Note: Descriptions are shown in the official language in which they were submitted.
~3~
4-181()5/~
HIV protease inhibitors and processes for their preparation
HIV (human immunodeficiency virus) is a retrovirus that causes the AIDS disease which
generally proves fatal in man. While it is today possible to alleviate the effects of AIDS
and to prolong patients' lives, there have hitherto been no pharrnaceutical compositions
that effectively combat this virus as the cause of AIDS. One possible aim in the treatment
of AII~S is to prevent the propagation of HIV without at the same time damaging the still
intact cell systems of the patient.
The genome of the two variants of HIV, HIV- 1 and HIV-2, that are known so far has a
region that codes for a "gag protease". In addition, both viruses comprise a region that
codes for the group specific antigens (gag). The corresyonding genes are expressed as
precursor protein and the actual gag proteins, the structural proteins of the virus nucleus
p 17, p24, p9 and p7, are freed proteolytically therefrom by the above-mentioned gag
protease. Gag protease itself is also excised from a precursor protein from which further
viral proteins, such as reverse transcriptase and integrase, originate. I'his process is
presumed to proceed autoproteolytically. It is also known that gag protease cleaves the
major core protein (p24) of HIV-l and HIV-2 preferentially N-terrninally at proline, for
exarnple at Phe-Pro, Leu-Pro or Tyr-Pro. A Pro-terlninal p24 thus shortened is then used
together with other viral structural proteins for ~he synthesis of the nucleocapsid and ~he
virus coat of HIV- 1 and HIV-2. It is also known that the HIV-l gag protease cleaves
Leu-Phe, Leu-Ala, Met-Met and Phe-Leu sequences with varying degrees of efficiency. It
is clear that, in addition to the amino acid sequence, the conformation contributes to the
specificity of gag protease. ~ ~
If the action of gag protease could be inhibited? neither functional structural proteins nor
the necessary viral enzymes would be available to the virus and its propagation would be
hindered, if not intelrupted altogether. There is therefore a need for complete or at least
partial inhibitors of gag protease. Such compounds would play an important role as
antiviral agents against AIDS or other retroviral diseases.
It has now surprisingly been found that the compounds according to the invention are
. . .
, , : . - ~ .
.. . . . . ~ : .
:
~:
4~;6i~3
suitable as gag protease inhibitors since they are capable of inhibiting gag proteases even
in the nanomolar concentration range.
The compounds according to the invention are the compounds of formula
~,N~J~ 3
V
wherein R1 is di-lower alkylamino, pyrrolidino, piperidino, morpholino, thiomorpholino,
S,S-dioxothiomorpholino, piperazino, N-lower alkylpiperazino, benzyloxy or benzyloxy
substituted in the phenyl ring by lower alkyl or by lower alkoxy, Al is a bivalent radical of
one of the amino acids valine and asparagine that is bonded N-terminally to the group
-(C=O) and C-terrninally to the group -NH, A2 is a bivalent radical of one of the amino
acids tyrosine and phenylalanine that is bonded N-terminally to the group -(C=O) and
C-terminally to the group -NH, and R3 is OR2 or NHR2 wherein R2 is hydrogen or lower
alkylr and also salts of those compounds insofar as one or more salt-forming groups is
(are) present.
The invention relates also to processes for ~he preparation of the said compounds, to
pharmaceutical compositions comprising those compounds and to the use of those
compounds as medicaments or for the preparation of pharmaceuticaI compositions.
In the description of the present invention, the term "lower" denotes that the groups or
radicals so defined, for example lower alkyl or lower alkoxy, comprise preferably from 1
to 7 carbon atoms and especially from 1 to 4 carbon atoms. The general terms and names
have the following meanings:
Lower alkyl has preferably from 1 to 7 carbon atoms and is, for example, methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl or n-hexyl. Lower alkyl R2 in OR2 is
especially methyl, ethyl or n-butyl, and in NHR2 is especially n-propyL Lower alkyl in
.. . . -. .. . : ~
" ; . . , ~,. ~ . . ,: ~
. , , ~ .
'', , ' : . ' ', :
'. ~ .
3 ~ ~ 3
di-lower alkylamino Rl is especially methyl or ethyl, more especially methyl. The
preferred lower alkyl substituent in benzyloxy R1 is methyl.
Lower alkoxy has preferably from 1 to 7 carbon atoms and is, for example, methoxy,
ethoxy, isopropoxy or tert-butoxy, especially methoxy.
S,S-dioxothiomorpholino denotes the radical, bonded by way of nitrogen, of the sulfone,
4-aza-1,1-dioxo-1-thiacyclohex-4-yl, derived from thiomorpholine.
The substituent lower alkyl or lower alkoxy in benzyloxy R1 is in the 2-, 3- or 4-position
of the phenyl ring, preferably in the 4-position, and may also occur several times. Substit-
uted benzyloxy Rl is, for example, 2-, 3- or 4-methylbenzyloxy, 2-, 3- or 4-methoxy-
benzyloxy, 2,4- or 3,5-dimethylbenzyloxy or 2,4-dimethoxybenzyloxy.
Valine (ol-aminoisovaleric acid) and asparagine (o~-aminosuccinic acid 4-amide) Al or
phenylalanine (~-amino-~-phenylpropionic acid) and tyrosine (a-amino-~-(p-hydroxy-
phenyl)propionic acid) A2 denote the corresponding L-ol-amino acids occurring in natural
proteins.
Salts are especially the pharrnaceutically acceptable non-toxic salts of compounds of
formula I that cornprise one or more salt-forrning acidic or basic groups.
Such salts are formed, for example, from compounds o~ formula I wherein R2 is hydrogen,
and are, especially, sui~able alkali metal salts, for example sodium or potassium salts, or
alkaline earth metal salts, for example rnagnesium or calcium salts, also zinc salts or
ammonium salts, and also those salts which are formed with organic amines, such as
unsubstituted or hydroxy-substituted mono-, di- or tri-alkylamines, for example with
diethylamine, di-t2-hydroxyethyl)amine, triethylamine, N,N-dimethyl-N-(2-hydroxy-
ethyl)amine, tri-(2-hydroxyethyl)amine or N-methyl-D-glucamine. The compounds offormula I having a basic group, for example an amino group, can form acid addition salts,
for example, with inorganic acids, ~or example hydrochloric acid, sulfuric acid or
phosphoric acid, or with organic carboxylic, sulfonic or sulfo acids, for example acetic
acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,
methylmaleic acid, fumaric acid, malic acid, ta~taric acid, cit~ic acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic
acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, also with
.
. . .
~3~
- 4 -
a-amino acids, and with methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethane-
sulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic
acid or naphthalene-2-sulfonic acid, or with other acidic organic compounds, such as
ascorbic acid. Compounds of formula I having acidic and basic groups can also form
internal salts.
For the purpose of isolation or purification, it is also possible to use pharmaceutically
unsuitable salts.
The compounds of the present invention exhibit gag protease-inllibiting effects. In
particular, they inhibit the action of the gag protease of HIV- 1 and HIV-2 in
concentrations as low as the nanomolar range and are accordingly suitable as agents
against diseases caused by these or related retroviruses, such as, for example, against
AIDS.
The ability of the compou~ds of forrnula I to inhibit the proteolytic activity of, for
example, HIV-1 protease can be demonstrated, for example, in accordance with themethod described by J. Hansen et ah, The EMBO Journal 7, 1785-1791 (1988). The
inhibition of the action of gag protease is measured on a substrate that is a fusion protein,
expressed in E. coli, of the gag precursor protein and MS-2. The substrate and its cleavage
products are separated by polyacrylamide gel electrophoresis and are rendered visible by
immunoblotting using monoclonal antibodies to MS-2.
In a test that is even simpler to carry out and perrnits exact quantitative statements to be
made, a synthetic icosapeptide that corresponds to the cleavage site of the gag precursor
protein is used as the substrate for the gag protease. This substrate and its cleavage
products can be measured by high pressure liquid chrvmatography (HPLC). In this test,
compounds of the present invention exhibit inhibitory effects in concentrations of
10-6 mol/l.
In another test it can be shown that the compounds of the present invention protect cells
that are normally infected by HIV against such infection, or at least retard such infection.
In this test, the human T-cell leukaemia cell line MT-2 (Science 22g, 563 (1985)), which
is extremely sensitive to the cytopathogenic effect of HIV, is incubated with HIV only or
with HIV in the presence of the compounds according to the invention and, after a few
days, the viability of the cells so treated is assessed. Compounds according to the
,, . :
,
.
3~3~3
invention exhibit infection-inhibiting effects in concentrations of 10-5 mol/l.
Preferred are compounds of formula I wherein R1 is di-lower alkylamino, piperidino,
morpholino, thiomorpholino, S,S-dioxothiomorpholino or benzyloxy, A1 is the bivalent
radical of the amino acid valine, A2 is the bivalenl radical of the amino acid tyrosine, and
R3 is OR2 wherein R2 is lower alkyl.
Also preferred are compounds of formula I wherein R1 is di-lower alkylamino, piperidino,
morpholino, thiomorpholino, S,S-dioxothiomorpholino or benzyloxy, A1 is the bivalent
radical of the amino acid asparagine or valine, A2 is the bivalent radical of the amino acid
phenylalanine or tyrosine, and R3 is OR2, wherein R2 is lower alkyl, or NHR2, wherein R2
is hydrogen or lower alkyl.
Very especially preferred are compounds of formula I wherein Rl is morpholino, thio-
morpholino, S,S-dioxothiomorpholino or benzyloxy, A1 is the bivalent radical of the
amino acid valine, A2 is the bivalent radical of the amino acid tyrosine, and R3 is OR2
wherein R2 is lower alkyl, especially methyl.
Also very especially preferred are the compounds of formula I wherein R1 is morpholino
or benzyloxy, A1 is the bivalent radical of the amino acid asparagine, A2 is the bivalent
radical of the amino acid tyrosine, and R3 is OR2 wherein R2 is lower alkyl, especially
methyl.
Likewise very especially preferred are compounds of formula I wherein R1 is morpholino,
A1 is the bivalent radical of the amino acid valine or asparagine, A2 is the bivalent radical
of the amino acid phenylalanine or tyrosine, and R3 is NHR2 wherein R2 is hydrogen or
lower alkyl, especially hydrogen or n-propyl.
