Note: Descriptions are shown in the official language in which they were submitted.
The invention relates to ~ater-soluble agents whioh are
e~fective a~ immunological adjuvants ~or promoting immunising
responses, or al~o as anti in*ectious age~ts.
~ he i~ent~on al~o relates to the medioinal compositio~s
which con~a~ these a~ent~ 7 as well as to the prooes~e~ ~or
their p~eparation~
The agent~ according to the inYention are the methyl~
ethyl a~d prop~l mo~o-ester~ in the ~ position, or the meth~l,
ethyl a~d prop~l diesters o~ 2-(2-acetamido-2-deox~-3-0-D
glucopyranosyl)~D pr~pionyl-I_alan~l D~glutamic acid, or al~o
the meth~l9 ethyl a~d propyl e3~er~ of 2-(2 acetamido-2-dsox~
3-O~D-~lucopyranosyl)-D-propionyl~-ala~yl D-iso~lutamine.
~he oompounds correspond to the general formula developed
below:
CE20
~,0~ ~
CH3
.E3C ~ CH - ao MH - I - clo ~ ~H - I ~ co~l
C~3 (I 2~2
~-- CC)R2
: ` -
D
in whi~h R1 is -Oa~H~n+1 or -~H2~ with n = 1, 2 or 3~ R2 i8
25 -OCpH2p~1, with p ~ 0, l~ 2 or 3, p not be~ equal to 0 when
R1 i~ ~H2~
A pxe~er~ed ~roup o~ compound~ accordin~ to the invention
is con~tituted b~ the mono- (in the ~C~position) or dlm~thyl
ester o~ the ~-(2-acetamid~-2-deox~-3-0-D-glucop~ranosyl)-~D~
propio~yl I_alanyl-D~lutamic acid a~d the methyl ester of the
35~
2-(2-acetamLdo-2 deo~y~0-D-glucopyranos~ D-propionyl-~
ala~ D-isoglutam-i~e, i.e~ the compound~ ~or which, in the
general ~ormula indicated above~ R1 i~ -Oa~H2~+1 with n = 1, 2
or 3 a~d p = 0 or 1, or R1 i9 -~E2 and p = 1.
~hese oompound~ wLll ~ubsequentl~ be denoted by the
abbreviatio~s
Mur-~Ac ~ Bla-D-~lu-~-O~H~
Mur-~Ae-~-Ala-D-Glu(OoH3)2
a~d Mur-~Ac~ la D~iso-Gl~(0~E3~0
In srder to pxepare the compound~ a~cording to the inven-
tio~, in a ~ir~t ~tage~ a~ e~ui~ale~t derivati~e o~ the
~ra~ment corre~ponding to the peptide ohain ~nd to that o~ the
~ra~me~t denoted by the abbreviat:L~n Mur~Ac i~ syn~hesised
the ~u~ot~onal grOUpB which mu~t ~lOt react bein~ -~irst prote~t-
e~ the~ ln a ~eeond ~tage, the oouplin~ o~ the~e two
deri~ati~e~ o~ these ~ragms~ts i~ e~Pected~ ~he protecting
group~ are ~inall~ removed, libsratin~ the functlons which
were previousl~ blocked.
It i~ al~o p~ssible to e~eo-t the synthe~i~ o~ these
oompounds by e~fectin~ the separate couplin~ o~ a deri~abive
the Mur-~Ao ~ith a d~rivatl~e ~ the ~-alanin~ then
¢oupling the re~ulting produot w~th the equiv~lent derivabive
o* the gl~tamic aaid or the i~glutami~e, acoordi~g ~o ~he
proce~ ge~erall~ use~ in pepbide ~y~thesis.
~he i~entio~ al~o relates to the medicinal composition~
co~taini~g the ~ald a~erlts and ser~i~g e~p~ci~lly to increa~e
the action o* tha weak immu~isin ~u~t~nce~ or al~o to the
treatme~t o~ i~*ect1ou~ eaæ~sc Mo~ part~¢ularly~ the
lnventlo~ ~oncer~ oompositlo~s co~taini~g the said a~e~t which
oan be u~ed ~or ~he i~un~sation ~r ~or the treatment o~ me~
and warm~blooded animals so as to preve~t or cure bacterial,
viral a~d parasitic in~ections, or to ~i~h~ against variou~
or~aniG ti~æue antigens O:e normal or pathologieal ori~inO
One of the interest~ o~ the new produot~ accordi~g to the
in~e~tion i~ in the ~act that it is not neGe~ry to u~e
partlcular media ~or their admlni~tration on ~hich the
mani~estatlo~ of their ph~macological activit~ would de~end,
especially the adju~ant actlon, and the vehicleæ ~ith wh~oh o~e
$3 lea to a~300iatc them ha~e only the object of ~aoilitating
the u3e 0~ these produot~. In parti¢~lar, it is not neoe~æary9
whe~ the~e products are injectea, to us~ ~or thi3 purpose a
composition oontai~ing an oi~ phaæe.
