Development of paramagnetic probes for molecular recognition studies in protein kinases

Article abstract of DOI:10.1021/jm800068w

We report on the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an effective chemical probe for second site screening against the protein kinase JNK using NMR-based techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of a bidentate compound spanning both the ATP site and the docking site. We show that the resulting compound has nanomolar affinity for JNK despite the relatively weak affinities of the individual fragments that constitute it. The approach demonstrates that targeting the docking site of protein kinases represents a valuable yet unexplored avenue to obtain potent kinase inhibitors with increased selectivity.

Full text of DOI:10.1021/jm800068w

3460
J. Med. Chem. 2008, 51, 3460–3465
Development of Paramagnetic Probes for Molecular Recognition Studies in Protein Kinases
Jesus Vazquez, Surya K. De, Li-Hsing Chen, Megan Riel-Mehan, Aras Emdadi, Jason Cellitti, John L. Stebbins,
Michele F. Rega, and Maurizio Pellecchia*
Burnham Institute for Medical Research, La Jolla, California
ReceiVed January 22, 2008
We report on the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an
effective chemical probe for second site screening against the protein kinase JNK using NMR-based
techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding
events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of
a bidentate compound spanning both the ATP site and the docking site. We show that the resulting compound
has nanomolar affinity for JNK despite the relatively weak affinities of the individual fragments that constitute
it. The approach demonstrates that targeting the docking site of protein kinases represents a valuable yet
unexplored avenue to obtain potent kinase inhibitors with increased selectivity.
Introduction
Hence, our hypothesis is that by tailoring a second-site JIP1-
mimic to an ATP-mimic, it should be possible to develop potent
and selective inhibitors of the therapeutically relevant JNK and
potentially to other MAPKs that contain specific docking sites.
An interesting approach to screen for second site binders was
recently reported by Jahnke and co-workers.19–21 This method
utilizes initial binders chemically labeled with organic nitroxide
radicals (“spin labels” such as the 2,2,6,6-tetramethylpiperidine
1-oxyl (TEMPO)) to perform second-site NMR spectroscopic
screens of fragment libraries. The binding of a second-site ligand
can be simply detected by measuring the relaxation enhancement
induced by the spin-labeled first ligand.19–21 We also recently
reported the use of furanyl-salicyl-nitroxide derivatives as
versatile probes for NMR-based second-site screening in protein
tyrosine phosphatases.22
Such chemical tools are then useful for the design and
synthesis of bidentate compounds with increased affinity but
also specificity for a given target. In fact, if specificity is a major
issue, second site ligands that are specific for a given protein
may be selected by performing the NMR screening against
counter targets. On the basis of these premises, we report herein
the synthesis and characterization of the indazole-nitroxide
derivative 9 (Figure 1), its use as a probe for structural
determination of docking-site binders, and the NMR-based
design of the very first bidentate, substrate competitive inhibitor
of JNK (Figure 2).
JNKs are a family of serine/threonine protein kinases
members of the superfamily of the mitogen-activated protein
kinases (MAPK).a Misregulation of JNK activation results in
several human disorders.1 JNK is associated to inflammatory
diseases modulating TNF-R secretion among other proteins,2,3
neurodegenerative diseases, where JNK activity is correlated
to neuronal apoptosis,4,5 metabolic diseases, where JNK inhibits
insulin signaling,6,7 and cancer, where several tumor cell lines
have been reported to possess active JNK.8–10 JNKs are therefore
very promising drug targets for the development of novel
therapies against a variety of diseases.
There are three JNK genes in mammals encoding JNK-1,
JNK-2, and JNK-3 proteins, which are very similar in structure
and composition to each other. JNK binds to scaffold proteins
and substrates containing a D-domain, which consensus se-
quence is R/KXXXXLXL.11,12 JNK-interacting protein-1 (JIP1)
is a scaffolding protein that enhances JNK signaling by creating
a proximity effect between JNK and upstream kinases.13 The
JNK-JIP1 interaction is mediated by a specific, high affinity
D-domain on JIP1. The overexpression of either the D-domain
of JIP1 or the full-length protein potently inhibits JNK signaling
in the cell. The minimal region of JIP1 consisting in the single
D-domain has been identified as retaining the JNK-inhibitory
property.14–16 This peptide, pepJIP1 (a peptide of sequence
RPKRPTTLNLF corresponding to the D-domain of JIP1),
inhibited JNK activity in vitro toward recombinant c-Jun, Elk,
and ATF2 and displayed a remarkable selectivity (no inhibition
of the closely related Erk and p38 MAPKs).17
Results and Discussion
The design of the paramagnetic probe was based on the
structure of the ATP-mimic 1 (Figure 1A),17,18 but in which
the keto group was removed to facilitate the subsequent synthetic
efforts (Figure 1B). The X-ray structure of the ternary complex
between JNK1, compound 1, and a JIP1 peptide18 suggests the
most convenient position to elongate the ATP mimic toward
the JIP1 site (Figure 1A,B). The length and nature of the linker
were investigated by molecular modeling studies, leading to the
design of compound 9, which is predicted to bring the unpaired
electron of the TEMPO just at the edge of the JIP1 binding site
(Figure 1A).
