution, with saturated aqueous NaHCO3
solution, and with brine. After drying over anhydrous sodium
sulfate, ethyl 3-(1-benzothiophen-5-yl)-3-oxopropanoate (6.22 g,
45%) was obtained. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.20
(t, J = 7.13 Hz, 3H), 4.14 (q, J = 7.07 Hz, 2H), 4.27 (s, 2H), 7.63
(d, J=5.37 Hz, 1H), 7.89-7.92 (m, 1H), 7.92-7.93 (m, 1H),
8.18 (d, J = 8.54 Hz, 1H), 8.56 (d, J = 1.59 Hz, 1H). Anal.
(C17H13N5OS) C, H, N, S.
From ethyl 3-(1-benzothiophen-5-yl)-3-oxopropanoate and
1-(2-amino-4-pyrimidinyl)-2-bromo-ethanone hydrobromide (2c),
the title compound was obtained in 17% overall yield. 1H NMR
(400 MHz, DMSO-d6) δ ppm 6.36 (bs, 2H), 6.84 (bs, 1H), 7.04
(d, J = 5.24 Hz, 1H), 7.31 (d, J = 2.56 Hz, 1H), 7.34 (bs, 1H),
7.50 (dd, J=5.49, 0.49 Hz, 1H), 7.62 (dd, J = 8.41, 1.71 Hz,
1H), 7.80 (d, J = 5.49 Hz, 1H), 8.01 (d, J = 8.41 Hz, 1H), 8.13
(d, J = 1.34 Hz, 1H), 8.21 (d, J = 5.37 Hz, 1H), 11.75 (bs, 1H).
ESI (þ) MS: m/z 336 (MHþ). HRMS (M þ H)þ calcd for
C17H13N5OS þ Hþ, 336.0914; found, 336.0918.
2-Piperidin-4-yl-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Dihydrochloride (26). Amide 10 (0.030 g, 0.08 mmol) was dis-
solved in methanol (5 mL), 2N HCl (1 mL) was added, and the
clear solution was warmed at 50 ꢀC under stirring for 5 h. The
precipitate was filtered and washed with methanol, yielding
1
the title compound as a white solid (0.025 g, 90%). H NMR
(400 MHz, DMSO-d6) δ ppm 1.96 (d, J = 13.05 Hz, 2H),
2.09-2.24 (m, 2H), 2.92-3.07 (m, 2H), 3.74-3.87 (m, 1H), 7.01
(bs, 1H), 7.49 (bs, 1H), 7.66 (s, 1H), 8.10 (bs, 2H), 8.56 (bs, 1H),
8.71 (d, J = 6.34 Hz, 2H), 8.83 (bs, 1H), 11.97 (bs, 1H). ESI (þ)
MS: m/z 271 (MHþ). HRMS (M þ H)þ calcd for C15H18N4O,
271.1554; found, 271.1550.
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-carboxylic
Acid Methylamide Hydrochloride (27). To a solution of 5-(2-
aminopyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-carboxylic acid 3
(0.023 g, 0.07 mmol) in anhydrous N,N-dimethylformamide,
1,10-carbonyldiimidazole (0.025 g, 0.14 mmol) was added and
the mixture stirred at 45 ꢀC for 1 h. After cooling to room
temperature, the solution was treated with 0.5 mL of 33%
methylamine in ethanol. The mixture was stirred overnight,
filtered, and the filtrate was poured into water. After extraction
with ethyl acetate (ꢀ2), the organic layer was concentrated,
dissolved in ethanol, and treated with excess 1.25 M HCl in
methanol. Diethyl ether was added, and the yellow crystalline
solid was filtered, washed with diethyl ether, and recovered. The
title compound was obtained (8 mg, 37% yield). 1H NMR (400
MHz, DMSO-d6) δ ppm 2.70 (d, J = 4.63 Hz, 3H), 7.36 (d, J =
6.58 Hz, 1H), 7.39-7.49 (m, 3H), 7.53 (d, J = 2.44 Hz, 1H),
7.64-7.70 (m, 2H), 7.84 (bs, 3H), 8.02 (q, J =4.51 Hz, 1H),
8.29 (d, J = 6.46 Hz, 1H), 12.20 (bs, 1H). ESI (þ) MS: m/z
294 (MHþ). HRMS (M þ H)þ calcd for C16H15N5O þ Hþ,
294.1349; found, 294.1345.
5-(2-Amino-pyrimidin-4-yl)-2-(1-methyl-1H-indol-2-yl)-1H-
pyrrole-3-carboxamide (24). A solution of 1-methylindole-2-
carboxylic acid (0.311 g, 1.71 mmol) and 1,10-carbonyldiimida-
zole (0.360 g, 2.22 mmol mmol) in anhydrous tetrahydrofuran
(10 mL) was stirred at room temperature for 1 h under argon
atmosphere. In another flask, to a cooled (0 ꢀC) solution of
3-ethoxy-3-oxopropanoic acid (0.522 g, 3.93 mmol) in anhydrous
tetrahydrofuran (10 mL), under stirring and argon atmosphere,
Mg(OEt)2 (0.255 g, 2.22 mmol) was added and the reaction
mixture was stirred for 2 h at room temperature. To this solu-
tion, kept at 0 ꢀC under stirring, the solution of imidazolide was
added dropwise and stirred at 0 ꢀC for 30 min. The mixture was
heated to 50 ꢀC and stirred at this temperature for 8 h and then
for 18 h at room temperature. After solvent removal, the residue
was dissolved in diethyl ether and washed with 1 N HCl, with
saturated aqueous NaHCO3 solution, and with brine. After
drying over anhydrous sodium sulfate, ethyl 3-(1-methyl-1H-
indol-2-yl)-3-oxopropanoate was isolated as a yellow oil
Analogously the following compounds were prepared:
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-carboxylic
Acid Isopropylamide (28). Yield 30%. 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.09 (d, J = 6.58 Hz, 6H), 3.93-4.06 (m,
1H), 6.36 (bs, 2H), 7.04 (d, J = 5.24 Hz, 1H), 7.23 (d, J = 2.44
Hz, 1H), 7.30-7.43 (m, 3H), 7.59-7.67 (m, 3H), 8.21 (d, J =
5.24 Hz, 1H), 11.61 (bs, 1H). ESI (þ) MS: m/z 322 (MHþ).
1
(0.412 g, quant). H NMR (400 MHz, DMSO-d6) δ ppm 1.21
(t, J = 7.13 Hz, 3H), 4.02 (s, 3H), 4.14 (q, J = 6.99 Hz, 2H),
4.14 (s, 2H), 7.17 (ddd, J = 7.99, 6.95, 0.91 Hz, 1H), 7.42 (ddd,

7310 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Menichincheri et al.
HRMS (M þ H)þ calcd for C18H19N5O þ Hþ, 322.1662; found,
322.1667.
4-(pyridin-4-yl)-4-oxo-butyric acid ethyl ester (33a) as an oil
(1.03 g, 4.4 mmol, 60%). 1H NMR (DMSO-d6/400 MHz) δ ppm
1.23 (t, J = 7.07 Hz, 3H), 3.87 (m, 2H), 4.22 (q, J = 7.07 Hz,
2H), 4.64 (t, J = 5.37 Hz, 1H), 7.88 (m, 2H), 8.86 (m, 2H).
ESI (þ) MS: m/z 233(MHþ). HRMS (M þ H)þ calcd for
C12H12N2O3 þ Hþ, 233.0921; found, 233.0922.
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-carboxylic
Acid Benzylamide (29). Yield 66%. 1H NMR (400 MHz,
DMSO-d6) δ ppm 4.37 (d, J = 6.10 Hz, 2H), 6.37 (bs, 2H),
7.05 (d, J = 5.24 Hz, 1H), 7.18-7.44 (m, 9H), 7.64 (d, J = 8.30
Hz, 2H), 8.21 (d, J = 5.24 Hz, 1H), 8.48 (t, J = 6.10 Hz, 1H),
11.70 (bs, 1H). ESI (þ) MS: m/z 370 (MHþ). HRMS (M þ H)þ
calcd for C22H19N5O þ Hþ, 370.1662; found, 370.1668.
5-(2-Amino-pyridin-4-yl)-2-phenyl-1H-pyrrole-3-carboxamide
(32). To a solution of 3-oxo-3-phenyl-propionic acid ethyl ester
(0.351 g, 2 mmol) in THF (45 mL) at 0 ꢀC, NaH (60% in oil,
0.182 g, 4.5 mmol) was added and the mixture was stirred for
20 min at 0 ꢀC. 2-Bromo-1-(2-chloro-4-pyridinyl)-ethanone
hydrobromide 2f (0.635 g, 2 mmol) was added, and the reaction
mixture was stirred at 0 ꢀC for 2 h, then at room temperature
for 2 h. After solvent removal, absolute ethanol (25 mL) and
ammonium acetate (0.505 g, 6.5 mmol) were added and the
mixture was stirred at room temperature for 20 h. The solvent
was removed, and the residue was taken up with ethyl acetate
and water. The organic phase was dried over anhydrous sodium
sulfate and purified by flash chromatography (dichloromethane/
methanol 95:5), affording 5-(2-chloro-pyridin-4-yl)-2-phenyl-
1H-pyrrole-3-carboxylic acid ethyl ester (30, 0.342 g, 50%).
