Novel erythromycin compounds

Novel erythromycin compounds of the formula ##STR1## wherein R.sup.1 is hydrogen or methyl, and a pharmaceutically acceptable salt thereof are disclosed. They exhibit excellent antibacterial activity against Gram-positive bacteria, acid stability and in vivo activity.

The present invention relates to novel and useful erythromycin compounds of 
the formula 
##STR2## 
wherein R.sup.1 is hydrogen or methyl, and a pharmaceutically acceptable 
salt thereof, having a strong antibacterial activity against Gram-positive 
bacteria. 
The present invention is based on the discovery that novel compounds of 
formula(I) exhibit significantly antibacterial activity against 
Gram-positive bacteria even when administered orally, contrary to other 
closely analogous compounds such as erythromycin A. That is, although 
erythromycin A is known to be a useful macrolide antibiotic having a 
strong activity against Gram-positive bacteria, this compound has an 
undesirable property that it loses rapidly the antibacterial activity by 
the acid in stomach when administered orally, whereupon its blood 
concentration remains at a low level. 
Accordingly, it is an object of the present invention to provide novel 
compounds of formula(I) valuable as medicines possessing not only 
excellent antibacterial activity against Gram-positive bacteria and acid 
stability but also remarkable in vivo activity. 
The compound of formula(I) may be prepared, for example, by the following 
processes. 
Namely, a compound of the formula 
##STR3## 
may be reacted with methyl iodide in the presence of a suitable base in a 
solvent to give a compound of the formula 
##STR4## 
wherein R.sup.1 is as defined above. 
In the reaction, 5-10 moles of methyl iodide and 1-2 moles of the base are 
employed per mole of the compound of formula(II). The reaction proceeds at 
temperature ranging from -78.degree. C. to room temperature, preferably 
from -15.degree. C. to 5.degree. C. 
Examples of the base are an alkali metal hydride (e.g., lithium hydride, 
sodium hydride or potassium hydride), an alkali metal amide (e.g., lithium 
amide, sodium amide or potassium amide), butyllithium or lithium 
diisopropylamide. 
Suitable solvents include polar aprotic solvents such as 
N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide or 
hexamethylphosphoric triamide, preferably N,N-dimethylformamide, 
dimethylsulfoxide or their mixture with tetrahydrofuran. 
Purification of the compound of formula(III) may be carried out by using 
conventional methods such as silica gel column chromatography. 
According to the method of E. H. Flynn et al. in Journal of the American 
Chemical Society, 77, 3104 (1955), the compound of formula(III) may be 
treated to remove benzyloxycarbonyl group by hydrogenolysis, and then 
subjected to the reductive methylation in the presence of excess amount of 
formaldehyde to give the compound of formula(I). 
Alternatively, the compound of formula(I) may be obtained by performing 
removal of benzyloxycarbonyl group and N-methylation of the compound of 
formula(III), at the same time. 
The pharmaceutically acceptable salts of the compounds of formula(I) 
include salts with organic acids such as an organic carboxylic acid (e.g., 
tartaric acid, citric acid, stearic acid or succinic acid), 
methanesulfonic acid, aminoethanesulfonic acid, an amino acid (e.g., 
aspartic acid or glutamic acid) or the like. These salts may be obtained 
by treating the compound of formula(I) with the corresponding acid by the 
conventional manners. 
The compound of formula(II) may be prepared according to the 
above-described method of E. H. Flynn et al. 
The compound of the present invention can be used as therapeutic agents 
against Gram-positive bacteria, mycoplasma and chlamydia in mammals. For 
these purposes, a compound of formula(I) may be administered orally or 
parenterally in a conventional dosage form such as tablet, capsule, 
powder, troches, dry mixes, ointment, suspension or solution prepared 
according to conventional pharmaceutical practices. 
These compounds of formula(I) can be administered at a dosage of from about 
1 mg/kg to about 1000 mg/kg of body weight per day. The preferred dosage 
range is from about 5 mg/kg to about 200 mg/kg of body weight per day. 
The compounds of the present invention have extremely low toxicity. The 
LD.sub.50 in mice is in excess of 5000 mg/kg of body weight.

The present invention is further illustrated by the following detailed 
examples . 
EXAMPLE 1 
(1) In a mixture of 50 ml of dry dimethylsulfoxide and 100 ml of dry 
tetrahydrofuran were dissolved 30 g of 
O,N-dibenzyloxycarbonyl-des-N-methylerythromycin A and 18 ml of methyl 
iodide. 
