Novel penicillin derivatives

Novel penicillin derivatives of the formula ##STR1## and pharmaceutically acceptable salts thereof, wherein R is hydrogen or hydroxy, are produced by acylating ampicillin, amoxicillin or a salt thereof with 4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid or its functional derivative. They have high anti-pseudomonas activity and other antibacterial activity against gram-positive and gram-negative bacteria, and are of extremely low toxicity.

BACKGROUND 
Some naphthyridine and quinoline derivatives of ampicillin possessing 
antibacterial activity are disclosed in U.S. Pat. No. 3,951,955. They are, 
however, not of sufficiently low toxicity. 
The present inventors have found that pyrazinoquinoline derivatives of 
penicillins of the formula (I) described hereinafter have excellent 
anti-pseudomonas activity and other antibacterial activity against 
gram-positive and gram-negative bacteria, and are of extremely lower 
toxicity as compared with the known penicillin derivatives.

DESCRIPTION AND PREFERRED EMBODIMENTS 
The present invention relates to noval penicillin derivatives that are 
useful as pharmacological agents. More particularly, the present invention 
concerns with novel penicillin derivatives of the formula(I) 
##STR2## 
and pharmaceutically acceptable salts thereof, wherein R is hydrogen or 
hydroxy. 
An object of the present invention is to provide some novel penicillin 
derivatives which are useful as antibacterial agents with extremely low 
toxicity, and are particular interest because of their activity against 
pseudomonas bacteria. 
The compound of the formula(I) may be prepared by acylating ampicillin, 
amoxicillin or a salt thereof with a novel compound 
4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid or its functional 
derivative in an organic solvent. The acylation temperature is suitably in 
the range of 0.degree. to 50.degree. C. 
Examples of the organic solvent are methylene chloride, chloroform, 
dioxane, acetonitrile, dimethylformamide, dimethyl sulfoxide, 
hexamethylphosphoric triamide and the like. 
Examples of suitable functional derivative of 4-hydroxypyrazino 
[2,3-f]quinoline-3-carboxylic acid are the corresponding acid halides 
(e.g., chloride and bromide), acid azide, and active esters (e.g., 
N-hydroxysuccinimide and N-hydroxyphthalimide). These functional 
derivatives may be prepared by reacting 4-hydroxy-pyrazino 
[2,3-f]quinoline-3-carboxylic acid with a corresponding halogenating 
agent, azide forming agent or ester forming agent in a known manner per 
se. 
When using 4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid as the 
reactant, the reaction may be carried out in the presence of a condensing 
agent, e.g., carbodiimides such as dicyclohexylcarbodiimide, 
1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline and isoxazolium salts 
such as Woodward's reagent. 
Suitable salts of ampicillin or amoxicillin include the corresponding 
organic base salts, for example, trimethylamine, triethylamine or pyridine 
salt. They are well documented in the prior art. 
4-Hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid used as the reactant 
may be prepared by the following reaction sequence. 
##STR3## 
In this reaction sequence, R.sup.1 and R.sup.2 are the same or different 
alkyl containing 1 to 3 carbon atoms. 
Condensation of 6-aminoquinoxaline(II) with a methylenemalonate (III) may 
be carried out with heating at 100.degree. to 150.degree. C. for 0.5 to 2 
hours. Examples of methylenemalonate(III) are diethyl 
ethoxymethylenemalonate, dimethyl ethoxymethylenemalonate, diisopropyl 
methoxymethylenemalonate, and diethyl methoxymethylenemalonate. 
Ring closure of N-(6-quinoxalyl)aminomethylenemalonate(IV) may be carried 
out in the presence of an organic solvent such as diphenyl, diphenyl ether 
or dibutyl phthalate, at the temperature of 250.degree. to 300.degree. C. 
4-Hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid(VI) may be obtained by 
hydrolysing a 4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid 
ester(V) in the presence of a caustic alkali such as potassium hydroxide 
or sodium hydroxide at 25.degree. C. to the boiling temperature, in a 
known manner per se. 
The pharmaceutically acceptable salts of the compounds of the formula(I) 
include the corresponding alkali metal salts such as sodium and potassium 
salts, alkali earth metal salts such as calcium, barium and magnesium 
salts, unsubstituted and substituted ammonium salts, and arginine salt. 
They are obtained by reacting the compounds of the formula(I) with the 
corresponding base in a known manner per se. 