1H NMR (400 MHz, DMSO-d6) δ ppm: 1.19 (t, J = 7.07 Hz,
3H), 4.14 (q, J = 7.07 Hz, 2H), 7.38-7.52 (m, 4H), 7.62-7.68
(m, 2H), 7.80 (d, J = 5.37, 1H), 7.97 (s, 1H), 8.33 (d, J = 5.37
Hz, 1H), 12.37 (bs, 1H). ESI (þ) MS: m/z 327 (MHþ). HRMS
(M þ H)þ calcd for C18H15ClN2O2 þ Hþ, 327.0895; found,
327.0900.
A solution of cyanoester 33a (1.02 g, 4.3 mmol), dissolved in
diethyl ether and dichloromethane, was added dropwise to a solu-
tion of HBr (33% in acetic acid, 13 mL, 43.1 mmol) at 0 ꢀC. The
reaction mixture was left for 20 min at 0 ꢀC and then at room
temperature until disappearance of the starting material (2.5 h).
The solid was filtered and washed with acetone and methanol.
The pyridinium salt was neutralized with 7N NH3 in methanol.
The solid was purified by flash chromatography (dichloromethane/
methanol 95:5) to give 0.805 g (62%) of 2-bromo-5-pyridin-4-yl-
1H-pyrrole-3-carboxylic acid ethyl ester (34a) as an orange solid.
1H NMR (400 MHz, DMSO-d6) δ ppm 1.31 (t, J = 7.12 Hz, 3H),
4.24 (q, J = 7.12 Hz, 2H), 7.26 (s, 1H), 7.71 (d, J = 6.22 Hz, 2H),
8.54 (d, J = 6.22 Hz, 2H), 12.85 (s, 1H). ESI (þ) MS: m/z 295-297
(MHþ). HRMS (ESI): calcd for C12H11BrN2O2 þ Hþ, 295.0077;
found, 295.0078.
Ester 34a (1.10 g, 3.74 mmol), dissolved in 8 mL of 4 M
aqueous NaOH and 8 mL of ethanol, was refluxed for 4 h. The
solution was cooled and neutralized with acetic acid. The pre-
cipitate was filtered and washed with water and acetone to
afford 0.850 g (85%) of 2-bromo-5-pyridin-4-yl-1H-pyrrole-3-
carboxylic acid as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ ppm 7.33 (s, 1H), 7.83 (d, J = 6.00 Hz, 2H), 8.58 (d, J = 6.00
Hz, 2H), 12.37 (bs, 1H), 12.91 (s, 1H). MS: m/z 265-267 [M - H].
The acid (0.450 g, 1.68 mmol) was dissolved in anhydrous
tetrahydrofuran (20 mL) in the presence of DIEA (1.27 mL, 7.30
A mixture of 30 (0.122 g, 0.37 mmol), t-butyl carbamate
(0.215 g, 1.84 mmol), Xantphos (0.016 g, 0.028 mmol), palla-
dium diacetate (0.004 g, 0.018 mmol), and cesium carbonate
(0.242 g, 0.73 mmol) in dioxane (3 mL) was stirred at 140 ꢀC in a
microwave cavity for 20 min. The crude material was taken up
with methanol, filtered through Celite, treated with ethyl acetate
and water, dried, and concentrated. The residue was purified
by flash chromatography (dichloromethane/methanol 95:5)
affording 5-(2-amino-pyridin-4-yl)-2-phenyl-1H-pyrrole-3-
carboxylic acid ethyl ester (31, 0.045 g, 40%). ESI (þ) MS: m/z
308 (MHþ).
mmol). To the solution, cooled to 0 ꢀC, EDC HCl (1.03 g, 5.5
3
mmol) and HOBt NH3 (0.810 g, 5.34 mmol) were added. The
3
reaction mixture was left overnight at room temperature. The
solvent was evaporated, water was added, and the slurry was
extracted with dichloromethane. The crude was purified by
flash-chromatography (dichloromethane/methanol 95:5) to yield
0.153 g (33%) of the title compound as a pale-yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.02 (s, 2H), 7.29 (s, 1H),
7.59 (d, J = 6.25 Hz, 2H), 8.52 (d, J = 6.25 Hz, 2H), 12.54 (s,
1H). ESI (þ) MS: m/z 266-268 (MHþ). HRMS (ESI): calcd for
C10H8BrN3O þ Hþ, 265.9923; found, 265.9935.
A solution of 31 (0.042 g, 0.15 mmol) in ethanol (0.5 mL) and
4 N NaOH (0.5 mL) was warmed at reflux for 2 h. The mixture
was acidified with acetic acid and stripped under reduced
pressure. The residue was dissolved in N,N-dimethylformamide
5-(3-Fluoro-pyridin-4-yl)-2-phenyl-1H-pyrrole-3-carboxamide
hydrochloride (39). Ethylcyanoacetate (715 μL, 6.7 mmol) was
added to a suspension of sodium metal (0.154 g, 6.7 mmol) in
20 mL of anhydrous ethanol at 0 ꢀC. The solution was stirred
until sodium dissolved completely. The solvent was evaporated,
and the solid was added to a solution of 2-bromo-1-(3-fluoro-4-
pyridinyl)-ethanone (2.04 g, 6.7 mmol) and DIEA (1.16 mL, 6.7
mmol) in 40 mL of anhydrous tetrahydrofuran. The mixture was
stirred overnight at room temperature. The solvent was evapo-
rated, and the residue was suspended in water and extracted with
dichloromethane. The organic extracts were dried over anhy-
drous sodium sulfate, and the crude material was purified
by flash chromatography (dichloromethane/methanol 98:2)
to afford 2-cyano-4-(3-fluoro-pyridin-4-yl)-4-oxo-butyric acid
(2 mL) and treated with HOBt NH3 (0.042 g, 0.27 mmol),
EDC HCl (0.052 g, 0.27 mmol), and DIEA (0.12 mL) for
3
3
3 days at room temperature (two more additions of the same
amount of reagents after one and two days). After concentration
and aqueous workup with ethyl acetate, the residue was purified
by reverse phase flash chromatography, affording the title
1
compound (0.033 g, 80%). H NMR (400 MHz, DMSO-d6) δ
ppm 6.37 (bs, 2H), 6.83 (bs, 1H), 7.02 (d, J = 5.78 Hz, 1H), 7.28
(d, J = 2.68 Hz, 1H), 7.32 (bs, 1H), 7.31-7.46 (m, 3H), 7.65 (d,
J= 8.17 Hz, 2H), 7.90 (d, J= 5.78 Hz, 1H), 11.64 (bs, 1H). ESI (þ)
MS: m/z 279 (MHþ). HRMS (M þ H)þ calcd for C16H14N4O þ
Hþ, 279.1241; found, 279.1245.
1
ethyl ester (33b) as an oil (1.05 g, 4 mmol, 60%). H NMR
(400 MHz, DMSO-d6) δ ppm 1.22 (t, J = 7.07 Hz, 3H), 3.78 (m,
2H), 4.20 (q, J = 7.07 Hz, 2H), 4.61 (t, J = 5.37 Hz, 1H), 7.81
(m, 1H), 8.64 ((dd, J = 5.00, 1.22 Hz, 1H), 8.82 (d, J = 2.56 Hz,
1H). ESI (þ) MS: m/z 251(MHþ). HRMS (M þ H)þ calcd for
C12H11FN2O3 þ Hþ, 251.0826; found, 251.0816.
2-Bromo-5-pyridin-4-yl-1H-pyrrole-3-carboxylic Acid Amide
(36a). Ethylcyanoacetate (0.79 mL, 7.4 mmol) was added to a
suspension of sodium metal (0.17 g, 7.4 mmol) in 25 mL of
anhydrous ethanol at 0 ꢀC. The solution was stirred until sodium
dissolved completely. The solvent was evaporated, and the solid
was added to a solution of 2-bromo-1-(4-pyridinyl)-ethanone
(2.08 g, 7.4 mmol) and DIEA (1.29 mL, 7.4 mmol) in 45 mL of
anhydrous tetrahydrofuran. The mixture was stirred overnight
at room temperature. The solvent was evaporated, and the
residue was suspended in water and extracted with dichloro-
methane. The organic extracts were dried over anhydrous
Na2SO4, and the crude material was purified by flash chroma-
tography (dichloromethane/methanol 95:5) to afford 2-cyano-
A solution of 33b (1.05 g, 4 mmol) in anhydrous diethyl ether
(3 mL) and dichloromethane (2 mL) was added to 33% HBr in
acetic acid (12 mL), cooled at 0 ꢀC. The reaction mixture was
stirred for 3 h, the precipitate was filtered, washed with acetone
and methanol, and neutralized with 7 N NH3 in methanol. The
solvent was evaporated to give 1.03 g (80%) of 2-bromo-5-
(3-fluoro-pyridin-4-yl)-1H-pyrrole-3-carboxylic acid ethyl ester
1
(34b) as a solid. H NMR (400 MHz, DMSO-d6) δ ppm 1.26

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7311
(t, J=7.07 Hz, 3H), 4.20 (q, J=7.07 Hz, 2H), 7.08 (d, J=3.53
Hz, 1H), 7.30 (bs, 1H), 7.83 (m, 1H), 8.39 (dd, J = 5.12, 0.85 Hz,
1H), 8.55 (d, J = 3.41 Hz, 1H). ESI (þ) MS: m/z 313-315
(MHþ). HRMS (M þ H)þ calcd for C12H10BrFN2O2 þ Hþ,
312.9982; found, 312.9976.
