Note: Descriptions are shown in the official language in which they were submitted.
WO 95/19956 1 ~ ~ ~ PCT/GB95/00111
1
IMetalloproteinase Inhibitors
The present invention relates to therapeutically active hydroxamic acid and
carboxylic acid derivative:>, to processes for their preparation, to
pharmaceutical
compositions containing them, and to the use of such compounds in medicine. In
particular, the compounds are inhibitors of metalloproteinases involved in
tissue
degradation, and in addition are inhibitors of the release of tumour necrosis
factor
from cells.
background to the Inventi~
Compounds which have the property of inhibiting the action of
metalloproteinases
involved in connective tissue bre<3kdown such as collagenase, stromelysin and
gelatinase (known as "matrix metalloproteinases". and herein referred to as
MMPs) are thought to be ~cotentially useful for the treatment or prophylaxis
of
conditions involving such tissue breakdown, for example rheumatoid arthritis,
osteoarthritis, osteopenias such as osteoporosis, periodontitis, gingivitis,
corneal
epidermal or gastric ulceration, and tumour metastasis, invasion and growth.
MMP
inhibitors are also of potential value in the treatment of neuroinflammatory
disorders, including those involving myelin degradation, for example multiple
sclerosis, as well as in the manac,~ement of angiogenesis dependent diseases,
which include arthritic conditions .and solid tumour growth as well as
psoriasis,
proliferative retinopathies" neovascular glaucoma, ocular tumours,
angiofibromas
and hemangiomas. However, the relative contributions of individual MMPs in any
of the above disease statE~s is not yet fully understood.
Metalloproteinases are characterised by the presence in the structure of a
zinc(//)
ionic site. It is now known that there exists a range of metalloproteinase
enzymes
that includes fibroblast collagenase (Type 1 ), PMN-collagenase, 72 kDa-
gelatinase, 92 kDa-gelatinase, stromelysin, stromelysin-2 and PUMP-1 (J.F.
Woessner, FASEB J, 1991, 5, 21 ~45-2154). Many known MMP inhibitors are
peptide derivatives, based on naturally occuring amino acids, and are
analogues
of the cleavage site in the collagen molecule. A recent paper by Chapman et.
al. (J.
WO 95/19956 2 '~ PCT~GB95100111
2
Med. Chem. 1993, 36, 4293-43U1 ) reports some general structure/activity
findings
in a series of N-carboxyalkyl peptides. Other known MMP inhibitors are less
peptidic in structure, ands may more properly be viewed as pseudopeptides or
peptide mimetics. Such compounds usually have a functional group capable of
binding to the zinc (II) site in the MMP,-and known classes include those in
which
the zinc binding group is a hydroxamic acid, carboxylic acid, sulphydryl, and
oxygenated phosphorus (eg phosphinic acid and phosphonamidate including
aminophosphonic acid) groups.
Two known classes of paeudopeptide or peptide mimetic MMP inhibitors have a
hydroxamic acid group and a carboxylic group respectively as their zinc
binding
groups. With a few exceptions. such known MMPs may be represented by the
structural formula (I)
O R, Ra
I
R. ~ N _Rs (I)
NH
O
Ri \X
in which X is the zinc binding hydroxamic acid (-CONHOH) or carboxylic acid
(-COOH) group and the groups R~ to R5 are variable in accordance with the
specific
prior art disclosures of such compounds. Examples of patent publications
disclosing such structurE~s are given below.
In such compounds, it is generally understood in the art that variation of the
zinc
binding group and the substituents R~, R2 and R3 can have an appreciable
effect on
the relative inhibition of i:he metalloproteinase enzymes. The group X is
thought to
interact with metalloproteinase Enzymes by binding to a zinc(II) ion in the
active
site. Generally the hydroxamic acid group is preferred over the carboxylic
acid
group in terms of inhibitory activity against the various metalloproteinase
enzymes.
However, the carboxylic acid group in combination with other substituents can
provide selective inhibition of gelatinase (EP-489,577-A). The R~, RZ and R3
groups
W0 95/19956 PCT~GB95/00111
3
are believed to occupy rs~spectively the Pt , P1' and P2' amino acid side
chain
binding sites for the natural enzyme substrate. There is evidence that a
larger R~
substituent can enhance activity against stromelysin, and that a (C~-C6)alkyl
group
(such as iso-butyl) at R2 may be preferred for activity against collagenase
whilst a
phenylalkyl group (such as phenylpropyl) at R2 may provide selectivity for
gelatinase over the other metalloproteinases.
Tumour necrosis factor (herein rEaferred to as 'TNF") is a cytokine which is
produced initially as a cell-associated 28kD precursor. It is released as an
active,
l7kD form, which can mE~diate a large number of deleterious effects in vivo.
When
administered to animals or humans it causes inflammation, fever,
cardiovascular
effects, haemorrhage, coagulation and acute phase responses, similar to those
seen during acute infections and shock states. Chronic administration can also
cause cachexia and anorexia. Accumulation of excessive TNF can be lethal.
There is considerable evidence from animal model studies that blocking the
effects
of TNF with specific antibodies can be beneficial in acute infections, shock
states,
graft versus host reactions and autoimmune disease. TNF is also an autocrine
growth factor for some rr~yeloma.s and lymphomas and can act to inhibit normal
haematopoiesis in patients with these tumours.
Compounds which inhibit the production or action of TNF are therefore thought
to
be potentially useful for the treatment or prophylaxis of many inflammatory,
infectious, immunological or malignant diseases. These include, but are not
restricted to, septic shock, haemodynamic shock and sepsis syndrome, post
ischaemic reperfusion injury, malaria, Crohn's disease, mycobacterial
infection,
meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia,
graft
rejection, cancer, autoimmune disease, rheumatoid arthritis, multiple
sclerosis,
radiation damage, toxicity following administration of immunosuppressive
monoclonal antibodies such as t'KT3 or CAMPATH-1 and hyperoxic alveolar
injury.
WO 95J19956 . PCTJGB95J00111
4
Since excessive TNF production has been noted in several diseases or
conditions
also characterised by MPvIP-mediated tissue degradation, compounds which
inhibit
both MMPs and TNF production may have particular advantages in the treatment
or
prophylaxis of diseases or conditions in which both mechanisms are involved.
Recently, WO 93/20047 disclosed a class of hydroxamic acid based MMP
inhibitors
which also are active in inhibiting TNF production.
As mentioned above, MAP inhibitors have been proposed with hydroxamic acid or
carboxylic acid zinc binding groups. The following patent publications
disclose
hydroxamic acid-based P,AMP inhibitors:
US 4599361 (Searle)
EP-A-0236872 (Roche)
EP-A-0274453 (Bellon)
WO 90/05716 (British Bio-technology)
WO 90/05719 (British Bio-technology)
WO 91/02716 (British Bio-technology)
EP-A-0489577 (Celltech)
EP-A-0489579 (Celltech)
EP-A-0497192 (Roche)
WO 92/13831 (British Bio-technology)
WO 92/17460 (SmithKline Beecham)
WO 92/22523 (Ress~arch Corporation Technologies)
WO 93/09090 (Yam:anouchi)
WO 93/09097 (SanN;yo)
WO 93/20047 (British Bio-technology)
WO 93/24449 (Celltech)
WO 93/24475 (Celltech)
EP-A-0574758 (Roche)
WO 95/19956 ~ . PCT~GB95100111
The following patent publications disclose carboxylic acid-based MMP
inhibitors:
EP-A-0489577 (Cellt~ech)
EP-A-0489579 (Cellt~ech)
WO 93/24449 (Cellt~ech)
WO 93124475 (Cellt~ech)
Brief DescrirJtion of the Inv n i n
This invention is based on the finding that in compounds of formula (I) above
wherein X is a hydroxamic acid or carboxylic acid group, an aromatic or
heteroaryl
R4 substituent has in general thE~ unexpected and desirable effect of
increasing
activity against stromely;>in relative to compounds of otherwise similar
structure but
with the usual R4 substituents, while maintaining activity against collagenase
and
gelatinase. This finding leads to compounds that are broad spectrum inhibitors
of
the known metalloproteinases. 'The class of compounds of the invention also
includes compounds in which the ability to inhibit TNF production is improved
relative to compounds of otherwise similar structure but with the usual R4
substituents. The class also includes compounds which are orally bioavailable.
In general, metalloproteinase inhibitors known in the art to possess good
activity
against stromelysin are compounds such as BB-94 (WO 90/05719 Example 2) that
possess relatively large substituents at R~. However, BB-94 and compounds with
large substituents at R~ tend to be less bioavailable when dosed orally than
compounds with smaller or no R~,~ substituent. A particular advantage of the
compounds of the present invention is that the combination of an aromatic or
heteroaryl R4 substituent with no R~ substituent or a small R~ substituent can
provide oral activity together with broad spectrum activity against the
metalloproteinase enzyrnes, including good potency against stromelysin.
The art does not appear to haves recognised the role of an aromatic or
heteroaryl R4
substituent in increasing the activity of hydroxamic acid and carboxylic acid
based
2-1 ~ 15'~ ~ -
6
pseudopeptide or peptide mimetic MMP inhibitors against stromelysin. Of the
publications relating to hydroxamic acid based MMP inhibitors listed above,
only
WO 93109097 (Sankyo) refer:. to the possibility of a phenyl group in the
relevant
position. In a structurally different series of MMP inhibitors with phosphinic
acid zinc
binding groups, WO 93/14112 (Merck) discloses compounds with certain aryl
groups in the equivalent position. In neither case is there any comment on the
role
of that group in the overall stn.rcture/activity relationships of the
disclosed
compounds. In the field of natural peptide analogues the publication by
Chapman
et. al. referred to above discloses that aryl groups in the corresponding
position to
the R4 position of compounds of this invention appear to be preferred for
stromelysin activity. V~JO 92121360 (Merck) also discloses natural peptide
analogues
having certain aryl groups in the corresponding position to the R4 position of
'
compounds of this invention. However it is not clear that structure/activity
relationships which are true in the field of natural peptide MMP inhibitors
will hold
true in the field of pseudopeptide or peptide mimetic MMP inhibitors with
which this
invention is concerned.
Detailed Descri tip on c~f the Invention ___
The present invention provides compounds of general formula I
O R3 Ra
RZ N~
H ~ R5 (I)
0
R~ ~X
wherein
X is a -COZH or -CONHC>H group;
R, is hydrogen; (C,-CB)alN;yl; (Cz-Cs)alkenyl; phenyl; substituted phenyl;
phenyl
A(tlENDED SHEET
WO 95/19956 2181 ~ ~ ~ PCT/GB95/00111
7
(C~-C6)alkyl); substituted phenyl(C~-C6)alkyl; heterocyclyl; substituted
heterocyclyl; heterocyclyl(C~-C6)alkyl; substituted heterocyclyl(C~-C6)alkyl;
a
group BSO~A- wherein n is 0, 1 or 2 and B is hydrogen or a (C~-C6) alkyl,
phenyl, substituted phenyl, heterocyclyl, (C~-C6)acyl, phenacyl or substituted
phenacyl group, and A represents (C~-C6)alkyl; amino; protected amino;
acylamino; OH; SH; (C~-C6)alkoxy; (C~-C6)alkylamino; di-(C~-C6)alkylamino;
(C~-C6)alkylthio; aryl (C~-C6)alkyl; amino(C~-C6)alkyl; hydroxy(C~-Cs)alkyl,
mercapto(C~-C6)alkyl or carboxy(C~-C6)alkyl wherein the amino-, hydroxy-,
mercapto- or carboxyl-group are optionally protected or the carboxyl- group
amidated; lower alkyl sub:;tituted by carbamoyl, mono(lower
alkyl)carbamoyl, di(lower ~alkyl)carbamoyl, di(lower alkyl)amino, or carboxy-
lower alkanoylamino:
R~ is a (C~-C6)alkyl, (~~2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C~-C6)alkyl,
heteroaryt(C~-C6)alkyl, cycloalkyl(C~-C6)alkyl or cycloalkenyl(C~-C6) alkyl
group, any one of 'which may be optionally substituted by one or more
substituents selected from (C~-C6)alkyl, -O(C~-C6)alkyl, -S(C~-C6)alkyl, halo
and cyano (-CN);
R3 is the characterising group of a natural or non-natural a amino acid in
which
any functional groups may be protected;
Ra is a phenyl or 5- or 6-membered heteroaryl ring wherein any ring nitrogen
atom may be oxidi;;ed as an N-oxide, which may be optionally fused to a
benzene ring or to a 5-, 6- or 7-membered heterocyclic ring, and wherein
any of the rings may be optionally substituted by:
(a) one or more substituents independently selected from hydroxyl,
halogen, -CN, -C02H, -C02(C~-C6)alkyl, -(C~-C6)alkyl-C02(C,-
C6)alkyl, -CONH2, -CONH(C~-C6)alkyl, -CON((C~-C6)alkyl)2, -CHO,
-CH20H, -(G~-C4)pE~rfluoroalkyl, -O(C~-C6)alkyl, -S(C~-Cs)alkyl,
WO 95/19956 2 ~ ~; PCT~GB95/00111
8
-SO(C~-C6);alkyl, -S02(C~-C6)alkyl, -N02, -NH2, -NH(C~-C6)alkyl,
-N((C~-C6)alkyl)2, and -NHCO(C~-C6)alkyl, or
(b) a group selected from (C~-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl,
(C3-C8)cycloalkyl, (C4-Ce)cycloalkenyl, phenyl, benzyl, heteroaryl or
heteroarylmethyl any of which groups may be optionally substituted
with one or more substituents selected from halogen, hydroxyl, amino,
carboxyl, (C;~-C4)perfluoroalkyl, (C~-C6)alkyl, -O(C~-Cs)alkyl or
-S(C,-C6)afkyl;
R5 is hydrogen or a (~~~-C6)alkyl group;
or a salt, hydrate or solv~ite therE~of.
As used herein the term "(C~-C6)alkyl" or "lower alkyl" means a straight or
branched
chain alkyl moiety having from 1 to 6 carbon atoms, including for example,
methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl and
hexyl.
The term "(C2-C6)alkenyl" means a straight or branched chain alkenyl moiety
having from 2 to 6 carbon atoms and having in addition one double bond of
either
E or Z stereochemistry vvhere applicable. This term would include, for
example,
vinyl, t-propenyl, 1- andl 2-butenyl and 2-methyl-2-propenyl.
The term "cycloalkyl" mE~ans a saturated alicyclic moiety having from 3-8
carbon
atoms and includes, for example, cyclohexyl, cyclooctyl, cycloheptyl,
cyclopentyl,
cyclobutyl and cyclopropyl.
The term "cycloalkenyl" means an unsaturated alicyclic moiety having from 3-8
carbon atoms and inclu~jes, for example, cyclohexenyl, cyclooctenyl,
cycloheptenyl, cyclopentenyl, cyclobutenyl and cyclopropenyl. In the case of
cycloalkenyl rings of from 5-8 carbon atoms, the ring may contain more than
one
double bond.
WO 95/19956 ~ PCTIGB95l00111
9
The unqualified term "heterocyclyl" or °heterocyclic" means (i) a 5-7
membered
heterocyclic ring containing one or more heteroatoms selected from S, N and O,
and optionally fused to a benzene ring, including for example, pyrrolyl,
furyl,
thienyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl,
pyrrolidinyl,
pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimidazolyl, maleimido,
succinimido, phthalimido, 1,2-dinnethyl-3,5-dioxo-1,2,4-triazolidin-4-yl, 3-
methyl-
2,5-dioxo-1-imidazolidinyl and 3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl, or
(ii) a
naphththalimido (ie 1,3-dihydro-1,3-dioxo-2H-benz[fJisoindol-2-yl), 1,3-
dihydro-1-
oxo-2H-bent[fJisoindol-2-yl, 1,3-dihydro-1,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-
yl,
or 2,3-dihydro-1,3-dioxo-1 H-bent[d,e]isoquinolin-2-yl group.
The term "5- or 6-membered heterocyclic ring" means such rings having 5 or 6
atoms in the ring, wherein the heteroatom(s) may be one or more nitrogen.
oxygen
or sulphur atoms, and includes heterocycles containing nitrogen, oxygen, or
sulphur alone or containing two nitrogen atoms, a nitrogen and an oxygen atom,
a
nitrogen and a sulphur atom, two nitrogen atoms and an oxygen atom, two
nitrogen
atoms and a sulphur.
The "heteroaryl" means a 5-7 membered substituted or unsubstituted aromatic
heterocycle containing one or more heteroatoms. Illustrative of such rings are
thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, pyrazolyl, isoxazolyl,
isothiazolyl, trizolyl,
thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and
triazinyl.
Unless otherwise specified in the context in which it occurs, the term
"substituted"
as applied to any moiety herein means substituted with up to four
substituents,
each of which independently may be (C~-C6)alkoxy, hydroxy, mercapto, (C~-
C6)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo),
trifluoromethyl,
vitro, -COOH, -CONH2 or -CONHRA wherein RA is a (C~-C6)alkyl group or the
residue of a natural alpha-amino acid.
The term "characteristic side chain of a natural alpha-amino acid" means the
WO 95119956 ~ PCT/GB95I00111 - _
characteristic side chain attached to the -CH(NH2)(COOH) moiety in the
following
amino acids: glycine, ala.nine, valine, leucine, isoleucine, phenylalanine,
tyrosine,
tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine,
lysine,
histidine, arginine, glutamic acid and aspartic acid.
Natural alpha-amino acids which contain functional substituents, for example
amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups in
their
characteristic side chains include arginine, lysine, glutamic acid, aspartic
acid,
tryptophan, histidine, ser~,ine, thre~onine, tyrosine, and cysteine. When R3
in the
compounds of the invention is one of those side chains, the functional
substituent
may optionally be protected.
The term "protected" whE~n used in relation to a functional substituent in a
side
chain of a natural alpha-,amino acid means a derivative of such a substituent
which
is substantially non-functional. Ins this context, protected amino groups
include
amido and acylamino, protected hydroxy or mercapto groups include ethers and
thioethers, protected carboxyl groups include esters, and imidazolyl, indolyl
or
guanidyl groups may be protected as t-butoxycarbonyl derivatives. These are
only
examples of the many protecting derivatives known in the art, and others will
be
known to the skilled man.
Salts of the compounds of the invention include physiologically acceptable
acid
addition salts for example hydrochlorides, hydrobromides, sulphates, methane
sulphonates, p-toluenesulphonates, phosphates, acetates, citrates, succinates,
lactates, tartrates, fumarates and maleates. Salts may also be formed with
bases,
for example sodium, potassium, magnesium, and calcium salts.
There are several chiral centres in the compounds according to the invention
because of the presence of asymmetric carbon atoms. The presence of several
asymmetric carbon atoms gives rise to a number of diastereomers with R or S
stereochemistry at each chiral centre. General formula (I), and (unless
specified
otherwise) all other formulae in i:his specification are to be understood to
include all.
WO 95/19956 PGT/GB95/00111
11
such stereoisomers and mixtures (for example racemic mixtures) thereof.
In the compounds of the invention, the preferred stereochemistry is in general
as
follows:
C atom carrying the R~ and X groups - S,
C atom carrying the R2 group - R,
C atom carrying the R3 group - S,
but mixtures in which th~a above configurations predominate are also
contemplated.
