Indolin-2-ones with high in vivo efficacy in a model for multiple sclerosis

Article abstract of DOI:10.1021/jm0504151

The known KDR inhibitor SU5416 and several analogues of the indolin-2-one family were surprisingly found to be highly efficacious in the EAE model, an established model for multiple sclerosis. The high in vivo effect could be correlated to in vitro inhibi

Full text of DOI:10.1021/jm0504151

5412
J. Med. Chem. 2005, 48, 5412-5414
Chart 1. Compounds 1, 14 and 16: Structure and
Activity in the EAE Model (ip Dosing)
Indolin-2-ones with High in Vivo Efficacy
in a Model for Multiple Sclerosis§
Lae¨titia Boue´rat,* Jef Fensholdt, Xifu Liang,
Sophie Havez, Simon F. Nielsen, Jens R. Hansen,
Simon Bolvig, and Christina Andersson*
LEO Pharma A/S, Industriparken,
DK-2750 Ballerup, Denmark
a n.s.: nonsignificant effect.
Scheme 1. Synthesis of Compounds 4 and 5a
Received May 2, 2005
Abstract: The known KDR inhibitor SU5416 and several
analogues of the indolin-2-one family were surprisingly found
to be highly efficacious in the EAE model, an established model
for multiple sclerosis. The high in vivo effect could be cor-
related to in vitro inhibition of the pro-inflammatory cytokine
IL-2. Activity following po administration was obtained with
several analogues and via the use of prodrugs.
Multiple sclerosis (MS) is a chronic autoimmune
disease of the central nervous system (CNS). Both
environmental and genetic factors are implicated in a
misdirected immune response against myelin antigens,
leading to axonal and nerve cell damage.1,2 Existing
treatments have limited efficacy on attack frequency
and on slowing down the disease progression.3-6 Be-
sides, progressing forms of MS without attacks are poor-
ly influenced by treatments and the sustained disability
caused by the disease progression is irreversible.7-9 For
all drugs with documented effect, the administration
mode is parenteral and no oral formulation, although
needed, is available. Today, there is an obvious need
for treatments of MS patients, which are more effective,
better tolerated, and easier to administer. The most
widely used animal model for MS is the rodent experi-
mental autoimmune encephalomyelitis (EAE) model,
where EAE can be induced by auto antigens such as
proteolipid protein, myelin basic protein, or other com-
ponents of the myelin sheaths.10,11 Screening low mo-
lecular weight compounds with potential to fulfill the
unmet clinical needs within MS, we found compound 1
to be very efficacious in the EAE model (up to 98%
inhibition, dosed daily 50 mg/kg, ip, Chart 1). Compound
1 is an indolin-2-one that has previously been developed
by Sugen as a tyrosine kinase inhibitor for the treat-
ment of cancer.12
a Reagents and conditions: i. NBS, benzoyl peroxide, CCl4,
reflux (92%); ii. indolin-2-one, piperidine (cat.), EtOH, reflux (92%);
iii. NBS, benzoyl peroxide, CCl4, reflux (26%); iv. SO2Cl2, CH2Cl2,
0 °C to room temperature (34%).
Scheme 2. Synthesis of Compounds 7 and 8a
a Reagents and conditions: i. 3,5-dimethyl-1H-pyrrole-2-car-
baldehyde, piperidine (cat.), EtOH, reflux (87%); ii. acetic anhy-
dride, Et3N, room temperature (71%).
tively, starting from the hydroxamic acid 6 prepared as
described in the literature14 (Scheme 2).
As compound 1 exhibited a high in vivo activity
despite a low plasma concentration, we investigated
whether reported primary and secondary metabolites15
showed any activity in the EAE model. Metabolites 12
(major metabolite, Scheme 3), 13 (Scheme 3), and 14
(Chart 1, X ) OH) have been isolated and characterized
by Sugen. To obtain compounds 12 and 13 in the
required quantities for testing in the EAE model, we
developed the synthesis of both compounds in moderate
to high yields: compound 12 was obtained in three steps
