An efficient large-scale synthesis of EDP-420, a first-in-class bridged bicyclic macrolide (BBM) antibiotic drug candidate

Article abstract of DOI:10.1021/op900228u

A multistep, practical, and cost-effective synthesis of novel bridged bicyclic macrolide drug candidate EDP-420 (1) is described. Starting from inexpensive and commercially available erythromycin A 9-oxime, the current chemical process involves a series of transformations: triacetylation, Pd-catalyzed O,O-bis-allylation (bridge formation), acid-catalyzed sugar cleavage, oxime reduction, acetylation, Os-catalyzed bridge olefin oxidative cleavage, Corey-Kim oxidation, bridge oxime formation, deprotection, and final purification. Multikilogram quantities have been synthesized.

Full text of DOI:10.1021/op900228u

Organic Process Research & Development 2010, 14, 504–510
An Efficient Large-Scale Synthesis of EDP-420, a First-in-Class Bridged Bicyclic
Macrolide (BBM) Antibiotic Drug Candidate
Guoyou Xu, Datong Tang,* Yonghua Gai, Guoqiang Wang, Heejin Kim, Zhigang Chen, Ly T. Phan, Yat Sun Or, and
Zhe Wang
Enanta Pharmaceuticals, Inc., 500 Arsenal Street, Watertown, Massachusetts 02472, U.S.A.
Abstract:
A multistep, practical, and cost-effective synthesis of novel bridged
bicyclic macrolide drug candidate EDP-420 (1) is described.
Starting from inexpensive and commercially available erythro-
mycin A 9-oxime, the current chemical process involves a series
of transformations: triacetylation, Pd-catalyzed O,O-bis-allylation
(bridge formation), acid-catalyzed sugar cleavage, oxime reduction,
acetylation, Os-catalyzed bridge olefin oxidative cleavage,
Corey-Kim oxidation, bridge oxime formation, deprotection, and
final purification. Multikilogram quantities have been synthesized.
Figure 1. Chemical structure of EDP-420 (1).
Introduction
EDP-420 (1, Figure 1),1 entered phase II clinical trials in
Like the classic macrolide drug erythromycin A,4 1 also
early 2006 for the treatment of community-acquired pneumonia.
features a polyfunctionalized 14-membered lactone ring. How-
First in its class, 1 is a bridged bicyclic macrolide (BBM) that
was designed and discovered by Enanta to have optimal
ever, 1 is a 3-keto macrolide molecule (known as a ketolide)
bearing a unique 6,11-O,O-three-carbon bridged (E)-oxime side-
pharmacokinetics (PK) and to provide broad treatment against
respiratory pathogens, including several multidrug resistant
strains for which traditional macrolides, penicillins, and fluo-
roquinolones are no longer effective.
chain subunit. The structural complexity constitutes a formidable
challenge for supplying the active pharmaceutical ingredient
(API) in high purity for preclinical toxicity studies and clinical
trials. Erythromycin A 9-oxime 2, derived from erythromycin
A via an oxime formation process and commercially available
in bulk quantity, was chosen as the starting material. The
construction of 1 requires the 6,11-bridge formation as the key
step, (Figure 2). Our process optimization focused on this key
step with the ultimate goal of pilot-plant production of 1 in
multikilogram quantities.
EDP-420 has broad activity against respiratory pathogens
including macrolide resistant Streptococcus pneumoniae, PRSP,
S. pyogenes, Haemophilus influenzae and atypicals. In addition,
this compound broadens the spectrum of existing macrolide
antibiotics by demonstrating excellent in Vitro activity against
many strains of multidrug resistant S. pneumoniae, including
strains resistant to the traditional macrolides, penicillins and
fluoroquinolones. Extensive comparative in ViVo efficacy studies
were performed between EDP-420 and telithromycin against
H. influenzae, S. pneumoniae, Staphylococcus aureus, and S.
pyogenes. In these studies, EDP-420 consistently performed
equal to or better than telithromycin.2 EDP-420 also possesses
an excellent PK profile across multiple species, with a half-life
2-3 times longer as compared to those of clarithromycin and
telithromycin.3 On the basis of these observations, we expect
EDP-420 will be a once daily dosed drug.
Results and Discussion
6,11-O,O-Bridge Formation. Using bis(Boc)-protected al-
lylic diol 3 as the dielectrophile, we developed a novel bridging
reaction for erythromycin A-derived macrolide Via a pal-
ladium(0)-catalyzed tandem 6-O and 11-O-allylic dialkylation
(Scheme 1).5 This sequence first required protection of the
otherwise reactive hydroxyls. Thus, commercially available 2
was first protected as its 9,2′,4′′-triacetate 5 by reacting with
Ac2O in THF in the presence of triethylamine and catalytic
amount of DMAP. Isolation and characterization of some of
the impurities established that there were one monoacetate (9-
oxime acetate), two diacetate (9,2′-diacetate and 9,4′′-diacetate),
and possibly one tetraacetate (9,2′,4′′,11-tetra-acetate). However,
* Author for correspondence. E-mail: dtang@enanta.com.
(1) (a) Scorneaux, B.; Arya, A.; Polemeropoulos, A.; Lillard, M.; Han,
F.; Amsler, K.; Sonderfan, A.; Wang, G., Wang, Y.; Peng, Y.; Xu,
G.; Kim, H.; Lien, T.; Phan, L. T., Or, Y. S. Abstract No. F-1191;
43rd Annual ICAAC, Chicago, IL, September 2003. (b) McAlpine,
J. B.; Yagisawa, M. Antibacterials for the Treatment of Gram Positive
Infections. Ann. Rept. Med. Chem. 2005, 40, 301-322.
(2) Revill, P.; Bolos, J.; Serradell, N. Drugs Future 2006, 31, 479.
(3) Jiang, L.-J.; Wang, M.; Or, Y. S. Antimicrob. Agents Chemother. 2009,
53, 1786.
