Pyrazole derived from (+)-3-carene; a novel potent, selective scaffold for sphingosine-1-phosphate (S1P1) receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2009.11.045

High throughput screening and hit to lead optimization led to the identification of 'carene' as a promising scaffold showing selective S1P₁ receptor agonism. In parallel to this work we have established a pharmacophore model for the S1P₁ receptor highlighting the minimal structural requirement necessary for potent receptor agonism.

Full text of DOI:10.1016/j.bmcl.2009.11.045

Bioorganic & Medicinal Chemistry Letters 20 (2010) 35–37
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazole derived from (+)-3-carene; a novel potent, selective scaffold for
sphingosine-1-phosphate (S1P1) receptor agonists
b
a
c,
Frédéric J. Zécri a, , Rainer Albert , Gregory Landrum , Klaus Hinterding , Nigel G. Cooke a, Danilo Guerini a,
*
Markus Streiff a, Christian Bruns a, Barbara Nuesslein-Hildesheim a
a Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland
b Gerbergässlein 8/III, CH-4051 Basel, Switzerland
c F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
High throughput screening and hit to lead optimization led to the identification of ‘carene’ as a promising
scaffold showing selective S1P1 receptor agonism. In parallel to this work we have established a pharma-
cophore model for the S1P1 receptor highlighting the minimal structural requirement necessary for
potent receptor agonism.
Received 12 October 2009
Revised 11 November 2009
Accepted 12 November 2009
Available online 15 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
S1P1 agonist
Carene
Pharmacophore model
FTY720 (fingolimod™) is a novel immunomodulator that is
highly effective in animal models of transplantation and autoim-
munity.1 Once daily oral treatment with FTY720 has been shown
to be effective in preventing relapsing–remitting multiple sclerosis
in a Phase II clinical trial and may represent a novel modality for
immunomodulator therapy.2
Detailed mechanistic studies have shown that FTY720 effec-
tively inhibits the egress of T-cells and B-cells from lymph nodes,
thereby reducing the number of activated cells that recirculate to
peripheral inflammatory tissues.3 More specifically, it was demon-
strated that FTY720 is acting via its phosphorylated form FTY720-
P, which is a potent agonist on 4 out of the 5 known S1P receptors.4
FTY720-P acts as a functional antagonist at the S1P1 receptor
inducing internalization of the receptor in lymphocytes resulting
in blockage of S1P directed migration of the lymphocytes out of
the lymph node (see Fig. 1).5
The understanding of the complex biology of the S1P receptors
has been facilitated by the development of tool compounds with
different selectivity and different pharmacokinetic profiles.
Numerous groups have used and reported medicinal chemistry ef-
forts using SEW28716 as a lead structure, leading to, for example,
AUY954 a selective S1P1 agonist with similar efficacy to FTY720
in rat heterotopic heart transplantation.7
High throughput screening of the Novartis Compound Library
was carried out by recording compound mediated Ca2+ mobiliza-
tion in CHO cells over-expressing the human S1P1 receptor.8 Pri-
mary hits were validated in a [35S]-GTP
cS binding assay using
cell membranes prepared from the hS1P1 CHO cells.7 This led to
the identification of 5 as an interesting S1P1 receptor agonist scaf-
fold. It is noteworthy that this pyrazole scaffold is derived from the
natural monoterpene (+)-3-carene and has found application in the
resolution of racemic cis-Permethric and cis-Z-cyhalothric acids.9
In the absence of ligand-bound X-ray co-crystal structures, con-
clusive information about the biologically active conformation of
S1P1 agonists is not available. A common solution to this problem
is the use of homology models and point-mutation experiments to
R
F3C
C8H17
S
O
O
N
N
NH2
Ph
CF3
HO
R=H, FTY720
R=PO3H2, FTY720-P
SEW2871
O
O
OH
F3C
Ph
O
N
N
O
N
H
S
* Corresponding author. Tel.: +41 61 3244759.
E-mail address: frederic.zecri@novartis.com (F.J. Zécri).
Present address.
AUY954
1
Figure 1. Representative S1P1 agonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.045

36
F. J. Zécri et al. / Bioorg. Med. Chem. Lett. 20 (2010) 35–37
The nitrogen in position 1 of pyrazole 4 can be reacted with a vari-
ety of electrophiles such as activated carboxylic acids, sulfonyl
chlorides, allyl or alkyl to yield analogues 5–19. The acylation
and alkylation reactions of
selectivity (see Scheme 1).
