Note: Descriptions are shown in the official language in which they were submitted.
3~2~
4-11611/1-3/-~/s
Canada
Processes for the manufacture of novel glucose derivatives
The invention relates to processes for the manu~
facture of N-alkylated muramyl peptides and derivatives
thereof~ which can be used for the manufacture of pharma-
ceutical preparations and in a method for the therapeutic
treatment of the human or animal body.
The invention relates especially to a process for
the manufacture of N-alkylated muramyl peptides of the
formula I
2 R6
~ ~-0
~ 7 ~I)
R3~CH (D) N-X-R2
\ 13
R8 110 IR11
CoN-cH-coN-cH-cH2cH-R12
R7 (L) Rg (D)
~335~
-- 2
in which
X represents a carbonyl group,
R represents hydrogen, alkyl, optionally
substituted benzyl or acyl,
R represents optionally substituted alkyl or
carbocyclic aryl,
R and R represent, independently of each other,
hydrogen, alkyl, optionally substituted
benzyl or acyl,
R represents hydrogen or alkyl,
at least one of the radicals
R7, R and R represents lower alkyl, especially
methyl,
and the others represent hydrogen,
R represents hydrogen, lower alkyl, free,
esterified or etherified hydroxy-lower alkyl,
free, esterified or etherified mercapto-lower
alkyl, free or acylated amino-lower alkyl,
cycloalkyl having 5 or 6 carbon atoms, cyclo-
alkyl-lower alkyl in which the cycloalkyl
radical contains 5 or 6 carbon atoms,
optionally substituted aryl or aralkyl,
nitrogen-containing heterocyclyl or hetero-
cyclyl-lower alkyl,
R and R together also represent alkylene having 3 or 4
carbon atoms,
and the radicals
10~ Rll and R12 represent, independently of one
another, an optionally esterified or amidated
carboxy radical and
Rll also represents hydrogen,
salts of such compounds having salt-forming groups, pro-
cesses for the manufacture of these compounds, pharmaceuti-
cal agents containing such compounds and their use as phar-
macologically active compounds.
The configuration of compounds in which R3, R
~L~83~2~
3 ~
and/or R are different from hydrogen is indicated in
formula I by ~D) and (L), respectively, at the respective
centres of asymmetry.
The term "muramyl peptide" represents, in the strict
sense, only compo~nds derived from muramic acid in which
R is methyl. I~owever, unless expressly indicated to
the contrary in the context, in this Application the term
"muramyl peptides" ls applied also to compounds in which
R represents hydrogen or an alkyl radical other than
methyl and which, in the strict sense, should be termed
normuramyl and ho~omuramyl peptide derivatives, respec-
tively.
Alkyl is straight-chained or branched, having up to
18 carbon atoms and bonded in any position, but is
especially lower alkyl.
As substituents of the optionally substituted alkyl
group there come into consideration - especially free
or functionally modified hydroxy or mercapto groups, such
as etherified or esterified hydroxy or mercapto groups,
for-example lower alkoxy or lower alkylmercapto groups,
or halogen atoms or free or functionally modified carboxyl
groups, such as lower alkoxycarbonyl or carbamoyl groups.
The substituted alkyl radical, such as the lower alkyl
radical, can contain one, two or more identical or different
substituents, especially free hydroxy groups or halogen
atoms.
Carbocyclic aryl radicals are especially monocyclic,
but also bicyclic, aryl radicals, especially phenyl, but
also naphthyl. They may optionally be mono-, di- or poly-
substituted, for example by lower alkyl groups, free,
etherified or esterified hydroxy, for example lower alkoxy
or lower alkylenedioxy, or halogen atoms, and/or trifluoro--
methyl groups.
Aralkyl is especially aryl-lower alkyl, in which aryl
has the meaning given above. Aryl-lower alkyl represents
especially benzyl or phenethyl, in which the phenyl nucleus
3S;~
may be mono-, di- or poly-substituted.
Optionally substituted benzyl radicals are especially
those benzyl radicals in which the aromatic nucleus is
optionally mono-, di- or poly-substituted, for example by
lower alkyl, free, etherified or esterified hydroxy or
mercapto groups, for example lower alkoxy, lower alkylene-
dioxy or lower alkylmercapto groups, or trifluoromethyl
groups and/or halogen atoms.
Nitrogen-containing heterocyclyl is especially the
radical of a 5- or 6-membered heterocyclic compound con-
taining one or two nitrogen atoms in the ring. It can be
unsaturated or saturated and, for example, contain a ~used-
on phenyl radical. Examples that may be mentioned are the
pyrrole, indane, pyridyl or imidazole ring.
An optionally esterified or amidated carboxyl group
is especially the carboxyl group itself or a carboxyl group
esterified by a lower alkanol, or a carbamoyl group which,
at the nitrogen atom, is unsubstituted or mono- or di-
substituted by alkyl, especially lower alkyl, aryl, espe-
cially phenyl, or aralkyl, such as benzyl. The carbamoyl
group can, however~ also carry an alkylene radical, such
as the tetramethylene or pentamethylene radical. A carb-
amoyl group R can also be substituted at the nitrogen
atom by the carbamoy:Lmethyl group.
Acyl is especially an acyl radical of an organic acid,
especially an organic carboxylic acid. ~hus, acyL is espe-
cially alkanoyl, especially having from 2 to 18 carbon
atoms, but more especially lower alkanoyl, or alternatively
aroyl, such as naphthoyl-l, naphthoyl-2 and especially
benzoyl, or benzoyl or naphthoyl substituted by halogen,
lower alkyl, lower alkoxy, trifluoromethyl, hydroxy or lower
alkanoyloxy, or alternatively an acyl radical of an organic
sulphonic acid, for example an alkanesulphonic acid, espe-
cially a lower alkanesulphonic acid, or an arylsulphonic
acid, especially a phenylsulphonic acid, such as benzene-
sulphonic acid or p-toluenesulphonic acid, optionally sub-
35;~l~
-- 5
stituted by lower alkyl or halogen, and also carbamoyl,for example unsubstituted carbamoyl, lower alkylcarbamoyl
or arylcarbamoyl, such as rnethyl- or phenyl-carbamoyl.
