WO2011159870A9

WO2011159870A9 - Methods and compounds for the preparation of fluorine-labeled deoxy-fty720

Info

Publication number: WO2011159870A9
Application number: PCT/US2011/040645
Authority: WO
Inventors: Rolf E. Swenson; Maria E. Estrella-Jimenez; Aldo Cagnolini
Original assignee: Bracco Imaging S.P.A.
Priority date: 2010-06-17
Filing date: 2011-06-16
Publication date: 2012-04-12
Also published as: WO2011159870A1

Abstract

Highly efficient processes for making radioactive and non-radioactive Fluorodeoxy- FTY720 are provided. Also provided are novel precursor compounds included in such processes. In addition the present invention prov ides compounds useful in the treatment and/or prevention of diseases and disorders associated with S1P receptors.

Description

METHODS AND COMPOUNDS FOR THE PREPARATION OF FLUORINE-LABELED

DEOXY-FTY720

Field of the Invention

[0001 ] The present invention is in the field of PET imaging and the treatment of diseases and conditions related to S I P receptors.

Background of the Invention

[00021 Immunosuppressants play an important role in organ and tissue transplantation as well as the treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis, Crohn's disease, and ulcerative colitis. The

immunosuppressive natural product ISP-I (myriocin) was isolated from the culture broth of 1 sari a sinclairii, a type of vegetative wasp. Its chemical modification yielded a new compound designated by Novartis as FTY720. This compound has been shown to have more potent immunosuppressive activity and less toxicity than ISP-I.

10003] FTY720 has also been shown to bind to four of the five receptor subtypes of Sphingosine- 1 -phosphate (SI P) receptors: S I Pi. S1P₃, S1P₄ and S1P₅. The different S I P receptor subtypes have been shown to be involved in angiogenesis, immune response and other critical biological systems,

[0004] FT Y 720 appears to be a unique immunosuppressant due to its ability to prevent lymphocytes from attacking transplanted organs without changing the body's ability to respond to other immune challenges. FTY720 reached phase III clinical trials for kidney transplantation although it was discontinued due to lack of benefits in comparison with the existing standard of care. However, FTY720 is currently in phase III clinical trials for the treatment of multiple sclerosis and may be useful in other sphingosine-1 -phosphate receptor-mediated diseases and conditions. 100051 Various methods of synthesis of FT Y720 have been reported since the discovery of this immunosuppressant. Such methods include regioselective opening of epoxides, Weinreb amide based strategy, Petasis reaction, iron-catalyzed cross-coupling reaction, and chiral synthesis. Due to the rapid growth of positron emission tomography (PET), there is increasing interest in synthetic methods for the preparation of tracers labeled with radioactive fluorine (¹⁸F). However, efficient labeling of organic molecules with ^{I 8}F is fraught with difficulties since such labeling must proceed rapidly and in good yield, due to the short half-life of this radionuclide. Summary of the Invention

100061 In one embodiment, the present invention provides highly efficient methods for making ¹⁸F labeled deoxy-FTY720.

10007] In another embodiment, the present invention provides novel intermediate, precursor compounds which are useful in such methods.

[0008 j In another embodiment, such methods include the production of an ¹⁸F labeled - deoxy-FTY720 enantiomcr that binds to the S 1 P i receptor and an enantiomer that does not. 10009] In another embodiment, the present invention provides methods of using ¹⁸F labeled deoxy-FTY720 according to the methods described herein in the imaging of diseases and conditions associated with S I P receptors including, S I P i, S1P₃, S1P₄ and SIP₅

|00010] In yet another embodiment, the present invention provides a non-binding nonradioactive fluorinated enantiomer which inhibits sphingosine kinase.

[00011] In another embodiment, the present invention provides methods of using nonradioactive fluorinated enantiomers of deoxy-FTY720 to treat conditions associated with sphingosine kinase 1 or 2. [00012] In still another embodiment the present invention provides novel methods for F-

1 8

labeling other compounds which require nitro reduction after F introduction as a synthesis step. Brief Description of the Drawings

[00013] Figures l A, I B and 1 C show a chromatography (HPLC) trace of compounds according to the present invention both before ( Fig. 1 A) and after chiral chromatographic separation into separate racemic components (Figs 1 B and 1 C)

Q

Figure 2 shows a rad iochrom atogram of HPLC purified F-Compound 9.

Detailed Description of the Invention

18

[00014] Disclosed herein are compositions and methods useful in the production of F- labeled FTY 720. FTY 720 has the following chemical structure:

[00015] The present invention also relates to non-radioactive synthesis processes and compounds useful in such processes. Also disclosed herein are compositions and methods for imaging and/or treatment or prevention of conditions associated with S I P receptors.

Non-Radioactive Synthesis

[00016] One aspect of the present invention includes a novel cyclic sulfate precursor

18

(compound 6) which is useful as a starting compound for making F labeled FTY720. Another aspect of the present invention is a non-radioactive synthesis process for making this novel cyclic sulfate precursor. A representative synthesis scheme is shown below. Scheme l a:

[00017] Methods for making the novel cyclic precursor (compound 6) include converting compound 1 to compound 2, compound 2 to compound 3, compound 3 to compound 4, compound 4 to compound 5 and compound 5 to compound 6 as shown above.

[00018] In one embodiment the alkyl bromide compound 1 may be nitrated to form compound 2. The aromatic ring of compound 2 may be functionalizcd. for example, by F re id el - Craft aeylation to form the ketone compound 3. The benzylic ketone of compound 3 may be completely reduced to form compound 4. Compound 4 the may be subjected to bisalkylation via a nitro-aldol to form compound 5. The diols of compound 5 then may be subjected to conditions which yield the cyclic sulfate compound 6.

[00019] In one embodiment the process for forming compound 6 may be carried out as follows: Scheme lb:

O

AgN0₂, Et₂0. CI C₇H₁₅

Br NO,

76% AICI_3j CICH₂CH₂CI

' 79%

1000201 Examples 1 -5 below provide more detail on how these steps may be carried out in certain embodiments of the present invention.

|000211 Compound 6 may be used as starting material for the production of ¹⁸F-labeled

FTY720 discussed in the Radiocative Chemistry section below.

[000221 Another embodiment f the present invention includes using Compound 6 as starting material to obtain tl uorodeoxy-FTY 720 and fl uorodeoxy- FT Y 720 Phosphate-(P) racemates. For example, starting with Compound 6 obtained as shown in Schemes 1 A and I B. tl o urodeo x y- FT Y 720 and flourodeoxy-FTY720-P racemates may be synthesized according to Scheme 2.