The solution was stirred under cooling at -12.degree.--10.degree. C. in a 
nitrogen stream and 2.4 g of 55-65% sodium hydride oily dispersion were 
added thereto in small portions. The mixture was stirred for a further one 
hour. After completion of the reaction, 50 ml of triethylamine were poured 
into the reaction mixture with stirring under ice-cooling, and the 
precipitates were filtered off. The obtained solid product was washed 
thoroughly with ethyl acetate, and the washings and the mother liquor were 
combined. The combined liquor was washed with a saturated aqueous sodium 
chloride solution and dried over anhydrous magnesium sulfate. The solvent 
was evaporated in vacuo and the crude product was applied onto a silica 
gel dry column (E. Merck Darmstadt; silica gel 60 for column 
chromatography, 70-230 mesh, .phi.5.4.times.60 cm), and eluted with a 
mixture of ethyl acetate and n-hexane (1:1). 
15 ml each of fraction was collected and analyzed by silica gel thin layer 
chromatography (E. Merk Darmstadt; precoated thin layer chromatography 
plate silica gel 60 F254) for the presence of the reaction product, 
developing in a mixture of ethyl acetate and n-hexane (1:1). The fractions 
having Rf value 0.16 were combined (c.f., Rf value of starting compound 
0.07) and the solvent was evaporated in vacuo, affording 12.2 g of a 
colorless froth. 
(2) In a mixture of 1.32 g of sodium acetate, 0.8 ml of acetic acid, 40 ml 
of water and 200 ml of ethanol were dissolved 10 g of the colorless froth 
obtained in item(1), and 1.0 g of palladium black was added to the above 
solution. Catalytic reduction was performed for 5 hours at room 
temperature under atmospheric pressure in a gentle hydrogen stream. 32 ml 
of 37% aqueous formaldehyde solution were poured into the reaction mixture 
and the catalytic reduction was continued for a further 7 hours. After 
completion of the reaction, the catalyst was filtered off and the filtrate 
was concentrated under reduced pressure approximately to a quarter volume. 
To the concentrate were added 100 ml of water, and the mixture was 
adjusted to about pH 10 with an aqueous sodium carbonate solution. The 
mixture was extracted thoroughly with chloroform and the extract was 
washed with water and dried. After evaporation of the solvent in vacuo, 
the residue was recrystallized from a mixture of chloroform and diethyl 
ether, giving 6 g of crystals. 
The crystals were stirred for 5 hours in 500 ml of diethyl ether and 
filtered off. The filtrate was concentrated to dryness and the residual 
substance was recrystallized from a mixture of chloroform and diethyl 
ether, giving 4.5 g of 6-O-methylerythromycin A in the form of colorless 
needles. 
(3) A chloroform solution of 1 g of the crystals obtained in item(2) was 
absorbed on 5 g of a reversed phase silica gel (E. Merck Darmstadt, silica 
gel 60 silanized for column chromatography, 70-230 mesh) and the solvent 
was evaporated under reduced pressure. The silica gel was placed on a 
column packed with the reversed phase silica gel (.phi.5.0.times.40 cm), 
and eluted with a mixture of methanol and a 0.1 M phosphate buffer 
solution (pH 7.0) (3:2) to collect each 15 ml of eluate. 
Each fraction was analyzed by thin layer chromatography (E. Merck 
Darmstadt. thin layer chromatography plate silica gel 60 silanized 
precoated, thickness 0.25 mm), developing in a mixture of methanol and a 
0.1 M phosphate buffer solution (pH 7.0) (3:2). The fractions having Rf 
value 0.42 (c.f., erythromycin A, Rf value 0.46) were combined and most of 
the methanol was evaporated in vacuo. The residue was made alkaline with 
sodium carbonate and extracted with chloroform. The chloroform layer was 
washed with water, dried and concentrated in vacuo. The crystals obtained 
were recrystallized from a mixture of chloroform and diisopropyl ether 
(1:2), giving 700 mg of pure 6-O-methylerythromycin A (formula(I), R.sup.1 
is hydrogen) in the form of colorless needles. 
m.p. 217.degree.-220.degree. C. (with decomposition). 
Elementary analysis for C.sub.38 H.sub.69 NO.sub.13 ; Calcd. (%); C: 61.02; 
H: 9.30; N: 1.87, Found (%): C: 60.37; H: 9.18; N: 1.71. 
Mass spectrum m/e: M.sup.+ =747. 
IR.nu..sub.max.sup.CHCl.sbsp.3 cm.sup.-1 =3500, 1730, 1690. 
.sup.1 H-NMR (CDCl.sub.3): .delta.=0.84 (dd, 3H), 1.40 (s, 3H), 2.28 (s, 
6H), 3.03 (s, 3H), 3.33 (s, 3H), 4.44 (d, 1H), 4.93 (dd, 1H), 5.06 (dd, 
1H). 