Further9 the~e compou~d~ wh:ich ma~ be u~ed ~or their
adju~ant or anti-~nfectious actiol~, ~y be admini~tered oral~;y
1~ or pare~terally, a~d e~peoially b~y injeotion~
The i~ention relate3 in particular to medi¢inal adj~nt
composition~ o~ immunit~ containi~g a ~rodu~t o~ the i~ention~
e~pecially Mur-NAc-~ ~la-D-Glu-c~-OoE~, M~r~Ac~ la-D~Glu-
(O~I3)2 or Mur-~Ac~ la-D i~o-~n~0~3)y i~ as~o~iatio~ with
a pharmaceutically ac~eptable ~ehiGl~. aompo~itl~n3 of thi~
type whi¢h are parti¢ularl~ pre~erred are oon~ti~uted b~
~ectab~e ~olutlo~ oontainl~g a~ e~eotiv~ do~e o~ the
produ~t o~ ~he în~e~tion. Steril~ ~olution~ i~ a~ aqueou~9
pre~era~ly i~otonic~ pha~e~ æuoh a~ ~aline i~otonio æolution~
or i~oto~lo ~olution~ treat9d wlth gluco~e, are advanbageou~ly
used ~or thiæ p~rp~e, ~hi~ 1~ o~ oouræe not re~trlo~iv~ a
~i~ple ~olutio~ in dlætillea waber ca~ al~o b~ u~edO
~ he ad~uvan-t medi¢lnal compo~ltiQ~ o~ the in~e~ti~ ma~
al~o be pre~e~ted in v~ri~u~ ~Orm~9 b~ u~ing ~r thi~ purp~e
~ehiole~ ~uitab~e ~or the ~elected method of admini~ration~
l?or example; Gompositions will be us~d iIl the ~orm of cache~s,
compressed ta~lets or gelatine-ooated pill~ or oral admi~i~-
tratio~, ~d ae~osol~ or gels for the applicatiQ~ to mucous
membra~es
The ad~uva~t age~ may al~o be ~ l~ophilised form 30 a~
to permit the eætemporaneou~ preparation of the adjuvant
medicill~.l compositiolls
~ pha~rma~uti~all~ ad~tageo~s form oomp~ises UXlit dose~
o:~ a~out 100 to 800/~ o~ the ad~u~ant produol; ac¢ord~g to
the inYentior;9 ~ pre~erabl~ o~ a~out 400JUg~
~he ~ve:Dtio:~L al~o i~¢lu~e~ medîc~al comp~itio:lls in
whi¢h the produ~ts of the iILT~ tion ~re as~oolated with a
~g a,~ent, e~pe~iall~r a wedlk ~ao~inating a~tige~O
Acc~rdi~g t~ a:nother a~peot~ the in~reIltio:~ al~o ~once~
medioir~al ~ompositio~ contai~g the product~ o:e the i:~ventio~
a:~d e~peciaLLy the M~ A~ la~3~-Glu-K-OG~I~;, the Mur~lo~
Qla-D-Glu(OaE~ 2 or the Mur A~ lla-^D-i~o-Glrl(OCH3~, and
u~e*~l ae anti-i~eotiou~ age~s. ~he~ prod~t~ Eaot" when
they are admix~l~tered alo~e, ~O ~ithout vaoeinati~g compo~i
tion, e~peoiall~ with~t a weak immunising age~t, have ~ho~
bhat they ma~i~e$t anti ~nfectiou~ propertie~ o~ the pre~sn~ive
~r ~ve~ curati:Ye type~ In oth~r word~, th~ anti-i~eotiou~
propertie~ arR f~una whel~ the~e product~ are administerea at
th~ ~ame tims that the contaminatio~ i~ accompl~h~d, or evs:~
~ub~eg~3r~ to thi g ~,
~hese a~ti~ sctious propertie~ are quite une2pected
~ak~g into a~oount that orle knew be~or~ha~d a~ the activit~ o~ -
the compou~de oapabls o~ incr~as~g the resi~ta~oe o~ the hoæ~.
~hus~ ~t i~ k~ow~ that o~e Ca~ ore~e the ~on speci~io
resi~tanee to an in~ectio~ b~ previousl~ in~e~tlng di~erent
i~m~nostimulants o~ bacterial origin, such as certain strai~s
of Cor~nebacterium, M~cobacterla a~d their "cord ~actor''g or
lipopolyosides (IPS) e~tracts o* gram-negati~e bacteriar This
protection i~ only ~a~ested, however, on conditio~ o~ respe~
ing certain intervals o~ time between the administratio~ of
the~e immuno~timula~t agent and the momen~ of the co~tamina-
tion. ~hu~ o~e has bee~ able to ~how e~perimentall~ that the
better pereentage~ o~ survi~al in the ca~e o~ mice i~ected
with Elebsiella are observed when the ad~inistration of the
immu~ostimula~t is e~ected abnut 14 da~s beforehand for the
B~G~ about 7 da~ be~ore Yor the Corynebacteria, 6 to 48 hours
before for the IPS. I~ all these ca~e~, the i~muno~timulatio~
b~ mea~ o~ the~e agents mu~t preo~de the in~eotionO For e~am-
ple, it is well known that, if the I2S i~ i~jected at the ~ame
time a~ the inoculum bacteria or a~ter, it produces a "negative
rea¢tio~'l whieh has a tende~y to diminl~h the resi3tance o~
the ho~t who oa~ ~ucoum~ a~ter the a~mi~i~tratio~ o~ a bacter~al
straiQ o~ even little virul~nce. On the other hand9 whe~
admini~terea ~nder good condition~, ~he~e treatment3 ~timulate
oon~iderably the no~-~peci~ ~m~nitg even with re~ard t~
StraiQ~ ren~ered re~istant to antibloti¢e bg mutation or b~
tr~n~r o~ plasmide~. Eowever, it is di~ ult or even i~pos-
sible to u~e the~e treatments on a~unt o~ secondar~ ef~ect~
observed a~ter the admini~tration~ strong do~e~ o~ aorynebac~
teria or o~ B~ and above all o~ account o* the to~i¢ e~ect
~ to ma~, oY the ~P~ which represent~ the to~io-an~igen o~ the
- gram-negat1ve bacterla~
0~ the baBi9 o~ the ~e3ult3 obtained by mean~ o~ the
adju~ant a~ent~ o~ ba~terial ori~inJ and e~peclall~ the te~t3
made with the ~PS, lt ~as there~ore quite surprising to ~ind
that the above-men-tioned synthetic adjuvants, acoordi~ to the
in~ention9 show in additiorl to -their adjuvant properties used
within the oompass of preventive immunis~ng treatmen-t~9 an anti-
infeotious a¢ti~ity which i$ mani~es-ted in a pre~enti~re, or
even curative way, without them bei~g associated with vacc~ne
antigens. The~e products also ha~e ~o mi-togen acti~ity (absence
OL blastic tra~sformation oP the lymphocytes). ~hey are not
antigenic; in ~act7 they do n~t release any retarded sen~ibili-
ty reac-tion in the oase o~ the guinea-pig previously æensitised
b~ means of the ~reu~d complete adju~ant. ~hey ha~e no hyper-
thermis~n~ action with the rabblt ~or doses m~ch greater than
tho~e ~or whiGh their a~ti-in~eotious action is shown. ~hey are
~egati~e to the ~im~lu~ test and -their injection does not cause
the death Q~ ~uprare~alectomised mice although these axe
rendered e~tremel~ ~en~itive to ~he lethal e~ec~ o~ the
endotoxlns by ~his operatio~. ~he~3e resul~æ ~how that these
compounds are~comple~el~ deprived o~ endotoxic character. ~he~
ha~e the ad~antage o~ be~n~ acti~3 con~idered as ~nti~ e~iou~
agents in the absence o~ an oil~ pha~e 9 whether thc admini~tra-
2~ tlo~ made p~rentera~ r orall~g al l;hou~h the ad~uvant~eu¢~ as bhe IæS ax~ onl~ active administerea parenterally.