The compounds of formula I according to the invention and salts of such compounds
having at least one salt-forming group are obtained in accordance with processes known
se, for example by
condensing a fragment of a compound of formula I having a terminal carboxy group or a
reactive acid derivative of that fragment with a fragment that is complementary to the
compound of formula I and has a free amino group or with a reactive derivative thereof
having an activated amino group to ~orm an amide bond, functional groups present in the
,
,:
:
. .,
3~3
reactants, with the exception of the groups participating in the reaction, optionally being in
protected form, and, if desired,
removing protecting groups present in an obtainable compound and/or converting an
obtainable compound of formula I having a salt-forming group into its salt or converting
an obtainable salt into the free compound or into a different salt and/or separating optio-
nally obtainable isomeric mixtures and/or converting a compound of formula I according
to the invention into a different compound of fonnula I according to the invention.
Fragments of a compound of formula I having a terminal carboxy group that can becondensed with a fragment complementary to the compound of formula I to form an
amide bond are, for example, compounds of formulae
Rl-COOH (II),
R1 A1 (III),
O H OH ~
R~JI\A~ \~ COOH
(IV).
/ \~ - (V), ~nd
,
~3~
O H OH ~
R~A~ \~ ~A2 (VI)
O ~
wherein the radicals are as defined above, the activated esters or reactive anhydrides
derived from those compounds, and also reactive cyclic amides. The reactive acid deriv-
: ~ atives can also be formed in situ.
Activated esters are especially esters that are unsaturated at the linking carbon atom of the
esterifying radical, for example of the vinyl ester type, such as vinyl esters (obtainable, for
example, by transesterifying a corresponding ester with vinyl acetate; activated vinyl ester
method), carbamoylvinyl esters (obtainable, for example, by treating the corresponding
acid with an isoxazolium reagent; 1,2-oxazolium or Woodward method), or 1-lower alk-
oxyvinyl esters (obtainable, for example, by treating the corresponding acid with a lower
alkoxyacetylene; ethoxyacetylene method~, or esters of the amidino type, such as N,N'-di-
substituted amidino esters (obtainable, for example, by treating the corresponding acid
with a suitable N,N'-disubstituted carbodiimide, for example N,N'-dicyclohexylcarbo-
dlimide; carbodiimide method), or N,N-disubstituted amidino esters~ (obtainable, for
example, by treating the corresponding acid with an N,N-disubstituted cyanamide; cyan-
amide method), suitable aryl esters, especially phenyl esters substituted by electron-
attracting substituents (obtainable, for example, by treating:the corresponding:acid with a
suitably substituted phenol, for example 4-nitrophenol, 4-methylsulfonylphenol, 2,4,5-~ri-
chlorophenol, 2,3,4,5,6-pentachlorophenol or 4-phenyldlazophenol, in the presence of a.
condensation agent, such as N,N'-dicyclohexylcarbodiimide; activated aryl esters. method), cyanomethyl esters (obtainable, for example, by treating the corresponding acid
with chloroacetonitrile in the presence of a base; cyanomethyl esters method), thio esters,
: especially phenylthio esters that are unsubstituted or substituted, for example, by nitro
(obtainable, for example, by treating the corresponding acid with thiophenols that are
unsubstituted or substituted, for example, by nitro, inter alia with the aid of the anhydride
or carbodiimide method; activated thio esters method), or especially aniino or amido
esters (obtainable, for exarnple, by treating the corresponding acid with an N-hydroxy-
. :' ' .~ .
. ; I ,. ~ ,~ `
s~
amino or N-hydroxyamido compound or activated derivatives thereof, for example N~
hydroxysuccinimide, N-hydroxypiperidine, N-hydroxyphthalimide, N-hydroxy-5-nor-
bornene- or -norbornane-2,3-dicarboxylic acid imide, 1-hydroxybenzotriazole or benzo-
triazol-l-yloxyphosphonium salts or O-benzotriazol-1-yluronium salts, or 3-hydroxy-
3,4-dihydro-1,2,3-benzo~iazin-4-one, for example according to the anhydride or carbo-
diimide method; activated N-hydroxy esters method).
Anhydrides of acids may be symmetrical or preferably mixed anhydrides of those acids,
for example anhydrides with inorganic acids, such as acid halides, especially acid
chlorides (obtainable, for example, by treating the corresponding acid with thionyl
chloride, phosphorus pentachloride or oxalyl chloride or optionally by treating an alcohol
or amine with phosgene; acid chloride method), azides (obtainable, 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 semi-esters, for
example carbonic acid lower alkyl semi-esters (obtainable, for example, by treating the
corresponding acid with chloroformic acid lower alkyl esters, or with a 1-lower alkoxy-
carbonyl-2-lower alkoxy-1,2-dihydroquinoline, for example 1-ethoxycarbonyl-2-ethoxy-
1,2-dihydroquinoline; mixed O-alkylcarbonic acid anhydrides method), anhydrides with
dihalogenated, especially dichlorinated, phosphoric acid (obtainable, for example, by
treating the corresponding acid with phosphorus oxychloride; phosphorus oxychloride
method), anhydrides with other phosphoric acid derivatives (for example those which can
be obtained with phenyl-N-phenylphosphoramidochloridate) or with phosphorous acid
derivatives, or anhydrides with organic acids, such as mixed anhydrides with organic
carboxylic acids (obtainable, for example, by treating the coIresponding acid with an
unsubstituted or substituted lower alkanecarboxylic acid halide or phenyl-lower alkane-
carboxylic acid halide, for example phenylacetic acid chloride, pivalic acid chloride or
trifluoroacetic acid chloride; mixed carboxylic acid anhydrides method) or with organic
sulfonic acids (obtainable, for example, by treating a salt, such as an alkali metal salt, of
the corresponding acid with a suitable organic sulfonic acid halide, such as a lower alkane-
sulfonic acid chloride or arylsulfonic acid chloride, for example methane- or p-toluene-
sulfonic acid chloride; mixed sulfonic acid anhydrides method) and symmetrical
anhydrides (obtainable, for example, by condensing the corresponding acid in the presence
of a carbodiimide or of l-diethylaminopropyne; symmetrical anhydrides method).
Sui~able cyclic amides are especially amides with five-membered diazacycles of aromatic
character, such as amides with imidazoles, for example imidazole (obtainable, for
2 ~
example, by treating the corresponding acid with N,N'-carbonyldiimidazole; imidazole
method), or pyrazoles, for example 3,5-dimethylpyrazole (obtainable, for example, by way
of the acid hydrazide by treatment with acetylacetone; pyrazolide method).
Fragments having a free amino group that are complementary to the compound of formula
I are, for example, compounds of formulae
H OH ~/ H O
A~ \~ - A2~ R3 (VII),
0/ 0 ~
OH ~/ H O
H2N~ ~A/
- 2 (VIII),
0/ ~
A~ 3 (IX),
A2 (X) and
,
:
r~
- 10-
H2N--R2 (XI),
wherein the radicals are as de~lned above.
The amino group that is present in a fragment complementary tO a compound of formula I
and participates in the reaction is preferably in free form, especially if the carboxy group
reacting therewith is in reac~ive forrn; it can also, however, itself be derivatised, for
example by reaction with a phosphite, such as diethyl chlorophosphite, 1,2-phenylene
chlorophosphite, ethyl dichlorophosphite, ethylene chlorophosphite or tetraethyl pyro-
phosphite. A derivative of such a complementary fragment having an amino group is, for
example, also a carbamic acid halide or an isocyanate, tlle amino group participating in the
reaction being substituted by halocarbonyl, for example chlorocarbonyl, or modi~ed in the
form of an isocyanate group.
Functional groups in starting materials, the reaction o which is to be avoided, especially
carboxy, amino or hydroxy groups, can be protected by suitable protecting groups that are
customarily used in the synthesis of peptide compounds, but also of cephalosporins and
penicillins. Those protectirlg groups may already be present in the precursors and are
intended to protect the functional groups in question against undesired side-reactions, such
as acylation, etherification, esterification, oxidation, solvolysis, etc.. Protecting groups
may, however, also be present in the end products. Compounds of formula I having pro-
tected functional groups can have a higher metabolic stability than can the corresponding
compounds having free functional groups.
: ~
The protection of funceional groups by such protecting groups, the protecting groups
themselves and the reactions by which they are removed are described, for example, in
standard worksj such as in J.F.W. McOmie, "Protective Groups in Organic Chemistry",
Plenum Press, London and New ~ork 1973, in Th. W. Greene, "Protective Groups in
Organic Synthesis", Wiley, New York 1981, in "The Peptides"; volume 3 (edi$ed by E.
Gross and J. Meienhofer), Academic Press, London and New ~ork 1981, and in
"Methoden der organischen Chemie", Houben-Weyl, 4th Edition, vol. 15/I, &eorg Thieme
Verlag, Stuttgart 1974.
.
,
.
~ .
.
- 11 -
A carboxy group is protected, for example, in the form of an ester group that is selectively
cleavable under mild conditions. A carboxy group protected in esteri~led form is esteri~led
especially by a lower alkyl group that is branched in the 1-position of the lower alkyl
group or substituted by suitable substituents in the 1- or 2-position of the lower alkyl
group.
A protected carboxy group esterified by a lower alkyl group that is branched in the
1-position of the lower alkyl group is, for example, tert-lower alkoxycarbonyl, for example
tert-butoxycarbonyl, or arylmethoxycarbonyl having one or two aryl radicals in which aryl
is unsubstituted phenyl or phenyl mono-, di- or tri-substituted, for example, by lower al-
kyl, for example tert-lower alkyl, such as tert-butyl, lower alkoxy, for example methoxy,
hydroxy, halogen, for example chlorine, and/or by nitro, for example benzyloxycarbonyl,
benzyloxycarbonyl substituted by the mentioned substituents, for example, 4-nitrobenzyl-
oxycarbonyl or 4-methoxybenzyloxycarbonyl, diphenylmethoxycarbonyl or diphenyl-
methoxycarbonyl substituted by the mentioned substituents, for example di-(4-methoxy-
phenyl)methoxycarbonyl .
