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

Article abstract of DOI:10.1021/acsmedchemlett.8b00616

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, wh

Full text of DOI:10.1021/acsmedchemlett.8b00616

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Letter
Discovery of soft drug topical tool modulators
of sphingosine-1-phosphate receptor 1 (S1PR1)
Mark Bell, David William Foley, Claire Naylor, Gavin Wood, Colin Robinson, Jennifer Riley, Ola
Epemolu, Lucy Ellis, Paul Scullion, Yoko Shishikura, Maria Osuna-Cabello, Liam Ferguson, Erika Pinto,
Daniel Fletcher, Elad Katz, W. H. Irwin Mclean, Paul G. Wyatt, Kevin D Read, and Andrew Woodland
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00616 • Publication Date (Web): 14 Feb 2019
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ACS Medicinal Chemistry Letters
Discovery of soft drug topical tool modulators of sphingosine-1-phosphate receptor 1 (S1PR1)
1
Mark Bell*†, David Foley§, Claire Naylor†, Gavin Wood†, Colin Robinson†, Jennifer Riley†, Ola
Epemolu†, Lucy Ellis ‡, Paul Scullion†, Yoko Shishikura†, Maria Osuna-Cabello†, Liam Ferguson†,
Erika Pinto†, Daniel Fletcher†, Elad Katz†, W. H. Irwin McLean‖, Paul Wyatt†, Kevin D Read†, Andrew
Woodland*†.
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†Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow
Street, Dundee. DDI 5EH.
§Medicines Discovery Institute, Cardiff University, Cardiff, CF10 3XQ, UK.
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‡New Modalities, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, SE-431 83, Sweden.
‖Dermatology and Genetic Medicine, Division of Molecular Medicine, University of Dundee, Dundee DD1 5EH, UK.
KEYWORDS. S1PR, soft-drug, plasma stability, topical tool compound.
ABSTRACT: 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.
Sphingosine-1-phosphate receptor (S1PR) agonists, such as
fingolimod and ponesimod (Figure 1), initially activate S1P
receptors, but subsequently trigger receptor internalisation and
down regulation of signalling; shutting down the sphingosine-1-
phosphate signalling pathway. Fingolimod was approved in 2010
for the treatment of relapsing/remitting multiple sclerosis and is the
only S1PR agonist approved to date.1 It is efficacious at low doses
(0.5 mg/day) and at low steady state systemic concentrations (Cmax
3.1 ng/mL). Recently the potential for the S1PR pathway to be of
therapeutic use in the treatment of a range of diverse inflammatory
skin diseases has emerged.2-6 Some studies have explored the skin
biology of S1PR agonists by topical application of these
compounds in various animal models of diseases such as atopic
dermatitis,4 allergic dermatitis5 and psoriasis.6 Topical application
can however also lead to systemic effects. Following penetration
through the stratum corneum, drugs will eventually distribute into
the vasculature. If the rate of absorption exceeds the rate of
elimination, topical dosing will lead to systemic drug exposure.
Topical dosing of potent drugs, such as fingolimod, may lead to
sufficient systemic drug concentrations to elicit measureable
biological effects, complicating the interpretation of such studies.
suited to treatment with topical soft drugs, which can safely engage
biological targets, previously shown to lead to adverse side effects,
following oral dosing.
In their paper describing ponesimod’s discovery, Bolli et al.
disclosed that phenols, such as compound 4a, although active were
unsuitable for progression, as an oral drug, due to high clearance in
both in vitro and in vivo experiments.10 The authors speculated that
the high clearance may be due to the fact that phenols are well-
known substrates for phase 2 metabolism conjugating enzymes.10
Glucuronidation is a common phase II metabolism pathway that
covalently conjugates glucuronic acid, in a base-catalysed process
from UDPGA (uridine-50-diphosphoglucuronic acid) to lipophilic
substrates via UGT enzymes (uridine-50-diphosphoglucuronosyl
transferases).11 Sulfation, another common phase II metabolism
pathway, covalently links a substrate to a sulfo group (SO3),
usually derived from 3'-phosphoadenosine-5'-phosphosulfate
(PAPS), via sulfotransferase enzymes.12 As the glucuronide and
sulfate metabolites are highly polar, and therefore water-soluble,
they subsequently undergo renal or biliary elimination. Due to their
affinity for phase II metabolism, phenols are commonly used
motifs when designing soft drugs.13,14 There is little evidence of
clinically relevant drug-related inhibition of glucuronidation or
sulfation, so the risk of drug-drug interactions is considered to be
low.15 Accordingly we set out to utilise phase II metabolism
pathways as the major routes of clearance for our S1PR agonist soft
drugs.
