Note: Descriptions are shown in the official language in which they were submitted.
.
WO 95/17903 ~ ; 2 1 7 ~ 8 4 PCT/US93112591
MODULAR DESIGI~ AND SYNTHESIS OF OXAZOLONE-
DERIVED MOLECULES
1. FTT .T n OF T~ ~IVET`~TION
The prescnt invention relates to the lo~ical
development of hioch.-mic~l and biopharm~re~r~ agents and
of new materials including f~hric~r~ materials such as fibers,
beads, films, and gels. Specifically, the invention relates lo the
development of molecular modules denved from oxazolone
(azlactone) and related structures, and to the use of these
modules in the assembly of simple and complex molecules,
polymers and fabricated materials with tailored properties;
where said properties can be planned and are ri~t~rmin~d 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,
enzvmes, ~enetic materials, and other chiral molecules, and are
thus of great interest in the fields of bioph~rr~e~ltir~lc,
separation and materials science.
'~ . BACKGROUND OFT~F INVET~
The discovery of new molecules has traditionall~
focused in two broad areas, biologically active mt l~cul~c, which
are used as drugs for the treatment of life-threatening
diseases, and neu ma~erials. which are used in commercial.
especiall~ hightechnological applications. In both areas. the
strate~ used to discover new molecules has involved tuo basic
operalionS: (i) a more or less random choice oi a molecular
candida~e~ prepared either ia chemical svnthesis or isola~ed
from na~ural sources, and (iij the ~es~in~ ol ~he molecul~r
WO 95/17903 ~ , ~ 2 1 7 9 9 8 4 PCT/US93112S9I
candidate for the property or properties of interest. This
discovery cycle is repeated indefinitely until a molecule
possessing the desirable 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 pre~l-min~n~ly 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 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, ~
biologically active molecule, referred to as a ligand. binds with
another molecule, usuall~ a macromolecule referred to as
ligand-acceptor (e.o., a receptor or ~n enzyme), and this
WO95117903 '- `, ." ` ~ `.. 2 1 79~84 PCTlllS93~1259
bindirlg 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. I Design and Synthesis of Nucleotides
Recent interest in gene therapy and manipulation of
gene expression has focused on the design of synthetic
oligonucleotides that can be used to block or suppress gene
expression via an antisense, ribozyme or triple helix
m,~h~ni~m. To this end, the sequence of the native target DNA
or RNA molecule is characterized and standard methods are
used to synthesize oligonucleotides representing the
complement of the desired target sequence (see, S. Crooke, The
FASEF~ ~ourr~l. Vol. 7, Ap~. 1993, p. ~33 and references cited
therein). Attempts to design more stable forms of such
oligonucleotides for use ~Qhave typically involved the
addition of various groups, e.g., halogens, azido, nitro, methyl,
keto, etc. to various positions of the ribose or deoxyribose
subunits (cf., The Organic 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 increasingly are being
viewed as the components of living systems with the
enormously complex structures required for the 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
IV~o nalurally occurring amino ~cids can be used by nalure to
-
` 2 1 7 9 PCT/US93/12~91
WO 9~/17903 , ~ _ 9 ~ 4
convey ~ fundamental molecular messages, i.e, via formation
of the two possible dipeptide structures, and four different
nucleotides convey '74 molecular messages, two different
monos~cch~ride subunits can giYe 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 discreet molecular messenger in a
given physiological system.
The gangliosides are examples of the 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 strateglc locations on
the cell wall: their lipid component enables them to anchor in
the hydrophobic interior of the cell wall, positioning their
hydrophilic component in the aqueous extracellular milieu.
Thus the gangliosides (like many other 5~r~h:~rj~1~c) have been
chosen to act as cellular sentries: they are involved in both the
inactivation of bacterial toxins and in contact inhibition, the
latter being the complex and poorly understood process by
which normal cells irlhibit 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
WO 95/17903 . ~ 2 1 7 9 9 8 4 PCT/U593112591
~ '~,
o~=
0~o ,~ ~
~0~ 0
~_
wo 9~/1 7903 A 2 ~ 7 , ~ 8 ~ PCT/1l5931 1 2s9 1
The olit~osaccharide componentS of the glycoproteins
(su~ar-protein composites) responsible for the human blood-
group antigens (the A, B, and O blood classes) are shown below:
HocH t
HOCHt ~ ~O~ ptcltein
H~Ht l~c \H
H OH
H~o
~ O
CH~
BLOOD GROUP O ANTIGEN, TYPE II
HOCH t
HO~O ~pn~tcin
H~ H Ht~AC
HOC H OH
H~--~ rlCOC ~eottp ~' Y-OH
H r CH~
~LOOD GROUP A AND ~ ANTIGENS
G
WO 95/17903 2 1 7 9 ~ 8 ~ iY~llL'.Yl
~.
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, deglycosylation of
yLill~Jpo~tin 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 ~P~iPm~hPr et al., Ann. Rev. Biochem
57, 78~ (1988); and glycosylation of 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 Biological Ligands
A currently favored strategy for the 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 b ological testing. Since most useful drugs have been
WO 9~/17903 ` I i ` PCT/lJS93112~91
2 1 7q984
discovered not through the ``rational" ~pproach 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 struetures 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 faeile hydrolysis in aeidie media or in the presenee 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., reeeptors or enzymes, whieh can be 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 tissues and
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 ~ basis. Using numerous redesign-
synthesis-screening cycles, peptidic ligands belonging to a
certain biochemic~l class h~ve been converted b-~ groups of
organic chemists and pharm~cologists to specific
peptidomimetics: however~ in the majoritv of c~ses the results
in one biochemical ~rea, e.g.~ peptidase inhibi~or design using~
g
wo 9511~9û3 ~ '- 2 1 7 9 9 ~ 4 PCT~S93112591
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 alpha-amino acids. Many of these
mimetics exhibit several of the troublesome features of native
peptides (which also comprise alpha-amino acids) and are,
thus, not favored for use as drugs. Recently, flln~i~m~rr~l
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 T~irsrhm~nn, R. et al., 1992 J. Am. Ch~m 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 idenrifir~rion of
lead-structures, and also the identification of useful drug
candidates through screening of randomly chosen 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 wi~hout removing them
from the pins. Houghton, (1985, Proc. N~tl Acad. Sci. USA
8':~131; and U.S. Patent No. 4,631,711) 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 usin~ solid phase synthesis
WO 95/17903 - _ 2 1 7 9 ~ ~ 4 PCT/US93/12591
techniques. The peptides produced are then recovered ~nd
tested individually. Fodor et al., (1991, Science ~51:Z67~
desc}ibed 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 en~in~Pprinv methods for expressing
huge peptide libraries on the surface of phages (Cwirla et al.,
1990, Ptoc. 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, then divided into portions each of which was
re-subjected to acylation with a second desirable amino acid
producing 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.
7~1cl~Prm~n 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, 199'2.
Proc. Natl. Acad. Sci. USA 89:5381).
Recently in an alternate strategy for the design of
therapeutically active mimetic ligands much ~ttention has been
focused on the construction and applic~tion of molecules which
possess the property of binding to nucleic ~cids. These
materials, whether they be direct Watson-Crick type
"~ntisense" nucleotide mimetics. Hoogstein-type binders or
1~
wo 95/1~903 : ~ ' 2 1 7 9 9 ~ ~ PCTIUS93/12~S9I
minor groove binding compounds such as those pioneered by
Dervan and coworkers, have employed a variety of derivatives
and variants of the naturally occuring sugar-phosphate
backbone. Polyamide backbones have also been employed to
support the base complements. While binding and desired
functionality is observed in virrO withthese systems. they have
inherent design drawbacks for in vivo use for hybridization
against a rogue gene or its insidious RNA. The two main
drawbacks of these polyamide systems are in (a) the persistent
reliance upon an amide bond which is susceptible to proteolytic
cleavage, and (b) the inability of the compound either as a
class, or even singularly show efficient membrane
permeability .
However, in the course of this work, a great amount
of knowledge has been amassed vis-a-vis 1.) the ability of a
synthetic scaffold to support a series of natural or designed
bases in such a manncr that tight binding to natural nucleic
acids is observed; 2.) the requirements for designed or
naturally occurring nucleotide bases other than guanosine,
cytosine, thymidine, adenine, or uridine, to efficiently hydrogen
bond (hybridize) to another, natural base or nucleotide. Among
these natural nucleotide mimetics are showdomycin ( I ) and
pseudouridine (~) and the synthetic compounds (3) and (4).
NH ~H HN--~ NHJ~ N
HO~ O~ HO oS~ HO O~J HO ~N
~)(1~ ~'~(2) -~(3~ h~O~
OH OH OH OH OH OH OH OH
It has been demonstrated that such unnatural or
modified bases can show efhcient hybridiz~tion if projected
from an effectlve scaffold as shown here for both tautomers of
5-bromouracil, which can bind to either adenine or guanine
1~
W095/17903 ~ 2 1 79~84 PCTll~S93/lZ591
N=\ N~\
,_H ~\N_5Uo "~ ,\N_5=o
Br~H"~ ~. ~ ,~,N
Suo Sug
The primary goal of any "antisense" or "gene
therapy" is to inactivate the archival rogue information
(deliterious DNA) or the messenger information (the
corrPps~n~iing. RNA) by very tight, specific hybridization.
As previously stated, there are a multitude of paths
by which the "anti-sense" agent may be metabolized or
destroyed outright, and as a result of these known obstacles,
chemists have pursued alternative backbones that might
enable their compounds to (a) survive the degradative
response of the immune and metabolic pathways, and (b)
transit the cellular and nuclear membranes to the site at which
hybridization may occur.
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 ~re~ of biological
WO 95117903 . ~ ' PCT)US93112C9l
chemistry depends on the effective utilization of sophisticated
separ~tion technologies.
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 m:~cc~ resulting in separation.
The development of substrates or supports to be
used in separations has involved either the polyme}ization-
crocclinkin~ 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. In the majority of cases,
prior art support materials have been developed to perform
specific separations or types of separations and are thus 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-
forminr materials have been used to modify commercially
avail~ble fabric~ted materials into articles with improved
proper~ies: however the success of this ~pproach remains tO be
seen.
13
WO 95/17903 2 ~ 7 ~ PCTIUS93/12591
If a chromatographic support is equipped with
molecules which bind specifically with a component of a
complex mixture, that component will be separated from the
mixture and may be released subsequently by changing the
rim~rt~l conditions (e.g., buffers. stringency, etc.) This
type of separation is appropriately called "affinity
chromatography'' and remains an extremely effective and
wide~y 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 which in order to attain their maximum
separating efflciency need to be used under conditions that are
damaging to biomolecules, e.g., conditions involving high
pressure, use of organic solvents and other d~n:~tl~rin~ agents,
etc.
The deYelopment of more powerful separation
technologies depends ~i~nific~ntly 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 te~ dtU-~ 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 poiymerization
reactions .
14
wo 95/17903 ., ~ , . 2 ~ 7 9 9 8 ~ PCTIUS93/1259~
2.2 ~xazolones
Oxazolones, or az~actones, are structures of the
general formula:
R
R
A--~/2 5 (CH2)n
\ 1 6/
0~
O
where A is a functional group and n is 0-3. Oxazolones
containing a fiYe-membered ring and a single substituent at
position 4 are typically encountered as transient
in~Prm.or~i~tl~S 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:
Ay~O A~ ~0
R1 ~1 R2
Chiral oxazolones possessin~ 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
WO95/17903 ~ 2 ~ 4 PCT~Sg31l259l
acylamino acyl structures, have been prepared and isol~ted
in the pure, crystalline state (Bodansky. M.; Klausner, Y. S.;
Ondetti, M. A. in "Pep~ide Synthesis", Second Edition, John
Wiley & Sons, New York, 1976, p. 14 and references cited
therein). 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, Synthesis, and Biology", Vol. 1, Gross, E.
& Meienhofer, J. editors, 1979, p. 315).
Rs~ mi7~lion 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 _
Vl shown below (see Atherton, E.; Sheppa}d, R. C. "Solid
Phase Peptide Synthesis. A Practical Approach", IRL Press
at Oxford UniYersity 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 (rV) and aminolysis of the racemic oxazolone
(V) producing acemic peptide products (VI).
.
/~
WO95/17903 , . ~ ,i 2 1 799~4 P~ S93112591
i " , ,
H
S~
Rl
Base SSs~ R2 Proton Donor
lV
55s H~ Rl H R3 arninolysis
V ~CO2--~
o ~ u Vl
Extensive research on the trapping of oxazolones
III (or of their activated acyl precursors II) to give
acylating ~gents which undergo little or no racemi~ation
upon aminolysis has been c~rried out, and successes in this
__
WO9S/17903 ~ 21 79984 PCT/~S93112591
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. 31~).
Thus, attempts to deal with the racemization
problem in peptide synthesis have involved suppressing .or
avoiding 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
N H N H
H
3 . SUMMARY OF T~F.~VE~TION
A new approach to the construction of novel
molecules is described. This approach involves the
development of oxazolone (azlactone) derived 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.
1~
WO 9~i/17903 ` ' ` ' ~- 2 1 7 9 ~ 8 ~ PCTJUS93112591
and/or interact with the binding sites of ~ n~tive 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 apparatus that performs a
specific operation wherein each module performs a specific
task contributing to the overall operation of the apparatus.
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 pi-type interactions,
hydrophobic and van der Waals forces, hydrogen bonds.
(3) A continuum of fabricated materials exiSls spanning a
dimensional range from about 100 Angstroms to I 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 may form during the synthesis of
peptides, would be ideal building bloclcs for constructing
backbones or scaffolds bearing the appropriate functional
groups that either mimic desired ligands. and/or interact
with qppropriate receptor binding sites. ~nd for carrying
out the synthesis of the various p~rts of the functionalized
sc~ffold orthogon~, provided th~t raCemiZ~Iion of the
19
wo g5,l7903 2 1 7 ~ 9 8 ~ PCT/US93/12591
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 nove~ molecules. Furthermore, 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.
Working examples describing the use of
oxazolone denved 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; carbohydrate, oligonucleotide and pharmacophore
mimetics and polymers formed via free-radical or
condensation polymerization of oxazolone-containing
monomers .
rn accordance with the present invention, the
oxazolone-derived molecules of interest possess the desired
stereochemistry and, when required. ~re obtained
enantiomerically pure. In addition to the synthesis of single
molecular entities, the synthesis of libraries of oxazolone-
derived moiecules. using the techniques described herein or
modifications thereof ~ hich are well known in the art to
WO95/17903 ' . 2 ~ 7 9 9 8 4 PCT/US93112!;91
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
tlic~hs~ir~rPd carbon at the 4-position, as well as synthetic
nonchiral oxazolones may be synthesized readily and used
as molecular modules capable of controlled reaction with a
Yariety 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 stereo~ hPmictry. Furthermore, according to
the present invention, 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 enzymo~ogical
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
cons~ruction 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 ox~zolone precursor in the enantiomerically pure
21
wo 95/17903 ~ 7 ~ 9 ~ 4 PCT~Sg3/12591
state, ox~zolone precursors which are not en~ntiomeric~lly
pure may be used for syntheses.
22
WO 95117903 . ` ~ ~ ~ r ~ 2 ~ 79 ~ Pc~msg3)~C91
The invention is also directed to a method of making a
polymer having a particular water solubility' comprising the
steps of; a) choosing a first monomer having the formula
R~..n
A--X - NH--C CO--Gl ~ ~ Y--
I
R~ n , n
wherein R and R' are the same or different and are chosen
from those organic moieties exhibiting hydrophobicity; b)
choosing a second monomer having the formula
R1..n
A--X ~ NH--C CO--G~ Y--E~
R.~...n . n
wherein R and R' are the same or different and are chosen
from those organic moieties exhibiting hydrophilicity; and c)
reacting said monomers to provide an effective amount of
each monomer in a developing polymer chain until a
polymer having the desired water solubility is created.
According to this method said hydrophobic organic moieties
can include those which do not have carboxyl, amino or
ester functionality. Also said hydrophilic moieties can
include those which do have carboxyl, amino or ester
functionality.
This invention is further directed to using said method
of preparing a synthetic compound to produce a compound
that mimics or complements the structure of a biologicall~
active compound of the formula. This method can be used
to produce pharmacaphores, peptide mimetics, nucleotide
mimetics, carbohydrate mimetlcs. and reporter compounds,
for example.
23
WO 95/17903 . 2 1 7 9 9 8 4 PCT/US93/12591
This invention is also fu}ther directed to a method of
preparing a combinatorial library which comprises: a)
preparing a compound having the formula;
R1,.n
A--X ' NH--C CO--Gl n ~ Y--B
R.1...n , rl
n 2 1; and b) c~ tin~ further reactions with the
compound to form a combinatorial library.
Still further this invention is directed to a method of
separating a desired COlu~v~ 1 from a plurality of
,olu~ouuds, which C~ ;ces, a) preparing a separator
compound having the formula:
R1 n
A--X ~ NH--C CO--Gl-n Y--B
R~ n , n
n> 1;
b) c~nt~rtin~ said separator compound with the plurality of
compounds; and c) differentiating said second compound
and the separated compounded from said plurality of
compounds.
24
wo s/l79n3 . - tt!~ 2 ~ 7 9 9 ~ 4 PCTIUS9311~591
9 , , .
,.
different ways. The compounds of the present invention can
be synthesized by many different routes. It is well known
in the art of organic syn~hesis that many different synthetic
protocols can be used to prepare a given compound.
Different routes can involve more or less expensive
reagents, easier or more difficult separation or purification
procedures, straightforward or cumbersome scale up, and
higher or lower yield. The skilled synthetic organic chemist
knows well how to balance the competing characteristics of
synthetic strategies. Thus the compounds of the present
invention are not limited by the choice of synthetic
strategy, and any synthetic strategy that yields the
compounds described below can be used.
4.1 Synthe~ic of Chir~l Substituted 0~:~7nlones
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.:
The scope of this invention is intended to encompass
each species of the aforementioned Markush genus. Thus,
for example, where there is a numeric designation in the
claim, that can be an integer, i.e. m or n, the scope of this
invention is intended to cover each species that would be
represented by every different integer.
WO95/17903 i ~ ` 2 t 7 ~ 9 8 4 PCT/U593/1~491 ~
ACOCI + R' R2 AJ~N~COzH
HzN C02H H
AC~O
~<X
Rz
When the substit~ 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-
type addition to an alkenyl oxazolone as follows:
A7~
Rz ~F~z
where X = S or NR and A' is a functional group.
