MXPA06008296A - Indol-alanine derivatives as selective s1p4-agonists - Google Patents

Abstract

The present invention relates to agonists of the S1P4 receptor, which are selective for the S1P4 receptor over one or more of the S1P1, S1P2, S1P3 or S1P5 receptors of at least 10 fold, in particular new indol-alanine derivatives of structure (I), process for their production, their uses, in particular in transplantation, and pharmaceutical compositions containing them (I) wherein R1 is phenyl or naphthyl, wherein phenyl is substituted by one or two of halogen, C1-6alkyl, C1-6alkoxy or phenylC1-6alkyl;and R2 is hydrogen or C1-6alkyl;in free or salt form.

Description

INDOL-ALANINE DERIVATIVES AS SELECTIVE S1P4 AGONISTS The present invention relates to organic compounds, a process for their production and pharmaceutical compositions containing them. In one aspect, the present invention provides a compound which is a sphingosine-1-phosphate (S1P4) receptor agonist, wherein the compound possesses selectivity for the S1P4 receptor of more than one or more of the S1P1, S1P2, S1P3 receptors. or S1P5. S1P receptors are described, for example, in WO 03/061567. Preferably the compound is selective for the S1P4 receptor on each of the above S1P receptors. The compound preferably exhibits a selectivity of at least 10 times, preferably 20 times, more preferably 100 times for the S1P4 receptor on one or more of the above S1P receptors. The selectivity can be measured by determining the ratio of the EC50 of the compound for the S1P4 receptor to the EC50 of the compound for the S1P1 receptor, S1P2, S1P3 or S1P5. The EC50 values can be obtained, for example, according to the GTP? 35S binding assay or calcium mobilization assay described below. In another preferred embodiment, the compound also shows an EC50 value for the S1P4 receptor of 1 μM or less in the GTP? 35S binding assay or calcium mobilization assay. In a preferred embodiment, the present invention relates to a compound of formula I wherein Ri is phenyl or naphthyl, wherein the phenyl is substituted by one or two of halogen; alkyl d-6, alkoxy C? -6 or phenyl-alkyl C-6; and R 2 is hydrogen or C 1-6 alkyl; in free form or salt. Any alkyl radical can be linear or branched. The C1-6 alkyl is preferably C1- alkyl. The C 1-6 alkoxy is preferably C 1-4 alkoxy. Halogen can be F, Cl, Br, or I. R1 is preferably a group of formula la: wherein one or two of R3, R4 and R5 is hydrogen; and one or two of R 3, R 4 and Rs, is C 1-6 alkyl, C 1-6 alkoxy or phenyl C 1-6 alkyl; or R3 and R4 or R and R5, together with the carbon atoms to which they are attached, together form a benzene ring. In formulas I and the following meanings are preferred independently, collectively or in any combination or sub-combination: a) one of R or R5 is otherwise hydrogen, more preferably R5 is otherwise hydrogen and R4 is hydrogen; b) R4 or R5 is benzyl, ethyl, butoxy, propyl, isopropyl or chloro, more preferably R5 is one of the aforementioned groups and R is hydrogen; b) R3 is hydrogen or chloro, more preferably hydrogen; c) R2 is hydrogen or methyl. The compounds of formula I can also form inorganic or organic acid or acid addition salts, for example hydrochloric, hydrobromic or maleic acid. The compounds of formula 1 can also form cationic salts derived from the carboxy group, more particularly alkali metal or alkaline earth metal salts, for example sodium, potassium, calcium or magnesium salt, and ammonium salts derived from ammonia or organic amines. The compounds of formula I contain a chiral center at the carbon atom that leads to the primary amino group. The compound of formula I will therefore exist in two enantiomeric forms. It is to be understood that the present invention comprises the racemate of the formula I and any enantiomeric form. The compounds of formula I can also be obtained in the form of their hydrates. Hydrates also form part of the invention. The present invention also provides a process for the production of the compounds of formula I, which comprises deprotecting a compound of formula 11 wherein R -i and R2 are as defined above, R6 is C1-6 alkyl or benzyl, R7 is an amino protecting group, and and if it is desired to convert the compound of formula I I into a salt thereof. The process can be carried out using conventional methods. Examples of suitable amino protecting groups are for example as described in "Protective Groups in Organic Synthesis" T.W. Greene, J.Wiley &; Sons NY, 2d0 is. , chapter 7, 1 991, and references thereto, for example, acyl, for example tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, trifluoroacetyl, trimethylsilylethanesulfonyl and the like. The deprotection can be effected, for example, by hydrogenolysis, when R7 is benzyloxycarbonyl and R6 is benzyl. The reaction