Note: Descriptions are shown in the official language in which they were submitted.
CA 02253796 2000-11-23
Inhibition of l~rlatrix Metalt~oproteases by acetylene containing compounds
BACKGR1~C''.rD 4F TFIE IIYV'ENTIOH
Field of the Invention
This invention relates to enzyne inhibitors. and more particularly, to novel
biaryl acetylene
containing compounds or derivatives thereof useful for inhibiting matrix
metalloproteases.
Description of the Related Art
The matrix metalloproteases (a.k.a. matrix metalloendo-proteinases or VL~tPs)
are a familu
of zinc endoproteinases which include, but are not limited to, interstitial
collagenase !a.k.a..
VIVtP-I ), stromelysin (a.k.a.. proteoglycanase, crattsin, or H~-3),
gelatinase .4 (a.k.a..
7~kDa-gelatinase or ~-2) and gelati.nase B (a.k.a.. 95kDa-gelatinase or
:vLl~iP-9). These VLVIPs
are secreted by a variety of cells including fibroblasts and chondrocytes,
along with natural
proteinaceous inhibitors known as TIlviPs (issue Inhibitor of
MGtallo,~roteinase).
All of these'.vffVIPs are capable: of destroying a variety of connective
tissue components of
1 ~ articular cartilage or basement membranes. 'Each MMP is secreted as an
inactive proenzyme which
must be cleaved in a subsequent step before it is able to exert its own
proteolytic activity. In
addition to the matrix destroying el:fect,' certain of these MMPs such as
MIV)P-3 have been
implicated as the in vivo activator for other MMPs such as MMP-1 and MMP-9
(Ito, et al., Arch
Biochem Biophys. ~, 211, 1988; Ogata., et a1.,1. Biol. Chem. ~7, 3581, 1992).
Thus, a cascade -
'0 of proteolytic activity can be initiated by an excess of MIvIP-3. It
foliows that specific IvllVlP-3
inhibitors should iimit the activity of other MMPs that are not directly
inhibited by such inhibitors.
It has also bees reported that M~Q'-3 can crave and thereby inactivate the
endogenous
inhibitors of other proteinases such as elastase (Winyard, et al., FENS Lens.
~Q, 1, 91, 1991).
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Inhibitors of 1~~P-3 could thus influence the activity of other destructive
proteinases by modifying
the level of their endogenous inhibitors.
.~ number of diseases are thought to be mediated by excess or undesired matrix-
destrovng
metalloprotease activity or by an imbalance in the ratio of the l~~iPs to the
TL~fPs. These include:
a~ osteoarthritis (Woessner, et al., J. Hiol.Chem. ?59(6). 3633, 1984; Phadke,
et al., J. Rheumatol.
t~. 8~'_'. 1983), bi rheumatoid arthritis (Mullins, et al.. Biochim. Biophys.
acts 69~, 117. 1983:
V'oolley, et al., arthritis Rheum. ?0, 1231, 1977; Gravallese, et al.,
Arthritis Rheum. 3~, 1076.
1991 ), c) septic arthritis (Williams, et al., arthritis Rheum. 3 533, 1990),
d) tumor metastasis
l Reich. et al.. Cancer Res. 48, 3307. 1988, and ;~fatrisian, et- al., Proc.
Nat'1. Acad. Sci.. USA 3
IO 9-11 3. 1986), e) periodontal diseases (Overall, et al., J. Periodontal
Res. 3, 81, 1987), f) corneal
ulceration Bums, et al.. Invest. Opthalmol. Vis. Sci. ~, 1569, 1989), g)
proteinuria (Baricos, et al.,
Biochem. J. 254, 609, 1988), h) coronary thrombosis from atherosclerotic
plaque rupture (Henney,
et al.. Proc. Nat'1. Acad. Sci., USA 8~, 8154-8158, 1991 ), l) aneurysmal
aortic disease (Vine, et aJ.,
Clin. Sci. 81, 333, 1991), j) birth control (Woessner, et al.. Steroids 54
491, 1989), k) dystrophobic
15 epidetmolysis bullosa (Kronberger, et al., J. Invest. Dermatol. 79 208,
1982), and 1) degenerative
cartilaee loss following traumatic joint injury, m) conditions leading to
inflammatory responses,
osteopenias mediated by MMP activity, n) tempero mandibular joint disease, o)
demyelating
diseases of the nervous system (Chantry, et al., J. Neurochem. 50 688, 1988).
The need for new therapies is especially important in the case of arthritic
diseases. The
'0 primary disabling effect of osteoarthritis (OA), rheumatoid arthritis (RA)
and septic arthritis is the
progressive loss of articular cartilage and thereby normal joint function. No
marketed
pharmaceutical agent is able to prevent or slow this cartilage loss, although
nonsteroidal anti-
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inflammatory drugs (NSA)Ds) have been given to control pain and swelling. The
end result of these
diseases is total loss of joint function which is only treatable by joint
replacement surgery. VtViP
inhibitors are expected to halt or reverse the progression of cartilage loss
and obviate or delay
surgical intewention.
Proteases are critical elements at several stages in the progression of
metastatic cancer. In
:his process. the proteoUtic degradation of structural protein in the basal
membrane allows for
:xpansion of a tumor in the primary site, evasion from this site as well as
homing and invasion in
distant, secondary sites. Also, tumor induced angiogenesis is required for
tumor growth and is
dependent on proteolytic tissue remodeling. Transfection experiments with
various apes of
prote3ses have shown that the matrix metalloproteases play a dominant role in
these processes in
particular gelatinases A and B (LLI~iP-2 and ~tMP-9, respectively). For an
overview of this field see
'tullins. et al.. Biochim. Biophys. ~cta 69~, 177, 1983; Ray, et al., Eur.
Respir. J. 7 2062, 1994;
Birkedal-Hansen, et al., Crit. Rev. Oral Biol. ~ted. 4, 197, 1993.
Furthermore, it was demonstrated that inhibition of degradation of
extracellular matrix by
1 ~ the native matrix metalloprotease inhibitor TIMP-2 (a protein) arrests
cancer growth (DeClerck, et
al.. Cancer Res. ~?. 701, 1992) and that TIMP-2 inhibits tumor-induced
angiogenesis in
experimental systems (Moses, et al. Science 2~8, 1408. 1990). For a review,
see DeCIerck, et al.,
.inn. V. Y. Acad. Sci. ~, 222, 1994. It was further demonstrated that the
synthetic matrix
metalloprotease inhibitor batimastat when given intraperitoneally inhibits
human colon tumor
growth and spread is an orthotopic model in nude mice (Wang, et al. Cancer
Res. ~, 4726, 1994)
and prolongs the survival of mice bearing human ovarian carcinoma xenografts
(Davies, et. al.,
3
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Cancer Res. ~~, 2087, 1993). The use of this and related compounds has been
described in Brown.
et al.. ~'~'O-9321942 A2.
There are several patents and patent applications claiming the use of
metalloproteinase
inhibitors ;or the retardation of metastatic cancer, promoting tumor
recession. inhibiting cancer cell
proliferation, slowing or preventing cartilage loss associated with
osteoarthritis or for treatment of
ether diseases as noted above (e.g. Lew, et al., WO-9519965 A l : Beckett. et
al.. WO-9519956 :~ l
Beckett. et al., WO-9519957 A1: Beckett, et al.. WO-9519961 A1; Brown. et al..
WO-9321942 A?:
Cnrnrttin. et al., WO-9421625 Al; Dickens. et al., U.S. Pat. Vo. 4,599.361:
Hughes, et al., LT.S. Pat.
~o. 5.190.937; Broadhurst, et al.. EP 574758 Al; Broadhurn, et al., EP 276436;
and Vlyers, et al.,
EP ~~0~73 Al. The preferred compounds of these patents have peptide backbones
with a zinc
complexing group (hydroxamic acid, thiol, carboxylic acid or phosphinic acid)
at one end and a
variety of sidechains. both those found in the natural amino acids as well as
those with more novel
functional groups. Such small peptides are often poorly absorbed, exhibiting
low oral
bioavailability. They are also subject to rapid proteolvtic metabolism, thus
having short half lives.
1 ~ As an example, batimastat, the compound described in Brown, et al., WO-
9321942 A2, can only be
given intra peritoneally.
Certain 3-biphenoylpropanoic and 4-biaryloylbutanoic acids are described in
the literature
as anti-inflammatory, anti-platelet aggregation, anti-phlogistic, anti-
proliferative, hypolipidemic,
antirheumatic, analgesic, and hypochoiesterolemic agents. In none of these
examples is a reference
made to MIvIF' inhibition as a mechanism for the claimed therapeutic effect.
Certain related
compounds are also used as intermediates in the preparation of liquid
crystals.
SUBSTITUTE SHEET (RULE 26~
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Specifically. Tomcufcik, et al., US patent 3.784,701 claims certain
substituted
bettzovlpropionic acids to treat inflammation and pain. These compounds
include
-biphenoylpropanoic acid (a.ic.a. fenbufen) shown below.
' 0
/ off
- o
Feabufen
Child. et a1..1. Phann. Sci. 66, 466, 1977 describes structure-activity
relationships of several
analoes of fenbuien. These include several compounds in which the biphenyl
ring system is
substituted or the propanoic acid portion is substituted with phenyl, halogen,
hydroxyl or methyl,
or the carboxylic acid or carbonyl functions are converted to a variety of
derivatives. Rio compounds
are described which contain a 4'-substituted biphenyl and a substituted
propanoic acid portion
combined in one molecule. The phenyl (compounds XLIX and LXXVIn and methyl
(compound
XL,VII) substituted compounds shown below were described as inactive.
1 ~ _ _ O ~ 0 CHI
/ ~ / OH ~ / ~ / OH
O O
~.IX 70.VII
O
/ OH
O
LXXVB
Kameo, et al., Chew. Phann. Bull. ~ø, 2050, 1988 and Totnizawa, et al.. JP
patent 62132825
A2 describe certain substituted 3-biphenoylpropionic acid derivatives aad
analogs thereof including
S
SUBSTfTUTE SHEET (RULE 26)
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PCT/US97/07921
the followine. Various compounds with other substituents on the propionic acid
portion are
described, but they do not contain biphenyl residues.
0
X : / \ / off
0
s
0
wherein X = H. ~'-Br. ~'-Cl. 4'-CHI, or 2'-Br.
Cousse, et al., Eur. J. Vied. Chem. ~, .~~, 1987 describe the following methyl
and methylene
substituted 3-biphenoyl-propanoic and -propenoic acids. The corresponding
compounds in which
the carbonyl is replaced with either CH,OH or CH, are also described.
_ O O
/ \ / OH X v / \ / OH
p v I O
0 ~ 0
/ \ / ~ OH X v / \ / OH
O 0
1 ~ whezetn X = H. C1. Hr. CHsO, F, or NHi.
Nichl, et al. DE patent 1957750 also describes certain of the above methylene
substituted
biphenovlpropanoic acids.
El-Hashash, et al., Revue Roum. Chim. ~3. 1581, 1978 describe produce derived
from
(3-aroyl-acrylic acid epoxides including the following biphenyl compound. No
compounds
'0 substituted on the biphenyl portion are described.
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
cH,1
\1
0
/ 0
CH,
OH
Kitamura. et al.. JP patent 60209539 describes certain biphenyl compounds used
as
intermediates for the production of liquid crystals including the following.
The biphenyl is not
substituted in these intermediates.
/ ~ o
off
R' p
wherein R' is an alkvi of l-l0 carbons.
Thyes. et al.. DE patent 2854475 uses the following compound as an
intermediate. The
biphenyl group is not substituted.
O
off
l~ O
Sammour, et al., Egypt J. Chem. ]"~, 311. 1972 and Couquelet, et al., Hull.
Soc. Chim. Fr.
9, 3196. 1971 describe certain dialkylamino substituted biphenoylpropanoic
acids including the
following. In no case is the biphenyl group substituted.
R' R=
O ~N
OH
~ / ~ /
O
wherein R', RZ = alkyl, benzyl, H, or, together with the nitrogen,
morpholinyl.
7
SUBSTITUTE SHEET (RULE 26~
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WO 97143245 PCT/US97/07921
Others have disclosed a series of biphenyl-containing carboxylic acids,
illusu~ated by the
compound shown below, which inhibit neural endopeptidase hiEP 24.11 ), a
membrane-bound unc
metalloprotease (Stanton. et al., Bioorg. :fed. Chem. Lett. .~, X39, 1994;
Lombaett. et al.. Bioore.
~ted. Chem. Lett. -t. .71 ~. 1994: Lombaert, et al.. Bioorg. Med. Chem. Lett.
=. 1.~5. 199: Lombaert.
et al.. Bioorg. Lied. Chem. Lett. ~, 1 ~ 1. 1995).
0 O
Ph0-P~ ~~ ~.~COZH
Ph0 1-I
It has been reported that N-carboxyalkyl derivatives containing a
biphenvlethylgiycine.
illustrated by the compound shown below, are inhibitors of stromelysin-t
(VC~iP-3), 72 kDA
gelatinise (:~-2) and collagenase (burette, et al.. WO-929689).
F
1~ I \
0 ~H~
P6. ~ ~~ ~~C~H
IiO
CHI CHI
CHI
.0 It would be desirable to have effective MMP inhibitors which possess
improved
bioavailability and biological stability relative to the peptide-based
compounds of the prior art, and
8
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
PCT/US97/07921
WO 97143245
which can be optimized for use against particular target ViMPs. Such compounds
are the subject
of t5e present application.
