S1P receptor mediated activity of FTY720 phosphate mimics

Article abstract of DOI:10.1016/j.bmcl.2010.01.118

Various carboxylic acids, phosphonic acids, sulfonic acids, tetrazoles as well as sulfonylhydantoins were prepared as phosphate mimics of the chiral aminophosphate 1-P to act as agonists on the S1P₁ receptor. It was found that amino phosphonate

Full text of DOI:10.1016/j.bmcl.2010.01.118

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1485–1487
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
S1P receptor mediated activity of FTY720 phosphate mimics
à
*
Klemens Högenauer , Klaus Hinterding , Peter Nussbaumer
Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Various carboxylic acids, phosphonic acids, sulfonic acids, tetrazoles as well as sulfonylhydantoins were
prepared as phosphate mimics of the chiral aminophosphate 1-P to act as agonists on the S1P1 receptor. It
was found that amino phosphonates and amino carboxylates are potent S1P1 binders. b-Amino acid 11
could be shown to reversibly reduce blood lymphocyte counts in rats after po administration.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 9 December 2009
Revised 20 January 2010
Accepted 21 January 2010
Available online 28 January 2010
Keywords:
FTY720
S1P receptor agonist
Phosphate mimic
FTY720 (fingolimod™) is a novel orally active immunomodula-
tor that has shown efficacy in the treatment of multiple sclerosis.1
The agent has a unique mode of action, namely modulating lym-
phocyte trafficking. Specifically, immunoreactive T-cells are
trapped in secondary lymphoid organs and cannot exert their
physiological role in plasma or tissue.2 On a molecular level, this
reversible reduction of blood lymphocyte count is thought to be
mediated by FTY720-P, generated in vivo, acting as an agonist on
sphingosine-1-phosphate receptor 1 (S1P1), thereby internalizing
the receptor and thus acting as functional antagonist.3
To allow for maximal overlap of the acidic moieties of these
mimics with 1-P, we initially designed molecules 3, 4, and 6 featur-
ing a 2-carbon spacer next to the chiral center (Scheme 1). Our syn-
thetic strategy made use of the availability of aldehyde 2 as a key
intermediate6 as well as Horner–Wadsworth–Emmons (HWE)
methodology. Thus, HWE reaction with ethyl (diethoxyphospho-
(CH2)7CH3
HO
The discovery that FTY720 is stereoselectively phosphorylated
by sphingosine kinases (SPHKs) in vivo to the active principle
(S)-FTY720-phosphate (Fig. 1)4 explained the activity of the chiral
amino alcohol 1 and the inactivity of its enantiomer.5 Amino alco-
hol 1 reversibly reduces blood lymphocytes in vivo to a similar ex-
tent as FTY720. The activity can be rationalized in vitro by the high
turnover of 1 to its phosphate 1-P by SPHKs as well as potent bind-
ing of 1-P to S1P1. In line with the mode of action, the enantiomer
of 1 is not a good substrate for SPHKs, and the enantiomer of 1-P
binds far less potently to S1P1.6 These findings encouraged us to
explore chiral phosphate mimics based on 1-P that would act with-
out the need for a phosphorylation step in vivo. This study comple-
ments the work of other groups,7 in particular investigations of
various phosphate replacements by Merck researchers.7a
HO
FTY720
SPHKs
(CH2)7CH3
NH2
O
P
OH
HO
O
HO
HO
NH2
FTY720-P
O
O
P
OH
(CH2)6CH3
O
NH2
1-P
We chose to investigate carboxylic acids, phosphonic acids, sul-
fonic acids, but also heterocyles like tetrazoles and sulfonylhydan-
toins for their potential to mimic the phosphate group of 1-P.
SPHKs
O
(CH2)6CH3
* Corresponding author. Tel.: +41 61 3246225; fax: +41 61 3246735.
E-mail address: klemens.hoegenauer@novartis.com (K. Högenauer).
Present address: F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland.
HO
NH2
1
à
Present address: Lead Discovery Center GmbH, Emil-Figge-Strasse 76a, D-44227
Dortmund, Germany.
Figure 1. Phosphorylation of FTY720 and amino alcohol 1 in vivo.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.118

1486
K. Högenauer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1485–1487
ryl)methanesulfonate, followed by hydrogenation of the double
bond and treatment with acid to simultaneously saponify the sul-
fonic ester and to remove the Boc group led to amino sulfonic acid
3. Amino phosphonic acid 4 was prepared by reaction of aldehyde
2 with dibenzyl methylenediphosphonate, followed by double
bond hydrogenation/debenzylation with H2/Pd-C and removal of
the Boc group. HWE reaction of aldehyde 2 with ethyl 2-(diethoxy-
phosphoryl)acetate followed by double bond hydrogenation and
LiOH mediated ester saponification gave carbamate 5. Acidic
O
(CH2)6CH3
a
HO
HN
Boc
8
O
(CH2)6CH3
O
N
9
S
O
Boc
O
b
deprotection finally liberated c-amino acid 6.
