Note: Descriptions are shown in the official language in which they were submitted.
"'O 92/06g66 ~ PCT~GB91/0174g
N-(2-ALKYL-3-MERCAPTOGLUTARYL)-AMINO DIAZA CYCLOALKANQNE ~.
DERIVATIVES AND THEIR USE AS CQLLAGENASE INHIBITORS.
The present invention relates to novel thiol carboxylic
acid derivatives, processes for their preparation and
their use in medicine. In particular, the present
invention relates to their use as inhibitors of enzymes of
the collagenase family of neutral metalloproteases, ~or
treating arthritic and other diseases.
The manunalian collagenase family of enzymes comprises a
number of proteases, exempli~ied by intersti~ial (type I)
collagenase itself, the stromelysins ~also known as
proteoglycanases or transins), fibroblast and
polymorphonuclear leucocyte yelatinases ~also known as
coll~gen-IV-ases), and 'pump~ pu~ative metalloprotease
1, uterine metalloprotease). Membership of the ~ ~-
mammalian collagenase family of proteases is evident by
possession of a number of highly characteristic and
experimentally veri~iable properties. [Goldberg et al.,
J. Biol. Chem. 2610, 6600, 1986; Whltham et al., Biochem.
J. 240, 913, 1986; Breathnach et al., Nucleic Acids Res.,
15, }139, l9B7; Muller et al., Biochem. J., 253, 187,
1988; Collier et al., J. Biol. Chem., 263, 6579, 1988;
Murphy et al., Biochem. J., 258, 463, 19~9; Quantin et
a}., Biochem. (N.Y.), 28, 5327, 1989; Birkedal-Hansen, J.
Oral Pathol., 17, 445, 1988~.
The range of therapeutic applications of the invention
described hereinafter reflects the fundamental role of
collagen and other proteinaceous substrates of the
collagenase family of enz~rmes in the connective tissue
matrix throughout the body. Applications extend to
clinical interventions in many diseases and phenomena
involving the destruction of collagen and other connec~ive
35 tissue components, and also normal or disordered tissue
remodelllng.
W092/06966 ~ 2- PCTtGB91/
Inhibltors of the collagenase family of enzymes are
considered to provide useful treatments for:
arthritic diseases, such as rheumatoid and osteo-
arthritis, soft tissue rheumatism, polychondritis and
tendonitis; bone resorption diseases, such as
osteoporosis, Paget's disease, hyperparathyroidism and
cholesteatoma; the enhanced collagen destruction that
occurs in association with diabetes; the recessive classes
of dystrophic epidermolysis bullosa; periodontal disease
and related consequences of gingival production of
collagenase, or of PMNL collagenase release following
cellular infiltration to inflamed gingiva, including by
combating the greater susceptibility of diabetes patients
to periodontal disease, corneal ulceration, e.g. that
induced by alkali or other burns, by radiation, by vitamin
E or retinoid deficiency; ulceration of the skin and
gastro-intestinal tract, and abnormal wound healing;
post-operative conditions, including colonic anastomosls,
in which collagenase levels are raised; cancer, where
members of the collagenase family of enzymes have been
implicated in the neovascularization required to support
tumoUr growth and survival [P. Basset et_al., Nature, 348,
699, 1990] in the tissue remodelling required to
ac~ommodate the growing primary and secondary tumours, and
in the penetration of tumour cells through the basement
membrane of the vascular walls during metastasis; and
demyelinating diseases of ~he central and peripheral
nervous systems, including syndromes in which myelin loss
is the primary pathological event and those in which
demyelination ~ollows axonal atrophy. The degrada~ion of
myelin in these diseases, exemplified by multlple
sclerosis, is mediated by members of the collagenase
family of enzymes.
2 09~ 41 1
-vo 92/Oli9t~6 PC~tGB91/01749
-3-
As a particular example of the therapeutic value of
inhibitors of the collagenase family of enzymes such as
are disclosed ln the present invention, chronic arthri~ic
diseases leading to extensive loss of the collagen,
proteoglycan and elastin components of the cartilage, bone
and tendons wlthin the joints, should be amenable to
treatment with inhibitors of the collagenases,
proteoglycanases (stromelysins) and gelatinases currently
thought to be the major enzymes involved.
These enzymes have been detected in extracts of synovial
and cartilage tissue, and have also been extensively
studied in tissue cultures of a wide range of connective
tissues. Apart from control of the biosynthesis,
secretion and acti~ation of the enzymes, the most
important natural regulation of these enzymes in normal
and diseased states, is considered to be the endogenous
production of inhibitors such as the family of Tissue
Inhibitor of Metalloproteases ~TIMPS), and alpha-2
macroglobulin. An imbalance between the local levels of
the proteolytic enzymes and natural inhibitors will allow
destruction of connective tissue components to occur.
The compounds described in the present invention, being
synthetic and low molecular weight inhibitors of this
family of enzymes, offer a therapeutically useful way in
which a more normal or non-pathological balance between
inhibition and enzymic activity can be restored: they thus
act to complement and supplement the endogenous enzyme
inhibitors. Indeed, because these enzymes usually act
only within restricted pericellular environments, before
being inactivated by inhibitors circulating in the blood
and present in most inflammatory exudates, the low
molecular weight inhibitors disclosed here may be more
effective than endogenous proteinaceous inhibitors that
are excluded by their size from the localized regions of
connective tissue destruction.
.. . . ..
~ r~
WO92/Q69~ PCT/GB91/0174
_9_
European Patent Application 0273689 ~Beecham Group)
discloses a class of thiol-carboxylic acid derivatives
having activi~y as inhibitors of collagenase and u~eful ln
the treatment of rheumatoid arthritis and related diseases
in which collagenolytic activity is a contributing factor.
A novel class of thiol-carboxylic acid deriva~i~es has now
been discovered, which are collagenase inhibitors and thus
of potential utility in the treatment of diseases in which :
activity of members of the collagenase family of neutral
metalloproteases is implicated. .
According to the present invention there is pro~ided a
compound of general formula (I), or a salt, solvate or
hydrate thereof:
R3 H O
O R2 R4~
(Il
in which,
Rl is -OH; alkoxy; aryloxy or aralkyloxy in each o~ which
the aryl group is optionally substituted; -NR6R7, where
each of R6 and R7 is independently hydrogen or alkyl, or
R6 and R7 together with ~he nltrogen atom to which they
are bonded ~orm a 5-, 6- or 7-membered ring with an
optional oxygen or sulphur atom or an optionally
substituted second nitrogen atom in the ring; or a group
:.. . . ... . . .
:.: . . . . . . , ... : - .
--'O 92/06g66 _5_ 2 0 9 2 '11/~ PCI/GB91/01749
-NH-CH-C--Rg
R~
where R8 is hydrogen; alkyl optionally substituted by
-OH, alkoxy, -NR6R7 as defined for Rl, guanidine, -CO2H,
-CONH2, -SH, or -S-alkyl; or -CH2 Ar where Ar is
optionally substituted aryl; and Rg is alkoxy; O~; or
-NR6R7 as defined for Rl;
R2 is hydrogen; C2_8 alkanoyl; or optionally substituted
aroyl;
R3 is C3-6 alkyl; and
R4 is ~(CH2)p~X~~CH2)q~ where p is an integer from l to 9,
q is an integer from 2 to l0, and the molety -(CH2)p- is
adjacent to the carbon atom marked with an asterisk in
formula (I), and X is -NR5- where R5is selected from
hydrogen, Cl 6 alkyl, C2_6 alkanoyl, Cl_6 alkoxY Y
and aroyl, aralkyl or aralkyloxycarbonyl in each of whlch
the aryl moiety is optionally substituted.
