Patent Application
20070167488
The present invention relates to the use of certain indolinone compounds in the prevention, treatment or amelioration of multiple sclerosis.
Multiple sclerosis is an auto-immune inflammatory disease of the central nervous system characterised by T-cell infiltration, demyelination of white matter and axonal injury. The disease mostly affects young adults with an onset at 20-40 years of age and affects twice as many women as men (A. Compton and A. Coles, The Lancet 359, 6 April 2002, pp. 1221-1231). Multiple sclerosis is more common in temperate climate zones and thus has a prevalence of 50-130 out of 100,000 in northern Europe and North America (N. Hellings et al., Immunologic Research 25(1), 2002, pp. 27-51). While the higher incidence and prevalence of multiple sclerosis in certain European populations has not been adequately explained, it is believed that increased genetic susceptibility in these populations is partly responsible. The presence of a genetic element in the etiology of the disease is supported by family studies showing that first-degree relatives of multiple sclerosis patients have a 20-40 times increased risk of developing the disease relative to the general population (J. H. Noseworthy et al., New England Journal of Medicine 343(13), 2000, pp. 938-952). Furthermore, it has been recognised that populations with a high frequency of for instance the HLA-DR2 allele have a significantly higher risk of developing multiple sclerosis (Hellings et al., supra; Noseworthy et al., supra). However, no single major susceptibility gene for multiple sclerosis has been identified so far, and the results of genome screens conducted to identify susceptibility genes rather point to multiple genes exerting a moderate effect (Hellings et al., supra).
Based on these studies, it would appear that genetic susceptibility is not enough in itself to provoke multiple sclerosis. This theory is given credence by the fact that the rate of prevalence of multiple sclerosis among people of European descent living outside Europe is half of that persisting in parts of northern Europe and that the low frequency of multiple sclerosis in Africans increases significantly among first-generation decendants living in Europe (Compton and Coles, supra). Environmental factors have therefore also been proposed as contributing to the development of multiple sclerosis.
In particular, it is believed that certain antigens present on pathogenic organisms such as viral or bacterial epitopes which structurally resemble autoantigenic epitopes of, for instance, myelin basic protein, proteolipid protein, myelin-associated glycoprotein or oligodendrocyte glycoprotein, which are all components of the myelin sheath, may lead to activation of T-cells that are reactive with such antigenic epitopes and initiating the inflammatory process eventually resulting in clinical manifestations of multiple sclerosis. This phenomenon is generally referred to as molecular mimicry (Hellings et al., supra; A. Bar-Or et al., J. Neuroimmunol. 100, 1999, pp. 252-259; A. Karni and H. L. Weiner, “Organ-Specific Inflammatory Diseases” Chapter 77 in Clinical Immunology; Principles and Practice, 2nd Ed. (R. R. Rich et al., Eds.), Mosby, London, 2001).
Multiple sclerosis is usually defined as either a relapsing-remitting or a progressive disease. The relapsing-remitting form with which 80% of the patients are initially afflicted (Compton and Coles, supra) is characterised by discrete attacks with full or partial recovery between relapses. In 40-50% of the patients, the disease eventually becomes progressive (secondary progressive stage). The disease may also be progressive from the outset (primary progressive form) characterised by a gradual decline in neurological function with no periods of remission. The clinical symptoms of the relapsing-remitting form of multiple sclerosis may vary widely from one patient to the other, but commonly affected individuals initially experience some degree of visual and sensory impairment, limb paresthesias, limb weakness, clumsiness, fatigue and gait ataxia, while in the later stages cognitive impairment, progressive quadriparesis, sensory loss, ataxic tremors, pain and spasticity are more common (Noseworthy et al., supra). The primary progressive form may initially manifest as one or more of these symptoms, gradually declining into quadriparesis, cognitive decline, visual loss, brainstem syndromes and cerebellar, bowel and bladder dysfunction (Noseworthy et al., supra).
Pathologically, multiple sclerosis is characterised by the presence of demyelinated plaques or sclerotic lesions where the myelin sheath surrounding the axons is destroyed. The inflammatory infiltrate in the lesions is composed of T-cells, B-cells, microglia and macrophages which interact with the myelin sheath and participate in the demyelinating process by local production of immune-related molecules such as adhesion molecules, cytokines and chemokines as well as demyelinating antibodies, oxygen free radicals and nitric oxide (Karni and Weiner, supra). While axonal destruction is not pronounced in the early stages of the disease (although more pronounced in patients suffering from the primary progressive form), demyelination of the axons results in slowing and blocking conductivity (Noseworthy et al., supra).
Regression of the symptoms may be associated with partial remyelination after the initial inflammation has subsided showing that oligodendrocytes (myelin-producing cells) are present in the lesions (Karni and Weiner, supra). In later stages, irreversible axonal injury, gliotic scarring and gradual loss of oligodendrocyte progenitor cells may result from repeated episodes of inflammatory attack and leads to permanent loss of neurological function (Noseworthy et al., supra).
While the immunopathogenesis of multiple sclerosis is still largely unknown, it has been shown that autoreactive T-cells specific for myelin basic protein and other antigens of the central nervous system exist in the periphery of healthy individuals as well as individuals who later develop multiple sclerosis (Bar-Or et al., supra; O'Connor et al., J. Clin. Immunol. 21(2), 2001, pp. 81-93). Thus, the presence of myelin-reactive T-cells in the periphery is not enough in itself to explain the development of multiple sclerosis. In multiple sclerosis patients, these T-cells become activated, possibly by cross-reactivity with bacterial or viral antigens that structurally resemble myelin antigens (i.e. the phenomenon known as molecular mimicry) and/or by bacterial superantigens, and persist in an enhanced state of activation (Hellings et al., supra). It has been found that the autoreactive T-cells are predominantly CD4+ T helper cells type 1 (Th1) producing interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumour necrosis factor (TNF-α) (B. Gran and A. Rostami, Current Neurology and Neuroscience Reports 1, 2001, pp. 263-270). In order for such proinflammatory T-cells to migrate to the central neurvous system, they express chemokine receptors, adhesion molecules and matrix metalloproteinases that enable them to cross the blood-brain barrier. Thus, it has been found that expression levels of the chemokines which are chemotactic for Th1 cells, IP-10 and RANTES, and their corresponding receptors, CXCR3 and CCR5, are elevated in sclerotic lesions and cerebrospinal fluid of multiple sclerosis patients (Bar-Or et al., supra). Altered levels of the adhesion molecules ICAM-1 and VCAM-1 have been identified on endothelial cells of multiple sclerosis lesions (O'Connor et al., supra). ICAM-1 and VCAM-1 are important for endothelial-leukocyte interactions and leukocyte extravasation. Matrix metalloproteinases expressed by activated T-cells, monocytes and astrocytes may disrupt the basement membrane of the blood-brain barrier and facilitate transmigration of T-cells and breakdown of the extracellular matrix (O'Connor et al., supra).
Once the T-cells have entered the central nervous system they become reactivated on encountering the autoantigen, e.g. myelin basic protein, presented by MHC class II expressing antigen presenting cells (microglia and dendritic cells), and the Th1 cells respond by producing proinflammatory cytokines such as TNF-α, IFN-γ and IL-2, while the Th2 cells produce anti-inflammatory cytokines such as IL-4, IL-5 and IL-10 (Bar-Or et al., supra). In turn, the inflammatory process leads to up-regulation of MHC class II expression and adhesion molecules on the blood-brain barrier endothelium, facilitating a further influx of T-cells, B-cells and macrophages and hence an amplification of the inflammatory response (Hellings et al., supra). This theory is supported by the finding that myelin basic protein reactive T-cell clones from multiple sclerosis patients were found to secrete increased amounts of different cytokines such as TNF-α, IL-2 and IL-10 (Hellings et al., supra). Demyelination (myelin destruction) is believed to be brought about by the combined effects of cytotoxic cells (macrophages and T-cells), oxygen free radicals, demyelinating autoantibodies and cytokine-induced toxicity (Hellings et al., supra).
Traditionally, corticosteroids such as prednisolone have been administered intravenously to multiple sclerosis patients during acute relapses in order to attenuate the inflammatory response. It has been found that treatment with corticosteroids during relapses reduces the duration of relapses and their short-term morbidity, but not the permanent disabilities resulting from repeated relapses (Compton and Coles, supra). Furthermore, treatment with potent corticosteroids at high doses has serious side effects, notably osteoporosis, aseptic bone necrosis, skin atrophy, striae cutis, insomnia, myopathy, posterior and capsular cataract and glaucoma as well as reactivation of the disease upon cessation of treatment. More recently, interferon-β (IFN-β) was introduced as a treatment of relapsing-remitting multiple sclerosis and was found to decrease the rate of relapse, increase the proportion of patients who were relapse free and reduce the number of patients who had moderate to severe relapses. On the other hand, INF-β treatment is extremely costly and its long-term efficacy has not been established. There is concern that the treatment may induce the formation of neutralising antibodies that may reduce the activity of IFN-β (Noseworthy et al., supra). Most of the patients initially experience flu-like symptoms when treated with IFN-β. Glatiramer acetate is another recent treatment based on a mixture of random synthetic peptides intended to mimic myelin basic protein. In a double-blind trial of relapsing-remitting multiple sclerosis, glatiramer acetate was found to decrease the rate of relapse. Glatiramer acetate is believed to be most effective for mildly disabled patients with a recent diagnosis of multiple sclerosis. Fewer treatment options exist for patients in the progressive phase of the disease. Immunosuppressive therapy, e.g. with cyclophosphamide or methotrexate, is frequently attempted, but it is generally recognised that once the disease enters the progressive stage treatment is very difficult. IFN-β has been in clinical trials for secondary progressive multiple sclerosis but the results did not show that the treatment slowed progression of disability and the benefits of this treatment in secondary progressive disease are controversial.
Thus, it would appear that there is a continued medical need for effective treatment of multiple sclerosis. It would also be beneficial if a medicament suitable for oral administration were to be developed.
In the course of research leading to the present invention, it was surprisingly found that certain indolinone compounds which have previously been shown to be inhibitors of receptor tyrosine kinases and suggested for the treatment of cancer exhibit a substantial level of activity in experimentally induced autoimmune encephalomyelitis (EAE) which is generally recognised as an animal model of multiple sclerosis. EAE may be induced by injection of antigenic peptides of myelin such as myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein. EAE is an inflammatory condition of the central nervous system characterised by T-cell infiltration and focal demyelination. EAE can also be induced by transfer of myelin reactive T-cells to normal individuals.
Accordingly, the present invention relates to the use of a compound of general formula I
wherein
R1, R2, R3 and R4 are the same or different and independently selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR10, —C(O)R10, —C(O)OR10, OC(O)R10, —NR10R11, —C(O)NR10R11, —NHC(O)R10, —SR10, —S(O)R10, —S(O)2R10, —S(O)2NR10R11 and —S(O)OR10, wherein R10 and R11 are the same or different and independently selected from the group consisting of hydrogen, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R10 and R11, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each of C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR12, —C(O)R12, —C(O)OR12, —OC(O)R12, —NR12R13, —C(O)NR12R13, —NHC(O)R12, —SR12, —S(O)R12, —S(O)2R12, —S(O)2NR12R13 and —S(O)OR12, wherein R12 and R13 are the same or different and independently selected from the group consisting of hydrogen, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R12 and R13, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl substituent being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4 alkoxycarbonyl, carboxy, —CONH2, S(O)NH2, aryl, heteroaryl, heterocyclyl or carbocyclyl, said aryl, heteroaryl, heterocyclyl or carbocyclyl being optionally substituted with one or more of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2 or —S(O)NH2;
X is O or S;
R5 is hydrogen, hydroxy, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, C1-6alkoxy, carbonyl, carboxy, amido, thioamido, guanyl, guanidinyl, ureidyl, sulfonyl, trihalomethanesulfonyl, —C(O)OR14, —C(O)R14, wherein R14 is hydrogen, C1-6alkyl, C2-6 alkenyl, C2-6 alkynyl, cycloalkyl or aryl;
R6 is hydrogen, C1-6 alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, halogen, —OR7, —C(O)R7, —C(O)OR7, —NR7R8, S(O)2NR7R8, wherein R7 and R8 are independently hydrogen, C1-6 alkyl, aryl or heterocyclyl, said C1-6 alkyl or heterocyclyl being optionally substituted by heterocyclyl, —OR7, —C(O)R7 or C(O)OR7, the zigzag line indicating that the group denoted R6 is present as the E- or Z-isomer;
A is phenyl or a monocyclic or bicyclic heteroaryl ring, optionally substituted at one or more positions with hydrogen, halogen, trihalomethyl, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR10, —C(O)R10, —C(O)OR10, OC(O)R10, —NR10R11, —C(O)NR10R11, —NHC(O)R10, —SR10, —S(O)R10, —S(O)2R10, —S(O)2NR10R11 and —S(O)OR10, wherein R10 and R11 are the same or different and independently selected from the group consisting of hydrogen, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R10 and R11, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each of C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR12, —C(O)R12, —C(O)OR12, —OC(O)R12, —NR12R13, —C(O)NR12R13, —NHC(O)R12, —SR12, —S(O)R12, —S(O)2R12, —S(O)2NR12R13 and —S(O)OR12, wherein R12 and R13 are the same or different and independently selected from the group consisting of hydrogen, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R12 and R13, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each C1-16-alkyl, C2-16-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl substituent being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4 alkoxycarbonyl, carboxy, —CONH2, S(O)NH2, aryl, heteroaryl, heterocyclyl or carbocyclyl, said aryl, heteroaryl, heterocyclyl or carbocyclyl being optionally substituted with one or more of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2 or —S(O)NH2; the zigzag line indicating that the group denoted A is present as the E- or Z-isomer;
or pharmaceutically acceptable salts thereof, for the preparation of a medicament for the prevention, treatment or amelioration of multiple sclerosis, or to delay of the onset of or reduce the relapse rate in multiple sclerosis.
In another aspect, the invention relates to a method of preventing, treating or ameliorating multiple sclerosis, or delaying the onset of or reducing the relapse rate in multiple sclerosis, the method comprising administering, to a patient in need thereof, a pharmacologically effective amount of a compound of general formula I as shown above.
Compounds of formula I are disclosed in, e.g., WO 96/40116 in which they are indicated to be inhibitors of tyrosine kinases and as such useful in the treatment of cancer, blood vessel proliferative disorders, fibrotic disorders, mesangial cell proliferative disorders and metabolic diseases. There is no suggestion that compounds within this group might have any utility in the treatment of multiple sclerosis.
Definitions
In the present context, the term “C1-12-alkyl” is intended to mean a linear or branched hydrocarbon group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, iso-propyl, cyclopropyl, butyl, tert-butyl, iso-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, etc. Analogously, the term “C1-10 alkyl” and “C1-6-alkyl” is intended to mean a linear, cyclic or branched hydrocarbon group having 1 to 10 or 1 to 6 carbon atoms, respectively, such as methyl, ethyl, propyl, iso-propyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the term “C1-4-alkyl” is intended to cover linear or branched hydrocarbon groups having 1 to 4 carbon atoms, e.g. methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl.
Similarly, the terms “C2-12-alkenyl”, “C4-12-alkadienyl”, and “C6-12-alkatrienyl” are intended to cover linear, cyclic or branched hydrocarbon groups having 2 to 12, 4 to 12, and 6 to 12, carbon atoms, respectively, and comprising one, two, and three unsaturated bonds, respectively. Examples of alkenyl groups are vinyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, heptadecaenyl. Examples of alkadienyl groups are butadienyl, pentadienyl, hexadienyl, heptadienyl, heptadecadienyl. Examples of alkatrienyl groups are hexatrienyl, heptatrienyl, octatrienyl, and heptadecatrienyl. Preferred examples of alkenyl are vinyl, allyl, butenyl, especially allyl.
Similarly, the term “C2-12-alkynyl” is intended to mean a linear or branched hydrocarbon group having 2 to 12 carbon atoms and comprising a triple bond. Examples hereof are ethynyl, propynyl, butynyl, octynyl, and dodecaynyl.
“Halogen” includes fluoro, chloro, bromo, and iodo.
In the present context the term “aryl” is intended to mean a fully or partially aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferred example.
The term “heteroaryl” is intended to mean a fully or partially aromatic carbocyclic ring or ring system, usually a mono- or bicyclic ring system comprising 5-12 ring atoms, where one or more of the carbon atoms have been replaced with heteroatoms, e.g. nitrogen (═N— or —NH—), sulphur, and/or oxygen atoms. Examples of such heteroaryl groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, coumaryl, furyl, thienyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzooxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl, phenoxazonyl. Particularly interesting heteroaryl groups are oxazolyl, isoxazolyl, oxadiazolyl, oxatriazolyl, thiazolyl, isothiazolyl, thiadiazolyl, thiatriazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, furyl, thienyl, quinolyl, triazolyl, tetrazolyl, isoquinolyl, indolyl in particular pyrrolyl, imidazolyl, pyridinyl, pyrimidinyl, thienyl, quinolyl, tetrazolyl, and isoquinolyl.
The term “carbocyclyl” is intended to indicate a cyclic hydrocarbon radical, which may be a saturated or unsaturated, non-aromatic, mono- or bicyclic ring comprising 5-12 ring atoms, such as C3-8 cycloalkyl, e.g. cyclopropyl, cyclopentyl, cyclohexyl or cyclooctyl, or a C3-8 cycloalkylene radical, e.g. cycloprop-2-enyl, cyclobut-2-enyl, cyclopent-2-enyl, cyclohex-3-enyl, cycloocta-4-enyl or cyclohex-3,5-dienyl.
The term “heterocyclyl” is intended to mean a non-aromatic ring or ring system, usually a mono- or bicyclic ring system comprising 5-12 ring atoms, where one or more of the carbon atoms have been replaced with heteroatoms, e.g. nitrogen (═N— or —NH—), sulphur, and/or oxygen atoms. Examples of such heterocyclyl groups are imidazolidine, piperazine, hexahydropyridazine, hexahydropyrimidine, diazepane, diazocane, pyrrolidine, piperidine, azepane, azocane, aziridine, azirine, azetidine, pyroline, tropane, oxazinane (morpholine), azepine, dihydroazepine, tetrahydroazepine, and hexahydroazepine, oxazolane, oxazepane, oxazocane, thiazolane, thiazinane, thiazepane, thiazocane, oxazetane, diazetane, thiazetane, tetrahydrofuran, tetrahydropyran, oxepane, tetrahydrothiophene, tetrahydrothiopyrane, thiepane, dithiane, dithiepane, dioxane, dioxepane, oxathiane, oxathiepane. The most interesting examples are imidazolidine, piperazine, hexahydropyridazine, hexahydropyrimidine, diazepane, diazocane, pyrrolidine, piperidine, azepane, azocane, azetidine, tropane, oxazinane (morpholine), oxazolane, oxazepane, thiazolane, thiazinane, and thiazepane, in particular imidazolidine, piperazine, hexahydropyridazine, hexahydropyrimidine, diazepane, pyrrolidine, piperidine, azepane, oxazinane (morpholine), and thiazinane.
The term “EAE” or “EAE model” is used herein to denote experimentally induced autoimmune encephalomyelitis (EAE) which is generally recognised as an animal model of multiple sclerosis. EAE may be induced by injection of antigenic peptides of myelin such as myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein. EAE is an inflammatory condition of the central nervous system characterised by T-cell infiltration and focal demyelination. EAE can also be induced by transfer of myelin reactive T-cells to normal individuals.
The term “alkoxy” is intended to indicate a radical of formula OR*, wherein R* is alkyl as defined above, e.g. methoxy, ethoxy, propoxy, butoxy, etc.
The term “alkylaryl” is intended to indicate an alkyl group covalently joined to an aryl group.
The term “sugar residue” is intended to indicate a glucuronide, e.g. hydroxyl or acyl glucuronide.
The term “halogen” is inteded to indicate fluoro, chloro, bromo or iodo.
The term “pharmaceutically acceptable salt” is intended to indicate salts prepared by reacting a compound of formula I with a suitable inorganic or organic acid, e.g. hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric, acetic, phosphoric, lactic, maleic, phthalic, citric, propionic, benzoic, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, sulfamic or fumaric acid.
The term “indolinone compound” (used synonymously with “oxindole compound” herein) is intended to include compounds of formula I, II, III or IV as shown herein as well as other, structurally related compounds, such as the compounds disclosed in WO 96/40116, U.S. Pat. No. 6,316,635, U.S. Pat. No. 6,225,335, WO 99/48868, WO 99/61422, WO 01/60814, WO 00/56709, WO 01/83450, EP 934 931, U.S. Pat. No. 5,834,504, WO 98/07695, WO 02/02551, WO 00/08202, WO 98/50356, WO 96/22976, WO 01/45689, WO 02/055517 and WO 01/94312 which are hereby incorporated by reference in their entirety. Methods of preparing the compounds are also disclosed in these publications.
The term “KDR” is understood to indicate a receptor tyrosine kinase which binds selectively to vascular endothelial growth factor (VEGF). The DNA and amino acid sequence of KDR as well as its proposed use to identify agonists and antagonists of VEGF action are disclosed in WO 92/14748.
The term “ameliorate” is intended to mean reducing the severity of the neurological symptoms during relapses of multiple sclerosis by administering an effective amount of an active compound whereby it may be possible to reduce or delay permanent disability resulting from neurological damage sustained during relapse, in particular demyelination.
The term “delay the onset of multiple sclerosis” is used to indicate a prophylactic administration of an effective amount of an active compound to prolong the period where no symptoms, or at least no severe symptoms, of multiple sclerosis are observed in susceptible individuals, e.g. in first-degree relatives of multiple sclerosis patients.
The term “reduce the relapse rate in multiple sclerosis” is intended to mean reducing the frequency with which relapses occur or, in other words, prolong the periods of remission. This may make it possible to reduce or delay the accumulation of disabilities resulting from the neurological damage sustained during each relapse, in particular demyelination which eventually leads to increasingly severe disability.
Preferred Embodiments of the Invention
In one embodiment, the invention relates to the use of a compound of general formula II
wherein R1, R2, R3, R4, R6 and X are as indicated for formula I,
R8 and R4′ are independently hydrogen, hydroxy, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, C1-6alkoxy, carbonyl, carboxy, amido, thioamido, guanyl, guanidinyl, ureidyl, sulfonyl, trihalomethanesulfonyl, —PO(OR)(OR′), wherein R and R′ are independently selected from hydrogen or C1-6 alkyl, —OR10, —C(O)R10, —C(O)OR10, OC(O)R10, OC(O)OR10, —NR10R11, —C(O)NR10R11, —OC(O)NR10R11, —NHC(O)R10, —SR10, —S(O)R10, —S(O)2R10, —S(O)2NR10R11, —S(O)OR10 and CH2-aryl-OR10, wherein R10 and R11 are the same or different and independently selected from the group consisting of hydrogen, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R10 and R11, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each of C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR12, —C(O)R12, —C(O)OR12, —OC(O)R12, OC(O)OR12, —NR12R13, —C(O)NR12R13, —OC(O)N12R13, —NHC(O)R12, —SR12, —S(O)R12, —S(O)2R12, —S(O)2NR12R13 and —S(O)OR12, wherein R12 and R13 are the same or different and independently selected from the group consisting of hydrogen, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R12 and R13, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl substituent being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2, —S(O)NH2, aryl, heteroaryl, heterocyclyl or carbocyclyl, said aryl, heteroaryl, heterocyclyl or carbocyclyl being optionally substituted with one or more of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2 or —S(O)NH2; —C(R24R25)—OR16 or —OC(O)R16, wherein R16 is hydrogen, C1-6 alkyl, aralkyl, acyl or —PO(OR)(OR′), —C(R24R25)—NR26R27, wherein R24 is hydrogen, C1-6 alkyl or aryl, R25 is hydrogen, and R26 and R27 are independently hydrogen or C1-6 alkyl or, together with the nitrogen atom to which they are attached, form a heteroaryl or heteroaryl ring optionally substituted with hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4 alkoxycarbonyl, carboxy, —CONH2 or —S(O)NH2; —NR20R21, —O(CH2)mNR20R21, —N(CH2)mNR20R21, —O(CH2)mC(O)R22, —N(CH2)mC(O)R22, wherein m is 0, 1, 2 or 3, R20 and R21 are the same or different and independently selected from the group consisting of hydrogen, C1-6 alkyl, cycloalkyl, aryl, carbonyl, acetyl, trihalomethylcarbonyl, carboxy, sulfonyl or trihalomethanesulfonyl, or R20 and R21 together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, and R22 is hydroxy, C1-6 alkoxy, aryloxy, amino, hydroxylamino, carboxy or —NR20R21, wherein R20 and R21 are as indicated above; and
R1′, R2′ and R3′ are the same or different and independently selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR10, —C(O)R10, —C(O)OR10, OC(O)R10, —NR10R11, —C(O)NR10R11, —NHC(O)R10, —SR10, —S(O)R10, —S(O)2R10, —S(O)2NR10R11 and —S(O)OR10, wherein R10 and R11 are the same or different and independently selected from the group consisting of hydrogen, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R10 and R11, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each of C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR12, —C(O)R12, —C(O)OR12, —OC(O)R12, —NR12R13, —C(O)NR12R13, —NHC(O)R12, —SR12, —S(O)R12, —S(O)2R12, —S(O)2NR12R13 and —S(O)OR12, wherein R12 and R13 are the same or different and independently selected from the group consisting of hydrogen, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R12 and R13, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each C1-6-alkyl, C2-16-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl substituent being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, hydroxy, C1-4alkyl, C1-4alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4alkylthio, C1-4alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2, S(O)NH2, aryl, heteroaryl, heterocyclyl or carbocyclyl, said aryl, heteroaryl, heterocyclyl or carbocyclyl being optionally substituted with one or more of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2 or —S(O)NH2; and
pharmaceutically acceptable salts thereof, for the preparation of a medicament for the prevention, treatment or amelioration of multiple sclerosis, or to delay the onset of or reduce the relapse rate in multiple sclerosis.