(4) For recent macrolide reviews, see: (a) Katz, L.; Ashley, G. W. Chem.
ReV. 2005, 105, 499. (b) Chu, D. T. W. Med. Res. ReV. 1999, 19,
497. (c) Bryskier, A. Expert Opin. InVest. Drugs 1999, 8, 1171.
(5) Wang, G.; Niu, D.; Qiu, Y.; Phan, L. T.; Chen, Z.; Polemeropoulos,
A.; Or, Y. S. Org. Lett. 2004, 6, 4455.
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10.1021/op900228u  2010 American Chemical Society
Published on Web 05/03/2010

Figure 2. Retrosynthetic analysis of EDP-420.
Scheme 1. Strategic 6,11-O,O-bridge construction via
Scheme 2. Formation of the major byproduct isolated in
the bridge formation step
Pd-catalyzed diallylationa
Like most of Pd (0)-catalyzed chemistry, the macrolide
bridge formation reaction was highly oxygen- and moisture-
sensitive. In addition, both the purity and dryness of the
triacetate 5 were critical for a high yield in this reaction.
Otherwise, up to 15% of a “dehydrated ketal” byproduct,
erythralosamine 2′-acetate 8, would be isolated after the
subsequent acidic sugar cleavage reaction. The byproduct 8 was
a well-known degradation product from erythromycins6 and was
formed, in this study, from the unreacted triacetate and its
impurities through 9-hydrolysis, 6,9- and 12,9-hemiketal forma-
tion, dehydration, spiroketal formation, and cladinose sugar
cleavage (Scheme 2). To minimize this unproductive pathway,
the crystallized triacetate 5 was first dried azeotropically from
toluene before the Pd-catalyzed bridge formation reaction. This
minimized the formation of 8. At 4 kg scale, the overall isolated
yield of bridged olefin 9-oxime 7 was 55-65% based on the
triacetate 2. The chemical purity of 7 was above 95 area % by
HPLC analysis.
a Conditions: (i) Ac2O, DMAP, TEA, rt, then crystallization from MeCN
(80%); (ii) 3, Pd2(dba)3, dppb, THF, reflux; (iii) 1 N aq HCl, 60 °C, then
crystallization from MeCN (60% from 5).
since the partial hydrate of the triacetate 5 was highly crystalline
when recrystallized from MeCN, the impurities were efficiently
removed in solution. The recrystallized chemical yield for
triacetylation on multikilogram scale in the plant was 70-90%.
Treatment of the resulting triacetate 5 with bis(Boc)-protected
diol 3 in the presence of tris(dibenzylideneacetone)dipal-
ladium(0) [Pd2(dba)3, 2 mol %] as catalyst and catalytic 1,4-
bis(diphenylphosphino)butane [dppb, 4 mol %] as ligand
smoothly effected a regioselective tandem diallylation at the
6,11-hydroxyl groups through the “Pd-π-allyl complex” transi-
tion state to afford the desired 6,11-O,O-bridged macrolide key
intermediate 6. Without isolation, the 3-cladinose sugar fragment
and the 9-oxime acetate were selectively hydrolyzed with 1 M
HCl to give the bridged olefin 9-oxime 7, which was easily
isolated by crystallization from acetonitrile on multikilogram-
scale preparation (Scheme 1).
The bridging reagent bis(Boc)-protected diol 3 was quanti-
tatively prepared on multikilogram scale by reacting 2-meth-
ylene-1,3-propanediol with Boc2O with phase-transfer catalysis
(PTC) in aqueous NaOH and CH2Cl2. The crude product was
typically contaminated with 5 mol % of unreacted Boc2O, which
did not affect the subsequent reaction. Therefore, the crude
product 3 was telescoped directly into the next stepsthe
macrolide bridge formation.
Oxime Reduction to Imine Acetamide Intermediate 10.
Aqueous TiCl3 is a useful reagent for the reduction of oximes
to imines.7 Bridged 9-oxime intermediate 7 was reduced with
20% TiCl3 solution in 3% aqueous HCl to 9-imine 9 (Scheme
3). Surprisingly, the imine functionality of intermediate 9 was
very stable under these aqueous acidic condition. Conventional
(6) Flynn, E. H.; Sigal, M. V.; Wiley, P. F.; Gerzon, K. J. Am Chem.
Soc. 1954, 76, 3121.
(7) (a) Timms, G. H.; Wildsmith, E. Tetrahedron Lett. 1971, 12, 195. (b)
Gasparski, C. M.; Ghosh, A.; Miller, M. J. J. Org. Chem. 1992, 57,
3546.
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Scheme 3. Bridged olefin 9-imine acetamide, 10a
Scheme 5. Bridge oxime formation to EDP-420a
a Conditions: (i) TiCl3 in aq HCl, EtOH, rt, then basification; (ii) Ac2O,
CH2Cl2, rt, then crystallization from EtOAc/heptanes (85% from 7).
a Conditions: (i) 4, HCl, EtOH/H2O, 0 to 5 °C; (ii) MeOH, rt, then
crystallization from EtOH/H2O (40% from 12).
Scheme 4. Late-stage intermediate diketone, 12a
It is well-known that the Dess-Martin oxidation is a mild, clean,
and usually high-yielding reaction. However, in addition to its
cost issue, we were uncomfortable about using it for a large-
scale process due to its potentially explosive nature. Instead,
we screened some commonly used oxidation reagents10 that
would be most suitable for a large-scale preparation, including
the TPAP [Pr4NRuO4]-NMO reagent and the “activated”
DMSO oxidations, i.e., the Swern and Moffatt reactions. These
reagents either led to incomplete conversions or significant side
reactions. However, the Corey-Kim reaction11 provided the
answer. Corey-Kim oxidation is a mild and selective reaction
particularly suitable for complex alcohols, such as macrolide
molecules, in which N-chlorosuccinimide (NCS) is first reacted
with dimethyl sulfide (DMS) to form the NCS-DMS complex
reagent. The stoichiometry of NCS [optimized as 1.30 equiv
to intermediate 11] was critical for the product purity. Excess
NCS resulted in 10-15% yields of 12-methythiomethyl (MTM)
ether byproduct formation. Because the preparation of diketone
12 was a two-step, one-pot process (Scheme 4) without isolation
of the intermediate 11, the solution yield (typically ∼78% for
olefin cleavage step) of 11 was determined by 1H NMR analysis.
