CA1098515A - Immunising and anti-infectious adjuvant agents constituted by esters of the n-acetyl-muramyl-l- alanyl-d-isoglutamine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to new water-soluable agents which are effective as immunising adjuvants. These adjuvant agents are the methyl, ethyl or propyl esters of 2-(2-acetamido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-isoglutamine and of 2-(2-aceramido-2-deoxy-3-0-D-glucopyranosyl)-D-propionyl-L-alanyl-D-glutamic acid. They are particularly useful to prepare adjuvant medicinal compositions, used for increasing the efficiency of vaccines.

Description

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:

~,0~ ~

.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 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$) o~ the p-toluene-sulphonate of -the benzyl die~ter of D-glu-tamic acid a~d 1 ml (9 mmole~) o* ~-meth~lmorpholine. ~he reaction mi~ture is left for 12 hours at the ambient temperature. It i~
the~ concentrated to dryness. The dry compound i~ taken up 5 50 ml o~ ethyl aoetate and wa~hed successively with a 10~o solution o~ citric acid, water, with a solution o~ 1~ sodium bicaxbonate7 and ~inally with water. The ethyl acetat~ phase i~
dried ~er MgS0~, filtered and ooncentrated. On crystalli~ing *rom a mlxture o~ ethyl acetate and hexane, 2.50 g (67.5%) are obtained o~ the de~ired product of which the phy~ical conætant~
are:
M~po 105~106C
o~ =+`~.3~
The eleme~tary a~aly~is of thi~ produot iæ:
C27E3407N2 (498~5) a % ~ % N %
oaloulated : 65 609 5~6 ~ou~d ~4!~85 7.0 5.5 b) Pre~aration o-~ the be~z l dies-ber o~ the ~2-benz~
acetamido-4~5-b~nzylidene-2 deo~y-3~0-D-~luco~ranos~
~ro~ionyl-(O~benzyl~ alan~l-D-glutami~ a~id ~B) ____~_ ______.__ _______._____ _ _ __ _______ 500 mg (1 mmole) o~ compound (A) are treated with 5 ml o~
a 1 E ~olutlon o~ h~drochloric acid in ~la¢ial a~etie acid.
~ter ~0 mlnute~, the rea~tio~ mixture i$ 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

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.

, ~ , .

~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 ~ 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.

.

.~ :

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

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 - ' . ,

Claims (8)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A process for the production of a compound of the formula:

in which R1 is -OCnH2n+1, with n = 1, 2 or 3, or -NH2, R2 is -OCpH2p+1, with p = 0, 1, 2 or 3, p being not equal to 0 when R1 is -NH2, which comprises reacting according to the techniques of protein synthesis the 2-acetamido-2-deoxy-3-0-(D-2-propionyl)-D-glucopyranose whose functional groups other than the propionyl are protected whenever necessary with A. in a first stage an L-alanyl derivative whose carboxyl is protected and then in a second stage with a glutanyl derivative of the formula:
H2N-CH(COR1)-CH2-CH2-COR2 in which R1 and R2 are as defined above or form protective groups; or B. with a derivative of the formula:
H2N-CH(CH3)-CO-NH-CH-(COR1)-CH2-CH2-COR2 in which R1 and R2 are as defined above or form protective groups and finally removing the protective groups when same are present.

2. The process of Claim 1, wherein R1 is methoxy and R2 is OH.

3. The process of Claim 1, wherein both R1 and R2 are methoxy.

4. The process of Claim 1, wherein R1 is -NH2 and R2 is methoxy.

5. A compound of the formula:

in which R1 is -OCnH2n+1, with n = 1, 2 or 3, or -NH2, R2 is -OCpH2p+1, with p = 0, 1, 2 or 3, p being not equal to 0 when R1 is -NH2, when prepared by the process defined in Claim 1 or by an obvious chemical equivalent.

6. The compound of Claim 5, wherein R1 is methoxy and R2 is OH, when prepared by the process defined in Claim 2 or by an obvious chemlcal equivalent.

7. The compound of Claim 5, wherein both R1 and R2 are methoxy, when prepared by the process defined in Claim 3 or by an obvious chemical equivalent.

8. The compound of Claim 6, wherein R1 is -NH2 and R2 is methoxy, when prepared by the process defined in Claim 4 or by an obvious chemical equivalent.