OH OH
N
OH
NH2
O
S
OH
N
Cl
O
Although 4a had been shown to be rapidly cleared, which was
confirmed in our hands (Table 1), the compound displayed poor
aqueous solubility. Aqueous solubility is an important parameter
for topically applied drugs as it can support use in a higher water
content formulations, such as a creams, which may be preferred by
patients over oily formulations like ointments. We therefore set out
to improve the aqueous solubility of 4a.
Fingolimod
Ponesimod
Figure 1. Selected S1PR modulators.
In order to remove the potential for systemic exposure, we decided
to develop a soft drug S1PR modulator.7,8 Soft drugs are locally
active, in this case in the skin, but are designed to undergo rapid
systemic metabolism to metabolites, which are either inactive or
rapidly cleared from systemic circulation.9 Due to ease of access
of the diseased organ, many dermatological diseases are ideally
Keeping the 3-chloro-4-hydroxybenzylidene motif from 4a
constant we synthesised a series of phenols with different
substituents to replace the 2-tolyl 4a motif with aromatic or
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aliphatic groups (Scheme 1). Using Method A the appropriate
an improvement in aqueous solubility (>250 µM) presumably due
1
2
3
4
5
6
7
8
aniline was reacted with 2-chloroacetyl chloride to give the
corresponding 2-chloro-N-phenylacetamide, which was condensed
with 1-isothiocyanatopropane to give the required thiazolidinone
to the addition of the polar hydroxyl-group and the commensurate
reduction in CHIlogD, but unfortunately the compound had a pIC50
of <6.0. The fact that 9a improves aqueous solubility and was
equipotent identifies the 2-oxetan-3-ylimino group as a potentially
useful change to incorporate in the design of future compounds.
core 2a,b.
Subsequent condensation with 3-chloro-4-
hydroxybenzaldehyde 3, generated compounds 4a,b. Compounds
4c-g with aliphatic R1 groups used Method B, where amines 1c-g
were reacted with 1-isothiocyanatopropane, then with 2-
bromoacetyl bromide in the same reaction vessel. The resulting
thiazolidinone cores 2c-g were condensed with 3-chloro-4-
hydroxybenzaldehyde 3 and the products 4c-g obtained using
preparatory HPLC. 4h was prepared by BBr3 demethylation of the
anisole 4b to give the corresponding phenol.
Having examined two of the vectors off the thiazolidin-4-one core
we turned our attention to the benzylidene substituent to optimise
activity, aqueous solubility and hepatic metabolism. For reason of
synthetic expediency, we kept the 2-tolyl and n-propyl groups in
place with the intention of combining the optimum substituents in
subsequent design rounds. We therefore synthesised a series of
phenols (9f-9l) using the method shown in Scheme 2. 9f-9l
contained a range of electron withdrawing and donating groups
ortho to the 4-phenol of the benzylidene substituent. 9f, 9g and 9i-
9k were largely equipotent to 4a, while 9h and 9l had a pIC50 of
<6.0, presumably in the case of 9l due to increased steric bulk
(Table 2). The trifluoromethyl group of 9g had low aqueous
solubility, while 9f and 9h-9l had acceptable solubility.
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Compounds 9a-9e replaced the n-propyl group of 4a with several
small N-linked aliphatic substituents, while compounds 9f-l looked
at effects of substituents on the 4-hydroxybenzylidene group
(Scheme 2). The appropriately substituted thiazolidin-4-one core
7a-d was synthesised utilising a one-pot, two step reaction. Alkyl
amines were reacted with 1-isothiocyanato-2-methylbenzene 5 to
give the resulting thiourea 6a-d which was condensed with 2-
bromoacetyl bromide, followed by addition of pyridine to furnish
the desired thiazolidin-4-one. The thiazolidin-4-one cores 6a-d
were condensed with the 4-hydroxybenzaldehyde 3 to give 9a-d.
9e was synthesised by treatment of 9c with BBr3. 2a was reacted
with 8f,g,i-l to furnish products 9f,g,i-l. 9h was synthesised using
a Negishi coupling with dicyanozinc and palladium tetrakis from
9f.
