Aryl-indolyl maleimides as inhibitors of CaMKIIδ. Part 1: SAR of the aryl region

Article abstract of DOI:10.1016/j.bmcl.2008.02.059

A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 34 nM to >20 μM and were dependant upon both the nature of the aryl group and the hydrogen bond donating potential of the maleimide ring. Key interactions with the kinase ATP site and hinge region were found to be consistent with homology modeling predictions.

Full text of DOI:10.1016/j.bmcl.2008.02.059

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2390–2394
Aryl–indolyl maleimides as inhibitors of CaMKIId.
Part 1: SAR of the aryl region
Daniel E. Levy,* Dan-Xiong Wang, Qing Lu, Zheng Chen, John Perumattam,
Yong-jin Xu, Albert Liclican, Jeffrey Higaki, Hanmin Dong, Maureen Laney,
Babu Mavunkel and Sundeep Dugar
Scios, Inc., 6500 Paseo Padre Parkway, Fremont, CA 94555, USA
Received 6 December 2007; revised 21 February 2008; accepted 22 February 2008
Available online 4 March 2008
Abstract—A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase.
Inhibitory activities against the enzyme ranged from 34 nM to >20 lM and were dependant upon both the nature of the aryl group
and the hydrogen bond donating potential of the maleimide ring. Key interactions with the kinase ATP site and hinge region were
found to be consistent with homology modeling predictions.
Ó 2008 Elsevier Ltd. All rights reserved.
H
N
Calcium signaling is a critical component of biological
pathways leading to cardiac1–3 and neuropathic2,4,5 re-
sponses. Additionally, calcium serves as an important
second messenger in processes including apoptosis, cell
cycle regulation, gene expression, and hormone signal-
ing.6 In order to induce these responses, calcium utilizes
calmodulin as a ubiquitous intracellular receptor. The
resulting calcium-calmodulin complex binds to and acti-
vates the family of Ca2+/calmodulin-dependant protein
kinases (CaMK)s.6
O
O
N
N
H
H2N
1
Figure 1. Lead aryl–indolyl maleimide.
The family of CaMKs consists of three types (CaMKI,
CaMKII and CaMKIV). Furthermore, CaMKII is
known to be a family of four isoforms (a, b, c, and d)
distributed in various tissue types. For example, CaM-
KIIa and b are found primarily in the brain,7 while
CaMKIId is found in the heart.8 Utilizing recombinant
Glu100
O
N
Secondary
H
H
Hydrophobic
CaMKIId, commercially available compound
1
N
O
O
Pocket
(Fig. 1)9 was shown to inhibit the enzymatic activity
with an IC50 of 380 nM.
N
N
As shown in Figrue 2, compound 1 was docked into a
homology model of CaMKIId and key interactions
within the ATP binding site and hinge region were iden-
tified. Specific interactions include dual hydrogen bonds
H
Primary
Hydrophobic
Pocket
H
N
H
H
O
Glu106
O
Keywords: Calmodulin; Kinase; Inhibitors; Maleimides.
*
Figure 2. Interactions between compound 1 and the CaMKIId
catalytic site as indicated by homology modeling.
Corresponding author. Tel.: +1 650 704 3051; e-mail:
del345@gmail.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.059

