Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
  • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
  • C07D271/06—1,2,4-Oxadiazoles; Hydrogenated 1,2,4-oxadiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4245—Oxadiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

• a modulator of the sphingosine-1 -phosphate receptor is provided for treatment of a malcondition for which activation of the same is medically indicated.
• the SIPi/EDGi receptor is a G-protein coupled receptor (GPCR) and is a member of the endothelial cell differentiation gene (EDG) receptor family.
• Endogenous ligands for EDG receptors include lysophospholipids, such as sphingosine- 1 -phosphate (SIP).
• SIP sphingosine- 1 -phosphate
• ligation of the receptor propagates second messenger signals via activation of G-proteins (alpha, beta and gamma).
• development of small molecule SIPi agonists and antagonists has provided insight into some physiological roles of the SIPi/SIP-receptor signaling system.
• SIP receptors are divided into five subtypes (i.e., SIPi, SIP2, SIP3, SIP4 and SIP5), which subtypes are expressed in a wide variety of tissues and exhibit different cell specificity.
• Agonism of the SIPi receptor perturbs lymphocyte trafficking, sequestering them in lymph nodes and other secondary lymphoid tissue. This leads to rapid and reversible lymphopenia, and is probably due to receptor ligation on both lymphatic endothelial cells and lymphocytes themselves (Rosen et al, Immunol. Rev., 195: 160-177, 2003).
• a modulator of the sphingosine-1 -phosphate receptor is provided for treatment of a malcondition for which activation of the same is medically indicated.
• a compound is provided in an isolated or purified form having the following structure (also referred to as“Compound 1” or“Cpd 1”): Compound 1
• a pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, in combination with a pharmaceutically acceptable carrier or diluent.
• Figure 1 shows the mean plasma concentration relative to lymphocyte count (CD4 + , CD8 + and B220 + ) for Compound 1.
• the present invention is directed to a compound and composition which modulates an SIP receptor.
• SIP receptors are divided into five subtypes (i.e., SIPi, SIP2, SIP3, SIP4 and SIP5), which subtypes are expressed in a wide variety of tissues and exhibit different cell specificity.
• the compound disclosed herein modulates one or more of these subtypes.
• the compound is an "SIPi" modulator as it modulates subtype 1 of a sphingosine-1 -phosphate receptor.
• the compound modulates subtype 1 and another subtype, such as subtype 5.
• an "SIPi modulator” is understood to encompass a compound that modulates the SIPi subtype alone, or modulates the SIPi subtype as well as one or more other subtypes. In one embodiment, the SIPi modulator modulates both the SIPi subtype and the SIP5 subtype.
• a“modulator” of the SIPi receptor is a compound which, when administered to a subject, provides the desired interaction with the target receptor, either by way of the compound acting directly on the receptor itself, or by way of a metabolite of the compound acting on the receptor. Upon administration to a subject, the compound of this invention modulates the SIPi receptor by activating on the receptor for signal transduction.
• Such compound is also referred to herein as an “agonist” or“SIPi agonist”.
• SIPi agonist can be selective for action on SIPi.
• the compound may be selective for action on SIPi at a lower concentration than on other subtypes of the SIP receptor family.
• a compound is provided in an isolated or purified form having the following structure (“Compound 1”):
• the compound is provided having a purity in excess of 90% (w/w), having a purity in excess of 95% (w/w), or having a purity in excess of 98% (w/w). In a further embodiment, the compound is provided having a purity in excess of 99% (w/w).
• a “salt” as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion.
• acids in their anionic form can form salts with cations such as metal cations, for example sodium, potassium, and the like; with ammonium salts such as NFLri or the cations of various amines, including tetraalkyl ammonium salts such as tetramethylammonium and alkyl ammonium salts such as tromethamine salts, or other cations such as trimethyl sulfonium, and the like.
• a “pharmaceutically acceptable” or “pharmacologically acceptable” salt is a salt formed from an ion that has been approved for human consumption and is generally non-toxic, such as a chloride salt or a sodium salt.
