AU597571B2 - Acylated hexose derivatives and processes for the manufacture thereof - Google Patents

Description

b jj S Farm COMMONWEALTH OF AUISTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION (OR IGINAL) Class tnt. Class Application Number: Lodged: J* 5 76 T/'6 Complete Specification Lodged: Accepted: Published: Priority: Related Art: amendments nu~de under Swctiob2 49, and Is orrn Ot f" prtn o 0*

U

NamBe of Applicant: o Address of Applicant: Actual Inventor: C C "Add~ress for Service: CIBA-GEIGY AG.

Klybeckstrasse 141, 4002 Basle, Sueitzerland.

OSKAR WACKER, ALBERT HARTMANN, JAROSLAV SAE'X11W0 BRUNO FEICHTIG AND GERHARD BASCHANG.-4 ,-EDWD-WAR-&-SNS,- U FF 150-QUEEN-S-T-REET,-MELBOURNE,AUSTRALIA, 3000.

04 0 Complete Specification for the invention entitled: ACYLATED HEXOSE DERIVATIVES AND PROCESSES FOR THE MANUFACTURE THEREOF.

0000

U

00 0

U

U

The following statement is a full description of this invention, including the best method of performing it known to us :I la- 4-15272/1+2/+ Acylated hexose derivatives and processes for the manufacture thereof The invention relates to 1,4,6-tri-O-acylated muramyl peptide derivatives and analogous D-mannose or D-galactose derivatives, processes for their manufacture, pharmaceutical preparations containing these derivatives, and their use as medicaments.

The invention relates especially to the compounds of the formula I 0 0 0 00 0 00 0 0 0 00 0 0." 0 00 00 O o 0 o o o0 0 0 00C 0 t e* 00 00 0 0

I

J:i Z- l 01 NH--t-'g R i i 2 in which the hexose moiety is derived from D-glucose, D-mannose or D-galactose, n represents 0 or 1, each of R, R 4 and R 6 independently of the others, represents lower alkanoyl or benzoyl, R 2 represents lower alkyl or phenyl, each of R 3

R

5 and R 7 independently of the others, represents hydrogen or lower alkyl, or R 5 and R 8 together represent trimethylene and R 7 represents hydrogen, R 8 represents hydrogen, or lower alkyl that is unsubstituted or is substituted by phenyl, hydroxy, mercapto or lower alkylthio, R 9 and R 12 independently of one another, each represents hydroxy, amino,

C

1 10 -alkoxy, aryl-ldwer alkoxy, alkanoyloxy-lower alkoxy having up to 16 carbon atoms, aroyloxy-lower alkoxy, 3-cholesteryloxy or 2-trimethylammonioethoxy,

R

10 represents hydrogen, carboxy, lower alkoxycarbonyl or aryl-lower alkoxycarbonyl and R represents hydrogen, or lower alkyl that is unsubstituted or is substituted by amino, hydroxy, lower alkanoylamino, lower alkanoyloxy, 2-benzyloxycarbonylamino-ethylsulphinyl, 2-benzyloxycarbonylamino-ethyl-sulphonyl, o2-lower alkoxycarbonylamino-ethyl-sulphinyl, 2-lower 0 0 alkoxycarbonylamino-ethyl-sulphonyl or by guanidino, with the proviso that at least one of the radicals R 9 and R 12 is other than hydroxy, amino and C1_7-alkoxy t. or R10 is other than hydrogen, carboxy and alkoxycarbonyl having up to 7 carbon atoms in the alkoxy moiety, and to salts of such compounds having at least one salt-forming group.

Preferably, the hexose moiety is derived from D-glucose.

In the case of asymmetrical substitution, the configuration at the atoms C-R 3

C-R

8 and C-CO-R 9 is and respectively, as indicated in formula I. The configuration at C-R 11 in the 3 case of asymmetrical substitution is or preferably Lower alkanoyl R 1

R

4 and R 6 is especially

C

2 -6-alkanoyl, for example n-hexanoyl, preferably

C

2 4 -alkanoyl, for example butyryl, propionyl or, preferably, acetyl.

Lower alkyl R is preferably C1_ 4 -alkyl, especially C1_ 2 -alkyl.

Lower alkyl R 3

R

5 or R 7 is preferably

C

1 3 -alkyl, especially methyl.

Unsubstituted lower alkyl R 8 or R 11 is preferably C1_ 4 -alkyl, for example ethyl, isopropyl, 2-methylpropyl, sec.-butyl or, especially, methyl.

Lower alkyl R 8 substituted by phenyl, hydroxy, mercapto, or lower alkylthio, such as, especially, methylthio, is preferably correspondingly substituted

C

1 -2-alkyl, for example benzyl, hydroxymethyl, r 1-hydroxyethyl, mercaptomethyl or 2-methylthioethyl.

Ak 9 12 Alkoxy R 9 or R is preferably C 1 _4-alkoxy, for example methoxy, n-butoxy or tert.-butoxy.

Aryl as a moiety of aryl-lower alkoxy, aryl-lower Salkoxycarbonyl or aroyloxy-lower alkoxy is especially phenyl or naphthyl that is unsubstituted or is substituted by one or more, for example from 1 to 3, of the aryl substituents mentioned below, but is i preferably unsubstituted phenyl.

r Aryl substituents are especially lower alkyl, for *example methyl, phenyl, halogen, hydroxy, lower alkoxy, 3 for example methoxy, lower alkanoyloxy, for example acetoxy, amino, mono- or di-lower alkylamino, for example mono- or di-methylamino, or lower alkanoylamino, for example acetylamino.

SAryl-lower alkoxy R and/or R 12 is lower alkoxy substituted by one or more, for example from 1 to 3, preferably 1 or 2, aryl radicals, for example aryl- -4methoxy, such as, especially, benzyloxy or benzhydryloxy.

Aryl-lower alkoxycarbonyl R 10 is lower alkoxycarbonyl substituted by one or more, for example from 1 to 3, preferably 1 or 2, aryl radicals, for example arylminethoxycarbonyl, such as, especially, benzyloxycarbonyl or benzhydryloxycarboniyl.

Lower alkoxycarbonyl R10 is preferably alkoxycarbonyl having up to 5 carbon atoms, for example methoxycarbonyl, n-butoxycarbonyl or tert.-butoxycarbonyl.

Lower alkyl R 11 substituted by amino or lower alkanoylamino is preferably 4-amino-n-butyl or 4-lower alkanoylamino-n-butyl. Lower alkyl R 1 substituted by 2-benzyloxycarbonylamino-ethyl-sulphinyl or -sulphonyl or by 2-lower alkoxycarbonylamino-ethyl-sulphinyl or -sulphonyl is preferably correspondingly substituted methyl, for example C 6 5 -CH 2 -O-C(=0)-NH-CH 2

-CH

2 -S(=0)-CH 2 or C 6 H 5

-CH

2 -O-C(=0)-NH-CH 2

-CH

2

-SO

2

-CH

2 Lower alkyl R11 substituted by guanidino is preferably 3-guanidino- Va n-propyl.

Alkanoyloxy-lower alkoxy R o R12 is especially lower alkanoyloxymethoxy, for example pivaloyloxymethoxy.

Aroyloxy-lower alkoxy R 9 or R 12 is especially 0aroyloxymethoxy, for example benzoyloxymethoxy.

lict Lower alkyl R 11 substituted by hydroxy or lower alkanoyloxy is especially correspondingly substituted h. C1- 2 -alkyl, for example hydroxymethyl, 1-hydroxyethyl, lower alkanoyloxymethyl or 1-lower alkanoyloxyethyl.

3-cholesteryloxy is the radical formed by ,abstraction of hydrogen from the hydroxy group of S* cholesterol.

*t The general terms used hereinbefore and hereinafter preferably have the following meanings: The prefix "lower" denotes radicals having up to 5 and including 7, especially up to and including 4, carbon atoms.

Halogen is especially chlorine or bromine, or also fluorine or iodine.

Salt-forming groups in a compound of the formula I are acidic groups, for example free carboxy groups, or basic groups, such as, especially, free amino or guanidino groups. Compounds of the formula I having a trimethylammonio group are present in salt form.

Depending on the type of salt-forming group, the compounds of the formula I form metal or ammonium salts or acid addition salts. Salts of a compound of the formula I are preferably pharmaceutically acceptable and non-toxic, for example alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or salts with ammonia or suitable organic amines aliphatic, cycloo.

.4 aliphatic, cycloaliphatic-aliphatic or araliphatic primary, secondary or tertiary mono-, di- or polyamines and also heterocyclic bases being especially suitable for the salt formation such as lower alkyl- 2° amines, for example triethylamine, hydroxy-lower alkylamines, for example 2-hydroxyethylamine, bis-(2hydroxyethyl)-amine, 2-hydroxyethyldiethylamine or tri- 44 (2-hydroxyethyl)-amine, basic aliphatic esters of carboxylic acids, for example 4-aminobenzoic acid 2diethylaminoethyl ester, lower alkyleneamines, for example 1-ethylpiperidine, cycloalkylamines, for example dicyclohexylamine, or benzylamines, for example N,N'-dibenzylethylenediamine, and also bases of the pyridine type, for example pyridine, collidine or quinoline. Compounds of the formula I having at least one basic group can form acid addition salts, for example with inorganic acids, such as hydrochloric acid, sulphuric acid or phosphoric acid, or with plr 6 6 suitable organic carboxylic or sulphonic acids, for example trifluoroacetic acid, and with amino acids, such as arginine and lysine. If several acidic or basic groups are present, mono- or poly-salts can be formed. Compounds of the formula I having an acidic group, for example a free carboxy group, and a free basic group, for example an amino group, may also be in the form of internal salts, that is to say in zwitterionic form, or one part of the molecule may be in the form of an internal salt and another part may be in the form of a normal salt.

For isolation or purification it is also possible to use pharmaceutically unacceptable salts. However, only the pharmaceutically acceptable non-toxic salts are used therapeutically and these are therefore preferred.

Muramyl peptides of the type of the compound N-acetylmuramyl-L-alanyl-D-isoglutamine are known from French Patent Application No. 74 22 909, publication number 2 292 486. These compounds are described as immunological adjuvants, that is to say S they can be used in admixture with vaccines to improve S" the success of the inoculation. Independent tests, for example in mice infected with influenza viruses, have shown that N-acetyl-muramyl-L-alanyl-D-isoglutamine S per se, that is to say without admixture of vaccines, *e is ineffective in the prophylaxis and treatment of S. virus infections. Also, the possibility of using Sstructurally more simple muramyl peptides and their analogues against virus infections has not hitherto o. been described.

The problem underlying the present invention is to provide muramyl peptide derivatives that are structurally relatively simple, and therefore relatively easy to manufacture, which, when administered to warm- 7 blooded animals, are highly effective for the prophylaxis and treatment of virus infections.

According to the invention it has surprisingly been found that the above-mentioned compounds of the formula I and their pharmaceutically acceptable salts are outstandingly suitable for both the prophylaxis and the treatment of virus infections, as is apparent, for example, from tests on animals as illustrated in the Examples. In these animal tests, animals such as mice or guinea pigs are infected with the most varied types of viruses at a dose that is lethal to all or to the great majority of the untreated (control) animals, for example LD 80 90 and the course of the infection observed in the untreated control animals is compared with that observed in animals treated with one of the above-mentioned compounds or a salt thereof before, simultaneously with or after infection.

It is found that a prophylactic effect is achieved even when the compounds of the formula I are admino ,istered several days up to some weeks, for example four o 'eeks, before infection, and that a therapeutic effect i is still achieved when the compounds are administered several days, for example one week, after infection.

The compounds of the formula I are effective in j as low a dosage range as from 0.0001 mg/kg to 0.1 mg/kg j, in the above-mentioned test in mice.

Also noteworthy is the broad spectrum of viruses against which the above-mentioned compounds are effective.

The compounds of the formula I can be used especially for the prophylaxis and treatment of diseases caused by the viruses specified below [for nomenclature see J.L. Melnick, Prog. med. Virol. 26, 214-232 (1980) and 28, 208-221 (1982)]: DNA viruses with cubic symmetry and naked ll- .«.,17lllllll.llllI r 8 a r o at 00** DoDi COo o.

*r o a 4 0 aI U.

nucleocapsid, DNA viruses with encapsulated virion and RNA viruses with cubic symmetry and those with helical symmetry of the capsid.

The compounds of the formula I are preferably used in the case of DNA viruses with encapsulated virion and cubic symmetry of the capsid, in the case of RNA viruses with cubic symmetry of the capsid and naked virion and in the case of RNA viruses with helical symmetry of the capsid, in which the nucleocapsid capsule is positioned at the surface membrane, but also in the case of adenoviridae, poxviridae and coronaviridae, such as, especially, human corona viruses.

The compounds of the formula I are used especially in the case of herpesviridae, picornaviridae and myxo viruses, but also in the case of mastadeno viruses, such as, especially, human adeno viruses, in the case of chordopoxvirinaek such as, chiefly, orthopox viruses, such as, especlally, for example, vaccinia viruses, in the case of reoviridae, above all (especially human) rota viruses, and in the case of caliciviridae and rhabdoviridae, such as, especially, vesiculo viruses in humans and also in horses, cattle and pigs.

The compounds of the formula I are used chiefly in the case of alpha-herpesvirinae, such as varicella viruses, for example human varicella-zoster viruses, rhino viruses, cardio viruses and ortho-myxoviridae, but also in the case of beta-herpesvirinae, such as, especially, human cytomegalo viruses, in the case of aphtho viruses, especially aphtho viruses in animals with cloven hooves, such as, especially, cattle, and in the case of para-myxoviridae, such as, especially, pneumo viruses, for example respiratory syncytial viruses in humans, and such as, also, morbilli viruses 4'r tlt i i# U. a 40 *0 *0 44 *4+ a, a *0S0So 4

B

1~ c !4 or para-myxo viruses, such as para-influenza viruses, for example human para-influenza viruses, including Sendai 7iruses, and in the case of arbo viruses or vesiculo viruses, for example Vesicular stomatitis viruses.

The compounds of the formula I are used most especially in the case of simplex viruses, for example human Herpes simplex viruses of types 1 and 2, in the case of human encephalomyocarditis viruses, in the case of influenza viruses, such as, especially, influenza A and influenza B viruses, in the case of vaccinia and para-influenza viruses and most especially in the case of the .viruses mentioned in the Examples.

The compounds of the formula I can be used for the prophylaxis and treatment of virus infections, especially in warm-blooded animals, including humans, by administering them enterally or parenterally, especially together with suitable adjuncts or carriers.

0 o°*o They are preferably applied to the mucous membranes, for example intranasally, rectally, vaginally, or to the conjunctiva of the eye, or orally. However, the anti- S* viral effect also occurs in the case of administration by other routes, for example subcutaneously, intravenously or intramuscularly, or in the case of application to normal skin.

1" The dosage of the active ingredient depends, O inter alia, on the species of warm-blooded animal, the organism's resistance, the method of administration and the type of virus. There is relatively little relationship between the dosage and the effect.

For prevention, a single dose of from approxi" imately 0.01 mg to approximately 10 mg, preferably from 0.05 to 1 mg, for example 0.2 mg, of active ingredient is administered to a warm-blooded animal of approximately 70 kg body weight, for example a human. The 10 prophylactic effect of this dose lasts for several weeks, If necessary, for example when there is an increased risk of infection, the administration of this dotse can be repeated.

The therapeutic dose for warm-blooded animals of approximately 70 kg body weight is from 0.1 mg to 25 mg, preferably from 0.1 to 1 mg, for example 0.5 mg, especially in the case of oral administration. The dose in the case of topical, especially intranasal, administration is up to ten times lower. If necessary, the administration of compounds of the formula I can be repeated until there is an improvement in the illness. Often, however, a single administration is sufficient.

The compounds of the formula I also have antitumour properties. These are based on their ability, for example when incorporated in multilamellar liposomes or in phosphate-buffered physiological sodium 0 1 chloride solution (PBS), to activate macrophages in 04 such a manner that these endogenous defence cells are o capable of killing tumour cells (cytotoxicity) or of hindering their growth (cytostasis). The inducement of 0 4 :0 tumoricidal and tumoristatic alveolar macrophages in rats in vitro and in situ can be demonstrated, for example, in the following test: SAlveolar macrophages are obtained by washing the I lungs with culture medium. These macrophages are activated either by injecting the test substances into the rats (intravenously or intranasally, in situ 0 0 activation) or by a 24-hour preliminary incubation with a compound of the formula I in a CO 2 incubator (in vitro activation). The macrophages activated in this manner are then incubated for a further 72 hours with tumour cells.

In order to measure tumoricidal activities of the macrophages, the tumour cells are labelled with 11 0 0 o 0.

:0604, a 6 o G *0 0 o 0 *0 0rV .44 0U 12 5 I-iododeoxyuridine before the 72-hour incubation.

The number of tumour cells that have not been killed can be measured, after washing away the radioactivity released by lysed tumour cells, on the basis of the radioactivity that remains.

In order to ascertain tumoristatic activities of the macrophages, 3H-thymidine is added to the cultures 8 hours before the end of the 72-hour incubation period, and afterwards the 3 H-thymidine incorporation into the tumour cells is measured. In vitro, the substances, both when dissolved in PBS and when incorporated in liposomes, are able to induce tumoricidal alveolar macrophages in rats in doses as low as 20 nanogram/0.2 ml of culture. In rats, a single intravenous administration of the compounds incorporated in liposomes at a dose of 160 ug/animal causes the inducement of tumoricidal and tumoristatic alveolar macrophages. In addition, a single intranasal admrnistration of the substances in PBS at a dose of 25 ug/rat causes the inducement of tumoricidal alveolar macrophages.

The compounds of the formula I can thus be used in warm-blooded animals, including humans, also for the treatment of tumour diseases, especially, for example, for avoiding the formation of metastases, for example in the case of surgical removal of a primary tumour.

The invention relates especially to those compounds of the formula I in which the hexose moiety is derived from D-glucose, D-mannose or D-galactose, n represents 0 or 1, each of R R 4 and R independently of the others, represents lower alkanoyl or benzoyl, R 2 represents lower alkyl or phenyl, each of R, R 5 and R 7 independently of the others, represents hydrogen or lower alkyl, or R 5 and R 8 together represent trimethylene and R 7 represents hydrogen, R 8 represents hydrogen, or lower alkyl that SI c Ij~r r o* 4 944, Cj 0 .I ti i *1 12 is unsubstituted or is substituted by phenyl, hydroxy, mercapto or lower alkylthio, R and R 12 independently of one another, each represents hydroxy, amino, C1_-1-alkoxy, aryl-lower alkoxy, alkanoyloxy-lower alkoxy having up to 16 carbon atoms, aroyloxy-lower alkoxy or 3-cholesteryloxy, R 10 represents hydrogen, carboxy, lower alkoxycarbonyl or aryl-lower alkoxycarbonyl and R 1 1 represents hydrogen, or lower alkyl that is unsubstituted or is substituted by amino or hydroxy, with the proviso that at least one of the radicals R 9 and R 12 is other than hydroxy, amino andC 1 7 -alkoxy or R 0 is other than hydrogen, carboxy and alkoxycarbonyl having up to 7 carbon atoms in the alkoxy moiety, and to salts of such compounds having at least one salt-forming group.

Preferred are the above-mentioned compounds of the formula I in which R and R 12 independently of one another, each represents hydroxy, amino, C1_ 10 -alkoxy, e alkanoyloxy-lower alkoxy having up to 16 carbon atoms, *3-cholesteryloxy, or phenyl- or benzoyloxy-lower alkoxy that is unsubstituted or substituted in each case in the phenyl moiety by lower alkyl, for example methyl, phenyl, halogen, hydroxy, lower alkoxy, for example methoxy, lower alkanoyloxy, for example acetoxy, amino, St C mono- or di-lower alkylamino, for example mono- or di-methylamino, or lower alkanoylamino, for example acetylamino, and R0 represents hydrogen, carboxy, lower alkoxycarbonyl, or phenyl-lower alkoxycarbonyl that is unsubstituted or substituted in the phenyl moiety by lower alkyl, for example methyl, phenyl, halogen, hydroxy, lower alkoxy, for example methoxy, lower alkanoyloxy, for example acetcxy, amino, monoor di-lower alkylamino, for example mono- or dimethylamino, or lower alkanoylamino, for example acetylamino, and the remaining substituents have the 13 meanings mentioned above, with the proviso that at least one of the radicals R 9 and R 1 2 is other than hydroxy, amino and lower alkoxy or R 10 is other than hydrogen, carboxy and lower alkoxycarbonyl, and salts of such compounds having at least one salt-forming group.

Especially preferred are compounds of the formula I in which the hexose moiety is derived from D-glucose or D-mannose, n represents 0 or 1, each of R 1

R

and R 6 independently of the others, represents

C

2 4 -alkanoyl or benzoyl, R 2 represents C 1 _4-alkyl or phenyl, each of 3

R

5 and R 7 independently of the others, represents hydrogen or methyl, or R and R 8 together represent trimethylene and R 7 represents hydrogen, R represents hydrogen,

C

1 4 -alkyl, or C 1 _2-alkyl substituted by phenyl, hydroxy, mercapto or methylthio, R 9 and R 12 independently of one another, each represents hydroxy, amino, lower alkoxy, phenyl-lower alkoxy, lower alkanoyloxy-lower alkoxy, benzoyloxy-lower alkoxy or 3-cholesteryloxy, R10 represents hydrogen, carboxy, s lower alkoxycarbonyl or phenyl-lower alkoxycarbonyl and

R

11 represents hydrogen, or C 1 i 4 -alkyl that is unsubstituted or is substituted by amino or hydroxy, with the proviso that at least one of the radicals R 9 Sc and R 12 is other than hydroxy, amino and lower alkoxy or R10 is other than hydrogen, carboxyand lower alkoxycarbonyl, and salts of such compounds having at least one salt-forming group.

Especially preferred are compounds of the formula I in which the hexose moiety is derived from D-glucose 9*4 or D-mannose, n represents 0 or 1, each of R R 4 and R 6 independently of the others, represents SC2_4-alkanoyl or benzoyl, R represents C1- 2 alkyl or phenyl, each of R 3

R

5 and R 7 independently of the others, represents hydrogen or methyl, 14

R

8 represents C 1 _4-alkyl, R 9 and 12 independently of one another, each represents hydroxy, amino, C1_ 4 -alkoxy, phenylmethoxy, lower alkanoyloxymethoxy, benzoyloxymethoxy or 3cholesteryloxy, R 10 represents hydrogen, carboxy, alkoxycarbonyl having up to 5 carbon atoms, or phenylmethoxycarbonyl and R 1 1 represents C 1 4 alkyl, with the proviso that at least one of the radicals R 9 and R 12 is other than hydroxy, amino and C1_ 4 -alkoxy or R 10 is other than hydrogen, carboxy and alkoxycarboryl having up to 5 carbon atoms, and salts of such compounds having at least one saltforming group.

Most especially preferred are compounds of the formula I in which the hexose moiety is derived from D-glucose, n represents 0 or 1, R R and R represent acetyl or butyryl, R 2 represents C1-2- Salkyl or phenyl, R 3 represents hydrogen or methyl,

R

5 and R 7 represent hydrogen, R 8 represents o 9

C

1 -3-alkyl, R represents amino, C1_ 4 -alkoxy, c10 pivaloyloxymethoxy or mono- or diphenylmethoxy, R represents hydrogen, R 11 represents methyl and R12 gal 6 represents mono- or diphenylmethoxy or 3cholesteryloxy.