Esterified or etherified hy~roxy is especially lower
alkoxy or lower acyloxy, such as lower alkanoyloxy.
Esterified or etherifled mercapto is especially lower
alkylmercapto or lower acylmercapto, such as lower alkanoyl-
mercapto.
Acylated amino is especially lower alkanoylamino or
carbamoylamino.
The radicals and compounds referred to as "lower" in
the context of the present description and the patent claims
contain preferably up to and including 7, and especially
up to and including 4, carbon atoms.
Hereinbefore and hereinafter the general terms can
have the following meanings:
Lower alkyl is, for example, n-propyl, n-butyl, iso-
butyl, sec.-butyl or tert.-butyl, also n-pentyl, n-hexyl,
isohexyl or n-heptyl and, especiaIly, methyl or ethyl.
In aryl-, cycloalkyl- or heterocyclyl-lower alkyl, the lower
alkyl radical is especially methyl or ethyl, the aryl,
cycloalkyl or heterocyclyl radical having the meanings given
above.
Lower alkoxy is, for example, n-propoxy, n-butoxy,
isobutoxy, sec.-butoxy or tert.-butoxy and, especially,
methoxy or ethoxy.
Lower alkylmercapto is, for example, n-propyl-,
n-butyl-, isobutyl-, sec.-butyl~ or tert.-butylmercapto
and, especially, methylmercapto or ethylmercapto.
Lower alkylenedioxy is especially methylenedioxy,
ethylenedioxy or propylenedioxy.
Halogen represents fluorine or bromine, but preferably
represents chlorine.
Lower alkanoyl is especially propionyl or butyryl,
but more especially acetyl.
The novel compounds of the present invention may be
~L835~
-- 6
in the form oE mi~tures oE isomers or in the form of pure
isomers.
Special mention should be made of compounds of the
formula I in which R , R and R represent hydrogen,
X represents carbonyl, R represents lower alkyl option-
ally substituted by hydroxy or by methoxy, or phenyl option-
ally substituted by hydroxy, methoxy, methyl, ethyl or by
halogen, R and R represent hydrogen or methyl, R
and R represent lower alkyl or hydrogen, with the
proviso that at least one of the radicals R , R and
R represents lower allcyl, R represents methyl, ethyl,
n-propyl, isopropyl, 2-methylpropyl, methylmercaptomethyl,
hydroxymethyl, hydroxyethyl, phenyl, benæyl or p-hydroxy-
benzyl~ Rlo' Rll and R represent carboxy, lower
alkoxycarbonyl or carbamoyl and R also represents hydro~
gen. Of these compounds there are preferred those in which
R represents lower alkyl, especially methyl, and R13
represents hydrogen.
The compounds preferred most of all are those
mentioned in the Examples and homologues thereof.
The compounds of the formula I can be manufactured
according to methods known per se. They can be obtained,
for example, by condensing, in a manner known per se, a
compound of the formula
CH2R6
O ~ vV-R1 (II)
~-X-R2
R3--CH (D) R
1 3
\
COOH
~1 ~8~5'~3
2 3 13 eanings given
above and R, RC' and ~ represent the radicals R ,
R and ~ , respectively, or represent a protecting group
that can be split off readily, or a derivative thereof,
with a compound of the formula
8 ~10 ~11
I - CH - COI - C~ICH2C~ - R12
R7 (L) R9 (D)
(III)
in which R an~ R have the meanings given above, R,
RC' , R and R have the meanings of R , R , 8
and R , with the proviso that carboxy groups and,
if desired, free hydroxyl groups present in these radicals
are protected by protecting groups that can be split off
readily, and splitting off optionally present protecting
groups.
In this case, condensation is carried out, for
example, by reacting the compound II in the form oE the
activated carboxylic acid ~ith the amino compound III or
by reacting the acid II with the compound III in which the
amino group is in activated form. The activated carboxyl
group can be, for example, an acid anhydride, preferably
a mixed acid anhydride, such as an acid azide, an acid
amide, such as an imidazolide or isoxazolide, or an
activated ester. ~ctivated esters to be given special
mention are cyanomethyl esters, carboxymethyl esters, p-
nitrophenylthio esters, p-nitrophenyl esters, 2,4,5-
trichlorophenyl esters, pentachlorophenyl esters, N-
hydroxysuccinimide esters, N-hydroxyphthalimide esters,
8-hydroxy~uinoline esters, 2-hydroxy-1,2-dihydro-1-ethoxy-
carbonylquinoline esters, N-hydroxypiperidine esters, or
enol esters which are obtained with N-ethyl-5-phenyl-isoxa-
zolium 3'-sulphonate. Activated esters can also be
obtained, if desired, with a carbodiimide with the addition
of N-hydroxysuccinimide or an unsubstituted or, for e~ample
halogen-, methyl- or methoxy-substituted, l-hydroxy-benzo-
triazole or 3-hydroxy-4-oxo~3,4-dihydro-benzo[d]1,2,3-tri-
azine.
The amino group is activated, for example, by reaction
with a phosphite amide.
Among the methods of reaction with activated esters
there should be mentioned especially those with N-ethyl-5-
phenyl-isoxazolium 3'-sulphonate (Woodward reagent K) or
2-ethoxy-1,2-dihydro-1-ethoxycarbonylquinoline or carbodi-
imide.
Protecting groups that can be split off readily are
those known from peptide and sugar chemistry. For carboxy
groups there should be mentioned especially tert.-butyl,
benzyl or benzhydryl, and for hydroxy groups especially
acyl radicals, for example lower alkanoyl radicals, such
as acetyl, aroyl radicals, such as benzoyl, and, more espe-
cially, radicals derived from carbonic acid, such as benzyl-
oxycarbonyl or lower alkoxycarbonyl, or alkyl, especially
tert.-butyl, benzyl or tetrahydropyranyl each optionally
substituted by nitro, lower alkoxy or halogen, or optionally
substituted alkylidene radicals that link the oxygen atoms
in the ~- and 6-position of the glucose moiety. Such
alkylidene radicals are especially lower alkylidene
radicals, especially ethylidene, isopropylidene or propyli-
dene radicals, or alternatively benzylidene radicals that
are optionally substituted, preferably in the p-position.