To assess the influence of the spacer length, we prepared carbox-
ylic acid derivatives 7 and 11 (Schemes 1 and 2). Pinnick oxidation
of aldehyde 2, followed by acidic deprotection of the amino group
O
(CH2)6CH3
c
NC
gave
a-amino acid 7. Sulfamidate 9, generated in two steps from
HN
amino alcohol 8 via the corresponding diastereomeric sulfami-
dites,8 served as the precursor for the one-carbon chain elongation
with sodium cyanide to give nitrile 10.9 Simultaneous saponifica-
tion and Boc removal led to formation of b-amino acid 11.
To generate the tetrazole, we converted acid 5 to amide 12
(Scheme 3). This amide was then transformed to tetrazole 13 using
TMSN3.10 Basic cleavage of the cyanoethyl group, followed by
acidic amine deprotection liberated tetrazole 14.
10
Boc
O
(CH2)6CH3
HO
O
NH2
11
Scheme 2. Reagents and conditions: (a) (i) SOCl2, pyridine, CH3CN, À40 °C?À10 °C,
86%; (ii) RuCl3 cat., NaIO4, CH3CN, H2O, rt, 60%; (b) NaCN, DMF, rt, 92%; (c) HCl,
dioxane, microwave, 160 °C, 45%.
Scheme 4 shows the preparation of sulfonylhydantoin 18.11
Reductive amination of aldehyde 2 with glycine methyl ester gave
amine 15. Sulfamoylation to 16 was achieved via in situ generation
of H2NSO2Cl. Cyclization with sodium methoxide generated carba-
mate 17 which was deprotected to give sulfonylhydantoin 18.
All phosphate mimics were tested for S1P isoform affinity in a
O
O
(CH2)6CH3
a
HN
CN
5
HN
12
Boc
ligand induced [c-
35S]GTP binding assay with membrane frag-
ments from CHO transfectants stably expressing human S1P recep-
tors.7g Although S1P3 has been shown to be responsible for
transient heart rate reduction in rodents,7b it could recently be
demonstrated that S1P1 mediates these heart rate lowering effects
in humans.12 In parallel to the in vitro binding affinity, reduction of
in vivo lymphocyte count was assessed 6 h after po application to
Lewis rats.7g
The biological results are summarized in Table 1 with FTY720-P
and 1-P as reference compounds. Sulfonic acid 3, tetrazole 14 as
well as sulfonylhydantoin 18 only exhibited potency above
100 nM on S1P1. Phosphonate 4 showed 20 nM potency on S1P1,
but weak selectivity over S1P3. For the carboxylic acid derivatives,
the S1P agonist profile was strongly dependent on the length of the
b
O
N
N
(CH2)6CH3
N
N
13
HN
Boc
c
CN
O
N
N
(CH2)6CH3
N
N
H
NH2
14
spacer between the chiral center and the acidic moiety. Whereas
amino acid 6 lacked significant S1P1 potency, the corresponding b-
amino acid 11 as well as -amino acid 7 were considerably more
c-
Scheme 3. Reagents and conditions: (a) NC(CH2)2NH2, EDC, HOAt, DMF, rt, 72%; (b)
Ph3P, DIAD, TMSN3, CH3CN, rt, 57%; (c) (i) DBU, CH2Cl2, rt, 90%; (ii) HCl, MeOH, rt,
77%.