Compounds of formula II are disclosed in, e.g. WO 99/61422 in which they are indicated to be inhibitors of protein tyrosine kinases and as such useful in the treatment of cancer, diabetes, hepatic cirrhosis, cardiovascular disease, such as atherosclerosis and angiogenesis, immune disease such as autoimmune disease and renal disease. There is no suggestion that compounds within this group might have any utility in the treatment of multiple sclerosis.
In another embodiment, the invention relates to the use of compounds of formula III
wherein R1, R2, R3, R4, R5, R6 and X are as indicated for formula I, and
R1″, R2″, R3″, R4″ and R5″ are the same or different and independently selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR10, —C(O)R10, —C(O)OR10, OC(O)R10, —NR10R11, —C(O)NR10R11, —NHC(O)R10, —SR10, —S(O)R10, —S(O)2R10, —S(O)2NR10R11 and —S(O)OR10, wherein OR10 and R11 are the same or different and independently selected from the group consisting of hydrogen, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R10 and R11, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each of C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, halogen, —OR12, —C(O)R12, —C(O)OR12, —OC(O)R12, —NR12R13, —C(O)NR12R13, —NHC(O)R12, —SR12, —S(O)R12, —S(O)2R12, —S(O)2NR12R13 and —S(O)OR12, wherein R12 and R13 are the same or different and independently selected from the group consisting of hydrogen, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R12 and R13, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each C1-16-alkyl, C2-16-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl substituent being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4 alkoxycarbonyl, carboxy, —CONH2, —S(O)NH2, aryl, heteroaryl, heterocyclyl or carbocyclyl, said aryl, heteroaryl, heterocyclyl or carbocyclyl being optionally substituted with one or more of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2 or —S(O)NH2;
and pharmaceutically acceptable salts thereof, for the preparation of a medicament for the prevention, treatment or amelioration of multiple sclerosis, or to delay the onset of or reduce the relapse rate in multiple sclerosis.
Compounds of formula III are disclosed in WO 96/40116 in which they are indicated to be inhibitors of tyrosine kinases and as such useful in the treatment of cancer, blood vessel proliferative disorders, fibrotic disorders, mesangial cell proliferative disorders and metabolic diseases. There is no suggestion that compounds within this group might have any utility in the treatment of multiple sclerosis.
In a further embodiment, the invention relates to the use of compounds of formula IV
wherein R1, R2, R3, R4, R5, R6 and X are as indicated for formula I,
R1″, R2″, R3″, R4″ and R5″ are the same or different and independently selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR10, —C(O)R10, —C(O)OR10, OC(O)R10, —NR10R11, —C(O)NR10R11, —NHC(O)R10, —SR10, —S(O)R10, —S(O)2R10, —S(O)2NR10R11 and —S(O)OR10, wherein R10 and R11 are the same or different and independently selected from the group consisting of hydrogen, C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R10 and R11, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each of C1-12-alkyl, C2-12-alkenyl, C4-12-alkadienyl, C6-12-alkatrienyl, C2-12-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, halogen, trihalomethyl, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, hydroxy, carboxy, formyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, amino, carbamoyl, cyano, guanidino, carbamido, —OR12, —C(O)R12, —C(O)OR12, —OC(O)R12, —NR12R13, —C(O)NR12R13, —NHC(O)R12, —SR12, —S(O)R12, —S(O)2R12, —S(O)2NR12R13 and —S(O)OR12, wherein R12 and R13 are the same or different and independently selected from the group consisting of hydrogen, C1-6-alkyl, C2-6-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl, or wherein R12 and R13, together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, each C1-16-alkyl, C2-16-alkenyl, C4-6-alkadienyl, C2-6-alkynyl, aryl, heteroaryl, carbocyclyl and heterocyclyl substituent being optionally substituted with one or more, same or different substituents selected from the group consisting of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4 alkoxycarbonyl, carboxy, —CONH2, —S(O)NH2, aryl, heteroaryl, heterocyclyl or carbocyclyl, said aryl, heteroaryl, heterocyclyl or carbocyclyl being optionally substituted with one or more of hydrogen, hydroxy, C1-4 alkyl, C1-4 alkoxy, nitro, cyano, amino, oxo, halogen, trihalomethyl, C1-4 alkylthio, C1-4 alkylamino, C1-4alkoxycarbonyl, carboxy, —CONH2 or —S(O)NH2; and
R6″ is hydrogen, heterocyclyl, heteroaryl, —C(O)R23, —S(O)2R23, —C(O)OR23 or C1-6 alkyl optionally substituted with heterocyclyl, heteroaryl or —C(O)OR23, wherein R23 is hydrogen, C1-6 alkyl, aryl, heteroaryl or heterocyclyl;
and pharmaceutically acceptable salts thereof, for the preparation of a medicament for the prevention, treatment or amelioration of multiple sclerosis, or to delay the onset of or reduce the relapse rate in multiple sclerosis.
Compounds of formula IV are disclosed in WO 98/07695 in which they are indicated to be inhibitors of tyrosine kinases and as such useful in the treatment of cell proliferative diseases such as cancer, atherosclerosis, arthritis and restenosis, and metabolic diseases such as diabetes. There is no suggestion that compounds within this group might have any utility in the treatment of multiple sclerosis.
In the compound of formula I,
X may be O or S;
R1, R2, R3 and R4 may be the same or different and independently selected from the group consisting of hydrogen, C1-10 alkyl, C1-10 alkoxy, aryl, heteroaryl, aryloxy, C1-10 alkylaryl, C1-10 alkylaryloxy, halogen, trihalomethyl, S(O)R18, S(O)2R18, S(O)2NR18R19, S(O)3R18, SR18, NO2, NR18R19, OH, CN, C(O)R18, C(O)OR18, OC(O)R18, NHC(O)R18, (CH2)nC(O)2R18 and C(O)NR18R19, wherein R18 is hydrogen, C1-6 alkyl, heteroaryl or aryl, said C1-6 alkyl, heteroaryl or aryl being optionally substituted with hydroxy or NR26R27, wherein R26 and R27 are independently hydrogen or C1-6 alkyl or, together with the nitrogen atom to which they are attached, form a heteroaryl or heterocyclic ring, R19 is hydrogen, C1-6 alkyl or aryl, and n is 0-3;
A may be phenyl or a monoclyclic or bicyclic heteroaryl ring selected from the group consisting of pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, tetrazole and indole, optionally substituted at one or more positions with C1-10 alkyl, C1-10 alkoxy, aryl, heteroaryl, aryloxy, C1-10 alkylaryl, C1-10 alkylaryloxy, halogen, trihalomethyl, a sugar residue, S(O)R18, S(O)2R18, S(O)2NR18R19, S(O)3R18, SR18, NO2, NR18R19, OH, CN, CH2OH, C(O)R18, C(O)OR18, OC(O)R18, NHC(O)R18, (CH2)nC(O)2R18 and C(O)NR18R19, wherein R18, R19 and n are as indicated above;
R5 may be hydrogen or C1-6 alkyl; and
R6 may be hydrogen.
More specifically, in the compound of formula I, R5 is preferably hydrogen; X is preferably oxygen; R1, R2, R3 and R4 are preferably the same or different and independently selected from hydrogen and C1-6 alkyl, R6 is preferably hydrogen or COOH; and/or A is pyrrole, phenyl or indole, said pyrrole, phenyl or indole being optionally substituted at one or more positions with C1-10 alkyl, C1-10 alkoxy, aryl, heteroaryl, aryloxy, C1-10 alkylaryl, C1-10 alkylaryloxy, halogen, trihalomethyl, a sugar residue, S(O)R18, S(O)2R18, S(O)2NR18R19, S(O)3R18, SR18, NO2, NR18R19, OH, CN, CH2OH, C(O)R18, C(O)OR18, OC(O)R18, NHC(O)R18, (CH2)nC(O)2R18 and C(O)NR18R19, wherein R18, R19 and n are as indicated above. In particular, A is pyrrole substituted at position 3 and 5 with C1-6 alkyl, or or at position 3 with C1-6 alkyl and at position 5 with CH2OH, COOH or a sugar residue, or at position 3 and 5 with C1-6alkyl and at position 4 with halogen, or at position 5 with C(O)O—C1-6 alkyl, and at position 5 with C1-6 alkyl. Alternatively, A is phenyl substituted at position 2 and 5 with C1-6 alkyl, C1-6 alkoxy, halogen, C1-6alkyl-NR26R27, NH—C1-6alkyl-NR26R27 or O—C1-6alkyl-NR26R27, wherein R26 and R27 are as indicated above.
In the compound of formula II, R1, R2, R3, R4, R6 and X are preferably as indicated above, and R1′, R2′ and R3′ are preferably the same or different and independently selected from the group consisting of with C1-10 alkyl, C1-10 alkoxy, aryl, heteroaryl, aryloxy, C1-10 alkylaryl, C1-10 alkylaryloxy, halogen, trihalomethyl, a sugar residue, S(O)R18, S(O)2R18, S(O)2NR18R19, S(O)3R18, SR18, NO2, NR18R19, OR18, CN, CH2OH, C(O)R18, C(O)OR18, OC(O)R18, NHC(O)R18, (CH2)nC(O)2R18 and C(O)NR18R19, wherein R18 is hydrogen, C1-6 alkyl, heteroaryl or aryl, said C1-6 alkyl, heteroaryl or aryl being optionally substituted with hydroxy or NR26R27, wherein R26 and R27 are independently hydrogen or C1-6 alkyl or, together with the nitrogen atom to which they are attached, form a heteroaryl or heterocyclic ring, R19 is hydrogen, C1-6 alkyl or aryl, and n is 0-3.
In a currently favoured embodiment of the compound of formula II, R8 and R4′ are independently hydrogen, hydroxy, —PO(OR)(OR′), —OR10, —C(O)OR10, —C(O)NR10R11, —C(O)R14, —C(R24R25)OR16, —OC(O)R16 or —C(R24R25)NR26R27, wherein R, R′, R10, R11, R14, R16, R24, R25, R26, R27 are as indicated above.
More specifically, in the compound of formula II, R1, R2, R3 and R4 are preferably the same or different and independently selected from hydrogen, halogen and C1-6 alkyl, or R2 may be hydroxy or heteroaryl, such as pyridyl, or a group C(O)R20, wherein R20 is heteroaryl, such as pyridyl or thienyl, and R1, R3 and R4 are hydrogen. Furthermore, in the compound of formula II, R1′, R2′ and R3′ are preferably the same or different and independently selected from hydrogen, halogen, C1-6 alkyl, C1-6 alkoxy, CH2OH or C(O)OR18 or C(O)NR18R19, wherein R18 and R19 are as defined above. By way of example, in the compound of formula II, R1′ and R3′ may both be C1-6 alkyl, in particular methyl, and R2′ may be hydrogen, or R1′ may be C1-6 alkyl and R3′ may be C1-6 alkoxy, CH2OH, C(O)OR18 or C(O)NR18R19, or R1′ and R3′ may both be C1-6 alkyl, in particular methyl, and R2′ may be halogen, in particular chloro or bromo, or R1′ may be C1-6 alkyl and R3′ may be C(O)O—C1-6 alkyl, or R1′ may be C1-6 alkyl and R3′ may be C(O)NH—C1-6 alkyl substituted with hydroxy.
Examples of compounds of formula II are selected from the group consisting of
In the compound of formula III, R2, R3, R4, R5, R6 and X may be as indicated above, and R1″, R2″, R3″, R4″ and R5″ are preferably the same or different and independently selected from the group consisting of with C1-10 alkyl, C1-10 alkoxy, aryl, heteroaryl, aryloxy, C1-10 alkylaryl, C1-10 alkylaryloxy, halogen, trihalomethyl, a sugar residue, S(O)R18, S(O)2R18, S(O)2NR18R19, S(O)3R18, SR18, NO2, NR18R19, OR18, CN, CH2OH, C(O)R18, C(O)OR18, OC(O)R18, NHC(O)R18, (CH2)nC(O)2R18 and C(O)NR18R19, wherein R18 is hydrogen, C1-6 alkyl, heteroaryl or aryl, said C1-6 alkyl, heteroaryl or aryl being optionally substituted with hydroxy or NR26R27, wherein R26 and R27 are independently hydrogen or C1-6 alkyl or, together with the nitrogen atom to which they are attached, form a heteroaryl or heterocyclic ring, R19 is hydrogen, C1-6 alkyl or aryl, and n is 0-3.
More specifically, in the compound of formula III, R2″ and R5″ are preferably the same or different and independently are C1-6 alkyl, in particular methyl, or C1-6alkoxy, in particular methoxy, or halogen, in particular chloro or bromo. By way of example, in the compound of formula III, R5 may be hydrogen, hydroxy or C(O)R14 or C(O)OR14, wherein R14 is as defined above.
Examples of compounds of formula III are selected from the group consisting of
In the compound of formula IV, R2, R3, R4, R5, R6 and X are preferably as indicated above, and R1″, R2″, R3″, R4″ and R5″ are preferably the same or different and independently selected from the group consisting of with C1-10 alkyl, C1-10 alkoxy, aryl, heteroaryl, aryloxy, C1-10 alkylaryl, C1-10 alkylaryloxy, halogen, trihalomethyl, a sugar residue, S(O)R18, S(O)2R18, S(O)2NR18R19, S(O)3R18, SR18, NO2, NR18R19, OR18, CN, CH2OH, C(O)R18, C(O)OR18, OC(O)R18, NHC(O)R18, (CH2)nC(O)2R18 and C(O)NR18R19, wherein R18 is hydrogen, C1-6 alkyl, heteroaryl or aryl, said C1-6 alkyl, heteroaryl or aryl being optionally substituted with hydroxy or NR26R27, wherein R26 and R27 are independently hydrogen or C1-6 alkyl or, together with the nitrogen atom to which they are attached, form a heteroaryl or heterocyclic ring, R19 is hydrogen, C1-6 alkyl or aryl, and n is 0-3; and R6″ is hydrogen, C1-6 alkyl, heteroaryl, heteroaryl-C1-6 alkyl, C(O)R18, C(O)OR18 or S(O)2R18, wherein R18 is as indicated above.
More specifically, in the compound of formula IV, R5″ may be hydrogen or C1-6 alkyl and/or R6″ may be hydrogen or C1-6 alkyl.
Examples of compounds of formula IV are selected from the group consisting of
Compounds of formula I are generally lipophilic in nature which makes the compounds sparingly soluble in water and consequently difficult to formulate in, for instance, parenteral, injectable compositions where isotonic saline is used as the solvent. To provide an adequate solubility of the compounds, they may advantageously be provided in the form of prodrugs. The term “prodrug” is intended to indicate a derivative of an active compound which does not, or does not necessarily, exhibit the physiological activity of the active compound, but which may be subjected to enzymatic cleavage such as hydrolysis in vivo so as to release the active compound on administration of the prodrug. In this particular instance, the prodrug comprises the active compound which in itself is highly lipophilic provided with a side chain with predominantly hydrophilic properties imparting improved solubility characteristics to the prodrug, thereby making it more suitable for parenteral administration in the form of a solution or for oral administration to obtain an improved bioavailability. Examples of prodrugs are compounds of formula II wherein R8, apart from being hydrogen or alkyl, is a group —PO(OR)(OR′), wherein R and R′ are independently selected from hydrogen or C1-6 alkyl, —C(O)OR10, —C(O)NR10R11, wherein R10 and R11 are as defined above, —CH2-aryl-OR14, —C(O)R14, wherein R14 is hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, cycloalkyl or aryl, —C(R24R25)—OR16 or —OC(O)R16, wherein R16 and R17 are the same or different and independently selected from the group consisting of hydrogen, C1-6 alkyl, aralkyl, acyl or —PO(OR)(OR′), or wherein R16 and R17 together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring, or —C(R24R25)—NR26R27, wherein R24 is hydrogen, C1-6 alkyl or aryl, R25 is hydrogen, and R26 and R27 are independently hydrogen or C1-6 alkyl or, together with the nitrogen atom to which they are attached, form a heteroaryl or heterocyclic ring. Such prodrugs and other, similar prodrugs may suitably be prepared by a procedure described in WO 01/90068, WO 01/90103, WO 01/90104 and WO 02/081466, the disclosures of which are hereby incorporated by reference in their entirety.
Currently favoured compounds of formula I are 3-(3,5-dimethyl-1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one (Compound 226), 3-(1H-indol-3-ylmethylene)-1,3-dihydroindol-2-one (Compound 57) and 3-(2,5-dimethoxy-benzylidene)-1,3-dihydroindol-2-one (Compound 110) as well as derivatives thereof, e.g. the compounds shown in Table 4 or Table 7 below. These compounds have surprisingly been found to exhibit certain properties making them particularly favourable for the present purpose: they are highly lipophilic (logP 4.7) which might enable them to cross the blood-brain barrier, and when tested on a number of different cytokines, they have been found to be potent inhibitors of the proinflammatory cytokines IL-2, IL-6, INF-γ and IL-17 (at a concentration of 10−10 M) while not inhibiting cytokines such as TNF-α. In case of the latter, it is interesting to note that a trial where anti-TNF antibodies were used in the treatment of multiple sclerosis actually led to an exacerbation of the symptoms (Karni and Weiner, supra). It would therefore appear to be an advantage to provide a treatment of multiple sclerosis which does not involve inhibition of TNF-α.
As indicated above, compounds of formula I have been described as tyrosine kinase inhibitors and, in particular, the compound 3-(3,5-dimethyl-1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one (Compound 226) has been in development as an inhibitor of the vascular endothelial growth factor receptor KDR for the treatment of cancer. The compound has also been found to inhibit the p60c tyrosine kinase (L. Sun et al., J. Med. Chem. 43, 2000, p. 2655). In this context, it is interesting to note that compounds derived from a benzylidene malononitrile scaffold, termed tyrphostins, have been found to to be effective in the EAE model. Tyrphostins are known tyrosine kinase inhibitors. In particular, tyrphostin B42 (also termed AG490) has been found to inhibit IL-12 induced tyrosine phsophorylation and activation of JAK-2 kinase (cf. J. J. Bright et al., J. Immunol. 162, 1999, pp. 6255-6262). Furthermore, tyrphostin AG490 has been found to be an effective inhibitor of lymphocyte adhesion to inflamed vessels (cf. G. Constantin et al., J. Immunol. 162, 1999, pp. 1144-1149). It is suggested that the ability of AG490 to block the development of EAE may be ascribed, at least in part, to its ability to inhibit lymphocyte adhesion.
It is recognised that tyrosine kinases play an important role in the regulation of cell signalling by phosphorylating tyrosine residues of proteins and peptides, and that excessive activation of tyrosine kinases may lead to the development of various diseases of the immune system. In this regards, members of the src kinase family have been found to be of interest, in particular the p56lck kinase which is only expressed in T-cells and which is crucial for T-cell receptor mediated signal transduction, eventually leading to production of proinflammatory cytokines, including IL-2. It has been found that T-cells which lack the p56lck kinase cannot signal through the T-cells receptor (D. B. Straus and A. Weiss, Cell 70, 1992, p. 585). As other tyrosine kinases are seemingly unable to compensate for blocking p56lck activity (M. R. Myers et al., Curr. Pharm. Design 3, 1997, p. 473), this kinase may be an attractive target for therapeutic intervention.
It is well established that T-cells contribute to the development of several chronic inflammatory and autoimmune diseases. Initially in the disease process, naïve T-cells are activated by antigens and produce the proinflammatory cytokine interleukin-2 (IL-2) leading to clonal expansion and production of other inflammatory cytokines involved in the generation of the inflammatory or autoimmune response. Excessive T-cell activity is involved in allergies and immunoinflammatory diseases such as asthma, psoriasis, rheumatoid arthritis and multiple sclerosis. IL-2 has been found to have an important role in promoting the growth of T-cells in that it is a growth factor for both CD4+ and CD8+ T-cells as well as natural killer cells. Furthermore, IL-2 influences the differentiation of T helper cells into Th1 and Th2 cells and potentiates the production of cytokines by each cell type. IL-2 appears to be initially produced by activated CD4+ T-cells, inducing proliferation of CD8+ T-cells and production of proinflammatory cytokines such as IL-1, IL-6 and TNF-α. Thus, IL-2 is a key factor in the primary cellular immune response and, as such, it may be an attractive target for therapeutic intervention, such as in antiinflammatory or immunomodulatory therapy.
Proinflammatory cytokines produced by activated T-cells in the central nervous system are important factors in the demyelination process characteristic of multiple sclerosis (cf. B. Gran and A. Rostami, supra). Proinflammatory cytokines are believed to participate directly in myelin destruction and axonal damage (O'Connor et al., supra) and also to play a role in the upregulation of MHC class II molecules on astrocytes and microglia as well as adhesion molecules on the blood-brain barrier endothelium, facilitating the further influx of T-cells, B-cells and macrophages in the central nervous system (Hellings et al., supra). Such cytokines may also be attractive targets for therapeutic intervention.
The present inventors have indeed established a correlation between in vitro IL-2 inhibition and significant activity of compounds of formula I in the EAE model. Out of 50 compounds tested in vitro in an IL-2 assay (e.g. as described in example 7 below), 45% of those compounds that inhibited IL-2 with a pIC50 (−log IC50) value of 6 (i.e. 10−6 M) or more were also found to exhibit significant inhibitory activity in the EAE model, 50% of those compounds that inhibited IL-2 with a pIC50 value of 7 (i.e. 10−7 M) or more were also found to exhibit significant inhibitory activity in the EAE model, and 61% of those compounds that inhibited IL-2 with a pIC50 value of 8 (i.e. 10−8 M) or more were found to exhibit significant inhibitory activity against EAE. Conversely, only 38% of all 50 tested compounds, when not sorted by IL-2 inhibitory activity, showed any significant inhibitory activity in the EAE model. None of the compounds that did not significantly inhibit IL-2 in the assay showed any significant inhibitory activity in the EAE model. Thus, the in vitro IL-2 assay may be a useful tool when screening compounds that might exhibit activity in vivo in the EAE model.
In a further embodiment, the invention therefore relates to the use of a compound of general formula I capable of inihibiting the production of proinflammatory cytokines, in particular IL-2, by T-cells or capable of blocking a cytokine receptor for the preparation of a medicament for the prevention, treatment or amelioration of multiple sclerosis, or to delay the onset of or reduce the relapse rate in multiple sclerosis. Examples of such compounds are 3-(3,5-dimethyl-1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one and 3-(1H-indol-3-ylmethylene)-1,3-dihydro-indol-2-one) which, as discussed above, have been found to inhibit expression of IL-2, IL-6, INF-γ and IL-17 (cf. example 6 and
When spleen cell cDNA derived from EAE mice treated with 3-(3,5-dimethyl-1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one and untreated EAE mice was tested in an oligonucleotide array of more than 5000 mouse genes (such as Atlas Plastic Mouse 5 K MicroArray, available from BD Biosciences, California, USA), a pattern emerged in cells from treated mice of downregulation of genes involved in the inflammatory response and upregulation of genes encoding adhesion molecules, suggesting that maturation and activation of inflammatory cells is retarded and consequently their migration into the CNS is reduced in the treated mice.