Scheme 1.
Cl
N
N
OH
Method A
Method B
v
S
S
NH2
R1
N
R1
R1
N
Cl
OH
O
O
O
1a-g
2a-g
3
4a-g
The configuration of the double bonds in ponisimod and 4a were
determined by X-ray crystallography.10 The HMBC and NOESY
data of ponesimod and 4a were compared with 9k and 10a (see
supporting information). The HMBC data for the alkene proton to
the carbonyl carbon (H9-C3 or H9’-C3’) in all cases was consistent
and suggested a Z double bond arrangement of the alkene bond (the
size of the 1H-13C coupling constant was estimated to be 6-7 Hz).
The only cross peaks observed in the NOESY experiments were
between the 2-tolyl and imine groups. These weak signals between
the respective methyl groups (see supporting information) were
also observed for ponesimod, 4a, 9k and 10a. It may be expected
that if the imine was in the E configuration that there would have
been cross peaks observed between the methyl of the 2-tolyl group
and the NCH2 protons of the imine group, however this was not
observed. Taken together, the data was consistent with the Z
configuration observed using X-ray crystallography but did not
confirm it. Based on the analysis of analogous compounds 4a-h,
9a-l and 10a-i were assigned to the Z,Z-isomer, unless stated
otherwise.
O
4a: R1 = 2-tolyl
4c: R1 = i-Pr
4e: R1
=
4g: R1 = cyclopropane
4h: R1 = 2-phenol
S
O
N
O
4b: R1 = 2-anisole 4d: R1
=
4f: R1
vi
=
O
Method A (i) 2-chloroacetyl chloride, TEA, THF, -78 ˚C to RT,
2h (ii) 1-isothiocyanatopropane, NaH, DMF, RT, 16h. Method B
(iii) 1-isothiocyanatopropane, CH2Cl2, RT, 2h (iv) 2-bromoacetyl
bromide, pyridine, CH2Cl2, 0 ˚C to RT, 1h (v) NaOAc, AcOH, 65
˚C, 16h (vi) BBr3, CH2Cl2, -70 ˚C to 0 ˚C, 3h.
Scheme 2.
R1
R1
R2
X
N
N
S
OH
i
ii
iii
S
C
S
S
N
R1
N
N
N
N
R2
H
H
O
O
OH
O
5
6a-d
7a-d, 2a
9a-l
X
3 or 8f,g,i-l
Compounds 4c-h and 9a-e were designed to improve solubility by
reducing logD or aromatic ring count.16 Although the CHIlogD
values were lower or equivalent for 4d, 4e, 4g and 9a-e, the
compounds did not show an improvement in aqueous solubility
(Table 1). Reducing the aromatic ring count in 4c-e and 4g also
failed to improve aqueous solubility, while 4f gave an improvement
in aqueous solubility 250 µM possibly due to a 3-log unit reduction
in CHIlogD, but had a pIC50 of <6.0. The addition of a 2-phenol
group into the R1 position, compound 4h, lowered the CHIlogD by
1.1 units and improved aqueous solubility to 220 µM. Two of the
changes to the 2-propylimino group showed an improvement in
aqueous solubility. 9a with a 2-oxetan-3-ylimino group moderately
increased solubility to 150 µM, compared to 79 µM for 4a. 9e gave
9a: R1
9b: R1
=
O
R2 = Cl, X = CH
R2 = Cl, X = CH
9f: R1 = n-Pr, R2 = Br, X = CH
9g: R1 = n-Pr, R2 = CF3, X = CH
9h: R1 = n-Pr, R2 = CN, X = CH
9i: R1 = n-Pr, R2 = H, X = N
(iv)
F
F
=
9c: R1 = CH2CH2OMe, R2 = Cl, X = CH
9d: R1 = CH2CH2CH2F, R2 = Cl, X = CH
9e: R1 = CH2CH2OH, R2 = Cl, X = CH
9j: R1 = n-Pr, R2 = H, X = CH
9k: R1 = n-Pr, R2 = Me, X = CH
9l: R1 = n-Pr, R2 = i-Pr, X = CH
(v)
(i) R2NH2, CH2Cl2, RT, 1h (ii) 2-bromoacetyl bromide, pyridine,
CH2Cl2, 0 ˚C to RT, 2h (iii) NaOAc, AcOH, 65 ˚C, 16h (iv)
dicyanozinc, Pd(PPh3)4, DMA 100 ˚C, 1.5h. (v) BBr3, DCM, -78
˚C, 3h then 0 ˚C, 3h.