As previously stated, the compounds of the invention are principally
distinguished
from the compounds disclosed in the prior art patent publications listed above
by
the identity of the group 1~4. Accordingly, the groups R~, R2, R3, and RS may
be any
of the groups which have been disclosed in the corresponding positions of
compounds disclosed in any of those prior art patent publications listed
above.
Without limiting the genE~rality of the foregoing, the following classes of
substituent
R3 have been disclosed in the corresponding position of prior art compounds,
and
are therefore suitable R~, groups for use in compounds of the present
invention:
(C~-C6)alkyl, benzyl, hydroxybenzyl, benzyloxybenzyl, (Ci-C6)alkoxybenzyl,
or benzyloxy(C~-C6)alkyl group; and
the characterisincl group of a natural a amino acid, in which any functional
group may be protected, any amino group may be acylated and any
carboxyl group present may be amidated; and
a group -[AIkj~Rs where Alk is a (C~-C6)alkyl or (C2-C6)alkenyl group
optionally interrupted by one or more -O-, or -S- atoms or -N(R~)- groups
[where R~ is a hydrogen atom or a (C~-C6)alkyl group], n is 0 or 1, and R6 is
an optionally substituted cycloalkyl or cycloalkenyl group; and
a benzyl group substituted in the phenyl ring .by a group of formula
WO 95!19956 ~ .~ PCTIGB95/00111
12
-OCH2COR8 where R8 is hydroxyl, amino, (C~-C6)alkoxy, phenyl(C,-
Cs)alkoxy, (C~-C6)alkylamino, di((C~-C6)alkyl)amino, phenyl(Ci-
C6)alkylamino, the residuE~ of an amino acid or acid halide, ester or amide
derivative thereof, said residue being linked via an amide bond, said amino
acid being selected from glycine, a or ~i alanine, valine, leucine,
isoleucine,
phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine,
asparagine, glutarnine, ly;>ine, histidine, arginine, glutamic acid, and
aspartic
acid; and
a heterocyclic((C~-C6)alkyl group, either being unsubstituted or mono- or di-
substituted in the ~heterocyclic ring with halo, vitro, carboxy, (C~-
C6)alkoxy,
cyano, (C,-C6)alkanoyl. trifluoromethyl (C~-C6)alkyl, hydroxy, formyl, amino,
(C~-C6)alkylamino, di-(C~~~C6)alkylamino, mercapto, (C~-C6)alkylthio,
hydroxy(C~-C6)al~;yl, merc:apto{C~-C6)alkyl or (C~-C6)alkylphenylmethyl;
R3 may also be a group -CRaRbIR~ in which:
each of Ra, Rb and R~ is independently hydrogen, (C~-C6)alkyl, (C2-
C6)alkenyl, (C2-C,;)alkynyl, phenyl(C~-C6)alkyl, (C3-C8)cycloalkyl, the
foregoing being subject to~ the proviso that Ra, Rb and R~ are not all
hydrogen; or
R~ is hydrogen, (C~-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C~-
C6)alkyl, or (C3-C,3)cycloalkyl, and Ra and Rb together with the carbon atom
to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-
membered hetero~cyclic ring; or
Ra, Rb and R~ together with the carbon atom to which they are attached form
a tricyclic ring (for example adamantyl); or
Ra and Rb are each independently (C~-C6)alkyl, (C2-C6)alkenyl, (C2-
WO 95/19956 . _ . PCT/GB95/00111
13
C6)alkynyl, phenyl(C~-C6)alkyl, or a group as defined for R~ below other than
hydrogen, or Ra and Rb together with the carbon atom to which they are
attached form a 3 to 8 membered cycloalkyl or a 3- to 8-membered
heterocyclic ring, and R~ is hydrogen, -OH, -SH, halogen, -CN, -C02H, (C~-
C4)perfluoroalkyl, -CH20H, -C02(C~-C6)alkyl, -O(C~-C6)alkyl, -O(C2-
C6)alkenyl, -S(C~-C6)alkyl, -SO(C~-Cs)alkyl, -S02(C~-C6) alkyl, -S(C2-
C6)alkenyl, -SO(C2-C6)alf;enyl, -S02(C2-C6)alkenyl or a group -C~-W wherein
C~ represents a bond or -O-, -S-, -SO- or -S02- and W represents a phenyl,
phenylalkyl, (C3-C8)cycloalkyl, (C3-Ca)cycloalkylalkyl, (C4-Ce)cycloalkenyl,
(C4-C8)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W
may optionally be substituted by one or more substituents independently
selected from, hydroxyl, halogen. -CN, -C02H, -C02(C~-C6)alkyl, -CONH2,
-CONH(C,-C6)alkyl, -CONH(C,-C~alkyl)2, -CHO, -CH20H. (C~-
C4)perfluoroalkyl, -O(C1-C;6)alkyl, -S(C~-Cs)alkyl, -SO(C~-C6)alkyi, -S02(C~-
C6)alkyl, -NOZ, -NH2, -NH(C~-C6)alkyl, -N((C~-C6)alkyl)2, -NHCO(C~-C6)alkyl,
(C~-C6)alkyl, (C2-t~s)alkenyl, (C2-C6)alkynyl, (C3-C8)cycloalkyl, (C4-
C8)cycloalkenyl, phenyl or benzyl.
More specifically with respect to the groups R~, R2, R3, R4 and R5 in
compounds of
the invention:
Examples of particular R~ groups include hydrogen, methyl, ethyl, hydroxyl,
allyl, thienylsulphanylmetlhyl, thienylsulphinylmethyl, thienylsulphonylmethyl
and phthalimidomethyl. Presently preferred are compounds in which R~ is
hydrogen, hydroxyl, allyl or phthalimidomethyl.
Examples of particular R2 groups include iso-butyl, n-pentyl, n-hexyl, n-
heptyl, n-octyl, n-nonyl, n~-decyl, cyclohexylpropyl, phenylpropyl, 4-
chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl,
phenylbutyl, propyloxymethyl and propylsulphanyl. Presently preferred are
compounds in whiich R2 is isobutyl, n-heptyl, or phenylpropyl.
WO 95/19956 ~ ~ ~ ,, PCTIGB95/00111 '""'
14
Examples of particular R3 groups include benzyl, iso-butyl or t-butyl, 1-
benzylthio-1-methylethyl, and 1-mercapto-1-methylethyl. Presently preferred
are compounds in which R3 is t-butyl or 1-mercapto-1-methylethyl.
Examples of R4 groups include optionally substituted phenyl, napthyl,
furanyl, thienyl, pyrrolinyl, tetrahydrofuranyl, imidazolyl, oxadiazolyl,
thiazolyl, thiadiazolyl, pyridinyl, pyridinyl N-oxides, piperazinyl, indolyl,
benzimidazolyl, benzotriazolyl, pyrazinyl, pyridazinyl, pyrimidinyl,
dithianyl,
benzo[b]thienyl, isoxazolyl or quinolinyl. Examples of particular R4 groups
include phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,4-
dimethoxyphenyl, 2-fluorophenyl. 3-fluorophenyl, 4-fluorophenyl, 2-
chlorophenyl. 3-ciilorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 3.5-
dichlorophenyl, 2-bromophenyl. 3-bromophenyl, 4-bromophenyl, 2-
iodophenyl, 3-iodophenyl, 4-iodophenyl, 2-methylphenyl. 3-methylphenyl, 4-
methylphenyl, 3,4-dimethyl, 2-t-butylphenyl, 3-t-butylphenyl, 4-t-butylphenyl,
4-t-butyl-2,6-dimethylphenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-
cyanophenyl, 3-cyanophe~nyl, 4-cyanophenyl, 2-acetylphenyl, 3-
acetylphenyl, 4-ac;etylphenyl, 2-methylsulphonylphenyl, 3-
methylsulphonylphenyl, 4-methylsulphonylphenyl, 2-trifluoromethylphenyl,
3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 3,5-ditrifluoromethylphenyl,
2-aminophenyl, 3-aminophenyl, 4-aminophenyl, 2-N,N-
dimethylaminophE~nyl, 3-N,N-dimethylaminophenyl, 4-N,N-
dimethylaminophE~nyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl,
2-napthyl, furan-2-yl, thien-2-yl, pyrrol-2-yl, tetrahydrofuran-2-yl, imidazol-
2-
yl, thiazol-2-yl, 4-ethoxycarbonylmethylthiazol-2-yl, 4-phenylthiazol-2-yl,
4,5-
dimethylthiazol-2-yl, 5-bromothiazol-2-yl, 4-Pert-butylthiazol-2-yl,
benzothiazol-2-yl, 1,2,4-oxadiazol-5-yl, 3-methyl-1,2,4-oxadiazol-5-yl , 3-
phenyl-1,2,4-oxadiazol-5-yl, 1,2,4-oxadiazol-3-yl, 1,3,4-oxadiazol-2-yl, 1,2,4-
thiadiazol-5-yl, 3-phenyl-1i ,2,4-thiadiazol-5-yl, 1,3,4-thiadiazol-2-yl, 5-
methyl-
1,3,4-thiadiazol-2-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, N-oxides of
pyridin-2-yl pyridin-3-yl and pyridin-4-yl, piperazin-1-yl, indol-2-yl,
PCT/GB95/00111
WO 95/19956
benzimidazol-2-yl, benzotriazol-2-yl, pyrazin-2-yl, 1,2-pyridazin-3-yl, 1,3-
pyrimidin-5-yl, 1,3-dithian-2-yl, benzo[b]thien-2-yl, isoxazol-5-yl, quinolin-
3-
yl. In general, compounds wherein R4 is a thiazolyl or substituted thiazolyl
group are preferred for their activity in inhibiting the release of TNF.
Presently preferred! are compounds in which R4 is phenyl, 3-methoxyphenyl,
pyridin-2-yl, pyridin-3-yl, and thiazol-2-yl, 4,5-dimethylthiazol-2-yl, 5-
bromothiazol-2-yl, 4-ethoxycarbonytmethylthiazol-2-yl, 5-methyl-1,3,4-
thiadiazol-2-yl or 4-terlbutylthiazol-2-yl. Particularly preferred at present
are
compounds wherein R4 is 3-methoxyphenyl, pyridin-2-yl, pyridin-3-yl,
thiazol-2-yl, 4-ethoxycarbonylmethylthiazol-2-yl, 5-methyl-1,3,4-thiadiazol-2-
yl and 4-tert-butylthiazol-2-yl.
Examples of particular RS groups include hydrogen, methyl and ethyl.
Presently preferred are compounds in which RS is hydrogen.
Compounds of the present invention which are currently preferred for their
combination of high intrin;>ic activity and good bioavailability when
administered
orally are:
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-2S-hydroxy-
5-methylhexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(5-mE~thyl-1,3,4-thiadiazol-2-ylcarbamoyl)-
propylcarbamoyl)-2S-hydroxy-5-methylhexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(3-methoxyphenyl)carbamoyl-propylcarbamoyl)-2S-
hydroxy-5-methylhE~xanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-pyridin-3-ylcarbamoyl-propylcarbamoyl)-2S-hydroxy-
WO 95119956 PCTIGB95/00111
16
5-methylhexanohydroxamic acid and
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
phthalimidomethylhexanohydroxamic acid,
and salts, solvates or hydrates thereof.
Compounds of the present invention which are currently preferred for their
activity
in inhibiting TNF releasE~ are:
3R-(2,2-Dimethyl-1 S-(thiazol-2-ylcarbamoyl)-propylcarbamoyl)-5-methyl-
2S-propen-2-yl-hexanohydroxamic acid,
3R-(2.2-Dimethy~-1 S-(thiazol-2-ylcarbamoyl)-propylcarbamoyl)-2S-hydroxy-
5-methylhexanohydroxarnic acid, and
3R-(2,2-Dimethyl-1 S-(4-ethoxycarbonylmethylthiazol-2-ylcarbamoyl)-
propylcarbamoyl;I-2S-hyciroxy-5-methylhexanohydroxamic acid,
and salts, solvates or hydrates thereof.
Further specific compounds of the invention are:
5-Methyl-3R-(2-phenyl-1 S-phenylcarbamoyl-ethylcarbamoyl)-
hexanohydroxamic acid,
3R-{2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-5-methyl-
hexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-
hexanohydroxamic acid,
WO 95119956 ~ PCT/GB95100111
17
3R-(2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
methylhexanohydroxamic acid,
2S-Hydroxy-3R-(;3-methyl-1 S-napth-2-ylcarbamoyl-butylcarbamoyl)-5-
methyl-hexanohydroxamic acid-
2S-Hydroxy-3R-(a-methyl-1 S-(4-methoxyphenyl)carbamoyl-
butylcarbamoyl)-5-methyl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(4-fe~rtbutyl-2,6-dimethylphenyl)carbamoyl-
propylcarbamoyl)-2S-hydroxy-5-methylhexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(4-methoxyphenyl)carbamoyl-propylcarbamoyl)-2S-
hydroxy-5-methylhexanoh~ydroxamic acid,
3R-(2,2-Dimethyl-1 S-pyridin-4-ylcarbamoyl-propylcarbamoyl)-2S-hydroxy-
5-methylhexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(4-hydroxyphenyl)carbamoyl-propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxamic acid,
3R-(2-Benzylthio-;?-methyl-1 S-(pyridin-2-ylcarbamoyl)propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
phenylhexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(4,5-dimethylthiazol-2-ylcarbamoyl)-propylcarbamoyl)-
2S-hydroxy-5-methylhexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(5-bromo-thiazol-2-ylcarbamoyl)-propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxamic acid,
PCT/GB9s/00111 " '
wo 9sn~s6
18
3R-(2,2-Dimethyl~-1 S-{4-phenyl-thiazol-2-ylcarbamoyl)-propylcarbamoyl)-
2S-hydroxy-5-methylhexanohydroxamic acid,
3R-(2,2-Dimethyl-~1 S-(4-t,ert-butylthiadiazol-2-ylcarbamoyl)-
propylcarbamoyl)-2S-hydroxy-5-methylhexanohydroxamic acid,
3R-{2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-~1 S-(4-nnethoxyphenylcarbamoyl)-propylcarbamoyl)-5-
methyl-2S-propen-2-yl-hE~xanohydroxamic acid,
3R-(2-Benzylthio-2-methyl-1 S-(pyridin-2-ylcarbamoyl)-propylcarbamoyl)-5-
methyl-2S-propen-2-yl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-~1 S-(pyridin-3-ylcarbamoyl)-propylcarbamoyl)-5-methyl-
2S-propen-2-yl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-~1 S-(4-hydroxyphenylcarbamoyl)-propylcarbamoyl)-5-
methyl-2S-propen-2-yl-hE~xanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(3-methoxyphenylcarbamoyl)-propylcarbamoyl)-5-
methyl-2S-propen-2-yl-hErxanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(pyridin-4-ylcarbamoyl)-propylcarbamoyl)-5-methyl-
2S-propen-2-yl-hE~xanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(2-methoxyphenylcarbamoyl)-propylcarbamoyl)-5-
methyl-2S-thien-2-ylsulfanylmethyl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(pyridin-2-ylcarbamoyl)-propylcarbamoyl)-5-methyl-
WO 95/19956 ~ PCTlGB95100111
19
2S-thien-2-ylsulfanylmethyl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(2-methoxyphenylcarbamoyl)-propylcarbamoyl)-5-
methyl-2S-thien-2-ylsulfinylmethyl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-(pyridin-2-ylcarbamoyl)-propylcarbamoyl)-5-methyl-
2S-thien-2-ylsulfinylmethyl-hexanohydroxamic acid,
3R-(2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
phenylhexanoic acid,
3R-(2,2-Dimethyl-1 S-(N-oxy-pyridin-2-yl)carbamoyl-propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxamic acid.
and salts, solvates or hydrates thereof.
Compounds according to the present invention in which X is a hydroxamic acid
group -CONHOH may beg prepared from corresponding compounds of the
invention in which X is a carboxylic acid group -COOH or from the
corresponding
protected hydroxamic acid derivatives. That process, which forms another
aspect of
the invention, comprises:
(a) causing an acid of general formula (II)
O R, Ri
I
R= ~ N-RS (II)
NH
O
R~ ~COOH
or an activated derivative thereof to react with hydroxylamine, O-protected
hydroxylamine, or an N,O-diprotected hydroxylamine, or a salt thereof, R~,
R2, R3, R4, and R5 being as defined in general formula (I) except that any
substituents in R, , R2, R3, R4, and RS which are potentially reactive with
WO 95/19956 PCT/GB95/00111
~~$~.~~~~ 20
hydroxylamine, O-protected hydroxylamine, the N,O-diprotected
hydroxylamine or their salts may themselves be protected from such
reaction, then removing any protecting groups from the resultant hydroxamic
acid moiety and from any protected substituents in R~, R2, R3, R4, and R5; or
(b) deprotecting a diprotected hydroxamic acid derivative of formula (Ilb)
O R3 Ra
R2 NH N'Rs(Ilb)
O
R' O
N
R,,,~ ~O'R,s
in which R~, R2, R3, R4, and RS are as defined in general formula (I), Ri4 is
an
amino protecting group and RCS is a hydroxyl protecting group.
For method (a) conversion of (II) to an activated derivative such as the
pentafluorophenyl, hydroxysuccinyl, or hydroxybenzotriazolyl ester may be
effected
by reaction with the appropriate alcohol in the presence of a dehydrating
agent
such as dicyclohexyl dicarbodiimide (DCC), N,N-dimethylaminopropyl-N'-ethyl
carbodiimide (EDC), or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ).
Protecting groups as referred to above are well known per se, for example from
the
techniques of peptide chE~mistry. Amino groups are often protectable by
benzyloxycarbonyl, t-butoxycarbo~nyl or acetyl groups, or in the form of a
phthalimido group. Hydroxy groups are often protectable as readily cleavable
ethers such as the t-butyl or benz:yl ether, or as readily cieavable esters
such as the
acetate. Carboxy groups are often protectable as readily cleavable esters,
such as
the t-butyl or benzyl ester.
Examples of O-protected hydroxylamines for use in method (a) above include O-
WO 95/19956 PCT/GB95/00111
21
benzylhydroxylamine, O-.4-methoxybenzylhydroxyiamine, O-
trimethylsilylhydroxylamine, and O-tert-butoxycarbonylhydroxylamine.
Examples of O,N-diprotecaed hydroxylamines for use in method (a) above include
N,O-bis(benzyl)hydroxylamine, N,O-big(4-methoxybenzyl)hydroxylamine, N-tert-
butoxycarbonyl-O-tert-butyldimethylsilylhydroxylamine, N-tert-butoxycarbonyl-O-
tetrahydropyranylhydroxylamine, and N,O-bis(tert-butoxycarbonyl)hydroxylamine.
For method (b) suitable protectingi groups Ri4 and R~5 are benzyl and
substituted
benzyl (eg 4-methoxybenzyl). Such protecting groups may be removed by
hydrogenolysis, while the 4-methoxybenzyl group may also be removed by acid
hydrolysis.
