Floxa.igsz.de

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United States Patent
5,053,407
Hayakawa , et al.
October 1, 1991
Optically active pyridobenzoxazine derivatives and anti-microbial use Abstract
An optically action pyridobenzoxazine derivative, a process for preparing the sameand a novel intermediate useful for preparing the optically active pyridobenzoxazinederivative are disclosed. The optically active pyridobenzoxazine derivative possessesincreased antimicrobial activity and reduced toxicity. The intermediate is useful forpreparing such optically active pyridobenzoxazine derivatives such as Ofloxacin andanolog compounds.
Inventors: Hayakawa; Isao (Tokyo, JP), Atarashi; Shohgo (Tokyo, JP), Imamura;
Masazumi (Tokyo, JP), Yokohama; Shuichi (Tokyo, JP), Higashihashi;
Nobuyuki (Tokyo, JP), Sakano; Katsuichi (Tokyo, JP), Ohshima;
Masayuki (Tokyo, JP)
Assignee: Daiichi Pharmaceutical Co., Ltd. (Tokyo, JP)
Appl. No.: 06/876,623
Filed:
June 20, 1986
Foreign Application Priority Data
Current U.S. Class:
514/230.2 ; 544/101
Current International Class:
498/06 (20060101); C07D 413/06 (20060101); 265/00 (20060101); C07D 413/00 (20060101); http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
Field of Search:
References Cited [Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Albrecht et al., Progress in Drug Research, vol. 21 (1971). .
Rohlfing et al., J. Antibacterial Chemotherapy (1985). .
Gerster et al., Proceedings of the North American Medicinal ChemistrySymposium, (1982). .
Hayakawa et al., Chemical & Pharmaceutical Bulletin, vol. 32, No. 13, 1984,pp. 4907-4913, Tokyo, JP. .
J. F. Gerster et al., J. Med. Chem., 1987, 30, 839. .
Hayakawa et al., "Synthesis and Antibacterial Activities of Optically ActiveOfloxacin", vol. 29, No. 1, Jan. 1986, pp. 163-164.
Primary Examiner: Raymond; Richard L.
Attorney, Agent or Firm: Sughrue, Mion, Zinn, Macpeak & Seas Claims
1. An S(-)-pyridobenzoxazine compound represented by the formula (VI) ##STR9##wherein X.sub.1 represents a halogen atom, R.sub.1 represents an alkyl group having1 to 4 carbon atoms, and R.sub.3 represents an alkyl group having 1 to 3 carbonatoms.
2. S(-)-9-Fluoro-3-methyl-10 -(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxa zine-6-carboxylic acid according to claim 1.
3. S(-)-9-Fluoro-3-methyl-10-(4-ethyl-1-pipe razinyl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid according to claim 1.
4. A process for treating a patient in need of an antimicrobial therapy which http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
comprises administering to said patient an antimicrobially effective amount of anS(-)-pyridobenzoxazine compound represented by the formula (VI) ##STR10##wherein X.sub.1 represents a halogen atom, R.sub.1 represents an alkyl group having1 to 4 carbon atoms, and R.sub.3 represents an alkyl group having 1 to 3 carbonatoms.
5. A process for treating a patient in need of an antimicrobial therapy as in claim 4which comprises administering to said patient an antimicrobially effective amount ofS(-)-9-Fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H-py rido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
6. A process for treating a patient in need of an antimicrobial therapy as in claim 4which comprises administering to said patient an antimicrobially effective amount ofS(-)-9-Fluoro-3-methyl-10-(4-ethyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H-pyr ido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.
Description
This invention relates to optically active pyridobenzoxazine derivatives and a processfor preparing the same and to novel intermediates useful for preparing suchderivatives. More particularly, it relates to optically active compounds of Ofloxacinand its analogs, a process for preparing the same and intermediates useful forpreparing the same.
Ofloxacin ((.+-.)-9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid) is known to be an excellentsynthetic antimicrobial agent as disclosed in Japanese Patent Application (OPI) No.
46986/82 (the term "OPI" used herein means "unexamined published patentapplication").
Ofloxacin has an asymmetric carbon atom at the 3-position thereof and is obtained asa racemate (specific rotation [.alpha.].sub.D =0.degree.) by known processes. Thepresent inventors obtained optically active compounds of the racemic Ofloxacin andfound that the S(-)-compound possesses an antimicrobial activity of about 2 timeshigher than that of the (.+-.)-compound and an acute toxicity (LD.sub.50) weaker thanthat of the (.+-.)-compound as determined in mice by intravenous administration. Onthe other hand, the present inventors found that the R(+)-compound exhibits anantimicrobial activity of only about 1/10 to 1/100 times that of the (.+-.)-compound,whereas it possesses an acute toxicity substantially equal to that of the(.+-.)-compound. That is, the S(-)-form of Ofloxacin has been found to have verydesirable properties, i.e., increased antimicrobial activity and reduced toxicity, and isexpected to be a very useful pharmaceutical agents as compared with the http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
(.+-.)-compound. Further, both the R(+)- and S(-)-compounds of Ofloxacin in the freeform have markedly high water-solubility as compared with (.+-.)-compound and ascompared with free compounds of this type, and can be used as injectablepreparations. These advantages will be apparent from the experimental data shownhereinafter.
As a result of investigations with the purpose of preparing, especially S(-)-form havinghigher activity, among the two isomers of Ofloxacin, it has now been found thatcompounds having the following formula (X) are useful as intermediates forsynthesizing an isomer of Ofloxacin as well as other isomers of pyridobenzoxazinederivatives having excellent antimicrobial activity: ##STR1## wherein X.sub.1 andX.sub.2, which may be the same or different, each represents a halogen atom, such asa fluorine atom, a chlorine atom, and preferably both X.sub.1 and X.sub.2 are fluorineatoms; R.sub.1 represents an alkyl groups having from 1 to 4 carbon atoms, such as amethyl group, an ethyl group, a propyl group, etc., and preferably a methyl group; Qrepresents a hydrogen atom or a group ##STR2## wherein R.sub.2 represents asubstituted sulfonyl group, an alkoxycarbonyl group or an aralkyloxycarbonyl group,such as a p-toluenesulfonyl group, a benzenesulfonyl group, a methanesulfonyl group,a t-butoxycarbonyl group, a benzyloxycarbonyl group, a p-methoxybenzyloxycarbonylgroup, etc., preferably a substituted sulfonyl group, and more preferably ap-toluenesulfonyl group; and n represents an integer of from 1 to 3, and preferably 1or 2.
An object of this invention is to provide optically active Ofloxacin and its analogs.
Another object of this invention is to provide a novel intermediate represented by theabove-described formula (X) which is useful for synthesizing optically active Ofloxacinand other pyridobenzoxazine derivatives.
A still another object of this invention is to provide a novel process for preparingoptically active Ofloxacin and its analogs by the use of the above-describedintermediate.
The optically active Ofloxacin and its analogs according to the present invention canbe represented by the formula (VI): ##STR3## wherein X.sub.1 and R.sub.1 are asdefined above, and R.sub.3 represents an alkyl group having 1 to 3 carbon atoms.