The compounds of the present invention have high antipseudomonas activity 
and other antibacterial activity against gram-positive and gram-negative 
bacteria. For example, 
D-.alpha.-(4-hydroxy-pyrazino[2,3-f]quinoline-3-carbonamido)benzylpenicill 
in sodium salt(TPC-20) and 
D-.alpha.-(4-hydroxy-pyrazino[2,3-f]quinoline-3-carbonamido)-p-hydroxybenz 
ylbenzylpenicillin sodium salt(TAC-20) give the minimal inhibitory 
concentrations as shown in Table 1, by the agar dilution method. 
TABLE 1 
__________________________________________________________________________ 
TPC-20 
TAC-20 
AMPICILLIN 
CARBENICILLIN 
__________________________________________________________________________ 
S. aureus 209 P 
0.1 1.6 &lt;0.05 0.4 
S. smith 0.4 0.8 &lt;0.05 0.4 
E. coli NIHJ 2 
1.56 3.1 6.2 6.2 
E. coli B &lt;0.05 
0.4 0.2 0.8 
Kleb. pneumo. 
0.8 1.6 12.5 50 
Kleb. pneumo. 309 
3.1 12.5 50 &gt;400 
Ent. cloacae IFO 13535 
3.1 6.2 .gtoreq.200 
25 
Proteus vulgaris 
0.8 1.6 0.8 0.8 
Serratia marc. IID 618 
0.4 3.1 12.5 3.1 
Sal. enteritidis 
0.8 0.8 0.2 0.8 
Ps. aeruginosa NC 5 
12.5 12.5 .gtoreq.200 
100 
Ps. aeruginosa GNB 1-1-1 
3.1 3.1 &gt;400 50 
Ps. aeruginosa IID 1052 
3.1 6.2 .gtoreq.200 
25 
__________________________________________________________________________ 
(Numbers:.mu.g/ml) 
The compounds of the present invention may be used as antibacterial agents 
in the same manner as other penicillins. For example, they may be used in 
mammals in an amount of about 1 mg to 100 mg/kg, daily, parenterally, in 
single or two to four divided doses to treat infections of bacterial 
origin, e.g., 10 mg/kg in mice. 
The compounds of the present invention are of low toxicity. That is, they 
hardly show any intraperitoneal acute toxicity in rats at a dose less than 
5000 mg/kg of body weight. 
The compounds of the present invention may be used alone or in combination 
as the active ingredients in any one or a variety of pharmaceutical 
preparations. They may be administered by parenteral injections such as 
subcutaneous, intramuscular or intravenous injection, in the form of 
solution or suspension in suitable media, e.g., sterile water, saline, 
glycols, oils, or as dry preparations suitable for the extempore 
preparation of injectable forms. In addition, the compounds of the present 
invention may be administered in the form of suppository in suitable 
media, e.g., stearic acid, its salt, talc, vegitable oils and glycols. 
The present invention is further illustrated by the following detailed 
examples which are not intended to limit the scope of the present 
invention. 
EXAMPLE 1 
D-.alpha.-(4-hydroxy-pyrazino[2,3-f]quinoline-3-carbonamido)-benzylpenicill 
in sodium salt 
Step A 
diethyl N-(6-quinoxalyl)aminomethylenemalonate 
A mixture of 6-aminoquinoxaline(19.2 g) and diethyl 
ethoxymethylenemalonate(34.8 g) was heated for an hour at 110.degree. C. 
After filtration, the crystals thus obtained were crystallized from 
ethanol to give diethyl N-(6-quinoxalyl)aminomethylenemalonate (37.5 g) as 
pale yellow needles, m.p. 112.degree.-114.degree. C. 
Step B 
ethyl 4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylate 
Diethyl N-(6-quinoxalyl)aminomethylenemalonate(37.5 g) was gradually added 
to diphenyl ether(300 ml) at 260.degree.-280.degree. C. The resulting 
mixture was heated for an additional hour at the same temperature. After 
cooling, the mixture was mixed with n-hexane (500 ml), and filtered off. 
The resulting solid was washed with n-hexane and acetone to give ethyl 
4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylate(28.8 g) as a colorless 
powder, m.p. 223.degree.-225.degree. C. 
Step C 
4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid 
A mixture of ethyl 4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylate(28.8 g) 
and 10% potassium hydroxide solution(350 ml) was heated under reflux for 
an hour. The resulting reaction solution was acidified with a concentrated 
hydrochloric acid. After filtration, the resulting solid was washed with 
water and acetone, and then dried over phosphorus pentoxide to give 
4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid(23.5 g) as pale 
yellow crystalline powders, m.p. &gt;300.degree. C. 
