Design and synthesis of selective and potent orally active S1P5 agonists

Article abstract of DOI:10.1002/cmdc.201000253

Putting the brakes on demyelination: Fingolimod (FTY720) was recently shown to significantly decrease relapse rates in patients with multiple sclerosis. This drug attenuates the trafficking of harmful T-cells entering the brain by regulating sphingosine-1-phosphate (S1P) receptors. We designed, synthesized, evaluated 2H-phthalazin-1-one derivatives (e.g., 1L) as selective S1P5 receptor agonists; these compounds are highly potent and selective, with good PK properties, and significant activity in oligodendrocytes. (Chemical Presented).

Full text of DOI:10.1002/cmdc.201000253

DOI: 10.1002/cmdc.201000253
Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists
Henri Mattes,*[a] Kumlesh Kumar Dev,[a, b] Rochdi Bouhelal,[a] Carmen Barske,[a] Fabrizio Gasparini,[a]
Danilo Guerini,[a] Anis Khusro Mir,[a] David Orain,[a] Maribel Osinde,[a] Anne Picard,[a] Celine Dubois,[a]
Engin Tasdelen,[a] and Samuel Haessig[a]
The immunomodulatory drug fingolimod (FTY720, 2-amino-2-
[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal
metabolite (ISP-1, myriocin), is phosphorylated in vivo by
sphingosine kinases to produce (R)-FTY720-phosphate
(FTY720-P).[1,2] FTY720-P activates sphingosine-1-phosphate
(S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar
concentrations and is inactive toward the S1P2 receptor.[1] The
FTY720-P-mediated activation of the S1P1 receptor on lympho-
cytes induces receptor internalization, which attenuates T-cell
response to S1P gradients, preventing their egress from secon-
dary lymphoid tissues.[3] In addition to playing a role in the
immune system, all S1P receptors except S1P4 are also found
differentially expressed in the central nervous system[4] and on
various tumor cell types.[5,6] Although the precise regulation of
these receptors by locally released S1P remains unclear, S1P re-
ceptors are thought to play a role in such events as astrocyte
migration,[7] oligodendrocyte differentiation, and cell survival[8,9]
and neurogenesis.[10,11] To assess the relevance of individual
S1P receptor subtypes for the activity of FTY720-P, selective ag-
onists are required. Because S1P5 receptors are expressed on
oligodendrocytes, and S1P5 receptors are thought to play a
role in oligodendrocyte differentiation and survival, we focused
on the development of S1P5 agonists. By using a high-
throughput screening calcium mo-
To guide the optimization process, homology models of all
S1P receptors were built from a crystal structure of bovine rho-
dopsin (PDB ID: 1F88).[13] Docking experiments of 1 into these
models revealed a possible location of the binding site, some
essential features of the interactions, and indicated potential
regions for gaining selectivity and improving potency. In these
complexes (Figure 1), 1 adopts a twisted conformation with
the aniline ring, ~708 out of the benzamide plane and stabi-
lized by a hydrogen bond between the aniline NH group and
the amide carbonyl. In the S1P5 receptor complex, the amide
group forms a hydrogen bond with OG1-Thr120. The benza-
mide phenyl ring lies in a large hydrophobic pocket surround-
ed by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and
Leu271. The aniline ring undergoes a T-shaped interaction with
Phe116 and hydrophobic contacts with Leu271 and Leu292.
The ortho-methyl substituents fill a small pocket formed by
Tyr89, Val115, and Leu292 on one side, and sit at the face of
Phe196 on the other side. Inspection of sequence alignments
(Figure 2) revealed two positions, one in transmembrane (TM)
helix TM3 (115, S1P5 sequence) and one in TM5 (192), where
S1P5 has smaller residues lining the binding site, thus creating
putative pockets. We hypothesized that filling these pockets
with atoms from our ligands should lead to high selectivity for
the S1P5 receptor. Position 2 on the benzamide core, which
was closest to the hypothesized pocket around Val115, was
therefore extensively modified.
bilization assay with GPCR priming
and FLIPR technology,[12] we dis-
covered benzamide 1, which has
good in vitro potency toward the
Syntheses of derivative 1A–L (Scheme 1) began with 3-fluo-
robromobenzene 2, which was converted into acid 3 by reac-
tion with lithium diisopropylamide (LDA) and carbon dioxide.