Most especially preferred are also compounds of I t the formula I in which the hexose moiety is derived from D-glucose, n represents 0 or 1, R R and R o* represent C 2 -6-alkanoyl, R 2 represents C 1 -4-alkyl or phenyl, each of R 3 R and R 7 independently of the others, represents hydrogen or methyl, R 8

R

9 represents C 1 -4-alkyl, R represents amino, lower alkoxy, pivaloyloxymethoxy, diphenylmethoxy, benzyloxy or 2-trimethylammonioethoxy, R 10 represents hydrogen or lower alkoxycarbonyl, R 11 represents C1_ 4 -alkyl, lower alkanoyloxymethyl or (2-benzyloxycarbonylamino- 15 ethyl)-sulphonyl-methyl and R 12 represents amino, lower alkoxy, pivaloyloxymethoxy, diphenylmethoxy, benzyloxy, 2-trimethylammonioethoxy, 3-cholesteryloxy or benzoyloxymethoxy, with the proviso that at least one of the radicals R 9 and R 12 is other than amino and lower alkoxy, and salts of such compounds that are capable of salt formation.

Most especially preferred are above all compounds of the formula I in which at least one of the radicals R and R 2 represents pivaloyloxymethoxy, diphenylmethoxy, benzyloxy, 2-trimethylammonioethoxy, 3-cholesteryloxy or benzoyloxymethoxy, and the other of the radicals R 9 and R12 has the above-mentioned meaning, and salts of such compounds that are capable of salt formation.

Preferred most of all are the compounds of the a formula I described in the Examples, The compounds of the formula I are manufactured in a manner known per se: a* They are manufactured, for example, as follows: a a) a compound of the formula II Hp2R o H-R2 a (II) Sc n

(L)

-16in which at least one of the radicals R R 2

R

4 and Ga R represents hydrogen and the remainder of these radicals have the meanings of R1, the group

R

4 and R 6 respectively, and the remaining substituents have the meanings mentioned above, any free functional groups present in a compound of the formula II, with the exception of groups that are to participate in the reaction, being protected, if necessary, by readily removable protecting groups, is reacted with an acylating agent transferring the radical R 1

R

2

R

4 or R 6 to be introduced, and any protecting groups present are, if necessary, removed, or b) a compound of the formula III 106 *LF H20R 0* -2

OR

H R

(II)

-C-R'

S i I in which the substituents have the meanings mentioned a above, or a reactive derivative thereof, is reacted with a compound of the formula IV R7- I

(IV)

X-H- H-CH2- HC- NH-H- R12

(D)

n 1. p '7 17 in which X represents a reactive esterified hydroxy group, and the remaining substituents have the meanings mentioned above, any free functional groups present in a compound of the formula IV, with the exception of X, being protected, if necessary, by readily removable protecting groups, and any protecting groups present are, if necessary, removed, or c) a compound of the formula V, R'Ov~ 0- VA1 R/-C NHv) P

(L)

in which each of L, s and t, independently of the others, represents 0 or 1, and in which the substituents have the meanings mentioned above, any free functional groups present in a compound of the formula V, with the exception of the group that is to participate in the reaction, being protected, if necessary, by readily removable protecting groups, or a reactive carboxylic acid derivative thereof, is reacted with a compound of the formula VI S S

S.

SS

S S

S.

S

S..

*4 SO 5 5

S

5* S S

S

S S~ 54 j 4( tC S S. S 555.55 S S 18 (D 2NHH--CH C }R 2 (VI in which each of u, v and x, independently of the others, represents 0 or I and the remaining symbols and substituents have the meanings mentioned above, any free functional groups present in a compound of the formula VI, with the exception of the group that is to 'participate in the reaction, being protected, if necessary, by readily removable protecting groups, and u, v and x representing I when a and t in the reactant of the formula V represent 0, or u representing 0 and v and x representing I when represents I and t represents 0, or u and v representing 0 and x representing 1 when r and °t represent I and s represents 0, or (for the manufacture of compounds of the formula I in which n ""represents 1) u and x representing 0 when a, r, s and t represent 1, or with a reactive derivative thereof, and any protecting groups present are, if necessary, removed, or d) for the manufacture of a compound of the formula I in which R 9 has one of the meanings mentioned above apart from hydroxy and amino and/or Ri 0 represents lower alkoxycarbonyl or aryl-lower alkoxycarbonyl and the remaining substituents have the meanings mentioned 19above, a compound of the formula VII H2 OR 6 *--0o 'NH- -R2

R

3

(VII)

Nfhi--H-C R 1 3 R2 n in which at least one of the radicals R 1 a and R 3 represents carboxy and the other of the radicals R 10a and R 13 has the meaning mentioned above for R 0 and for the group R 9 respectively, and in which the remaining substituents have the meanings mentioned o*o* above, any free functional groups present in a compound of the formula VII, with the exception of the groups that are to participate in the reaction, being o protected, if necessary, by readily removable protecting groups, or a reactive carboxylic acid derivative thereof, is esterified and any protecting groups present are, if necessary, removed, or e) in a compound of the formula I in which at least one of the radicals R 8

C(=O)-R

9

R

10

R

11 and C(=-R12 is present in a protected form which does not correspond to the definition of the desired end product the corresponding protecting group(s) is (are) removed, or iti -I 20 f) for the manufacture of a compound of the formula I in which R 9 represents amino and the remaining substituents have the meanings mentioned above, a compound of the formula VIII, R( 0

(VIII)

NH-CH RI 2 3 o o f o 4* 04 0 4 4 It 4 t t I,

I

I t 4 4 in which the radical R 14 represents carboxy and the remaining substituents have the meanings mentioned above, any free functional groups present in a compound of the formula VIII, with the exception of the groups that are to participate in the reaction, being protected, if necessary, by readily removable protecting groups, or a reactive carboxylic acid derivative thereof, is amidated, and any protecting groups present are, if necessary, removed, and, if desired, after carrying out one of the processes a a resulting compound of the formula I having at least one salt-forming group is converted into its salt or a resulting salt of a compound of the formula I is converted into the free compound, and/or a resulting isomeric mixture is separated.

4,44 4 4

*I

4r*~ 4 21- Preferred are processes a, c, d and e, and also process f.

The execution of the above-mentioned process variants is explained in detail in the following: Process a: Preferably, process a) is used to introduce an acyl radical as R 4 and/or R 6 The starting materials used are preferably compounds of the formula II in which the radical R 2 a represents the group

R

2 Free functional groups that may be present in a compound of the formula II, which are preferably protected by readily removable protecting groups, are especially free hydroxy or mercapto in radical R 8 free carboxy R 9

R

10 or R 12 as well Sas free amino, hydroxy or guanidino in radical R 1 1 The protection of free carbamoyl R 9 or R12-C(=0)- is optional.

oa Protecting groups and the manner in which they are S* introduced and removed are described, for example, in "Protective Groups in Organic Chemistry", Plenum Press, 0 London, New York 1973, and in "Methoden der organischen Chemie", Houben-Weyl, 4th Edition, Vol. 15/1, Georg- Thieme-Verlag, Stuttgart 1974 and in Theodora W. Greene, "Protective Groups in Organic Synthesis", John Wiley Sons, New York 1981. It is characteristic of protecting groups that they can be readily removed, that is to say without undesired secondary reactions taking place, for example by solvolysis, reduction, photolysis or alternatively under physiological conditions.

Hydroxy-protecting groups are, for example, acyl radicals, such as optionally substituted, for example halo-substituted, lower alkanoyl, such as 2,2-dichloroacetyl, or acyl radicals of carbonic acid semi-esters, -:1 00 0* o 00 00 0* to 090* 0 0* *r 4 09 0 9r 712 22 especially tert.-butoxycarbonyl, optionally substituted benzyloxycarbonyl or diphenylmethoxycarbonyl, for example 4-nitrobenzyloxycarbonyl, or 2-halo-lower alkoxycarbonyl, such as 2,2,2-trichloroethoxycarbonyl, also trityl or formyl, or organic silyl or stannyl radicals, also readily removable etherifying groups, such as tert.-lower alkyl, for example tert.-butyl, 2-oxa- or 2-thia-aliphatic or -cycloaliphatic hydrocarbon radicals, especially 1-lower alkoxy-lower alkyl or 1-lower alkylthio-lower alkyl, for example methoxymethyl, 1-methoxyethyl, 1-ethoxyethyl, methylthiomethyl, 1-methylthioethyl or 1-ethylthioethyl, or 2-oxa- or 2-thia-cycloalkyl having 5 or 6 ring atoms, for example tetrahydrofuryl or 2-tetrahydropyranyl or corresponding thia analogues, and also optionally substituted 1-phenyl-lower alkyl, such as optionally substituted benzyl or diphenylmethyl, there being suitable as substituents of the phenyl radicals, for example, halogen, such as chlorine, lower alkoxy, such as methoxy, and/or nitro.

Carboxy groups are customarily protected in esterified form, such ester groupings being readily cleavable under mild conditions. Carboxy groups protected in this manner contain as esterifying groups especially lower alkyl groups that are branched in the 1-position or suitably substituted in the 1- or 2position. Preferred carboxy groups in esterified form are, inter alia, tert.-lower alkoxycarbonyl, for example tert.-butoxycarbonyl, arylmethoxycarbonyl having one or two aryl radicals, these being phenyl radicals optionally mono- or poly-substituted, for example, by lower alkyl, such as tert.-lower alkyl, for example tert.-butyl lower alkoxy, such as methoxy, hydroxy, halogen, for example chlorine, and/or by nitro, such as benzyloxycarbonyl optionally substi- 23 tuted, for example, as mentioned above, for example 4methoxybenzyloxycarbonyl or 4-nitrobenzyloxycarbonyl, or diphenylmethoxycarbonyl optionally substituted, for example, as mentioned above, for example diphenylmethoxycarbonyl or di-(4-methoxyphenyl)-methoxycarbonyl, 1-lower alkoxy-lower alkoxycarbonyl, such as methoxymethoxycarbonyl, l-methoxyethoxycarbonyl or 1ethoxymethoxycarbonyl, 1-lower alkylthio-lower alkoxycarbonyl, such as 1-methylthiomethoxycarbonyl or 1ethylthioethoxycarbonyl, aroylmethoxycarbonyl in which the aroyl group represents benzoyl optionally substituted, for example, by halogen, such as bromine, for example phenacyloxycarbonyl, 2-halo-lower alkoxycarbonyl, for example 2 ,2,2-trichloroethoxycarbonyl, 2bromoethoxycaOoonyl or 2-iodoethoxycarbonyl, or 2-(tritoo substituted silyl)-ethuxycarbonyl in which each of the substituents, independently of the others, represents an aliphatic, araliphatic, cycloaliphatic or aromatic hydrocarbon radical that is optionally substituted, for to example, by lower alkyl, lower alkoxy, aryl, halogen ,and/or by nitro, such as corresponding optionally SOo substituted lower alkyl, phenyl-lower alkyl, cycloalkyl or phenyl, for example 2-tri-lower alkylsilylethoxyearbonyl, 2-trimethylsilylethoxycarbonyl or 2-(di-n- E butylmethylsilyl)-ethoxycarbonyl, or 2-triarylsilylethoxycarbonyl, such as 2-triphenylsilylethoxycarbonyl.

The organic silyl or stannyl radicals mentioned hereinbefore and hereinafter preferably contain lower alkyl, especially methyl, as substituent of the silicon or tin atoms. Corresponding silyl or stannyl groups 6 are especially tri-lower alkylsilyl, especially 4 trimethylsilyl, also dimethyl-tert.-butylsilyl, or correspondingly substituted stannyl, for example tri-nbutylstannyl.

Preferred protected carboxy groups are tert.-lower 24 alkoxycarbonyl, such as tert.-butoxycarbonyl, and especially benzyloxycarbonyl or diphenylmethoxycarbonyl optionally substituted, for example, as mentioned above, such as 4-nitrobenzyloxycarbonyl, and more especially 2-(trimethylsilyl)-ethoxycarbonyl.

A protected amino group can be, for example, in the form of a readily cleavable acylamino, arylmethylamino, etherified mercaptoamino, 2-acyl-lower alk-1enylamino, silylamino or stannylamino group or in the form of an azido group.

In a corresponding acylamino group, acyl is, for example, the acyl radical of an organic carboxylic acid having, for example, up to 18 carbon atoms, especially an alkanecarboxylic acid optionally substituted, for example, by halogen or aryl, or of benzoic acid optionally substituted, for example, by halogen, lower o alkoxy or by nitro, or of a carbonic acid semi-ester.

Such acyl groups are, for example, lower alkanoyl, such o as formyl, acetyl or propionyl, halo-lower alkanoyl, 0 ,6 such as 2-haloacetyl, especially 2-chloro-, 2-bromo-, 2- Siodo-, 2,2,2-trifluoro- or 2,2,2-trichloro-acetyl, benzoyl optionally substituted, for example, by halogen, lower alkoxy or by nitro, for example benzoyl, .t 4-chlorobenzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl, or lower alkoxycarbonyl that is branched in the 1-position of the lower alkyl radical or suitably substituted in the 1- or 2-position, especially tert.-lower alkoxycarbonyl, for example tert.-butoxycarbonyl, arylmethoxycarbonyl having one or two aryl radicals that ?are preferably phenyl optionally mono- or poly- 4 substituted, for example, by lower alkyl, especially tert.-lower alkyl, such as tert.-butyl, lower alkoxy, such as methoxy, hydroxy, halogen, for example chlorine, and/or by nitro, such as optionally substituted benzyloxycarbonyl, for example 4i 25 25 nitrobenzyloxycarbonyl, or substituted diphenylmethoxycarbonyl, fj)r exai-iple benzhydryloxycarbonyl or di-(4-methoxyphenyl)-methoxycarbonyl, arol1methoxycarbonyl in which the aroyl group preferably represents benzoyl optionally substituted, for example, by halogen, such as bromine, for example phenacyloxycarbonyl, 2-halo-lower alkoxycarbonyl, for example 2,2,2-trichloroethoxycarbonyl, 2-bromoethoxycarbonyl or 2-iodoethoxycarbonyl, or 2-(tri-substituted silyl)ethoxycarbonyl in which each of the substituents, independently of the others, represents an aliphatic, araliphatic, cycloaliphatic or aromatic hydrocarbon radical that has up to 15 carbon atoms and is optionally substituted, for example, by lower alkyl, lower alkoxy, aryl, halogen or by nitro, such as corresponding optionally substituted lower alkyl, phenyl-lower alkyl, cycloalkyl or phenyl, for example 2tri-lower alkylsilylethoxycarbonyl, such as 2-trimethylsilylethoxycarbonyl or 2-(di-n-butylmethylsilyl)-

OS

ethoxycarbonyl, or 2-triarylsilylethoxycarbonyl, such os Co as 2-triphenylsilylethoxycarbonyl.

Further acyl radicals coming into consideration as amino-protecting groups are also corresponding radicals of organic phosphoric, phosphonic or phosphinic acids, t such as di-lower alkylphosphoryl, for example dimethylphosphoryl, diethylphosphoryl, di-n-propylphosphoryl or diisopropylphosphoryl, dicycloalkylphosphoryl, for S.0 0 example dicyclohexylphosphoryl, optionally substituted diphenylphosphoryl, for example diphenylphosphoryl, di-(phenyl-lower alkyl)-phosphoryl that is optionally 4 substituted, for example, by nitro, for example dibenzyl- 0 phosphoryl or di-(4-nitrobenzyl)-phosphoryl, optionally substituted phenoxyphenylphosphonyl, for example phenoxyphenylphosphonyl, di-lower alkylphosphinyl, for example diethylphosphinyl, or optionally substituted i A 26 diphenylphosphinyl, for example diphenylphosphinyl.

In an arylmethylamino group that is a mono-, dior especially tri-arylmethylamino group, the aryl radicals are especially optionally substituted phenyl radicals. Such groups are, for example, benzylamino, diphenylmethylamino and especially tritylamino.

An etherified mercapto group in an amino group protected by such a radical is especially arylthio or aryl-lower alkylthio in which aryl is especially phenyl that is optionally substituted, for example, by lower alkyl, such as methyl or tert.-butyl, lower alkoxy, such as methoxy, halogen, such as chlorine, and/or by nitro. A corresponding amino-protecting group is, for example, 4-nitrophenylthio.

In a 2-acyl-lower alk-1-en-l-yl radical that may be used as an amino-protecting group, acyl is, for example, the corresponding radical of a lower alkanecarboxylic acid, of benzoic acid that is optionally substituted, for example, by lower alkyl, such as methyl or tert.-butyl, lower alkoxy, such as methoxy, halogen, such as chlorine, and/or by nitro, or 2 especially of a carbonic acid semi-ester, such as a carbonic acid lower alkyl semi-ester. Corresponding protecting groups are especially 1-lower alkanoylprop-1en-2-yl, for example 1-acetylprop-1-en-2-yl, or 1-lower ,alkoxycarbonylprop-1-en-2-yl, for example 1-ethoxycarbonylprop-1-en-2-yl.

An amino group can also be protected in protonated form; as corresponding anions there come into consideration especially those of strong inorganic I acids, such as hydrohalic acids, for example the chlorine or bromine anion, or organic sulphonic acids, such as p-toluenesulphonic acid.

Preferred amino-protecting groups are acyl radicals of carbonic acid semi-esters, especially 27 tert.-butoxycarbonyl, or benzyloxycarbonyl or diphenylmethoxycarbonyl optionally substituted, for example as indicated, for example 4-nitrobenzyloxycarbonyl, or 2halo-lower alkoxycarbonyl, such as 2,2,2-trichloroethoxycarbonyl, also trityl or formyl.

A mercapto group, such as, for example, in cysteine, can be protected especially by S-alkylation with optionally substituted alkyl radicals, by thioacetal formation, S-acylation or by establishing asymmetrical disulphide groupings. Preferred mercapto-protecting groups are, for example, benzyl optionally substituted in the phenyl radical, for example by methoxy or nitro, such as 4-methoxybenzyl, diphenylmethyl optionally substituted in the phenyl moiety, for example by methoxy, such as 4,4'-dimethoxydiphenylmethyl, triphenylmethyl, trimethylsilyl, benzylthiomethyl, tetrahydropyranyl, acylaminomethyl, benzoyl, benzyloxycarbonyl or aminocarbonyl, such as ethylaminocarbonyl.

Primary carboxylic acid amide groups (carbamoyl S, groups, -CONH 2 are protected, for example, in the form of N-(9-xanthenyl) derivatives or in the form of N-(mono-, di- or tri-arylmethyl) derivatives, aryl representing especially unsubstituted phenyl or phenyl C substituted by up to 5 identical or different I substituents. Such phenyl substituents are preferably lower alkyl, such as methyl, or lower alkoxy, such as 4 methoxy. There may be mentioned as examples of such arylmethyl protecting groups 4-methoxybenzyl, 2,4,6trimethoxybenzyl, diphenylmethyl, di-(4'-methoxyphenyl)methyl and di-(4-methylphenyl)-methyl.

The protection of carbamoyl groups is optional, that is to say under suitable reaction conditions, for example if using suitable condensing agents, it is not absolutely necessary.

28 Guanidino groups are protected, for example, in the form of an acid addition salt, especially in the form of the hydrochloride or toluenesulphonate.

An acylating agent transferring the radical R i

R

2

R

4 or R 6 is especially the corresponding carboxylic acid, that is Ri-OH, R 2 -COOH, R 4 -OH or

R

6 -OH, or preferably a reactive acid derivative of the same, it being possible for the activation of the carboxylic acid used as acylating agent also to be carried out in situ in the presence of the compound of the formula II.

The invention relates especially to those embodiments of process a) in which an acyl radical R R 4 and/or R 6 is introduced.

Activated carboxylic acid derivatives that can be used as acylating agents are especially reactive activated esters or reactive anhydrides, and also reactive cyclic amides.

Activated esters of acids are especially esters that are unsaturated at the linking carbon atom of the *esterifyi.gj radical, for example of the vinyl ester type, such as actual vinyl esters (which can be obtained, for example, by transesterification of a corresponding ester with vinyl acetate; activated vinyl ester method), carbamoylvinyl esters (which can 1 be obtained, for example, by treating the corresponding acid with an isoxazolium reagent; 1,2-oxazolium or Woodward method), or 1-lower alkoxyvinyl esters (which Scan be obtained, for example, by treating the corresponding acid with a lower alkoxyacetylene; Si* ethoxyacetylene method), or esters of the amidino type, such as N,N'-disubstituted amidino esters (which can be obtained, for example, by treating the corresponding acid with a suitable N,N'-disubstituted carbodiimide, for example N,N'-dicyclohexyl carbodiimide; 29 carbodiimide method), or N,N-disubstituted amidino esters (which can be obtained, for example, by treating the corresponding acid with an N,N-disubstituted cyanamide; cyanamide method), suitable aryl esters, especially phenyl esters suitably substituted by electron-attracting substituents (which can be obtained, for example, by treating the corresponding acid with a suitably substituted phenol, for example 4nitrophenol, 4-methylsulphonylphenol, 2,4,5trichlorophenol, 2,3,4,5,6-pentachlorophenol or 4phenyldiazophenol, in the presence of a condensation agent, such as N,N'-dicyclohexyl carbodiimide; activated aryl esters method), cyanomethyl esters (which can be obtained, for example, by treating the corresponding acid with chloroacetonitrile in the presence of a base; cyanomethyl esters method), thioi esters, especially phenylthioesters optionally substituted, for example, by nitro (which can be obtained, for example, by treating the corresponding acid with o* thiophenols that are optionally substituted, for 84 04 example, by nitro, inter alia with the aid of the anhydride or carbodiimide method; activated thioesters method), or amino or amido esters (which can be obtained, for example, by treating the corresponding s acid with an N-hydroxyamino or N-hydroxyamido compound, for example N-hydroxysuccinimide, N-hydroxypiperidine, N-hydroxyphthalimide or 1-hydroxybenzotriazole, for S* *example according to the anhydride or carbodiimide method; activated N-hydroxy esters method), or silyl esters (which can be obtained, for example, by treating the corresponding acid with a silylating agent, for .U example hexamethyldisilazane, and which readily react with hydroxy groups but not with amino groups).

Anhydrides of acids may be symmetrical or, preferably, mixed anhydrides of these acids, for I 30 example anhydrides with inorganic acids, such as acid halides, especially acid chlorides (which can be obtained, for example, by treating the corresponding acid with thionyl chloride, phosphorus pentachloride or oxalyl chloride; acid chloride method), azides (which can be obtained, for example, from a corresponding acid ester by way of the corresponding hydrazide and the treatment of the latter with nitrous acid; azide method), anhydrides with carbonic acid semiderivatives, such as with corresponding esters, for example carbonic acid lower alkyl semi-esters (which can be obtained, for example, by treating the corresponding acid with haloformic acid lower alkyl esters, such as chloroformic acid lower alkyl esters, or with a 1-lower alkoxycarbonyl-2-lower alkoxy-1,2-dihydroquinoline, for example 1-lower alkoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline; mixed 0-alkylcarbonic acid anhydrides method), or anhydrides with dihalogenated,

S.

especially dichlorinated, phosphoric acid (which can be obtained, for example, by treating the corresponding acid with phosphorus oxychloride; phosphorus oxychloride method), or anhydrides with organic acids, Go such as mixed anhydrides with organic carboxylic acids (which can be obtained, for example, by treating the corresponding acid with an optionally substituted lower alkanecarboxylic acid halide or phenylalkanecarboxylic S" acid halide, for example phenylacetic acid chloride, pivalic acid chloride or trifluoroacetic acid chloride; mixed carboxylic acid anhydrides method) or with organic sulphonic acids (which can be obtained, for example, by treating a salt, such as an alkali metal salt, of the corresponding acid with a suitable S organic sulphonic acid halide, such as lower alkanesulphonic acid chloride or arylsulphonic acid chloride, for example methane- or p-toluene-sulphonic acid Qk_; 31 chloride; mixed sulphonic acid anhydrides method), and symmetrical anhydrides (which can be obtained, for example, by condensing the corresponding acid in the presence of a carbodiimide or of 1-diethylaminopropine; symmetrical anhydrides method).

Suitable cyclic amides are especially amides having five-membered diazacycles of aromatic character, such as amides with imidazoles, for example imidazole (which can be obtained, for example, by treating the corresponding acid with N,N'-carbonyldiimidazole; imidazolide method), or pyrazoles, for example dimethylpyrazole (which can be obtained, for example, by way of the acid hydrazide by treatment with acetylacetone; pyrazolide method).

