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Importance of Sphingosine Kinase (SphK) as a Target in Developing Cancer Therapeutics and Recent Developments in the Synthesis of Novel SphK Inhibitors Miniperspective Daniel Plano,†,‡ Shantu Amin,† and Arun K. Sharma*,† †
Department of Pharmacology, Penn State Hershey Cancer Institute, CH72, Penn State College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033, United States ‡ Department of Organic and Pharmaceutical Chemistry, University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain ABSTRACT: Sphingosine kinase (SphK) is an oncogenic lipid kinase that regulates the sphingolipid metabolic pathway that has been shown to play a role in numerous hyperproliferative/inflammatory diseases. The SphK isoforms (SphK1 and SphK2) catalyze the conversion of the proapoptotic substrate D-erythrosphingosine to the promitogenic/migratory product sphingosine 1-phosphate (S1P). Accumulation of S1P has been linked to the development/progression of cancer and various other diseases including, but not limited to, asthma, inflammatory bowel disease, rheumatoid arthritis, and diabetic nephropathy. SphK therefore represents a potential new target for developing novel therapeutics for cancer and other diseases. This finding has stimulated the development and evaluation of numerous SphK inhibitors over the past decade or so. In this review, we highlight the recent advancement in the field of SphK inhibitors including SphK1 and SphK2 specific inhibitors. Both sphingolipid based and nolipidic small molecule inhibitors and their importance in treatment of cancer and other diseases are discussed.
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INTRODUCTION Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes the conversion of the sphingolipid sphingosine to sphingosine 1-phosphate (S1P). The two SphK isoforms (SphK1 and SphK2) regulate this sphingolipid metabolism; however, SphK1 is more closely linked to signaling pathways associated with cancer cell proliferation/survival, metastasis, and multidrug resistance (MDR).1−5 SphK1 regulates the fate of cells by catalyzing the formation of promitogenic S1P at the expense of proapopototic ceramide. Overexpression of SphK1 is observed in many tumor tissues.6,7 Importantly, studies have shown that high SphK1 levels are a prognostic factor for decreased metastasis-free survival and overall poor outcome of patients.8−10 On the basis of these and many related studies,11−15 inhibition of SphK1 is considered a novel approach for the treatment of cancers including metastatic cancer and/or MDR. Therefore, there has been extensive focus on the development of effective SphK inhibitors (SKIs) during the past 10 years. The classes of compounds identified include those derived from sphingolipids, natural products, and nolipidic small druglike molecules. The structure−activity relationship (SAR) studies using analogues of the lipid substrates of SphK, namely, sphingosine and 1 (FTY720), have been one of the main focuses of drug design.16−18 The quest to identify a small druglike inhibitor started with a library screening reported in the year 2003,19 and several optimization studies have been reported thereafter with variable success. So © 2014 American Chemical Society
far, the development of SKIs has been hampered by the lack of a crystal structure of SphK1, and therefore, rational drug design was impractical. The recent report by Wang et al.20 describing the crystal structure of SphK1 (Figure 1) is expected to provide a new direction to SKI design, which may lead to more effective and specific inhibitors in the near future. This review focuses on the importance of targeting SphK, particularly in the treatment of cancer, highlights recent advances in the development of SKIs, and speculates on the future promise of these inhibitors.