286 (MHþ). HRMS (M þ H)þ calcd for C13H11N5OS þ Hþ,
286.0757; found, 286.0753. Anal. (C13H11N5OS) C, H, N, S.
5-Pyridin-4-yl-2-p-tolyl-1H-pyrrole-3-carboxamide (43). Acid
35a (0.503 g, 1.87 mmol) was loaded on Rink amide MBHA
resin (1.38 g, 0.935 mmol, theoretical loading 0.68 mmol/g) by
The ester 34b (1.0 g, 3.2 mmol) was dissolved in 8 mL of
ethanol and 8 mL of 1 M aq NaOH and heated at 100 ꢀC for 6 h.
The product was precipitated with acetic acid, and the solid was
filtered and washed with acetone to afford 0.722 g (77%) of
2-bromo-5-(3-fluoro-pyridin-4-yl)-1H-pyrrole-3-carboxylic acid
(35b). 1H NMR (400 MHz, DMSO-d6) δ ppm 6.98 (d, J =
5.12 Hz, 1H), 7.81 (q, J = 5.12 Hz, 1H), 8.13 (d, J = 5.60 Hz,
1H), 8.25 (d, J = 4.02 Hz, 1H). MS: m/z 283-285 [M - H].
HRMS (M þ H)þ calcd for C10H6BrFN2O2 þ Hþ, 284.9669;
found, 284.9670.
stirring with DIEA (0.65 mL, 3.74 mmol), EDC HCl (0.537 g,
3
2.8 mmol), and HOBt (0.379 g, 2.8 mmol) in 20 mL of N,N-
dimethylformamide at room temperature overnight. The sub-
stitution rate was 78%, and the resin had been previously
cleaved with 20% piperidine in N,N-dimethylformamide. The
supported amide (0.112 g, 0.052 mmol) was heated at 100 ꢀC
overnight with 4-methylphenylboronic acid (0.035 g, 0.26
mmol), LiCl (0.011 g, 0.26 mmol), Cs2CO3 (0.085 g, 0.26 mmol),
and (PhP3)2PdCl2 (0.007 g, 0.01 mmol) in 2 mL of deoxygenated
N,N-dimethylformamide and 0.1 mL of deoxygenated water.
The Rink amide was cleaved with trifluoroacetic acid/dichloro-
methane 95:5 at room temperature. The solution was concen-
trated and the crude was purified by preparative HPLC to afford
the title compound (yield: 54%). 1H NMR (400 MHz, DMSO-d6)
δ ppm 2.37 (s, 3H), 7.00 (bs, 2H), 7.25 (d, J = 8.00 Hz, 2H), 7.34
(d, J = 2.56 Hz, 1H), 7.58 (d, J = 8.00 Hz, 2H), 7.79 (d, J = 6.22
Hz, 2H), 8.56 (d, J = 6.22 Hz, 2H), 11.85 (s, 1H). ESI (þ) MS: m/
z 278 (MHþ). HRMS (M þ H)þ calcd for C17H15N3O þ Hþ,
278.1288; found, 278.1289.
The acid 35b (1.68 mmol) was dissolved in anhydrous tetra-
hydrofuran (20 mL) in the presence of DIEA (1.27 mL, 7.30
mmol). To the solution, cooled to 0 ꢀC, EDC HCl (1.03 g, 5.5
3
mmol) and HOBt NH3 (0.81 g, 5.34 mmol) were added. The
3
reaction mixture was left overnight at room temperature. The
solvent was evaporated, water was added, and the slurry was
extracted with dichloromethane. The crude was purified by flash
chromatography (dichloromethane/methanol 98:2) to yield
2-bromo-5-(3-fluoro-pyridin-4-yl)-1H-pyrrole-3-carboxamide
(36b) as a yellow solid (48% yield). 1H NMR (400 MHz, DMSO-d6)
δ ppm 7.09 (s, 2H), 7.35 (s, 1H), 7.98 (d, J = 4.83 Hz, 1H), 8.47
(d, J = 4.84 Hz, 1H), 8.61 (d, J = 0.91 Hz, 1H), 12.05 (s, 1H).
ESI (þ) MS: m/z 284-286 (MHþ). HRMS (M þ H)þ calcd for
C10H7BrFN3O þ Hþ, 283.9829; found, 283.9832.
Analogously the following compounds were prepared:
5-Pyridin-4-yl-2-m-tolyl-1H-pyrrole-3-carboxamide (42). Yield
52%. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.20 (s, 3H), 6.90
(bs, 2H), 7.20-7.40 (m, 5H), 7.70 (d, J = 6.00 Hz, 2H), 8.55 (d,
J = 6.00 Hz, 2H), 11.88 (s, 1H). ESI (þ) MS: m/z 278 (MHþ).
HRMS (M þ H)þ calcd for C17H15N3O þ Hþ, 278.1288; found,
278.1294.
To a solution of 36b (0.227 g, 0.8 mmol) in deoxygenated
toluene/ethanol 1:1 (10 mL), deoxygenated 1 M aqueous
Na2CO3 (2.2 mL, 2.2 mmol), LiCl (2.7 mmol), phenyl boronic
acid (1.4 mmol), and (Ph3P)2PdCl2 (0.006 g) were added and the
mixture was stirred at 100 ꢀC until disappearance of the starting
material. The solvent was evaporated under reduced pressure,
and the crude was purified by flash chromatography (eluant:
dichloromethane/methanol 95:5). When required, the product
was dissolved in ethanol and treated with 2N HCl in diethyl
ether until precipitation of the hydrochloride salt occurred. The
precipitate was filtered, affording 0.188 g of the title compound
2-(3-Methoxy-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carbox-
amide (44). Yield 49%. 1H NMR (400 MHz, DMSO-d6) δ ppm
3.83 (s, 3H), 6.66 (bs, 2H), 7.28 (m, 3H), 7.40 (m, 2H), 8.24
(d, J=6.82 Hz, 2H), 9.11 (d, J=6.82 Hz, 2H), 12.32 (s, 1H).
ESI (þ) MS: m/z 294 (MHþ). HRMS (M þ H)þ calcd for
C17H15N3O2 þ Hþ, 294.1237; found, 294.1238.
2-(2-Nitro-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
1
(45). Yield 50%. H NMR (400 MHz, DMSO-d6) δ ppm 6.96
(bs, 2H), 7.37 (s, 1H), 7.58-7.65 (m, 4H), 7.79 (m, 1H), 8.07 (dd,
J = 8.17, 1.22 Hz, 1H), 8.55 (d, J = 6.22 Hz, 2H), 12.19 (s, 1H).
ESI (þ) MS: m/z 309 (MHþ). HRMS (M þ H)þ calcd for
C16H12N4O3 þ Hþ, 309.0982; found, 309.0980.
1
(74% yield). H NMR (400 MHz, DMSO-d6) δ ppm 6.95 (bs,
2H), 7.34 (m, 4H), 7.69 (m, 2H), 8.10 (m, 1H), 8.5 (d, J = 5.37
Hz, 1H), 8.71 (d, J = 4.02 Hz, 1H), 12.01 (bs, 1H). ESI (þ) MS:
m/z 282 (MHþ). HRMS (M þ H)þ calcd for C16H12FN3O þ Hþ,
282.1037; found, 282.1033.
2-(3-Nitro-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
1
(46). Yield 53%. H NMR (400 MHz, DMSO-d6) δ ppm 7.30
(bs, 2H), 7.35 (d, J = 2.69 Hz, 1H), 7.76 (m, 3H), 8.15 (m, 1H),
8.22 (m, 1H), 8.58 (dd, J = 4.63, 1.58 Hz, 2H), 8.61 (t, J = 1.81
Hz, 1H), 12.07 (s, 1H). ESI (þ) MS: m/z 309 (MHþ). HRMS (M þ
H)þ calcd for C16H12N4O3 þ Hþ, 309.0982; found, 309.0978.
