P. Ferraboschi et al. / Tetrahedron Letters 50 (2009) 4384–4388
4387
12.23,24 The presence of this compound is common to other depro-
tection methods, for example, hydrofluoric acid in acetonitrile,4 in
variable elevated amounts.
lective lipase-catalyzed steps, can be useful not only for the
preparation of pimecrolimus but also for the preparation of other
32-substituted derivatives of ascomycin.21,27
Overall yields (29%) of pimecrolimus 1 from ascomycin 2, were
comparable to these observed when we prepared intermediate 4
through the reported bissilylation–monodesilylation process.4
In order to unambiguously ascertain the outcome of lipase-cat-
alyzed transformations a careful high field NMR study, through 1D
and 2D experiments, was performed. This investigation has lead to
the complete assignment of proton resonances of both rotamers of
monoacetates 5,7 and diacetate 7, in the moiety directly involved
in the enzymatic transesterification and alcoholysis.
2.2. NMR study of acetates 5–7
To ascertain the structure of 5–7 a more accurate NMR study25
was done and, through 1D and 2D COSY, HSQC, and HMBC exper-
iments, it was possible to unambiguously establish the acetyl posi-
tions in these compounds. The experiments were carried out in
Pyd5 at 323 K. In fact using these experimental conditions the spec-
tra showed a good spread of the proton resonances and, especially,
of all the methyl groups. Two sets of signals are identified, since the
macrolactam system exists as a mixture of two rotamers in a 60/40
ratio, as in the case of ascomycin 2.12 The study started with 24,32-
diacetate 6 and was focused on the signals of ascomycin moiety
carrying the acetyl groups. As an entry point for the study of 6,
the well resolved characteristic C-25-linked methyl doublet at
1.12 and 1.11 ppm (major and minor rotamers) was selected. Start-
ing from that signal, H-25 (2.18 and 2.42 ppm, major and minor
rotamers), H-24 (5.50 and 5.65 ppm, major and minor rotamers),
and H-26 (5.47 and 5.52 ppm, major and minor rotamers) were as-
signed by COSY. Long range HMBC correlations between H-24 and
a carbonyl 13C resonance (169.5 ppm) and between this carbonyl
and a methyl singlet at 2.05 and 2.07 ppm (major and minor rota-
mers) confirmed the presence of an acetoxy group linked at C-24.
From H-26 it was also possible to assign, by COSY, H-28 (5.40
and 5.51 ppm, major and minor rotamers) because of an allylic
coupling between the two protons and, consequently, H-29
(2.42 ppm, major and minor rotamers), H-30a (1.20, major and
minor rotamers), H30b (2.15 ppm, major and minor rotamers),
H-31 (3.34 ppm, major and minor rotamers), and H-32
(4.97 ppm, major and minor rotamers). The identities of H-31
and H-32 were proved, also in this case, by heteronuclear experi-
ments. In particular, the resonances assigned to H-31 and to a
methoxyl (3.36 and 3.35 ppm singlets, major and minor rotamers)
were shown to be correlated to the same carbon (C-31, 80.5 ppm)
by means of HSQC and HMBC, respectively. Also H-32 was shown
to be linked to a carbon carrying an acetyl group from the presence
of HMBC cross peaks between H-32 and a carbonyl at 169.7 ppm,
and between the same carbonyl and an acetyl methyl singlet reso-
nance at 2.02 ppm (major and minor rotamers).
Acknowledgments
This work was partially financially supported by Università de-
gli Studi di Milano. We thank Dr. Shahrzad Reza-Elahi and Mr Ric-
cardo Monti for technical assistance and Professor Fiamma
Ronchetti for helpful discussions.
References and notes
1. (a) Bornhövd, E. C.; Burgdorf, W. H. C.; Wollenberg, A. Curr. Opin. Invest. Drugs
2002, 3, 708–712; (b) Gupta, A. K.; Chow, M. J. Eur. Acad. Dermatol. 2003, 17,
493–503; (c) Stuetz, A.; Baumann, K.; Grassberger, M.; Wolff, K.; Meingassner,
J. G. Int. Arch. Allergy Immunol. 2006, 141, 199–212; (d) Wollina, U.; Hansel, G.;
Koch, A.; Abdel-Naser, M. B. Expert Opin. Pharmacother. 2006, 7, 1967–1975; (e)
Day, I.; Lin, A. N. J. Cutan. Med. Surg. 2008, 12, 17–26.
2. Hebert, A. A. Clin. Ther. 2006, 28, 1972–1982.
3. Arai, T.; Koyama, Y.; Suenaga, T.; Honda, H. J. Antibiot. Ser. A. 1962, 15, 231–232.
4. Baumann, K.; Emmer, G. E.P. 0, 427, 680, A1, 1991.
5. Baumann, K.; Bacher, M.; Damont, A.; Högenauer, K.; Steck, A. Tetrahedron
2003, 59, 10075–10087.
6. (a) Degueil-Castaing, M.; De Jeso, B.; Drouillard, S.; Maillard, B. Tetrahedron Lett.
1987, 28, 953–954; (b) Wang, Y. F.; Lalonde, J. J.; Momongan, M.; Bergbreiter, D.
E.; Wong, C.-H. J. Am. Chem. Soc. 1988, 110, 7200–7205.
