First scale-up synthesis of FU-23, a novel water-soluble pleuromutilin-derived antibiotic

Article abstract of DOI:10.1021/op100063h

A first scale-up synthesis of FU-23 (1), a potent and effective water-soluble pleuromutilin-derived antibiotic, is described. The original synthesis from the medicinal chemistry group provided 1 in seven steps and 10.9% overall yield, required four chroma

Full text of DOI:10.1021/op100063h

Organic Process Research & Development 2010, 14, 815–819
First Scale-Up Synthesis of FU-23, a Novel Water-Soluble Pleuromutilin-Derived
Antibiotic
Liqiang Fu, Xin Liu, Jianjun Cheng, Huili He, Zhan Cai, Xingsheng Guo, Shi Ding, and Yushe Yang*
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, P.R. China
Abstract:
A first scale-up synthesis of FU-23 (1), a potent and effective water-
soluble pleuromutilin-derived antibiotic, is described. The original
synthesis from the medicinal chemistry group provided 1 in seven
steps and 10.9% overall yield, required four chromatographies
and employed expensive reagents such as AgOCN and 4-acetyl-
salicoyl chloride. The optimized synthetic route for the preparation
of phosphate salt 1 consists of seven linear steps with a 42.8%
overall yield. Significant cost reduction and more robust reaction
conditions have been developed with no chromatography required
at any stage.
Figure 1. Chemical structure of FU-23 (1).
Results and Discussion
The initial medicinal chemistry approach to the synthesis
of the title compound 1 is depicted in Scheme 1, which provided
1 in 10.9% overall yield. The key intermediate 7 was prepared
successfully, following the method reported by Jeremy D.
Hinks.8 The synthesis of 7 started with commercially available
pleuromutilin 2, which was treated with trimethyl orthoformate
and concd H2SO4 in methanol at 35 °C to give 3, followed by
the subsequent hydrolysis to afford 4. AgOCN was suspended
in a solution of 4-acetoxybenzoyl chloride 5 in dry dichlo-
romethane under an atmosphere of argon and stirred at reflux
for 4 h; then, 4 was added at room temperature for 1 h to give
compound 6, which was obtained in high yield after chromato-
graphic isolation. Then, 6 in dioxane was treated with a saturated
solution of zinc chloride in concd HCl to afford the intermediate
7 in 27% yield after chromatographic purification.8 The regio-
selective phosphorylation of the phenolic hydroxyl group of
Introduction
Owing to the rapid emergence of multidrug-resistant Gram-
positive bacteria, there is an urgent need to identify and develop
new antibacterial agents with novel mechanisms of action
against drug-resistant bacterial strains. Pleuromutilin was first
isolated in 1951 from basidiomycetes Pleurotus and P. passeck-
erianus and displayed modest activity against Gram-positive
organisms in Vitro.1 Further studies have shown that this class
of antibiotics interferes with bacterial protein synthesis via a
specific interaction with the 23S rRNA of the 50S bacterial
ribosome subunit.2 These compounds display a distinct anti-
bacterial profile and show no cross-resistance with any other
class of antibiotics.3,4 In 2007, GlaxoSmithKline’s novel pleu-
romutilin analogue retapamulin, with excellent activity in Vitro,
was first approved for human use as a topical antimicrobial
agent to treat skin infections.5,6 The emergence of FU-23 (1)
as a promising water-soluble pleuromutilin-derived preclinical
candidate within our lab discovery program led us to seek a
practical and scalable route to synthesize mutigram quantities
required for regulatory toxicology studies and preclinical
evaluation.7 Herein is described the first scale-up synthesis of
FU-23 (1).
7
9,10 and hydrogenolysis of the benzyl and vinyl moieties of 8
provided the free acid of 1. Methanol and water were then added
to the free acid of 1, and the pH was adjusted to 9 by addition
of 10% Na2CO3 solution. The salt was purified by chromatog-
raphy (C18), eluting with a gradient of 0-100% methanol in
water to give 1 in about 92% yield for two steps.7
As noted above, the original synthesis was judged not
suitable for scale-up, as it involved several chromatographic
purifications and provided very low overall yield (10.9%).
Moreover, it also required the use of 4-acetoxybenzoyl chloride
and AgOCN which are expensive and have processing issues.
