Flipped out: Structure-guided design of selective pyrazolylpyrrole ERK inhibitors

Article abstract of DOI:10.1021/jm061381f

The Ras/Raf/MEK/ERK signal transduction is a key oncogenic pathway implicated in a variety of human cancers. We have identified a novel series of pyrazolylpyrroles as inhibitors of ERK. Aided by the discovery of two distinct binding modes for the pyrazoly

Full text of DOI:10.1021/jm061381f

1280
J. Med. Chem. 2007, 50, 1280-1287
Flipped Out: Structure-Guided Design of Selective Pyrazolylpyrrole ERK Inhibitors‡
Alex M. Aronov,* Christopher Baker, Guy W. Bemis, Jingrong Cao, Guanjing Chen, Pamella J. Ford, Ursula A. Germann,
Jeremy Green, Michael R. Hale, Marc Jacobs, James W. Janetka, Francois Maltais, Gabriel Martinez-Botella,
Mark N. Namchuk, Judy Straub, Qing Tang, and Xiaoling Xie
Vertex Pharmaceuticals Inc., 130 WaVerly Street, Cambridge, Massachusetts 02139-4242
ReceiVed NoVember 30, 2006
The Ras/Raf/MEK/ERK signal transduction is a key oncogenic pathway implicated in a variety of human
cancers. We have identified a novel series of pyrazolylpyrroles as inhibitors of ERK. Aided by the discovery
of two distinct binding modes for the pyrazolylpyrrole scaffold, structure-guided optimization culminated
in the discovery of 6p, a potent and selective inhibitor of ERK.
Introduction
Cancer has surpassed heart disease as the leading cause of
death in the U.S. for persons younger than 85.1 For most types
of cancer, chemotherapeutic treatments have achieved limited
success to date and highlight the need for more efficacious,
better-tolerated therapy. Improved understanding of cancer
Figure 1. Structure of ERK2 screening hit 1.
biology at the molecular level has led to a shift in treatment
paradigm from the traditional cytotoxics toward more rationally
designed targeted therapies. These target molecular lesions that
are deregulated in cancer, primarily oncoproteins and oncogenic
pathways. Examples of molecular targets in cancer include
overexpression of receptor tyrosine kinases (RTKsa) or mutants
thereof (such as BCR-ABL,2 EGFR,3 ErbB2,4 and FLT35), as
well as activating mutations in the Ras GTPase proteins6 or
B-Raf.7
A common feature of many deregulated molecular lesions
in cancer is the activation of the Ras/Raf/MEK/ERK signal
transduction pathway (hereafter referred to as the ERK pathway).
This pathway controls a number of fundamental cellular
processes including cell survival, proliferation, motility, and
Figure 2. Crystal structure of ERK2 bound to screening hit 1.
differentiation.8-12 It is constitutively activated in cancers of
the lung, colon, pancreas, kidney, and ovary. Furthermore, some
of the best characterized oncogenic mutations (e.g., Ras, B-Raf)
This report describes our efforts in combining X-ray crystal-
reside in the ERK pathway. A number of companies have
lography, molecular modeling, and medicinal chemistry toward
targeted ERK pathway blockers. Sorafenib (Nexavar),13 an
the discovery and optimization of pyrazolylpyrroles as a novel
inhibitor of Raf kinase along with a number of RTKs, was
selective series of ERK1,2 inhibitors.
approved in late 2005 for the treatment of renal cell carcinoma.
In the course of an initial high throughput screen pyra-
Two highly selective ATP-noncompetitive inhibitors of ERK-
zolylpyrrole 1 (Figure 1) was discovered as a micromolar
activating kinase MEK, PD-0325901 (Pfizer),14 and ARRY-
inhibitor of ERK (KI ) 2.3 µM). Kinase activity of pyra-
142886/AZD6244 (Array/AstraZeneca)15 are currently under-
zolylpyrroles has been reported previously.16-18 We initiated
going clinical development.13
crystallographic studies of 1 complexed to ERK to further our
ERK is pivotal in the pathway downstream of Ras, Raf, and
structural understanding of the key binding elements responsible
MEK, acting as a central point where multiple signaling
for its ERK inhibition. The resulting X-ray structure (Figure 2)
pathways coalesce to drive transcription. ERK1 and ERK2 share
revealed that the pyrazole ring of 1 was critical to its activity,
making two hydrogen bonds to the hinge region of ERK. The
amide carbonyl of 1 formed a hydrogen bond with the catalytic
Lys52, and the pyrazole-linked phenyl made a hydrophobic
88% sequence identity, and the residues in the ATP site are
conserved. Our goal was to design novel and selective ATP
competitive inhibitors of ERK that would enable control over
the penultimate step in this key signal transduction pathway.
interaction with Val37 of the glycine-rich loop. Finally, the
pyrrole NH was involved in a hydrogen bond with the side-
‡ The atomic coordinates for ERK2 and JNK3 complexes have been
chain carbonyl of the gatekeeper residue Gln103. The gatekeeper
deposited in the Protein Data Bank under accession numbers 2OJG, 2OJI,
residue has a well-established role in determining the selectivity
of kinase inhibitors.19 Based on this structural information, we
chose to vary substituents on the phenyl and the amide, where
additional space was available, while keeping the pyrazolylpyr-
role amide core of 1 intact.
2OJJ, and 2OK1.
* To whom correspondence should be addressed. Telephone: (617) 444-
6100. Fax: (617) 444-6566. E-mail: alex_aronov@vrtx.com.
a Abbreviations: ATP, adenosine triphosphate; ERK, extracellular signal-
regulated kinase; JNK, c-Jun N-terminal kinase; PKA, protein kinase A;
RTK, receptor tyrosine kinase.
10.1021/jm061381f CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/15/2007

Structure-Guided Design of ERK2 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1281
Scheme 1a
a Reagents: (i) AlCl3, CH2Cl2; (ii) t-BuOCH(NMe2)2, DMF; (iii) (a) N2H4, DMF; (b) 50% NaOH in EtOH; (iv) EDCI, HOBT, DIEA, DMF, R1R2NH.