The required chiral amino acid ~ ,u~ for
oxazolone synthesis may be produced using stereoselective
reactions that employ chiral auxiliaries. An example of
such a chiral auxiliary is (~)-(-)- I -dimethoxymethyl-2-
methoxymethylpyrrolidine (SMPD) (~ iebiF's Ann Chern
1668 (1983)) as shown below,
,: ~
WO95~179~13 ~ 7',!~ ~ ~' 2 1 7998~ PCTIUS93111~i91
~j~H R'f~NH.
lo o COOCH3
C H3 N \C H
~\ C H30 0 C R~
~X/LDA
~t CH I -~8C
\N H
~\HCOOH ~H
~" hydrolysis b o
H H O O C "~R3 1
0 ~2 CH300C--7\R3
CH3 R2
wherein R2 = CH3, i-Bu, or benzyl; and R3 = CH3, CHF2,
C2Hs, n-Bu, or benzyl. A second example involves 5H,10~-
Hoxazolo[3,2-c][1,3]benzoxazine-2(3H),5-diones (55 J. Org.
Chem. 5437 (1990)),
~1
WO 9r/17903 , : ., 2 t 7 9 9 8 4 PCT11~593/12~r91
CHO
/OH \~,H + COCI~ CO3
H~N~ co2 CHCI~
R~
R'X ~ , LiOH \~ ~
O ~ H3N' CO2
wherein Rl = phenyl or i-Pr; and R2 = CH3, C2Hs, 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 }eactions, as shown below for a case involving
a commercially-available organism (~3 J. Org. Ch- m. 1826
(1988)),
R\ /NH2 mycob~ctenum R~ ~NH~ NHz
/C\ oeo2uJrum /C\ + /C\
F12 C O N H2 ATCC25795 ~ ~ C O O e~ 2 C O N H2
L-Acid D-Amide
wherein Rl = i-Pr, i-Bu, phenyl, benzyl, p-methoxybenzyl,
or phenethyl; and R = CH3 ~r CoH~.
Wo 95~17903 . ~ - , 2 ~ 7 9 ~ 8 ~ PCT/US93J12591
Racemic mixtures of 4,4'-disubstituted
oxazolones may be prepared from monosubstituted
oxazo~ones by alkylation of the 4-position, as in the
following transformation (Svnthesis Commun.. Sept. 1984,
at 763; 23 Tetrahedron Lett. 4259 (1982)):
Ph
Ph~ H + >_ r~. Ph--</
Resolution of racemic mixtures of oxazolones
may be effected using chromatography or chiral supports
under suitable conditions which are well known in the art;
using fractional crys-~lli7~inn of stable salts of oxazolones
with chiral acids; or simply by hydrolyzing the racemic
oxazolone to the amino acid derivative and resolving the
racemic modification using standard analytical techniques.
A wide variety of 4-monosllhs~it~ d 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 ~
Chem. :ZSO~ (1984): 4~8 SyD-hesis Comm~lnica~lons (1984))
74
~VO 9~/17903 ~ 2 ! ~ 9 ~ 8 4 PCT/11593/12591
Ph~N~COOH ~CHO ~N\~o H
H~
Ph~ H
o
The hydrogenation may be carried out using a
stereospecific hydrogenation catalyst to produce an
enantiomerically pure product. This product may
subsequently be stereoselectively alkylated to produce the
enantiomeric disubstituted oxazolone module. An example
of this is given below for the synthesis of an
enantiomerically pure adenine-derivatized nucleotide
mimetic oxazolone module:
~c
WO 95/179~3 . ' '~ 2 ~ 7 9 ~ ~ ~ PCT/I~S93112591
Step 1. - ~rtachment of ~ Carbonyl-terrninal Spacer
NHBZ
;~CN 1 l~ N~--3C >
Step 2. - Coupling to the Oxazolone 4-Position
NH8~ NH8~
b~X > ~ ~t N PhH 50 ~ N~ ~Ph
Step 3. - Stereospecific Reduction and Phase Transfer
Methyiation
NHO- NH8~
~ 2 M~l Cù,mn Eu N OH' ~N o~Ph
Thus, there are numerous chemical and
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 and
oligonucleotides, mimics and variants of these,
carbohydrate ~nd pharmacophore variants and mimetics, or
any other side chain substituent which can be ~ttached to a
scaffold or a backbone to produce a desired interaction with
a target system.
31
` i~ 9
WO 95117903 ~ 1 7 9 9 ~ 4 PCTIUS93/1
4.~ Chiral Reco~nition
"Chiral recognition" is a process whereby individual chiral
enantiomers display differenial binding energies with an
enantiomerically pure chiral target or recognition agent. This agent
may be attached to a surface to produce a chiral stationary phase
(CSP) for chromatographic use or may be used to form
diastereomeric complexes with the racemic target. These complexes
have differing physiochemical propereties which allow them to be
separated using standard unit processes, such as fractional
crystallization.
Two steps are necessary for this recognition process to
occur with a CSP; 1.) absorption and 2.) energetic differentiation
between the enantiome}s. The absolute binding energies between the
enantiomers and the surface determine the tightness of the binding.
The difference in energy between the complexes d~t~rminps the
selectivity. This is represented in the following diagram
Energy
Ener~y of R and S Isomers
R-R Compie~
R-S Compie~
3Z
~ wo 95117903 2 1 7 9 ~ 8 4 PCTIUS93112591
The interaction of the enantiomeric R and S species with
the CSP can be envisioned as a "three point interaction". This does not
mean that three actual points of :~tt~hm.~nt or association are
necessary, but rather that any three kinds of attractive or repulsive
interactions within the diastereomeric complexes can serve to
differentiate ("recognize") the enantiomers. Greater differentiation
("recognition") betwen the complexes is promoted by multiple
combinations of attractive and/or repulsive interactions, including
hydrogen bonding, ionic interactions, dipole interactions,
hydrophobic, pi-pi interactions and steric interactions between the
two chiral species. The larger the number and the more varied the
types of these interactions, the greater the resulting energy
differences between the complexex and the greater the degree of
"recognition" per interaction.
This is figuratiYely illustrated below:
"Three point interaction"
\~ "~N~,
~ ~ ~ ~, N
NOz
33
WO 9~/17903 ;~ 7 9 9 8 4 PCTIUS93112591
The possible modes of interaction which can
participate in such "three point interactions" is depicted below
for a enantiomerically pure oxazolone derivative:
C~r~ c Ir2ul~st r Inter2 cbons
Et-sonds
HydropAob~
~-- -- ~H~/J`\N02
0 H~C CH20H
Et-Botlds
4.3 Syr~th~tic Tr~ncform ~tiol-c of ('hiral Oxazolsnes
4.3.1 Reactions with One or Two Nucleophiles Producing
Conjugates
Chiral oxazolones may be subjected to ring
opening reactions with a variety of nucleophiles producing
chiral molecules as shown below:
~R A J~ N ~ \
R1 ~ o
In the structure ~bove, Y represents an oxygen, sulfur, or
nitrogen atom. R I and R'~ differ from one ~nother and
taken ~lone each signifies one of the following: alkyl
34
WO 95/17903 2 t ~ ~ 9 ~ 4 PCT/US93112591
.., . ~ , ~ ~
inc~uding carbocyclic and substituted forms thereof; aryl,
ara~kyl, alkaryl, and substituted or heterocyclic versions
thereof; preferred forms of R1 and R'' are the side chain
substituents occurring in native polypeptides,
oligonucleotides, variants or mimetics of these,
carbohydrates, pharmacophores, variants or mimetics of
these, or any other side chain substituent which can be
attached to a scaffold or a backbone to produce a desired
interaction with a target system.
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 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 must be temporarily protected using
suitable orthogonal protection strategies based on the many
protecting groups known in the art; cf., e.g., Protective
Groups in Or~nic 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:
I ) Amino acid derivatives of the form (AA)N,
which would include, for example, natural and synthetic amino
acid residues (N= I ) including all of the naturally occuring alpha
amino acids, especially alanine, arcinine, asparagnine, aspartic
acid, cysteine, glutamine, glutamic acid, clycine, histidine,
isoleucine, leucine, Iysine, methionine. phenylalanine, proline.
serine, threonine, tryptophan. tyrosine; the naturally occuring
disubs~itu~ed ~mino acids, such as ~mino isobutyric acid, ~nd
isovaline, etc.; a varie~y of synthe~ic ~mino acid residues,
3~
WO 95/17903 , , l~ PCT/US93112591
21 79984
including alpha-disubstituted variants, species with olefinic
substitution at the alpha position, species having derivatives,
variants or mimetics of the naturally occuring side chains; N-
Substituted glycine residues; natural and synthetic species
known to functionally mimic amino acid residues, such as
statine, bestatin, etc. Peptides (N=2-30~ constructed from the
amino acids listed above. such as angiotensinogen and its
family of physiologically important angiotensin hydrolysis
products, as well as derivatives, variants and mimetics made
from various combinations and permutations of a~l the natural
and synthetic residues listed above. Polypeptides (N=3 1-70).
such as big endothelin, pancreastatin, human growth hormone
releasing factor and human pancreatic polypeptide.
Proteins (N>70) including structural proteins such
as collagen, functional proteins such as hemoglobin, regulatory
proteins such as the dopamine and thrombin receptors.
2) Nucleotide dcliv~ a of the form (NUCL)N,
which includes natural and synthetic nucleotides (N=l) such as
~IAPrlo~in.~, thymine, guanidine, uridine, cystosine, derivatives
of these and a variety of variants and mimetics of the purine
ring, the sugar ring, the phosphate linkage and combinations of
some or all of these. Nucleotide probes (N=2-25) and
oligonucleotides (N>25) including all of the various possible
homo and heterosynthetic combinations and permutations of
the naturally occuring nucleotides, derivatives and variants
cnnt~inin~ synthetic purine or pyrimidine species or mimics of
these, various sugar ring mimetics, and a wide variety of
alternate backbone analogues including but not limited to
phosphodiester, phosphorothionate, phosphorodithionate.
phosphoramidate, alkyl phosphotriester, sulfamate, 3'-
thioformacetal, methylene(methylimino), 3-N-carbamate.
morpholino carbamate and peptide nucleic acid analogues.
3 ) Carbohydrate derivatives of the form (CH)n.
This would include natural physiologically active carbohydrates
such as including related compounds such as glucose. galactose.
sialic ~cids, beta-D-glucosvlamine and nojorimycin which ~re
36
wo 95/179113 - - 2 1 7 9 q 8 4 PCTIUS931~cgl
j .
both inhibitors of g~ucosldase, pseudo sugars, such as 5a-carba-
2-D-galactopyranose~ which is known to inhibi~ the growth of
Klebsiella pneumonia (n= I ), synthetic carbohydrate residues
and derivatiYes of these (n=l) and all of the complex oligomeric
permutations of these as found in nature, including high
mannose oligosaccharides, the known antibiotic streptomycin
(n>l).
4 ) A naturally occurring or synthetic organic
structural motif. This term is defined as meaning an organic
molecule having a specific structure that has biological activity,
such as having a complementary structure to an enzyme, for
instance. This term includes any of the well known base
structures of ph~rm~elltic~l compounds including
pharmacophores or metabolites thereof. These include beta-
lactams, such as pennicillin, known to inhibit bacterial cell wall
biosynthesis; iihen7~7ppines~ known to bind to CNS receptors,
used as antidepressants; polyketide macrolides, known to bind
to bacterial ribosymes, etc. These structural motifs are
generally known to have specific desirable binding properties
to ligand acceptors.
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 ~he groups
without adversely interfering with the reporting functionality
of the group. Preferred reactive groups are amino, thio,
hydroxy, carboxylic acid, carboxylic acid ester, particularly
methyl ester, 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
undercoinc condensation polymerization or copol~ merization.
Suit~ble oroups include vinyl groups, oxirane oroups carboxylic
acids, ~cid chlorides, esters, amides, lactones ~nd l~ctams.
37
WO 95/17903 ~ . 2 1 7 ~ 9 8 4 PCT/US93112591
Other organic moiety such ~s those defined for R and R' may
also be used. - - -
7 ) A macromolecular component, such as amacromolecular 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
(l,otP~min~d or limited by the macromolecule. This includes
porous and non-porous inorganic macromolecular components,
such as, for example, silica, alumina, zirconia, titania ar~d the
like, as commonly used for various applications, such as normal
and reverse phase chromatographic separations, water
purification, pigments for paints, etc.; porous and non-porous
organic macromolecular components, including synthetic
components such as styrene-divinyl benzene beads, various
methacrylate beads, PVA beads, and the like, commonly used
for protein purification, water softening and a variety of other
applications, natural components such as native and
functionalized celluloses, such as, for example, agarose and
chitin, sheet and hollow fiber membranes made from nylon,
polyether sulfone or any of the materials mentioned above. 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-lOOOAngstroms ), latex
particles (dp= I OOO-SOOOAngstroms), porous or non-porous
beads (dp=O.S- 1000 microns), membranes, gels, macroscopic
surfaces or functionalized or coated versions or composites of
these.
A and/or B may be a chemical bond to a suitable
organic moiety, ~ hydrogen atom, an organic moiet~ which
contains a suitable electrophilic group, such ~s an ~Idehyde,
ester. allc~ l halide. I;etone, nitrile, epoxide or the lil;e. a suit~ble
nucleophilic oroup. such as a hvdroxyl, ~mino, c~rbox~ late,
~mide, carb~nion, ure~ or the like, or one of the 1~ oroups
de~ined b~lo~ . In Iddi[ion. .~ ~nd B ma~ join u~ form ~ rino or
WO gS117903 ` ` 2 1 7 9 9 ~ 4 PCrlUs9311~cgl
.... .
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 moie~ies.
A more generalized structure of the composition of this
invention is defined by the following formula:
Fl1...n
A--X NH--C CO--Gl n ~ Y--E3
~ .1..n , n
wherein:
a. At least one of A and B are as defined above
aQd 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
represents B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy,
thio, straight or branched chain alkyl, carbocyclic aryl and
substituted or heterocyclic derivatives 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;
As used herein, the phrase linear chain or branched
chained alkyl groups means any substituted or unsubstituted
acyclic carbon-containing compounds, including alkanes,
alkenes and alkynes. Alkyl groups having up to 30 carbon
atoms are preferred. Examples of alkyl groups include lower
alkyl, for example, meth~ l, ethyl, n-propyl, iso-propyl, n-butyl,
iso-butyl or tert-butyl; upper alk~i, for example. cotyl, nonyl,
3~
WO g5117903 P !~ 2 1 7 9 9 8 4 PCTII~S93/12591
.....
decyl, and the like; lower alkylene, for example, ethylene,
propylene, propyldiene, butylene, butyldiene; upper alkenyl
such as l-decene, I-nonene, 6-dimethyl-5-octenyl, 6-ethyl-
~-octenyl or heptenyl, and the like; alkynyl such as l-ethynyl,
2-butynyl, I-pentynyl and the like. The ordinary skilled
artisan is familiar with numerous linear and branched alkyl
groups, which are within the scope of the present invention.
In addition, such alkyl group may also contain
various substituents in which one or more hydrogen atoms has
been replaced by a functional group. Functional groups include
but are not limited to hydroxyl, amino, carboxyl, amide, ester,
ether, and halogen (fluorine, chlorine, bromine and iodine), to
mention but a few. Specific suhstit--t~d alkyl groups can be, for
example, alkoxy such as methoxy, ethoxy, butoxy, pentoxy and
the like, polyhydroxy such as 1,2-dihydroxypropyl, 1,4-
dihydroxy- I -butyl , and the like; methylamino, ethylamino,
dimethylamino, diethylamino, triethylamino, cyclopentylamino,
benzylamino, dibenzylamino, and the like; propanoic, butanoic
or pentanoic acid groups, and the like; formamido, acetamido,
butanamido, and the like, methoxycarbonyl, ethoxycarbonyl or
the like, chloroformyl, bromoformyl, I, I -chloroethyl, bromo
ethyl ,and the like, or dimethyl or diethyl ether groups or the
like.
As used herein, substituted and unsubstituted
carbocyclic groups of up to about 20 carbon atoms means cyclic
carbon-containing compounds, including but not limited to
cyclopentyl, cyclohexyl, cycloheptyl, admantyl, and the like.
such cyclic groups may also contain various substituents in
which one or more hydrogen atoms has been replaced by a
functional group. Such functional groups include those
described above, and lower alkyl groups as described aboYe
The cyclic groups of the invention may further comprise a
heteroatom. For example. in a specific embodiment, R~ is
cycohexanol.
As used herein. substituted and unsubstituted arYI
groups means a hydrocarbon ring be~ring a system of
~a
WO 95117903 . ` 2 1 7 9 9 8 4 l'CTlUS93112591
conjugated double bonds, usually comprising an even number
of 6 or more (pi) electrons. Examples of aryl groups include~
but are not limited to, phenyl, naphthyl, anisyl, toluyl, xy~enyl
and the like. According to the present invention, aryl also
includes aryloxy, aralkyl, aralkyloxy and heteroaryl groups,
e.g., pyrimidine, morpholine, piperazine, piperidine, benzoic
acid, toluene or thiophene and the like. These aryl groups may
also be substituted with any number of a variety of functional
groups. In addition to the functional groups described above in
connection with s~stilllt~d alkyl groups and carbocylic groups,
functional groups on the aryl groups can be nitro groups.
As mentioned above, R2 can also represent any
combination of alkyl, carbocyclic or aryl groups, for example, 1-
cyclohexylpropyl, benzylcyclohexylmethyl. 2-cyclohexyl-
propyl, ~,2-methylcyclohexylpropyl, ,2methylphenylpropyl,
2,2-methylphenylbutyl, and the like.
d. G is a chemical bond or a connecting group
and G may be different in adjacent n units; and
e. n is equal to or greater than 1.
Preferably, if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and if n is I and X and Y are chemical bonds, R and R'
are the same, A and B are different and one is other than H or
R
Under certain circl~mct~nc~c~ A and/or B may be
a chemical bond to a suitable organic moiety, a hydrogen
atom, an organic moiety which contains a suitable
electrophi~ic group, such as an aldehyde, ester, alkyl halide,
ketone, nitrile, epoxide or the like, a suitable nucleophilic
group. such as a hydroxyl, amino, carbox,vlate, aminde,
carbanion. urea or the like, or one of the R groups defined
below. In addition, A and B may join to form ~ ring or
4/
WO 9S/17903 ~ , 2 1 7 9 ~ 8 4 PCT/lJS93/12S9I
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
R1.. n ~ ~
A--X ~ NH--C CO--Gl n Y--
Rlt. n , n
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 selected from the group consisting of
A, B, cyano, nitro, halogen, oxygen, hydroxy,
alkoxy, thio, straight or branched chain alkyl,
carbocyclic, aryl and substituted or heterocyclic
derivatives 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. n2 1.
Preferably, (I) if n is 1, and X and Y are
chemical bonds, A and B ~re different and one is other than
chemic~l bond, H or R; (') if n is I and Y is a chemical
4~
~ wo ss/17so3 s~ ? '~ 9 ~ 8 ~ 9~ 53~
bond~ G includes a NH, OH or SH terminal group for
connection to the carbonvl group and G-B is other than an
amino acid residue or a peptide; (3) if n is I 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.