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SPHINGOSINE KINASE AS A THERAPEUTIC TARGET The potential of the sphingolipid metabolic pathway for the development of therapeutic targets for cancer has been recognized for years. The de novo sphingolipid synthesis pathway is a multistep process that begins with the condensation of serine and palmitoyl-CoA catalyzed by serine palmitoyl tranferase and yields 3-ketosphinganine.21 The complex regulation of sphingolipid metabolism is well documented22−25 and is not the focus of this paper. The most important Cer-to-Sph-to-S1P conversion steps involve deacylation of ceramide by ceramidases to generate sphingosine and the subsequent conversion of sphingosine to S1P catalyzed Received: July 31, 2013 Published: January 28, 2014 5509
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can guide the cell toward either an apoptotic process or a survival process depending on the relative position of the rheostat. A shift toward ceramide can be achieved by stress signaling such as radiation and chemotherapy treatment or by using SKIs, driving cancer cells to undergo apoptosis and antiproliferation.22 On the other hand, a shift toward S1P accumulation drives cells to prosurvival, antiapoptosis, and/or chemoresistance conditions.27 The Cer/S1P rheostat is functional not only in cancer disease but also in other diseases such as inflammatory, cardiovascular, and fibrotic diseases.22,24 SphKs, particularly SphK1, play a central role in the sphingolipid metabolic pathway as the key enzymes regulating the equilibrium between proapoptotic ceramide/sphingosine (Cer/Sph) and promitogenic/prosurvival (S1P) (Figure 2). Therefore, there is a strong rationale for selectively targeting SphK1 as an anticancer therapeutic strategy. Thus, given its role in the Cer/S1P rheostat, the fact that overexpression of SphK1 is sufficient to promote the transformation of normal cells,5 and the accumulating evidence linking its overexpression to development/progression of numerous cancers,19,28 SphK1 is a novel target for development of anticancer therapeutic strategies. However, the sphingosine rheostat concept could be considered simplistic. Although biosynthetically ceramide lies upstream of sphingosine and S1P, there are conflicting reports regarding the effects of SphK1 inhibitors and Cer/S1P levels. Some of the reports have demonstrated no accumulation of Cer/S1P associated with inhibition of SphK1.29,30
Figure 1. Cartoon representation of the crystal structure of SphK1 in three different orientations (adapted from Protein Data Bank, PDB reference 3VZB).
by SphKs (Figure 2). There are two isoforms of SphKs known to catalyze this transformation: SphK1, which is found in the cytosol of eukaryotic cells and migrates to the plasma membrane upon activation; SphK2, which is localized to the nucleus. The S1P thus formed can be dephosphorylated by S1P phosphatases to generate sphingosine, which can be acylated to ceramide by ceramide synthase (CerS), closing the loop of the Cer/S1P interconversion (Figure 2). The only exit point for this cycle is the irreversible cleavage of S1P to hexadecenal and phosphoethanolamide mediated by S1P lyase. Finally, S1P binds to a family of five G-protein-coupled receptors, termed S1PRs (S1P1−5), and can be transported outside of the cell. The dynamic balance between the two signaling arms of sphingolipids with opposite effects (i.e., S1P and its precursors, ceramide and sphingosine) is an important factor that determines cell fate.26 This dynamic balance between ceramide and S1P signaling, known as the Cer/S1P rheostat (Figure 2),
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DEVELOPMENT OF SKIs AND THEIR APPLICATIONS IN CANCER TREATMENT The SphK/S1P pathway is involved in multiple cellular processes and is implicated in the inhibition, maintenance, and progression stages of several diseases. Hence, SphK/S1P system is now considered to be a novel and innovative target in the treatment of several diseases. During the past decade, there has been a growing interest in the development of novel inhibitors of the SphK/S1P pathway. In this section, we will analyze different structural modulations carried out to date on known SKIs, based on different structure classes, in order to increase the activity and/or selectivity for one of the SphKs. Spingosine-Based. Three well differentiated chemical regions are located in the sphingosine molecule: (1) a polar
