5412
`
`J. Med. Chem. 2005, 48, 5412-5414
`
`Indolin-2-ones with High in Vivo Efficacy
`in a Model for Multiple Sclerosis§
`
`Chart 1. Compounds 1, 14 and 16: Structure and
`Activity in the EAE Model (ip Dosing)
`
`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.
`
`Received May 2, 2005
`
`Scheme 1. Synthesis of Compounds 4 and 5a
`
`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
`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.
`
`i. NBS, benzoyl peroxide, CCl4,
`a Reagents and conditions:
`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
`
`i. 3,5-dimethyl-1H-pyrrole-2-car-
`a Reagents and conditions:
`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
`
`10.1021/jm0504151 CCC: $30.25 © 2005 American Chemical Society
`Published on Web 07/27/2005
`
`Apotex v. Novartis
`IPR2017-00854
`NOVARTIS 2029
`
`

`

`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
`
`i. (chloromethylene)dimethylam-
`a Reagents and conditions:
`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
`IC50 IL-2
`IC50 KDR
`EAE
`(10-6 M)
`(10-6 M)
`significanceb
`inhib, %a
`compd
`p < 0.0001
`-98
`1 (SU5416)
`0.005
`0.23
`p ) 0.0014
`-78
`4
`0.003
`0.32
`p < 0.0001
`-96
`5
`0.004
`1.0
`p ) 0.0008
`-67
`7
`0.018
`0.50
`p ) 0.0262
`-48
`8
`0.011
`0.40
`p ) 0.0019
`-44
`11
`0.014
`8.0
`p ) 0.0002
`-57
`12 (SU9838)
`0.05
`0.32
`>1
`>10
`-23
`13 (SU6595)
`n.s.
`-12
`14 (SU6689)
`1.0
`0.10
`n.s.
`>10
`p < 0.0446
`-41
`15
`0.03
`>1
`-9
`16
`0.14
`n.s.
`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
`
`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-(cid:231), 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
`
`

`

`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
`
`Chart 4. po Activity in the EAE Model of Compound
`20, a Prodrug of 1
`
`-49%. This was surprising since all described in vivo
`active analogues exhibited both a poor aqueous solubil-
`ity (<0.1 mg/mL) and a low oral absorption. To improve
`the oral bioavailability of the indolin-2-one family, we
`designed a series of prodrugs.20,21 The Mannich base
`type prodrug 20 was synthesized as described in the
`literature22 and tested in the EAE model. This prodrug
`of compound 1 showed significant po efficacy (-56%) as
`compared with the parent compound 1 which was not
`orally active (Chart 4).
`In conclusion, our results suggest that the in vivo
`efficacy of the indolin-2-one family in the EAE model
`might be related to the inhibition of the pro-inflamma-
`tory cytokine IL-2. The IL-2 PBMC assay was success-
`fully used as a screening assay to find indolin-2-ones
`exhibiting in vivo activity. Additionally, it was shown
`that po efficacy could be obtained by preparing prodrugs
`of active compounds. Further investigations should
`address the therapeutic dosing of orally active com-
`pounds in the EAE model as positive results could have
`important implications for the future treatment of MS.
`
`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.
`
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`JM0504151
`
`