Discovery of super soft-drug modulators of sphingosine-1-phosphate receptor 1

Article abstract of DOI:10.1016/j.bmcl.2018.07.044

The oral S1PR1 agonist ponesimod demonstrated substantial efficacy in a phase II clinical trial of psoriasis. Unfortunately, systemic side effects were observed, which included lymphopenia and transient bradycardia. We sought to develop a topical soft-drug S1PR1 agonist with an improved therapeutic index. By modifying ponesimod, we discovered an ester series of S1PR agonists. To increase metabolic instability in plasma we synthesised esters described as specific substrates for paraoxonase and butyrylcholinesterases, esterases present in human plasma.

Full text of DOI:10.1016/j.bmcl.2018.07.044

Accepted Manuscript
Discovery of super soft-drug modulators of sphingosine-1-phosphate receptor
1
Mark Bell, David Foley, Claire Naylor, Colin Robinson, Jennifer Riley, Ola
Epemolu, Paul Scullion, Yoko Shishikura, Elad Katz, W.H. Irwin McLean, Paul
Wyatt, Kevin D Read, Andrew Woodland
PII:
S0960-894X(18)30639-5
https://doi.org/10.1016/j.bmcl.2018.07.044
BMCL 25975
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 May 2018
19 July 2018
29 July 2018
Please cite this article as: Bell, M., Foley, D., Naylor, C., Robinson, C., Riley, J., Epemolu, O., Scullion, P.,
Shishikura, Y., Katz, E., McLean, W.H.I., Wyatt, P., Read, K.D., Woodland, A., Discovery of super soft-drug
modulators of sphingosine-1-phosphate receptor 1, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.07.044
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Discovery of super soft-drug modulators of sphingosine-1-phosphate receptor 1.
a
a
a
a
a
a
Authors: Mark Bell* , David Foley , Claire Naylor , Colin Robinson , Jennifer Riley , Ola Epemolu ,
a
a
a
b
a
a
Paul Scullion , Yoko Shishikura , Elad Katz , W. H. Irwin McLean , Paul Wyatt , Kevin D Read , and
a
Andrew Woodland*
a
The Drug Discovery Unit, Biological Chemistry and Drug Discovery, University of Dundee, Dundee,
DD1 4HN, UK
b
Dermatology and Genetic Medicine, Biological Chemistry and Drug Discovery, University of
Dundee, Dundee DD1 5EH, UK
*
Corresponding authors: Dr Andrew Woodland and Dr Mark Bell, The Drug Discovery Unit, Biological
Chemistry and Drug Discovery, University of Dundee, Dundee, DD1 4HN. awoodland@dundee.ac.uk
and m.u.bell@dundee.ac.uk.
Abstract
The oral S1PR1 agonist ponesimod demonstrated substantial efficacy in a phase II clinical trial of
psoriasis. Unfortunately, systemic side effects were observed, which included lymphopenia and
transient bradycardia. We sought to develop a topical soft-drug S1PR1 agonist with an improved
therapeutic index. By modifying ponesimod, we discovered an ester series of S1PR agonists. To
increase metabolic instability in plasma we synthesised esters described as specific substrates for
paraoxonase and butyrylcholinesterases, esterases present in human plasma.
Graphical abstract
Keywords
S1PR1, soft-drug, plasma stability, psoriasis, topical
1
Psoriasis is a common chronic inflammatory skin disease that affects 2% of the population. 52.3%
of patients were dissatisfied with current treatments in a recent survey from the National Psoriasis
1
Foundation in the US. Recently approved biological drugs targeting disease relevant receptors, such
2
3
4
as secukinumab or ixekizumab for interleukin (IL)-17 and ustekinumab for IL-12/23 have brought
great benefit to patients with severe symptoms of the disease. However, a need remains for safe,
convenient, efficacious therapies for mild and moderate psoriasis.
Sphingosine-1-phosphate receptor (S1PR) agonists are of interest to the pharmaceutical industry,
due to their potential to treat diseases of the immune system such as psoriasis and multiple sclerosis
5
,6
as well as cancer. S1PR agonists, such as fingolimod and ponesimod (Figure 1), initially activate
sphingosine-1-phosphate receptors, but subsequently trigger receptor internalisation. This shuts
down the sphingosine 1-phosphate signalling pathway, which then prevents the maturation and
7
migration of lymphocytes. In 2010 fingolimod was approved for the treatment of
relapsing/remitting multiple sclerosis and is the only S1PR1 agonist approved to date.
8

