Note: Descriptions are shown in the official language in which they were submitted.
~21~34994
~echnieal rield
~hit ~nvent$on i~ direeted to temperature table emi-
~ynthetic cephalo~porin calt~ whoee preparation ba~ not been
de~cribed in the literature, to the preparation of ~uch salts,
and to admixtures containing thece calte
Backqround Of ~he lnvention
Aburaki et al ~ S Patent No. 4,406,899 di~closes 7-
I~-(2-aminothiazol-4-Yl)~ 2)-nethoximinoacetamido~-3-
~ methyl-1-pyrrolidinio)-methyl]-3-cephem-4-carboxyl~te in the
zwitterion form and mentionc corresponding cid addition ~alts
(which are pre~ent in the 2witterion form in injectable composi-
tions) and chows t~Dt the zwitterion form has broader spectru~
activity than ceftazidime nd cefotaxime.
However, the aforementioned A~uraki et al cephalosporins
re table only for a few hour~ a~ ~njectable composition~ and
the zwitterion form even as a dry powder i~ un6table at room
temperature and 106e~ 30~ or more of it~ activity on ~torage at
clevated temperature~ (e g. 45 deg C nd above) for even one
week and therefore reguires ~pecial in-ulated packaging and/or
refrigeration nd i~ t a p-ckag~ng and torage di-advantaqe
comparcd to ceftazidime ~nd cefot~xime.
While Aburaki et al. mentions acid addition calts, the
patent does not ctate how to make tbe~e or rtate which if any of
these ~alts have good rtability in dry powder form ~Xessler et
al., ~Compari~on of a New Cephalo~porin, BMY 28142, with ~ther
Bro~d-Spectrum ~-Lactam A~tibiotic~, Antimicrob~al Aqents and
Chemothera~v, Vol 27, No. 2, pp. 207-216, February l9B5 mentions
the ~ulfatc ~alt, but doe- not di~clo~e how to prepare ruch or
th~t thi~ ~alt has room temperaturc tability nd good elevated
temperature rta~illty in ~ry powder form
~ ` lZ84994
~umm~rv Of The Inventlon
It ha- been d~-covered herein that certain eryctalline acid
ddition zalt~ of ~-[~-~2-aminothiazol-4-yl)-o-~z)-
methox~1m1no~cet~1do~-3-l~l~methYl-l-pyrrolidinio~-mcthyll-
3-cephem-~-carboxylate in dry powder form have excellent room
temperature ~tability and bave euperior elevatea temperature
etability eompared to the zwitterion form The term ~dry powder
form~ a5 used herein means a moisture content of leee than 5~ by
weight
The~e acid addition calt- are the cry~talline eolt~ of
7-l~-(2-aminothiazol-4-yl~-~-(Z)-methox~iminoacetamido~-3-
t~l-metbyl-l-pyrrolidinio)~methyll-3-cephem-4-carboxylate
eelected from the groùp consi-ting of the ulfuric, di-nitric,
mono-bydrochloric, ~nd di-hydrocbloric acid addition ~alt~ and
_; orthophosphoric cid ddition ealte (l 5-2 molee of
orthophospboric cid per mole of alt, e g range of from the
~c~qui- to the di-orthophosphoric acid ralte), or ~olvate~
thereof The term ~cry~talline~ is ueed berein to mean at least
come characterizing rrangement of molecule~ While the
:D rulfuric, di-nitric, di-bydrochloric and ortbopho~pboric acid
ddition ealte herein are prepared in clearly cryetalline form
~-e evidenced by birefringence under polarizinq micro~cope)
with precire rrangement of molccules, the mono-hydrochloric acid
daition ealt hae been prepared only with ome regularity in tbe
~~ arrangement of ite moleeules ~ae evidenced by poor birefringence
under polarizing microecope) ana not preci~e predictable
arrangement and thue ie ~poorly~ cryetalline The term
~cryetalline~ ie ueed herein to embrace not only the clearly
cryetalline ralt- but leo the ~poorly~ appearing cry~talline
_D ~ono-hydrochloric cid addition ~alt
~ he acid ddition altc herein when formed into queou~