As mentioned, derivatives of acids, which are used as acylating agents, can also be formed in situ. For example, N,N'-di-substituted amidino esters can be formed in situ by reacting a mixture of the starting oo* material of the formula II and the acid used as acylating agent in the presence of a suitable N,No disubstituted carbodiimide, for example N,N'-dicyclohexyl carbodiimide. It is also possible to form amino Sor amido esters of the acids used as acylating agents in the presence of the starting material of the formula II to be acylated by reacting a mixture of the corresponding acid and amino starting materials in the presence of an N,N'-disubstituted carbodiimide, for example N,N'-dicyclohexyl carbodiimide, and an Nhydroxyamine or N-hydroxyamide, for example N-hydroxysuccinimide, optionally in the presence of a suitable base, for example 4-dimethylaminopyridine.

The reaction can be carried out in a manner known per se, the reaction conditions depending especially on whether and how the carboxy group of the acylating agent has been activated, customarily in the presence i\ 32 of a suitable solvent or diluent or a mixture thereof and, if necessary, in the presence of a condensation agent which, for example when the carboxy group taking part in the reaction is in the form of an anhydride, may also be an acid-binding agent, while cooling or heating, for example in a temperature range of from approximately -30 0 C to approximately +150 0

C,

especially approximately from 0 C to +100 0 C, preferably from room temperature (approximately +20 0 C) to 0 C, in a closed reaction vessel, and/or in the atmosphere of an inert gas, for example nitrogen.

Customary condensation agents are, for example, carbodiimides, for example N,N'-diethyl-, N,N-dipropyl-, N,N'-dicyclohexyl- or N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide, suitable carbonyl compounds, for example carbonyldiimidazole, or 1,2-oxazolium compounds, for example 2-ethyl-5-phenyl-1,2-oxazolium- 3'-sulphonate and perchlorate, or a suitable acylamino compound, for S. example 2-ethoxy-l-ethoxycarbonyl-1,2dihydroquinoline. Customary acid-binding condensation agents are, for example, alkali metal carbonates or bicarbonates, for example sodium or potassium carbonate or bicarbonate (customarily together with a sulphate), or organic bases, such as customarily sterically Sr i hindered tri-lower alkylamines, for example N,Ndiisopropyl-N-ethylamine.

S The removal of the protecting groups, for example the carboxy-, amino-, hydroxy- or mercapto-protecting groups, that are not constituents of the desired end product of the formula I is carried out in a manner known per se, for example by means of solvolysis, especially hydrolysis, alcoholysis or acidolysis, or by means of reduction, especially hydrogenolysis or chemical reduction, optionally in stages or simul- 33 taneously, it also being possible to use enzymatic methods.

Thus, tert.-lower alkoxycarbonyl, or lower alkoxycarbonyl substituted in the 2-position by an organic silyl group or in the 1-position by lower alkoxy or lower alkylthio, or optionally substituted diphenylmethoxycarbonyl can be converted into free carboxy, for example, by treatment with a suitable acid, such as formic acid or trifluoroacetic acid, optionally with the addition of a nucleophilic compound, such as phenol or anisole. Optionally substituted benzyloxycarbonyl can be freed, for example, by hydrogenolysis, that is to say by treatment with hydrogen in the presence of a metallic hydrogenation catalyst, such as a palladium catalyst. In addition, suitably substituted benzyloxycarbonyl, such as 4-nitrobenzyloxycarbonyl, can also be converted into free carboxy by chemical reduction, for example by treatment with an alkali metal dithionite, for example sodium dithionite, or with a reducing S' metal, for example zinc, or a reducing metal salt, such as a chromium(II) salt, for example chromium(II) chloride, usually in the presence of an agent that yields hydrogen and that, together with the metal, is capable of producing nascent hydrogen, such as an acid, Ir especially a suitable carboxylic acid, such as a lower r alkanecarboxylic acid optionally substituted, for example, by hydroxy, for example acetic acid, formic i acid, glycolic acid, diphenylglycolic acid, lactic acid, mandelic acid, 4-chloromandelic acid or tartaric acid, or an alcohol or thiol, water preferably being added. By treatment with a reducing metal or metal salt, as described above, it is also possible to convert 2-halo-lower alkoxycarbonyl (optionally after conversion of a 2-bromo-lower alkoxycarbonyl group into a corresponding 2-iodo-lower alkoxycarbonyl group) or -rr,,-nuLr 34 aroylmethoxycarbonyl into free carboxy, it being possible to cleave aroylmethoxycarbonyl also by treatment with a nucleophilic, preferably salt-forming, reagent, such as sodium thiophenolate or sodium iodide.

Substituted 2-silylethoxycarbonyl can also be converted into free carboxy by treatment with a salt of hydrofluoric acid yielding the fluoride anion, such as an alkali metal fluoride, for example sodium or potassium fluoride, in the presence of a macrocyclic polyether ("Crown ether"), or with a fluoride of an organic quaternary base, such as tetra-lower alkylammonium fluoride or tri-lower alkylarylammonium fluoride, for example tetraethylammonium fluoride or tetrabutylammonium fluoride, in the presence of an aprotic polar solvent, such as dimethyl sulphoxide or N,N-dimethylacetamide.

Esterified carboxy can also be cleaved 'enzymatically; for example esterified lysin, for example lysin methyl ester, can be cleaved by means of trypsin.

SA protected amino group is freed in a manner known S. per se and, depending on the nature of the protecting groups, by various methods, but preferably by solvolysis or reduction. 2-halo-lower alkoxycarbonyl- 4 amino (optionally after conversion of a 2-bromo-lower alkoxycarbonylamino group into a 2-iodo-lower alkoxycarbonylamino group), aroylmethoxycarbonylamino or 4-nitrobenzyloxycarbonylamino can be cleaved, for example, by treatment with a suitable chemical reducing agent, such as zinc in the presence of a suitable carboxylic acid, such as aqueous acetic acid. Aroylmethoxycarbonylamino can also be cleaved by treatment with a nucleophilic, preferably salt-forming, reagent, such as sodium thiophenolate, and 4-nitrobenzyloxycarbonylamino also by treatment with an alkali metal

-K.

C- 'I; 48 35 dithionite, for example sodium dithionite. Optionally substituted diphenylmethoxycarbonylamino, tert.-lower alkoxycarbonylamino or 2-trisubstituted silylethoxycarbonylamino can be cleaved by treatment with a suitable acid, for example formic acid or trifluoroacetic acid, optionally substituted benzyloxycarbonylamino can be cleaved, for example, by hydrogenolysis, that is to say by treatment with hydrogen in the presence of a suitable hydrogenation catalyst, such as a palladium catalyst, optionally substituted triarylmethylamino or formylamino can be cleaved, for example, by treatment with an acid, such as a mineral acid, for example hydrochloric acid, or an organic acid, for example formic, acetic or trifluoroacetic acid, optionally in the presence of water, and an amino group protected by an organic silyl group can be freed, for example, by hydrolysis or alcoholysis. An amino group protected by "2-haloacetyl, for example 2-chloroacetyl, can be freed by treatment with thiourea in the presence of a base, or with a thiolate salt, such as an alkali metal thiolate, of thiourea, and by subsequent solvolysis, such as alcoholysis or hydrolysis, of the resulting condensation product. An amino group protected by 2substituted silylethoxycarbonyl can also be converted Sinto the free amino group by treatment with a salt of I hydrofluoric acid yielding fluoride anions, as indicated above in connection with the freeing of a correspondingly protected carboxy group.

Amino protected in the form of an azido group is converted into free amino, for example, by reduction, for example by catalytic hydrogenation with hydrogen in the presence of a hydrogenation catalyst, such as platinum oxide, palladium or Raney nickel, or alternatively by treatment with zinc in the presence of an acid, such as acetic acid. The catalytic hydrogenation 1 36 is preferably carried out in an inert solvent, such as a halogenated hydrocarbon, for example methylene chloride, or alternatively in water or a mixture of water and an organic solvent, such as an alcohol or dioxan, at approximately from 20 0 C to 25 0 C, or alternatively while cooling or heating.

A hydroxy or mercapto group protected by a suitable acyl group, an organic silyl group or by optionally substituted 1-phenyl-lower alkyl is freed analogously to a correspondingly protected amino group.

Hydroxy protected by optionally substituted 1-phenyllower alkyl, for example benzyl, is freed preferably by catalytic hydrogenation, for example in the presence of a palladium-on-carbon catalyst. A hydroxy or mercapto group protected by 2,2-dichloroacetyl is freed, for example, by basic hydrolysis, and a hydroxy or mercapto group etherified by tert.-lower alkyl or by a 2-oxa- or s' 2-thia-aliphatic or -cycloaliphatic hydrocarbon t. radical is freed by acidolysis, for example by treatment with a mineral acid or a strong carboxylic acid, 'o 4 for example trifluoroacetic acid. Two hydroxy groups that are together protected by a preferably substituted methylene group, such as by lower alkylidene, for example isopropylidene, cycloalkylidene, for example cyclohexylidene, or benzylidene, can be freed by acid S. solvolysis, especially in the presence of a mineral acid or a strong organic acid. Hydroxy etherified by :an organic silyl radical, for example trimethylsilyl, can also be freed with a salt of hydrofluoric acid that yields fluoride anions, for example tetrabutylammonium fluoride.

If several protected functional groups are present, the protecting groups are preferably so chosen that more than one such group can be removed at the same time, for example by acidolysis, such as by 37 treatment with trifluoroacetic acid or formic acid, or by reduction, such as by treatment with zinc and acetic acid, or with hydrogen and a hydrogenation catalyst, such as a palladium-on-carbon catalyst. If the desired end products contain protecting groups, for example if

R

12 represents arylmethoxy, such as benzyloxy, those protecting groups which are to be removed when the reaction is complete are so selected that they can be removed again regioselectively, for example hydroxy etherified with an organic silyl radical in the radical R or R 11 can be freed with fluoride, an arylmethoxy protecting group being retained in the radical R 9 or R 12 The starting materials of the formula II in which

R

2a represents hydrogen are manufactured, for example, from corresponding compounds in which R 2a represents an amino-protecting group, for example B benzyloxycarbonyl.

S e Process b: A reactive derivative of a compound of the formula 4 4 III is, for example, a metal oxy compound, as can be obtained, for example, by reacting a compound of the formula III with a suitable base, such as an alkali metal hydride or amide.

Reactive esterified hydroxy X is, for example, hydroxy esterified by a strong inorganic or organic 4 acid, such as a mineral acid, for example a hydrohalic acid, such as hydrochloric, hydrobromic or hydriodic acid, also sulphuric acid or a halosulphuric acid, for example fluorosulphuric acid, or with a strong organic sulphonic acid, such as a lower alkanesulphonic acid optionally substituted, for example, by halogen, such as fluorine, or an aromatic sulphonic acid, for example a benzenesulphonic acid optionally substituted by lower

IF.

r 38 alkyl, such as methyl, halogen, such as bromine, and/or by nitro, for example a methanesulphonic, trifluoromethanesulphonic or p-toluenesulphonic acid, and is preferably a chloride, bromide or iodide.

Free functional groups in a compound of the formula IV that are advantageously protected by readily removable protecting groups are especially hydroxy, mercapto or carboxy groups.

The reaction is preferably carried out in an inert organic solvent at a temperature of from -30 0 C to +150 0 C, especially from 0 C to +100 0 C, for example from +20°C to +70 0 C, if necessary in the presence of an acid-binding agent.

The removal of protecting groups that are not constituents of the desired end product of the formula I is carried out as described in process a).

Process c: A reactive carboxylic acid derivative of a compound of the formula V is especially a reactive ester, a reactive anhydride or a reactive cyclic amide in which the carboxy group is activated analogously to the reactive acylating agents described in process a), it also being possible for the activation to be carried I' 'out in situ.

Functional groups that may be present in compounds of the formula V or VI, which are preferably protected by readily removable protecting groups, are especially T1 amino and guanidino in the radical R 1 1 carboxy as radical R i0 and hydroxy as radical R 9 or R 12 but in addition also hydroxy or mercapto in the radical •49 R 8 or hydroxy in the radical R 11 4 In a reactive derivative of a compound of the formula VI the amino group is activated, for example, by reaction with a phosphite, for example diethyl 39 chlorophosphite, 1,1-phenylene chlorophosphite, ethyl dichlorophosphite, ethylene chlorophosphite or tetraethyl pyrophosphite, or by bonding to a halocarbonyl, for example chlorocarbonyl, or is activated in the form of an isocyanate group. Preferably, the reaction is carried out by reacting a reactive carboxylic acid derivative of a compound of the formula V with a compound of the formula VI, the amino or hydroxy group participating in the reaction being present in free form.

The reaction and the subsequent removal of the protecting groups that are not constituents of the desired end product are carried out in a manner analogous to that described in process a).

The esterification of a compound of the formula V in which r and t represent 1 can also be carried 4 *,out by means of the esterifying agents described in process d).

00 O oProcess d: o Free functional groups that may be present in a o 4 compound of the formula VII, which are protected by readily removable protecting groups, are especially carboxy groups that are not to be esterified, and also *hydroxy, mercapto, guanidino and amino groups.

I Suitable protecting groups and the removal thereof f are described in process a).

A reactive carboxylic acid derivative of a compound of the formula VII is especially a reactive ester, a reactive anhydride or a reactive cyclic amide in which the carboxy group is activated analogously to the reactive acylating agents described in process a), p it also being possible for the activation to be carried out in situ. Preferably, the reaction is carried out by reacting a reactive carboxylic acid derivative of a 40 compound of the formula VII, for the esterification of carboxy R 13 with a compound of the formula H-Rga in in which R 9a has the above-mentioned meanings of R with the exception of hydroxy and amino or, for the esterification of carboxy R 1 a, with a lower alkanol or aryl-lower alkanol.

Alternatively, a compound of the formula VII having a free carboxy group can be esterified by reaction with a reactive derivative of the alcohol desired as esterification component.

Suitable agents for the esterification are, for example, corresponding diazo compounds, such as optionally substituted diazo-lower alkanes, for example diazomethane, diazoethane, diazo-n-butane or diphenyldiazomethane. These reagents are used in the presence of a suitable inert solvent, such as an aliphatic, cycloaliphatic or aromatic hydrocarbon, such as hexane, o 00 cyclohexane, benzene or toluene, a halogenated 4* o aliphatic hydrocarbon, for example methylene chloride, or an ether, such as a di-lower alkyl ether, for example diethyl ether, or a cyclic ether, for example tetrahydrofuran or dioxan, or in the presence of a solvent mixture and, depending on the diazo reagent used, while cooling, at room temperature or while heating slightly, also, if necessary, in a closed £,eg vessel and/or under an inert gas atmosphere, for example a nitrogen atmosphere.

Further suitable agents for the esterification are esters of corresponding alcohols, especially those with strong inorganic or organic acids, such as mineral acids, for example hydrohalic acids, such as hydrochloric acid, hydrobromic acid or hydriodic acid, also sulphuric acid, or halosulphuric acid, for example fluorosulphuric acid, or strong organic sulphonic acids, such as lower alkanesulphonic acids that are I- r 41 optionally substituted, for example, by halogen, such as fluorine, or aromatic sulphonic acids, such as, for example, benzenesulphonic acids optionally substituted by lower alkyl, such as methyl, halogen, such as bromine, and/or by nitro, for example methanesulphonic, trifluoromethanesulphonic or p-toluenesulphonic acid.

Such esters are, inter alia, lower alkyl halides, dilower alkyl sulphates, such as dimethyl sulphate, also fluorosulphonic acid esters, such as fluorosulphonic acid lower alkyl esters, for example fluorosulphonic acid methyl ester, or optionally halo-substituted methanesulphonic acid lower alkyl esters, for example trifluoromethanesulphonic acid methyl ester. They are customarily used in the presence of an inert solvent, such as an optionally halogenated, such as chlorinated, aliphatic, cycloaliphatic or aromatic hydrocarbon, for example methylene chloride, an ether, such as dioxan or tetrahydrofuran, or a mixture thereof. There are preferably also used suitable condensation agents, such as alkali metal carbonates or bicarbonates, for example sodium or potassium carbonate or bicarbonate (usually together with a sulphate), or organic bases, such as Stri-lower alkylamines which are customarily sterically C, 4 hindered, for example N,N-diisopropyl-N-ethylamine S(preferably together with halosulphonic acid lower alkyl esters or optionally halo-substituted methanesulphonic acid lower alkyl esters), the operation being carried out while cooling, at room temperature or while heating, for example at temperatures of from approximately -20 0 C to approximately 50 0 C, and, if necessary, in a closed vessel and/or in an inert gas atmosphere, for example a nitrogen atmosphere.

Further agents for the esterification are corresponding tri-substituted oxonium salts (so-called i 42 Meerwein salts) or di-substituted carbenium or halonium salts in which the substituents are the etherifying radicals, for example tri-lower alkyloxonium salts and di-lower alkoxycarbenium or di-lower alkylhalonium salts, especially the corresponding salts with complex fluorine-containing acids, such as the corresponding tetrafluoroborates, hexafluorophosphates, hexafluoroantimonates or hexachloroantimonates. Such reagents are, for example, trimethyloxonium or triethyloxonium hexafluoroantimonate, hexachloroantimonate, hexafluorophosphate or tetrafluoroborate, dimethoxycarbenium hexafluoro-phosphate or dimethylbromonium hexafluoroantimonate. These agents are preferably used in an inert solvent, such as an ether or a halogenated hydrocarbon, for example diethyl ether, tetrahydrofuran or methylene chloride, or in a mixture thereof, if necessary in the presence of a base, such as an organic base, for example a tri-lower alkylamine which is preferably sterically hindered, for example N,N-diisopropyl-N-ethylamine, and while cooling, at room temperature or while heating slightly, 0 .o for example at from approximately -20 0 C to approximately 50 0 C, if necessary in a closed vessel and/or in an inert gas atmosphere, for example a nitrogen atmosphere.

S A preferred embodiment of process d) is the f PP reaction of a caesium salt of a compound of the formula VII with the alcohol desired as esterification component, in which the hydroxy group is present in reactive esterified form.

4. 0 Process e: A compound of the formula I in which at least one of the radicals R 8

R

9

R

10

R

11 and R 12 is present in a protected form which does not correspond to the I 43 43 definition of the desired end product is especially a compound of the formula I in which a hydroxy or mercapto group in the radical R 8 an amino or hydroxy group in the radical R 1 1 free hydroxy R 9 or R 12 and/or carboxy R 10 is(are) protected by a readily removable protecting group that is not contained in the desired end product.

Readily removable protecting groups are especially those mentioned in process The removal of protecting groups is carried out in a manner analogous to that described in process a).

Process f: Free functional groups that may be present in a compound of the formula VIII and that are nrotected by readily removable protecting groups are especially S* carboxy groups that are not to be amidated, and also hydroxy, mercapto, guanidino and amino groups.

Suitable protecting groups and the removal thereof are described in process a).

4 reactive carboxylic acid derivative of a compound of the formula VIII is especially a reactive ester, a reactive anhydride or a reactive cyclic amide, in which the carboxy group is activated analogously to the reactive acylating agents described in process a), 7 it also being possible for the activation to be carried Sout in situ. Preferably, the reaction is carried out by reacting a reactive carboxylic acid derivative of a compound of the formula VIII with ammonia. Alternatively, it is also possible to react a compound of the s* formula VIII having a free carboxy group with a reactive ammonia derivative, the activation of the amino group being carried out, for example, in a manner analogous to that described in process c).

The reaction and the subsequent removal of the -2 r 44 protecting groups that are not constituents of the desired end product is carried out in a manner analogous to that described in process a).

Additional operations: Salts of compounds of the formula I having salt-forming groups can be manufactured in a manner known per se. For example, salts of compounds of the formula I having acidic groups can be formed by reaction with a suitable base, for example by treatment with suitable metal compounds, such as alkali metal salts of suitable organic carboxylic acids, for example the sodium salt of a-ethylcaproic acid, or with suitable inorganic alkali or alkaline earth metal salts, especially those derived from a weak and preferably volatile acid, for example sodium bicarbonate, or with ammonia or a suitable organic amine, preferably stoichiometric amounts or only a small excess of the salt-forming agent being used. Acid addition salts of compounds of the formula I are obtained in customary manner, for example by treatment with an acid or a suitable anion exchange reagent. Internal salts of compounds of the formula I that contain, for example, a free carboxy group and a free amino group, can be formed, for example, by neutralising salts, such as acid addition salts, to the isoelectric point, for example with weak bases, or by treatment with liquid ion exchangers.

Salts can be converted into the free compounds in customary manner; metal and ammonium salts can be converted, for example, by treatment with suitable acids, and acid addition salts, for example by treatment with a suitable basic agent.

Mixtures of isomers can be resolved into the individual isomers in a manner known per se, for C V.* CC C ca+ 5.

I*

C IS r SC i 9. d* SC h I. 0 CC S

SCCI&

4 0o~

I

45 example by fractional crystallisation, chromatography etc.

The processes described above, including the processes for removing protecting groups and the additional process steps, are, unless indicated otherwise, carried out in a manner known per se, for example in the presence or absence of preferably inert solvents and diluents, if necessary in the presence of condensation agents or catalysts, at reduced or elevated temperature, for example in a temperature range of from approximately -20 0 C to approximately +150 0 C, especially from approximately 0°C to approximately +70 0 preferably from approximately 0 °C to approximately +40 0 C, above all at room temperature, in a closed vessel and/or in an inert gas atmosphere, for example a nitrogen atmosphere.

Taking into account all the substituents in the molecule, there should be used, if necessary, for example if readily hydrolysable radicals are present, particularly mild reaction conditions, such as short reaction times, mild acidic or basic agents in a low concentration and stoichiometric quantity ratios, and suitable catalysts, solvents, temperature and/or pressure conditions should be selected.

The invention also relates to those embodiments of the process in which a compound obtainable as intermediate at any stage of the process is used as starting material and the remaining process steps are carried out or the process is interrupted at any stage or a starting material is formed under the reaction conditions or is used in the form of a reactive derivative or salt. The starting materials used are preferably those which, according to the process, result in the compounds described above as being especially valuable.

B £4 4* 4 S.t 044 ot 0 04 0 5 r I 70 Lt I C

I

46 The present invention also relates to novel starting materials and/or intermediates and to processes for their manufacture. The starting materials used and the reaction conditio.,s chosen are preferably those which result in the compounds described in this Application as being especially preferred.

The invention also relates to pharmaceutical preparations that contain an effective amount of the active ingredient, especially an amount that is effective for the prophylaxis or treatment of virus infections or for the treatment of tumour diseases, together with pharmaceutically acceptable carriers, which are suitable for topical, for example intranasal, enteral, for example oral or rectal, or parenteral administration, and which may be inorganic or organic and solid or liquid. For example, there are used tablets or gelatine capsules that contain the active ingredient together with diluents, for example lactose, S e dextrose, sucrose, mannitol, sorbitol, cellulose and/or S* glycerine, and/or lubricants, for example silica, talc, S stearic acid or salts thereof, such as magnesium or Da calcium stearate, and/or polyethylene glycol. Tablets may also contain binders, for example magnesium .r aluminium silicate, starches, such as corn, wheat or Sr. rice starch, gelatine, methylcellulose, sodium carboxymethylcellulose and/or polyvinyloyrrolidone, S and, if desired, disintegrators, for example starches, agar, alginic acid or a salt thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, 2 colourings, flavourings and sweeteners. The a pharmacologically active compounds of the present invention can also be used in the form of parenterally administrable preparations or in the form of infusion solutions. Such solutions are preferably isotonic 1 47 aqueous solutions or suspensions, it being possible, for example in the case of lyophilised preparations that contain the active ingredient alone or together with a carrier, for example mannitol, for these to be manufactured before use. The pharmaceutical preparations may be sterilised and/or contain adjuncts, for example preservatives, stabili4ers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers. The present pharmaceutical preparations which, if desired, may contain other pharmacologically active ingredients, such as antibiotics, are manufactured in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising processes, and contain approximately from 0.001% to especially from approximately 0.01% to approximately 10%, more especially from 0.1% to of 0 o4 the active ingredient(s), an active ingredient concentration of less than 1% being especially suitable for preparations that are to be applied topically.

