JPS63152318A - Anti-mycoplasmal agent - Google Patents

Abstract

PURPOSE:To obtain an anti-mycoplasmal agent useful not only as a drug, animal drug but also as an agent preventing mycoplasma contamination in cell culture and a removing agent, containing a pyridonecarboxylic acid or a salt thereof as an active ingredient. CONSTITUTION:A pyridonecarboxylic acid shown by formula [R1 is 1-piperazinyl or 1-pyrrolidinyl; R2 is cyclopropyl or phenyl; A is N, C-H or C-Y (Y is halogen); X is H, amino or F] or a salt thereof is blended with a drug carrier and pharmaceutically manufactured by a conventional procedure to give the aimed substance. A dose is 10mg-3g, preferably 50-1,000mg/day and administered once - several times dividedly. The compound shown by the formula in the form of usually reagent is added to a medium before or during cultivation in such a way that the concentration is 0.1-10mug/ml, preferably 0.4-3mug/ml and used for preventing mycoplasma contamination or as a removing agent.

Description

[Detailed description of the invention] Fields of use in industry The present invention relates to anti-My=1 plasma agents.

The anti-mycoplasma agent of the present invention is useful not only as a medicine or veterinary drug but also as an agent for preventing or removing mycoplasma contamination in cell culture.

Prior Art and Problems to be Solved Mycoplasma (lllycoplasma) lacks a cell wall and is surrounded by a three-layered cell membrane, and is a type of bacteria (Shizomycetes network) and viruses (Mi
crotatobiotes) [Maedo Biological Encyclopedia, 3rd edition, p. 1240 (
1985), Maedo Shoten].

Mycoplasma causes pneumonia and arthritis in humans, mastitis in cattle,
It causes chronic respiratory diseases in chickens.

There are few drugs that are effective against such mycoplasma infections. Ofloxacin has been reported as a pyridonecarboxylic acid anti-mycoplasma agent (Japanese Patent Application Laid-Open No. 60-184014'
) has been done. However, its anti-mycoplasma action is not necessarily strong enough.

Additionally, many cultured animal cells are contaminated with mycoplasma. Animal cells contaminated with mycoplasma undergo morphological, genetic, and physiological changes. The mycoplasma contamination rate in large-scale or long-term culture of animal cells is said to reach 80-90%. vaccine.

When producing physiologically active substances such as hormones, antibodies, and enzymes, it is required to use animal cells that are not contaminated with mycoplasma.

Therefore, the minimum inhibitory concentration (M
It is possible to contaminate or prevent mycoplasma by using minomycin or diamulin, which have a low IC). However, these drugs only act in a static mycoplasma-like manner and do not act in a mycoplasmicidal manner, and therefore, mycoplasma whose growth is once inhibited regenerates after a certain period of time (that is, becomes recontaminated). Therefore, once contaminated, there is no effective way to remove it, so the reality is that there is no other suitable method other than to discard the culture loft and start culturing anew.

Therefore, the present inventors have searched for various agents useful as agents for removing or preventing mycoplasma contamination in pharmaceuticals and veterinary medicines, and have completed the present invention.

The present invention relates to the general formula (where RI means a 1-piperazinyl group or a 1-pyrrolidinyl group which may be substituted with one or two substituents R).

The substituent R shown here means a fluorine atom, an amino group, a lower alkyl group having 1 to 5 carbon atoms, or a lower alkyl group having 1 to 5 carbon atoms which may be substituted with a 1-ruamino group, Alternatively, it means an amino group or a fluorine atom. R2 means a cyclopropyl group or a tuunyl group optionally substituted with one or two halogen atoms. A is N or C-H or C-Y
, and Y here means a halogen atom. X means a hydrogen atom, an amine group or a fluorine atom. ) The present invention relates to an anti-mycoplasma agent containing pyridonecarboxylic acid represented by () or pyridonecarboxylic acid as an active ingredient.

