WO1995004740A1 - 7α-AMINO-1,1-DIOXOCEFEM L-VALYLAMIDES - Google Patents

Abstract

L-valylamides and L-homovalylamides of 7α-amino-1,1-dioxo-3-cefem of formula (I), the process for obtaining them and the pharmaceutical compositions which contain them are described. The above-mentioned compounds are active in chronic bronchitis, in emphysema, in ARDS, in cystic fibrosis and in rheumatoid arthritis.

Description

  
 



   Description    7α-amino-1,1-dioxocefem L-valylamides   
The present invention relates to   7'-amino-l,l-dioxocefem    Lvalylamides,   the    method for their preparation and the pharmaceutical compositions containing them.



  More particularl the present invention refers to L-valyl and   L-homovalylamide    compounds of the formula (I):
EMI1.1     
 wherein R is hydrogen, a conventional protecting group of the
 amino group   of-α-aminoacids    and an arylcarbonyl
 radical;
 R is a straight or branched alkoxy radical,
   cont;li.ning    1-4 carbon atoms,   benzhydryloxy    radical;
 4-substituted piperazin-l-yl or the group NR R
 45
 where R4 is hydrogen and   P5    represents the rest of
 a   L-α

  ;-aminoacid    or R4 and R5 together with the
 nitrogen to which they are attached form a 5 or 6
 atom ring selected from 2-carboxypyrrolidyl and
   4-carboxypiperidyl,    the carboxy groups optionally
   being    all at the same time   esterified    preferably as
 tert-butyl and/or benzhydryl esters or in the form
 of pharmaceutically acceptable salts of an
 inorganic cation and/or ammonium;
 R is a straight or branched   alkyl    radical, containing
 2
 1-4 carbon atoms;  
   P    is a straight alkyl radical containing 1-2 carbon
 3
 atoms; and to the salts with pharmaceutically acceptable acids of the compounds (I) in which R is hydrogen.



  Examples of   conventional    protecting groups of the   α-aminoacid      amino    groups are benzyloxycarbonyl in which the benzene ring is not substituted or 4-methoxy or 2,6-dimethyl substituted; methoxy-, ethoxy-, allyloxy- carbonyl;   2,2, 2-trichloroe thoxycarbonyl;    9-fluorenylmethoxycarbonyl.



  Among them benzyloxycarbonyl is preferred.



  Examples of arylcarbonyl radicals are radicals of formula:
EMI2.1     
 wherein X is selected from the group consisting of -NH-CO- and -NH- and P is selected from the group consisting of hydrogen, chlorine and methyl, preferably being chlorine.



  Examples of   4-substituted-piperazin-l-yl    are 4-methyl   piperazin-1-yl,      4-ethylpiperazin-1-yl,    4-(2-dimethylamino) ethylpiperazin-l-yl, 4-(2-diethylamino)ethylpiperazin-l- yl.



  Examples of residues of   L-x-aminoacids    are those deriving from mono-and   di-cark)oxylic    -aminoacids optionally substituted with a S atom   ci    with a SO2 group, among which are preferred those residues of a C3-C6 dicarboxylic   L-oc-aminoacids,    namely such as aspartic, glutamic, adipic, 4-thiaadipic, 4,4-dioxo-4-thiaadipic acid among   which    4-thiaadipic acid is preferred..



     P    is preferably methyl.



   2   P    is preferably methyl.



   3
Examples of straight or branched alkoxy radical, containing 1-4 carbon atoms are methoxy, ethoxy, isopropyloxy, butoxy and  tert-butoxy; among them tert-butoxy is preferred.



  Examples of straight or branched alkyl radical, containing 1-4 carbon atoms, are   methyl,    isopropyl and tert-butyl; among them methyl is   preferre(].   



  The preferred compound of the invention is: 7o(-[(N-benzyloxycarbonyl)-L-valylamido]-1,1-dioxo-3-acetoxy-methyl-4-tertbutoxycarbonyl-3-cefem.



  Other preferred   compounds    of the invention are: 7   α    -[(N-benzyloxycarbonyl)-L-isoleucylamido]-1,1-dioxo-3 acetoxymethyl-4-tert-butoxycarbonyl-3-cefem;    7 α -[(N-(9-fluorenylmethoxy)carbonyl)-L-valylamido]-1,1-dioxo-    3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem ; 7   α    -[(N-(9-fluorenylmethoxy)carbonyl)-L-isoleucylamido]-1,1dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem ; 7   c(    - [(N-benzyloxycarbonyl)-L-valylamido]-1,1-dioxo-3-acetoxy- methyl-4-benzhydryloxycarbonyl-3-cefem; 7   ot    - [(N-benzyloxycarbonyl)-L-isoleucylamido]-1,1-dioxo-3- acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem;    7α-[(N-(9-fluorenylmethoxy)carbonyl)-L-valylamido]-1,1-dioxo3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem;

   7α-[(N-(9-fluorenylmethoxy)carbonyl)-L-isoleucylamido]-1,1dioXo-3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem;    7   α    -[(N-4-(4-chlorophenylsulphonylamino)phenyl)carbonyl)-Lvalylamido]-1,1-dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3cefem;    7α-[(N-4-(4-chlorophenylsulphonylaminocarbonyl)phenyl)carbonyl -L-valylamido0-1,1-dioxo-3-acetoxymethyl-4-tert-butoxy-    carbonyl-3-cefem; 7   α    -[(N-4-(4-chlorophenylsulphonylamino)phenyl)carbonyl)-L valylamido]-1,1-dioxo-3-acetoxymethyl-4-benzhydryloxy  carbonyl-3-cefem;

   7   α   -[(N-4-(4-chlorophenylsulphonylaminocarbonyl)phenyl)   carbonyl-L-valylamido3 -l , l-dioxo-3-acetoxymethyl-4-benzhy-    dryloxycarbonyl-3-cefem;
N-(2S)-4-thiahexane-1,6-dioic,   7α-[(N-benzyloxycarbonyl)-L-      valylamido1-l,l-dioxo-3-acetoxymethyl-3-cefem-4-carboxy-    amide;    1-methyl, 4[-[(7 α

   -[(N-benzyloxycarbonyl)-L-valylamido]-1,1-    dioxo-3-acetoxymethyl-3-cefem-4-carbonyl)] piperazine ;
The compounds (I) of the invention may be prepared by oxidization of cefem derivatives of formula:
EMI4.1     
 wherein R' represents a conventional protecting group of   α-aminoacid    amino groups and an arylcarbonyl radical and R1,
R and   P    have the above meaning, with the proviso that
 2 3 when   R    is a straight or branched C1-C4 alkoxy radical the compounds (III) have, preferably, the cef-3-em structure, and when   P    is the benzhydryloxy radical the compounds (III) have, preferably, the cef-2-em structure, to give a cef-3-em compound of formula:

  :
EMI4.2     
  wherein R', R1, R2 and R3 have the above-mentioned meaning, which may optionally be submitted to a selective removal of the conventional R' amino protecting group so giving the corresponding compounds wherein R is hydrogen, which compounds, if   desired,    are salified with a pharmaceutically acceptable acid or optionally transformed into other compounds (Ia) by restoring the R' substituent.



  In alternative, compounds (Ia) wherein R is a   C -C    straight
 1   14    or branched alkoxy radical, a 4-substituted piperazin-l-yl radical or Che above-defined   NR4R5      hay    be prepared from compounds (Ia) wherein R represents a benzhydryloxy or a tert-butoxy radical by methods known in the art which include the selective removal of the benzhydryloxy or tert-butoxy radical to give the corresponding 4-carboxylic acid
EMI5.1     
 which is then   processed    as following described.