UV.lambda..sub.max.sup.CHCl.sbsp.3 nm (.epsilon.): 288 (27.9). 
EXAMPLE 2 
(1) In 150 ml of dry N,N-dimethylformamide were dissolved 39.6 g of 
O,N-dibenzyloxycarbonyl-des-N-methylerythromycin A and 17 ml of methyl 
iodide. To the solution were added with stirring, 2.3 g of 50-55% sodium 
hydride oily dispersion, in small portions, under cooling at 
0.degree.-5.degree. C. in a nitrogen stream. The mixture was stirred for a 
further one hour. 
After completion of the reaction, 50 ml of triethylamine were poured into 
the reaction mixture, under ice-cooling, and the resulting precipitates 
were filtered off. The solid substance thus obtained was washed thoroughly 
with ethyl acetate and the washings were combined with the mother liquor. 
The combined solution was washed with a saturated aqueous sodium chloride 
solution and dried over anhydrous magnesium sulfate. The solvent was 
evaporated in vacuo and the resulting product was purified by silica gel 
column chromatography and detected by thin layer chromatography (E. Merck 
Darmstadt, precoated thin layer chromatography plate silica gel 60 F254), 
developing in a mixture of ethyl acetate and n-hexane (2:1). The fractions 
having Rf value 0.33 (c.f., Rf value of starting compound 0.20) were 
combined and the solvent was evaporated in vacuo, giving 10 g of 
6,11-di-O-methyl-O,N-dibenzyloxycarbonyl-des-N-methylerythromycin A in the 
form of colorless needles. 
m.p. 108.degree.-110.degree. C. 
NMR (CDCl.sub.3): .delta.=2.81 and 2.85 (3H), 3.07 (s, 3H), 3.57 (s, 3H), 
5.07-5.28 (4H), 7.32-7.50 (10H). 
(2) In a mixture of 0.4 ml of acetic acid, 20 ml of water and 100 ml of 
ethanol were dissolved 1.8 g of 
6,11-di-O-methyl-O,N-dibenzyloxycarbonyl-des-N-methylerythromycin A and 
0.66 g of sodium acetate, and catalytic reduction was performed at room 
temperature under atmospheric pressure in a gentle hydrogen stream, by 
using 0.2 g of palladium black. After completion of the reaction, the 
catalyst was filtered off, and the filtrate was concentrated under reduced 
pressure approximately to a quarter volume. 50 ml of water were added to 
the concentrate and the mixture was adjusted to about pH 10 with an 
aqueous sodium carbonate solution, then extracted with chloroform. The 
chloroform layer was washed with water, dried over anhydrous magnesium 
sulfate, and the solvent was evaporated in vacuo. The residue was 
recrystallized from a mixture of chloroform and ethyl ether, affording 
0.93 g of 6,11-di-O-methyl-des-N-methylerythromycin A in the form of 
colorless needles. 
m.p. 224.5.degree.-227.degree. C. 
NMR (CDCl.sub.3): .delta.=2.42 (s, 3H), 3.10 (s, 3H), 3.32 (s, 3H), 3.57 
(s, 3H). 
(3) To 50 ml of methanol were added 0.4 g of 
6,11-di-O-methyl-des-N-methylerythromycin A and 1 ml of 37% aquous 
formaldehyde solution. 
After the mixture was allowed to stand for one hour at room temperature, 
catalytic reduction was performed at room temperature under atmospheric 
pressure in a gentle hydrogen stream, by using 0.2 g of 5% palladium on 
charcoal. After completion of the reaction, the catalyst was filtered off 
and most of the methanol was evaporated from the filtrate in vacuo. 50 ml 
of water were added to the residue and the mixture was made alkaline by 
addition of sodium carbonate. The mixture was extracted with 
dichloromethane and the extract was washed with water and dried. The 
solvent was evaporated in vacuo and the residue was recrystallized from a 
mixture of chloroform and diethyl ether, giving 0.4 g of 
6,11-di-O-methyl-erythromycin A (formula(I), R.sup.1 is methyl) in the 
form of colorless needles. 
m.p. 251.degree.-253.degree. C. (with decomposition). 
Elementary analysis for C.sub.39 H.sub.71 NO.sub.13 : Calcd. (%): C: 61.47; 
H: 9.39; H: 1.84, Found (%): C: 61.48; H: 9.29; H: 1.72. 
Mass spectrum: M.sup.+ =761. 
IR.nu..sub.max.sup.CHCl.sbsp.3 cm.sup.-1 =3500, 1720, 1705. 
