Note: Descriptions are shown in the official language in which they were submitted.
1~156~Z
This invention relates to derivatives of cephalosporin
and more particularly, to oxygen analogs of 7-aminocephalosporanic
acid and biologically active derivatives thereof.
This application is a divisional application of copend-
ing application No. 232,812 filed August 5, 1975.
Following the discovery of the penicillins and their
synthesis, perhaps one of the most important advances in medical
research was the discovery of the cephalosporin antibiotics and
their use in clinical medicine. The cephalosporin antibiotics,
though not penicillins, have a structure quite similar to the
structure of the penicillins and the two can be coproduced in the
fermentation of a cephalosporium organism. Because of this
similarity in structure and a similarity in chemical reactivity,
considerable research has been devoted to the formation of deriva-
tives of cephalosporins using, to a large extent, chemical reac-
tions useful for the formation of penicillin derivatives. For
example, 7-aminocephalosporanic acid (7-ACA) may be obtained by
mild acid hydrolysis of Cephalosporin C. The 7-ACA compound is
then available for formation of a multitude of derivatives. For
example, reacylation of 7-ACA with phenylacetyl chloride gives
a derivative that has antibacterial activity approximately 100-
fold greater than Cephalosporin C. Many other reactions of the
amino group of 7-ACA are known and reported in the literature.
Thus, acyl groups, isocyanates, isothiocyanates, halogen compounds
methylisoureas, ethylene oxide, ethylene imine and the like have
been introduced into the 7-amino group of 7-ACA to form both
biologically active and biologically inactive derivatives.
- 2 -
1~1563~
In addition to the above, there have been reactions
of both the ~-lacta~ ring system and the dihydrothiazine ring
system of the cephalosporins. ~or example, with regard to the
~-lactam ring system, C-7 epimers may be formed by treatment of
cephalothin sulfoxide with triethylamine in refluxing chloroform.
With regard to the dihydrothiazine ring system, there is the
possibility of reaction of the double bond, the C-3 substituents
and the carboxyl group to form a vast number of derivatives.
Reactions of the cephalosporins, as described above,
are reported in part by R. B. Morin and B. G. Jackson, "Chemistry
of Cephalosporin Antibiotics", Progress in the Chemistry of
Organic Natural Products XXVIII, Wein, Springer-Verlag, 1970.
For brevity, the commonly accepted abbreviation "7-ACA"
will be used for the term 7-aminocephalosporanic acid throughout
the balance of this specification.
Summary of the Invention
The present invention provides a wide variety of new
derivatives of the cephalosporins and is based upon the discovery
of certain esters of 7-oxocephalosporanic acid and methods for
the formation of said esters. The esters of this invention are
intermediates useful for the formation of the biologically active
oxygen analog (7~-hydroxycephalosporanic acid) of 7-ACA. This
oxygen analog may be used to form a wide variety of biologically
active derivatives analogous to the derivatives of the 7-ACA.
Thus, the invention provides novel esters of 7-oxocephalosporanic
acid, the oxygen analog of 7-ACA, derivatives of said oxygen
analog and methods for the formation of the aforesaid.
The esters of 7-oxocephalosporanic acid are formed by
esterifyino the acid group of 7-ACA with a pharmaceutically
acceptable hlocking group, diazotization of the amino group, and
contact of the diazo compound so formed with a hypohalous acid
and a base in a water miscible organic solvent.
1~1569~
The oxygen analog of 7-aminocephalosporanate is formed
by reducin~ the a~esaid ester to a corresponding 7~-hydroxy-
cephalosporanate, Thereafter, derivatives of the oxygen analog
can be formed by any suitable hydroxyl group modification reaction
such as acylation or other derivatizations analogous to those of
7-aminocephalosporanate. The pharmaceutically acceptable blocking-
group can then be removed regeneratiny the free ac~d.
Description of the Preferred Embodiments
The first step in the formation of the esters of 7-oxo-
cephalosporanic acid is the formation of an ester of 7-ACA by an
esterification reaction whereby the carboxyl group is protected
with a pharmaceutically acceptable blocking group. This is neces-
sary to prevent reaction through the reactive carboxyl group which
could interfere with the desired reaction.
