Note: Descriptions are shown in the official language in which they were submitted.
CA 02466358 2004-05-06
WO 03/040162 PCT/EP02/12234
Description
Method for the production of desclarithromycin, and intermediate products
The present invention describes a method for the production of
desclarithromycin via the novel intermediates of erythromycin N-oxide and
6-O-methylerythromycin N-oxide which are protected with silyl groups. In
addition, the novel intermediate of desclarithromycin N-oxide is described.
Desclarithromycin (II) is a building block for new types of macrolide
antibiotics. The production of desclarithromycin (II) starting from
clarithromycin (I), which can be produced from erythromycin A (III) by
various synthetic routes, is known (see, for example, Abbott Laboratories
WO 97/36912). In this case, desclarithromycin (II) is obtained by acid
treatment of clarithromycin (I). There is in this case selective elimination
of
the cladinose, and the resulting desclarithromycin (II) is obtained (J. Med.
Chem. 1998, 41, 4080 - 4100) (formula scheme I)
HCr
~ OH OH
OCH3
Clarithromycin (t) Desctarithromycin (II)
Formula scheme I
A further possibility is to produce desclarithromycin (II) directly from
erythromycin A (III) in accordance with the following synthetic sequence:
Firstly, erythromycin A (III) is oximated by the action of hydroxylamine (see,
for example, Abbott Laboratories WO 97/38000). The cladinose is
eliminated and removed by extraction from the resulting erythromycin
CA 02466358 2004-05-06
2
a
oxime by acid treatment (formula scheme II). Decladinosed erythromycin
oxime (IV) is obtained.
,OH
NHzOH ~ HCI
OH
Erythromycin A (III) Decladinosed erythromycin oxime (IV)
Formula scheme II
Subsequently (as disclosed by Bonnet et al. United States Patent
5,969,161), the oxime group is protected with methoxypropene under
slightly acidic conditions and the hydroxyl groups are protected by the
action of trimethylchlorosilane under basic conditions (formula scheme III).
OCH3 Trimethyl
/~ chlorosilane
Decladinosed erythromycin oxime (IV)
Disilylated decladinosed erythromycin
oxime acetonide (V)
Formula scheme III
CA 02466358 2004-05-06
3
The protected compound is methylated in the 6-O position by the action of,
for example, methyl iodide and a strong base (e.g. potassium hydroxide)
and subsequently converted by acid elimination of the protective groups
into desclarithromycin oxime (VI) (formula scheme IV).
KOH, HCI
Disilylated decladinosed erythromycin oxime acetonide (V)
Desclarithromycin oxime (VI)
Formula scheme IV
Desclarithromycin (II) is then obtained by the action of sodium metabisulfite
on desclarithromycin (VI) (formula scheme V).
NazS205
OH
Desclarithromycin oxime (VI) Desclarithromycin (Il)
i=ormelschema V
Formula scheme V
CA 02466358 2004-05-06
A disadvantage of this method is the production of polymethylated by-
products which, besides the methylation in the 6-O position, also exhibit '
methylated hydroxyl groups in the 11 andlor 12 position of the molecule
(e.g. formulae (VII) and (VIII)). They interfere with further processing of
desclarithromycin (II) to macrolide antibiotics and must therefore be
removed beforehand by elaborate purification processes. A further
disadvantage is the use of oximated intermediates because, in this case,
E/Z isomers occur and have different physical properties (e.g. solubility)
and therefore lead to losses of yield during reworking.
4
H
OH
Formula (VII) Formula (VIII)
It is an object of the present invention to find a method for the production
of
desclarithromycin (II) which avoids the disadvantages described above.
This can be achieved by following the synthetic route, what is characterized
by new types of intermediate compounds, as follows:
The present invention relates to a method for the production of
desclarithromycin in which erythromycin A (III) is initially silylated in the
2'-
O position and 4"-O position by silylation with basic agents,
trialkylchlorosilane and trialkylsilylimidazole are preferred) in a known
manner Y. Kawashima et al., Chem. Pharm. Bull. 38, 1485-1489, 1990 (IX).
The conditions described in Y. Kawashima et al., Chem. Pharm. Bull. 38,
1485-1489, 1990, are preferred (formula scheme VI).