An lmpo~tant adYanta~e o~ the anti-in~e~tiou~ use accord-
in to the in~e~ti9n o~ the co~pound~ deno~ed above is the
po~ibility o~ ac~ion against t~e pathogenic germ~ whi~h ha~e
2~ become ~esi~ta~ to antibiotic~ ~ollowing treatm~nts by the
traditional antibiotic method3~
It mu~t al~9 be ~ndicated that the anti-in~ectlou~ curative
act~ity o~ the~e compounds is all the more remarkable and
une~pected ~ince the te~t~ show that tha~ ha~e no bactericidal
or bao~erio~tat~c activit~ $n vitso.
As ~or the compositions intended to promote the im~unising
responses, the medicinal compositions containing the above-
mentioned methyl esters 9 and used in an~i-infectious therapeu-
tiCS9 can take ~ery varied ~orms sinoe th.e properties o~ the
products are shown whe~ the administration is oral or parente-
ral. ~he medici~al compositions in particular could be in the
form o~ e¢table solutions ~especially in the form of isoto-
nic aqueous solutions), drin~able solutions~ cachets, ~elat~
coated pills 9 aeroæols, gels~ etcO
Other characteri.stios o~ the in~ention will appear during
the descrip~ion o~ examples o~ preparation o~ products accord
~n~ to the in~entio~9 as well as tests which re~eal the
pharmacological properties of the~e product~
In the course of this a~count~ th~ abbreviations used ha~e
the ~ollowing meanings:
Mur-~Ac : 2~a~etamids~-2 deo2~-3-0-tD-2-~ropion~
D-glucopyra~o~e
Ala : alanine
Glu : ~lutamic acid
2~ iso~G~ : lso~lutami~e
4,6~0-bæi : 496-0-benzylidè~e
~~bz~ benzyl
BOC : t-butylo~ycarbonyl
O~zl . benzyl e~ter
25 O~u ~ suoci~imlde ester
Bzl : ben~yl ether
~
a) t~but ~ ox~carbon~ alan~l D-~lutami-c OC-ben~l ester ~
4 g (14 mmoles) o* the succinimide ester o~ BOa~ alanine,
prepared in the manner described by ~ Schnabel (Justus
~iebl~'s Ann. Chem. l~, 18~ (1967~, are dissol~ed in 15 ml of
tetrahydrofura~0 ~his solutio~ is added to an a~ueous solution
(25 ml) of 373 g (14 mmoles) o~ the r-benz~l ester o~ D-
glutamiG acid, obtained by ~ollowing the m~thod o~ ~ Guttmannand R.A. ~ois~enas (Helv. Chim. Acta, 41, 1864 ~1958)), and
1,4 ~ (14 mmole~) of potaæsium bicarbonateO After one night,
the pE i3 adju~ted to 8.5 a~d the reaction mixture i~ extracted
with ethyl a¢etate~ ~he a~ueous phase ~ acidified in the cold7
to p~ 3.5 with a 4-~ solution o~ h~drochloric acid, then it i~
e~tra~ted with ethyl aoetate~ ~he organic phase is then washea
with water, dried and ooncentrated~ ~he produot is cr~tallised
: ~rom an ethyl-acetate-petrol ether miæture. 4,77 g o~ produ¢t
are obtained, i.e~ a ~ield o~ 870~%~ It3 physical co~a~t~ are:
1S M~po 68~70C r~J ~ =12D (ml~thanol)
~he elementar~ anal~ gi~e~:
7~2 (408,45~ a ~0 H % ~ %
cal~ulated : 58~81 6g9 6.85
fou~d : 58.7 6~4 6.8
b) t-but~lo~ycarbo~ alan~l D-~lu~amio ~e~meth~l es~er, r_
benz~l e~ter ~II)
____ _____ _ __
408 mg o~ (I) (1 mmole) are solubili~ed in 100 ml o~
anhydrou~ ~ethanol. An ethereal solution o~ diazomethane (about
10 mmole~) is added in 15 minute~ at ooa. After 2 hours at
ordinar~ temperature, the reaotio~ mi~ture i~ co~¢entrated to
dry~e~s and ta~en up in 25 ml o~ ethyl acetate. ~he orga~ic
phase i~ washed su~ces~lvely with a 10~o solution o~ citric acid,
water, a 1 M ~olutio~ of s~dium bic~rbo~ate, and ~ater until a
neutral p~ i~ obtained. ~he ethyl acetate phase is dried over
~V M~S04, ~iltered and conce~trat~d. An uncrgstalli~able oil is
obtai~ed (385 mg, i.eO a yield o~ 91%)o
c) Ihe h~drochloride o~ L_alan~ -D-~lutamio G~-meth~l ester~
~-ben~l est9~ I2
385 ~g o~ (II) (O~91 mmole) are treated with 3 ml o~ an
~ ~olution o~ hydrochloric acid i~ glacial acetio acid ~or 30
mi~ute~ ~he reaction mixture is con¢entrated to dryne~s and
the oil obtained is dried (330 mg, i~e. a yield o~ 10~%).
d) 2~(benz2~-2_acetamido ~,6 Q-benz~idene-~-deo~_3-0- ~-D-
~1UC~ ranos~ D-~ro~ion l-~-alanyl~D~lutamic c~-meth~l
ester~ ~-benæyl e~ter ~I ~
473 mg (1 mmole~ o~ benzyl-2-acetamido-4,6-ben~ylidene-3-
O-(D-¢arbo~.yethyl)-2~de~xy- ~-D-g~.uoopyra~oside, prepared în
the wa~ desoribed by ~lowers and R.W. Jea~Lloz (J~ Org. Chem.