In the above-described formula (VI), X.sub.1 preferably represents a fluorine atom,and R.sub.1 preferably represents a methyl group, and R.sub.3 preferably representsa methyl group or an ethyl group.
The optically active Ofloxacin and its analogs of the invention can be prepared by anyof Processes A, B and C shown below: ##STR4## wherein X.sub.1, X.sub.2 andR.sub.3 are as defined above.
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More specifically, the (.+-.)-3-hydroxymethyl compound (I) is treated with3,5-dinitrobenzoyl chloride, etc. to obtain its derivative, such as the (.+-.)-3,5-dinitrobenzoate compound (II), which is then optically resolved into two opticallyactive compounds by an appropriate method, such as high performance liquidchromatography (HPLC). The subsequent procedures can be applied to either of theS(-)-compound and the R(+)-compound to lead to the respective final product, but inview of the purpose of the present invention, the final product (VI') is shown asS(-)-form.
The resulting optically active compound is treated with sodium hydrogencarbonate,etc. to selectively hydrolyzing the benzoate moiety to form the hydroxymethylcompound (III). The hydroxymethyl compound is converted into the 3-iodomethylcompound (IV) by using a iodinating reagent, which is then reduced with n-tributyltinhydride, etc. to prepare the 3-methyl compound. This compound, without beingisolated and purified, can be hydrolyzed as such under an acidic condition to obtainthe 3-methyl-6-carboxylic acid (V'). The carboxylic acid is then reacted with anN-alkylpiperazine by, for example, heating while stirring to obtain the 10-(4-alkylpiperazinyl) compound (VI') as a final product.
The compounds of the formula (VI) wherein R.sub.1 represents an alkyl group otherthan the methyl group can also be prepared in the same manner from an appropriate(.+-.)-3-hydroxyalkyl compound of the formula (I). ##STR5## wherein X.sub.1,X.sub.2 and R.sub.3 are as defined above.
As described previously, 7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine havingthe formula (X) wherein X.sub.1 and X.sub.2 represent fluorine atoms, Q represents ahydrogen atom and R.sub.1 represents a methyl group is an important intermediatefor synthesizing Ofloxacin. The inventors have conducted various investigations on anadvantageous process for preparing an optical isomer of this compound expectingthat such would be a useful starting material for synthesizing the S(-)-isomer ofOfloxacin.
As a result, it has been found that when a racemic 7,8-dihalogeno-2,3-dihydro-3-acetoxymethyl-4H-[1,4]benzoxazine (VII), as a substrate, is hydrolyzed with anappropriate enzyme, such as a certain kind of lipase, to form a 7,8-dihalogeno-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine (VIII), there is a difference in therate of hydrolysis between the (+)-isomer and the (-)-isomer.
For example, the compound (VII) was reacted with lipoprotein lipase (LPL Amano 3derived from Pseudomonas aeruginosa, produced by Amano Seiyaku K.K.) or lipase(derived from Porcine pancreas, produced by Shigma Chemical Company (U.S.A.);derived from Candida cylindracea, produced by Shigma Chemical Company; orderived from Rhizopus delemar, produced by Seikagaku Kogyo Co., Ltd.), and thereaction change with time was determined through HPLC (column: TSK gelODS-120A, 4.6.times.250 mm; solvent: acetonitrile/water=1/1 by volume; velocity: 1ml/min). When the rate of hydrolysis reached about 55%, the compound (VII) wasrecovered and led to a 3,5-dinitrobenzoyl derivative (IX). The resulting reaction http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
product was quantitatively determined by HPLC (column: Sumipacks OA-4200,4.0.times.250 mm; solvent: n-hexane/1,2-dichloroethane/ethanol=92/6.4/1.6 byvolume; velocity: 1.6 ml/min) to obtain a ratio of the (+)-isomer/(-)-isomer of thecompound (VII). The results obtained were shown in Table 1 below.
TABLE 1 ______________________________________ Rate of Enzyme Hydrolysis (Origin)(%) (+)/(-) ______________________________________ LPL Amano 3 54.7 23.0/77.0 (P.
aeruginosa) Lipase 53.6 42.6/57.4 (R. delemar) Lipase 54.5 61.9/38.1 (C. cylindracea)Lipase 55.2 56.8/43.2 (P. pancreas) ______________________________________ These results lead to a conclusion that the optically active compounds (VII) and (VIII)can be obtained by utilizing the so-called asymmetric hydrolysis with these enzymes.
Process B according to the present invention comprises reacting a racemic7,8-dihalogeno-2,3-dihydro-3-acetoxymethyl-4H-[1,4]benzoxazine (VII) with anasymmetric hydrolytic enzyme to recover a mixture comprising the startingcompound (VII) rich in either one of optical isomers and a 3-hydroxymethyl compound(VIII), separating the mixture into each compound, dinitrobenzoylating either one orboth of these compounds and further acetylating the benzoylated 3-hydroxymethylcompound (VIII) to obtain the compound (IX), separating the compound into aracemate and an optical isomer by crystallization, subjecting the resulting opticalisomer to deacylation and dehydroxylation by means of known chemical processes toobtain an optically active 7,8-dihalogeno-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine(X') and then obtaining therefrom optically active Ofloxacin or its analog (VI') byknown processes.
Process B of the invention will further be illustrated in some detail referring tospecific examples.
The racemic compound (VII) is dissolved in a 0.1M phosphoric acid buffer (pH 7.0),and lipoprotein lipase (LPL Amano 3) is added to the solution to cause enzymaticreaction at 37.degree. C. By this reaction, the (+)-compound is preferentiallyhydrolyzed to thereby form a mixture of the compound (VII) rich in the (-)-isomer andthe compound (VIII) rich in the (+)-isomer. The reaction mixture is recovered in anappropriate stage by extraction with an organic solvent, such as ethyl acetate.
The above enzymatic reaction may also be carried out in an appropriate organicsolvent, such as a mixed solvent of benzene and n-hexane, by using a hydrophilicresin, e.g., DEAE-Toyopearl 650M or Toropearl HW-40, etc., or Celite as a dispersingagent or by using a resin, such as Amberlite XAD-7, Butyl-Toyopearl 650M, etc., as anadsorptive fixing agent. In addition, use of inclusive fixing agents, such as photo-crosslinked resins, urethane prepolymers, etc., is also considered to make it possibleto effect the enzymatic reaction in an organic solvent.
The reaction in an organic solvent with an aid of appropriate dispersing agents orfixing agents as described above is advantageous in that the substrate can be reactedat higher concentrations than in an aqueous solution and that post-treatments afterthe reaction can be simplified. Actually, in the case of performing the reaction in an http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
organic solvent, the reaction mixture can be recovered in high yields simply byfiltering the dispersing agent or fixing agent in an appropriate stage andconcentrating the filtrate. Moreover, the fixing agents can be repeatedly reused toadvantage.
