Field of the Invention

The present invention relates to novel cephalosporin derivatives of the formula( I ) and pharmaceutically acceptable non-toxic salts thereof; and more particularly, to processes for preparing these compounds and to pharmaceutical compositions containing the same as active ingredients, which have broad antibacterial activities against both Gram-positive and Gram-negative bacteria.

Summary of the Invention

An object of the present invention is to provide the novel cephalosporin derivatives of the formula( I ) and pharmaceutically acceptable non-toxic salts thereof.

wherein R _la is a hydrogen atom or an amino protecting group;

Rib is a hydrogen atom, G-4 alkyl group, C2-4 alkenyl group, C2-4 alkynyl group, fluoro-substituted C1-3 alkyl group or a substituted or unsubstituted carboxyalkyl group (preferably methyl group, ethyl group, allyl group, propargyl group, fluoromethyl group, 2-fluoroethyl group,

-C(CH3) ₂ COOH group, -CH2COOH group);

Q is CH or N; R2 and R3 may be the same or different and mean individually a group selected from C1-3 alkyl group, C2-4 alkenyl group, C2-4 alkynyl group

and hydroxyl-substituted C1-3 alkyl group; and

wherein R5 is a hydrogen atom, C1-3 alkyl group or a hydroxyl-substituted C1-3 alkyl group;

Rc is a hydrogen atom, C1-3 alkyl group, C2-4 alkenyl group or an amino group;

R7 is a C1-3 alkyl group or a hydroxyl-substituted C1-3 alkyl group; Y is O or S or N; and

Z is a hydrogen atom, d-3 alkyl group, chlorine or fluorine atom.

Detailed Description of the Invention

The cephalosporin compounds of the formula( I ) can be prepared by the displacement reaction of the compounds of the formula(ILT) with the compounds of the formula(IV).

The compounds of the formula(ILT) can be prepared in accordance with known methods(J. of. Antibiotics., 43, 5, 533, 1990. European Patent No. 0333154).

I Isomerization

wherein Rι _a , Rib, R2, R3, R4 and Q are the same as defined above;

Rβ is a hydrogen atom or a carboxyl protecting group; and

L is a leaving group.

Also, the cephalosporin compounds of the formula( I ) can be prepared by the acylation reaction of the compounds of the formula(VDI) with the activated form of the compounds of the formula(IX).

The compounds of the formula(LX) can be prepared in accordance with known methods (J. of. Antibiotics., 36, 8, 1020, 1983. J. of. Antibiotics.,

45, 5, 709, 1992).

( V ) (VI)

wherein Rι _a , Rib, R2, R3, R4, Rs, Q and L are the same as defined above; R9 is an acyl group, formyl group, salicylaldehyde group or benzaldehyde group; and X is a halogen atom or an acid residue.

Specific examples of the compounds of formula( I ) provided by this invention are shown below:

7 β -[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-[(E )- 3-[(4-methylthiazol-5-yl)-methyldimethylammonio]-l-propen-l - yl]3-cephem-4-carboxylate or pharmaceutically acceptable non-toxic salts thereof.

7 β -[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-[(E )-3- [(l-carbamoyl-l-propen-3-yl)-3-ethylmethylammonio]-l -propen- l - yl]3-cephem-4-carboxylate or pharmaceutically acceptable non-toxic salts thereof.

7 ^ -[(Z)-2-(2-aminothiazol-4-yl)-2-fluoroethoxyiminoacetamido]- 3- [(E)-3-[(4-methylthiazol-5-yl)-methyldimethylammonio]-l-prop en- l -yl]3-cephem-4-carboxylate or pharmaceutically acceptable non-toxic salts thereof.

7 β -[(Z)-2-(2-aminothiazol-4-yl)-2-fluoroethoxyiminoacetamido]- 3- [(E)-3-[(l-carbamoyl-l-propen-3-yl)-3-ethylmethylammonio]-l- propen-l-yl]3-cephem-4-carboxylate or pharmaceutically acceptable non-toxic salts thereof.

7 β -[(Z)-2-(5-amino-l,2,4-tMaώazol-3-yl)-2-methoxyirranoacetam ido]-3- [(E)-3-[(l-carbamoyl-l-propen-3-yl)-3-ethylmethylammonio]-l- propen-l-yl]3-cephem-4-carboxylate or pharmaceutically acceptable non -toxic salts thereof.

7 β -[(Z)-2-(5-ammo-l,2,4-u iacliazol-3-yl)-2-methoxyiminoacetamido]-3- [(E)-3-[(4-methylthiazol-5-yl)-methyldimethylammonio]-l-prop en- l -yl]3-cephem -4-carboxylate or pharmaceutically acceptable non -toxic salts thereof.

7 β -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-fluoroethoxyimino- acetamido]-3-[(E)-3-[(4-methylthiazol-5-yl)-methyldimethyl- ammonio]-l-propen-l-yl]3-cephem-4-carboxylate or pharmaceutically acceptable non-toxic salts thereof.

7 ^' β -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-fluoroethoxyimino- acetamido]-3-[(E)-3-[(l-carbamoyl-l-propen-3-yl)-3-ethylmeth yl- ammonio]-l-propen-l-yl]3-cephem-4-carboxylate or pharmaceutically acceptable non -toxic salts thereof.

The following preparations and examples are provided for the purpose

of illustrating certain aspects of the present invention only; and are not to be construed as limiting the scope of the present invention in any way.