Alternative Approaches to 1. We first looked at the
possibility of a more efficient process for the synthesis of 1.
As a result, the alternative approach shown in Scheme 2 was
implemented.
* Author for correspondence. Telephone: (86-21) 50806786. Fax: (86-21)
50806786. E-mail: ysyang@mail.shcnc.ac.cn.
(1) Kavanagh, F.; Hervey, A.; Robbins, W. J. Proc. Natl. Acad. Sci. U.S.A.
1951, 37, 570.
(2) (a) Long, K. S.; Hansen, L. H.; Jakobsen, L.; Vester, B. Antimicrob.
Agents Chemother. 2006, 50, 1458. (b) Davidovich, C.; Bashan, A.;
Auerbach, N. T.; Yaggie, R. D.; Gontarek, R. R.; Yonath, A. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 429.
(8) (a) Hinks, J. D.; Takle, A. K.; Hunt, E. Chem. Abstr. 1997, 127,
161997. Hinks, J. D.; Takle, A. K.; Hunt, E. WO/97/25309, 1997. (b)
Brooks, G.; Burgess, W.; Colthurst, D.; Hinks, J. D. Bioorg. Med.
Chem. 2001, 9, 1221.
(3) Fukuda, Y. Drug Future 2009, 34, 127.
(4) Phillips, O. A.; Sharaf, L. H. Expert Opin. Ther. Pat. 2007, 17, 429.
(5) Parish, L. C.; Parish, J. L. Drugs Today. 2008, 44, 91.
(6) Daum, R. S.; Kar, S.; Kirkpatrick, P. Nat. ReV. Drug DiscoVery 2007,
6, 865.
(9) Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron Lett. 1996,
37, 771.
(7) Fu, L. Q.; Jiang, Z.; Cai, Z.; Liu, X.; He, H.; Yang, Y. S. Bioorg.
Med. Chem. Lett. 2009, 19, 5407.
(10) Silverberg, L. J.; Dillon, J. L.; Vemishetti, P.; Sleezer, P. D.; Discordia,
R. P.; Hartung, K. B. Org. Process Res. DeV. 2000, 4, 34.
10.1021/op100063h  2010 American Chemical Society
Published on Web 05/12/2010
Vol. 14, No. 4, 2010 / Organic Process Research & Development
•
815

Scheme 1. Medicinal chemistry synthesis of compound 1a
a Reagents and conditions: (a) (CH3O)3CH, MeOH, H2SO4, 75%; (b) NaOH, MeOH, H2O, chromatography, 90%; (c) CH2Cl2, AgOCN (4.6 equiv), chromatography,
80%; (d) dioxane, concd HCl, ZnCl2, 27%; (e) (BnO)2POH, DIPEA, DMAP, CCl4, CH3CN, chromatography, 82%; (f) 10%Pd/C, MeOH; (g) Na2CO3, 92% for 2
steps.
As reported by Joseph Sisko,11 there were clear benefits in
the conversion of 2 to 4 without isolation of 3 with good overall
yields. However, intermediate 4 was difficult to crystallize,
probably owing to impurities derived from fermentation product
2. Despite this, we refocused our efforts on the purification of
intermediate 4 by recrystallization and found that 4 could be
crystallized in petroleum ether at 0 °C. After a wash with cold
petroleum ether, compound 4 was isolated as a fine crystalline
solid in high purity (98%, HPLC). This method avoids the need
for isolating the intermediate 3 and offers advantages in terms
of practicality.
To avoid 4-acetoxybenzoyl chloride and AgOCN owing to
cost and processing issues, we turned our attention to 4-ben-
zyloxybenzamide 9 as an alternative starting material. This
compound is readily available in bulk quantities.12 4-Benzy-
loxybenzoyl isocyanate 10 was prepared by treating amide 9
with oxalyl chloride in dichloromethane at 50 °C in almost
quantitative yield.13 Without any isolation, slow addition of 4
in dichloromethane to the solution of 10 generated intermediate
11 in high yield (92%) and high purity (>98%).