Scheme 2a:

1000231 One aspect of the invention provides nov el compounds 7, 8, 9, 10 and methods for making these compounds. For example, compound 6, may be converted to compound 7, compound 7 to compound 8, compound 8 to compound 9 and compound 9 to compound 10.

[00024] In one embodiment fluorine may be added to compound 6 by. for example, nucleophilic fluorination to form compound 7. The sulfate of compound 7 may be hydrolyzed to form compound 8. The formation of compound 8 may be performed under acidic conditions. The nitro of compound 8 may then be reduced to from the amine-containing compound 9. In one embodiment the nitro group may be reduced prior to hydrolyzing the sulfate group. After obtaining compound 9 it may be phosphorylated to form compound 10.

1000251 In one embodiment the process may be carried out as indicated in the following scheme.

Scheme 2b

8 9 10 1000261 In a further embodiment f the present invention Compound 9 may be separated, for example, by chiral separation, into compounds 9a (shown as peak 2 in Figure 1) and 9b (shown as peak 1 in Figure 1). Compound 9b was unexpectedly and surprisingly found to exhibit sphingosine inhibition activity, particularly with respect to sphingosine kinase 2 as shown in Example 1 5.

[00027] Compounds 9a and 9b may be further converted to compounds 10a and 10b, respectively, as shown below in Scheme 3.

Scheme 3a:

Assignment of Chirality of F-deoxy-FTY720

[00028] The introduction of fluorine (whether radioactive or non-radioactive) to the achiral substrate generates two enantiomers of the final product Fluorodeoxy-FTY720. The two structures are shown below, in which F may refer to radioactive or non-radioactive fluorine.

(S-enantiomer) (Compound 9b ) (R-enantiomer) (Compound 9a)

[00029] After chiral separation, chemical phosphorylation produced the analogs below, one of which bound and was internalized by the S I P) receptor.

(S-enantiomer) (Compound 10b) (R-enantiomer) (Compound 1 0a)

1000301 In order to assign the configuration of the active enantiomer vibrational circular dichroism of the alcohol corresponding to the active phosphorylated analog of Fluorodeoxy- FTY720 was performed. The VCS study provided strong evidence for the assignment f absolute configuration to the R-con figuration.

[00031] Two literature precedents (V. Brinkmann, et. al. J. Biol. Chem. 2002, 277,

21453 ) sustain the findings. In the first, the R enantiomer was found to be enzymatically phosphorylated and not the S enantiomer (see below).

(S-enantiomer) (Compound 1 l b) ( R-cnantiomer) (Compound 1 1 a)

[00032] In addition, further clarification was obtained by the enantioselective synthesis of the phosphate ester of FT Y 720 (Y.Y. Lu, R. Bittman, Tetrahedron Lett. 2006, 47, 825). where the S-enantiomer was found to be active.

( R-enantiomer) (Compound 1 2b) (S-enantiomer) (Compound 1 2a)

10003 1 Based on these two closely related compounds, the R-enantiomer of Fluorodcoxy-

FTY720 would be expected to bind and internalize the S I P receptors (SI Pi, S I P;,, S 1P₄, and S l P ). one embodiment the process may be carried out as follows

[00035] Compound 9, fl uorodeoxy- FT Y 720 and Compound 10, fluorodeoxy-FTY720-P raeemates may be synthesized in moderate yields when utilizing the processeses described above.

[00036] Compound 9 as a racemic mixture f Compound 9a and 9b may be used as starting material for separation into its constituent enantiomers.

[00037] After separation. Compound 9b may be used as a sphingosine kinase inhibitor and is thus useful in pharmaceutical compositions for treating and preventing diseases and conditions associated with sphingosine kinase receptors.

[00038] After separation. Compound 9a, labeled with a pet isotope 18 fluorine, may be used as an imaging agent.

[00039] Compound 10b is a chemically phosphorylated enantiomer derived from

Compound 9b.

[00040] Compound 10a is a chemically phosphorylated enantiomer derived from

Compound 9a. As described in Example 18, compound 1 Oa was shown to internalize the S I Pi receptor. Radioactive Chemistry

18

[00041] The present invention also prov ides methods for preparing F-labeled FTY720 and useful intermediate precursors associated with such methods.

[00042 J As described in more detail in Example 9 below, testing with the use of MS/MS instruments with non-radioactive reagents unexpectedly allowed fo the estimation of the concentration and conditions useful for radioactive labeling.

18

[00043] A representative scheme used for the preparation of F-compound 9 is outlined below starting with compound 6 described above. Throughout this section and in Schemes 4a and 4b, F or fluorine refers to radioactive fluorine ( 18 F). Scheme 4a:

8 9

In one embodiment of the present invention compound 6 may be converted to compound 7, compound 7 may be converted to compound 8, compound 8 may be converted to compound 9.

In another embodiment of the present invention fluorine may be added to compound 6 by, for example, nucleophilic fluorination to form compound 7. The sul fate of compound 7 may be hydrolyzed to form compound 8. The formation of compound 8 may be performed under acidic conditions. The nitro of compound 8 may then be reduced to form the amine-containing compound 9. In one embodiment the nitro group may be reduced prior to hydrolyzing the sulfate group. After obtaining compound 9 it may be phosphorylated to form compound 10.

In one embodiment the process may be carried out as indicated in the following scheme. Scheme 4b:

1000441 The fluorine is introduced with KF/Kryptofix in acetonitrile and then hydrolyzed with aqueous trifluoroacetic acid. The nitro is reduced with samarium diiodidc in a solution of THF and methanol.

Pharmaceutical Preparations

1000451 The compounds described above in schemes 1 -4 may be formulated into conventional pharmaceutical preparations and are useful for imaging or treating conditions associated with S I P receptors. Therapeutic Agents

[00046] Therapeutic agents include compound 9b with a non-radioactive fluorine.

Compounds that can be readily converted into compound 9b, such as compound 6 and nonradioactive fluorine embodiments of compounds 7, 8 and 9 also have utility in that they can be used as starting materials for the efficient formation of compound 9b. Thus, such precursor compounds may be included in kits which may be employed to make therapeutic compounds accordi g to the present invention.

[000471 Therapeutic compounds may be formulated into pharmaceutical preparations and are useful treating or preventing conditions associated with sphingosine kinase.

1000481 These compounds may be generally administered in association with a pharmaceutically acceptable diluent or carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to sterile water, saline solution, buffered saline (including bu ffers like phosphate or acetate), alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose. magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxynethyl cellulose, po 1 y vi n yl pyrro 1 i do n e and the like.

