Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
NOVEL SUCCINIC ACID METALLO-BETA-LACTAMASE INHIBITORS AND
THEIR USE IN TREATING BACTERIAL INFECTIONS
BACKGROUND OF THE INVENTION
The present invention relates to compounds which have metallo-(3-
lactamase inhibitory characteristics. The invention also relates to methods of
preparing, pharmaceutical compositions and uses of the compounds.
Metallo-(3-lactamases are bacterial enzymes which confer resistance to
virtually all clinically relevant ~i-lactam antibiotics, including carbapenems
and
jeopardize the future use of all such agents. The increased treatment of
infections with
carbapenems and other (3-lactam antibiotics may lead to the proliferation of
clinical
bacterial strains which are able to produce metallo-(3-lactamases and thus
resist the
effects of ~3 -lactam antibiotics. In fact, metallo-~i-lactamases have now
been
identified in a number of pathogenic bacterial species including Bacillus
cereus,
Bacteroides fragilis, Aeromonas hydrophila, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Shigella
flexneri,
Legionella gormanii, Chryseobacterium meningosepticum, Chryseobacterium
indologenes, Acinetobacter baumannii, Citrobacter freundii, and Aeromonas
veronii.
Accordingly, there is an increasing need for agents which when
combined with a (3-lactam antibiotic, e.g. imipenem, will restore the
effectiveness of
the [3-lactam antibiotics and which are at the same time relatively free from
undesirable side effects.
WO 98/17639, 97/30027, 98/40056,98/39311 and 97/10225 teach
certain beta-thiopropionyl-amino acid derivatives and their use as inhibitory
agents
against metallo-(3-lactamases. Goto et. al., Biol. Pharm. Bull. 20, 1136
(1997), Payne
et. al., FEMSMicrobiology Letters 157, 171 (1997), Payne et al., Antimicrob.
Agents
Chemother. 41, 135 (1997), Page et. al., Chem. Commun. 1609 (1998) and Page et
al.,
Biochem. J. 331, 703 (1998) also disclose certain thiols and thioesters as
metallo-(3-
lactamase inhibitors. Additionally, Toney et al., Chemistry and Biology 5, 185
(1998), Fastrez et al., Tetrahedron Lett. 36, 9313 (1995), Schofield et al.,
Tetrahedron
53, 7275 (1997), Schofield et. al., Bioorg. & Med. Chem. Lett. 6, 2455 (1996)
and
WO 97/19681 disclose other metallo-(3-lactamase inhibitors. However, the above
noted references do not teach the compounds of the instant invention.
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Other references which disclosed the general state of the art are Bush et
al., Antimicrob. Agents Chemother. 41, 223 (1997); Livermore, D. M. J.
Antimicrob.
Chemother. 1998, 41 (Suppl. D), 25; Bush, K. Clin. Infect. Dis. 1998, 27
(Suppl 1),
548; Livermore, D. M. J. Antimicrob. Chemother. 1997, 39, 673 and Payne, D. J.
J.
Med. Microbiol. 1993, 39, 93.
SUMMARY OF THE INVENTION
This invention relates to novel substituted succinic acid metallo-(3-
lactamase inhibitors, which are useful potentiators of (3-lactam antibiotics.
Accordingly, the present invention provides a method of treating bacterial
infections
in animals or humans which comprises administering, together with a (3 -lactam
antibiotic, a therapeutically effective amount of a compound of formula I:
2 R1
M20 ~OM1
O O
I
including pharmaceutically acceptable salts, prodrugs, anhydrides, and
solvates
thereof, wherein:
M1 and M2 are independently selected from:
(a) Hydrogen,
(b) Pharmaceutically acceptable canon, and
(c) Pharmaceutically acceptable esterifying group;
R1 and RZ are independently selected from the following:
(a) Hydrogen, provided that Rl and R2 are not hydrogen at the same time;
(b) a C1 to C~6 straight, branched or unsaturated alkyl group optionally
substituted
with 1 to 3 RX groups and optionally interrupted by one of the following O, S,
502, -C(O)-, -C(O)-NRa-, -COZ-;
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(c) a group of the formula:
(RX)o-s
wherein
-A- represents a single bond, C1 to Cg straight, branched or unsaturated alkyl
group
optionally substituted with 1 to 2 Rx groups and optionally interrupted by one
of the
following O, S, S02, -C(O)-, -C(O)-NRa-, -CO2-;
~ represents:
( 1 ) a C6 to C 14 aryl group;
(2) a C3 to Clo alicyclic group;
1 S (3) a C3 to C14 heteroaryl group, which contains 1 to 3 heteroatoms, 0 to
3 of which
heteroatoms are nitrogen and 0 to 1 of which are oxygen or sulfur;
(4) a C3 to Clo heterocyclic group, which contains 1 to 2 heteroatoms, 0 to 1
of which
heteroatoms are nitrogen, and 0 to 2 of which are oxygen or sulfur;
(d) a group of the formula:
A' c
(RX)o-2 (RX)o-2
wherein:
-A- is as defined above;
A' is a single bond, O, S, or a C1 to C( straight, branched or unsaturated
alkyl group
optionally substituted with 1 to 2 Rx groups and optionally interrupted by one
of the
following groups O, S, 502, -C(O)-, -C(O)-NRa-, -COZ-;
O and
are independently selected from:
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( 1 ) a C6 to C ~ o aryl group;
(2) a C3 to Cg alicyclic group;
(3) a C2 to C9 heteroaryl group, which contains 1 to 3 heteroatoms, 0 to 3 of
which
heteroatoms are nitrogen and 0 to 1 of which are oxygen or sulfur;
(4) a C3 to Cg heterocyclic group, which contains 1 to 2 heteroatoms, 0 to 1
of which
heteroatoms are nitrogen, and 0 to 2 of which are oxygen or sulfur;
where each RX is independently selected from the group consisting of
(a) F, Cl, Br, I ,
(b) CF3,
(c) ORb,
(d) CN,
(e) -C(O)-Rc ,
(~ -s(02)-Rf
(g) -C(O)-ORa
(h) -O-C(O)-Rc,
(i) -s-Rb
~) -N(Ra)-C(O)-Rc~
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NORa
-C-R~ ,
/ORb
CI) _C_N\
Ra
/ Rd
(m) -O-C_N\
Re
,
O d
R
O) _C_N/
Re
,
O d
) OS~N/R
\Re
,
Ra O Rd
~P) -N-CI-N/
Re
,
(c~ -N(Ra)-C(O)-ORf
(r) -S(O)-Rf
(s) -N(Ra)-S(02)-Rf
(t) N02, and
(u) C1 to Cg straight, branched or unsaturated alkyl optionally substituted
with one of
the substituents (a) through (t) above;
(v) -CH2-aryl wherein the aryl is optionally substituted with one of the
substituents
(a) through (t) above;
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or two adjacent RX groups on an aromatic ring may consist of the following
divalent
moiety, -O-CH2-O-;
wherein:
Ra is H, C 1 to C6 alkyl optionally substituted with RY;
Rb is H, C1 to C6 alkyl optionally substituted with RY, CH2_aryl, or aryl,
said aryls
optionally substituted with 1-2 RY groups;
Rc is H, C1 to C6 alkyl optionally substituted with RY, CF3, or aryl, said
aryl
optionally substituted with 1 to 2 RY groups;
Rd and Re are independently hydrogen, C1 to C4 alkyl optionally substituted
with RY
1 S or Rd and Re taken together may represent a 3 to 5-membered alkyl radical
to form a
ring, or Rd and Re taken together may represent a 2 to 4-membered alkyl
radical
interrupted by O, S, SO or S02 to form a ring;
Rf is C1 to C6 alkyl optionally substituted with RY, or aryl, said aryl
optionally
substituted with 1 to 2 RY groups; and
RY is -OH, -OCH3, OCONH2, OCOCH3, CHO, COCH3, C02CH3, CONH2, CN,
SOCH3, S02CH3, S02NH2, F, Cl, Br, I or CF3.
The invention is intended to include all of the isomeric forms of the
compounds of formula I, including racemic, enantiomeric and diastereomeric
forms.
Also included in this invention are compositions containing the
compounds of formula I and method of treatments using the same.
DETAILED DESCRIPTION OF THE INVENTION
The invention is described herein in detail using the terms defined
below unless otherwise specified.
The term "alkyl" refers to a monovalent alkane (hydrocarbon) derived
radical containing from 1 to 16 carbon atoms unless otherwise defined. It may
be
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straight or branched. Preferred alkyl groups include methyl, ethyl, propyl,
isopropyl,
butyl and hexyl. When substituted, alkyl groups may be substituted with up to
3
substituent groups, selected from RX as defined, at any available point of
attachment.
When the alkyl group is said to be substituted with an alkyl group, this is
used
interchangeably with "branched alkyl group". When the alkyl chain is
interrupted by
a group, eg. O, this may occur between any two saturated carbons of the alkyl
chain.
The term unsaturated alkyl refers to "alkenyl" or "alkynyl". The term
"alkenyl" refers to an unsaturated alkyl such as a hydrocarbon radical,
straight or
branched containing from 2 to 16 carbon atoms and at least one carbon to
carbon
double bond. Preferred alkenyl groups include propenyl, hexenyl and butenyl.
The
term "alkynyl" refers to an unsaturated alkyl such as a hydrocarbon radical
straight or
branched, containing from 2 to 16 carbon atoms and at least one carbon to
carbon
triple bond. Preferred alkynyl groups include propynyl, hexynyl and butynyl.
The term "alicyclic" refers to non-aromatic monocyclic or bicyclic
C3-C10 hydrocarbons, including unsaturated, which can be substituted with 0-3
groups of Rx. Examples of said groups include cycloalkyls such as cyclohexyl,
cyclopentyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]hepta-2,5-dienyl,
bicyclo[2.2.2]octyl,
bicyclo[2.2.2]octa-2,5-dienyl.
The term "alkylidene" refers to an alkyl group which is attached
through two bonds on the same carbon atom of the alkyl group to a single
attachment
atom, Examples of said groups include methylene, ethylidene, isopropylidene
and the
like.
Examples of when Rd and Re are taken together along with the
adjacent nitrogen atom to represent a 3 to 5 membered alkyl radical forming a
ring or
a 2 to 4 membered alkyl radical interrupted by O, S, SO, 502, to form a ring
are
pyrrolidinyl, piperidinyl, morpholinyl and the like.
The term "heterocyclic" refers to a monocyclic non-aromatic moiety
containing 3-8 ring atoms or a bicyclic non-aromatic moiety containing 6-10
ring
atoms, at least one of which ring atoms is a heteroatom selected from
nitrogen,
oxygen and sulfur and where one additional ring atom may be oxygen or sulfur.