 

  The oxidization of compounds (III) for obtaining compounds (Ia) is carried out using conventional methods, as for example, reacting (III), in the presence of an alkaline bicarbonate, with at least two mole equivalents, preferably from 2.2 to 2.5, of an organic peracid such as peracetic, perbenzoic,   m-chloroperbenzoic,    monoperphtalic acid, in a solvent as   dichloromethane,    ethyl acetate and/or their mixtures, at   a    temperature between -20 and   +2O0C,    but preferably at   0 C,    for a time varying from ten minutes or so  to few hours.   Ai    bicarbonate, the sodium bicarbonate is preferred.



  The reaction is usually completed after 3 hours.



  The removal of the conventional R' protecting groups is carried out by   using    conventional and well known methods in the art.



  In the case of a protection carried out with   2,2,2-trichloroethnxycarbonyl    such removal is carried out with
Zn and aqueous acetic acid according to T.A. Montzka et al.,
Tetrahedr. Lett., 1325, 1974.



  When the protecting group is represented by 9-fluorenylmethoxycarbonyl radical the selective removal is carried out by treatment with at least the equimolar amount, but preferably an excess amount of piperidine in a suitable aprotic solvent.



  The restoration of conventional protecting groups as well as arylcarbonyl groups R' in the compounds of   formula (I), in    which R is hydrogen, for obtaining other compounds (Ia) is carried out using conventional methods, well   nown    in the art, such as the reaction of amino group with the chloride of a suitable carbonic acid monoester or with the chloride of the selected arylcarboxylic acid or from the arylcarboxylic acid by reaction with a carbodiimide optionally in the presence of
N-hydroxy succinimide and /or hydroxybenzotriazole.



  The compounds   (Ill)    are obtained by a multistep reaction which comprises: - reacting a   7CA-      aminocefem    of formula:  
EMI7.1     
 wherein   R2    has the above-described meaning and R1''' is selected among benzhydryloxy and tert-butoxy radicals with a
L-valyl or a   homotalyl    derivative of formula:
EMI7.2     
 in which R' and   P    have the above described meaning and X
 3 represents an hydroxy group or a carbonyl activating group, to give a cefem derivative of formula (IIIa') and (IIIb') respectively:

  :
EMI7.3     
 wherein   R',R2,R3    and R''' have the above-defined meanings and
R'''' is benzhydryloxy radical; - optionally removing the protecting benzhydryloxy or tert-butoxy and following acylation of the so formed carboxylic group to provide the compounds (III ).



  Suitable activating groups of compound (V) are those normally used for the formation of the peptide bond; among these, in  particular, azide, imidazolide and N-hydroxysuccinimide. The condensation reaction between the derivative of formula (V) and   7o(-aminocefem    derivative of formula (IVa,b) is carried out using the conventional, well known methods of the peptide synthesis which also foresee a condensation between the carboxylic group of compounds of formula (V) where X is OH and the aminocefem in the presence of condensing agents as carbodiimides.



  The removal of the carboxy protecting benzhydryl and tert-butoxy groups in compounds   (IIIa')    and (IIIb') is preferably carried out by reaction with a molar excess of trifluoroacetic acid and the so obtained free acids are acylated using the well known methods such as the esterification by means of a diazoalkane or formation of an amidic bond,   such    as by activation of the carboxy group by means of   N-iiydroxysuccinimide,    N-hydroxybenzotriazole optionally in the presence of a   carbodiimide    followed by reaction with Che selected primary or secondary amine.



  Activation of the carboxy group may also be performed through achievement of   iiitermediates    such as imidazolides or mixed anhydrides following the process described for   7 > -methoxycefem    analogues by   P.E.Finke    in J.Med.Chem.35,3731,1992.



  The compounds of formula:
EMI8.1     

EMI8.2     
 wherein   P    has the above-described meaning, are obtained by
 2 epimerization of the carbon in 7 of   7ss-aminocefem    of formula:  
EMI9.1     
 wherein   R2    has the above-defined meanig and   R1 " '    represents the benzhydryloxy or the tert-butoxy radical, following the method of   JB.l)oherty    et al., J.Med.Chem., 33,2513,1990 in accordance with   (.V.Kim    et al.,J.of   Antibiotics,27,881,(1974).   



  In alternative tie epimerization of a 7ss-aminocefem of formula (VI) to give a compound of formula:
EMI9.2     
 wherein   R2    and R1   P      '    are as above-defined, may be performed throught diazotation of compound (VI) with an excess of sodium nitrite to give the corresponding diazoderivate which is reacted with an excess of azido ions (N ) and gives the
 3 corresponding   7α-azidocefem    derivative which is mildly reduced with triphenylphosphine to the compound (IVa) according to the following reaction schema:

  :
EMI9.3     
  
EMI10.1     

The compounds of formula (VI) are derivatives of the   7ss-aminocephalosporanic    acid and may be prepared from said acid according to methods well known in the art, e.g., from   7ss-aminocephalosporanic    acid and isobutene (see abovementioned   J.B.l)oherty    et al article) or by reaction of a   7ss-ammonium    or metansulphonate or p-toluensulphonate salt of said acid in a solvent such as acetonitrile in the presence of an excess of a diazoderivate.



  As part of a study of inhibitors of the human elastase of leucocytes (HLE), in the above-mentioned article the synthesis of thienyl-, formyl- and methoxy-carbonylamides of 7ss-amino-1,1-dioxo-3-acetoxymethyl-4-tert-butyloxycarbonyl3-cefem is also described near the one of the corresponding   7α    -amino epimer and, finally, the preparation of   7α    -(N-trifluoroacetamino)-1,1-dioxo-3-acetoxymethyl-4-tert-butox   ycarbonyl-3-cefem.   

 

  The   713-amides    resulted as being compounds substantially lacking   antielastase    activity while the   7α-amides    are defined as constituting a new class of inhibitors of the elastase even if less potent because of the rigidity and polarity of the amide bond which would mediate against the optimum of interaction of   these    molecules with the enzyme S1 site.



  To support this hypothesis, attention is drawn by Authors to how only small   oc-oriented    amides have a certain activity: in active compounds the IC50 vary from 2   oW    (trifluoacetamino) to  4 uM (formamide, methoxycarbonylamide) while more cumbersome amides (such as thienylamide) result practically inactive with
IC around 40 uM- so that they decided for a very scanty
 50 interest for such a class of compounds.



  The   7 -L-valylamids    (I) object of the present invention have unexpectedly proved to be effective inhibitors of the human elastase with   I    values inferior to 1 uM. Moreover, the same
 50
L-valylamides   (t)    show a greater affinity and, therefore, better selectivity, towards the human neutrophile elastase (HLE) rather than towards type III porcine pancreatic elastase (PPE) and cathepsin G of human neutrophiles   (HCG).   



  In fact 7   α    -[N-benzyloxycarbonyl)-L-valyl]-1,1-dioxo-3acetoxymethyl-4-tert-butoxycarbonyl-3-cefem, in a concentration ratio to the enzyme of 25:1, inhibits the HLE in a   90,0    proportion while it only inhibits in a 30% and 15-20% proportion the PPE and the HCG respectively. Incubation with serum does not substantially alter the inhibitory activity of compounds (I), object of the invention.



  It should   iurther    be observed that the epimer 7ss-(N-benzyloxycarbonyl)-L-valylamide shows contrasting activities since at concentrations which inhibit PPE in a   30%    proportion it appears to be completely inactive against HLE.



  This peculiar affinity and selectivity of the compounds of the invention on   HLF:    makes them to be particularly useful in the treatment of   pattiological    diseases such as chronic bronchitis, emphysema,   ARDS    (acute respiratory distress syndrom), cystic fibrosis and rheumatoid arthritis in which an efficient enzyme inhibitor is supposed to contribute to the arrest and prevention of   these    pathological diseases becoming acute once again.  