On the basis of its solution yield, the NCS stoichiometry was
adjusted accordingly.
For multikilogram preparation, we also screened the solvent
and temperature. The optimal Corey-Kim conditions were
using methylene chloride at -15 to -20 °C. In the plant, 4 kg
batches (16 batches) achieved a 55% overall average yield (two
steps) after crystallization from EtOH in 95 area % purity (NMR
and HPLC analysis).
Bridge Oxime Formation to EDP-420 (1). The final
operational step was a two-step, one-pot process (Scheme 5)
without isolation of the bridge oxime E/Z mixture 13. The oxime
was formed by treating the diketone 12 with hydroxylamine 4
in EtOH. After aqueous workup (basification and extraction),
the E/Z mixture (∼4:1) of the oximes was telescoped into the
next step: methanolysis for deprotection at the 2′ position. The
oxime mixture was then purified by crystallization from aq
EtOH to provide crude EDP-420. On typical kilogram scale,
overall isolated yields for crude 1 were 40-42% in 95 area %
HPLC purity. Most of the undesired Z-isomer remained in the
mother liquor and was thus easily removed by filtration during
recovery of the crystallized 1.
a Conditions: (i) OsO4, NaIO4, acetone/H2O; (ii) NCS, DMS, CH2Cl2, -20
°C, then TEA, crystallization from EtOH (55% from 10).
hydrolysis leading to the corresponding 9-ketone under various
conditions investigated in our laboratories proved to be difficult.
We believe that the stability of this imine group is due to the
constrained conformation of the macrocycle that results in 8,10-
methyl groups blocking one side of the imine while the bridge
blocks the other side. Due to the presence of the neighboring
“dimethyl amine” group on the desosamine sugar moiety,
aqueous acid conditions prevented the otherwise expected
hydrolysis of the 2′-acetate.
Subsequent basification and extraction produced the 9-imine
9 as a solution in EtOAc. This imine intermediate solution was
solvent exchanged to CH2Cl2 and treated with Ac2O to produce
the bridged olefin 9-imine acetamide 10. For a typical 4-kg-
scale operation, the overall yield for this one-step process was
85-90% after crystallization from EtOAc/heptanes.
Late-Stage Intermediate Diketone 12. Diketone 12 was
prepared from bridged olefin 9-imine acetamide 10 Via a 2-step
one-pot process, i.e., oxidative cleavage of bridge olefin to
intermediate 11 followed by oxidation at the 3 position, (Scheme
4). If the reactions were reversed, overall yield was lower. The
exocyclic olefin’s oxidative cleavage reaction8 was conducted
by using OsO4 (0.28 mol %)-catalyzed NaIO4 in aqueous
acetone to give ∼85% yield (solution NMR analysis). Alter-
natively, ozonolysis in EtOAc/MeOH at low temperature with
a Me2S reductive workup also worked very well in comparable
product yield and purity. Although ozone is more typically used
for olefin cleavages,9 we chose OsO4-catalyzed NaIO4 cleavage
for the multikilogram-scale reaction primarily due to equipment
limitations in our kilolab.
3-Oxidation was first carried out by a Dess-Martin perio-
dinane oxidation by our discovery medicinal chemistry team.
(8) Pappo, R.; Allen, D. S., Jr.; Lemieux, R. U.; Johnson, W. S. J. Org.
Chem. 1956, 21, 478.
EDP-420 has the E-oxime configuration on the 6,11-bridge.
It was obvious that the ability to generate a high E/Z ratio for
(9) (a) Bailey, P. S. Ozonation in Organic Chemistry; Academic: New
York, 1978; Vol. 1. Bailey, P. S. Ozonation in Organic Chemistry;
Academic: San Diego, 1982; Vol. 2. (b) Razumovskii, S. D.; Zaikov,
G. E. Ozone and Its Reactions with Organic Compounds; Elsevier:
Amsterdam, 1984.
(10) For a recent review about alcohol oxidation by activated DMSO and
related reactions, see: Tidwell, T. T. Synthesis 1990, 857–870.
(11) Corey, E. J.; Kim, C. U.; Misco, P. F. Org. Synth. 1978, 58, 122.
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Table 1. Acid-catalyzed formation of bridge oxime 13: E/Z
In conclusion, we have developed a practical 10-step
chemical process for the multikilogram scale preparation of
EDP-420 API. All isolated intermediates were obtained by
crystallization and filtration. This efficient process has been
successfully demonstrated at the pilot plant scale.
ratioa
entry
acid
noneb
L-malic acid
MeSO3H
p-TsOH
H3PO4
CSA
BF3 or ZnCl2
HCl
pKa
3.4
oxime E/Z ratio by HPLC
1
0.7
1.2
1.6
1.6
1.9
2.5
3.0
4.0
4.0
4.3
8.0
2
3
-2
4
-2.8
2.1
Experimental Section
5
General Methods. All solvents were purchased from
Aldrich. All reagents were used as purchased. MS experiments
were carried out on an Agilent 6210 Time-of-Flight in ESI +
mode using direct flow injection. Mobile phases are A: water
+ 0.1% formic acid and B: ACN + 0.1% formic acid. 1H and
13C NMR spectra were recorded on a Varian INOVA 500
spectrometer operating at 500 and 125 MHz, respectively. Two-
dimensional (2D) NMR spectra were acquired using standard
pulse sequences. HPLC method used for determination of the
purity of 1: L-column ODS, 5 µm, 4.6 mm × 250 mm, a
constant temperature of about 40 °C; detector 210 nm; mobile
phase A is a mixture of 0.005 mol/L KH2PO4 buffer ( pH 8.0)
and acetonitrile (3:2). Mobile phase B is a mixture of 0.012
mol/L KH2PO4 buffer ( pH 8.0) and acetonitrile (1:3). Gradient
program: HPLC program: 0-5 min (70% A); 5-40 min (70
to 0% A); 40-65 min (0% A); 65-65.01 min (0 to 70% A);
65.01 to 80 min (70% A). Flow rate 1.0 mL/min. tR for 1 ≈ 30
min.