The development of efficacious l~~iP inhibitors would afford new therapies for
diseases
mediated by the presence of, or an excess of VL'uP activity, including
osteoarthritis. rheumatoid
arthritis. septic arthritis. tumor metastasis, periodontal diseases, corneal
ulcerations, and proteinuria.
Several inhibitors of VtMPs have been described in the literature, including
thiols (Beszant, et al..
J. ~ted. Chem. ~, 4030, 1993), hydroxamic acids (Wahl, et al. Bioorg. died.
Chem. Lets. ~. 349.
199: Conway. et al. J. Exp. Vied. 1~2_, 449. 1995; Porter, et al., Bioorg.
Vied. Chem. Lett. 4,'_7.1,
1994: Tomczuk. et al., Bioorg. Vted. Chem. Lett. ~, 343, 1995; Castelhano, et
al.. Bioorg. Vied.
Chem: Lztt. ~, 141, 1995), phosphorous-based acids (Bird, et al. 3. Med. Chem.
37, 1~8, 1994;
l~torphy. et al.. Bioorg. Med. Chem. Left. .1, 2747, 1994; Komlewicz, et al..
J. Med. Chem. 3~. 263.
1990), and carboxylic acids (Chapman, et al. J. Vied. Chem. ,3~, -X293, 1993:
Brown, et al.. J. Vted.
Chem. 37, 674. 1994; Morphy, et al., Bioorg. Vied. Chem. Lett. ~ 2747, 1994;
Stack, et al.. :arch.
Biochem. Biophys. ?~7, 240, 1991: Ye. et al., J. Vied. Chem. 7 206, 1994;
Grobelny, et al.,
! ~ Biochemistry 2~, 6145, 1985; Mookhtiar, et al., Biochemistry 27, 4299.
1988). However, these
inhibitors eenerally contain peptidic backbones, and thus usually exhibit low
oral bioacuvity due
to poor absorption and short half lives due to rapid proteolysis. Therefore,
there remains a need for
improved MMP inhibitors.
=0 SUMMARY OF THE INVENTION
This invention provides compounds having matrix metalloprotease inhibitory
activity. These
compounds are useful for inhibiting matrix metalloproteases and, therefore,
combating conditions
9
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to which VfVtP's contribute. Accordingly, the present invention also provides
pharmaceutical
compositions and methods for treating such conditions.
The compounds described relate to a method of treating a mammal comprising
administering
to the mammal a matrix metalloprotease inhibiting amount of a compound
according to the invention
sufficient to:
~a) alleviate the effects of osteoarthritis, rheumatoid arthritis, septic
arthritis. periodontal
disease, corneal ulceration, proteinuria, aneurysmal aortic disease,
dvstrophobic
epidermolysis. bullosa, conditions leading to inflammatory responses,
osteopenias mediated
by ~tl~tP activity, tempero mandibular joint disease, demyelating diseases of
the nervous
system;
(b) retard tumor metastasis or degenerative cartilage loss following traumatic
joint injury;
(c) reduce coronary thrombosis from athrosclerotic plaque rupture; or
(d) effect birth control
The compounds of the present invention are also useful scientific research
tools for studying
l ~ functions and mechanisms of action of matrix metalloproteases in both in
vtvo and in virro systems.
Because of their N(lv>P-inhibiting activity, the present compounds can be used
to modulate MMl'
action, thereby allowing the researcher to observe the effects of reduced MMP
activity in the
experimental biological system under study.
This invention relates to compounds having matrix metalloprotease inhibitory
activity and
?0 the generalized formula:
TxA-B-D-E-G . (L)
io
SUBSTITUTE SHEET {RUtE 26)
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In the above generalized formula IL). T~A represents a substituted or
unsubstituted aromatic
6-membered ring or heteroaromatic ~ - 6 membered ring containing 1 - '_ atoms
independently
selected from the group of N, O, or S. T represents a substituted acerylenic
moiety.
In the generalized formula (L), B represents an aromatic 6-membered ring or a
heteroaromatic ~ - 6 membered ring containing 1 - 2 atoms independently
selected from the group
of ~. O. or S. It is referred to as the B ring or B unit. When N is employed
in conjunction with
either S or O in the B ring, these heteroatoms are separated by at least one
carbon atom.
In the generalized formula (L), D represents
\ \ \ \.H \,H
~C=O ~C='dOH GCS~ j 'OH ~ ~H
l~
In the generalized formula (L), E represents a chain of n carbon atoms bearing
m substituents
R° in which the R6 groups are independent substituents, or constitute
spiro or nonspiro rings. Rings
may be formed in two ways: a) two groups R6 are joined, and taken together
with the chain atoms)
to which the two R6 ~oup(s) are attached, and any intervening chain atoms,
constitute a 3 - 7
l . membered ring, or b) one group R° is joined to the chain on which
this one group R6 resides, and
taken toeether with the chain atoms) to which the Rb group is attached, and
any intervening chain
atoms, constitutes a 3 - 7 membered ring. The number n of carbon atoms in the
chain is 2 or 3, and
the number m of R6 substituents is an integer of 1 - 3. The number of carbons
in the totality of R°
groups is at least two.
30 Each group R° is alkyl, alkenyl, alkynyl, heteroaryl, non-aromatic
cyclic, and combinations
thereof optionally substituted with one or more heteroatoms as dexribed more
fully below. In the
//
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97143245 PCT/US97107921
generalized formula (L), E is a substituted mono- or bicyclic moiety
optionally substituted ~zth one
or more heteroatoms.
In the eeneralized formula (L). G represents -PO,H, . -of
O ~ 0 R ~ 3 ~-'.~1
C N H ~t ' ~ H H ~t . or
in which Vi represents -CO,H. -COi'1(R"l, or -CO,R'=, and R" represents any of
the side chains of
the ! 9 noncyclic naturally occurring amino acids.
"lie most preferred compounds of the invention are:
O R~6
!0 R~s-~=0 ~ / ~ / OH
0
where R's is selected tom the group comprising: HOCH2, VteOCH:, (n-Pr):NCH,.
CH,CO:CH,,
EtOCO,CH,. HO(CH,),, CH,CO:(CH,),, HO:C(CH2)~, OHC(CH),, HO(CH~, Ph, 3-HO-Ph,
and
PhCH,OCH,; and R'b is
0
Ph
1 ~ ~ or /~ N
O
Pharmaceutically acceptable salts of these compounds are also within the scope
of the
tnventton.
In most related reference compounds of the prior art, the biphenyl portion of
the molecule
~4 is unsubstituted, and the propanoic or butanoic acid portion is either
unsubstituted or has a single
methyl or phenyl group. Presence of the larger phenyl group has beea reported
to cause prior art
compounds to be inactive as anti-inflammatory analgesic agents. See, for
example, Child, et a1.,1.
IL
SUBSTITUTE SHEET (RULE 26)
CA 02253796 2002-12-16
Pharm. Sci. ~~, X66 (19??). By contrast, it has now beta fotmd drat compounds
which c~thibit
Fotent Vf~tP inhibitory activity contain a substituent of significant size on
the propanoic or butanoic
portion of the molecule. The biphenyl portions of the best 1~1V~ inhibitors
also preferably contain
a substituent on the ~' position, althou~ah when the propanoic or butanoic
portions ate optimally
substituted, the unsubstituted biphenyl compourats of the invention have
sufficient activity to be
considercd realistic drug candidates.
The fot~egoing merely summarizes certain aspects of the peesmt invention and
is not
t0
inc~d. nor should it be construed, to limit the invcnaon in any way.
DESCRIPTION OF THE PREFERRED EMBODIME~y'fS
More particularly, the compounds of the present invention are materials having
matrix
metalloprotease inhibitory activity and the generalized formula:
T~.~,-&D-tr-G (L)
1~ in which T,.~ represents a substituted or unsubst'ttuted aromatic or
heteroaromatic moiety selected
from the group consisting of:
/ /
T ~~ T ~~ T ~~.1
T=
T /N Ni
T /O Tx Ni Tx S
R
T ~ T. . ~ T; ~N J T.
" N. J NON N N
N
!3
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
T
W I
in which R' represents H or alkyl of 1 - 3 carbons.
ThrouQttout this application. in the displayed chemical structures, an open
bond indicates the
point at ~chich the structure joins to another soup. For example,
Rso
/I
where R'' is
l0
is the structure
I
In these structures, the aromatic ring is referred to as the A ring or A unit,
and T represents
1 ~ a substituent group, referred to as a T group or T unit. T is a
substituted acetylenic moiety and x is
The B ring of generalized formula (L) is a substituted or unsubstitutcd
aromatic or
heteroaromatic ring, in which any substituents are groups which do not cause
the molecule to fail
to fit the active site of the target enzyme, or disrupt the relative
conformations of the A and B rings,
'0 such that they would be detrimental. Such substituents may be moieties such
as lower alkyl, tower
alkoxy, C'~I, NOi, halogen, etc., but are not to be limited to such groups.
In the generalized formula (L), H represents an aromatic or heteroaromatic
ring selected from
the group consisting of:
/'
SUQSTlTUTE SHEET (RULE 26)
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/ / ~~ / ~ ~ /
0
R l y ~ ~ / ~ '~1 R' -
' ~~ N ~ / N
i~
R
--~ N~-
0
R,
0
R R N
j R~
N~ l=N / /
N~ '' , I
~I , ~N~ ~N~ ~ N
--y---
N N
~'J~ N' I N II / I /
W N /~ ~N ~ .N
N N
in which R' is defined as above. These rings are referred to as the B ring or
B unit.
In the generalized formula (L), D represents the moieties:
H H
~C=O ~ =NOH ~ =S % OOH ~ ~H
Ln the generalized formula (L), E represents a chain of n carbon atoms bearing
m substituents
R°, referred to as R6 groups or R °units. The R °groups
are independent substituents, or constitute
spiro or nonspiro rings. Rings may be formed in two ways: a) two groups
R° are joined, and taken
together with the chaia atoms) to which the two R6 groups) are attached, and
any intervening chain
atoms, constitute a 3 - 7 membered ring, or b) one group R6 is joined to the
chain on which this one
group Rb resides, and taken together with the chain atoms) to which the R6
group is attached, and
/S
SUBSTITUTE SHEET (RULE 26)
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any intervening chain atoms, constitutes a 3 - 7 membered ring. The number n
of carbon atoms in
the chain is ? or 3, and the number m of R6 substituents is an integer of 1 -
3. 'Ihe number of
carbons in the totality of R° stoups is at least two.
Each soup R6 is independently selected from the group consisting of the
substituents listed
s below as items 1 ) - 14).
1 ) alkyl of 1 - 10 carbons, provided that if the A unit is phenyl, the B unit
is
phcnylene, m is 1. n is ?. and the alkyl group is located on the alpha carbon
relative to the
D unit. then x is 1 or 3:
.) aryl of 6 - 10 carbons, provided that if the A unit is phenyl, the B unit
is
pheny lene, the aryl group is phenyl, n is 2, and m is 1 or ?, then x is l or
2;
3) hcteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom;
~) arylalkyl in which the aryl portion contains 6 - 10 carbons and the alkyl
portion
contains 1 - 8 carbons;
~) heteroaryl-alkyl in which the heteroaryi portion comprises 4 - 9 carbons
and at
1 ~ least one N, O, or S heteroatom, and the alkyl portion contains 1 - 8
carbons;
6) alkenyl of 2 - 10 carbons;
7) aryl-alkenyl in which the aryl portion contains 6 - 10 carbons and the
alkenyl
portion contains 2 - S carbons;
8) heteroaryl-alkenyl in which the heteroaryl portion comprises 4 - 9 carbons
and
30 at least one N, 0, or S heteroatom and the alkenyl portion contains 2 -5
carbons;
9) alkynyl of 2 - 10 carbons;
16
SUBSTITUTE SHEET (RULE 26)
CA 02253796 2000-11-23
10) aryl-alfcrnyi in which the aryl portion contains 6 - 10 carbons and the
allc~rvl
portion contains ? - ~ carbons;
1 1 ) heteroaryl-alkynyl in which the heteroaryt portion comprises 4 - 9
carbons and
at feast one ~. O, or S heteroatom and the alkynyi portion contains ? - ~
carbons:
1?)-(CH,),R' in which t is 0 or an integer of 1 - ~ and R' is selected from
the group
consisting of:
0 O
w ~ l
-N I ) -N I _N
r r
O 0
O
r ~ w O\\ / O
w r _N N Rt
II Rt _N
0 ~~ N'~O 0
I
O O O
NRt
_N _'N I NRt
O O O
t t 1 1
R O R t R O -N_O-Rt
-N_C-N_Fi .-N_C_ORt
-Nt Rt r ~ ~~~)u
N
as well as corresponding hevtcroaryl moieties in which the aryl portion of as
aryl-containing
R' group comprises 4 - 9 carbons and at least one N, 0, or S heteroatom. In
such R' groups,
Y represents O or S; F;' is as defined above, and a is 0, 1, or 2 provided
that
when R' is
CA 02253796 2000-11-23
t
/-'/. fR )~ w'.~1, R1
Y
or
and the A unit is phenyl, the 13 unit is phenylene, m is l, n is 2, and t is
0, rhea x is 1 or 2.
