Synthesis of chiral analogues of FTY720 and its phosphate

Article abstract of DOI:10.1055/s-2003-40883

Efficient and versatile protocols for the synthesis of chiral analogues of the novel immunomodulator FTY720 and its phosphate are described. These synthetic procedures allow for broad structural variation and deliver essential tools to further elucidate FTY720's novel mechanism of action.

Full text of DOI:10.1055/s-2003-40883

PAPER
1667
Synthesis of Chiral Analogues of FTY720 and its Phosphate
S
ynthesis of FT
Y
7
l
20 Ana
a
logues us Hinterding,*a,1 Sylvain Cottens,a,1 Rainer Albert,a,1 Frederic Zecri,a Peter Buehlmayer,a Carsten Spanka,a
Volker Brinkmann,a Peter Nussbaumer,b Peter Ettmayer,b Klemens Hoegenauer,b Nathanael Gray,c Shifeng Panc
a
Novartis Institutes for Biomedical Research, S507.409, 4002 Basel, Switzerland
Fax +41(61)3246735; E-mail: klaus.hinterding@pharma.novartis.com
b
Novartis Forschungsinstitut GmbH, Brunner Str. 59, 1235 Wien, Austria
c
Novartis Institute for Functional Genomics Inc,, 10675 John Jay Hopkins Drive, San Diego CA 92121, USA
Received 16 May 2003
herein describe experimental details of a practical and ver-
satile stereoselective synthesis of chiral FTY720 ana-
logues 3, 5, 7, 9 and their corresponding phosphates 4, 6,
8, 10.7
The Schöllkopf protocol8 was chosen as the centerpiece of
our synthesis, because a) both Val-Gly- and Val-Ala-de-
rived auxiliaries are commercially available in either R-
and S-configuration9 and b) the protocol allows for broad
structural variations at the quaternary center of the hydro-
philic head-group, the central aromatic ring, and the lipo-
philic side-chain of FTY720 (Figure 1).
Abstract: Efficient and versatile protocols for the synthesis of
chiral analogues of the novel immunomodulator FTY720 and its
phosphate are described. These synthetic procedures allow for
broad structural variation and deliver essential tools to further elu-
cidate FTY720’s novel mechanism of action.
Key words: phosphates, sphingolipids, stereoselective synthesis,
drugs, chiral auxiliaries
FTY720 (Figure 1) is a novel immunomodulator which
acts via S1P-receptor agonism and is highly effective in
animal models of organ transplantation and autoimmuni-
ty.2 Additionally, in a recently completed Phase II clinical
trial, the drug has proven efficacious in preventing kidney
allograft rejection in humans.3 Unlike any other immuno-
suppressant currently on the market, FTY720 does not in-
hibit T- and B-cell proliferation and activation at
therapeutically relevant concentrations; instead it leads to
a sequestration of lymphocytes from the periphery into
secondary lymphoid organs.4 According to our current un-
derstanding, the phosphorylated molecule FTY720-Phos-
phate 2 (Figure 1), which is generated in vivo via a
sphingosine-kinase, signals as an agonist through four of
five sphingosine-1-phosphate (S1P) receptors (formerly
known as EDG-receptors).5
Generation of the Quaternary Center
The generation of the quaternary center was accomplished
by sequential double alkylation of the Gly-derived
Schöllkopf-auxiliary (Scheme 1, steps a and b). Both
enantiomeric forms of the target molecules were available
by either choosing the appropriate configuration of the
auxiliary or by adjusting the sequence of alkylation steps.
While, in general, the first alkylation proceeded unevent-
fully irrespective of the size of the alkylating reagent, the
overall yield was higher, if the sterically smaller residue
was introduced last. Iodides as alkylating reagents were
preferred, but satisfactory results and yields (ca. 65% per
alkylation step) were also obtained with bromides.
Chiral analogues of FTY720 and its phosphate played an
important role in the initial understanding of FTY720’s
mode of action.6 They are also invaluable tools to further
clarify the pharmacology of single S1P-receptors.5 We
Preferred building blocks for the first alkylation (step a)
were benzyl-protected iodides like 12, since they allowed
Figure 1 Structures of the immunomodulators FTY720, its phosphate and structural analogues
Synthesis 2003, No. 11, Print: 05 08 2003.
Art Id.1437-210X,E;2003,0,11,1667,1670,ftx,en;C03403ss.pdf.
© Georg Thieme Verlag Stuttgart · New York

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K. Hinterding et al.