A protected carboxy group esteri~led by a lower alkyl group that is substituted by suitable
substituents in the 1- or 2-position of the lower alkyl group is, for example, 1-lower
alkoxy-lower alkoxycarbonyl, for example methoxymethoxycarbonyl, 1-methoxye~hoxy-
carbonyl or 1-ethoxyethoxycarbonyl, 1-lower alkylthio-lower alkoxycarbonyl, for example
1-methylthiomethoxycarbonyl or 1-ethylthioethoxycarbonyl, aroylmethoxycarbonyl, for
example phenacyloxycarbonyl, 2-halo-lower alkoxycarbonyl, for example 2,2,2-trichloro-
ethoxycarbonyl, 2-bromoethvxycarbonyl or 2-iodoethoxycarbonyl, and also 2-tri-lower
alkylsilyl-lower alkoxycarbonyl, for example 2-trimethylsilylethoxycarbonyl.
A carboxy group can also be protected in the fonn of an organic silyloxycarbonyl group.
An organic silyloxycarbonyl group is, for example, a tri-lower alkylsilyloxycarbonyl
group, for example trimethylsilyloxycarbonyl.
A protected carboxy group is preferably tert-lower alkoxycarbonyl, for example tert-
butoxycarbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl or diphenylmethoxy-
carbonyl.
An amino group can be protected, for example, in thc form of an acylamino, arylmethyl-
amino, etheri~led mercaptoamino or silylamino group.
s .,. . .. ; ~ :,
- 12-
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 of a lower alkane-
carboxylic acid that is unsubstituted or substituted, for example, by halogen or aryl, or of
benzoic acid that is unsubstituted or substituted, for example, by halogen, lower alkoxy or
nitro, or preferably of a carbonic acid semi-ester. Such acyl groups are, for example, lower
alkanoyl, such as formyl, acetyl, propionyl or pivaloyl, halo-lower alkanoyl, for example
2-haloacetyl, such as 2-chloro-, 2-bromo-, 2-iodo-, 2,2,2-trifluoro- or 2,2,2-trichloro-
acetyl, benzoyl that is unsubstituted or substituted, for example, by halogen, lower alkoxy
or nitro, for example benzoyl, 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, for example tert-lower alkoxycarbonyl, such as
tert-butoxycarbonyl, arylmethoxycarbonyl having one or two aryl radicals which are
phenyl that is unsubstituted or mono- or poly-substituted, for example, by lower alkyl, for
example tert-lower alkyl, such as tert-butyl, lower alkoxy, such as methoxy, hydroxy,
halogen, such as chlorine, and/or by nitro, for example benzyloxycarbonyl, 4-nitro-
benzyloxycarbonyl, diphenylmethoxycarbonyl, di-(4-methoxyphenyl)methoxycarbonyl or
9-fluorenylmethoxycarbonyl, aroylmethoxycarbonyl, for example phenacyloxycarbonyl,
2-halo-lower alkoxycarbonyl, for example 2-chloroethoxycarbonyl, 2,2,2-trichloroethoxy-
carbonyl, 2-bromoethoxycarbonyl or 2-iodoethoxycarbonyl, 2-tri-lower alkylsilyl-lower
alkoxycarbonyl, for example 2-trimethylsilylethoxycarbonyl, or 2-triarylsilyl-lower
alkoxycarbonyl, for example 2-triphenylsilylethoxycarbonyl.
An arylmethylamino group is, for example, mono-, di- or especially tri-phenylmethyl-
amino, for example benzyl-, diphenylmethyl- or trityl-amino. In an etherif1ed mercapto-
amino group, the etherified mercapto group is especially substituted arylthio, for e-xample
4-nitrophenylthio. A silylamino group is, for example, a tri-lower alkylsilylamino group,
for example trimethylsilylamino.
Preferred amino-protecting groups are acyl radicals of carbonic acid semi-esters,
especially tert-butoxycarbonyl, unsubstituted or substituted benzyloxycarbonyl, for
example 4-nitrobenzyloxycarbonyl, diphenylmethoxycarbonyl, 2-halo-lower alkoxy-
carbonyl, for example 2,2,2-trichloroethoxycarbonyl, and also trityl and formyl.
A hydroxy group can be protected, for example, by a lower alkanoyl group substituted by
halogen, for example chlorine, for example 2,2-dichloroacetyl, or especially by a carbonic
,
acid semi-ester acyl radical mentioned for protected amino groups. A preferred hydroxy-
protecting group is, for example, 2-chloroethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl,
4-nitrobenzyloxycarbonyl or diphenylmethoxycarbonyl. A hydroxy group can also be pro-
tected by tri-lower alkylsi]yl, for example trimethylsilyl or, pre~erably, tert-butyl-di-
methylsilyl, a readily removable alkyl group, such as tert-lower alkyl, for example tert-
butyl, an oxa- or a thia-aliphatic or -cycloaliphatic hydrocarbon radical, for example
1-lower alkoxy-lower alkyl or l-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 from 5 to 7 ring atoms, for example 2-tetrahydro-
furyl or 2-tetrahydropyranyl, or a corresponding thia analogue, or also by 1-phenyl-lower
alkyl, for example benzyl, diphenylmethyl or trityl, it being possible for the phenyl
radicals to be substituted, for example, by halogen, for example chlorine, lower alkoxy, for
example methoxy, and/or by nitro.
The condensation for the production of the amide bond can be carried out in a manner
known per se, for example as described in standard works, such as Houben-Weyl, "Metho-
den der organischen Chemie", 4th edition, vol. 15/II, Georg Thieme Verlag, Stuttgart
1974, "The Peptides" (edited by E. Gross and J. Meienho~er), volumes 1 and 2, Academic
Press, London and New York, 1979/1980, or M. Bodanszky, "Principles of Peptide Syn-
thesis", Springer-Verlag, Berlin 1984.
The condensation of a free carboxylic acid with the corresponding amine can be carried
out in the presence of one of the customary condensation agents. Customary condensation
agents are, for example, carbodiimides, for example diethyl-, dipropyl- or N-ethyl-
N'-(3-dimethylaminopropyl)carbodiimide or especially dicyclohexylcarbodiimide, also
suitable carbonyl compounds, for example carbonyldiimidazole, 1,2-oxazolium com-pounds, for example 2-ethyl-5-phenyl-1,2-oxazolium 3'-sulfonate and 2-tert-butyl-
S-methylisoxazolium perchlorate, or a suitable acylamino compound, for example
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or activated phosphoric acid den-
vatives, for example diphenylphosphoryl azide, diethylphosphoryl cyanide, phenyl-
N-phenylphosphoramidochloridate, bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride or
1-benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate.
If desired, an organic base is added, for example a tri-lower alkylamine having buLky
radicals, for example ethyldiisopropylamine, or a heterocyclic base, for example pyridine,
4-dimethylaminopyridine or, preferably, N-methylmorpholine.
.
.
.
- 14-
The condensation of activated esters, reactive anhydrides or reactive cyclic amides with
the corresponding amines is customarily carried out in the presence of an organic base, for
example simple tri-lower alkylamines, for example triethylamine or tributylamine, or one
of the above-mentioned organic bases. If desired, a condensation agent, as described for
free carboxylic acids, is also used.
The condensation of acid anhydrides with amines can be effected, for example, in the
presence of inorganic carbonates, for example alkali metal carbonates or hydrogen
carbonates, such as sodium or potassium carbonate or sodium or potassium hydrogen
carbonate (customarily together with a sulfate).
Carboxylic acid chlorides, for example the chlorocarbonic acid derivatives derived from
the acid of formula II, are condensed with the corresponding amines preferably in the
presence of an organic amine, for example the above-mentioned tri-lower alkylamines or
heterocyclic bases.
The condensation is preferably carried ou~ in an inert, polar, aprotic, preferably anhydrous
solvent or solvent mixture, for example in a carboxylic acid amide, for example form-
amide or dimethylformamide, a halogenated hydrocarbon, for example methylene chlo-
ride, carbon tetrachloride or chlorobenzene, a ketone, for example acetone, a cyclic ether,
for example tetrahydrofuran, an ester, for example ethyl acetate, or a nitrile, for example
acetonitrile, or in mixtures thereof, optionally at reduced or elevated temperature, for
example within a temperature range of from approximately -40C to approximately
+100 C, preferably from approximately -10C to approximately +50C, and optionally
under an inert gas atmosphere, for example a nitrogen atrnosphere.
Reactive acid derivatives can also be formed In situ. Thus, for example, N,N'-disub-
stituted amidino esters can be formed in situ by reacting the mixture of the fragment
having a free carboxy group and the complementary fragment ha~!ing an amino group in
the presence of a suitable disubstituted carbodiimide, for example dicyclohexyl-carbodiimide. Amino or amido esters of such acids can also be formed in the presence of
the amino component to be acylated, by reacting the mixture of the corresponding acid
and amino starting materials in the presence of a disubstituted carbodiimide, for example
dicyclohexylcarbodiimide, and an N-hydroxylamine or N-hydroxyamide, for example
N-hydroxybenzotriazole, N-hydroxysuccinimide or N-hydroxynorbornane-2,3-dicarbox-
.
, ~
~f~3~3
ylic acid imide, optionally in the presence of a suitable base, for example 4-dimethyl-
aminopyridine, N-methylmorpholine or ethyldiisopropylamine.
The condensation of a carboxylic acid with the corresponding fragment that is
complementary to the coMpound of fo~mula I and has a free amino group can also be
achieved in a manner known ~ se using enzymes, for example as described by H.-D.Jakubke et ah in Angewandte Chemie 97, 79 (1985). Suitable enzymes are, for example,
thermolysine, carboxypeptidase Y, papain, chymotrypsin, trypsin or pepsin. The reaction
is preferably carried out in water or in mixtures of water with organic solvents, for
example with lower alkanols, such as ethanol, dimethylformamide, dimethyl sulfoxide,
eehers, such as tetrahydrofuran, dioxane or 1,2-dimethoxyethane, acetone, acetonitrile or
polyalcohols, for example ethylene glycol or di-, tri- or poly-ethylene glycol, but it can
also be carried out with non-miscible organic sol~ents, for example methylene chloride or
ethyl acetate, at a pH of from S to 8, preferably at approximately the neutral point, at
temperatures of from 0C to 50C. The solvents and the reaction conditions are preferably
so chosen that the desired compound precipitates or is extracted into the non-miscible
organic phase and thus withdrawn from the reaction equilibrium. It is also possible to
carry out the condensation with enzymes, such as those mentioned above, immobilised on
a suitable carrier, in the mentioned organic solvents in admixture with a small amount of
water.