The formation of such an ester is a well known procedure
and is common practice in the art. It ~s used in the formation of
derivatives of 7-ACA as well as in the formation of derivatives
of 6-amino penicillanic acid (6-APA). Preferably, for purposes
set forth herein, the benzhydryl ester is formed by reaction with
diphenyldiazomethane, though any other pnarmaceutically acceptable
blocking group may be used provided the group is read;ly removable
when desired.
Using the benzhydryl ester for purposes of illustration
only, the ester of 7-ACA is diazotized by contact for between lO
and 60 minutes with nitrous acid, generated most conveniently by
addition of a nitrite salt to an acidified solution of the amine.
Common nitrite salts, MN02, include but are not limited to salts
where M is potassium, sodium, ammon;um or the like. Most common
acids including perchloric, sulfuric, sulfonic, haloic
~D
l~lS63Z
and tetrafluoroboric, etc. have found use in this acidification.
Alkyl nit~ites such as i~soamyl nitrite alone or in combination with
trifluoroacetic acid are employed as d~azotization agents ln an-
hydrous organic media. This reaction is performed in a solution
~referably cooled below ambient temperature, more preferably to
about 0C. The diazo compound is isolable by extraction followed
by drying and concentrating the extract until only the oily diazo
compound remains.
The diazo compound is converted to the ketone -- e.g. a
7-oxocephalsporanate by contact with about an equimolar amount of
a hypohalous acid dissolved in a water miscible organic solvent
containing aqueous base. Preferably, the reaction is cooled down
to a temperature of no more than room temperature and more
preferably, to a temperature within the range of from 0VC to
-25C. The time of reaction should not exceed two hours and typi-
caly requires from about 15 minutes to 45 minutes.
Hypohalous acids for use in the above transformation
may be conveniently generated in situ via hydrolysis of N-halo-
amines. The N-haloamide used preferably conforms to the formula
O R
Il I
R - C - N - X
where X is halogen, preferably chlorine or bromine, and most
preferably bromine. Iodine and fluorine are uncommon in this
reaction and consequently less preferred. R and Rl are not
critical and may be selected from the group of hydroaen, a hydro-
carbon radical having up to about 8 carbon atoms such as methyl,
ethyl, propyl and the like, aryl or acylradicals together, or
R and R' may form part of a heterocyclic ring system having up to
a total of six atoms. Examples of N-haloamides within the scope
~r - 5 -
~ 156~2
of the invention include N-bromoacetamide, N-chloroacetamide,
N-bromosuccinamide, N-chlorosuccinamide and the like.
The mechanism of this reaction is proposed to be as
follows:
O R O R
R - C - N - X + H2O > R - C - NH2 + HOX
HO-X
B-: ~ R' -BB ~ ~ R'
C2R" C2R"
~ ~ R'
C2R" C2R"
~ -HX (HX + B~ BH + X ~)
H
O~ ~S~
O ~ ~ N
CO2
Hypohalous acid is generated upon hydrolysis of an
N-haloamide. This reactant is both a source of halonium ion to
effecta halogenation and also of hydroxide ion for solvolytic
displacement of N2 . The unstable hydroxyhalo intermediate
is transformed to the 7-oxocephalosporanate via rapid elimination
~S692
of hydrohalic acid. This acid is neutralized by the base
present in the reaction mixture.
The base employed in this neutralization can be any
common organic or inorganic base known in the art. Examples of
bases used within the scope of this invention include sodium
bicarbonate, sodium carbonate, pyridine, dimethyl aniline and the
like.
In the aforesaid reaction, the reactants are dissolved
in a suitable organic solvent to which water in an amount of at
least 5%, and preferably from 10 to 35%, by volume of soivent
has been added. The water is necessary for formation of the
ketone. The organic solvent is not critical provided it is a
solvent for the reactants, is water miscible to the extent that
water is present, and is non-reactive with the reactants.
Organic solvents such as but not limited to dimethylformamide,
dimethyl sulfoxide, acetone, tetrahydrofuran and dioxane are
suitable solvents for this purpose.
