Synthesis of sphingomyelin difluoromethylene analogue

Article abstract of DOI:10.1016/j.tetlet.2006.02.018

As a new sphingomyelinase inhibitor, a novel sphingomyelin CF₂ analogue was designed and synthesized. One key step of this synthesis is the very mild hydrolysis of the oxazolidinone ring, a suitable intermediate for the introduction of a diethy

Full text of DOI:10.1016/j.tetlet.2006.02.018

Tetrahedron Letters 47 (2006) 2627–2630
Synthesis of sphingomyelin difluoromethylene analogue
Toshikazu Hakogi,a Tetsuya Yamamoto,a Shinobu Fujii,b
Kiyoshi Ikedab and Shigeo Katsumuraa,*
aSchool of Science and Technology, Kwansei Gakuin University, 2-1, Gakuen, Sanda, Hyogo 669-1337, Japan
bDepartment of Biochemistry, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Osaka 569-1041, Japan
Received 24 September 2005; revised 30 January 2006; accepted 6 February 2006
Available online 28 February 2006
Abstract—As a new sphingomyelinase inhibitor, a novel sphingomyelin CF2 analogue was designed and synthesized. One key step
of this synthesis is the very mild hydrolysis of the oxazolidinone ring, a suitable intermediate for the introduction of a diethyl
difluoromethylphosphonate group, by utilizing the characteristic electron withdrawing nature of the nosyl group at the oxazolid-
inone ring in an alcoholic solvent to produce the corresponding carbonate attaching at the secondary hydroxy group. The
synthesized CF2 analogue inactivated toward B. cereus sphingomyelinase with nearly the same attitude as the nitrogen analogue
that we previously reported.
ꢀ 2006 Elsevier Ltd. All rights reserved.
In our project to develop new sphingomyelinase
(SMase) competitive inhibitors, which act at the cata-
lytic site of the enzyme, we have already reported the
syntheses of carbon,1 nitrogen,2 and sulfur analogues3
that possess a shorter and saturated backbone skeleton
than natural sphingomyelin. These backbone skeleton
were designed based on the previous results of the initial
hydrolytic velocity of sphingomyelin analogues toward
B. cereus sphingomyelinase.4,5 In these analogues, one
oxygen atom, at which the sphingomyelin was hydro-
lyzed by SMase, was replaced with a carbon, a nitrogen,
or a sulfur atom. In general, SMase is regarded as a key
enzyme of the sphingolipid metabolism6 and catalyzes
the hydrolysis of sphingomyelin to produce ceramide,
recognized as an essential signal transduction factor in
cell differentiation and in programmed cell death (apop-
tosis) derivation (Fig. 1).7 Strong and competitive inhib-
itors against SMase would be valuable for the
elucidation of the SMase hydrolytic mechanism.8
Sphingomyelinase
O
O
C15H31 NH
C13H27
O
P O
O
C15H31 NH
O
C13H27
OH
NMe3
OH
OH
Ceramide
Sphingomyelin
Figure 1. Sphingomyelin catabolite.
O
C5H11
C7H15
NH
F
F
O
P
NMe3
O O
OH
1
Figure 2. Difluoromethylene analogue.
In this letter, we report the synthesis and inhibitory
activity of sphingomyelin analogue 1, which possesses
a difluoromethylene group in the place of the hydro-
lyzed oxygen atom (Fig. 2). The difluoromethylene
group is well regarded as an isoster of the oxygen atom
due to similar steric, electrical, and polar properties.9
The retrosynthesis of sphingomyelin CF2-analogue 1 is
described in Figure 3. Difluoromethylphosphonate 3
would be a suitable intermediate for the synthesis of 1,
and be obtained by the introduction of
a dif-
Keywords: Sphingomyelin CF2 analogue; Sphingomyelinase inhibitor.
luoromethylphosphonate group10 to the corresponding
*
Corresponding author. Tel.: +81 79 565 8314; fax: +81 79 565
9077; e-mail: katsumura@ksc.kwansei.ac.jp
triflate of alcohol 2, which would be prepared in the
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.018

2628
T. Hakogi et al. / Tetrahedron Letters 47 (2006) 2627–2630
O
O
O
R
R
N
PMB
O
N
O
ii
F
F
F
F
O
N
1
OEt
OEt
OX
C7H15
P
C7H15
P
C7H15
O OEt
O OEt
3
2 : X = H
10 : X = Tf
3
i
N3
O
3 : R = PMB
12 : R = H
iii
iv
R
O
N
O
C7H15
OH
13 : R = Boc
14 : R = Ns
v
OH
C7H15
4
2
Ns
O
Figure 3. Retrosynthesis of CF2 analogue 1.