The process can also be carried out in automated form in a manner known ~ se, for
example in accordance with the technique known as solid phase synthesis which was
originated by R. Merrifield and is described, for example, in Angew. Chem. 97, 801-812
(1985), Naturwissenschaften 71, 252-258 (1984) or in R.A. Houghten, Proc. Natl. Acad.
Sci., 82, 5131-5135 (1985).
The fragments of the compound of for.-nula I which are condensed with one another
according to said process can also be used in the form of racemates or in the form of
diastereoisomeric mixtures. Diastereoisomeric mixtures that comprise compounds of
formula I are forrned and have to be subjected to separation of the diastereoisomers in
accordance with the subsequent treatment described hereinafter.
In an obtainable compound of formula I a carboxy group COOR2 present in free or
reactive forrn can be esterified, and an esterified carboxy group (~OOR2 can be converted
into a free carboxy group.
::
' ' '` '
- 16-
For the esterification of the carboxy group in a compound of formula I the free acid can be
used or the free acid can be converted into one of the above-mentioned reactive
derivatives and reacted witll an alcohol, or the free acid or a reactive salt, for example the
caesium salt, can be reacted with a reactive derivative of an alcohol. For example, the
caesium salt of a carboxylic acid can be reacted with the halide or sulfonic acid ester
corresponding to the alcohol. The esterification of the carboxy group can also be effected
with other customary aLlcylating agents, for example with diazomethane, Meerwein salts or
1-substituted 3-aryltriazenes.
One of the methods described hereinafter in connection with the removal of the
carboxy-protecting groups, or, if desired, alkaline hydrolysis under the reaction conditions
mentioned in Organikum9 17th edition, VEB Deutscher Verlag der Wissenschaften, Berlin
(East) 1988, can be used to convert an esterified carboxy group in a compound of formula
I into a free carboxy group.
In a compound of formula I an esterified carboxy group can be converted into a carb-
oxamide group of formula -CO-NHR2 by aminolysis with ammonia or a lower alkylamine.
The aminolysis can be ef~ected under the reaction conditions mentioned for such reactions
in Organikum, 15th edition, VEB Deutscher Verlag der Wissenschaften, Berlin ~ast),
1976 or preferably by reaction in an inert solvent or especially without the addition of a
solvent, at normal or elevated pressure, for example in a bomb tube7 at temperatures of
from 0 to 100C, preferably from 20 to 80C.
In an obtainable compound of formula I the ~hio group of thiomorpholino R1 can be
oxidised to a sulfonyl group.
The oxidation to the sulfonyl group can be carried out with most of the customary
oxidising agents. It is preferable to use those oxidising ag7ents that oxidise the thio ~roup
selectively in the presence of other functional groups, for example the amide function and
the hydroxy group, of the compound of formula I, for example aromatic or aliphatic
peroxycarboxylic acids, for example perbenzoic acid, monoperphthalic acid, m-chloroper-
benzoic acid, peracetic acid, performic acid or trifluoroperacetic acid. The oxidation with
peroxycarboxylic acids is carried out in the customary solvents suitable for the purpose,
for example chlorinated hydrocarbons, for example methylene chloride or chloroform,
diethyl ether, ethyl acetate or the like, at temperatures of from -78C to room temperature,
,
2 ~ 3
for example at from -20C to +10C, preferably at approximately 0C. The peroxy-carboxylic acid can also be formed in situ, -for example with hydrogen peroxide in acetic
acid or formic acid, each of which optionally contains acetic anhydride, for example with
30 % or 90 % hydrogen peroxide in acetic acid/acetic anhydride. Other peroxo com-
pounds are also suitable, for example potassium peroxomonosulfate in mixtures of lower
alkanol and water, for example methanol and water or ethanol and water, or in aqueous
acetic acid, at temperatures of from -70C to +30C, for example from -20C to room
temperature, also sodium metaperiodate in methanol or mixtures of methanol and water at
temperatures of from 0~C to 50C, for example at approximately room temperature.
If desired, the sulfonyl group of S,S-dioxothiomorpholino ~1 in an obtainable compound
of formula I can be reduced to a thio group, for example using diisobutylaluminium
hydride in diethyl ether or tetrahydrofuran.
In an obtainable compound of formula I in which one or more functional groups are
protected, those groups, for example carboxy, amino or hydroxy groups, can be freed in a
manner known ~_ se, optionally in stages or simultaneously, by means of solvolysis,
especially hydrolysis, optionally enzymatic hydrolysis, alcoholysis or acidolysis, or by
means of reduction, especially hydrogenolysis, or chemical reduction. The removal of the
protecting groups is described in the standard works mentioned hereinbefore in the section
"protecting groups".
For example, protected carboxy, for example tert-lower alkoxycarbonyl, lower alkoxy-
carbonyl substituted in the 2-position by an organic silyl group or in the 1-position by
lower alkoxy or lower alkylthio, or unsubstituted or substituted diphenylmethoxycarbonyl,
can be converted into free carboxy by treatment with a suitable acid, ~or example formic
acid or trifluoroacetic acid, optionally with the addition of a nucleophilic compound, for
example phenol or anisole. Unsubstituted or substituted benzyloxycarbonyl can be freed,
for example, by means of hydrogenolysis, that is to say by treatment with hydrogen in the
presence of a metallic hydrogenation catalyst, such as a palladium catalyst. Suitably
substituted benzyloxycarbonyl, such as 4-nitrobenzyloxycarbonyl, can also be converted
into -free carboxy by reduction, for example by treatment with an alkali metal dithionite,
for example sodium dithionite, or with a reducing metal, for example zinc, or a reducing
metal salt, such as a chromium(II) salt, for example chromiumaI) chloride, customarily in
the presence of a hydrogen donor that together with the metal is capable of producing
nascent hydrogen, such as an acid, especially a suitable carboxylic acid, such as a lower
.:
: ..;
;
~. .
- 18-
alkanecarboxylic acid that is unsubstituted or substituted, for example, by hydroxy, for
example acetic acid, formic acid, glycolic acid, diphenylglycolic acid, lactic acid,
mandelic acid, 4-chloromandelic acid or tartaric acid, or an alcohol or thiol, with water
preferably being added. It is also possible to convert 2-halo-lower alkoxycarbonyl
(optionally after converting a 2-bromo-lower alkoxycarbonyl group into a corresponding
2-iodo-lower alkoxycarbonyl group) or aroylmethoxycarbonyl into free carboxy by
treatment with a reducing metal or a reducing metal salt, as described above. Aroyl-
methoxycarbonyl can also be cleaved by treatment with a nucleophilic, preferablysalt-forming, reagent, such as sodium thiophenolate or sodium iodide. 2-tri-lower
alkylsilyl-lower alkoxycarbonyl can also be converted into free carboxy by treatmen~ with
a salt of hydrofluoric acid yielding the fluoride anion, such as an alkali metal fluoride, for
example sodium or potassium fluoride, optionally in the presence of a macrocyclic poly-
ether ("crown ether"), or with a fluoride of an organic quaternary base, such as tetra-lower
alkylammonium fluoride or tri-lower alkylaryl-lower alkylammonium fluoride, for ex-
ample tetraethylammonium fluoride or tetrabutylammonium fluoride, in ehe presence of an
aprotic, polar solvent, such as dimethyl sulfoxide or N,N-dimethylacetamide. Carboxy
esteri~1ed by an organic silyl group, such as tri-lower alkylsilyl, for example trimethylsilyl,
can be freed in customary manner by solvolysis, for example by treatment with water, an
alcohol or an acid, or also a fluoride, as described above. Esterified carboxy can also be
cleaved enzymatically, ~or example esterified arginine or Iysine, such as Iysine methyl
ester, can be cleaved by means of trypsin.
A protected amino group is freed in a manner known ~ se and, depending on the nature
of the protecting groups, by various methods, but preferably by solvolysis or reduction.
2-halo-lower alkoxycarbonylamino (optionally after converting a 2-bromo-lower alkoxy-
carbonylamino group into a 2-iodo-lower alkoxycarbonylamino group), aroylmethoxy-
carbonylamino or 4-nitrobenzyloxycarbonylamino can be cleaved, for example, by treat-
ment with a suitable 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-fo~ing, reagent, such as sodium thio-
phenolate, and 4-nitrobenzyloxycarbonylamino also by treatment with an alkali metal
dithionite, for example sodium dithionite. Unsubstituted or substituted diphenyl-
methoxycarbonylamino, tert-lower alkoxycarbonylamino or 2-tri-lower alkylsilyl-lower
alkoxycarbonylamino can be freed by treatment with a suitable acid, for example formic
acid or trifluoroacetic acid, unsubstituted or substituted benzyloxycarbonylamino can be
freed, for example, by hydrogenolysis, i.e. by treatment with hydrogen in the presence of a
3~3
- 19-
suitable hydrogenation catalyst, such as a palladium catalyst, unsubstituted or substituted
triarylmethylamino or formylamino can be freed, for example, by treatment with an acid,
such as a mineral acid, for example hydrochloric acid, or an organic acid, for example
formic acid, acetic acid 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 substitution product. An amino group protected
by 2-tri-lower alkylsilyl-lower alkoxycarbonyl can also be converted into the free amino
group by treatment with a salt of hydrofluoric acid yielding fluoride anions, as indicated
above in connection with the freeing of a correspondingly protected carboxy group. Silyl,
such as trimethylsilyl, that is bonded directly to a hetero atom, such as nitrogen, can also
be removed by means of fluoride ions.
