Expedient and practical synthesis of CERT-dependent ceramide trafficking inhibitor HPA-12 and its analogues

Article abstract of DOI:10.1021/ol2001057

The practical stereodivergent route to both syn- and anti-diastereomers of 1-substituted 3-aminobutane-1,4-diols based on the crystallization-induced asymmetric transformation (CIAT) approach was completed. This led to the revision of the reported stereoc

Full text of DOI:10.1021/ol2001057

ORGANIC
LETTERS
2011
Vol. 13, No. 7
1642–1645
Expedient and Practical Synthesis of
CERT-Dependent Ceramide Trafficking
Inhibitor HPA-12 and Its Analogues
†
Andrej Duris, Tomas Wiesenganger, Daniela Moravcıkova, Peter Baran,
‡
ꢀ †
ꢁꢀ
Jozef Kozısek, Adam Daıch, and Dusan Berkes*
ꢀ
´
ꢁ †
ꢀ
§
,†
ꢀ ꢀ
´
ꢀ
ꢀ
¨
†
ꢁ
Department of Organic Chemistry, Slovak University of Technology, Radlinskeho 9,
81237 Bratislava, Slovakia, ‡Department of Chemistry, Juniata College, 1700 Moore
Street, Huntingdon, Pennsylvania 16652, United States, §Department of Physical
ꢁ
Chemistry, Slovak University of Technology, Radlinskeho 9, 81237 Bratislava, Slovakia,
and URCOM, EA 3221, INC3M CNRS FR-3038, UFR Sciences & Techniques,
University of Le Havre, 25 rue Philippe Lebon, B.P. 540, F-76058 Le Havre Cedex,
France
dusan.berkes@stuba.sk
Received January 13, 2011
ABSTRACT
The practical stereodivergent route to both syn- and anti-diastereomers of 1-substituted 3-aminobutane-1,4-diols based on the crystallization-
induced asymmetric transformation (CIAT) approach was completed. This led to the revision of the reported stereochemistry of the first inhibitor
of CERT-dependent ceramide trafficking HPA-12 from (R,R)-anti- to the (R,S)-syn-enantiomer. Due to the expeditiousness of production and
inexpensive conditions developed, a series of alkyl- and aryl-substituted analogues of HPA-12 is also reported.
Sphingolipids are ubiquitous components of eukaryotic
cell membranes where they play very pivotal roles in
intracellular signaling and in membrane structure. Even
though the biochemical pathway of sphingolipid synthesis
and its compartmentalization between the endoplasmic
reticulum (ER) and Golgi apparatus have been known for
many years, the transport mechanistic paradigms in this
pathway have only recently been elucidated. One of the
most exciting developments in this field over the past few
years was the identification of a protein, CERT,1 a cyto-
solic 68 kDa protein, which mediates the transport of
ceramide from the ER to the Golgi apparatus in a non-
vesicular manner. Sphingolipid misregulation contri-
butes to the development of variety of disease states.
CERT works as one of the mediators of sphingolipids
homeostasis2 and was found likely to be relevant to Good-
pasture autoimmune disease.1b
(1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenyl-
propyl)dodecamide (commonly known as HPA-12) has
been used to determine the role of CERT in sphingolipids
biosynthesis. It was identified as the first specific inhibitor
for sphingomyelin (SM) synthesis in mammalian cells and
a potential drug that inhibits the CERT-dependent path-
way of ceramide trafficking in intact cells. As was further
demonstrated, the (1R,3R)-HPA-12 isomer, but not other
stereoisomers, inhibits the CERT-mediated intermem-
brane transfer of ceramide in a cell-free assay system,
(1) (a) Hanada, K. Proc. Jpn. Acad., Ser. B 2010, 86, 426–437. (b)
Hanada, K.; Kumagai, K.; Tomishige, N.; Yamaji, T. Biochim. Biophys.
Acta 2009, 1791, 684–691. (c) Perry, R. J.; Ridgway, N. D. Biochim.
Biophys. Acta 2005, 1734, 220–234. (d) Kumagai, K.; Yasuda, S.;
Okemoto, K.; Nishijima, M.; Kobayashi, S.; Hanada, K. J. Biol. Chem.
