Leonard et al.
reaction completion (2.5 h). The reaction was quenched and the
product precipitated by addition of NaOAc‚3H2O (38.3 g, 282
mmol, 1.0 mol/mol triflic acid) in 333 mL of water. The suspension
was warmed 17 °C, maintained for 2 h, and then cooled to 0 °C.
The product was isolated by filtration and washed with 1:9 (v/v)
H2O/CH3CN to give 7 (93.4 g, 94 wt % purity by HPLC vs a pure
standard) in 88% yield: 1H NMR (399.87 MHz, CD3OD) δ 7.57-
7.55 (om, 2H), 7.29-7.24 (om, 3H), 7.13 (m, 2H), 6.74 (m, 2H),
5.55 (d, J ) 2.0, 1H), 5.05 (d, J ) 3.2, 1H)), 4.96 (d, J ) 3.2, 1H),
4.57 (dd, J ) 11.2, 7.2, 1H), 4.52-4.49 (om, 2H), 4.44 (dd, J )
12.9, 4.8, 1H), 4.38-4.34, om, 2H), 4.29 (dd, J ) 8.0, 1.2, 1H),
4.26 (d, J ) 8.0, 1H), 4.22 (d, J ) 2.8, 1H), 4.18 (ddd, J ) 10.0,
5.6, 2.0, 1H), 4.00-3.89 (om, 3H), 3.76-3.66 (om, 2H), 2.76 (dd,
J ) 15.7, 4.0, 1H), 2.45 (dd, J ) 15.7, 9.6, 1H), 2.40 (om, 1H),
2.16-1.95 (om, 6H), 1.83 (m, 1H), 1.54 (m, 2H), 1.49-1.18 (om,
15H), 1.11 (d, J ) 6.4, 3H), 1.10-1.00 (om, 2H), 0.91 (t, J ) 6.8,
1H), 0.86 (t, J ) 7.6, 3H), 0.84, (d, J ) 6.8, 3H), 0.83 (d, J ) 6.4,
3H); 13C NMR (101 MHz, CD3OD) 176.9, 175.7, 174.3, 173.4,
172.45, 172.43, 171.68, 168.9, 158.4, 134.83, 134.78 (2C), 133.1,
129.9 (2C), 129.7 (2C), 128.8, 116.2 (2C), 77.0, 75.8, 74.5, 71.3,
70.6, 70.5, 69.8, 68.2, 62.5, 61.6, 58.6, 57.1, 56.1, 55.7, 51.1, 46.8,
45.9, 39.7, 38.5, 38.1, 36.8, 36.0, 34.7, 32.9, 31.24, 31.16, 30.7,
30.6, 30.4, 30.3, 28.0, 27.1, 20.7, 20.2, 19.5, 11.6; IR (KBr) 3800-
purify it from echinocandin process impurities including the 2-â
diastereomer, a ring-opened byproduct, and several impurities
resulting from fermentation analogues. The same C18 reversed-
phase adsorbent and column that was used for the HPLC
purification of amine 10 was employed in this final purification.
The worked up reaction solution containing 1:5 MeOH/water
was loaded on the column to capture the product 2. The column
was eluted with 22:78 v/v acetonitrile/aqueous acetic acid, and
the fraction containing 2 of desired purity was collected in 90%
recovery.31
Final Salt Formation. On small scale, the HPLC fractions
of suitable quality for crystallization were lyophilized to a
powder. On a larger scale, the HPLC fractions were concentrated
from the acetonitrile/aqueous acetic acid eluent using nanofil-
tration and solvent switched to a mixture of ethanol/water (9:1
v/v) by diafiltration. Acetic acid was added to ensure equiva-
lency, and the solution was subjected to an ultrafiltration to
remove endotoxins.32 Ethyl acetate was added to initiate
crystallization of 2 as the diacetate salt. After a seed bed was
established, additional ethyl acetate was added to complete the
crystallization in 95% recovery. The crystallization was em-
ployed to produce a stable crystalline solid and to slightly reduce
several echinocandin process impurities. Removal of residual
ethanol from the crystalline solid required passing humid
nitrogen through the filter cake. This treatment also controlled
the residual water level and minimized the formation of product
degradates. The final hygroscopic crystalline solid contains 6-8
wt % residual water. The recovery across the column, nanofil-
tration, and crystallization was typically 86% giving an overall
isolated yield of 69-74% for the last step.
2300, 3345, 2925, 2853, 1633, 1516, 1439, 1234, 1086 cm-1
;
HRMS (HPLC/MS ESI-TOF) calcd for C56H84N8O16S 1157.5799
(M + H+), found 1157.5839 (M + H+).
Amine 10. To THF (3 L) were added the phenylthioaminal 7
(75.7 g, corrected for residual H2O as assayed by KF titration, 64.8
mmol) and then phenylboronic acid (8.69 g, 71.3 mmol). The
suspension was brought to reflux and azeotropically dried (to <35
mol % H2O) by passing the refluxate through molecular sieves,
3A (250 mL). The solution was cooled and maintained at 20 °C
where BSTFA (52 mL, 194 mmol) was added and the mixture
stirred for 1 h. The solution was cooled to -5 to 0 °C, and BH3‚
THF (1 M in THF, 195 mL, 195 mmol) was added. The mixture
was maintained at -5 to 0 °C for 2 h. (Caution: Hydrogen gas is
released during the addition, reaction, and workup of BH3‚THF.)
