CN101119714A - Sphingosine-1-phosphate agonists containing cycloalkyl and 5-membered heterocycles substituted by amino and phenyl groups - Google Patents

Abstract

The present invention provides sphingosine-1-phosphate analogs that are potent and selective agonists of one or more S1P receptors, particularly of the _S1P1 receptor type. The compounds of the present invention include compounds having a phosphate moiety and compounds having hydrolysis resistant phosphate substitutes such as phosphonates, alpha-substituted phosphonates and thiophosphates.

Description

Sphingosine-1-phosphate agonists containing cycloalkyl and 5-membered heterocycles substituted by amino and phenyl groups

Cross-references to related applications

This application claims priority to provisional applications 60/652,642, filed February 14, 2005 and 60/669,616, filed April 8, 2005, and 60/692,760, filed June 22, 2005, all of which The content is incorporated by reference into this application in its entirety.

US government rights

This invention was made with US Government support under Grant Nos. RO1 GM067958 and RO1 GM052722 awarded by the National Institutes of Health. The US Government has certain rights in this invention.

technical field

The present invention relates to novel sphingosine-1-phosphate analogs that are active at one or more sphingosine-1-phosphate receptors.

Background technique

Sphingosine 1-phosphate (S1P) is a lysophospholipid modulator that elicits a variety of cellular responses by stimulating five members of the endothelial differentiation gene (EDG) receptor family. The EDG receptor is a G protein-coupled receptor (GPCR) and when stimulated transmits by activating the heterotrimeric G protein alpha (G _α ) subunit and beta-gamma (G _βγ ) dimer Second messenger signal.

Sphingosine 1-phosphate (S1P) elicits many responses from cells and tissues. Chief among these are anti-apoptosis, changes in cell morphology, cell migration, cell division, angiogenesis and regulation of the immune system by altering lymphocyte trafficking. Thus, the S1P receptor is a target for the treatment of eg neoplastic diseases, autoimmune diseases and rejection of tissue allografts. The sphingosine-1-phosphate moiety signals to cells through a group of G protein-coupled receptors named _S1P1 , _S1P2 , _S1P3 , _S1P4 and _S1P5 . These receptors share 50-55% amino acid identity and are clustered with three other receptors ( _LPA1 , _LPA2 and _LPA3 ) for structurally related lysophosphatidic acid (LPA).

When a ligand binds the receptor, a conformational change in the G protein-coupled receptor (GPCR) is induced, resulting in displacement of GDP by GTP on the associated G protein alpha subunit and subsequent release of the G protein into the cytoplasm. The alpha subunits then dissociate from the beta gamma subunits, each of which can then associate with effector proteins, which activates second messengers that lead to cellular responses. Finally, GTP on the G protein is hydrolyzed to GDP and the subunits of the G protein reassociate with each other and then with the receptor. Amplification reactions play a major role in the usual GPCR pathway. Binding of one ligand to one receptor results in the activation of many G proteins, each of which is capable of associating with many effector proteins leading to an amplified cellular response.

S1P receptors are good drug targets because each receptor is both tissue specific and response specific. Tissue specificity of the S1P receptor is desirable because the development of a selective agonist or antagonist of a receptor focuses the cellular response on the tissue containing the receptor, limiting unwanted side effects. Response specificity of the S1P receptor is also important because it allows the development of agonists or antagonists that initiate or inhibit certain cellular responses without affecting others. For example, the responsive specificity of the S1P receptor may allow the development of S1P mimics that initiate platelet aggregation without affecting cell morphology.

Sphingosine-1-phosphate is formed as a metabolite of sphingosine in its reaction with sphingosine kinase and is abundantly stored in platelet aggregates in the presence of high levels of sphingosine kinase and lack of S1P lyase. S1P is released during platelet aggregation, accumulates in serum, and is also found in malignant ascites. The reversible biodegradation of S1P is thought to be via hydrolysis by exophosphoesterases such as S1P phosphohydrolase, by which S1P is irreversibly degraded.

The physiological significance of stimulation of individual SlP receptors is largely unknown, in part due to the lack of receptor-type selective ligands. Isolation and characterization of S1P analogs with potent agonist or antagonist activity at S1P receptors has been limited due to synthetic complexity resulting from the lack of solubility of S1P analogs.

Currently, there is a need for new, potent and selective formulations of S1P receptor agonists. There is also a need for pharmacological tools for further investigation of physiological processes associated with agonism of S1P receptors.

Contents of the invention

The present invention provides sphingosine-1-phosphate analogs that are potent and selective agonists of one or more S1P receptors, particularly the _S1P1 receptor type. Compounds of the invention include compounds having a phosphate moiety and compounds having hydrolysis resistant phosphate substitutes such as phosphonates, a-substituted phosphonates (especially where the a-substituent is a halogen), and thiophosphates. In addition, the present invention provides prodrugs, such as primary alcohol-containing compounds, which are activated or converted (eg, phosphorylated) in vitro by, for example, sphingosine kinase, especially sphingosine kinase type 2 (SPHK2).

Accordingly, the present invention provides a sphingosine-1-phosphate analogue of formula (I) or (II) or a pharmaceutically acceptable salt or ester thereof:

或

or

Wherein, R ⁴ and R ⁷ are independently CH or CH ₂ ; R ⁵ is C, CH or N, R ⁶ is CH, CH ₂ , O, S or NR ³ ; R ³ is hydrogen or alkyl;

X is selected from hydroxyl (-OH), phosphate (-OPO ₃ H ₂ ), phosphonate (-CH ₂ PO ₃ H ₂ ), α-substituted phosphonate;

R ¹ is selected from hydrogen, halogen, trifluoromethyl, (C ₁ -C ₁₀ ) alkyl, (C ₁ -C ₁₀ ) alkyl substituted by halogen, hydroxyl, alkoxy or cyano;

R ² is selected from (C ₁ -C ₂₀ ) alkyl, cycloalkyl substituted alkyl, (C ₂ -C ₂₀ ) alkenyl, (C ₂ -C ₂₀ ) alkynyl, aryl, alkyl substituted aryl A group consisting of radical, aralkyl group and aryl-substituted aralkyl group; wherein one or more carbon atoms in the ^R2 group can be independently replaced by non-peroxide oxygen, sulfur or ^NR8 ; wherein ^R8 is hydrogen or (C ₁ -C ₁₀ ) alkyl;

代表1、2或3，任选的双键。wherein the alkyl, alkenyl and alkynyl groups in ^R are optionally substituted by oxo; n is 0, 1, 2 or 3; and

Represents 1, 2 or 3, optionally a double bond.

The present invention also provides esters, such as phosphate esters, of any compound of formula (1) or formula (II), wherein ester functionality may be added to form prodrugs with increased oral availability.

The present invention also provides compounds of formula (I) or formula (II) for use in medical therapy.

On the other hand, the present invention also provides:

Compounds of the invention that may be prodrugs, ie, they may be activated by phosphorylation in a subject, for example to form a monophosphorylated analog following administration of a primary alcohol. The activated forms of certain compounds of the present invention are agonists of S1P1-type receptors, and thus induce lymphopenia for about 7 days or more when introduced into an animal;

Compounds of the present invention that can be selective agonists of S1P ₁ , S1P ₄ and S1P ₅ receptors and have a long duration of action, such as a longer action time than FTY-720 (fingolimod);

A pharmaceutical composition containing a compound of formula (I), formula (II) or a mixture thereof or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable excipient;

A method of preventing or treating an autoimmune disease such as uveitis, type I diabetes, rheumatoid arthritis, inflammatory bowel disease, and more particularly multiple sclerosis, the method Including administering an effective amount of a compound of formula (I), formula (II) or a pharmaceutically acceptable salt thereof to a mammal (such as a human) in need of such treatment;

A method of preventing or treating progressive dementia or brain degenerative disease;

Alteration of lymphocyte trafficking as a method for prolonging the survival of allografts, such as solid organ transplantation, treatment of graft-versus-host disease, bone marrow transplantation, etc.;

Preventing cancer progression by inhibiting autotoxins, for example by preventing or inhibiting angiogenesis in tumors; or

Use of a compound of formula (I), formula (II) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or inhibiting autoimmune diseases or angiogenesis in mammals (such as humans) or tumors.

The present invention also provides novel intermediates and processes disclosed herein for the preparation of compounds of formula (I) or formula (II), including general and specific intermediates and synthetic methods described in the Figures and Examples herein .

Brief description of the drawings

Figure 1 provides synthetic pathways for the preparation of VPC01091 (6) and for the preparation of VPC01211 (7).