The following are preferred as forms of administration to be applied topically: creams, ointments or pastes having an active ingredient content of from 0.001% to especially from 0.01% to for example 0.05%, for example ointments for intranasal ct application or lipsticks, or aqueous solutions having an active ingredient content of from 0.001% to 1%, especially from 0.05% to for example 0.1%, S" preferably isotonic, sterile and physiologically tolerable solutions, for example eye drops, preferably in microcontainers for once-only use, or sprays for use in the mouth and throat area.

The pharmaceutical preparations described in the Examples are especially suitable.

Creams are oil-in-water emulsions that contain ,t

J

48 more than 50% water. There are used as oily base especially fatty alcohols, for example lauryl, cetyl or stearyl alcohol, fatty acids, for example palmitic or stearic acid, liquid to solid waxes, for example isopropyl myristate, wool wax or beeswax, and/or hydrocarbons, for example petroleum jelly (petrolatum) or paraffin oil. Suitable as emulsifiers are surfaceactive substances having predominantly hydrophilic properties, such as corresponding non-ionic emulsifiers, for example fatty acid esters of polyalcohols or ethylene oxide adducts thereof, such as polyglycerine fatty acid esters or polyoxyethylene sorbitan fatty acid esters (Tweens), also polyoxyethylene fatty alcohol ethers or polyoxyethylene fatty acid esters, or corresponding ionic emulsifiers, such as alkali metal salts of fatty alcohol sulphates, for example sodium lauryl sulphate, sodium cetyl sulphate or sodium stearyl sulphate, which are usually used in the presence of fatty alcohols, for example cetyl alcohol or stearyl alcohol. Additives to the aqueous phase are, inter alia, agents that reduce the drying-out of the creams, for example polyalcohols, such as glycerine, sorbitol, propylene glycol and/or polyethylene glycols, also preservatives, perfumes, etc..

Ointments are water-in-oil emulsions that contain up to 70%, but preferably from approximately 20% to approximately 50%, water or aqueous phase. Suitable as the fatty phase are especially hydrocarbons, for example petroleum jelly, paraffin oil and/or hard paraffins, which, in order to improve the water-binding capacity, preferably contain suitable hydroxy compounds, "I such as fatty alcohols or esters thereof, for example cetyl alcohol or wool wax alcohols, or wool wax.

Emulsifiers are corresponding lipophilic substances, 49 0 0 00 p 0000 0U o 0 000 *d 00 p ow *i 0 0-0. 0 such as sorbitan fatty acid esters (Spans), for example sorbitan oleate and/or sorbitan isostearate. Additives to the aqueous phase are, for example, humectants, such as polyalcohols, for example glycerine, propylene glycol, sorbitol and/or polyethylene glycol, and also preservatives, perfumes, etc..

Fatty ointments are anhydrous and contain as base especially hydrocarbons, for example paraffin, petroleum jelly and/or liquid paraffins, also natural or partially synthetic fats, for example coconut fatty acid triglyceride, or preferably hardened oils, for example hydrogenated groundnut or castor oil, also fatty acid partial esters of glycerine, for example glycerine mono- and di-stearate, and also, for example, the fatty alcohols increasing the water-absorption capacity, emulsifiers and/or additives mentioned in connection with the ointments.

Pastes are creams and ointments having secretion-absorbing powder constituents, such as metal oxides, for example titanium oxide or zinc oxide, also talc and/or aluminium silicates, the purpose of which is to bind any moisture or secretions present.

Foams are administered from pressurised containers and are liquid oil-in-water emulsions in aerosol form; halogenated hydrocarbons, such as chlorofluoro-lower alkanes, for example dichlorodifluoromethane and dichlorotetrafluoroethane, are used as propellants.

There are used as the oily phase, inter alia, hydrocarbons, for example paraffin oil, fatty alcohols, for example cetyl alcohol, fatty acid esters, for example isopropyl myristate, and/or other waxes. There are used as emulsifiers, inter alia, mixtures of emulsifiers having predominantly hydrophilic properties, such as polyoxyethylenesorbitan fatty acid esters (Tweens), and emulsifiers with predominantly 00 *r 0 0*40 00 0* *J 0 00

I

-1 m(.

50 lipophilic properties, such as sorbitan fatty acid esters (Spans). The customary additives, such as preservatives, etc., are also added.

Tinctures and solutions generally have an aqueousethanolic base to which are added, inter alia, polyalcohols, for example glycerine, glycols and/or polyethylene glycol, as humectants for reducing evaporation, and fat-restoring substances, such as fatty acid esters with lower polyethylene glycols, that is to say lipophilic substances that are soluble in the aqueous mixture, as a replacement for the fatty substances removed from the skin by the ethanol, and, if necessary, other adjuncts and additives.

The manufacture of the topically administrable pharmaceutical preparations is carried out in a manner known per se, for example by dissolving or suspending the active ingredient in the base or, if necessary, in a part thereof. When processing the active ingredient as a solution, it is generally dissolved in one of the two phases before emulsification; when processing the active ingredient as a suspension, it is mixed with part of the base after emulsification and then added to the rest of the formulation.

44 4 p,

S.

*i S 4540 4.

4 o 055 o S 0 .i4 0 P"s i *4 4i 1440 54 44 4

UMW.&

51 The following Examples illustrate the invention without limiting it in any way. The Rf values are ascertained on thin layer silica gel plates (Merck, Darmstadt, Germany). The ratio of eluants in the eiuant mixtures used is in proportions by volume and temperatures are given in degrees Celsius. The concentratlrOn, c, of the substance in the solvent (or solvent mixture) is in the case of optical rotation given as a percentage (weight/volume).

Abbreviations: abs. absolute Boc tert.-butoxycarbonyl HV high vacuum i. vac. in vacuo Me methyl MeOH methanol PTFE polytetrafluoroethylene, Teflon® RT room temperature m.p. melting point So decomp. decomposition Example 1: 11.4 g (16.77 mmol) of N-propionyl- S demethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester (a,8-mixture), which contains 1.17 mol of water, are dissolved in 120 ml of absolute pyridine. 7.6 ml (80.3 mmol) of acetic anhydride are added to the resulting solution and the whole is stirred for 92 hours at room temperature. Subsequently the yellowish solution is concentrated by evaporation in a high vacuum at 400. The resulting crude product is triturated with 100 ml of diethyl ether and the resulting suspension is stirred for 2 hours at room l/ I- I- 1 i p.

t" 1

I

52 .4 44 4 o 4 4 4.

0 44 II

'I

4t 44 4 temperature. The crystals that have formed are then filtered off with suction and subsequently washed with diethyl ether.

After recrystallisation twice from ethyl acetate/ isopropanol/diethyl ether (100:5:40) a first fraction of chromatographically pure 1,4,6-tri-O-acetyl-Npropionyl-demethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester (a,8-mixture) is obtained in the form of colourless crystals, m.p. 165-1660 The mother liquors from the recrystallisations are combined and concentrated by evaporation in vacuo.

The resulting residue is purified by column chromatography on 600 g of silica gel (type 60, high purity, Merck; 0.063-0.2 mm) in the system chloroform/ethanol (10 ml fractions).

Fractions 217-310 are combined and concentrated by evaporation in vacuo. A second fraction of chromatographically pure 1,4,6-tri-O-acetyl-N-propionyldemethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester (a,3-mixture) is obtained which, together with the first fraction, is dissolved in 200 ml of absolute methanol. The resulting slightly turbid solution is then filtered through a millipore filter (Fluoropore, PTFE, 0.2 pm) and the clear filtrate is concentrated by evaporation in vacuo at 400. The residue is then dissolved in 50 ml of absolute ethyl acetate, which has previously been filtered through a millipore filter (Fluoropore, PTFE, 0.2 um), and is crystallised by the addition of 20 ml of absolute diethyl ether, which has also been filtered through a millipore filter, and is subsequently washed with filtered absolute diethyl ether. 1,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl- L-alanyl-D-isoglutamine benzhydryl ester -mixture) is obtained in the form of colourless crystals, m.p. 165-1660, which still contain 0.27 mol of water.

4444 I SI- I

I

I I I i ;4 53 C38H48N4014 0.27 H 2 0 (789.68) Calc. C 57.80 H 6.22 N 7.10 0 28.91 H 2 0 0.62 Found C 57.91 H 6.20 N 7.11 0 28.77 H 2 0 0.62 32.7 2.10 (c 0.486, methanol), R 0.28 D f (chloroform:methanol Rf 0.70 (chloroform: methanol 4:1).

A further product is obtained from fractions 105-140 of the above-described column chromatography.

The yellow residue (0.55 g, foam) obtained after concentration by evaporation in vacuo is dissolved hot in cyclohexane/diethyl ether/methylene chloride (10:30:5) and the resulting solution is cooled to room temperature. A yellow oil separates off in the course of cooling which, after the solvent has been decanted off, is stirred for 1/2 hour with 50 ml of absolute diethyl ether. The resulting crystals are filtered off o* with suction and subsequently washed with absolute diethyl ether.

o The crystals are then dissolved in 100 ml of e t) *tert.-butanol/water The resulting solution is filtered through a millipore filter (Fluoropore, PTFE, 0.2 um) and lyophilised.

la,4,6-tri-0-acetyl-2-deoxy-2-propionylamino-Dbenzhydryl ester is obtained in the form of a colourless powder S..containing 1.11 mol of water.

ar"C38 48 40 4 1.11 H 2 0 (804.31) Calc. C 56.71 H 6.31 N 6.96 0 30.03 H 2 0 2.48 SFound C 56.94 H 6.36 N 7.22 0 30.15 H 2 0 2.48 20 2o 5.5 2.70 (c 0.365, methanol), Rf 0.49 (chloroform:methanol Rf 0.79 (chloroform: methanol 4:1).

54 The starting material is obtained as follows: Stage 1.1: 16.0 g (31.63 mmol) of N-propionyldemethylmuramyl-L-alanyl-D-isoglutamine containing 0.74 mol of water are dissolved in 300 ml of methanol/ dimethoxyethane and then 9.2 g (47.4 mmol) of diphenyldiazomethane are added. The whole is stirred for 20 hours at room temperature. After 20, 44 and 68 hours, in each case a further 4.6 g (23.7 mmol) of diphenyldiazomethane is added. When the reaction is complete (90 hours), the red solution is concentrated to dryness by evaporation in vacuo at 400, 300 ml of absolute diethyl ether are added to the resulting red resin and the whole is stirred for 1/2 hour at room temperature.

Colourless crystals are obtained, which are filtered off with suction and subsequently washed with 0 diethyl ether.

Recrystallisation from 400 ml of acetone yields N-propionyl-demethylmuramyl-L-alanine-D-isoglutamine S* benzhydryl ester in the form of colourless crystals, m.p. 188-1900 (decomp.), containing 1.17 mol of water.

C32H42N4011 1.17 H 2 0 (679.78) Calc. C 56.55 H 6.60 N 8.24 O 28.63 H 2 0 3.09 SFound C 56.67 H 6.67 N 8.43 O 28.65 H 2 0 3.09 7.5 0.1 (c 0.898, methanol), Rf 0.62 (chloroform:methanol:water 70:30:5), Rf 0.62 (chloroform:methanol 7:3).

Example 2: 2.33 g (3.8 mmol) of N-propionyldemethylmuramyl-L-alanyl-D-isoglutamine benzyl ester, which still contains 1.59 mol of water, are dissolved in 24 ml of absolute pyridine, then 1.4 ml (14.8 mmol) of acetic anhydride are added and the resulting

SB-

solution is stirred for 16 hours at room temperature.

Subsequently the colourless solution is concentrated by evaporation in a high vacuum at 300. A slightly yellow foam is obtained, which is purified by column chromatography on 400 g of silica gel (type 60, highpurity, Merck; 0.063-0.2 mm) in the system chloroform/ ethanol (10 ml fractions), the B-anomer, the a,B-anomeric mixture and the a-anomer of 1,4,6-tri-Oacetyl-N-propionyl-demethylmuramyl-L-alanyl-D-isoglutamine benzyl ester being eluted in succession.

Fractions 112-126 (B-anomer), 127-190 (amixture) and 191-260 (a-anomer) are in each case combined and concentrated by evaporation in a high vacuum at The residue of fractions 112-126 is crystallised from 60 ml of chloroform:diethyl ether 1(,4,6tri-O-acetyl-N-propionyl-demethylmuramyl-L-alanyl-Disoglutamine benzyl ester is obtained in the form of 8.S colourless crystals having a melting point of 189-1900.

0a The crystals are subsequently dissolved in 100 ml of o twice-distilled water/tert.-butanol The resulting solution is filtered through a millipore filter (Fluoropore, PTFE, 0.2 um) and lyophilised in a high vacuum. 18,4,6-tri-O-acetyl-N-propionyl-demethyl- Smuramyl-L-alanyl-D-isoglutamine benzyl ester is obtained in the form of a colourless powder containing 1.21 mol of water.

C32H44N4014 1.21 H 2 0 (730.52) Calc. C 52.62 H 6.43 N 7.67 0 33.30 H 2 0 2.98 Found C 52.97 H 6.27 N 7.86 0 33.24 H 2 0 2.98 00 2 20 o t] 14.3 4.2' (c 0.237, dimethylformamide), Rf 0.25 (chloroform:ethanol Rf 0.67 (chloroform:ethanol Rf 0.45 (chloroform: methanol 9.1).

i-r 56 The residue of fractions 127-190 is crystallised from 120 ml of chloroform/diethyl ether 1,4,6tri-O-acetyl-N-propionyl-demethylmuramyl-L-alanyl-Disoglutamine benzyl ester (a,B-mixture, according to H-NMR ca:B 6:1) is obtained in the form of colourless crystals, m.p. 156-1570 The crystals are then dissolved in 150 ml of twice-distilled water/tert.-butanol The resulting solution is filtered through a millipore filter (Fluoropore, PTFE, 0.2 jim) and lyophilised in a high vacuum. 1,4,6-tri-0-acetyl-N-propionyl-demethylmuramyl- L-alanyl-D-isoglutamine benzyl ester (a,B-mixture) is obtained in the form of a colourless powder containing 1.00 mol of water.

C

3 2 H44N 4 014 1.00 H 2 0 (726.73) S.4 Calc. C 52.89 H 6.41 N 7.71 0 33.02 H 0 2.48 2 SFound C 53.18 H 6.42 N 7.49 0 32.81 H 0 2.48 ao 2 20+ o S[a]D0 56.0 1.10 (c 0.948; dimethylformamide), 0*4- [ci]D 6 04) S* R 0.17 0.24 (chloroform:ethanol 9:1; double spot), Rf 0.45 (chloroform:methanol Rf 0.67 *(chlotoform:methanol 4:1).

The residue of fractions 191-260 is crystallised from 90 ml of chloroform/diethyl ether lca,4,6tri-0-acetyl-N-propionyl-demethylmuramyl-L-alanyl-Disoglutamine benzyl ester is obtained in the form of colourless crystals, m.p. 147-1490 The crystals are then dissolved in 100 ml of twicedistilled water/tert.-butanol The resulting £4 r t solution is filtered through a millipore filter (Fluoropore, PTFE, 0.2 pm) and lyophilised in a high vacuum.

la,4,6-tri-0-acetyl-demethylmuramyl-N-propionyl-L-alanyl- D-isoglutamine benzyl ester is obtained in the form of a colourless powder still containing 1.12 mol of water.

57 C 32

H

44 N 4 0 14 .1.12 H 2 0 (728.89) Caic. C 52.74 H 6.43 N 7.69 0 33.18 H 2 0 2.76 Found C 52.96 H 6.54 N 7.62 0 33.15 H 2 0 2.76 36.9 1.70 (c 0.593; methylene chloride: methanol Rf 0.17 (chloroform:ethanol Rf 0.45 (chloroform:methanol Rf =0.67 (chloroform:methanol Example 3: Analogously to Example 1 there is obtained from 1.19 g (1.54 mmol) of N-propionyldemethylmuramyl-L-alanyl--D-isoglutaminyl-L-alanine benzhydryl ester (c,B-mixture) containing 2.24 mol of water, with 0.7 ml (7.4 mmol) of acetic anhydride in 12 ml of pyridine (20 hours, room temperature), 1,4,6tr i-0-acetyl-N-propionyl-demethylmuramyl-L-alanyl-Disoglutaminyl-L-alanine benzhydryl ester (c,B-mixture) in the form of a colourless powder containing 1.33 mol 0 of water.

a0 ~0 0 %1 fo0C 41

H

53 N 5 0 15 *1 .33 H 2 0 (879.85) Calc. C 55.97 H 6.40 N 7.96 0 29.69 H 2 0 2.72 Found C 56.14 H 6.36 N 7.98 0 29.56 H 2 0 2.72 o [a 2 23.8 2.10 (c 0.478; dimethylformamide), Rf 0.36 (chloroform:methanol Rf 0.69 IC (chloroform:methanol Rf 0.87 (chloroform: I methanol 7:3).

a The starting material is obtained as follows: Stage 3.1: Analogously to Example 1, there is obtained from 2.11 g (4.07 mmol) of N-propionyl- 0:80.:demethylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine containing 1.56 mol of water, with a total of. 2.14 g (11.06 mmol) of diphenyldiazomethane in 42 ml of methanol/dimethoxyethane (18 hours, room temper- -58 ature), N-propionyl-demethylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine benzhydryl ester in the form of a colourless powder containing 2.24 mol of water.

C 3H47N 50 12*2.24 H 20 (770.14) Caic. C 54.58 H 6.76 N 9.09 0 29.59 H 2 0 5.25 Found C 54.85 H 6.86 N 9.11 0 29.21 H 2 0 5.25 [a 2 2.9 t 20 (c =0.478; dimethylformamide), Rf 0.57 (chloroform:methanol Rf =0.65 (chloroform:methanol:water =70:30:5).

Example 4: Analogously to Example 1 there is obtained from 0.767 g (1.09 mmol) of N-propionyldemethylmuramyl-L-alanyl-D-glutamic acid -methyl ester-(C Y)-benzhydryl ester still containing 1.42 mol of water, with 0.49 ml (5.2 mmol) of acetic anhydride in 8.7 ml of pyridine (44 hours, room temperature), 00 1,4 6 -tri-0-acetyl-N-propionyl-demethylmuramy.L.alany..

*'004 D-glutamic acid (C,)-methyl ester-(C Y)-benzhydryl 0 ad*ester (c,B-mixture) in the form of a colourless powder V"0 containing 0.71 mol of water.

0 *4 C 3 9

H

49 N 3 0 15 0.71 H 2 0 (812.62) Caic. C 57.65 H 6.28 N 5.17 0 30.92 H 2 0 1.57 Found C 58.09 H 6.23 N 5.28 0 30.71 H 2 0 1.57 [a]I2 53.9 1.9' (c 0.519; dimethylformamide), Rf 0.55 (chloroform:methanol Rf 0.87 0 t(chloroform:methanol Rf 0.95 (chloroform: obtaied fom 1.2 g 2.5 m~~~df N-rpnydethmethanol 7:3).

Stage 4.1: Analogously to Example 1 there is murayl--alnylD-gutaic cid(Ca)-methyl ester still containing 0.68 mol of waterp~ with a total of 1.4 g (7.2 mmol) of diphenyldiazomethane in 27 ml of -59methanol/dimethoxyethane (18 hours, room temperature) N-propionyl--demethylmuramyl--L-alanyl- D-alutamic acid (Ce) -methyl ester- (C Y)-benzhydryl ester in the form of a colourless powder containing 1.42 mol of water.

C33 H43 N3 012 *1.42 H 20 (699.30) Calc. C 56.69 H 6.63 N 6.01 0 30.70 H 2 0 3.65 Found C 56.75 H 6.73 N 6.21 0 30.74 H 2 0 3.65 10.3 2.30 (c =0.435; water), 0.7 [aID Rf 07 (chloroform:methanol:water 70:30:5), Rf =0.91 (chloroform:methanol 7:3).

Example 5: Analogously to Example 1 there is obtained from 1.15 g (1.57 mmol) of N-propionyldemethylmuramyl-L-alanyl-D-glutamic acid -n-butyl 0.ester-(C Y)--benzhydryl ester still containing 0.99 mol o of water, with 0.70 ml (7.4 mmol) of acetic anhydride o a.

00 0D in 12 ml of pyridine (16 hours, room temperature), '00 146-tri-0-acetyl-N-propionyl-demethylmuramyl-L-alanyl- 0 0 D-glutamic acid (C )-n-butyl ester-(C )-benzhydryl 00:ester (a,B-mixture) in the form of a colourless powder containing 0.73 mol of water.

C 42C H 55N 30 15 0.73 H 20 (855.08) ItotCalc. C 58.99 H 6.60 N 4.91 0 29.50 H 2 0 1.54 to0 Found C 58.96 H 6.72 N 4.76 0 29.49 H 2 0 1.54 0 ~~20 +126+23 -26t23 (c 0 .4 43; chloroform), Rf 0.72 (chloroform:methanol Rf =0.96 (chloroform: methanol 4:1).

The starting material is obtained as follows: Stage 5.1: Analogously to Exapmle 1 there is obtained from 1.9 g (3.3 mmol) of N-propionyl-demethyl- -v

I

muramyl-L-alanyl-D-glutamic acid (C a)-n-buy ester containing 1.03 mol of water, with a total of 2.5 g (12.85 inmol) of diphenyldiazomethane in 40 ml of methanol/dimethoxyethane 70 hours, room temperature) N-propionyl-demethylmuramyl-L-alanyl-D-glutamic acid )-n-butyl ester- (C )-benzhydryl ester in the form of a colourless powder containing 0.99 mol of water.

C36 H 49 N 3 0 12 0.99 H 2 0 (733.62) Calc. C 58.94 H 7.03 N 5.73 0 28.32 H 2 0 2.43 Found C 59.24 H 7.06 N 5.59 0 28.08 H 2 0 2.43 [a 2 12.5 t 1.90 (c 0.522; methanol), Rf 0.46 (chlorcform:methanol Rf 0.63 (chloroform: methanol Rf =0.88 (chloroform:methanol 7:3).

Example 6: Analogously to Example 1 there is obtained from 1.0 g (1.43 mmol) of crude N-acetylmuramyl-L-alanyl-D--glutamic acid -pivaloyloxymethyl ester-(C )-benzyl ester and 0.57 ml (6.0 mmol) 0 go4a0 60,040of acetic anhydride in 10 ml of absolute pyridine (20 hours, room temperature), N-acetyl-1 ,4,6-tri-0- 001acetyl-muramyl-L-al~anyl-D-glutamic acid (C.-pivaloyloxymethyl ester-(C Y)-benzyl ester (a,B-mixture) in the t form of a colourless powder containing 0.79 mol of water.

C C 38

H

53 N 3 0 17 *0.79 H 2 0 (838.08) Calc. C 54.47 H 6.60 N 5.02 0 33.96 H0 1.69 Found C 54.52 H 6.36 N 5.12 0 33.71 H 2 0 1.69 [a 0= 68.5 4.10 (c 0.241; dimethylformamide), Rf 0.52 (chloroform:methanol Rf 0.55 (chloroforni:ethanol 9:1).

The starting material is obtained as follows: I' I I- 61 Stage 6.1: 20.2 g (60 mmol) of N-tert.-butoxycarbonyl-D-glutamic acid (C )-benzyl ester are dissolved in a mixture of 300 ml of tetrahydrofuran and ml of water. There are added to this solution 19.55 g (60 mmol) of caesium carbonate (purum, Fluka), dissolved in 100 ml of water, and the whole is left to stand for 1/2 hour at room temperature. Subsequently, the whole is concentrated by evaporation in a high vacuum at 300 and the resulting residue is evaporated in succession, in a high vacuum at 400, with 200 ml of methanol and twice with 200 ml each time of dimethylformamide.