1. )-(CH:),ZR' in which v is an interger of 1 to 4, Z represents -S-. -S(0)-. -
S0,-. or
-O-, and R' is selected from the group consisting of alkyl of 1 to 12 carbons,
aryl of 6 to 10
carbons, heteroaryl comprising .~ to 9 carbons and at least one ~. 0. or S
heteroatom:
awlalkyl in which the aryl portion contains 6 to 12 carbons and the alkyl
portion contains
1 to -f carbons: heteroarytalkyl in which the aryl portion contains 6 to 12
carbons and at least
one N. O, or S heteroarom and the alkyl portion contains ! to 4 carbons; -
C(O)R9 in which
the R9 represents alkyl of 2 to 5 carbons, aryl of 6 to 10 carbons, heteroaryl
comprising 4 to
9 carbons and at least one N, 0, or S heteroatom, and arylalkyl in which the
aryl portion
contains 6 to 10 carbons or is a heteroaryl comprising 4 to 9 carbons and at
least one ~I, O,
or S heteroatom, and the alkyl portion contains 1 to 4 carbons with the
provisos that when
R8 is -C(O)R9, Z is -S- or-O-; when Z is -O--, Rg may also be -(C,,HZ,,O)tR9
in which v, t and
R9 are as defined above: and when the A unit is phenyl, the B unit is
phenylene, m is 1, n is
2. and v is 0, then x is 1 or ~; and
1.~~{CH:)",SiR'°~ in which w is an integer of 1 to 3, aad R'°
represeau alkyl of 1 to
carbons.
In addition, aryl or hcteroaryl portions of any of the T or Rb groups
optionally may bear up
30 to two substitueats selected from the group consisting of -
(CH~,C(R"xR'i)OH, -(CH~~,OR",
-(CH,)ySR", -(CH~~,S(O)R", -(CH~)yS(O~R". -(CH=~,SO~N(R"}r, -{CHI)a,N(R"~,
-(CH,)"N(R")COR'i, -OC(R"~O- in which both oxygen atoms arc connected to the
aryl ring,
~8
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
-~CH,)~COR". -(CH,hCON(R"),, -ICH,)~CO,R". -(CH,)~OCOR".-halogen. -CHO. -CF;. -
NO:.
-C~. and -R'Z. in which v is 0 - 4: R" represents H or alley! of I - .t
carbons: and R'= represents alkyl
of f --I carbons.
In the eeneralized formula (L). G represents -PO,H, -Vh
0 ~ 0 R" N-N
-C-N-H-~t . -C-~-~-M , or ~~ .N
N
in which VI represents -CO,H. -CON(R" ),, or -CO,R'2, and R" represents anv of
the side chains of
the 19 noncyclic naturally occurring amino acids.
Pharmaceutically acceptable salts of the compounds falling within the
generalized formula
I 0 ( L ) are also within the invention.
The G unit is most preferably attached to the E unit at the carbon ~i to the D
unit and is
preferably a carboxylic acid group.
It is to be understood that as used herein, the term "alkyl" means straight,
branched, cyclic,
and polycyclic materials. The term "haloalkyl" means partially or fully
halogenated alkyl groups
1 ~ such as -1 CH,):C1. -CF, and -C6F,3, for example.
In one of its embodiments, the invention relates to compounds of generalized
formula (L)
in which at least one of the units A, B, and R° comprises a
heteroaromatic ring. Preferred
heteroaromatic ring-containing compounds are those in which the heteroaryl
groups are heteroaryl
of ~ - 9 carbons comprising a 5 - 6 membered heteroaromatic ring containing O,
S, or NR' when the
?0 ring is 5-membered, and N when said ring is 6-membered. Particularly
preferred heteroaromatic
ring-containing compounds are those in which at least one of the A and B units
comprises a
thiophene ring. When A unit is thiophene, it is preferably connected to B unit
at position 2 and
/9
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97!43245 PCT/L1S97107921
caries one substituent croup T on position ~. When B unit is thiophene, it is
preferably connected
throueh positions 2 and 5 to D and A units respectively.
In the generalized formula (L), the A and B rings are preferably phenyl and
phenvlene.
respectively, the .W ing preferably bears at least one substituent group T
preferably located on the
position furthest from the position of the A ring which is connected to the B
ring, the D unit is
preferably a carbonyl group, and the G unit is preferably a carboxyl group.
In another embodiment. the invention relates to compounds of generalized
formula (L), in
the E unit of which n is 2 and m is 1. These compounds thus possess two carbon
atoms between the
D unit and the G unit, and carry one substituent on this two-carbon chain.
In another of iu embodiments, the invention relates to compounds of
generalized formula
(L) in which the A ring is a substituted or unsubstituted phenyl group, the H
ring is p-phenylene, and
aryl portions of any aryl-containing Rb moieties contain only carbon in the
rings. These compounds
thus contain no heteroaromatic rings.
In another of its embodiments, the invention relates to compounds of
generalized formula
I ~ ~ L ) in which m is l and Rb is an independent substituent. These
compounds are materials which
contain only a single substituent R° on the E unit, and this
substituent in not involved in a ring.
Preferred compounds within this subset have the formula
0
/ ~ / C CHiCHR6COOH
T
?0 in which x is 1 aad the substituent group T is located on the 4- position
of the A ring, relative to the
point of attachment between the A and B rings. The para substituent group T of
this subset is more
preferably acetylene containing moities selected from the following group:
MeOCH2C=C-, (»-
i~
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
Pr),VCH,C=C-, CH~CO:CH:C=C-, EtOCO,CH,C=C-. HOCH:C=C-, HO(CH,~,C=C-.
CH..CO=(CHIC=C-. HO=C(CH~C=C-. OHC(CH~~C=C-. HO(CH,),C=C-. PhC=C-. 3-HO-PhC=C
and PhCH,OCH,C=C-.
Other compounds of general formula (L) in which Rb is -(CH=),R' have t as an
integer of 1-~.
Preferred compounds of general formula (L) in which R6 is -(CH:)~ZR' have v as
an integer of 1-~l
and Z as -S- or -O-. additional compounds of general formula (L) in which R6
is alkyl contain ~
or more carbons in said alkyl and those in which R6 is an~lalkyl contain 2-3
carbons in the alkyl
portion of said arylalkyl.
In another of its embodiments. the invention relates to compounds of
generalized formula
IO (L) in which the number of substituents m on the E unit is 2 or 3; and when
m is 2, both groups R6
are independent substituents, or together constitute a spiro ring, or one
group R6 is an independent
substituent and the other constitutes a spiro ring; and when m is 3, two
groups R6 are independent
substituents and one group R6 constitutes a ring, or two groups R6 constitute
a ring and one group
R° is an independent substituent, or three groups Rb are independent
substitucnts. This subset
l ~ therefore contains compounds in which the E unit is di- or tri-
substituted, and in the disubstituted
case any rings formed by one or both R6 groups are spiro rings, and in the
trisubstituted case, the R°
groups may form either spiro or nonspiro rings.
in another of its embodiments, the invention relates to compounds of
generalized formula
(L) in which the number of substituents m on the E unit is 1 or 2; and when m
is 1, the group R6
30 constitutes a nonspiro ring; and when m is 2, both groups R6 together
constitute a nonspiro ring or
one group R6 is an independent substituent and the other constitutes a
nonspiro ring. 'Ibis subset
=1
SUBSTITUTE SHEET (RULE 26~
CA 02253796 2002-03-25
therefore contains compounds in Which the E unit carries one or two
substituents R6, and at least one
of these substituents is involved in a nonspiro ring.
More particularly, representative compounds of generalized formula (L) in
which one or
more of the substituent groups 1t6 are involved in formation of nonspiro rings
have E units of the
following structures:
- (fh.aR°~) (CR°.~t.~a-"
a
(Ht.eR°~)---(CR°~t.~?
~ (R,~Jr , ~'(lit.eR°eC~~R°~t.~-
(C.~..~ \ttR~°)~C
- (HZaR6aC) (CRsoH~.Ja""
tHt.bR°dC~'t~ Rae~'tt.~1
~lRt~
- (HZaR°,C) (CR°Wale"'~
(R°~. ~a
(Ht-eR°dC)----.--I °~t.e)
tR~Jt -(Ht-aR°~~~(CR°eHt.~-
(G~-r~ \ ~ tRt4h~
_. (~
- (HZ.,R°,C) (CRa,,HZ.~dw'
....
( \ _..
t~
(R )k
- (H2.aR°aC) (CR°,H2-a~a""'
(H~.eR°dC?w'- I~ et'rt~l
( -
"-r"'_'~'(Ru~c , 8ttd
m
22
CA 02253796 2002-03-25
inwhichais0, l,or2;bis0or i;cis0or l;dis0orl;c+dis0or leis 1-S;fis 1-4;gis3
- S; h is 2 - 4; i is 0 - 4; j is 4 - 3; k is 0 - 2; the total number of
groups R6 is 0, 1, or 2; U represents
O, S, or NR'; and z is 1 or 2; each group R'4 is independently selected from
the group consisting of
alkyl of 1 - 9 carbons; arylalkyl in which the alkyl portion contains 1 - 7
carbons and the aryl portion
contains 6 - 10 carbons; alkenyl of 2 - 9 carbons; aryl-substituted alkenyl in
which the alkenyl
portion contains 2 - 4 carbons and the aryl portion contains 6 -10 carbons;
alkynyl of 2 - 9 carbons;
aryl-substituted alkynyl in which the alkynyl portion contains 2 - 4 carbons
and the aryl portion
contains 6 - 10 carbons; aryl of 6 - 10 carbons; -CORi; -COZR3; -CON(Ri~; -
(CH~rR' in which t is
0 or an integer of 1 - 4; and -(CHZ),,ZR' in which v is 0 or an integer of 1
to 3, and Z represents -S-
or -O-. R', R', and R' have been defined above.
Preferred compounds of generalized formula (L) in which one or more of the
substituent
groups R6 are involved in the formation of nonspim rings have E units of the
following structures:
- (HziR°,C) ~ R°~2-~Id'-' - (H2.aR°sC1 (CR°aH2-
a)d'
(H~.bR° C)---(CR°eH~.~ (H~.bR°eCy--'(CR°n~'~~.~
l~~Jr , -~ -(R"h
(C,H~ (CoH~.y~
_" (H2.vR°sC) (CR°~2.Jd-'
~R°
(H~.eR ~( et'i~.~
lx"),,
(C;Hx.~t
in which a, b, c, d, (c + d), e, g, i, k, the total number of groups R6, U,
and R" are as defined above.
'?2a
CA 02253796 2002-03-25
The substituent group 'T is preferably sn acetylene containing moiety with the
general
formula:
R'°(CH~aC=C-
where n is 1-4 and R~° is selected from the group consisting of HO-,
Me0-, (n-Pr~N-, CH3C02-,
CH3CHiOC02-, HOzC-, OHC-, Ph-, 3-HO-Ph- and PhCHZO-, provided that when
R~° is Ph or 3-HO-
Ph,n=0.
23
CA 02253796 1998-11-OS
WO 97!43245 PCT/US97/07921
Vtost preferably, T is: ~teOCH,C=-C-. (n-Pr),NCH:C=C-. CH,CO,CH,C=C-.
EtOCO:CH,C=C-. HOCH,C=C-. HO(CH,),C=C-. CH,CO,(CH:),C=C-. HO,C(CH,)_C=C-.
OHCiCH:),C=C-, HO(CH,),C=C-, PhC=C-, 3-HO-PhC=C- and PhCH,OCH,C=C-.
The subscript x. which defines the number of T substituents, is preferably 1
or '_', most
preferably 1. and when x is 1 the T is preferably on the 4- position of ring
A.
The A ring is preferably a phenyl or thiophene ring, most preferably phenyl.
The B ring is preferably a 1,-1-phenylene or ?.~-thiophene ring, most
preferably
1.-~-phenylene.
The D unit is most preferably a carbonyl group.
In the E soup. R6 is preferably:
1 ) arylalkyl wherein the aryl portion contains 6 - 10 carbons and the alkyl
portion
contains 1 - 8 carbons;
'_' ) -(CH=),R' wherein t is 0 or an integer of 1 - ~ and R' is an imidoyl
group
containing an aromatic residue: or
l ~ 3 ) -(CH=)~ZRa wherein v is 0 or an integer of 1 - :~ , Z is S or 0, and
R' is aryl of 6
- 10 carbons or arylalkyl wherein the aryl portion contains 6 to 12 carbons
and the alkyl
portion contains 1 to 4 carbons.
The group R° is most preferably the following, wherein, any aromatic
moiety is preferably
substituted:
'_0 1 ) arylalkyl wherein the aryl portion is phenyl and the alkyl portion
contains 1 - 4
carbons;
~ZS~
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCTlUS97/07921
2) -(CH:)~R' wherein t is an integer 1 - 3, and R' is ~1-(1.?-naphthalene-
dicarbox-
imidoyl), N-(2.3-naphthalene-dicarboximidoyl), or ~-(1.8-naphthalene-
dicarboximidovl);
or'.~I-phthalimidoyl.
-! CH:),ZRB wherein v is an integer of 1 - 3. Z is S, and R~ is phenyl.
The more preferred compounds of generalized formula (L) have R° units
of the following
structures:
0
Ph
or /~ N I I
0
l0 Those skilled in the art will appreciate that many of the compounds of the
invention exist in
enantiomeric or diastereomeric forms, and that it is understood by the art
that such stereoisomers
generally exhibit different activities in biological systems. This invention
encompasses all possible
stereoisomers which possess inhibitory activity against an wimp, regardless of
their stereoisomeric
designations, as well as mixtures of stereoisomers in which at least one
member possesses inhibitory
1~ activity.