4 proceed with complete regio-
As described in our pharmacophore analysis, it was clear that
carene analogues 5 represented a good starting point for hit to lead
optimization as it met the minimal structural requirement for S1P1
potency and offered a high level of rigidity. This rigidity is helpful
for achieving high selectivity across the S1P receptor isoforms.
Table 1 shows the importance of the H-bond acceptor and linker
geometry of the carene analogues. Deletion of the key acceptor
oxygen atom from the amide bond of 5 to yield amine 6, results
in a significant decrease in agonism across the S1P receptor panel
as predicted by the pharmacophore model. Replacing the amide of
5 with a sulfonamide, 7, leaves the key acceptor intact but substan-
tially changes the shape of the ligand, resulting in a loss of activity.
Reduction of rigidity via hydrogenation of the double bond of 5 to
analogue 8 demonstrates that increasing the flexibility of the li-
gand has a detrimental effect on potency. Finally, analogues 9–11
highlight the importance of the appropriately positioned phenyl
group and the overall ligand shape.
Table 2 summarizes our efforts to further improve S1P1 receptor
agonism while retaining overall S1P receptor selectivity. We kept
the hydrophobic and the H-bond acceptor linker moieties constant
and studied the effect of phenyl group modification on the S1P
receptors panel. Within the carene series, the phenyl group
tolerates a range of different substitution patterns ranging from
3,4-dimethoxy 12, 2-chloro 13, to more polar substituents such
as trifluoromethyl 14, sulfonamide 15, 16, or various heterocycles
17–19. Introduction of a trifluoromethyl substituent on the carene
phenyl group 14, mimicking the trifluoromethyl-phenyl group of
SEW2871, does not lead to substantial change in S1P1 agonism,
though it does increase activity on S1P5. Increasing polarity in
the meta position by replacing the phenyl ring with a pyridine or
adding polar substituents increases agonism on both S1P1 and
S1P5. Moving the polar substituent to the para position, such as
sulfonamide 16, substantially decreases agonism on S1P1 and
S1P5. The polar head groups in meta could be involved in interac-
tions with Arg120 or Glu12110c an interaction which is lost when
the substituent is moved to para. The 4-pyridine 17, on the other
hand shows increased agonism across the panel.
The variety of polar substituents tolerated in the carene series
offer a handle to tune ADME properties such as solubility by
increasing PSA or introducing charged groups. In conclusion we
have optimized carene series via phenyl group modification yield-
ing analogue 19: a subnanomolar S1P1 agonist with micromolar
activity on S1P3 and 250-fold selectivity over S1P4 and S1P5.
In vitro potent and selective S1P1 agonists 17 and 19 were fur-
ther evaluated in an in vivo PK/PD model, where lymphocyte count
is measured in Lewis rat blood at different time points. Both after
3 mg/kg po and 0.3 mg/kg iv application no significant reduction in
lymphocyte count was observed correlating with a lack of blood
exposure. Further studies revealed that the compounds were
unstable at pH >7 with a t1/2 of approximately 3 h for analogues
17. Characterization of the decomposition product revealed that
the amide bond was cleaved to yield amine 4 and the correspond-
Figure 2. Top: FieldTemplater alignment of the S1P1 agonists SEW2871 (magenta),
1 (cyan), and carene 5 (yellow). Bottom: A pharmacophore describing the common
features of the ligands. The features are a planar hydrophobic group (cyan), an
aromatic ring (magenta), and a hydrogen-bond acceptor (blue). The distances
indicated are derived from these three structures.
rationalize observed SAR.10 Another approach to obtaining approx-
imate information to help understand details of the SAR landscape
is to flexibly align several known agonists with consistent SAR. We
performed such an alignment of SEW2871, 1,11 and carene ana-
logue 5 using the FieldTemplater software.12 This procedure results
in a very good alignment, volume similarity of 0.75, and overall
Cresset similarity of 0.66 (see Fig. 2).