Table 1. Activity of Pyrrole Carboxamide Analogs of 1
Chemistry. Pyrazolylpyrrole carboxamides were prepared in
a four step procedure (Scheme 1). A Friedel-Crafts acylation
of commercially available ethyl pyrrole-2-carboxylate produced
ketone 2. Treatment of 2 with Bredereck’s reagent followed by
cyclization with hydrazine afforded carboxylic acid 4 after
hydrolysis of the ethyl ester. A standard coupling procedure
with EDCI furnished the desired analogs 5 and 6.
Results and Discussion
We first proceeded to introduce small, mostly lipophilic
substituents into the phenyl ring of 1 to optimize the interaction
with the glycine-rich loop (data not shown). 3-Chlorophenyl
afforded a 3-fold improvement in activity over 1, and the
subsequent work described in the present communication
includes this substituent throughout.
The pyrrole carboxamide portion of the scaffold provided a
straightforward entry for diversity generation from a list of
available amines. The amide projects into the salt bridge area
of the ATP site between Lys52 and Asp165, making ample
space available to accommodate the amide substituents. Cy-
clization of the amide fragment in 5b and 5c did not provide
any advantage over uncyclized tertiary amides, such as 5a.
Surprisingly, introduction of a basic N-ethylpyrrolidine moiety
(5e) resulted in a complete loss of ERK activity, despite its
special proximity to carboxylate of Asp165. Aliphatic amides
were preferred over aromatics, with anilide 5f losing 10-fold
relative to pyrrolidine amide 5b, likely due to the steric clash
with Asp165 incurred by the planar amide extension. The
biggest ERK KI improvement was observed in the case of
benzylamide 5g (KI ) 86 nM). A number of modifications to
the benzyl fragment were detrimental to ERK activity to varying
degrees. The m/p-picolyl amides 5h and 5i were 3- and 2-fold
less active than 5g, respectively, likely due to unfavorable
interactions with the hydrophobic pocket bounded by the
glycine-rich loop. Amide methylation of 5g resulted in a 5-fold
drop in potency (5j). Both rigidification of benzylamide to
tetrahydroisoquinoline 5m and elongation of the linker to
hydroxamate ester 5o led to a loss in activity. However,
hydrophobic substitution of the benzyl moiety (2,3-difluoro-,
5k) led to a further 2-fold potency improvement.
The activity of the pyrazolylpyrroles against JNK3 was
established early as the main selectivity concern for the series.
As seen in Table 1, most pyrazolylpyrrole carboxamides
afforded a selectivity window of less than 10-fold between
ERK2 and JNK3. The selectivity increased to approximately
20-fold in the case of 5k, and we chose to collect structural
information that would allow to better differentiate between
a ND ) not determined.
ERK and JNK.

1282 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
AronoV et al.
Figure 4. Crystal structure of selective ERK2 inhibitor 6p bound to
ERK2. (A) Hydrogen-bonding pattern in the structure of 6p bound to
ERK2. (B) Steric fit of 6p within the ERK2 active site.
be particularly beneficial for the ERK-bound conformation of
5g. The side chain carboxylate of Asp165 and the amide
carbonyl of Asn152 both place their sp2 oxygens in the vicinity
of the benzylic position and would be expected to form a
favorable interaction with a donor moiety. In contrast, the
benzylic carbon of 5g is in Van der Waals contact with main
chain atoms of Asp150 in the JNK3-bound structure, with
branching predicted to be detrimental for JNK activity. We
expected (R)-stereochemistry to be preferred for the benzylic
carbonsit is positioned within 3.7 Å of the Asp165 side chain
on the pro-(S) side. To confirm the desired stereochemistry at
the benzylic position, chiral methyl benzamides 6a and 6b were
prepared (Table 2). Indeed, the (R)-enantiomer 6a was equi-
potent with 5g, while (S)-methyl led to a 7-fold drop in activity.
Importantly, selectivity of 6a against JNK3 improved to 30-
fold, compared to only 6-fold for 5g. Validating our structural
observations, the hydroxyl donor-containing (S)-phenylglycinol
6c led to a further 3-fold improvement in ERK KI relative to
6a, from 100 nM to 35 nM. As expected, placement of a
carboxylate in proximity to Asp165 resulted in a loss of ERK
activity for 6d. Reintroduction of hydrogen bond donors as the
primary amide (6e) regained some of the activity lost by 6d,
but additional steric bulk (6f) predictably interfered with
hydrogen bond formation. Rigidification of the glycinol moiety
into indane 6g led to 100-fold loss in ERK activity.
The final optimization step involved exploration of substituent
effects on the phenyl moiety of phenylglycinol, using (S)-
phenylglycinol 6c (KI ) 35 nM) as a benchmark. As seen from
our previous efforts around 5g (Table 1), hydrophobic sub-
stitutions such as 3,4-difluoro (5k) were expected to be preferred
for phenylglycinols as well. At the para-position of phenyl, the
4-chloro substituent in 6i was optimal (KI ) 14 nM), with
both a smaller 4-fluoro and a larger 4-CF3 dropping off by
3- to 4-fold in potency. The ortho-substituent was not well
tolerated in 6k, while introduction of 3-fluoro afforded 6l (KI
) 24 nM). Dihalogenated phenylglycinols 6m-o provided
the most active compounds in the series. Finally, the (S)-
enantiomer 6p was the most potent compound synthesized, with
ERK KI ) 2 nM, 43-fold more potent that 5g. Remarkably, it
maintained its selectivity over JNK3 (KI > 4 µM). The
Figure 3. Structural characterization of the binding of 5g to kinases.
(A) Crystal structure of 5g bound to ERK2. (B) Crystal structure of 5g
bound to JNK3. (C) Overlay of the binding modes of 5g in ERK2
(yellow) and JNK3 (white).
The crystal structure of 5g bound to ERK2 (Figure 3A) was
largely consistent with the previously obtained structure of 1.