The reaction sequence for the formation of the ester
of 7-oxocephalosporanic acid, using the benzhydryl ester as an
example, for purposes of illustration only, is as follows:
~156~
H H
H2N ~/ S
0 " N ~ R'
C02H
~ Ph2CH-N- N:
H H
2 ~ ~
"L___N ~ R'
C02CH Ph2
¦ NaN02
~ HC104
H H
~N
R~
o
20 CO2CH Ph2
CH3CONHBr
aq- cH3cOcH3
, ~ NaHC03
~S
~ N ~ R'
C02CHPh2
30R' in the above reaction sequence will be defined below.
As noted above, the aforesaid reaction sequence for
the formation of the benzhydryl 7-oxocephalosporanate may be used
for the formation of other esters of 7-oxocephalosporanic acid by
different pharmaceutically acceptable blocking group. In this
respect, for purposes of this invention, a general formula for the
ester of 7-oxocephalosporanic acid is as set forth below:
(I) ~ Lr s ~
~ 2
CO R"
: where R' is an organic nucleophile selected from the group of
hydrogen, halogen hydroxyl, alkoxy, aryloxy, alkylamino, aryl-
amino, carboxyl, organic carbonyl, organic sulfonyl, carbamyl,
thiocarboxyl and other analogous functionalities. R" represents
a pharmaceutically acceptable, readily removable protective group.
Such groups include: (1) alkyl, cycloalkyl, aryl, alkaryl and aral-
kyl as illustrated by me-thyl, benzyl, p-nitrobenzyl, p-methoxy-
benyyl, benzhydryl and ~ -trichloroethyl, (2) phenacyl with
or without substitution on the ring such as p-methoxyphenacyl and
2,5-dimethoxyphenacyl, (3) salts such as sodium, potassium, N-
ethylpiperidine and dicyclohexylamine and (4) organo silicon groups
such as trimethyl silyl. It should.be understood that some of the
aforesaid groups may be more difficult to remove than others,
but most are groups heretofore used as protective groups in ana-
logous reactions for both penicillins and cephalosporins and are
removed in accordance with recognized procedures dependent upon
the particular group involved.
~s~
The 7~-oxy~en analog o~ 7-ACA is ~ormed by reduct~on of
the ester of 7-~xocephalosporanic acid (~) above, Reduction of
such a reactiye ~ diketone can '~e accomplished by myriad techni-
ques well known in the art. Such reducing agents include potassium
borohydride, sodium borohydride, alkylated borohydrides, lithium
aluminum hydride and its alkylated derivatives, well known hydro-
genation catalysts, zinc dust in acetic acid and the like. The
reduction is preferably carried out in an aqueous alcoholic solu-
tion at room temperature of below -- e.g., down to about 0C.
The blocking group can be readily removed there~y forming the free
acid by such procedures as hydrogenation of hyrolysis with tri-
fluoracetic acid (TFA) or by using other methods known to the art.
The reaction sequence for forming the 7~ hydroxy-cephalosporic
acid from 7-oxocephalosporanate ester for illustration purposes
only is as follows:
- 10-
~lS~
o~
0 ~ LN ~ ~1~/ R
C2R"
1 4/aq alcohol
IlO '~ Y~ S
II "
C02R"
1TFA/H 2 0
H H
III HO - C~/~RI
CO 2H
The free acid (III) is the oxygen analog of 7-ACA. It
is biologically active which is unexpected since prior art had
taught that a ~ Nitrogen substituent was essential to this activity.
Both the ester of 7~-hydroxycephalosporanic acid (II)
and the acid itself(III) can be used for the formation of other
biologically active derivatives of 7-ACA. In this respect, a
wide variety of funct.ional groups can be introduced into the
-- 1 1 --
1~156~
hydroxyl group thus making it ~os~ible to pxoduce ~ wide variety
of oxy~en an~ s o~ ce~hal~sp4rin, In this respect, typical
side chain modificat~ons'include ~or example,' formyl, acetyl,
phenylacetyl, phenoxyacetyl, carbo~ethoxy, carbobenzyloxy, p-
nitrocarbobenzyloxy, carbophenoxy, p-chIorocarbophenoxy, methane-
sulfonyl, benzylsulfonyl, p-chlorobenzylsulfonyl~ phenylsul~onyl,'
or p-aminophenylsulfonyl. Although the halide, especially chloride
and bromide, or anhydride of the functionalizing agent'isparticu-
larly suitable for this modification, other agents may also be
used. Such agents include mixed anhydrides, acid azides, lactones,
particularly ~-lactones, "activated esters" such as thiol esters
and phenolic esters, carboxylic acids with carbodiimides or alkoxy-
acetylenes, thiolactones, particularly ~-thiolactones, and acylated
enols.