28, 2983 (1963)~9 are di~sol~ed irL 5 ml o~ dim~thylformamide
cooled to -1 soa. 0.11 ml (1 mmole~ of ~-me~h~lmorpholln~ a~d
0.13 ml (1 mmole) o~ isobut~l chlo~ocarbonate are ~uccessively
added~
To thi~ reaGtion mi~ture i~ added a ~olutio~ o~ 330 ~g
: (079 mmole) o~ (III) a~Ld 0.1 ~1 (0.9 mmole~ o~ ~ met~l morpho~
line in 5 ml o~ dimeth~l~ormamid~ previousl~ cvoled to -15C.
A~ter one ni ht at -15~ 1 ml o~ a 2~5 ~ solution o*
pota~ium bicarbonate is a~dedO A~ter 30 minute~, the produ¢t
1~ precipitated by additio~ o~ 40 ml o~ distilled water,
~iltered o~ and driedO 667 mg o~ product are obtained, i~e.
25 a yield o~ 95.5%~ :
e) 2 ~ 2~acetamido 2-deox~ 0-D~luou~ranose?~ ro~io~yl-T~-
ala~yl~ lutamic 3~methyl ester (
305 ~g o~ (IV) (0~39 ~m~le) are h~drogenated ~or 15 hour~
i~ ~olu~ion in 50 ml o~ glacial aceti~ a~id, in the p~e~ence o~
~00 ~g o~ 5% palladium on charcoal. ~ter ~iltration o~ the
1U
catalyst~ then concen-tration to dryness o~ the acetic acid, the
product is precipitated from methanol-acetone-ether, the~
centrifuged. 140 m~ are obtained~ i.e~ a yleld o~ 70~0. ~he
produo~ is purified by chromatography on a column (2 x 10 cm)
*illed with an ion~exchanger resin~ commer¢ialised under the
name AG1g-2 bg the ~IOR~D Company (acetate ~orm). It is eluted
with a 0.2 M solution o~ acetic a¢id~ the intere~ti~g ~ra~tions
are u~ited a~d lyophilised. 117 mg ~re recovered9 iOe. a yield
: of 83.5%~ o~ a produ~t o~' which the rotatory power is rc7 ~ :
- ~39 (methanol)~ ~he product is ~inally obtained a~ter
pas~age o~er a colum~ (2 ~ 80 cm)~ ~illed with ion-e~han~er
co~mercialised under the ~ame o~ ~PHADEX G.15 by PHARMACI~
UPSAIA (elution with aceti¢ acid 0.~ M) a~d lyophilisatlon o~
th~ ~raotlo~ o~ interest. A~ the end 94 ~ o~ produ~t (V) are
obtained9 i,e~ a ~ield o~ 80~ ~he rotabory power remain~ at
~7 2~ = ~393 ~methanol~9 and the eleme~tary a~alyeie thereo~
i~:
2~3312N3~ 1H~0 (5~5-50) ~ % ~ ~0 ~ %
calculated : 45~7 6.7 7~99
found 4~993 6~2 7.91
a ~ir~t ~t~ge~ the Mur-~AG-~-Ala~D Glu i~ prepared in
the ~ollowin~ way:
a~ Pre~ar~tion o* the be~l diester o-~ th~ ~Oa-~-alan~l-D-
___ __________________ _____~____~___________ _ _~ ____
~lutamiG a¢id ~
_____________ _
2.3 g ~8 mmolee) o~ sucoinimide ester o~ t-butyloxy-
carbonyl-~-alanine, the ami.ne ~unction o~ whieh i~ protected by
the t-butylo~yca~bon~:L group (~OC~ la-O~u~, are aQded ~ith
~tirring to a eolution in dimethyl~ormamide o~ 415 g ~9 m~ole$)
1 1
o~ the p-toluene-sulphonate of -the benzyl die~ter of D-glu-tamic
acid a~d 1 ml (9 mmole~) o* ~-meth~lmorpholine. ~he reaction
mi~ture is left for 12 hours at the ambient temperature. It i~
the~ concentrated to dryness. The dry compound i~ taken up
5 50 ml o~ ethyl aoetate and wa~hed successively with a 10~o
solution o~ citric acid, water, with a solution o~ 1~ sodium
bicaxbonate7 and ~inally with water. The ethyl acetat~ phase i~
dried ~er MgS0~, filtered and ooncentrated. On crystalli~ing
*rom a mlxture o~ ethyl acetate and hexane, 2.50 g (67.5%) are
obtained o~ the de~ired product of which the phy~ical conætant~
are:
M~po 105~106C
o~ =+`~.3~
The eleme~tary a~aly~is of thi~ produot iæ:
C27E3407N2 (498~5) a % ~ % N %
oaloulated : 65 609 5~6
~ou~d ~4!~85 7.0 5.5
b) Pre~aration o-~ the be~z l dies-ber o~ the ~2-benz~
acetamido-4~5-b~nzylidene-2 deo~y-3~0-D-~luco~ranos~
~ro~ionyl-(O~benzyl~ alan~l-D-glutami~ a~id ~B)
____~_ ______.__ _______._____ _ _ __ _______
500 mg (1 mmole) o~ compound (A) are treated with 5 ml o~
a 1 E ~olutlon o~ h~drochloric acid in ~la¢ial a~etie acid.
~ter ~0 mlnute~, the rea~tio~ mixture i$ oonoen~rated to dry-
ne~ he oil obtai~ed i~ take~ up i~ 25 ml o-~ an acetonitrile
25 di~eth~l~or~amide mixture (2/1, ~/~ ~ ~he mi~ture i~ cooled to
0~ a~d 0.141 ml (1 mmole) oi triethylamin~ i~ added. The
~olution prepared i~ po~red with stirring at 0~ i~to a
~u~pen~io~ prepared 105 hour~ before and ~ormed ~rom 472 m~ (1
mmole) of ben~yl-2 acetamido-4,6-O~benz~lidene~3-0-(D~1
carbo~ethyl)-2 deo~y~ D-~lucopyrarloside and 0.141 ml (1mmole)
~8~
of -triethylamine in 25 ml of the acetonitrile-d.imethylformamide
mixture (2~ /v).