Examples of heterocyclic groups are furanyl, pyranyl, morpholinyl, dioxanyl
and
quinuclidinyl:
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O O O N
O
N .O
furan pyran H dioxane quinuclidine
morpholine
Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and the
like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl
fluorenonyl and
the like. An aryl group thus contains at least one ring having at least 6
atoms, with up
S to three such rings being present, containing up to 14 atoms therein, with
alternating
(resonating) double bonds between adjacent carbon atoms. The preferred aryl
groups
are phenyl, naphthyl, and fluorenone. Aryl groups may likewise be substituted
as
defined. Preferred substituted aryls include phenyl, fluorenonyl and naphthyl.
The term "heteroaryl" (Het) refers to a monocyclic aromatic group
having 5 or 6 ring atoms, a bicyclic aromatic group having 8 to 10 atoms, or
tricyclic
having 12-14 ring atoms, containing at least one heteroatom, O, S or N, in
which a
carbon or nitrogen atom is the point of attachment, and in which one or two
additional
carbon atoms is optionally replaced by a heteroatom selected from O or S, and
in
which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen
1 S heteroatoms, said heteroaryl group being optionally substituted as
described herein.
Examples of this type are pyrrole, pyridine, oxazole, thiazole, dibenzofuran,
dibenzothiophene, carbazole, phenanthrene, anthracene, dibenzothiophene
sulfone,
fluorenone, quinoline and oxazine. Additional nitrogen atoms may be present
together with the first nitrogen and oxygen or sulfur, giving, e.g.,
thiadiazole.
Examples include the following:
_g_
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N N S
I I /~ /
CO C N
N
pyrrole imidazole thiazole
O O S
I / I /
N
oxazole furan thiophene
O
N N~ I / N
/N,
N
pyrazole isoxazole
triazole
S \ N
I /N
CO
N N
isothiazole pyridine
pyrazine
I \~ ~ N NON
N: N , I J , ~ J ,
N N
pyridazine pyrimidine triazine
/ I
I \ \ / I w
Ni / \ N J
quinoline ' phenanthridine
\ \ \ \
I I and I
O / / S
dibenzofuran dibenzothiophene
The term "heteroatom" means O, S or N, selected on an independent
basis.
Halogen and "halo" refer to bromine, chlorine, fluorine and iodine.
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The term "pro-drug" refers to compounds with a removable group
attached to one or both of the carboxyl groups of compounds of formula I (e.g.
biolabile esters). Groups which are useful in forming pro-drugs should be
apparent to
the medicinal chemist from the teachings herein. Examples include
S pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, and
others described in detail in U.S. Pat. No. 4,479,947. These are also referred
to as
"biolabile esters.
The term "hydrate" is used in the conventional sense to include the
compounds of formula I in physical association with water.
When a group is termed "substituted", unless otherwise indicated, this
means that the group contains from 1 to 3 substituents thereon.
A bond terminated by a wavy line is used herein to signify the point of
attachment of a substituent group. This usage is illustrated by the following
example:
H02C C02H
/
/ H02C C02H
When a functional group is termed "protected", this means that the
group is in modified form to preclude undesired side reactions at the
protected site.
Suitable protecting groups for the compounds of the present invention will be
recognized from the present application taking into account the level of skill
in the art,
and with reference to standard textbooks, such as Greene, T. W. et al.
Protective
Groups in Organic Synthesis Wiley, New York (1991). Examples of suitable
protecting groups are contained throughout the specification.
In some of the compounds of the present invention suitable protecting
groups represents hydroxyl-protecting or carboxyl-protecting groups. Such
conventional protecting groups consist of groups, which are used to
protectively block
the hydroxyl or carboxyl group during the synthesis procedures described
herein.
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These conventional blocking groups are readily removable, i.e., they can be
removed,
if desired, by procedures which will not cause cleavage or other disruption of
the
remaining portions of the molecule. Such procedures include chemical and
enzymatic
hydrolysis, treatment with chemical reducing or oxidizing agents under mild
conditions, treatment with a transition metal catalyst and a nucleophile and
catalytic
hydrogenation.
Examples of carboxyl protecting groups include
allyl, benzhydryl, 2-naphthylmethyl, benzyl, silyl such as
t-butyldimethylsilyl (TBDMS), phenacyl, p-methoxybenzyl,
o-nitrobenzyl, p-methoxyphenyl, p-nitrobenzyl, 4-pyridylmethyl
and t-butyl.
Examples of suitable hydroxyl protecting groups include triethylsilyl,
t-butyldimethylsilyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
benzyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl, 2,2,2-
trichloroethyloxycarbonyl and the like.
The compounds of the present invention are useful per se and in their
pharmaceutically acceptable salt and ester forms are potentiators for the
treatment of
bacterial infections in animal and human subjects. The term "pharmaceutically
acceptable ester, salt or hydrate", refers to those salts, esters and hydrated
forms of the
compounds of the present invention which would be apparent to the
pharmaceutical
chemist. i.e., those which are substantially non-toxic and which may favorably
affect
the pharmacokinetic properties of said compounds, such as palatability,
absorption,
distribution, metabolism and excretion. Other factors, more practical in
nature, which
are also important in the selection, are cost of the raw materials, ease of
crystallization, yield, stability, solubility, hygroscopicity and flowability
of the
resulting bulk drug. Conveniently, pharmaceutical compositions may be prepared
from the active ingredients in combination with pharmaceutically acceptable
carriers.
Thus, the present invention is also concerned with pharmaceutical compositions
and
methods of treating bacterial infections utilizing as an active ingredient the
novel
metallo-beta-lactamase inhibitors of formula I.
The pharmaceutically acceptable salts referred to above also include
acid addition salts. Thus, the Formula I compounds can be used in the form of
salts
derived from inorganic or organic acids. Included among such salts are the
following:
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate,
butyrate,
citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,
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dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-
hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-
naphthalenesulfonate,
nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate,
picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and
undecanoate.
The pharmaceutically acceptable cations which can form a salt with
one or both of the carboxyls (C02M1 and C02M2) of the compounds of formula I
are
known to those skilled in the art. Examples include those where M1 and M2
independently can be alkali metals such as sodium, potassium and the like,
ammonium and the like or hydrogen. It is noted that the compounds claimed in
the
instant invention are, as necessary, charged balanced in accordance with the
knowledge of those skilled in the art.
The pharmaceutically acceptable esterifying groups are such as would
be readily apparent to a medicinal chemist, and include, for example, those
described
in detail in U.S. Pat. No. 4,309,438. Included within such pharmaceutically
acceptable esters are those which are hydrolyzed under physiological
conditions, such
as pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl,
and
others described in detail in U.S. Pat. No. 4,479,947. These are also referred
to as
"biolabile esters".
Biolabile esters are biologically hydrolizable, and may be suitable for
oral administration, due to good absorption through the stomach or intenstinal
mucosa, resistance to gastric acid degradation and other factors.
Some of the compounds of formula I may be crystallized or
recrystallized from solvents such as organic solvents. In such cases solvates
may be
formed. This invention includes within its cope stoichiometric solvates
including
hydrates as well as compounds containing variable amounts of solvents such as
water
that may be produced by processess such as lyophilization. The compounds of
formula I may be prepared in crystalline form by for example dissolution of
the
compound in water, preferably in the minimum quantity thereof, followed by
admixing of this aqueous solution with a water miscible organic solvent such
as a
lower aliphatic ketone such as a di-(C1-() alkyl ketone, or a (C1-() alcohol,
such as
acetone or ethanol.
A subset of compounds of formula I which is of interest relates to those
compounds where Rl and RZ are not hydrogen and all other variables are as
described
above.
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A subset of compounds of formula I which is of interest relates to those
compounds where Rl and RZ are not the same and all other variables are as
described
above.
Another subset of compounds of formula I which is of interest relates
to those compounds where Rl and/or RZ represents a C~ to C16 straight,
branched or
unsaturated alkyl group optionally substituted with 1 to 3 Rx groups and
optionally
interrupted by one of the following O, S, 502, -C(O)-, -C(O)-NRa-, -COZ- and
all
other variables are described as above. A subset of this invention is realized
when Rl
and/or RZ is a CS to C16 alkyl, preferably C7 to C16 alkyl and are not the
same.
A subset of compounds of formula I which is of interest relates to those
compounds where M1 and M2 are independently hydrogen, sodium or potassium and
all other variables are as described above.
Another subset of compounds of formula I which is of interest relates
to those compounds where Rl and/or R2 represents CS-16, preferably C~_16
straight,
branched or unsaturated alkyl optionally substituted with 1 to 2 RX , wherein
all
variables are as described above.
Another subset of compounds of formula I which is of interest relates
to those compounds where one of Rl or RZ is benzyl or substitued benzyl and
all
variables are as originally described. A further subset of this invention is
realized
when the substituted benzyl is substituted with -OH, -OCH3, OCH2Phenyl, or
OCH20.
Another subset of compounds of formula I which is of interest relates
to those compounds where Rl and/or R2 represents
(C) ~Rx)o-3
wherein all other variables are described as above.
Another subset of compounds of formula I which is of interest relates
to those compounds where R' and/or RZ represents
a A~ c
(d) ~Rx)0-2 ~Rx)0-2
wherein all other variables are described as above.
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Another subset of compounds of formula I which is of interest relates
to those compounds wherein one of Rl or RZ is a benzyl or substituted benzyl
and the
-A B A~ C
other of Rl and R2 is (Rx)o-2 (Rx)o-z
wherein all other variables are described as above.
Another subset of compounds of formula I which is of interest relates
to those compounds where the relative and absolute stereochemistry is:
R~
,.
H02C C02H
Still another subset of compounds of formula I which is of interest
relates to those compounds where RI and/or RZ represents a group of the
formula:
A
~Rx)o-2
wherein A is (CH2)1-5 ~d
is phenyl, naphthyl, cyclohexyl or dibenzofuranyl.
Still another subset of compounds of formula I that is of interest relates
to those compounds where R' and/or RZ represents a group of the formula
-A B A~ C
(Rx)0-2 (Rx)0-2
wherein A is (CH2)1-3, A' is a bond, -O- or (CH2)1-2 and
and
independently represent phenyl, thienyl, pyridyl, furanyl or
cyclohexyl.
Still another subset of compounds of formula I that is of interest relates
to those compounds where one of R1 or R2 is:
/ \ /
~''~ ~~RX~o-2 and all other variables are as originally
defined.
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Still another subset of compounds of formula I that is of interest relates
to those compounds of the following structure:
(RX o-~ ~RX)o-2
M202C C02M~
wherein RX, M2, and M1 are as originally defined.
Another subset of compounds of formula I which is of interest relates
to those compounds where R1 and R2 are not the same and neither is hydrogen.