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. Examples 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:
Rl
A--C O--N H--C C O--~--(Sur~ace)
F'~2
rn 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:
r ~ ~-
Wo 95117903 21 7 9 9 8 4 ~ g~
~Surfæe)--C O--N H--C--C O--Y--~
In the description tha~ follows, Rn 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 ~minimill.o moiety. This moiety may
be introduced, for example by reacting the oxazolone with
an asymmetrically substituted hydrazine and alkylating the
resulting hydrazide, (e.g., by reaction with an alkyl halide,
or epoxide). An example of such a surface is shown below.
R1 R
(Su~face)--C O--N H--C C O N N R4
R2 Rs
A~other embodiment of the invention relates to
an oxazolone ring having the structure
R
N~ R'
A--</ (cH2)
0~ ~
where A, R and R' are as descrlbed above and q is zero or 1.
Preferably, Y is a chemical bond This ring is useful for
preparing the desired oxazolone deri~atives.
f~
WO 95/17903 `,; ~ ' 2 ~ 7 9 9 8 4 PCTIUS9311259
A further embodiment of the invention exploi~s
the capability of ox~zolones with suitable substituents at
the 2-position to act as re~ctive agents. Appropriate
substituents include vinyl groups, which make the
oxazolone ~ 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 ~-
vinyl azlactone derivative~ is as follows:
+ AXH ' --
N N~
Rt R2 R, R2
~YH
XA ~--\ N ~ YB
r
,L5'
WO95117903 ' ;~ ~ 7 ~ PCTIUS93112591
wherein X can represent a sulfur, oxygen or
nitrogen atom; Y can represent 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
amino acids, oligopeptides, polypeptides and proteins,
nucleotides, oligonucleotides, ligand mimetics.
carbohydrates, aminimides, 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 the nucleophile,
AXH, and the oxazolone in a suitable solvent, such as
to~uene, ethyl acetate, dimethyl formamide, an alcohol, or
the like. 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. The selectivities of the Michael and oxazolone
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 usually
require. acidic catalysts (e.g., BF3); thus. X should not
normally be oxygen.
Likewise, primary amines tend to add via ring-
opening, ~nd X should therefore not be NH. Secondary
amines readily add to the double bond under appropriale
re~ction conditions, but many can also cause
4~
-
~ WO 95117903 ~ 2 1 7 9 ~ 8 4 PCT/US931~2591
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., in the presence of ~ (non-oxazolone-reactive)
base strong enough to remove the SH proton; on the other
hand, such sulfur containing nucleophiles will exclusively.
add via Michael reaction under non-ionizing, i..e., neutral or
mildly 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 .
Sllmm~ri7in~ AXH can be a secondary amine or
a thiol, and BYH can be a primary or secondary amine, a
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
nonporous 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, etc. The result of this reaction sequence is a
structure such as the one shown below:
N ~/ (Surfac~)
H
o
41
-
WO 9~117903 ~ 2 1 7 9 9 8 4 PCT/U593112591
In another variant, the roles of AXH and BYH
are reversed, so that BYH is the substituent selected from
the list aboYe and AXH represents a functionalized surface.
.
Other important bifunctionally reactive
oxazolone derivatives include:
And ~
These are produced by acylation of an
alpha,alpha-disubstituted amino acid residue with the
apropriale functionalized acid chloride, followed by
cyclization to the oxazolone.
Alternatively, oxazolones posessing reactive
groups at the 2-position may be produced~ia suitable
acylation reactions, as shown for the specific example of a
benzoyl chloride oxazolone derivative containing a reactive
p-benzyl group:
X- CH2~C--Cl + R~R
X CH2~C--N~COH ~ X-CH2~< R
In the case where X is part of a group whose
reactivity is orthogonal to that of the oxazolone ring, such ~s
in the case of a benzyl chloride group, ring-opening addition
with BYH may be carried out ~nd followed by reaction
wo g5/17903 . . ~ 2 7 7 9 9 8 ~ PCTJIlS93/12591
with an appropriate AXH l~roup, e.g. a primary amine, to
give the product shown:
CI CH2~(/ ~R' + BYH Cl CH2~l0l ~ 1l
ANH.
H ~ 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 the benzylic electrophile.
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 CH2C1 ), and the resulting product is then reacted
with an appropriate electrophile~ e.g., A-CH-~-Br, thus
introducing substituent A into the molecule.
B~NH-CH~ R' Y " B~NHCH2~ H -
Deprotec~
kCH~-HCH~ H H.~CH.~C~ C-C-BY
~9
wo 95/1~903 ,~ , 2 ~ 7 9 q 8 4 PCT/US93/12S9I
4.3. Catenation of Chiral Oxa~olones Producing Chiral
Oligomers and Polymers
By choosing oxazolone-derived building blocks
possessing functional groups capable of establishing predictable
binding interactions with target molecules, and using synthetic
techniques such as those broadly described above to effect
catenation (~inking) of the bui~ding blocks, it is possible to
construct sequences of oxazolone-derived subunits mimicking
se~ected native oligomers or polymers, e.g. peptides and
polypeptides, oligonucleotides, carbohydrates as well as any
other biologically active species whose three dimensional
birtding geometry can be mimicked by various combinations of
oxazolone derivitive containing scaffolds and side chains. This
may be accomplished using a wide variety of side chain
recognition group substituents, including, but not limited to the
stlbstir~ rlt~ found in the side chains of naturally occuring
amino acids; purine and pyrimidine groups as well as
derivatives and variants of these; natural and synthetic
carbohydrate recognition groups, such as sialic acids; groups
containing organic structures with known pharmacological
activities, such as beta lactam antibiotic moities, which are
known to be efficient inhibitors of bacterial cell wall
biosynthesis, to produce structures which have highly specific
activities. These moities may be attached, arranged and spaced
in a position-specific manner along a scaffold whose basic
geometry, spacing, rigidity and other properties can be
designed and locallv tuned to= functionally mimic the natural
scaffolds found in peptides, proteins~ oligonucleotides or
carbohydrates; or which can simply serve Io array sequences
or combinations of these side chain recognition groups in
appropriate structural relationships to the scaffold and to each
other to produce species with highlv specific and selective
activity. In addition. because of the improved h~drolytic and
enz,~matic stabilit~ proper~ies of the oxa2010ne-derived
5~1
W09~117903 2 1 7g~û~ PCT)US93112591
Iinkages, these designed functional molecules will have better
stability and pharmacokinetic properties than those of the
native specieS. The integrated modularity of the cherni~trie5
allows the constrUction of this wide variety of molecules to be
carTied Out in a manDer analogous to the desicn of an electronic
device by combining component subsystems using a relatively
small number of interchangable reaCtive modules and
protocols. This is figuratively outlined below:
¦ MODULAR DEStGN AND ASSEMBLY FLOW CHAR~ ¦
~ ~U.,...~U..
MûDULE
Carbohydtt te moi~ty.
Pyntnidire mciety, etc.)
RECoGNmON = SUBUNIT
. MODULE ASSt,MBLY
CONNECTED Tû
SPACER MODULE
SPACER
MODULE t~r;~ v ~
h otthagonal RECOGNlT.tON
ues) BUtLDI RG BLûC~
MO ~ULE
ûXAZûLûNE
CONTAINING ûR cAnENAnoN
suBuNrr = ~ORMING ~ PRûCESSES
ASSEMBL~t lN~rERMEDtATE AND
PRûTOCOL5
I-lrh~EnC/ ¦
AGE.~ r
~ ^ MODULAR COMPONE~
This approach is illustrated belou for the
introduction of ~ generic "base" (purine or p~rimidine) group
into ~n oxazolone-derived scaffold connected ~ a c~rbonvl-
~1
WO 9_/17903 ' ` ~ 7 q 9 8 4 PCTIU593/1259
terminal spacer. While the example uses a base as therecognition group, it should be kept in mind that this group can
be any group which will provide the desired end product, such
as, for example, a carbohydrate, a pharmacophore moiety or a
designed synthetic recognition element.
The following speciflc sequence illustrates the .
construction of a ligand having bases attached to every
other oxazolone-derived module. Alternatively, species may
be constructed with bases attached to each sequential
oxazolone module. The s~stlt~ntc on the recognition
group-bearing modules may all posess the same chirality,
may have regularly alternating chirality or may be racemic,
depending on the desired structural relationships between
the individual recognition groups and between each
recognition group and the backbone scaffold.
Alternatively, Other variations may be
constructed, including those employing non-hydrogenated
modules, which produces derivatives with the bases
attached to the scaffold via double bonds. In these
structures, the assembled ligand may be subjected to
multiple cim~llt~n~ous stereospecific hydrogenation of this
unsaturated linkage producing derivatives with alpha-
hydrogen substituents in a stereocontrolled manner, and
avoiding the racemization problems involved in
constructing these ligands via alpha- hydrogen containing
oxazolones. as outlined above.
~Z
j ~ 21 79984
WO 95/179~13 i PCTllJS93/12591
SPACER MODtJLE
OMe
~ BASE + C~ ~ OMo -- - ,ASECH2CHO
RECOGNITION \ N
MODULE r ~
BUILDING
BLOC~ \\
fMODUI,E o
CHCH 8ASE 1. H2/CnL ~ ~e
--~ RXIP.T.
fr--CATLNATION
H2NC(CH3)2C02Li
EASE
J~ ,X~ Li CIC02E~ ~ R~
CYCLIZATION ~X~
ctc. H3 CH3
E~A'E ~'/R NH2
Ph ~ ~o H,C C~,
33
WO g~/17903 2 ~ 4 PCTIUS93112591
4 3.~ 1 Catenation Via Alternating Sequences of
Nucleophilic Oxazolone-Ring-Opening Addition Reactions
Followed by Oxazolone-Forming Cyclization Reactions
a. Alpha,Alpha-Disubstituted Sequences
According to this approach, oxazolone modules
are catenated via ring-opening nucleophilic attack by the
amino group of a (chiral) alpha,alpha-disubstituled amino
acid derivative, usually a lithium salt; the resulting adduct
is subsequently recyclized to form a terminal oxazolone
(with retention of chirality). This oxazolone is then
subjected to another nucleophilic ring-opening catenation
reaction sequence, producing a growing chiral chain, as
shown below. This procedure is repeated until the desired
polymer is obtained.
WO 95~17903 ,~ " `, 2 1 7 ~ ~ 8 4 PCTII~S93112591
I' N~\ CO M ~/
~2N CO~M H \~R~
A~N~ N~2M
sHx
o ~ ~ " N~/ B
Wherein M is an alkali metal; each member of
the substituent pairs Rl and R2, R3 and R4, R5 and R6 and
Rn and Rn-~ differs from the other and, taken alone, each
signifies alkyl, cycloalkyl, or substituted versions thereof,
ar~ l~ aralkyl or alkaryl, or substituted and heterocyclic
versions thereof; these substituent pairs can also be joined
in~o a carbocyclic or heteroc~clic ring; preferred forms of
R~ and R' are the side chain substituents occuring in
native polypeptides, oli~onucleotides, carbohydrates~
~5
r ~ -
WO 95/17903 ~ 2 ~ 7 9 9 ~ 4 PCTIIJS93/12591
pharmacophores, Yariants or mimetics of these, or any
other side chain substituent which can be attached to a
scaffold or backbone to produce a desired interaction with a
target system. i X represents an oxygen, sulfur, or nitrogen
atom; and A and B are the su~srirllenr~ described above.
A chiral oxazolone derivative containirlg a
blocked terminal amino group may be prepared from a
blocked, disubstituted dipeptide, that was prepared by
standard techniques known to those skilled in the art, as
shown:
B~ ~ ~, N ~ COOH R'~
R~ R'
wherein B 1 is an appropriate protecting group, such as Boc
(t-butoxycarbonyl) or Fmoc (fluorerlyl- methoxy carbonyl).
One may then use this oxazolone to acylate an amine,
hydroxyl, or sulfhydryl-group in a linker structure or on a
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
reaactively 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
polymeric structure, as shown below:
!~6
t- t ~.' 2 1 7 q ~ Q~ 4 931~2591
WO 95117903 ~ S
AX!I ~ ~ O A~H~N
deblock
G ~ ~ R
C ~ 6
C ~ ~
A~ N ~ 3~?~ ~h " ~ C
In the reaction shown, Y is a lirlker, such as, for
example, a functionalized aryl group; X is a nitrogen of
suitable structure, an oxygen or a sulfur atom; each
member of the substituent pairs Rl and R~, R3 and R~, Rn'
I and Rn differs from the other and, taken alone, each
signifies alkyl, carbocyclic, or functionalized versions
thereof, ~ryl, aralkyl or alkaryl or
functionalities, including heterocyclic versions thereof,
preferred forms of Rl and R~ ~re the side-chain
substituents occuring in n~tive polypeptides.
57
WO 9S/17903 ' -, r -~ r ^ 2 1 7 9 9 ~3 4 PCT/US93112S9I
oligonucleotides, carbohydrates, pharmacophores, variants
or mimetics of these, or any other side-chain substituent
which can be attached to a scaffold or a backbone to
produce a desired interacrion with a target system;
substituent R can also be part of a carbocyclic or
heterocyclic ring; A is a substituent as described above; C. is
a substituent selected from the set of structures for A; and
B I is a blocking or protecting group.
It can be seen that the above catenation
involves introduction of two amino acid resldues per
polymer-elongation cycle and therefore produces ligands
witb 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 denvative as
shown below, prepared via standard synthesis.
H
R~ R'
,, ~.
R' R~ R; R~ O
H J~ ~ C
In the polymers described above each
individual module may c;3rry ~ reco~ition group
substituen~, ~s is the c~se for ligands designed to mimic
peptides. Alternativelv, this se~uence of re~ctions can be
~g
~ wo gS/17903 ~ 7 9 9 ~ 4 PCr/US93)125gl
used to construct ligands with continuollsly vari~ble
stepwise control of the periodicity and the stereochemistry
of each of the attached substituent groups and,
consequently, of the resulting structural and functional
properties of the ligand. This can be done by separating the
recognition group-bearing modules from each other by on.e
or more modules which do not carry recognition groups.
These intervening modules may be achiral, alpha, alpha-
disubstituted or, in cases where chirality is not important,
they may be standard hydrogen-bearing alpha amino acid
modules. These may serve as spacers, to regulate the
periodicity of substitution or may serve various other co-
functions, such as limiting the flexibility of the ligand. The
substituents on the recognition group-bearing modules may
be constructed to all posess the same chirality, may have
regularly altrrnatin~ chirality or may be racemic,
depending on the desired structural relationship between
the individual recognition groups and between each
recognition group and the backbone scaffold.
Alternatively, modular "sub assemblies" capable
of corlferring higher order structural properties may be
pre- constructed and assembled together using these same
reaction sequences in a manner which allows control of the
higher order structure. This is illustrated for the c~se of a
polymer formed witha repeating pattern of alternating
modules of the type:
~3'
/--SO3
" H ~
S9
WO95117903 ' - ~ ' ' S 2 1 7998~ PCI-/IJS93/12591
This polymer will form 3-10 helices. driven by the
conformational restrictions imposed by the repetitive
viscinal disubstitution. This triadic periodicity results in the
formation of a helical superstructure which has charged
sulfonate g}oups lined up regularly along one side of the
helix:
This helix-forming phenomenon has been observed
with naturally occuring peptides which contain sequences
of adjacent aminoisobutyric acid (aib) residues, a naturally
occuring achiral alpha, alpha-~i~ubstitllt~d amino acid.
Examples of this include certain naturaily occuring peptide
antibiotics, such as alemethicin and sll7llkicillin whose aib-
derived helical structures have been postulated to be an
important constituent in the cell wall-disrupting mode of
action of these antibiotics.
b. Other Bifunctionally Reactive Elements
At any point in the polymer syntheses shown
above, a structura~ species, possessing ( I ) a terminal OH,
-SH or -NH~ group c~pable of ring-opening addition to the
oxazolone and (2) another ~erminal group capable of
re~cting with the amino group of a chiral alpha,
alpha'disubstituted amino acid. may be inserted in the
polymer backbone as shown below;
~c
WO 95117903 . ' I 2 1 7 9 ~ 8 4 PCT117S93/lZ~9I
C O O H D
I ' 2
H~ ~CO~
H 11
~R'X~\ N/ -- ~R~"R'
,~H/~ <~
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 each individual step of the synthesis.
The experimental procedures described above
for oxazolone formation and for the use of oxazolones as
acylating agents are expected to be useful in these
oxazolone-directed catenations. Solubility and coupling
problems that may arise in specif1c 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 dipol~r aprotic solvents (e.g.,
dimeth~l formamide, DMF, dimethyl sulfoxide, DMSO. l~T-
methyl pyrolidone, etc.) and chaotropic (molecular
aggregatebreaking) agents (e.g., urea) will be very useful ~s
c~tenations produce prooressively l~rger molecules.
C~/
WO 95/17903 ~ ~ r~' tl 1, C` 2 1 7 9 9 8 4 PCT/US93112591
4.3 2.2 Catenations Using Bifunctionally Reactive
Oxazolones
When the substituent at the 2-position of ~he
oxazolone (azlactone) ring is capable of undergoing an
addition reaction that proceeds with retention of the
chirality at the 4-position, this addition reaction may be
combined with a rin~-opening acylation to produce chiral
polymeric sequences. This is shown for the case of alkenyl
azlactones
below .
GZ
; - r:`
W0 951S7903 ~ "~ 2 ? 7 9 9 8 ~ PCT~S93)~259~
<~O f ~O
N BXH N HNU ZNU-H f~h""~
Rl R2 R. R2 ~\
(C) )=
~,0~o HNU2ZNU'
N~!, r
~NU~ ~NU~O ~'
O BX ~ O
R~ S"R,
HNU3ZINU~H R~
BX~ $NU~ ,NU~N ~ I~NU~ NU~
O NU~W R~=
N H
¦ NH3
K ~ H~ Z I~ ;~ NUI NU '~, GH2~ Jl~ ~ NHZ
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-
amino propane-3-thiol, and so on; groups Nul, Nu~ Nu3
and Nu1 need not be identical ~nd Z is a linker structure ~s
described ~bove.
C3
wo 95117903 ~ ' . 2 f 7 ~ 9 ~ 4 PCT111593/12591
Structure HNu I -Z-Nu2H may contain two
nucleophilic aroups of differential reactivity as stated
above, or if Nu I and Nu2 are of comparable reactivity one
of the nucleophilic groups is protected to prevent it from
competing with the other and is deprotected selectively
following ~cylation; 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
HNu I -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 simila} to those described above for related
polymers .
The above types of oligomers are highly useful
biochemically because of their structural similarity to
biological scaffolds, particularly polypeptide scaffolds. The
substituents R can be chosen to tailor the steric, charge or
hydrophobicity characteristics of the oligomer such that a
versatile mimetic results.