The recently determined X-ray structure of JNK1 in complex
with pepJIP1 and the ATP-mimic SP600125 (1)17,18 reveals a
close proximity between the ATP and the JIP1 binding sites,
suggesting the possibility to obtain high affinity and selective
compounds by designing appropriate bidentate molecules.
* To whom correspondence should be addressed. Phone: (858) 646-3159.
Fax: (858) 713-9925. E-mail: mpellecchia@burnham.org. Address: Maurizio
Pellecchia, Burnham Institute for Medical Research, 10901 North Torrey
Pines Road, La Jolla, California, 92037.
a Abbreviations: JNK, C-Jun N-terminal protein kinase; MAPK, mitogen-
activated protein kinases; JIP1, JNK-interacting protein-1; ILOE, interligand
Overhauser effect; TEMPO, 2,2,6,6-tetramethylpiperidine 1-oxyl; WSC,
water soluble carbodiimide; DELFIA, dissociation-enhanced lanthanide
fluorescent immunoassay.
Thus, the synthesis of compound 9 starts from the 1-azaindole
(Figure 1B). After the iodination of the 1-azaindole with iodide
and its protection with tert-butyloxy anhydride, a Suzuki
coupling between 4 and 4-methoxycarbonylphenylboronic acid
10.1021/jm800068w CCC: $40.75
2008 American Chemical Society
Published on Web 05/22/2008

Paramagnetic Probes for Molecular Recognition Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3461
Figure 1. (A) Docking of compound 9 in JNK. The surface of the protein is displayed to show the cavities and the compound is displayed in
capped sticks without protons to better visualize its structure. The chemical structure of 1 is also reported. (B) Synthesis of the paramagnetic probe
9: (i) I2, KOH, DMF, 1 h, r.t.; (ii) Boc2O, DMAP, CH3CN, 10 h, r.t.; (iii) 4-methoxycarbonylphenyl boronic acid, Pd(dppf)Cl2, Na2CO3, toluene/
ethanol (10:1), 12 h, 80 °C; (iv) LiOH, THF/MeOH (5:1), 18 h, r.t.; (v) methyl 4-aminobutanoate, EDC, HOBt, DIEA, DMF, 20 h, r.t; (vi)
4-aminoTEMPO, EDC, HOBt, DIEA, DMF, 20 h, r.t.. (C) Amide region of T1F experiments (100 ms spin lock time) of peptide Ac-RPTTLNL-OH
(1 mM) in the presence of 200 µM compound 9 (black), in the presence of 10 µM of protein JNK (blue), and in the presence of both 200 µM of
compound 9, and 10 µM of protein JNK (red). (D) Docking of compound 9 and peptide Ac-RPTTLNL-OH in JNK. The surface of the protein is
displayed to highlight the cavities, the compound is displayed in capped sticks without protons to better visualize the structure and the peptide is
displayed in sticks with a translucent surface in a gradient from red to grey coding for the effect of the paramagnetic probe in each residue: red,
more affected; grey, less affected. Peptide pose correspond to that obtained directly from the X-ray structure PDB-ID 1UKI.
yielded compound 5. This compound was then saponificated
with lithium hydroxide and the Boc group was sequentially
eliminated in presence of 3N HCl.
An amide bond was formed between the resulting compound
6 and the methyl 4-aminobutanoate, obtaining compound 7 with
a 62% yield. The ester was then saponificated with lithium
hydroxide. The free acid 8 was used to anchor the 4-amino
TEMPO through an amide bond employing water-soluble
carbodiimide (WSC).
