C H A P T E R Synthesis and Characterization of 4-Thiobutyl-triphenylphosphonium-pantetheine, a Pantetheine Derivative

Pantetheine, a low molecular weight thiol, has been found to ameliorate symptoms in various disease models but specifically in Pantothenate Kinase-Associated Neurodegeneration (PKAN). Pantetheine is usually administered in its disulfide form (i.e. pantethine) since pantethine is commercially available and is reduced to pantetheine in biological systems. The applicability and efficacy of pantethine (therefore also pantetheine) as a clinical therapeutic however is hampered since both forms can be degraded by pantetheinases present in the body. Here, we report the synthesis of a masked form of pantetheine, namely 4-thiobutyltriphenylphosphonium-pantetheine (TBTP-pantetheine), following our hypothesis that this pantetheine-derivative might be more stable in the presence of pantetheinases than pantetheine itself. Higher stability would enhance transport into the cytoplasm where TBTP-pantetheine is metabolized into pantetheine which can subsequently execute its medicinal action. We find that TBTP-pantetheine is stable in aqueous solution, however it was found to be less stable in 10% fetal calf serum (which contains pantetheinases) compared to pantethine, the commercially availabile disulfide of pantetheine. We show that TBTP-pantetheine has improved lipophilicity, but equal passive membrane permeability/diffusion, as compared with pantethine.


INTRODUCTION
Pantethine, the disulfide of pantetheine (scheme 1a, 1), has been investigated in the context of biological processes involved in several diseases, including hypercholesterolaemia [1], cataract disease [2] and cerebral malaria [3].In addition, preclinical research has shown beneficial effects of pantethine on a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mouse model for Parkinson's disease [4,5] and pantethine also prevented neuronal degeneration in animal models of Parkinson's disease [6,7].In general, pantethine is investigated as possible treatment since it is commercially available, however the beneficial effects are due to the reduced form (i.e.pantetheine, 1), which is formed in vivo.Recently, pantethine has been investigated as a potential rescue agent for Pantothenate Kinase-Associated Neurodegeneration (PKAN), a neurodegenerative disease caused by mutations in the human PANK2 gene (one of the four human pantothenate kinase genes (PANK1-4known) [8,9].The PANK2 protein is mainly localized in the brain [8,9].It is believed that mutations in the human PANK2 gene render the enzyme pantothenate kinase, which is responsible for the pivotal, rate limiting step in the biosynthesis of coenzyme A (CoA) during which pantothenate (2, Vit B5, precursor for CoA) is phosphorylated to 4'phosphopantothenate, inactive and therefore impairs the pathway to produce CoA which is an essential cofactor in all organisms.Pantethine has shown potential as rescue agent in a Drosophila model for PKAN where it restored CoA levels, rescued brain degeneration, mitochondrial dysfunction and locomotor disabilities [8].
Although pantethine has been shown to be effective in Drosophila PKAN models, a possible clinical implementation may be limited by the fact that pantethine is rapidly hydrolysed when administered orally [10].Vanin proteins (also known as pantetheinases) present in humans contain pantetheinase activity and have the ability to degrade pantetheine (1), its natural substrate, to form pantothenate (2) and the antioxidant cysteamine (3) (scheme 1a).Since these enzymes are present in the gastrointestinal mucosa and human serum [11], they limit the use of pantethine (which has also been shown to be degraded by pantetheinases [12]) as a possible rescue agent for PKAN.Vanin proteins have also been shown to be an important implication in the use of pantothenamides as antimalarials, since this specific class of pantothenate derivatives has the same core structure as 1 and pantetheinases can hydrolyze pantothenamides with a wide range of structural modifications on the cysteamine moiety [13][14][15][16].Inhibitors for pantetheinases have been described [17] and combining pantothenate derivatives (i.e.pantethine or pantothenamides) with such inhibitors has been proposed as a strategy to overcome the obstacle formed by pantetheinases [18].Alternatively, small modifications in the core structure of pantothenamides led to increased stability towards pantetheinase activity and increased antiplasmodial activity towards the malaria parasite Plasmodium falciparum [16].