The four hydrogen bonds observed for 1 were all present. As
expected, the 3-chloro substituent was in contact with Val37.
Finally, the benzyl moiety of 5g pointed toward the glycine-
rich loop, while making favorable hydrophobic contacts with
the Cδ of Lys52 side chain, which likely explains its higher
ERK activity. Open space near the m/p-positions of the benzyl
explained the activity of 5k, pointing toward a potential for more
favorable interactions in that region of the active site.
The crystal structure of 5g bound to JNK3 was sought to
explain the rather consistent inhibition of JNK by the pyra-
zolylpyrroles and to provide guidance for eliminating this
undesirable activity. To our surprise, the binding mode of 5g
in the active site of JNK3 (Figure 3B) was dramatically different
from the previously observed ERK binding mode (Figures 2
and 3A). Indeed, the conformation and orientation of 5g bound
to JNK was almost exactly opposite of the ERK-bound
conformation (Figure 3B,C). Two hydrogens bonds were
observed between the pyrazole core of 5g and the hinge region
of JNK, however, both were made by the alternate tautomer of
the pyrazole ring. Correspondingly, the pyrrole and the chlo-
rophenyl substituents effectively swapped places within the ATP
site. The chlorophenyl was observed in proximity to Lys52,
while the pyrrole carboxamide straddled the hinge of JNK3 out
toward solvent.
Based on the difference between the two binding modes, we
hypothesized that the addition of a polar group containing a
hydrogen bond donor to the benzylic methylene of 5g should

Structure-Guided Design of ERK2 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1283
Table 2. Activity of Pyrrole Carboxamide Analogs of 5g
a ND ) not determined.
corresponding (R)-enantiomer 6q was approximately 70-fold less
active than 6p.
bonds observed previously for 1 and 5g were also present, such
that six hydrogen bonds now anchor 6p within the ERK2 active
site. The halogen substituents on the phenylglycinol fill the
hydrophobic cavity (Figure 4B) and provide favorable interac-
tions with the glycine-rich loop of ERK. The favorable
stereochemistry of (S)-phenylglycinol is determined by the
geometry of the sites(R)-stereochemistry at this position would
interfere with the side chain of Asp165, which lies within 4 Å
of the benzylic carbon of 6p. It appears that the combination
of hydrogen bonds to the hinge and the salt bridge portion of
the ERK2 active site, coupled to the interactions with the
glycine-rich loop provided by the three halogens in 6p, and the
additional hydrogen bond to the gatekeeper Gln103 are respon-
sible for the potency and ERK selectivity displayed by 6p.
The kinase counterscreening profile of 6p is shown in Table
3. The compound is about 200-fold selective against PKA, the
smallest selectivity window of all kinases tested, and is active
in the Colo205 cell proliferation assay (IC50 ) 0.54 µM).
Encouraged by the ERK-selective kinase activity profile of 6p,
we pursued the crystal structure of the 6p-ERK2 complex as
further validation of our structure-guided optimization strategy.
The structure shown in Figure 4A confirmed the presence of
two additional hydrogen bonds made by the hydroxyl of (S)-
phenylglycinol in the salt bridge area of the ATP site to the
carboxylate oxygen of the Asp165 as well as the carboxamide
oxygen of the Asn152 side chain. In addition, four hydrogen

1284 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Table 3. Kinase Counterscreening Data and Cellular Activity for 6p
AronoV et al.
mL/min; detection diode array. (iii) Method C: Water Symmetry
C18 column, 4.6 × 50 mm; linear gradient from 2% to 90% CH3-
CN in H2O over 3 min (2% ammonium formate); flow rate 1.5
mL/min; detection diode array. (iv) Method D: YMC ODS-AQ 5
µm 120A column, 3.0 × 150 mm; linear gradient 10% to 90%
CH3CN in H2O over 8 min (0.1% trifluoroacetic acid); flow rate
1.0 mL/min; 214 nM. (vi) Method E: YMC ProC18 column, 2.0
× 50 mm; linear gradient from 10% to 90% CH3CN in H2O over
5 min (0.2% formic acid); flow rate 1.5 mL/min; detection diode
array. (v) Method F: YMC ODS-AQ 5 µm 120A column, 3.0 ×
150 mm; linear gradient from 10% to 90% CH3CN in H2O over
22 min (0.1% trifluoroacetic acid); flow rate 1.0 mL/min; 214 nM.
(vi) Method G: YMC ODS-AQ 5 µm 120A column, 3.0 × 150
mm; linear gradient from 15% to 90% CH3CN in H2O over 10
min (0.2% trifluoroacetic acid); flow rate 1.5 mL/min; detection
assay
KI, µM
ERK2
PKA
FLT3
SRC
0.002
0.39
0.75
1.1
LCK
1.2
AKT3
GSK3
KDR
1.8
3.3
3.9
1
diode array. H NMR spectra (δ, ppm) was recorded either using
a Bruker DRX-500 (500 MHz) or a Bruker Avance II-300 (300
MHz) instrument. Elemental analyses were performed by Quantita-
tive Technologies, Inc. Column chromatography was performed
using Merck silica gel 60 (0.040-0.063 mm). Preparative reversed-
phase chromatography was carried out using a Gilson 215 liquid
handler coupled to a UV-vis 156 Gilson detector, an Agilent
Zorbax SB-C18 column, 21.2 × 100 mm, a linear gradient from
10% to 90% CH3CN in H2O over 10 min (0.1% trifluoroacetic
acid); flow rate 20 mL/min. 2-(3-Chlorophenyl)acetyl chloride was
synthesized following literature procedures.24 Most amines are
commercially available, except for the following: (i) the amine in
6f, which was obtained following literature procedures25 and (ii)
the amines in 6j, 6o, 6p, 6q were obtained from the corresponding
substituted phenylalanines after reduction with borane26 (data not
shown).
JNK3
AURA
CDK2
MEK1
P38R
4
>4
>4
>4
>4
Colo205
0.54a
a IC50 in the Colo205 proliferation assay (see Supporting Information
for details).