can then be carried out in an organic solvent such as tetrahydrofuran, methylene chloride or dioxane at room temperature using palladium-carbon as a catalyst. If R6 is C1-6 alkyl, the process is conveniently carried out in two stages. In the first step the carboxy group of a compound of formula II can be deprotected by light hydrolysis with for example aqueous NaOH or a basic ion exchange resin in an organic solvent such as tetrahydrofuran, dioxane, methanol or ethanol at room temperature. In the second step the amino group is deprotected with for example iodotrimethylsilane in methylene chloride. If R7 is benzyloxycarbonyl, hydrogenolysis can be used. Stage two of the process is suitably used when one or two of R3 to R5 are halogen. The optional formation of a salt can be carried out conventionally. A racemic mixture of the compound of formula I can be resolved in a known manner, for example using an optically active acid as a resolving agent. Alternatively, a pure enantiomeric form can be produced using optically active starting materials. The compounds of formula I I used as starting material can be prepared with for example, by reacting a compound of formula I I I wherein R6 and R are as defined above, with a compound of formula IV wherein Ri and R2 are as defined above, and X is a leaving group. The reaction can be carried out in a conventional manner. For example, the reaction can be carried out in an organic solvent such as methylene chloride at a temperature between -1.5 ° to 25 °. The leaving group X is, for example, halogen, especially chlorine or hydroxy. Conveniently an acid binder, for example a tertiary amine such as pyridine, is present. As far as the production of the starting materials for the above processes is not particularly described, they can be produced in an analogous manner by known compounds or the processes described herein. In the following Examples all temperatures are given degrees centigrade and are uncorrected. The values [a] D20 are also uncorrected. Example 1 : 3- (4- (2-eti Ifen il) -2-carboxamido-idol) -D-alanine a) 4- (2-ethylphenyl) -indole-2-carboxylic acid To a mixture of 0.28 of 4-acid bromo-indole-2-carboxylic acid and 0.069 of tetrakistriphenylphosphine palladium in 1 ml of toluene and 2 ml of 2M soda is added a solution of 0.300 of 2-ethylphenylboronic acid in 3 ml of ethanol. This mixture was refluxed for 16 h, filtered and the aqueous phase was acidified with 2N HCl and extracted with ethyl acetate. The concentration of the organic phase gave the product as a brownish powder, m.p. 230 - 233 °, sufficiently pure for the next stage. b) 3- (4- (2-ethylphenyl) -2-carboxamido-indole) -N (2) -ZD-alanine methyl ester To a solution of 2.1 1 g of 4- (2-ethylphenyl) -indoI-2 acid - carboxylic acid in 32 ml of pyridine was added 1.441 g of carbonyldiimidazole. After finishing the gas evolution, 2.29 of N (2) -Z-D-2,3-diaminopropionate methyl were added and the mixture was stirred at room temperature for 4 days. After evaporation of the solvent the residue was extracted with water / ethyl acetate, the organic phase was dried, concentrated and the crude product was chromatographed on silica with isopropyl acetate / toluene 4: 1, yielding the desired compound as an amorphous powder yellow. c) 3- (4- (2-Ethylphenyl) -2-carboxamido-indole) -D-alanine A mixture of 2.03 g of 3- (4- (2-eti-phenyl) -2-carboxamido-indole) methyl ester) -N (2) -ZD-alanine, 4.1 ml of 2N NaOH and 10 ml of dioxane were stirred at room temperature for 2 h. After working in a conventional manner, the acid obtained was dissolved in 22 ml of methylene chloride and treated with 1.03 ml of iodotrimethylsilane at 0 ° C. After stirring for 50 min. , the mixture was concentrated to dryness and the residue was taken in water. The crude product was precipitated and collected by filtration, washed with water and recrystallized from water / isopropanol under pH adjustment to 6 by the addition of 0.2N NaOH, yielding the title compound as a white powder, pf 258-265 ° C. The compounds of formula I wherein R-i and R2 are as defined in Table 1, can be prepared by following one of the above procedures but using the appropriate starting materials.

TABLE 1 The compounds described herein which are selective S 1 P 4 receptor agonists (hereinafter referred to as the compounds of the invention), for example the compounds of formula I, in free form or in pharmaceutically acceptable salt form , exhibit valuable pharmacological properties, for example modulating properties of lymphocyte recirculation, for example as indicated in in vitro and in vivo tests and are therefore indicated for therapy. In particular, the compounds of the invention are useful as functional agonists of human S1 P4 receptors (EDG6), as demonstrated in the following tests.