Yet another subset of compounds of formula I, that is of interest relates
to those compounds where one of R1 or R2 is a group of the formula:
A
(RX)o-2
where A is (CH2)1-2 ~d
is phenyl or cyclohexyl and the other of R1 or R2 is a group of the
formula:
-A B A' C
(Rx)o-z (Rx)o-z
where A is (CH2)1-2, A' is a single bond,
is phenyl or cyclohexyl and ~ is phenyl, thienyl or pyridyl.
Still another subset of compounds of formula I that is of interest relates
to those compounds where:
R1 is CS-7 alkyl substituted with 0 to 2 RX groups,
~RX)o-2 °~ ~ ~ ~RX)o-2
R2 is C~_10 alkyl substituted with 0 to 2 Rx groups,
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O
or / ~ \~ (RX)o-2
' \ /
- ~RX)o-z
and all other variables are as described above.
A preferred subset of Rx is RY.
The compounds of the invention, which are succinic acids or
derivatives thereof, can be formulated in pharmaceutical compositions by
combining
the compound with a pharmaceutically acceptable carrier. Examples of such
carriers
are set forth below. The compounds of formula I have metallo-(3-lactamase
inhibitory
properties, and are useful when combined with a (3-lactam antibiotic for the
treatment
of infections in animals, especially mammals, including humans. The compounds
may be used, for example, in the treatment of infections of, amongst others,
the
respiratory tract, urinary tract and soft tissues and blood.
The compounds may be employed in powder or crystalline form, in
liquid solution, or in suspension. They may be administered by a variety of
means;
those of principal interest include: topically, orally and parenterally by
injection
(intravenously or intramuscularly).
Compositions for injection, a preferred route of delivery, may be
prepared in unit dosage form in ampules, or in multidose containers. The
injectable
compositions may take such forms as suspensions, solutions, or emulsions in
oily or
aqueous vehicles, and may contain various formulating agents. Alternatively,
the
active ingredient may be in powder (lyophillized or non-lyophillized) form for
reconstitution at the time of delivery with a suitable vehicle, such as
sterile water. In
injectable compositions, the carrier is typically comprised of sterile water,
saline or
another injectable liquid, e.g., peanut oil for intramuscular injections.
Also, various
buffering agents, preservatives and the like can be included.
Topical applications may be formulated in carriers such as hydrophobic
or hydrophilic bases to form ointments, creams, lotions, in aqueous,
oleaginous or
alcoholic liquids to form paints or in dry diluents to form powders.
Oral compositions may take such forms as tablets, capsules, oral
suspensions and oral solutions. The oral composions may utilize carriers such
as
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conventional formulating agents, and may include sustained release properties
as well
as rapid delivery forms.
The compounds of the instant invention are metallo-(3-lactamase
inhibitors, which are intended for use in pharmaceutical compositions.
Accordingly,
it is preferable that the metallo-(3-lactamase inhibitors are provided in
substantially
pure form, for example at least about 60% to about 75% pure, preferably about
85%
to about 95% pure and most preferably about 98% or more pure (% are on a
weight
for weight basis). Impure preparations of the compounds may be used for
preparing
the more pure forms used in pharmaceutical compositions.
The dosage to be administered depends to a large extent upon the
condition and size of the subject being treated, the route and frequency of
administration, the sensitivity of the pathogen to the particular compound
selected, the
virulence of the infection and other factors. Such matters, however, are left
to the
routine discretion of the physician according to principles of treatment well
known in
the antibacterial arts. Another factor influencing the precise dosage regimen,
apart
from the nature of the infection and peculiar identity of the individual being
treated, is
the molecular weight of the compound.
The compositions for human delivery per unit dosage, whether liquid
or solid, may contain from about 0.01 % to as high as about 99% of active
material,
the preferred range being from about 10-60%. The composition will generally
contain
from about 15 mg to about 2.5 g of the active ingredient; however, in general,
it is
preferable to employ dosage amounts in the range of from about 250 mg to 1000
mg.
In parenteral administration, the unit dosage will typically include the pure
compound
in sterile water solution or in the form of a soluble powder intended for
solution,
which can be adjusted to neutral pH and isotonic.
The invention described herein also includes a method of treating a
bacterial infection in a mammal in need of such treatment comprising
administering to
said mammal a compound of formula I in conjunction with a (3-lactam antibiotic
such
as a carbapenem, penicillin or cephalosporin in an effective combination.
The preferred methods of administration of the Formula I compounds
include oral and parenteral, e.g., i.v. infusion, i.v. bolus and i.m.
injection.
The compounds of formula I may suitably be administered to the
patient at a daily dosage of from 0.7 to 50 mg/kg of body weight. For an adult
human
(of approximately 70 kg body weight), from 50 to 3000 mg, preferably from 100
to
1000 mg, of a compound according to the invention may be administered daily,
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suitably in from 1 to 6, preferably from 2 to 4, separate doses. Higher or
lower
dosages may, however, be used in accordance with clinical practice.
The compounds may be used in combination with antibiotic agents for
the treatment of infections caused by metallo-(3-lactamase producing strains,
in
addition to those infections which are subsumed within the antibacterial
spectrum of
the antibiotic agent. Metallo-~3-lactamase producing strains include: Bacillus
cereus,
Bacteroides fragilis, Aeromonas hydrophila, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Shigella
flexneri,
Legionella gormanii, Chryseobacterium meningosepticum, Chryseobacterium
indologenes, Acinetobacter baumannii, Citrobacter freundii, and Aeromonas
veronii.
In accordance with the instant invention, it is generally advantageous to
use a compound of formula I in admixture or conjuction with a carbapenem,
penicillin, cephalosporin or other (3-lactam antibiotic or prodrug. It also
advantageous
to use a compound of formula I in combination with one or more (3-lactam
antibiotics
1 S because of the metallo-(3-lactamase inhibitory properties of the
compounds. In this
case, the compound of formula I and the (3-lactam antibiotic can be
administered
separately or in the form of a single composition containing both active
ingredients.
Carbapenems, penicillins, cephalosporins and other (3-lactam
antibiotics suitable for co-administration with the compounds of Formula I,
whether
by separate administration or by inclusion in the compositions according to
the
invention, include both those known to show instability to or to be otherwise
susceptible to metallo-(3-lactamases and also known to have a degree of
resistance to
metallo-/3-lactamase.
When the compounds of Formula I are combined with a carbapenem
antibiotic, a dehydropeptidase (DHP) inhibitor may also be combined. Many
carbapenems are susceptible to attack by a renal enzyme known as DHP. This
attack
or degradation may reduce the efficacy of the carbapenem antibacterial agent.
Inhibitors of DHP and their use with carbapenems are disclosed in, e.g.,
(European
Patent 0007614, filed July 24, 1979 and application number 82107174.3, Fled
August
9, 1982. A preferred DHP inhibitor is 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-
dimethylcyclopropanecarboxamide)-2-heptenoic acid or a useful salt thereof.
Thus,
compounds of the present invention in combination with a carbapenem such as
imipenem and a DHP inhibitor such as, cilastatin is contemplated within the
scope of
this invention.
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A serine (3-lactamase inhibitor such as clavulanic acid, sulbactam or
tazobactam may also be co-administered with the compound of the invention and
(3-
lactam antibiotics, either by separate administration, or co-formulation with
one, other
or both of the compounds of the invention and the (3-lactam antibiotic.
Examples of carbapenems that may be co-administered with the
compounds of formula I include imipenem, meropenem, biapenem, (4R, SS, 6S)-3-
[3S, SS)-5-(3-carboxyphenyl-carbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1-
hydroxyethyl]-
4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, (1S, SR, 6S)-2-
(4-
(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl)-ethyl(1,8-
naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-
carboxylate
chloride, BMS181139 ([4R-[4alpha,5beta,6beta(R*)]]-4-[2-
[(aminoiminomethyl)amino] ethyl]-3-[(2-cyanoethyl)thio]-6-( 1-hydroxyethyl)-7-
oxo-
1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), B02727 ([4R-3[3S*,SS*(R*)],
4alpha,Sbeta,6beta(R*)]]-6-( 1-hydroxyethyl)-3-[ [5-[ 1-hydroxy-3-
(methylamino)propyl]-3-pyrrolidinyl]thio]-4-methyl-7-oxo-1-azabicyclo[3.2.0]
hept-
2-ene-2-carboxylic acid monohydrochloride), E 1 O 10 (( 1 R, S S, 6S)-6-[ 1
(R)-
hydroxymethyl]-2-[2(S)-[1(R)-hydroxy-1-[pyrrolidin-3(R)-yl] methyl]pyrrolidin-
4(S)-
ylsulfanyl]-1-methyl-1-carba-2-penem-3-carboxylic acid hydrochloride) and
S4661
((1R,SS,6S)-2-[(3S,SS)-5-(sulfamoylaminomethyl) pyrrolidin-3-yl]thio-6-[(1R)-1-
hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylic acid), (1S,SR,6S)-1-methyl-2-
f 7-
[4-(aminocarbonylmethyl)-1,4-diazoniabicyclo(2.2.2)octan-1 yl]-methyl-fluoren-
9-on-
3-yl}-6-(1R-hydroxyethyl)-carbapen-2-em-3 carboxylate chloride.
Examples of penicillins suitable for co-administration with the
compounds according to the invention include benzylpenicillin,
phenoxymethylpenicillin, carbenicillin, azidocillin, propicillin, ampicillin,
amoxycillin, epicillin, ticarcillin, cyclacillin, pirbenicillin, azloccillin,
mezlocillin,
sulbenicillin, piperacillin, and other known penicillins. The penicillins may
be used
in the form of pro-drugs thereof; for example as in vivo hydrolysable esters,
for
example the acetoxymethyl, pivaloyloxymethyl, a-ethoxycarbonyloxy-ethyl and
phthalidyl esters of ampicillin, benzylpenicillin and amoxycillin; as aldehyde
or
ketone adducts of penicillins containing a 6-a-aminoacetamido side chain (for
example hetacillin, metampicillin and analogous derivatives of amoxycillin);
and as a-
estsers of carbenicillin and ticarcillin, for example the phenyl and indanyl a-
esters.
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Examples of cephalosporins that may be co-administered with the
compounds according to the invention include, cefatrizine, cephaloridine,
cephalothin,
cefazolin, cephalexin, cephacetrile, cephapirin, cephamandole nafate,
cephradine, 4-
hydroxycephalexin, cephaloglycin, cefoperazone, cefsulodin, ceftazidime,
cefuroxime, cefinetazole, cefotaxime, ceftriaxone, and other known
cephalosporins,
all of which may be used in the form of pro-drugs thereof.