[00049] Such compositions may further comprise conventional excipients: i.e..

pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds. The pharmaceutical preparations can be sterilized and i f desired, mixed with auxiliary agents, e.g. lubricants, preservatives, stabilizers, wetting agents, em ul si tiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, and/or aromatic substances and the like which do not deleteriously react with active compounds. The pharmaceutical composition may be prepared by any of the known procedures as described in Remington's Pharmaceutical Sciences, Mack Publishing Co. Eaton, Pa. 1 6th Ed, 1980.

1000501 The pharmaceutical compositions may be in various forms such as tablets or solutions and may be administered by various routes including, but not limited to, parenterally (including intravenously, intramuscularly, subcutaneously and intraperitoneally), orally, topically (including ocular administration) nasally and via inhalation.

[00051 | For oral administration, particularly suitable are tablets, dragrees or capsules having talc and/or a carbohydrate carrier binder or the like, the carrier preferably being lactose and/or com starch and/or potato starch. Syrups, elixirs or the like may be used as sweeteners. Sustained release compositions may be formulated and include those wherein the active component is protected with di ferentially degradable coatings, e.g.. by microencapsulation, multiple coatings and the like. Other methods of delaying release of the active component also may be employed.

[00052] It will be appreciated that the actually preferred amount of active compounds used will vary according the specific compound being utilized, the particular composition

formulated, the mode of application and the part icular site of administration. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art, using conventional dosage determination tests conducted with regard to the foregoing guidelines.

[00053] Accordin to the present invention, a "therapeutically effective amount" of a pharmaceutical composition is an amount which is sufficient to achieve the desired

pharmacological effect. [00054] Generally the dosage required to provide an effective amount of the composition, and which can be adjusted by one of ordinary skill in the art, will vary, depending upon the age, health, physical conditional, sex, weight and extent of disease, of the recipient. Additional ly, the dosage may be determined by the frequency of treatment and the nature and scope of the desired effect. Appropriate dosages will be determined by those of ordinary skill in the art, using routine methods.

1 00551 Typically, the dose range is from 0.001 to 100 mg of active compound per kilogram body weight. Preferably, the range is from 0.01 to 50 mg f active substance per kilogram body weight. A preferred composition of the invention is for example, one suitable for oral administration in unit dosage form, for example a tablet or capsule which contains from 1 microgram to 500 mg, more preferably from 10 to 100 mg, of the active component in each unit dose, such that a daily oral dose is from 1 nanogram to 50 milligram per kg of body weight, more preferably from 0. 1 to 25 mg/kg, is thereby achieved. Another preferable compositio is one suitable for parenteral administration which contains from 0.5 to 100 mg of active compound per ml, more preferably from 1 to 10 mg of compound per ml of solution, such that a daily parenteral dose of from 1 nanogram to 1 0 mg per kg of body weight, more preferably from 0. 1 to 10 mg/kg, is thereby achieved

Imaging Agents

[00056] Imaging agents in rad i opli arm a ceuti cal formulations include compound 9a labeled with radioactive fluorine. Compound 6 and radioactive fluorine embodiments of compounds 7, 8 and 9 may be used as starting materials for the efficient formation of compound 9a. Certain precursor compounds may be included in kits which may be used to make imaging agents according to the present invention. Such formulations may contain less than about 5 g of imaging agent per mL of diluent. In certain embodiments formulations of the invention contains about 4 ii of imaging agent per mL of diluent, with a total reconstitution volume between about 0.5 mL and about 1 mL. The present invention also includes

radiopharmaceutical formulations that may be prepared and administered to a subject without any purification. In addition, rad ioph arm aceuti cal formulations of the invention may have a radiochemical purity (RCP) of greater than or equal to 90% at about 15 minutes after preparation (i.e.. reconstitution). Most preferred rad i opharm aceuti cal formulations of the invention have a RCP that is greater than or equal to about 90% at about six hours after preparation.

[00057] Diluents used to reconstitute the radi opharm aceuti cal formulations of the present inv ention may be any combination of water, normal saline and/or ethanol. In certain

embodiments the diluent is a mixture f water, normal saline and ethanol in which the percentage of ethanol may range from about 20% to about 40% (v/v), and in other embodiments is about 30%. In certain embodiments the pH of the formulation after reconstitution may be between 2.8 and 4.0, and in other embodiments is about pH 3.0.

[00058] Radio-labeled formulations of the present invention may contain one or more stabilizers chosen from maltose, ascorbic acid, gentisic acid or pharmaceutically acceptable salts of these acids. In certain embodiments sodium salt is preferred. This reagent in certain embodiments may be present at a concentration of about 10 to about 25 mg/mL, and in other embodiments about 20 mg/mL.

[00059] A detergent may be optionally present in the formulation of the present invention to help improve recovery of the radioactive product from the vial. Such detergents can include non-ionic, cationic, anionic and zwitterionic surfactants including, for example, sodium dodecyl sulfate, N-dodecyl sulaine, Tween™ 80, cetyltrimethylamnionium bromide, cyclo-n-methyl-B- D-malloside and n-hexyl-B-D-glucopyranoside. Non-ionic detergents are particularly preferred in certain embodiments, with cyclo-n-methyl-B-D-malloside and n-hexyl-B-D-glucopyranoside especially preferred. In certain embodiments detergent may be present in an amount in the range of about 5- 100 mg/ml.

[00060] Compounds of the present invention can be used to prepare radiopharmaceutical formulations at any temperature between room temperature and 100°C. If prepared by heating to 100°C, optimal yield of the desired compound (>90% RCP) is obtained when the three steps f compound formation are heated for about 5 minutes to about 30 min, most preferably between about 5 to about 15 min.

[000611 Solubilization aids, such as cyclodcxtrins (e.g. alpha, beta or gamma cyclodextrin or hydroxypropyl-.gamma.-cyclodextrin) can optionally be present in the formulations of the present invention. Gamma cyclodextrins are preferred, and h y dro ypropyl - .gamma.- eyelodextrin is most preferred. The concentration of cyclodextrin may be from about 0.1 to about 10 mg/niL, preferably between about 2.5 and about 5 iiig/mL.