The most prefered compounds of the present invention are as indicated and
named in the Iist
below:
I) RIS 4'-(3-hydroxy-1-propynyl~y-oxo-a-(3-phenylpropyl)-[i,l'-biphenyl)-~-
butanoic acid,
tI) S-4'-(3-hydroxy-1-propynyl)-y-oxo-a-{3-phenylpropyl)-{1,1'-biphenyl)-
butanoic acid,
?0 III) R-4'-(3-hydroxy~1-propynyl)-y-oxo-a-(3-phenyipropyl)-[1,1'-biphenyl]-4-
butanoic acid,
N) 4'-(3-methoxy-1-propynyl)-y-oxo-a-{3-phenylpropyl)-[1,1'-biphenyl]-4-
butanoic acid,
V) y-oxo-a-(3-phenytpropyir4'-{3-propyl-1-hexynyl~[1,1'-biphenyl)-4-butanoic
acid,
ZS
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97143245 PCT/US97107921
VI) ~'-[3-(acetyloxy)-1-propvnyl]-y-o!co-a-(3-phenyipropyl)-[1,1'-biphenyl]-.~-
butanoic acid.
~'1T1 ~'-[3-[(ethoxvcarbonvl)oxy]-1-propvnyl]-y-oxo-a-(3-phenylpropyli-[l.l'-
biphenyl]--t.
butanoic acid.
~'iII~ ~'-~-~-hydroxy-1-butv~nyi)-y-oxo-a-(3-phenylpropyl)-[l.l'-biphenyl]--~-
butanoic acid.
3 L~C) -1'-[3-(acetyloxy)-1-propy~yl]-y-oxo-a-(3-phenylpropyl)-[1.1'-biphenyl]-
~-butanoic acid,
Y) .~'-(~-carboxy-1-butvnyl)-y-oxo-a-(3-phenylpropyl)-[1,1'-biphenyl]-i-
butanoic acid,
YI) y-oxo--t'-(3-oxo-1-penrynyl)-a-(3-phenylpropyl)-[1.1'biphenyl]-4-butanoic
acid.
~CII) ~'-(6-hydroxy-1-henynyl)-y-oxo-a-(3-phenylpropyl)-[1,1'-biphenyl]-i-
butanoic acid,
lIII) y-oxo-~'-(phenylethynyl)-a-(3-phenyipropyl)-[1.1'-biphenyl]-~-butanoic
acid and
~CIV) .i'-[3-hydroxyphenyl)ethvnyl]-y-oxo-a-(3-phenylpropyl)-[I.1'-biphenyl]-
.~-butanoic acid,
yCV) 1,3-dihydro-1.3-dioxo-a-[2-oxo-2-[4'-[3(phenylmethoxy)-1-propvnvl][1,1'-
biphenyl]-.~-
yl]ethyl]-2H-isoindole-2-butanoic acid, and
XVn 1.3-dihydro-a-[2-[4'-(hydroxyethynyl)[1,1'-biphenyl]-4-yl]-2-oxoethyl]-I.3-
dioxo-?N
isoindole-2-butanoic acid.
1~
General Preparative Methods:
The compounds of the invention may be prepared readily by use of known
chemical
reactions and procedures. Nevertheless, the following general preparative
methods are presented
to aid the reader in synthesizing the inhibitors, with more detailed
particular examples being
'_0 presented below in the experimental section describing the working
examples. :a,.ll variable groups
of these methods are as described in the generic description if they are not
specifically defined
below. The variable subscript n is independently defined for each method. When
a variable group
SUBSTITUTE SHEET {RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
with a given syTrtbol (i.e R9) is used more than once in a given structure, it
is to be understood that
zach of these groups may be independently varied within the range of
definitions for that svtnbol.
Geaera! Method A - '/he compounds of this invention in which the rings A and B
are
substituted phem~l and phenylene respectively are conveniently prepared by use
of a Friedel-Crafrs
reaction of a substituted biphenyl VIII with an activated acyl-containing
intermediate such as the
succinic or glutaric anhydride derivative ViIII or acid chloride MIV in the
presence of a Lewis acid
catalyst such as aluminum trichloride in an aprotic solvent such as i,l.'_.2-
tetrachloroethane. 'Ihe
well known Friedel-Crafrs reaction can be accomplished with use of many
alternative solvents and
acid catalysts as described by Berliner. Org. React., ~, '_'?9, 1949 and
Heaney, Comp. Org. Synth.
~ ?3.1991.
If the anhydride :vIIII is monosubstituted or multiply-substituted in an
unsymmetrical way,
the raw product VtI-A often exists as a mixture of isomers via attack of the
anhydride from either
of the two carbonyls. The resultant isomers can be separated into pure forms
by crystallization or
chromatography using standard methods known to those skilled in the art.
1 ~ When they are not commercially available, the succinic anhydrides ;~tIII
can be prepared via
a Stobbe Condensation of a dialkyl succinate with an aldehyde or ketone
(resulting in side chain R°),
followed by catalytic hydrogenation, hydrolysis of a hemiester intermediate to
a diacid. and then
com~etsion to the anhydride IvtIIII by reaction with acetyl chloride or acetic
anhydride. Alternatively,
the hemiester intermediate is convened by treatment with thionyl chloride or
oxalyl chloride to the
acid chloride NllV. For a review of the Stobbe condensation, including lists
of suitable solvents and
bases see Johnson and Daub, Org. React. 6_, 1, 1951.
2~
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-05
WO 97/43245 PCT/US97/07921
This method, as applied to the preparation of 1~LQT (R6 = H, isobuyt and H. n-
pentvt), has
been described Wolanin- et al.. C; S Patent 4,77 t .038.
~Iet6od a
O a ut~
Lewis acid - - '
iT)~% / \ / + O O Soi~~;% / \ / ~ Re Rb 0
~n
R~R6
~I n
!vQII
n=2or3
or
O OR~z ~ OR's
C1 R6 R6 0 ~~ % / \ / ~Rb Rs 0
/n ' - ' /n
~V :vt1-A-2
n=2or3
- - + O p O 0
/ \ / Lewis acid - - CO H
(T)x~ (CHI" ~~ Solvent ~~% / \ /
R (CH~~ R ~,
~I IvQII-A ~-p-33
1~ nsa-3
Base
O
CO H
('>~x / \ /
(~z)~ Ru
Ivll-A-4
Method A is especially useful for the preparation of cyclic compounds such as
MI-A-3, in
which two R6 groups are connected in a methylene chain to form a 3-7 member
ring. Small ring (3-5
ZS
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
member) anhydrides are readily available only as cis isomers which yield cis
invention compounds
ELI-.~-~. The traps compounds ~-A--t are then prepared by treatment of W-A-3
with a base such
as DBL' in THF. The substituted four member ring starting material anhydrides
such as ~-A-1
are formed in a photochemical ?-? reaction as shown below. This method is
especially useful for
the preparation of compounds in which R" is acetoxy or acetoxymethylene.
:after the subsequent
Friedel-Crafts reaction the acetate can be removed by basic hydrolysis and the
carboxyl protected
by conversion to 2-(trimethylsilyl)ethyl ester. The resultant intermediate
with R" = CH,OH can
be converted to invention compounds with other R'j groups by using procedures
described in
General Method G.
p\~p 0 ~ 0
UV tight
acaouiuzle
-1 Ri4
The Friedel-Crafts method is also useful when double bonds are found either
between C-2
and C-3 of a succinoyl chain (from malefic anhydride or l-cyclopentene-1,2-
dicarboxylic anhydride,
1 ~ for e~cample) or when a double bond is found in a side chain, such as in
the use of itaconic anhydride
as starting material to yield products in which two Rb groups are found on one
chain carbon together
to form an exo-methylene (=CHI) group. Subsequent uses of these compounds are
described in
Methods D.
General Method B - Altemativeiy the compounds MI can be prepared via a
reaction
sequence involving mono-alkylation of a dialkyl malonate MVI with an alkyl
halide to form
intermediate MVB, followed by alkylation with a haiomethyl biphenyl ketone
MVIZI to yield
intermediate NBC. Compounds of s~uucture IvBX are then hydrolyzed with aqueous
base and heated
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97143245 PCT/US97/07921
to decarboxyiate the malonic acid intermediate and yield VII-B-2 (Method B-1
). By using one
equivalent of aqueous base the esters V1I~B-2 with R'= as alkyl are obtained,
and using more than
m~o equivalents of base the acid compounds (R'= = H) are obtained. Optionally,
heat is not used
and the diacid or acid-ester III-B-1 is obtained.
Alternatively, the diester intermediate ~~(I~C can be heated with a strong
acids such as
concentrated hydrochloric acid in acetic acid in a sealed tube at about 110 'C
for about 2:~ hr to
yield ViI-B-1 (R'= = H). Alternatively, the reaction of MVI with MVIII can be
conducted before
that with the alkyl halide to yield the same MIX (Method 8-2).
Alternatively, a diester intermediate .W~IX, which contains R'= = allyl, can
be exposed to
Pd catalysts in the presence of pvrrolidine to yield MI-B-? (R'~ = H) (Dezeil,
Tetrahedron Lett. ~,
X371. 1990.
Intermediates MVII are formed from biphenyls MB in a Friedel-Craft reaction
with
haloacetyl halides such as bromoacetyl bromide or chioroacetyl chloride.
Alternatively, the
biphenyl can be reacted with acetyl chloride or acetic anhydride and the
resultant product
1 ~ halogenated with, for example. bromine to yield intermediates MVIII (X =
Br).
vlethod B has the advantage of yielding single regio isomers when Method A
yields
mixtures. Method B is especially useful when the side chains R6 contain
aromatic or hetemaromatic
rings that may participate in intramolecular acylation reactions to give side
products if Method A
were to be used. This method is also very useful when the R° group
adjacent to the carboxyl of the
?0 final compound contains hetematoms such as oxygen, sulfur, or nitrogen, or
more complex
functions such as imide rings.
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97107921
METHOD B
0 X
X~R6 X~BrotCl
~x
ewis acid 0
O~ H OR~~
I) ' I'R6 OR°i
X R6 H
Lewis acid 0 0 ~tVI
X
21 Halogenation ' /
. R6 R6 ~ Aah. base
c-r)~~
vtvt~ B- I ) o
B-2)
6
s O R ORis
X R 0 R6 0 ORiZ ~. H ORti
,"QX - - 0 base 0 'vtVfI
I 0 m ~ / ~ / R6 Rs
Saong acid
1 ) Aqueous base Heat or whoa
2) Acid Rli , ~yl, Pd(Ph3P)a~
0 pyrrolidine
0 R6 OH a 0 R6 H a
_ OR _ OR
/ 6 R6 O ~ / R6 R6 O
~x R
I ~ ~-B.I ~-B-2
When R6 contains selected functional groups Z, malonate MVII can be prepared
by
alkylating a commercially available unsubstituted malonate with prenyl or
allyl halide, subject this
product to ozonalysis with reductive work-up, and the desired z group can be
coupled via a
'_0 ~Iitsunobu reaction (Mitsunobu, Synthesis 1, 1981 ). Alternatively, the
intermediate alcohol can be
subjected to alkylation conditions to provide malonate MVII containing the
desired Z group.
3~
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
1 ) NaH, TFff . they
isoprenyl bromide
i ~' 2) 0~, CH:CI: then Rs
R O~R ;~a;gH,, VieOH R~iO~OR~'
~0 ~O 3) Mitsuaobu ~O ~0
3) CBr,, or Ph~P ,
4) Alkylanoa R6 - (CH:)=Z
General Method C - Especially useful is the use of chiral I~PLC to separate
the enantiomers
of racemic product mixtures (see, for example, Arit, et al., Chem. Int. Ed.
Engl. ,~, 30, i 991 ). The
compounds of this invention can be prepared as pure enantiomers by use of a
chiral auxiliary route.
See. for example, Evans. Aldtzchimica Acts. 1 ~(2), ~3, 1982 and other similar
references known
to one skilled in the art.
General rlethod D - Compounds in which R6 are alkyl- or aryl- or heteroaryl-
or acyl- or
heteroarylcarbonyl-thiomethylene are prepared by methods analogous to those
described in the
patent WO 90/05719. Thus substituted itaconic anhydride MXVI (n = 1) is
reacted under
Friedel-Crafts conditions to yield acid MI-D-1 which can be separated by
chromatography or
1 ~ crystallization from small amounts of isomeric MI-D-5. :alternatively, MI-
D-Ss are obtained by
reaction of invention compounds MI-D-4 (from any of Methods A through C) with
formaldehyde
in the presence of base.
Compounds MI-D~1 or MI-D-5 are then reacted with a mercapto derivative MXVII
or
VIXVIII in the presence of catalyst such as potassium carbonate,
ethyldiisobutylamine,
?0 tetrabutylamtnonium fluoride or free radical initiators such as
azobisisobutyronitrile (AIBI~ in a
solvent such as diethylfotinamide or tetrahydrofuran to yield invention
compounds MI-D-2, MI-D-3,
MI-D-6, or MI-D-7.