CA 02466358 2004-05-06
Rssicuaase
~H R = CH3, I
__
Erythromycin A (III) Silylated erythromycin A (IX)
5 Formula scheme VI
The silylated erythromycin A is subsequently converted by oxidation with
customary oxidizing agents, hydrogen peroxide or m-chloroperbenzoic acid
are preferred, into the silylated erythromycin A N-oxide (X) (formula
scheme VII).
H202 or m-CPi3
R = CH3, C2Hs
R'= H or Sins
__
__..3
Silylated erythromycin A (IX) Silylated erythromycin N-oxide (X)
Formelschema VII
Formula scheme VII
It is optionally possible also to change the sequence of the first two steps
(that is to say firstly to N-oxidize erythromycin and then to introduce the
silyl
protective groups), or the two steps can be linked in a one-pot reaction.
CA 02466358 2004-05-06
6
The protected compound is then methylated selectively in the 6-O position
with a methylating agent, methyl iodide or dimethyl sulfate are preferred, '
under basic conditions, basification by addition of potassium hydroxide is
preferred (formula scheme VIII).
CH31 or (CH3)2SO,MOH
0
+_-
R3Si0 O N~ 0
R = CH9, C2H5
OCH OR, R' = H or SiR3 --
s
Silylated erythromycin N-oxide (X) Sifyated 6-O-methylerythromycin N-oxide
(XI)
Formelschema (VIII)
Formula scheme (VIII)
The cladinose and the silyl protective groups are eliminated from the
silylated 6-O-methylerythrornycin N-oxide (XI) by acid treatment, the
addition of HCI is preferred (formula scheme (IX). This results in
desclarithromycin N-oxide (XII).
HCI
Silylated 6-O-methylerythromycin N-oxide (XI)
Desclarithromycin N-oxide (XII)
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7
Formula scheme (IX)
Desclarithromycin N-oxide (XII) is then reduced by known methods,
preferably catalytically with palladiumlcarbon in the presence of hydrogen
or of a hydrogen donor, preferably with cyclohexene), Raney nickel and
hydrogen or sodium bisulfate, to desclarithromycin (II) (formula scheme X).
irbon and hydrogen or
y nickel and hydrogen or
m bisulfate
iui~~ u~au~~i~c
OH
Desciar'tthromycin N-oxide (XII) Desclarithromycin (la)
Formeaschema (X)
Formula scheme (X)
It is optionally possible also to change the sequence of the last two steps
(formula schemes IX and X) (that is to say firstly to reduce the N-oxide to
the amine and then to eliminate the cladinose and the protective groups),
or the two steps can be linked in a one-pot reaction, e.g. by stirring the
solution of desclarithromycin N-oxide (XII) obtained as shown in formula
scheme (IX) with an aqueous sodium bisulfate solution until (XII) is reduced
to desclarithromycin (II), and isolating the latter from the reaction mixture,
for example by crystallization.
The described reaction steps, which are illustrated by formula schemes (VI)
to (X) may proceed under various conditions. It is sensible to vary the
reaction conditions, taking account of generally known relevant prior art
methods, in order to find the best embodiment. According to the current
state of knowledge, the following reaction conditions are preferred:
The reaction depicted in formula scheme VI takes place in an organic
solvent, preferably ethyl acetate, butyl acetate, dichloromethane, MTB
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ether, THF, toluene, especially ethyl acetate. The reaction can take place
at various temperatures, the procedure at room temperature is preferred.
The reaction depicted in formula scheme VII takes place in an organic
solvent, preferably dichloromethane, ethyl acetate, butyl acetate, DMF,
N,N-dimethylacetamide, NMP, especially dichloromethane. The reaction
can take place at various temperatures, the procedure at about 0°C is
preferred.
The reaction depicted in formula scheme VIII takes place in an organic
solvent, preferably dimethyl sulfoxide, tetrahydrofuran, DMF, N,N-
dimethylacetamide, NMP, dimethyltetrahydropyrimidinone (DMPU),
especially a mixture of preferably equal parts of dimethyl sulfoxide and
tetrahydrofuran. The reaction can take place at various temperatures, the
procedure at room temperature is preferred.
The reaction depicted in formula scheme IX preferably takes place in
aqueous phase. The reaction can take place at various temperatures, the
procedure at room temperature is preferred.