Here, the salts of pyrido7carboxylic acid #[I] include, for example, salts with inorganic acids such as hydrochloric acid and phosphoric acid; acetic acid, lactic acid,
Oxalic acid, succinic acid, methanesulfonic acid, maleic acid,
Salts with organic acids such as malonic acid and gluconic acid; Salts with acidic amino acids such as aspartic acid and glutamic acid; Salts or compounds with basic amino acids such as lysine and arginine [I]
of sodium, potassium, calcium, magnesium,
Examples include metal salts such as. Compound [I] also exists as a hydrate. Furthermore, the substituent R in RI is 2
When there is a cis isomer and a trans isomer, such isomers are also included in the general formula [I].

Substituent R in R1 of general formula [I] may be bonded to any position of the 1-pyrrolidinyl group or 1-piperazinyl group, but is preferably bonded to the 3- or 4-position. Examples of the substituent R are methyl, ethyl, and
Lower alkyl groups having 1 to 5 carbon atoms such as pyru, butyl, and bemethyl; Lower alkyl groups substituted with a fluorine atom or amine group such as fluoromedyl, 2-fluoroethyl, and aminomethyl; Lower alkyl groups such as methylaminomethyl Amino lower alkyl group; examples include fluorine atom and amino group.

Next, specific examples of compounds included in general formula [I] will be given. In addition, compounds 2,9゜IO and 15 in the following are described in JP-A-214773, JP-A-59-130880, JP-A 0O-2581 (i3 and IO-228479), and the others are compounds not described in the literature. Compounds not described in the literature can be easily produced by the method described in Reference Example 1 below, the method described in the above-mentioned patent publication, or a method analogous thereto.

Among these, compounds in which X is an amine group, salts thereof, or hydrates thereof are particularly useful as anti-mycoplasma agents.

Test Examples The various properties of these compounds and control compounds (hereinafter also collectively referred to as test drugs) as anti-mycoplasma agents will be explained in more detail by giving test examples below.

Test Example 1 Anti-mycoplasma activity (MIC) The minimum inhibitory concentration (MIC: μg7) of the test drug was determined by the agar dilution method.
ml) was measured.

(1) Preparation of mycoplasma solution for inoculation Mycoplasma cryopreservation solution 0.2-0.3mj! [Mycoplasma pneumoniae (M.

pneumoniae) and Acholeplasma laidlawi (8 choleplasma Iaidlaw).
fi), 0.5% glucose added Chanok (
Chanock) liquid medium pl+7.8 (Bacteriology Series 2
, “Isolation and Identification of Human, Animal, and Plant Mycoplasma”, Japanese Society of Bacteriology Education Committee, p. 8 (1982), Natane Publishing), Mycoplasma hyolainis (M, hy
In the case of Mycoplasma hyorinis (M, hyorhini+s), the liquid medium pi-
17.8 (daily magazine 32.34-38 (+979))
, for other Mycoplasma species, Chanock liquid medium supplemented with 0.2% arginine pt
l (3,81 2 to 3 kaku β) and serially diluted 10 times to 10,000 times using this stock solution in the preculture medium. After culturing for several days, among those in which discoloration of the medium was observed due to mycoplasma proliferation, the one with the highest dilution ratio was diluted 100 times with a preculture medium, and this was used as a mycoplasma solution for inoculation.

(2) Preparation of agar plate containing test drug
Prepare 000 μg 7 ml water + 8 liquids (if the test drug is poorly soluble, add equimolar amount of sodium hydroxide to dissolve it.

Same below). Prepare a 2-fold serial dilution series using this as a stock solution, and add 1 ml each to a Plus Deck petri dish. Chanock agar medium [Mycoplasma hymi1 lineis (M, hy.
In the case of M. orhinis), 0.5% Muji/additive was dissolved by heating, kept at 55°C, and dispensed in 9 lp portions into the test drug-containing Petri dishes and thoroughly mixed with the test drug. Prepare an agar plate containing the test drug.