  Example 1 7   0'   -   C(N-benzyloxycarbony1 )-L-valylamiddJ -1, l-dioxo-3-acetoxy-      methyl-4-tert-butrxycarbonyl-3-cefem.   



  To a solution of 1.53 g (6.08 mmole) of
N-benzyloxycarbonyl-L-valine in   15    ml anhydrous tetrahydrofuran, maintained under nitrogen, stirred magnetically and cooled to -100C, is added drop-wise in the course of 5 minutes a solution of 0.84 ml (6.0 mmole) of triethylamina in I ml anhydrous tetrahydrofuran, and then,in a drop-wise manner in the course of 5 minutes, a solution of 0.58 ml (6.08 mmole) of ethyl chloroformate in 1 ml anhydrous tetrahydrofuran is added. After 30 minutes under stirring at the same temperature, the formed solid is removed by filtration under vacuum and the organic solution is evaporated under vacuum to dryness.

  The oily residue is dissolved in 20 ml of anhydrous   methylene    chloride and the resulting solution is added drop-wise in the course of 15 minutes to a solution of 1 g (3.04 mmole)   -amino-3-acetoxymethyl-4-tert-butyloxycarbonyl-3-cefem    in 20 ml of anhydrous methylene chloride cooled to   -lO0C    and stirred under nitrogen
The addition being completed, the mixture is brought to   OOC    and stirred under nitrogen for 2 hours, then washed on a separating funnel in the sequence: twice with 10% acetic acid and crushed ice, twice with an aqueous sodium bicarbonate saturated solution and with a sodium chloride saturated solution till neutrality, then filtered and evaporated at reduced pressure.

  Grams 2.42 of an oily residue is obtained which is submitted to chromatography on silica gel column at medium pressure eluting with a 2:1 hexane:methylethylketone  mixture and   collecting    15 ml fractions. From the central fractions, collected together and evaporated, 0.450 g of   7α    -[(N-benzyloxycarbonyl)
L-valylamido]-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem as an oil are obtained.



  1H NMR (CDCl1)   #    0.870-1.01 1m. 6H CH (CH) 7. 1.55 [s. 9H.



      3   
C(Ch3)3], 2.06 (s, 3H, CH3CO), 2.14 [m, 1H, CH(Ch3)2], 3.24 e 3.60 (2d, 2H, J   -    18.3 Hz,   SCH ),    4.06'(dd, 1H, J = 5.9 e 8.5
 2
Hz, NHCHCONH), 4.64-5.05 (m, 6H), 5.31 (d, 1H, J = 8.5 Hz,
OCONH), 7.06 (d, lH, J = 7.0 Hz,   NHcHcHS),    7.34 (brs, 5H,
C6H5). IR   (CCl4) #    1789 cm-Ú (C=O ss-lactame).



  Elementary analysis for   C H N 0 S   
 27 35 3 8
 C H N
Calculated % 57.74 6.28 7.48
Found   %    57.82 6.31 7.40
To a   solution    of 0.39 g (0.69 mmole) 7 oC    
F( N-benzyloxycarbonyl ) L-valylamidoj -3-acetoxymethyl-4-tert-bu    toxycarbonyl-3-cefem in 14 ml methylene chloride cooled to 0 C and magnetically stirred, 0.143 g (1.7 mmole) of solid sodium bicarbonate are added and then, in one portion, 0.420 g (1.70 mmole) of 70% m-chloroperoxybenzoic acid. The resulting mixture is stirred, at 0 C for three hours, washed on a separating funnel, in the sequence: three times with a 10% aqueous thiosulphate solution, twice   with    an aqueous saturated sodium bicarbonate solution and three times with water, filtered and evaporated under reduced pressure.

  Grams 0.33 g of a vitreous residue consisting of practically pure   7α      - E( N-benzyloxycarbonyl ) -L-valylamidoJ -1, l-dioxo-3-acetoxy    methyl-4-tert-butoxycarbonyl-3-cefem (TLC) are obtained.



  1H NMR (CDCl3)   #    0.85-0.91 [m, 6H CH(CH3)2], 1.48 [s, 9H,  
C(CH3)3], 2.02 (s, 3H, CH3CO), 3.60 e 3.96 (2d, 2H, J = 18.4
Hz SCH ), 4.01 (dd, 1H, J = 6.0 e 7.6 Hz, NHCHCONH), 4.60-5.12
 2 (m, 6H), 5.86 (d, 1H, J = 7.6 Hz, OCONH), 7.28 (brs, 5H,
C6H5), 7.75 (d, 1H, J = 6.6 Hz, NHCHCHS). IR   (CCl4) # 1811    cm-Ú (C=O ss-lactame).



  Elementary analysis for C27H35N3O10S
 C H N
Calculated %   54.63    5.94 7.08
Found % 53.89 6.07 6.62
Example 2-5
Operating in a   similar    manner as described above and using the corresponding protecting groups are obtained at first: -   7α-[(N-4-methoxybenzyloxycarbonyl)-L-valylamido]-3-    acetoxymethyl-4-tert-butoxycarbonyl-3-cefem; -   7α-[(N-2,6-dimethylbenzyloxycarbonyl)-L-valylamido]-3-    acetoxymethyl-4-tert-butoxycarbonyl-3-cefem ; -   7o(- E(N-tert-butoxycarbonyl )-L-valylamido3 -3-acetoxymethyl-      4-tert-butoxycarbnyl-3-cefem; 

  ;    -   7Cy- C(N-2,2,2-tri chloroethoxycarbony1 )-L-valylamidol -3-    acetoxymethyl-4-tert-butoxycarbonyl-3-cefem and then, after oxidization according to the above cited
Example, respectively: -   7d- C( N-4-methoxybenzyloxycarbonyl )-L-valylamidol      -l,l-dioxo-    3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem.



  -   7α    -[(N-2,6-dimethylbenzyloxycarbonyl)-L-valylamido]-1,1dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem.



  -   7α    -[(N-tert-butoxycarbonyl)-L-valylamido]-1,1-dioxo-3acetoxymethyl-4-tert-butoxycarbonyl-3-cefem.



     - 7α -[(N-2,2,2-trichloroethoxycarbonyl)-L-valylamido]-1,1dioxo-3-acetoxymet yl-4-tert-butoxycarbol3yl-3-cefem.     



  Example 6 7ss-[(N-benzyloxycarbonyl)-L-valylamido]-1,1-dioxo-3acetoxymethyl-4-tert-butoxycarbonyl-3-cefem.



  According to the procedure of Example 1 by reacting 3.07 g (12.22 mmole) N- benzyloxycarbonyl -L-valine with 2.0 g (6.1 mmole) 7ss-amino-3-acetoxymethyl-4-tert-butoxycarbonyl-3- cefem 2.06 g of practically pure 7ss-[(N-benzyloxycarbonyl) -L-valylamido]-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem melting at 92-940C are obtained.



  1H NMR (CDCl3)   #    0.90-1.02 [m, 6H CH(Ch3)2], 1.53 [s, 9H,
C(CH3)3], 2.07 (s, 3H, CH3CO), 2.10 [m, 1H, CH(CH3)2], 3.23 e 3.58 (2d, 2H, J = 18.8 Hz, SCH ), 4.07 (dd, 1H, J = 6.1 e 8.8
 2
Hz, NHCHCONH), 4.69-5.11 (m, 6H), 5.16 (d, 1H, J = 8.8 Hz,
OCONH), 5.78 (dd, 1H, J = 4.8 e 8.8 Hz, CHCHS), 6.75 (d, 1H, J = 8.8 Hz, NHCHCHS), 7.34 (brs, 5H, C6H5). IR (CCl4)   #    1803 cm-Ú (C=O ss-lactame).