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ACS Medicinal Chemistry Letters
Table 1. Optimisation of the thiazolidinone core.
1
R2
2
3
4
Cl
N
OH
S
1 N
R
5
6
O
7
8
9
Compound
R1
R2
CHIlogDb
Kinetic Solubility
(µM)c
d
H S1PR1 pIC50
4a
4b
4c
4d
2-tolyl
2-anisole
i-Pr
n-Pr
n-Pr
n-Pr
n-Pr
3.8
3.3
79
79
20
70
7.4
7.2
6.8
7.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
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60
>4.3
3.7
O
O
4e
n-Pr
n-Pr
2.6
0.6
75
6.7
S
O
4fa
>250
<6.0
N
O
4g
4h
9a
cyclopropane
2-phenol
2-tolyl
n-Pr
n-Pr
3.4
2.7
2.6
79
6.2
6.6
7.3
220
150
O
F
F
9b
2-tolyl
3.6
79
6.7
9c
9d
9e
2-tolyl
2-tolyl
2-tolyl
CH2CH2OMe
CH2CH2CH2F
CH2CH2OH
2.9
3.4
1.9
110
20
6.3
7.6
>250
<6.0
aracemic mixture. bReverse-phase HPLC method to determine the chromatographic hydrophobicity index (CHI): n of 1. The aqueous
kinetic solubility of the test compounds was measured using laser nephelometry: n of 1.dHuman S1PR1 activity was measured using a human
PathHunter β-Arrestin recruitment assay. All pIC50s reported in this table correspond to n ≥ 2, reported as their geometric mean.
c
As soft drugs must be rapidly cleared systemically and phenols
commonly undergo phase 2 metabolism, we used human
hepatocytes (H Heps) to study this potential route of metabolism.
We sought to obtain clearance rates of greater than 85% human
liver blood flow (>4.8 mL/min/g); data shown in Table 2. We then
measured intrinsic clearance in human liver microsomes (HLM) to
determine if phase 1 metabolism was contributing to the observed
intrinsic clearance in hepatocytes. As glucuronidation is a base-
catalyzed process, where conserved carboxylate and histidine
residues facilitate the deprotonation of the phenol, we expected to
see an effect of the pKa of the phenolic hydrogen on the rate of
hepatic clearance.14 We explored the effect of the phenol pKa on
hepatic clearance with a set of ortho-substituents and a meta-
pyridine (Table 2). Electron-withdrawing groups did reduce the
pKa of the phenol 4a and 9f-i have increased hepatic clearance rates
vs unsubstituted 9j. However, weakly electron-donating groups
demonstrated an even greater increase in hepatic clearance rates
(9k and 9l) despite the expected increase in pKa. For this phenolic
scaffold ortho-substituents led to an increase in glucuronidation
rate in all cases and was independent of phenolic pKa.
Table 2. Effect of substituents on the phenol.
R1
N
OH
S
N
X
O
Compound
R1
X
H
S1PR1
pIC50
Kinetic
pKac
HLM Cld
H Heps
Cle
Stabilityf
CHI
logDg
Solubility
(µM)b
a
4a
9f
Cl
Br
CH
CH
CH
CH
7.4
7.7
79
79
6.8
6.4
6.6
-
1.6
1.6
1.0
-
8.4
6.0
6
3.8
3.9
3.8
-
16
10
20
9g
9h
CF3
CN
7.5
14
4.2
<6.0
220
<0.5
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9i
9j
H
H
N
7.0
7.1
110
79
7.1
8.3
8.5
8.6
2.8
2.4
2.8
-
7.9
1.7
31
4
0
3
3
3.0
3.5
3.8
4.3
1
2
3
4
CH
CH
CH
9k
9l
Me
i-Pr
7.6
78
<6.0
79
12
5
6
7
8
aHuman S1PR1 activity was measured using a human PathHunter β-Arrestin recruitment assay. All pIC50s reported in this table
correspond to n ≥ 2, reported as their geometric mean. bThe aqueous kinetic solubility of the test compounds was measured using laser
nephelometry: n=1. pKa was determined using a potentiometric fast UV-metric titration method: n=1. Intrinsic clearance in human liver
microsomes (mL/min/g): n=1. eIntrinsic clearance in human liver hepatocytes (mL/min/g): n=1. f% decrease in purity when stored in DMSO
solution for 28 days: n=1. gReverse-phase HPLC method to determine the chromatographic hydrophobicity index (CHI): n=1.