Fig. 2 is a graph illustrating the results of a measurement that the compound VPC01091 has no effect on the heart rate of mice. In this assay, test compounds are administered intravenously in a vehicle of 2% cyclodextrin.

Figure 3 graphically illustrates total blood lymphocyte counts following administration of a single dose of VPC01091 or vehicle to mice. Five mice per group, 10-11 weeks old, svl29 x C57B1/6 line. The ordinate indicates lymphocytes (K/μl). The abscissa represents time (days).

Figure 4 graphically illustrates lymphopenia following oral administration (gavage) of different concentrations of VPC01091 (vehicle control, 0.1, 0.3, 1.0 and 3.0 mg/kg) in 2% hydroxypropyl β-cyclodextrin The measurement results of each group of 3 mice (same line as in Fig. 3) were detected at 24 hours (the left bar of each group) and 48 hours (the right bar of each group).

Figure 5 ^graphically illustrates total lymphocyte counts (k/ ^μl ), wild Type mice received only vehicle (hydroxypropyl β-cyclodextrin). Disruption of the SPHK2 gene by insertion of exon-trap elements into both alleles.

Figure 6 graphically illustrates the results of a binding assay of disrupted ^GTPy35S cells to the human _S1P1 receptor, testing S1P and three other compounds. Compound "CA5-P" is VPC01211. Compounds VPC01214(CA6-P) and VPC01222(CA4-P) are the corresponding cyclohexyl and cyclobutyl compounds. The ordinate represents the percentage of GTPγ ³⁵ S binding, and the abscissa represents the log molar concentration of phospholipids.

Figure 7 graphically illustrates the results of a calcium mobilization assay using CHO-K1 cells expressing recombinant human _S1P2 receptor. Compound "CA5-P" is VPC01211. Compounds CA6-P and CA4-P are the corresponding cyclohexyl and cyclobutyl compounds. The ordinate represents the percentage of maximum calcium mobilization, and the abscissa represents the log molar concentration of phospholipids.

Figure 8 graphically illustrates the results of a binding assay of disrupted ^GTPy35S cells to the human _S1P2 receptor. Compound "CA5-P" is VPC01211. Compounds CA6-P and CA4-P are the corresponding cyclohexyl and cyclobutyl compounds. The ordinate represents the percentage of GTPγ ³⁵ S binding, and the abscissa represents the log molar concentration of phospholipids.

Figure 9 graphically illustrates the results of a calcium mobilization assay using CHO-K1 cells expressing recombinant human _S1P3 receptor. Compound "CA5-P" is VPC01211. Compounds CA6-P and CA4-P are the corresponding cyclohexyl and cyclobutyl compounds.

Figure 10 graphically illustrates the results of a binding assay of disrupted ^GTPy35S cells to the human _S1P4 receptor. Compound "CA5-P" is VPC01211. Compounds CA6-P and CA4-P are the corresponding cyclohexyl and cyclobutyl compounds.

Figure 11 graphically illustrates the results of a binding assay of disrupted ^GTPy35S cells to the human _S1P5 receptor. Compound "CA5-P" is VPC01211. Compounds CA6-P and CA4-P are the corresponding cyclohexyl and cyclobutyl compounds.

Figure 12 illustrates the Schild curve of the S1P dose response curve results for the human _S1P3 receptor for the indicated concentrations of the test compound VPC01222. The rightward shift of the S1P dose response curve indicates that VPC01222 acts as an overcome antagonist of this receptor type.

Figure 13 illustrates the Schild curves of the S1P dose response curve results for the _S1P3 receptors of the test compound VPC01211 at the indicated concentrations. The rightward shift of the S1P dose response curve indicates that VPC01211 acts as an overcome antagonist of this receptor type.

Figure 14 illustrates the Schild curves of the S1P dose response curve results for the _S1P3 receptors of the test compound VPC01214 at the indicated concentrations. The rightward shift of the S1P dose response curve indicates that VPC01214 acts as an overcome antagonist of this receptor type.

Figure 15 illustrates the results of a calcium mobilization assay in T24 cells overexpressing the human _S1P3 receptor in response to S1P and S1P in the presence of 10 μM VPC01211. Shift to the right indicates surmountable antagonism of the _S1P3 receptor by VPC01211.

Figure 16 illustrates the ability of the phosphorylated isoform VPC01211 (phosphorylated VPC01091) to agonize the _S1P1 receptor.

Figure 17 illustrates the ability of the phosphorylated isoform VPC01211 (phosphorylated VPC01091) to agonize the _S1P3 receptor.

Figure 18 illustrates SPHK2 activity with four VPC01091 isoforms.

Figure 19 graphically illustrates total lymphocyte counts (k/μl) at 24 hours (left bar for each group) and 96 hours (right bar for each group) after oral doses of VPC01091 isomers were administered to mice.

Detailed ways

Acronym

The following abbreviations are used in this text: S1P, sphingosine-1-phosphate; GPCR, G protein-coupled receptor; SAR, structure-activity relationship; EDG, endothelial cell differentiation gene; EAE, experimental autoimmune encephalomyelitis; NOD, non-obese diabetes; TNFα, tumor necrosis factor alpha; HDL, high-density lipoprotein; RT-PCR, reverse transcription polymerase chain reaction.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section.

In describing and claiming the invention, the following terminology will be used in accordance with the following definitions.

For the purpose of describing the present invention, the articles ("a" and "an") are used herein to refer to one (species) or more than one (species) (ie at least one (species)) substitute. By way of example, "an element" means an element(s) or more than one element.

As used herein, an "analogue" of a chemical compound is, for example, a compound that is structurally similar to another compound but not necessarily an isomer (eg, 5-fluorouracil is an analog of thymine).

A "control" cell, tissue, sample or subject is a cell, tissue, sample or subject of the same type as the cell, tissue, sample or subject being tested. For example, the control can be detected at exactly or nearly the same time as the test cells, tissue, sample or subject is detected. For example, the control can also be tested at a time remote from the time when the cells, tissue, sample or subject being tested, and the results of the control recorded so that the recorded results can be correlated with the cells, tissue, sample or subject tested by the test. The obtained results are compared. A control may be obtained from another source or a similar source than the test group or subject of the test, where the test sample is obtained from a patient suspected of having the disease or condition for which testing is being performed.

A "test" cell, sample or subject is the cell, sample or subject being tested.

A "pathologically indicative" cell, tissue or sample is one that indicates that the animal in which the cell, tissue or sample is located (or from which the tissue was obtained) is afflicted by a disease or disorder. For example, the presence of one or more mammary cells in lung tissue of an animal indicates that the animal is affected by metastatic breast cancer.

A tissue "normally includes" a cell if one or more cells are present in the tissue of an animal not affected by the disease or condition.

As used herein, a "derivative" of a compound refers to a chemical compound that can be produced from another compound of similar structure in one or more steps, such as substitution of hydrogen by an alkyl, acyl or amino group.

The word "detection" and its grammatical variants mean non-quantitative measurement of a species, while the word "determination" or "measurement" used in conjunction with its grammatical variants means quantitative measurement of a species. The terms "detect" and "identify" are used interchangeably herein.

As used herein, "detectable label" or "reporter molecule" refers to an atom or molecule that allows specific detection of a compound in the presence of a similar compound without the label. Detectable labels or reporter molecules include, but are not limited to, radioisotopes, antigenic determinants, enzymes, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that provide a change. Molecules that fluoresce through polarization or changes in light scattering.

As used herein, "effective amount" refers to an amount sufficient to produce the selected effect. A "therapeutically effective amount" of a compound is that amount of the compound sufficient to provide a beneficial effect to the subject to whom the compound is administered.

As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to convey the usefulness of a composition of the invention for its intended use. The instructional material of the kit of the invention may, for example, be attached to or shipped with the container containing the composition. Alternatively, the instructional material may be shipped separately from the container, for the purpose of the instructional material and the composition being used jointly by the recipient.

As used herein, the term "purified" and like terms refer to the enrichment of a molecule or compound with other components that are normally associated with the molecule or compound in its natural environment. The term "purified" does not necessarily indicate that complete purification of a particular molecule has been achieved during the procedure. As used herein, a "highly purified" compound refers to a compound that is greater than 90% pure.

As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier known in the art, such as phosphate buffered saline solution, water, emulsions (such as oil/water or water/oil emulsions), and Various types of humectants. The term also includes any formulation for use in animals, including humans, approved by a regulatory agency of the United States Federal Government or listed in the United States Pharmacopoeia.