The resulting caesium salt of N-tert.-butoxycarbonyl-D-glutamic acid (C )-benzyl ester (28.1 g; mmol) is suspended in 500 ml of dimethylformamide.

18.0 g (17.4 ml; 120 mmol) of pivalic acid chloromethyl ester (purum, Fluka) and 18.0 g (120 mmol) of sodium iodide are added to this suspension at room temperature.

The resulting mixture is stirred for 17 hours at room temperature. It is subsequently filtered and the filtrate is concentrated by evaporation in a high vacuum at 400. The resulting residue is taken up in 500 ml of ethyl acetate and washed in succession in each case three times with 150 ml of 0.1N sodium thiosulphate solution and water. After drying the ethyl acetate phase over sodium sulphate, concentration by evaporation in vacuo is carried out again. The still faintly yellow residue is recrystallised from 450 ml of methanol/water N-tert.-butoxycarbonyl- D-glutamic acid (C.)-pivaloyloxymethyl ester-(C benzyl ester is obtained in the form of colourless crystals, m.p. 76-77°.

1 @4 lo4 0 r 0 04 00 0 4040 00 0 1.04 04 00 04* O 0 0 04~ 00 0 *Js0 0 4 *1 90 4 0; 00l O 0 00 00 0 O01 i I r 62

C

23

H

33

NO

8 (451.52) Calc. C 61.18 H 7.37 N 3.10 0 28.35 Found C 61.08 H 7.25 N 3.33 0 28.58 [a]2 18.4 0.1° (c 1.034; ethyl acetate), Rf 0.85 (methylene chloride:methanol 9:1).

Stage 6.2: 20.0 g (44.3 mmol) of N-tert.-butoxycarbonyl-D-glutamic acid (Ca,-pivaloyloxymethyl ester- (C )-benzyl ester are stirred for 1.5 hours at 00 in a mixture of 400 ml of trifluoroacetic acid/methylene chloride The reaction solution is then concentrated by evaporation at 300 in a high vacuum and the resulting residue is repeatedly taken up in methylene chloride and concentrated by evaporation again. A yellow oil is obtained which is taken up in a mixture of 100 ml of water and 300 ml of diethyl ether and cooled to 00. 80 ml of IN sodium hydroxide solution (pH 7.5) are added to the solution while stirring vigorously and the ethereal phase is separated off. The ethereal solution is extracted once with 100 ml of water and the aqueous phase once with 200 ml of diethyl ether. The ethereal phases are combined, dried over sodium sulphate and filtered. Then, 8.43 g (44.3 mmol) of 2 -toluenesulphonic acid monohydrate (puriss., Fluka) dissolved in 100 ml of diethyl ether are then added to the reddish filtrate and the whole is stirred for 1 hour at room temperature then for 1/2 hour at The resulting crystals are filtered off with suction and washed with diethyl ether. The resulting crude product is recrystallised from 400 ml of ethyl acetate. D-glutamic acid (C))-pivaloyloxymethyl ester-(C )-benzyl ester tosylate is obtained in the form of colourless crystals, m.p. 135-1370.

0 a'4 4 0 0 44 0 44( *a 4

IO~

o I o 4 S It LI Il 40 6* I I 63

C

25

H

33

NO

9 S (523.60) Calc. C 57.35 H 6.35 N 2.68 0 27.50 S 6.12 Found C 57.39 H 6.52 N 2.72 0 27.53 S 6.12 [a]D2 8.8 0.10 (c 0.924; methylene chloride), Rf 0.78 (methylene chloride:methanol 9:1; double spot), Rf 0.84 (methylene chloride:methanol 5:1; double spot).

Stage 6.3: 2.6 g (6.5 mmol) of the sodium salt of N-acetyl-muramyl-L-alanine containing 0.63 mol of water are dissolved in 50 ml of dimethylformamide. 1.47 g (7.14 mmol) of N,N-dicyclohexylcarbodiimide, 0.74 g (7.14 mmol) of N-hydroxysuccinimide and 3.41 g mmol) of D-glutamic acid (C,)-pivaloyloxymethyl ester-(Cy)-benzyl ester tosylate are added to this solution at room temperature and the resulting clear colourless solution is stirred for 14 hours at room n *0o d temperature.

The crystals (N,N-dicyclohexyl urea) precipitated O* during the reaction are then filtered off with suction a. and the filtrate is concentrated by evaporation at 0 0• 400 in a high vacuum. The residue is taken up in 300 ml of ethyl acetate, filtered, and the resulting solution is washed in succession four times with 100 ml of water each time, three times with 100 ml of saturated aqueous sodium sulphate solution each time and twice with 100 ml of water each time.

The ethyl acetate phases are combined, dried over 0' sodium sulphte, filtered and concentrated by evaporation again.

1 A slightly yellow foam is obtained, which is purified by column chromatography on 440 g of silica gel (type 60, high-purity, Merck, 0.063-0.200 mm) in the system acetone/ethyl acetate (10 ml fractions). Fractions 21-70 are combined and concen- 64 trated by evaporation at 400 in vacuo. Slightly contaminated N-acetyl-muramyl-L-alanyl-D-glutamic acid (C )-pivaloyloxymethyl ester-(C )-benzyl ester is obtained in the form of a colourless foam, which is processed without further purification.

Rf 0.74 (chloroform:methanol:water 70:30:5), Rf 0.85 (chloroform:methanol 7:3).

Example 7: 10 g (15.86 mmol) of N-benzoyl-demethylmuramyl-L-alanyl-D-isoglutamine benzyl ester are dissolved in 100 ml of pyridine and 9 ml of acetic anhydride are added. After 24 hours at room temperature the whole is concentrated by *ation in a high vacuum, the residue is taken up ii .ethylene chloride and the organic phase is extracted by shaking in succession with 300 ml each of 1N hydrochloric acid, o water, 5% NaHCO 3 solution and water. The aqueous *o phases are extracted twice with 150 ml of methylene chloride each time. The combined organic phases are :e dried with sodium sulphate and concentrated by evaporation.

For the purpose of crystallisation, the residue is dissolved in 20 ml of ethanol, and 30 ml of ethyl acetate are added followed by diethyl ether until the u mixture turns turbid ml). Colourless crystals of

C

cta,-1,4,6-tri-O-acetyl-N-benzoyl-demethylmuramyl-Lalanyl-D-isoglutamine benzyl ester, m.p. 168-1690, are obtained. Further crystals can be recovered from the mother liquor.

20 44.9° 1.2° (c 0.813, CHC13 D 3 Rf 0.54 (CHCl 3 :methanol 9:1).

0 Example 8: 3.7 g (19.1 mmol) of diphenyldiazomethane in 30 ml of acetonitrile are added to 4.5 g (6.4 mmol) of 1, 4 i~l \c 65 alanyl-D-isoglutamine dissolved in 60 ml of acetonitrile. After 5 hours at room temperature the reaction is complete; the whole is concentrated to dryness by evaporation in vacuo, and the residue is extracted four times with 20 ml of diethyl ether each time and purified by chromatography on 250 g of silica gel (Merck, 0.04-0.6 nun). Elution is carried out in succession with 1 litre of methylene chloride/acetone fraction per fraction in each case 500 ml of

CH

2 Cl 2 /acetone fractions 2 and 3) and per fraction in each case 1 litre of CH 2 Cl 2 /acetone fractions 4, 5 and There is thus obtained from fraction 1 impure a-anomer, from fraction 3 pure a-anomer and from fractions 4 and 5 an a,8-anomeric mixture of 1,4,6-tri-O-acetyl-N-benzoyl-demethylmuramyl- L-alanyl-D-isoglutamine benzhydryl ester.

a-anomer: M.p. 108-1100 [a]2 36.3 1 (c 1.01, 0 0 D D CHCl 3 Rf 0.27 (CHCl 3 :acetone 1:1).

o The starting material, 1,4,6-tri-O-acetyl-N- 00. benzoyl-demethylmutamyl-L-alanyl-D-isoglutamine, is Soobtained by catalytic hydrogenation of the corresponding benzyl ester (Example 7) with 5% palladium-oncarbon in dimethoxyethane, in the form of an anomeric :t mixture. M.p. 114-1200, [a]20 51.90 1 (c 0.963,

D

SH

2 Rf 0.16 (CHCl 3 :methanol:H 2 0 85:15:2).

S ,tO Example 9: 1.5 g (2 mmol) of N-benzoyl-(ia,8),4,6tri-0-butyryl-demethylmuramyl-L-alanyl-D-isoglutamine are reacted analogously to Example 8 with diphenyldiazomethane. After working up in analogous manner a crude product is obtained which is chromatographed on g of silica gel (Merck, 0.04-0.6 mm). Elution is carried out in succession with 1 litre of CH 2 C12/ acetone fraction per fraction in each case 1 litre of CH 2 Cl 2 /acetone fractions 2 and 3) -o -66and per fraction in each case 500 ml of CH 2 Cl 2 acetone fractions 4 and The following are obtained: from fraction 2 c-anomer and from fraction 4 impure B-ainomer of N-benzoyl-1,4,6-tri-O-butyryl-demethylmuramyl-L-alanyl- D-isoglutamine benzhydryl ester. The a-anomer is obtained in the form of crystals by dissolving in acetone (0.7 ml/g) and adding diethyl ether (22 ml/g).

M.p. 109-113O0, a 20 45.10 1.10 (c 0.92, C~ R f 0.62 (CHCl 3 :acetone The a-anomer is obtained in the form of crystals by dissolving in 8 ml of acetone at 500 and adding ml of diethyl ether at room temperature.

M.p. 169-171 0 20 13.80 1.3 0 (c =0.741, C~ aD -Cl 3 methanol Rf =0.52 (CHCl 3 :acetone 00 The starting material is obtained as follows: 44 04Stage 9.1: N-benzoyl-(l±,) ,4,6-tri-O-butyryldemethylmuramyl-L-alanyl--D-isoglutamine benzyl ester is obtained in known manner from N-benzoyl-demethylmuramyl- L-alanyl-D-isoglutamine benz:Tl ester in pyridine with 6 equivalents of butyric acid anhydride with the addition of catalytic amounts of 4-dimethylaminopyridine. After t 1z 4working up in customary manner and triturating with diethyl ether, crystals having a melting point of 166-1710 00 are obtained; [a]ID 43.20 1 (c 0.954, CHCl 3 a-anomer: Rf 0.42 (CHCl 3 :acetone=1:) 0444 -anomer: Rf 0.28 (CHCl 3 :acetone f. 3 o 9Stage 9.2: By catalytic hydrogenation of N-benzoyl- (lcx,8) ,4,6-tri-O-butyryl-demethylmuramyl-L-alanyl-Disoglutamine benzyl ester with 5% palladium-on-carbon in tetrahydrofuran, N-benzoyl-(lt,B) ,4,6-tri-O-butyryl- 67 demethylmuramyl-L-alanyl-D-isoglutamine is obtained in the form of a hemihydrate, m.p. 100-1050.

20 o [a]D 54.2 1 (c 0.96, methanol), R 0.45 (CHCl 3 :methanol 8:2).

Example 10: Analogously to Example 1, 2.04 g mmol) of acetic anhydride are reacted (1 hour, room temperature) with 3.69 g (5.6 mmol) of N-acetyldemethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester dissolved in 40 ml of abs. pyridine. The clear solution is substantially concentrated at 300, the residue is taken up in 150 ml of ethyl acetate and the solution is extracted twice with 50 ml of water each time. After drying and evaporating the solvent, N-acetyl-(la,8),4,6-tri-O-acetyl-demethylmuramyl-Lalanyl-D-isoglutamine benzhydryl ester remains.

*0 *20 o+ o [a] D 33 1 (c 1.159, methanol), R 0.70 S. (chloroform:methanol:water 70:30:5), Rf 0.48 (n-butanol:acetic acid:water 75:7.5:21) and R t'e, 0.81 (ethyl acetate:n-butanol:pyridine:acetic acid: Swater 42:21:21:6:10).

SThe N-acetyl-demethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester can be manufactured as follows: 4 t Stage 10.1: 11.6 g (60 mmol) of diphenyldiazomethane are added to a solution of 22.8 g (40 mmol) of N-acetyldemethylmuramyl-L-alanyl-D-isoglutamine in 500 ml of a 1:1 mixture of 1,2-dimethoxyethane and methanol and the solution is stirred for 16 hours at room temperature.

The red suspension is concentrated by evaporation at 200 under reduced pressure and the residue is triturated repeatedly with diethyl ether until an almost colourless product is obtained. This is dissolved in 100 ml of methanol and crystallised by the addition, in i 68 portions, of a 2:1 mixture of diethyl ether and petroleum ether. After stirring for several hours, first at room temperature, then in an ice bath, the crystal mass is filtered off and dried under reduced pressure. In this manner N-acetyl-demethylmuramyl-Lalanyl-D-isoglutamine benzhydryl ester is obtained in the form of cube-shaped crystals. M.p. 1700 (with decomposition), []20 14 10 (c 1.5, methanol), Rf 0.40 (chloroform:methanol:water 70:30:5), and Rf 0.66 (ethyl acetate:n-butanol:pyridine:glacial acetic acid:water 42:21:21:6:10).

Example 11: Analogously to Example 10 there is obtained from 0.55 g (0.85 mmol) of N-acetyldemethylmuramyl-L-a-aminobutyryl-D-isoglutamine benzhydryl ester, dissolved in 5 ml of abs. pyridine, and 0.281 g (2.76 mmol) of acetic anhydride o minutes, room temperature), N-acetyl-(la,8),4,6-tri-Oacetyl-demethylmuramyl-L-a-aminobutyryl-D-isoglutamine o benzhydryl ester. Rf 0.63 (chloroform:methanol: water 70:30:5) and Rf 0.82 (ethyl acetate:nbutanol:pyridine:acetic acid:water 42:21:21:6:10).

Example 12: From 2.10 g (2.54 mmol) of N-acetyl- C demethylmuramyl-L-a-aminobutyryl-D-glutamic acid dibenzhydryl ester and 1.053 ml (11.43 mmol) of acetic anhydride in 6 ml of absolute pyridine the peracetyl compound is obtained analogously to Example 1, after leaving to stand for 12 hours at room temperature. Purification is carried out on 450 g of silica gel (type 60, Merck; particle size 0.063-0.200 mm; ml fractions) first with chloroform, then, from fraction 20 onwards, with chloroform:isopropanol The material contained in fractions 43-106 is collected, dissolved in 5 ml of chloroform, after the addition of

'I(

1 69 ml of abs. dioxan filtered through a millipore filter (Fluoropore, PTFE, 0.2 um) and then lyophilised.

N-acetyl-(la,8), 4 6 -tri-O-acetyl-demethylmuramyl-L-aaminobutyryl-D-glutamic acid dibenzhydryl ester is obtained in the form of a loose, colourless powder containing 0.37 mol of water.

C51H N3015 0.37 H 2 0 (958.69) Calc. C 63.90 H 6.09 N 4.38 H 2 0 0.69 Found C 63.6 H 6.1 N 4.6 H 2 0 0.70 29.6 1.70 (c 0.577; methanol), R 0.69 (n-butanol:acetic acid:water 75:7.5:21) and Rf 0.93 (ethyl acetate:n-butanol:pyridine:acetic acid:water 42:21:21:6:10).

The starting material is obtained analogously to Example 1, in the following manner: Excess diphenyldiazomethane is added at room temperature, while stirring, to 3.00 g (6.1 mmol) of W-acetyldemethylmuramyl-L-a-aminobutyryl-D-glutriic acid dissolved in 30 ml of a 1:1 mixture of dimethylformamide and methanol. After 3 days, the red suspension is concentrated by evaporation in a high vacuum at SO.s 300, the red residue is triturated repeatedly with diethyl ether/petroleum ether and the supernatant is decanted. The solid residue is purified first by chromatography on 600 g of silica gel (type 60, Merck) in the system chloroform/methanol/water (70:30:5; inter alia for the purpose of removing semiester), then on a second column (100 g) first with ethyl acetate, then ethyl acetate/methanol 5 ml fractions).

The collected material is dissolved in 4.5 ml of methanol and, after the addition of 70 ml of abs.

dioxan and customary filtration through a millipore filter, lyophilised. N-acetyl-demethylmuramyl-L-a- L 70 aminobutyryl-D-glutamic acid dibenzhydryl ester is obtained in the form of a colourless powder containing 0.93 mol of water.

C

4 5

H

5 1

N

3 0 12 0.93 H 2 0 (842.67) Calc. C 64.14 H 6.34 N 4.99 H 2 0 1.99 Found C 64.1 H 6.5 N 4.9 H 2 0 20 2 11.6 2.90 (c 0344; methanol), R 0.55 =a 11.6 -R (ethyl acetate:methanol 4:1) and Rf 0.77 (chloroform:isopropanol Example 13: Analogously to Example 1, there is obtained from 0.67 g (1 mmol) of N-acetyl-demethylmuramyl-L-valyl-D-isoglutamine benzhydryl ester and 0.367 g (3.6 mmol) of acetic anhydride in 5 ml of abs.

pyridine (30 minutes, room temperature), N-acetyl-(la, 6*9 8 4 o isoglutamine benzhydryl ester.

[]20 350 1 (c 0.594; methanol), R 0.92 o D f (chloroform:methanol:water 70:30:5) and Rf 0.80 (ethyl acetate:n-butanol:pyridine:acetic acid:water 42:21:21:6:10).

The starting material is obtained analogously to Example 1 in the following manner: K Stage 13.1: 1.60 g (3.15 mmol) of N-acetyl-demethylmnuramyl-L-valyl-D-isoglutamine, dissolved in 30 ml of methanol, are esterified with excess diphenyldiazomethane (2 hours, room temperature). The product is 69 purified by repeated precipitation from methanol/ c diethyl ether N-acetyl-demethylmuramyl-L-valyl- D-isoglutamine benzhydryl ester is obtained.

r

*P

71 13° 10 (c 1.067; methanol), R 0.56 D f (chloroform:methanol:water 70:30:5), Rf 0.42 (n-butanol:acetic acid:water 75:7.5:21) and Rf 0.72 (ethyl acetate:n-butanol:pyridine:acetic acid: water 42:21:21:6:10).

Example 14: 0.75 ml of acetic anhydride is added, while stirring, to a solution of 1.2 g of N-acetylmuramyl-L-valyl-D-isoglutamine benzhydryl ester in 12 ml of absolute pyridine and the whole is left to stand for 24 hours at room temperature. The reaction mixture is then poured onto 40 ml of ice water, the product crystallising out. The crystals are filtered off with suction, washed with water and dried.

Recrystallisation from ethyl acetate and diethyl ether S' yields N-acetyl-1, 4,6-tri-O-acetyl-muramyl-L-valyl-Do* isoglutamine benzhydryl ester, Rf 0.3 (methylene chloride: methanol 5:1).

o The starting material used is manufactured as follows: Stage 14.1: 1.1 g of diphenyldiazomethane is added to o a solution of 2.0 g of N-acetyl-muramyl-L-valyl-Disoglutamine in 25 ml of methanol and 25 ml of 1,2- S dimethoxyethane and the whole is stirred for 20 hours at room temperature. The reaction mixture is Sconcentrated to dryness by evaporation and extracted with diethyl ether. The residue is suspended in water, stirred, filtered off and dried over NaOH pellets. The resulting N-acetyl-muramyl-L-valyl-D-isoglutamine benzhydryl ester melts at 1850 (decomp.), Rf (chloroform:methanol:water 14:6:1), [al20 380 (c 0.912; methanol).

1 11 72 Example 15: 1.7 ml of acetic anhydride are added, while stirring, to a solution of 2.0 g of N-acetylmuramyl-L-alanyl-D-isoglutamine-benzhydryl ester in ml of absolute pyridine and the whole is left to stand for 24 hours at room temperature. The reaction mixture is then poured onto 50 ml of ice water. The material that separates out is taken up to 100 ml of ethyl acetate, washed with dilute hydrochloric acid and semi-saturated sodium chloride solution and dried over magnesium sulphate. After concentrating the solvent by evaporation, N-acetyl-1,4,6-tri-O-acetyl-muramyl-Lalanyl-D-isoglutamine benzhydryl ester is obtained.

Rf 0.7 (methylene chloride:methanol 5:1).

The starting material used is manufactured as follows: s Stage 15.1: 0.42 g of diphenyldiazomethane is added to 0 a solution of 1.1 g of N-acetyl-muramyl-L-alanyl-Disolgutamine in 12.5 ml of methanol and 12.5 ml of 1,2- 0 o. dimethoxyethane and the whole is stirred for 20 hours at 40 0 C. After concentrating the solvent by evaporation, the residue is extracted thoroughly with diethyl ether and water and dried over NaOH pellets.

N-acetyl-muramyl-L-alanyl-D-isoglutamine benzhydryl ester is obtained in the form of white crystals.

SRf 0.6 (methylene chloride:methanol:water 14:6:1).

Example 16: 0.73 g (7.15 mmol) of acetic anhydride are added to 1.45 g (1.99 mmol) of N-acetyl-muramyl-L-alanyl- D-isoglutaminyl-L-alanine benzhydryl ester dissolved in a a ml of abs. pyridine and the whole is left to stand for 35 hours at room temperature. The yellowish solution is concentrated to dryness by evaporation at 300 in a high vacuum, abs. dioxan is added to the 4 73 residue and lyophilisation is carried out. The freezedried material is taken up in 10 ml of chloroform/ methanol and precipitated (3 times) with 100 ml of abs. diethyl ether. The resulting powder is dissolved in 100 ml of abs. dioxan/water filtered in customary manner through a millipore filter (PTFE; 0.2 pm) and lyophilised. N-acetyl-(1a,8),4,6-tri- O-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine benzhydryl ester is obtained in the form of a colourless powder containing 1.42 mol of water and 0.7 mol of dioxan.

C41H51N5015 0.7 C4H 02 1.42 Calc. C 55.90 H 6.37 N 7.44 H20 2.72 Found C 55.98 H 6.42 N 7.76 H20 2.71 25.5 2.10 (c 0.475; dimethyl sulphoxide), Rf 0.39 (n-butanol:acetic acid:water 75:7.5:21), Rf 0.83 (chloroform:methanol:water 70:30:5) and Rf 0.85 (chloroform:methanol:water:acetic acid 70:40:9:1).

0@ S S Se The starting material is obtained as follows: Stage 16.1: 3.25 g (5.6 mmol) of N-acetyl-muramyl-Li alanyl-D-isoglutaminyl-L-alanine are dissovled in 50 ml St, of methanol and, while stirring, esterified with excess diphenyldiazomethane. After 6 hours the violet solution is concentrated by evaporation and the yellowish pr~ residue is purified by chromatography on 400 g of silica gel (type 60, Merck) in the system chloroform: methanol:water (70:30:5) (13 ml fractions). The material contained in fractions 42 to 60, after first runnings of 600 ml, is collected, taken up in 100 ml of chloroform, filtered through a millipore filter (PTFE, 0.2 um) and concentrated by evaporation. The residue is taken up in 150 ml of abs. dioxan and lyophilised.

i- L I A y^ 7.11 -74- N-acetyl-muramyl--L-alanyl-D-isoglutaminyl-L-alanine benzhydryl ester is obtained in the form of a loose colourless powder.

C3H47N5 012 *1.51 H 20 (756.99) Caic. C 55.54 H 6.69 N 9.25 H 2 0 3.59 Found C 55.68 H 6.39 N 9.04 H 2 0 3.59 []20 16.3 10 0.979; dimethyl sulphoxide), [aD Rf 0.31 (n-butanol:acetic acid:water 75:7.5:21), Rf 0.57 (chloroform:methanol:water 70:30:5) and Rf =0.33 (chloroform:methanol:water:acetic acid- 75:27:5:0.5).