The reaction depicted in formula scheme X takes place in an organic
solvent, preferably dichloromethane, ethyl acetate, butyl acetate, THF,
DMF, N,N-dimethylacetamide, NMP, especially dichloromethane. The
reaction can take place at various temperatures, the procedure at room
temperature is preferred.
For the reactions shown in formula schemes VI to X it may be helpful to
carry out the reaction under a protective gas atmosphere. The resulting
products can be isolated in various ways, such as, for example, filtration,
extraction, by chromatography etc.
The compounds of the formulae X, XI and XII have not previously been
disclosed and, like the methods for producing them, the present invention
likewise relates thereto. Said compounds are particularly suitable as
intermediate products in chemical synthesis, especially for the production
of desclarithromycin.
The following exemplary embodiments are intended to explain the present
invention in detail without the invention being restricted to the embodiment
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9
described in the examples. The individual features of the examples stand
for specific embodiments which can be combined with generalized features
as disclosed in the descriptive text and/or in the claims.
TLC analyses were carried out on coated glass plates (5 x 20 cm, silica gel
60 F2~ from Merck Darmstadt) in ascending mode, the gas phase being
saturated with eluent vapor. The staining {detection of the separated
reaction products) after development of the TLC plate and drying with a
hot-air blower took place by briefly immersing the TLC plate in a solution of
25 g of molybdatophosphoric acid and 10 g of cerium(IV) sulfate in 940 ml
of water and 60 ml of conc. sulfuric acid, allowing the TLC plate to drip dry
and finally heating it at about 160°C on a DESAGA thermoplate ST"". 'H-
NMR spectrum and '3C-NMR spectrum were recorded using a Bruker 400
UItraShieIdT"" spectrometer. For the interpretation of the '3C-NMR spectra,
primary, secondary, tertiary and quaternary carbon atoms were
differentiated by recording DEPT 135° spectra. However, no
multidimensional spectra ('H-'H or 'H-'3C correlation) were recorded. It
therefore cannot be precluded that signal assignments need to be
interchanged, especially for protons or'3C atoms of the same multiplicity.
Example 1:
Synthesis of 2',4"-O-bis(trimethylsilyl)erythromycin A (formula IX, R = CH3)
[modified on the basis of Y. Kawashima et al. Chem. Pharm. Bull. 38, 1485-
1489 (1990)]
Erythromycin A from Abbott Laboratories, which contain 94.0 HPLC area
percent of erythromycin A, was employed in this approach. The water
content according to Karl-Fischer titration was 0.5% by weight.
A clear solution of 36.7 g (50.0 mmol tel qel, 47.0 mmol content) of
erythromycin A was prepared in 1000 ml of ethyl acetate in a 2 I flask with
mechanical stirrer, thermometer and dropping funnel under a nitrogen
atmosphere. While maintaining the temperature at 20°C (waterbath) a
solution of 8.15 g (74.3 mmol) of trimethylchlorosilane and 10.52 g
(72.7 mmol) of N-(trimethylsilyl)imidazole in 50 ml of ethyl acetate was
added dropwise over the course of 30 min. The reaction was exothermic.
15 min after starting the dropwise addition, a precipitate formed and
initially
formed lumps but subsequently became a well-dispersed suspension. TLC
monitoring (CH2C12 / MeOH 9:1 plus 1 % 25% strength ammonia solution)
after 0.25 h showed complete conversion of the erythromycin A (Rf = 0.43)
CA 02466358 2004-05-06
. to the title compound (ca. 70%; Rf = 0.67) and the monosilyl
product
(ca.
30%; Rf = 0.54). After 2.5 h, the monosilyl intermediate product
had been
converted into the title compound, apart from about 5% remaining.
The
suspension was poured into a magnetically stirred ice-cold solution
of 15 g
5 (178.6 mmol) of sodium bicarbonate into 285 ml of water. The
aqueous
phase was separated off and the organic phase was vfiashed first
with
300 ml of water and then with 300 ml of saturated brine. The organic
phase
was dried over magnesium sulfate, filtered, evaporated to dryness
in vacuo
at a bath temperature of 40C and the crystalline residue was dried
under
10 high vacuum (HV) (44.6 g, 101.5% of theory of crude product).