A hydroxy group protected by a suitable acyl group, an organic silyl group or byunsubstituted or substituted l-phenyl-lower alkyl is freed analogously to a corres-
pondingly protected amino group. A hydroxy group protected by 2,2-dichloroacetyl is
freed, for example, by basic hydrolysis, while a hydroxy group protected by tert-lower
alkyl or by a 2-oxa- or 2-thia-aliphatic or -cycloaliphatic hydrocarbon radical is freed by
acidolysis, for example by treatment with a mineral acid or a strong carboxylic acid, for
example trifluoroacetic acid. A silyl group, for example a trimethylsilyl group or a
tert-butyldimethylsilyl group, is likewise removed by acidolysis, for example by a mineral
acid, preferably hydrofluoric acid, or a strong carboxylic acid. 2-halo-lower
alkoxycarbonyl is removed by the above-mentioned reducing agenes, for example reducing
metals, such as zinc, reducing metal salts, such as chromium(II) salts, or by sulfur
compounds, for example sodium dithionite or, preferably, sodium sulfide and carbon
disulfide.
Salts of compounds of formula I having salt-forming groups can be prepared in a manner
known ~ se. For example, salts of compounds of formula I having acidic groups can be
formed, for example, by treatment with metal compounds, such as alkali metal salts of
suitable organic carboxylic acids, for example the sodium salt of 2-ethylhexanoic acid, or
with inorganic alkali metal or alkaline earth metal salts, for example sodium hydrogen
carbonate, 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
.
,
3~
- 20 -
of compounds of 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 formula
I that comprise, 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 ion exchangers.
Salts can be converted in customary manner into the free compounds: metal and
ammonium salts, for example, by treatment with suitable acids, and acid addition salts, for
example, by treatment with a suitable basic agent.
Stereoisomeric mi~tures, especially diastereoisomeric mixtures, can be separated into the
individual isomers in a manner known Per se, for example by fractional crystallisation,
chromatography, etc..
Racemates can be split in a manner known ~ se, for example after converting the optical
antipodes into diastereoisomers, for example by reaction with optically active acids or
bases.
The invention relates also to those forms of the process in which a compound obtainable
as intermediate at any sta~e is used as starting material and the remaining steps are carried
out or in which the process is discontinued at any stage or in which a compound obtain-
able in accordance with the process according to the invention is produced under the pro-
cess conditions and further processed in s,tu.
Pharmaceutical compositions:
The pharmacologically acceptable compounds of the present invention, especially the
compounds of formula I obtained in accordance with the above processes, can be used, for
example, for the manufacture of pharmaceutical compositions that comprise an effective
amount of the active ingredient together or in admixture with a significant amount of
inorganic or organic, solid or liquid, pharmaceutically acceptable carriers.
The pharmaceutical compositions according to the invention are for enteral, such as nasal,
rectal or oral, administration or for parenteral, such as intramuscular or intravenous,
administration to warm-blooded animals (humans and animals), which comprise an effec-
tive dose of the pharmacological active ingredient on its own or together with a signi~lcant
~,
, ~,
, ~
~3~
amount of a pharmaceutically acceptable carrier. The dose of Ihe active ingredient
depends on the species of warm-blooded animal, the body weight, age and individual con-
dition, on the disease to be treated and also on the mode of administration.
The invention relates also to a method of treating diseases caused by retroviruses, for
example ~IDS, which comprises administering an amount of compounds of formula I
according to the invention that is therapeutically effective against such diseases. The doses
to be administered to warm-blooded animals, especially warm-blooded animals suffering
from diseases caused by retroviruses, for example humans of approximately 70 kg body
weight, are from approximately 3 mg to approximately 3 g, preferably from approximately
10 mg to approximately 1.5 g, for example approximately from 300 mg to 1000 mg, per
person per day, divided into pre~erably from l to 3 single doses which may, for example,
be of equal size. Children usually receive half the adult dose.
The novel pharmaceutical compositions comprise from approximately 1 % to
approximately 95 %, preferably from approximately 20 % to approximately 90 ~/O, active
ingredient. Pharrnaceutical compositions according to the invention may, for example, be
in unit dose form, such as ampoules, vials, suppositories, dragées, tablets or capsliles.
The pharrnaceutical compositions of the present invention are produced in a manner
known ~ se, for example by rneans of conventional dissolving, lyophilising, mixing,
granulating or confectioning processes.
There are preferably used solutions of the active ingredient, and also suspensions,
especially isotonic aqueous solutions or suspensions, it being possible, for example in the
case of lyophilised compositions which comprise the active ingredient on its own or
together with a carrier, for example mannitol, to prepare these before use. The
pharmaceutical compositions may be sterilised and/or comprise excipients, for example
preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for
regulating the osmotic pressure and/or buffers, and are prepared in a manner known ~ se,
for example by means of conventional dissolving or Iyophilising processes. The solutions
or suspensions mentioned may comprise substances that increase the viscosity, such as
sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpylTolidone or
gelatin.
Suspensions in oil comprise as oily component the vegetable, synthetic or semi-synthetic
- ': ' '
,
- 22 -
oils customary for injection purposes. There may be mentioned as such especially liquid
fatty acid esters that comprise as acid component a long-chained fatty acid having from 8
to 22, especially from 12 to 22, carbon atoms, such as, for example, lauric acid, tridecylic
acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic
acid, behenic acid, or corresponding unsaturated acids, such as, for example, oleic acid,
elaidic acid, erucic acid, brassidic acid or linoleic acid. The alcohol component of these
fatty acid esters has at most 6 carbon atoms and is a mono- or poly-hydric, for example
mono-, di- or tri-hydric, alcohol, for example methanol, ethanol, propanol, butanol or
pentanol or their isomers, but especially glycol or glycerol. There may therefore be
mentioned as examples of fatty acid esters: ethyl oleate, isopropyl myristate, isopropyl
palmitate, "Labrafil M 2735" (polyoxyethylene glycerol trioleate manufactured byGattefossé, Paris), "Myglyol 812" (triglyceride of saturated fatty acids of chain length C8
to Cl2, manufactured by Chemische Werke Witten/Ruhr, Gerrnany), but especially
vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean
oil and, especially, groundnut oil.
The manufacture of the injection compositions is effected in customary rmanner under
sterile conditions, as is the introduction thereof into ampoules or vials and the sealing of
the containers.
Pharmaceutical compositions for oral administration can be obtained by combining the
active ingrediene with solid carriers, optionally granulating a resulting mixture and, if
desired or necessary after the addition of suitable excipients, processing the mixture or
granulate into tablets or dragée cores. They can also be incorporated into plastics carriers
which Telease the active ingredients, or allow them to dif~use, in a controlled manner.
Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose,
mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example
tricalcium phosphate or calcium hydrogen phosphate, also binders, such as starch pastes
using, for example, corn, wheat, rice or potato starch, gelatin, tragacanth, meehylcellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinyl-
pyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also
carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt
thereof, such as sodium alginate. Excipients are especially flow-regulating agents and
lubricants, for example silica, talc, stearic acid or salts thereof, such as magnesium
stearate or calcium stearate, and/or polyethylene glycol. Dragée cores are provided with
suitable coatings that may be resistant to gastric juices, there being used, inter alia,
concen~ated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone,
polyethylene glycol and/or titanium dioxide, lacquer solutions in suitable organic solvents
or solvent mixtures or, for the production of coatings that are resistant to gast~ic juices,
solutions of suitable cellulose preparations, such as ethylcellulose phthalate or hydroxy-
propylmethylcellulose phthalate. Colourings or pigments can be added to the tablets or
dragée coatings, for example for identification purposes or to indicate different doses of
active ingredient.
Startin~ materials:
The starting materials used and the reaction conditions chosen are preferably such that the
compounds mentioned as being preferred are obtained.
~he starting materials for canying out the above-mentioned process are known or can be
prepared according to processes that are known ~ se, for example from the relevant
amino acids by condensation in a manner analogous to that of the process described
herein~efore. For example, a compound of formula IV, V, VI, VII or VIII can be
manufactured analogously to the process described in European Patent Application EP
143 746 (published on 05.06.1985) or
EP 258 183 (published on 02.03.1988).
The following Examples serve to illustrate the invention but do not limit the scope thereof
in any way.
Temperatures are given in degrees Celsius. The Rf values are determined on silica gel
thin-layer plates in the following solvent systems:
A ethyl acetate/n-hexane 1:1
B ethyl acetate/n-hexane 1 :2
C ethyl acetate/n-hexane 1:4
D ethyl acetate/n-hexane 1:6
E ethyl acetate/n-hexane l:g
F methylene chloride/methanol 19:1
G methylene chloride/methanol 9:1
H methylene chloride/methanol 4:1
-
- .
- 24 -
methylene chloride/methanol/water 300:10:1
J methylene chloride/diethyl ether 4:1
K methylene chloride/methanol/water 14:6:1
L chloroform/methanol/water/glacial
acetic acid 170:26:3:1
For example, the abbreviation "Rr(A)" denotes that the R~ value has been determined in
system A. The ratio of the solvents to one another is given in parts by volume.
The same abbreviations are used for the eluant systems in the flash chromatography and
the medium-pressure chromatography.
The values for infrared spectroscopy (IR) are given in cm-l. The values for proton nuclear
magnetic resonance spectroscopy (lH-N~lR) are given in ppm (parts per million) with
reference to tetramethylsilane as the internal standard. s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, dd = double doublet, br = broad. In the fast atom bombardment
mass spectrometry (FAB-MS), the values are given for the protonated mass (M+H)+.
The radical referred to as -Cha c Val-- denotes the bivalent radical of (2S,4S,5S)-
5-amino-6-cyclohexyl-4-hydroxy-2-isopropylhexanoic acid and has the formula
H HO
--N~
o/6 0
The radical referred to as -Cha cx Val- is derived from the radical -Cha c Val-- by
bridging NH and OH by an isopropylidene group and has the formula
.
~:,
. ~ ,,
. . .
.
3 ~ ~ 3
~o
/N ~
o ..
The abbreviations conventionally used in peptide chemistry are used to refer to bivalent
radicals of natural a-amino acids. Tyrosine radicals that are esterified or etherified at the
phenolic hydroxy group by the radical R are referred to as Tyr(R).