Assay for determining the in vitro pharmacology of the S1 P4 compounds a) Expression vector encoding the human S1 P receptors An expression vector for the human S1 P4 gene (HSEDG4; GenBank Accession Number AJ000479) fused to the peptide tag c -myc tag in the C-terminal is obtained as described in Van Brockiyn et al. 2000, Blood 95 (8), 2624. The coding DNA for the S1 P4-myc fusion protein is cloned into the mammalian expression vector pRc / CMV (Invitrogen), which confers the G418 resistance for cell selection stable mammals. The sequence of Insert DNA S1 P4 is determined in both strands. No nucleotide difference leading to the amino acid changes is found with respect to the GenBank entry for HSEDG4. The S1 P4-myc cDNA is inserted into the pRc / CMV vector using Hindl l / Xbal.

The following vectors are used. The human S1 P1 (GenBank ™ access number M31210) and S 1 P3 (GenBank ™ accession number X83864) of the cDNA containing the myc sequence in front of the human S 1 P 1 and S 1 P 3 sequences are inserted into the cDNA 3.1 vector. The vectors are sequenced to finish and confirm the correctness of the construct. Human S1 P2 (Gl: 4090955, TREM BL: 01 951 36) is cloned by a PCR-based method. The lung cDNA (Marathon cDNA) is obtained (BD Biosciences Chontech, Palo Alto, CA 94303, USA). The following oligonucleotides are used for the PCR reaction: direct CAC primer CAT GGG CAG CTT GTA CTC GGA GTA CCT GAA CCC CAA CAA GGT CCA G (1 to 45, GenBank ™ accession number AF034780) and reverse primer 5'-GAT TCA GAC CAC CGT GTT GCC CTC CAG (1 062 to 1 039, GenBank ™ access number AF034780), translation initiation (ATG) and termination codon are underlined. PCR is performed in the presence of 0.2-0.4 μg of cDNA, 1 x reaction buffer (PfuTurbo 1 0x DNA polymerase buffer, La Jolla, CA 92037, USA), 0.5 μM of primers, 0.25 mM dNTP's and 2.5 units PfuTurbo DNA polymerase (Stratagene), a first stage at 95 ° C for 2 minutes, 30 cycles (30 seconds at 95 ° C, 30 seconds at 60 ° C and 90 seconds at 72 ° C) and the final stage of 10 minutes at 72 ° C. The product amplified around 1 1 00 bp is analyzed by gel-electrophoresis of standard agarose. After cloning the PCR product into the cDNA 3.1 Topo V (Invitrogen Corporation) the DNA inserts from different bacterial colonies are sequenced. The final sequence is assembled from three independent plasmid preparations that are sequenced in both directions. The human S 1 P5: (Gl: 30171332 or GenBank ™ access number AY262689, TREMBL: Q9H228) is cloned by a method based 1 in PCR. The lung and spleen (Marathon cDNA) are obtained from BD Biosciences Chlontec (BD Biosciences Chlontech, Palo Alto, CA 94303, USA); Genomic DNA is isolated from HeLa cells using standard procedures. The following oligonucleotides are used for the PCR reaction: the direct CAC CAT GGA GTC GGG GCT GCT GCG (-4 to 20, GenBankTM access number AY262689) and reverse primer 5'-TCA GTC TGC AGC CGG TTC TGA TAC CAG AGT C (1 1 97 to 1 1 31, AY262689), initiation of translation ( ATG) and the termination codon are underlined. PCR is performed in the presence of 0.2-0.4 μg of cDNA, 1 x reaction buffer (10x DNA polymerase buffer PfuTurbo, Stratagene, La Jolla, CA 92037, USA), 0.5 μM of primers, 0.25 mM dNTP's and 2.5 units of PfuTurbo DNA polymerase (Stratagene), a first stage at 95 ° C for 2 minutes , 30 cycles (30 seconds at 95 ° C, 30 seconds at 60 ° C and 90 seconds at 72 ° C) and the final stage of 10 minutes at 72 ° C. The amplified product of about 1 100 bp is analyzed by standard agarose gel-electrophoresis. After the cloning of the PCR products in the cDNA 3.1 Topo V (Invitrogen Corporation) the DNA inserts of different bacterial colonies are sequenced. The final sequence is assembled from three independent plasmid preparations that are sequenced in both directions. b) Development of a