Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D207/12—Oxygen or sulfur atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
  • C07F9/572—Five-membered rings

Definitions

• the invention is generally directed to azacyclic constrained analogs of FTY720, medicaments formed from these analogs, and methods for the treatment of disorders using such therapeutics.
• Sphingosine-1 phosphate receptors are found on the surface of many cell types. S1 P receptors are activated by binding sphingosine-1 phosphate. There are five types of S1 P receptors, each of which triggers distinct signal transduction pathways. S1 P binding to S1 P receptors may activate different cellular functions, including cell proliferation and differentiation, cell survival, cell invasion, lymphocyte trafficking, and cell migration.
• FTY720 is an immunosuppressant prodrug that functions by antagonizing S1 P receptors. In its active, phosphorylated state, FTY720 binds four of the five S1 P receptors. FTY720 binding to S1 P1 causes receptor activation and subsequent down- regulation trapping lymphocytes in secondary lymphoid organs.
• FTY720 is marketed to treat relapsing-remitting multiple sclerosis (MS).
• MS multiple sclerosis
• the invention is directed to small molecules in the nature of azacyclic constrained analogs of FTY720, medicaments formed from these small molecules, and methods for the treatment of disorders using such therapeutics are disclosed.
• aspects of the invention are directed to compounds having the following molecular formula:
• Ri is an optional functional group selected from an alkyl chain, (CH 2 ) n OH, CHOH-alkyl, CHOH-alkyne, and (CH 2 ) n OMe;
• R2 is an aliphatic chain (Ce - C10)
• R3 is a mono-, di-, tri- or quad- aromatic substituent comprising hydrogen, halogen, alkyl, alkoxy, azide (N 3 ), ether, NO2, or cyanide (CN); R is an optional alcohol (CH 2 OH) with R ⁇
• L is O-CH 2 ;
• Me is an alkyl, alkene or alkyne
• n is an independently selected whole integer selected from 1 to 3; and wherein the phenyl can be moved along the R2 or R 3 carbon chain.
• the compound stimulates PP2A activity.
• the stereochemistry of the compound is selected from the group consisting of S at position 2 and R at position 4, R at position 2 and S at position 4, R at position 2 and R at position 4, and S at position 2 and S at position 4.
• the functional groups attached to the pyrrolidine group are in a cis relative orientation.
• the functional groups attached to the pyrrolidine group are in a trans relative orientation.
• Ri is an alkyl chain having 1 to 6 carbons.
• the compound is in the form of a salt.
• the salt is a pharmaceutically acceptable salt.
• the compound is phosphorylated. In some such embodiments the compound is phosphorylated at any hydroxymethyl group.
• the compound shows reduced activity against binding S1 P receptors when compared to FTY720. In some such embodiments, the compound shows reduced activity against binding S1 P1 and S1 P3 receptors when compared to FTY720. In some embodiments, the compound has activity in down-regulating cellular nutrient transport.
• R 2 is CsHi / and Ri is CH 2 OH.
• aspects of the invention are directed to a medicament for the treatment of a disorder including: a pharmaceutical formulation containing a therapeutically effective amount of one or more azacyclic constrained FTY720 analog small molecule compounds.
• the medicament is directed toward the treatment of a disorder selected from the group consisting of cancer, leukemia, diabetes and obesity.
• the medicament is formulated for a form of administration selected from the group consisting of oral, parenteral, and transdermal.
• the compound stimulates PP2A activity.
• the compound shows reduced activity against binding S1 P receptors when compared to FTY720.
• the compound shows reduced activity against binding S1 P1 and S1 P3 receptors when compared to FTY720.
• aspects of the invention are drawn to a method of treating disease in a patient including:
• azacyclic constrained FTY720 analog small molecule compounds effective in down-regulating cellular nutrient transport.
• FIG. 1 provides a molecular structure of FTY720.
• FIGs. 2a to 2d provide molecular structures of therapeutic small molecule analogs in accordance with embodiments of the invention.
• FIGs. 3a to 3I provide reaction pathways for the production of therapeutic small molecule analogs in accordance with various embodiments of the invention.