D. E. Levy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2390–2394
2391
OH
OBn
between the Glu100 hinge backbone and the maleimide
O
O
as well as a salt bridge between Glu106 and the terminal
amine. Additional hydrophobic pockets with varying
geometry were noted.
(a)
N
N
H
H
In order to develop our understanding of the structural
requirements necessary to inhibit CaMKIId, chemistry
was employed where an aryl glyoxalate was reacted with
an aryl acetamide to yield the desired maleimides under
basic conditions.10 Because the glyoxalate and acetam-
ide components were interchangeable, we were able to
utilize a diverse menu of commercially available starting
materials.
6
7
(b)
NH2
OH
OBn
O
O
O
(d)
(c)
N
N
N
With the aryl component serving as the structural vari-
able in this study, enablement of the SAR depended
upon the availability of the required 3-acetamido and
3-methylglyoxalyl indoles. Preparation of the glyoxa-
late, as shown in Scheme 1, began by treating 3-bromo-
10
9
8
BocHN
BocHN
BocHN
Scheme 2. Reagents: (a) BnBr, Cs2CO3, CH3CN, 96%; (b) 3, NaH,
DMF, 60%; (c) H2, 10%Pd/C, MeOH, 87%; (d) CDI, THF then NH3
(0.5 M in MeOH), 94%.
propylamine hydrobromide
2
with di-tert-butyl
dicarbonate and diisopropylethylamine giving the de-
sired 3-bromopropyl-tert-butylcarbamate 3. Compound
3 was then coupled with commercially available methyl
3-indoleglyoxalate 4 giving the required substituted
indoleglyoxalate 5.
mides, 13. All compounds 13 were converted to their
corresponding primary amine hydrochlorides 14 under
acidic conditions. The chemistry utilized in these
transformations is illustrated in Scheme 3. In order
to study the importance of the basic amine, as shown
in the homology model, examples of compounds 13
were assayed in addition to all of the deprotected ana-
logs 14.
Preparation of the acetamide, as illustrated in Scheme 2,
began with the conversion of commercially available 3-
indole acetic acid 6 to its corresponding benzyl ester 7
utilizing benzyl bromide and cesium carbonate. Com-
pound 7 was then coupled with compound 3 utilizing
conditions already described, and yielding compound
8. The benzyl group was removed via catalytic hydroge-
nation giving carboxylic acid 9. Final conversion to
acetamide 10 was achieved utilizing carbonyldiimidazole
and ammonia.
Finally, in order to verify the importance of the hydro-
gen bond associated with the maleimide NH, N-methyl
maleimides were prepared. As shown in Scheme 4,
examples of compounds 13 were treated with methyl
Continuing with compound 10, commercially available
methyl arylglyoxalates 11 were utilized in the prepara-
tion of target aryl-indole maleimides 13. Alternatively,
commercially available arylacetic acids were converted
to their corresponding arylacetamides 12 utilizing
chemistry identical to conditions (d) shown in Scheme
2. These aryl acetamides were combined with com-
pound 5 yielding additional target aryl-indole malei-
OCH3
O
H2N
O
O
+
Aryl
H
N
12
N
O
O
(a)
(a)
Aryl
5
BocHN
NH2
H3CO
O
O
O
N
Br
Br
+
Aryl
(a)
13
(b)
BocHN
HBr . H2N
BocHN
11
N
2
3
H
N
10
BocHN
OCH3
O
O
O
OCH3
O
O
O
Aryl
3
+
N
N
(b)
N
H
HCl . H2N
BocHN
4
5
14
Scheme 1. Reagents: (a) (Boc)2O, DIEA, CH2Cl2, 100%; (b) NaH,
DMF, 78%.
Scheme 3. Reagents: (a) tBuOK, THF, 40–80%; (b) HCl, dioxane,
MeOH, 100%.