c
d
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Although several compounds shown in Table 2 satisfy the rapid
clearance requirements of a soft drug and retain primary activity,
none were suitable for progression into in vivo studies due to
chemical stability liabilities. Analysis of the originally pure
compounds shown in Table 1, after being in DMSO solution for 28
days showed a range of purities. Compounds with electron-
withdrawing groups ortho to the phenol (4a, 9f-h) were the least
stable with a 6-20% impurity formed over 28 days. Compounds
with neutral or donating groups in the ortho position (9j-l) were
more stable, in some cases giving compounds which were stable
over a 28 day period (9j). The instability in solution represented a
major development hurdle as topical drugs are usually stored in
solution or suspensions (cream, ointment, paste, lotion or gels),
rather than in solid form, as is the case for oral drugs. We turned
our attention to identifying the impurity and preventing its
formation.
Scheme 3.
10a: R1 = Me, R2 = Me
10b: R1 = Br, R2 = Me
10c: R1 = CF3, R2 = Me
10d: R1 = Me, R2 = CH2OMe
10e: R1 = Me, R2 = CH2NMe2
10f: R1 = Me, R2 = CH2CH2OH
10g: R1 = Me, R2 = CH2CH2OMe
10h: R1 = Me, R2 = i-Pr
R1
R1
N
N
i or ii
R2
OH
O
S
N
S
N
O
O
O
9k: R1 = Me
9f: R1 = Br
9g: R1 = CF3
10i: R1 = Me, R2 = t-Bu
(i) Acid chloride (1eq), DMAP (0.05 eq), TEA (1.2eq) in CH2Cl2
at rt, 16h (ii) 2,2-dimethylpropanoic acid (1eq), DCC (1.2eq),
DMAP (0.2eq), DMF, 40 ˚C, 16h.
We were delighted to discover that acylation blocked isomerisation
and 10a,f shown in Table 3 did not isomerise after being in a
DMSO solution for 28 days. Electron-withdrawing groups at R1
(10b,c) did lead to a slight decrease in purity (6.3 and 1.4%
respectively) over the 28 day duration of this experiment, but the
degradation product was due to hydrolysis of the ester to the phenol
rather than isomerisation of the double bond. Decomposition
We conducted NMR studies of compound 9h after incubation in
DMSO-d6 for 6 months (see supporting information for HMBC and
NOESY spectra). In that time the impurity had increased from 20%
to 32% of the mixture based on the integration of H18 vs H18’ in the
1H NMR spectrum. The NOESY spectrum of the mixture indicated
no changes in the arrangement of the imine (no correlation was
observed between H11-H19 or H11’-H19’). HMBC experiments
measuring the three bond coupling constant between H9-C3 and
H9’-C3’ were analysed and confirmed that the double bond in the
major component (68%) had a coupling constant of 6.4 Hz
indicating a Z arrangement, while in the minor component (32%)
the coupling was measured at 11.9 Hz indicating an E arrangement.
1
studies used H-NMR to monitor the increase in the acetic acid
methyl group peak over 28 days (see supporting information). 10a
showed no hydrolysis or isomerisation. However, the chemical
stability was poor when heteroatoms were alpha to the carbonyl of
the acetate group 10d,e and these compound degraded on standing
within 24h, preventing full characterisation.
instability was not an issue when the heteroatoms were in the beta
Chemically,
position 10f,g.
We expected the ester to be unstable in skin, which would liberate
the phenol to engage the receptor in the target tissue. Determining
skin stability using human skin S9 fraction (Table 3) demonstrated
compounds 10a-c,f,g underwent rapid metabolism. Bulking out the
ester with i-Pr 10h or t-Bu 10i gave longer half-lives as expected.
As we expected them to be significantly less active due to the
orientation of the phenol group, no examples of (Z,E) compounds
were isolated.
Unfortunately acylation of 9k led to a decrease in aqueous
solubility, for example 10a (39 µM), 9k (79 µM). This could be
improved by adding polar groups as in 10f (79 µM), however
solubility only improved to the level of phenol 4a, and is still lower
than what is desirable in a topical drug. Our identification of the
propensity for the phenol series to isomerise meant they were
unsuitable for development as tool compounds or potential drugs.