Figure 2. Role of SphK1/2 in Cer/S1P rheostat: effects on cancer cells and therapeutic intervention by SKIs treatment. 5510
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varies with the assay used and may not be compared between the publications. However, they do provide an idea of the selectivity of the novel compounds. Compound 2, the synthetic threo stereoisomer of the naturally occurring D-erythro-dihydrosphingosine, has been shown to induce cancer cell death through apoptosis32 and autophagy33,34 in vitro in various cancer types. In addition, 2 has excelled as a promising agent for use in combination with other chemotherapeutic drugs.35−39 Compound 2 potentiates the cytotoxic effects of several chemotherapeutic agents, such as doxorubicin, irinotecan, cisplatin, and mitomycin C, in vitro and in vivo in various types of cancer.35−38 Recently, a phase I clinical trial in patients with advanced solid tumors has indicated that 2 can be safely administered in combination with cisplatin, showing only a reversible dose-dependent hepatic toxicity.39 Nevertheless, none of these effects can be attributed only to its SphK inhibition, since it has also been shown to inhibit PKC. In fact, many of the synergetic effects shown by 2 seem to be mediated by the PKC inhibition.40 A recent study has shown that a novel liposomal formulation of 2 is able to extend the median survival time in an acute myeloid leukemia murine model.41 Compound 3 has also been shown to induce apoptosis in a variety of human cancer cell lines42,43 and acted synergetically with N-(4-hydroxyphenyl)retinamide in an ovarian carcinoma cell line.44 Ghosh et al. have reported the synthesis of a variety of coumarin derivatives of 3, although no improvement in the cytotoxicity was achieved with these derivatives.45 De Jonghe and co-workers46 reported the synthesis and SphK inhibitory effects of several short-chain sphinganine and 3-fluorosphingosine analogues (Figure 4). The results allowed them to establish an SAR: (1) a shortened lipophilic backbone (C12) was as potent as 3 in inhibiting sphingosine kinases; (2) the 4,5-trans double bond in the short-chain analogues had a significant inhibitory effect; (3) the replacement of the hydrocarbon backbone by an aromatic residue in the short-
head, comprising an hydroxyl group, which can be phosphorylated; (2) a lipophilic tail, formed by a long hydrocarbon chain; (3) a linker that connects the polar head and the lipophilic tail, which is composed of a hydroxyl and an amine group (Figure 3). The different structural modifications of sphingosine
Figure 3. Sphingosine structure and its key chemical regions.
reported in the literature can be divided into two groups: (1) those modifications that leave the polar headgroup unchanged and (2) the modifications that alter the polar head. Unchanged Polar Head. Some of the first SKIs reported emerged from minor modifications of the sphingosine molecule, i.e., the removal of the 4,5-trans double bond presented in the lipophilic tail, resulting in 2 (safingol), and the methylation of the amine group located in the linker, giving 3 (DMS) (Figure 4).24 Neither 2 nor 3 can be considered as a specific SKI. Compound 2 is a competitive inhibitor of SphK1 (Ki ≈ 3−6 μM),31 although it acts as substrate for SphK2 and can be incorporated into the sphingolipid metabolic pathway.24 Compound 3 is an inhibitor of both SphK isoforms, presenting Ki values of 5 and 12 μM for SphK1 and SphK2, respectively.24 Furthermore, they show inhibitory effects on important cellular kinases, such as protein kinase C (PKC), ceramide kinase (CK), phosphatidylinositol kinase (PI3K), mitogen-activated protein kinase (MAPK), and epidermal growth factor receptor (EGFR).24 It is noted here that the calculation of Ki values
Figure 4. Chemical structures of sphingosine-based SKIs grouped by the various modifications carried out over the sphingosine structure. 5511
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Figure 5. Structures of 1 and its analogues.