2-(4-Nitro-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Analogously the following compounds were prepared:
2-(2-Methoxy-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Hydrochloride (37). From 2-bromo-1-pyridin-4-yl-ethanone hy-
1
drobromide (2a, 32% yield). H NMR (400 MHz, DMSO-d6)
δ ppm 3.76 (s, 3H), 6.95 (bs, 2H), 7.06 (t, J = 8.05 Hz, 1H),
7.16 (d, J = 8.05 Hz, 1H), 7.40 (dd, J = 7.44, 1.71 Hz, 1H),
7.46 (m, 1H), 7.73 (s, 1H), 8.15 (d, J = 7.00 Hz, 2H), 8.71 (d, J =
7.00 Hz, 2H), 12.42 (s, 1H). ESI (þ) MS: m/z 294 (MHþ).
HRMS (M þ H)þ calcd for C17H15N3O2 þ Hþ, 294.1237;
found, 294.1233.
2-(4-Methoxy-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
Hydrochloride (38). From 2-bromo-1-pyridin-4-yl-ethanone
hydrobromide (2a, 42% yield). 1H NMR (400 MHz, DMSO-d6)
δ ppm 3.84 (s, 3H), 7.07 (d, J = 8.90 Hz, 2H), 7.33 (bs, 2H),
7.67 (d, J = 8.90 Hz, 2H), 7.73 (s, 1H), 8.22 (d, J = 6.50 Hz,
2H), 8.72 (d, J = 6.50 Hz, 2H), 12.28 (s, 1H). ESI (þ) MS: m/z
294 (MHþ). HRMS (M þ H)þ calcd for C17H15N3O2 þ Hþ,
294.1237; found, 294.1234.
1
(47). Yield 35%. H NMR (400 MHz, DMSO-d6) δ ppm 7.40
(bs, 2H), 7.70 (s, 1H), 8.02 (d, J = 8.78 Hz, 2H), 8.19 (d, J = 6.20
Hz, 2H), 8.32 (d, J = 8.78 Hz, 2H), 8.77 (d, J = 6.20 Hz, 2H),
12.57 (bs, 1H). ESI (þ) MS: m/z 309 (MHþ). HRMS (M þ H)þ
calcd for C16H12N4O3 þ Hþ, 309.0982; found, 309.0981.
2-(2-Cyano-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
(48). Yield 44%. 1H NMR (400 MHz, DMSO-d6) δ ppm
6.88 (bs, 2H), 7.39 (s, 1H), 7.59 (m, 1H), 7.64 (m, 3H), 7.75
(t, J = 7.19 Hz, 1H), 7.88 (d, J = 7.08 Hz, 1H), 8.56 (d, J = 4.75
Hz, 2H), 12.21 (s, 1H). ESI (þ) MS: m/z 289 (MHþ). HRMS (M þ
H)þ calcd for C17H12N4O þ Hþ, 289.1084; found, 289.1085.
2-(3-Cyano-phenyl)-5-pyridin-4-yl-1H-pyrrole-3-carboxamide
1
(49). Yield 48%. H NMR (400 MHz, DMSO-d6) δ ppm 6.99
5-(2-Amino-pyrimidin-4-yl)-2-thiophen-3-yl-1H-pyrrole-3-
carboxamide (40). From 1-(2-amino-4-pyrimidinyl)-2-bromo-
ethanone hydrobromide (2c, 27% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 6.36 (bs, 2H), 6.87 (bs, 2H), 7.01 (d, J = 5.24
Hz, 1H), 7.26 (d, J = 2.44 Hz, 1H), 7.54 (dd, J = 5.00, 2.93 Hz,
1H), 7.65 (dd, J = 5.00, 1.22 Hz, 1H), 8.11 (dd, J = 2.93, 1.22 Hz,
1H), 8.20 (d, J = 5.24 Hz, 1H), 11.52 (bs, 1H). ESI (þ) MS: m/z
(bs, 2H), 7.32 (d, J = 2.56 Hz, 1H), 7.64 (t, J = 7.56 Hz, 1H),
7.69 (d, J = 6.10 Hz, 2H), 7.82 (m, 2H), 8.03 (m, 2H), 8.14 (m,
1H), 8.56 (d, J = 6.10 Hz, 2H), 11.95 (s, 1H). ESI (þ) MS: m/z
289 (MHþ). HRMS (M þ H)þ calcd for C17H12N4O þ Hþ,
289.1084; found, 289.1082.
5-(2-Amino-pyrimidin-4-yl)-2-naphthalen-1-yl-1H-pyrrole-
3-carboxamide (50). Yield 42%. 1H NMR (400 MHz, DMSO-d6)

7312 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Menichincheri et al.
δ ppm 6.33 (bs, 2H), 6.65 (bs, 2H), 6.96 (d, J = 5.27 Hz, 1H),
7.40-7.60 (m, 5H), 7.98 (m, 2H), 8.17 (d, J=5.27 Hz, 1H), 11.95
(bs, 1H). ESI (þ) MS: m/z 330 (MHþ). HRMS (M þ H)þ calcd
for C19H15N5O þ Hþ, 330.1349; found, 330.1349.
starting material. The catalyst was removed by filtration
through a Celite pad, and the filtrate was concentrated. Satu-
rated aqueous NaHCO3 was added, and the slurry was extracted
with ethyl acetate, affording 0.310 g (63%) of 5-pyridin-4-yl-1H-
pyrrole-3-carboxylic acid ethyl ester 56 as a white solid. 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.30 (t, J = 7.07 Hz, 3H),
4.24 (q, J = 7.07 Hz, 2H), 7.20 (m, 1H), 7.63 (m, 1H), 7.68 (m,
2H), 8.52 (m, 2H), 12.27 (bs, 1H). ESI (þ) MS: m/z 217 (MHþ).
HRMS (ESI): calcd for C12H12N2O2 þ Hþ, 217.0971; found,
217.0972.
5-(2-Amino-pyrimidin-4-yl)-2-naphthalen-2-yl-1H-pyrrole-
3-carboxamide (51). Yield 47%. 1H NMR (400 MHz, DMSO-d6)
δ ppm 6.37 (bs, 2H), 6.87 (bs, 1H), 7.05 (d, J=5.24 Hz, 1H),
7.32 (d, J = 2.32 Hz, 1H), 7.39 (bs, 1H), 7.51-7.59 (m, 2H),
7.78 (dd, J=8.41, 1.71 Hz, 1H), 7.88-7.97 (m, 3H), 8.16 (d,
J = 1.22 Hz, 1H), 8.23 (d, J = 5.24 Hz, 1H), 11.80 (bs, 1H).
ESI (þ) MS: m/z 330 (MHþ). HRMS (M þ H)þ calcd for
Ester 56 (0.200 g, 0.92 mol) in 4 M aq NaOH (4.6 mL) and
ethanol (4 mL) was heated at 100 ꢀC for 1 h. The reaction
mixture was cooled to 0 ꢀC, and the product was precipitated by
adding concentrated HCl. The solid was filtered to obtain 0.160 g
(78%) of 5-pyridin-4-yl-1H-pyrrole-3-carboxylic acid hydro-
C
19H15N5O þ Hþ, 330.1349; found, 330.1350.
5-(2-Amino-pyrimidin-4-yl)-2-benzofuran-2-yl-1H-pyrrole-
3-carboxamide (52). Yield 51%. 1H NMR (400 MHz, DMSO-
d6) δ ppm 6.53 (bs, 2H), 7.07 (bs, 1H), 7.11 (d, J = 5.12 Hz,
1H), 7.27 (td, J = 7.56, 0.98 Hz, 1H), 7.34 (td, J = 7.56, 0.98
Hz, 1H), 7.42 (s, 1H), 7.61 (dd, J = 7.56, 0.98 Hz, 1H), 7.65
(bs, 1H), 7.70 (dd, J = 7.56, 0.98 Hz, H), 7.87 (d, J = 0.98 Hz,
1H), 8.26 (d, J = 5.12 Hz, 1H), 11.61 (bs, 1H). ESI (þ) MS:
m/z 320 (MHþ). HRMS (M þ H)þ calcd for C17H13N5O2 þ Hþ,
320.1142; found, 320.1142.
1
chloride as a white solid. H NMR (400 MHz, DMSO-d6) δ
ppm 7.60 (m, 1H), 7.81 (m, 1H), 8.14 (m, 2H), 8.72 (m, 2H),
12.70 (s, 1H). MS: m/z 187 [M - H].
To a solution of the acid (0.137 g, 0.61 mmol) in DIEA
(213 μL, 1.22 mmol) and anhydrous tetrahydrofuran (8 mL),
cooled at 0 ꢀC, EDC HCl (0.175 g, 0.9 mmol) and HOBt NH3
3
3
5-(2-Amino-pyrimidin-4-yl)-2-benzo[b]thiophen-2-yl-1H-pyrrole-
3-carboxamide (53). Yield 47%. 1H NMR (400 MHz, DMSO-d6) δ
ppm 6.42 (bs, 2H), 6.99 (bs, 1H), 7.08 (d, J = 5.12 Hz, 1H), 7.32 (s,
1H), 7.33-7.41 (m, 2H), 7.56 (bs, 1H), 7.84 (bs, 1H), 7.91-8.03
(m, 2H), 8.25 (d, J = 5.12 Hz, 1H), 11.82 (bs, 1H). ESI (þ) MS:
m/z 336 (MHþ). HRMS (M þ H)þ calcd for C17H13N5OS þ Hþ,
336.0914; found, 336.0918.