Unless otherwise specified, each alkyl or alkoxy group is
a Cl_8 group, more preferably a Cl_6 group, and may be
straight chain or branched.
Values for aryl groups include naphthyl and phenyl,
preferably phenyl.
Optional substituents for aryl groups may be selected from
-OH, Cl_6 alkyl, Cl_6 alkoxy and halogen.
- . ~ .. .. .
~: ' ' . " - ' " ': .' : . .. ' '. ..... .: . ' . .. . . ..... .
W~9~ 9~ 9~ -6- pcl/GBsl/ol74^
Values for Rl include hydroxy; Cl_6 alkoxy, such as
methaxy, ethoxy, iso-propoxy or t-butyloxy; benzyloxy; and
-NR6R7 in which R6 is hydrogen, and R7 is hydrogen or Cl_B
alkyl such as methyl or ethyl, or -NR6R7 is
N'-methyl-N-piperazlnyl or N-morpholinyl~
Rl is preferably hydroxy, Cl_~ alkoxy or Cl_6 alkylamino.
Most preferably Rl is hydroxy, methoxy, iso-propoxy or
methylamino.
~0
O
R2 is preferably hydrogen, acetyl or Ph-C- in which Ph is
an optionally substituted phenyl group. Most preferably
R2 is hydrogen or acetyl.
R3 is preferably a C4 alkyl group, such as n-butyl,
iso-butyl or sec-butyl. Most preferably R3 is iso-butyl.
R4 is preferably ~(CH2)p~X~~CH2)q~ where p and q have
values such that R4 forms part of an ll- to l6-membered
lactam structure, and X is a group -NR5- where R5 is
hydrogen, methyl, benzyl, t-butoxycarbonyl or
benzyloxycarbonyl.
Most preferably R4 is ~(CH2)p~X~(CH2)q~ whera p ls 4 and q
is 5 or p is 4 and q is 6 and X is a group -N~5- where R5
is hydxogen.
The compounds of formula (I) may form salts with bases
e.g. sodium hydroxide. When a basic nitrogen atom is
present, the compounds of formula ~I) may form acid
addition salts e.g. with hydrochloric acid. Such
compounds form part of the present invention.
.... .. ?
v~g2/~966 ~ 09 2 'I 7 ~ PCT/GB91/01749
The compounds of formula (I~ have at least three
asymmetric centres and therefore exist in more than one
stereoisomerio form. The invention extends to all such
forms and to mixtures thereof, including racemates, and
dlastereoisomeric mixtures.
Where compounds of formula (I), or pharmaceutically
acceptable salts thereof, form solvates such as hydrates,
these also form an aspect of the invention.
: .
Preferred isomers are those having the ~S)-configuration
at the chiral centre marked with an asterisk in formula
(I).
The compounds of formula (I) and their pharmaceutically
acceptable salts are preferably in substantially pure
form.
A substantially pure form will generally contain at least
50% by weight, preferably 75%, more preferably 90% and
still more preferably 95% or 99% or more of the compound
of formula ~I) or its pharmaceutically acceptable salt.
The present invention provides the compounds of formula
~I) or pharmaceutically acceptable salts thereof for use
as active therapeutic agents, particularly as agents for
treatment of musculo-skeletal disorders resulting from
collagenolytic activity, particularly arthritic diseases,
and tissue remodelling.
Compounds of ~ormula II) also have potential utility in
the treatment of cancer; for preventing myelin degradation ~;
in the central and peripheral nervous system; and in other
conditions in which members of the collagenase family of
neutral metalloproteases have pathological or other roles.
,
.,.,'; : ~' ' . ~
W092/~6 ~ PCT/GB91/0174
q~ 8-
The present ~nvention also provides a process ~or th~
preparation of a compound of formula (I), which process
comprises ~he reaction of a compound o~ formula (II):
R3 H
~ ~ N
1 \ J '" R 3
~ 4
(II)
wherein Rl, R3 and R4 are as defined in formula ~I), with
a thiol of formula (III~:
L - SH (III)
wherein L ls a conventional sulphur protection group, to
give a compound of formula (IV):
:.
R3
l ~ ~ N
S-L
-R , .
4~_, (IV)
2i5
wherein Rl, R3 and R4 are as defined in formula (I) and L
is as defined in formula (III); and subsequently as
necessary
cleaving the group L and~or R5 to give a compound of
formula ~I) in which R2 and/or R5 is hydrogen;
,:
,
-V092/06g66 PCT/G891/01749
converting the group R2 in a compound of formula (I)
into another group R2;
where appropriate converting the group R5 in a
5compound of formula (I) into another group R5.
Typically a sulphur protection group L is a substituted
benzyl group, such as alkoxybenzyl, for example
4-methoxybenzyl, or an aliphatic or aryl acyl group such
as acetyl or benzoyl. When L is an acyl group which is
C2_8 alkanoyl or optionally substituted aroyl it is of
course identical to R2, so that compounds of formula (IV)
in which L=R2 are themselves compounds of the invention.
When L is a substituted benzyl sulphur protection group,
such as 4-methoxybenzyl, then L may be removed by
treatment with mercury acetate in trifluoroacetic acid
containing anisole, followed by reaction with hydrogen
sulphide in dimethylformamide, in a procedure analogous to
that described in Chem. Pharm. Bull 1576, 26, ~1978).
When L is an acyl group it may be removed by treatment
with a base, for example aqueous ammonia or dilute aqueous
sodium hydroxide, or by treatment with an acid, for
example methanolic hydrochloric acid.
:'~
Other conventional methods ~or removing sulphur protection
groups may also be used.
Compounds o~ the formula ~ n which R~ is hydrogen may
be converted to compounds of formula (I) in which R2 is
C2_8alkanoyl using standard acylation procedures.
, ... . ,, ;,.- . . .
. .. :. . . . : . . . .: ~ . . .
:~ .;, . - . ., , ~ . .. . :
W092/06966 ~ 10- PCT/GB91tOl7
Compounds of formula ~IV) can be converted to further
compounds of formula tIV) while retaining the same group
L, which ~roup can in turn be cleaved to form compounds of
the inven~ion in which R2 is hydrogen.
For example, those compounds of formula ~IV) in which R1
is -OH may be prepared under acid conditions by hydrolysis
of compounds in which R1 is alkoxy, aryloxy or aralkyloxy
or by hydrogenolysis of compounds in which R1 is benzyloxy
or substituted benzyloxy in the presence of a catalyst
such as palladium black.
Those compounds of formula (IV) in which R1 is -NR6R7 may
be prepared from compounds in which R1 is -OH by treating
the latter compounds with an amine of formula NHR6R7 in
the presence of a coupling agent such as
N,N-dicyclohexylcarbodiimide or
N-ethyl-N'-dimethylaminopropylcarbodiimide.
Compounds of formula ~IV) in which Rl is -NH-CH~R8)-CORg
may be similarly prepared from compounds in which R1 is OH
by treatment with amine derivatives of formula
NH2CHtR8)CORg where Rg is an alkoxy or amine group,
followed by hydrolysis to give an Rg hydroxy group, if
desired.