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Scheme 1 Synthesis of FTY720 amino alcohol analogues 3, 5, 7, 9 and hydrolysis of the bislactim ether
for broad structural variation of the lipophilic side-chain tection completed the synthetic sequence to deliver amino
after benzylic deprotection (step f) and phenol ether for- alcohols 3, 5, 7 and 9.
mation (step g).
Synthesis of phosphates
Hydrolysis of the Bislactim Ether
Due to the difficult physicochemical properties of amino-
For Me-substituted chiral centers, the hydrolysis of R- phosphates, in particular their low solubility, and due to
Val-derived bislactim ethers 14 (step c) proceeded in good lack of commercially available sphingosine kinases, their
yields of ca. 60–80% by stirring a homogenous solution of biochemical synthesis and isolation is tedious. We there-
starting material in mixtures of either dioxane or THF fore developed straightforward chemical approaches for
with trifluoroacetic acid or aqueous HCl overnight. The the preparation of phosphates 4, 6, 8, 10, which do not re-
final concentration of acid was adjusted to 0.1–0.5 M. In- quire a purification step of the final products (Scheme 2).
creasing the steric bulk at the quaternary center only The nature and lability of substituents R2 and R3 deter-
slightly, e.g. by substitution of ethyl for methyl, already mined whether trivalent phosphorylation agent A,11 which
required significantly longer reaction times of 5–7 days. is deprotected via hydrogenation, or B,12 which is depro-
Nevertheless, satisfactory yields (50–60%) were still ob- tected via acid treatment, was used. Both methods deliv-
tained, if trifluoroacetic acid was used at 0.5–0.7 M in ered the final products 4, 6, 8, 10 with similar high yields
acetonitrile. The use of higher concentrations of various of 65–80% over all steps starting from Boc-protected ami-
acids (HCl, TFA, HBr) in different solvents (dioxane, no alcohols 17. The only chromatographic purification of
THF, EtOH) did not result in a reduction of reaction times this sequence was performed after oxidation on fully pro-
but in an increased formation of amido ester and dike- tected phosphates 18 and 19. While the use of A required
topiperazine side-products and thereby lower yields.10 It is an additional deprotection step (hydrogenolysis, step j) its
of importance to note that the configuration of the asym- reaction products 18 where somewhat more stable than
metric centers in bislactim ethers 14 had a significant in- the ones of B (19) under standard work-up and chroma-
fluence on the optimal conditions of hydrolysis: in the tography conditions.13
case of the S-Val stereoisomer of 14 a 1:1 mixture of
TFA–water was used to obtain a good yield of 72% after
stirring at room temperature for 64 hours.
Critical steps of the reaction sequence are exemplified by the syn-
thesis of amino alcohol 9 and phosphate 10.
Second Alkylation of Schöllkopf-Auxiliary (Step b); (2R,5R)-2-
[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]-3,6-diethoxy-2-ethyl-
5-isopropyl-2,5-dihydropyrazine (14; R1 = OMe, R2 = Et); Typ-
ical Procedure
To a solution of monoalkylated Schöllkopf-adduct (2R,5R)-2-[2-(4-
benzyloxy-3-methoxyphenyl)ethyl]-3,6-diethoxy-5-isopropyl-2,5-
dihydropyrazine (14; R1 = OMe, R2 = H; 1.5 g, 3.3 mmol) in THF
(4 mL) was added a solution of n-BuLi in hexanes (1.6 M) at
–75 °C. After stirring for 30 min at this temperature, a solution of
EtI (618 mg, 4.0 mmol) in THF (1 mL) was added dropwise. The
stirring was continued at –75 °C for 30 min, before warming to 0 °C
and stirring for another 60 min. Quenching with sat. aq NaHCO3
was followed by extraction with EtOAc (3 × 15 mL), drying of the
Completion of Amino Alcohol Synthesis
After reduction of the ethyl ester functionality (step d), the
amino-group was protected as its Boc-derivative (step e).
