WO2014118556A2 - Selective inhibitors and allosteric activators of sphingosine kinase - Google Patents
Selective inhibitors and allosteric activators of sphingosine kinase Download PDFInfo
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- WO2014118556A2 WO2014118556A2 PCT/GB2014/050264 GB2014050264W WO2014118556A2 WO 2014118556 A2 WO2014118556 A2 WO 2014118556A2 GB 2014050264 W GB2014050264 W GB 2014050264W WO 2014118556 A2 WO2014118556 A2 WO 2014118556A2
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- 0 CC(C(*)O)N(C)C Chemical compound CC(C(*)O)N(C)C 0.000 description 10
- MZSCOUBLAZADTI-UHFFFAOYSA-N CCCCCCCCc1c[n](-c(cc2)ccc2O)nn1 Chemical compound CCCCCCCCc1c[n](-c(cc2)ccc2O)nn1 MZSCOUBLAZADTI-UHFFFAOYSA-N 0.000 description 1
- KVOMIWOWXGXJRR-UHFFFAOYSA-N CCCCCCCCc1ccc(CCN2CCN(CCO)CC2)cc1 Chemical compound CCCCCCCCc1ccc(CCN2CCN(CCO)CC2)cc1 KVOMIWOWXGXJRR-UHFFFAOYSA-N 0.000 description 1
- WWORABZRQUPBET-UHFFFAOYSA-N CCCCCCCCc1ccc(CC[N+]2(C)CCCCCC2)cc1 Chemical compound CCCCCCCCc1ccc(CC[N+]2(C)CCCCCC2)cc1 WWORABZRQUPBET-UHFFFAOYSA-N 0.000 description 1
- NVSUDBRRQVZHFB-VRCFUYIHSA-N C[C@H]([C@H]1O)OCC1NC(Nc(c(F)c(c(F)c1F)F)c1F)=S Chemical compound C[C@H]([C@H]1O)OCC1NC(Nc(c(F)c(c(F)c1F)F)c1F)=S NVSUDBRRQVZHFB-VRCFUYIHSA-N 0.000 description 1
- BXPMPILJMCJWGX-UHFFFAOYSA-N O=S(CCCCCCc1ccc(CCN2CCCCC2)cc1)(c1ccccc1)=O Chemical compound O=S(CCCCCCc1ccc(CCN2CCCCC2)cc1)(c1ccccc1)=O BXPMPILJMCJWGX-UHFFFAOYSA-N 0.000 description 1
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Definitions
- Sphingosine kinase catalyzes the transfer of a phosphate group of ATP to sphingosine (Sph), forming sphingosine 1-phosphate (SIP).
- S IP is a bioactive lipid that mediates inflammation and regulates cell proliferation and cell motility.
- SK plays an important role in the balance between S IP, which is anti-apoptotic, and the pro-apoptic sphingolipid precursors sphingosine and ceramide.
- Sphingosine kinase exists as two isoforms: sphingosine kinase 1 (SKI) and sphingosine kinase 2 (SK2).
- the isoforms are encoded by distinct genes and differ in their biochemical properties, subcellular localization, and function.
- SK is elevated in many human diseases, including cancers, pulmonary fibrosis, inflammatory diseases such as asthma and atherosclerosis, and infectious diseases.
- Transforming growth factor-beta2 upregulates sphingosine kinase- 1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression.
- Kidney International 76, 857-867 SKI and Connective Tissue Growth Factor (CTGF) are up-regulated in podocytes from streptozotocin-induced diabetic mice and the disease is exacerbated in SKI -deficient mice, as evidenced by enhanced albuminuria and CTGF expression compared to wild type mice (Ren et al. 2009).
- CGF Connective Tissue Growth Factor
- Inhibitors of SK2 are also therapeutically indicated for induction of autophagic death in cancer cells (see: Watson, D., Tonelli, F., Al-Osaimi, M., Williamson, L., Chan, E., Gorshkova, I., Berdyshev, E., Bittman, R., Pyne, N.J., and Pyne, S. (2013) The role of sphingosine kinase 1 and 2 in regulating the Warburg effect in prostate cancer cells. Cellular Signalling 25, 1011-1017).
- the present invention provides compounds, methods for their preparation, compositions containing the compounds, and methods of use of the compounds to selectively inhibit either of the two SK isoforms, to induce proteasomal degradation of SKI, to inhibit DNA synthesis in mammalian pulmonary smooth muscle cells and cancer cells, to induce apoptosis in these cells, and to activate SKI for indication as an anti- fibrotic agent.
- the present invention also provides methods of use of the compounds for the treatment of disorders and diseases associated with the activities of sphingosine kinase isoforms 1 and 2.
- the present invention also provides for therapeutic agents for cancer, vascular remodeling in pulmonary hypertension, and fibrotic disease through the modulation of the activity of sphingosine kinases.
- Also provided in this invention are compounds that activate SKI and methods for treatment of disorders such as fibrosis, where intracellular S IP is anti-fibrotic (see: Pyne, S., Dubois, G., and Pyne, N.J. (2013) Role of sphingosine 1-phosphate and
- the present invention provides for a compound of formula I:
- R 1 is a hydrogen, lower alkyl, or lower alkoxy
- R 2 and R 3 are independently hydrogen, Q-Qo alkyl, or -C(X)NHAr
- X is oxygen or sulfur
- Ar is aryl or heteroaryl group
- R 4 is a hydrogen, or hydroxyl
- R 5 is a C m H2 m+ i straight-chain or branched alkyl, C 2 -C 2 o-alkenyl, C 2 -C 20 -alkynyl, or Ci-C 20 -alkoxy
- m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
- bond A is a single or double bond
- R 1 is not hydroxymethyl (CH 2 OH).
- R is C3-C12 alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl, having a single cyclic ring or multiple condensed rings, quaternary ammonium group, or
- R is straight-chain or branched alkyl C m H2 m+ i, C 2 - C 2 o-alkenyl, C 2 -C 2 o-alkynyl, Ci-C 2 o-alkoxy, or C 2 -C 2 o-alkyl-substituted heterocycle; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; n is 0, 1, 2, 3, 4, or 5.
- W is -CH 2 or oxygen; and X and Y are independently hydrogen, Ci-C4-alkyl, or X and Y taken together are oxygen or sulfur.
- the present invention provides for a compound of formula
- R 1 is a C m H 2m+ i straight-chain or branched alkyl, C 2 -C 2 o-alkenyl, C 2 -C 20 - alkynyl, or Ci-C 20 -alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
- R 2 is hydrogen, hydroxyl, or Ci-C 2 o-alkoxyl
- R 3 is oxygen or sulfur
- R 4 is aryl or heteroaryl
- X and Y are independently NH or oxygen.
- the present invention provides a method for selectively inhibiting SKI in a cell by administering the compounds described above.
- the present invention provides a method for selectively inhibiting SK2 in a cell by administering the compounds described above.
- the present invention provides a method for selectively activating SKI in a cell by administering the compounds described above.
- the present invention provides a method of inducing apoptosis in a cell by administering the compounds described above. In yet another aspect, the present invention provides a method of selectively inhibiting SKI in a cell by administering a compound of formula IV:
- FIG. 1 depicts the effect of compounds RB-001 - RB-022 on SKI or SK2 activity.
- SKI activity was measured using 3 ⁇ sphingosine and 250 ⁇ ATP.
- RB series compounds were used at 50 ⁇ .
- FIG. 2 depicts the effect of compounds on SKI or SK2 activity.
- SKI activity was measured using 3 ⁇ sphingosine and 250 ⁇ ATP.
- FIG. 3 depicts the effect of compounds RB-023-RB-065 on SKI or SK2 activity.
- SKI activity was measured using 3 ⁇ sphingosine and 250 ⁇ ATP.
- FIG. 4 depicts the evaluation of compounds as putative substrates of SKI and SK2.
- Control activity using Sph alone (3 ⁇ for SKI and 10 ⁇ for SK2) and is represented as 100%, against which each compound alone is compared.
- FIG. 5 depicts the effect of inhibitors on SKI or SK2 activity.
- SKI activity was measured using 3 ⁇ sphingosine and 250 ⁇ ATP.
- FIG. 6 depicts the evaluation of compounds as putative substrates of SKI and SK2.
- Control activity using Sph alone (3 ⁇ for SKI and 10 ⁇ for SK2) and is represented as 100% against which each compound alone is compared.
- FIG. 8 depicts the effects of azido alcohol and azido fluoro analogues of (S)-FTY720 vinylphosphonate on SKI activity.
- the control is set at 100% and represents the SKI activity against sphingosine alone.
- FIG. 9 depicts the effects of 55-21 (A), F-02 (B) and RB-005 (C) on SKI expression.
- Pulmonary arterial smooth muscle cells (PASMC) were treated with or without MG132 (10 ⁇ , 30 min) before 55-21, F-02, or RB-005 (all at 10 ⁇ , 24 h).
- Cell lysates were western blotted with anti-SKl and -actin antibodies. Results are representative of three experiments.
- the present invention provides compounds, methods for their preparation, compositions containing the compounds, and methods of use of the compounds to selectively inhibit either of the two SK isoforms, to induce proteasomal of SKI, to inhibit DNA synthesis in mammalian pulmonary smooth muscle cells and cancer cells, to induce apoptosis in these cells, and to activate SKI for indication as an anti-fibrotic agent.
- the present invention provides for the compound of formula I: (I)
- R 1 represents a hydrogen, lower alkyl, or lower alkoxy
- R 2 and R 3 independently represent hydrogen, Ci-Cio alkyl, or -C(X)NHAr, in which X is oxygen or sulfur
- alkyl refers to a saturated, linear or branched hydrocarbon moiety, such as -CH 3 or -CH(CH 3 ) 2 .
- lower alkyl refers to straight or branched chain moiety having up to eight carbon atoms.
- alkoxy refers to the group “alkyl-O-" which includes, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, i-butoxy, sec-butoxy, n-pentoxy, and the like.
- Ar refers to aryl or heteroaryl group.
- Aryl refers to an aromatic carbocyclic group having at least one aromatic ring or multiple condensed rings in which at least one ring is aromatic.
- Heteroaryl refers to an aromatic ring system containing at least one ring heteroatom selected from, for example, oxygen (O), nitrogen (N), sulfur (S), silicon (Si), and selenium (Se).
- the heteroaryl rings typically comprise a four, five, six, seven, or eight membered aromatic ring, which may however be bonded to additional rings, so as to form a polycyclic ring system. At least one of the rings present in the ring system is aromatic and contains at least one ring heteroatom.
- Polycyclic heteroaryl groups include those having two or more heteroaryl rings fused together, as well as those having at least one monocyclic heteroaryl ring fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, and/or non- aromatic cycloheteroalkyl rings.
- a heteroaryl group as a whole, can have, for example, 5 to 24 ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 membered heteroaryl group).
- the heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure.
- heteroaryl rings do not contain O-O, S-S, or S-0 bonds.
- one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S,S- dioxide).
- heteroaryl moieties include furyl, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, quinolyl, isoquinolyl and indolyl.
- Alkyl alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,
- heterocycloalkenyl, aryl, and heteroaryl mentioned herein include both substituted and unsubstituted moieties, unless specified otherwise.
- Possible substituents on cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl include, but are not limited to, Ci-Cio alkyl, C 2 -Cio alkenyl, C 2 -Cio alkynyl, C 3 -C 2 o cycloalkyl, C 3 -C 2 o cycloalkenyl, Ci-C 2 o heterocycloalkyl, Ci-C 2 o heterocycloalkenyl, Ci-Cio alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, Ci-Cio alkylamino, Ci-C 2 o dialkylamino, arylamino, diarylamino, Ci-Cio alky
- Cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl also include those fused with one or more additional rings.
- the aryl group is optionally substituted with one or more of a halogen, or CF 3 .
- suitable heteroaryl groups include perfluorophenyl, pyridyl, piperidyl, or pyrrolyl.
- R 4 represents a hydrogen, or hydroxyl.
- R 4 represents hydroxyl when A is a single bond.
- R 5 represents a C m H 2m+ i straight-chain or branched alkyl, C 2 -C 2 o-alkenyl, C 2 -C 2 o-alkynyl, or Ci-C2o-alkoxy; m is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
- Bond A is a single bond or a double bond.
- R 4 represents hydroxyl when A is a single bond.
- alkynyl refers to a linear or branched hydrocarbon moiety that contains at least one triple bond, such as -C ⁇ C-CH 3 .
- R 1 is not methyl
- R 1 is not hydroxymethyl (CH 2 OH).
- R 1 is not hydroxymethyl
- the present invention provides a compound of formula I having the following structure: Scheme A
- the present invention provides a compound of formula I having the following structure:
- the invention provides a compound having formula II: (II)
- R is C3-C 12 alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl, having asingle cyclic ring or multiple condensed rings, quaternary ammonium group, or
- the azido group (N 3 ) may be converted to a triazole-containing group.
- the triazole group may be substituted or non- substituted.
- heterocyclyl groups examples include piperidyl, pyrrolidyl, pyridyl, pyrrolyl, pyrimidinyl, furyl, imidazolyl, tetrazolyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolinyl, and isoquinolinyl, or an acyclic nitrogen- containing group.
- heteroaryl groups can be, for example, the 5- or 6- membered monocyclic and 5- or 6- membered bicyclic ring.
- R is straight-chain or branched alkyl C m H2 m+ i, C2-C2o-alkenyl, d-Cio-alkynyl, Ci-C2o-alkoxy, or C2-C2o-alkyl-substituted heterocycle group.
- the heterocyclic group can be triazole, oxadiazole, oxazole, or thiazole; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; n is 0, 1, 2, 3, 4, or 5; when n is 0, the adjacent atoms are linked by a single bond.
- the heterocyclic group can be triazole, oxadiazole, oxazole, or thiazole.
- W is -C3 ⁇ 4 or oxygen.
- X and Y are independently hydrogen, Ci-C4-alkyl, or X and Y taken together are oxygen or sulfur.
- the triazole ring may be replaced by one of the following rings:
- the present invention provides a compound of formula II having the following structure: Scheme C
- R is hydrogen or hydroxyl
- m is 6, 8, 9, 10, 11, 12, 13, 14, or 15
- n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
- the present invention provides a compound of formula II having the following structure: Scheme D
- R is a C m H 2m+ i straight-chain or branched alkyl, C 2 -C 2 o-alkenyl, C 2 -C 2 0- alkynyl, or Ci-C 20 -alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; X is -OH, F, Br, CI, or I; n is 0, 1, 2, 3, 4, 5, or 6; and W is oxygen or carbon. When n is 0, the adjacent atoms are connected by a single bond.
- the azido group (N 3 ) may be converted to a triazole-containing group.
- the triazole group may be substituted or non-substituted.
- the present invention provides for a compound having formula III:
- R 1 is a C m H 2m+ i straight-chain or branched alkyl, C 2 -C 2 o-alkenyl, C 2 -C 2 0- alkynyl, or Ci-C 20 -alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
- R 2 is hydrogen, hydroxyl, or 3 4
- Ci-C 2 o-alkoxyl is oxygen or sulfur; R is aryl or heteroaryl; and X and Y are independently NH or oxygen.
- the present invention provides a compound of formula III having the following structure: Scheme E
- the present invention provides a method of selectively inhibiting SKI in a cell by administering a compound of formula IV:
- the above-described compounds may be synthesized by any known method. Examples of synthesis schemes are provided in the examples below.
- the synthetic methods described herein may additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups, or to introduce additional substituent groups in order to ultimately allow synthesis of the compounds disclosed herein.
- This invention provides a method of inhibiting SK by administering the compounds described above. Accordingly, these compounds may be used to treat cells having elevated levels of S IP, and may therefore have therapeutic effect. Without wishing to be bound by theory, it is thought that cancer cells and pulmonary smooth muscle cells of people having pulmonary arterial hypertension have abnormally elevated levels of S IP.
- these compounds can be used as anti-proliferative and pro- apoptotic/autophagic agents, and therefore have promise in the treatment of pulmonary arterial hypertension and cancer.
- some of the compounds in the present invention stimulate the degradation of one of the isoforms of SK (SKI) by the proteasome of cancer and pulmonary smooth muscle cells.
- Some of the compounds in the invention inhibit or activate one of the two isoforms of SK and not the other isoform; these compounds can be used to analyze the roles of the two isoforms of SK, which are called SKI and SK2.
- the isoform-selective SK inhibitors in the present disclosure are useful in establishing the role of SKI and SK2 in cancer and vascular biology.
- the compounds of this invention that induce allosteric activation of SKI are of use in treatment of fibrosis.
- the present invention provides a method to selectively inhibit SKI or SK2. Selectively inhibits means that one isoform is inhibited more than the other isoform.
- the present invention provides a method of selectively inhibiting SKI by administering compounds described in Scheme A, Scheme C, or Scheme E to a cell.
- the present invention provides a method of selectively inhibiting SK2 by administering compounds described in Scheme B to a cell.
- the present invention provides a method of selectively activating SKI by administering compounds described in Scheme D to a cell.
- the present invention provides a method of inducing apoptosis in a cell by administering compounds described in Scheme A, Scheme C, or Scheme E to a cell.
- compositions described herein including salts (including amine salts, salts of mineral acids including but not limited to hydrochloride salts, phosphate-containing salts, and sulfate salts, salts of organic acids, and various alkali and alkali earth metal salts), esters, solvates, hydrates, and prodrugs of the compounds described herein.
- prodrug is intended to include any covalently bonded carriers of the disclosed compounds, which release the active compound on metabolism when the compound is administered to a living mammalian organism.
- the compounds described herein may contain one or more chiral centers, in which case the compounds may exist as stereoisomers. These structures include all stereoisomers. Accordingly, the chemical structures depicted herein encompass all of the possible stereoisomeric forms, including the stereochemically pure form and
- a pharmaceutical composition containing an effective amount of at least one compound described herein and a pharmaceutical acceptable carrier.
- this disclosure includes a method of administering an effective amount of one or more of the compound described herein to a patient having a disease (e.g., an inherited or acquired disease).
- diseases that can be treated by the compounds disclosed above include hyper-proliferative diseases, such as cancer and vascular remodeling in pulmonary arterial hypertension.
- An effective amount refers to the amount of an active compound described herein that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.
- composition having one or more compound described herein can be administered parenterally, orally, nasally, rectally, topically, or buccally.
- parenteral refers to
- a sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol.
- a non-toxic parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol.
- acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution.
- fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides).
- Fatty acid such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
- oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
- These oil solutions or suspensions can also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersing agents.
- Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.
- a composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions.
- commonly used carriers include lactose and corn starch.
- Lubricating agents such as magnesium stearate, are also typically added.
- useful diluents include lactose and dried corn starch.
- a nasal aerosol or inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation.
- such a composition can be prepared as a solution in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
- composition having one or more active compound described herein can also be administered in the form of suppositories for rectal administration.
- the carrier in the pharmaceutical composition must be "acceptable” in the sense that it is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
- One or more solubilizing agents can be utilized as pharmaceutical excipients for delivery of an active compound described herein.
- examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.
- the compound described herein can be preliminarily screened for their efficacy in treating an above-described disease by an in vitro assay and then confirmed by animal experiments and clinic trials. Other methods will also be apparent to those of ordinary skill in the art.
- each member may be combined with any one or more of the other members to make additional sub-groups.
- additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
- Fig. 1 shows that these modifications reduced inhibition of both SKI and SK2. Relocation of the quaternary nitrogen functionality to an exocyclic position as in RB-016 - RB-018 also afforded nonselective SK inhibitors (Fig. 1).
- RB-020 is less efficiently phosphorylated by SKI than sphingosine, and probably overlaps the catalytic site of SKI to inhibit phosphorylation of sphingosine (Table 2). However, this is not the case for SK2 where phosphorylation of sphingosine and RB-20 appear mutually exclusive (Table 2).
- RB-019 is a very weak substrate for SK2 (10% of the activity against sphingosine, data not shown) and inhibits SKI activity with sphingosine as the substrate (Fig. 1). Both RB-019 and RB-020 contain a hydroxyl group that is likely to be phosphorylated by SKI and SK2 (Table 2).
- RB- 020 has a primary hydroxyl group attached to the heterocyclic ring through a 4-CH 2 group
- RB-019 has a secondary hydroxyl group directly attached to C-3 of the heterocyclic ring; therefore, the latter hydroxyl group may be too far removed from the catalytic determinants of SKI to be phosphorylated, but probably overlaps the substrate binding site to inhibit SKI activity.
- RB-008 and RB-009 have a second nitrogen atom in the heterocyclic ring, and also possess a -(CH 2 ) 2 OH and -(CH 2 ) 3 0H group, respectively.
- RB-008 and RB-009 are not substrates for SKI or SK2 (data not shown) but are inhibitors of SKI (Fig. 1).
- the aliphatic chain at the para position of the benzene ring of FTY720 is C 8 H 17 , which is known to be optimal for the action of FTY720 on its targets such as S IP receptors.
- the inhibitory activities of RB-026 (which has a methyl group as the alkyl substituent), RB-027 (which has a n-hexyl group), RB-005 (which has a n-octyl group), and RB-028 (which has a n-dodecyl group) were examined.
- RB-065 is a highly selective SKI inhibitor, whereas the other ten triazole analogues, all of which lack the 4-hydroxypiperidyl group, were inactive.
- the S enantiomers RB-041 and RB-043 and RB-037 are substrates for SK2 (Fig. 4).
- Example 5
- the structures of the 1-deoxy sphingoid bases 55-21, 55-22, 77-7, and 77- 13; the thiourea- PHS derivatives 67-301, 67-306, 67-310, and the urea-PHS derivative 67-311 ; the thiourea-sphinganine bases F01 and F02; and the thiourea-pachastrissamine derivative 67-341 are shown. Also displayed is the structures of the 4-sphingenine (sphingosine) adducts 67-320 and 67-330. (2S,3R)-1 -Deoxysphinganine derivatives (2S,3R)-Sphinganine derivatives
- the dose-dependent inhibition analysis revealed that F-02 inhibited SK2 activity with an IC 5 0 value of 21.8 + 4.2 ⁇ but only very weakly inhibited SKI activity (with an IC 5 0 value of 69 + 5.5 ⁇ ) (Fig. 2G, H).
- 1-Deoxysphinganine analogue 55-21 and its N,N-dimethyl derivative 55-22 are selective SKI inhibitors (Fig. 5).
- 55-21 inhibited SKI activity with an IC 50 value of 7.1 + 0.75 ⁇ and SK2 activity with an IC 50 value of 766 + 133 ⁇ (Fig. 21, J)
- 77-7 inhibited SKI activity with an IC 50 value of 27.8 + 3.2 ⁇ and SK2 activity with an IC 50 value of 300 + 62.3 ⁇ (data not shown).
- 77-7 inhibited SKI activity with an IC 50 value of 27.8 + 3.2 ⁇ and SK2 activity with an IC 50 value of 300 + 62.3 ⁇ (data not shown).
- 77-7 inhibited SKI activity with an IC 50 value of 27.8 + 3.2 ⁇ and SK2 activity with an IC 50 value of 300 + 62.3 ⁇ (data not shown).
- 77-7 inhibited SKI activity with an IC 50 value of 27.8 +
- SK substrates was examined.
- FOl, 77-13, 67-341, and 67-302 are weak substrates of SKI (Fig. 6), but probably overlap the sphingosine binding site in SKI, thereby inhibiting catalytic phosphorylation of sphingosine.
- F02 and FOl were very weak substrates of SK2, but 67-302 (czs-sphingosine) was efficiently
- Figure 7 shows a comparison of the effects of two known, highly potent SKI inhibitors, PF-543 and VPC96091, with 55-21 on the growth of pulmonary arterial smooth muscle cells (PASMC).
- This comparison which is based on [ H] -thymidine incorporation into DNA in PASMC, shows that the highly potent PF-543 and VPC96091 compounds were ineffective in inhibiting the growth of PASMC, whereas the less potent compound 55-21 of this invention was effective.
- the compounds disclosed here including but not limited to 55-21, which have a moderate potency on inhibition of SKI enzymatic activity, may possess a more favorable profile than highly potent inhibitors in terms of selectively abrogating SKI function without exhibiting Off-target' effects on sphingosine/ceramide metabolizing enzymes.
- 55-21 recapitulates siRNA knockdown and genetic studies in terms of reducing cell growth; thus 55-21 is expected to have utility in unraveling the functions of SKI in hyperproliferative disorders.
- azido-alcohol changed to an azido-fluoro derivative also gave a compound that activates SKI at low concentrations. Both of these compounds induced a 30-60% stimulation of SKI activity at low concentrations (Fig. 8).
- the structural difference in these azide-containing compounds concern fluorine, which can only accept hydrogen bonds, and a hydroxyl group, which can both accept and donate hydrogen bonds.
- the azido group is critical in this activity; the amino-fluoro derivative does not affect SKI activity.
- concentrations above 50 ⁇ the % activation in response to this compound diminished markedly (Fig. 2K).
- This biphasic response suggests that the compound might bind to the catalytic site (or alter its conformation) to inhibit SKI activity at these higher concentrations.
- proteasomal degradation of SKI in response to SKi reduces intracellular S IP and increases C22:0-ceramide levels in prostate cancer cells, thereby promoting apoptosis (see: Loveridge, C, Tonelli, F., Leclercq, T., Lim, K.G., Long, S., Berdyshev, E., Tate, R.J., Natarajan, V., Pitson, S.M., Pyne, N.J. & Pyne, S.
- FIG. 9A shows that treatment of pulmonary arterial smooth muscle cells (PASMC) with the SK1- selective inhibitor 55-21 (10 ⁇ , 24 h) reduced the expression of SKI; this was reversed by pre-treatment of the cells with the proteasomal inhibitor MG132. In contrast, treatment of PASMC with the SK2-selective inhibitor F-02 was without effect on SKI expression (Fig.
- Scheme 1 outlines the preparation of 1-deoxysphinganine analogues 55-21 and 55-22 via cyclic sulfate intermediates of (2S,3 ?)-2-azidosphinganine.
- Azidoester 1 was prepared by asymmetric dihydroxylation of ethyl hexadecenoate using AD-mix- ⁇ , followed by conversion to a cyclic sulfate intermediate and regioselective azidation with sodium azide in aqueous acetone.
- Reagents and conditions (a) NaI0 4 , THF/H 2 O, 0 °C - rt; (b) (EtO) 2 P(0)CH 2 C0 2 Et, K 2 C0 3 , 2-PrOH/H 2 0 (1: 1) , 0 °C - rt, overnight; (c) AD-mix ⁇ , MeS0 2 NH 2 , i-BuOH/H 2 0 (1: 1), rt; (d) SOCl 2 , py, CH 2 C1 2 , 0 °C; (e) cat.
- Ethyl 4-Tetradecyloxy-2(E)-butenonate (6) To a solution of NaI0 4 (4.12 g, 19.2 mmol) in 25 mL of water was added a solution of 1-Otetradecyl-rac-glycerol (4, 4.27 g, 14.8 mmol) in 25 mL of THF at 0 °C, followed by stirring at rt. After the oxidative glycol cleavage was completed (about 2 h at rt), the mixture was concentrated under reduced pressure in order to remove THF and the formaldehyde formed, providing aldehyde 5.
- the tertiary amines shown in Table 1 were prepared in good yields by displacement of mesylate ion from 4-(octylphenethyl) methanesulfonate (prepared as displayed in Scheme 4) with amines in acetonitrile.
- Some of the quaternary ammonium salts were prepared by N-alkylation of the tertiary amines (and the secondary amine RB- 006) with an excess of Mel and K 2 C0 3 in acetonitrile whereas others (RB-011, RB-012, RB-017, and RB-018) were prepared by N-alkylation with tertiary N-methylamines.
- the azepane derivative RB-003 was prepared from 4-octylphenethyl
- Compound RB-004 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-001, using
- Compound RB-019 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-005,
- Compound RB-020 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-005,
- 4-(Octylphenyl)methanol was prepared in two steps from 4-iodobenzyl alcohol; first, a Sonogashira reaction with 1-octyne afforded (4-(oct-l-ynyl)phenylmethanol as a yellow oil, and then catalytic hydrogenation of the triple bond provided 4-(octylphenyl)methanol as a colorless oil.