These protecting groups can be split off in a manner
known per se. ~hey can be removed by hydrogenolysis, for
example with hydrogen in the presence of a noble metal
catalyst, such as a palladium or platinum catalyst, or by
acid hydrolysis.
~35;~
_ 9 _
The starting materials used are known or can be manu-
factured in a manner known per s
Another method of manufacturing these novel starting
materials comprises condensing, in a mannee known per se,
a compound of the formula IV
CH2R6
,1--
~ ~ rO-R1 (IV)
N-X-R2
R3-CH (D) R o
I '
CON- CH - COOH
R7 (L)
in which Rl, R2, R3, R, R, R and R have
the meanings given above, with a compound of the formula
R R
I 10 1 11
I -CH - CH2CH - R12
Rg (D)
(V)
in which R , R , R and R have the meanings
9 lO ll 12
given above, with the proviso that carboxyl groups present
n the radicals R , R and R and, if desired,
~l 12
free hydroxy groups are protected by protecting groups that
can be split off readily, and splitting off optionally pre-
sent protecting groups.
The condensation is carried out in this case, for
example, by reacting the compound IV in the form of the
activated carboxylic acid with the amino compound V or by
reacting the acid IV with the compound V in which the amino
group is in activated form. The activated carboxyl group
~3S;~3
- 10 - .
can be, for example, an acid anhydride, preferably a mixed
acid anhydride, an acid amide or an activated ester. As
such, there come into consideration especially the acid
anhydrides, amides or esters mentioned above. The amino
group is activated, for example, by reaction with a
phosphite amide.
The protecting groups that can be split of~ readily
also correspond to those mentioned above. They can be split
off in a manner known per se, for example by hydrogenolysis,
for example with hydrogen in the presence of a noble metal
catalyst, such as a palladium or platinum catalyst, or by
aci~ hydrolysis.
The starting materials can be obtained in a manner
known per se. For example, corresponding su~ars that are
unsubstituted in the 3-position can be reacted with a halo-
R -acetamido-R-acetic acid, or a compound of the
formula II can be reacted, in the manner described above,
with an amino-R -acetic acid in which the carboxyl group
is protected, and the protecting group split off.
A further method of introducing the side chain located
in the 3-position of the sugar moiety is to react a compound
of the formula
CH2 OR6
/ ~
R~O~ ~ O-Rl (VI)
~--X--~
R13
in which ~, R2, Rl, RC4, R6 and R13 have the
meanings given above and hydroxy yroups optionally present
therein are protected by a protecting group that can be
split off readily, with a cornpound of the formula
33~;~8
11 --
(D) R8 1lO 1ll
z fH CONCH - CON - C~ - CH2CH ~ R12 ~V~I)
R3 R7 Rg
in which Z represents a reactive esterified hydroxy group
3 7 8' 9' Rlo' Rll and R have
the meanings given above, and to split off optionally pre-
sent protecting groups.
A reactive esterified hydroxy group is especially
a hydroxy group esterified by a strong inoeganic or organic
acid, especially one esterified by a hydrohalic acid, such
as hydrochloric, hydrobromic or hydriodic acid.
The protecting groups that can be split off readily
correspond to those already mentioned above. They can be
split off in a manner kno~n per se, for example by hydro-
genolysis, for example with hydrogen in the presence of
a noble metal catalyst, such as a palladium or platinum
catalyst, or by acid hydrolysis.
Depending on the nature of their substituents, the
present novel compounds are neutral, acidic or basic. If
acid groups are present in excess, they form salts with
~ases, such as ammonium salts or salts with alkali metals
or alkaline earth metals, for example sodium, potassium,
calcium or magnesium. If, however, basic groups are pre-
sent in excess, they form acid addition salts.
~ cid addition salts are especially pharmaceutically
acceptable non-toxic acid addition salts, such as those
with inor~anic acids, for example hydrochloric, hydrobromic,
nitric, sulphuric or phosphoric acids, or with organic
acids, such as organic carboxylic acids, for example acetic
acid, propionic acid, glycolic acid, succinic acid, maleic
~ 83~
- 12 -
acid, hydroxymaleic acid, methylmaleic acid, fumaric acid,
malic acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, 4-arninosali-
cylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,
embonic acid, nicotinic acid or isonicotinic acid, or
organic sulphonie acids, for example methanesulphonic acid,
ethanesulphonic acid, 2 hydroxyethanesulphonic acid, ethane-
1,2-disulphonic acid, benzenesulphonic acid, p-toluenesul-
phonic acid or naphthalene-2-sulphonic acid, and also other
acid addition salts which ean be used, or example, as
intermediates, for example for purifying the free com~ounds
or for the manufacture of other salts, and for characterisa-
tion~ such as, for example, those with picric acid, picro-
lonic acid, flavianic acid, phosphotungstic-acid, phospho-
molybdic acid, chloroplatinic acid, Reinecke's acid or per-
chlorie acid.
The muramyl peptides of the formula I according to
the invention can be used both as intermediates and as end
produets. For example, they are used for the manufaeture
of the muramyl peptide~antigen conjugates deseribed in
European Patent Application No. 79100513.5 (Publication
Number 0003833).
Irhe muramyl peptides of the formula I as such are
pharmacologically active and can be used as medicaments
for the treatment of the human or animal body. In parti-
eular, they have an immunomodulatory action, as can be
demonstrated inter alia by the tests described below:
1. Potentia~ion of cellular immunity in vivo: intensifi-
eation of delayed hyper-sensitivity to ovalbumin in guinea
pigs _ _ -----
Pirbright guinea pigs are immunised on day 0 withn mg of ovalbumin in complete Freund's adjuvant by injec-
ting 0.1 ml of an antigen-adjuvant mixture into each hind
paw. 4 weeks-later, skin reactions are triggered by intra-
~ILi835~28
- 13 -
cutaneous injection of 100 ~g of ovalbumin in 0.1 mL of
buffered physiological salt solution and are quantified
on the basis of the reaction volume calculated 2~ hours
thereafter by mean~ of the surface area of the erythema
and the increase in skin thickness. The antigen-specific
increase in the reaction volume observed after 2~1 hours
(delayed reaction) is considered as a measure of cell-
mediated immunity. Ovalburnin is too weak an immunogen to
induce a delayed reaction on its own or in a water-oil
emulsion with incomplete Freund's adjuvant (10 parts of
ovalbumin solution in 0.9 % NaCl mixed with ~.5 parts of
Bayol F and 1.5 part of Arlacel A) and, for effective
immunisation, has to be administered in complete Freund's
adjuvant to which mycobacteria have been added (5 mg of
dead and lyophilised M butyricum per 10 ml of Bayol
F/Arlacel A). To demonstrate the immunopotentiating action
of the test substances, the latter can be added to the anti-
gen-oil mixture, in doses of from 10 to 100 ~g, instead of
the mycobacteria.