5-(2-Amino-pyrimidin-4-yl)-2-benzo[b]thiophen-3-yl-1H-pyrrole-
3-carboxamide (54). Yield 52%. 1H NMR ((400 MHz, DMSO-d6)
δ ppm 6.35 (bs, 2H), 6.76 (bs, 1H), 6.99 (d, J = 5.24 Hz, 1H), 7.20
(s, 1H), 7.35-7.42 (m, 3H), 7.51-7.57 (m, 1H), 7.90 (s, 1H),
8.00-8.06 (m, 1H), 8.20 (d, J = 5.24 Hz, 1H), 11.91 (bs, H).
ESI (þ) MS: m/z 336 (MHþ). HRMS (M þ H)þ calcd for
C17H13N5OS þ Hþ, 336.0914; found, 336.0910.
(0.137 g, 0.9 mmol) were added and the solution was stirred
overnight at room temperature. The solution was concentrated,
water was added, and the product was extracted with dichloro-
methane. The organic layer was washed with water, dried over
anhydrous sodium sulfate, and concentrated to give a solid that
was triturated with diethyl ether and filtered to afford 0.052 g
(44%) of the title compound. 1H NMR (400 MHz, DMSO-d6) δ
ppm 6.98 (bs, 2H), 7.61 (bs, 1H), 7.79-7.82 (m, 1H), 8.06 (d, J =
6.58 Hz, 2H), 8.71 (d, J = 6.83 Hz, 2H), 12.57 (s, 1H). ESI (þ)
MS: m/z 188 (MHþ). HRMS (ESI): calcd for C10H9N3O þ Hþ,
188.0818; found, 188.0820.
5-(2-Amino-pyrimidin-4-yl)-1-methyl-2-phenyl-1H-pyrrole-3-
carboxamide (58). Methyl iodide (0.015 g, 0.107 mmol) was
added to a solution of 5-(2-aminopyrimidin-4-yl)-2-phenyl-1H-
pyrrole-3-carboxamide 18 (0.030 g, 0.107 mmol) and Cs2CO3
(0.070 g, 0.215 mmol) in dry N,N-dimethylformamide (0.5 mL),
and the reaction mixture was stirred at room temperature for
24 h. To achieve completion, a further addition of methyl iodide
(0.009 g, 0.064 mmol) was required and the reaction mixture was
stirred at 80 ꢀC for 12 h. The solvent was removed under reduced
pressure and the residue was taken up into dichloromethane
(2 mL, with 2% methanol), which was washed with a saturated
solution of sodium bicarbonate (2 mL) and dried over anhy-
drous Na2SO4. The filtrate was evaporated to dryness to give the
crude product, which was purified by flash chromatography
over silica gel (dichloromethane/methanol/33% ammonium
hydroxide 95/0.5/0.1), affording the title compound as a white
5-Pyridin-4-yl-1H-pyrrole-3-carboxylic Acid Amide (57). To a
suspension of sodium (0.081 g, 3.5 mmol) in 10 mL of anhydrous
ethanol, ethyl cyanoacetate (0.37 mL, 3.5 mmol) was added at
0 ꢀC. The solution was stirred until sodium was completely
dissolved. The solvent was evaporated to obtain a white
solid that was added portionwise to a stirred solution of
4-(bromoacetyl)pyridine hydrobromide 2a (1.04 g, 3.5 mmol)
in anhydrous tetrahydrofuran (20 mL) and DIEA (0.6 mL, 3.5
mmol). The reaction mixture was stirred overnight at room
temperature. The solvent was removed, and the residue was
suspended in water and extracted with dichloromethane. The
organic extracts were combined, dried (anhydrous sodium sul-
fate), and concentrated. The crude product was purified by
flash chromatography (dichloromethane/methanol 95:5) to give
0.710 g (87%) of 2-cyano-4-oxo-4-pyridin-4-yl-butyric acid
1
solid (0.020 g, 63%). H NMR (400 MHz, DMSO-d6) δ ppm
3.70 (s, 3H), 6.53 (s, 2H), 6.73 (bs, 1H), 6.81 (bs, 1H), 6.83 (d,
J=5.40 Hz, 1H), 7.25 (s, 1H), 7.35-7.40 (m, 2H), 7.41-7.51
(m, 3H), 8.20 (d, J = 5.20 Hz, 1H). ESI (þ) MS: m/z 294 (MHþ).
HRMS (M þ H)þ calcd for C16H15N5O þ Hþ, 294.1350; found,
294.1348.
1
ethyl ester 33a as a reddish oil. H NMR (400 MHz, DMSO-
d6) δ ppm 1.23 (t, J = 7.07 Hz, 3H), 3.88 (d, J = 5.25 Hz, 2H),
4.21 (q, J = 7.07 Hz, 2H), 4.64 (t, J = 5.25 Hz, 1H), 7.89 (d, J =
6.00 Hz, 2H), 8.85 (d, J = 6.00 Hz, 2H). ESI (þ) MS: m/z 233
(MHþ). HRMS (ESI): calcd for C12H12N2O3 þ Hþ, 233.0921;
found, 233.0921.
To a solution of 33a (0.550 g, 2.37 mmol) in diethyl ether
(1 mL) at 0 ꢀC, 4 N HCl in dioxane (6 mL, 23.7 mmol) was added
dropwise. The reaction mixture was left for 10 min at 0 ꢀC and
then stirred at room temperature for 6 h. The solid was filtered
and washed with diethyl ether to yield 0.510 g (84%) of 2-chloro-
5-pyridin-4-yl-1H-pyrrole-3-carboxylic acid ethyl ester hydro-
chloride 55 as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ
ppm 1.34 (t, J = 7.07 Hz, 3H), 4.27 (q, J = 7.07 Hz, 2H), 7.73 (s,
1H), 8.25 (d, J = 5.61 Hz, 2H), 8.78 (m, 2H), 13.5 (bs, 1H). ESI
(þ) MS: m/z 251 (MHþ). HRMS (ESI): calcd for C12H11ClN2O2 þ
Hþ, 251.0582; found, 251.0581.
5-(2-Amino-pyrimidin-4-yl)-1-trifluoroethyl-2-phenyl-1H-
pyrrole-3-carboxamide (59). To a solution of 18 (0.063 g,
0.215 mmol) in acetonitrile (6 mL), 18-crown-6 (0.085 g, 0.322
mmol), K2CO3 (0.059 g, 0.403 mmol), and 2,2,2-trifluoroethyl
trifluoromethanesulfonate (46 μL, 0.320 mmol) were added and
the suspension was heated at 80 ꢀC for 5 h. The reaction mixture
was taken up in water (10 mL) and extracted with ethyl acetate
(3ꢀ10 mL). The combined organic phases were washed with
brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. After preparative HPLC, the title compound
was obtained as a white solid (70 mg, 90% yield). 1H NMR (400
MHz, DMSO-d6) δ ppm 5.49 ppm (bs, 2H), 6.66 (s, 2H), 6.86 (d,
J = 5.20 Hz, 1H), 6.89 (bs, 1H), 6.97 (bs, 1H), 7.33-7.40 (m,
3H), 7.43-7.53 (m, 3H), 8.24 (d, J = 5.20 Hz, 1H). ESI (þ) MS:
m/z 362 (MHþ). HRMS (M þ H)þ calcd for C17H14F3N5O þ
Hþ, 362.1223; found, 362.1228.
A mixture of 55 (0.630 g, 2.2 mmol) in 30 mL of methanol,
ammonium formate (1.26 g, 19.8 mmol), and 10% Pd/C (0.633 g)
was stirred at room temperature until disappearance of the

Article
5-(2-Aminopyrimidin-4-yl)-1-(2-hydroxyethyl)-2-phenyl-1H-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7313
To a suspension of the acid (0.142 g, 0.33 mmol) in 6 mL
of dimethylacetamide and diisopropylethylamine (338 μL, 1.98
mmol), O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium tetra-
fluoroborate (0.138 g, 0.43 mmol) and ammonium chloride
(0.106 g, 1.98 mmol) were added to the mixture. The reaction
was heated at 80 ꢀC for 16 h. The solvent was then removed
under reduced pressure, and the residue was dissolved with
dichloromethane and washed with NaHCO3 saturated aqueous
solution. The organic layer was then dried over anhydrous
sodium sulfate and evaporated to dryness. The crude material
was purified by flash chromatography on a silica gel column
(dichloromethane/methanol 95:5), giving 0.065 g (47%) of tert-
butyl {2-[5-(2-aminopyrimidin-4-yl)-3-carbamoyl-2-phenyl-1H-
pyrrol-1-yl]ethyl}carbamate (63a). 1H NMR (400 MHz, DMSO-
d6) δ ppm 1.30 (s, 9H), 3.17 (q, J = 6.06 Hz, 2H), 4.22 (t, J =
6.83 Hz, 2H), 6.58 (s, 2H), 6.61 (bs, 1H), 6.71 (bs, 1H), 6.83 (t,
J = 5.97 Hz, 1H), 6.86 (d, J = 5.37 Hz, 1H), 7.34 (s, 1H), 8.18 (d,
J = 5.37 Hz, 1H). ESI (þ) MS: m/z 423 (MHþ). HRMS(M þ H)þ
calcd for C22H26N6O3 þ Hþ, 423.2139; found, 423.2138.