- RB-035 To a solution of RB-005 (25 mg, 0.080 mmol) in CH 2 C1 2 (3 mL) at 0 °C was added pyridinium chlorochromate (PCC, 25 mg, 0.12 mmol). After being stirred at rt for 4 h, the reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, evaporated, and dried.
- PCC pyridinium chlorochromate
- reaction mixture was stirred at 50 °C for 12 h.
- the reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. Purification by silica gel chromatography, eluting with
- reaction mixture was stirred at 50 °C for 12 h.
- the reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. Purification by silica gel chromatography, eluting with
- the catalyst was removed by filtration through a pad of Celite, which was rinsed with hexanes/EtOAc (3: 1). 3-((4- (Oct-l-ynyl)phenyl)ethynyl)pyridine (53 mg, 0.18 mmol) was dissolved in EtOAc (8 mL), and 10% Pd/C (53 mg, 100 wt %) was added. The reaction mixture was
- the latter compound was synthesized from 4-chloropyridine hydrochloride (0.20 g, 1.3 mmol) in dry acetonitrile (4 mL). N,N-Diisopropylethylamine (DIPEA, 0.7 mL, 4.0 mmol) was added, followed by 4-methylpiperidine (0.16 mL, 1.3 mmol). The reaction mixture was subjected to microwave irradiation at 160 °C for 1 h. After the reaction mixture was cooled rt, EtOAc was added, and the solution was washed with water and brine, dried (Na 2 S0 4 ), and concentrated in vacuo.
- DIPEA N,N-Diisopropylethylamine
- the aryl amine was converted to the corresponding aryl azide by the reaction of 4-(oct-l-ynyl)aniline (157 mg, 0.78 mmol) in 2 mL of 10% aqueous HC1 with NaN0 2 (65 mg, 0.94 mmol) in 1 mL of water at 0 °C. After the solution was stirred for 30 min, NaN 3 (61 mg, 0.94 mmol) in 1 mL of water was added at 0 °C, with stirring for another hour. The reaction mixture was warmed to 25 °C, diluted with EtOAc, washed with water and brine, dried (Na 2 S0 4 ), and concentrated in vacuo, affording the aryl azide.
- RB-056 2-(4-(4-Octyl-lH-l,2,3-triazol-l-yl)phenyl)ethanol (RB-056) was prepared by a click reaction as follows. To a solution of 4-(azidophenyl)-2-ethanol (200 mg, 1.23 mmol) and 1-decyne (508 mg, 3.68 mmol) in i-BuOH/H 2 0 (6 mL, 1: 1) were added CuS0 4 (196 mg, 1.23 mmol) and sodium ascorbate (243 mg, 1.23 mmol). The reaction mixture was stirred at rt for 12 h and then was diluted with EtOAc and washed with brine.
- reaction mixture was stirred for 1 h at 0 °C, quenched with saturated aqueous NH 4 C1 solution, and extracted with EtOAc. The organic layer was dried (MgS0 4 ) and concentrated under reduced pressure. The residue was purified by chromatography (elution with EtOAc) to give 750 mg (94%) of the desired dimethyl phosphonate product.
- HEK 293 cells stably over-expressing GFP-SK1 (30-fold increase in SKI activity versus vector-transfected cells) were cultured in DMEM supplemented with 10% European fetal calf serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 1%
- Sphingosine Kinase Activity Assays In order to measure SK2 activity, sphingosine (Sph) was complexed with fatty acid free bovine serum albumin (final concentration, 0.2 mg/mL) in buffer A containing 20 mM Tris (pH 7.4), 1 mM EDTA, 1 mM Na 3 V0 4 , 40 mM ⁇ -glycerophosphate, 1 mM NaF, 0.007% (v/v) ⁇ -mercaptoethanol, 20% (v/v) glycerol, 10 ⁇ g/mL aprotinin, 10 ⁇ g/mL soybean trypsin inhibitor, 1 mM PMSF, 0.5 mM 4-deoxypyridoxine, and 400 niM KCl.
- SK2 assays were performed using 37 ng of purified SK2 and incubating the assay for 30 min at 30 °C in the presence of 10 ⁇ Sph, 250 ⁇ [ ⁇ - 32 ⁇ ] ⁇ in 10 mM MgCl 2 , and varying concentrations of the inhibitors dissolved in DMSO or control (5% v/v DMSO).
- Sph was solubilized in Triton X-100 (final concentration, 0.063% w/v) and combined with buffer A without KCl.
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Abstract
Sphingosine 1-phosphate (S1P) is involved in hyper-proliferative diseases, such as cancer and vascular remodeling in pulmonary arterial hypertension. Inhibitors of sphingosine kinase 1 and 2 (SK1 and SK2), which catalyze the synthesis of S1P, may be useful anti- proliferative agents. We have synthesized a series of sphingosine-based inhibitors of SK and SK2. Also provided in this invention are compounds that activate SK1 which can be used in diseases such as fibrosis, where intracellular S1P is anti-fibrotic.
Description
SELECTIVE INHIBITORS AND ALLOSTERIC ACTIVATORS OF
SPHINGOSINE KINASE
This invention was supported by a grant from National Institutes of Health, grant number R24 HL083187, and British Heart Foundation grant number 29476. The United States government has rights in this invention
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to Provisional Application No. 61759393, filed January 31, 2013, the disclosure of which is incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Sphingosine kinase (SK) catalyzes the transfer of a phosphate group of ATP to sphingosine (Sph), forming sphingosine 1-phosphate (SIP). S IP is a bioactive lipid that mediates inflammation and regulates cell proliferation and cell motility. SK plays an important role in the balance between S IP, which is anti-apoptotic, and the pro-apoptic sphingolipid precursors sphingosine and ceramide. Sphingosine kinase exists as two isoforms: sphingosine kinase 1 (SKI) and sphingosine kinase 2 (SK2). The isoforms are encoded by distinct genes and differ in their biochemical properties, subcellular localization, and function. SK is elevated in many human diseases, including cancers, pulmonary fibrosis, inflammatory diseases such as asthma and atherosclerosis, and infectious diseases.
Therefore, reduction of the levels of S IP can prevent hyperproliferation of cells that lead to pulmonary arterial hypertension and cancer. On the other hand, there is evidence that raising the level of intracellular S IP by activation of SKI in response to Transforming Growth Factor-β has a potential anti-fibrotic effect in kidney (see: Ren, S., Babelova, A., Moreth, K., Xin, C, Eberhardt, W., Doller, A., Pavenstadt, H., Schaefer, L., Pfeilschifter, J., Huwiler, A. (2009) Transforming growth factor-beta2 upregulates sphingosine kinase- 1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF
expression. Kidney International 76, 857-867). SKI and Connective Tissue Growth Factor (CTGF) are up-regulated in podocytes from streptozotocin-induced diabetic mice and the disease is exacerbated in SKI -deficient mice, as evidenced by enhanced albuminuria and CTGF expression compared to wild type mice (Ren et al. 2009).
Inhibitors of SK2 are also therapeutically indicated for induction of autophagic death in cancer cells (see: Watson, D., Tonelli, F., Al-Osaimi, M., Williamson, L., Chan, E., Gorshkova, I., Berdyshev, E., Bittman, R., Pyne, N.J., and Pyne, S. (2013) The role of sphingosine kinase 1 and 2 in regulating the Warburg effect in prostate cancer cells. Cellular Signalling 25, 1011-1017).
As SKI and SK2 are potential and promising targets for cancer chemoprevention, a number of SK inhibitors have been prepared in order to reduce cancer cell survival but in general only very few have been found to be isoform selective or are metabolically stable. Therefore, there is an unmet need for selective inhibitors of either SKI or SK2, but not both.
SUMMARY OF THE INVENTION
The present invention provides compounds, methods for their preparation, compositions containing the compounds, and methods of use of the compounds to selectively inhibit either of the two SK isoforms, to induce proteasomal degradation of SKI, to inhibit DNA synthesis in mammalian pulmonary smooth muscle cells and cancer cells, to induce apoptosis in these cells, and to activate SKI for indication as an anti- fibrotic agent.
The present invention also provides methods of use of the compounds for the treatment of disorders and diseases associated with the activities of sphingosine kinase isoforms 1 and 2.
The present invention also provides for therapeutic agents for cancer, vascular remodeling in pulmonary hypertension, and fibrotic disease through the modulation of the activity of sphingosine kinases.
Also provided in this invention are compounds that activate SKI and methods for treatment of disorders such as fibrosis, where intracellular S IP is anti-fibrotic (see: Pyne, S., Dubois, G., and Pyne, N.J. (2013) Role of sphingosine 1-phosphate and
lysophosphatidic acid in fibrosis. Biochimica Biophysica Acta 1831, 228-238).
In one aspect, the present invention provides for a compound of formula I:
(I)
in which R 1 is a hydrogen, lower alkyl, or lower alkoxy; R 2 and R 3 are independently hydrogen, Q-Qo alkyl, or -C(X)NHAr; X is oxygen or sulfur; Ar is aryl or heteroaryl group; R4 is a hydrogen, or hydroxyl; R5 is a CmH2m+i straight-chain or branched alkyl, C2-C2o-alkenyl, C2-C20-alkynyl, or Ci-C20-alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; bond A is a single or double bond; with the proviso that when R 2 and R 3 are hydrogen, R 1 is not methyl; and with the proviso that when A is a trans-double bond, and when the configuration at C-2 is S and when the configuration at C-3 is R, R1 is not hydroxymethyl (CH2OH).
In another aspect, the present invention provides for a compound of formula II:
2
in which Z is -OH, F, Br, CI, or I. R is straight-chain or branched alkyl CmH2m+i, C2- C2o-alkenyl, C2-C2o-alkynyl, Ci-C2o-alkoxy, or C2-C2o-alkyl-substituted heterocycle; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; n is 0, 1, 2, 3, 4, or 5. W is -CH2 or oxygen; and X and Y are independently hydrogen, Ci-C4-alkyl, or X and Y taken together are oxygen or sulfur.
In yet another aspect, the present invention provides for a compound of formula
(III)
in which R1 is a CmH2m+i straight-chain or branched alkyl, C2-C2o-alkenyl, C2-C20- alkynyl, or Ci-C20-alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20; R 2 is hydrogen, hydroxyl, or Ci-C2o-alkoxyl; R 3 is oxygen or sulfur; R 4 is aryl or heteroaryl; and X and Y are independently NH or oxygen. In another aspect, the present invention provides a method for selectively inhibiting SKI in a cell by administering the compounds described above.
In another aspect, the present invention provides a method for selectively inhibiting SK2 in a cell by administering the compounds described above.
In another aspect, the present invention provides a method for selectively activating SKI in a cell by administering the compounds described above.
In another aspect, the present invention provides a method of inducing apoptosis in a cell by administering the compounds described above.
In yet another aspect, the present invention provides a method of selectively inhibiting SKI in a cell by administering a compound of formula IV:
(IV)
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the effect of compounds RB-001 - RB-022 on SKI or SK2 activity. SKI activity was measured using 3 μΜ sphingosine and 250 μΜ ATP. SK2 activity was assayed using 10 μΜ sphingosine and 250 μΜ ATP (n = 3 for each compound, results expressed as % of the control ± S.D). RB series compounds were used at 50 μΜ.
FIG. 2 depicts the effect of compounds on SKI or SK2 activity. SKI activity was measured using 3 μΜ sphingosine and 250 μΜ ATP. SK2 activity was assayed using 10 μΜ sphingosine and 250 μΜ ATP (n = 3 for each compound, results expressed as % of the control ± S.D).
FIG. 3 depicts the effect of compounds RB-023-RB-065 on SKI or SK2 activity. SKI activity was measured using 3 μΜ sphingosine and 250 μΜ ATP. SK2 activity was assayed using 10 μΜ sphingosine and 250μΜ ATP (n = 3 for each compound, results expressed as % of the control ± S.D).
FIG. 4 depicts the evaluation of compounds as putative substrates of SKI and SK2. SKI and SK2 activity was measured using 50 μΜ compound and 250 μΜ ATP in the absence of Sph (n = 3 for each compound). The results are expressed as % of control ± S.D. Control = activity using Sph alone (3 μΜ for SKI and 10 μΜ for SK2) and is represented as 100%, against which each compound alone is compared.
FIG. 5 depicts the effect of inhibitors on SKI or SK2 activity. SKI activity was measured using 3 μΜ sphingosine and 250 μΜ ATP. SK2 activity was assayed using 10 μΜ sphingosine and 250 μΜ ATP (n = 3 for each compound, results expressed as % of the control ± S.D).
FIG. 6 depicts the evaluation of compounds as putative substrates of SKI and SK2. SKI and SK2 activity was measured using 50 μΜ compound and 250 μΜ ATP in the absence of Sph (n = 3 for each compound); results are expressed as % control ± S.D. Control = activity using Sph alone (3 μΜ for SKI and 10 μΜ for SK2) and is represented as 100% against which each compound alone is compared.
FIG. 7 depicts the assessment of the effects of PF-543 (10 nM and 100 nM, 24 h), VPC96091 (300 nM, 24 h), and 55-21 (100 nM and 1 μΜ, 24 h) on [3H] -thymidine incorporation into DNA in PASMC. Results are expressed as % control ± S.D. of control (n = 3); the control is set to 100%. *** p <0.05 versus control.
FIG. 8 depicts the effects of azido alcohol and azido fluoro analogues of (S)-FTY720 vinylphosphonate on SKI activity. The substrates were 3 μΜ sphingosine (which corresponds to the Km of SKI) and 250 μΜ ATP (n = 3, results expressed as % control ± S.E.). The control is set at 100% and represents the SKI activity against sphingosine alone.
FIG. 9 depicts the effects of 55-21 (A), F-02 (B) and RB-005 (C) on SKI expression. Pulmonary arterial smooth muscle cells (PASMC) were treated with or without MG132 (10 μΜ, 30 min) before 55-21, F-02, or RB-005 (all at 10 μΜ, 24 h). Cell lysates were western blotted with anti-SKl and -actin antibodies. Results are representative of three experiments.
DETAILED DESCRIPTION
The present invention provides compounds, methods for their preparation, compositions containing the compounds, and methods of use of the compounds to selectively inhibit either of the two SK isoforms, to induce proteasomal of SKI, to inhibit DNA synthesis in mammalian pulmonary smooth muscle cells and cancer cells, to induce apoptosis in these cells, and to activate SKI for indication as an anti-fibrotic agent.
In one aspect, the present invention provides for the compound of formula I: (I)
in which R 1 represents a hydrogen, lower alkyl, or lower alkoxy; R 2 and R 3 independently represent hydrogen, Ci-Cio alkyl, or -C(X)NHAr, in which X is oxygen or sulfur; The term "alkyl" refers to a saturated, linear or branched hydrocarbon moiety, such as -CH3 or -CH(CH3)2. The term "lower alkyl" refers to straight or branched chain moiety having up to eight carbon atoms. The term "alkoxy" refers to the group "alkyl-O-" which includes, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, i-butoxy, sec-butoxy, n-pentoxy, and the like. The term "Ar" refers to aryl or heteroaryl group. Aryl refers to an aromatic carbocyclic group having at least one aromatic ring or multiple condensed rings in which at least one ring is aromatic. Heteroaryl refers to an aromatic ring system containing at least one ring heteroatom selected from, for example, oxygen (O), nitrogen (N), sulfur (S), silicon (Si), and selenium (Se). The heteroaryl rings typically comprise a four, five, six, seven, or eight membered aromatic ring, which may however be bonded to additional rings, so as to form a polycyclic ring system. At least one of the rings present in the ring system is aromatic and contains at least one ring
heteroatom. Polycyclic heteroaryl groups include those having two or more heteroaryl rings fused together, as well as those having at least one monocyclic heteroaryl ring fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, and/or non- aromatic cycloheteroalkyl rings. A heteroaryl group, as a whole, can have, for example, 5 to 24 ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 membered heteroaryl group). The heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O-O, S-S, or S-0 bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S,S- dioxide). Examples of heteroaryl moieties include furyl, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, quinolyl, isoquinolyl and indolyl.
Alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,
heterocycloalkenyl, aryl, and heteroaryl mentioned herein include both substituted and unsubstituted moieties, unless specified otherwise. Possible substituents on cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl include, but are not limited to, Ci-Cio alkyl, C2-Cio alkenyl, C2-Cio alkynyl, C3-C2o cycloalkyl, C3-C2o cycloalkenyl, Ci-C2o heterocycloalkyl, Ci-C2o heterocycloalkenyl, Ci-Cio alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, Ci-Cio alkylamino, Ci-C2o dialkylamino, arylamino, diarylamino, Ci-Cio alkylsulfonamino, arylsulfonamino, Cl-Cio alkylimino, arylimino, Ci-Cio alkylsulfonimino, arylsulfonimino, hydroxyl, halo, thio, Ci-Cio alkylthio, arylthio, Ci-Cio alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl,
aminothioacyl, amidino, guanidine, ureido, cyano, nitro, nitroso, azido, acyl, thioacyl, acyloxy, carboxyl, and carboxylic ester. Cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl also include those fused with one or more additional rings.
In one embodiment the aryl group is optionally substituted with one or more of a halogen, or CF3. Examples of suitable heteroaryl groups include perfluorophenyl, pyridyl, piperidyl, or pyrrolyl. R4 represents a hydrogen, or hydroxyl. Optionally, R4 represents hydroxyl when A is a single bond. R5 represents a CmH2m+i straight-chain or branched
alkyl, C2-C2o-alkenyl, C2-C2o-alkynyl, or Ci-C2o-alkoxy; m is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Bond A is a single bond or a double bond. Optionally, R4 represents hydroxyl when A is a single bond. When the bond denoted as "A" is a double bond, both E and Z configurations have been contemplated. The term "alkenyl" refers to a linear or branched hycrocarbon moiety that contains at least one double bond, such as -CH=CH-CH3. The term "alkynyl" refers to a linear or branched hydrocarbon moiety that contains at least one triple bond, such as -C≡C-CH3.
When R 2 and R 3 are hydrogen, R 1 is not methyl.
When A is a trans-double bond, and when the configuration at C-2 is S and when the configuration at C-3 is R, R1 is not hydroxymethyl (CH2OH).
In another emobodicment, with the proviso that when A is a double bond, R1 is not hydroxymethyl.
In another preferred embodiment, the present invention provides a compound of formula I having the following structure: Scheme A
In another preferred embodiment, the present invention provides a compound of formula I having the following structure:
10
In another aspect, the invention provides a compound having formula II: (II)
in which Z is -OH, F, Br, CI, or I. Optionally, the azido group (N3) may be converted to a triazole-containing group. The triazole group may be substituted or non- substituted.
Examples of suitable heterocyclyl groups include piperidyl, pyrrolidyl, pyridyl, pyrrolyl, pyrimidinyl, furyl, imidazolyl, tetrazolyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolinyl, and isoquinolinyl, or an acyclic nitrogen- containing group.
In some embodiments, heteroaryl groups can be, for example, the 5- or 6- membered monocyclic and 5- or 6- membered bicyclic ring.
Examples of suitable R groups are shown below:
When n is 0, the adjacent atoms are connected by a single bond.
2
R is straight-chain or branched alkyl CmH2m+i, C2-C2o-alkenyl, d-Cio-alkynyl, Ci-C2o-alkoxy, or C2-C2o-alkyl-substituted heterocycle group. In some embodiments, the heterocyclic group can be triazole, oxadiazole, oxazole, or thiazole; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; n is 0, 1, 2, 3, 4, or 5; when n is 0, the adjacent atoms are linked by a single bond. In some embodiments, the heterocyclic group can be triazole, oxadiazole, oxazole, or thiazole. W is -C¾ or oxygen. X and Y are independently hydrogen, Ci-C4-alkyl, or X and Y taken together are oxygen or sulfur.
The following compound is exemplary of an alkyl-substituted triazolium:
In another preferred embodiment, the present invention provides a compound of formula II having the following structure:
Scheme C
, in which R is hydrogen or hydroxyl; m is 6, 8, 9, 10, 11, 12, 13, 14, or 15; and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In another preferred embodiment, the present invention provides a compound of formula II having the following structure: Scheme D
in which R is a CmH2m+i straight-chain or branched alkyl, C2-C2o-alkenyl, C2-C20- alkynyl, or Ci-C20-alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; X is -OH, F, Br, CI, or I; n is 0, 1, 2, 3, 4, 5, or 6; and W is oxygen or carbon. When n is 0, the adjacent atoms are connected by a single bond. Optionally, the azido group (N3) may be converted to a triazole-containing group. The triazole group may be substituted or non-substituted.
In another aspect, the present invention provides for a compound having formula III:
(III)
19, or 20; R 2 is hydrogen, hydroxyl, or 3 4
Ci-C2o-alkoxyl; R is oxygen or sulfur; R is aryl or heteroaryl; and X and Y are independently NH or oxygen.
In another preferred embodiment, the present invention provides a compound of formula III having the following structure: Scheme E
In yet another aspect, the present invention provides a method of selectively inhibiting SKI in a cell by administering a compound of formula IV:
(IV)
The above-described compounds may be synthesized by any known method. Examples of synthesis schemes are provided in the examples below. The synthetic methods described herein may additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups, or to introduce additional substituent groups in order to ultimately allow synthesis of the compounds disclosed herein. This invention provides a method of inhibiting SK by administering the compounds described above. Accordingly, these compounds may be used to treat cells having elevated levels of S IP, and may therefore have therapeutic effect. Without wishing to be bound by theory, it is thought that cancer cells and pulmonary smooth muscle cells of people having pulmonary arterial hypertension have abnormally elevated levels of S IP. Therefore, these compounds can be used as anti-proliferative and pro- apoptotic/autophagic agents, and therefore have promise in the treatment of pulmonary arterial hypertension and cancer. In addition to blocking the enzymatic activity of SK, some of the compounds in the present invention stimulate the degradation of one of the isoforms of SK (SKI) by the proteasome of cancer and pulmonary smooth muscle cells. Some of the compounds in the invention inhibit or activate one of the two isoforms of SK and not the other isoform; these compounds can be used to analyze the roles of the two isoforms of SK, which are called SKI and SK2. The isoform-selective SK inhibitors in the present disclosure are useful in establishing the role of SKI and SK2 in cancer and vascular biology. In addition, the compounds of this invention that induce allosteric activation of SKI are of use in treatment of fibrosis. Until the present invention, nothing was known about the allosteric effects of chiral derivatives of FTY720 on the allosteric activation or allosteric inhibition of SKI.
In another aspect, the present invention provides a method to selectively inhibit SKI or SK2.
Selectively inhibits means that one isoform is inhibited more than the other isoform.
In another aspect, the present invention provides a method of selectively inhibiting SKI by administering compounds described in Scheme A, Scheme C, or Scheme E to a cell.
In another aspect, the present invention provides a method of selectively inhibiting SK2 by administering compounds described in Scheme B to a cell.
In another aspect, the present invention provides a method of selectively activating SKI by administering compounds described in Scheme D to a cell. In another aspect, the present invention provides a method of inducing apoptosis in a cell by administering compounds described in Scheme A, Scheme C, or Scheme E to a cell.
Also provided in this invention are pharmaceutically acceptable derivatives, including salts (including amine salts, salts of mineral acids including but not limited to hydrochloride salts, phosphate-containing salts, and sulfate salts, salts of organic acids, and various alkali and alkali earth metal salts), esters, solvates, hydrates, and prodrugs of the compounds described herein. The term "prodrug" is intended to include any covalently bonded carriers of the disclosed compounds, which release the active compound on metabolism when the compound is administered to a living mammalian organism.
The compounds described herein may contain one or more chiral centers, in which case the compounds may exist as stereoisomers. These structures include all stereoisomers. Accordingly, the chemical structures depicted herein encompass all of the possible stereoisomeric forms, including the stereochemically pure form and
stereoisomeric mixtures, which may be resolved using routine methods.
Also within the scope of this disclosure is a pharmaceutical composition containing an effective amount of at least one compound described herein and a pharmaceutical acceptable carrier. Further, this disclosure includes a method of administering an effective amount of one or more of the compound described herein to a
patient having a disease (e.g., an inherited or acquired disease). Examples of diseases that can be treated by the compounds disclosed above include hyper-proliferative diseases, such as cancer and vascular remodeling in pulmonary arterial hypertension. "An effective amount" refers to the amount of an active compound described herein that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.
To practice the pharmaceutical composition described, a composition having one or more compound described herein can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term "parenteral" as used herein refers to
subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique. A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.
A composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. In the case of tablets, commonly used carriers include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
A nasal aerosol or inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation. For example, such a composition can be prepared as a solution in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
A composition having one or more active compound described herein can also be administered in the form of suppositories for rectal administration.
The carrier in the pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. One or more solubilizing agents can be utilized as pharmaceutical excipients for delivery of an active compound described herein. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.
The compound described herein can be preliminarily screened for their efficacy in treating an above-described disease by an in vitro assay and then confirmed by animal experiments and clinic trials. Other methods will also be apparent to those of ordinary skill in the art.
In this specification, groups of various parameters containing multiple members are described. Within a group of parameters, each member may be combined with any one or more of the other members to make additional sub-groups. For example, if the members of a group are a, b, c, d, and e, additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
EXAMPLES
The following examples are illustrative and not intended to be limiting. General Experimental Considerations
Synthetic Procedures
General experimental methods. All chemicals were reagent grade and were used as purchased. Reactions were carried out under a dry nitrogen atmosphere using oven-dried glassware and magnetic stirring. The solvents were dried as follows: THF was heated at reflux over sodium benzophenone ketyl; toluene was heated at reflux over sodium;
CH2CI2 were dried over CaH2. The progress of the reactions was monitored by thin-layer chromatography analysis using aluminum-backed silica gel 60 F254 plates of 0.2-mm thickness. The spots were visualized with short wavelength ultraviolet light or by charring after spraying with 15% H2SO4. Flash chromatography was performed on silica gel grade 60 (230-400 ASTM mesh). 1H NMR and 13C NMR spectra were recorded in δ units relative to deuterated solvents (CDCI3 δ = 7.26 ppm for 1H NMR and 77.00 ppm for 13C NMR); CD3OD δ = 4.78, 3.31 ppm for 1H NMR and 49.1 ppm for 13C NMR), which served as an internal reference, at 400 or 500 (for 1H NMR) and 100 MHz (for 13C NMR), respectively. The purity of the products was >95% based on proton NMR spectra. High-resolution mass spectra (HRMS) were recorded on an Agilent Technologies
G6520A Q-TOF mass spectrometer using electrospray ionization (ESI). Optical rotations were recorded on a digital polarimeter at the sodium-D line at rt.
Example 1
Enzymatic Activity - Inhibition of SKI by RB-005 and Its Analogues
Previous work has established SKI and SK2 assays using sphingosine/[ 32 P]-ATP with over-expressed recombinant SKI in HEK 293 cell lysates or purified SKI or SK2
(see: Lim, K.G., Tonelli, F., Li, Z., Lu, X., Bittman, R., Pyne, S., Pyne, N.J. (2011) FTY720 analogues as sphingosine kinase 1 inhibitors: Enzyme inhibition kinetics, allosterism, proteasomal degradation and actin rearrangement in MCF-7 breast cancer cells. Journal of Biological Chemistry 286, 18633-18640; also see: Lim, K.G., Sun, C, Bittman, R., Pyne, N.J., Pyne, S. (2011) (tf)-FTY720 methyl ether is a specific sphingosine kinase 2 inhibitor: effect on sphingosine kinase 2 expression in HEK 293 cells and actin rearrangement and survival of MCF-7 breast cancer cells. Cellular Signalling 23, 1590-1595) ; also see Tonelli, F., Lim, K.G., Loveridge, C, Long, J., Pitson, S.M., Tigyi, G., Bittman, R., Pyne, S & Pyne N.J. (2010) FTY720 and (S)- FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promotes its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen- independent prostate cancer cells. Cellular Signalling 22, 1536-1542.).
Select compounds of the invention are listed in Table 1.