cancer cell lines in vitro and markedly reduced tumor growth in xenograft models.12,49 These studies suggest that selective inhibition of SphK1 may be important in glioblastoma and leukemia, and thus, SphK1 can be considered as a promising target for treatment of these cancer types. One of the most studied sphingosine-based analogues is compound 1 (Figures 4 and 5), which acts through Tlymphocyte-specific immunosuppression.50 Compound 1 is phosphorylated by SphK2, and the fingolimod phosphate (FTY720-P) thus generated (but not parent compound 1) acts as an agonist at four of the five S1PRs (excluding S1P2), with EC50 values in the nanomolar range.51,52 Compound 1 has been shown to prevent organ transplant rejection and used in the treatment of several autoimmune diseases, for which it is currently undergoing multiple clinical trials. Compound 1 has also been demonstrated to reduce tumor growth and prevent cancer progression in murine models of several types of cancer.53−57 Several recent reviews address its mechanism of action in depth and describe ongoing clinical trials of 1 for different diseases.58−60 The U.S. Food and Drug Administration (FDA) approved 1 (Gylenia, Novartis) on September 22, 2010, to reduce relapses and delay disability progression in patients with relapsing forms of multiple sclerosis (MS) in the dose of 0.5 mg.61 The key structural features of 1 are (1) the aminodiol group, which is phosphorylated by SphK2; (2) the 1,4-disubstituted phenyl ring, which acts as a rigid linker group; and (3) the lipophilic tail, which is important for interactions with the hydrophobic binding pocket of the S1PRs.62,63 Two structurally related derivatives of 1 are the methyl ether 8 (FTY720-OCH3) (Figure 5), in which one of the prochiral hydroxyl groups of 1 is replaced by a methoxy group in order to block the site that is phosphorylated by SphK2,64 and 9 (FTY720-vinylphosphonate) (Figure 5).65 The methyl ether derivative 8 is a specific competitive inhibitor of SphK2 (with respect to sphingosine), the inhibition being enantioselective for the (R)-FTY720-
chain derivatives resulted in an increased inhibitory potency; (4) the replacement of hydroxyl group presented in the linker by a fluorine atom increases the inhibitory effect for the sphingosine and C12-sphingosine analogues. Another modification strategy involved coupling biologically active phenethyl isothiocyanate (PEITC) to sphinganine (4, PEITC-Sa) and sphingosine (5, PEITC-So) (Figure 4) by the substitution on the amino group of the linker.47 Although the authors did not report the inhibitory effect on SphK, both isothiocyanate derivatives exhibited higher cytotoxicity than sphingosine and sphinganine in the human myeloid leukemia cell line HL-60. Furthermore, 4 and 5 increased the therapeutic efficacy of 1-(β-D-arabinofuranosyl)cytosine (araC).47 Recently, a patent application (WO2010078247) disclosed the development of novel sphingoguanidine derivatives. These derivatives combined a sphingolipid backbone, or a related analogue, with a guanidine moiety introduced at the sphingolipid C2 position. The purpose of these modifications was 2-fold: (1) the guanidine moiety can generate increased hydrogen bonding interactions with the catalytic site of SphK; (2) these compounds can act as dual site/dual mode SKIs, since guanidine is believed to be able to interact directly with ATP, impeding the phosphorylation reaction.48 The most effective agent, 6 (LCL351) (Figure 4), exhibited IC50 values of 40 and 300 nM for inhibition of SphK1 and SphK2, respectively, thus exhibiting selectivity for SphK1 at lower doses.48 Compound 6 showed an inhibitory effect on cell migration in DU145 (human prostate cancer) cells.48 In 2008, Paugh et al. reported a new water-soluble sphingosine-based analogue 7 (SK1-I) (Figure 4).49 An aromatic ring and a methyl group were incorporated into the lipophilic tail and to the amine group in the linker, respectively. Compound 7 was shown to be a competitive and selective inhibitor of SphK1 (IC50 = 10 μM) without affecting SphK2, PKC, or several other protein kinases.49 Compound 7 exerted a cytotoxic effect toward human leukemia and glioblastoma 5512
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Figure 6. Structures of amidine derivatives obtained after various chemical modulations.