Pharmaceutical Compositions
For use in the present invention, the active ingredient may be formulated into a pharmaceutical composition together with a pharmaceutically acceptable vehicle and optionally one or more other therapeutic ingredients. The vehicle must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulation may be in a form suitable for oral or parenteral (including subcutaneous, intramuscular, interperitoneal, intraarticular and intravenous) administration.
The formulations may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy, e.g. as disclosed in Remington, The Science and Practise of Pharmacy, 20th Ed., 2000. All methods include the step of bringing the active ingredient into association with the vehicle which constitutes one or more auxiliary constituents. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid vehicle or a finely divided solid vehicle or both, and then, if necessary, shaping the product into the desired formulation.
The term “dosage unit” is understood to mean a unitary, i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active ingredient as such or a mixture of it with solid or liquid pharmaceutical vehicle materials.
Unlike IFN-β and glatiramer acetate which are peptidic in nature and only suitable for parenteral administration, such by injection, the compounds of formula I are small organic molecules and may therefore be administered orally. This represents a clear benefit for the patient as it permits self-medication and is less painful than for instance injections of IFN-β which often cause pain at the site of injection. Compounds of formula I, in particular compounds of formula II and III, have surprisingly exhibited an acceptable oral bioavailability and EAE inihibitory activity, cf. Table 7 below, an may therefore be suitable for oral administration.
Formulations suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid, such as ethanol or glycerol; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. Such oils may be edible oils, such as e.g. cottonseed oil, sesame oil, coconut oil or peanut oil. Suitable dispersing or suspending agents for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose and polyvinylpyrrolidone. The active ingredient may also be administered in the form of a bolus, electuary or paste.
A tablet may be prepared by compressing or moulding the active ingredient optionally with one or more auxiliary constituents. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient(s) in a free-flowing form such as a powder or granules, optionally mixed by a binder, such as e.g. lactose, glucose, starch, gelatine, acacia gum, tragacanth gum, sodium alginate, carboxymethylcellulose polyethylene glycol, waxes or the like; a lubricant such as e.g. sodium oleate, sodium stearate, magnesium steatrate, sodium benzoate, sodium acetate, sodium chloride or the like; a disintegrating agent such as e.g. starch, methyl cellulose, agar, bentonite, xanthan gum or the like or dispersing agent. Moulded tablets may be made by moulding, in a suitable machine, a mixture of the powdered active ingredient and suitable carrier moistened with an inert liquid diluent.
Formulations suitable for parenteral administration may conveniently comprise a sterile oily or aqueous preparation of the active ingredients, which is preferably isotonic with the blood of the recipient, e.g. an isotonic saline, isotonic glucose solution or buffer solution. Liposomal formulations may also be used to present the active ingredient for parenteral administration. The formulation may conveniently be sterilised by for instance filtration through a bacteria retaining filter, addition of sterilising agent to the formulation, irradiation of the formulation or heating of the formulation.
Alternatively, the formulation may be provided as a sterile, solid preparation, e.g. a freeze-dried powder, which is readily dissolved in a sterile media immediately prior to use.
In addition to the aforementioned ingredients, the formulations comprising a compound of formula I may include one or more additional ingredients such as diluents, buffers, flavouring agents, colourants, surface active agents, thickeners, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like.
In addition to the formulations described above, compounds of formula I may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the active ingredient may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in a pharmaceutically acceptable oil), or an ion exchange resin.
In a still further aspect, the present invention relates to a method of preventing, treating or ameliorating multiple sclerosis, or delaying the onset of or reducing the relapse rate in multiple sclerosis, the method comprising administering, to a patient in need thereof, a pharmacologically effective amount of a compound of general formula I. The invention further relates to a method of preventing, treating or ameliorating multiple sclerosis, or delaying the onset of or reducing the relapse rate in multiple sclerosis, the method comprising administering, to a patient in need thereof, a pharmacologically effective amount of a compound capable of inhibiting the production of one or more proinflammatory cytokines, such as IL-2, by CD4+ Th1 cells, as discussed above.
For systemic treatment according to the present invention, daily doses of from 0.001-100 mg/kg body weight, preferably from 0.002-15 mg/kg body weight, for example 0.003-10 mg/kg of a compound of formula I or II are administered, typically corresponding to a daily dose for an adult human of from 0.2 to 750 mg of the active ingredient. Oral compositions are formulated, preferably as tablets, capsules, or drops, containing from 0.05-250 mg, preferably from 0.1-125 mg, of a compound of formula I per dosage unit.
The invention is further described in the following examples which are not in any way intended to limit the scope of the invention as claimed.
The compounds of general formula I can be prepared in a number of ways well known to those skilled in the art of organic synthesis. The compounds of formula I can be synthesised using the methods outlined below, together with methods known in the art of synthetic organic chemistry, or variations thereof as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The compounds of formula I may be prepared using the reactions and techniques described in this section. The reactions are performed in solvents appropriate to the reagents and materials employed and suitable for the transformations being effected. Also, in the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of experiment and work-up procedures, are chosen to be conditions of standard for that reaction, which should be readily recognised by one skilled in the art. It is understood by one skilled in the art of organic synthesis that the functionalities present on various portions of the starting molecules in a reaction must be compatible with the reagents and reactions proposed. Not all compounds of formula I falling into a given class may be compatible with some of the reaction conditions required in some of the methods described. Such restrictions to the substituents which are compatible with the reaction conditions will be readily apparent to one skilled in the art and alternative methods can be used.
The compounds of formula I can be prepared by techniques and procedures readily available to one of ordinary skill in the art, for example by following the procedures as set forth in the following Schemes. These Schemes are not intended to limit the scope of the invention in any way. All substituents, unless otherwise indicated, are previously defined. The reagents and starting materials are readily available to one of ordinary skilled in the art.
General Method A
Some of the compounds of general formula II can be prepared according to the general method A described below.
Some of the compounds of general formula I can be prepared according to the general method B described below.
Some of the compounds of general formula I can be prepared according to the general method C described below.
Smith and 1. March chapter 10-23 p. 484, chapter 10-26 p. 488 and chapter 10-28 p. 490) such as acid catalysed esterification (e.g. using sulphuric acid as a catalyst in MeOH), or such as base catalyzed esterification (e.g. using chlorosulfates as reagents under phase transfer conditions, see Synthetic Communications, 14 (9), 857-864, 1984) or such as using diazomethane in a suitable solvent such as Et2O or EtOH, or using trimethylsilyldiazomethane in a suitable solvent such as toluene, or using an electrophilic reactant (as for example benzyl bromide or methyliodide) in the presence of a base such as K2CO3 or Cs2CO3 in a solvent such as DMF or acetonitrile.
General Method D
Some of the compounds of general formula II can be prepared according to the general method D described below.
Some of the compounds of general formula IV can be prepared according to the general method E described below.
Some of the compounds of general formula III can be prepared according to the general method F described below.
The alkyl- or dialkyl aminomethyl-benzaldehydes can be prepared in two steps:
Some of the compounds of general formula III can be prepared according to the general method G described below.
Some of the compounds of general formula I can be prepared according to the general method H described below.
Some of the compounds of general formula I can be prepared according to the general method I described below.
General Method J
Some of the compounds of general formula I can be prepared according to the general method J described below.
Thurston, I., Synthetic Communication, 20 (14), 2133-2138 (1990)].
General Method K
Some of the compounds of general formula II can be prepared according to the general method K described below.
Some of the compounds of general formula I can be prepared according to the general method L described below.
For 1H nuclear magnetic resonance (NMR) spectra (300 MHz) and 13C NMR (75.6 MHz) chemical shift values (δ) (in ppm) are quoted for dimethyl-d6 sulfoxide (DMSO-d6) solutions relative to internal tetramethylsilane (δ=0) standard. The value of a multiplet, either defined (doublet (d), triplet (t), quartet (q)) or not (m) at the approximate mid point is given unless a range is quoted, (bs) indicates a broad singlet. The ES mass spectra were obtained on a VG Quattro II triple quadrapole mass spectrometer (Micromass, Manchester, UK) operating in either positive or negative electrospray mode with a cone voltage of 30V.
The compounds of the present invention can exist in two isomeric forms: the Z and the E isomeric forms. The NMR data characterize the isomer forms that are present in the solvent used to record the NMR spectrum and determine their molar ratio. For NMR solutions where both the E-isomer and the Z-isomer are present in equal or close to equal amounts, the chemical shifts of both forms are given. For NMR solutions where the equilibrium is shifted in favour of one form, the chemical shifts of the dominating form are given.
The organic solvents used were anhydrous unless otherwise specified. Flash chromatography was performed on silica gel.
The following abbreviations have been used throughout:
To a solution of (chloro-methylen)-dimethyl-ammonium-chloride (1.64 g, 12.8 mmoles) in dry DCE (6 mL) was added dropwise under argon atmosphere a solution of 4-methyl-1H-pyrrole-2-carboxylic acid ethyl ester (1.31 g, 8.6 mmoles) in DCE (5 mL). The reaction mixture was heated slightly to dissolve all solid particules and stirred at room temperature overnight. TLC indicated that there was no starting material left. The pH of the reaction mixture was adjusted to 10 by addition of NaOH (2 N). The crude mixture was extracted with CH2Cl2 (3×10 mL). The combined organic phases were washed once with H2O, once with brine, dried over MgSO4 and filtered. After removal of the solvent, 1.49 g of the title compound (99% yield) were obtained as a pale red/orange solid and used in the next step without further purification.
1H NMR (DMSO-d6) δ 12.6 (br, 1H), 9.80 (s, 1H), 6.70 (s, 1H), 4.27 (q, 2H), 2.30 (s, 3H), 1.30 (t, 3H).
A solution of aldehyde (1 eq.), 1,3-dihydro-indol-2-one (1 eq.) and piperidine (catalytic amount) in EtOH is heated to reflux overnight. The crude mixture is allowed to come to room temperature, cooled in an ice bath. A precipitate is obtained which is filtered and washed with cold EtOH. If no precipitation occurs, the solvent is removed and the product is purified by flash chromatography.
The general procedure 1 was followed using a mixture of 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid ethyl ester (0.93 g, 7 mmoles), 1,3-dihydro-indol-2-one (1.27 g, 7 mmoles) and piperidine (few drops) in EtOH (25 mL). After filtration, 1.98 g of the title compound as orange crystals were obtained (99% yield).
13C-NMR (DMSO-d6, Z isomer) δ 169.0, 159.7, 139.4, 129.9, 128.8, 128.0, 124.5, 124.3, 122.5, 121.4, 120.7, 119.6, 116.4, 109.7, 60.3, 14.2, 11.0.
A mixture of 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid ethyl ester (0.46 g, 1.57 mmoles) K2CO3 (0.35 g, 2.52 mmoles) and ethanolamine (28 mL, 466 mmoles) in acetonitrile (14 mL) was heated at 105° C. for 1 hour (the reaction was followed by TLC). The mixture was allowed to come to room temperature, poured into H2O, extracted with EtOAc (3×20 mL). The combined organic phases were washed once with H2O, once with brine and dried over MgSO4. After filtration and removal of the solvent, 0.42 g of the title compound were obtained as an orange solid (85% yield).
1H NMR (DMSO-d6) δ 13.64 (s, 1H), 10.89 (br, 1H), 8.32 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.01 (t, 1H), 6.88 (d, 1H), 6.83 (d, 1H), 4.73 (t, 1H), 3.50 (m, 2H), 3.3 (m, 2H), 2.35 (s, 3H).
To a dry solution of 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid ethyl ester (1 g, 3.38 mmoles) in dry toluene (20 mL) was added under argon and at −78° C. a solution 1 M of DIBAL-H in toluene (34 mL). The reaction was followed by TLC. After 1.5 hours, extra amounts (17 mL and 10 mL) of the solution of DIBAL-H (1 M in toluene) were added at −78° C. and the temperature was allowed to raise to −40° C.
MeOH (60 mL) was added at −75° C. and the mixture was allowed to come to room temperature. After addition of saturated aqueous NH4Cl solution (40 mL), the crude mixture was extracted with EtOAc (3×60 mL). The combined organic phases were washed once with H2O, once with brine and dried over MgSO4. After filtration and removal of the solvent, 0.88 g of 3-(5-hydroxymethyl-3-methyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one. After crystallization from EtOAc and petroleum ether, 0.58 g of pure title compound were obtained (68% yield) as orange crystals.
13C-NMR (DMSO-d6, Z isomer) δ 169.2, 140.0, 138.3, 130.7, 126.4, 125.9, 125.7, 123.3, 120.7, 118.1, 113.6, 110.2, 109.2, 56.6, 11.3.
To a solution of 3-(5-hydroxymethyl-3-methyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (50 mg, 0.2 mmole) in pyridine (0.5 mL) cooled in an ice bath was added dropwise 4-methyl-benzenesulfonyl chloride (41.7 mg, 0.22 mmole). The mixture was stirred at 0° C. for 2 hours. H2O (3 mL) was added to the crude mixture and the aqueous phase was extracted with EtOAc (3×3 mL). A precipitate formed in the aqueous phase which was filtered to afford 35 mg of the title compound as an orange solid (50% yield).
13C-NMR (DMSO-d6, Z isomer) δ 169.2, 146.1, 144.7, 138.9, 129.7, 129.1, 128.4, 128.4, 127.0, 124.9, 123.2, 121.1, 119.0, 117.1, 113.7, 109.6, 56.7, 11.1.
A solution of the ester (1 eq.) in a 1:1 mixture of THF or MeOH and aqueous solution of LiOH is heated under reflux. The reaction is followed by TLC. When TLC indicates full conversion the pH of the solution is adjusted to acidic pH (6-7) by addition of HCl (1N). A precipitate forms which is filtered and dried furnishing the desired product.
The general procedure 2 was followed using a solution of 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid ethyl ester (2.8 g, 9.5 mmoles) in a mixture of THF (150 mL) and an aqueous solution of LiOH (1N). After heating at reflux for 4 hours, the pH of the reaction was adjusted to acidic pH by addition of an aqueous solution of HCl 1 N (about 150 mL). A bright orange precipitate formed which was filtered to afford the title compound as an orange solid (2.47 g, 97% yield).
13C-NMR (DMSO-d6, Z isomer) δ 169.2, 161.3, 139.6, 129.7, 129.0, 128.0, 125.6, 124.8, 122.7, 121.5, 120.5, 119.6, 116.4, 109.8, 11.1.
To a mixture of 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (1 eq.), EDCI (1.5 eq.), HOBt (1.6 eq.), Et3N (2 eq. or 4 eq.) in DMF is added the corresponding amine as a free base or as a salt (2 eq.). The crude mixture is then stirred or shaked at room temperature overnight. After partial removal of the solvent, a saturated aqueous solution of Na2CO3 is added slowly. A precipitate forms. It is filtered and if necessary purified by flash chromatography.
The title compound was obtained by following the general procedure 3 using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (0.94 g, 3.5 mmoles), EDCI (1.01 g, 5.25 mmoles), HOBt (0.756 g, 5.6 mmoles), Et3N (1 mL, 7 mmoles) in DMF (12 mL) and adding N,N-diethyl-ethane-1,2-diamine (1 mL, 7 mmoles). After filtration 0.45 g of the title compound were obtained as an orange solid (35% yield).
13C-NMR (DMSO-d6, Z isomer) δ 169.0, 159.4, 139.2, 129.5, 129.1, 128.2, 127.4, 124.9, 122.8, 121.1, 119.2, 118.6, 112.1, 109.5, 51.4, 46.7, 37.0, 11.7, 11.3.
The title compound was obtained by following the general procedure 3 using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (0.94 g, 3.5 mmoles), EDCI (1.01 g, 5.25 mmoles), HOBt (0.766 g, 5.7 mmoles), Et3N (1 mL, 7 mmoles) in DMF (12 mL) and adding 2-methoxy-ethylamine (0.61 mL, 7 mmoles). The reaction was followed by LC/MS. After addition of H2O (10 mL), a precipitate formed. After filtration, the solid was suspended in water and extracted with CH2Cl2 (3×20 mL). The organic phases were combined and concentrated in vacuo to obtain 0.68 g of the title compound as an orange solid (60% yield).
1H NMR (DMSO-d6) δ 13.65 (s, 1H), 10.91 (s, 1H), 8.40 (t, 1H), 7.80 (d, 1H), 7.65 (s, 1H), 7.17 (t, 1H), 7.00 (t, 1H), 6.89 (d, 1H), 6.84 (d, 1H), 3.43 (m, 4H), 3.27 (s, 3H), 2.35 (s, 3H).
To a mixture of 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (1 eq.), EDCI (72 mg, 0.37 mmole, 1.5 eq.), HOBT (51 mg, 0.37 mmole, 1.5 eq.), Et3N (0.07 mL or 0.14 mL, 0.5 or 1 mmole, 2 or 4 eq.) in DMF (0.8 mL) is added the corresponding amine as a free base or a salt (2 eq.). The crude mixture is shaked or stirred at room temperature overnight. After removal of DMF under high vacuum, the crude is purified by flash chromatography using MeOH/CH2Cl2 mixtures as eluent.
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), N-formyl-4-(3-aminopropyl)-piperidin; hydrochloride (103 mg, 0.5 mmole) and Et3N (0.14 mL, 1 mmole, 4 eq.). After purification by flash chromatography, 87 mg of the title compound were obtained as an orange solid (84% yield).
1H NMR (DMSO-d6) δ 13.63 (s, 1H), 10.91 (s, 1H), 8.33 (t, 1H), 7.96 (s, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.00 (t, 1H), 6.88 (d, 1H), 6.81 (d, 1H), 4.14 (m, 1H), 3.64 (m, 1H), 3.23 (q, 2H), 2.98 (m, 1H), 2.56 (m, 1H), 2.35 (s, 3H), 1.70 (m, 2H), 1.52 (m, 3H), 1.25 (m, 2H), 1.10-0.80 (m, 2H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 4-amino-butyric acid methyl ester; hydrochloride (77 mg, 0.5 mmole) and Et3N (0.14 mL, 1 mmole, 4 eq.). After purification by flash chromatography, 60 mg of the title compound were obtained as an orange solid (65% yield).
1H NMR (DMSO-d6) δ 13.64 (s, 1H), 10.91 (s, 1H), 8.36 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.17 (t, 1H), 7.01 (t, 1H), 6.89 (d, 1H), 6.81 (d, 1H), 3.59 (s, 3H), 3.26 (q, 2H), 2.37 (t, 2H), 2.35 (s, 3H), 1.77 (m, 2H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 6-amino-hexan-1-ol (59 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 47 mg of the title compound were obtained as an orange solid (51% yield).
1H NMR (DMSO-d6) δ 13.63 (s, 1H), 10.91 (s, 1H), 8.31 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.00 (t, 1H), 6.88 (d, 1H), 6.81 (d, 1H), 4.32 (t, 1H), 3.38 (q, 2H), 3.23 (q, 2H), 2.35 (s, 3H), 1.6-1.2 (m, 8H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), aminomethyl-cyclohexane (57 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 24 mg of the title compound were obtained as an orange solid (26% yield).
1H NMR (DMSO-d6) δ 13.62 (s, 1H), 10.91 (s, 1H), 8.30 (t, 1H), 7.79 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.00 (t, 1H), 6.88 (d, 1H), 6.85 (d, 1H), 3.09 (t, 2H), 2.35 (s, 3H), 1.8-0.8 (m, 11H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 4-amino-1-butanol (45 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 33 mg of the title compound were obtained as an orange solid (39% yield).
1H NMR (DMSO-d6) δ 13.63 (s, 1H), 10.91 (s, 1H), 8.32 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.00 (t, 1H), 6.88 (d, 1H), 6.81 (d, 1H), 4.40 (t, 1H), 3.42 (q, 2H), 3.24 (q, 2H), 2.35 (s, 3H), 1.6-1.35 (m, 4H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 6-amino-hexanoic acid ethyl ester; hydrochloride (98 mg, 0.5 mmole) and Et3N (0.14 mL, 1 mmole, 4 eq.). After purification by flash chromatography, 78 mg of the title compound were obtained as an orange solid (76% yield).
1H NMR (DMSO-d6) δ 13.63 (s, 1H), 10.91 (s, 1H), 8.31 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.00 (t, 1H), 6.88 (d, 1H), 6.81 (d, 1H), 4.04 (q, 2H), 3.23 (q, 2H), 2.35 (s, 3H), 2.29 (t, 2H), 1.53 (m, 4H), 1.31 (m, 2H), 1.17 (t, 3H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), tetrahydro-furfurylamine (51 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 64 mg of the title compound were obtained as an orange solid (73% yield).
1H NMR (DMSO-d6) δ 13.65 (s, 1H), 10.91 (s, 1H), 8.41 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.01 (t, 1H), 6.89 (m, 2H), 3.96 (m, 1H), 3.78 (m, 1H), 3.63 (m, 1H), 3.30 (t, 2H), 2.34 (s, 3H), 2.0-1.5 (m, 4H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 2-(1H-indol-3-yl)-ethylamine (80 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 34 mg of the title compound were obtained as a red solid (33% yield).
1H NMR (DMSO-d6) δ 13.67 (s, 1H), 10.92 (s, 1H), 10.80 (s, 1H), 8.49 (t, 1H), 7.80 (d, 1H), 7.65 (s, 1H), 7.59 (d, 1H), 7.34 (d, 1H), 7.18 (m, 2H), 7.10-6.95 (m, 3H), 6.89 (d, 1H), 6.81 (d, 1H), 3.54 (q, 2H), 2.94 (t, 2H), 2.35 (s, 3H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 3-phenyl-propylamine (68 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 72 mg of the title compound were obtained as a yellow/orange solid (75% yield).
1H NMR (DMSO-d6) δ 13.64 (s, 1H), 10.91 (s, 1H), 8.36 (t, 1H), 7.80 (d, 1H), 7.65 (s, 1H), 7.33-7.11 (m, 6H), 7.00 (t, 1H), 6.89 (d, 1H), 6.83 (d, 1H), 3.27 (q, 2H), 2.63 (t, 2H), 2.35 (s, 3H), 1.82 (m, 2H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 4-phenyl-butylamine (75 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 72 mg of the title compound were obtained as a yellow/orange solid (74% yield).
1H NMR (DMSO-d6) δ 13.63 (s, 1H), 10.90 (s, 1H), 8.32 (t, 1H), 7.79 (d, 1H), 7.64 (s, 1H), 7.32-7.10 (m, 6H), 7.01 (t, 1H), 6.89 (d, 1H), 6.80 (d, 1H), 3.27 (q, 2H), 2.61 (t, 2H), 2.34 (s, 3H), 1.68-1.44 (m, 4H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 5-amino-1-pentanol (52 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 48 mg of the title compound were obtained as a yellow/orange solid (55% yield).
1H NMR (DMSO-d6) δ 13.63 (s, 1H), 10.90 (s, 1H), 8.31 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.18 (t, 1H), 7.00 (t, 1H), 6.89 (d, 1H), 6.82 (d, 1H), 4.34 (t, 1H), 3.39 (q, 2H), 3.23 (q, 2H), 2.35 (s, 3H), 1.6-1.25 (m, 6H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), 4-amino-butyric acid ethyl ester; hydrochloride (84 mg, 0.5 mmole) and Et3N (0.14 mL, 1 mmole, 4 eq.). After purification by flash chromatography, 75 mg of the title compound were obtained as a yellow/orange solid (79% yield).
1H NMR (DMSO-d6) δ 13.64 (s, 1H), 10.91 (s, 1H), 8.36 (t, 1H), 7.80 (d, 1H), 7.64 (s, 1H), 7.17 (t, 1H), 7.01 (t, 1H), 6.88 (d, 1H), 6.82 (d, 1H), 4.05 (q, 2H), 3.26 (q, 2H), 2.35 (s, 3H), 2.35 (t, 2H), 1.77 (m, 2H), 1.18 (t, 3H).