Com ound * Compound
Table 1. Structures of RB-OOl-RB-020 27
By using sphingosine concentrations of 3 μΜ and 10 μΜ, which correspond to the Km values of SKI and SK2, respectively, the results of these assays demonstrate that RB- 001-003, RB-005, RB-007-009, RB-019, and RB-021 (Table 1) are selective inhibitors of SKI over SK2. SKI activity was measured using 3 μΜ sphingosine and 250 μΜ ATP. SK2 activity was assayed using 10 μΜ sphingosine and 250 μΜ ATP (n = 3 for each compound); results expressed as % control ± S.D. The control is 100% and equals activity against sphingosine alone. Small changes in the structure of this tertiary amine result in large changes in selectivity, arguing for the hypothesis that RB-005 is a selective inhibitor of SKI (Fig. 1). To investigate the role of the hydroxyl group in the heterocyclic ring in the inhibition of SKI, the 4-hydroxy group was replaced with a 4-methyl group to afford RB-004, which is a moderate inhibitor of both SKI and SK2 (Fig. 1); the 3 -hydroxy regioisomer RB-019 is a selective but less potent SKI inhibitor. To investigate the effect of the size of the heterocycle and the charge on the ring, heterocyclic amines RB-003 - RB-006 and quaternary ammonium compounds RB-013 - RB-016 were prepared and assayed. Fig. 1 shows that these modifications reduced inhibition of both SKI and SK2. Relocation of the quaternary nitrogen functionality to an exocyclic position as in RB-016 - RB-018 also afforded nonselective SK inhibitors (Fig. 1). The data in Fig. 1 show that RB-005 has the highest selectivity for SKI over SK2 (15-fold) and Fig. 2A shows that RB-005 exhibits and IC50 = 3.6 ± 0.38 μΜ for SKI.
Example 2
Examination of RB-005 Analogues as Possible SK Substrates
Furthermore, the possibility that the six inhibitors that bear a hydroxyl group may also serve as SK substrates was examined. At 50 μΜ, none of the compounds with the exception of RB-020 is a substrate for SKI or SK2 (Table 2).
SKI activity SK2 activity
(% control) (% control)
Sph 100 + 4.4 100 + 12.8
Sph + RB-020 34.4 + 4.1 315 + 5.1
RB-020 27.5 + 3.0 228 + 17.9
5
Table 2. RB-020 (50 μΜ) is a substrate of SKI and SK2 and an inhibitor of SKI activity against sphingosine (Sph). Sph was used at 3 and 10 μΜ for SKI and SK2, respectively. Results are represented as % control ± SD (n = 3) of control.
RB-020 is less efficiently phosphorylated by SKI than sphingosine, and probably overlaps the catalytic site of SKI to inhibit phosphorylation of sphingosine (Table 2). However, this is not the case for SK2 where phosphorylation of sphingosine and RB-20 appear mutually exclusive (Table 2). RB-019 is a very weak substrate for SK2 (10% of the activity against sphingosine, data not shown) and inhibits SKI activity with sphingosine as the substrate (Fig. 1). Both RB-019 and RB-020 contain a hydroxyl group that is likely to be phosphorylated by SKI and SK2 (Table 2). It is noteworthy that RB- 020 has a primary hydroxyl group attached to the heterocyclic ring through a 4-CH2 group, while RB-019 has a secondary hydroxyl group directly attached to C-3 of the heterocyclic ring; therefore, the latter hydroxyl group may be too far removed from the catalytic determinants of SKI to be phosphorylated, but probably overlaps the substrate binding site to inhibit SKI activity. RB-008 and RB-009 have a second nitrogen atom in the heterocyclic ring, and also possess a -(CH2)2OH and -(CH2)30H group, respectively. However, RB-008 and RB-009 are not substrates for SKI or SK2 (data not shown) but are inhibitors of SKI (Fig. 1).
Example 3
Enzymatic Activity - Inhibition of SKI by RB-023 -RB-065
Compound Structure Compound Structure
RB-030 R1 = N3, R2 = C8H17
RB-031 R-i = NH2, R2 = CH3
To investigate the structural determinants that result in selective SKI inhibition, a series of analogues bearing a 4-hydroxypiperidinyl group was prepared in which the linker length between the aryl group and the piperidyl ring was varied. The effect of linker length on potency was assessed by comparing the % inhibition of SKI and SK2 obtained with RB-023 (which has a one-carbon tether), RB-024 (three-carbon tether), and RB-025 (four-carbon tether). The linker length did not significantly alter the ability of RB-023, RB-024, and RB-025 to inhibit SKI activity (Fig. 3). RB-023 - RB-025 also retained selectivity for SKI over SK2 (Fig. 3).
The aliphatic chain at the para position of the benzene ring of FTY720 is C8H17, which is known to be optimal for the action of FTY720 on its targets such as S IP receptors. To examine the role of the alkyl substituent on the benzene ring of RB-005, and thus the lipophilicity of the molecule, the inhibitory activities of RB-026 (which has a methyl group as the alkyl substituent), RB-027 (which has a n-hexyl group), RB-005 (which has a n-octyl group), and RB-028 (which has a n-dodecyl group) were examined. SKI inhibition was decreased by more than 6-fold in RB-026 compared with RB-023 (Fig. 3). The almost complete lack of inhibition displayed by RB-026 against SKI indicates that a larger alkyl group than a methyl group is required for inhibitory activity.
The importance of the 4-hydroxyl group of RB-005 was examined by replacing it with an azido, amino, fluoro, keto, or methoxy group (RB-029 - RB-036). Azido replacement (RB-029, RB-030) reduced SKI inhibition markedly, while replacement of the 4-hydroxyl group with an amino group (RB-032) diminished the potency of SKI inhibition. RB-032 inhibited SKI activity with an IC50 = 16.9 ± 1.6 μΜ (Fig. 2B). The isoform selectivity of SKI over SK2 was retained for RB-032 (Fig. 3), suggesting that the amino group replacement maintains efficient binding to SKI.
Replacement of the 4-hydroxyl group of RB-005 with a fluoro (RB-034) or methoxy group (RB-036) eliminated inhibitory activity against SKI, while replacement with a keto group to produce RB-035 increased inhibition of SK2 and maintained inhibition of SKI but eliminated the isoform selectivity (Fig. 3).
To investigate the role of the piperidyl group in inhibition of SK, experiments were carried out in which the piperidyl group was replaced with a pyrrolidine ring; the hydroxyl-containing substituent was retained (as either a chiral hydroxyl or a chiral hydroxymethyl group) but its orientation was varied, as shown in compounds RB-037 - RB-043. RB-037 and RB-038 retained inhibitory activity against SKI despite having opposite configurations at C-3 of the pyrrolidin-3-ol group. Stereoisomers RB-040 and RB-042, which differ in the length of the aliphatic chain (CgHn vs. C12H5) but possess the R configuration at C-2 of the 2-hydroxymethyl pyrrolidinyl group, were equipotent inhibitors of SKI and SK2. RB-040 inhibits SKI activity with an IC50 = 2.2 μΜ, and SK2 with an IC50 = 5.2 ± 0.82 μΜ (Fig. 2C, D). RB-042 inhibits SKI activity with an IC50 = 5.3 ± 0.5 μΜ and SK2 with an IC50 = 5.0 ± 1.3 μΜ (Fig. 2E, F). The
corresponding S enantiomers RB-041 and RB-043 were much less active (Fig. 3).
Experiments were performed to test the concentration-dependence of RB-041 and RB-043 inhibition of SKI and SK2 activity. At a higher concentration of each (100 μΜ, compared to the 50 μΜ concentration data shown in Fig. 3), the inhibition of SKI and SK2 activity with RB-041 was 72.2 ± 5.9% and 45.7 ± 2.6%, respectively, whereas with RB-043 the inhibition of SKI and SK2 activity was 49.9 ± 6.2% and 49.7 ± 7%, respectively. These findings indicate that RB-041 and RB-043 can inhibit SKI and SK2,
but that the sensitivity of inhibition compared with RB-040 and RB-042 is considerably reduced.
To further examine the influence of the length of the alkyl substituent on the benzene ring on SK activity, the extent of SK inhibition afforded by pyrrolidine derivatives RB-039, RB-042, and RB-043 was assessed. The ability of the compound to inhibit SKI is abolished in RB-039 and RB-043 (Fig. 3), which have a methyl and a n- dodecyl group in the lipophilic tail, respectively. Replacing the methylene linker between the aryl group and the heterocycle with a keto group produced the benzamide analogues RB-044 - RB-050. Inhibition of SKI was effectively abolished (Fig. 3) in these analogues, and also in the pyridine derivatives (RB-048 and RB-051). A series of triazole analogues of RB-005 (RB-054 - RB-065) was prepared and tested for SK inhibitory ability. As shown in Fig. 3, RB-065 is a highly selective SKI inhibitor, whereas the other ten triazole analogues, all of which lack the 4-hydroxypiperidyl group, were inactive.
Example 4
Examination of RB-023 - RB-065 as Possible SK Substrates
The S enantiomers RB-041 and RB-043 and RB-037 are substrates for SK2 (Fig. 4). Example 5
Enzymatic Activity - Inhibition of SKI or SK2 Enzymatic Activity by Analogues of 1- Deoxysphinganine, Sphingosine Derivatives, Dihydrosphingosine Derivatives,
Phytosphingosine Derivatives, and Pachastrissamine Derivatives
The structures of the 1-deoxy sphingoid bases 55-21, 55-22, 77-7, and 77- 13; the thiourea- PHS derivatives 67-301, 67-306, 67-310, and the urea-PHS derivative 67-311 ; the thiourea-sphinganine bases F01 and F02; and the thiourea-pachastrissamine derivative 67-341 are shown. Also displayed is the structures of the 4-sphingenine (sphingosine) adducts 67-320 and 67-330.
(2S,3R)-1 -Deoxysphinganine derivatives (2S,3R)-Sphinganine derivatives
F-02, Z
(2S,3R,4S)-Phytosphingosine (2S,3R)-Sphingosine (2S,3S,4R)-Pachastrissamine derivatives derivatives derivative
67-301 , X = S, Z = F, Y = H
67-310, X = S, Z= CF3, Y = H
67-306, X = S, Z = F, Y = F
67-311 , X = 0, Z = CF3, Y = H
The effects of fluorine and trifluoromethyl substitution in the benzene ring of these putative inhibitors were examined. Although the /^-fluorophenyl thiourea-PHS derivative 67-301 is a weak and nonselective SK inhibitor, the activity is improved by insertion of five fluorine atoms into the benzene ring to afford 67-306, which shows a moderate selectivity for inhibition of SKI (Fig. 5). Thiourea 67-310 and urea 67-311, which are both /?-trifluoromethylphenyl PHS derivatives, are moderately effective SK2 inhibitors (64.5 + 4.9% and 53.9 + 0.9% inhibition at 50 μΜ, respectively, n = 3). The p fluorophenyl thiourea- sphingo sine derivative 67-320 is a highly selective SK2 inhibitor (79.2 + 1.9% inhibition at 50 μΜ, n = 3) whereas its /?-trifluoromethylphenyl analogue 67-330 is a weak inhibitor of both SK isoforms (Fig. 5). The 4-e/?z'-pachastrissamine pentafluorophenyl thiourea derivative 67-341 is a selective SKI inhibitor (64.7 + 5.3% inhibition at 50 μΜ, n = 3) (Fig. 5).
The sphinganine thiourea derivative F-02 is a highly selective SK2 inhibitor (80 2% inhibition at 50 μΜ, n = 3), but its analogue F-01 is less selective (Fig. 5). The dose-
dependent inhibition analysis revealed that F-02 inhibited SK2 activity with an IC50 value of 21.8 + 4.2 μΜ but only very weakly inhibited SKI activity (with an IC50 value of 69 + 5.5 μΜ) (Fig. 2G, H).
1-Deoxysphinganine analogue 55-21 and its N,N-dimethyl derivative 55-22 are selective SKI inhibitors (Fig. 5). 55-21 inhibited SKI activity with an IC50 value of 7.1 + 0.75 μΜ and SK2 activity with an IC50 value of 766 + 133 μΜ (Fig. 21, J), whereas 77-7 inhibited SKI activity with an IC50 value of 27.8 + 3.2 μΜ and SK2 activity with an IC50 value of 300 + 62.3 μΜ (data not shown). Thus, insertion of an oxygen atom into the aliphatic chain afforded 77-7, which is also a selective SKI inhibitor. However, the N,N,N-trimethylammonium salt 77-13 is a nonselective SK isoform inhibitor.
Example 6
Examination of FOl, F02, 55-21, 55-22, 77-7, 77-13, 67-341, 67-330, 67-320, 67-311, 67-310, 67-306, 67-302, and 67-301 as Possible SK Substrates The possibility that the compounds that bear a hydroxyl group may also serve as
SK substrates was examined. At 50 μΜ, FOl, 77-13, 67-341, and 67-302 are weak substrates of SKI (Fig. 6), but probably overlap the sphingosine binding site in SKI, thereby inhibiting catalytic phosphorylation of sphingosine. At 50 μΜ, F02 and FOl were very weak substrates of SK2, but 67-302 (czs-sphingosine) was efficiently
phosphorylated by SK2 (Fig. 6). None of the other compounds were substrates.
Example 7
Examination of Effect of 55-21 on Growth of Pulmonary Arterial Smooth Muscle Cells
Figure 7 shows a comparison of the effects of two known, highly potent SKI inhibitors, PF-543 and VPC96091, with 55-21 on the growth of pulmonary arterial smooth muscle cells (PASMC). This comparison, which is based on [ H] -thymidine incorporation into DNA in PASMC, shows that the highly potent PF-543 and VPC96091
compounds were ineffective in inhibiting the growth of PASMC, whereas the less potent compound 55-21 of this invention was effective. Thus, the compounds disclosed here, including but not limited to 55-21, which have a moderate potency on inhibition of SKI enzymatic activity, may possess a more favorable profile than highly potent inhibitors in terms of selectively abrogating SKI function without exhibiting Off-target' effects on sphingosine/ceramide metabolizing enzymes. In this regard, 55-21 recapitulates siRNA knockdown and genetic studies in terms of reducing cell growth; thus 55-21 is expected to have utility in unraveling the functions of SKI in hyperproliferative disorders. Example 8
Examination of (S)-FTY720 Vinylphosphonate Analogues as Allosteric Activators of SKI
(S)-FTY720 vinylphosphonate 3-azido-3-fluoromethyl analogue 3-azido-3-hydroxymethyl analogue of (S)-FTY720 vinylphosphonate of (S)-FTY720 vinylphosphonate
(SJ-FTY720 vinylphosphonate (at 50 μΜ) inhibits SKI activity by 62.7 ± 0.9% (n = 3) using 3 μΜ sphingosine (Sph) as the substrate (see: Liu, Z., MacRitchie, N., Pyne, S., Pyne, N.J. & Bittman, R. (2013) Synthesis of (S)-FTY720 vinylphosphonate analogues and evaluation of their potential as sphingosine kinase 1 inhibitors. Bioorganic Medicinal Chemistry 21, 2503-2510). (SJ-FTY720 vinylphosphonate is an uncompetitive inhibitor (with sphingosine) of SKI with a Kiu = 14.5 ± 4.4 μΜ. (S)-FTY720
vinylphosphonate, which is not a substrate for SKI, competes with ATP for the ATP- binding site, but flips to bind to an allosteric site when ATP binds to the catalytic site. Therefore, the -P(0)(OH)2 group appears to be essential for binding of (S)-FTY720 vinylphosphonate to both the catalytic and allosteric sites. This invention shows that replacement of the amino group with an azido group (N3) produces an azido-alcohol derivative that activates of SKI at low micromolar concentrations (and is not a substrate,
data not shown) (Fig. 8). Moreover, changing the azido-alcohol to an azido-fluoro derivative also gave a compound that activates SKI at low concentrations. Both of these compounds induced a 30-60% stimulation of SKI activity at low concentrations (Fig. 8). The structural difference in these azide-containing compounds concern fluorine, which can only accept hydrogen bonds, and a hydroxyl group, which can both accept and donate hydrogen bonds. The azido group is critical in this activity; the amino-fluoro derivative does not affect SKI activity.
At concentrations below 50 μΜ, the azido-alcohol compound stimulated SKI activity with an EC50 of 8.3 ± 3.0 μΜ, n = 3 (Fig. 2K), indicative of binding to an allosteric site. At concentrations above 50 μΜ, the % activation in response to this compound diminished markedly (Fig. 2K). This biphasic response suggests that the compound might bind to the catalytic site (or alter its conformation) to inhibit SKI activity at these higher concentrations. Similar results were obtained with the azido- fluoride compound (EC50 of 5.7 ± 5.5 μΜ, n = 3, data not shown). The biphasic curves found with both compounds indicate that the azido group and not the fluoro or hydroxyl group is responsible for this phenomenon.
Example 9
Inducing the Proteasomal Degradation of SKI in Pulmonary Arterial Smooth Muscle Cells with Compounds 55-21 and RB-005
Without wishing to be bound by theory, it is believed that proteasomal degradation of SKI in response to SKi (2-( ?-hydroxyanilino)-4-( ?-chlorophenyl)thiazole) reduces intracellular S IP and increases C22:0-ceramide levels in prostate cancer cells, thereby promoting apoptosis (see: Loveridge, C, Tonelli, F., Leclercq, T., Lim, K.G., Long, S., Berdyshev, E., Tate, R.J., Natarajan, V., Pitson, S.M., Pyne, N.J. & Pyne, S. (2010) The sphingosine kinase 1 inhibitor 2-( ?-hydroxyanilino)-4-(p- chlorophenyl)thiazole induces proteosomal degradation of sphingosine kinase 1 in mammalian cells. Journal of Biological Chemistry 285, 38841-38852). Figure 9A shows that treatment of pulmonary arterial smooth muscle cells (PASMC) with the SK1-
selective inhibitor 55-21 (10 μΜ, 24 h) reduced the expression of SKI; this was reversed by pre-treatment of the cells with the proteasomal inhibitor MG132. In contrast, treatment of PASMC with the SK2-selective inhibitor F-02 was without effect on SKI expression (Fig. 9B), suggesting that changes in the ceramide-sphingosine-S IP rheostat regulated by SK2 is not accessible to the proteasome and, therefore, does not regulate SKI turnover. The ability of RB-005 to promote the proteasomal degradation of SKI in cells was also examined. As shown in Fig. 9C, treatment of PASMC with RB-005 (10 uM, 24 h) reduced the expression of SKI. The effect of RB-005 on SKI expression was reversed by pre-treatment of the cells with the proteasomal inhibitor MG132.
Example 10
Synthesis of the compounds described above
Synthetic Procedures
General experimental methods. All chemicals were reagent grade and were used as purchased. Reactions were carried out under a dry nitrogen atmosphere using oven-dried glassware and magnetic stirring. The solvents were dried as follows: THF was heated at reflux over sodium benzophenone ketyl; toluene was heated at reflux over sodium;
CH2CI2 were dried over CaH2. The progress of the reactions was monitored by thin-layer chromatography analysis using aluminum-backed silica gel 60 F254 plates of 0.2-mm thickness. The spots were visualized with short wavelength ultraviolet light or by charring after spraying with 15% H2SO4. Flash chromatography was performed on silica gel grade 60 (230-400 ASTM mesh). 1H NMR and 13C NMR spectra were recorded in δ units relative to deuterated solvents (CDCI3 δ = 7.26 ppm for 1H NMR and 77.00 ppm for 13C NMR); CD3OD δ = 4.78, 3.31 ppm for 1H NMR and 49.1 ppm for 13C NMR), which served as an internal reference, at 400 or 500 (for 1H NMR) and 100 MHz (for 13C
NMR), respectively. The purity of the products was >95% based on proton NMR spectra. High-resolution mass spectra (HRMS) were recorded on an Agilent Technologies
G6520A Q-TOF mass spectrometer using electrospray ionization (ESI). Optical rotations were recorded on a digital polarimeter at the sodium-D line at rt.
Preparation of Compounds 55-21, 55-22, and 77-13
Scheme 1 outlines the preparation of 1-deoxysphinganine analogues 55-21 and 55-22 via cyclic sulfate intermediates of (2S,3 ?)-2-azidosphinganine. Azidoester 1 was prepared by asymmetric dihydroxylation of ethyl hexadecenoate using AD-mix-β, followed by conversion to a cyclic sulfate intermediate and regioselective azidation with sodium azide in aqueous acetone. Reduction of ester 1 with sodium borohydride in THF/MeOH (100: 1) gave 2-azido-l,3-diol 2, which was converted to the cyclic sulfate intermediate 3 by reaction with SOCl2 in the presence of pyridine followed by oxidation of resulting cyclic sulfite with catalytic Ru04. Without further purification, 3 was subjected to reduction with sodium borohydride in DMF in the presence of sodium iodide, which removed the primary hydroxyl group, affording 55-21 in 79% yield. The reaction of 55-21 with formaldehyde in the presence of NaBH3CN in MeOH furnished the N,N-dimethylamino derivative 55-22 in 82% yield. N-Methylation of 55-22 with methyl tosylate in THF gave the N,N,N-trimethylammonium salt, 77-13.
f
77-13
Scheme 1. Synthesis of 1-deoxy sphingoid derivatives 55-21, 55-22, and 77-13 by removal of the primary hydroxyl group from sphinganine via cyclic sulfate chemistry. Reagents and conditions: (a) NaBH4, THF, MeOH, -78 °C - rt; (b) SOCl2, py, CH2C12, - 78 °C - rt; (c) cat. RuCl3-3H20, NaI04, MeCN/H20 (5: 1), rt, 2 h; (d) NaBH4 (2 equiv.), Nal (1 equiv.), DMF, 0 °C - rt, then aq. HC1 (79%); (e) CH20 (10 equiv.), NaBH3CN (11 equiv.), MeOH, 0 °C - rt (82%); (f) MeOTs-/?, THF, rt, overnight (100%).
(2S,3/?)-2-Azidooctadecane-l,3-diol (2). This compound was prepared by reduction of azidoester 1 with an excess of NaBH4 in THF/MeOH (100: 1); 1H NMR (CDC13) 6 0.88 (t, 7 = 6.6 Hz, 3H), 1.26 (m, 26H), 1.5-1.58 (m, 2H), 3.42 (q, 7 = 5.2 Hz, 1H), 3.90 (d, J = 1.2 Hz, 2H); 13C NMR (CDCI3) δ 14.1,22.7, 25.6, 29.3, 29.48, 29.52, 29.56, 29.62, 29.68, 31.9, 33.7, 62.5, 66.8, 72.1.
(25,3/?)-l-Deoxy-2-amino-3-octadecanol (55-21). To a solution of azide 2 (1.50 g, 4.58 mmol) in 50 mL of CH2C12 was added 430 μΐ. (5.92 mmol) of SOCl2, followed by 950 μ-h (11.7 mol) of pyridine at -78 °C. The reaction mixture was stirred at -78 °C for 2 h, then at rt for 2 h, and was filtered through a pad of silica gel, which was washed with hexane/EtOAc (10: 1). The filtrate was concentrated to give a cyclic sulfite intermediate. To a solution of the cyclic sulfite in 25 mL of MeCN were added 1.30 g (6.07 mmol) of crystalline NaI04 and 30 mg (0.14 mmol) of RuCl3- 3H20 in 5 mL of H20. The mixture was stirred at rt for 2 h, and then was diluted with 250 mL of Et20 and washed with H20. The ether layer was dried over Na2S04 and concentrated to give 2-azido-l,3-cyclic sulfate 3. To a solution of 3 in 25 mL of DMF were added 380 mg (10.0 mmol) of NaBH4 and 760 mg (5.07 mmol) of Nal at 0 °C. The mixture was stirred for 48 h at rt, and then was diluted with 200 mL of Et20, treated with 100 mL of 1 M of aqueous HC1 solution for 4 h, and neutralized with 5 M of aqueous NaOH solution. The organic layer was separated, dried over Na2S04, and concentrated. The product was purified by column chromatography on silica gel (eluting with CHCl3/MeOH/concd NH4OH 130:25:4) to afford 1.04 g (79%) of 55-21. The product was dissolved in a minimum volume of CHC13 and passed through a Cameo filter to remove dissolved silica gel: [OC]D +5.0 (c 0.40, MeOH); 1H NMR (CDCI3) δ 0.88 (t, 3H, 7 = 6.4 Hz), 1.01 (d, 2H, 7 = 6.4 Hz), 1.26 (m,
26H), 1.35 (m, 1H), 1.73 (br s, 3H), 2.98 (m, 1H), 3.44 (m, 1H); liC NMR (CDC13) δ 14.1, 22.7, 26.5, 29.34, 29.60, 29.61, 29.64, 29.69, 29.8, 31.9, 33.8, 63.4, 70.8.
(25,3/?)-2-N,N-Dimethylamino-3-octadecanol (55-22). To a mixture of 55-21 (457 mg, 1.60 mmol) and paraformaldehyde (500 mg, 16.6 mmol) in 50 mL of MeOH was added NaBH3CN (1.10 g, 17.5 mmol) at 0 °C. After the mixture was stirred at rt for 48 h, it was diluted with 200 mL of EtOAc and was washed with brine. The organic layer was dried over Na2S04 and concentrated. The product was purified by column chromatography on silica gel (eluting with hexane/ EtOAc 1 : 1) to afford 412 mg (82%) of compound 55-22: [cc]D +4.9 (c 0.35, MeOH); 1H NMR (CDC13) δ 0.88 (t, 3H, J = 6.8 Hz), 0.98 (d, 3H, J = 6.8 Hz), 1.26 (m, 26H), 1.53 (m, 1H), 2.21 (m, 2H), 2.30 (s, 6H), 3.72 (m, 1H); 13C NMR (CDC13) δ 9.6, 14.1, 22.7, 26.5, 29.35, 29.61, 29.62, 29.65, 29.68, 29.8, 31.9, 33.8, 42.8, 63.6, 70.8.
(25,3/f)-N,N,N-Trimethyl-3-hydroxy-2-octadecanaminium ?-toluenesulfonate (77-13). A mixture of 55-22 (81 mg, 0.26 mmol) and methyl /?-toluenesulfonate (63 mg, 0.34 mmol) in 5 mL of THF was stirred overnight at rt. After the N-methylation reaction was completed, the mixture was diluted with 5 mL of hexane. Filtration provided 125 mg (100%) of compound 77-13: 1H NMR (CDC13) δ 0.88 (t, 3H, = 6.8 Hz), 0.98 (d, 3H, = 6.8 Hz), 1.26 (m, 26H), 1.53 (m, 1H), 2.21 (m, 2H), 2.30 (s, 9H), 3.72 (m, 1H); 13C NMR (CDC13) δ 9.6, 14.1, 22.7, 26.5, 29.35, 29.61, 29.62, 29.65, 29.68, 29.8, 31.9, 33.8, 42.8, 63.6, 70.8; HRMS (M+) calcd for mlz C2iH48NO+ 328.3574, found 328.3579.
Preparation of 77-7. As shown in Scheme 2, the synthesis of oxyspisulosine analogue 77-7, which contains an oxygen atom in the aliphatic chain, started with rac-1- Otetradecylglycerol (4) using the same strategy as described in the preparation of 55-21. Oxidative cleavage of vicinal diol 4 with NaI04 afforded aldehyde 5. Because the EIZ stereoselectivity of the Horner- Wadsworth-Emmons (HWE) reaction in organic solvents is influenced by an oxygen-containing group adjacent to the aldehyde' s formyl group, (E)- , -unsaturated ester 6 was prepared by the HWE reaction of 5 with
(EtO)2P(0)CH2C02Et in aqueous 2-propanol the presence of K2C03, affording ester 6 with good E selectivity. This method has the advantage that the wet aldehyde 5 can be
subjected to the HWE reaction. Asymmetric dihydroxylation of ester 6 with AD-mix-β proceeded smoothly, affording the desired chiral 2,3-diol ester 7 in 89% yield.
