Note: Descriptions are shown in the official language in which they were submitted.
wo g~/18186 ~ 2 1 7 9 9 ~ 3 r. ~ Y.,i126l2
MODULAR DESIGN AND SYNTEIESIS OF AMINIMIDE
CONTAINING MOLECULES
S "
1. FIFT .n OF T~F INVF.NTION
The present invention relates to the logical
10 development of biochemical and bioph-. ~r~ r~l agents and
of new materials including fahri~tPd materials such as fibers;
beads, films, and gels. Specifically, the invention relates to the
dc~ p..l~ of molecular modules based on ~minimirlP. and
related structures, and to the use of these modules in the
lS assembly of simple and complex m-~lPcl~lPs~ polymers and
fAhricatPd materials with tailored properties; where said
properties can be planned and are d~ by the
contributions of the individual building modules. The
molecular modules of the invention are preferably chiral, and
20 can be used to ~y~ iL.. , new C~ ul ' arld f~hric~t~d
materials which are able to recognize biol~gir~l receptors,
enzymes, genetic m~tP.ri~lc, and other chiral molecules, and are
thus of great interest in the fields of biû~ c.,lir~lc,
separation and materials science.
2 . BA~'TCt~Tl~OUl~n OF TElF INVF:~ON
The discovery of new molecules has traditionally
focused in two broad areas, biologically active mcl 1PC, which
are used as drugs for the treatment of life-threatening
diseases, and new m~ri~lc, which are used in commercial,
especially hi~htP~hr~rlogical applir~tin~c In both areas, the
strategy used to discover new m~lPclllps has involved two basic
.,l;.~--- (i) a more or less random choice of a molecular
c~n~ tP, prepared either via chemical synthesis or isolated
from natural sources, and (ii) the testing of the molecular
candidate for the property or ~ .lics of interest. This
SU`~TiTUTE S~E~T (R~LE 26)
W095118186 ~'''-) ' '~ 2 1 79983 PCT/US93/12612 ~
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
S 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
10 result, the discovery of new functional molecules, being ~hQ~
in nature and relying predominantly on serendipity, has been
an extremely time-co~c~min~, laborious, unpredictable, and
costly enterprise.
A brief account of the strategies and tactics used in
lS the discovery of new molecules is described below. The
emphasis is on biologically interesting molecules; however, the
technical problems ~ ,.cd in the discovery of biologically
active molecules as outlined here are also illustrative of the
problems ecncuu..tc.cd in the discovery of molecules which can
20 serve as new materials for high technological applications.
Furthermore, as discussed below, these problems are also
illustrative of the problems ~..cuu..t.,l~,d in the de~,lo~...cnt of
fabricated materials for high technological applications.
2~ 2.1 Drug Design
Modern theories of biological activity state that
biological activities, and therefore physiological states, are the
result of mo~ r recognition events. For example, ~ t;~l~s
can form c~ base pairs so that COI.I~ILII~
30 single-stranded moiecules hybridize resulting in double- or
triple-helical structures that appear to be involved in
regulation of gene expression. In another example, a
biologically active molecule, referred to as a ligand, binds with
another molecule, usually a macrom~l~c~ o referred to as
3S ligand a~ceptor (e.g., a receptor or an enzyme), and this
binding elicits a chain of molecular events which ultimately
SUBST~TUTE SHEET (RULE 26)
Wo 95/18186 - ; ~ 2 1 7 9 9 8 3 r.llU:~Y~llZ612
gives rise to a physiological state, e.g.. normal cell growth and
differentiation, abnormal cell growth leading to carcino~enesis,
blood-pressure }egulation, nerve-impulse-generation and
-propagation, etc. The binding between ligand and ligand-
S acceptor is geometrically characteristic and extraordinarily
specific, involving appropriate three-dimensional structural
arrangements and chemical interactions.
2.1.1 Design and Synthesis of Nucleotides
Recent interest in gene therapy and 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-~rhqni~m To this end, the sequence of the native target DNA
lS or RNA molecule is cl~ ,t~ d and standard methods are
used to ~y..ll.e~i~,c oligonucleotides representing the
complement of the desired target sequence (see, S. Crooke, The
PASER Jollrnql Vol. 7, Apr. 1993, p. 533 and .~E~ cited
therein). Attempts to design more stable forms of such
20 nlis -l~Qtides for use in vivo have 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 ~'~ y of Nucleic Acids, Y. Mizuno.
Elsevier Science Publishers BV, Amsterdam, The Neth~lqn-lc.
2S 1987).
2.1.2 Glycopeptides
As a result of recent advances in biological
carbohydrate chemistry, c~..l,ohyd.~.tes increasingly are bein~
30 viewed as the co.ll~o.l~llt~ of living systems with the
en~rmr,~ly cQmplex structures required for the encoding of
the massive amounts of information needed to ~I~,he~ tc the
processes of life, e.g., cellular recognitinn immunity, embryonic
development, c~l.,...ot~ ;..s and cell-death. Thus, whereas
3S two naturally occurring amino acids can be used by nature to
convey 2 filr~iqm.-ntql molecular m-oSsq~e~ i.e., via formation
SUB~TITUT~: Sh~ET (~.ULE ~6~
WO 95/18186 2 1 7 9 9 8 3 PCT/U593/12612 ~
of the two possible dipeptide structures, and four different
nucleotides convey 24 molecular messages, two different
monoc:~rch~ride subunits can give rise to 11 unique
disaccharides, and four dissimilar monosaccharides can give
5 rise to up to 35,560 unique tetramers, each capable of
functioning as a flln~isml-nt:ll discreet molecular messenger in a
given physiological system.
The gangliosides are examples of the versatility and
effect with which organisms can use saccharide structures.
10 These molecules are glycolipids (sugar-lipid Cv~ O~ s) and as
such are able to position themselves at strategic locations on
the cell wall: their lipid CC1111~JV..CI1l enables them to anchor in
the hydrophobic interior of the cell wall, positioning their
hydrophilic colllL,o~nt in the aqueous extracellular milieu.
15 Thus the gangliosides tlike many other csrchsriri~os) have been
chosen to act as cellular sentries: they are involved in both the
irlactivation of bacterial toxins and in contact inhibition, the
latter being the complex and poorly understood process by
which normal cells inhibit the growth of adjacent cells, a
20 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 p~ AIl. ;c structure
is shown below.
.
3~ .,
SU~T~U s, ~c ~ ~RULE 2~
wo9S/18186 ; ' ~ ~ ', 21 79983 r~ 26l2
_ ,~
o--~_
0 T
~T
lS ~
o = o o
--~ ~ C
2S ~=
~[=
3S
s
~UBSTITUTE SHEET (RULE 26)
WO95/18186 1 ~ ' 2 1 79983 ~ sll2612 ~
The oligosaccharide components of the glycoproteins
(sugar-protein composites) responsible for the human blood-
group antigens (the A. B, and O blood classes) are shown below:
HOCH~
HO~;~O
0 H~\2~ H HN~C
H
lS ~o
CH,
BLOOD GROUP O ANTIGEN, TYPE II
HOCH2
HO~ pr~lein
H~ H HN~C
2S H
HOCH~ ~ O Blco~ ~roup ~: Y~NH~"
~ ~ Blo~ ~roup t: Y~OH
CH~
BLOOD GROUP A AND B ANTIGENS
SU9ST~TUTE SHE~T ~RULE 26)
WO95/18186 ',~ , 2 1 7 r~l~u~y~ D
Interactions involving complementary proteins and
glycoproteins on red blood cells belonging to in~omr ~tihle
5 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
10 covalent linking with sugars). Thus, deglycosylation of
erythropoetin causes loss of the hormone's biological activity;
deglycosylation of human gonadotropic hormone increases
receptor binding but results in almost complete loss of
biological activity (see ~ m~rh~r et al., Ann. Rev. Biochem
lS 57, 785 (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.
20 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
2S discovery of forms of ligands of biological receptors, enzymes,
or related ma~ r~ ul~c 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
30 screening of molecules (produced through chemical synthesis
or isolated from natural sources), or by using a so-called
"rational" approach involving id~ntific~tion of a lead-structure,
usually the structure of the native ligand, and o~ ion of
its properties through numerous cycles of structural redesign
3S and biological testing. Since most useful drugs have been
discovered not through the "rational" approach but through the
~;llBST~T~TF SY~F, ~ ~RU~F 26
WO 95/18186 ; 2 ~ 7 9 ~ 8 3 PCT/US93112612
screening of randomly chosen compounds, a hybrid approach to
drug discovery has recently emerged which is based on the use
of combinatorial chemistry to construct huge libraries of
randomly-built chemical structures which are screened for
S 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,
' 10 especial~y the small ones, are relatively unstable in
physiological fluids, due to the tendency of the peptide bond to
undergo facile hydrolysis in acidic media or in the presence of
peptitl~c~s Thus, such ligands are decisively inferior in a
ph~rmorokinetic sense to nonreptirlir compounds, and are not
1~ favored as drugs. An qrltlitinnsl limitation of small peptides as
drugs is their low affinity for ligand zrceptr~rs This
phc.lo..,cl.on is in sharp contrast to the affinity demonstrated
by large, folded polypeptides, e.g., proteins, for specific
orcpptnrS, e.g., receptors or enzymes, which can be in the
20 sl~hr~ -lar range. For peptides to become effective drugs,
they must be transformed into nonpeptidic organic ~l-u~lu-~S.
i.e., peptide mimetics, which bind tightly, preferably in the
nanomolar range, and can withstand the chemical and
~iorh~mic~l rigors of c~o~ . with biological tissues and
2S fluids.
Despite numerous incremental advances in the art
of pepti~l~....;,.. ti~ design, no general solution to the problem of
converting a polypeptide-ligand structure to a peptidomimetic
has been defined. At present, "rational" peptid~mimPti~ design
30 is done on an ~Q~ basis. Using numerous redesign-
synthesis-screening cycles, peptidic ligands belonging to a
certain hio.~h~.mi~l class have been converted by groups of
organic chemists and rhsrmsrologictc to specific
peptidomim~ticc; however, in the majority of cases the results
35 in one h;o 1~- "~ area, e.g., peptidase inhibitor design using
the enzyme substrate as a lead, cannot be L~ rc.~l~d fo} use
SUBST~TUTE ~HE~T (~ULE 26)
wo 95/18186 - ~ ' ; 2 1 7 9 9 8 3 r~ Y~I26l2
in another area, e.g, tyrosine-kinase inhibitor design using the
kinase substrate as a lead.
In many cases, the pepti~lnmim~tics that result
from a peptide structural lead using the "rational" approach
5 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, fundamental
research on the use ~f nonpeptidic scaffolds, such as steroidal
10 or sugar structures, to anchor specific receptor-binding groups
in fixed geometric relationships have been described (see for
example T~;.~. l.,..-..,. R. et al., 1992 J. Am Chem. Soc..
114:9699-9701; ~jr~rh~nn R. et al., 1992 J. Am ('h.om Soc..
114:9217-9218); however, the success of this approach
15 remains to be seen.
In an attempt to accelerate the i~ ntifir~tion of
lead-structures, and also the i~l~.ntifir~inn of useful drug
candidates through screening of randomly chosen compounds,
l~,ae~ hc.a have developed ~ n~n~t~d methods for the
20 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 mnrlifir~til~, of Merrifi~l~l peptide
25 synthesis, wherein the C-terminal ar~ino 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
30 peptides are then screened for activity without removing them
from the pins. Houghton, (1985, Proc. ~tl Acad. Sci. USA
82:5131; and U.S. Patent No. 4,631,211) utilizes individual
polyethylene bags ("tea bags") - e C-terminal amino
acids bound to a solid support. These are mixed and coupled
35 with the requisite arnino acids using solid phase synthesis
t~rhniq--~s The peptides produced are then .~cov~,..,d and
SUB~TITUT~ L~ 26~
WO95/18186 ~ 2 ~ 79983 P~ Y~ll26l2
tested individually. Fodor et al., (1991, Science 251:767)
described light-directed, spatially addressable parallel-peptide
synthesis on a silicon wafer to generate large arrays of
addressable peptides that can be directly tested for binding to
5 biological targets. These workers have also developed
recombinant DNA/genetic engineering methods for expressing
huge peptide libraries on the surface of phages (Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87:6378).
In another. cnmhin~tori~l 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
15 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.
7.1lr~rmq~ et al., (1992, Int. J. Pe~tide Prot~oin 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 d~.iv.~ , called "peptoids", which
2~ are screened for activity against a variety of ~:c~' r
targets. (See also, Symon et al., 1992, Proc. ~qtl Acad. Sci. USA
89:9367). E~ncoded cnmhin-~rriql chemical syntheses have
been described recently (S. Brenner and R.A. Lerner, 1992,
Proc. ~51tl Acad. Sci. USA 89:5381).
Recently in an alternate strategy for the design of
ther~re~tirally active mimetic ligands much attention has been
focused on the construction and application of molecules which
possess the property of binding to nucleic acids. These
m~trriqlc whether they be direct Watson-Crick type
3S "antisense" nllrl~oti~l~ mim.~tirS, Hoogstein-type bindérs or
minor gro~ve binding C~ UI1JI~5 such as those pioneered by
SUB~T~TUTE SHE~r (RULE 26)
wo 9S/18186 2 1 7 ~ ~ ~ 3 PCTIUS93/126~2
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
S 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
10 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
lS synthetic scaffold to support a series of natural or designed
bases in such a manner that tight binding to natural nucleic
acids is observed; 2.) the ~ ui~ L~ for designed or
naturally occurring nucleotide bases other than guanosine,
cytosine, thymidine, adenine, or uridine, to efficiently hydrogen
20 bond (hybridize) to another, natural base or nucleotide. Among
these natural n~ otiri~ mimetics are showdomycin (I) and
pseudollriliin~ (2) and the synthetic compounds (3) and (4).
O~J HO_~ HO_~J HO_ $
CH OH OH OH OH CH CH CH
It has been demonstrated that such unnatural or
modified bases can show efficient hy~ri~li7~tiQn if projected
from an effective scaffold as shown here for both tautomers of
5-bromouracil, which can bind to either adenine or guanine
11
SUBST~TU~ ~HE~ ~R~LE 26)
W095118186 ~ 2 ~ 799~3 r.,-lu~ 612
N~\ N~
,H ~/ --Sug ", ~\N--sU9
"N~N ¢~ N
SU9 SU9
The primary goal of any "antisense" or "gene
the}apy" is to inactivate the archival rogue information
10 (deliterious DNA) or the messenger information (the
correpsonding 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,
15 chemists have pursued alternat~ve 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 m~rnhr~n.~s to the site at which
hybrirli7~ion 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, ofteh in conjunction with molecular
m~ pllin&~ lrulr~ti~ S, 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-concl~min~, laborious,
and ~ ,..si~,. Success in this important area of biological
12
SU~S~iT~TE ~HEET tRVLE 2~)
~ W0 95/18186 ~ ` 2 1 7 9 ~ 8 3 P~ u~YJ/l~612
chemistry depends on the effective utilization of sophisticated
separation technologies.
Crystallization can be valuable as a separation
- technique but in the majority of cases, especially in cases
S 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 co~ on.,..~ of a mixture as the
mixture moves on an active natural, synthetic, or semisynthetic
10 surface; tight-binding components in the moving mixture leave
the surface last en m~Cc~ resu~ting in separation.
The development of substrates or supports to be
used in separations has involved either the polymerization-
crocslinkin~ of mnr~ m~riC molecules under various conditions
lS to produce fqlhri~t--d materials such as beads, gels, or films, or
the chemical modification of various commercially available
f~hri~t~d 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
20 specific separations or types of separations and are thus of
limited utility. Many of these materials are inrnmrqtihl~ with
biological macromolecules, e.g., reverse-phase silica frequentl~
used to perform high pressure liquid chromatography can
denature hyd-u~hobic proteins and other polypeptides.
2S Furthermore, many supports are used under conditions which
are not cnmrqtihl~o with sensitive hi~mc~ As such as
proteins, enzymes, gl~o~.uleins, etc., which are readily
~enst~ hle and sensitive to extreme pH's. An additional
difficulty with separations carried out using these supports is
30 that the separation results are often support-batch ~l~p~nden~.
i.e. they are irreproducible.
Recently a variety of coatings and C~ r-
forming materials have been used to modify Cu..llll~. .,ially
available f~h-ir~tAd materials into articles with improved
3S properties; however the success of this approach remains to be
seen.
13
SUBST,TUTt Sl~tT (F~U~ 263
wo 95/18186 ~ 2 1 7 9 9 8 3 E~ Y~ 26I2 ~
If a chromatographic support is equipped with
molecules which bind specifically with a ~U~ o~ t of a
complex mixture, that component will be separated from the
mixture and may be released subsequently by changing the
S experimental conditions (e.g., buffers, stringency, etc.) This
type of separation is appropriately called "affinity
chromatography" and remains an extremely effective and
widely used separation technique. It is certainly much more
selective than traditional chromatographic techniques, e.g
10 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 m7Yimllm
separating efficiency need to be used under conditions that are
damaging to biomolecules, e.g., conditions involving high
lS pressure, use of organic solvents and other ~n7~1lrin~ agents,
etc.
The development of more powerful separation
technologies depends ~i~nifirAntly on breakthroughs in the
field of materials science, sperifir~lly in the design and
20 construction of materials that have the power to recognize
specific molecular shapes under eYr-orim~ntAl conditions
resembling those found in physiological media, i.e., these
experim~ntAl c~ ' onC must involve an aqueous medium
whose ~ ,.d~UlC and pH are close to the physiological levels
2S and which contains none of the agents known to damage or
denature bir mol~c~lps The construction of these "intelligent"
materials frequently involves the introduction of small
molecules capable of sperifir~lly l~ iUg others into
existing materials, e.g. surfaces, films, gels, beads, etc., by a
30 wide variety of chemical mo~lifirA-io~; alternatively molecules
capable of recognition are converted to III~J..JIIIe. :~ and used to
create the "intPlli~Pnt" materials through polymrri7Atinn
reactions .
3S
14
SUBSTITUT~ Si~T ~R~LE 26)
~ wo 95118186 ~ ? ~ 2 1 7 9 9 ~ 3 Pcr~sg3/l2612
3 . SUMM~RYOF T~ ~NTION
A new approach to the construction of novel
molecules is described. This approach involves the
S development of :Iminimif~ based 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 novel
~minimi~1--derived molecules which are the subject of this
invention have the following structure:
~3R~ ~
A-X- CO-N- IN-G -Y-B
R~ n
wherein:
a. A and B are the same or different, and
each .eull~a~ a chemical bond; hydrogen; an electrophillic
20 group; a n-lrl~ophillic group; R; R'; an amino acid derivative; a
nucleotide d~ ati~,; a carbohydrate derivative; an organic
structural motif; a reporter element; an organic moiety
cont~inin~ a pol~-u~.iL..ble group; and a macromolecular
c~ uorlcn~t wherein A and B are optionally cQnn~ct.od to each
2S other or to other structures and R and R' are as defined below;
b. X and Y are the same or different and
each l~,~JICS~ a a chemical bond or one or more atoms of
carbon, nitrogen, sulfur, oxygen, phOayllu1uua~ silicon or
30 combinations thereof;
c. R and R' are the same or different and
each Ic~lcaE~ a A, B, cyano, nitro, halogen, oxygen, hydroxy,
alkoxy, t_io, straight or branched chain alkyl, .,~..I,o.,yclic aryl
35 and substi~ut.-d or h.,t~,.o."~,lic derivatives thereof, wherein R
SU~ST~TUTE S~EET ~RUI E 26~
t r 2 1 7 q 9 8 3
WO 9S/18186 i i - PCTN593J12612
and R', may be the same or different in adjacent n units and
have a selected stereochemical arrangement about the carbon
atom to which they are attached;
d. G is a chemical bond or a connecting
g}oup that includes a terminal carbon atom for ~tt~hment to
the quarternary nitrogen and G may be different in adjacent n
units; and
e. n is greater than or equal to 1;
provided that, (I) if G is a chemical bond, Y
includes a terminal carbon atom for ~ rnt to the
u,u~u~ uy nitrogen; and (2) if n is 1, X and Y are chemical
lS bonds and R and R' are the same, A and B are different and one
is other than H or R.
3.1 Physical and Chemical P.~ ,s of the
~minimid~ Functional Group
Aminimides are zwitterionic structures described
by the resonance hybrid of the two energetically comparable
Lewis ~.uclul.,s shown below:
R2 R2
2S Rl--ICI--N--N+-R4 . . R~--C=N--N+-R,,
o- R3
The tetr~ t~ d nitrogen of the ~minimid~
30 group can be asymetric rendering ~minimiri~s chiral as shown
by the two Pn~nti~m~rs below:
' ~ DR2 ~ D
3~ R4 N/ R 3 R4 N/ R 3
16
SUBST~TI ~T~ S!~ET (~ULE ~6)
~ ~ {~
i ~
~ W0 95118186 2 1 7 9 ~ 8 3 r ~ .t/12612
As a result of the polarity of their structures, but
lack of net charge, simple ~minimitlPs are freely soluble in both
- water and (especially) organic solvents.
Dilute aqueous solutions of :~minimi~ilos are neutral
and of very low conductivity; the conjugate acids of simple
~minimid~Ps are weakly acidic, pKa of ca. 4.5. A striking
property of aminimides is their hydrolytic stability, under
acidic, basic, or enzymatic conditions. For example, boiling
trimethyl amine ben7:lmi~lP in 6 N NaOH for 24 hrs leaves the
aminimide llnch~n~e~l Upon thermolytic treatment, at
tC~ Uli,S excee~lin~ 180_C, ~minimi~lP5 decompose to give
isocyanates as follows.
lS lR2 R~
Rl--IC=N--N+--R4 Rl--N=C=O + IN--R4
o- R3 R3
The aminimirlP building blocks of the invention can
be used to synthesize novel molecules designed to mimic the
three-dimensional structure and function of native ligands,
and/or interact with the binding sites of a native reeeptor.