OCH3 enantiomer, with a Ki value of 27 μM.24 By contrast, (S)-FTY720-vinylphosphonate is an uncompetitive inhibitor (with respect to sphingosine) and a mixed inhibitor (with respect to ATP) of SphK1.65 A number of reviews have reported data on the effects of 1 and its analogues in the inhibition of SphKs and their cancer therapeutic effects.24,66,67 Another analogue of 1, the compound 10 (SG-12) (Figure 5), maintains the lipophilic tail but incorporates a modified linker in which the OH group has been moved from the carbon bearing the amine to the adjacent carbon.68 Compound 10 was shown to be a SphK2-selective inhibitor,68 and it was reported that 10 accelerates Fas-mediated cell death in the murine Blymphoma-derived cell line A20/2J.69 Furthermore, 10 induces apoptosis via phosphorylation by SphK2 in SphK2 transfected A20/2J cells.70 A potent, selective, and orally active S1P1 agonist 11 (CS0777) (Figure 5)71 represents another promising derivative of 1. Compound 11 is phosphorylated in vivo, and its phosphate analogue (CS-0777-P) acts as a selective S1P1 modulator, presenting ∼320-fold greater agonist activity for human S1P1 relative to S1P3.71 Although no anticancer activity is reported for 11 yet, it is currently in clinical trials for the treatment of multiple sclerosis (MS). Compound 11 was shown to be a safe and well-tolerated compound in an open-label pilot study conducted in 25 MS patients.72 In 2013, several novel derivatives of 1 were reported in which the polar head and/or linker was modulated. A series of tertiary amine and quaternary ammonium salt derivatives of 1 were also developed.17 Nine of the 20 novel analogues were selective inhibitors of SphK1 over SphK2. For example, 12 (RB-005) and 13 (RB-019) (Figure 5) showed the highest selectivity for SphK1 over SphK2 (15.0- and 6.1-fold, respectively).17 Some preliminary SARs were pointed out for compounds 12 and 13. The hydroxyl group in the heterocyclic ring was found to be important for the inhibitory effect of 12, since the replacement of the 4-OH group with a 4-methyl group compromised its Sphk1 activity and led to a moderate inhibitor of both SphK1 and SphK2. The size of the heterocyclic ring was also shown to be important, the seven member ring derivative being less selective and less potent than the six member ring present in 12.17 A series of analogues of 1 wherein the lipophilic region was modulated were also reported in the same year.18 The structure of these analogues contained an extra substituted aromatic ring in the lipophilic tail. These new analogues of 1 were evaluated in vitro against human hepatoma SMCC-7721 and human promyelocytic leukemia HL-60 cell lines. The authors found that the length of the lipophilic region plays a critical role in the activity, medium-length substituents being the most active ones.18 Changed Polar Head. The effectiveness of sphingoid bases seems to be limited by their phosphorylation by SphKs.
Therefore, the compounds that cannot be phosphorylated would be more effective in suppressing cancer than the naturally occurring sphingoid base sphingosine. Considering that the hydroxyl group located in the polar head is responsible for phosphorylation, it seems logical to think that the removal of this hydroxyl group could be a good approach to obtain SKIs with better anticancer activities. This strategy was used to obtain 14 (enigmol) (Figure 4), which is a structural analogue of sphingosine and 2.73 The only structural change over sphingosine is the displacement of the hydroxyl group from position 1 to 5. Compound 14 is not phosphorylated and inhibits both SphK and CerS, albeit 14 did not alter the amount of cellular S1P.73,74 Furthermore, 14 demonstrated potent anticancer activity in cells derived from multiple types of cancers, as well as a significant oral efficacy in rodent models of colon and prostate cancers with no evidence of host toxicity at effective doses.74 In order to gain further knowledge of the structural features required for its bioactivity, Garnier-Amblard et al. examined the pharmacological roles of stereochemistry and N-methylation in the 14 structure.75 The four diastereomers synthesized by manipulating the C-3 and C-5 positions were shown to be active in vitro against prostate cancer cell lines.75 Furthermore, two diastereomers, 2S,3S,5R-enigmol and 2S,3R,5S-enigmol, also caused statistically significant inhibition of tumor growth in nude mouse xenograft models of human prostate cancer.75 Nevertheless, the N-methylenigmol showed significantly less oncolytic activity in vivo.75 Another structural modification conducted recently is the fluorination of 14. One of the two analogues obtained was more effective on inhibiting tumor growth rates in PC3 xenografts than 14.76 To recapitulate, 14 is equally effective as standard prostate cancer therapies (androgen deprivation or docetaxel), and considering the anticancer effects of some of its analogues, 14 can be considered to be a very interesting scaffold for further development as a promising therapeutic potential. Several N-arylamide phosphonates (e.g., VPC44152 in Figure 4) were reported in 2007.77 These derivatives are structurally close-related analogues of S1P and present a potent and a subtype-selective agonistic and antagonistic activity against S1PRs (S1P1−5).77 Foss et al. concluded that the replacement of the phosphorus−oxygen bond present in S1P with a phosphorus−carbon bond provides bioactive agents with putative resistance to degradative phosphatase activity. Later, the same authors designed metabolically more stable S1PRs agonist prodrugs using the structures of these N-arylamide moieties as a starting point (Figure 4).78 These prodrugs, a series of 2-amino-2-heterocyclic propanols, present two different structural features: (1) removal of the phosphate group in the polar head and (2) introduction of some heterocycles (imidazole, oxazole, and oxadiazole) in the linker part.78 The oxazole-derived structure was most active for human SphK2.78 5513
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Figure 7. Chemical structures of nolipidic small molecule SphK inhibitors.