2005, 280, 6488–6495. (e) Hanada, K.; Kumagai, K.; Yasuda, S.; Miura,
Y.; Kawano, M.; Fukasawa, M.; Nishijima, M. Nature 2003, 426, 803–
809.
(2) (a) Breslow, D. K.; Weissman, J. S. Mol. Cell 2010, 40, 267–279.
(b) Lev, S. Nature Rev. Mol. Cell Biol. 2010, 11, 739–750.
r
10.1021/ol2001057
Published on Web 02/25/2011
2011 American Chemical Society

indicating this diastereomer to be a direct antagonist of
CERT.1d,3 The crystal structures of the CERT START
domain, in complex with (1R,3R)-HPAs of varying acyl
chain lengths outlining the more significant role of stereo-
chemistry of the inhibitors, have been published recently.4
its aryl-substituted analogues, and also the (1R,3S)-HPA-
12 together with some 1-alkyl-substituted analogues of the
same stereochemistry.
Our strategy is based on the use of an expedient
straightforward transformation of enantiomerically and
diastereomerically pure oxoamino acids easily accessible
via the CIAT process from available substrates (Scheme 1).11
The application of 1-phenylethylamine (PEA) as a chiral
mediator in such a transformation is especially favorable
since both (R)- and (S)-enantiomers of PEA are commer-
cially available, with both antipodes of the final compounds
attainable. The required (1R,3R)-anti-relationship of 3-ami-
no-1-phenylbutane-1,4-diols was accomplished using (R)-
PEA and another CIAT process in tandem with reversible
acid-catalyzed lactonization.11a
Scheme 1. Crystallization-Induced Asymmetric Transforma-
tions (CIAT) Approaches to the Starting Oxoamino Acid
Derivatives
Scheme 2. Expedient Approach to the cis-Butanolide 2a
The oxoamino acids and their derivatives are now
recognized as the crucial intermediates for the HPA synthe-
sis, although other approaches have been presented.5 The
first synthesis of HPA-12 was published in 2001 by the
Kobayashi group.6 The asymmetric Mannich protocol
was improved by the same authors in the following years.7
While the C-N bond was introduced with high selectivity,
the diastereoselectivity of C-OH bond formation was
modest. For the last step of the synthesis, anti-stereoselec-
tive reduction of γ-oxoamino acid derivatives, both K-Se-
lectride and LiBEt3H have been used to give the expected
aminodiol in 9/91 (syn/anti) selectivity. Interestingly, the
selectivity dropped to 40/60 with traces of moisture in the
reaction media.8 Other strategies for the anti-1,3-amino
alcohol moiety in the same or related aminodiols provide
low stereoselectivity as well.9 Elsewhere, the stereoselective
synthesis of the anti-1,3-amino alcohol moiety via the
improved oxazolidine route in the racemic aminobutane-
diols was published only recently.10 In this paper, we
introduce viable scalable synthesis of (1R,3R)-HPA-12,
Sincein our previouspaper we describedthe preparation
of the trans-lactone 20f,12 we are now investigating mod-
ified conditions to reach the required (2R,5R)-derivative
(2a, Scheme 2). We are pleased to notice that a simple
raising of the reaction temperature from 25 up to 80 °C
under otherwise unchanged conditions (8 M HCl, 4 h) is
sufficient to achieve the transformation of trans-lactone 20f
to the thermodynamically more stable cis-butanolide 2a in
84% yield. Finally, optimal conditions for the required cis-
lactone 2a were realized in two steps-a one-pot procedure
starting from oxoamino acid 1a using a nonstereoselective
reduction with sodium borohydride (dr 2/1) followed by
acid-catalyzed lactonization under the formerly described
conditions.11a Under these conditions, the lactonization
occurred smoothly and the desired product 2a started to
seed within 30 min of the reaction.
(3) Yasuda, S.; Kitagawa, H.; Ueno, M.; Ishitani, H.; Fukasawa, M.;
Nishijima, M.; Kobayashi, S.; Hanada, K. J. Biol. Chem. 2001, 276,
43994–44002.