Preparation 1:

Preparation of 5-diπιethylaιninomethyl-4-methyloxazole a) Preparation of 4-methyl-5-oxazolecarboxylic acid ethyl ester

To ethyl-2-chloroacetoacetate(5.6g) was added formamide(4.6g). The reaction mixture was stirred at 120 ^° C for 12 hours, cooled to 0"C and aqueous potassium carbonate was added thereto. The mixture was extracted with benzene, dried with anhydrous magnesium sulfate, and concentrated in vacuo to obtain the target product(2.1g). NMR(CDC1 ) δ (ppm) : 1.40(t,3H), 2.50(s,3H), 4.35(q,2H), 7.84(s,lH)

b) Preparation of 5-hydroxymethyl-4-methyloxazole

After 4-methyl-5-oxazolecarboxylic acid ethyl ester(3g) was dissolved in ethyl alcohol (50ml), sodiumborohydride(3g) was added thereto and refluxed for 17 hours. The reaction mixture was concentrated under reduced pressure, water was added to the concentrated solution. After extraction with ethyl acetate dried with anhydrous magnesium sulfate, and then concentrated to obtain the target product(0.9g). NMR(CDC1 ₃ ) δ (ppm) : 2.18(s,3H), 3.45(s,lH), 4.65(s,2H), 7.80(s,lH)

c) Preparation of 5-dimethylaminomethyl-4-methyloxazole To a solution of 5-hydroxymethyl-4-methyloxazole(2g) dissolved in dichloromethane(20ml) were added thionylchloride(3.9ml) at 0 ^° C. After stirring at room temperature for 4 hours, ice- water (15ml) were added thereto. The seperated organic layer was dehydrated and concentrated. The residue were added ethylalcohol(25ml) dimethylamine hydrochloride (2.5g) and potassium carbonate(6.3g). After stirring at room temperature for 4 hours, the solid was filtered off. The filtrate was concentrated under reduced pressure, water and dichloromethane added thereto. The separated organic layer was dehydrated, and concentrated to obtain the target product(0.9g). NMR(CDCl3) δ (ppm) : 2.12(s,3H), 2.24(s,6H), 3.42(s,2H), 7.78(s,lH)

Preparation 2-

Preparation of 5-dimethylaminomethyl-4-methylthiazole a) Preparation of 4-methyl-5-thiazolecarboaldehyde.

To a solution of pyridinium dicromate(164.19g) in dichloromethane (600ml), water(20ml) was added. A solution of 4-methyl- 5-thiazoleethanol(10g) in dichloromethane(500ml) was added dropwise to the mixture and the resulting mixture stirred at room temperature for 24 hours. The solid was filtered off and washed with water. The separated organic layer was dehydrated, and concentrated to obtain the target product(6g).

NMR(CDC1 ₃ ) δ (ppm) : 2.79(s,3H), 9.02(s,lH), 10.18(s,lH)

b) Preparation of 4-methyl-5-thiazolemethanol

To sodiumborohydride(2.32g) was added tetrahydrofurane(35ml)at 0 ^° C. A solution of 4-methyl-5-thiazolecarboaldehyde(6g) in tetrahydrofurane (35ml) was added dropwise to the mixture and the resulting mixture stirred at room temperature for 3 hours. To the reaction mixture was added ice-water(60ml) and saturated brine(30ml). The separated organic layer was dehydrated, and concentrated to obtain the target product (5g). NMR(CDC1 ₃ ) δ (ppm) : 2.35(s,3H), 4.79(s,2H), 8.61(s,lH)

c) Preparation of 5-dimetiιylaminomethyl-4-methylthiazole

To a solution of 4-methyl-5-thiazolemethanol(1.5g) in chloroform(50ml), thionylchloride (1.7ml) was added, and the mixture was refluxed for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue were added ethylalcohol(25ml), dimethylamine hydrochloride (1.33g) and potassium carbonate(2.3g). After refluxing for 5 hours, the solid was filtered off. The filtrate was concentrated under reduced pressure, water and chloroform added thereto. The separated organic layer was dehydrated, and concentrated to obtain the target product(lg). NMR(CDCb) δ (ppm) : 2.24(s,6H), 2.41(s,3H), 3.63(s,2H), 8,62(s,lH)

Preparation 3: Preparation of 4-ethytaethylaιnmo-2-butenylamide a) Preparation of 4-bromo-2-butenenitrile To a solution of ally ley anide(40g) dissolved in tert-butanol(60ml) and petroleum ether(273ml) was added bromine (30.6ml) dissolved in

tert-butanol(60.8ml) at 15°C, and then stirred at room temperature for 15 — 30 minutes. To sodium ethoxide(222.6ml, 21%) was added dropwise to the mixture and the solid was filtered off. The filtrate was distilled under reduced pressure to obtain the target product(55g) b.p : 80~85t (12mm).

NMR(CDC1 ₃ ) δ (ppm) :4.10(s,2H), 5.54(m,lH), 6.81 (m,lH)

b) Preparation of 4-bromo-2-butenylamide

To a solution of 4-bromo-2-butenenitrile(20g) suspended in water (2.3ml) was added slowly sulfuric acid(7.8ml) at 40 TJ, and then stirred at 80~90°C for an hour. The reaction mixture was cooled to 40 ^° C, and then added ice(40g) and ammonia solution(19.8ml). The resulting precipitate was collected by filtration and recrystallized from ethylacetate to obtain the target product(7.7g). NMR(CDCl3) δ (ppm) : 4.15(d,2H), 5.8~6.2(br m,lH, NH ₂ ), 6.92(m,lH)

c) Preparation of 4-ethylmethylamino-2-butenylamide

To a solution of 4-bromo-2-butenylamide(5.4g) in acetonitrile(55ml), N-ethylmethylamine(5.7ml) was added at 0 ^° C. The reaction mixture was stirred for an hour and the resulting precipitate was collected by filtration to obtain the target product(3.1g).