The compounds (VII) and (VIII) in the reaction mixture can be separated and purifiedby a usual method of separation, such as silica gel column chromatography. The thusseparated compound (VII) is treated, for example, with 3,5-dinitrobenzoyl chloride intetrahydrofuran in the presence of pyridine to form the 3,5-dinitrobenzoyl derivative(IX), which is then recrystallized from an appropriate solvent, e.g., a mixed solvent ofethyl acetate and n-hexane, whereby the racemate is preferentially crystallized. Theracemic crystals are separated by filtration, and the (-)-7,8-dihalogeno-2,3-dihydro-3-acetoxymethyl-4H-[1,4]benzoxazine 3,5-dinitrobenzoyl derivative (IX) having a highoptical purity is obtained from the filtrate.
The compound (IX) is then subjected to de-acylation treatment by, for example,hydrolysis under an alkaline condition to form the (-)-isomer of the compound (VIII).
This compound is dissolved in pyridine and treated with thionyl chloride, and theproduct is further subjected to dehydroxylation in a usual manner, such as reductionwith sodium borohydride in dimethyl sulfoxide, to thereby obtain the (-)-7,8-dihalogeno-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (X') having an optical purity of99% or higher.
When the racemic compound (VII) is treated with lipase (derived from Candidacylindracea or Porcine Pancreas), the (-)-compound is preferentially hydrolyzed toobtain the compound (VIII) rich in the (-)-isomer. The resulting compound is led to acompound in the form of the compound (IX), which is then treated in accordance withthe procedures as described above, such as separation by crystallization, to form the(-)-isomer of the compound (VIII). The (-)-isomer of the compound (X') can then beprepared from this product in the same manner as described above with a high purity.
Other asymmetric hydrolyses which can achieve the object of the present invention inaddition to the above-recited enzymes can be found based on the above-mentionedelucidation. Further, when it is intended to obtain (+)-compounds, the sameprocedures as described above can be followed based on the above elucidation.
The (-)-compound of Ofloxacin and analogs thereof can be prepared from the novelintermediates of the present invention having the formula (X') in accordance withProcess C as illustrated below. ##STR6## wherein n, X.sub.1, X.sub.2, R.sub.1,R.sub.2 and R.sub.3 are as defined above, and X.sub.3 is a carboxyl group or areactive derivative thereof, for example, an active ester, a halide or an acid anhydrideof the carboxylic acid.
In Process C, the 7,8-dihalogeno-1,4-benzoxazine derivative (X') is condensed with acyclic amino acid or a reactive derivative thereof (XIV) through amide linkageformation to produce the compound (X"). The condensation reaction can be effectedby either one of the active ester method, the acid anhydride method or the DCCmethod, but, generally, the compound (X') and an acid chloride (XIV) wherein X.sub.3 http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
is --COCl are reacted in an organic solvent such as halogenated hydrocarbons, e.g.,dichloromethane, in the presence of an acid acceptor such as pyridine, triethylamineor potassium carbonate, at room temperature while stirring. The reaction product canbe isolated and purified in a conventional manner, such as crystallization, columnchromatography, etc.
In this condensation reaction, when either one of the two isomers of the cyclic aminoacid or its reactive derivative (XIV), i.e., an S-compound or an R-compound, is used,separation of the diasteromeric mixture of the compound (X") can be carried outeasily. More specifically, derivatives (XIV), such as S- or R-proline, S- or R-pipecolicacid (piperidine-2-carboxylic acid), etc., are suitably used. The most preferredcompounds (XIV) include (S)-N-benzenesulfonylproline and (S)-N-p-toluenesulfonylproline.
The diastereomeric mixture of the compound (X") can be separated by fractionalcrystallization, chromatography using silica gel, etc. as a carrier, or a combinationthereof.
The thus separated diastereomer is hydrolyzed, usually under a basic condition, toform a 7,8-dihalogeno-3-(S or R)-lower alkyl-[1,4]benzoxazine (X). This compound canbe led to a 9,10-dihalogeno-3-(S or R)-lower alkyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid (V) through known reactions, which can thenbe converted to a 10-(4-alkylpiperazinyl) compound (VI).
Of the above-described Processes A, B and C, the particularly preferred is Process C.
The antimicrobial activities of the optical isomers [(-) and (+)] of Ofloxacin and ananalog compound to various microorganisms were compared with that of Ofloxacin(racemate), and the results are shown in Table 2 below. The test method was inaccordance with the standard method specified by The Japan Society ofChemotherapy.
TABLE 2 __________________________________________________________________________Minimum Inhibitory Concentration (MIC; .mu.g/ml) S(-)- Racemic R(+)- Compound ofMicroorganism Ofloxacin* Ofloxacin Ofloxacin Example 17__________________________________________________________________________ E. coli, NIHJ<0.1 <0.10 0.39 <0.1 K. pneumoniae, Type 2 <0.1 0.10 1.56 <0.1 Ser. marcescens,10100 <0.1 0.10 1.56 <0.1 Ps. aeruginosa, 32104 0.39 0.78 12.5 0.39 Ps. aeruginosa,32121 0.10 0.20 6.25 0.10 Ps. maltophilia, IID1275 0.39 0.78 12.5 0.10 S. aureaus,Smith 0.10 0.20 6.25 <0.1 S. aureaus, 209P 0.20 0.39 25 0.10 Str. pyogenes, G-360.78 1.56 <100 1.56__________________________________________________________________________ The acute intravenous toxicity of (.+-.), R(+) and S(-)-forms of Ofloxacin in male mice http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
TABLE 3 ______________________________________ Dose Numbers Day after treatmentCompounds (mg/kg) of mice 1 2 3 Mortality ______________________________________(.+-.) 100 5 0 0 0 0/5 200 5 2 0 0 2/5 400 5 5 0 0 5/5 R(+) 100 5 0 0 0 0/5 200 5 3 0 03/5 400 5 5 0 0 5/5 S(-) 100 5 0 0 0 0/5 200 5 0 0 0 0/5 400 5 5 0 0 5/5______________________________________ LD.sub.50 (i.v. in mice) (.+-.)form 203 mg/kgS(-)form 244 mg/kg The solubility of (.+-.), R(+) and S(-)-forms of Ofloxacin in water at a temperature inthe range of from 23.degree. to 26.degree. C. is shown in Table 4 below.
TABLE 4 ______________________________________ Compounds Water-Solubility(.mu.g/ml) ______________________________________ (.+-.) 2,400 R(+) 25,800 S(-) 22,500______________________________________ The conversion of the intermediates of the formula (X) into the desired Ofloxacin oran analog thereof can be carried out by a well known process as disclosed, forexample, in U.S. Pat. No. 4,382,892, EPC Patent 47005, Japanese Patent Application(OPI) Nos. 29789/83 and 43977/82.
This invention will now be illustrated in greater detail with reference to the followingexamples, but it should be understood that they are not intended to limit the presentinvention. Examples 1 to 7 describe preparation of Ofloxacin according to Process A;Examples 8 to 11 describe preparation of Ofloxacin according to Process B; andExamples 12 to 17 describe preparation of Ofloxacin and analog compoundsaccording to Process C.