• FIG. 4a provides molecular structures of therapeutic small molecule analogs in accordance with various embodiments of the invention.
• FIG. 4b provides data plots summarizing studies of the stereochemistry influence of ether relative to hydroxymethyl on the ability of embodiments of therapeutic small molecule analogs in accordance with the invention to kill leukemia cells.
• FIG. 5a provides molecular structures of therapeutic small molecule analogs in accordance with various embodiments of the invention.
• FIG. 5b provides data plots summarizing studies of the effect of pyrrolidine ring orientation on anticancer activity of embodiments of therapeutic small molecule analogs in accordance with the invention.
• FIG. 6a provides molecular structures of therapeutic small molecule analogs in accordance with various embodiments of the invention.
• FIG. 6b provides data plots summarizing studies of the effect of the loss of phosphorylation sites on the hydroxymethyl group on the efficacy of embodiments of compound 5 therapeutic small molecule analogs in accordance with the invention.
• FIG. 6c provides data plots summarizing studies of the effect of the loss of phosphorylation sites on the hydroxymethyl group on the efficacy of embodiments of compound 6 therapeutic small molecule analogs in accordance with the invention.
• FIG. 7a provides molecular structures of therapeutic small molecule analogs in accordance with various embodiments of the invention.
• FIG. 7b provides data plots summarizing studies of the effect of the length of aliphatic chains on the efficacy of anticancer activity of embodiments of therapeutic small molecule analogs in accordance with the invention.
• FIG. 8 provides data plots summarizing studies of the ability of embodiments of therapeutic small molecule analogs in accordance with the invention to trigger nutrient transporter loss in Sup-B15 leukemia cells.
• FIGs. 9a and 9b provide a molecular structure of compound 6 and its phosphate in accordance with the invention.
• FIGs. 10a to 10o provide data plots providing the results of studies on the efficacy and activity of an exemplary therapeutic small molecule analog in accordance with the invention.
• small molecules capable of treating disorders, including cancer, from a variety of therapeutic mechanisms including triggering cellular nutrient transporter down-regulation, medicaments formed from these small molecules, and methods for the treatment of disorders using such therapeutics are disclosed.
• the small molecules are azacyclic constrained analogs of FTY720. Additional embodiments of the small molecules are o-benzyl pyrrolidines.
• Embodiments can exist in a pure compound form or in the form of pharmaceutically effective salts. Some embodiments inhibit cellular nutrient transport by stimulating PP2A activity.
• formulations and medicaments are provided that are directed to the treatment of disease.
• these formulations and medicaments target cancers, such as, for example, leukemia, and potentially other diseases.
• Therapeutic embodiments contain a therapeutically effective dose of one or more small molecule compounds, present either as pharmaceutically effective salt or in pure form.
• Embodiments allow for various formulations, including, but not limited to, formulations for oral, intravenous, or intramuscular administration.
• Other additional embodiments provide treatment regimes for disorders using therapeutic amounts of the small molecules.
• the small molecules, delivery systems and dosage regimes are directed to the treatment of cancers, such as, for example, leukemia and potentially other diseases, including diseases in which nutrient transport down-regulation is therapeutically effective.
• Sphingosine-1 phosphate (S1 P) is formed in cells in response to diverse stimuli and plays an important role in cell signaling.
• S1 P receptor is any receptor that binds molecules including, but not limited to, S1 P, FTY720, and any analogs of FTY720 or S1 P. This class of receptors includes any of the known G-protein coupled types of S1 P receptors.
• Nutrient transport refers to a cell's capacity to regulate the import and export of metabolically relevant chemical compounds including but not limited to amino acids, glucose, and iron.
• P2A is a serine/threonine phosphatase that plays a role in inactivating signal transduction pathways, antagonizes the action of Bcl-2 family members including Bcl-2 and Bad, and in regulating many other cellular processes.