As used herein, "sample" preferably refers to a biological sample from a subject, including but not limited to normal tissue samples, diseased tissue samples, biopsies, blood, saliva, stool, semen, tears, and urine. A sample can also be any other source of material obtained from a subject that contains cells, tissues or fluids of interest. Samples can also be obtained from cell or tissue cultures.

As used herein, the term "standard" refers to the material used for comparison. For example, it may be a known standard formulation or a compound administered or added to a control sample and used to compare the results when said compound is measured in a test sample. A standard may also refer to an "internal standard," such as a preparation or formulation that is added to a sample in known amounts prior to measurement of a marker of interest and that can be used to determine items such as purification or recovery when the sample is processed or subjected to purification or extraction procedures. compound.

A "subject" to be analyzed, diagnosed or treated is an animal. Said animals include mammals, preferably humans.

A "subject" for diagnosis or treatment is an animal, including a human.

A "therapeutic" treatment is one that is administered to a subject exhibiting signs of pathology to alleviate or eliminate those signs.

A "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to the subject to whom the compound is administered.

As used herein, the term "treating" includes prophylaxis of a particular disorder or disorder, or alleviation of symptoms associated with a particular disorder or disorder, and/or prevention or alleviation of symptoms. "Prophylactic" treatment is administration to a subject showing no signs of disease, but only early signs of disease, to reduce the risk of developing pathology associated with the disease.

As used herein, the term "receptor agonist" is defined as a compound that mimics the action of SlP on its receptor but with different potency and/or efficacy.

As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate-buffered saline, hydroxypropyl beta-cyclodextrin (HO-propyl beta-cyclodextrin), water, Emulsions (such as oil/water or water/oil emulsions), and various types of humectants. The term also includes any formulation for use in animals, including humans, approved by a regulatory agency of the United States Federal Government or listed in the United States Pharmacopoeia.

As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention and are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts through the presence of amino and/or carboxyl groups or similar groups thereof.

As used herein, the term "treating" includes prophylaxis of a particular disorder or disorder, or alleviation of symptoms associated with a particular disorder or disorder, and/or prevention or alleviation of symptoms.

As used herein, "effective amount" refers to an amount sufficient to produce the selected effect. For example, an effective amount of a S1P receptor agonist is an amount that reduces the cell signaling activity of the S1P receptor.

As used herein, "instructional material" includes publications, records, diagrams or any other method of expression that may be used to convey the usefulness of a compound of the invention for its intended use. The instructional material of the kit of the invention may, for example, be attached to or shipped with the container containing the composition. Alternatively, the instructional material may be shipped separately from the container, for the purpose of the instructional material and the composition being used jointly by the recipient.

The methods of the invention include kits comprising an inhibitor identified in the invention and instructional material describing the administration of the inhibitor or a composition comprising the inhibitor to a cell or animal. This is to be construed as including kits of other embodiments known to those skilled in the art, for example comprising solvents (preferably sterile) suitable for dissolving or suspending the compounds of the invention prior to administration of said compounds to cells or animals kit. Preferably, the animal is a human.

Those skilled in the art will understand that compounds of the present invention having chiral centers can exist and be isolated in optically active and racemic forms. Certain compounds can exhibit pleomorphism. It is to be understood that the present invention includes any racemic, optically active, polymorphic or stereoisomeric form of a compound of the invention, or mixtures thereof, which possess the useful properties described herein, how the optically active form is prepared (e.g., by Resolution of racemic forms, recrystallization techniques, synthesis from optically active starting materials, chiral synthesis or separation by chromatography using chiral stationary phases) and determination using standard test methods described herein or using other similar test methods known in the art S1P agonist activity is well known in the art.

In cases where the compounds are sufficiently acidic or basic to form acid or base salts, the use of the compounds as salts is appropriate. Examples of acceptable salts are organic acid addition salts with acids forming physiologically acceptable anions, such as tosylate, methanesulfonate, acetate, citrate, malonate, tartaric acid Salt, Succinate, Benzoate, Ascorbate, Alpha-Ketoglutarate, Alpha-Glycerophosphate. Suitable inorganic salts may also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates and carbonates.

Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Inorganic acids from which salts are derived include hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Organic acids from which salts are derived include acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic , Methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, Di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dienylamines, trienylamines, substituted alkenylamines, di(substituted alkenyl)amines, Tri(substituted alkenyl)amine, cycloalkylamine, di(cycloalkyl)amine, tri(cycloalkyl)amine, substituted cycloalkylamine, disubstituted cycloalkylamine, trisubstituted cycloalkane Cycloalkenyl amines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amines, trisubstituted cycloalkenyl amines, aryl amines , diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines, of which At least two substituents on the amine are different and selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, A group consisting of aryl, heteroaryl, heterocycle, etc. Also included are amines in which two or three substituents together with the amino nitrogen form a heterocycle or heteroaryl. Examples of suitable amines include, by way of example only, isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, aminobutylamine Triols, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamine, Theobromine, purine, piperazine, piperidine, morpholine, N-ethylpiperidine, etc. It should also be understood that other carboxylic acid derivatives may be used in the practice of this invention, such as carboxylic acid amides, including carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and the like.

Acceptable salts may be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid affording a physiologically acceptable anion. Alkali metal (eg sodium, potassium or lithium) or alkaline earth metal (eg calcium) salts of organic acids (eg carboxylic acids) can also be prepared.

A process for the preparation of a compound of formula (I), formula (II) or an intermediate for the preparation of a compound of formula (I) or formula (II) is provided as a further embodiment of the invention. Intermediates for the preparation of compounds of formula (I) or formula (II) are also provided as further embodiments of the present invention.

chemical definition

As used herein, the term "halogen" or "halo" includes bromine, chlorine, fluorine and iodine.

As used herein, the term "haloalkyl" refers to an alkyl group bearing at least one halogen substituent, eg, chloromethyl, fluoromethyl, or trifluoromethyl, and the like.

As used herein, the term "alkyl or C ₁ -C ₁₀ alkyl" means a branched or straight chain alkyl group having 1 to 6 carbon atoms. Typically, C ₁ -C ₁₀ alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl base, octyl, etc.

As used herein, the term "alkenyl or C ₂ -C ₁₀ alkenyl" denotes an ethylenically unsaturated branched or straight chain group having 2 to 10 carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, 1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl, and the like.

The term "alkynyl or C ₂ -C ₁₀ alkynyl" refers to an unsaturated branched or straight chain group having 2 to 10 carbon atoms and at least one triple bond, examples of such groups include, but are not limited to 1 -propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl and the like.

The term "C ₃ -C ₈ cycloalkyl" means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

As used herein, the term "optionally substituted" refers to zero to four substituents, wherein each of the substituents is independently selected. Each independently selected substituent may be the same as or different from the other substituents.

As used herein, the term "aryl" refers to a monocyclic or bicyclic _C5 - _C10 carbocyclic ring system having one or two aromatic rings, including, but not limited to, phenyl, benzyl, naphthyl, tetrahydro Naphthyl, indanyl, indenyl, etc.

As used herein, "optionally substituted aryl" includes aryl compounds having zero to four substituents, and substituted aryl includes aryl compounds having one to three substituents, wherein the substituents include, for example Alkyl, halogen or amino substituents.

The term "aralkyl" refers to any aryl group attached to the parent moiety through an alkyl group, eg, aryl(C ₁ -C ₈ alkyl). Thus, the term (C ₅ -C ₆ aryl)(C ₅ -C ₈ alkyl) refers to a five or six membered aromatic ring attached to the parent moiety through a C ₅ -C ₈ alkyl.

The term "heterocyclic group" refers to an optionally substituted monocyclic or bicyclic carbocyclic ring system containing one to three heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen.

As used herein, the term "heteroaryl" refers to an optionally substituted monocyclic or bicyclic carbocyclic ring system having one or two aromatic rings containing one to three heteroatoms, including but not limited to furyl, thienyl , pyridyl, etc.

The term "bicyclic" denotes an unsaturated or saturated stable 7- to 12-membered bridged or fused bicyclic carbocycle. The bicyclic ring can be attached at any carbon atom that provides a stable structure. The term includes, but is not limited to, naphthyl, dicyclohexyl, dicyclohexenyl, and the like.

The compounds of the present invention may contain one or more asymmetric centers in the molecule. According to the present invention, any structure for which no stereochemistry is assigned is understood to include all various optical isomers and racemic mixtures thereof.

The compounds of the present invention may exist in tautomeric forms, and the present invention includes both mixtures and separated individual tautomers. For example, the following structure:

is understood to mean a mixture of the following structures:

和

and

The term 16:0, 18:0, 18:1, 20:4 or 22:6 hydrocarbon refers to a branched or straight chain alkyl or alkenyl group, where the first integer represents the total number of carbons in the group and the second The integers represent the number of double bonds in the group.