NMR(DMSO-dc)) δ (ppm) : 1.01(t,2H), 2.13(s,3H), 2.35(q.2H), 3.04(d,2H), 5.99(d,lH), 6.55(m,lH)

Example 1

Synthesis of 7 ? -[(Z)-2-(2-ammothiazol-4-yl)-2-methoxyiτιιino- acetamido]-3- [(E)-3- [(4-methylthiazol-5-yl)-methyldimethyl- ammonio] - 1 -propen- 1 -yl]3-cephem-4-carboxylate

To a solution of jo-methoxybenzyl 7 iS -[(Z)-2-methoxyimino-2-(2- tritylammothiazol-4-yl)acetamido]-3-[(Z)-3-chloro-l-propen-l -yl]3- cephem-4-carboxylate(1.5g) dissolved in acetone (30ml), sodium iodide(0.82g) was added thereto under ice-cooling. The resulting solution was stirred for lδminutes under ice-cooling and for additional 60minutes at room temperature. The solvent was distilled off, and the residue was extracted with ethyl acetate. The extract was washed with a 10%-sodium thiosulfate and with a saturated brine, followed by

drying with addition of anhydrous magnesium sulfate. The dried extract was concentrated under reduced pressure and the resulting precipitates were collected by filtration to obtain 1.2g of p-methoxy benzyl 7 β -[(Z)-2-methoxyimino-2-(2-tritylaminothiazol- 4-yl)acetamido]-3-[(E)-3-iodo-l-propen-l-yl]3-cephem-4-carbo xylate. The powder was dissolved in N,N-dimethylformamide(2ml), and 5-dimethylaminomethyl-4-methylthiazole(0.31g) was added thereto at -10 ^° C . The resulting solution was stirred for 4 hours at -10"C. 10%-sodium thiosulfate(70ml) was added to the reaction solution, and the resulting precipitates were collected by filtration to obtain the yellowish brown powder. The powder was stirred in 88% formic acid( 1.8ml) for an hour at 40 "C, and acetone(400ml) was added thereto. The resulting precipitates were collected by filtration and washed with acetone. The precipitates were chromatographed over silicagel [acetonitrile : distilled water 4:1 (v/v)], Sephadex LH-20(or Amberlite XAD-2), and then lyophilized to obtain the target product (250mg) as white solid, mp : 181 ^C C (dec.) IR(KBr,cm ^_1 ) : 1762, 1663, 1608, 1530 NMR(DMSO-de) δ(ppm) : 2.43(s,3H), 2.94(s,6H), 3.42(s,2H), 3.82(s,3H), 4.01(d,2H), 4.62(s,2H), 5.04(d,lH), 5.58(d,lH), 5.62(br d,lH), 7.1 ~7.4(d,lH. s,lH. s,lH), 9.62(d,lH)

Example 2 : Synthesis of 7i? -[(Z)-2-(2-ammoι^azol-4-yl)-2-methoxyimino- acetaπύdo]-3-[(E)-3-[(5-methylimidazol-4-yl)-meΛyldimethy l- ammonio]-l-propen-l-yl]3-cephem-4-carboxylate

The same procedures as described in Example 1 were repeated using

0.17g of 4-dimethylaminomethyl-5-methylimidazole in place of 5-dimethylaminomethyl-4-methylthiazole to obtain the target product(lθθmg). mp : 178 ^° C (dec.)

IR(KBr,cm ^_1 ) : 1760, 1670, 1610, 1531

NMR(DMSO-de) δ(ppm) : 2.25(s,3H), 2.98(s,6H), 3.38(s,2H), 3.80(s,3H), 4.11(d,2H), 4.59(s,2H), 5.01(d,lH), 5.53(d,lH), 5.59(br d,lH), 7.2-

7.84(d,lH. s,lH. s,lH), 9.66(d,lH)

Example 3 :

Synthesis of 7>? -[(Z)-2-(2-ammotJιiazol-4-yl)-2-methoxyimino- ace1^ιmido]-3--[(E)-3-[(2-aminothiazol-4-yl)-methyldimethyl - ammonio]-l -propen-l-yl]-3-cephem-4-carboxylate

The same procedures as described in Example 1 were repeated using

0.21g of 4-dimethylaminomethyl-2-aπύr othiazole in place of

5-dimethylaminomethyl-4-methylthiazole to obtain the target product(150mg). mp : 192 °C (dec.) m(KBr,cm ^_1 ) : 1760, 1672, 1600, 1530

NMR(DMSO-de) δ(ppm) : 2.12(s,6H), 3.35(s,2H), 3.83(s,3H), 4.01(d,2H),

4.55(s,2H), 5.02(d,lH), 5.58(d,lH), 5.62(br d,lH), 6.72(br,2H), 7.1 -

7.5(d,lH. s,lH. s,lH), 9.60(d,lH)

Example 4 :

Synthesis of 7 S -[(Z)-2-(2-ammothiazol-4-yl)-2-methoxyimino- acetamido]-3-[(E)-3-[(thiopen-2-yl)-methyldimethylammonio]-

1 -propen- 1 -yl]3-cephem-4-carboxylate The same procedures as described in Example 1 were repeated using

0.18g of 2-thiopenmethyldimethylamine in place of 5-dimethylamino- methyl-4-methylthiazole to obtain the target product (170mg). mp : 186 ^° C(dec.)