Examples of (3-lactam antibiotics other than penicillins and
cephalosporins that may be co-administered with the compounds according to the
invention include aztreonam, latamoxef (Moxalactam-trade mark), and other
known
(3-lactam antibiotics such as carbapenems like imipenem, meropenem or (4R, SS,
6S)-
3-[(3 S,5 S)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-( 1 R)-1-
hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]kept-2-ene-2-carboxylic acid,
all of
which may be used in the form of pro-drugs thereof.
Preferred carbapenems are imipenem, meropenem and (4R, SS, 6S)-3-
[(3S,SS)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1-
hydroxyethyl]-
4-methyl-7-oxo-1-azabicyclo[3.2.0]kept-2-ene-2-carboxylic acid.
Particularly suitable penicillins for co-administration with the
compounds according to the invention include ampicillin, amoxycillin,
carbenicillin,
piperacillin, azlocillin, mezlocillin, and ticarcillin. Such penicillins may
be used in
the form of their pharmaceutically acceptable salts, for example their sodium
salts.
Alternatively, ampicillin or amoxycillin may be used in the form of fine
particles of
the zwitterionic form (generally as ampicillin trihydrate or amoxycillin
trihydrate) for
use in an injectable or infusable suspension, for example, in the manner
described
herein in relation to the compounds of formula I. Amoxycillin, for example in
the
form of its sodium salt or the trihydrate, is particularly preferred for use
in
compositions according to the invention.
Particularly suitable cephalosporins for co-administration with the
compounds according to the invention include cefotaxime, ceftriaxone and
ceftazidime, which may be used in the form of their pharmaceutically
acceptable salts,
for example their sodium salts.
When the compositions according to this invention are presented in
unit dosage form, each unit dose may suitably comprise from about 25 to about
1000
mg, preferably about from 50 to about 500 mg, of a compound according to the
invention. Each unit dose may, for example, be 62.5, 100, 125, 150, 200 or 250
mg of
a compound according to the invention.
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When the compounds of formula I are co-administered with a
penicillin, cephalosporin, carbapenem or other (3-lactam antibiotic, the ratio
of the
amount of the compounds of formula I to the amount of the other (3-lactam
antibiotic
may vary within a wide range. The said ratio may, for example, be from 100:1
to
1:100; more particularly, it may for example, be from 2:1 to 1:30. The amount
of
carbapenem, penicillin, cephalosporin or other (3-lactam antibiotic according
to the
invention will normally be approximately similar to the amount in which it is
conventionally used.
The claimed invention also includes the use of a compound of formula
I, a pharmaceutically acceptable salt, ester, prodrug, anhydride or solvate
thereof, in
the manufacture of a medicament for the treatment of bacterial infections.
The claimed invention also includes the use of a compound of formula
I as a metallo-~3-lactamase inhibitor.
The claimed invention further includes a method of treating bacterial
infections in humans or animals which comprises administering, in combination
with
a (3-lactam antibiotic, a therapeutically effective amount of a metallo-(3-
lactamase
inhibitor of formula I.
The claimed invention further includes a method of treating bacterial
infections in humans or animals which comprises administering, in combination
with
a carbapenem antibiotic, a therapeutically effective amount of a metallo-~i-
lactamase
inhibitor of formula I.
The claimed invention also includes a composition comprising a
metallo-~3-lactamase inhibitor of formula I together with a ~3-lactam
antibiotic and a
pharmaceutically acceptable carrier.
The claimed invention also includes a composition comprising a
metallo-(3-lactamase inhibitor of formula I together with a carbapenem
antibiotic and
a pharmaceutically acceptable carrier.
The compositions discussed above may optionally include a serine (3-
lactamase inhibitor as described above as well as a DHP inhibitor.
The compounds of the present invention are synthesized using the
general conditions shown in the accompanying flow charts (A through E).
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FLOW SHEET A
/C02P'
~
1.
NaN(TMS~,
THF
1.
tBuCOCI,
Et3N
HO~R~ O
~ R~
N~R~ ~
-.
~
'
2. O
O 2.
BrCH2COzP
A1 A3
~N'u~
A2
~/
-Ph
Remove
Chlral
Auxilliary
1.
Strong
Base
(2-3
eq)
R R~
THF,
low
temp
P'OZC P'OZC
COzH COZH
2.
Alkylating
agent
AS A4
(R2X)
Remove
carboxyl
protecting
group
02 Pt = carboxyl
R, protecting
group
X = displaceablegroup
HOZC leaving
COzH
A6
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FLOW SHEET B
1. Strong Base OH
R~ THF, low temp Ar~~~R~
P~02 C02H 2. Ar~CHO P~02C~C02H
A4 B1
cyclize
Are O O
Ar~~R~ reduction
P~02C~'C02H P~OzC R'
B3 B2
Remove carboxyl
protecting group
Ar~~R~ P' = carboxyl protecting group
H02C C02H Ari = optionally substituted
B4 aryl or heteroaryl group
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FLOW SHEET C
R~ 1. Strong base, THF Y-Ar2-(CH2)n \R~
PLO C CO H PLO ~ O H
2 2 2. Alkylating agent 2 2
A4 [ Y-Arz-(CH2)r; X J C1
Protect carboxyl
group
R3- Ar2- (CH2)n 3 Y-Ar2- (CH2)n
~\R~ R -Met ~~R
PLO C CO P2 ~ Palladium catalyst P~02C~---~C02P2
2 2
C3 solvent, 0 C2
Remove carboxyl
protecting groups X = displaceable leaving group
2
Ar optionally substituted aryl
= or heteroaryl
R3-Ar2-(CH2)n group
R~
Y = iodide, bromide, chloride
H02C C02H or protected hydroxy
C4 n = 1, 2, 3 or 4
_ 3
R optionally substituted alkenyl,
= alkynyl,
aryl or heteroaryl group
Met boronic acid or trialkyltin
= moiety
P1 carboxyl protecting group
=
P2 carboxyl protecting group
=
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FLOW SHEET D
(~ 1. Strong ~R~
~R~ base ' ~
THF, low
tem
p
MO~ HOZC COZH
D1 2. Oxidizing D2
agent
3. Acidic
work-up
4. Remove
ester
group if
present
M = H or esterifying group
FLOW SHEET E
R 1. Strong base
HO~R~ 1. tBuCOCI, Et3N ~R~ THF, low temp
E1 2, '-', 2. Oxidizing agent
~Li E2 =Ph
'' =Ph
R' ~t~ Remove R' .ft~ O
Chiral Auxilliary ~ X* - p~N~~
X* ~X* ' HOzC COZH
d~O
E4 -Ph
E3
The 2,3-disubstituted succinic acid compounds of the present invention
can be prepared by the general methods described in Flow Sheets A-E. When one
or
more Rx substituents are present in a compound synthesized according to the
Flow
Sheets, it is sometimes advantageous that they be carried through the
syntheses in
protected or precursory form and then be deprotected or elaborated at or near
the end
of the synthesis. For example, if a desired Rx group is incompatible with the
reaction
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conditions of the synthesis being employed, said Rx group may be introduced
initially
in a protected form and then be deprotected at the end of the synthesis.
The synthesis of Flow Sheet A is based on a known literature
procedure (M. J. Crimmin et. al., SynLett 1993, 137). Referring to Flow Sheet
A, the
R1-substituted acetic acid starting materials A1 are readily available from
commercial
sources or are readily prepared by a variety of methods known in the art.
Briefly, the
starting material A1 is alkylated with an ester derivative of bromoacetic
acid,
employing a chiral auxiliary group to achieve stereoselectivity in the
reaction. After
removal of the chiral auxiliary to give A4, the R2-alkyl group is introduced
stereoselectively by an alkylation reaction to give A5. Removal of the
carboxyl
protecting group of AS provides the final compound A6.
The first step of Flow Sheet A is introduction of the chiral auxiliary. A
suggested method is as follows. A mixed anhydride is formed between the
starting
carboxylic acid A1 and pivalic acid by treating A1 with a tertiary amine base
such as
triethylamine and pivaloyl chloride in a suitable ethereal solvent such as
tetrahydrofuran at reduced temperature such as between -78°C and
0°C. After a
suitable reaction time, such as from 30 min to 3 hours, the resulting
activated
intermediate is then reacted with a freshly prepared solution of lithio-(4R)-
benzyl-2-
oxazolidinone in tetrahydrofuran at reduced temperature such as between -
78°C and
0°C. After conventional isolation and purification, intermediate A2 is
obtained.
Intermediate A2 is deprotonated with a strong base such as sodium
hexamethyldisilazide in a solvent such as tetrahydrofuran at reduced
temperature such
as between -78°C and -70°C. The resulting enolate is alkylated
by addition of
BrCH2C02P1, where P1 is a removable carboxyl protecting group. After an
appropriate reaction period, such as from 1 to 3 hours, compound A3 is
obtained by
conventional isolation and purification techniques. Suitable removable ester
derivatives of bromoacetic acid for this alkylation reaction are t-butyl
bromoacetate,
allyl bromoacetate or benzyl bromoacetate.
The oxazolidinone chiral auxiliary group of A3 is removed by a
hydrolysis reaction. Aqueous lithium hydroxide and aqueous hydrogen peroxide
are
employed for this reaction along with an organic co-solvent such as
tetrahydrofuran.
The reaction is carried-out at a temperature of from 0°C to 30°C
for a reaction time of
from 30 min to 4 hours. After acidification, conventional isolation and
purification
provides intermediate A4.
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An alternative method of removing the chiral auxilliary consists of
reacting A3 with lithium benzyloxide (LiOCH2Ph) followed by cleavage of the
resulting benzyl ester to give A4. The reaction of A3 with lithium benzyloxide
is
carried-out in tetrahydrofuran as solvent at a temperature of from -
78°C to 30°C for a
reaction time of from 30 min to 4 hours. Cleavage of the resulting benzyl
ester is
accomplished in conventional fashion, eg by hydrogenolysis employing a
suitable
catalyst such as palladium on carbon in an appropriate solvent such as ethanol
at 1-2
atmospheres pressure of hydrogen. After conventional isolation and
purification,
compound A4 is obtained.
Alkylation of A4 to give AS is accomplished by deprotonating A4 with
>2 equivalents of a strong hindered base to give a dianion, which is then
reacted with
an alkylating agent R2X to give A5, where R2 is as defined above and X is a
displaceable leaving group such as iodide, bromide or
trifluoromethanesulfonate. The
reaction proceeds with high stereoselectivity to give predominately the
stereoisomer
1 S shown in Flow Sheet A. The deprotonation reaction is carried-out in a
suitable
solvent such as tetrahydrofuran at a temperature of from -78°C to -
70°C for a reaction
time of from 30 min to 3 hours. Preferred bases for this reaction are lithium
bis(trimethylsilyl)amide and lithium diisopropylamide. After addition of the
alkylating agent, the reaction is allowed to proceed at a temperature of from -
78°C to
25°C for a reaction time of from 1 to 12 hours. Progress of the
reaction can be
monitored by conventional analytical methods, eg HPLC and TLC. Preferred
alkylating agents for this reaction are alkyl iodides and alkyl bromides.