The residue
was mixed with 150 ml of n-heptane and slowly heated. A clear
colorless
solution formed at 84C. It was allowed to cool, removing the heating
bath,
and at 65C was seeded with crystals of the title compound. It
was allowed
to cool further with mechanical stirring (320 rpm) to room temperature,
and
was then cooled to 15C and stirred at this temperature for a further
min. The precipitate was filtered off with suction on a G4 glass
frit, washed
with 20 ml of n-heptane and then dried in vacuo in a stream of
nitrogen at
40C. 31.0 g of white crystals were obtained. The mother liquor
was
concentrated to one third of the volume under weak vacuum, whereupon
this solution became slightly cloudy. It was cooled to 10C and
stirred at
this temperature for 15 min. The precipitate was filtered off
with suction,
washed with 10 ml of n-heptane and then dried under HV. 4.4 g
of white
crystals were obtained. Total yield: 35.4 g (40.3 mmol, 85.7%
of theory),
melting point: 213-215C (Lit. 194-197C). 'H-NMR (400 MHz, CDC13):
8 =
4.98 (dd, 1 H, 13-H), 4.83 (d, 1 H, 1 "-H), 4.39 (d, 1 H, 1'-H),
4.22 (m, 1 H, 5"-
H), 4.16 (d, 1 H, 3-H), 3.83 (1 H, 11-OH), 3.80 (1 H, 11-H), 3.59
(m, 1 H, 5'-
H), 3.56 (d, 1 H, 5-H), 3.30 (s, 3H, 3"-OMe), 3.18 (m, 1 H, 2'-H),
3.17 (d, 1 H,
4"-H), 3.11 (qua, 1 H, 10-H), 3.01 (s, 1 H, 12-OH), 2.80 (qui,
1 H, 2-H), 2.74
(m, 1 H, 8-H), 2.53 (m, 1 H, 3'-H), 2.37 (d, 1 H, 2"-H), 2.23
(br s, 6H, NMe2),
1.97-1.82 (m, 3H, 14-H, 4-H, 7-H), 1.72-1.60 (m, 4H, 7-H, 6-OH,
4'-H),
1.55-1.45 (m, 2H, 2'-H, 14-H), 1.44 (s, 3H, 6-Me), 1.23-1.10 (22H,
6"-Me, 8-
Me, 3"-Me, 4'-H, 6'-Me, 12-Me, 10-Me, 2-Me), 1.09 (d, 3H, 4-Me),
0.87 (t,
3H, 15-H), 0.16 (s, 9H, 4"-OSiMe3), 0.10 (s, 9H, 2'-OSiMe3). '3C-NMR
(100
MHz, CDC13): 8 = 221.3 (C-9), 176.4 (C-1 ), 102.9 (C-1'), 96.8
(C-1 "), 81.7
(C-5), 81.0 (C-4"), 79.6 (C-3), 77.1 (C-13), 75.3 (C-6), 75.0
(C-12), 73.3 (C-
2'), 73.1 (C-3"), 69.0 (C-11 ), 67.8 (C-5'), 65.2 (C-3'), 65.1
(C-5"), 49.8 (3"-
OMe), 44.9 (C-2), 44.4 (C-8), 41.0 (NMe2), 40.5 (C-4), 39.0 (C-7),
38.7 (C-
10), 35.9 (C-2"), 29.8 (C-4'), 27.3 (6-Me), 22.2 (5'-Me), 21.7
(3"-Me), 21.4
(C-14), 19.4 (5"-Me), 18.3 (8-Me), 16.4 (12-Me), 15.6 (2-Me),
11.8 (10-Me),
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11
10.9 (C-15), 9.7 (4-Me), 1.02 [2'-OSi(CH3)3], 0.96 [4"-OSi(CH3)3]. MS (ESI):
[M+H]+ m/z = 878 (Cq3H83NO13s~2)~
The product can also be recrystallized / reprecipitated in high yield and
purity from acetone/water instead of n-heptane.