in 67% overall yield using DIBAL-H for the reduction
of the ester function of the intermediate 11. Compound
13 was prepared in three steps from commercially
available pyrrole 9 in 95% overall yield. In an attempt
to increase solubility and to investigate the role of the
substituents of the pyrrole ring, compounds 15 and 16,
bearing a polar amide at position 5 of the pyrrole and
no substituent on the pyrrole ring,16 respectively, were
prepared. Compound 15 was prepared in one step from
intermediate 11 using ethanolamine as both reactant
In this paper, we describe the synthesis of a series of
indolin-2-ones and analogues and their evaluation in the
EAE model. Analogues of compound 1 were first inves-
tigated. Among them, compounds 4 and 5 were synthe-
sized (Scheme 1). Both compounds were prepared in one
step starting from compound 1 using respectively NBS
and SO2Cl2 as halogenating agents. Alternatively com-
pound 4 could be prepared in two steps starting from
the pyrrole 213 in 84% overall yield. Compounds 7 and
8 were prepared in 87% and 62% overall yields, respec-
* To whom correspondence should be addressed. For L.B., Dept. of
Marketing Healthcare: phone, (0045) 72262435; Fax, (0045) 44945823;
e-mail: laetitia.bouerat@leo-pharma.com. For C.A., Dept. of Pharma-
cology: phone, (0045) 72263614; Fax, (0045) 72263335; e-mail,
christina.andersson@leo-pharma.com.
§ Dedicated to Lise and Ernst Binderup on the occasion of their
retirement in December 2004.
10.1021/jm0504151 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/27/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5413
Scheme 3. Synthesis of the Primary and Secondary
Metabolites of Compound 1: 12 and 13; Synthesis of
Compounds 11 and 15a
Chart 2. Activity of a Known KDR Inhibitor in the
EAE Model
illustrated by compounds 11 and 15, respectively, that
both exhibit more than 40% inhibition.
Indolin-2-ones have primarily been developed as KDR
inhibitors and have been shown to have an anti-
angiogenic effect. It has recently been suggested that
angiogenesis would be an interesting approach for the
treatment of MS.17 Thus all new compounds and me-
tabolites were tested for their ability to inhibit the KDR
activity in an in vitro assay (Table 1). However, no
obvious correlation was found between the in vivo
efficacy in the EAE model and the in vitro potency in
the KDR assay. For example the most potent compounds
(14 and 16) did not exhibit any effect in the EAE model
while one of the less in vitro active analogues, 5,
exhibited a high in vivo activity in the EAE model.
Furthermore, no significant effect was obtained when
mice were dosed with another known KDR inhibitor,
which is currently undergoing clinical evaluation (com-
pound 17,18 Chart 2). In conclusion, a mechanism of
action other than KDR and angiogenesis inhibition or
possibly a combination of several mechanisms accounts
for the effect of compound 1 and its analogues in the
EAE model.
Cytokines are known to play an important role in the
regulation of disease expression in EAE and in tolerance
to disease induction. It is believed that the disease
outcome can be modulated by tipping the balance
between pro-inflammatory cytokines produced by Th1
cells (IL-2, IFN-γ, TNF-R) and antiinflammatory cyto-
kines produced by Th2 cells (IL-4, IL-5, IL-10).2,19 In
EAE, the Th2 myelin specific cells suppress the disease
whereas Th1 cells promote it.10 To investigate the
mechanism of action of indolin-2-ones, we tested the
effect of compound 1 and analogues on the production
of the pro-inflammatory cytokine IL-2 in a PBMC in
vitro assay (Table 1). A clear correlation was found
between the inhibition of the production of IL-2 and the
in vivo activity in the EAE model: the most in vivo