proved that (1) a bulky substitution at the tail terminus, such as cyclohexane, increases the potency and selectivity regardless of amide orientation, (2) the ideal cLogP value is around 4.2, and (3) the restriction of the dihedral angle between the position of the amide and that of the amidine should have an effect on selectivity and potency. After these SAR studies, an optimized amidine analogue was identified with Ki = 75 nM for SphK1 and an 80-fold selectivity index for this kinase versus the SphK2 subtype.80 Additionally, they generated a SphK1 homology model from the crystal structure of DGKB and used it for the in silico design of novel amidine derivatives. This in silico design led to the synthesis of an oxazole derivative 16 (Figure 6) with Ki = 47 nM and a 180-fold selectivity ratio for SphK1.80 The most potent and selective amidine derivatives were validated in human leukemia U937 cells, where they significantly reduced endogenous S1P levels at nanomolar concentrations.80 Later, the same research group used all the SAR properties obtained previously for the amidine analogues79,80 and designed enantiomeric pairs of rigid proline analogues (Figure 6) with an amidine group in the polar head.29 Both enantiomers showed Ki values in the submicromolar range for SphK1 and decreased the S1P levels in both cultured cells and mice.29 The latest addition to this class of SKIs is 17 (SLR080811) (Figure 6) emerged by modifying the chemical structure of one of these enantiomers.81 The replacement of the amidine and the amide groups in proline analogues by guanidine and oxadiazole groups, respectively, in 17 entailed a dual effect: (1) enhanced half-life compared with the proline analogue81 and more surprisingly (2) reversed selectivity for SphK isotypes, 17 being selective (approximately 1 order of magnitude more potent) for SphK2.81 Kharel et al. demonstrated that administration of 17 to wild-type mice triggered a rapid increase in blood S1P levels,
Furthermore, a stereochemical preference of the quaternary carbon was crucial for phosphorylation by the kinases and altered binding affinities at the S1PRs.78 While in vitro data were initially quite promising, these new prodrugs did not prove to be viable therapeutic candidates after in vivo analysis of lymphocyte levels.78 In 2010, Mathews et al. reported for the first time the synthesis, evaluation, and SAR properties of a new class of amidine-based sphingosine analogues (Figures 4 and 6).79 Most of these compounds acted as competitive inhibitors of SphKs with varying degrees of enzyme selectivity and Ki values in the submicromolar range for both SphKs.79 These amidines decreased the S1P levels and initiated growth arrest in cultured vascular smooth muscle cells.79 The authors modulated various structural features in the three different chemical regions of these analogues, allowing them to establish some SAR properties: (1) in the polar head; substituting the carboxylic acid and the primary amide by an amidine group suggested that the unique electrostatic properties associated with amidines in aqueous environment and its direct interaction with ATP γphosphate are critical for the activity of these compounds;79 (2) in the linker region; the elimination of chirality and the increase of steric bulk seem to improve the activity of these analogues as dual inhibitors, and furthermore, the inversion of benzamide seems to improve their selectivity for SphK1;79 (3) in the lipophilic tail; varying linear hydrocarbon sizes revealed that the potency of these amidines increased at a length of 12 carbons for the alkyl group but decreased with 14 and 16 carbons.79 In a second approach, Kennedy et al.80optimized the structure of the most selective SphK1 inhibitor 15 (Figure 6) synthesized by Mathews et al.,79 by modulating several structural features in this compound to improve its potency and selectivity. They 5514
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Figure 8. Structures of nonlipidic small molecule SphK inhibitors.