Example 2:
Synthesis of 4"-O-(trimethylsilyl)erythromycin A N-oxide (formula X, R=CH3,
R'=H)
a) Preparation of 99% pure 3-chloroperoxybenzoic acid from commercial
77% pure 3-chloroperoxybenzoic acid (Aldrich):
400 ml of phosphate buffer of pH 7 (Riedel 10240) were introduced into a
1 I round-bottomed flask with KPG stirrer, thermometer and calibrated pH
electrode under a nitrogen atmosphere. A pH of 7.5 was adjusted by
adding a total of 8.47 g of disodium hydrogen phosphate. 50.16
(223.8 mmol) of 77% pure 3-chloroperoxybenzoic acid were added all at
once thereto. A suspension formed. The pH fell, rapidly at first and then
more slowly, until it came to a stop at pH 6.42. The pH was raised again to
6.95 by adding 13.92 g of disodium hydrogen phosphate. The suspension
was filtered with suction. The solid was washed with water which had
previously been adjusted to pH 7, and was then dried under HV in a
desiccator. 31.9 g of white crystals (83% of theory based on the content of
the commercial material employed) were obtained. The water content
(K.F.- titr.) was 0.27%.
b) 4"-O-(Trimethylsilyl)erythromycin A N-oxide:
A clear solution was prepared from 13.18 g (15.0 mmol) of
disilylerythromycin A (from example 1 ) in 25 ml of dichloromethane
(0.025% water content according to K.-F. titrat.) in a 250 ml flask with
mechanical stirrer, thermometer and dropping funnel under a nitrogen
atmosphere, and 2.27 g (27.0 mmol) of dry sodium bicarbonate were
added. The suspension was cooled with an icebath to 0°C. A solution of
3.12 g (17.9 mmol) of the above approx. 99% pure 3-chloroperoxybenzoic
acid in 50 ml of dichloromethane was added dropwise thereto. The cooling
bath was removed and the mixture was allowed to warm to 23°C and
stirred at this temperature for 1 h, during which a thick suspension formed.
TLC (CH2C12 / MeOH 9:1 plus 1 % 25% strength ammonia solution) of a
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12
' ~ sample which had been removed by filtration with suction from the
precipitate showed clean quantitative conversion of the precursor (Rf =
0.67) into the product (Rf = 0.25). The suspension was cooled in the
icebath at 2°C and, after addition of 40 ml of half-saturated aqueous
sodium bicarbonate solution, vigorously stirred. The mixture was filtered
with suction and washed with 2 X 10 ml of cold half-saturated sodium
bicarbonate solution, sucked dry and then dried under HV over phosphorus
pentoxide. 11.8 g (14.4 mmol, 95.6% of theory) of colorless crystals were
obtained, melting point 204 - 205°C (composition). 'H-NMR (400 MHz,
CDC13): 8 = 5.03 (dd, 1 H, 13-H), 4.89 (d, 1 H, 1 "-H), 4.68 (d, 1 H, 1'-H),
4.18
(m, 1 H, 5"-H), 3.98 (d, 1 H, 3-H), 3.88 (s, 1 H, 11-OH), 3.84 (m, 1 H, 5'-H),
3.80 (m, 1 H, 11-H), 3.74 (dd, 1 H, 2'-H); 3.58 (d, 1 H, 5-H), 3.47 (m, 1 H,
3'-
H), 3.36 (s, 3H, 3"-OMe), 3.18 and 3.17 (2 x s, 2 x 3H, N(O)Me2), 3.16
(concealed, 1 H, 4"-H), 3.09 (qua, 1 H, 10-H), 3.05 (s, 1 H, 12-OH), 2.90
(qui,
1 H, 2-H), 2.68 (m, 1 H, 8-H), 2.38 (d, 1 H, 2"-H), 2.36 (s, 1 H, 6-OH), 2.06-
1.83 (m, 4H, 4'-, 4-, 7-, 14-H), 1.71 (d, 1 H, 2'-OH), 1.57 - 1.45 (m, 3H, 4'-
,
2"-, 14-H), 1.45 (s, 3H, 6-Me), 1.30 (m, 1 H, 7-H), 1.24 - 1.08 (24H, 8 x Me),
0.85 (t, 3H, 15-H), 0.15 (s, 9H, 4"-OSiMe3). '3C-NMR (100 MHz, CDC13): b
= 221.7 (C-9), 175.9 (C-1 ), 101.8 (C-1'), 96.2 (C-1 "), 83.1 (C-5), 80.7 (C-
3),
79.3 (C-4"), 76.6 (C-13), 75.8 (C-3'), 74.8 and 74.7 (C-6, C-12), 73.2 (C-3"),
72.8 (C-2'), 68.9 (C-11 ), 66.1 (C-5'); 65.0 (C-5"), 58.8 [N(O)-CH3], 51.8
(N(O)-CH3], 49.7 (3"-OCH3), 45.1 (C-2), 44.6 (C-8), 39.3 (C-4), 38.5 (C-7),
37.8 (C-10), 35.6 and 35.0 (C-2", C-4'), 26.8 (6-CH3), 22.2 (5'-CH3), 21.6
(3"-CH3), 21.1 (C-14), 19.3 (5"-CH3), 18.3 (8-CH3), 16.1 (12-(H3), 15.9 (2-
CH3), 12.0 (10-CH3), 10.6 (C-15), 9.1 (4-CH3), 0.9 [4"-OSi(CH3)3]. MS (ESI)
m/z = 822 (C4pH75NO14S~).