6
1.2
c
7
-
8
-7
9
HBr/HI
H2SO4
-9/-10
-9
10
11
d
HClO4
-10
a Experiments were carried out on 2 g scale in EtOH/H2O at 0 to 5 °C for 2 h
except where noted. b In toluene at rt for 2 days, <65% conversion. c In EtOAc or
EtOH/THF. d In aq EtOH/MeCN at -35 °C for 7 h.
2-Methylene-1,3-propanediol bis(tert-butyl Carbonate)
(3). A solution of Boc2O (6.6 kg, 30.24 mol) in CH2Cl2 (15 L)
was treated with 2-methylene-1,3-propanediol (1.0 kg, 11.35
mol) and followed by (n-Bu)4NHSO4 (0.641 kg, 1.888 mol).
This was agitated vigorously and cooled to 12-15 °C. A
solution of 6 N aq NaOH (13.2 L) was added over 2 h at 25-30
°C. The resulting two-phase mixture was agitated at 25 °C for
2-3 h. The aqueous layer was separated, and additional (n-
Bu)4NHSO4 (64 g, 0.1888 mol, 10% of the initial amount),
Boc2O (0.66 kg, 3.024 mol, 10% of the initial amount), and
CH2Cl2 (2 L) were added. A solution of 6 N aq NaOH (1.32
L, 10% of the initial amount) was added over 0.5-1 h at 25-30
°C. This was agitated at 20-28 °C for 3-4 h. After separation
of phases, the organic phase was washed with H2O (3 × 8 L)
and concentrated in Vacuo until water content (Karl Fischer)
of the residue was NMT 100 ppm. (If necessary, anhydrous
EtOAc (5 L) was added and distilled in Vacuo to dry the residue
azeotropically) The crude 3 was obtained as an oil in quantitative
yield. 1H NMR (CDCl3, 500 MHz): δ 5.21 (s, 2 H), 4.50 (s, 4
H), 1.39 (s, 18 H). 13C NMR (CDCl3, 125 MHz): δ 153.4,
138.5, 117.5, 82.2, 66.3, 26.8.
Erythromycin A 9-Oxime 9,2′,4′′-triacetate (5). A solution
of erythromycin A 9-oxime (2) (1.0 kg, 1.335 mol) in EtOAc
(4 L) was treated with TEA (0.744 L, 5.338 mol) followed by
DMAP (48.9 g, 0.40 mol). This was agitated vigorously and
Ac2O (0.441 L, 4.674 mol) was added over a period of 1-2 h
at below 40 °C. This was agitated for 2 h at 20-28 °C.
Additional TEA (74.4 mL, 0.5338 mol, 10% of initial amount)
and additional Ac2O (44.1 mL, 0.4674 mol, 10% of initial
amount) was added over 30 min at <35 °C. This was further
agitated at 20-28 °C for 1.5-2 h. After the reaction was
complete as indicated by HPLC analysis (conversion >97%),
it was diluted with EtOAc (6 L), quenched with saturated aq
NaHCO3 (3 L), and agitated at 20-28 °C for 10 min. The
Figure 3. SEM crystal image of polymorphic form I for EDP-
420 API, 1.
the oxime formation step would translate into overall higher
yields and in general be a superior process. We found that the
oxime E/Z ratio for diketone 12 was dependent on the pKa of
the acid or Lewis acid catalysts (Table 1). The highest E/Z ratio
(8:1, 89% E) was achieved by using perchloric acid as catalyst.
Unfortunately, in addition to being a hazardous reagent, it also
required low temperature (-35 °C) and longer reaction time.
Aqueous HCl as the acid catalyst for the bridge oxime formation
(E/Z ratio 4:1, 80% E) was still a better fit in our facilities for
our large-scale preparations in spite of the inherent yield loss.
The osmium level was measured by inductively coupled
plasma (ICP) spectroscopy in the crude EDP-420 as usually
<1 ppm, which attained our acceptance critieria for GMP clinical
material. In cases when osmium level was high (up to 10 ppm),
we found that washing the crude API solution in EtOAc with
3% sodium metabisulfite in aqueous saturated NaHCO3 fol-
lowed by recrystallization in EtOH/water effectively reduced
the Os level to <1 ppm.
Final Purification of EDP-420. Conventional recrystalli-
zation from various solvents proved insufficient to achieve the
limit of >98% HPLC purity, required for clinical use. Instead,
we developed a purification process using the L-malic acid salt
after screening numerous acids. Our optimized process involved
L-malic acid salt formation in EtOAc/IPA, recrystallization, salt
release, and then final recrystallization from aq EtOH to afford
1 as a monohydrate in >98% HPLC area % purity and in
75-80% overall yield based on crude API. Our largest plant
scale for the production ofAPI 1 was 3 kg per batch. While
EDP-420 possesses multiple polymorphs, this technique yielded
the desired form (Figure 3) reproducibly.
Vol. 14, No. 3, 2010 / Organic Process Research & Development
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aqueous phase was separated and discarded. The organic phase
was washed with saturated aq NaHCO3 (3 L), followed by 15%
aq NaCl (3 L), and concentrated in Vacuo to 3-4 L of remaining
volume. The resulting solution was taken up in acetonitrile (4
L) and concentrated in Vacuo to 3-4 L of remaining volume.
Acetonitrile (2 L) was added and the solution was concentrated
in Vacuo until crystallization occurred (final volume 3-4 L).