• FY720 (2-Amino-2-[2-(4-octylphenyl)ethyl]propane 1 ,3-diol hydrochloride), shown diagrammatically in FIG. 1 , is a synthetic immunomodulatory agent bearing an aminodiol functionality on an aromatic moiety bearing a hydrophobic aliphatic chain. It is presently marketed under the trade name GilenyaTM for the treatment of relapsing- remitting multiple sclerosis.
• Selective refers to a compound ligand directed toward a specific receptor site. At the binding site, the compound can act to agonize or antagonize the target molecule. This can be done directly or indirectly by affecting a step in a signal transduction pathway that regulates the activity of a specific target protein.
• phosphate precursor and "phosphate precursor analog” refer to substituent moieties in invention compounds that may be directly phosphorylated in vivo.
• Phosphate derivative refers to substituent moieties in invention compounds that contain a phosphate or phosphate ester group.
• Prodrug refers to a compound that becomes biologically active in vivo only after phosphorylation.
• Acyl phosphate means an acyl group bonded to a phosphate, RCO2PO3 2" .
• Alcohol means a compound with an -OH group bonded to a saturated, alkane-like compound, (ROH).
• Alkyl refers to the partial structure that remains when a hydrogen atom is removed from an alkane.
• Alkane means a compound of carbon and hydrogen that contains only single bonds.
• Alkyne refers to a hydrocarbon structure that contains a carbon-carbon triple bond.
• Alkoxy refers to a portion of a molecular structure featuring an alkyl group bonded to an oxygen atom.
• Aryl refers to any functional group or substituent derived from an aromatic ring.
• Amine molecules are compounds containing one or more organic substituents bonded to a nitrogen atom, RNH 2 , R2NH, or R 3 N.
• amino acid refers to a difunctional compound with an amino group on the carbon atom next to the carboxyl group, RCH(NH 2 )CO2H.
• Cyanide refers to CN.
• Ester is a compound containing the -CO 2 R functional group.
• Ether refers to a compound that has two organic substituents bonded to the same oxygen atom, i.e., R-O-R'.
• Halogen or "halo” means fluoro (F), chloro (CI), bromo (Br), or iodo (I).
• Hydrocarbon means an organic chemical compound that consists entirely of the elements carbon (C) and hydrogen (H).
• Phosphate means a compound containing the elements phosphorous (P) and oxygen (O).
• R in the molecular formula above and throughout are meant to indicate any suitable organic molecule.
• FTY720 is a well-known immunosuppressant, and has been the subject of intensive research. When employed as an immunosuppressant, FTY720 is a pro-drug. In vivo phosphorylation leads specifically to the pro-S-phosphate ester isomer. Once phosphorylated, FTY720 acts as a functional antagonist binding to S1 P receptors, which stimulates lymphocytic migration to secondary lymphoid tissues causing circulating lymphocytes to be sequestered. In other words, FTY720 suppresses the immune system by taking immune cells out of circulation.
• FTY720 for use as an anticancer agent.
• Suggested approaches include: (1 ) designing FTY720 with selective activity toward specific S1 P receptors; and (2) the use of FTY720 to promote PP2A activation, resulting in down-regulation of the oncogenic Bcl-2 family proteins.
• FTY720 analogs directed to a specific subset of S1 P receptors can mitigate the harsh side effects associated with high doses of FTY720.
• FTY720 itself, not any particular analog, can be used to treat leukemia by stimulating the activity of PP2A, a serine/threonine phosphatase, despite the molecule's inherent activity to S1 P receptors.
• PP2A and other phosphatases play important roles in inactivating signal transduction pathways and antagonizing Bcl-2 family proteins.
• azacyclic constrained analogs of FTY720 described herein employ several mechanisms to fight cancer without the lethal effects inherent to approaches taken by others in the field, and which make the use of FTY720 as an anticancer agent effectively untenable. Accordingly, presented below are embodiments of small molecule azacyclic constrained analogs of FTY720, therapeutics based on such small molecules, and treatment regimes incorporating such therapeutics for use in treating cancer and other disorders.
• FIG. 1 A chemical compound in accordance with embodiments of the invention is illustrated in FIG. 2 and pictured below.
