Contribution of indazolinone tautomers to kinase activity

Article abstract of DOI:10.1016/j.bmcl.2012.06.009

The design and synthesis of indazolinone containing kinase inhibitors are reported. Regioisomers that showed profound potency variation in previously-reported isoindolinone and aminoindazole systems were surprisingly found to have similar potencies in the case of the indazolinone chemical series. An interpretation using differential hinge hydrogen bonding and tautomeric equilibrium of indazolinone ring system is supported by quantum mechanics calculations. The equipotent inhibition of a representative kinase (KDR) by regioisomeric indazolinones 4 and 5 is clear evidence that in case of the indazolinone hinge, both tautomers are equally favored, and should be considered in design of inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.06.009

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4502–4505
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Contribution of indazolinone tautomers to kinase activity
⇑
Anil Vasudevan , Mary K. Verzal, Clara I. Villamil, Kent D. Stewart, Cele Abad-Zapatero, Tetsuro Oie,
Stevan W. Djuric
Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of indazolinone containing kinase inhibitors are reported. Regioisomers that
showed profound potency variation in previously-reported isoindolinone and aminoindazole systems
were surprisingly found to have similar potencies in the case of the indazolinone chemical series. An
interpretation using differential hinge hydrogen bonding and tautomeric equilibrium of indazolinone
ring system is supported by quantum mechanics calculations. The equipotent inhibition of a representa-
tive kinase (KDR) by regioisomeric indazolinones 4 and 5 is clear evidence that in case of the indazolinone
hinge, both tautomers are equally favored, and should be considered in design of inhibitors.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 1 May 2012
Revised 31 May 2012
Accepted 4 June 2012
Available online 9 June 2012
Keywords:
Kinase inhibitors
Tautomers
Kdr
Indazolinones
Analysis of the many available crystal structures of kinase/
inhibitor complexes reveals conserved interactions that are impor-
tant for the recognition of small heterocyclic molecules by the ATP
binding site of this class of enzymes. Prominent among these are
hydrogen bond interactions that inhibitors make with the back-
bone of the amino acid stretch that connects the kinase N- and
C-terminal domains, the so-called ‘hinge’ loop. These hydrogen
bonds position the inhibitors in an orientation that allow them to
make multiple favorable contacts with the side chains of hydro-
phobic residues that normally comprise the environment of the
adenine ring of ATP in the pocket. Given the importance of the
hinge hydrogen bonds and the associated hydrophobic interactions
to obtain binding affinity for the ATP pocket, one strategy to find
new kinase inhibitors is based on the design of molecular scaffolds
targeting these interactions.
Inspired by the hinge interaction in Staurosporine (1), Curtin et
al. reported the elegant design and synthesis of isoindolinone ureas
(2) as inhibitors of KDR (Fig. 1).1 An overlay model of KDR and
CDK2 with bound 2 indicated that the lactam N–H and C@O make
interactions with the hinge carbonyl Glu915 and amide Cys917
respectively. Subsequently, Dai et al. reported 3-amino indazole
containing biarylureas (3) wherein the 3-amino group and the
N-2 nitrogen participate in hinge interactions, a maneuver which
also resulted in potent KDR inhibitors.2 In this communication,
we report the design and synthesis of indazolinone inhibitors 4
and 5.
The design strategy of this work relied on the potential ability of
the indazolinone core to tautomerize between keto and hydroxy
forms, as shown in Figure 2. It was anticipated that this tautomeriza-
tion would allow the indazolinone hinge-binder 6 to make similar
hydrogen bond interactions with the backbone hinge similar to
either or both the isoindolinone core present in 2 and aminoindazole
core in 3. With no literature information on indazolinone tautomer
preference as a guide, computer modeling studies were carried out
on several test molecular systems.