To characterize the affinity of the compounds, we tested their
inhibitory activity against JNK. The paramagnetic compound
9 showed an inhibition constant of 1.2 µM. Compound 8, which
lacks the 4-amino TEMPO attached, had an IC50 of 1.4 µM,
showing that the addition of the paramagnetic moiety does not
affect the inhibitory properties of the indazole derivative.
Subsequently, we monitored the effect of the paramagnetic
compound on the NMR spectra of pepJIP1 or its fragments.
The peptides we used were Ac-RPTTLNL-OH and Ac-LNL-
OH. The affinities of these peptides for JNK were determined
by employing a displacement assay based on the DELFIA
(dissociation-enhanced lanthanide fluorescent immunoassay)
platform in which a biotin-linked pepJIP1 is adsorbed onto a
streptavidin-coated plate followed by incubation with GST-
JNK1. Detection of the pep-JIP1/GST-JNK complex is facili-
tated by a highly fluorescent anti-GST Eu-antibody conjugate
(Perkin-Elmer). In this assay, the peptide Ac-RPTTLNL-OH
showed an IC50 of 53 µM, whereas the peptide Ac-LNL-OH
had no appreciable displacement up to 200 µM. These values
are in good agreement with the JNK inhibition studies previously
reported for pepJIP1 and related peptides.11,12 Subsequently, we
1
recorded H NMR T1F experiments with these peptides in the
presence of JNK and in presence and absence of the paramag-
netic probe 9. When the paramagnetic probe was in the sample
mixture, the proton NMR signals of both peptides are largely
attenuated in these experiments (Figures 1C and 2A) due to
their proximity to the unpaired electron of compound 9. The
unpaired electron possesses a gyromagnetic ratio 657 times
higher than a hydrogen nucleus and thus produces a very
efficient distance-dependent relaxation effect via dipole-dipole
interactions with a given nucleus. This observation has two
important implications. First, only compounds that bind to JNK
at the JIP1 site will experience the relaxation enhancement effect
of the paramagnetic probe. Second, analysis of the differential
signal reduction within the test molecule provides crude but
significant information on the relative orientation of such a
molecule with respect to the indazole-TEMPO compound.
Accordingly, in examining the signal intensity reduction induced
by the indazole-TEMPO on the pepJIP peptides, it is evident

3462 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Vazquez et al.
Figure 2. (A) Amide region of T1F experiments (100 ms spin lock time) of peptide Ac-LNL-OH (1 mM) in the presence of 200 µM compound
9 (black), in th epresence of 10 µM of protein JNK (blue), and in the presence of both 200 µM of compound 9 and 10 µM of protein JNK (red).
(B) Docking of compound 9 and peptide Ac-LNL-OH in JNK. The surface of the protein is displayed to highlight the cavities, the compound is
displayed in capped sticks without protons to better visualize the structure, and the peptide is displayed in sticks with a translucent surface in a
gradient from red to grey coding for the effect of the paramagnetic probe in each residue: red, more affected; grey, less affected. Peptide pose
correspond to that obtained directly from the X-ray structure PDB-ID 1UKI. (C) Synthesis of the bidentate compound 12: (i) tert-butyl
3-aminopropylcarbamate, EDC, HOBt, DIEA, DMF, 16 h, r.t.; (ii) TFA/DCM (1:1), 2 h, r.t.; (iii) Ac-LNLGG-OH, EDC, HOBt, DIEA, DMF, 16 h,
50 °C; (D) IC50 curves obtained for compound 12 in the kinase assay (left) and in the DELFIA displacement assay (right).
that the various residues experience different relaxation en-
hancement effects (Figures 1C and 2A). For the peptide Ac-
RPTTLNL-OH, the C-terminal leucine is the most affected
residue, being the closest to the unpaired electron of TEMPO,
while the acetylated N-terminal arginine is the less affected one,
being the most distant from the paramagnetic probe (Figure 1D).
Furthermore, by monitoring the displacement of the signal
reduction of a known reference molecule (the pepJIP1 for
example) by a test compound, it is possible to estimate its
inhibitory constant (Supporting Information).23 Again, these data
suggest that compound 9 can be used to characterize binding
and to rapidly gather structural information about the relative
orientation of a test molecule in the bound state, which
constitutes fundamental information for the design of bidentate
inhibitors.
One advantage of the fragment-based approach is that the
covalent linkage of even weakly interacting fragments can lead
to high affinity compounds.24–27 To illustrate the usefulness of
compound 9 in such endeavors, we report on the synthesis and
characterization of compound 12.