NH
F
F
F
HC
F
vi
C7H15
BnO
OEt
O
P
P
optically pure form from known 49 by regioselective
azide introduction followed by oxazolidinone ring
formation.11
O OEt
OEt
EtO
11
17
O
Scheme 2. Introduction of CF2 group and nosyl group. Reagents and
conditions: (i) Tf2O, 2,6-lutidine, CH2Cl2, ꢀ78 ꢁC; (ii) 11, LDA,
HMPA, ꢀ78 ꢁC, 72% for two steps; (iii) CAN, CH3CN, H2O, 0 ꢁC,
74%; (iv) Boc2O, DMAP, Et3N, DMF, quant.; (v) n-BuLi, NsCl,
ꢀ78 ꢁC, 78%; (vi) BnOH, K2CO3, 18-crown-6, 94%.
Thus, epoxide ring opening of 4 selectively proceeded at
the C-2 position in a 6:1 ratio by treatment with sodium
azide in the presence of phenylboric acid to give phenyl
boronate 5,12 which was hydrogenated in the presence of
di-tert-butyl dicarbonate in methanol to give the pro-
tected amine 6.13 Desired oxazolidinone alcohol 2 was
obtained in excellent yield by a sequence of the deprotec-
tion of boronate 6, the protection of the resulting pri-
mary alcohol of 7, formation of the oxazolidinone
ring, the introduction of a p-methoxybenzyl group at
the nitrogen atom of 8, and the subsequent removal of
the tert-butyldimethylsilyl group. Compound 2 was eas-
ily purified by recrystallization (Scheme 1).
generated from 11.9b,15 After reinvestigating the reaction
conditions, we successfully realized the modification of
the reaction conditions to obtain the desired 3 in a
72% yield even on a 5-g scale by the following proce-
dure: a pre-cooled THF solution of the anion derived
from 11 at ꢀ100 ꢁC was quickly added to a mixture of
10 and HMPA in THF at ꢀ78 ꢁC, and then the resulting
mixture was rapidly poured into a mixture of 2 N hydro-
chloric acid and ethyl acetate.
With desired oxazolidinone 2 in hand, the introduction
of the diethyl difluoromethylphosphonate group at the
C-1 position was examined (Scheme 2). The reaction
of triflate 10, which was prepared from 2 by treatment
with trifluoromethanesulfonic anhydride and 2,6-
Next is the conversion of 3 to 17. The PMB group of 3
was replaced by a Boc group to activate the oxazolidi-
none ring for hydrolysis based on a previous meth-
od.1b,16 Unfortunately, the chemoselective hydrolysis
of the oxazolidinone ring of 13 was unsuccessful, and
unidentified compounds having higher polarity were
produced. A nosyl group was then introduced into oxa-
zolidinone 12 by treatment with n-butyllithium and no-
syl chloride in THF.17 Fortunately, the oxazolidinone
ring of 14 was cleanly hydrolyzed within 5 min at room
temperature by treatment with potassium carbonate in
methanol to produce the desired methyl carbonate 17.
To the best of our knowledge, this is one of the mildest
conditions for hydrolyzing an oxazolidinone ring.16,18
Then, we studied this hydrolysis by using other alcohols,
which led to the alcoxy part of the resulting carbonate.