Alternatively, compounds of formula (IV) in which Rl is
-NR6R7 may be prepared ~rom compounds of formula ~IV) in
which R1 is alkoxy by aminolysis of the latter compound
with an amine of formula NHR6R7 in the presence of a
cataly~ic amount of cyanide. Aminolysis procedures are
described by Hogber, T. et al., J. Org. Chem. 19~7, 52,
2033-2036; De Ferand, R.J. et al., J. Org. Chem. 1963, 28,
2915-2917.
.. . . .. .
~'092/~966 2 0 9 ~ PCT/GB91/0l74g ~.
In addition, compounds of formula ~IV) in which L is an
acyl group can be converted to compounds of ~he invention
with interconversion of Rl and concomitan~ cleavage of the
acyl group to give compounds of formula (I) in which R2 is
hydrogen.
It will be appreciated ~hat the above transformations for
compounds of formula (IV) will also be applicable for
compounds of formula (IV) in which L=R2, i.e. compounds of
formula (I).
For example, those compounds of formula (I) in which Rl is
-OH and R2 is hydrogen may be prepared by hydrolysis of -
compounds of formula (IV) in which Rl is alkoxy, aryloxy
or aralkyloxy and L is acyl, under basic conditions such
as treatment with dilute sodium hydroxide.
The intexmediate compounds of formula (II) may be prepared
by treating a compound of formula (V):
R3
I OH
R~
O ~V) ,'`
in which Rl and R3 are as defined in formula ~I), with a
compound of formula (VI):
O
R
R4
(VI)
wherein R4 is as defined in formula (I).
. . : . . --: . - . : :: -. . : : - . . . : . . : : : : : . . : . . .: . :
:; ':" :' .~' :.:,:: '~ .: ,.. -:::,. : . ::: . , ,' ,: :' :' :' ' :, : " . " ' . ":
W0~2/~ ~ PCT/GB91/017
~12-
The reaction is suitably carried out in the presence of a
coupling agent, such as 1,1'-carbonyldiimldazale.
Where R5 in R4 is hydrogen, the secondary amine is
suitably in protected form, for example as a
benzyloxycarbonyl derivative.
Compounds of formula (VI) may be prepared by oxidising the
primary alcohol function in a compound of formula (VII):
Y
H NH H
Z-N-(CH2)P -- N-~cH2)q-oH
(~II)
wherein p and q are as defined for R4 in formula (I), Y is
a nitrogen protection group, and Z is R5, to give the
corresponding aldehyde, fQllowed by removal of Z when R5
is an acyl group; cyclisation and reduction; and
thereafter, as necessary, removing the nitrogen protection
group Y and interconverting R5.
Suitable values for Y inc~ude t-butoxycarbonyl (BOC) and
benzyloxycarbonyl groups, preferably t-butoxycarbonyl.
The oxidation may be carried out uslng pyridinium
chlorochromate or under Swern oxidlsing conditions, for
example by treatment with dimethylsulphoxide and an acyl
halide ~ollowed by triethylamine, as described by D. Swern
et al., J. Org. Chem., 43, 2480 (1978). The cyclisation
and reductive amination step may be effected by catalytic
hydrogenation over a su$table noble metal catalyst, for
example palladium on carbon, or by reaction with sodium
cyanoborohydride or sodium borohydride.
`/092/06966 -13 2 ~ 9 ~ PCT/GB9l/0~749
Nitxogen protection groups may be removed by standard
methods. When Y or Z are t-butoxycarbonyl groups these
may be removed by treatment with trifluoroacetic acid at
reduced temperature. When Y or Z are benzyloxycarbonyl
S groups these may be removed by conventional hydrogenation
over palladium on carbon, or by treatment with either
formic acid-methanol and palladium black, or with HBr and
glacial acetic acid. The group Z may be selected to
undergo concomitant cleavage during the cyclisation
reaction to give a compound in which R5 is hydrogen. For
example, when Z is a benzyloxycarbonyl group, it will be
readily removed by catalytic hydrogenation.
An R5 hydrogen in compounds of formulae (I), (II), (IV),
1~ (VI) and ~VII) may be interconver~ed to an R5 Cl_6 alkyl,
aralkyl or acyl group. The nitrogen atom in R4 may be
alkylated, for example methylated to form an R5 methyl
group, or acylated to form an R5 C1_6 alkoxycarbonyl or
aralkyoxycarbonyl group.
Methylation procedures are described by E. Askitoglu et
al.,,Helv. Chim. Acta., 68, 750, (1985); E. Engler et al.,
Helv. Chim. Acta., 68, 789, (1985); and M. Lennon et al.,
J. Chem. Soc. ~Perkin I), 622, (1975).
Compounds of formula (VII) may be prepared by reacting a
compound of formula (VIII):
H NH
Z-N-(CH2)p--~ ~ OH
(VIII)
. ... . . . . . :, .. ;, ,. . . .. -. .. . . . .
wO 92/06966 ~9 r PCT/GB91/0174~
wherein p, Y and Z are as defined for formula ~VII), with
a compound of formula (IX):
H2N~(CH2)q~OH tIX)
wherein q is as defined for formuLa (VII).
The reaction may be carried out using standard procedures
for forming an amide from a carboxylic acid and an amine,
for example using a coupling agent such as
l,l'-carbonyldiimidazole, l,3-dicyclohexylcarbodiimide or
N-ethyl-N'-dimethylaminopropylcarbodiimide.
Compounds of formula (VIII) are di-aminoalkanoic acid
derivatives. These are known compounds or may be prepared
from known starting materials by standard methods.
For example the compound of formula ~VI) in which R4 is
- ~(CH2)p~X~(CH2)q~ where p is 3, q is 6 and X is -NH- is
prepared from a compound of formula (VIII) derived from
ornithine which is commercially available.
The compound of formula (VI) in which R4 is
~(CH2)p~X~(CH2)q~ where p is 4, q is 5 and X is -NH- is
prepared from a compound of formula (VIII) derived from
the amino acid lysine. The compound of formula (VIII),
derived from (S)-lysine, in which Y is t-butoxycarbonyl
and Z is benzyloxycarbonyl, is commercially available. ~``
Similarly, the compound of ~ormula ~VI) in which R4 is
-~CH2)p-X-~CH2)q- where p is l, q is 8 and X is -NH may
be prepared from 2,3-diaminopropionic acid.
0~2/~966 -l5- 2 0 9 2 ~ CT/GB91/01749
The thiols of formula ~III) and the amino alcohols of
formula (IX) are known compounds or may be prepared from
known compounds by known methods.
Intermediate compounds of formulae ~II) and ~IV) disclosed
herein are novel compounds and form an aspec~ of the
present invention.
The preparation of certain compounds of formula ~V) is
described in EP-A-0273689.
Where obtainable, pharmaceutically acceptable salts of the
compounds of formula (I) may be formed conventionally ~y
reaction with the appropriate acid or base. Solvates may
be formed by crystallization from the appropriate solvent.
As mentioned previously, the compounds of formula (I)
exist in more than one diastereoisomeric form. Where the
processes of the invention produce mixtures thereof, the
individual isomers may be separated one from another by
chromatography, e.g. column chromatography or HPLC.
Alternati~ely, separate diastereoisomeric compounds of
formula ~I) can be obtained by using stereoisomerically
2~ pure starting materials or by separating desired isomers
of intermediates at any stage in the overall synthetic
process, and converting these intermediates to compounds
of formula (I).