The tripeptide Ac-LNL-OH has no appreciable displacement
up to 200 µM in the DELFIA assay. However, it binds weakly
in the JIP pocket as evident by the NMR data in the presence
of the paramagnetic probe 9 (Figure 2), suggesting that it can
be a suitable fragment for the design of a higher affinity
compound when tethered with the ATP-mimic. The design of
a proper linker between Ac-LNL-OH and the indazole derivative
was determined based on the paramagnetic compound 9 and
the differential relaxation effect it induced on the tripeptide.
Two glycine residues were added to the peptide in the
C-terminal end to link the peptidyl fragment and the ATP mimic.
The synthesis (Figure 2C) was accomplished starting from
compound 6. The tert-butyl 3-aminopropylcarbamate was then
attached by an amide bond, and the elimination of the Boc
protecting group led to compound 11. The peptide Ac-LNLGG-
OH was finally anchored by an amide bond, obtaining the
“bidentate” compound 12 in 61% yield.
This compound was subsequently tested against JNK in the
kinase activity and pepJIP1 DELFIA displacement assays.
Compound 12 showed an IC50 of 0.295 µM in the kinase assay
in a substrate competitive manner. In addition, the compound
also showed an IC50 of 0.020 µM in displacing pepJIP1, as
measured by the DELFIA assay. This is particularly relevant
because the peptide Ac-LNL-OH or the peptide Ac-LNLGG-
OH did not show any displacement, up to 200 µM, in the same
assay. These data suggest that by tailoring an ATP mimic to a
weak docking site binder, it is possible to obtain potent, substrate
competitive kinase inhibitors with increased selectivity. In fact,
no appreciable inhibitory activity was detected up to 100 µM
for compound 12 against the closely related MAPK p38R.
In conclusion, we report here on the synthesis and charac-
terization of the indazole spin-labeled derivative 9 that can be
used as a probe for NMR-based second-site screening in proteins
of the MAPK family such as JNKs. We demonstrated that this
paramagnetic probe is able to detect binders in the substrate
docking site of JNK, providing rapid and useful information

Paramagnetic Probes for Molecular Recognition Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3463
(3 mL). The resulting reaction mixture was stirred at room
temperature for 18 h. After completion of the reaction, the reaction
mixture was acidified with 3 N HCl and stirred for 2 h to remove
the boc-group at the same pot. The reaction mixture was extracted
with CH2Cl2 (3 × 50 mL). The combined organic layers were dried
over MgSO4 and concentrated in vacuo. The residue was chro-
matographed over silica gel (5% methanol in dichloromethane) to
afford the acid 6 (242 mg, 90%).
on the relative orientation of such molecules. This data is critical
for the design of bidentate compounds with higher affinity and
presumably higher selectivity. We designed and synthesized
compound 12 as proof-of-concept in support to this hypothesis,
resulting in the first substrate competitive JNK inhibitor with
nanomolar activity and no appreciable activity against the most
closely related kinase p38R. We are currently working on the
use of this probe to detect additional nonpeptide small molecule
binders in the JIP1 binding site. Such fragments will be used
to synthesize new bidentate compounds, which presumably will
have higher affinity, selectivity, and better drug-like properties.
1H NMR (300 MHz, DMSO-d6) δ: 7.25 (t, J ) 7.5 Hz, 1 H),
7.43 (t, J ) 6.9 Hz, 1 H), 7.63 (d, J ) 8.1 Hz, 1 H), 8.02-8.18
(m, 5 H). MS m/z 239 (M + H)+, 201, 158, 129, 102, 84, 56.
HRMS calcd for C14H11N2O2 (M + H) 239.0821, found 239.0820.
Synthesis of Methyl-4-(4-(1H-indazol-3-yl)benzamido)butanoate
(7). To a solution of 6 (170 mg, 0.714 mmol) in DMF (3 mL) were
added EDC (163 mg, 0.856 mmol), HOBt (120 mg, 0.0.856 mmol),
DIEA (0.38 mL, 2.142 mmol), and amine (121 mg, 0.785 mmol)
at room temperature. The resulting reaction mixture was stirred at
room temperature for 20 h. The reaction mixture was diluted with
water (40 mL) followed by extraction with ethyl acetate (3 × 40
mL). The combined organic layers were washed with saturated
NaHCO3 solution (2 × 30 mL), water (3 × 30 mL), and brine (30
mL) successively, dried (MgSO4), and then concentrated in vacuo.