concd HCl without zinc chloride was used, a longer reaction
time was needed, and the reaction stalled at 90-95% conver-
sion. When a temperature greater than 30 °C was employed,
more than 10% of impurity formation was observed, and further
recrystallization from a variety of solvents did not remove the
impurity. Finally, we found that a half-saturated solution of zinc
chloride in concd HCl (250 g ZnCl2 in 500 mL concd HCl) at
room temperature effected the conversion of 11 to 12 in good
yield with low levels of impurity formation (Table 1). Further-
more, under these reaction conditions the poor solubility of 12
could be used advantageously in the isolation. In fact 12 was
obtained directly from the reaction mixture by simple addition
of a 1/3 equivalent volume of water followed by filtration and
washing with water and ethanol, giving 12 as a colourless solid
in high yield (93.4%) and high purity (>99.0%). Hydrogenation
of the double bond and debenzylation to deprotect the hydroxy
group could be done simultaneously to afford alcohol 13 in
near quantitative yield.
The regioselective phosphorylation of the phenolic hydroxyl
group of 13 at the C-14 side chain without protecting the C-11
skeleton hydroxyl group relied on the higher acidity of the
phenolic proton and the steric hindrance of the C-11 hydroxyl
group.9 Treatment of 13 with N,N-dimethylaminopyridine
(DMAP), carbon tetrachloride, N,N-diisopropylethylamine
(DIPEA) and dibenzyl phosphite (BnO)2P(O)H successfully
produced the phosphoric acid dibenzyl ester compound 14 in
high yield and high purity after recrystallization from hexane
and ethyl acetate. Various conditions were tested before the
suitable reaction conditions were found. The results from the
screening studies are shown in Table 2. A critical parameter in
the reaction is to control the reaction temperature between -20
and -25 °C (entries 5-9, Table 2). At this low temperature,
impurity formation was greatly reduced. The number of
equivalents of (BnO)2P(O)H is also very important. The reaction
was best run using 1.15 equiv of (BnO)2P(O)H at -25 °C, under
which conditions the byproducts were minimised (entry 7,
Table 2).
The skeletal rearrangement of intermediate 11 to generate
12 proved to be more difficult than expected. When a solution
of 11 in dioxane was treated with a saturated solution of zinc
chloride in concd HCl, up to 10% of impurities formation was
observed, and the major one is amide 9 as identified by HPLC
and NMR. We speculated that the presence of zinc chloride
accelerated the decomposition, perhaps owing to the high
concentration of nucleophilic chloride ion. However, when
(11) Andemichael, Y.; Chen, J.; Clawson, J. S.; Dai, W.; Diederich, A.;
Downing, S. V.; Freyer, A. J.; Liu, P.; Oh, L. M.; Patience, D. B.;
Sharpe, S.; Sisko, J.; Tsui, J.; Vogt, F. G.; Wang, J.; Wernersbach,
L.; Webb, E. C.; Wertman, J.; Zhou, L. Org. Process Res. DeV. 2009,
13, 729.
(12) Compound 9 was readily prepared in high yield from commercially
available 4-benzyloxybenzoic acid by sequential treatment with oxalyl
chloride and concentrated aqueous ammonia.
(13) (a) McGrew, L. A.; Sweeny, W.; Campbell, T. W.; Foldi, V. S. J.
Org. Chem. 1964, 29, 3002. (b) Anchoori, R. K.; Kortenhorst, M. S.;
Hidalgo, M.; Sarkar, T.; Hallur, G.; Bai, R.; Van Diest, P. J.; Hamel,
E.; Khan, S. R. J. Med. Chem. 2008, 51, 5953.
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Vol. 14, No. 4, 2010 / Organic Process Research & Development

Scheme 2. First scale-up synthesis of compound 1a
a Reagents and conditions: (a) (CH3O)3CH, MeOH, H2SO4, 35 °C; (b) NaOH, MeOH, H2O, 65 °C, 81% for 2 steps; (c) ClCOCOCl, CH2Cl2, rt to 50 °C; (d) CH2Cl2,
rt, 92% for 2 steps; (e) dioxane, concd HCl, ZnCl2, 25 °C, 93%; (f) H2, 10% Pd/C, THF, rt, 95%; (g) (BnO)2P(O)H, DIPEA, DMAP, CCl4, CH3CN, -25 °C, 82%;
(h) H2, 5% Pd/C, THF, rt, 96%; (i) H2O, MeOH, Na2CO3, i-PrOH, 82%.