This logical approaeh to molecular construction is,applicable to
the synthesis of all types of molecules, ineluding but not
2S lirnited to mimeties of peptides, proteins, oli~ Pot~ Ps,
earbohydrates, lipids, polymers and to f~l rir~tPd materials
useful in materials seience. It is analogous to the modular
construetion of a mPrh~nic~l deviee that performs a speeifie
operation wherein eaeh module performs a speeifie task
co~ ing to the overall operation of the deviee.
The invention is based, in part, on the following
insights of the discovc.~,l. (I) All ligands share a single
universal ~,l,;~ -1 feature: they consist of a seaffold
strueture, made e.g., of amide, earbon-earbon, or
3S phosphodiester bonds whieh support several funetional groups
17
SU~STiT~TE SI~ET (~'L' 26~
~ ! 1 7 7~SJ
WOg5/18186 ,~,";;; ~ '7qq~`3 PCIIUS93112612
in a precise and relatively rigid ~eometric arrangement. (~)
Binding modes between ligands and receptors share a single
uniYersal feature as well: they all involve attractive
inte}actions between complementary stru~tural elements, e.g
charge- and pi-type interactions~ hydrophobic and Van der
Waals forces, hydrogen bonds. (3) A continuum of fabricated
materials exists spanning a dimensional range from 100
Angstroms to 1 cm in diameter, comprising various materials of
varied construction, geometries, morphologies and functions, all
of which possess the common feature of a functional surface
which is presented to a biologically active molecule or a
mixtu}e of molecules to achieve recognition between the
molecule (or the desired molecule in a mixture) and the
surface. And (4) ~minimid~ structures, which have remained
relatively unexp~ored in the design and synthesis of
1~ biologically active CUIII~U_ ~C and especially of drugs, would be
ideal building blocks for constructing backbones or scaffolds
bearing the appropriate functional groups, that either mimic
desired ligands and/or interact with appropriate receptor
binding sites; furthermore, aminimi-~ modules may be utilized
in a variety of ways across the ~ illUIIIII of fAhrir~Pd
materials described above to produce new materials capable o!
specific molecular recognition. These Aminimid~ building block-
may be chirally pure and can be used to ~ molecules
that mimic a number of biologically active molecules, includinc
but not limited to peptides, proteins, olig~ -'e~tides,
polynucleotides, CalbUII~dl~ a~ lipids, and a variety of
polymers and fAhrirA~.od materials that are useful as new
- -lc, including but not limited to solid supports useful in
column chromatography, catalysts, solid phase imm
drug delivery vehicles, films, and "in~ " materials
designed for use in selective s~alatiot~s of various componen~
of complex mixtures.
Examples describing the use of ~minimi~-based
modules in the mûdular assembly of a variety of molecular
3S structures are given. The molecular structures include
18
SUBSTiTU I E ~HFI~T (~UI ~ 26~
2~ 79983
WO 95/18186 ` ' r~ 93/lJi612
functionalized silica surfaces useful in the optical resolution of
racemic mixtures; peptide mimetics which inhibit human
elastase, protein-kinase. and the HIV protease: polvmers
formed via free-radical or condensation polymerization of
aminimide-containing monomers; and lipid-mimetics useful in -
the detectlon, isolation, and purification of a variety of
receptors. Accordingly, the present invention relates to a novel
class of aminimide compounds and their use in the construction
of simple and complex molecules and macromolecular
combinations of molecules.
The present invention also.relates to the use of said
aminimide compounds in biochemical and biopharmaceutical
applications as well as their use in materials such as fibers~
beads, films and gels.
The present invention also relates to the use of the
lS inventive class of compounds to logically develop intelligent
molecules and fi~hric^~ materials which are able to recognize
biological receptors, enzymes, genetic materials and chiral
molecules.
Still further, the present invention relates to the
synthesis of libraries of ~minimiAP-based molecules employing
tl~hniql~s herein disclosed or other techniques well known to
those skilled in the art.
In addition, the present invention relates to chirally
pure C~ 4 -Ac, that may be synthesized chirally pure and
can be used to recognize other chiral c4l r '-
Yet still further, the present invention relates to a
class of aminimiA~ COIIl~_ '~ that can be used as mimetics for
UUIIIC.~5 biologically active agents.
The present invention also relates to aminimi~Af~
molecules which posess enhanced hydrolytic and enzymatic
5t~h;1iti.o5, and in the case of ' ~ , n~lly active materials, are
L~ ls~ul~,d to target ligand ?~c~p~or macromolecules in vivo
without causing serious side effects.
19
SLI~ST~ ~ lJTE SH~FT (P.l~LE 2~)
WO95/18186 1`"'~ ` 21 79q83 Pcr~ss3/126l2
The invention is also directed to a method of makin~ a
polymer having a particular water solubility comprising ~he
steps of; a) choosing a first monomer having the formula
A--X~ I I--N--Nt--(G) I n~ Y--B
wherein R and R' are the same or different and are chosen
from those organic moieties exhibiting hydrophobicity; b)
choosing a second monomer haYing the formula
I ...n
A--X~ll--N--N~ G)I---nl_Y--B
wherein R and R' are the same or different and are chosen
from those organic moieties exhibiting hydrophilicity; and c)
reacting said ~uollolu.,. ~ 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
2!i can include those which do not have carboxyl, amino or
ester funrtiQ~slity. 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 c~,r~ the structure of a biologically
active compound of the formula. This method can be used
to produce pl~srrl~s~rhnres, peptide mimetics, nucleotide
mimrt;cc ca~l,ohy~l~.L~ mimetics, and reporter compounds,
3S for example.
SU~TlTUTe S~EET (RUL~ 26)
2 1 79983
wo 95118186 ~ Vi~Y.3/l~C12
This invention is also further directed to a method of
preparing a combinatorial library which comprises: a)
preparing a compound having the formula;
S Rl.. n
A--X~C--Nii--N~--(G)I n~Y--B
O R,.n n
n > l; and b) conducting further reactions with the
compound to form a combinatorial library.
Still further this invention is directed to a method of
separating a desired c~ o, 1 from a plurality of
compounds, which c~ a) preparing a separator
compound having the formula:
Rl...n
A--X--+ICI--N--N+--(G)I n~Y--B
n> I;
b) CQrltP^tin~ said separator ~u~ ou~d with the plurality of
c~ c and c) differentiating said second compound
and the separated compounded from said plurality of
compounds.
~ .
21
SI~B~TITUTE ~HI ~r (Rl)LE 2~
W095/18186 .,~ 2 1 799~3 I_l/LI~Y.~ 612
1. DET~n .Fn DESCR n'TlON OF T~F INVFNTIO~
4.1. I Use of Ihe ~minimi~l~ Group as a
Mimetic of the Amide Group
S
Th~ aminimide group mimics several key structural
features of the amide group~ such as overall geometry (e.g.,
both functional groups contain a planar carbonyl unit and a
tetrahedral atom linked to the acylated nit}ogen) and aspects
0 of charge distribution (e.g., both functional groups contain a
carbonyl with significant negative charge development on the
oxygen). These structural relationships can be seen below,
where the resonance hybrids of the two groups are drawn.
lS C R~N~R , ~C~ ,N~5SS
1I R~ H C~) Rl~H
~,~ NH ~5S ' ~C~N~ 55S
Being hydrolytically and enzymatically more stable
than amides and ~rJScPccin~ novel solubility ~IU~ due to
their zwitterionic structures, Aminimiti~s are valuable building
blocks for the construction of mimetics of biolr,gicAlly active
5 with superior pharmacological properties. For the
purposes of tbis invention the term biological activity is
defined as having a beneficial biological effect. For the
construction of these mimetics, the aminimi~e backbone is used
as a scaffûld for the geometrically precise ~-t~ of
structural units possessing desired ~,lco~h~ ir~l and t
electronic features, such as suitable chiral atoms, hydrogen-
bonding centers, hy.l-~ 'r. and charged groups, pi-systems,
22
SU~ST5T~E S! ~'F. T ~)LE 26)
WO 95/18186 2 1 7 9 9 8 3 P~ 612
etc. Furthermore, multiple aminimide units can be linked in a
variety of modes, using likers of diverse structures, to produce
polymers of a great variety of structures. Specific molecular
forms are chosen for screening and further study using several
criteria. In one instance, a certain sminimi~i~ structure is
chosen bec~use it is novel and has never been tested for
activity as a bioph~rm~reutical agent or as material for device
construction. In a preferable instance, an Rminimi~lP ligand is
chosen because it incorporates structural features and
properties suggested by a certain bioch~mic~l mPch~icm In
another preferable case, the aminimide structure is the result
of assembly of molecular modules each making a specific
desirable contribution to the overall properties of the
aminimide-containing molecule.
Sllmmsri7in~, ~minimi~iP5 are functional groups
with unusual and very desirable physiochemical properties.
which can be used as molecular modules for the construction of
molecular ~u~,lu~cS that are useful as biopharn~greutir~l
agents and as new materials for high technological application~.
4.2 General Synthetic Routes to ,~minimirlps
,~minimirlP$ can be synthesized in a variety of
different ways. The compounds of the present invention can b~
synthesized by many routes. It is well known in the art of
organic synthesis that many different synthetic protocols can
2~ be used to prepare a given ~ Different routes can
involve more or less ~ ,G~i~,e reagents, easier or more
difficult separation or purification p~UC~ul~s, straightforw~r~
or ~ lUb..l:lU...C, scale-up, and hi$her or lower yield. The skilled
synthetic organic chemist knows well how to balance the
cnmre~in~ s of co~ strategies. Thus, the
c~,...l.ù~n ls of the present invention are not limited by the
- choice of synthetic strategy and any synthetic strategy that
yields the .~..I.u~ described above can be used.
23
SU~T~TlîîrE ~.~E~ L~ 2~
~o 95/18186 2 1 7 q ~ 8 3 PCTIUS93/12612 ~
Th,e 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.
lS
^` 20
2S
~_. ..... .
24
SUBSTITLITE StlEET (RU~ E ?6)
;` i:`; :;
WO 95118186 ' 2 1 7 9 9 8 3 Pc~rfusg3~l26l2
4.2.1 Aminimides via Alkylation of N,N-Disubstituted
S Hydrazides
Alkylation of a hydrazide followed by
neutralization with a base produces an ~minimi~l
Rl~N C (I) R3X Rl~+,R3 8
R2' `N' ~R4 (2) n.~l~tral;7~ n R2~ N' R4
H
This alkylation is carried out in a suitable solvent, such as a
hydroxylic solvent, e.g., water, ethanol, i~oulu~ 1 or a dipolar
aprotic solvent, e.g., DMF, DMSO, ~r~t-~nitril~, usually with
heating. An example of this reaction is the synthesis of the
trifluoroacyl-analide dipeptide elastase inhibitor mimetics
shown in the examples below.
The hydrazide to be used in the above synthesis is
produced by the reaction of a 1,1-disubstituted hydrazine with
an activated acyl deriYative or an iSOI~allal~:, in a suitable
organic solvent, e.g., methylene chloride, toluene, ether, etc. in
the presence of a bâse such as ~ lllylalllil~ to neutralize the
hâloacid generated during the acylation. This reaction is
ese"ted as follows:
2,N--NH2 + C . 2--N`N'C`R4
Activated acyI derivatives include acid ~hl~ri~l~c
chlOlueal~ ~r~5, chlorothiocarbonates, etc.; the acyl derivative
may also be replaced with a suitable carboxylic acid and a
3S c~ ~l- C ng agent such as N,N-dicyclohe~yl-,a u~ f (DCC).
SUBSTITl~TE ~H~rT (RULE 2~)
wO 95118186 , ; I . 2 ~ 7 9 9 8 3 ~ Y.~1l26l2
.,, --
The alkylating agent R3X used in the hydrazide
alkylation may be an alkyl halide (X = Cl, Br, I), a tosylate (X =
OTs), or some other suitable reactive species~ such as an
epoxide.
The desired I, I -disubstituted hydrazines may be
readily prepared in a number of ways well known in the art:
for example, the reaction of a secondary amine with NH~CI in
an inert organic solvent.
Rl -- Rl
NH + H~N--Cl ~ N--NH HCI
~ Ri 2
A second synthetic route for the preparation of
hydrazines is alkylation of monoalkyl hydrazines, shown
below for methyl hydrazine:
N--NH2 + lleutr, N--NH2
Detailed r~r~ri~^~'~lc for the synthesis of a
number of l,l-~ "i~ d hydrazines via this reaction are
2S set forth in the e~amples below.
The above route to ~minimirlPs is broadly
applicable and allows the in~ ,olation of a wide variety of
aliphatic, aromatic and heterocyclic groups into variouS
positions in the aminimid~ structure.
4.2.2 ~minimi~lps via Acylation of l,l,1-Trialkyl Hyd~ iull,
Salts
Acylation of a suitable trialkyl ~ a~;ll;l".. salt by
an acyl d~ a~ . or iSO~,~a~ht~, in the presence of a strong
26
SUBST~ . S~FET ~F~ULE 26!
WO 95118186 ~ 2 1 ~ 9 9 8 3 Pc~/Uss3n26l2
base in a suitable organic solvent, e.g. dioxane, ether,
acetonitrile, etc. produces good yields of aminimides.
R~ O . R- O
R~ H, X + R~--C--oR5 base R~ C--R~ -
S . R3 R3
The acyl derivatives for the acylation reaction are
the same as those required for the synthesis of the hydrazides
outlined above.
This hydrazinium salt synthesis method can be
subject to the possibility of rearrangements and side reactions
which compete with the formation of the ~minimi~i~ The
conditions under ~vhich these rearrangements can take place
are highly fl~p.on~ nt on the specific 51~hstitl-- t~ on the
lS quarternary nitrogen and, thus, the application of this.. synthetic
route for the production of ~mi-~mi~-derived species needs
to take into consideration the specific nature of the desired R
groups at this position.
Two basic rearrangements are possible:
a. Migration of a _' - group from the
quaternary nitrogen to form a hydrazide (Wawazo~.,k
r~ ng~ n~nt - cf. ~'h.~m. Rev.~ 73, 255, 1972; In-i E~. Ch~m
Prod. Res. Devel.. 19, 338, 1980).
30 (~)
~1 0
R--N--NH ~ RNE~(CH3)2
c~3
27
SUBSTiT~TE S~l.ET fRUL~ 2~'
WO 95/18186 ,~ 2 1 7 9 9 8 3 PCT/l~S93tl2612
The facilitv of this rearrangement is highly dependent on the
nature of the substituent group. Alk~ l and arvl substituents. as
well as substituents attached to the quaternary nitrogen with
alkyl or aryl connecting groups require vigorous reaction
conditions, while allylic substituents migrate under much
milder conditions. Thus, I-benzyl- I, I-dimethyl hydrazinium
chloride requires heating with powdered potassium hydroxide
at 200-300C to effect rearrangement to the hydrazide, while
I -allyl- I, I -dimethyl hydrazinium chloride rearranges in I M
aqueous sodium hydroxide in 3 hours at 60C (Chem Ber., 103.
2052, 1970) and l,l-dimethyl-l-phenacyl hydrazinium
bromide has been reported to rearrange in n-propanol at reflux
(Tetrqh~ron Lett.. 38, 3336, 1977)
b. Flimi~q~inn reaction with s~lhstitll~ntc posessing
a beta hydrogen:
~ H / ~ 2 ~F=
NH2--NR 2
This reaction has been ~osrlll ' to be responsible
2S for the gen~qtion of cyclo~-Y~n~ from dimethylcyclohexyl
hydrazinium chloride and a mixture of butene isomers from
dimethyl s-butyl llydl~Lilliunl chloride on refluxing the
hydrazinium salts with potassium t-butoxide in refluxing t-
butanol (J. Or~ h~nn 39, 1588, 1974).
While these rearrangements do not present any
filr~- 1 problem in the syntheses which are the subject o~
the present invention, they must be kept in mind in selecting
synthetic strategies and reaction con~itinnC for the assembly of
ligands via hydrazinium int~rm~ iqr~S bearing ~ rnt
3S groups which may be subject to these n~ side-reactionS-
28
SUBST~ T~ S~EET ~U~E ~6)
WO 95/18186 ~ 2 1 7 9 9 8 3 ~ Y~ 612
In some cases. it may be appropriate to select other
synthetic pathways for a specific assembly step involving such
a module. Alternatively, the reaction may be carried out usin~
mild reaction conditions to avoid any rearrangement
The required hydrazinium salts may be prepared
by routine alkylation of a l, l -disubstituted hydrazines or by
treatment of a tertiary amine with a haloamine (see 78 J. Am.
Chem Soc. 1211 (1956)). Alternatively, a tertiary amine may
be reacted with hydroxylamine-O-sulfonic acid (prepared by
the method of Goesl and Meuwsen; Chem. Ber., 92, 2521, 1959),
as shown:
R3N + H2NOSO3H R3N-NH2 Cl
This *action is carried out by reacting a suspension
of the tertiary amine in a vigorously stirred cold aqueous
solution of an equivalent amount of poths~iulll carbonate
sesquihydrate, cc ~ a small amount of EIDTA, with a cold
solution of an equivalent amount of hydroxylamine-O-sulfonic
acid in water, added over a I hour period. Methanol is added,
and the precipitated K2SO4 is removed by filtration. The
filtrate is adjusted to pH 7 with hydrochloric acid and the
solvent is removed on a rotary ~v~pul~tOr. The hydrazinium
salt is isolated by ~ alion from the thick glassy residue by
the addition of acetone.
H~ 1~,.7;.. ;.. salts, being chiral at nitrogen, may be
resolved, e.g., by treatment with a chiral acid followed by
separation of the di~ L.~ (e.g., using chromatography or
fractional cryst~lli7~tinn and the resulting enantiomers used in
stereoselective syntheses of ~minimirl-~s.
When one of the alkyl groups in a hydra2inium salt
is an ester group, the ester may be e~ronifi~d efficiently using
LiOH in a m xture of methanol and water, producing a useful -
29
S~I~ST~U ~ IEET (F~L~ 2~)
WO g5/18186 , ~ 217 9 9 8 3 r~ 2612
hydrazinium acid after neutralization of the reaction mix~ure
~ith an acid.
3 1. LiOH 3
R~ R MeOH-H,O R~ R
N+ ,COORI ~ . N~ ,COO.
S H,N CH, 2. ~ H2N CH,
X
Suitably protected hydrazinium carboxylates may
10 be used in condensation reactions to produce :IminimitirS,
Procedures analogous to those known to be useful in effecting
peptide bond formation are expected to be useful; e.g. DCC or
other carbodiimides may be used as cnn~ rlcin~ agents in
solvents such as DMF.
lS /
Rl NH R4 Rs Rl ~ N~ NHB
~)~ + N ' ,COOR 5~ N~
R2 R3 B~NH CH2 R2 R3 o R4 Rs
~~ ~
Alternatively, the hy~laLilliulll carboxylate units may be
coupled with alpha-amino-acids or with other nllcl~ophil~s,
such as amines, thiols, alcohols, etc., using standard techniques,
to produce tnr~ '5 of wide utility as ligand mimetics and
2S new materials for high technological applications.
The alpha-hyJI~Lilliu~l. esters may, in turn, be
produced by the alkylation of a l,l-~ bstit---~d hydrazine
with a haloester under standard reaction cor~ ionC~ such as
those given above for the alkylation of hydrazides.
R2 R; ~
N--NH + Cl CO2Me . ,N' ,CO2Me
2 ~ ~ H2N CH2
SU~ST~rU~t S~'ET (RUL~ 26)
WO95118186 ; ~ t; 2 1 7 9 9 8 3 PCTNS93112612
hydrazinium acid after neutralization of the reaction mixture
with an acid.
R~ R 1. LiOH R~ R
.Nr+ ,COORZ MeOH H,O Sl~ ,COo-
H~N CH2 2. r, ~ H2N CH,
X
Suitably protected hydrazinium carboxylates may
be used in condensation reactions to produce ~minimides
Procedures analogous to those known to be useful in effecting
peptide bond formation are expected to be useful; e.g. DCC or
other carbodiimides may be used as condensing agents in
solvents such as DMF.
o
D ~< + ~< ~COOR _ R~ N--Rs
RZ R3 B ~1 ~ Hz RZ2 R3 - X~3 NHal
I x
Alternatively, the hydrazinium carboxylate units may be
coupled with alpha-ami~ c or with other nucleophiles,
such as amines, thiols, alcohols, etc., using standard Pr hnirlllr 5
2S to produce mr~lPclllPs of wide utility as ligand mimetics and
new materials for high technological applir ~tir.)n~
The alpha-ll~dla~iZliull. esters may, in turn, be
produced by the alkylation of a I, I -disubstituted hydrazine
wiZ~h a haloester under standard reaction con~litir)nc~ such as
those given above for the alkylation of hydrazides.
R2 i~ RZ~
)i--NH2 + C CO2ME ~ ~<~CO2M3
31
SZ~æSTllTU~E SHZ-ET !~ZuZ E 26)
wo 95118186 - 2 1 7 9 9 8 3 PCTIUS93/12612
Alternatively, these hydrazinium esters may be produced by
standard alkylation of the appropriate alpha-hydrazino ester.
NH / XCO2R3 RJX NH2~ >~ 3
The required I, I-disubstituted hydrazine for the
above reaction may be obtained by acid or base hydrolysis of
the col.~J~ ding hydrazone (see 108 J. Am ('h~m Soc. 6394
(1986)); the alkylated hydrazone is produced from the
monosubstituted hydrazide by the method of Hinman and
Flores (24 J. Org. Chem. 660 (1958)).
~CH2OC--NHNHCI Co2R3 N OE ~CH2-o--c--N--NHc--co R3
H EtOH H
, 1 1 2 /2H
2S H2N H~C~co R3
The monosubstituted hydrazided required above
may be obtained by reduction of the Schiff base formed from
an alpha-keto ester and a suitable hydrazide. This reduction
may also be carried out stereoselectively, if desired, using
DuPHOS-Rhodium catalysis (114 J. Am (~h.-m Soc. 6266 (1992):
259 ~ 479 (1993)), ~ hown:
Sll~STITU~ S'~ T ~nULE 26~
2 1 79~83
W095/18186 ' !-` ' ` ' r~l~u~ 261~
H
,C=O + H,~i C~ R300C 1(- \~
- H~ N o ~ [Ph (El-DupHos)]
R200C, R- \~
H
~.