Example 17: Analogously to Example 16, the peracetyl compound is obtained from 1.00 g (1.53 mmol) of N-acetyl--muramyl-L-alanyl-D-isoglutaminyl-L-alanine benzyl ester, dissolved in 18 ml of abs. pyridine, and 0.66 g (5.97 mmol) of acetic anhydride (6 hours, room temperature) Purification is carried out by chromatography on 150 g of silica gel (type 60, Merck) in the system chloroform/methanol/water (70:30:5; 5 ml **~*fractions). N-acetyl-(lx,8) ,4,6-tri-0-acetyl-muramyl- L-alanyl-D-isoglutaminyl-L-alanine benzyl ester is obtained in the form of a colourless lyophilisate containing 1.84 mol of water.

C H N 0 .1.84 H 0 (812.94) 49 5 152 Calc. C 51.72 H 6.56 N 8.62 H 2 0 4.07 Found 7 51.5 H 6.9 N 8.3 H 0 4.1 [aLID 38.5 3.50 (c 0.282; dimethylformamide), Rf 0.63 (acetonitrile:water Rf 0.39 :(n-butanol:acetic acid:water Rf 0.83 (ethyl acetate:n-butanol:pyridine:acetic acid:water= 42:21:21:6:10).

Example 18: 1.45 g (1.5 mmol) of N-acetyl-muramyl-Lalanyl-D-isoglutaminyl-L-alanine-cholesteryl-3-ester 75 0 d 040 0 44 4 44.4 *a 4 44 and 0.72 g (7.1 mmol) of acetic anhydride are dissolved in 16 ml of a mixture of abs. pyridine and dimethylformamide and the whole is left to stand at room temperature for 22 hours. The clear solution is concentrated to approximately 4 ml in a high vacuum at 300, the resulting jelly-like residue is triturated with 50 ml of ethyl acetate and the supernatant is decanted off (4 times). The solid residue is taken up in 5 ml of dimethylformamide, 50 ml of abs. dioxan are added, and the whole is filtered through a millipore filter (PTFE, 0.2 um) and lyophilised. N-acetyl-(la,6), 4,6-tri-0-acetyl-muramyl-L-alanyl-D-isoglutaminyl-Lalanine-cholesteryl-3-ester is obtained in the form of a colourless powder containing 0.62 mol of water.

C

55 H87N515 0.62 H 2 0 (1069.49) Calc. C 61.77 H 8.33 N 6.55 H20 1.04 Found C 61.8 H 8.4 N 6.8 H 2 7.7 1 (c 0.977; dimethyl sulphoxide), Rf 0.41 (n-butanol:acetic acid:water 75:7.5:21), Rf 0.87 (ethyl acetate:n-butanol:pyridine:acetic acid:water 42:21:21:6:10).

Example 19: Analogously to Example 1, N-acetyl-1,4,6tri-0-acetyl-muramyl-L-alanyl-D-glutamine pivaloyloxymethyl ester is obtained from N-acetyl-muramyl-Lalanyl-D-glutamine pivaloyloxymethyl ester and acetic anhydride in absolute pyridine.

The starting material is obtained as follows: Stage 19.1: 3.0 g (8.54 mmol) of N-benzyloxycarbonyl- L-alanyl-D-glutamine are dissolved in a mixture of 300 ml of tetrahydrofuran and 20 ml of water. 1.39 g (4.27 mmol) of caesium carbonate (purum, Fluka), dissolved in 7 ml of water, are added to this solution and the whole is left to stand for 1/2 hour at room temperature. Subsequently, the whole is concentrated ra 4 *r C~r 44 *r .4

I

Ii 76 by evaporation at 300 in a high vacuum and the resulting residue is evaporated off in a high vacuum at 400 in succession with 250 ml of methanol and twice with 250 ml of dimethylformamide each time.

The resulting caesium salt of N-benzyloxycarbonyl- L-alanyl-D-glutamine is suspended in 300 ml of dimethylformamide. 2.57 g (2.48 ml; 17.08 mmol) of pivalic acid chloromethyl ester (purum, Fluka) and 2.56 g (17.08 mmol) of sodium iodide are added to this suspension at room temperature. The resulting mixture is stirred for 42 hours at room temperature.

Concentration by evaporation is then carried out at 400 in a high vacuum. The resuling yellow crystalline residue is taken up in 500 ml of ethyl acetate (suspension) and washed 3 times with 150 ml of L water each time. The clear ethyl acetate phases are combined, dried over sodium sulphate and concentrated by evaporation in vacuo at 30". The residue r' crystallises from ethyl acetate:n-pentane 1:15 (20:300 ml). Recrystallisation twice yields N-benzyloxycarbonyl-L-alanyl-D-glutamine pivaloyloxymethyl ester in the form of colourless crystals, m.p. 109-1100.

C

22

H

3 1

N

3 0 8 (465.50) Calc. C 56.76 H 6.71 N 9.03 0 27.49 Found C 56.42 H 6.69 N 8.82 0 27.73 5.5 0.10 (c 0.973; methanol), R 0.62 D f (methylene chloride:methanol Rf 0.86 (methylene chloride:methanol:water 70:30:5).

Stage 19.2: A solution of 6.7 g (14.4 mmol) of N-benzyloxycarbonyl-L-alanyl-D-glutamine pivaloyloxymethyl ester in 200 ml of dimethoxyethane is hydrogenated at a constant pH value of 5.5 (titration with IN hydrochloric acid) with 1.0 g of paladium-on-carbon as catalyst for 40 minutes at room 77 temperature under normal pressure.

Then, the catalyst is filtered off and the filtrate is concentrated by evaporation in a high vacuum at 300. The resulting residue is suspended in ml of diethyl ether. The resulting colourless crystals are filtered off with suction and subsequently washed with diethyl ether. L-alanyl-D-glutamine pivaloyloxymethyl ester hydrochloride is obtained in the form of colourless crystals, m.p. 110-1110 (decomp.).

C

14

H

26

CIN

3 0 6 (367.83) Calc. C 45.72 H 7.13 C1 9.64 N 11.42 0 26.10 Found C 45.51 H 7.36 Cl 9.34 N 11.47 0 26.40 [a]2 25.5 0.10 (c 1.128; methanol), R 0.07 (methylene chloridemethanol R 0.37 (methylene chloride:methanol:water 70:35:1), Rf 0.37 (methylene chloride:methanol:water 70:30:5), Rf 0.59 (methylene chloride:methanol:water 5:5:1).

4 g 4 *4 S4 Stage 19.3: 5.18 g (13.0 mmol, 2.52 mmol/g) of the .oo. sodium salt of N-acetyl- 4 -0,6-0-isopropylidene-muramic '"oo acid are suspended in 150 ml of dimethylformamide.

Then, at room temperature, 4.8 g (13.0 mmol) of L-alanyl-D-glutamine pivaloyloxymethyl estez hydrochloride, 2.95 g (14.3 mmol) of dicyclohexylcarbodiimide and 1.64 g (14.3 mmol) of -hydroxysuccinimide are added. The resulting slightly yellow suspension is stirred for 22 hours at room temperature.

Subsequently, most of the resulting dicyclohexyl urea is filtered off with suction and the resulting filtrate is concentrated by evaporation in a high vacuum at ::400. The yellow oil remaining is taken up in 20 ml of methanol, a further fraction of dicyclohexyl urea is filtered off with suction, and the filtrate is again concentrated by evaporation in a high vacuum.

The resulting crude product, which also still contains a small amount of dicyclohexyl urea, is 78 purified by column chromatography on 1000 g of silica gel (type 60, high-purity, Merck; 0.063-0.2 mm) in the system methylene chloride:methanol 9:1 (10 ml fractions).

Fractions 380-1210 are combined, and from fraction 850 elution is carried out with the system methylene chloride:methanol:water 70:30:5.

After concentrating the eluates by evaporation in a high vacuum at 300, a mixture consisting of chiefly N-acetyl-4-0,6-O-isopropylidene-muramyl-L-alanyl-Dglutamine pivaloyloxymethyl ester and some N-acetylmuramyl-L-alanyl-D-glutamine pivaloyloxymethyl ester is obtained in the f6rm of a faintly yellow foam, which is processed without further purification.

Rf 0.33 [N-acetyl-muramyl-L-alanyl-D-glutamine 0 pivaloyloxymethyl ester] and 0.66 [N-acetyl-4-0,6-Oc isopropylidene-muramyl-L-alanyl-D-glutamine pivaloyl- 4 0 "oxymethyl ester] (methylene chloride:methanol:water o 70:30:5).

a 0 Sa 2 Stage 19.4: A solution of 5.5 g (approximately mmol) of crude N-acetyl-4.0,6-O-isopropylidenemuramyl-L-alanyl-D-glutamine pivaloyloxymethyl ester in 100 ml of glacial acetic acid/water is stirred 0 for 4.5 hours at room temperature. Then, the slightly Syellow solution is concentrated by evaporation in a S high vacuum at 400 and the resulting residue is purified by column chromatography on 500 g of silica gel (type 60, high-purity, Merck; 0.063-0.2 mm) in the system methylene chloride:methanol:water (70:30:5) ml fractions). Fractions 52-65 are combined and concentrated by evaporation in a high vacuum at 300.

N-acetyl-muramyl-L-alanyl-D-glutamine pivaloyloxymethyl ester is obtained, which is dissolved in 60 ml of bidistilled water. The solution is filtered through a iii 79 millipore filter (Nalgene S; 0.2 um) and then lyophilised in a high vacuum. N-acetyl-muramyl-Lalanyl-D-glutamine pivaloyloxymethyl ester is obtained in the form of a colourless powder still containing 1.21 mol of water.

C

2 5

H

42 N40 1 3 .1.21 H 2 0 (628.50) Calc. C 47.78 H 7.16 N 8.91 0 36.18 H 2 0 3.48 Found C 48.09 H 7.15 M 9.02 0 36.01 H 2 0 3.48 39.5 0.10 (c 0.443; water), Rf 0.61 (methylene chloride:methanol:water 70:30:5).

Example 20: The following compounds are obtained in accordance with the processes described in this application: a) N-acetyl-1,4,6-tri-O-acetyl-muramyl-L-N-methylalanyl-D-isoglutamine benzhydryl ester, b) N-acetyl-1,4,6-tri-0-acetyl-muramyl-a-aminoisobutyryl-D-isoglutamine benzhydryl ester and t4a c) N-acetyl-1 ,4,6-tri-0-acetyl-demethylmuramyl-L- 4 alanyl-D-y-methoxycarbonyl-isoglutamine benzhydryl ester.

Example 21: Female MF-2f SPF mice with a body weight of from 14-16 g are infected intranasally, under light narcosis with a mixture of equal parts of diethyl f ether, ethanol and chloroform, with lethal doses (approximately LD 80 9 0 1-4 plaque forming units in the form of in each case 0.05 ml of a suspension of Influenza A/Texas/1/77 (virus A) or of Influenza B/Hong Kong 15/72 (virus B) viruses (mouseadapted strains).

Each mouse, from groups of in each case 10 of these mice, is, at the time given below (days) related to the day of infection, administered once (single 80 dose), in the manner indicated in Table 1, the amount mentioned in Table 1 of the active substance concerned in 0.05 ml for intranasal administration, or 0.2 ml for oral administration, of a 0.005 by weight solution of the sodium salt of carboxymethylcellulose in twicedistilled pyrogen-free water.

In each case 20 of the above-mentioned infected mice are used as a control, that is to say they are given a placebo (0.005 by weight solution of the sodium salt of carboxymethylcellulose).

The intranasal administration of the active substance is carried out under light narcosis with a mixture of equal parts of diethyl ether, ethanol and chloroform.

Compound I 1,4,6-tri-O-acetyl-N-propionyldemethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester.

air

D

4

I

a a I 9 t r r t i t 1 L .i 0 00 00 0 04.4.

0 0 zoo 000 000 0 0 a Table 1 I I Active Mode of Virus substance I administration Time of administration [days] Percentage of mice still living 23 days after infection as a function of the amount of active substance [mg/kg], stat P :5 0.05, 01 n.t. not tested istical significance (VTierfelder test), 0 0 .1 0.01 0.00 1 0.0001 control A I oral +7 n. t. 100** 70(* 100** n. t. n.t. oral +7 80** 70** 90** n.t. n.t. n.t. intranasal -7 n.t. 77* 100** 80** n.t. n.t. B I oral +7 n.t. n.t. n.t. 50** 0 10 0 intranasal -7 n.t. n.t. n.t. '100** 100** 90* p.

82 Example 22: Non-aqueous single-dose for nasal administration Composition: 1,4 ,6-tri-O-acetyl-n-propionyl-demethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester Miglyol 812 0.03 mg 30.00 mg Manufacture: 0.03 mg of the active ingredient are dissolved under aseptic conditions in 29.97 mg of Miglyol.

This solution is introduced into a customary commercial single administration nasal applicator, which before use is mounted onto a propellant can.

Example 23: Nose drops abbe be* 'Go*

S

5 0 5.

Composition: 1,4 ,6-tri-O-acetyl-N-propionyldemethylmuramyl-L-alanyl-Disoglutamine benzhydryl ester thiomersal sodium monohydrogen phosphate.2H 2 0 sodium dihydrogen phosphate.12H 2 0 benzalkonium chloride disodium salt of ethylenediaminetetraacetic acid (EDTA) sodium chloride demineralised water 0.15 0.02 0.30 10.10 0.10 mg 0.30 mg 10.10 mg 0.10 mg 0.50 mg 4.50 mg 987.60 mg 0.50 mg 3.70 mg 988.30 mg Manufacture: Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, thiomersal and EDTA 83 disodium salt are dissolved at room temperature, while stirring, in a portion of the above-mentioned amount of dimineralised water.

The active ingredient is then dissolved in this solution and the rest of the demineralised water is added.

The solution or a portion or itiultiple thereof is filtered through a membrane filter and introduced into cleaned containers. Suitable containers are, for example: a) glass or plastics containers (5 or 10 ml) that have a glass or plastics pipette with an elastomeric pipette suction bulb, b) compressible plastics bottles with an ascending tube and a spray head of plastics material, o°o single dose containers of plastics material (content ,2-3 drops) or d) glass or plastics bottles provided with a standardised pump-metered spray of plastics material (without propellant gas).

*4 40 0 Example 24: Nasal ointment 0 Composition: 22E_ i tlo 1,4,6-tri-O-acetyl-N-propionyl-dem thyl- S a muramyl-L-,alanyl-D-isoglutamine benzhydryl ester 0.03 g S paraffin oil, viscous 20.00 g white petroleum jelly 30.00 g •"wool fat, anhydrous 40.00 g demineralised water 19.97 g Manufacture: The fatty phase, consisting of paraffin oil, petroleum jelly and wool fat, is melted together. The k 6 -n l 84 aqueous solution of the active ingredient is incorporated into the fatty phase at approximately 50 0

C.

Example 25: Manufacture of 1000 tablets, containing active ingredient Composition per 1000 tablets: 1,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester 0.5 g lactose, ground 43.0 g corn starch 52.0 g Pharmacoat 603 (hydroxypropylmethylcellulose, containing 28-30% methoxy groups, supplied by Shinetsu Chemical Company, Toio, Japan) 3.0 g Aerosil' (colloidal silica, supplied by Degussa, Frankfurt, Federal Republic of Germany) 1.0 g magnesium stearate 0.5 g 0 Manufacture: The active ingredient and 15 g of lactose are mixed first. The resulting mixture is mixed with 28 g t" of lactose and 47 g of corn starch. A mass ready for er. S granulation is produced from the mixture so obtained and an aqueous solution of the Pharmacoat, and this mass is granulated, dried and ground. 5 g of corn starch, Aerosil and magnesium stearate are added to this and the mixture is compressed into 1000 tablets each weighing 100 mg.

The compacts can be lacquered in a manner known per se so as to be resistant to gastric juice.

I~-

~1

C;

at- 85 Example 26: The following compounds are obtained in accordance with the processes described in this Application: N-acetyl-1 ,4,6-tri-O-acetyl-muramyl-L-alanyl-Disoglutaminyl-L-a- ([2-benzyloxy-carbonylainino-ethyl]suiphonyl-methyl) -glycine benzyl ester, N-acetyl-1 ,4 ,6-tri-O-acetyl-muramyl--L-alanyl-Disoglutaminyl)-L-O-acetyl-serine benzyl ester, 1 ,4 ,6-tri-O-acetyl-N-benzoyl-demethylmuramyl-L-alanyl- D-glutamic acid dicholine ester, N-propionyl-1 ,4,6-tri-O-propionyl--demethylmuramyl-Lalanyl-D-isoglutamine benzhydryl ester, 1 ,4 ,6-tri-O-acetyl-N-propionyl--demethylmuramyl-Lalanyl-D-glutlamic acid c-n-butyl ester-y-benzyl ester, 1 ,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L-alanyl- '410 D-glutamic acid dibenzyl ester, :000" -acetyl-1 ,4,6-tri-O-propionyl-muramyl-L-ct-aminobutyryl- D-isoglutamine benzhydryl ester, ,4,6-tri-o-acetyl-N-benzoyl-demethylmuramyl-L-alanyl-Disoglutamine benzyl ester and 1 ,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L-alanyl D-glutamic acid dipivaloyloxymethyl ester.

sees Example 27: Analogously to Example Ithere is obtained from 250 mg (circa 0.098 mmol) of crude 4,6-di- O-acetyl-N-propionyl--demethylmuramyl-L-alanyl-Da a glutamic acid dipivaloyloxymethyl ester (c,g-mixture) and 2.0 ml (21.0 mmol) of acetic anhydride in 10 ml of absolute pyridine (18 hours, room temperature), 1,4,6a tr i-O-acetyl-N-propionyl-demethylmuramyl-L-alanyl-Dgltutamic acid dipivaloyloxymethyl ester (c,B-mixture); Rf 0.43 (chloroform:ethanol =95:5), Rf 0.56 (chloroform:methanol 9:1), Rf 0.72 (chloroform:methanol 86 The starting material is obtained as follows: Stage 27.1: 15.9 g (34.1 mmol) of D-glutamic acid (Ct)-pivaloyloxymethyl ester-(C )-benzyl ester tosylate and 3.75 ml (34.1 mmol) of N-methylmorpholine are added at room temperature, while stirring, to a solution of 9.78 g (34.1 mmol) of N-tert.-butoxycarbonyl-L-alanine-N-hydroxysuccinimide ester in 200 ml of absolute N,N-dimethylformamide. After stirring for 18 hours at room temperature, the whole is concentrated by evaporation in vacuo at 400. The resulting crude product (yellow oil) is purified by column chromatography on 800 g of silica gel (type 60, highpurity, Merck; 0.063-0.2 mm) in the system methylene chloride/ethyl acetate (85:15) (10 ml fractions).

Fractions 126-230 are combined and concentrated by evaporation in vacuo at 300. N-tert.-butyloxycarbonyl-L-alanyl-D-glutamic acid (C )-pivaloyloxymethyl ester-(C )-benzyl ester is obtained in the o form of a yellowish oil; ]20 -10.6 0.10 (c 1.035; methylene chloride),

[]D

Rf 0.29 (methylene chloride:ethyl acetate 85:15), Rf 0.73 (methylene (hloride:methanol 9:1), Rf 0.78 (methylene chloride:methanol 5:1), Stage 27.2: 100 ml of approximately 5N hydrochloric i acid in ethyl acetate are added at 00 to a solution of r 4.0 g (7.65 mmol) of N-tert.-butoxycarbonyl-L-alanyl-Dglutamic acid -pivaloyloxymethyl ester-(Cy)-benzyl ester in 50 ml of absolute ethyl acetate and the whole is stirred for 1 hour at 00. Subsequently the whole is concentrated by evaporation in a high vacuum at 300 and the resulting residue is repeatedly taken up in ethyl acetate and concentrated by evaporation again.

A colourless foam is obtained, which is dissolved in

R__

Y 87 ml of ethyl acetate. After the addition of 250 ml of pentane and cooling to -100, L-alanyl-D-glutamic acid (C,)-pivaloyloxymethyl ester-(C )-benzyl ester hydrochloride is obtained in the form of colourless crystals, m.p. 73-74o, still containing 0.63 mol of water; +23.5 0.10 (c 1.150; methanol), S= 0.20 (methylene chloride:ethanol 9.1), Rf 0.49 (methylene chloride:methanol 5:1), Rf 0.49 (methylene chloride:methanol 5:1), Rf 0.87 (methylene chloride:methanol:water 70:30:5).

Stage 27.3: There are added in succession to a suspension of 2.99 g 5.0 mmol, still containing sodium chloride; 1.673 mmol/g) of the sodium salt of 1-a-O-benzyl-4,6-0,0-isopropylidene-N-propionyldemethylmuramic acid in 50 ml of absolute N,N-dimethylformamide, at room temperature and while stirring, 1.34 g (6.5 mmol) of N,N-dicyclohexylcarbodiimide, 1.03 g (6.5 mmol) of 1-hydroxybenzotriazole and 2.35 g go 40 0 (5.0 mmol) of L-alanyl-D-glutamic acid (C,)-pivaloyloxymethyl ester-(C )-benzyl ester hydrochloride and the whole is then further stirred for 22 hours at room temperature. The suspension is subsequently filtered (N,N-dicyclohexyl urea) and the filtrate is concen- :i trated by evaporation in a high vacuum at 400. The residue, a yellowish foam, is taken up in 100 ml of ethyl acetate, and the resulting solution is washed in succession with 50 ml each of 2N citric acid, sodium bicarbonate solution and water. The ethyl acetate phases are combined, dried over sodium sulphate and concentrated by evaporation in vacuo. The resulting crude product is purified by column chromatography on 400 g of silica gel (type 60, high-purity, Merck; 0.063-0.2 mm) first with chloroform (first runnings 2 litres), then in the system chloroform/- I 88 ethanol (95:5) (10 ml fractions). Fractions 93-122 are combined and concentrated by evaporation in vacuo.

Still slightly impure 1-c--benzyl-4,6-O,O-isopropyl idene-N-propionyl-demethylmuramyl-L-alanyl-Dglutamic acid (C,)-pivaloyloxymethyl ester-(C Y)_ benzyl ester is obtained in the form of a colourless foam, which is processed without further purification; Rf 0.55 (chloroform:ethanol Rf 0.63 (chloroform:methanol Stage 27.4: 1,5 ml of trifluoroacetic acid is added at 00 to a solution of 830 mg (1.0 mmol) of benzyl-4 ,6-O,O-isopropylidene-N-propionyl-demethylmuramyl-L-alanyl-D-glutamic acid (C,)-pivaloyloxymethyl ester-(C Y)-benzyl ester in 28.5 ml of .44..absolute methylene chloride and the whole is stirred for 1.5 hours at 00. Subsequently, the resulting colourless solution is concentrated to dryness by evaporation in a high vacuum at 400. The residue 4 9crystallises from chloroform/methanol/diethyl ether 4* 1-c--benzyl-N-propionyl-demethylmuramyl- L-alanyl-D-glutamic acid (Ca) -pivaloyloxymethyl :ester-(C Y)-benzyl ester is obtained in the form of colourless crystals; m.p. 148-1490.

f=03 clrf6mmtao 0 0 6 46Rf 0.67 (chloroform:methanol 4:1), .110 Rf=06 4lrfrmmtao 4 Rf 0.90 (chloroform:methanol:water 70:30:5).

Stage 27.5: 400 mg (0.5 mmol) of 1-c-O-benzyl-N- 00. 0 propionyl-demethylmuramyl-L-alanyl-D-glutamic acid 00,306:(C )-pivaloyloxymethyl ester- (C )-benzyl ester are 4 01a hydrogenated at room temperature under normal pressure in 80 ml of tetrahydrofuran/methanol with 200 mg of 10% palladium-on-carbon as catalyst. When hydrogenation is complete (1 hour), the catalyst is filtered.

r s I I l -i I ~ir;at*n~ 1 89 P. 0 9 0 1 0**0 0 *0* 04 0 a t a *4 0« SI I 0 4 0 99 off and the filtrate is concentrated by evaporation in a high vacuum. The residue, a colourless foam, is dissolved in a mixture of 30 ml of tetrahydrofuran and 1 ml oE water, and a solution of 96 mg (0.25 mmol) of caesium carbonate in 0.38 ml of water is added to the resulting solution at room temperature. After standing for half an hour at room temperature, the colourless solution is concentrated by evaporation in vacuo. The residue is then treated twice with 100 ml of methanol each time and once with 100 ml of N,N-dimethylformamide and concentrated by evaporation again. Still slightly impure caesium salt of demethylmuramyl-L-alanyl-D-glutamic acid (C,)-pivaloyloxymethyl ester is obtained in the form of a colourless foam, which is processed without further purification.