The mixture is left for 12 hour~ at the ambient tempera-
ture; lt is the~ concentrated and the residue i~ precipitated
i~ a 10% solutio~ o~ citric aci~. ~he precipitate is filtered
of~, wa~hed copiou~ly with water and dri~dO 800 mg (9~%) are
obtained o~ -the desired product the constants o~ which are:
M~p. 1~8-199oa
cr ~ - 4.92 (dimethyl~ormamide)
After recry~tall~sation ~rom ethanol, the melting point
is established at 220~C.
~he elementary analysis of thi~ product is~
C47H53012~3 (851.96) a % H % ~ %
calculated : 66.26 6.27 4.93
found : 66.34 6.~5 4.92
c) Pre~aratio~ o~ the 2 (2-a~etam~;do-2-deo ~3-0-D~luco~a-
no~ D ~ro~ion~ alan~ lutamic acid 5a) or ~Iur-~Ac~
__~
la~D-Glu
700 mg (0~8 mmole) o~ the compound (~) are breated with
20 40 ml of a 60% solution o~ aceti~ acid on a boilin~ water-~a~,h
for 1 hour. ~he reaotion mixture is then concentrated to dry-
~ess (the~ dried over M~S04). ~he residue i3 taken up i~ 1 ml
of a chloro~orm-methanol mixture (3/3, v/v) a~d placed on a
siliGa column (35 g) pre~iously equili~rated with the ~ame
~5 ~olvent mlxture. The fractions containi~g the produot are
oolleoted and concentratea to dryness ~their ho~ogeneit~ is
tested by chromatography on a thi~ layer of sili~a gel in the
same mixture of solve~ts). 185 ~g (30~) o~ deri~ative are
obtai~ed~
76 mg~ o~ this derivati~e are dissolved in 15 ml of glacial
~cetic acid; then subjeoted to a hydro~enation in the presence
of 5~ palladium on chareoalO After filtration~ the mixture is
concentrated to dryness and precipi-tated in a methanol-acetone-
ether mixture. 45 mg (92%) of the desired product are thus
obtained 9 of whi.ch the constants are:
M.p~ 150-155C
oc~ = +~30 (glacial acetic aoid)
~he elementary a~alysi~ of this product is:
C19H311 ~31X2o (511.48) a % H % ~ %
calcu:La$ed : 44.6 8.2 6.5
found : 44~7 8.1 6.4
In a seco~d stage~ the previousl~ prepared Mur-~c-I~LLa-
D-Glu s esteri~iedO
100 m~ (0.2 ~mole) o~ Mur-~o-~-LLa~D-~lu are dissol~ed in
10 ml o~ absolute methanol. In 15 ~inutes, 10 ~L of an ethereal
solut1on o~ diazomethane ~about 0,,7 mm~l~ per ~L) are added.
A~ter 90 minutes~ a drop of aoetio acid is added a~d the
~ reactiQn mixt~re is concentrat~d -to dryness. The residue
; obtained is puri~ied on a column o~ silioa gel (1 x 16 cm),
with -for solvent the mlxture chloroform~methanol (6/2, v/v)0
The pure ~raction3 are united and c~neentrateaO ~he pro~u~ is
: ~recipltated from a meth~nol-aceto~e-ether mixture. 83 ~g ~
produ~t (yield 80~o) are obtained~ o~ which the constant~ are:
: M.p. 137~142C
~ 31~6 (~lacial acetio acid)
~he elementary analysis is as ~ollow~:
C21~351~3 (521-53) C ~0 H ~0 ~ ~0
caloulate~ : 48.36 6.76 8.57
~ound 4800 7.0 8.2
30 ~!SYl~ ~o ~L
14
For the preparation o~ this product, the Mur-~Ac-~-Ala~D-
iso-Gln obtained in the way described in the ~rench Pate~t
~pplication ~oO 74 22909 is u~ed~
100 mg (0~2 mmole) o~ Mur-~c~ la-D-iso-Gln are treated
i~ the wa~ previou~l~ de~cribed for the esterificatio~ o~ the
Mur-~Ac ~-Ala~D Glu. The purificatio~ 1~ ef~ected by mean~ of
the chlor~fo~-methanol mixture (5/5, v/v)~ 80 mg o~ produ~t
are obtai~ed (80 % of yield) o~ whi~h the co~tants are:
M~po ~01~
25 = ~4402 (glaolal acetic acid)
~he elementary a~alysls of thi~ produ~t i~:
a2~34011~4 (506.52) a % H ~o ~ ~
caloulated : 47~52 6.76 11~06
foun~ . 47 6.5 lOo~
GO~ ~ pRG7~rll~s
1) ~oxi¢i~
___ __ _
~ he to:gioit~ o~ the proaucts a~cording to the inVe:~tion
ha~ b~en ~tudied b~ parenteral admi:~istra~lon t~ e a:~d
:~ 20 ~abb~ tB ~ It was ~ou~d that the to~ic dose5 are of an order of
magnitude much greater than that o~ the do~es at whl¢h these
produots shb~ their activity~ ~husg the~e produo~ ar~ well
tolerated by mioe at dose~ equal to or greater than 10~ mg/k~
o~ animal9 a~d by rabbits at doses e~ual to or greater than
25 5 mg/kg o~ animalO
2) ~dluvant character of the Mur~o~ la-D-Glu-OC-OaH L ~
the Mur-:~Ac-~ Ala-D-~lu(OCH ~ d o~ the ~ A¢- ~-Ala~
~ 3 2
iso~ ,O~E l ~ a~ouæ ~ha~s
In the ~eries o~ tests of which the re~ults are indicated
here~ter, the i~luenca o~ the aotive principle aocordlng to
.. ..
. .
the invention on the proportion of the anti-album~n antibody
has been studied under the follo~ing condition~.