pyrrole-3-carboxamide (60). To a solution of 5-(2-aminopyrimi-
din-4-yl)-2-phenyl-1H-pyrrole-3-carboxamide 18 (0.162 g, 0.57
mmol) in 5 mL of dry N,N-dimethylformamide, cesium carbo-
nate (0.741 g, 2.28 mmol) and iodoethanol tetrahydropyranyl-
ether (0.292 g, 1.14 mmol) were added under stirring. The mix-
ture was heated at 80 ꢀC for 8 h. The solvent was then removed
under reduced pressure, the residue taken up with water, and
the product extracted with dichloromethane and dried over
anhydrous sodium sulfate. By trituration with diethyl ether
5-(2-aminopyrimidin-4-yl)-2-phenyl-1-[2-(tetrahydro-2H-pyran-
2-yloxy)ethyl]-1H-pyrrole-3-carboxamide (0.163 g; 69%) was
obtained. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.20-1.70 (m,
6H), 3.21-3.58 (m, 4H), 4.28 (t, J = 2.93 Hz, 1H), 4.49-4.66
(m, 2H), 6.52 (s, 2H), 6.73 (s, 2H), 6.84 (d, J = 5.24 Hz, 1H), 7.29
(s, 1H), 7.35-7.49 (m, 5H), 8.20 (d, J = 5.36 Hz, 1H). ESI (þ)
MS: m/z 408 (MHþ). HRMS (M þ H)þ calcd for C22H25N5O3 þ
Hþ, 408.2030; found, 408.2033.
To a solution of 5-(2-aminopyrimidin-4-yl)-2-phenyl-
1-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1H-pyrrole-3-carbo-
xamide (0.158 g, 0.39 mmol) in 5 mL of dry dichloromethane,
2 mL of trifluoroacetic acid were added. The solution was stirred
at room temperature for 5 h in a closed bottle. After solvent
evaporation, the residue was taken up with water, neutralized
with saturated aqueous NaHCO3, extracted with dichloro-
methane, and dried over anhydrous sodium sulfate. The crude
product was then purified by flash chromatography on a silica
gel column (dichloromethane/methanol 9:1) and afforded 0.123 g
(95%) of the title compound. 1H NMR (400 MHz, DMSO-d6) δ
ppm 3.32 (m, 2H), 4.35 (t, J = 6.58 Hz, 2H), 4.66 (t, J = 5.85 Hz,
1H), 6.54 (s, 2H), 6.62-6.75 (m, 2H), 6.85 (d, J = 5.24 Hz, 1H),
7.29 (s, 1H), 7.35-7.40 (m, 2H), 7.45-7.49 (m, 3H), 8.20 (d, J =
5.24 Hz, 1H). ESI (þ) MS: m/z 324 (MHþ). HRMS (M þ H)þ
calcd for C17H17N5O2 þ Hþ, 324.1455; found, 324.1450.
To a solution of carbamate 63a (0.065 g, 0.15 mmol) in 15 mL
of dry dioxane, 4 mL of 4 M HCl in dioxane were added. The
resulting solution was stirred at room temperature in a closed
bottle overnight. The solvent was removed under reduced
pressure, and toluene was added and evaporated again, giving a
residue that was triturated with diethyl ether then collected by
1
filtration, giving 0.050 g (86%) of the title compound 64. H
NMR (400 MHz, DMSO-d6) δ ppm 4.55 (t, J = 7.32 Hz, 2H),
6.83 (bs, 1H), 6.94 (bs, 1H), 7.18 (d, J = 6.46 Hz, 1H), 7.88 (s,
1H), 8.08 (bs, 3H), 8.23 (d, J = 6.58 Hz, 1H). ESI (þ) MS: m/z
323 (MHþ). HRMS (M þ H)þ calcd for C17H18N6O þ Hþ,
323.1615; found, 323.1614.
1-(3-Aminopropyl)-5-(2-aminopyrimidin-4-yl)-2-phenyl-1H-pyr-
role-3-carboxamide Dihydrochloride (65). As reported for the
synthesis of 64, ethyl 5-(2-aminopyrimidin-4-yl)-1-{3-[(tert-
butoxycarbonyl)amino]propyl}-2-phenyl-1H-pyrrole-3-carboxy-
late 62b was prepared (98%). 1H NMR (400 MHz, DMSO-d6) δ
ppm 1.00 (t, J = 7.07 Hz, 3H), 1.32 (s, 9H), 1.54-1.69 (m, 2H),
2.63-2.71 (m, 2H), 3.98 (q, J = 6.99 Hz, 2H), 4.19-4.37 (m,
2H), 6.56 (bs, 2H), 6.63 (t, J=5.55 Hz, 1H), 6.97 (d, J = 5.37
Hz, 1H), 7.23 (s, 1H), 7.31-7.40 (m, 2H), 7.42-7.53 (m, 3H),
8.20 (d, J = 5.24 Hz, 1H). ESI (þ) MS: m/z 466 (MHþ). HRMS
(M þ H)þ calcd for C25H31N5O4 þ Hþ, 466.2449; found,
466.2444.
1-(2-Aminoethyl)-5-(2-aminopyrimidin-4-yl)-2-phenyl-1H-pyr-
role-3-carboxamide Dihydrochloride (64). Cesium carbonate
(1.03 g, 3.25 mmol) and tert-butyl (2-bromoethyl)carbamate
(0.364 g, 1.63 mmol) were added to a solution of ethyl 5-(2-
aminopyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-carboxylate 61 (0.25g,
0.81 mmol) in 5 mL of dry N,N-dimethylformamide. The mix-
ture was stirred at 85 ꢀC for 6 h. The solvent was removed in
vacuo and the residue taken up with dichloromethane and
washed with water. The organic layer was then dried over
anhydrous sodium sulfate and concentrated under reduced
pressure. The residue was crystallized from di-iso-propyl ether
to yield 0.363 g (98%) of ethyl 5-(2-aminopyrimidin-4-yl)-1-{2-
[(tert-butoxycarbonyl)amino]ethyl}-2-phenyl-1H-pyrrole-3-car-
As reported for the synthesis of 64, 5-(2-aminopyrimidin-4-yl)-
1-{3-[(tert-butoxycarbonyl)amino]propyl}-2-phenyl-1H-pyrrole-
3-carboxylic acid was prepared similarly (37%). 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.32 (s, 9H), 1.55-1.66 (m, 2H),
2.62-2.71 (m, 2H), 4.23 (t, J = 7.44 Hz, 2H), 6.53 (bs, 2H), 6.62
(t, J = 5.91 Hz, 1H), 6.94 (d, J = 5.24 Hz, 1H), 7.20 (s, 1H),
7.32-7.48 (m, 5H), 8.19 (d, J = 5.37 Hz, 0H), 11.48 (s, 1H).
ESI (þ) MS: m/z 438 (MHþ). HRMS (M þ H)þ calcd for
C23H27N5O4 þ Hþ, 438.2136; found, 438.2137.
1
boxylate 62a. H NMR (400 MHz, DMSO-d6) δ ppm 1.00 (t,
J = 7.07 Hz, 3H), 1.32 (s, 9H), 1.55-1.68 (m, 2H), 2.61-
2.73 (m, 2H), 3.98 (q, J = 6.99 Hz, 2H), 4.19-4.34 (m,
2H), 6.56 (bs, 2H), 6.63 (t, J = 5.79 Hz, 1H), 6.97 (d, J =
5.37 Hz, 2H), 7.23 (s, 1H), 7.33-7.38 (m, 2H), 7.44-7.52 (m,
3H), 8.20 (d, J = 5.24 Hz, 2H). ESI (þ) MS: m/z 452 (MHþ).
HRMS (M þ H)þ calcd for C24H29N5O4 þ Hþ, 452.2293;
found, 452.2305.
To a solution of ester 62a (0.363 g, 0.80 mmol) in a mixture
of tetrahydrofuran/methanol/water 8/1/1, lithium hydroxide
monohydrate (0.268 g, 6.4 mmol) was added under stirring.
The reaction was heated at 80 ꢀC for 16 h. The solvent was then
evaporated under reduced pressure and the residue taken up
with water and neutralized with 5% KHSO4 aqueous solution.
The resulting precipitate was collected by filtration and washed
with water and acetone, giving 0.262 g (77%) of 5-(2-aminopyr-
imidin-4-yl)-1-{2-[(tert-butoxycarbonyl)amino]ethyl}-2-phenyl-
1H-pyrrole-3-carboxylic acid. 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.32 (s, 9H), 3.12-3.22 (m, 2H), 4.19 (t, J = 6.89 Hz, 2H),
6.46 (s, 2H), 6.80 (bs, 1H), 6.83 (d, J = 5.37 Hz, 1H), 7.06 (s, 1H),
7.27-7.39 (m, 5H), 8.08 (d, J = 5.49 Hz, 1H). ESI (þ) MS: m/z
424 (MHþ). HRMS (M þ H)þ calcd for C22H25N5O4 þ Hþ,
424.1980; found, 424.1982.