As used herein, "S1P modulator" refers to a compound or composition capable of inducing a detectable change in S1P receptor activity (e.g., an increase or decrease of at least 10% in S1P activity), either in vivo or in vitro, that is regulated by S1P Detectable changes in body activity are measured by a given assay, such as a bioassay described in the Examples and known in the art. As used herein, "S1P receptor" refers to all S1P receptor subtypes (eg, S1P receptors _S1P1 , _S1P2 , _S1P3 , _S1P4 , _S1P5 ), unless a specific subtype is specified.

As used herein, the term " _EC50 of a formulation" refers to binding of a sphingosine ligand or other ligand at a given activity including S1P receptor and/or functional activity (e.g. signaling activity) of S1P receptor Concentration of formulation that is 50% maximally active for S1P receptors. Described differently, the _EC50 is the concentration of the formulation that provides 50% activity, where 100% activity is set as the amount that adds more ligand/agonist without increasing S1P receptor activity, and 0% is set as the amount in the absence of The amount of activity in the assay of the added ligand/agonist.

As used herein, the terms "phosphate analogs" and "phosphonate analogs" include analogs of phosphate and phosphonate wherein the phosphorus atom is in the +5 oxidation state and one or more oxygen atoms are replaced by non-oxygen moieties, Including, for example, the phosphate analogues phosphorothioate, phosphorodithioate, selenophosphate, diselenophosphate, aniline thiophosphate, aniline phosphate, phosphoramidite, borophosphate, etc., including Relevant counterions such as hydrogen, _NH4 , Na, etc. if such counterions exist.

The present invention relates to sphingosine-1-phosphate (S1P) analogs which are active as receptor agonists at one or more S1P receptors, in particular of the _S1P1 , _S1P4 and _S1P5 receptor types. Compounds of the present invention include compounds having a phosphate moiety and those having hydrolysis-resistant phosphate substitutes such as phosphonates, α-substituted phosphonates (particularly when the α-substituent is a halogen), and thiophosphates. compound.

In one embodiment of the S1P receptor agonist or a pharmaceutically acceptable salt thereof, it has the general structure of formula (IIA):

Wherein, n is 0, 1, 2 or 3; X is selected from hydroxyl (-OH), phosphate (-OPO ₃ H ₂ ), phosphonate (-CH ₂ PO ₃ H ₂ ), α-substituted phosphonate ( including -CHFPO ₃ H ₂ , -CF ₂ PO ₃ H ₂ , -CHOHPO ₃ H ₂ , -C=OPO ₃ H ₂ ),

wherein R ¹ is selected from the group consisting of hydrogen, halogen (where F or C1 is preferred halogen), (C ₁ -C ₆ )alkyl such as methyl, ethyl and propyl, or halogen, hydroxy, alkoxy , cyano-substituted (C ₁ -C ₆ )alkyl, eg trifluoromethyl.

The ^R2 group is selected from the group consisting of alkyl, alkenyl, alkynyl, alkyl-substituted aryl, alkyl-substituted cycloalkyl, aralkyl, and aralkyl-substituted aryl. In ^R2 , a chain length of 5-8 carbon atoms is preferred.

The present invention also provides esters of any compound of formula (II), such as phosphate esters, wherein ester functionality may be added to form prodrugs with increased oral availability.

In a preferred embodiment, said compound of formula (II) may have R ¹ selected from the group consisting of H, halogen (preferably F or Cl), methyl, trifluoromethyl, ethyl, propyl Or other lower alkyl (C ₁ -C ₆ ), or lower alkyl substituted by halogen, hydroxyl, alkoxy, cyano; and R ² is selected from alkyl, alkenyl, alkynyl, alkyl (optionally substituted aryl), alkyl (optionally substituted cycloalkyl), aralkyl, and aralkyl (optionally substituted aryl) preferably having a length of 5 to 8 carbon atoms.

Potential uses of S1P receptor antagonist prodrugs (preferably _S1P1 receptor type selective antagonists) include, but are not limited to:

Alteration of lymphocyte trafficking as a treatment for autoimmune diseases such as uveitis, type I diabetes, rheumatoid arthritis, inflammatory bowel disease, and more particularly multiple sclerosis. "Treatment" of multiple sclerosis includes multiple forms of the disease, including relapsing-remitting, chronic progressive, etc., and S1P receptor antagonists may be used alone or in combination with other agents that relieve the signs and symptoms of the disease as well as prophylaxis sexual use.

In addition, the compounds of the invention are useful for altering lymphocyte trafficking as a method of prolonging the survival of allografts, for example for solid organ transplantation, treatment of graft versus host disease, bone marrow transplantation, etc. .

In addition, the compounds of the invention are useful for inhibiting autotaxins. Autotaxin, a serum phosphodiesterase, has been shown to undergo end-product inhibition. Autotaxin hydrolyzes several substrates to produce lysophosphatidic acid and sphingosine-1-phosphate and is implicated in cancer progression and angiogenesis. Therefore, S1P receptor agonist prodrugs such as VPC01091 can be used to inhibit autotaxin. This activity may be combined with or independent of agonism of the S1P receptor.

In addition, the compounds of the invention are useful for inhibiting S1P lyases. S1P lyases are intracellular enzymes that irreversibly degrade S1P. Inhibition of S1P lyase disrupts lymphocyte trafficking with concomitant lymphopenia. Therefore, S1P lyase inhibitors can be used to modulate immune system function. Therefore, prodrugs such as VPC01091 can be used to inhibit S1P lyase. The inhibitory effect may be consistent with S1P receptor activity, or may be independent of activity at any S1P receptor.

"Treatment" of multiple sclerosis includes multiple forms of the disease, including relapsing-remitting, chronic progressive, etc., and S1P receptor agonists may be used alone or in combination with other agents that relieve the signs and symptoms of the disease and prevent sexual use.

The invention also includes pharmaceutical compositions comprising the compounds of the invention. More specifically, the compounds can be formulated into pharmaceutical compositions using standard pharmaceutically acceptable carriers, fillers, solubilizers and stabilizers known to those skilled in the art. For example, compositions containing a compound of the invention, or an analog, derivative or modification thereof, as described herein are used to administer the compound to a subject as appropriate.

The compound of the present invention can be used for the treatment of diseases or disorders, comprising administering a therapeutically acceptable amount of a compound of formula (I) to a subject in need thereof, or containing a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable Carrier pharmaceutical composition.

A specific value of lower alkyl is ethyl or propyl.

Specific values for halogen are fluorine or chlorine.

The specific value of X is hydroxyl or OPO ₃ H ₂ .

Alpha-substituted _{phosphonates include -CHFPO3H2 , -CF2PO3H2 , -CHOHPO3H2} , -C= _OPO3H2 or _{phosphorothioate} ( _{OPO2SH2 )} .

The specific value of ^R1 is hydrogen.

The specific value of ^R2 is an alkyl group with a length of 5-8 carbon atoms.

More specific values for R ² are heptyl, octyl, nonyl, -O-heptyl, -C(=O)heptyl or CH ₃ -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ - _CH2 -O-.

The more specific value of the alkyl group in ^R2 is octyl or -O-heptyl.

The more specific value of the alkyl group in ^R2 is octyl.

The specific value of n is 1 or 2.

Specific cycloalkyl groups having a double bond include:

Particular compounds of the invention have the ^R2 group positioned para to the cycloalkyl ring.

Particular compounds of the invention have an ^R1 group located in the ortho or meta position to ^R2 .

Particular compounds of the invention have an ^R2 group located in the para position (ie 1,4) to the benzylcycloalkyl.

Non-limiting examples of esters of compounds of the invention include compounds wherein the X group is:

成的组；以及wherein Y is selected from O, CH ₂ , CHOH, CHF, CF ₂ and

into groups; and

R ⁹ and R ¹⁰ are independently selected from alkoxy, alkenyloxy, alkynyloxy, aryloxy,

以及

组成的组； OR ¹¹ ,

as well as

composed of groups;

Wherein, R ¹¹ is selected from the group consisting of C ₁ -C ₄ alkyl, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and optionally substituted aryl. Particularly preferred R ⁹ and R ¹⁰ are alkoxy,

as well as

Synthetic routes for the preparation of VPC01091 and VPC01211 are provided in the scheme in Figure 1 . Other compounds of formula (I) or formula (II) can be prepared by those skilled in the art using known techniques modified from the procedures of the schemes described and detailed in the specific examples herein.