Conclusion
We have designed a series of selective and potent inhibitors
of ERK kinase by starting with a micromolar lead compound
1, followed by several rounds of structure-guided optimization.
In particular, selectivity against JNK3 was optimized following
the discovery of two alternative binding modes assumed by the
pyrazolylpyrrole inhibitors. Stabilizing the ERK-bound orienta-
tion of 5g by introducing additional functionality, we were able
to ultimately design 6p, a selective nanomolar ERK2 inhibitor.
This study is one of only a few examples in the literature of
compounds assuming different binding modes when bound to
two distinct kinases. The best known case to date is the ability
of imatinib to inhibit ABL20 and SYK21 by binding to these
kinases in two drastically different conformations/orientations.
Both imatinib binding conformations have been shown to be
important in the recognition of other protein kinasessimatinib
binds to KIT in the ABL-like mode,22 while its des-methyl
analog assumes the SYK-like conformation for binding to Src.23
In our experience, this observation is not uncommon, and we
expect future crystallographic studies with known inhibitors to
produce more examples of the “multiple binding modes”
phenomenon.
Ethyl 4-(2-(3-Chlorophenyl)acetyl)-1H-pyrrole-2-carboxylate
(2). To a solution of 2-(3-chlorophenyl)acetyl chloride (19.9 g, 105
mmol) and ethyl 1H-pyrrole-2-carboxylate (11.3 g, 81 mmol) in
dichloromethane (100 mL) at 0 °C, AlCl3 (32 g, 243 mmol) was
added in small batches. The reaction mixture was allowed to
warmed up to room temperature overnight. The crude was poured
onto ice and extracted with dichloromethane. After drying over
anhydrous sodium sulfate and removing the solvent under reduced
pressure, the crude product was purified by flash chromatography
on silica gel, eluting with dichloromethane and methanol (3%). The
title compound 2 was isolated as a solid (23 g, 99%). HPLC
(Method F) tR ) 10.07 min; 1H NMR (500 MHz, CDCl3) δ 10.15
(br s, 1H), 7.5 (d, 1H), 7.35 (s, 1H), 7.30-7.15 (m, 3H), 7.1 (d,
1H), 4.35 (q, 2H), 4.1 (s, 2H), 1.4 (t, 3H).
Ethyl 4-(2-(3-Chlorophenyl)-3-(dimethylamino)acryloyl)-1H-
pyrrole-2-carboxylate (3). To a solution of 2 (23 g, 79 mmol) in
DMF (40 mL), tert-butoxy-N,N,N′,N′-tetramethanediamine (29.6
g, 170 mmol) was added, and the resulting mixture was stirred
overnight at room temperature. The crude product was dissolved
in ethyl acetate and washed with water. After drying over anhydrous
sodium sulfate, the solvent was removed under reduced pressure,
and the resulting oil 3 was dried under vacuum overnight and used
in the next step without further purification. HPLC (Method F)
tR ) 13.77 min.
Experimental Section
General Experimental Details. All commercial reagents and
anhydrous solvents were obtained from commercial sources and
were used without further purification, unless otherwise specified.
Mass samples were analyzed on a Micro Mass ZQ, ZMD, Quattro
LC, or Quatro II mass spectrometer operated in a single MS mode
with electrospray ionization. Samples were introduced into the mass
spectrometer using flow injection (FIA) or chromatography. The
mobile phase for all mass analysis consisted of acetonitrile-water
mixtures with either 0.2% formic acid or ammonium formate. The
high-resolution mass spectrum was measured using a 9.4T APEX
III FTMS Bruker Daltonics instrument. The following HPLC
methods were used to obtain the reported retention times. (i)
Method A: YMC ProC18 column, 2.0 × 50 mm; linear gradient
from 0% to 90% CH3CN in H2O over 4 min (0.1% trifluoroacetic
acid); flow rate 0.8 mL/min; 214 nM/254 nM. (ii) Method B: Water
Symmetry C18 column, 4.6 50 mm; linear gradient from 10% to
90% CH3CN in H2O over 3 min (0.2% formic acid); flow rate 1.5
Ethyl 4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrole-2-
carboxylate (7). To a solution of 3 (79 mmol) in DMF (40 mL),
hydrazine hydrate (4.05 g, 81 mmol) was added, and the resulting
mixture was warmed at 60 °C overnight. The mixture was diluted
in ethyl acetate and washed with water. After removing the solvent
under reduced pressure, the crude was purified by flash chroma-
tography on silica gel, eluting with dichloromethane and methanol
(3%), yielding the title compound 7 (20 g, 78%). HPLC (Method
F) tR ) 10.25 min; LC-MS (Method G) tR ) 7.4 min, 316.2 (M +
1), 314.2 (M - 1); 1H NMR (500 MHz, CDCl3) δ 10.3 (br s, 1H),
7.75 (s, 1H), 7.45 (s, 1H), 7.30-7.20 (m, 3H), 7.1 (d, 1H), 7.05
(d, 1H), 4.35 (q, 2H), 4.1 (s, 2H), 1.3 (t, 3H).
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrole-2-carbox-
ylic Acid (4). To a solution of 7 (2.3 g, 7.2 mmol) in ethanol (50
mL), aqueous NaOH (50%; 1 mL) was added, and the resulting
mixture was warmed at 60 °C overnight. The solvent was removed

Structure-Guided Design of ERK2 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1285
under reduced pressure, and the crude was dissolved in ethyl acetate
and water. The water layer was washed with ethyl acetate (3 × 50
mL). The aqueous layer was then acidified to pH ) 3 and then
extracted with ethyl acetate. The organic fraction was dried over
magnesium sulfate, and the solvent was removed under reduced
pressure. The desired acid 4 was isolated and used without further
purification (1.72 g, 83%). HPLC (Method F) tR ) 8.59 min; LC-
MS (Method B) tR ) 2.79 min, 288.4 (M + 1), 286.3 (M - 1); 1H
NMR (300 MHz, DMSO-d6) δ 12.9 (br s, 2H), 11.9 (br s, 1H),
7.88 (s, 1H), 7.40-7.27 (m, 4H), 7.0 (s, 1H), 6.78 (s, 1H); HRMS
calcd for C14H10ClN3O2+H, 288.0534; found, 288.0534.