stable CHO-K1 cell line expressing S 1 P4 To develop stable cell lines expressing the S 1 P4 receptor labeled c-myc 5 μg of plasmid pRc / CMV myc-S 1 P4 is cut using the restriction of the Pvul endonuclease, which dissociates the plasmid once. The linearized plasmid is then precipitated using a final concentration of 0.3M sodium acetate, pH5.0 and 66% ethanol. After centrifugation and washing, the DNA pellet is dissolved in 30 μl of water. For transfection, CHO-K1 cells (ATCC number: CCL-61) are grown in plates in MEM medium (modification of minimal essential alpha medium) containing 1 0% FCS (fetal calf serum) the day before transfection at a cell density of 1 x 106 cells per well of the 100-mm cell culture dish (Falcon) and incubate at 37 ° C in 5% of a CO2 atmosphere for 24 hours. On the day of transfection, 5 μg of linearized pRC / CMV myc-S1 P4 plasmid is added to 270 μl of RPMl medium. After adding 60 μl of SuperFect Transfection Reagent (Qiagen) to the DNA solution and mixing for 10 seconds, the sample is incubated for 10 minutes to leave the complex formation. The DNA-SuperFect complex is mixed with an additional 3 ml of RPM / 10% FCS and then transferred to the cell monolayer. After 3 hours of incubation, the transfection medium is removed and the cells are washed with PBS. The fresh RPMl medium supplemented with 10% FCS is added and the cells are incubated for 72 hours at 37 ° C and 5% CO2. The selection for the transformed clones is made after adding 500 μg / ml of G41 8 (Lifetechnologies). After approximately 12-14 days, the cell clones obtained by sorting individual cells according to their forward and reverse scattering characteristics in individual wells of a 96-well cell culture plate using a FACStar Plus cell separator (Becton Dickinson) . The individual clones growing after the sorting and selection procedure are expanded and a clone is finally selected based on the expression level of S 1 P 4 mRNA determined by the quantitative TaqMan PCR assay. c) Cell culture maintenance The parental CHO-K1 cells are maintained in the RPMl medium (Lifetechnologies), supplemented with 10% FB'S and 10 μg / ml gentamicin (Lifetechnologies). The S1 P4 / myc cell transfected CHO-K1, including a finally selected clone is maintained in alpha MEM medium, supplemented with 10% FBS, 10 μg / ml gentamicin and 0.5 mg / ml G418 (Lifetechnologies). d) Determination of transcription levels of S1 P4 in stable clones of CHO The total RNA of cells is isolated with the help of! RNeasy Mini kit (Qiagen). After disruption of cells (grown in a monolayer) directly in culture dishes (for a 35 mm dish, 350 μl of buffer are used, prepared according to the Qiagen protocol), the lysate is pipetted onto a Qiashredder rotating column and centrifuged for 2 minutes at 21 O00 xg. 350 μl of 70% ethanol are added to continuous flow, from the mixed well, applying to a RNeasy Mini column, and centrifuged at 1 5 seconds at 1 0O00 xg. The continuous flow is eliminated, 700 μl of RW1 buffer (Qiagen) are added to the RNeasy column and centrifuged for 15 seconds to clarify the column. The column is further washed by adding twice 500 μl of RPE buffer (Qiagen) and centrifuging for 1 5 seconds at 1 0000 xg. The RNA is eluted by adding 30-50 μl of RNAse-free water in the RNeasy column and centrifuging for 1 minute at 10O00 xg.

The RNA is reverse transcribed with the Omniscript kit from Qiagen (Qiagen). About 2 μg of RNA are mixed with 2 μl of 1 0 x buffer, 2 μl of dNTP of Mix, 1 μl of RNase inhibitor (10 units / μl), 0.5 μg of random hexamer, 1 μl of Omniscript Reverse Transcriptase and RNase-free water at a final volume of 20 μl, and incubated at 37 ° C for 60 minutes and an additional 30 minutes at 42 ° C. The reaction is inactivated for 5 minutes at 93 ° C, cooled on ice and stored at -20 ° C until use.