In parallel to the synthesis of 64, tert-butyl {3-[5-(2-amino-
pyrimidin-4-yl)-3-carbamoyl-2-phenyl-1H-pyrrol-1-yl]propyl}-
carbamate 63b was prepared (68%). 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.31 (s, 9H), 1.59 (quin, J = 7.50 Hz, 2H),
2.66 (q, J = 6.50 Hz, 2H), 4.21 (t, J = 7.19 Hz, 2H), 6.52 (bs,
2H), 6.61 (t, J = 5.49 Hz, 1H), 6.71 (bs, 2H), 6.84 (d, J = 5.37
Hz, 1H), 7.29 (s, 1H), 8.20 (d, J = 5.37 Hz, 1H). ESI (þ) MS: m/z
437 (MHþ). HRMS (M þ H)þ calcd for C23H28N6O3 þ Hþ,
437.2296; found, 437.2300.
Similarly, the title compound 65 was prepared (81%). 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.78 (quin, J = 7.70 Hz, 2H),
2.53-2.62 (m, 2H), 4.37 (t, J= 7.38 Hz, 2H), 6.89 (bs, 2H), 7.14 (d,
J = 6.46 Hz, 1H), 7.74 (bs, 3H), 7.78 (bs, 1H), 7.95 (bs, 3H), 8.24
(d, J = 6.46 Hz, 1H). ESI (þ) MS: m/z 337 (MHþ). HRMS (M þ
H)þ calcd for C18H20N6O þ Hþ, 337.1772; found, 337.1771.
5-(2-Amino-pyrimidin-4-yl)-1-ethyl-2-phenyl-1H-pyrrole-3-
carboxamide (67). To a solution of ethyl benzoylacetate (1.34 g,
7 mmol) in anhydrous tetrahydrofuran (100 mL) at 0 ꢀC, sodium

7314 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Menichincheri et al.
hydride (60% in mineral oil, 0.711 g, 17.5 mmol) was added
under argon with stirring. After 5 min, 1-(2-amino-4-pyrimidi-
nyl)-2-bromo-ethanone hydrobromide 2c (2.52 g, 8.4 mmol) was
added and the mixture was stirred at room temperature for 3 h.
The solvent was evaporated, and the residue was dissolved
in acetic acid (30 mL) and 2 M ethylamine in tetrahydrofuran
(8.7 mL, 17.5 mmol). The mixture was subjected to microwave
irradiation at 170 ꢀC for 5 min, then diluted with ethyl acetate
and washed with NaHCO3 saturated aqueous solution. The
water layer was extracted with ethyl acetate, and the combined
organic layers were washed with water, dried over anhydrous
sodium sulfate, and concentrated under reduced pressure. The
residue was purified by flash chromatography (dichloromethane/
ethanol/acetone 96:2:2), thus affording 0.723 g of 5-(2-amino-
pyrimidin-4-yl)-1-ethyl-2-phenyl-1H-pyrrole-3-carboxylic acid
Hz, 1H). ESI (þ) MS: m/z 310 (MHþ). HRMS (M þ H)þ calcd
for C17H15N3O3 þ Hþ, 310.1186; found, 310.1177.
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-thiophene-3-carboxylic
1
acid ethyl ester (70). H NMR (400 MHz, DMSO-d6) δ ppm:
1.14 (t, J = 7.1 Hz, 3H), 4.16 (q, J = 7.1 Hz, 2H), 6.73 (bs, 2H),
7.20 (d, J = 5.2 Hz, 1H), 7.43-7.55 (m, 5H), 8.18 (s, 1H), 8.30
(d, J = 5.2 Hz, 1H). ESI (þ) MS: m/z 326 (MHþ). HRMS (M þ
H)þ calcd for C17H15N3O2S þ Hþ, 326.0958; found, 326.0965.
To a solution of ester 69 (0.225 g, 0.71 mmol) in water/ethanol
1:1 (9 mL), 4 M aqueous NaOH (10 equiv) was added and the
reaction mixture was stirred at 100 ꢀC for 1 h. After cooling to
room temperature, the solution was acidified with 2 M HCl,
yielding 5-(2-amino-pyrimidin-4-yl)-2-phenyl-furan-3-carbox-
ylic acid as a white solid which was filtered, washed with water,
and dried under reduced pressure (quantitative). 1H NMR (400
MHz, DMSO-d6) δ ppm 6.75 (bs, 2H), 7.06 (d, J = 5.2 Hz,1H),
7.51 (m, 3H), 8.0 (m, 2H), 8.33 (d, J=5.2 Hz,1H), 13.00 (bs,
1H). ESI (-) MS: m/z 280 [M - H]. HRMS (M þ H)þ calcd for
C15H11N3O3 þ Hþ, 282.0873; found, 282.0872.
1
ethyl ester (66, 29% yield). H NMR (400 MHz, DMSO-d6) δ
ppm 0.97-1.04 (m, 6H), 3.97 (q, J = 7.07 Hz, 2H), 4.38 (q, J =
6.95 Hz, 2H), 6.56 (s, 2H), 6.97 (d, J = 5.37 Hz, 1H), 7.23 (s, 1H),
7.34-7.41 (m, 2H), 7.46-7.54 (m, 3H), 8.20 (d, J = 5.37 Hz,
1H). ESI (þ) MS: m/z 337 (MHþ), HRMS (M þ H)þ calcd for
C19H20N4O2 þ Hþ, 337.1659; found, 337.1664.
To a mixture of the acid (0.172 g, 0.61 mmol) and DIEA
(213 μL, 1.22 mmol) in dry tetrahydrofuran (4 mL), EDC HCl
(0.143 g, 0.92 mmol) and HOBt NH3 (0.145 g, 0.92 mmol) were
3
To a suspension of the ester 66 (0.711 g, 2.08 mmol) in 95%
ethanol (8 mL), 4 M aq NaOH (8 mL) was added and the
mixture was stirred for 1 h at 100 ꢀC. The solvent was removed
under vacuum, and the aqueous residue, when acidified with
37% HCl to pH 5, led to precipitation of the product. The
mixture was filtered, the solid was washed with a little cold water
and dried, thus affording 0.662 g of 5-(2-amino-pyrimidin-4-yl)-
1-ethyl-2-phenyl-1H-pyrrole-3-carboxylic acid that was used in
the next step without further purification. MS: m/z 307 [M - H].
To a suspension of the acid (0.413 g, 1.31 mmol) in 10 mL of
tetrahydrofuran and 600 μL of DIEA (3.52 mmol), cooled in
3
added at 0 ꢀC. The reaction mixture was stirred at room temperature
overnight. After solvent evaporation, the residue was taken up with
dichloromethane and washed with water. The aqueous phase was
extracted with ethyl acetate, and the organic layer was dried over
anhydrous sodium sulfate and concentrated under reduced pressure
to give the title compound 5-(2-amino-pyrimidin-4-yl)-2-phenyl-
1
furan-3-carboxamide (71) as a yellow solid (0.083 g, 48%). H
NMR (400 MHz, DMSO-d6) δ ppm: 6.69 (bs, 2H), 7.01 (d, J = 5.2
Hz, 1H), 7.41-7.48 (m, 4H), 7.53 (s, 1H), 7.92 (bs, 1H), 8.05 (m,
2H), 8.33 (d, J = 5.2 Hz,1H). ESI (þ) MS: m/z 281 (MHþ). HRMS
(MþH)þ calcd for C15H12N4O2 þ Hþ, 281.1033; found, 281.1035.
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-thiophene-3-carboxamide
(72). Analogously to the previously reported synthesis, from
ester 70, 5-(2-amino-pyrimidin-4-yl)-2-phenyl-thiophene-3-car-
an ice bath, 0.336 g of EDC HCl (1.75 mmol) and 0.267 g of
3
HOBt NH3 (1.75 mmol) were added and the mixture was stirred
3
overnight at room temperature. Ethyl acetate and water were
added and the organic layer was washed with 1 M aqueous
NaOH and water and then dried over anhydrous sodium sulfate
and concentrated. Flash chromatography (dichloromethane/
methanol/acetone 90:5:5) afforded 0.343 g (83% yield) of the
desired amide 67. 1H NMR (400 MHz, DMSO-d6) 0.89 (t, J =
7.14 Hz, 3H), 4.30 (q, J = 7.14, 2H), 6.51 (s, 2H), 6.66 (s, 1H),
6.77 (d, J = 5.09 Hz, 1H), 6.78 (s, 2H), 7.08 (m, 2H), 7.42 (tt, J =
6.83, 1.64 Hz, 1H), 7.55 (m, 2H), 8.18 (d, J = 5.09 Hz, 1H).