In a variation of the synthesis given above~ ome~a-
halo aminimides are prepared using an omega-halo acyl halide
(prepared by acylation of the trisubstituted hydrazinium
tosylate salt with a haloalkyl acyl halide, such as CICH2COCl)
lS These halo stminimid~s may be reacted with nucleophiles
c~nt~jnjn~ reactive hydroxyl, thio, or amino groups to give
~minimid~ derivatized molecules.
4.2.3 ~minimi-l~s via the Hydrazine-Epoxide-Ester Reaction
A very useful and Yersatile synthesis of aminimid~
involves the one-pot reaction of an epoxide, an asymetricall~,
~ ,h~ rd hydrazine, and an ester in a hydroxylic solvenl.
usually water or an alcohol, which is allowed to proceed usu~
2S at room temperature over several hours to several days.
R2
Rt_C~7H2 + N--NH2 + R~--COORs
R3
R2
- R1--CH--CHy ~ C--R4 + R50H
O H R3 o
3S
33
SU~S ~ iT~ S~ir~T ~ ' 26)
W09S/18186 ~ S 21 7q983 I~ Y,3112612
In the equation above. R I, R~ and R3 are selected from a set of
diverse structural types (e.~. alkyl. carbocyclic, aryl, aralkyl.
alkaryl or many substituted versions thereof). and R4 and R5
are alkyl,, carbocyclic. cycloalkyl, aryl or alkaryl.
The rates for the above reaction increase with
increasin~ electrophilicity of the ester component. Generally, a
mixture of 0.1 mol of each of the reactants in ~0-lO0 ml of an
appropriate solvent is stirred for the required period at room
temperature (the reaction may be monitored by thin layer
chromatography). At the end of this period, the solvent is
removed in vacuo to give the. crude product.
If Cut-stir~Pnt R4 of the ester co.l-~on~ in the
above aminimide formation contains a double bond, an
~minimirl~ with a terminal double bond results which may be
epoxidized, e.g. using a peracid under standard reaction
con~litionc, and the resulting epoxide used as starting rnaterial `
for a new Aminimirl~ formation. Thus, a structure cont~inine
two ~minimi(~-o subunits results. If the ~minimi~ formation
and eFn~itistiotl sequence is repeated n times, a structure
cl~nt~inin~ n ~minimirl~ subunits results; thus when R4 is
propene, n repetition of the sequence results in the structure
shown below:
R2 R~ '' O
2~ RloHcHz--N---N-- {Cll--CH2-CI11 CHl 11 --N--~ -CCHzC'H=CH2
OH R3 O OH R~ n
where the ~ Cigrstinnc R2 and R3 are used to illustrate the
manner in which the hydrazine s~lhstit- R2 and R3 can be
varied in each polymerization step to produce oligomers or
polymers of diverse structures. This is described in examples
which follow below.
A related ~minimit~ poly~-ri7~tinn sequence
utilizes an ester moie~y bonded directly to the epoxide group.
34
SUB~ E .~t-~ F 26~
Gr` ~ 2 1 79~83
W0 95/18186 ~ ! ` ` ' PC'rNS93/~2612
An additional related polymerization sequence
involves the use of bifunc~ional epoxides and esters of the
following form
CH~ /H (Y~ CH~C 2 d RO ~ fOR
O O O O
so as to produce polymers of the following structure (shown for
the case of reaction with l,l-dlmethylhydrazine):
O O R R2
{--C~ C N N ~--C H2--C H--~--C H--C H2 - l--N-- -
1~ R1 1H 1H 12
where X and Y are alkyl, .,~uI,o,,,~.,lic, aryl, aralkyl or alkaryl
linkers.
These polymers, which are produced by reacting
5tni~hiom~tric amounts of the reactants neat or dissolved in
lower alcohols or alcohol/water mixtures at temperatures from
20 C to 80 C have been utilized for the case where R = R' as
int~rm~ t~ thermally activated (>150 C) isocyanate
~.. ul~ul~ for use as crosCIirbin~ materials (cf. U.S. patents
3,565,868 and 3,671,473), herein crerifi~lly incorporated by
reference .
Another ~minimir~ synthesis is the reaction of 2,4-
dilliilùyhcllyl pyridinium salts ("Zinke salts") with
IIIU-- b, ~ . z , as shown below (US pat.
4,563 ,467):
3s
SU~ST~TUTE S~;,EET (R~LE 2~!
WO 95118186 ~ ' 2 1 7 9 ~ 8 3 PCTIIJS93112612
Rs
~R ~ ~NH2
~ N2 H
N02
' 10
R3~
R2 R, ~3
This reaction is carried out by heating equimolar
amounts of the pyridinium salt and the hydrazine with a slight
excess of triethylamine in an alcohol, such as ethanol, for 12
hours. The desired product is obtained from the precipitated
solid produced by extraction with iinY --~W~t~ removal of
the dioxane in vacuo, followed by ~ci~'r'--- with HCI,
filtration to remove salts and neutralizetion with NaOH. The
2S mixture is cooled, the crystallized product is collected by
filtration and purified by recryst~lli7~tio~ from ethanol (yields
65-85%). Although this ~eaction suffers from the disadvantage
of requiring the removal of the dinitro analine by-product by
extraction, it is very useful in forming ~;di"iu... ~minimi~lP
C ' ' ~ m~ 5 co ~ ~tes, mono=~r~ .od sc~ffolds.
SIJBST~ ,' U~ Et~ ~RULE ~&~
wo 95/18186 ~ 2 1 7 9 9 8 3 PCT/l~S93112612
4.~.1 Synthesis of Enantiomerically-Pure Aminimides
Enantiomerically-pure aminimides may be
produced by acylation of chiral hydrazinium salts as shown in
the example below.
~ C'H5C02CH3 C ~C ~
Chirally-pure hydrazinium salts may be obtained
by resolution of the racemates; resolution can be effected by
lS forming salts with optically pure acids, e.g. tartaric acid, and
separating the resulting diastereomers by means of
chromatography or fractional crysrAlli7q.~inn (see, e.g., 103 L
('h~m Soc. 604 (1913)); alternatively the racemic modification
is resolved by subjecting it to chromatographic separation
using a chiral SrA~ chromatographic support, or if
feasible, by the use of a suitable enzyme system.
Enantiomerically-pure qminimi~ps may also be
obtained by resolution of the racemic mn-lifi~qti~ - using one
of the tP~hniq~ $ described above for the resolution of racemic
h~dlaLilliu~LI salts (for an example, see 28 J. Or~ Ch~m 2376
( 1 963)).
An q~l~irionql approach to the synthesis of chiral
qminimirlPs involves chiral synthesis; an example is provided
by the reaction of (S)-(-)-propylene oxide with I, I -
dimethylhydrazine and methyl-(R)-3-hydroxy-butyrate, all of
which are com~nercially available.
37
SUB~ ur,- SHE~T ~R~ ~ 2~'
Wo 95/18186 ; P~l/u~ 2612
3~ + ~N--NH, + HO,"~ CH3
S 3H``` i N+ ~CH3
HO H3C CH3 o HO H
A variety of chiral epoxides, prod,uced by chiral
epoxidations such às those developed by Sharpless (Asvmm. .
~L, J.D. Morrison ed., Vol. 5; Ch. 7 + 8, Acad. Press, New York.
N.Y., 1985), and chiral esters, produced by standard
plV~.~dUI~,S, may be used to produce a wide variety of chiral
aminimides .
lS Chirally-pure aminimi~lP molecular building block-s are
especially preferred since they can be used to produce a vast
array of m~lPclllps useful as new materials for high
technological applications and as molecular recognition agents.
including biological ligand mimetics which can be used as
drugs, ~liaEnoStics. and separation agents.
4.4 Synthesis of Specific Classes of ~minimi~iPS
4.4.1 Synthesis of Chiral ~minimi~ip-cont~inin~ Conjugates
2S ~he synthetic routes outlined above may be utiliz~d
to produce a wide variety of chiral aminimi~iP conjugates of ~h~
following general structure:
o Rl
A--X--C--N--N+-Y--B
R2
3S
38
SligS~ !E~T ~F~UI ~
~ - l ` ' 21 79983
WO 95/18186 PCT/U593/12612
The substituents A and B may be the same or different and
may be of a variety of structures and may differ marked~y in
their physical or functional properties, or may be the same;
they may also be chiral or symmetric. A and B are preferably
S selected from:
I ) amino acid derivatives of the form (AA)N.
which would include, for example, natural and synthetic amino
acid residues (N=1) including al~ of the naturally occuring alpha
amino acids, especially alanine, arginine, asparagnine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, Iysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine; the naturally occuring
disubstituted amino acids, such as amino isobutyric acid, and
isovaline, etc.; a variety of synthetic amino acid residues,
lS 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-
Sllhstitllt~d glycine residues; natural and synthetic species
known to fi-nrti~ -lly mimic amino acid residues, such as
statine, bestatin, etc. Peptides (N=2-30) COIIaL~ ,t~,~ from the
amino acids listed above, such as ~n~ ngcl~ and its
family of physiologically important angiotensin hydrolysis
products, as well as d~,.iv~ti-~s, variants and mimetics made
from various combinations and p. ~ of all the natural
2S and synthetic residues listed above. Polypeptides (N=3 1-70),
such as big endothelin, parl.,lca~l~tin, human growth hormone
releasing factor and human pancreatic polypeptide.
Proteins (N>70) including structural proteins such
as collagen, functional proteins such as hemoglobin, regulatory
30 proteins such as the dopamine and thrombin receptors.
2) a tl~CI~O~ derivative of the form (NUCL)N,
which includes natural and synthetic nl~rl.-oti~i~s (N=l) such as
z,i.~.nsin~, thymine, gll~ni~ir-, uridine, cystosine, derivatives
of these and a variety of variants and mimetics of the purine
3S nng, the sugar ring, the Fh- ~ ' linkage and combinations of
39
SUB~ JT~.E~ ~UL~263
i, ~ i `, 2 ~ 79983
W0 95/18186 ~ Y~ 612
some or all of these. Nucleotide probes (N=2-~5) and
oligonucleotides (N>'5) including all of the various possible
homo and heterosynthetic combinations and permutations of
the naturally occuring nucleotides, derivatives and variants
containing 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 ) a ~albo~lydlate derivative of the form (CH)n.
This would include natural physiologically active carbohydrates
such as including related cu~ ol '- such as glucose, galactose,
sialic acids, beta-D-glucosylamine and nojorimycin which are
both inhibitors of gi ~osidqc~, pseudo sugars, such as 5a-carba-
2-D-gal~ u~ ai~ose~ which is known to inhibit the growth of
Rl~bci~ pn~umrniq. (n=l), synthetic carbohydrate residues
and d,,l;~ati~.,s of these (n=1) and all of the complex oligomeric
pl~rmU~qrior~C of these as found in nature, including high
mannose oligosqc c i~q~id~5, 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,
2S such as having a c~, r ' ,y structure to an enzyme, for
insîance. This term includes any of the well known base
structures of pharmaceutical compounds including
r~ --G~.hores or metabolites thereof. These include beta-
lactams, such as p~nnirillin known to inhibit bacterial cell wall
biosynthesis; ~ 7~ ' 5, 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 bindirlg properties
to ligand acceptors.
3~
.
SU~ST~UTE SHE~T (RULE 2~)
2 1 799~3
WO 9SIIR186 PCI'IUS93112612
5 ) a reporter e!ement 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 aminimide structure or
reaction scheme and may be attached through the groups
without adversely interfering with the reporting functionality
of the group Preferred reactive groups are amino, thio,
hydroxy, carboxylic acid, 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 filr~tinnqiities capable of
undergoing condensation polymerization or copolymerization
Suitable groups include vinyl groups, oxirane groups, carboxylic
acids, acid chlorides, esters, amides, lactones and lactams.
Other organic moiety such as those defined for R and R! may
also be used.
7 ) a macromnlpclllqr c~ nl~ l such as a
macromolecular surface or structures which may be attached to
the qminimi~ir 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 i~ activity of the attached f -~ lity is
d~tr~mir~^d or limited by the macromolecule. This includes
porous and non-porous inorganic .lla.,.~ -Iccular cv...l,on~nts.
such as, for example, silica, alumina, zirconia, titania and the
like, as commonly used for various applirP~in-~c such as normal
and reverse phase chromatographic separations, water
~ -rqtion pigments for paints, etc.; porous and non-porous
organic macromolecular c~ ,on...l~, including synthetic
co."po tc such as styrene-divinyl benzene beads, various
methacrylate beads, PVA beads, and the like, commonly used
for protein pllrifirqtinn water softening and a variety of other
applications, natural ~,u~ O.~_uL~ such as native and
fllnrtionqli7rd c~ ns~s, such as, for example, agarose and
3~ chitin, sheet and hollow fiber membranes made from nylon,
41
SU~E i ~ 26)
;` . ' ~' '' 1 ',~, 2 ~ 79983
WO 95118186 PCT/US93112612
polyether sulfone or any of the materials mentioned above. The
molecular weight of these macromolecules may range from
about 1000 Daltons to as hi8h as possible. They may take the
form of nanoparticles (dp=100-lOOOAngstroms ), latex
particles (dp= I 000-5000Angstroms), porous or non-porous
S beads (dp=0.5-1000 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, a hydrogen atom, an organic moiety which
contains a suitable electrophilic. group, such as an aldehyde,
ester, alkyl halide, ketone, nitrile, epoxide or the like, a suitable
nucleophilic group, such as a hydroxyl, amino, carboxylate,
amide, carbanion, urea or the like, or one of the R groups
defined below. In addition, A and B may join to form a ring or
lS structure which connects to the ends of the repeating unit of
the COIll~Ol ~ defined by the preceding formula or may be
separately corln~ct~d to other moieties.
A rnore generalized structure of the comrocition of this
invention is defined by the following formula:
Rl...n
A--X ~ CO--N--N~-GI n~Y--B
2S ~ Rl. 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
30 cOmpounds;
b. X and Y are the same or different and each
~ JICS~ a chernical bond or one or more atoms of carbon,
3S nitrogen, sulfur, o~cygen or comhin~ - thereof;
42
S~TI ~ LE 26)
WO 9~;/t8186 2 1 7 9 ~ 8 3 PCI'IUS93/12612
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 s~bsti~lt~od 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, methyl, ethyl, n-propyl, iso-propyl, n-but~ l.
iso-butyl or tert-butyl; upper alkyl, for example, cotyl, nonyl,
decyl, and the like; lower alkylene, for example, ethylene,
propylene, propyldiene, butylene, butyldiene; upper alkenyl
such as l-decene, l-nonene, 2,6-dimethyl-5-octenyl, 6-ethyl-
5-octenyl or heptenyl, and the like; alkynyl such as l-ethynyl.
2-butynyl, l-pentynyl and the like. The ordir~ary 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 ~ - r l~ in which one or more hydrogen atoms h~
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 ~ ~,,lil..~. d alkyl groups can be. t(lr
example, alkoxy such as methoxy, ethoxy, butoxy, pentoxy ~n-l
the like, polyhydroxy such as 1,2-di~ ytJlu,u~l, 1,4-
dill~LUAy-l-butyl, and the like; methylamino, ethylamino.
dimethylamino, diethylamino, triethylamino, cyclopentylamino.
benzylamino, dib~,lLyla~illo, and the like; propanoic, butanoic
or pentanoic acid groups, and the like; fnrm^~nitln, ?~et~mi~r!.
bl-t~n~mi-1r. and the like, methoAy.,a.l.ullyl, clllvAy~,~L.onyl or
the like, chl~lur~llllyl, bromoformyl, l,l-chloroethyl, bromo
43
S~lBST~Li~ SHEE ~ (RULE 26)
wo95/18186 ' `- ` 21 7q983 r~ s~ 6l2
eth~ l ,and the like. or dimethyl or diethyl ether groups or the
Iike.
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 s~lbstit~-~nts 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 above.
The cyclic groups of the invention may further comprise a
heteroatom. For example, in a specific embodiment, R2 is
cycohexanol .
As used herein, substituted and unsubstituted aryl
groups means a hydlu~,al bUII ring bearing a system of
conjugated double bonds, usually comprising an even riumber
of 6 or more (pi) electrons. Examples of aryl groups include,
but are not limited to, phenyl, naphthyl, anisyl, toluyl, xylenyl
and the like. According to the present invention, aryl also
includes aryloxy, aralkyl, aralkyloxy and ~t.,.u~l groups,
e.g., pyrimidine, morpholine, pi~ a~ C, pirPri~iinr, benzoic
acid, toluene or thiophene and the like. These aryl groups may
also be s~bstirlltPd with. any number of a variety of functional
groups. In addition to the functional groups described above in
connection with s~bs~it~ d alkyl groups and carbocylic groups,
functional groups on the aryl groups can be nitro groups.
As mPntiûnpd above, R2 can also represent any
comhirPti- L of alkyl, Laubu~ ,lic or aryl groups, for example, 1-
cyclohexylpropyl, benzylcyclohexylmethyl, 2-cyclohexyl-
propyl, 2,2-methylcyclohexylpropyl, 2,2methylphenylpropyl,
2,2-methylphenylbutyl, and the like.
d. G is a chemical bond or a connecting group
that includes a terminal carbon atom for ~t-PrhmPnt to the
~!u~t~,lualy nitrogen and G may be different in adjacent n units;
and
44
SUF~T~ EI ~RULE~6!
WO 9S/18186 i ! ~ ` 2 ~ 7 ~ ~ & 3 F~ 111612
d G is a chemical bond or a connecting group
that includes a terminal carbon atom for attachment to the
quaternary nitrogen 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 ~tr~-~hm~n~ to the quaternary
nitrogen; and if n is 1 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. Also, when A is a sl~hstit~ d benzene ring, the meta position
will not be s~hs-ir-lt~d with an So2NH2 group when n =1, X is a
C-C bond and R and R' together form a trimethyl suhstitl-tPd
pyridine ring.
In one embodiment of the invention, at least one of
lS A and B l~t,l.,i.. ,.. ~ an organic or inorganic macromole~ular
surface. Examples of preferred macromolecular surfaces
include ceramics such as silica and alumina, porous and
nonporous beads, polymers such as a latex in the form of
beads, membranes, gels, Il.a.,.uscu~.:c surfaces or coated
versions or composites or hybrids thereof. This functionalized
surface may be ~ s~..t~d as follows:
Rl O
Il
(SURFACE)--Y--Ni +-N--C--X--A
R2
In a further emhs~im~nt of the invention, the
above roles of A and B are reversed, so that B is the svbstitl-l~nt
selected from the foregoing list and A l~ ,5~ a
fllr^ti "-li7~d surface, as shown below:
O Rl
(SURFACE)--X--C--N--N+-Y--B
SU~T~TUT~ ' T (RULE 263
Wo 95118186 . ' '', 2 1 7 9 9 8 3 PCI/U593/12612
In a third preferred embodiment of the invention.
either A, B, or both contain one or more double bonds capable
of undergoing free-radical polymerization or copolymerization
. to produce achiral or chiral oligomers, polymers, copolymers
etc
Another embodiment of the invention relates to a
composition having the structure:
R
A--Y--G~ W
R~ ~3
lS wherein A, Y, R, Rl and G are as defined above and
W is -H or -H2X- where X- is an anion, such as a halogen or
tosyl anion
Yet another aspect of the invention relates to a lipid
mimetic composition having the structure
,Q I
Q--N~-N--C--Q
Q
2S wherein Q is a chemical bond; hydrogen; an electrophilic group:
a n~lrl~ophilir group; R; an amino acid derivative; a nllrl-otiri
derivative; a c~.l,Ghyd.ale d.,.iv~lti-~,; an organic structural
motif; a reporter element; an organic moiety co~inin~ a
polymerizable group; a macromolecular cc....~orn,..~; or the
~ X(T) or X(T)2; wherein R is an alkyl, carbocyclic,
aryl, aralkyl or alkaryl group or a s~bs~it--t-~d or heterocyclic
derivative thereof, and T is a linear or branched llyd-uc~
having between 12 and 20 carbon atoms some of which are
optionally ~ hsl;~ with oxygen, nitrogen or sulfur atoms or
3S
46
SU~T~TUTE SHEET (RULE 26)
.- `.i` i 2179983
wo gS/18186 PCTlUSg3~12612
by an aromatic ring; and provided that at least ~wo T
substituents are present in the structure of the composition
In the description that follows, Rn where n is an
integer will be used to designate a group from the definition of
R and Rl.
Another aspect of the invention relates to
functionalized polymers having the structure:
SURFACE) RI ' ~ R2 R2~1 1
CHCH2 ~ N '--N--fi--x--fi---N - N C H2 CH--Y - CH--CH2--N--N--C X CO,R
OH Rl R2 OH OH RZD~I o
,n
or
P~l ~2
(SURFACE~ c--~ N - ~1 CH~ Cl --Y - CIH--CH2 -1` ~--N---ICI - X - C- ~--OR
o ~ ~, OH OH ~ 2 o ~ n
wherein
a. X and Y are ¢ groups;
b. Rln or R'ln (where n = an integer) each
represent alkyl, cycloalkyl, aryl, aralkyl and alkaryl;
c. (SURFACE) is a .. ac.~ r CU.
and
d. n is equal to or greater than 1.
These functionalized polymers may be made
according to known techniques for attaching terminal groups to
the surface or by attaching a monomer unit to the surface and
then building the polymer, for instance.
The invention also ~ ".r~c~r s various methods of
producing an ~minimitlP-functional support. One method
3S c.. ~ the steps of reacting a polymer or oligomer
47
SU~ST~UTE SHEET (RULE 26)
: `;`; `, ' S 2 1 7~983
WO ~5/18186 f ~ 3/1~612
containing pendant moieties of OH, NH or SH with a compounJ
of the formula:
S Cl--CH~--C-- N--r ~I~R3
Il R2
wherein-RI and R2 each represent alkyl,
carbocyclic, aryl, aralkyl or alkaryl, and R3 is an amino acid
derivative; a nucleotide derivative; a carbohydrate derivative:
an organic structural motif; a reporter element; an organic
moiety containing a polymerizable group; or a macromolecul~r
component; coating the reacted polymer or oligomer onto a
lS support to form a film thereon; and heating the coated . support
to crosslink the film.