The residue was chromatographed over silica gel (60% ethyl acetate
in hexane) to give the pure product 7 (151 mg, 62%).
Experimental Section
General. Unless otherwise indicated, all anhydrous solvents were
commercially obtained and stored in Sure-Seal bottles under
nitrogen. All other reagents and solvents were purchased as the
highest grade available and used without further purification. NMR
spectra were recorded on Varian 300 or Bruker 600 MHz instru-
ments. Chemical shifts (δ) are reported in parts per million (ppm)
referenced to 1H (Me4Si at 0.00). Coupling constants (J) are reported
in Hz throughout. Mass spectral data were acquired on an Esquire
LC00066 for low resolution, an Agilent ESI-TOF for high resolu-
tion, or a JEOL LC-mate tuned for either low resolution or high
resolution. Retention time for product 8 was obtained in a HPLC
Breeze from Waters Co. using an Atlantis T3 3 µm 4.6 mm × 150
mm reverse phase column. The progress of reactions was monitored
by TLC. The purity of the key compounds 9 and 12 was determined
by HPLC (Supporting Information), resulting in a purity greater
than 95%.
1H NMR (300 MHz, DMSO-d6) δ: 1.82 (quintet, J ) 7.5 Hz, 2
H), 2.39 (t, J ) 7.5 Hz, 2 H), 3.31 (q, J ) 6 Hz, 2 H), 7.24 (t, J
) 7.5 Hz, 1 H), 7.42 (t, J ) 6.9 Hz, 1 H), 7.62 (d, J ) 8.4 Hz, 1
H), 7.99 (d, J ) 8.4 Hz, 2 H), 8.05-8.16 (m, 3 H), 8.58 (t, J ) 5.1
Hz, 1 H, NH). MS m/z 360 (M + Na)+, 338 (M + H)+, 266, 221,
186, 175, 102, 49. HRMS calcd for C19H20N3O3 (M + H) 338.1499,
found 338.1505.
Synthesis of 3-Iodo-1H-indazole (3). The compound 2 (inda-
zole) was commercially available, which was iodinated according
to the reported procedures to give product 3.28,29
Synthesis of 4-(4-(1H-Indazol-3-yl)benzamido)butanoic acid (8).
To a solution of compound 7 (140 mg, 0.415 mmol) in THF (3
mL) and methanol (1 mL) was added LiOH solution (102 mg, 4.15
mmol) in water (2 mL). The resulting reaction mixture was stirred
at room temperature for 18 h. After completion of the reaction, the
reaction mixture was acidified with 3 N HCl followed by extraction
with CH2Cl2 (3 × 50 mL). The combined organic layers were dried
over MgSO4 and concentrated in vacuo. The residue was chro-
matographed over silica gel (10% methanol in dichloromethane)
to afford the acid 8 (122 mg, 91%).
Synthesis of tert-Butyl-3-iodo-1H-indazole-1-carboxylate
(4). To a solution of 3 (3.66 g, 15 mmol) in CH3CN (30 mL) were
added Et3N (3.13 mL, 22.5 mmol) and DMAP (90 mg, 0.75 mmol,
5 mol%) at room temperature under nitrogen atmosphere. After 10
min, (Boc)2O (3.59 g, 16.5 mmol) was added to the reaction
mixture. The resulting reaction mixture was stirred at room
temperature for 10 h, then the solvent and triethyl amine were
removed in vacuo. The residue was extracted with ether (200 mL),
and washed with brine (2 × 50 mL), dried (MgSO4), and then
concentrated. The residue was chromatographed over silica gel (5%
ethyl acetate in hexane) to give the pure product 4 (5.14 g, 92%).
1H NMR (300 MHz, CDCl3) δ: 1.72 (s, 9 H), 7.37 (t, J ) 8.1
Hz, 1 H), 7.49 (d, J ) 8.1 Hz, 1 H), 7.58 (t, J ) 7.5 Hz, 1 H), 8.11
(d, J ) 8.7 Hz, 1 H). MS m/z 367 (M + Na)+, 345 (M + H)+,
310, 289, 244, 124, 74, 56. HRMS calcd for C12H14IN2O2 (M +
H) 345.0100, found 345.0095.