• Embodiments comprise the molecule as illustrated in FIG. 2a, phosphates of such molecules as illustrated in FIG. 2b, phosphonates of such molecules as illustrated in FIG. 2c, or a pharmaceutically acceptable salt thereof, wherein:
• R 2 is an aliphatic chain (Ce - C10)
• R3 is a mono-, di-, tri- or quad- aromatic substituent comprising hydrogen, halogen, alkyl, alkoxy, azide (N 3 ), ether, NO 2 , or cyanide (CN);
• R 4 is an optional alcohol (CH 2 OH) with Ri ;
• L is O-CH 2 ;
• Me is an alkyl, alkene or alkyne
• n is an independently selected integer selected from 1 , 2, or 3;
• the O-benzyl group can be moved to position 3 or 4, where the position not occupied by the O-benzyl group is now H (i.e., CH 2 ), as shown in FIG. 2d, and reproduced below.
• alkyl, CH 2 OH, or (CH 2 ) n OH groups can be added to position 5.
• the R 2 and R3 substituents can have different combinations around the phenyl ring with regard to their position.
• the Ri may be an alkyl having 1 to 6 carbons.
• compounds in this invention may exist as stereoisomers, including phosphate, phosphonates, enantiomers, diastereomers, cis, trans, syn, anti, solvates (including hydrates), tautomers, and mixtures thereof, are contemplated in the compounds of the present invention. (See, e.g., FIGs. 2b to 2c, 4a, 5a, 6a and 7a, for example.)
• the claimed invention can also be included in/relate to pharmaceutically acceptable salts.
• a "pharmaceutically acceptable salt” retains the desirable biological activity of the compound without undesired toxicological effects.
• Salts can be salts with a suitable acid, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, benzoic acid, pamoic acid, alginic acid, methanesulfonic acid, naphthalenesulphonic acid, and the like.
• incorporated cations can include ammonium, sodium, potassium, lithium, zinc, copper, barium, bismuth, calcium, and the like; or organic cations such as tetraalkylammonium and trialkylammonium cations.
• organic cations such as tetraalkylammonium and trialkylammonium cations.
• acidic and cationic salts include salts of other acids and/or cations, such as salts with trifluoroacetic acid, chloroacetic acid, and trichloroacetic acid.
• azacyclic constrained FTY720 analogs as well as modified azacyclic constrained FTY720 analogs, suitable for practice of the present invention will be apparent to the skilled practitioner, and include any O-benzyl pyrrolidine compound that may employ several mechanisms including the inhibition or down-regulation of nutrient transport and/or the up-regulation of PP2A activity, without inducing toxic S1 P receptor activity, even if not structurally identical to the compounds shown above.
• the small molecule azacyclic constrained FTY720 analogs are formulated into a therapeutic medicament for treatment of disorders, such as, for example, cancers susceptible to the inhibition of nutrient transport, or to PP2A activation.
• the modes of administration for the therapeutics include, but are not limited to, oral, transdermal, transmucosal (e.g., sublingual, nasal, vaginal or rectal), or parenteral (e.g., subcutaneous, intramuscular, intravenous, bolus or continuous infusion).
• the actual amount of drug needed will depend on factors such as the size, age and severity of disease in the afflicted individual.
• the actual amount of drug needed will also depend on the effective inhibitory concentration ranges of the various azacyclic constrained analogs of FTY720. Different analogs have different effective inhibitory concentration ranges, as shown and described in greater detail in FIGs. 5 to 7, below.
• Embodiments of therapeutics may be administered at dosages and for periods of time effective to reduce, ameliorate or eliminate the symptoms of diseases or pathological conditions susceptible to such treatment, such as, for example, cancers like leukemia.
• Other indications for use may include type-2 diabetes and obesity.
• using various embodiments of the FTY720 small molecule compound may be used to restrict caloric uptake and/or extend lifespan.
• Dose regimens may be adjusted for purposes of improving the therapeutic or prophylactic response of the compound. For example, several divided doses may be administered daily, one dose, or cyclic administration of the compounds to achieve the desired therapeutic result.