The reaction mixture was quenched with 2 N aq HCl (178 mL)
and stirred for 2.5 h at -5 to 5 °C. HPLC assay showed 85%
conversion and 76% reaction yield of the amine salt (54.7 g).
The reaction mixture was diluted with chilled water (3 L) while
maintaining the solution at <10 °C. Chromatographic purification
was carried out on Kromasil C-18 adsorbent, eluting sequentially
with 10:90 CH3CN/H2O, 29:71 CH3CN/0.014 M aq HCl, and 29:
71 CH3CN/H2O.
Conclusion
The antifungal agent caspofungin acetate (2) was prepared
in three chemical steps from the fermentation product pneu-
mocandin B0 (1) in 45% overall yield. This synthesis demon-
strates that subtle features of a complex substrate reacting with
appropriate reagents and conditions can provide exquisitely
chemo-, regio-, and stereoselective reactions.
Regioselective in situ formation of the cyclic monoboronate
derivative of 1 prior to formation of the phenylthioaminal 7
suppressed the formation of undesired the bis(phenyl sulfide)
8, and the use of TfOH/MeCN led to a highly stereoselective
reaction in 87-91% isolated yield.
A chemoselective borane reduction of the primary amide in
7 was demonstrated, and the conversion was increased by in
situ derivitization with phenylboronic acid and BSTFA giving
an overall yield of 75-80% for amine 10.
Finally, reaction of the diastereomerically pure 10 with
ethylenediamine gave stereoselective substitution on the unac-
tivated phenylthioaminal producing the desired product 2 in 69-
74% yield after chromatographic purification and crystallization.
These reactions combine to form a robust synthesis that is
currently used as the manufacturing process for caspofungin
acetate.
The fractions containing 10 were concentrated by nanofiltration
and diafiltered against MeOH. The MeOH solution contained 10
(35.8 g by HPLC assay) in 99% recovery across the column/
nanofiltration/diafiltration. The MeOH solution was used as is in
the next reaction.
A portion of the solution was evaporated to dryness to obtain
the following spectroscopic data: 1H NMR (399.87 MHz, CD3-
OD) δ 7.58-7.54 (om, 2H), 7.29-7.26 (om, 3H), 7.11 (m, 2H),
6.74 (m, 2H), 5.36 (d, J ) 2.0, 1H), 4.95 (d, J ) 3.2, 1H), 4.90 (d,
J ) 6.0, 1H), 4.61-4.53 (om, 3H), 4.37 (dd, J ) 12.9, 4.8, 1H),
4.30-4.28 (om, 2H), 4.23 (dd, J ) 8.0, 1.6, 1H), 4.20 (d, J ) 4.0,
1H), 4.16 (ddd, J ) 10.0, 5.6, 2.0, 1H), 4.05-3.99 (om, 2H), 3.96
(dd, J ) 11.2, 3.2, 1H), 3.87-3.73 (om, 3H), 3.04 (t, J ) 7.2,
2H), 2.42 (dd, J ) 13.3, 7.2, 1H), 2.15-1.99 (om, 7H), 1.93-1.76
(om, 2H), 1.57-1.19 (om, 17H), 1.14 (d, J ) 6.4, 3H), 1.18-1.03
(om, 2H), 0.92 (t, J ) 7.2, 1H), 0.86 (t, J ) 7.2, 3H), 0.84 (d, J )
6.4, 6H); 13C NMR (101 MHz, CD3OD) 176.3, 174.3, 173.5, 172.6,
172.5, 171.7, 168.8, 158.6, 135.2 (2C), 134.9, 133.0, 130.0 (2C),
129.6 (2C), 129.0, 116.2 (2C), 77.4, 75.5, 74.4, 72.5, 71.4, 70.3,
69.5, 68.3, 62.7, 61.9, 58.3, 57.1, 56.5, 55.9, 51.2, 46.9, 45.9, 39.1,
Experimental Section
Phenylthioaminal 7. To CH3CN (3 L) were added finely divided
pneumocandin B0 (1) (100 g, corrected for residual H2O as assayed
by KF titration, 94.0 mmol), phenylboronic acid (22.9 g, 188 mmol),
and thiophenol (29.0 mL, 282 mmol). The suspension was cooled
and maintained at -15 °C where triflic acid (24.9 mL, 282 mmol)
was added and the reaction maintained until HPLC assay indicated
(31) Oram, P. D.; Seibert, K, Leonard, W.; Dovletoglou, A. J. Liq.
Chromatogr. Relat. Technol. 2001, 24, 781-798.
(32) Grau, J. P.; Tung, H.-H. PCT Patent, WO 2003028858.
2342 J. Org. Chem., Vol. 72, No. 7, 2007