Examples

[00062] For non-radioactive chemistry processes referenced above, solvents for reactions were purchased from Sigma-Aldrich (St. Louis, MO) and solvents for chromatographic purifications were ACS grade solvents from VWR Corporation (West Chester, PA). Thin-layer chromatography was performed using silica gel 60 F₂₄ precoatcd plates (250 μ m thickness). Automated column chromatography purifications were performed in an SP1 model from Biotage ®. NMR spectra were obtained on a Varian 500 MHz spectrometer; chemical shifts are reported in d units relative to the tetram ethyl si lane (TMS) signal at 0.00 ppm. Coupling constants are reported in Hz. Low resolution mass spectroscopy was obtained in HP 1 100 model Mass Spectrometer. High resolution mass spectrometry was obtained from UC Irvine mass spectrometry services. Analytical HPLC data were obtained using a Shimadzu HPLC employing a Waters XTerra MS-C l 8 4.6 x 50 mm column (particle size: 5 μ; 120 A pore size), and gradient elution system using water (0.1 % TFA) as eluent A and C¾CN (0.1 % TFA) as eluent B. Detection of compounds was accomplished using UV at 220 and 254 nm. Chiral HPLC separation was obtained from a Chiralpack AD 4.6 mm i.d. x 250 mm column using isocratie conditions with 95 % A and 5% B at a flow rate of 0.8 mL/min and monitoring at a wavelength of 254 nm. The column was maintained at 35°C. Mobile phase A was hexanes with 0.2 % diethylamine. Mobile phase B was 50:50 methanol/ethanol with 0.2 % diethylamine.

[00063] Examples 1-8 relate to non-radioactiv e chemistry processes described above.

Example 1 Formation of (3-nitropropyl)benzene (Compound 2)

[00064] A solution of nitro 1 -bromo-3-phenylpropane in 10 niL of ether was added dropwise to a suspension of AgN( , (7.0 g, 35 mmol) in ether (75 ml_) at 0°C. The reaction was allowed to stir for 4 h at 0°C then at room temperature for 1 8h. Upon completion the mixture was filtered through celite eluding with EtOAc, and concentrated. The residue was then puri fied by automated silica gel column chromatography with gradient elution of ethyl acetate-hexanes (0 to 20 %). Puri fication afforded 4.42 g of desired product (76 % yield). Ή NMR (500 MHz.

CDC1₃): d 7.33-7.30 (2 H, m), 7.25-7.22 (1 H, m), 7.19-7.17 (2 H, m), 4.37 (2 H, t, J = 7.0 Hz),

2.73 (2 H, t, J = 7.3 Hz), 2.33 (2 H, quintet, J = 7.3 Hz).

Example 2 l-(4-(3-nitropropyl)phenyl)octan-l -one (Compound 3)

[00065] A solution of ( 3-nitropropyI (benzene (4.5 g, 27 mmol ) and oetanoyl chloride

(4.39 g, 27 mmol, 4.6 ml.) in 1 , 2-dichloroethane (10 niL) was added dropwise to a suspension of AICI₃ (5.5 g, 41 mmol) in 1 , 2-dichloroethane (80 niL) at 0°C over a period of 20 min. After stirring at room temperature for 12 h, the mixture was cooled with ice and water (50 mL) was added slowly. The layers were separated and aqueous layers were extracted with EtOAc, filtered, and concentrated. The residue was then purified by automated silica gel column chromatography using gradient elution of ethyl acetate-hexanes (7 to 50 %). Purification afforded 6.20 g as a white solid, 79 % yield. Ή NMR (500 MHz, CDC1₃): d 7.91 (2H, d, J = 8.2 Hz), 7.28 (2 H, d, J = 8.2 Hz), 4.39 (2 H, t, J = 6.9 Hz), 2.94 (2 H, t, J = 7.3 Hz), 2.79 (2 H, t, 7.3 Hz), 2.35 (2 H, q, 7.3 Hz), 1 .73 (2H, q, 7.3 Hz), 1 .37- 1 .28 (8 H, m), 0.89 (3 H, t, J = 6.9 Hz); ¹³C NMR ( 125 MHz, CDCI₃): d 200.2. 145.0, 135.8, 128.8 (2C), 128.7 (2C), 74.7. 38.8, 32.4, 31.9, 29.5, 29.3. 28.6, 24.6. 22.8, 14.2: MS ( EI) m/z 29(⁾.1 ( M-H ) ^' , 292.2 (M+Hf .

Example 3 l-(3-nitropropyl)-4-octylbenzene (Compound 4)

[00066] Triethylsilane (4.9 g, 42 mmol) was added to a solution of 1

nitropropyDphenyl )octan- 1 -one (6. 1 g, 21 mmol) in trifluoroacetic acid (25 mL). After stirring at room temperature for 5 h, the mixture was concentrated and coevaporated with EtOAc (30 mL) 4 times. The mixtures was concentrated and purified by automated silica gel column chromatography using gradient elution of ethyl acetate-hexanes (0 to 50 %). Purification afforded 5.5 g of product as a pale yellow oil (95 % yield). Ή NMR (500 MHz, CDC1₃): d 7.10 (4H, dd. J = 2 1 .3, 8. 1 Hz). 4.35 (2H, t, J = 7.0 Hz), 2.67 (2 H. t, J = 4.4 Hz). 2.57 (2 H, t, J = 7.7 Hz), 2.30 (2 H, q, J = 7.7 Hz), 1.59 (2 H, q, J = 7.3 Hz), 1 .32- 1 .26 (10 H, m), 0.88 (3H, t, 7.0 Hz). ^,3C NMR (125 MHz, CDC1₃): d 141 .5. 1 36.8. 128.9 (2C), 128.5 (2C), 74.9, 35.8. 32. 1 , 3 1 .8. 29.7. 29.5. 29. 1 . 22.9, 14.3.

Example 4 Compound 6

[00067] A) A mixture of 1 -(3-nitropropyl )-4-octylbenzene 4 (5.5 g, 19.8 mmol) in EtOH

(30 mL) and dioxane (10 mL), 1 N NaOH (0.4 niL). and 37 % aqueous formaldehyde (4.0 mL, 49.6 mmol) was stirred at room temperature for 16 h. The reaction mixture was evaporated and diluted with a mixture of EtOAc and water. The layers were separated and the aqueous layers were extracted with EtOAc. The organic layers were dried over MgS0₄, filtered and

concentrated producing 2-nitro-2-(4-oetylphenethyl (propane- 1 ,3-diol 5 as a white solid in 94% yield. 6.28 g. Ή NMR ( 500 MHz, CDC1₃): d 7.09 (4H, dd, J = 22.2. 8. 1 Hz), 4.26 (2 H, d, J = 12.2 Hz), 4.05 (2 I I , d. J = 12.5 Hz), 2.60-2.55 (2 H, m ), 2.21 -2. 1 7 (2 H, m ). 1.59 (2 H, q, J = 5.9 Hz), 1 .30- 1 .27 (10 H, m), 0.89 (3H, t, 7.0 Hz). ^{I 3}C NMR (125 MHz, CDC1₃): d 141 .3. 137. 1 . 1 28.8 (2C), 1 28.2 (2C), 94.0. 64.3. 36.0, 35.7, 34.8, 32.0, 3 1 .7, 29.6, 29.5, 29.4. 29.3. 22.8, 14.3.* Product was used in the next reaction step without further purification.