Conversion of diol 7 to cyclic sulfate intermediate 8, followed by regioselective azidation gave azidoester 9, and reduction of the ester functionality in 9 with NaBH4 in
THF/MeOH (100: 1) gave 2-azidol.3-diol 10. An attempted removal of the primary hydroxyl group from 1,3-diol 10 by the same method as shown in Scheme 1 did not succeed; when the cyclic sulfate of 10 was reduced with NaBH4 a mixture of primary and secondary alcohols was formed that was difficult to purify. Therefore, a route was devised involving a dibutylstannane intermediate to synthesize 77-7 (Scheme 2).
Monotosylation by reaction of 10 with dibutyltin oxide followed by treatment of 11 with /?-tosyl chloride gave intermediate 12, which was converted to 77-6b in two steps and 66% overall yield from 10. In contrast to the reduction of 3, the azido group was not completely reduced even in DMF at elevated temperature. Therefore, catalytic hydrogenolysis was necessary to complete the reduction of azide 12.
6 (91 %) 7 (89%)
Scheme 2. Synthesis of 1-deoxy sphingoid derivative 77-7 by removal of the primary hydroxyl group from (2S,3 ?)-oxysphinganine 10 via dibutylstannane-mediated monotosylation of 1,3-diol followed by NaBH4 reduction. Reagents and conditions: (a) NaI04, THF/H2O, 0 °C - rt; (b) (EtO)2P(0)CH2C02Et, K2C03, 2-PrOH/H20 (1: 1) , 0 °C
- rt, overnight; (c) AD-mix β, MeS02NH2, i-BuOH/H20 (1: 1), rt; (d) SOCl2, py, CH2C12, 0 °C; (e) cat. RuCl3-3H20, NaI04, MeCN/H20 (5:2); (f) NaN3 (3 equiv), Me2CO/H20 (2: 1), then Et20, aq. H2S04; (g) NaBH4, THF/MeOH (100: 1), 0 °C - rt; (h) Bu2SnO, toluene, reflux; (i) /?-TsCl, CH2C12, 0 °C - rt; (j) NaBH4, THF, 0 °C - rt; (k) Pd(OH)2/C, MeOH, rt; (1) CH20, NaBH3CN (12.5 equiv), MeOH, 0 °C - rt (80%).
Ethyl 4-Tetradecyloxy-2(E)-butenonate (6). To a solution of NaI04 (4.12 g, 19.2 mmol) in 25 mL of water was added a solution of 1-Otetradecyl-rac-glycerol (4, 4.27 g, 14.8 mmol) in 25 mL of THF at 0 °C, followed by stirring at rt. After the oxidative glycol cleavage was completed (about 2 h at rt), the mixture was concentrated under reduced pressure in order to remove THF and the formaldehyde formed, providing aldehyde 5. To the residue of crude 5 was added a solution of triethyl phosphonoacetate (4.68 g, 20.9 mmol) in 50 mL of 2-propanol, followed by dropwise addition of a solution of K2C03 (26.0 g, 187 mmol) in 50 mL of water at 0 °C. The reaction mixture was gradually warmed to rt. After the mixture was stirred overnight at rt, the olefination product 6 was extracted with Et20 (3 x 50 mL). The combined organic layers were washed with brine, dried (Na2S04), and concentrated. The product was purified by flash column chromatography on silica gel (elution with hexane/EtOAc 25: 1) to give 4.40 g (91%) of compound 6: 1H NMR (CDC13) δ 0.88 (t, = 7.0 Hz, 3H), 1.25 (s, 22H), 1.31(t, J = 1.2 Hz, 3H), 1.57-1.63 (m, 2H), 3.46 (t, = 6.6 Hz, 2H), 4.13 (dd, = 2.0, 4.3 Hz, 2H), 4.21 (q, = 7.2 Hz, 2H), 6.08 (dt, = 2.0, 15.7 Hz, 1H), 6.97 (dt, = 4.3, 15.7 Hz, 1H); 13C NMR (CDC13) δ 14.1, 14.2, 22.7, 26.1, 29.3, 29.45, 29.56, 29.58, 29.63, 29.64, 29.66, 31.9, 60.3, 69.3, 71.2, 121.1, 144.7, 166.3; HRMS (M + H)+ calcd for mlz
C20H39O3 + 327.2894, found 327.2893.
Ethyl (2/?,3/?)-4-Tetradecyloxy-2,3-dihydroxybutanonate (7). After a solution of 14.0 g of AD-mix-β in 100 mL of i-BuOH/H20 (1: 1) was stirred vigorously at rt for 1 h, 950 mg (10.0 mmol) of MeS02NH2 was added, and stirring was continued for an additional 10 min. After 3.27 g (10.0 mmol) of compound 6 was added, the mixture was allowed to warm to rt. After the ,β-unsaturated ester was completely consumed (TLC), the reaction was quenched by the addition of sodium sulfite (1.5 g, 14.6 mmol). The
product was extracted with EtOAc. The combined extracts were dried (Na2S04) and concentrated. Chromatography on silica gel (elution with hexane/EtOAc 2: 1) gave 3.07 g (89%) of compound 7: [cc]D +7.8 (c 1.61, CHCl3/MeOH 1: 1); 1H NMR (CDC13) δ 0.87 (t, 7 = 6.6 Hz, 3H), 1.24 (s, 7 = 22H), 1.31 (t, 7 = 7.2 Hz, 3H), 1.54-1.57 (m, 2H), 2.61 (d, 7 = 7.4 Hz, 1H), 3.27 (d, 7 = 5.9 Hz, 1H), 3.47 (t, 7 = 6.7 Hz, 2H), 3.51-3.62 (m, 2H), 4.06- 4.13 (m, 1H), 4.23 (dd, 7 = 2.1, 5.8 Hz, 1H), 4.27 (q, 7 = 7.2 Hz, 2H); 13C NMR (CDC13) δ 14.08, 14.10, 22.7, 26.0, 29.3, 29.4, 29.53, 29.56, 29.58, 29.62, 29.63, 29.65, 31.9, 62.0, 70.8, 71.0, 71.2, 71.7, 173.1; HRMS (M + H)+ calcd for mlz C20H3iO5 + 361.2949, found 361.2952. Ethyl (25,3/f)-4-Tetradecyloxy-2-azido-3-hydroxybutanonate (9). To a solution of 2.89 g (8.01 mmol) of compound 7 in 50 mL of CH2C12 was added 1.3 mL (16.1 mmol) of pyridine at rt. After the mixture was stirred and chilled to 0 °C, 760 \L (10.4 mmol) of SOCl2 was added slowly. The reaction mixture was stirred for 30 min, and then was filtered through a pad of silica gel in a sintered glass funnel. The pad was washed with hexane/EtOAc 10: 1, and the filtrate was concentrated and further dried under high vacuum (~1 torr) for 2 h. To a solution of the crude cyclic sulfite in 20 mL of MeCN was added 2.57 g (12.0 mmol) of NaI04. The heterogeneous mixture was stirred vigorously while a solution of 2.8 mg (0.080 mmol) of RuCl3-3H20 in 8 mL of H20 was added. After the full consumption of the starting cyclic sulfite was observed (~1 h), the reaction mixture was diluted with 250 mL of Et20 and washed with brine. The organic phase was dried (Na2S04) and passed through a small pad of silica gel. Concentration of the filtrate gave crude cyclic sulfate 8, which was used without further purification. To a solution of 8 in 30 mL of acetone was added 1.56 g (24 mmol) of NaN3, followed by 15 mL of H20. The reaction mixture was stirred at rt until 8 was fully consumed. After acetone and water were removed, the residue was dissolved in 100 mL of Et20, and the solution was treated with 50 mL of 20% aqueous H2S04 in a fume hood with vigorous stirring of the heterogeneous mixture until the hydrolysis was completed. The layers were separated, and the aqueous layer was extracted with Et20 (2 x 50 mL). The combined organic layers were treated with anhydrous K2C03 (-100 mg) to remove the dissolved H2S04 and then were dried (Na2S04). After concentration, the product was purified by
chromatography on silica gel (elution with hexane/EtOAc 10: 1) to give 2.44 g (79%) of azide 9: [cc]D +40.9 (c 0.40, CHC13); 1H NMR (CDC13) δ 0.88 (t, J = 6.7 Hz, 3H), 1.26 (s, 22H), 1.33 (t, = 7.2 Hz, 3H), 1.54-1.57 (m, 2H), 2.77 (d, = 7.4 Hz, 1H), 3.41-3.51 (m, 2H), 3.52-3.61 (m, 2H), 4.03-4.13 (m, 2H), 4.28 (q, = 7.2 Hz, 2H); 13C NMR (CDC13) δ 14.08, 14.10, 22.7, 26.0, 29.3, 29.4, 29.56, 29.59, 29.63, 29.67, 31.9, 62.0, 63.2, 70.3, 70.8, 71.8, 168.7; HRMS (M + Na)+ calcd for mlz C2oH39N3Na04 + 408.2833, found 408.2837.
(2S,3/?)-4-Tetradecyloxy-2-azido-l,3-butanediol (10). To a solution of 2.43 g (6.30 mmol) of compound 9 in 100 mL of THF was added 380 mg (10.0 mmol) of NaBH4, followed by 1 mL of MeOH at 0 °C. The reaction mixture was stirred vigorously while the reaction mixture was allowed to warm to rt until the disappearance of 9
(monitored by TLC). After THF and water were removed, the residue was dissolved in 200 mL of EtOAc, and then was washed with brine and water. The organic layer was dried (Na2S04) and concentrated. The product was purified by column chromatography on silica gel (elution with hexane/EtOAc 10: 1, 8: 1, and 6: 1) to give 1.11 g (51%) of 10:
[cc]D +3.5 (c 0.40, CHC13); 1H NMR (CDC13) δ 0.87 (t, = 6.7 Hz, 3H), 1.26 (s, 22H), 1.53-1.63 (m, 2H), 2.51 (t, = 5.7 Hz, 1H), 2.78 (d, = 6.0 Hz, 1H), 3.40-3.65 (m, 5H), 3.77-3.87 (m, 2H), 3.88-3.97 (m, 1H); 13C NMR (CDC13) δ 14.1, 22.7, 26.0, 29.51, 29.56, 29.59, 29.63, 29.65, 29.67, 31.9, 62.7, 64.0, 70.7, 71.2, 71.8; HRMS (M + H)+ calcd for mlz C20H39O3 + 327.2894, found 327.2893.
(25,3/?)-4-Tetradecyloxy-2-amino-3-butanol (77-6b). A mixture of 10 (382 mg, 1.11 mmol) and n-Bu2SnO (280 mg, 1.12 mmol) in 25 mL of toluene was heated at reflux until a clear solution was formed. The solvent was removed under reduced pressure and the residue (11) was dried further under high vacuum for 2 h. After the dry residue was dissolved in 25 mL of CH2C12, 220 mg (1.15 mmol) of /?-toluenesulfonyl chloride was added at 0 °C. After the mixture was stirred overnight at rt, the reaction was quenched by addition of H20 (20 \L, 1.11 mmol). The mixture was filtered through a pad of Celite, which was washed with 100 mL of Et20. The filtrate was washed with brine, aqueous saturated NaHC03 solution, and water. The organic layer was dried (Na2S04) and
concentrated to give crude tosylate 12. To a solution of 12 in 25 niL of THF was added 100 mg (2.64 mmol) of NaBH4 at 0 °C. The mixture was stirred for 48 h at rt, diluted with 200 mL of Et20, and washed with brine. The ether layer was dried (Na2S04) and concentrated. To the residue was added Pearlman's catalyst (50 mg) in 25 mL of MeOH, and the mixture was stirred overnight at rt under a hydrogen atmosphere. After the catalyst was removed by filtration, the product was purified by column chromatography on silica gel (eluting with CHCl3/MeOH/conc. NH4OH 130:25:4) to afford 241 mg (66%) of 77-6b. The product was dissolved in a minimum volume of CHC13 and passed through a Cameo filter to remove dissolved silica gel: [cc]D +4.5 (c 0.40, MeOH); 1H NMR (CDC13) 6 0.88 (t, J = 6.4 Hz, 3H), 1.10 (d, J = 6.4 Hz, 2H), 1.26 (m, 26H), 1.57 (m, 2H), 2.45 (br s, 3H), 3.05 (m, 1H), 3.43-3.49 (m, 4H), 3.62 (m, 1H); 13C NMR (CDCI3) δ 14.1,
18.6, 22.7, 26.1, 29.33, 29.44, 29.56, 29.58, 31.9, 48.8, 71.7, 71.8, 73.6; HRMS (M + H)+ calcd for mlz Ci8H40NO2 + 302.3054, found 302.3057.
(25,3R)-4-Tetradecyloxy-2-(N^V-dimethylamino)-3-butanol (77-7). To a mixture of paraformaldehyde (50 mg, 1.6 mmol) and 77-6b (43 mg, 0.14 mmol) in 10 mL of MeOH was added NaBH3CN (110 mg, 1.75 mmol) at 0 °C. The mixture was stirred at rt for 48 h, and then was diluted with 200 mL of EtOAc and washed with brine. The organic layer was dried over Na2S04 and concentrated. The product was purified by column chromatography on silica gel (eluting with CHC13, CHCl3/MeOH 25: 1, and then CHCl3/MeOH/NH4OH 130:25:4) to afford 38 mg (80%) of product: 1H NMR (CDC13) δ 0.88 (t, 3H, J = 6.6 Hz), 1.02 (d, 3H, J = 6.7 Hz), 1.26 (m, 26H), 1.58 (q, J = 6.7 Hz, 2H), 2.27 (s, 6H), 2.49 (q, J = 6.6 Hz, 1H), 3.37 (dd, J = 7.9, 9.5 Hz, 1H), 3.46 (t, J = 6.6 Hz, 2H), 3.54 (dd, J = 3.6, 9.5 Hz, 1H), 3.87-3.82 (m, 1H); 13C NMR (CDCI3) δ 8.5, 14.1,
22.7, 26.1, 29.35, 29.46, 29.59, 29.64, 29.67, 31.9, 41.7, 61.2, 70.8, 71.5, 73.1 ; HRMS (M + H)+ calcd for mlz C20H44NO2 + 330.3367, found 330.3365.
General Procedure for Preparation of N-Arylthiourea and N-Arylurea Derivatives. To a solution of the amino- sphingoid base (1.02 mmol) in 30 mL of CHCl3/MeOH (1 : 1) was added the aryl isothiocyanate or aryl isocyanate (XC6H4NCS or XC6H4NCO, 1.00 mmol) at rt. The mixture was stirred overnight, and then was
concentrated under reduced pressure. The product was purified by column
chromatography on silica gel.
Preparation of 67-320. 67-330, 67-301, 67-310. 67-311. 67-306. F-02, F-01, and 67- 302
Scheme 3. Synthesis of thiourea derivatives 67-320, 67-330, 67-301, 67-310, 67-311, 67- 306, F-01, and F-02 by the reaction of sphingosine, D-ribo-phytosphingosine (PHS), or sphinganine with an aryl isothiocyanate or an aryl isocyanate.
Synthesis of 67-341.
67-341
Data for 67-341. Yield: 84%; 1H NMR (CDC13/CD30D) δ 0.88 (t, = 3.7 Hz, 3H), 1.26 (m, 24H), 1.41-1.49 (m, 2H), 3.62 (t, = 9.4 Hz, 1H), 3.73-3.78 (m, 1H), 4.00- 4.05 (m, 1H), 4.22-4.34 (m, 1H), 4.77 (br s, 1H), 7.33 (s, 1H); 13C NMR (CDCI3/CD3OD) δ 13.9, 22.5, 25.6, 29.2, 29.42, 29.46, 29.52, 29.55, 31.8, 33.3, 56.7, 70.0, 74.0, 82.5, 85.2, 114.1, 136.4, 138.9, 141.7, 142.9, 145.3, 183.4; HRMS (M + H)+ mlz calcd. for
525.2569, found 525.2570.
Synthesis of czs-Sphingosine (67-302).
67-304 67-302
Data for carbamate 67-304. 1H NMR (CDC13) δ 0.88 (t, = 6.6 Hz, 3H), 1.26 (m, 20H), 1.31-1.40 (m, 2H), 1.98-2.14 (m, 2H), 3.64 (d, = 4.7 Hz, 2H), 3.86 (dt, = 4.7, 8.1 Hz, 1H), 5.46 (t, = 8.6 Hz, 1H), 5.57-5.63 (m, 1H), 5.75-5.82 (m, 1H), 6.64 (s, 1H); 13C NMR (CDCI3) δ 14.1, 22.7, 27.9, 29.2, 29.32, 29.34, 29.42, 29.5, 29.64, 29.67, 31.9, 57.3, 61.6, 75.2, 122.0, 137.7, 160.6; HRMS (M + Na)+ mlz calcd. for Ci9H35NNa03+ 348.2509, found 348.2509.
Data for 67-302 (cw-Sphingosine). 1H NMR (CDCI3/CD3OD) δ 0.89 (t, = 7.0 Hz, 3H), 1.27 (m, 22H), 2.00-2.22 (m, 2H), 3.19-3.24 (m, 1H), 3.65-3.86 (m, 2H), 4.69-4.75 (m, 1H), 5.30-5.45 (m, 1H), 5.63-5.74 (m, 1H); 13C NMR (CDCI3/CD3OD) δ 13.5, 22.2, 27.4,
28.9, 29.08, 29.13, 29.18, 29.21, 29.24, 31.5, 67.1, 57.8, 64.5, 125.7, 135.2; HRMS (M + H)+ mlz calcd. for Ci8H38N02 + 300.2897, found 300.2899.
Preparation of Compounds RB-001 through RB-010, and of RB-019, RB-020, and
RB-011 through RB-018
General Procedure for Preparation of Tertiary Amine Analogues of FTY720.
The tertiary amines shown in Table 1 were prepared in good yields by displacement of mesylate ion from 4-(octylphenethyl) methanesulfonate (prepared as displayed in Scheme 4) with amines in acetonitrile. Some of the quaternary ammonium salts were prepared by N-alkylation of the tertiary amines (and the secondary amine RB- 006) with an excess of Mel and K2C03 in acetonitrile whereas others (RB-011, RB-012, RB-017, and RB-018) were prepared by N-alkylation with tertiary N-methylamines.
12 h, rt 3 h, rt t x , xl_ , reflux, 6 h
2-phenyethyl acetate 4-octanoylphenethyl acetate 4-octylphenethyl acetate
4-Octanoylphenethyl Acetate. To a suspension of A1C13 (1.2 g, 9.1 mmol) in 1,2- dichloroethane (DCE, 25 mL) was added dropwise caprylyl chloride (1.03 mL, 6.1 mmol). After the reaction mixture had stirred at rt for 1 h, a solution of phenethyl acetate (1.0 g, 6.1 mmol) in DCE (5 mL) was added. The mixture was stirred for 12 h at rt, poured into 1 N NaOH, and extracted with EtOAc. The extract was washed with brine, dried, and evaporated. Silica gel chromatography, eluting with hexanes/EtOAc (8: 1), gave 4-octanoylphenethyl acetate (1.06 g, 60%) as a yellow waxy solid; 1H NMR (400
MHz, CDCI3) δ 0.88 (t, = 6.9 Hz, 3H), 1.24-1.37 (m, 8H), 1.59-1.67 (m, 2H), 2.04 (s, 3H), 2.94 (td, = 7.4, 2.4 Hz, 2H), 3.00 (t, = 6.9 Hz, 2H), 4.27-4.32 (m, 2H), 7.30 (d, = 8.2 Hz, 2H), 7.91 (d, =8.3 Hz, 2H).
4-Octylphenethyl Acetate. To a solution of 4-octanoylphenethyl acetate (1.0 g, 3.4 mmol) in trifluoroacetic acid (10 mL) was added triethylsilane (1.1 mL, 6.9 mmol). The reaction mixture was stirred at rt for 3 h, concentrated, and diluted with EtOAc. The solution was washed with 1 N NaOH and brine, dried, and evaporated. Silica gel chromatography, eluting with hexanes/EtOAc (15: 1), gave the product (780 mg, 82%) as a yellow liquid; 1H NMR (400 MHz, CDC13) δ 0.88 (t, J = 6.8 Hz, 3H), 1.22-1.31 (m, 10H), 1.58-1.60 (m, 2H), 2.04 (s, 3H), 2.57 (td, = 7.6, 1.5 Hz, 2H), 2.88-2.93 (m, 2H), 4.24-4.29 (m, 2H), 7.11 (s, 4H).
4-Octylphenethyl Alcohol. To a solution of 4-octylphenethyl acetate (500 mg, 1.81 mmol) in MeOH (10 mL) was added sodium methoxide (195 mg, 3.61 mmol). The mixture was heated at reflux for 6 h, evaporated, and partitioned between EtOAc and water. The organic layer was separated and washed with brine. The solution was dried over Na2S04 and evaporated to provide 4-octylphenethyl alcohol (390 mg, 92%) as a yellow oil; 1H NMR (400 MHz, CDCI3) δ 0.88 (t, J = 6.8 Hz, 3H), 1.22-1.30 (m, 10H), 1.55-1.63 (m, 2H), 2.57 (t, J = 7.8 Hz, 2H), 2.83 (t, J = 6.6 Hz, 2H), 3.84 (t, J = 6.6 Hz, 2H), 7.11 (s, 4H). 4-Octylphenethyl Methanesulfonate (Scheme 4).
To a solution of 4-octylphenethyl alcohol (200 mg, 0.853 mmol) and
triethylamine (1.19 mL, 8.53 mmol) in CH2C12 (8 mL) at 0 °C was added
methanesulfonyl chloride (0.33 mL, 4.27 mmol). After being stirred at rt for 5 h, the reaction mixture was evaporated, diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. Purification by silica gel chromatography, eluting with hexanes/EtOAc (5: 1), gave 253 mg (95%) of the
mesylate as a yellow liquid; 1H NMR (400 MHz, CDC13) δ 0.87 (t, J = 6.9 Hz, 3H), 1.26- 1.30 (m, 10H), 1.58 (m, 2H), 2.57 (t, = 7.7 Hz, 2H), 2.83 (s, 3H), 3.02 (t, = 7.0 Hz, 2H), 4.40 (t, = 7.0 Hz, 2H), 7.14 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3 (2C), 29.5, 31.6, 31.9, 35.3, 35.6, 37.3, 70.6, 128.8, 128.9, 133.4, 141.9; ESI-HRMS (M + Na)+ mlz calcd for Ci7H28Na03S 335.1657, found 335.1655.
Preparation of l-(4-Octylphenethyl)pyrrolidine (RB-001)
To a solution of 4-octylphenethyl methanesulfonate (15 mg, 0.040 mmol) in 3 mL of acetonitrile was added pyrrolidine (80 L, 0.90 mmol). The reaction mixture was stirred at 50 °C for 24 h and concentrated. Purification by silica gel chromatography, eluting with CH2Ci2/MeOH (5: 1), gave 11 mg (80%) of RB-001 as a slightly yellow waxy solid; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.8 Hz, 3H), 1.20-1.29 (m, 10H), 1.56-1.60 (m, 2H), 2.03-2.06 (m, 4H), 2.13-2.19 (m, 2H), 2.56 (t, = 7.8 Hz, 2H), 3.03- 3.10 (m, 6H), 7.10-7.15 (m, 4H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.7, 23.4, 29.3, 29.5, 31.0, 31.5, 31.9, 32.7, 35.6, 53.4, 53.9, 57.4, 128.5, 128.8, 134.3, 141.7; ESI-HRMS (M + H)+ mlz calcd for C20H34N 288.2691, found 288.2689.
Preparation of l-(4-Octylphenethyl)piperidine (RB-002)
To a solution of 4-octylphenethyl methanesulfonate (100 mg, 0.32 mmol) in 5 mL of acetonitrile was added 4-hydroxypiperidine (162 mg, 1.60 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH (5: 1), gave 87 mg (86%) of RB-002 as a colorless oil; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.8 Hz, 3H), 1.23-1.29 (m, 10H), 1.54-1.60 (m, 4H), 1.77-1.80 (m, 4H), 2.56 (t, = 7.8 Hz, 2H), 2.71-2.78 (m, 6H), 2.91- 2.95 (m, 2H), 7.09-7.14 (m, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 23.6, 25.8,
29.3, 29.4, 29.5, 31.6, 31.9, 35.6, 54.1, 60.5, 128.6, 128.7, 136.1, 141.0; ESI-HRMS (M + H)+ mlz calcd for C2iH36N 302.2847, found 302.2842.
Preparation of l-(4-Octylphenethyl)azepane (RB-003)
The azepane derivative RB-003 was prepared from 4-octylphenethyl
methanesulfonate according to a coupling procedure similar to that described for compound RB-001, using hexamethyleneimine. Yield = 76%; 1H NMR (400 MHz, CDCl3) 5 0.88 (t, = 6.8 Hz, 3H), 1.22-1.32 (m, 10H), 1.54-1.61 (m, 2H), 1.69-1.84 (m, 4H), 1.92-2.13 (m, 4H), 2.56 (t, J = 7.7 Hz, 2H), 3.14-3.24 (m, 6H), 7.11-7.15 (m, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 23.5, 26.9, 29.3, 29.4, 29.5, 30.3, 31.5, 31.9, 35.5, 54.5, 58.8, 128.6, 129.0, 133.3, 142.1; ESI-HRMS (M + H)+ mlz calcd for C22H38N 316.3004, found 316.3002. Preparation of 4-Methyl-l-(4-octylphenethyl)piperidine (RB-004)
Compound RB-004 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-001, using
4-methylpiperidine. Yield = 73%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.8 Hz, 3H), 0.99 (d, = 6.2 Hz, 3H), 1.25-1.32 (m, 10H), 1.52-1.66 (m, 5H), 1.74-1.77 (m, 2H), 2.31-2.36 (m, 2H), 2.56 (t, = 7.8 Hz, 2H), 2.81-2.85 (m, 2H), 2.98-3.02 (m, 2H), 3.25- 3.28 (m, 2H), 7.11 (dd, = 8.2, 10.6 Hz, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 19.8, 21.3, 22.7, 25.5, 29.3, 29.4, 29.5, 31.5, 32.5, 35.6, 53.4, 128.6, 128.7, 132.7, 141.3; ESI- HRMS (M + H)+ mlz calcd for C22H38N 316.3004, found 316.3003.
Preparation of l-(4-Octylphenethyl)piperidin-4-ol (RB-005)
To a solution of 4-octylphenethyl methanesulfonate (30 mg, 0.096 mmol) in 5 mL of acetonitrile was added 4-hydroxypiperidine (49 mg, 0.48 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH (5: 1), gave 27 mg (90%) of RB-005 as a colorless oil; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.8 Hz, 3H), 1.22-1.30 (m, 10H), 1.55-1.68 (m, 6H), 1.94-1.97 (m, 2H), 2.24 (t, = 9.1 Hz, 2H), 2.54-2.61 (m, 4H), 2.76-2.80 (m, 2H), 2.84-2.89 (m, 2H), 3.71-3.75 (m, 1H), 7.08-7.12 (m, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 33.4, 34.4, 35.6, 51.0, 60.6, 128.4, 128.6, 137.4, 140.7; ESI-HRMS (M + H)+ mlz calcd for C2iH36NO 318.2797, found 318.2793.
Preparation of 2-(4-Octylphenyl)-N-((tetrahvdrofuran-2-yl)methyl)ethanamine (RB- 006)
The secondary amine derivative RB-006 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-001, using tetrahydrofurfurylamine. Yield = 87%; 1H NMR (400 MHz, CDC13) 5 0.89 (t, = 6.7 Hz, 3H), 1.26-1.39 (m, 10H), 1.49-1.60 (m, 4H), 1.84-1.91 (m, 2H), 2.56 (t, = 7.7 Hz, 2H), 2.68-2.78 (m, 2H), 2.81-2.90 (m, 2H), 2.94-2.97 (m, 2H), 3.73 (dd, / = 7.0, 14.0 Hz, 1H), 3.83 (dd, = 7.0, 14.4 Hz, 1H), 4.02-4.06 (m, 1H), 7.11 (dd, / = 8.3, 10.7 Hz, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 25.7, 29.3, 29.4, 29.5, 29.7, 31.6, 31.9, 35.4, 35.6, 51.3, 53.9, 128.5, 128.6, 136.6, 140.9; ESI-HRMS (M + H)+ mlz calcd for C2iH36NO 318.2797, found 318.2795.
reparation of l-(4-Octylphenethyl)piperazine (RB-007)
Compound RB-007 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-001, piperazine. Yield = 72%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.8 Hz, 3H), 1.23- 1.32 (m, 10H), 1.55-1.60 (m, 2H), 2.49-2.64 (m, 8H), 2.75-2.79 (m, 2H), 3.01 (t, J = 4.6 Hz, 4H), 7.07-7.12 (m, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 32.9, 35.6, 45.2, 53.1, 60.9, 128.4, 128.5, 137.1, 140.8; ESI-HRMS (M + H)+ mlz calcd for C2oH35N2 303.2800, found 303.2797.