Table 1. Optimization of skeletal rearrangement conditions
reaction
mixture
composition (%)
other impurities
entry
reaction conditions
12
impurity 9
yield (%)
1
2
3
4
concd HCl, 10 h, 35 °C
85.0
86.2
93.0
96.5
10.0
8.0
4.3
2.0
5.0
5.8
2.7
1.5
73.9
81.5
86.8
93.4
saturated ZnCl2-concd HCl, 2.5 h, 30 °C
half-saturated ZnCl2-concd HCl, 3 h, 30 °C
half-saturated ZnCl2-concd HCl, 5 h, 25 °C
Table 2. Effects of (BnO)2P(O)H and temperature on
screened various solvent systems such as methanol, methanol/
THF, methanol/ethyl acetate, and THF for the hydrogenolysis
of 14. In methanol the rate of reaction was fast; however, the
purity of product 15 was less than 98.0%. Ultimately, it was
determined that the preferred reaction solvent was THF, and
further recrystallization of 15 from water and CH3CN gave a
purity over 99.2%.
For salt formation, 15 was dissolved in methanol and water,
and a solution of Na2CO3 (1.05 equiv) was added. After a small
quantity of insoluble material was removed by filtration, the
filtrate was concentrated under reduced pressure, and the residue
was recrystallized from methanol/isopropanol to give 1 in 82%
yield with >99.5% purity and no single impurity >0.1%.
formation of phosphate 14
reaction condition
(BnO)2P(O)H impurity yield puritya
entry T (°C)
(equiv)
(%)
(%)
(%)
1
2
3
4
5
6
7
8
9
-5
1.5
15.0
13.2
10.3
9.1
55.2
66.1
72.8
75.0
75.1
77.5
82.0
76.5
73.6
90.0
93.0
93.8
95.0
97.1
98.0
98.8
98.0
96.5
-10
-15
-20
-20
-25
-25
-25
-40
1.5
1.5
1.5
1.2
8.0
1.2
6.0
1.15
1.10
1.15
4.2
7.8
10.6b
a The single recrystallization purity by HPLC. b Including unreacted starting
material 13 (4.0%).
Conclusion
In conclusion, we have developed an improved and more
efficient process for the synthesis of 1, which offers distinctive
advantages over the published procedures.7,8 The present
Hydrogenolysis of the benzyl moieties of 14 smoothly gave
15 in excellent yield. As a part of our optimization study, we
Vol. 14, No. 4, 2010 / Organic Process Research & Development
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817

synthesis is free of column chromatography in all stages and
greatly improves the overall yield from 10.9% to 42.8%. This
process is amenable for the large-scale laboratory production
of FU-23 (1).
these manipulations. Heating was discontinued after 2 h, and
the mixture allowed to cool to room temperature. 4 (267.5 g,
0.8 mol) dissolved in dichloromethane (500 mL) was added
dropwise to the above mixture, resulting in a mild exotherm.