Anal. Calcd. for C.sub.11 H.sub.7 N.sub.3 O.sub.3 : C, 59.75; H, 2.93; N, 
17.42. Found: C, 59.40; H, 3.17; N, 17.23. 
Step D 
N-hydroxysuccinimide ester of 
4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid 
4-Hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid(2.41 g) was refluxed 
with thionyl chloride(15 ml) for an hour, and then concentrated in vacuo. 
To the residue were added N-hydroxysuccinimide(1.27 g), 
N,N-dimethylformamide(50 ml) and pyridine (2 ml). The resultant mixture 
was stirred for 2 hours at room temperature. The solid product was 
collected, washed with N,N-dimethylformamide and acetone, and then dried 
over phosphorus pentoxide to give N-hydroxysuccinimide ester of 
4-hydroxy-pyrazine[2,3-f]quinoline-3-carboxylic acid(2.34 g), m.p. 
261.degree.-263.degree. C.(decomp.). 
Step E 
D-.alpha.-(4-hydroxy-pyrazino[2,3-f]quinoline-3-carbonamide)benzylpenicilli 
n sodium salt 
A solution of ampicillin trihydrate(403 mg), triethylamine (0.42 ml) and 
methylene chloride(10 ml) was dried over anhydrous magnesium sulfate. The 
dried solution was added to a stirred suspension of N-hydroxysuccinimide 
ester of 4-hydroxy-pyrazino[2,3-f] quinoline-3-carboxylic acid(338 mg) in 
hexamethylphosphoric triamide (10 ml) at room temperature. The resulting 
mixture was gently stirred for 6 hours at the same temperature. After 
addition of methylene chloride(50 ml), the reaction mixture was acidified 
to pH 2 with 10% hydrochloric acid. The organic layer was separated and 
dried over anhydrous magnesium sulfate. After removal of the solvent in 
vacuo, the residue was dissolved in ethyl acetate(50 ml) and added to a 
30% n-butanol solution of sodium 2-ethylhexanoate. After filtration, the 
separated crystals were washed with ethyl acetate and ether, and then died 
over phosphorus pentoxide to give 
D-.alpha.-(4-hydroxy-pyrazino[2,3-f]quinoline-3-carbonamido)benzylpenicill 
in sodium salt(494 mg), m.p. 235.degree. C. (decomp.) 
IR: .nu..sub.max.sup.KBr 1765 cm.sup.-1 (.beta.-lactam C=0) 
NMR: (DMSO-d.sub.6), .delta.1.48 (3H, s), 1.60 (3H, s), 4.05 (1H, s), 
5.30-5.70 (2H, m), 6.02 (1H, d, J=8 Hz), 7.10-7.70 (5H, m), 8.14 (2H, s), 
8.82 (1H, s), 8.89 (1H, d, J=2 Hz), 8.90-9.30 (1H, broad), 9.07 (1H, d, 
J=2 Hz), 11.31 (1H, d, J=8 Hz). 
EXAMPLE 2 
D-.alpha.-(4-hydroxy-pyrazino[2,3-f]quinoline-3-carbonamido)-p-hydroxybenzy 
lpenicillin sodium salt 
A solution of amoxicillin trihydrate(419 mg), triethylamine (0.42 ml), 
methylene chloride(7 ml) and hexamethylphosphoric triamide (7 ml) was 
dried over anhydrous magnesium sulfate. The dried solution was added 
dropwise to a suspension of N-hydroxysuccinimide ester of 
4-hydroxy-pyrazino[2,3-f]quinoline-3-carboxylic acid(338 mg) in 
hexamethylphosphoric triamide. The reaction mixture was treated by a 
manner similar to that described in Example 1, Step E to give 
D-.alpha.-(4-hydroxy-pyrazino[2,3-f]quinoline-3-carbonamido)-p-hydroxy-ben 
zylpenicillin sodium salt (385 mg), m.p. 223.degree.-226.degree. C. 
(decomp.). 
IR: .nu..sub.max.sup.KBr 1763 cm.sup.-1 (.beta.-lactam C=0) 
NMR: (DMSO-d.sub.6), .delta.1.49 (3H, s), 1.61 (3H, s), 4.05 (1H, s), 
5.34-5.52 (2H, m), 5.86 (1H, d, J=7 Hz), 6.72 (2H, d, J=8 Hz), 7.33 (2H, 
d, J=8 Hz), 8.16 (2 H, s), 8.82 (1H, s), 8.85-9.15 (1H, broad), 8.92 (1H, 
d, J=2 Hz), 9.10 (1H, d, J=2 Hz), 11.12 (1H, d, J=7 Hz).