General Method for the Synthesis of Amides 5a-5o and 6a-
6q. The acid 4 (30 mg, 0.10 mmol), EDCI (24 mg, 0.12 mmol),
HOBt (17 mg, 0.12 mmol), DIEA (73 µL, 0.4 mmol), and the
desired amine (1.2 equiv) were combined in dichloromethane (2
mL) and stirred at room temperature for 2-16 h. The solvent was
then removed under reduced pressure, and the resulting crude was
dissolved in ethyl acetate, washed with 0.5 N aqueous HCl,
saturated aqueous sodium bicarbonate, and brine, and then dried
over anhydrous magnesium sulfate. After removing the solvent
under reduced pressure, the crude product was purified by reversed-
phase chromatography. Samples were isolated as TFA salts after
lyophilizing.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-ethyl-N-methyl-
1H-pyrrole-2-carboxamide (5a). HPLC (Method A) tR ) 3.23
min; LC-MS (Method B) tR ) 3.0 min, 329.4 (M + 1), 327.4
(M - 1); LC-MS (Method C) tR ) 2.7 min, 329.1 (M + 1), 327.2
(M - 1); HRMS calcd for C17H17ClN4O+H, 329.1164; found,
329.1166.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrol-2-yl)(pyr-
rolidin-1-yl)methanone (5b). HPLC (Method A) tR ) 3.27 min;
LC-MS (Method B) tR ) 3.0 min, 341.5 (M + 1), 339.4 (M - 1);
LC-MS (Method C) tR ) 2.7 min, 341.1 (M + 1), 339.2 (M - 1);
1H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1H), 7.75 (s, 1H), 7.47
(br t, 1H), 7.39-7.37 (m, 2H), 7.32-7.28 (m, 1H), 6.99 (m, 1H),
6.70 (br t, 1H), 3.58 (br t, 2H), 3.47 (br t, 2H), 1.92-1.81 (m,
4H); HRMS calcd for C18H17ClN4O+H, 341.1164; found, 341.1165.
Anal. Calcd for C20H18ClF3N4O3‚0.5H2O: C, 51.79; H, 4.13; N,
12.08. Found: C, 51.56; H, 4.03; N, 11.91.
(300 MHz, DMSO-d6) δ 12.0 (br s, 1H), 9.8 (s, 1H), 7.72 (d, 3H),
7.46 (s, 1H), 7.38-7.29 (m, 6H), 7.15 (br s, 1H), 7.04 (t, 1H);
HRMS calcd for C20H15ClN4O+H, 363.1007; found, 363.1005.
N-Benzyl-4-(4-(3-chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrole-
2-carboxamide (5g). HPLC (Method F) tR ) 10.1 min; MS calcd,
1
377.8; found, 377.4; H NMR (500 MHz, CDCl3) δ 7.7 (s, 1H),
7.4 (s, 1H), 7.3 (m, 5H), 7.25 (m, 3H), 6.95 (s, 1H), 6.8 (s, 1H),
4.55 (s, 1H).
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-((pyridin-3-yl)-meth-
yl)-1H-pyrrole-2-carboxamide (5h). HPLC (Method A) tR ) 2.69
min; LC-MS (Method B) tR ) 2.1 min, 378.5 (M + 1), 376.5
(M - 1); LC-MS (Method C) tR ) 2.5 min, 378.2 (M + 1), 376.3
1
(M - 1); H NMR (300 MHz, DMSO-d6) δ 11.7 (br s, 1H), 8.75
(t, 1H), 8.70 (br s, 1H), 8.61 (d, 1H), 8.05 (br d, 1H), 7.79 (s, 1H),
7.66 (br t, 1H), 7.43 (s, 1H), 7.38 (d, 2H), 7.29-7.26 (m, 1H),
6.98 (br s, 1H), 6.93 (br t, 1H), 4.5 (d, 2H); HRMS calcd for C20H16-
ClN5O+H, 378.1116; found, 378.1116. Anal. Calcd for C24H18-
ClF6N5O5‚1.5H2O: C, 45.55; H, 3.34; N, 11.07. Found: C, 45.30;
H, 3.02; N, 10.88.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-((pyridin-4-yl)-meth-
yl)-1H-pyrrole-2-carboxamide (5i). HPLC (Method F) tR ) 7.5
min; LC-MS (Method G) tR ) 4.9 min, 378.4 (M + 1), 376.3
(M - 1); 1H NMR (500 MHz, CDCl3) δ 8.5 (d, 2H), 8.65 (s, 1H),
7.45 (s, 1H), 7.3 (m, 5H), 6.95 (s, 1H), 6.85 (s, 1H), 4.6 (s, 2H).
N-Benzyl-4-(4-(3-chlorophenyl)-1H-pyrazol-3-yl)-N-methyl-
1H-pyrrole-2-carboxamide (5j). HPLC (Method A) tR ) 3.59 min;
LC-MS (Method B) tR ) 3.26 min, 391.5 (M + 1), 389.3 (M -
1); HPLC (Method C) tR ) 3.0 min, 391.3 (M + 1), 389.4 (M -
1); HRMS calcd for C22H19ClN4O+H, 391.1320; found, 391.1320.
N-(3,4-Difluorobenzyl)-4-(4-(3-chlorophenyl)-1H-pyrazol-3-
yl)-1H-pyrrole-2-carboxamide (5k). HPLC (Method A) tR
) 3.56
min; LC-MS (Method B) tR
(M - 1); LC-MS (Method C) tR
(M - 1); HRMS calcd for C21H15ClF2N4O+H, 413.0975; found,
) 3.2 min, 413.5 (M + 1), 411.5
) 3.0 min, 413.2 (M + 1), 411.3
413.0975.