a
potent. Interestingly, 7 also showed pronounced selectivity over
S1P3. Comparing these results to those of an earlier Merck study7a
shows the influence of the quaternary stereogenic center at the
carbon attached to the amino group on S1P1 potency. The racemic
Merck b- and c-amino acids 19 and 20 (Fig. 2) were found to be
poor S1P1 binders assessed by inhibition of [33P]-S1P1 binding
O
O
H
(CH2)6CH3
O
(CH2)6CH3
O
(CH2)6CH3
a
d
HO3S
O
HO
HN
O
2
NH2
NH2
O
3
7
O
b
c
O
O
P
OH
(CH2)6CH3
(CH2)6CH3
HO2C
HO
5:
6:
HN
R = Boc
R = H
NH2
4
R
Scheme 1. Reagents and conditions: (a) (i) EtO3SCH2P(O)(OEt)2, n-BuLi, THF, À78 °C?rt, 74%; (ii) Pd-C, H2, EtOAc, rt, 61%; (iii) HCl, Et2O, rt, 53%; (b) (i)
(BnO)2P(O)CH2P(O)(OBn)2, n-BuLi, À78 °C?rt, 53%; (ii) Pd-C, H2, EtOAc, rt; (iii) HCl, MeOH, rt, 56% over two steps; (c) (i) EtO2CCH2P(O)(OEt)2, n-BuLi, THF, À78 °C?rt,
80%; (ii) Pd-C, H2, EtOAc, rt, 89%; (iii) LiOH, MeOH, THF, rt, 99%; (iv) TFA, CH2Cl2, rt, 99%; (d) (i) NaClO2, 2,3-dimethyl-2-butene, KH2PO4, t-BuOH, rt, 96%; (ii) TFA, CH2Cl2, rt,
50%.

K. Högenauer et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1485–1487
1487
derivatives.14 However, these remained undetermined in this sin-
gle time point PD experiment.
In summary, we have shown that the phosphate group of 1-P
can be mimicked by various functional groups in a potency range
O
(CH2)6CH3
a
2
MeO2C
N
R
HN
Boc
on S1P1 between 20 nM and 1 lM. b-Amino acid 11 also showed
15:
16:
R = H
R = SO2NH2
b
efficacy on peripheral lymphocyte count in rats after 6 h, illus-
trating the potential of amino carboxylates as oral S1P mod-
ulators.
c
Acknowledgements
O
(CH2)6CH3
O
O
N
We thank R. Kühr and M. Kraus for their synthetic contributions
toward phosphate mimics. Practical support in blood lymphocyte
count determination by C. Pally as well as practical support in
the S1P binding assays by K. Welzenbach, D. Guerini, M. Streiff,
S. Schmutz, D. Fehlmann, and C. Bourquin is highly appreciated.
We are grateful for the overall project support of B. Nüsslein-Hil-
desheim, N. G. Cooke, and C. Bruns.
S
HN
HN
R
O
17: R = Boc
18: R = H
d
Scheme 4. Reagents and conditions: (a) H2NCH2CO2MeÁHCl, NaCNBH4, pH 5 buffer,
rt, 29%; (b) ClSO2NCO, HCOOH, rt, then add 15, Et3N, CH2Cl2, rt, 93%; (c) NaOMe,
MeOH, rt, 35%; (d) HCl, Et2O, rt, 90%.
References and notes
1. Kappos, L.; Antel, J.; Comi, G.; Montalban, X.; O’Connor, P.; Polman, C. H.; Haas,
T.; Korn, A. A.; Karlsson, G.; Radue, E. W. N. Engl. J. Med. 2006, 355, 1124.
2. Brinkmann, V. Pharmacol. Ther. 2007, 115, 84.
3. (a) Matloubian, M.; Charles, C. G.; Cinamon, G.; Lesneski, M. J.; Xu, Y.;
Brinkmann, V.; Allende, M. L.; Maria, L.; Proia, R. L.; Cyster, J. G. Nature 2004,
427, 355; (b) Schwab, S. R.; Pereira, J. P.; Matloubian, M.; Xu, Y.; Huang, Y.;
Cyster, J. G. Science 2005, 309, 1735.
Table 1
[c-
35S]GTP binding assay results of phosphate mimics
Compound
S1P1
S1P3
S1P4
S1P5
EC50 (nM)
a
a
a
a
EC50 (nM)
EC50 (nM)
EC50 (nM)
4. For recent reviews see: (a) Cooke, N. G.; Zécri, F. Annu. Rep. Med. Chem. 2007, 42,
245; (b) Marsolais, D.; Rosen, H. Nat. Rev. Drug Disc. 2009, 8, 297.
5. Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens, S.; Hof, R.; Bruns,
C.; Prieschl, E.; Baumruker, T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K.
R. J. Biol. Chem. 2002, 277, 21453.
FTY720-P
1-P
3
4
6
0.14
0.85
110
20
240
44
19
960
460
2.5
5.4
nd
151
3100
>22,000
300
6400
2400
0.70
5.7
nd
0.46
2.2
nd
19
nd
280
200
3600
1000
19
800
140
120
670
1700
6. Högenauer, K.; Billich, A.; Pally, C.; Streiff, M.; Wagner, T.; Welzenbach, K.;
Nussbaumer, P. Chem. Med. Chem. 2008, 3, 1027.