injectable compo~ition- provide the zwitterion in ~olution The
zwitterion bae the rtrueture C~ :~ S
N ~ ~ C~2-N
~ O-C~,~ C~De C~
-3-
1284994
The bro-d cpee~rum utility gainst variou~ organicms of t~e
zwitterion form, nd thu- ~f queouc compo6ition~ made up from
t~c ~alt~ ~ere~n, is ihown by the data in Abura~i et al ~ S
4,~06,899
S Aqueous compocitions made up from the acid addition fialts
hercin cimply by the addition of cterile water provide acidic
~olutione whic~ provoke unacceptable irritati~n on intravenous
dministration to rabbitc ~nd unacceptably painful eensation on
intr muscular administration to rabbits ~he culfuric acid and
di-nitric acid addition calts bave reduced colubilities which are
insufficient for typical injectable compositions It bas been
found herein that these objectionable characteri~tics are
overcome by utilizing t~e ~alts herein in pbysical admixture
(that ic as an admixture of colids) with a pharmaceutically
cceptable n~n-toxic organic or inorganic base in proportions to
provide a pH of about 3 5 to abDut 7 on dilution with water to a
rwitterion activity of from 1 mg/nl to 400 mg/ml, normally 250
~g/nl (as determined by high pe~formance liquid chromatography,
~ereinafter HPLC)
A preferred Eialt herein is the crystalline culfuric aeid
ddition calt It is preferred because its lo~ colubility in
water ~25 mg/ml) allows high recovery from aqueous medium on
cryctallization
~he crystalline ~ulfurie acid addition calt is readily
preparcd by a process compri~ing the teps of la) forming an aqueous
admixture of ~i) at least 1 molar equivalent of culfuric acid and
(ii) zwitterion in an a~ount co as to be present in t~e adnuxture
~t a concentration greater tban 25 mgtml, (b) causing
cryctallization of the culfuric acid addition calt to occur, and
(c) ~colating erystalline culfuric acid addition calt
`_ "~,!
12R4994
Detailed DeccriDtion
The cryctallihe c~ltc herein Ihereinafter referred to cimply
c the salt~ ~erein) have excellent etability at room temperature
~nd bave ~ potency loss las determined by HPLC), of le~s than 1
on ztorage for a mont~ at room temperature ~hese ealt~ also
bave excell nt stability ot elevated temperatures ~nd have a
potency loss (a~ determined by HPLC) of less than 15~ on stor~ge
for a month at ~5-56 deg C
The culfuric acid additiDn alt is a preferred ~alt herein
lD It has a potency loss of less t~an 10~ on ~torage for a month a
~5-56 deg C Very importantly, it has a low olubility in
water, i e ~bout 25 mg/ml, and therefore i5 crystallized from
water with minimized residual loss.
~ he di-nitric acid addition ~alt herein alco has a 1DW
colubility in water, i e about 60 mg/ml, ~nd therefore also
provides low residual loss on crystallization from water
The mono-hydrochloric, di-hydrochloric ~nd sesqui- or
di-orthophotphoric acid addition calts have water colubilities
greater than 200 mg/ml , ~nd therefore are preferably crystallized
from orqanic ~olvents, rather than from water, in order to obtain
goDd yieldc
We turn now to the preparation of the sslts herein
As previously indicated the ~ulfuric acid addition calt
berein is prepared by a process comprising the teps of (a)forming
an aqueous admixture of (i) at least one molar equivalent of
zulfuric acid and (ii) zwitterion corresponding to caid calt in
an ~mount ~o ac to be present in the admixture at ~ concentration
of greater than 25 mg/nl, (b) causing erystallization to occur,
~nd (c) ~solating crystalline culfuric acid ~ddition salt
-5-
~21~9~4
.