3y
SUBSTITUTE SMEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
Viethod D
o a
- ~R6 O Lev~ns~c~d
/ ' ~R6 ~ O SOIV~ / / ~ ~ ~ 6 6
'R R Jo 0
-D-1
NOM
o - ! or 2 - ~Q~D-5
0 R~ R~ 0 R6 R~
~ OH Fo_ rm~dehyd ~ - - o OH
/ O Bite mij, / ~
0
'.vQ-D-4
NQ-D-S
O SRS
OH
Vii, / ~ / RR61
H . S . R, ~I NQ-D-2
'D"! or O
OII ;vOMII 0 S-~-R9
HS~ R9 OH
~R R~, O
NQ-D-3
Re R6 OH
0
/n \\
H.S.Rs NaCVII ('17,r~ ~ ~ ~ ~ O
M-I-D-5 S' R~
0 ~ :vt-I-D-6
~~ R9 or
Rs R6 OH
0
In \~
O
'_'0
'.vi-I-D-7 O
R9
;3
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCTILTS97107921
General Method E - Biaryi compounds such as those of this application may also
be prepared by
Suzuki or Stille cross-coupling reactions of aryl or heteroaryl metallic
compounds in which the
metal is zinc. tin. magnesium. lithium, boron, silicon, copper, cadmium or the
like with an aryl or
heteroay 1 halide or triflate (trifluoromethane-sulfonate) or the like. In the
equation below either
Vlet or X is the metal and the other is the halide or triflate (OTf). Pd(com)
is a soluble complex of
palladium such as tetrakis(triphenylphosphine)-palladium(O) or bis-
Itriphenylphosphine)-
palladium(I)1) chloride. These methods are well known to chose skilled in the
art. See, for example.
Suzuki. Pure Appl. Chem. 63, Z13, 1994; Suzuki, Pure Appl. Chem. ø~3, 419.
1991; and Farina and
Roth. "Metal-Organic Chemistry" Volume 5 (Chapter 1 ), 1994.
The starting materials :~ixXBII (B = 1..~-phenylene) are readily formed using
methods
analogous to chose of methods A, B, C, or D but using a halobenzene rather
than a biphenyl as
starting material. When desired, the materials in which X is halo can be
converted to those in which
X is metal by reactions well known to those skilled in the art, such as
treatment of a bromo
intermediate with hexamethylditin and palladium tetrakistriphenylphosphine in
toluene at reflux to
1 ~ yield the trimethyltin intetTrtediate. The starting materials 1v00~ (B =
heteroaryl) are most
conveniently prepared by method C but using readily available heteroaryl
rather than biphenyl
starting materials. The intermediates MXXII are either commercial or easily
prepared from
commercial materials by methods well known to those skilled in the art.
Method E
?0 (T)tA-Met + X-B-E-G -i ('I~;A-B-D~E-G
VIXXII MXXIII Pd(com) MI-E
T, x. A, B, E and G as in Structure (L)
Met = Metal and X = Halide or Triflate
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
or
stet = Halide or Triflate arid X = Metal
These general methods are useful for the preparation of compounds for which
Friedel-Crafts
s reactions such as those of Methods A, B, C, or D would lead to mixtures with
various biaryf
acylation patterns. Lfethod E is also especially useful for the preparation of
products in which the
aryl soups. ~ or B, contain one or more heteroatoms (heteroaryls) such as
those compounds that
contain thiophene, furan, pyridine, pyrrole, oxazoie, thiazole, pyrimidine or
pyrazine rings or the
like instead of phenyls.
General ytethod F - When the Rb groups of method F form together a 4 - 7
member carbocyclic
ring as in Intermediate VQ~CV below, the double bond can be moved out of
conjugation with the
ketone soup by treatment with two equivalents of a strong base such as lithium
diisopropylamide
or lithium hexamethylsilylamide or the like followed by acid quench to yield
compounds with the
structure MXXVI. Reaction of :vQ~XVI with mercapto derivatives using methods
analogous to
1 ~ those of General Method D then leads to cyclic compounds MI-F-I or MI-F-2.
?0
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
Method F
0 O 0 0
\ - _ - t~W
/ \ o off
iT)x - \ ~ fCHJ" Solvent (T)=
HII \ ~ ( CH=)"-
W~QN
n - I-3
0
/ \ O OH
H.S. Ri (T~~ - \ ~ (CHI" \
O
O II
/ \ 0 OH SH~ R9 LXVIII
(T)X ~~ ~ (CHI" ~ S O
0
-F-1 R~ / \ OH
\ / (CHJn
Ci7~ -
S~O
:vQ-F-2
R9
General Method G - The compounds of this invention in which two Rb groups are
joined
to form a substituted 5-member ring are most conveniently prepared by method
G. In this method
l~ acid CII (R = H) is prepared using the protocols described in Tetrahedron
~7, Suppl., 411, 1981.
The acid is protected as an ester [eg. R = benryl (8n) or 2-
(trimethyisilyl)ethyl (TMSE)J by use of
coupling agents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride and
procedures well known to those skilled in the art. Substituted bromobiphenyl
CIa is converted to
its Grignard reagent by treatment with magnesium and reacted with CI to yield
alcohol CVI.
?0 alcohol CVI is eliminated via base treatment of its mesylate by using
conditions well known to
those skilled in the art to yield olefin CVII. Alternatively CITI is converted
to a uimethyltin
intermediate via initial metallation of the bromide with n-butyllithium at low
temperature (-78 °C)
36
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97143245 PCT1US97/07921
followed by treatment with chloronimethyltin and CI is converted to an
enoltriflate (C~ by reaction
wsth ?- (~1 ~l-bis(ttifluoromethylsulfonyl)amino)-~-chloropvridine in the
presence of a strong aprotic
base. The tin and enoltriflate intermediates are then coupled in the presence
of a Pd° catalyst. CuI
and AsPh, ~o yield directly intermediate CVII. Ozonoiysis of CVB (workup with
methylsufide)
~~ields aldehyde CVm. Alternatively treatment with OsO, followed by HIO,
converts CVII to
cVm.
Viethod G ~ ~1
w ,
ROC RFC I ~ _
BrMg / ~ \ \ ~ ~ CVI
(T~ OH
Cl CV 1) MsC!
C>PyN.I.~ 2) elimination
Mg
ROiC _ ROiC
'~ OTf X
/ + ~ ~\~ Pd(0~ /
1) HuLi ~ CILI: X = Br C~ 1) Ozone
2) CISnMe~ CN: X = SnMe~
2) MtSMt
1~
O CO=R O COiR
J~ ,,,cHO
,,vCHiOH
m~~ ~ ~~ m=%J cviB
or ~u~bu.
TsC1
acytatioa or
alkylation
o ~o=~ o co=R
?0
,,.CHzOTs ~ ~ ~~ ,,.CHZZ
alkytanon ~ ~ W
mi J cx m~ ~ c~a
3~
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/L1S97I07921
Conversion of key intermediate CVI>I to the targeted patent compounds is
accomplished in
several ways depending on the identity of side chain function Z. Reaction of
CVIII with Vl%ittig
reagents followed by hydrogenation yields products in which Z is alkyl and or
arylalkyl. Selective
reduction of aldehyde CVTII with a reducing agent such as lithium tris [(3-
ethyl-
3pentyl)oxy~altnninurtt hydride (LTEPA) yields alcohol C13C. The alcohoi is
converted to phenyl
ethers or a variety of heteroatom substituted derivatives used to generate
sidechain Z via the
Llitsunobu conditions well known to those skilled in the art (see Mitsunobu,
Synthesis. l, 1981).
Alternatively the alcohol of CL~C is convened to a leaving group such as
tosylate (C3~ or bromide
by conditions well known to those skilled in the art and then the leaving
group is displaced by an
appropriate nucleophile. Several examples of this type of reaction can be
found in Norman, et al.,
J. Died. Chem. 37, 252. 1994. Direct acylation of the alcohol CIX yields
invention compounds in
which Z = OAcyt and reaction of the alcohol with various alkyl halides in the
presence of base
yields alkyl ethers. In each case a final step is removal of acid blocking
group R to yield acids (R
= H) by using conditions which depend on the stability of R and Z, but in all
cases well known to
1 ~ those skilled in the art such as removal of benzyl by base hydrolysis or
of 2-ltrimethylsilyl)ethyl by
treatment with tetrabutylammonium fluoride.
General il~tet6od H - Amides of the acids of the invention compounds can be
prepared from
the acids by treat;rtent in as appropriate solvent such as dichloromethane or
dimethylfotmamide with
a primary or secondary amine and a coupling agent such as
dicyclohexylcarbodiimide. These
?0 reactions are well known to those skilled in the art. The amine component
can be simple alkyl or
arylalkyi substituted or can be amino acid derivatives in which the carboxyl
is blocked and the
amino group is free.
38
SUBSTITUTE SHEET (RULE 2~
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
General Method I - The compounds of this invention in which (T); is an
alky~nyl or
substituted alkynyl are prepared according to general method I (Austin. J.
Org. Chem. -~6, =X80.
1981 ). Inurmediate VfX is prepared according to methods A. B, C. D or G by
starting with
commercial VfItI IR' = 9r). Reaction of W~C with substituted acetylene 1~LYI
in the presence of
Cu(i) % palladate reagent gives invention compound VtI-I-1. In certain cases.
R' may be an alcohol
blocked as trialkylsilyl. In such cases the silyl group can be removed by
treatment with acids such
as tritluoroacetic acid or HF - pyridine reagent.
Method I
/Rs ~ /Rs
C (CH:),
1l7 Q fCHi). R3(~:~-C=CH / \ OH
,/ \ " ~ OH
/ (EthN Rs(CHih-C=Cue- \ /
~'t1. C Cul nao~~ W-I-1
dichlorob~~atp6myl
pbosphme pailadate
Suitable pharmaceutically acceptable salts of the compounds of the present
invention include
addition salts formed with organic or inorganic bases. The salt forming ion
derived from such bases
t ~ can be metal ions, e.g., aluminum, alkali metal ions, such as sodium or
potassium, alkaline earth
metal ions such as calcium or magnesium, or an amine salt ion, of which a
number are known for
this purpose. Examples include ammonium salts, arylalkylamines such as
dibenzylamine and
.V..~'-dibenrylethylenediamine, lower alkylamines such as methylamine, r-
butylamine, procaine,
lower alkylpiperidines such as N ethylpiperidine, cycloalkylamines such as
cyclohexylamine or
?0 dicyclohexylamine, 1-adarnantyiamine, benTathine, or salts derived from
amino acids like. arginine,
lysine or the like. The physiologically acceptable salts such as the sodium or
potassium salts and
the amino acid salts can be used medicinally as described below and are
preferred.
39
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
These and other sale which are not necessarily physiologically acceptable are
useful in
isolating or purifying a product acceptable for the purposes described below.
For example, the use
of commercially available enantiomerically pure amines such as (+)-cinchonine
in suitable solvents
can yield salt crystals of a single enatiomer of the invention compounds,
leaving the opposite
enantiomer in solution in a process often referred to as "classical
resolution." As one enantiomer
of a given invention compound is usually substantially greater in
physiological effect than its
antipode, this active isomer can thus be found purified in either the crystals
or the liquid phase. The
salts are produced by reacting the acid fotin of the invention compound with
an equivalent of the
base supplying the desired basic ion in a medium in which the salt
precipitates or in aqueous
medium and then lyophilizing. The free acid form can be obtained from the salt
by conventional
neutralization techniques, e.g.. with potassium bisulfate, hydrochloric acid,
etc.
The compounds of the present invention have been found to inhibit the matrix
metalloproteases l~tNiP-3. GIMP-9 and MMP-2, and to a lesser extent MMP-1, and
are therefore
useful for treating or preventing the conditions referred to in the background
section. As other
1 ~ Vil~lPs not listed above share a high degree of homology with those listed
above, especially in the
catalytic site, it is deemed that compounds of the invention should also
inhibit such other MMPs to
varying degrees. Varying the substituenu on the biaryl portions of the
molecules, as well as those
of the propanoic or butanoic acid chains of the claimed compounds, has been
demonstrated to affect
the relative inhibition of the listed MMPs. Thus compounds of this general
class can be "tuned" by
selecting specific substituenu such that inhibition of specific MIvIF'(s)
associated with specific
pathological conditions can be enhanced while leaving non-involved MMPs less
affected.
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97107921
The method of treating matrix metalloprotease-mediated conditions may be
practiced in
mammals. including humans, that exhibit such conditions.
The inhibitors of the present invention are contemplated for use in veterinary
and human
applications. For such purposes, they will be employed in pharmaceutical
compositions containing
active in~edient(s) plus one or more pharmaceutically acceptable carriers,
diluents, fillers, binders.
and other excipients. depending on the administration mode and dosage form
contemplated.
Administration of the inhibitors may be by any suitable mode known to those
skilled in the
art. Examples of suitable parenteral administration include intravenous,
intraarcicular, subcutaneous
and intramuscular routes. Intravenous administration can be used to obtain
acute regulation of peak
t 0 plasma concentrations of the drug. Improved half life and targeting of the
drug to the joint cavities
may be aided by entrapment of the drug in liposomes. It may be possible to
improve the selectivity
of tiposomal targeting to the joint cavities by incorporation of ligands into
the outside of the
liposomes that bind to svnovial-specific macromolecules. Alternatively
intramuscular, intraarticular
or subcutaneous depot injection with or without encapsulation of the drug into
degradable
1 ~ microspheres e.g., comprising poly(DL-lactide-co-glycolide) may be used to
obtain prolonged
sustained drug release. For improved convenience of the dosage form it may be
possible to use an
i.p. implanted reservoir and septum such as the Percuseal system available
from Pharmacia.