• a single azacyclic constrained FTY720 analog small molecule compound may be administered, or combinations of various azacyclic constrained FTY720 analog small molecule compounds may also be administered.
• the claimed compounds can be formulated with one or more adjuvants and/or pharmaceutically acceptable carriers according to the selected route of administration.
• adjuvants and/or pharmaceutically acceptable carriers for oral applications, gelatin, flavoring agents, or coating material can be added.
• carriers may include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles can include sodium chloride and potassium chloride, among others.
• intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers and the like.
• Preservatives and other additives can also be present. (See generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, (1980), the disclosure of which is incorporated herein by reference.)
• Biological data supports the use of the aforementioned azacyclic constrained analogs of FTY720 in a variety of embodiments to treat disease (cancer, obesity, diabetes). Above, are described embodiments incorporating small molecule compounds, medicaments, and as part of treatment regimes. Previous studies have established that chemical modifications to the flexible aminodiol portion of FTY720 influence the selective binding to S1 P receptors. (Clemens, J. J. et al., cited above.) It is noted that embodiments of azacyclic constrained analogs of FTY720 in accordance with the disclosure kill cells at least in part by starvation with reduced activity toward the binding of the S1 P receptors, thereby avoiding lethal side effects, like bradycardia.
• the expected therapeutic efficacy of the azacyclic constrained FTY720 analog small molecule embodiments stems from its demonstrated biological activity in preliminary studies using Sup-B15 leukemia cells. As discussed below, minor chemical and structural modifications, including changes to stereochemistry, O-benzyl chain position, loss of phosphorylation sites, and length of aliphatic chain, have a slight effect on FTY720 small molecule analog activity, but all analogs still show therapeutic advantages over the FTY720 control.
• Embodiments include enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines starting with appropriately substituted 2- and 4-hydroxy D- or L- prolines.
• Some listed embodiments of the azacyclic constrained FTY720 analog small molecule compound originate from similar reactions.
• For compounds 5, 6, 7, 8, 13, 14, and 15 molecular precursors can be purchased in place of carrying out the full synthesis reaction.
• the aforementioned precursors are all known compounds and spectral data were in agreement with the proposed structures and matched those in literature.
• compound 5 As illustrated in Fig. 3a, the synthesis of compound 5 is a six step process. However, compounds 5b, 5c, and 5d are all known compounds consistent with the precursors mentioned above. As such, relevant synthesis reactions begin with compound 5e.
• compound 5e ((2S, 4R)-1 -Boc-4-((4-bromobenzyl)oxy)-2- (terf-butyldimethylsilyloxymethyl)-hydroxypyrrolidine)
• compound 5d (495 mg, 1 .5 mmol) is dissolved in dry THF (10ml_), purged with Argon and cooled to 0°C before NaH (60% in mineral oil, 180 mg, 4.5 mmol).
• the reaction mixture is refluxed with vigorous stirring overnight. The mixture was cooled down to room temperature and a brine solution was added. The mixture is extracted three times with Et2O and the combined organic layers were dried over Na 2 SO4 and filtrated. The solvent is removed under reduced pressure and the residue is purified by flash chromatography (hexane: EtOAc, 12:1 to 9:1 ) to give a slight yellow oil. This oil is then dissolved in EtOAc (140 ml_) and Pd/C (10 %, 745.0 mg, 0.7 mmol) was added. The air is pumped out of the flask and replaced by H 2 . Upon completion as indicated by TLC, the reaction is stopped and the reaction mixture is filtered through cotton and Celite.
• synthesis of compound 14 involves three steps. First, compound 6g (100 mg, 0.238 mmol) is dissolved in CH2CI2 (0.8 ml_) and Et3N (66 ⁇ _, 0.476 mmol) is added. Then, the solution is cooled to 0°C before MsCI (28 ⁇ _, 0.357 mmol) is added and the solution is run over night. The reaction mixture is poured into water and extracted with EtOAc. The combined organic phases are dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue is purified by flash chromatography (hexane: EtOAc, 3:1 ) to give compound 14a (1 12 mg, 95 %) as a slight yellow oil.