Preferably the ~witterion is used ~n step ~a) ~n an um~unt eD a~
to be pre-ent in the dmixture t concentration ranging from
ab~ut 100 mg/ml to b9ut 200 mg/ml, nd tep (b) ls carried out
~n an aqueou~ medium free of organic solvent. Normally no more
than 2 molar cquivalent6 of eulfuric ac~d are utilized in ctep
la). Normally rwitterion ic u5ed in etep (a) in an amount so as
to be present in the admixture at a concentration less than 500
mg/ml.
Step (a) ie readily c~rried out either by addin~ solid
zwitterion to ~ulfuric acid solution (e.g. lN H2S~4) with rapid
etirring to form a ~olution. Alternatively step (a) can be
carried by diseolving solid zwitterion in water and slowly adding
~ulfuric acid with stirring to form a ~olution.
Step (b) ie carried out by inducing crystallization,
preferably by ~eeding, and then slurrying, preferably for 15
minutes to 2 ho~rs. It is preferred that this crystallization
etep be carried out in aqueous medium, free of organic ~olvent,
~nd in such ea~e purities greater than 98~ are normally obtained.
While the pre6ence of organic olvent, euch as acetsne, fosters
crystalliiation and increases yield by lowering the ~olubility of
the formed sulfurie acid addition ealt in the cryst~llization
medium, it also can foeter precipitation of impurities resulting
in decreased purity. When the zwitterion i~ used in step (a) in
an amount o as to be present in the admixture in an amount less
than 25 mg/ml, orqanic solvent, preferably acetone, must be
included in the cry~tsllization medium to provide reasonable
recovery. When acetone is used, it i5 appropriately used in
amounte of 0.5 to 10 volumes per volume of ague~us
cry~tallization medium.
Step (c) i~ carried out by eeparating the cry~tals from the
crystallization ~edium, preferably by vacuum filtration, then
~ashing e.g. with acetone/water followed by acetone alone or O.lN
~ulfuric acid (e.g. 1/10 volume) followed by acetone le.g. 1/4
` - ~2~ 3i9~
volume), ~nd then drying, g by v-cuu~ ~rying t 30-50 de~ C
for ~-20 hour~
The ~et~Dd herein ~or forming the ~ulfuric ~cid ddition
~lt rc~ults in the purificati~n of the ~itterion form because
S of the limited ~olubility ~ the au~furic acid eddition ~lt
compared to the zwitterion form nd can be used to purify
~witterion without icolating it ~c ~olid If it i~ desired to
obtain ~ubstantially pure zwitterion (free-~ase) from the formed
~ulfuric acid addi~ion calt, thi~ can be carried out by
di~o~ving the ~alt in water, edaing ~a~H)2 8H2O in an am~unt of
90-100~ of theory ~t a p~ of le-~ than 6 5 to precipitate ~aS~4,
filtering to remove the BaSO4 nd recovering the iltrate
containing the rwitterion di~solvea therein and utilizing it as a
rolution or i~olating ~olid zwitterion (free-base) by
lyophilizinq it or by dding ~cetone to precipitate amorphous
z~itterion followed by i~olating ~olid zwitterion by vacuum
filtration, washing e g wit~ aeetone, and vacuum dryinq
_ Alternatively, the ~ulfuric acid ~daition alt i- converted to
the free-base ùtilizing ion exchange re~ins, g Dowex WGR (a
weak ba~e anion exc~ange re~in) and Dowex XU-40090 01 (a ~trong
acid cation exchange resin) with ~ub~eguent lyophilization
Turning now to the preparation of thc cryttalline di-nitric
cid addition alt herein, thic ~ obtained by admixinq ~i) at
lea~t two molar equivalen~ of nitric cid and lii) zwitterion
corre~ponding to taid ~alt ~o to be pre~ent in the admixture at a
concentration greater than 100 mg/ml, and then indueinq
cry~talli~ation by ~eeding or rub~ing with a glass rod, dilutinq
with 2-propanol ~nd eooling The cry~talline di-nitric acid
ddition ~alt i~ recovered e g by filtering, wa~ing
requentially, e g with 2-propanol-h2O ISD~ v/v), 2-propan~l, and
ether, and then vacuum drying ~t 50 deg C for 2 hour~
The mono-hydrochlorie cid ddition ~alt ~erein i~ prepared
~y di~solvinq ~witterion in ~pproximately one molar eguivalent of
_?_
*Trade Mark
!