(3) Measurement of MIC The mycoplasma solution for inoculation prepared in section (1) is inoculated in 3μβ portions onto an agar plate containing the test drug using a Cassara replicator (3W Sono pin). Mycoplasma pneumoniae = x (M, pneumoniae) + Mycoplasma alki = −−=−(M, arg; n
tni), Acholeplasma leidolorai (8, 1
The plates inoculated with Mycoplasma hyorinis (M, hyorh; n; s) were placed in a plastic bag and sealed together with a Plasdec petri dish containing water-soaked absorbent cotton. Mycoplasma pneumoniae (M.

pneumoniae) for 7 days, Mycoplasma arginini (M, arginini) and Acholeplasma laidlawii (^, Iaidlawii)
) is aerobically cultured at 37° C. under high humidity conditions for 2 days. For other mycoplasma species, perform anaerobic culture for 2 days using a gas bag anaerobic system (1) 13L). After culturing, the cells were observed using an Olympus stereomicroscope at a magnification of 20 to 100 times, and the minimum concentration of the test drug at which mycoplasma proliferation was not observed was defined as the MIC.

The results are shown in Tables 1 and 2 below.

(Left below) 7 Mycoplasma species AI Mycoplasma Nicomoni x (M, pneum
oniae) MacB: Mycoplasma orala (M, orala) Cto19299C, Mycoplasma hominis (M, hominis) PC-21
DI Mycoplasma fermentans (M, ferm.
entans) P[+-+8E: Mycoplasma
Salivarium (11,5allvar+um) PG
-21F, Mycoplasma buccal (M, buccal)
e) CH-20247G; Acholeplasma arginini (^laidlawN) PC-8H; Mycoplasma arginini (M, arginini)
G-2301; Mycoplasma hyolainis (M, h
yorh+n+s) BST-7 However, A-F are human-derived, G and 11 are bovine-derived, and I is pig-derived.

(Left below) As shown in Tables 1 and 2, the anti-mycoplasmal activity (MIC) of the compound of general formula CI is stronger than ofloxacin, which is the same pyrido/carboxylic acid.

Test Example 2 Anti-Mycoplasma Effect (MIC, MMC) Minimum inhibitory concentration CM I C by liquid dilution method.

μg/-β) and minimum mycoplasmacidal concentration (MMC
, μIH/wJ) was measured. In addition, MIC in this section
The measurement method is different from that in Test Example 1.

A 1,000 μg/ml aqueous solution of the test drug is prepared, diluted 5 times with preculture medium, and then a 2-fold dilution series is further prepared. Dispense 50 μβ of this diluted solution into wells of a microplate. Note that wells to which no test drug was added were used as controls.

A mycoplasma solution for inoculation prepared in the same manner as in Test Example 1 is dropped in an amount of 50 μβ into each of the test drug-free wells using a micropipette. Seal the microplate with a plate seal and incubate at 37°C for 5 minutes after storing the microplate.
Incubate for days. In the control without the addition of the test drug, discoloration of the medium due to mycoplasma proliferation was observed after 2 days of culture.

At this point, 3 μl of the liquid culture solution was added to Chanoc (Chanoc) containing no test drug using a Cassara replicator.
k) Inoculate on agar medium. The above liquid medium and agar medium were further cultured at 37°C for 3 days [anaerobic culture in the case of Mycoplasma orale (M, orale), Acholeplasma laidlawii and Mycoplasma arginin
>, perform aerobic culture. From the observation results on the 5th, M
Determine IC and MMG. That is, the minimum concentration of the test drug at which mycoplasma proliferation was not observed in a liquid medium was defined as MIC, and the minimum concentration of the test drug at which mycoplasma proliferation was not observed in an agar medium was defined as MMC.

The results are shown below.

As shown in Table 3, the MIC values and MMC values of Compound 1 and Compound 2 are the same. That is, Compound 1 and Compound 2 act mycoplasmicidal at the MIC value. On the other hand, minocytalline acts in a mycostatic manner at the MIC value and does not act in a mycoplasmicidal manner.

Test Example 3 Effect of removing and preventing mycoplasma contamination Test drug was applied to animal cells experimentally contaminated with mycoplasma.
The cells were allowed to act for one day, and then subcultured in a medium without the test drug added to see if mycoplasma would grow again (that is, recontaminate).