The glucosamine peptides according to the invention
are capable of imitating the effect of the mycobacteria
in the described test and of quantitatively surpassing that
effect.
A significant potentiation of the delay2d reactivity
to ovalbumin can also be achieved by administering the com-
pounds of the described kind subcutaneously in sodium
chloride solution, in doses of from 10 to 100 ~g per animal,
for a few days after immunisation (for example, on day 0, 1,
2, 5, 6 and 7), rather than incorporating them into the
antigen-oil mixture.
This shows that compounds of the described kind are
capable of considerably increasing cellular immunity both
in admixture with the antigen itself (adjuvant effect in
the narrower sense) and when administered separately at
a different time and at a different site from the antigen
injection (systemic irnmunopotentiation).
3~i2~
2. Potentiation of humoral immunity ln vivo increase in
the production of antibodies to bovine serumalbumin ~BS~)
in mice
. _ .
N~RI mice are immunised on day O by intraperitoneal
(i.p.) injection of lO ~Lg of precipitate-free BSA. 9, 15
and 29 days later, serum samples are taken and examined
for their content of anti-BSA-antibodies by means of a
passive haemagglutination technique. In the dosage used,
soluble BSA is sub-immunogenic for the recipient animals,
i.e. it is unable to initiate anyl or is able to initiate
only a very insignificant, production of antibodies. Addi-
tional treatment of the mice with certain immunopotentiating
substances before or after the administration of antigen
results in an increase in the antibody titre in the serum.
The effect of the treatment is expressed by the score value
achieved, that is to say, by the sum of log titre
differences on the three days on which blood samples were
taken. Compounds of the present invention are capable of
significantly increasing the production of antibodies to
BSA when administerecl intraperitoneally or subcutaneously
(s.c.) at a dosage of 100 - 300 mg/kg/animal on five suc-
cessive days (day O to 4) after immunisation with BSA.
~ he immunostimulating effect of the mentioned com-
pounds is, in contrast to that of other bacterial immuno-
leptics (for example, LPS from E. coli), antigen-dependent:
injection of the novel compounds results in an increase
in the anti-BSA titre only in BSA-immunised and not,
however, in non-immunised mice. Remarkably, s.c. admini-
stration of the mentioned compounds is just as effective
as i.p. administration, that is to say, the observed immuno-
potentiating action is systemic and is not dependent on
the stimulant being administered by the same route as the
antigen or in admixture with the antigen, as is the case
with conventional adjuvants.
The described tests show that compounds of the kind
~ ~35;~8
- 15 -
described are also capable of speciEically increasing
humoral immunity, that they improve the immunological
response to stimulus and that their immunopotentiating
effects are due to a systemic activation of the immune
mechanism.
3. Potentiation of humoral immunity in vitro: T-cell- -
substituting effect in the antibody response of mice
splenocyt~s to sheep erythrocytes (SE)
To induce an antibody response, in many cases
lymphocytes originating from the thymus (T-cells) are
required. These cells co-operate with the precursors of
antibody-forming lymphocytes (B-cells) and help them to
react to stimulation by so-called T-dependent antigens with
proliferation, differentiation and antibody synthesis.
Splenocyte suspensions of congenitally athymic nu/nu mice
do not contain any fun-ctional T-cells and, for example,
are not capable of forming any antl-SE-antibodies in vitro
in the presence of SE. The compounds of the present
invention, surprisingly, are capable of functionally
replacing T-cells in such cultures and~making possible an
antibody response to SE. The addition of these substances
to nu/nu splenocyte cultures in the presence of SE resultsr
within 4 days, in a considerable increase in the number of
antibody-forming cells~ The findings show that the
mentioned compounds are capable of increasing humoral anti-
body formation ln vltro and of compensating a clefect of
the T-cell system.
4. Selective mitogenicity for B-cells: proliferation-
promotinq effect in B-lymphocyte cultures
SUSpQnSions of highly enriched B-lymphocytes (lymph
node cells of congenitally athymic nu/nu mice~ and sub-
stantially pure, immature and mature T-lymphocytes (thymus
3S~
- lG -
cells and cortisone-resistant thymus cells, i.e. those per-
sisting 48 hours after a cortisone injection, of Balb/c
mice) are incubated in the presence of the test substances
for 3 days. The incorporation of M -thymidine into the
lymphocytes during the last 18 hours of the culture period
is considered as a measure of the prolifeeation activity.
The compounds according to the invention are mitogenic
for B-lymphocytes (i.e. for the precursors of the antibody-
forming cells) but not for 'r-lymphocytes.
They are thus capable of stimulating the proliferation
of lymphocytes involved in the humoral immune response.
5. Tolerability
By comparison with the muramyl peptides of the formula
I belonging to the state of the art in which R , R
and R represent hydrogen, the compounds of the formula
I according to the invention, especially those in which
the radical R represents lower alkyl, especially methyl,
and more especially those in which R represents lower
alkyl and R represents methyl, are distinguished by a
greatly reduced pyrogenic action or, rather, one which is
undetectable or tolerable in the dosages that come into
consideration for practical applications, while the immuno-
modulating properties remain roughly the same or are
altered, usually increased, to a minor extent. These
findings are of great practical significance because the
pyrogenic action of the hydrophilic muramyl peptides known
before the present invention in mammalsother than and as
opposed ~ roden-ts is considerable and their therapeutic range
is therefore too narrow to allow them to be used safe]y without
const~nt medical supervision. In particular, the pyrogenic
side effect could, in unfavourable circumstances, lead to
thermal shock and, for that reason, certain forms of admini-
stration, such as intravenous administration, are out oE
the question.
sz~
- 17 -
The muramyl peptides according to the invention are
not toxic even when 300 mg/kg are administered i.p. to mice
5 times.