In method (a) in the special case where R~ in compound (I) is hydroxy, a
particularly useful technique may be reaction of hydroxylamine with a
dioxalone of
formula (Ila):
O R, R~
I
R,, N-RS
~fH (I la)
O' O
O
R,, -
Ri~O
wherein the groups R~2 and R~3 are derived from a dioxalone forming reagent,
and
may be, for example, hydrogen, alkyl, phenyl or substituted phenyl. The
dioxalone
ring is opened on reaction with hydroxylamine to give the required hydroxamic
acid
derivative of formula (I). -
Compounds according to the present invention in which X is a carboxylic acid
group -COOH may be prepared by a process comprising: coupling an acid of
formula (III) or an activated derivative thereof with an amine of formula (IV)
PCTlGB95/00111 ~'"
WO 95119956
22
O
R' OH (III) R~ Ny R' (IV)
H,N
R, COOR" O
wherein R~ R2, R3, R4, and RS are as defined in general formula (I) except
that any
substituents in R~, R2, R3, R4, and R5 which are potentially reactive in the
coupling
reaction may themselves be protected from such reaction, and R> > represents a
hydroxy protecting group, and subsequently removing the protecting group R»
and
any protecting groups frorn R~ R2, R3, R4, and R5.
Compounds of formula (Ilb) may be prepared by a process comprising: causing an
acid of formula (Illa) or an activated derivative thereof to react with an
amine of
formula (IV)
0
R2
~OH
(Illa) R~ R~ (IV)
O
R~ N _Rs
H=N
R~a/N.O_R~s O
wherein R~ R2, R3, R4, and RS are as defined in general formula (I) except
that any
substituents in R~, R2, R3, R4, and R5 which are potentially reactive in the
coupling
reaction may themselves be protected from such reaction, R~4 is an amino
protecting group and R~5 is a hydroxyl protecting group as referred to in
connection
with formula (Ilb) above, and subsequently removing any protecting groups from
R~
R2, R3, R4, and Rs.
Active derivatives of acid:; (III) and (Illa) include activated esters such as
the
WO 95/19956 PCT/GB95/00111
23
pentafluorophenyl ester, acid anhydrides and acid halides, eg chlorides.
Suitable
hydroxy protecting group:. R» may be selected from those known in the art.
Amine intermediates of formula (IV) are either known compounds or may be
prepared from known amino acid starting materials using standard methods and
by
analogy with the specific ~preparavive examples herein.
In the special case where R~ in compound (III) or (Illa) is hydroxy, it too
may be
protected during the coupling of compounds (III) or (Illa) and (IV). In the
case
where R~ is hydroxy in compound (III) a particularly useful technique may be
simultaneous protection o~f the two hydroxy groups as a dioxalone of formula
(V):
0
R,, ~
,OH (V)
O'
~ O
R1,~~~
Ru
wherein the groups R~2 and R~3 are derived from a dioxalone forming reagent,
and
may be, for example, hydrogen, alkyl, phenyl or substituted phenyl.
As mentioned above, compounds of formula (I) are useful in human or veterinary
medicine since they are active as inhibitors of MMPs, and a further advantage
lies
in their ability to inhibit the release of tumour necrosis factor (TNF) from
cells.
Accordingly in another asF~ect, this; invention concerns:
(i) a method of management (by which is meant treatment or prophylaxis) of
diseases or conditions mediated by MMPs and/or TNF in mammals, in particular
in
humans, which method comprises administering to the mammal an effective
amount of a compound as defined with respect to formula (I) above, or a
PCTIGB95/00111
WO 95!19956 2181 ~ 7
24
pharmaceutically acceptable salt thereof; and
(ii) a compound as defin~sd with respect to formula (I) for use in human or
veterinary
medicine, particularly in the management (by which is meant treatment or
prophylaxis) of diseases or conditions Tnediated by MMPs and/or TNF; and
(iii) the use of a compound as defined with respect to formula (I) in the
preparation
of an agent for the management (by which is meant treatment or prophylaxis} of
diseases or conditions mediated by MMPs and/or TNF.
Diseases or conditions mediated by MMPs include those involving tissue
breakdown such as bona resorption, inflammatory and neuroinflammatory
diseases, dermatological conditions, solid tumour growth and tumour invasion
by
secondary metastases, and angiogenesis dependent diseases, in particular
rheumatoid arthritis, ostE~oarthritis, periodontitis, gingivitis, corneal
ulceration, solid
tumour growth and tumour inva:>ion by secondary metastases, neovascular
glaucoma, multiple sclerosis, and psoriasis. Diseases or conditions mediated
by
TNF include inflammation, fever, cardiovascular effects, haemorrhage,
coagulation
and acute phase responae, cachexia and anorexia, acute infections, shock
states,
graft versus host reactions and autoimmune disease.
In a further aspect of the invention there is provided a pharmaceutical or
veterinary
composition comprising .a compound of formula (I) together with a
pharmaceutically
or veterinarily acceptablE~ excipiE~nt or carrier. Included within this aspect
of the
invention is a pharmaceutical or veterinary composition comprising a compound
of
formula (I) together with a pharmaceutically or veterinarily acceptable
excipient or
carrier, characterised in that the composition is adapted for oral
administration.
One or more compounds. of general formula (I) may be present in the
composition
together with one or more excipient or carrier.
The compounds with which the invention is concerned may be prepared for
WO 95119956 c~ PCTlGB95/00111
administration by any route consistent with their pharmacokinetic properties.
The
orally administrable comiaosition;s may be in the form of tablets, capsules,
powders,
granules, lozenges, liquid or gel preparations, such as oral, topical, or
sterile
parenteral solutions or suspensions. Tablets and capsules for oral
administration
may be in unit dose presentation form~and may contain conventional excipients
such as binding agents, 'for example syrup, acacia, gelatin, sorbitol,
tragacanth, or
polyvinyl-pyrrolidone; fillE~rs for example lactose, sugar, maize-starch,
calcium
phosphate, sorbitol or glycine; ta~bletting lubricant, for example magnesium
stearate, talc, polyethylene glycol or silica; disintegrants for example
potato starch,
or acceptable wetting agents such as sodium lauryl sulphate. The tablets may
be
coated according to methods well known in normal pharmaceutical practice. Oral
liquid preparations may be in the form of, for example, aqueous or oily
suspensions, solutions. emulsions, syrups or elixirs, or may be presented as a
dry
product for reconstitution with water or other suitable vehicle before use.
Such
liquid preparations may contain nonventional additives such as suspending
agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin
hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan
monooleate, or acacia; non-aqus~ous vehicles (which may include edible oils),
for
example almond oil, fractionated coconut oil, oily esters such as glycerine,
propylene glycol, or ethyl alcohol; preservatives, for example methyl or
propyi p-
hydroxybenzoate or sorbic acid, and if desired conventional flavouring or
colouring
agents.
The dosage unit involved in oral .administration may contain from about 1 to
250mg, preferably from about 25 to 250mg of a compound of the invention. A
suitable daily dose for a rnammal may vary widely depending on the condition
of
the patient. However, a dose of a compound of general formula I of about 0.1
to
300mg/kg body weight, particularly from about 1 to 100mg/kg body weight may be
appropriate.
For topical application to l:he skin, the drug may be made up into a cream,
lotion or
ointment. Cream or ointment formulations which may be used for the drug are
WO 95/19956 ~ ~ ~ PCTIGB95I00111
26
conventional formulations well known in the art, for example as described in
standard textbooks of pharmaceutics such as the British Pharmacopoeia.
For topical application to the eye, the drug may be made up into a solution or
suspension in a suitable sterile aqueous or non aqueous vehicle. Additives,
for
instance buffers such as sodium metabisulphite os disodium edeate;
preservatives
including bactericidal and fungicidal agents such as phenyl mercuric acetate
or
nitrate, benzalkonium chloride or chlorhexidine, and thickening agents such as
hypromellose may also be included.
The dosage for topical administration will of course depend on the size of the
area
being treated. For the eyes, each dose may typically be in the range from 10
to
100mg of the drug.
The active ingredient may also be administered parenterally in a sterile
medium.
Depending on the vehicle and concentration used, the drug can either be
suspended or dissolved in the vehicle. Advantageously, adjuvants such as a
local
anaesthetic, preservativE~ and buffering agents can be dissolved in the
vehicle.
For use in the treatment of rheumatoid arthritis, the drug can be administered
by the
oral route or by injection intra-articularly into the affected joint. The
daily dosage for
a 70kg mammal may be in the range 1 Omgs to 1 gram.
The following Examples illustrate embodiments of the invention:
The amino acids used in the examples were commercially available or were
prepared according to lit~srature procedures.
The following abbreviations have been used throughout:
DIPE Diisopropyl ether
DMF N,N-Dimethylformamide
WO 95/19956 PCT/GB95/00111
27
HOBt 1-Hydroxyb~enzotriazole
LDA Lithium diisopropylamide
mCPBA m-Chlorope~rbenzoic
acid
NMM N-Methylmorpholine
THF Tetrahydrof~uran
TFA Trifluoroacetic acid
TLC Thin layer chromatography
EDC N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride
~ H and ~3C NMR spectra were recorded using a Bruker AC 250E spectrometer at
250.1 and 62.9 MHz, respectively. Elemental microanalyses were performed by
CHN Analysis Ltd. (Alpha House, Countesthorpe Road, South Wigston, Leicester
LE8 2PJ, UK) or Medac Ltd. (Department of Chemistry, Brunel University,
Uxbridge,
Middlesex UB8 3PH).
EXAMPLE 1
5-Methyl-3R-(2-phenyl-1:i-phenylcarbamoyl-ethylcarbamoyl)-hexanohydroxamic
acid
0
w H '~~~ H NH
y H ~w
CONHOH O
T P
N-(4-Methylpentanoyl)-4S.-phenyl methyl-oxazolidin-2-one
A dry 500 ml flask equippE~d with a magnetic stirrer was charged with 4S-
phenylmethyl-oxazolidin-2-one (1 i'.72 g, 100 mmol), this was capped with a
rubber
septum and flushed with nitrogen. Anhydrous THF (300 ml) was added via a
WO 95119956 ~ ~ ~ ~ ~~ ~ ~ PCTIGB95/00111
28
cannula and the resulting solution was cooled to -78°C in an
acetone/dry-ice bath.
A solution of 1.47 M n-butyllithium in hexane (68.4 ml, 101 mmol) was
transferred
via cannula to a dry, septum-stoppered 100 ml dropping funnel. This was added
dropwise to the THF solution over 10 minutes.
4-Methylvaleric acid chloride (14.80 g, 110 mmol) was added in one portion by
syringe after completion of the addition of n-butyllithium. The resulting
solution was
stirred at -78°C for 30 minutes and then allowed to warm to ambient
temperature
over 30 minutes. Excess acid chloride was quenched by the addition of aq.
ammonium chloride (60 ml) and the bulk of the solvent was removed under
reduced pressure. The resulting slurry was extracted with dichloromethane (2 x
80
ml). The combined organic extracts were washed with 1 M sodium hydroxide (75
ml), brine (75 ml), dried (;anhydrous sodium sulphate) and filtered. The
solvent was
removed to yield a yellow oil (29.20 g, including residual solvent) which was
used
directly in Step B. ~H-NMR; 8 (CDC13), 7.34 - 7.19 (5H, m), 4.73 - 4.63 (1 H,
m), 4.25
- 4.16 (2H, m), 3.30 (1 H, dd, J = 3.3 Hz), 3.05 - 2.85 (2H, m), 2.78 (1 H,
dd, J = 9.5
Hz), 1.76 - 1.53 (3H, m) and 0.9T (6H, d, J = 6.2 Hz).
TEP B:
N-(4-(tert-Butyl)-2R-isobutyl-but.an-1,4-dioyl)-4S-phenylmethyl-oxazolidin-2-
one
N-(4-Methylpentanoyl)-4S-phenylmethyl-oxazolidin-2-one (20 g, 72.6 mmol) was
placed in a dry 1 litre 3-necked flask to which was added dry THF (400 ml).
The
mixture was kept under a stream of argon and cooled to -78°C (dry
ice/acetone).
Sodium bis(trimethyl)silylamide (1 M solution in THF, 72.6 ml, 72.6 mmol) was
added dropwise through a dropping funnel. After stirring for 20 minutes, terf
butyl
bromoacetate (21.02 g, ~I5.8 ml, 109 mmol) was added dropwise over 1 minute,
to
give an orange solution. The mixture was kept at -78°C and allowed to
warm to
-50°C over 2 hours (after' which time it turned pink). The reaction was
then
quenched by adding acetic acid (10.90 g, 10.4 ml, 182 mmol) in ether (50 ml)
at
-50°C, whereupon the solution became colourless. The solvent was
removed
PC'TIGB95/00111
WO 95/19956
29
under reduced pressure and the resulting slurry was partitioned between ethyl
acetate and brine. The ethyl acetate layer was washed once with brine and the
original brine layer was back-extracted with ethyl acetate. The combined
organic
layers were dried and the solvent removed, giving a yellow oil which
crystallised on
cooling overnight to yield the title compound as a crystalline solid (21.36 g,
76%).
~H-NMR; 8 (CDC13), 7.38 - 7.24 (5H, m), 4.67 (1 H, m), 4.27 (1 H, m), 4.18 -
4.16 (2H,
m), 3.36 (1 H, dd, J = 3.3 Hz), 2.72 (1 H, dd, J = 2.3 Hz), 2.49 (1 H, dd, J =
4.6 Hz),
1.72 - 1.24 (3H, m), 1.44 (9H, s) .and 0.91 - 0.96 (6H, dd, J = 4.5 Hz).
[a]25p = + 66.9°
(c=1, MeOH).
TEP
2R-Isobutyl-butan-1,4-dioic acid-~4-ten-butyl ester
N-(4-(tent Butyl)-2R-isobutyl-but;an-1,4-dioyl)-4S-phenylmethyl-oxazolidin-2-
one
(15.30 g, 39 mmol) was placed in a 1 litre flask with a stirrer bar and to it
was added
a mixture of THF (600 ml) and water (150 ml). The solution was stirred and
cooled
to 0°C (ice/acetone bath) then 60% aq. hydrogen peroxide (4.5 ml, 157
mmol) was
added via syringe over 5 minutes, followed by lithium hydroxide (2.65 g, 63
mmol)
in 100 ml water. The reaction mixture was stirred for 1 h at 0 °C. TLC
analysis
(10% methanol in dichloromethane) showed complete reaction (product gave a
yellow spot on TLC on staining vvith bromocresol green and heating). The
reaction
mixture was quenched with sodium nitrite (10.88 g, 157 mmol), the final pH was
12-
13. THF was removed in-vacuo and the aqueous layer was extracted with
dichloromethane (3 x 200 ml) to recover the chiral auxiliary. The organic
extracts
were dried (anhydrous magnesium sulphate), filtered and the solvent removed in-
vacuo and the resulting :>olid chiral auxiliary (7.05 g, 39 mmol, 100%)
recrystallised
from ethyl acetate-hexane (2:1 ). [a]25p = - 13.0° (c=1, MeOH)
The aqueous layer was cooled in an ice bath and acidified to pH 5-6 with 2M
hydrochloric acid. The rE~sulting cloudy solution was extracted with ethyl
acetate (4
WO 95!19956 Z ~ g . PCTlGB95/00111
x 200 ml), readjusting the pH to 5-6 in between extractions. The combined
organic
extracts were dried over rnagnesiium sulphate, filtered and the solvent was
removed to yield the title compound as a pale yellow oil (8.21 g, 91%). ~H-
NMR; b
(CDC13), 2.85 (1 H, m), 2.59 (1 H, dd, J = 16, 9 Hz), 2.38 (1 H, dd, J = 16, 5
Hz), 1.64
(1 H, m), 1.43 (9H, s), 1.28 (1 H, m) and 0.93 (6H, dd, J = 7, 8 Hz). [a)25o =
+ 10.4°
(c=1, MeOH)
T P
Na-Benzyloxycarbonyl-L-phenylalanine-N-phenylamide
Na-Benzyloxycarbonyl-L-phenylalanine (4.95 g, 16.5 mmol) was dissolved in
dichloromethane (70 ml) and the solution was cooled to 0°C and stirred
during the
addition of pentafluorophenol (3.35 g, 18.2 mmol), followed by EDC (3.49 g,
18.2
mmol). The mixture was allowed to warm to room temperature, stirred for a
further
1 hour then cooled back to 0°C. ,Aniline (3.85 g, 41.4 mmol) was added
dropwise
and the mixture was warrn~ed to room temperature then stirred overnight. The
solution was washed twicE~ with 1.M sodium carbonate, twice with 1 M
hydrochloric
acid and finally with brine before drying over anhydrous magnesium sulphate.
The
solution was filtered and evaporated to a white solid which was recrystallised
from
ethyl acetate-hexane. Yielld: 2.57 g (41 %). ~ H-NMR; b (CDC13), 7.87 (1 H, br
s),
7.43 - 7.03 (15 H, br m), 5.62 (1 H, m), 5.08 (2H, s), 4.59 (1 H, s) and 3.15
(2H, s).
T P
L-Phenylalanine-N-phenylamide
Na-Benzyloxycarbonyl-L-phenylalanine-N-phenylamide (2.50 g, 6.68 mmol) was
dissolved in ethanol (20 ml) and cyclohexene (5 ml) and 10% palladium on
charcoal (250 mg) was added. The mixture was heated at reflux for 1 hour after
which time no starting material was detectable (as indicated by TLC analysis).
The
- WO 95/19956 PCT/GB95/00111
31
catalyst was removed by filtration and the solvent evaporated to leave the
title
compound contaminated with reaidual ethanol (1.74 g). ~H-NMR; 8 (CD30D), 7.45
(2H, m), 7.18 (7H, m), 7.I)4 (1 H, m), 3.56 (1 H, m), 3.04 (1 H, dd, J = 6.4,
13.3 Hz)
and 2.85 (1 H, dd, J = 7.2, 13.3 Hz).
STEP F:
5-Methyl-3R-(2-phenyl-1 S-phenylcarbamoyl-ethylcarbamoyl)-hexanoic acid tert
butyl ester
2R-Isobutyl-butan-1,4-dioic acid-4-tert-butyl ester (1.17 g, 5.11 mmol),
prepared
according to the method describE~d in STEP C, was dissolved in DMF (30 ml) and
the solution was cooled in an ice bath. HOBt (0.76 g, 5.62 mmol) and EDC (1.07
g,
5.62 mmol) were added and the reaction mixture was stirred for 90 minutes at
0°C
and 30 minutes at room temperai:ure. The mixture was cooled back to
0°C, L-
phenylalanine-N-phenylamide (1 .60 g, 6.65 mmol) was added and the reaction
mixture was allowed to warm to room temperature with stirring overnight. TLC
analysis indicated that all of the carboxylic acid precursor had been
consumed.
The solvent was removed and the residue was taken up in diethyl ether and
washed successively with water, 1 M sodium carbonate, 1 M hydrochloric acid
and
brine. The organic phasE~ was dried (anhydrous magnesium sulphate), filtered
and
evaporated to leave an o1ff-white solid. Recrystallisation from ethyl acetate-
hexane
afforded the title compound (1.23 g, 53%). ~ H-NMR; b (CDC13), 8.16 (1 H, s),
7.39
(2H, m), 7.28 (7H, m), 7.08 (1 H, m), 6.53 (1 H, d, J = 7.7 Hz), 4.82 (1 H,
dd, J = 7.2,
14.5 Hz), 3.22 (2H, m), 2.66 (1 H, m), 2.52 (1 H, dd, J = 8.6, 16.4 Hz), 2.37
(1 H, dd, J
= 4.9, 16.4 Hz), 1.45 (2H, m), 1.43 (9H, s), 1.20 (1 H, m), 0.86 (3H, d. J =
6.4 Hz) and
0.81 (3H, d, J = 6.2 Hz).