The acylated series does not have the desired solubility of a
potential drug, but is suitable for use as a topical tool.
The association between electron withdrawing groups and the rate
of the isomerisation could be explained by the requirement for a
base catalysed isomerisation mechanism (see supporting
information for proposed mechanism). We hypothesised that
protecting the phenol via alkylation (as in ponesimod) or acylation
would remove the ability of the conjugated pi-system to isomerise
the double bond from Z to E. To test this theory we synthesised
compounds 10a-i via esterification of the parent phenol (Scheme
3). Reaction of phenol 9k,f,g with the corresponding acid chloride
gave compounds 10a-h.
Reaction of phenol 9k with
10a was selected for further study, due its ease of synthesis,
stability to degradation in solution and instability in skin.
dimethylpropanoic acid and DCC gave compound 10i.
Table 3. Effect of substitution at R1 and R2 on skin S9 and chemical stability.
R1
N
R2
O
S
N
O
O
Compound
Ponesimod
R1
Cl
R2
-
H Skin S9
Stability
(% decrease)c
(half-life min)b
>180
0
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10a
10b
10c
10d
10e
10f
10g
10h
10i
Me
Br
Me
Me
7.3
8.8
8.3
-
0
1
2
3
4
5
6
7
8
6.3
1.4
CF3
Me
Me
Me
Me
Me
Me
Me
a
CH2OMe
CH2NMe2
CH2CH2OH
CH2CH2OMe
i-Pr
-
a
-
-
8.2
4.8
21
0
-
-
9
10
11
t-Bu
>180
-
aUnstable after 24h in DMSO solution: n=1. bStability measured in skin S9 over 180 mins in the presence of enzymatic cofactors: n=1.
c% loss in purity when stored in DMSO solution for 28 days: n=1.
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The selectivity of (Z,Z)-10a and (Z,Z)-9k across S1PR1-4 was
determined (Table 4). As with ponesimod,10 both (Z,Z)-10a and
(Z,Z)-9k were most active against S1PR1, with >40-fold and >80-
fold selectivity respectively over the other S1PR isoforms
measured. (Z,Z)-10a and (Z,Z)-9k were equipotent. The reactivity
of the exocyclic double bond of (Z,Z)-10a was examined using a
glutathione trapping experiment in human liver microsomes; no
evidence of glutathione adducts or derivatives was observed (see
supporting information).
Metabolite identification of (Z,Z)-10a using incubation with human
skin S9 fraction confirmed that the expected phenol (Z,Z)-9k was
obtained after hydrolysis of the ester group: no other metabolites
were observed (Figure 2a). Based on the stability of (Z,Z)-9k in
DMSO over 28 days (Table 2) it is likely this hydrolysis is
enzymatically driven. We then performed metabolite identification
studies using (Z,Z)-9k in human hepatocytes to confirm the routes
of clearance of our S1PR1 modulators. As before (Z,Z)-9k
isomerises into (Z,E)-9k in solution; Figure 2b shows the
disappearance of parent phenol (both (Z,Z)-9k orange and (Z,E)-9k
green isomeric forms) and identifies the glucuronide conjugation
product, hydroxylation products and hydroxylation with sulfation
metabolites.
Table 4. Selectivity against S1PR1-4.a
Compound
S1PR1
pIC50
S1PR2
pIC50
S1PR3
pIC50
S1PR4
pIC50
In conclusion, we have used a fast follower approach to identify
several highly cleared and active phenolic S1PR1 modulators.
Many of the phenol soft drugs were unstable in solution due to
isomerisation. We were able to prevent this isomerisation by
acylation of the phenol, to deliver chemically stable chemical tools.
The strategy underpinning our S1PR1 soft drug modulators is
illustrated in Figure 2c. When (Z,Z)-10a is applied to the skin of
mice it should be enzymatically hydrolysed to give (Z,Z)-9k. At
this point 9k can bind to S1PR1 in the epidermis causing receptor
internalisation and degradation. (Z,Z)-9k will also start to slowly
isomerise to (Z,E)-9k. The mixture of isomers of 9k would then
enter the blood stream and be distributed to the liver, where it
would be rapidly metabolised and cleared. The hepatic intrinsic
clearance rate for phenol (Z,Z)-9k, 31 mL/min/g (Table 2), would
correspond to 97% liver blood flow if there is a good in vitro to in
vivo correlation, predicting that a single pass through the liver could
eliminate the majority of the drug, greatly reducing the risk of
systemic on-target toxicities, which to date have limited the use of
S1PR modulators.
