Note: Descriptions are shown in the official language in which they were submitted.
Wo 94/~K09 2 1 3 9 3 5 0 PCT/US93/06~0
"OXAZOLONE DERIVED MATERIALS".
1. FIELD QF THE INVENTION
The present invention relates to the logical
development of biochemical and biopharmaceutical agents
and of new materials, including fabricated materials such
as fibers, beads, films and gels. Specifically, the
invention relates to the development of molecular modules
derived from oxazolone (azlactone) and related
structures, and to the use of these modules in the
assembly of molecules and fabricated materials with
tailored properties, which are determined by the
contributions of the individual building modules. The
molecular modules of the invention are preferably chiral,
and can be used to synthesize new compounds and
fabricated materials which are able to recognize
biological receptors, enzymes, genetic materials, and
other chiral molecules, and are thus of great interest in
the fields of biopharmaceuticals, separation and
materials science.
-
2. BACKGROUND OF THE INVENTION
The discovery of new molecules hastraditionally focused in two broad areas, biologically
active molecules, which are used as drugs for the
treatment of life-threatening diseases, and new
materials, which are used in commercial, especially high-
technological applications. In both areas, the strategy
used to discover new molecules has involved two basic
operations: (i) a more or less random choice of a
molecular candidate, prepared either via chemical
synthesis or isolated from natural sources, and (ii) the
testing of the molecular candidate for the property or
properties of interest. This discovery cycle is repeated
indefinitely until a molecule possessing the desirable
W094/0~ ~ PCT/VS93/06~0
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- 2 -
properties is located. In the majority of cases, the
molecular types chosen for testing have belonged to
rather narrowly defined chemical classes. For example,
the discovery of new peptide hormones has involved work
with peptides; the discovery of new therapeutic steroids
has involved work with the steroid nucleus; the discovery
of new surfaces to be used in the construction of
computer chips or sensors has involved work with
inorganic materials, etc. As a result, the discovery of
new functional molecules, being ad hoc in nature and
relying predominantly on serendipity, has been an
extremely time-consuming, laborious, unpredictable, and
costly enterprise.
A brief account of the strategies and tactics
used in the discovery of new molecules is described
lS below. The emphasis is on biologically interesting
molecules; however, the technical problems encountered
in the discovery of biologically active molecules as
outlined here are also illustrative of the problems
encountered in the discovery of molecules which can serve
as new materials for high technological applications.
Furthermore, as discussed below, these problems are also
illustrative of the problems encountered in the
development of fabricated materials for high
technological applications.
2.1 Drug Design
Modern theories of biological activity state
that biological activities, and therefore physiological
states, are the result of molecular recognition events.
For example, nucleotides can form complementary base
pairs so that complementary single-stranded molecules
hybridize resulting in double- or triple-helical
structures that appear to be involved in regulation of
gene expression. In another example, a biologically
active molecule, referred to as a ligand, binds with
WOg4/~K09 PCT/US93/06~0
2l3s3sn
another molecule, usually a macromolecule referred to as
ligand-acceptor (e.g. a receptor or an enzyme), and this
binding elicits a chain of molecular events which
ultimately gives rise to a physiological state, e.g.
normal cell growth and differentiation, abnormal cell
growth leading to carcinogenesis, blood-pressure
regulation, nerve-impulse generation and propagation,
etc. The binding between ligand and ligand-acceptor is
geometrically characteristic and extraordinarily
specific, involving appropriate three-dimensional
structural arrangements and chemical interactions.
2.1.1 Design and Synthesis of Nucleotides
Recent interest in gene therapy and the
manipulation of gene expression has focused on the design
1~ of synthetic oligonucleotides that can be used to block
or suppress gene expression via an antisense, ribozyme or
triple helix mechanism. To this end, the sequence of the
native target DNA or RNA molecule is characterized and
st~d~rd methods are used to synthesize oligonucleotides
representing the complement of the desired target
sequence (see, S. Crooke, The FASEB Journal, Vol. 7, Apr.
1993, p. 533 and references cited therein). Attempts to
design more stable forms of such oligonucleotides for use
in vivo have typically involved the addition of various
functional groups, e.g., halogens, azido, nitro, methyl,
keto, etc. to various positions of the ribose or
deoxyribose subunits cf., The Or~anic ChemistrY of
Nucleic Acids, Y. Mizuno, Elsevier Science Publishers BV,
Amsterdam, The Netherlands, 1987.
2.1.2 GLYCOPEPTIDES
As a result of recent advances in biological
carbohydrate chemistry, carbohydrates are being
increasingly viewed as the components of living systems
with the enormously complex structures required for the
W094/~ ; PCT/US93/~ ~0
213935~ - 4 ~
encoding of the massive amounts of information needed to
orchestrate the processes of life, e.g., cellular
recognition, immunity, embryonic development,
carcinogenesis and cell-death. Thus, whereas two
S naturally occurring amino acids can be used by nature to
convey 2 fundamental molecular messages, i . e ., via
formation of the two possible dipeptide structures, and
four different nucleotides convey 24 molecular messages,
two different monosaccharide subunits can give rise to 11
unique disaccharides, and four dissimilar monosaccharides
can give rise to up to 35,560 unique tetramers each
capable of functioning as a fundamental molecular message
in a given physiological system.
The gangliosides are examples of the
S versatility and effect with which organisms can use
saccharide structures. These molecules are glycolipids
(sugar-lipid composites) and as such are able to position
themselves at strategic locations on the cell wall:
their lipid component enables them to anchor in the
hydropholic interior of the cell wall, positioning their
hydrophilic component in the aqueous extracellular
mil l ieu . Thus the gangliosides (like many other
saccharides) have been chosen to act as cellular
sentries: they are involved in both the inactivation of
bacterial toxins and in contact in~ibition, the latter
being the complex and poorly understood process by which
normal cells inhibit the growth of adjacent cells, a
property lost in most tumor cells. The structure of
ganglioside GM, a potent inhibitor of the toxin secreted
by the cholera organism, featuring a branched complex
pentameric structure is shown below.
SUBSTITUTE SHEET
WO 94/OO!iO9
2 I 3 9 3 ~ O pcr/us93/o624o
.
0~ o /r~)
~ I I
oU_~
I--~
2 0 ) I O ~: 1 r~ ~0
~<I r ~I
O ~I
O~I
I
0~o
3 ~ I J
SU~3STITUTE SHEE~
W094/~K~ PCT/US93/~ ~0
2139350
The oligosaccharide components of the
glycoproteins (sugar-protein composites) responsible for
the human blood-group antigens (the A, B, and O b-lood
classes) are shown below.
S HOCH2
~~ ~ pro~cin
H HNAC
~
H OH
HOCH"
L~_O H ~ \ o Bl~g~upa:Y=NHAc
HO~) I ~ Bl~g~upb:Y=OH
CH3
BLOOD GROUP A AND B ANTIGENS
HOCH2
H~ ~pro~ein
L/IOH H~
CH,
BLOOD GROUP O ANTIGEN, TYPE II
~~ ~Qc~TIT~JT~ S~EET
W094/0~ 2 1 3 9 3 5 0 PCT/US93/06~0
-5J~-
~ ~ t $
Interactions involving complementary proteins
and glycoproteins on red blood cells belonging to
incompatible blood classes cause formation of aggregates,
or clusters and are the cause for failed transfusions of
human blood.
Numerous other biological processes and
macromolecules are controlled by glycosylation (i.e., the
covalent linking with sugars). Thus, glycosylation of
erythropoetin causes loss of the hormone's biological
activity; deglycosylation of human gonadotropic hormone
increases receptor binding but results in almost complete
loss of biological activity (see Rademacher et al., Ann.
Rev. Biochem 57, 785 (1988); and glycosylation o~ three
sites in tissue plasminogen activating factor (TPA)
produces a glycopolypeptide which is 30~ more active than
the polypeptide that has been glycosylated at two of the
sites.
2.1.3 Design and Synthesis of Mimetics of
Bioloqical Liqands
SUBSTlTUTE StlEEl
W094/0~9 ~ PCT/US93/06~0
i ~, . ~. .
2 139350 _ 6 -
A currently favored strategy for development of
agents which can be used to treat diseases involves the
discovery of forms of ligands of biological receptors,
enzymes, or related macromolecules, which mimic such
ligands and either boost, i.e., agonize, or suppress,
i.e., antagonize, the activity of the ligand. The
discovery of such desirable ligand forms has
traditionally been carried out either by random screening
of molecules (produced through chemical synthesis or
isolated from natural sources), or by using a so-called
"rational" approach involving identification of a lead-
structure, usually the structure of the native ligand,
and optimization of its properties through numerous
cycles of structural redesign and biological testing.
Since most useful drugs have been discovered not through
'5 the "rational" approach but through the screening of
randomly chosen compounds, a hybrid approach to drug
discovery has recently emerged which is based on the use
of combinatorial chemistry to construct huge libraries of
randomly-built chemical structures which are screened for
specific biological activities. (S. Brenner and R.A.
Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381)
Most lead-structures which have been used in
"rational" drug design are native polypeptide ligands of
receptors or enzymes. The majority of polypeptide
ligands, especially the small ones, are relatively
unstable in physiological fluids due to the tendency of
the peptide bond to undergo facile hydrolysis in acidic
media or in the presence of peptidases. Thus, such
ligands are decisively inferior in a pharmacokinetic
sense to nonpeptidic compounds, and are not favored as
drugs. An additional limitation of small peptides as
drugs is their low affinity for ligand acceptors. This
phenomenon is in sharp contrast to the affinity
demonstrated by large, folded polypeptides, e.g.
proteins, for specific acceptors, e.g. receptors or
WOg4/~ 2 1 3 g 3 ~ 0 PCT/US93/06~0
enzymes, which is in the subnanomolar range. For
peptides to become effective drugs, they must be
transformed into nonpeptidic organic structures, i.e.,
peptide mimetics, which bind tightly, preferably in the
nanomolar range, and can withstand the chemical and
biochemical rigors of coexistence with biological fluids.
Despite numerous incremental advances in the
art of peptidomimetic design, no general solution to the
problem of converting a polypeptide-ligand structure to a
peptidomimetic has been defined. At present, ~rational"
peptidomimetic design is done on an ad hoc basis. Using
numerous redesign-synthesis-screening cycles, peptidic
ligands belonging to a certain biochemical class have
been converted by groups of organic chemists and
pharmacologists to specific peptidomimetics; however, in
the majority of cases the results in one biochemical
area, e.g. peptidase inhibitor design using the enzyme
substrate as a lead, cannot be transferred for use in
another area, e.g. tyrosine-kinase inhibitor design using
the kinase substrate as a lead.
In many cases, the peptidomimetics that result
from a peptide structural lead using the ~rational"
approach comprise unnatural ~-amino acids. Many of these
mimetics exhibit several of the troublesome features of
native peptides (which also comprise ~-amino acids) and
are, thus, not favored for use as drugs. Recently,
fundamental research on the use of nonpeptidic scaffolds,
such as steroidal or sugar structures, to anchor specific
receptor-binding groups in fixed geometric relationships
have been described (see for example Hirschmann, R. et
al., 1992 J. Am. Chem. Soc., 114:9699-9701; Hirschmann,
R. et al., 1992 J. Am. Chem. Soc., 114:9217-9218);
however, the success of this approach remains to be seen.
In an attempt to accelerate the identification
of lead-structures, and also the identification of useful
drug candidates through screening of randomly chosen
W094/~09 PCT~US93/06~0
21393~0
compounds, researchers have developed automated methods
for the generation of large combinatorial libraries of
peptides and certain types of peptide mimetics, called
~peptoids", which are screened for a desirable biological
activity. For example, the method of H. M. Geysen, (1984
Proc. Natl. Acad. Sci. USA 81:3998) employs a
modification of Merrifield peptide synthesis wherein the
C-terminal amino acid residues of the peptides to be
synthesized are linked to solid-support particles shaped
as polyethylene pins; these pins are treated individually
or collectively in sequence to introduce additional
amino-acid residues forming the desired peptides. The
peptides are then screened for activity without removing
them from the pins. Houghton, (1985, Proc. Natl. Acad.
Sci. USA 82:5131; and U.S. Patent No. 4,631,211) utilizes
individual polyethylene bags ("tea bags") containing
C-terminal amino acids bound to a solid support. These
are mixed and coupled with the requisite amino acids
using solid phase synthesis techniques. The peptides
produced are then recovered and tested individually.
Fodor et al., (1991, Science 251:767) described light-
directed, spatially addressable parallel-peptide
synthesis on a silicon wafer to generate large arrays of
addressable peptides that can be directly tested for
binding to biological targets. These workers have also
developed recombinant DNA/genetic engineering methods for
expressing huge peptide libraries on the surface of
phages (Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA
87:6378).
In another combinatorial approach, V. D.
Huebner and D.V. Santi (U.S. Patent No. 5,182,366)
utilized functionalized polystyrene beads divided into
portions each of which was acylated with a desired amino
acid; the bead portions were mixed together and then
split into portions each of which was subjected to
acylation with a second desirable amino acid producing
W094/~ 2 1 3 9 3 5 0 PCT/US93/06~0
.
_ g _
dipeptides, using the techniques of solid phase peptide
synthesis. By using this synthetic scheme, exponentially
increasing numbers of peptides were produced in uniform
amounts which were then separately screened for a
biological activity of interest.
Zuckerman et al., (1992, Int. J. Peptide
Protein Res. 91:1) also have developed similar methods
for the synthesis of peptide libraries and applied these
methods to the automation of a modular synthetic
chemistry for the production of libraries of N-alkyl
glycine peptide derivatives, called ~peptoids~, which are
screened for activity against a variety of biochemical
targets. (See also, Symon et al., 1992, Proc. Natl.
Acad. Sci. USA 89:9367). Encoded combinatorial chemical
syntheses have been described recently (S. Brenner and
R.A. Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381).
In addition to the lead structure, a very
useful source of information for the realization of the
preferred ~rational" drug discovery is the structure of
the biological ligand acceptor which, often in
conjunction with molecular modelling calculations, is
used to simulate modes of binding of the ligand with its
acceptor; information on the mode of binding is useful in
optimizing the binding properties of the lead-structure.
However, finding the structure of the ligand acceptor, or
preferably the structure of a complex of the acceptor
with a high affinity ligand, requires the isolation of
the acceptor or complex in the pure, crystalline state,
followed by x-ray crystallographic analysis. The
isolation and purification of biological receptors,
enzymes, and the polypeptide substrates thereof are time-
consuming, laborious, and expensive; success in this
important area of biological chemistry depends on the
- effective utilization of sophisticated separation
technologies.
W094/0~ ~ PCT/US93/06~0
, ,1 ~. . ,; .~,, , ,,~
2139350 - lO
Crystallization can be valuable as a separation
technique but in the majority of cases, especially in
cases involving isolation of a biomolecule from a complex
biological milieu, successful separation is
chromatographic. Chromatographic separations are the
result of reversible differential binding of the
components of a mixture as the mixture moves on an active
natural, synthetic, or semisynthetic surface; tight-
binding components in the moving mixture leave the
surface last en masse resulting in separation.
The development of substrates or supports to be
used in separations has involved either the
polymerization crosslinking of monomeric molecules under
various conditions to produce fabricated materials such
as beads, gels, or films, or the chemical modification of
various commercially available fabricated materials,
e.g., sulfonation of polystyrene beads, to produce the
desired new materials. Prior art support materials have
been developed to perform specific separations or types
of separations and are of limited utility. Many of these
materials are incompatible with biological
macromolecules, e.g. reverse-phase silica frequently used
to perform high pressure liquid chromatography can
denature hydrophobic proteins and other polypeptides.
Furthermore, many supports are used under conditions
which are not compatible with sensitive biomolecules,
such as proteins, enzymes, glycoproteins, etc., which are
readily denaturable and sensitive to extreme pH's. An
additional difficulty with separations carried out using
these supports is that the separation results are often
support-batch dependent, i.e., they are irreproducible.
Recently a variety of coatings and composite-
forming materials have been used to modify commercially
available fabricated materials into articles with
improved properties; however the success of this approach
remains to be seen.
WO94/~K09 PCT/US93/06240
2139350
-- 11 --
If a chromatographic surface is equipped with
molecules which bind specifically with a component of a
complex mixture, that component will be separated from
the mixture and may subsequently be released by changing
the experimental conditions, (e.g. buffers, stringency,
etc.) This type of separation is appropriately called
affinity chromatography and remains an extremely
effective and widely used separation technique. It is
certainly much more selective than traditional
chromatographic techniques, e.g chromatography on silica,
alumina, silica or alumina coated with long-chain
hydrocarbons, polysaccharide and other types of beads or
gels, etc., which in order to attain their maximum
separating efficiency need to be used under conditions
that are damaging to biomolecules, e.g. conditions
involving high pressure, use of organic solvents and
other denaturing agents, etc.
The development of more powerful separation
technologies d~p~n~s significantly on breakthroughs in
the field of materials science, specifically in the
design and construction of materials that have the power
to recognize specific molecular shapes under experimental
conditions resembling those found in physiological media,
i.e. these experimental conditions must involve an
aqueous medium whose temperature and pH are close to the
physiological levels and which contains none of the
agents known to damage or denature biomolecules. The
construction of these ~intelligent" materials frequently
involves the introduction of small molecules capable of
specifically recognizing others into existing materials,
e.g. surfaces, films, gels, beads, etc., by a wide
variety of chemical modifications. Alternatively,
molecules capable of recognition are converted to
monomers and used to create the ~intelligent" materials
through polymerization reactions.