The specific compound of formula (II) of the present invention is VPC01091, wherein X is OH, R ¹ is hydrogen, R ² is octyl (C ₈ H ₁₇ ), n is 2, and the R ² group is at the para position of the benzene ring. The formula is:

The specific compound of formula (II) of the present invention is VPC02162, wherein X is OH, R ¹ is hydrogen, R ² is octyl (C ₈ H ₁₇ ), n is 2, and the R ² group is at the meta-position of the benzene ring, The formula is:

The present invention also includes the following isomers:

以及

as well as

These compounds can be formulated as a mixture and separated by chromatographic methods. The suitable conditions for separation are as follows: column: Chiralpak AD 4.6 mmID x 250mm; mobile phase: Hex/EtOH/MeOH/DEA=95/2.5/2.5/0.03; flow rate: 1mL/min; detector: UV 220nm; column temperature: 40 °C; or column temperature: 25 °C. After isolation, both isoforms were found not to be phosphorylated by the SPHK2 enzyme in vitro. However, when tested for prior phosphorylation, the phosphorylated compounds were found to be potent agonists of the S1P receptor.

Another specific compound of formula (II) of the present invention is VPC01211, wherein X is OPO ₃ H ₂ , R ¹ is hydrogen, R ² is octyl (C ₈ H ₁₇ ), n is 2, and the R ² group is in benzene The paraposition of the ring, the formula is:

Another specific compound of formula (II) of the present invention is VPC02164, wherein X is OPO ₃ H ₂ , R ¹ is hydrogen, R ² is octyl (C8H17), n is 2, and the R ² group is between the benzene ring bit, the formula is:

Other examples of compounds of the invention that include heteroatoms (e.g., N, S, O) and/or double bonds in the cycloalkyl ring include the following structures:

or

Other compounds of formula (I) or formula (II) having general formula (III) are described below. The specific changes are described in Table 1:

Table 1

compound drawing number R n x VPC02004 - C ₇ H ₁₅ 2 OH VPC02007 - C ₇ H ₁₅ 2 OPO ₃ H ₂ VPC01091 CA5 C ₈ H ₁₇ 2 OH VPC01211 CA5-P C ₈ H ₁₇ 2 OPO ₃ H ₂ VPC02031 - C ₉ H ₁₉ 2 OH VPC02033 - C ₉ H ₁₉ 2 OPO ₃ H ₂ VPC01289 - C ₁₀ H ₂₁ 2 OH VPC01292 - C ₁₀ H ₂₁ 2 OPO ₃ H ₂ VPC01220 CA4 C ₈ H ₁₇ 1 OH VPC01222 CA4-P C ₈ H ₁₇ 1 OPO ₃ H ₂ VPC01213 CA6 C ₈ H ₁₇ 3 OH VPC01214 CA6-P C ₈ H ₁₇ 3 OPO ₃ H ₂

The present invention also provides esters of compounds of formula (I) or formula (II), wherein formation of the ester converts the compound into a prodrug that enhances administration, eg increases oral availability. In addition, the present invention also provides a pharmaceutically acceptable salt of the compound of formula (I) or formula (II). In addition, the present invention provides all possible isomers of the structure described in formula (I) or formula (II), note: when n is 1 (cyclobutanyl), the compound is symmetrical and lacks Chiral center, but cis and trans forms exist.

Pharmaceutical compositions containing one or more compounds of the present invention may be administered to a subject in need of such treatment by any number of routes and methods including, but not limited to, topical, oral, oral, intravenous , intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ocular, pulmonary, or rectal routes. The oral route will generally be used in most situations where the compounds of the invention are required. Administration by intravenous injection or infusion is preferred for acute treatment. For maintenance regimens, oral or parenteral, eg intramuscular or subcutaneous routes are preferred.

According to one embodiment, there is provided a composition containing the compound of the present invention or its analog, derivative or modified form and albumin, more particularly, the composition contains the compound of the present invention, a pharmaceutically acceptable carrier and 0.1 -1.0% Albumin. Albumin acts as a buffer and improves the solubility of the compounds. In one aspect, no albumin is added.

In one embodiment, the administration of pharmaceutical compositions useful in practicing the invention may be performed to deliver doses from 1 ng/kg/day to 100 mg/kg/day. In another embodiment, the administration of pharmaceutical compositions useful in practicing the invention can be performed to deliver doses from 1 ng/kg/day to 100 g/kg/day.

Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The pharmaceutical compositions can be prepared, packaged or sold in the form of sterile injectable solutions or oily suspensions or solutions. Such suspensions or solutions may be prepared according to known techniques and may contain, in addition to the active ingredient, other ingredients such as dispersing, wetting or suspending agents as herein described. Such sterile injectable preparations can be prepared using nontoxic parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, fixed oils such as synthetic mono- or diglycerides.

Compounds identified using any of the methods described herein can be formulated and administered to a subject for the treatment of any of the diseases and conditions described herein. However, the use of the compounds of the present invention should not be construed to include only the diseases and conditions described herein. Preferably, the subject is a human.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or later developed in the art of pharmacy. In general, these methods of preparation involve bringing the active ingredient into association with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product in the desired unit-dose or multi-dose form. unit.

Although the description of pharmaceutical compositions provided herein is in principle directed to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that these compositions are generally suitable for administration to all species of animals.

The modification of pharmaceutical compositions suitable for human administration in order to render the compositions suitable for administration to various animals is well known, and one of ordinary skill in veterinary pharmacology can devise and carry out such compositions using only ordinary experimentation. modified. Contemplated subjects for administration of the pharmaceutical compositions of this invention include, but are not limited to, humans and other vertebrates and mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats and dog.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in bulk as a single unit dose or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete quantity of a pharmaceutical composition containing a predetermined quantity of an active ingredient. The amount of said active ingredient is usually equal to the dose of active ingredient administered to the subject or a suitable fraction of such a dose, for example 1/2 or 1/3 of such a dose.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any other ingredients of the pharmaceutical compositions of this invention will vary according to their nature, size and condition of the subject being treated and also according to the route of administration of the composition. . For example, the composition may contain from 0.1% to 100% (w/w) active ingredient.

In addition to the active ingredient, the pharmaceutical compositions of the present invention contain one or more other pharmaceutically active ingredients. Other ingredients specifically contemplated include antiemetics and scavengers such as cyanide scavengers and cyanate scavengers.

Controlled or sustained release formulations of the pharmaceutical compositions of the invention can be prepared using conventional techniques.

In some cases, the dosage form used may be used as a sustained or controlled release of one or more active ingredients such as hydroxypropyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, Multiple coatings, microparticles, liposomes or microspheres or combinations thereof are provided to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can readily be selected for use with the pharmaceutical compositions of the invention. Accordingly, the invention includes single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps and caplets, which are adapted for controlled release.

Most controlled release formulations are designed to initially release the amount of drug that rapidly produces the desired therapeutic effect, and to gradually and continuously release other amounts of drug to maintain that level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

Controlled release of an active ingredient can be stimulated by various triggers, such as pH, temperature, enzymes, water or other physiological conditions or compounds.

Powder and granule preparations of the pharmaceutical preparations of the present invention can be prepared using known methods. These formulations can be administered directly to a subject, the formulations can be used, for example, to form tablets, fill capsules or to prepare aqueous or oily suspensions or solutions by the addition of aqueous or oily vehicles. Each of these formulations may further contain one or more dispersing or wetting agents, suspending agents and preservatives. Other excipients such as fillers and sweetening, flavoring or coloring agents may also be included in these formulations.

As used herein, an "oil" liquid is a liquid that contains liquid molecules comprising carbon and exhibits the characteristic of being less polar than water.

Formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged or sold as discrete solid dosage units including, but not limited to, tablets, hard or soft capsules, cachets, capsules containing Tablets, or lozenges, each contain a predetermined amount of active ingredient. Other formulations suitable for oral administration include, but are not limited to, powder or granule formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, oral douches, or emulsions . The terms oral douche and mouthwash are used interchangeably herein.

A tablet containing the active ingredient may be prepared, for example, by compressing or molding the active ingredient, optionally with one or more other ingredients. Compressed tablets may be prepared by mixing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granule formulation, optionally with one or more binders, lubricants, excipients, surface active and dispersing agents. Prepared by pressing. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier and at least sufficient liquid to wet the mixture. Pharmaceutically acceptable excipients for the preparation of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binders and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrants include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized cornstarch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silicon dioxide and talc.