EKKBr.cm ^"1 ) : 1762, 1670, 1600, 1530 NMR(DMSO-d _c ) δ(ppm) : 2.98(s,6H), 3.32(s,2H), 3.88(s,3H), 4.01(d,2H),

4.49(s,2H), 5.02(d,lH), 5.55(d,lH), 5.62(br d,lH), 6.70(br d,2H. IH), 7.1-7.

7(d,lH. s,lH. s.lH), 9.60(d,lH)

Example 5 : Synthesis of 7 # -[(Z)-2-(2-ammot iazol-4-yl)-2-methoxyLτmno- acetamido] -3- [(E)-3- [(4-methyloxazol-5-yl)-methyldimethyl- ammonio] - 1 -propen- 1 -yl]3-cephem-4-carboxylate

The same procedures as described in Example 1 were repeated using 0.31g of 5-dimethylaminomethyl-4-methyloxazole in place of 5-dimethylaminomethyl-4-methylthiazole to obtain the target product (300mg).

mp : 199 ^° C (dec.) mCKBr-cm ^"1 ) : 1762, 1668, 1610, 1533

NMR(DMSO-do) δ(ppm) : 2.25(s,2H), 2.95(s,6H), 3.42(s,2H), 3.84(s,lH), 4.01(d,2H), 4.62 5 (s,2H), 5.04(d,lH), 5.58(d,lH), 5.62(br d,lH), 7.1 -7.3(d,lH. s,lH. s,lH), 9.58(d,lH)

Example 6 :

Synthesis of 7^ -[(Z)-2-(2-ammothiazoI-4-yl)-2-methoxyimino- 10 acetamido]-3-[(E)-3-[(l-carbamoyl-l-propen-3-yl)-3-ethyl- methylammonio] - 1 -propen - 1 -y l]3-cephem-4 -carboxylate

The same procedures as described in Example 1 were repeated using

0.23g of 4-ethylmethylamino-2-butenylamide in place of

5-dimethylaminomethyl-4-methylthiazole to obtain the target product 15 (190mg). mp : 189 ^° C (dec.) m(KBr,cm ^_1 ) : 1761, 1675, 1608, 1530

NMR(DMSO-d ₆ ) δ(ppm) : 1.29(t,3H), 2.98(s,3H), 3.82(s,3H), 3.85(q,2H),

3.90(d,2H), 3.94 20 (d,2H), 4.75(s,2H), 5.08(d,lH), 5.62(d,lH), 5.68(br d,lH), 6.75(d,lH),

6.99-7.12(br d,lH), 7.21 ~7.38(d,lH. s,lH), 9.59(d,lH)

Example 7 :

Synthesis of 7/S -[(Z)-2-(2-ammothiazol-4-yl)-2-methoxyimino- 25 acetamido]-3-[(E)-3-[(l-carbamoyl-l-propen-3-yl)-3-dimethyl- ammonio]-l-propen-l-yl]3-cephem-4-carboxylate

The same procedures as described in Example 1 were repeated using

0.25g of 4-dimethylamino-2-butenylamide in place of

5-dimethylaminomethyl-4-methylthiazole to obtain the target 30 product(200mg). mp : 198°C(dec.)

HKKBr.cm ^"1 ) : 1763, 1670, 1600, 1530

NMR(DMSO-d6) δ(ppm) : 3.11(s,6H), 3.88(s,3H), 3.92(d,2H), 3.95(d,2H),

4.07(s,2H), 5.05(d,lH), 355.62(d,lH), 5.69(br d,lH), 6.73(d,lH), 7.01-7.14(br d,lH), 7.18~7.32(d,lH. s,lH), 9.57(d,lH)

Example 8 :

Synthesis of 7 /? -[(Z)-2-(2-aminot^azol-4-yl)-2-meΛoxyimino- acetamido]-3-[(E)-3-[(4-methyltlιiazol-5-yl)-metiιylethylm ethyl- ammonio]-l-propen-l-yl]3-cephem-4-carboxylate

The same procedures as described in Example 1 were repeated using 0.35g of 5-ethylmethylaminomethyl-4-methylthiazole in place of 5-dimethylaminomethyl-4-methylthiazole to obtain the target product (300mg). mp : 194 T, (dec.) πKKBr.cm ^"1 ) : 1762, 1662, 1610, 1530

NMR(DMSO-d ₆ ) δ(ppm) : 1.33(t,3H), 2.44(s,3H), 2.92(s,3H), 3.43(s,2H), 3.85(q,2H), 3.87(s,3H), 4.01 (d,2H), 4.60(s,2H), 5.05(d,lH), 5.56(d,lH), 5.67(br d,lH), 7.18-7.32(d,lH. s,lH. s,lH), 9.60(d, IH)

Example 9 :

Synthesis of 7/? -[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-

2-oxyrnιino)acetamido]-3-[(E)-3-[(l-carbamoyl-l-propen-3 -yl)-

3-ethylmethylammonio]-l-propen-l-yl]3-cephem-4-carboxylat e p-methoxybenzyl 7 5 -[(Z)-2-(2-carboxyprop-2-oxyimino)-2-(2-trityl- aminotlιiazol-4-yl)acetamido]-3-[(Z)-3-chloro-l-propen-l-yl ]3-cephem- 4-carboxylate(1.5g), sodium iodide (0.75g) and

4-ethylmethylamino-2-butenylamide (0.28g) were reacted in the same manner as described in Example 1 to obtain the target product(210mg). mp : 204 ^* C (dec.) lR(KBr, cm-1) : 1760, 1663, 1610, 1530.