WO94/~K~ ~ ~ PCT7US93/06~0
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- 12 -
2.2 Oxazolones
Oxazolones, or azlactones, are structures of
the general formula: R
N ~"'R'
A ~ / (CH~
o~o
where A is a functional group and n is O or 1 and
typically 1-3. Oxazolones containing a five-membered
ring and a single substituent at position 4 are typically
encountered as transient intermediates which cause
problematic racemization during the chemical synthesis of
peptides. An oxazolone can in principle contain one or
two substituents at the 4-position. When these
substituents are not equivalent, the carbon atom at the
4-position is asymmetric and two non-superimposable
oxazolone structures (azlactones) result:
AyO~O AyO>~5
N 7 ", N ~ R2
Chiral oxazolones possessing a single 4-
substituent (also known as 5(4H)-oxazolones), derived
from (chiral) natural amino acid derivatives, including
activated acylamino acyl structures, have been prepared
and isolated in the pure, crystalline state (Bodansky,
M.; Klausner, Y.S.; ondetti, M.A. in ~Peptide Synthesis~,
Second Edition, John Wiley & Sons, New York, 1976, p. 14
and references cited therei`n). The facile, base-
catalyzed racemization of several of these oxazolones has
been studied in connection with investigations of the
serious racemization problem confronting peptide
synthesis (see Kemp, D.S. in ~The Peptides, Analysis,
WO94/~K09 2 1 3 9 3 5 0 PCT/US93/06~0
.~
- 13 -
Synthesis, and Biology~, Vol. 1, Gross, E. & Meienhofer,
J. editors, 1979, p. 315).
Racemization during peptide synthesis becomes
very extensive when the desired peptide is produced by
aminolysis of activated peptidyl carboxyl, as in the case
of peptide chain extension from the amino terminus, e.g.
I - VI shown below (see Atherton, E.; Sheppard, R.C.
"Solid Phase Peptide Synthesis, A Practical Approach",
IRL Press at Oxford University Press, 1989, pages 11 and
12). An extensively studied mechanism describing this
racemization involves conversion of the activated acyl
derivative (II) to an oxazolone (III) followed by facile
base-catalyzed racemization of the oxazolone via a
resonance-stabilized intermediate (IV) and aminolysis of
the racemic oxazolone (V) producing racemic peptide
products (VI).
~ N=C=N
SSs~N $N~oH
2 o H o R2 H "activation"
~ H R
2s sss H~ R H ~0) 1~ ) sSS~N~N~
H~o R2~ N--H H 0
~b
H"" Rl
Base ~s~N~ ~R2 Proton Donor
3s H 0.
rv
W094/0~09 PCT/US93/06~0
2139350 - 14 -
co~_ ~ aminolysis
H Rl H H R3
Extensive research on the trapping of
oxazolones III (or of their activated acyl precursors II)
to give acylating agents which undergo little or no
racemization upon aminolysis has been carried out, and
successes in this area (such as the use of N-
hydroxybenzotriazole) have greatly advanced the art of
peptide synthesis (Kemp, D.S. in ~The Peptides, Analysis,
Synthesis, and Biology~, Vol. 1, Gross, E. & Meienhofer,
J. editors, 1979, p. 315).
Thus, attempts to deal with the racemization
problem in peptide synthesis have involved suppressing oravoiding the formation of oxazolone intermediates
altogether.
Furthermore, certain vinyl oxazolones having a
hydrogen substituent at the 4-position can also undergo
thermal rearrangements (23 Tetrahedron 3363 (1967)),
which may interfere with other desired transformations,
such as Michael-type additions.
~0 ~0
W094/0~09 PCT/US93~06~0
- 2139350
- 15 -
3. SUMMARY OF THE INVENTION
A new approach to the construction of novel
molecules is described. This approach involves the
development of oxazolone (azlactone) derivative molecular
building blocks, containing appropriate atoms and
functional groups which may be chiral and which are used
in a modular assembly of molecules with tailored
properties; each module contributing to the overall
properties of the assembled molecule. The oxazolone
derivative building blocks of the invention can be used
to synthesize novel molecules designed to mimic the
three-dimensional structure and function of native
ligands, and/or interact with the binding sites of a
native receptor. This logical approach to molecular
construction is applicable to the synthesis of all types
of molecules, including but not limited to mimetics of
peptides, proteins, oligonucleotides, carbohydrates,
lipids, polymers and to fabricated materials useful in
materials science. It is analogous to the modular
construction of a mechanical device that performs a
specific operation wherein each module performs a
specific task contributing to the overall operation of
the device.
The invention is based, in part, on the
following insights of the discoverer. (1) All ligands
share a single universal architectural feature: they
consist of a scaffold structure, made e.g. of amide,
carbon-carbon, or phosphodiester bonds which support
several functional groups in a precise and relatively
rigid geometric arrangement. (2) Binding modes between
ligands and receptors share a single universal feature as
well: they all involve attractive interactions between
complementary structural elements, e.g., charge- and ~-
- type interactions, hydrophobic and van der Waals forces,
hydrogen bonds. (3) A continuum of fabricated materials
exists spanning a dimensional range from about 100 A to
W094/~09 ~ ~ - PCT/~S93/06~0
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- 16 -
1 cm in diameter comprising various materials of
construction, geometries, morphologies, and functions,
all possessing the common feature of a functional surface
which is presented to a biologically active molecule or a
mixture of molecules to achieve recognition between the
molecule (or the desired molecule in a mixture) and the
surface. And (4) Oxazolone derivative structures,
heretofore regarded as unwanted intermediates which form
during the synthesis of peptides, would be ideal building
blocks for constructing backbones or scaffolds bearing
the appropriate functional groups that either mimic
desired ligands, and/or interact with appropriate
receptor binding sites, and for carrying out the
synthesis of the various parts of the functionalized
scaffold orthogonally, provided that racemization of the
lS oxazolone structures is prevented or controlled. Thus,
the invention is also based, in part, on the further
recognition that such derivatives of ozaxolones, which do
not racemize, can be used as universal building blocks
for the synthesis of such novel molecules. ~urthermore,
oxazolone derivatives may be utilized in a variety of
ways across the continuum of fabricated materials
described above to produce new materials capable of
specific molecular recognition. These oxazolone
derivatives may be chirally pure and used to synthesize
molecules that mimic a number of biologically active
molecules, including but not limited to peptides,
proteins, oligonucleotides, polynucleotides,
carbohydrates and lipids, and a variety of other polymers
as well as fabricated materials that are useful as new
materials, including but not limited to solid supports
useful in column chromatography, catalysts, solid phase
immunoassays, drug delivery vehicles, films, and
"intelligent" materials designed for use in selective
separations of various components of complex mixtures.
WO94/~K09 2 1 3 9 3 5 o PCT/US93/06~0
- 17 -
Working examples describing the use of
oxazolone-derived modules in the modular assembly of a
variety of molecular structures are given. The molecular
structures include functionalized silica surfaces useful
in the optical resolution of racemic mixtures; peptide
mimetics which inhibit human elastase, protein-kinase,
and the HIV protease; and polymers formed via free-
radical or condensation polymerization of oxazolone-
containing monomers.
In accordance with the present invention, the
oxazolone-derived molecules of interest possess the
desired stereochemistry and, when required, are obtained
enantiomerically pure. In addition to the synthesis of
single molecular entities, the synthesis of libraries of
oxazolone-derived molecules, using the techniques
described herein or modifications thereof which are well
known in the art to perform combinatorial chemistry, is
also within the scope of the invention. Furthermore, the
oxazolone-derived molecules possess enhanced hydrolytic
and enzymatic stabilities, and in the case of
biologically active materials, are transported to target
ligand-acceptor macromolecules in vivo, without causing
any serious side-effects.
According to the present invention, chiral
oxazolones, in which the asymmetric center is a
4-disubstituted carbon, as well as synthetic nonchiral
oxazolones may be synthesized readily and used as
molecular modules capable of controlled reaction with a
variety of other molecules to produce designed chiral
recognition agents and conjugates. These chiral
oxazolones may also be linked together, using
polymerizing reactions carried out either in a stepwise
or chain manner, to produce polymeric biological ligand
- mimics of defined sequence and stereochemistry.
Furthermore, according to the present invention,
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- 18 -
4-disubstituted chiral oxazolones are extremely useful in
the asymmetric functionalization of various solid
supports and biological macromolecules and in the
production of various chiral polymers with useful
properties. The products of all of these reactions are
surprisingly stable in diverse chemical and enzymological
environments, and uniquely suitable for a variety of
superior pharmaceutical and high-technological
applications.
For applications in which the 4 position of the
oxazolone precursor does not need to be chiral, e.g., the
construction of certain polymeric materials, the use of
oxazolones in the construction of linkers for the joining
of two or more pharmaceutically useful or, simply,
biologically active ligands, etc., symmetric or nonchiral
oxazolones are used in chemical syntheses. Furthermore,
if the oxazolone-derived product does not need to
incorporate the 4-position of the oxazolone precursor in
the enantiomerically pure state, oxazolone precursors
which are not enantiomerically pure may be used for
syntheses-
-
4. DETAILED DESCRIPTION OF THE I~v~ ON
To the extent necessary to further understand
any portion of the detailed description, the following
earlier filed U.S. patent applications are expressly
incorporated herein by reference thereto: DISUBSlllu
OXAZOLONE COMPOSITIONS AND DERIVATIVES THEREOF (Serial
No. 07/906,756 filed June 30, 1992); and DIRECTED CHIRAL
LIGANDS, RECOGNITION AGENTS AND FUNCTIONALLY USEFUL
MATERIALS FROM SUBSTITUTED OXAZOLONES AND DERIVATIVES
CONTAINING AN ASY.~ KIC CENTER (Serial No. 08/041,562
filed April 2, 1993).
4.1 SYnthesis of Chiral Substituted Oxazolones
WO94/~K09 2 I 3 9 3 5 0 PCT/US93/06~0
~.,
-- 19 --
Chiral 4,4'-disubstituted oxazolones may be
prepared from the appropriate N-acyl amino acid using any
of a number of standard acylation and cyclization
techniques well-known to those skilled in the art, e.g.:
G
ACOCl + R x~R R ~R
H2N CO2H H
A~O
A ~ ~ O
N ~
If the substituent at the 2-position is capable
of undergoing addition reactions, these may be carried
out with retention of the chirality at the 4-position to
produce new oxazolones. This is shown for the Michael
addition to an alkenyl oxazolone as follows:
A'XH + ~ ~ A~O~O
R~R2 R;;~R2
where X = S or NR and A' is a functionalized alkyl group.
The required chiral amino acid precursors for
oxazolone synthesis may be produced using stereoselective
reactions that employ chiral auxiliaries. An example of
such a chiral auxiliary is (5)-(-)-1-dimethoxymethyl-2-
Wog4/~K09 ~ PCT/US93/06~0
~ 20 -
methoxymethylpyrrolidine (SMPD) (Liebiq's Ann. Chem. 1668
(1983)) as shown below,
S ~H R2CHNH2 ~XH
olo \ COOCH3 ~ O\
¦ ¦ CH3 I CH3
CH300C R2
R3X/LDA
~s ^ -78C
CH3I
NaH ~
20HCOOH ~H
25~\ HOOC/~ "~R ~ hydrolysis \\
wherein R2 = CH3, i-Bu, or benzyl; and R3 = CH3, CHF2, C2H5,
n-Bu, or benzyl. A second example involves 5H,10bH-
oxazolo[3,2-c][1,3]benzoxazine-2(3H),5-diones (55 J. Orq.
Chem. 5437 (1990)),
WO94/~K09 2 1 3 9 3 5 0 PCT/US93/06240
- 21 -
CHO
+ \.~`~ + CQ2 K2CO3
CHCI3
~ H3N~ CO'
R ~ R ~
0 ~ ~ R'X ~ ~ ~OH ~ R'
H bææ H
~ ~N~ CO2-
wherein Rl = phenyl or i-Pr; and R2 = CH3, C2H5, or
CH2=CH-CH2 -
Alternatively, the desired chiral amino acid
may be obtained using stereoselective biochemical
transformations carried out on the racemate, synthesized
via standard reactions, as shown below for a case
involving a commercially-available organism (53 J. or~.
Chem. 1826 (1988)),
R ~ / NH2mycobacl~,iu~ 3 + R~ 2
/ \ n~a~m ~ ~ ~ ~
R2~ CONH2ATCC25795 R1 COO~ R2 CON~2
L-Acid D-Amide
wherein R~ Pr, i-Bu, phenyl, benzyl, p-methoxybenzyl,
or phenethyl; and R = CH3 or C2H5.
Racemic mixtures of 4,4'-disubstituted
oxazolones may be prepared from monosubstituted
WO94/~K09 ~ 1~393~S; PCT~US93/06~0
. ..
- 22 -
oxazolones by alkylation of the 4-position, as in the
following transformation (Synthesis Commun., Sept. 1984,
at 763; 23 Tetrahedron Lett. 4259 (1982)):
<oIo > rh </~<
Resolution of racemic mixtures of oxalolones
may be effected using chromatography or chiral supports
under suitable conditions which are well known in the
art; using fractional crystallization of stable salts of
oxazolones with chiral acids; or simply by hydrolyzizing
the racemic oxazolone to the amino acid derivative and
resolving the racemic modification using standard
analytical techniques.
A wide variety of 4-monosubstituted azlactones
may be readily prepared by reduction of the corresponding
unsaturated derivatives obtained in high yield from the
condensation reaction of aldehydes, ketones, or imines
with the oxazolone formed from an N-acyl glycine (49 J.
orq. Chem. 2502 (1984); 418 SYnthesis Communications
(1984))
0~
30 Ph~N~COoH ~ ~ Ph
CHO O
WOg4/0~09 PCT/US93/06~0
213'g'3~'0
- 23 -
11~ " -'
C \~ Ph~ H
o o
Thus, the art provides a wealth of chemical and
S biochemical methods which can be used to produce a wide
variety of enantiomeric, multifunctionalized oxazolones
whose substituents may be tailored to mimic any desirable
form of the side chains of native polypeptides.
4.2 SYnthetic Transformations of Chiral Oxazolones
4.2.1 Reactions with One or Two Nucleophiles
Producinq Coniuqates
Chiral oxazolones may be subjected to ring-
opening reactions with a variety of nucleophiles
lS producing chiral molecules as shown below:
~ ~ O A ~ N ~ B
.' ~R2 H
Rl ~
In the structure above, Y represents an oxygen, sulfur,
or nitrogen atom. Rl and R2 differ from one another and
taken alone each signifies one of the followng: alkyl
including cycloalkyl and substituted forms thereof; aryl,
aralkyl, alkaryl, and substituted or heterocyclic
versions thereof; preferred forms of Rl and R2 are
structures mimicking the side chains of naturally-
occurring amino acids as well as various ring structures.
The above ring-opening reaction can be carried
out either in an organic solvent such as methylene
chloride, ethyl acetate, dimethyl formamide (DMF) or in
water at room or higher temperatures, in the presence or
W094/0~09 PCT/US93/06~n
21393S '
- 24 -
absence of acids, such as carboxylic, other proton or
Lewis-acids, or bases, such as tertiary amines or
hydroxides, serving as catalysts. If structure BYH
contains nucleophilic functional groups which may
interfere with the ring-opening acylation, these groups
S must be temporarily protected using suitable orthogonal
protection strategies based on the many protecting groups
known in the art; cf., e.g., Protective Grou~s in Orqanic
SYnthesis, 2ed., T.W. Greene and P.G.M. Wuts, John Wiley
& Sons, New York, N.Y., 1991.
The substituents A and B shown may be of a
variety of structures and may differ markedly in their
physical or functional properties, or may be the same;
they may also be chiral or symmetric. A and B are
preferably selected from:
1) an amino acid derivative of the form
(AA)o~ which would include natural and synthetic
amino acid residues (n=1), peptides (n=2-30),
polypeptides (n=31-70) and proteins (n>70).
These derivatives are generally connected to
the amine of the amino acyl structure used to
form the oxazolone through a carbonyl group,
although other reactions which are known to
functionalize terminal amino groups may be
employed. It is recognized that certain amino
acid derivatives would already contain the
necessary connecting group, such as a carbonyl,
so that a direct chemical bond can be obtained
to the product of the oxazolone ring opening
reaction without the use of a connecting group.
2) a nucleotide derivative of the form
(NUCL)D, which would include natural and
synthetic nucleotides (n=1), nucleotide probes
(n=2-25) and oligonucleotides (n>25) including
both deoxyribose (DNA) and ribose (RNA)
variants-
W094/~09 2 1 ~ 9 3 5 0 PCT/US93/06240
, ': 1 . ~
- 25 -
3) a carbohydrate derivative of the form
(CH) n' This would include natural
physiologically active carbohydrates (glucose,
galactose, etc.) including related compounds
such as sialic acids, etc. (n=1), synthetic
5 carbohydrate residues and derivatives of these
(n=1) and all of the complex oligomeric
permutations of these as found in nature (n>1)
cf. Scientific American, January 1993, p. 82.