One gram of (.+-.)-9,10-difluoro-3-hydroxymethyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de] [1,4]benzoxazine-6-carboxylic acid ethyl ester (I) and 500 mg ofpyridine were suspended in 100 ml of anhydrous tetrahydrofuran (THF), and 1.6 g of3,5-dinitrobenzoyl chloride was added thereto, followed by refluxing at 90.degree. C.
The suspension was once dissolved, and a colorless precipitate was then formed. Thereaction was continued for 1.5 hours. After cooling, the precipitate was collected byfiltration, washed with methanol and diethyl ether and dried to obtain 1.2 g of(.+-.)-9,10-difluoro-3-(3,5-dinitrobenzoyloxy)methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid ethyl ester (II) as a colorlesspowder having a melting point of 240.degree.-242.degree. C.
NMR (CDCl.sub.3 /5% DMSO-d.sub.6) .delta.(ppm); 1,30 (3H, t, J=7.0Hz, --CH.sub.2CH.sub.3), 4.26 (2H, q, J=7.0Hz, --CH.sub.2 CH.sub.3), 4.4-4.5 (5H, m), 7.76 (1H, dd,J=11.0Hz, 7.0Hz, C.sub.8 --H), 8.8 (1H, s, C.sub.5 --H), 9.0 (2H, d, J=3.0Hz, aromatic http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
ring proton) and 9.2 (1H, t, J=3.0Hz, aromatic ring proton) Six milligrams of the benzoyloxy compound as obtained in Example 1 was dissolved inabout 0.6 ml of dimethylformamide (DMF) which had been purified by distillation. Thesolution was filtered through a millipore filter and subjected to HPLC using a columnof Sumipacks OA-4200 (2 cm.times.25 cm) and a solvent of n-hexane/1,2-dichloroethane/ethanol=6/3/1 (by volume) at a velocity of 8 ml/min.
Since the initial fractions (fractions of the (+)-compound) contained a slight amountof the racemic compound (I) due to partial hydrolysis when dissolved in DMF, theywere further purified by silica gel chromatography using chloroform to 10%methanol/chloroform as an eluent. These purification procedures were repeated tothereby obtain 250 mg each of the two optically active compounds [(-)-isomer and(+)-isomer] from 600 mg of the benzoyloxy compound (II).
(+)-Isomer: retention time: 56-76 mins. (column temperature: 22.degree. C.); meltingpoints: 235.degree.-240.degree. C.; [.alpha.].sub.D.sup.23 =+90.8.degree. (c=0.852,DMF) (-)-Isomer: retention time: 78-98 mins. (column temperature: 22.degree. C.); meltingpoints: 244.degree.-249.degree. C.; [.alpha.].sub.D.sup.23 =-92.5.degree. (c=0.889,DMF) Preparation of Ethyl (-)-9,10-Difluoro-3-Hydroxymethyl-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1, 4]Benzoxazine-6-Carboxylate (III) In a mixture of 10 ml of ethanol and 4 ml of a saturated aqueous solution of sodiumbicarbonate, 120 mg of the optically active benzoyloxy compound [(-)-isomer] wassuspended, and the suspension was heated at 50.degree. to 60.degree. C. for 2 hourswhile stirring. After concentration, water was added to the reaction mixture, and anyinsoluble material was collected by filtration, washed successively with water, 95%ethanol and diethyl ether to obtain 68 mg of an optically active 3-hydroxymethylcompound [(III), (-)-isomer] as a colorless crystal having a melting point of235.degree.-240.degree. C.
Elementary Analysis for C.sub.15 H.sub.13 F.sub.2 NO.sub.5 : Calcd. (%): C 55.39, H4.03, N 4.31. Found (%): C 55.44, H 4.01, N 4.49.
[.alpha.].sub.D.sup.23 =-125.9.degree. (c=0.918, DMF) In the same manner as described above, a (+)-3-hydroxymethyl compound wassynthesized from the (+)-benzoyloxy compound. Melting point: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
231.degree.-234.degree. C. [.alpha.].sub.D.sup.23 =+125.9.degree. (c=0.715, DMF).
Preparation of Ethyl (-)-9,10-Difluoro-3-Iodomethyl-7-oxo-2,3-Dihydro-7H-Pyrido-[1,2,3-de][1,4] Benzoxazine-6-Carboxylate (IV) In 12 ml of anhydrous DMF was suspended 63 mg of the (-)-3-hydroxymethylcompound (III), and the suspension was heated at 70.degree. to 80.degree. C. withstirring to form a solution, followed by allowing to cool to room temperature. To thesolution was added 340 mg of triphenylphosphite methiodide, followed by stirring for1.5 hours. The solvent was removed by distillation under reduced pressure, and theresidue was dissolved in chloroform. The solution was partitioned with a sodiumthiosulfate aqueous solution and then with a saturated sodium chloride aqueoussolution. The chloroform layer was dried over anhydrous magnesium sulfate, and thesolvent was removed by distillation. To the residue was added diethyl ether, followedby stirring, and the precipitated solid was collected by filtration, washed with diethylether and dried under reduced pressure to obtain 78 mg of an iodomethyl compound(IV) as a white powder having a melting point of 214.degree.-217.degree. C.
Elementary Analysis for C.sub.15 H.sub.12 F.sub.2 INO.sub.4 : Calcd. (%): C 41.40, H2.78, N 3.22 Found (%): C 41.16, H 2.58, N 2.99 The (+)-compound was obtained in the same manner as described above.
Preparation of S(-)-9,10-Difluoro-3-Methyl-7-Oxo-2,3-Dihydro-7H-Pyrido-[1,2,3-de][1,4]-Be nzoxazine-6-Carboxylic Acid (V') In 18 ml of absolute ethanol was suspended 78 mg of the iodomethyl compound (IV),and the suspension was heated at 60.degree. to 70.degree. C. with stirring to form asolution, followed by allowing to cool to room temperature. To the resulting solutionwas added 0.2 ml of n-tributyltin hydride, and the mixture was stirred at 50.degree. to60.degree. C. for 1 hour and then at room temperature for 1 hour. The solvent wasremoved by distillation, and the residue was subjected to column chromatographyusing 8 g of silica gel as a carrier and chloroform:methanol (40:1 by volume) as aneluent to obtain a crude methyl compound. The crude product was dissolved in 2 mlof glacial acetic acid, and 4 ml of concentrated hydrochloric acid was added thereto.
After heating at reflux for 40 minutes, the reaction mixture was concentrated. Waterwas added to the concentrate, and the thus precipitated crystal was collected byfiltration, washed successively with water, ethanol and diethyl ether and dried underreduced pressure to obtain 22 mg of crystals of a S(-)-compound (V') having a meltingpoint of 300.degree. C. or higher.