(1) Preparation of Mycoplasma 19-stained cells The mycoplasmas listed in Table 4 below are precultured as in Test Example 1 (1), and two volumes of the culture solution β are centrifuged at 1,500×g for 30 minutes to separate the mycoplasma.

Eagle's minimum essential medium supplemented with 10% bovine blood and 1 ml of 100 units of penicillin G, i.e. 2 Ill of 10% FI3S-MEM medium.
.

Suspend the isolated mycoplasma in the solution. On the other hand, mouse fibroblasts L-929 were suspended in 10%-FBS-MEM medium to a concentration of 2.5XIO' cells/l, and 4 ml of each was placed on two circular cover glasses (diameter 1 A tissue culture Petri dish (diameter 6
cm), and then further add the aforementioned My':J plasma turbid iffl1wj2. This at 37°C, 5%
After culturing in carbon dioxide gas for 3 days, subculture is performed twice.

Mycoplasma contamination was confirmed by the DNA binding/fluorochrome staining method (see [Isolation and Identification of Human and Animal Mycoplasma], pp. 95-97), and the cells were determined to be mycoplasma-contaminated cells.

[2) Mycoplasma Iη stain removal experiment Logarithmically grown mycoplasma-contaminated L-929 cells were cultured as in the previous section. A 1,000 μg/ml aqueous solution of the test drug is prepared, diluted with Eagle's minimum essential medium to a predetermined concentration, and added to the medium. Two Petri dishes were used for each test drug concentration, one with two circular cover glasses for Myco1 plasma testing, and the other one without a cover glass for subculture. And so. Cultivation in the test drug-containing medium was carried out for 4 days, and then 3 days.
Culture is continued for a week in a medium containing no test drug. Medium exchange is performed every 3-4 times, and subculture is performed once every week.

Tests for mycoplasma will be performed on the 3rd and 71st days, and every week thereafter. Mycoplasma is detected using DNA binding and fluorescent dye staining.

The results are shown below.

'The same Mycoplasma species as G and I-1 in Table 1 As shown in Table 4, in the system with addition of Compound 1 and Compound 2, Mycoplasma became negative after 3 days of culture, and after that, subculture without addition of the test agent was carried out. Even after culturing, no regrowth of mycoplasma was observed. On the other hand, even in the My7cytalin-added system, mycoplasma became negative after 3 days of culture, but after that, significant mycoplasma regrowth was observed in the test drug-free system.

X # ('jl-4-my;I plasma contamination removal/
Preventive effect Naturally infected human lung carcinoma cells PC=
10 and human amnion-derived cells 1-IM V-2 were tested for removal and prevention of Mycoplasma lTi staining according to Test Example 3, and the results shown in Table 5 were obtained.

The test drug addition period was 13 days for PC-10;
-140 for IMV-2. The mycoplasma species that infected the cells is unknown.

(Left below) Test drug concentration: 2μg/m11H for PC-10 cells
MV band and -; Same as Table 4.

Test Example 5 Effect on cultured cells It was investigated whether the test drug inhibited cell proliferation.

1,000 μg/■l test drug aqueous solution was added to Eagle (E
agle) minimum essential medium.

Mouse fibroblast L-929 or human amnion-derived cells PL were added 2x to 10% FBS-MEM medium 3.13 mA+.
Suspend 104 cells and place in a tissue culture dish (diameter 00 m)
Seed the seeds and culture in 15% carbon dioxide gas at 37°C for 48 hours.

Add 0.4 liters of the previously prepared test drug solution to each petri dish to obtain a predetermined concentration, and culture for an additional 3 days. After culturing, the medium was removed and Dulbecc
o) Wash once with phosphate buffer (p I-17,4). Mixture of 2.5% trypsin and 0.02% EDT (
Add 0.2 mA of 9) to
After appropriately diluting with FI3S-MIcM medium, the number of cells is measured using an Elsima hematology abacus. The cell proliferation rate is defined as 100% by subtracting the average cell number on the 2nd day from the 50th average cell number of the IQ group without the test drug added, and the percentage of the cell number of the test drug added group calculated in the same way as a1. Find and calculate. 50% cell growth inhibitory concentration (ICso: μg/■
The value is calculated from 17 straight lines around Ω degrees - percentage.