ESI (þ) MS: m/z 308 (MHþ). HRMS (M þ H)þ calcd
for C17H17N5O þ Hþ, 308.1506; found, 308.1508. Anal.
(C17H17N5O) C, H, N.
1
boxylic acid was obtained in 90% yield. H NMR (400 MHz,
DMSO-d6) δ ppm 7.04 (bs, 2H), 7.28 (d, J = 5.2 Hz, 1H),
7.43-7.48 (m, 3H), 7.54-7.58 (m, 2H), 8.25 (s, 1H), 8.31 (d, J =
5.2 Hz, 1H), 12.86 (bs, 1H). ESI (-) MS: m/z 296 [M - H].
HRMS (M þ H)þ calcd for C15H11N3O2S þ Hþ, 298.0645;
found, 298.0643.
From the carboxylic acid, the corresponding 5-(2-amino-
pyrimidin-4-yl)-2-phenyl-thiophene-3-carboxamide (72) was
prepared (48% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm:
6.69 (bs, 2H), 7.10 (d, J = 5.2 Hz,1H), 7.38-7.44 (m, 4H), 7.56
(m, 2H), 7.71 (bs, 1H), 8.01 (s, 1H), 8.29 (d, J = 5.2 Hz,1H).
ESI (þ) MS: m/z 297 (MHþ). HRMS (M þ H)þ calcd for
C15H12N4OS þ Hþ, 297.0805; found, 297.0804.
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-furan-3-carboxamide (71).
To a stirred solution of 3-oxo-3-phenyl-propionic acid ethyl-
ester (2.10 g, 10 mmol) and sodium hydride (60% in mineral oil,
1.03 g, 25 mmol) in dry tetrahydrofuran (200 mL) at 0 ꢀC, 1-(2-
amino-4-pyrimidinyl)-2-bromo-ethanone hydrobromide 2c
(3.56 g, 12 mmol) was added. The reaction mixture was stirred
at 0 ꢀC for 30 min, then additional ethanone 62c (1.48 g) was
added and the reaction mixture was stirred at room temperature
overnight. After solvent removal, the residue was diluted in dichlo-
romethane and washed with brine and dried over anhydrous
sodium sulfate, and the solvent removed under reduced pressure
to give 4-(2-amino-pyrimidin-4-yl)-2-benzoyl-4-oxo-butyric acid
ethyl ester (68, 3.27 g, 97%). ESI (þ) MS: m/z 328 (MHþ).
A mixture of 68 (3.25 g, 10 mmol) and Lawesson’s reagent
(2.43 g, 6 mmol) in toluene (100 mL) were refluxed under argon
for 4 h. After solvent removal, the residue was taken up in
dichloromethane, filtered, and purified by flash chromato-
graphy (dichloromethane/methanol 99:1) to give the furan deri-
vative 69 (0.236 g, 8%) and the thiophene derivative 70 (0.255 g,
8%) as yellow solids.
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-4-vinyl-1H-pyrrole-3-
carboxamide (74). To a stirred solution of 5-(2-amino-pyrimidin-4-
yl)-4-iodo-2-phenyl-1H-pyrrole-3-carboxamide (73) (0.052 g,
0.125 mmol) in dioxane (5 mL) and N,N-dimethylformamide
(0.5 mL), 2,6-dimethyl-4-tert-butyl phenol (0.005 g), palladium
tetrakis (0.005 g), and tributylvinyl stannane (145 μL) were
added. The mixture was warmed at 110 ꢀC for 8 h, cooled and
concentrated under reduced pressure. The crude product was
purified by flash chromatography (dichloromethane/methanol
20:1) affording the title compound as a pale-yellow solid (24%
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm: 5.15 (dd, J =
11.46, 1.95 Hz, 1H), 5.64 (dd, J = 17.93, 1.95 Hz, 1H), 6.49 (bs,
2H), 6.97 (d, J= 5.24 Hz, 1H), 7.31 (bs, 1H), 7.34 (m, 1H), 7.44 (m,
2H), 7.51 (dd, J = 17.95, 11.50 Hz, 1H),7.63 (bs, 1H), 7.69 (m,
1H), 8.23 (d, J = 5.24 Hz, 1H), 11.40 (bs, 1H). ESI (þ) MS: m/z
306 (MHþ). HRMS (M þ H)þ calcd for C17H17N5O þ Hþ,
306.1349; found, 306.1353.
5-(2-Amino-pyrimidin-4-yl)-2-phenyl-furan-3-carboxylic acid
2. Kinase Assays. Kinase assays were performed as previously
1
ethyl ester (69). H NMR (400 MHz, DMSO-d6) δ ppm: 1.29
(t, J = 7.1 Hz, 3H), 4.27 (q, J = 7.1 Hz, 2H), 6.76 (bs, 2H), 7.07
(d, J = 5.2 Hz, 1H), 7.52 (m, 4H), 8.03 (m, 2H), 8.34 (d, J = 5.2
described.12
3. High-Throughput Solubility. Solubility at pH 7 was per-
formed as previously described.29

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7315
4. Cell Permeability. PAMPA cell permeability assays were
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Kelly, K.; Carew, J.; Giles, F. Cdc7 kinase;a new target for drug
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performed as previously described.30
5. Plasma Protein Binding. Plasma protein binding was
(12) Vanotti, E.; Amici, R.; Bargiotti, A.; Berthelsen, J.; Bosotti, R.;
Ciavolella, A.; Cirla, A.; Cristiani, C.; D’Alessio, R.; Forte, B.; Isacchi,
A.; Martina, K.; Menichincheri, M.; Molinari, A.; Montagnoli, A.;
Orsini, P.; Pillan, A.; Roletto, F.; Scolaro, A.; Tibolla, M.; Valsasina,
B.; Varasi, M.; Volpi, D.; Santocanale, C. Cdc7 kinase inhibitors:
pyrrolopyridinones as potential antitumor agents. 1. Synthesis and
structure-activity relationships. J. Med. Chem. 2008, 51, 487–501.
(13) Menichincheri, M.; Bargiotti, A.; Berthelsen, J.; Bertrand, J.; Bossi,
R.; Ciavolella, A.; Cirla, A.; Cristiani, C.; Croci, V.; D’Alessio, R.;
Fasolini, M.; Fiorentini, F.; Forte, B.; Isacchi, A.; Martina, K.;
Molinari, A.; Montagnoli, A.; Orsini, P.; Orzi, F.; Pesenti, E.;
performed as previously described.29
6. In Vivo Pharmacokinetics. The pharmacokinetic profile of
the compounds was investigated in nu/nu mice as previously
described.13 Pharmacokinetic parameters were derived as pre-
viously described.13
7. Inhibition of Cell Proliferation. Inhibition of cell prolifera-
tion was performed as previously described.12
8. Cell Cycle Analysis and BrdU Determination. Data were
derived as previously reported.13
ꢀ
Pezzetta, D.; Pillan, A.; Poggesi, I.; Roletto, F.; Scolaro, A.; Tato,
9. Western Blot Analysis. Data were obtained as previously
reported.13
10. In Vivo Pharmacology. Evaluation of antitumor efficacy
was performed as previously described.13
M.; Tibolla, M.; Valsasina, B.; Varasi, M.; Volpi, D.; Santocanale,
C.; Vanotti, E. First Cdc7 kinase inhibitors: pyrrolopyridinones as
potent andorallyactiveantitumor agents. 2. Lead discovery. J. Med.
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Montagnoli, A.; Pillan, A.; Rainoldi, S.; Riccardi Sirtori, F.; Sola,
F.; Thieffine, S.; Tibolla, M.; Valsasina, B.; Volpi, D.; Santocanale,
C.; Vanotti, E. Cell Division Cycle 7 Kinase Inhibitors: 1H-
Pyrrolo[2,3-b]pyridines, Synthesis and Structure-Activity Rela-
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pyrroles: a challenge in regioselectivity and chemical reactivity.
Synthesis 2007, 20, 3095–3110.
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Rondinone, C. M.; Trevillyan, J. M.; Abad-Zapatero, C.; Fry, E. H.;
Sham, H. L.; Liu, G. Discovery of a new class of 4-anilinopyrimidines
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Acknowledgment. We are grateful to the group of Bio-
chemical Screening and to Nilla Avanzi and the group of
Assay Development of Nerviano Medical Sciences. We thank
Alessandra Badari, Davide Carenzi, and Gabriella Traquandi
for their skilled synthetic chemistry support. We thank Federico
Riccardi Sirtori for mass determinations and Franco Ciprandi
for HPLC purifications. Finally, we thank Rodrigo Bravo and
Nicola Mongelli for discussion and support, and Daniele
Donati for critical and helpful reading of the manuscript.
Note Added after ASAP Publication. This manuscript was
released ASAP on September 27, 2010 with incorrect numbering
of compounds in Tables 1, 3, 4, 6, 7, and 8. The correct version
was posted on October 1, 2010.
Supporting Information Available: Elemental analyses results
of compounds 18, 20, 22, 23, 40, 67, and 73. This material is
available free of charge via the Internet at http://pubs.acs.org.
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