NMR (CDCl.sub.3): .delta.=1.42 (s, 3H), 2.33 (s, 6H), 3.12 (s, 3H), 3.35 
(s, 3H), 3.60 (s, 3H). 
UV.lambda..sub.max.sup.CHCl.sbsp.3 nm (.epsilon.): 292 (23.6). 
EXAMPLE 3 
In a mixture of 0.8 ml of acetic acid, 40 ml of water and 200 ml of ethanol 
were dissolved 9.4 g of 
6,11-di-O-methyl-O,N-dibenzyloxycarbonyl-des-N-methylerythromycin A and 
1.32 g of sodium acetate. Catalytic reduction was performed for 5 hours at 
room temperature under atmospheric pressure, by using 1 g of palladium 
black. Into the reaction mixture were poured 32 ml of 37% aqueous 
formaldehyde solution and catalytic reduction was continued for a further 
7 hours. After completion of the reaction, the catalyst was filtered off 
and the filtrate was concentrated in vacuo approximately to a quarter 
volume. 100 ml of water were added to the concentrate and the mixture was 
adjusted to about pH 10 with an aqueous sodium carbonate solution. The 
mixture was extracted with chloroform and the extract was washed and 
dried. The solvent was evaporated in vacuo and the residue was 
recrystallized from a mixture of dichloromethane and petroleum ether, 
affording 6 g of 6,11-di-O-methylerythromycin A (formula(I), R.sup.1 is 
methyl) in the form of colorless needles. 
The physicochemical properties of the compound coincided with those of the 
compound obtained in Example 2(3). 
Experiments made on pharmaceutical properties of the compounds of the 
present invention are summerized below. In these experiments, "TE-031" and 
"TE-032" refer to 6-O-methylerythromycin A and 
6,11-di-O-methylerythromycin A of the present invention, respectively. 
EXPERIMENT 1 
Acid stability 
The compound of the formula(I) was treated in the Clark-Lubs's buffer 
solution at pH 2 for the prescribed period of time. 
Thereafter, the remaining antibacterial activity was determined by a disc 
method, using Staphylococcus aureus FDA 209P. Erthromycin A was also 
tested for the control. The results are shown in Table 1. 
TABLE 1 
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Acid stability 
Time.sub.(min.) 
Remaining activity (%) 
Compound 0 15 30 60 120 
______________________________________ 
TE-031 100 100 95 80 65 
TE-032 100 100 95 85 65 
Erythromycin A 
100 1.5 0.5 0 0 
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EXPERIMENT 2 
Antibacterial activity 
The compound of the formula(I) was tested for the antibacterial activity by 
the agar plate dilution method, using erythromycin A for the control. The 
results, indicated as the minimum inhibitory concentrations (MIC), are 
shown in Table 2. 
TABLE 2 
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Antibacterial spectrum 
MIC value (mcg/ml) 
Compound 
Erythro- 
Microorganism TE-031 TE-032 mycin A 
______________________________________ 
Staphylococcus aureus FDA 209P 
0.05 0.1 0.2 
Staphylococcus aureus Smith 
0.2 0.1 0.4 
Staphylococcus aureus Terashima 
0.2 0.4 0.4 
*Staphylococcus aureus TPR-5 
0.2 0.4 0.4 
**Staphylococcus aureus TPR-7 
0.2 0.4 0.4 
***Staphylococcus epidermidis TPR-14 
0.2 0.4 0.4 
Staphylococcus epidermidis IID866 
0.2 0.4 0.4 
Micrococcus luteus ATCC 9341 
.ltoreq.0.05 
.ltoreq.0.05 
.ltoreq.0.05 
Bacillus subtilis ATCC 6633 
0.05 0.05 0.1 
Bacillus cereus ATCC 9634 
0.2 0.4 0.4 
Streptococcus faecalis ATCC 8043 
0.05 0.05 0.05 
______________________________________ 
*Strain resistant to sulfonamides and penicillins 
**Strain resistant to sulfonamides and tetracyclines 
***Strain resistant to sulfonamides, penicillins and chloramphenicol 
EXPERIMENT 3 
In vivo activity 
Male ddY mice (body weight, 18-22 g; 14 mice per group) were inoculated 
with Staphylococcus aureus Smith No. 4. The compound of the formula(I) was 
administered orally one hour after the inoculation, and the number of mice 
surviving for seven days after the administration was noted. The results 
are shown in Table 3. 
TABLE 3 
______________________________________ 
In vivo activity 
The number of surviving mice 
Dose Compound 
(mg/kg) TE-031 TE-032 Erythromycin A 
______________________________________ 
400 14 14 14 
100 14 14 11 
25 13 7 1 
6.25 3 4 0 
1.56 1 0 0 
0.40 0 0 0 
______________________________________