The results are shown below.

(Margin below) As shown in Table 6, the 50% cell growth inhibition concentration (IC50; μg/Simplified β) of the test drugs is higher than these MICs. Therefore, the test drug does not have any adverse effect on cell proliferation at the MIC value.

Test Example 6 Acute Toxicity The oral toxicity of the test drug to ddY-3 male mice was investigated by a conventional method, and the LD50 value (-g/kg) was calculated by the Behrens-Kerber method.

The results are shown below.

Table 7 Acute Toxicity 1 > 2,000 2 > 2,000 3 > 2,000 4 > 2,000 5 > 2,000 6 > 1,700 The active ingredient [I] of the present invention is shown in Table 1 above. As shown in Table 3, it has a strong anti-mycoplasma effect, and as shown in Table 7, it has low toxicity, so it is useful as an anti-mycoplasma agent for animals including humans. The dosage of the anti-mycoplasma agent of the present invention varies depending on age, body weight, symptoms, and administration route, but it is usually 10-3 g as the active ingredient.
It is preferably administered for 50 to 101,000 days, and is administered once or in divided doses. The anti-mycoplasma agent of the present invention is generally administered orally. If active ingredient [I] of the present invention or a salt thereof or a hydrate thereof is usually administered in the form of a preparation prepared by mixing it with a pharmaceutical carrier. Specific examples of formulations include tablets, capsules, and granules.

Examples include syrups. These formulations can be prepared by conventional methods using conventional pharmaceutical carriers.

In addition, as shown in Tables 1 to 6, the active ingredient [I] of the present invention
has a strong anti-mycoplasma effect and an excellent effect of removing and preventing mycoplasma contamination, and does not inhibit the growth of cultured cells. Therefore, the anti-mycoplasma agent of the present invention is extremely useful as an agent for removing or preventing mycoplasma contamination of cultured cells. Compound [I], a salt thereof, or a hydrate thereof, which is an agent for removing and preventing mycoplasma contamination, has a concentration of 0.1 to 10 μg/box, preferably 0.4 to 10 μg/box.
Usually in the form of a reagent so that the concentration is 3tty/111,
It is added to the medium before or during culture. Although such a reagent may be in the form of a solid preparation that can be dissolved at the time of use, it is convenient to handle it if it is in the form of a liquid solution such as an aqueous solution. When the reagent is a cryogenic agent, it is best to store it frozen. In addition to the active ingredient [1], the reagent may further contain a buffer such as a phosphate, a solubilizing agent such as an alkali, a component of a cell culture medium, and the like.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples and Reference Examples.

Example 1 (Manufacture of capsules) Compound 1 or Compound 2 125g starch
50g lactose 35g talc
15g of the ingredients listed in item 1 are mixed well and made into granules, which are then filled into 1,000 capsules by a conventional method.

Example 2 (manufacture of tablets) Compound 1 or 2 125g starch
54g Calcium Carboxomable Seltos
40g Microcrystalline cellulose 50g Magnesium stearate 6g Mix the above ingredients well and press into tablets using a conventional method to obtain 1.000 g
Prepare individual tablets.

Example 3 (Manufacture of reagent) A 2 g/ml aqueous solution of Compound 1 or Compound 2, which has been sterilized using a microfilter, is dispensed into Ioml volume amber vials and stored frozen. 200 mg of Compound 1 or 2 is contained in O, lm1t of this solution.

Reference Example 1 (Production of Compound 1) (1) Known compound ethyl pentafluorobenzoylacetate [J, Org, Chem, 35.930 (+9
A mixture of 225 g of edyl orthoformate, 20 g of acetic anhydride, and 23 g of acetic anhydride was heated under reflux for 2 hours, and then the reaction mixture was concentrated to dryness under reduced pressure for 15 minutes. The residue was dissolved in gel ether and reacted with 5.1 g of cyclopropylamine at room temperature to obtain 28 g of 2-(pentafluorobenzoyl)-3-cyclopropylamine ethyl acrylate.

m, p, 89°C.