N-(4-Methoxybenzyl)-4-(4-(3-chlorophenyl)-1H-pyrazol-3-yl)-
1H-pyrrole-2-carboxamide (5l). HPLC (Method A) tR ) 3.43 min;
LC-MS (Method B) tR ) 3.1 min, 407.6 (M + 1), 405.5 (M - 1);
LC-MS (Method C) tR ) 2.9 min, 407.2 (M + 1), 405.3 (M - 1);
HRMS calcd for C22H19ClN4O2+H, 407.1269; found, 407.1268.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrol-2-yl)(3,4-
dihydroisoquinolin-2-(1H)-yl)methane (5m). HPLC (Method A)
tR ) 3.68 min; LC-MS (Method B) tR ) 3.3 min, 403.6 (M + 1),
401.5 (M - 1); LC-MS (Method C) tR ) 3.1 min, 403.2 (M + 1),
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrol-2-yl)(mor-
pholino)methanone (5c). HPLC (Method A) tR ) 3.07 min; LC-
MS (Method B) tR ) 2.7 min, 357.5 (M + 1), 355.5 (M - 1);
LC-MS (Method C) tR ) 2.5 min, 357.2 (M + 1), 355.3 (M - 1);
1H NMR (500 MHz, CDCl3) δ 10.3 (br s, 1H), 7.7 (s, 1H), 7.4 (s,
1H), 7.3 (m, 4H), 7.1 (s, 1H), 6.5 (s, 1H), 3.8 (m, 4H), 3.65 (m,
4H); HRMS calcd for C18H17ClN4O2+H, 357.1113; found, 357.1112.
Anal. Calcd for C18H17ClN4O2‚0.5CF3CO2H‚H2O: C, 52.85; H,
4.55; N, 12.97. Found: C, 52.92; H, 4.40; N, 12.70.
1
401.3 (M - 1); H NMR (300 MHz, DMSO-d6) δ 11.75 (s, 1H),
7.77 (s, 1H), 7.47 (br s, 1H), 7.40 (br d, 2H), 7.34-7.31 (m, 1H),
7.19 (s, 4H), 7.03 (br m, 1H), 6.67 (br s, 1H), 4.78 (s, 2H), 3.86
(br t, 2H), 2.86 (t, J ) 5.7 Hz, 2H); HRMS calcd for C23H19-
ClN4O+H, 403.1320; found, 403.1319.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-((tetrahydrofuran-
2-yl)methyl)-1H-pyrrole-2-carboxamide (5d). HPLC (Method A)
tR ) 3.14 min; LC-MS (Method B) tR ) 2.8 min, 371.5 (M + 1),
369.5 (M - 1); LC-MS (Method C) tR ) 2.6 min, 371.2 (M + 1),
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(2-(pyridin-3-yl)-
ethyl)-1H-pyrrole-2-carboxamide (5n). HPLC (Method A) tR )
2.7 min; LC-MS (Method B) tR ) 2.0 min, 392.6 (M + 1), 390.5
(M - 1); LC-MS (Method C) tR ) 2.5 min, 392.2 (M + 1), 390.3
(M - 1); 1H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.72 (s,
1H), 8.69 (d, J ) 4.8 Hz, 1H), 8.25 (d, J ) 7.8 Hz, 1H), 8.16 (br
t, 1H), 7.84-7.79 (m, 2H), 7.42 (d, 1H), 7.36-7.32 (m, 2H), 7.30-
7.27 (m, 1H), 6.94 (br s, 1H), 6.81 (br t, 1H), 3.52 (q, J ) 6.3 Hz,
2H), 2.97 (t, J ) 6.6 Hz, 2H); HRMS calcd for C21H18ClN5O+H,
392.1273; found, 392.1271.
1
369.3 (M - 1); H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1H),
8.12 (t, J ) 5.1 Hz 1H), 7.78 (s, 1H), 7.43 (br s, 1H), 7.34 (br d,
2H), 7.32-7.25 (m, 1H), 6.94 (br m, 1H), 6.90 (br t, 1H), 3.92 (m,
1H), 3.8 (m, obscured by H2O), 3.22 (q, 2H), 1.93-1.86 (m, 3H),
1.61-1.51 (m, 1H); HRMS calcd for C19H19ClN4O2+H, 371.1269;
found, 371.1271.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-((1-ethylpyrrolidin-
2-yl)methyl)-1H-pyrrole-2-carboxamide (5e). HPLC (Method A)
tR ) 2.78 min; LC-MS (Method B) tR ) 2.3 min, 398.6 (M + 1);
LC-MS (Method C) tR ) 2.6 min, 398.2 (M + 1), 396.3 (M - 1);
1H NMR (300 MHz, DMSO-d6) δ 11.8 (br s, 1H), 8.42 (br t, 1H),
7.8 (s, 1H), 7.43 (br s, 1H), 7.40-7.32 (m, 2H), 7.32-7.27 (m,
1H), 7.00 (br s, 1H), 6.91 (s, 1H), 6.90 (br t, 1H), 3.16-3.0 (m,
5H), 1.97-1.81 (m, 4H), 1.24 (t, J ) 7.2 Hz, 3H); HRMS calcd
for C21H24ClN5O+H, 398.1742; found, 398.1742.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-phenyl-1H-pyrrole-
2-carboxamide (5f). HPLC (Method A) tR ) 3.54 min; LC-MS
(Method B) tR ) 3.2 min, 363.5 (M + 1), 361.5 (M - 1); LC-MS
(Method C) tR ) 3.0 min, 363.1 (M + 1), 361.3 (M - 1); 1H NMR
N-(Benzyloxy)-4-(4-(3-chlorophenyl)-1H-pyrazol-3-yl)-1H-
pyrrole-2-carboxamide (5o). HPLC (Method A) tR ) 3.41 min;
LC-MS (Method B) tR ) 3.0 min, 393.6 (M + 1), 391.5 (M - 1);
LC-MS (Method C) tR ) 2.9 min, 393.2 (M + 1), 391.3 (M - 1);
HRMS calcd for C21H17ClN4O2+H, 393.1113; found, 393.1112.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-((R)-1-phenylethyl)-
1H-pyrrole-2-carboxamide (6a). HPLC (Method A) tR ) 3.57
min; LC-MS (Method B) tR ) 3.2 min, 391.6 (M + 1), 389.5
(M - 1); LC-MS (Method C) tR ) 3.0 min, 391.2 (M + 1), 389.4
1
(M - 1). H NMR (300 MHz, DMSO-d6) δ 11.6 (s, 1H), 8.4 (d,
J ) 8.1 Hz, 1H), 7.8 (s, 1H), 7.45 (br s, 1H), 7.37-7.18 (m, 9H),
7.05 (s, 1H), 6.94 (br m, 1H), 5.13 (p, J ) 7.5 Hz, 1H), 1.44 (d,

1286 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
AronoV et al.