T P
5-Methyl-3R-(2-phenyl-1 ~>-phenylcarbamoyl-ethylcarbamoyl)-hexanoic acid
WO 95/19956 ~ ~ ~ PCT/GB95/00111
32
5-Methyl-3R-(2-phenyl-1 S-phenylcarbamoyl-ethylcarbamoyl)-hexanoic acid terf
butyl ester (1.22 g, 2.70 mmol) was dissolved in dichloromethane (7.5 ml) and
TFA
(7.5 ml) and the solution was stored overnight at 4°C. The solvents
were removed
in vacuo, the residue was dissohved in ethyl acetate and washed twice with
water to
remove residual TFA. The organic phase was dried (anhydrous magnesium
sulphate), filtered and evaporated to leave a foam (1.07 g, including residual
solvent). ~ H-NMR; b (CD~3 OD), i'.41 (2H, m), 7.18 (7H, m), 7.02 (1 H, m),
4.70 (1 H,
dd, J = 7.0, 8.1 Hz), 3.17 (1 H, dd, J = 7.0, 13.7 Hz), 3.03 (1 H, dd, J =
8.1, 13.7 Hz),
2.76 (1 H, m), 2.44 (1 H, dd, J = 8.4, 16.5 Hz), 2.28 (1 H, dd, J = 5.9, 16.5
Hz), 1.40
(2H, m), 1.21 (1 H, m), 0.83 (3H, d, J = 6.3 Hz) and 0.76 (3H, d, J = 6.2 Hz).
TS EP H:
O-Benzyl-5-Methyl-3R-(~?-phenyl-1 S-phenylcarbamoyl-ethylcarbamoyl)-
hexanohydroxamate
5-Methyl-3R-(2-phenyl-1 S-phenylcarbamoyl-ethylcarbamoyl)-hexanoic acid (1.00
g, 2.52 mmol) was dissolved in DMF (5 ml) and cooled to 0°C in an ice
bath. HOBt
(0.41 g, 3.03 mmol), EDC (0.58 c~, 3.03 mmol) and NMM were added and the
mixture was stirred for 1 hour at 0°C followed by a further 2 hours at
room
temperature. The reaction was cooled back to 0°C during the addition of
O-
benzylhydroxylamine (0.47 g, 3.78 mmol) and then allowed to warm to room
temperature with stirring overnight. The solvent was removed in vacuoto leave
an
oil which crystallised on stirring with diethyl ether and water. Trituration
with ethyl
acetate gave the required product (0.63 g, 50%) which was used in STEP I
without
further purification. ~H-NMR; 8 (CD3OD), 7.45 (2H, m), 7.38 -7.08 (12H, m),
7.04
(1 H, m), 4.76 (2H, m), 4.E~9 (1 H, rn), 3.19 (1-H, dd, J = 6.7, 13.8 Hz),
3.00 (1 H, dd, J =
8.6, 13.8 Hz), 2.75 (1 H, m), 2.16 (1 H, dd, J = 8.0, 14.5 Hz), 2.02 (1 H, dd,
J = 6.7,
14.5 Hz), 1.38 (2H, m), 1.18 (1 H, m), 0.81 (3H, d, J = 6.3 Hz) and 0.75 (3H,
d, J =
6.2 Hz).
T PI:
WO 95/19956 PCTIGB95100111
33
5-Methyl-3R-(2-phenyl-1 S-phenylcarbamoyl-ethylcarbamoyl)-hexanohydroxamic
acid
Hydrogen gas was bubbled through a-slurry of O-benzyl-5-Methyl-3R-(2-phenyl-
1 S-phenylcarbamoyl-ethylcarbamoyl)-hexanohydroxamate (0.62 g, 1.23 mmol)
and 10% palladium on charcoal (0.12 g) in ethanol (30 mi) for 90 minutes,
after
which time no starting material remained (as indicated by TLC analysis). The
catalyst was removed by filtration and the filtrate was evaporated to leave a
white
solid. Recrystallisation from ethanol-ethyl acetate afforded the title
compound (0.37
g, 73%). m.p. 183 - 184~'C. ~H-NMR: 8 (CD3 OD), 7.45 (2H, m), 7.20 (7H, m),
7.05
(1 H, m), 4.70 (1 H, dd, J = 7.0, 8.2 Hz), 3.17 (1 H, dd, J = 7Ø 13.7 Hz),
2.99 (1 H,
dd, J = 8.3. 13.7 Hz), 2.76 (1 H, rn), 2.16 (1 H, dd. J = 7.4, 14.5 Hz), 2.04
(1 H, dd, J
= 7.2, 14.5 Hz), 1.41 (2H, m). 1.08 (1 H. m), 0.83 (3H, d, J = 6.4 Hz) and
0.77 (3H,
d, J = 6.4 Hz). ~3C-NMR; 8 (CD3 OD), 177.3, 171.7, 170.6, 139.3, 138.5, 130.9,
129.7, 129.5, 127.7, 12:1.5, 121.8, 56.8, 42.5, 42.4, 39.0, 36.9, 26.9, 23.4
and 22.4.
Found: C 65.64, H 6.96, N 9.91 °ro; C2oH3~ N3O5 . 0.5 H20 requires: C
65.70, H 7.19,
N 9.99%.
The following additional compounds were prepared according to the methods of
Example 1:
EXAMPLE 2
3R-(2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-5-methyl-
hexanohydroxamic acid
0
H '~'~ H H
N N \
H
O /
CONHOH
WO 95/19956 ~ ~ . PCTIGB95/00111
34
White powder. m.p. 151 - 153°C. ~ H-NMR: 8 (CD30D), 7.48 (2H, d, J =
7.8 Hz),
7.26 (2H, t, J = 7.6 Hz), 7.05 {1 H, t, J = 7.3 Hz), 4.40 (1 H, s), 2.95 {1 H,
m), 2.31
{1 H, dd, J = 7.8, 14.6 Hz), 2.15 (1 H, dd, J = 6.6, 14.6 Hz), 1.51 (2H, m),
1.20 (1 H,
m), 1.03 (9H, s), 0.86 (3H, d, J = 6.4 Hz) and 0.81 (3H, d, J = 6.3 Hz). ~3C-
NMR; 8
(CD30D), 177.2, 171.2, 170.6, 139.3, 129.8, 125.5, 121.6, 62.7, 42.5, 41.9,
37.1,
35.8, 27.2, 27.0, 23.5 and 22.6. Found: C 62.86, H 8.29, N 10.71 %; C2oH3i
N304 .
0.3 H20 requires: C 62.74, H 8.:12, N 10.97%
EXAMPLE 3
3R-(2.2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-
hexanohydroxamic acid
O
H .~~~ H N N
H /
O
CONHOH
Pale grey solid. m.p. 125 - 126°C. ~ H-NMR; 8 (CD30D), 8.26 (1 H, m),
8.04 (1 H,
d), 7.72 (1 H, dt, J = 1.9, 5.5 Hz), 7.07 (1 H, m), 4.47 (1 H, s), 2.97 (1 H,
m), 2.30 (1 H,
dd, J = 7.8, 14.5 Hz), 2.'18 (1 H, dd, J = 6.6, 14.5 Hz), 1.51 (2H, m), 1.15
(1 H, m),
1.03 (9H, s), 0.86 (3H, cl, J = 6.5 Hz), and 0.80 (3H, d, J = 6.4 Hz). ~3C-
NMR; 8
(CD30D), 177.3, 171.6, 170.6, '152.6, 149.1, 139.5, 121.1, 115.7, 62.8, 42.5,
41.9,
37.0, 35.7, 27.2, 27.0, 23.5 and 22.5. Found: C 60.11, H 7.90, N 14.79%;
Ci9H3oN404 requires: C 60.30, I~ 7.99, N 14.80%.
EXAMPLE 4
3R-(2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
methylhexanohydroxamic acid
PCT/GB95/00111
WO 95/19956
O
H ~H H
N .,~ N
H I
HO~ ' CONHOH O
H
TEP A:
2S-Hydroxy-3R-isobutenyl-butan-1,4-dioic acid diisopropyl ester
2S-Hydroxybutan-1,4-dioic acid diisopropyl ester (50 g, 230 mmol) was added to
a
solution of LDA [from N,N-diisopropylamine (80 ml, 570 mmol) and 10 M n-
butyllithium (48.1 ml, 481 mmol)] in dry THF (500 ml) whilst maintaining the
temperature at -70°C. When addition was complete the reaction was
warmed to
15°C and stirred for 8 hours. ThE~ reaction mixture was cooled to -
70°C and
methallyl iodide (46 g, 252 mmol) was added slowly, ensuring that the
temperature
did not exceed -65°C. Ths~ mixture was warmed to -40°C and
stirred for 18 hours
before quenching at -15°C with citric acid. The organic layer was
separated and
washed with 10% sodium hydrogen carbonate solution (500 ml) and brine (300 ml)
then dried (anhydrous magnesiurn sulphate). The solution was filtered and
concentrated in vacuo to give a brown oil (64 g) which was purified by column
chromatography (silica gel, 1 kg, gradient elution with 20 to 35% diethyl
ether in
hexane). The desired product was isolated as a colourless oil (30.9 g, 49%)
which
was found to be a 17:1 mixture of diastereoisomers by NMR. ~ H-NMR; b (CDC13,
major diastereoisomer), 5.06 (1 H, septet, J = 6.3 Hz), 4.97 (1 H, septet, J =
6.3 Hz),
4.78 (2H, d, J = 7.1 Hz), 4.16 (1 H, m), 3.20 (1 H, d, J = 6.2 Hz), 3.00 {1 H,
m), 2.50
(1 H, dd, J = 7.0, 14.5 Hz), 2.35 (1 I~, dd, J = 8.7, 14.4 Hz), 1.72 (3H, s)
and 1.24 -
1.16 (12H, 2m).
STEP B
2S-Hydroxy-3R-isobutyl-butan-t ,4-dioic acid diisopropyl ester
WO 95/19956 ~ PCT~GB95/00111 --
36
2S-Hydroxy-3R-isobutenyl-butan-1,4-dioic acid diisopropyl ester (7.14 g, 26.2
mmol) was dissolved in ethanol (80 ml), and stirred overnight with 10%
palladium
on charcoal catalyst (1.0 g) under an atmosphere of hydrogen. The catalyst was
removed by filtration and the filtrate was evaporated to dryness to leave the
product
as a clear oil (7.03 g, 98°/<>). ~H-NMR; 8 (CDC13), 5.06 (1 H, septet,
J = 6.3 Hz), 4.97
(1 H, septet, J = 6.3 Hz), 4.17 (1 H, br s,), 3.24 (1 H, br s), 2.83 (1 H, m),
1.68 (2H, m),
1.44 (1 H, m), 1.24 (6H, d, J = 6.2 Hz), 1.t8 (6H, d, J = 6.2 Hz) and 0.89
(6H, m).
T P
2S-Hydroxy-3R-isobutyl-butan-1,4-dioic acid
2S-Hydroxy-3R-isobutyl-butan-1,4-dioic acid diisopropyl ester (7.0 g, 25.6
mmol)
was dissolved in dioxane (15 ml) and water (15 ml), a solution of potassium
hydroxide (4.29 g) in water (22 ml) was added and the mixture was heated at
90°C
overnight. The solution was allovved to cool and then passed through an ion
exchange resin (Dowex 50X4-400, 200 ml) and evaporated to yield the title
compound (4.82 g, 99%). ~H-NMR; 8 (CDC13), 8.70 (2H, br s), 4.32 (1 H, br s),
3.10
(1 H, m), 1.85 - 1.55 (3H, m) and 1).96 (6H, m).
T P
2R-(2,2-Dimethyl-4-oxo-1,3-dioxalan-5S-yl)-4-methylpentanoic acid
2S-Hydroxy-3R-isobutyl-butan-1,4-dioic acid (5.19 g, 27.3 mmol) was dissolved
in
2,2-dimethoxypropane (150 ml) and DMF (40 ml) and stirred overnight at
30°C in
the presence of a catalytic amount of p-toluene sulphonic acid. The solvent
was
removed to give the title compound contaminated with solvent (6.87 g, crude).
~H-
NMR; 8 (CDC13), 4.41 (1 H, d, J = 4.8 Hz), 2.91 (1 H, m), 1.69 (3H, m), 1.54
(3H, s),
1.48 (3H, s) and 0.88 (6H, m).
PCTlGB95/00111
WO 95/19956
37
TEP
2R-(2,2-Dimethyl-4-oxo-1,3-diox:alan-5S-yl)-4-methyl pentanoic acid
pentafluorophenyl ester -
2R-(2,2-Dimethyl-4-oxo-'1,3-dioxalan-5S-yl)-4-methylpentanoic acid (558 mg,
2.4
mmol) was taken up in dichloronnethane (10 ml) and cooled to 0°C before
adding
pentafluorophenol (670 mg, 3.6 mmol) and EDC (560 mg, 2.9 mmol). The reaction
was stirred at 0°C for 2 hours thE~n the solution was washed with 1 M
sodium
carbonate (50 ml) and brine (20 ml). The organic layer was dried (magnesium
sulphate), filtered, evaporated to dryness and purified by column
chromatography
(silica gel, dichloromethane} to dive the activated ester (552 mg, 58%). ~ H-
NMR; 8
(CDC13), 4.57 (1 H, d, J = ~5.5 Hz), 3.32 (1 H, m), 1.86 (3H, m), 1.67 (3H,
s), 1.58 (3H,
s) and 1.03 (6H, m).
T P F:
L-tert-leucine-N-phenylarnide
The title compound was prepared from N-benzyloxycarbonyl-L-teri-leucine by
methods analogous to those described in Example 1 (STEPS D and E). ~ H-NMR;
8 (CDC13), 7.53 (2H, m), 7.28 (2H, m), 7.06 (1 H, m), 3.13 (1 H, s) and 1.00
(9H, s).
T P
N2-[2R-(2,2-Dimethyl-4-o:KO-1,3-clioxalan-5S-yl)-4-methylpentanoyl)-L-terf
leucine-
N-phenylamide
2R-(2,2-Dimethyl-4-oxo-1,3-dioxalan-5S-yl)-4-methylpentanoic acid
pentafluorophenyl ester (1.72 g, 5.77 mmol) and L-tert leucine-N-phenylamide
(1.25 g, 6.06 mmol) were dissolved in DMF (150 ml) and the mixture was stirred
WO 95/19956 ~ 1 . ~ PCTlGB95/00111 ---
38
overnight at room temperature. The solvent was removed to give an oil which
dissolved in diethyl ether and the solution was washed twice with 1 M sodium
carbonate and then with brine. 'The organic phase was dried (anhydrous
magnesium sulphate), filtered and evaporated to an oil. Crystallisation from
ethyl
acetate-hexane afforded the desired product as a white solid (1.55 g, 64%). ~H-
NMR; 8 (CDC13), 8.53 (1 H, s), 7.49 (2H, m), 7.26 {2H, m), 7.09 (1 H, m), 7.00
(1 H, d),
4.58 (1 H, d), 4.52 {1 H, d), 2.84 ('I H, m), 1.78 - 1.47 (3H, br m), 1.64
(3H, s), 1.54
(3H, s), 1.09 (9H, s), 0.88 (.3H, d, J = 5.9 Hz) and 0.83 (3H, d, J = 6.0 Hz).
STEP H:
3R-(2,2-Dimethyl-1 S-phenyicarbamoyl-propylcarbamoyl)-2S-hydroxy-5-methyl-1-
hexanohydroxamic acid
To a solution of hydroxylamine hydrochloride (0.93 g, 13.36 mmol) in methanol
(10
ml) was added sodium methoxide (0.72 g, 13.36 mmol) and the mixture was
stirred
at room temperature for .2 hours, after which time the precipitated solid was
removed by filtration. The filtrate was cooled in an ice bath prior to the
addition of
N2-[2R-(2,2-dimethyl-4-oxo-1,3-~dioxalan-5S-yl)-4-methylpentanoyl)-L-tert
leucine-
N-phenylamide (1.40 g, 3.34 mmol) which was added in portions. The mixture was
stirred at 0°C for 10 minutes then allowed to warm to room temperature
and stirred
overnight. The solvent v~ras removed under reduced pressure and the residue
was
purified by column chromatography {acid washed silica, 5% methanol in
dichloromethane) followE~d by crystallisation from ethyl acetate-hexane.
Yield: 0.89
g (68%). m.p. 122 - 124°C. ~ H-NMR; 8 (CD30D), 7.51 (2H, d, J = 7.8
Hz), 7.27
(2H, m), 7.06 (1 H, m), 4.40 (1 H, s), 4.02 (1 H, d, J = 6.1 Hz), 2.86 (1 H,
m), 1.64
(1 H, m), 1.51 (1 H, m), 1.26 (1 H, m), 1.03-(9H, s), 0.89 (3H, d, J = 6.4
Hz), and
0.84 (3H, d, J = 6.4 Hz). ~3C-NMR; 8 {(CD3)ZSO), 172.6, 169.2, 168.8, 138.7,
128.6, 123.3, 119.3, 71.4., 60.5, 47.7, 37.3, 34.5. 26.6, 25.3, 23.5 and 21.8.
Found:
C 60.08, H 7.97, N 10.44% ; C2oH3~ N305 . 0.4 H20 requires: C 59.95, H 8.00, N
10.49%.
PCTIGB95/00111
WO 95/19956
39
The following additional compounds were prepared according to the methods of
Example 4:
EX-AMPLE 5
2S-Hydroxy-3R-(3-methyl-1 S-napth-2-ylcarbamoyl-butylcarbamoyl)-5-methyl-
hexanohydroxamic acid
0
H _~~~ H
~~ N NH
H I
HO- ' CONHOH O
H
Off-white crystalline solid. m.p. 186°C. ~H-NMR; b (CD30D), 8.24 (0.5H,
d, J = 7.8
Hz), 8.15 (1 H, s), 7.80 - 7.64 (3h1, m), 7.54 (1 H, br d, J = 7.4 Hz), 7.43 -
7.29 (2H,
m), 4.70 - 4.50 (1 H, m), 4.03 (1 HI, d, J = 6.8 Hz), 2.90 - 2.74 (1 H, m),
1.83 - 1.41
(5H, m), 1.27 - 1.10 (1 H, m), 0.9!5 (3H, d, J = 5.2 Hz), 0.93 (3H, d, J = 5.4
Hz), 0.89
(3H, d, J = 6.4 Hz), and 0.82 (3H, d, J = 6.4 Hz). ~3C-NMR; 8 (CD30D), 176.0,
173.3, 171.5, 161.7, 136.9, 135.1, 132.1, 129.5, 128.5, 127.4, 126.0, 121.4,
118.2,
73.1, 54.1, 54.0, 41.8, 39..1, 26.9, 25.8, 23.8, 23.6, 22.2 and 22Ø I R; vm~
(KBr),
3422, 2917, 2850, 2363 and 1636.