Testing for pyrogenicity was carried out on rabbits
in the manner prescribed in European Pharmacopoeia, vol. 2,
1971, pages 56-59. ~ccording to this, for example even
when a h gh dose, such as 10 mg/kg, of N-acetyl-muramyl-L-
(~-methyl)-~-aminobutyryl-D-isoglutamine is a~ministered,
no pyrogenic effect can be observed.
In the light of the state of the art, the high adju-
vant activity of the compounds according to the invention
in comparison with their greatly reduced pyrogenic action
is surprising since, according to previous investigations,
high adjuvant activity W3S always associated with a high
pyrogenicity and vice versa (cf. Sh~ Kotani et al, Biken
Journal 19, 9-13[1976]).
The compounds according to the invention are capable,
on the one hand in admixture with an antigen, of increasing
the immunogenicity of the latter and, on the other hand
when administered systemically, of intensifying the immuno-
logical reactivity of the treated organism. In so doing,
the mentioned substances are able to promote both the
cellular and the humoral immunity and to activate the
lymphocytes responsible for the formation of antibodies.
The novel compounds can therefore be used as adjuvants
in admixture with vaccines to improve the success of vac-
cination and to improve the protection against infection
mediated by humoral antibodies and/or cellular immunity
against bacterial, viral or parasitic causative organisms.
Finally, the described compounds in admixture with
a wide variety of antigens are suitable as adjuvants in
the experimental and industrial manufacture of antisera
for therapy and diagnostics and in the induction of immuno-
logically activated lymphocyte populations for cell trans-
fer processes.
Furthermore, the novel compounds can be used also
s~
- 18 -
without the simultaneous administration of antigens to pro-
mote immune reactions already proceeding subliminally in
humans and animals. The compounds are accordingly suitable
especially for the stimulation of the body's own defences,
for example in chronic and acute infections or in the case
of selective (antigen-speciEic) immunological defects, and
in hereditary and also in acquired general (i.e. not
antigen-specific) immunologically defective conditions,
such as occur in old age, in the course of serious primary
illnesses and especiaLly arter therapy with ionising radia-
tion or with hormones having an immunosuppressive action.
The mentioned substances can therefore be administered,
preferably also in combination with antibiotics admini-
stered to combat infections, with chemotherapeutic agents
and other pharmaceutical remedies, to counteract immunolo-
gical damage. Finally, the described substances are suit-
able also for the general prophylaxis of infectious diseases
in humans and animals.
The present invention relates also to pharmaceutical
preparations that contain compounds of the formula I. The
pharmaceutical preparations according to the invention are
those suitable for enteral, such as oral or rectal, admini-
stration, and for parenteral administration to warm-blooded
animals, which contain the pharmacological active sub-
stance on its own or together with a pharmaceutically accep~
table carrier. The dosage of the active substance depends
on the species of warm-blooded animal, the age and
individual condition and also on the method of administra-
tion and the nature of the illness treated or the nature
of the desired effect.
Accordingly, the daily doses to warm-blooded animals
of 70 kg body weight lie, in the case of parenteral
administration, between 0.5 and 100 mg, preferably between
l and 50 mg, for example 5 mg, and, in the case of oral
administration, between 1 and 1500 mg, preferabLy between
5 and 750 mg, for example 50 mg~
~352~3
- 19 -
An appropriate fraction of the amount mentioned ~bove
is administered one or more times daily, preEerably from
one to three times.
The novel pharmaceutical preparations contain from
approximately 10 % to approximately 95 ~, preferably from
approximately 20 % to approximately 90 ~, of the active
substance. Pharmaceutical preparations according to the
invention can, for example, be in unit dose form, such as
deagées, tablets, capsules, suppositories or ampoules.
The pharmaceutical preparations of the present
invention are prepared in a manner known per se, Eor example
by means of conventional mixing, granulating, coating, dis-
solving or lyophilising processes~ Pharmaceutical prepara-
tions for oral use can be obtained by combining the active
substance with solid carriers, if desired granulating a
resulting mixture and processing the mixture or granulate,
if desired or necessary after the addition of suitable
adjuncts, to form tablets or dragée cores.
Suitable carriers are especially fillers, such as
sugars, for example :Lactose, saccharose, mannitol or
sorbitol, cellulose preparations and/or calcium phosphates,
for example tricalcium phosphate or calcium hydrogen
phosphate, also binders, such as starch pastes, using, for
example, corn, wheat, rice or potato starch, gelatin,
tragacanth, methylcellulose, hydroxypropylmethylcellulose,
sodium carboxymethylcellulose and/or polyvinylpyrrolidone,
and/or, if desired, disintegrators, such as the above-
mentioned starches, also carboxymethyl starch, crosslinked
polyvinylpyrrolidone, agar, alginic acid or a salt thereof,
such as sodium alginate. Adjuncts are especially flow-
regulators and lubricants, for example silica, talc, stearic
acid or salts thereof, such as magnesium or calcium
stearate, and/or polyethylene glycol. Dragée cores are
provided with suitable coatings that may be resistant to
gastric juice, there being used, inter alia, concentrated
sugar solutions that optionally contain gum arabic, talc,
- 20 --
polyvinylpyrrolidone, polyethylene glycol and/or titanium
dioxide, lacquer solutions in suitable organic solvents
or solvent mixtures or, for the manufacture of coatings
that are resistant to gastric juice, solutions of suitable
cellulose preparations, such as acetylcellulose phthalate
or hydroxypropylmethylcellulose phthalate. Colouring sub-
stances or pigments may be added to the tablets or dragée
coatings, for example for identification or for indicating
different doses of active substance.
In certain cases, liposomal forms of administration
are especially suitable.
The following Examples illustrate the invention
without limiting it in any form. The R values were
ascertained on silica gel thin-layer plates manufactured
by Merck. Temperatures are given in degrees Centigrade.