hydrocbloric cid nd c~using cry~tallization by ddinq cetone
witb stirring and eontinuing to ctir, followed by i-31ating
cry-talc, e.g. by vacuum filtration followed by ~a-hinq with
cetone nd vacuum drying. Alternatively the mono-hydrochloric
cid ddition salt is formed from the di-hy~rocblorlc cid
ddition salt by slurrying the di-hydrochloric cid adaition salt
in methylene chloride ~n~ ~dding 1 mole eguivalent of
triethylamine followed by slurrying to form the mono-~ydrochloric
cid addition talt ~hich is isolated, e.g. by vacuum filtration,
followed by washing wit~ met~ylene cbloride and vacuum drying.
The crystalline di-hydrochloric acid eddition c~lt herein is
prepared by dissolvins zwitterion in at least t~o molar
equivalents of hydrochloric acid, then causing crystallization by
adding acetone, then isolating crystal~ e.g. by vacuum
filtration, wa~hing with ~cetone and vacuum dryinq.
~ he crystalline di-orthophospboric acid addition salt herein
is prepared by dissolving the zwitterion in at least 2 molar
equivalents of phosphoric acid, causing crystallization by ~dding
acetone, ~nd isolating crystals by e.g. by vacuum filtration
followed by washing first with acetone and tben ~itb ether and
then vacuum drying. The crystalline eesqui-orthopbosphoric acid
addition salt i5 formed by this same procedure except that about
1.5 molar equivalents of phosphoric acid is used.
~he salts berein are formed into injectable compositions by
diluting witb sterile water ~nd buffering to a pH of 3.5-7 to
form an injectable concentration of 1 mg/ml up to 400 mg/ml of
zwitterion. Suitable buffering agentC include, for example,
tri~odium ortbopbosp~ate, sodium bicarbonate, sodium eitrate,
N-metbylglucamine, L~) lysine ~nd L(~) arginine. For
intramuscular or intravenous administration to ~n sdult human, a
total dossge of from ~bout 750 to about 3000 mg per day in
divided dose~ ic norm~lly sufficient.
-8-
9a~
~ he c~lt6 herein re not desirably formed into ln~cctable
compo~itions simply by the addition of terile wates becau-e the
sulfuric ~nd di-nitric acid addition altc are not ~ufficiently
soluble to form compositions of normal concentration for admini~-
tr-tion and because the caltc herein when dit~olved provide very
low pH compositions (1.8-2.5) which provide painful eensation on
injection. As indicated above, it ~a8 been found herein that
these chortcomings are overcome by forming the calts herein into
a phy~ical, i.e. colid, admixture with pharm~ceutically
acceptable, norm3lly colid non-toxic organic or inorganic bases
in proportions to provide a pH ranging from a~out 3.5 to ab~ut ~,
preferably from about 4 to about 6, on dilution of the admixture
with water to injectable concentration of 1 mg/ml up to ~00 mg/ml
of zwitterion, e.g. zwitterion activity of 250 mg/ml as
determined by HPLC assay.
~ he exact proportior.s of ingredients in the physical
admixture vary from lot to lot of the salt since the purity of
the calt varies from lot to lot. The proportions of ingredients
are established for a particular lot by pretitrating in respect
to a cample to obtain a selected pH within the aforementioned
range.