Improved convenience and patient compliance may also be achieved by the use of
either injector
pens (e.g. the Novo Pin or Q-pen) or needle-free jet injectors (e.g. from
Bioject, Mediject or Becton
?0 Dickinson). Prolonged zero-order or other precisely controlled release such
as pulsatile release can
also be achieved as needed using implantable pumps with delivery of the drug
through a cannula
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97/43245 PCT/I1S97/07921
into tile svnovial spaces. Examples include the subcutaneously implanted
osmotic pumps available
from ALZA, such as the ALZET osmotic pump.
~iasat delivery may be achieved by incorporation of the drug into bioadhesive
particulate
carriers 1_'00 um) such as those comprising cellulose, polyacrylate or
polycarbophil, in conjunction
with suitable absorption enhancers such as phospholipids or acylcarnitines.
Available systems
include those developed by DanBiosys and Scios Nova.
.~ noteworthy attribute of the compounds of the present invention in contrast
to those of
various peptidic compounds referenced in the background section of this
application is the
demonstrated oral activity of the present compounds. Certain compounds have
shown oral
bioavailabiliry in various animal models of up to 90 - 98 %. Oral delivery may
be achieved by
incorporation of the drug into tablets, coated tablets, dragees, hard and soft
gelatine capsules,
solutions, emulsions or suspensions. Oral delivery may also be achieved by
incorporation of the
drug into enteric coated capsules designed to release the drug into the colon
where digestive protease
activity is low. Examples include the OROS-CT/OsmetTM and PULSINCAPT'" systems
from ALZA
1 ~ and Scherer Drug Delivery Systems respectively. Other systems use azo-
crosslinked polymers that
are degraded by colon specific bacterial azoreductases, or pH sensitive
polyacrylate polymers that
are activated by the rise in pH at the colon. The above systems may be used in
conjunction with a
wide range of available absorption enhancers.
Rectal delivery may be achieved by incorporation of the drug into
suppositories.
''0 The compounds of this invention can be manufactured into the above listed
formulations by
the addition of various therapeutically inert, inorganic or organic carriers
well known to those skilled
in the art. Examples of these include, but are not limited to, lactose, corn
starch or derivatives
!f t
SUBSTITUTE SHEET (RULE 2b~
CA 02253796 1998-11-OS
WO 97/43245 PCTILTS97107921
thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene
glycol, water, saccharose,
alcohols, glycerin and the like. Various preservatives, emulsifiers,
dispersants, flavorants, wetting
aeents. antioxidants, sweeteners, colorants, stabilizers, salts, buffers and
the like are also added, as
required to assist in the stabilization of the formulation or to assist in
increasing bioavailability of
the active ingredients) or to yield a formulation of acceptable flavor or odor
in the case of oral
dosing.
The amount of the pharmaceutical composition to be employed will depend on the
recipient
and the condition being treated. The requisite amount may be determined
without undue
experimentation by protocols known to those skilled in the art. Alternatively,
the requisite amount
l 0 may be calculated, based on a determination of the amount of target enzyme
which must be inhibited
in order to treat the condition.
The matrix metalloprotease inhibitors of the invention are useful not only for
treatment of
the physiological conditions discussed above, but are also useful in such
activities as purification
of metalloproteases and testing for matrix metalloprotease activity. Such
activity testing can be both
1 s in vitro using natural or synthetic enzyme preparations or in vivo using,
for example, animal models
in which abnormal destructive enzyme levels are found spontaneously (use of
genetically mutated
or transgenic animals) or are induced by administration of exogenous agents or
by surgery which
disrupts joint stability.
,p E~,4MPLES
The following examples are offered for illustrative purposes only and are not
intended, nor
should they be construed, to limit the invention in any way.
9~3
SUE3STITUTE SHEET (RULE 26)
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General Procedt:res:
All reactions were performed in flame-dried or oven-dried glassware under a
positive
pressure of argon and were stirred magnetically unless otherwise indicated.
Sensitive liquids and
solutions were transferred via syringe or cannula and were introduced into
reaction vessels through
rubber septa. Reaction product solutions were concentrated using a Buchi
evaporator unless
otherwise indicated.
Viaterials:
Commercial grade reagents and solvents were used without further purification
except that
diethyl ether and tetrahydrofuran were usually distilled under argon from
benzophenone keryl, and
methylene chloride was distilled under argon from calcium hydride. Many of the
specialty organic
or organometallic starting materials and reagents were obtained from Aldrich.
1001 West Saint Paul
Avenue. Milwaukee, WI X3233. Solvents are often obtained from EM Science as
distributed by
V WR Scientific.
1 ~ Chromatography:
Analytical thin-layer chromatography (TLC) was performed on Whatmans pre-
coated
glass-backed silica gel 60 A F-254 250 ~cm plates. Visualization of spots was
effected by one of the
following techniques: (a) ultraviolet illumination, (b) expostue to iodine
vapor, (c) immersion of
the plate in a 10% solution of phosphomolybdic acid in ethanol followed by
heating, and (d)
?0 immersion of the plate in a 3% solution of p-anisaldehyde in ethanol
containing 0.5% concentrated
suifiu~ic acid followed by heating.
Column chromatography was performed using 230-400 mesh EM Sciences silica gel.
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
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Analytical high perfotmancc liquid chromatography (I-~LC) was performed at t
mL min''
on a 4.6 x 250 mm Microsorb~ cohunn monitored at 288 nm. and semi-preparative
HPLC was
performed at 24 mL min' on a 21.4 x 250 mm Microsorb~ column monitored at 288
nm.
Instrumentation:
Melting points (mp) were determined with a Thomas-Hoover melting point
apparatus and
are uncorrected.
Proton ('H) nuclear magnetic resonance (NMR) spectra wire measured with a
General
Electric GN-OMEGA 300 (300 MHz) spectrometer, and carbon thirteen ("C) NMR
spectra were
measured with a General Electric GN-OMEGA 300 (75 MHz) spectrometer. 'Most of
the
compounds synthesized in the experiments below were analyzed by nmr, and the
spectra were
consistent with the proposed strucnues in each case.
Mass spectral (MS) data were obtained on a Kratos Concept 1-H spectrometer by
liquid-cesium secondary ion (LCIMS), an updated version of fast atom
bombardment (FAB). Most
1 ~ of the compounds synthesized in the experiments below were analyzed by
mass spectroscopy, and
the spectra were consistent with the proposed structures in each case.
General Comments:
For mufti-step procedures, sequential steps are noted by numbers.
~5
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
Examyle 1- Preyaration of Com~ionnd I
0
v ~ ~OEt
0 OEt
Step 1 A dry 2-L, three-necked, round-bottomed flask was equipped with a stir
bar. a pressure
equalizing addition funnel, an argon inlet and a thermometer. The flask was
charged with a
suspension of sodium hydride (8.4 g of 95% NaH; 0.33 moi) in dry THF (700 mL)
and was cooled
with an ice water bath. Diethyl malonate (48.54 g, 0.30 mol) was added
dropwise from the addition
funnel over 25 min. Stirring was continued for 1.5 h before adding 1-bromo-3-
phenylpropane (47
I O mL. -61 g, -0.30 mol) over 10 min via the addition funnel. Rinses of the
addition funnel (THF. 3
x 10 mL) were added to the reaction mixture and stirring was continued for 30
min. The addition
funnel and thermometer were replaced with a reflex condenser and stopper, and
the reaction was
heated at reflex for 19 h. The mixture was cooled to room temperature and then
with an ice water
bath. Distilled water (400 mL) was slowly added with stirring. The layers were
separated and the
1 ~ aqueous phase was extracted with chloroform ( 100 mL). The combined
organics were washed with
10% HCl (250 mL) and the separated aqueous phase was back-extracted with
chloroform ( 100 mL).
The combined organics were washed with saturated NaHCO, (250 mL) and the
separated aqueous
phase was back-eacu~acted with chloroform ( 100 mL). The organics were dried
(Na=SO,) and
concentrated to yield a yellow oil which was purified by distillation through
a Vigreux column at
20 reduced pressure (0.4 torr). The fiaction boiling at 124-138 'C was cleaa
desired product (57.21 g,
0.206 mol; 68% yield). TLC (50% hexanes-dichloromethane): Rt= 0.32.
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97143245 PCT/US97107921
O
Br
Br
Step 2 A 1-L, one-necked, round bottom flask was equipped with a rubber septum
and an argon
inlet. The flask was charged with a solution of commercially available 4-
bromobiphenyl (~O.OOg,
0.'_ 1 ~ mol) in dichloromethane (100mL). Hromoacetyl bromide (2l.OmL. -t8.7g,
0.230 mot) was
added via syringe and the solution was cooled with an ice water bath to 0 JC,
while A1C13 (34.3g,
0.'_' ~ 8 mol) was added portionwise. Gas evolved >iom the opaque olive green
reaction mixture.
l0 After 2~h at room temperature, the reaction mixture was cautiously poured
into a cold saturated
aqueous NaHCO~ solution. The resulting mixture was extracted with three 200mL
portions of ethyl
acetate, and the combined organic layers were dried over Na,.SO, and
concentrated to afford the
desired pmduct as a yellow solid in quantitative yield. TLC (30%
dichloromethane-hexanes), Ri
= 0.30.
l~
20 Step 3 A dry 2-L, three-necked, round-bottomed flask was equipped with a
magnetic stir bar, as
argon inlet, and a pressure equalizing addition funnel. The flask was charged
with a solution of the
product of step 1 {63.0 g, 0.227 mol) in THF (500 mL). "Ihe reaction vessel
was cooled with an ice
y~
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97143245 PCT/US97/07921
water bath while soditun hydride (5.40 g of 9S% NaH, 0.214 mol) was added
slowly in portions.
The reaction mvetvre was stirred for 1 h at 0 °C, and a solution of the
product of step 2 (80.0 g, 0.31 ~
mol) in dt~.~ THF (300 mL) was added via addition funnel over ca. 20 min. The
deep orange reaction
mi~cture was stirred at room temperature under argon for 3 h. The reaction
vessel was cooled in an
ice water bath while distilled water ( 1 SO mL) was added cautiously. The
aqueous phase was
exnacted with three 300 mL portions of ethyl acetate, the combined organic
phases were dried over
VtgSO" and concentrated to afford 124 g of a dark orange oil. This material
was used in the
following operation without purification.
The orange oil was dissolved in 400 mL of 1:1 THF:methanol, and added to an
aqueous
~aOH solution (4 N, S00 mL. '_'.00 mot). The reaction mixture was stirred for
24 h at room
temperature, 48 h at SO °C, and 24 hotus at room temperature. The
majority of MeOH was removed
in vacuo and the residue extracted with a 200 mL portion of 1:1 ethyl
acetate:hexanes and a 200 mL
portion of hexanes. The aqueous phase was acidified with HCI, extracted with
two 200 mL portions,
and three 100 mL portions of ethyl acetate. The combined organic phases were
dried over MgSO,
1 ~ and concenuated to afford a quantitative yield of diacid. TLC ( 10%
methanol-chloroform with 1
acetic acid): R.r=0.45.
P~
Step 4 The unpurified diacid from step 3 was dissolved in 1,4-dioxane (500 mL)
aad heated to
reflex for 24 h. The solvent was removed in vacuo, and a 10 g portion of the
residue
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
chromatographed on silica gel (gradient elution with 10-50% ethyi acetate-
hexanes containing 1
acetic acid) to afford 0.840 g ( 10%) of the desired product as a yellow
solid. MP 174 °C.
0 Pb
HOCHtC=C ~ ~ ~ ~ OH
I
0
7
Step 5 - .a one-necked, 15-mL, round-bottomed flask equipped with a rubber
septum and an argon
needle inlet was charged with '_'.6 mL of diethylamine, the product of step 4
(0.300 g, 0.667 mmol).
propargyl alcohol ( 1.0 mL, 0.96 g, 17 mmol), copper (I) iodide (0.0220 g,
0.11 ~ mmol), and
craps-dichlorobis(triphenylphosphine)palladate (0.110 g, 0.157 mmol). The
resulting mixture was
stirred for .~ d at room temperature. The reaction mixture was concentrated
(290 mg residue) and
part of the residue (90 mg) was purified via column chromatography on 50 g of
silica get {20% ethyl
acetate-hexanes with 0.5% acetic acid) afforded the coupling product as a
white solid (0.035 g, 40%)
of coupling product as a white solid. MP 130 °C.
~xampte 2 and Example 3 - Preparation of Compounds II and I~
Example 2 and Example 3 were prepared by chiral separation of Example 1 on a
Chiralcel
~.D r column (2 cm x 25 em) using 5% EtOH. 4.75% H,O and 0.095% HOAc in CH,CN,
flow rate
?0 mLmin.
Example 2: First off Chiralcel AD ~ column; ' H NMR (300 N~iz, CDCI,) b 8.02
(d, J = 8.4 Hz,
?0 2H),7.67(d,J=8.7Hz,2H),7.58(d,J=8.7Hz,2H),7.53(d,J=8.4Hz,2H),7.17-7.33(m,5
H), 4.54 {s, 2 H), 3.46 (dd, J = 8.1, 16.8 Hz, 1 H), 3.14-3.02 (m, 2 H), 2.65
(t, J = 7.2 Hz, 2 H),
1.64-1.84 (m, 4 H).
g9
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97107921
Esampie 3: Second off Chiralcel AD ~ column; 'H NMR (300 MHz, CDCI,) E 8.02
(d. J = 8.4 Hz
' H).7.67(d,J=8.7Hz,2 H).7.58(d.J=8.7Hz,2H).7.53(d,J=8.4Hz2H),7.17-7.33 (m.~
H).-~.p4(s,2H),3..~6(dd,J=8.I, 16.8 Hz, 1 H),3.14-
3.02(m,2H),2.65(t,J=7.2Hz,2H),
1.64-1. 84 ( m. 4 H ).