4.3.3 Functionalization of Peptides and Proteins Using
Oxazolones
In a further embodiment of the invention, the
nucleophilic ring-opening of asymmetrically disubstituted
oxazolones may be utilized to introduce ~ chiral residue or
sequence in selected positions in peptides or proieins to
produce hvbrid molecu~es with improved hydrolytic and
enzymatic stability properties
G4
WO 95/17903 : i 2 1 7 9 9 8 4 PCTIUS93J12591
The reaction of a chiral azlactone with the amino
terminus of a synthetic tripeptide attached to a Merrifield
support is shown below.
R~ R3 R
~CO--C--NH--CO--C--NH--CO--C N-12 + --~R~
H H H
A
Rl R2 R3 ~4
~CO--C--NH--CO--C--NH--CO--C--NH--CO--C--NH--CO--3
_ . 2
H H H R~
The oxazolone used in the above aminolysis may
contain a blocked arnino terminus which, after the
arninolysis, is deblocked and used for further elongation via
acylation. This synthetic Yariation is shown below (B I
stands for a suitable blocking group as described above).
R'
N~ J~ NH2 + ~ /
O R~ *~
~H~H~ ~HJ~
R' H o R~ H o R~ R~
~W~ ~ NJ~ ~ NJj~ ~ ", ,,~ 9,
~S
WO 95/17903 ; " ,. 2 ~ 7 ~ 9 g 4 PCTIUS93/12591
After the desired oxazolone units have been
used to elongate a given polypeptide, the polypeptide
synthesis may be continued, if desired, using standard
peptidesynthesis techniques.
The structure below illustrates a short polymer
containing nine subunits prepared as ~bove and detached
from the solid phase synthesis support.
R H o~ H J~H ~ ~R ~ ~H i~ H ~ H
In the polyamide structure shown above, each
of the R groups signifies alkyl, carbocyclic, or substituted
versions thereof; aryl, aralkyl. alkaryl, or substituted
versions thereof, including heterocyclic versiorls; the R
groups can also define a carbocyclic or heterocyclic ring;
preferred structures for the R groups in this application 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:
y~NH2 ~ ~ N
G,6
WO 95117903 - ` ' ' 2 1 7 q 9 ~ 4 PCl`~S93112~91
, . .
b. Detachmen~ of the peptide prepared by
solid phase synthesis from the support. with reb~ocking of
the N-terminus if necessa~, followed by cyclization
producing the oxazolone as shown below:
N ~ O
Rn H N
c. ~ynthesis of a second desired peptide
sequence on a solid support:
j~X N J~
d. Coupling of the peptides produced in steps
(b) and (c) above, under suitable reaction conditions.
producing a rlovel peptide containing unnatural residues.
shown below after detachment of the peptide from the
support and removal of all protecting groups used during
its synthesis.
HOOC~N~ ~N~N~ ~ NH2
I
ID~
WO 95/17903 ,` " ~ 2 1 7 q ~ 8 4 PCTIUS93/12591
In the structure above, e~ch 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 struclural 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:
H~ + ~(Z)A
s,~
H ~ X A
X=O~121H~S
The above ~cylation product may be coupled
with a peptide to produce novel chir~l hybrlds; two
coupling routes may be used
~ WO 9~i117903 ~ ' .r -.i, ;~ . 2 1 7 9 ~ 8 4 PCIIUS93112S91
( I ) 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 a 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.
8~ ~, ",,. N~ ?~ ~COOH + ll,N Pep~de.COOR
DCC D~block
"H?~D~ H
(2) If A is a suitable nucleophilic group (e P.,
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:
~9
WO 95/17903 '\ '` .'` r . ~ - 2 1 7 9 9 8 ~ PCT/US93/12591
B ~ ~ N~/ \ N~SH +
Rr H ~ \RZ
B~ ~ ~ " N~ ~ d~blxk
O RZ H
H2N~ N$ ~N~~N , ~,NH2
The aboYe 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 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,
( I ) Nucleophilic attack on an azlactone~ tha
was previously derivatized via a Michael addition using a
nucleophile of general structure AXH, with a peptide amine:
~ WO 95~17903 ~ ,' !. , ' 2 1 7 9 9 : ~CT~Sg3112591
Peptide-~ + AX N~R2
O o
AX N~/ Peptide
( 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 furthe} modifications; this sequence produces polymeric
molecules of a variety of structures as shown below:
WO 95/17903 . ~ t-~ 2 1 7 9 9 8 4 PCT/US93/1~591 ~
Pept idel5~ + ~~0 Peptidel-S
""'~2
P e'~O
Pepude I -S ~ H2NZCOOH ¦
oS~\ t~H Pepude - S ~
p2~<
)= o ~'\ I`IH
Ht~ . p 2~
p~ptid~2 ~= o
Pepodel-S
Pcpdde2 N~ HOOC
O~t~H ~/ r~c;c
,e~l
Ft2~
~(~
P~ptid~
4.3 4. Other Mimetics
Oxazolone-derived mimetics can be produced, using
the oxazolone-forming and catenation chemistries outlined
above, so as to produce backbones having natural or synthetic
recognition~ groups, such as purine or pyrimidine bases,
carboh~drates, ph~rmacophores, etc., attached ~s side chain
72
~ WO95/17903 ` ` 2 ~ 79984 Pr~ 31~
substituents via appropriate sp~cers, i.e. R or R' in the general
structural formulas described above represents a recognition
group-spacer sub assembly.
This may be accomplished, for example, via the
following general synthesis scheme:
~ x~ ,~
L' ~ L~y
L~--L cO ~ N--~A
- Monomer
L CatenaDOns
~ - ~0
N--~A
Monomer
c L = modifiable function group
L = modified function group
'\,~ R = Recognition Group
O R' = Alkyl, aryl, H, F
~R"1 11 Y = masked carbonyl
I ~ /`N '~ l G = A connecting group or = O
5 ~ G ~ H J n n = any number > 1
S = rtL.~ may be
(R), (S) or
racemic for each value of n
.3.4.1. Synthesis of Oligonucleotide Mimetics
As discussed previously, much ~tlention has been
focused on the construction ~nd application of molecules which
possess the propertv of binding to nucleic ~cids. In the course
7~
wo ss/l7so3 : . 2 1 7 9 9 8 4 PCT/US93/125~
of wori~ in this area, a gre~t amount of knowledge has been
amassed vis-a-vis 1.) the abilily oF a synthetic scaffolding to
support a series of natural or designed bases in such a manner
that tight binding to natural nucleic acids is observed; 2.) the
requirements for designed or naturally occuring bases other
than cuanocine, cytosine, thymidine, adenosine or uridine to
efficiently bind (hybridize j to another natural base or
nucleotide. It has been demonstrated that even unnatur~l or
modified bases can show efficient hybridization if projected
from an effeclive scaffold. Our strategy, disclosed herein, is to
append natural and/or unnatural bases (e.g. thymine,
guanidine, 5-fiuorouricil(5FU)) onto oxazolone backbones to
form an antisense strand, or nucleotide mimetic. The resulting
linkages and backbones are superior in their resistance to base,
acid and proteolytic/phospholytic activity. The bases can be
attached using appropriate spacers and the stereochemistry
and periodiocity of substitution geometry and rigidity of the
backbone scaffold can be designed such that the bases are
~rrayed and projected in space to provide the optimum
arrangement and orientation of the bases to hybridize with
their targeted counterparts. Specific examples of the synthesis
of oxazolone-derived oligonucleotide mimetics are given below.
4.3.4.2. Synthesis of Carbohydrate Mimetics
As mentioned previously, carbohydrates
increasingly are being viewed as the components of living
systems with the enormously complex structures required for
the encoding of the massive amounts of information needed to
orchestrate the processes of life, e.g., cellular recognition,
immunity, embr,vonic development, c~rcinogenesis and cell-
death. This information is contained and utilized through highi!
specific bindin~ interactions mediated by the detailed three
dimensional-topolocical form of the specific carbohydrate. ~t is
of gre~t v~lue to be able to ~rr~nge and to connect these
moities i various arrays in controlled manner. This ma~ be
~ WO g~/17903 ~ r ~. 2 1 7 9 9 ~ 4 PCr~S93112591
done either by connecting c~rbohydr~te recognition groups
along an oligomeric backbone, as done by for random vinyl
copolymers containing functionalized sialic ~cid groups, which
were shown to Inhibit hemagluttinin binding (J. Am. Ch~m Soc..
I 13, 686, 1991 ) or by arranging multiple ~arbohydrate groups
with appropriate spacers on a suitable structural scaffold so
that the carbohydrate groups ~re oriented in space in such ~
way that they can bind selective~y to the target (cf., eg., ~. Am.
Chern Soc.. 113, 5865, 1991; ibid., 5865). Oxazolone-derived
carbohydrate mimetics may be synthesized from carbohydrate
modules containing functional groups, such as carboxylic acid
halides, carboxylic acids, alcohols, thiols, amines, aldehydes,
ketones, together with any other groups which are compatible
with the oxazolone-forming and catenating reactions outlined
above, thus ailowing the carbohydrates to be attached to a
basic scaffold, or to be arrayed along a backbone in a precise
controlled manner. ~xamples for the synthesis of such
carbohydrate modules are outlined below.
Module 1.
OH COOH OH COOH
HO~--OH I OHC,~OH
HO HO 13
¦ b
OH ~I! COOCH3
ACH = OAc
r,) ~COCI)~. DMS0, E~3N. CH.CI" -60 C
(b) Ac70. Pvridine, CH~CI., n
15'
WO9!"17903 ` 2 ~ 7 9 9 ~ 4 PCT/US93/12'i91
Module '.
OAo Cl
~~t OOCH~ r b ~b CHO
AcO
)2{?-Hht~yldrToHyFetl2y~ 3-dio~tne~A~-sr~licy~ THF~n ~ r
C OH
7~-hydto~yl~tbyl~1~3.diotrn-
Module 3
6 CHrO H~ - A~
7 8 NH~C
(~ (COCI)2~ t~MSQ Et]N, CH2C12~ -50 C
(b) Ac2C\, Pyndire, CH~CI~. n
Module 4.
CH20TMS CHtH
rMSO ~ a.b HO ~7
TMSO~ HO_~/
TMSO OTlpS HO OH ¦
NH2 16 H~ ~CHO
(t ) ~-(?-bromo~thvl)- 1~3 diox THF n
(o) HCI, THF. tt rne,
3r
'-(?-brotr oethyl)- 1.3-dio~n~
t'G
~ WO 95117903 ` ~ 2 1 7 9 9 8 ~ r~ y~JlL;~l
4.3.4.3. Synthesis of Pharmacophore Mimetics
B ackground
The physical principle governing the binding of a
natural ligand or substrate to a receptor or active site of an
enzyme, nucleotide or carbohydrate are the same principles
governing the binding of non-peptide, non-nucleotide and non-
carbohydrate compounds (competitive inhibitors or agonists).
The modification of a known biologically active compound as a
lead or prototype, then synthesizing and testing its structural
congers, homologues or analogues is a basic strategy for the
development of new therapeutic agents. Several advantages of
this method are:
Greater probability of theses modified
derivatives to possess physiological properties
most similar to those of the prototype than those
tested at random.
Possibility of obtaining pharmacologically
superior agents.
Economical production of a new drug.
Structure-activity relationships can be
established to assist in further developments.
The objectives of ~ny drug discovery program are:
(a) to obtain drugs that have more desirable properties than
the prototype in potency, specificity, stabilit~. pharmalogic~l
duration. toxicity, ease of administration and cost of production:
(b) the discovery of fe~tures of the molecule ~ hich imp~rt
WO 9~/17903 ''. :1 .` ~ ' `.. ~ 2 ~ 7 9 9 8 4 PCT/US93112591
pharmalogical action. The term pharmacophore is used to
describe these key fea~ures Ihat imparts this pharmalooical
action .
Several technologies exist where a biologically
active compound~ for example a protein or polypeptide, is
attached to a solid support, such as a resin or glass surface.
These linked compounds show diverse inhibitory activity, an
indication that the ability of linked molecules to retain its
binding properties despite the partial loss of mobility.
There are a wide variety of general
pharmacophores known which display specific known modes of
activity, e.g., f~-lactam actibacteric, interfering with bacterial
cell wall; piperidine and peperizine, which can act as
psychotropic agents or anticholinergics; and xanthines as
stimulants. The following general schemes outline the
synthesis of pharmacophore molecules, for inclusion in the
various aoxazolone-derived polymer backbone forming
reactions described above. Detailed examples are given below.
1. SYNTHESIS OF DA-AMINO-(N-(4-
(OXOMl~THYL)BENZYL)BENZYL-PENICLLIN:
W ~/ TNr ~ U ~ r ~
2. SYNTHESIS OF 4-HYDROXY-N-(2-(1,3-DIOXYL)-ETHYL)-4-
PHENYLPIPERIDINE:
7~
Wos~/l7go3 ; ~ i 2 1 7~q84 PCrrUSs3)1~91
A solution of 4-Hydroxy-/Y-(~-(l,3-dioxyl)-ethyl)- 1-
phenylpiperidine (x mg, x mmol), dissolved in an appropri~te
solvent such as methanol / water or THF l water, with an
equimolar amount of aqueous 0.5N HCI is stirred at ~0 C for 4
hours. The reaction mixture is diluted in a suitable solvent
such as methylene chloride or diethyl ether, and extraceted
with saturated aqueous NaHC03 to neutralize the acid, followed
by brine. The solvent is removed on a rotary evaporator to
afford a solid (x g, x%). A portion is recrystallized to yeild a
sample for analysis.
3. SYNTHESIS OF 5~-5-((1,3-DIOXAN-2-YL)-2-ETHENYL)-
Dl13EN70[A,D]CYCLOElEPTENE:
" ~ h ~,Ch
--J Th~r~O~
4.4 F~bric~tion of 07~Yolone-Perived
Macromslecul~r S~ructtlres Capable
of S~ecific Molec~ r Reco~nition
In an embodiment of the invention oxazolone-
derived molecular building blocks may be utilized to construct
new macromolecular structures capable of recognizing speciflc
molecules ("intelligent macromolecules"). The "intelligent
macromolecules" may be represented by the following general
formula:
~9
wo 95/1~903 i 2 ~ 7 9 9 8 4 PCT/US9311~91
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. Y
Structure R may be a native ligand or 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, etc.
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 is well known to those
skilled in the art. This coating element may be
1) a thin crosslinked polymeric film 10 - 50
Angstroms 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 below, the controlled microporosity gel may be
engineered to completely fill the porous structure of the
support platform. The polymeric coatings mav 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 re~ction conditions. such ~s temper~ture,
~0
WO 95/17903 : ` =. i . 2 1 7 9 ~ 8 4 PCTIUS93112591
agitation, e~c., in ~ m~nner that is well ~nown to those skilled
in the ar~
The support platform P may be a pel~icular material
having a diameter (dp) from 100 Angstroms to 1000 microns, a
latex particle (dp 0.1 - 0.~ microns), a microporous bead (dp I -
1000 microns), a porous membrane, a gel, a fiber, or a
continuous macroscopic surface. These may be commercially
available polymeric materials, such as silica, polystyrene,
polyacrylates, polysulfones, agarose, cellulose, etc. or synthetic
oxazolone-containing polymers such as those described below.
Any of the elements P, C, L, or R containing an
oxazolone-derived structure is derived from a form of the
element containing a precursor to the oxa2010ne-derived
structure. 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", and "structure" refer to either P, P linked to C or P
linked to C and L as defined above.
4.~ Chir~1 AlkPnvl A71slctone Mnnomers
~n(1 polylnpri7~tion 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 pol~mers, and
may be further crosslinked to produce chiral beads,
membranes, gels, coa[ings or composites of these materials
8/
WO95/17903 - ~ 2 1 7 ~ 9~ 4 PCT/IIS93/12591
ti~ n , ~ ~ 3
,~ ?~
CO\~
~'~Se
Other useful monomers, which may be used to
produce chiral crosslinkable polymers, may be produced by
rlucleophilic opening of a chiral 2-vinyl oxazolone with a
suitable amino alkene or other unsaturated nucleophile.
c~
Virlyl polymerization ~nd polymer-crosslinking
techniques are well-known in the art (see, e.g., U.S. Patent
No. ~,981,933) and are applicable to the above preferred
processes .
C~ ~ C~O~
~,
WO 9S/179~3 . ~ 9 ~ ~ ~ PCT~Sg3122~sg]
~ ; . . . ~ . ,
4.6 CombinAn-rial Libr~ries ~erived From 0~7r~10ne
Modules
The synthetic transformations of oxazolones
outlined above may be readily carried otlt 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 Syn~hesis, D. Rickwood & B.D.
Hames eds., IRL Press ed. Oxford U. Press, 1989). Since the
assembly of the oxazolonederived structures is modular, ~. e.,
the result of serial combination of molecular subunits, huge
combinatorial libraries of oxazolone-derived oligomeric
struceures may be readily prepared using suitable solid-
phase chemical synthesis techniques, such as those of
descnbed by Lam (K.S. Lam. et al. ~ 354, 82 (1991))
and 7-1ckerm~nn (R.N. 71~ rm~nn et al. Proc. Natl. Acad.
Ser. USA. 89, 4505 (1992); J.M. Kerr, et al., J. ~m Ch~rn Soc.
115, ''529 (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 krlown 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) 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
wo 95117903 '` ' ` 2 1 7 9 9 8 4 PCTIUS93112~91 ~
phosphatase) whose activity can give rise to color prodution
thus staining library support particles which contain actlve
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 Zucker`mann 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
oligorlucleotide or peptide), that is readily decipherable
(e.g., by sequencing using traditiorlal analytical methods), in
paralle~ with the synthesis of the li~sn~1c~n-1idates 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
impossible to elucidate using traditional analytical methods.
Coding schemes for construction of combinatorial libraries
have been described recently (for example, see S. ~3renner
and R.A. Lerner, Proc. Natl. Acad. Sci. USA 89, 5381 (1992);
J.M. Kerr, et al. J. Am Chem. Soc. 115, 25~9 (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 ~1~ the random Incorporation of 20
ox~zolones into pentameric structures, wherein each of the
i~
WO 95117903 ` ' '~ 2 1 7 9 9 8 4 PCT/US93J12~i91
fiYe subunits in ~he pent~mer is derived from one of the
oxazolones, produces a library of 20~ = 3,200,000
peptidomimetic ligandcandidates, each ligand-candidate is
attached to one or more solid-phase synthesis support
particles and each such particle cont~ins a single ligand-
canditate type. This libr~ry can be construc~ed 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 support via a succinoyl linker is giYetl as an example.
~CCH3 ~ XCO~H H `q~
( I ) A suitable solid phase synthesis support. e.g., the
chloromethyl resin of Merrifield, is split into three
equal portions.