Reaction of 1.0 equivalent of disilylerythromycin (Example 1 ) with 1.25
equivalents of commercial 77% pure 3-chloroperoxybenzoic acid in
dichloromethane at 0°C (formation of a two-phase mixture) likewise
results
in clean formation of the N-oxide, which can be isolated in a yield of 90-
93% of theory.
Example 3:
Methylation of 4"-O-(trimethylsilyl)erythromycin A N-oxide to 4"-O-
(trimethylsilyl)clarithromycin N-oxide [6-O-methyl-4"-O-(trimethylsilyl)-
erythromycin A N-oxide; formula XI, R = CH3, R' = H]
CA 02466358 2004-05-06
r
' 13
5.48 g (6.66 mmol) of 4"-O-(trimethylsilyl)erythromycin A N-oxide (from
Example 2) was stirred in 25 ml of dimethyl sulfoxide and 25 ml of
tetrahydrofuran under a nitrogen atmosphere in a 100 ml flask with
mechanical stirrer, thermometer and septum to give a thin suspension. This
was cooled to 0°C with an icebath. 559 mg (8.47 mmol) of 85% pure
potassium hydroxide powder were added ali at once. A deep yellow, cloudy
solution formed, to which were added, while stirring at 0°C, 1.04 ml
(16.40 mmol) of methyl iodide, during which the reaction temperature rose
from +2 to +4°C. The reaction mixture was allowed to warm to room
temperature while stirring over the course of half an hour, during which it
became pale yellow. After stirring for a further 2 hours at room temperature,
180 ml of ethyl acetate and 120 ml of ice-water were added. The aqueous
phase was separated, and the organic phase was washed with 120 ml of
water and then with 50 ml of water. The combined aqueous wash phases
were immediately back-extracted with 50 ml of ethyl acetate, and this
extract was washed with 20 ml of water/5 ml of saturated brine. The
combined organic phases were dried over sodium sulfate, filtered and
concentrated in vacuo, and the residue was dried under HV to give a solid
foam. 5.23 g (6.25 mmol, 94% of theory) of crude product were obtained, of
which about 50-60% consisted of the title compound. 'H-NMR (400 MHz,
CDC13): 8 = 3.37 (s, 3H, 3"-OMe), 3.22 [2 x s, 6H, 3'-N(O)Me2], 3.04 (s, 3H,
6-OMe), 0.15 (s, 9H, 4"-OSiMe3). ~3C_NMR (100 MHz, CDCI3): 8 = 221.5
(C-9), 176.2 (C-1 ), 102.6 (C-1'), 58.6 [N(O)CH3], 52.5 [N(O)CH3], 51.0 (6
OCH3), 49.9 (3"-OCH3, 0.9 [4"-OSi(CH3)3]. MS (ESI): [M+H]+ m/z = 836
(C4~H»N0~4Si).
Example 4:
Cladinose- and silyl-elimination to give desclarithromycin N-oxide [6-O-
methylerythromycin A N-oxide; formula XII]
A solution of 3.2 ml of 12 N hydrochloric acid (38.4 mmol HCI) in 32 ml of
water was added to 5.2 g (6.22 mmol) of the crude product from Example 3
under a nitrogen atmosphere and with icebath cooling in a 100 ml flask with
mechanical stirrer. The reaction mixture was stirred for 2 h at room
temperature, then saturated with sodium chloride, adjusted to pH8
with aqueous ammonia solution and extracted with 5 x 50 ml of ethyl
acetate. The combined extracts were dried over sodium sulfate, filtered,
CA 02466358 2004-05-06
a
14
. evaporated to dryness in vacuo and dried under HV. 4.7 g of pale brown
solid were obtained.