(4) Kudo, N.; Kumagai, K.; Matsubara, R.; Kobayashi, S.; Hanada,
K.; Wakatsuki, S.; Kato, R. J. Mol. Biol. 2010, 396, 245–251.
(5) Raghavan, S.; Rajender, A. Tetrahedron 2004, 60, 5059–5067.
(6) Ueno, M.; Kitagawa, H.; Ishitani, H.; Yasuda, S.; Hanada, K.;
Kobayashi, S. Tetrahedron Lett. 2001, 42, 7863–7865.
(7) (a) Kobayashi, S.; Matsubara, R.; Nakamura, Y.; Kitagawa, H.;
Sugiura, M. J. Am. Chem. Soc. 2003, 125, 2507–2515. (b) Manabe, K.;
Kobayashi, S. Chem.;Eur. J. 2002, 8, 4095–4101. (c) Hamada, T.;
Manabe, K.; Kobayashi, S. Chem.;Eur. J. 2006, 12, 1205–1215.
(8) Nakamura, Y.; Matsubara, R.; Kitagawa, H.; Kobayashi, S.;
Kumagai, K.; Yasuda, S.; Hanada, K. J. Med. Chem. 2003, 46, 3688–
3695.
(9) (a) Barfoot, C. W.; Harvey, J. E.; Kenworthy, M. N.; Kilburn,
J. P.; Ahmed, M.; Taylor, R. J. K. Tetrahedron 2005, 61, 3403–3417. (b)
Jorda-Gregori, J. M.; Gonzalez, R.; Cava-Montesinos, P.; Sepulveda-
Arques, J.; Galeazzi, R.; Orena, M. Tetrahedron: Asymmetry 2000, 11,
3769–3777. (c) Gonzalez-Rosende, M.; Jordagregori, J.; Sepulveda-arques,
J.; Orena, M. Tetrahedron: Asymmetry 2004, 15, 419–422. (d) Yan, G.; Wu,
Y.; Lin, W.; Zhang, X. Tetrahedron: Asymmetry 2007, 18, 2643–2648.
(10) (a) Partridge, K. M.; Guzei, I. A.; Yoon, T. P. Angew. Chem., Int.
Ed. 2010, 122, 942–946. (b) Morita, N.; Fukui, K.; Irikuchi, J.; Sato, H.;
Takano, Y.; Okamoto, I.; Ishibashi, H.; Tamura, O. J. Org. Chem. 2008,
73, 7164–7174.
ꢀ
ꢀ
(11) (a) Berkes, D.; Kolarovic, A.; Manduch, R.; Baran, P.;
ꢀ
Povazanec, F. Tetrahedron: Asymmetry 2005, 16, 1927–1934. (b)
ꢀ
ꢀ
ꢀ
Jakubec, P.; Berkes, D.; Kolarovic, A.; Povazanec, F. Synthesis
ꢀ
ꢀ
ꢀ
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2006, 4032–4040. (c) Berkes, D.; Korenova, A.; Safar, P.; Horvathova,
ꢁ
ꢁ
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H.; Pronayova, N. Cent. Eur. J. Chem. 2007, 5, 688–705. (d) Berkes,
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D.; Jakubec, P.; Winklerova, D.; Povazanec, F.; Daıch, A. Org.
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¨
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Biomol. Chem. 2007, 5, 121–124. (e) Jakubec, P.; Petras, P.; Duris,
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A.; Berkes, D. Tetrahedron: Asymmetry 2010, 21, 69–74.
(12) Berkes, D.; Kolarovic, A.; Povazanec, F. Tetrahedron Lett. 2000,
41, 5257–5260.
ꢀ
ꢀ
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Org. Lett., Vol. 13, No. 7, 2011
1643

Scheme 3. Synthesis of Inhibitor of CERT-Dependent Ceramide Trafficking HPA-12 and its Alkyl and Aryl Analogues
Having in hand a series of the required cis-lactones
expanded by the selected trans-butanolides (20 f 2)11b
with tethered alkyl substituents, the simple and straight-
forward three-step synthesis of HPA-12 and their ana-
logues was then achieved very easily (Scheme 3).