The obtained results are shown in Table 1. In the case
of benzyl alcohol, the addition of 18-crown-6 ether in
THF was very effective to obtain the corresponding car-
bonate in excellent yield. For further elaboration, we
chose benzyl carbonate 17, because the benzyl group
could be removed by hydrogenolysis and the resulting
alcohol was obtained by the procedure of simple
filtration.
lutidine, with
a
lithium anion of diethyl dif-
luoromethylphosphonate 11, gave the desired 3 in about
50% yield on a small scale according to the reported pro-
cedure.10 On a 1-g scale, however, the yield of 314 was
significantly decreased due to the instability of the anion
Boc
N3
NH
i
C7H15
ii
C7H15
4
O
O
O
O
B
B
5
6
Ph
Ph
O
Boc
NH
R2
iii
iv
O
N
C7H15
OR1
C7H15
OH OH
7
8 : R1 = TBS, R2 = H
9 : R1 = TBS, R2 = PMB
2 : R1 = H, R2 = PMB
v
vi
The remaining issues for the synthesis of difluoromethyl-
ene analogue 1 were the introduction of an acyl group
and then a choline group. The acylation of 17 to lead
19 was successful by reaction with hexanoyl chloride
in the presence of triethylamine in Et2O in 83% yield.19
Scheme 1. Synthesis of the difluoromethylene analogue. Reagents and
conditions: (i) NaN3, PhB(OH)2, DMF, 86%; (ii) Boc2O, H2, Pd–C,
MeOH, 86%; (iii) H2O2, MeOH, 0 ꢁC, 95%; (iv) (a) TBSCl, imidazole,
DMF, 0 ꢁC; (b) NaH, THF, 30 ꢁC, quant. for two steps; (v) PMBCl,
NaH, TBAI, THF, rt; (vi) 2 N HCl, MeOH, rt, quant. for two steps.

T. Hakogi et al. / Tetrahedron Letters 47 (2006) 2627–2630
2629
Table 1. Hydrolysis of oxazolidinone ring
O
O
R
Alcohol
Yield
Products
C5H11
C7H15
N
F
F
iii
i
OEt
17
Ns
P
NH
F2
C
O OEt
19 : R = Ns
20 : R = H
BnO
O
C7H15
O
OEt
OEt
Ethanol
90
P
ii
O
O
Me
15
O
O
O
O
Ns
O
C5H11
NH
F
F
NH
C5H11
C7H15
NH
F
iv
F2
C
F
C7H15
BnO
O
OH
P
C7H15
O
OEt
O
P
P
NMe3
Allyl alcohol
94
O OH
OEt
O
O O
OR
Allyl
21
22 : R = COOBn
1 : R = H
16
O
v
Ns
O
Scheme 3. Synthesis of difluoromethylene analogue 1. Reagents and
conditions: (i) C5H11COCl, Et3N, Et2O, 0 ꢁC, 85%; (ii) PhSH, DBU,
CH3CN, 0 ꢁC, 86%; (iii) TMSBr, CH2Cl2, then MeOH, 82%; (iv) (a)
CCl3CN, HO(CH2)2NHBoc, pyridine, quant.; (b) TFA, CH2Cl2, 0 ꢁC,
71%; (c) MeI, K2CO3, CHCl3, 96%; (v) H2, Pd/C, MeOH, 83%.
NH
F2
C7H15
O
C
OEt
OEt
69a
94b
P
Benzyl alcohol
O
Bn
17
O
Ns
O
NH
F2
C
110.0
90.0
70.0
50.0
30.0
10.0
C7H15
O
OEt
P
p-Methoxybenzylalcohol
77
OEt
O
PMB
18
O
a THF was used as a solvent.
b 18-Crown-6 was added as an additive.
The nosyl group was then removed by treatment with
benzenethiol and DBU in acetonitrile17 to produce cer-
amide derivative 20. Treatment of 20 with bromotri-
methylsilane produced the corresponding silyl ester,
which was hydrolyzed by continuously stirring in meth-
anol to produce acid 21 in 82% yield. A choline group
was successfully introduced by three-step procedures.
Thus, the reaction of 21 with Boc-protected aminoetha-
nol in the presence of trichloroacetonitrile in pyridine at
60 ꢁC, the removal of the Boc group by treatment with
TFA, and then the methylation of the resulting primary
amine produced the desired choline compound 22 in
68% yield. Pure 22 was obtained by using reverse phase
HPLC. Finally, the benzyl carbonate group was re-
moved by hydrogenolysis with palladium on carbon
under a hydrogen atmosphere to produce desired 1.20
Thus, we achieved synthesis of difluoromethylene ana-
logue 1 (Scheme 3).