active compounds were also the most in vitro potent
analogues (compounds 1, 4, 5) and the non in vivo active
compounds were also the less potent in vitro (compounds
11, 15, 16). The IL-2 PBMC in vitro assay was then used
as a tool for the selection of different indolin-2-ones for
testing in the EAE model. Two other classes of indolin-
2-ones were found to be both active in the IL-2 in vitro
assay and in the EAE model (Chart 3): the indole-
substituted analogues represented by compound 18 and
the aryl-substituted analogues such as compound 19.
Furthermore, compounds 18 and 19 were found orally
active, showing an activity of, respectively, -32% and
a Reagents and conditions: i. (chloromethylene)dimethylam-
monium chloride (Vilsmaier reagent), DCE; ii. NaOH (99%); iii.
indolin-2-one, piperidine (cat.), EtOH, reflux (99%); iv. LiOH (1
N), THF (97%); v. DIBAL-H (1 M in toluene), toluene (68%); vi.
ethanolamine (30 equiv), K2CO3, CH3CN (85%).
Table 1. In Vivo and in Vitro Activities of Selected Analogues
of Compound 1 in the EAE Model, the KDR Assay, and the
IL-2 PBMC Assay, Respectively
EAE
IC50 KDR IC50 IL-2
compd
inhib, %a significanceb
(10-6 M)
(10-6 M)
1 (SU5416)
-98
-78
-96
-67
-48
-44
-57
-23
-12
-41
-9
p < 0.0001
p ) 0.0014
p < 0.0001
p ) 0.0008
p ) 0.0262
p ) 0.0019
p ) 0.0002
n.s.
0.23
0.32
1.0
0.50
0.40
8.0
0.005
0.003
0.004
0.018
0.011
0.014
0.05
>1
1.0
0.03
>1
4
5
7
8
11
12 (SU9838)
13 (SU6595)
14 (SU6689)
15
0.32
>10
0.10
>10
0.14
n.s.
p < 0.0446
n.s.
16
a Inhibition in the EAE model; SJL/J mice were immunized for
EAE induction and assessed clinically for 21 days. Area under the
curve (AUC) of the disease score was calculated for all mice. Drug-
treated groups were compared to the vehicle-treated group.
Compounds were dosed ip, 50 mg/kg. b Significance; AUC of all
groups was compared using the Kruskal-Wallis test. When p <
0.05, the Mann-Whitney test was used to compare drug-treated
groups with the vehicle treated group (p < 0.05).
and solvent. The overall yield for the three-step syn-
thesis of compound 15 was 83%.
The described analogues of compound 1 were all
tested ip in the EAE model (Table 1). High in vivo
efficacy was obtained with related analogues such as
compounds 4 and 5 bearing a halogen substituent at
position 4 of the pyrrole ring. A particularly high activity
was observed with compound 5 (-96%). The amide
function of the indolin-2-one scaffold was successfully
replaced by a free or acetylated hydroxamic acid func-
tion while maintaining the in vivo activity as shown
with compounds 7 (-67%) and 8 (-48%). The major
metabolite 12 (hydroxylation of the methyl at position
5 of the pyrrole ring) exhibited in vivo efficacy (-57%).
However, compound 12 was less active than its parental
compound 1. Ester and amide functions at position 5 of
the pyrrole ring led to moderately active compounds as

5414 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Letters
Chart 3. Activity of Two Examples of Indole and Aryl
Substituted Indolin-2-ones in the EAE Model
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Acknowledgment. We are grateful to Scilla Latini
for providing in vitro data (KDR assay) and Malene
Mohr for providing HRMS data. We thank Manja
Møbjerg, Priscila Madsen, Bettina Borreschmidt Hans-
en, Pamela Lomholdt, Tina Skov Lundager, Anett Ravn
Møller, Mette Friis Knudsen, Birgitte Davidsen, Lone
Lerbech Dolleris, and Karin Kryger for excellent techni-
cal support.
Supporting Information Available: experimental pro-
cedures and spectral data for the preparation and character-
ization of compounds 3-5, 7, 8, 10-13, 15, 18, and 19,
descriptions of the in vitro and in vivo models and in vivo
results obtained with compound 1 are available free of charge
at http://pubs.acs.org.
JM0504151