J ) 6.9 Hz, 3H); HRMS calcd for C22H19ClN4O+H, 391.1320;
found, 391.1319. Anal. Calcd for C24H20ClF3N4O3‚1.5H2O: C,
54.19; H, 4.36; N, 10.53. Found: C, 54.36; H, 4.56; N, 10.31.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-((S)-1-phenylethyl)-
1H-pyrrole-2-carboxamide (6b). HPLC (Method A) tR ) 3.57
min; LC-MS (Method B) tR ) 3.2 min, 391.6 (M + 1), 389.5
(M - 1); LC-MS (Method C) tR ) 3.0 min, 391.2 (M + 1), 389.4
(M - 1); 1H NMR (300 MHz, DMSO-d6) δ 11.6 (s, 1H), 8.43 (d,
J ) 8.1 Hz, 1H), 7.80 (s, 1H), 7.45 (s, 1H), 7.37-7.28 (m, 8H),
7.23-7.19 (m, 1H), 7.05 (s, 1H), 6.94 (br m, 1H), 5.13 (p, J ) 7.8
Hz, 1H), 1.44 (d, J ) 6.9 Hz, 3H); HRMS calcd for C22H19-
ClN4O+H, 391.1320; found, 391.1319.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-((S)-2-hydroxy-1-
phenylethyl)-1H-pyrrole-2-carboxamide (6c). HPLC (Method A)
tR ) 3.23 min; LC-MS (Method B) tR ) 2.9 min, 407.6 (M + 1),
405.5 (M - 1); LC-MS (Method C) tR ) 2.7 min, 407.1 (M + 1),
405.3 (M - 1); HRMS calcd for C22H19ClN4O2+H, 407.1269;
found, 407.1270.
(S)-2-(4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrole-2-
carboxamido)-2-phenylacetic acid (6d). HPLC (Method D) tR )
6.2 min; MS 421.0 (M + 1), 419.0 (M - 1).
N-((S)-Carbamoyl(phenyl)methyl)-4-(4-(3-chlorophenyl)-1H-
pyrazol-3-yl)-1H-pyrrole-2-carboxamide (6e). HPLC (Method A)
tR ) 3.15 min; LC-MS (Method B) tR ) 2.8 min, 420.6 (M + 1),
418.5 (M - 1); LC-MS (Method C) tR ) 2.6 min, 420.1 (M + 1),
418.3 (M - 1); HRMS calcd for C22H18ClN5O2+H, 420.1222;
found, 420.1220.
N-((S)-(Methylcarbamoyl(phenyl)methyl)-4-(4-(3-chlorophe-
nyl)-1H-pyrazol-3-yl)-1H-pyrrole-2-carboxamide (6f). HPLC
(Method D) tR ) 6.0 min; LC-MS (Method E) tR ) 3.0 min, 434.0
(M + 1), 432.0 (M - 1).
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(2,3-dihydro-2-hy-
droxy-1H-inden-1-yl)-1H-pyrrole-2-carboxamide (6g). HPLC
(Method A) tR ) 3.3 min; LC-MS (Method B) tR ) 2.9 min, 419.4
(M + 1), 417.3 (M - 1); LC-MS (Method C) tR ) 2.7 min, 419.2
(M + 1), 417.3 (M - 1); 1H NMR (300 MHz, DMSO-d6) δ 11.65
(s, 1H), 8.39 (d, J ) 8.4 Hz, 1H), 7.79 (s, 1H), 7.44 (s, 1H), 7.36-
7.34 (m, 2H), 7.27-7.25 (m, 1H), 7.09 (br d, 1H), 6.98 (br s, 2H),
5.25 (t, J ) 7.8 Hz, 1H), 4.33 (q, J ) 7.2 Hz, 1H), 3.20-3.11 (m,
1H), 2.76-2.71 (m, 1H); HRMS calcd for C23H19ClN4O2+H,
419.1269; found, 419.1270.
11.70 (s, 1H), 8.36 (d, J ) 8.1 Hz, 1H), 7.80 (s, 1H), 7.45-7.42
(m, 2H), 7.37-7.35 (br d, 2H), 7.30-7.26 (m, 2H), 7.18-7.11 (m,
2H), 7.08 (s, 1H), 6.94 (br s, 1H), 5.34 (q, J ) 7.5 Hz, 1H), 3.80
(CH2 obscured by H2O); HRMS calcd for C22H18ClFN4O2+H,
425.1175; found, 425.1171. Anal. Calcd for C26H20ClF7N4O6‚
0.5H2O: C, 47.18; H, 3.20; N, 8.46. Found: C, 47.28; H, 3.23; N,
9.04.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(1-(3-fluorophenyl)-
2-hydroxyethyl)-1H-pyrrole-2-carboxamide (6l). HPLC (Method
D) tR ) 5.6 min; LC-MS (Method E) tR ) 2.9 min, 425.1 (M +
1), 423.1 (M - 1).