• a “zwitterion” is an internal salt such as can be formed in a molecule that has at least two ionizable groups, one forming an anion and the other a cation, which serve to balance each other. For example, amino acids such as glycine can exist in a zwitterionic form.
• a “zwitterion” is a salt within the meaning herein.
• the compounds of the present disclosure may take the form of salts.
• the term “salts" embraces addition salts of free acids or free bases which are compounds of the disclosure.
• Salts can be “pharmaceutically-acceptable salts.”
• pharmaceutically acceptable salt refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compounds of the disclosure.
• Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
• inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
• Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4 hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethane
• Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
• Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N' dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
• Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts.
• salts may be useful, for example as intermediates in the synthesis of compounds, for example in their purification by recrystallization. All of these salts may be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
• pharmaceutically acceptable salts refers to nontoxic inorganic or organic acid and/or base addition salts, see, for example, Gould et al., Salt Selection for Basic Drugs (1986), Int J Pharm ., 33, 201- 217, incorporated by reference herein.
• Non-limiting examples of potential salts of this disclosure include but are not limited to hydrochloride, citrate, glycolate, fumarate, malate, tartrate, mesylate, esylate, cinnamate, isethionate, sulfate, phosphate, diphosphate, nitrate, hydrobromide, hydroiodide, succinate, formate, acetate, dichloroacetate, lactate, p-toluenesulfonate, pamitate, pidolate, pamoate, salicylate, 4-aminosalicylate, benzoate, 4-acetamido benzoate, glutamate, aspartate, glycolate, adipate, alginate, ascorbate, besylate, camphorate, camphorsulfonate, camsylate, caprate, caproate, cyclamate, laurylsulfate, edisylate, gentisate, galactarate, gluceptate, glu
• a "homolog" of a compound of the disclosure is a compound having one or more atoms of the compound replaced by an isotope of such atom.
• homologs include compounds with deuterium in place of one or more hydrogen atoms of the compound such as compounds of the disclosure in which the methyl groups of the isopropoxy moiety of Formulas I-R and I-S are fully or partially deuterated ( e.g ., (D3C)2CHO-).
• Isotopic substitutions which may be made in the formation of homologs of the disclosure include non-radioactive (stable) atoms such as deuterium and carbon 13, as well as radioactive (unstable) atoms such as tritium, carbon 14, iodine 123, iodine 125, and the like.
• a "hydrate” is a compound that exists in a composition with water molecules. The composition can include water in stoichiometric quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. As the term is used herein a "hydrate” refers to a solid form, i.e., a compound in water solution, while it may be hydrated, is not a hydrate as the term is used herein.
• a “solvate” is a similar composition except that a solvent other that water replaces the water.
• a solvent other that water replaces the water.
• methanol or ethanol can form an “alcoholate”, which can again be stoichiometric or non-stoichiometric.
• a “solvate” refers to a solid form, i.e., a compound in solution in a solvent, while it may be solvated, is not a solvate as the term is used herein.
• a pharmaceutical composition comprising a compound having the following structure (Compound 1):
• compositions may take a variety of different forms, including (but not limited to) forms suitable for oral or intravenous administration.
• Pharmaceutically acceptable carriers and excipients are known in the art, such as those disclosed in Remington: The Science and Practice of Pharmacy, 22 nd Edition, Allen, Lloyd V., Jr. Ed. (2012) (incorporated herein by reference).
• Compound 1 can be prepared by techniques known to one skilled in the art, as well as by the procedures disclosed in the following Examples. EXAMPLES
• the gradient was 20-100% with mobile phase B over 2.5 min then held at 100% for 2.5 mins.
• the flow rate was 1 mL/min.
• Method 1 40-95% over 0.5 min, hold at 95% for 8.5 min, then return to 40% over 2 min, with a flow rate of 1 mL/min.