1000681 B) Thionyl chloride (0.5 ml ) was added to a solution of 2-nitro-2-(4- octylphenethyl)propanc- 1 ,3-diol (0.93 g, 2.8 mmol) in anhydrous CLLCf (10 m L) at room temperature. The mixture was allowed to stir for 5 h. The solvent was then evaporated and the residue was coevaporated with CJ-fCf to a white solid. The solid was diluted with EtOAc and washed with saturated solution of sodium bicarbonate. The organic layers were collected, dried over MgS0₄, filtered and concentrated to a white solid, 1 .02 g, directly used in the next reaction. C) The previous solid was dissolved in CH₃CN/ ¾0/ CH₂C1₂ (13 :7:5 niL) and NaI0₄ (890 mg, 4.0 mmol) and RuCl₃ (60 mg, 0.27 mmol) were added at room temperature. The reaction reached completion in 2 h. The mixture was then filtered through ceiitc and concentrated. The residue was diluted with EtOAc and the layers were separated. The EtOAc layers were dried over MgS0₄, filtered and concentrated. The residue was then purified by automated silica gel column chromatography using gradient elution of ethyl acetate-hexanes (0 - 40 %). Purification afforded 860 mg of desired product as a while solid. 81 % yield. Ή NMR (500 MHz, CDCh): d 7.13 (2 H. d, J = 8.0 Hz), 7.01 (2 H, d, J = 8.0 Hz), 5.30 (21 1, dt, J - 12.8, 1 .8 Hz), 4.81 (211, dt, J = 12.8. 1.8 Hz), 2.58-2.54 (4H, m), 2.14-2. 1 1 (2H, m), 1 .61 - 1 .55 (2H, m), 1 .29- 1 .26 ( 10 H, m ). 0.88 (3 H, t, J = 6.6 Hz); ¹³C NMR ( 125 MHz, CDCh ): d 142.5, 134.9, 1 29.3. 128.2. 84.2., 74.7. 35.7. 34.3. 32. 1 , 3 1 .7, 29.7, 29.5. 29.4, 29. 1 , 22.9, 14.3 ; HRMS (ESI with NaHCOO): mass calculated for C₁₉H₂₉N0₆SHCOO [M+HCOO]^" 444. 1 692. found 444. 17 1 .

Example 5 Formation of Compound 7

[00069] Fluorine addition to compound 6 was achieved with tetramethylammonium fluoride in reflux ing acetonitrile. The reaction was monitored by HPLC. and within 10 min, the cyclic sulfate was converted to acylic sulfate derivative compound 7.

Example 5 2-(fluoromethyl)-2-nitro-4-(4-octylphenyl)butan-l -ol (Compound 8)

[00070] A mixture f compound 6 (270 mg, 0.68 mmol) and tetram ct h y 1 am m on i um fluoride ( 126 mg. 1 .35 mmol) in anhydrous acetonitrile (2 niL) was refluxed for 45 min. Upon reaction completion, as monitored by HPLC, 1 M hydrochloric acid solution (3 mL) was added and the mixture was refluxed for 1 h. The solvents were evaporated and the residue was diluted in water/ethyl acetate mixture. The layers were separated and the organic layers were dried over MgS0₄, filtered and concentrated. The residue was purified by automated silica gel column chromatography using gradient elution (7-50 % ethyl acetate/ hexanes). Purification afforded

183 mg of product as a pale yellow oil (80 % yield). Ή NMR (500 MHz, CDC1₃): d 7.10 (2 H, d, J = 8.1 Hz), 7.06 (2 H, d, J = 8.1 Hz), 4.93 (1 H, dd, J = 76.0, 10.3 Hz), 4.84 (1 H, dd, J = 76.0, 10.3 Hz), 4.15 (1 H, ddd, J = 12.0, 6.0, 2.0 Hz), 4.06 (1 H, ddd, J = 12.0.6.0, 2.0 Hz), 2.65-2.53 (4 H, m) 2.26-2.16 (2 H, m), 2.10 (1 It. s), 1.61-1.55 (2 H, m), 1.30-1.26 (10 H, m), 0.88 (t, 3H, J = 7.0 Hz); ¹³C NMR (125 MHz, CDC1₃): d 141.6, 136.8.128.9, 128.3.92.3 (d. J = 18.3 Hz), 81.1 (d, J= 170.6 Hz), 62.6 (d, J = 3.7 Hz), 35.7.33.6 (d, J = 2.9 Hz), 32.1,31.7, 29.7, 29.54, 29.47, 29.2, 22.9, 14.3; HRMS (ESI with NaHCOO): mass calculated for C₁₉H₃₀FNO₃HCOO [M+HCOO]^" 384.2186, found 384.2187.

Example 7 2-amino-2-(fluoromethyl)-4-(4-octylphenyl)butan-l-ol (Compound 9)

1000711 2-(tluoromethyl)-2-nitro-4-(4-octy]phenyl )butan-l-ol (50 mg, 0.15 mmol) was dissolved in ethanol (3 mL), mixed with 20 mol % of 10% Pd/C (50% water) and hydrogenated at 2.5 atm of hydrogen for 18 h. Then the mixture is filtrated through a pad of celite, and concentrated. The residue was purified by automated silica gel column chromatography using gradient elution (0-10 % methanol/^' methylene chloride). Purification afforded 41 mg of product as a white solid (91 % yield). Ή NMR (500 MHz. CDC1₃): d 7.10 (4 H, s), 4.33 (2 H, d, J = 47.6 Hz), 3.55 (1 H, d, J 10.2 = Hz), 3.47 (1 H, dd, J = 10.8, 2.7 Hz), 2.65-2.62 (2H, m), 2.56 (211, t, 7.9 Hz), 1.79-1.72 (4 H, m), 1.61-1.55 (2 H, m), 1.31-1.24 (10 H, m).0.88 (3 H, t, J = 7.0 Hz); ¹³C NMR (125 MHz, CDC1₃): d 140.9, 139.0, 128.7.128.3, 86.4 (d, .1 = 173.6 Hz), 65.3 (d, J = 4.4 Hz), 55.8 (d, J = 16.8 Hz), 36.6 (d, J = 2.6 Hz), 35.7, 32.1 , 3 1 .8. 29.7, 29.6, 29.5. 29.2, 22.9, 14.3 : MS (El ) m/z 310.2 (M+H)⁺. ¹²

Example 8 2-amino-2-(fluoromethyl)-4-(4-octylphenyl)butyl dihydrogen phosphate

(Compound 10)