('2) ~ach portion is coupled to one and one o~ the glycines
shown above after conversion to the acylated t-butyl
ester derivative: -
R.~ >el H' H c- I ~S K~
H ~ co ~ :, N~l.ccllo~ H ~ k-- HO C--laK:~-C--~H ao--
aq._a o ~
e~CH -O,C-(CH~-
@ ~ p~lys~yr:~
R,,R . n, Ch). C:. ~H~. (C~lkCH
8S'
WO 95117903 ~ t ~ 2 1 7 9 9 8 4 PCT/US93/12591
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 miYture of TFA and CH2C12, to
remove the t-Bu blocking group. Th~ resulting acyl
amino acid resin is treated with ethyl chloroformate
as described above producing the oxazolone resin.
2)'~2'o2C~ ~ C~H~ 2-o2c-~l~-t~ ~
(4) The three oxazolone resin portions are thoroughly
mixed and the resultinv mixture is split into three
equal portions.
'' (5) Each of the resin portions is coupled to a different
glycine protected as t-butyl es~er using the conditions
described above; the amide product is deprotected as
described above, for each of the resin portions and
cyclized to the oxazolone usina the reac~ion with e~hy
chloroformate.
86
WOgS/17903 ~ r ~ 2 ~ 7~ ~ 8~ PCTIUS9315~S9I
~Chl-OIC-~CHss~C-- ; R,~ Collplsslt (~CN,-O,C-~Ch",-C Nh~ ,h
o R r B R-
h,COCOCI , ~Chl-OIC-~Chs~s--C--Nh~N 1'
Rl,RI.R,,RI-il.CHl.C~sCH~.C}~(CH~ ~0~
(6) The resulting resin portions are mixed thoroughly and
then split again into three equal portions.
(7) Each of the resin portions is coupled ~o 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 contairling 27 types of resin
beads, each type containing a single oxazolonederived
tripeptide analog linked to t~e 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.
al~C h, - OIC - (Q~s~s - C--hh ~ N ~ N ~XCO h
Many modifications of this general scheme are
envisioned, including the direct ~tt~chm~rlt of the ligand
candidates via a C-N bond using a benzhydryl support,
which would allow the straight forward d~t~rhm~nt of the
ligand candidates from the support via acidolysis for
further study ("orle-head, one-peptide-analog synthesis").
The ability of these chemistries to be modularl~
connected in a structure directed manner gives us a unique
ability to produce directed combinatorial libraries by
systemalically v"rying structural elements around a structura~
motif. This is exemplifved for the generation of a matrix of 16
~7
WO 95117903 ~ t,~ "~ ` 2 1 7 q ~ ~ ~ PCT/US93/12591
molecules around the following aryl-heterocvcle-alicyclic
amine structural theme.
Them~:
¦ Aryl Group ~N ~\ ~ Acyclic Amme ¦
He~erocYcle ¦
This was done by reacting the 2-phenyl and 2-(2-naphthyl)-S-
oxazolones (produced by reacting the lithium salt of glucine
with the aryl acid chlorides, followed by cyclization with ethyl
chloroformate at 0 C) with 2-furfural, 3-fufural, 2-thiophenal
and 3-thiophenyl to produce oxazolones functionalized at the
4-position, followed by ring-opening addition of 4-(3-
aminopropylmorpholine and 1-(3-aminopropyl)-2-pipicoline to
form the adducts shown. this was accomplished by carrying out
the reactions in individual vials such that each vial contained
one pure final compound as follows:
1.) equimolar quantities. of the oxazolone and the
aldehyde dissolved in dry benzene (25ml/gm reactants) were
heated to 75 C for 15 minutes; 2.) the reaction mixture was
cooled to 10 C and the amine was added dropwise with stirring;
3.) the mixture was re-heated to 75 C for 20 minutes and 4.)
the solvent was removed in vacuo to give the crude solid
product.
WO 9~i/17903 ' 2 1 7 9 q 8 4 PCT/l~S93112591
; , .
CHO ~
R
~NH2/--NJ\
1l 1 ~/Y
~ N ~\ , ~ ~
O ~X~ ~Y
Ar X / isomer R / Y
Pb O 2- H O
Pb S 2 H O
Ph O 2 CH3 CH2
Ph S Z- CH3 CH2
Nrphrhyl O 1 H O
N~phrhyl S 2- H O
Naphrhyl O ~ CH3 CH2
Nrphrhyl 5 2- CH3 CH~
Ph O 2- H O
Ph S Z- H O
Ph O ~- CH3 CHZ
Ph S 2- CH3 CH2
Naphrhyl O '- H O
Nrphrh~l S ' H O
Nrphrhyl O '- CH3 CH2
N~phrhyl S ~- CH3 CH2
WO9S/17903 ` ` !; 2 1 79~84 PCTIUS93112591
4.7 Desi~n and Synthesis of Oxazolone-Derived
Glvcopeptide Mimetics _
A great variety of saccharide and
polysaccharide structura~ motifs incorporating oxazolone-
derived structures are contemplated including but not
limited to the following.
( I ) 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
Co~rrehen~ive Or~nic Chemistry. Sir Derek Barton,
Chairman of Editorial Board, Vol. 5, E. Haslam, Ed., pp. 687-
815; A. Streitwieser, C.H. Heathcock, E. Kosower,
rn~rodUction to or~aniC 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 3glucosides
using well-known experimental conditions. The resulting
sugar, blocked at all positions except position '. can be used
acid c~talyst such as BF3 in a ~suitable inert orctanic solvent
to open a suitable oxazolone using the reaction conditions
described above, e.g., in the absence or presence of a Lewis
(e.g., EtOAC, dioxane, etc.).
~ WOg5/17903 i~ ~ t ~ 2 1 7 ~ 9 ~ 4 PCTlUS93~1~s91
ACO
hydnde
Similarly the sugar that results from reaction of
D-glucose with benzaldehyde c;ln be readily blocked at
positions 1 and 6, by sequential reactions with an alcohol in
the presence of acid, and trityiation using techniques well
known in the art of carbohydrate chemistry. The resulting
sugar, with position 3 unblocked can be used selec~ively as
described above tO derivatize a desired oxazolone struceure.
CH OH CH,OH
HO~H + C,H,CHO ~H
1.4-O xD.:ylide~c ~
A suitable oxazoione can also be ring-opened by
a sugar containing reactive amino substituents, i.e., an
aminosaccharide or polyaminos~crh~ri~ For example,
reaction with muramic acid is expected to proceed as
follows.
OH
=~H
COOH ~< Y
g/
WO 95117903 ~ . . ` 2 1 7 ~ ~ 8 4 PCT/US93112591
Similar treatment which is shown below, of the
structurally interesting ambecide paromomycin, with I to
equivalents of a tailored oxazolone is expected to produce a
series of novel structures in which a branched
~etrasaccharide scaffold supports peptidomimetic structures
derived from oxazolones in a geometrically defined manner.
~_NH2
c~_
/~NI~
\~NH,
\~o~
r411 J~;l.
( 3 ) Use of oxazolone-derived structures as
replacements of glycosidic linkages.
Selecuve 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 oxazo~one; this can then be ring~-opened~
by e.g., the anomeric hydroxyl of a sugar to give a novel
sacch~ride after deprotection.
~2
~ woss/17903 . ' ~ 1 7 9 9 8 4 PCTIUS9311~S91
G~ ?
H,CJ<CH, H,CJ<CH, A H, J<CH,
~11 , ' ~
4.8 Desi~n and Svnth~ of
0~7010ne-Derived Qli~onucleotide Mimetics
The art of nucleotide and oligonucleotide
synthesis has provided a great variety of suitably blocked
and activa~ed furanoses and other intermediates ~vhich are
expected to be very useful in the construction of
oxazolonederived mimetics (Co~ nrehensive Or~nic
Cherni~try. Sir Derek Barton, Chairman of l~ditorial Board.
Vol. 5, E. Haslam. Editor, pp. '3-176).
A great ~ arie~y of nucleotide and
oligonucleotide structur~l motifs incorporatino
ox~zolonederived structures ~re contempl~ted including.
bu~ not iimi~ed to, the follovvino.
( I ) For the s~nthesis of olioonucleotides
cont~inino pep~idic ox~zolone-derived linkers in pl~ce of
~he phosphate diester oroupinos found in n~ive
93
WO 95/17903 , ~ . , , 2 1 7 ~ ~ 8 ~ PCT/US93112591
oligonucleotides, the following approach is one of many that
is expected to be useful.
ro_ ., O
~ ~_r
SH ~:
10_ ~
S ~Nl l~ or,
~,o_ 0~ o~ ~o
;~' ~N ~j = S
0 1~ ~
H~r~
0"~0 ~
(2) For the synthesis of structures in which an
oxazolone-derived grouping is used to link complex
oligonucleotidederived units, an approach such as the
following is expected to be useful.
~-_ T o O ' ~ ,
_~o~ o ~
_~ H' ~I ,~1 o-r~o
o. ~\,
~34
~ WO 95/17903 ~ 21 7 9 ~ ~ ~ PCTIUS93112591
EXAMPLES
In order to exemplify the results achieved using
the oxazolone derivatives of the present invention, the
following examples are provided without any attempt to
limit the scope of the instant invention to the discussion
therein, all parts and percentages are by weight, unless
otherwise indicated.
EXAMPLE 1.
CHARACTERIZATION OF THE
ENANT~OMFI RIC PURrI'Y OF OXFENACINE
This example teaches the use of the ring
opening reaction of the pure chiral isomer azalactone (S)-~-
)-4difluoromethyl-4-benzyl-2-vinyl-5-oxazolo~e (1) with
racemic mixtures of the methyl esters of (R)- and (S)-
phydroxyphenylglycine to form the diastereomeric
conjugates (2) and (3), as sho~vn:
H r ~2~
--~3 I CHF2 ~} OH
D6~ o
~+~ + ~ +
HF2 ~5~ R~
O ¦ CHF2 ~e~ OH
OH ~ ~
~r
WO95/17903 ~, ~ t ~ 2 1 7 ~ ~ 8 4 PCTIUS93112591
These diastereomers can be separated by
standard HPLC methods on normal-phase silica to
quantitatively assay the enantiomeric composition of the
starting phydroxyphenylglycines from which the esters are
produced .
The (S)-isomer of p-hydroxyphenylg~ycine
(oxfenacine) is an efFective therapeutic agent for promoting
the oxidation of carbohydrates=when this process is
depressed by high fatty acid utilization levels (such as
occurs in ischemic heart disease), 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.
RESOLI lTION OF RACFMIC P-HYDROXYPHENYL GLYCINE
ESTERIFICATION OF P-HYDROXYPHENYL GLYCINE
0.3 g (0.2 ml) thionyl chloride was added
dropwise to S 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 remoYed at
room temperature under aspirator vacuum ( 10 torr) on a
rotary evaporator and a solid was obtained. This solid was
dissolved in 10 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 ~t 10 C 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%).
9(~
-
~ WO95/17903 ~ 2 ~ 7 ~ ~ ~ 4 PCT/US93112591
RlNG-OPr-NING ADDrTION.
0.181 g (0.001 mol) of the esterified ~-
hydroxyphenylglycine prepared as outlined above was
dissolved in 10 ml of peroxide-free dry dioxane. To thi~
mixture was added 0.~51 g (0.001 mol) of (S)-
4difluoromethyl-4-benzyl-2-vinyl-5-oxazolone, and the
resulting solution heated at reflux for ~ hours. The dioxane
was removed by rotary evaporation and 0 43 g (IooCo) of
the pale yellow solid amide residue was isolated.
HPLC ANALYSIS.
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 ul 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 containing
varying ratios of the two pure enantiomers. The pure
Lisomer was purchased from Schweizerhall Inc. The pure
Disomer was prepared from the commerciall~ available
D,Lracemate obtained from MTM Research
Chemicals/Lancaster Synthesis Inc. by the method of ClarL;,
Phillips and Steer (J. Chem. Soc.. Perkinc Tr;~n~ I at 475
[ 1 976])
c)1
wo 95/17903 ` ` ~ r t ~ ~ 2 1 --I ~ q 8 4 P~ Y.~
(S)- l-D~FLUOROMETHYL, 4-BENZYL-2-VINYL-5-OXAZOLONE
~/ XCHF 7~C~.
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 ceased ( 1.5 hours) . The triethylamine
hydrochloride was removed by filtration, the cake was
s~urried 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 vacuo to give 10.05 g
(80%) of (S)-4-diflupromethyl-4benzyl-2-vinyl azlactone.
NMR (CDC13); CH2 = CH - chemical shifts, splitting pattern in
6 ppm region and integration ratios diagnostic for structure.
FTIR (mull) strong azlactone CO band a~ 1820 cm~l.
SYNl~SIS OF N-ACRYLOYL-(S)-2-DIFLUOROMETHYL
PHENYLALANINE.
21.5 g (0.1 mol) (S)-2-difluoromethyl
phenylalanine, prepared using the method described by
Kolb and Barth (Liebi~s Ann Chem. 1668 (1983)), was
added with stirring to a solution of 8.0 g (0.2 mol) of
sodium hydfoxide in 100 ml water and stirred at this
temper~ture until complete solubiliz~tion was achieved.
9.05 g (0.1 mol) ~cryloyl chloride waS then added dropwise
with stirring, keeping the temperature ;~t 10-15C with
e.~tern~l cooling. After ~ddition w~s complete, stirring w~s
9~
WO 95117903 ~ ' 2 ~ 7 q 9 8 4 PCT11~593112~91
continued for 30 min. To this solution 10.3 ml (0.1''5 mol)
of concentrated hydrochloric acid was added over a 1 0-min.
period, keeping the temperature at 15C. After addition
was complete, the reaction mixture 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-difiuoromethyl phenylalanine. NMR
(CDCl3): chemical shifts, CH2 = CH - splitting pattern and
integration ratios diagnostic for structure
EXAMPLE 2.
PREPARATION OF CHIRAL CHROMATOGRAPE~C
STATIONARY PHASE RING OPENING FORi~,IATlON OF
CONJUGATE WITH AMINOPROPYL SILICA
O
C2Hs
N02
$ ~ H \ J
~ .0 g of aminopropyl-func~ionalized silica waS
slurried in 100 ml benzene in a three-necked fiask
equipped ~ ith a stirrer, a heating bath, ~ refiux condenser
and a Dean-Stark trap The mixture he~ted to refiux and
the water removed azeotropicaliy 3.69 g (0.01 mol) of (S)-
9~
WO 95/17903 ~ z 2 ~ 7 ~ ~ 8 4 PCT/US93/12591
4-ethyl, 1-benzyl-2-(3',5'-dinitrophenyl)-50xazolone 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 l 00 ml methanol
and refiltered a total of four times The resultin~ product
was dried in a vacuum oven set for 30" and 60C to yield
4.87 g functionalized silica. The bonded phase was packed
into a 25 cm x 0.46 cm stainlesssteel HPLC column from
methanol, and successfully used to separate a series of
mandelic acid derivatives using standard conditions.
SYNTHESIS OF (S)-4-ETHYL,4-BENZYL-2-(3',5'-
DINlTROPHENYL)-5-OXAZOLONE
Ph NOl
~\ ~N~2H )~ 5
NOI C~ h
l.09 g (0.0l mol) of ethyl chloroformate was
added with' stirring to 3.87 g (0.0l mol) N-3,5-
dinitrobenzoyl(S)-2-ethyl phenylalanine in 75 ml dry
acetone at room temperature. l.4 ml (0.0l mol) of
triethylamine was added dropwise over a l 0-min. period
and the miXIure was stirred at room temperature until gas
evolution ceased ( 1.5 hours). The trie~hylamine
h~ drochloride was removed bv filtration and the cake was
slurried with '5 ml acetone and refiltered. The combined
filtrates were concentrated to 50 ml on a rotary evaporator,
refiltered. cooled to 30"C and the crystallized roduct was
collected by filtration and dried in vac~o to ~ield '.88 g
(78~o) of (S)-i-ethyl- I-benzyl-~-(3',5'-
/co
~ wo 95/17903 ~ I - 2 ~ 7 9 ~ 8 4 PCTIUS93/12591
dinitrophenyl)az!actone. NMR (CDC13): Frequencies and
integration ratios diagnostic for structure. FTIR: srrong
azlactone band at ca 18~0 cm~ 1.
N-3,5-DINITROBENZLOYL-(S)-2-ETHYLPHENYLALANINE
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 L
Org. Chem 5437 (1990)) was 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 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 concentrated HCI over a 10
min. period, again keeping the l~,.u~ ul~ 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 0_C 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 '7.1 g (70%) N-3,5-
dinitrobenzoyl-(S)-2ethyl phenylalanine.
EXAMPLE 3.
SYNTHESIS OF PREPARATION OF AMINOPROPYL-
FUNCTIONALIZED SLICA.
200 g 015M Spherosil (IBF Corporation) waS
added to 500 ml toluene in a one-liter three-necked
roundbottomed flask equipped with a Teflon paddle stirrer.
a thermometer and a vertical condenser set up with a
DeanSt~rk trap through a cl~isen i3daptor. The slurry was
stirred, heated to a bath temperature of l lOC and the
/~/
WO 95117903 ~ 2 l 7 9 ~ 8 4 PCTIUS93/12591
water azeotropically removed by distillation and collected
in the Dean-Stark trap. The loss in to~uene volume was
measured and compensated for by the addition of
incrementa~ dry to~uene. ~ 5.0 g of 3-aminopropy~
trimethoxysilane was added carefu~ly 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 ~0C and the resu~ting functionali~ed si~ica
collected on a Buechner filter. The si~ica was then washed
twice with 50 ml to~uene sucked dry res~urried in 250 ml
to~uene refi~tered res~urried irl 250 m~ methano~ and
refiltered a total of three times. The resulting methano~ wet
cake was dried in a vacuum oven set for 30 at 60C to
yield 196.4 g aminopropyl si~ica.
EXAMPLE 1
RING-OPENING CONJUGATION OF (S)-l-
(lNAPHTHYL)ETHYLAM~ WITH THE MICHAEL-
ADDITION PRODUCT OF AMINOMERCAPTO-
FUNCTIONALIZED SILICA AND (S)-4ETHYL 1-BENZYL-
2-ACRYLoyL-s-oxAzoLoNE TO PRODUCE A CHIRAL
CHROMATOGRAPH[C STATIONARY PHASE
FORMATION OF CONJUGATE WITH (S)-(l~
( INAPHTHYL)ETHYLAMINE
/~z
WO 95/17903 2 ~ 7 ~ 9 8 4 PCT/US93J12591
CH~
H", ~ NH,
O I i ~CH~S ~ H~
Ph
~OSi(CH~)~S ~H~NH_C H
C~
10.0 g (S)-4-ethy~-4-benzyl-2-(ethylthiopropyl
silica)-5-oxazolone was slurried in 100 ml benzene in a
three-necked flask equipped with a stirrer, a heating bath,
a reflux condenser and a Dean-Stark trap. The mixture was
heated to reflux arld the water was removed azeotropically.