The mixture was stirred for 2 h at room temperature before
quenching with saturated brine (600 mL). The aqueous layer
was washed with dichloromethane (500 mL), and the combined
organic extracts were dried over anhydrous Na2SO4. Dichlo-
romethane was removed by vacuum distillation to a final
volume of 300 mL. Acetonitrile (2.0 L) was added, and the
resulting solution was heated to 60 °C for 1 h, and then the
mixture was cooled to 25 °C. The white precipitated crystals
were filtered off, washed with cold acetonitrile, and air dried
to afford 423 g (92.2%) of the title product, mp 80-81 °C; 1H
NMR (300 MHz, CDCl3): δ 7.87 (1H, s), 7.79 (2H, d, J ) 8.8
Hz), 7.28-7.42 (5H, m), 7.05 (2H, d, J ) 8.8 Hz), 6.74 (1H,
dd, J ) 11.0, 10.8 Hz), 5.84 (1H, d, J ) 9.8 Hz), 5.30 (1H, d,
J ) 10.8 Hz), 5.13 (2H, s), 5.05 (1H, d, J ) 17.1 Hz), 3.46
(1H, m), 3.23 (3H, s), 2.90 (1H, q, J ) 6.6 Hz), 2.51 (1H, m),
2.20 (1H, m), 1.90-2.05 (2H, m), 1.74 (2H, dd, J ) 5.8, 9.7
Hz), 1.4-1.6 (3H, m), 1.35 (1H, m), 1.30 (3H, s), 1.25 (1H,
m), 1.20 (3H, s), 1.05 (1H, m), 1.02 (3H, d, J ) 6.4 Hz), 0.95
(3H, d, J ) 6.6 Hz). 13C NMR (100 MHz, DMSO-d6): δ 214.3,
165.0, 161.8, 151.3, 140.5, 136.5, 130.6, 128.5, 127.9, 127.8,
125.6, 117.8, 114.6, 114.4, 82.3, 72.2, 69.4, 63.1, 56.3, 53.3,
47.0, 44.7, 44.2, 43.5, 42.9, 30.1, 28.9, 28.3, 25.3, 20.5, 16.3,
15.4. Anal. Calcd for C36H45NO6: C, 73.57; H, 7.72; N, 2.38.
Found: C, 73.66; H, 7.70; N, 2.41. MS (ESI) m/z 610.1 (M +
Na)+; 586.3 (M - H)-.
Mutilin 14-[N-(4-Benzyloxybenzoyl)carbamate] (12). A
solution of 11 (364 g, 0.62 mol) in dioxane (2.1 L) was cooled
to 10 °C and treated with a half-saturated solution of zinc
chloride in concd HCl (910 mL, 450 g ZnCl2) keeping the
temperature <15 °C. Then the mixture was heated at 25 °C for
5 h, and water (600 mL) was added slowly. Prior to completion
of the addition of water, a white precipitate began to form. After
completion of the addition, the heterogeneous mixture was
stirred at room temperature for 1 h. The white precipitate was
filtered, washed with water (2 × 300 mL) and ethanol (300
mL), then dried under vacuum to afford 335 g (93.4%) of 12
as a white solid, mp 186-187 °C. 1H NMR (300 MHz, CDCl3):
δ 7.83 (1H, s), 7.75 (2H, d, J ) 8.81), 7.28-7.43 (5H, m),
7.03 (2H, d, J ) 8.81), 6.55 (1H, dd, J ) 10.9, 10.8 Hz), 5.83
(1H, d, J ) 8.5 Hz), 5.38 (1H, d, J ) 11.0 Hz), 5.20 (1H, d, J
) 17.3 Hz), 5.12 (2H, s), 3.37 (1H, d, J ) 6.4 Hz), 2.35 (1H,
t, J ) 6.1 Hz), 2.30 (2H, m), 2.10 (2H, m), 1.60-1.80 (4H,
m), 1.50 (3H, s), 1.35-1.45 (3H, m), 1.20 (3H, s), 1.05 (1H,
m), 0.93 (3H, d, J ) 6.4 Hz), 0.90 (3H, d, J ) 6.6 Hz). 13C
NMR (100 MHz, DMSO-d6): δ 217.2, 164.9, 161.7, 151.0,
140.9, 136.5, 130.5, 128.5, 127.9, 127.8, 125.6, 115.2, 114.4,
72.7, 70.1, 69.4, 66.3, 57.4, 44.9, 43.9, 41.6, 36.4, 34.0, 30.1,
28.3, 26.6, 24.5, 16.0, 14.9, 11.5. Anal. Calcd for C35H43NO6:
C, 73.27; H, 7.55; N, 2.44. Found: C, 73.19; H, 7.70; N, 2.41.
MS (ESI) m/z 572.6 (M - H)-.
Experimental Section
General Methods. All reagents and solvents were com-
mercially available and were used without further purification
unless otherwise stated. NMR spectra of the intermediates were
recorded on a Bruker 300 NMR spectrometer using TMS as
an internal standard. EI-MS spectra were obtained on a Finnigan
MAT 95 mass spectrometer and ESI-MS spectra were obtained
on a Kratos MS 80 mass spectrometer. Elemental analysis was
obtained using a vario EL spectrometer. HPLC analysis of the
intermediates and reaction monitoring was performed on an
Agilent 1100 liquid chromatograph equipped with a Dikma
Technologies ODS 250 mm ×4.6 mm 5 µM column; flow rate
1 mL/min, wavelength: 210 nm, temperature: 25 °C. Standard
mobile phase composition: 30:70 to 10:90 gradient of water/
acetonitrile. Product 1 (FU-23) mobile phase composition: 30%
water with 0.1% TFA, 70% methanol, flow rate 1 mL/min.