Other suitable
alkylating agents are well known in the art and include alkyl
trifluoromethanesulfonates, alkyl methanesulfonates and alkyl tosylates. After
conventional isolation and purification, intermediate AS is obtained. The
minor
stereoisomer produced in this reaction can often be separated from AS at this
stage by
conventional chromatographic techniques. However, it is often preferable to
carry-out
this separation at the stage of A6, after removal of the carboxyl protecting
group as
described below.
Removal of the carboxyl protecting group of AS by standard methods
gives the final compound A6. When P 1 is t-butyl, this is accomplished by
treating AS
with a strong acid such as trifluoroacetic acid in a suitable solvent such as
dichloromethane. The reaction is carried-out at a temperature of from
0°C to 30°C for
a reaction time of from 1 to 8 hours. The final compound A6 is then isolated
by
conventional techniques. Other methods of removing tert-butyl ester groups are
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known in the art and may also be employed (see e.g. Greene, T. W., et al.
Protective
Groins in Or anic S tin hesis, John Wiley & Sons. Inc., 1991).
It will be apparent to one skilled in the art that employing a chiral
auxiliary of the opposite absolute configuration [eg. lithio-(4S)-benzyl-2-
oxazolidinone] in the first step of Flow Sheet A will make possible the
synthesis of
compound A3 with the alternative stereochemistry at the newly created
stereocenter.
This will in turn make possible the synthesis of the final compounds A6 of
Flow
Sheet A, with the opposite absolute configuration. Other chiral auxiliary
groups are
also known in the art and may also be employed.
Flow Sheet B illustrates a variation of Flow Sheet A which is preferred
in certain cases, for example when Arl is a heteroaryl group such as pyridyl.
1n this
synthesis the second substituent on the succinic acid is introduced by an
aldol reaction
instead of an alkylation reaction. The synthesis begins with compound A4,
which is
prepared as described in Flow Sheet A. Compound A4 is deprotonated with >2
equivalents of a strong hindered base to give a dianion which is then reacted
with an
aldehyde ArlCHO to give B15, where Arl is an optionally substituted aryl or
heteroaryl group, terms which are defined above. The deprotonation reaction is
carried-out in a suitable solvent such as tetrahydrofuran at a temperature of
from -
78°C to -70°C for a reaction time of from 30 min to 3 hours.
Preferred bases for this
reaction are lithium bis(trimethylsilyl)amide and lithium diisopropylamide.
After
addition of the aldehyde, the reaction is allowed to proceed at a temperature
of from -
78°C to 25°C for a reaction time of from 1 to 12 hours. After
conventional isolation
and purification, intermediate B 1 is obtained.
Compound B 1 is next cyclized to the lactone B2. Suitable conditions
for this cyclization reaction would be exposure of B 1 to acetic anhydride and
triethylamine in an inert solvent such as dichloromethane. Reductive opening
of
lactone B2, such as by hydrogenolysis over palladium on carbon in a suitable
solvent
such as methanol, provides compound B3. Removal of the carboxyl protecting
group
of B3 by conventional methods then gives the final compound B4.
Flow Sheet C illustrates an extension of the synthesis of Flow Sheet A
which makes possible the introduction of a variety of preferred biaryl-type R2
substituents. Briefly, starting with compound A4 from Flow Sheet A, alkylation
with
Y-Ar2-(CH2)n-X by the method described in Flow Sheet A gives intermediate C1;
where X is a displaceable leaving group such as iodide, bromide or
trifluoromethanesulfonate, n is 1,2,3 or 4, Ar2 is an optionally substituted
aryl or
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heteroaryl group as defined above, and Y is iodide, bromide, chloride or a
protected
hydroxyl group which can be converted to a trifluoromethanesulfonate group by
known methods. Protection of the free carboxyl group of C 1 with a removable
protecting group P2 gives C2. When Y is a protected hydroxyl group it is
deprotected
and converted to a trifluoromethanesulfonate group at this point. A palladium
catalyzed organometallic cross-coupling reaction between C2 and an
organometallic
reagent R3-Met gives compound C3; where Met is a boronic acid or trialkyltin
moiety
and R3 is an optionally substituted alkenyl, alkynyl, aryl or heteroaryl group
as
defined above Removal of the two carboxyl protecting groups of C3 then
provides
the final compound C4.
The P2 carboxyl protecting group is introduced in conventional
fashion. A preferred P2 group is p-methoxybenzyl which can be introduced
employing p-methoxybenzyl alcohol, a carbodiimide reagent such as 1,3-
diisopropylcarbodiimide and N,N-dimethylaminopyridine catalyst in a suitable
inert
solvent such as dichloromethane. Other suitable ester protecting groups known
in the
art could also be employed (see e.g. Greene, T. W., et al. Protective Groups
in
Organic Synthesis, John Wiley & Sons. Inc., 1991).
The palladium catalyzed cross-coupling reaction between C2 and R3-
Met is carned-out by procedures known in the scientific and patent literature.
When
Met is a boronic acid moiety [-B(OH)2] the reaction is commonly known as a
Suzuki
reaction (see Suzuki, Chem. Rev. 1995, 95, 2457). Compound C2 is combined with
the boronic acid R3-B(OH)2 in a coupling solvent such as 1,2-dimethoxyethane,
N,N-
dimethylformamide or toluene, optionally with water as a co-solvent, with a
base such
as sodium carbonate and a palladium catalyst such as
tetrakis(triphenylphosphine)palladium(0). The reaction is carried-out at a
temperature
of from 20 °C to 125 °C for a reaction time of from 1 to 48
hours. The coupled
product C3 is then isolated by conventional techniques. When Met is a
trialkyltin
moiety, the reaction is commonly known as a Stille reaction and the cross-
coupling is
carried-out by procedures well known in the literature (T. N. Mitchell,
Synthesis 1992,
803).
Removal of the carboxyl protecting groups of C3 by standaxd methods
provides the final compound C4. It is often convenient for the protecting
groups P1
and P2 to be selected such that they can both be removed under the same
reaction
conditions. For example, when P1 is tert-butyl and P2 is p-methoxybenzyl, both
esters of C3 can be removed in a single step by treating C3 with a strong acid
such as
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trifluoroacetic acid in a suitable solvent such as dichloromethane. It is
sometimes
advantageous to include a trapping agent such as triethylsilane or anisole in
the
reaction mixture. The reaction is carned-out at a temperature of from 0
°C to 30 °C
for a reaction time of from 1 to 8 hours. The final compound C4 is then
isolated by
conventional techniques. Other methods of removing tert-butyl and p-
methoxybenzyl
ester groups are known in the art and may also be employed (see e.g. Greene,
T. W.,
et al. Protective Groups in Organic Synthesis, John Wiley & Sons. Inc., 1991).
Flow Sheet D illustrates an alternative synthesis of compounds of the present
invention. The R1-substituted acetic acid starting materials D1 (M = H) and
the
esterified derivatives thereof (M = esterifying group) are readily available
from
commercial sources or are readily prepared by a variety of methods known in
the art.
The synthesis of Flow Sheet D is based on known literature procedures (see for
example J. L. Belletire and D. F. Fry, J. Org. Chem. 1987, 52, 2549). Briefly,
starting
material D1 is deprotonated with a strong base and the resulting dianion (M =
H) or
anion (M = esterifying group) is oxidatively coupled with a suitable oxidizing
reagent.
In the case of M = H, acidic work-up and conventional isolation and
purification gives
the final compound D2. In the case of M = esterifying group, an additional
deprotection step is also needed. A preferred strong base for the
deprotonation
reaction is lithium diisopropylamide. Suitable oxidizing agents for the
synthesis of
Flow Sheet D include iodine, copper(II) salts such as CuBr2, and titanium
tetrachloride.
Since the synthesis of Flow Sheet D is based on a dimerization-type
reaction, it is best suited for the synthesis of symmetrically 2,3-
disubstituted succinic
acids. For this reason, it is generally less preferred than the syntheses of
Flow Sheets
A, B and C. The synthesis of Flow Sheet D also generally produces a racemic
mixture of stereoisomers. However, it is possible to employ a chiral auxiliary
in the
synthesis of Flow Sheet D in order to achieve high stereoselectivity and
optical purity
(see for example N. Kise et. al. J. Org. Chem. 1995, 60, 1100). Such use of a
chiral
auxiliary is illustrated in Flow Sheet E.
The invention is further described in connection with the following
non-limiting examples.
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PREPARATION 1
THF
LiN O -7p °C P
PhJ
CI I / O N I /
O O O 1
Compound 1
A solution of (4R)-benzyl-2-oxazolidinone (2.44 g, 13.77 mmol) in 100 mL of
THF
was cooled to -70°C and metalated by the dropwise addition of a 2.5M
solution of n-
butyllithium in hexanes (5.52 mL, 13.77 mmol). After 20 min, neat
hydrocinnamoyl
chloride (2.05 ml, 13.79 mmol) was added. After 15 min, the reaction mixture
was
warmed by placing in an ice bath and kept at 0°C for 1 hr. The reaction
was
hydrolyzed by the addition of sat. aqueous NH4C1 and most of the THF was
removed
by rotary evaporation. The residue was partitioned between ethyl acetate and
sat.
aqueous NH4Cl and the organic phase was washed with sat. aqueous NaHC03, water
and brine. The organic layer was dried over Na2S04 and evaporated in vacuo to
give
a solid. Flash chromatography through 500 g of silica gel (50:40:10
hexane/CH2C12
/EtOAc) yielded 3.89 g of the title compound as a white solid.
1H-NMR (S00 Mz, CDC13): 8 2.79 (dd, J = 13.5, 9.4 Hz, 1H), 3.02-3.13 (m, 2H),
3.24-3.41 (m, 3H), 4.16-4.21 (m, 2H), 4.65-4.74 (m, 1H), 7.16-7.40 (m, 10H).
MS (CI): m/z = 385.2 (MH+).