• Final compounds were checked for purity using Method 2: 5% for 1 min, 5-95% over 9 min, then hold at 95% for 5 min, with a flow rate of 1 mL/min. Enantiomeric excess was determined by integration of peaks that were separated on a Chiralpak AD-H, 250 x 4.6 mm column, 5 pm particle size.
• Chiral Method 1 Chiralpak AY- H, 250 x 4.6 mm column, 5 pm particle size. Flow rate of 1 mL/min and an isocratic mobile phase.
• Chiral Method 2 Chiralcel OZ-3, 250 x 4.6, 3 pm particle size at a flow rate of 0.75 ml/min.
• the pyridine, dichloromethane (DCM), tetrahydrofuran (THF), and toluene used in the procedures were from Aldrich Sure-Seal bottles kept under nitrogen (N2).
• Step 1 Synthesis of 3-ethoxy-lH-indene-7-carbonitrile (Int 2): A stirred mixture of l-oxo-2,3-dihydro-lH-indene-4-carbonitrile (Int 1)
• the crude oil was then purified by filtration through a silica gel plug eluting with 15% EtO Ac/hexane. The pure fractions were combined and utilized for the next step.
• Step 3 Synthesis of N-(Y3-cvano-4-isopropoxybenzovDoxy)-3-ethoxy-lH-indene-7- carboximidamide (Tnt 4):
• Step 4 Synthesis of 5-(3-(3-ethoxy-lH-inden-7-vD-L2.4-oxadiazol-5-vD-2- isopropoxybenzonitrile (Int 5)
• Int 5 (30 g, 75.57 mmol) is suspended in 4: 1 IPA/H2O (300 mL).
• Catalytic H2SO4 (0.1 mL, 0.19 mmol) is added, and the resulting mixture is heated to reflux for 12 h.
• the slurry is cooled to ambient temperature and stirred for 1 h.
• the product is isolated by filtration and washed with 4: 1 IPA/H2O (100 mL). After drying on the filter for 1 h under vacuum, the wet cake is charged back to the reactor and suspended in EtOAc (300 mL). The mixture is heated to reflux for 3 h, then cooled to ambient temperature and stirred for 1 h.
• SlPiR is a GPCR that couples exclusively to the inhibitory G protein (God), decreasing adenylate cyclase activity and lowering cyclic adenosine monophosphate (cAMP levels) in the cell.
• the potency of Compound 1 was tested against the S1P1 receptor (S IPIR) and related family members.
• S IPIR S1P1 receptor
• the potency and SIP receptor selectivity were measured using two independent in vitro assays, [ 35 S]guanosine-5'-0-(3-thio)triphosphate ([ 35 S]GTPyS) and b-lactamase gene expression and activity.
• S IPIR-S IPSR The GTPyS binding to membranes prepared from stable mammalian cells lines expressing recombinant human SIP receptors 1-5 (S IPIR-S IPSR) was used to pharmacologically characterize ligand interaction with the SIP receptor family members.
• Membranes were incubated at room temperature (RT) with test compound (10-point serial half-log dose-range with the highest concentration of 300 nM-10 mM depending upon the potency of each compound) and wheat germ agglutinin- coated scintillation proximity assay beads on a plate shaker for 30 minutes before the addition of [ 35 S]GTPyS and an additional incubation of 40 minutes at RT.
• Membrane- bead complexes were then pelleted by centrifugation before the bound radioactivity was quantified with a MicroBeta2 ® microplate scintillation counter.
• the radioactive counts relative to DMSO vehicle were calculated and the half maximal effective concentration (EC50) was determined using non-linear regression.
• Up-regulation of b-lactamase gene expression and activity were used as a whole-cell functional readout of SIP receptor activation.
• Stable mammalian cells lines expressing recombinant human S1P1R-S1P5R designed to couple to the gene expression of b-lactamase were pre-seeded into 384 well microplates before incubation with test compound (10-point serial 1 :4 dose range with the highest concentration of 100 nM-10 pM depending upon the potency of the compound) for 4-5 hours at 37°C in a tissue culture incubator.