(20-48 h), and furthermore, a high extent of epimeriza-
tion to another diastereomer in the arylsubstituted series
was observed (anti/syn ≈ 90/10 for products 4a-e; syn/
anti ≈ 80/20 for product 40f). Interestingly, the addition
of 2 equiv of acetic acid shortened the reaction time (only
3-4 h are necessary for 4a-e instead of 24 h for 40f), and
it also improved the diastereomeric purity of the product
(dr >95/5).
As shown in Scheme 3, the ultimate chemoselective
N-acylation of the diamino diols 4 and their epimers 40
was accomplished under neutral conditions with suc-
cinimide dodecanoate at room temperature. After 1-2
days of the reaction, the resulting acylated products
5a-d and 50f-h were purified by silica gel column
chromatography.
To distinguish which stereogenic center is unstable
under debenzylation conditions, a mixture of syn/anti
diastereomers obtained from N-substituted amino diol 3a
and 30f was chromatographically separated to their pure
stereoisomers. After their N-acylation, the optical rotation
of final derivatives was compared with the literature data.
The major diastereomer (from the syn-amino diol product
30f) provided the product 50f with [R]D = -34, while the
minor epimer resulted in the formation of the derivative 5a
with [R]D = þ10. In an analogous manner, the N-acylated
product with [R]D = þ10 was isolated from the anti-
diastereoisomer 3a and the isolated minor diastereomer
(dr =95/5) showed [R]D = -33.
Moreover, the dextrorotatory nature of the major
diastereomer was also confirmed in the series of aro-
matic anti-(1R,3R)-analogues 4b-e and in the aliphatic
syn-aminodiols 40g,h; the levorotatory nature of the
major diastereomer was in agreement with the deriva-
tive 40f.
Figure 1. Molecular structure of product 3d showing the atom-
labeling scheme and displacement ellipsoids at the 30% prob-
ability level. For clarity, only one of the two symmetry-independent
molecules is shown.
Along this line, the reduction of lactones 2 and 20
proceeds with high yield and fortunately without erosion
of stereochemistry (Scheme 3). The relative configura-
tion of the N-substituted aminobutanediols 3 as well as
30 was secured without ambiguity on the basis of the
results of X-ray crystallographic analysis of product 3d
as a crystalline hemihydrate (Figure 1).13 The further
chemoselective catalytic N-debenzylation was accom-
plished under standard conditions using Pd(OH)2 in
methanol. However, an unusually long reaction time
was required for full conversion of starting material
However, the published [R]D value for HPA-12 in
the literature was -36.3 (c 0.505, CHCl3)8 and -35.1
(c 0.8, CHCl3),6 respectively, and was in stark contrast
to those measured for the synthesized (1R,3R)-5a which
(13) The crystallographic coordinates have been deposited with
the Cambridge Crystallographic Data Centre; deposition nos.
CCDC 806264 for 3d (Figure 1) and 805528 for 4c (Figure 2). These
data can be obtained via https://www.ccdc.cam.ac.uk/services/
structure_deposit/.
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Org. Lett., Vol. 13, No. 7, 2011

Table 1. Key 1H and 13C NMR Data for Comparisons (δ in ppm)a
(1R,3R) HPA-128
(1R,3S) HPA-1214
(1R,3R) 5a
(1R,3S) 50f
N-H
H-1
CdO
C-1
6.41 (d,J = 6.5 Hz)
6.27 (d,J = 8.3 Hz)
6.31 (d,J = 8.3 Hz)
6.40 (d,J = 6.3 Hz)
4.81 (dd, J = 3.4,8.3 Hz)
4.67 (brd, J = 10.5 Hz)
4.66 (dd, J = 2.7,10.3 Hz)
4.82 (dd, J = 3.4,8.8 Hz)
174.3
174.9
175.0
174.3
71.8
50.4
70.4
48.7
70.4
48.8
72.2
50.7
C-3
a All spectra were recorded in CDCl3.
is [R]D =þ10.5 (c 0.44, CHCl3). This value, however, is
in agreement with that obtained for (1S,3R)-50f, which is
[R]D = -34.4 (c 0.36, CHCl3), and therefore, a revision of
the previously proposed structure seems to be necessary.