Tablets may be uncoated, or may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. For example, glyceryl monostearate or glyceryl distearate may be used to coat tablets. Also by way of example, tablets may be coated to form osmotically controlled release tablets using the methods described in US Patent Nos. 4,256,108, 4,160,452 and 4,265,874. Tablets may also contain sweetening agents, flavoring agents, coloring agents, preservatives or some combination thereof to provide pharmaceutically aesthetically pleasing preparations.

Hard capsules containing the active ingredient may be prepared using physiologically degradable compositions such as gelatin. Such hard capsules contain the active ingredient and may also contain other ingredients including, for example, inert solid diluents such as calcium carbonate, calcium phosphate or kaolin.

Soft capsules containing the active ingredient can be prepared using physiologically degradable compositions such as gelatin. Such soft capsules contain the active ingredient which may be mixed with a water or oil base such as peanut oil, liquid paraffin or olive oil.

Liquid preparations of the pharmaceutical compositions of the present invention suitable for oral administration can be prepared, packaged or sold in liquid form or dry preparations to be reconstituted with water or other suitable vehicle before use.

Injections can be prepared, packaged, or sold in unit dosage form such as ampoules or in multi-dose containers with a preservative. Formulations for parenteral administration include, but are not limited to, suspensions in oily or aqueous vehicles, solutions, emulsions, pastes and implantable sustained-release or biodegradable formulations. The formulations may also contain one or more other ingredients including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of the formulation for parenteral administration, the active ingredient is provided in dry form (i.e. powder or granules) which is reconstituted with a suitable vehicle (e.g. sterile pyrogen-free water), The reconstituted composition is then administered parenterally.

The pharmaceutical composition of the present invention can be prepared, packaged or sold in a form suitable for oral administration. Suitable formulations may be prepared, for example, in the form of tablets or lozenges using conventional methods of administration, and may contain, for example, 0.1-20% (w/w) active ingredient, a balanced solution containing an orally dissolvable or degradable composition and any Optionally one or more other ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise powders or aerosolized or sprayed solutions or suspensions containing the active ingredient. These powder, aerosolized or spray formulations, when dispersed, preferably have an average particle or droplet size in the range of about 0.1 to about 200 nanometers, and may also contain one or more other ingredients described herein.

As used herein, "other ingredients" include, but are not limited to, one or more of the following ingredients: excipients, surfactants, dispersants, inert diluents, granulating and disintegrating agents, binders, lubricants agent, sweetener, flavoring agent, coloring agent, preservative, physiologically degradable composition such as gelatin, water carrier and solvent, oil carrier and solvent, suspending agent, dispersing agent or wetting agent, emulsifying agent, viscosifying agent , buffers, salts, thickeners, fillers, emulsifiers, antioxidants, antibiotics, antifungal agents, stabilizers and pharmaceutically acceptable polymeric or hydrophobic materials. See Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, incorporated herein by reference.

The compound may be administered to the subject frequently several times a day, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, e.g. Every few months or even yearly or less frequently. The frequency of dosing will be readily apparent to those skilled in the art and will depend on any number of factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the subject, and the like.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of the invention. According to one embodiment, a kit for treating a subject in need of immunomodulation is provided. Preferably, the subject is a human. In one embodiment, the kit includes one or more S1P analogs of the invention and may also include one or more known immunosuppressants. These pharmaceutical formulations can be packaged in a variety of containers, such as vials, test tubes, microtiter microplates, vials, and the like. Additional reagents can be included in separate containers and provided using a kit, eg, positive control samples, negative control samples, buffers, cell culture media, and the like. Preferably, the kit also includes instructions for use.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

Those skilled in the art will readily understand that the present invention will be well adapted to carry out the stated ends and obtain the mentioned ends and advantages as well as those inherent therein. The present invention may be embodied in other specific forms without departing from its spirit or essential properties.

Example

The invention will now be described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention should in no way be construed as limited to these examples, but rather should be construed to encompass any and all variations which may become apparent as a result of the teachings provided herein.

Example 1: (1-amino-3-(4-octylphenyl)cyclopentyl)methanol (6)

A: 3-(4-iodophenyl)cyclopentanone (1)

0.23g palladium(II) acetate ( _0.1eq ) and 0.23g antimony(III) chloride (0.1eq) were added to 80mL containing 0.82g (10mmol) 2-cyclopent-1-one, 2.48g (10mmol) 4-iodophenylboronic acid and 1.6g (20mmol) sodium acetate in acetic acid. After stirring at 25°C for 24 hours, the black precipitate was removed by filtration and the filtrate was diluted with 250 mL of brine and extracted twice with 50 mL of dichloromethane. The organic extract was stirred with saturated NaHCO ₃ solution for 30 min, then washed with brine and dried over MgSO ₄ . Removal of solvent gave a yellow oil which was further purified using flash column (chloroform) to give 1.92 g (67%) of the product as a white solid. J. Org. Chem., 1995, 60, 883-888.

¹ H NMR (CDCl ₃ ) δ7.63 (d, 2H, ArH), 7.00 (d, 2H, ArH), 3.35 (m, 1H, ArCHCC), 2.7-1.8 (m, 6H, cyclopentyl);

¹³ C NMR (CDCl ₃ ) δ 218, 143, 138, 129, 95, 46, 42, 39, 31.

B.: 3-(4-oct-1-ynyl)phenyl)cyclopentanone (2)

1.1 g (10 mmol) 1-octyne was added to a flame-dried 25 mL flask containing 10 mL of a THF solution containing 1.43 g (5 mmol) 1. After degassing for 30 min, 2 mL of triethylamine, 5 mg of CuI and 10 mg of Pd(PPh ₃ ) ₄ were added under N2 protection. The reaction was complete within 6 hours, and after removal of solvent and volatile reagents, the mixture was column purified using chloroform to afford 1.34 g (99%) of a yellow oil.

¹ H NMR (CDCl ₃ ) δ7.35 (d, 2H, ArH), 7.15 (d, 2H, ArH), 3.37 (m, IH, ArCHCC), 2.7-2.2 (m, 6H, cyclopentyl), 1.95 (m, 2H, CCCH ₂ CH ₂ ), 1.6-1.2 (m, 8H, CH ₂ ), 0.89 (t, J=6Hz, 2H, CH ₃ );

¹³ C NMR (CDCl ₃ ) δ 220, 143, 132, 127, 122, 91, 80, 46, 42, 39, 32, 31, 29, 29, 23, 20, 14.

C: 3-(4-octylphenyl)cyclopentanone (3)

A few drops of formic acid and a catalytic amount of 5% Pd/C were added to a 25 mL flask containing 10 mL methanol and 1.34 g (5 mmol) 2 . The reaction vessel was flushed 3 times with H ₂ and then filled with a H ₂ balloon. After two days of hydrogenolysis, the washed solutes were filtered through a pad of silica and concentrated to a yellow oil. 1.32 g (98%) of product collected.

¹ H NMR (CDCl ₃ ) δ7.18 (s, 4H, ArH), 3.38 (m, IH, ArCHCC), 2.60 (t, 2H, CCCH ₂ CH ₂ ), 2.45-1.91 (m, 6H, cyclopentyl ), 1.64-1.15 (m, 12H, CH ₂ ), 0.90 (t, 3H, CH ₃ );

¹³ C NMR (CDCl ₃ ) δ 220, 142, 140, 129, 127, 46, 42, 39, 36, 32, 32, 32, 30, 30, 29, 23, 14.

D.: 1-Amino-3-(4-octylphenyl)cyclopentanenitrile (4)

3.20 g (11.8 mmol), 1.15 g (23.5 mmol) of sodium cyanide and 1.25 g (23.5 mmol) of ammonium chloride were added to 20 mL of ammonium hydroxide. After stirring the mixture vigorously overnight, it was extracted twice with 10 mL of dichloromethane. The organic extract was dried and concentrated to give 3.30 g of a yellow oil. The crude product was used in the next step without further purification. J. Med. Chem., 1986, 29, 1988-1995.

E.: 1-Amino-3-(4-octylphenyl)cyclopentanecarboxylic acid (5)

3.3 g (11.2 mmol) and 50 mL of concentrated hydrochloric acid were heated to 70 °C and stirred overnight. The resulting clear aqueous solution was evaporated to dryness. Add 10 mL of water and dry again. Repeat the process several times. The crude product was washed with water and acetone to give a fine white powder. Yield 1.7 g (45%).

¹ H NMR (d ⁶ -DMSO) δ7.25-7.06 (m, 4H, ArH) ₉ 3.21 (m, IH, ArCHCC), 2.38-1.62 (m, 6H, cyclopentyl), 1.49-1.20 (m, 14H, CH ₂ ), 0.81 (t, J=6Hz, 3H, CH ₃ );

¹³ C NMR (d ⁶ -DMSO) δ 175, 141, 140, 64, 51, 46, 45, 44, 36, 35, 35, 34, 32, 32, 29, 29, 23, 15.