• the respective b-lactamase activity was then determined using a fluorescence resonance energy transfer (FRET)-based b-lactamase fluorescent substrate (LiveBLAzerTM-FRET B/G Loading Kit CCF4-AM) according to manufacturer’s instructions, and incubated with cells for 2 hours at RT. Plates were read using a SpectraMax M5 Multi-Mode microplate reader and the relative response to DMSO vehicle calculated for the EC50 determination using non-linear regression.
• FRET fluorescence resonance energy transfer
• Compound 1 is a potent and selective S IPIR and S IPSR agonist in both b- lactamase activity and GTPyS binding assays, having an EC50 of 2.1 nM for GTPyS binding and 0.04 nM for b-lactamase activity. Compound 1 is also active at S IPRS with EC50 values of 17.9 and 6.9 nM for GTPyS binding and b-lactamase activity, respectively.
• Compound 1 has minimal activity on SIP2-4 receptors, showing greater than 1000-fold selectivity for S IPIR over SIP2R, SIP3R and S1P4R using the [ 35 S]GTPyS binding activity readout, and greater than 200-fold selectivity for S IPSR over SIP2R, SIP3R and S1P4R using both assay readouts (see Table 1).
• Table 1
• Plasma samples 50 pL were aliquoted into a 96 deep-well polypropylene plate (2 mL/well) and 5 pL of dimethyl sulfoxide (DMSO) was added. Samples expected to be above the upper limit of quantitation (ULOQ) were diluted with plasma. Standard curves were prepared by mixing 5 pL of the test compound in DMSO at 10 times the concentration which was added to 50 pL of plasma in a 96 deep-well polypropylene plate. For example, for a 0.3 pM standard, the 10 X DMSO concentration of the test compound was 3 pM. Standard curves were prepared using analytical standards of Compound 1.
• DMSO dimethyl sulfoxide
• Protein was precipitated from study samples and standards by the addition of 150 pL of acetonitrile. The plate was vortexed to ensure complete precipitation of protein. The precipitated protein was pelleted by centrifugation at 4,000 rpm for 10 min at 20 °C and the clear supernatant was transferred to a clean 96-well plate and centrifuged again under the same conditions to pellet any transferred solid material.
• Samples (7 pL) were introduced to a Shimadzu HPLC (LC-20ADXR) with a SIL-30ACMP autosampler (Shimadzu).
• the mobile phase was a gradient using 0.1% formic acid in water and 0.1% formic acid in acetonitrile.
• the column used was a Phenomenex Kinetic C18 100A 2.6 p 30 x 3 mm (PN 946975-906).
• the CTO-20AC column oven (Shimadzu) was set at 40°C.
• Prepared samples were usually analyzed in order of lowest to highest concentration for standards and reverse order of time-points for study samples. Study samples were bracketed by full standard curves. Typically at least 6 standards were used for quantitation with a percent accuracy of +/- 15% for all standards except at the lower level of quantitation (LLOQ) where a percent accuracy of +/- 20% was allowed.
• LLOQ lower level of quantitation
• Rats are housed in an ALAAC accredited facility and the research approved by the facilities Institutional Animal Care and Use Committee (IACUC). The animals are acclimated to the laboratory for at least 48 h prior to initiation of experiments.
• IACUC Institutional Animal Care and Use Committee
• Compound 1 was formulated in 5%DMSO/5%Tween20 and 90% purified water (intravenous infusion) or 5%DMSO/5%Tween20 and 90% 0.1N HCL (oral gavage). The concentration of the dosing solutions is verified by HPLC-UV.
• blood is collected at eight time-points after dosing with the final sample drawn 24 h post dose.
• Calibration curve standards are prepared by spiking 5pL compound stock in DMSO into freshly collected EDTA rat blood. An eight point standard curve spanning a range of 5 nM to 10,000 nM is included with each bio-analytical run. The standards are processed identically to the rat pharmacokinetic samples.