  Elementary analysis for C27H35N308S
 C H N
Calculated % 57.74 6.28 7.48
Found % 57.47 6.22 7.50
The oxidation of 0.90 g (1.6 mmole)   7ss-E(N-      benzyloxycarbonyl ) -L-valylamido3 -3-acetoxymethyl-4-tert-    butoxycarbonyl-3-cefem as previously described, affords 0.72 g of an oily residue consisting of practically pure 7ss-[(N-benzyloxycarbonyl)-L-valylamido]-1,1-dioxo-3-acetoxy-me thyl-4-tert-butoxycarbonyl-3-cefem. Yield 76%.



   1H NMR (CDCl3)   #    0.88-1.02 [m, 6H CH(CH3)2], 1.53 [s, 9H,
C(CH3)3], 2.08 (s, 3H, CH3CO), 2.15 [m, 1H, CH(CH3)2], 3.62 e 3.97 (2d, 2H, J = 18.6 Hz, SCH ), 4.09 (dd, 1H, J = 5.6 e 8.8
 2
Hz, NHCHCONH),   4.()8    (d, 1H, J = 8.8 Hz, OCONH), 4.83-5.31 (m,  5H), 6.06 (dd, 1H, J = 5.0 e 10.3 Hz, CHCHS), 7.17 (d, 1H, J = 10.3 Hz, NHCHCHS), 7.33 (brs, 5H, C6H5). IR (CCl4)   #    1811 cm-Ú (C=O   13-lactame).   



  Elementary analysis for C27H35N3O10S
 C H N
Calculated   %    54.63 5.94 7.08
Found % 54.29 6.03 6.67
Examples 7
To a stirred and cold water bath cooled solution of 28.5 g of p-toluensulfonic acid monohydrate in 0.5 1 of dioxane is added to   Al    g of 7-ACA and then maintaining the internal temperature around 12-140C, is added a cooled 0.8   N    diphenyldiazomethane solution in 0.6 1 of dioxane. The reaction is stirred for 4 hours at room temperature and evaporated in vacuo at a temperature lower than 500C. The residue is triturated with n-hexane to give a solid material that is collected by filtration and then dissolved with 0.4 1 of dichloromethane.



  The organic layer is washed twice with 2 x 50 ml saturated aqueous sodium bicarbonate solution and with water, dried over sodium sulfate, and evaporated to a small volume. The solid material, that precipitates by dilution with 60-800C petroleum ether, is filtered and dried on vacuum to give 16.5 g of   7ss-amino-3-acetoxymethyl-4-      benzhydryloxycarbonyl-3-    cefem', melting point 119-1210C,   
Fi = 2.9 (CH C1 ).



   D 22   
According to this procedure, isonipecotic acid benzhydrylester and L-carboxymethylcysteine di-benzhydryl ester are prepared from their free acids.



  Example 8
To a solution of 2 g of   7ss-amino-3-acetoxymethyl-4-     benzhydryloxycarbonyl-3-cefem in dichloromethane are added 0.7 g of p-nitro-benzaldehyde and 3 g of anhydrous Na2SO4. After 4 hours, the   mixture    is filtered and evaporated to dryness on vacuum, affording 2.2 g of 7ss-(p-nitro- benzilyden-amino)-3-acetoxymethyl-4-benzhydryloxycarbonyl-3cefem. A green solution of the latter compound in 70 ml of DMF is cooled to 15 C and treated   witi    a cooled   30,0    (v/v) triethylamine solution in 10 ml DMF to give a blue solution that is furtherly stirred for 30 minutes, and then diluted with cold aqueous 0,015 N HCl solution.

  The aqueous phase is extracted with 2 x 250 ml ethyl ether, the combined organic extracts are washed with aqueous 5% dihydrogen sodium phosphate, brine, dried over sodium sulfate, and evaporated to dryness. The crude mixture is submitted to preparative HPLC chromatography (porasil silica 15-20 micron, n-hexane/AcOEt 75:25) to obtain respectively:

   a) 7ss-(p-nitro-benzyliden-amino)-3-acetoxymethyl-4-benz hydryloxycarbonyl-2-cefem T = 12.77;
 R b) 7   i      -( p-rlitro-benzyliden-amino ) -3-acetoxymethyl-4-benz-    hydryloxycarbonyl-3-cefem   TP    = 17.89; c)   713-(p-iiitro-benzyliden-amino)-3-acetoxymethyl-4-benz-    hydryl- oxycarbonyl-3-cefem T = 23.9;    P    d) 7   CA    -(p-nitro-benzyliden-amino)-3-acetoxymethyl-4-benz- hydryloxycarbonyl-2-cefem   TP    = 29.2; as pure substances.



  Example 9
Procedure A : Under vigorous stirring, 0.51 ml aqueous 2N H2SO4 is added dropwise to a double phase mixture of aqueous   NaNO2    0.047 g in   H 0    5 ml and 0.3 g of   713-amino-   
 2 3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem (5 ml) of dichloromethane solution. After 1 hour, the aqueous phase is  separated, extracted with 2 x 2 ml of dichloromethane; the combined organic extracts are washed with brine and dried over anhydrous sodium sulfate. The crude and dried solution of 7-diazo-3-acetoxynlethyl- 4-benzhydryloxycarbonyl-3-cefem (TLC on SiO2, eluent CHCl3-Et2O Rf= 0.62) is cooled at 0 C and treated with 5% HN solution in 12ml of chloroform.

  After 2
 3 hours at   OOC,      the    solution is maintained for a night at room temperature, washed with aqueous saturated sodium bicarbonate, brine and dried over sodium sulfate. After removal of solvents under vacuum, the residue is chromatographed to afford   7t;(-      azido-3-acetoxyniethyl-4-benzhydryloxycarbonyl-3-cefem    (TLC on
SiO2, eluent CHCl3-Et2O Rf= 0.72), (HPLC Spherisorb SW5, eluent CH2Cl2/iPrOH retention time 5'.66) I.R. 2115.6 cm-l (azide), NMR (CDCl3, TMS ppm) H-7 4.65 (J=1.9Hz), H-6 4.52 (J=1.9Hz).

  By treatment of the azide with equimolar amounts of   triphenylphoaphine    in wet THF at room temperature   7  -      amino-3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem    (HPLC
Spherisorb SW5, eluent   CH Cl /iPrOH    retention time 7'.83) is    22    prepared.

 

  Procedure B: 1 g 7   α    -(p-nitro-benzyliden-amino)-3acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem and 0.9 g of
Girard's T reagent are dissolved at room temperature in 25 ml of MeOH. The solution is stirred for 2 hours then is poured into 600 ml of 1:1 AcOEt-water, shaken, and separated. The organic phase is washed with water, dried over sodium sulfate and evaporated to dryness affording 0.69 g of   7α      -amino-3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem.   



  The geometric isomer   2-cephem-7α-(p=nitro-benzyliden-amino)    (1.35 g) when used in this procedure, yields 0.82 g of   7 -    amino-3-acetoxymethyl-4-benzhydryloxycarbonyl-2-cefem TR =  7.9 (Spherisorb SW5 mm 4.6x250 CH C1 -iPrOH 95.5 flow - 0.5    22    ml/min) that   i;    reacted with 0.52 g of Z-Val-OH in dry dichloromethane, at   OOC,    in the presence of WSCDI, 0.5 g of
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and some drops of triethylamine.

  The reaction mixture is stirred for 3 hours at room temperature, diluted with water to give, after tie usual work-up and flash chromatography    (hexane/AcOEt 3:1), 0.92 g 7 i -[(N-benzyloxycarbonyl)-Lvalylamide -3-acetoxymethyl-4-benzhydryloxycarbonyl-2-cefem Rf    = 0.16 (hexane/AcOEt 2:1).