Synthesis of tert-Butyl-3-(4-(methoxycarbonyl)phenyl)-1H-in-
dazole-1-carboxylate (5). A mixture of 4 (344 mg, 1 mmol),
4-methoxycarbonylphenyl boronic acid (271 mg, 1.5 mmol),
Pd(dppf)Cl2 (82 mg, 0.1 mmol), and saturated aqueous Na2CO3
solution (4 mL) in ethanol (1 mL) and toluene (10 mL) was stirred
at 80 °C for 12 h. After completion of reaction (TLC), the reaction
mixture was extracted with CH2Cl2 (3 × 50 mL). The combined
organic layers were washed with saturated NaHCO3 solution (50
mL), water (50 mL), and brine (50 mL), dried (MgSO4), and
concentrated in vacuo. The residue was chromatographed over silica
gel (10% ethyl acetate in hexane) to give the pure product 5 (228
mg, 65%).
1H NMR (300 MHz, DMSO-d6) δ: 1.78 (quintet, J ) 7.2 Hz, 2
H), 2.30 (t, J ) 7.2 Hz, 2 H), 3.29 (q, J ) 6 Hz, 2 H), 7.24 (t, J
) 7.5 Hz, 1 H), 7.40 (t, J ) 7.8 Hz, 1 H), 7.62 (d, J ) 8.7 Hz, 1
H), 7.98 (d, J ) 8.1 Hz, 2 H), 8.02-8.16 (m, 3 H), 8.56 (t, J ) 5
Hz, 1 H, NH). MS m/z 346 (M + Na)+, 324 (M + H)+, 221, 186,
130, 83. HRMS calcd for C18H18N3O3 (M + H) 324.1343, found
324.1351.
Synthesis of N-(4-(1-Oxo-2,2,6,6-tetramethylpiperidin-4-ylamino)-
4-oxobutyl)-4-(1H-indazol-3-yl)benzamide (9). To a solution of
compound 8 (50 mg, 0.15 mmol) in DMF (3 mL) were added EDC
(45 mg, 0.23 mmol), HOBt (24 mg, 0.15 mmol), DIEA (0.075 mL,
0.45 mmol), and 4-aminoTEMPO (28 mg, 0.165 mmol) at room
temperature. The resulting reaction mixture was stirred at room
temperature for 20 h. The reaction mixture was diluted with water
(40 mL) followed by extraction with ethyl acetate (3 × 40 mL).
The combined organic layers were washed with saturated NaHCO3
solution (2 × 30 mL), water (3 × 30 mL), and brine (30 mL)
successively, dried (MgSO4), and then concentrated in vacuo. The
residue was treated with dichloromethane, observing the formation
of a precipitate. This precipitate was filtered and dried, resulting in
the pure product 9 (49 mg, 68%).
1H NMR (300 MHz, CDCl3) δ: 1.76 (s, 9 H), 4.97 (s, 3 H),
7.40 (t, J ) 7.5 Hz, 1 H), 7.58 (t, J ) 8.1 Hz, 1 H), 7.99 (d, J )
8.1 Hz, 1 H), 8.10 (d, J ) 8.1 Hz, 2 H), 8.15 ) 8.26 (m, 3 H). MS
m/z 375 (M + Na)+, 353 (M + H)+, 311, 297, 253, 241, 163, 122,
74, 56. HRMS calcd for C20H21N2O4 (M + H) 353.1501, found
353.1491.
Synthesis of 4-(1H-Indazol-3-yl)benzoic acid (6). To a solution
of compound 5 (400 mg, 1.136 mmol) in THF (5 mL) and methanol
(1 mL) was added LiOH solution (272 mg, 11.36 mmol) in water
1H NMR (after treatment with HClaq to destroy the radical, 600
MHz, DMSO-d6) δ: 1.30 (s, 6 H, CH3), 1.43 (s, 6 H, CH3),
1.70-1.85 (m, 3.5 H, CH2), 1.85-1.94 (m, 1.5 H, CH2), 1.95-2.03
(m, 1.5 H, CH2), 2.08-2.20 (m, 3.5 H, CH2), 3.00-3.10 (m, 0.5
H, CH), 3.45-3.55 (m, 0.5 H, CH), 7.22 (dd, J1 ) 7.3 Hz, J2 )
7.4 Hz, 1H, CH arom), 7.41 (dd, J1 ) 7.3 Hz, J2 ) 7.7 Hz, 1H,
CH arom), 7.61 (d, J ) 8.3 Hz, 1H, CH arom), 7.96-8.04 (m, 2H,
CH arom), 8.04-8.08 (m, 2H, CH arom), 8.1 (d, J ) 8.1 Hz,
1H, CH arom), 8.15 (d, J ) 6.7 Hz, 0.5 H, CONH), 8.48 (d, J )

3464 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Vazquez et al.