It wiLl be appreciated that although the absolute
configuration at a particular chiral centre may not be
known, it is possible to characterise a given
diastereoisomer relative to its epimer or to another
diastereoisomer using NMR spectroscopy or optical
rotation.
::~ .. - , . :
WOg2/06966 ~ ~ l6- PCT/~B
The present invention further provides a phar~aceutical
composition, which comprises a compound of formula (I), or
a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier.
A composition of this invention is useful i~ the treatment
of musculo-skeletal disorders, particularly arthritic
diseases and for modulation of tissue remodelling.
A composition of the invention, which may be prepared by
admixture, may contain a diluent, binder, filler,
disintegrant, flavouring agent, colouring agent, lubricant ~`
or preser~ative in conventional manner. These
conventional excipients may be employed in conventional
manner, for example as in the preparation of compositions
of related peptide enzyme inhibitors, such as the ACE ?`
inhibitor captopril.
A composition of the invention may be adapted for oral,
topical, rectal or parenteral administration but oral
administration is preferred. Parenteral compositions may
be administered e.g. intravenously, intramuscularly or ~
intra-articularly. Preferably compositions are in unit ~ ;
dosage form or in a form that a patient can administer to
himself in a single dose.
Pre~erably, a pharmaceutical composition of the inventlon
is in unit dosage form and in a form adapted for use in
the medical or veterinarial fields. For example, such
preparations may be in a pack ~orm accompanied by written
or printed instructions for use as an agent in the
treatment or prophylaxis of any of the disorders mentioned
above.
' :''
'0~2/06966 PCT/GB91/01749
2 0 9 ~
Compositions may, for example, be in the form of tablets,
capsules, sachets, vials, powders, granules, lozenges,
reconstitutable powders~ or liquid preparations, for
example solutions or suspensions, or suppositories.
The compositions, for example those suitable for oral
administration, may contain conventional excipients such
as binding agents, for example syrup, acacia, gelatin,
sorbitol, tragacanth, or polyvinylpyrrolidone; fillers,
for example lactose, sugar, maize-starch, calcium
phosphate, sorbitol or glycine; ~abletting lubricants, for
example magnesium stearate; disintegrants, for example
starch, polyvinylpyrrolidone, sodium starch glycollate or
microcrys~alline cellulose; or pharmaceu~ically acceptable
wetting agents such as sodium lauryl sulphate.
Solid compositions may be obtained by conventional methods
of blending, filling, tabletting or the like. Repeated
blending operations may be used to distrlbute the active
agent throughout those compositions employing large
quantities of fillers. When the composi~ion is in the
form of a tablet, powder, or lozenge, any carrier suitable
for formulating solid pharmaceutical compositions may be
used, examples being magnesium stearate, starch, glucose,
lactose, sucrose, rice flour and chalk. Tablets may be
coated according to methods well known in normal
pharmaceutical practice, in particular with an enteric
coating. The composition may also be in the form of an
ingestible capsule, for example o~ gelatin containing the
compound, if desired with a carrier or other excipients.
For example, a hard gelatin capsule containing the
required amount of a compound of the invention in the form
of a powder or granula~e in intimate mixture with a
lubricant, such as magnesium stearate, a filler, such as
microcrystalline cellulose, and a disintegrant, such as
sodium starch glycollate.
:.~ . , .
WO 92J06g66 c~ PCI`/GB91/0174r
~ -18-
Compos1tions for o~al administration as liquids may be in
the form of, for example, emulsions, syrups, or elixirs,
or may be presented as a dry product for reconstitution
with water or other suitable vehicle before use. Such
liquid compositions may contain conventional additives
such as suspending agents, for example sorbitol, syrup,
methyl cellulose, gelatin, hydroxyethylcellulose,
carboxymethylcellulose, aluminium stearate gel,
hydrogenated edible fats; emulsifying agents, for example
lecithin, sorbitan monooleate, or acacia; aqueous or
non-aqueous vehicles, which include edible oils, such as
almond oil and fractionated coconut oil, oily esters, for
example esters of glycerine, propylene glycol, ethyl
alcohol, glycerine, water or normal saline; preservatives,
for example methyl or propyl p-hydroxybenzoate or sorbic
acid; and if desired conventional flavouring or colouring
agents.
The compounds of this invention may also be administered
by a non-oral route. In accordance with routine
pharmaceutical procedure, the compositions may be
formulated, for example for rectal administration as a
suppository or for parenteral administration in an
injectable form. Fox injection, for example by
intra-articular injection or by injection into the
cerebro-spinal fluid or via other routes which will gain
access to sites of demyelination, as Preely soluble
solutions or as poorly dispersed depot stores, the
compounds of the invention may be presented in an aqueous
or non-aqueous solution, suspension or emulsion in a
pharmaceutically acceptable liquid, e.g. sterile `
pyrogen-free water or a parenterally acceptable oil or a
mixture of liquids, which may contain bacteriostatic `
agents, anti-oxidants or other preservatives, buffers or
solutes to render the solution isotonic with the blood,
-~092~966 -l9- 2 ~ 9 2 ~ 1 ;7
thickening agents, suspending agents or other
pharmaceutically acceptable additives. Such forms will be
presented in sterile unit dose form such as ampoules or
disposable injection devices or in multi-dose forms such
as a bottle from which the appropriate dose may be
withdrawn or a solid form or concentrate which can be used
to prepare an injectable formulation.
For topical and percutaneous administration, the
preparations may also be presented as an ointment, cream,
lotion, gel, spray, aerosol, wash, skin paint or patch.
The suitable dosage range for the compounds of the
invention may vary from compound to compound and may
depend on the condition to be treated. It will also
depend, inter alia, upon the relation of potency to
absorbability and the mode of administration chosen.
A unit dose for treating diseases and physiological
phenomena in which enzymes from the collagenase family are
involved will generally contain from lO to lO00 mg and
preferably will contain from lO to 500 mg, in particular
lO, 50, lO0, 150, 200, 250, 300, 350, 400, 450 or 500 mg.
The composition may be administered once or more times a
day, for example 2, 3 or 4 times daily, so that the total
daily dose for a 70 kg adult will normally be in the range
lO to 3000 mg. Such a dose corresponds to approximately
0.15 to 50 mg/kg per day. Alternatively, in particular
for injection, the unit dose will suitably contain from
to 20 mg of a compound of the invention and be
administered in multiples, if desired, to give the desired
daily dose.
The present invention additionally provides a method of
treating conditions in which degradation of connective
tissue and other proteinaceous components of the body
. - . . ~ : .
. . . .. , . , . ~ , ~ . .
WO92/Ofi966 ~ 20-- PCT/GB91/017~-
occurs, such as rheumatism and~or arthri~ic conditions in
mammals, such as humans, which comprises administering to
the mammal in need of such treatment an effective amount
of a compound of formula (I) or a pharmaceutically
acceptable salt thereof.
The present invention also provides the use of a compound
of formula (I) or a pharmaceutically acceptable salt
thereof, for the manufacture of a medicament fox use in
the treatment of conditions in which degradation of
connective tissue and other proteinaceous components of
the body occurs sueh as rheumatism and/or arthritic
conditions.
The following Description and Examples illustrate the
preparation of compounds of the invention.
. .. - ., .: . . .
