Discovery of clinical candidate GSK1842799 As a selective S1P1 receptor agonist (prodrug) for multiple sclerosis

Article abstract of DOI:10.1021/ml400194r

To develop effective oral treatment for multiple sclerosis (MS), we discovered a series of alkyl-substituted biaryl amino alcohols as selective S1P₁ modulators. One exemplar is (S)-2-amino-2-(5-(4-(octyloxy)-3- (trifluoromethyl)phenyl)-1,3,4-th

Full text of DOI:10.1021/ml400194r

Letter
pubs.acs.org/acsmedchemlett
Discovery of Clinical Candidate GSK1842799 As a Selective S1P1
Receptor Agonist (Prodrug) for Multiple Sclerosis
Hongfeng Deng,†,§ Sylvie G. Bernier,‡ Elisabeth Doyle,‡ Jeanine Lorusso,‡ Barry A. Morgan,†
William F. Westlin,‡ and Ghotas Evindar*,†,§
‡
†Department of Medicinal Chemistry and Department of Preclinical Research, Praecis Pharmaceuticals Incorporated, 830 Winter
Street, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: To develop effective oral treatment for multiple
sclerosis (MS), we discovered a series of alkyl-substituted
biaryl amino alcohols as selective S1P1 modulators. One
exemplar is (S)-2-amino-2-(5-(4-(octyloxy)-3-
(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)propan-1-ol
(10, GSK1842799). Upon phosphorylation, the compound
(10-P) showed subnanomole S1P1 agonist activity with >1000× selectivity over S1P3. The alcohol 10 demonstrated good oral
bioavailability and rapid in vivo conversion to 10-P. Dosed orally at 0.1 mg/kg, 10 significantly reduced blood lymphocyte counts
6 h postdose, and at 3 mg/kg, 10 achieved efficacy equivalent to FTY720 in the mouse EAE model of MS. Further
pharmacokinetic/pharmacodynamic (PK/PD) study with cynomolgus monkeys indicated that, after oral dosing of 10 at 3.8 mg/
kg, the active phosphate reached plasma levels that are comparable to FTY-720 phosphate (FTY-P) revealed in human clinical
pharmacokinetics studies. On the basis of the favorable in vitro ADME and in vivo PK/PD properties as well as broad toxicology
evaluations, compound 10 (GSK1842799) was selected as a candidate for further clinical development.
KEYWORDS: S1P1 modulator, biaryl aminoalcohol, prodrug, multiple sclerosis, mouse EAE model
hronic demyelination and scarring of the neuronal cells in
prodrug FTY-720 to its active drug FTY-720-monophosphate is
Cbrain, spinal cord, and central nervous system lead to a facilitated by sphingosine kinase 2 (SphK2).12 One major
challenge in the prodrug strategy in S1P activation is its in vivo
phosphorylation of the prodrug to the desired phospho-drug
since this process requires prodrug phosphorylation with either
SphK1 or SphK2, while the phospho-drug could be converted
back to the starting prodrug through the action of sphingosine-
1-phosphate phosphatase 1 (S1PP1), sphingosine-1-phosphate
phosphatase 2 (S1PP2), or a potential lipid phosphate
phosphatase.13 In our most recent prodrug publication,10 we
reported on preclinical candidate PPI-4955 (Table 1) and its
phosphorylation outcome in rodent models. In an effort to
establish a backup molecule to PPI-4955 with improved profile
especially in in vivo phosphorylation, we carried out further
structure−activity relationship (SAR) studies with a focus on
sphingosine kinase activity and in vivo phosphorylation
relationships. Since the PPI-4955 series are structurally
homologous to FTY-720 and human SphK2 is known to
phosphorylate FTY-720 30-fold better than SphK1,14 our
structural modifications were mainly driven by SphK2 activity.