Nucleophilic substitution of the fluorine atom with trimethyla-
niline at ꢀ788C yielded 4. This intermediate was then used in
various ways. Copper-catalyzed nucleophilic substitution of the
bromine atom with various alcohols yielded ethers 5D–N,
which were amidated with ammonia using chlorodimethoxy-
triazine for activation to yield 1D–J. Palladium-catalyzed substi-
tution of the bromine atom in acid 4 with various alkylstan-
nanes yielded 6A–C, which were amidated as described above
to yield 1A–C. Alternatively, palladium-catalyzed substitution
of the bromine atom with tributyl-(1-ethoxyvinyl)stannane
yielded 9, which was cyclized to 1L by reaction with hydrazine.
Acid 4 was also amidated with allylamine, using chlorodime-
thoxytriazine for activation, to yield allylamide 10. Palladium-
catalyzed cyclization of this intermediate led to 1K.
S1P5 receptor (EC50 =270 nm), but
has modest selectivity against
S1P1 (EC50 =3140 nm) and S1P4
(EC50 =100 nm). Herein we report
our studies of various benzamide modifications carried out to
improve the selectivity, bioactivity, pharmacokinetic properties,
and ancillary profile of 1, ultimately resulting in the discovery
of potent and very selective S1P5 agonists.
[a] Dr. H. Mattes, Prof. Dr. K. K. Dev, Dr. R. Bouhelal, Dr. C. Barske,
Dr. F. Gasparini, Dr. D. Guerini, Dr. A. K. Mir, Dr. D. Orain, M. Osinde,
A. Picard, C. Dubois, E. Tasdelen, S. Haessig
Novartis Institute for Biomedical Research
WKL-122 4002 Basel (Switzerland)
Fax: (+41)61 696 2455
E-mail: henri.mattes@novartis.com
All compounds were assayed for S1P5 activation in GTPgS
assays,[13] which gave more reliable structure–activity results
than the FLIPR assays, at concentrations up to 10 mm. EC50
values were determined for all compounds (Table 1). Disrupt-
ing the intramolecular hydrogen bond by introducing small
alkyl substituents at position 2 (compounds 1A–C), led to a
[b] Prof. Dr. K. K. Dev
Molecular Neuropharmacology, Department of Physiology
Trinity College Institute of Neuroscience (TCIN) Medical School
Trinity College Dublin, Dublin 2 (Ireland)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000253.
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Scheme 1. Reagents and conditions: a) LDA (1.1 equiv), CO2, THF, ꢀ788C!
RT; b) trimethylaniline (2.1 equiv), LDA (3 equiv), THF, ꢀ788C!RT; c) NaH
(3 equiv), Cu (0.4 equiv), ROH/DMSO 5:1, 1008C; d) chlorodimethoxytriazine
(1.2 equiv), NMM (3 equiv), NH4OH (10 equiv), THF, RT; e) RSnBu3 (1.5 equiv),
Pd(PPh3)4 (0.05 equiv), dioxane, 858C; f) 1. TMSCH2N2, MeOH, THF, RT;
2. tributyl-(1-ethoxyvinyl)stannane (1.5 equiv), Pd(PPh3)4 (0.05 equiv), diox-
ane, 858C; g) NH2NH2 (2 equiv), EtOH, 958C; h) chorodimethoxytriazine
(1.2 equiv), NMM (3 equiv), allylamine (1.5 equiv), THF, RT; i) Pd(OAc)2
(0.05 equiv), tricyclohexylphosphine (0.1 equiv), N,N-dicyclohexylmethyla-
mine (4 equiv), DMA, 608C, 24 h.
Table 1. S1P receptor profiles for selected derivatives of 1.