WO 95119956 PCTIGB95/00111
21~~~~7t~
EXAMPLE 6
2S-Hydroxy-3R-(3-methyl-1 S-(4-methoxyphenyl)carbamoyl-butylcarbamoyf)-5-
methyl-hexanohydroxamic acid
O
,,A H NH
N
. " o ~ I
HO - CONHOH OMe
H
White powder. m.p. 192°C. ~H-NMR: b (CD30D), 7.33 (2H, d, J = 8.8 Hz),
7.30 -
7.10 (5H, m), 6.80 (2H, d, J = 8.E! Hz), 4.67 (1 H, br dd 7.4, J = 7.3 Hz),
4.01 (1 H, d,
J = 5.9 Hz), 3.72 (3H, s)., 3.19 (1 H, dd. J = 6.3, 13.6 Hz), 3.02 (1 H, dd, J
= 8.1, 13.9
Hz), 2.79 - 2.60 (1 H, m), 1.58 - 'I .40 (1 H, m), 1.40 - 1.19 (1 H, m), 1.20 -
1.06 (1 H,
m), 0.80 (3H, d, J = 6.5 i-Iz) and 0.77 (3H, d, J = 6.6 Hz). ~3C-NMR; 8
(CD30D),
175.6, 171.5, 158.2, 138..4, 132.1, 130.4, 129.5, 127.8, 123.6, 114.8, 72.9,
56.7,
55.8, 39.2, 39.0, 26.7, 2~~.8 and ;?2.1. IR; vm~ (KBr), 3420, 2343, 1653 and
1636
cm-~ .
EXAMPLE 7
3R-(2,2-Dimethyl-1 S-(4-tnrtbutyl~~2,6-dimethylphenyl)carbamoyl-
propylcarbamoyl)-
2S-hydroxy-5-methylhexanohydnoxamic acid
O Me
H ~ 'H H
N .,~ N
H I
O
HO~~ CONHOH Me ~ tBu
H
PCT/GB95100111
WO 95119956
41
White solid. m.p. 209 - 210°C. ~ I-i-NMR; b (CD30D), 7.07 (2H, s), 4.53
(1 H, s),
3.98 (1 H, d, J = 7.2Hz), ;?.87 (1 HI, m), 2.16 (6H, s), 1.64 (1 H, m), 1.51
(1 H, m),
1.25 (9H, s), 1.16 (1 H, m), 1.10 (9H, s), and 0.87 (6H, m). ~3C-NMR; 8
(CD30D),
175.5, 171.8, 171.7, 151.4, 136.2, 132.7, 126.2, 73.3, 62.1, 39.4, 35.6, 35.1,
31.7,
27.4, 26.9, 24.0, 22.2 and 19.3. Found: C 64.23, H 8.91, N 8.69% ; C26H~N305 .
0.5 H20 requires: C 64.17, H 9.11, N 8.63%.
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
methylhexanohydroxamic acid
O
H ,,,.v H N N
H ( \
HO~ - CONHOH O
H
White powder. m.p. 132..5 - 134.5 °C. ~ H NMR; 8 (CDC13), 9.97 (1 H,
s), 9.52 (1 H,
s), 8.73 (1 H, d, J =11.0 f-Iz), 8.18 (2H, m), 7.81 (1 H, m), 7.13 (1 H, m),
4.78 (1 H, d,
J =10.9 Hz), 4.19 (1 H, m), 2.95 ('I H, m), 2.00 (1 H, m), 1.53 (1 H, m), 1.42
(1 H, m),
1.10 (9H, s), 0.86 (3H, d, J = 6.2 Hz) and 0.85 (3H, d, J = 6.1 Hz). ~3C NMR;
8
(CDC13), 174.4, 170.1, 1 fi9.0, 152.0, 146.3, 139.9, 120.1, 116.2, 73.8, 63.2,
42.0,
39.8, 34.8, 27.0, 25.8, 23.0 and 21.8. Found: C 57.33, H 7.53, N 13.84% ;
C26H~N305 . 0.2 H20 requires: C 57.33, H 7.70, N 14.07%.
WO 95/19956 ~ ~ . PCTIGB95/00111 ...
42
EXAMPLE 9
3R-(2,2-Dimethyl-1 S-(4-methoxyphenyl)carbamoyl-propylcarbamoyt)-2S-hydroxy-
5-methylhexanohydroxarnic acid
O
H ~ .,av H N
H
HO 'CONHOH O ~ OMe
H
Pale brown solid. m.p. 118-120°C. ~H NMR; 8 (CD30D), 7.39 (2H, d, J =
9.4 Hz),
6.81 (2H, d. J = 9.4 Hz), 4.37 (1 H, s), 4.15 (1 H. d, J = 6.9 Hz), 3.73 (3H,
s), 2.91 -
2.79 (1 H, m), 1.70 - 1.44 (2H, m), 1.33 - 1.13 (1 H, m), 1.03 (9H, s), 0.89
(3H, d, J =
6.3 Hz), and 0.85 (3H, d, J = 6.3 Hz). ~3C NMR; 8 (CD30D), 175.6, 171.5,
171.0,
158.2, 132.1, 123.6, 115.0, 73.1, 62.4, 55.9, 39.7, 35.9, 30.9, 27.2, 27.0,
23.6 and
22.4. IR, vmax (KBr) 341!3, 2923, 2361, 1654, 1512, 1458, 1245, 1036 cm-~.
Found:
C 57.97%, H 7.73%, N 9.31 %; C~2~ H3gNgOg . 0.6 H20 requires C 58.08%, H
7.94%,
N 9.67%.
EXAMPLE 10
3R-(2,2-Dimethyl-1 S-pyridin-4-ylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
methylhexanohydroxamic acid
PCT/GB95/00111
WO 95119956
43
O
H , ~~ H H
N , N
H O ~ iN
HO CO_NHOH
H
White solid. m.p 120 - 1 c'S°C. ~ H-NMR; 8 ((CD3)2S0, 9:1 mixture
of
diastereoisomers), 10.62 (1 H, s), 10.59 (0.9H, s), 10.43 (0.1 H, s), 8.40
(2H, d, J =
6.2 Hz), 7.80 (0.9H, d, J =: 8.6 Hz}, 7.76 (0.1 H, d, J = 6.3 Hz), 7.60 (2H,
d, J = 6.2
Hz), 5.60 (0.1 H, s), 5.29 (0.9H, s), 4.44 (0.9H, d, J = 8.7 Hz), 4.31 (0.1 H,
d, J = 8.5
Hz), 3.72 (1 H, br d), 2.90 (1 H, m),. 1.49 (3H, m), 1.04 (9H, s) and 0.81
(6H, m).
~3C-NMR; 8 ((CD3)2S0, !a:1 mixture of diastereomers), 173.5, 172.9, 170.7,
170.6,
168.7, 150.1, 150.0, 145..3, 145.:3, 113.5, 113.2, 71.2, 61.0, 60.8, 47.5,
46.6, 37.2,
36.1, 34.3, 34.1, 26.6, 26.4, 25.2, 23.8, 23.4, 21.7 and 21.4.
EXAMPLE 11
3R-(2,2-Dimethyl-1 S-pyridin-3-ylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
methylhexanohydroxamic; acid
O
''~~ H N
~N
i: O ~ /
HO CONHOH
I~
White solid. m.p 108-112°C. ~ H-IVMR; 8 ((CD3)2S0), 10.60 (1 H, s),
10.26 (1 H, s),
8.90 (1 H, s), 8.74 (1 H, s), 8.26 (1 H, d, J = 4.4 Hz), 8.05 (1 H, d, J = 8.2
Hz), 7.77
(1 H, d, J = 8.7 Hz), 7.36 (1 H, m), 5.32 (1 H, d, J = 8.2 Hz), 4.45 (1 H, d,
J = 8.9 Hz),
3.75 (1 H, m), 2.81 (1 H, m), 1.40 (3H, m), 0.98 (9H, s) and 0.80 (6H, dd, J =
12.5,
6.2 Hz). ~3C-NMR; 8 (CD;3)2S0, '172.7, 169.8, 168.7, 144.2, 140.9, 135.3,
126.2,
WO 95119956 ~ ~ PCT/GB95/00111
44
123.5, 71.2, 60.5, 47.6, 3'7.3, 34.3, 26.5, 25.3, 23.4 and 21.7. Found: C
54.46, H
7.46, N 13.42%; C~9H3oN40$ . 1.2 H20 requires C 54.85, H 7.85, N 13.46%.
EXAMPLE 12
3R-(2,2-Dimethyl-1 S-(4-hydroxyphenyl)carbamoyl-propylcarbamoyf)-2S-hydroxy-
5-methylhexanohydroxamic acid
O
H ~ ,~~~~ H H
N N
H I
HO _ ~CONHOH O ~ QH
H
White solid. m.p. 138-147°C. ~ H-NMR; 8 ((CD3)2S0), 10.59 (1 H, s),
9.71 (1 H, s),
9.17 (1 H, s), 8.88 (1 H, s).. 7.61 (1 H, d, J = 9.2 Hz), 7.36 (2H, d, J = 8.7
Hz), 6.69
(2H, d, J = 8.7 Hz), 5.29 (1 H, d, J = 8.7 Hz), 4.40 (1 H, d, J = 9.2 Hz),
2.80 (1 H, m),
1.49 (2H, m), 1.29 (1 H, m), 0.95 (9H, s) and 0.80 (3H, d, J = 6.4 Hz) and
0.75 (3H,
d, J = 6.4 Hz). ~3C-NMR; 8 ((CD,3)2S0), 172.4, 168.7, 168.4, 153.3, 130.3,
121.1,
114.9, 71.3, 60.3, 47.7, 37.2, 34.7, 26.6, 25.3, 23.4 and 21.7. Found: C
57.28%, H
7.67%, N 10.17%; C2oH3~ N306 . ().5 H20 requires C 57.40%, H 7.71 %, N 10.04%.
EXAMPLE 13
3R-(2,2-Dimethyl-1 S-(3-methoxyphenyl)carbamoyl-propylcarbamoyl)-2S-hydroxy-
5-methylhexanohydroxamic acid
PCT/GB95100111
WO 95/19956
O
H ~ _~~~ H H
N N ~ OMe
H I
HO CONHOH O
H
White solid. m.p. 97 - 102°C. ~ H-NMR; 8 ((CD3)2S0), 10.58 (1 H, s),
9.96 (1 H, s),
8.87 (1 H, s), 7.67 (1 H, d, J = 9.2 Hz), 7.29 (1 H, s), 7.20 (2H, m), 6.64 (1
H, d, J = 7.8
Hz), 5.29 (1 H, d, J = 7.9 Hz), 4.43 (1 H, d, J = 9 Hz), 3.72 (3H, s), 2.77 (1
H, m}, 1.50
(3H, m), 0.97 (9H, s), 0.81 (3H, d, J = 6.4 Hz) and 0.78 (3H, d, J = 6.4 Hz).
~3C-NMR; b ((CD3)ZSO), 172.6, 169.3, 168.7, 159.4, 139.8, 129.4, 111.6, 108.6,
105.2, 71.3, 60.5, 54.9. 47.6, 37.3. 34.4, 26.5. 25.3. 23.4 and 21.8. Found: C
57.96%, H 7.58% N 9.39%; C2~ H33N3~6 . 0.7 H20 requires C 57.84%, H 7.95%, N
9.63%.
EXAMPLE 14
3R-(2-Benzylthio-2-methyl-1 S-(pyridin-2-ylcarbamoyl)propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxa~mic acid
/I
S
O
H ,,,~~ H N N
H_ I w
HO~- CONHOHO /
H
White foam. m.p. 112 - 115°C. ~ H-NMR; 8 (CD30D), 8.26 (1 H, m), 8.06
(1 H, d, J =
8.4 Hz), 7.73 (1 H, m), 7.'I 1-7.29 (5H, m), 7.07 (1 H, m), 4.68 (1 H, s),
4.15 (1 H, d, J =
WO 95/19956 2 ~ g 1 =) ~ ~ PCTIGB95100111
46
4.7 Hz), 3.80 (2H, m), 2.93 (1 H, m), 1.55-1.71 (1 H, m), 1.48 (3H, s), 1.44
(3H, s),
1.47-1.23 (2H, br m), 0.90 (3H, d, J = 6.4 Hz) and 0.84 (3H, d, J = 6.3 Hz).
~3C-NMR; 8 (CD30D), 1'75.7, 171.7, 170.5, 152.3, 149.0, 139.8, 138.8, 130.3,
129.4, 128.0, 121.4, 115.9, 73.0, 61.4, 49.5, 49.3, 39.8, 34.2, 32.8, 26.9,
26.9,
26.2, 23.6 and 22.4. Found: C 59.78%, H 6.97%, N 10.86%; C25H34N4O5S
requires C 59.74%, H 6.E32% N 11.15%
EXAMPLE 15
3R-(2,2-Dimethyl-1 S-(thiazol-2-ylcarbamoyl)-propylcarbamoyl)-2S-hydroxy-5-
methylhexanohydroxamic acid
O
H .,~~~ H H
N~S
. _: O N
HO CONHOH
H
White solid. m.p. 125-128°C. ~ H-NMR; 8 ((CD3)2S0), 10.60 (1 H, s},
8.86 (1 H, s),
7.80 (1 H, d, J = 8.4 Hz), 7.47 (1 H, d, J = 3.5 Hz), 7.22 (1 H, d, J = 3.5
Hz), 5.22 (1 H,
d, J = 7.9 Hz), 4.52 (1 H, d, J = 8.3 Hz), 3.74 (1 H, m), 2.83 (1 H, m), 1.45
(3H, m), 0.96
(9H, s) 0.79 (3H, d, J = 6.2 Hz) and 0.72 (3H, d, J = 6.2 Hz). ~3C-NMR; b
((CD3)2S0), 172.9, 169.3, 168.7, 157.4, 137.6, 113.5, 71.3, 59.9, 47.4, 37.2,
34.1,
26.4, 25.2, 23.5 and 21.7.
EXAMPLE 16
3R-(2,2-Dimethyl-1 S-phE~nylcarb~amoyl-propylcarbamoyl)-2S-hydroxy-5-
phenylhexanohydroxamic acid
pGT/GB95/00111
WO 95/19956
47
O
_H - ,,.w H H
N N
H
/ .: O /
HO CONHOH
H
Mixture of diastereoisomers (4:1, SRS:RRS).
White solid. m.p. 98 °C. ~ H-NMR; b (CD30D) 7.55 - 7.47 (2H, m), 7.28 -
7.21 (2H,
m), 7.12 - 7.01 (6H, m), 4.47 - 4.44 (0.8H, m), 4.39 - 4.35 (0.2H, m), 4.26
(0.2H, d,
J = 4.8 Hz), 4.06 (0.8H, d, J = 6.5Hz), 2.87 - 2.83 (1 H, m), 2.57 - 2.48 (2H,
m),
1.81 - 1.52 (4H, m), and 'I .03 (9H, s). ~3C-NMR; 8 (CD30D), 175.7, 171.5,
171.3,
143.3, 139.2, 129.6, 129.4, 129.3, 126.7, 125.5, 121.8, 73.0, 70.1, 62.6,
51.0, 36.7,
35.6, 30.3, 27.3 and 23.1. Found: C 65.05, H 7.57, N 8.78%; C25HaaNsOs . 4.3
H20
requires C 65.06%, H 7.5'7%, N 8.78%.
EXAMPLE 17
3R-(2,2-Dimethyl-1 S-(4,5-dimethylthiazol-2-ylcarbamoyl)-propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxarnic acid
O
H ~ ~~~ H H
N S
N
H
HO - ~CONHOH O N
H
White solid. m.p. 191.5 - 192°C. ~ H-NMR; b ((CD3)2S0), 11.84 (1 H,
s), 10.62
WO 95/19956 ~ ~ ~ ~ y ~ ~ PCT/GB95100111
48
(1 H, s), 8.87 (1 H, s), 7.T3 (1 H, d, J = 8.5 Hz), 5.23 (1 H, d, J = 7.9 Hz),
4.48 (1 H, d,
J = 8.5 Hz), 3.70 (1 H, m), 2.79 (1 H, m), 2.49 (3H, s), 2.22 (3H, s), 1.41
(2H, m),
0.94 (10H, s and br m), 0.72 (3H,. d, J = 6.2 Hz) and 0.78 (3H, d, J = 6.2
Hz).
~3C-NMR; S ((CD3)pS0), 171.5, 167.6, 167.4, 151.7, 140.4, 117.4, 70.1, 58.6,
46.2, 35.9, 32.9, 25.1, 24.0, 22.2, 20.5, 12.9 and 8.9. IR (KBr disk), vm~
1654 and
1535 cm-~ .
EXAMPLE 18
3R-(2,2-Dimethyl-1 S-(4-~ethoxycarbonylmethylthiazol-2-ylcarbamoyl)-propyl-
carbamoyl)-2S-hydroxy-5-methylhexanohydroxamic acid
O
H ~ .~~~~H H
N~S
HO CONHOH O N
H
C02Et
White solid. m.p. 104.5 - 110.5°C. ~ H-NMR; 8 ((CD3)2S0), 12.17 (1 H,
s), 10.61
(1 H, s), 8.87 (1 H, s), 7.T5 (1 H, cl, J = 7.9 Hz), 6.97 (1 H, s), 5.23 (1 H,
d, J = 7.9 Hz),
4.48 (1 H, d, J = 8.3 Hz), 4.06 (2H, q, J = 7.2 Hz), 3.67 (3H, m), 2.85 (1 H,
m), 1.40
(2H, m), 1.18 (3H, t, J = 'l.0 Hz), 0.96 (10H, s and br m), 0.78 (3H, d, J =
6.3 Hz)
and 0.72 (3H, d, J = 6.3 Hz). ~3C;-NMR; 8 ((CD3)2S0), 182.4, 178.9, 178.2,
166.6, 153.2, 119.9, 80.9, 69.8, X69.6, 56.9, 46.1, 45.9, 43.6, 35.9, 34.7,
33.0, 31.2
and 23.6. IR (KBr disc); vmaX 1735, 1644, 1549 cm-~. Found: C 51.44, H 7.06, N
11.14%; C2~ H34N40~S . ~0.2 H20 requires C 51.46, H 7.07, N 11.43%.
0 PCT/GB95100111
WO 95/19956
49
EXAMPLE 19
3R-(2,2-Dimethyl-1 S-(5-bromo-l;hiazol-2-ylcarbamoyl)-propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxamic acid
O
,~~~ H H
~N N
H II S Br
y O N
HO CONHOH
H
White solid. m.p. 196 - 198°C. ~ H-NMR; b ((CD3)2S0), 8.74 (1 H, s),
7.70 (1 H, d,
J = 8.2 Hz), 7.41 (1 H, s), 5.09 (1 H, d, J = 6.3 Hz), 4.36 (1 H, d. J = 8.2
Hz), 3.63
3.52 (1 H, m), 2.73 - 2.60 (1 H, m), 1.35 - 1.14 (2H, m), 0.93-0.72 (1 H, m),
0.81 (9H,
s), 0.65 (3H, d, J = 6.3 t-Iz) and 0.58 (3H, d, J = 6.3 Hz). ~3C-NMR; b
((CD3)2S0),
173.1, 170.0, 168.7, 157.7, 138.7, 101.7, 67.3, 59.9, 47.4, 37.2, 34.0, 26.4,
25.3,
23.5 and 21.8. IR (KBr ~jisc); v~,ax 3236, 2966, 1659 and 1534 cm-~.