Elementary Analysis for C.sub.13 H.sub.9 F.sub.2 NO.sub.4 : Calcd. (%): C 55.52, H3.23, N 4.98 Found (%): C 55.79, H 3.20, N 4.91 http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
[.alpha.].sub.D.sup.23 =-65.6.degree. (c=0.950, DMSO) Preparation of S(-)-9-Fluoro-3-Methyl-10-(4-Methyl-1-Piperazinyl)-7-Oxo-2,3-Dihydro-7H-Py rido[1,2,3-de][1,4]Benzoxazine-6-Carboxylic Acid In 3 ml of anhydrous dimethyl sulfoxide were dissolved 21 mg of the S(-)-9,10-difluoro-3-methyl-6-carboxylic acid (V') and 30 mg of N-methylpiperazine, and thesolution was stirred at 130.degree. to 140.degree. C. for 1 hour. The solvent wasremoved by distillation, and to the residue was added 2 ml of ethanol. The thusprecipitated solid was collected by filtration and washed successively with a smallamount of ethanol and diethyl ether. The resulting powder weighing 14 mg waspassed through a column of 5 g of silica gel and eluted with a lower layer solution ofchloroform-methanol-water (7:3:1 by volume) to obtain the titled compound, 10-(4-methyl-1-piperazinyl) compound (VI'). The mother liquor left after the filtration wassubjected to thin layer chromatography (silica gel; 20 cm.times.20 cm, 5 mm (t)) anddeveloped with a lower layer solution of chloroform-methanol-water (15:3:1 byvolume). Both the purified products were combined to yield 14 mg of the titledcompound as a crystal having a melting point of 220.degree.-228.degree. C. (withdecomposition).
Elementary Analysis for C.sub.18 H.sub.20 FN.sub.3 O.sub.4 : Calcd. (%): C 59.82, H5.58, N 11.63 Found (%): C 60.01, H 5.69, N 11.53 [.alpha.].sub.D.sup.24 =-68.8.degree. (c=0.711, 0.05N NaOH) NMR (CDCl.sub.3) .delta.(ppm): 1.63 (3H, d, C.sub.3 --CH.sub.3), 2.38 (3H, s,N--CH.sub.3), 2.54-2.6 (4H, m, 2.times.CH.sub.2 N), 3.4-3.44 (4H, m,2.times.CH.sub.2 N), 4.35-4.52 (3H, m, CH and CH.sub.2), 7.76 (1H, d, aromatic ringC.sub.8 --H) and 8.64 (1H, s, C.sub.5 --H) The (+)-compound was obtained in the same manner as described above. Meltingpoint: 218.degree.-226.degree. C. (with decomposition). [.alpha.].sub.D.sup.24=+68.7.degree. (c=0.560, 0.05N NaOH). MS (m/e): 361 (M.sup.+).
Preparation of S(-)-9-Fluoro-3-Methyl-10-(4-Methyl-1-Piperazinyl)-7-Oxo-2,3-Dihydro-7H-Py rido[1,2,3-de][1,4]Benzoxazine-6-Carboxylic Acid (VI') In 30 ml of diethyl ether was suspended 281 mg of the (-)-9,10-difluoro-3-methyl-6-carboxylic acid (V') as obtained in Example 5, and a large excess of boron trifluorideethyl etherate was added thereto while stirring at room temperature, followed byallowing the mixture to react for 45 minutes. The precipitate formed was collected byfiltration, washed with diethyl ether and dried under reduced pressure. The resulting http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
chelate compound weighing 310 mg was dissolved in 6 ml of dimethyl sulfoxide, and0.32 ml of triethylamine and 0.13 ml of N-methylpiperazine were added to thesolution. The mixture was stirred at room temperature for 17 hours, followed byconcentration to dryness under reduced pressure. The residue was washed withdiethyl ether and then dissolved in 20 ml of 95% ethanol containing 0.5 ml oftriethylamine, and the solution was heated at reflux for 8 hours. After cooling, thereaction mixture was concentrated to dryness under reduced pressure. The residuewas dissolved in 5% diluted hydrochloric acid, and the solution was distributedbetween chloroform and water. The aqueous layer was adjusted to a pH of 11 with 1Nsodium hydroxide and then to a pH of 7.4 with 1N hydrochloric acid. The solution wasextracted three times with 50 ml portions of chloroform, and the extract was driedover sodium sulfate. The chloroform was removed by distillation. Recrystallization ofthe resulting powder from ethanol-diethyl ether gave 120 mg of the titled compoundas a transparent fine needle-like crystal having a melting point of225.degree.-227.degree. C. (with decomposition). [.alpha.].sub.D.sup.24=-76.9.degree. (c=0.385, 0.05N NaOH) Elementary Analysis for C.sub.18 H.sub.20 FN.sub.3 O.sub.4.1/2H.sub.2 O: Calcd.
(%): C 58.37, H 5.72, N 11.35 Found (%): C 58.17, H 5.58, N 11.27 Preparation of (.+-.)-3-Acetoxymethyl-7,8-Difluoro-2,3-Dihydro-4H-[1,4]Benzoxazine##STR7## In 1.0 liter of acetone was dissolved 60.0 g of 2,3-difluoro-6-nitrophenol, and 70.0 g of1-acetoxy-3-chloro-2-propane and then 33.1 g of potassium carbonate were added tothe solution while stirring at room temperature. After stirring for an addirional 30 kminutes, 6.6 g of potassium iodide was added thereto, and the mixture was refluxedfor 4 hours. After allowing to cool, the reaction mixture was filtered, and the filtratewas concentrated under reduced pressure. The concentrate was dissolved in 4.0 litersof a mixed solvent of ethyl acetate:benzene (1:1 by volume). The resulting solutionwas washed with water, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The concentrate was subjected to column chromatography using1.2 Kg of silica gel and benzene/ethyl acetate (10/1) as an eluent to obtain 32.8 g ofCompound (A) as an oily product. Compound (A) was dissolved in 300 ml of methanol,and 115 ml of Raney nickel was added thereto to effect catalytic reduction underatmospheric pressure. The reaction mixture was filtered, and the filtrate wasconcentrated under reduced pressure. The concentrate was purified by columnchromatography by using 400 g of silica gel and benzene/ethyl acetate (10/1 byvolume) as an eluent, and the product was recrystallized from benzene-n-hexane toobtain 17.9 g of (.+-.)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine(VII) as a colorless crystal having a melting point of 73.degree.-74.degree. C.
Elementary Analysis for C.sub.11 H.sub.11 F.sub.2 NO.sub.3 : Calcd. (%): C 54.32, H4.56, N 5.76 Found (%): C 54.09, H 4.42, N 5.76 http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
Preparation of (-)-3-Acetoxymethyl-7,8-Difluoro-2,3-Dihydro-4H-[1,4]Benzoxazine-3,5-Dinit robenzoyl Derivative (IX) a) Ten grams of (.+-.)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine(VII) as a substrate was dissolved in 1.00 liter of a mixed solvent of benzene/n-hexane(4/1 by volume). A resin in a wet state which was prepared by suspending 100 ml ofDEAE-Toyopearl 650M in a 0.05M phosphoric acid buffer (pH 7.0) followed byfiltration by suction and 200 mg of lipoprotein lipase (LPL Amano 3) were added tothe above-prepared substrate solution. The reaction system was allowed to react at37.degree. C. for 6 hours under stirring. The reaction mixture was filtered by suction,and the resin was washed with 200 ml of benzene. The filtrate and the washing werecombined and concentrated under reduced pressure. The concentrate weighing 9.68g was subjected to column chromatography using 200 g of silica gel as a carrier andbenzene/ethyl acetate (10/1 by volume) as an eluent to obtain 4.67 g of3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.