4) a naturally occurring or synthetic
organic structural motif. This term includes
any of the well known base structures of
pharmaceutical compounds including
pharmacophores or metabolites thereof. These
structural motifs are generally known to have
specific desirable binding properties to ligand
acceptors of interest and would include
structures other than those recited above in
1), 2) and 3).
5) a reporter element such as a natural
or synthetic dye or a residue capable of
photographic amplification which possesses
reactive groups which may be synthetically
incorporated into the oxazolone structure or
reaction scheme and may be attached through the
groups without adversely interfering with the
reporting functionality of the group.
Preferred reactive groups are amino, thio,
hydroxy, carboxylic acid, acid chloride,
isocyanate alkyl halides, aryl halides and
oxirane groups.
6) an organic moiety containing a
polymerizable group such as a double bond or
other functionalities capable of undergoing
condensation polymerization or
copolymerization. Suitable groups include
W094/~K~ PCT/US93/~
213935 -26-
vinyl groups, oxirane groups, carboxylic acids,
acid chlorides, esters, amides, lactones and
lactams.
7) a macromolecular component, such as a
macromolecular surface or structures which may
be attached to the oxazolone modules via the
various reactive groups outlined above in a
manner where the binding of the attached
species to a ligand-receptor molecule is not
adversely affected and the interactive activity
of the attached functionality is determined or
limited by the macromolecule. The molecular
weight of these macromolecules may range from
about 1000 Daltons to as high as possible.
They may take the form of nanoparticles (dp=100-
lOOOA), latex particles (dp=lOOOA-5000A), porous
or non-porous beads (dp=0.5~-1000~), membranes,
gels, macroscopic surfaces or functionalized or
coated versions or composites of these.
Under certain circumstances, A and/or B may be a chemical
bond to a suitable organic moiety, a hydrogen atom, an
organic moiety which contains a suitable electrophilic
group, such as an aldehyde, ester, alkyl halide, ketone,
nitrile, epoxide or the like, a suitable nucleophilic
group, such as a hydroxyl, amino, carboxylate, aminde,
carbanion, urea or the like, or one of the R groups
defined below. In addition, A and B may join to form a
ring or structure which connects to the ends of the
repeating unit of the compound defined by the preceding
formula or may be separately connected to other moeities.
A more generalized presentation of the
composition of the invention is defined by the structure
~1 JQ,CT!T'~ S~
WO g4/~K~ 2 1 3 9 3 S O PCT/US93/06~0
--2 7
wherein:
a. at least one of A and B are as
defined above and A and B are optionally
connected to each other or to other compounds;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a connecting group or a chemical
bond which may be different in adjacent n
units; and
e. n > 1.
Preferably, (1) if n is 1, and X and Y are chemical
bonds, A and B are different and one is other than a
chemical bond, H or R; (2) if n is 1 and Y is a chemical
bond, G includes a NH, OH or SH terminal group for
connection to the carbonyl group and G-B is other than an
amino acid residue or a peptide; (3) if n is 1 and X, Y,
and G each is a chemical bond, A and B each is other than
a chemical bond, an amino acid residue or a peptide; and
(4) if n is 1, either X or A has to include a CO group
for direct connection to the NH group.
These compositions may be used to mimic various
compounds such as peptides, nucleotides, carbohydrates,
pharmaceutical compounds, reporter compounds,
polymerizable compounds or substrates.
Another composition is defined by the formula:
SUBSTlTUTE Sl lEE~
WO94/~K~ PCT/US93/06~0
213 9~5:0 ; -28-
k - X ~ C CO -G ~Y 8
1, .n
where A, B, X, Y and G are as defined above.
S In one embodiment of the invention, at least
one of A and B represents an organic or inorganic
macromolecular surface functionalized with hydroxyl,
sulfhydryl or amine groups. ~xamples of preferred
macromolecular surfaces include ceramics such as silica
and alumina, porous or nonporous beads, polymers such as
a latex in the form of beads, membranes, gels,
macroscopic surfaces, or coated versions or composites or
hybrids thereof. A general structure of a chiral form of
these materials is shown below:
R1
A CO Nl I C CO - Y (Surface)
R2
In another embodiment of the invention, the
roles of A and B in the structure above are reversed, so
that B is a substituent selected from the list given
- above and A represents a functionalized surface as shown
for one of the enantiomeric forms:
2s
(Su~aoe)- CO NH C CO Y - B
R2
In the description that follows, R" where n = an
integer will be used to designate a group from the
definition of R and Rl.
In a preferred embodiment, group A or B in the
above structure is an aminimide moiety. This moiety may
be introduced, for example by reacting the oxazolone with
an asymmetrically substituted hydrazine and alkylating
3s
SU~T~T~ U~l-T
W094/0~09 PCT/US93/06~0
2139350`
- 29 -
the resulting hydrazide, (e.g., by reaction with an alkyl
halide, or epoxide). An example of such a surface is
shown below.
R' R3
(Surfaoe)- CO- NH C CO--N N~ R4
R2 R5
Preferred aminimides are described in a PCT application
entitled MODULAR DESIGN AND SYNTHESIS OF AMINIMIDE-BASED
~ MOLECULES USEFUL AS MOLECULAR RECOGNITION AGENTS AND NEW
POLYMERIC MATERIALS (attorney docket no.: 5925-005-228)
and filed of even date herewith, the content of which is
expressly incorporated herein by reference thereto.
Another embodiment of the invention relates to
~5 an oxazolone ring having the structure
R
N~R'
A--y_</ (cH2)q
o~o
where A, R, R' and Y are as described above and q is zero
or 1. Preferably, Y is a chemical bond [see claim 36].
This ring is useful for preparing the desired oxazolone
derivatives.
A further embodiment of the invention exploits
the capability of oxazolones with suitable substituents
at the 2-position to act as alkylating agents.
Appropriate substituents include vinyl groups, which make
the oxazolone a Michael acceptor, haloalkyl and alkyl
sulfonate-ester and epoxide groups. For example, Michael
addition to the double bond of a chiral 2-vinyloxazolone
followed by a ring opening reaction results in a chiral
conjugate structure. This general reaction scheme,
illustrated for the case of a 2-vinyl azlactone
derivative, is as follows:
WOg4/~K09 PCT/US93/06~0
,. . .
` ~13935~
- 30 -
~1 R'~j~2
AX ~ R1 F~2 BYH
N~8
o
wherein X represents a sulfur or nitrogen atom; Y
represents a sulfur, oxygen, or nitrogen atom; and
substituents A and B, as described above, may adopt a
variety of structures, differing markedly in their
physical or functional properties or being the same, may
be chiral or achiral, and may be preferably selected from
5 amino acids, oligopeptides, polypeptides and proteins,
nucleotides, oligonucleotides, ligand mimetics,
carbohydrates, aminimides, or structures found in
therapeutic agents, metabolites, dyes, photographically
active chemicals, or organic molecules having desired
steric, charge, hydrogen-bonding or hydrophobicity
characteristics, or containing polymerizable vinyl
groups.
The Michael reaction described above is usually
carried out using stoichiometric amounts of nucleophile
AXH and the oxazolone in a suitable solvent, such as
toluene, ethyl acetate, dimethyl formamide, an alcohol,
and the li~e. The product of the Michael addition is
preferably isolated by evaporating the reaction solvent
in vacuo and purifying the material isolated using a
technique such as recrystallization or chromatography.
Gravity- or pressure-chromatography, on one of a variety
of supports, e.g., silica, alumina, under normal- or
reversed-phase conditions, in the presence of a suitable
solvent system, may be used for purification.
~5 The selectivities of the Michael and oxazolone
WOg4/~K09 2 I 3 ~ 3 50 PCT/US93/06240
- 31 -
ring-opening processes impose certain limitations on the
choice of AXH and BYH nucleophiles shown above.
Specifically, nucleophiles of the form ROH tend to add
primarily via the ring-opening reaction, and often
require acidic catalysts (e.g., BF3); thus, X should not
be oxygen. Likewise, primary amines tend to add only via
ring-opening, and X should therefore not be NH.
Secondary amines readily add to the double bond under
appropriate reaction conditions, but many can also cause
ring-opening; accordingly, X or Y can be N provided A or
B are not hydrogen. Nucleophiles of the form RSH will
exclusively add via ring-opening if the sulfhydryl group
is ionized (i.e., if the basicity of the reaction mixture
corresponds to pH ~ 9); on the other hand, such
nucleophiles will exclusively add via Michael reaction
~5 under non-ionizing (i.e., neutral or acidic) conditions.
During the Michael addition, it is important to limit the
presence of hydroxylic species in the reaction mixture
(e.g., moisture) to avoid ring-opening side-reactions.
Summarizing, AXH can be a secondary amine or
thiol, and BYH can be a primary or secondary amine or
thiol, or an alcohol.
In one variant of the Michael-ring-opening
sequence given above, A is a substituent selected from
the foregoing list and BXH comprises an organic or
inorganic macromolecular surface, e.g., a ceramic, a
porous or non-porous bead, a polymer such as a latex in
the form of a bead, a membrane, a gel or a composite, or
hybrid of these; the macromolecular surface is
functionalized with hydroxyl, sulfhydryl or amine groups
which serve as the nucleophiles in the ring-opening
reaction. The reaction sequence is carried out under
conditions similar to those given for the nonpolymeric
cases; purification of the final product involves
techniques used in the art to purify supports and other
surfaces after derivatization, such as washing, dialysis,
WO94/~K09 PCT/~S93/06~0
2l393so
etc. The result of this reaction sequence is a structure
such as the one shown below:
N ~ ~ (Su~ace)
u
In another variant, the roles of AXH and BYH
are reversed, so that BYH is the substituent selected
from the list above and AXH represents a functionalized
surface.
Alternatively, reactive groups may be
introduced at the 2-position of the oxazolone ring via
suitable acylations, as shown for the specific example of
a benzoyl chloride derivative:
20 X-CH2 ~ C-- ~ + H2N ~ CO2H
-
X (,.H2~C--N~CO H X-CH2~--<N
In the case where X is part of a group whose reactivity
is orthogonal to that of the oxazolone ring, such as in
the case of a benzyl chloride group, ring-opening
addition with BYH may be carried out and followed by
reaction with an appropriate AXH group, e.g. an amine
ANH2, to give the product shown:
WO 94/00509 PCI`/US93/06240
213~3~
-~.
CI-CH2~<N--......... "R. ~ Cl-CH~IO R O
ANH2
~=~ O R O
A--N-CH2~C--N--C--C--YB
If in the above sequences the benzylic electrophile
competes with the oxazolone ring for the nucleophile BYH,
a suitable protecting group, shown as Bl below, may be
used to block an existing benzylic amino group in the
oxazolone; subsequent to the ring-opening addition of BYH
the protected group is removed using standard techniques
(e.g., if the protecting group is Boc, it is removed by
using dilute TFA in CH2Cl2), and the resulting prQduct is
reacted with an appropriate electrophile, e.g., A-CH2-Br,
thus introducing substituent A into the molecule.
Bl-NH-CH2~N~ ~ Bl-NH-cH2~c--N--c--c--gY
2 5 Deprotect
A-cH2-N-cH2~c--N--c--c--BY H2N-cH2~ R o
WO 94/00509 PCI`/~:lS93/06240
,
21393SO
- 34 -
4.2.2 Catenation of Chiral Oxazolones
Producinq Chiral Polymers
By selecting appropriate oxazolone building
blocks and catenating (linking) them in one of a variety
of ways, it is possible to produce polymeric
functionalized scaffolds, of varying length and
complexity, each of which mimicks a biologically
important ligand and moreover possesses features which
are desired of potent drugs, such as stability in
physiological media, superior pharmacokinetics, etc. The
oxazolones selected for catenation contain functional
groups which, when part of the oxazolone-derived
scaffold, will make specific contributions to the ligand-
acceptor binding interaction, as determined by previousstructural studies on the binding interaction.
Alternatively, by the judicious insertion of
one or more oxazolone-derived units into a sequence of a
peptide or protein, that is susceptible to hydrolysis or
to enzymatic degradation, a hybrid molecule may be
produced which has improved stability properties. These
structures may be represented through the general
conjugate structure given above; A and B represent the
polypeptide sequences flanking the inserted oxazolone-
derived unit or units.
W O 94/00509 2 1 3 9 3 S O PC~r/US93/06240
The polymeric, oxazolone-derived ligand
sequences may be constructed in one of three ways as
outlined below.
4.2.2.1 Polymerization Via Sequences of
Nucleophilic Oxazolone-Ring-Opening
Followed by Oxazolone-Forminq CYclization
According to this approach, the oxazolone ring
is opened via nucleophilic attack by the amino group of a
chiral ~ disubstituted amino acid; the resulting amide
may be recyclized to the oxazolone, with retention of
chirality, and subjected to a further nucleophilic ring-
opening reaction, producing a growing chiral polymer as
shown below:
-
WO 94/00S09 . , PCI'/US93/06240
2~3g3S0 - 36 -
N~ HyN~COzM R3
0 Rs~Rs oJ~ R3
H2N CO2M A N ~eN
~R1~"
~o~R1~ ~ R6
2 0
2 5 BHX
~ofR'~ $ R~R~
WO94/~K~ 2 1 ~ 9 3 5 0 PCT/US93/06~
- 37 -
wherein M is an alkali metal; each member of the
substituent pairs Rl and R2, R3 and R4, and R5 and R6
differs from the other and taken alone each signifies
alkyl, cycloalkyl, or substituted versions thereof, aryl,
aralkyl or alkaryl, or substituted and heterocyclic
versions thereof; these substituent pairs can also be
joined into a carbocyclic or heterocyclic ring; preferred
versions of these substituents are those mimicking side-
chain structures found in naturally-occurring amino
acids; X represents an oxygen, sulfur, or nitrogen atom;
and A and B are the substituents described above.
At any point in the polymer synthesis shown
above, a structural species, possessing (l) a terminal -
OH, -SH or -NH2 group capable of ring-opening addition to
the oxazolone and (2) another terminal group capable of
reacting with the amino group of a chiral ~
disubstituted amino acid, may be inserted in the polymer
backbone as shown below:
A~O~O
2 o N~7( H2N~\COOH
R R2
J~ ` H R3~R' DCC
A N ~N ~COOH + H2N CO2H
H ¦¦ _
O
H~N-- ~R4~R3
o
A ~ N~ ~\\N~
3 5 R~ 'R3
W094/0~ PCT/US93/06~0
.
21 3g350 - 38 -
This process may be repeated, if desired, at
each step in the synthesis where an oxazolone ring is
produced. The bifunctional species used may be the same
or different in the steps of the synthesis.
The experimental procedures described above for
oxazolone formation and use of oxazolones as acylating
agents are expected to be useful in the oxazolone-
directed catenations. Solubility and coupling problems
that may arise in specific cases can be dealt with
effectively by one with ordinary skill in the art of
polypeptide and peptide mimetic synthesis. For example,
special solvents such as dipolar aprotic solvents (e.g.,
dimethyl formamide, DMF, dimethyl sulfoxide, DMSO, N-
methyl pyrolidone, etc.) and chaotropic (molecular
aggregate-breaking) agents (e.g., urea) will be very
useful as catenations produce progressively larger
molecules.
4.2.2.2 Polymerizations Using Bifunctional
Oxazolones Containinq a Nucleo~hilic Grou~
Alternatively, a chiral oxazolone derivative
containing a blocked terminal amino group may be prepared
~ from a blocked, disubstituted dipeptide, that was
prepared by standard techniqueE known to those skilled in
the art, as shown:
H 3~ H>~
WO9J/00509 2 1 3 9 3 5 0 PCT/US93/06~0
- 39 ~
wherein B~ is an appropriate protecting group, such as Boc
(t-butoxycarbonyl) or Fmoc (fluorenylmethoxycarbonyl).
One may then use this oxazolone to acylate an amine,
hydroxyl, or sulfhydryl-group in a linker structure or
functionalized solid support, represented generically by
AXH, using the reaction conditions described above. This
acylation is followed by deblocking, using standard amine
deprotection techniques compatible with the overall
structure of the amide (i.e., the amine protecting group
is orthogonal with respect to any other protecting or
functional groups that may be present in the molecule),
and the resulting amino group is used for reaction with a
new bifunctional oxazolone generating a growing chiral
~5 polymeric structure, as shown below:
.
-
PCI`/~S93/06240
WO 94/00509~ .
2139350 - 40 -
5 AXH + Bl ~N~ ~N
N~ "", R3
~bb~
Rs~< ~;?S;- ~2N~2
A~X~ f X ~H
~yyo~o
Rn~;7 'Rn
J~; ~6
WOg4/00509 2 1 3 9 3 5 0 PCT/US93/06~0
- 41 - ~
In the reaction shown above, Y is a linker (preferably a
functionalized alkyl group); X is a nitrogen of suitable
structure; an oxygen or a sulfur atom; each member of the
substituent pairs Rl and R2, R3 and R4, R~l and R differs
from the other and taken alone each signifies alkyl,
cycloalkyl, or functionalized versions thereof; aryl,
aralkyl or alkaryl or functionalized including
heterocyclic versions thereof (preferably, these R
substituents mimick the side-chain of naturally occurring
amino acids); substituent R can also be part of a
carbocyclic or heterocyclic ring; A is a substituent as
described above; and C is a substituent selected from the
set of structures for A; and B~ is a blocking or
protecting group.