Herein, we report our compound design and synthesis as well
as in vitro and in vivo activity data that led to the discovery of
clinical development candidate GSK1842799.
debilitating neurodegenerative autoimmune disorder known as
multiple sclerosis (MS). This disease has an onset usually in
young adults and more commonly in females, affecting lives of
hundreds and thousands of people across the globe.1 This
inflammatory disorder poses diverse neurological symptoms
often leading to physical and cognitive disability.2 The
emergence of FTY-720 (Fingolimod) as a novel class of orally
active immune-modulating agents for multiple sclerosis and
solid organ transplant rejection prevention has established the
sphingosine-1-phosphate (S1P) receptor class of G-protein
coupled receptors (GPCRs) as a competitive and promising
field of research and development. FTY-720 is a prodrug that,
upon in vivo phosphorylation, activates the S1P1 receptor and
sequesters lymphocytes in lymph nodes, preventing them from
trafficking to lymphoid tissues and contributing to autoimmune
reactions.3 FTY-720 reduces both relapses and disability
progression in relapsing-remitting multiple sclerosis (RRMS)
patients.4−7 However, aside from the clinical benefits of FTY-
720, a number of adverse side effects such as bradycardia, skin
cancer, liver injury, infections, and increased blood pressure
were attributed to activity on S1P3, leaving a window of
opportunity for exploration of alternative chemotypes with
improved overall profiles.
Our SAR study started with the modification of the head-
piece amino-alcohol and the impact on both human SphK2
Recently, we published a number of novel S1P1 agonists, a
few of which fall within the same class of prodrug as FTY-720
that require in vivo phosphorylation in order to trigger any
effects on S1P receptors (Table 1).8−11 The phosphorylation of
Received: May 18, 2013
Accepted: August 27, 2013
© XXXX American Chemical Society
A
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Table 1. Effects of Head-Piece Modification on SphK2 Activity, in Vivo Phosphorylation, and S1P1 Activity and Selectivity
a
a
b
phosphate
R
S1P1 (nM)
S1P3 (nM)
selectivity (S1P1/ S1P3)
prodrug
mSphK2 (%FTY) hSphK2 (%FTY) % in vivo PO4 (mice)
FTY-720-P
PPI-4955-P
1-P
1.20
0.83
0.30
2.3
13.3
11
FTY-720
100
49.6
5.2
100
7.8
2.8
1.9
70
59
3.0
20
CH3
7352
650
8862
2166
PPI-4955
H
1
2-P
CH2OH
>10000
>4348
2
1.9
a
b
[33P] binding activity on S1P1 and S1P3−5 receptor subtypes. Percent conversion of alcohol (prodrug) to phosphate (drug) at 6 h dose
administration upon 10 mg/kg oral (PO) administration of the alcohol.
(hSphK2) and mouse SphK2 (mSphK2) activity with respect
to PPI-4955. The biological data for PPI-4955 and head-piece
modified analogues 1 and 2 as well as FTY-720 (as control) are
reported in Table 1. In pursuit of better understanding PPI-
4955 and analogue activity as substrates of SphK2 and the
correlation of this data with in vivo phosphorylation, all the
compounds were tested for in vitro activity against mSphK2
and hSphK2 as well as in vivo conversion to the corresponding
phosphates in mouse upon oral administration. In comparison
to FTY-720 phosphate (FTY-720-P), PPI-4955 phosphate
(PPI-4955-P) showed better potency and much improved
selectivity for S1P1 over S1P3. Setting FTY-720 activity against
mSphK2 and hSphK2 at 100%, PPI-4955 performed reasonably
well in mSphK2 activity (about 50%) correlating to mouse in
vivo phosphorylation of 59% compared to FTY-720 of 70%.
However, activity of PPI-4955 against hSphK2 was low (7.8%)
with respect to activity of FTY-720, indicating a difference
between mouse and human SphK2 at the substrate binding site
and a potential point for further improvement in SphK2
activity. The first attempt at modification of the head-piece,
where the R group was changed either into a proton (1) or a
hydroxymethyl (2), led to decreased SphK2 activities. However,
phosphates of both compounds still showed potent S1P1
binding activity and good selectivity against S1P3.
Chemical modification of the PPI-4955 imidazole ring was
then explored. Similarly, all of the corresponding compounds
were tested for in vitro activity as potential substrates for
mSphK2 and hSphK2 as well as in vivo phosphorylation in
mouse upon oral administration. A quick profiling of the azole
compounds generated provided insight and guidance in further
SAR exploration (Table 2). As S1P receptor subtype binding
data indicated, all the corresponding phosphates for com-
pounds 3−5 and 6−8 maintained excellent S1P1 binding
activity and analogous selectivity for S1P1 over S1P3 as PPI-
4995-phosphate. For sphingosine kinase activity, while the
added 4-methyl group on the imidazole ring (3) was
detrimental for both mouse and human SphK2 enzymes, the
corresponding 5-methyl oxazole analogue (4) gave a similar
profile as that for PPI-4955. The 2,4-disubstituted thiazole (5)
and 2,5-disubsituted oxazole (6) analogues were less active than
imidazole-containing PPI-4955. Interestingly, the 2,5-substi-
tuted thiazole (7) and 1,3,4-thiadiazole (8) showed much
improved activity against both mSphK2 and hSphK2 enzymes,
Table 2. Structural Modification of PPI-4955 Imidazole Ring
Effects on Agonist Profile
a
[33P] binding activity on S1P1 and S1P3−5 receptor subtypes.