Grou~s o~ 8 ~Wi9S mice aged two months receive by subcu-
taneous in~ection (~C) or orally (PO) 0~5 mg of ~ntigen
constituted by the albumin o~ bovine serum (~S~) with or
~ithout the su~stanoe tested in an isotonic saline solution.
~hi~ large dose of a~tige~, sinoe i.t is situated at the limit
o~ tha paralysi~g dose with respect to the immunisi~g respon~e7
on a~oount of this ~act has a weak respon~e or no response to
the antig~n alone in the case of the controls; it there~ore
constitute~ a se~ere criteri~n *o~ ~howin~ th~ activity o~ an
ad~u~ant sub~tanceO ~hirty d~y~ later~ the mice receive, by the
~ame method of administration, a ~urther dose containing Ool mg
o~ the same a~tigen.
~he proportion o~ antibody is determined by pas~ive
hemag~lutination by usi~g the red blood corpusoles o~ ~heep
treat~d with ~or~alin ~nd recover~sd ~rom the a~ti~e~ ~tudied
; according to the method de~ribed by A~o ~irata ~n~ M~Wo
B~and~s tJ. Imm~nol~, ~Q~, 641-648~ 1968~. The taki~g o* the
20 blo~d oocurred 149 28, 34 ~nd 36 day~ a~ter the ~irs~ inj~otio~
By wa~ o~ compari~on7 mice receiv~ instead of the produ~t
accordin~ to the invention~ elther~lipop~ a¢ch~ride~ (IP~)
(oxtra~t o~ ~L~E}~ di~ by the wa~er~phenol method)~ ~r the
adj~ant denoted by th~ name '1WS~" a~d described ~ ~dam e~ al.
25 ~ n~eo~ Immuno (1973) 7~ 855-86 ~ ~ ~he oontrol m1ce onl~
receive the antigen~
~he results o~ the~e te~t~ are gi~en i~ the ~ollowing
~a~le. ~he proportion~ o~ antiboay expre~ the maximum ~erum
dilution which a~gluti~ate~ a give~ tity o~ red blood
corpu~¢lea o* ~heep.
16
, ~ , .
~able l
! i~dmin-i Prop~rtion of a~tibo~y !
~ li3tra ~ ~ I
5 i ; ; da~ j day j d~ i day ;
! i i i i ! i
: ! B~ co~trol~ I ~.C. ! ~ 3 ! 3 ! 3 ! 20 !
I BS~ ~ IæS (100/ug) 1 ~0~. s ~ 3 ! 6 ! 50 !1310 !
: 3 ~SA ~ WS~(300~ug) ! ~Oa~ I ~ 3 I c 3 1 ~ 3 ! 6 !
S ! 7
113 1 I I ! I ! !
:! BS~ ~ Mur-~Ac;~ la~D-Gl~- ! ! ! ! I I
! cc-OoE3 (t~0/ug) 1 ~.C~ ! 12 ! 12 ! 200 ' 400 !
! ! I ! I ~ !
!:BSA ~ ~ur-~Ac~ la-D-alu- ! :! ! 7
;! o~Oo~3 (10/ug) ! ~a. 1 6 ~6 ! 200 ! 400 !
ux-~e-I~Ala-D-~lu~ i ! !! ! :!:
0~0 ~ : t2000/ug) ~ PØ ~ 6 ~6 ~ 50 ' 200 !
~A ~ Mur-~Ac-~-Ala D-i~o~ I ! ! ! I :
:~ ~ln(OoH33: tlOO/u~ C~ ! 12 } :12 1 100 ! 800 !
A:~ ~ur-~c~ D-Glu~
~'~ ~: ': : ' =
:
Dose~ BSA 0.5~mg/a~imal
hese re3ult~ how ~hat the~produ~t~ of the~in~ntio~,
admini~ered in~isotoni~ ~aline~eolu~lon, cau~ a~ e i~area~
e~o~ ha ~roportion o~ a~tibod~ ~rmed, e~en in~he ~a~e
~:~ wher~ the adjuvant is o~ally.ad~ni~ered.:::
~he acti~ prin~iples aoo~rding t~ the:i~nti~ en e~der
re~pon~e~ whioh may ~e co~sidered in a gen~ral w~y as
comparable to those that are obtai~ed~with the I~LpgllJ but it
must be remmr~dd that, ~o~rary to the latter~ they have no
t~x~cit~. : .
~0 ~) Ad~uva~t character o~ ~he Mur-~Ac;~-Ala-D-Glu~ ~-oo~ ~ o~
,
the Mur~:WAc=L-Ala-D~Glu(OCE ) a~d of the Mur-~c~-Ala-D-iso--
G~n(OCH ~. in the ;~resence o~ a~ oil~ ;2hase
In these test;s, the increa~e o~ the propoxtion o:E antibody
3peei~ic to æ gi~en anti,~en is followed whsn the latter i~
5 injected, with or without the ad juvant compouna acoordi~ to
the in~Tention, in a water-i~-oil emulsion.
The tests are e~ected on batches o~ 6 Hartley guinea-
pi~s, ~emale~, of 350 g. ~he adm~i~tration ls made b~ intra-
dermal inje~tion in the planter paa 0~ each o~ the hind ~eet~
10 ~he o~albumin (constituting the antigen) at the rate o~ 1 mg or
0.5 mg is ~epared in 0.1 ml o:E a}l e~ ion o~ ~aline isotonio
~oll;Ltion, in an oily p~aqe ~on~tituted either by the :Fre~uld
incomplete ad~uvant (~IA) or b~ the oomplete adiu~ant (~a~)
~o~med by the ~IA to which i~ ad~ed Ool mg o~ entire cell~ of
M~cobacterium ~megmatis. The compound a~¢ordi~g to the in~en-
tio~ is admi~is~ered at the rate o~ Ool mg ~ontained in the
emulsion containin~ the ~IA~
~ ighteen day~ a-~ter thi~ immunisation~ ~e loo~s ~or
po~sible ~eactio~ o~ retarded hyper~ensltivity in the a~tige~
on injecting indradermally 0.01 mg or 5/u~ o~ ovalbumln on ~he
side o~ the aaimal~5 and 48 hours a~tex, the reaotio~ at the
point ffl i~jeotiGn ls obserqed~ ~he diameter i~ mm o~ the
reaotio~ thus oaused i~ mea3ured.