It can be seen that the above polymerization
involves introduction of two amino acid residues per
polymer-elongation cycle and therefore produces ligands
with an even number of residues. To obtain ligands
containing an odd number of residues, a preliminary step
may be carried out with a suitable amino acid derivative
as shown below, prepared via standard synthesis.
-
WO 94/00509 PCl`~US93/06240
2139350 42-
~o ~?~NH2 B,~O AX~,N~,B,
~/
~ Y~
AX~ R~R
W094/0~ PCT/US93/06~0
2139350
. .
4.2.2.3 Polymerization Using Bifunctional Oxazolones
Containinq an Additional Electrophilic Group
When the substituent at the 2-position.of the
oxazolone (azlactone) ring is capable of undergoing an
addition reaction, that proceeds with retention of the
chirality of the 4-position, the addition reaction may be
combined with a ring-opening acylation to produce chiral
polymeric sequences. This is shown for the case of
alkenyl azlactones below. A~O
N~ HNu~ZNu2H R ~"",.
Rl R2 R2~
f(B) ~ o
~6~~ HNu2ZNu
N$ `Z~ ~0
HNu3ZNu4H R ~",* ~P
R2~=
- 25 A~ j~Nu ~Nu~N~Nu3 NU~z o
,.,Nu4H R4~?=
NH
0=~
NH3
.~
35 RX~ $NU1 ~Nu2 ~ ~Nu3 N5J4 ~ ~ 2~CH2J~N~
SUBSTITUTE SHEET
W094/0~ ~ PCT/US93/06~0
. --
2i393SO - 44 -
In the above sequence of reactions, A denotes a structure
of the form described above and HNul-Z-Nu2H represents a
structure containing two differentially reactive
nucleophilic groups, such as methylamino-ethylamine, 1-
S amino propane-3-thiol, and so on; groups Nul, Nu2, Nu3 and
Nu4 need not be identical and Z is a linker structure as
described above.
Structure HNu'-Z-Nu2H may contain two
nucleophilic groups of differential reactivity, as stated
above, or if Nul and Nu2 are of comparable reactivity one
of the nucleophilic groups is protected to prevent it
from competing with the other and deprotected selectively
following acylation; protecting groups commonly used in
the art of peptide synthesis (e.g., for the nucleophilic
groups such as amino, hydroxyl, thio, etc.) are useful in
the protection of one of the Nu substituents of the
structure HNul-Z-Nu2H. The product of the acylation
reaction with HNul-Z-Nu2H (after Nu-deprotection, if
necessary) is further reacted with a new oxazolone unit
in a Michael fashion, and this addition is followed by
ring-opening acylation with an additional dinucleophile;
repetition of this sequence of synthetic steps produces a
growing polymeric molecule. Reaction conditions for
carrying out these processes are similar to those
described above for related polymers.
The above types of oligomers are highly useful
biochemically because of their structural similarity to
polypeptides. The substituents R can be chosen to tailor
the steric, charge or hydrophobicity characteristics of
the oligomer such that a versatile polypeptide mimetic
results.
4.2.3 Functionalization of Peptides
and Proteins Usinq Oxazolones
WOg4/OK~9 PCT/US93/~
213935~
-45-
In a further embodiment of the invention, the
nucleophilic ring-opening of asymmetrically disubstituted
oxazolones may be utilized to introduce a chiral residue
or sequence in selected positions in peptides or proteins
to produce hybrid molecules with improved hydrolytic
stability or other properties.
The reaction of a chiral azlactone with the
amino terminus of a synthetic tripeptide attached to a
Merrifield support is shown below.
~CO--C 1111 CO--C ~111 CO~
~ R~ Ri7 RJ R~
2 0--~CO--C rJI I CO--C ~111 CO--C ~JI I CO--C rll I CO--B
_~ -- -- _ _
H H H R5
The oxazolone used in the above aminolysis may
contain a blocked amino terminus which, after the
aminolysis, is deblocked and used for further elongation
via acylation. This synthetic variation is shown below
(B~ stands for a suitable blocking group as described
above).
SUBSTITUTE SHEET
WO 94/00509 PCI'/ US93/06240
2,~.393S~ - 46 -
G~=
10 Cll~
~5 ~ 0~ 0~
~ : _ G~
2 0 z T Z = Z r
IG~ =O TG~ eO I~
IZ IZ IZ
=~T =>~T ~T
WO94/~K09 PCT/US93/06240
21393~
., . ., I
- 47 - ~
After the desired oxazolone units have been
used to elongate a given polypeptide, the polypeptide
synthesis may be continued, if desired, using standard
peptide-synthesis technigues.
The structure below illustrates a short polymer
containing nine subunits prepared as above and detached
from the solid phase synthesis support.
~100C h~ ~N~ ~N~ ~X~;~
0 ~ N ~s T
In the polyamide structure shown above, each of the R
groups signifies alkyl, cycloalkyl, or substituted
version thereof; aryl, aralkyl, alkaryl, or substituted
including heterocyclic versions thereof; the R groups can
also define a carbocyclic or heterocyclic ring; preferred
structures for the R groups are those mimicking the
structures of the side-chains of naturally-occurring
amino acids.
The syntheses outlined above may be carried out
using procedures similar to those described previously
for related molecules and macromolecules.
Alternatively, disubstituted chiral azlactones
may be utilized to introduce a variety of novel,
unnatural residues into peptides or proteins using the
following multistep procedure:
a. Synthesis of a peptide whose carboxyl
terminal residue is chiral and disubstituted, preferably
via solid phase synthesis:
~NI12 ~ "I;XN~
W094/00509 ` ~ PCT~US93/06240
2~3935 48
b. Detachment of the peptide prepared by
solid phase synthesis from the support, with reblocking
of the N-terminus if necessary, followed by cyclization
producing the oxazolone as shown below:
RnY~H NX
c. Synthesis of a second desired peptide
sequence on a solid support:
H ~
~ H~NHz
d. Coupling of the peptides produced in steps
(b) and (c) above, under suitable reaction conditions,
producing a novel peptide containing unnatural residues,
shown below after detachment of the peptide from the
support and removal of all protecting groups used during
its synthesis.
R~ $ ~ ~J~ ' ~NHz
W094/~09 21~93~ PCT/US93/06~0
- 49 -
In the structure above, each of the R groups signifies
alkyl, cycloalkyl, aryl, aralkyl or alkaryl, or
substituted or suitably heterocyclic versions thereof;
the R groups may also define a carbocyclic or
heterocyclic ring; preferably the R groups are structural
mimetics of the side-chains of naturally-occurring amino
acids.
Again, the reactions shown in steps a-d above
are carried out using the conditions described above for
related cases. Couplings of peptide segments on a
support or in solution are carried out using the
traditional techniques from the field of peptide
synthesis.
In a variation of the above synthesis, the
oxazolone peptide produced in step (b) above may be
reacted with a variety of bifunctional nucleophilic
molecules to give acylation products as shown below:
R~ ~ +~Z)A
B~ ?~ X~ ~A
. X=O,NH,S
The above acylation product may be coupled with a
peptide to produce novel chiral hybrids; two coupling
routes may be used.
W094/00~09 PCT~US93/06~0
213~350
- 50 -
(1) If A is a group which can be condensed
with an amino group, the condensation reaction is used
for coupling. For example, if A is a carboxyl group,
condensation with a peptide amine using DCC or similar
reagent produces the desired product. Reaction
conditions and suitable (orthogonal) protecting groups
well-known in the art, such as those described above, are
expected to be useful.
0 R3 H 0
~N~ N~X~ ~COOH + H~r1 P~-C!OOR
DCC Deblock ~ _
?~ ~' `N'~N~ N~
(2) If A is a suitable nucleophilic group
(e.g., hydroxyl, amino, thio, etc.) it may be used to
open a peptide oxazolone containing a protected amino
terminus. In the case shown below, groups Y, A and Z of
the general structure shown above have been defined as
follows: Y = NCH3, A = SH and Z = CH2CH2:
WO 94/OXH~ 2 1 ~ ~ 3 5 0 PCT/US93/06~
S u~H O P~CH3 SH +
R H ~J ~-
B, ~ ~ ~' U ;~ ~ deblock
" N~
5~ H o R R2 CH3 0 H '~
The above reactions are run under conditions,
similar to those described above for related peptide
syntheses. A great variety of molecules possessing
nucleophilic hydroxyl, thio, amino and other groups,
e.g., carbohydrates, may be conjugated with peptidic and
2S related frameworks using reactions with suitable
oxazolones as outlined above.
Alternatively, residues may be attached to or
inserted into peptide chains using oxazolones with
reactive groups attached at the 2-position of the ring.
This may be accomplished in either of two ways, as
illustrated below for the case of 2-alkenyl azlactones.
(1) Nucleophilic attack on an azlactone, that
was previously derivatized via a Michael addition using a
nucleophile of general structure AXH, with a peptide
amine:
SUBSTITUTE SHEET
W094/00509 PCT/US93/06240
2 139 350 _ 52 -
Peptide-NH2 + ~ ~ RZ
N~N~Pep~de
o
(2) Michael addition of a peptide nucleophile,
e.g., a sulfhydryl group, to the double bond of a 2-vinyl
oxazolone, followed by nucleophilic attack on the
oxazolone ring by another peptide nucleophile, e.g., an
amine followed by further modifications; this sequence
~oduces polymeric molecules of a variety of structures
as shown below:
~5
~,~
WO 94/00509 2 1 3 9 3 5 0 PCr/US93/06240
-- 53 --
PeptidelSH + ~ ~ ~ Pcptidcl-S
s R~ R2 N~O
PeptidC2 NH/ ~O
~ R2
Pep~del-S~ ~ H2
O~NH Peptidel-S_~
Rl_ /
R2~
~=0 0~ NH
HN R1
\r~ R2~
HN
Pepndel-S / \z
Pcp~ide2 NH2 /
HOOC
~ DCC
O NH
2 5 R~
R2
~=O
HN
Z
0~
NH
Peptide2
W094/~5~ PCT~US93/06240
`
2139350
- 54 -
4.2.4 Fabrication of Ozaxolone-Derived
Macromolecular Structures Capable
of Specific Molecular Recoqnition
In an embodiment of the invention oxazolone
molecular building blocks may be utilized to construct
new macromolecular structures capable of recognizing
specific molecules ("intelligent macromolecules"). These
"intelligent macromolecules" may be represented by the
following general formula:
P - C - L - R
where R is a structure capable of molecular
recognition;
L is a linker;
P is a macromolecular structure serving as
a supporting platform;
C is a polymeric structure serving as a
coating which surrounds P.
Structure R may be a native ligand of a
biological ligand-acceptor, or a mimetic thereof, such as
those described above.
Linker L may be a chemical bond or one of the
linker structures listed above, or a sequence of subunits
such as amino acids, aminimide monomers, oxazolone-
derived chains of atoms or the like.
Polymeric coating C may be attached to the
supporting platform either via covalent bonds or "shrink
wrapping," i.e., the bonding that results when a surface
is subjected to coating polymerization well known to
those skilled in the art. This coating element may be
1) a thin crosslinked polymeric film 10 - 50 A in
thickness, 2) a crosslinked polymeric layer having
controlled microporosity and variable thickness, or 3) a
controlled microporosity gel. When the support platform
is a microporous particle or a membrane, as described
WOg4/~09 PCT/US93/06~0
213935U
below, the controlled microporosity gel may be engineered
to completely fill the porous structure of the support
platform. The polymeric coatings may be constructed in a
controlled way by carefully controlling a variety of
reaction parameters, such as the nature and degree of
coating crosslinking, polymerization initiator, solvent,
concentration of reactants, and other reaction
conditions, such as temperature, agitation, etc., in a
manner that is well known to those skilled in the art.
The support platform P may be a pellicular
material having a diameter (dp) from 100 ~ to 1000 ~, a
latex particle (dp 0.1 - 0.2 ~), a microporous bead
(dp 1 - 1000 ~), a porous membrane, a gel, a fiber, or a
continuous macroscopic surface. These may be
commercially availsble polymeric materials, such as
silica, polystyrene, polyacrylates, polysulfones,
agarose, cellulose, etc.
The multisubunit recognition agents above are
expected to be very useful in the development of targeted
therapeutics, drug delivery systems, adjuvants,
diagnostics, chiral selectors, separation systems, and
tailored catalysts.
In the present specification the terms
"surface", "substrate" or "structure" refer either to P,
P linked to C or P linked to C and L as defined above.
W094/0
PCT/US93/06
2I39350- -
-56-
4.2.4.1 Chiral Alkenyl Azlactone Monomers
and Polymerization Products
When used on an alkenyl azlactone, the
azlactone ring-opening addition reaction discussed above
may be used to directly produce a wide variety of chiral
vinyl monomers. These may be polymerized or
copolymerized to produce chiral oligomers or polymers,
and may be further crosslinked to produce chiral beads,
membranes, gels, coatings or composites of these
materials.
R~R2 H~
~ /~iL~ho
NH
R2_/
R1~"
~0
B
Other useful monomers, which may be used to
produce chiral crosslinkable polymers, may be produced by
nucleophilic opening of a chiral 2-vinyl oxazolone with a
suitable amino alkene or other unsaturated nucleophile.
~ + ~
SIJ~CT!TUTC ~ F
WO94/~K~ 2 1 3 ~ 3:5 0 PCT/US93/06~0
Vinyl polymerization and polymer-crosslinking
techniques are well-known in the art (see, e.g., U.S.
Patent No. 4,981,933) and are applicable to the above
preferred processes.
A~F~
t,
R1
(surlaoe~--CO r~l I C co Y--E3
R2
4.2.5 Combinatorial Libraries of Peptidomimetics
Derived From Oxazolone Modules
The synthetic transformations of oxazolones
outlined above may be readily carried out on solid
supports in a manner analogous to performing solid phase
peptide synthesis, as described by Merrifield and others
(see for example, Barany, G., Merrifield, R.B., Solid
Phase Peptide Synthesis, in The Peptides Vol. 2, Gross
E., Meienhofer, J. eds., p. 1-284, Acad. Press, New York
1980; Stewart, J.M., Yang, J.D., Solid Phase Peptide
Synthesis, 2nd ed., Pierce Chemical Co., Rockford,
Illinois 1984; Atherton, E., Sheppard, R.C., Solid Phase
Peptide Synthesis, D. Rickwood ~ B.D. Hames eds., IRL
Press ed. Oxford U. Press, 1989). Since the assembly of
the oxazolone-derived structures is modular, i.e., the
result of serial combination of molecular subunits, huge
combinatorial libraries of oxazolone-derived oligomeric
structures may be readily prepared using suitable solid-
phase chemical synthesis techniques, such as those of
W094/0~ ~ PCTiUS93/06~0
:.... ..
21~9~50
- 58 -
described by Lam (K.S. Lam, et al. Nature 354, 82 (1991))
and Zuckermann (R.N. Zuckermann, et al. Proc. Natl. Acad.
Ser. USA, 89, 4505 (1992); J.M. Kerr, et al., J. Am Chem.
Soc. 115, 2529 (1993)). Screening of these libraries of
compounds for interesting biological activities, e.g.,
binding with a receptor or interacting with enzymes, may
be carried out using a variety of approaches well known
in the art. With "solid phase" libraries (i.e.,
libraries in which the ligand-candidates remain attached
to the solid support particles used for their synthesis)
lo the bead-staining technique of Lam may be used. The
technique involves tagging the ligand-candidate acceptor,
e.g., an enzyme or cellular receptor of interest, with an
enzyme (e.g., alkaline phosphatase) whose activity can
give rise to color prodution thus staining library
support particles which contain active ligands-candidates
and leaving support particles containing inactive ligand-
candidates colorless. Stained support particles are
physically removed from the library (e.g., using tiny
forceps tht are coupled to a micromanipulator with the
aid of a microscope) and used to structurally identify
the biologically active ligand in the library after
removel of the ligand acceptor from the complex by e.g.,
washing with 8M guanidine hydrochloride. With "solution-
phase" libraries, the affinity selection techniques
described by Zuckermann above may be employed.
An especially preferred type of combinatorial
library is the encoded combinatorial library, which
involves the synthesis of a unique chemical code (e.g.,
an oligonucleotide or peptide), that is readily
decipherable (e.g., by sequencing using traditional
analytical methods), in parallel with the synthesis of
the ligand-candidates of the library. The structure of
the code is fully descriptive of the structure of the
ligand and used to structurally characterize biologically
active ligands whose structures are difficult or
WO94/~K09 2 1 ~ 9 3 S O PCT/US93/06~0
- 59 -
impossible to elucidate using traditional analytical
methods. Coding schemes for construction of
combinatorial libraries have been described recently (for
example, see S. Brenner and R.A. Lerner, Proc. Natl.
Acad. Sci. USA 89, 5381 (1992); J.M. Kerr, et al. J. Am.
Chem. Soc. 115, 2529 (1993)). These and other related
schemes are contemplated for use in constructing encoded
combinatorial libraries of oligomers and other complex
structures derived from oxazolones.