Preparation of 2-(4-(4-Octylphenethyl)piperazin-l-yl)ethanol (RB-008)
Compound RB-008 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-001, using l-(2-hydroxyethyl)piperazine. Yield = 63%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.9 Hz, 3H), 1.26-1.30 (m, 10H), 1.55-1.62 (m, 2H), 2.49-2.62 (m, 14H), 2.76-2.80 (m, 2H), 3.63 (t, J = 5.4 Hz, 2H), 7.08-7.13 (m, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 33.2, 35.6, 52.8, 53.2, 57.7, 59.2, 60.6, 128.5, 128.6, 137.2, 140.8; ESI-HRMS (M + H)+ mlz calcd for C22H39N20 347.3062, found 347.3061. Preparation of 3-(4-(4-Octylphenethyl)piperazin-l-yl)propan-l-ol (RB-009)
Compound RB-009 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-001, l-(3-hydroxypropyl)piperazine. Yield = 75%; 1H NMR (400 MHz, CDC13) δ (t, J = t Hz, 3H), 1.25-1.32 (m, 10H), 1.54-1.62 (m, 2H), 1.75 (q, J = 5.5 Hz, 2H), 2.56 (t,
Hz, 4H), 2.59-2.64 (m, 4H), 2.69 (t, J = 5.7 Hz, 4H), 2.75-2.79 (m, 4H), 3.81 (t, J = 5.2 Hz, 2H), 7.09 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 26.9, 29.3, 29.4, 29.5, 29.7, 31.6, 31.9, 33.0, 35.6, 52.8, 53.1, 58.6, 60.3, 128.4, 128.5, 137.0, 140.8; ESI-HRMS (M + H)+ mlz calcd for C23H4iN20 361.3219, found 361.3217.
Preparation of l'-(4-Octylphenethyl)-l<4'-bipiperidine (RB-010)
Compound RB-010 was prepared from 4-octylphenethyl methanesulionate according to a coupling procedure similar to that described for compound RB-001, using l,4'-bipiperidine. Yield = 70%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.8 Hz, 3H), 1.26-1.29 (m, 10H), 1.42-1.45 (m, 2H), 1.54-1.70 (m, 8H), 1.81 (d, = 12.3 Hz, 2H), 1.99 (dt, = 11.7, 1.6 Hz, 2H), 2.30-2.36 (m, 1H), 2.53-2.58 (m, 8H), 2.74-2.79 (m, 2H), 3.07 (d, = 11.6 Hz, 2H), 7.07-7.12 (m, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 24.8, 26.3, 27.6, 29.3, 29.4, 29.5, 31.6, 31.9, 33.5, 35.6, 50.1, 53.6, 60.8, 62.9, 128.4, 128.6, 137.5, 140.6; ESI-HRMS (M + H)+ mlz calcd for C26H45N2 385.3582, found 385.3577.
Pre aration of l-Methyl-l-(4-octylphenethyl)pyrrolidinium Methanesulfonate (RB-
To a solution of 4-octylphenethyl methanesulfonate (10 mg, 0.032 mmol) in 3 mL of acetonitrile was added 1-methylpyrrolidine (34.1 μί, 0.32 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. The residue was washed with hexane to give 12 mg (92%) of RB-011 as a yellow liquid; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.8 Hz, 3H), 1.26-1.30 (m, 10H), 1.54-1.58 (m, 2H), 2.22 (m, 4H), 2.55 (t, = 7.8Hz, 2H), 2.75 (s, 3H), 3.01 (t, = 8.2 Hz, 2H), 3.25 (s, 3H), 3.70-3.77 (m, 6H),
7.13 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H); liC NMR (100 MHz, CDC13) δ 14.1, 21.5, 22.7, 29.3 (2C), 29.5, 30.1, 31.5, 31.9, 35.5, 48.2, 64.4, 64.6, 128.9, 129.1, 132.2, 142.3; ESI-HRMS (M)+ mlz calcd for C2iH37N+ 303.2926, found 303.2875. Preparation of l-Methyl-l-(4-octylphenethyl)piperidinium Methanesulfonate (RB-
012)
To a solution of 4-octylphenethyl methanesulfonate (10 mg, 0.032 mmol) in 3 niL of acetonitrile was added 1-methylpiperidine (38.9 μί, 0.32 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. The residue was washed with hexane to give 12 mg (90%) of RB-012 as a yellow liquid; 1H NMR (400 MHz, CDC13) δ 0.88 (t, J = 6.8 Hz, 3H), 1.26-1.29 (m, 10H), 1.57 (t, 7 = 7.3 Hz, 2H), 1.72-1.88 (m, 6H), 2.55 (t, = 7.7 Hz, 2H), 2.75 (s, 3H), 3.00-3.05 (m, 2H), 3.30 (s, 3H), 3.52-3.56 (m, 2H), 3.64- 3.70 (m, 4H), 7.15 (d, = 8.0 Hz, 2H), 7.12 (d, = 8.0 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 20.2, 20.7, 22.7, 28.2, 29.3 (2C), 29.5, 31.5, 31.9, 35.5, 39.7, 48.5, 61.0, 128.9, 129.1, 132.1, 142.3; ESI-HRMS (M)+ mlz calcd for C22H39N+ 317.3082, found 317.3032.
Scheme 5. Synthesis of compounds RB-013-016.
Preparation of l-Methyl-l-( -octylphenethyl)azepanium Iodide (RB-013)
To a solution of RB-003 (10 mg, 0.032 mmol) in MeCN (3 mL) was added K2CO3 (22 mg, 0.16 mmol) at rt. After the suspension was stirred for 10 min, Mel (10 μί, 0.16 mmol) was added. The reaction mixture was stirred overnight at rt. The reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. The residue was washed with hexane to give 12 mg (84%) of RB-013 as a colorless oil; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.8 Hz, 3H), 1.24-1.32 (m, 10H), 1.54-1.57 (m, 2H), 1.72-1.77 (m, 4H), 1.91-1.96 (m, 4H), 2.54 (t, J = 7.7 Hz, 2H), 3.11-3.15 (m, 2H), 3.42 (s, 3H), 3.71-3.73 (m, 4H), 3.77- 3.81 (m, 2H), 7.12 (d, = 8.0 Hz, 2H), 7.30 (d, = 8.0 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 14.2, 22.1, 22.7, 27.3, 29.0, 29.3, 29.5, 29.7, 31.5, 31.9, 35.6, 51.5, 65.1, 65.6, 129.1, 129.2, 131.9, 142.3; ESI-HRMS (M+) mlz calcd for C23H40N+ 330.3161, found 331.3153. Preparation of l,4-Dimethyl- -(4-octylphenethyl)piperidinium Iodide (RB-014)
RB-014
Compound RB-014 was prepared from RB-004 according to a coupling procedure similar to that described for compound RB-013. Yield = 90%; 1H NMR (400 MHz, CDCI3) δ 0.88 (t, = 6.8 Hz, 3H), 1.02 (d, = 6.5 Hz, 3H), 1.22-1.35 (m, 10H), 1.51-1.64 (m, 5H), 1.80-1.84 (m, 2H), 2.53 (t, J = 7.8 Hz, 2H), 3.08-3.12 (m, 2H), 3.26 (s, 3H), 3.60-3.82 (m, 4H), 4.04-4.10 (m, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.2, 20.9, 22.7, 28.1, 29.3, 29.4, 29.7, 31.5, 31.9, 35.6, 44.9, 61.0, 67.9, 129.1, 129.4, 131.4, 142.2; ESI-HRMS (M+) mlz calcd for C23H40N+ 330.3161, found 330.3156.
Preparation of 4-Hvdroxy-l-methyl-l-(4-octylphenethyl)piperidinium Iodide (RB- 015)
Compound RB-015 was prepared from RB-005 according to a coupling procedure similar to that described for compound RB-013. Yield = 84%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, J = 6.7 Hz, 3H), 1.25-1.28 (m, 10H), 1.50-1.57 (m, 2H), 2.02- 2.11 (m, 3H), 2.23-2.28 (m, 2H), 2.51 (t, J = 6.9 Hz, 2H), 3.04-3.11 (m, 2H), 3.37 (d, / = 9.2 Hz, 3H), 3.62-3.76 (m, 6H), 4.18-4.24 (m, IH), 7.13 (dd, J = 2.1, 7.2 Hz, 2H), 7.29 (dd, J = 2.2, 6.5 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 27.8, 28.2, 28.4,
28.7, 29.3, 29.4, 29.5, 31.6, 31.9, 35.6, 57.6, 58.4, 129.1, 129.2, 131.9, 142.3; ESI-HRMS (M+) mlz calcd for C22H38NO+ 332.2953, found 332.2946.
Preparation of N V-Dimethyl-2-(4-octylphenyl)-N-((tetrahvdrofuran-2- yl)methyl)ethanaminium Io -016)
RB-016
Compound RB-016 was prepared from RB-006 according to a coupling procedure similar to that described for compound RB-013. Yield = 88%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, J = 6.7 Hz, 3H), 1.26-1.29 (m, 10H), 1.55-1.67 (m, 6H), 1.89- 1.99 (m, 2H), 2.25-2.54 (m, 2H), 2.56 (t, J = 7.7 Hz, 2H), 3.03-3.13 (m, 2H), 3.47 (s,
3H), 3.48 (s, 3H), 3.81-3.88 (m, IH), 3.93-3.99 (m, IH), 4.34-4.39 (m, IH), 7.13 (d, J = 8.0 Hz, 2H), 7.22 (d, / = 8.0 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 24.9, 29.2, 29.3, 29.5, 30.4, 31.5, 31.9, 35.6, 52.4, 60.2, 69.3, 72.9, 129.0, 129.2, 131.6, 142.4; ESI-HRMS (M+) mlz calcd for C2 H40NO+ 346.3110, found 346.3102.
Preparation of N V-Dimethyl-N-(4-octylphenethyl)cvclohexanaminium Mesylate (RB-017)
RB-017
MsO"
Compound RB-017 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-011, using N,N-dimethylcyclohexylamine. Yield = 76%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, J = 6.7 Hz, 3H), 1.26-1.29 (m, 10H), 1.35-1.45 (m, 5H), 1.51-1.58 (m, 2H), 1.96 (d, J = 12.1 Hz, 2H), 2.20 (d, J = 11.6 Hz, 2H), 7.55 (t, J = 7.7 Hz, 2H), 2.73 (s, 3H), 3.04-3.08 (m, 2H), 3.25 (s, 6H), 3.48 (m, 1H), 3.58-3.62 (m, 2H), 7.12 (d, J = 7.9, 2H), 7.22 (d, = 7.9, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 24.7, 24.8, 24.9, 25.3, 26.2, 26.6, 28.6, 29.3, 29.5, 31.5, 31.9, 35.3, 39.7, 48.5, 63.1, 72.2, 128.9, 129.1, 132.4, 142.2; ESI- HRMS (M+) mlz calcd for C24H42N+ 344.3317, found 344.3313.
Preparation of N V-Dimethyl-N-(4-octylphenethyl)butan-l-aminium Mesylate (RB- 018)
Compound RB-018 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-011, using N-n-butyldimethylamine. Yield = 65%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.7 Hz, 3H), 0.97 (t, = 7.3 Hz, 3H), 1.21-1.29 (m, 10H), 1.27-1.40 (m, 2H), 1.54-1.58 (m, 2H), 1.59-1.68 (m, 2H), 2.54 (t, = 7.7 Hz, 2H), 2.72 (s, 3H), 3.01-3.05 (m, 2H), 3.30 (m, 3H), 3.45-3.49 (m, 2H), 3.58-3.62 (m, 2H), 7.12 (d, = 7.9Hz, 2H), 7.21 (d, = 8.0 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 14.1, 19.6, 22.6, 24.6, 28.8, 29.2, 29.3, 29.4, 31.5, 31.9, 35.5, 39.7, 51.0, 63.7, 64.2, 128.9, 129.1, 132.2, 142.2; ESI-HRMS (M+) mlz calcd for C22H40N+ 318.3161, found 318.3156.
reparation of l-(4-Octylphenethyl)piperidin-3-ol (RB-019)
Compound RB-019 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-005,
3-hydroxypiperidine. Yield = 82%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.9 Hz, 3H), 1.20-1.30 (m, 10H), 1.57-1.60 (m, 5H), 1.86-1.89 (m, 1H), 2.39-2.43 (m, 1H), 2.54-2.67 (m, 7H), 2.78-2.81 (m, 2H), 7.10 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 21.4, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 32.7, 35.6, 53.6, 60.1, 60.3, 66.0, 128.5, 128.6, 136.9, 140.8; ESI-HRMS (M + H)+ mlz calcd for C2iH36NO 318.2797, found 318.2792.
Preparation of (l-(4-Octylphenethyl)piperidin-4-yl)methanol (RB-020)
Compound RB-020 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for compound RB-005,
4-piperidine methanol. Yield = 87%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.8 Hz, 3H), 1.21-1.32 (m, 10H), 1.54-1.61 (m, 2H), 1.69-1.75 (m, 3H), 1.89-1.92 (m, 2H), 2.39-2.48 (m, 2H), 2.56 (t, = 7.8 Hz, 2H), 2.88-2.92 (m, 2H), 3.01-3.05 (m, 2H), 3.36 (d, = 11.0 Hz, 2H), 3.56 (d, = 5.4 Hz, 2H), 7.10-7.14 (m, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 27.0, 29.3, 29.4, 29.5, 31.3, 31.5, 31.9, 35.6, 37.4, 53.1, 59.7, 66.7, 128.6, 128.8, 135.0, 141.5; ESI-HRMS (M + H)+ mlz calcd for C22H38NO 332.2953, found 332.2948.
CH2CI2 RB-021 (R : OH), 78%
RB-022 (R : H), 83%
Scheme 6. Synthesis of RB-021 and RB-022. (Hex-5-ynylsulfonyl)benzene. To a solution of 6-chloro-l-hexyne (100 mg, 0.86 mmol) in 12 mL of THF/DMF (2: 1) was added benzenesulfinic acid sodium salt (422 mg, 2.6 mmol) in a sealed tube. The reaction mixture was stirred at 80 °C for 3 d. The reaction mixture was then diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. Purification by silica gel chromatography, elution with hexane/EtOAc (3: 1), afforded 17 mg (56%) of hex-5- ynylsulfonyl)benzene as a colorless oil; 1H NMR (400 MHz, CDCI3) δ 1.61 (quin, J = 7.3 Hz, 2H), 1.81-1.89 (m, 2H), 1.95 (t, J = 2.6 Hz, 1H), 2.19 (dt, J = 6.9, 2.6 Hz, 2H), 3.11— 3.15 (m, 2H), 7.56-7.60 (m, 2H), 7.65-7 '.69 (m, 1H), 7.90-7.92 (m, 2H); 13C NMR (100 MHz, CDCI3) δ 17.9, 21.8, 26.8, 55.6, 69.3, 83.0, 128.0, 129.3, 133.7, 139.0, 128.0, 129.3, 133.7, 139.0; ESI-HRMS (M + Na)+ mlz calcd for Ci2Hi4Na02S 245.0612, found 245.0612.
2-(4-(6-(Phenylsulfonyl)hex-l-ynyl)phenyl)ethanol. To a deaerated solution of 2-(4- bromophenyl)ethanol (100 mg, 0.50 mmol), bis(triphenylphosphine)palladium dichloride (29 mg, 0.025 mmol), and copper(I) iodide (4.8 mg, 0.025 mmol) in anhydrous triethylamine (TEA, 10 mL) was added (hex-5-ynylsulfonyl)benzene (221 mg, 0.99 mmol) at rt. The reaction mixture was heated at 50 °C for 8 h. After saturated aqueous ammonium chloride solution was added, the mixture was extracted with EtOAc. The combined solution was washed with water, brine, and dried. Flash column
chromatography with hexanes/EtOAc (1:2) as the eluent gave the product (114 mg, 67%) as a yellow oil; 1H NMR (400 MHz, CDC13) δ 1.63 (br s, 1H), 1.68 (quin, / = 7.3 Hz, 2H), 1.87-1.95 (m, 2H), 2.41 (t, = 6.9 Hz, 2H), 2.85 (t, = 6.6 Hz, 2H), 3.14-3.18 (m, 2H), 3.84 (dd, = 6.1, 10.9 Hz, 2H), 7.14 (d, = 8.2 Hz, 2H), 7.26 (d, = 8.1 Hz, 2H), 7.53-7.57 (m, 2H), 7.63-7.67 (m, 1H), 7.91-7.93 (m, 2H); 13C NMR (100 MHz, CDC13) δ 19.0, 22.0, 27.1, 39.0, 55.9, 63.5, 81.4, 88.4, 121.7, 128.1, 129.0, 129.3, 131.7, 133.8, 138.4, 139.1; ESI-HRMS (M + Na)+ mlz calcd for C2oH22Na03S 365.1187, found
365.1183. Preparation of 2-(4-(6-(Phenylsulfonyl)hexyl)phenyl)ethanol
2-(4-(6-(Phenylsulfonyl)hex-l-ynyl)phenyl)ethanol (40 mg, 0.12 mmol) was dissolved in EtOAc (5 mL), and 10% Pd/C (40 mg, 100 wt %) was added. The reaction mixture was hydrogenated at rt for 12 h. The catalyst was removed by filtration through a pad of Celite and rinsed with EtOAc. The product was obtained without purification as a colorless oil; 1H NMR (400 MHz, CDC13) δ 1.22-1.41 (m, 4H), 1.56 (quin, / = 7.6 Hz, 2H), 1.65 (br s, 1H), 1.66-1.74 (m, 2H), 2.54 (t, = 7.6 Hz, 2H), 2.83 (t, = 6.6 Hz, 2H), 3.05-3.09 (m, 2H), 3.84 (t, = 6.6 Hz, 2H), 7.08 (d, = 8.0 Hz, 2H), 7.13 (d, = 8.0 Hz, 2H), 7.55-7.59 (m, 2H), 7.64-7.68 (m, 1H), 7.89-7.91 (m, 2H); 13C NMR (100 MHz, CDC13) δ 22.6, 28.1, 28.6, 31.0, 35.3, 38.8, 56.2, 63.7, 128.0, 128.6, 129.0, 129.6, 133.7, 135.8, 139.1, 140.5; ESI-HRMS (M + Na)+ mlz calcd for C20H26NaO3S 369.1500, found 369.1499.
Preparation of 4-(6-(Phenylsulfonyl)hexyl)phenethyl Methanesulfonate
The mesylate was prepared from the alcohol in 92% yield; 1H NMR (400 MHz, CDC13) δ 1.24-1.42 (m, 4H), 1.55 (quin, = 7.6 Hz, 2H), 1.67-1.74 (m, 2H), 2.55 (t, = 7.6 Hz, 2H), 2.85 (s, 3H), 3.02 (t, = 7.0 Hz, 2H), 3.05-3.09 (m, 2H), 4.40 (t, = 7.0 Hz, 2H), 7.09 (d, = 8.2 Hz, 2H), 7.13 (d, = 8.1 Hz, 2H), 7.53-7.59 (m, 2H), 7.64-7.68 (m, 1H), 7.89-7.91 (m, 2H); 13C NMR (100 MHz, CDC13) δ 22.6, 28.1, 28.6, 31.0, 35.3, 37.3,
56.2, 70.5, 128.0, 129.0, 129.3, 133.6, 133.7, 139.2, 141.2; ESI-HRMS (M + Na)+ mlz calcd for CiiHisNaOsSi 447.1276, found 447.1275.
Preparation of l-(4-( -(Phenylsulfonyl)hexyl)phenethyl)piperidin-4-ol (RB-021)
RB-021 was prepared according to a coupling procedure similar to that described for RB- 005. Yield = 78%; 1H NMR (400 MHz, CDC13) δ 1.26-1.32 (m, 4H), 1.38 (quin, J = 7.4 Hz, 2H), 1.55 (quin, J = 7.5 Hz, 2H), 1.66-1.78 (m, 5H), 2.08-2.18 (m, 2H), 2.53 (t, J = 7.6 Hz, 2H), 2.78-2.83 (m, 2H), 2.92-2.94 (m, 2H), 2.98-3.08 (m, 4H), 3.96-3.98 (m, 1H), 7.06 (d, = 8.0 Hz, 2H), 7.11 (d, = 8.0 Hz, 2H), 7.55-7.59 (m, 2H), 7.64-7.68 (m, 1H), 7.89-7.90 (m, 2H); 13C NMR (100 MHz, CDCI3) δ 22.6, 28.1, 28.5, 31.0, 33.2, 34.4, 35.3, 50.4, 51.1, 56.2, 60.1, 67.7, 128.1, 128.6, 129.3, 133.7, 139.1, 140.6; ESI-HRMS (M + H)+ mlz calcd for C25H36NO3S 430.2416, found 430.2413. Preparation of l-(4-( -(Phenylsulfonyl)hexyl)phenethyl)piperidine (RB-022)
RB-022 was prepared according to a coupling procedure similar to that described for compound RB-002. Yield = 83%; 1H NMR (400 MHz, CDCI3) δ 1.25-1.42 (m, 6H), 1.51-1.59 (m, 4H), 1.66-1.74 (m, 4H), 1.89-1.93 (m, 4H), 2.53 (t, = 7.6 Hz, 2H), 2.95- 3.08 (m, 6H), 7.07 (d, = 8.0 Hz, 2H), 7.12 (d, = 8.0 Hz, 2H), 7.56-7.59 (m, 2H), 7.64- 7.68 (m, 1H), 7.89-7.91 (m, 2H); 13C NMR (100 MHz, CDC13) δ 22.6, 23.0, 24.0, 28.2, 28.6, 29.7, 31.0, 35.3, 39.5, 53.9, 54.4, 56.3, 59.4, 128.1, 128.4, 129.3, 133.7, 193.2; ESI- HRMS (M + H)+ mlz calcd for C25H36NO2S 414.2466, found 414.2468.
Preparation of l-(4-Octylbenzyl)piperidin-4-ol (RB-023)
RB-023
4-(Octylphenyl)methanol was prepared in two steps from 4-iodobenzyl alcohol; first, a Sonogashira reaction with 1-octyne afforded (4-(oct-l-ynyl)phenylmethanol as a yellow oil, and then catalytic hydrogenation of the triple bond provided 4-(octylphenyl)methanol as a colorless oil. To a solution of 4-(octylphenyl)methanol (37 mg, 0.17 mmol) and triethylamine (0.23 mL, 1.7 mmol) in CH2CI2 (5 mL) at 0 °C was added methanesulfonyl chloride (40 μί, 0.50 mmol). After being stirred at rt for 3 h, the reaction mixture was evaporated, diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. To a solution of the reaction mixture (0.17 mmol) in MeCN (3 mL) was added 4-hydroxypiperidine (86 mg, 0.85 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH (5: 1), gave 35 mg (69%, 2 steps) of RB- 023 as a colorless oil; 1H NMR (400 MHz, CDCI3) δ 0.88 (t, = 6.8 Hz, 3H), 1.23-1.30 (m, 10H), 1.56-1.73 (m, 4H), 1.97-2.06 (m, 2H), 2.35-2.47 (m, 2H), 2.59 (t, = 7.7 Hz, 2H), 2.85-2.90 (m, 2H), 3.64 (s, 2H), 3.78-3.81 (m, 1H), 7.15 (d, = 7.9 Hz, 2H), 7.27 (d, = 8.0 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.5, 31.9, 33.2, 35.7, 50.1, 62.2, 128.5, 129.7, 137.2, 142.8; ESI-HRMS (M + H)+ mlz calcd for C20H34NO 304.2640, found 304.2637.
Preparation of l-(3-(4-Octylphenyl)propyl)piperidin-4-ol (RB-024)
RB-024
This product was prepared from l-bromo-4-n-octylbenzene in three steps. First, 3-(4- octylphenyl)prop-2-yn-l-ol was prepared from l-bromo-4-n-octylbenzene and propargyl alcohol by a Sonogashira reaction procedure; yield = 82%; 1H NMR (400 MHz, CDC13) δ 0.86 (t, = 6.8 Hz, 3H), 1.22-1.30 (m, 10H), 1.57-1.60 (m, 2H), 2.59 (t, = 7.7 Hz, 2H), 4.49 (s, 2H), 7.11 (d, = 7.8 Hz, 2H), 7.34 (d, = 7.7 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.7, 29.2, 29.4, 31.2, 31.9, 35.9, 51.7, 85.9, 86.5, 119.6, 128.4, 131.6, 143.7; ESI-HRMS (M + H)+ mlz calcd for Ci7H250 245.1905, found 245.1903. Then, catalytic hydrogenation provided 3-(4-octylphenyl)propan-l-ol ; 1H NMR (400 MHz, CDC13) 6 0.88 (t, = 6.8 Hz, 3H), 1.24-1.30 (m, 10H), 1.59 (quin, / = 7.4 Hz, 2H), 1.88 (quin, = 6.5 Hz, 2H), 2.56 (t, = 7.7 Hz, 2H), 2.67 (t, = 7.7 Hz, 2H), 3.66 (t, = 6.4 Hz, 2H), 7.10 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.7, 31.9, 34.3, 35.6, 62.4, 128.3, 128.4, 138.9, 140.5; ESI-HRMS (M + H)+ mlz calcd for Ci7H290 249.2218, found 249.2210. Finally, RB-024 was prepared from 3-(4- octylphenyl)propan-l-ol according to a procedure similar to that described for RB-023; yield = 73%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.8 Hz, 3H), 1.25-1.30 (m, 10H), 1.55-1.65 (m, 4H), 1.79-1.92 (m, 4H), 2.16-2.20 (m, 2H), 2.40-2.43 (m, 2H), 2.53-2.63 (m, 4H), 2.80-2.83 (m, 2H), 3.67-3.69 (m, 1H), 7.08 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 28.5, 29.3, 29.4, 29.5, 31.6, 31.9, 33.3, 34.0, 35.6, 50.9, 57.9, 128.2, 128.4, 138.9, 140.4; ESI-HRMS (M + H)+ mlz calcd for C22H38NO 332.2953, found 332.2951.