Relative tautomer energies of structurally-simplified regioiso-
meric phenyl-substituted indazolinone structures were calculated
and are given in Table 1.3 Structures 7 and 8 match the core units
of compounds 4 and 5, respectively. Both 7 and 8 give, at most, a
1.7 kcal/more energy difference between the keto and hydroxy
tautomers in various environments. Strikingly, there was a reversal
of tautomer preference under conditions of low or high dielectric,
with the keto form being preferred at high dielectric. Regardless
of the exact environment, the calculations suggest that the keto
and hydroxy tautomers are close enough in energy that under
the varied conditions of dissolution in aqueous solution or being
bound within the active-site, compounds 7 and 8 will have both
tautomeric forms present to some extent.
The synthesis of 4 involved cyclization of the product of the
Suzuki cross coupling reaction 10 with hydrazine hydrate to afford
the indazolinone intermediate 11 (Scheme 1). Urea formation
using meta trifluoromethyl phenylisocyanate afforded 4. Similar
synthetic elaboration of 12 afforded the regioisomer 5.
Models of the binding modes of 4 and 5 in complex with KDR
are shown in Figure 3a–b.4 The previously reported model of
isoindolinone 2 was based on Staurosporine and satisfactorily
⇑
Corresponding author.
E-mail address: Anil.Vasudevan@abbott.com (A. Vasudevan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.009

A. Vasudevan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4502–4505
4503
NH2
Br
F
F3C
NH
NH
a,b
F3C
NH
NH
HO
O
O
F
O
O
N
O
N
O
NH
H
N
H2N
O
9
10
O
H
N
N
N
H
c
O
1
2
3
NH2
d
4
F3C
NH
NH
F3C
NH
H
N
O
O
NH
HN
O
H
N
O
11
F
H
N
H
N
N
H
b,c,d
O
O
5
5
4
O
I
12
Figure 1. Staurosporine, representative biaryl urea inhibitors and target com-
pounds from the present study.
Scheme 1. Reagents and conditions: (a) CH3I, Cs2CO3, DMF, 3 h, 67%; (b)
4-anilinophenyl boronic acid, PdCl2dppf, Na2CO3, DME:CH3CH2OH:H2O, wave,
l
160 °C, 20 min, 65%; (c) N2H4, pTsOH, 130 °C, 20 min, 25–40%; (d) 3-CF3phenyliso-
cyanate, DMF, 4 h, 70–75%.
O
O
Design
correlated with the SAR reported in the study.1 This model was
used as a template in modeling compound 4 which possesses the
same regiochemical substitution pattern as 2 (Fig. 3a). As men-
tioned previously, in this model, the inhibitor carbonyl accepts a
H-bond from Cys917 and the 2-NH donates a H-bond to Glu915.
In an analogous exercise, the previously reported model of amino-
indazole 3 was based on a thienopyrimidine hinge,5 and satisfacto-
rily correlated with the SAR.2 This model was used as a template in
modeling compound 5 which possesses the same regiochemical
substitution pattern as 3. (Fig. 3b) In this model, the inhibitor hy-
droxy group donates a H-bond to the backbone carbonyl of
Glu915 and the 5-membered ring nitrogen accepts a H-bond from
the backbone NH of Cys917. Both models of 4 and 5 gave satisfac-
tory structural fit within the active site. The models are consistent
with compound 4 bound in the keto-tautomeric form and com-
pound 5 bound in the hydroxy-tautomeric form.
NH
NH
N
H
Isoindolinone hinge
6 (Keto-tautomer of hinge
present in 2
interaction present in 4)
Tautomerism
OH
NH2
N
Design
N
N
H
N
H
6 (Hydroxy-tautomer of hinge
interaction present in 5)
3-aminoindazole hinge
present in 3
Figure 2. Design strategy – structural similarity of the indazolinone hinge 6, to the
isoindolinone and 3-aminoindazole hinge.
Compounds 4 and 5 were evaluated for their KDR kinase
activity (Table 2)
Table 1
Relative energies of tautomers in gas and aqueous phase, calculated at HF/6-31G⁄
H
N
H
N
level of quantum mechanics.3
e
= dielectric constant,
D
E = energy difference for the
tautomeric equilibrium, kcal/mole
Ar
=
O
R1
R2
R1
O
O
R
NH
NH
As depicted in Table 2, both 2 and 3 were potent inhibitors of KDR,
whereas the regioisomeric biarylureas 13 and 14 were devoid of
KDR activity. In contrast, both regioisomers of 4 and 5 were deter-
mined to be potent inhibitors of KDR with IC50 values of 0.04
and 0.01 M, respectively.