Twenty-one days a~ter the injectio~, the ani~als are bled.
~5 On th~ ~e~um colleoted l~ measurea the content O~ a~tibody
~pe~i~ic to the ovalb~mi~ by precipitation o~ the complex
a~tibod~antigen i~ the ~one of equivalence. The quantit~ o~
protein nitrogen contained in ~hi~ precipitate i~ evaluated
aooordlng to the method o~ ~oli~O ~he a~erage value~ o~ the
content~ o~ a~tibody are i~dicated in the ~able of re~ult~.
1~
These values e~pre~ the quantlty~ in microgrammes, o~ nitrogen
which can be preoipitated by the anti~en; per ml o~ ~erum. In
~ome cases, the antibody level ha~ been de-termined too b~
pa~ive hemagglu-tination (PHA) a~ abo~e indioated.
The re~ult~ o~ the~e test~ are reported in the ~ollowing
~able 20
~able 2
i ISerum anti~ody7
I Compositio~ of the emulsi.o~ I ~ ; Cutaneous
oontaining tha antige~ ; pi~a~ a ; e~ ;
; tio~ ; tion j(di~meter in mm);
i--i~i ,,
! ~valbumin (1 mg) ~ PI~ ! ~ 500 ! - ! 0 7
t I I !
l~~ ~~~~~~~~~ ~ ~ ~ i i . !
I ovalbumi~ (1 mg) ~ ~CA ~100/ug) ! 3aoo ! - ! 10 ~ 1.5
I ~ ! ! t
! ova~bumin t1 mg) + ~IA ~ Mur~ i 2600 ~ ! 6 ~ 3
! ~o ~-Ala~ lu~ OCH3(100/ug) !
! ovalbumin t0~5 mg) -~ FI~! ~ 500 ! 900 ! 0
!
o~al~umin ~0.5 mg) ~ A tlOO/ug)! 2100 !3600 ! 13.5
t
1, ovalbumin (0.5 mg) ~ ~qA ~ Mur~ 10OO I 1~ !
~J~c ~-Ala-D~lso~(OCX~ ) ( 100 /ug)lI ~ ! I
i
! ovalbuml~ (0~5 m~ IA + Mur~ !950 1 ~60~ ! 6 2
NA¢-~-Ala-D-Glu.( OaEI3 ) 2 ~1 00/~ )
~hese re~ult~ show that bhe compou~d9 o~ the invention,
admini~tered in an oily emul~i~n, ha~e an in*luence on the
proportio~ o* antibod;y ~ormed in respon~e to the injectio~ o~
anti~e~, and that ib induce~ a reactio~ o~ retarded hgper~e~-
~itivity with re~pect to the ~ame anti~e~
4) Anti-in~eotious character
19
~ he following tests illustrate the anti-infectious
properties of the Mur~NAc~ Ala-D-~lu-Cr~OCH3 t of the Mur~c~
~Ala-D-~lu(OoH~)2 and of the Mur-NAc~-Ala-D-i~o-Gln(OCE3~.
In the prellminar~ tests, an e~perimental method wa3
establi~hed permitting the anti-in-~eotious charac~e~ of the
product~ to be.shown~ It has thu~ been sho~ that a dose o~
104 Klebsie~1a pneumoniaep inj~cted intramuscularly in mice,
produced the pro~ressive decease of a large par-t~ if not the
whole7 of the animals in the week ~ollowin~ ~he i~oculation~
~ter eight days 9 the survival of the animal~ is ~inally
ascertained.
~ he ~l~rvi~al o~ ~roups of miG~ inoculated under the
Gondition~ indicat~d abo~a a~d treated b~ mea~ of ths methyl
e~ter considered was follo~ed.
~y way o~ compari~on; batche~3 o~ mice ha~e been treated
with BCG and ~PSO '~his latterg as is known, is a~ e~tremel~
aotive lmmun~timuIant when it is admini~tered 24 houræ be~ore
the in~ection~
~or the~e test~? hgbrid mioe (as7~1/6 ~ AER)~19 rearad at
the P~T~UR I~TI~UTE9 ~rom ~trains~ooming from the breedin~
the G~R.S, at Orlea~s 9 were u~edc '~he endotoxin or I2S ~a~
extracted by the phenol~water me~hod *rom Salmonella e~teri*idi~
~riat~ Dan~s~ ~o. 5629,PA~'~EUR I~S~ U~E). ~he B~G comes ~rom
the ~raln Myoobaoterium tuberoulo~is v~, bo~ o. 1173 P2
25 OI the PAS'~:~UR INS'~Ir~U'~9), oultivated on Sau~on me~ium a~d kill-
ed by a ~olution o~ 2~ ~f phenol.
'~he ir~eotion by ~ Leb~iella p~eumoniae, 3train OI cap~ular
type. 2, biot~pe d, is made ~rom a culture o~ 16 hours in a
medium Ior pneumoco~cu~ (~o~ 53515" PA~llR INS~ U'~)" ~he
~C) preparation~ injected be~ore or at the moment ~ the in~ection
are always diluted in apyrogen.ic ph~siological solution, at
the rate of 0.2 ml ~or parenteral administration a~d On5 ml -for
oral administratlon, the controls receivi~g the solution alone~
In the tests of which the results are reported in the
~ables 3 and 4~ the influence of the treatment by ~arying the
methods, the doses and the time o~ administration of the
produots studied has be~n determi~ed. ~he per~entage of pro- ,,
teotion e~p,resseæ the difference of the percenta~e~ of
survivor3 in the group of treated animals with respect to the
¢orrespond~ cont~ol group.-
The re~ult~ show that the products studied ha~e an anti-
infectiou~ ac-tivity~ ~hether they are administe,red paren~er~
or ora~1~. On the co~trary, the ~P~ ~s inacti~e whe~ give~
orally, even for bhe very lar~e ao~e~ (1 oo/ug ~ T.PS represen-t
100QO time~ the an~i-infeotious do~e taken parenterally).