Preparation of l-(4-(4-Octylphenyl)butyl)piperidin-4-ol (RB-025)
RB-025
Compound RB-025 was prepared from 4-(4-octylphenyl)butan-l-ol according to a procedure similar to that described for compound RB-023; yield = 67% (2 steps); 1H NMR (400 MHz, CDC13) δ 0.88 (t, 7 = 6.9 Hz, 3H), 1.24-1.33 (m, 10H), 1.55-1.68 (m, 4H), 1.80-1.88 (m, 4H), 2.21-2.26 (m, 2H), 2.56 (t, 7 = 7.8 Hz, 2H), 2.61 (t, 7 = 7.5 Hz, 2H), 2.71-2.89 (m, 4H), 3.11 ( 7 = 9.0 Hz, 2H), 3.97-4.01 (m, 1H), 7.05 (d, 7 = 8.4 Hz, 2H), 7.09 (d, 7 = 8.4 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.7, 28.9, 29.3, 29.4, 29.5, 31.6, 31.9, 34.9, 35.6, 57.5, 128.: 128.5, 138.6, 140.7; ESI-HRMS (M + H)+ mlz calcd for C23H40NO 346.3110, found 346.3107 Preparation of RB-026 - RB-033
Products RB-026, RB-027, and RB-028 were prepared in three steps from 2-(4- bromophenyl)ethanol. First, 2-(4-(hex-l-ynyl)phenyl)ethanol was prepared from 2-(4- bromophenyl)ethanol and 1-hexyne by a Sonogashira procedure; yield = 60%; 1H NMR (400 MHz, CDC13) 6 0.95 (t, 7 = 7.3 Hz, 3H), 1.43-1.52 (m, 2H), 1.55-1.62 (m, 2H), 2.40 (t, 7 = 7.0 Hz, 2H), 2.85 (t, 7 = 6.5 Hz, 2H), 3.84 (t, 7 = 6.5 Hz, 2H), 7.14 (d, 7 = 8.1 Hz, 2H), 7.34 (t, 7 = 8.1 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 13.7, 19.1, 22.0, 29.7, 30.9, 39.0, 63.5, 80.3, 90.2, 122.3, 128.9, 131.7, 137.9; ESI-HRMS (M + H)+ mlz calcd for
C14H19O 203.1436, found 203.14333. Similarly, 2-(4-(dodec-l-ynyl)phenyl)ethanol was prepared from 2-(4-bromophenyl)ethanol and 1-decyne by a Sonogashira reaction; yield = 62%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, 7 = 6.8 Hz, 3H), 1.23-1.30 (m, 12H),
1.40-1.45 (m, 2H), 1.59 (quin, 7 = 7.3 Hz, 2H), 2.38 (t, 7 = 7.1 Hz, 2H), 2.81 (t, 7 = 6.6 Hz, 2H), 3.79 (t, 7 = 6.5 Hz, 2H), 7.11 (d, 7 = 8.2 Hz, 2H), 7.32 (d, 7 = 8.1 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 19.4, 22.7, 24.9, 28.8, 29.0, 29.2, 29.4, 29.6, 31.9, 39.0, 63.4, 80.4, 90.2, 122.3, 128.6, 131.4, 138.0; ESI-HRMS (M + H)+ mlz calcd for C20H3iO
287.2375, found 287.2371. Catalytic hydrogenation of these alkynes afforded the corresponding saturated alcohols, 2-(4-hexylphenyl)ethanol and 2-(4- dodecylphenyl)ethanol. Data for 2-(4-hexylphenyl)ethanol: 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.6 Hz, 3H), 1.22-1.37 (m, 6H), 1.55-1.63 (m, 2H), 2.57 (t, = 7.8 Hz, 2H), 2.84 (t, = 6.6 Hz, 2H), 3.84 (t, = 6.5 Hz, 2H), 7.13 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.6, 29.0, 31.5, 31.7, 35.6, 38.8, 63.8, 128.6, 128.9, 135.5, 141.2; ESI- HRMS (M + H)+ mlz calcd for Ci4H230 207.1749, found 207.1725. Data for 2-(4- dodecylphenyl)ethanol: 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.8 Hz, 3H), 1.23-1.31 (m, 18H), 1.57-1.60 (m, 2H), 2.56 (t, = 7.8 Hz, 2H), 2.81 (t, = 6.6 Hz, 2H), 3.81 (t, = 6.6 Hz, 2H), 7.12 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.4, 29.6, 29.7, 31.6, 31.9, 35.6, 38.8, 63.7, 128.6, 128.9, 135.5, 141.2; 13C NMR (100 MHz, CDC13) δ 21.0, 32.3, 33.1, 39.5, 50.5, 59.9, 66.0, 128.6, 129.3, 135.9, 136.0; ESI-HRMS (M + Na)+ mlz calcd for C20H34ONa 313.2507, found 313.2502.
Preparation of l-(4-Methylphenethyl)piperidin-4-ol (RB-026)
RB-026
Compound RB-026 was prepared from 2-(4-methylphenyl)ethanol according to a procedure similar to that described for RB-023; yield = 69%; 1H NMR (400 MHz, CDC13) δ 1.69-1.77 (m, 2H), 1.99-2.04 (m, 2H), 2.31 (s, 3H), 2.48-2.52 (2H), 2.72-2.76 (m, 2H), 2.79 (s, IH), 2.84-2.88 (m, 2H), 2.98-3.03 (m, 2H), 3.78-3.84 (m, IH), 7.10 (s, 4H); ESI-HRMS (M + H)+ mlz calcd for Ci4H22NO 220.1701, found 220.1699.
Preparation of l-(4-Hexylphenethyl)piperidin-4-ol (RB-027)
RB-027
Compound RB-027 was prepared from 2-(4-hexaphenyl)ethanol according to a procedure similar to that described for RB-023; yield = 79%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, 7 = 6.5 Hz, 3H), 1.26-1.36 (m, 6H), 1.58 (quin, = 7.4 Hz, 2H), 1.75- 1.80 (m, 2H), 2.08-2.13 (m, 2H), 2.56 (t, J = 7.7 Hz, 2H), 2.74-2.77 (m, 2H), 2.90-2.94 (m, 2H), 3.00-3.04 (m, 2H), 3.86-3.90 (m, 1H), 7.11 (s, 4H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.6, 29.0, 31.5, 31.7, 35.6, 50.1, 59.9, 128.5, 128.6, 141.2; ESI-HRMS (M + H)+ mlz calcd for C19H32NO 290.2484, found 290.2478.
Preparation of l-(4-Dodecylphenethyl)piperidin-4-ol (RB-028)
RB-028
Compound RB-028 was prepared from 2-(4-dodecylphenyl)ethanol according to a procedure similar to that described for RB-023; yield = 75%; 1H NMR (400 MHz, CDC13) 6 0.88 (t, = 6.6 Hz, 3H), 1.23-1.33 (m, 18H), 1.56-1.60 (m, 2H), 1.67-1.72 (m, 2H), 1.98-2.01 (m, 2H), 2.34-2.39 (m, 2H), 2.56 (t, = 7.4 Hz, 2H), 2.64-2.68 (m, 2H), 2.81-2.84 (m, 2H), 2.91-2.95 (m, 2H), 3.75-3.79 (m, 1H), 7.10 (s, 4H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.7, 29.4, 29.5, 29.6, 29.7, 31.6, 31.9, 32.8, 35.6, 50.6, 60.3, 128.5, 136.7, 140.9; ESI-HRMS (M + H)+ mlz calcd for C25H44NO 374.3423, found 374.3414.
Preparation of 4-Azido-l-(4-methylphenethyl)piperidine (RB-029)
RB-029
To a solution of RB-026 (115 mg, 0.52 mmol) and triethylamine (0.73 mL, 5.24 mmol) in CH2CI2 (5 mL) at 0 °C was added methanesulfonyl chloride (0.12 mL, 1.57 mmol). After being stirred at rt for 4 h, the reaction mixture was evaporated, diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, evaporated, and dried. To a solution of reaction mixture in 5 mL of DMF was added sodium azide (170 mg, 2.62 mmol). The reaction mixture was stirred at 80 °C for 12 h and then concentrated. The residue was dissolved in EtOAc and the organic phase was evaporated and dried. Purification by silica gel chromatography, eluting with
hexane/EtOAc (1/1), gave 79 mg (62%) of RB-029 as a colorless oil; 1H NMR (400 MHz, CDC13) 5 1.69-1.78 (m, 2H), 1.96-1.99 (m, 2H), 2.30-2.35 (m, 2H), 2.31 (s, 3H), 2.60-2.65 (m, 2H), 2.76-2.80 (m, 2H), 2.86-2.89 (m, 2H), 3.44-3.48 (m, 1H), 7.09 (s, 4H); 13C NMR (100 MHz, CDC13) δ 21.0, 29.4, 30.5, 33.0, 50.9, 57.3, 60.4, 128.6, 129.2, 132.4, 135.7, 136.7; ESI-HRMS (M + H)+ mlz calcd for C14H21N4 245.1766, found 245.1763.
Preparation of 4-Azido-l-(4-octylphenethyl)piperidine (RB-030)
RB-030
Compound RB-030 was prepared from RB-005 according to a procedure similar to that described for RB-029; yield = 71%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.8 Hz, 3H), 1.24-1.31 (m, 10H), 1.58 (quin, = 7.2 Hz, 2H), 1.67-1.76 (m, 2H), 1.93-1.97
(m, 2H), 2.24-2.29 (m, 2H), 2.54-2.61 (m, 2H), 2.75-2.79 (m, 2H), 2.84-2.90 (m, 2H), 3.40-3.46 (m, 1H), 7.10 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 29.7, 30.3, 30.7, 31.6, 31.9, 33.2, 35.6, 51.1, 57.6, 60.5, 128.5, 137.2, 140.8; ESI- HRMS (M + H)+ mlz calcd for C21H35N4 343.2862, found 343.2857. Preparation of l-(4-Methylphenethyl)piperidin-4-amine (RB-031)
RB-031
To a solution of RB-029 (30 mg, 0.12 mmol) in MeOH/CH2Cl2 (3/1, 3 niL) was added 10% Pd/C (50 wt %). The reaction mixture was hydrogenated at rt for 12 h. The catalyst was removed by filtration through a pad of Celite, which was rinsed with
MeOH/CH2Cl2 (3/1). The residue was washed with EtOAc/hexane (1/1), evaporated, and dried. RB-031 was obtained as a white solid; 1H NMR (400 MHz, CD3OD) δ 1.82-1.90 (m, 2H), 2.17 (d, = 10.2 Hz, 2H), 2.28 (s, 3H), 2.34 (t, = 10.6 Hz, 2H), 2.68-2.71 (m, 2H), 2.79-2.83 (m, 2H), 3.13 (d, = 11.2 Hz, 2H), 3.13-3.20 (m, 1H), 7.07 (s, 4H); 13C NMR (100 MHz, CDCI3) δ 21.0, 30.7, 32.4, 48.2, 51.4, 59.8, 128.6, 129.2, 135.8, 136.0; ESI-HRMS (M + H)+ mlz calcd for Ci4H23N2 219.1861, found 219.1858.
Preparation of l-(4-Octylphenethyl)piperidin-4-amine (RB-032)
RB-032
Compound RB-032 was prepared from RB-030 according to a procedure similar to that described for RB-031; 1H NMR (400 MHz, CD3OD) δ 0.91 (t, = 6.8 Hz, 3H), 1.28-1.34 (m, 10H), 1.61 (quin, = 6.5 Hz, 2H), 2.06-2.15 (m, 2H), 2.32 (d, = 13.2 Hz, 2H), 2.60 (t, = 7.52 Hz, 2H), 3.08-3.14 (m, 2H), 3.24 (t, = 11.5 Hz, 2H), 3.35-3.38
(m, 2H), 3.51-3.58 (m, 1H), 3.76 (d, J = 11.8 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 7.23 (d, = 8.0 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 14.4, 23.7, 30.3, 30.4, 30.6, 31.1, 32.7, 33.0, 36.5, 36.9, 129.8, 129.9, 134.7, 143.2; ESI-HRMS (M + H)+ mlz calcd for C2iH37N2 317.2957, found 317.2951. Preparation of l-(4-Dodecylphenethyl)piperidin-4-amine (RB-033)
RB-033
To a solution of RB-028 (20 mg, 0.050 mmol) and triethylamine (70 L, 0.54 mmol) in CH2C12 (3 mL) at 0 °C was added methanesulfonyl chloride (10 μί, 0.15 mmol). After being stirred at rt for 4 h, the reaction mixture was evaporated, diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, evaporated, and dried. To a solution of the reaction mixture in 3 mL of DMF was added sodium azide (10 mg, 0.16 mmol). The reaction mixture was stirred at 100 °C for 12 h, and then concentrated. The residue was dissolved in EtOAc and the organic phase was evaporated and dried. To a solution of residue in MeOH/CH2Cl2 (3/1, 3 mL) was added 10% Pd/C (50 wt %). The reaction mixture was hydrogenated at rt for 12 h. The catalyst was removed by filtration through a pad of Celite, which was rinsed with MeOH/CH2Cl2 (3/1). The residue was washed with EtOAc/hexane (1/1), evaporated, and dried, affording RB-033 as a white solid; 1H NMR (400 MHz, CD3OD) δ 0.89 (t, = 6.9 Hz, 3H), 1.27- 1.32 (m, 18H), 1.58-1.62 (m, 2H), 2.00-2.10 (m, 2H), 2.29 (d, J = 13.2, 2H), 2.58-2.62 (m, 2H), 3.05-3.09 (m, 2H), 3.15 (d, J = 13.6 Hz, 2H), 3.28-3.33 (m, 2H), 3.46-3.54 (m, 1H), 3.71 (d, = 10.9 Hz, 2H), 7.17 (d, = 7.9 Hz, 2H), 7.22 (d, = 7.9 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 14.5, 23.8, 29.9, 30.3, 30.5, 30.6, 30.7, 30.8, 30.9, 31.3, 32.8, 33.1, 36.5, 36.9, 129.9, 130.1, 133.1, 143.1 ; ESI-HRMS (M + H)+ mlz calcd for C25H45N2 373.3583, found 373.3576.
Preparation of 4-Fluoro-l-(4-octylphenethyl)piperidine (RB-034)
RB-034
To a solution of RB-005 (12 mg, 0.040 mmol) in CH2C12 (3 mL) at 0 °C was added diethylaminosulfur trifluoride (DAST, 15 μί, 0.12 mmol). After being stirred at rt for 5 h, the reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, evaporated, and dried. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH (10: 1), gave 11 mg (90%) of RB-034 as a colorless oil; 1H NMR (400 MHz, CDC13) δ 0.87 (t, J = 6.8 Hz, 3H), 1.25-1.30 (m, 10H), 1.54-1.62 (m, 2H), 1.90-2.00 (m, 4H), 2.48-2.63 (m, 6H), 2.76-2.80 (m, 2H), 4.61-4.66 (m, 0.5H), 4.74-4.78 (m, 0.5H), 7.10 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 29.7, 33.2, 35.6, 49.4, 60.5, 128.5, 137.2, 140.8; ESI-HRMS (M + H)+ mlz calcd for C2iH35FN 320.2754, found 320.2751.
Preparation of l-(4-Octylphenethyl)piperidin-4-one (RB-035)
RB-035 To a solution of RB-005 (25 mg, 0.080 mmol) in CH2C12 (3 mL) at 0 °C was added pyridinium chlorochromate (PCC, 25 mg, 0.12 mmol). After being stirred at rt for 4 h, the reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, evaporated, and dried. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH (3: 1), gave 17 mg (70%) of RB-035 as a colorless oil; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 6.6 Hz, 3H), 1.22-1.30 (m,
10H), 1.58-1.60 (m, 2H), 2.47-2.52 (m, 4H), 2.57 (t, = 7.7 Hz, 2H), 2.72-2.74 (m, 2H),
2.81-2.86 (m, 6H), 7.12 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 29.7, 31.6, 31.9, 33.7, 35.6, 36.0, 41.2, 53.1, 59.4, 128.6, 137.0, 141.0, 178.0; ESI- HRMS (M + H)+ mlz calcd for C2iH34 NO 316.2640, found 316.2635.
Preparation of 4-Methoxy-l-(4-octylphenethyl)piperidine (RB-036)
RB-036
To a solution of 4-octylphenethyl methanesulfonate (17 mg, 50 μιηοΐ) in MeCN (3 mL) was added at rt. After the suspension was stirred for 10 min, 4-methoxypiperidine (19 mg, 0.16 mmol) was added. The reaction mixture was stirred at 50 °C for 12 h. The solvent was evaporated and the residue was purified by silica gel chromatography, eluting with CH2Cl2/MeOH (5: 1), to give 14 mg (79%) of RB-036 as a yellow liquid; 1H NMR (400 MHz, CDC13) 5 0.87 (t, = 6.5 Hz, 3H), 1.23-1.31 (m, 10H), 1.56-1.61 (m, 2H), 1.67-1.72 (m, 2H), 1.95-2.00 (m, 2H), 2.30-2.37 (m, 2H), 2.56 (t, = 7.7 Hz, 2H), 2.62-2.64 (m, 2H), 2.79-2.85 (m, 4H), 3.25-3.30 (m, 1H), 3.34 (s, 3H), 7.10 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 35.6, 50.8, 55.6, 60.5, 128.5, 128.6, 140.8; ESI-HRMS (M + H)+ mlz calcd for C22H38NO 332.2953, found 332.2948.
Preparation of (5)-l-(4-Octylphenethyl)pyrrolidin-3-ol (RB-037)
RB-037
To a solution of 4-octylphenethyl methanesulfonate (20 mg, 0.064 mmol) in MeCN (4 mL), K2C03 (44 mg, 0.32 mmol) was added at rt. After the suspension was stirred for 10 min, (S)-pyrrolidine-3-ol hydrochloride (79 mg, 0.64 mmol) was added.
The reaction mixture was stirred at 50 °C for 12 h. The reaction mixture was diluted with
water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. Purification by silica gel chromatography, eluting with
CH2Cl2/MeOH (3: 1), gave 17 mg (86%) of RB-037 as a yellow liquid; 1H NMR (400 MHz, CDCl3) 5 0.88 (t, 7 = 6.8 Hz, 3H), 1.22-1.32 (m, 10H), 1.58 (quin, 7 = 7.3 Hz, 2H), 1.85 (quin, 7 = 6.7 Hz, IH), 2.19-2.28 (m, IH), 2.46-2.54 (m, 2H), 2.56 (t, 7 = 7.8 Hz, 2H), 2.68 (dd, 7 = 5.1, 10.4 Hz, IH), 2.78-2.88 (m, 4H), 2.91 (d, 7 =10.4 Hz, IH), 3.07- 3.13 (m, IH), 4.38-4.41 (m, IH), 7.12 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 34.3, 34.7, 35.6, 52.6, 57.8, 62.9, 71.1, 128.5, 128.6, 136.5, 141.0; ESI-HRMS (M + H)+ mlz calcd for C20H34NO 304.2640, found 304.2639. Preparation of (R)-l-(4-Octylphenethyl)pyrrolidin-3-ol (RB-038)
RB-038
Compound RB-038 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for RB-036, using (R)- pyrrolidine; yield = 72%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, 7 = 6.7 Hz, 3H), 1.26- 1.29 (m, 10H), 1.56-1.59 (m, 2H), 1.94-2.01 (m, IH), 2.22-2.31 (m, IH), 2.56 (t, 7 = 7.9 Hz, 2H), 2.74-2.76 (m, IH), 2.91-2.99 (m, 4H), 3.14-3.24 (m, 2H), 3.30-3.35 (m, IH), 4.47-4.50 (m, IH), 7.12 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.6, 29.3, 29.4, 29.5, 31.5, 31.9, 33.4, 34.2, 35.6, 52.9, 57.9, 62.7, 70.4, 128.5, 128.7, 135.2, 141.5; ESI- HRMS (M + H)+ mlz calcd for C20H34NO 304.2640, found 304.2637. Preparation of (R)-(l-(4-Methylphenethyl)pyrrolidin-2-yl)methanol (RB-039)
RB-039
Compound RB-039 was prepared from 2-(4-methylphenyl)ethanol according to a coupling procedure similar to that described for RB-037, using D-prolinol; yield = 62%;
1H NMR (400 MHz, CDC13) δ 1.84-1.91 (m, 1H), 1.97-1.21 (m, 1H), 2.03-2.13 (m, 2H), 2.31 (s, 3H), 2.83-2.89 (m, 1H), 2.96-3.08 (m, 2H), 3.13-3.21 (m, 1H), 3.34-3.41 (m, 1H), 3.49-3.56 (m, 1H), 3.72-3.77 (m, 1H), 3.85 (d, J = 5.3 Hz, 2H), 7.12 (dd, / = 1.9, 8.4 Hz, 4H); 13C NMR (100 MHz, CDC13) δ 21.0, 23.6, 26.5, 32.0, 39.4, 50.7, 55.0, 57.9, 61.1, 69.5, 128.6, 129.5, 133.7, 136.7; ESI-HRMS (M + H)+ mlz calcd for Ci4H22NO 220.1701, found 220.1698.
Preparation of (/?)-(l-(4-Octylphenethyl)pyrrolidin-2-yl)methanol (RB-040)
Compound RB-040 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for RB-037, using D-prolinol; yield = 59%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.8 Hz, 3H), 1.22-1.31 (m, 10H), 1.54-1.62 (m, 2H), 1.84-2.05 (m, 4H), 2.56 (t, = 7.8 Hz, 2H), 2.64-2.70 (m, 1H), 2.85- 3.00 (m, 3H), 3.08-3.13 (m, 1H), 3.32-3.36 (m, 1H), 3.57-3.62 (m, 1H), 3.67 (dd, / = 5.5, 13.7, Hz, 1H), 3.78 (dd, = 3.2, 12.2 Hz, 1H), 7.11 (s, 4H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.7, 23.8, 26.9, 29.3, 29.5, 29.7, 31.0, 31.5, 31.9, 35.6, 54.5, 56.9, 61.4, 66.3, 128.5, 128.7, 135.5, 141.5; ESI-HRMS (M + H)+ mlz calcd for C2iH36NO 318.2797, found 318.2792.
Preparation of (5)-(l-(4-Octylphenethyl)pyrrolidin-2-yl)methanol (RB-041)
Compound RB-041 was prepared from 4-octylphenethyl methanesulfonate according to a coupling procedure similar to that described for RB-037, using L-prolinol; yield = 54%; 1H NMR (400 MHz, CDCI3) δ 0.88 (t, = 6.7 Hz, 3H), 1.22-1.31 (m, 10H), 1.58 (quin, = 7.3 Hz, 2H), 1.80-1.96 (m, 4H), 2.52-2.55 (m, 1H), 2.56 (t, = 7.8 Hz,
2H), 2.73 (td, 7 = 11.6, 2.7 Hz, IH), 2.86-2.96 (m, 3H), 3.18-3.25 (m, IH), 3.47 (quin, 7 = 4.7 Hz, IH), 3.56 (dd, 7 = 4.4, 11.7 Hz, IH), 3.70 (dd, 7 = 3.2, 11.8 Hz, IH), 7.11 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 23.8, 27.1, 29.3, 29.4, 29.5, 29.7, 31.5, 31.9, 33.7, 35.6, 54.3, 57.0, 61.5, 66.9, 128.5, 128.8, 135.8, 141.2; ESI-HRMS (M + H)+ mlz calcd for C2iH36NO 318.2797, found 318.2791.
Preparation of (R)-(l-(4-Dodecylphenethyl)pyrrolidin-2-yl)methanol (RB-042)
Compound RB-042 was prepared from 2-(4-dodecylphenyl)ethanol according to a coupling procedure similar to that described for RB-037, using D-prolinol; yield = 62%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, 7 = 6.6 Hz, 3H), 1.22-1.31 (m, 18H), 1.56-1.60 (m, 2H), 2.00-2.16 (m, 4H), 2.56 (t, 7 = 7.7 Hz, 2H), 2.82-2.88 (m, IH), 3.03-3.09 (m, 2H), 3.22-3.30 (m, IH), 3.35-3.39 (m, IH), 3.46-3.53 (m, IH), 3.75-3.80 (m, IH), 3.86 (dd, 7 = 6.5, 12.7 Hz, IH), 3.95 (dd, 7 = 2.1, 12.0 Hz, IH), 7.12 (s, 4H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 24.0, 26.5, 29.3, 29.4, 29.5, 29.6, 29.7, 31.5, 31.8, 31.9, 35.5, 54.7, 58.0, 60.9, 70.2, 128.5, 128.9, 133.6, 142.0; ESI-HRMS (M + H)+ mlz calcd for Q5H44NO 374.3423, found 374.3418.
Preparation of (S)-(l-(4-Dodecylphenethyl)pyrrolidin-2-yl)methanol (RB-043)
Compound RB-043 was prepared from 2-(4-dodecylphenyl)ethanol according to a coupling procedure similar to that described for RB-037, using L-prolinol; yield = 55%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, 7 = 6.6 Hz, 3H), 1.23-1.30 (m, 18H), 1.56-1.62 (m, 2H), 1.93-2.11 (m, 4H), 2.56 (t, 7 = 7.8 Hz, 2H), 2.98-3.06 (m, 2H), 3.14-3.22 (m, IH), 3.28-3.32 (m, IH), 3.41-3.49 (m, IH), 3.65-3.68 (m, IH), 3.71-3.73 (m, IH), 3.79-
3.83 (m, 1H), 3.89 (dd, J = 2.5, 12.0 Hz, 1H), 7.12 (s, 4H); 1JC NMR (100 MHz, CDC13) δ 14.1, 22.7, 23.9, 26.8, 29.4, 29.5, 29.6, 29.7, 31.5, 31.9, 35.6, 54.5, 57.5, 61.1, 70.1,
128.5, 128.8, 134.1, 141.7; ESI-HRMS (M + H)+ mlz calcd for C25H44NO 374.3423, found 374.3415. Preparation of Compounds RB-044 - RB-050
These products were prepared from 4-iodobenzoic acid in three steps. First, 4- (oct- l-ynyl)benzoic acid was prepared by a Sonogashira reaction. To a deaerated solution of 4-iodobenzoic acid (500 mg, 2.02 mmol), bis(triphenylphosphine)palladium dichloride (116 mg, 0.10 mmol), and copper(I) iodide (19 mg, 0.10 mmol) in anhydrous
triethylamine (15 mL) was added 1-octyne (0.89 mL, 6.05 mmol) at rt. The reaction mixture was heated at 60 °C for 12 h. After saturated aqueous ammonium chloride solution was added, the product was extracted with EtOAc. The combined solution was washed with water, brine, and dried. Purification by silica gel chromatography, eluting with hexane/EtOAc (3: 1), gave 395 mg (85%) of the alkyne product as a yellow liquid; 1H NMR (400 MHz, CDC13) δ 0.90 (t, = 6.7 Hz, 3H), 1.31-1.35 (m, 4H), 1.43-1.50 (m, 2H), 1.58-1.66 (m, 2H), 2.43 (t, = 7.1 Hz, 2H), 7.47 (d, = 8.4 Hz, 2H), 8.02 (d, = 8.4 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 13.8, 19.3, 22.3, 28.3, 31.1, 79.8, 94.4, 127.6,
129.6, 129.7, 131.3, 171.8; negative-ion ESI-HRMS (M - H)~ mlz calcd for Ci5Hi702 ~ 229.1234, found 229.1237. Then, catalytic hydrogenation afforded 4-octylbenzoic acid as a yellow solid without purification; 1H NMR (400 MHz, CDCI3) δ 0.88 (t, = 6.8 Hz, 3H), 1.22-1.32 (m, 10H), 1.61-1.64 (m, 2H), 2.64 (t, = 7.7 Hz, 2H), 7.25 (d, = 8.1 Hz, 2H), 8.02 (d, = 8.2 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 31.1, 31.9, 36.1, 126.9, 128.6, 130.3, 149.6, 172.6; negative-ion ESI-HRMS (M - H)~ mlz calcd for C 15H2i 02 " 233.1547, found 233.1548.