• Concentrations in the rat pharmacokinetic samples are determined using a standardized HPLC-LC/MS/MS method relative to the eight point standard curve.
• the system consists of a Leap CTC Pal injector, Agilent 1200 HPLC with binary pump coupled with an Applied Biosystems 3200 QTrap. Compounds are chromatographed on a Phenomenex Synergy Fusion RP 20x2mm 2um Mercury Cartridge with Security Guard.
• a gradient method is used with mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile at flow rates varying from 0.7 to 0.8 mL/min.
• Ions are generated in positive ionization mode using an electrospray ionization (ESI) interface.
• MRM Multiple reaction monitoring
• the heated nebulizer is set at 325°C with a nebulizer current of 4.8 mA.
• Collision energies are used to generate daughter ions ranged between 29 and 39 V.
• Peak area ratios are obtained from MRM of the mass transitions specific for each compound used for quantification.
• the limit of quantification of the method is typically 5 nM. Data are collected and analyzed using Analyst software version 1.4.2.
• Compound 1 was administered in a formulation of 10% volume/volume (v/v) DMSO and 5% v/v Tween 20 in water by IV administration (10 mL/kg) or 0.5% weight/volume (w/v) CMC in water by oral gavage (10 mL/kg). Animals received 0.2 mg/kg as the IV dose or 2 mg/kg/day orally as a single administration. Blood samples were collected at 1, 5, and 30 minutes, 2, 4, 8, 10, 24, 24, 48, and 72 hours after IV dosing and 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, and 72 hours following oral dosing. Blood was collected into tubes containing the anti-coagulant K2EDTA and processed to plasma. Plasma concentrations for each analyte were determined using a qualified LC- MS/MS method. The PK parameters are summarized in Table 2 below.
• Total lymphocyte counts were determined in a standard hematology panel on a calibrated analyzer (Test #416; Horiba Medical ABX Pentra XL 80) and by in-house flow cytometry using fluorescent- activated cell sorting (FACS) analysis to determine the number of CD4+, CD8+ and B220+ lymphocytes (ThermoFisher Attune NxT flow cytometer). Data are expressed as follows for absolute lymphocyte counts (hematology analyzer), the individual lymphocyte subtypes (CD4+, CD8+ and B220+) and the sum of the individual lymphocyte subtypes (flow cytometry).
• FACS fluorescent- activated cell sorting
• the oral dose level of 2 mg/kg of Compound 1 administered to the animals was intended to induce a significant reduction ( ⁇ >60%) in the number of circulating lymphocyte.
• blood samples were serially collected from the jugular vein of conscious Sprague Dawley rats prior to dosing and 3 hours, 6 and 24 hours after administration.
• the absolute number of circulating lymphocytes was quantified in whole blood samples using a hematology analyzer and the CD4 + , CD8 + and B220 + lymphocyte subtypes by flow cytometry.
• the plasma concentrations for each compound were quantified with liquid chromatography, tandem mass spectrometry.
• Compound 1 significantly reduced total circulating lymphocyte counts (total number of CD4 + , CD8 + and B220 + ) by 52% relative to pre-dose values (see Figure). The effects on individual lymphocyte subtypes were similar with CD4 + , CD8 + and B220 + lymphocyte populations reduced by 68%, 62% and 35%, respectively. Furthermore, measurement in a hematology analyzer also gave similar results (44% fewer lymphocytes relative to pre-dose values). In general, the maximum effect was observed by six hours after administration. Compound 1 was without significant effect at the 24 hour time-point. Lymphocyte counts reflected the plasma concentrations of Compound 1 after administration, demonstrating a clear pharmacokinetic/pharmacodynamic (PK/PD) relationship.
• PK/PD pharmacokinetic/pharmacodynamic
• Serial blood PK samples were collected on Days 1 and the last day of dosing.
• PK parameters for ozanimod and its metabolites including Compound 1 were estimated using non-compartmental analysis and actual PK collection times.

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