Scheme 1. Metabolism of pantetheine (1) and TBTP-pantetheine (4)
a) Natural pantetheine recycling through degradation of pantetheine ( 1) by pantetheinases to produce pantothenate (2) and cysteamine (3).As a result, 2 can be taken up by the cell and be converted to coenzyme A. b) Proposed mechanism of action of TBTP-pantetheine ( 4).The TBTP-moiety of 4 will enable transport of 4 into the cytoplasm where it can be reduced by glutathione reductase to release pantetheine (1) as rescue agent.The TBTP cation (5) will dispense to the mitochondria where it will slowly be released from the cell We set out to prepare the novel pantetheine derivative, 4-thiobutyltriphenylphosphoniumpantetheine (TBTP-pantetheine, 4, scheme 1b) in order to protect pantetheine against degradation by pantetheinases, so it can function as a better rescue agent for PKAN.Since the core pantetheine structure of the molecule must be intact in order to be converted to CoA, we envisioned to couple pantetheine (1) and 4-thiobutyltriphenylphosphonium (TBTP, 5), a lipophilic cation, to construct the pantetheine-derivative 4 with supposedly improved pharmacokinetic properties as compared to pantetheine [19,20].Pantetheinases seem to be promiscuous towards the cysteamine moiety of pantetheine derivatives, and therefore we assumed that some degradation of 4 would still take place.Despite the latter, the TBTPmoiety might reduce the affinity of pantetheinases for 4 compared to that of 1 and also improve the cellular uptake, as was previously shown for other TBTP-linked compounds [21].This might lead to shorter exposure of 4, as compared to 1, in the compartments where pantetheinases are abundant.Moreover, the TBTP-moiety of 4 may facilitate blood brain barrier crossing, as permeability depends highly on a compound's lipophilicity.Once TBTPpantetheine (4) enters the cell it should be reduced by the glutathione pool present in the cytoplasm to release pantetheine (1) as rescue agent of the impaired CoA biosynthesis pathway present in PKAN patients.The TBTP cation5 will dispense to the mitochondria where it will slowly be released from the cell [20].Although TBTP-linked molecules have been found to accumulate in internal organs and the central nervous system, these compounds are generally well tolerated and have previously been used in clinical trials [21][22][23].This study describes a simple synthesis of TBTP-pantetheine (4) in three chemical steps and shows that 4 is stable over a prolonged period in aqueous solution at various temperatures.We also compared the stability of TBTP-pantetheine (4) to pantethine (disulphide of 1) in the presence of fetal calf serum, and investigated the membrane permeability of both these compounds.

MATERIALS AND METHODS
All chemicals were purchased from Sigma-Aldrich Chemical Co. and solvents were purchased from Biosolve or Sigma-Aldrich Chemical Co.Fetal calf serum (FCS) was obtained from Greiner Bio-one and Dulbeccos Phosphate Buffered Saline (PBS) was from Invitrogen.Centrifugal devices for protein removal were from PALL.Gentest pre-coated PAMPA plates were from BD Biosciences.UV 96-well flat-bottomed plates were from Greiner Bio-one.
UV absorbance was determined using a SPECTROstar Omega plate reader from BMG Labtech.NMR analyses were performed on a Varian Inova 200 and 300 machine at the NMR Center, University of Groningen.Chemical shifts (δ) are reported in parts per million (ppm).Mass Spectrometry was performed using Orbitrap high resolution mass spectrometry (HRMS) by the Mass Spectrometry core facility, University of Groningen.
High performance liquid chromatography (HPLC) analysis was performed on a Shimadzu LC-10AC system using a SCL-10A system controller, SIL-10AC automatic sample injector, and LC-10AT solvent delivery system.The peaks of interest were detected at 205 nm and/or 267 nm with a Shimadzu SPD-M10A photodiode array detectorafter injection of 25 µl of each sample.Separation was achieved by using a Synergi 4u Hydro-RP 80A (150 × 4.60 mm, 4 µm) C18 column from Phenomenex at 30°C while maintaining a flow rate of 1.0 ml/min.HPLC method: The column was equilibrated in solution A (20 mM KH 2 PO 4 , 0.1% hexanesulfonic acid, pH 3) and the sample was injected.After 5 min elution with 100% A, the following linear gradients were used: a linear gradient increasing solution B (acetonitrile) to 30% (5-18 min), a linear gradient increasing solution B to 50% (18-21 min), isocratic elution at 50 % solution B (21-23 min), a linear gradient decreasing solution B to 0% (23-24 min) and finally isocratic elution in 100% solution A (23-30 min).