335;~
- 21 -
Example 1
7.12 g (20 mmol) of the sodium salt of benzyl-2-
acetylamino-3-0-carboxymethyl-2-deoxy-4,6-0-isopropyli-
dene-a-D-glucopyranoSide are suspended in 60 ml oE
dimethylformamide. After the addition of 6.46 g (20 mmol)
of L-(N-methyl)-alanyl-D~isoglutamine-~-tert.-butyl ~ster
hydrochloride, 5.91 g (24 mmol) of ~EDQ (2-ethoxy-N-
ethox~carbonyl-1,2-dihydro-quinoline) are added to the
mixture and the whole is stirred for 24 hours at room
temperature. The suspension is concentrated by evaporation
at 30 in vacuo, the residue is taken up in 100 ml of
ethyl acetate and extracted successively, at low
temperature, with 2~ potassium bicarbonate solution and
2N citric acid solution, i5 finally washed neutral with
water and, after drying over sodium sulphate, concentrated
by evaporation. ~he by-products still present are remoYed
by chromatography over silica gel (toluene/acetone = 7/3).
Having been concentrated by evaporation, the eluate yields
7.7 9 (57 %) of henzyl-2-acetylamino-~,6-O~isopropylidene-3-
O-{~L-1-(D-l~carbamoyl-3-tert.-butoxycarbonyl-propyl)-
carbamoyl-ethyl]-N-methyl-carbamoylmethyl}-2-deoxy-a-D-
glucopyranoside.
[~]D = +66 (c = 1.1; methanol),
R = 0.81 (ethyl acetate/n-butanol/pyridine/acetic acid/
water = 42/21/21/6/lO),
R = Or 56 (n~butanol/acetic acid/water = 75/7.5/21),
R = ~.30 tchloroform/isopropanol/acetic acid = 70/8/2)o
C H N O calc. C 58.39 1~ 7.42 ~ 8.25
33 50 ~ 11
(678.80) found C 58.01 H 7.50 N 8.20
The protecting grou?s are split off from the resulting
product as follows:
7.60 g (11.1 mmol) of benzyl-2-acetylamino-4,6-0-iso-
propylidene-3-0-{[L-l-(D-l-carbamoyl-3-tert.-butoxycarbonyl-
- 22 -
propyl)-carbamoyl-ethyl]-N-methyl-carbamoylmethyl}-2-
deoxy-~-D-glucopyranoside are dissolved in 80 ml of cold
95 ~ trifluoroacetic acid, the solu~ion is stirred for 15
minutes at ~ C and then 1.1 litre of ether/petroleum ether
= 9/1 is added. The oily precipitate is triturated several
times with fresh mixture until a solid deposit forms. The
latter is dissolved in 30 ml of water and filtered over
20 ml of weakly basic ion-exchanger (Dowex-3) in acetate
form to remove the trifluoroacetic acid. The column is
subsequently rinsed with water and the combined eluates
are lyophilised. 4.95 9 (85 %) of benzyl-2-acetylamino-3-0-
{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-N-
methyl-carbamoylmethyl}-2--deoxy-~-D-glucopyranoside are
obtained.
[a]20 = +~5 (c = 1-2; H20),
R - 0.28 (n-butanol/acetic acid/water - 75/7.5/21),
R = 0.10 (chloroform/methanol/water=70/30/5).
4.65 g ~80 mmol) of the resulting benzyl-2-acetyl-
amino-3-0-{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-
ethyl]-~-methyl-carbamoylmethyl}-2-deoxy-a-D-gluco~yranoside
are dissolved in 100 ml of methanol, 4.6 9 of palladium-on-
carbon (10 %) are added and the mixture is treated exhaus-
tively with hydrogen Eor 2 days. The catalyst is Eiltered
off and the filtrate is concentrated by evaporation at room
temperature. The residue is dissolved in 50 ml of water,
extracted several times with n-butanol and, after evapora-
ting the butanol, the aqusous phase is lyophilised. 3.2 9
(81.4 %) oE 2-acetylamino-3-0-{[L-l-(~-l-carbamoyl-3-
carboxy-propyl)-carbamoyl-ethyl~-N-methyl-carbamoylmethyl}-
2~deoxy-D-glucose are obtained.
[a]DO = -19 (c = 1.1; methanol),
f = 0.20 (ethyl acetate/n-butanol/pyridine/acetic acid/
water = 42/21/21/6/10),
~ = 0.10 (n-butanol/acetic acid/water = 75/7.5/21),
Rf = 0.45 (acetonitrile/water = 3/1)
S28
- 23 -
C H N 0 .1.2H 0 calc. C ~ 36 ~ 6.78 N 10.~9
19 32 ~ 11 2
(514.46) found C 44.32 H 6.75 ~ 10.79
The coupling components (muramic acid derivative and
dipeptide) used as starting materials are obtained as
follows:
1.2 g of sodium hydride pract~ (Fluka) is added to
a solution of 8.0 g of benzyl-2-acetylamino-2-deoxy-4,6-0-
iSopropylidene-a-D-glucopyranoside in 80 ml of acetonitrile
and the mixture is stirred at 40 C for 2 hours. The reac-
o otion mixture is subsequently cooled to from -5 to -10
and 5.2 g of bromoacetic acid methyl ester are added. The
mixture is stirred for a further 30 minutes at -5 C, 10 ml
of methanol are added and, after a further 10 minutes, the
mixture is neutralised with acetic acid. The reaction mix-
ture is concentrated to dryness by evaporation and the resi-
due is partitioned between distilled water and ether. The
ether solution is washed with distilled water, dried over
magnesium sulphate and concentrated to dryness by evapora-
tion. The resulting residue is crystallised from ether/
petroleum ether. Benzyl-2-acetylamino-3-0-methoxycarbonyl-
2-deoxy-4,6-0-isopropylidene-a-D-glucopyranoside is
obtained.
[~]20 = +150 ~ ~ (c = 0.875, C~C13),
m.p. 122-3 C.
15 ml of lN sodium hydroxide solution are added to
a solution of 4.2 g of benzyl-2-acetylamino-3-0-methoxy-
carbonylmethyl-2-deoxy-4,6-0-isopropylidene-a-D~gluco-
pyranoside in 20 ml of methanol and the resulting solution
is left to stand at room temperature for one hour. Then,
5 ml of lN hydrochloric acid are added dropwise, while
stirring, and the solution is concentrated to dryness by
evaporation. The resulting po~der is dried at a high tem-
perature under a high vacuum and constitutes 10 mmol of the
33~
- 2~ -
~sodium salt of ben~yl-2-acetylamino-3-0-carboxy,nethyl-2-
deoxy-4,6-0-isopropylidene~~-D-glucopyranoside.