Another method cr,...l,..c~s the steps of coating a
mixture of multifunctional esters and multifunctional epoxides
onto a support to form a film thereon; and reacting the coated
support with I, I '-dialkylhydrazine to crosslink the film.
A third method comprises the steps of coating a
mixture of an ~minimirl~-functional vinyl monomer, a
difunctional vinyl monomer and a vinyl polymerization
initiator onto a support to form a film thereon; and heating th~
2~ coating support to form a crosslinked film.
The ~minim~ functionalized support prepared
according to the previous methods are another aspect of the
invention.
The ability to derivatize an ~minimirl~ scaffold in
numerous ways using the synthetic techniques outlined abo~
as well as those given below, offers a vast array of structure~
capable of ,~ ; specific molecular entities via
~5t~hlichm~nt of specific types of binding interactions. Thus
the ~minimiri~o shown below is in principle capable of
establishing the following interactions~ stacking involving
48
S! )~ TE ~ T (RULE 26)
WO9S/18186 ;'" '~ 21 7~q8~ Pcrn~ss3JI26l2
the phenyl group; hydrogen bonds: acid-base interactions
involving the anionic nitrogen: salt bridges involving the
quaternary nitrogen: steric interactions with the bulk~
isopropyl substituent: and hydrophobic interactions involving
the hydrocarbon chain.
Chiral Recognition
"Chiral recognition" is a process whereby chiral
enantiomers display differential 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 dia~ l.,.ic complexes with the racemic
target. These complexes have differing phycioch~rnic~l
propereties which allow them to be separated using standard .
unit processes, such as fractional crystallization.
Two steps are necessary for the recognition process
to occur with a CSP; 1.) absu.~liù~ and 2.) energetic
differentiation between the en~n~iom~rs. The absolute binding
energies between the enantiomers and the surface determine
the tightness of the binding. The dirr.,.~ e in energy between
the complexes d~f~mi- the sele~,livily. This is represented
in the following diagram
2S The ;- t .,~l;u;~ of the ~n 'rnn~rir R and S species
with the CSP can be envisioned as a "three point interaction".
This does not mean that three actual points of ~ t or
~9
S1!e~T~Tl~ F ~- (RULE 26)
t , 2 1 7 9 ~ ~ 3
WO 95/18186 I ~ 612
Energy
... .---- -.. ----- Energy o~ F( r,nd S Isomers
S
i~
R-R Comple:~
/\ '
~ R-S Comple~
lS a
slcSorir~inn 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
2S species. The larger the number and the more varied the types
of these interactions, the greater the resulting energy
.lirr.,..,.lccs between the complexex and the greater the degree
of "recognition" per interaction.
.
3S
SU~ LITESi~rI (~LILE26)
wo95/18186 , ~;s ~ ~ 2 1 7q983 ~ Y~/l26l2
"Three point interaclion
NO2 ~ H u
NO2
The possible modes of interaction which can participate in such
"three point inyteractions" is depicted below for a
enantiomerically pure ~minimi~
H-bonds
~O~¢
N~ Stenc
Ch / ~ CH~CloH~3
1 ~
Charge HYdlU~IJ~I,;C
As a further example, possible interactions between
35 a recognition target and a ~pecific supported ~minimitl~ are
51
SUBSTlTUTt ~HEET (RULE 26~
wo g~/18186 ' ;, . r ~ 2 ~ 7 9 9 8 3 PCI/U593/12612
sho~ n below. Experimental procedures for the synlhesis of
specific chiral aminimides are given below.
S Cbarge/
[~ Acid-Base
`~ ~ ~5
H o H3C CH3 H OH
lS H-bonds
3S
52
SUB~ ~ IT~Tt S~ T (RU~ E 2~,
. ~. . . 2 1 79983
W095118186 r_l~U:~Y3/12612
.
Sequential Catenation of Aminimide Subunits Producing
Sequences of Various Sizes
By choosing aminimide building blocks possessing
S functional groups capable of establishing predictable binding
interactions with target molecules, and by using synthetic
techniques such as those broadly described above to effect
catenation (linking) of the building blocks, it is. possible to
construct sequences.of ~minimid~P subunits mimicking selected
native oligomers or polymers; e.g.l peptides and polypeptides.
oligonucleotides, ~albûhydlates, as well as any other
biologically active species whose three dimensional binding
geometry can be mimickPd by various combinations of
~minimid~P-containing scaffolds and side chains. This may be
lS accnmrliehPd using a wide variety of side chain recognition
group substituents including, but not limited to, the
sll~stitllPnts found in the side chains of naturally occuring
amino acids; purine and pyrimidine groups, as well as
de.iv~ ,s and variants of these; natural and synthetic
câlL.Ot~yd~al~ 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 specifi~
2S activities. These moieties 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 ~nri Inr~lly tuned to rl ~i -lly mimic the natural
scaffolds found in peptides, proteins, oligonucleotides or
carbohydrates; or which can simply serve to array sequences
or cu...bil.à~ions of these side chain recognition groups in an
appropriate structural rel~tionehirs to the scaffold and to e~ch
other to produce species with highly specific and selective
activity. In addition, because of the inherent hydrolytic and
3~ enzymatic stability and solllbili7in~ ~IU~ s of the
53
SU~S i ~ ' E ~ 26 ~
wo 95/18186 ~ ` 2 1 7 9 9 8 3 PCTiUS93/12612
properties of the aminimide linkage. these designed functional
molecules will have better stability and pharmacokinetic
properties than those of the native species. The integrated
modularity of the chemistries allows the construction of this
wide variety of molecules to be carned out in a manner
analogous to the design of an electronic device by combining
component subsystems using a relatively smal~ number of
interchangab~e reactive modules and protocols. This is
figuratively outlined below.
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
~he scope and spirit of the present invention. Hence, the
invention should not be limited by the description of the
specific emb~di-ll.,...~ disclosed herein but only by the folowing
claims.
S4
SU~ ~ LT (~iiE 2~`
WO 95/18186 2 1 7 9 ~ 8 3 PCT/13593~12612
¦ MODULAR DESIGN AND ASSEMELY FLOW CHAR~
S
RECOGNITION
MODULE
'Carbohydra~e moiely.
Pynmidin~ moiety, etc.)
. RECOGNITION = SUBUNIT
. MODULE ASSEMBLY
CONNECTED TO
SPACE~ MODULE
SPACER
MODULE L~ntVll~lJ
(with o~thagonal ~ ' RECOGNI~ON
reacdvitdes) BUILDNG BLOCK
r ''~,~IIT .F
lS
AMINIMIDE~
SUBUNIT FORMING CATENATION
ASSEMBLY = (monoalkyl hydraz~ne PROCESSES
e-g-) PPflTOCOl.C
M~IETIC/ ¦
AGENT
~ = MODULAR COMPONENT
2S
The generic concept is illl-~tr~t-~d below for the introduction of
a generic "base" (purine or pyrimidine) into an aminimi~f~
scaffold as a hydrazine equivalent conn~ct~d Yia a spacer.
While the example uses a base as the recognition group, it
should be kept in mind that this group could e4ually well be a
carbohydrate, a pharmacophore moiety or a designed synthetic
recognition element.
3~
S~STITiJ~ Si~E1 ~g~.E 2~!
W0 95/18186 ' ~ t 7 q ~ ~ 3 I ~ tY~3/126I2
SP.~ÇER MODULE
Base + ~7 Base-CH~CH20H TsOH Base-CH CH~OTs
~) CH3NHNH'
S RECOGNITION
MODULE ~ A~IINIMIDE
FORMING
OH ~)' NH9 ' 3 ~N NH INTERi~lEDl.~TE
Bas~J Base~ '~
- BUILDING
X` /~ BLOCK
COOCH3 / cATENATloN MODULE
PROCESS
H3C~
N--NH2
J--N~X~ Bw~ Bas~J o OH ~H
~" etc.
~ etc.
2S MIMETIC
Specific syntheses of multisubunit aminimi~s are outlined
below:
4.4.2.1 C~ n~ ~. of ~minimit~ Subunits
via Acylation/Alkylation Cycles
56
SUBST~; IJ i-t ~i~t~T iRULE ~6)
wo 95/18186 , 2 1 7 ~ ~ 8 3 PCI'IUS93112612
The following steps are involved in this synthesis:
1. Acylation of a chiral hydrazinium salt.
prepared as described above. with a molecule capable of
functioning both as an acylating and as an alkylating agent
producing an ~minimi~ BrCH~COCI and other bifunc~ional
species, such as bromoalkyl isocyanates, 2-bromoalkyl
oxazolones, etc., may be used as acylating agents under the
reaction conditions given above.
2. Further reaction of the product from the
above reaction with an asymmetrically disubstituted hydrazine
10 to form a diastereomeric mixture of aminimi~lP hydrazinium
salts under reaction conditions similar to those described
above .
3. Isolation of the diaa~ .v.~ a produced in
Step 2 as described above, e.g., by fractional crystallization or
by chromatography using techniques familiar to those skilled
in the art.
4. Acylation of the desired di,lD~.co...c. from
Step 3 with a ~.r ~;cr-l acyl derivative âimilar to those listed
in Step 1 above producing a dimeric type structure.
5. Repetition of Steps 2, 3 and 4 the required
number of times to build the desired ,tminimitlP subunit
sequence.
6. Capping of the assembled sequence, if desired,
for example, by reaction with an acylating agent, such as acetyl
2S chloride.
The ~ tlt~l conditions (e.g. reaction-solvent,
temperature and time, and purification p.-,cedu.cD for
products) for all of the above reactions were described above
and are also well-known and practiced in the art. As the
molecular weight of the products increases (e.g. in step 5
above) solubility and reaction-rate problems may develop if
the reactions are run under the cor~liti~nc that successfully
gave products of smaller molecular weight. As is well known
from the art of peptide synthesis, these problems are probably
3S due to cour~ l (folding) effects and to a~;-c~,ltio~
57
~3ST!TUTE SHEET (RU~E 26)
WO 9~/18186 PCTNS93/12~12
phenomena. and procedures found to wo}k in the related
peptide cases are expected to be very useful in the case of
aminimide catenations. For example. reaction sol-ents such ~s
DMF. or N-methyl pyrollidone. and chaotropic (aggregate-
breaking) agents, such as urea, are expected to be helpful in
alleviating reactivity problems as the molecular-weight of the
product increases.
0 ~ R n " " " ~ N~ CH~0
4.4.2.2 Catenation of ~minimidP Subunits via
Alkylation/Acylation Cycles
lS
The following steps are involved in this synthesis;
rxrPrimPnt~l ~or' onc for running the reactions are similar to
those given for the cu..c,~ liug steps in the above catenation
scheme .
1. Alkylation of .an asymmetrically disubstituted
hydrazide, prepared as outlined above, with a molecule capabl~
of functioning both as an alkylating and an acylating agent to
form a racemic mixture of ~minimirlPs as before the use of
BrCH2COCI is shown below, but other bifunctional species, such
2~ as bromoalkyl isocyanates, 2-bromoalkyl oxazolones, etc. ma~
also be used.
2. Reaction of the racemate from above with an
asymmetrically dic~lbstit~tPd hydrazine to form the hydrazid~
3. Resolution of ttte racemic modification from
th~ previous step as described above.
4. Alkylation of the product from step 3 with
bifunctional molecule capable of alkylation and acylation,
which may be the same as that used in step I or different, to
form a mixture of dia~ minimi~lPs
58
SUBSTITUT~ SH~ET (RIJ~E 26)
,~ ~ ! ' 2 1 79983
WO 9511R186 ' r~ 1~t'~Y.~ 612
5. Reaction of the diastereomers from step ~
with a suitable asymmetrically disubstituted hydrazine to form
the diastereomeric hydrazides. as shown:
o
3~ 'N "`~ ~N ~CH,~ N N~ R7
Rs o
R3~ N+~N ~CH21~N_N-R
lS 6. Separation of the diaa~ ,o~ as described .
above .
7. Repetition of steps 4, 5 and 6 to build the
desired sequence of ~minimiriC~ subunits.
8. Capping of the sequence, if desired, using e.g.
20- methyl bromide to produce a sequence such as shown below.
R3~N'N; R; ~ N~N+ CH
4.4.2.3 Catenation of .Aminimi-lt- Subunits Using
HytllaLillolysis of an Ester in the Presence of an
Epoxide
The following steps are involved in this synthesis;
e~cperimental ct~ c for running the reactions are given
- above.
1. Formation of an ~ F from the reaction
35 of an 1,1-, ay Lically liCllhstit~lt~od hydrazioe with an
59
Sl3B,ST~T,U' !~:_ SHE,~T ~ L.~ 26)
WO95/18186 ~`' '~ ~' ('! '~- r ~ 2 1 79 983 P~ Y~/I2612
epoxide; the reaction is illustrated for a chiral epoxide below
(the chiral epoxide may be obtained by e.g. a Sharpless
epoxidation):
\~7 + 3,N--NH2 ' ~ -NH Rl ,NH
The AminiminP. is normally not isolated, but used directly for
the following reactioh.
2. The Aminimi~P is reacted with an ester-
epoxide to give an A~ ; for the mixture of
diastereomeric Aminimi~PS above and the ester-epoxide shown
below, the following is obtained.
' H2C
H~R ''R~ ~ R~R, R~f ~r
+ ~ C02Me
~--N,, H`~--`
3. Separation of the diaD~ c~ .ic aminimitlPs
as described above.
4. Reaction of the desired dia~t~ ---e~ic
Aminimiti~ with an a~yllll-.ctlically ~iicllbsti~l~tpd hydrazine to
30 form diastereomeric ~minimi~ Aminiminps
3S
SUBSrlTUTE SHEET (RULE 26)
79983
WO 9~i/18186 ~ PC'r/US~3J~2612
Rl +N,N~ U R
S OH R2 O
+
~-N-" 3~ A~ H
S R~petitinn of steps 2, 3 and 4 above
using the appropriate hydrazines and epoxy-esters in each step
lS to produce the desired aminimitl~ sequence.
6. "Capping" of the final sequence, if
desired, by acylation with a simple ester, such as methyl
acetate, to produce the designed ~minimid~ ligand shown:
H~--'N~ 3~ " 3
4.4.2.4 Catenation of alpha-Hyd~ illiulll Esters
or Carboxylic Acids
The following steps are involved in this synthesis;
c~ 1itinn~ for running the reaction are given
above.
l. Treatment of a chirally-pure hydrazinium salt
(produced as described above) with a strong base, such as
3S NaOMe in an alcohol solvent, to form the imino ~nion:
61
S~BSTITUTE S~IEET (Rl ILE 26)
wo 95/18186 2 1 7 ~ 9 ~ 3 r ,/~J~Y3/1~612
-"R3 X ~ R3
R2 R2
S 2. Addition of an Alpha-Hydrazinium ester
(again produced as discussed above) to an appropriately
blocked imino-anion-containing mixture from step I to form
the hydrazinium-Rminimide. as shown.
0 ~ NH
\(3N/ ~\ ~ NH~, ~ N ~ 01 F's
lS
In the equatlon above, B I IS an appropriate
protecting group such as BOC (~-butyl carbamate), particularly
suitable for this purpose, readily cleaved by acid hydrolysis;
2,4-dichlorobenzene carbamate, cleaved by acid hydrolysis, but
more stable than BOC; 2-(biphenylyl)isopropyl carbamate,
cleaved more easily than BOC by dilute acid; FMOC (9-
fluorenylmethyl carbamate), cleaved by B-elimination with
base; isonicotinyl ca~ e, cleaved by reduction with zinc in
acetic acid; I-adamantyl carbamate, readily cleaved by
2S trifluoroacetic acid; 2-phenylisopropyl carbamate, cleaved by
acid hydrolysis but slightly more stable than BOC; imines and
erqmin.oS, readily cleaved by acid hydrolysis; mono and bis
trialkylsilyl derivatives, cleaved by heating in water or in the
presence of fluoride ion; phosphinamides and some
30 sulfenqmid~os. which are cleaved by mild acid hydrolysis; and
alkylsulfonamides, cleaved by strong acid hydrolysis.
3. Removal of Bl followed by repetition of steps
I and 2 the required number of times to obtain the desired
3S ~minimirl~ sequence, followed by a "capping" step, using a
simple ester as acylating agent.
62
SllBSTITl~TE SHEr~T ~ i' E 2~)
WO 9S118186 ~ ; ' C 2 1 7 9 9 8 3 pCTlUS93/12612
~ N
Alternatively, the alpha-hydra2inium carboxylic
acids may be obtained by treatment of the esters with LiOH in
10 MeOH/H20 at room tc~ lul~, as described above, and
coupled with each other using~ con~nC~tir)n reactions promoted
by DCC or other agents. Protecting groups used in traditional
peptide synthesis are expected to be useful here as well.
lS An alternate strategy is to catenate seq~l~on~es of
substituted hydrazides to obtain ligands with the desired side-
chain sl-hstitl-tir~n patterns, and s~bsrq~ -ly convert all of the
hydrazide groups to ~minimi~ s by multiple cimlllt:~neous
alkylation followed by neutralization. This approach, which is
outlined below, does not allow ~t~l~ o~ 1 control of the
chiral center and, as a result, each :~ ninimi~l~ center forrned
will exist as a racemic mixture. However, the hydrazide
oligomers themselves may, in fact, serve as useful binding
ligands.
2S
-
3S
63
SU~ST~TUTE SHEET (RULF 26~
i.,l; :c 217~83
W09!5/18186 ' - ' PCI/US93112612
R DCC R
ACO2H + H2NI~ ~ ACON~
co2su-t COz5u-t
¦ trifluorod2cetic 4
R
H2N~_
R COzBu-t R
ACONH~ R ~ ACONH~_
CONHN~ C02H
\--COzBu t
0
acid
tt
~ cozsu-t
lS etc., etc.
1 H2NNI~B
20 { } b~-e { st ~ O }
Re~l~,O~ examples of ilccrn~hl~e of a
hydrazinium-based scaffold via iterative hydrazide
2~ homologation and i~Lh,~ alkylation are set forth in
examples below.
4.4.3 Synthesis of ~minimidP-Containing
Peptides and Proteins
~minimid~ subunits may be inLIotl~ d into any
position of a polypeptide via chemical synthesis, using one of
the procedures outlined above, i~cluding the techniques for
dealing with ~ubh,~ tic reactions of high molecular weight
species. The resulting hybrid ~ l~c~lrs have improved
properties over the native molecules; for example, the
64
SUiB~TlTUTE SHE~T (RULE 26)
` "` ~ 2 1 7~83
W0 95~18186 P~~ YJII26IZ
aminimide group can confer greater hydrolytic and enzymalic
stability to the hybrid molecule over its native counterpart.
As an example of a synthesis of an aminimide-
modified peptide, the modification of a peptide attached to a
Merrifield solid phase synthesis support by alkylation with
5 aminimide-containing molecule is shown below.
~:x" ~
If moiety B contains a functional group which can
be used to link ~ ition~l ~m;nimi~i~o and natural or unnatural
amino acid subunits, e.g. via acylation reactions, complex
hybrid ~llu~,~u~,s may be obtained using the .oYr.~rim~r~l
15 procedures outlined above.
~ o$R~ $~
20 4 4 4 Synthesis of Oligv~ rv~ Mimetics
As discussed previously, much attention has been
focused on the construction and application of molecules which
possess the property of binding to nucleic acids. In the course
of work in this area, a great amount of knowledge has been
2S amassed vi~-a-vic 1.) the ability of a synthetic scaffold to
support a series of natural or designed bases in such a manner
that tight binding to natural nucleic acids is observed; 2.) the
ilCll.CllL~ for designed or naturally occuring bases other
than guanocine, cytosine, thymidine, p~ n~in~ or uridine to
30 efficiently bind (hybridi2e) to another natural base or
nucleotide. It has been demonstrated that even unnatural or
modified bases can show efficient hybririi7~-inn if projected
from an effective scaffold. Our strategy, disclosed herein, is to
append natural and/or unnatural bases (e.g. thymine,
3S guanidine, 5-fluorouricil(5FU)) onto ~minimi~l~ backbones to
SlJ~STITUTE SHEET (RUL~ 26~
2 ~ 79983
WO 95/18186 T ~ ~ Y~ 612
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
5 and periodiocity of substitution geometry and rigidity of the
backbone scaffold can be designed such that the bases are
geometrically arrayed and projected to provide the optimum
arrangement and orientation of the bases to hybridize with
their targeted counterparts.
,~minimi~l~ oligonucleotide mimetics can be
produced using the ~minimid~ forming and c~tl-n~tion
~hPmictri~c outlined above so as to produce ~minimi~
backbones having natural or synthetic bases attached as side
15 chain sllhstitllpntc to the backbone via appropriate spacers, i.e.
R or R' in the general structural formulas described above
i~rqt~s the Base-spacer grouping.
This may be ~rcv~ via the following general
20 synthesis schemes:
I. Ceqn-nti~l Acylation/Alkylation Reactions Using
Base-Flln~tinnqli7~d Hyd~ lcs - This is outlined for the
Acylationlalkylation case below:
66
SUBSTIT~TE ShlEET (RUL~ 26~
~ WO95/18186 ~ S 21 7q~83 r.~ Y~ll26l2
1. Sequential Acylation/Alkylation Reactions Using Base-r, li7~d Hydra7ines
5 J~C I Nl- Base ACONH\ ~Base
/Br ~X)COCI
~) ~/ (X = Determinant of
ACON \~3~Base backoone spacing and
geometry)
H3C \ (x)cOCl
Base
H2N~
~ CH3
ACON~8~ Bas~3
H3C (X)CON~ ~Bas~
Br(X)COCI
~ etc., etc.