1000721 2-amino-2-(fluoromethyl)-4-(4-octylphenyl)butan-l-ol ( 144 mg, 0.46 mmol) was dissolved in trim ethyl phosphate ( 1 .5 m L) employing gentle heating. Phosphorus oxychloride (213 mg, 1.39 mmol) was slowly added to the solution at 0 °C. After 4 h, ice was added to the mixture, and neutralized with 5M NaOH solution. The solution turned cloudy and ethyl acetate was added. The layers were separated and the organic layers were dried over MgS0₄, filtrated and concentrated. The residue was purified by preparative HPLC. Purification afforded 70 mg of product as a white solid after lyophilization (39 %). Ή NMR (500 MHz, TFA-d): d 7.30 (2

H, d, 8.0 Hz), 7.24 (2 H, d, J = 8.0 Hz), 4.91 (2 I I, dd, J = 1 5.0, 1 1 Hz), 4.81 (1 I I, dd, J = 1 5.0. 1 1 Hz), 4.62-4.55 (2 H, m), 2.98-2.88 (2H, m ). 2.72 (2H, t, 8.0 Hz), 2.43 (2 H, t, J = 8.0 Hz),

I .77- 1 .71 (2 H, m), 1 .47- 1 .40 (10 H. m), 0.99 (3 H, t, J = 7.0 Hz). HRMS (ESI with NaHCOO): mass calculated for C₁ I½.FNChNa [M+Na]⁺ 412.2029, found 412.2029.

Experimental Section for Radioactive Chemistry Processes

[00073] For the Radioactive Chemistry processes described herein, all reagents, unless otherwise specified, were of analytical grade and commercially available.

[00074] No-carrier-added [' ⁸F] fluoride, produced by the ¹⁸0(p,n)¹⁸F irradiation of an ^{, 8}0- enriched water target was obtained from IB A Molecular. Sep-Pak Light C 18 cartridges were purchased from Waters (Mil ord, MA). F syntheses were performed using an automated Tracerlab FX-FN synthesizer (GE Healthcare, Chalfont St. Giles, UK).

1000751 Semi-preparative HPLC puri fication of ¹⁸F-compound 9 was conducted on a

Sykam Model S 1 122 isocratic Pump interfaced with the Tracerlab FX-FN software. HPLC analysis and characterization of reaction mixtures and final products was conducted with an Agilent 1 100 Series quaternary pump gradient system driven by Agilent ChemStation software, Model No. G1319A, Rev. A.09.01 and lilted with a UV detector (A280 nm ) and a Canberra Nal detector (Model 802-2x2 W) with high voltage power supply (Model 3102D), single channel analyzer (Model 201 5 A) and a linear/logarithmicratemeter (Model 148 1 LA).

1000761 Examples 8-15 refer to experimental procedures associated with Radioactive

Chemistry processes referenced above.

Example 9 LC-MS/MS studies

1000771 LC-MS/MS analysis were conducted with an API 3000 triple quadrupole mass spectrometer with heated nebulizer interface (Applied Biosystems, Foster City, CA) and Agilent 1 100 pumps, detector and autosampler (Agilent Technologies, Wilmington, DE).

Radiofluorination conditions were investigated by running non-radioactive reactions at the fluoride concentration and stoichiometry used in the radioactiv e ones. The amount of fluoride added to the reaction mixture (0.095g/5 nmol) mimicked that used in a radiofluorination reaction run using 1850 MBq (50 mCi) of ¹⁸F with an estimated specific activity of 10 Ci/mol. The nonradioactiv e reactions were run in the automated Tracerlab FX-FN synthesizer with the same program later used for the radiofluorination and the product of interest was quantified by LC- MS/MS. An LC-MS/MS method that could resolve starting material compound 6 from compound 7, compound 8 and compound 5 (possible nitro diol formed upon hydrolysis of compound 6) was developed. The LC-MS/MS was used to create a calibration curve for the quantification of compound 8 and compound 9 over a range from 1 % to 100% yield (r² > 0.99). The programs shown in Table 1 were used.

Table 1

[00078] A splitter (164 mm long, 0.18 mm ID) reduced the flow rate to the mass spectrometer from 2000 LiL/min to 200 μ ΐ./min. Example 10 Synthesis of ¹⁹F compound-8

10007 1 A 9.5 μ ί aliquot of fluoride standard (VWR, 0.01 mg fluoride/mL, 0.095 pg fluoride) corresponding to the mass of F^" present in 1 .85 GBq ( 50 mCi) f ^{I 8}F at the specific activity of 10 Ci/μηιοι) was mixed with 1.8 mg of K₂C0₃ dissolved in 600

of H₂0 and 13 mg of Kryptofix dissolved in 500 μL· of CH₃CN. The fluoride was dried under vacuum at 120°C. A solution prepared by dissolving cyclic sulfate compound 6 ( 5 mg, 12.5 μιη ΐ) in 1 niL of dry CH₃CN was added to the dry fluoride and the reaction was heated at 85° C for 10 min. I N HCl (1 m l_) was added and the solution was heated at 100°C for 5 min to form compound 8. After being cooled to 30° C, the reaction mixture was analyzed by LC-MS/MS using the following conditions:

100080] Bonus RP column (4.6 x 250 mm) eluted with a 30 min gradient from 40% H₂0

(0.05% formic acid. v/v)/60% CH₃CN(0.05% formic acid, v/v) to 100% CH₃CN(0.05% formic acid, v/v) at a flow rate of 2 mL/min. Retention time: 22 min.

[000811 Parent mass: 384 ([M - 1 1]^"), fragment detected: 288.

Example 11 Synthesis of '''F-compound 9

1000821 A 9.5 μ ΐ. aliquot of 0.01 mg/m L fluoride standard (0.095 μ«, corresponding to the mass of fluoride present in 1 .85 GBq of ¹⁸F at a specific activity of 10 Ci/μιηοΐ) was mixed with 2.3 mg of K₂C0₃ dissolved in 140 μΐ. of H₂0 and 1 3 mg of Kryptofix dissolved in 860 iL of CH₃CN. The fluoride was dried under vacuum at 120°C. A solution prepared by dissolving 5 mg of compound 6 in 1 inL of dry CH₃CN was added to the dry fluoride and the solution was heated at 85°C for 10 min. CH₃CN (400 iL), TFA (50 μΐ ) and H₂0 (50 μΐ) were added and the mixture was heated at 100°C for 5 min. The temperature was lowered to 60°C and the solution evaporated by applying vacuum and a stream of helium for 5 min. then dried under vacuum at 100° C for 5 minutes.