An analytical sample was obtained by flash chromatography of a sample
(200 mg) of this crude product through 40 g of silica gel 60 (Merck, 0.040 -
0.063 mm) with the eluent dichlurorr~eihane i methanol 8:2. 95 mg of white
crystals were obtained, melting point 209 - 210°C (decomposition), >97%
pure according to HPLC analysis (LiChroCART 125 X 4 mm LiChrospher
100 RP18e, 5 ~.m, Det. 210 nm, 25°C, flow 0.5 ml/min, eluent A: CH3CN
CF3COZH 1000 : 0.5, eluent B: H20 / CF3C02H 1000 : 0.5; linear gradient
from 30% A / 70% B on injection to 50% A / 50% B after the
chromatography had lasted 10 minutes; retention time: 8.55 min), single
spot according to TLC analysis (CH2C12 / CH30H 8:2 plus 1 % 25% strength
ammonia solution, Rf = 0.34).'H-NMR (400 MHz, CDC13): 8 = 5.17 (dd, 1 H,
13-H), 4.48 (d, 1 H, 1'-H), 3.88 (s, 1 H, 11-OH), 3.86 (d, 1 H, 11-H), 3.80
(dd,
1 H, 2'-H), 3.72 (s, 1 H, 5-H), 3.64 (m, 1 H, 5'-H), 3.57 (d, 1 H, 3-H), 3.38
(m,
1 H, 3'-H), 3.27 (s, 1 H, 12-OH), 3.18 [s, 3H, N(O)Me], 3.15 [s, 3H, N(O)Me],
3.01 (m, 1 H, 10-H), 2.97 (s, 3H, 6-OMe), 2.65 (m, 1 H, 2-H), 2.57 (m, 1 H, 8-
H), 2.09 (qua, 1 H, 4'-H), 2.02 - 1.87 (m, 3H, 4-, 7-, 14-H), 1.53 (d, 1 H, 2'-
OH), 1.49 (m, 1 H, 14-H), 1.40 (d, 1 H, 4'-H), 1.37 (s, 3H, 12-Me), 1.31 (d,
3H, 5'-Me), 1.27 (d, 3H, 2-Me), ca. 1.26 (m, concealed, 1 H, 7-H), 1.19 (s,
3H, 6-Me), 1.16 (d, 3H, 8-Me), 1.15 (d, 3H, 10-Me), 1.13 (d, 3H, 4-Me),
0.84 (t, 3H, 15-H). '3C-NMR (100 MHz, CDC13): b = 220.4 (C-9), 175.2 (C-
1 ), 106.1 (C-1'), 89.0 (C-5), 78.7 (C-3), 78.0 (C-6), 76.5 (C-13), 75.7 (C-
3'),
74.2 (C-12), 72.1 (C-2'), 69.7 (C-5'), 68.2 (C-11 ), 59.0 [N(O)-CH3], 51.9
[N(O)-CH3], 49.4 (6-OCH3), 45.4 (C-2), 44.6 (C-8), 38.7 (C-7), 37.6 (C-4),
36.0 (C-10), 34.4 (C-4'), 21.4 (C-14), 20.9 (5'-CH3), 18.7 (6-CH3), 17.7 (8-
CH3), 16.2 (12-CH3), 15.3 (2-CH3), 12.5 (10-CH3), 10.3 (C-15), 8.3 (4-CH3).