Example 6 - PreQaration of Compound VI
.~ one-necked, 10-mL, round-bottomed flask equipped with a rubber septum and
an argon
needle inlet was charged with 0.~ mL of pyridine, Example 1 (0.0070 g, 0.014
mmol), and acetic
anhydride (0.020 mL. 22 mg, 0.21 mmol). The reaction mixture was stirred for 2
h at room
temperature, and then added to 30 mL of 1N HC1. The resulting mixture was
extracted with three
30 mL portions of ethyl acetate, the combined organic phases were dried over
~gSO,, and
concentrated. Purification via HPLC (2.5% ethyl acetate-dichloromethane with
0.01%
trifluoroacetic acid) afforded 3 mg (38%) of Example 6. MP 137 °C.
Example 7 - Preparation of Compound VII
1 ~ ~ one-necked, 15-mL. round-bottomed flask equipped with a rubber septum
and an argon
needle inlet was charged with 2 mL of triethylamine, 2 mi of THF, compound I
(0.0570 g, 0.134
mmol), and ethyl chlorofotmate (0.032 mL. 36 mg, 0.34 mmol). The reaction
mixture was stirred
for 16 h at room temperature and then added to 50 mL of 1N HCI. The resulting
mixture was
extracted with three 50 mL portions of ethyl acetate, the combined organic
phases were dried over
?0 VigSO,, and concentrated. Column chromatography on 10 g silica gel (40%
ethyl acetate-hexanes
with 0.~% HOAc) followed by purification via HPLC (1.5% ethyl acetate-
dichloromethane with
0.01% trifluoroacetic acid) afforded 1 mg (1.5%) of Example 7. MS (FAB-LSIMS)
499 [M+H)-.
So
SUBSTITUTE SHEET (RULE 26)
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ExamRie 11- Preyaration of Compo~pd XI
:~ one-necked, 2~-mL. round-bottomed flask equipped with a rubber septum and
an argon
needle inlet was charged with 1 mL of CH:CI=, Example 12 (0.012 g, 0.026
mmol), and the
Dess-Martin reagent (16 mg, 0.038 mmol) prepared according to Dess, et al.. J.
Org. Chem. 48.
4166. 1983. The resulting mixture was stirred for 30 min at 0 °C,
diluted with 30 mL of ethyl
acetate, and washed with two 20 mL portions of 1N HCI. The organic layer was
dried over ~IgSO"
and concentrated. Purification via HPLC ( 1.6% ethyl acetate-dichloromethane
with 0.01
trifluoroacetic acid) afforded 1 mg (9%) of Example 11. 'H NMR (300 MHz.
CDCI,) b 9.70 (t. J
= 1.3 Hz. 1 H), 8.05 (d, J = 8..~ Hz. 2 H), 7.70 (d, J = 8.4 Hz, 2 H), 7.65
(d. J = 8.4 Hz, 2 H). 7..~4
(d. J= 8.4 Hz. 2 H). 7.16-7.33 (m. ~ H), 3.46 (dd. J= 8.1, 16.8 Hz, 1 H), 3.14-
3.02 (m. 2 H), 2.67
(t. J = 7.'_ Hz. 2 H), 2.48 (t, J = 7.5 Hz. 2 H). 2.41 (dt. J = 1.3 Hz and 6.3
Hz, 2 H), 1.96 (m, 2 H),
1.64-1.84 (m. 4 H).
The above methods for the preparation of Example 1, Example 2, Example 6,
Example 7,
and lrxample 11 were used to prepare the following series of biphenyl
containing products.
I3
23
5I
SUBSTITUTE SHEET RULE 26~
CA 02253796 1998-11-OS
WO 97!43245 PCT/LJS97/07921
rABLE I
0 Ph
I ~ I ~ OH
0
comp R isomer m.p.(C)lother
characterization
I HOCH,C=C R.S 130
II HOCH,C=C S 'H NVtR (300
IvBiz. CDCI,)
b 8.02 (d,
J =
8.4 Hz, 2 H),
7.67 (d, l
= 8.7 Hz.
2 H), 7.58
!d. J = 8.7
Hz, 2 H),
7.53 {d, J
= 8.4 Hz.
2 H),
7.17-7.33 (m,
~ H), 4.54
(s, 2 H),
3.46 (dd,
J
= 8.1.16.8
Hz, 1 H),
3.02-3.14
(m, 2 H),
2.65
(t, J = 7.2
Hz. 2 H),
1.64-1.84
(m, 4 H).
III HOCH,_C=C R 'H NMR (300
IvB-Iz, CDCI,)
b 8.02 (d,
J =
8.4 Hz. 2 H),
7.67 (d, J
= 8.7 Hz,
2 H), 7.58
(d. J = 8.7
Hz, 2 H),
7.53 (d, J
= 8.4 Hz.
2 H),
7.17-7.33 (m,
~ H), 4.54
(s, 2 H),
3.46 {dd.
J
= 8.1,16.8
Hz, 1 H),
3.02-3.14
(m, 2 H),
2.65
(t, J = 7.2
Hz, 2 H),
1.64-1.84
(m, 4 H).
N MeOCH,C=C RS 136
V (-Pr),NCH,C=C R.S ' MS (FAB-LSIMS)
510 [M+H)'
VI CH,CO,CH~C=C RS 137
VII EtOCO:CH:C=C R,S leis (FAB-LSIMS)
499 [M+H]'
VIII HO(CH=)zC=C R,S 124
IX CH,CO:(CH~)~C=CR,S MS (FAB-LSIMS)
483 [M+H)-.
X HOzC(CH~C=C R,S 184
XI OHC(CHZ),C=C R,S 'H NMR
(300 MHz,
CDCh) b 9.70
(t, J=
1.3 Hz, 1 H),
8.05 (d, J
= 8.4 Hz,
2 H), 7.70
(d, J = 8.4
Hz, 2 H),
7.65 (d, J
= 8.4 Hz,
2 H),
7.44 (d, J=
8.4 Hz, 2
H), 7.15-7.35
(m, 5 H),
3.46 (dd, J
= 8.1, 16.8
Hz, 1 H),
3 .14-3.02
(m, 2 H), 2.67
(t, J a 7.2
Hz, 2 H),
2.48 (t, J
= 7.5 Hz, 2
H), 2.41 (dt,
J = 1.3 Hz
and 6.3
Hz, 2 H), 1.96
(m, 2 H),
1.64-1.84
(m, 4 H).
fZ
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97143245 PCTIUS97107921
XII HO(CH,),C=C R.S 123
PhC=C R,S 154
XIV 3-HO-PhC=C RS 237
Eramgle 15 - Prenaratioe of Compound XV
0' ~ N' ~ 0
J
~0 0
0 0
Step 1 A solution of sodium hydride (4.35 g, 181 mmol) in freshly distilled
THF (100 mL) was
cooled to 0 °C and treated with commercially available diallyl malonate
(35.0 g, 190 mmol) over
:~0 min via a dropping funnel. After stirring at room temperature for 30 min.
:W(2-bromoethyl)phthalimide {43.9 g, 247 mmol) was added to the solution in
one portion and the
1 ~ mixture was heated to reflux. After 48 h the solution was cooled to 0
°C, quenched with 2N HCl
and concentrated to about 20% of its original volume. The concentrate was
diluted with ethyl
acetate (300 mL) and washed successively with saturated aqueous solutions of
K:CO~ and NaCI.
The organic layer was dried over MgSO" filtered and concentrated under reduced
pressure.
Purification by flash column chromatography (gradient elution with 5-25% ethyl
acetate-hexanes)
afforded diallyl 2-phthalimidoethylmalonate (41.2 g, 64%) as a colorless oil.
'H NMR (300 MHz,
CDCI,) b 7.82 (m, 2H), 7.72 (m, 2H), 5.85 (m, 2H), 5.30 (m. 2H), 5.22 (m, 2H),
4.60 (m, 4H), 3.80
(t, J = 6.6 Hz, 2H), 3.46 (t, J = 7.2 Hz, 1 H), 2.30 (dd, J = 13.8, 6.9 Hz,
2H).
53
SUBSTITUTE SHEET (RULE 26)
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0 CO=Allyl 0
CO,Allyl
\ /
0
Br
Step 2 A solution of the product of step 1 (x.20 g, 1.x.6 mmol) in treshly
distilled THF (100 mL~
was cooled to 0 °C, while NaH (385 mg, 16.1 mmol) was slowly added.
After 40 minutes. the
reaction mixture was warmed to room temperature, the product of Example l,
step 2 (.~.>j g, 1.x.6
mmoi) was added in portions, and the miacture was stirred for 24 h. The
reaction mixture was cooled
to 0 'C, quenched slowly with 2~I HC1 (300 mL), extracted with one 150 mL
portion of
dichloromethane and two 100 mL portions of dichloromethane. The combined
organic phases were
dried over MgSO" filtered and concentrated to afford 6.~0 g (71%} of the
desired product which was
used in step 3 without purification. 'TLC (30% ethyl acetate.hexanes): Rf=
0.4.
O
N
1> > \ /
Step 3 A solution of the product of step 2 (6.50 8,10.4 mmol) in 1,4-dioxane (
100 mL) was cooled
to 0 ~C, while tetrakis(triphenylphosphine)paliadium (0.180 g, 146 mmol) and
pyrrolidine (2.40 mL,
29.'? mmol) were added sequentially. ABer stirring for 2 h at 0 'C and 4 h at
room temperature, the
reaction mixttae was poured into 2N HCl ( 100 mL). The resulting mixture was
extracted with four
100 mL portions of dichloromethane, the combined organic phases were dried
over MgSO,, and
concentrated to give the diacid as a yellow solid (9.70 g). A 3.8 g sample of
this material was
dissolved in 1,4-dioxane (150 mL) and heated at reflex for 1 h. After cooling
to room temperature,
sy
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
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WO 97143245
the solution was concentrated and the residue was chromatographed on 300 g
silica gel (gradient
with ~%-15% methanol-dichloromethane) to give the desired acid (0.300 g) which
was further
punned ~1a recrystallization to afford 0.170 g (59% overall yield from step 21
of the desired product
as a white cn~stalline solid. MP 209-210 °C.
j Ph~ 0
~--C=CH
Step -t A one-necked, 100-mL. round-bottomed flask equipped with a rubber
septum and an argon
needle inlet containing 2 ml of THF was charged with NaH (435 mg, 17.2 mmol)
and cooled to 0
'C while propargyl alcohol {1.0 mL, 0.963 g, 17.2 mmol) was added via syringe
over ca. S min. The
resulting mixture was stirred at 0 °C for 10 min and at room
temperature for 30 min. Henzylbromide
( 1.8 ml. 2.59 g, 15.1 mmol) was added, the reaction mixture was stirred at
room temperature for 36
h, poured into pentane (150 mL), and washed with a 100 mL portion of brine.
'Ihe solvent was
removed via distillation and the residue (3.5 g of a yellow oil) was used in
step 5 directly. 'H ~iR
(300 \~iHz, CDCI,) 5 7.36-7.31 (m. 5 H), 4.61 (s, 2H), 4.17 (d, J=2.4 Hz, 2
H), 2.47 (t, J= 2.4 Hz,
1 H).
\ /
O .N. ,0
O
Ph~O~C-C / \ / \ OH
O
Step 5 "f he procedure of Example 1, step 5 was used to prepare Example I 5
using the product from
step 4 and the product of step 3 as starting materials. MP 151 ~C.
SUBSTITUTE SHEET (RULE 26~
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WO 97/43245 PCT/US97/07921
xamp]e 16 - PreQaration of Compound XVI
t-BuM~SiO
~--C=CH
Step 1 ~ one-necked, 100-mL, round-bottomed flask equipped with a rubber
septum and an argon
needle inlet was charged with propargyl alcohol (1.0 mL. 0.963 g, 17.2 mmol),
ether f20 ml). and
cooled to 0 °C while NaH (435 mg, 17.2 mmol) was added siowly. The
resulting mixture was
stirred at room temperature for 1 h, and t-buryldimethylsilyl chloride (2.60
g, 17.2 mmol) was added.
The reaction mixture was stirred at room temperature for 6 h, poured into
hexane ( 150 mL), and
washed with 1 N HCI. The organic phase was dried over :vIgSO, and concentrated
to atTord 2.88
g of a yellow oil which was used in step 2 without purification. 'H NMR (300
biz, CDCh) b 4.29
~d. J = ~.1 Hz. 2 H), 2.37 (t. J=2.1 Hz, 1 H), 0.89 (s, 9 H), 0.11 (s, 3 H).
0
~ \ \ /
Step 2 The procedure of Example l, step 2 was used to prepare the desired
acetyl biphenyl using
commercially available 4-iodobiphenyl and aceryi chloride. TLC ( 10% ethyl
acetate-hexanes): R~-
1 ~ 0.3.
0
t-suMe=sio~ / \ / \
c-_-c
Step 3 The procedure ofExample 1, step 5 was used to prepare the desired
biphenyl acetylene using
the product of step 1 and the product of step 2. TLC ( 10% ethyl acetate-
hexanes): Rf- 0.4.