~I! BX
ACO ~ \~~Bas3 ~
H3C '~)~CO-- ~N3~Base
-
67
Sl~BST~ ~ UTE SHE~ L~
WO 95/18186 ` ' ~ 2 ~ 7 9 9 8 3 PCT/U593J12612
II. Sequential L~ r~,./ll~B.~I.~ Reactions
a. Bi~nctional Epo~ide-Esters With Bar,e~ Hydrazines
~7 + H2Nj~~ HOCH2CH2\(3~Base
CH3 H3C NH3
/\ / (X)COOCB
~/ ~
HOCH2CH2 ~3~ Base J
~N~ .. (X - Determinanl of
H3 ~Co(x~7 backbone spacing and
o ger~metry)
H2NN~B~se
CH3
~Tr~7r~7 ~3 ~~Base
H3C NCO(X)--~
OH N Br~se
H3C ~H
(X)COOCH3
O
etc., etc.
~ BX
~r~T7r~7 ~~ Base
H3C ~1~ CO(X)CH(OH)C~
- n\~Base
H3C ~COB
3!i
.
68
Sl I~STiT~lTC S~!~ET (RLILF ?6~
2 1 79983
Wo 9S/~8186 ~ P~ Y~ 6~2
I, ~. BASE-Fls'~CI ION.~lIZED C.~RBOXYESTER-HYDRAZINE
S
~7 H2NI~ HOCH~CH~ \~3~32s~
0 A A NH(3
HOCH2C~ \~3 ~ Base ~H~ ~ 82se
lS A N~) (1CO2CH3
~1~ ,,, B2se ~
NH2 \ / (X = D~r~rrnin~nt OI
\ \ / b~ckbo!lcs~2cin~ a~d
\~ geomer~y)
HOCH2CH2 \~3/~ 8ase
A N~3
etc. I
BCO2CH3\ ~ Base ~ 3 3ase
(X)CO2CH3 OH
30 /8ase
3S H/~ B
69
SU~STIT~)TF SH~ET (~
wo gS/18186 2 ~ 7 9 9 8 3 r~ Y~I26l2
Alternatively, these reactions may be carried out in a concerted
manner with mixtures of base-functionalized hydrazines to
produce random oligonucleotide sequences which can be
S screened for activity, as outlined: a.)
r1aso
H2N~--
CH3
+
~Baso2
H2N~ ~ \ / (X)COOCH3
CH3 O
lS
~Base n
H2Nlj~
CH3 J
x MIXTURES
N~ , OF
H3C~ ~-co(x)cH(oH)cH2 RANDO~
2S -
-
3S
SUBSTITUTE SHEET (RUL~ 263
~ . 2 1 7 ~ 9 8 3
WO 95/18186 PCrNS93112612
b.)
Base
H2N~
(X)C02CH3
Base2 1' o ~Basen
' 10 (X)C~cH~ o . 1 ~N--
+ ~OH ~3 ~ n
.
~ Basen MIX~URES
lS r RANDOM
(X)CO2CH3 ~ SEOUENCES
4.4.5 Syntbesis of Call,~ly-Ldle Mimetics
As nl~ontiom~d previously, ca-l,ohydldt.,s
increasingly are being viewed as tbe ~ r 7nt of living
systems with the enormously complex ~L.u~lul~;. required for
the encoding of the massive amounts of information needed to
~..,I.e~Lldl~ the processes of life, e.g., cellular recognition,
2S illllllU~iLy, embryonic d.,~clo~. I,n~, Cdl~' ~o '- and c~
death. This information is contained and utilized through highly
specific binding interactions mediated by tbe detailed three
dimensional-topological form of the specific calboll~dte. It is
of great value to be able to arrange and to connect tbese
30 moities in various arrays in a controlled manner. This may be
done either by c~nn~ctinE carbohydrate recognition groups
along an I-liEom~ir l,dc}.l,.n~, as done by for random vinyl
copolymers cont~ininE funrti~ li7~d sialic acid groups, which
were shown to inhibit hP~glllttinin binding (J. Am (~h~m Soc..
3S 113, 686, 1991) or by arranging multiple ~,dlbo~y~ e groups
with appropriate spacers on a suitable structural scaffold so
71
SllBST~T~!TE S.~E~ T (F~ULE 26~
- ` ` 7 9 9 8 3
WO 9~118186 PCIIUS93/12612 ,~
carbohydrate groups are oriented in space in such a way that
they can bind selectively to the target (cf., eg., J. Am. Chem.
Soc., 113, 5865, 1991; ibid., 5865). ~minimi~ derived
carbohydrate mimetics may be synthesized from carbohydrate
5 derivatives containing functional groups, such as epoxide
groups, ester groups, hydrazine groups or alkylating groups,
which are compatible with the ~minimitl~ forming and
c~n~in~ reactions outlined above, thus allowing the
carbohydrates to be attached to a basic scaffold, or to be
arrayed along a backbone in a precise controlled manner.
Examples for the synthesis of such carbohydrate derivatives
are outlined below.
Carbohydr~te mimetics-synthesis of aminimide
lS c g
Scheme 1
HO OH COOH ~o COOH
~ OH a,b ~--~--OH
HOl HO 2
c
o ~ COOCH3
~J_OAC
AcO
(a) ~TsCI (1 eq), Pyridine, rt
(b) DBU, diethyl ether. rt
(c) Ac20, Pyridine, CH2CI2, rt
72
~liBSTiTllTE SHEET (RULE 26)
f ~ _ .
~wo 95/18186 2 ~ 7 ~ 9 8 3 ~ u~ fl6~Z
Scheme 2
~ .
SACO~ ~ COOCH3 a ACO~_OCH
4 ACO
0 (a) Glycidol, Ag-Salicylate, C6H6, n
Scheme 3
lS
~CH2OH CHO CHO
HO~ a HO~ b ACO~
HO~V HO~_/ ACO~
HO OH I HO OH I ACO OAC1
6 7 j C NHAC
ACO ~ d ACO
2S . ACO ~/ ACO _~3/
NHAC 9 NHAC
(a) (COCI~, DMSO, E~3N, CH2CI2, 60 C
(b~ Ac2O, Pyridine, CH2CI2, tt
30 (c) Ph3PCH21, PhLi, THF, n
(d) m-CPBA, CH2CI2, tt
3S
73
SU~rlTUTE SHEET (Rll~E 26)
2 1 7 9 9 83
WO 95/18186 ; PCT/US93/12612
Scheme 4
CH2oH CH2OTMS
HO ~ a TMSO
HO _~/ TMSO _~/
6 OHNH2
NH2
(a) TMSCI, CH2CI2, Et3N, rt b, c
(b) Ethylene oxide, CH2CI2, rt, p-TsCI, pyridine
CH20TMS
TMSO ~~
lS TMSO_~(
TMSO OT~IS
12 HN ~~OTs
4.4.6 Synthesis of Pha.---aco~hu-c Mimetics
Background
The physical principle governing the binding of a
natural ligand or substrate to a receptor or active site of an
2~ enzyme, nucleotide or carbohydrate are the same principles
governing the binding of non-peptide, non~ and non-
carbohydrate compounds (competitive inhibitors or agonists).
The mrJ~lifir~tion of a known hic logir~lly active compound as a
lead or ~ tOty~." then ay.lll~F-;,;..p and testing its structural
30 congers, homologues or analogues is a basic strategy for the
development of new ~ lir agents. Several advantages of
this method are: -
74
SU~ST5TUTE SHEET (~llLE ~`
wo gSrl8l86 ~ ` 2 1 7 9 ~ 8 3 1 ~I/U Y~ 61Z
' ` .
Greater probability of theses modified
derivatives to possess physiologica~ propertieS most similar to
those of the prototype than those tested at random.
Possibility of obtaining pharmacologically
S superior agents.
Economical production of a new drug.
Structure-activity relationships can be
established to assist in further developments~
The objectives of any drug discovery program are:
(a) to obtain drugs that have more desirable properties than
the prototype in potency, specificity, stability, pharmalogical
duration, toxicity, ease of administration and cost of production;
(b) the discovery of features of the molecule which impart
lS pharmalogical action~ The term pharmacophore is used to
describe these key features that impart this pharmalogical
action.
Several technologies exist where a biologically
active compound, for example a protein or polypeptide, is
20 attached to a solid support, such as a resin or glass surface.
These linked compounds show diverse inhibitory activity, an
indication that linked molecules are able to retain their binding
properties despite the partial loss of mobility.
There are a wide variety of general
2S pharmacopLc .~i.. known which display specific known modes of
activity, e.g., B-lactam actibacteric, interfering with bacterial
cell wall; piperidine and peperizine, which can act as
psychotropic agents or anticholinergics; and x~nthines as
s~ilT lll~ntc The following general schemes outline the
30 synthesis of pharmacophore molecules, for inclusion in the
various ~minimid~ polymer backbone. The following scheme
3S outlines the gener~l approach:
SU~STI~(~T~ St~~
WO 95/18186 ~ ' ~ 2 t 7 9 9 ~ 3 PCTIUS93/12612 ~
~3X X_ , ~N--R ~ - N-R~
5 ,~ NH2 ~ R' \
Moncm~r Polym~r
(~ Ll . modi~l-blo lunc~ion group
L~ L . moddlod tundion ~noup
R . Phrlrm~copbon~
\,~ R~ . hydn zino proi-div~ gnoup
0 (~ N~ n . n)r n=2 1
~ 0~ 0~ 0~
lS ~7 N J~--Nl`N~ NtNJ~--Nt~
t~mpl- m . ~, n . 3
The polymer can be arranged so as to be
20 homogeneous, that is, the entire polymer is made from the
samè ~ t.~u~ , or he,t~,l o5~.lc(, s, that is, the polymer can be
made with any varying sequences of monomers in a
controllable fashion. The length of the linker, the molecular
fragment that connects the pharmacophoric portion to the
2S quatenary nitrogen of the Aminimit1~ polymer, can be of
various lengths and shapes, such as but not limited to a linear
alkyl chain. As such, the ArrAn~r~n~ and the geometric
configuration of the ~ ro~ ,s on the backbone polymer
can be controlled.
The following figures are general examples of
ph~rmsrophores that are illustrative of the approach:
3!i
76
SUB~T~T~ i ~ S~ErT ~UL~
Wo 95118186 i;', (.' ~ 7 9 9 8 3 PC~/US93/12612
Antibactenals, e.~.,
OH
~ ~ e ,~,
H H
O R,
,N--R,
0 ~h-- HO ~ \ /~o H H
lS
3S
77
SUBST~TUTE S~iE~T (RULE 26)
t ~.` 2 t 7 q 9 ~ 3 PCT/US93/1261~ ~
WO 95118186
Anal~esics~AI,l;~,.. ,.~ù~ ,a/Fs, `,ui,u,u;~,s, e.~.,
~N~ Mepen~ine
N NR ~N~
0 HO N~ 1, II~N--
or
5 ~ ~ N-NH2
'N;N O
~N~
N-NH2
3~
78
SU~ITU~E S~E~T ~RULt 26~
-
~ WO 95/18186 - ~ ` 2 1 7 9 g 8 3 PCT~US93~1261~
Al I r e.g.,
N~P 13~zapm~xide
5 a H
~ N_ ~a ~ _~, ~N~ R~
A" ' ' _ e.g.,
cr
T~d:he~llthyl Chlo~de
~N
OH
~ ~N~N-R~
4.4.7 Synthesis and pOlym~ri7~tioD of Chiral
Aminimide-Containing Monomers
The C~ ;OLI of many of the ~minimi~1~
2~ structures ~IPsrrihed above into monomer building blocks
which can be polymerized to give novel ~1&~ 5. which
are useful in a v,qriety of high technological ~rplirstinnc. is
contemplated, The following synthetic ~.~ q ' -~ are
expected to be very useful in the production of new materials.
(a) Free-Radical Poly...~ tion
of Vinyl ~minimi~src
Chiral (as well as achiral) vinylgminimi~
monomers of the general structures shown below may be
35 readily prepared, following the ~JlU~.Cdulcs outlined above, and
79
SlJBSTIT~ITE ~h'~T (i~ULE 26)
wo9~18186 ~ t ~ 2 1 799~3 P~ 261~ ~
used in free-radical polymerizations, according to experimenta
procedures well-known in the art, to produce a vast array of
novel polymeric materials.
S A~ +,X and ~XJ~T~N`A
Additional monomeric structures useful in
10 preferred free radical polymerizations include those shown
below; they produce polymeric chains capable of being
crosslinked into more rigid structures. The monomers shown
below may be prepared using the synthetic procedures
outlin~d above, and the polymerizationfcros~linkin~ reactions
may be run using standard polymerization techniques. See, for
example, Practical Macromolecular Organic Chemistry, Braun,
Cherdron and Kern, trans. by K. Ivin, 3ed., Vol Z, Harwood
Academic Publishers, New York, N.Y. 1984.
~R
D~ J~ ~N
2S
The monomers shown above may be polymerized
with other alkenes or dienes, which are either commercially
available or readily prepared using standard synthetic
reactions and techniques, to furnish copolymers with novel
structures and molecular recognition characteristics.
3~ ~ ~ m
H3C~ N_C2Hj
CH~CH,OH
SUBSTITUTE SHEET (RULE 26)
~ WO 95118186 `~ 2 1 7 9 9 8 3 ~ JbyJ/ll6l2
S
- (b) Condensation Polymerizations Producing
Aminimide-Containing Macromolecules
Sequential condensations of aminimide-forming
molecules may be used to produce a variety of novel polymers
10 of controlled size. An example involving dimeric epoxides and
esters is given below; processes involving trimeric and more
complex epoxides and esters are also contemplated; and
experimental conditions for running these polymerizations
(including techniques for resolving experimental ~iiffi~`-lltj~5 as
lS product molecular weight increases) have been described
above .
CO2Et
Y H~ OH H3C~
~H H~Y HO~ H H3C
CO2Et
H H 3 ~ ~CH3
~ N - N+ "
Alternatively, con~ nC~ion polymerization may be
carried out by reacting alpha carboxyester derivatized
hydrazines (prepared as outlined above) with chiral epoxides
3S to produce the novel polymers shown:
81
SUBSTlrUT~ S~EET (~l~L~ 26)
WO 95118186 - -- 2 ~ 7 9 9 8 3 PCINS93~12612 ~
{~ + M~
When the poly~ iLdtion reaction is carried out with molecules
immnbili7Pd on a support, e.g. silica, a support capable of
specific mo~ecular recognition is produced; an example of such
10 a support is given below:
FORMULA 8B
lS #4, p. 82
20 4.4.8 Lipid Mimetics
~ minimiriP conjugate structures c~ntAinin~ a single
long-chain hydlvc..lbo.. group can be used as :Imrhirhilli~
surface active materials which have great utility as delivery
2S systems for the a.l ~ hd~ion of drugs. The ~ hrnPnt of a
"rPcogni~ n group" to the ~minimiriP moiety gives a material
which is highly compatible with lipophillic structures, such as
cell wall ..~ .-..,s, and which itself will form mi~ellul~r
~h, ~,;. in water with the ~ecognition group pointed out or
30 "displayed " on the surface of the micelle. This may be
represented by the general sch~Pm~ic shown:
3S
82
SUBST~TUTE SHEET ~R! 5L~ 26)
2t 79983
W0 9S/18186 ~ /u~ 612
¦ RECOGNlTION GROUP ¦l AMINIMIDE ~ HYDROCARBONTAIL ¦
MOETY
Il
-WATER
lS H20 (~) H20
H20(~ )H20
(~vv~
2S
Examples of the synthesis of these COI.J~ ., are given below.
,~minimid~ structures possessing two long-chain
alkyl groups capable of producing bilayer m~mhr~n~ structures
30 are preferred embodi~ ts of the present invention. Because
of the presence of the double tail on the ~mrhirhilir group,
these molecules prefer to form cr,ntinll bilayer m.-mhra~P
structures, such as those found in cell wall ~ S rather
than micelles. As such they may function as "cell wall-
3~ mimirl~in~"' c~ vl,- ~t~ This is s~h~mstir~lly illustrated
below:
83
SUBSTITO' j~ S~E~T ~ULF 2~!
r ~
WO 95/18186 2 1 ~ 9 ~ 8 3 PCr/US93/12612
SUBSTITUENT/ V,¦ HYDROCARBON TAIL ¦
RECOGNITION AMINIMIDE
GROUP MOETY ~ HYDROCARBON TAIL ¦
(~)~
~ATER
EI20
(~)(~)O(~)
H20
-
Among the many uses for these unique compounds
are the isolation and stabilization of biologically-active
molecules from the cell-wall, the ~ _ on of affirlity
chrnm~togr~rhy supports for the isolation and purification of
~mrhirhilir macrnmnl~cul~s~ e.g., receptors, enzymes, etc., and
3S the effective delivery~ tion of drugs.
~4
SUBSTITUT~ ~;EET ~ 5L~
- j ! 2 1 7 9 9 8 3
WO 95/18186 . I ~./V:~Y~jl2612
The structure of one preferred lipid mimetlc is
shown below. Substituents R may be chosen from a variety of
structures of various sizes including structures of ligands of
biological receptors or enzymes; a preferred combination of
substituents involves sterically small groups for Rl and R~ and
a group such as A or B described above for R3; the long-chain
alkyl groups are 4-30 carbons in length; group X is a linker
composed of atoms chosen from the set of C, H, N, O, S, P and Si.
0 R2 CHAIN
1~3 ~3 /
R1 Nl N ICl (X)
R3 CHAIN
lS
A further desirable Yariation of the surface-active
structure shown above is as follows:
.
CHAIN R2
(X) ~ N C R,
CElAlN/ R3
2S
In the above structure, X is a linker group (e.g., CH);
one or more _~ R are chosen from the group of
structures A and B described above and the remaining
~vbs~it~ ~(s) in preferably a sterically small group, e.g., H, or
CH3. An æ~lriitinn~l desirable ~mrhirhilir structure is shown
~elow: ~ubs~ n~ s~mclures ;re simil.r ~o ~hose lis~ed above.
SU~STIT!JTE SH~FT ~F~U~E 26}
~0 95/18186 2 1 7 9 9 ~ 3 PCTIUS93112612
CHAIN
I
R, N N C R,
CHAIN
Lipid mimetics are illust}ated in the Examples that follow.
4.4.9 Fabrication of Aminimide-Containing Macromolecular
Structures Capable of Specific Molecular Recognition
In an embodiment of the invention aminimide
molecular building blocks may be utilized to construct new
0 macromolecular structures capable of recognizing specific
molecules ("intelligent macromolecules"). The "intelligent
macromolecules" may be represented by the following gener~l
formula:
P-C-L-R
where, R is a structure capable of molecular
recognition;
L is a linker;
P is a .lla.,lul..ol~.,ular structure serving as
supporting platform;
C is a polymeric structure serving as a coatino
which surrounds P.
Structure R may be a native ligand or a biological
ligand-acceptor or a mimctic thereof, such as those described
~5 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, ~minimitl~ mon~m~ nY l7~10n~-derived ch~in~
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 polym~i7~inn is well known to those
skilled in the art. This coating element may be
86
SUBST~TUT_ ~h~E~ 6)
W095~18186 .~ r. . ~ 21 7q983 P~l/v~Y~/~26l2
I) a thin crosslinked polymeric film 10 - 50
Angstroms in thickness;
') a crosslinked polymeric layer having controlled
microporosity and variable thickness, or
3) a controlled microporosity gel. When the
S 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 may be constructed
in a controlled way by carefully controlling a variety of
reaction ~ald~ t~l~ such as the nature and degree of coating
crosclinlcin~, polymerization initiator, solvent, concentration of
reactants, and other reaction conditions, such as ~ la~ulc,
agitation, etc., in a manner that is well known to those skilled
in the art.
lS The support platform P may be a pellicular materiâl
having a diameter (dp) from 100 Angstroms to 1000 microns, a
latex particle (dp 0.1 - 0.2 microns), a microporous bead (dp I -
1000 microns), a porous mP nhrPnP. a gel, a fiber, or a
c - Illa~.loscu~:c surface. These may be commercially
avâilable polymeric m~t.~ri~lc. such as silica, polystyrene,
pOlya.,lylat_s, polysulfones, agarose, cellulose, etc. or synthetic
~minimi~lP-crJnt~inin~ polymers such as those described below.
Any of the elements P, C, L, or R cn~tqinin~ an
~minimi~ based structure is derived from a form of the
element cont~inin~ a precursor to the ~minimid~P-based
structure. The mlllti '_ recognitirJn agents above are
expected to be very useful in the d_~_lv~ of targeted
th~r~reutirc. drug delivery systems, adjuvants, rliq~nosti
chiral selectors, separation systems, and tailored catalysts.
In the present specifir~tion the terms "surface",
"substrate", and "structure" refer to either P, P linked to C or P
- linked to C and L as defined above.
Thus, another aspect of the invention relates to a
three-~ I crosslinked random copolymer cr)~t~inin~, in
3~ copolymerized form about I to 99 parts of a free-radically
87
SUBSTITUTE ~ ULE 26~
WO 95/18186 2 ~ 7 9 ~ 8 3 pCr/US93J12612
polymerizable monomer containing an :lminimide group; up to
98 parts of a free-radically addition-polymerizable comonomer:
and about l to S0 parts of at least one crosslinkinE monomer
The comonomer used in this copolymer may be
water-soluble or water-insoluble, and the copolymer is
S fashioned into a water-insoluble bead, a water-insoluble
membrane or a latex particle, or can be a swollen aqueous gel
suitable for use as an elc~,lopl~lc;.is gel.
This copolymer is preferably the reaction product
of about I to 99 parts of a condensation-polymerizable
monomer containing a moiety cluster selected from the group
consisting of ( I ) at least three epoxy groups, (2) at least three
ester groups, (3) at least one epoxy and at least two ester
groups and (4) at least one ester and at least two epoxy groups;
about I to 99 parts of a second co~lt ~ Qn-polymerizable
lS monomer containing a moiety cluster selected from the- group
cn~ of (I) at least two ester groups, (2) at least two
epoxy groups and (3) at least one ester and one epoxy group;
and an amount of 1, I-dialkylhydrazine equivalent, on a molar
basis, ~ lly equal to the total molar conterlt of epoxy
groups.