The power of combinatorial chemistry in
generating screenable libraries of chemical compounds
e.g., in connection with drug discovery, has been
described in several publications, including those
mentioned above. For example, using the "split solid
phase synthesis" approach outlined by Lam et al, the
random incorporation of 20 oxazolones into pentameric
structures, wherein each of the five subunits in the
pentamer is derived from one of the oxazolones, produces
a library of 205 = 3,200,000 peptidomimetic ligand-
candidates, each ligand-candidate is attached to one or
more solid-phase synthesis support particles and each
such particle contains a single ligand-canditate type.
This library can be constructed and screened for
biological activity in just a few days. Such is the
power of combinatorial chemistry using oxazolone modules
to construct new molecular candidates.
The following is one of the many methods that
are being contemplated for use in constructing random
combinatorial libraries of oxazolone-derived compounds;
the random incorporation of three oxazolones derived from the
amino acids glycine methyl-ethyl-glycine ,and isopropyl methyl
glycine to produce~27 trimeric structures~linked to the
su~o-L via a succinoyl linker is given as an example.
3~
W094/O~g PCT/US93/06~W
~13935~ -60-
H3C ~ CH3 H3C, ~ H -H
H2N CO2H H2N CO2H H2N~<Coi~H
s
(1) A suitable solid phase synthesis support, e.g., the
chloromethyl resin of Merrifield, is split into
three equal portions.
(2) Each portion is coupled to one and one of the
glycines shown above after conversion to the
acylated t-butyl ester derivative:
~\0
i5 R~<2 i . >e/ H ~R2 ~--6
H2N CO2H 2. Neut~li7~ion H2N C02
1l H, ~R2
HO2C--(CH2)2-C--NH C02
~CH2 - C, 8 R~,~ 2
H2-O2C-(CH2)2-C--NH co2
2 S CsCO3
= ~Iysty~nt
RI~R2 = H, CH3,C~i2CH3.(CH3)2cH
The conditions for carrying out the above
transformations are well known and used routinely in
the art of peptide synthesis as described in the
references given above.
(3) Each amino acyl resin portion is treated with an
acid solution such as neat trifluoroacetic acid
(TFA), or preferably, a 1:1 mixture of TFA and
~ !QC!T!TI lT~ C~L~t'C'T
WO94/~K09 PCT/US93/06~0
2139350
-61-
CH2Cl2, to remove the t-Bu blocking group. The
resulting acyl amino acid resin is treated with
ethyl chloroformate as described above producing the
oxazolone resin.
(~cH2-o2c-(cH2)2-c--NH ~C 1- TFA
2 CH30CCl
(~CH2-o2c-(cH2)2--C=N
db~Rl
o R2
(4) The three oxazolone resin portions are thoroughly
lS mixed and the resulting mixture is split into three
equal portions.
(S) Each of the resin portions is coupled to a different
glycine protected as t-butyl ester using the
conditions described above; the amide product is
deprotected as described above, for each of the
resin portions and cyclized to the oxazolone using
the reaction with ethyI chloroformate.
R 2" I coupl~ng
(~cH2-o2c-(cH2)2-lc=N\ R, + H2N~co~
2s ~5 o ~
o 2
(~CH2-02C-(CH2)2-C--NH ~_ H,~CO2H
o R2
H3COCOCI (~CH2-02C-(CH2~2--c--NH
35 R~ R2~ R 1~ R2 = H, CH3, CH2CH3, CH(CH3)2 o
SUBSTITUTE S~IEET
WO94/~K~ PCT/.US93/~
-62-
213~35~
(6) The resulting resin portions are mixed thoroughly
and then split again into three equal portions.
(7) Each of the resin portions is coupled to a different
glycine, containing a carboxyl protected as the
t-butyl ester, and the product is deprotected using
TFA as described above; the resin portions are mixed
producing a library contA;n;ng 27 types of resin
beads, each type contA;ning a single oxazolone-
derived tripeptide analog linked to the support via
a succinoyl linker; this linker may be severed using
acidolysis to produce a "solution-phase" library of
peptides whose N-terminus is succinoylated.
R'2"~R'I
-o2c-(cH2)2-c-NH $ -N Rl 1. H2N co2
~ ~2 ~ TFA
~ CH2-O2C-(CH2)2-C NH $ ~ N ~ C02H
Many modifications of this general scheme are
envisioned, including the direct attachment of the ligand
candidates via a C-N bond using a benzhydryl support,
which would allow the straight forward detachment of the
ligand candidates from the support via acidolysis for
further study ("one-head, one-peptide-analog synthesis").
SUBSTITUTE S~
W094/~09 PCT/US93/06240
2139350
- 63 -
4.2.6.1 Design and Synthesis of Oxazolone-Derived
GlycoPeptide Mimetics
A great variety of saccharide and
polysaccharide structural motifs incorporating oxazolone-
derived structures are contemplated including but not
limited to the following.
(1) Oxazolone-derived structures which mimic
native peptide ligands capable of binding to saccharide
and polysaccharide receptors using the design and
synthesis techniques that are described above.
(2) Oxazolone-derived structures linking mono-
, oligo- or polymeric saccharides with each other or with
other structures capable of recognizing a ligand
acceptor.
A wealth of chemical methods for synthesis of
the above saccharides are available. The art of
carbohydrate chemistry describes numerous sugars of
variety of sizes with selectively blocked functional
groups, which allows for selective reactions with
oxazolone and related species producing the desired
products (see ComDrehensive organic ChemistrY, Sir Derek
Barton, Chairman of Editorial Board, Vol. 5, E. Haslam,
Ed., pp. 687-815; A. Streitwieser, C.H. Heathcock, E.
Kosower, Introduction to Orqanic Chemistry, 4th Edition,
MacMillan Publ. Co., New York, pp. 903-949.
For example, Brigl's anhydride shown below can
be reacted with unhindered alcohols to produce ~-
glucosides using well-known experimental conditions. The
resulting sugar, blocked at all positions except position
2, can be used to open a suitable oxazolone using the
reaction conditions described above, e.g., in the absence
or presence of a Lewis acid catalyst such as BF3 in a
suitable inert organic solvent (e.g., EtOAC, dioxane,
etc.).
WO g4/00509 PCl'~US93/06240
- 64
213~3~
I' A~OH ~1A
Bngl's~ ~idc O
Similarly the sugar that results from reaction
of D-glucose with benzaldehyde can be readily blocked at
positions 1 and 6, by sequential reactions with an.
alcohol in the presence of acid, and tritylation using
te~hnjques well known in the art of carbohydrate
chemistry. The resulting sugar, with position 3
unblocked can be used selectively as described above to
derivatize a desired oxazolone structure.
H
CH20H C~l2oH
HO~O ~ L~ OH
~ H + C6H5CHO
HO O
0~ 0
2 0 H C6~1s
2,4-0-bcnzylidcnc-D-glucosc
A suitable oxazolone can also be ring-opened by
a sugar containing reactive amino substituents, i.e., an
aminosaccharide or polyaminosaccharide. For example,
reaction with muramic acid is expected to proceed as
follows.
m uamic acid R~ 1
S~ T~T~
W094/~09 PCT/US93/06~0
21393~5~
.~
- 65 -
Similar treatment which is shown below, of the
structurally interesting ambecide paromomycin, with 1 to
5 equivalents of a tailored oxazolone is expected to
produce a series of novel structures in which a branched
tetrasaccharide scaffold supports peptidomimetic
structures derived from oxazolones in a geometrically
defined manner.
NH2
~ -NH2
HO H~--(
~ NH2
\~NH2
\,~OrH
OH
Paromomycin
(3J Use of oxazolone-derived structures as
replacements of glycosidic linkages.
Selective blocking of all but one hydroxyl in a
sugar allows the selective oxidation of the hydroxyl to
the carbonyl-derivative, which can then be used in an
aldol-type condensation reaction with a methylene
oxazolone to produce an alkene oxazolone; this can then
be ring-opened, by e.g., the anomeric hydroxyl of a sugar
to give a novel saccharide after deprotection.
W094/00509 PCT/US93/06~0
. .
~393S~ 66 -
HX' HXH, HX'~
HO~ of ~ o ~
H3C CH3 H3C CH3 A H3C CH3
OH
HO--~ O
OR OR OH \~OH
RO~ Il~vg~ H20 ~OH -- OJ~A
ZO
OTTGONUCLEOTIDES
4.2.7 Design and Synthesis of
Oxazolone-Derived Oliaonucleotide Mimetics
The art of nucleotide and oligonucleotide
synthesis has provided a great variety of suitably
blocked and activated furAno~s-c and other intermediates
which are expected to be very useful in the construction
of oxazolone-derived mimetics (Comprehensive Organic
ChemistrY, Sir Derek Barton, Chairman of Editorial Board,
Vol. 5, E. Haslam, Editor, pp. 23-176).
A great variety of nucleotide and
oligonucleotide structural motifs incorporating
oxazolone-derived structures are contemplated including,
but not limited to, the following.
WO ~/~9 PCT/US93/06~
21393~o
(1) For the synthesis of oligonucleotides
containing peptidic oxazolone-derived linkers in place of
the phosphate diester groupings found in native
oligonucleotides, the following approach is one of many
S that is expected to be useful.
f_ O~ ~0
~o~`o
, " ~ z
~L ~Z
``c~
/o' o
,~,
~ -
..
~o~
~Z~ o ~ O~
o )~' '`~ ~
+ ~ o
~ ~? ~?
o o o
c ~
- SU~S~TUTF SH~T
W094/0~9 PCT~US93/~ ~0
`' :.
213 93~
(2) For the synthesis of structures in which
an oxazolone-derived grouping is used to link complex
oligonucleotide-derived units, an approach such as the
following is expected to be useful.
~7~
~ ~~ 1-o/~
o o
o~t
/
X
~ ~
~
~ T ~
0~ ~Z
+
r ~
S~JBsTlT-lTE SU~T
-
W094/O~Og 21 39 3~o PCT/US93/06~0
- 68 -
5. Example: Characterization of the
Enantiometric PuritY of Oxfenacine
This example teaches the use of the ring
opening reaction of the pure chiral isomer azalactone
(S)-(-)-4-difluoromethyl-4-benzyl-2-vinyl-5-oxazolone (1)
with racemic mixtures of the methyl esters of (R)- and
(S)-p-hydroxyphenylglycine to form the diastereomeric
conjugates (2) and (3), as shown:
s) ~ ~ (s) (s)
- ^O~e .H
HzN ~ O ~ ~O
2 0 F'h A~HFz ~
(~ N~"~ OH
OH J
These diastereomers can be separated by standard HPLC
methods on normal-phase silica to quantitatively assay
the enantiomeric composition of the starting p-
hydroxyphenylglycines from which the esters are produced.
The (S)-isomer of p-hydroxyphenylglycine
W094/0~9 PCT~US93/06~0
213935 - 69 -
(oxfenacine) is an effective therapeutic agent for
promoting the oxidation of carbohydrates when this
process is depres6ed by high fatty acid utilization
levels (such as occurs in ischemic heart di~ease), and is
also an important chiral intermediate in the production
of penicillin, amoxicillin and several other
semisynthetic antibiotics, including the cephalosporins.
Oxfenacine is prone to racemization, and the assay for
chiral purity described in this example therefore
represents a useful development and quality-control tool.
6. Example: Resolution of Racemic p-Hydroxyphenyl
GlYcine Esterification of D-hydroxY~henYl qlycine
0.3 g (0.2 ml) thionyl chloride was added
dropwise to 5 ml of a stirred solution of 0.4 g of the
stereoisomeric mixture of 4-hydroxyphenylglycine
enantiomers to be characterized in methanol and the
temperature of the mixture kept between 10 and 20C with
ice cooling. The reaction was allowed to proceed at room
temperature for 1 hour. The methanol was then removed at
room temperature under aspirator vacuum (10 torr) on a
rotary evaporator and a solid was obtained. This solid
was dissolved in lO ml of deionized water and the pH
adjusted to 9.2 with 0.88 M ammonium hydroxide. The
solution was then stirred for 1 hour at 10C and the
precipitated solid ester mixture was filtered off, washed
with deionized water and dried at 45C under vacuum to
give 0.41 g of product (94%).
Rinq-O~eninq Addition. 0.181 g (O.OOl mol) of the
esterified 4-hydroxyphenylglycine prepared as outlined
above was dissolved in 10 ml of peroxide-free dry
dioxane. To this mixture was added 0.251 g (0.001 mol)
of (S)-4-difluoromethyl-4-benzyl-2-vinyl-5-oxazolone, and
the resulting solution heated at reflux for 2 hours. The
dioxane was removed by rotary evaporation and 0.43 g
W094/~09 PCT/US93/06240
213~350
_ 70 _
(100%) of the pale yellow solid amide residue was
isolated.
HPLC Analvsis. A solution of the diastereomeric amides
was prepared in methylene chloride at a concentration of
7 mg/ml. This solution was injected into a DuPont Model
830 liquid chromatograph equipped with a detector set at
254 nm using a 20 ~1 loop valve injection system. The
sample was chromatographed on a 25 cm x 0.4 cm stainless
steel HPLC column packed with 5~ Spherisorb S5W silica
gel using a 98/1/1 cyclohexane/n-butanol/isopropanol
mobile phase at a flow rate of 0.9 ml/min. The
enantiomeric amide conjugates were then quantitated using
a calibration curve generated with a series of synthetic
mixtures ContA i ni ng varying ratios of the two pure
enantiomers. The pure L-isomer was purchA~e~ from
Schweizerhall Inc. The pure D-isomer was prepared from
the commercially available D,L-racemate obtained from MTM
Research Chemicals/Lancaster Synthesis Inc. by the method
of Clark, Phillips and Steer (J. Chem. Soc., Perkins
Trans. I at 475 tl976])
rS)-4-difluoromethyl. 4-benzyl-2-vinYl-5-oxazolone
CHF~ ~ N
'h
Ph
5.43 g (0.05 mol) of ethyl chloroformate was
added with stirring to 13.46 g (0.05 mol) of N-acryloyl-
(S)-2-difluoromethyl phenylalanine in 75 ml of dry
acetone at room temperature. 7.0 ml (0.05 mol) of
triethylamine were then added dropwise over a period of
10 min., and the mixture was stirred at room temperature
until gas evolution c A~ 5 hours). The
WOg4/~09 j~ ~ PCT~US93/06~0
213~350 - 71 -
triethylamine hydrochloride was removed by filtration,
the cake was slurried in 25 ml of acetone and refiltered.
The combined filtrates were concentrated to 50 ml on a
rotary evaporator, refiltered, cooled to -30C and the
crystallized product was collected by filtration and
dried in v~cuo to give 10.05 g (80%) of (S)-4-
difluoromethyl-4-benzyl-2-vinyl azlactone. NMR (CDCl3);
CH2 = CH - chemical shifts, splitting pattern in 6 ppm
region and integration ratios diagnostic for structure.
FTIR (mull) strong azlactone CO band at 1820 cm-l.
N-AcrYloYl-(S~-2-difluoromethyl Dhenvlalanine.
21.5 g (0.1 mol) (S)-2-difluoromethyl
phenylalanine, prepared using the method described by
Kolb and Barth (T~ebias Ann. Chem. 1668 (1983)), was
added with stirring to a solution of 8.0 g (0.2 mol) of
sodium hydroxide in 100 ml water and stirred at this
temperature until complete solubilization was achieved.
9.05 g (0.1 mol) acryloyl chloride was then added
dropwise with stirring, keeping the temperature at 10-
15C with external cooling. After addition was complete,stirring was continued for 30 min. To this solution 10.3
ml (0.125 mol) of concentrated hydrochloric acid was
added over a 10-min. period, keeping the temperature at
15C. After addition was complete, the reaction mixture
2S was stirred an additional 30 min., cooled to 0C, and the
solid product was collected by filtration, washed well
with ice water and pressed firmly with a rubber dam. The
resulting wet cake was recrystallized from ethanol/water
to yield 18.8 g (70%) of N-acryloyl-(S)-2-difluoromethyl
phenylalanine. NMR (CDCl3): chemical shifts, CH2 = CH -
splitting pattern and integration ratios diagnostic for
structure
7. Example: Preparation of Chiral Chromatographic
stationary Phase Ring Opening Formation of
coniuqate with A~inoproDyl Silica
W094/00~09 2 1 3 ~ 3 5 0 PCrfUSg3/06240
- 72 -
NO2
o--Si(CH2)3NH2 ~
~) 1 o2N/~/~<~
. N~(,
~ ~C2Hs
Ph
NOz
~ ~rrO--~Si(CH~3 Nl ~ ~ ~ N2
5.0 g of amino~u~yl-functionalized silica was
slurried in 100 ml benzene in a three-necke~ flask
eq~ipped with a stirrer, a heating bath, a reflux
condenser and a Dean-Stark trap. The mixture heated to
reflux and the water removed azeotropically. 3.69 g
(0.01 mol) of (S)-4-ethyl,4-benzyl-2-(3',5'-
dinitrophenyl)-5-oxazolone was added and the mixture was
heated at reflux for 3 hours. The mixture was
subsequently cooled, and the silica collected on a
Buechner filter and washed with 50 ml benzene. The wet
cake was reslurried in 100 ml methanol and refiltered a
total of four times. The resulting product was dried in
a vacuum oven set for 30" and 60OC to yield 4.87 g
functionalized silica. The bonded phase was packed into
a 25 cm x 0.46 cm stainless-steel HPLC column from
methanol, and successfully used to separate a series of
mandelic acid derivatives using standard conditions.