. " : ~, -
: . . .: . :. ~ . ,
~9~/06966 -21- 2 ~ 1 PCT/GB91/0l749
DescriPtion 1
4-Isopro~oxycarbonyl-2-t2-methvlpropvl)bu~-2-enoic acld
~L
~ / ~ OH
O O
A solution of 2-(2-methylpropyl)pent-2-enedicarboxylic
anhydride (prepared as in EP-A-273689) in 2-propanol
- 15 ~100 ml) was heated under reflux for 8h. The solvent was
evaporated in vacuo to give the title compound as a brown
oil (31.7g, 98%). ~CDCl3):
0.88 (6H,d, J=7Hz), 1.25 (6H,d,J=7Hz), 1.80 ~lH,
septuplet, J=7Hz), 2.18 (2H,d,J=7Hz), 3.60 ~2H, d,
J=7Hz), 5.05 ~lH, septuplet, J-7Hz) and 6.30
(lH,t,Ja7Hz).
DescriPtion 2
26
N-BenzvloxvcarbonYl-Na-tert-butoxvCarbOnYl-(S)-lYsine-15-
hvdroxy)pen~ylamide ~D2)
O
H3C CEI3O ~ OH
NH~,~ OCH2Ph
O
- ~ -. .... ,: ,:., . ;. .
WOg2~966 ~ ~ J~ 22- PCT~GB91/0l7
To a solution of N~-benzyloxycarbonyl-Na-tert-
butoxycarbonyl-~S)-lysine t7.8g, 21 mmol) in anhydrous
dichloromethane tl5oml) maintained at 0C was added
1-t3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (4.3g, 22.5 mmol) and l-hydroxybenzotriazole
t3.6g, 26.5 mmol). The mixture was stirred for 0.5h at ~
0C, 5-aminopentan-1-ol t2.3g, 22.5 mmol) added and -
stirring continued at room temperature. After 3h the
mixture was washed with saturated aqueous NaHC03 ~60 ml),
dried over anhydrous magnesium sulphate and evaporated in
vacuo to afford a viscous oil. Purification by flash
chromatography [(CHC13:MeOH) (20:1) v/v] gave the title
compound (D2) as a clear oil (8.01g).
(Found C, 61.54; H, 8.52; N, 9-18- C24H396N3 re~uires C~
61.91; H, 8.44; N, 9.02%). Observed (M+H)+466. C24H3906N3
requires M 465.
DescriPtion 3
N-BenzYloxYcarbonvl-N~-tert-butoxvcarbonyl- ~-lysine-(4-
formyl)butylamide ~D3~
: :
H
H3C X O ~ N ~ ,--~_,'~_~
N~OCH2Ph ~ ~
O ' '.
To a stirred solution of oxalyl chloride ~1.47g, 12 mmol)
in anhydrous dichloromethane (40 ml) maintained under an
atmosphere of nitrogen at -60C was added
) 92/06g6~i 9 ~ PCr/GB91/01749
dimethylsulphoxide (1.21q, 15 mmol) dropwise, such that
the temperature remained below -50C. The mix~ure was
left stirrin~ at -60C for 15 minutes, alcohol ~D2) (3.6g,
7.7 mmol) diluted in anhydrous dichloromethane ~10 ml) was
added, and allowed to warm up to -25C over lh. The
mixture was then cooled down to -60C, triethylamine
(4.7g, 46 mmol) added slowly such that the internal
temperature remained below -50C. On completion of
addition, the mixture was gradually warmed up to room
10 temperature, washed with water (30 ml) and sat. aq. NaCl ~`
(30 ml). The aqueous washes were back extracted with
dichloromethane (2x30 ml) and the combined organic
fractions were dried over anhydrous magnesium sulphate and
evaporated ln vacuo to yield a viscous clear oil.
Purification by flash chromatography
[~EtOAc:MeOH)(20:1)v/v] afforded the title compound (D3)
as an oil (2.8g).Observed ~M+H) 464. C24H3706N3 re~uires
M 463.
DescriPtion 4
(S)-~-(N-tert-ButoxYcarbonyl)amino-8-(N-
ben~yloxycarbonYl)-1,8-diazacyclotridecan-2-one (D4~ -
H
H3C CH30 ~
OCH~Ph
::: . . . .. ... . . ... . .
W09V06gS6 ~ PCr/G~9ltO17
Method A
The aldehyde (D3) ~l.Bg, 3.88 mmol) was dlssolved ln
ethanol (180 ml) and hydrogenated over 10% palladium on
charcoal (200 mg) at atmospherlc pressure and 35C for
72h. The su~pension was flltered through Keiselguhr and
evaporated in vacuo to ~ive crude ts)-3-~N-tert-butoxy-
carbonyl)amino-1,8-diazacyclotridecan-2-one. The crude
amine was dissolved in a mlxed solvent system of -~
tetrahydrofuran/water, (6:20 ml) v/v,coo~ed to 0C and
treated with benzyl chloroformate tO.66g, 3.88 mmol) and
excess sodium carbonate to maintain a pH between 10 and
11. The mixture was left stirring at room temperature
overnight, washed with ethyl acetate ~3x25 ml), and the
combined organic fractions dried over anhydrous magnesium
sulphate and evaporated in vacuo to afford a clear oil.
Purification by flash chromatography~EtOAc:MeOH)
~20:1)v/v] yielded the title compound (D4) as a white `~
solid (0.2g).0bserved M+ 447. C24H370sN3 requires M 447-
"`'`
Method B
The aldehyde (D3) was hydrogenated at about lOOpsi of
pressure over 10% palladium on charcoal in methanol,and
then in acidic methanol to afford crude tS)-3-
tN-tert-butoxycarbonyl)amino~ diazacyclotridecan-2-one.
The amine was treated with benzyl chloroformate and
purified as described in Method A, to yield the identlcal
title compound.
; :, .. .. : ~ .
~92/~966 PCT/GB91/Ot749
~5- 20~
Descri~tion 5
ts)-3-Amiino-B~ lN-benzyloxycarbQnyl~
diazacvclotridecan-2-one, trifluoroacetate ~alt_(D5)
O
T F ~ . H2N ~
O OCH2Ph -
A cooled (0C) solution of the lactam ~D4) (0.5gg, 1.28
mmol) in dichloromethane ~10 ml) was treated with
trifluoroacetic acid ~Sml). The mixture was stirred for
lh at 0C, warmed up to room temperature and left stirring
overnight. The solvent was evaporated under reduced
pressure, to afford crude title compound (D5) as the
trifluoroacetate salt. This was used as such without
~urther purification.
pescriPtion 6
6-Methvl-4-E[8-~N-benzyloxvcarbonvl)-l~8-
diazacvclotridecan-2-one-3-Yl1aminocarbonYllhePt-2tand 3)-
enoic acids. isoPropvl esters (D6)
. .
L
~ o~,H
O O N
O
Ph `
WO 92~966 q~ 26- PCT/GBgl/017
A solution of 4-isopropoxycarbonyl-2-(2-methylpropyl)
but-2-enoic acid (Dl) ~3.7g, 16.1 mmoll in anhydrous
dichloromethane (10 ml) under nitrogen was caoled to 0C
in an ice bath and then treated wlth l,l,-carbonyldl-
imidazole (3.03g, 18.7mmol) in one portion. A~ter lh at
0C the mixture was sequentially treated with ~.