• LiBHEt 3 (1 M solution in THF, 0.644 ml_, 0.644 mmol) is slowly added to an ice-cold solution of compound 14a (80 mg, 0.161 mmol) in THF (0.16 mL). The solution warms to room temperature. After 2 hours of stirring, no more starting material should be visible on TLC. The reaction is then quenched with water and poured into EtOAc. The water phase and the EtOAc phase are separated and the water phase was extracted with EtOAc. The combined organic phases were washed with brine, dried over MgSO 4 , filtered and concentrated under reduced pressure.
• Compound 15 is synthesized in a single 2-step reaction where compound 6g is the chemical precursor.
• compound 6g ((2R,4S)-2-(methoxymethyl)-4- ((4-octyl benzyl )oxy)-pyrrol id ine hydrochloride salt)
• compound 6g 50 mg, 0.12 mmol
• dry THF 1 mL
• Argon purged with Argon
• cooled to 0 °C before NaH 60 % in mineral oil, 9.6 mg, 0.24 mmol
• Mel 15 ⁇ , 0.24 mmol
• the reaction is allowed to warm to room temperature and run over night.
• synthesis of compound 7 requires several steps, many of which can be eliminated by using known molecular precursors. In particular, synthesis can occur using connpound 7b or connpound 7c as precursors. Both are known compounds and spectral data are in agreement with the proposed structures and matched those reported in the literature. (See Watanabe, A et a!,, J. Heterocyclic Chem. 2011 , 48, 1 132-1 139, the disclosure of which is incorporated herein by reference.) Starting with compound 7c, compound 7d is obtained as a colorless oil. Next, compound 7e is obtained over two steps (enumerated in FIG. 3d) from compound 7d. Finally, compound 7 was obtained as a yellow solid/oil from compound 7e.
• compound 8 can be synthesized according to the procedure for synthesizing compounds 5 and 7.
• Compounds 8b and 8c are known compounds and special data were in agreement with the proposed structures and matched those reported in the literature. (See Watanabe, J. Heterocyclic Chem. 201 1 , 48, 1 132-1 139., the disclosure of which is incorporated herein by reference.)
• Compound 8d was obtained as a colorless oil from 8c.
• Compound 8e was obtained as a colorless oil from 8d.
• Compound 8 is a salt, obtained as a yellow oil over two steps from 8e.
• synthesis of compound 9 involves several intermediate reactions. Precursors to compound 9 (including compound 9d) are produced according to the procedure described by Evano et al. (See Toumi, M., et al., Angew. Chem. Int. Ed. 2007, 46, 572-575, the disclosure of which is incorporated herein by reference). Compound 9e is obtained as a slight yellow oil from compound 9d. Next, compound 9f was obtained as a slight yellow oil from compound 9e. Compound 9 is obtained as a yellow solid from compound 9f.
• synthesis of compound 1 1 is a several step process. It implicates compound 9d, as produced by the Evano et al. process discussed above.
• NaHCO 3 134 mg, 1 .60 mmol
• CH 2 CI 2 1 .0 mL
• Dess- Martin periodinane 134 mg, 0.32 mmol
• synthesis of compound 13 is a multistep process.
• compound 13 several precursors can be used.
• Known precursors include compounds 13b-f.
• compounds and spectral data is in agreement with the proposed structures and matches those reported in the literature.
• compound 13g is obtained as a colorless oil.
• compound 13h is obtained as a colorless oil over two steps from 13g.
• compound 13 is synthesized by dissolving compound 13h (100 mg, 0.25 mmol) in 4 M HCI in dioxane (6 ml_) and stirring overnight. TLC analysis of the crude mixture should show only the desired compound. The solvent is evaporated and the residue is dissolved in pure dioxane and the solvent evaporated again. The residue was purified by flash chromatography (EtOH:DCM, 1 :9) to give compound 13 (62 mg, 73%) as a slightly yellow solid.