NMR(DMSO-d6) δ(ppm) : 1.29(t,lH), 1.49(s,6H), 2.98(s,3H), 3.85(q,2H), 3.90(d,2H), 3.94(d,2H), 4.75(s,2H), 5.08(d,lH), 5.62(d,lH), 5.68(br d,lH), 6.75(d,lH), 6.99~7.12(br d,lH), 7.21 ~7.38(d,lH. s,lH), 9.59(d,lH)

Example 10 :

Synthesis of 7 /S -[(Z)-2-(2-aιi-inothiazol-4-yl)-2-(2-carboxyprop-

2-oxyiιn o)acetemido3-3-[(E)-3-[(4-methylthiazol-5-yl)-methyl- όimethylammonio]-l-pτopen-l-yl]3-cephem-4-carboxylate p-methoxybenzyl 7 ^ -[(Z)-2-(2-carboxyprop-2-oxyimino)-2-(2-trityl- aminothiazol-4-yl)acetamido] -3-[(Z) -3-chloro- 1 -propen- 1 -yl]3-ceρhem-

4-carboxylate(1.5g), sodium iodide (0.75g) and 5-dimethylaminomethyl- 4-methylthiazole(0.31g) were reacted in the same manner as described in Example 1 to obtain the target product(180mg). mp : 198 ^° C (dec.) IR(KBr, cm-1) : 1762, 1665, 1610, 1525.

NMR(DMSO-d6) δ(ppm) : 1.50(s,6H), 2.43(s,3H), 2.91(s,6H), 3.41(s,2H), 4.03(d,2H), 4.60(d,2H), 4.60(s,2H), 5.05(d,lH), 5.57(d,lH), 5.65(br d,lH), 7.1 ~7.4(d,lH. s,lH. s,lH), 9.62(d,lH)

Example 11 :

Synthesis of 7 ^ -[(Z)-2-(2-aminothiazol-4-yl)-2-fluoroethoxy- iminoacetamido]-3-[(E)-3-[(4-methylthiazol-5-yl)-methyl- dimethylammonio]-l-propen-l-yl]3-cephem-4-carboxylate p-methoxybenzyl 7 β [(Z)-2-fluoroethoxyimino-2-(2-tritylaminothiazol- 4-yl)acetamido]3-[(Z)-3-chloro-l-propen-l-yl]3-cephem-4-carb oxylate

(1.3g), sodium iodide(0.68g) and 5-dimethylaminomethyl-4-methyl- thiazole(0.27g) were reacted in the same manner as described in

Example 1 to obtain the target product (300mg). mp : 196"C (dec.) IRd Br.cm ^"1 ) : 1765, 1665, 1610, 1530

NMR(DMSO-d ₆ ) δ(ppm) : 2.41 (s,3H), 2.95(s,6H), 3.42(s,2H), 4.04(d,2H),

4.25(t,2H), 4.53(t,2H), 4.79(s,2H), 5.04(d,lH), 5.56(d,lH), 5.61 (br d,lH), 7.1

~7.4(d,lH. s,lH. s,lH), 9.65(d, IH)

Example 12 :

Synthesis of 7 ? -[(Z)-2-(2-aminothiazol-4-yl)-2-fluoroethoxy- iminoacetamido]-3-[(E)-3-[(l-carbamoyl-l-propen-3-yl)-3-ethy l- methylammonio] - 1 -pτopen-1 -yl]3-cephem-4-carboxy late p-methoxybenzyl 1 β -[(Z)-2-fluoroethoxyimino-2-(2-tritylaminothiazol- 4-yl)acetamido]-3-[(Z)-3-chloro-l-propen-l-yl]3-cephem-4-car boxylate

(1.3g), sodium iodide(0.68g) and 4-ethylmethylamino-2-butenylamide

(0.25g) were reacted in the same manner as described in Example 1 to obtain the target product(230mg). mp : 191 ^C C (dec.) IRdOBr.cπT ¹ ) : 1760, 1672, 1600, 1530

NMR(DMSO-d ₆ ) δ(ppm): 1.27(t,lH), 2.99(s,3H), 3.85(q,2H), 3.90(d,2H),

3.94(d,2H), 4.27(t,2H), 4.52(t,2H), 4.75(s,2H), 5.09(d,lH), 5.60(d,lH), 5.69(br d,lH), 6.74(d,lH), 7.19-7.35(d,lH. s,lH), 9.61(d, IH)