4.4.9.1 AminimiA~ Cr,n~Ainin~ Support Materials
C ~ially available or readily obtainable
chromatographic support materials for chromatographic and
2S other Ar?lir~ior~c. as well as other '`hbllC~tL,;I materials can be
derivatized with tailored aminimiA~ moieties, through chemical
mrAifir~ion, J,- ~ novel materials capable of recognizing
specific molecular structures.
These are IcJlcsclltcd by the following general
structureS:
R
~ (y)~SURFACE)
3~ ~2
88
SU~TIT~JT~ S~T ~RULE 26)
95118186 ~ 2 1 7 9 ~ 8 3 I ~_I/U~YJII.I6I2
S and
Rl
(SURFACE)--(Y~f j~ --(X)--A
. ~2
lS
In the structures above, A is selected from the group consisting
of amino acids, oligopeptides, polypeptides and proteins,
nucleotides, oligo~l~lrl~oti~ c polynucleotides, carbohydrates,
molecular ~l~uclu~cis ~csoci~ted with therapeutic agents,
metabolites, dyes, photo~rArhir~lly active chPrnic~lc and
organic structures having desired steric, charge, hydrogen-
bonding or l-y~u~hoi ~/ elements; X and Y are chemical
bonds or groups cr~ncictin~ of atoms selected from the set of C,
H, N, O, S; Rl and R2 are chosen from t_e group of alkyl,
2S carbocyclic, aryl, aralkyl, alkaryl and, preferably, structures
mimir~in~ the side-chains of naturally-occurring amino acids.
Surfaces and other structures function~li7~d with
multiple ~minimid~ subunits are also preferred; general
~l~uclu.. is are shown below.
3S
89
SUBSTITUTE SHEET ~RULt 26)
WO 95/18186 ` 2 1 7 9 9 8 3 PCTIUS93112612
Rl...n
A--X I C--N--N+~ (SURFACE)
o R l...n
, n
+ ~
Rl...n
A--X ~ N+-N- C--(Y) ~(SURFACE)
I I.. n a
- n
In the above structures Rl...n and R'l...n are used to illustrate
the manner in which the hydrazine 51l1,"' . Rl and R2 can
lS be Yaried in each polymerization step described above to
produce a functional supported oligomer or polymer.
The following chemical mr~riifir~ m~ can be used to
prepare Aminimi~P-functionalized surfaces.
4.4.9.1. I F~nrtinn~ Ation of Ester and Epoxy Surfaces
A surface bearing ester groups can be treated with
an epoxide, cr---Ainin~ desired group B, and a .1;~ ;tl~d
hydrazine to form an: i,lP surface as follows:
2S a
(SURFACE3--COOR' + ~NH2 +
P~
(SURFACE~&
R3
3S OH
CU~ ~TJTUTE SHE~T (RU~E 26)
~ wo 95118186 ' 2 1 7 9 9 ~ 3 PCT/IJ59:~12612
To carry out the above reaction, the surface is
treated with a solution containing a 10C~c molar excess of the
epoxide (based on the calculated number of reactive ester
groups of the surface), and a stoichiometric amount of the
hydrazine (with respect to the amount of the epoxide) in an
appropriate solvent, such as an alcohol, with shaking. The
mixture is then allowed to stand at room temperature for I
week with occasional shaking. At the end of this period, the
solvent is removed by decantation, and the surface is
thoroughly washed with fresh solvent and air dried.
This approach allo~vs the func~iorl~li7~rion of
readily available supports containing ester groups.
The above reaction sequence can also be employed
with an epoxide-functionalized surface:
lS Rl ' R'
(SURrACE)-CH-CH~ N--c-cH2 cH cHl . N:--N--C-CHl CH CH~
OH R ~ ' O OH R'~l O
To carry out the above reaction, the surface is
treated with a solution con~Aininp a 10% molar excess of the
ester (based on the calculated number of reactive epoxide
groups of the support), and a strirhinm~trir amount of the
hydrazine (with respect to the amount of the ester used), in ~n
~y~up~iato solvent, such as an alcohol, with shaking. The
mixture is then allowed to stand at room t~ aLul~ for l
week with ~ ~. - I shaking. At the end of this period, the
solvent is removed by ti.~rAntAtir,n and the surface is
thoroughly washed with fresh solvent and air dried.
The foregoing reaction can be modified by utilizin~
an ester whose s~lhstitl ~ B contains a double bond. After
completion of the reaction shown above, the double bond of th~
ester can be epoxidized using one of a variety of reactions
including the asymetric epr,sir~ ir~n of Sharples (e.g., utilizing
peracid under suitable reactiot~ cQ~ ionc well-known in the
art), and the product used as the epoxide in a new repetition of
91
SUB~TITUTE SH~ET (R~ILE 26)
WO 95/18186 ~ A 7 9 9 ~ 3 PCI/U593J12612
the aminimide-forming reaction. The overall process can be
repeated to form oligomers and polymers.
For example, using ~-butenoic ncid methyl ester ~s
the ester, n repetitions of the above reaction sequence
produces a compound of the form:
Rl n Rn'
~SURFACE~eHi --CH~N'--~--C--CH2-fH-cH2 r N~--N--Ci--CH~-CH CH~
OH Rl.n o OH R''l O
,n
10 where the designations R2.. n and R3.. n are used to illustrate
the manner in which the hydrazine s~sti~uPn~s R2 and R3 are
varied in each polymerization step, if desired, to produce an
oligomer or polymeF.
The foregoing reactions can be carried oul using
1~ hifunr~ n~l esters of the form ROOC-X-COOR'. where X is a
linker and R and R' are alkyl groups as defined above, and/or
bifunctional epoxides of the form shown below,
3S
92
SUBST!TUT~ SHEET (RVLE 2~!
WO 95/18186 ~ . I r~ 2 1 7 ~ ~ 8 3 Pc!r/r~s93~26l2
H-C~ CH--Y--CH\--C~i
O O
wherein Y is a linker as defined above, to form desirable
polymers. If an ester-functionalized surface is reacted with
bifunctional esters and epoxides, the resulting surface will have
the following general structure.
Rl R2
(SURFACE)--IC~N-N~ CH~CIH-~Y) Cl CH2 ~N -N-c-(x)-c~oR
0 O ~ RI OH OH R- O Jn
If an epoxide-functionalized surface is reacted as
above the derivatized surface will have the following general
structure .
lS
(SURF~ CE) Rl lR2 R2~1
CH-CH2--N~-N--S--X-C---N-~NCH2CIH Y CIH-CH7--N~-N--C--X-C--OR
OH Rl R2 OH OH Q R~l O O
4.4.9.1.2 F~ i7~rinn of Amine Surfaces
An amine-functionalized surface can be converted
to an ester-bearing surface by reaction with an acrylic ester as
shown in sequence (a) below. This reaction is followed by
reaction with hydrazine and an epoxide as shown in sequence
2S (b)
93
S V~ J ~ _ ~J-i ~L ~ i E "~
WO 95/18186 r i i ; 2 1 7 9 9 ~3 3 PCTNS93/126~2 ~
O D
SLRFACE~--.`.H, + H2C:CH C--O--CH3 ~SI RFACE~-~tH. CH C-O-CH
~; / ,N H `
R2 X C~H .
~ B,CH o
O Rl
(SURFAC10-NIicHl'CH2`c-N-N;CH2CH-9
R2 OH
For reaction (a), a 10% molar excess of methyl
acrylate (based on the number of reactive amino groups the
surface as ~ d by a titration with acid) is dissolved in
an appropriate solvent, such as an alcohol, and added to the
surface. After addition is complete, the mixture is shaken at
room t~ alul~ for 2 days. The solvent is then removed by
d~csnt~ti~~ and the surface is washed thoroughly with fresh
solvent in preparstion for the next step.
For reaction (b) the stoichiometric amount of a 1:1
mixture of the hydrazine and the epoxide, is combined in an
appropriate solvent, such as an alcohol, and quickly added to
the solvent-wet surface from reaction (a). The mixture is
shaken at room t~,.-.~. . for 3 days. The solvent is then
2~ removed by d~c~nt~ion and the surface is washed thoroughly
with fresh solvent and dried.
The above reaction sequence can also be employed
with an epoxide-f~lnrtionsli7~d surface, in which case
S~h5tir~ nt B in the structure above l~ .S~ht~ the surface and
the desired functional group bears lhe amine moiety. One way
of obtaining such a surface is to react a silica surface with a
silicic ester cont~inin~ an epoxide group to produce a so-called
"epoxy silica". as shown below.
94
SU~SrlTU 1~ SHEET (RU~ 26~
~` 2 1 7 ~ 9 8 3 pCr~US93/l26l2
WO 9sllgl86 o
OH + ICH10)3 si~cH7-cH~-cH~-o-cH~--CH--CH.
--Si--O--Si CH~-CH,-CH-- ~ CH- CH--CH.
("Epoxy Silica")
lS
2S
- 95
SU~ST,T~ r~-T (~UL~
wo ss/ls l86 ~ i ? ~ 7 9 ~ 8 3 PCT/US93~1 2G t 2
4.4.9.1.3 Functionalization of Carboxylic-Acid-Containing
Surfaces
A surface functionalized with a carboxylic acid
group can be reacted with an 1, I -dialkylhydrazine and a
coupling a~ent. such as dicyclohexyl carbodiimide (DCC). to
S form a hydrazone-containing surface as shown in step (a)
below. This surface can then be coupled with a desired ~roup B
bearing a sllbstittl~nt capable of alkylating the hydrazone to
give an ~Iminimi(1P structure (after treatment with base), as
shown in step (b):
(SURFACE)--C--OH + H2N--N~ (SURFACE) ICl ~R~
'/
~/ B--CH2X
Rl
(SURFACE)--fi--N--:N CH2-B
R2
S~1.s~ B is a surface fUn~tio^~li7Pd with an
alkylating agent capable of reacting with a hydl _z ~ - .
To perform the above chemical m~ fir~ion of a
carboxyl-bearing surface, the surface is treated with a 10%
molar exccss equimolar amounts of the N,N-dimethylhydrazin~
and DCC in a suitable solvent, such as lu~ chloride, and
the mixture is shaken for 2 hours at room 1~ . The
slurry is then removed by ~ ;on and the surface is
washed Lhol- ~hly with fresh solvent to remove any residu~l
1~ d di~ loh_~yl urea. The surface is then treated
with a ' - c amount of the alkylating agent in a
suitable solvent, warmed to 70 _C and held at this t~ ldlUI~:
3S for 6 hours. The m~xture is then cooi~d, the solvent is removed
96
SUBST~TLI i-E ~HE~ Ul.~ .~6,
2 1 79q83
~ WO 95118186 . '- r~ YJ/l~cl2
by decantalion, and the surface is washed wltn fre~h solvent
and dried.
1.4.9.1.4 Funtionalization of Surfaces
Capable of Hydrazide Alkylation
A surface bearing a ~roup capable of alkylating acyl
hydrazones can be functionalized to contain aminimide groups
as follows:
Rl Rl
0 ~N--NH--C--W--3 + (SUREAC_)--Z--X ~ (SUi~FACE)--Z--N'--N'--C--W--3
In the equation above. Z and W are linkers composed of atoms
selected from the set of C, N, H, O, S, and X is a suitable leaving
group, such as a halogen or tosylate.
lS A hydlazo~ bearing a desired group B is produced
by reacting the appropriate 1, I '-dialkylhydrazihe with any of a
variety of d~,.ivali~s c~-tqinin~ B via reactions that are well-
known in the art. These d~.ivdii~.,;. may be acid halides,
a7lq~tc-rl~s (oYq.7oll ~), isocyanat~s~ chloroformates, or
20 chlorothioformates..
4.4.9.1.5 F~ln~tinnqli7qtion of Surface Bearing -NH, -SH,
or -OH Groups with Chl~.u...cillyl ~minimiri~5
Surfaces r - -lj7~d with -NH2, -SH, or-OH
2S groups can be f -nrti~ -li7~d by treating them with
chl~.u...~ lyl qminimirl~s in the presence of strong base using
the .~Yrl~rim~ntql conditions outlined above:
O Rl O Rl
30 (SURFACB)--XH + Ci--CHl--C--N--N'--B -- (SURF~CE)--X--CHl--C--N--N'--3
R~ Rl
The required chlolull.~,.llyl qminimitl~s can be
3S prepared by known literature ~lucedulcs (See, e.g., 21 J.
97
SUBSTI ~ UT~ S~EET ~RiiJLE 26)
WOg5/18186 ~ ~ 2 1 ~99~3 P~ Y~II26I2 ~
Polymer Sci.. Polvmer Chem. Ed. 1159 (1983)). or by usin~ the
techniques described above.
- 4.4.9.1.6 Functionalization of Oxazolone-Containin~ Surfaces
Oxazolone-containing surfaces can be functionalized
by first reacting them with ,1, I '-dialkylhydrazine as shown in
step (a) below followed by alkylation of the resulting
llydlaz<J:le with an alkylating agent B-CH2-X as shown in step
(b); reaction conditions similar, to those described above are
expected to be effective in carrying out these modifications.
Rl (a) R3 Rl
,N--NH2 + (SURFACE~--A2 (SURFACE)--C - NH - c--C--NH--N
R2 o R~ B ~R2
~/
~/ B--CH2X
R3 Rl
(S URFACE)--C--Ni~--C--C--N--N~--CH B
O R~ R2 2
In the structures above, R3 and R4 are derived from the five
membered azlactone ring denoted by Az.
Although the previous tlicc~ccionc are specically
directed to the filnrrjonqli7iA~ion of surfaces, these reactions
can also be used to construct AminAmi~i~ linkages to the other
spcciesof A and B which are described in this application.
4.4.10 Preparation of ~minimi~ir-Based
Coatings for Support Materials
It is possible to produce - ' ' '~I~ filnr~i~ -1i7~d
CO ~.r.~ support materials by coating various soluble
aminimi~l~ fn.rmlllqti( - on the surfaces of e~isting supports,
98
SU~ST3TUTE ~HEET (~ULE ~6)
~ 7998~
~ W0 95~18186 . ' ` P~ YJ~I~6I2
and subsequently crncclinkin~ the resulting oati~g~ in place to
form mechanically stable surfaces. The coating may be
engineered for a particular application (e.g.. to take the form of
a thin non-porous film or to possess localized microporosity for
enhanced surface area) by judicious selection of process
conditions. monomer loading levels, the crocclinkin~
mechanism and the amount of crosslinker.
For example, any of the foregoing reactions can be
carried out with a vinyl ~minimi~,~ in contact with a selected
surface, which is p`olymerized according to well-known
techniques (see, e.g., U.S. Patent No. 4,737,560). The
polymerization results in a surface coated with a polymer
containing ~minimi-l~ side-chains. Other coating ~.uc~du.~s
employing ~minimi~.- functional groups are described below in
greater detail.
lS
H2C
Epoxy Silica + N--NH2 + (C2H,)2--N-cH2-cH2-cooc2H5
H3C
--si--o--Si CHrCH2 CH2-O-CH2 CH CH2 N -N--C--CH2--CH2-N(C2H~).
OH CH,
4.4.11 Synthesis of ~mini~i~P-containing Materials Via
2S Polymerizations of Aminimi-lP-Based Molecules
In addition to utilizing ~minimi-3r ~ hy to
rhPmir~lly rnodify commercially available or readily obtainable
surfaces, new surfaces and other materials can be f:~hrir~-Pd de
novo from aminimi~i~ pl~,~ulavl~ bearing polymerizable groups
by poly-ll.,.i~alions and/or COpOIymPri7~tiQnc in the presence
or absence of crocclinkin~ agents. Depending upon the
properties for the desired material, various comhi~tin~c of
mnnnmPrc crosslinkers, and ratios thereof may be employed.
The resultant support materials may be latex particles, porous
3S or non-porous beads, e b ~s, fibers, gels, clc~,hu,uh~ is
99
SllB~TirUT~ SHEET (RULE 26?
wo 95/18186 2 1 7 q 9 ~ 3 ~ Y~ 2612
gels, or hybrids thereof. Furthermore, the monomers and
crocclinl~inc agents may or may not all be aminimides.
Vinyl or condensation polymerizations may be
advantageously employed to prepare the desired aminimide-
S containing materials. Vinyl polymerization can include use of
one or more monomers of the form CH2=CH-X that are
copolymerizable with s~minimi(~S suitable examples include
styrene, vinyl acetate, and acrylic monomers. If desired,
compatible non-~minimi~ll crosslinkers, such as divinyl
benzene, may be employed (either singly or in combination as
the other such agents).
Condensation polymerization may be accomplished
using multifunctional epoxides and multifunctional esters with
the appropriate amounts of an I, I '-dialkylhydrazine, using the
reaction con-litionc described above. Either the ester
lS cu~ orellt or the epoxide co---pc - - should be at least--
trifunctional to obtain three-dimensionally crosslinked polymer
~LI U~,LUl_S; preferably, both cr~ rsn~ are trifunctional.
The nature and co. ' ~ ~ of processing, the ratio of
the various monomers and the ratio of crosslinker to total
monomer content can be varied to produce a variety of product
structures (e.g., beads, fibers, membranes, gels, or hybrids of
the foregoing) and to tailor the mPrh~nir~l and surface
properties of the final product (e.g., particle size and shape,
porosity, and surface area). Appropriate p~ t~ for a
2S particular application are readily selected by those skilled in
the art.
4.4.12 Combinatorial Libraries of Peptidûmimetics
Derived From ~minimiflP Modules
The synthetic transformations of Iminimi~lP5
outlined above may be readily carried out on solid supports in
a manner analogous to performing solid phase peptide
synthesis, as described by ~IP~rifiPIA and others (see for
35 example, Barany, G., MPrrifiPI-I. R.B., Solid Phase Peptide
100
SU~TITU~ ~rT ~RU' E 2~
WO 95/18186 ` " ~ ; , 2 1 7 9 ~ 8 3 PCT,'U593/1261Z
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 Svnthesis, D. Rickwood & B.D. Hames eds., IRL Press ed.
Oxford U. Press, 1989). Since the assembly of the ~minimid
S derived structures is modular, i.e., the result of serial
combination of molecular subunits, huge combinatorial libraries
of aminimide-based oligomeric structures may be readily
prepared using suitable solid-phase chemical synthesis
techniques, such as those of described by Lam (K.S. Lam~ et al.
Nature 354, 82 (1991)) and Zuckermann (R.N. 7~1rl~Prmqnn et
al., Proc. Natl. Acad. Sci. USA. 89, 4505 (1992); J.M. Kerr, et al.. L
Am Chem Soc.. 115, 2529 (1993)). Screening of these libraries
of compounds for interesting biological activities, e.g., binding
with a receptor or interacting with enzymes, may be carried
lS out using a variety Of ~ ' well known in the art.- With
"solid phase" libraries (i.e., libraries in which the ligand-
c~n~lirl~trs remain attached to the solid support particles used
for their synthesis) the bead-staining technique of Larn 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 rhosph~c~) whose activity can gi-~
rise to color production thus staining library support particles
which conuin active ligand-c~n~ ps and leaving support
particles cont~inin~ inactive ligand-c~r~ rs colorless.
2S Stained support particles are physically removed from the
library (e.g., using tiny forceps that are coupled to a
mi.,.v~ with the aid of a microscope) and used to
structurally identify the biologically active ligand in the libr~r~
after removal of the ligand acceptor from the complex by e.~
washing with 8M guanidine hydrorhlori~ With "solution-
phase" libraries, the affinity selection techniques described b~
7-~, L.. ~nn above may be employed.
An especially preferred type of r~ mhir ~ rial
library is the encoded comhir~nri~l library, which involves the
3S synthesis of a unique chemical code (e.g., an oli~ '-DtrlP or
101
SU~ITU I ~ S~ET (RULE 26)
WO 95/18186 2 ~ 7 9 9 8 3 r~ Y.sll261Z ~
pep~ide). tha~ is readily decipherable (e.g, by sequenCIllg uslng
traditional analytical methods), in parallel with the synthesis of
the ligand-~An~ tPs of the library. The structure of the code
is` fully descriptive of the structure of the ligand and used to
5 structurally characterize biologically active ligands whose
structures are difficult or impossible to elucidate using
traditional analytical methods. Coding schemes for constrùction
of combinatorial libraries have been described recently (for
example, see S. BrenneF and R.A. Lerner, Proc. Natl. Acad. Sei.
USA . 89, 5381 (1992); J. M. Kerr, et al. J. Am C~Pnn Soc. 115,
2529 (1993)). These and other related schemes are
contemplated for use in Cor.~l u~ g encoded combinatorial
libraries of oligomers and other eomplex structures derived
from aminimide units.
lS The power of e~mhinqr-)riAl chemistry in generating
screenable libraries of ehemieal eomrol~n.lc e.g., in eonneetion
with drug diseovery, has been deseribed in several
publieations, ineluding those m.~nti~ ~~ above. For example,
using the "split solid phase synthesis" approaeh outlined by
20 Lam et al., the random il~,ul~ tion of 20 different AminimitiP
units into pentameric structures, wherein eaeh of the five
subunits in the pentamer is derived from one of the Aminimi~iP
units, produces a library of 205 = 3,200,000 peptidomimetic
ligand-cAr~ Atps~ each ligand-eandidate is attached to one or
25 more solid-phase synthesis support particles and each such
particle contains a single ligand-eandidate type. This library
ean be corla~lL~l~d and sereened for biologieal activity in just a
few days. Sueh is the power of co~ . ;Al chemistry using
~minimi~iP modules to eonstruet new moleeular ~An~i~qr~s
An example of one of the many methods for use in
constructing random combinatorial libraries of Aminimides-
based compounds; the random i~- ~ola~ion of three
Aminimi~lP.s derived from alpha-ehloroacetyl chloride and the
hydrazines shown below to produce 27 trimeric structures
linked to the support via a succinoyl linker is given below.
102
SU~S l ITU i-E SHEET ~U~E 26)
-
wo g5rl8l86 : ~ 2 ~ 7 g ~ 8 3 PcTnrsg3/l~6lz
.