• compound 16b was obtained as a colorless oil (1 .3 g, 100 %) from (R)-3- Pyrrolidinol (539 mg, 6.19 mmol). Spectral data were in agreement with the proposed structures and matched those reported in the literature. (See Kucznierz, R. et. al., J. Med. Chem. 1998, 41 , 4983-4994.) Compound 16c was obtained as a colorless oil from compound 16b. Compound 16e was obtained as a colorless oil over two steps from compound 16c. Finally, compound 16 was obtained as a yellow oil from compound 16e.
• FIG. 3I demonstrates that synthesis of compound 17 uses compound 16b as a precursor and overall, the synthesis process is similar to that of compound 16.
• compound 17a is synthesized by dissolving compound 16b (400 mg, 2.14 mmol) in THF (8 ml_). PPh3 (1 .18 g, 4.49 mmol) and 4-nitrobenzoic acid (750mg, 4.49 mmol) are sequentially added. The solution is cooled to 0 °C before DIAD (0.88 ml_, 4.49 mmol) is added. The reaction is stirred at room temperature overnight. TLC should show no more starting material and the reaction mixture is diluted with EtOAc and washed with water and saturated solution of NaHCO 3 .
• Leukemia cell assays Effects of chemical changes on ability to kill cells as observed in cell viability assays using the BCR-ABL positive human acute lymphoblastic leukemia (ALL) cell line, Sup-B15 are described. Assays using the Sup-B15 cell line were designed to determine efficacy of azacyclic constrained small molecule analogs of FTY720 in killing leukemia cells. Analogs were generated using the synthesis methods mentioned above.
• Sup-B15 cells were maintained at 2-3million/mL in RPMI 1640 (by MediatechTM) supplemented with 10% fetal calf serum (by Sigma-AldrichTM), 10mM Hepes (by MediatechTM), 55 ⁇ ⁇ -mercaptoethanol (by Sigma-AldrichTM), 2 mM L- glutamine (by MediatechTM), and antibiotics.
• BV173, Nalm-6, and Blin-1 were maintained at 1 -2 million/mL and CCRF-CEM at less than 500,000/mL in the same medium.
• BMp190 cells were created by transducing murine bone marrow cells with pMIC-p190 which expresses the p190 isoform of BCR-Abl and human CD4 from an IRES; these cells were kept at 1 -2 million/mL in RPMI supplemented as above.
• Flow cytometry Much of the biological activity presented is shown through flow cytometry data. Flow cytometry data shows how many cells in culture were killed by FTY720 and analogs and levels of cell surface receptor expression. Here, IC 50 flow cytometry assays were used. In them, viability was determined at 72 hours by vital dye exclusion [propium iodide or DAPI (4', 6 - diamindino-2-phenylindole)].
• cell culture assays were carried out to demonstrate the killing capabilities of different small molecule diastereomers in accordance with embodiments.
• compounds 5-8 (shown in FIG. 4a), provide members of a diastereomeric series, and the assays indicate that the three-dimensional orientation of the ether appendage relative to the hydroxymethyl group influence the ability of compounds to kill cancer cells.
• FIG. 4b even slight variations in the compound's stereochemistry can affect the potency of the small molecule analogs.
• certain compounds have greater killing ability than others.
• compound 8 has an 8-fold decrease in activity relative to compound 6, and is 2-fold weaker than its enantiomer (compound 5).
• stereochemistry can either substantially increase the anti-leukemic activity of certain embodiments of the azacyclic constrained FTY720 analog, or it can play a trivial role that does not undermine the analogs overall ability to kill leukemia cells. Accordingly, modifying the stereochemistry of the analog embodiments has the potential to affect azacyclic constrained FTY720 analog efficacy, but with respect to some embodiments, and particularly SupB15 cells, does not decrease anti-cancer efficacy compared to conventional compounds.
• Nalm-6, Blin-1 , and CCRF-CEM are also ALL cell lines but do not express the oncogenic BCR-ABL fusion protein. In these three human leukemia cell lines, 6 no longer exhibited increased potency relative to other compounds in the series. The effect of compound 6 and its diastereoisomeric congeners on the prostate cancer cell lines PC3 and DU145 was also determined. Compounds 5 and 6 induced cell death to a similar extent, and the potency of the constrained analogs was slightly reduced relative to FTY720. Table 1. Mean IC50 (in ⁇ +/- SEM) of analogs in cell viability assays in a range of human cancer cell lines and BCR-Abl-expressing murine bone marrow (BM).