Synthesis of S-acetyl-thiobutyltriphenylphosphonium bromide (8)
The synthesis of 8 was carried out according to a previously published method [19].Briefly, 7 (2.14 g, 10.1 mmol) was dissolved in dry toluene (6 ml) under nitrogen followed by the addition of triphenylphosphine (2.66 g, 10.1 mmol).The reaction mixture was refluxed for 2 hours under nitrogen after which the heat was turned down and the reaction mixture left in the heating mantle to cool down slowly overnight.An upper colourless toluene layer and lower yellow layer formed.The mixture was stored at -20ºC for a few hours until a white precipitate started to form.The upper toluene layer was removed with a pipet followed by washing of the bottom layer three times with pentane followed by three times with diethyl ether.Finally, 8 was collected as a white precipitate which was dried In vacuo (3.01 g, 63%).

Stability of TBTP-pantetheine (4) in Water
A solution of TBTP-pantetheine (4, 1 mM) was prepared in water and kept at different temperatures (-20ºC, 4ºC, 25ºC and 37ºC) for 14 days.Samples were injected on HPLC (method already described) on day 1, day 7 and day 14 in order to determine the concentration of 4 in solution, based on a standard curve of known concentrations prepared for TBTP-pantetheine (4).The experiment was performed in triplicate and the indicated errors are standard deviations.

Stability of TBTP-pantetheine (4) in Fetal Calf Serum
Solutions of TBTP-pantetheine (4, 1 mM) or D-pantethine (1 mM) were prepared in 10% FCS (pre-incubated at 37ºC) in PBS in a final volume of 150 µl.Corresponding control samples either contained no FCS, no pantethine or no TBTP-pantetheine (4).The samples were incubated at 37ºC for 60 min and degradation action of the enzyme stopped at specific timepoints (5, 10, 20, 45 and 60 min) by flash freezing in liquid nitrogen followed by centrifugation for 45 minutes at 14 000 rpm in 3 Kcentrifugal filters at 4ºC.The experiment was performed in triplicate and each sample was analyzed by the already described HPLC method.The amount of pantothenate formed was calculated by correlation to a standard curve of known pantothenate (2) concentrations.The amount of pantothenate (2) formed upon degradation of pantethine, was divided by 2in order to compensate for the 2:1 pantetheine (1) ratio between pantethine and TBTP-pantetheine (4).The errors bars indicate standard deviations.

Membrane Permeability of TBTP-pantetheine (4)
To determine the membrane permeability potential of 4, a previously reported In vitro model was used [24].Experiments were carried out according to the manufacturer's instructions.The donor well was filled with 300 μl of a solution of the test compound (either D-pantethine or 4) in PBS and the acceptor wells contained 200 μl PBS.Two different concentrations were tested: A low concentration of 800 μM and high concentration of 2 mM, with every condition tested in The Parallel Artificial Membrane Permeability Assay (PAMPA) plate system was assembled and incubated for 5 hours at room temperature without agitation.After incubation, 100 μl of each donor and acceptor well solution was transferred to a UV 96-well plate where the absorbance was measured between 220 and 400 nm using a SPECTROstar Omega plate reader.The concentration of compound present in each solution was determined by comparison with standard curves determined for each compound.Caffeine and amiloride were measured as positive and negative control for membrane permeation, respectively [5].Permeability and mass retention were determined.