4:4.13 Design and Synthesis of
Aminimide-Based Glycopeplide Mimetics
A great variety of saccharide and polysaccha~ide
structural motifs incorporating ~minimiri.o structures are
contemplated including, but not limited to, the following.
( I ) Replacement of certain glycosidic linkages by
~minimitl~ backbones using reactions well known in the art of
PO o
H100-- 1 Tsa H.N--N~ H~O\lO X
I~NH~ R~ ~OP H A
H~o~op R2 H~O\OP Y--Y
H OP H OP
PO-- R
21H~o~0 N ¦ ~
PO~H H~o~OP Rcmoval of protec~ng group P
H P po~H
2S H OP
HO R
;~~0 N ¦ ~
HO~H HL/--O~OH
H OH ~OH HA
HO~H
3S H OH
103
S~BST~TUTE SHE~,T (RuLr 26~
c
W0 95118186 ' ~ - ' 2 1 7 9 ~ 8 3 F~ Y3/126l2 ~
(~) Use of ~minimi(l,~ structures as linkers
holding in place a sugar derivative and a tailored mime~ic, or
ano~her sugar.
~CO~C
H ~ pO~P
HV~O~OP H oP
pO~H 2. D~IULC~UUI
H OP
R~ O
N~--N---C
lS R
H~o~lOH HV~ OH
HO~H HO~H
H OH H OH
4.4.14 Design and Synthesis of
,~minimi~ Cont~inin~ Oligo~ r~tirl~ Mimetics
The art of nl~rl~oti~ and oli" ~ synthesis
has provided a great variety of suitably blocked and activated
2S furanoses and other i.~ t- S which are expected to be
very useful in the construction of ~minimirl~-based _imetics.
(Cu.ll~chcr. ,i~., Organic Chemistry, Sir Derek Barton, Chairrnan
of Editorial Board, Vol. 5, E. Haslam, Editor, pp. 23-176).
A great variety of nl-rl- Oti~ and oligon-lrl~-o-itl,-
30 structural motifs incorporating ~minimi~-based structures are
Cû~llclllylr-L~d ~ in~ but not limited to, the following.
( I ) For the synthesis of oli3~ s
c~n-~inin~ peptidic ~rninimi~i~-based linkers in place of the
pho~ diester groupings found in native oligo~l~rl~o~ s
3S the following approach is one of many that can be used.
104
SUB~T~TUTE S~IEET (RULE 26)
~ W095~18186 21 7~9~3 r~ Y~/l26lz
P O~T ROOC~ T
R,--N+-RI oP2
.~
PlO~;~T ' ~s O //
15 N~ R.--. I-RI
OP2
~O--P ~ O
(2) For the synthesis of structures in which an s55`
~ ninimi~l~ grouping is used to link complex olie~j"~ ^I. oti~
derived units, arl approach such as the following can be very
llseful .
.
-
105
SUBSTITlJT~ S' 5 -.T ~RULF 2~'
. `; c 2 1 79983
WO 9S/18186 PCT/IJ593/12612 ~
OTms
Rl O I 1. couplin~,
11 + X-(CH.)~ --C--O j
TmsO ~ H I ~. H
3. R,-X
O
H3CO--C T
OJ~N R~ ~P~
(CH2)n + IN--l~H2
R2
lS
H`NJ~CH3 several steps
20 ~CH2)n IN--N~T
R2
O--P
2SH~ CH3
(CH2)n N--N~T
R2
'O--P=O
0~555
3S
106
SUBSTITU ~T~ ~r~ JL~ 2~
r; `
~ WO 95/18186 2 ~ 7 ~ 9 8 3 1 ~, I f V:~Y f~126~2
EXAMPLES
In order to exemplify the results achieved using the
aminimides of the present invention, the following examples
are provided without any intent to limit the scope of the
instant invention to the discussion therein, all parts and
percentages are by weight unless otherwise indicated.
EXAMPLE I
Synthesis of a vinyr ~minimifif~ monomer
This example illustrates the alkylation of 1,1-
dimethyl-2-acryloylhydrazide by treatment with an equimolar
amount of methyl iodide in acetonitrile.
~/ \N/ + CH31 ~`~f ~(CH3,3
CH3 O
This reaction is carried out with eqUimf~l~r quantities of the
reactants dissolved in acetonitrile (0.1 mol ea,'l00 mL) under
gentle reflux overnight. The mixture is concentraud on a
rotary ~ pu.~u., methanol is added and the pH is adjusted to
the ~ lphth~ in end point with methanolic KOH. The
solvents are removed in Vf3CUO, the residue is dissolved in the
2S minimum amount of benzene, the precipitated salts are
removed by filtration and the crude product is isolated by
removal of solvents to dryness. Purified monomer is obtained
by recrystallizaiion from ethyl acetate.
.
107
SUBSTITUT~ ~,t!EE ~ ' 2`
2 1 799~3
~, G`~ i C
WO 9!i118186 ' ` ' ' I ~ 9J/lZ61Z ~
EXAMPLE ~ ~
Synthesis of a trifluoroacyl dipeptide analide elastase inhibitor
peptidomimetic:
S o
O --Jl~NH
J~H3C~,,N~ ~CH3 li'
0 R
CF3
o
Il H,C~ ~CH3
lS ~o/H~C ~Oa/ ~CF3
An eqllimol~r mixture of an a-halo carbonyl
compound (such as 2-bromoacetyl-4'-isopropylanilide) ar a
dialkylhydrazide (such as N-(2-trifluoroacetamidoisobutyryl)-
N'-benzyl-methylhydrazine) in acetonitrile; at a final
c~Jn~ ion of ca. 0.1M is heated at reflux for periods ranging
from 1-6 days, r~rper.~line on the solvent employed, with the
2S progress of the reaction being llw~i1u~ by in-process TLCs.
On .,~ r' on of the reaction the mixture is cooled and the
solvent removed in vacuo. In the case of the aqueous reaction
con~iiti~ ~ the mixture is partitioned between water and a
suitable organic solvent to dissolve the ~minqmir~r (e. g.
chloroform). The solvent is removed in vacuo and the residue
is recrystallized from a solvent such as ethyl acetate to afford
crystals of the ~min~nir~f~ In reactions which did not employ
water as a cosolvent, the residue is treated with one equivalent
of 1.0M KOH in MeOH and gently warmed for 10-lS minutes to
ensure complete formation of the ylide. The methanol is
108
SUBSTlTU~.rS~E~ (Rl3L~
! 2 1 79983
~ WO 95/18186 ` PCTnJ593/12612
removed in vacuo and the residue triturated with THF and
filtered to remove the KBr formed. The residue is
recrystallized as for the aqueous case above from a solvent
such as eIhyl acetate to afford crystals of the aminamide. The
yields of the aminamide from the aqueous solvent systems ~re-
S superior. providing cleaner crude reaction mixtures and
superior yields. Using this method N-isobutyl-N-methyl-N-(2-
acetyl-(4'-isopropylanilide))-amin-N'-(2-
trifluoroacetamidoisobutyramide, N-benzyl-N-methyl-N-(2-
acetyl-(4'-trifluoromethylanilide))-amin-N'-(2-
trifluoro~cet:~mi~oisobutyramidel and N,N-dimethyl-N-(2-
acetyl-(4'-trifluoromethylanilide))-amin-N'-(2-
trifluoroacetamido isobutyramide are synth~si7.-~l These
mimetic ligands are useful as inhibitors of human leucocyte
lS elastase and porcine pancreatic elastase.
EXAMPLE 3
Synthesis of trifluoromethyl hydrazide modules:.
F3C~aCXC 3 H NN f J~H~ ~ ~N~
O F!
N-(2-trifluoroacetamidoisobutyryl)-N'-isobutyl-
2S methylhydrazine (TFA-AIB isot ~ eLllylhy~-azide) - A
solution of 2-trifl-- ~ ~el~l..;doicobt~tyric acid (796 mg, 4.0
mmol) in dry THF (15 ml) is stirred while dicyclohexyl-
calbO~ o (824 mg, 4.0 mmol) is added. The reaction is
51 bse,~ J strirred for three minutes, after which 1-
isobutyl-l-l...,tllyllJ~d-aLille (408 mg, 4.0 mmol) is added neat.
Dicyclohexylureâ ~ ,italLd ir~m~ t~ly~ The resultant
5l~cr~ncion is stirred for one hour, filtered to remove the
insoluble urea and the solvent is removed on a rotary
ev~u.alu. to afford an off white solid (1.11 g, 98%) which
3S
109 ,
SUBST~T~T~ S~ ! gP.I~L~ 2~)
2 1 79983
wo 95fl8186 Pcrfuss3fl26l2
exhibited spectral properties consistenl with those expected for
N-(2-trifluoroacetamidoisobutyryl)-,~f '-isobutyl-N'-methyl-
hydrazine .
In a similar manner, N~
~rifluoroacetamidoisobutyryl)-N'-benzyl-methylhydrazine. N-
('-trifluoroacetamidoisobutyryl)-N'.N'-dimethylhydrazine. and
N-( ' -trifluoroacetamidoisobutyryl)-N'.N'-pentamethylene-
hydrazine are prepared in comparable yields from 2-
trifluoroacetamido-isobutyric acid and the respective I, I -
dialkylhydrazines .
EXAMPLE 4
Synthesis of 2-bromacet-4.-trifluoromethylanalide
Br Jl~
NH2 NH
\J~ B r ~ ~
To a biphasic mixture conciC~in~ of diethyl ether
(300 ml), 4-trifl~ u~llcthylaniline (aminobenzotrifluoride,
25.0g, 0.155 mole) and aqueous NaOH (IM, 200 ml) cooled to 0
C is added, with vigorous stirring, a solution of l,lu.lloa~,e~yl
bromide (37.6 g, 16.2 ml, 0.186 mole) in diethyl ether (150 ml)
over one hour. The reaction is stirred an ~ n~l ten
minutes at 0C and the layers are then S~p~rA~ The aqueous
phase is extracted with ether (200 ml) and the combined
organic layer are dried (sat'd aq NaCI, Na2SO4) and
' to afford 47 g of a yellow oil. Crys~lli7~ion from
ethyl acetate afforded two crops of pale yellow rod-like
crystals (27.9, then 11.3 g, 89%) which exhibited spectral
110
SUBST~TU ~ E ~ (P~U~L 2&,
2 1 79~83
~ W0 9S~18186 1~ U~Y.~I~16I2
properties consistent with those expected for 2-bromoacet-~'-
trifluoromethylanilide.
In the same fashion 7-bromoacet-~'-
isopropylanalide was made (74.9g. 79~) and characterized
EXA~fPLE 5.
Synthesis of I -substituted- I-methyl hydrazine modules
C~3 C~3
N--NHz + RX ~ N--NH2
H R
l-benzyl- I -methylhydrazine - A solution of
lS methylhydrazine (46 g, I mole) in THF (200 ml) is coole.d at
0C and a solution of benzyl bromide (57.01g, 0.3 mole) in THF
(100 ml) is added dropwise with stirring over a period of 30
minutes. The reaction is stirred at 0 ooC for another 15
minutes, then heated to reflux and held at reflux for two hours.
A water-cooled d~ ~d .,J '-- is set up and
~pyl~ lla~ely half of the solvent is removed by distillation.
The residue is poured into water (200 ml), which is then made
basic by the addition of con~ aqueous NaOH. The
layers are s~Fo~At~ the aqueous phase (ca. 250 ml) is
extracted with ether (2 x 200 ml) and the combined organic
phases are washed (I x 100 ml H2O), dried (sat'd aq NaCI,
MgSO4) and ' ' by ~ictillori~n to give 54 g of a
yellow oil. Distillation at reduced pressure afforded l-benz~ l-
:- ylhydlaLine as a colorless liquid (b. p. 103-107, 16 mm
Hg, 19.8 g, 48%), which exhibited spectral ~ ies consisten~
with those reported previously.
In a similar manner, I -isopropyl- I -
methylhyd.aLi~c 12.3g, 42%); I-(t~r~-butyl 2-acetyl)-1-
methylhydrazine (3.40g, 42%); l-isobutyl-l-methylhydrazine
(9.80g, 29%), ~nd 1-(2-(3-indolyl)-ethyl)-1-methylhydrazine
111
SUBST~ T ~R~LE 2~)
. r, ~ !, ~ ' . t j 2 1 7 9 9 ~
~VO 9~/18186 PCr/US93112612 ~
il.3~g. 69%) are prepared from the respective alkyl bromides
and characterized.
EXAMPLE 6
Synthesis of a vinyl oxazolone-derived aminimide monomer: -
+ C6Hs(CH3)2NNH2 t-3uOK H O H3C~ CH3
0 o~~~cl~ OTs ~uOH O~H3C CH3 ~3
This reaction is carried out by stirring equimolar
amounts of the I, I ,1 -trialkylhydrazinium tosylate (prepared
from l-methyl-1-phenyl hydrazine and p-toluenesulfonic acid
lS in toluene) in t-butanol at room t~ U.~ overnight. An
equimolar amount of 2-vinyl-4,4-dimethylazlactone (SNPE
Chemical Inc.) is added and, the solution is stirred an additional
6 hours. An equal volume of toluene is added. The system is
filtered and the filtrate is col1cc..lr~t~,d in vacuo on a rotary
t:V~l~Ul~llUI to yield the product as a thick oil. Pure crystalline
product is obtained by crystallization from acetone.
EXAMPLE 7
Preparation of ~minimitl~ r~ 1i7~d agarose:
This example is ill ' for filr^ti~mi7in~
c~ ..,ially available 6% crosslinked agarose with l-benzyl-
I,l-dimethyl chl~-u--.etl~yl aminimi~ (prepared from 1-
benzyl- I, I -dimethylhydrazinium chloride [from I, I -
dimethylhydrazine and benzyl chloride in toluene] to produce
the ~minimi~l~ functionalized agarose, useful as a hydrophobic
interaction support material for the chromatographic
separation of proteins .
112
S~iBST~TUTE SH~T (R~LE 26)
~ wo 95~8186 2 1 7 9 ~ 8 3 PCT/US93112612
~ ; base
[AGAIROSE] + CICH'CO~ (CH3)~ [AGA~OSE]
OH ~ OCH'N~N(CH3)'
b~
S .
This reaction is carried out by steeping the agarose with
potassium t-butoxide in a mixture of t-butanol and DMF under
10 nitrogen for one hour at room temperature. The hydrazinium
salt is added, and the mixture is stirred for 24 hours. The
functionalized agarose is collected by filtration, washed with ~-
butanol, methanol and finally with water. This material is
stored in water for future use.
lS
EXAMPLE 8.
Construction of a trimeric species using an epoY~ ion
iteration: An example of stepwise poly...~ri7~ D with MW + I
Dalton.
A mixture of styrene oxide (12.02 g, 0.1 mole), 1,1-
dimethylhydrazine (6.01 g, 0.1 mole). and methyl 4-pentl~no~t,~
(11.41 g, 0.1 mole) in methanol (150 mL) is stirred at room
t~ ,la~Ul~: for four days. The solvent is removed in vacuo to
afford a white solid (26.4 g, 101%, >95% pure).
2S This solid is dissolved in methylene chloride (300
mL) and cooled to 0 C while a solution of m-CPBA (51.8 g, 50-
60%, ca. 0.15 mole) in methylene chloride (200 mL) is added.
The mixture is stirred until the alkene is c~r~c~m~od (this
reaction is followed by IH-NMR). The mixture is extracted with
1.0 M NaOH solution (500 mL) and the organic layer is dried
(saturated NaCI, anhydrous Na2S04) and co~lc.,.l~lalr,d to afford
a cream-colored solid (29.3g, 106%) which is recrystallized
from methanol to afford the epoxy ,....;..;...:~ (26.3g, 95%).
This epoxide (0.095 mole) is treated with I,1-
di~u.,;~lylL~ ~ (5.73 g, 0.095 mole) in methanol (100 mL
113
SUBSTITliT~ S~t~T ~UL~ 26)
~ t ~q983
W0 95118186 ' ' ` ~ /U:~Y~ 6I2
and is refluxed for eight hours. The mixture is cooled and
methyl l-pentenoate (10.87 g, 0.09~ mole) in methanol (loo
mL) is added. The resultant solution is stirred at room
temperature for 48 h. The solvent is removed to provide a
pale yello~ solid (45.6 g, 114%). Treatment of this material
with m-CPBA (ca. I.S eq) in methylene chloride provides~ after
recrystallization, colorless crystals of the epoxy~iqminimide
(38.2 g, 0.091 mole, 96%).
The epoxide is treated with I, I -dimethylhydrazine
(5.47 g, 0.091 mole) in methanol (100 mL) at room
temperature and the ylide which is formed in situ is acylated
by the addition of methyl 4 pPn~n~qtP (10.39 g, 0.091 mole).
Treatment of the crude reaction mixture with excess m-CPBA
in methylene chloride affords the epoxide (47.64 g, 0.08 mole).
Purification and iteration of the previous steps can
lS provide a polymer which has an exact molecular weight of 120
+ N(158) Da where N is the number of ~ ' ~ steps.
EXAMPLE 9.
Construction of a functionalized surface via hydrazine ester
~; ~Pncotion with epoxy silica.
A slurry of epoxy silica (10.0 g, 15 m Exsil C-200
silica, vide infra) in methanol (100 mL) is treated with 1,1-
dimethylhydrazine (6.01 g, 0.1 mole), and stirred at room
.,latu~c for two hours (mPrl~qni~l stirring provides a
2S more efficient ~ ldtiVC ~1. ' c, as well as a superior
product). To this slurry is added methyl 4_p~..r. rr.~ (11.41
g, 0.1 mole) and the resultant mixture is n~ocl~nir~lly stirred
for five days. The r, ~ 1i7Pd silica is collected by filtration
and washed by repeatedly s~-~F: "n~ in methanol and
filtering to removed the soluble material. After six washings,
the solid obtained is dried overnight in a vacuum oven (60
C/O.I mm Hg) to afford 9.86 g of product.
This material is s r '~~ in methylene chloride
and treated with m-CPBA (51.8 g, 50-60%, ca. 0.15 mole). The
3~ 5.1cppncir~n is stirred - ~ r~lly overnight at room
114
SUB~T~TUTE SffEET ~RU~ E 26)
. ` ' '~ 21 7~983
~ WO ~>5/18186 I ~ U:~Y~ 6I2
temperature and washed with methanol, as before. to remove
the unreacted and spent reagents. The solid is dried overnight
in a vacuum oven (60 C/0.1 mm Hg) to afford 9.83 g of
produc t.
This homologous epoxy silica is slurried in methanol
(100 mL) and treated with l.l-dimethylhydrazine (6.01 g, 0.1
mole). The suspension is stirred at room temperature for two
hours and methyl 4-Fer~t~no~t~ (11.41 g, 0.1 mole) is added.
After five days, the material is collected by filtration and
washed repeatedly with methanol as above. Drying this
material overnight in a vacuurA oven (60 C/0. I mm Hg)
affords 9.88g of the alkene-functionalized surface.
Further iteration of the epoxidation and hydrazine-
ester condensations provides silica beads with known
lS filn~ tion~l-ty (or functionalities) and size (or sizes).
EXAMPLE 10.
Acylation of S- 1 -ethyl- I -methyl- I -phenylhydrazinium iodide
with bromoacetyl chloride:
C2H5 ~N,J~ + ~ 2 ~N,~
2S H2N CH3 I- Br ~O
A solution of S-l-ethyl-l-methyl-l-phenyl-
lly-LaLilliulu iodide (2,78 g, 10 mmol) in benzene (50 mL) and
pyridine (2,38 g, 30 mmol, 2.42 mL) is cooled to 0 C, then
treated with a solution of blulllO~tyl chloride (1.73 g, 11
mmol, 0.91 mL) in benzene (10 mL). The mixture is stirred at
0 C for one hour, then room ~ I- al- ~ for two hours.
During the course of the reaction, the pyridinium salts
3S precipitate and s~bs~q~ n~iy are removed by filtrabon,
115
SUBSTITUTE S~ ' t ~)
:, `` ; .' ~ 2 1 79983
WO 95118186 PCT/US93/12612
Following removal of the solvent, the residue is recrystallilze~d
from ethyl acetate to afford pale yellow crystals of ~-
bromoacetyl-S- I -ethyl- I -methyl- I -phenylhydrazinium inner
salt ('.24 g, 78%). Alternatively, Amberlite IR-45 resin can be
used in place of pyridine to extract the acidic protons. The
resin is conveniently removed by filtration.
EXAMPLE 11.
Homologation of 2-bromoacetyl-S- I -ethyl- I -methyl- I -
phenylhydrazinium inner salt by alkylation of an hydrazine
followed by acylation.
~N.. ? + N--NH~
~N H3C~
Br N ~ + Br ~,N~--o~N~
A mixture of 2-bromoacetyl-S-I-ethyl-l-methyl- I -
phenylhy.~ inner salt (2.24 g, 7.8 mmol) in THF (100
mL) is cooled to O C while a solution of l-ethyl-l-methyl-
hydrazine (6.96 g, 0.94 mmol) in THF (25 mL) is added
dropwise. The mi~ture is stirred for 15 minutes at O C, then
room t~ ..rlLulc overnight. The resultant s~Cp~nci~rl is
filtered. The ~c.,i~ t-,d diaa~ ullcla are isolated as a whlt~
powder (2.36 g, 84%).
The ~ la from the above reaction are separate~
on a C-18 reverse phase silica media with an acetonitrile-water
gradient. The fractions c~ inin~r the desired liaa~lc~ r are
pooled, and the product is isolated by removal of the solvents
in vacuo.
116
Sl~BST~T~ ~ ~ S~-L~ (~ULE 2B~
~ WO 9SI18186 2 1 7 ~ 9 8 3 PCTIU5931126IZ
~r /~ C-Hs
O HsC. CH3 0 CH~0
The resulting dried powder from the above reaction is
dissolved in benzene and pyridine ( 1.59 g, 20 mmol, 1.61 mL
or Amberlite IR-45 resin, vide supra). then cooled to 0 C while
a solution of bromaacetyl chloride ( I .13 g, 7.2 mmol, 0.59 mL)
in benzene (10 mL) is added The mixture is stirred overnight
at room ~cl.lp~lalul~. The pyridinium salts are subsequently
removed by filtration and the filtrate is concentrated to afford
a pale yellow solid (1.87 g). The pure material is obtained by
recrystallization of the yellow solid from ethyl acetate.