PREPARATION 2
1. NaN(TMS~ P t-BuO,
~O
THF, 78 °C
2. BrCH2C02t-Bu
1 O v 2
Compound 2
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A stirred solution of compound 1 (3.283 g, 10.612 mmol) in 35 mL of
THF was cooled to -78°C and a 1.0M solution of NaN(TMS)2 in THF
(11.67 mL,
11.67 mmol) was added dropwise during 15 min. After 30 min, a solution of t-
butyl
bromoacetate (2.04 mL, 13.82 mmol) in 2 mL of THF was added dropwise during 5
min. The solution was stirred at -78°C for 1 h and then the ice bath
was removed and
stirnng was continued for 1 h. The reaction was hydrolyzed by the addition of
sat.
aqueous NH4C1 and most of the THF was removed by rotary evaporation. The
residue was partitioned between ethyl acetate and sat. aqueous NH4C1 and the
organic
phase was washed with water and brine. The organic layer was dried over Na2S04
and evaporated in vacuo to give a solid. Flash chromatography through 410 g of
silica
gel (35:60:5 hexane/CH2C12/EtOAc) yielded 2.86 g of the title compound as a
white
foam.
1H-NMR (500 Mz, CDC13): 8 1.43 (s, 9H), 2.41 (dd, J = 17.0, 4.1 Hz, 1H), 2.64-
2.80
(m, 2H), 2.88 (dd, J = 17.0, 11.0 Hz, 1H), 3.04 (dd, J = 13.0, 6.3 Hz, 1H),
3.34 (dd, J
= 13.5, 3.2 Hz, 1 H), 3 .95 (t, J = 8.4 Hz, 1 H), 4.08-4.12 (m, 1 H), 4.5-4.6
(m, 2H),
7.21-7.40 (m, 10H).
MS (ESI): m/z = 441.3 (M+NH4+)
PREPARATION 3
Ph t-Bu0
~O
\ LiOH / HOOH
THF, H20, 0 °C t-Bu0 CO H
2 2
O O
2 3
Compound 3
A stirred solution of compound 2 (2.860 g, 6.753 mmol) in 70 mL of
4:1 THF/H20 was cooled to 0°C and 30% aq. hydrogen peroxide (2.8 mL,
27.01
mmol) was added dropwise during 5 min. After 5 min, a 1.0M solution of
LiOH~H20
in H20 (13.51 ml, 13.51 mmol) was added dropwise during 10 min. The reaction
was kept at 0°C for 1.75 hr. and then a 1.5M solution of Na2S03 in H20
(18.0 ml,
27.01 mmol) was added. The ice bath was removed and the reaction mixture was
allowed to warm towards room temperature over 30 min. A solution of 1.0N
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NaHC03 in H20 was added until the reaction mixture had a pH=9 by pH paper (~S
ml). Most of the THF was removed by rotary evaporation and the residue was
partitioned between CH2C12 and H20. The aqueous layer was washed 3 x CH2C12
and then acidified with 2N HCl until pH=3 by pH paper. The aqueous layer was
extracted 4 x CH2Cl2 and the combined organic extracts were dried over Na2S04
and
evaporated in vacuo to give an oil. Flash chromatography through 100 g of
silica gel
(94:6 CH2C12/MeOH + 0.5% HOAc) yielded 1.74 g of the title compound as a white
solid.
1H-NMR (500 Mz, CDC13): 8 1.45 (s, 9H), 2.38 (dd, J = 16.6, 4.4 Hz, 1H), 2.58
(dd,
J = 16.6, 8.5 Hz, 1H), 2.80 (dd, J = 15.3, 10.3 Hz, 1H), 3.10-3.20 (m, 2H),
7.20-7.40
(m, SH), 11.99 (bs, 1H).
MS (ESI): m/z = 430.2 (M+NH4+)
PREPARATION 4
1. CICOt-Bu, Et~N P
THF, -70 °C
HOzC I ~ O N
THF
Li ~ _7p °C O O 4
Ph-'
Compound 4
To a stirred solution of 3-(4-biphenyl)-propionic acid ( 1.805 g, 7.977
mmol) in 40 mL of THF was added Et3N (1.28 mL, 9.17 mmol) and the solution was
cooled to -70°C. Neat pivaloyl chloride (1.0 ml, 8.1 mmol) was added
and a thick
white suspension resulted. After 15 min, the reaction mixture was warmed by
placing
in an ice bath and kept at 0°C for 40 min. The mixture was then re-
cooled to -70°C.
In a separate flask, a solution of (4R)-benzyl-2-oxazolidinone (1.44 g, 8.13
mmol) in
mL of THF was cooled to -70°C and metalated by the dropwise addition of
a 2.5M
solution of n-butyllithium in hexanes (3.25 mL, 8.13 mmol). The resulting
anion
solution was added to the re-cooled suspension via a cannula, rinsing with an
additional 3 mL of THF. After 1 S min, the reaction mixture was warmed by
placing
30 in an ice bath and kept at 0°C for 30 min. The reaction was
hydrolyzed by the
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addition of sat. aqueous NH4Cl and most of the THF was removed by rotary
evaporation. The residue was partitioned between ethyl acetate and sat.
aqueous
NH4Cl and the organic phase was washed with sat. aqueous NaHC03, water and
brine. The organic layer was dried over Na2S04 and evaporated in vacuo to give
a
solid. Flash chromatography through 240 g of silica gel (CH2C12) yielded 2.45
g of
the title compound as a white solid.
1H-NMR (500 Mz, CDCl3): b 2.79 (dd, J = 13.3, 9.4 Hz, 1H), 3.05-3.15 (m, 2H),
3.25-3.41 (m, 3H), 4.15-4.25 (m, 2H), 4.65-4.75 (m, 1H), 7.15-7.65 (m, 14H).
MS (E1): m/z = 385.2 (M+).
PREPARATION 5
P / I 1. NaN(TMS)2 P t-Bu~
O
THF, -78 °C ~ _
O N I / O N I /
2. BrCHzCOzt-Bu
O O 4 O O 5
Compound 5
A stirred solution of compound 4 (1.000 g, 2.594 mmol) in 40 mL of
THF was cooled to -78°C and a 1.0M solution of NaN(TMS)2 in THF (2.85
mL, 2.85
mmol) was added dropwise during 5 min. After 30 min, a solution of t-butyl
bromoacetate (0.500 mL, 3.37 mmol) in 4 mL of THF was added dropwise during 5
min. The solution was stirred at -78°C for 1 h and then the reaction
was hydrolyzed
by the addition of sat. aqueous NH4Cl. The reaction mixture was partitioned
between
ethyl acetate and sat. aqueous NH4Cl and the organic phase was washed with
water
and brine. The organic layer was dried over Na2S04 and evaporated in vacuo to
give
a solid. Flash chromatography through 150 g of silica gel (65:30:5
hexane/CH2C12/EtOAc) yielded 1.12 g of the title compound as a white solid.
1H-NMR (500 Mz, CDCl3): b 1.43 (s, 9H), 2.45 (dd, J = 16.9, 4.1 Hz, 1H), 2.65-
2.80
(m, 2H), 2.89 (dd, J =16.9, 10.8 Hz, 1H), 3.07 (dd, J = 13.0, 6.1 Hz, 1H),
3.34 (dd, J
= 13.5, 3.0 Hz, 1H), 3.94 (t, J = 8.4 Hz, 1H), 4.08-4.11 (m, 1H), 4.5-4.6 (m,
2H),
7.25-7.60 (m, 14H).
MS (ESI): m/z = 517.5 (M+NH4+).
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PREPARATION 6
t-Bu / t-Bu
LiOBn
\ ~ \
THF, -78 °C Bn0 I /
O O
6
Compound 6
S
A stirred solution of compound 5 (0.907 g, 1.815 mmol) in 10 mL of
THF was cooled to -70°C and a freshly prepared 0.27 M solution of LiOBn
in THF
(10 mL, 2.7 mmol) was added dropwise during 10 min. The reaction was allowed
to
warm gradually to -10°C during 2h and was then placed in an ice bath
and kept at 0°C
for 50 min. The reaction mixture was partitioned between ethyl acetate and
sat.
aqueous NH4Cl and the organic phase was washed with water and brine. The
organic
layer was dried over Na2S04 and evaporated in vacuo to give an oil. Flash
chromatography through 125 g of silica gel (75:20:5 hexane/CH2C12/EtOAc)
yielded
0.696 g of the title compound as a white solid.
1H-NMR (500 Mz, CDC13): b 1.42 (s, 9H), 2.43 (dd, J = 16.6, 5.1 Hz, 1H), 2.67
(dd,
J = 16.6, 9.1 Hz, 1 H), 2.85 (dd, J = 13.6, 7.9 Hz, 1 H), 3.07 (dd, J = 13.5,
6.9 Hz, 1 H),
3.15-3.25 (m, 1 H), 5.09 (d, J = 12.4 Hz, 1 H), 5.1 S (d, J = 12.4 Hz, 1 H),
7.20-7.65 (m,
14H).
MS (El): m/z = 430.2 (M+).
PREPARATION 7
t-BuQ
\ ~ H2, Pd/C
Bn0
EtOH, THF t-Bu02C COzH
O
6 7
Compound 7
A solution of compound 6 (0.696 g, 1.617 mmol) in 10 mL of EtOH
and 5 mL of THF was hydrogenated at atmospheric pressure at room temperature
over
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70 mg of 10% PdIC. After 20 h, the mixture was filtered and evaporated to give
a
solid. Flash chromatography through 50 g of silica gel (5:2:2:1
hexane/CH2Cl2/EtOAc/MeOH + 0.05% HOAc) yielded 0.540 g of the title
compound as a white solid.
1H-NMR (500 Mz, CDCl3): 8 1.45 (s, 9H), 2.43 (dd, J = 16.6, 5.1 Hz, 1H), 2.62
(dd,
J = 16.8, 8.6 Hz, 1H), 2.84 (dd, J = 15.5, 10.4 Hz, 1H), 3.1-3.2 (m, 2H), 7.25-
7.60 (m,
9H).
MS (E)]: m/z = 340.2 (M+).
EXAMPLE 1
STEP A
1. LiN(TMSh
THF, -70 °C
/ 2. / \ ~ /
t-BuOZC COZH I \ / t-BuOZC COZH
3 I 8
TFA
CHZCI2 STEP B
/
HOZC COzH
9
Compound 9
STEP A:
A stirred solution of compound 3 from Preparation 3 (1.011 g, 3.825
mmol) in 15.5 mL of THF was cooled to -70°C and a 1.0M solution of
LiN(TMS)2 in
hexane (8.42 mL, 8.42 mmol) was added dropwise. After 1 h, a freshly prepared
1.14M solution of p-iodobenzyl iodide in THF (6.0 mL, 6.84 mmol) was added
dropwise. The solution was stirred at -70°C for 30 min and was then
allowed to warm
gradually to 10°C during 90 min. The reaction was hydrolyzed by the
addition of sat.
aqueous NH4Cl and most of the THF was removed by rotary evaporation. The
residue was partitioned between ethyl acetate and sat. aqueous NH4C1 and the
organic
phase was washed with water and brine. The organic layer was dried over Na2S04
and evaporated in vacuo to give a solid. Flash chromatography through 450 g of
silica
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gel (98:2 CH2C12/MeOH + 0.1 % HOAc) yielded 1.78 g of compound 8 as a ~6:1
mixture of (S,S:R,S) diastereomers (major isomer depicted).