To determine the relative transcription levels of S1 P4 mRNA in the selected clones, real-time PCR analysis (polymerase chain reaction) is performed. The optimal primer and test concentrations are determined as described in the User's bulletin provided by the PE Biosystems (Perkin / Elmer) using a human S1 P4 containing the plasmid as a template. The human S1 P4 oligonucleotides as well as their concentrations optimized for real-time PCR are the following (based on HSEDG4; accession number GenBank AJ000479): The first strand of cDNA is then used for quantitative PCR in the TaqMan AB1 7700 machine (Biosystems PE). As an internal control of the amount of RNA present in each sample, the 18S ribosomal RNA is used by multiplexing the reaction of S1 P4 PCR with the ribosomal RNA PCR in the same tube using the control reagents of the TaqMan with the ribosomal RNA (PE Biosystems, P / N430831 0, test marked VIC). Although the similar efficacy of the two independent amplifications for S 1 P4 and ribosomal RNA is not formally determined, the semi-quantitative determination of the transcription levels of S1 P4 in different cell clones is sufficient for the selection of cell clones for also be characterized in functional tests. e) Membrane preparation Membrane proteins are prepared from wild type CHO-K1 and CHO cell clones expressing S1 P4. human. To obtain 1 0-30 mg of membrane proteins, the cells are grown in a large culture dish (500 cm2) per cell clone at 80 and 90% confluence. The culture medium is removed and the cells are harvested on ice from the dish, scraping in 20 ml of cold 10 mM HEPES (pH 7.5) supplemented with 0.1% fatty acid free bovine serum albumin (BSA) and a cocktail of inhibitors. of protease (one tablet per 50 ml, Roche Diagnostics, Rotkreuz). Cells are centrifuged at 750xg for 10 minutes at 4 ° C and resuspended in 10 ml of cold membrane buffer (20 mM HEPES, pH 7.4, 1 mM NaCl, 1.0 mM MgCl2, 1 mM EDTA, 0.1 % BSA and cocktail of protease inhibitors). The cell suspension is homogenized on ice, using a Polytron homogenizer at 25000 rpm in three intervals of 20 seconds each. The homogenate is centrifuged at 26'900 xg for 30 minutes at 4 ° C and the pellet of the membrane protein is resuspended by vortexing in 2 ml of cold membrane buffer. The protein concentration is determined using the Bio Rad Protein Assay and human IgG as standard. The suspension volume of the membrane protein is adjusted to give a final concentration of 2 to 3 mg protein / ml. The solution is then homogenized again (Polytron) on ice at 25,000 rpm for 20 seconds before being aliquoted into Eppendorf tubes in the volume of 0.8-1 ml. f) Activity measurements of cells expressing hS1 P4: f1) GTP agglutination assay 35S The selection of the clones to be tested in the GTP? S assay is based on real-time PCR assays. CHO clones expressing large amounts of human S 1 P 4 are used in these experiments. The membrane proteins are prepared as described above. The basic protocol of the GTP? 35S agglutination assay used is described in a recent publication (Brinkmann et al. 2002, J. Biol. Chem, 277, 21453) with the modifications described herein below. To characterize the agglutination of GTP? 35S for the membrane proteins of CHO cells expressing S1 P, WGA-coated PVT granulates are used (SPA-bead, Amersham Biosciences). Because the ß-rays of GTP? 35S have significant penetration into the water solution, the dishes are centrifuged to minimize non-specific effects caused by non-binding of GTP? 35S. The assay is performed in 96-well Optiplates (Packard, Cat. No. 6005190) in final volume of 225 μl / well. After a short homogenization, the membrane proteins are resuspended at different concentrations (between 25 to 50 μg / ml) in 50 mM of HEPES, 100 mM of NaCl, 10 mM of MgCl2, 20 μg / ml of saponin (Riedel-de -Haen: Cat. No. 161 09), 0.1% fat free BSA (Sigma Cat. No. A0281) pH 7.4. The membrane proteins are mixed with 1 mg / well of pellet SPA, 10 μM of G DP, different concentrations of agonists and incubated for 1 0-1 5 minutes at room temperature. The GTP? 35S agglutination reaction is initiated by the addition of 200 pM of GTP? 35S (Amersham, Cat. No. SJ 1 308, >1 000 Ci / mmol). The Optiplates is sealed and incubated at room temperature for 1 10-120 minutes with constant agitation. The plates are then centrifuged for 10 minutes at 2000 rpm and equipped with a TopCount instrument (Packard). EC50 calculations are performed with a non-linear regression adjustment program such as that available in the Origin 7 RS2 software package (Origin Lab Corporation, One Roundhouse Plaza, Northampton, MA 01060, USA). f2) Calcium mobilization assay CHO cells are grown in plates on a black Costar plate (96 or 384 wells, 50O00 cells or 12,500 cells, respectively) in a MEM with FCS and cultured for 20-24 h at 37 ° C in a CO2 incubator. After removal of the culture medium, the cells are incubated in the HBSS medium containing 2 μM, Flu04AM (Molecular Probes, Cat. No, F-1241, 1 mg / ml material in DMSO), 5 mM probenicide for 1 at 37 ° C, rinsing with HBSS buffer, 2.5 mM probenicide and coating with the same medium (75 μl for 96-well dishes, 50 μl for 384 dishes). The dishes are transferred to the FLI PR. After measuring the baseline for 40 seconds, the agonist in HBSS is added and the fluorescence is measured at 2-second intervals for 3 to 5 minutes. In some cases the cells are pre-treated for 5 h with 50 ng / ml of pertussis toxin (Sigma, Cat. Nr P2980). 2-Aminoethoxydiphenylborate (2-APB, Calbiochem, Juro Supply, Bleicherstr. 1 1, Luceme, Switzerland, Cat. No. 1 00065) a blocker of calcium release from the endoplasmic reticulum (Ascher-Landsberg et al., 1999, Biochem.

