Discovery of Soft-Drug Topical Tool Modulators of Sphingosine-1-phosphate Receptor 1 (S1PR1)

In order to study the role of S1PRs in inflammatory skin disease, S1PR modulators are dosed orally and topically in animal models of disease. The topical application of S1PR modulators in these models may, however, lead to systemic drug concentrations, which can complicate interpretation of the observed effects. We set out to design soft drug S1PR modulators as topical tool compounds to overcome this limitation. A fast follower approach starting from the drug ponesimod allowed the rapid development of an active phenolic series of soft drugs. The phenols were, however, chemically unstable. Protecting the phenol as an ester removed the instability and provided a compound that is converted by enzymatic hydrolysis in the skin to the phenolic soft drug species. In simple formulations, topical dosing of these S1PR modulators to mice led to micromolar skin concentrations but no detectable blood concentrations. These topical tools will allow researchers to investigate the role of S1PR in skin, without involvement of systemic S1PR biology.


Kinetic Solubility
The aqueous solubility of the test compounds was measured using laser nephelometry. Compounds were subject to serial dilution from 10 mM to 0.5 mM in DMSO. An aliquot was then mixed with MilliQ water to obtain an aqueous dilution plate with a final concentration range of 250 -12 µM, with a final DMSO concentration of 2.5%. Triplicate aliquots were transferred to a flat bottomed polystyrene plate which was immediately read on the NEPHELOstar (BMG Lab Technologies). The amount of laser scatter caused by insoluble particulates (relative nephelometry units, RNU) was plotted against compound concentration using a segmental regression fit, with the point of inflection being quoted as the compounds aqueous solubility (µM).
All values reported correspond to n of 1.
pKa Sirius T3 (Sirius Analytical Inc, UK) instrument has been used for pK a determination of the compounds. The pK a determination is based on acid-base titration and the protonation/deprotonation of the molecule is measured either by UV spectroscopy or potentiometrically. The pK a value is calculated from the pH where the 50-50% of the protonated and unprotonated form of the molecules are present. The UV-metric method provides pK a results for samples with chromophores whose UV absorbance changes as a function of pH. It typically requires 5 µl of a 10 mM solution of the samples and the UV absorbance is monitored over 54 pH values in a buffered solution in about 5 min. When the ionization centre is far from the UV chromophore pHmetric method based on potentiometric acid-base titration is used. The pH of each point in the titration curve is calculated using equations that contain pK a , and the calculated points are fitted to the measured curve by manipulating the pK a . The pK a that provides the best fit is taken to be the measured pK a . Usually 0.5 -1 mg of solid material is required for the measurements. When the compound precipitates at some point during the pH titration co-solvent method using methanol is applied using various concentration of co-solvent. The pK a in water is calculated using the Yasuda-Shedlovsky extrapolation method.
All values reported correspond to n of 1.

Human Liver Microsome (HLM) stability experiments
Test compound (0.5 μM) was incubated with female CD1 mouse liver microsomes (Xenotech LLC TM; 0.5 mg/mL 50 mM potassium phosphate buffer, pH 7.4) and the reaction started with addition of excess NADPH (8 mg/mL 50 mM potassium phosphate buffer, pH 7.4). Immediately, at time zero, then at 3, 6, 9, 15 and 30 min an aliquot (50 uL) of the incubation mixture was removed and mixed with acetonitrile (100 μL) to stop the reaction. Internal standard was added to all samples, the samples centrifuged to sediment precipitated protein and the plates then sealed prior to UPLCMSMS analysis using a Quattro Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard at each timepoint. The rate of intrinsic clearance (CLi) of each test compound was then calculated using the following calculation: CLi (mL/min/g liver) = k x V x Microsomal protein yield Where V (mL/mg protein) is the incubation volume/mg protein added and microsomal protein yield is taken as 52.5 mg protein per g liver. Verapamil (0.5 μM) was used as a positive control to confirm acceptable assay performance. Experiments were performed using a single timecourse experiment.
All values reported correspond to n of 1.

Human Liver Hepatocyte (HLH) stability experiments
Cryopreserved vials of human cryopreserved hepatocytes, supplied by Life Technologies, were thawed according to manufacturer's instructions and cells resuspended in Williams Medium E (WME) containing cell maintenance supplement pack (CM4000, Life Technologies). Hepatocytes were incubated in suspension (0.5 million cells/mL) in 48 well non-collagen coated cell culture plates for 10 minutes at 37 ˚C, 5% CO 2 . Upon addition of an equal volume of supplemented WME containing 1 μM test compound, an aliquot of incubation solution was removed to acetonitrile containing internal standard (final concentration 0.5 μM test compound and a cell density of 0.25 million cells/mL). Similarly, aliquots were removed at 3, 6,9,15,30,45,60,90 and 120 minutes. 100 μL of 80:20 water:acetonitrile was added to all samples and the analysis plate was centrifuged for 10 min at room temperature prior to injection and analysis of samples by UPLC-MS/MS. The response (area ratio of test compound to internal standard) was plotted against time using an exponential decay model and rate of disappearance calculated.
All values reported correspond to n of 1.