Percent conversion of alcohol (prodrug) to phosphate (drug) at 6
b
h dose administration upon 10 mg/kg oral (PO) administration of the
alcohol.
suggesting the SphK2 enzyme activity is very sensitive to the
azole moiety modifications where a 2,5-disubstituted thiazole
(7) and thiadiazole (8) orientations are analogous to a para-
subtituted phenyl moiety, while the 2,4-disubstituted thiazole
(5) and 2,5-disubsituted oxazole (6) analogues are closer to a
meta-substituted phenyl group. Accordingly, the corresponding
in vivo phosphorylation levels were also significantly increased
(89% and 90%, respectively), a demonstration of the
correlation between SphK2 enzyme activity and the in vivo
phosphorylation.
The findings in 2,5-disubstituted thiazole 7 and disubstituted-
1,3,4-thiadiazole 8 provided the basis and rationale for the next
generation agonist design. Previously, it was observed that both
FTY-720 and a number of prodrugs with the alkyl tail group,
B
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analogous to FTY-720, have demonstrated good mSphK2 and
in vivo lymphopenia activity. It was hypothesized that
replacement of the tail group in both 7 and 8 with an alkyl
group might lead to further improvement in SphK2 activity. As
per earlier reports,8 the best alkyl tail group with improved lead
prodrug profile was octane ether; thus, a series of azole
analogues with the n-octyl tail group were synthesized as shown
in Table 3. Investigation of SphK2 activity of the prodrugs with
In order to confirm agonist activity and selectivity of the
corresponding phosphates, compounds 9-P and 10-P were
further investigated in a [γ-35S]GTP functional assay15 as
reported in Table 4. The two compounds showed analogous
Table 4. [γ-35S]GTP Functional Activity of 9-P and 10-P on
all S1P Receptor Subtypes
Table 3. mSphK2 and hSphK2 Activity of Prodrug
Analogues Containing Octyl Ether Tail
hS1P1 hS1P2
hS1P3
EC50
(nM)
hS1P4 hS1P5
EC50
EC50
EC50
EC50
agonist
X
(nM)
(nM)
(nM)
(nM) S1P3/S1P1
S1P
9-P
4.25
0.44
0.26
1.3
3.94
13
1.8
0.9
CH
N
>3000
>3000
3.60
4.57
2.00
0.66
>6800
>11500
10-P
activity profiles toward all of the S1P receptor subtypes with
subnanomolar agonist affinity on S1P1. Neither of the agonists
had any binding effect on S1P3, demonstrating an excellent
selectivity for S1P1 over S1P3. Notably, both compounds also
had potent agonist activity on S1P4 and S1P5.
The lead prodrugs 9 and 10 were evaluated in an in vivo
dose−response analysis when orally administered in mice
(Figure 1). At the lowest dose of 0.1 mg/kg, both prodrugs
a
[33P] binding activity on S1P1 and S1P3−5 receptor subtypes.
Percent lymphopenia observed in mouse 6 h postdose upon 10
b
mg/kg oral (PO) administration of the corresponding alcohol in
c
comparison to oral administration of 1 mg/kg of FTY-720. Percent
conversion of alcohol (prodrug) to phosphate (drug) at 6 h dose
administration upon 10 mg/kg oral (PO) administration of the
alcohol.