[00083] The temperature was lowered to 30° C. Anhydrous MeOH (50 μΙ anhydrous

THF (100 μΙ_ ) and Sml₂ (4 niL of a 0. 1 M solution in THF) were added to reduce the nitro group f compound 8 to an amine. After 1 5 min at room temperature. H₂0 (500 iiL) was added to destroy the excess Sml_2. and the solution was analyzed by LC -MS/MS using the following conditions: Macherey-Nagel Nucleosil 100-10 C I 8 column (4.6 x 250 mm ) eluted with a gradient from 40% H₂0 (0.05% formic acid, v/v)/60% CH₃CN(0.05% formic acid, v/v) to 100% CH₃CN with 0.05% formic acid (v/v) in 30 min at a flow rate of 2 m L/min. Retention time: 1 1 .2 minutes Parent mass 310 ([M + H]⁺):, fragment detected: 1 05.

Example 12 Synthesis of ¹⁸F for comparison to LC-MS/MS results

[00084] A solution of ¹⁸F (740 MBq) was loaded on a Cromafix Sep-Pak by applying

1 8

vacuum. The F was eluted from the Sep-Pak using a solution prepared by mixing 2.3 mg of 2C0₃ dissolved in 140 iiL of H₂0 and 13 mg of Kryptofix dissolved in 860 μ L of CH₃CN. This solution was taken to dryness at 120°C under vacuum.

[00085] Compound 6 (5 mg in 1 mL of dry CH₃CN) was added and the resulting solution heated at 100°C. After 10 min, CH₃CN (400 μί), TFA (50 μΐ.) and H₂0 (50 μί) were added and the solution was heated for additional five min. The temperature was lowered to 60°C and the solution evaporated by applying vacuum and a stream of helium for 5 min. The solution was then taken to dryness at 100° C by applying vacuum for 5 min.

[00086] The temperature was lowered to 30° C and MeOH (100 μΜ, THF (50 μΙ_) and

Sml_{2 (}4 mL of a 0.1 M solution in THF) was added. After 15 min, the temperature was increased to 60°C and vacuum was applied for 10 min. The residual was dissolved with 5 mL of 75%CH₃CN (0.1 % TFA) /25%H₂0 (0.1 % TFA) and purified by HPLC using the following conditions: MN Nucleosil 100-7 CI 8 column (20 x 250 mm ) eluted with 75%CH₃CN (0.1 % TFA) /25%¾0 (0.1% TFA ). Flow rate 5 mL/min; retention time 13 min. The total synthesis time was 1 h and 33 minutes.

1000871 The solution was analyzed by HPLC using the following conditions: Macherey-

Nagel Nucleosil 1 0- 1 C18 Column (4.6 x 250 mm) eluted with a gradient from 40% in FLO with 0.1 TFA (v/v)/60% CH₃CN with 0.1 % TFA (v/v) to 100% CH₃CN with 0.1 % TFA (v/v) in 30 min. Flow rate 1 mL/min. Retention time 1 0.4 min.

Example 13 Separation of F-compound 9 isomers

[00088] The synthesis of ^{l s} F-compound 9 leads to the formation of a chiral center and ¹⁸F- compound 9 is present in solution as a racemic mixture. A chiral separation of the two stereoisomers was performed using the following method: Chiralpak AD-H column (4.6 x 250 mm) eluted with a 95/5 mixture of A/B, where A = 95% Hexane(0.2%DEA) and B =50% EtOH(0.2%> DEA):50% MeOH (0.2% DEA). Flow rate 0.8 mL/min. A solution containing the two stereoisomers of ¹⁸F-9 ( 1 3.9 MBq) was evaporated to dryness in a speed vacuum and redissolved in 500 μ L of mobile phase.

[00089 J This solution was injected and two peaks (4.2 and 4.6 MBq) during at 25.4 and

30.6 min. were collected. The reinject ion of these two peaks in the analytical system described in the radiochemistry section confirmed their identity, relative to standards of each isomer.

Example 14 Non Radioactive Enzymatic Phosphorylation of Racemic Mixture

[00090] Since such compounds must be phosphorylated before they can be internalized, the enzymatic phosphorylation of a racemic mixture of compound 9 isomers and of the two pure isomers were studied. To a 1 mL solution containing 0.5M HEPES buffer, 1 50 mM NaCl, 5 mM MgCl₂, 0.003 μΜ sodium orlhovanadate, 1 niM DDT, 0.5M ATP and 0.75 ^i of human recombinant sphingosine kinase 2 (Cayman Chemicals, Cat. No. 10009237) was added 2 iiL of a 1 mg/mL solution of compound 9 racemic mixture or 2 g of the resolved isomers of compound 9. These solutions were incubated for 1 hour at 37°C.

[00091] The formation of the phosphorylated product compound 10 was monitored by

LC-MS/MS, relative to a quantitative calibration curve prepared with authentic compound 10. The following method was used: Macherey-Nagel Nucleosil 100- 10 C 18 column (4.6 x 250 mm) eluted with a gradient from 100% in H₂O(0.05% Formic acid,v/v) to 100% CH₃CN(0.05% Formic acid, v/v) over 30 min. Flow rate 2 mL/min. Retention time: 1 1 .2 min. Parent mass: 390.2 ([M + H]⁺):, fragment detected 275.3. The mass spectrometer methods for quantification of compound 10 are shown in Table 2 below.

Table 2

Ql mass for compound 10 390.2

Q3 mass for compound 10 275.3

Dwell(msec) 200

Example 15 Inhibi t ion of Sphingosine Kinase 2 with Isomer 2 of compound 9 (compound 9b)

1000921 The inhibition of sphingosine kinase 2 with the non-radioactive isomer 2 of compound 9 (compound 9b) was studied. The formation of the sphingosine phosphate was monitored by LC-MS. relative to a quantitative calibration curve prepared with authentic sphingosine phosphate.

10009 1 The amount of sphingosine phosphate obtained in presence of isomer 2 of compound 9 was compared to the amount formed in a control reaction where compound 9 was not present

1000941 To a 1 m L solution containing 0.5 H EPES buffer, 1 50 niM NaCl, 5 niM MgCk

0.003 μΜ sodium orthovanadate, 1 niM DDT. 0.5M ATP, 0.75 ,ug of human recombinant sphingosine kinase 2 (Cayman Chemicals. Cat. No. 10009237) and 4 uM D-erithrosphingosine was added 3 μ L of a 10 jig/mL solution of compound 9 isomer 2 (Ι ΟΟηΜ) in the inhibition experiment or 3 uL of DM SO in the control. These solutions were incubated for 1 hour at 37°C, 1 mL of CH₃CN was added to terminate the reactio and the solutions were analyzed by LC-MS.

[00095] The following method was used: Macherey-Nagel Nucleosil 1 00- 1 0 CI 8 column

(4.6 x 250 mm) eluted with a gradient from 100% in H₂0 (0.05% Formic acid,v/v) to 100% CTLCN (0.05% Formic acid, v/v) over 30 min. Flow rate 2 mL/min. Retention time: 1 7.1 min. Mass: 424 ([M - H]^").