1H-NMR (500 Mz, CDC13): 8 1.32 (s, 9H), 2.74-3.05 (m, 6H), 6.89 (d, J = 8.2
Hz,
2H), 7.12-7.28 (m, SH), 7.56 (d, J = 8.4 Hz, 2H).
S MS (E>]: m/z = 480.4 (M+).
STEP B:
To a solution of compound 8 (182.0 mg, 0.3789 mmol) in 0.6 mL of
CH2Cl2 was added neat trifluoroacetic acid (0.2 mL). The solution was stirred
at
room temperature for 4 h, and was then evaporated in vacuo to give an oil.
Separation
by reverse phase medium pressure chromatography on RP-18 (40:60 MeCN/0.1%
aqueous TFA) gave after lyophilization 103.7 mg of the title compound as a
white
solid.
1H-NMR (500 Mz, CD30D): b 2.84-2.91 (m, 2H), 2.96-3.06 (m, 4H), 6.89 (d, J =
8.2
Hz, 2H), 7.11-7.25 (m, SH), 7.55 (d, J = 8.2 Hz, 2H).
MS (E>]: m/z = 424.2 (M+).
EXAMPLE 2
STEP A
1. LiN(i-Pr)z
THF, -70 °C
> / ~ : \ / ~ /
t-BuOz COZH 2. BrCH2Ph t_guOzC , COZH
7 10 (S,S):(R,S)-8:1
TFA STEP B
CHZCiz
/ ~ ;: \ / \ / + / ~ .; \ / ~ /
HOz COZH HOZC COZH
12 11
Compounds 11 & 12
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STEP A:
A stirred solution of compound 7 (26.2 mg, 0.0770 mmol) in 0.7 mL of
THF was cooled to -70°C and a freshly prepared l .OM solution of LiN(i-
Pr)2 in THF
(0.17 mL, 0.17 mmol) was added dropwise. After 1 h, neat benzyl bromide (0.015
mL, 0.12 mmol) was added dropwise. The solution was stirred at -70°C
for 20 min
and was then allowed to warm gradually to 10 °C during 90 min. The
reaction was
hydrolyzed by the addition of sat. aqueous NH4C1. The reaction mixture was
partitioned between ethyl acetate and sat. aqueous NH4C1 and the organic phase
was
washed with water and brine. The organic layer was dried over Na2S04 and
evaporated in vacuo to give an oil. Purification by preparative layer
chromatography
on silica gel (93:7 CH2Cl2/MeOH + 0.1 % HOAc) yielded 28 mg of compound 10 as
an ~8:1 mixture of (S,S:R,S) diastereomers (major isomer depicted).
1H-NMR (500 Mz, CDCl3): b 1.33 (s, 9H, isomer B, minor), 1.37 (s, 9H, isomer
A,
major), 2.85-3.20 (m, 6H, isomers A & B), 7.10-7.65 (m, 14H, isomers A & B).
1 S MS (E~: m/z = 430.3 (M+)
STEP B:
To a solution of compound 10 from Step A (10.3 mg, 0.0239 mmol) in
0.3 mL of CH2C12 was added neat trifluoroacetic acid (0.1 mL). The solution
was
stirred at room temperature for 4 h, and was then evaporated in vacuo to give
an oil.
Separation by reverse phase medium pressure liquid chromatography on RP-18
(45:55
MeCN/0.1 % aqueous TFA) gave after lyophilization 5.0 mg of compound 11 and
0.7
mg of compound 12 as white solids.
Compound 11:
1H-NMR (500 Mz, CD30D): 8 2.90-2.97 (m, 2H), 3.0-3.1 (m, 4H), 7.14 (d, J = 7.1
Hz, 3H), 7.15-7.25 (m, SH), 7.41 (t, J = 7.7 Hz, 2H), 7.52 (d, J = 8.0 Hz,
2H), 7.58 (d,
J = 7.8 Hz, 2H).
MS (En: m/z = 374.2 (M+).
Compound 12:
1H-NMR (500 Mz, CD30D): 8 2.85-3.00 (m, 6H), 7.19 (d, J = 6.8 Hz, 3H), 7.24-
7.32
(m, SH), 7.30 (t, J = 7.4 Hz, 1H), 7.41 (dd, J = 7.5, 8.0 Hz, 2H), 7.49 (d, J
= 8.0 Hz,
2H), 7.58 (d, J = 7.6 Hz, 2H).
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MS (E1): m/z = 374.2 (M+)
EXAMPLE 3
STEP A
PMB-OH
CHzCiz, 0 °C T
/ DMAP, DIC
t-BuO2C COZH t-BuO2C COZPMB
8 13
STEP B
Pd(PPh 3)a
NazC03, H20
Me0 ~ ~ B(OH)Z DME, 100 °C
STEP C
Me0 ~ ~ ~ ~ ~ ~ Me0
CHZCIZ ~ /
HOZC CO2H t-BuO2C C02PMB
15 14
Compound 15
STEP A:
A stirred solution of compound 8 (830.1 mg, 1.728 mmol) and p-
methoxybenzyl alcohol (0.54 ml, 4.33 mmol) in 14 mL of CH2C12 was cooled to
0°C,
and a 1.0M solution of N,N-dimethylaminopyridine in CH2C12 (0.259 ml, 0.259
mmol) was added, followed by neat 1,3-diisopropylcarbodiimide (0.541 ml, 3.46
mmol). After 1 hr, the cooling bath was removed. The reaction mixture was
stirred an
additional 180 min, and was then hydrolyzed by the addition of sat. aqueous
NH4C1.
The reaction mixture was partitioned between ethyl acetate and sat. aqueoues
NH4C1
and the organic phase was washed with water and brine. The organic layer was
dried
over Na2S04 and evaporated in vacuo to give a semi-solid. This crude material
was
triturated with 10 ml CH2Cl2 and filtered through a sintered-glass funnel.
Evaporation of the filtrate in vacuo gave an oil. Flash chromatography through
160 g
of silica gel (73:20:7 hexane/CH2C12/EtOAc) yielded 921.6 mg of compound 13 as
a
white solid.
1H-NMR (500 Mz, CDCl3): b 1.37 (s, 9H), 2.8-3.1 (m, 6H), 3.83 (s, 3H), 4.98
(dd, J
= 43.5, 11.9 Hz, 2H), 6.76 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 7.07
(d, J =
6.6 Hz, 2H), 7.16-7.27 (m, 5H), 7.53 (d, J = 8.1 Hz, 2H).
MS (ESI): m/z = 623.2 (M+Na+).
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STEP B:
To a stirred solution of compound 13 (76.6 mg, 0.1276 mmol) and
tetrakis(triphenylphosphine)palladium(0) (7.4 mg, 0.0064 mmol) in 1.1 ml DME
was
added a solution of 4-methoxybenzeneboronic acid (29.1 mg, 0.192 mmol) in 0.2
ml
DME. After 10 min, a 2.0M solution of Na2C03 in H20 (0.130 ml, 0.260 mmol)
was added and the reaction mixture was heated to 100°C for 3.5 hr. The
reaction
mixture was allowed to cool to RT and then hydrolyzed by the addition of sat.
aqueous NH4C1. The reaction mixture was partitioned between ethyl acetate and
sat.
aqueous NH4C1 and the organic phase was washed with sat. aqueous NaS2O3,
water,
and brine. The organic layer was dried over Na2S04 and evaporated in vacuo to
give
an oil. Flash chromatography through 18 g of silica gel (73:20:7
hexane/CH2C12BtOAc) yielded 37.1 mg of compound 14 as a white solid.
1H-NMR (500 Mz, CDC13): 8 1.36 (s, 9H), 2.90-3.07 (m, 6H), 3.82 (s, 3H), 3.87
(s,
3H), 4.98 (dd, J = 34.8, 11.9 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.99 (d, J =
8.7 Hz,
2H), 7.09 (d, J = 7.8 Hz, 4H), 7.16-7.26 (m, SH), 7.42 (d, J = 8.0 Hz, 2H),
7.52 (d, J =
8.5 Hz, 2H).
MS (ES)]: m/z = 603.3 (M+Na+)
STEP C:
To a solution of compound 14 (37.1 mg, 0.064 mmol) in 0.6 mL of
CH2Cl2 was added neat trifluoroacetic acid (0.2 mL). The solution was stirred
at
room temperature for 4 h, and was then evaporated in vacuo to give an oil.
Separation
by reverse phase medium pressure chromatography on RP-18 (45:55 MeCN/0.1
aqueous TFA) gave after lyophilization 9.3 mg of the title compound as a white
solid.
1H-NMR (500 Mz, CD30D): 8 2.89-2.96 (m, 2H), 3.01-3.07 (m, 4H), 3.81 (s, 3H),
6.97 (d, J = 8.9 Hz, 2H), 7.11-7.25 (m, 7H), 7.43 (d, J = 8.2 Hz, 2H), 7.51
(d, J = 8.7
Hz, 2H).
MS (ESn: m/z = 427.1 (M+Na+).
EXAMPLES 4-141
Employing the procedures described herein, additional compounds of
the present invention were prepared. Additional examples of representative
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compounds of the present invention are described in Tables 1-5, which include
characterizing data.