Benzylic deprotection via hydrogenolysis (step f) was fol-
lowed by the incorporation of the lipophilic side chain via
phenol alkylation using bromides or mesylates in the pres-
ence of either K2CO3 or Cs2CO3 (step g). The use of io-
dides in this alkylation step led to predominant
elimination and alkene formation. Uneventful Boc-depro-
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Synthesis of FTY720 Analogues
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Scheme 2 Synthesis of FTY720 phosphate analogues 4, 6, 8, 10
organic phase (MgSO4) and evaporation of the solvent. Chromato-
graphy on silica gel using hexanes–EtOAc (99:1 to 95:5) yielded
the desired product as a colorless oil (984 mg, 62%).
lized from toluene, 0.6 mmol, 42 mg) in anhyd THF (2 mL) was
added phosphoamidite A (0.3 mmol, 72 mg), dissolved in anhyd
THF (0.5 mL). After stirring at r.t. under argon for 1 h, H2O2 was
added (30%, 240 L, ca. 2 mmol) and the stirring was continued for
30 min. Quenching with aq sat. Na2S2O3 and extraction with EtOAc
(3 × 10 mL) was followed by drying (Na2SO4), evaporation of sol-
vent and chromatography on silica gel using Et2O–CH2Cl2 (1:1) to
yield the fully protected phosphate 18 [R1 = OMe, R2 = Et,
R3 = (CH2)4Ph, 118 mg] as a colorless oil. This intermediate was
dissolved in MeOH (10 mL) and stirred together with Pd/C (10%,
50 mg) for 2 h under an atmosphere of H2. After filtration over
Celite and washing with MeOH and CH2Cl2, the solvent was re-
moved in vacuo and the residue dissolved in AcOH (2 mL) and HCl
(37%, 0.5 mL). After 16 h at r.t., the solvent was removed by lyo-
philization to yield the desired phosphate 10 [R1 = OMe, R2 = Et,
R3 = (CH2)4Ph, 72 mg, 80%] as a white solid.
Hydrolysis of Double-Alkylated Bislactim Ether (Step c); (R)-2-
Amino-4-(4-benzyloxy-3-methoxyphenyl)-2-ethylbutyric Acid
Ethyl Ester (15; R1 = OMe, R2 = Et); Typical Procedure
A homogenous solution of (2R,5R)-2-[2-(4-benzyloxy-3-methoxy-
phenyl)ethyl]-3,6-diethoxy-2-ethyl-5-isopropyl-2,5-dihydropyra-
zine (14, R1 = OMe, R2 = Et, 984 mg, 2.0 mmol) in MeCN (43 mL)
and 2 M trifluoroacetic acid in H2O (21 mL) was stirred for 5 d at
r.t. After addition of aq sat. NaHCO3 (1.5 ml), the reaction mixture
was extracted with EtOAc (3 × 20 mL). Drying (MgSO4) and evap-
oration of the solvent was followed by chromatography on silica gel
using EtOAc–hexanes (1:2) to yield the desired ethyl ester as a col-
orless oil (395 mg, 52%).
Completion of Amino Alcohol Synthesis (Steps d–h); (R)-2-
Amino-2-ethyl-4-[3-methoxy-4-(4-phenylbutoxy)phenyl]butan-
1-ol (9); Typical Procedure
Spectral Data of Selected Final Products
(S)-2-Amino-2-[2-(4-heptyloxyphenyl)ethyl]pent-4-yn-1-ol (3)
1H NMR (400 MHz, DMSO-d6): = 0.90 (t, J = 7 Hz, 3 H, CH3),
1.23–1.45 [m, 8 H, (CH2)4], 1.72 (p, J = 7 Hz, 2 H, OCH2CH2), 1.86
(dd, J = 7, 11 Hz, 2 H, Cq-CH2), 2.54–2.65 (m, 4 H, Cq-CH2,
PhCH2), 3.16 (s, 1 H, C CH), 3.57 (d, J = 6 Hz, 2 H, CH2OH), 3.94
(t, J = 7 Hz, 2 H, OCH2), 5.65 (t, J = 6 Hz, 1 H, OH), 6.88 (d, J = 8
To a solution of ester 15 (R1 = OMe, R2 = Et, 395 mg, 1.1 mmol) in
anhyd THF (10 ml) was added a solution of LiAlH4 in THF (1 M,
1.7 mmol, 1.7 mL). Stirring at r.t. for 1 h was followed by quench-
ing the mixture with aq sat. Na2SO4 solution and extraction with
EtOAc (3 × 15 mL). After drying (Na2SO4) and evaporation of the
solvent, the crude material (385 mg) was dissolved in CH2Cl2 (15
mL) and treated with Boc2O (1.7 mmol, 371 mg) at r.t. and the mix-
ture was stirred for 16 h. Evaporation of the solvent and chromatog-
raphy using EtOAc–hexanes (2:3) afforded alcohol 16 (R1 = OMe,
R2 = Et, 1.0 mmol, 402 mg) in 95% yield. The alcohol 16 was dis-
solved in EtOH (25 mL) and stirred together with 10% Pd/C (90
mg) for 1 h at r.t. under an atmosphere of H2. Filtration over Celite
and evaporation of the solvent yielded the phenol in quantitative
yield as an oil, which solidified. A mixture of this phenol (313 mg,
1.0 mmol), K2CO3 (415 mg, 3.0 mmol), phenylbutyl bromide (426
mg, 2.0 mmol) in EtOH (9 mL) and DMF (3 mL) was stirred at
55 °C for 16 h. Addition of EtOAc (50 mL) was followed by wash-
ing with H2O (25 mL) and brine (25 mL). Drying (Na2SO4) and
evaporation of solvent was followed by chromatography on silica
gel using Et2O–hexanes (4:1) to give Boc-protected product 17
(R1 = OMe, R2 = Et, 0.7 mmol, 330 mg) in 72% yield as white solid.