Biophys. Res. Commun. 264, 979) at 50 μM and / or 1 50 μM is added directly to the cell medium 20-40 minutes before measurements. The EC50 calculations are performed using a non-linear regression adjustment program provided in the Origin 7 RS2 software package (Origin Lab Corporation) available from Novartis.

In vitro determination of the specificity and selectivity of the S1P4 agonist: The compounds mentioned in this application are tested for specificity, for example the activity they have in the previous transformation of the parental cell line with the S1P4 cDNA and for selectivity in CHO cell lines transformed with the S1P1, S1P2, S1P3 and S1P5. The generation of stable cell lines for the other S1P receptors is done as described for S1P4, using published human DANc sequences as described above. Preferably the compounds with a selectivity and specificity of 100x are chosen. For example, the specific S1P4 and selective compounds may have an EC50 (apparent EC50) measured in S1P4 that is 10x, preferably 100x lower than is measured in wild-type CHO cells or in the cell expressing any of the other four recipients of S1P.

For example if EC50 of 200 nM is measured for S1P4, the EC50's in CHO cells or in CHO cells expressing S1P1, 2, 3, 5 is preferably = 20 μM. The compound of Example 1 has an EC50 for S1P4 in this 432 nM assay.

The EC50 values (in μM) for the compound of Example 2 in CHO cells or in CHO cells expressing several S1P receptors is shown in Table 2 below: The compounds of the invention are, therefore, useful in the treatment and / or prevention of diseases or disorders mediated by lymphocyte interactions, for example in transplants, such as acute or chronic rejection of cell, tissue or allo- or xenografts of organs or late graft function, graft-versus-host disease, autoimmune diseases, for example rheumatoid arthritis, systemic lupus erythematosus, hashimoto thyroid, multiple sclerosis, myasthenia gravis, type I or II diabetes and the disorders associated therewith, vasculitis, pernicious anemia, Sjoegren's syndrome, uveitis, psoriasis, Graves' ophthalmopathy, alopecia areata and others, allergic diseases, for example allergic asthma, atopic dermatitis, rhinitis / allergic conjunctivitis, allergic contact dermatitis, inflammatory diseases optionally with fundamental aberrant reactions, example inflammatory bowel disease, Crohn's disease or ulcerative colitis rativa, intrinsic asthma, inflammatory liver injury, inflammatory glomerular lesion, atherosclerosis, osteoarthritis, irritant contact dermatitis and other eczematous dermatitis, seborrheic dermatitis, cutaneous manifestations of immunologically mediated disordersinflammatory disease of the eye, keratoconjunctivitis, myocarditis or hepatitis, ischemia / reperfusion injury, for example myocardial infarction, sudden attack, ischemia of the intestine, renal failure or hemorrhagic shock, traumatic shock, others, cancer, for example cell lymphomas T or T cell leukemias, infectious diseases, for example toxic shock (for example induced superantigen), septic shock, adult respiratory distress syndrome or viral infections, for example AIDS, viral hepatitis or chronic bacterial infection. Examples of cell, tissue or transplants of solid organs include for example pancreatic islets, stem cells, bone marrow, corneal tissue, neuronal tissue, heart, lung, combined heart-lung, kidney, liver, intestine, pancreas, trachea or esophagus. For the above uses the required dosage will of course vary depending on the mode of administration, the particular condition to be treated and the desired effect. In general, satisfactory results are indicated to be obtained systemically in daily dosages from about 0.03 to 2.5 mg / kg per body weight. An indicated daily dosage in the largest mammal, human, is in the range of from about 0.5 mg to about 1000 mg, administered suitably, for example, in divided doses up to four times a day or in a delayed manner. Suitable unit dosage forms for oral administration comprise from about 1 to 50 mg of the active ingredient. The compounds of the invention can be administered by any conventional route, in particular enterally, for example orally, for example in the form of tablets or capsules, or parenterally, for example in the form of injectable solutions or suspensions, topically, for example in the form of lotions, gels, ointments or creams, or in a nasal form or suppository. Pharmaceutical compositions comprising a compound of the invention in free form or in pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing with a pharmaceutically acceptable carrier or diluent. The compounds of the invention can be administered in free form or in pharmaceutically acceptable salt for example as indicated above. Such salts can be prepared in a conventional manner and exhibit the same order of activity as the free compounds. In accordance with the above the present invention further provides: 1. A method for preventing or treating lymphocyte-mediated disorders or diseases, for example as indicated above, in a subject in need of such treatment, which method comprises administering to the subject an effective amount of a compound of the invention, for example a compound of formula I, or a pharmaceutically acceptable salt thereof; 1.2 A method for preventing or treating rejection of chronic or acute transplantation or inflammatory or autoimmune diseases mediated by T cell, for example as indicated above, in a subject in need of such treatment, which method comprises administering to the subject a effective amount of a compound of. the invention, for example a compound of formula I, or a pharmaceutically acceptable salt thereof; 2. A compound of the invention, for example a compound of formula I, in free form or in a pharmaceutically acceptable salt form for use as a drug, for example in any of the methods as indicated under point 1 .1 or 1 .2 previous. 