5.9 Hz, 0.5 H, CONH), 8.58-8.64 (m, 0.5 H, CONH), 8.64-8.70
(m, 0.5 H, CONH), 9.34 (ws, 0.5 H, NOH), 9.45 (ws, 0.5 H, NOH).
RT (solvent A: H2O with 0.1% formic acid; solvent B: ACN with
0.1% formic acid; linear gradient at 1 mL/min from 95% A and
5% B to 5% A and 95% B in 15 min, λ ) 220 nm): 11.65 min.
HRMS calcd for C27H36N5O3 (M + H) 477.2734, found 477.2735.
Synthesis of tert-Butyl-3-(4-(1H-indazol-3-yl)benzamido)propy-
lcarbamate (10). To a solution of 6 (155 mg, 0.421 mmol) in DMF
(3 mL) were added EDC (96 mg, 0.505 mmol), HOBt (68 mg,
0.505 mmol), DIEA (0.19 mL, 1.052 mmol), and mono-Boc-1,3-
diamino propane (82 mg, 0.463 mmol). The reaction mixture was
stirred at room temperature for 16 h. The reaction mixture was
diluted with water (40 mL), followed by extraction with ethyl
acetate (3 × 40 mL). The combined organic layers were washed
with saturated NaHCO3 solution (2 × 30 mL), water (3 × 30 mL),
and brine (30 mL) successively, dried (MgSO4), and then concen-
trated in vacuo. The residue was chromatographed over silica gel
(50% ethyl acetate in hexane) to give the pure product 10 (175
mg, 79%).
JNK Kinase Activity Assay. The kinase assay was performed
based on the LanthaScreen platform from Invitrogen using a time-
resolved fluorescence resonance energy transfer assay (TR-FRET)
in 384 well plates. Each well received JNK1 (35 ng/mL), ATF2
(400 nM), and ATP (0.2 mM) in 50 mM HEPES, 10 mM MgCl 2,
1 mM EGTA and 0.01% Brij-35, pH 7.5, and test compounds. The
kinase reaction was performed at room temperature for 1 h. After
this time, the terbium-labeled antiphospho-ATF2 antibody and
EDTA were added into each well. After an additional hour
incubation, the signal was measured at 520/495 nm emission ratio
on a fluorescence plate reader (Victor 2, Perkin-Elmer).
Modeling Studies. Docking studies were performed with
GOLD30–32 and analyzed with Sybyl (Tripos, St. Louis). Molecular
surfaces were generated with MOLCAD.33 The X-ray coordinates
of JNK1/pepJIP1/compound 1, PDB-ID 1UKI, were used to dock
the compounds. Molecular models were generated with CON-
CORD34 and energy minimized with Sybyl. For each compound,
10 solutions were generated and subsequently ranked according to
Chemscore.32 Top solutions were used to represent the docked
geometry of the azaindole derivative. Peptide poses reported in
Figures 1 and 2 of the manuscript correspond to those obtained
directly from the X-ray coordinates.
Ki Determination. The paramagnetic compound 9 can also be
used as a tool for IC50 determination for a given compound in the
JIP site when compared against a reference molecule. This is shown
in the NMR data obtained for peptide Ac-RPTTLNL-OH when
titrated with a JIP-mimic compound BI78D3 (Figure S1, Supporting
Information).35 Because the peptide is being displaced from JNK
upon titration of BI78D3, its signals become less affected by the
paramagnetic probe 9. These changes can finally provide an IC50
value for BI78D3.
1H NMR (300 MHz, CD3OD) δ: 1.47 (s, 9 H), 1.87 (quintet, J
) 6.6 Hz, 2 H), 3.18 (t, J ) 6.6 Hz, 2 H), 3.48 (t, J ) 6.6 Hz, 2
H), 7.28 (t, J ) 7.2 Hz, 1 H), 7.47 (t, J ) 6.9 Hz, 1 H), 7.61 (d,
J ) 8.4 Hz, 1 H), 8.01 (d, J ) 8.4 Hz, 2 H), 8.08 (d, J ) 8.4 Hz,
1 H), 8.10 (d, J ) 8.4 Hz, 2 H). EIMS m/z 395 (M + H)+, 339,
295, 221, 83. HRMS calcd for C22H27N4O3 395.2078 (M + H),
found 395.2077.