0.000
0.050
0.100
0.150
0.200
0.250
0.300
Concentration of analogues (mM)
O
O
C5H11
C7H15
NH
C5H11
C7H15
NH H
H
H
O
O
P
N P
NMe3
NMe3
O O
O O
OH
OH
23
24
O
O
C5H11
C7H15
C5H11
C7H15
NH H
NHF
O O
P
H
F
NMe3
O
P
O
NMe3
O O
H H
25
OH
OH
1
The inhibitory ability of the synthesized CF2 analogue 1
was tested against SMase from B. cereus under the same
experimental conditions as the measurements of the
methylene, ethylene, and nitrogen analogues.1a,2,21 As
shown in Figure 4, CF2 analogue 1 showed nearly the
same inhibitory ability to that of nitrogen analogue 23
and showed stronger ability than methylene 24 and eth-
ylene 25 analogues. The IC50 values of CF2 analogue 1
and nitrogen analogue 23 were approximately 57 and
53 lM, compared with 120 and 78 lM of the methylene
and ethylene analogues, respectively.
Figure 4. Inhibitory abilities of both 1 and the compounds previously
synthesized.
synthesis, we found an extremely mild condition to
hydrolyze the oxazolidinone ring by utilizing the strong
electron withdrawing effect of a nosyl group. In addi-
tion, a choline group was introduced into the phos-
phonic acid moiety by convenient three-step
procedures in excellent yield. Analogue 1 apparently
inhibited the hydrolytic ability of B. cereus sphingomy-
elinase with nearly the same attitude to that of nitrogen
analogue 24.2
In conclusion, we achieved the synthesis of difluoro-
methylene analogue 1 in an optically pure form. In this

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T. Hakogi et al. / Tetrahedron Letters 47 (2006) 2627–2630
Respondek, M.; Lieverknecht, A.; Werner, J.; Fischer, P.
References and notes
Synthesis 1989, 256; (c) Hatakeyama, S.; Matsumoto, H.;
Fukuyama, H.; Mukugi, Y.; Irie, H. J. Org. Chem. 1997,
62, 2275.
1. (a) Hakogi, T.; Monden, Y.; Taichi, M.; Iwama, S.; Fujii,
S.; Ikeda, K.; Katsumura, S. J. Org. Chem. 2002, 67, 4839;
(b) Hakogi, T.; Monden, Y.; Iwama, S.; Katsumura, S.
Org. Lett. 2000, 2, 2627.
2. Hakogi, T.; Taichi, M.; Katsumura, S. Org. Lett. 2003, 5,
2801.
3. Hakogi, T.; Fujii, S.; Morita, M.; Ikeda, K.; Katsumura,
S. Bioorg. Med. Chem. Lett. 2005, 15, 2141.
4. Murakami, M.; Iwama, S.; Fujii, S.; Ikeda, K.; Katsu-
mura, S. Bioorg. Med. Chem. Lett. 1997, 7, 1725.
5. Weis, A. L. Chem. Phys. Chem. 1999, 102, 3.
12. Hayakawa, H.; Okada, N.; Miyazawa, M.; Miyashita, M.
Tetrahedron Lett. 1999, 40, 4589.
23:0
13. Spectra data of 6. ½aꢁD 25.9 (c 0.99, CHCl3); IR (KBr
disk) 3329, 2930, 2106, 1601, 1522, 1318, 1256, 1167 cmꢀ1
;
1H NMR (CDCl3, 400 MHz) d 7.80 (md, J = 6.6 Hz, 2H),
7.44 (m, 1H), 7.36 (mtd, J = 6.8, 7.6 Hz, 2H), 4.93 (br d,
J = 8.1 Hz, 1H), 4.26 (dd, J = 2.9, 11.0 Hz, 1H), 3.90–4.02
(m, 2H), 3.84 (m, 1H), 1.58–1.69 (m, 2H), 1.40–1.52 (m,
2H), 1.44 (s, 9H), 1.23–1.40 (m, 8H), 0.89 (t, J = 7.1 Hz,
3H); 13C NMR (CDCl3, 100 MHz) d 155.2, 134.9, 133.9,
130.9, 127.6, 90.0, 74.9, 62.7, 49.7, 35.4, 31.8, 29.4, 29.2,
28.3, 25.3, 22.6, 14.1.