(3R)-3-Deoxo-11-deoxy-3-methoxy-11-oxo-4-epi-mutilin
(4). Pleuromutilin (400 g, 1.06 mol) was dissolved in methanol
(1.5 L) and trimethyl orthoformate (504 g, 520 mL, 4.75 mol)
at 20 °C. Concentrated sulphuric acid (198 g, 100 mL, 2.06
mol) was added over 10 min at <30 °C. The mixture was heated
to 30 °C and stirred for 8 h. A solution of NaOH (320 g, 8.0
mol) in water (350 mL) was added to the methanol mixture,
and the contents were heated to 60-63 °C for 2 h. The mixture
was cooled to 40 °C, and methanol was removed by vacuum
distillation to a final volume of 500 mL. After cooling to room
temperature, water (350 mL) was added, and the reaction
mixture was extracted with ethyl acetate (1000 mL). The
aqueous layer was further extracted with ethyl acetate (500 mL),
and the combined organic extracts were dried over anhydrous
Na2SO4 and concentrated under reduced pressure to give the
crude oily mixture (300 mL). Petroleum ether (500 mL) was
added to the crude oily mixture at 0 °C, and a thick, white slurry
resulted within 10 min. The slurry was stirred for 1 h. The white
precipitate was filtered and washed with cold petroleum ether
to give 285.6 g (81.0%) of compound 4 as a crystalline solid,
mp 80-81 °C; 1H NMR (300 MHz, CDCl3): δ 6.05 (1H, dd,
J ) 11.0, 10.8 Hz), 5.26 (2H, m), 4.61 (1H, d, J ) 9.2 Hz),
3.48 (1H, m), 3.23 (3H, s), 2.92 (1H, q, J ) 6.6 Hz), 2.45 (1H,
dd, J ) 15.3, 10.0 Hz), 2.18 (1H, m), 2.01 (2H, m), 1.86 (1H,
d, J ) 15.4 Hz), 1.73 (1H, d, J ) 11.4 Hz), 1.58 (2H, s), 1.46
(1H, m), 1.32 (1H, m), 1.18 (3H, s), 1.67 (3H, s), 1.16 (1H,
m), 1.09 (1H, m), 1.01 (3H, d, J ) 6.4 Hz), 0.95 (3H, d, J )
6.6 Hz). Anal. Calcd for C20H34O3: C, 75.41; H, 10.25. Found:
C, 75.38; H, 10.16. MS: 235 (M + 1).
(3R)-3-Deoxo-11-deoxy-3-methoxy-11-oxo-4-epi-mutilin
14-[ N-(4- Benzyloxybenzoyl)carbamate] (11). To a well-
stirred suspension of dry 4-benzyloxybenzamide (181.6 g, 0.80
mol) in dry dichloromethane (1.2 L), freshly distilled oxalyl
chloride (83.7 mL, 111.7 g, 0.88 mol) was added in dropwise
fashion at 25 °C in 15 min. The mixtures agitated for a further
30 min at 25 °C, then were heated at 65 °C for 1 h, and the
suspended solid disappears completely. Extreme care was taken
to prevent the introduction of moisture into the sample during
19,20-Dihydromutilin 14-[N-(4-Hydroxybenzoyl)carbam-
ate] (13). To a solution of compound 12 (270 g, 0.47 mol) in
THF (2.0 L) was added 10% Pd-C (30 g, 50% water) and the
mixture stirred under H2 atmosphere employing 3 bar of
818
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Vol. 14, No. 4, 2010 / Organic Process Research & Development

pressure at 25 °C. The reaction mixture was hydrogenated until
no further H2 was consumed. The reaction mixture was filtered,
and the filtrate was concentrated to give crude solid 13. Acetone
(200 mL) was added to the crude solid, and the slurry was stirred
for 1 h. The white precipitate was filtered and washed with
ethanol (300 mL) to afford 217.0 g (95.2%) of compound 13
as a white solid, mp 187-189 °C; 1H NMR (300 MHz, DMSO-
d6): δ 10.47 (1H, s), 10.25 (1H, s), 7.74 (2H, d, J ) 8.8 Hz),
6.81 (2H, d, J ) 8.8 Hz), 5.53 (1H, d, J ) 7.9 Hz), 4.40 (1H,
d, J ) 6.4 Hz), 2.35 (1H, t, J ) 6.1 Hz), 2.0-2.30 (3H, m),
1.45-1.80 (6H, m), 1.42 (3H, s), 1.20-1.40 (3H, m), 1.05 (1H,
m), 0.95 (3H, s), 0.90 (3H, d, J ) 6.4 Hz), 0.60-0.80 (6H, m).