In a phase 2 study in plaque psoriasis, a 40 mg oral dose of ponesimod, led to a 75% reduction in
9
psoriasis area and severity index (PASI) score in 77% of the patients. This level of efficacy is
1
0
11
competitive with other small molecules in development to treat psoriasis (apremilast , tofacitinib
1
2
and sotrastaurin ). Ponesimod was not however progressed into phase 3, possibly due to safety
concerns. Oral use of pan-S1PR and S1PR1 selective modulators is associated with several severe
side effects such as lymphopenia, bradycardia and dyspnoea, which limits their utility as therapies
5
for non-life threatening chronic diseases.
In mice, topical application of the S1PR agonist fingolimod led to a reduction in the number of
1
3
Langerhans cells migrating to the lymph node. Work by Griffiths and co-workers has shown that
Langerhans cell migration is higher in involved areas of patient’s skin than in non-involved areas of
14, 15
skin.
We hypothesised that local inhibition of Langerhans cell migration, through the effects of
topical S1PR agonists, could restore a normal skin phenotype to patients with psoriasis.
Figure 1. Selected S1PR1 modulators
Due to the potential of S1PR agonists to act as efficacious therapies, for the treatment of psoriasis,
we decided to embark on developing a topical S1PR1 modulator using a fast-follower approach,
inspired by ponesimod. Topical therapies apply drugs directly to the site of action and can lead to a
reduction in the systemic exposure of a drug when compared to oral dosing. However, due to the
chronic nature of psoriasis and the need to be able to treat large body surface areas (>10%), there is
a danger that topical application could lead to biologically relevant systemic drug concentrations. To
1
6, 17
maximise the therapeutic index we decided to develop a soft drug.
Soft drugs are locally active,
in this case in the skin, but then undergo rapid systemic metabolism, to metabolites, which are
1
8
either inactive or rapidly cleared from the systemic circulation.
1
9
Esters can be metabolised in liver by esterases and cytochrome P450 enzymes. However, most
tissues are capable of metabolising an ester. Importantly, human blood contains a wide range of
2
0
esterases and the rate of ester hydrolysis in blood can lead to ultra-rapid clearance, what we term
a super-soft drug. Because S1PR agonists induce transient bradycardia, and as blood returning to
circulation from skin passes through the heart before reaching the liver, we felt that a very rapid
blood clearance mechanism could potentially discharge the cardiotoxicity risk. To select compounds
which would be likely to demonstrate the desired pharmacokinetic profile, we targeted a human
plasma stability half-life of <5 minutes. We chose to first develop benzoate ester analogues of
ponesimod to determine if they could demonstrate potential as super-soft drugs. The desired
compound would have S1PR pXC50 of >7 and a logP in the range of 2-4 which is believed to be
2
1
optimal for skin penetration.
Compounds 4a and 5a-d were synthesised as shown in Scheme 1. The (Z)-2-(propylimino)-3-(o-
tolyl)thiazolidin-4-one core 2 was synthesised utilising a one-pot, two step reaction. Only the Z
isomer was observed. N-propylamine was reacted with 1-isothiocyanato-2-methylbenzene 1 to give
the resulting thiourea, which was condensed with 2-bromoacetyl bromide followed by addition of
pyridine to furnish the desired thiazolidin-4-one 2. This core was then condensed with the
appropriately substituted benzaldehyde 3a-b to furnish 4a and 5a. Finally, esterification or