7. 32-O-Acetyl-ascomycin 5. To a solution of ascomycin (0.1 g; 0.126 mmol) in
toluene (8 mL) vinyl acetate (0.473 g, 5.5 mmol) and lipase from Candida
antarctica (CAL B, Novozym 435, FLUKA, 0.140 g, 2 U/mg) were added. The
reaction progress was monitored by TLC (hexane/acetone 65/35). After stirring
at 40 °C for 80 h the enzyme was filtered off and the recovered filtrate was
concentrated under reduced pressure. The residue was crystallized from
acetone/water (0.1 g; 94%). Differential Scanning Calorimetry (DSC)
endothermic peak of fusion 134.25 °C; ½
a 2D0
ꢀ ꢁ74.2 (c 0.5 CHCl3); IR mmax
3484.245, 2935.287, 1735.331, 1649.741, 1450.039, 1372.278 cmꢁ1; MS (ESI+)
(m/z) 856.4 [M+Na]+.
8. Petuch, B. R.; Chen, S.-S. T.; Arison, B. H. E.P. Patent 0, 464, 895, A2, 1992. The
32-acetylation was performed with acetic anhydride in benzene and the yields
were in the range of 20–30%.
9. Tacrolimus is the 21-allyl analogue of ascomycin.
10. 24,32-Di-O-acetyl-ascomycin 6. To
a stirred solution of ascomycin (1 g;
1.26 mmol) in pyridine (12.5 mL), kept at 0 °C, DMAP (0.680 g) and acetic
anhydride (0.570 g, 5.6 mmol) were added. After 1.5 h, under stirring at 0 °C,
the reaction mixture was diluted with water and extracted with ethyl acetate
(3 ꢂ 25 mL); collected organic phases were washed with 0.5 N HCl (5 ꢂ 10 mL),
dried over Na2SO4, and evaporated at reduced pressure. The residue was
crystallized from acetone/water (0.985 g; 90%). A sample was purified for
analytical purposes by silica gel column chromatography (hexane/acetone 8/2
Using the same NMR strategy, the identity of 32- and 24-mono-
acetates 5, and 7 was then confirmed.26
as eluant). DSC endothermic peak of fusion 234.10 °C; ½a D20
ꢁ100.0 (c 0.5
ꢀ
CHCl3); IR mmax 3462.749, 2935.824, 1734.403, 1650.739, 1449.091,
1371.079 cmꢁ1; MS (ESI+) (m/z) 898.4 [M+Na]+.
11. 24-O-Acetyl-ascomycin 7. To
a solution of 24,32-di-O-acetyl-ascomycin, 6
3. Conclusion
(0.5 g; 0.57 mmol) in TBME (25 mL), n-octanol (4.5 equiv; 0.371 g), and lipase
from Candida antarctica (CAL B, Novozym 435, 1.1 g) were added. The reaction
progress was monitored by TLC (hexane/acetone 65/35). After stirring at 40 °C
for 100 h the enzyme was filtered off and the recovered filtrate was
concentrated under reduced pressure. The residue was crystallized from
acetone/water (0.44 g; 93%). A sample was purified for analytical purposes by
column chromatography on silica gel (hexane/acetone 7/3 as eluant). DSC
Through a chemoenzymatic approach the synthesis of pime-
crolimus 1, from ascomycin 2, was realized with satisfactory yields,
considering the very high value of the final product; moreover dur-
ing the regioselective CAL B-catalyzed steps, performed in mild
conditions, only the desired products are obtained in absence of
rearrangements or degradation processes very frequently observed
in the case of sensitive molecules as ascomycin and related com-
pounds, containing a large number of functionalities.5,15–17 The
same sample of immobilized CAL B can be easily recycled for either
transesterification or alcoholysis, without loss of activity. Use of
polymer-bound triphenyl phosphine, for the introduction of
chlorine at position 32, in addition to high yields, allows a simple
recovery of reagent that can be regenerated by treating with tri-
chlorosilane.13 Key intermediate 4, obtained through two regiose-
endothermic peak of fusion 134.68 °C; ½a D20
ꢁ102.7 (c 0.5 CHCl3); IR mmax
ꢀ
3491.528, 2935.860, 1744.728, 1710.227, 1652.310, 1448.662, 1371.335 cmꢁ1
;
MS (ESI+) (m/z) 856.4 [M+Na]+.
12. Or, Y. S.; Clark, R. F.; Xie, Q.; McAlpine, J.; Whittern, D. N.; Henry, R.; Luly, J. R.
Tetrahedron 1993, 49, 8771–8786.
13. Regen, S. L.; Lee, D. P. J. Org. Chem 1975, 40, 1669–1670.
14. 24-O-Acetyl-pimecrolimus 9. DSC endothermic peak of fusion 231.67 °C; ½a D20
ꢀ
ꢁ75.2 (c 0.5 CHCl3); 1H NMR (CDCl3) selected data of major rotamer d (ppm)
2.06 (s, CH3CO), 4.50–4.60 (m, H-2 and H-32), 5.22 (m, H-24). IR
m
max 3464.941,
2934.360, 1738.993, 1650.366, 1450.424, 1371.557 cmꢁ1
; MS (ESI+) (m/z)
874.3 [M+Na]+.
15. Zimmer, R.; Grassberger, M. A.; Baumann, K.; Schulz, G.; Haidl, E. Tetrahedron
1994, 50, 13655–13670.