The observation of this high level of potency for both 4 and 5 is
striking given that the biaryl urea moiety is attached at diametri-
cally opposite 4- and 7-positions on the benzo ring of the indazoli-
none ring system. In fact, the two compounds overlay within the
active site with a similar orientation (schematized in Fig. 4). We
N
H
N
H
R2
7
8
lM
l
R1
R2
e
D
E
Preferred tautomer
H
H
Ph
Ph
Ph
Ph
H
1
80
1
À0.49
+1.67
À0.90
+0.66
7, Hydroxy
7, Keto
8, Hydroxy
8, Keto
H
80

4504
A. Vasudevan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4502–4505
F3C
NH
NH
F3C
NH
NH
O
O
HO
Glu 915
Cys 917
H
N
Glu 915
Cys 917
HN
N
N
H
O
4
5
Figure 4. Bioactive hinge interactions of 4 and 5.
Figure 3a. Compound 4 bound to ATP-binding site of KDR kinase in the DFG-out
conformation.
postulate that this overlay is feasible because of the potential for
tautomerism of the indazolidinone ring system and the different
hydrogen bonding arrangements with the hinge (Fig. 4). In sharp
contrast to the indazolinone systems reported here, the isoindo-
lines (2, 13) and aminoindazoles (3, 14) are expected to be over-
whelmingly in one tautomeric form and activity is observed for
only one regioisomer in each case. Additional SAR on the similar ki-
nase inhibition profile on close analogs of 4 and 5 are representa-
tive of a larger analoging effort (data not shown here).
In this Letter, we have reported on a new class of kinase inhib-
itors based on an indazolinone ring system.6 We believe that two
tautomers of this ring system exist, that permit two different
chemical series to be generated, here represented by compounds
4 and 5. The concept of tautomerism in kinase hinge interactions
has been discussed previously, as have the challenges of differ-
ences between tautomerization states and conformation states in
binding free-energy calculations.7 This study demonstrates that
in case of the indazolinone hinge, there exists the possibility of
multiple tautomers that provide additional options in analog de-
sign. Therefore, both tautomers should be considered in design of
kinase inhibitors.8 For heterocycles that can exist in tautomeric
forms with reasonable abundance, kinase inhibitor design can ex-
ploit unique features of each tautomer to discover novel hinge-
binding units.
Figure 3b. Compound 5 bound to ATP-binding site of KDR kinase in the DFG-out
conformation.
Table 2
KDR IC50 (1 mM ATP) of 4, 5 and comparator analogs
Compound
Structure
R
KDR IC50
0.007
(lM)
O
NH
Acknowledgements
2
CF3
Ar
Ar
We thank Niru Soni, Peter Kovar, Dr. Eric Johnson and Dr. Pat
Marcotte for the biological evaluation of 4 and 5, and Dr. Michael
Curtin for valuable discussions.
NH
13
3
CH3
CF3
>1000
0.009
O
References and notes
H
N
1. Curtin, M. L.; Frey, R. R.; Heyman, H. R.; Sarris, K. A.; Steinman, D. H.; Holmes, J.
H.; Bousquet, P. F.; Cunha, G. A.; Moskey, M. D.; Ahmed, A. A.; Pease, L. J.; Glaser,
K. B.; Stewart, K. D.; Davidsen, S. K.; Michaelides, M. R. Bioorg. Med. Chem. Lett.
2004, 14, 4505.
2. Dai, Y.; Hartandi, K.; Ji, Z.; Ahmed, A. A.; Albert, D. H.; Bauch, J. L.; Bouska, J. J.;
Bousquet, P. F.; Cunha, G. A.; Glaser, K. B.; Harris, C. M.; Hickman, D.; Guo, J.; Li,
J.; Marcotte, P. A.; Marsh, K. C.; Moskey, M. D.; Martin, R. L.; Olson, A. M.;
Ostering, D. J.; Pease, L. J.; Soni, N. B.; Stewart, K. D.; Stoll, V. S.; Tapang, P.;
Reuter, D. R.; Davidsen, S. K.; Michaelides, M. R. J. Med. Chem. 2007, 50, 1584.