3. A pharmaceutical composition, for example for use in any of the methods as in 1 .1 or 1 .2 above, comprising a compound of the invention, for example a compound of formula I, in free form or salt form pharmaceutically acceptable in association with a pharmaceutically acceptable diluent or carrier therefore. The compounds of the invention, for example a compound of formula 1 can be administered as the sole active ingredient or together with, for example as an adjuvant to, or other drugs for example immunosuppressive or immunomodulatory agents or other anti-inflammatory agents, for example for the treatment or prevention of acute or chronic rejection of the allo-or xenograft or inflammatory or autoimmune disorders, or chemotherapeutic agent, for example a malignant cell antiproliferative agent. For example, the compounds of the invention can be used in combination with a calcineurin inhibitor, for example cyclosporin A or FK 506; an mTOR inhibitor, for example rapamycin, 40-O- (2-hydroxyethyl) -rapamycin, CC1779 or ABT578; an ascomycin that has immunosuppressive properties, for example ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprene; methotrexate; lefluno ida; mizoribin; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualin or an immunosuppressive homologue, analog or derivative thereof; immunosuppressive monoclonal antibodies, for example, monoclonal antibodies to leukocyte receptors, for example, MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, for example a recombinant binder molecule having at least a portion of the extracellular domain of CTLA4 or of mutant thereof, for example in at least the extracellular portion of CTLA4 or a mutant thereof linked to a sequence of protein without CTLA4, for example CTLA4lg (for example ATCC 68629) or a mutant thereof, for example LEA29Y; molecular adhesion inhibitors, for example LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent, for example paclitaxel, cisplatin, gemcitabine, doxorubicin or 5-fluorouracil. Where the compounds of the invention, for example a compound of formula I, are administered in combination with other immunosuppressive / immunomodulatory, anti-inflammatory or chemotherapeutic therapy, dosages of the co-administered immunosuppressant, immunomodulatory, anti-inflammatory or chemotherapeutic compound will of course vary depending on the type of co-drug used, for example if it is a spheroid or a calcineurin inhibitor, in the specific drug used, in the condition being treated and so on. In accordance with the foregoing, the present invention still provides an additional aspect: 4. A method as defined above comprising the co-administration, e.g. concomitantly or in sequence, of a non-toxic, therapeutically effective amount of a compound of the invention, for example a compound of formula 1 and at least one second substance of the drug, for example an immunosuppressant, immunomodulatory, anti-inflammatory or chemotherapeutic drug, for example as indicated above. 5. A pharmaceutical combination, for example a kit, comprising a) a first agent that is a compound of the invention, for example a compound of formula I, as described herein, in free form or in pharmaceutically salt form acceptable, and b) at least one co-agent, for example an immunosuppressant, immunomodulatory, anti-inflammatory or chemotherapeutic drug. The kit can comprise the instructions for its administration. The terms "co-administration" or "combined administration" or the like as used herein means understanding the administration of the selected therapeutic agents to a single patient, and it is desired to include treatment regimens in which the agents are not effective. necessarily administered by the same administration route or at the same time. The term "pharmaceutical combination" as used herein means a product resulting from the mixture or combination of more than one active ingredient and includes the fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, for example a compound of the invention and a co-agent, are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, for example a compound of the invention and a co-agent, are both administered to a patient as separate entities simultaneously, concurrently or sequentially with nonspecific time limits, in wherein such administration provides therapeutically effective levels of the 2 compounds in the patient's body. The latter also applies to cocktail therapy, for example the administration of 3 ingredients or more active ingredients.