  When in   accor(lance    to the above   procedure,    one of the followings:
Z-Ile-OH, Fmoc-Val, Fmoc-Ile-OH, p-Cl-Phe-SO2NH-Phe-CO-Val
OH, p Cl-Phe-SO2NH-CO-Phe-CO-Val-OH, p-Cl-Phe-SO2NH-Phe-CO
Ile-OH, p-Cl-Phe-SO2NH-CO-Phe-CO-Ile-OH is used to acylate a 7   -amino-cephem-b'tzhydryl    ester, the following 3-cefem- and 2-cefem-3-acetoxymethyl-4-benzhydryloxycarbonyl ; -   7α-[(N-benzyloxycarbonyl)-L-iso-leucylamide],    -   7α-[(N-Fmoc)-L-iso-leucylamide],    -   7α-[(N-Fmoc)-L-valylamide],    -   7α-[(N-p-Cl-Phe-SO2NH-Phe)carbonyl-L-iso-leucylamide],    -   7α-[(N-p-Cl-Phe-SO2NH-CO-Phe)carbonyl-L-iso-leucylamide],    -   7α

  ;-[(N-p-Cl-Phe-SO2NH-Phe)carbonyl-L-valylamide],    - 7   o(    - [(N-p-Cl-Phe-SO2NH-CO-Phe)carbonyl-L-valylamide] are prepared.



  Example 10
A 30 ml solution of dichloromethane of 1.04 g of   benizyloxycarbonyl )-L-valylamide3 -3-acetoxynethyl-4-benzhy-    dryloxycarbonyl-2-cefem is oxidized in the presence of 0.6 g
NaHCO3 with m-CPBA 55%, 1.36 g of m-chloroperbenzoic acid, by  
 stirring at room temperature for 16 hours. After the usual
 work up (treatment with aqueous NaHCO3 solution containing
 sodium sulfite Lo destroy any excess of m-CPBA), pyridine (few
 drops) is added to the final organic layer, when is dried on
 sodium sulfate. The solution is stirred up to obtain complete
 isomerization of any residual   2-cefem-compound    (about 1 hour),
 then is   filtered    and evaporated to dryness.

  The residue is   
 purified on SiO LC (eluent hexane/AcOEt 2:1) to give 0.72 g
 2   
 of 7   y      -f(N-benzyl-       oxycarbonyl )-L-valylamide3 -l , l-dioxo-3-acetoxymethyl-4-ben-   
 zhydryloxycarbonyl-3-cefem,   Rf    = 0.44 (hexane/AcOEt 1:1).



   Using in the same procedure a cefem-4- benzhydryloxycarbonyl-7   α-amide    of example 9, the following
 1,1-dioxo-3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem :
 -   7α-[(N-Fmoc-L-iso-leucylamide],       - 7α-[(N-Fmoc-L-valylamide],   
 -   7α-[(N-p-Cl-Phe-SO2NH-Phe)carbonyl-L-iso-leucylamide],       - 7α-[(N-p-Cl-Phe-SO2NH-CO-Phe)carbonyl-L-iso-leucylamide],   
 -   7α-[(N-benzyloxycarbonyl)-L-iso-leucylamide],       - 7α-[(N-p-Cl-Phe-SO2NH-Phe)carbonyl-L-valylamide],   
 - 7 or -[(N-p-Cl-Phe-SO2NH-CO-Phe)carbonyl-L-valylamide] are
 prepared.



   Example 11
 To 0.25 g of   7α-[(N-benzyloxycarbonyl)-L-valylamide]-3-   
 acetoxymethyl-4-tert-butoxycarbonyl-3-cefem   [α]D    + 27.8 (c=1%
 MeOH) is added a cooled mixture of 0.04 ml of anisole and 0.65
 ml of TFA. After 2 hours, the ice-water bath is removed; the
 reaction   mixture    is maintained for 3 hours at room
 temperature,   Lhen    it is diluted with 40 ml of
 water:ice:dichloromethane 1:1:2, and stirred. The organic  layer, after   the    usual work-up, is extracted with 3x8 ml of aqueous saturaLed   NaHCO    solution. The combined alkaline
 3 extracts are acidified with 2N   HOl    and extracted with AcOEt.



  The combined AcOEt extracts are dried over sodium sulfate and evaporated to dryness and give 0.12 g of   7   - (N-      benzyloxycarbonyl) -L-valylamidel -3-acetoxymethyl-3-cefem-4-    carboxylic acid (TLC SiO2AcOEt/AcOH 99:1 Rf=0.27).



  To an ice bath cooled solution of 0.1 g of the above acid in 2 ml of   dioxane    are sequentially added 0.03 g of   N-Oil-succinimide    and 0.06 g of DDC. The reaction is stirred at room temperature for 30 minutes and then recooled in an ice bath. 0.6 ml of triethylamine is added; after stirring for 30 minutes at   OOC    0.22 g of L-glutamic acid di-tert-butyl ester dibenzenesulfimide salt is added all at once. The mixture is maintained for 3 hours at room temperature, then, after dilution with   2U    ml of diethylether, is filtered. The filtrate is poured in ice-water containing 2N HCl; the aqueous phase is separated, washed with diethylether. The combined organic extracts are washed with water and aqueous sodium hydrogen carbonate.

  After the usual work-up, the crude residue is purified by preparative LC (SiO2, n-hexane/AcOEt 1:1) to give 0.1 g of 7   K      - E( N-benzyloxycarbonyl ) -L-valylamide 3-acetoxymethyl-4    carboxamide-3-cephem N-L-glutamyl-di-tert-butyl ester.



  The following m-CPBA oxidation affords: 7   α-[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3-acetoxy-    methyl-4-carboxamide-3-cefem   N-L-glutamyl-di-tert-butyl    ester.



  When, in accordance to the above procedure, a tert-butylester as: 7   o(      -(N-benzyloxycarbonyl )-L-valylamideJ-3-acetoxymethyl-4-     tert-butoxycarbonyl-3-cefem, 7   α    -[(N-benzyloxycarbonyl)-L-iso-leucylamide]-3-acetoxymethyl-4-tert-butoxycarbonyl-3-oefem, 7       -   E(N-(   4-(4-Cl-phenylsulfonylamino)phenyl)carbonyl)-L-   valylamideC-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem,    7   or      -E(N-(4-(4-(;1-phenylsulfonylaminocarbonyl)phenyl)carbonyl)      -L-valylamide -3-acetoxymethyl-4-tert-butoxycarbonyl-3-    cefem, is cleaved, one of the following acids:

   7   o(    -   E( N-benzyloxycarbonyl ) -L-iso-leucylamide -3-acetoxy-      methyl-3-cefem-'i-carboxylic    acid 7   α    -[(N-(4-(4-Cl-phenylsulfonylamino)phenyl)carbonyl)-Lvalylamide]-3-acetoxymethyl-3-cefem-4-carboxylic acid, 7   α    -[(N-(4-(4-Cl-phenylsulfonylaminocarbonyl)phenyl)carbonyl)   -L-valylamidel -3-acetoxymethyl-3-cefem-4-carboxylic    acid, is obtained,   thal;,    Lhen, likely as: 7   α    -[(N-benzyloxycarbonyl)-L-valylamide]-3-acetoxymethyl3-cefem-4-carboxylic acid by reaction with an'   amine    (or ammonium salt) selected in the group consisting of: 

   isonipecotic acid benzhydrylester and ethylester,
L-2-aminoadipic acid di-tert-butyl ester dibenzonosulfimide salt,
L-aspartic acid di-tert-butyl ester dibenzenesulfimide salt,
L-glutamic acid di-tert-butyl ester dibenzenesulfimide salt gives rise to   tile    corresponding: isonipecotoyl benzhydrylesters:
L-2-aminoadipoyl di-tert-butyl ester,
L-aspartyl di-tert-butyl ester,
L-glutamyl di-tert-butyl ester 4-carboxyamides, that are coverted into their related 1,1-dioxo compounds by m-CPBA oxidation:   -[(N-benzyloxycarbonyl)-L-valylamide] -3-acetoxymethyl-4-carboxyamido-3-cefem N-isonipecotic acid benzhydryl ester   [α]D=-18.9    (c= 9 ;

  MeOh) Rf = 0.13 (hexane-AcOEt 1:1]    -7α-[(N-benzyloxycarbonyl)-L-valylamide]-3-acetoxy-    methyl-4-carboxyamide-3-cefem N-isonipecotic acid ethyl ester   Rf    = 0.1 (hexane-AcOEt 1:1)   [ JD    = -28 (c= 10; MeOH).