The resulting compound was dissolved in 200 ml of tetrahydrofuran, and 5.76 g of3,5-dinitrobenzoyl chloride and 3.3 ml of pyridine were added thereto, followed byheating at 60.degree. C. for 3 hours. The reaction mixture was concentrated underreduced pressure, and the concentrate was dissolved in 400 ml of ethyl acetate,washed successively with diluted hydrochloric acid, an aqueous solution of sodiumbicarbonate and water, dried over anhydrous sodium sulfate and concentrated underreduced pressure. Addition of n-hexane to the concentrate caused precipitation ofpale yellow crystals of a racemate. After sufficient precipitation, the precipitate wasseparated by filtration, and the filtrate was concentrated by dryness to obtain 3.93 gof a 3,5-dinitrobenzoyl derivative of the (-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (IX).
b) To about 2.0 ml of Amberlite XAD 7 was added 2.0 ml of a 0.05M phosphoric acidbuffer (pH 7.0) having dissolved therein 20 mg of lipoprotein lipase (LPL Amano 3),and the system was allowed to stand at room temperature for 18 hours to therebyadsorb the enzyme onto the resin. The resin was filtered by suction and washed with10 ml of a 0.05M phosphoric acid buffer (pH 7.0). A solution of 250 mg of (.+-.)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine as a substrate in 25 ml ofa mixed solvent of benzene and n-hexane (4:1 by volume) was added to the thusprepared resin in a wet state, followed by allowing to react at 37.degree. C. for 4hours under stirring. The reaction mixture was worked-up in the same manner asdescribed in a) above to obtain 117 mg of optically active 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[ 1,4]benzoxazine. In the same manner, there wasobtained 65 mg of a 3,5-dinitrobenzoyl derivative of the (-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.
c) To 3.60 liters of a 0.1M phosphoric acid buffer (pH 7.0) was added 3.60 g of(.+-.)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine as a substrate,and the mixture was stirred at 37.degree. C. for 18 hours to form a solution. To theresulting solution was added 50 mg of lipoprotein lipase (LPL Amano 3), followed byallowing the system to react at 37.degree. C. for 190 minutes while stirring. The http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
reaction mixture was extracted three times with 2.0 liter portions of ethyl acetate.
The combined extract was washed with water, dried over anhydrous sodium sulfateand concentrated under reduced pressure. The concentrate was subjected to columnchromatography using 70 g of silica gel as a carrier and chloroform as an eluent toobtain 1.07 g of optically active 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine. The resulting compound was treated in the same manner asdescribed in a) above to obtain 0.9 g of a 3,5-dinitrobenzoyl derivative of the (-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.
d) To 3.70 liters of a 0.1M phosphoric acid buffer (pH 7.0) was added 3.70 g of(.+-.)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine as a substrate,followed by stirring at 37.degree. C. for 3.5 hours to form a solution. To the resultingsolution was added 2.22 g of lipase (derived from Candida cylindracea), followed byallowing the system to react at 37.degree. C. for 7.5 hours while stirring. The reactionmixture was extracted three times with 2.0 liter portions of ethyl acetate. Thecombined extract was washed with water, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The concentrate was subjected to columnchromatography using 70 g of silica gel and developed first with benzene/ethylacetate (5/1 by volume) to elute 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine and then with benzene/ethyl acetate (1/1 by volume) to elute7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine.
The latter eluate weighing 1.31 g was dissolved in 60 ml of tetrahydrofuran, and 1.70g of 3,5-dinitrobenzoyl chloride was added thereto, followed by heating at 37.degree.
C. for 20 hours. The reaction mixture was concentrated under reduced pressure, andthe concentrate was dissolved in 400 ml of ethyl acetate, washed successively with asodium bicarbonate aqueous solution and water, dried over anhydrous sodium sulfateand concentrated to dryness under reduced pressure to obtain 2.52 g of a reactionproduct. The reaction product was dissolved in 10 ml of pyridine, and 10 ml of aceticanhydride was added thereto, followed by heating at 37.degree. C. for 20 hours. Thereaction mixture was worked-up and recrystallized in the same manner as describedin a) above to remove the racemate crystals to thereby increase optical purity. Sincethe product still contained slight amounts of the reaction by-products, it was furtherpurified by Toyo-pearl HW-40-column chromatography (column: 2.5.times.95 cm;developing solvent: methanol/acetonitrile=1/1 by volume) and then silica gel columnchromatography (column: 1.8.times.34 cm; developing solvent;chloroform/acetone=20/1 by volume) to finally obtain 0.44 g of a 3,5-dinitrobenzoylderivative of (-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.
.sup.1 H-NMR (CDCl.sub.3, 200 MHz) .delta.(ppm): 2.14 (3H, s, --OCOCH.sub.3), 4.26(2H, d, J=7.0 Hz, --CH.sub.2 OCOCH.sub.3), 4.45 (1H, dd, J=3.0 Hz, 12.0 Hz, C.sub.2--H), 4.71 (1H, d, J=12.0 Hz, C.sub.2 --H), 4.94 (1H, m, C.sub.3 --H), 6.60 (2H, m,aromatic ring proton), 8.73 (2H, d, J=2.0 Hz, aromatic ring proton) and 9.19 (1H, t,aromatic ring proton) http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
Preparation of (-)-7,8-Difluoro-2,3-Dihydro-3-Hydroxymethyl-4H-[1,4]Benzoxazine(VIII) In 135 ml of tetrahydrofuran was dissolved 3.03 g of a 3,5-dinitrobenzoyl derivative of(-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine, and 135 ml ofethanol and 30 ml of 1.0N potassium hydroxide were added to the solution. After thereaction mixture was stirred at room temperature for 30 minutes, 3 ml of acetic acidwas added thereto for neutralization. The mixture was concentrated under reducedpressure, and the concentrate was dissolved in 400 ml of chloroform, washedsuccessively with a sodium bicarbonate aqueous solution and water, dried overanhydrous sodium sulfate and concentrated to dryness under reduced pressure. Thesolid was subjected to column chromatography using 40 g of silica gel and eluted withchloroform/methanol (50/1 by volume) to obtain 1.17 g of (-)-7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine.
[.alpha.].sub.D.sup.22 =-14.1.degree. (c=1.80, CHCl.sub.3) .sup.1 H-NMR (CDCl.sub.3, 200 MHz) .delta.(ppm): 3.5-4.4 (5H, m), 6.3-6.42 (1H, m,aromatic ring proton) and 6.54-6.74 (1H, m, aromatic ring proton) Preparation of (-)-7,8-Difluoro-2,3-Dihydro-3-Methyl-4H-[1,4]Benzoxazine (X') In 20 ml of pyridine was added 1.17 g of (-)-7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine, and 2.77 g of thionyl chloride was added thereto dropwiseunder ice-cooling, followed by stirring at 50.degree. to 60.degree. C. for 40 minutes.