3.42 g (0.02 mol) (S)-(-)- (Inaphthyl)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 100 ml benzene. The we~ cake was
reslurried in 100 ml methanol and refiltered a total of four
times. The product was dried in a vacuum oven set for 30"
and 60C to give 9.72 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 pi-acceptor amine derivatives using
standard conditions described in the Chromatography
Cata~og distributed by Regis Chemical, Morton Grove. 111.
600~3 (e.g., the 3,5dinitro benzoyl derivatives of r~cemic '-
amino- I-butanol + alpha methvl benzyl amine).
~3
wo 95/17903 ~ ~ ~ ` '' 2 ~ 7 9 9 8 ~ Pc~;~usg3/l25g
MICHAEL .~DDITION BY MERCAPTOPROPYL SLICA
~0--$i~CH2)3SH + y :
C2H5
Ph
I
--Si(CHz)3S _6,0~ G
~.~
~ C
20 g ~ tU~lUUyl silica was added to 200 ml
benzene in a 500 ml three-necked rûund-bottomed flask
equipped with a Teflon paddle stirrer, a th~rmom~t~r and a
vertical condenser set up with a Dean-Stark trap through a
claisen adaptor. The slurry was sti}red, heated to a bath
temperature of 140C and the water azeotropically
remoYed by distillation and collected in the Dean-Stark
trap. The loss in benzene volume was measured and
compensated for by the addition of incremental dry
benzene. 6.88 g (0.03 moi) of (S)-4-ethyl,4-benzyl-2-vinyl-
5-~xazolone 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 m~ of methanol and refiltered
a total of four time. The resulting methanol wet cake was
dried in a vacuum oven set for 30" at 60C to yield 19.45 g
oxazolone-functionalized silica.
io~
~ WO95117903 2 t 7 ~ 9 8 ~ PCT/US93~12591
SYNTHESIS OF (S)-1-ETHYL-1'-BENZYL-~-
ACRYLOYL-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 ~50 ml dry acetone at room
temperature. 14 ml (0.1 mol) of triethylamine was added
dropwise over a 1 0-min. perlod 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 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 vaCvo to yield 19.5 g (85%) (S)-4ethyl-4-benzyl-2-
vinyl-5-azlactone, NMR 9CDCl): chemical shifts, CH2 = CH -
splitting pattern in 6 ppm region + integration ratiosdiagnostic for structure. FTIR + (mull): strong azlactone CO
band in 1820 cm~ 1 region.
PREPARATION OF MERCAPTOPROPYL-
FUNCTIONALIZED SrLICA
200 g of l0u (80A) Exsil silica (Exmere 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-
Stark trap through a claisen adaptor. The slurry was stirred,
heated to a bath temperature of 140C and the water was
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 dr~
toluene. 110.0 g of 3-merc~ptopropyl trimethoxysilane was
added carefully through a funnel and the mi.Yture was
s~irred and refluxed for 3 hours wi~h ~he ba~h temperature
se~ at 1~0C. The reaction mixture was then cooled to abou~
1 0C. The resulting functionalized silica was collec~ed on a
/os-
WO 95/17903 . . ' `~ ` , 2 1 7 ~ ~ 8 ~ PCTIUS93112591
Buechner iilter, washed twice with 50 ml toluene, sucked
dry, reslurried in '250 ml toluene, refiltered, reslurried in
250 ml methanol and refiltered ~ 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.
EXAMPLE 5.
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.
SYNTHESIS OF N-TRIFLUOROACETYL-(S~-2-METHYL
LEUCYL-(S)-2-ETHYLPHENYLALANYLP-ISOPROPYLANL~E
0.135 g (0.001 mol) 4-isopropyl arlaline is
dissolved in the minimum amount of an appropriate
solvent, such as acetorlitrile, and 0.384 g (0.001 mol) of 2-
(Ntrifluoroacetyl-(S)-2-methyl leucyl)-(S)-4-methyl-4-
benzyl5-oxazolone dissolved in the minimum amourlt of the
same solvent is 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-
methylleucyl-(S)-2-ethylphenylalanyl analide, useful as a
competitive inhibitor of human elastase in essentially
quantitative yield.
/o~
WO 95/1~903 . ~ 2 1 7 9 9 8 ~ PCTll~S93112591
2-(N-TRIFLUOROACETYL-(S)-2-METHYLLEUCYL)-
(S)-4-METHYL-4BENZYL-5-OXAZOLONE
4.1 g (0.01 mol) N-trifluoroacetyl-(S)-2-
methylleucyl(S)-2-methylphenylalanine lilhium salt is
slurried in 50 ml of an approprlate 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 atld the cake is triturated with benzene and
refiltered. The combined filtrates are stripped using a pot
Lulc of 40C to yield 3.50 g (90%) of crude
oxazolone. The product was purified by recrystallization
from acetone at -30-C. FTIR (mull): Strong azlactone CO
band in 1820 cm~1 region.
SYNTHESIS OF N-TRIFLUORACETYL-(S)-2-METHYLLEUCYL-
(S)-2METHYLPHENYLALAN~NE.
2.23 g (0.01 mol) 2-trifluoroacetyl-(S)-4-
methyl4-isobutyl-5-oxazolone is dissolved with stirring in
the minimum amount of an appropriate solvent, such as
~e~nnitril~, 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. Or~. Ch~m 543'7 (1990))
with one equivalent of LiOH in an appropriate solvent, such
as ethanol, followed by removal of the solvent ~n vacuo.
/c~
WO 95/17903 ~ .. . 2 1 7 9 9 8 ~ PCTII~S93/12591
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 solYent is then
removed in vacuo to yield the solid Ntrifluoroacetyl-(S)-~-
methylleucyl-(S)-2methylphenylalanine lithium salt in
nearly quantitative yield.
SYNTHESIS OF 2-TR'FLUOROACETYL-(S)-4-
MET~YL 1 ISOPROPYL-5-OXAZOLONE.
12.05 g (0.05 mol) of N-trifuoroacetyl-(S)-
2methyl-leucine was stirred at room t~ll.p.,ldtUl~ 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 evd~oldLu, 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-trifuoroacetyl-5-oxazolone. FTIR (mull): strong
azlactone CO band in 1820 cm~l region.
SYNTE~SIS OF
N-T~FLUOROACETYL-(S)-2-MET-YL-LEUCrNE
14.5 g (0.1 mol) of (S)-2-methyl-leucine,
prepared from D,L-leucine methyl ester hydrochloride
using the method of Kolb and Barth (Liebi~'s Ann Chern 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
1 0C, and the mixture stirred at this temperature until
complete solubilization was achieYed. 13.25 g (0.1 mol)
trifuoroacetyl chloride was then added d}opwise with
~o8
wo ss/l7so3 ~ ` 2 ~ 7 9 9 8 ~ PcT~uss3~ 9l
.
stirring, keeping the ~emperature at 10C with ex~ernal
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
hydrochloric 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 vacuo
to give 17.4 g (72%) of N-trifluoroacetyl-(S)-2methyl-
leucine which was used directly in the following step in the
sequence (above).
EXAMPLE 6.
SYNTHESIS OF A PEPSTATIN MIMETIC
This example teaches the synthesis of an
oxazolonederived mimetic of the known aspartyl protease
inhibitor, pepstatin, which has the structure shown:
This mimetic is useful as a competitive inhibitor for
proteases inhibited by pepstatin.
~-isovalervl-(S)-2-methvlvalervl-(3S,4S)-statvl-(S)-
~melh~yl-alanvl-(3S.4S)-statine.
The Boc-protected lithium salt prepared as
described below cimlllr~n~ ously 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 I hour.
The solvent then stripped in vacuo to give a quantitative
WO 95/17903 .... 2 1 7 9 5 8 ~ PCTIUS93/12591
yield of N-isovaleryl-(S)-2-methylvnlyl-(3S,4S)-s~atyl(S)-2-
methylalanyl-(3S,4S)-statine, useful as a pepstatinmimetic
competitive inhibitor for aspartyl proteases which are
inhibited by pepstatin (see, ~3 J. Med. Chem '7 (1980) and
references cited therein). NMR (d6 DMSO): chemical shifts,
integrations and D2O exchange experiments diagnostic for
structure .
N-Boc-(3S .4S)-statvl-(S~-2-methylalanyl-(3S .4S)-sta~ine
lilhil-m 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 sa~t
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 quantitative yield of N-Boc-(3S,4S)-statyl-(S)2-
methylalanyl-(3S,4S)-statine lithium salt.
Boc-protected (3S,4S)-statine, [(3S,4S)-4-
amino3-hydroxy-6- methylheptanoic acid] was produced
from the commercially available amino acid, coupled with
2methylalanine using standard peptide synthesis methods
and converted to the lithium salt using the method
described below. 18.30 g (0.05 mol) of this derivative was
stilred 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
/~
WO 95117903 ` . . ' ` 2 t 7 9 ~ 8 ~ PCTII~S93112591
(CDC13) - chemical shifts and splitting patterns diavnostic
for structure. FTIR (mull): shows a strong azlactone CO
band in the 1820 cm~l region.
N-isovalervl-(S)-2-methvlvalyl-(S)-4-methvl-4-isopropvl-
Sox~7010ne.
13.46 g (0.04 mol) of 2-isovaleryl-(S)-2-
methylYalyl(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 ceased ( 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
recrystallization from acetone at -30C. NMR (CDC13):
chemical shifts and splitting patterns diagnostic for
structure. FTIR (mull): shows strong azlactone CO band in
the 1820 cm~ I region.
N-isovalervl-(S)-2-rnethv~v31vl-(5)-2-methyl v~line lithium
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 I hour. The solvent was then stripped
in vaC~lo to oiV~ a 98% yield of N-isovaleryl-(S)2-
///
WO 95/17903 ;`: : f~ ~ 2 1 7 9 9 ~ ~ PCTIU593/12591
methylvalyl-(S)-2-methyl valine lithium salt. This salt was
used directly in the next step.
2-isovalerv]-(S)-4-methyl-4-iso~ropvl-5-oxazolone.
2-(S)-methylva~ine was prepared from (S)-
valine by the method described by Kolbe and Barth (~
Ann. Chern. at 1668 ~1983)), and was acylated with
isovaleryl chloride using standard acy~ation methods to
produce Nisovaleryl-(S)-methylvaline, this was
subsequently treated with one equivalent of LiOH in
ethanol, followed by removal of the solvent in vacllo 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 ceased ( 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 and the filtrate was again
stripped on a rotary evaporator to yield 17.4 g (85%) of
crude 2-isovaleryl-(S)-4-methyl-4-isopropyl-50xazolone.
Analytically pure material was obtained by recrystallization
from acetone at -30C. FTrR Imull): shows a strong
azlactone CO band in the 1820 cm~1 region. NMR (CDC13):
chemical shifts and splitting patterns diagnostic for
structure.
EXAMPLE 7.
SYNTHESIS OF A MrMETIC
rf~HrBITOR 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
i/~
wo9sll7903 ~ 2 1 79qi~ P( ~
.
substrate, Ac-Ser-Leu-Asn-Phe-Pro-lle-ValOMe See. e.g.,
33 J. Med, Chem 1285 (1990) and references cited therein.
0.341 g (1 mmol) of HN-(L)-Pro-(L)-Ile-(L)-
ValOMe prepared using standard peptide-synthesis
techniques, is dissolved in the minimum amount of DMF. To
this mixture is added 0.229 g (I mmol) 2-acryloyl-(S)-4-
ethyl4-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
lcr~.~,n~s 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 standard C18
reverse-phase chromatography to yield the protected
peptide. The sidechain blocking groups are subsequently
removed usi~g standard peptide deprotection techniques to
yield the product MeO-D-Ser-D-Leu-D-Asn-NH-CO-(S)-Phe-
[Me]-NH-COCH2-CH2-L-N-Pro-L-lle-L-Val-OMe, useful as a
competitive inhibitor for the HIV protease.
EXAMPLE 8.
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.
0.82 g (I mmol) of the uracil derivative, whose
preparation is described below, is coupled through the free
proline carboxylic acid group to 0.244 g ( I mmol) of lle-
//3
-
WO 95/17903 . ~ 2 1 7 9 9 8 4 PCr/US93/12591
.
Val-OMe using standard peptide coupling methods. The
p}oduct is purified by standard C 18 reverse-phase
chromatography to yield the protected peptide. The Bzl
side-chain blocking group is then removed using standard
deprotection techniques to yie~d the product shown above,
useful as a competitive inhibitor for the HIV protease.
0.47 g (I mmol) of the (S)-(S)-
prolinevinylazlactone Michael adduct is dissolved in the
minimum amount of DMF. 0.488 g (I mmol) of MeO-D-Ser-
(Bzl)-D-LeuD-Asn-NH2, prepared from the BOC-protected
amino acid via standard peptide synthesis techniques (see,
e.g., 33 J. Med. Che-m 1285 (1990) and references cited
therein) is then added and the mixture is heated to 60C
and stirred at this Ltlu~ a~ulG for 1~ hours. The DMF is
then removed under high vacuum to yield 0.95 g of crude
product.
2.33 g (5 mmol) of L-proline is dissolved in the
minimum amount of DMF, 1.75 g (5 mmol) of racemic
uracilfunctionalized azlactone is added and the mixture is
stirred at room te~ GldLù.G until the Michael addition
reaction proceeds to c~lmrl~ti~n (as monitored by TLC). The
DMF is then removed under high vacuum and the
diastereomeric mixture is purified by standard
normalphase chromatography to give the desired (S)-(S)-
Michael adduct.
3.69 g (0.01 mol) racemic N-acryloyl-2-
methyl(3'methyluracil)-5'-alanine is stirred with 50 ml of
dry ace~one 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 hydrochloride is
removed by filtration and the cake was slurried with 20 ml
of acetone and refiltered. The combined filtrntes are
concentrated to 50 ml on a rotary evaporator, cooled to
30C ~nd the crystallized product collected by filtration and
~f4
WO gS/179~3 ; = ~ l 7 q q 8 ~ PCTIUS93Jl~59
dried in vacllo to yield racemic 4-(2-methyl-5'-
r3 methyluracil])4-me~hyl-2-vinylazlactone.
17.15 g (0.05 mol) of the racemic ''-(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 is 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 O_C, 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.
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 Ter~hedron Lett. 4259
(1982)) and 17.4 g (0.1 mol) of 3-methyl-5chloromethyl
uracil in the mimim~lm 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 C6HsCH2NEt3Cl 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 IN HCI/Et20 at
0C for 3 hours to yield 29.5 g (86%) of the racemic alpha-
methyl amino acid ester.
,~/5'
Wo 95/17903 ', ^i C, ' ~ 2 ~ 9 9 8 4 PCT/U593112591
SYNTHESIS OF 3-METHYL-5-CHLOROMETHYLURACIL
A. 74.0~ g ( 1 mol) of N-methyl urea ~nd
216.2 g (I mol) of diethylethoxymethylenemalonate are
heated together at 1 22C for ~4 hours, followed by 1 70_C
for 12 hours to yield the 3-methyluracil-5-car~oxylic acid
ethyl ester in 35% yield, following recrystallization from.
ethyl acetate.
B. 30 g 3-methyluracil-5-carboxylic acid
ethyl ester was saponified with 1 0~o NaOH to give the free
acid in 92% yield, after standard 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.
EXAMPLE 9.
PREPARATION OF A C~RAL
CROSSLlNKlNG CONJUGATE MONOMER
4.59 g (0.02 mol) (S)-4-ethyl,4-benzyl-2-
vinylS-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
croddlinked chiral gels, beads, membranes and composites
for chiral separations.
~J~
WO95117903 ~ 2 1 79q84 PCIIUS93J12591
EXAMPLE 10.
SYNIXESIS OF CONJUGATE USEFUL IN ISOLATION AND
PURrFICATION OF SEROTONIN-B~DING RECEPTORS
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 ~ lul~ 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 solvent is added over a 30-min period. The
reaction mixture is allowed to come to room
~,IUp~ UlC 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 which is useful as a ligand for the
stabilization and isolation of serotonin-binding
membrane receptor proteins.
EX~MPLE 11.
SYNTHESIS OF A CONJUGATE USEFUL
rN THE ISOLATION AND PURIFICATION OF T~E
MORPE~NE RECEPTOR
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 (Il) in 10 ml of
the same solvent. The resulting solution is heated to 70C
and held at this temperature for 10 hours. At the end of
this time the solvent is removed under vacuum to yield
//r
_
WO 95/17903 ` ~ ` 2 1 7 9 ~ 8 4 PCT/US93/12591
0.42 g of the Michael adduct (III). 0.~1 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 m~ of a 1:1 mixture of water and an appropriate
solvent, such as acetone, ~djusted to pH 7.5. at 0C under a
nitrogen blanket. The reaction mixture is stirred at 0C for
I hour and then allowed to come to room temperature. The
mixture is then stirred at room temperature under ~
nitrogen blanket for 7 days. The solvent is removed under
vacuum and the water is removed by freeze dryirig to give
the product (V). (V) is useful as a probe for the study of
receptor proteins that bind morphine and its derivatives.
EXAMPLE 12.
SYNTE~SIS 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 2vinyl-
4,4'-dimethyl-5-azlactone in 10 ml of the same solvent.
The mixture is heated to 70C and stirred at this
temperature for 4 hours. The solvent is completely
removed under vacuum and the product (VI) is redissolved
in 200 ml of dimethyl fnrrn~ 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, dissoived 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 I hour, allowed to come to room temperature amd
stirred for 12 hours. The mixture is then added to I liter of
25% NaCI in water at 0C and stirred for 15 min; then 100
ml of 10M hydrochloric acid is added with stirring and
cooling, and the blue precipitate is collected by filtration,
reslurried in I liter of water and refiltered. This extraction
~f8
-
O95117903 ~ 2 1 799~4 PCIIIJS93112'~;91
,: -
procedure is repeated two more times. The product (VIII)is dried at 60C in a vacuum oven at 30" of v~cuum. (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
transmembrane processes involving proteins that bind to
the dye function.
Pteparation of Cib:~ron Blue Derivative (VTTT)
40.0 g (0.05 mol) of Cibacron Blue F3 GA is
dissolved in 1 liter of DMF at 40C with stirring. To this
solution is added 26.5 g (0.23 mol) of hexamethylene
diamine with stirring, followed by 4.0 g (0.05 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
10M hydrochloric acid and 940 g of NaCI. 3.5 liters of water
are added to precipitate the modified dye. The mixture is
stirred for 1 hour and the dye is collected by filtration. The
cake is washed with an additional 3.5 liters of water at pH
2.0 water and dried at 7ûC in.a vacuum oven at 30" of
vacuum to yield 34.0 g of the aminofunctionalized dye
(VII) .
EXAMPLE 13.