Upon formation of crystals, the slurry was agitated at 10-15
°C for at least 2 h. The crystals were collected by filtration and
dried under vacuum until constant weight to afford crystalline
triacetate5 with a typical yield of 80%. 1H NMR (CDCl3, 500
MHz): δ 5.15 (1 H, 13-OCH), 4.98 (1 H, 1′′-OCHO), 4.79 (1
H, 2′-OCH), 4.68 (2 H, 1′-OCHO and 4′′-OCH), 4.33 (1 H,
11-OH), 4.31 (1 H, 5′′-OCH), 3.89 (1 H, 3-OCH), 3.77 (1 H,
11-OCH), 3.74 (1 H, 5′-OCH), 3.67 (1 H, 8-CH), 3.45 (1 H,
5-OCH), 3.35 (3 H, 3′′-OCH3), 3.19 (1 H, 12-OH), 2.88 (1 H,
2-CH), 2.78 (1 H, 10-CH), 2.74 (1 H, 3′-NCH), 2.42 and 1.66
(2 H, 2′′-CH2), 2.29 (6 H, 3′-NCH3 × 2), 2.17 (3 H,
9dNOCOCH3), 2.11 (3 H, 4′′-OCOCH3), 2.06 (3 H, 2′-
OCOCH3), 1.95 and 1.48 (2 H, 14-CH2), 1.93 (1H, 4-CH), 1.72
and 1.34 (2 H, 4′-CH2), 1.51 (2 H, 7-CH2), 1.43 (3 H, 6-CH3),
1.27 (3 H, 10-CH3), 1.19 (6 H, 2-CH3 and 5′-CH3), 1.18 (3 H,
12-CH3), 1.14 (3 H, 3′′-CH3), 1.13 (3 H, 5′′-CH3), 1.11 (3 H,
8-CH3), 0.95 (3 H, 4-CH3), 0.85 (3 H, 15-CH3). 13C NMR
(CDCl3, 125 MHz): δ 178.7, 175.4, 170.4, 170.0, 168.3, 100.6,
96.3, 83.4, 79.4, 79.0, 77.4, 74.9, 74.0, 72.6, 72.2, 70.1, 67.7,
63.6, 63.4, 49.5, 45.0, 40.7, 39.2, 37.4, 35.7, 34.5, 31.5, 28.6,
26.9, 21.7, 21.7, 21.5, 21.4, 21.1, 20.2, 18.7, 18.6, 16.7, 16.1,
15.0, 10.9, 9.3. HRMS Calcd for C43H74N2O16 (MH+) 875.5111.
Found 875.5141.
3-Descladinose-6,11-O,O-bridged Erythromycin A 9-Oxime
2′-acetate (7). A solution of 5 (1.0 kg, 1.143 mol) in anhydrous
THF (5 L) was treated with a solution of 3 (0.62 kg, 2.150
mol) in anhydrous THF (2 L). This was degassed by bubbling
a steady stream of dry N2 gas for 5-10 min. 1,4-Bis(diphe-
nylphosphino) butane (dppb) (19.5 g, 45.7 mmol, 4 mol %)
and tris(dibenzylideneacetone)dipalladium(0) [Pd2(dba)3] (20.8
g, 22.7 mmol, 2 mol %) were added sequentially. The resulting
mixture was immediately degassed as previously described. This
was then heated, while being agitated, to reflux over the period
of about 2 h and then held at the reflux temperature for 6 h at
which point the reaction was complete as indicated by HPLC
analysis (conversion >97%). After cooling to room temperature,
the reaction mixture was filtered through a 2 in pad of silica
gel (0.1-0.25 kg) to remove most of the Pd catalyst, phosphine
ligand, and other polar impurities. The silica gel pad was washed
with fresh THF (1 L). The combined filtrate was concentrated
in Vacuo to 1.5-2 L of remaining volume to afford a solution
of 6 in THF. This was treated with 1 N HCl (8 L), heated to
60 °C over a period of 1-2 h and agitated at 58-62 °C for
additional 2 h at which point the reaction was complete as
indicated by HPLC analysis (conversion >97%). After cooling
to room temperature, this was washed with MTBE (2 × 4 L).
The aqueous solution was diluted with EtOAc (2 L) and 50 wt
% aq K2CO3 (about 0.8 kg of solid K2CO3 in H2O) was added
at 22-28 °C to pH 9-10. The resulting mixture was extracted
with EtOAc (2 × 4 L). The combined organic phase was
washed with H2O (4 L) and concentrated in Vacuo to 1.5-2 L
of remaining volume. MeCN (4-6 L) was added and the
solution was concentrated in Vacuo to 1.5-2 L of remaining
volume. The concentrated residue was agitated at 40-45 °C
under atmospheric pressure until crystallization occurred. The
resulting slurry was cooled to 0-4 °C over 3-4 h and held for
at least 2 h. The crystals were collected by filtration, washed
with chilled MeCN (0.25 L) and dried under vacuum until
constant weight to afford crystalline 7. The typical yield for
1
this 2-step one-pot process was 50-65%. H NMR (CDCl3,
500 MHz): δ 5.09 and 4.98 (2 H, 19dCH2), 4.97 (1 H, 13-
OCH), 4.78 (1 H, 2′-OCH), 4.74 (1 H, 1′-OCHO), 4.43 (1 H,
11-OCH), 4.39 and 4.07 (2 H, 16-CH2), 3.90 and 3.67 (2 H,
18-CH2), 3.84 (1 H, 8-CH), 3.74 (1 H, 5-OCH), 3.50 (1 H,
5′-OCH), 3.43 (1 H, 3-OCH), 2.82 (1 H, 12-OH), 2.79 (1 H,
3′-NCH), 2.73 (1 H, 2-CH), 2.62 (1 H, 10-CH), 2.43 (1 H,
4-CH), 2.31 (6 H, 3′-NCH3 × 2), 2.08 (3 H, 2′-OCOCH3), 2.03
and 1.46 (2 H, 14-CH2), 1.77 and 1.35 (2 H, 4′-CH2), 1.59 and
1.32 (2 H, 7-CH2), 1.38 (3 H, 6-CH3), 1.24 (3 H, 5′-CH3), 1.23
(3 H, 2-CH3), 1.22 (6 H, 10-CH3 and 12-CH3), 0.97 (6 H, 4-CH3
and 8-CH3), 0.89 (3 H, 15-CH3). 13C NMR (CDCl3, 125 MHz):
δ 175.0, 170.1, 166.1, 143.5, 119.2, 99.6, 81.5, 79.5, 78.3 (3-
OCH and 13-OCH), 77.2, 76.0, 73.9, 71.7, 68.9, 65.7, 63.4,
44.1, 40.9, 37.5, 36.1, 34.2, 31.3, 25.8, 23.3, 21.8, 21.4, 20.1,
19.5, 17.3, 15.6, 14.6, 11.9, 7.9. HRMS Calcd for C35H60N2O11
(MH+) 685.4270. Found 685.4409.