EXAMPLE 20
3R-(2,2-Dimethyl-1 S-(4-phenyl-thiazol-2-ylcarbamoyl)-propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxamic acid
O
H .~~~~H H
N~S
HO ~ - CON HOH O N
H Ph
White solid. m.p. 158 - 1 ~60°C. ~H-NMR; b ((CD3)2S0), 8.74 (1 H, s),
7.80-7.64
WO 95/19956 ~ ~ $ PCT~GB95100111
(3H, m), 7.45 (1 H, s), 7.33-7.12 (3H, m), 4.42 (1 H, d, J = 8.3 Hz), 5.39 (1
H, d, J =
8.6 Hz), 2.77-2.63 (1 H, rn), 1.35/-1.16 (2H, m), 0.84 (9H, s), 0.94-0.73 (1
H, m),
0.67 (3H, d, J = 6.4 Hz) and 0.59 (3H, d, J = 6.4 Hz). ~3C-NMR; b ((CD3)2S0),
173.0, 169.8, 168.8, 157.4, 148.9, 134.3, 128.7, 127.7, 125.7, 108.1, 71.4,
60.1,
47.5, 37.2, 34.1, 26.5, 25.3, 23.5 and 21.8. IR (KBr disc); vmax 3310, 2956,
1653,
1541 cm-~. Found: C 55.69, H 6.82, N 11.15%; C23H32N4O5S . 1.1 H20 requires C
55.65, H 6.94, N 11.29%.
EXAMPLE 21
3R-(2,2-Dimethyl-1 S-(5-methyl- t ,3,4-thiadiazol-2-ylcarbamoyl)-
propylcarbamoyl)-
2S-hydroxy-5-methyfhexanohyd~roxamic acid
O
.~~~H H
N S
N
H
HO '~CONHOH O N1 N
H
White solid. m.p. 159 - 161 °C. ~ H-NMR; 8 ((CD3)2S0), 8.73 (1 H, m),
7.69 (1 H, d, J
= 8.2 Hz), 5.10 (1 H, m), 4..38 (1 H, d, J = 8.0 Hz), 3.58 (1 H, m), 2.69 (1
H, m), 2.45
(3H, s), 1.36 - 1.12 (2H, nn), 0.94 - 0.73 1 H, m), 0.82 (9H, s), 0.65 (3H, d,
J = 6.3 Hz)
and 0.58 (3H, d, J = 6.3 Hz). ~3C-NMR; S ((C03)2S0), 173.1, 169.6, 159.4,
157.9,
71.3, 60.2, 47.4, 37.2, 34.0, 28.4, 25.3, 23.5, 21.8 and 14.7. IR (KBr disc);
vmax
3260, 2959, 1655, 1540 .and 1311 cm-~. Found: C 46.71, H 7.24 N 16.02%;
C»H29N505S . 1.2 H20 requires C 46.71, H 7.24, N 16.02%.
~ ~ s ~. ~ 7 a pCT1GB95100111
WO 95119956
51
EXAMPLE 22
3R-(2,2-Dimethyl-1 S-(4-teri butylthiadiazol-2-ylcarbamoyl)-propylcarbamoyl)-
2S-
hydroxy-5-methylhexanohydroxamic acid
O
w ?~ ''~~ H N
N ~S
H O N
HO . CONHOH
H
White solid. m.p. 169 - 171 °C. ~ f~-NMR; 8 ((CD3)2S0), 8.73 (1 H, s),
7.60 (1 H, d,
J = 8.3 Hz), 6.58 (1 H, s), 5.09 (1 H, d. J = 7.9 Hz), 4.34 (1 H, d, J = 8.3
Hz), 3.56
(1 H, dd, J = 8.3 Hz), 3.74 - 2.60 (1 H, m), 1.39 - 1.03 (2H, m), 1.11 (9H,
s), 0.92 -
0.70 (1 H, m), 0.82 (9H, s), 0.65 (3H, d, J = 6.3 Hz) and 0.58 (3H, d, J = 6.3
Hz).
~3C-NMR; 8 ((CD3)2S0), 172.9, 169.4, 168.7, 160.2, 156.7, 104.7, 71.4, 60.1,
47.4, 37.2, 34.1, 34.0, 29.13, 26.5, 25.2, 23.6 and 21.7. IR (KBr disc); vm~
3270,
2962, 1668, 1557, 1368, 1270 cm-~. Found: C 53.74, H 8.18, N 11.45%;
C2~ H36N405S. 0.9 H20 requires C 53.35, H 8.06, N 11.85%.
R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-propen-
2-yl-hexanohydroxamic acrid
o
E~ ,,.v H H
N ~N
N
H O
CONHOH
H
WO 95119956 ~, PCT/GB95100111
52
T P A:
3R,S-Allyl-2R-isobutyl-butan-1,4-dioic acid-4-tert-butyl ester (1:9, RS:RR)
To a stirred solution of 2R-isobutyl-1,4-dioic acid-4-tent butyl ester (5 g,
21.7 mmol)
in dry THF (100 ml), und~ar an argon atmosphere, at -78°C, was added
1.5M LDA
(31.8 ml, 47.7 mmol) dropwise via cannula. After stirring the solution at -
78°C for 1
hour, allyl bromide (2.44 ml, 28.2 mmol) was added dropwise via syringe. The
resulting solution was allowed to warm to room temperature over a 2 hour
period.
Methanol (10 ml) was added ands the solution stirred at room temperature.
After 30
minutes the reaction mixture was concentrated under reduced pressure. The
residue was taken up in dichloromethane (100 ml) and washed with 1 M
hydrochloric acid (100 mI~) and brine (100 ml). The dichloromethane layer was
dried over anhydrous ma~gnesiurn sulphate, filtered and solvent removed under
reduced pressure to give the title compound as a golden oil (5.6 g, 97%) (1:9,
RS:RR) ~H-NMR; 8 (CDC;13, major diastereoisomer), 5.78 - 5.63 (1 H, m), 5.01 -
5.11
(2H, m), 2.57 - 2.72 (2H, rn), 2.37 (2H, m), 1.52 -1.67 (2H, m), 1.42 (9H, s),
1.37
(1 H, m) and 0.90 (6H, d, .J = 6.3 Hz). ~3C-NMR; S (CDC13, major
diastereoisomer)
181.1, 172.9, 134.6, 117.3, 81.2, 47.8, 44.3, 38.4, 27.9, 25.9, 23.5, and
21.5.
STEP B:
3R,S-Allyl-2R-isobutyl-1,4.-dioic acid-4-terf butyl ester (3:1, RS:RR)
(i) To a stirred solution of 3R.,S-allyl-2R-isobutyl-1,4-dioic acid-4-tert-
butyl ester
(1:9, RS:RR) (5.11 g, 18.51 mmol) in dry THF (100 ml) under argon at -
78°C was
added 1.5M LDA (27.7 ml, 41.6 mmol) via cannula. The reaction mixture was
warmed to room temperature over a 2 hour period then cooled back to -
78°C and
methanol (8 ml) was addE~d via syringe. The reaction was then allowed to warm
to
room temperature for a further 2 hours. The solvent was removed under reduced
pressure. The residue was taken up in dichloromethane (150 ml) and washed with
WO 95/19956 PCT/GB95/00111
53
1 M hydrochloric acid (150 ml) and brine (150 ml). The dichloromethane layer
was
dried over anhydrous magnesium sulphate, filtered and the solvent removed
under
reduced pressure to yield the title compound (3:2, RS:RR), as a brown oil (4.7
g,
92%).
(ii) Utilising the epimerisation procedure described in Example 23B (i), but
employing a reaction temperature of -78°C after addition of LDA in lieu
of allowing
the reaction mixture to warm to room temperature yielded the title compound,
as
the major diastereoisomer as a brown oil (4.6 g, 98%) (3:1, RS:RR). ~ H-NMR; 8
(CDC13, major diastereoisomer), 11.60 (1 H, br s), 5.75 - 5.61 (1 H, br m),
5.06 - 4.96
(2H, br m), 2.70 - 2.52 (2'H, br m), 2.36 - 2.19 (2H, br m), 1.65 - 1.44 (2H,
br m), 1.40
(9H, s), 1.13 (1 H, m) and 0.86 (E',H, dd, J = 4.4, 2.1 Hz). ~3C-NMR; b
(CDC13, major
diastereoisomer) 180.7, 172.2, 134.6. 1 17.1, 81Ø 48.6, 45.7, 38.9, 34.8,
33.4, 27.9,
26.2 and 21.2.
TEP
3R,S-Allyl-2R-isobutyl-1,4-dioic acid-1-pentafluorophenyl-4 tent-butyl diester
(3:1,
RS:RR)
To a stirred solution of 3R,S-allyl-2R-isobutyl-1,4-dioic acid-4 ten-butyl
diester (4.60
g, 17.2 mmol) (3:1, RS:FIR) in dichloromethane (50 ml) was added
pentafluorophenol (6.13 g, 33.3 mmol). The reaction mixture was cooled to
0°C
and NMM (2.02 g, 20.0 nnmol) and EDC (3.94 g, 20.0 mmol) were added. The
reaction mixture was allowed to warm to room temperature and stirred for 12
hours.
The solvent was removed under reduced pressure. The residue was taken up in
dichloromethane (50 ml) and washed with 1 M hydrochloric acid (3 x 50 ml),
saturated sodium hydrogen carbonate (3 x 50 ml) and brine (50 ml). The
dichloromethane layer was dried over anhydrous magnesium sulphate, filtered
and
the solvent removed under reduced pressure to give a brown oil. Column
chromatography (silica gel, dichloromethane) yielded the title compound as a
WO 95/19956 ~ ~ ~ PCT/GB95100111
54
golden oil (5.47 g, 74%) (3:1, RS:SR). b (COC13, major diastereoisomer), 5.85 -
5.67 (1 H, br m), 5.17 - 5.05 (2H, br m), 3.10 - 3.01 (1 H, m), 2.79 - 2.69 (1
H, m), 2.51
- 2.29 (2H, br m}, 1.88-1.61 (2H, br m), 1.46 {9H, s), 1.37-1.24 (1 H, m) and
0.96 (6H,
dd, J = 4.0, 4.5 Hz). ~3C~~NMR; 8~ (CDC13, major diastereoisomer}, 171.5,
170.3,
134.1, 117.5, 81.4, 48.8, 45.8, 39.5, 35.0, 27.9, 26.3, 23.5, and 21Ø
TS EP D:
L-tent leucine-2-pyridylarnide
The title compound was prepared from Na-benzyloxycarbonyl-L-iert-leucine by
methods analogous to those described in Example 1 (Steps D and E). ~ H-NMR; b
(CDC13), 8.26 (1 H, m). 8. ? 0 (1 H. m). 7.74 (1 H. m), 7.06 (1 H, m), 3.25 (1
H, s), 1.00
(9H, s).
T P
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanoic acid-1-teri-butyl ester
The products from 3R,S-allyl-2R-isobutyl-1,4-dioic acid-1-pentafluorophenyl-4
Pert
butyl diester (3:1, RS:RR) (5.59 c,~, 12.8 mmol) and L-tert leucine-2-
pyridylamide
(2.91 g, 14.1 mmol) were dissolved together in DMF (50 ml) and stirred at
30°C for
40 hours. TLC revealed that all of the pentafluorophenyl ester had been
consumed. The solvent 'was removed and the residue was purified by column
chromatography {silica gel, ethyl acetate-hexane, 1:1 ) and crystallisation
from ethyl
acetate-hexane. Yield: 1.41 g (24%, 5:1 mixture of SRS:RRS diastereoisomers).
~ H-NMR; b (CDC13, major diastereoisomer}, 9.58 (1 H, m), 8.52 (1 H, m), 8.19
(1 H, d,
J = 8.3 Hz), 7.73 {1 H, m), 7.12 (1 H, m), 6.49 (1 H, d, J = 9.2 Hz), 5.76 (1
H, m), 5.05
(1 H, m), 4.62 (1 H, d, J = 9.2 Hz), 2.68 (1 H, m), 2.53 {1 H, m), 2.29 (2H,
m), 1.73 (1 H,
m), 1.47 (1 H, m), 1.45 (9H, s), 1.15 (1 H, m), ?.02 (9H, s), 0.87 (3H, d, J =
6.5 Hz)
PCT/GB95/00111
WO 95/19956
and 0.79 (3H, d, J = 6.5 Hz).
STEP F:
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanoic acid
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanoic acid -1-ten! butyl ester (1.74 g, 3.78 mmol) was
deprotected
by TFA acidolysis according to the method described in Example 1 (Step G),
affording the title compound contaminated with residual TFA. Yield: 1.61 g
(contains solvent), 5:1 mixture of diastereoisomers. ~H-NMR; b (CD30D, major
diastereoisomer). 8.29 (1 H, m), i .90 (2H, m). 7.23 (1 H, m), 5.72 (1 H, m),
4.99 (2H,
m), 4.46 (1 H. s). 2.80 (1 H, dt. J = 3.3, 10.8 Hz), 2.52 (1 H, dt, 4.3. 10.2
Hz), 2.40 -
2.12 (2H, br m), 1.62 (1 H, m), 1.42 (1 H, m), 1.10 (2H, m), 1.08 (9H, s),
0.86 (3H, d, J
= 6.5 Hz) and 0.75 (3H, d, J = 6.6 Hz).
STEP G
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanoic acid (1.5 g, 3.71 mmol) was dissolved in DMF (5 ml) and
the
solution was cooled to 0°C during the addition of HOBt (0.60 g, 4.46
mmol) and
EDC (0.86 g, 4.46 mmol). The mixture was stirred at 0°C for 1 hour then
at room
temperature for 2 hours to ensurs~ complete formation of the active ester. The
solution was cooled back to 0°C, hydroxylamine hydrochloride (0.39 g,
5.57 mmol)
was added followed by NIV1M (0.56 g, 5.57 mmol) and the reaction mixture was
allowed to warm to room temperature then stirred overnight. The solvent was
removed in vacuo and the residue was triturated with a mixture of diethyl
ether (25
ml) and water (25 ml) and left to stand for 2 hours. The resulting solid was
WO 95119956 ~ ~. ~ ~. ~ !~ ~ PCT/GB95100111
56
collected by filtration, recrystallised from methanol-DIPE and dried under
high
vacuum at 60°C for 24 hours to ,afford the title compound (0.62 g, 40%;
single
diastereoisomer) containing ca. 0.4 mol DIPE (NMR). m.p. 221 - 223°C.
~H-NMR;
8 (CD30D), 8.26 (1 H, m), 8.04 (1 H, d, J = 8.3 Hz), 7.72 (1 H, dt, J = 1.9,
5.5 Hz),
7.07 (1 H, m), 5.64 (1 H, m), 4.96 (2H, m), 4.52 (1 H, s), 2.72 {1 H, m), 2.26
(2H, m),
2.10 (1 H, m), 1.50 (1 H, m), 1.39 (1 H, m), 1.06 (9H, s), 1.07 (1 H, m), 0.84
(3H, d, J =
6.4 Hz) and 0.73 (3H, d, J = 6.5 I-iz). ~3C-NMR; 8 (CD30D), 176.8, 172.4,
171.4,
152.7, 149.1, 139.4, 136.0, 121.1, 117.5, 115.7, 63.1, 48.2, 47.8, 41.8, 36.4,
35.3,
27.3, 27.0, 24.4 and 21.9. Found: C 63.68, H 8.66, N 12.08%; C22H34N4O4 . 0.4
C6H~40 requires: C 63.79, H 8.69, N 12.20%.
The following additional compounds were prepared according to the methods of
Example 23:
EXAMPLE 24
3R-(2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-5-methyl-2S-propen-2-
yl-hexanohydroxamic acid
O
H .,.w H N
N
_ H O
CONHOH
H
White solid. m.p. 211.5-;?12.5°C. ~H NMR; 8 (CD30D), 7.45 (2H, m), 7.26
(2H, m),
7.05 (1 H, m), 5.66 (1 H, m), 4.9Ei (2H, m), 4.46 (1 H, s), 2.73 (1 H, m),
2.26 (2H, m),
2.11 (1 H, m), 1.49 (1 H, m), 1.48 (1 H, m), 1.08 (1 H, m), 1.06 (9H, s), 0.84
(3H, d, J
= 6.4 Hz) and 0.75 (3H, d, J = 6.5 Hz). ~3C- NMR; 8 (CD30D), 176.7, 173.0,
171.0;
PCT/GB95/00111
WO95/19956 ~~~~-~n~~
57
139.3, 136.1, 129.8, 125.8, 121.6, 117.5, 62.9, 48.2, 41.8, 36.4, 35.4, 27.3,
27.1,
24.4 and 21.9. Found: C 63.68, IH 8.55 N 9.69%; C23H35N3O4 . 0.9 H20 requires
C
63.69, H 8.55, N 9.69%.
3R-(2,2-Dimethyl-1 S-(thiazol-2-ylcarbamoyl)-propyicarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid
O
H ,av H N
H II S
O N
CONHOH
H
White solid. m.p. > 300°C. ~H-NIVIR; S ((CD3)2S0), 12.10 (1 H, s),
10.46 (1 H, s),
8.77 (1 H, s), 8.12 (1 H, d, J = 7.9 Hz), 7.45 (1 H, d, J = 3.5 Hz), 7.20 (1
H, d, J = 3.5
Hz), 5.62 (1 H, m), 4.93 (2H, m), 4.51 (1 H, d, J = 4.5 Hz), 2.82 (1 H, m),
2.30 (1 H,
m), 2.22 (2H, m), 1.41 (3H, m), 0.98 (9H, s), 0.88 (3H, d, J = 6.3 Hz) and
0.78 (3H,
d, J = 6.3 Hz). ~3C-NMR; b ((CD;3)2S0), 174.1, 169.3, 169.2, 157.4, 137.7,
135.8,
116.2, 113.5, 60.3, 45.9, .45.3, 40.2, 34.9, 33.5, 26.5, 25.2, 24.0 and 21.7.
Found:
C 56.30%, H 7.70% N 12.70%; C2pH32N404S . 0.2 H20 requires C 56.10%, H
7.63% N 13.09%.
EXAMPLE 26
3R-(2,2-Dimethyl-1 S-(4-rnethoxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid
WO 95/19956 PCT/GB95/00111 '-'~
218~:'~7t~
58
O
H ~ .,~~~H H
N N \
H I
H ~CONHOH O ~ OMe
White crystaline solid. m.p. 213 ~- 228°C. ~ H-NMR; b (CD30D), 8.13 (1
H, d, J = 9.0
Hz), 7.33 (2H, d, J=9.0 I-iz), 6.8'1 (2H, d, J = 9.0 Hz), 5.63 (1 H, m), 4.97
{2H, m),
4.45 (1 H, t, J = 5.4, 3.6 Hz), 3.72 (3H, s), 2.73 (1 H, m), 2.20 (3H, m),
1.43 (2H, m),
1.10 (1 H, m), 1.06 (9H, ~;), 0.84 (3H, d, J = 6.4 Hz), and 0.75 (3H, d. J =
6.5 Hz).
~3C-NMR; 8 (CD30D), 176.7, 172.4, 170.8. 158.1, 136.0, 132.1, 123.5, 117.5,
115.0, 62.9, 55.9, 48.1, 41.9, 36.4. 35.4. 27.4. 27.1, 24.4 and 22Ø IR (KBr
disc); v
max 3308, 2958, 1644. 1513, 1245, 1036 and 830 cm-~. Found C 63.39, H 8.26, N
9.30%. C24H3~N305 . 0.4 H20 requires C 63.39, H 8.38, N 9.24%.