Other groups can also be introduced into the hydroxy
group of (II) or (III) to provide additional types of oxygen ana-
logs by means of such reagents as: isothiocyanates, for example,
phenylisothiocyanate and ethylisothiocyanate, to convert the
hydroxy group to a substituted thiocarbamate, reactive halogen
compounds, such as triphenylmethyl chloride which forms the trityl
ether derivative; methylisourea which converts the hydroxyl group
to an isourea group; ethylene oxide and ethyleneimine, which add
to the hydroxyl group with ring opening andothers known to the
art. Further exemplification of the akove and additional groups
can be found by reference to Naylor, Proc. R. Soc. Lond, B 179,
pp. 357-367, 1971, wherein reactions of 6-aminopenicillanic acid
are described. These are very analogous to the reactions of 7-
hydroxycephalosporanic acid.
~ - 12 -
1~15~ Z
With further reference to the above reaction scheme, it
should be noted that the free acid (III) can be esterified in
conventional manner to further alter the structure of the
derivatives such as by formation of the methyl ester by reaction
with diazomethane. Thus, by selection of the appropriate
functionalization agent for reaction with the hydroxyl group and
with the carboxyl group of 7~-hydroxycephalosporanic acid (III),
a multitude of derivatives of the ~ygen analogs can be formed
having the formula:
H H S
R"' O ~ ~
(IV) ~ l l R'
N ~/
C2R
where Rl and R" are as above defined. R"'is an organic electro-
phile produced during the ~-hydroxy modifications defined above.
Specific examples of R' and R"'are set forth below
in the following Table:
-
-H, -C l, -Br, -OH, -O-CH 3,
--o-CH2-CH3 ~ --- @~
0 ~31 CH 3
~+
_ o - C - NH2
¦¦ CH3
- o - C - N<H
l
- S - C - CH3
O
- S - C - C6H5
INH 2
- S -C - NH2
N N
Il 11 .
- S~S ~ CH
-,~ -14-
6~ 1
".
i' o .
_ C - CH2 - C6H5
,., ,o, .
, 5 - C - CH2 - O - C6H5
. - C - CH - C6H5
Ot CO~H
~0 11 ,0~
~5
- O ---- -
- C - CH2 - CH
,f ' ' O -' . .
- C~- C~2 - S--
~ ~o - --N N
,' . ..... _~ot ,_ ~ ,,, ,_ .
- C - H, -C - CH3,
--O ' '~
,5 ~ H2, - ~ ~n
O
_ ~ ~X -C~I3, _ ~ - ~H3
-15-
~156~:
Example 1
Benzhydryl 7-aminocephalosporanate -- A suspension of
7-ACA (13.6g, 0 05 mole3 in methanol (20 ml) and dichloromethane
(70 ml) was stirred overnight with diphenyldiazomethane (15 7g)
s prepared accordin~ to the method set forth in Fieser and Yieser,
~'~ Organic Reagents, pg. 338, Wiley Interscience, 1967. The violet
color was discharged at the end of the reaction. Ethyl ether
(200 ml) was added to precipitate the unreacted 7-ACA. Filtration
and evaporation of the solvents gave a crystalline product which
was recrystallized from a mixture of dichloromethane and ethyl
ether. The first crop weighed 9.5 g (43~) and had the following
properties: mp. 128.5-129.5; nmr (DCC13,ppm): 7.39 (S,lOH), 7.00
(S, lH), 5.19-4.62 (M, 4H), 3.50 (D, 2H), 2.05 (S, 3H), 1.84
(S, br, 2H); ir (film, cm 1): 3400, 1770, 1730, 1655, 1390, 1225.
Example 2
Benzhydryl 7-diazocephalosporanate -- senzhydryl 7-amino-
cephalosporanate prepared as in Example 1 (3g, 6.85 mole) was dis-
solved in dichloromethane(90 ml) and stirred at 0C. Sodium nitrite
(0.7g, 1.5eg), dissolved in H2O (10 ml), was added to the cooled
stirred solution and 1.01 N perch]oric acid (10.5 ml, 1.5 eg.)
was àdded dropwise. The mixture was stirred at 0~C for 1 hr. and
diluted with additional dichloromethane, washed twice with ice
cold water and once with an ice cold sodium chloride solution.