2392
D. E. Levy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2390–2394
Table 2. SAR based on monocyclic aryl and heteroaryl groups (R1)
CH3
N
H
N
O
O
H
N
O
O
O
O
Aryl
Aryl
R1
N
(a)
N
N
R2
13
BocHN
15
(b)
BocHN
Compound
R1
R2
IC50 (lM)
CH3
N
19
13a
NH2
NHBoc
0.38 (n = 1)
3.81 0.25 (n = 2)
N
H
O
O
Aryl
14g
13g
NH2
NHBoc
1.35 0.64 (n = 2)
>20 (n = 2)
N
N
HCl . H2N
14h
14i
NH2
NH2
NH2
NH2
1.58 (n = 1)
16
Scheme 4. Reagents: (a) MeI, Cs2CO3, CH3CN, 90–100%; (b) HCl,
Dioxane, MeOH, 100%.
0.54 (n = 1)
N
iodide and cesium carbonate to form the methylated
analogs 15. Final cleavage of the carbamate under
acidic conditions yielded the desired primary amines
as their hydrochloride salts 16.
14j
1.60 (n = 1)
N
14k
1.49 0.47 (n = 2)
O
S
Table 1. SAR based on fused bicycloaryl groups (R1)
H
N
14l
13l
NH2
NHBoc
1.19 (n = 1)
>20 (n = 1)
O
O
R1
N
IC50 values11 for all tested compounds are listed in the
following tables. The data contained in Tables 1–4 re-
fer to the general structure shown in Figure 1. As
shown in Table 1, multiple fused bicycloaryl groups
were studied in an effort to understand the importance
of the indole moiety. As the data suggest, fused bicy-
cloaryl groups were tolerated with IC50 values ranging
from 0.38 to 0.63 lM. Of particular interest is the low-
er potency noted for compound 14f. This supports the
possibility of significant lone-pair/lone-pair repulsion
between the quinoline nitrogen and the adjacent malei-
mide carbonyl thus raising the energy of the bound
conformation.
R2
Compound
R1
R2
IC50 (lM)
19
13a
NH2
NHBoc
0.38 (n = 1)
3.81 0.25 (n = 2)
N
H
14b
13b
NH2
NHBoc
0.36 0.01 (n = 2)
>20 (n = 2)
S
14c
14d
NH2
NH2
0.29 (n = 1)
In an effort to expand the SAR beyond fused bicyclo-
aryl groups, monocyclic aryl and heteroaryl modifica-
tions were studied. As shown in Table 2, monocyclic
substituents were less tolerated than the bicyclic groups
shown in Table 1. In general, these modifications re-
sulted in a 3- to 4-fold loss in potency compared to
compound 1. A notable exception is the 3-pyridyl ana-
log 14i suggesting the possibility of a new interaction
within the active site. However, evidence did not exist
that these relatively small groups were sufficiently capa-
ble of taking full advantage of the active site geometry.
Therefore, a series of substituted monocyclic aryl
groups was studied.
0.50 0.05 (n = 2)
14e
14f
NH2
NH2
0.63 0.04 (n = 2)
N
N
>20 (n = 2)

D. E. Levy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2390–2394
2393
Table 3. SAR based on substituted monocyclic aryl and substituted
monocyclic heteroaryl groups
Table 4. SAR based on modifications to the indole ring system (R1)
H
N
O
O
H
N
O
O
R1
R
N
N
R2
H2N
Compound R1
R2
IC50 (lM)
Compound
R
IC50 (lM)
19
13a
NH2 0.38 (n = 1)
NHBoc 3.81 0.25 (n = 2)
N
H
19
0.38 (n = 1)
N
H
N
14q
NH2
NH2
4.56 (n = 1)
0.31 (n = 1)
14i
0.54 (n = 1)
N
14r12
14m
14n
14o
14p
0.18 0.06 (n = 3)
0.28 0.07 (n = 2)
1.32 0.26 (n = 2)
1.68 (n = 1)
Br
N
CH3
CH3
14s
13s
NH2
NHBoc 7.61 (n = 1)
0.75 0.31 (n = 2)
H3C
N
H
Br
14t
13t
NH2
NHBoc 7.54 (n = 1)
0.034 0.009 (n = 3)
O
N
H
Table 5. SAR based on methylation of the maleimide nitrogen
R2
N
Based on the relatively higher potency noted for the 3-
pyridyl analog 14i compared to all other monocyclic
derivatives, a series of meta-substituted phenyl analogs
were prepared. The data, summarized in Table 3, indi-
cate that small groups such as bromo 14m or methyl
14n can further enhance potency through more optimal
hydrophobic interactions. However, larger groups such
as phenyl 14o or phenoxy 14p caused reductions in po-
tency. This indicated that the steric capacity of the
hydrophobic pocket is too small to accommodate such
groups.
O
O
R1
N
H2N
Compound
R1
R2
H
IC50 (lM)
19
13a
0.38 (n = 1)
3.81 0.25 (n = 2)
N
H
CH3
With the promising results noted resulting from place-
ment of small groups on the phenyl ring, similar studies
were applied to the parent indole compound 1. As
shown in Table 4, with the exception of inverting the ori-
entation of the indole ring, substitutions on this group
were generally well tolerated. Of particular note was
the 5-bromoindole analog 14t which, according to the
homology model, makes highly efficient use of the
hydrophobic site geometry shown in Figure 2.
14b
16b
H
CH3
0.36 0.01 (n = 2)
2.32 (n = 1)
S
14g
16g
H
CH3
1.35 0.64 (n = 2)
15.02 (n = 1)
Br
Having probed the indole-region hydrophobic site
geometry, attention was directed to the predicted hydro-
gen bonding interactions between the maleimide and the
hinge region. As shown in Table 5, the N-methyl malei-
mide variants of several analogs were prepared and as-
sayed. In all the cases, a significant reduction in
potency was noted. The apparent imbalance between
the 10-fold reduction noted for compounds 16a, 16b,
14t
16t
H
CH3
0.034 0.01 (n = 3)
>20 (n = 1)
N
H
and 16g as compared to 16t can be rationalized based
on the methyl group forcing a shift in the binding geom-
etry. The larger substituted indole fits very well when the