3-Descladinose-6,11-O,O-bridged Erythromycin A 9-Imi-
ne Acetamide 2′-Acetate (10). A solution of 7 (1.0 kg, 1.460
mol) in EtOH (2 L) was treated with 20% TiCl3 solution in
3% aqueous HCl (2.847 kg or 2.33 L, 3.691 mol) over 1 h at
25-35 °C. This was agitated at 25-30 °C for 3 h or until the
reaction was complete by HPLC analysis (conversion >97%).
The reacton mixture was diluted with 8-12 °C H2O (15 L)
and treated with 50 wt % aq NaOH (0.466 L) over 0.5-1 h at
<35 °C to pH 6-7, and then was further basified with saturated
aq K2CO3 (about 0.666 L) at <35 °C to pH 9-10. This was
extracted with CH2Cl2 (4 × 4 L). The combined extracts were
concentrated in Vacuo to 2-2.5 L of remaining volume. The
resulting concentrated solution of 9-imine 9 in CH2Cl2 was
treated with Ac2O (0.277 L, 2.936 mol), and the 9-acetylation
reaction was complete at ambient temperature within 2 h as
indicated by HPLC analysis (conversion >97%). The solvent
was exchanged by repeated EtOAc addition (2 or 3 × 3 L)
and distillation in Vacuo until crystallization occurred at 1.5-2
L of remaining volume. Heptanes (1.5 to 2 L) was added at
40-45 °C. The slurry was cooled to 0-4 °C over 2 h and held
for 2 h. The crystals were collected by filtration, washed with
chilled EtOAc/heptanes (1:2, 0.3 L), and dried under vacuum
at 45 °C until constant weight to afford crystalline 10. The
average yield (2 kg/batch, 10 batches) for this two-step, one-
pot process was 84%. 1H NMR (CDCl3, 500 MHz): δ 5.18 (2
H, 19dCH2), 4.93 (1 H, 13-OCH), 4.75 (1 H, 2′-OCH), 4.74
(1 H, 1′-OCHO), 4.59 (1 H, 11-OCH), 4.13 and 3.60 (2 H,
18-CH2), 4.08 and 4.52 (2 H, 16-CH2), 3.74 (1 H, 5-OCH),
3.48 (1 H, 5′-OCH), 3.43 (1 H, 3-OCH), 2.84 (1 H, 12-OH),
2.73 (1 H, 2-CH), 2.72 (1 H, 3′-NCH), 2.66 (1 H, 10-CH), 2.55
(1 H, 8-CH), 2.43 (1 H, 4-CH), 2.26 (6 H, 3′-NCH3 × 2), 2.12
(3 H, 9dNCOCH3), 2.07 (3 H, 2′-OCOCH3), 2.02 and 1.46 (2
H, 14-CH2), 1.73 and 1.33 (2 H, 4′-CH2), 1.69 and 1.39 (2 H,
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7-CH2), 1.31 (3 H, 6-CH3), 1.26 (3 H, 10-CH3), 1.23 (9 H,
2-CH3, 12-CH3 and 5′-CH3), 1.10 (3 H, 8-CH3), 0.97 (3 H,
4-CH3), 0.91 (3H, 15-CH3). 13C NMR (CDCl3, 125 MHz): δ
184.3, 176.4, 174.5, 169.7, 141.8, 122.3, 99.6, 81.5, 78.6, 78.0
(3-OCH and 13-OCH), 76.3, 75.9, 73.8, 71.7, 68.9, 65.8, 63.2,
43.7, 40.9, 39.9, 39.2, 38.5, 35.5, 31.1, 25.6, 23.3, 21.7, 21.2,
19.8, 19.5, 17.2, 15.8, 14.6, 11.8, 7.8. HRMS Calcd for
C37H62N2O11 (MH+) 711.4426. Found 711.4431.
2-CH), 3.65 (1 H, 5′-OCH), 3.44 (1 H, 4-CH), 2.87 (1 H, 3′-
NCH), 2.83 (1 H, 10-CH), 2.67 (1 H, 12-OH), 2.64 (1 H, 8-CH),
2.36 (6 H, 3′-NCH3 × 2), 2.10 (3 H, 9dNCOCH3), 2.08 and
1.57 (2 H, 14-CH2), 2.07 (3 H, 2′-OCOCH3), 1.84 and 1.38 (2
H, 4′-CH2), 1.79 and 1.49 (2 H, 7-CH2), 1.35 (3 H, 6-CH3),
1.33 (3 H, 2-CH3), 1.32 (3 H, 12-CH3), 1.29 (3 H, 10-CH3),
1.28 (3 H, 5′-CH3), 1.27 (3 H, 4-CH3), 1.17 (3 H, 8-CH3), 0.92
(3 H, 15-CH3). 13C NMR (CDCl3, 125 MHz): δ 205.5, 204.9,
184.2, 175.8, 169.8, 169.4, 99.8, 79.9, 79.2, 79.0, 75.9 (12-OC
and 16-CH2), 74.9, 71.2, 69.2, 68.9, 63.7, 51.1, 45.3, 40.7, 39.7,
38.8, 36.8, 30.9, 25.3, 23.1, 21.6, 21.4, 20.1, 19.7, 17.1, 15.6,
14.3, 12.9, 11.6. HRMS Calcd for C36H58N2O12 (MH+) 711.4063.