Preparation of (/?)-(4-Dodecylphenyl)-(2-(hvdroxymethyl)pyrrolidin-l- vDmethanone (RB-044)
RB-044
To a solution of 4-(n-dodecyl)benzoic acid (10 mg, 0.034 mmol) in CH2CI2 (3 mL), thionyl chloride (0.25 mL, 0.34 mmol) was added at rt. The reaction mixture was heated at reflux for 12 h. The reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. To a solution of residue in MeCN (3 mL) was added K2CO3 (24 mg, 0.17 mmol) at rt. After the suspension was stirred for 10 min, D-prolinol (10 mg, 0.10 mmol) was added. The reaction mixture was stirred at 50 °C for 12 h. The reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. Purification by silica gel chromatography, eluting with
CH2Cl2/MeOH (10: 1), gave 9 mg (72%) of RB-044 as a yellow liquid; 1H NMR (400 MHz, CDCI3) δ 0.88 (t, = 6.6 Hz, 3H), 1.23-1.34 (m, 18H), 1.59-1.64 (m, 4H), 1.70- 1.77 (m, 1H), 1.84-1.89 (m, 1H), 2.14-2.21 (m, 1H), 2.62 (t, 7 = 7.6 Hz, 2H), 3.46-3.59 (m, 2H), 3.71-3.82 (m, 2H), 4.39-4.44 (m, 1H), 7.20 (d, 7 = 7.9 Hz, 2H), 7.43 (d, 7 = 7.9 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 25.1, 28.6, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 31.3, 31.9, 35.8, 51.3, 61.6, 67.6, 127.2, 128.3, 133.8, 145.5, 172.5; ESI- HRMS (M + H)+ mlz calcd for C24H40NO2 374.3059, found 374.3055. Preparation of (5)-(4-Dodecylphenyl)(2-(hvdroxymethyl)pyrrolidin-l-yl)methanone
(RB-045)
RB-045
Compound RB-045 was prepared from 4-(n-dodecyl)benzoic acid according to a coupling procedure similar to that described for RB-044, using L-prolinol instead of D- prolinol; yield = 65%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, J = 6.7 Hz, 3H), 1.23-1.30 (m, 18H), 1.58-1.64 (m, 4H), 1.70-1.77 (m, IH), 1.84-1.89 (m, IH), 2.14-2.21 (m, IH), 2.62 (t, = 7.7 Hz, 2H), 3.47-3.59 (m, 2H), 3.71-3.82 (m, 2H), 4.38-4.44 (m, IH), 7.20 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 7.9 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 25.1, 28.6, 29.2, 29.4, 29.5, 29.6, 29.7, 31.2, 31.9, 35.8, 51.3, 61.6, 67.5, 127.2, 128.3, 133.8, 145.5, 172.5; ESI-HRMS (M + H)+ mlz calcd for C24H40NO2 374.3059, found
374.3058. Preparation of (4-Hvdroxypiperidin-l-yl)(4-octylphenyl)methanone (RB-046)
RB-046
Compound RB-046 was prepared from 4-(n-dodecyl)benzoic acid according to a coupling procedure similar to that described for RB-044, using 4-hydroxypiperidine; yield = 70%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, J = 6.7 Hz, 3H), 1.26-1.30 (m, 10H), 1.51-1.62 (m, 4H), 1.80-1.97 (m, 2H), 2.63 (t, J = 7.7 Hz, 2H), 3.21-3.36 (m, 2H), 3.67- 3.76 (m, IH), 3.93-4.00 (m, IH), 4.18-4.26 (m, IH), 7.19 (d, / = 8.2 Hz, 2H), 7.31 (d, = 8.2 Hz, 2H), 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.2, 29.3, 29.4, 31.3, 35.8, 67.4, 126.9, 128.5, 130.2, 133.2, 144.8, 170.7; ESI-HRMS (M + H)+ mlz calcd for C2oH32N02 318.2428, found 318.2432. Preparation of (4-Dodecylphenyl)(4-hvdroxypiperidin-l-yl)methanone (RB-047)
RB-047
Compound RB-047 was prepared from 4-(n-dodecyl)benzoic acid according to a coupling procedure similar to that described for RB-044, using 4-hydroxypiperidine; yield = 81%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, J = 6.7 Hz, 3H), 1.22-1.37 (m, 18H), 1.58-1.63 (m, 4H), 1.84-1.95 (m, 2H), 2.61 (t, J = 7.7 Hz, 2H), 3.21-3.34 (m, 2H), 3.67- 3.75 (m, 1H), 3.95 (sep, = 3.9 Hz, 1H), 4.18-4.23 (m, 1H), 7.19 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.2, 22.7, 29.3, 29.4, 29.5, 29.6, 29.7, 31.3, 31.9, 35.8, 67.2, 126.9, 128.5, 133.1, 144.9, 170.8; ESI-HRMS (M + H)+ mlz calcd for C24H40NO2 374.3059, found 374.3055.
Preparation of 4-Octyl-N-(pyridin-4-ylmethyl)benzamide (RB-048)
RB-048
Compound RB-048 was prepared from 4-(n-dodecyl)benzoic acid according to a coupling procedure similar to that described for RB-044, using 4-(aminomethyl)pyridine; yield = 69%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.8 Hz, 3H), 1.07-1.30 (m, 10H), 1.59-1.63 (m, 2H), 2.65 (t, = 7.7 Hz, 2H), 4.63 (d, = 6.0 Hz, 2H), 6.97 (t, = 5.6 Hz, NH), 7.24 (d, = 8.0 Hz, 4H), 7.74 (d, = 8.2 Hz, 2H), 8.51-8.56 (m, 2H); 13C NMR
(100 MHz, CDC13) δ 14.1, 21.1, 22.7, 29.2, 29.4, 29.7, 31.2, 31.8, 35.8, 42.7, 60.4, 127.1, 128.5, 128.7, 131.2, 147.4, 147.8, 149.9, 167.7, 171.2; ESI-HRMS (M + H)+ mlz calcd for C21H29N2O 325.2274, found 325.2277.
Preparation of N-(4-Hvdroxyphenyl)-4-octylbenzamide (RB-049)
RB-049
Compound RB-049 was prepared from 4-(n-dodecyl)benzoic acid according to a coupling procedure similar to that described for RB-044, using 4-aminophenol; yield = 55%; 1H NMR (400 MHz, CDC13) δ 0.88 (t, J = 6.8 Hz, 3H), 1.11-1.29 (m, 10H), 1.54- 1.60 (m, 2H), 2.67 (t, J = 7.1 Hz, 2H), 6.83 (d, J = 7.8 Hz, 2H), 7.28 (d, J = 7.8 Hz, 2H), 7.43 (d, = 6.8 Hz, 2H), 7.80 (d, = 6.8 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.3, 22.9, 27.6, 29.6, 29.7, 30.0, 30.4, 31.6, 32.2, 36.2, 123.2, 127.5, 129.0, 132.5, 147.6, 167.8; ESI-HRMS (M + H)+ mlz calcd for C21H28NO2 326.2115, found 326.2118.
Preparation of N-(4-(2-Hydroxyethyl)phenyl)-4-octylbenzamide (RB-050)
Preparation of RB-051 and RB-052 These compounds were prepared from l-bromo-4-iodobenzene. First, l-bromo-4-
(oct- l-ynyl)benzene was prepared from l-bromo-4-iodobenzene by a Sonogashira reaction; yield = 70%; 1H NMR (400 MHz, CDC13) δ 0.90 (t, = 7.1 Hz, 3H), 1.29-1.34 (m, 4H), 1.40-1.47 (m, 2H), 1.59 (quin, = 7.3 Hz, 2H), 2.37 (t, = 7.1 Hz, 2H), 7.24 (dt, = 8.3, 2.0 Hz, 2H), 7.39 (dt, = 8.6, 2.0 Hz, 2H); 13C NMR (100 MHz, CDC13) δ
14.1, 19.5, 22.6, 28.8, 31.4, 79.6, 91.8, 121.5, 123.1, 131.4, 133.0; ESI-HRMS (M)+ mlz calcd for Ci4Hi7Br 264.0514, found 264.0508.
Preparation of 4-(4-Octylphenethyl)pyridine (RB-051)
RB-051 To a deaerated solution of l-bromo-4-(oct-l-ynyl)benzene (100 mg, 0.38 mmol), bis(triphenylphosphine)palladium dichloride (22 mg, 0.010 mmol), and copper(I) iodide (4 mg, 10 μιηοΐ) in anhydrous triethylamine (8 mL) was added 3-ethynylpyridine (78 mg, 0.75 mmol) at rt. The reaction mixture was heated at 80 °C for 3 d. After saturated ammonium chloride solution was added, the product was extracted with EtOAc. The combined solution was washed with water, brine, and dried. The catalyst was removed by filtration through a pad of Celite, which was rinsed with hexanes/EtOAc (3: 1). 3-((4- (Oct-l-ynyl)phenyl)ethynyl)pyridine (53 mg, 0.18 mmol) was dissolved in EtOAc (8 mL), and 10% Pd/C (53 mg, 100 wt %) was added. The reaction mixture was
hydrogenated at rt for 12 h. The catalyst was removed by filtration through a pad of Celite, which was rinsed with EtOAc. Flash column chromatography with
hexanes/EtOAc (1: 1) as eluent gave RB-051 (48 mg, 88%) as a yellow oil; 1H NMR (400 MHz, CDCl3) 5 0.88 (t, / = 6.8 Hz, 3H), 1.27-1.34 (m, 10H), 1.59 (quin, = 7.3 Hz, 2H), 2.56 (t, = 7.8 Hz, 2H), 2.86-2.92 (m, 4H), 7.05 (d, = 8.1 Hz, 2H), 7.09 (d, = 8.1 Hz, 2H), 7.17 (dd, = 4.8, 7.7 Hz, 1H), 7.43 (dt, = 7.8, 1.8 Hz, 1H), 8.42-8.44 (m, 2H); 13C NMR (100 MHz, CDC13) δ 14.2, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 35.0, 35.6, 37.1,
123.2, 128.3, 128.5, 135.9, 137.0, 140.8, 147.5, 150.0; ESI-HRMS (M + H)+ mlz calcd for C2iH30N 296.2378, found 296.2374.
Preparation of l-Methyl-4-(4-octylphenethyl)pyridinium Iodide (RB-052)
RB-052
To a solution of RB-051 (32 mg, 0.11 mmol) in MeCN (5 mL) was added K2C03 (75 mg, 0.54 mmol). After the suspension was stirred at rt for 10 min, Mel (30 μί, 0.54 mmol) was added. The reaction mixture was stirred overnight at rt. The reaction mixture was diluted with water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated. The residue was washed with hexane to give 38 mg (80%) of RB-052 as a yellow solid; 1H NMR (400 MHz, CDC13) δ 0.88 (t, = 6.9 Hz, 3H), 1.26-1.30 (m, 10H), 1.56 (quin, J = 7.3 Hz, 2H), 2.54 (t, J = 7.8 Hz, 2H), 3.02 (t, J = 7.6 Hz, 2H), 3.17 (t, J = 7.6 Hz, 2H), 4.60 (s, 3H), 7.07 (s, 4H), 7.92 (dd, J = 6.2, 7.7 Hz, 1H), 8.06 (d, / = 8.0 Hz, 1H), 9.12 (d, J = 6.5 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 34.4, 35.5, 35.7, 49.2, 127.6, 128.6, 128.7, 136.1, 141.4, 142.8, 143.0, 145.1, 145.2; ESI-HRMS (M)+ mlz calcd for C22H32N+ 310.2535, found 310.2533. Preparation of 4-(4-Methylpiperidin-l-yl)-l-(4-octylbenzyl)pyridinium bromide
(RB-053)
Br
First, l-(bromomethyl)-4-(oct- l-ynyl)benzene was prepared from 4-iodobenzyl bromide and 1-octyne by a Sonogashira reaction; yield = 69%; 1H NMR (400 MHz, CDC13) 5 0.87 (t, J = 6.9 Hz, 3H), 1.27-1.30 (m, 8H), 2.38 (t, J = 6.8 Hz, 2H), 4.44 (s,
2H), 7.30 (d, = 8.0 Hz, 2H), 7.33 (d, = 8.0 Hz, 2H); 13C NMR (100 MHz, CDC13) δ
14.1, 19.3, 22.4, 28.4, 28.7, 31.2, 33.3, 80.0, 91.2, 124.3, 128.9, 131.8, 136.9; ESI-HRMS (M + H)+ mlz calcd for Ci5H20Br 279.0748, found 279.0730. Then, this compound was subjected to N-alkylation with 4-(4-methylpiperidin-l-yl)pyridine. The latter compound was synthesized from 4-chloropyridine hydrochloride (0.20 g, 1.3 mmol) in dry acetonitrile (4 mL). N,N-Diisopropylethylamine (DIPEA, 0.7 mL, 4.0 mmol) was added, followed by 4-methylpiperidine (0.16 mL, 1.3 mmol). The reaction mixture was subjected to microwave irradiation at 160 °C for 1 h. After the reaction mixture was cooled rt, EtOAc was added, and the solution was washed with water and brine, dried (Na2S04), and concentrated in vacuo. Ether (3 mL) was added to the resulting crude oil, and the inorganic precipitate was removed by filtration. Evaporation of the solvents afforded 4-(4-methylpiperidin-l-yl)pyridine. To a solution of l-(bromomethyl)-4-(oct-l- ynyl)benzene (100 mg, 0.35 mmol) in 5 mL of 2-butanone was added 4-(4- methylpiperidin-l-yl)pyridine (124 mg, 0.71 mmol) in a sealed tube. The reaction mixture was stirred at 100 °C for 3 d and concentrated. The residue was washed with EtOAc to give 125 mg (78%) of 4-(4-methylpiperidin-l-yl)-l-(4-(oct-l- ynyl)benzyl)pyridinium bromide (RB-053) as a slightly yellow solid; 1H NMR (400 MHz, CDC13) 5 0.86 (t, = 7.0 Hz, 3H), 0.98 (d, = 6.4 Hz, 3H), 1.16-1.33 (m, 10H), 1.55-1.60 (m, 4H), 1.72-1.78 (m, 1H), 1.84 (d, = 13.8 Hz, 2H), 2.58 (t, = 7.6 Hz, 2H), 3.12 (td, = 12.5, 2.4 Hz, 2H), 4.08 (d, = 13.5 Hz, 2H), 5.47 (s, 2H), 7.02 (d, = 7.6 Hz, 2H), 7.18 (d, = 8.0 Hz, 2H), 7.43 (d, = 8.0 Hz, 2H), 8.46 (d, = 7.6 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.2, 19.4, 21.3, 22.6, 28.6, 29.7, 30.5, 31.3, 33.5, 47.4, 60.3, 79.8, 92.2, 108.4, 125.3, 128.9, 132.4, 133.1, 143.0, 155.2; ESI-HRMS (M + H)+ mlz calcd for C26H36N2 + 376.2873, found 376.2871. Catalytic hydrogenation provided 4-(4- methylpiperidin-l-yl)-l-(4-octylbenzyl)pyridinium bromide (RB-053) in 87% yield; 1H NMR (400 MHz, CDC13) δ 0.86 (t, = 7.0 Hz, 3H), 0.98 (d, = 6.4 Hz, 3H), 1.16-1.33 (m, 10H), 1.55-1.60 (m, 4H), 1.72-1.78 (m, 1H), 1.84 (d, = 13.8 Hz, 2H), 2.58 (d, = 7.6 Hz, 2H), 3.13 (td, / = 12.5, 2.4 Hz, 2H), 4.08 (d, = 13.5 Hz, 2H), 5.47 (s, 2H), 7.02 (d, = 7.6 Hz, 2H), 7.18 (d, = 8.0 Hz, 2H), 7.34 (d, = 8.0 Hz, 2H), 8.46 (d, = 7.5 Hz, 2H); 13C NMR (100 MHz, CDC13) δ 14.1, 21.3, 22.7, 29.2, 29.3, 29.4, 29.7, 30.3, 30.5, 31.4, 31.9, 33.5, 35.7, 47.5, 60.8, 108.5, 128.8, 129.5, 130.9, 142.8, 144.5, 155.2; ESI- HRMS (M)+ mlz calcd for C26H39N2 + 379.3113, found 379.3108.
Preparation of 3-(1-(4-θ€ΐν1ρΗ6ην1)-1Η-1,2,3-1 ζο1-4-ν1)ρν (1ίη6 (RB-054)
RB-054
This compound was prepared from 4-iodoaniline in three steps. First, 4-(oct- l- ynyl)aniline was prepared from 4-iodoaniline and 1-octyne; yield = 62%; 1H NMR (400 MHz, CDC13) δ 0.90 (t, 7 = 7.0 Hz, 3H), 1.26-1.35 (m, 4H), 1.40-1.47 (m, 2H), 1.58 (quin, 7 = 7.4 Hz, 2H), 2.37 (t, 7 = 7.1 Hz, 2H), 6.57 (d, 7 = 8.6 Hz, 2H), 7.19 (d, 7 = 8.6 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 14.1, 19.5, 22.6, 28.6, 29.0, 31.4, 80.7, 87.9, 113.7, 114.8, 132.7, 145.9; ESI-HRMS (M + H)+ mlz calcd for Ci4H20N 202.1596, found 202.1589. The aryl amine was converted to the corresponding aryl azide by the reaction of 4-(oct-l-ynyl)aniline (157 mg, 0.78 mmol) in 2 mL of 10% aqueous HC1 with NaN02 (65 mg, 0.94 mmol) in 1 mL of water at 0 °C. After the solution was stirred for 30 min, NaN3 (61 mg, 0.94 mmol) in 1 mL of water was added at 0 °C, with stirring for another hour. The reaction mixture was warmed to 25 °C, diluted with EtOAc, washed with water and brine, dried (Na2S04), and concentrated in vacuo, affording the aryl azide. Then, a Cu(I)-catalyzed azide-alkyne 1,3-dipolar (click) reaction was carried out. Without purification, the aryl azide (43 mg, 0.19 mmol) and 3-ethynylpyridine (39 mg, 0.38 mmol) were dissolved in i-BuOH/H20 (3 mL, 1 : 1), and CuS04 (30 mg, 0.19 mmol) and sodium ascorbate (37 mg, 0.19 mmol) were added at rt. The reaction mixture was stirred for 2 days and then was diluted with EtOAc and washed with brine. The aqueous layer was extracted with EtOAc. The combined organic layers were dried (MgS04) and concentrated in vacuo. Purification by silica gel chromatography, eluting with
hexanes/EtOAc (1 : 1), gave 50 mg (67%, 2 steps) of 3-(l-(4-(oct-l-ynyl)phenyl- lH-l,2,3- triazol-4-yl)pyridine as a white solid; 1H NMR (400 MHz, CDC13) δ 0.92 (t, 7 = 6.9 Hz, 3H), 1.29-1.38 (m, 4H), 1.47 (quin, 7 = 7.3 Hz, 2H), 1.63 (quin, 7 = 7.3 Hz, 2H), 2.44 (t, 7 = 7.1 Hz, 2H), 7.41 (dd, 7 = 4.8, 7.9 Hz, 1H), 7.57 (d, 7 = 8.6 Hz, 2H), 7.74 (d, 7 = 8.6 Hz, 2H), 8.27-8.30 (m, 2H), 8.61-8.63 (m, 1H), 9.08 (s, 1H); 13C NMR (100 MHz,
CDCI3) δ 14.1, 19.5, 22.6, 28.5, 28.6, 31.4, 79.3, 93.0, 117.8, 120.2, 123.1, 123.9, 125.2, 126.4, 133.0, 133.3, 135.6, 139.5, 145.4, 147.1, 149.6, 153.2, ESI-HRMS (M + H)+ /z calcd for C21H23N4 331.1923, found 331.1919. Catalytic hydrogenation afforded 3-(l-(4- octylphenyl)-lH-l,2,3-triazol-4-yl)pyridine (RB-054) in 82% yield; 1H NMR (400 MHz, CDCl3) 5 0.89 (t, = 6.8 Hz, 3H), 1.25-1.35 (m, 10H), 1.62-1.68 (m, 6H), 2.69 (t, = 7.7 Hz, 2H), 7.36 (d, = 8.3 Hz, 2H), 7.42 (dd, = 4.8, 7.8 Hz, 1H), 7.69 (d, = 8.3 Hz, 2H), 8.25 (s, 1H), 8.30 (d, = 7.9 Hz, 1H), 8.61-8.63 (m, 1H), 9.08 (s, 1H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.7, 29.2, 29.3, 29.4, 29.7, 31.4, 31.9, 35.5, 118.1, 120.6, 123.9, 126.6, 129.8, 133.2, 134.7, 144.4, 145.2, 147.1, 149.4; ESI-HRMS (M + H)+ mlz calcd for C21H27N4 335.2236, found 335.2232.
Preparation of 4-(4-Octyl-lH-1.2.3-triazol-l-yi)phenol (RB-055)
RB-055
4-(4-Octyl-lH-l,2,3-triazol-l-yl)phenol (RB-055) was prepared from 4- aminophenol according to a procedure similar to that described in the above click reaction; yield = 70%; 1H NMR (400 MHz, CDC13) δ 0.86 (t, = 6.7 Hz, 3H), 1.23-1.38 (m, 10H), 1.72 (t, = 8.6 Hz, 2H), 2.79 (t, = 8.5 Hz, 2H), 7.11 (d, = 7.6 Hz, 2H), 7.54 (d, = 7.6 Hz, 2H), 7.68 (s, 1H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.6, 25.5, 29.2, 29.3, 29.4, 31.8, 116.7, 119.6, 122.3, 129.7, 148.8, 157.7; ESI-HRMS (M + H)+ mlz calcd for C16H24N3O 274.1914, found 274.1918. Preparation of 2Α Α -θαν\ΛΗΛ 3 νΪΆτο\Λ )Ό^ην\)^Άηο\ (RB-056)
RB-056
2-(4-(4-Octyl-lH-l,2,3-triazol-l-yl)phenyl)ethanol (RB-056) was prepared by a click reaction as follows. To a solution of 4-(azidophenyl)-2-ethanol (200 mg, 1.23 mmol) and 1-decyne (508 mg, 3.68 mmol) in i-BuOH/H20 (6 mL, 1: 1) were added CuS04 (196 mg, 1.23 mmol) and sodium ascorbate (243 mg, 1.23 mmol). The reaction mixture was stirred at rt for 12 h and then was diluted with EtOAc and washed with brine. The aqueous layer was extracted with EtOAc. The combined organic layers were dried (MgS04) and concentrated in vacuo. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH (10: 1), gave 296 mg (80%) of RB-056 as a white solid; 1H NMR (400 MHz, CDC13) 5 0.88 (t, = 6.9 Hz, 3H), 1.27-1.42 (m, 10H), 1.71 (quin, = 7.5 Hz, 2H), 2.76 (t, = 7.7 Hz, 2H), 2.92 (t, = 6.6 Hz, 2H), 3.89 (t, = 6.6 Hz, 2H), 7.35 (d, = 8.5 Hz, 2H), 7.61 (d, = 8.5 Hz, 2H), 7.70 (s, 1H); 13C NMR (100 MHz, CDC13) δ 14.1, 22.6, 25.6, 29.1, 29.2, 29.3, 29.4, 31.8, 38.7, 63.1, 118.9, 120.4, 130.2, 135.6, 139.7, 149.1; ESI-HRMS (M + H)+ mlz calcd for Ci8H28N30 302.2227, found 302.2230.
Preparation of RB-057 via Triazole Intermediates.
4-( 4-Butyl- 1H- 1,2,3-triazol- l-yl)phenethyl Methanesulfonate
To a solution of 4-(azidophenyl)-2-phenethyl alcohol (200 mg, 1.23 mmol) and 1- hexyne (0.42 mL, 3.68 mmol) in te/ -BuOH/H20 (6 mL, 1: 1) were added CuS04 (196 mg, 1.23 mmol) and sodium ascorbate (243 mg, 1.23 mmol). The reaction mixture was stirred at rt for 12 h and then was diluted with EtOAc and washed with brine. The aqueous layer was extracted with EtOAc. The combined organic layers were dried (MgS04) and concentrated in vacuo, affording the triazole without purification, as a yellow liquid. To a solution of triazole (1.23 mmol) and triethylamine (0.86 mL, 6.15 mmol) in CH2C12 (10 mL) at 0 °C was added methanesulfonyl chloride (0.29 mL, 3.69 mmol). After being stirred at rt for 5 h, the reaction mixture was evaporated, diluted with water, and the product was extracted with EtOAc. The extract was washed with brine,
dried, and evaporated. Purification by silica gel chromatography, eluting with
hexanes/EtOAc (1:2), gave 253 mg (66%, 2 steps) of 4-(4-butyl-lH-l,2,3-triazol-l- yl)phenethyl methanesulfonate as a white solid; 1H NMR (400 MHz, CDC13) δ 0.96 (t, = 7.3 Hz, 3H), 1.43 (sex, / = 7.5 Hz, 2H), 1.72 (quin, / = 7.6 Hz, 2H), 2.80 (t, = 7.7 Hz, 2H), 2.92 (s, 3H), 3.12 (t, = 6.7 Hz, 2H), 4.45 (t, = 6.7 Hz, 2H), 7.38 (d, = 8.5 Hz, 2H), 7.69 (d, = 8.5 Hz, 2H), 7.73 (s, 1H); 13C NMR (100 MHz, CDC13) δ 13.9, 22.3, 25.3, 31.5, 31.6, 35.1, 37.4, 69.7, 118.8, 120.6, 130.3, 136.3, 136.9, 149.2; ESI-HRMS (M + H)+ mlz calcd for Ci5H22N303S 324.1382, found 324.1382.
Preparation of 4-(4-Pentyl-lH-l,2,3-triazol-l-yl)phenethyl Methanesulfonate This compound was prepared according to a coupling procedure similar to that described for 4-(4-butyl-lH-l,2,3-triazol-l-yl)phenethyl methanesulfonate , using 1- heptyne; yield = 58% (2 steps); 1H NMR (400 MHz, CDC13) δ 0.91 (t, = 7.1 Hz, 3H), 1.36-1.40 (m, 4H), 1.74 (quin, / = 7.5 Hz, 2H), 2.79 (t, = 7.7 Hz, 2H), 2.93 (s, 3H), 3.12 (t, = 6.7 Hz, 2H), 4.46 (t, = 6.7 Hz, 2H), 7.38 (d, = 8.5 Hz, 2H), 7.69 (d, = 8.5 Hz, 2H), 7.74 (s, 1H); 13C NMR (100 MHz, CDC13) δ 14.0, 22.4, 25.6, 29.1, 31.4, 31.6, 35.1, 37.4, 60.4, 69.7, 118.8, 120.6, 130.3, 136.2, 136.9, 149.3; ESI-HRMS (M + H)+ mlz calcd for Ci6H24N303S 338.1538, found 338.1536.
Preparation of 1-(4-(4-Β^ν1-1^1,2,3-ΐ 3ζο1-1-ν1)ρΗ6η6ΐΗν1)ρίρ6 (1ίη6 (RB-057)
RB-057
To a solution of 4-(4-pentyl-lH-l,2,3-triazol-l-yl)phenethyl methanesulfonate (50 mg, 0.15 mmol) in 3 mL of acetonitrile was added piperidine (150 \L, 1.54 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH (5: 1), gave 11 mg (75%) of RB-057 as a slightly yellow waxy solid; 1H NMR (400 MHz, CDC13) δ 0.96 (t, = 7.4 Hz, 3H), 1.42
(sex, J = 7.4 Hz, 2H), 1.51-1.55 (m, 2H), 1.72 (quin, J = 7.6 Hz, 2H), 1.80 (quin, J = 5.4 Hz, 4H), 2.74-2.83 (m, 8H), 3.04-3.08 (m, 2H), 7.36 (d, / = 8.4 Hz, 2H), 7.63 (d, / = 8.4 Hz, 2H), 7.71 (s, 1H); 13C NMR (100 MHz, CDC13) δ 13.9, 22.3, 23.5, 24.7, 25.3, 29.7, 31.5, 31.9, 54.1, 60.0, 118.8, 120.5, 130.0, 135.7, 139.6, 149.1; ESI-HRMS (M + H)+ /z calcd for C19H29N4313.2392, found 313.2385.