6. (a) Bernardo, K.; Krut, O.; Wiegmann, K.; Kreder, D.;
Micheli, M.; Schafer, R.; Sickman, A.; Schmidt, W. E.;
Schroder, J. M.; Meyer, H. E.; Sandhoff, K.; Kronke, M.
J. Biol. Chem. 2000, 275, 7641; (b) Hofmann, K.; Tomiuk,
S.; Wolff, G.; Stoffel, W. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 5895; (c) Chatterjee, S.; Han, H.; Rollins, S.;
Cleveland, T. J. Biol. Chem. 1999, 274, 37407; (d) Tomiuk,
S.; Hofmann, K.; Nix, M.; Zumbansen, M.; Stoffel, W.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3638.
7. For recent reviews on signal transduction mediated by the
sphingolipids, see: (a) Hannun, Y. A.; Luberto, C.;
Argraves, K. M. Biochemistry 2001, 40, 4893; (b) Kolter,
T.; Sandhoff, K. Angew. Chem., Int. Ed. 1999, 38, 1532,
and references cited therein.
8. A few SMase inhibitors were reported: (a) Nara, F.;
Tanaka, M.; Hosoya, T.; Suzuki-Konagai, K.; Ogita, T. J.
Antibiol. 1999, 52, 525; (b) Nara, F.; Tanaka, M.;
Matsuda-Inoue, S.; Yamamoto, Y.; Doi-Yoshioka, H.;
Suzuki-Konagai, K.; Ogita, T. J. Antibiol. 1999, 52, 531;
(c) Uchida, R.; Tomoda, H.; Dong, Y.; Omura, S. J.
Antibiol. 1999, 52, 572; (d) Taguchi, M.; Sugimoto, K.;
Goda, K.; Akama, T.; Yamamoto, K.; Suzuki, T.;
Tomishima, Y.; Nishiguchi, M.; Arai, K.; Takahashi,
K.; Kobori, T. Bioorg. Med. Chem. Lett. 2003, 13, 1963;
(e) Taguchi, M.; Goda, K.; Sugimoto, K.; Akama, T.;
Yamamoto, K.; Suzuki, T.; Tomishima, Y.; Nishiguchi,
M.; Arai, K.; Takahashi, K.; Kobori, T. Bioorg. Med.
Chem. Lett. 2003, 13, 3681.
9. (a) Yokomatsu, T.; Murano, T.; Akiyama, T.; Koizumi,
J.; Shibuya, S.; Tsuji, Y.; Soeda, S.; Shimeno, H. Bioorg.
Med. Chem. Lett. 2003, 13, 229; (b) Yokomatsu, T.;
Shibuya, S. J. Synth. Org. Chem. Jpn. 2002, 60, 740; (c)
O’Hagan, D.; Rzepa, H. S. Chem. Commun. 1997, 645; (d)
Thacher, G. R. J.; Campbell, A. S. J. Org. Chem. 1993, 58,
2272; (e) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J.
Chem. Soc., Perkin Trans. 1 1984, 1119, and references
cited therein.