tail group and azole ring modifications proved to be
instrumental in directing the research effort toward the next
generation of the lead molecules. As demonstrated in Table 3,
replacement of the [1,1′-biphenyl]-4-ylmethyl ether group with
an octyl ether in both 7-P and 8-P gave compounds 9-P and
10-P, which showed a good improvement in S1P1 binding
activity, while still maintaining excellent selectivity for S1P1
over S1P3. At the same time, both prodrugs 9 and 10 showed a
substantial improvement in both mSphK2 (2-fold) and hSphK2
(5-fold) over the predecessors 7 and 8. Both prodrugs 9 and 10
gave analogous lymphopenia profile as FTY720 with
quantitative in vivo phosphoryaltion when administered orally
in mouse. Further modification of the azole system to
oxadiazole 11-P, pyrazole 12-P, isoxazole 13-P, oxadiazole 14-
P, and imidazole 15-P provided comparable S1P1 binding
profile as the compounds 9-P and 10-P, while no further
improvement was observed in SphK2 activity, especially
hSphK2 activity. It was interesting to see that, while the 1,3-
thiazole (9) and 1,3,4-thiadiazole (10) analogues gave the best
SphK2 activity profile, the 1,3,4-oxodiazole (11) and imidazole
(15) analogues were poor substrates for SphK2 in the series,
suggesting that the insertion of a sulfur atom in the azole ring
might have changed the geometry of the molecule significantly.
Figure 1. Dose response lymphopenia of lead compounds 9 and 10
relative to the vehicle.
showed a mild reduction in lymphocyte counts, while a
significant lymphopenia was observed in all three higher doses.
Both compounds showed excellent dose responsiveness when
administered orally at doses of 0.1, 0.3, 1.0, and 3.0 mg/kg. The
prodrugs 9 and 10 were further evaluated in the MOG peptide
experimental autoimmune encephalitis (EAE) mouse multiple
sclerosis disease model (Figure 2).16 In this experiment, both
compounds demonstrated efficacy equivalent to FTY-720 when
administered orally once a day at 3 mg/kg, nearly completely
reversing the signs and symptoms of disease (Figure 2).
In vitro ADME profiles of 9 and 10 met general criteria for a
development candidate with reasonable aqueous solubility and
protein binding, high permeability in Caco-2 with no efflux, no
CYP450 inhibition, and good metabolic stability (Table S1,
Supporting Information). An in vivo dog PK study suggested
phosphate drug exposure, half-life, and oral bioavailability
(Table S2, Supporting Information) that support once a day
dosing. When dosed orally (single-dose) in multiple species
C
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the head-piece (20) was first converted to amide then to
thioamide (23) by Lawesson’s reagent under THF refluxing
condition. The thioamide further reacted with bromo-
acetophenone (17, R1 = H) to form 2,4-disubstituted thiazole
(23), which afforded the desired compound 5 after removal of
the protecting group.
In Scheme 2, the intermediate 25, prepared from bromo-
acetophenone (17) through azide and amine intermediates
Scheme 2. Synthesis of Oxazole, Thiazole, Thiadiazole, and
a
1,3,4-Oxadiazole Analogues 6−11
Figure 2. Effect of lead S1P1 agonists 9 and 10 in the MOG peptide
EAE model of multiple sclerosis.
(Table S3, Supporting Information), the active phosphate of
10-P reached plasma levels that ranged ∼10−50 ng/mL. In
cynomolgus monkeys administered a dose of 3.8 mg/kg, a
plasma Cmax = 50.9 ng/mL was achieved and a lymphocytosis
EC90 = 16.2 ng/mL, which is comparable to FTY-720
phosphate levels revealed in human clinical pharmacokinetics
studies.17,18 After broad toxicology evaluations, compound 10
(GSK1842799) was selected as a candidate for further
development.
Syntheses of the above compounds are outlined in Schemes
1−3. In Scheme 1, analogously to the synthesis of PPI-4955,10
Scheme 1. Synthesis of Imidazole, Oxazole, and Thiazole
a
Analogues (1−5 and 15)
a
Reagents and conditions: (a) (i) CuBr2, EtOAc/CHCl3 (1:1), reflux;
(ii) NaN3, DMF; (b) H2 (gas), 5% Pd−C, MeOH, HCl; (c) 20,
HATU, DIPEA, DCM/DMF; (d) R2CH2OH, KOtBu, THF, 70 °C;
(e) hydrazine, HATU, DIPEA, DCM/DMF; (f) Lawesson’s reagent,
toluene, 120 °C; (g) Lawesson’s reagent, DCM; (h) PPh3 (5 equiv),
hexachloroethane (C2Cl6, 2.5 equiv), TEA (10 equiv); (i) PPh3,
CCl3CN, CH3CN, MW, 120 °C, 20 min; (j) 6 N HCl, dioxane or
TFA, DCM; or TsOH, MeOH, reflux.