Example 13 : Synthesis of 7/S -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2- methoxyiminoacetamido] -3 -[ (E ) -3- [ (1 -carbamoyl- 1 -propen-3 - yl)-3-ethylmethylammonio]-l-propen-l-yl]3-cephem-4- carboxylate To a solution of p-methoxybenzyl 7£ [(Z)-2-(5-amino-l,2,4-thiadiazol- 3-yl)-2-methoxyiminoacetamido]-3-[(Z)-3-chloro-l-propen-l-yl ]3- cephem-4-carboxylate(1.5g) dissolved in acetone (20ml), sodium iodide (0.82g) was added thereto under ice-cooling. The resulting solution was stirred for 15 minutes under ice-cooling and for additional an hour at room temperature. The solvent was distilled off, and the residue was extracted with ethyl acetate. The extract was washed with a 10% -sodium thiosulfate and with a saturated brine, followed by drying with addition of anhydrous magnesium sulfate. The dried extract was concentrated under reduced pressure and the resulting precipitates were collected by filtration to obtain lg of p-methoxybenzyl Iβ -[(Z)- 2-(5-amino-l,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3 -[(E)-3- iodo-l-propen-l-yl]3-cephem-4-carboxylate. The powder was dissolved in N,N-dimethylformamide(2.5ml), and 4-ethylmethylamino-

2-butenylamide(0.2g) was added thereto at -10 ^° C. The resulting solution was stirred for 4 hours at -10°C. 10%-sodium thiosulfate(70ml) was added to the reaction solution, and the resulting precipitates were collected by filtration to obtain the yellowish brown powder. The powder was stirred in 88%-formic acid(1.3ml) for 3 hours at room temperature, and acetone(400ml) was added thereto. The resulting precipitates were collected by filtration and washed with acetone. The precipitates were chromatographed over silicagel[acetonitrile: distilled water 4 : 1 (v/v)], Sephadex LH-20(or Amberlite XAD-2), and then lyophilized to obtain the target product (180mg) as white solid, mp : 202 TJ (dec.) IR(KBr,cm ^_1 ) : 1765, 1670, 1600, 1520 NMR(DMSO-do) δ(ppm): 1.29(t,3H), 2.99(s,3H), 3.83(s,3H), 3.84(q,2H), 3.91 (d,2H), 3.94(d,2H), 4.73(s,2H), 5.07(d,lH), 5.66(d,lH), 5.69(br d,lH),

6.59(dd,lH), 6.99~7.15(br d,lH), 7.28(dd, IH), 8.63(d,lH), 9.59(d,lH)

Example 14 •

Synthesis of 7^ -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2- fluoroethoxyiminoacetamido]-3-[(E)-3-[(4-methylthiazol-5-yl) - methyldimethylammonio]-l-propen-l-yl]3-cephem-4-carboxylate p-methoxybenzyl 1 β -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-fluoro- ethoxyiminoacetamido] -3-[(E)-3-iodo-l -propen-1 -yl]3-cephem-4- carboxylate(l.lg) and 5-dimethylaminomethyl-4-methylthiazol (0.36g) were reacted in the same manner as discribed in Example 13 to obtain the target product(330mg). mp : 188 ^° C (dec.) πKKBr.cm ^'1 ) : 1763, 1670, 1595, 1522

NMR(DMSO-de) δ(ppm) : 2.40(s,3H), 2.93(s,6H), 3.42(s,2H), 4.04(d,2H), 4.28(t,2H), 4.55(t,2H), 4.78(s,2H), 5.03(d,lH), 5.57(d,lH), 5.61(br d,lH), 7.1

~7.32(d,lH. s.lH), 9.63(d, IH)

Example 15 '■

Synthesis of 7iϊ -[(Z)-2-(5-amino-l,2 _f 4-thiadiazol-3-yl)-2-fluoro- ethoxyiιιιmoacetømido]-3-[(E)-3-[(l-carbamoyl-l-propen-3 -yl)-3- ethylmethylammonio]-l-propen-l-yl]3-cephem-4-carboxylate p-methoxybenzyl 1 β -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-fluoro- ethoxyiminoacetamido]-3-[(E)-3-iodo-l-propen-l-yl]3-cephem-4 - carboxylate(lg) and 4-ethylmethylamino-2-butenylamide(0.3g) were reacted in the same manner as described in Example 13 to obtain the target product(290mg). mp : 196'C (dec.)

IR(KBr,cm ^~l ) : 1762, 1680, 1600, 1520

NMR(DMSO-de) δ(ppm): 1.27(s,lH), 2.98(s,3H), 3.83(q,2H), 3.91(d,2H), 3.94(d,2H), 4.27(t,2H), 4.55(t,2H), 4.76(s,2H), 5.11(d,lH), 5.60(d,lH),

5.70(br d,lH), 6.75(d,lH), 7.03-7.17(d,lH), 9.59(d, IH)

Example 16 :

Synthesis of 7>? -[(Z)-2-(5-aιnino-l,2,4-thiadiazol-3-yl)-2-fluoro- ethoxyiminoacetamido]-3-[(E)-3-[(l-carbamoyl-l-propen-3-yl)- 3-dimethylammonio] -1 -propen-1 -yl]3-cephem-4-carboxylate

p-methoxybenzyl 7 β -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2-fluoro- ethoxyiminoacetamido]-3-[(E)-3-iodo-l-propen-l-yl]3-cephem-4 - carboxylate(1.2g) and 4-dimethylamino-2-butenylamide(0.32g) were reacted in the same manner as described in Example 13 to obtain the target product(230mg). mp : 184 "C (dec.)