The reaction mixture was concentrated under reduced pressure, and the concentratewas dissolved in 300 ml of chloroform and washed with 100 ml of a sodiumbicarbonate aqueous solution. The washing was extracted twice with 200 ml portionsof chloroform. The combined chloroform layers were washed with water, dried overanhydrous sodium sulfate and concentrated under reduced pressure. The concentratewas subjected to column chromatography using 40 g of silica gel and eluted withchloroform to obtain 1.18 g of the reaction product as a colorless oily product. Thisproduct was dissolved in 30 ml of dimethyl sulfoxide, and 0.41 g of sodiumborohydride was added thereto, followed by heating at 80.degree. to 90.degree. C. for1 hour. The reaction mixture was dissolved in 500 ml of benzene, washed with waterto remove the dimethyl sulfoxide, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The concentrate was subjected to columnchromatography using 40 g of silica gel and eluted with benzene to obtain 0.80 g of(-)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine as a colorless oily product.
[.alpha.].sub.D.sup.25 =-9.6.degree. (c=2.17, CHCl.sub.3) .sup.1 H-NMR (CDCl.sub.3, 200 MHz) .delta.(ppm): 1.20 (3H, d, J=6.0 Hz,--CH.sub.3), 3.53 (1H, m, C.sub.3 --H), 3.81 (1H, dd, J=8.0 Hz, 10.0 Hz, C.sub.2 --H),4.31 (1H, dd, J=3.0 Hz, 10.0 Hz, C.sub.2 --H), 6.24-6.36 (1H, m, aromatic ring proton) http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
and 6.52-6.7 (1H, m, aromatic ring proton) The product was led to the 3,5-dinitrobenzoyl derivative and quantitativelydetermined by HPLC using a column of Sumipacks OA-4200; 4.0.times.250 mm and amixed solvent of n-hexane/1,2-dichloroethane/ethanol (90/9.1/0.9 by volume) at avelocity of 1.5 ml/min.
To 1.13 g of (-)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine was added 1.58g of diethyl ethoxymethylenemalonate, and the mixture was stirred at 130.degree. to140.degree. C. for 70 minutes. The reaction mixture was subjected as such to columnchromatography using 50 g of silica gel and eluted with chloroform to obtain 2.47 g ofdiethyl [(-)-7,8-difluoro-3-methyl-2,3-dihydro-4H-[1,4]benzoxazin-4-yl]methylenemalonate. This product was dissolved in 5 ml of acetic anhydride, and 10 ml of a mixtureof acetic anhydride and concentrated sulfuric acid (2/1 by volume) with stirring underice-cooling, followed by stirring at 50.degree. to 60.degree. C. for 40 minutes. To thereaction mixture were added ice and an aqueous solution of sodium bicarbonate, andthe reaction product was extracted three times with 150 ml portions of chloroform.
The combined extract was washed with water, dried over anhydrous sodium sulfateand concentrated under reduced pressure. When a solid began to precipitate, a smallamount of diethyl ether was added thereto, and the precipitate was collected byfiltration. The precipitate was washed with a small amount of diethyl ether to yield1.32 g of (-)-ethyl 9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazi ne-6-carboxylate.
In 12 ml of acetic acid was dissolved 1.20 g of the resulting compound, and 25 ml ofconcentrated hydrochloric acid was added to the solution, followed by refluxing at120.degree. to 130.degree. C. for 90 minutes. Upon allowing the reaction mixture tostand at room temperature, colorless needle-like crystals were precipitated, whichwere collected by filtration and washed successively with a small amount of water,ethanol and diethyl ether to obtain 0.96 g of (-)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzo xazine-6-carboxylic acid.
In 30 ml of diethyl ether was suspended 324 mg of the resulting compound, and alarge excess of boron trifluoride ethyl etherate was added thereto, followed bystirring at room temperature for 30 minutes to form a chelate compound. The productwas collected by filtration and washed with a small amount of diethyl ether to obtain373 mg of a powder. The powder was dissolved in 7 ml of dimethyl sulfoxide, and 136mg of N-methylpiperazine and 228 mg of triethylamine were added thereto, followedby stirring at room temperature for 17 hours. The reaction mixture was concentratedto dryness under reduced pressure, and to the solid were added 15 ml of 95%methanol and 0.31 ml of triethylamine. The resulting mixture was refluxed for 3hours. The reaction mixture was concentrated under reduced pressure, and the http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
residue was filtered and washed successively with a small amount of ethanol anddiethyl ether to obtain 350 mg of a white powder. Recrystallization from a mixedsolvent of ethanol and thick aqueous ammonia gave 230 mg of S(-)-Ofloxacin.
Melting Point: 225.degree.-227.degree. C. (with decomposition).
[.alpha.].sub.D.sup.23 =-76.9.degree. (c=0.39, 0.05N NaOH) .sup.1 H-NMR (CDCl.sub.3, 200 MHz) .delta. (ppm): 1.63 (3H, d, C.sub.3 --CH.sub.3),2.38 (3H, s, N--CH.sub.3), 2.54-2.60 (4H, m, 2.times.CH.sub.2 N), 3.4-3.44 (4H, m,2.times.CH.sub.2 N), 4.35-4.52 (3H, m, CH and CH.sub.2), 7.76 (1H, d, aromatic ringC.sub.8 --H) and 8.64 (1H, s, C.sub.5 --H) Preparation of 3S-(+)-7,8-Difluoro-2,3-Dihydro-3-Methyl-4-[(S)-N-para-toluenesulfonylprol yl]-4H-[1,4]Benzoxazine (X") A solution of an acid chloride, which was prepared from 61.9 g of (S)-N-p-toluenesulfonylproline and thionyl chloride, in 350 ml of dried dichloromethane wasslowly added dropwise to a solution of 32.8 g of (.+-.)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine and 28 ml of pyridine in 300 ml of drieddichloromethane at room temperature under stirring. The stirring was furthercontinued for an additional 4 hours at room temperature. The reaction mixture waswashed successively with 10% hydrochloric acid, a saturated aqueous solution ofsodium hydrogencarbonate and a saturated aqueous solution of sodium chloride anddried over anhydrous magnesium sulfate. The dichloromethane was removed bydistillation, and the oily residue was dissolved in 200 ml of ethyl acetate. To thesolution was slowly added dropwise 750 ml of n-hexane while stirring wherebycrystals ((-)-isomer of the compound (X") wherein X.sub.1 =X.sub.2 =F; R.sub.1=CH.sub.3 ; R.sub.2 =p-toluenesulfonyl; and n=1) precipitated immediately. Theprecipitated crystals were separated by filtration, and the filtrate was concentrated todryness under reduced pressure. The residue was subjected to columnchromatography using 500 g of silica gel and eluted with benzene/ethyl acetate(50/1-25/1 by volume) to obtain an oily product. The oily product was dissolved in 500ml of ethanol, and the solution was allowed to stand at room temperature for 1 day,thereby to precipitate crystals. The ethanol was distilled off, and to the thusrecovered crystals were added diethyl ether and n-hexane, followed by filtration. Thesolid was dried under reduced pressure to obtain 33.4 g of 3S-(+)-7,8-difluoro-2,3-dihydro-3-methyl-4-[(S)-N-p-toluenesulfonylprolyl] -4H-[1,4]benzoxazine((+)-isomer of the compound (X") wherein X.sub.1 =X.sub.2 =F; R.sub.1 =CH.sub.3 ;R.sub.2 =p-toluenesulfonyl; and n=1) having a melting point of107.degree.-108.degree. C.