N,N-diisopropylethylamine (2.59g, 20.1 mmol) and the crude
diazalactam (D5). The solution was stirred at O~C for lh,
warmed up to room temperature, and stirring continued
overnight. The solution was washed successi~ely with
water, 10% citric acid, 10% NaHC03, brine, dried o~er
anhydrous magnesium sulphate and finally evaporated ln
vacuo to remove the solvent. The product was subjected to
flash-column chroma~ography on silica gel, eluting with
15 ethyl acetate-pentane tl:l) v/v to afford the title ~:-
compound ~D6) as a white solid (3.04g) m.p. 112-117C.
(Found: C, 66.56; H, 8.32; N, 7.52. C31H47O6N3 requires
66.76; H, ~.49; N, 7.53%~.
: 20 DescriPtion 7
Na-tert-ButoxYcarbonyl-NE-benzvloxYcarbonYl-tS)-lYsine-(6- -
hvdroxY)hexYlamide tD7)
H C
3 ~ O~N ~ N ~ OH
CH3 ~ N ~ OCH2Ph
O ,
A solution of Na-tert-butoxycarbonyl-N-benzyloxycarbonyl-
tS)-lysine tl5.Bg, 0.042 mol) in anhydrous dichloromethane
(200 ml) maintained at 0C, was treated sequentially with
, ~, ~ ,,:, .. . . ................................ .
~92/U6966 PCT/GB~1/01749
2 ~ 9 ~ ~ I L~
1-(3-dimethylaminopropyl)-3-ethylcarbQdiimide
hydrochloride ~9.96g, 0.051 mol) and 1-hydroxy-
benzotriazole ~7.0g, 0.051 mol). The solution was stirred
at 0C for lh, treated with 6-aminohexan-1-ol ~4.7g, 0.046
mol~, and left stirring overnight at room temperature.
The mixture was then washed with saturated aqueous NaHC03, -
dried over anhydrous magnesium sulphate, and evaporated in
acuo to afford a viscous oil. Puri~ication by flash
chromatography [~CHCl3:MeOH) ~20:1) v/vJ gave the title
compound ~D7) as a clear oil ~16g), which on standing
solidified to a white solid.
Obse~ved (M+H)+ 480- C25H41N36 requires M 479.
DescriPtion 8
Na-tert-ButoxYcarbonYl-N-benzvloxYcarbonyl-~S)-lvsine-~5-
formy-l)~entvlamide-(D8L
H3C o N
2~
A stirred solution of dimethyl sulphoxide ~3.63g, 0.046
mol) in anhydrous dich~oromethane (100 ml) maintalned at
-60C, was treated with oxalyl chloride (2.58g, 0.0198
mol) diluted in dichloromethane (10 ml) at such a rate, so
as to ensure temperature remalned below -50C. After
stirring for 20 mlnutes, the alcohol (D7) (6.35g, 0.013
mol) dissolved in dichloromethane (50 ml) was added
: .: , , .. ,, . . , .. , : ,
w092/~ ~ 28- PC~/GB91/
dropwise over S mi~s. The mlxture was stirred at -60C
for 15 mins) warmed up to -35C, stirred for a further 10
mins then cooled down to -60C. The solu~ion was treated
with trlethylamine (8g, 0.08 mol), warmed up to room ~ .
5 temperature, washed with water ~2x100 ml), dried over ~;
anhydrous magnesium sulphate and sol~ent evaporated under ~
reduced pressure to afford a viscous oil. Purifica~ion by ~ :
flash chromatography [~EtOAc:MeOH) (30:1) v/v] gave the
title compound (D8) as an oil (5g), which on standing ~:
solidified to a white solld.
Observed (M+H) 478- C25H39N3O6 requires M 477-
Description 9
(S)-3-(N-tert-ButoxvcarbonYl)amino-8-~N-
benzvloxvcarbonYl?-1,8-diazacyclotetradecan-2-one (D9)
H O
H~C X o~N ~ N
C CH O H ~
OCEI2Ph
Method A
The aldehyde ~D8) (5.0g) in methanol ~450 ml) was treated
with 5% palladium on charcoal ~S.5g). The suspension was
hydrogenated at 190 psi and ambient temperature for 48h,
3Q treated with 2.SM aqueous hydrochloric acid ~3 ml1 and
hydrogenation continued at the said pressure for a further
~92JO69~ PCT/GB91/01749
_zg_
24h. The suspension was filtered throu ~ ~Kiesel~uhr and
evaporated ~ 35~ to give crude (S)-3-(N-tert-butoxy-
carbo~yl)amino-l,a-diazacyclotetradecan-2-one. The crude
amine was dissolved ln a mixed solvént system of
tetrahydrofuran/water, ~10:90 ml) v/v cooled to 0C and
treated with benzyl chloroformate (2.85g) and excess
sodium carbonate to maintain a pH between 10 and 11. The
mixture was left stirring at room temperature for 4h,
solvent partially evaporated in ~acuo and the residue
extracted with dichloromethane (3x100 ml). The organic
fraction was dried over anhydrous magnesium sulphate and
evaporated in vacuo to afford a clear oil. Purification
by flash chromatography [(EtOAc:MeOH) (50:1) v/v] yielded
the title compound (D9) as a white solid (2.0g) m.p.
131.5-134.0C.
Observed M~ 461. C2sH3gN3Os requires ~; 461-
Method B
The aldehyde ~D8) (5.8~) in methanol ~600 ml) was treated
with 5% palladium on charcoal (6g)and hydrogenated at 50
psi for 18h. The suspension was filtered through
Kieselguhr and the solvent partly evaporated in vacuo.
The concentrated solution (- 200 ml) was treated with 4A
molecular sieves and NaBH3CN (1.5g). The pH was ad~usted
to 6 by the addition of concentrated HCl then left
stirring overnight. The suspension was filtered through
Kieselguhr, and acidified to pH3. The solvent was
evaporated in vacuo, and the residue taken up in
dichloromethane (100 ml) and washed with a~. lM NaOH (2 x
60 ml). The organic fraction was dried and th,e solvent
removed under reduced pressure to afford a white solid.
The solid was disisiolved in THF/H20 ~100 : 30 ml) mixture,
treated with benzyl chloro~ormate (1.37g) and excess
W092/06966 - ~ PCT/GB91/017
-30-
Na2C03. The mixture was stirred for 4h, then the solvent .
partially evaporated in vacuo. The residue was extracted
with dichloromethane (3 x 80 ml), dried and evaporated
under reduced pressure to give a white solid. Trituration
S with diethyl ether and pentane gave the title compound
(D9) as a white solld.
DescriPtlon 10
(S?-3-Amino-8-(N-benzvloxYcarbonv~
diazacyc otetradecan-2-one, trifluoroacetate salt ~D10)
H O
H ~
OC~2Ph
A cooled (0C) solution of the lactam ~D9'~ ~9g~ in
dichloromethane llSOml) was treated with trifluoroacetic
acid ~50 ml). After lh the solvent was evaporated under
reduGed pressure, to afford crude title compound (D10) as
an oil. This was used as such without further
purification.
' :: ~, :. . ., - . , : . . : : :
)92/06966 PCT/GB91/01749
2 0 9 ~
Des criDt i on
6-Methvl-4-~L8-(N-benzyloxycarbony~ 8-
diazac~clotetradecan-2-on~ C{ "~L~11heDt-2(and
5 3)-enoic aclds, meth~l este3~ a~
:,
~ H O
CH3-O ~ N ~ N
V ~ N ~
OCH2Ph
A solution of 4-methoxycarbonyl-2-(2-methylpropyl)but-2-
enoic acid tprepared as in EP-A-273689) (5.08g) in
anhydrous dichloromethane (200 ml) maintained at 0C was
treated with 1,1,-carbonyldiimidazole (4.12g) in one
portion. After lh at 0C the mixture was sequentially
treated with N,N-diisopropylethylamine (5g) and the crude
diazalactam (D10) (0.019 mol) dissolved in dlchloromethane
(20 ml). The solution was stirred at 0C for lh, warmed ;
up to room temperature, and stirring continued overnight.