The dichloromethane layer was then dried and evaporated to a yel-
low oil. It had the following properites: nmr (DCC13,ppm):
7.39 (S,lOH), 7.00 (S, lH), 5.40 (S, lH), 5.08-4.52 (Q, 2H), 3.35
(S, br, 2H), 1.98 (S, 3H); ir (film cm 1): 2090, 1780, 1735, 1235.
Example 3
_ . .
i B ~ 1_7-oxocephalosporanate -- Benzhydryl 7-diazo-
cephalosporanate prepared from 3g of benzhydryl 7-amino-cephalo-
sporana~e in the manner of Example 2 was used without further puri-
fication. It was dissoved in a 10% aa. acetone (90 ml) solution
- 16 -
, ~D
1~156~;~
and cooled in an ice-acetone~odium chIoride bath ~-15C). Sodium
bicarbonate (3~] and N-bromoacetamide (0,945g) were pouxed into
the stirred cold solution. A~ter 45 ~inutes, the reaction mix-
ture was diluted with dichloromethane and water. Extraction with
dichloromethane was repeated three`times. The organic layer was
washed once with cold water and once with cold sodium chloride
solution. Dryiny and evaporation of solvent gave about 3g of
yellow oil which was purified by column chromatography on silicic
acid and eluted with 1:9 ethyl ether-dichloromethane. The yield
was 50~ from benzhydryl 7-amino-cephalosporanate. The properites
were as follows: nmr (DCCl3,ppm): 7.39 (S, lOH), 7.00 (s~lH)~
5.21 (S, lH), 5.10-4.65 (Q, 2H), 3.48 (Q, 2H), 2.01 (S, 3H);
ir (film, cm 1): 1825, 1785, 1730, 1230.
Example 4
Benzyhydryl 7~-hydroxycephalosporanate -- Crude benzhydryl
7-oxocephalosporanate (2.95g) was dissolved in ethanol (150 ml).
~o this cooled and stirred solution, there was added a solution
of potassium borohydride (0.74g) in a 1:1 ethanol-water mixture
(150 ml). The reaction was quenched after 2 minutes by addition
of lN HCl to pH 2. The reaction mixture was diluted with water
and extracted twice with dichloremethane. The organic layer was
washed once with sodium bicarbonate solution and once with
sodium chloride solution. Drying and evaporation gave a yellow
oil which was chromatographed to give 1.2g solid product. The
product was recrystallized from benzene. Its properties are as
follows: mp. 122-3; nmr (DCC13,ppm): 7.39 (S, lOH), 7.00 (S, lH)
5.29 (d, lH, J = 4.5), 5.20-4.62 (m, 3H, J=4.5 and 13), 3.90
(S, br., lH); 3.45 (d, 2H), 2.04 (S, 3H); ir (CH2C12, cm ): 3540,
1785, 1735, 1225.
Example 5
7~-hydroxycephalosporanic acid -- Benzhydryl 7~-hydroxy-
cephalosporanate (0.3g, 0.68 m moles) was dissolved at 0C in a
~I~ - 17 -
~1156~2
mixture of tr~fluoroacetic acid (7 ml) and anisole (1 ml). After
, 1 hr., the solvents ~ere eva~orated under vacuum. The residual
;~ yellow material was washed with petroleum ether and then dissolved
s in ethyl acetate. Treatment with charcoal for half an hour and
evaporation of the solvent gave a solid produce, 0.17~ (99%). It
was recrystallized from ethyl acetate. Its properties were as
follows: mp 132 (decomp). nmr (acetone d6,ppm): 5.40 (d, lH,
J=4.8Hz), 5.15-4.80 (m, 3H, J=4.8 and 13 Hz), 3.55 (d, 2H), 2.04
s (S, 3H); ir (Kbr, cm ): 3439, 3100, 1780~1700, 1625, 1380, 1220.Bioassay results (minimum inhibitory concentration in Mg~ml):
S.aureus (75), B subtilis (75) E.coli(50), K. Pneumoniae (200).