2394
D. E. Levy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2390–2394
8. Edman, C. F.; Schulman, H. Biochim. Biophys. Acta 1994,
1221, 89.
9. Compound 1 was obtained from Calbiochem–Novabio-
chem Corporation, catalog # 203294.
10. Faul, M. M.; Winneroski, L. L.; Krumrich, C. A. J. Org.
Chem. 1998, 63, 6053.
hinge interaction is intact but fits very poorly if the ori-
entation of the inhibitor in the active site is shifted.
In summary, novel inhibitors of CaMKIId were prepared.
SAR efforts supported homology model predictions
pointing to critical hydrogen bonds between the primary
amine and Glu106 as well as between the maleimide and
Glu100 of the hinge region. These data are supported by
the decreased potency associated with NHBoc and N-
methyl maleimide groups. Inhibitory activity was opti-
mized through incorporation ofa 5-bromoindole generat-
ing compound 14t possessing an IC50 of 34 nM.
11. Assays were performed with inhibitor or suitable control
solvent added 10 ll per well in a 96-well microtiter plate
(Corning, NY). CaMKIId was diluted in enzyme buffer
(50 mM PIPES pH7, 0.2 mg/ml BSA, 1 mM DTT) and
added 10 ll per well. Reactions were initiated with 30 ll
reaction buffer (62.5 mM PIPES pH7, 0.25 mg/ml BSA,
33.3 mM MgCl2, 83 lM ATP, 0.4 mM CaCl2, 8.3 lg/ml
calmodulin, 25 lM [His 5] Autocamtide-2, 120 nM [g-
33P]ATP) and incubated at rt for 3 min. Reactions were
terminated by transferring 25 ll to a UNIFILTER 96-well
P81microplate (Whatman, UK), pre-wet with 15 ll 1%
phosphoric acid. After 10 min, the plate was washed 3
times with 1% phosphoric acid and one time with 95%
ethanol on a BiomekFX (Beckman Coulter, CA) equipped
with a vacuum manifold. Plates were dried for approxi-
mately 60 min, scintillant was added to the wells, and the
plates were read on a TopCount NXT Microplate
Scintillation and Luminescence Counter (Perkin-Elmer,
MA).
References and notes
1. Thorneloe, K. S.; Nelson, M. T. Can. J. Physiol.
Pharmacol. 2005, 83, 215.
2. Rossier, M. F. Recent Res. Dev. Biochem. 2003, 4, 13.
3. Takano, H.; Zou, Y.; Akazawa, H.; Nagai, T.; Mizukami,
M.; Toko, H.; Komuro, I. Prog. Exp. Cardiol. 2003, 7, 85.
4. Verkhratsky, A. Physiol. Rev. 2005, 85, 201.
5. Putney, J. W. Cell Calcium 2003, 34, 339.
6. Hook, S. S.; Means, A. R. Annu. Rev. Pharmacol. Toxicol.
2001, 41, 471.
12. Compound 14r was obtained from Calbiochem–Novabio-
chem Corporation, catalog # 557508.
7. Hudmon, A.; Schulman, H. Biochem. J. 2002, 364, 593.