potential to create novel drugs for cancers as well as several other diseases. Nolipidic Small Molecules. In 2003, a chemical library screening by French et al. revealed four different types of small molecules [19 (SKI-I), 20 (SKI-II), SKI-III, and SKI-IV] (Figure 7) with SphK inhibitory effect at submicromolar to micromolar concentrations.19 These four derivatives were shown to be selective toward human SphK compared with a panel of human lipid and protein kinases, and they were not competitive inhibitors of the ATP-binding site of SphK.19 Furthermore, they showed an antiproliferative action toward a panel of tumor cell lines and inhibited the endogenous human SphK activity in intact cells.19 Further studies on three of these compounds (19, 20, and SKI-V) (Figure 7) demonstrated their antitumor activity in mice without systemic toxicity.83 Among these SKIs, the most extensively studied compound 20 (Figure 7) is orally bioavailable and showed a significant inhibition of tumor growth in mice and a growth inhibitory effect in vitro toward several cancer cell lines.83 Compound 20 induced Bcl-2independent apoptosis in prostate adenocarcinoma cells,84 inhibited colony formation,85 and activated caspase-3 in both temozolomide-sensitive and resistant glioblastoma multiforme cells.85 In 2010, reports suggested novel mechanisms of action for 20 other than the direct inhibition of the enzymatic activity of SphK1 described by French et al.19 Loveridge et al. showed that 20 induced proteosomal degradation of SphK1 in several cancer cell lines, and this is likely mediated by ceramide as a consequence of catalytic inhibition of SphK1.86 Also, a new post-translational mechanism has been proposed for the activity of 20.87 Ren et al. reported that 20 acts by stimulating SphK1 protein degradation and consequently by this mechanism reduces SphK1 activity.87 This degradation of SphK1 occurred by a lysosomal route involving cathepsin B.87 These new mechanisms of action strongly suggest that additional targets of 20 may exist other than SphK1. In this regard, Antoon et al. have proposed a dual mechanism of action for 20 in human breast cancer: inhibition of SphK and inhibition of estrogen receptor (ER) signaling.88 Compound 20 dose-dependently decreased estrogen-stimulated estrogen response element transcriptional activity and diminished mRNA levels of the ER-regulated genes progesterone receptor and steroid derived factor 1.88 Furthermore, 20 binds to ER directly in the antagonist ligand-bind domain.88 Different approaches have been used to improve some parameters of 19 and/or 20. We have synthesized the prodrugs of both SKIs using the aspirinyl (Asp) backbone.89 The compounds 21 (SKI-I-Asp) and 22 (SKI-II-Asp) (Figure 7)
in contrast to SphK1 specific inhibition that lowers the circulating S1P levels.81 Many of the SKIs, having a thiazolidine-2,4-dione structure, have been shown to inhibit ERK and Akt pathways as a downstream event of SphK inhibition. One of these thiazolidine-2,4-dione analogues, 18 (K145) (Figure 4),82 presents two structural features that make it a very attractive scaffold: (1) the amino terminal group, which may be able to mimic the aminohydroxyl sphingosine base and (2) a phenyl ring with an alkyloxy chain. Liu et al. reported the synthesis and biological characterization of 18 and showed it to be a selective SphK2 inhibitor, with a Ki value of 4.30 μM and no inhibitory effect on SphK1 at concentrations of up to 10 μM.82 The treatment of human leukemia U903 cells with 18 caused a decrease of total S1P without significant effects on ceramide levels, as well as inhibitory and apoptotic effects.82 Compound 18 also significantly inhibited tumor development in various mice models upon both intraperitoneal and oral administrations.82 Since the window of SphK2 to SphK1 (10−4.3 μM) inhibition is very low, it would be difficult to achieve a concentration in vivo to claim that the antitumor effect is due to exclusive inhibition of SphK2. To summarize, classes of sphingosine-based SKIs with both changed and unchanged polar heads, compounds specific to SphK1 and SphK2, and nonspecific inhibitors with varying efficacy have been reported. Taking into account all modifications carried out in the sphingosine-based SphKIs and the biological effects that these modifications entailed, several conclusions can be pointed out: (1) N-substitutions in the sphingosine structure (e.g., 3, 5, and 7) do not seem to adversely affect the cytotoxic potency of the new derivatives. In fact, modifications in the linker region of sphingosine are generally well tolerated. (2) The introduction of a phenyl ring in the lipophilic region of sphingosine (e.g., in 1, 7, and 18) typically does not reduce the inhibitory or anticancer effects of the sphingosine-based SKIs. By contrast, this phenyl ring increases their bioavailability, presenting a better druglike profile. (3) The substitution of the hydroxyl group in the polar head in order to prevent its phosphorylation by other polar groups (e.g., amidines and 18) appears to be a valid approach in the design of novel sphingosine-based SKIs. Among these new polar groups, the SKIs with an amidine group in the polar head have been shown to be very promising molecules to further develop. Many of the sphingosine-based SKIs have been found to be effective against multiple diseases both in the preclinical studies and in the clinic. This modification strategy thus has the 5515
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Figure 9. Chemical structures of 28 and its analogues.