Ponesimodb
8.2
7.6
8.0c
<5.0
<5.0
<5.0
7.0
6.0
6.1
6.0
10a
9k
<5.0
<5.0
aS1PR1-4 activity was measured using a human PathHunter β-
Arrestin recruitment assay (n=2). bS1PR1-4 activity reported in the
literature using a GTPγS assay.9 cThe potency of 9k on S1PR1
slightly shifted to a higher value in this experiment, which is
independent to the experiments performed to establish the SAR
(Table 2).
To demonstrate (Z,Z)-10a is a suitable tool for in vivo experiments,
a topical pharmacokinetic experiment in mice (see supporting
information) using 22.5 µL of 1% propylene glycol/ethanol 7/3
formulation was carried out. At 2 and 8 h time points, (Z,Z)-10a
concentrations in blood were below the lower limit of
quantification (LLoQ). (Z,Z)-10a concentrations in blood were 8.8
µM (2h) and 4.9 µM (8h). (Z,Z)-9k concentrations were below the
LLoQ in blood at both time points and 134 µM (2h) and 101 µM
(8h) in the skin. (Z,Z)-9k is present in the skin of mice at >10,000-
fold above the IC50 demonstrating that the modulator is likely to be
present at sufficient concentration to inhibit local S1PR1. (Z,Z)-
10a is also present in the skin of mice at >350-fold above the IC50
and will also be able to locally inhibit S1PR1.
(Z,Z)-10a provides the community with a valuable new tool that
will enable targeted studies of S1PR biology in skin, lung or other
suitable tissues.
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Figure 2. (a) Metabolite identification of (Z,Z)-10a in human skin S9 fraction (n=1). (b) Metabolite identification of (Z,Z)-9k and (Z,E)-9k
in human hepatocytes (n=1). (c) Depiction of the enzymatic hydrolysis of (Z,Z)-10a and the hepatic metabolism of (Z,Z)-9k and (Z,E)-9k.
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(5) Reines, I.; Kietzmann, M.; Mischke, R.; Tschernig, T.; Luth, A.;
Kleuser, B.; Baumer, W. Topical application of sphingosine-1-
phosphate and FTY720 attenuate allergic contact dermatitis reaction
through inhibition of dendritic cell migration. J. Invest. Dermatol.
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
Experimental and characterization data for all new
2009, 129, 54-59.
Compounds and all biological and DMPK methods (PDF).
(6) Schaper, K.; Dickhaut, J.; Japtok, L.; Kietzmann, M.; Mischke,
R.; Kleuser, B.; Baumer, W. Sphingosine-1-phosphate exhibits anti-
proliferative and anti-inflammatory effects in mouse models of
psoriasis. J. Dermatol. Sci. 2013, 71, 29-36.
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AUTHOR INFORMATION
Corresponding Authors
* Dr Andrew Woodland, Drug Discovery Unit, Wellcome Centre
for Anti-Infectives Research, School of Life Sciences, University
of Dundee, Dow Street, Dundee. DDI 5EH.
(7) Bodor, N.; Buchwald, P. Retrometabolic drug design: Principles
and recent developments. Pure Appl. Chem. 2008, 80, 1669-1682.
(8) Mark B.; David F.; Claire N.; Colin R.; Jennifer R.; Ola E.; Paul
S.; Yoko S.; Elad K.; W.H. Irwin M.; Paul W.; Kevin D. R.; Andrew
W. Discovery of super soft-drug modulators of sphingosine-1-
phosphate receptor 1. Bioorg. Med. Chem. Lett. 2018 28, 3255-3259
(9) Felding, J.; Sorensen, M. D.; Poulsen, T. D.; Larsen, J.;
Andersson, C.; Refer, P.; Engell, K.; Ladefoged, L. G.; Thormann, T.;
Vinggaard, A. M.; Hegardt, P.; Sohoel, A.; Nielsen, S. F. Discovery
and Early Clinical Development of 2-{6-[2-(3,5-Dichloro-4-
awoodland@dundee.ac.uk.