R[a]
EC50 [nm][b]
Figure 1. A) Longitudinal and B) transversal views of 1 (orange) docked into
Compd
the S1P5 homology model. Residues Val115 and Ala192 are shown in green.
hS1P5
hS1P1
hS1P4
1A
1B
1C
1D
1E
1F
1G
1H
1I
Me
Pr
Pentyl
OMe
OEt
685 (77) >10000 (62) >10000 (67)
1155 (68) >10000 (4)
>10000 (22)
>10000 (0)
1466 (73)
>10000 (0)
100 (79)
>10000 (0)
4067 (82)
72 (76) >10000 (26) >10000 (19)
50 (75) >10000 (21) >10000 (16)
OiPr
O(CH2)2OH
O(CH2)3OH
O(CH2)2OMe
6 (99)
71 (93)
113 (53)
404 (78)
1001 (67)
5500 (63) >10000 (32)
151 (126)
4529 (119)
1J
7 (95)
318 (85) 36 (152)
[a] See Scheme 1 for structures. [b] Values in parentheses indicate percent
efficacy relative to FTY720-P; see Ref. [7] for details of GTPgS assays.
Figure 2. Aligned sequences of S1P receptors. Position 115 (TM3, S1P5 se-
quence) and 192 (TM5) are highlighted in bold.
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loss of agonist activity at all S1P receptors, in line with the con-
formational hypothesis delineated above. Conversely, introduc-
ing small alkoxy substituents at position 2 (compounds 1D–J,
1M, and 1N), which would further stabilize an in-plane confor-
mation of the amide, led to an improvement in agonist activity
of the compounds at the S1P5 receptor. In line with our
pocket hypothesis, slightly increasing the size of the alkoxy
substituent also dramatically enhanced selectivity over other
S1P receptors. Compounds 1E and 1F represent potent and
exquisitely selective S1P5 agonists. Inverting the intramolecular
hydrogen bond network by introducing an aniline at position 2
completely abolished agonist activity at all S1P receptors. Our
models suggested that introducing hydrogen bond donors
into the alkoxy substituents may yield additional binding affini-
ty by interacting with OG-Ser122 or OD1-Asn128. This led to
some very potent S1P5 agonists such as the hydroxyethoxy an-
alogue 1G and its stereochemically defined and conformation-
ally constrained version, 1J. However, such compounds dis-
played a slightly lower selectivity than the parent compound
1. Given the necessity to keep the amide group in plane with
the phenyl ring, a number of cyclic analogues of 1 were syn-
thesized and tested (Table 2). In line with our models, the 3,4-
Table 3. S1P receptor profiles for selected derivatives of 1.
Compd
R2
R3
EC50 [nm][a]
hS1P5
hS1P1
hS1P4
1M
1N
1O
1P
OMe
OMe
H
Me
Cl
Me
iPr
2 (101)
13 (77)
17 (86)
8 (90)
476 (64)
283 (84)
122 (95)
124 (91)
373 (110)
480 (117)
45 (106)
NT
H
1Q
H
1 (115)
10 (106)
NT
[a] Values in parentheses indicate percent efficacy relative to FTY720-P;
see Ref. [7] for details of GTPgS assays; NT: not tested.
Based on their overall S1P receptor profiles, 1F and 1L were
chosen for further profiling. Both compounds have good selec-
tivity profiles with no apparent binding to a panel of more
than 60 receptors and enzymes (data not shown). The com-
pounds showed a low risk of hERG interaction, with IC50 values
>30 mm in the hERG cell-based assay. They also showed no
substantial inhibition of all tested cytochrome P450 enzymes
up to 10 mm, making in vivo drug–drug interactions unlikely
(data not shown).
Table 2. S1P receptor profiles for selected cyclic analogues of 1.
Compd[a]
EC50 [nm][b]
hS1P5
hS1P1
hS1P4
607 (69)
Compound 1F has rather low water solubility, ranging from
0.007 gLꢀ1 at pH 1 to 0.009 gLꢀ1 at pH 6.8, whereas moderate
solubility, ranging from 0.058 gLꢀ1 at pH 1 to 0.037 gLꢀ1 at
pH 6.8, was observed for 1L. Furthermore, both compounds
display good permeability as determined in the PAMPA and
Caco2 assays, ranking them as class II compounds. Data re-
garding oral absolute bioavailability and disposition pharmaco-
kinetics (PK) for 1F and 1L, as well as their in vivo brain pene-
tration, were collected (Table 4). When tested in rats
(10 mgkgꢀ1 p.o.), 1F displayed low oral bioavailability, a rela-
tively short half-life, moderate-to-high clearance, and good
brain penetration. On the other hand, 1L exhibited good PK
properties, with a reasonable AUC (24354 pmolmLꢀ1 hꢀ1) and a
good brain/plasma ratio of 3.8. In a study with a dose of
3 mgkgꢀ1 i.v., a rat plasma half-life of 7.4 h was observed, with
a moderate volume of distribution (Vdss =2.4 Lkgꢀ1) and a low
1K
1L
41 (101)
51 (87)
357 (52)
>10000 (41)
>10000 (79)
[a] See Scheme 1 for structures. [b] Values in parentheses indicate percent
efficacy relative to FTY720-P; see Ref. [7] for details of GTPgS assays.