Stirring Boc-urethane 17 (0.5 mmol, 230 mg) for 16 h at r.t. in di-
oxane containing 4 M HCl (5 mL), yielded the desired amino alco-
hol 9 (0.4 mmol, 158 mg) as a white solid after precipitation with
Et2O, filtration and drying.
+
Hz, 2 Harom), 7.12 (d, J = 8 Hz, 2 Harom), 8.00–8.20 (br, 3 H, NH3 ).
MS (ES+): m/z = 318.5 (MH)+.
Phosphoric Acid Mono-{(S)-2-amino-2-[2-(4-heptyloxyphe-
nyl)ethyl]pent-4-ynyl} Ester (4)
1H NMR (400 MHz, DMSO-d6): = 0.90 (t, J = 7 Hz, 3 H, CH3),
1.25–1.45 [m, 8 H, (CH2)4], 1.72 (p, J = 7 Hz, 2 H, OCH2CH2), 1.91
(m, 2 H, Cq-CH2), 2.58 (m, 2 H, Cq-CH2C CH), 2.67 (m, 2 H,
PhCH2), 3.19 (s, 1 H, CCH), 3.93 (m, 4 H, PhOCH2, CH2OP), 6.86
(d, J = 8 Hz, 2 Harom), 7.13 (d, J = 8 Hz, 2 Harom).
MS (ES+): m/z = 398.3 (MH)+, 795.3 (2 M + H)+.
MS (ES–): m/z = 396.3 (M – H)–, 397.3 (M–), 793.3 (2M – H)–.
Phosphoric Acid Mono-{(S)-2-amino-2-[2-(4-heptyloxyphe-
nyl)ethyl]-5-hydroxypentyl} Ester (6)
1H NMR (400 MHz, DMSO-d6): = 0.90 (t, J = 7 Hz, 3 H, CH3),
1.25–1.53 [m, 10 H, CH2, (CH2)4], 1.65–1.85 (m, 6 H, 2 × Cq-CH2,
OCH2CH2), 2.53 (m, 2 H, PhCH2), 3.43 (m, 2 H, CH2OH), 3.88 (d,
J = 7 Hz, 2 H, CH2OP), 3.94 (t, J = 7 Hz, 2 H, PhOCH2), 6.86 (d,
J = 8 Hz, 2 Harom), 7.12 (d, J = 8 Hz, 2 Harom).
MS (ES+): m/z = 418.3 (MH)+, 873.4 (2 M + K)+.
MS (ES–): m/z = 416.4 (M – H)–, 417.3 (M–), 833.5 (2 M – H)–.
Phosphate 10 [R1 = OMe, R2 = Et, R3 = (CH2)4Ph] ; Typical
Procedure
To a solution of Boc-protected amino alcohol 17 [R1 = OMe,
R2 = Et, R3 = (CH2)4Ph, 0.2 mmol, 100 mg] and tetrazole (recrystal-
Synthesis 2003, No. 11, 1667–1670 © Thieme Stuttgart · New York

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K. Hinterding et al.
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(R)-2-Amino-4-[3-methoxy-4-(4-phenyl-butoxy)-phenyl]-2-me-
thyl-butan-1-ol (7)
(2) Brinkmann, V.; Lynch, K. R. Curr. Opin. Immunol. 2002,
14, 569.
1H-NMR (400 MHz, DMSO): = 0.98 (s, 3 H, CH3), 1.52 (q, J = 7
Hz, Cq-CH2), 1.74 [m, 4 H, (CH2)2], 2.53 (m, 2 H, PhCH2), 2.68 (m,
2 H, Ph-CH2), 3.18 (d, J = 6 Hz, 2 H, CH2OH), 3.78 (s, 3H, OCH3),
3.94 (s, 2 H, OCH2), 4.57 (t, J = 6 Hz, 1 H, OH), 6.69 (d, J = 8 Hz,
1 Harom), 6.80 (s, 1 Harom), 6.84 (d, J = 8 Hz, 1 Harom), 7.15–7.35 (m,
5 H, 5 Harom).
(3) Tedesco, T.; Kahan, B.; Mourad, G.; Vanrenterghem, Y.;
Grinyo, J.; Weimar, W.; Pellet, P.; Chodoff, L.; Sablinski, T.