[.alpha.].sub.D =+70.7.degree. (c=0.953, chloroform) http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
IR .nu.sub.max.sup.KBr (cm.sup.-1): 1685, 1510, 1490 Elementary Analysis for C.sub.21 H.sub.22 F.sub.2 N.sub.2 O.sub.4 S: Calcd. (%): C57.79, H 5.08, N 6.42 Found (%): C 58.05, H 5.14, N 6.47 Preparation of S-(-)-7,8-Difluoro-2,3-Dihydro-3-Methyl-4H-[1,4]Benzoxazine (X) In 1 liter of ethanol was dissolved 32.8 g of the (+)-isomer as obtained in Example 12,and 300 ml of 1N sodium hydroxide was added thereto, followed by refluxing for 3hours. The ethanol was removed by distillation, and the oily residue was extractedwith benzene. The extract was washed with a saturated aqueous solution of sodiumchloride, dried over sodium sulfate and distilled to remove benzene. The residue wassubjected to column chromatography using 200 g of silica gel as a carrier andbenzene as an eluent to obtain 12.7 g (yield: 91.4%) of S-(-)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine as an oily product.
[.alpha.].sub.D =-9.6.degree. (c=2.17, chloroform) The absolute configuration of this compound was decided to be an S-configuration byX-ray analysis on its hydrochloride.
Preparation of Ethyl (S)-(-)-9,10-Difluoro-3-Methyl-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]B enzoxazine-6-Carboxylate (XII) To 15.8 g of the (S)-(-)-benzoxazine derivative as obtained in Example 13 was added24.0 g of diethyl ethoxymethylenemalonate, and the mixture was stirred at130.degree. to 140.degree. C. for 1 hour under reduced pressure. After cooling, thereaction mixture was dissolved in 50 ml of acetic anhydride, and 80 ml of a mixture ofacetic anhydride and concentrated sulfuric acid (2:1 by volume) was slowly addeddropwise to the solution while stirring under ice-cooling. After continuing the stirringfor additional one hour at room temperature, the reaction mixture was stirred in a hotbath of 50.degree. to 60.degree. C. for 30 minutes. Ice-water was added to thereaction mixture, and powdery potassium carbonate was added thereto forneutralization. The mixture was extracted with chloroform, and the extract waswashed successively with a saturated aqueous solution of sodium hydrogencarbonateand a saturated aqueous solution of sodium chloride and dried over sodium sulfate.
The chloroform was removed by distillation, and to the residue was added diethylether. The crystals thus formed were collected by filtration to give 20.0 g of the titledcompound having a melting point of 257.degree.-258.degree. C.
[.alpha.].sub.D =-68.1.degree. (c=0.250, acetic acid) http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&.
Preparation of S-(-)-9,10-Difluoro-3-Methyl-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]Ben zoxazine-6-Carboxylic Acid In 150 ml of acetic acid was dissolved 19.5 g of the ester compound obtained inExample 14, and 400 ml of concentrated hydrochloric acid was added thereto,followed by refluxing for 3 hours. After cooling, the precipitated crystals werecollected by filtration, washed successively with water, ethanol and diethyl ether anddried to obtain 16.2 g of the corresponding carboxylic acid having a melting point of300.degree. C. or higher.
[.alpha.].sub.D =-65.6.degree. (c=0.985, DMSO) Preparation of S-(-)-9-Fluoro-3-Methyl-10-(4-Methyl-1-Piperazinyl)-7-oxo-2,3-Dihydro-7H-P yrido[1,2,3-de][1,4]Benzoxazine-6-Carboxylic Acid (VI) (S-(-)-Isomer ofOfloxacin In 600 ml of diethyl ether was suspended 14.3 g of the carboxylic acid obtained inExample 15, and 70 ml of boron trifluoride diethyl etherate was added thereto,followed by stirring at room temperature for 5 hours. The supernatant liquid wasremoved by decantation, and to the residue was added diethyl ether, followed byfiltration. The solid was washed with diethyl ether and dried. The product wasdissolved in 100 ml of dimethyl sulfoxide, and 14.2 ml of triethylamine and 7.3 ml ofN-methylpiperazine were added to the solution. After the mixture was stirred at roomtemperature for 18 hours, the solvent was removed by distillation. Diethyl ether wasadded to the residue, followed by filtration. The collected yellow powder wassuspended in 400 ml of 95% methanol, and 25 ml of triethylamine was added thereto.
The mixture was heated at reflux for 25 hours. The solvent was distilled off underreduced pressure, and the residue was dissolved in 500 ml of 10% hydrochloric acidand washed three times with chloroform. The washed solution was adjusted to a pH of11 with a 4N sodium hydroxide aqueous solution and then to a pH of 7.3 with 1Nhydrochloric acid. The solution was extracted three times with 2 liter portions ofchloroform, and the combined extract was dried over sodium sulfate. The chloroformwas removed by distillation, and the resulting crystal was recrystallized fromethanol/diethyl ether to obtain 12.0 g of the titled compound having a melting point of226.degree.-230.degree. C. (with decomposition).
[.alpha.].sub.D =-76.9.degree. (c=0.655, 0.05N NaOH) Preparation of (S)-(-)-9-Fluoro-3-Methyl-10-(4-Ethyl-1-Piperazinyl)-7-Oxo-2,3-Dihydro-7H- Pyrido[1,2,3-de][1,4]Benzoxazine-6-Carboxylic Acid (VI) In the same manner as described in Example 16 except that N-ethylpiperazine wasused in place of N-methylpiperazine, the titled compound was obtained having amelting point of 229.degree.-230.degree. C. (with decomposition).
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Elementary Analysis for C.sub.19 H.sub.22 FN.sub.3 O.sub.4 : Calcd. (%): C 60.79 H5.91 N 11.19 Found (%): C 60.97 H 5.91, N 11.30 [.alpha.].sub.D =-67.0.degree. (c=0.585, H.sub.2 O) NMR (CDCl.sub.3) .delta. (ppm): 1.16 (3H, t, J=7 Hz, --CH.sub.2 CH.sub.3), 1.63 (3H,d, J=7 Hz, CH.sub.3), 2.53 (2H, q, J=7 Hz, CH.sub.2 CH.sub.3), 2.57-2.69 (4H, m,2.times.CH.sub.2), 3.4-3.53 (4H, m, 2.times.CH.sub.2), 4.32-4.58 (3H, m, CH andCH.sub.2), 7.77 (1H, d, J=12 Hz, C.sub.8 --H), 8.67 (1H, S, C.sub.5 --H) While the invention has been described in detail and with reference to specificembodiments thereof, it will be apparent to one skilled in the art that various changesand modifications can be made therein without departing from the spirit and scopethereof.

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