The alignment shows that the central core of all three ligands
have a similar shape and suggest a common pharmacophore,
shown in Figure 2 below for carene 5. This common pharmaco-
phore, the minimal structural requirement for activity in these ser-
ies, is defined by the following features: a planar hydrophobic (or
aromatic) group, a hydrogen-bond acceptor, and an aromatic ring.
This pharmacophore is consistent with the SAR reported here for
the carenes, SEW2871 and its derivatives, as well as the S1P1 ago-
nists discovered via HTS by Schürer et al., and finally the large ser-
ies of S1P1 agonists built around a series of five-membered
aromatic heterocycles reported by Vachal et al.6h This minimal
pharmacophore reflects the fact that a polar interaction with the
S1P1 receptor, (cf. FTP-720-P and AUY954) is not required for
agonism. This has been demonstrated by Schürer et al.11 and is fur-
ther reinforced by the results presented below. In this model it is
not necessary to invoke ion–dipole interactions via the trifluoro-
methyl group in order to explain the activity of SEW2871.
Following the protocol developed by Tkachev,13 it is possible to
prepare large quantities of 4 via the spontaneous cyclization of
readily available keto ester 2 to afford bis-ketone 3, which can be
reacted with hydrazine to generate the tri-cyclic pyrazole core 4.
H
H
H
a
b
c
N
H
OH
H
N
NH
O
O
O
N
H
O
H
H
O
2
3
4
5
Scheme 1. General synthetic route for the preparation of acyl carene. Reagents and conditions: (a) NaOMe–MeOH, reflux, 15 min, (60–80%); (b) NH2NH2ÁH2O, ethylene glycol,
reflux, 6 h (70–85%); (c) EDCI, HOBt, CH2Cl2, rt, 16 h (82%).

F. J. Zécri et al. / Bioorg. Med. Chem. Lett. 20 (2010) 35–37
37
Table 1
ing carboxylic acid. It is hypothesized that the acyl carene is acting
as an activated carboxylic acid when subjected to various nucleo-
philes and that amine 4 is acting as a leaving group.
Structure–activity relationship of H-bond acceptor linker on S1P receptor panel
H
In summary we have discovered and optimized pyrazoles de-
rived from (+)-3-carene, as a novel class of S1P1 agonists with excel-
lent selectivity against all the other S1P isoforms. During the course
of this work, we have established a common pharmacophore with
existing S1P agonists and defined the minimal structural require-
ments needed for S1P1 agonism across chemical series.
N
H
N
R
*
*
*
*
Compds
R
EC50
hS1P1
EC50
hS1P3
EC50
EC50
hS1P4
hS1P5
O
*
*
5
6
7
0.034 (82) >1 (94)
>10 (À2)
1.76 (90)
Acknowledgments
>10 (45)
>10 (7)
>10 (14) >10 (10)
>10 (10)
We wish to thank C. Simeon and H. Knecht for their practical
support in the synthesis of carene analogues and C. Pally and C.
Beerli for their practical support in the PK/PD experiment.
O
O
S
>10 (À2) >10 (À19) >10 (À1)
*
*
O
References and notes
8
9
0.086 (55) >10 (38) >10 (0)
>10 (26)
>10 (2)
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O
O
>10 (25)
0.28 (76)
>10 (44) >10 (À6)
*
*
10
11
>10 (8)
>10 (3)
>10 (1)
>10 (22)
>10 (29)
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O
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Cyster, J. G. Science 2005, 309, 1735.
*
>10 (18)
>10 (15)
EC50 obtained in [35S] GTP
c
S binding assays7 are reported in
lM. Value in bracket
*
represents the Emax, % inhibition at 10
lM compared to S1P. Values reported are the
mean of two experiments.
Table 2
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Mils, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G. J.;
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Chun, J.; Gray, N.; Rosen, H. J. Biol. Chem. 2004, 279, 13839; (c) Anari, M. R.;
Creighton, M. D.; Ngui, J. S.; Tschrret-Guth, R. A.; Teffera, Y.; Doss, G. A.; Tang,
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Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G.-J.;
Chrebet, G.; Parent, S. A.; Bergstrom, J.; Card, D.; Forrest, M.; Quackenbush, E. J.;
Wickham, L. A.; Vargas, H.; Evans, R. M.; Rosen, H.; Mandala, S. J. Med. Chem.