followed by coupling with the orthogonally protected α-methyl
serine (20) or from 3-trifluoromethyl-4-fluorobenzoic acid (26)
via hydramide formation and coupling reaction with 20, was
cyclized using Lawesson’s reagent either in toluene or
dichloromethane to afford thiazole and thiadiazole analogues
7, 8, 9, and 10 after deprotection with TFA. This synthetic
route was developed into a fully telescoped process synthesis
for compound 10 at multikilogram scale.19 Alternatively, the
intermediates 25 were converted to oxazole 6 or 1,3,4-
oxadiazole 11 using triphenylphosphine and hexachloroethane
or triphenylphosphine and trichloroacetonitrile correspond-
ingly after deprotection with TFA.
In Scheme 3, the head piece building block 20 was converted
to methylketone 29 through Weinreb ketone synthesis. The
methylketone 29 was then reacted with acid 26 through acid
chloride intermediate to give diketone 30, which further
condensed with either hydrazine or hydroxylamine followed by
deprotection with TFA affording the desired pyrazole and
isoxazole analogues 12 and 13. Meanwhile, using 4-fluoro-3-
(trifluoromethyl)benzonitrile (32) as starting material, the
1,2,4-oxadiazole analogue 14 was prepared through the
formation of hydroxylamidine intermediates 33 and 34
followed by cyclization and Boc deprotection. All of the
phosphates were synthesized from corresponding alcohols
analogously to the synthesis of PPI-4955-P.10
a
Reagents and conditions: (a) CuBr2, EtOAc/CHCl3 (1:1), reflux; (b)
Cs2CO3, DMF, 18, 19, or 20; (c) NH4OAc, toluene, reflux; (d) 6 N
HCl, THF or TFA, DCM; or TsOH, MeOH, reflux; (e) 22, THF,
reflux.
the substituted bromo-acetophenones (17) were converted to
corresponding esters (21) by reacting with different head-piece
moieties including Boc-serine−OH, 5-(tert-butoxycarbonylami-
no)-2,2-dimethyl-1,3-dioxane-5-carboxylic acid (18), 5-((tert-
butoxycarbonyl)amino)-2,2-dimethyl-1,3-dioxane-5-carboxylic
acid (19), and (S)-3-(tert-butoxycarbonyl)-2,2,4-trimethylox-
azolidine-4-carboxylic acid (20). Using NH4OAc under toluene
refluxing conditions, these esters were cyclized to substituted
imidazole or oxazole intermediates, which upon acidic treat-
ment provided final compounds 1, 2, 3, 4, and 15. Meanwhile,
D
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Notes
Scheme 3. Synthesis of Pyrazole, Isoxazole, and 1,2,4-
Oxadiazole Analogues 12−14
a
The authors declare no competing financial interest.
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In summary, replacement of the imidazole ring of the novel
S1P1 prodrug PPI-4955 with a series of bioisosteric hetero-
cycles (azoles) and switching the biphenyl moiety with an n-
octyl tail led to the identification of several potent and selective
prodrugs for modulating S1P1 receptors. Among them,
compound 10 demonstrated good in vitro ADME and in
vivo PK properties. Upon phosphorylation, it showed
subnanomole S1P1 agonist activity with >1000× selectivity
over S1P3. Dosed orally at 0.1 mg/kg, 10 significantly reduced
blood lymphocyte counts 6 h postdose and achieved in vivo
efficacy analogous to FTY720 in the mouse EAE model of MS.
Further pharmacokinetic/pharmacodynamic (PK/PD) study
with monkeys indicated that oral dosing of 10 at 3.8 mg/kg led
to active phosphate species that reached plasma levels
comparable to FTY-720 phosphate revealed in human clinical
pharmacokinetics studies. Compound 10 (GSK1842799) was
selected as a candidate for clinical development.
ASSOCIATED CONTENT
* Supporting Information
Experimental details for the synthesis of all the compounds and
in vitro ADME and in vivo PK/PD data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
*E-mail: ghotas.x.evindar@gsk.com.
■
Present Address
§GlaxoSmithKline, Platform Technology & Science, MDR
Boston, 830 Winter Street, Waltham, Massachusetts 02451,
United States.
E
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