F.: (1-Amino-3-(4-octylphenyl)cyclopentyl)methanol (6)

63.4 mg (0.2 mmol) of 5 and 27 mg (0.6 mmol) of sodium borohydride were dissolved in 3 mL of THF. After cooling the solution to 0 °C, 51 mg (0.2 mmol) _I2 was dissolved in 1 mL THF and added dropwise. Then, the vessel was equipped with a condenser and the reaction mixture was refluxed under _N2 for 5 hours. Excess _NaBH4 was quenched with methanol. After removing the solvent, 2 mL of water and 5 mL of dichloromethane were added, and the mixture was stirred for about 1 hour until the organic layer became clear. The organic phase was collected and the aqueous phase was extracted two more times with dichloromethane. The combined organic extracts were dried and concentrated to give 43 mg (71%) of crude product. Further purification on TLC using methanol/chloroform (5:95) afforded 13 mg of a clear oil. J. Org. Chem., 1993, 58, 3568-3571.

¹ H NMR (CD ₃ OD) δ7.11 (m, 4H, ArH), 3.80 (t, J=7.5Hz, IH, cyclopentyl-CH ₂ O), 3.67 (t, J=7.5Hz, IH, Cyclopentyl-CH ₂ O), 3.01 (m, IH, ArCHCC) ₅ 2.55 (t, J=7.5Hz, 2H, ArCH ₂ ), 2.29-1.69 (m, 6H, cyclopentyl), 1.57 (m, 2H, ArCH ₂ CH ₂ ), 1.38-1.28 (m, 10H, CH ₂ ), 0.89 (t, J=7.5Hz, 3H, CH ₃ );

¹³ C NMR (CD ₃ COCD ₃ ) δ141, 128, 127, 96, 45, 44, 43, 35, 35, 33, 33, 32, 32, 29, 29, 29, 23, 13.

Example 2: (1-amino-3-(4-octylphenyl) cyclopentyl) methyl dihydrogen phosphate (7)

(1-Amino-3-(4-octylphenyl)cyclopentyl)methyl dihydrogen phosphate (7)

1 mL of 85% H ₃ PO ₄ was slowly added dropwise into 0.5 g of P ₂ O ₅ , and then the acid-anhydride mixture was heated at 100° C. for 1 hour under the protection of N ₂ . An additional 0.5 g of P ₂ O ₅ and 30 mg of 6 were added to the polyphosphoric acid and heated at 100° C. for 5 hours. After cooling to room temperature, 10 mL of ice water was added to the reaction mixture. The product precipitated out as a white solid. The product was collected and washed with water. 31 mg (82%) of the green product were collected after vacuum drying. MS only two peaks: M+1 = 384.4 and 304.4 (hydrolyzed back to 6).

Example 3: GTPγS-35 binding assay

This assay demonstrates agonist activity of G protein-coupled receptors (GPCRs) in isolation. This assay induces recombinant GPCRs (such as S1P1-5 receptors) and three subunits of heterotrimeric G proteins (typically α1, β2, γ3) by transfecting HEK293T cells with four plasmid DNAs encoding each protein Concomitant expression of each in said cells. Approximately 60 hours after transfection, cells were harvested, disrupted, and nuclei discarded. This allows the preparation of coarse microsomes from the residue. Stimulation with agonists (eg, S1P) of the G protein receptor complex on microsomes results in a dose-dependent conversion of GTP to GDP on the alpha subunit. To detect the GTP-bound alpha subunit, a GTP analog (GTPγS-35), a radionuclide (sulfur-35) labeled phosphorothioate that is not hydrolyzed to GDP, was used. Microsomes with attached G protein were collected by filtration and bound GTPγS-35 was quantified in a liquid scintillation counter. Relative potencies ( _EC50 values) and maximal effects (efficacy, _Emax ) were determined from the assay. Antagonist activity is measured as a right shift in the agonist dose-response curve in the presence of a fixed amount of antagonist. If the antagonist acts competitively, the affinity of the receptor/antagonist pair (Kj) can be determined.

In this assay, phosphorylated forms of all VPC01091 isoforms, as well as phosphorylated VPC01091 (VPC01211, CA5-P) were negative by themselves, or were inverse agonists of S1P ₃ receptors, and were S1P ₁ receptors relative to S1P partial agonists (i.e. not fully effective). Inverse agonists are antagonists, ie, they interfere with binding of the agonist ligand, but do not stimulate activation of the receptor.

VPC01211 (CA5-P) _{and cyclohexyl (CA6-P) analogs are partial agonists at the S1P 1 receptor (} Fig. Inactive. These compounds are full agonists of the _S1P4 receptor (Figure 10) and partial agonists of the _S1P5 receptor (Figure 11). Cyclohexyl compounds (CA4-9) have very little agonist activity at the _S1P1 receptor, but have similar activity to cyclopentyl and cyclohexyl compounds.

The effects of sphingosine 1-phosphate (S1P), VPC01211 (phosphorylated VPC01091 ) and four component isomers of VPC01211 on recombinant human type 1 S1P (S1P ₁ ) receptors were evaluated ( FIG. 16 ). The assay was performed as described in Davis, MD., JJ. Clemens, TLMacdonald and KRLynch (2005) "S1P Analogs as Receptor Antagonists" Journal of Biological Chemistry, vol.280, pp.9833-9841. The order of potency ( _EC50 ) of the compounds in this experiment was: Isomer 1 > Isomer 3 > Isomer 4 > Isomer 2 > S1P > VPC01211. Isomers were prepared by chemical phosphorylation of each isomer of VPC01091.

The effects of sphingosine 1-phosphate (S1P), VPC01211 (phosphorylated VPC01091) and four component isomers of VPC01211 on recombinant human S1P type ₃ (S1P3) receptors (Figure 17) were evaluated. The assay was performed as described in Davis, MD, JJ. Clemens, TLMacdonald and KRLynch (2005) "S1P Analogs as Receptor Antagonists" Journal of Biological Chemistry, vol.280, pp.9833-9841. The potency rank order ( _EC50 ) of the compounds in this experiment was: S1P>Isomer 2>VPC01211>Isomer 4>Isomer 1>Isomer 3. Isomers were prepared by chemical phosphorylation of each isomer of VPC01091.

Example 4: Lymphopenia Assay

Prodrug compounds (ie primary alcohols such as VPC01091) are dissolved in 2% hydroxypropyl [beta]-cyclodextrin and introduced into mice by oral gavage at a dose of 0.01-30 mg/kg body weight. After 24 hours (or multiples thereof), the mice were gently anesthetized and 0.1 ml of blood was drawn from the orbital sinus. Use a Hemavet blood analyzer to measure the number of lymphocytes (expressed in thousands per ml of blood, normally 4-11 thousand). In the histogram, showing the four isomers of VPC01091, the 100% values for vehicle-treated mice were 7.5 at 24 hours and 5 at 96 hours. Three mice per group whose germline was mixed svl29 x C57BL/6. The active compound (eg VPC01211) was dissolved at 20 mM in acidified DMSO and diluted 1:20 in 2% aqueous hydroxypropyl [beta]-cyclodextrin with stirring. The solution was introduced into mice by intraperitoneal (i.p.) injection at a dose of 0.01-10 mg/kg body weight.

When dissolved in 2% aqueous hydroxypropyl [beta]-cyclodextrin (vehicle) and administered orally (gavage) to mice, VPC01091 induced a deep and long-lasting lymphopenia (Figure 3). Figure 3 depicts total blood lymphocyte counts following a single dose of VPC01091 or vehicle. A single ED ₉₅ dose of VPC01091 can cause lymphopenia for a week or more. Five mice per group, male, 10-11 weeks old, svl29/C57B16 line.

The dose response curve for VPC01091 in the lymphopenia assay is given in FIG. 4 . Figure 4 schematically summarizes the oral administration of VPC01091 in 2% hydroxypropyl [beta]-cyclodextrin to 3 mice per group (syngeneic as in Figure 3).

This phosphorylation of these compounds is thought to be catalyzed in vivo by sphingosine type 2 (SPHK2), which is encoded by the SPHK2 gene in mice. When VPC01091 was introduced into mice without a functional SPHK2 gene, no lymphopenia was observed (Figure 5).

In Figure 18, the ability of the SPHK2 enzyme to phosphorylate four VPC01091 isoforms is depicted.