Am. J. Transplant. 2001, 1, S243.
(4) Chiba, K.; Yanagawa, Y.; Yasubuchi, Y.; Kataoka, H.;
Kawaguchi, T.; Ohtsuki, M.; Hoshino, Y. J. Immunol. 1998,
160, 5037.
(5) (a) 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. (b) Mandala, S.; Hajdu, R.;
Bergstrom, J.; Quakenbush, E.; Xie, J.; Milligan, J.;
Thornton, R.; Shei, G. J.; Card, D.; Keohane, C.; Rosenbach,
M.; Hale, J.; Lynch, C. L.; Rupprecht, K.; Parsons, W.;
Rosen, H. Science 2002, 296, 346.
(6) Kiuchi, M.; Adachi, K.; Kohara, T.; Minoguchi, M.;
Hanano, T.; Aoki, Y.; Mishina, T.; Arita, M.; Nakao, N.;
Ohtsuki, M.; Hoshino, Y.; Teshima, K.; Chiba, K.; Sasaki,
S.; Fujita, T. J. Med. Chem. 2000, 43, 2946.
MS (ES+): 358.3 (MH)+
(R)-2-Amino-2-ethyl-4-[3-methoxy-4-(4-phenylbutoxy)phe-
nyl]butan-1-ol (9)
1H NMR (400 MHz, DMSO-d6): = 0.85 (t, J = 9 Hz, 3 H, CH3),
1.35 (dq, J = 9, 5 Hz, 2 H, CH2CH3), 1.50 (t, J = 8 Hz, 2 H, Cq-CH2),
1.71 (m, 4 H, OCH2CH2CH2) 2.48 (m, 2 H, PhCH2), 2.65 (m, 2 H,
PhCH2), 3.20 (s, 2 H, CH2OH), 3.76 (s, 3 H, OCH3), 3.93 (t, J = 7
Hz, 2 H, PhOCH2), 4.50 (br, 1 H, OH), 6.67 (d, J = 8 Hz, 1 Harom),
6.78 (s, 1 Harom), 6.83 (d, J = 8 Hz, 1 Harom), 7.18–7.33 (m, 5 Harom).
MS (ES+): m/z = 372.3 (MH+).
(7) Hinterding, K.; Albert, R.; Cottens, S. Tetrahedron Lett.
2002, 43, 8095.
(8) (a) Schöllkopf, U. Pure Appl. Chem. 1983, 55, 1799.
(b) Schöllkopf, U.; Groth, U.; Deng, C. Synthesis 1981, 737.
(9) Commercially available as OMe-ethers from Merck and
Fluka.
Phosphoric Acid Mono-{(R)-2-amino-2-ethyl-4-[3-methoxy-4-
(4-phenylbutoxy)phenyl]butyl} Ester (10)
1H NMR (400 MHz, DMSO-d6): = 0.92 (t, J = 8 Hz, 3 H, CH3),
1.65–1.85 (m, 8 H, CH2), 2.55 (m, 2 H, PhCH2), 2.65 (m, 2 H,
PhCH2), 3.75 (s, 3 H, OCH3), 3.88 (d, J = 7 Hz, 2 H, CH2OP), 3.94
(t, J = 7 Hz, 2 H, PhOCH2), 6.70 (d, J = 8 Hz, 1 Harom), 6.85 (m, 2
(10) Schöllkopf, U.; Westphalen, K. O.; Schroeder, J.; Horn, K.
Liebigs Ann. Chem. 1988, 781.
Harom), 7.18–7.33 (m, 5 Harom).
(11) Watanabe, Y.; Komoda, Y.; Ebisuya, K.; Ozaki, S.
Tetrahedron Lett. 1990, 31, 255.
MS (ES–): m/z = 450.5 (M – H).
(12) Perich, J. W.; Johns, R. B. Synthesis 1988, 142.
(13) See, e.g.: Burger, A.; Tritsch, D.; Biellmann, J. F.
Carbohydr. Res. 2001, 332, 141.
References
(1) These authors contributed equally to this study.
Synthesis 2003, No. 11, 1667–1670 © Thieme Stuttgart · New York