,
Other abbreviations:
abs. = absolute (anhydrous)
DCCI = dicyclohexylcarbodiimide
DCU = dicyclohexylurea
DMF = dimethylformamide
DMSO = dimethyl sulfoxide
ether = diethyl ether
Fmoc = 9-fluorenylmethoxycarbonyl
HOBt = l-hydroxybenzotriazole
Me = methyl
min. = minute(s)
m.p. = melting point
Tcp = 2,4,5-trichlorophenyl
THF = tetrahydrofuran
Z = benzyloxycarbonyl
Example 1: S~S-dioxothiomorpholinocarbonyl-Val Cha-CVal-Val-Tyr-OMe
34.8 ~,11 of triethylamine and then 16.6 mg (0.083 mmol~ of S,S-dioxothio-
morpholinocarbonyl chloride in 2 ml of methylene chloride are added at room ~emperature
to 45 mg (0.069 mmol) of H-Val-ChaC- Val-Val-Tyr-OMe dissolved in 2 ml of D~rF. The
mixture is stirred for 2 hours at room temperature and then concentrated and the residue is
digested in a small amount of diisopropyl ether. The cMde product is purified bychromatography on silica gel (eluant G). Rf(L) = 0.42; FAB-~IS: (M+H)+ = 808.
-
., : , - ~ .
', ' ~ ~ ' ' ' ' ' ' .
;
' :
- 26 -
The starting materials are prepared in the following manner:
a) 2(S)-BenzyloxYcarbonylamino-3-cYclohexYI-propionic acid ethYI ester: 243 g of2(S)-benzyloxycarbonylamino-3-cyclohexyl-propionic acid (manufacture: Helvetica
Chimica Acta 57, 2131 tl974)) are placed in 600 ml of toluene and 900 ml of ethanol.
The reaction mixture is cooled to 0 and 88.3 g of thionyl chloride are added dropwise
over a period of 30 min. The cooling is removed and the mixture is stirred for 18 hours.
The reaction mixture is filtered and the ~1ltrate is concentrated. The residue is separated
by means of flash chromatography (2 kg of silica gel 60,40-63 ~lm, eluant E). The
fractions containing the product are combined, concentrated by evaporation and dried
under a high vacuum. The title compound is obtained in the form of a slightly yellowish
oil. Rf(E) = 0.2; Rf(B) = 0.52.
b) 2(S)-Benzyloxycarbonylamino-3-cyclohexvl-propanal: 116.1 g of 2(S) benzyloxy-carbonylamino-3-cyclohexyl-propionic acid ethyl ester are placed in 2.2 litres of toluene
and cooled to -65. ~36 ml of diisobutylaluminium hydride are added dropwise at -65
over a period of 30 min. and the mixture is then stirred for 20 min. Then 84.2 ml of
methanol are added dropwise over a period of 10 min. at -65 and subsequently 825 ml of
aqueous potassium sodium tartrate solution without cooling. The reaction m;xture is
discharged onto 3 litres of potassium sodium tartrate solution/ice and extracted with 5
litres of ether. The ethereal phase is washed with 2 litres of water, then immediately
poured into a solution consisting of 106 g of semicarbazide hydrochloride and 156.5 g of
sodium acetate in 620 rnl of water and 620 ml of ethanol. The reaction mixture is then
stirred at room temperature for 1 hour and subsequently separated in a separating funnel,
and the aqueous phase is extracted with 2 x 1.5 litres of ether. The organic phase is dried
over magnesium sulfate and concentrated by evaporation. The crude product is puri~led
by means of flash chromatography (2 kg of silica gel 60, 40-63 llm, eluant A).
Concentration by evaporation of the combined product-containing fractions yields the
semicarbazone of the title compound, Rf(G) = 0.51. 130 g of this semicarbazone are
dissolved in 1 litre of THF, and 282 ml of 37 % forrnaldehyde solution and then, at 10,
143 ml of O.SN HCl are added thereto. The reaction mixture is stirred at room
temperature for 2 hours and is filtered, and the filtrate is washed with 0.5 Iitre of water, 0.5
litre of NaHC~:)3 and 0.5 Iitre of water. The aqueous phases are extracted with 600 ml of
ether. The ethereal phases are dried over magnesium sulfate and concentrated by
evaporation. 100 ml of toluene are added to the residue and the batch is concentrated by
,
.
3~
- 27 -
evaporation to yield the title compound. The latter is further processed immediately.
c) (l(S)-BenzvloxvcarbonYlamino-2-cYclohexyl-ethyl)-oxirane: 18.9 g of sodium hydride
dispersion (55 % in oil) are freed of oil in a dry sulfonating flask under argon by stirring
three times in 50 ml of petroleum ether (b.p. 40-60) and subsequently decanting off the
solvent each time. After drying under a high vacuum, a grey powder is obtained which is
placed in 500 ml of THF; 55.6 g of trimethylsulfoxonium iodide are added thereto, the
temperature increasing to approximately 40. The grey suspension is boiled under re~lux
for 1 hour and then, over a period of 50 min. at -70, a solution of 108.6 g of
2(S)-benzyloxycarbonyl-amino-3-cyclohexyl-propanal in 250 ml of THF is added. The
yellow suspension is stirred at 0 for 2 hours. The yellowish turbid solution is poured onto
500 g of ice. The aqueous solution is extracted with 2.5 litres of ether, and the organic
phase is washed with water and, after being dried over sodium sulfate, is concentrated by
evaporation. The oily residue is separated by means of flash chromatography (2.5 kg of
silica gel 60,40-63,um, eluant C). The fractions containing the product are combined,
concentrated by evaporation and dried under a high vacuum. The title compound
(diastereoisomeric mixture, approximately 4:1) is obtained in the form of a slightly
yellowish oil. R3(I~ = 0.71; Rf(C) = 0.16.
d) 3(S)-Benzyloxycarbonvlamino-4-cyclohexYI-1-iodo-butan-2(R,S)-ol: 42.3 g of
(l(S)-benzyloxycarbonylamino-2-cyclohexyl-ethyl)oxirane are taken up in 200 ml of
acetonitrile and the resulting solution is cooled to 0. After the addition of 20.9 g of
sodium iodide, there are added dropwise at 0~, over a period of 30 min., 17.7 ml of
trimethylchlorosilane. The mixture is stirred at 0 to 3 for 40 minutes and then poured
into 700 ml of ice-cold water. The aqueous mixture is extracted with ether and the organic
phase is washed with 7S0 ml of a 5 % aqueous sodium~ thiosulfate solution and 750 ml of
saturated, aqueous sodium chloride solution. After drying over sodium sulfate and
concentrating by evaporation, an oily mixture of the title compound is obtained which is
further processed directly.
e) 3-Benzvloxycarbonyl-4(S)-cvclohexvlmethvl-2.2-dimethyl-5 (R)-iodomethyl- 1 ~3-
oxazolidine: 49.3 g of the compound of Example ld) and 1.07 g of p-toluenesulfonic acid
monohydrate are stirred in 140 ml of 2,2-dimethoxypropane and 450 ml of methylene
chloride for 3 hours at room temperature. The mixture is extracted by shaking between 1
litre of methylene chloride and 500 ml of saturated, aqueous sodium hydrogen carbonate
solution. The organic phase is washed with water, dried over sodium sulfate and
~.
-
~3~3
- 28 -
concentrated by evaporation. The crude product is purified by means of flash chromato-
graphy (3 kg of silica gel 60, 40-63 ~lm, eluant D). Concentration by evaporation of the
combined, product-containing fractions yields the title compound in the form of a slightly
yellowish oil. R,(C) = 0.55; Rf(D) = 0.46.
f) 2(R~S)-(3-Benzylox~Lcarbonyl-4(S)-cyclohexylmethyl-2~2-dimethvl-l~3-oxazolidin
S(S)-methyl)-3-methxl-butvric acid methYl ester: 14.3 ml of diisopropylamine aredissolved under argon in 200 ml of abs. THF and cooled to 0. Then, at 0 to 5, 65.8 ml of
a 1.6M solution of n-butyllithium in hexane are added dropwise to the mixture over a
period of 20 min. and the batch is stirred for 20 min. Then, at from -70 to -75, 13.3 ml of
isovaleric acid methyl ester are added dropwise and the mixture is stirred for l.S hours at
-75. At from -60 to -75, 320 ml of hexamethylphosphoric acid triamide are added
dropwise with stirring. The resulting suspension is stirred for 10 minutes and, ~lnally, at
from -70 to -75, a solution of 43.4 g of the compound of Example le) in 110 ml of
tetrahydrofuran is added dropwise over a period of S min. The reaction mixture is stirred
at room temperature for 2.5 hours and finally poured onto a mixture of 1 litre of saturated,
aqueous ammonium chloride solution and 500 g of ice. The aqueous phase is extracted
with 2 litres of ethyl acetate, and the organic phase is washed with water and dried over
sodium sulfate. After concentration by evaporation, the diastereoisomeric mixture of the
title compound is obtained in the form of a yellow oil. R~{C) = 0.36; Rf(E) = 0.21 (values
for the less polar component).
g) 2(R,S)-(3-Benzyloxycarbonyl-4(S)-cyclohexylmethyl-2,2-dimethvl-1,3-oxa-
zolidinyl-5(S)-methYl)-3-methYl-butvric acid: 1.77 ml of water are added at approximately
5 to 16.5 g of potassium tert-butanolate in 250 ml of ether. The white suspension is
stirred in an ice bath for a further 10 minutes and then 35.8 g of the compound of E~xample
lf) (diastereoisomeric mixture) in 250 ml of ether are added thereto, the temperature being
maintained below 10. The reaction mixture is then stirred at room temperature for 18
hours and finally poured into 500 ml of saturated, aqueous ammonium chloride solution.
The aqueous phase is extracted with ethyl acetate, and the organic phase is washed with
saturated, aqueous sodium chloride solution, dried over sodium sulfate and concentrated
by evaporation. The oily crude product is separated by flash chromatography (2.5 kg of
silica gel 60, 40-63 ,um, eluant C). :Z;-Cha--Val-OH , the less polar component of the
title compound having the desired configuration of the carbon atom bonded to theisopropyl group (S-configuration), is obtained in the form of a yellow oil. ~(I) = 0.20;
Rf(J) = 0.35.