The physical admixture is readily stored and shipped in
colid form thereby taking advantage of the ~tability of the salts
herein and i~ readily converted into an injectable composition
simply by addition of water, e.g. by a nurse or doctor,just prior
to use.
~ he physical admixture is prepared by blending the calt and
the base into a uniform blend, e.g. utilizing a ~tandard blender
$n a dry atmosphere, ~nd is then preferably filled into a vial or
other container, all under aseptic conditions.
The bases for u~e in the admixture include, for example,
tri~odium orthophosphate, codium bicarbonate, codium citrate,
N-methylglucamine, Ll+~ lysine and L(+) arginine. L(+) lysine
_9_
9~
and L(~) a~ginine are preferred ince admixture~ eontaining t~ese
re reconstituted to provide injectable composition6 which on
~njection provide ~es~ pain in animals than compo~itions derived
from admixture~ containing other ba~e~. ~he L(~) ly~ine i6 very
prcfer~bly utili2ed in a proportion to provide pH of 3.5-6 on
dilution of the admixture with water to provide a composition
with a zwitterion activity of 250 ns/ml (as determined by HPLC
assay).
~he salts herein and ~ubstantially dry physical admixtures
containing them can be ~tored wit~out refrigeration Dr insul2ted
packaging and still retain bigh potency.
In several of the preparations herein the unsta~le
zwitterion i~ used as the tarting material. ~he preparation of
this is described in Examples 1-3 of Aburaki et al. ~.S.
4,406,899. ~he zwitterion is referred to in Aburaki et al. as
7-l(2)-2-methoxyimino-2-(2-aminothia201-4-yl)acetamido-3-l(l-
met~yl-l-pyrrolidinium)methyl]-3-cephem-4-carboxylate.
~he invention i5 illustrated in the following w~rking
examples.
Example I
PreParation of the Sulfuric Acid Addition Salt
1.5 9 of zwitterion ~re added 610wly to 10 ml o~ rapidly
6tirred lN ~254 (1.59 molar equivalents) at 20-26 deg. C. A
solution is obtained. Crystalliz~tion i6 then induced by seeding
with crystalline 6ulfuric ~cid addition calt and the cryctalline
mass is elurried for 0.5 hours. ~he cry~tals ~re then separated
by vacuum Siltration, washed with 3 ml of 50~ aeetone/water ~V/V)
~nd with two 5 nl portions of acetone, ~nd vacuum dried at 40-50
deg. C overnight.
--10--
~ ~ i
A typ~l y1eld 1~ 1.3 9 of ulfuric cid addition alt.
Analys~s Calculated for ClgH2~N6O5S2 H2SO4 ~C, 39.44; ~H~
4.53; ~N, 14.52; ~S, 16.62~ ~H2O, none. Found: ~C, 38.91; ~H,
~.57s -N, 14.64; tS, 16.71s ~H2O, 1.42.
ExamDle II
Pre~aration of the Sulfuric Acid Addition Salt
1.5 9 of 2witterion ~re dissDlved in 5 ~1 of ~ter. 5 ~1 of
IM 112S04 ~re slowly ~dded to this solution with stirrinq.
Crystallizntion i~ then induced by seeding Wlth crystalline acid
addition salt and the crystalline mass is lurried for 0.5 hours.
The crystals are then eparated by vacuum filtration, washed with
~ ml of 50~ acetone/water (V/V) and with two 5 ~1 portions of
acetone, and vacuum ~ried at 40-50 deg. C overnight.
A typical yield i~ 1.3 9 of Eulfuric acid addition salt.
lS Example III
PreParation of the (HNO~)~ Acid Addition Salt
300 mg of zwitterionare dissolved in 2N nitric acid (0.5
ml). The solution i~ ru~bed with a qlass rod, diluted with
2-propanol (0.4 ml) and cooled. The crystalline title compound
is collected and i~ ~equentially washed with 0.4 ml of 2-propanol
H2O ~1:1), 2-propanol ~nd then ether to afford 127 mgs of the
dinitrate salt.