In Figure 19, the results of an assay administered by oral gavage using the phosphorylated isoform of VPC01091 are graphically illustrated. Total lymphocyte counts (k/μl) were recorded 24 hours and 96 hours after IV administration of the phosphorylated VPC01091 isoform.

Example 5: Sphingosine Kinase Assay

Recombinant sphingosine kinase type 2 (SPHK2) was produced by transfecting the relevant plasmid DNA into HEK293T cells to induce expression of mouse or human recombinant enzymes. After about 60 hours, the cells were harvested, disrupted and the microsomal-free (ie soluble) fraction was retained. The disrupted cell suspension containing the recombinant enzyme was mixed with the test compound (VPC01091, sphingosine, etc.) (5-50 micromole) and γ-32P-ATP and incubated at 37°C for 0.5-2.0 hours. The liposomes in the reaction mixture were extracted into an organic solvent and visualized by normal phase thin layer chromatography. Radiolabeled bands were detected by autoradiography, scraped from plates and quantified by scintillation counting. In the histograms shown, sphingosine was 15 μM, VPC01091 and its isomers were 50 μM, and the incubation time was 0.5 h.

Example 6: Calcium mobilization assay

Hamster CHOK1 cells were transfected with human S1P ₂ or human S1P ₃ receptor DNA, and clonal populations showing ectopically expressed receptors were isolated and expanded. To detect calcium mobilization in response to agonist stimulation, cells were plated into 96-well culture plates, loaded with calcium sensing dye Fluo-4AM, and cells were exposed to different concentrations of agonists for 3-5 minutes. Fluorescence changes associated with intracellular calcium mobilization were detected using a FlexStation Fluorometer. Each agonist concentration was tested in triplicate. This scheme is described in detail in Davis, MD, JJ. Clemens, TLMacdonald and KRLynchSphingosine 1-phosphate analogs as receptor antagonists. J. Biological Chemistry 280, 9833-9841 (2005). The results are depicted in Figures 7 and 9.

Figure 7: CHOK1 cells transfected with _S1P2 receptor DNA.

Figure 9: CHOK1 cells transfected with _S1P3 receptor DNA.

Embodiment 7: heart rate measurement

Mice were administered with VPC01091 (intravenously, 3 mg/kg) or vehicle (2% hydroxypropyl β-cyclodextrin), and heart rate was measured at indicated times after administration. The heart rate of uninhibited, conscious animals was captured using the ECGenie ^™ system.

The results are depicted in Figure 2.

Headings are included in this article for reference and to help locate certain sections. These headings are not intended to limit the scope of the concepts described below them, and these concepts may apply throughout the rest of the specification.

Other methods used but not described herein are well known and within the ability of those of ordinary skill in the fields of clinical, medical, cytology, histochemistry, biochemistry, molecular biology, microbiology, and recombinant DNA technology.

Abbreviations used herein have their traditional meanings in the fields of chemistry and biology. All publications, patents, and patent documents cited in the specification are herein incorporated by reference as if each were incorporated by reference. In case of any inconsistency, the content of the present invention, including any definitions therein, shall prevail. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made within the spirit and scope of the invention.

Claims (30)

1. chemical compound or its pharmaceutically acceptable salt or ester with following formula:

Or

Wherein, R ⁴And R ⁷Be CH or CH independently ₂R ⁵Be C, CH or N, R ⁶Be CH, CH ₂, O, S or SR ³R ³Be hydrogen or (C ₁-C ₁₀) alkyl;

X is selected from the phosphonate radical of hydroxyl, phosphate radical, phosphonate radical, alpha-substituted;

R ¹Be selected from hydrogen, halogen, trifluoromethyl, (C ₁-C ₁₀) alkyl, by halogen, hydroxyl, (C ₁-C ₁₀) (the C that replaces of alkoxyl or cyano group ₁-C ₁₀) group formed of alkyl; And

R ²Be selected from (C ₁-C ₂₀) alkyl, the alkyl of cycloalkyl substituted, (C ₂-C ₂₀) thiazolinyl, (C ₂-C ₂₀) group formed of the aralkyl that replaces of alkynyl, aryl, the alkyl aryl, aralkyl and the aryl that replace; R wherein ²One or more carbon atoms in the group can be independently by non-peroxide oxygen, sulfur or NR ⁸Replace;

R wherein ⁸Be hydrogen or (C ₁-C ₁₀) alkyl;

R wherein ²In alkyl, thiazolinyl and alkynyl optional replaced by oxo; N is 0,1,2 or 3; And Table 1,2 or 3 optional two keys.

2. chemical compound as claimed in claim 1 or its pharmaceutically acceptable salt, it has following formula (II):

Wherein, X is selected from the phosphonate radical of hydroxyl, phosphate radical, phosphonate radical, alpha-substituted;

R wherein ¹Be selected from hydrogen, halogen, (C ₁-C ₆) alkyl, by (the C of halogen, hydroxyl, alkoxyl, cyano group replacement ₁-C ₆) group formed of alkyl;

R ²Be selected from the group that aryl that aryl that alkyl, thiazolinyl, alkynyl, alkyl replace, cycloalkyl, aralkyl and aralkyl that alkyl replaces replace is formed; And n is 0,1,2 or 3.

3. chemical compound as claimed in claim 1 or 2, wherein said R ¹Be fluorine or chlorine.

4. as each described chemical compound of claim 1-3, wherein said X is hydroxyl or OPO ₃H ₂

5. chemical compound as claimed in claim 4, wherein said X is OPO ₃H ₂

6. chemical compound as claimed in claim 4, wherein said X is a hydroxyl.

7. chemical compound as claimed in claim 1 or 2, the phosphonate radical of wherein said alpha-substituted is-CHFPO ₃H ₂,-CF ₂PO ₃H ₂,-CHOHPO ₃H ₂,-C=OPO ₃H ₂Or-OPO ₂SH ₂

8. chemical compound as claimed in claim 7, the phosphonate radical of wherein said alpha-substituted is-CHFPO ₃H ₂,-CF ₂PO ₃H ₂,-CHOHPO ₃H ₂, or-C=OPO ₃H ₂

9. as each described chemical compound of claim 1-8, wherein said R ¹Be hydrogen.

10. as each described chemical compound of claim 1-9, wherein said R ²It is alkyl with 5,6,7 or 8 carbon atoms.

11. chemical compound as claimed in claim 10, wherein said R ²Be heptyl, octyl group, nonyl or-the O-heptyl.

12. chemical compound as claimed in claim 11, wherein said R ²It is octyl group.

13. as each described chemical compound of claim 1-12, wherein said n is 1 or 2.

14. as each described chemical compound of claim 1-13, wherein said R ²Group is positioned at the para-position of described cycloalkyl ring.

15. chemical compound as claimed in claim 1 or 2, wherein said cycloalkyl has following formula:

Or

16. as each described chemical compound of claim 1-15, wherein said R ¹Group is positioned at described R ²An ortho position or a position.

17. as each described chemical compound of claim 1-15, wherein said R ²Group is positioned at the para-position of described benzyl rings alkyl.

18. chemical compound as claimed in claim 1 or 2, it has following formula:

Or

19. method that is used to prevent or treat mammiferous pathologic conditions or symptom, the activity that wherein relates to 1-phosphoric acid sphingol receptor, and described active excitement expects, described method comprise deliver medicine to described mammal effective dose as each described chemical compound of claim 1-18.

20. method as claimed in claim 19, wherein said pathologic conditions is an autoimmune disease.

21. method as claimed in claim 20, wherein said autoimmune disease are uveitis, type i diabetes, rheumatoid arthritis, inflammatory bowel or multiple sclerosis.

22. method as claimed in claim 21, wherein said autoimmune disease is a multiple sclerosis.

23. being the lymphocyte transportations, method as claimed in claim 19, wherein said pathologic conditions change.

24. method as claimed in claim 23, wherein said treatment are to change the lymphocyte transportation.

25. method as claimed in claim 23, wherein said lymphocyte transportation provides the time-to-live of the allograft that prolongs.

26. method as claimed in claim 23, wherein said allograft is used for transplanting.

27. method that is used to prevent or treat mammiferous pathologic conditions or symptom, wherein relate to S1P lyases activity, and the inhibition of S1P lyases is required, it comprise deliver medicine to described mammal effective dose as each described chemical compound of claim 1-18.

28. each described chemical compound of claim 1-18 is used for the purposes of therapeutic treatment.

29. each described chemical compound of claim 1-18 is used to prepare the purposes that is used to prevent or treat the medicine of mammal pathology disease or symptom, wherein relates to the activity of 1-phosphoric acid sphingol receptor.

30. purposes as claimed in claim 28, wherein said medicine contains liquid-carrier.

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