* Dr Mark Bell, Drug Discovery Unit, Wellcome Centre for Anti-
Infectives Research, School of Life Sciences, University of
Dundee, Dow Street, Dundee. DDI 5EH. m.u.bell@dundee.ac.uk.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
pyridyl)acetyl]-2,3-dimethoxyphenoxy}-N-propylacetamide
(LEO
29102), a Soft-Drug Inhibitor of Phosphodiesterase 4 for Topical
Treatment of Atopic Dermatitis. J. Med. Chem. 2014, 57, 5893-5903.
(10) Bolli, M. H.; Abele, S.; Binkert, C.; Bravo, R.; Buchmann, S.;
Bur, D.; Gatfield, J.; Hess, P.; Kohl, C.; Mangold, C.; Mathys, B.;
Menyhart, K.; Muller, C.; Nayler, O.; Scherz, M.; Schmidt, G.; Sippel,
V.; Steiner, B.; Strasser, D.; Treiber, A.; Weller, T. 2-Imino-
thiazolidin-4-one Derivatives as Potent, Orally Active S1P(1) Receptor
Agonists. J. Med. Chem. 2010, 53, 4198.
(11) Fisher, M. B.; Paine, M. F.; Strelevitz, T. J.; Wrighton, S. A.
The role of hepatic and extrahepatic UDP-glucuronosyltransferases in
human drug metabolism. Drug Metab. Rev. 2001, 33, 273-297.
(12) Falany, C. N. Human Cytosolic Sulfotransferases. In Drug
metabolism and transport : molecular methods and mechanisms; 1st
ed.; Lash, L. H., Ed.; Humana Press: Totowa, New Jersey, 2005; pp
341-378.
(13) Jones, P.; Storer, R. I.; Sabnis, Y. A.; Wakenhut, F. M.;
Whitlock, G. A.; England, K. S.; Mukaiyama, T.; Dehnhardt, C. M.;
Coe, J. W.; Kortum, S. W.; Chrencik, J. E.; Brown, D. G.; Jones, R.
M.; Murphy, J. R.; Yeoh, T.; Morgan, P.; Kilty, I. Design and Synthesis
of a Pan-Janus Kinase Inhibitor Clinical Candidate (PF-06263276)
Suitable for Inhaled and Topical Delivery for the Treatment of
Inflammatory Diseases of the Lungs and Skin. J. Med. Chem. 2017, 60,
767-786.
(14) Glossop, P. A.; Watson, C. A.; Price, D. A.; Bunnage, M. E.;
Middleton, D. S.; Wood, A.; James, K.; Roberts, D.; Strang, R. S.;
Yeadon, M.; Perros-Huguet, C.; Clarke, N. P.; Trevethick, M. A.;
Machin, I.; Stuart, E. F.; Evans, S. M.; Harrison, A. C.; Fairman, D. A.;
Agoram, B.; Burrows, J. L.; Feeder, N.; Fulton, C. K.; Dillon, B. R.;
Entwistle, D. A.; Spence, F. J. Inhalation by design: novel tertiary
amine muscarinic M(3) receptor antagonists with slow off-rate binding
kinetics for inhaled once-daily treatment of chronic obstructive
pulmonary disease. J. Med. Chem. 2011, 54, 6888-6904.
Funding Sources
This work was supported by the Wellcome Trust
[098439/Z/12/Z].
ABBREVIATIONS
S1PR, sphingosine-1-phosphate receptor; IL, interleukin; PASI,
psoriasis area and severity index; UDPGA, uridine-50-
diphosphoglucuronic
diphosphoglucuronosyl transferase; HPLC, high pressure liquid
chromatography; rt, room temperature; TEA, triethylamine; THF,
tetrahydrofuran DMF, dimethyl formamide; CHI, chromatographic
hydrophobicity index; HLM, human liver microsomes; DMSO,
dimethylsulfoxide; Hz, hertz; DMAP, dimethylaminopyridine;
acid;
UGT
uridine-50-
DCC,
N,N'-Dicyclohexylcarbodiimide;
DMA,
Dimethylacetamide; HMBC, Heteronuclear Multiple Bond
Correlation; HSQC, Heteronuclear Single Quantum Correlation;
NOESY, Nuclear Overhauser Effect Spectroscopy.
REFERENCES
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1
2
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N
N
Cl
OH
OH
O
O
S
S
N
N
O
5
6
O
O
7
8
9
Ponesimod
pIC50 8.0
Soft-drug group
(phenol) rapidly
cleared in human
hepatocytes
Ester rapidly
cleared in human
skin S9 to active
phenol (pIC50 7.6)
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