Analysis: Calculated for ClgH24N6O552~2H~O3 ~C, 37.62;
~H, 4.32; ~N, 18.47; ~S, 10.57. ~ound: ~C, 36.92; ~H, 4.10;
~N, 18.08; ~S, 10.67; ~H2O content 0.90~).
--1 1--
.
$~34
. .
Example IV
Preparation of the Monohvdrochloride Acid Addition Salt
1 g of 2witterion is dis~olved in 2.08 ml of l~ HCl (1 molar
equivalent) at 20-25 deg. C. Thirty ml of aceto~e are added with
rapid rtirring over a 15 minute period whereby crystal5 form.
Stirring is continued for 1 hour. ~he crystals are isolated by
v~cuum filtration, washed with 10 ml of acetone and va~uum dried
at 50 deg. C for 2 hour~.
A typical yield is 0.9 9 of crystalline monohydrochloride
~alt. Analysis: Calculated for ClgH24N60552.HCl: ~C, 41.37;
~H, 4.75; ~N, 15.2; ~S, 11.63; ~Cl, 12.B6. Eound: ~C, 39.32;
~H, 4.88; ~-N, 13.95; ~S, 11.28; ~Cl, 12.44; ~H20, 4.5.
(Corrected for H2~: tC, 41.17; ~, 14.61; ~S, 11.82; ~Cl,
13.03).
Exam~le V
~reparation of the Dihydrochloride Acid Addition Salt
and Preparation of the Monohydrochloride Acid
Addition Salt Erom It
350 mg of zwitterion are dissolved in 2 ml of IN-HCI. 10 ml
of acetone are added to the resultant solution, with rapid stirrinq
and over a 5 ~inute interval, whereby crystals form. Stirrinq is
continued for 5 additional minutes. Then 10 additional ml of
acetone are added and stirrin~ is carried out for 0.5 hours. The
cry~tals are removed ~y vacuum filtration, washed with two 5 ml
portions of acetone ~nd vacuum dried at 40-45 deg. C for 24
~ours.
A typical yield iS 300 my of crystalline dihydrochloride
acid addition ~alt. Analysi~ Calculated for
--1 2--
128499a~
C19~2~N6O5S2.2HCl: ~C, 41.38; H, 4.~5; ~N, 15.2: tS, 11.62
~Cl, 12.B. Fo~nd: ~C, 40.78; ~H, 4.98; ~N, 14.7; ~S, 11.25;
~H20, 1. 2S. (Corrected for H2O: ~C, ~1.1; ~N, 14.8B:
~S, ~1.39; ~Cl, 11.94)
1 9 of dihydrochloride s21t prepared as aboYe is lurried in
20 ml of methylene chloride at 20-25 deg. C in a sealed flask and
0.28 ml of triethylamine is added over a 15 minute inter~ he
cryst~lline mass is then slurried for 5 hours. ~he resultant
monohydrochloride crystals are then isolated by vacuum
filtration, washed with two 5 ml p~rtions of methylene ehloride
and vacuum dried ~t 50 deg. C for 2 hours. A typical yield i6
800 m~.
ExamPle Vl
Preparation of the Di-orthop~osphoric
Acid Addition Salt
1 g of zwitterion is dissolved in 3.4 ml ~f 144 mg/ml
PO4 ~2.2 m~lar eguivalents) at 15 deg. C. The resulting
solution is suitably filtered to clarify it. 12 ml of acetone
are ~dded to the clarified solution, with raDid stirrino and over a
10 minute period,whereby crystal5 form. Stirring is continued
for 10 minutes. ~hen 30 ml of acetone a-eadded over a 10 minute
period, and stirring is continued for an additional 15 minutes.
~he crystals are collected by vacuum filtration, washed with two
5 ml portions of acetone and two 5 ml portions of ether and dried
~nder high va~uum for 16 hours.