Example 6
Benzhydryl 7~-phenoxyacetoxycephalosporanate -- Benzyhydryl
7B-hydroxycephalosporante (0.8g), (1.82 m moles) and phenoxy-
acetyl chloride (0.42g, 1.5eq.) were dissolved in dichlormethane
(50 ml). Pyridine (0.15 ml, 1.5eq.) was added to the cooled stirred
solution. After stirring for three hours at room temperature, the
dichloromethane solution was washed with water, sodium ~icarbonate
solution, and sodium chloride solution. Drying and evaporation
gave a yellow oily product which was chromatographed on silicic
acid using a 1:20 ethyl ether/dichloromethane mi~ture to yield
0.85g of a pale yellow oil, 88% yield. Its properties are as
follows: nmr(DCC13,ppm): 7.54-6.80 (m, 16H), 6.10 (d, lH, J=4.8Hz)
5.20-4.65 (q, 3H, J=4.8 and 14hZ), 4.75 (S, 2H), 3.38 (s, br., 2H),
1.98 (S, 3H); ir (film, cm 1): 1785, 1730, 1600, 1495, 1380, 1225.
Example 7
7~ phenoxyacetoxycephalosporanic acid -- The procedure of
Example 5 was repeated with a product yield of 93%, the product
having the following properties: nmr (acetone-d6,ppm): 8.10
(br, IH), 7.42-6.87 (m, 5H), 6.32(d, +H, J=4.8Hz), 5.25 (d, lH,
J=4.8Hz), 5.28-4.70 (q, 2H, J=14Hz), 4.92 (S, 2H), 3.60 (d, 2H),
2.02 (S, 3H): ir (film, cm 1):3580, 3520-2500, 1785-1690, 1635,
- 18 -
~iS~i~2
... .
1600, 1495, 138Q~ 1230 Bloasaay results (minimum inhibitory
concentxation In Mg/~l): S.aureus (I2~5), s.subtilis (25), E. Coli
(200)~ K. pneumoniae (200).
Example 8
Benzhydryl 7~-(2-thienyl)'a'cetoxycephalosporana'te --
Benzhydryl 7~-hydroxycephalosporanate (0.45g, 1.02 m moles), 2-
thienylacetic acid (0.21g, 1.5eq.), and pyridine (0.1 ml~l.2eq.)
~ were dissolved in dichloromethane (50 ml) at 0. To this solution
,~ was added diisopropyl carbodiimide (0.13g~1eq.). The cold solu-
tion was stirred one hour and then allowed to stand 17 hours under
refrigeration. The solid urea formed was separated by filtration
and the filtrate was diluted with dichloromethane and washed with
cold dilute hydrochloric acid, sodium bicorbonate solution, and
sodium chloride solution. Drying and evaporation gave a yellow
oil which was chromatographed on solicic acid in a 1:20 mixtùre
of ethyl ether and dichloromethane to yield 0.6g product (>95~)
having the following properties: nmr. (DCC13ppm): 7.54-6.90 (m,
14H), 6.05 (d, lH, J=4.8Hz), 5.20-4.60 (d on q, 3H, J=4.8Hz and
14 Hz), 3.91 (S, 2H), 3.36 (S, br, 2H), 1.98 (S, 3H); ir (film,
cm 1): 1785, 1330, 1360, 1235.
Example 9
7~ (2-thienyl)acetoxycephalosporanic acid -- The proce-
dure of Example 5 was used to yield a productobta'ined by freeze
drying from benzene. The yield was 98~ and the product had the
following properties: nmr (DCC13,ppm): 7.73(S, br, lH), 7.40-7.20
(m, 2H), 7.00 (d, lH), 6.19(d, lH, J=4.8Hz), 5.38-4.82(d on q, 3H,
J=4.8Hz and 15Hz), 4.00 (S, 2H), 3.48 (S, br., 2H), 2.13 (S, 3H);
ir (film, cm ); 3560-2540, 1780, 1725, 1380, 1225. Bioassay
results (minimum inhibitory concentration in Mg/ml): S~aureus
(12.5), S. Fecalis (200), B, subtilis (6.25), P. Mirabilis (200),
P. vulgaris (200), K pneumoniae (100).
~ -- 19 --