2005, 48, 6169; (e) Colandrea, V. J.; Legiec, I. E.; Huo, P.; Yan, L.; Hale, J. J.; Mills,
S. G.; Bergstrom, J.; Card, D.; Chebret, G.; Hajdu, R.; Keohane, C. A.; Milligan, J.
A.; Rosenbach, M. J.; Shei, G.-J.; Mandala, S. M. Bioorg. Med. Chem. Lett. 2006, 16,
2905; (f) Yan, L.; Budhu, R.; Huo, P.; Lynch, C. L.; Hale, J. J.; Mills, S. G.; Hajdu, R.;
Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G.-J.; Chrebet, G.;
Bergstom, J.; Card, D.; Mandala, S. M. Bioorg. Med. Chem. Lett. 2006, 16, 3564; (g)
Yan, L.; Huo, P.; Doherty, G.; Toth, L.; Hale, J. J.; Mills, S. G.; Hajdu, R.; Keohane,
C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G. J.; Chrebet, G.; Bergstrom, J.; Card,
D.; Quackenbush, E.; Wickham, A.; Mandala, S. M. Bioorg. Med. Chem. Lett. 2006,
16, 3679; (h) Vachal, P.; Toth, L.; Hale, J. J.; Yan, L.; Mills, S. G.; Chrebet, G.;
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Structure–activity relationship of head group on S1P receptor
H
N
N
R
H
*
*
*
*
Compds
R
EC50
EC50
EC50
EC50
hS1P1 hS1P3
hS1P4
hS1P5
O
*
O
O
0.025 >10
>10
(30)
0.90
(80)
12
(89)
(46)
O
O
O
O
Cl
0.016 >1
>10
(42)
*
*
*
*
13
14
15
>10 (À19)
(89)
(69)
0.026 >10
>10
(17)
0.325
(56)
CF3
(65)
(46)
0.011 1.35
(92) (84)
0.116
(21)
0.082
(76)
SO2NH2
0.119 >1
>10
(16)
16
>10 (À22)
(68)
(68)
SO2NH2
8. Ullrich, T.; Ghobrial, M.; Peters, C.; Billich, A.; Guerini, D.; Nussbaumer, P. Chem.
Med. Chem. 2008, 3, 356.
O
O
O
0.007 0.345
0.10
(32)
0.13
(93)
*
*
*
17
18
19
9. Popov, S. A.; Tkachev, A. V. Tetrahedron: Asymmetry 1995, 6, 1013.
10. (a) Deng, Q.; Clemas, J. A.; Chrebet, G.; Fischer, P.; Hale, J. J.; Li, Z.; Mills,
S. G.; Bergstrom, J.; Mandala, S.; Mosley, R.; Parent, S. A. Mol. Pharmacol.
2007, 71, 724; (b) Fujiwara, Y.; Osborne, D. A.; Walker, M. D.; Wang, D.-A.;
Bautista, D. A.; Liliom, K.; Van Brocklyn, J. R.; Parrill, A. L.; Tigyi, G. J. Biol.
Chem. 2007, 282, 2374; (c) Jo, E.; Sanna, M. G.; Gonzalez-Cabrera, P. J.;
Thangada, S.; Tigyi, G.; Osborne, D. A.; Hla, T.; Parrill, A. L.; Rosen, H.
Chem. Biol. 2005, 12, 703.
(92)
(83)
N
N
0.017 >1
0.10
(30)
0.345
(87)
(90)
(61)
0.002 0.995
0.560
(28)
0.505
(130)
NH
(90)
(84)
11. Schürer, S. C.; Brown, S. J.; Gonzalez-Cabrera, P. J.; Schaeffer, M.-T.; Chapman,
J.; Jo, E.; Chase, P.; Spicer, T.; Hodder, P.; Rosen, H. Chem. Biol. 2008, 3, 486.
12. FieldTemplater v2.1.0. http://www.cresset-bmd.com.
*
EC50 obtained in [35S] GTP
c
S binding assays7 are reported in
l
M. Value in bracket
represents the Emax, % inhibition at 10
mean of two experiments.
lM compared to S1P. Values reported are the
13. Popov, S. A.; Denisov, A. Y.; Gatilov, Y. V.; Bagryanskaya, I. Y.; Tkachev, A. V.
Tetrahedron: Asymmetry 1994, 5, 479.