Table 1 R
H02C C02H
Example No. R m/z
4 499.1 (M-H+); ESI-Neg
OMe
499.1 (M-H+); ESI-Neg
Me0
6 ~ ~ ~ ~ 404.2 (M+); EI
H O ~.r
Me
7 ~ ~ ~ ~ 387.2 (M-H+); ESI-Neg
5
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R
Table 1 (Cont'd)
H02C COZH
Example No. R m/z
\ ~ ~ w
353.1 (M-H+); ESI-Neg
322.2 (M+-H20); EI
11
Ph--~
12 O
O
Phi
13 ~ I 312.3 (M+); EI
14
/ \ / ~ ~ 399.6 (M-H+); ESI-Neg
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R ~ \ /
Table 1 (Cont'd)
H02C C02H
Example No. R m/z
16 HO ~ 345.4 (M-Na+); ESI
17 HO \ / 312.3 (M+ - H20); EI
HO
18 \ / 328.1 (M+); EI
Me0
/ \ \ / ~ 401.5 (M-H+); ESI-Neg
20 / \ O \ / 404.2 (M+); EI
21 ~. 315.05 (M+Na+); ESI
22 \ / 406.0 (M+-H20); EI
I
\ \
23 ~ / / '~ 387.4 (M-H+); ESI-Neg
O
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R
Table 1 (Cont'd)
H02C C02H
Example No. R m/z
24
371.20 (M+Na+); ESI
26
,,.,~' 298.2 (M+); EI
27
28
348.1 (M+); EI
29 O
342.1 (M+); EI
O
308.1 (M+-H20); EI
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R
Table 1 (Cont'd)
H02C C02H
Example No. R m/z
31 ~ ~ 355.1 (M-H+); ESI-Neg
Me02C
32 ~ ~ 366.3 (M+); EI
F3C
33 ~~ 306.1 (M+-H20); EI
34 HO ~ ~ 314.1 (M+); EI
35 ~ ~ 348.1 (M+); EI
36 Me-(CH2)~ ~-CH2-
37 ~ ~ 316.3 (M+); EI
F
38 ~~ 299.0 (M+Na+); ESI
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R
Table 1 (Cont'd)
H02C C02H
Example No. R m/z
39
40 287.15 (M+Na+); ESI
HO
41 ~ / 357.2 (M+H+); ESI
42 Me ~ ~ 312.2 (M+); EI
43 HO ~ ~ 346.2 (M+NH4+); ESI
44
374.2 (M+); EI
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R
Table 1 (Cont'd)
H02C C02H
Example No. R m/z
46
47
Me0
48
357.0 (M-H+); ESI-Neg
Me0
F
49
334.2 (M+); EI
F
323.2 (M+); EI
NC
51 NC
323.2 (M+); EI
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R
Table 1 (Cont'd)
H02C C02H
Example No. R m/z
52 MeO
O
53
54
56 Me-C=C
283.05 (M+Na+); ESI
57
Me~ 236.2 (M+); EI
5$ Me02C
279.0 (M-H+); ESI-Neg
59
Me 222.1 (M+); EI
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R '~ \
Tabie 1 (Cont'd)
H02C C02H
Example No. R m/z
F3C
60 O
O ~
61 N \
300.1 (M+H+); ESI
62 N ~
300.1 (M+H+); ESI
63 ~ N 300.1 M+H+ ' ESI
( ),
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Table 2 H02C 02H
Example No. R m/z
64
66 ~ ~ 353.1 (M-H+); ESI-Neg
Ph--~
67 O
O
Phi
348.1 (M+); EI
68
F
69 ~ ~ 334.4 (M+); EI
F
~ ~ ~ ~ ~ 400.5 (M+); EI
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Table 2 (Cont'd) Ho2 co2H
Example No. R m/z
71 ~~ 323.1 (M-H+); ESI-Neg
/
72 ~ 294.2 (M+-H20); EI
73 ~ ~ 258.3 (M++NH4+); EI
74 ,.r 356.2 (M+-H20); EI
75 355.1 (M-H+); ESI-Neg
Me02C
76 348.1 (M+); EI
77
328.1 (M+); EI
Me0
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R v
Table 2 (Cont'd) Ho2 co2H
Example No. R m/z
78 O
342.2 (M+); EI
O
79
316.3 (M+); EI
F
80 ~ ~ O
~ 404.2 (M+); EI
81
366.5 (M+); EI
F3C
82
323.2 (M+); EI
NC
83
298.2 (M+); EI
84 Me
235.1 (M-H+); ESI-Neg
85 NC
323.2 (M+); EI
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Table 3
Example No. Compound m/z
86 ~ \ ~ \ Me
\ ~ 388.2 (M+); EI
H02C ~C02H
87 ~ \
391.5 (M+Na+); ESI
H02C C02H
~O O
88 O ~ \ ~ \ ~ O
H02C C02H
89 ~ \
\ ~ 362.2 (M+-2H20); EI
OH
H02C C02H
90 / \ Me
\ ~ 294.1 (M+-H20); EI
H02C '~~C02H
91 ~ \ ~ \
\ ~ \ ~ 450.2 (M+); EI
H02C C02H
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Table 3 (Cont'd)
Example No. Compound m/z
92 ~ \
\ ~ 298.1 (M+); EI
H02C C02H
~O O
93 O ~ \ ; \ ~ O 387.2 (M+H+); CI
H02C C02H
94 Me ', \ / \ ~ 298.2 (M+); EI
H02C C02H
95 ~ \ Me'
\ ~ 312.2 (M+); EI
H02C .~COzH
96 ~ 337.2 (M+Na+); ESI
H02C C02H
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Table 3 (Cont'd)
Example No. Compound m/z
97 / \ / \ \ / \ / 432.2 (M+-H20); EI
':
H02C COzH
98 / \ Me
\ / 294.1 (M+-H20); EI
H02C~' .~C02H
/ \
99 ~ 349.1 (M+Na+); ESI
H02C C02H
100 / \ Me;
\ / 312.2 (M+); EI
H02C~ ~C02H
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R '~ ~
Table 4
H02C C02H
Example No. R
101
S f,.r
102
S ~
103 N ~
N-
104
105
O r,r
106
O ~
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R
Table 4 (Cont'd)
H02C C02H
Example No. R
/
107 /
S
108 \ I S
-N
109
N-
110
//
S
111
S
112
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R
Table 4 (Cont'd)
H02C C02H
Example No. R
S
113
O
114 /
/ O
115
116
H
/ N
117
OS O
118
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R
Table 4 (Cont'd)
H02C C02H
Example No. R
119 HO
120
O
O
121
H2N
F3C
122
F
123
F
124 Me02C ~ ~ ~
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R
Table 4 (Cont'd)
H02C C02H
Example No. R
125
126
HO
127 ACC~
128 H2N
O
129
HO
130
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Table 5
H02C C02H
Example No. R
131
132
133
134
Me0
135 /
136
137 SL=
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R '~ ~ / ~
Table 5 (Cont'd)
H02C C02H
Example No. R
138
139
F
140 F
O
H2N
141
BIOLOGICAL ACTIVITY
IMP-1 metallo-13-lactamase lacking the N-terminal 18 hydrophobic
amino acids which encode the putative periplasmic signal sequence (EMBL access
code PACATAAC6) was PCR amplified from plasmid DNA prepared from a
carbapenem-resistant strain of Pseudomonas aeruginosa (CL5673). The PCR
product was cloned into pET30a+ (Novegen) and expressed in E.coli BL21(DE3)
after induction with 0.5 mM IfTG for 20 hours at room temperature in minimal
media
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supplemented with casamino acids and 348 ~.M ZnS04. Soluble IMP-1 was purified
from cell extracts by SP-Sepharose (Pharmacia) ion exchange and Superdex 75
(Pharmacia) size-exclusion chromatography.
The ICSO of succinate derivatives of Formula I was determined
following a 15 minute incubation at 37°C with IMP-1 (0.75nM in 50mM
MOPS, pH
7). Using initial velocity as a measure of activity, inhibition was monitored
spectrophotometrically at 490nm in a Molecular Devices SPECTRAmaxTM 250 96-
well plate reader employing nitrocefin as the reporter substrate at
approximately Km
concentration (60~M).
A laboratory strain of E.coli engineered to express IMP-1 was used to
evaluate the ability of succinate derivatives of Formula I to reverse metallo-
13-
lactamase-mediated carbapenem resistance in bacteria. Native IMP-1, which
included
the N-terminal periplasmic signal sequence, was PCR amplified from CNA
isolated
from a carbapenem resistant P. aeruginosa clinical isolate, CL56673, and
cloned into
the pET30a vector. The basal (uninduced) level of IMP-1 expressed when pET30a-
IMP-1 was introduced into E. coli BL21(DE3) resulted in 4-, 64- or 500-fold
reduced
sensitivity to impenem, meropenem or (1S,5R,6S)-1-methyl-2-{7-[4-
(aminocarbonylmethyl)-1,4-diazoniabicyclo (2.2.2)octan-1-yl] methyl-fluoren-9-
on-3-
yl}-6-(1R-hydroxyethyl)-carbapen-2-em-3-carboxylate chloride (a carbapenem
synthesized at Merck Research Laboratories) respectively. For example, the
minimum inhibitory concentration (MIC) of (1S,5R,6S)-1-methyl-2-{7-[4-
(aminocarbonylmethyl)-1,4-diazoniabicyclo(2.2.2)octan-1-yl]methyl-fluoren-9-on-
3-
yl},-6-(1R-hydroxyethyl)-carbapen-2-em-3-carboxylate chloride, was typically
increased from 0.06-0.12 ~,g/ml to 16-32 ~g/ml by the expression of IMP-1. To
evaluate IMP-1 inhibitors, an overnight culture of E. coli BL2(DE3)/pET30a-IMP-
1,
grown 35°C in LB broth (Difco) or Mueller Hinton broth (BBL)
supplemented with
kanamycin (50 ~M/ml), was diluted to a final concentration of 105 cells/ml in
Mueller Hinton broth (BBL) containing a subinhibitory concentration (0.25x
MIC) of
the carbapenem, (1S,5R,6S)-1-methyl-2-{7-[4-(aminocarbonylmethyl)-1,4-
diazoniabicyclo(2.2.2)octan-1-yl]methyl-fluoren-9-on-3-yl}-6-(1R-hydroxyethyl)-
carbapen-2-em-3-carboxylate chloride. Various concentrations of compounds of
Formula I were added to the bacterial growth medium and their capacity to
effect a
four-fold or greater increase in sensitivity to the carbapenem was monitored.
The
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readout for antibacterial activity showed no visible growth after 20 hours
incubation at
35°C.
Representative compounds of Formula I were tested as inhibitors
against purified IMP-1 metallo-13-lactamase and found to be active in an ICSO
range of
from about 0.2nM to about SOOpM. The ability of representative compounds of
Formula I to potentiate the activity of the carbapenem antibiotic (1 S,SR,6S)-
1-methyl-
2- {7-[4-(aminocarbonylmethyl)-1,4-diazoniabicyclo(2.2.2)octan-1 yl]-methyl-
fluoren-
9-on-3-yl)-6-(1R-hydroxyethyl)-carbapen-2-em-3-carboxylate chloride against an
1MP-1 producing laboratory strain E. coli BL21(DE3)/pET30a-IMP-1 was tested.
Compounds of Formula I in the concentration range of from about 0.002p,M to
about
100pM. were found to produce 4-fold increase in sensitivity to the carbapenem
antibiotic (1S,SR,6S)-1-methyl-2-{7-[4-(aminocarbonylmethyl)-1,4-
diazoniabicyclo(2.2.2)octan-1-yl]-methyl-fluoren-9-on-3-yl) -6-( 1 R-
hydroxyethyl)-
carbapen-2-em-3-carboxylate chloride in an IMP-1 producing laboratory strain
E. coli
BL21(DE3)/pET30a-IMP-1.
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