amidation of 4a gave the desired products. Based on the configurational analysis by X-ray, which
2
2
characterised crystalline analogues of ponesimod as Z,Z , all structurally related compounds 4a, 5a-
d, 11, 12a-f and 15a,b were assigned to the Z,Z-isomer.
Scheme 1. Reagents and conditions for preparation of compounds 4a and 5a-d: (a) n-PrNH
CH Cl , rt (b) 2-bromoacetyl bromide (1eq) and pyridine (2eq), CH Cl , 0 ˚C to rt (two steps) (c)
aldehyde (1eq), NaOAc (2eq), AcOH, 65 ˚C (d) alcohol (5eq), HOAt (1.5eq), EDC (1.5eq), DIPEA
2.5eq), CH Cl , rt. eq: equivalent; rt: room temperature; HOAt: 1-Hydroxy-7-azabenzotriazole; EDC:
-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; DIPEA: N,N-Diisopropylethylamine.
2
(1eq),
2
2
2
2
(
2
2
1
Table 1
Effect of R substitution on S1PR1 activity, kinetic aqueous solubility and human plasma stability.
2
1
2
Compound
Ponesimod
R
R
Kinetic
CHIb
logD
S1PR1
pIC50
H Plasma
Stability
(half-life min)
>180
c
solubility
a
e
(
µM)
110
39
-
-
3.1
>4.4
>4.4
1.0
3.4
2.3
7.9 (0)
8.6 (0.1)
6.5 (0)
<6 (0)
6.9 (0)
7.0 (0)
5a
Cl
H
H
H
H
OMe
OMe
OH
CH
CH
>180
>180
-
5
4
b
a
28
163
110
5c
OCH
NHCH
2
2
OH
OH
>180
5d
2
2
>250
>180
a
b
The aqueous solubility of the test compounds was measured using laser nephelometry. Reverse-
phase HPLC method to determine the chromatographic hydrophobicity index (CHI). All pIC50
c
s
reported in this table correspond to n of 2, reported as their geometric mean. The range of pIC50
d
values is provided in brackets. Incubated in human plasma at 37 ᵒC.
We selected human plasma stability as a model for human blood metabolism. Esters 5a-c and amide
5
d demonstrated half-lives of >180 minutes in human plasma, far greater than the <5 minutes we
2
3
were aiming for. S1PR1 activity was measured using a PathHunter β-Arrestin recruitment assay.
All of the ester and amide compounds in Table 1 were active and had pIC50 values ranging from 6.5
to 7.0, with the exception of compound 5a where a chlorine ortho to the ester provided 100-fold
improvement in activity compared to 5b. Gratifyingly, the parent carboxylic acid 4a, which is the

desired metabolite of our esters/amides was less active, giving a pIC50 of <6 and meeting our
targeted criteria for producing inactive metabolites.
Aqueous solubility is an important parameter for topically applied drugs. Increasing aqueous
2
4
solubility, should directly increase skin penetration rates. Increasing aqueous solubility also
increases the range of formulation options that can be used in pre-clinical development. We were
2
pleased to see that introduction of a hydrogen bond donor at R in 5c led to a 4-fold increase in
solubility relative to 5b. Changing the ester linker in 5c to an amide in 5d further improved solubility
to >250 µM, probably due to improvements in lipophilicity (3.4 vs 2.3 respectively).
Encouraged by the solubility and activity data, we focused our efforts on optimising the metabolic
profile of the esters. At this time, we identified that switching from the o-tolyl group used in
ponesimod to an o-phenol 6 increased aqueous solubility 2-fold, presumably due to the reduction in
lipophilicity (Figure 2). As 6 was equipotent to ponesimod (pIC50 7.9) and to maximise compound
solubility, subsequent compounds incorporated the o-phenol group.
Figure 2. Improvements in aqueous kinetic solubility from introduction of o-phenol group
Compounds 11, 12a-c, 15a and 15b were synthesised using the route shown in Scheme 2. 12a-c
were more soluble than ponesimod, probably due to significant improvements in CHI logD (Table 2).
As expected a clear trend can be seen between low lipophilicity and improved solubility.
Homologation of the ester group (15a,b) showed increased lipophilicity (3.2 and 3.5 respectively)
and a commensurate reduction in solubility. However, as with 5a-d, amide 12a and esters 12b-c also
showed a lack of metabolism in our human plasma stability assay (Table 2).

Scheme 2. Reagents and conditions for preparation of compounds 11, 12a-f and 15a,b: (a) n-PrNH
1eq), CH Cl , rt (b) 2-bromoacetyl bromide (1eq) and pyridine (2eq), CH Cl , 0 ˚C to rt (two steps) (c)
aldehyde (1eq), NaOAc (2eq), AcOH, 85 ˚C (d) BBr (8eq), CH Cl , -78 ˚C to rt (e) For 12a-c. amine
3eq) or alcohol (5eq), HOAt (1.5eq), EDC (1.5eq), DIPEA (2.5 eq), CH Cl , rt (f) For 12d-f. alcohol,
alkyl chloride or alkyl tosylate (1.1-1.5eq) K CO (1.5eq), NaI (0.1eq), DMF, 70 ˚C (g) NEt (3eq), EDC
1.1eq), HOAt (1.1eq), alcohol (1.5eq) rt (h) BBr (5eq), CH Cl , -78 ˚C to rt. eq: equivalent; rt: room
temperature; HOAt: 1-Hydroxy-7-azabenzotriazole; EDC: 1-Ethyl-3-(3-
2
(
2
2
2
2
3
2
2
(
2
2
2
3
3
(
3
2
2
dimethylaminopropyl)carbodiimide; DIPEA: N,N-Diisopropylethylamine; DMF: dimethyl formamide.
Table 2
1
Effect of R substitution on kinetic aqueous solubility and human plasma stability.
1
b
Compound
Ponesimod
n
R
Kinetic
CHI logD
H Plasma
solubility
Stability (half-
a
c
(
µM)
110
219
183
life min)
-
-
3.1
1.9
2.3
1.8
>180
>180
>180
>180
1
2a
2b
2c
0
0
0
NHCH
OCH
OCH CH(±CH
2
CONMe
2
1
2
CH
2
CH
2
OH
OH)OH
1
2
2
>250