Human Skin S9 stability experiments
An incubation mix was prepared containing 50 mM potassium phosphate buffer, pH 7.4), 0.3 mg/mL human skin S9 (Sekisui Xenotech), NADPH (final concentration 0.8 mg/mL), UDPGA (final concentration 0.16 mg/mL) and warmed to 37 ˚C for 5 minutes. The reaction was initiated upon addition of test compound (final concentration 0.5 μM). Immediately, at time zero, then at 3, 6, 15, 30, 60, 120 and 180 minutes, an aliquot (50 μL) of the incubation mixture was removed and mixed with acetonitrile (100 μL) to terminate the reaction. Internal standard was added to all samples, the samples centrifuged to sediment precipitated protein and the plates then sealed prior to UPLCMSMS analysis using a Quattro Premier XE (Waters corporation, USA).
Grafit (Erithacus Ltd) was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard at each timepoint. The half life (T ½ ) of each test compound was determined using the following equation: All values reported correspond to n of 1.

In vivo pharmacokinetics
All regulated procedures on living animals in the University of Dundee were carried out under the authority of a project licence issued by the Home Office under the Animals (Scientific Procedures) Act 1986, as amended in 2012 (and in compliance with EU Directive EU/2010/63). Licence applications will have been approved by the University's Ethical Review Committee (ERC) before submission to the Home Office. The ERC has a general remit to develop and oversee policy on all aspects of the use of animals on University premises and is a sub-committee of the University Court, its highest governing body.
A 22.5 µL single dose of compound (10a) at a 1% w/v concentration in a propylene glycol:ethanol 7:3 formulation was applied topically to a 1.5 cm 2 area of shaved skin on female Balb/c mice. The skin was shaved 24 h prior to treatment. Two hours post application (n=3 mice) or eight hours post application (n=3 mice) a blood sample was taken from each mouse and diluted with nine volumes of sterile water. The mice were then humanely killed. The exposed skin at the application site on each mouse was cleaned with vehicle to remove excess dose and the skin dissected. The diluted blood samples and skin samples were stored frozen prior to analysis of compound 10a levels by UPLC/MS/MS. Experimental procedures and analysis for compounds 4a-h, 9a-l and 10a-i Sodium acetate (5.5 g, 67 mmol) was dissolved in AcOH (75 ml) and the 3-chloro-4-hydroxybenzaldehyde (5.2 g, 33.5 mmol) was added to the stirred solution. The mixture was stirred for 3 hours until a homogeneous solution was obtained. In a separate flask, the 1-isothiocyanato-2-methylbenzene (5 g, 33.5 mmol) was dissolved in DCM under nitrogen and n-propylamine (2 g, 33.5 mmol) added dropwise at room temperature (exotherm to 35 °C). The mixture was stirred for 15 minutes at room temperature then cooled in an ice-salt bath to -2 °C. The addition funnel was rinsed with DCM and bromoacetyl bromide (6.8 g, 33.5 mmol) added dropwise at such a rate as to maintain the temperature below 5 °C over 15 minutes. The mixture was stirred for 15 minutes with cooling before again rinsing the addition funnel with DCM and the dropwise addition of pyridine (5.4 g, 68.7 mmol), again keeping the temperature below 5 °C. After the addition was complete, the resulting suspension was stirred with cooling for 45 minutes then allowed to warm to room temperature. The mixture was stirred a further 60 minutes. The mixture was then heated to 60 °C and approximately 45 ml of DCM distilled off under a stream of nitrogen. The AcOH solution of the benzaldehyde and sodium acetate was added in a steady stream and the resulting yellow suspension heated overnight at 60 °C. Approximately 25 ml of solvent was removed under vacuum. The remaining mixture was heated at 60 °C and water (40 ml) added dropwise over 45 minutes. The yellow suspension was stirred for 30 minutes with heating, then allowed to cool to room temperature. The mixture was filtered and the collected pale yellow solid washed with 2:1 AcOH/H2O (25 ml) followed by water (25 ml To a solution of 2-methoxyaniline (1 g, 8 mmol) in THF (35 ml) was added triethylamine (1.2 g, 12 mmol) and the reaction cooled to -70 °C. To this was added 2-chloroacetyl chloride (0.9 g, 8 mmol) dropwise and the reaction slowly warmed to room temperature. The reaction was diluted with water, extracted with ethyl acetate and the organics dried (MgSO 4 ), filtered and concentrated to give 2-chloro-N-(2-methoxyphenyl)acetamide as a red oil (1.6 g, 98% yield).