The solution was washed successively with water, 10%
citric acid, 10% NaHC03 and brine, dried over anhydrous
magnesium sulphate and evaporated in vacuo. The residue
was purified by ~lash-column chromatography on silica gel,
- eluting with ethyl acetate-pentane ~1:1 v/v) to a~ord the
title compound ~D11) as a white solid ~5.lg).
Observed M~ 543.3307. C30H45O6N3 requires M 543.3~05. ~;
~`~
,~'-,
- . . .:: . . , . ;,: .: , , , ., . . : , i . . . .
, .-; . .. ,.: : , ... . . ~ .. ".: ~.. . ., ,,:- :
wV92/06s66 ~ -32- PCT/GB9~tO
Descr1Dtion 12
3-Acetylmercapto-6-methyl-4- L~ N-benzyloxvcarbonyl)-1,8-
diazacycIotetr~decan-2-one-3-yllaminocarbonyl]h E~tanoic ~
acid, methYl ester !D12) L ~:
~ H O
CH30 ~ N ~
OCH2Ph
A solution of the esters ~Dll) t5g) in thiolacetic acid
~35 ml) was set aside at room temperature for 18 days and
then evaporated to dryness in vacuoO The product was
subjected to flash-column chromatography on silica gel
using diethyl ether, followed by diethyl e~her - ethyl
acetate ~4:1 v/v) as the eluent. The first fractions to
; contain solids on evaporation of solvent were combined and
triturated with diethyl ether-pentane to afford the tltle
compound (D12) as a single diastereoisomer (Isomer A~.
Later fractions contained mixtures of diastereoisomers.
Observed M+ 619. C32H49N307S requires M 619
)92/06966 _3~g 9 2 ~1 ~ PCTt~B91/01749
Description 13
3-MercaPto-6-methyl-4-~8-(N~benzvloxycarbonv~ 8- :
diazacYclotetradecan-2-one-3-yl1aminocarbonYlL~evtanoic
acid, methYl ester LD13)
CH3O ~ N ~
OCH2Ph
An ice-cooled solution of the diastereoisomeric mixture of
esters (D12) (0.215g) in ni~rogen-purged methanol (20 ml)
was treated with 35% aqueous ammonia (5 ml), and the
reaction mixture was stirred under nitrogen for 2h and
then evaporated to dryness in vacuo to afford a white
solid. The crude product was used without ~urther
purification in the preparation of examples 5 and 6.
Observed M+ 577. C30Hq7O6N3S requires M 577
The above procedure may be repeated with Isomer A ~D12) to
afford single diastereoisomer (D13). This may then be
converted to single diastereoisomers (E5) and (E6).
'':
::,, - . . .: : . : :. . . . . . . .
.. .. . - . : . ., , : .:. .- .. .
WO 92/06~6 ~ ~9 ~ 34 PCT/GB9l/0l7~
Descrlp~ion 14
3-Mercapto-6-meth~1-4-~8-(N-benzvloxycarbonvl)-1,8-
diazacvclotetradecan-2-one-3-yl1Aml.nooarhonyllhe~tanoic
S acid, methyl amide ~D14)
H
~o ~
OCH2Ph ~::
A solution of the methyl ester ~D13) (0.04g) was heated in
. nitrogen-purged methanol (3 ml) containing excess N-
;: methylamine and sodium cyanide (0.013g) at 65C for 42h in
1~ a sealed vessel. The reaction mixture was evaporated to
- dryness in vacuo and chromatographed on silica gel.
Elution with 5% me~hanol in CHC13 gave the title compound
(0.025g) as a white solid.
Observed (M+H)+ 577. C30H4805N4S requires M 576.
:
, .
,;.- . . . .
~92/06966 9~ PCT/GB9l/01749
Example 1
3-Acetylmercapto-5~met~y~ 8-(N-benzvloxycarbonyl)-1,8-
diazacyclotridecan-2-one-3-vllaminocarbonvllheptanolc
acld, isoproPYl ester (El~
~o~H_~,
Q ~ O :~
~Ph
A solution of the esters (D6) (3g, 5.38 mmol) in
thiolacetic acid (35 ml) was set aside at room temperature
for 18 days and then evaporated to dryness in vacuo. The
product was subjected to flash-column chromatography on
silica gel using diethyl ether, followed by diethyl
ether-ethyl acetate (4:1) v/v as the eluent~ The first
fractions to contain solids on evaporation of solvent were
combined and recrystallised from diethyl ether-pentane to
afford the title compound ~E1) (0.32g), m.p. 135-138C. :~ :
(Found: C, 62.29; H, 8.03; N, 6.59. C33H5107N3S qu
C, 62.53; H, 8.11; N, 6.63%).
: 25
W092/~966 ~ 36- PCT/GB91/~17 ~.
ExamPle--?
3-Acetylmercapto-6-me
one-3-Yl)aminocarb~ny1lhe~tanoi~c acid, isoproPYl ester
(E2)
10~ O ~ N ~ ~ ~
O SAc o N ~-
H :
A solution of the lactam (El~ (0.lSg, 0.24mmol) in 4.5%
formic acid/methanol (4 ml) was added under nitrogen, to a
stirred suspension of palladium black (160 mg) in methanol
(10 ml). After 2h the mixture was filtered through
Kieselguhr and evaporated in vacuo to afford an oil which
on standing over pentane-diethyl ether gave the title
compound (E2) (0.llg). This was used as such wlthout
further purification.
I
Observed M+ 499. C25H4505N3S requires M 499.
ExamPle 3
3-Mercapto-6- ~ cyclotridecan-2-one-3-
yl)aminocarbonvllhePtanoic acid, isopropvl ester
hvdrochloride_salt tE3
. ,
~ o ~ ~ N ~ ~ HCI
o SH O N
.
.- ,~:: . . .: . - . , -
.. , :. ~ - . :. . .:. . . . :. . . . .. : ..
~92/~ ~6 PCT/GB91/Ot749
~37~ 2 ~2~
An ice-cooled solution of the amine ~E2)~0.1g) in
nitrogen-purged methanol (20 ml) was treated with 35%
aqueous ammonia ~5 ml), and the reaction mixture was
stirred under nitrogen for 2h and then evaporated to
dryness ln vacuo. The product was chromatographed on
silica gel using ethyl acetate-chloroform (1:1) v/v then
methanol-chloroform ~l:S) v/v as the eluent to afford the
title compound free base as a clear oil. The product was
dissolved in ethanol and treated with a few drops of 1
etheral RCl to afford, on removal of the solvent, the
title compound hydrochloride salt (E3) as a white solid
(0.08g), m.p. 110-115C.
~(CD30D) 0.89 (3H, d, ~=6Hz), 0.91, (3H, d, J=6Hz), 1.23
(6H, d, J=6Hz), 1.3-1.9 (15H, m), 2.42 (lH, dd, J-10,
16Hz), 2.52 (lH, m), 2.71 ~lH, dd, J=16, 3Hz), 2.93 (lH,
m), 2.99-3.3 (3H, m), 3.1-3.23 (2H, m), 3.65 (lH, m), 4.35
(lH, m), 5.0 (lH, m).