• BM murine bone marrow
• Viability was measured by vital dye exclusion and 1 low cytometry at 72 h. When compared to FTY720 using a t test (two-tailed): * p ⁇ 0.05; ** p ⁇ ( ).01 ; *** p ⁇ 0.001 .
• the enhanced potency of 6 over its diastereoisomers is a characteristic associated with hematologic but not prostate cancers and may be linked to expression of the BCR-ABL fusion protein.
• BCR- ABL dependent signaling drives the survival and proliferation of chronic myelogenous leukemias and a subset of ALLs.
• embodiments of the analog compounds with activity against BCR-ABL positive leukemias could have particular clinical utility.
• FIGs. 9a and 9b The molecular formula for this compound and its phosphate are shown in FIGs. 9a and 9b.
• FIGs. 10a to 10o A summary of the results are provided, below, along with a number of correlated data graphs (FIGs. 10a to 10o).
• compound 6 is labeled with the number "177”
• its phosphate is labeled with the number "1062”
• its enantiomer is labeled "39”.
• FTY720 itself cannot be used in cancer patients because phosphorylation of FTY720 by SphKs makes FTY720-P, which acts at S1 P receptors to cause bradycardia and immunosuppression (on-target effect for primary use).
• FIG. 10a telemetry was used to measure mouse heart rate for 24 h after administration of anti-cancer dose of FTY720 and a dramatic drop in heart rate is observed.
• FTY720 also causes lymphocyte sequestration, as measured by the number of circulating lymphocytes 12 h after dosage.
• compound 6, according to embodiments of the invention does not cause immunosuppression (FIG. 10b), nor does it decrease heart rate at the same doses. (Note, both single doses of 10 mg/kg and multiple doses at 30 mg/kg of compound 6 and its phosphate were administered in this study with similar results.)
• compound 6 has a specific and very high activity to BCR-Abl+ leukemias compared to other cell types. This trend is shown in FIG. 10d by calculating the ratio of IC50 for compound 6 (also called 177) and its enantiomer compound 5 (also called 39). If the drugs are similarly effective a ratio near 1 would be expected, and if compound 6 works better (has a lower IC50), a ratio greater than one as with SupB15s would be expected.
• the ratio of phosphorylated to unphosphorylated compound 6 in the spleen and bone marrow is close to 1 :1 after i.p. administration. Normalization to total protein suggests that compound 6 accumulates in tissues to higher levels than are present in blood.
• FIGs. 10n and 10o provide data results from a study of the effectiveness of treating BCR-Abl+ SFO2 cells (10n), and also in BCR-Abl- LAX7R cells (10o), which is a late-stage leukemia resistant to chemotherapy.
• combining a cytostatic concentration of the MEK1 inhibitor trametinib with a cytostatic concentration of compound 6 kills patient-derived leukemia cells without killing the irradiated stromal cell layer.
• Some BCR-Abl negative patient leukemia samples (FIG. 10o) are sensitive to this drug combination although they are not sensitive to compound 6 as a single agent. Similar to the BCR-Abl negative leukemia results shown in FIG.
• trametinib and compound 6 can kill human colon cancer cells that do not die when treated with compound 6 or trametinib as single agents.
• elevated ERK activity confers resistance to compound 6 and that inhibiting MEK, and thus ERK, signaling with trametinib sensitizes BCR-Abl-positive and - negative cancer cells to compound 6.
• FTY720 functions as an immunosuppressant due to its effect on sphingosine- 1 -phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has anti-neoplastic actions. It has now been determined that FTY720's anti-cancer activity depends in part on its ability to induce nutrient transporter down- regulation. Embodiments of compounds that trigger nutrient transporter loss but lack FTY720's S1 P receptor-related, dose-limiting toxicity are presented that can be used as effective and selective anti-tumor agents.

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