The general procedure 4 was followed using 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole), C-[1-(4-chloro-phenyl)-cyclopropyl-methylamine (91 mg, 0.5 mmole) and Et3N (0.07 mL, 0.5 mmole, 2 eq.). After purification by flash chromatography, 91 mg of the title compound were obtained as a yellow/orange solid (84% yield).
1H NMR (DMSO-d6) δ 13.60 (s, 1H), 10.91 (s, 1H), 8.28 (t, 1H), 7.79 (d, 1H), 7.63 (s, 1H), 7.32 (s, 4H), 7.17 (t, 1H), 7.00 (t, 1H), 6.88 (d, 1H), 6.85 (d, 1H), 3.52 (d, 2H), 2.33 (s, 3H), 0.99 (m, 2H), 0.77 (m, 2H).
To a solution of 4-methyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (67 mg, 0.25 mmole, 1 eq.) in DMF (5 mL) is added K2CO3 in one portion followed by the corresponding bromide. The mixture is shaken or stirred at room temperature overnight. After addition of few drops of H2O and cooling in an ice bath, a precipitate forms which is filtered and if necessary purified by flash chromatography.
The general procedure 5 was followed using bromomethyl-benzene (45 μL, 0.37 mmole, 1.5 eq.) and K2CO3 (53 mg, 0.37 mmole, 1.5 eq.). After purification, 33 mg of the title compound were obtained as an orange solid (37% yield).
1H NMR (DMSO-d6) δ 14.09 (s, 1H), 11.06 (s, 1H), 7.85 (d, 1H), 7.71 (s, 1H), 7.5-7.3 (m, 5H), 7.22 (t, 1H), 7.03 (t, 1H), 6.91 (d, 1H), 6.84 (d, 1H), 5.35 (s, 2H), 2.35 (s, 3H).
To a suspension or solution of the starting pyrrole (1 eq.) in CCl4 is added NBS (1.05 eq.) followed by benzoyl-peroxide (0.025 eq.) under argon. The mixture is then heated to reflux and the reaction is followed by LC/MS. The crude mixture is then allowed to come to room temperature and cooled in an ice bath. A precipitate forms which is filtered and purified by flash chromatography. If no precipitation occurs the solvent is removed under reduced pressure and the crude is purified by flash chromatography.
The general procedure 6 was followed using 3,5-dimethyl-1H-pyrrole-2-carbaldehyde (8 g, 65 mmoles), NBS (12.16 g, 68.3 mmoles) and benzoyl-peroxide (394 mg, 1.63 mmole) in CCl4 (300 mL).
After purification 11.9 g of 4-bromo-3,5-dimethyl-1H-pyrrole-2-carbaldehyde were obtained as a dark solid (92% yield).
1H NMR (DMSO-d6) δ 12.06 (br, 1H), 9.50 (s, 1H), 2.22 (s, 3H), 2.19 (s, 3H).
The general procedure 6 was followed using 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (2 g, 8.4 mmoles), NBS (1,57 g, 8.8 mmoles) and benzoyl-peroxide (51 mg, 0.21 mmole) in CCl4 (80 mL).
After purification 0.7 g of the title compound were obtained as an orange/brown solid (26% yield).
1H NMR (DMSO-d6) δ 13.79 (s, 1H), 10.90 (s, 1H), 7.77 (s, 1H), 7.63 (s, 1H), 7.14 (t, 1H), 6.99 (t, 1H), 6.88 (d, 1H), 2.32 (s, 3H), 2.28 (s, 3H).
To a suspension of 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (2 g, 8.4 mmoles) in dry CH2Cl2 (30 mL) was added under argon at 0° C. sulfonyl chloride (0.84 mL, 8.4 mmoles). The dark mixture was stirred for 70 minutes at 0° C. and then allowed to come to room temperature for 15 minutes. After removal of the solvent and purification by flash chromatography, 770 mg of the title compound were obtained as an orange solid (34% yield).
1H NMR (DMSO-d6) δ 13.72 (s, 1H), 10.90 (s, 1H), 7.77 (d, 1H), 7.61 (s, 1H), 7.13 (t, 1H), 7.00 (t, 1H), 6.88 (d, 1H), 2.32 (s, 3H), 2.28 (s, 3H).
To a suspension of NaH (60% dispersion in mineral oil, 1.5 eq.) in DMF is added a solution of aldehyde (1 eq.) in DMF. After stirring at room temperature for 30 minutes, the mixture is cooled down in an ice bath and the chloroester (1 eq.) is added dropwise. The crude mixture is then allowed to come to room temperature and left under stirring. The reaction is followed by TLC. The reaction mixture is poured into ice and extracted with Et2O. The combined organic phases are washed once with brine and concentrated in vacuo together with silica gel. The residue is purified by chromatography.
General procedure 7 was followed using NaH (0.49 g, 20.5 mmoles) in DMF (5 mL), 1H-indole-3-carbaldehyde (1.99 g, 13.7 mmoles) in DMF (15 mL), and chloro-acetic acid methyl ester (1.05 mL, 13.7 mmoles). Flash chromatography on silica gel using a gradient of EtOAc/Petroleum ether from 1:3 to 3:1 furnished the title compound (2.1 g, 71%).
1H NMR (DMSO-d6) δ 9.95 (s, 1H), 8.28 (s, 1H), 8.12 (m, 1H), 7.54 (m, 1H), 7.29 (m, 2H), 5.31 (s, 2H), 3.71 (s, 3H).
General procedure 1 was followed using 1,3-dihydro-indol-2-one (0.14 g; 1.84 mmoles), (3-formyl-indol-1-yl)-acetic acid methyl ester (1.87 g, 1.83 mmoles) and piperidine (10 drops). After filtration a mixture of [3-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-indol-1-yl]-acetic acid methyl ester (45%) and [3-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-indol-1-yl]-acetic acid ethyl ester (55%) was obtained as an orange solid (2.27 g, 80% yield).
1H-NMR (DMSO-d6, compound 24) δ 10.55 (s, 1H), 9.41 (s, 1H), 8.21 (m, 1H), 8.13 (s, 1H), 7.91 (d, 1H), 7.53 (m, 1H), 7.29 (m, 2H), 7.16 (t, 1H), 7.00 (t, 1H), 6.86 (d, 1H), 5.37 (s, 2H), 3.72 (s, 3H). 1H-NMR (DMSO-d6, compound 25) δ 10.55 (s, 1H), 9.41 (s, 1H), 8.21 (m, 1H), 8.13 (s, 1H), 7.91 (d, 1H), 7.53 (m, 1H), 7.29 (m, 2H), 7.16 (t, 1H), 7.00 (t, 1H), 6.86 (d, 1H), 5.35 (s, 2H), 4.19 (q, 2H), 1.23 (t, 3H).
General procedure 2 was followed using a 45:55 mixture of [3-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-indol-1-yl]-acetic acid methyl ester and [3-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-indol-1-yl]-acetic acid ethyl ester (1.76 g, about 8 mmoles) in a 1:1 mixture of an aqueous solution of LiOH 1N (25 mL) and THF (25 mL). After heating to reflux for 6 hours, HCl (1N) was added and the title compound was obtained as an orange solid (1.37 g, 53% yield).
1H-NMR (DMSO-d6, Z isomer) δ 13.19 (br, 1H), 10.55 (s, 1H), 9.40 (s, 1H), 8.21 (m, 1H), 8.13 (s, 1H), 7.90 (d, 1H), 7.54 (m, 1H), 7.29 (m, 2H), 7.15 (t, 1H), 7.00 (t, 1H), 6.86 (d, 1H), 5.24 (s, 2H).
General procedure 7 was followed using NaH (0.41 g, 10.3 mmoles) in DMF (5 mL), 1H-indole-3-carbaldehyde (1.0 g, 6.9 mmoles) in DMF (15 mL), and 3-chloro-propionic acid ethyl ester (0.86 mL, 6.9 mmoles). Flash chromatography on silica gel using a gradient of EtOAc/Petroleum ether from 1:3 to 3:1 furnished the title compound (1.49 g, 88% yield).
1H NMR (DMSO-d6) δ 9.91 (s, 1H), 8.30 (s, 1H), 8.11 (d, 1H), 7.66 (d, 1H), 7.30 (m, 2H), 4.54 (t, 2H), 4.03 (q, 2H), 2.93 (t, 2H), 1.11 (t, 3H).
General procedure 1 was followed using 1,3-dihydro-indol-2-one (0.46 g, 3.43 mmoles), 3-(3-formyl-indol-1-yl)-propionic acid ethyl ester (0.84 g, 3.42 mmoles) and piperidine (10 drops). After filtration the title compound was obtained as a yellow solid (1.08 g, 88% yield).
13C-NMR (DMSO-d6, Z isomer) δ 10.52 (s, 1H), 9.45 (s, 1H), 8.20 (m, 1H), 8.11 (s, 1H), 7.88 (d, 1H), 7.64 (m, 1H), 7.29 (m, 2H), 7.15 (t, 1H), 6.99 (t, 1H), 6.85 (d, 1H), 4.58 (t, 2H), 4.04 (q, 2H), 2.92 (t, 2H), 1.12 (t, 3H).
General procedure 2 was followed using 3-[3-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-indol-1-yl]-propionic acid ethyl ester (0.62 g, 1.73 mmoles) in a 1:1 mixture of an aqueous solution of LiOH 0.2N (30 mL) and MeOH (30 mL). After heating to reflux for 4 hours, the pH was adjusted to acidic pH by addition of HCl (1N) and the title compound was obtained as orange crystals (0.57 g, 100% yield).
1H-NMR (DMSO-d6) δ 12.46 (br, 1H), 10.49 (s, 1H), 9.47 (s, 1H), 8.19 (m, 1H), 8.10 (s, 1H), 7.88 (d, 1H), 7.64 (m, 1H), 7.29 (m, 2H), 7.15 (t, 1H), 6.99 (t, 1H), 6.85 (d, 1H), 4.55 (t, 2H), 2.86 (t, 2H).
General procedure 1 was followed using commercially available 1-(2-chloro-thiazol-5-ylmethyl)-1H-indole-3-carbaldehyde (310 mg, 1.1 mmoles, from Bionet) and 1,3-dihydro-indol-2-one (149 g, 1.1 mmoles) as a yellow powder (127 mg, 29% yield).
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 10.55 (s, 1H), 9.50 (s, 1H), 8.23 (dd, 1H), 8.12 (s, 1H), 7.90 (d, 1H), 7.86 (s, 1H), 7.75 (m, 1H), 7.30 (m, 2H), 7.16 (t, 1H), 7.00 (t, 1H), 6.86 (d, 1H), 5.87 (s, 2H).
Under dry conditions is prepared a solution of KOH (4 eq., 2-3 N) in DMSO. After stirring at room temperature for 5 minutes is added the starting indole (1 eq.) under argon atmosphere and the mixture is stirred an additional 45 minutes.
The alkylating agent (1.1 eq.) is then added dropwise at 0° C. and the mixture is allowed to come to room temperature. The reaction is followed by TLC. H2O is added and the aqueous phase is extracted with Et2O (3×). The combined organic phases are washed once with H2O, once with brine, dried over MgSO4. Removal of solvent under vacuum affords the expected compound which can be used without further purification.
General procedure 8 was followed using KOH (15.46 g, 0.28 mole) in DMSO (100 mL), 1H-indole-3-carbaldehyde (10 g, 69 mmoles) and iodomethane (4.73 mL, 76 mmoles). The title compound was obtained as a yellow/brown solid (7.2 g, 66% yield).
1H-NMR (DMSO-d6) δ 9.90 (s, 1H), 8.26 (s, 1H), 8.11 (d, 1H), 7.58 (d, 1H), 7.30 (m, 2H), 3.90 (s, 3H).
General procedure 8 was followed using KOH (1.58 g, 28 mmoles) in DMSO (15 mL), 1H-indole-3-carbaldehyde (1 g, 7.0 mmoles) and iodopropane (0.74 mL, 7.6 mmoles). The title compound was obtained as an orange oil (1.3 g, 100% yield).
13C NMR (DMSO-d6) δ 184.4, 140.7, 137.0, 124.6, 123.4, 122.3, 120.9, 116.9, 111.0, 47.7, 22.6, 10.9.
The title compound was obtained as a yellow powder following the general procedure 1 and using 1-methyl-1-H-indole-3-carbaldehyde (159 mg, 1 mmole) and 1,3-dihydro-indol-2-one (161 mg, 1.2 mmoles): 205 mg, 75% yield.
1H-NMR (DMSO-d6) δ 10.52 (s, 1H), 9.41 (s, 1H), 8.20 (m, 1H), 8.13 (s, 1H), 7.88 (d, 1H), 7.58 (m, 1H), 7.30 (m, 2H), 7.14 (t, 1H), 6.99 (t, 1H), 6.85 (d, 1H), 3.95 (s, 3H).
The title compound was obtained as orange crystals following the general procedure 1 using 1-propyl-1-H-indole-3-carbaldehyde (217 mg, 1.16 mmoles) and 1,3-dihydro-indol-2-one (154 mg, 1.16 mmoles): 330 mg, 94% yield.
3C-NMR (DMSO-d6, Z isomer) δ 168.0, 139.1, 135.9, 135.8, 128.7, 126.7, 126.5, 125.6, 122.5, 120.9, 120.4, 119.0, 118.6, 110.7, 110.4, 108.9, 47.8, 22.9, 11.1.
To a solution of (3-bromo-4-methoxy-benzyl)-dimethylamine (2.5 g, 10.2 mmoles) in dry THF (46 mL) at −78° C. was added n-BuLi (1.6 M, 7.0 mL, 11.3 mmoles) keeping the temperature below −70° C. The mixture was stirred for 60 minutes and dry DMF (1.6 mL, 20 mmoles) was added in one portion. The mixture was stirred for 2 h at −78° C., the cooling bath was removed and the mixture was allowed to warm to 0° C. Aqueous NaOH (2M, 20 mL) was added and the mixture was extracted with Et2O (3×100 mL). The organic phase was dried over Na2SO4 and the solvent was removed under reduced pressure leaving a yellow oil. Distillation (100° C., 1 mbar) afforded the product as a colourless oil in 35% yield (0.7 g, 3.6 mmoles).
1H-NMR (CDCl3): 10.45 (1H, s), 7.80 (1H, d J=2.3 Hz), 7.54 (1H, dd J=8.8 Hz, 2.3 Hz), 6.96 (1H, d J=8.8 Hz), 3.93 (3H, s), 3.39 (2H, s), 2.22 (6H, s).
The title compound was prepared following the general procedure 1 using 1,3-dihydro-indol-2-one (0.36 g, 2.6 mmoles) and 5-dimethylaminomethyl-2-methoxy-benzaldehyde (0.5 g, 2.6 mmoles). Yield: 72% (0.58 g, 1.9 mmoles) as a yellow oil.
13C-NMR (DMSO-d6, E isomer) δ 168.5, 156.7, 142.7, 132.0, 131.6, 130.5, 129.8, 127.2, 122.5, 122.3, 121.1, 120.8, 111.2, 109.9, 62.4, 55.6, 44.7
To a stirred solution of 2-fluoro-5-methoxy-benzaldehyde (10.6 mmoles) in dry DMF (15 mL) was added K2CO3 (1.8 g, 12.7 mmoles) and N,N,N′-trimethylethylendiamine (1.7 mL, 12.7 mmoles). After stirring at 100° C. overnight, the reaction mixture was cooled to room temperature. Water was added (50 mL) and the aqueous phase was extracted with ethyl acetate (3×50 mL). The combined organic phases were washed with water and dried (Na2SO4). Evaporation in vacuo and purification by chromatography gave the pure title product in 13% yield (0.33 g, 1.4 mmol) as a brown oil.
1H-NMR (CDCl3): 10.41 (1H, s), 7.31 (1H, d J=3.0 Hz), 7.19 (1H, d J=8.8 Hz), 7.10 (1H, dd J=8.8 Hz; 3.0 Hz), 3.82 (3H, s), 3.11 (2H, dd J≈6 Hz; 8 Hz), 2.82 (3H, s), 2.42 (2H, dd J≈6 Hz; 8 Hz), 2.31 (6H, s).
The title compound was prepared following the general procedure 1 using 1,3-dihydro-indol-2-one (0.12 g, 0.9 mmole) and 2-[(2-dimethylamino-ethyl)-methyl-amino]-5-methoxy-benzaldehyde (0.21 g, 0.9 mmole). Yield: 87% (0.28 g, 0.8 mmole) as yellow oil.
13C-NMR (CDCl3, E isomer) δ 170.3, 154.3, 147.3, 141.3, 136.7, 129.6, 129.5, 126.1, 122.9, 122.1, 121.7, 120.3, 117.0, 114.2, 110.1, 57.4, 55.9, 55.8, 45.6, 42.5.
The title compound was prepared following the general procedure 1 using 1,3-dihydro-indol-2-one (0.085 g, 0.6 mmole) and 4-[(2-dimethylamino-ethyl)-methyl-amino]-3′,5′-dimethyl-biphenyl-3-carbaldehyde (0.2 g, 0.6 mmole, synthesis described in patent WO-03097576). Yield: 50% (0.14 g, 0.3 mmole) as yellow oil.
1H-NMR (CDCl3) δ 7.96 (d, 1H), 7.90 (s, 1H), 7.87 (br, 1H), 7.76 (d, 1H), 7.60 (dd, 1H), 7.3-7.1 (m, 4H), 7.0-6.8 (m, 3H), 3.18 (t, 2H), 2.88 (s, 3H), 2.60 (t, 2H), 2.35 (s, 6H), 2.19 (s, 6H).
The title compound was prepared following the general procedure 1 using 1,3-dihydro-indol-2-one (0.77 g, 5.8 mmoles) and 2-dimethylaminomethyl-benzaldehyde (0.93 g, 5.8 mmoles, synthesis described in Journal of Organic Chemistry (1969), 34(8), 2482-4). Yield: 16% (0.26 g, 0.9 mmol) as yellow crystals.
13C-NMR (DMSO-d6, E isomer) δ 168.3, 142.7, 138.0, 135.1, 134.3, 130.0, 129.7, 129.1, 128.5, 127.7, 127.1, 122.1, 121.2, 120.9, 109.9, 61.3, 44.9
To a solution of 2-hydroxy-5-methoxy-benzaldehyde (1 eq.) in DMF is added Cs2CO3 (5 eq.), NaI (2.5 eq.) and the chloroalkyl-amine as a free base or as a salt (2.5 eq.). The suspension is stirred at 110° C. during 4 hours and the reaction is followed by LC/MS. The reaction mixture is then filtered, evaporated in high vacuo, and chromatographed to afford the expected product.
To a solution of 2-hydroxy-5-methoxy-benzaldehyde (2.5 g, 16.6 mmoles) in DMF (100 mL) were added Cs2CO3 (27 g, 82.9 mmoles), NaI (6.2 g, 41.4 mmoles) and (2-chloro-ethyl)-diethyl-amine; hydrochloride (7,1 g, 41.4 mmoles), following the general procedure 9. The title compound was obtained as a yellow oil (2.89 g, 69% yield).
13C-NMR (CDCl3) δ 189.7, 156.2, 153.7, 125.2, 123.5, 114.6, 110.2, 68.3, 55.8, 51.8, 48.0, 12.0.
To a solution of 2-hydroxy-5-methoxy-benzaldehyde (2.5 g, 16.6 mmoles) in DMF (100 mL) were added Cs2CO3 (27 g, 82.9 mmoles), NaI (6.2 g, 41.5 mmoles) and 4-(2-chloro-ethyl)-morpholine; hydrochloride (7,7 g, 41.5 mmoles), following the general procedure 9. The title compound was obtained as a brown oil (3.3 g, 75% yield).
13C-NMR (DMSO-d6) δ 189.0, 155.5, 153.2, 124.9, 123.0, 116.1, 109.8, 67.1, 66.1, 56.8, 55.4, 53.4.
To a solution of 2-hydroxy-5-methoxy-benzaldehyde (2.5 g, 16.6 mmoles) in DMF (100 mL) were added Cs2CO3 (27 g, 82.9 mmoles), NaI (6.2 g, 41.5 mmoles) and 1-(2-chloro-ethyl)-piperidine; hydrochloride (7.8 g, 42.4 mmoles), following the general procedure 9. The title compound was obtained as a brown oil (2.26 g, 52% yield).
13C-NMR (DMSO-d6) δ 189.0, 155.6, 153.2, 124.9, 123.0, 116.2, 109.7, 67.4, 57.2, 55.5, 54.2, 25.6, 23.8.
The title compound was obtained following the general procedure 1 using 1,3-dihydro-indol-2-one (0.8 g, 6 mmoles), 2-(2-diethylamino-ethoxy)-5-methoxy-benzaldehyde (1.53 g, 6 mmoles) and piperidine (0.35 mL) in EtOH (20 mL). After chromatography the title compound was obtained as a hygroscopic solid (1.54 g, 70% yield)
13C-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 168.5, 152.5, 151.1, 142.7, 131.6, 129.8, 127.5, 123.7, 122.4, 121.1, 120.9, 116.8, 114.3, 113.7, 109.9, 67.6, 55.4, 51.0, 47.0, 11.9.
To a solution of 3-[2-(2-diethylamino-ethoxy)-5-methoxy-benzylidene]-1,3-dihydro-indol-2-one (1.5 g, 4 mmoles) in EtOAc (3 mL) was added a solution of HCl in dioxane (4N, 2 mL, 8.2 mmoles). After addition of Et2O, a precipitate formed which was filtered and washed with Et2O to afford the title compound (1.32 g, 80% yield).
13C-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 168.3, 153.1, 149.9, 142.8, 131.0, 130.0, 128.0, 123.8, 122.4, 121.0, 120.8, 116.5, 114.5, 113.5, 110.1, 66.3, 63.3, 55.5, 47.2, 8.4.
The title compound was obtained following the general procedure 1 using 1,3-dihydro-indol-2-one (0.74 g, 5.6 mmoles), 5-methoxy-2-(2-morpholin-4-yl-ethoxy)-benzaldehyde (1.50 g, 5.6 mmoles) and piperidine (0.35 mL) in EtOH (20 mL). After chromatography the title compound was obtained as an orange solid (2.04 g, 96% yield)
13C-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 168.5, 152.7, 151.0, 142.7, 131.6, 129.8, 127.5, 124.0, 122.4, 121.0, 120.9, 116.8, 114.3, 114.2, 109.9, 66.8, 66.0, 56.8, 55.4, 53.5.
The title compound was obtained following the general procedure 1 using 1,3-dihydro-indol-2-one (0.76 g, 5.7 mmoles), 5-methoxy-2-(2-piperidin-1-yl-ethoxy)-benzaldehyde (1.52 g, 5.8 mmoles) and piperidine (0.35 mL) in EtOH (20 mL). After chromatography the title compound was obtained as an orange solid (2.07 g, 96% yield)
13C-NMR (DMSO-d6, mixture of the E and Z isomers) δ 168.5, 167.0, 152.6, 152.2, 151.9, 151.1, 142.7, 131.6, 130.2, 129.8, 128.8, 127.5, 126.0, 123.9, 123.1, 122.4, 121.0, 120.9, 119.0, 118.1, 116.8, 116.2, 114.1, 113.6, 109.9, 109.3, 67.4, 67.0, 57.3, 57.1, 55.4, 55.3, 54.3, 25.6, 25.5, 23.8.
1—To a solution of 1,3-dihydro-indol-2-one (1 g, 7.57 mmoles) in acetonitrile (50 mL) were added DMAP (100 mg, 7.57 mmoles) and (Boc)2O (1.82 g, 8.33 mmoles). The reaction was followed by TLC. The crude mixture was concentrated in vacuo and CH2Cl2 was added (50 mL). The organic phase was washed once with a 10% aqueous solution of KHSO4 (50 mL), once with an aqueous saturated solution of NaHCO3 (50 mL), twice with H2O (2×50 mL) and was then concentrated in vacuo together with silica gel. The residue was purified by flash chromatography using a gradient of petroleum ether/EtOAc from 100:00 to 80:20, furnishing 2-oxo-2,3-dihydro-indole-1-carboxylic acid tert-butyl ester (619 mg, 35%).