   D
By m-CPBA oxidation, the following tert-butyl esters: 7        -[(N-benzyloxycarbonyl)-L-valylamide]-1, l-dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem, 7   α    -[(N-benzyloxycarbonyl)-L-iso-leucylamide]-1,1-dioxo-3acetoxymethyl-4-tert-butoxycarbonyl-3-cefem,    7α-[(N-(4-(4-Cl-phenylsulfonylamino)phenyl)carbonyl)-L-valyl-    amide]-1,1-dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem,    7 α -[(N-4-(4-Cl-phenylsulfonylaminocarbonyl)phenyl)carbonyl) -L-valylamidel -1, l-dioxo-3-acetoxymethyl-4-tert-butoxy-    carbonyl-3-cefem are also prepared.



  Example 12
To a stirred (and cooled at 0 C)   solui;ion    of 13.4 g of   7α-      amino-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem    and 14.8 g of Fmoc-L-valine in 35 ml of dichloromethane is added all at once 7.9 g of WSCDI. The mixture is allowed for 8 hours at room temperature. After the usual work-up (wahings with water, 5% aqueous NaHCO3, 2N HCl, brine, drying over sodium sulfate, removal of solvents on vacuum) 25 g of the crude residue is purified by SiO2 chromatography to give 7   α       E(N-Fmoc)-L-valylamidoJ -3-acetoxymethyl-4-tert-butoxycarbon-    yl-3-cefem (22 g ; TLC SiO2 hexane-MEC 2/1 Rf=0.21).



  To a stirred solution of 3.8 g of this compound in 120 ml of dry dichloromethane, cooled at 0 C, are added 1.5 g of NaHCO3  and all at once 4.5 g of 70% m-CPBA. The stirring is maintained for 2 hours at   OOC,    then at room temperature for 4.5 hours before dilution with AcOEt-water. After the usual work-up, the crude residue is purified by elution on florisil column (hexane,   methylethylketone    (MEK) 2:1) to obtain 2.19 g of pure 7   o(    -[(N-Fmoc)-L-valyamide]-1,1-dioxo- 3-acetoxymethyl-4-Lert-butoxycarbonyl-3-cefem (TLC   SiO2,    hexane : MEC 2:1 Rf=0.23).

  In similar way : -   7α-[(N-Fmoc)-L-iso-leucylamide]-1,1-dioxo-3-acetoxymethyl    4-tert-butoxycarbonyl-3-cefem, and -   7α-[(N-benzyloxycaronyl)-L-iso-leucylamide]-1,1-dioxo-3-    acetoxymethyl- 4-tert-butoxycarbonyl-3-cefem   [α]    = + 8.8 (c=1% MeOH), - 7   α      - C( N-Fmoc) -L-valylamido) -1, l-dioxo-3-acetoxymethyl-4-      benzhydryloxycarbonyl-3-cefem,    are also prepared.



  Example 13
To a solution of 1.5 g of   7c?-f(N-Fmoc)-L-valylamide -      1, 1-di oxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem    in 10 ml of dichloromethane is added 0.8 ml of piperidine. After 30 minutes at 20-230C, the reaction mixture is diluted with 25 ml of 10% aqueous citric acid solution and stirred for a night at   5-lO0C.    The aqueous phase is separated, cautiously basified up to pH 9 by addition of 4N NaOH (by external cooling at   5-lO0C)    and then extracted with 3xlOml of AcOEt. The combined organic extracts are dried on   Na2SO4,    evaporated to dryness to give 0.8 g of 7   0?    valylamide)-1,1-dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl]-3cefem.



  To a stirred and cooled (about 0 C) solution of 0.5 g of 4-(4-chlorophenylsulphonylaminocarbonyl)benzoic acid in (3 ml  of DMF are added 0.4 g of   l-OH-benzotriazole    hydrate. After 5 minutes, to it   is    added a cooled solution of 0.5 g of 7 -(L-valylamido)-1,1-dioxo-acetoxymethyl-4-tertbutoxycarbonyl-3-cefem in 2 ml of DMF containing 0.22 ml of triethylammine. At the end, to the stirred mixture 0.35 g of
DCC is added and   Ihe    stirring is continuted for 3 hours at 0 C and 3 hours at room temperature. The mixture is diluted with diethylether and filtered, partitioned with cooled water.

  The aqueous phase is washed with AcOEt; the combined organic extracts after the usual work-up (washings with aqeous   10%    citric acid, saturated NaHC03 and brine) and drying over sodium sulfate are evaporated to dryness to give after LC purification 0.62 g 7   i      -5(N-(4-(4-chlorophenyl-    sulfonylaminocarbonyl)phenyl)carbonyl)-L-valylamide]-1,1dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem.



  According to the above procedure, the   1, l-dioxo-3-acetoxymethyl-3-cefem-4-benzhydryloxycarbonyl    and   4-tert-butoxycarbonyl-7α-[(N-Fmoc)-L-valylamides]    and   - (N-Fmoc)-L-iso-leucylamidesg    produce by reaction with piperidine the corresponding 7(x -(L-valylamides) and   7α    -(L-iso-leucylamides), that if desired are acylated by reaction with a preferred convenient arylcarboxylic acid of the invention.



  Example 14
To 1 g of   7α-[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-    3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem is added a cold mixture of 2.4 ml of trifluoroacetic acid (TFA) and 0.2 ml of anisole. The mixture is maintained at 0 C for 2 hours, at room temperature for 3 hours, and then is partioned between 100 ml of 1:1 dichloromethane and ice-water. The aqueous layer is  extracted with   2xlO    ml of   dichlorometllane,    the combined organic layers are washed with water and then repeatedly extracted with aqueous saturated sodium hydrogen carbonate solution. The basic extracts are combined, acidified to pH 2 and extracted with 3x6 ml of AcOEt.

  The combined AcOEt extracts are dried over sodium sulfate and evaporated to dryness to give 0.73 g of 7   d      ll(N-    benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3-acetoxymethyl-3-cefem-4-carboxy acid   EJ    + 16.7 (MeOH)   Rf    = 0.06
 D (AcOEt-AcOH 98:2).



  A solution of the compound and 0.24 g of l-hydroxybenzotriazole hydrate in 3 ml of DMF is cooled in an ice bath, and 0.6 g DDC is added portion wise over 5 minutes.



  The reaction is stirred at room temperaLure for 2 hours and then recooled at   -5      -gOC.    0.42 g of L-Proline tert-butyl ester is added all at once and the reaction is stirred at room temperature for 2.5 hours.

 

  The mixture is diluted with diethylether and filtered and the filtrate is poured in ice-water containing 2N HCl. The aqueous phase is separated, washed with ether; the combined organic extracts are washed with water,   sodium    hydrogen carbonate solution and brine, dried over sodium sulfate and evaporated.



  The product is purified by preparative LC (SiO2, n-hexane/AcOEt   55:45)    to give 0.8 g of   7α-[(N-benzyloxy-    carbonyl ) L-valylamideJ -l , l-dioxo-3-acetoxymethyl-4carboxamide-3-cefem N-L-prolyl-tert-butyl ester.