3S
(S)-4-ethYl,4-benzyl-2-(3'.5'-dinitro~henYl)-5-oxazolone
WO94/~K09 PCT7US93/06~0
1393~50
- 73 -
Ph NO2
-~N~CO2H ~3y
NOz C2Hs \Ph
1.09 g (0.01 mol) of ethyl chloroformate was
added with stirring to 3.87 g (0.01 mol) N-3,5-
dinitrobenzoyl-(S)-2-ethyl phenylalanine in 75 ml dry
acetone at room temperature. 1.4 ml (0.01 mol) of
triethylamine was added dropwise over a 10-min. period
and the mixture was stirred at room temperature until gas
evolution ceased (1.5 hours). The triethylamine
hydrochloride was removed by filtration and the cake was
slurried with 25 ml acetone and refiltered. The combined
filtrates were conc~ntrated to 50 ml on a rotary
evaporator, refiltered, cooled to -30C and the
crystallized roduct was collected by filtration and dried
in vacuo to yield 2.88 g (78~) of (S)-4-ethyl-4-benzyl-2-
(3',5'-dinitrophenyl)azlactone. NMR (CDCl3): Freguencies
and integration ratios diagnostic for structure. FTIR:
2S strong azlactone band at ca. 1820 cm~~.
N-3,5-dinitrobenzloYl-lSl-2-ethYl~henylalanine
19.3 g (0.1 mol) of (S)-2-ethylphenylalanine,
prepared from (S)-phenylalanine and ethyl iodide using
the method described by Zydowsky, de Lara and Spanton (55
J. Ora. Chem. 5437 (1990)) wàs added with stirring to a
solution of 8 g (0.2 mol) sodium hydroxide in 100 ml
water and cooled to about 10C. The mixture was then
stirred at this temperature until complete solubilization
was achieved. 23.1 g (0.1 mol) 3,5-dinitrobenzoyl
W094/0~9 PCT/US93/06~0
21393-50~ ~
- 74 -
chloride was then added dropwise with stirring, keeping
the temperature at 10-15C with external cooling. After
this addition was complete, stirring was continued for 30
min. To this solution was added 10.3 ml (1.25 mol) of
conc~ntrated HCl over a 10 min. period, again keeping the
temperature at 15C. During this addition a white solid
formed. After the addition was complete, the reaction
mixture was stirred for an additional 30 min., cooled to
0C and the white solid was collected by filtration,
washed well with ice water and pressed firmly with a
rubber dam. The resulting wet cake was recrystallized
from ethanol/water and dried in a vacuum oven set for 30"
at 60C to yield 27.1 g (70~) N-3,5-dinitrobenzoyl-(S)-2-
ethyl phenylalanine.
PreDaration of Amino~roDyl-Functionalized Silica.
200 g 015M Spherosil (IBF Corporation) was
added to 500 ml toluene in a one-liter three-necked
round-bottomed flask eguipped with a Teflon paddle
stirrer, a thermometer and a vertical condenser set up
with a Dean-Stark trap through a claisen adaptor. The
slurry was stirred, heated to a bath temperature of 140C
and the water azeotropically removed by distillation and
collected in the Dean-Stark trap. The loss in toluene
volume was measured and compensated for by the addition
of incremental dry toluene. 125.0 g 3-
aminopropyltrimethoxysilane was added carefully through a
funnel and the mixture stirred and refluxed for 3 hours
with the bath temperature set at 140C. The reaction
mixture was cooled to about 40C and the resulting
functionalized silica collected on a Buechner filter.
The silica was then washed twice with 50 ml toluene,
sucked dry, reslurried in 250 ml toluene, refiltered,
reslurried in 250 ml methanol and refiltered a total of
three times. The resulting methanol wet cake was dried
3S
WO94/~K~ PCTrUS93/06~0
' 213935~''' ''`
- 75 -
in a vacuum oven set for 30" at 60C to yield 196.4 g
aminG~G~yl silica.
8. Example: Ring-O~ning Conjugation of (S)-1-(1-
naphthyl)ethylamine With The ~ichael-Addition
Product Of Aminomercapto-Functionalized Silica And
(S)-4-Ethyl-4-benzyl-2-acryloyl-5-oxazolone To
Produce A Chiral Chromatoqra~hic Stationary Phase
Formation Of Coniu~ate With rS~ -nA~hthYl)-
ethylamine
CH3
H~" ~NH2
~o~~ )Js~
Ph
Ph
~ o~(~JS ~ N ~ HN
o ~,~
10.0 g (S)-4-ethyl-4-benzyl-2-(ethylthiopropyl
silica)-5-oxazolone was slurried in 100 ml benzene in a
three-n~cke~ flask equipped with a stirrer, a heating
bath, a reflux condenser and a Dean-Stark trap. The
mixture was heated to reflux and the water was removed
azeo~o~ically. 3.42 g (0.02 mol) (S)-(-)- (1-
naphthyl)ethylamine was added and the mixture was heated
at reflux for 6 hours. The mixture was then cooled, the
silica collected on a Buechner filter and washed with loo
ml benzene. The wet cake was reslurried in 100 ml
WO94/~K09 PCT/US93/06~0
213935{) ~
- 76 -
methanol and refiltered a total of four times. The
product waC dried in a vacuum oven eet for 30" and 60C
to give 9.72 g functionalized silica. The bonded phace
was packed into a 25 cm x 0.46 cm stainless-steel HPLC
column from methanol and sl)ccecsfully used to separate a
series of ~-acceptor ~mine derivatives using standard
conditions described in the Chromatography Catalog
distributed by Regis Chemical, Morton Grove, Ill. 60053
(e.g., the 3,5-dinitro benzoyl derivatives of racemic 2-
amino-l-butanol + alpha methyl benzye amine).
Michael Addition bY Merca~toDropvl Silica
15 ~--li(CH2)~SH +
~'
Ph
~ I
~ ~0--Si(cH2)3 - ~o>oo
~ ~ C2Hs
20 g mercapto~o~yl silica was added to 200 ml
benzene in a 500 ml three-necked round-bottomed flask
equipped with a Teflon paddle stirrer, a thermometer and
a vertical condenser set up with a Dean-Stark trap
through a claisen adaptor. The slurry was stirred,
heated to a bath temperature of 140C and the water
azeotropically removed by distillation and collected in
the Dean-Stark trap. The loss in benzene volume was
W094/0~ ~ PC17US93/06~0
?.,~393 i
- 77 -
measured and compensated for by the addition of
incremental dry benzene. 6.88 g (0.03 mol) of (S)-4-
ethyl,4-benzyl-2-vinyl-5-oxazolone was added and the
mixture was stirred and refluxed for 16 hours. The
reaction mixture was then cooled to about 40C. The
resulting functionalized silica was collected on a
Buechner filter, washed with 50 ml benzene, sucked dry,
reslurried in 100 ml of methanol and refiltered a total
of four time. The resulting methanol wet cake wa8 dried
in a vacuum oven set for 30" at 60C to yield 19.45 g
oxazolone-functionalized silica.
(5)-4-ethYl-4'-benzyl-2-acrvloyl-5-oxazolone.
10.9 g (0.1 mol) of ethyl chloroformate was
added with stirring to 24.7 g (0.1 mol) of N-acryloyl-
(S)-2-ethyl phenylalanine in 250 ml dry acetone at room
temperature. 14 ml (0.1 mol) of triethylamine was added
dropwise over a 10-min. period and the mixture was
stirred at room temperature until gas evolution ce~ce~
(1.5 hours). The triethylamine hydrochloride was removed
by filtration and the cake was slurried with 50 ml of
acetone and refiltered. The combined filtrates were
concentrated to 150 ml on a rotary evaporator,
refiltered, cooled to -30C and the crystallized product
was collected by filtration and dried in vacuo to yield
19.5 g (85~) (S)-4-ethyl-4-benzyl-2-vinyl-5-azlactone.
NMR 9CDCl): chemical shifts, CH2 = CH - splitting pattern
in 6 ppm region + integration ratios diagnostic for
structure. FTIR + (mull): strong azlactone CO band in
1820 cm-l region.
Preparation of Mercapto~roYl-~unctionalized Silica. 200
g of 10~ (80A) Exsil silica (Exnere Ltd.) was added to
500 ml toluene in a one-liter three-necked round-bottomed
flask equipped with a Teflon paddle stirrer, a
thermometer and a vertical condenser set up with a Dean-
WOg4/~K09 PCT/US93/06~0
213~3SO
- 78 -
Stark trap through a claisen adaptor. The slurry was
stirred, heated to a bath temperature of 140C and the
water was azeGL~o~ically removed by distillation and
collected in the Dean-Stark trap. The loss in toluene
volume was measured and compensated for by the addition
of incremental dry toluene. 110.0 g of 3-
mercaptopropyltrimethoxysilane was added carefully
through a funnel and the mixture was stirred and refluxed
for 3 hours with the bath temperature set at 140C. The
reaction mixture was then cooled to about 40C. The
resulting functionalized silica was collected on a
Buechner filter, washed twice with 50 ml toluene, sucked
dry, reslurried in 250 ml toluene, refiltered, reslurried
in 250 ml methanol and refiltered a total of three times.
The resulting methanol wet cake was dried in a vacuum
oven set for 30" at 60C to yield 196.4 g of
mercaptopropyl silica.
Chiral azlactone conjugates may similarly be
produced using a variety of azlactone derivatives
containing at the 2-position other yLo~ capable of
undergoing addition (and sequential ring-opening)
reactions. Examples of these groups include
hydroxyalkyl, haloalkyl and oxirane groups.
9. Example: Synthesis of a Mimetic
of Known Human Elastase Inhibitor
This example teaches the synthesis of a
competitive inhibitor for human elastase based on the
structure of known N-trifluoroacetyl dipeptide analide
inhibitors - see, e.g., 107 Eur. J. Biochem. 423 (1980);
162 J. Mol. Biol. 645 (1982) and references cited
therein.
WO 94/00509 PCl'iUS93/06240
~3935o
CF3C~)CI + ~CH, ~
Cl ~ OC2~s
0 Ph
H~ ~CH, ~_
)\ H2N CO2H
Cl ~OC2Hs
2 0
CF~ $CH~ ~ CF~ $N~
N-tri f luoroacetvl- ( S ~ -2 -methY 1 1 eucyl - ( S ~ -2 -
35 ethyl~henYlalanyl-~-isopropylanlide.
W094/~ PCT/US93/06~0
21333Sl~
- 80 -
0.135 g (0.001 mol) 4-isG~o~yl analine ie
dissolved in the minimum amount of an appropriate
solvent, such as acetonitrile, and 0.384 g (0.001 mol) of
2-(N-trifluoroacetyl-(S)-2-methyl leucyl)-(S)-4-methyl-4-
benzyl-5-oxazolone dissolved in the minimum amount of the
same solvent i~ added gradually to the stirred solution
with cooling. Following addition, the reaction mixture
is allowed to come to room remperature and is stirred at
room temperature for 36 hours. The solvent is then
removed in vacuo to yield the solid N-trifluoroacetyl-
(S)-2-methyl-leucyl-(S)-2-ethylphenylalanyl analide,
useful as a competitive inhibitor of human elastase in
essentially quantitative yield.
2-(N-trifluoroacetYl-(S~-2-methYlleucYl)-(S)-4-methYl-4-
~5 benzvl-5-oxazolone.
4.1 g (0.01 mol) N-trifluoroacetyl-(S)-2-
methylleucyl-(S)-2-methylphenylalanine lithium salt is
slurried in 50 ml of an appropriate solvent, such as dry
benzene, in a three-necked round-bottomed flask equipped
with a stirrer, heating bath, claisen head, downward
condenser, thermometer and dropping funnel. The system
is heated to 65C, and 1.09 g (0.01 mol) of ethyl
chloroformate dissolved in 10 ml dry benzene is added
over a 10-min. period. Addition is accompanied by the
vigorous evolution of gas and the distillation of a
benzene/ethanol azeotrope. Following the completion of
the addition, heating is continued for 30 min. The
heating bath is then removed and the slurry is stirred
for an additional 15 min. The precipitated lithium
chloride is carefully removed by filtration and the cake
is triturated with benzene and refiltered. The combined
filtrates are stripped using a pot temperature of 40C to
yield 3.50 g (90%) of crude oxazolone. The product was
purified by recrystallization from acetone at -30C.
FTIR ~mull): 8trong azlactone C0 band in 1820 cm-l region.
W094/0~09 - PC~/US93/06~0
21393~0
- 81 -
N-trifluoracetYl-(S~-2-methYlleucYl-(S)-2-
methvlphenylalanine.
2.23 g (0.01 mol) 2-trifluoroacetyl-(S)-4-
methyl-4-isobutyl-5-oxazolone is dissolved with stirring
in the minimum amount of an appropriate solvent, such as
acetonitrile, and 1.85 g (0.01 mol) of the lithium salt
of (S)-2-methyl phenylalanine in the minimum amount of
the same solvent is added gradually, and with cooling.
This salt is obtained by treatment of (S)-2-
methylphenylalanine (produced from (S)-phenylalanine and
methyl iodide using the method of Zydoski et al., 55 J.
Orq. Chem. 5437 (1990)) with one equivalent of LioH in an
appropriate solvent, such as ethanol, followed by removal
of the solvent in vacuo. After addition of the lithium
salt, the reaction mixture is allowed to warm to room
temperature and is stirred at room temperature for 36
hours. The solvent is then removed in vacuo to yield the
solid N-trifluoroacetyl-(S)-2-methylleucyl-(S)-2-
methylphenylalanine lithium salt in essentially
quantitative yield.
2-trifluoroacetyl-(S~-4-methyl-4-iso~G~vl-5-oxazolone.
12.05 g (0.05 mol) of N-trifluoroacetyl-(S)-2-
methyl-leucine was stirred at room temperature in 100 ml
dry acetone and 5.43 g (0.05 mol) ethyl chloroformate was
added. 7.0 ml (0.05 mol) of triethylamine was added
dropwise over a period of 10 min. and the mixture was
stirred at room temperature until gas evolution ceased
(1.5 hours). The triethylamine hydrochloride was removed
by filtration and the cake was slurried with 25 ml of
acetone and refiltered. The combined filtrates were
concentrated to 75 ml on a rotary evaporator, refiltered,
cooled to -30C and the crystallized product was
collected by filtration and dried in vacuo to yield 10.6
g (88%) of (S)-4-methyl-4-isobutyl-2-trifluoroacetyl-5-
WO94/~K~ PCT/US93/06~0
2139350
- 82 -
oxazolone. FTIR (mull): strong azlactone CO band in 1820
cm-l region.
N-trifluoroacetvl-(S)-2-methYl-leucine.
14.5 g (0.1 mol) of (S)-2-methyl-leucine,
S prepared from D,L-leucine methyl ester hydrochloride
using the method of Kolb and Barth (Liebiq's Ann. Chem.
at 1668 (1983)) was added with stirring to a solution of
8 g (0.2 mol) of sodium hydroxide in 20 ml water, cooled
to 10C, and the mixture stirred at this temperature
until complete solubilization was achieved. 13.25 g (0.1
mol) trifluoroacetyl chloride was then added dropwise
with stirring, keeping the tempe~ature at 10C with
external cooling. After the addition was complete,
stirring was continued for 30 min. To this solution was
added, over a 10-min. period, 10.3 ml (0.125 mol) of
concentrated hyd o~hloric acid, again keeping the
temperature at 15C During the addition, a white solid
formed. After the addition was complete, the reaction
mixture was stirred for an additional 30 min. and cooled
to 0C. The white solid was collected by filtration,
washed well with ice water and pressed firmly with a
rubber dam. The resulting wet cake was recrystallized
from ethanol/water and dried in ~acuo to give 17.4 g
(72%) of N-trifluoroacetyl-(S)-2-methyl-leucine which was
2S used directly in the following step in the sequence
(above).
11. Example: Synthesis of a PeDstatin Mimetic
This example teaches the synthesis of an
oxazolone-derived mimetic of the known aspartyl protease
inhibitor, pepstatin, which has the structure shown:
W O 94/00509 PC~r/US93/06240
213~350
~ COOH
This mimetic is useful as a competitive inhibitor for
proteases inhibited by pepstatin.
15 ~o + l`~CH, >~~i
/~2C~5
~0~
~'
H2N CC~zU
1"~CH
,1, (~2C2~5
~
~0~0
WO 94~00509 PCI`/US93/06240
2~3935 - 84 -
~ + H~N~CO2H ~ CH~
/ ~CO,Ii
H ~ s ~ 11 3 ~ ~ 3 H ~" ~
~N~ l CO2U
~ ~CH3
' ~ ?=
\~ ~CH3 HO" H H3 ~CH3 H ,S~s
>~ ~H~ ~ COOH
3S
W094/0~ PC~/US93/06~0
21393~0 - 85 -
N-isovalervl-(S)-2-methYlvaleryl-(3S.4S)-statyl-(S)-2-
methvl-alanYl-(3S.4S)-statine.