Found 711.4066.
(9E)-9-(Acetylimino)-3-de[(2,6-dideoxy-3-C-methyl-3-O-
methyl-r-L-ribo-hexopyranosyl)oxy]-9-deoxo-3-oxo-6,11-O-
[(2E)-2-({[6-(1H-pyrazol-1-yl)pyridin-3-yl]methoxy}imino)-
propane-1,3-diyl]erythromycin, EDP-420 (1). A solution of
hydroxylamine 412 (308 g, 1.619 mol) in EtOH (2.76 L) was
treated with 1 N aq HCl (2.76 L) at <25 °C by adjusting the
addition rate. The mixture was cooled to 0-5 °C and 12 (1.0
kg, 1.407 mol) was added at 0-5 °C. The mixture became a
clear solution and was agitated for 0.5-1 h, at which point the
reaction was complete as indicated by HPLC analysis (conver-
sion >97%). This was warmed to 15-20 °C, diluted with
EtOAc (7 L) and washed with saturated aq NaHCO3 (6 L).
The aqueous phase was back-extracted with EtOAc (3 L). The
combined organic extracts were concentrated in Vacuo to 1-1.5
L to give a solution of 13 (oxime E/Z ratio ∼4:1 by HPLC) in
EtOAc. The solvent was swapped to MeOH by charging MeOH
(4 L) and continuously concentrating in Vacuo to 1.5-2 L.
Additional MeOH (2 L) was added, and the resulting solution
was filtered. The filtrate (13 in MeOH) was agitated at ambient
temperature for 15-18 h, after which the 2′-deacetylation
reaction was complete as indicated by HPLC analysis (conver-
sion >97%). The reaction mixture was concentrated in Vacuo
to 1-2 L. The solvent was swapped to EtOH by charging EtOH
(5 L) and concentration to 2.5-3 L. H2O (3 L) was slowly
added until crystallization occurred while maintaining an internal
temperature of 70-80 °C. The hot slurry was gradually cooled
to 20-22 °C over a period of 3-4 h. The crystals were collected
by filtration, washed with EtOH/H2O (1:2, 0.3 L), and dried
under vacuum at 50-70 °C to afford crystalline crude 1 [typical
yield: 65%, typical purity of the desired E-oxime product: >90
area % by HPLC, oxime E/Z ratio >30:1].
3-Descladinose-6,11-O,O-bridged Ketone Erythromycin
A 9-Imine Acetamide 2′-Acetate (12). A solution of 10 (4.0
kg, 5.628 mol) in acetone (7 L) was treated with NaIO4 (2.648
kg, 12.38 mol) and followed by H2O (14 L). A 4 wt % aq OsO4
(96 mL, 15.72 mmol, 0.28 mol %) solution was added during
a period of 5 to 10 min. (Caution: OsO4 is highly poisonous,
even at low exposure levels, and must be handled with
appropriate precautions!) This was an exothermic reaction
and the temperature gradually rose to 40-45 °C over the period
of 1 h., The reaction was complete after additional 2-3 h at
35-42 °C as indicated by HPLC (conversion >97%). If the
diol intermediate persisted, additional 10% NaIO4 (264 g, 1.236
mol) was added and the reaction mixture was agitated for 1 h.
After cooling to 20-25 °C, the reaction mixtue was diluted
with EtOAc (40 L), treated with saturated aq NaHCO3 (16 L),
and the resulting two-phase mixture was agitated vigorously
for 15 min. The organic phase was separated and the remaining
aq suspension was further extracted with EtOAc (2 × 16 L).
The combined organic extracts were treated with a mixed
solution of Na2S2O5 (0.40 kg, 2.104 mol) in H2O (1.5 L) and
saturated aq NaHCO3 (16 L) to remove remaining oxidizing
reagents. After agitation and separation, the organic phase was
washed with 15% aq NaCl (16 L) and concentrated in Vacuo
to 7-8 L of remaining volume. Toluene (8 L) was added and
distilled in Vacuo to dry the material azeotropically. CH2Cl2
(16 L) was added and concentrated again in Vacuo to 7-8 L
of remaining volume to give a solution of 11 in CH2Cl2., The
typical yield for this olefin oxidative cleavage step was 78% as
determined by 1H NMR analysis. A suspension of NCS (752
g, 5.632 mol) in CH2Cl2 (12 L) was agitated and cooled to -15
°C. Me2S (580 mL, 7.88 mol) was added dropwise over 25
min at -13 to -15 °C. After 15 min, the mixture was cooled
to -20 °C. This was treated with the above concentrated CH2Cl2
solution of 11 over 1 h at -18 to -20 °C. After agitating for
3 h, Et3N (784 mL, 5.624 mol) was added over 20 min at -15
to -18 °C by controlling the addition rate. After 1 h, the reaction
mixture was warmed to 5-10 °C and then diluted with EtOAc
(64 L) and washed with saturated aq NaHCO3 (2 × 20 L) and
15% aq NaCl (20 L). The organic phase was concentrated in
Vacuo to 5-6 L of remaining volume. The solvent was swapped
to EtOH by charging EtOH (about 10 L) while continuously
concentrating the organic solution in Vacuo until crystallization
occurred. The resulting slurry was gradually cooled to 0-4 °C
and held for 2 h. The crystals were collected by filtration,
washed with chilled EtOH (0.6 L at 5 °C), and dried under
vacuum at 30 °C until at constant weight to afford 2.08 kg (52%
overall yield for two steps) of 12. 1H NMR (CDCl3, 500 MHz):
δ 4.93 (1 H, 13-OCH), 4.78 (1 H, 2′-OCH), 4.63 (1 H, 11-
OCH), 4.53 (1 H, 1′-OCHO), 4.41 and 3.56 (2 H, 18-CH2),
4.34 and 3.95 (2 H, 16-CH2), 4.24 (1 H, 5-OCH), 4.00 (1 H,
Purification of (9E)-9-(Acetylimino)-3-de[(2,6-dideoxy-3-
C-methyl-3-O-methyl-r-L-ribo-hexopyranosyl)oxy]-9-deoxo-
3-oxo-6,11-O-[(2E)-2-({[6-(1H-pyrazol-1-yl)pyridin-3-
yl]methoxy}imino)propane-1,3-diyl]erythromycin, EDP-420
(1). Crude 1 (1.26 kg, 1.5 mol) was dissolved in ethyl acetate
(3.2 L) at 60-70 °C and was treated with a solution of L-malic
acid (0.24 kg, 1.79 mol) in ethyl acetate (3.2 L) at 55-65 °C.