  To an ice bath cooled sample of this 0.8 g compound, 7.5 ml of
TFA and 0.5 ml   of'    anisole are added. The reaction is stirred for 1 hour and then evaporated in vacuo at 30 C ; the residue is taken up twice in dichloromethane and evaporated to remove  the TFA excess. By flash chromatography (eluent hexane-AcOEt 30:70, then 0.5%   AcOIl-AcOEt)    0.52 g of   704      -E(N-    benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3    acetoxymethyl-4-[(2(S)-carboxypyrrolidino)carbonyl] -3-cefem Rf = 0.27 (AcOEt-AcOH 1%) , are obtained.   



  Using in the above procedure, L-carboxymethylcysteine di-tert-butyl ester and carboxymethylcysteine di-benzhydrylester, the following: - 7   o(    -[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3- acetoxymethyl-4-carboxamide-3-cefem N-L-carboxymethylcysteinyl di-tert-butyl ester, - 7   α    -[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3acetoxymethyl-4-carboxamide-3-cefem N-L-carboxymethylcysteinyl di-benzhydryl ester, that, after cleavage of the protective ester group, each of them produces: 7   o4    -   C( N-benzyloxycarbonyl )-L-valylamide¯S -1 ,l-dioxo-3-    acetoxymethyl-4-carboxamide-3-cefem N-L-carboxymethylcysteinyl.



  Example 15
The l,l-dioxo-3-acetoxymethyl-3-cephem-4-carboxyamides Nisonipecotinoyl di-tert-butyl esters, N-L-aspartyl di-tert-butyl esters, and N-L-glutamyl di-tert-butyl esters of example 11 are converted in the related free acids including: 7   α    -[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3acetoxymethyl-4-carboxamide-3-cefem N-isonipecotic acid   Pf      =    0.7 (AcOEt-AcOH 98:2),    7 α -[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3-    acetoxymethyl-4-carboxamide-3-cefem N-aspartic acid, 7   α    -[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3  acetoxymethyl-4-carboxamide-3-cefem N-glutamic acid by cleavage of the tert-butyl and benzhydryl protective groups when treated in accordance to the procedure of example 14.



  Example 16
In accordance with the procedure of example 14 the protective tert-butyl ester and benzhydrylester groups of the following esters: -   7α -[(N-benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3-    acetoxymethyl-4-tert-butoxycarbonyl-3-cefem,    - 7 α

   - -|(N-benzyloxycarbonyl)-L-iso-leucylamide]-l,l-dioXo-3-    acetoxymethyl-4-tert-butoxycarbonyl-3-cefem, -   7α-[(N-(4-(4-phenylsulfenylamino)phenyl)carbonyl)-L-valyl-    amide]-1,1-dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3cefem, -   7α-[(N-(4-(4-phenylsulfenylaminocarbonyl)phenyl)carbonyl)-   
L-valylamide]-1,1-dioxo-3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem, - 7   d    -[(N-benzyloxycarbonyl)-L-iso-leucylamide]-1,1-dioxo-3- acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem, - 7   R      -E(N-benzyloxycarbonyl)-L-iso-leucylamideJ-ll-dioXo-3-    acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem, -   7α

  ;-[(N-(p-Cl-Phe-SO2NH-Phe)carbonyl-L-iso-leucylamide]-    1,1-dioxo-3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem, -   7α-[(N-(p-Cl-Phe-SO2NH-CO-Phe)carbonyl-L-iso-leucylamide]-    1,1-dioxo-3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem, - 7   α      -r(l\J-(benzyloxycarbonyl)-L-valylamide0-l,l-dioXo-3-    acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem, -   7α-[(N-(p-Cl-Phe-SO2NH-Phe)carbonyl-L-valylamide]-1,1-    dioxo-3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem, -   7α-[(N-(p-Cl-Phe-SO2NH-CO-Phe)carbonyl-L-valylamide-1,1-        dioxo-3-ace toxyme I-hy l-4-benzhydryloxycarbonyl-3-cefeml    are removed to produce the 4-carboxy acids:

  : - 7   d    -[(N-(benzyloxycarbonyl)-L-valylamide]-1,1-dioxo-3- acetoxymethyl-3-cefem-4-carboxy acid, - 7   α      -E(N-(4-(4-Cl-phenylsulfonylamino)-phenyl)carbonyl)-L-      valylamide3 -1, l-dioxo-3-acetoxymethyl-3-cefem-4-carboxy    acid, - 7   α    -[(N-(4-(4-Cl-phenylsulfonylaminocarbonyl)-phenyl)   carbonyl ) -L-valylamide3 -1, l-dioxo-3-acetoxymethyl-3-cefem-    4-carboxy acid, -   7 i      -E(N-benzyloxycarbonyl)-L-iso-leucylamide3-1,l-dioxo-3-    acetoxymethyl-3-cefem-4-carboxy acid   [α]D    = +13.07 (c=13 ;
MeOH)   Rf    = 0.12 (AcOEt-AcOH 98:

  :2), -   7α-[(N-(p-Cl-Phe)carbonyl-L-iso-leucylamide]-1,1-    dioxo-3-acetoxymethyl-3-cefem-4-carboxy acid, -   7α-[(N-(p-Cl-Phe-SO2NH-CO-Phe)carbonyl-L-iso-leucylamide]-    1,1- dioxo-3-acetoxymethyl-3-cefem-4-carboxy acid,
Example 17
In according to   procedure    of example 11, to an ice bath cooled solution of 0.74 g of   7α-[(N-4-(4-Cl-phenylsulfonyl-    aminocarbonyl)-phenyl)carbonyl)-L-valylamide]-1,1-dioxo-3   acetoxymethyl-3-cefem-4-carboxy    acid in 4 ml of dioxane is sequentially added 0.12 g of N-OH-succinimide and 0.38 g of
DCC.

  The mixture is stirred at room temperature for 30 minutes and then recooled in an ice bath. 0.2 ml of triethylamine is added and after further stirring for 30 minutes 0.37 g of   4-(2-diethylaminoethyl)piperazine    is added all at once. After 3.5 hours, the reaction mixture is diluted with diethylether, filtered and evaporated to dryness. The residue is dissolved in cold 0.5 N sulfuric acid, the aqueous phase is repeatedly extracted with AcOEt and the combined organic extract are  discarded.



  The aqueous phase is then basified to pH 9 and extracted with 3x9 ml of AcOEt; the usual work-up on the combined organic phase, and LC   pulification      (AcOEt-MeOH-Nii )    yields 0.82 g of
 3   
N-(2-diethylaminoethyl) ,N'-E7 cC -E(N-(4-(4-Cl- phenylsulfonylaminocarbonyl )-phenyl)carbonyl) -L-valylamidk3 -    1,1-dioxo-3-acetoxymethyl-3-cefem-4-carbonyl]-piperazine.



  Example 18
By reaction, in the procedure of the example 17, of a   1, l-dioxo-3-acetoxymethyl-3-cefem-4-carboxy    acid, prepared in accordance of the procedure of the example 16, with a piperazine selected from the group consisting of 4-methylpiperazine, 4-ethyl-piperazine, 4-(2-dimethylamino)piperazine and 4-(2-diethylamino)-piperazine, the related piperazine 1,1-dioxo-3-acetoxymethyl-3-cefem-4-carboxy- amides are prepared, including:   N-methyl-N'- E7      cd    - [(N-benzyloxycarbonyl)-L-iso-leucylamide]-   1, 1-di oxo-3-acetoxymethyl-3-cefem-4-carbonyl3 -piperazine    Rf = 0.52   (CHC1 -MeOIl-cyclohexane-NH OH    68:15:15: 

  :2)
 3 4
Example 19 2.3 equiv. of t-BuOLi (by treatment of equimolar amount of tert-BuOH in dry THF with n-BuLi) are reacted with 1 equiv. of teraphtoyl chloride; the teraphtoyl di-tert-butylester is converted in the monoester by treatment with 1.05 equiv. of
KOH in t-BuOH. 'J'he salt is neutralized; a dichloromethane solution of the resulting mono tert-butylester is reacted with p-C1-phenylsolfonamide in the presence of DMAP and equimolar amounts of WSDCI to obtain tert-butyl 4-(p-Cl-phenyl-sulfonyl-amino-carbonyl)benzoate.