In order to clarify this issue, we were looking for
independent facts to resolve the observed discrepancy.
We were delighted to observe that also the spectroscopic
data for product 5a closely matched those of (1S,3R)-
HPA-12 diastereomer14 and vice versa, while the measured
product 50f data nearly matched the literature data of
(1R,3R)-HPA-128 (see Table 1).
favorable conformations afforded similar coupling con-
stants for both H2R and H2β protons in the N-substituted
and unsubstituted amino diols enantiopure 3a-e and
4a-e, respectively. Finally, since the similarities in the
1H NMR spectral data are evident in the both series of
anti-amino diols (products 3a-e and 4a-e; see the
Supporting Information for more details), the anti-re-
lationship established with X-ray analysis could be pre-
dicated also for the phenyl derivatives 3a and 4a.
Thus, we suspect that the stereochemical assignment of
the HPA-12 structure based on the original published data
of Ueno et al.6 might be incorrect, and we thus recommend
a revision of the HPA-12 stereochemistry from the
(1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)-
dodecamide (5a) to the (1R,3S)-50f diastereomer.
The absolute configuration of HPAs is crucial for their
biological activities with respect to the CERT protein. In their
recent crystallographic study, Kudo et al.4 invoked interac-
tions of the CERT START domain with three out of four
possible stereoisomers of HPAs, and on the basis of their
previous study16 they favored (1R,3R)-HPA over (1R,3S)-
HPA. Our findings herein demonstrate that they have been
indeed using (1R,3S)-HPA. As a consequence, revision of
some other stereochemical transformations can be expected.5
Insummary, the demonstrated expeditious practicaland
inexpensive approach represents a short stereodivergent
elegant route to both diastereomers of 1-aryl-3-aminobu-
tane-1,4-diols (4a-e and 40f), which opens the door to the
multigram synthesis of the first inhibitor of CERT-depen-
dent ceramide trafficking and its analogues.
Figure 2. Molecular structure of 4c AcOH showing atom labeling
3
scheme and displacement ellipsoids at the 30% probability level.
The hydroxy group (O2-H) is disordered over two positions.
Only a dominant position with an occupation factor of 0.71 is
shown. The corresponding hydroxy group hydrogen atom could
not be located because of the disorder.
Finally we succeeded in the preparation of suitable
crystals for crystallographic analysis from the derivate
Acknowledgment. Financial aid by the Slovak grant
agency 1/0629/08 and NMR measurements provided by
the Slovak state program project No.2003SP200280203
are gratefully acknowledged. We are grateful also to Dr.
RaphaelG. Raptis, from the Universityof Puerto Rico, for
providing access to the X-ray diffractometer.
4c. Thus, the X-ray analysis of 4c AcOH15 confirmed
the anti-relationship between both stereogenic centers.
3
1
Furthermore, from an H NMR standpoint, the large
coupling constants between H2 atoms in antiperiplanar
relationships were evidenced in spectra of N-substituted
syn-amino diols 30f and 30g recorded in CDCl3. Inaddition,
in nonprotic solvent, a strong intramolecular hydrogen bond
could be anticipated due to stabilizing effects of the R1 group
as large substituents in equatorial positions. In the anti
derivatives, such stabilizing effects do not exist, and the
Supporting Information Available. Experimental proce-
dures and characteristics of all new compounds. 1H and 13
C
NMR spectra for all novel compounds. This material is
available free of charge via the Internet at http://pubs.acs.org
(14) Kobayashi, S.; Hamada, T.; Manabe, K. J. Am. Chem. Soc.
2002, 124, 5640–5641.
(15) See ref 12 for CCDC deposition number for product 4c.
(16) Kudo, N.; Kumagai, K.; Tomishige, N.; Yamaji, T.; Wakatsuki,
S.; Nishijima, M.; Hanada, K.; Kato, R. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 488–493.
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