A typical yield for this type of preparation was 1.1 9 of
cryctalline di-orthophosphoric acid ~ddition salt. Analyeis:
Calculated for ClgH24N6~5S2 3 4
~, 12.42. ~ound: ~C, 33.43; ~H, 4.65; ~N, 12.02; ~H2O, 1.82.
(Corrected for H2O: ~C, 34.0; tN, 12.2).
~2134994
~ e ~esqui-ort~opho-p~oric cid ddition alt ic formea as
above xcept that 1.5 olar equivalents of H3P~4 are u5ed inttead
of 2.2 ~olar equivalentc.
S ExamPle VII
Stabilities at Elevated TemDeratures
Elevated temperature stabilities were determined by toring
the preparations in dry container~ at temperatures and for time
periods as denoted below and potency losses or gains were
determined by ~PLC. A ~ potency qain is indicated by a plus sign
in front of a figure. A less than 10~ potency loss over a 2 to 4
week period at 45-56 deg. C i~ usually indic~tive of less than
10~ potency loss over a 2-3 year period at room temperature.
9~3~
PERCENT LOSS
J5 Deg. C 56 Deg. C 100 Deo . C
(Weeks) IWeeks) (Days)
Form 1 ~ _ 6 1 2 4
zwitterion 3~ ~ 71 _ 57 _ _100
~2SO4 Salt 12.q to ~. ~ ~S 1 45 ~o ~6 ~3 0 to ~6 0-10
(NNO3)2 Salt8.8 3.4 0.6810.3 3.7 2.4 _
_
HCl Salt 4.~ ~ 6 0 6 4 6.4 _ _
lHCl)2 Salt 0 ~ 7 ~ _ 0 _ 7 2 12.4
I0 (H3P4)2 Salt j ~ ~ _ 2.7 5.C _
-' ExamPle VIII
Testinq of Physical Admixtures
Physical admuxtures were made up of crystalline sulfuric
acid ~alt with (a) trisodium orthophosphate, (b) sodium
bicarbonate, Ic) L(~) lysine, and (d) L(~) arginine. The bases
were added in proportions to provide pH'~ on dilution of the
dmixture with water to a zwitterion activity of 250 mg/ml (as
determined by NPLC acsay) as follows: trisodium orthophosphate
(to provide a pH of 6.0): codium bicarbonate (to provide a pH of
6.0); L(~) lysine (to provide a pH of 6.0); L(l) arginine (to
34
provide pH of 6.0). ln~ect-ble compo~itiont were made up by
rcconttituting witb sterile water to rwitterion activity of 250
mg/ml ac determ$ned ~y BPLC scay. There were no solubility
problems. Injections ~100 mg/kg) were carried out
intramuscularly ~n rabbits with pain within acceptable
threcholds. The least pain was with the arginine containing
compo~ition.
Similar results of good ~olubility and acceptable pain on
intramuseular injection are obtained on use of the other ~alts
herein in the physical admixtures with the above bases.
~ igure 1 i~ the infra red absorption spectrum of the
cry~talline ~ulfate ~alt prepared as described in Examples I or
II pelletized in the crystalline form with potassium bromide.
The X-ray powder diffraetion pattern of the crystalline
~ulfate salt of 7-la-(2-~minothiazol-4-yl)-a-(z)-methoxyimuno-
acet~mido~-3-[(1-methyl-1-pyrrolidinio)-methyl~-3-cephem-q-
carboxylate prepared as de~cribed in Example I or II was
determined with a Rigaku Powder Diffractometer using a copper
target X-ray tube, a nickel filter, and the sample contained in a
glass dich. The scan rate was 2 deg./min. over the range from 5
deg. to 40 deg. and a chart was mechanically recorded to ~h3w the
ngles of maximum diffraction. ~rom this the (d) spacings and
rclative intensities (I/I) were calculated. ~hey are listed
bel~w.