After 10 min 2-propanol (6.4 L) was added. This was cooled
to 18-23 °C over the period of 2-3 h and held for at least
1 h. The crystals were collected by filtration, washed with ethyl
acetate/2-propanol (1:1, 0.6 L), and dried under vacuum at
40-45 °C until at constant weight. This was dissolved in ethyl
(12) Tang, D.; Qiu, Y.; Kim, H.; Or, Y. S.; Wang, Z. U.S. Patent 7,538,225,
2009.
Vol. 14, No. 3, 2010 / Organic Process Research & Development
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509

acetate (6.4 L) at 70-80 °C. 2-Propanol (6.4 L) was added at
60-65 °C. The hot slurry was cooled to 18-23 °C over 2-3
h and held for 1 h. The crystals were collected by filtration,
washed with ethyl acetate/2-propanol (1:1, 0.6 L), and dried
under vacuum at 40-45 °C until at constant weight. A
suspension of this crystalline solid in ethyl acetate (10 L) was
slowly treated with saturated aqueous sodium bicarbonate
solution (6.0 L) at 18-25 °C. After 20 min, the layers were
separated. The aqueous solution was extracted with ethyl acetate
(7.0 L). The combined organic extracts were washed with brine
(6.0 L), concentrated in Vacuo to 4 L of remaining volume,
and filtered. The filtrate was concentrated in Vacuo to 1-2 L.
The solvent was swapped to EtOH by charging ethanol (5.0 L)
and concentrating to 2.5-3 L of remaining volumes. H2O (3
L) was slowly added over 0.5 h until crystallization occurred
while maintaining an internal temperature of 70-80 °C. The
hot slurry was gradually cooled to 20-22 °C over a period of
3-4 h and held for at least 2 h. The crystals were collected by
filtration, washed with EtOH/H2O (1:1, 0.5 L), and dried under
vacuum at 40-45 °C to afford 0.8 kg (63% overall yield) of 1
as a white crystalline solid in >98 area % purity by HPLC
analysis. 1H NMR (CDCl3, 500 MHz): δ 8.58 (1 H, -CHdN
in pyrazole), 8.42 (1 H, -CHdN in pyridine), 7.96 (1 H,
-CHd in pyridine), 7.85 (1 H, -CHd in pyridine), 7.75 (1
H, -CHdN in pyrazole), 6.47 (1 H, -CHd in pyrazole), 5.31
(2 H, ArCH2O), 4.72 (1 H, 11-OCH), 4.64 and 4.51 (2 H, 16-
CH2), 4.63 (1 H, 13-OCH), 4.47 (1 H, 5-OCH), 4.35 (1 H, 1′-
OCHO), 3.99 (1 H, 2-CH), 3.98 and 3.55 (2 H, 18-CH2), 3.61
(1 H, 5′-OCH), 3.46 (1 H, 4-CH), 3.18 (1 H, 2′-OCH), 2.80 (1
H, 10-CH), 2.75 (1 H, 12-OH), 2.62 (1 H, 8-CH), 2.49 (1 H,
3′-NCH), 2.28 (6 H, 3′-NCH3 × 2), 2.11 (3 H, 9dNAc), 2.01
and 1.46 (2 H, 14-CH2), 1.81 and 1.68 (2 H, 7-CH2), 1.69 and
1.24 (2 H, 4′-CH2), 1.41 (3 H, 4-CH3), 1.38 (3 H, 6-CH3), 1.33
(6 H, 2-CH3 and 10-CH3), 1.29 (3 H, 12-CH3), 1.26 (3 H, 5′-
CH3), 1.16 (3 H, 8-CH3), 1.00 (3 H, 15-CH3). 13C NMR (CDCl3,
125 MHz): δ 205.8, 184.2, 177.8, 167.7, 153.9, 151.1, 148.3,
142.2, 139.3, 131.2, 127.3, 112.3, 108.0, 103.0, 79.4, 79.1, 76.5,
75.6, 74.7, 73.2, 70.6, 69.7, 66.2, 63.0, 62.9, 50.7, 46.2, 40.6,
40.5, 38.7, 37.2, 28.6, 25.4, 23.9, 21.6, 20.3, 19.6, 18.0, 15.1,
14.1, 13.8, 13.0. HRMS Calcd for C43H64N6O11 (MH+)
841.4706. Found 841.4704.
Acknowledgment
We thank our macrolide discovery group for discussions and
suggestions. We thank Dr. Emil Lobkovsky of Cornell Uni-
versity for EDP-420 X-ray structure determination and Dr.
Harry Li of Wilmington PharmaTech for SEM crystal image
of EDP-420 polymorphous form I.
Supporting Information Available
1H and 13C NMR spectra and assignments for all new
isolated compounds 1, 7, 10-13. Two-dimensional NMR
spectra (COSY, HMBC, HMQC, NOESY, TOCSY) for 1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Received for review August 27, 2009.
OP900228U
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