19:5
14. Spectra data of 3. ½aꢁD 17.43 (c 0.53, CHCl3); IR (NaCl
neat) 2930, 2859, 1755, 1514, 1273, 1250, 1177, 1032 cmꢀ1
;
1H NMR (CDCl3, 400 MHz) d 7.22 (md, J = 8.8 Hz, 2H),
6.86 (md, J = 8.8 Hz, 2H), 4.72 (d, J = 15.4 Hz, 1H), 4.45
(m, 1H), 4.18–4.30 (m, 4H), 4.04 (d, J = 15.4 Hz, 1H), 3.97
(dt J = 2.4, 7.8 Hz, 1H), 3.80 (s, 3H), 2.24–2.56 (m, 2H),
1.53–1.65 (m, 3H), 1.38 (t, J = 7.1 Hz, 3H), 1.37 (t,
J = 7.1 Hz, 3H), 1.20–1.35 (m, 9H), 0.87 (t, J = 7.1 Hz,
3H); 13C NMR (CDCl3, 100 MHz) d 159.3, 158.0, 129.5,
127.9, 120.0 (dt, JC–P, JC–F = 215.9, 260.5 Hz), 114.2, 77.5,
64.8, (JC–P = 6.6 Hz), 55.2, 51.6 (m), 45.7, 31.7, 31.5 (dt,
JC–P, JC–F = 14.1, 19.9 Hz), 29.9, 29.2, 29.0, 25.6, 22.6,
16.4 (JC–P = 5.0 Hz), 14.0.
15. Burton, D. J.; Yang, Z.-Y. Tetrahedron 1992, 48, 189.
16. Ishizuka, T.; Kunieda, T. Tetrahedron Lett. 1987, 28,
4185.
17. Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett.
1995, 36, 6373.
18. (a) Evans, D. A.; Britton, T. C.; Ellman, J. A. Tetrahedron
Lett. 1982, 28, 6141; (b) Damon, R. E.; Coppola, G. M.
Tetrahedron Lett. 1990, 31, 2849.
19. When THF was used as a solvent, the desired 20 was
produced in 43% yield, while in DMF only an unidentified
product was produced.
26:0
20. Spectra data of 1 ½aꢁD 6.10 (c 0.32, MeOH); IR (KBr
disk) 3395, 2926, 1649, 1561, 1092, 970 cmꢀ1 1H NMR
;
(CD3OD, 400 MHz) d 4.36–4.44 (m, 2H), 4.21 (ddd,
J = 1.7, 5.4, 10.0 Hz, 1H), 3.61–3.65 (m, 2H), 3.49 (m,
1H), 3.22 (s, 9H), 2.49 (m, 1H), 2.22 (m, 1H), 2.16 (t,
J = 7.6 Hz, 2H), 1.56–1.60 (m, 2H), 1.47–1.53 (m, 2H),
1.23–1.40 (m, 14H), 0.91 (t, J = 6.8 Hz, 3H), 0.90 (t,
J = 6.8 Hz, 3H); 13C NMR (CD3OD, 100 MHz) d 175.5,
124.0 (dt, JC–P, JC–F = 201.0, 260.5 Hz), 75.0, 67.8 (m),
61.2 (JC–P = 5.8 Hz), 54.74, 54.71, 54.68, 49.6 (m), 37.3,
34.5, 34.2 (dt, JC–P, JC–F = 14.1, 19.9 Hz), 33.0, 32.6, 30.7,
30.4, 27.0, 26.7, 23.7, 23.5, 14.4, 14.3.
10. (a) Berkowitz, D. B.; Eggen, M.; Shen, Q.; Sloss, D. G. J.
Org. Chem. 1993, 58, 6174; (b) Berkowitz, D. B.; Shen, Q.;
Maeng, J.-H. Tetrahedron Lett. 1994, 35, 6445; (c)
Berkowitz, D. B.; Sloss, D. G. J. Org. Chem. 1995, 60,
7047; (d) Berkowitz, D. B.; Eggen, M.; Shen, Q.; Shoe-
maker, R. K. J. Org. Chem. 1996, 61, 4666.
11. For recent studies on selective opening of 2,3-epoxyalco-
hol, see: (a) Paquette, L. A.; Kesselmayer, M. A.; Kunzer,
H. J. Org. Chem. 1988, 53, 5185; (b) Schmidt, U.;
21. We tested the ability of CF2 analogue 1 to inhibit SMase
from B. cereus. Enzyme activity was measured three times
at 37 ꢁC and ionic strength 0.2 with a buffer of 50 mM
Tris–HCl buffer (pH 7.5) in the presence of 10 mM MgCl2.
The concentration of SMase and 2-hexadecanoylamino-4-
nitrophenylphosphocholine used as the substrate were
1.0 · 10ꢀ9 M and 1.0 mM, respectively.