5G
-SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
0
r-HuMeZSiO~ ~ ~ ~ ~ ~ 8r
C=C
Step ~i .a one-necked. ~0-mL. round-bottomed flask equipped with a rubber
septum and an argon
s needle inlet was charged with ~ mL of THF, the product of step 3 (1.06 g,
?.94 mmol), and cooled
to -78 'C while potassium hexamethyldisilazide (617 mg. ?.94 mmol) was added
dropwise via
svrinee. The reaction mixture was stirred at -78 °C for 30 min,
trimethylsilyl chloride (0.374 mL.
0.3.0 g, '_.94 mmol) was added dropwise via syringe, and the resulting mixture
was stirred at -78
'C for 3 h. The reaction mixture was warmed to 0 °C for 1 h. N
bromosuccinimide (0.540 g, 2.94
I 0 mmoll was added, and the mixture was allowed to warm to room temperature
and stirred 16 h. The
reaction mixture was poured into a 100 mL portion of aqueous saturated NH,CI,
and extracted with
three ~0 mL portions of dichloromethane. The combined organic phases were
dried over MgSO,
and concenuated. Column chromatography on 200 g of silica gel (gradient
elution with 0- 5% ethyl
acetate-hexanes) afforded 0.264 g (20%) of the bromomethyl ketone. 'TLC ( 10%
ethyl
1 ~ acetate-hexanes): R~ 0.5.
O _N_ ,O
0
t-BuMezSiO - OH
~-C=C / \ ~ /
0
ss
SUBSTITUTE SHEET (RULE 26~
CA 02253796 1998-11-OS
PCT1US97107921
WO 97/43245
Step 5 The procedures of Example 13, steps 2-3 were used to prepare the
desired biphenyl
phthalimide using the product of step 4. TLC (~0% ethyl acetate-hexanes with 1
% acetic acid): R=
0.3.
\ /
0 -N- ' 0
0
HO - OH
~-C'-__C / \ \ /
0
Step 6 A one-necked. ~0-mi.. round-bottomed flask equipped with a rubber
septum and an argon
needle inlet was charged with 10 mL of CH_Cl,, the product from step ~ (0.040
g, 0.067 mmol), and
'_ mL of 1-ff'-pyridine. The resulting mixture was stirred for 10 minutes at
room temperature, diluted
with a 7~ mL portion of water, and extracted with a 75 mL portion of CH=C1:.
The organic phase
was dried over MgSO, and concentrated. Column chromatography on 5 g of silica
gel (25% ethyl
acetate-hexanes with 1 % HOAc) afforded 6 mg ( 19%) of Example 16. MP 145
°C.
1 ~ Example 17
Biological Assays of Invention Compounds
P218 Quenched Fluorescence Assay for MMP Inhibition:
The P218 quenched fluorescence assay (Microfiuorometric Profiling Assay) is a
modification of that originally described by Knight, et al.. FEBS Lett. ~,
263, 1992 for a related
substance and a variety of matrix metalloproteinases (MMPs) in cuvettes. The
assay was run with
each invention compound and the three MMPs, Mmp-3, MMP-9 and Nlivlp-2,
analyzed in parallel,
adapted as follows for a 96-well microtiter plate and a Hamilton A'T~
workstation.
58
SUBSTITUTE SHEET (RULE 26~
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WO 97143245
P218 Fluoro~~stTate: P218 is a synthetic substrate containing a -~-acetyl-7-
methoxycoumarin ( I~iCA) group in the N-terminal position and a 3-(2. -~-
dinitrophenvl)-L-2.3-
diaminopropionyl (DPA) group internally. This is a modification of a peptide
reported by Knight
( 199'_'1 that was used as a substrate for matrix metalloproteinases. Once the
P'_18 peptide is cleaved
putative clip site at the Ala-Leu bond), the fluorescence of the MCA group can
be detected on a
tluorometer with excitation at 328 rtm and emission at 393 nm. P218 is
currently being produced
BACHE~t exclusively for Bayer. P218 has the structure:
H-VIC?~-Pro-Lys-Pro-Leu-Ala-Leu-DPA-Ala-Arg-NH2 (MW 1332.2)
Recombinant Hutnan CHO Stromelvsin l:~:HP-31
Recombinant Human CHO Pro-;t-f.'l-fP-3: Human CHO pro-stromelysin-257 (pro-
~L\~iP-3)
was expressed and purified as described by Housley, et al., J. Biol. Chem.
?~,$, 4481. 1993.
.-lcrivation ojPro-~yfMP-3: Pro-MMP-3 at 1.72 ~M ( 100 ~g/mL) in 5 mM Tris at
pH 7.5, 5
mNt CaCI,. ~5 mM NaCI, and 0.005% Brij-35 MMP-3) activation buffer) was
activated by
incubation with TPCK (N-tosyl-(L)-phenylalanine chloromethyl ketone) trypsin
(1:100 wlw to pro-
Vi:~tP-3) at 25 'C for 30 min. The reaction was stopped by addition of soybean
trypsin inhibitor
(SBTI; 5:1 Ww to trypsin concentration). This activation protocol results in
the formation of 45 kDa
active ~L~LP-3. which still contains the C-terminal portion of the enzyme.
Preparation of Human Rer~mbinant Pro-Gelatinase A IMMP-21:
Recombinant Human Pro-~~fMP-2: Human pro-gelatittase A (pro-MMP-2) was
prepared
?0 using a vaccinia expression system according to the method of Fridman, et
al., J. Biol. Chem. 2~7,
15398, 1992.
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
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PCT/US97107921
activation of Pro-~l~LtfP-2: Pro-MMP-2 at 252 mglmL was diluted 1:5 to a final
concentration of SO p.glmL solution in 25 mM Tris at pH 7.5, ~ mM CaCI,. 1 ~0
mM NaCI, and
0.005°'° Brij-35 (1~-2 activation buffer). p-Aminophenylmercuric
acetate ( APMA) was prepared
in 10 m-~t c3.~ mymL) in 0.05 NaOH. The AP'v1A solution was added at l;'20 the
reaction volume
for a final AMPA concentration of 0.~ mM, and the enzyme was incubated at 37
°C for 30 min.
:activated MMP-2 (l5 mL) was dialyzed twice vs. 2 L of MMP-2 activation buffer
(dialysis
membranes were pre-treated with a solution consisting of 0.1 % BSA in bLl~tP-2
activation buffer
for 1 min. followed by extensive H,O washing). The enzyme was concentrated on
Centricon
concentrators (concentrators were also pre-treated a solution consisting of
0.1% BSA in MMP-2
activation buffer for 1 min.. followed by washing with H,O. then MMP-2
activation buffer) with re-
dilution followed by re~oncentration repeated twice. The enzyme was diluted to
7.5 mL (0.5 times
the original volume) with MNLP-2 activation buffer.
Preparation of Human Recombinant Pro-Gelatinase B (l4iMP-9)~
Recombinant Human Pro-~~fMP-9: Human pro-gelatinase B (pro-I~iMP-9) derived
from
1 ~ L;937 cDNA as described by Wilhelm, et al. J. Biol. Chem. ~~,~4, 17213,
1989 was expressed as the
full-length form using a baculovirus protein expression system. The pro-enzyme
was purified using
methods previously described by Hibbs, et a1.1. Biol. Chem. 2~Q, 2493, 1984.
Activation ojPro-MMP-9: Pro-MMP-2 20 pg/mL in 50 mM Tris at pH 7.4, l OmM
CaCI,,
1 ~0 mM NaCI, and 0.005% Btij-35 (I~-9 activation buffer) was activated by
incubation with 0.5
30 mMp-aminophetrylmercuric acetate (APMA) for 3.5 h at 37 °C. The
enzyme was dialyzed against
the same buffer to revmove the A.PMA.
SUBSTITUTE SNEET (RULE 26~
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he strumeatatios:
Hamiltion .~ficrolab .~T Plus: The :vL'viP-Profiling Assay is performed
robotically on a
Hamilton ~LicroLab AT PlusD. The Hamilton is programmed to: ( 1 ) serially
dilute up to 11 potential
inhibitors automatically from a 2.~ mM stock in 100% DMSO; (2) distribute
substrate followed by
inhibitor into a 96 well Cytofluor plate; and (3) add a single enzyme to the
plate with mixing to start
the reaction. Subsequent plates for each additional enzyme are prepared
automatically by beginning
the program at the substrate addition point, remixing the diluted inhibitors
and beginning the
reaction by addition of enzyrrte. In this way, all M~ assays were done using
the same inhibitor
dilutions.
.tfillipore Cvtofluor 1l. Following incubation, the plate was read on a
Cy~tofluor II
fluorometric plate reader with excitation at 340 nM and emission at 395 nM
with the gain set at 80.
Buffe
1.licrofluoromerric Reaction Buffer (MRB): Dilution of test compounds,
enzymes, and P~ 18
substrate for the microfluorometric assay were made in microfluorometric
reaction buffer consisting
1 ~ of ~0 mM 2-(tai-morpholinokthanesulfonic acid (IvIES) at pH 6.5 with 10 mM
CaCI:, 150 mM NaCI,
0.005% Brij-3~ and 1% DMSO.
a h s:
~I~f~NP .'I~ficrofluorometric Prof ling Assay. The assay is done with a final
substrate
concentration of 6 p.M P218 and approximately .~ to .8 nM'.~LMP with variable
drug concentrations.
'_'0 The Hamilton is programmed to serially dilute up to 11 compounds from a
2.5 mM stock (100%
DMSO) to lOx the final compounds concentrations in the assay. Initially, the
instrument delivers
various amounts of microfluoromentric reaction buffer (MRB) to a 96 tube rack
of 1 ral Marsh
61
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97!43245 PCT/ITS97/07921
dilution tubes. the instrument then picks up 20 ~1 of inhibitor (2.5 mM) from
the sample rack and
mixes it with a buffer in row A of the Marsh rack, resulting in a ~0 ~tM drug
concentration. The
inhibitors are then serially diluted to 10, 5, 1. .2, .OS and .01 ~M. Position
1 on the sample rack
contains only DViSO for the "enzyme-only" wells in the assay, which results in
no inhibitor in
column 1, rows A through H. The instrument then distributes 107 u! of P? 18
substrate (8.2 uM in
VtRB) to a single 96 well cvtofluor microtiter plate. The instrument re-mixes
and loads 14.~ ~1 of
diluted compound from rows A to G in the Marsh rack to corresponding rows in
the microtiter plate.
( Row H represents the "background" row and 39.5 ~l of MR.B is delivered in
placed of drug or
enzyme). The reaction is started by adding 25 ~1 of the appropriate enzyme (at
5.86 times the final
enzyme concentration) from a BSA treated reagent reservoir to each well,
excluding Row H, the
"background" row. (The enzyme reservoir is pretreated with 1% HSA in ~0 m:'vf
Tris, pH 7.5
containing 150 mM NaC 1 for 1 hour at room temp., followed by extensive H,O
washing and drying
at room temp.).
~~frer addition and mixing of the enzyme, the plate is covered and incubated
for 25 min. at
1 ~ 37 'C. Additional enzymes are tested in the same manner by beginning the
Hamilton program with
the distribution of P218 substrate to the microtiter plate, followed by re-
mixing and distribution of
the drug from the same Marsh rack'to the microtiter plate. The second (or
third, etc.) MMP to be
tested is then distributed from a reagent rack to the microtiter plate with
mixing, prior to covering
and incubation. This is repeated for all additional MMP's to be tested.
lCSO Determination in Micro~luorornetric Assay: Data generated on the
Cytofluor II is
copied from an exported ".CSV" file to a master Excel spreadsheet. Data from
several different
MMPs (one 96 well plate per MMP) were calculated simultaneously. The percent
inhibition is
SUSSTIT'UT"E SNEET (RULE 2'6~
CA 02253796 1998-11-OS
WO 97/43245 PCTNS97/07921
determination for each drug concentration by comparing the amount of
hydrolysis (fluorescence
units generated over '_'~ minutes of hydrolysis) of wells containing compound
with the wertzyme
only" wells in column 1. Following subtraction of the background the percent
inhibition was
calculated as:
((Control values - Treated values)/Control values) x 100
Percent inhibitions were determined for inhibitor concentrations of 5, l, 0.~,
0.1, 0.02. 0.00 and.
0.001 ~M of drug. Linear regression analysis of percnet inhibition versus log
inhibitor
concentration was used to obtain IC,o values.
Tab a
COMP.i~ MMP-3 Fluorogenic MMP-9 FluorogenicMMP2 Fluorogenic
IC-50 IC-50 IC-50
I 21 106 4
II 2184 I = 35% 252
III 5 37 I
IV 38 704 21
V 327 2630 235
VI 36 834 14
VII 67 '460 103
VUI 32 122 6
Dt 57 542 37
X 203 I = 27% 108
XI 1730 I = 24% 596
XII 56.6 614 36
XIII 405 245
63
SUBSTITUTE SHEET (RULE 26)
CA 02253796 1998-11-OS
WO 97/43245 PCT/US97/07921
XIV 125 I = 46% 85
~f V 11
~CVt 4 2 1
Other embodiments of the invention will be apparent to those skilled in the
art from a
consideration of this specification or practice of the invention disclosed
herein. It is intended that
the specification and examples be considered as exemplary only, with the true
scope and spirit of
the invention being indicated by the following claims.
d~
SUBSTITUTE SHEET (RUtE 26)