.
Observed FAB (M+H)+ 458 (free base~.
C23H43N304S requires M 457-
ExamPle 4
3-MercaPtO-6-methvl-4- r (1, 8-diazacvclotetradecan-2-one-3-
vl~aminocarbonvllhePtanoic acid. methvl ester
hvdrochloride salt (E4)
CH30 ~ N
,~
WO!)~/~6966 ,~ 3a- pcr/GBs
A solution of the lactam tD12) (Isomer ~, 0.22g) in
glacial acetic acid (3 ml) was ~reated with 45~ HBr ln
glacial acetic acid t3 ml) and left stirring for 45
minutes at room temperature. The solvent was removed by
evaporation in vacuo, and the residue dissolved in
methanol ~20 ml~. The solution was purged with nitrogen
for 10 minutes, cooled in an ice bath, then treated with
35% aqueous ammonia ~5 ml). The mixture was allowed to
stir at 0C for 0.5h then at room temperature for 3h. The
solvent was evaporated under reduced pressure to a~ford a
viscous oil. The residue was diluted with anhydrous
methanol (1 ml~ and treated dropwise with excess lM
ethereal HCl to afford the title compound as a white solid
(0.12g).
Observed M+ 443 (free base~- c22H4lN3o4s requireS M 443-
(CD30D): 0.6-0.85 (6H, m), 1.2-1.8 (17H, m), 2.32-3.18
(9H, m), 3.5-3.62 (4H, m), 4.25 (lH, m).
Example 5
3-Mercapto-6-methYl-4-~(1.8-diazacYclotetradecan--2-one-3
yl)aminocarbonYllheptan ~ drochloride salt L~5?
HO ~ N N
0 SH O ~
To a suspension of methyl ester (D13) (0.1 g) in
isopropanol (5 ml), previously purged with nitrogen, was
092/~966 _39_ 2~ PCT/GB91/01749
added a solu~ion of sodium hydroxide (0.02g) in water
( 1 ml ) and the resulting solution was stirred at room
temperature, under nitrogen for 18h. The ~olution was
acidified with an excess of ethereal~HCl and then
evaporated to dryness in vacuo. The residue wa~ diluted
with lN aqueous HC1 (4 ml) and extracted with CHC13
(3 x 5 ml). The combined organic fraction was dried and
evaporated to leave a viscous oil which on trituration
with ether gave a white solid. The solid was dissolved in
glacial acetic acid (5 ml) and treated with 45% hydrogen
bromide in glacial acetic acid (2 ml). The mixture was
stirred for lh and then treated with ether to precipitate
an oil. The solvent was decanted and residue dissolved in
anhydrous methanol (20 ml) and treated with excess lM
ethereal HCl to form a salt. The HCl salt was triturated
with ether to give the title compound as a white solid.
Observed ~M)+-H2S 395 (free base)- C2lH39N3o4s require9 M
429.
~ (CD30D): 0.9 ~6H, m), 1.2-2.0 ~17H, m), 2.45-3.3 ~9H,
m), 3.65 ~lH, m), 4.35 (lH, m).
Example _6
3-MercaPto-6-methvl-4-~(1,8-diazacvclotetradecan-2-one-3-
vl~aminocarbonYllheptanoic aci~ g~ _ amide
hYdrochloride salt (E6)
1 '
o
CH3NH ~ ,N~ .HCI
WO 92/06~ r PCI/CBgl/017 ~~
~,9 ~ ~ 4 0 ~
A solution of amide (D14) (0.02g) in glaclal acetic acid
(3 ml) was treated with 45% hydrogen bromide in glacial
acetic acid (l ml). The mixture was stirred for lh and
then t~eated with ether to precipitate a solid. The
solvent was decanted, and the residue dissolved in
anhydrous methanol (2 ml) and treated with excess lM
ethereal HCl. The HCl salt that formed was triturated
with ether to give the tltle compound as a yellow solid.
Observed (MH)+-H2S 409 (free base). C22H42N403S requires .-
M 442.
~ ~CD30D): 0.85 (6H, m), 1.25-~.0 (17H, m), 2.4-3.3 (12H,
m), ~.65 (lH, m), 4.35 (lH, m).
~5
ExamPle 7
Pharmaceutical compositions ~or oral administration are
prepared by combining the following:
1) Solid Dosa~e Formulation
% w/w
Compound of Example 1 10$
Magnesium stearate 0.5%
Starch 2.0%
HPM cellulose 1.0%
Microcrystalline cellulose 86.5%
The mixture may be compressed to tablets, or filled
into hard gelatin capsules.
. ~ . . ." .,; .. ,. . " , .... . , . . . . . . ~ :
: . . , .: :;,: : ~ . : . . ~ , : . .
, !, ~ , ` ~, ~ ` ' `
~92/06966 PCT/GB91/01749
-~1-2~
The tablet may br2 coated by applying a suspension of
film former ~e.g. HPM cellulose), pi~ment (e.g.
titanium dloxide) and plasticiser (e.g. diethyl
phthalate) and drying the film by evaporation of the
solvent. The film coat can comprise 2.0% to 6.0% of
the tablet weight, preferably about 3.0%.
2) CaPsule
%w/w , ,
Compound of Example 1 20%
Polyethylene glycol 80%
The medicinal compound is dispersed or dissolved in
the liquid carrier, with the thickening agent added,
if present. The formulation is then enclosed in a
soft gelatin capsule by suitable technology.
Example 8
A pharmaceutical composition for parenteral administration
is prepared by combining the following:
Compound of Example 1 1.0%
Saline 99.0%
The solution is sterilised and sealed in sterile
c~ntainers.
:. . : .. ~: : : : .- : . ., - , ; . :
WO 92/06966~9Ç~ 42- PCI'/GB91/017
COLI~AGENASE INHI:BITOP~ ASSP-Y
The t~st is performed essentially as in Cawsto~ and
Barrett, Anal. Biochem. 99, 34Q-345 ~1979). Compounds
for testing are dissolved in methanol by sonication and
added to collagenase (purified from culture supernatants
from the human lung fibroblast cell line, WI-38) in
buffer. To ensure that thiol collagenase inhibitors
remain unoxidised, ~-mercaptoethanol may be incorporaked
in the methanol solvent and~or the diluent buffers to gi~e
a final concentration of 9.6 x 10 5M. The minimal direct
effect of ~-mercaptoethanol on the degradation of collagen
by human collagenase is controlled for. After a 5 min
pre-incubation at 37C, the assay tubes are cooled to 4C
1~ and 3H-acetylated rat skin type I collagen is added. The
assay tubes are incubated at 37C overnight. The 3H-
collagen forms insoluble fibrils, which are the substrate
for the enzyme.
To terminate the assay, the assay tubes are spun at 12000
rpm for 15 minutes. Undigested 3H-collagen is pelleted,
wh~le digested 3H-collagen is ~ound as soluble peptides in
the supernatant. A sample of the supernatant is taken
for liquid scintillation counting.
The activity of collagenase inhibitors (IC50: 50
inhibitory concentration) is expressed as that
concentration of compound that inhibits a known ~standard)
concentration of enzyme by 50%.
The compounds o~ Examples E1-E6 had IC50 values ln the
range 4.7 x 10 6 _ 9.7 x 10-BM.