SYNTHESIS OF A PHOTOREACTIVE
CONJUGATE~ USEFUL ~N THE ISOLATION AND
PUR~ICATION OF ~-N-ACr TYLGLUCOSAMTDASE~
3.63 g (0.01 mol) of 2-acetamido-2-deoxy-1-
thiob-D-glucopyranose-3,4,6-triacetate (IX) and 1.39 g of
2vinyl-4,4'-dimethylazlactone are dissolved with stirring in
100 ml of an appropriate solvent, heated to 70C and held
at this temperature with stirring for 1'7 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
//7
WO 95/1~903 s ~ ; 2 1 7 9 9 ~ 4 PCT/US93112~91
min period The temperature is the allowed to r1se 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 Bio~hys. Acta. 437
(1974)). The dopamineconnected catechol functionality is a
photographic developer, capable of photographic
amplification by means of standard techniques.
EXAMPLE:14.
SYNTHESIS OF A LIGAND OF PROTEIN KINASE
100 mg of the 20-mer cysteine variant, Cys-
ThrTyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-
Arg-AsnAla-Ile-His-Asp, of a protein kinase natural
binding peptide ligand PK (5-24) (See, 253 ~i~cç 414
(1991)), synthesized by standard peptide synthesis
techniques, is shaken with 7 mg of 2-vinyl-4,4'-dimethyl
azlactone in 0.5 ml of an appropriate 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 Lc.~ tulc~ 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 binding affinity of
competitive ligands for protein kinases and structurally
similar proteins.
/2
WO 95/17903 ~ 2 1 7 9 9 8 4 PCTIUS931~2~91
~ , . , ~
EgAMPLE IS.
SYNTHESIS OF A 4-METHYLENYL-S-O~AZOLOI~IE DER~VED
DIMl~RIC OXAZOLONE OLIGOMI~R
NHB2
NH92 ~ OMe Nd~N
N~-- ~ J 1. Humg ~ E~ I ~
N Cl 2. T OH. MoOH-H20 ~N~ N ~o
A solution of 6-benzoylamidopurine ( 3.9 g, 0.10 mole)
and diisopropylethylamine (14.22 g, 0.11 mole, 19.16 mL) in
acetonitrile (200 mL) is cooled at 0C while 4-
chlorobutryaldehyde ( 15.26 g, 0.10 mole) is added dropwise.
The mixture is stirred at room temperature for 12 hours and
the diisopropylethylamine hydrochloride is removed by
filtration. The filtrate is concentrated in vacuo and
recrystallized from ethyl acetate to afford white, powder
crystals of the product (22.37 g, 0.063 mole, 63%).
This material is dissolved in methanol (400 mL) to which
water (25 mL) and p-to~ n~slllfonic acid (0.5 g) is added. The
mixture is heated at reflux to exhaustion of the acetal. The
reaction mixture is concentrated in vacuo and the residue
partitioned between THF and an aqueous solution of sodium
bicarbonate (10 % w/v, 300 mL). The aqueous phase is
extracted with THF and the combined orgaincs are dried (sat'd
aq NaCI, MgSO4), filtered and concentrated to afford, after
recrystallization, 6-benzoylamido-9-(4-oxobutyl)purine (16.38
g, 0.053 mole, 84%).
NHs2 NHE~
El3N, Ph~, 50 -C N~ ~Ph
Triethylamine (0.101 g, 1.0 mmol) is added to a solu~ion
of 2-phenyl-5-oxazolone ( 1.61 g, 10 mmol) and 6-
/~
WO 95/17903 , 2 1 7 9 9 8 4 Pt~T/US93112591
benzoylamido-9-(4-oxobutyl)purine ((3.09 g, 10 mmol) in
benzene (20 ml). The resultant mixture is heated to 50C for
10 minutes and, after cooling to room temperature, the solvent
is removed in vacuo. The residual pasty mass is triturated
with ethanol to afford a solid which is subsequently
recrystallized from ethanol to afford off-white crystals of
oxazolidinone-linked benzoyladenine (~.84 g, 63%).
NH3~ NHB~
N~--~) ~ t H tl ~m) Pd C N~(~ O~Ph
bNlN~ THF-1M ~q N~OH ~,~ N
A suspension of the adeninyl oxazolone (4.52 g, 10 mmol)
and 10% palladium on carbon (106 mg, 1 mol%) in ethyl acetate
(100 mL) is sparged with dry hydrogen gas until the exocyclic
methylene is fully reduced (1 equivalent). The catalyst is
removed by filtration through a pad of celite and tne filtrate is
concentrated. The residue is dissolved in tetrahydrofuran ( 100
mL) and aq NaOH (1.0 M, 100 mL), tetra-n-butylammonium
hydroxide (0.26 g, 1.0 mmol) and quinine (0.324 g, 1.0 mmol)
was added. The mixture is cooled at 0C while methyl iodide
(3.53 g, 25 mmol, 1.55 mL) is added. the mixture is stirred
until the oxazolone is exhaustiYely alkylated. The organic
phase is separated and the aqueous phase extracted with ether
(2 x 100 mL). The combined organics are dried (sat'd aq NaCI,
MgSO) and .,o~ ,L~.Led to afford a solid (4.98 g) which is
chromatographed to afford 4-(4-benzoyladeninylbutyl)-4-
methyl-5-oxazolone (3.87 g, 8.27 mmol, 83 %).
NHB2 NHB~
N~N~_~ 2 ClCO2Et El,N. PhH ~N~
/2?
; ,; 2 ~ 7 9 ~ 8 4 PCTlUS93112591
wo gS/17gO3 . ,j
.,
The 4-(4-benzoy~adeninylbutyl)-~-methyl-~-oxazolone
(3.87 g, 8.27 mmol) is dissolved in methanol (100 mL) and
glycine ~ithium salt (1.01 g, 12.41 mmol)) is added. The
mixture is warmed at 50C for three hours. After the ring-
opening reaction is complete water ( 100 mL) is added ~nd
acidified to pH = 5.0 with dil. HCl. The resultant solution is
concentrated in vacuo and the solid is dissolved in benzene
with the addition of a small percentage of ethyl acetate. The
solution is cooled in an ice bath while ethyl chloroformate ( 1.31
g, 12.41 mmole, 1.18 mL) and tnethylamine (1.26 g, 12.41
mmol, 1.32 mL) are added. Following cessation of the gas
evolution the salts are removed by suction filtration and the
filtrate is concentrated in vacuo. The residue is recrystallized
from ethanol to afford a 79% yield of the 2-(5-
benzoyladeninyl-2-benzamidoyl-2-methylpentyl)-5-
oxazolidinone (4.54 g, 8.58 mmol)
NIIB2 ~ CHJCN, r~lu~ BzH ~
CI J-- 2. T~OH. M~IOH-H20 N O
4-Chlorobutryaldehyde (15.26 g, 0.10 mole) is added
dropwise to an ice cooled solution of 4-
benzoylamidopyrimidinone (21.5 g, 0.10 mole) anddiisopropylethylamine (14.22 g, 0.11 mo~e, 19.16 mL) in
acetonitrile (200 mL). The bath is removed and the mixture
stirred overnight at room temperature. Removal of the salts by
filtration and the solvent in vacuo, followed by rec}ystallization
from ethyl acetate affords the desired product (23.8 g, 0.072
mole, 72%).
To a solution of this acetal dissolved in methanol (400
mL), p-toluenesulfonic acid (0.5 g) and water (25 mL) are
added, and the mixture refluxed to exhaustion of the acetal.
The reaction mixture is concentrated in vac~to and the residue
partitioned between THF and an aqueous solution of sodium
bicarbonate (10 % w/v, 300 mL). The aqueous phase is
/2 ~
WO 95/17903 J~ 2 1 7 9 9 8 4 PCT/US93/12591
extracted with THF and the combined organics are dried (sat'd
aq NaCI, MgSO4), filtered and concentrated to afford, after
recrysta~lization, 4-benzoylamido- 1-(4-oxobutyl)pyrimidinone
(16.38 g, 0.053 mole, 84%).
D
1-- B~CyUdin ~
B Ad~nin~ q N~ lm) Pd C E~OAc ~<
O O THF 1M ~q N~Olt B Ad~nin~
E-CyEdin~ o
A solution of 4-benzoylamido- 1-(4-
oxobutyl)pyrimidinone (0.57 g, 2.0 mmol), the previously
prepared 2-(5-(benzoyladeninyl)-2-benzamidoyl-2-
methylpentyl)-5-oxazolidinone ( 1.06 g, 2.0 mmol), and
triethylamine (138 mL, 10 mg, 0.1 mmol as catalyst.) in
benzene (20 mL) is warmed at 50C for two hours, to
exhaustion of the starting materials. Removal of the solvent i n
vac~o, followed by trituration with, then recrystallization from,
ethanol affords the product (0.99 g, 1.39 mmol, 70%).
A Sllcp~nci,ln of the adeninyl oxazolone (0.99 g, 1.39
mmol) and 10% palladium on carbon (15 mg, 1 mol%) in ethyl
acetate (15 mL) is sparged with dry hydrogen gas until the
exocyclic methylene is fully reduced (1 equivalent). The
catalyst is removed by filtration through a pad of celite and the
filtrate is concentrated. The residue is dissolved in
tetrahydrofuran (15 mL) and aq NaOH (1.0 M, 15 mL), tetra-n-
butylammorlium hydroxide (4 mg, 0.15 mmol) and quinine (45
mg, 0.15 mmol) was added. The mixture is cooledat 0C while
methyl iodide (0.49 g, 3.5 mmol, 0.22 mL) is added. The
mixture is stirred until the oxazolone is exhaustively alkylated.
The organic phase is separated and the aqueous phase
extracted with ether (2 x 20 mL). The combined organics are
dried (sat'd aq NaCI, MgS04) and concentrated to afford a solid
( 1.23 g) which is chromatographed to afford 2-(5-
(benzoyladeninyl)-2-benzamidoyl-2-methylpentyl)-4-(4-
/24
wo g~/17903 ~ 2 1 ~ 9 ~ ~ 4 PCTIUS93112'~591
ben20ylcytidinylbutyl)-4-methyl-5-oxazolone (0.87 g, 1.
mmol, 86 %).
J~
D~nidln ~ _?
N~< ,L CICO~EL EbN, PhH 3~Cytdin . NH
C~Ad~ni_ ~N~," Ph
// 3~Ad~nir NH
O o~
Ph
A solution of 2-(5-(benzoyladeninyl)-2-benzamidoyl-2-
methy~pentyl)-4-(4-benzoylcytidinylbutyl)-4-methyl-5 -
oxazolone (0.87 g, 1.2 mmol) and glycine li~hium salt (150 mg,
1.8 mmol)) in methanol is warmed at 50C for three hours.
After the ring-opening reaction is complete water (10 mL) is
added and acidified to pH = 5.0 with dil. HCI. The resultant
solution is ~,oncellt.dt~d in vacuo and the solid is dissolved in
benzene with the addition of a small percentage of ethyl
acetate. The so~ution is cooled in an ice bath whi~e ethyl
chloroformate (190 mg, 1.8 mmole, 0.17 mL) and triethylamine
(183 mg, 1.8 mmol, 0.19 mL) are added. After three hours the
salts are removed by suction filtration and the filtrate is
concentrated in vacuo. The residue is recrystallized from
ethanol to afford a 62% yield of the 2-(2-(5-(benzoyladeninyl)-
2-benzamidoyl-2-methylpentanoyl amido)-2-(4-
benzoylcytidinyl)-2-methy~pentyl)-5-oxazolone (585 mg, 0.74
mmol)
The Erlerlmeyer products also may be left unreduced to
provide an alternative scaffolding from which to present the
recognition groups. Whereas this provides a flat structure it
will also provide a different spacing and presentation of those
groups. Shown below is an experimental sequence to provide
the seminal units for such a molecule.
WO 95/17903 ' ''i ~ '-. 2 1 7 9 9 8 4 PCT/US93/lZ591
NHB: NHB:
N~N~ ~Ph1,Li,Gy~in ,M OH ~ , \>,~ N~Ph
The adeninyl oxazolone (~.84 g, 6.3 mmol) is dissolved in
methanol (10 mL) and glycine lithium salt (0.77 g, 9.45 mmol))
is added. The mixture is warmed at 50C for three hours.
After the ring-opening reaction is complete water ( 100 mL) is
added and acidified to pH = S 0 with dil. HCI. The resultant
solution is concentrated in vacuo and the solid is dissolved in
benzene with the addition of a small percentage of ethyl
acetate. The solution is cooled in an ice bath while ethyl
chloroformate ( 1.03 g, 9.45 mmole, 0.9 mL) and triethylamine
(0.96 g, 9.45 mmol, 1.32 mL) are added. Following cessation of
the gas evolution the salts are removed by suction filtration
and the filtrate is concentrated in vacuo. The residue is
recrystallized from ethanol to afford a 67% yield of the
nlr~7r)~ innn~. (2.12 g, 4.24 mmol)
112Cytldin~ ~ B~Cytldln ~
11:Ad~n~n ~_N~Ph B Ad~nln-~ N~Ph
A solution of 4-benzoylamido- 1-(4-
oxobutyl)pyrimidirlone (0.57 g, 2.0 mmol), the previously
prepared oxazolidinone-linked benzoyladenine ( 1.18 g, 2.0
mmol), and triethylamine (138 mL, 10 mg, 0.1 mmol as
catalyst.) in benzene (20 mL) is warmed at 50C for two hours,
to exhaustion of the starting materials. Removal of the solvent
~n ~acuo, followed by trituration with, then recrystallization
from, ethanol affords the product (0.90 g, 1.16 mmol, 58~o).
/26
wo 95/17903 ' 2 1 7 9 9 8 4 PC~ 93)1~591
.
)
8rCylidin t~
~'~~o :. Li-Gycin~, M~OH NH
N~o< lClCO~,E2~N,PhH BZCYtidin~ o=~
3~Ad~nine ~Ph HN) O
"~
'd~d~nine NH
~(Ph
This product is dissolved in methanol ( 15 mL) and
treated with glyci~e lithium salt (0.14 g, 1.74 mmol)) is added.
The mixture is warmed at 50C for three hours. After the ring-
opening reaction is complete water ( 10 mL) is added and
acidified to pH = 5.0 with dil. HCI. The resultant solution is
concentrated in vacuo and the solid is dissolved in benzene
with the addition of a small percentage of ethyl acetate. The
solution is cooled in an ice bath while ethyl chloroformate ( 18 9
mg, 1.74 mmole, 165 mL) and triethylamine (176 mg, 1.74
mmol, 242 mL) are added. Following cessation of the gas
evolution the salts are removed by suction filtration and the
filtrate is concentrated in vacuo. The residue is recrystallized
from ethanol to afford a 51% yield of the oxazolidinone (493
mg, 0.59 mmol)
-
I2?
. .. ` ;` - 2 1 7998~ 3/12591
wo 95/17903 PcrraS9
EXAMPLE 16.
SYNTHESIS OF CARBOHYDRATE MODULE I
OH COOH OH COOH
AcH~OH
~ b
OAc COOCH3
Ac~OAc
AcO
(a) (COCI)2, DMSO~ Et3N, CH.C12, -60 ~C
(b) Ac~O, Pyridine, CH~CI~, rt
A three neck round-bottom flask is charged with 5
mL of a suitable solvent such as CH2C12 and 337 mL (3.9 mmol,
1.2 equiv) oxalyl chloride. The solution is stirred and cooled at
-60 C as 460 mL (505 mg, 6.5 mmol, 2 equiv) of DMSO in 5
mL dichloromethane is added dropwise at a rapid rate. After 5
min, compound 1 ( I g, 3.23 mmol, 1.0 equiv) is added dropwise
oYer 10 min m~inr:~inin~ the temperature at -60 C. After
another 15 min, triethylamine (4.5 mL, 32.3 mmol, 10 equiv) is
added dropwise while keeping the termperature at -60 C.
Stirring is continued for 5 min, after which time the mixture is
allowed to warm to room temperature and water is added. The
aqueous layer is seperated and extracted with a somewhat
polar solvent such as ethyl acetate. . The organic layers are
combined, washed with 1% HCI until it is no longer basic and
washed again with saturated sodium chloride and dried over
anhydrous magnesium sulfate. The filtered solution is
/2~
wo95/17903 .., . ~.' t C~ 2 ~ ~9~84 pcTlus93ll2s
concentrated by rotary evaporation to otain the aldehyde 2
(900 mg, 91~o).
To the aldehyde 2 (500 mg, 1.63 mmol) in pyridine
(1.32 mL, 16.3 mmol, 10 equiv) is added acetic anhydride (996
mg, 9.8 mmol, 6 equiv). The reaction mixture is heated on a
steam bath for 6 h. The excess pyridine, acetic anhydride and
the acetic acid are removed at reduced pressure. The resulting
residue is purified by column chromatography to otain the
pure product 3 (758 mg, 95%).
EXAMPLE 17.
SYNTHESIS OF CARBOHYDRATE MODULE II
AcO ~ COOCH~ a b AcO ~ CHO
(a) 2-(2-hydroxylethyl)- 1 .3-dioxane, Ag-Salicylate, THF, n
(b) aq EICI. THF. rt.
C~~~~ OH
2-(2 '.,~LvA~1.,~.,~)-1,3-dio1:ane
To compound 4 (500 mg, 0 98 mmol) in a suitable
solvent such as THF (5 mL) is added Ag-Salicylate (265 mg,
1.08 mmol, 1. I equiv). After 10 min at room temperature, 2-
(2-hydroxylethyl)-1,3-dioxane (130 mg, 0.98 mmol, 1.0 equiv)
is added to the mixture. The reaction mixture is stirred at
room tell~pcld~Lu~ for 2 h. lN aqueous HCI (j mL) is added to
,^ the reaction mixture, and the reaction is continued for another
30 min. Water is then added to the re~ction. The aqueous
/29
wo 95~17903 ~ 9 8 4 PCTIUS93~12591
phase is extracted several times with ethyl acetate The
combined organic extract is washed with saturated aqueous
NaCI. dried with MgSO4, filtered and concentrated. Purification
with column chromatography gives a pure product ~ (483 mg,
90%).
EXAMPLE 18.
SYNTHESIS OF CARBOHYDRATE MODULE m
H20H CHO CHO
H~ 0~ b ~
6 7 H2 8 NHAc
(a) (COCI)2, DMSO, El3N, CH2CI2, -60 C
(b) Ac20, Pyridin~. CH2CI2, rl
A three neck round-bottom flask is charged with 10
mL of a suitable solvent such as CH2C12 and 540 mL (6.2 mmol,
1.2 equiv) oxalyl chloride. The solution is stirred and cooled at
-60 C as 740 mL (810 mg, 10.4 mmol, 2 equiv) of DMSO in 5
mL dichloromethane is added dropwise at a rapid rate. After 5
min, compound 6 (1 g, 51.8 rnmol, 1.0 equiv) is added dropwise
over 10 min m:~int~inin~ the temperature at -60 C. After
another 15 min, triethylamine (7.2 mL, 51.8 mmol, 10 equiv) is
added dropwise while keeping the termperature at -60 C.