The Boc-protected lithium salt prepared as
described below simultaneously converted to the acid form
and deprotected by treatment with acid under standard
deprotection conditions. 5.17 g (0.01 mol) of N-
isovaleryl-(S)-2-methy derivative added to 100 ml dry
acetonitrile, stirred at room temperature and 3.17 g
(0.01 mol) of the valyl-(S)-4-methyl-4-isopropyl-5-
oxazolone was added with cooling. Once addition was
complete, the mixture was heated to reflux and held at
reflux for 1 hour. The solvent then stripped in vacuo to
give a quantitative yield of N-isovaleryl-(S)-2-
methylvalyl-(3S,4S)-statyl-(S)-2-methylalanyl-(3S,4S)-
statine, useful as a pepstatin-mimetic competitive
1~ inhibitor for aspartyl proteases which are inhibited by
pepstatin (see, 23 J Med. Chem. 27 (1980) and references
cited therein). NMR (d6 DMSO): chemical shifts,
integrations and D2O exchange experiments diagnostic for
structure.
N-Boc-(3S.4S)-statyl-(S)-2-methvlalanyl-(3S.4S)-statine
lithium salt.
6.84 g (0.02 mol) of the Boc-protected
oxazolone prepared below stirred in 100 ml of dry
acetonitrile at room temperature and 3.62 g (0.02 mol) of
the lithium salt of (3S,4S)-statine, prepared from
statine using the method outlined below, was added with
cooling. once addition was complete, the mixture was
heated to reflux and held at reflux for 1 hour. The
solvent was then stripped in vacuo to give a guantitative
yield of N-Boc-(3S,4S)-statyl-(S)-2-methylalanyl-(3S,4S)-
statine lithium salt.
Boc-protected (3S,4S)-statine, [(3S,4S)-4-
amino-3-hydroxy-6- methylheptanoic acid] was produced
from the commercially available amino acid, coupled with
W094/0~ ~ PCT/US93/06~0
Zl39370 86 - ~
2-methylalanine using st~nr~rd peptide synthesis methods
and converted to the lithium salt using the method
described below. 18.30 g (0.05 mol) of this derivative
was stirred in 150 ml dry acetonitrile at room
temperature, 5.45 g (0.05 mol) of ethyl chloroformate and
7.0 ml (0.05 mol) of triethylamine were sequentially
added with stirring and the mixture was stirred at room
- temperature until gas evolution ceased (1.5 hours). The
mixture was then stripped to dryness on a rotary
evaporator, the residue was triturated with 100 ml of
benzene, filtered to remove salts, and the filtrate was
again stripped on a rotary evaporator to yield 16.4 g
(96%) of crude 2-BOC-(3S,4S)-statyl-4,4-dimethyl-5-
oxazolone. Analytically pure material was obtained by
recrystallization from acetone at -30C. NMR (CDCl3) -
chemical shifts and splitting patterns diagnostic forstructure. FTIR (mull): shows a sL~c,ny azlactone CO band
in the 1820 cm-l region.
N-isovalervl-(S)-2-methvlvalyl-(S)-4-methYl-4-isoDropvl-
5-oxazolone-
13.46 g (0.04 mol) of 2-isovaleryl-(S)-2-
methylvalyl-(S)-2- methyl valine lithium salt, as
prepared below, was stirred in 150 ml of dry acetonitrile
at room temperature. 4.36 g (0.04 mol) of ethyl
chloroformate and 5.6 ml (0.04 mol) of triethylamine were
then sequentially added with stirring, and the mixture
was stirred at room temperature until gas evolution
ce~ (1.5 hours). The mixture was then stripped to
dryness on a rotary evaporator, the residue was
triturated with 100 ml benzene, filtered to remove salts,
and the filtrate was again stripped on a rotary
evaporator to yield 12 g (96%) of crude N-isovaleryl-(S)-
2-methylvalyl-(S)-4-methyl-4-isopropyl-5-oxazolone.
Analytically pure material was obtained by
3S recrystallization from acetone at -30C. NMR (CDCl3):
W094/~09 PC~/US93/06~0
2139350 87 -
chemical shifts and splitting patterns diagnostic for
structure. FTIR (mull): shows strong azlactone CO band
in the 1820 cm-~ region.
N-isovaleryl-~S)-2-methylvalYl-(S)-2-meth~l valine
lithium salt.
6.85 g (0.05 mol) of (S)-2-methylvaline lithium
salt, prepared from (S)-methyl valine by the method
described below, was stirred in 150 ml dry acetonitrile
at room temperature and 9.93 g (0.05 mol) of the
oxazolone prepared below was added portionwise with
cooling. Once addition was complete, the mixture was
heated to reflux and held at reflux for 1 hour. The
solvent was then stripped in vacuo to give a 98% yield of
N-isovaleryl-(S)-2-methylvalyl-(S)-2-methyl valine
lithium salt. This salt was used directly in the next
step (above).
2-isovaler~l-(S)-4-methyl-4-iso~ro~yl-5-oxazolone.
2-(S)-methylvaline was prepared from (S)-valine
by the method described by Kolbe and Barth (Tiebiqs Ann.
Chem. at 1668 (1983)), and was acylated with isovaleryl
chloride using stAn~rd acylation methods to produce N-
isovaleryl-(S)-methylvaline, this was subsequently
treated with one equivalent of LioH in ethanol, followed
by removal of the solvent in vacuo to yield the N-
isovaleryl-(S)-methylvaline lithium salt. 22.3 g (0.1
mol) of this Li salt was stirred in 150 ml of dry
acetonitrile at room temperature, 10.9 g (0.01 mol) of
ethyl chloroformate and 14 ml (0.1 mol) of triethylamine
were sequentially added with stirring, and the mixture
was stirred at room temperature until gas evolution
c~A~ed (1.5 hours). The mixture was then stripped to
dryness on a rotary evaporator, the residue was
triturated with 150 ml benzene, filtered to remove salts
3S and the filtrate was again stripped on a rotary
WO94/~K09 2 1 3 9 3 5 0 PCT/US93/06~0
- 88 -
evaporator to yield 17.4 g (85%) of crude 2-isovaleryl-
(S)-4-methyl-4-isopropyl-5-oxazolone. Analytically pure
material was obtained by re-crystallization from acetone
at -30C. FTIR (mull): shows a strong azlactone CO band
in the 1820 cm-l region. NMR (CDCl3): chemical shifts and
splitting patterns diagnostic for structure.
12. Example: Synthesis of a Mimetic
Inhibitor of the HIV Protease
This example teaches the synthesis of a
competitive inhibitor for the HIV protease, based on the
insertion of a chiral azlactone residue into a
strategically important position in the scissile position
of the known substrate, Ac-Ser-Leu-Asn-Phe-Pro-Ile-Val-
OMe. See, e.g., 33 J. Med. Chem. 1285 (1990) and
references cited therein.
~\<~ + HNQ DMF
20 Ph- ~ CO-L-lle-L-Val-OMe
O ~ N Q Md}D-S~kl~D-~-~A~-NH
25 ~
~I C~L-lle-L-Val-OMe
Ph~ "
Ph
~ / O
McaD-Ser-D-l~u-D-~ HN~N~Q
C~lle-Val-OMe
WOg4/~09 PCT/US93/06~0
2139350- 89 -
0.341 g (1 mmol) of HN-(L)-Pro-(L)-Ile-(L)-Val-
OMe prepared using stAn~Ard peptide-synthesis techniques,
is dissolved in the minimum amount of DMF. To this
mixture is added 0.229 g (1 mmol) 2-acryloyl-(S)-4-ethyl-
4-benzyl-5-oxazolone described above, and the mixture is
stirred at room temperature until the Michael addition
reaction has proceeded to completion (as monitored by
TLC). 0.393 g (1 mmol) of MeO-D-Ser(Bzl)-D-Leu-D-Asn-NH2,
prepared from the BOC-protected D-amino acids using
standard peptide protection and coupling chemistries
(see, e.g., J. Med. Chem. 1285 (1990) and references
cited therein) is then added and the mixture is heated to
60C and stirred at this temperature for an additional 12
hours. The DMF is then removed under high vacuum and the
residue is purified by stAn~Ard C18 reverse-phase
chromatography to yield the protected peptide. The side-
chain blocking y~OU~ are subsequently removed using
st~n~rd peptide deprotection terhniques to yield the
product MeO-D-Ser-D-Leu-D-Asn-NH-CO-(S)-Phe-[Me]-NH-CO-
CH2-CH2-L-N-Pro-L-Ile-L-Val-OMe, useful as a competitive
inhibitor for the HIV protease.
13. Example: Synthesis of a Mimetic
Inhibitor for the HIV Protease
This example teaches the synthesis of another
competitive inhibitor for the HIV protease. In this case
the phenyl substituent is replaced with a uracil
derivative.
WO9~/0~ ~ PCT/US93/06~0
2139350
~N~O
H2~Va~ ~ /~ ~CH3
~CH~NH-Asn(~Leu-~(Bz,~)-Ser-~OMe
CO2H H
o
0
CH3
N NH
~ G
Me~D-Ser-D-Leu-D-Asr~NH~CH ~
2 0 CC II' I I'e Val-OMe
0.82 g (1 mmol) of the uracil derivative, whose
preparation is described below, is coupled through the
free proline carboxylic acid group to 0.244 g (1 mmol) of
25 Ile-Val-OMe using stAnAArd peptide coupling methods. The
product is purified by standard C18 reverse-phase
chromatography to yield the protected peptide. The Bzl
side-chain blocking group is then removed using standard
deprotection techniques to yield the product shown above,
useful as a competitive inhibitor for the HIV protease.
WO94/00509 - ` pclius93/o624n
2139350 91-
~N~ 0~70
CO2H~N ~4~ ~ H~ Bzl)-OMe
N~o
H
- ~ H O
CO2H N~ o
H \ H
~N Asn-D-Leu-D-Ser(Bz~)-OMe
0.47 g (1 mmol) of the (S)-(S)-proline-
vinylazlactone Michaei adduct is dissolved in the minimum
amount of DMF. 0.488 g (1 mmol) of MeO-D-Ser-(Bzl)-D-
Leu-D-Asn-NH2, prepared from the BOC-protected amino acid
via st~n~Ard peptide synthesis techniques (see, e.g., 33
J. Med. Chem. 1285 (1990) and references cited therein)
is then added and the mixture is heated to 60C and
stirred at this temperature for 12 hours. The DMF is
then removed under high vacuum to yield 0.95 g of crude
product.
W094/0~09 2 1 3 9 3 ~ O PCT/US93/06~0
- 92 - ~ `-
~<`F
~, 'JH -t N--~""
_ CH3H ~ ~/CH3
~N~o
CO2H N ~ , G
CH3 CH ~ N~CH3
H
2.33 g (5 mmol) of L-proline is dissolved in
the minimum amount of DMF, 1.75 g (5 mmol) of racemic
uracil-functionalized azlactone is added and the mixture
is stirred at room temperature until the Michael addition
reaction proce~A~ to completion (as monitored by TLC).
The DMF is then removed under high vacuum and the
diastereomeric mixture is purified by ~t~nAArd normal-
phase chromatography to give the desired (S)-(S)-Michael
adduct.
30 ~ N y CO2H ~ ~
~ N/CH3 CH7( Cl~ ~CH3
N ~ ~ N ~0
W094/00509 ~ PCT/US93/06240
2l3935~ - 93 ~
3.69 g (o.01 mol) racemic N-acryloyl-2-methyl-
(3'methyluracil)-5'-alanine is stirred with 50 ml of dry
acetone and 1.09 (0.01 mol) of ethyl chloroformate was
added. 1.4 ml (0.01 mol) of triethylamine is added
dropwise over a period of 10 min. and the mixture is
stirred at room temperature until the evolution of gas
ceases (1.5 hours). The triethylamine hyd.o~hloride is
removed by filtration and the cake was slurried with 20
ml of acetone and refiltered. The combined filtrates are
concentrated to 50 ml on a rotary evaporator, cooled to -
30OC and the crystallized product collected by filtration
and dried in vacuo to yield racemic 4-(2-methyl-5'-
[3'methyluracil])-4-methyl-2-vinylazlactone.
W094/0~09 2 1 3 9 ~ ~ PCT/US93/06~0
- 94 -
N ~ /N~ Cl
CH~/ CO2Et
H
\N~c/ ~-
CH~ CO2Et o
o~N~
H
17.15 g (0.05 mol) of the racemic 2-(3'-
methyluracil)-5'-methylalanine ethyl ester is added with
stirring to a solution of 4.0 g (0.1 mol) sodium
hydroxide in 100 ml water. The mixture is stirred until
complete solubilization i~ achieved, and then cooled to
10C. 0.05 g 2,6-di-t-butyl-p-cresol is added as a
polymerization inhibitor followed by 4.52 g (0.05 mol)
acryloyl chloride, which is added dropwise with stirring,
keeping the temperature at 10-15C with external cooling.
To this solution is then added over a 10-min. period 5.7
ml (0.0625 mol) concentrated hydrochloric acid, again
keeping the temperature at 15C. After the addition is
complete, the reaction mixture is stirred for an
additional 30 min., cooled to 0C, and the solid product
is collected by filtration, washed well with ice water
and pressed firmly with a rubber dam. The resulting wet
cake is recrystallized from ethanol/water, and the wet
cake is hydrolized with 6N HCL to yield 12.91 g (70%) of
racemic N-acryloyl-(3'-methyluracil)-5'-methylalanine.
W094/0~09 PCTiUS93/06~0
21 39~ 5~ - 95 ~
CH
\N ~ CI H\ ~N- CH~ p ~ ~o~y~ H30
~ J + CH ~ CO2Et K
O~ N
H
\N ~ /N~2
~ J CH / CO2Et
20.5 g (0.1 mol) of the Schiff base prepared
from the ethyl ester of alanine and benzaldehyde
according to the method of O'Donnell et al. (23
Terahedron T~tt. 4259 (1982)) and 17.4 g (0.1 mol) of 3-
methyl-5-chloromethyluracil in the mimimum amount of
methylene chloride is added dropwise with stirring to a
mixture of finely powdered potassium hydroxide and a
catalytic amount (0.01 eq) of the phase-transfer reagent
C6H5CH2NEt3Cl in the same solvent at 0C. Following
addition, the mixture is stirred at 10C until the
starting material is consumed (approximately 2 hours).
An aqueous workup is followed by mild acid hydrolysis of
the crude with lN HCl/Et2O at 0C for 3 hours to yield
29.5 g (86%) of the racemic ~-methyl amino acid ester.
SYnthesis of 3-methyl-5-chloromethYluracil
A. 74.08 g (1 mol) of N-methyl urea and 216.2
g (1 mol) of diethylethoxymèthylenemalonate are heated
together at 122 C for 24 hours, followed by 170C for 12
hours to yield the 3-methyluracil-5-carboxylic acid ethyl
ester in 35% yield, following recrystallization from
ethyl acetate.
W O 94/00509 PC~r/US93/06240
2139350
- 96 - - ~
B. 30 g 3-methyluracil-5-carboxylic acid
ethyl ester was ~aponified with 10% NaOH to give the free
acid in 92% yield, after stAn~Ard work-up and
recrystallization from ethyl acetate.
C. 20 g of 3-methyluracil-5-carboxylic acid
was decarboxylated at 260C to give a quantitative yield
of 3-methyluracil.
D. 3-methyluracil-5-carboxylic acid was
treated with HCL and CH2O using standard chloromethylation
conditions to yield 3-methyl-5-chloromethyluracil in 52%
yield, following standard work-up and recrystallization
from ethyl acetate.
.
WO 94/00509 PCr/US93/06240
213g350 97
J~ +EIO~< (A) CH",J¦3~CO EI
CH3NH NH2 C02~ 2.170C tl2h J'~HN
O (B~
~NJ¦~/CO2H ~/10% NaOH
H
(C) 260C
CH3 ~ J¦~
o~NHJI
( I) ~ CH20/
2 5 ~,
\N ~\CI
0
W094/~09 PCT/US93/~ ~0
2139350
- 98 -
14. Example: Preparation of a Chiral
Crosslinkinq Coniuaate Monomer
~yO~O
S N ~ ~ H4N O PhC ~ N
4.59 g (0.02 mol) (S)-4-ethyl,4-benzyl-2-vinyl-
5-oxazolone as prepared in Example 3.3.3 above was added
portionwise to a stirred solution of 1.14 g (0.02 mol)
allyl amine in 75 ml of methylene chloride cooled to 0C
with an ice bath. After 15 min. the mixture was allowed
to warm to room temperature, and was then stirred at room
temperature for 4 hours. The solvent was stripped under
aspirator vacuum on a rotary evaporator to yield 5.7 g of
crude monomer, identified by NMR and FTIR analyses. The
product was recrystallized from ethyl acetate to yield
pure white crystalline monomer, useful for fabricating
crosslinked chiral gels, beads, membranes and composites
for chiral separations.
15. Examples: Synthesis of Conjugate Useful in
Isolation and Purification of Serotonin-Binding
ReceDtors
28.6 g (0.1 mol) of sieve-dried octadecane
thiol and 13.9 g (0.1 mol) of 2-vinyl-4,4'-
dimethylazlactone are mixed in a dry round-bottomed flask
equipped with a magnetic stirrer and a drying tube filled
with Drierite and cooled in an ice bath. After 1 hour
the mixture is allowed to come to room temperature and is
held at room temperature for four days. The product is
then dissolved in 250 ml of a suitable solvent, the
system cooled in an ice bath, and a chilled solution of
17.62 g (0.1 mol) of serotonin in 250 ml of the same
W094/00509 PCTiUS93/06~0
21393~0
solvent i8 added over a 30-min period. The reaction
mixture is allowed to come to room temperature over a 2-
hour period and stirred at room temperature for a further
4 hours. The solvent is then removed by freeze drying to
yield 60 g of the derivative
s
CH3(CH2)"SCH2CH2CONHC(CH3)2CONHCH2CH
¢~
~0 ~
OH
which is useful as a ligand for the stabilization and
isolation of serotonin-bin~ing membrane receptor
~5 proteins.