100096] A 55% inhibition of sphingosine phosphate formation was observed when a

1 OOnM concentration of compound 9 isomer 2 (compound 9b) was added to the reaction. This indicates that this compound may be used in vivo to inhibit sphingosine kinase and is useful for the treatment or prevention of diseases and conditions associated therewith.

Example 16 Enzymatic Phosphorylation of F- compound 9

1000971 The enzymatic phosphorylation of a 18 F-9 racemic mixture was studied as follows. ¹⁸F-9 (747 μΠ) in a test tube was taken to dryness by speed vacuum. To the tube was added a 1 ml, solution containing 0.5M HOPES buffer, 150 mM Nad. 5 mM MgCl₂, 0.003 μΜ sodium orthovanadate, 1 mM DDT. 0.5M ATP and 3.15 μg of human recombinant sphingosine kinase 2 (Cayman Chemicals, Cat. No. 10009237). This solution was incubated at 37°C. After 30 min, an aliquot was analyzed by 11 PLC.

After 1 h. CH₃CN (500 μΕ) was added to stop the reaction and the solution was analyzed by HPLC.

[00098] Formation of the phosphorylated product ¹⁸F-compound 10 was monitored by

HPLC using the following method: Macherey-N agel Nucleosil 100- 10 C I S column (4.6 x 250 mm) eluted with a gradient from 90% H₂0 (0.1% TFA, v/v)/10% CH₃CN (0.1% TFA, v/v) to 100% CH₃CN (0.1% TFA. v/v) over 30 min. Flow rate 2 mL/min. Retention time: ^{l s}F- l (): 17 min. ¹⁸F-compound 9: 18.7 min. The % of ¹⁸F-10 formed was 28% at 30 min and 19% at 1 h.

The decrease of 18 F-com pound 10 percentage at 1 h is probably due to an increase in 18 F- compound 9 solubility due to the CH₃CN addition.

Example 17 In Vivo Phosphorylation of 18 F- compound 9

[00099] ¹⁸F-compound 9 (RCP = 99.6 %) was diluted to 2.9 mCi/mL using 10% ethanol, and 294 μθ, 0.1 ml, was injected into a lateral tail vein of a CD-I normal male mouse, Charles River Laboratories, ~8 weeks old, 35 g). The mouse was sacrificed after 1 h and the blood was taken from the descending aorta and stored on ice. Within 10 min, it was centrifuged at 4 °C for 15 min at 3300 x g to remove the cellular fraction, and the plasma fraction was removed and kept on ice. An accurate al iquot of the plasma was vorte ed on ice with an equal volume of 50% EtOH containing 0.1 % TFA (v/v) and 0.05 nig/ml, EDTA, treated with 3 parts of ice-chilled CH₃CN and centrifuged at 4 °C for 15 min at 20,000 x g.

[000100] The blood extract was analyzed using the II PLC method described in the ¹⁸F- compound 9 enzymatic phosphorylation experimental. The HPLC analysis showed 49.8% of ¹⁸F-compound 10 indicating in vivo phosphorylation of ¹⁸F-compound 9. This indicates that this compound is active in vivo and may thus be used in vivo to image patients with or suspected of having a disease or condition associated with sphingosine kinase receptors, including SI Pi, S1P₂, S1P₃, SlP₄ and SlP₅.

Example 18

lOOOlO lj Internalization assay: HEK 293-S1P1-GFP cells were grown in 10% FBS-DMEM in 5% CC 37° C incubator. Cells were starved with 2% Charcoal-stripped serum in DMEM for 48 hours. They were pre-incubated in 0% scrum in DMEM for 2 hours. Then cells were treated with the compound of interest or S 1 P or FTY720-P for 1 hr. Cells were fixed with

paraformaldehyde and internalized S I Pi receptors were examined by con focal microscope.

[000102] Compound 10, and compound 1 Oa were shown to internalize.

Claims

What is claimed is:

1. A compound selected from the group consisting of:

wherein F refers to radioactive fluorine.

2. A method for making

comprising the steps of:

3. The method of claim 2 wherein compound 1 is nitrated to form compound 2, compound 2 is acylated to form compound 3, compound 3 is reduced to form compound 4, compound 4 is subjected to bisalkylation to form compound 5 and compound 5 is subjected to conditions to form compound 6.

4. A method for making

comprising the steps of

wherein F refers to radioactive or non-radioactive fluorine.

A method for making

comprising the steps of

9, wherein F refers to radioactive or non-radioactive fluorine.

6. The method of claim 5 wherein compound 6 is converted to compound 7 by nucleophilic fluorination, compound 7 is hydrolyzed to form compound 8, compound 8 is reduced to form compound 9 and compound 9 is phosphorylated to form compound 10.

7. A method for making

comprising the steps of:

9a feeak ¾ wherein F refers to radioactive fluorine.

8. A method for making

comprising the steps of:

9b ipsM } wherein F refers to non-radioactive fluorine.

9. A method for making

comprising the steps of:

6

9 a (peal: 2) 10 a wherein F refers to radioactive or non-radioactive fluorine ( F).

A method for making

comprising the steps of:

9t> (psaM } 10b

11. A method of making ¹⁸F labeled FTY720 comprising:

wherein F refers to radioactive fluorine.

12. The method of claim 10 wherein compound 6 is converted to compound 7 by nucleophilic fluorination, compound 7 reduced to form compound 8 and compound 8 is phosphorylated to form compound 9.

13. An imaging agent comprising F-compound 9 or F-compound 9a.

14. A pharmaceutical composition for treating or preventing diseases or conditions associated with sphingosine kinase comprising non-radioactive compound 9b or compound 9.

15. A kit for the preparation of a radiopharmaceutical, the kit comprising a compound selected from the group consisting of: compound 6, compound 7, compound 8, compound 9, compound 9a and compound 10a.

16. A kit for the preparation of a radiopharmaceutical, the kit comprising a compound selected from the group consisting of: compound 9 and compound 9b.

17. A compound selected from the group consisting of:

Wherein F refers to a non-radioactive fluorine.

18. A sphingosine kinase inhibitor selected from the group consisting of non-radioactive compounds 9 and 9b.

19. A method of treating or preventing diseases or conditions associated with sphingosine kinase receptors comprising administering to a subject a sphingosine kinase inhibitor selected from the group consisting of compound 9, compound 9b, and combinations thereof.

20. A method of imaging a subject comprising administering to the subject an imaging

18 18

agent selected from the group consisting of F-compound 9, F-compound 9a, and combinations thereof.