7
11
14
18
7. Earlier studies describing phosphonates and carboxylates: (a) Hale, J. J.; Neway,
W.; Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M.; Milligan, J.; Shei, G.;
Chrebet, G.; Bergstrom, J.; Card, D.; Koo, G. C.; Koprak, S. L.; Jackson, J. J.; Rosen,
H.; Mandala, S. Bioorg. Med. Chem. Lett. 2004, 14, 3351; (b) Hale, J. J.; Doherty,
G.; Toth, L.; Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M.; Milligan, J.;
Shei, G.; Chrebet, G.; Bergstrom, J.; Card, D.; Forrest, M.; Sun, S.; West, S.; Xie,
H.; Nomura, N.; Rosen, H.; Mandala, S. Bioorg. Med. Chem. Lett. 2004, 14, 3501;
(c) Hale, J. J.; Lynch, C. L.; Neway, W.; Mills, S. G.; Hajdu, R.; Keohane, C. A.;
Rosenbach, M. J.; Milligan, J. A.; Shei, G.; Parent, S. A.; Chrebet, G.; Bergstrom, J.;
Card, D.; Ferrer, M.; Hodder, P.; Strulovici, B.; Rosen, H.; Mandala, S. J. Med.
Chem. 2004, 47, 6662; (d) 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.; Mandala, S. M. Bioorg. Med. Chem. Lett.
2006, 16, 2905; (e) Foss, F. W.; Snyder, A. H.; Davis, M. D.; Rouse, M.; Okusa, M.
D.; Lynch, K. R.; Macdonald, T. L. Bioorg. Med. Chem. 2007, 15, 663; (f) Lu, X.;
Sun, C.; Valentine, W. J.; Shuyu, E.; Liu, J.; Tigyi, G.; Bittman, G. J. Org. Chem.
2009, 74, 3192; (g) Pan, S.; Mi, Y.; Pally, C.; Beerli, C.; Chen, A.; Guerini, D.;
Hinterding, K.; Nuesslein-Hildesheim, B.; Tuntland, T.; Lefebvre, S.; Liu, Y.; Gao,
W.; Chu, A.; Brinkmann, V.; Bruns, C.; Streiff, M.; Cannet, C.; Cooke, N.; Gray, N.
Chem. Biol. 2006, 13, 1227.
a
All compounds shown are full agonists.
(CH2)7CH3
( )n
HO2C
19:
20:
n=1
n=2
NH2
Figure 2. Achiral Merck amino carboxylates 19 and 20.
(IC50 = 1900 nM for 19, 710 nM for 20, and 0.16 nM for FTY720-P as
reference). In contrast, various racemic phosphonates of the Merck
series were shown to retain high affinity for S1P1.
8. Meléndez, R. E.; Lubell, W. D. Tetrahedron 2003, 59, 2581.
The compounds in Table 1 were also tested for S1P2 activity, but
all of them showed an IC50 >22,000 nM.
9. Elongation with standard CN displacement of the mesylate failed because of
competing cyclic carbamate formation: Curran, T. P.; Pollastri, M. P.; Abelleira,
S. M.; Messier, R. J.; McCollum, T. A.; Rowe, C. G. Tetrahedron Lett. 1994, 35,
5409.
10. Duncia, J. V.; Pierce, M. E.; Santella, J. B., III J. Org. Chem. 1991, 56, 2395.
11. Campbell, A. D.; Birch, A. M. Synlett 2005, 834. and references cited therein.
12. Gergely, P.; Wallstroem, E.; Nuesslein-Hildesheim, B.; Bruns, C.; Zécri, F.;
Cooke, N.; Traebert, M.; Tuntland, T.; Rosenberg, M.; Saltzman, M. Mult. Scler.
2009, 15, S125.
Comparing the S1P1 potency of the phosphate mimics to amino
phosphate 1-P, it is not too unsurprising that none of them dis-
played pronounced activity in reducing blood lymphocyte counts
in vivo. For compound throughput reasons, we assessed this by
measuring the ED50 at a single time point (6 h). The ED50 for b-ami-
no acid 11 was determined to be 3.3 mg/kg, all other derivatives
did not show significant reduction of lymphocyte count after a
po dose of 3 mg/kg.13 It should be noted that it is quite likely that
there are significant differences in the PK parameters of these
13. ED50 of FTY720 in that assay is 0.09 mg/kg.
14. For assay throughput and project requirement reasons, compounds were
routinely tested using po administration. Only amino acid 7 was also dosed ip
to give an ED50 of ca. 10 mg/kg.