13C NMR (100 MHz, DMSO-d6): δ 217.3, 164.9, 161.5, 151.4,
130.7, 123.8, 114.9, 73.7, 69.6, 57.5, 44.9, 40.3, 36.4, 36.3,
34.4, 33.9, 30.2, 26.7, 26.4, 24.4, 20.2, 16.3, 14.9, 11.5, 8.1.
Anal. Calcd for C28H39NO6: C, 69.25; H, 8.09; N, 2.88. Found:
C, 69.31; H, 8.00; N, 2.78. MS (ESI) m/z 508.1 (M + Na)+;
484.2 (M -H)-.
evaporated and tert-butyl methyl ether was added. A white solid
was collected by filtration and washed twice with tert-butyl
methyl ether. The crude product was dissolved in CH3CN (1.6
L), and water (32 mL) was added slowly into the solution at
ambient temperature. The solution was then cooled to 0 °C and
was seeded while cooling. After 2 h, the white crystals were
collected by filtration and washed with CH3CN (500 mL). The
solid was dried under high vacuum at 35 °C to yield 145.0 g
(95.6%) of 15. 1H NMR (300 MHz, CD3OD): δ 0.81 (3H, d,
J ) 6.6 Hz), 0.88 (3H, d, J ) 7.0 Hz), 1.10-1.81 (12H, m),
1.15 (3H, s), 1.51 (3H, s), 2.09-2.26 (2H, m), 2.38 (1H, bs),
2.40 (1H, q, J ) 6.9 Hz), 3.45 (1H, d), 5.78 (1H, d, J ) 8.5
Hz), 7.30 (2H, d, J ) 8.8 Hz), 7.90 (2H, d, J ) 8.8 Hz). 13
C
NMR (100 MHz, CD3OD): δ 220.4, 168.2, 157.0, 156.9, 153.6,
131.8, 131.2, 121.8, 121.7, 77.2, 72.5, 60.0, 47.3, 43.7, 42.3,
42.0, 38.6, 36.5, 35.8, 32.0, 28.6, 27.3, 26.3, 21.9, 17.4, 16.0,
12.3, 9.1. Anal. Calcd for C28H40NO9P: C, 59.46; H, 7.13; N,
2.48. Found: C, 59.41; H, 7.15; N, 2.46. MS (ESI) m/z 565.2
(M - H)-.
19,20-Dihydromutilin 14-[N-(4-Phosphoric Acid Dibenzyl
Ester Benzoyl)carbamate ] (14). 13 (172.8 g, 0.36 mol) and
dimethylaminopyridine (4.4 g, 0.036 mol) were dissolved in
anhydrous CH3CN (2.0 L) under argon. The reaction mixture
was cooled to -25 °C, and CCl4 (160 mL, 1.66 mol) and
diisopropylethylamine (134.5 mL, 0.77 mol) were added. After
10 min, dibenzylphosphite (94.4 mL, 0.43 mol) was added over
a 30 min period. The mixture was then stirred at -20 °C for
2 h. During this time, the starting material dissolved. KH2PO4
(0.5 M, 600 mL) was added, and the solution was allowed to
warm to room temperature and stirred for 1 h. The aqueous
mixture was extracted with ethyl acetate (3 × 350 mL) and
then washed with water (2 × 150 mL). The organic layer was
dried over Na2SO4 and concentrated in Vacuo to a volume of
500 mL. Hexane (750 mL) was added. Solvent (500 mL) was
removed in Vacuo, and the solid precipitated during this process.