Stirring is continued for 5 min, after which time the mixture is
allowed to warm to room temperature and water is added. The
aqueous layer is seperated and extracted with a somewhat
polar solvent such as ethyl acetate. The organic layers are
combined, washed with 1% HCI until it is no longer basic and
washed again with saturated sodium chloride ar;d dried over
anhydrous m~ni:cillm sulfate. The filtered solution is
concentrated by rotary evaporation to otain the aldehyde 7
(890 mg, 90%).
/30
wo 95/~7903 ; ~ ~ ! 7 q ~ 8 4 pcTnlsg3/l~ssl
.
To the aldehyde 7 (800 mg, 4.2 mmol) in pyridine
(3.4 mL, 42 mmol, 10 equiv) is added acetic anhydride (3 g,
29.3 mmol, 7 equiv). The reaction is stirred at room
temperature for 12 h. The excess pyridine, acetic anhydride
and acetic acid are removed at reduced pressure. The residue is
purified by column chromatography to give the desired.
product 8 (1.48 g, 88%).
EXAMPLE l9.
SYNTHESIS OF CARBOHYDR~TE MODULE IV
..
CH20H CH20TMS
HO ~ a TMSO
HO_~ TMSO~
OHNH2 TMSO 1T,PS
NH2
(a) TMSCI, CH2Cl~ Et3N, r~ ¦ b, c
(b) 2-(2-bromrxthyl)- 1 ,3-oioxarle, THF, rt
(c) HCI, T~, rt CH20H
HO~
O~ sr HO_~(
C HO OHI
2-(2-brOmOethYI)-I~3-dIOX2ne 10 HN~~CHO
To amine 6 (500 mg, 2.59 mmol) in a suitable
solYent such as CH2C12 (5 mL) is added TMSCl (1.55 g, 14.2
mmol, 5.5 equiv) followed by triethylamine (2.9 mL. 20.7
mmol, 8 equiv). The reaction mixture is stirred at room
temperature for 6 h. Water is added to quench the reaction.
The organic layer is washed with water and saturared NaCI and
dried oYer anhydrous magnesium sulfate. The filtered solution
/3/
r
WO 95/17903 ~ ~ ~ . 2 1 ~7 9 9 8 ~ PCT/US93/12591
is concentrated by rotary ev~por~tion to otain the silylated
product 9 ( 1.3 g, 91%).
To compound 9 ( I g~ 1.8 mmol) in a suitable solvent
such as THF (8 mL) is added 2-(2-bromoethyl)- 1,3-dioxane
(387 mg, 1.98 mmol, 1.1 equiY). After 2 h at room
temperature, lN aqueous HCI ( 10 mL) is added to the reaction
mixture and the reaction is continued at room temperature for
another 30 min. Water is then added to the reaction. The
aqueous phase is extracted several times with ethyl acetate.
The combined organic extract is washed with saturated
aqueous NaCI, dried with MgSO4, filtered and concentrated.
Purification with column chromatography gives a pure product
10 (400 mg, 89%).
EXAMPLE 20.
SYNTHESIS OF DA-AMINQ-(N-(4-
(OXOMETHYL)BENZYL)BENZYL-PENIt~lT J IN:
~ c ~ ~ ~ ~
Synthesis of Da-Amino-(N-(4-(oxomethyl)benzyl)benzyl- penicillin:
A solution of Da-Amino-(N-(4-(diethoxymethyl)benzyl)benzyl-
penicillin, methyl ester (32.5 g, 58.7 mmol), dissolved in an
appropriate solvent such as methanol / water or THF / water
(100 mL), with an equimolar amount of aqueous 0.5 N HCI is
stirred at 50 C for 4 hours. The solvent is evaporated
lyophilized to afford a solid (29.5 g, 99%). A portion is
recrystallized to ~fford a sample for analysis.
t3z
wo g~/17903 ~ 2 1 7 9 9 8 4 PCTIUSg3Jl2591
. .i
Synthesis of Da-Amino-~N-(4-(diethoxymethyl)benzyl)benzyl-
penicillin, Methyl ester:
A solution of Da-aminobenzylpenicillin. methyl
ester (36.3 g, 99.9 mmol, synthesized from the reaction of D(-)-
a-aminobenzylpenicillin in as solution of anhydrous methanol
in the presence of a resin-supported super acid, such as Nafion)
and 4-(diethoxymethyl)benzaldehyde (21.2 g, 101.8 mmol) in
an anhydrous solvent such as THF or methanol (400 mL) under
an inert atmosphere such as argon or nitrogen is stirred,
typically overnight, until the imine intermediate is formed and
the starting reagents consumed as shown by thin layer
chromatography (TLC). The reaction mixture is cooled to 0C.
Sodium cyanoborohydride (7.63 g, 121.4 mmol) is added, and
the mixture is stirred at 0C for at least 15 min. until the imine
intermediate is consumed, as shown by TLC. The solvent is
partially eYaporated by rotary evaporation. The residual is
dissolved in a suitable solvent such as methylene chloride or
diethyl ether, extracted with saturated aqueous NaHCO3 (2 x
200 mL) followed by brine (I x 100 mL), and dried over
anhydrous Na2S O 4 . The solvent is removed on a rotary
evaporator to yield arl off white solid (52.8 g, 9~%). A portion
is recrystallized to afford a sample for analysis.
EXAMPLE 21.
SYNTHESIS OF 4-HYDROXY-N-(2-( 1 ,3-DIOXYL)-ETHYL)-4-
PHENYLPlPERIDrNE:
?~N~ ~ ~CN '~ ~3CN
nyl~n~
/~3
-
WO 95/17903 ' ` 2 ~ 7 9 ~ 8 4 PCT/I~S931~2591
Synthesis of 4-Hydroxy-N-((1,3-dioxan-'-yl)-ethyl)-4-
phenylpiperidine:
A solution of 4-hydroxy-4-phenylpiperidine (5.00
g, 28.2 mmol) and 2-(2-bromoethyl~-1,3-dioxane (5.53 g. '8.4
mmol) in a suitable solvent such as xylenes or
dimethylformamide (DMF) (100 mL) under an inert
atmosphere such as argon or nitrogen is gently refluxed, for
several hours to overnight, in the presence of K2CO3 or another
appropriate inorganic base. The reaction mixture is cooled to
room temperature, diluted in a suitable solvent such as
methylene chloride or diethyl ether, extracted with saturated
aqueous NaHC03 (2 x 100 mL) followed by brine (I x 100 mL)
and dried over Na2S 04. The solvent is removed on a rotary
evaporator to afford an off white solid (7.31 g, 89%). A portion
is recrystallized to yield a sample for analysis.
Synthesis of 4-Hydroxy-N-(3-oxopropyl)-4-phenylpiperidine:
A solution of 4-hydroxy-N-((1,3-dioxan-2-yl)-
ethyl)-4-phenylpiperidine (5.30 g, 18.2 mmol), dissolved in an
appropriate solvent such as methanol / water or THF / water
(100 mL), with a 1.5 x molar excess of aqueous 0.5 N HCI is
stirred at 50 C for 4 hours. The reaction mixture is diluted in
a suitable solvent such as methylene chloride or diethyl ether,
extracted with saturated aqueous NaHC03 (2 x 100 mL) to
neutrali~e the acid, followed by brine (I x 100 mL) and dried
over MgS04. The solvent is removed on a rotary evaporator to
afford an off white solid (4.04 g, 95%). A portion is
recrystalli~ed to yield a sample for analysis.
EXAMPL~ 22.
SYNTHESIS OF 5H-5-((1,3-DIOXAN-2-YL)-2-ETHENYL)-
DIBENZO[A,D]CYCLOHE~NE:
/34'
WO 9~17903 , ' ~ . ~ ` 2 1 7 9 q 8 ~ PC~US93112S9
. .
rHf 1~ ~.OH ~ =
HC ~
A solution of 2-(1,3-dioxan-2-yl)-
ethyltriphenylphosphonium bromide (27.8 g, 60.8 mmol) in a
suitable anhydrous solvenl, such as THF (300 mL) is cooled at 0
C while an equimolar amount of a strong base, such as a
solution of n-butyllithium (2.5 M in hexanes / 25.0 mL) is
added dropwise with stirring over a period of 30 minutes. The
reaction is stirred at room temperature for another hour or
more to ensure the anion formation. A solution of
dibenzosuberenone (12.5 g, 60,6 mmol) in an appropriate
anhydrous solvent such as THF (100 mL) is added dropwise
with stirring over a period of 30 minutes. Stirring continues at
0 C for another two hours. The reaction is quenched with the
addition of water (50 mL). The solvent is partially evaporated
by rotary evaporation. The residual is dissolved in a suitable
solvent such as methylene chloride or diethyl ether, extracted
with saturated aqueous NaHCO3 (2 x 200 mL) followed by brine
(1 x 100 mL), and dried over anhydrous Na2SO4. The organic
solvent is concentrated by rotary evaporation to afford 39 g of
a colored oil. The crude material is purified with column
chromatography on a suitable stationary phase such as normal
phase silica gel and eluted with an appropriate mobile phase
such as hexanes / ethyl acetate mixtures, to afford the desired
compound (15.7 g, 85%). A portion is repurified to yield a
sample for analysis.
.
Synthesis o~ 5H-5-( 1 -oxo-3-propenyl)-
dibenzo[a,d]cycloheptene:
/3S
wo 95117903 .~ 2 1 7 9 9 8 4 PCTNS93112~91
A solution of 5H-5-(( 1 ,3-Diox~n-2-yl)-2-ethenyl)-
dibenzo[a,d]cycloheptene (14.3 g, 47.0 mmol), dissolved in an
appropriate solvent such as methanol / water or THF / water
(l00 mL), with a 1.5 x molar excess of aqueous 0.5 N HCI is
stirred at 50 C for 4 hours. The reaction mixture is diluted in
a suitable solvent such as methylene chloride or diethyl. ether,
extracted with saturated aqueous NaHCO3 (2 x 100 mL) to
neutralize the acid, followed by brine (l x 100 mL) and dried
oYer anhydrous MgSO4. The solvent is concentrated by rotary
evaporation to afford 13 g of a colored oil. The crude material
is purified with column chromatography on a suitable
stationary phase such as normal phase silica gel and eluted
with an appropriate mobile phase such as hexanes / ethyl
acetate, to afford the desired compound (ll.l g, 96riO). A
portion is repurified to yield a sample for analysis.
SYNTBSIS OF 5H-5-((~-(2,2-DIMETHOXYETHYL)- I -
AMINO-3- PROPENYL)-D~3ENZO[A,D]CYCLOHEPTENE:
A solution of 5H-5-(1-oxo-3-propenyl)-dibenzo[a,d]-
cycloheptene (9.80 g, 39.8 mmol) and ~minr~ret~ hyde
dimethyl acetal (4.21 g, 40.0 mmol) in an anhydrous solvent
such as THF or methanol (100 mL) under an inert atmosphere
such as argon or nitrogen is stirred, typically overnight, until
the imine int~rm.-rii~r~ is formed and the starting reagents are
consumed as shown by thin layer chromatography (TLC). The
reaction mixture is cooled to 0 C. Sodium cyanoborohydride
(3.06 g, 48.7 mmol) is added, and the mixture is stirred at 0 C
for at least 30 minutes until the imine intermediate is
consumed, as shown by TLC. The solvent is partially
evaporated by rotary evaporation. The residual is dissolved in
a suitable solvent such as methylene chloride or diethyl ether,
extracted with saturated aqueous NaHCO3 (2 x 100 mL)
followed by brine (I x 100 mL), and dried over Na2SO4. The
solvent is removed on a rotary evaporator to yield a solid
/3G
WO 95~17903 `- ' ~ 2 1 7 ~ ~ 8 4 pCTnlS93112591
. . . .
(1~.02 g, 90%). A portion is recrystallized to afford ~ sample
for ana~ysis.
SYNTHESIS OF 5H-5-((N-DIMETHYL-N-(2-OXOETHYL~-I-
AMINO-3-PROPENYL)-DIBhNZO[A,D]CYCLOl~PTENE:
A solution of 5~I-5-((N-(~,2-dimethoxyethyl)- 1 -amino-3-
propenyl)-dibenzo[a,d]cycloheptene (9.71 g, 28.9 mmol) and
one equiYalent of a non-nucleophilic base such as pyridine,
dissolved in an appropriate solvent such as methanol or THF
(100 mL) is added iodomethane (8.35 g, 58.8 mmol). The
reaction is gently refluxed for several hours to overnight, until
the starting material is consumed and the quartenary-
s~bslir~ d amino compound is formed as shown by TLC. A 10
x molar excess of aqueous 1.0 N HC1 is added, and stirring
continues at 50 C for 4 hours. The solvent is partially
evaporated by rotary evaporation. The residual is dissolved in
a suitable solvent such as methylene chloride or diethyl ether,
extracted with saturated aqueous NaHCO3 (100 mL) to
neutralize the acid, followed by brine (100 mL), then dried
over MgSO4. The solvent is removed on a rotary evaporator
and vacuum pump to yield a solid (8,16 g, 80%). A portion is
recrystallized to afford a sample for analysis.
EXAMPLE 23.
SYNTHESIS OF MATERIALS USEFUL AS COATrNGS
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-methylethyle~ min~ were dissolved in 75 ml
methylene chloride with stirring and cooled to 0C In an ice
bath. Then, 13.9 g (0.10 mol) of dimethylvinylazlactone
(the starting species illustrated in Eq. 3 with R2 = R3 = CH3)
pre-cooled to 0C were added to the methylene chloride
137
wo 95/17903 i - 2 1 7 9 9 8 4 P~ ~ Y~ Y I
mixture such that the temperalure 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 0C. 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) spectroscopy as follows: NMR (CDC13): CH3-
N/gem (CH3)2 ratio 1:2; CH2 = CH - splitting pattern in 6
ppm region, integration ratios and D2O exchange
experiments diagnostic for structure. FTIR (null): azlactone
CO band at 1820 cm~ I absent; strong amide bands present
in 1670 - 1700 cm~l region.
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 tr ~ cr~,Lul~:. 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 ~atio 1:4; CH2 = CH -
splitting pattern in 6 ppm region, integration ratios and D2O
exchange experiments diagnostic for structure. FTIR (null):
strong azlactone CO band at 1800 cm~ 1
In the final synthetic step, 3.5 g (0.01 mol) of
(II) and 1.61 g ~0.01 mol) of H2N(CH2)3CH(OC2Hs)2 were
dissolved in 50 ml acetone chilled to 0C and stirred for 4 h
at 0C. The solution was allowed to come to room
temperature and to stand for ' days. The resulting
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,
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WO 95/17903 ~ 2 1 7 9 9 8 ~ PCTNS93~12591
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 dryinc in a vacuum oven adiusted for a 30"
vacuum at room temperature overnight. The final product
was identified by NMR and FTIR spectroscopy as follows:
NMR (CDC13): CH2 = CH - splitting pattern in 6 ppm region,
integration ratios and D2O exchange experiments diagnostic
for structure. FTIR (mull): azlactone CD band at 1820 cm~ I
absent.
EXAMPLE 24.
PREPARATION OF COATED SLICA
SUPPORTS USEFUL IN AFF~ITY CHROMATOGRAPHY
This example describes preparation of an
affinity coating from compound (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 50 ml
methanoi, after which I.11 ml water were added. To this
solution were added 5 g of glycidoxypropyl
trimethoxysilanefunctionalized silica ("Epoxy Silica"). The
mixture was stirred in a rotary at room temperature for 15
min and then stripped, using a bath temperature of 44C, to
a volatiles content of 15% as measured by weight loss (from
25-200C With a sun gun). The silica, coated as a result of
exposure to the mixture of ingredients, was slurried in 50
ml isooctane containing 32.0 mg VAZO-64 (i. 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 70C 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
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WO 95/17903 .; " ~ ~ 2 1 7 9 9 ~ 4 PCT/US93112S91
heated at 120C for two hours to cure the coating and yield
5.4 g of coated silica The silica contained the following
attached groups:
1.5 g of the coated silica beads were shaken
with 20 ml aqueous HCI (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 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:
Repligen Protein A was coupled to the aldehyde
packing using the standard conditions given for the
~rt~rhm~n~ 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
ProteinA functionalized material and loaded with human
IgG from PBS buffer (pH = 7.4) at a flow rate of 1.6 mLlmin.
The IgG was eluted in 0.01M NaOAc (pH = 3.0). The IgG was
then collected and the amount measured
spectrophotometrically using standard calibration curves.
The measured capacity of the packing was 12 mg IgG per
ml of column volume.
EXAMPLE ~5.
FUNCTIONALIZATrON OF
AZLACTONE-CONTA~ING POLYMERS
It is possible to procure 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
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WO 95117903 ' ~ . 2 1 7 ~ 9 ~ ~ PCTILTS93112591
successive reactions with dinucleophilic species of the form
HNu I -Z-Nu2H and suitable azlactones. a surface of the form
(SURFACE)-(X)-Az,
where X is a linker and Az slands for axlactone, can be
transformed into the species
(SURFACE)-(X)-CONHC(CH3)2CONu I (Z)Nu2CH2CH2-Az
which may be linked, if desired, to a biomolecule to form
the following conjugate:
(SllRFACE)
(X)-
CONHC(CH3)2CONu 1 (Z)Nu2CH2CH2CONHC(CH3)2COBiomolecu
le
A suitable Pxr~rim~ntr~l procedure is as follows.
The a~lactone-functional support is slurried in a suitable
solvent, such as CHC13, and cooled to 0C. An amount of the
bifunctional nucleophile equivalent on a molar basis to the
total number of surface azlactone groups present, is
dissolved in the same solvent and added with shaking. The
mixture is then shaken at 0C for 6 hours, allowed to come
to room L~ .,.dlul~, and shaken at room temperature
overnight. The support is collected by filtration, washed
with fresh solvent, re-slurried in an appropriate solvent
and one equivalent of vinylazlactone, dissolved in the same
solvent, is added thereto. The mixture is then shaken,
heated to 70C 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 support is then washed
thoroughly with fresh solvent and dried in vacllo.
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wo gs/l7903 - : : ; 2 1 7 9 9 ~ 4 PCT/US93/12591
EXAMPLE 26.
PREPARATION OF A SUPPORT USEFUL
IN THE PURIFICATION OF HUMAN IGG FROM SERUM
The functional beads prepared as above are
suspended in pH 7.5 aqueous phosphate buffer. A solut~on
of protein A (Repligen) in 10~mM phosphate buffer (pH 7.0)
and at a concentration of 10 mg/900 Ul is added, and the
mixture is then gently shaken at room t~ p~ UI~ for 3
hours. The beads are concentrated by centrifugation, the
supernate decanted off and the beads washed five times
with pH 7.5 aqueous phosphate buffer. The beads are then
loaded into a 0.46 cm inner-diameter glass column and
used to purify human IgG from serum using standard
affinity purification techniques.
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.
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