Claims (10)

1. A compound which is an S1P4 receptor agonist, wherein the compound possesses a selectivity for the S1P4 receptor of more than one or more of the S1P1, S1P2, S1P3 or S1P5 receptors of at least 10 times as measured by the ratio of the EC50 of the compound for the S1P4 receptor of the EC50 of the compound for the S1P1, S1P2, S1P3 or S1P5 receptor, in free form or in pharmaceutically acceptable salt form.

2. A compound of formula I wherein R-, is phenyl or naphthyl, wherein the phenyl is substituted by one or two of halogen; C 1-6 alkyl, C 1-6 alkoxy or phenyl C 1-6 alkyl; and R2 is hydrogen or C1-6 alkyl; in free form, hydrate or salt.

3. A compound according to claim 1 or claim 2, which is selected from 3- (4- (2-ethylphenyl) -2-carboxamide-indole) -alanine, 3- (4- (2-benzyl) phenyl) -2-carboxamido-indole) -alanine, 3- (4- (naphthalen-2-yl) -2-carboxamido-lndol) -alanine, 3- (4- (naphthalen-1-yl) -2- carboxamido-indole) -alanine, 3- (4- (2-butoxy-phenyl) -2-carboxamido-indole) -alanine, 3- (4- (2-propyl-phenyl) -2-carboxamido-indole) -lanine , 3- (4- (2-isopropyl-phenyl) -2-carboxamido-indole) -alanine and 3- (4- (2,4-dichloro-phenyl) -2-carboxamido-indole) -alanine, or a salt pharmaceutically acceptable thereof.

4. A compound according to claim 3, which is 3- (4- (2-ethylphenyl) -2-carboxamido-indole) -D-alanine, in free form or in a pharmaceutically acceptable salt form.

5. A compound according to any of claims 1 to 4, in free form or in a free form or in a pharmaceutically acceptable salt form, for use as a drug.

6. A pharmaceutical composition comprising a compound as defined in any of claims 1 to 4, in free form or in a pharmaceutically acceptable salt form, in association with a pharmaceutically acceptable diluent or carrier accordingly.

7. Use of a compound according to any of claims 1 to 4, in free form or in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 4 in the manufacture of a medicament for the treatment or prevention of disorders or diseases mediated by 'lymphocytes, rejection of acute or chronic transplantation, inflammatory or autoimmune diseases mediated by the T cell, diabetes, allergic diseases, myocarditis, hepatitis, ischemia / reperfusion injury, renal failure, hemorrhagic shock, traumatic shock, cancer or infectious diseases.

8. A pharmaceutical combination comprising a compound according to any of claims 1 to 4, in free form or in a pharmaceutically acceptable salt form and another agent selected from immunosuppressive, immunomodulatory, anti-inflammatory and chemotherapeutic agents.

9. A process for the production of the compound according to claim 2, which process comprises deprotecting a compound of formula II wherein R ^ and R2 are as defined in claim 2, R6 is C1-6 alkyl or benzyl, R7 is an amino protecting group, and and optionally converting the compound of formula I obtained in free form to a salt form or vice versa .

10. A method for treating or preventing lymphocyte-mediated disorders or diseases, rejection of acute or chronic transplantation, inflammatory or autoimmune diseases mediated by the T cell, diabetes, allergic diseases, myocarditis, hepatitis, ischemia / reperfusion injury, renal failure, hemorrhagic shock, traumatic shock, cancer or infectious diseases, in a subject in need of such treatment, which method comprises administering to the subject an effective amount of a compound according to any of claims 1 to 4, or a pharmaceutically acceptable salt of the same.