2—The general procedure 1 was followed using 2-oxo-2,3-dihydro-indole-1-carboxylic acid tert-butyl ester (564 mg, 2.42 mmoles), 1H-indole-3-carbaldehyde (351 mg, 2.42 mmoles) and piperidine (3 drops). The pure title compound was obtained after filtration (482 mg, 55% yield).
1H-NMR (DMSO-d6, Z isomer) δ 12.17 (br, 1H), 9.36 (s, 1H), 8.31 (s, 1H), 8.24 (m, 1H), 8.06 (d, 1H), 7.75 (d, 1H), 7.56 (m, 1H), 7.34-7.16 (m, 4H), 1.63 (s, 9H).
To a suspension of NaH (60% oil dispersion, 209 mg, 5.25 mmoles) in dry DMF (30 mL) was added under argon at 0° C. a solution of 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (1.2 g, 5 mmoles) in DMF (46 mL) followed by 1-chloromethyl-4-methoxy-benzene (2.1 mL, 15 mmoles). The reaction was left under stirring and allowed to come to room temperature. The reaction evolution was followed by TLC. After completion of the reaction a saturated aqueous solution of NaHCO3 (25 mL) was added followed by H2O (25 mL). The aqueous phase was extracted with EtOAc (3×50 mL). The combined organic phases were washed once with a saturated aqueous solution of NaHCO3 (50 mL) and dried over MgSO4. After filtration and removal of the solvent, a brown solid was obtained. crystallization from EtOH and H2O afforded the pure title compound as a yellow solid (1.08 g, 60% yield).
13C-NMR (DMSO-d6, Z isomer) δ 167.4, 158.4, 138.2, 136.3, 132.4, 128.8, 128.6, 126.6, 125.5, 124.8, 123.7, 121.4, 117.9, 113.9, 112.7, 111.1, 108.7, 54.9, 42.1, 13.5, 11.2.
To a suspension of NaH (60% oil dispersion, 151 mg, 3.8 mmoles) in dry DMF (20 mL) was added under argon at 0° C. a solution of 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (741 mg, 3.1 mmoles) in DMF (28 mL) followed by iodomethane (0.24 mL, 3.8 mmoles). The reaction was stirred and allowed to come to room temperature. The reaction evolution was followed by LC/MS. H2O (25 mL) was added and the mixture was stirred until a thick orange precipitate formed. The solid was filtered. Crystallization from MeOH and H2O afforded the pure title compound as a yellow solid (504 mg, 63% yield).
13C-NMR (DMSO-d6, Z isomer) δ 167.4, 139.2, 135.9, 131.9, 126.5, 125.6, 124.7, 123.3, 121.3, 117.7, 112.5, 111.4, 108.1, 25.9, 13.4, 11.2.
To a suspension of 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one (2.02 g, 8.5 mmoles) and Cs2CO3 (11.05 g, 34 mmoles) in CH3CN (200 mL) was added acetic acid chloromethyl ester (3.3 mL, 34 mmoles). The reaction mixture was heated at 40° C. and left under stirring overnight. The reaction evolution was followed by LC/MS. The mixture was then concentrated in vacuo together with silica gel. The residue was purified by chromatography using a gradient of petroleum ether/ethyl acetate from 85:15 to 50:50, furnishing the title compound (488 mg, 19% yield) as a deep orange solid.
1H-NMR (DMSO-d6, Z isomer) δ 13.01 (s, 1H), 7.81 (d, 1H), 7.65 (s, 1H), 7.26-7.05 (m, 3H), 6.07 (d, 1H), 5.89 (s, 2H), 2.35 (s, 3H), 2.33 (s, 3H), 2.04 (s, 3H).
To a solution of 3-(2,5-dimethoxy-benzylidene)-1,3-dihydro-indol-2-one (422 mg, 1.5 mmoles) in DMF (10 mL) was added NaH (60% dispersion in mineral oil, 60 mg, 1.5 mmoles). The yellow solution was stirred at room temperature for 15 minutes and turned red. Acetic anhydride (284 μL, 3 mmoles) was added. The solution turned back to yellow and a precipitate formed. After 2 hours the solid was filtrated, washed with water and dried in vacuo to afford 449 mg of the title compound as a yellow solid (92% yield).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant isomer) δ 8.21 (s, 1H), 7.80 (s, 1H), 7.59 (d, 1H), 7.39 (t, 1H), 7.23 (d, 1H), 7.19-7.05 (m, 3H), 3.81 (s, 3H), 3.73 (s, 3H), 2.66 (s, 3H).
1) 2-Nitrophenylacetic acid (7.10 g, 39.2 mmoles) was hydrogenated in the presence of DMSO (5.8 mL) and 5% Pt/C (0.58 g) in EtOH (350 mL) at room temperature under 1 atmosphere for 24 hours. Then the catalyst was filtered off and washed with EtOH. The filtrate was concentrated in vacuo. The residue was redissolved in AcOH. The obtained solution was stirred at room temperature for 40 hours and concentrated in vacuo. The residue was taken up in toluene. The obtained mixture was concentrated in vacuo to remove the residual acetic acid, giving crude 1-hydroxy-1,3-dihydro-indol-2-one.
2) Crude 1-hydroxy-1,3-dihydro-indol-2-one was taken up in CH2Cl2 (50 mL). To the obtained mixture were added imidazole (6.00 g, 88.1 mmoles) and tert-butyldimethylsilyl chloride (5.90 g, 39.2 mmoles) at room temperature. After being stirred at room temperature for 2 hours, the reaction solution was washed twice with H2O, dried over MgSO4, and concentrated in vacuo. The residue was purified by chromatography, providing 1.90 g (18% yield based on 2-nitrophenylacetic acid) of 1-(tert-butyidimethylsilyloxy)-1,3-dihydroindol-2-one as a reddish solid.
13C-NMR (DMSO-d6) δ 169.7, 143.2, 127.7, 124.5, 122.3, 121.4, 107.0, 33.2, 25.6, 17.9, −4.8.
3) A mixture of 1-(tert-butyidimethylsilyloxy)-1,3-dihydroindol-2-one (0.80 g, 3.0 mmoles), 3,5-dimethyl-1H-pyrrole-2-carbaldehyde (0.80 g, 6.5 mmol), and piperidine (0.80 mL) in EtOH (20 mL) was heated to reflux for 5 h. The mixture was then concentrated in vacuo together with silica gel. The residue was purified by chromatography first with petroleum ether/ethyl acetate 1:1 and then with ethyl acetate/MeOH 10:1 as eluents, furnishing the title compound (0.61 g, 79% yield based on 1-(tert-butyidimethylsilyloxy)-1,3-dihydroindol-2-one) as a red solid.
13C-NMR (DMSO-d6, Z isomer) δ 163.1, 137.4, 136.2, 132.3, 126.3, 125.7, 123.8, 121.4, 121.0, 117.7, 112.6, 109.0, 106.5, 13.4, 11.2.
The general procedure 1 was followed using 4-bromo-3,5-dimethyl-1H-pyrrole-2-carbaldehyde (0.63 g, 3.1 mmoles), 1-hydroxy-1,3-dihydro-indol-2-one (0.46 g, 3.1 mmoles) and piperidine (10 drops) in EtOH (5 mL). After filtration, 0.80 g of the title compound were obtained (77% yield).
1H-NMR (DMSO-d6, Z isomer) δ 13.65 (s, 1H), 10.95 (br, 1H), 7.84 (d, 1H), 7.69 (s, 1H), 7.23 (t, 1H), 7.06 (t, 1H), 7.00 (d, 1H), 2.34 (s, 3H), 2.28 (s, 3H).
In a sealed glass vessel, a mixture of 1H-indole-3-carbaldehyde (1.95 g, 13.4 mmoles), 1-hydroxy-1,3-dihydro-indol-2-one (1.00 g, 6.7 mmoles), and piperidine (1.14 g, 13.4 mmoles) in EtOH (40 mL) was stirred at 100° C. for 8 hours. The reaction mixture was then concentrated in vacuo. The residue was purified by flash chromatography (silica gel, CH2Cl2/AcOH 40:1), providing the title compound (0.90 g, 49%) as a yellow solid.
13C-NMR (DMSO-d6, Z isomer) δ 162.2, 138.5, 135.8, 133.7, 128.2, 128.1, 126.7, 122.5, 121.1, 121.0, 120.8, 118.4, 118.3, 115.5, 112.2, 111.1, 106.2.
A mixture of 2,5-dimethoxybenzaldehyde (2.23 g, 13.4 mmoles), 1-hydroxy-1,3-dihydro-indol-2-one (1.00 g, 6.7 mmoles), and piperidine (1.14 g, 13.4 mmoles) in EtOH (40 mL) was treated as described in preparation 57. Flash chromatography (silica gel, using a gradient of eluents CH2Cl2/AcOH from 100:0 to 40:1) furnished the title compound (1.14 g, 57% yield) as a yellow solid.
13C-NMR (DMSO-d6, E isomer) δ 162.9, 152.5, 151.8, 142.1, 132.3, 130.0, 124.6, 123.0, 122.0, 121.8, 117.3, 117.2, 114.4, 112.7, 107.3, 55.9, 55.5.
The title compound was prepared following the general procedure 1 using 1-methoxy-oxindole (0.49 g, 3 mmoles, prepared as described in Journal of Enzyme Inhibition and Medicinal Chemistry 2003, 18(3), 243-252) and 3,5-dimethyl-1H-pyrrole-2-carbaldehyde (0.37 g, 3 mmoles). Yield: 61% (0.49 g, 1.8 mmoles) as orange crystals.
3C-NMR (DMSO-d6, Z isomer) δ 162.8, 137.1, 134.6, 133.3, 126.5, 125.7, 124.6, 122.0, 121.2, 118.1, 113.0, 108.0, 106.5, 63.6, 13.4, 11.3.
The title compound was prepared following the general procedure 1 using 1-methoxy-oxindole (0.49 g, 3 mmoles) and 2,5-dimethoxybenzaldehyde (0.5 g, 3 mmoles). Yield: 63% (0.59 g, 1.9 mmoles) as yellow crystals.
13C-NMR (DMSO-d6, mixture of the Z and E isomers, signals of the predominant form) δ 162.4, 152.5, 151.9, 139.4, 133.2, 130.1, 123.9, 122.7, 122.5, 122.4, 117.5, 117.4, 114.3, 112.7, 107.4, 63.5, 55.9, 55.5.
To a suspension of 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1-hydroxy-1,3-dihydro-indol-2-one (416 mg, 1.64 mmoles) in acetonitrile (12 mL) were added acetic anhydride (2.5 mL) and triethylamine (2.5 mL). The suspension turned to a solution, which was stirred for 1 hour at room temperature. Concentration in vacuo and purification by flash chromatography (using a gradient of petroleum ether/EtOAc from 100:0 to 85:15) afforded 348 mg of the title compound (71% yield).
1H NMR (d6-DMSO, Z isomer) δ 12.77 (s, 1H), 7.87 (d, 1H), 7.72 (s, 1H), 7.20 (t, 1H), 7.12 (t, 1H), 7.04 (d, 1H), 6.08 (d, 1H), 2.45 (s, 3H), 2.34 (s, 6H).
To a solution of 1-hydroxy-1,3-dihydro-indol-2-one (1 g, 6.7 mmoles) and 2-(3-bromo-propyl)-isoindole-1,3-dione (2 g, 7.4 mmoles) in CH3CN (12 mL) was added Cs2CO3 (4.36 g, 13.4 mmoles). The reaction mixture was stirred under argon at 30° C. The reaction evolution was followed by LC/MS. The mixture was then concentrated in vacuo together with silica gel. The residue was purified by chromatography to afford the pure title compound (1.25 g, 56% yield).
13C-NMR (DMSO-d6) δ 169.4, 167.9, 141.5, 134.2, 131.7, 127.7, 124.7, 122.9, 122.4, 121.1, 107.0, 73.5, 34.5, 33.4, 27.1.
The title compound was obtained following the general procedure 1 using 2-[3-(2-oxo-2,3-dihydro-indol-1-yloxy)-propyl]-isoindole-1,3-dione (0.63 g, 1.9 mmoles) and 3,5-dimethyl-1H-pyrrole-2-carbaldehyde (0.23 g, 1.9 mmoles, synthesis described in J. Chem. Soc. Perkin trans I, 1988, p. 1195 (448 mg, 54%).
13C-NMR (DMSO-d6, Z isomer) δ 12.94 (s, 1H), 7.91-7.78 (m, 5H), 7.63 (s, 1H), 7.21 (t, 1H), 7.12 (d, 1H), 7.07 (t, 1H), 6.05 (s, 1H), 4.28 (t, 2H), 3.83 (t, 2H), 2.35 (s, 3H), 2.31 (s, 3H), 2.12 (m, 2H).
1,3-Dihydro-indol-2-one (100 mg, 0.76 mmole), phenyl-p-tolyl-methanone (164 mg, 0.83 mmole) and LiCl (cat.) were suspended in dry ethanol (1 mL). 2-Dimethylamino ethylamine (0.42 mL, 3.8 mmoles) was added. The flask was flushed with argon, sealed and exposed to microwave irradiations (125 W, 120° C.) for 1 hour. After concentration in vacuo and purification by flash chromatography (using a gradient of petroleum ether/EtOAc from 1:0 to 2:3) the title compound was obtained as a red-orange oil (43 mg, 18% yield).
13C NMR (CDCl3, mixture of E and Z isomers) δ 168.3, 168.3, 155.4, 141.6, 140.5, 140.3, 140.1, 139.6, 139.5, 138.4, 136.8, 130.6, 130.4, 129.6, 129.5, 129.2, 128.9, 128.6, 128.5, 127.8, 124.4, 124.4, 124.1, 124.0, 123.3, 121.2, 109.4, 109.3, 21.5, 21.5.
NaH (50% dispersion in oil, 50 mg, 1 mmole) was added in one portion to a solution of 1,3-dihydro-indol-2-one (130 mg, 1 mmole) in dry THF (5 mL). When the resulting gas evolution ceased bis-(4-methoxy-phenyl)-methanone (290 mg, 1.2 mmoles) was added in one portion. The reaction mixture was heated to reflux for about 3 days. Extra portions of NaH (in total 75 mg, 1.5 mmoles) were added over the next 2 days. H2O was then added (10 mL) and the pH of the crude was adjusted to 6-7 by addition of cold acetic acid. Crystals formed which were filtered and recrystallized from MeOH to afford 200 mg of the pure title compound (55% yield)
1H NMR (DMSO-d6) δ 10.37 (s, 1H), 7.18 (m, 4H), 7.04 (m, 3H), 6.88 (d, 2H), 6.75 (d, 1H), 6.59 (t, 1H), 6.27 (d, 1H), 3.83 (s, 3H), 3.79 (s, 3H).
To a solution of 1,3-dihydro-indol-2-one (1.0 g, 7.5 mmoles) and 1-(2,5-dimethoxy-phenyl)-ethanone (2.43 g, 13.5 mmoles) in EtOH (5 mL) was added diethyl-amine (1 mL). After 5 days at reflux the mixture was allowed to come to room temperature, diluted with MeOH/EtOH and filtered. The filtrate was concentrated in vacuo and purified by flash chromatography using a mixture of petroleum ether and EtOAc as eluent. The title compound was obtained as a yellow powder (300 mg, 13% yield).
13C-NMR (DMSO-d6, Z isomer) δ 168.7, 153.7, 150.7, 148.7, 140.6, 131.6, 128.1, 124.0, 122.8, 122.0, 120.5, 114.1, 113.3, 113.1, 109.1, 56.0, 55.5, 21.0.
In a flask equipped with a Dean Stark apparatus was heated to reflux a solution of sodium hydroxide (1.6 g, 40 mmoles), 1,3-dihydro-indol-2-one (2.62 g, 19.7 mmoles) and (1-methyl-1H-indol-3-yl)-oxo-acetic acid (4.0 g, 19.7 mmoles) in toluene (70 mL) overnight. After removal of the solvent in vacuo and addition of water (50 mL), the solution was acidified by addition of an aqueous solution of HCl (1.5 N) at 0° C. A precipitate formed which was filtered and purified by flash chromatography to afford the pure title compound (1.41 g, 22% yield).
13C-NMR (DMSO-d6) δ 169.6, 167.5, 142.2, 137.1, 137.0, 132.4, 129.0, 125.0, 123.0, 122.4, 121.9, 120.7, 120.6, 120.5, 111.0, 109.5, 108.2, 33.0.
The following compounds were obtained following general procedure 1 and corresponding starting materials:
13C-NMR (DMSO-d6 mixture of E and Z isomers) δ 169.6, 168.0, 141.6, 139.1, 136.3, 135.8, 133.4, 129.7, 128.1, 127.6, 127.1, 126.7, 125.6, 122.6, 122.4, 122.2, 122.1, 121.3, 120.8, 120.7, 120.4, 119.0, 118.8, 118.6, 118.3, 112.4, 112.2, 111.2, 110.4, 109.4, 108.9.
1H-NMR (DMSO-d6) δ 12.28 (s, 1H), 10.56 (s, 1H), 7.70 (m, 3H), 7.57 (m, 3H), 7.48 (t, 1H), 7.28 (m, 1H), 7.20-7.07 (m, 3H), 6.90 (d, 1H), 6.75 (m, 2H).
13C-NMR (DMSO-d6, E isomer) δ 169.2, 144.0, 141.9, 137.7, 130.8, 129.7, 129.1, 128.6, 128.4, 128.0, 124.4, 124.1, 124.1, 122.6, 122.0, 121.6, 120.6, 120.3, 111.2, 109.2, 31.5.
1H-NMR (DMSO-d6) δ 12.32 (s, 1H), 10.56 (s, 1H), 7.69 (d, 2H), 7.64 (d, 2H), 7.57 (d, 1H), 7.28 (m, 1H), 7.19-7.07 (m, 3H), 6.88 (d, 1H), 6.72 (m, 2H).
13C-NMR (DMSO-d6, E isomer) δ 169.2, 142.0, 140.6, 136.8, 132.7, 132.5, 128.8, 128.5, 128.5, 128.2, 128.1, 127.6, 126.9, 125.9, 125.5, 125.0, 124.4, 122.8, 122.1, 121.4, 120.4, 120.2, 112.3, 109.2, 108.4.
1H-NMR (DMSO-d6, Z isomer) δ 12.09 (br, 1H), 10.65 (s, 1H), 9.48 (s, 1H), 8.30 (m, 2H), 8.08 (d, 1H), 7.53 (m, 1H), 7.25 (m, 2H), 7.15 (dd, 1H), 6.84 (d, 1H).
1H-NMR (DMSO-d6, mixture of E and Z isomers) δ 11.90 (s, 1H), 10.49 (s, 0.6H), 10.46 (s, 0.4H), 9.42 (d, 0.6H), 8.20 (s, 0.4H), 8.13 (s, 0.6H), 7.98 (s, 0.6H), 7.89 (s, 0.6H), 7.87 (s, 0.4H), 7.80 (d, 0.4H), 7.48 (s, 0.4H), 7.42 (t, 1H), 7.24-6.80 (m, 4H), 2.48 (s, 1.8H), 2.43 (s, 1.2H).
1H-NMR (DMSO-d6, Z isomer) δ 12.18 (br, 1H), 11.02 (s, 1H), 9.52 (s, 1H), 8.32 (s, 1H), 8.26 (m, 1H), 7.80 (dd, 1H), 7.55 (m, 1H), 7.27 (m, 2H), 7.05 (t, 1H).
1H-NMR (DMSO-d6, Z isomer) δ 12.08 (br, 1H), 10.65 (s, 1H), 9.48 (s, 1H), 8.30 (s, 1H), 8.28 (m, 1H), 8.20 (d, 1H), 7.53 (m, 1H), 7.31-7.20 (m, 3H), 6.80 (d, 1H).
1H-NMR (DMSO-d6, E isomer) δ 11.99 (br, 1H), 10.62 (br, 1H), 8.22 (s, 1H), 7.86 (s, 1H), 7.73 (dd, 1H), 7.64 (d, 1H), 7.55 (d, 1H), 7.30-7.15 (m, 2H), 6.80-6.65 (m, 2H).
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 12.08 (br, 1H), 10.66 (br, 1H), 9.46 (s, 1H), 8.23 (s, 1H), 8.20 (m, 1H), 7.87 (d, 1H), 7.53 (m, 1H), 7.25 (m, 2H), 7.17 (dd, 1H), 6.99 (d, 1H).
1H-NMR (DMSO-d6, mixture of E and Z isomers) δ 11.99 (br, 1H), 10.20 (s, 0.5H), 10.16 (s, 0.5H), 9.44 (s, 0.5H), 8.92 (br, 1H), 8.14 (s, 1H), 8.00 (s, 0.5H), 7.82 (s, 0.5H), 7.68 (d, 0.5H), 7.52 (m, 1H), 7.34-7.15 (m, 3H), 6.72-6.54 (m, 2H).
1H-NMR (DMSO-d6, E isomer) δ 13.00 (br, 1H), 10.63 (s, 1H), 9.46 (s, 1H), 7.95 (s, 1H), 7.56 (d, 1H), 7.19 (t, 1H), 7.00 (t, 1H), 6.86 (d, 1H).
1H-NMR (DMSO-d6, Z isomer) δ 12.03 (s, 1H), 10.54 (s, 1H), 9.43 (s, 1H), 8.20 (dd, 1H), 8.13 (s, 1H), 7.90 (d, 1H), 7.33 (dd, 1H), 7.2-7.05 (m, 2H), 6.99 (t, 1H), 6.85 (d, 1H).
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 12.95 (br, 1H), 10.67 (s, 1H), 8.16 (dd, 1H), 8.08 (d, 1H), 7.80-7.60 (m, 6H), 7.17 (m, 1H), 6.91 (d, 1H), 6.72 (m, 2H).
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 12.13 (br, 1H), 10.81 (s, 1H), 9.50 (s, 1H), 8.24 (s, 1H), 8.21 (m, 1H), 7.93 (d, 1H), 7.55 (d, 1H), 7.32 (d, 1H), 7.26 (m, 2H), 6.96 (t, 1H).
1H-NMR (DMSO-d6, E isomer) δ 11.87 (br, 1H), 10.50 (s, 1H), 9.38 (s, 1H), 8.12 (s, 1H), 8.05 (d, 1H), 7.87 (d, 1H), 7.31 (s, 1H), 7.13 (t, 1H), 7.07 (d, 1H), 6.98 (t, 1H), 6.84 (d, 1H), 2.45 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 11.99 (br, 1H), 10.50 (s, 1H), 9.46 (s, 1H), 8.13 (s, 1H), 7.99 (d, 1H), 7.87 (d, 1H), 7.2-7.0 (m, 4H), 6.86 (d, 1H), 2.54 (s, 3H).
13C-NMR (DMSO-d6, E isomer) δ 169.2, 141.8, 140.5, 135.8, 128.3, 127.7, 125.7, 123.9, 122.8, 122.3, 121.4, 120.4, 120.3, 119.8, 111.4, 109.1, 108.2, 12.6.
1H-NMR (DMSO-d6, Z isomer) δ 10.55 (s, 1H), 9.43 (s, 1H), 8.10 (dd, 1H), 8.09 (s, 1H), 7.92 (d, 1H), 7.60 (dd, 1H), 7.2-7.1 (m, 2H), 6.99 (t, 1H), 6.85 (d, 1H), 3.95 (s, 3H).
1H-NMR (DMSO-d6, Z isomer) δ 12.07 (s, 1H), 10.53 (s, 1H), 9.48 (s, 1H), 8.12 (s, 1H), 8.07 (dd, 1H), 7.92 (d, 1H), 7.53 (dd, 1H), 7.2-6.95 (m, 3H), 6.84 (d, 1H).
13C-NMR (DMSO-d6, mixture of E and Z isomers) δ 169.7, 168.1, 155.0, 154.7, 141.6, 139.0, 134.0, 131.2, 130.8, 130.2, 129.1, 128.1, 127.9, 127.6, 126.5, 125.9, 122.3, 120.8, 120.4, 118.7, 118.4, 113.3, 112.9, 112.4, 111.4, 110.5, 109.5, 108.9, 100.8, 55.6.
13C-NMR (DMSO-d6 mixture of E and Z isomers) δ 169.6, 168.1, 154.0, 153.8, 141.6, 139.0, 137.5, 134.0, 131.4, 130.9, 130.2, 129.1, 128.3, 128.0, 127.7, 127.6, 127.5, 127.4, 127.3, 126.5, 125.8, 122.3, 122.1, 120.7, 120.3, 118.6, 118.4, 113.4, 113.2, 112.9, 112.7, 111.3, 110.4, 109.4, 108.8, 102.3, 70.0, 69.8.