IR(KBr,crn ^_1 ) : 1762, 1675, 1598, 1520

NMR(DMSO-d6) δ(ppm): 3.10(s,6H), 3.88(d,2H), 3.92(d,2H), 3.93(d,2H), 4.25(t,2H), 4.52(t,2H), 4.77(s,2H), 5.15(d,lH), 5.59(d,lH), 5.71 (br d,lH), 6.72(d,lH), 7.01 ~7.22(br d,lH), 7.15~7.34(d,lH), 9.62(d,lH)

Example 17 :

Synthesis of p-methoxybenzyl 7)S -benzylideneamino-3-[(E)-3- [(l-carbamoyl-l-prorien-3-yl)-3-ethylmethylammonio]-l-propen - l-yl]3-cephem-4-carboxylate iodide

To a solution of p-methoxybenzyl 7/3 -benzyUdeneamino-3-[(Z)-3- chloro-l-propen-l-yl]3-cephem-4-carboxylate (1.6g) dissolved in acetone(lθml), sodium iodide(1.5g) was added thereto under ice-cooling. The resulting solution was stirred for 3 hours at room temperature. The solvent was distilled off, and the residue was extracted with ethyl acetate. The extract was washed with a 10%-sodium thiosulfate and with a saturated brine, followed by drying with addition of anhydrous magnesium sulfate. The dried extract was concentrated to about 20ml. To the concentrate was added 4-ethylmetlιyl-amino-2-butenylamide (0.57g) in ethyl acetate(30ml). The mixture was stirred for 2 hours at 0 "C, and ethyl ether(50ml) was added thereto. The resulting precipitates were collected by filtration to obtain the target product(1.5g). IR(KBr,cnT) : 1762, 1678, 1600 NMR(DMSO-dc) δ(ppm): 1.27(t,3H), 3.14(s,3H), 3.41 ~3.82(m,4H), 3.73(s,3H), 3.95(br,2H), 4.26(m,2H), 5.23(d,lH), 5.44(d,lH), 5.74(br d,lH), 6.17(m,lH), 6.79(d,lH), 7.01 ~7.19(br d,lH), 7.28(d,lH), 7.3~7.9(m,9H), 8.51(br s,lH)

Example 18 : Synthesis of 7^ -amino-3-[(E)-3-[(l-carbamoyl-l- propen-3-yl)-3-ethylmethylammonio]-l-propen-l-yl]3-cephem-4- carboxylate hydrochloride

The compound(1.5g) prepared in Example 17 was added 90%-formic acid(4.2ml) and 35% -hydrochloric acid(0.85ml). The resulting mixture was stirred for 90 minutes at room temperature, and acetone(500ml) was added thereto. The resulting precipitates were collected by filtration to obtain the target product(0.6g). IR(KBr,cm ^_1 ) : 1786, 1688

NMR(DMSO-d6) δ(p _pm ): 1.27(t,3H), 3.11 (s,3H), 3.24~4.35(m,6H), 4.08(br,2H), 5.22(br,2H), 6.0~6.5(m,lH), 7.04(d,lH), 7.27(d,lH),

7.36(m,lH)

Example 19 :

Synthesis of 7 /? -[(Z)-2-(5-amino-l,2,4-thiadiazol-3-yl)-2- fluoroethoxyiminoacetamido]-3-[(E)-3-[(l-carbamoyl-l-propen- 3- yl)-3-ethylmethylammonio]-l-propen-l-yl]3-cephem-4- carboxylate

The compound(0.6g) prepared in Example 18 was added water ^' methyl alcohol(l:6, 26ml), sodium acetate(0.61g) and (Z)-2-(5-amino-l,2,4- thiadiazol-3-yl) -2-fluoroethoxyiminoaceticacidchloride hydrochloride

(0.43g). The resulting mixture was stirred for 3 hours at 25— 30 "C. After the mixture cooled to 0 ^° C, insoluble materials were filtered off. The filtrate was chromatographed over silicageltacetonitrile :distilled water 4:1 (v/v)], Sephadex LH-20(or Amberlite XAD-2), and then lyophilized to obtain the target product(420mg) as white solid, mp, LR, NMR : Identical with Example 14

Experimental Tests

1. in vitro activity test In order to illustrate the surprisingly superior antibacterial activity of the compounds of the present invention, the minimal inhibitory concentrations(MIC) of the compounds synthesized in the above Examples were determined and compared with Cefotaxime and Cefpirome which were used as the control compounds. These MIC values were determined by agar dilution method : that is, two-fold dilutions of each of the test compounds were made and dispersed in a

Mueller-Hinton Broth medium. Standard test strain which had the 10 ⁶ CFU per mi was inoculated on the medium, and was incubated at 37 ^° C for 18 to 20 hours. The results of the MIC tests are shown in Table 1.

Table 1. Minimal Inhibitory Concentration (MIC) of test compounds ( g/ml)