18.5 g t43.9 mmol) of N-benzyloxycarbonyl-N-rllethyl-
L-alanyl-D-isoglutamine-y-tert.-butyl ester are dissolved
in 150 ml of methanol, 4 g of palladium-on-carbon tlO ~)
are added and the mixture is hydrogenated at pE~ 4.5 tpH-
stat) in the presence of 0.65N methanolic hydrochloric acid.
The catalyst is filtered off, the filtrate is concentrated
by evaporation in vacuo and the residue is crystallised
from 25 ml of dimethox~ethane and 100 ml of ether. 11.6 g
(82 ~) of L-(N methyl)-alanyl-D-isoglutamine-y-tert.-butyl
ester hydrochloride are obtained.
m.p. 210-12 ,
~]~ = ~5 tc = 986; methanol),
R = 0.15 (n-butanol/acetic acid/water = 75/7.5/21),
C H ClN O calc. C 48.22 H 8.10 Cl 10.95 N 12.98
13 26 3 4
t323.82) found C 48.20 H 8010 C1 10~70 N 12.90
11.6 g t50 rnmol) of N-benzyloxycarbonyl-N-methyl-L-
alanine tJ. R. Coggins and N. L. Benoiton, Canad. J~ Chem,
49, 1968 [1971]), 10.1 g t50 mmol) of D-isoglutamine-y-
tert.-butyl ester and 14.8 g t60 mmol) of E~DQ are dissolved
in 110 ml of dimethoxyethane and the solution is stirred at
room temperature for 20 hours. The reaction solution is
concentrated by evaporation, the residue is taken up in
250 ml of ethyl acetate and extracted at low temperature
with 2~ 2otassium bicarbonate solution and 2~ citric acid
solution. The ethyl acetate phase is washed neutral with
water, deied over sodium sulphate and concentrated by eva-
poration. After recrystallisation of the solid residue from
dimethoxyethane/petroleum ether, 18.4 g t86 %) of N-benzyl-
oxycarbonyl-L-t~-methyl)-alanyl-D-isoglutamine-y-tert.-butyl
ester are obtained.
m.p. 83-85 ,
835Z~3
25 -
[~ = -8 (c = 1.1; methanol),
R = 0.63 (n-butanol/acetic acid/water = 75/7.5/21),
C H N 0 calc. C 60.12 fl 6.96 N 10.01
21 31 3 6
(~21.49) ~ound C 60.10 H 6.90 N 9.95
Example 2: The ~ollowing are obtained in an analogous
. . _
manner:
2-acetylamino-3-0-{[(D-l-carbamoyl-3-carboxy-propyl)-
carbamoylmethyl]-N-methyl-carbamoylmethyl}-2-deoxy-D-
glucose,
[a] = +6 (c = 1, methanol), R = 0.23 (aceto~iteile/
water = 3/1),
2-benzoylamino-3-0-{[tD-l-carbamoyl-3-carboxy-propyl)-
carbamoylmethyl]-N-methyl-carbamoylmethyl}-2-deoxy-D-
glucose,
2-acetylamino-3 0-{[(L-l-(D-1-carbamoyl-3-carboxy-propyl)-
carbamoyl-ethyl]-N-ethyl-carbamoylmethyl}-2-deoxy-D-glucose,
2-acetylamino-3-0-{[L-1-(D-l-carbamoyl-3-carboxy-propyl)-
carbamoyl-propyl]-N-methyl-carbamoylmethyl}-2-deoxy-D-
glucose,
2-acetylamino-3-0-{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-
carbamoyl-propyl~-N-ethyl-carbamoylmethyl}-2-deoxy-D-glucose,
D = -23 (c - 0.9; water), R = 0.37 (aceto-
nitrile/~ater = 3/1),
2-benzoylamino-3-0-{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-
carbamoyl-propyl]~N-ethyl-carbamoylmethyl}-2-~eoxy-D-glucose,
2-acetylamino-3-0-{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-
carbamoyl-ethyl]-N-propyl-carbamoylmethyl}-2-deoxy-D-glucose,
2-acetylamino-3-0-{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-N-
methyl-carbamoyl-ethyl]-carbamoylmethyl}-2-~eoxy-D-glucose,
2-acetylamino-3-0-{D-l-[(D-l-carbamoyl-3-carboxy-propyl)-
carbamoyl-methyl]-N-methyl-carbamoyl-ethyl}-2-deoxy-D-
glucose,
2-benzoylamino-3 0-{D-l-[(D-l-c d rbamoyl-3-carboxy-propyl)-
352~3
- 26 -
carbamoyl-methyl]-N-methyl-carbamoyl-ethyl}-2-deoxy-D-
glucose,
2-acetylamino-3-0-{D-l-[L-l-(D-l-carbamoyl-3-carboxy-
propyl)-carbamoyl-ethyl]-N-ethyl-carbamoyl-ethyl}-2-deoxy-
D-glucose,
2-acetylamino-3-0-{D-l-[L-1-tD-l-caebamoyl-3-carboxy-
propyl)-carbamoyl-propyl]-N-methyl~carbamoyl-etllyl}-2-deoxy-
D-glucose,
2-acetylamino-3-0- f D-l-[L-l-carbamoyl-3~carboxy-propyl)-car-
bamoyl-propyl~-N-ethyl-carbamoylmethyl}-2-deoxy-D-glucose,
2-benzoylamino-3-0-{D-l-[L-l-(D-l-carbamoyl-3-carboxy-
propyl)-carbamoyl-propyl]~N-ethyl-carbamoyl-ethyl}-2-deoxy-
D-glucose,
2-acetylamino-3-0-~D-l-[L-l-(D-l-carbamoyl~3-carboxy-
propyl3-carbamoyl-ethyl]-N-propyl-carbamoyl-ethyl}-2-deoxy-
D-glucose,
2-acetylamino-3-0-{D-l-[L-l-(D-l-carbamoyl-3-carboxy-
propyl)-W-methyl-carbamoyl-ethyl]-carbamoyl-ethyl}-2-deoxy-
D-glucose and
2-aaetylamino-3-0-{[L-l-(D-1,3-dicarboxy-propyl)-N-methyl-
carbamoyl-ethyl]-carbamoylmethyl}-2-deoxy-D-glucose.