Rf 0.15 (chloroform:methanol) 4:1), Rf 0.35 (chloroform:methanol) 7:3), Rf 0.45 (chloroform:methanol:water) 70:30:5).

Stage 27.6: 150 mg (145 ul, 1.0 mmol) of pivalic acid chloromethyl ester and 150 mg (1.0 mmol) of sodium iodide are added at room temperature, while stirring, to a solution of 420 mg (0.5 mmol) of the caesium salt of D-glutamic acid (C,)-pivaloyloxymethyl ester in 15 ml of N,N-dimethylformamide. The resulting mixture is stirred for 18 hours at room temperature, then again 145 ul (1.0 mmol) of pivalic acid chloromethyl ester and 150 mg (1.0 mmol) of sodium iodide are added and the whole is stirred for a further 48 hours.

Subsequently the mixture is concentrated by evaporation in a high vacuum at 300. The crude product is purified by column chromatography twice on 200 and 250 g of silica gel (type 60, high purity, Merck; 0.063-0.2 mm) in the system chloroform/methanol (9:1) 0000 0 *0 9 0 *04000 0 0 ij and chloroform:methanol (95:5) respectively. The fractions containing the desired product are in each case combined and concentrated by evaporation in vacuo. 1 -c--benzyl-N-propionyl-demethylmuramy±.-Lalanyl-D-glutamic acid dipivaloyloxymethyl ester is obtained; Rf 0.25 (chloroform:methanol 9:1), Rf 0.35 (chloroform:ethanol Rf 0.70 (chloroform:methanol 4:1).

Stage 27.7: There is obtained analogously to Example 1 from 80 mg (0.098 mmol) of 1-c--benzyl-N-propionyldemethylmuramyl-L-alanyl-D-glutamic acid dipivaloyloxymethyl ester and 0.8 ml (8.4 mmol) of acetic anhydride in 8 ml of absolv."-e pyridine (18 hours, room temperature), 4,6-di-O-accy--t-O-benzyl-N-propionyldemethylmuramyl-L-alanyl-D-glutamic acid dipivaloyloxy- 66 methyl ester; 00 Rf 0.50 (chloroform:ethanol 95:5), a f Rf 0.65 (chloroform:ethanol 9:1), Rf 0.77 (chloroform:methano. 9:1).

Stage 27.8: 140 mg (0.098 mmol) of 4,6-di-O-acetyl- 1 -ct--benzyl-N-propionyl-demethylmuramyl-L-alanyl-Dglutamic acid dipivaloyloxymethyl ester are hydrogenated for 20 hours at room temperature under normal 9 pressure in 80 ml of dimethoxyethane/water with palladium-on-carbon (Degussa, E 101 N) as catalyst.

'sO fhltratels is sus~eqentby evaportiof ind hg Thetcatals is subeqentlyiteed prtoff nd heg vacuum at 300. crude 4,6-di-O-acetyl-N-propionyldemethylmuramyl-L-alanyl-D-glutamic acid dipivaloyloxymethyl ester (a,B-mixture) is obtained, which is processed without further purification; 91 -f 0.38 (chloroform:methanol 9:1), Rf 0.6438 (chloroform:methanol:water 70:30:5),9:1), Rf 0.64 (chloroform:methanol:water 70:30:5), Rf 0.68 (chloroform:methanol 4:1).

Example 28: Analogously to Example 1, from 174.0 mg (0.25 mmol) of N-propionyl-demethylmuramyl-L-alanyl-Disoglutaminyl-L-alanine benzoyloxymethyl ester, which still contains traces of N-methylmorpholine hydrochloride, in 1.7 ml of absolute pyridine with 0.1 ml (1.04 mmol) of acetic anhydride, with the addition of a small amount of 4-dimethylamino-pyridine, there is obtained 1, 4 6 -tri-0-acetyl-N-propionyl-demethylmuramy- L-alanyl-D-isoglutaminyl-L-alanine benzoyloxymethyl ester (a,B-mixture) in the form of colourless crystals; Pe m.p. 177-1780 (from metlhylene chloride:diethyl ether 1:1),

R

R g 0.45 (chloroform:methanol 4:1), 44*p s Rf 0.64 (chloroform:methanol 7:3).

Pr P 4 The starting material is obtained as follows: **p Stage 28.1: 14.9 ml (0.1 mol) of 1,8-diazabicyclo- [5.4.0]undec-7-ene and 36.2 ml (0.45 mol) of diiodomethane are added to a solution of 18.9 g 0" mol) of N-tert.-butoxycarbonyl-L-alanine in 50 ml of N,N-dimethylacetamide and the whole is stirred for 7 hours at 220. The solution is concentrated by evaporation in a high vacuum and a suspension of 36.6 g mol) of benzoic acid, 6.1 g (0.05 mol) of 4-dimethylaminopyridine and 37.3 ml of 1,8-diazabicyclo- [5.4.0]undec-7-ene in 75 ml of N,N-dimethylacetamide is added to the residue. The clear solution obtained after 40 minutes is left to stand for 16 hours at 220. The mixture is shaken with a mixture of 0.3 molar dipotassium hydrogen phosphate solution and ethyl I i r 92 acetate, and the organic layer is separated off, washed with further phosphate solution, dried over sodium sulphate and concentrated by evaporation. Digestion of the residue with pentane, then pentane/diet.yl ether yields N-tert.-butoxycarbonyl-L-alanine benzoyloxymethyl ester; Rf 0.68 (chloroform:ethyl acetate 9:1).

Stage 28.2: 3.72 g (11.5 mmol) of N-tert.-butoxycarbonyl-L-alanine benzoyloxymethyl ester are dissolved until clear at 0° in 100 ml of a 5 molar hydrogen chloride solution in ethyl acetate and the whole is left to stand for 2 hours at 00. At 00, the solution is added to 200 ml of diethyl ether/pentane and, after stirring for 20 minutes at 00, the precipitated jelly-like crude product is filtered off.

It is stirred for 2 hours at 220 in 360 ml of ethyl acetate/diethyl ether/pentane then filtered.

L-alanine benzoyloxymethyl ester hydrochloride is o. obtained; m.p. 99-1000.

Stage 8.3: 268 mg (1.3 mmol) of N,N-dicyclohexylcarbodiimide and 207 mg (1.3 mmol) of 1-hydroxybenzotriazole are added at room temperature, while stirring, to a solution of 506 mg (1.0 mmol) of N-propionyldemethylmuramyl-L-alanyl-D-isoglutamine, containing 0.77 mol of water, in 10 ml of absolute N,N-dimethylformamide. After 2 hours there are added in succession to the resulting colourless suspension 269 mg (1.0 mmol) of L-alanine benzoyloxymethyl ester hydrochloride and 110 ul (1.0 mmol) of N-methylmorpholine and-the S whole is subsequently further stirred at room temperature. After 22 hours, precipitated N,N-dicyclohexyl urea is filtered off and the filtrate is concentrated by evaporation in a high vacuum at 300.

93 The resulting faintly yellow-coloured residue (foam) is suspended in 25 ml of ethyl acetate and this suspension is stirred for 1 hour at room temperature. The crude product, obtained in the form of colourless crystals, is further purified by column chromatography on 150 g of silica gel (type 60, high-purity, Merck; 0.063-0.2 mm) in the system chloroform/methanol/water (70:30:5) (5 ml fractions). Fractions 25-35 are combined and concentrated by evaporation in a high vacuum Almost pure N-propionyl-demethylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine benzoyloxymethyl ester (a,R-mixture) is obtained in the form of a colourless foam which still contains traces of N-methylmorpholine hydrochloride, and is processed without further purification.

R 0.36 (chloroform:methanol 7:3), f Rf 0.41 (chloroform:methanol:water 70:30:5), R 0.74 (acetonitrile:water 3:1).

•f f Example 29: 1.95 g (2.18 mmol) of N-acetyl-muramyl- L-alanyl-D-isoglutaminyl-L-(Ca)-([2-benzyloxycarbonylamino-ethyl]-sulphonyl-methyl)-glycine benzyl ester are dissolved in 40 ml of abs. pyridine and peracetylated with 30 times the molar amount of acetic anhydride *t *t hours, RT). The jelly-like mass is brought into solu- .tion with a small amount of water, and the whole is Sconcentrated to a small volume in a rotary evaporator at 300 in a high vacuum and, after the addition of r abs. dioxan, lyophilised. The crude product is subjected to flash chromat'ography on silica gel (particle size 0.04-0.063 mm) first with chloroform, then with chloroform/methanol mixtures (98:2 to 1:1) (145:1; 25 ml fractions; 0.5 bar). The fractions containing the product are collected, dissolved in 90 dioxan (80 ml) and, after sterile-filtration (0.2 um, i 94 PTFE), lyophilised. N-acetyl-1,4,6-tri-O-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-(Ca)-([2-benzyloxycarbonylamino-ethyl]l-sulphonyl-methyl)-glycine benzyl ester (a,8-mixture) is obtained in the form of a faintly yellowish powder containing 1.27 mol of water; +36.6 10 (c 1.030; dimethylformamide),

D

Rf 0.15 (chloroform:methanol:water 70:30:5), Rf 0.53 (n-butanol:acetic acid:water 75:7.5:21), Rf 0.61 (acetonitrile:water 3:1).

The starting material is nbtained as follows: Stage 29.1: 21.5 g (100 mmol) of a 31.4 hydrogen peroxide solution are added dropwise within a period of minutes, at RT while stirring, to a solution of Sf. 8.50 g (20 mmol) of Ne-benzyloxycarbonyl-L-thialysine- :4benzyl ester hydrochloride. After stirring for hours, 1.5 g of palladium-on-carbon (5 are added to decompose excess peroxide, a strong evolution of gas occurring. After stirring for 3 hours, the catalyst is *0 0 removed and the reaction solution is evaporated to dryness at 300 in a rotary evaporator. The oily residue is dissolved in 11 ml of acetone and crystallised by the addition of 20 ml of diethyl ether.

Recrystallisation from the same mixture yields N -benzyloxycarbonyl-L-thialysine S,S-dioxide benzyl ester hydrochloride in the form of colourless needles; m.p. 141-1420 (decomp.); m.p. of the p-toluenesulphonate 130-134o (decomp.); R 0.50 (chloroform:methanol:water 70:30:5), Rf 0.89 (methyl ethyl ketone:pyridine:water 65:5:20), Rf 0.71 (n-butanol:glacial acetic acid:water 3:1:1).

Stage 29.2: 2.95 g (6 mmol) of N-acetyl-muramyl-Lalanyl-D-isoglutamine and 2.74 g (6 mmol) of Ne-benzyl- 95 oxycarbonyl-L-thialysine S,S-dioxide benzyl ester hydrochloride are linked with one another in customary manner (see Stage 30.1) in accordance with the dicyclohexylcarbodiimide-1-hydroxybenzotriazole method. After stirring for a total of 51 hours at RT, 200 ml of ethyl acetate are added to the reddish suspension and, after stirring for 1 hour, insoluble material is filtered off and the filtrate is evaporated to dryness in a rotary evaporator at 300 in a high vacuum. 100 ml each of ethyl acetate and water are added to the residue and the whole is stirred for one hour. The jelly-like material is filtered off, dried and then partitioned between in each case 80 ml of upper and lower phase of mutually saturated n-butanol/water After stirring for one hour the insoluble material is filtered off with suction, washed and dried. N-acetyl-muramyl-L-alanyl-Disoglutaminyl-L-(Ce)-([2-benzyloxycarbonylamino- $to ethyl]-sulphonyl-methyl)-glycine benzyl ester (a,Bgo mixture) is obtained in the form of a white powder containing 1.63 mol of water; Rf 0.60 (.chloroform:methanol:water 70:30:5), Rf 0.83 (acetonitrile:water 3:1).

Example 30: 1.50 g (2.24 mmol) of N-acetyl-muramyl-Lalanyl-D-isoglutaminyl-L-serine benzyl ester are I r suspended in 40 ml of abs. pyridine and peracetylated S" with 40 times the molar amount of acetic anhydride.

The jelly-like material obtained after stirring for 24 hours at RT is brought into solution by the addition of 4 5 ml of water, and the whole is concentrated to approximately 5 ml in a rotary evaporator in a high vacuum (300), twice-distilled water is added and lyophilisation is carried out. The crude product is taken up in 50 ml of warm ethyl acetate and precipitated by the addition of five parts by volume of diethyl ether 96 (twice). The precipitate is dissolved in chloroform/ methanol sterile-filtered (0.2 Mm, PTFE) and, after the evaporation of the solvent, taken up in abs.

dioxan and freeze-dried. N-acetyl-1,4,6-0-triacetylmuramyl-L-alanyl-D-isoglutaminyl-L-O-acetyl-serine benzyl ester hydrate (a,8-mixture) is obtained in the form of a colourless powder; 0 +26.5 0.9 0 (c 1.096; chloroform:methanol 1:1); Rf 0.10 (chloroform:isopropanol 7:2), Rf 0.69 (chloroform:methanol:water 70:30:5), Rf 0.33 (n-butanol:acetic acid:water 75:7.5:21).

The starting material is obtained as follows: 9 Stage 30.1: 5.00 g (10.15 mmol) of N-acetyl-muramyl- L-alanyl-D-isoglutamine, 2.00 g (13.2 mmol) of 1hydroxybenzotriazole (containing 12 water) and 2.35 g (10.15 mmol) of L-serine benzyl ester *;ydrochloride are 9 dissolved in 50 ml of dimethylformamicv. 1.17 ml S(10.66 mmol) of N-methylmorpholine and then 2.72 g (13.2 mmol) of N,N'-dicyclohexylcarbodiimide are added thereto. After stirring for 24 hours at RT, 100 ml of 1 ethyl acetate are added to the reddish suspension and -the whole is stirred for 1 hour. The insoluble dicyclohexyl urea is filtered off and the filtrate is 4 concentrated by evaporation in a high vacuum in a rotary evaporator at 300. The resulting yellow oil t.,O jis purified on silica gel (60:1; 15 ml fractions) in the system chloroform:methanol:water 70:30:5. The pure fractions containing the product are collected and, after evaporation of the solvent, lyophilised from tert.-butanol/water N-acetyl-muramyl-L-alanyl-Disoglutaminyl-L-serine benzyl ester (a,8-mixture) is 3 i I irc~- i- -r _I I I 97 obtained in the form of a colourless powder containing 1.87 mol of water; 54620 nm +12.4 1 (c 0.978; water), Rf 0.41 (chloroform:methanol:water 70:30:5), Rf 0.59 (ethyl acetate:n-butanol:pyridine:acetic acid:water 42:21:21:6:10).

Example 31: 0.36 g (0.535 mmol) of N-acetyl-muramyl- L-N-methyl-alanyl-D-isoglutamine benzhydryl ester (a,8-mixture) is dissolved in 4 ml of abs. pyridine and, after the addition of 0.15 ml (16 mmol) of acetic anhydride, left to stand for three hours at RT. The yellowish solution is concentrated by evaporation in a high vacuum at 300, taken up in 10 ml of water and lyophilised. The crude product is purified by chromatography on silica gel (1:180) in the system chloroform/isopropanol (0.8 ml fractions). The homogeneous fractions are collected and lyophilised a* from abs. dioxan. N-acetyl-1,4,6-tri-O-acetyl-muramyl- S' L-N-methyl-alanyl-D-isoglutamine benzhydryl ester (a,-mixture) is obtained in the form of a colourless powder; R 0.61 (n-butanol:acetic acid:water 75:7.5:21), Rf 0.20 (chloroform:isopropanol:acetic acid 70:8:2).

Cr C The starting material is obtained as follows: Stage 31.1: 0.50 g (0.99 mmol) of N-acetyl-muramyl-L- N-methyl-alanyl-D-isoglutamine (a,8-mixture), dissolved in methanol, is esterified with excess diphenyldiazomethane. After standing for two hours, the whole is evaporated to dryness and the residue is repeatedly triturated with petroleum ether. Filtration with suction is carried out, the residue is taken up in a small amount of methanol, and the material is precipi- -98tated with ten times the amount of diethyl ether:- Petroleum ether N-acetyl-muramyl-L-N-methylalanyl-D-isoglutamine benzhydryl ester is obtained in the form of a colourless powder; Rf 0.76 (acetonitrile:water 3:1), Rf 0.63 (chloroform:methanol:water 70:30:5).

Example 32: 0.50 g (0.99 mmol) of N-acetyl-muramyl-aaminoisobutyryl-D-isoglutamine (c,B-mixture) are esterified analogously to Stage 31.1 with diphenyldiazomethane. The crude product [Rf =0.48 (chloroform: methanol:water 70:30:5)] is peracetylated analogously to Example 31. The reaction solution is evaporated to dryness in vacuo, and the residue is taken up in water and lyophilised. Purification is carried out on silica gel (1:100) in chloroform/methanol 1 ml fractions) The pure fractions are combined, taken up *in 5 ml of chloroform, filtered (PTFE; 0.2 um) and then evaporated to dryness. The residue is taken up in abs.

dioxan and lyophilised. N-acetyl-1 ,4,6-tri-0-acetylmuramyl-ct-aminoisobutyryl-D-isoglutamine benzhydryl ester (c,$-mixture) is obtained in the form of a colourless powder containing 0.73 mol of water; 10 (c 0.993; methanol), RfD08 clrfrnmtao~ae 03:) Rf 0.32 (chloroform:methanolwae 90:1),) .:Rf 0.50 (n-butanol:acetic acid:water =75:7.5:21).

Example 33: 33 mg of N-acetyl-demethylmuramyl-Lalanyl-D-y-methoxycarbonyl-isoglutamine benzhydryl 0 ester (a,B-mixture) are peracetylated analogously to 0 0 Example 31. Lyophilisation from abs. dioxan yields N-acetyl- 1 6-tr i-O-acetyl-demethylniuramyl-L-alanyl- D-y-methoxycarbonyl-isoglutamine benzhydryl ester (c,8-mixture) in the form-T of a colourless powder; 01- 99 Rf 0.67 (chloroform:methanolwater 70:30:5), Rf 0.35 (n-butanol:acetic acid:water 75:7.5:21).

The starting material is obtained as follows: Stage 33.1: 0.216 g (0.40 mmol) of N-acetyl--demethylmuramyl-L-alanyl--D-y-carboxy-isoglutamine (a,9-mixture) is esterified analogously to Stage 31.1 with diphenyldiazomethane (3 equivalents). The light reddish suspension is filtered after stirring for 3 hours, and the filtrate is evaporated to dryness. The mixture, consisting of educt, mono- and di-benzhydryl ester and decomposition products originating from diphenyldiazomethane, is separated by chromatography on silica gel (1:100) in the system chloroform:methanol: water (70:30:5). Chromatography twice yields N-acetyldemethylmuramyl-L-alanyl-D-y-benzhydryloxycarbonylisoglutamine benzhydryl ester (ca,8-mixture) in the form of a colourless resin with the following Rf values Rf 0.67 (chloroform:methanol:water 70:30:5) and Rf 0.46 (n-butanol:acetic acid:water 75:7.5:21) and N-acetyl-demethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester with the Rf value Rf 0.13 (chloroform:methanol:water =70:30:5).

Stage 33.2: N-acetyl-demethylmuramyl-L-alanyl-Disoglutamine benzhydryl ester is esterified in methanolic solution in customary manner with a solution of diazomethane in diethyl ether. Concentration by evaporation yields N-acetyl-demethylmuramyl-L-alanyl-Dy-methoxycarbonyl-isoglutamine benzhydryl ester (a,8-mixture) in the form of a colourless resin; Rf 0.56 (chloroform:methanol:water 70:30:,5).

o 6 4 .4 4.

0 *4 4 *66 0* 44 4 46 9 9 4 tt 4. S 9* 94 4444 4 4. 6 4 496*44 4 9 100- Example 34: 1.70 g (2.53 mmol) of N-acetyl-muramyl- L-ct-aminobutyryl-D-isoglutamine benzhydryl ester (c,$-mixture), dissolved in 25 ml of abo. pyridine, are perpropionylated analogously to Example 31 equivalents of anhydride). After standing for three hours at RT, 30 ml of water are added a~nd the whole is evaporated to dryness in HV at 300 The resinous residue is taken up in 80 ml of abs. dioxan, filtered (PTFE; 0.2 Pam) and lyophilised. N-acetyl-1,4,6-tri- O-propionyl-muramyl-L-ca-aminobutyryl-D-isoglutamine benzhydryl ester (c,f-mixture) is obtained in the form of a colourless powder containing 0.79 mol of water; 2 +5.t.0(c 0.270; methanol),

D

Rf 0.67 (acetonitrile:water Rf 0.32 (chloroform:isopropanol:acetic acid= 70:8:2), Rf 0.87 (ethyl acetate:n-butanol:pyridine:acetic ~9 acid:water 42:21:21:6:10).

taeThe starting material is obtained as follows: Sae34.1; 2.03 g (4 mmol) of N-acetyl-muramyl-Lc-aminobutyryl-D-isoglutamine (ca,8-mixture) are converted into the benzhydryl ester analogously to Stage 31.1. Customary working up yields N-acetylmuramyl-L-ci-aminobutyryl-D-isoglutamine benizhydryl ester (c,8-mixture) in the form of a colcourless powder; *20 0 D =+41.8-1.8 (c 0.548; methanol), Rf =0.73 (chloroform:methanol:water 70:30:5), R f 0.78 (acetonitrile:water 3:1).

Analogously to Example 1 there is obtained from 2.0 g (2.94 nimol) of N-propionyldemethylmuramyl-L-alanyl-D-isoglutamine benzhydryl ester (c,B-mixture), containing 1.17 mol of water, in 101 em C C me em m e *00*

C.

C

.me me Ce *0 4

C

4* 0 0 4*4 C em ft ft fttt te i ft ft ft ft.

OC C C C m 44 ft C me me C 4.4444 C C ml of absolute pyridine, with 1.68 nil (13.06 nimol) of propionic acid anhydride (purissimum; d 1.012), 1 ,4,6-tri-o-propionyl-N-propionyl-demethylmuramyl-Lalanyl-D-isoglutamine benzhydryl ester (ca,8-mixture) in the form of a colourless lyophilisate still containing 0.68 mol of water; m.p. 156-1570, +39.0±2.10 (c 0.467; methanol), Df=04 clrfr~tao Rf 0.48 (chloroform:ethanol R f 0.88 (chloroform:methanol 9 Example 36: There is obtained analogously to Example 1, from 2.0 g (3.1 nimol) of N-propionyl--demethylmuramyl-L-alanyl-D-glutamic acid )-n-butyl ester (C Y)-benzyl ester (mainly c-anomer), still containing 0.41 mol of water, in 20 ml of absolute pyridine with 1.14 ml (12.0 nimol) of acetic anhydride, 1,4,6-tri-Oacetyl-N-propionyl-demethylmuramyl-L-alanyl-D-glutamic acid (Cc)-n-butyl ester-(C Y)-benzyl ester in the form of a colourless lyophilisate still containing 0.73 mol of water. According to the IH-NMR spectrum (360 MHz), the compound is almost exclusively in the form of the a-anomer; 11mepe" 62-640, (a]I2 +36.4±3.60 (c 0.280; methanol), Rf 0.43 (chloroform:methanol Rf 0.55 (chloroform:ethanol Rf 0.85 (chloroform:methanol 4:1).