Synthesis
We set out to prepare TBTP-pantetheine (4) following our hypothesis that this pantetheinederivative exhibits enhanced bioavailability and pharmacokinetic properties as compared to pantetheine itself.TBTP (5) was synthesized according to known literature procedures by firstly preparing the acetylated form of TBTP (8, Fig. 1a), which prevented oxidation of the thiol under synthesis conditions [19].Prior to coupling TBTP (i.e. a thiol (5) which is the nonacetylated form of compound 8) to pantetheine (1) to produce the required disulfide 4, the acetyl group of 8 was removed by means of base hydrolysis in a one-pot synthesis procedure.This resulted in the successful synthesis and purification of TBTP-pantetheine (4, Fig. 1a).The overall yield of this simple three-step synthesis is low (overall yield of 5%), mainly due to two factors.First, the reported preparation of 8 in this manner constitutes low yields (30%) [19] and we found similar results with an overall yield of 17%.An alternative route for the preparation of TBTP ( 5) has been reported and utilizes commercially available 4-bromobutyltriphenylphosphonium bromide in combination with hydrosulphide exchange resin to deliver TBTP in one step in a yield of 95% [20].This method might prove much more effective in future strategies for TBTP-linked molecules.Secondly, synthesis involving pantothenate derivatives proves to be challenging if the 4'-hydroxyl group is not protected, since these compounds are usually water-soluble which impairs the use of aqueous work-up procedures before purification.This, in combination with the presence of water soluble TBTP and bis-TBTP in the reaction mixture, complicates purification of the final product.Nevertheless, despite these obstacles we were still successful in the preparation and purification of 4 and the final purity of the compound was determined as >83% pure by HPLC analysis (assuming identical Ɛ-values of the contaminant(s) as compared to 4).

Stability of TBTP-pantetheine
The aqueous stability of 4 was determined after 1, 7 and 14 days at various temperatures (-20ºC, 4ºC, 25ºC and 37ºC).No degradation was observed at low temperatures (i.e.-20ºC and 4ºC); however, mild decay (<15%) was observed for samples stored in water at 25ºC and 37ºC (Fig. 1b).Degradation resulted in the formation of pantetheine and TBTP.The data therefore suggest that decay of 4, when measured in a biological matrix, is not due to aqueous instability of the compound.
Next, we set out to determine the stability of TBTP-pantetheine (4) in the presence of fetal calf serum which exhibits pantetheinase activity.Pantethine and TBTP-pantetheine were incubated in the presence of 10% fetal calf serum for 60 minutes, and the amount of pantothenate (2, the degradation product) formed was measured by HPLC at different time intervals.The stability of pantetheine bearing a TBTP-moiety (4) appeared not to be higher than the stability of pantethine.In fact, from Fig. 1c it is clear that more pantothenate( 2) is formed in 60 minutes from 4 than from pantethine.This illustrates that, similar to previous studies, pantetheinases are very promiscuous in accepting pantetheine derivatives with modifications on the cysteamine moiety of the molecule.

Membrane Permeability of TBTP-pantetheine (4)
It is of particular clinical interest to study whether the derivatization of pantetheine (1) by attaching a TBTP-moiety (5) leads to an increase in membrane permeability.In general, it is assumed that passive diffusion is the most important mode of permeability involved in the blood brain barrier [24].A Parallel Artificial Membrane Permeability Assay (PAMPA) was used to investigate the passive diffusion behavior of TBTP-pantetheine (4) compared to pantethine (the disulphide of 1) [24].By using this assay, 4 and pantethine were tested for their ability to permeate through the artificial membrane and their mass (or membrane) retention was determined.The negative logarithm of permeability is shown (Fig. 2a) and indicates that compounds with a high tendency to permeate through the membrane will have a low value, such as caffeine which was used as positive control, whereas a high value will indicate little diffusion as seen for amiloride which was used as negative control for permeation [24].The permeability of pantethine and TBTP-pantetheine (4) was found to be similar (Fig. 2a), indicating poor diffusion through the membrane by both these compounds.However, the mass retention of TBTP-pantetheine (4, 20%) was found to be significantly higher than that for pantethine (~2%) (Fig. 2b, Student's t-test, two-sided, P = 0.0075).These results show that TBTP-pantetheine ( 4) is indeed more lipophilic than pantethine (the disulphide of 1) but that passive permeability of the compound is not increased.This assay does not include the possibility of active transport of either TBTP-pantetheine (4) or pantethine by carriers present in the blood brain barrier.It is believed that the involvement of these active transport processes in permeability of drugs have been underestimated and might be processes which warrant further investigation [24].