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(1-(3,4-difluorophe-
nyl)-2-hydroxyethyl)-1H-pyrrole-2-carboxamide (6m). HPLC
(Method A) tR ) 3.36 min; LC-MS (Method B) tR ) 3.0 min, 443.6
(M + 1), 441.5 (M - 1). LC-MS (Method C) tR ) 2.7 min, 443.2
1
(M + 1), 441.3 (M - 1). H NMR (500 MHz, acetone-d6) δ 7.9
(s, 1H), 7.65 (s, 1H), 7.5 (br d, 1H), 7.4 (br d, 2H), 7.35 (br d,
1H),7.3 (t, 2H), 7.25 (t, 1H), 7.15 (br s, 1H), 7.1 (br s, 1H), 5.2 (br
t, 1H), 3.8 (d, 2H); HRMS calcd for C22H18ClF2N4O2+H, 443.1081;
found, 443.1083.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(1-(3,5-dichlorophe-
nyl)-2-hydroxyethyl)-1H-pyrrole-2-carboxamide (6n). HPLC
(Method D) tR ) 7.1 min; LC-MS (Method E) tR ) 3.5 min, 476.8
(M + 1), 474.8 (M - 1).
N-(1-(3-Chloro-4-fluorophenyl)-2-hydroxyethyl)-4-(4-(3-chlo-
rophenyl)-1H-pyrazol-3-yl)-1H-pyrrole-2-carboxamide (6o). HPLC
(Method D) tR ) 6.5 min; LC-MS (Method E) tR ) 3.3 min, 458.9
(M + 1), 457.0 (M - 1).
Compounds 6p and 6q were isolated after chromatography using
a chiral column: ChiralPack AD column, 4.5 × 250 mm; eluting
with a 40% mixture of isopropanol and hexanes over 18 min run
time; flow rate 1 mL/min; 254 nM. Compound 6p eluted first at
8.4 min, followed by 6q at 12.6 min.
N-((S)-1-(3-Chloro-4-fluorophenyl)-2-hydroxyethyl)-4-(4-(3-
chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrole-2-carboxamide (6p).
HPLC (Method A) tR ) 3.47 min; LC-MS (Method B) tR ) 3.1
min, 459.5 (M + 1), 457.5 (M - 1); LC-MS (Method C) tR ) 2.8
min, 459.1 (M + 1), 457.3 (M - 1); 1H NMR (300 MHz, DMSO-
d6) δ 11.70 (s, 1H), 8.35 (d, J ) 8.1 Hz, 1H), 7.80 (s, 1H), 7.56 (d,
J ) 7.5 Hz, 1H), 7.45 (s, 1H), 7.37-7.35 (m, 4H), 7.30-7.26 (m,
2H), 7.06 (s, 1H), 6.94 (br s, 1H), 5.04 (q, J ) 7.8 Hz, 1H), 3.80
(CH2 obscured by H2O); HRMS for calcd for C22H17Cl2FN4O2+H,
459.0785; found, 459.0783. Anal. Calcd for C24H18Cl2F4N4O4‚
H2O: C, 48.75; H, 3.41; N, 9.47. Found: C, 48.58; H, 3.41; N,
9.26.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(1-(4-fluorophenyl)-
2-hydroxyethyl)-1H-pyrrole-2-carboxamide (6h). HPLC (Method
A) tR ) 3.31 min; LC-MS (Method B) tR ) 2.9 min, 425.6 (M +
1), 423.5 (M - 1); LC-MS (Method C) tR ) 2.7 min, 425.1 (M +
1
1), 423.3 (M - 1); H NMR (300 MHz, DMSO-d6) δ 11.65 (s,
N-((R)-1-(3-Chloro-4-fluorophenyl)-2-hydroxyethyl)-4-(4-(3-
chlorophenyl)-1H-pyrazol-3-yl)-1H-pyrrole-2-carboxamide (6q).
HPLC (Method A) tR ) 3.47 min; LC-MS (Method B) tR ) 3.1
min, 459.5 (M + 1), 457.5 (M - 1); LC-MS (Method C) tR ) 2.8
min, 459.1 (M + 1), 457.3 (M - 1).
1H), 8.31 (d, J ) 8.4 Hz, 1H), 7.80 (s, 1H), 7.45 (br s, 1H), 7.42-
7.35 (m, 4H), 7.31-7.26 (m, 1H), 7.13 (t, 2H), 7.05 (br s, 1H),
6.94 (br m, 1H), 5.06 (q, J ) 6.9 Hz, 1H), 3.70 (CH2 obscured by
H2O); HRMS calcd for C22H18ClFN4O2+H, 425.1175; found,
425.1172. Anal. Calcd for C24H19ClF4N4O4‚0.5H2O: C, 52.61; H,
3.68; N, 10.23. Found: C, 52.46; H, 3.37; N, 10.18.
Acknowledgment. The authors thank Nigel Ewing and Hong
Tsao for help in generating the supporting data.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(1-(4-chlorophenyl)-
2-hydroxyethyl)-1H-pyrrole-2-carboxamide (6i). HPLC (Method
A) tR ) 3.43 min; LC-MS (Method B) tR ) 3.1 min, 441.5 (M +
1), 439.5 (M - 1); LC-MS (Method C) tR ) 2.8 min, 441.1 (M +
1), 439.3 (M - 1); HRMS calcd for C22H18Cl2N4O2+H, 441.0879;
found, 441.0880.
4-(4-(3-Chlorophenyl)-1H-pyrazol-3-yl)-N-(1-(4-(trifluoro-me-
thyl)phenyl)-2-hydroxy-ethyl)-1H-pyrrole-2-carboxamide (6j).
HPLC (Method A) tR ) 3.54 min; LC-MS (Method B) tR ) 3.1
min, 475.6 (M + 1), 473.5 (M - 1); LC-MS (Method C) tR ) 2.9
min, 475.2 (M + 1), 475.3 (M - 1); HRMS calcd for C23H18-
ClF3N4O2+H, 475.1143; found, 475.1145.
Supporting Information Available: Experimental details on
compound characterization; biochemical and biological procedures;
crystallization conditions; and data collection statistics. This material
is available free of charge via the Internet at http://pubs.acs.org.
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