EXAMPLE 27
3R-(2-Benzylthio-2-methyl-1 S-(pyridin-2-ylcarbamoyl)-propylcarbamoyl)-5-
methyt-
2S-propen-2-yl-hexanohydroxamic acid
I
O S
H .aH N N
I W
CONHOH O
H
PCT/GB95100111
WO 95/19956
59
White solid. m.p. 214 - 214.5°C. ~ H-NMR; 8 (CD30D at 338 K), 8.36 (1
H, m), 8.02
(1 H, m), 7.72 (1 H, m), 7.32 - 7.12 (5H, br m), 7.05 (1 H, m), 5.62 - 5.53 (1
H, br m),
4.96 - 4.82 (3H, br m), 3.87 (2H, s), 2.69 (1 H, m), 2.37 - 2.21 (3H, m), 1.61
- 1.34
(2H, br m), 1.51 (3H, s), 1.45 (3H, s), 1.13 (1 H, br m), 0.82 (3H, d, J = 6.4
Hz) and
0.73 (3H, d, J = 6.4 Hz). ~3C-NMR; 8 ((CD3)2S0), 172.8, 168.1, 167.9, 146.8,
136.9,
136.4, 134.9, 128.0, 127.0, 125.4, 118.4, 114.7, 112.5, 56.5, 47.2, 44.8,
44.4, 33.6,
31.2, 25.3, 24.2, 22.7, 22.64 and 20.4.
EXAMPLE 28
3R-(2,2-Dimethyl-1 S-(pyridin-3-ylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid
.,~~ H N
~N
NHOH ~
White solid. m.p. 220 - 224°C. ~ H-NMR; b ((CD3)2S0), 10.30 (1 H, s),
10.00 (1 H,
s), 8.73 (1 H, s), 8.26 (1 H., d, J = 4.1 Hz), 7.99 (1 H, d, J = 8.2 Hz), 7.81
(1 H, d, J =
8.5 Hz), 7.34 (1 H, m), 5.E~5 (1 H, rn), 4.93 (2H, m), 4.43 (1 H, d, J = 5.3
Hz), 2.74
(1 H, m), 2.50 - 2.21 (3H, rn), 1.SCI (3H, m), 1.04 (9H, s), 0.83 (3H, d, J =
6.4 Hz),
and 0.77 (3H, d, J = 6.3 H:z). ~3C-NMR; 8 ((CD3)2S0), 174.0, 169.7, 169.2,
144.2,
140.7, 135.8, 135.4, 126.1), 123.6, 116.1, 61.0, 45.9, 45.5, 34.9, 33.7, 26.6,
25.3,
24.0 and 21.7.
WO 95119956 ~ ~ ~ ~ PCT/GB95100111 '"'
EXAMPLE 29
3R-(2,2-Dimethyl-1 S-(4-hydroxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid
O
H .~~~~ H H
\ _~ N N \
H O I /
H CONHOH OH
Yellow solid. ~ H-NMR; 8 ((CD3)2S0), 10.46 (1 H, s), 9.70 (1 H, s). 9.17 (1 H,
s), 8.77
(1 H, s), 8.00 (1 H, d, J = E1.7 Hz), 7.31 (2H, d, J = 8.8 Hz), 6.70 (2H, d, J
= 8.8 Hz),
5.67 (1 H, m), 4.93 (2H, m), 4.38 (1 H, d, J = 8.7 Hz), 2.70 (1 H, m), 2.31-
2.19 (3H, m),
1.44 (3H, m), 0.98 (9H, s), 0.81 (3H, d, J = 6.3 Hz) and 0.73 (3H, d, J = 6.3
Hz).
~3C-NMR; 8 ((CD3)2S0)" 173.8, 169.3, 168.4, 153.3, 135.9, 130.4, 121.0, 116.1,
114.9, 60.8, 45.8, 45.7, 3.4.9, 33.9, 26.7, 25.3, 24.0 and 21.7.
EXAMPLE 30
3R-(2,2-Dimethyl-1 S-(3-;methoxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic .acid
O
H ~ ,,,~vH N OMe
N \
H I
CONHOH O /
H
WO 95/19956 ~ . PCTIGB95J00111
61
White solid. ~ H-NMR; 8 ((CD3)2:50), 10.45 (1 H, s), 9.96 (1 H, s), 8.77 (1 H,
s), 7.99
(1 H, d, J = 8. Hz), 7.40 (1 H, s), 7.26 (2H, m), 6.60 (1 H, d, J = 7.8 Hz),
5.60 (1 H, m),
4.93 {2H, m), 4.40 (1 H, d, J = 8.5 Hz), 3.72 (3H, s), 2.81 (1 H, m), 2.10 -
2.49 (2H,
m), 1.99 (1 H, m), 1.44 (;2H, m), 1.09 (9H, s), 0.99 (1 H, m), 0.82 (3H, d, J
= 6.3 Hz)
and 0.72 (3H, d). ~3C-NMR; b ((CD3)2S0), 173.9, 169.3, 159.5, 140.0, 135.9,
129.5, 116.2, 111.6, 1 Oi3.5, 105.1, 61.1, 55.0, 45.9, 45.6, 40.3, 34.9, 33.9,
26.7,
25.3, 24.1 and 21.7.
EXAMPLE 31
3R-(2,2-Dimethyl-1 S-(p~yridin-4-ylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
propen-2-yl-hexanohydroxamic acid
H
,av H
N N \
H O ~ iN
~NHOH
Pale pink solid. m.p. 210°C (dec:.). ~H-NMR; 8 ((CD3)2S0), 10.15 (2H,
s), 8.28
(2H, d, J = 3.9 Hz), 7.73~ (1 H, d, J = 8.1 Hz), 7.44 (2H, d, J = 5.1 Hz),
5.51 (1 H, m),
4.81 (2H, m), 4.26 (1 H, d, J = 8.~4 Hz), 2.75 (1 H, m), 2.36 (2H, m), 2.11 (1
H, m),
1.24 (2H, m), 0.88 (9H, s), 0.84 (1 H, m), x.68 (3H, d, J = 6.4 Hz) and 0.59
(3H, d, J
= 6.4 Hz). ~3C-NMR; b ((CD3)2S0), 172.8, 169.3, 167.9, 148.6, 144.3, 134.5,
114.8, 111.9, 60.1, 44.E~, 44.1, ;33.6, 32.3, 25.2, 23.9, 22.7 and 20.3.
WO 95/19956 PCT/GB95/00111 ~'°
62
3R-(2,2-Dimethyl-1 S-(2-rnethoxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
thien-2-ylsulfanylmethyl-hexanohydroxamic acid
OMe
H .,~~~H H
N N \
H
CONHOH O
S
T P A'
2-Benzyloxycarbonyl-3R-(2,2-dimethyl-1 S-(2-methoxyphenylcarbamoyl)-
propylcarbamoyl)-5-methyl-hexanohydroxamic acid benzyl ester
2-Benzyloxycarbonyl-3R-~;,arboxy-5-methylhexanoic acid benzyl ester (prepared
by
the method described in E:P 0 446 267) (20.1 g, 50.5 mmol) and L-Pert-leucine-
N~-
(2-methoxyphenyl)amide (prepared according to the method described in Example
1, Steps D and E) (14.3 g, 60.6 mmol) were dissolved together in ethyl acetate
(300
ml). HOBt (8.2 g, 60.6 mrnol) anc! EDC (11.6 g, 60.6 mmol) were added and the
mixture was stirred and h~aated at reflux overnight, after which TLC analysis
showed that the reaction was cornplete. The solution was cooled and washed
successively with 5% aq. sodium hydrogen carbonate (2 x 200 ml), 5% citric
acid (2
x 200 ml) and brine (1 x 2'00 mi), dried over anhydrous magnesium sulphate,
filtered and evaporated to give a brown oil (32.75 g, contained residual
solvent)
which was used in Step B without further purification. ~H-NMR; 8 (CDC13), 8.07
(1 H, s), 7.40 - 7.22 (10H, br m), 7.15 (1 H, m), 6.96 (1 H, m), 6.75 (1 H,
m), 6.65 (1 H,
m), 5.12 - 5.04 (3H, br m), 4.37 (1.H, d, J = 9.0 Hz), 3.85 (1 H, d, J = 9.6
Hz), 3.76
PCT/GB95/00111
WU 95/19956
63
(3H, s), 3.08 (1 H, m), 1.80 - 1.37 (3H, br m), 1.06 (9H, s), 0.79 (3H, d, J =
6.4 Hz),
0.71 (3H, d, J = 6.5 Hz).
T P
3R-(2,2-Dimethyl-1 S-(2-methoxyphenylcarbamoyl)-propylcarbamoyl)-2-methylene-
5-methyl-hexanohydroxamic acid
2-Benzyloxycarbonyl-3R-(2,2-dimethyl-1 S-(2-methoxyphenylcarbamoyl)-
propylcarbamoyl)-5-methyl-hexanohydroxamic acid benzyl ester (28.7 g, 45.40
mmol) was dissolved in ethanol (200 ml) and the solution was placed under a
blanket of argon. 10% F~alladiunn on charcoal was added and a fine stream of
hydrogen gas was passE~d through the suspension for 3h with stirring. TLC
showed that all the starting material had been consumed. The system was purged
with argon and the catalyst was removed by filtration. The filtrate was cooled
and
stirred in an ice bath and treatecl with piperidine (4.04 g, 47.4 mmol) which
was
added dropwise, followed by 37~% formaldehyde solution (32.3 ml, ca. 430
mmol).
The reaction mixture wa;~ allowed to warm slowly to room temperature, then
stirred
overnight. The solvents were removed under reduced pressure and the residual
oil was partitioned betwE~en ethyl acetate (300 ml) and 1 M hydrochloric acid
(300
ml). The organic layer Nras separated, washed with 1 M hydrochloric acid and
brine, dried over magnesium sulphate and evaporated to dryness. The remaining
pale brown foam (18.94 g, crude) contained a number of minor impurities but
was
used in Step C without purification. ~ H-NMR; 8 (CD30D), 7.55 (1 H, m), 7.09
(3H,
m), 6.95 (1 H, m), 6.59 (1 H, m), 6.26 (1 H, s), 5.72 (1 H, s), 4.30 (1 H, d,
J = 9.2 Hz),
3.67 (1 H, s), 3.65 (1 H, m), 1.70 (1 H, m), 1.41 (2H, m), 0.91 (9H, s), 0.81
(3H, d, J =
6.3 Hz), 0.76 (3H, d, J = 6.3 Hz).
T P
3R-(2,2-Dimethyl-1 S-(2-~methoxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
thien-2-ylsulfanylmethyl-hexanoic acid
WO 95119956 PCTIGB95/00111
21$~.~i0
64
3R-(2,2-Dimethyl-1 S-(2-~methoxyphenylcarbamoyl)-propylcarbamoyl)-2-methyiene-
5-methyl-hexanohydroxamic acid (20.43 g, 48.7 mmol) was dissolved in methanol
and placed under a blanket of argon prior to addition of 2-mercaptothiophene
(20
ml). The mixture was stirred overnight at 60°C under argon with the
exclusion of
light. The solvent was removed under reduced pressure to leave an oil to which
was added cold diethyl Ether (2170 ml). The product precipitated on standing
in an
ice bath and was removed by fil~~tration and washing with thoroughly with cold
diethyl ether. The product was further purified by trituration with hot ethyl
acetate
and column chromatography (silica gel, gradient elution, 0-X20% methanol in
dichloromethane). Fracl;ions were combined and evaporated to yield the title
compound as an off-white foam (18.80 g. 72%). ~ H-NMR; b (CDC13), 7.33 (1 H,
m),
7.14 - 7.09 (2H, m), 7.10 (1 H, m), 6.95 - 6.85 (2H, br m), 6.58 (1 H, m),
4.31 (1 H, s),
3.66 (3H, s), 2.99 - 2.58 (4H, br m), 1.60 - 1.48 (1 H, m), 1.37 - 1.18 (1 H,
m), 1.01
(1 H, m), 0.95 (9H, s), 0.75 (3H, d, J = 6.5 Hz), 0.68 (3H, d, J = 6.5 Hz).
STEP D:
3R-(2,2-Dimethyl-1 S-(2-methoxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
thien-2-ylsulfanylmethyl-hexanohydroxamic acid
To a ice-cooled solution of 3R-(2,2-dimethyl-1 S-(2-methoxyphenylcarbamoyl)-
propylcarbamoyl)-5-methyl-2S-thien-2-ylsulfanylmethyl-hexanoic acid (20.43 g,
38.1 mmol) in DMF (100 ml) was added HOBt (6.18 g, 45.7 mmol) followed by EDC
(8.77 g, 45.7 mmol). The mixture was stirred at 0°C for 1 h then at
room
temperature for a further 2h, cooled back to 0°C and treated with
hydroxylamine
hydrochloride (3.97 g, 5i'.2 mmol) and NMM (5.78 g, 57.2 mmol). The reaction
mixture was stirred overnight at room temperature then evaporated to an oil
which
was triturated with 1:1 diethyl ether / water (240 ml) and left to stand for
1.5 h. The
resulting precipitate was collected by filtration and washed thoroughly with
cold
diethyl ether. HOBt was removed by crystallisation from ethyl acetate and the
WO 95/19956 c~ PCTlGB95/00111
mother liquors were evaporated to give the title compound as an orange foam
(4.96 g, 24%). m.p. 191 ~~ 195°C. ~ H-NMR; b (CD30D), 8.07 (1 H, d, J =
8.8 Hz),
7.31 (1 H, m), 7.11 (2H, rn), 7.02 (1 H, m), 6.92 (1 H, m), 6.86 (1 H, m),
6.56 (1 H, m),
4.28 (1 H, m), 3.66 (3H, s), 3.00 (1 H, m), 2.75 (1 H, m), 2.62 (1 H, m), 2.35
(1 H, m),
1.43 (1 H, m), 1.26 {1 H, rn), 1.03 {1 H, m), 0.94 (9H, s), 0.75 (3H, d, J =
6.4 Hz) and
0.67 (3H, d, J = 6.6 Hz). ~3C-NMR; 8 ((CD3)2S0), 176.2, 171.2, 165.4, 161.9,
140.8, 135.2, 131.0, 130.9, 128.0, 114.0, 111.3, 107.7, 63.4, 56.0, 46.2,
42.0, 40.8,
35.8, 27.7, 27.4, 24.7 and 22.2. IR (KBr disk) vmax 3294, 3087, 2959, 1771,
1644,
1547, 1493, 1466, 1430, 1386 and 1369 cm-1. Found: C 57.80, H 6.99, N 7.74%;
C26H3~NZOSS2 . 0.3H20 ~~equires C 57.71, H 7.00, N 7.77%.
The following additional compound was prepared according to the methods of
Example 32:
EXAMPLE 33
3R-(2,2-Dimethyl-1 S-(pyridin-2-ylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
thien-
2-ylsulfanylmethyl-hexanohydroxamic acid
O
H .,a~H N N
CONHOH O
S S
White crystalline solid. m.p. 199 - 200°C. ~H-NMR; S ((CD3)2S0), 8.82
(1 H, s),
8.15 (1 H, m), 7.89 (1 H, rn), 7.86. (1 H, m), 7.60 (1 H, m), 7.44 (1 H, m),
6.93 (2H, m),
6.87 (1 H, m), 4.36 (1 H, d, J = 8.3 Hz), 3.04 {1 H, m), 2.75 (1 H, m), 2.56
(1 H, m),
2.22 (1 H, m), 1.29 (1 H, rn), 1.12' (1 H, m), 0.90 (1 H, m), 0.82 (9H, s),
0.65 (3H, d, J
WO 95/19956 ~ ~ ~ ~ ~ PCTIGB95/00111
66
= 6.4 Hz) and 0.55 (3H., d, J = 6.5 Hz). ~3C-NMR; b ((CD3)2S0), 173.1, 170.0,
167.9, 151.5, 148.0, 138.0, 133.8, 132.6, 129.6, 127.8, 119.3, 113.6, 60.7,
48.6,
46.0, 45.6, 33.6, 26.6, 25.2, 23.9 and 21.5. IR (KBr diSC); vrt,ax 3256, 2890,
2871,
1651, 1579, 1520, 146E~, 1435, 1369, 1298, 1217, 1152, 1054 and 1002 cm-~ .
EXAMPLE 34
3R-(2,2-Dimethyl-1 S-(2-methoxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
thien-2-ylsulfinylmethyl-hexanohydroxamic acid
O OMe
H .,~~~ H H
N N \
" O ~ i
~CONHOH
~S ,OOH
3R-(2,2-Dimethyl-1 S-(2-methoxyphenylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
thien-2-ylsulfanylmethyl-hexanohydroxamic acid (prepared in Example 32) (1.00
g,
1.81 mmol) was dissolved in methanol (20 mi) and cooled to 0°C in an
ice bath
during addition of mCPE3A (0.34. g, 2.00 mmol). The reaction mixture was
allowed
to warm to room temperature, stirred for 4 h, and the solvent was removed
under
reduced pressure. The residue was triturated with diethyl ether leaving the
crude
product as a solid which was co~ilected by filtration. Recrystallisation from
ethyl
acetate afforded the title compound as a white powder in two crops (304 mg,
30%).
m.p. 210 - 211 °C. ~ H-NMR; 8 (CD30D), 7.31 (1 H, m), 7.01 (2H, m),
7.00 (1 H, m),
6.81 (1 H, m), 6.80 (1 H, rn), 6.56 (1 H, m), 4.29 (1 H, s), 3.65 (3H, s),
2.95 (1 H, m),
2.73 (1 H, m), 2.63 (1 H, rn), 2.37 (1 H, m), 1.43 (1 H, m), 1.26 (1 H, m),
1.05 (1 H, m),
0.95 (9H, s), 0.75 (3H, d, J = 6.4 Hz) and 0.68 (3H, d, J = 6.5 Hz). ~3C-NMR;
8
(CD30D), 175.5, 170.7, 170.6, 161.1, 140.0, 134.5, 130.3, 130.2, 128.2, 113.3,
WO 95/19956 ~ PCT/GB95/00111
67
110.6, 107.0, 62.6, 55.3, 41.3, 40.1, 35.1, 27.0, 26.7, 24.0 and 21.5. IR (KBr
disc)
vmax 3292, 1614, 1538, 1489, 1428, 1371, 1232, 1170 and 1047 cm-~. Found: C
55.70, H 6.68, N 7.39%; C26H3~P~306S2 . 0.5 H20 requires C 55.69, H 6.83, N
7.49%.
The following additional c:ompou~nd was prepared according to the methods of
Example 34:
EXAMPLE 35
3R-(2,2-Dimethyl-1 S-(pyridin-2-ylcarbamoyl)-propylcarbamoyl)-5-methyl-2S-
thien-
2-ylsulfinylmethyl-hexanohydroxamic acid
O
H ,,.~~H N N
H
CONHOH C
S S,D
White powder. m.p. 166 -166.5°C. ~H-NMR; 8 ((CD30D), 8.18 {1 H, m),
7.94 (1 H,
m), 7.82 (1 H, m), 7.64 (1 H, m), 7.50 (1 H, m), 7.10 (1 H, m), 7.00 (1 H, m),
4.42 (1 H,
m), 3.53 (1 H, m), 2.99 (1 H, m), 2.77 (1 H, m), 2.19 (1 H, m), 1.53 (1 H, m),
1.34
(1 H, m), 1.13 (1 H, m), 0.98 (9H, s), 0.77 (3H, m), and 0.66 (3H, m). ~3C-
NMR; 8
((CD30D), 175.4, 171.2, 169.9, 1'~ 52.7, 149.2, 144.8, 139.4, 134.2, 133.5,
128.6,
121.1, 115.6, 63.2, 61.2, 59.8, 4;3.2, 41.2, 35.3, 27.4, 27.0, 24.3 and 21.8.