Hexane (500 mL) and ethyl acetate (300 mL) were added, and
the mixture was heated to reflux until all solid dissolved. The
solution was cooled to room temperature and then to 0 °C for
2 h. A white solid was collected, washed twice with cold
hexane, and dried in Vacuo to yield 220.0 g (82.0%) of 14, mp
88-90 °C; HPLC tR ) 12.8 min; 1H NMR (300 MHz, DMSO-
d6): δ 0.81 (3H, d, J ) 6.6 Hz), 0.88 (3H, d, J ) 7.0 Hz),
1.10-1.81 (12H, m), 1.15 (3H, s), 1.51 (3H, s), 2.09-2.26 (2H,
m), 2.38 (1H, bs), 2.40 (1H, quintet J ) 6.9 Hz), 3.41 (1H, d),
5.10-5.16 (4H, d, J ) 8.8 Hz), 5.57 (1H, dd, J ) 11.0, 1.5
Hz), 7.20 (2H, d, J ) 8.8 Hz), 7.30-7.40 (10H, m), 7.90 (2H,
d, J ) 8.8 Hz), 10.80 (1H, s). 13C NMR (100 MHz, CDCl3): δ
217.2, 163.9, 153.9, 151.1, 135.0, 134.9, 129.8 129.7, 128.8,
128.6, 128.0, 120.0, 77.3, 77.0, 76.7, 71.6, 70.3, 70.2, 58.4,
45.5, 41.9, 40.8, 36.6, 34.4, 34.3, 30.2, 26.8, 26.3, 24.8, 20.6,
16.5, 15.0, 14.1, 11.0, 8.2. Anal. Calcd for C42H52NO9P: C,
67.64; H, 7.03; N, 1.88. Found: C, 67.84; H, 6.87; N, 1.81.
MS (ESI) m/z 768.80 (M + Na)+.
Disodium 19,20-Dihydromutilin 14-[N-(4-Phosphoric acid
benzoyl)carbamate] (1). The acid 15 (120 g, 0.21 mol) was
dissolved in methanol (200 mL) and cooled to 0 °C. A solution
of Na2CO3 (23.3 g, 0.22 mol) in distilled water (50 mL) was
added dropwise to the methanol solution over 10 min. After
the addition was complete, the clear reaction mixture was stirred
for an additional 2 h at room temperature. After filtration to
remove some unsoluble material, the solution volume was
reduced by 50% (100 mL) on a rotary evaporator, and the
residue was recrystallised from methanol/isopropanol (600 mL,
1:2) giving 1 as a white precipitate which was filtered off and
washed with cold isopropanol (100 mL). The filter cake was
dried under vacuum at 35 °C to afford 111.5 g (82.3%) of 1 as
white solid, mp 200-202 °C. HPLC tR ) 6.58 min, >99.5%
purity. 1H NMR (300 MHz, D2O): δ 0.79 (6H, m), 0.88 (5H,
m), 1.12 (1H, t), 1.40-1.71 (12H, m), 1.75-1.90 (3H, m),
2.20-2.36 (2H, m), 2.40 (1H, q, J ) 6.9 Hz), 2.50 (1H, bs),
3.54 (1H, d), 5.55 (1H, d, J ) 8.5 Hz), 7.32 (2H, d, J ) 8.8
Hz), 7.80 (2H, d, J ) 8.8 Hz). 13C NMR (100 MHz, D2O): δ
224.0, 169.2, 161.8, 158.4, 152.3, 129.8, 126.0, 119.9, 119.8,
76.0, 71.4, 58.4, 45.5, 41.6, 40.1, 39.3, 36.7, 34.5, 34.1, 29.8,
26.3, 25.5, 24.2, 20.0, 15.7, 14.5, 10.7, 7.3. Anal. Calcd for
C28H38NNa2O9P: C, 55.17; H, 6.28; N, 2.30. Found: C, 55.16;
H, 6.32; N, 2.31.
Acknowledgment
We are grateful to National Science and Technology Major
Project for the support of this research. The project described
was supported by Key New Drug Creation and Manufacturing
Program, China (Number: 2009ZX09301-001).
Supporting Information Available
Copies of the NMR spectra of all intermediates and final
product as well as HPLC conditions and retention times. This
material is available free of charge via the Internet at
http://pubs.acs.org.
19,20-Dihydromutilin 14-[N-(4-Phosphoric acid benzoyl)-
carbamate] (15). To a solution of 14 (200 g, 26.82 mmol) in
THF (1.5 L) was added 5% Pd-C (45 g), and the mixture was
hydrogenated at ambient temperature and 15 psig hydrogen for
5 h. The mixture was filtered through Celite-521 and a 0.45
µm membrane and was rinsed with THF. The solvent was
Received for review March 6, 2010.
OP100063H
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