dihydro-2H-naphthalen-1-one derivatives and indan-1-one de-
rivatives respectively exhibited low or no agonistic activity at
the S1P5 receptor, highlighting the importance of the amide
NH group. On the other hand, the 2H-isoquinolin-1-one deriva-
tive 1K and the 2H-phthalazin-1-one derivative 1L proved to
be potent S1P5 agonists, with 1L displaying very high selectiv-
ity over all other S1P receptors. Due to the significant increase
in selectivity observed for benzamides with 2-alkoxy substitu-
ents, additional analogues were designed to further investigate
substituents projecting into a hydrophobic pocket, which, ac-
cording to our models, is located close to position 3 (Table 3).
Incorporation of a 3-methyl or a 3-chloro group into 1D dra-
matically increased potency, with 1M and 1N having respec-
tive EC50 values of 2 and 13 nm at the S1P5 receptor. Both
compounds still display good selectivity over the other S1P re-
ceptors. Further increasing the size of substituents at posi-
tion 3 led to progressive loss of agonist activity at S1P recep-
tors (data not shown). All compounds have lower selectivity
than 1D, a fact that can be explained by the apparent conser-
vation of this pocket in all S1P receptors. Likewise, removing
the substituent at position 2, while keeping one at position 3,
led to some very potent S1P5 agonists such as 1O–Q, which
lack selectivity versus all other S1P receptors.
Table 4. Detailed in vivo bioavailability and PK profiles for novel S1P5 ag-
onists in rats.
Compounds:
1F
1L
AUC [pmolmLꢀ1 hꢀ1]:[a]
Cmax [pmolmLꢀ1]:[a]
Brain/plasma ratio (4 h i.v.):[b]
t1/2 [h]:[b]
234
340
3.1
2.8
68.1
3.7
3
24354
4343
3.8
7.4
12.3
2.4
CL [mLminꢀ1 kgꢀ1]:[b]
Vdss [Lkgꢀ1]:[b]
F [%]:
48
[a] 10 mgkgꢀ1 p.o. in NMP/Solutol/PEG-300 (5:10:85 v/v/v). [b] 3 mgkgꢀ1
i.v. in NMP/PEG (5:95 v/v).
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clearance of CL=12.3 mLminꢀ1 kgꢀ1. These properties make 1L
a good candidate for further biological profiling.
then FTY720-P and the novel S1P5 agonists described herein
may directly promote myelination. Importantly, these S1P5 ag-
onists may therefore be beneficial in the treatment of demyeli-
nating disorders such as multiple sclerosis.
From the five S1P receptors, S1P5 is best studied in oligo-
dendrocytes, where it is thought to be involved in regulating
oligodendrocyte differentiation and survival. The mRNA for
S1P5 is predominantly expressed in the white matter tracts of
the brain, including the cerebellum, corpus callosum, and the
spinal cord.[14–19] S1P5 receptors are preferentially expressed in
oligodendrocytes at all stages of development: progenitors
and mature lineages.[9,17,20] The distinct distribution pattern of
S1P5 suggests it may play an important role in regulating mye-
lination. A recent study proposed that S1P treatment increases
survival of mature oligodendrocytes via a pertussis-toxin-sensi-
tive, Akt-dependent pathway.[9] S1P5 plays a role in these pro-
survival effects of S1P, as S1P-mediated cell protection is atte-
nuated after oligodendrocytes are treated with RNAi against
S1P5.[9]
Keywords: agonists
·
FTY720
·
myelination
·
oligodendrocytes · receptors · sphingosine
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motes oligodendrocyte survival in vivo, as observed in vitro,
Received: June 15, 2010
Revised: July 26, 2010
Published online on September 2, 2010
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