-16-
~2~4994
d ~Pac~nq (A) I/I 1-)
9. 20 lOD
6.80 50
5.50 2B
5.09 22
q.50 38
4.41 ~4
.lg 63
3. 78 38
103 . 64 ~4
3.39 ?s
3.31 31
3.15 47
--1 7--
~z1~4994
EXAMPLE IX
PREPARATION OF THE SESOUIPHOSPHATE SALT
The zwitterion, 0.70 g., is dissolved with rapid
stirring in from 2.2 to 2.4 ml. of 85% phosphoric acid ~2.1
to 2.2 molar equivalents) which has been diluted 1:10 (v/v)
with water. The solution is clarified by filtration through
a 0.22-0.45 micron pore-size membrane filter. From 5 to 7
parts by volume (15-20 ml) of methanol is added to the
filtrate with rapid stirring during a 30 to 60 min. period.
Crystals form during this operation, and rapid stirring is
continued for 1.5 to 2 hours. The crystalline product is
recovered by vacuum filtration. The product is washed on
the filter first with 6 to 8 ml of 1:1 (v/v)
methanol:acetone taking care to maintain a tightly packed
filter cake, and then with acetone. The product is dried in
vacuo at 50 C for 2 hours; typical yield 0.7 to 0.75 g.
Infrared Interpretation (See Figure 2)
(IR, KBr pellet)
Peak Position (cm 1) Functional GrouP
2800-3400 NH,NH3 ,carboxyl OH
1780 ~-lactam C=O
1680 Carboxyl C=O
1660 Amide C=O
1630 C=N,C=C
1550 Amide OH
980,1040 PO4=
- 18 -
~2~4~94
.
Behavior On Heatinq
An exotherm is shown at 171.8C in the differential scanning
calorimeter tracing.
X-Rav Diffraction Pattern
The X-ray powder diffraction pattern of the foregoing
sesquiphosphate salt was measured with a Rigaku Powder
Diffractometer in the same fashion as described above with
respect to sulfate salt with the following results.
d I/I~
l1.0~ - 32
9~2 - 16
7.89 - 24
7.02 - 42
6.7 - 32
5.5 - 26
4.64 - ]00
4.45G - S3
4.3 - 58
3 88 - 2G
3 75 - ~9
3.56 - ~]
3.3] - 2G
3.05 - ~G
~8~
N ~ Interpretation
( H 90 MHz NMR, D2O solution)
2-_ .
N ~OC~,
~ C~2 '
Chemical Shift
(PPm ~ vs. TSP) DescriPtion Inteqral Assiqnment
2.0-2.4 Multiplet 4 14CH2, 14'CH2
3.04 Singlet 3 12CH3
3.3-3.6 Multiplet 5 ~ 2' 13 CH2
3.94 Doublet 1 2CH
4.12 Singlet 3 20CH3
4.12 Doublet 1 llCH
4.8 Doublet 1 llCH
5.42 Doublet 1 6CH
5.88 Doublet 1 7CH
7.21 Singlet 1 18CH
- 20 -
~4~94
.
Stability
Time-Tcmperature ~ I,oss
1 day; 100C - 10.9
3 days; 70C - o
7 days; 70C - 1.9
1 week; 56C - 1 0
2 weeks; 56C - l q
4 wecks; 56C - 0
1 week; q5C - 0
2 weeks; qSC - 1 4
4 weeks; 45C - 0 7
8 weeks; 45C - 1 6
1 month; 37C - 2 5
Elemental Analvsis ~percent bv weiqht)
Found Drv Basis Theory (SesquiPhosPhate)
C 35.44 36.3 ~6.4
H 4.66 4.41 . 4.7
N 12.88 13.2 13.4
H2O 2.29* -monohydrate = 2.8% H2O
H3PO4 23.06 23.6 23.6
*Karl Fischer Method
- 21 -