~3~3
- 29 -
h) HCl-H-Val-Tyr-OMe: 10.0 g of Z-Val-Tyr-OMe are hydrogenated under normal
pressure and at room temperature in 200 ml of methanol and 24 ml of lN HCl in the
presence of 1.0 g of palladium-on-carbon (10 % Pd) until saturation is reached. The
reaction mixture is filtered, and the filtrate is concentrated by evaporation and dried.
Rf(K) = 0.64.
i) Z-Cha CxVal-Val-Tyr-OMe:: A mixture of 2.06 g of HCl-H-Val-Tyr-OMe,3.12 g of
Z-Cha CxVal-OH , 1.74 g of DCCI, 1.22 g of HOBt, 0.75 g of N-methylmorpholine and
50 ml of DMF is stirred at room temperature for 16 hours. The DCU is filtered off, and
the filtrate is concentrated and dried under a high vacuum. The residue is purified by
means of flash chromatography (100 g of silica gel 60, eluant B).
Rf(A) = 0.36.
j) H-Cha c Val-Val-Tyr-OMe: 3.6 g of Z-Cha CxVal-Val-Tyr-OMe are hydrogenated
under normal pressure and at room temperature in 80 ml of methanol/water 9: 1 in the
presence of 360 mg of palladium-on-carbon (10 % Pd) until saturation is reached. The
reaction mixture is filtered, and the filtrate is diluted with 30 ml of water and stirred at
room temperature for 5 hours. After evaporating off the solvent, the title compound is
lyophilised from dioxane/water 9: 1. R~(H) = 0.10.
k) Fmoc-Val-Cha c Val-Val-Ty -OMe: 3.0 g (5.48 mmol) of H-Cha c Val-Val-Tyr-OMe
are dissolved in 75 ml of DMF. 3.69 g (7.12 mmol) of Fmoc-Val-OTcp and then 477 mg
(6.03 mmol) of ethyldiisopropylamine are added to the solution. The mixture is stirred for
5 hours at room temperature and then concentrated by evaporation and the residue is
digested in diethyl ether in an ultrasound bath. The product is isolated by ~lltration and
then washed with diethyl ether and dried. IR (KBr): 3300, 1695, 1645; FAB-MS:
(M+H)+ = 870; R~(L) = 0.5.
1) H-Val-Cha c Val-Val-TYr-OMe:: 70 ml of piperidine are added to a suspension of 3.62 g
(4.17 mmol) of Fmoc-Val-Cha c Val-Val-Tyr-OMe in 70 ml of DMF. The mixture is
stirred for 2 hours at room temperature, a clear solution being formed. The solution is
concentrated by e~aporation, the residue is digested in diisopropyl ether, the product is
isolated by filtration and then washed with diisopropyl ether and dried. IR (KBr): 2g20,
1750, 1650; FAB-MS: (M+H)+ = 647.
. . .
,:
.
,
.
~36~
- 30 -
m) N-BenzyloxYcarbonYlthiomorpholine: 1.74 g (16 mmol) of chloroformic acid benzyl
ester are added dropwise, with stirring, over a period of S min. to 1.03 g (10 mmol) of
thiomorpholine in 30 ml of water. The heterogeneous mixture is stirred for 30 min~ during
which time the pH is maintained at a constant level of 9-10 by the continuous addition of
lN NaOH. The product is extracted with ethyl acetate, the organic phase is washed with
water, dried over MgSO4 and concentrated by evaporation and the residue is
chromatographed on silica gel. IR (CH2CI2): 16~2, 1415.1H-NMR (CDCI3): 2.50-2.76(m,4H); 3.70-3.84 (m,4H); 5.15 (s,2H); 7.38 (s,5H).
n) N-BenzyloxYcarbonylthiomorpholine-S,S-dioxide: 1.84 g (3.0 mmol) of Oxone(~
(potassium monopersulfate triple salt, Fluka) in 10 ml of water are added dropwise at from
5 to 8 over a period of 15 min. to 510 mg (2.15 mmol) of N-benzyloxycarbonylthio-
morpholine in 11 ml of methanol. The mixture is stirred for 30 min. at room temperatur~,
diluted with water and extracted with ethyl acetate. The organic extracts are dried over
MgSO4 and concentrated by evaporation, the title compound being obtained in the form of
a crystalline solid. FAB-MS: (M+H)+=270.1H-NMR (CDCI3): 2.9-3.1 (m,4~); 3.95-4.06
(m,4H); 5.15 (s,2H); 7.38 (s,5H).
o) Thiomorpholine-S,S-dioxide: 544 mg (2.02 mmol) of N-benzyloxycarbonylthio-
morpholine-S,S-dioxide are hydrogenated for 18 hours at room temperature in 30 ml of
methanol in the presence of palladium-on-carbon (5 % Pd). The catalyst is filtered off, the
filtrate is concentrated by evaporation and the residue is recrystallised from ether/benzene.
M.p. 66-68.
p) S.S-Dioxothiomorpholinocarbon~l chloride: 246 mg (1.82 mmol) of thiomorpholine-
S,S-dioxide dissolved in 1~) ml of toluene are added dropwise at from S to 20 to a solution
of 3.83 mmol of phosgene 20 % in toluene. The mixture is stirred for 1 hour at room
temperature. The resulting hydrochloride of thiomorpholine-S,S-dioxide is isolated by
filtration and then washed with toluene. The filtrate is concentrated by evaporation, in the
final stages under a high vacuum, and the title compound is used further directly.
IR (CH2Cl2): 1725, 1320, 1120.1H-NMR (CDCI3): 3.09-3.20 (m,4H); 4.05-4.30 (m,4H).
Example 2: Thiomorpholinocarbonvl-Val-Cha c Val-Val-Tvr-OMe
23.2 ~,11 of triethylamine and then a solution of 9.2 mg (0.055 mmol) of
thiomorpholinocarbonyl chloride (prepared from thiomorpholine and phosgene
- 31 -
analogously to Example lp)~ in 1 ml of methylene chloride are added at room temperature
to 30 mg (û.046 mmol) of H-Val-Cha c Val-Val-Tyr-OMe (Example 11) dissolved in 1 ml
of DMF. The mixture is stirred for 6 hours at room temperature, concentrated completely
by evaporation and the residue is digested in a small amount of diisopropyl ether. The
crude product is purified by chromatography on silica gel (eluant F). R~(L) = 0.43;
FAB-MS: (M+H)+ = 776.7.
Example3: Morpholinocarbonyl-val-cha-cval-val-Tvr-oMe
23.2,ul of triethylamine and then a solu~ion of 8.3 mg (0.055 mmol) of
morpholinocarbonyl chloride (prepared from morpholine and phosgene analogously to
Example lp)) in 1 ml of methylene chloride are added at room temperature to 30 mg
(0.046 mmol) of H-Val-Cha c Val-Val-Tyr-OMe (Example 11)) dissolved in 1 ml of
DMF. The mixture is stirred for 3 hours at room tempera~ure, concentrated completely by
evaporation and the residue is digested in a small amount of diisopropyl ether. The crude
product is purified by chromatography on silica gel (eluant F). Rf(L) = 0.48; FAB-MS:
(M+H)+ = 760.6.
Example 4: Z-Val-Chac Val-Val-Tyr-OMe
25.8 ml of triethylamine and 10.6111 of benzyloxycarbonyl chloride (chloroformic acid
benzyl ester, Z-CI) are added to 40 mg of H-Val-Cha c Val-Val-Tyr-OMe (Example 11))
in 1 ml of DMF. The reaction mixture is stirred overnight at room temperature and then
concentrated by evaporation. The crude product is purified over silica gel with eluant F.
The product-containing fractions are concentrated by evaporation and the residue is
digested with diisopropyl ether and dried. IR (KBr): 3300, 2910, 1640; FAB-MS
(M+H)~ = 781.
Example 5: Morpholinocarbonvl-Val- Cha-Val -Val-Tvr-NH-n-Propvl: 65 mg of the title
compound of Example 3 are dissolved in 5 ml of n-propylamine and stirred first for 18
hours at room temperature and then for 24 hours in a bomb tube at 65C. After
concentration using a rotary evaporator under reduced pressure, the crude product is
purifled by column chromatography on silica gel (eluant L). R~(L) = 0.41; FAB-MS:
(M+H)+=787.
Example 6: Morpholinocarbonyl-Val- Cha-Val -Val-Phe-NH2
2 ml of a solution of 53.5 mg of morpholinocarbonyl-Val-OH in DMF, 102.7 mg of bis-
., . . , , ~ . .
:, : : `;
,:
~3~3
(2-oxo-3-oxazolidinyl)phosphinic acid chloride and 53.9 mg of triethylamine are added in
succession to a solution of 100 mg of H- Cha-Val -Val-Phe-NH2 in 5 ml of DMF. The
solution is stirred for 30 min. at room temperature and then concentrated under a high
vacuum at 40~C. The residue is digested in diisopropyl ether. The crude product is purified
by column chromatography (eluant L) on silica gel. R~(L) = 0.43; FAB-MS: (M+H)+ =
72~.
The starting materials are prepared in the following manner:
a) N-(5(S)-Azido-6-cvclohexvl-4(S)-tert-butyldimethYlsilvloxY-2(S)-isopropvl-hexan-
ovl)-Val-Phe-NH2: 192 mg of 5(S)-azido-6-cyclohexyl-4(S)-tert-butyldimethylsilyl-
oxy-2(S)-isopropyl-hexanoic acid (J. Org. Chem. 54, 1178 (1989)) are dissolved in 4 ml of
DMF, and 227 mg of bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride, 69.3 mg of HOBt
and 154111 of N-methylmorpholine are added in succession thereto. After stirring for 20
min. at room temperature,200 mg of H-Val-Phe-NH2 (hydrochloride, Bachem,
Switzerland) are added to the mixture. After stirring for 30 min. at room temperature,
concentration is carried out using a high vacuum pump and the residue is then taken up in
methylene chloride and washed twice with saturated aqueous sodium hydrogen carbonate
solu~ion. After back-extracting the aqueous extracts once with methylene chloride, the
combined organic phases are dried over sodium sulfate and concentrated by evaporation.