(2) Dissolve 28 g of this compound in anhydrous tetrahydrofuranic acid and 3.85% 60% sodium hydride at room temperature.
g to react with 1-cyclopropyl-5,6,7,8
18.4 g of ethyl -tetrafluoro r+ -1,4-dihydro-4-oxoquinoline-3-carboxylate are obtained.

m, 9.170-171°C9 (3) 28.2 g of this compound, benzylamine 9.
8 horses l.

Anhydrous potassium carbonate 23.0g, acetonitrile 140■
1 of the mixture was heated at 100-110°C for 1 hour to
-benzylamino-1-cyclopropyl-6,7,8-
21.4 g of ethyl refluoro1)-1,4-dihydro-4-oxoquinoline-3-carboxylate are obtained. Recrystallize from ethanol. m, p, 134-135°C.

(4) Dissolve 20 g of this compound in 1001 acetic acid and 150 μl of ethanol, and dissolve 0.5 g of 5% palladium-carbon.
Hydrolysis in the presence of 5-amino-1-Schiff “1
14.
Obtain 1g. Recrystallize from 110 form-ethanol. +n, p, 230-237°C.

(5) A mixture of 12.0 g of this compound, 80 liters of acetic acid, 50 ml of water and 9 ml of sulfuric acid was heated to 100-110°C.
5-amino-1-cyclopropyl-6,7,8-trifluoro-1,4-dihydro-4
11.1 g of -oxoquinoline-3-carboxylic acid are obtained.

Recrystallize from chloroform-ethanol.

m, p, 294-295°C.

(6) A mixture of 1.25 g of the above compound, 2.0 g of 3-medylpiverazine, and pyridine 13-1 was added to 105~
Heat and stir at 110'C for 1 hour. Concentrate to dryness under reduced pressure,
Add water to the residue and extract with chloroform. After drying the extract, chloroform is distilled off, ethanol is added to the residue, and the crystals are collected by filtration. Recrystallized from chloroform-ethanol to give 5-amino-1-cyclopropyl-6,8-
Difluoro-7-(3-methyl-1-piperazinyl)-
1.4 g of 1,4-dihydro-4-oxoquinoline-3-carboxylic acid (compound 1) is obtained. m, p, 25
1-253℃.

(7) Add 700 mg of this compound to 7 l of 20% hydrochloric acid.
and concentrated to dryness under reduced pressure. Ethanol was added to the residue, the crystals were collected by filtration, and recrystallized from water-ethanol.
-amino-1-cyclopropyl-6,8-difluoro-
425 mg of 7-(3-methyl-1-piperazinyl)-1,4-dihydro-4-oxoquinol 7-3-carbo/hydrochloride (hydrochloride of compound 1) is obtained.

m, p, 300℃ or higher” -0

Claims (4)

[Claims] (1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R_1 means a 1-piperazinyl group or a 1-pyrrolidinyl group that may be substituted with one or two substituents R. The substituent R herein means a fluorine atom, an amino group, or a lower alkyl group having 1 to 5 carbon atoms which may be substituted with a lower alkylamino group having 1 to 5 carbon atoms, or an amino group or a fluorine atom. R_2 means a cyclopropyl group or a phenyl group optionally substituted with 1 or 2 halogen atoms. A is N or C-H
or C-Y, where Y means a halogen atom. X means a hydrogen atom, an amino group or a fluorine atom. ) An anti-mycoplasma agent containing pyridonecarboxylic acid or its salt as an active ingredient. (2) The anti-mycoplasma agent according to claim 1, wherein the active ingredient is pyridonecarboxylic acid or a salt thereof in which X is an amino group. (3) Claims in which R_1 is a 1-pyrrolidinyl group optionally substituted with one or two substituents R, and X is an amino group, the active ingredient being pyridonecarboxylic acid or a salt thereof. The anti-mycoplasma agent according to item 1. (4) Claims in which R_1 is a 1-piperazinyl group optionally substituted with one or two substituents R, and X is an amino group, the active ingredient being pyridonecarboxylic acid or a salt thereof. The anti-mycoplasma agent according to item 1.