  The following cleavage of the tert-butyl ester group (TFA 0 C) gives  4-(p-Cl-phenylsulfonyl-amino-carbonyl)benzoic acid that is coupled with   l,-valine    and L-isoleucine methyl and/or tert-butyl ester to obtain: - 4-(p-Cl-phenyl-sulfonyl-amino-carbonyl)benzoyl-L-valine   [α]D=    +10 (c= 10 ; MeOH) Rf =0.28 (CHCl3-MeOH 6:4) m.p. >  300 C and -   4-(p-Cl-phenyl-sulfonyl-amino-carbonyl)benzoyl-L-iso-    leucine as methyl ester and/or tert-butylester the ester functions of which are cleaved with N NaOH in MeOH and TFA respectively to yield the free acids.



  The coupling of p-Cl-phenylsulfonyl chloride with p-amino benzoic acid methyl and/or tert-butylester affords the methyl or tert-butyl: 4-(p-Cl-phenylsulfonylamino)benzoates. Cleavage of the ester functions and following coupling with L-valine and L-isoleucine methyl and/or tert-butyl ester allows to obtain: - 4-(p-Cl-phenyl-sulfonyl-amino)benzoyl-L-valine   [α]D=    (c= 10; MeOH)   Rf    = 0.24 (CHCl3-MeOH-AcOH 94:5:1) and -   4-(p-Cl-phenyl-sulfonyl-amino) benzoyl-L-iso-leucine    as methyl ester and/or tert-butylester, the ester functions of which are cleaved with N NaOH in MeOH and TFA respectively to produce the free acid.



  The compounds of the present invention can be appropriately formulated in pharmaceutical compositions obtained according to known technics, for example those described in "Remington's
Pharmaceutical Sciences Handbook", Hack Publishing Co, U.S.A.

 

  In particular the compositions suitable for oral administration and by aerosol are preferred, such as solid and liquid compositions, as capsules, pills, tablets, suspensions, emulsions, syrups, drops or vials.  



  A suitable dose of the compounds of formula (I) varies according to the nature and intensity of the affection, of the way of   administralion,    the age, weight and state of gravity of the patient.



  In particular   tle    quantity of the active principle orally administered, as total dose, varies from 5 to 1500 mg a day subdivided into repeated administrations. The dose of active principle administered by aerosol, as total dose, varies from 0.05 to 50 mg a day. 

Claims

1. L-valyl- and homovalyl-amides of 7α-amino-1,1-dioxo-3-cefem of formula EMI33.1 wherein: R is hydrogen, a conventional protecting group of α-aminoacid amino groups and an aryl radical; P is a straight or branched alkoxy radical, containing 1-4 carbon atoms, benzhydryloxy radical;

4-substituted piperazin-l-yl or the group NR R where P is hydrogen and P represents the 4b 4 5 rest of a L-α-aminoacid or R4 and R5 together with the nitrogen to which they are attached form a 5 or 6 atom ring selected from 2-carboxypyrrolidyl and 4-carboxypiperidyl, the carboxy groups optionally being all contemporarily esterified preferably as tert-butyl and/or benzhydryl esters or in the form of pharmaceutically acceptable salts of an inorganic cation and/or ammonium; P is a straight or branched alkyl radical, 2 containing 1-4 carbon atoms; R3 is a straight alkyl radical containing 1-2 carbon atoms;

and the salts with pharmaceutically acceptable acids of the compounds (I) in which R is hydrogen.

2. L-valylamides of 7d-amino-l, l-dioxo-3-cefem according to claim 1, characterized by the fact that the conventional d-arninoacid amino protecting group is selected from the group consisting of benzyloxycarbonyl optionally substituted at the benzene ring with 4-methoxy or 2,6-dimethyl radical; methoxy-, ethoxy- and allyloxy-carbonyl; 2,2,2-trichloroethoxycarbonyl and 9-fluorenylmethoxycarbonyl.

3. L-homovalylamides of 74-amino-1, l-dioxo-3-cefem according to claim 1, characterized by the fact that the conventional Saminoacid amino protecting group is selected from the group consisting of benzyloxycarbonyl optionally substituted at the benzene ring with 4-methoxy or 2,6-dimethyl radical; methoxy-, ethoxy- and allyloxycarbonyl; 2,2, 2-trichloroethoxycarbonyl and 9-fluorenylmethoxycarbonyl.

4. L-valyl- and homovalyl-amides according to claim 1, characterized by the fact that the arylcarbonyl radical is selected from the group consisting of p-chlorophenylsulphonylaminocarbonylphenylcarbonyl and p-chlorophenylsulphonylaminophenylcarbonyl radicals.

5. Valyl- and homovalyl-amides according to claim 1, characterized by the fact that the 4-carbonyl group of the cefem nucleus carries an amine residue selected from the group consisting of the residue of a L-o(-aminoacid, 4-substituted piperazine and the 2-carboxypyrrolidyl and 2-carboxypiperidyl radical.

6. 7 o( -[(N-benzyloxycarbonyl)-L-valylamido]-1,1-dioxo-3- acetoxymethyl-4-tert-butoxycarbonyl-3-cefem.

7. 7 o( - (N-benzyloxycarbonyl)-L-isoleucylamido -1,1-dioxo- 3-acetoxymethyl-4-tert-butoxycarbonyl-3-cefem.

8.7α-[(N-benzyloxycarbonyl)-L-valylamido]-1,1-dioxo-3-acetoxy methyl-4-benzhydryloxycarbonyl-3-cefem.

9. 7 S -l (N-benzyloxycarbonyl)-L-isoleucylamido -1,1-dioxo- 3-acetoxymethyl-4-benzhydryloxycarbonyl-3-cefem.

10. 7α-[(N-4-(4-chlorophenylsulphonylamino)phenyl)carbonyl)- L-valylamido]-1,1-dioxo-3-aceroxymethyl-4-tert-butoxycarbonyl3-cefem.

11. 7α-[(N-4-(4-chlorophenylsulphonylaminocarbonyl)phenyl) carbonyl)- L-valylamido i-l,l-dioxo -3-acetoxymethyl -4-tertbutoxycarbonyl-3-cefem.

12. 7o( - i ( N-4-(4-chlorophenylsulphonylamino)phenyl)carbonyl) L-valylamido -1,1- dioxo -3- acetoxymethyl - 4-benzhydryloxycarbonyl-3-cefem.

13. 7α-[(N-4-(4-chlorophenylsulphonylaminocarbonyl)phenyl)- carbonyl)-L-valylamido]-1,1-dioxo-3-acetoxymethyl-4-benzhydryl oxycarbonyl-3-cefem.

14. N-(2S)-4-thiahexane-1,6-dioic, 7α-[(N-benzyloxycarbonyl)-L -valylamido]-1,1-dioxo-3-acetoxymethyl-3-cefem-4-carboxyamide.

15. 1-methyl, 4-[(7α-[(N-benzyloxycarbonyl)-L-valylamido]-1,1- dioxo-3-acetoxymethyl-3-cefem-4-carbonyl)]piperazine.

16. A pharmaceutical compositon active in the cure of chronic bronchitis, emphysema, ARDS, cystic fibrosis and rheumatoid arthritis, characterized by containing a therapeutically effective amount of a compound according to claims 1-15 in admixture with suitable pharmaceutically acceptable diluents.