3. Structures were geometry optimized at the HF/6-31G⁄ level (gas phase) using
Gaussian 98 software (Gaussian, Inc., Pittsburgh, PA). The gas phase energy
N
NH2
NH2
Ar
Ar
N
14
CH3
>1000
N
H
O
difference between tautomeric structures,
31G⁄ level using the geometry obtained at the HF/6-31G⁄ level. The solution
energy E(s) = E(g) + E(solv), where the solvation energy E(solv) was
DE(g), was calculated at the MP2/6-
NH
D
D
4
5
CF3
CF3
0.04
0.01
N
H
calculated at the B3LYP/6-31G⁄ level using the same geometry and the
conductor-like polarizability continuum model (CPCM). The estimated error in
Ar
Ar
these energy differences was +/À 0.5 kcal/mole at
e = 1 and +/À 1.0 at e = 80.
O
4. Previous modeling indicated that diaryl urea compounds such as 4 and 5 bind to
a DFG-out form of KDR kinase [Ref.2 and3]. DFG-out protein from entry 1YWN
(PDB database, BMCL, 2005, 15, 2203) was selected and models of isoindolinone
inhibitor 21 and aminoindazole inhibitor 32 were overlaid using a least-squared
superposition of the alpha-carbon protein coordinates. Models of compounds 10
and 11 were built from structures 2 and 3, respectively (Insight II software,
NH
N
H

A. Vasudevan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4502–4505
4505
Accelrys, San Diego, California). Energy minimization to eliminate close contacts
provided the final structures illustrated in Figure 4.
Michaelides, M. R.; Rafferty, P.; Sowin, T. J.; Stewart, K. D.; Xia, Z.; Zhang, H. Q.
Bioorg. Med. Chem. Lett. 2006, 16, 4266; (b) Ho, C. H.; Ludovici, D. W.; Maharoof,
U. S. M.; Mei, J.; Sechler, J. L.; Tuman, R. W.; Strobel, E. D.; Andraka, L.; Yen, H. K.;
Leo, G.; Li, J.; Almond, H.; Lu, H.; DeVine, A.; Tominovich, R. M.; Baker, J.;
Emanuel, S.; Gruninger, R. H.; Middleton, S. A.; Johnson, D. L.; Galemmo, R. A. J.
Med. Chem. 2005, 48, 8163; (c) Furet, P.; Meyer, T.; Strauss, A.; Raccuglia, S.;
Rondeau, J. M. Bioorg. Med. Chem. Lett. 2002, 12, 221.
5. Dai, Y.; Guo, Y.; Frey, R. R.; Ji, Z.; Curtin, M. L.; Ahmed, A. A.; Albert, D. H.; Arnold,
L.; Arries, S. S.; Barlozzari, T.; Bauch, J. L.; Bouska, J. J.; Bousquet, P. F.; Cunha, G.
A.; Glaser, K. B.; Guo, J.; Li, J.; Marcotte, P. A.; Marsh, K. C.; Mosley, M. D.; Pease, L.
J.; Stewart, K. D.; Stoll, V. S.; Tapang, P.; Wishart, N.; Davidsen, S. K.; Michaelides,
M. J. Med. Chem. 2005, 48, 6066.
6. A recent patent claims kinase inhibitors based on the indazolinone hinge.
US7262200.
8. An ab initio study on the contribution of tautomeric CDK2 inhibitors has been
described Duca, J. S.; Madison, V. S. Biopolymers 2005, 80, 312.
7. (a) Dinges, J.; Ashworth, K. L.; Akritopoulou-Zanze, I.; Arnold, L. D.; Baumeister,
S. A.; Bousquet, P. F.; Cunha, G. A.; Davidsen, S. K.; Djuric, S. W.; Gracias, V. J.;