X~ additlon, the ~esul~ are the same if the products are
adminl~tered 24 hour~ or only 1 hour before the i~feotan~
injeot~o~, admi~i~tered intram~cularly.
.
.~ :
21
5~
Table 3
~nti-infe~tlou~ prot~ction with respect to an intramus~ular
i~oculation o~ 104 K.pneumoniae
~reatment 24 hour~ be~ore th~ in~eotio~
~o ~ 7j~umber ~ ~ercPanimalsl -
; 0~ ; ; o~ jsurvivingon dayi ~r~ ;
jtreat-; ;a~ ; 3 ~ 5 1 8 itePctioni
; me~t i ;~atedi
! ! Control! 24 ! 1~ ! 8 ! 2 !
PS1/ug ~. 24 t 24 ~ 22 , 22 ~ 8
i ~ Controli 24 ! 11 ! 9 i 6 i
! ! ~aG 100/ug:! 24 ! 24 ! 24 ! 21 ! 63
t 7
i i i i I ! ! !
: ! ! Control ! 24 t 15 l 11 ~ 7 !
i Mur-~A~-I-Ala-D-Glu~
i ! or~a~ jwg i 24 i 24 j ~4 j 23 i 67
i i ~ontrol~ i 24 i 13 j 9 i 4 ~ 7
: ~ I: ! Mur~A¢-L ~la;D-Gln-: ! I ! I ! !
tI ~H2 100 /Ug ! 24 ! 21 ! 13 ! 11 1 29
! - i ! ! ! ! ! t
~ tI aO~trOl ! 24 ! 1~ 1 9 ! 4:i
M~-NAo-I-Ala-D~Glu~
20 ~ (ocH3)2 ~ 10 ~ ! 24 ! 2i ;l 19 7 15 i 46
oDtrol : !~ 12~ ~ 6~ 4 , 2 , ~!
Mur-N~c-:L-A1a-D-~lu ! I ! ~ 7 ! ~ !
: I ! ~-~C~3 2000,ug~! 12 ! 11 ~ 9 ! 6 ! 40~5 1
~ ~~i ! I ' ' 7
! aontrol: ~ 6 1 5 ! 2 ~ : !
lper 0~ Mu~A¢~ a ~Gl
i ~ 2 /ug i12~ i 10 i 9 ! 8 i 5~ ~
! ! a0n~rol i24 ~i 14 , 10 ! 7 ~ 7
! ! IæS : lOOjug ! 2~ ! 1C ! 8 !
.
~2
s~s
~able 4
Anti~in~ectious protection with respec~ to an intramusGular
inoeulation o~ 104 K.pneumoniae
~reatment 1 hour after the infection
jMethod; ;~umber ~ bero~ a~imalæ- ~ 0~ -
; Of ; ; o~ jsur~ib~g on day! ~ro- i
~treat i ~trea~edi 3 ~; 5 1; 8 itecti~ni
i i i i i i i !
! ! Control t 16 ! 6 ! 2 ! 1 !
; i Ml~r NAc-~-Ala-D-Glu~
i i c~-Oo~3 100/Ug i 16 i 16 i 16 ! 14 1 ~ i
! I.V. ~ G0ntrol ~ 8 ~ 6 1 5
: ~ ~ Mur--N~c~ la D-Glu
i i ( oaH3 3210 ~ u~ ! 8 1 8 !8 !8 ! 88
J
~ ! ! i ! ~ I I
! ! C~ntrol ~ 16 ! 10 !6 !1 !
: 15 ~ ! ~aG 100/u~ ~ 16 ~ 14 ~ 13 1 10 ~ 50
- .
In anobher æerieæ of te~ts, the ~nti-in~ectiou~ properties
~; o~ the produ¢t~ were ~tudied with respect to a ver~ ~iolent
infection Gaused by the intravenou~ in~ection (and not intra
~ .
mus~ular) o~ tO~ K.pneumoniae~ :
IY1 these te~ts, al~o eff'eobe~ o~ mioe" the treatme~t~ axe
e~fected 24 hours before the inoculation. ~he~ methods a~d the
doses are indicated i~ ~able 5 i~L which the r esults o~ these
tests a~e reportedO
23
s~
Table 5
~nti-imectious protectio~ with re~pect to an intravenous
i n~culatio~ o* 103 l~ Op~eumoniae
Treatment 24 hour~ be~ore the ~ ec-tion
M~thod ~ l~umber'~m~er ~ anim?1
~f ~SU~ving al day~ % ~
treat~ pr~-
mant j ; t~aated~ 3 ~ 5 ~ 8 ~tection ~
! Mur-~Ac-~Ala-D-Glu- ! ! I ! I !
I cV., ~ oC OaH 100 ~u~ ! 16 ! 16 ! 16 ! 1 2 ! 65
3 / ! ~ ! ! ! !
10 ~ ! M~ Ac~ Ala-D-Glu- I ~ ! ! I !
IoV~ I (Cl(~ );~!1Ç)0/u~ ! 16 ! 1~ ! 12 1 1Q I52~,5 !
3 ~ ! ! ! ! ! !
II~Vo I ICPS 1 ~ug 1 16 9 16 ! 16 ! 16 3 90
To ! ~G 100~u~ 1 16 ! 16 ! 16 ~ 16 ! 90
!per~ o~! :BC~ 2000/~ ! 16 ! 3 ! 0 !
: :
The re~ult7 ~hGW a ~ignifica~t proteot~ o~ the ca~e o~
mi~e trea;Sed b~ mea;n~ of the pr~du¢t~ according to the
ve~tio~
hu~ ing ~o the invention, there i~ pr~ided a new
0 adjuvant ags~;t OI im~nity soluble ~ l,1ater a~d active even in
the~bsenoe ~f~ oil~ pha~, as Well as~ age~ts ~or the :
tréatmen~i; o~ eGtious disease~,~ which ha~e no to:~:ioit~ and
.
ca~ ~e ad~inis~ered pare~terally or orally and ara active even
~or the antibi~tiG~-resista;nt pathogeni~ genes.
~:
.
~4
- ' . ,