Example 37: There is obtained analogjously to Example 1, from 0.57 g (0.83 mrnol) of N-propionyldemethylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester (a,B-mixture), containing 0.89 mol of water, in 8 ml of absolute pyridine, with 0.36 ml (3.807 mmol) of acetic 102anhydride, 1,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester (a,8mixture) in the form of colourless crystals; m.p. 141-1420 (from chloroform:diethyl ether 1:10), +22.7-2.2° (c 0.454; methylene chloride), Rf 0.42 (chloroform:methanol 9:1), Rf 0.46 (chloroform:ethanol 9:1), Rf 0.70 (chloroform:methanol 4:1).

The starting material is obtained as follows: Stage 37.1: 0.74 g (1.95 mmol) of the sodium salt of 4,6-O,O-isopropylidene-N-propionyl-demethylmuramic acid is suspended in 20 ml of N,N-dimethylformamide, subsequently 0.342 g (2.145 mmol) of 1-hydroxybenzotriazole, 0.442 g (2.145 mmol) of N,N-dicyclohexylcarbodiimide and 0.85 g (1.95 mmol) of L-alanyl-Dglutamic acid dibenzyl ester hydrochloride are added and the whole is stirred overnight at room temperature.

Subsequently, the precipitated N,N-dicyclohexyl urea is filtered off and the resulting filtrate is concentrated to dryness by evaporation in a high vacuum at 300.

The residue is taken up in 150 ml of ethyl acetate and washed in succession with 50 ml each of saturated sodium bicarbonate solution, 2N citric acid, saturated Ssodium bicarbonate solution and water. The organic :phases are combined, dried over sodium sulphate and S' concentrated by evaporation in a high vacuum at 300.

4,6-O,0-isopropylidene-N-propionyl-demethylmuramyl-Lalanyl-D-glutamic acid dibenzyl ester (a,8-mixture) is obtained, which contains some N-propionyl-demethylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester (a,8-mixture) and N,N-dicyclohexyl urea, and is processed without further purification.

103 Stage 37.2: 3.3 g of crude 4,6-0,0-isopropylidene-Npropionyl-demethylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester (a,8-mixture) are left to stand overnight in 30 ml of 50% acetic acid. The resulting solution is then filtered and the filtrate is concentrated by evaporation in a high vacuum at 300.

The residue is purified by column chromatography on 150 g of silica gel (type 60, high-purity, Merck; 0.063-0.2 mm) in the system chloroform/ethanol (9:1; ml fractions). Fractions 56-95 are combined and concentrated by evaporation in a high vacuum. The residue is then dissolved in 80 ml of methanol and the resulting, slightly turbid solution is filtered through a millipore filter (Fluoropore, PTFE, 0.2 um).

The clear filtrate is concentrated by evaporation in vacuo at 400. The residue is then dissolved in 10 ml of absolute methanol, which has beforehand been filtered through a millipore filter (Fluoropore, PTFE, 0.2 um), and crystallised by the addition of 100 ml of a. absolute diethyl ether, which has also been filtered Sthrough a millipore filter, and subsequently washed with filtered absolute diethyl ether. N-propionyldemethylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester c (a,8-mixture) is obtained in the form of colourless crystals containing 0.89 mol of water; l m.p. 146-147°, +1462.1 0 (c 0.478; methanol), Rf 0.20 (chloroform:methanol 9:1), Rf 0.65 (chloroform:methanol 4:1), Rf 0.81 (chloroform:methanol 7:3).

S" Example 38: Analogously to Example 1 there is obtained with pyridine and acetic anhydride, from N-benzoyl-demethylmuramyl-L-alanyl-D-glutamic acid dicholine ester dichloride, 1,4,6-tri-O-acetyl-N-

U

benzoyl-demEthylmuramyl-L-alanyl-D-glutamic acid dicholine ester dichloride.

The starting material is obtained as follows: Stage 38.1: D-glutarnic acid dicholine ester dichloride hydrochloride is obtained in a manner analogous to that described in Synthesis 1982, 138, from D-glutamic acid dimethyl ester hydrochlorL4Je and excess choline chloride using titanium tetraethyl ester as catalyst in acetonitrile at 00.

Stage 38.2: N-benzoyl-demethylmuramyl-L-alanyl-Dglutamic acid dicholine ester dichloride is obtained analogously to Stage 6.3 from N-benzoyl-demethylmuramyl- L-alanine and D-glutamic acid dicholine ester dichloride hydrochloride with dicyclohexylcarbodiimide.

Example 39: There is obtained analogously to Example 1 from N-benzoyl-demethylmuramyl-L-alanyl-D-iso- *.glutamine benzhydryl ester with n-hexanoyl chloride, :4 pyridine and 4-dimethylaminopyridine, N-benzoyl-1 ,4,6tri-0-n-hexanoyl-L-alanyl-D-isoglutamine benzhydryl ester; m.p. 95-980, 20D49O±18 (c =0.552; chloroformn), *R f 0.38 (chloroform: acetone

Claims (22)

1. Compounds of the formula I MHWOR' 4 H RNH-C-R. R 3 -C R' D)(D) n in which the hexose moiety is derived from D-glucose, a 6 0 D-mannose or D-galactose, n represents 0 or 1, each 1 446 5 8* 044 of R 1 R 4 and R independently of the others, represents lower alkanoyl or benzoyl, R2 represents lower alkyl or phenyl, each of R 3 R 5 and RU independently of the others, represents hydrogen or lower alkyl, or R and R together represent trimethylene and R 7 represents hydrogen, R 8 represents hydrogen, or lower alkyl that is unsubstituted or is substituted by phenyl, hydroxy, mercapto or lower alkylthio, R 9 and R 12 independently *4 of one another, each represents hydroxy, amino, C 1 10 -alkoxy, aryl-lower alkoxy, alkanoyloxy-lower alkoxy having up to 16 carbon atoms, aroyloxy-lower alkoxy, 3-cholesteryloxy or 2-trimethylammonioethoxy, 10 Ri represents hydrogen, carboxy, lower alkoxycarbonyl or aryl-lower alkoxycarbonyl and R 1 1 represents 106 hydrogen, or lower alkvl that is unsubstituted or is substituted by amino, hydroxy, lower alkanoylamino, lower alkanoyloxy, 2-benzyloxycarbonylamino-ethyl- sulphinyl, 2-benzyloxycarbonylamino-ethyl-sulphonyl, 2 -lower alkoxycarbonylamino-ethyl-sulphinyl,

2-lower alkoxycarbonylamino-ethyl-sulphonyl or by guanidino, with the proviso that at least one of the radicals R 9 and R 12 is other than hydroxy, amino and C1 7 alk ok y 10 or R 1 is other than hydrogen, carboxyand alkoxy- carbonyl having up to 7 carbon atoms in the alkoxy moiety, the prefix "lower" used hereinbefore denoting radicals having up to and including 7 carbon atoms, and salts of such compounds having at least one salt-forming 99 group. *9 S 2. Compounds of the formula I according to claim 1 in Swhich the hexose moiety is derived from D-glucose, D-mannose or D-galactose, n represents 0 or 1, each of R I R 4 and R 6 independently of the others, S represents lower alkanoyl or benzoyl, R 2 represents Slower alkyl or phenyl, each of R, R 5 and R 7 S independently of the others, represents hydrogen or lower alkyl, or R 5 and R 8 together represent tri- methylene and R 7 represents hydrogen, R 8 represents "hydrogen, or lower alkyl that is unsubstituted or is substituted by phenyl, hydroxy, mercapto or lower Salkylthio, R 9 and R 12 independently of one another, S each represents hydroxy, amino, C1_ 10 -alkoxy, aryl- lower alkoxy, alkanoyloxy-lower alkoxy having up to 16 carbon atoms, aroyloxy-lower alkoxy or 3-cholesteryloxy, R1 0 represents hydrogen, carboxy, lower alkoxycarbonyl or aryl-lower alkoxycarbonyl and R 11 represents hydrogen, or lower alkyl that is unsubstituted or is substituted by amino or hydroxy, with the proviso that -o L i :1 107 at least one of the radicals R 9 and R 12 is other than hydroxy, amino and lower alkoxy or R 10 is other than hydrogen, carboxy andlower alkoxycarbonyl, and salts of such compounds having at least one salt- forming group.

3. Compounds according to claim 1 or 2, in which R 9 and R independently of one another, each represents hydroxy, amino, C1_ 10 -alkoxy, alkanoyloxy-lower alkoxy having up to 16 carbon atoms, 3-cholesteryloxy, or phenyl- or benzoyloxy-lower alkoxv, and P. represents o hydrogen, carboxy, lower alkoxycarbonyl, or phenyl-lower .alkoxycarbonyl, and the remaining substituents have the °o meanings mentioned above, with the proviso that at least 9 12 one of the radicals R and R is other than hydroxy, amino Sand lower alkoxy or R IO is other than hydrogen, carboxy 4 and lower alkoxycarbonyl, and salts of such compounds having at least one salt-forming group. o 4

4. Compounds according to claim 1 in which the hexose moiety is derived from D-glucose or D-mannose, n 'represents 0 or 1, each of R I R 4 and R 6 independ- ently of the others, represents C 2 _4-alkanoyl or benzoyl, R 2 represents C 1 4 -alkyl or phenyl, each of R 3 R 5 and R 7 independently of the others, represents hydrogen or methyl, or R 5 and R together represent trimethylene and R 7 represents hydrogen, R 8 repre- sents hydrogen, C1_ 4 -alkyl, or C 1 _2-alkyl substituted by phenyl, hydroxy, mercapto or methylthio, R 9 and R 12 independently of one another, each represents hydroxy, 108 amino, lower alkoxy, phenyl-lower alkoxy, lower alkanoyloxy-lower alkoxy, benzoyloxy-lower alkoxy or 3 -cholesteryloxy, R 10 represents hydrogen, carboxy, lower alkoxycarbonyl or phenyl-lower alkoxycarbonyl and R 1 1 represents hydrogen, or C 1 _4-alkyl that is unsubstituted or is substituted by amino or hydroxy, with the proviso that at least one of the radicals R and R 12 is other than hydroxy, amino and lower alkoxy or R 10 is other than hydrogen, carboxy and lower alkoxycarbonyl, and salts of such compounds having at least one salt-forming group. Compounds according to claim 1 in which the hexose moiety is derived from D-glucose or D-mannose, n represents 0 or 1, each of R I R 4 and R 6 inde- pendently of the others, represents C 2 _4-alkanoyl or Ss" benzoyl, R 2 represents C1_ 2 -alkyl or phenyl, each o of R 3 R 5 and R 7 independently of the others, 8 o represents hydrogen or methyl, R 8 represents C 1 4 o Q14 alkyl, R and R 1 independently of one another, S.o each represents hydroxy, amino, C 1 _4-alkoxy, phenyl- methoxy, lower alkanoyloxymethoxy, benzoyloxymethoxy or 3-cholesteryloxy, R1 represents hydrogen, carboxy, alkoxycarbonyl having up to 5 carbon atoms, or tt t 11 S phenylmethoxycarbonyl and R 11 represents C1- 4 S" alkyl, with the proviso that at least one of the radic- O als R 9 and R1 2 is other than hydroxy, amino and y- C 1 -4-alkoxy or R10 is other than hydrogen, carboxy and alkoxycarbonyl having up to 5 carbon atoms, and salts of such compounds having at least one salt- b forming group. 0 ;I 109

6. Compounds according to claim 1 in which the hexose moiety is derived from D-glucose, n represents 0 or 1, R R and R 6 represent acetyl or butyryl, R 2 represents C1_2-alkyl or phenyl, R 3 repre- sents hydrogen or methyl, R 5 and R 7 represent hydrogen, R 8 represents C 1 _3-alkyl, R 9 represents amino, C 1 _4-alkoxy, pivaloyloxymethoxy or mono- or di-phenylmethoxy, R 10 represents hydrogen, R 11 represents methyl and R 12 represents mono- or di- phenylmethoxy or 3-cholesteryloxy.

7. Compounds according to claim 1 in which the hexose moiety is derived from D-glucose, n represents 0 or 1, R 4 and R 6 represent C2_6-alkanoyl, R 2 represents C 1 4 -alkyl or phenyl, each of R 3 R 5 and R 7 independently of the others, represents hydrogen or methyl, R 8 represents C 1 4 -alkyl, R represents amino, lower alkoxy, pivaloyloxymethoxy, diphenyl- •methoxy, benzyloxy or 2-trimethylammonioethoxy, R 1 0 r, represents hydrogen or lower alkoxycarbonyl, R 1 9 represents C1- 4 -alkyl, lower alkanoyloxymethyl or (2-benzyloxycarbonylaminoethyl)-sulphonyl-methyl and R 12 represents amino, lower alkoxy, pivaloyloxy- 4 methoxy, diphenylmethoxy, benzyloxy, 2-trimethylammonio- ethoxy, 3-cholesteryloxy or benzoyloxymethoxy, with the proviso that at least one of the radicals R 9 and S R 12 is other than amino and lower alkoxy, and salts of such compounds that are capable of salt formation.

8. Compounds according to claim 1 or 7 in which at R 9 12 least one of the radicals R and R represents pivaloyloxymethoxy, diphenylmethoxy, benzyloxy, 2- trimethylammonioethoxy, 3-cholesteryloxy or benzoyloxy- methoxy, and the other of the radicals R 9 and R12 has the above-mentioned meaning, and salts of such 1 ~Yr -1101 compounds that are capable of salt formation.

9. 1,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L- alanyl-D-isoglutamine benzhydryl ester according to claim 1. 1 ,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L- alanyl-D-isoglutamine benzyl ester according to claim 1.

11. 1 ,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L- alanyl-D-glutamic acid (C )-n-butyl ester (CY) benzhydryl ester according to claim 1.

12. N-acetyl-1 ,4,6-tri-O-acetyl-muramyl-L-alanyl-D- *glutamic acid (C,)-pivaloyloxymethyl ester (C Y)- POP benzyl ester according to claim 1. a P (P 413. 1 ,4,6-tri-O-acetyl-N-benzoyl-demethylmuramyl-L- alanyl-D-isoglutamine benzyl ester according to claim a 1.

14. 1 ,4,6-tri-O-acetyl-N-benzoyl--demethylmuramyl-L- alanyl-D-isoglutamine benzhydryl ester according to claim 1. a 15. N-benzoyl-1 ,4,6-tri-O-butyryl-demethylmuramyl-L- alanyl-D-isoglutamine benzhydryl ester according to claim i.

16. N-acetyl-(la,B) ,4,6-tri-O-acetyl-demethylmuramyl-L- c-amino-butyryl-D-isoglutamine benzhydryl ester according to claim 1.

17. N-acetyl-(lt,) ,4,6-tri-O-acetyl-demethylmuramyl-L- a-amino-butyryl-D-glutamic acid dibenzhydryl ester according to claim 1.

18. N-acetyl-(l±,i) ,4,6-tri-o-acetyl-demethylmuramyl-L- valyl-D-isoglutamine benzhydryl ester according to claim 1.

19. N-acetyl-(lct,B), 4 ,6-tri-O-acetyl-muramyl-L-alanyl- D-isoglutaminyl-L-alanine cholesteryl-3-ester according to claim 1. N-acetyl-1,4,6-tri-O-acetyl-muramyl-a-amino- isobutyryl-D-isoglutamine benzhydryl ester according to claim 1. *21. N-acetyl-1 ,4,6-tri-O-acetyl-demethylmuramyl-L- :0 alanyl-D-y.-methoxycarbonyl-isoglutamine benzhydryl 00; ester according to claim 1.

22. N-acetyl-1,4,6-tri-O-acetyl-muramyl-L-alanyl-D- isoglutaminyl-L-a( [2-benzyloxy-carbonylamino-ethyll *e sulphonyl-methyl)-glycine benzyl ester according to I Iclaim 1. to 1. 23. 1 ,4,6-tri-O-acetyl-N-benzoyl-demethylmuramyl-L- 0 so alanyl-D-glutamic acid dicholine ester according to claim 1. '00:16 of* 24. N-propionyl-1 ,4 ,6-tri-O-propionyl-demethylmuramyl- *0 L-alanyl-D-isoglutamine benzhydryl ester according to claim 1. -112 1 ,4,6-tri-0-acetyl-N-propionyl-demethylmuramyl-L- alanyl-D-glutamic acid c-n-butyl ester y-benzyl ester according to claim 1.

26. 1 ,4,6-tri-0-acetyl--N-propionyl-demethylmuramyl-L- alanyl-D-glutamic acid dibenzyl ester according to claim 1..

27. 1 ,4,6-tri-0-acetyl--N-benzoyl-demethylmuramyl-L- alanyl-D-isoglutamine benzyl ester according to claim 1.

28. 1 ,4,6-tri-0-acetyl-N-propionyl-demethylmuramyl-L- alanyl-D-glutamic acid dipivaloyloxymethyl ester according to claim 1. *29. 1 ,4,6-tri-O-acetyl-N-propionyl-demethylmuramyl-L- alanyl-D-isoglutaminyl-L-.alanine benzoyloxymethyl ester :000 according to claim 1. 000 0 30. Pharmaceutical preparations that contain a compound according to any one of claims 1 to 29 together with a pharmaceutical carrier.

31. Use of a compound according to any one of claims 1 to 29 for the manufacture of pharmaceutical pre- 0 parations for the prophylaxis and treatment of virus infections. 0000 :32. Use of a compound according to any one of claims I to 29 for the prophylaxis and treatment of virus in- fections that are caused by influenza, parainfluenza, herpes, encephalomyocarditis or vaccinia, viruses.

33. Process for the manufacture of compounds of the formula 1, 113 HzORG OR' -0 I) N~H--R' R 3 -C 0 -n L) in which the hexose moiety is derived from D-glucose, D-mannose or D-galactose, n represents 0 or 1, each of R1, R 4 and R 6 independently of the others, represents lower alkanoyl or benzoyl, R 2 represents 0 007 lower alkyl or phenyl, each of R 3 R 5 and R independently of the others, represents hydrogen or 0000 00,lower alkyl, or R5 and R 8 together represent 0 00 trimethylene and R 7 represents hydrogen, R 8 represents hydrogen, or lower alkyl that is 00 0 unsubstituted or is substituted by phenyl, hydroxy, mercapto or lower alkylthio, R 9 and R 12 independently S. of one another, each represents hydroxy, amino, C 1 10 -alkoxy, aryl-lower alkoxy, alkanoyloxy-lower alkoxy having up to 16 carbon atoms, aroyloxy-lower 0 alkoxy, 3-cholesteryloxy or 2-trimethylammonioethoxy, RIO represents hydrogen, carboxy, lower alkoxycarbonyl or aryl-lower alkoxycarbonyl and R 11 represents *e hydrogen, or lower alkyl that is unsubstituted or is substituted by amino, hydroxy, lower alkanoylamino, 0 lower alkanoyloxy, 2-benzyloxycarbonylamino-ethyl- sulphinyl, 2-benzyloxycarbonylamino-ethyl-sulphonyl, 2-lower alkoxycarbonylamino-ethyl-sulphinyl, 2-lower alkoxycarbonylamino-ethyl-sulphonyl or by guanidino, 114 with the proviso that at least one of the radicals R 9 and R 12 is other than hydroxy, amino and C1-7-alkoxy or R is other than hydrogen, carboxy and alkoxy- carbonyl having up to 7 carbon atoms in the alkoxy moiety, the prefix "lower" used hereinbefore and hereinafter denoting radicals having up to and including 7 carbon atoms, and salts of such compounds having at least one salt-forming group, characterised in that a) a compound of the formula II aa R* R1a (II) NH-R2 *7 Z a 4 4.44 n a in which at least one of the radicals R la R 2 a R 4a and R represents hydrogen and the remainder of these radicals have the meanings of R 1 the group SR 4 and R 6 respectively, and the remaining 4 substituents have the meanings mentioned above, any free functional groups present in a compound of the formula II, with the exception of groups that are to participate in the reaction, being protected, if necessary, by readily removable protecting groups, is reacted with an acylating agent transferring the radical R R 2 R 4 or R 6 to be introduced, and any protecting groups present are, if necessary, removed, or OFF1C~ -i 1 1 i i -115 b) a compound of the formula III H zOR *-0 R' OOsws* H .VVOR i (III) o flH- -RI in which the substituents have the meanings mentioned above, or a reactive derivative thereof, is reacted with a compound of the formula IV R o 099 3 (D)H n 0 v S(IV) in which X represents a reactive esterified hydroxy group, and the remaining substituents have the meanings mentioned above, any free functional groups present in a compound of the formula IV, with the exception of X, being'protected, if necessary, by readily removable S ,protecting groups, and any protecting groups present are, if necessary, removed, or *0 0 1- 116 c) a compound of the formula V, SHzOR -o *'Iow 7 8v- (v) R 3 -C (D) q i(D) r (t (L) in which each of q, r, s and t, independently of the others, represents 0 or 1, and in which the substituents have the meanings mentioned above, any free functional groups present in a compound of the formula V, with the exception of the group that is to o 00 participate in the reaction, being protected, if necessary, by readily removable protecting groups, or a o reactive carboxylic acid derivative thereof, is reacted with a compound of the formula VI 60 0 a *9 bs h ee o b H R. IA. I n a (VI) (VI) 0 in which each of u, v and x, independently of the others, represents 0 or 1 and the remaining symbols and substituents have the meanings mentioned above, any free functional groups present in a compound of the 117 formula VI, with the exception of the group that is to participate in the reaction, being protected, if necesriry, by readily removable protecting groups, and u, v and x representing 1 when and t in the reactant of the formula V represent 0, or u representing 0 and v and x representing 1 when represents 1 and t represents O~ Or u and v representing 0 and x representing 1 when g, r and t represent 1 and s represents 0, or (for the manufacture of compounds of the formula I in which n represents 1) u and x representing 0 when r, s and t represent 1, or with a reactive derivative thereof, and any protecting groups present are, if necessary, removed, or d) for the manufacture of a compound of the formula I in which R 9 has one of the meanings mentioned above apart from hydroxy and amino and/or R 10 represents 0 lower alkoxycarbonyl or aryl-lower alkoxycarbonyl and the remaining substituents have the meanings mentioned above, a compound of the formula VII 46 0* *-0 C H OR 6 R' -dC (D) v(D) 6 (VII) 118 in which at least one of the radicals R 10 a and R 13 represents carboxy and the other of the radicals R 10a and R 13 has the meaning mentioned above for R i o and for the group R 9 respectively, and in which the remaining substituents have the meanings mentioned above, any free functional groups present in a compound of the formula VII, with the exception of the groups that are to participate in the reaction, being protected, if necessary, by readily removable protect- ing groups, or a reactive carboxylic acid derivative thereof, is esterified and any protecting groups present are, if necessary, removed, or e) in a compound of the formula I in which at least one of the radicals R 8 9 10 R 1 1 and C(0)-R 1 2 4 Poo is present in a protected form which does not corres- pond to the definition of the desired end product the corresponding protecting group(s) is (are) removed, or 49 0 f) for the manufacture of a compound of the formula I in which R 9 represents amino and the remaining substituents have the meanings mentioned above, a compound of the formula VIII, 4 A k I H 2 OR6 Ik~ RI O R R 3 -C (VIII) H H- RI 2 SL n 119 in which the radical R 14 represents carboxy and the remaining substituents have the meanings mentioned above, any free functional groups present in a compound of the formula VIII, with the exception of the groups that are to participate in the reaction, being protected, if necessary, by readily removable protect- ing groups, or a reactive carboxylic acid derivative thereof, is amidated, and any protecting groups present are, if necessary, removed, and, if desired, after carrying out one of the processes a a resulting compound of the formula I having at least one salt-forming group is converted into its salt or a resulting salt of a compound of the formula I is converted into the free compound, and/or a 3o 0* resulting isomeric mixture is separated. o 4

34. Compounds of the formula shown in claim 1, said compounds substantially as herein described with reference to any one of the Examples. t a A method for the prophylaxis or treatment of virus diseases or for the treatment of tumour diseases in a subject which comprises administering to said subject an amount of a compound defined in any of claims 1 to 29 or 34 effective for said prophylaxis or treatment. DATED this 15th day of February 1990. SCIBA-GEIGY AG By Its Patent Attorneys ARTHUR S. CAVE f CO.