13C-NMR (DMSO-d6, mixture of E and Z isomers) δ 169.7, 168.1, 156.5, 141.7, 139.1, 137.2, 136.7, 132.6, 128.8, 128.2, 127.9, 127.3, 126.7, 125.8, 122.3, 121.3, 120.9, 120.5, 119.7, 119.1, 119.0, 118.6, 111.4, 110.8, 110.6, 109.5, 109.0, 95.4, 55.3.
13C-NMR (DMSO-d6, mixture of E and Z isomers) δ 169.7, 168.0, 155.3, 155.0, 141.5, 139.0, 137.2, 133.6, 131.9, 131.6, 129.6, 128.4, 128.0, 127.2, 126.8, 126.5, 125.8, 122.3, 120.8, 120.3, 118.6, 118.2, 112.8, 112.2, 111.6, 111.4, 110.3, 109.3, 108.8, 101.0, 55.7, 55.4, 33.5, 33.3.
13C-NMR (DMSO-d6, mixture of E and Z isomers) δ 169.6, 167.9, 156.7, 141.6, 139.1, 137.8, 137.4, 136.0, 132.3, 128.1, 127.0, 126.6, 125.7, 122.6, 122.3, 122.2, 121.8, 120.8, 120.4, 119.6, 119.2, 118.9, 118.6, 110.7, 110.5, 109.7, 109.4, 108.9, 94.2, 55.5, 33.3, 33.1.
13C-NMR (DMSO-d6, mixture of E and Z isomers) δ 167.7, 153.8, 138.8, 137.6, 137.2, 137.0, 132.9, 131.7, 130.0, 128.8, 128.6, 128.3, 128.2, 127.3, 126.7, 126.3, 125.8, 123.5, 123.3, 121.0, 120.7, 120.3, 118.3, 117.2, 116.4, 112.0, 108.9, 105.9, 102.9, 69.9, 69.1.
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 12.26 (br, 1H), 10.84 (br, 1H), 9.56 (s, 1H), 8.41 (s, 1H), 8.24 (m, 1H), 8.11 (d, 1H), 7.55 (m, 1H), 7.45 (d, 1H), 7.27 (m, 2H), 7.18 (s, 1H).
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 11.76 (br, 2H), 9.50 (s, 1H), 8.34 (s, 1H), 8.22 (m, 2H), 7.6-7.45 (m, 2H), 7.27 (m, 2H), 7.14 (t, 1H).
1H-NMR (DMSO-d6, Z isomer) δ 12.15 (br, 1H), 11.02 (br, 1H), 9.49 (s, 1H), 8.46 (s, 1H), 8.41 (s, 1H), 8.27 (m, 1H), 7.55 (m, 2H), 7.27 (m, 2H), 7.00 (d, 1H).
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 12.08 (br, 1H), 10.98 (br, 1H), 9.49 (s, 1H), 8.24 (s, 1H), 8.20 (m, 1H), 7.76 (d, 1H), 7.54 (m, 1H), 7.25 (m, 2H), 7.00 (m, 2H).
1H-NMR (DMSO-d6, mixture of E and Z isomers, signals of the predominant isomer) δ 12.15 (br, 1H), 10.78 (br, 1H), 9.54 (s, 1H), 8.38 (s, 1H), 8.24 (m, 1H), 8.12 (d, 1H), 7.55 (m, 1H), 7.34 (d, 1H), 7.27 (m, 2H), 7.08 (s, 1H).
1H-NMR (DMSO-d6) δ 10.85 (br, 1H), 9.57 (s, 1H), 8.40 (s, 1H), 8.22 (br, 1H), 8.13 (br, 1H), 7.55 (br, 2H), 7.26 (br, 3H), 3.20 (s, 3H).
1H-NMR (DMSO-d6, Z isomer) δ 12.14 (br, 1H), 10.89 (br, 1H), 9.51 (s, 1H), 8.44 (s, 1H), 8.37 (s, 1H), 8.32 (m, 1H), 7.54 (m, 1H), 7.47 (d, 1H), 7.26 (m, 2H), 7.00 (d, 1H).
1H-NMR (DMSO-d6, Z isomer) δ 11.87 (br, 1H), 10.24 (s, 1H), 9.36 (s, 1H), 8.20 (m, 1H), 7.99 (s, 1H), 7.58 (s, 1H), 7.50 (m, 1H), 7.22 (m, 2H), 6.49 (s, 1H), 3.83 (s, 3H), 3.77 (s, 3H)
1H-NMR (DMSO-d6, Z isomer) δ 12.19 (br, 1H), 10.76 (br, 1H), 9.48 (s, 1H), 8.18 (d, 1H), 7.84 (m, 1H), 7.56 (m, 1H), 7.27 (m, 2H), 7.16 (m, 1H), 6.65 (dd, 1H)
1H-NMR (DMSO-d6, Z isomer) δ 11.99 (br, 1H), 10.28 (br, 1H), 9.45 (s, 1H), 8.22 (m, 1H), 8.15 (s, 1H), 7.57 (s, 1H), 7.51 (m, 1H), 7.24 (m, 2H), 6.73 (m, 2H), 3.81 (s, 3H)
1H-NMR (DMSO-d6, Z isomer) δ 12.06 (br, 1H), 10.65 (br, 1H), 9.44 (s, 1H), 8.21 (s, 1H), 8.18 (m, 1H), 7.91 (d, 1H), 7.53 (m, 1H), 7.25 (m, 2H), 7.03 (dd, 1H), 6.86 (d, 1H)
13C-NMR (DMSO-d6, mixture of E and Z isomers) δ 169.3, 166.7, 151.2, 149.8, 141.6, 139.6, 135.0, 134.7, 128.0, 127.5, 126.8, 125.6, 124.2, 123.9, 122.2, 122.2, 121.1, 120.7, 120.3, 120.2, 120.1, 119.9, 118.5, 110.1, 109.2, 109.0, 108.7, 100.5, 99.5, 55.1, 51.0, 50.3, 45.8, 29.6.
13C-NMR (DMSO-d6, mixture of E and Z isomers) δ 169.0, 167.3, 156.3, 155.3, 142.4, 139.9, 137.6, 136.8, 133.4, 130.2, 129.2, 127.6, 125.6, 125.5, 125.0, 124.5, 124.4, 123.6, 122.7, 121.8, 121.4, 120.9, 120.7, 118.8, 109.8, 108.9, 16.6, 16.4.
13C-NMR (DMSO-d6) δ 169.0, 156.0, 142.5, 138.8, 137.8, 137.5, 130.2, 129.3, 126.9, 125.4, 124.4, 121.9, 121.3, 120.6, 109.9, 34.6, 30.1.
1H-NMR (DMSO-d6) δ 10.62 (br, 1H), 7.72 (d, 2H), 7.66 (d, 2H), 7.56 (s, 1H), 7.48 (d, 1H), 7.24 (t, 1H), 6.86 (m, 2H).
1H-NMR (DMSO-d6) δ 10.60 (br, 1H), 7.68 (s, 1H), 7.54 (d, 1H), 7.40-7.05 (m, 5H), 6.86 (d, 1H), 6.77 (t, 1H), 2.30 (s, 3H).
1H-NMR (DMSO-d6) δ 10.57 (br, 1H), 7.64 (s, 1H), 7.47 (d, 1H), 7.30-7.05 (m, 4H), 6.85 (d, 1H), 6.78 (t, 1H), 2.34 (s, 3H), 2.27 (s, 3H).
1H-NMR (DMSO-d6) δ 10.7 (br, 1H), 7.65 (d, 2H), 7.53 (dd, 1H), 7.42 (s, 1H), 7.23 (t, 1H), 6.80 (t, 1H), 6.66 (d, 1H), 6.48 (d, 1H).
1H-NMR (DMSO-d6) δ 10.64 (br, 1H), 7.95 (d, 1H), 7.78 (d, 1H), 7.70 (dd, 1H), 7.56 (s, 1H), 7.41 (d, 1H), 7.25 (t, 1H), 6.86 (m, 2H).
1H-NMR (DMSO-d6) δ 10.62 (br, 1H), 7.73 (d, 2H), 7.58 (m, 3H), 7.48 (d, 1H), 7.23 (t, 1H), 6.86 (m, 2H).
13C-NMR (DMSO-d6, E isomer) δ 168.3, 142.6, 139.1, 136.8, 134.7, 131.0, 130.7, 129.8, 128.3, 127.7, 126.4, 122.2, 121.1, 121.0, 109.9, 20.8, 19.3.
13C-NMR (DMSO-d6, E isomer) δ 168.3, 142.8, 134.8, 134.8, 133.6, 133.5, 130.3, 129.9, 128.5, 128.3, 122.2, 121.1, 121.1, 110.0, 20.4, 18.9.
1H-NMR (DMSO-d6) δ 10.47 (br, 1H), 7.43 (m, 2H), 7.16 (t, 1H), 6.80 (m, 5H), 3.75 (s, 6H).
1H-NMR (DMSO-d6) δ 10.60 (br, 1H), 7.70 (d, 2H), 7.64 (s, 1H), 7.50 (m, 4H), 7.23 (t, 1H), 6.85 (m, 2H).
1H-NMR (DMSO-d6) δ 10.54 (br, 1H), 7.71 (d, 2H), 7.65 (d, 1H), 7.58 (s, 1H), 7.22 (t, 1H), 7.09 (d, 2H), 6.87 (m, 2H), 3.85 (s, 3H).
13C-NMR (DMSO-d6, E isomer) δ 168.8, 162.6, 159.3, 142.4, 131.6, 130.5, 129.3, 125.2, 121.9, 121.3, 120.9, 115.3, 109.8, 105.3, 98.4, 55.7, 55.5.
1H-NMR (DMSO-d6) δ 10.59 (br, 1H), 7.59 (d, 1H), 7.56 (s, 1H), 7.23 (t, 1H), 6.90-6.83 (m, 4H), 6.61 (m, 1H), 3.79 (s, 6H).
13C-NMR (DMSO-d6, E isomer) δ 168.6, 152.5, 151.8, 142.8, 131.4, 129.9, 127.5, 123.3, 122.4, 121.0, 121.0, 116.8, 114.4, 112.6, 110.0, 55.9, 55.5.
1H-NMR (DMSO-d6) δ 10.56 (br, 1H), 7.59 (d, 1H), 7.57 (s, 1H), 7.46 (m, 2H), 7.29 (d, 1H), 7.20 (t, 1H), 6.85 (m, 2H), 2.30 (s, 3H), 2.29 (s, 3H).
1H-NMR (DMSO-d6) δ 10.63 (br, 1H), 8.28 (s, 1H), 8.06 (d, 1H), 8.03-7.97 (m, 2H), 7.82 (m, 2H), 7.64-7.52 (m, 3H), 7.24 (t, 1H), 6.90 (d, 1H), 6.85 (t, 1H).
1H-NMR (DMSO-d6) δ 10.67 (br, 1H), 8.07 (m, 3H), 7.93 (m, 1H), 7.83 (d, 1H), 7.66 (d, 1H), 7.61 (m, 2H), 7.18 (t, 1H), 6.89 (m, 2H), 6.68 (t, 1H).
1H-NMR (DMSO-d6) δ 10.60 (br, 1H), 7.64 (s, 1H), 7.36 (d, 1H), 7.26-7.17 (m, 4H), 6.82 (m, 2H), 3.87 (s, 3H), 3.75 (s, 3H).
1H-NMR (DMSO-d6) δ 10.71 (br, 1H), 9.39 (s, 1H), 8.64 (d, 1H), 8.27 (dd, 1H), 7.97 (s, 1H), 7.76 (m, 2H), 7.26 (t, 1H), 7.03 (t, 1H), 6.85 (d, 1H).
1H-NMR (DMSO-d6) δ 10.65 (br, 1H), 7.76 (t, 1H), 7.54 (m, 2H), 7.37 (m, 2H), 7.24 (m, 2H), 6.86 (m, 2H).
13C-NMR (CDCl3) δ 169.9, 138.4, 137.9, 134.8, 130.5, 129.9, 129.8, 128.6, 126.4, 123.1, 121.9, 110.0, 21.4.
13C-NMR (CDCl3, E isomer) δ 170.3, 162.8, 142.0, 137.0, 135.6, 130.4, 130.3, 128.7, 125.1, 123.2, 122.0, 121.3, 116.5, 115.9, 110.5.
1H-NMR (DMSO-d6) δ 10.46 (br, 1H), 7.77 (d, 1H), 7.63 (d, 2H), 7.51 (s, 1H), 7.19 (t, 1H), 7.03 (d, 2H), 6.87 (m, 2H).
1H-NMR (DMSO-d6) δ 10.74 (br, 1H), 8.78 (s, 1H), 8.35 (s, 1H), 8.21 (d, 2H), 7.98 (d, 2H), 7.62-7.48 (m, 4H), 7.07 (t, 1H), 6.84 (d, 1H), 6.39 (t, 1H), 5.65 (d, 1H).
1H-NMR (DMSO-d6) δ 10.58 (s, 1H), 7.72 (d, 1H), 7.56 (s, 1H), 7.46 (dd, 1H), 7.38 (d, 1H), 7.23 (t, 1H), 6.94 (d, 1H), 6.88 (m, 2H).
1H-NMR (DMSO-d6) δ 10.53 (s, 1H), 10.16 (br, 1H), 7.69 (s, 1H), 7.62 (d, 1H), 7.50 (d, 1H), 7.38-7.12 (m, 2H), 7.00-6.78 (m, 4H).
13C-NMR (CDCl3, mixture of the E and Z isomers) δ 170.2, 167.8, 152.7, 151.8, 139.9, 139.6, 139.3, 137.9, 132.6, 131.8, 131.2, 129.7, 129.3, 128.2, 127.3, 127.1, 127.0, 126.6, 125.8, 124.4, 123.3, 123.0, 119.4, 111.0, 110.5, 34.3, 34.2, 23.8, 23.7.
1H-NMR (DMSO-d6) δ 10.58 (br, 1H), 7.72 (d, 1H), 7.64 (d, 2H), 7.60 (s, 1H), 7.23 (dd, 1H), 6.86 (m, 3H), 3.05 (s, 6H).
1H-NMR (DMSO-d6) δ 10.84 (br, 1H), 8.83 (s, 1H), 8.23 (d, 1H), 7.92 (s, 1H), 7.78 (m, 1H), 7.33 (s, 1H), 7.28 (d, 1H), 6.85 (d, 1H).
1H-NMR (DMSO-d6) δ 10.73 (br, 1H), 7.69 (s, 1H), 7.39 (d, 1H), 7.28 (dd, 1H), 7.23 (d, 1H), 7.15 (m, 2H), 6.88 (d, 1H), 3.81 (s, 3H), 3.75 (s, 3H)
1H-NMR (DMSO-d6) δ 10.82 (br, 1H), 7.77 (m, 1H), 7,68 (m, 2H), 7.54 (m, 2H), 7.30 (dd, 1H), 7.01 (d, 1H), 6.90 (d, 1H).
1H-NMR (DMSO-d6) δ 10.75 (br, 1H), 7.71 (s, 1H), 7.28-7.22 (m, 5H), 6.88 (d, 1H), 3.88 (s, 3H), 3.75 (s, 3H).
1H-NMR (DMSO-d6) δ 10.55 (br, 1H), 7.70 (d, 2H), 7.57 (m, 2H), 7.31 (dd, 1H), 6.91 (d, 1H), 6.39 (d, 1H).
1H-NMR (DMSO-d6) δ 7.78 (s, 1H), 7.59 (dd, 1H), 7.42 (d, 1H), 7.33 (t, 1H), 7.25 (dd, 1H), 6.99 (d, 1H), 6.94 (t, 1H), 6.88 (d, 1H), 5.3 (br, 2H).
1H-NMR (DMSO-d6) δ 10.70 (br, 1H), 7.73 (s, 1H), 7.68 (d, 2H), 7.57 (d, 1H), 7.28 (dd, 1H), 7.12 (d, 2H), 6.89 (d, 1H), 3.86 (s, 3H).
1H-NMR (DMSO-d6) δ 10.80 (br, 1H), 8.42 (d, 1H), 7.94 (s, 0.5H), 7.85 (s, 0.5H), 7.74 (d, 1H), 7.68 (s, 0.5H), 7.62 (, 1H), 7.55 (d, 1H), 7.38 (d, 0.5H), 7.25 (m, 1H), 6.89 (d, 0.5H), 6.83 (d, 0.5H).
1H-NMR (DMSO-d6) δ 10.82 (br, 1H), 8.18 (s, 1H), 8.10 (m, 2H), 7.91 (d, 1H), 7.84 (d, 1H), 7.73-7.57 (m, 3H), 7.24 (dd, 1H), 6.89 (d, 1H), 6.77 (d, 1H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.75 (br, 1H), 8.33 (d, 1H), 7.92 (s, 0.5H), 7.85 (s, 0.5H), 7.76 (d, 1H), 7.67 (m, 2.5H), 7.38 (d, 0.5H), 7.29 (m, 1H), 6.90 (d, 0.5H), 6.83 (d, 0.5H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.72 (br, 1H), 8.33 (d, 1H), 7.90 (s, 0.5H), 7.85 (s, 0.5H), 7.69 (s, 0.5H), 7.61 (d, 1H), 7.50 (d, 0.5H), 7.37 (d, 1H), 7.28 (m, 2H), 6.89 (d, 0.5H), 6.82 (d, 0.5H), 2.40 (s, 1.5H), 2.38 (s, 1.5H).
1H-NMR (DMSO-d6) δ 10.89 (br, 1H), 8.82 (s, 1H), 8.47 (s, 1H), 8.23 (d, 2H), 7.97 (d, 2H), 7.62-7.54 (m, 4H), 7.13 (dd, 1H), 6.86 (d, 1H), 5.52 (d, 1H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.78 (br, 1H), 8.86 (s, 0.5H), 8.60 (d, 0.5H), 8.29 (s, 0.5H), 8.18-7.74 (m, 5H), 7.63 (m, 2H), 7.48 (d, 0.5H), 7.28 (m, 1H), 6.92 (d, 0.5H), 6.86 (d, 0.5H).
1H-NMR (DMSO-d6) δ 10.65 (br, 1H), 7.69 (s, 1H), 7.65 (d, 1H), 7.43 (d, 1H), 7.25 (dd, 1H), 6.87 (d, 1H), 6.73 (m, 2H), 3.87 (s, 6H).
1H-NMR (DMSO-d6) δ 10.84 (br, 1H), 7.88 (m, 1H), 7.81 (d, 1H), 7.63 (dd, 1H), 7.59 (d, 1H), 7.30 (t, 1H), 7.05 (d, 1H), 6.91 (d, 1H).
1H-NMR (DMSO-d6) δ 10.66 (br, 1H), 8.68 (d, 1H), 7.89 (s, 1H), 7.80 (d, 1H), 7.36 (m, 1H), 7.20 (dd, 1H), 7.09 (d, 1H), 6.83 (d, 1H), 3.86 (s, 3H), 3.84 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.74 (br, 1H), 7.89 (s, 0.5H), 7.83 (d, 0.5H), 7.75 (d, 1H), 7.64 (s, 0.5H), 7.56 (s, 0.5H), 7.29 (dd, 0.5H), 7.25 (dd, 0.5H), 6.86 (m, 2H), 6.64 (t, 1H), 3.80 (s, 6H).
1H-NMR (DMSO-d6) δ 10.64 (s, 1H), 8.44 (s, 1H), 7.68 (s, 1H), 7.6 (br, 1H), 7.54 (s, 2H), 7.26 (dd, 1H), 6.88 (d, 1H), 1.43 (s, 18H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.71 (br, 1H), 8.04 (s, 1H), 7.91 (s, 0.5H), 7.80 (d, 0.5H), 7.73 (d, 0.5H), 7.65 (s, 0.5H), 7.29 (dd, 0.5H), 7.26 (dd, 0.5H), 7.10 (s, 1H), 6.89 (d, 0.5H), 6.85 (d, 0.5H), 3.85 (s, 3H), 3.83 (s, 3H), 3.76 (s, 3H)
1H-NMR (DMSO-d6, mixture of the E and Z isomers) 10.75 (br, 1H), 8.38 (m, 0.5H), 7.95 (s, 0.5H), 7.86 (d, 0.5H), 7.71 (m, 2H), 7.57-7.47 (m, 3H), 7.40 (d, 0.5H), 7.29 (dd, 0.5H), 7.25 (dd, 0.5H), 6.89 (d, 0.5H), 6.83 (d, 0.5H).
1H-NMR (DMSO-d6) δ 10.71 (br, 1H), 9.77 (br, 1H), 7.64 (s, 1H), 7.49 (d, 1H), 7.35 (t, 1H), 7.27 (t, 1H), 7.07 (m, 2H), 6.89 (m, 2H).
1H-NMR (DMSO-d6) δ 10.72 (br, 1H), 7.73 (s, 1H), 7.65 (dd, 1H), 7.53 (t, 1H), 7.28 (s, 1H), 7.27 (dd, 1H), 7.20 (dd, 1H), 7.11 (t, 1H), 6.88 (d, 1H), 3.86 (s, 3H).
1H-NMR (DMSO-d6) δ 10.75 (br, 1H), 7.78 (s, 1H), 7.53 (d, 1H), 7.37 (m, 3H), 7.26 (dd, 1H), 6.98 (d, 1H), 6.88 (d, 1H), 2.30 (s, 3H).
13C-NMR (DMSO-d6, mixture of the E and Z isomers) δ 168.3, 167.2, 152.5, 151.8, 143.0, 140.5, 136.0, 133.8, 133.1, 133.0, 130.2, 128.8, 128.6, 128.5, 127.5, 125.9, 124.8, 121.9, 121.1, 121.0, 120.6, 119.5, 111.7, 111.0, 110.2, 109.3.
13C-NMR (DMSO-d6, mixture of the E and Z isomers) δ 168.3, 167.1, 150.3, 149.8, 149.4, 148.7, 143.1, 140.9, 134.2, 133.2, 132.0, 131.6, 130.5, 130.0, 129.3, 128.6, 127.5, 126.4, 124.5, 122.4, 121.2, 121.2, 120.4, 120.0, 119.9, 118.4, 118.0, 117.2, 110.2, 109.5.
13C-NMR (DMSO-d6, E isomer) δ 168.4, 153.7, 142.4, 131.3, 130.9, 129.6, 129.4, 129.2, 128.5, 127.6, 127.0, 124.0, 123.6, 123.1, 121.7, 120.7, 118.1, 113.7, 109.4.
1H-NMR (DMSO-d6, E isomer) δ 10.57 (br, 1H), 7.60 (m, 4H), 7.34 (d, 2H), 7.23 (t, 1H), 6.86 (m, 2H), 2.39 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.54 (br, 1H), 8.68 (d, 1H), 7.84 (dd, 1H), 7.75 (s, 1H), 7.67 (d, 1H), 7.18 (t, 1H), 7.07 (d, 1H), 6.98 (t, 1H), 6.83 (d, 1H), 3.85 (s, 3H), 3.84 (s, 3H).
13C-NMR (DMSO-d6, mixture of the E and Z isomers) δ 168.5, 167.0, 159.2, 158.8, 142.9, 140.7, 136.6, 135.7, 135.5, 135.2, 130.1, 129.8, 129.1, 128.9, 127.7, 126.9, 124.8, 122.5, 121.4, 121.0, 120.8, 119.7, 116.5, 116.4, 115.5, 114.2, 110.1, 109.3, 55.1.
1H-NMR (DMSO-d6, E isomer) δ 10.67 (br, 1H), 7.77 (m, 1H), 7.66 (m, 1H), 7.59 (s, 1H), 7.51 (m, 2H), 7.24 (t, 1H), 7.14 (d, 1H), 6.88 (d, 1H), 6.81 (t, 1H).
1H-NMR (DMSO-d6, E isomer) δ 10.65 (br, 1H), 7.74 (s, 1H), 7.68 (m, 1H), 7.59 (s, 1H), 7.55 (m, 2H), 7.41 (d, 1H), 7.25 (t, 1H), 6.87 (m, 2H).