Preparation of 1-(4-(4-Ρ6η1ν1-1^1,2,3-1 ζο1-1-ν1)ρΗ6η6ΐΗν1)ρίρ6 (1ίη6 (RB-058)
RB-058
Compound RB-058 was prepared from 27 according to a coupling procedure similar to that described for RB-057; yield = 81%; 1H NMR (400 MHz, CDC13) δ 0.91 (t, J = 6.9 Hz, 3H), 1.36-1.43 (m, 4H), 1.47-1.52 (m, 2H), 1.67-1.77 (m, 6H), 2.53-2.61 (m, 4H), 2.65-2.69 (m, 2H), 2.78 (d, = 7.7 Hz, 2H), 2.92-2.96 (m, 2H), 7.34 (d, = 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H); 13C NMR (100 MHz, CDC13) δ 14.0, 22.4, 24.0, 25.5, 25.7, 29.1, 31.5, 32.7, 54.4, 60.7, 118.8, 120.5, 129.9, 135.6, 140.6, 149.1; ESI-HRMS (M + H)+ mlz calcd for C20H3iN4 327.2549, found 327.2543. Preparation of 1- 4- 4-θ€Ϊν1-1^1,2,3-ΐ 3ζο1-1-ν1)ρΗ6η6ΐΗν1)ρίρ6 (1ίη6 (RB-059)
RB-059
To a solution of RB-056 (50 mg, 0.17 mmol) and triethylamine (116 \L, 0.83 mmol) in CH2C12 (5 mL) at 0 °C was added methanesulfonyl chloride (39 μί, 0.51 mmol). After being stirred at rt for 5 h, the reaction mixture was evaporated, diluted with
water, and the product was extracted with EtOAc. The extract was washed with brine, dried, and evaporated to afford the mesylate as a yellow liquid. To a solution of 65 mg (0.17 mmol) of the mesylate (without purification) in 3 mL of acetonitrile was added piperidine (168 μί, 1.70 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. Purification by silica gel chromatography, eluting with CH2Cl2/MeOH
(5: 1), gave 11 mg (66%) of RB-059 as a slightly yellow waxy solid; 1H NMR (400 MHz, CDC13) 5 0.88 (t, J = 6.8 Hz, 3H), 1.25-1.39 (m, 10H), 1.58-1.62 (m, 2H), 1.72 (quin, / = 7.3 Hz, 2H), 1.89 (quin, / = 5.1 Hz, 4H), 2.78 (t, = 7.7 Hz, 2H), 2.96-3.04 (m, 6H), 3.14-3.18 (m, 2H), 7.40 (d, = 8.1 Hz, 2H), 7.64 (d, = 8.1 Hz, 2H), 7.67 (s, 1H); 13C NMR (100 MHz, CDC13) δ 14.0, 22.6, 22.8, 23.9, 25.6, 29.1, 29.2, 29.3, 30.9, 31.8, 53.8, 59.1, 118.9, 120.6, 130.0, 135.8, 138.5, 149.1, 173.3; ESI-HRMS (M + H)+ m/z calcd for C23H37N4 369.3013, found 369.3015; (M + H - N2)+ m/z calcd for C23H35N2 341.2951, found 341.2954.
Preparation of 1-(4-(4-Βυ1ν1-1^1,2,3-1 ζο1-1-ν1 Η6η6ΐΗν1 -1-ιη6ΐΗν1 ί 6 (1ίιιίυιιι Methanesulfonate (RB-060)
To a solution of 4-(4-butyl-lH-l,2,3-triazol-l-yl)phenethyl methanesulfonate (15 mg, 46 μιηοΐ) in 3 mL of acetonitrile was added 1-methylpiperidine (23 mg, 0.23 mmol). The reaction mixture was stirred at 50 °C for 12 h and concentrated. The residue was washed with hexane to give 16 mg (82%) of RB-060 as a yellow oil; 1H NMR (400 MHz, CDC13) 5 0.95 (t, = 7.3 Hz, 3H), 1.41 (sex, = 7.5 Hz, 2H), 1.66-1.74 (m, 4H), 1.80- 1.87 (m, 4H), 2.77 (s, 3H), 2.74-2.84 (m, 2H), 3.13-3.17 (m, 2H), 3.57 (t, = 5.5 Hz, 4H), 3.71-3.76 (m, 2H), 7.54 (d, = 8.5 Hz, 2H), 7.62 (d, = 8.5 Hz, 2H), 7.76 (s, 1H); 13C NMR (100 MHz, CDC13) δ 13.9, 20.2, 20.8, 21.5, 22.3, 22.8, 25.3, 27.8, 31.5, 39.6,
44.2, 55.4, 61.0, 119.1, 120.6, 120.7, 136.1, 136.2, 149.2; ESI-HRMS (M)+ mlz calcd for C2oH3iN4 + 327.2549, found 327.2546.
Preparation of l-Methyl-l-(4-(4-pentyl-lH-1.2.3-triazol-l- yl)phenethyl)piperidinium Methanesulfonate (RB-061)
Compound RB-061 was prepared according to a coupling procedure similar to that described for RB-060; yield = 77%; 1H NMR (400 MHz, CDC13) δ 0.90 (t, = 7.2 Hz, 3H), 1.34-1.39 (m, 4H), 1.68-1.75 (m, 4H), 1.81-1.86 (m, 4H), 2.76 (s, 3H), 2.74- 2.83 (m, 2H), 3.13-3.18 (m, 2H), 3.28 (s, 3H), 3.59 (t, = 5.5 Hz, 4H), 3.75-3.79 (m, 2H), 7.55 (d, = 8.5 Hz, 2H), 7.64 (d, = 8.5 Hz, 2H), 7.76 (s, 1H); 13C NMR (100 MHz, CDC13) δ 14.0, 20.2, 20.8, 21.5, 22.4, 22.8, 25.6, 27.9, 29.1, 31.5, 39.7, 44.2, 55.3, 61.0, 119.0, 120.6, 120.8, 130.3, 130.8, 136.0, 136.2, 149.3; ESI-HRMS (M)+ mlz calcd for C2iH33N4 + 341.2705, found 341.2704.
Preparation of 1-Μ6ΐΗν1-1-(4-(4-θ€Ϊν1-1^1,2,3-ΐ 3ζο1-1-ν1)ρΗ6η6ΐΗν1)ρίρ6Γί(1ίηίυηι methanesulfonate (RB-062)
Compound RB-062 was prepared according to a coupling procedure similar to that described for RB-060; yield = 71%; 1H NMR (400 MHz, CDC13) δ 0.87 (t, = 7.1 Hz, 3H), 1.27-1.49 (m, 10H), 1.71-1.78 (m, 4H), 1.85-1.89 (m, 4H), 2.76 (s, 3H), 2.80- 2.90 (m, 2H), 3.14-3.18 (m, 2H), 3.35 (s, 3H), 3.48 (t, = 5.5 Hz, 4H), 3.67-3.85 (m,
2H), 7.60 (d, = 8.5 Hz, 2H), 7.68 (d, = 8.5 Hz, 2H), 7.83 (s, 1H); 13C NMR (100 MHz,
CDCI3) δ 14.2, 20.2, 20.5, 20.9, 21.4, 22.7, 23.0, 25.7, 27.9, 28.9, 29.1, 29.3, 29.4, 29.5, 31.9, 39.6, 44.0, 55.0, 61.1, 119.1, 120.4, 120.8, 130.2, 130.8, 136.1, 136.3, 149.3; ESI- HRMS (M)+ m/z calcd for C24H39N4+ 383.3169, found 383.3174.
Preparation of 1-(4-(4-Β^ν1-1^1,2,3-1 ζο1-1-ν1)ρΗ6η6ΐΗν1)ρίρ6 (1ίιι-4-ο1 (RB-063)
Compound RB-063 was prepared according to a coupling procedure similar to that described for RB-057, using 4-hydroxypiperidine; yield = 65%; 1H NMR (400 MHz, CDC13) δ 0.96 (t, = 7.4 Hz, 3H), 1.42 (sex, = 7.4 Hz, 2H), 1.60-1.75 (m, 4H), 1.93- 1.97 (m, 2H), 2.26 (t, = 9.4 Hz, 2H), 2.60-2.65 (m, 2H), 2.79 (t, = 7.7 Hz, 2H), 2.85- 2.89 (m, 4H), 3.75 (sep, = 4.4 Hz, 1H), 7.33 (d, = 8.4 Hz, 2H), 7.62 (d, = 8.4 Hz, 2H), 7.69 (s, 1H); 13C NMR (100 MHz, CDC13) δ 13.9, 22.3, 25.3, 29.7, 31.5, 33.3, 34.4, 51.1, 60.0, 67.6, 118.8, 120.4, 129.9, 135.5, 141.0, 149.1; ESI-HRMS (M + H)+ mlz calcd for Ci9H29N40 329.2341, found 329.2336.
Preparation of 1- 4- 4-Ρ6ηΐν1-1^1,2,3-ΐ 3ζο1-1-ν1)ρΗ6η6ΐΗν1)ρίρ6Γί(1ίη-4-ο1 (RB- 064)
Compound RB-064 was prepared according to a coupling procedure similar to that described for RB-057, using 4-hydroxypiperidine; yield = 70%; 1H NMR (400 MHz, CDC13) 5 0.90 (t, = 7.1 Hz, 3H), 1.34-1.42 (m, 4H), 1.61-1.77 (m, 4H), 1.93-1.97 (m, 2H), 2.26 (t, = 9.5 Hz, 2H), 2.61-2.65 (m, 2H), 2.78 (t, = 7.7 Hz, 2H), 2.85-2.89 (m, 4H), 3.75 (sep, / = 4.2 Hz, 1H), 7.33 (d, = 8.4 Hz, 2H), 7.62 (d, = 8.4 Hz, 2H), 7.69
(s, 1H); 13C NMR (100 MHz, CDC13) δ 14.0, 22.4, 25.6, 29.1, 31.4, 33.3, 34.4, 51.0, 60.0, 67.6, 118.8, 120.4, 129.9, 135.5, 141.0, 149.1; ESI-HRMS (M + H)+ mlz calcd for C20H31N4O 343.2498, found 343.2495.
Preparation of 1-(4-(4-θ€ΐν1-1^1,2,3-ΐ ζο1-1-ν1) Η6η6ΐΗν1) ί 6Γί(1ίιι-4- ΐιιίη6 (RB- 065).
Compound RB-065 was prepared according to a coupling procedure similar to that described for RB-057, using 4-hydroxypiperidine; yield = 63%; 1H NMR (400 MHz, CDCI3) δ 0.87 (t, 7 = 6.7 Hz, 3H), 1.27-1.39 (m, 12H), 1.71 (quin, 7 = 7.5 Hz, 2H), 1.77- 1.84 (m, 2H), 2.77 (t, 7 = 7.7 Hz, 4H), 2.91-2.95 (m, 2H), 3.05-3.07 (m, 2H), 3.09-3.14 (m, 2H), 3.89-3.93 (m, 1H), 7.37 (d, 7 = 8.4 Hz, 2H), 7.63 (d, 7 = 8.4 Hz, 2H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.6, 25.6, 29.1, 29.2, 29.3, 29.4, 31.7, 50.0, 58.9, 118.9, 120.6, 130.0, 135.8, 138.9, 149.2; ESI-HRMS (M + H)+ m/z calcd for C23H37N4O
385.2962, found 385.2965.
Preparation of (5^)-3-azido-3-(hvdroxymethyl)-5-(4-octylphenyl)pent-l- enylphosphonic acid and (5,E)-3-azido-3-(fluoromethyl)-5-(4-octylphenyl)pent-l- enylphosphonic acid
(S)-2-(4-octylphenethyl)oxirane- 2-carbaldehyde
3-azido-3-hydroxymethyl derivative of (S)-FTY720 vinylphosphonate
3-azido-3-fluoromethyl derivative of
(S)-FTY720 vinylphosphonate
Scheme 5. Preparation of (S,E)-3-azido-3-(hydroxymethyl)-5-(4-octylphenyl)pent-l- enylphosphonic acid and (S,E)-3-azido-3-(fluoromethyl)-5-(4-octylphenyl)pent- l- enylphosphonic acid
Data for (5)-2-(4-octylphenethyl)oxirane-2-carbaldehyde: 1H NMR (500 MHz, CDC13) δ 0.83-0.92 (m, 3H), 1.22-1.36 (m, 10H), 1.56-1.64 (m, 2H), 2.01-2.08 (m, 1H), 2.20- 2.28 (m, 1H), 2.58 (t, = 7.8 Hz, 2H), 2.72 (t, = 8.2 Hz, 2H), 2.99 (d, = 4.6 Hz, 1H), 3.04 (d, = 4.6 Hz, 1H), 7.10-7.13 (m, 4H), 8.89 (s, 1H); 13C NMR (125 MHz, CDC13) δ 14.1, 22.6, 29.2, 29.3, 29.5, 29.9, 30.2, 31.5, 31.9, 35.5, 49.8, 60.9, 128.1, 128.5, 138.0, 140.8, 198.8; ESI-HRMS (M + Na)+ m/z calcd for CigHisNaC^ 311.1982, found
311.1986.
(E)-Dimethyl 2-[(/?)-2-(4-octylphenethyl)oxiran-2-yl]vinylphosphonate. To a mixture of NaH (57-63% oil dispersion, 240 mg, 6.00 mmol) and 50 mL of THF was added a solution of 1.41 g (6.08 mmol) of CH2[(P(0)(OMe)2] in 10 mL of THF at 0 °C. After the
mixture had been stirred for 30 min, a solution of (S)-2-(4-octylphenethyl)oxirane-2- carbaldehyde (585 mg, 2.03 mmol) in 10 mL of THF was added. The reaction mixture was stirred for 1 h at 0 °C, quenched with saturated aqueous NH4C1 solution, and extracted with EtOAc. The organic layer was dried (MgS04) and concentrated under reduced pressure. The residue was purified by chromatography (elution with EtOAc) to give 750 mg (94%) of the desired dimethyl phosphonate product. 1H NMR (400 MHz, CDCI3) δ 0.83-0.92 (m, 3H), 1.20- 1.38 (m, 10H), 1.53-1.64 (m, 2H), 1.90-2.01 (m, IH), 2.08-2.18 (m, IH), 2.53-2.59 (m, 2H), 2.62-2.77 (m, 3H), 2.87 (t, J = 5.4 Hz, IH), 3.72 (d, J = 5.5 Hz, 3H), 3.74 (d, = 5.5 Hz, 3H), 5.95 (dd, / = 17.2, 19.4 Hz, IH), 6.83 (dd, = 17.2, 22.2 Hz, IH), 7.05-7.13 (m, 4H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.6, 29.2, 29.3, 29.4, 30.6, 31.5, 31.8, 35.2, 35.5, 52.38 (d, = 5.4 Hz), 52.41 (d, = 5.4 Hz), 55.9, 58.2 (d, = 24.0 Hz), 116.5 (d, = 189.6 Hz), 128.0, 128.5, 137.9, 140.8, 151.6 (d, = 6.5 Hz); 31P NMR (162 MHz, CDC13) δ 20.6; ESI-HRMS (M + H)+ m/z calcd for
C22H3604P+ 395.2346, found 395.2346.
(S,E)-Dimethyl 3-azido-3-(hydroxymethyl)-5-(4-octylphenyl)pent-l- enylphosphonate. Ti(0-z'-Pr)4 (2.4 mL, 8.02 mmol) and TMSN3 (2.2 mL, 16.6 mmol) were added to anhydrous toluene (50 mL), and the mixture was heated at reflux (85 °C) under N2 for at least 5 h. A solution of the epoxide (670 mg, 1.70 mmol) in anhydrous toluene (10 mL) was added to the above solution in one portion. The mixture was stirred for 15 min at 85 °C and was then cooled to rt. The solvent was removed under reduced pressure. Et20 (20 mL) was added, followed by 10% HC1 (40 mL). The solution was stirred at rt until two clear phases appeared. The aqueous phase was extracted with Et20. The organic phase was dried over MgS04, filtered, and concentrated under reduced pressure. Purification by flash chromatography (elution with EtOAc) afforded the target 3-azido-3-hydroxymethyl dimethyl phosphonate ester (500 mg, 68%) as a yellow oil. 1H NMR (500 MHz, CDC13) δ 0.83-0.92 (m, 3H), 1.20-1.36 (m, 10H), 1.54-1.62 (m, 2H), 1.90- 1.99 (m, IH), 2.03-2.11 (m, IH), 2.51-2.67 (m, 4H), 3.01 (s, IH, OH), 3.70-3.80 (m, 8H), 6.03 (dd, = 17.1, 19.3 Hz, IH), 6.72 (dd, = 17.2, 22.7 Hz, IH), 7.06-7.12 (m, 4H); 13C NMR (100 MHz, CDCI3) δ 14.1, 22.6, 29.2, 29.3, 29.46, 29.50, 31.6, 31.9, 35.5,
36.0, 52.53 (d, J = 5.5 Hz), 52.55 (d, = 5.5 Hz), 67.4, 69.0 (d, = 19.4 Hz), 118.1 (d, = 186.9 Hz), 128.1, 128.6, 137.9, 140.9, 151.0 (d, = 6.3 Hz); 31P NMR (162 MHz, CDC13) δ 20.5; ESI-HRMS (M + H)+ m/z calcd for CiiHseNsC P* 438.2516, found 438.2519. (5^)-3-Azido-3-(hydroxymethyl)-5-(4-octylphenyl)pent-l-enylphosphonic acid. To a solution of the dimethyl phosphonate ester (10 mg, 0.023 mmol) in 2 mL of dry CH2CI2 at rt was added 30 mL (0.23 mmol) of TMSBr. After the reaction mixture had been stirred for 6 h, the solvent was removed, and the residue was dried and dissolved in 2 mL of 95% MeOH with stirring for 1 h. Removal of the solvent afforded 9 mg (100%) of the product. 1H NMR (500 MHz, CDCI3) δ 0.83-0.92 (m, 3H), 1.20- 1.36 (m, 10H), 1.52-1.63 (m, 2H), 1.85- 1.94 (m, 1H), 1.97-2.07 (m, 1H), 2.52-2.68 (m, 4H), 3.67-3.74 (m, 2H), 6.09-6.22 (m, 1H), 6.45-6.59 (m, 1H), 7.07-7.13 (m, 4H); 13C NMR (125 MHz, CDC13) δ 13.7, 22.4, 28.98, 29.04, 29.1, 29.2, 29.4, 31.3, 31.6, 35.2, 36.0, 68.4 (d, = 18.4 Hz), 127.8, 128.4, 138.1, 140.5, 146.7; 31P NMR (202 MHz, CDC13) δ 15.5; ESI-HRMS (M + H)+ m/z calcd for C22H33N304P+ 410.2203, found 410.2206.
(S,E)-Dimethyl 3-Azido-3-(fluoromethyl)-5-(4-octylphenyl)pent-l-enylphosphonate.
To a solution of alcohol (S,E) -dimethyl 3-azido-3-(hydroxymethyl)-5-(4- octylphenyl)pent- l-enylphosphonate (75 mg, 0.171 mmol) in anhydrous CH2C12 (1.0 mL) was added diethylaminosulfur trifluoride (DAST) (68 mL, 0.515 mmol) at -78 °C. The reaction mixture was stored at -78 °C overnight, and then was stirred at rt for 3 h. The mixture was poured into aqueous saturated NaHC03 solution, the aqueous phase was extracted with CH2C12, and the combined CH2C12 layers were dried (Na2S04), filtered, and concentrated under reduced pressure. Purification by flash chromatography (elution with PhMe/EtOAc, 1 : 1 to 100% EtOAc) afforded 23 (49 mg, 65%), along with 10 mg (13%) of recovered alcohol. 1H NMR (500 MHz, CDCI3) δ 0.83-0.92 (m, 3H), 1.20- 1.36 (m, 10H), 1.53- 1.63 (m, 2H), 1.88-2.02 (m, 1H), 2.12-2.22 (m, 1H), 2.51-2.60 (m, 3H), 2.64-2.72 (m, 1H), 3.34-3.53 (m, 2H), 3.77 (d, 7 = 11.1 Hz, 6H), 6.12 (dd, J = 17.1, 18.8, 1H), 6.73 (ddd, = 17.1, 21.4, 22.8 Hz, 1H), 7.03-7.13 (m, 4H); 13C NMR (100 MHz,
CDCI3) δ 14.1, 22.6, 28.6, 28.7, 29.2, 29.3, 29.4, 31.5, 31.9, 35.5, 37.4 (d, J = 21.9 Hz), 52.5 (t, = 5.5 Hz), 56.5 (d, = 23.9 Hz), 97.5 (dd, = 19.6, 184.9 Hz), 118.0 (dd, = 9.6, 187.8 Hz), 128.0, 128.6, 137.5, 141.0, 149.5 (dd, = 5.8, 20.4 Hz); 31P NMR (162 MHz, CDCI3) δ 19.6; 19F NMR (376 MHz, CDC13) δ -164.5; ESI-HRMS (M + H)+ m/z calcd for CiiHseFNsOsP* 440.2473, found 440.2472.
(5^)-3-Azido-3-(fluoromethyl)-5-(4-octylphenyl)pent-l-enylphosphonic acid. To a solution of (S,E) -dimethyl 3-azido-3-(fluoromethyl)-5-(4-octylphenyl)pent- l- enylphosphonate (10 mg, 0.023 mmol) in 2 mL of dry CH2CI2 at rt was added 30
(0.23 mmol) of TMSBr. After the reaction mixture had been stirred for 6 h, the solvent was removed, and the residue was dried and dissolved in 2 mL of 95% MeOH with stirring for 1 h. Removal of the solvent afforded 10 mg (100%) of the product as a white solid. 1H NMR (400 MHz, CDCI3/CD3OD/CD3CO2D 80:20: 1) δ 0.83-0.92 (m, 3H), 1.20- 1.36 (m, 10H), 1.52- 1.63 (m, 2H), 1.87-2.05 (m, 1H), 2.10-2.24 (m, 1H), 2.50-2.69 (m, 4H), 3.40-3.50 (m, 2H), 6.16-6.28 (m, 1H), 6.49-6.68 (m, 1H), 7.04-7.14 (m, 4H); 13C NMR (125 MHz, CDCI3/CD3OD/CD3CO2D 80:20: 1) δ 13.7, 22.3, 28.3, 29.0, 29.2, 31.3, 31.6, 35.2, 37.1, 37.3, 56.3 (d, = 24.4 Hz), 97.2 (d, = 184.2 Hz), 127.8, 128.3, 137.7, 140.6, 145.2; 31P NMR (162 MHz, CDCI3/CD3OD/CD3CO2D 80:20: 1) δ 15.2; ESI- HRMS (M + H)+ m/z calcd for C2oH32FN03P+ 386.2255, found 386.2256.
ASSAYS
Cell Culture. HEK 293 cells stably over-expressing GFP-SK1 (30-fold increase in SKI activity versus vector-transfected cells) were cultured in DMEM supplemented with 10% European fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 1%
nonessential amino acids, and 0.8% geneticin at 37 °C in 5% CO2.
Sphingosine Kinase Activity Assays. In order to measure SK2 activity, sphingosine (Sph) was complexed with fatty acid free bovine serum albumin (final concentration, 0.2 mg/mL) in buffer A containing 20 mM Tris (pH 7.4), 1 mM EDTA, 1 mM Na3V04, 40 mM β-glycerophosphate, 1 mM NaF, 0.007% (v/v) β-mercaptoethanol, 20% (v/v) glycerol, 10 μg/mL aprotinin, 10 μg/mL soybean trypsin inhibitor, 1 mM PMSF, 0.5 mM
4-deoxypyridoxine, and 400 niM KCl. SK2 assays were performed using 37 ng of purified SK2 and incubating the assay for 30 min at 30 °C in the presence of 10 μΜ Sph, 250 μΜ [γ-32Ρ]ΑΤΡ in 10 mM MgCl2, and varying concentrations of the inhibitors dissolved in DMSO or control (5% v/v DMSO). To measure SKI activity, Sph was solubilized in Triton X-100 (final concentration, 0.063% w/v) and combined with buffer A without KCl. 30 μg of recombinant SKI was incubated for 30 min at 30 °C, in the presence of 3 μΜ Sph, 250 μΜ [γ-32Ρ]ΑΤΡ in 10 mM MgCl2 with or without inhibitor dissolved in DMSO or control (5% v/v DMSO). Both assay reactions were terminated by the addition of 500 μΐ^ of 1-butanol. After 1 mL of 2 M KCl was added, with mixing, two phases were formed. The lower (aqueous) phase, which contains unreacted [γ- 32 P]ATP, was removed and discarded. The organic phase containing [ 32 P]-S 1P was extracted by washing twice with 2 M KCl (1 mL each time) before quantification by Cerenkov counting. To evaluate the test compounds as putative substrates of SKI and SK2, the assay was conducted in the presence of 50 μΜ of the test compound (but in the absence of Sph) and radioactivity in the 1-butanol phase was quantified.
Claims
A compound having formula I:
(I)
wherein
R1 is a hydrogen, lower alkyl, or lower alkoxy;
R 2" and R 3J are independently hydrogen, Q-Qo alkyl, or -C(X)NHAr; wherein
X is oxygen or sulfur;
Ar is aryl or heteroaryl group; R4 is a hydrogen, or hydroxyl;
R5 is a CmH2m+i straight-chain or branched alkyl, C2-C2o-alkenyl, C2-C2o-alkynyl, or Ci-C2o-alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;
wherein bond A is a single bond or a double bond;
with the proviso that when R 2 and R 3 are hydrogen, R 1 is not methyl; and with the proviso that when A is a double bond, R1 is not hydroxymethyl.
2.) The compound according to claim 1, wherein the heteroaryl group is mono- or poly-halophenyl.
3.) The compound according to claim 1, selected from the group consisting of:
, and
A compound having formula II:
R1 is C3-C12 alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl, having a single cyclic ring or multiple condensed rings, quaternary ammonium group, or
wherein Z is -OH, F, Br, CI, or I;
R is straight-chain or branched alkyl CmH2m+i, C2-C2o-alkenyl, C2-C2o-alkynyl, Ci- C2o-alkoxy, or C2-C2o-alkyl- substituted heterocycle; m is 1, 2, 3,
4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; n is 0, 1, 2, 3, 4, or 5;
W is -CH2 or oxygen; and
X and Y are independently hydrogen, Ci-C4-alkyl, or X and Y taken together are oxygen or sulfur.
6.) The compound according to claim 5, wherein R1 is selected from the group consisting of:
n is 0, 1, 2, 3, 4, or 5; and
R is C3-C7 -alkyl.
7. ) The compound according to claim 5, wherein the heterocycle is selected from the group consisting of triazole, oxadiazole, oxazole, and thiazole.
8. ) The compound according to claim 5, selected from the group consisting of:
wherein
R is hydrogen or hydroxyl; mis 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and nis 0, 1,2,3,4,5,6, 7, 8,9, or 10.
A compound according to claim 5, wherein said compound comprises:
wherein R is a CmH2m+i straight-chain or branched alkyl, C2-C2o-alkenyl, C2-C2o-alkynyl, or
Ci-C2o-alkoxy; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;
X is -OH, F, Br, CI, or I; n is 0, 1, 2, 3, 4, 5, or 6; and W is oxygen or carbon.
A compound having formula III:
9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;
R is hydrogen, hydroxyl, or Ci-C2o-alkoxyl; R is oxygen or sulfur;
R4 is aryl or heteroaryl; and
X and Y are independently NH or oxygen.
11.) The compound according to claim 10, wherein the heteroaryl group is mono- or poly-halophenyl.
A compound according to claim 10, selected from the group consisting of:
13) A pharmaceutical formulation comprising a compound according to any preceding claim, together with a pharmaceutically acceptable excipient.
14) A compound of claim 3 or claim 8 or claim 12 for use in a method of selectively inhibiting SKI in a cell.
15) A compound for use in the method according to claim 14 for use in treating a disease associated with abnormal cell proliferation, such as a cancer and pulmonary arterial hypertension. 16) A compound of claim 4 for use in a method of selectively inhibiting SK2 in a cell.
A compound for use in the method according to claim 16 for use in treating disease associated with abnormal cell proliferation, such as a cancer and pulmonary arterial hypertension.
18) A compound of claim 9 for use in a method of selectively activating SKI in a cell.
A compound for use in the method according to claim 18 for use in treating fibrosis.
20) A compound of claim 3, claim 8 or claim 12 for use in a method of inducing apoptosis in a cell.
21) A compound of formula IV, or czs-sphingosine for use in a method of selectively inhibiting SKI in a cell:
(IV)
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| US10583115B2 (en) | 2014-10-08 | 2020-03-10 | University Of South Australia | Modulators of 14-3-3 functionality and uses thereof |
| WO2016085933A1 (en) | 2014-11-24 | 2016-06-02 | The Board Of Trustees Of The University Of Illinois | Method of preventing or treating a pulmonary disease or condition |
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| US11026918B2 (en) | 2014-11-24 | 2021-06-08 | The Board Of Trustees Of The University Of Illinois | Method of preventing or treating a pulmonary disease or condition |
| US11999693B2 (en) | 2015-09-24 | 2024-06-04 | The Regents Of The University Of California | Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment |
| WO2017129769A1 (en) | 2016-01-28 | 2017-08-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for enhancing the potency of the immune checkpoint inhibitors |
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