MS (ESI): [M+H]+ m/z = 606 (C3pH55N011)~
Example 5:
Reduction of the crude N-oxide to desclarithromycin [6-O-methyl-
erythromycin A; formula II]
4.5 g of the crude desclarithromycin N-oxide from Example 4 were mixed
with 50 ml of dichloromethane and the solution of 1.5 g (7.9 mmol) of
sodium metabisulfite (Na2S205) in 15 ml of water in a 100 ml flask with
mechanical stirrer and the two-phase mixture was vigorously stirred at
room temperature under a nitrogen atmosphere. It was possible to follow
CA 02466358 2004-05-06
the reduction using the HPLC system described in Example 4 (retention
time of II = 7.37 min) and using the TLC system described in Example 4 (Rf
II = 0.52), and it was complete after 3 hours. The aqueous phase was
separated off and extracted with 20 ml of dichloromethane. The combined
5 organic phases were concentrated to about 15 ml in vacuo, and then 45 ml
of water were added, and the pH was adjusted to 1.0 witH 36% strength
hydrochloric acid. The organic phase was separated off, and remaining
cladinose and the secondary product thereof were washed out of the acidic
aqueous phase by extraction with 5 x 10 ml of dichloromethane. It was
10 possible to follow this procedure using the TLC system described in
Example 4. The aqueous phase was then adjusted to pH 5.3 with 25%
strength aqueous ammonia, the stirrer was switched off, a layer of 5 ml of
methyl isobutyl ketone (MIBK) was put on top of the aqueous solution, and
the two-phase mixture was stirred at a very low speed (about 20 rpm) with
15 negligible phase mixing at 20°C for 15 minutes. The MIBK phase
(containing impurities) was separated off in a separating funnel, and the
aqueous phase was slowly adjusted with 25% strength aqueous ammonia
while stirring vigorously at 25°C (slight cooling) to pH 9.5, the
solution being
seeded with pure product crystals at pH 7.5, and the product crystallizing
out from about pH 8.3 onwards. The suspension was then stirred at 25°C
for 30 minutes and at 15-20°C for a further 30 minutes. The precipitate
was
filtered off with suction, washed with 30 ml of water, sucked dry and dried
under HV at 40°C for 16 hours. 1.8 g of white crystals were obtained
(3.05 mmol, 46% of theory based on the 4"-O-(trimethylsilyl)erythromycin
N-oxide employed in Example 3). Taking account of the 200 mg removed in
Example 4 to obtain the analytical sample, the overall yield for the reactions
described in Examples 3 to 5 is 48% of theory), melting point 154 -
155°C.
'H-NMR (400 MHz, CDC13): 8 = 5.18 (dd, 1 H, 13-H), 4.38 (d, 1 H, 1'-H),
3.92 (s, 1 H, 11-OH), 3.87 (br s, 1 H, 3-OH), 3.86 (d, 1 H, 11-H), 3.68 (s, 1
H,
5-H), 3.55 (m, 2H, 3- and 5'-H), 3.26 (s, 1 H, 12-OH), 3.24 (dd, 1 H, 2'-H),
3.01 (qua, 1 H, 10-H), 2.97 (s, 3H, 6-OMe), 2.66 (m, 1 H, 2-H), 2.58 (m, 1 H,
8-H), 2.47 (m, 1 H, 3'-H), 2.26 (s, 6H, NMe2), 2.12 (m, 1 H, 4'-H), 1.94 (m,
2H, 4- and 7-H), 1.66 (d qua, 1 H, 14-H), 1.56 (dd, 1 H, 4'-H), 1.49 (m, 1 H,
14-H), 1.37 (s, 3H, 12-Me), 1.26 (d, 6H, 5'-Me and 2-Me), ca. 1.25 (m,
concealed, 1 H, 7-H), 1.18 (s, 3H, 6-Me), 1.13 (d, 6H, 8-Me and 10-Me),
1.12 (d, 3H, 4-Me), 0.84 (t, 3H, 15-H). '3C-NMR (100 MHz, CDC13): 8 =
220.6 (C-9), 175.0 (C-1 ), 106.6 (C-1'), 88.2 (C-5), 78.9 (C-3), 78.1 (C-6),
76.6 (C-13), 74.2 (C-12), 70.7 (C-2'), 70.2 (C-11 ), 69.8 (C-5'), 65.6 (C-3'),
49.5 (6-OCH3), 45.5 (C-2), 44.5 (C-8), 40.2 [3'-N(CH3)2], 38.7 (C-7), 37.5
CA 02466358 2004-05-06
16
(C-4), 35.9 (C-10), 28.1 (C-4'), 21.4 (C-14), 21.2 (5'-CH3), 18.8 (6-CH3),
17.7 (8-CH3), 16.2 (12-CH3), 15.2 (2-CH3), 12.6 (10-CH3), 10.4 (C-15), 8.2
(4-CH3). MS (ESI): [M+H]+ m/z = 590 (C3pH55N~10)~
The patent claims which follow and which form part of the contents of the
description contribute to the aisciosure of the invention.