1H-NMR (DMSO-d6, E isomer) δ 10.69 (br, 1H), 7.83 (d, 1H), 7.79 (d, 1H), 7.58 (dd, 1H), 7.51 (s, 1H), 7.25 (t, 1H), 7.14 (d, 1H), 6.88 (d, 1H), 6.83 (t, 1H).
1H-NMR (DMSO-d6, E isomer) δ 10.66 (br, 1H), 7.95 (d, 1H), 7.79 (d, 1H), 7.71 (dd, 1H), 7.56 (s, 1H), 7.42 (d, 1H), 7.26 (t, 1H), 6.87 (m, 2H).
1H-NMR (DMSO-d6, E isomer) δ 10.63 (br, 1H), 7.87 (s, 1H), 7.69 (m, 2H), 7.59 (s, 1H), 7.49 (t, 1H), 7.40 (d, 1H), 7.25 (t, 1H), 6.87 (m, 2H).
1H-NMR (DMSO-d6, E isomer) δ 10.51 (br, 1H), 10.13 (br, 1H), 7.70 (d, 1H), 7.62 (d, 2H), 7.54 (s, 1H), 7.21 (t, 1H), 6.89 (m, 4H).
1H-NMR (DMSO-d6, E isomer) δ 10.58 (br, 1H), 9.72 (br, 1H), 7.59 (d, 1H), 7.55 (s, 1H), 7.33 (t, 1H), 7.23 (t, 1H), 7.10 (m, 2H), 6.87 (m, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.43 (br, 1H), 10.08 (br, 2H), 7.69 (s, 1H), 7.63 (d, 1H), 7.55 (d, 1H), 7.18 (t, 1H), 6.86 (m, 2H), 6.44 (d, 1H), 6.38 (dd, 1H).
1H-NMR (DMSO-d6, E isomer) δ 10.71 (br, 1H), 7.69 (s, 1H), 7.39 (d, 1H), 7.28 (dd, 1H), 7.24 (d, 1H), 7.11 (m, 2H), 6.89 (d, 1H), 3.81 (s, 3H), 3.75 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.57 (br, 1H), 8.02 (s, 2H), 7.76 (s, 1H), 7.68 (d, 1H), 7.21 (t, 1H), 7.00 (t, 1H), 6.85 (d, 1H), 3.85 (s, 6H), 3.76 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.59 (br, 1H), 7.59 (d, 1H), 7.56 (s, 1H), 7.23 (t, 1H), 6.93-6.81 (m, 4H), 6.61 (t, 1H), 3.79 (s, 6H).
1H-NMR (DMSO-d6, E isomer) δ 10.60 (br, 1H), 7.64 (s, 1H), 7.37 (d, 1H), 7.27-7.15 (m, 4H), 6.85 (m, 2H), 3.87 (s, 3H), 3.75 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.62 (br, 1H), 8.11 (d, 1H), 7.98 (m, 1H), 7.83 (s, 1H), 7.61 (m, 2H), 7.43 (m, 2H), 7.12 (t, 1H), 6.84 (d, 1H), 6.59 (t, 1H), 6.20 (d, 1H), 3.93 (s, 3H)
13C-NMR (DMSO-d6, E isomer) δ 168.5, 157.6, 142.7, 131.6, 131.6, 129.8, 129.4, 127.3, 122.9, 122.2, 121.1, 121.0, 120.2, 111.5, 110.0, 55.6.
1H-NMR (DMSO-d6, E isomer) δ 10.50 (br, 1H), 8.69 (d, 1H), 7.74 (m, 1H), 7.69 (s, 1H), 7.65 (d, 1H), 7.16 (t, 1H), 7.0-6.8 (m, 3H), 3.86 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.52 (br, 1H), 9.37 (br, 1H), 7.73 (d, 1H), 7.50 (s, 1H), 7.20 (m, 3H), 7.06 (d, 1H), 6.88 (m, 2H), 3.85 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.72 (br, 1H), 7.68 (s, 1H), 7.54 (d, 1H), 7.40 (dd, 1H), 7.24 (d, 1H), 7.11 (m, 2H), 6.84 (d, 1H), 3.81 (s, 3H), 3.76 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.71 (br, 1H), 7.67 (s, 1H), 7.36 (d, 1H), 7.20 (d, 1H), 7.08 (m, 3H), 7.01 (d, 1H), 3.80 (s, 3H), 3.73 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 10.85 (br, 1H), 7.69 (s, 1H), 7.43 (m, 2H), 7.21 (d, 1H), 7.09 (m, 2H), 6.84 (t, 1H), 3.81 (s, 3H), 3.73 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.76 (br, 1H), 7.59 (s, 1H), 7.43 (m, 1H), 7.20 (d, 1H), 7.09 (m, 2H), 6.70 (m, 2H), 3.80 (s, 3H), 3.74 (s, 3H).
13C-NMR (DMSO-d6, E isomer) δ 168.4, 152.6, 151.9, 145.8, 133.6, 127.0, 126.1, 122.5, 121.4, 121.3, 118.8, 118.4, 113.8, 113.0, 110.2, 55.8, 55.5.
1H-NMR (DMSO-d6, E isomer) δ 10.08 (s, 1H), 7.51 (s, 1H), 7.22 (d, 1H), 7.06 (m, 2H), 6.87 (d, 1H), 6.56 (d, 1H), 6.47 (dd, 1H), 4.66 (br, 2H), 3.81 (s, 3H), 3.74 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 11.10 (br, 0.5H), 10.81 (br, 0.5H), 7.86 (s, 0.5H), 7.73 (d, 1H), 7.25-6.88 (m, 3.5H), 5.00 (s, 1H), 4.88 (s, 1H), 3.81 (s, 3H), 3.75 (s, 3H).
13C-NMR (DMSO-d6, E isomer) δ 168.5, 152.5, 151.7, 135.1, 131.0, 128.1, 123.3, 121.6, 116.9, 116.4, 114.0, 112.6, 110.3, 109.9, 55.9, 55.4
1H-NMR (DMSO-d6, E isomer) δ 11.01 (br, 1H), 8.21 (s, 1H), 7.88 (d, 1H), 7.73 (s, 1H), 7.31 (d, 1H), 7.12 (m, 2H), 6.98 (d, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.77 (s, 3H).
13C-NMR (DMSO-d6, E isomer) δ 169.1, 142.6, 140.3, 140.1, 137.8, 129.5, 127.5, 126.4, 125.0, 124.9, 122.7, 122.4, 122.1, 121.6, 121.5, 121.0, 120.7, 119.6, 110.0, 109.6, 109.4, 37.3, 13.8.
1H-NMR (DMSO-d6, E isomer) δ 10.57 (s, 1H), 9.41 (s, 1H), 7.70 (s, 1H), 7.51 (d, 1H), 7.21 (m, 2H), 7.08 (d, 1H), 6.88 (m, 3H), 3.86 (s, 3H).
13C-NMR (DMSO-d6, E isomer) δ 166.1, 152.5, 152.0, 143.6, 137.6, 135.9, 122.1, 118.8, 116.7, 116.5, 111.8, 105.0, 56.2, 55.3.
13C-NMR (DMSO-d6, E isomer) δ 168.2, 150.8, 142.9, 130.3, 130.0, 129.8, 128.8, 127.6, 125.5, 122.8, 122.6, 122.4, 121.1, 120.6, 110.1.
1H-NMR (DMSO-d6, E isomer) δ 10.56 (br, 1H), 7.62 (s, 1H), 7.45 (d, 1H), 7.21 (m, 2H), 7.1-6.95 (m, 2H), 6.86 (m, 2H), 4.06 (q, 2H), 3.98 (q, 2H), 1.31(t, 3H), 1.27 (t, 3H).
13C-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 168.9, 149.4, 142.6, 132.7, 129.6, 126.1, 122.6, 121.5, 121.3, 121.0, 119.0, 116.9, 114.8, 109.9.
1H-NMR (DMSO-d6, E isomer) δ 10.52 (br, 1H), 7.66 (s, 1H), 7.61 (d, 1H), 7.28 (s, 1H), 7.20 (t, 1H), 6.88 (m, 2H), 6.82 (s, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.71 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.58 (s, 1H), 8.57 (s, 1H), 8.21 (d, 1H), 7.91 (dd, 1H), 7.86 (s, 1H), 7.77 (m, 2H), 7.67 (d, 1H), 7.53 (t, 1H), 7.27 (t, 1H), 7.23 (t, 1H), 6.89 (m, 2H), 3.95 (s, 3H).
13C-NMR (DMSO-d6, E isomer) δ 168.4, 155.4, 142.9, 139.9, 130.4, 130.1, 127.8, 124.2, 122.3, 122.2, 122.0, 120.9, 120.8, 117.0, 110.1.
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant isomer) δ 11.44 (br, 1H), 10.56 (br, 1H), 7.98 (s, 1H), 7.79 (m, 2H), 7.49 (m, 3H), 7.21 (t, 1H), 6.88 (d, 2H), 6.56 (d, 1H).
1H-NMR (DMSO-d6) δ 11.44 (br, 1H), 10.62 (br, 1H), 7.94 (s, 1H), 7.56 (d, 1H), 7.49 (m, 2H), 7.38 (d, 1H), 7.25 (d, 1H), 7.20 (t, 1H), 6.88 (d, 1H), 6.79 (t, 1H), 6.42 (s, 1H).
1H-NMR (DMSO-d6, E isomer) δ 11.45 (br, 1H), 10.62 (br, 1H), 7.98 (s, 1H), 7.70 (d, 1H), 7.53 (d, 1H), 7.39 (t, 1H), 7.31 (d, 1H), 7.18 (m, 2H), 6.88 (d, 1H), 6.78 (t, 1H), 6.56 (m, 1H).
1H-NMR (DMSO-d6, E isomer) δ 11.52 (br, 1H), 10.58 (br, 1H), 8.21 (d, 1H), 7.96 (s, 1H), 7.60 (d, 1H), 7.46 (m, 2H), 7.31-7.10 (m, 3H), 6.87 (d, 1H), 6.77 (t, 1H), 2.80 (s, 3H), 2.57 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.52 (br, 1H), 8.94 (s, 0.5H), 8.00 (s, 0.5H), 7.69 (s, 0.5H), 7.62 (d, 0.5H), 7.54 (m, 1H), 7.48-7.31 (m, 4H), 7.28 (s, 0.5H), 7.18 (m, 1H), 7.0-6.8 (m, 3.5H), 5.28 (s, 1H), 5.23 (s, 1H), 3.86 (s, 3H), 3.77 (s, 1.5H), 3.71 (s, 1.5H).
13C-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 167.9, 143.4, 135.0, 132.0, 131.5, 131.5, 131.0, 130.8, 130.6, 129.9, 129.7, 122.6, 121.5, 120.4, 110.5.
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 11.53 (br, 1H), 7.75 (s, 1H), 7.67 (d, 1H), 7.59 (d, 1H), 7.20 (d, 1H), 7.15-7.06 (m, 2H), 7.03 (t, 1H), 3.80 (s, 3H), 3.73 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 10.92 (br, 1H), 7.82 (s, 1H), 7.65 (d, 1H), 7.45 (d, 1H), 7.32 (s, 1H), 7.24 (s, 1H), 7.18-7.03 (m, 2H), 3.82 (s, 3H), 3.75 (s, 3H), 3.20 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 11.09 (br, 1H), 7.76 (s, 1H), 7.70 (dd, 1H), 7.66 (m, 1H), 7.25 (d, 1H), 7.15-7.08 (m, 2H), 7.03 (d, 1H), 3.81 (s, 3H), 3.76 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 10.85 (br, 1H), 7.79 (s, 1H), 7.62 (d, 1H), 7.26 (m, 2H), 7.16-7.03 (m, 3H), 3.82 (s, 3H), 3.74 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 11.08 (br, 1H), 7.70 (s, 1H), 7.29 (d, 1H), 7.22 (d, 1H), 7.15 (m, 1H), 7.10 (m, 2H), 6.89 (m, 1H), 3.81 (s, 3H), 3.74 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 10.90 (br, 1H), 7.81 (s, 1H), 7.56 (d, 1H), 7.35 (dd, 1H), 7.21 (m, 2H), 7.12 (m, 2H), 3.81 (s, 3H), 3.74 (s, 3H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 10.72 (br, 1H), 7.64 (s, 1H), 7.42 (d, 1H), 7.20 (d, 1H), 7.11-7.05 (m, 2H), 6.94 (dd, 1H), 6.88 (d, 1H), 3.80 (s, 3H), 3.73 (s, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.70 (br, 1H), 7.89 (d, 1H), 7.76 (d, 1H), 7.62 (dd, 1H), 7.43 (s, 1H), 7.25 (t, 1H), 6.99 (d, 1H), 6.84 (t, 1H), 6.83 (d, 1H).
1H-NMR (DMSO-d6, E isomer) δ 10.60 (br, 1H), 7.77 (d, 1H), 7.62 (dd, 1H), 7.53 (s, 1H), 7.29 (d, 1H), 7.23 (t, 1H), 7.13 (d, 1H), 6.86 (m, 2H), 4.12 (q, 2H), 1.28 (t, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.60 (br, 1H), 7.77 (d, 1H), 7.65 (dd, 1H), 7.52 (s, 1H), 7.28 (d, 1H), 7.23 (t, 1H), 7.15 (d, 1H), 6.86 (m, 2H), 3.85 (s, 3H).
1H-NMR (CDCl3, E isomer) δ 8.07 (br, 1H), 7.77 (s, 1H), 7.49 (d, 1H), 7.24-7.14 (m, 2H), 7.11 (t, 1H), 6.98-6.86 (m, 3H), 3.80 (s, 3H).
1H-NMR (CDCl3, mixture of the E and Z isomers, signals of the predominant E isomer) δ 7.90 (br, 1H), 7.70 (s, 1H), 7.40 (m, 1H), 7.30-7.01 (m, 4H), 6.89 (m, 2H).
1H-NMR (DMSO-d6, E isomer) δ 10.64 (s, 1H), 8.97 (d, 1H), 8.24 (dd, 1H), 7.84 (d, 1H), 7.82 (s, 1H), 7.76 (d, 1H), 7.29 (t, 1H), 7.02 (t, 1H), 6.85 (d, 1H).
1H-NMR (CDCl3, E isomer) δ 7.97 (m, 3H), 7.88 (m, 1H), 7.83 (d, 1H), 7.24 (t, 1H), 6.88 (m, 2H), 6.80 (t, 1H).
1H-NMR (DMSO-d6, Z isomer) δ 10.61 (br, 1H), 8.15 (d, 1H), 7.72 (m, 3H), 7.46 (dd, 1H), 7.26 (t, 1H), 7.00 (t, 1H), 6.82 (d, 1H).
13C-NMR (DMSO-d6) δ 159.8, 155.5, 147.7, 146.6, 142.3, 130.6, 129.1, 120.1, 119.8, 118.8, 116.9, 115.6, 115.1, 110.6, 55.8.
13C-NMR (DMSO-d6, mixture of the E and Z isomers) δ 169.0, 167.4, 142.5, 139.9, 139.3, 138.3, 135.5, 129.8, 129.4, 129.1, 128.9, 128.1, 127.8, 127.1, 126.8, 125.6, 124.9, 124.4, 123.3, 121.8, 121.4, 120.9, 120.8, 120.7, 120.1, 119.3, 119.0, 115.9, 113.2, 109.9, 109.0, 101.7.
13C-NMR (DMSO-d6, E isomer) δ 168.4, 157.1, 152.6, 151.8, 139.1, 133.0, 127.3, 122.8, 122.0, 117.2, 116.1, 114.3, 112.8, 110.7, 109.3, 55.9, 55.5.
13C-NMR (DMSO-d6) δ 168.8, 159.3, 157.1, 146.9, 142.6, 137.1, 135.7, 134.3, 131.4, 129.8, 129.7, 128.5, 127.9, 127.2, 127.1, 126.6, 125.8, 122.0, 121.0, 121.0, 119.6, 115.1, 114.5, 109.9, 70.9.
1H-NMR (DMSO-d6) δ 8.49 (d, 1H), 8.08-7.86 (m, 4H), 7.78-7.48 (m, 6H), 7.29-7.10 (m, 3H), 6.85 (m, 2H), 5.38 (s, 2H).
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.51 (br, 1H), 8.49 (d, 0.5H), 8.35 (d, 0.5H), 8.29 (d, 0.5H), 7.98-7.82 (m, 2.5H), 7.80-7.50 (m, 4.5H), 7.46 (s, 0.5H), 7.35 (d, 0.5H), 7.20 (t, 1H), 7.09 (d, 0.5H), 6.89-6.72 (m, 2H), 5.29 (s, 1H), 5.23 (s, 1H).
1H-NMR (DMSO-d6) δ 13.11 (br, 1H), 10.54 (s, 1H), 7.70 (d, 2H), 7.64 (d, 1H), 7.57 (s, 1H), 7.22 (t, 1H), 7.02 (d, 2H), 6.87 (m, 2H), 4.96 (q, 1H), 1.54 (d, 3H).
13C-NMR (DMSO-d6, mixture of the E and Z isomers) δ 168.6, 167.1, 147.9, 147.5, 144.0, 143.4, 143.0, 140.6, 138.7, 138.6, 136.5, 135.4, 133.1, 132.5, 130.1, 128.9, 128.2, 128.0, 127.6, 126.9, 125.8, 124.9, 123.5, 122.9, 122.6, 121.2, 121.0, 120.7, 119.8, 119.4, 115.6, 114.9, 113.5, 110.1, 109.2, 42.7, 42.5, 32.6, 32.4, 30.1, 19.2, 19.1, 13.5, 13.5.
ES+(MSQ14914):[M+H]+32 496; ES−(MSQ14914):[M−H]−=494.
1H-NMR (DMSO-d6, mixture of the E and Z isomers) δ 10.63 (br, 1H), 8.62 (d, 0.5H), 8.01 (d, 0.5H), 7.82 (s, 0.5H), 7.77 (d, 0.5H), 7.59 (d, 0.5H), 7.55 (s, 0.5H), 7.51 (m, 1.5H), 7.38-7.12 (m, 5.5H), 7.07-6.96 (m, 1.5H), 6.95-6.80 (m, 3.5H), 6.32 (m, 1H), 6.20 (d, 0.5H), 6.10 (d, 0.5H), 5.78 (t, 0.5H), 5.56 (t, 0.5H), 4.36 (t, 2H).
1H-NMR (DMSO-d6) δ 10.59 (br, 1H), 8.49 (d, 1H), 7.97 (d, 1H), 7.85 (s, 1H), 7.78 (d, 1H), 7.35-7.10 (m, 5H), 7.00 (m, 2H), 6.93-6.78 (m, 3H), 5.22 (q, 1H), 2.74 (d, 3H).
1H-NMR (DMSO-d6, E isomer) δ 10.60 (br, 1H), 8.77 (s, 1H), 8.23 (s, 1H), 7.85 (s, 1H), 7.77 (d, 1H), 7.54 (br, 2H), 7.06 (m, 6H), 7.00 (t, 1H), 6.85 (d, 1H), 4.35 (s, 2H), 4.01 (q, 2H), 1.17 (t, 3H).
1H-NMR (DMSO-d6, E isomer) δ 13.04 (br, 1H), 10.58 (br, 1H), 7.74 (s, 1H), 7.71 (d, 1H), 7.45 (m, 2H), 7.22 (t, 1H), 7.09 (m, 2H), 6.84 (m, 2H), 4.82 (s, 2H).
1H-NMR (DMSO-d6, E isomer) δ 10.55 (br, 1H), 7.74 (m, 3H), 7.65 (d, 1H), 7.58 (s, 1H), 7.34 (d, 1H), 7.22 (m, 2H), 7.18 (d, 2H), 6.87 (m, 2H), 5.22 (s, 2H), 2.50 (s, 3H).
1H-NMR (DMSO-d6) δ 10.63 (br, 1H), 8.11 (s, 1H), 7.68-7.60 (m, 4H), 7.25 (dd, 1H), 7.13 (d, 2H), 6.88 (d, 1H), 3.51 (m, 4H), 3.45-3.34 (m, 4H).
13C-NMR (CDCl3, mixture of the E and Z isomers) δ 170.2, 167.8, 152.7, 151.8, 139.9, 139.6, 139.3, 137.9, 132.6, 131.8, 131.2, 129.7, 129.3, 128.2, 127.3, 127.1, 127.0, 126.6, 125.8, 124.4, 123.3, 123.0, 119.4, 111.0, 110.5, 34.3, 34.2, 23.8, 23.7.
13C-NMR (DMSO-d6, E isomer) δ 169.2, 161.0, 151.5, 142.4, 136.6, 131.6, 129.2, 124.1, 124.0, 121.9, 121.5, 120.9, 114.7, 109.9, 48.0, 46.8, 44.3. 13C-NMR (DMSO-d6, Z isomer) δ 167.5, 161.0, 151.7, 139.9, 137.3, 134.4, 127.6, 125.8, 124.5, 122.2, 120.7, 118.8, 113.9, 109.0, 47.7, 46.5, 44.3.
1H-NMR (DMSO-d6, E isomer) δ 11.14 (br, 1H), 7.75 (s, 1H), 7.25 (t, 1H), 7.21 (d, 1H), 7.12 (m, 2H), 7.00 (dd, 1H), 3.81 (s, 3H), 3.74 (s, 3H).
1H-NMR (DMSO-d6) δ 10.52 (s, 1H), 8.88 (d, 1H), 8.41 (d, 1H), 8.09 (d, 1H), 7.83 (t, 1H), 7.72 (t, 1H), 7.67-7.53 (m, 4H), 7.50 (s, 1H), 7.20 (t, 1H), 7.07 (d, 2H), 6.83 (m, 2H), 6.44 (q, 1H), 1.75 (d, 3H).
1H-NMR (DMSO-d6) δ 10.55 (br, 1H), 8.61 (d, 2H), 7.72 (d, 2H), 7.63 (d, 1H), 7.58 (s, 1H), 7.47 (d, 2H), 7.23 (t, 1H), 7.18 (d, 2H), 6.87 (m, 2H), 5.29 (s, 2H).
The title compound was obtained as a brown solid following the general procedure 5 using 4-bromomethyl-pyridine; hydrobromide (63 mg, 0.25 mmole, 1 eq.) and K2CO3 (70 mg, 0.5 mmole, 2 eq.): 45 mg, 50% yield.
1H NMR (DMSO-d6, Z isomer) δ 14.25 (s, 1H), 12-10 (bs, 1H), 8.60 (m, 2H), 7.85 (d, 1H), 7.72 (s, 1H), 7.47 (m, 2H), 7.21 (bt, 1H), 7.03 (bt, 1H), 6.95-6.88 (m, 2H), 5.40 (s, 2H), 2.37 (s, 3H).
The title compound was obtained as an orange/red solid following the general procedure 5 using bromomethyl-benzene (30 μL, 0.25 mmole, 1 eq.) and K2CO3 (53 mg, 0.37 mmole, 1.5 eq.): 56 mg, 61% yield.
13C NMR (DMSO-d6) δ 169.4, 166.9, 138.4, 136.2, 133.9, 132.8, 130.5, 129.3, 128.5, 128.4, 126.6, 122.6, 122.4, 121.1, 120.0, 115.4, 109.9, 109.6, 68.2, 13.3, 12.9.
To a hot dark red solution of 5-amino-3-(2,5-dimethoxy-benzylidene)-1,3-dihydro-indol-2-one (0.70 g, 2.4 mmoles) in THF (35 mL) was added methanesulfonic acid (0.15 mL, 2.4 mmoles) and the formation of a yellow precipitate was observed. The precipitate was filtered and dried to afford the title compound as a yellow/orange solid (0.83 g, 90% yield).
13C-NMR (DMSO-d6, mixture of the E and Z isomers, signals of the predominant E isomer) δ 168.4, 152.7, 151.8, 142.3, 133.3, 126.6, 124.8, 124.6, 122.8, 121.9, 117.9, 117.2, 113.7, 112.9, 110.6, 55.9, 55.5, 39.6.
Materials and Methods
Compounds: Compound 226=3-(3,5-dimethyl-1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one; Compound A=3-[2,4-dimethyl-5-(oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid; Compound B=a semisynthetic analogue of fumagillol, CAS RN 129298-91-5.