Example 3
A solution of 4.2 g o~ benzyl-2-(N-acetyl-N-methyl)-
amino-3-0-{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-
ethyl]-carbamoyl-methyl}-2-deoxy-D-glucopyranoside in 75 ml
of methanol/water 1/1 is hydrogenated at 45 C and normal
pressure in the presence of 0.5 g of 10 % palladium-on-
carbon. The catalyst is filtered off and the filtrate is
concentrated to dryness by evaporation. The residue is
dissolved in a little distilled water and freeze-dried.
Thus, 2-(N-acetyl-N-methyl)-amino-3-0-{[L-l-(D-l-carbamoyl-
3-carboxy-propyl)-carbamoyl-ethyl]-carbamoylmethyl}-2-deoxy-
D-glucose is obtained in the form of a white powder.
The starting material used is manufactured as follows:
0.75 g of sodium hydride (Fluka, pract.) is added,
5Z~
while stirring and excluding moisture, under a nitrogen
atmosphere, to a solution of 8.5 g of benzyl-2-acetylamino-
2-deoxy-4,6-0-isopropylidene-3-0-methoxycarbonylmethyl--
D-glucopyranoside in 30 ml of absolute acetonitrile and
the mixture is stirred at 40 C for one hour. The reaction
mixture is then cooled to room temperature and a solution of
6.0 g of methyl iodide in 50 ml of absolute acetonitrile is
added dropwise in the course of ~ hours. AEter a further 3
hours, the reaction mixture is filtered and the filtrate is
concentrated to dryness by evaporation. The residue is
dissolved in ethyl acetate, the solution is washed with
water, dried over sodium sulphate and concentrated to dry-
ness by evaporation. Thus, benzyl-2-(N-acetyl-N-methyl)-
amino-2-deoxy-4,6-0-isopropylidene-3-0-methoxycarbonyl-
methyl-~-D-glucopyranoside is obtained in the form of a
yellow oil.
R = 0.45 (methylene chloride/ethyl acetate = 85/15).
A solution of ~.4 g of benzyl-2-(N-acetyl-N-methyl)-
amino-2-deoxy-4,6-0-isopropylidene-3-0-methoxycarbonyl-
methyl-a-D-glucopyranoside in 60 ml of methanol and 15 ml
of lN sodium hydroxide solution is left to stand at room
temperature for one hour, 5 ml of lN hydrochloric acid are
added and the mixture is concentrated to dryness by evapora-
tion. The resulting sodium salt of benzyl-2-(N-acetyl-N-
methyl)-amino-3-0-carboxymethyl-2-deoxy-4,6-0-isopropyli-
dene-~-D-glucopyranoside is dissolved in 50 ml of N,N-
dimethylformamide and condensed with 3.2 g of L-alanyl-D-
isoglutamine-tert.-butyl ester hydrochloride in the presence
of 2.5 g of ~EDQ. The solution is concentrated to dryness
by evaporation in vacuo and the residue is dissolved in
ethyl acetate. This solution is washed in succession with
water, ice-cold lN hydrochloric acid, water, a saturated
sodium bicarbonate solu~ion and water, is dried over mag-
nesium sulphate and concentrated to dryness by evaporation.
Thus, benzyl-2-(N-acetyl-N-methyl)-amino~3-0-{[L-l-(D-l-
carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-
~i~335Z~
~ 2~ -
methyl}-2-deoxy-~,6-0-isopropylidene-~-D-glucopyranoside-
tert.-butyl ester is obtained in the ~orm o~ a yellowish
foam. ~his product is dissolved in ~5 ml of ~5 ~ trifluoro-
acetic acid that has been pre-cooled to O C and the solu-
tion is stirred at O C for one hour. The reaction mix-
ture is poured onto 400 ml of absolute ether, the precipi-
tated product is filtered with suction, washed with ether
and dried. By treating this substance with the ion-
exchange resin Dowex-3 (acetate form), trifluoroacetic
acid-~ree benzyl-2-(N-acetyl-N-methyl)-amino-3-0-{[L-l-(D-
l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-
methyl}-2-deoxy-a-D-glucopyranoside is obtained in the form
of a white powder.
The following are manufactured in an analogous manner:
2-(N-acetyl-N-methyl)-amino-3-0-{D-l-[L-l-(D-l-carbamoyl-3-
carboxy-propyl)-carbamoyl-ethyl~-carbamoyl-ethyl}-2-deoxy-D-
glucose,
2-(N-acetyl-N-methyl)-amino-3-0-{D-l-[L-l-(D-l-carbamoyl-3-
carboxy-propyl)-carbamoyl-propyl]-carbamoyl-ethyl}-2-deoxy-
~-glucose,
2-(N-acetyl-N-methyl~-amino-3-0-{[L-l-(D-l-carbamoyl-3-
carboxy-propyl)-carbamoyl-propyl]-carbamoylmethyl}-2-deoxy-D-
glucose,
2-(N-acetyl-N-methyl)-amino-3-0-{[L-l-(D-l-carbamoyl-3-
carboxy-propyl)-carbamoyl-2-methyl-propyl]-carbamoylmethyl}-
2-deoxy-D-glucose,
2-(N-acetyl-N-methyl)-amino 3-0-{D-l-[L-l-(D-l-carbamoyl-3-
carboxy-propyl)-carbamoyl-2'~methyl-propyl]-carbamoyl-ethyl}-
2-deoxy-D-glucose,
3-0-{[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-
carbamoylmethyl}-2-deoxy 2-(N-propionyl-N-methyl)-amino-D-
glucose,
2-(~-butyryl-N-methyl)-amino-3-0-{[L-l-(D-l-carbamoyl-3-
carboxy-propyl)-carbamoyl-ethyl]-carbamoylmethyl}-2-deoxy-D-
glucose.