Example Example Example

Test Strain CTX CPR 1 6 12

1. Streptococcus pyogenes TIA 0.006 0.012 0.025 0.012 0.012

2. Streptococcus faecium MD 8b 50 100> 100> 100> 100

3. Staphylococcus aureus SG511 0.4 0.8 0.8 1.6 0.8

4. Staphylococcus aureus 285 0.4 0.4 0.8 1.6 0.4

5. Staphylococcus aureus 503 0.8 0.8 0.8 3.1 0.8

6. Escherichia coli 078 <0.006 0.12 0.025 0.025 0.025

7. Escherichia coli DC 0 0.025 <0.006 <0.006 0.012 0.012

8. Escherichia coli DC 2 0.05 0.05 0.05 0.05 0.1

9. Escherichia coli TEM 0.05 0.05 0.05 0.05 0.05

10. Escherichia coli 1507E 0.025 0.05 0.05 0.025 0.025

11. Pseudomonas aeruginosa 9027 12.5 6.3 12.5 25 6.3

12 Pseudomonas aeruginosa 1592E 3.1 3.1 6.3 25 3.1

13. Pseudomonas aeruginosa 1771 6.3 3.1 6.3 12.5 3.1

14. Pseudomonas aeruginosa 1771M 0.4 0.4 0.8 0.4 0.8

15. Salmonella typhimurium 0.1 0.1 0.4 0.1 0.1

16. Klebsiella aerogenes 1082E 6.3 3.1 3.1 6.3 6.3

17. Klebsiella aerogenes 1522E 0.05 0.1 0.1 0.1 0.1

18. Enterobacter cloace P99 0.4 1.6 1.6 100> 3.1

19. Enterobacter cloace 1321E 0.012 0.025 0.05 0.05 0.025

8? CTX : Cefotaxime X CPR : Cefpirome

Table 1. (continued)

Example Example Example

Test Strain CTX CPR 13 14 15

1. Streptococcus pyogenes 77A 0.025 0.05 0.025 0.012 0.012

2. Streptococcus faecium MD 8b 100> 100> 100> 100> 100

3. Staphylococcus aureus SG511 0.8 0.4 0.8 1.6 0.8

4 Staphylococcus aureus 285 0.8 0.8 0.8 1.6 0.4

5. Staphylococcus aureus 503 0.8 0.8 0.8 3.1 0.8

6. Escherichia coli 078 0.05 0.012 0.025 0.025 0.025

7. Escherichia coli DC 0 0.025 <0.006 0.012 0.012 0.012

8. Escherichia coli DC 2 0.2 0.05 0.1 0.05 0.1

9. Escherichia coli TEM 0.05 0.025 0.05 0.05 0.05

10. Escherichia coli 1507E 0.1 0.025 0.1 0.025 0.025

11. Pseudomonas aeruginosa 9027 3.1 12.5 3.1 25 6.3

12 Pseudomonas aeruginosa 1592E 1.6 3.1 3.1 25 3.1

13. Pseudomonas aeruginosa 1771 1.6 3.1 3.1 12.5 3.1

14. Pseudomonas aeruginosa 1771M 0.4 0.4 0.8 0.4 0.8

15. Salmonella typhimurium 0.2 0.1 0.2 0.1 0.1

16. Klebsiella aerogenes 1082E 3.1 3.1 3.1 6.3 6.3

17. Klebsiella aerogenes 1522E 0.2 0.025 0.05 0.1 0.1

18 Enterobacter cloace P99 1.6 0.4 0.8 100> 3.1

19. Enterobacter cloace 1321E 0.05 0.012 0.05 0.05 0.025

CTX : Cefotaxime

* CPR : Cefpirome

As can be seen from Table 1, the cephalosporin compounds of the present invention possess potent and broad antibacterial activities as compared with the known broad spectrum cephalosporin antibiotics, Cefotaxime and Cefpirome. More specifically, MICs of Example 1, 6, 12, and 14 against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were superior to that of Cefotaxime and similar to that of Cefpirome. Also, MICs of Example 13 against Pseudomonas aeruginosa was superior to that of Cefpirome.

2. Acute toxicity studies

In order to illustrate usefulness of the compounds of the present invention, acute toxicity test of the compounds synthesized in the above Examples were carried out Each dose of the compounds dissolved in saline and buffer solution (PH=7.0) was administered by intravenous or subcutaneous injection. Mortalities of the animals was recorded seven days latter. The results of the acute toxicity studies are shown in Table 2.

Table 2.

LDsomg/kg

Compound

I.V s.c

Example 1 >3000 >4000

Example 2 >2000 >3000

Example 6 >3000 >3000

Example 12 >2000 >3000

Example 13 >2000 >3000

Example 15 >2000 >3000

^ Mouse : Male ICR strain, 4 weeks

The compounds of the Example 1, 2, 6, 12, 13 and 15 are shown high stability as an antimicrobial medicament from >2000mg/Kg in intravenous routes and >3000mg/Kg in subcutaneous routes. Accordingly, the compounds of the present invention can be used in the therapeutic treatment of human beings or animals infected with variety of Gram-positive or Gram-negative bacteria.

The compounds of the present invention may be administered 1 — 3 times in an amount ranging from 50— 5000mg/day. The present invention also includes within its scope pharmaceutical compositions comprising one or more of the compound( I ) and their derivatives as active ingredients, in association with pharmaceutically acceptable carriers, excipients or other additives, if necessary. The compositions may be formulated into various forms such as tablets, capsules, solution, injection, syrup, ointment, cream, suppositories, which may contain conventional additives such as a dispersant, suspending agent, stabilizer and the like.

Formulation examples are described below.

Formulation example 1

The compound of prepared Example 1 lOOmg

Lactose lOOmg

Corn starch 50mg Talc 45mg

Magnesium stearate 5mg

The above compositions are formulated into tablets by the conventional tablets preparation method.

Formulation example 2

The compound of prepared Example 6 lOOmg

Lactose 50mg

Con starch lOOmg Talc 20mg

Magnesium stearate 5mg

The above compositions are formulated into capsules by the conventional capsules preparation method.

Formulation example 3

The compound of prepared Example 13 250mg Sodium chloride 12.5mg

The above compositions are formulated into injection by the conventional injection preparation method.