1
5a
1
2
OMe
OMe
155
79
3.2
3.5
>180
>180
15b
a
b
The aqueous solubility of the test compounds was measured using laser nephelometry. Reverse-
c
phase HPLC method to determine the chromatographic hydrophobicity index (CHI). Incubated in
human plasma at 37 ᵒC.
As simple esters had shown themselves to be stable to human plasma, we turned our attention to
ester functional groups which had been shown to be unstable in human plasma in the literature
2
0
(
half-life <5mins). The most successful studies focused on optimising metabolism by the esterases
2
5
26
paraoxonase and butyrylcholinesterases , which are mainly expressed in blood plasma.
We synthesised compounds 11 and 12d-f using the route shown in Scheme 2, which all contain ester
groups that are unstable in human plasma when attached to other scaffolds. Although 12d or 12e
did not show sufficient clearance in human plasma we were pleased to identify compound 12f
containing a (2-oxo-1,3-dioxolan-4-yl)methyl ester which is rapidly degraded in human plasma. The
half-life of this ester in human plasma was 9 minutes and metabolite identification confirmed the
conversion to the inactive parent acid 11 (Figure 3).
1600
1400
1200
1000
800
600
400
200
0
1
2f
1
1
0
10
20
30
40
50
60
Time
Figure 3. Profile of compound 12f being hydrolysed to 11 in human plasma. 12f was incubated in
human plasma at 37 ᵒC. The experiment was conducted as an n of one to provide a qualitative
analysis.
Unfortunately 12f was also rapidly metabolised in human skin S9 fraction with a half-life of just 23
minutes, far faster than the desirable half-life of >6h that we target for once daily dosing. In fact,
there are suggestions of a correlation between the relative stability of 12d-f in human plasma and
skin S9 fraction. It may be that the esterases responsible for the metabolism of 12f are present in
both human plasma and skin S9 fraction or that skin and plasma esterases have similar substrate
affinities. Unfortunately, 12f was also inactive as a S1PR1 modulator.

Table 3
1
Effect of R substitution on kinetic aqueous solubility and human plasma stability.
1
Compound
R
H Plasma
Kinetic
CHI
H Skin S9 S1PR1
c
e
Stability (half- solubility logD
(half-life
min)
pIC50
a
b
d
life min)
>180
(µM)
-
11
OH
-
-
<6 (0)
-
12d
180
117
3.0
152
1
2e
61
9
79
79
2.8
3.3
40
23
-
1
2f
<6 (0)
a
b
Incubated in human plasma at 37 ᵒC. The aqueous solubility of the test compounds was measured
c
using laser nephelometry. Reverse-phase HPLC method to determine the chromatographic
hydrophobicity index (CHI). Stability measured in skin S9 over 180 mins in the presence of
enzymatic cofactors. All pIC50s reported in this table correspond to n of 2, reported as their
d
e
geometric mean. The range of pIC50 values is provided in brackets.
In summary, using a fast follower approach, we identified a series of ester S1PR1 modulators.
Further optimisation led to improved solubility and identification of ester 12d that is plasma
unstable. Unfortunately, the ester is inactive and is metabolised in human skin models. Further
efforts will focus on identifying an active, skin stable compound that is rapidly metabolised
systemically will be disclosed in due course.
Acknowledgements
The Centre for Dermatology and Genetic Medicine is supported by a Wellcome Trust Strategic Award
(reference 098439/Z/12/Z).
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Highlights



Identification of soft drug S1PR agonists
Development of a process for the evaluation of topical esterase soft drugs
Study skin and plasma esterase SAR