stituent on the amide group of 24 by a dihydroxyphenyl ring, leading to 25 (ABC294735) (Figure 8), affected the selectivity of the compound.96 The new analogue 25, thus formed, is a sphingosine-competitive inhibitor for both SphK1 and SphK2, with Ki values of 3.1 and 4.2 μM, respectively.96 Although 24 and 25 distinctly affect SphKs, being SphK2 selective and dual SphK1/SphK2 inhibitors, respectively, both of them have been demonstrated to cause a comparable delay in the tumor growth in two xenograft models of pancreatic and kidney cancers.95 The tumor growth inhibition is potentiated by the coadministration of sorafenib for both compounds.95 Recently, a new nolipidic SphK1 selective inhibitor 26 (PF543) (Figure 8) was developed.30 Its structure maintains the 1,2-amino alcohol motif presented in sphingosine, although the nitrogen in 26 is tertiary amine, forming part of a pyrrolidine ring. Compound 26 is a sphingosine-competitive inhibitor with Ki = 4.3 nM and is more than 100-fold selective for SphK1 over the SphK2 isoform.30 It also decreased the level of endogenous S1P 10-fold with proportional increase in the level of sphingosine.30 Nevertheless and despite the dramatic change in the cellular S1P/sphingosine ratio, 26 did not show any effect on the proliferation and survival of 1483 human head and neck squamous cell carcinoma cells.30 These observations contradict the reports that selective inhibition of SphK1 alone, to decrease S1P levels and enhance sphingosine levels, is responsible for anticancer properties. A simultaneous inhibition of other signaling pathways, yet unknown, by several reported SKIs may be responsible for their activity. Compound 26 can be considered an ideal candidate to test the effects of the inhibition of SphK1 in vitro, but its effects in vivo have not been reported. Another example that proves the success of applying more than one medicinal chemistry technique in the design of novel SKIs is 27 (Figure 8).97 Initially, a thionicotinamide was identified as a SKI using a high throughput screening (HTS) approach.97 After further structural modifications and the establishment of the SAR properties, Pennington et al.97 replaced the sulfur atom of this thionicotinamide by an oxygen atom to yield 27, which lacks a polar head. It has been shown to be a S1P1 agonist (EC50 = 35 nM) and to possess a >100-fold selectivity over S1P2−5 subtypes.97 Furthermore, 27 is orally bioavailable and shows a dramatic reduction of circulating lymphocytes in rats 24 h after a single oral dose.97 However,
were designed to take advantage of the fact that apart from an easy release of the parent compound by hydrolysis of the ester linkage by esterases on administration in vivo, aspirin will be released as a side product.89 Both the parent SKIs (19 and 20) and their Asp derivatives (21 and 22) showed a similar cytotoxicity in vitro in most cancer cell lines tested, as well as equally potent inhibition of SphK1 at low doses (i.e.,