The crude product is stirred in n-hexane/ethyl acetate 1/1 (v/v), filtered off with suction
and then washed with n-hexane/ethyl acetate. Rf(L) = 0.65; FAB-MS: (M+H)+ = 657.
b) H- Cha-Val -Val-Phe-NH2 860 mg of the title compound of Example 6a) are
dissolved in 100 ml of methanol; 430 mg of palladium-on-carbon (S % Pd~ are added and
the batch is reduced with hydrogen. A~ter 4 hours at room temperature, the catalyst is
~lltered off, the solution is concentrated and the residue is digested in ether. After ~lltering
off and drying, the title compound is obtained. Rf(L) = 0.83; FAB-MS: (M+H)+ = 517.
Example 7: Z-Asn- C ha-Val -Val-Tvr-OMe:
0.25 ml of ethyl diisopropylamine and 232.4 mg of Z-Asn-ONP (ONP = ortho-nitrophenyl
ester) are added in portions and in succession to a solution of 274 mg of
c
H- Cha-Val -Val-Tyr-OMe (Example lj)) in S ml of DMF. After stirring ~or 1 hour at
room temperature, the batch is concentrated under reduced pressure and the residue is
,
~36~3
digested in ether and ~11tered off. Then the solid material is again stirred in methanol,
isolated by filtration and washed with methanol and ether. After drying under a high
vacuum, the title compound is obtained. R~(G) = 0.57; FAB-MS: (M+H+) = 796.
Examp!e 8: Morpholinocarbonyl-Asn- 5ha-Va! -Val-Tvr-OMe:
200 mg of H-Asn- Cha-Val -Val-Tyr-OMe in DMF are converted into the title compound
analogously to Example 3 with 90.4 mg of morpholinocarbonyl chloride and 0.15 ml of
triethylamine. The crude product is purified by chromatography on silica gel (eluant L).
Rf(G) = 0.45; FAB-MS: (M+H)+ = 775.
The starting material is prepared in the following manner:
a) H-Asn- Cha-Val -Val-TYr-OMe: 1022 mg of the title compound from Example 7 aredissolved in 150 ml of methanol, and 200 mg of palladium-on-carbon (5 % Pd) are added
and the batch is reduced with hydrogen. After 2.5 hours at room temperature, the catalyst
is filtered off, the solution is concentrated and the residue is treated with ether in an
ultrasound bath. After filtering off and washing again with ether, drying is carried out
under a high vacuum. Rf(L) = 0.07; FAB-MS: (M+H)+ = 662.
Example 9: Morpholinocarbonyl-Asn~ Cha-Val -Val-Tyr-NH-n-propYI:
80 mg of the title compound from Example 8 are stirred for 30 hours at from 65 to 70C in
S ml of N-propylamine and 2 ml of DMF in a bomb tube. After cooling and concentrating
under a high vacuum, the residue is digested in ether, isolated by filtration and purified by
column chromatography (eluant L) on silica gel. ~f(L) = 0.21; FAB-MS: (M+H)~ = 802.
Example 10: Gelatin solution
An aqueous solution of
S,S-dioxothiomorpholinocarbonyl-Val-Cha -CVal-Val-Tyr-(:)Me is sterile-filtered and
mixed with a sterile gelatin solution comprising phenol as preservative, while heating
under aseptic conditions, in such amounts that 1.0 ml of solution has the following
composition:
S,S-dioxothiomorpholinocarbonyl-Val-Cha-CVal-Val-Tyr-OMe 3 mg
gelatin 15û.0 mg
, -
.
.. ,
, ~ ,
,
- 34 -
phenol 4.7 mg
distilled water up to 1.0 ml
The mixture is introduced into 1.0 ml vials under aseptic conditions.
Example 11: Sterile dry sllbstance for injection
5 mg of S,S-dioxothiomolpholinocarbonyl-Val-Cha -CVal-Val-Tyr-OMe are dissolved
in 1 ml of an aqueous solution with 20 mg of mannitol. The solution is sterile-filtered and
introduced under aseptic conditions into a 2 ml ampoule, deep-frozen and Iyophilised.
Before use, the Iyophilised substance is dissolved in 1 ml of distilled water or 1 ml of
physiological saline solution. The solution is administered intramuscularly or
intravenously. This forrnulation may also be introduced into double-chamber syringe
ampoules.
Example 12: Nasal spraY
500 mg of finely ground (~5.0 ,um)
S,S-dioxothiomorpholinocarbonyl-Val-Cha -GVal-Val-Tyr-OMe are suspended in a
mixture of 3.5 ml of Myglyol 812(g) and 0.08 g of benzyl alcohol. The suspension is
introduced into a containeI having a metering valve. 5.0 g of Freon(~) 12 are introduced
into the container under pressure through the valve. By shaking, the Freon~) is dissolved
in the MyglyoVbenzyl alcohol mixture. The spray container contains approximately 100
single doses which may be administered singly.
Example 13: Film-coated tablets
The following constituents are processed for the preparation of 10,000 tablets each
comprising 100 mg of active ingredient:
S,S-dioxothiomorpholinocarbonyl-Yal-Cha -GVal-Val-Tyr-OMe 1000 g
corn starch 680 g
colloidal silica 200 g
magnesium stearate 20 g
stearic acid 50 g
sodium carboxymethyl starch 250 g
water q.s.
A mixture of the S,S-dioxothiomorpholinocarbonyl-Val-Cha -CVal-Val-Tyr-OMe , 50
.
.
` ' ` `' ' ', ' ` `
- 35 -
g of corn starch and the colloidal silica is processed with a starch paste consisting of 250 g
of corn starch and 2.2 kg of demineralised water to forrn a moist mass. This mass is
forced through a sieve of 3 mm mesh size and dried for 30 min. at 45 in a fluidised-bed
drier. The dry granulate is pressed through a sieve of 1 mm mesh size, mixed with a
previously sieved mixture (1 mm sieve) of 330 g of corn starch, the magnesium stearate,
the stearic acid and the sodium carboxymethyl starch and compressed to form slightly
biconvex tablets.
The compressed tablets are coated in a coating vessel of 45 cm diameter by uniform
spraying for 30 min. with a solution of 20 g of shellac and 40 g of hydroxypropyl-
methylcellulose llow viscosity) in 110 g of methanol and 1350 g of methylene chloride;
drying is effected by simultaneously blowing in air at 60.
Instead of the active ingredients mentioned in Examples S to 8, it is also possible to use in
these l~xamples the same amount of a different active ingredient from the preceding
Examples.
Example 14: Inhibition of isolated HIV- 1 ~a~ protease
10 ',ll of a solution of ~IIV-1 gag protease (acetone extract of a gag protease expressed in
E. coli according to J. Hansen et al., The EMBO Journal 7, 1785 (1988)) and 190 ,ul of
~-morpholinoethanesulfonic acid-buffer solution pH 6 containing 0.01 %
2-amino-4-nitrophenol as the internal standard are pre-incubated at 37. 10 ,ul of a 0.24
mM DMSO solution of the substrate H-Arg-Arg-Ser-Asn-Gln-Val-Ser-Gln-Asn-Tyr-
Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly-Arg-Arg-OH (icosapeptide according to J. Schneider et
aL, Cell 54, 363 (1988), prepared in an automated peptide synthesising apparatus with
Fmoc-protected amino acid units) and 10 111 of a DMSO solution of the compound ~o be
investigated for its inhibitory action, in a concentration of 22 x 104M or 22 x 10-6M, are
then added simultaneously. After one hour, 50 111 of reaction solution are taken, 5 ~11 of
0.3M perchloric acid are added thereto and the batch is centrifuged. The amount of
unconsumed substrate and the cleavage products in the supernatant solution are
determined by HPLC and the percentage inhibition at 10-4M and 10-6M is calculated
therefrom.
For the HPLC analysis there is used a 125 x 4.6 mm reversed phase Clg Nucleosil~) S ~,
column; gradient 10 % acetonitrile/0.1 % trifluoroacetic acid in water ~ 25 % aceto-
nitrile/0.08 % trifluoroacetic acid in water in 30 min., flow rate 1.5 ml/min.
~, :
,
. . . : .
. '
~3~
- 36 -
The compounds of the preceding Examples have, at concentrations of 104M, an inhibitory
action of more than 80 % and, at 10-6M, an inhibitory action of more than 50 ~o.
Example 15: Protection against ~IIV infection in a cell test
The cell line MT-2 which is used is a human T-cell leukaemia that has been transformed
with HTLV-l and continuously produces HTLV-1, which renders the cells extremely
sensitive to the cytopathogenic effect of HIV (Science 229, 563 (1985)). The MT-2 cells
are cultivated in RPMI 1640 medium comprising 12 % heat-deactivated foetal calf's
serum (Seromed Biochrom KG, Berlinj Federal Republic of Germany), glutamine and
standard antibiotics. The cells are kept at 37 in a humidified atmosphere of 5 % CO2 in
air and are used in the logarithmic growth phase for the cell test.
HIV LAV-03 (AIDS Research and Reference Reagent Program, NIH, Bethesda, MD,
USA) is cultured in A 3.01 cells. The titre is determined in a reverse transcriptase assay.
For the charge used it is 2 x 107 lU/ml.
40,000 exponentially growing MT-2 cells in 50 ~1 of culture medium are introduced into
each of the wells in a 96 round-base titre plate. The compounds to be investigated are
added in the given concentration in 50 ,ul of culture medium and, immediately thereafter,
the HIV in 100 111 of culture medium is added. lO0 ~1 of culture medium without HISI are
added to comparison samples. The titre plates are incubated for six days. The via~ility of
the HIV-infected cells and the comparison cells is then tested in an MTT assay: the MTT
assay relies on the reduction of yellow-coloured 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St.Louis, USA) by
mitochondrial dehydrogenases of metabolically active cells to a blue formazan which can
be measured spectrophotometrically at 540 nm (J. Virological Methods 20, 309 (1988)).
The viability of comparison cells and HIV-infected cells is also determined
microscopically in a haemocytometer according to the trypan blue exclusion method.
The investigated compounds oi the preceding Examples exhibit a protective action against
HIV infection at concentrations of 10-5 mol/litre.
-
.