IR (KBr
disc); vmax 3259, 2959, 1647, 1578, 1529, 1467, 1435, 1369, 1297, 1224, 1152
and
1034 cm-~. Found: C 53.ti4, H 6.:52 N 10.33%; C24HsaNaOsS2 . 0.8 H20 requires
C
53.67, H 6.68, N 10.43%.
PCTlGB95/00111
WO 95119956
68
EXAMPLE 36
3R-(2,2-Dimethyl-1 S-phenylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
phenylhexanoic acid
O
H .,~~~ H H
/ N N \
\ I H O I /
HO C02H
H
A solution of N2-[2R-(2,'.2-dimethyl-4-oxo-1,3-dioxalan-5S-yl)-6-
phenylhexanoyl)-L-
tert leucine-N~-phenylarnide (prepared by a method analogous to that described
in
Example 4) (1.00 g, 2.08 mmol) in THF (15 ml) was cooled to 0°C
and 1 M
hydrochloric acid (15 ml) was added. The mixture was stirred at room
temperature
until TLC indicated that all of the starting material had been consumed
(several
days). The solvents were removed under reduced pressure to leave a pale yellow
foam which was dissolved in ethyl acetate and filtered through a pad of
silica. The
product was purified further by extraction into 1 M sodium carbonate solution,
re-
acidification with 1 M hydrochloric acid and back extraction into ethyl
acetate. The
ethyl acetate layer was dried over magnesium sulphate, filtered and evaporated
under reduced pressure to afford the title compound as an off-white foam (320
mg,
35%; 3:1 mixture of dias~tereoisomers). m.p. 159°C. ~ H-NMR; 8 (CD30D),
7.51 -
7.48 (2H, m), 7.26 - 7.00 (8H, m), 4.39 (1 H, s), 4.25 (0.25H, d, J = 3.7 Hz),
4.18
(0.75 H, d, J = 2.7 Hz), 2.90 (1 H, br s), 2.56 - 2.51 (2H, br m), 1.74-1.56
(4H, m),
and 1.02 (9H, s). ~3C-NMR; 8 (CD30D), 179.3, 176.9, 171.1, 143.4, 139.2,
129.7,
129.4, 129.3, 126.7, 125.5, 121.8, 73.6, 63.1, 50.9, 36.6, 35.6, 30.5 and
27.4.
Found: C 66.07, H 7.21, N 5.98%: C25Hs2N20s . 0.8H20 requires C 66.00, H 7.44,
N
6.16%.
PCT/GB95/00111
WO 95/19956
69
EXAMPLE 37
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2S-
phthalimidomethylhexanohydroxamic acid
O
H ,~~~ H H
N N
H
CONHOH O
H
O N, O
TS EP A:
2-Benzyloxycarbonyl-3R-tent-butoxycarbonyl-5-methyl-2-phthalimidomethyl-
hexanoic acid benzyl e:>ter
To an ice-cooled solution of 2-benzyloxycarbonyl-3R-tert-butoxycarbonyl-5-
methylhexanoic acid benzyl ester (prepared by the method described in EP 0 446
267) (39.4 g, 86.78 mm~ol) in dr'y DMF (400 ml) was added sodium hydride (60%
dispersion in mineral oil, 3.83 g, 95.46 mmol) with stirring. The reaction
mixture
was maintained at 0°C for 20 rains then allowed to warm to room
temperature and
stirred for a further 2.5 h. After cooling to 0°C, N-
bromomethyl)phthalimide (25 g,
104.1 mmol) was added and the mixture was stirred for 0.5 h at 0°C then
at room
temperature overnight. The soilvent was removed under reduced pressure to
leave
an oil which was extracted with diethyl ether (400 ml) and the solid residues
were
removed by filtration. The filtrate was washed successively with water (300
ml), 1 M
PCT/GB95/00111 "'
W095119956 ~1~1~~~
hydrochloric acid (300 ml) and brine (300 ml), dried over anhydrous magnesium
sulphate and filtered. The solution was concentrated in vacuo to leave a
yellow oil
which was purified by column chromatography (silica gel, 50% diethyl ether in
hexane) to afford the title compound as a colourless oil (26.24 g, 49%). ~ H-
NMR; 8
(CDC13), 7.78 (2H, m), 7.Ei7 (2H, m), 5.28 - 5.05 (4H, br m), 4.54 - 4.35 (2H,
br m),
3.03 (1 H, m), 1.86 (1 H, m), 1.68 (1 H, m), 1.50 (9H, s), 1.49 (1 H, m), 0.82
(3H, d, J =
6.6 Hz) and 0.78 (3H, d, J - 6.5 I-Iz).
STEP B
3R-teri Butoxycarbonyl-5-methyl-2-phthalimidomethyl-hexanoic acid
2-Benzyloxycarbonyl-3R-Pert-butoxycarbonyl-5-methyl-2-phthalimidomethyl-
hexanoic acid benzyl ester (26.24. g, 42.8 mmol) was deprotected by catalytic
hydrogenolysis in ethanol, according to the method described in Example 32
(Step
B). The solvent was removed urnder reduced pressure, the residue was dissolved
in toluene (250 ml) and NMM (4.33 g, 42.8 mmol) was added. The mixture was
heated under reflux for 2 h. Solvents were evaporated and the remaining oil
was
dissolved in ethyl acetate and the solution was washed with
5% citric acid (2 x 200 ml,'~ and brine (200 ml), dried over anhydrous
magnesium
sulphate and filtered. Ths~ solvent was removed, leaving the desired product
as a
yellow foam (16.58 g, including residual solvent) which was used directly in
Step
C. ~ H-NMR; 8 (CDC13), 7.83 (2H, m), 7.72 (10H, m), 4.12 (1 H, m), 3.83 (1 H,
m),
3.21 (1 H, m), 2.72 (1 H, m), 1.81 - 1.55 (2H, br m), 1.48 (9H, s), 1.31 (1 H,
m) and
0.92 (6H, m).
3R-tert-Butoxycarbonyl-5-methyl-2-phthalimidomethyl-hexanoic acid benzyl
ester
PCTIGB95/00111
WO 95/19956
71
3R-tent Butoxycarbonyl-5-methyl-2-phthalimidomethyl-hexanoic acid (16.58 g,
42.56 mmol) was dissolved in dry DMF and placed under a blanket of argon. The
solution was cooled in an ice bath, benzyl bromide (5.56 ml, 46.82 mmol) and
anhydrous sodium carbonate (4.96 g, 46.82 mmol) were added and the mixture
was left to stir overnight at room temperature. The solvent was removed under
reduced pressure and the residual oil was dissolved in diethyl ether (300 ml)
and
washed successively with water (2 x 200 ml), 1 M hydrochloric acid (2 x 200
ml) and
brine (200 ml). The organic phase was dried (anhydrous magnesium sulphate),
filtered and evaporated i:o a crude yellow oil which was purified by column
chromatography (silica del, gradient elution, 30-X50% diethyl ether in
hexane).
The desired product was isolated as a pale yellow oil (18.2 g, 89%; 3:2
mixture of
diastereoisomers). ~H-NMR; 8 (CDC13), 7.78 (2H, m), 7.67 (2H, m), 7.24 (5H,
m),
5.05 (2H, m), 4.18 - 4.04 (1 H, br m), 3.81 (1 H, br m), 3.15 (1 H, m), 2.73
(1 H, m),
1.72 - 1.53 (2H, br m), 1.50 (5.4f~, s), 1.41 (3.6H, s) , 1.11 (1 H, m) and
0.90 (6H, m).
T P
3R-Carboxy-5-methyl-2-phthalimidomethyl-hexanoic acid benzyl ester
3R-tent Butoxycarbonyl-5-methyl-2-phthalimidomethyl-hexanoic acid benzyl ester
was deprotected by acid~olysis with TFA according to the procedure described
in
Example 1 (Step G). The product was isolated as a pale yellow oil (16.54 g,
including residual solvent) and was used in Step E without further
purification. ~ H-
NMR; 8 (CDC13, 3:2 mixture of diastereoisomers), 8.28 (1 H, br s), 7.78 (2H,
m), 7.68
(2H, m), 7.25 (5H, m), 5.08 (2H, m), 4.15 (1 H, m), 3.89 (1 H, m), 3.25 (1 H,
m), 2.88
(1 H, m), 1.82 - 1.52 (2H, br m), 1.25 (1 H, m), and 0.89 (6H, m).
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2RS-
phthalimidomethylhexanoic acid benzyl ester
PCTIGB95/00111
WO 95/19956
72
3R-Carboxy-5-methyl-2-phthalimidomethyl-hexanoic acid benzyl ester was
dissolved in ethyl acetate (200 ml), HOBt (5.21 g, 38.58 mmol) and EDC (7.40
g,
38.58 mmol) were added and the reaction mixture was stirred for 3.5 h to
ensure
complete formation of the activated ester. L-tent Leucine-N-(2-pyridyl)amide
(7.32
g, 35.36 mmol) was added and the reaction mixture was heated at reflux
overnight.
The solution was cooled before washing successively with 5% aq. sodium
bicarbonate, (2 x 200 ml) and water (2 x 200 ml). The organic phase was dried
(anhydrous magnesium sulphate), filtered and evaporated under reduced pressure
to leave a yellow foam. Column chromatography (silica gel, gradient elution,
20-X50% ethyl acetate in hexane) gave the desired product as an inseparable
mixture of diastereoisom~ers (16.89 g, 86%). ~H-NMR; S (CDC13), 9.76 (0.6H,
s),
9.58 (0.4H, s), 8.45 (1 H, m), 8.2E~ (1 H, m), 7.85 - 7.60 (5H, br m), 7.23
(4H, m), 7.08
(2H, m), 5.05 (2H, m), 4.7 6 (0.6H, m), 4.57 (0.4H, m), 4.03 (2H, m), 3.33
(0.6H, m),
3.22 (0.4 H, m), 2.85 (0.6H, m), 2.70 (0.4H, m), 1.78 (1 H, m), 1.55 (2H, m),
1.10
(5.5H, s), 1.05 (3.5H, s) and 0.93 - 0.67 (6H, br m).
TEP F:
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2RS-
phthalimidomethylhexanoic acid
3R-(2,2-Dimethyl-1 S-pyriidin-2-yl'carbamoyl-propylcarbamoyl)-5-methyl-2RS-
phthalimidomethylhexancric acid benzyl ester (5.08 g, 8.3 mmol) was
deprotected
by catalytic transfer hydrogenolysis in ethanol, according to the method
described
in Example 1 (Step E). The product was isolated as a white solid (4.34 g 75%)
following solvent evaporation and was used without further purification. ~ H-
NMR; b
(CD30D, 3:2 mixture of diastereoisomers), 8.31 (1 H, m), 8.08 (1 H, m), 7.81
(5H, m),
7.11 (1 H, m), 4.61 (0.6H, s), 4.52 (0.4H, s), 4.11 (0.6 H, m), 3.99 (0.4H,
m), 3.76 (1 H,
m), 3.14 - 2.85 (2H, br m)" 1.73 (1 H, m), 1.53 (2H, m), 1.14 (5.5H, s), 1.08
(3.5H, s),
0.92 (3.6H, m) and 0.82 (;?.4H, m).
PC'TIGB95100111
WO 95!19956
73
T P
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2RS-
phthalimidomethylhexanohydroxamic acid
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-5-methyl-2RS-
phthalimidomethylhexanoic acid was converted to the corresponding hydroxamic
acid by the method described in Example 32 (Step D). The solvent was removed
under reduced pressure and the residue was dissolved in ethyl acetate and
washed in 5% aq. sodium hydrogen carbonate, 5% citric acid and brine, dried
(anhydrous magnesium ;~ulphate~), filitered and evaporated. The product was
purified by column chromatography (acid-washed silica, gradient elution 0-5%
methanol in dichloromethane). Yield: 2.10 g (63%). ~ H-NMR: b ((CD3)2S0, 2:1
mixture of diastereoisomers), 8.42 - 8.15 (2H, br m), 7.65 (5H, m), 6.99 (1 H,
m),
4.68 (0.75H, m), 4.55 (0.25H, m), 4.07 (1 H, m), 3.52 (1 H, m), 2.99 - 2.72
(2H, br m),
1.34 - 1.24 (3H, br m), 1.17 (6H, s), 0.95 (3H, s), 0.79 (3H, m) and 0.68 (3H,
m).
3R-(2,2-Dimethyl-1 S-(N-oxy-pyridin-2-yl)carbamoyl-propylcarbamoyl)-2S-hydroxy-
5-methylhexanohydroxamic acid)
O O-
H ,~~~ H H
N N N~
. : H O ~ /
HO CONHOH
H
PCTIGB95/00111
WO 95/19956
74
3R-(2,2-Dimethyl-1 S-pyridin-2-ylcarbamoyl-propylcarbamoyl)-2S-hydroxy-5-
methylhexanohydroxamic; acid (E=xample 8) (100 mg, 0.25 mmol) was dissolved in
dichloromethane (10 ml) and mCPBA (48 mg, 0.28 mmol) was added. The
reaction mixture was stirred at room temperature for 3 h, after which TLC
analysis
revealed that all of the starting materiavhad been converted to a more polar
ferric
chloride positive compound. The solvent was removed under reduced pressure
and the residue was purified by column chromatography (acid-washed silica gel,
10% methanol in dichloro~methane) to afford the title compound as a glassy
solid
(60 mg, 58%). m.p. 150 -152°C. ~ H-NMR; 8 ((CD3)2S0), 8.42 - 8.26 (2H,
m), 8.19
(1 H, d, J = 7.9 Hz), 7.53 - 7.43 (1 I-i, m), 7.16 - 7.06 (1 H, m), 4.47 (1 H,
d, J = 7.8 Hz),
4.02 (1 H, d, J = 6.1 Hz), 2.96 - 2.84 (1 H, m), 1.63 - 1.38 (2H, m), 1.26 -
1.07 (1 H, m),
0.99 (9H, s), 0.82 (3H, d, ,J = 6.4 Hz) and 0.75 (3H, d, J = 6.4 Hz). ~3C-NMR;
8
((CD3)2S0}, 173.7. 170.3, 168.7. 143.2, 137.4. 127.2, 119.5, 114.5, 71.2,
62.1,
47.5, 37.2, 33.6, 26.6, 25.2, 23.6 ~3nd 21.8. IR (KBr diSO); vmax, 3258, 2958,
1652,
1510 and 1430 cm-~ .
BIQLOGICAL EXAMPLE
The following table compares the in vitro potencies of compounds of the
present
invention against those of similar compounds known in the art where R4 = Me
(Comparators 1 to 5).
Comparator 1: 5-Methyl-;3R-(1 S-methylcarbamoyl-2-phenylethylcarbamoyl)-
hexanohydroxamic acid.
Comparator 2: 3R-(2,2-Dimethyl-1 S-methyJcarbamoyt-propylcarbamoyl)-2S-
hydroxy-5-methylhexanohydroxamic acid.
Comparator 3: 3R-(2,2-Dimethyl-1 S-methylcarbamoyl-propylcarbamoyl)-5-
methylhexanohydroxamic acid.
CA 02181570 2005-O1-17
WO 95119956 PCTlGB95/(10111
Comparator 4: 2S-Hydroxy-3R-(1S-methylcarbamoyl-2-phenylethylcarbamoyl)-5-
methylhexanohydroxamic acid.
Comparator 5: 2S-Hydroxy-3R-(1 S-methylcarbamoyl-3-methyl-butylcarbamoyl}-5-
methylhexanohydraxamic acid.
The potency of compounds of the invention as inhibitors of collagenase was
determined by the procedure of Cawston and Barrett, (Anal. iochem., 99, 340-
345, 7 979), whereby a 1 mM solution of tree
compound being tested, or a dilution thereof, was incubated at 37' for 16
hours
with collagen and collagenase (buffered with 25mM Hepes, pH 7.5 containing
5mM CaCl2, O.OS% Brij 35 and 0.02% NaN3). The collagen was acetylated ~4C
collagen prepared by the method of Cawston and Murphy, (Methods in
Enzvm,~gy, 80, 711, 1981 ). The samples were
centrifuged to sediment undigested collagen, and~an aliquot of the radioactive
supernatant removed for assay on a scintillation counter as a measure of
hydrolysis. The collagenase activity in the presence of 1 mM of the test
compound,
or a dilution thereof, was compared to activity in a control devoid of
inhibitor and
the result reported below as that of inhibitor concentration effecting 50%
inhibition
of the collagenase activity (lCSO).
The potency of compounds of the invention as inhibitors of stromelysin was
determined by the procedure of Cawston et al, ( i h m~J., 195, 159-165, 1981),
whereby a 1 mM solution of the compound being
tested, or a dilution thereof, was incubated at 37' for 16~ hours with
stromelysin and
~aC acetylate casein {buffered with 25mM Hepes, pH T.S containing 5mM CaCl2,
0.05% Brij 35 and 0.02% NaN3). The casein was acetylated ~4C casein prepared
by the method of Cawston et al (ibid}. The stromelysin activity in the
presence of
1 mM of the test compound, or a dilution thereof, was compared to activity in
a
control devoid of inhibitor and the result reported below as that of inhibitor
concentration effecting 50% inhibition of the stromelysin activity (lCSq).
WO 95119956 s~ ~ ~ ~ ~ ~ ~ PCT~GB95100111
76
The potency of compounds of the invention as inhibitors of 72 kDa gelatinase
was
determined by a procedure basE~d on the method of Sellers et. al, Siochem. J.,
171, 493-496 (1979). 72' kDa gE~latinase, derived from RPMI-7951 cells was
purified by gelatin-agarose chromatography. The enzyme was activated by
incubation with aminophenyl mercuric acetate and approximately 0.05 units was
incubated with 50pg [14C;]-radiolabellet gelatin in an appropriate buffer for
16 hours
at 37°C. At the end of the incubation 50pg bovine serum albumin,
together with
trichloroacetic acid (final concentration 16%) were added to stop the reaction
and
to precipitate any undegraded substrate. The reaction tubes were placed on ice
for
15 minutes before centrifugation at 10,OOOg for 15 minutes to sediment the
precipitated substrate. A 200p1 aliquot of the reaction supernatant was
removed
and the radioactivity determined by liquid scintillation counting. The effect
of the
inhibitors was determined by ref~arence to a dose response curve. The IC5o
(the
concentration of inhibitor required to cause a 50% decrease in enzyme
activity)
was obtained by fitting a curve to the data and computing the concentration of
inhibitor required to achieve 50°io inhibition of the enzyme. For each
ICSo
determination, the effect on gelatinase activity of at least 8 concentrations
of the
inhibitor were examined. The inhibitors were dissolved and diluted in DMSO.
WO 95119956
77
PCT/GB95/00111
In vitro inhibitory activity
IC~o nM
TEST Collagf;nase
72 kDa
Stromelysin
COMPOUND Gelatinase
Example 1 20 60 10
Comparator 1 15 10 350
Example 4 2 5 9
Example 8 I 6 15 30
Example 17 2 30 20
Comparator 2 5 6 200
,
Example 2 30 30 15
Example 3 40 30 150
Comparator 3 I 1 ~D 8 700
Example 6 ~ 3~0 8 3
Comparator 4 1.5 3 200
Example 5 30 6 5
Comparator 5 I 8 - 90