Synthesis of N-(3-Aminopropyl)-4-(1H-indazol-3-yl)benzamide
(11). To a solution of compound 10 (41 mg, 0.104 mmol) in CH2Cl2
(2 mL) was added TFA (1 mL). The resulting reaction mixture
was stirred at room temperature for 2 h. TFA and dichloromethane
were removed in vacuo to give 11. This compound was used for
the next step without further purification.
Synthesis of N1-(1-(4-(1H-Indazol-3-yl)phenyl)-16-methyl-1,7,10,13-
tetraoxo-2,6,9,12-tetraazaheptadecan-14-yl)-2-(2-acetamido-4-eth-
ylpentanamido)succinamide (12). To a solution of 11 (30 mg, 0.102
mmol) in DMF (2 mL) were added EDC (20 mg, 0.104 mmol),
HOBt (14 mg, 0.103 mmol), DIEA (0.5 mL), and Ac-LNLGG-
OH (44 mg, 0.085 mmol) at room temperature. The reaction mixture
was stirred at 50 °C for 16 h. After completion of reaction, DMF
and DIEA were removed in vacuo to give crude reaction mixture.
The final product was obtained by HPLC purification (41 mg, 61%).
1H NMR (300 MHz, CD3OD) δ: 0.72-0.88 (m, 12 H),
1.10-1.22 (m, 2 H), 1.40-1.1.46 (m, 2 H), 1.50-1.62 (m, 4 H),
1.74 (quintet, J ) 6.3 Hz, 2 H), 1.88 (s, 3 H), 2.56-2.79 (m, 2 H),
3.37 (q, J ) 4.8 Hz, 2 H), 3.72-3.89 (m, 4 H), 4.10-4.23 (m, 2
H), 4.52-4.59 (m, 2 H), 7.15 (t, J ) 7.5 Hz, 1 H), 7.30-7.41 (m,
2 H), 7.49 (d, J ) 8.1 Hz, 1 H), 7.62 (d, J ) 7.8 Hz, 1 H, NH),
7.76 (br s, NH), 7.90 (d, J ) 8.1 Hz, 2 H), 7.94-8.01 (m, 3 H),
8.48 (br s, NH). EIMS m/z 813 (M + Na)+, 791 (M + H)+, 663,
571, 550, 522, 448, 409, 338, 270, 221, 130, 83. HRMS calcd for
C39H55N10O8 (M + H) 791.4199, found 791.4186.
pepJIP1 DELFIA Displacement Assay. To each well of 96-
well streptavidin-coated plates (Perkin-Elmer), 100 µL of a 100
ng/mL solution of biotin-labeled pep-JIP1 (Biotin-lc-KRPKRPT-
TLNLF, where lc indicates a hydrocarbon chain of 6 methylene
groups) was added.
After 1 h incubation and elimination of unbound biotin-pep-JIP1
by 3 washing steps, 87 µL of Eu-labeled anti-GST antibody solution
(300 ng/mL;1.9 nM), 2.5 µL DMSO solution containing test
compound, and 10 µL solution of GST-JNK2 for a final protein
concentration of 10 nM was added. After 1 h incubation at 0 °C,
each well was washed 5 times to eliminate unbound protein and
the Eu-antibody if displaced by a test compound. Subsequently,
200 µL of enhancement solution (Perkin-Elmer) was added to each
well and fluorescence measured after 10 min incubation (excitation
wavelength, 340 nm; emission wavelength, 615 nm). Note that
measurements are made in time-resolved mode given the relaxation
properties of Eu. Controls include unlabeled peptide and blanks
receiving no compounds. Protein and peptide solutions were
prepared in DELFIA buffer (Perkin-Elmer).
We can also determinate the Ki for compound BI78D3 employing
the Cheng-Prusoff equation (Figure S2, Supporting Information).36
Accordingly, with this equation, our experimental NMR data
estimates that the Ki for compound BI78D3 against JNK is 1.3 µM.
This data is in agreement of that obtained previously in our
laboratory.35
Acknowledgment. Financial support was obtained thanks
to NIH grants DK073274 and DK080263 to M.P.
Supporting Information Available: Spectroscopic characteriza-
tion of the compounds, HPLC traces of key compounds, and
supporting figures. This material is available free of charge via the
Internet at http://pubs.acs.org.
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