Peptide The following peptide from myelin proteolipid protein was used; PLP139-153 H-HCLGKWLGHPDKFVG-OH. The peptide was synthesized by Fmoc chemistry (Schafer-N, Copenhagen, Denmark). Purity (>95%) was verified by reversed-phase HPLC and integrity by mass spectrometry.
Mice Female SJL/J (H-2s) inbred mice purchased from Charles River.
Immunization The SJL/J mice (about 8 weeks old) were immunized on day 0 with the PLP139-153 peptide (dissolved in sterile NaCl) emulsified 1:1 (vol/vol) in Complete Freund's Adjuvant (5 mg Mycobacterium tuberculosis/ml) (SSI, Copenhagen, Denmark). Intradermal injections corresponding to 100 μg peptide and 125 μg Mycobacterium tuberculosis were given at the base of the tail in a total volume of 50 μl. The mice were additionally given an i.v. injection with 100 ng pertussis toxin (Sigma) dissolved in sterile NaCl on day 0 and day 2, injection volume was 100 μl.
Compound treatment Groups of 10 mice were dosed daily with compound 226 (from 4 mg/kg to 50 mg/kg), compound A (from 50 mg/kg to 200 mg/kg) or compound B (5 mg/kg to 10 mg/kg) in suspension vehicle (4 g Tween-80, 2 g Carboxy-methyl cellulose 7H4XF, 8 g NaCl, 1 liter H2O), starting on day 0 unless otherwise specified. Control groups were given either suspension vehicle or dexamethasone (Dexadreson Vet, Intervet, Holland). Suspension vehicle was given from day 0 i.p. whereas dexamethasone (0.5 or 1 mg/kg) was given p.o. from day 1.
Clinical evaluation Mice were weighed and assessed clinically daily from day 5 p.i. according to the following criteria: 0, no disease; 1, tail paralysis; 2, clumsy gait/poor righting ability and limb weakness; 3, moderate or total hind limb paralysis; 4, moribund state or dead.
Statistics
Area-under-curve (AUC) of the disease score was calculated for all mice. The medians of AUC of all groups were compared using Kruskal-Wallis test. When P<0.05 in the Kruskal-Wallis test, the Mann-Whitney test was used to compare drug treated groups with the suspension vehicle treated control group (P<0.05). AUC were calculated from day 0 to the termination of the individual experiment (day 21, day 28 or day 35).
Results
In four separate experiments, compound 226 (50 mg/kg) was shown to significantly inhibit EAE induced in SJL/J mice with a peptide from myelin proteolipid protein when compared with the suspension vehicle group (table 1,
From the data obtained in experiment 3 (table 1,
Area-under-curve of the disease score for four individually experiments. In each experiment a suspension vehicle group was included. AUC of the disease score were calculated for all groups and the Mann-Whitney test was used to compare the drug treated groups with the suspension vehicle group.
*This experiment was terminated on day 21.
**This experiment was terminated on day 21.
***This experiment was terminated on day 28.
****This experiment was terminated on day 35.
#AUC for the experiments are calculated from day 0 to termination of the individual experiment.
Inhibition of IL-2 Production in Spleen Cells of Mice Dosed with Compound 226
Materials and Methods
Peptide The following peptide from myelin proteolipid protein was used; PLP139-153 H-HCLGKWLGHPDKFVG-OH. The peptide was synthesized by Fmoc chemistry (Schafer-N, Copenhagen, Denmark). Purity (>95%) was verified by reversed-phase HPLC and integrity by mass spectrometry.
Mice Female SJL/J (H-2s) inbred mice purchased from Charles River.
Immunization The SJL/J mice (about 8 weeks old) were immunized on day 0 with the PLP139-153 peptide (dissolved in sterile NaCl) emulsified 1:1 (vol/vol) in Complete Freund's Adjuvant (5 mg Mycobacterium tuberculosis/ml) (SSI, Copenhagen, Denmark). Intradermal injections corresponding to 100 μg peptide and 125 μg Mycobacterium tuberculosis were given at the base of the tail in a total volume of 50 μl. The mice were additionally given an i.v. injection with 100 ng pertussis toxin (Sigma) dissolved in sterile NaCl on day 0 and day 2, injection volume was 100 μl.
Compound treatment A group of 6 mice were dosed i.p. daily for 10 days with compound 226 (50 mg/kg), in suspension vehicle (4 g Tween®-80, 2 g carboxymethylcellulose 7H4XF, 8 g NaCl, 1 liter H2O), starting on day 0. A control group was given suspension vehicle daily.
Clinical evaluation Mice were weighed and assessed clinically daily from day 5 p.i. according to the following criteria: 0, no disease; 1, tail paralysis; 2, clumsy gait/poor righting ability and limb weakness; 3, moderate or total hind limb paralysis; 4, moribund state or dead.
In vitro restimulation Ten days after immunization individual spleens were collected and single-cell suspensions were prepared in RPMI (Bio Whittaker) supplemented with 1% mouse serum, 1% penicillin/streptomycin (Gibco) and 1% L-glutamine (Gibco). The erythrocytes were lysed by 0.83% NH4Cl treatment. Subsequently the cells were washed and plated out (5×105 cells/well) on 96-well flat-bottomed microtiter plates (Nunclon, NUNC, Denmark). Different concentrations of the PLP139-153 peptide were added to the wells. Control wells did not contain the PLP139-153 peptide. Cells from each mouse were plated with or without 10−7M of compound 226 on the microtiter plate. The plates were incubated at 37° C. for 3 days. Afterwards 100 μl of the supernatant from each well was transferred to a corresponding 96-well flat-bottomed microtiter plate (Maxisorp, NUNC, Denmark) coated with 50 μl/well of an anti-IL-2 antibody (2 μg/ml) (PharMingen, Becton Dickinson) in coating buffer (sodium bicarbonate, 0.1 mol/L, pH 9.5). The Maxisorp plates were blocked with blocking buffer (10% FCS in PBS) and washed with (0.05% Tween®-20 in PBS) before addition of the supernatant. After incubation overnight at 4° C. the plates were washed and 100 μl of biotinylated anti-IL-2 antibody (1 μg/ml) (PharMingen, Becton Dickinson), diluted in blocking buffer, was added to each well. After 45 min. incubation at room temperature, the plates were washed and incubated for 2 hrs. at room temperature on a plate-shaker with 100 μl/well of Eu3+-labeled streptavidin (Wallac, Perkin Elmer) diluted 1/1000 in assay buffer (Wallac, Perkin Elmer). After washing, the plates were incubated for 10 min. on a plate-shaker with 180 μl enhancement solution (Wallac, Perkin Elmer) which releases europium from streptavidin and forms a highly fluorescent micellar solution. Finally, fluorescence was measured in a time-resolved fluorometer VICTOR (Wallac, Perkin Elmer).
Conclusion
Two separate in vitro restimulation experiments (one example is given) show that the IL-2 production is markedly inhibited in vivo after dosing with compound 226 (50 mg/kg) for 10 days (
Inhibition of EAE in a Second Model for EAE
Materials and Methods
Compounds: Compound 226=3-(3,5-dimethyl-1H-pyrrol-2-yl-methylene)-1,3-dihydro-indol-2-one
Peptide The following peptide from myelin oligodendrocyte glycoprotein was used; MOG35-55 H-MEVGWYRSPFSRVVHLYRNGK-OH. The peptide was synthesized by Fmoc chemistry (Schafer-N, Copenhagen, Denmark). Purity (>95%) was verified by reversed-phase HPLC and integrity by mass spectrometry.
Mice Female C57BL/6JBom (H-2b) inbred mice purchased from Taconic M&B, Denmark.
Immunization The C57BL/6JBom mice (about 8 weeks old) were immunized at day 0 with the MOG35-55 peptide (dissolved in sterile NaCL) emulsified 1:1 (vol/vol) in Complete Freund's Adjuvant (5 mg Mycobacterium tuberculosis/ml) (Statens Serum Institut, Copenhagen, Denmark). Intradermal injections corresponding to 200 μg peptide and 250 μg Mycobacterium tuberculosis were given at the base of the tail in a total volume of 50 μl. The mice were additionally given an i.v. injection with 100 ng Pertussis Toxin (Sigma) dissolved in sterile NaCl at day 0 and day 2, injection volume was 100 μl.
Compound treatment Groups of 10 mice were dosed daily with compound 226 (50 mg/kg i.p.) in suspension vehicle (4 g Tween-80, 2 g carboxy-methyl cellulose 7H4XF, 8 g NaCl, 1 liter H2O) starting at day 0. Control groups were given either suspension vehicle or dexamethasone (Dexadreson Vet, Intervet, Holland). Suspension vehicle was given from day 0 i.p. whereas dexamethasone (1 mg/kg) was given p.o. from day 1.
Clinical evaluation Mice were weighed and assessed clinically daily from day 5 post immunization (p.i.) according to the following criteria: 0, no disease; 1, tail paralysis; 2, clumsy gait/poor righting ability and limb weakness; 3, moderate or total hind limb paralysis; 4, moribund state or dead.
Statistics
Area under curve (AUC) of the disease score was calculated for all mice. The medians of AUC of all groups were compared using Kruskal-Wallis test. When p<0.05 in the Kruskal-Wallis test, the Mann-Whitney test was used to compare drug treated groups with the suspension vehicle treated control group (p<0.05). AUC were calculated from day 0 to the termination of the experiment (day 21).
Results
The results presented in table 2 demonstrate that compound 226 has a significant effect on disease development in C57BL/6 mice immunized with a peptide from myelin oligodendrocyte glycoprotein, indicating that the effect of compound 226 on EAE is independent of the mouse strain used in the experiment and the immunization protocol used for the induction of EAE.
#Area-under-curve of the disease score. AUC of the disease score were calculated for all groups and the Mann-Whitney test was used to compare the treated groups with the suspension vehicle group.
Inhibition of EAE with Analogues of Compound 226
Materials and Methods
Compounds: Compound 57=3-(1H-indol-3-ylmethylene)-1,3-dihydro-indol-2-one) and Compound 110=3-(2,5-dimethoxybenzylidene)-1,3-dihydro-indol-2-one.
Peptide The following peptide from myelin proteolipid protein was used; PLP139-153 H-HCLGKWLGHPDKFVG-OH. The peptide was synthesized by Fmoc chemistry (Schafer-N, Copenhagen, Denmark). Purity (>95%) was verified by reversed-phase HPLC and integrity by mass spectrometry.
Mice Female SJL/J (H-2s) inbred mice purchased from Charles River or Taconic M&B (Denmark).
Immunization The SJL/J mice (about 8 weeks old) were immunized at day 0 with the PLP139-153 peptide (dissolved in sterile NaCL) emulsified 1:1 (vol/vol) in Complete Freund's Adjuvant (5 mg Mycobacterium tuberculosis/ml) (Statens Serum Institut, Copenhagen, Denmark). Intradermal injections corresponding to 100 μg peptide and 125 μg Mycobacterium tuberculosis were given at the base of the tail in a total volume of 50 μl. The mice were additionally given an i.v. injection with 100 ng Pertussis Toxin (Sigma) dissolved in sterile NaCl at day 0 and day 2, injection volume was 100 μl.
Compound treatment Groups of 10 mice were dosed daily with compound 57 (50 mg/kg) or compound 110 (50 mg/kg) in suspension vehicle (4 g Tween-80, 2 g Carboxy-methyl cellulose 7H4XF, 8 g NaCl, 1 liter H2O), starting at day 0. Control groups were given either suspension vehicle or dexamethasone (Dexadreson Vet, Intervet, Holland). Suspension vehicle was given from day 0 i.p. whereas dexamethasone (1 mg/kg) was given p.o. from day 1.
Clinical evaluation Mice were weighed and assessed clinically daily from day 5 p.i. according to the following criteria: 0, no disease; 1, tail paralysis; 2, clumsy gait/poor righting ability and limb weakness; 3, moderate or total hind limb paralysis; 4, moribund state or dead.
Statistics
Area under curve (AUC) of the disease score was calculated for all mice. The medians of AUC of all groups were compared using Kruskal-Wallis test. When p<0.05 in the Kruskal-Wallis test, the Mann-Whitney test was used to compare drug treated groups with the suspension vehicle treated control group (p<0.05). AUC were calculated from day 0 to the termination of the experiment (day 21).
Results
Analogues of the potent compound 226 were tested in the EAE model in order to further evaluate the in vivo effect of this group of compounds. The results are shown in table 3 from which it appears that the two analogues, compound 57 and compound 110, both show a clear and significant inhibition of the disease. Furthermore, in another experiment compound 110 showed 90% inhibition of EAE when dosed with 50 mg/kg and a clear dose-response course was observed when compound 110 was given in different doses (50 mg/kg, 25 mg/kg, 10 mg/kg, 4 mg/kg), data not shown. There was significant inhibition of EAE by dosing compound 110 as 50 mg/kg, 25 mg/kg and 10 mg/kg with inhibition of disease ranging from 42% with the lowest dose to 90% with the highest dose.
#Area-under-curve of the disease score. A suspension vehicle group was included. AUC of the disease score were calculated for all groups and the Mann-Withney test was used to compare the treated groups with the suspension vehicle group.
Inhibition of EAE with Further Oxindole Compounds
Materials and Methods
Compounds: The compounds shown in Table 4 below dosed in an amount of 50 mg/kg.
The immunization and treatment of the mice was otherwise carried out as described in example 4.
Results
A series of oxindole compounds were tested in the EAE model to further evaluate the in vivo effect of this group of compounds. The results are shown in table 4. The inhibitory effects shown in Table 4 are all significant (p<0.05). The oxindole compounds inhibit EAE with varying effects ranging from 34% to 96% inhibition.
*Area-under-curve of the disease score. A suspension vehicle group was included in all experiments. AUC of the disease score were calculated for all groups and the Mann-Withney test was used to compare the treated groups with the suspension vehicle group.
Inhibition of Pro-inflammatory Cytokines
Materials and Methods
Peptide The following peptide from myelin proteolipid protein was used; PLP139-153 H-HCLGKWLGHPDKFVG-OH. The peptide was synthesized by Fmoc chemistry (Schafer-N, Copenhagen, Denmark). Purity (>95%) was verified by reversed-phase HPLC and integrity by mass spectrometry.
Mice Female SJL/J (H-2s) inbred mice purchased from Charles River.
Immunization The SJL/J mice (about 8 weeks old) were immunized at day 0 with the PLP139-153 peptide (dissolved in sterile NaCl) emulsified 1:1 (vol/vol) in Complete Freund's Adjuvant (5 mg Mycobacterium tuberculosis/ml) (Statens Serum Institut, Copenhagen, Denmark). Intradermal injections corresponding to 100 μg peptide and 125 μg Mycobacterium tuberculosis were given at the base of the tail in a total volume of 50 μl. The mice were additionally given an i.v. injection with 100 ng Pertussis Toxin (Sigma) dissolved in sterile NaCl at day 0 and day 2, injection volume was 100 μl.
Compound treatment Groups of 4 mice were dosed i.p. daily for 10 days with compound 226 (50 mg/kg) or compound 57 (50 mg/kg) in suspension vehicle (4 g Tween-80, 2 g Carboxy-methyl cellulose 7H4XF, 8 g NaCl, 1 liter H2O), starting at day 0. A control group were given suspension vehicle daily.
Clinical evaluation Mice were weighed and assessed clinically daily from day 5 p.i. according to the following criteria: 0, no disease; 1, tail paralysis; 2, clumsy gait/poor righting ability and limb weakness; 3, moderate or total hind limb paralysis; 4, moribund state or dead.
In vitro restimulation Ten days after immunization individual spleens were collected and single-cell suspensions were prepared in RPMI (Bio Whittaker) supplemented with 1% mouse serum, 1% penicillin/streptomycin (Gibco) and 1% L-glutamine (Gibco). The erythrocytes were lysed by 0.83% NH4CL treatment. Subsequently the cells were washed and plated out (5×105 cells/well) on 96-well flat-bottomed microtiter plates (Nunclon, NUNC, Denmark). Different concentrations of the PLP139-153 peptide were added to the wells. Control wells were without the PLP139-153 peptide. The plates were incubated at 37° C. for 3 days. Afterwards the supernatants from each mouse were pooled within the respective group and frozen for later analysis. For analysis the supernatants were transferred to a 96-well flat-bottomed microtiter plate (Maxisorp, NUNC, Denmark) coated with 50 μl/well of an anti-cytokine antibody (table 5) in coating buffer (natriumbicarbonate, 0.1 mol/L, pH 9.5). The Maxisorp plates were blocked with blocking buffer (10% FCS in PBS) and washed with (0.05% Tween-20 in PBS) before addition of the supernatant. After incubation O/N at 4° C. the plates were washed and 100 μl of biotinylated anti-cytokine antibody (table 5) diluted in blocking buffer was added to each well. After 45 min. incubation at room temperature, the plates were washed and incubated for 2 hr. at room temperature on a plate-shaker with 100 μl/well of EU3+-labeled streptavidin (Wallac, Perkin Elmer) diluted 1/1000 in assay buffer (Wallac, Perkin Elmer). After washing, the plates were incubated for 10 min. on a plate-shaker with 180 μl enhancement solution (Wallac, Perkin Elmer) which releases europium from streptavidin and forms a highly fluorescent micellar solution. Finally, fluorescence was measured in a time-resolved fluorometer VICTOR (Wallac, Perkin Elmer).
Results
It appears from
Inhibition of IL-2 by Oxindole Compounds
Peripheral blood mononuclear cells (PBMCs) were isolated from human blood. The lymphocytes, a fraction of the PBMCs, were activated to secrete IL-2 using the polyclonal mitogen phytohemagglutinin. The test compounds were added in concentrations from 10−5 to 10−10, and the cells (106/ml) were incubated in microtiter wells at 37° C. for approximately 46 hours.
After 46 hours the cells were centrifuged down for 25 minutes at 1000×g and the supernatants were transferred to transferred to microtiter wells precoated with a monoclonal antibody against human IL-2. The IL-2 concentration in the supernatants was determined by a sandwich ELISA. Microtiter plates were coated (1 μg/ml) with a monoclonal antibody (R&D, UK) against human IL-2, washed 4 times, blocked with 1% casein buffer for 2 hours and washed 2 times. 100 μl sample was added to each well and incubated overnight. All samples were tested in triplicate. 100 μl IL-2 standards (R&D, UK) at a concentration range of 10000-0 pg/ml were tested in triplicate. After incubation the plates were washed and incubated with biotinylated polyclonal secondary antibody against human IL-2 (R&D, UK) for 45 minutes and thereafter washed 4 times. 100 μl enzyme conjugate, horseradish peroxidase conjugated streptavidin diluted 1:4000 (Zymed, USA), was added to all wells and incubated for 30 minutes. 100 μl OPD substrate (KEM EN TEC-DK) was added and the enzyme/substrate reaction stopped after 10 minutes at room temperature with 50 μl 1 M H2SO4. The colour development (optical density—OD) was determined at 492 nm on an ELISA reader and the background OD at 620 nm subtracted).
The results are shown in Table 6 below, including the tested oxindole compounds that may subsequently be subjected to testing in the EAE model as described above in examples 1, 3, 4 or 5. The results are expressed as a percentage of the control (PHA+DMSO) and the potency of the test compound was expressed as the concentration resulting in 50% inhibition of the response of the control stimulated cells (pIC50).
Differential Up- and Downregulation of Genes in EAE Mice Treated with Compound A Compared to Untreated Controls
The mouse spleen is one of the organs where autoimmune cells accumulate, differentiate and maturate prior to entering the CNS via the bloodstream. The process of activation and maturation of inflammatory cells, primarily T-cells, involves activation of antigen presenting cells, e.g. macrophages, which present the antigen to a T-cell receptor and thereby activate a cascade of adhesion molecules, proteases, cytokines and chemokines (O. Neuhaus et al., Trends Pharmacol. Sci. 24, 2003, pp. 131-138). Oligonucleotide DNA arrays have been used to obtain an image of the differences in gene expression in inflammatory cells in the spleen with or without treatment with Compound 226.
RNA Extraction, Probe Synthesis and Hybridization
SJL mice immunized with an encephalitogenic epitope of the proteolipid protein (PLP139-153) were dosed daily with compound 226 from day 0 as described in example 1. Control mice were given suspension vehicle as described in example 1. On day 7 after treatment, the mice (both treated and control) were sacrificed and their spleens were excised, submerged into the RNA storage solution RNA-later (Sigma-Aldrich, St. Louis, USA) and stored at −80° C. Total RNA was extracted from the mouse spleens using the TRIZOL extraction procedure according to the manufacturer's protocol followed by DNAse treatment (Invitrogen, Carlsbad, Calif., USA). The RNA quality was visualized by agarose gel electrophoresis. Ten μg total RNA was converted into purified 33P-labelled cDNA probe according to the Atlas Pure Total RNA labelling protocol (BD Biosciences, Palo Alto, Calif., USA).
Labelled probes (10×106 CPM) were denatured at 95° C. for 2 minutes and cooled at 4° C. for 2 minutes prior to hybridization to oligonucleotide arrays comprising about 5.000 mouse genes in duplicate dots (Atlas Plastic Mouse 5K MicroArray, BD Biosciences, Palo Alto, Calif., USA). Arrays were hybridized for 16 h at 60° C. with rotation according to the manufacturer's protocol. After hybridization and appropriate washing steps, the arrays were imaged using phospho imager technology together with a STORM 860 scanner (Molecular Dynamics, Sunnyvale, Calif., USA). Subsequently, the images were analysed using the Atlas Image 2.7 software (BD Biosciences, Palo Alto, Calif., USA).
Results
Approximately 1.5% of the genes (75 genes) located on the Atlas MicroArray were differentially expressed when arrays from the spleen cDNA of treated and control mice were compared. Several immune response genes were found to be upregulated (class II major histocompatilibility complex [NM—010379, NM—010388]), immunoglobulin-associated beta [NM—008339], transforming growth factor beta-3 [NM—009368], Fc receptor (IgE, high affinity I, gamma polypeptide [NM—010185]) and beta-2 microglobulin [NM—009735]) in the treated group. Genes encoding adhesion molecules (lymphocyte antigens 6 locus D [NM—010742], lymphocyte antigens 6 locus E [NM—008529] and lymphocyte antigens 84 [NM—010743]) were found to be upregulated in the treated group. Genes encoding the interleukin 1 receptor [M20658] and the interleukin 10 receptor alpha [L12120] together with genes encoding tumor necrosis factor related proteins (TNF receptor [M59378] and TNF receptor-associated factor 1 [NM—009421]) were downregulated in the treated group. Upregulation of genes encoding adhesion molecules as well as downregulation of genes involved in the inflammatory response (interleukin and TNF related genes) suggests that maturation and activation of inflammatory cells in the spleen is reduced as is their migration into the bloodstream and further into the CNS. This is in agreement with the observation that treatment with Compound 226 delays the onset of EAE.
Genes related to anti-apoptosis (Bcl-2 like protein [U51279] and elastase [NM—015779]) were found to be downregulated in the treated group allowing apoptosis of inflammatory cells to take place.
Genes having a proposed role in re-myelination were found to be differentially expressed (complement factor [NM—009777], myelin transcription factor [AF004294], fibroblast growth factor 15 [AF007268]) in treated and control mice, indicating a role of compound 226 in the regression of symptoms of EAE and consequent remyelination of axons.
The figures in square brackets above refer to the GenBank Accession No. of the respective genes.
Activity of Oxindole Compounds on Oral Administration
Materials and Methods
Compounds: The compounds shown in Table 7 below.
All compounds were dosed p.o. at 50 mg/kg. In other respects, immunization and treatment of the mice was carried out as described in example 4.
Results
A series of oxindole compounds were administered orally in the EAE model. The results are shown in Table 7 below. The inhibitory effects shown in the table are significant (p<0.05). The oxindole compounds inhibit EAE with varying effects ranging from 32% to 67% inhibition.
Conclusion
Several oxindole analogues have the potential to inhibit EAE disease when given orally.
*Area-under-curve of the disease score. A suspension vehicle group was included in all experiments. AUC of the disease score were calculated for all groups and the Mann-Withney test was used to compare the treated groups with the suspension vehicle group.
**In these experiments compounds 110 and 211 were dosed in a suspension vehicle adjusted to pH 4 by addition of aqueous HCl.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DK04/00875 | 12/16/2004 | WO | 1/3/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60529630 | Dec 2003 | US |