CONCLUSION AND FUTURE OUTLOOK
Here we described the derivatization of pantetheine in an attempt to improve its pharmacological activity as a rescue agent for PKAN.We were successful in preparation and sufficient purification of TBTP-pantetheine (4) and showed that the TBTP-pantetheine shows increased membrane affinity, but not increased passive membrane permeability, as compared to pantethine.TBTP-pantetheine ( 4) is stable in aqueous solution, however it is degraded by pantetheinases present in fetal calf serum.This effect might be overcome when using this compound in combination with known pantetheinase inhibitors; although, pantetheinase inhibitors may introduce toxicity and alter some biological processes [17].
Even though TBTP-pantetheine ( 4) is being degraded faster than pantethine In vitro, future work will be of value to test this novel derivative of pantetheine In vivo on insect and human cells in a PKAN model to determine if it has superior rescue potential over pantethine.There is also potential to extend 4 further as a drug for other diseases.Among other uses, pantethine has been found to ameliorate MPTP induced toxicity in a murine model of Parkinson's disease.However, effective delivery to the drug target of the compound is still lacking [5].The exact mode of action of pantethine is still unknown.Pantethine is reduced In vivo to pantetheine and subsequently the rescue potential of this molecule might be due to its metabolites cysteamine or pantothenate.Although cysteamine is being used to treat cystinosis, it has also been found to have side effects when treating this disease [25].In contrast a study on the toxicity of pantetheine shows that this compound has low toxicity in mice [26,27].Therefore, treatment with pantethine or pantetheine might be a safer alternative for cysteamine, because pantethine is less toxic and still will be reduced to pantetheine (1) and then further degraded by pantetheinases to cysteamine, which is the active rescue compound for cystinosis treatment.It is currently unclear why cysteamine shows increased toxicity compared to pantethine.Addition of the TBTP-moiety (5) to pantetheine (1), as presented and investigated here, could possibly act as carrier to either deliver pantetheine or cysteamine to its specific target in various diseases.The applicability of TBTP (5) in this regard however needs to be further investigated.

Fig. 1 .
Fig. 1.Synthesis and stability of TBTP-pantetheine (4) a) Synthesis route for TBTP-pantetheine in three steps starting from 4-bromo-1-butene (6).b) Stability of TBTP-pantetheine in water at different temperatures (-20ºC in black, 4ºC in dark grey, 25ºC in light grey, 37ºC in white) over two weeks.Experiment was performed in triplicate and error bars denote standard deviations.c) Pantothenate formation by the degradation of TBTP-pantetheine (triangles) vs. pantethine (circles) in 10% FCS over 60 min.Experiment was done in triplicate and error bars denote standard deviations

Fig. 2 .
Fig. 2. Membrane diffusion of TBTP-pantetheine (4) compared to pantethine (disulphide of 1)a) Membrane permeability of TBTP-pantetheine (4) compared to pantethine (disulphide of 1) using a PAMPA test.Caffeine and amiloride are included as positive and negative control, respectively.No significant difference is denoted as "ns" and was determined with a Mann-Whitney U-test (two-sided, P = 0.4166).Note that the negative logarithm of permeability is shown.b) Membrane retention of TBTPpantetheine (4) compared to pantethine (disuphide of 1) using the same experiment that generated data shown in Fig.2a.The significant difference observed between TBTP-pantethine (4) and pantethine (disulphide of 1) is indicated by ** (Student's t-test, two-sided, P = 0.0075).Experiments were done in quadruplicate with error bars indicating standard error mean.