PRODUCT]
16. Example: Synthesis of a Conjugate Useful
in the Isolation and Purification of the
MorDhine ReceDtor
W094/0~9 PCT/US93/06~0
- loo 21393SO
CH30 ~ CH3O ~
0~ + ~<~ ~ 0~
~ CH3 3 ~ ~~3
(I) ' ~, (III)
/ `N 2
/ O ~ N O
/ LiO3S ~ 503L
CH30 ~
SC~Li
~ N ~ OCH~3CH3H 8
H o H H ~
- SO3Li
To a solution of 0.285 g (0.001 mol) of
norcodeine (I) dissolved in 50 ml of the appropriate
solvent, such as benzene, is added a solution of 0.139 g
(0.001 mol) of 4,4'-dimethylvinylazlactone (II) in 10 ml
of the same solvent. The resulting solution is heated to
70 C and held at this temperature for 10 hours. At the
end of this time the solvent is removed under vacuum to
yield 0.42 g of the Michael adduct (III). 0.21 g (0.0005
mol) of this adduct is added portionwise over a 30 minute
period, with stirring, to 0.23 g (0.0005 mol) of lucifer
yellow-CH (IV) in 50 ml of a 1:1 mixture of water and an
appropriate solvent, such as acetone, adjusted to pH 7.5.
W094/~509 PCT/US93/06~0
2139350
at 0 C under a nitrogen blanket. The reaction mixture
is stirred at 0 C for 1 hour and then allowed to come to
room temperature. The mixture is then stirred at room
temperature under a nitrogen blanket for 7 days. The
solvent is removed under vacuum and the water is removed
by freeze drying to give the product (V). (V) is useful
as a probe for the study of receptor proteins that bind
morphine and its derivatives.
17. Example: Synthesis of Conjugate Useful
in the Isolation and Purification of
Proteins Binding Cibacron Blue
To 4.03 g (0.01 mol) of a stirred solution of
thiocholesterol in 100 ml of an appropriate solvent, such
as benzene, is added a solution of 1.39 g (0.01 mol) of
2-vinyl-4,4'-dimethyl-5-azlactone in 10 ml of the same
solvent. The mixture is heated to 70 C and stirred at
this temperature for 4 hours. The solvent is completely
removed under vacuum and the product (VI) is r~AisFQlved
in 200 ml of dimethyl formamide. This solution is cooled
in an ice bath and 8.5 g (0.01 mol) of the Cibacron Blue
derivative (VII), prepared as described below, dissolved
in 250 ml of DMF and 100 ml of triethylamine is added
over a 30 min period. The reaction mixture is stirred
with cooling for 1 hour, allowed to come to room
temperature amd stirred for 12 hours. The mixture is
then added to 1 liter of 25% NaCl in water at 0 C and
stirred for 15 min; then 100 ml of lOM hydrochloric acid
is added with stirring and cooling, and the blue
precipitate is collected by filtration, reslurried in 1
liter of water and refiltered. This extraction procedure
is repeated two more times. The product (VIII) is dried
at 60 C in a vacuum oven at 30" of vacuum. (VIII) is
useful for inserting and positioning the Cibacron Blue
functionality, which is a broadly versatile affinity
recognition ligand in cell membranes for the study of
W O 94/00509 PC~r/US93/06240
- 2139350
- 102 - .
transmembrane ~o~c~c~F involving proteins that bind to
the dye function.
O ~ 2
10 1~ S3
O NH
-03S~
NH
HN N 1 N ~ NH2
. I H
-O3S ~
VrI
WO 94/00509 PCr~US93/06240
2i393S~ - 103 -
~ SO3
O NH
-o3S
NH
HNlN lN~-- H O
H H3C~
2 0 ~ H3C NH
-O3S~J =S
H <S
H= l l
CH3 < l CH3
H3C ~
3 o CH3
VIII`
W094/0~9 PCT/US93/06~0
- 104 - 21393~
Preparation of Cibacron Blue Derivative rVIII)
40.0 g (0.05 mol) of Cibacron Blue F3 GA is
dissolved in 1 liter of DMF at 40 C with stirring. To
this solution is added 26.5 g (0.23 mol) of hexamethylene
diamine with stirring, followed by 4.0 g (o.Os mol) of
pyridine. The reaction mixture is allowed to stir
overnight and the pH is adjusted to 2.0 by the addition
of 80 ml of lOM hydrochloric acid and 940 g of NaCl. 3.s
liters of water are added to precipitate the modified
dye. The mixture is stirred for 1 hour and the dye is
0 collected by filtration. The cake is washed with an
additional 3.5 liters of water at pH 2.0 water and dried
at 70 C in a vacuum oven at 30" of vacuum to yield 34.0
g of the amino-functionalized dye (VII).
18. Example: Synthesis of a Photoreactive
Conjugate Useful in the Isolation and
Purification of B-N-AcetYlglucosamidase
3.63 g (0.01 mol) of 2-acetamido-2-deoxy-1-
thio-b-D-glucG~r nO-? 3,4,6-triacetate (IX) and 1.39 g
f 2-vinyl-4,4~-dimethylazlactone are dissolved with
stirring in 100 ml of an a~G~riate solvent, heated to
70 oc and held at this temperature with stirring for 12
hours. At the end of this time the mixture is cooled to
room temperature and 1.53 g (0.01 mol) of dopamine,
dissolved in 50 ml of the same solvent is added, with
cooling and stirring, over a 30 min period. The
temperature is the allowed to rise to room temperature
and the reaction mixture is stirred overnight. The
solvent is then removed by freeze drying to produce 6.5 g
of the product (X) which is useful for the study of beta-
N-acetylglucosamidase and related proteins of similar
specificity, since the carbohydrate functionality can
bind to these proteins (See 350 Biochim. Biophys. Acta.
437 (1974)). The dopamine-connected catechol
functionality is a photographic developer, capable of
W094/~ PCTiUS93/06~0
2139~50 105 -
photographic amplification by means of standard
tDchniques.
~ N
AcOH2C AcOH2C J H ~
A ~ AcO N~LAc ~ OH
OH
~5 19. ExamDle: SYnthesis of a T-i qand of Protein Kinase
100 mg of the 20-mer cysteine variant, Cys-Thr-
Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-
Ala-Ile- His-Asp, of a protein k; nAC~ natural binding
peptide ligand PK (5-24) (See, 253 Science 414 (1991)),
synthesized by s~n~rd peptide synthesis tec~niques, is
shaken with 7 mg of 2-vinyl-4,4'-dimethyl azlactone in
0.5 ml of an a~o~Liate solvent at room temperature for
6 days. At the end of this period 23 mg of Lucifer
Yellow CH in 0.5 ml of water is added, and the mixture is
shaken at room temperature for 6 hours. The solvents are
removed by freeze drying to yield 130 mg of the
bifunctional adduct (XI), which is useful as a ligand for
competitive evaluation of the bin~ing affinity of
competitive ligands for protein kinases and structurally
similar proteins.
W094/~09 PCT/US93/06~0
" 213935~ - 106 -
H H ~ CH3 ~ S ~ T~-A~-Asp-Phe-~e-A~r
O ~ N ~ O O A-g
LiO3S ~ SO3Li HO-Asp-EGs-lle-Ala-Asn-Asg-A~g
20. ~Y~mple: Svnthesis of Materials Useful as Coatinas
This example describes preparation of a coating
by a ring-opening reaction followed by Michael-addition.
In the first synthetic step, 8.82 g (0.113 mol)
of 95% N-methylethylenediamine were dissolved in 75 ml
methylene chloride with stirring and cooled to 0 C in an
ice bath. Then, 13.9 q (0.10 mol) of
dimethylvinylazlactone (the starting species illustrated
in Eq. 3 with R2 = R3 = CH3) pre-cooled to O C were added
to the methylene chloride mixture such that the
temperature remained below 5 C. The solution was then
stirred at room temperature. After approximately 15 min
a white precipitate began to form. The mixture was
stirred for an additional 2 h at 0 C. A white solid was
collected on a Buechner funnel, washed twice with 25 ml
methylene chloride and air dried to yield 13.92 g of the
ring-opened adduct, identified by nuclear magnetic
resonance (NMR) and Fourier transform infrared reflection
(FTIR) spe~L~c-copy as follows: NMR (CDCl3): CH3-N/gem
(CH3)2 ratio 1:2; CH2 = CH - splitting pattern in 6 ppm
regioin, integration ratios and D2O exchange experiments
diagnostic for structure. FTIR (null): azlactone CO band
at 1820 cm-l absent; strong amide bands present in 1670 -
1700 cm-l region.
W094/OOS09 PCTiUS93/06240
- 107 -
2139350
CH3
H2C=CH-C-NH-C--C-NH-CH2-CHrNH-CH3
(I)
In the next synthetic step, 6.39 g (0.3 mol) of
(I) and 4.17 g (0.3 mol) of dimethylvinylazlactone were
dissolved in 50 ml of benzene and heated to 70 C for 4
h. The flask was cooled to room temperature, stoppered
and allowed to stand for 3 days at room temperature. The
solvent was then decanted off from the thick oil that had
formed. This oil was dissolved in 50 ml acetone and
stripped to produce another thick oil. This latter oil
was pumped on at 1 torr overnight to yield 3.53 g of a
white crystalline solid, identified by NMR and FTIR
spectroscopy as follows: NMR: CH3-N/gem (CH3) 2 ratio 1:4;
CH2 = CH - splitting pattern in 6 ppm region, integration
ratios and D2O exchange experiments diagnostic for
structure. FTIR (null): s~oll~ azlactone CO band at 1800
cm~l .
H2C=CH-fi-NH-C- C-N-CH2CH2-N-CH2CH2 ~/ - CH3
O H3C o (II) . O
In the final synthetic step, 3.5 g (0.01 mol)
of (II) and 1.61 g (0.01 mol) of H2N(CH2)3CH(OC2H5)2 were
dissolved in 50 ml acetone chilled to 0 C and stirred
for 4 h at 0 C. The solution was allowed to come to
35 room temperature and to stand for 2 days. The resulting
WOg4/~K~ PCT/US93/~ ~0
2 1~ 9350 ~ 108 -
yellowish solution was stripped and pumped on at 1 torr
at room temperature overnight to produce 5.0 g of a white
solid. 4.5 g of this solid were dissolved in hot ethyl
acetate, brought to the cloud point with hot hexane and
allowed to crystallize at room temperature overnight.
3.54 g of a white crystalline solid were obtained after
collection by filtration and drying in a vacuum oven
adjusted for a 30" vacuum at room temperature overnight.
The final product was identified by NMR and FTIR
spectroscopy as follows: NMR (CDCl3): CH2 = CH -
~
splitting pattern in 6 ppm region, integration ratios and~0 exchange experiments diagnostic for structure. FTIR
(mull): azlactone C0 band at 1820 cm-~ absent.
CH3 H CH3 H CH3 H ~O~H5
H2C-C.H-~C~ C--`~C~--N~H2-N CH~H2 C--N--C--C--N-CH~ H2 CH
o H3C O ~ o H3C 0 H5
21. Example: Preparation of Coated Silica
Supports Useful in AffinitY Chromatographv
This example describes preparation of an
affinity coating from compou~d (III) as prepared in the
previous example.
1.76 g (0.0034 mol)-of (III) and 0.328 g
(0.0032 mol) of n-methylol acrylamide were dissolved in
S0 ml methanol, after which 1.11 ml water were added. To
this solution were added S g of
glycidoxypropyltrimethoxysilane-functionalized silica
("Epoxy Silican). The mixture was stirred in a rotary at
room temperature for lS min and then stripped, using a
bath temperature of 44 C, to a volatiles content of 15%
as measured by weight loss (from 25-200 C with a sun
3S gun). The silica, coated as a result of exposure to the
SUBST~ruTE ~HE~r
WO94/O~W PCT/~S93/~
-109-
21~9350
mixture of ingredients, was slurried in 50 ml isooctane
containing 32.0 mg VAZO-64 (l .e., the polymerization
catalyst 2,2'-azobisisobutyronitrile dissolved in 0.5 ml
toluene that had been de-aerated with nitrogen. The
slurry was then thoroughly de-aerated with nitrogen and
subsequently stirred at 70 C for 2 h. The coated silica
was then collected by filtration and washed three times
in 50 ml methanol, and air dried. Finally, the silica
was heated at 120 C for two hours to cure the coating
and yield 5.4 g of coated silica. The silica contained
the following attached groups:
CH3 H CH3 H CH3 H ,OC2H5
(SILICA)--C-NH-C--C--N-CH2CH2-N;CH~H2-C~--N--~C--~C,--N-CH~H2~H2 CH
1. 5 g of the coated silica beads were shaken
with 20 ml aqueous HCl (pH = 3.0) for 4 h at room
temperature. The course of the reaction was followed by
testing for the generation of free aldehyde with
ammoniacal silver nitrate (Tollens test). The resulting
25 solid was collected on a Buechner filter, then reslurried
and recollected until the wash water was neutral. The
silica particles were then air dried to yield 1.25 g of
aldehyde packing, the terminal methoxy groups having been
replaced with a single aldehyde group as follows:
CH3 H CH3 H CH3 H
(SILICA)--C-NH-C--C--N-CH2~H2-N-CH2~ H2-C--N--C--C--N-CH2~2 H2-CHO
O H3C O O H3C O
Sl~ iTITU, E Sl IEET
W094/~09 PCT/US93/06~0
21~9350 - 110 -
Repligen Protein A was coupled to the aldehyde
packing using the stAn~rd conditions given for the
attachment of Bovine Serum Albumin in the accompanying
instructions (Technical Note No. 4151) from Chromatochem
Inc., Missoula, MT.
A one-cm glass column was packed with the
Protein-A functionalized material and loaded with human
IgG from PBS buffer (pH = 7.4) at a flow rate of 1.6
ml/min. The IgG was eluted in 0.01M NaOAc (pH = 3.0).
The IgG was then collected and the amount measured
~e~L~G~hotometrically using stA~Ard calibration curves.
The measured capacity of the packing was 12 mg IgG per ml
of column volume.
22. Example: Functionalization of
Azlactone-Containina Polymers
It is possible to p~O~L e existing azlactone-
functionalized polymeric surfaces (e.g., as described in
U.S. Patent No. 4,737,560) and to functionalize them
according to the steps outlined above. For example, by
using successive reactions with dinucleophilic species of
the form HNul-Z-Nu2H and suitable azlactones, a surface of
the form
(SURFACE)-(X)-Az,
where X is a linker and Az stands for axlactone, can be
transformed into the species
W094/~09 PCT/US93/06~0
2139350 - 111
(SURFACE)-IX)-CONHC(CH3)2CONul(Z)Nu2CH2CH2-Az
which may be linked, if desired, to a biomolecule to form
the following conjugate:
5 (SURFACE)
(X)-CONHC(CH3)2CONu~(Z)Nu2CH2CH2CONHC(CH3)2CO-Biomolecule
A suitable experimental procedure is as
follows. The azlactone-functional support is slurried in
a suitable solvent, such as CHCl3, and cooled to 0 C. An
amount of the bifunctional nucleophile eguivalent on a
molar basis to the total number of surface azlactone
~L OU~ present, is dissolved in the same solvent and
added with chAk;ng. The mixture is then shaken at o oc
for 6 hours, allowed to come to room temperature, and
ch~n at room temperature overnight. The support is
collected by filtration, washed with fresh solvent, re-
slurried in an a~lG~riate solvent and one equivalent of
vinylazlactone, dissolved in the same solvent, is added
thereto. The mixture is then shaken, heated to 70 C and
held at this temperature for 12 hours. At the end of
this time, the mixture is cooled and the support
collected by filtration. The ~u~o~ is then washed
thoroughly with fresh solvent and dried in vacuo.
23. Example: Preparation of a Support Useful
in the Purification of Human IqG from Serum
The functional beads prepared as above are
suspended in pH 7.5 aqueous phosphate buffer. A solution
of protein A (Repligen) in 10 mM phosphate buffer (pH
7.0) and at a concentration of 10 mg/900 ~l is added, and
the mixture is then gently shaken at room temperature for
3 hours. The beads are concentrated by centrifugation,
the supernate decanted off and the beads washed five
3S times with pH 7.5 agueous phosphate buffer. The beads
are then loaded into a 0.46 cm inner-diameter glass
WOg4l00509 PCT/US93/06~
21333~ 112 -
column and used to purify human IgG from serum using
stAn~Ard affinity-purification t~c~niques.
It should be apparent to those skilled in the
art that other compositions and processes for preparing
the compositions not specifically disclosed in the
instant specification are, nevertheless, contemplated
thereby. Such other compositions and processes are
considered to be within the scope and spirit of the
present invention. hence, the invention should not be
limited by the description of the specific embodiments
disclosed herein but only by the following claims.
2S