The material from the above reaction is dissolved in THF
(50 mL), then treated with I -ethyl- I -methylhydrazine (0.46 g,
6.2 mmol) in THF (10 mL) at 0 C. The mixture is stirred
overnight at room t~ .,lalul~. The volume of the reaction
mixture is reduced by a~loxilllâtely half, then the precipitate
is filtered and washed with cold ether to afford a white powder
(1.36 g, 72%). The di~st~,lc~lll,,la are again separated on a C-18
reverse phase silica media with an acetonitrile-water gradient.
The fractions ~ 1~ 'nin~ the desired dia~t.,lcG..-.,l are pooled
and the product is isolated by removal of the solvents ~n vacuo.
The product from the above step is dissolved in benzene
(20 mL) and pyridine (0.71 g, 9 mmol, 0.73 mL or Amberlite
IR-45 resin. vide supra), then cooled to 0 C while a solution of
acetyl chloride (0.35 g, 4.4 mmol, 0.31 mL) in benzene (5 mL)
is added. The resultant mixture is stirred overnight at room
~ al~ . The mixture is filtered and the solvent is
removed in vacuo to afford an orange gum (2.13 g).
Cryst~lli7~io~ of this material from ethyl acetate gave the
trimeric ~minimi~lP stereoisomer shown below:
117
SU~STITUT~ S~E~ L~ ~63
2 1 79983
WO 95/18186 I ~~ Y.5112612
EXAMPLE 12.
Synthesis of an hydrazinium backbone via hydrazide
homologation .
AoCI ~ H N~OX 0C-nT J~uox
Acetyl chloride (8.64 g, 0.11 mole) is added to an
lS ice-cooled solution of N-amino-N-methylglycine tert-butyl
ester (11.6 g, 0.10 mole) in pyridine (10 mL) and THF (250
mL). The mixture is stirred at 0 C for 30 minutes, then room
t~.,.r,...~.l...~ for three hours. The mixture is COIIC.i...latLd on a
rotary ~;va~lalol and the remaining volatiles are ~emoved in
vacuo. The residue is recrystallized from ether to afford the
hydrazide ester (14.S4 g, 0.092 mole).
2S H O ~ 1N~N~OX
H~N ~ ~0~<
3 The product is dissolved in THF (300 rnL) and
treated with ~ ,ace~ic acid (0.1 mL, 148 mg, 1.3 mmoles).
This mixturc is stirred at room t~.y~ for two hours, then
a solution of N,N'-dicyclohexyh,~ul,o.~ (19.22 g, 0.093
mole) in THF (100 mL) is added followed by tne addition of a
118
SUBSTITlJ I E SH~T (Rll~ F 26)
W095/18186 ~ 2 ~ 79983 PCI~U593/12~12
solution of N-amino-N-(2-methylpropyl)-~lycine rerr-butyl
ester (1:~.71 g. 0.094 mole) in THF (100 mL). The precipitate
(dicyclohexylurea) is removed by filtration and the filtrate is
concentrated on a rotary evaporator to afford an amorphous
mass. which yields white crystals (27.06 g. 89~, 0.08~ mole
after recrystallization from ethyl acetate.
N--N ~ 1 Ahl, E~,o, aiU~ N--N'~
,~ N~O N--N~O
A solution of this bis-hydrazide (27.06 g, 0.082
mole) in diethyl ether (300 mL) is treated with methyl- iodide
(17.3 g, 0.12 mole) and refluxed for twelve hours. The reaction
mixture is cu~ ated to remove excess methyl iodide, and
dissolved in is~lulu~u101 (200 mL). Amberlite IR-45 resin is
added and the solution is stirred at room ~ u..dtUlc for ei~ht
hours. The solids are removed by filtration and the solvent is
reduced to saturation. This saturated solution is cooled to -20
C for 36 hours, ar~d the resultant crystals are collected by
filtration to afford bis-l~y~ Li~illlll inner salt (21.42 g, then
5.87 g, 93%) as a racemate.
EXAMPLE 13.
Tr ,uul~.tion of an ~mi- ~lidinium functionality into an
aminimide backbone.
A solution of l-amino-4-pyri~in -~rboxylic tert-butyl
ester iodide (3.22 g, 10 mmol) in THF (25 mL) is added to a
solution of N-benzoyl-N'-acetate-N'-isobutyl-N'-
methyllly~l,,.,;..;,..- inner salt (2.64 g, 10 mmol) and N,N'-
di~ lo~_,.yl~o~ (2.06 g, 10 mmol) in THF (100 mLl
and stirred for two hours at room lr- ~ ...c. The
118d
SU~STiTUTE S~ T ~ LL- 2~
t (~ 2 1 79983
WO 9S118186 ~ 3II26I2
suspension is treated with Amberlite rR-45 (or an equivalent
basic resin) for three hours at room temperature, then filtered
to remove both the resin and precipitated dicyclohexylurea.
The filtrate is concentrated, and the residue is recrystallized
from ethyl acetate to afford the bis hydrazinium inner salt
(3.56 g, 78S~).
H,N~N,~
10 ~ ~><
Ph~N~ ~OH J~ ~N~JI~ ,N~
lS The entirety of this material is dissolved in
acetonitrile ( 150 mL), and Amberlite IR- 118 is added. The
mixture is refluxed until the ester is completely : - '
The resin is removed by filtration and the solution is treated
with N,N"-di-~,loh~ylcarbodiimide (1.61 g, 7.8 mmol) in
P~etonitril~ (25 mL). After three minutes of stirring, I-benzyl-
I-methylhydrazine (1.38 g, 9.36 mmol) is added neat, and the
resultant sl~cp~ncion is stirred at room t~..a~ul~ for two
hours. Removal of the precipitated di~ .,lol~ylu.~d by
filtration, and Cull~ ha~ion of the filtrate, affords a solid (5.01
2S g), which is dissolved in isopropyl alcohol (100 mL). Propylene
oxide (0.542 g, 9.36 mmol) is added. The mixture is refluxed
for seven hours, then the volatile ~.G..I~ ' are removed in
vacl~o. Cryst~lli7~tion of the residue from ethyl acetate
provides the tris-ylide (2.95 g, 5.30 mmol, 68 %).
119
~UBST~U ~ HE~r ~RU~E 26~
2 1 79983
~ Wl) 95/18186 I .~ Y3/126I2
N N~-- ' ) 'N
N~ ~; N~,~ N~
o~f HN_N~Ph --N~ Ph
l l
EXAMPLE 14.
Synthesis of an hydrazine tethered pyrimidinone via
quaternization.
lS A mixture of 2,4-diethoxypyrimidine (16.8 g, 0.1
mole) in ~ (250 mL) is cooled in an ice bath while a
solution of 3-bromo-1-tert-butyldimethylsilyloxypropane ( 5.3
g, 0.1 mole) in s~ t-`";tril~' (150 mL) is added. The rate of
addition is adjusted so that the internal reaction ~ ,.a~ure
20 does not exceed 10 C. The mixture is stirred at 0 C for two
hours, then refluxed for 10 h. The solvent is removed in vacuo
The residue is dissolved in THF (200 mL), and a solution of
tetra-n-l,~ /' fluoride in THF is added (1.0 M, 100
mL). The orange solution is stirred at room t,~ a~lc for
25 one hour, then is poured into brine (300 mL). The layers are
separated and the aqueous phase is extracted with ether (2 x
200 mL). The combined organic extracts are dried over sodium
sulfate, filtered and ~ .,d to afford an orange oil (36.7
g). This oil is C~ glalJIIcd on silica gel (gradient elution
with EtOAc-Hexanes) to provide the pure alcohol (13.1 g, 0.066
mole, 66%).
3S
120
SIJB~TUT~ ~'E~T 'F~ULE 76~
~ ;"; ` S 2179983
WO 95/18186 r~I~U~Y~I126I2
/~ o~
~N then r~iU~ N
N O~\ Br/~/\OTBDMS TBAF. TH 1~ j~O
--OH
This alcohol is dissolved in THF (200 mL), and
treated with m.~th~ln~sulfonyl chloride (9.07 g, 0.079 mole,
6.12) and 1,8-diazabicyclo[5.4.0]undec-7-ene (12.05 g~ 0.079
mole, 11.84 mL) at 0 C for two hours. The mixture is
Ll~uar~ d via cannula into an ice-cooled solution of
methylhydrazine (15.2 g, 0.33 mole, 17.6 mL) in THF (100 mL).
The reaction is stirred at 0 C for four hours, then room
t~ p~.a~ulc for two hours. This mixture is poured into 200
1~ mL of a solution of I M Na2CO3 saturated with NaCI. The
layers are separated, and the aqueous phase is extracted with
diethyl ether (2 x 200 mL). The combined organics are dried
over sodium sulfate and ~c ~ ~ILd to afford an amber
syrup. Column chromatography on silica gel (gradient elution
20 with chloroform-methanol) provided the pyrimidylhydrazine
in 48% yield (6.77g).
o~\ ~ I o~
2S ¢~N MsCL DBU THF ¢~ N
--OH --OM- ~ IN~ NH,
A solution of this hydrazine (2.14 g, 10 mmol) in
1.0 M NaOH (30 mL) is warmed gently to eYhP~ti- n of the
starting material. The reaction mixture is l~vl' li7~1 and the
resulting powder is triturated with THF to dissolve the
1~,' Filtration of the salts and c~ A~ n of the
3~
121
~U~STITUTE S~EET (RULE 26)
~ ~ j ( 21 79983
WO 95/18186 PCTNS93/12612
filtrate affords nearly pure 1-(3-( I-uridyl)-propyl)- I -
methylhydrazine ( 1.80 g, 97~c).
In another flask. the pyrimidyl hydrazine ('. 11 g.
10 mmol) is treated with an excess of anhydrous ammonia in
methanol at 0 ~C. then stirred at room temperature overnight.
Removal of the solvent on a rotary evaporator afforded a
residue which was chromatographed on silica gel (gradient
elution with chloroform-methanol), providing the pure 1-(3-(1-
cytidyl)-propyl)- I -methylhydrazine ( 1.62 g, 8~%).
O~ N~OH. H20 ~ IN O
lS ¢~ N~ N*
~NH~ MeOH ~ ~ ~1
¦ IN ~o
~N,NH2
I
EXAMPLE 15.
25 Stepwise ~c5~mhl~. of a modular scaffold which presents a
known sequence of nucleotides to a desired target.
A solution of 1-(3-(1 -uridyl)-propyl)- I -
methylhydrazine (186 mg, 1.0 mmol) in THF (10 mL) is treated
with acetyl chloride (79 mg, 1.0 mmol, 72 mL). The resultant
30 solution is stirred at room ~ alul~ for three hours. This
mixture is L.~ rL..~ via cannula into an ice-cooled solution of
tert-butyl 1;,., -- (195 mg, 1.0 mmol, 161 mL) in THF (5
mL). The hydrazinium bromide is converted to the inner salt
by treatment of the SllSp~r~cit~n with Amberlite IR-45 resin.
35 The volatile . , are remove in vacuo, and the residue
122
SUBSnTUT~ SHE~T (RULE 26)
~ ~ 7 ~ 9 9 8 3
WO 95/18186 PCT/US93/12612
is recrvstallized from ethyl acetate to afford the 2-acetyl-1-(3-
( I-uridyl)-propyl)- I -(tert-butyl 2-aceto)-1-
methylhydrazinium inner salt (265 mg, 7s5rc).
¢~NH l.Acc~ylchlonde,ll{F ~NH
~0 3 Ambc;LIc;R~15
0 --N,NH2 . ~/\N\ ~I'
This material was dissolved in methanol (10 mL)
and three drops of trifluoroacetic acid are added. After 10
minutes at room ~ atul~, the mixture is concc..tlated on a
lS rotary ~v~.,...tv,. To a solution of this crude acid in THF (10
mL) is added N,N'-dicyclohexylca l,o~ (ISS mg, 0.75
mrnol). The resultant mixture is treated with 1-(3-(1-cytidyl)-
propyl)-l-methylhydrazine (140 mg, 0.75 mmol) in THF (10
mL). The white sllcr~ncir)n is stirred for two hours, filtered to
20 remove the ~ d urea, and the filtrate concentrated.
The residue is recrystallized from methanol to afford the
hydrazide (220 mg, 0.48 mmol, 65%).
)cO 'N
N 1. lPA. McOH \
~ 2. CCC. 71~: N~
~= N~o H~cO
~< /~ ~NH2
123
SUB~T~r~Tr SH,~ ~ (R~E 2~
` t ~ 2 1 7q~3
W09Srl8186 ' r~,O~y~ 6~2
This material is treated with tert-butyl
bromoacetate (94 mg, 0.48 mmol, 77 mL) in THF (S mL). The
hydrazinium bromide is converted to the inner salt by
treatment of the suspension with Amberlite IR-45 resin. The
volatile components are remove in vacuo, and the residue is
purified by column chromatography on RP C- 18 silica (gradient
elution with MeOH-Water) to afford the bis-hydrazinium inner
salt (208 mg, 0.38 mmoles).
~o
\ . N
~=O \.~
N N/~eo 1 3.CH~:0 1.3~ ~e N~0
lS ~ Amber~r~ ~NH~
~N )~
Deprotection and reiteration of the above steps with
the uridyl s~hstitl~t.-d hydrazine provides the tris-hydrazinium
inr~er salt, which presents the sequence U-C-U as a recognition
sequence for the RNA codon A-G-A.
2S ~0 Ntlz O
~ / /
J~N~N~N~ ~N~N-~10'
3S
124
SUB~TI~-U~~r S~-c ~ U. E ;2~
., ,: .,., ", ~ C, 21 7g
WO 95/18186 ~ 3 PCrlUS93112612
As can be seen, this series of reaction can be
repeated, substituting the five natural bases as well as other
bases for each step as the desired sequence dictates or
warrants. This materiai also can be elongated using silyl
protected purines. which prevents inter- and intramolecular
binding of the bases. In some cases, the ring amine of the
cytidyl hydrazine is protected as well, by a trialkylsilyl group
prior to incorporation into the backbone.
EXAMPLE 16.
Synthesis of carbohydrate modules for incorporation into
tlminimitlt~ backbone scaffolds
Module I
.
OH COOH COOH
OH a.b ~ OH
Ic
COOCH3
2S ~~o ~~ OAc
At O /_~
AcO
(a) p-TsCI ( I et~), Pynt~ine, n
(b) DBU, t~iethyl ether, n
(c) Ac20; Pyntiine, CH2C12, n
To a solution of sialic acid (l g, 3.23 mmol) in
pyridine (2.9 mL, 37 mmol, ll equiv) is added p-
Toluenesulfonyl chloride (620 mg, 3.23 mmol, l.0 equiv). The
3S reactioQ rrlixture is stirred at room t. ~l.. ,.l,.. ~; for 12 h. The
125
SIJB~TITUTE SHE~T (RULE 26)
.S 2t 79983
~ WO 95/181X6 PCTIUS93/12612
crude mixture is quenched with water, then extract with
diethyl ether several times. The combined organic extract is
washed ~i~h I N HCI~ dried with M~SO1, filtered and
concentrated on a rotary evaporator to give the crude product
(1.4 g, 95qc).
To the tosylate from the above reaction (I g. ~ 16
mmol) in a suitable solvent, such as diethyl ether (10 mL), is
added DBU (821 mg, 5 4 mmol, 2.5 equiv). The mixture is
stirred at room temperature for 5 h. The crude mixture is
washed with I N HCI, then saturated NaCI solution, dried over
MgSO4, filtered and concentrated to obtain epoxide 2 (566 mg,
90%)-
To a solution of epoxide 2 (500 mg, 1.7 mmol) in
pyridine (695 mL, 8.6 mmol, 10 equiv) is added acetic
anhydride ( 1.05 g, 10.3 mmol, 6 equiv). The reaction mixture
15 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 obtain pure 3 (683 mg, 92%).
Module Il
~COOCH3 ~1 AcO~
(~) Glycidol. Ag-S liqlale. C6H~,
To a solution of 4 (500 mg, 0.98 mmol), in a
suitable solvent such as benzene (6 mL), is added Ag-Salicylate
(265 mg, 1.08 mmol, 1.1 equiv). After 10 min at room
t~ ., glycidol (73 mg, 0.98 mmol, 1.0 equiv) is added to
- 35 the mi~cture. The reaction mixture is stirred at room
126
SVBST'TU 1~.- S;4~ LE Z6)
' ` 21 79983
W0 95118186 r~ Y.~/I26l2
temperature for ~ h. Water is added to quench the reaction.
The organic solution is then washed with saturate~ aqueous
NaCI, dried over MgSO4, filtered and concentrated. Purification
with column chromatography gives 5 (483 mg, 90C~c).
Module 111
CHzOH CHO CHO
0 H~ HO ~ AcO
HO OH H HO OH AcO OA~¦
6 7 8 NHAc
Ic
d
1~ NHAc 9 NHAc
(a) (COCI)z, DMSO. Et3N, CH2Cl2, -60 C
(bl Ac20. Pyridin~, CH~CIz, n
(c) Ph3PCH21, PhLi. THF. n
(d) m-CPBA. CHlCli, n
A three neck round-bottom flask is charged with 10
mL of a suitabb solvent, such as CH2C12, and oxalyl chloride
(540 mL, 6.2 mmol, 1.2 equiv). The solution is stirred and
cooled to -60 C as DMSO (740 IlL, 810 mg, 10.4 rnmol, 2 equiv)
in dichl~-ul--.,.hd-.c (5 mL) is added dropwise at a rapid rate.
After 5 min, 6 (1 g, 51.8 mmol, 1.0 equiv) is added dropwise
over 10 min period, rn9int~inin~ the ~ ...c at -60 C.
After an P~ltli~in-sl 15 min, trieLII~' - (7.2 mL, 51.8 mmol,
10 equiv) is added dropwise, keeping the t~...,.. Irl~c at -60
C. Stirring is continued for 5 min. The mixture is warmed to
room ~ c, and water is added. The aqueous layer is
separated and extractcd with a polar solvent, such as ethyl
127
SUBSTI~U~ E,~ ~F~L-- 26~
t ~. 2 ~ 79983
o 95118186 P~ 112612
acetate. The organic layers are combined, washed with I ~'c HCI
. until acidic, then washed again with saturated sodium chloride
and dried over anhydrous magnesium sulfate. The filtered
solution is concentrated by rotary evaporation to obtain the
aldehyde 7 (890 mg, 90%).
To a solution of 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
t~ UlC 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 8 (1.48 g, 88%).
A 50 mL. three-neck, round bottom flask, equipped
with a pressure-equalizing dropping funnel, thermometer,
magnetic stirring bar, and serum caps, is charged with
~ tllyllfi~lJ.nylphosphonium iodide (I.lg, 2.74 mmol, 1.1
3L~ equ;v) and THF (10 mL), then flushed with argon. The flask is
cooled in an ice bath, and the s~crPnci~n is stirred under a
positive pressure of argon while 5 ~LL to 14 llL of 1.8 M
phenyllithium in 30:70 ether:cycl~lh~S~nP is added dropwise
until the sl~cper~cion develops a pPrml - yellow color. 1.6
mL of 1.8 M phenyllithium is added dropwise over 10 min.
The ice bath is removed, and the orange sllcp~ncion corlt~inin~
excess phosphonium salt is stirred at room t~ Lul~ for 30
min. The reaction mixture is stirred and cooled to 0-5 C.
Compound 8 (I g, 2.49 mmol, 1.0 equiv) in 5 mL of THF is
added dropwise over 10 min. The dropping funnel is rinsed
with a small amount of THF. The mixture is stirred at room
t~ ... e for 2 h. The light orange mixture is hydrolyzed
by adding methanol (I mL). Most of the solvent is removed on
a rotary ~ until a slurry results. The slurry is diluted
with petroleum ether (20 mL), and the SllrPr~q~nr solution is
decanted and filtered. The filtrate is washed with water, dried
over MgSO4, filtered and cor.c~ ' to give the desired
product (900 mg, 90%).
3S
128
SUBST~T~Tr ~i5 LF, ~ ULE 2~
W0 95118186 ; ~ t S 2 ~ 7 9 9 8 3 r~ Y~/l26l2
To compound 9 (800 mg~ 2.0 mmol) in CH2CI~ (10
mL) is added m-CPBA (410 mg, 2. l mmol, 1.~ equi~ ). Stirring
is continued at room temperature for ~ h. The resulting
mixture is washed with 10% Na2so3~ water and saturated NaCI.
The organic layer is dried over MgSO4, filtered and
concentrated. Purification by column chromatography gives
the desired product (740 g, 89%).
Module IV
H20H CH20TMS
lS HO ~ TM~
(a) TMSCI, CH2CI2, Et3 N, rt ¦ b, c
(b) Ethylene oxide, CH2CI2, rt, ~TsCI, pyridine
CH2OTMS
TM
TMSO _~/
TMSO OT91S
12 HN ~~ OTs
2S
To a solution of amine 6 (500 mg, 2.59 mmol), in a
suitable solvent such as CH2cl2 (5 mL), is added trimethylsilyl
chloride (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, saturated NaCI and dried over anhy~luils m~ Sillm
sulfate. The filtered solution is co~ cd by rotary
evaporation to obtain the silylated product, 11 (1.3 g, 91%).
3S
129
SI~B~TiTlJTE SlttET (RULE 26)
c 2 t 79983
wo 951~8186 r~l,o~Y~tl26l2
To a solution of 11 (I g, 1.8 mmol). in a suitable
Solvent such as CH2C12 (10 mL), is added ethylene oxide (87
mg, 1.98 mmol, 1.1 equiv). After stirring ~ h at room
temperature, p-toluenesulfonyl chloride (340 mg, 1.8 mmol. 1.0
S equiv) and pyridine (~80 mg, 3.6 mmol, 2 equiv) are added to
the reaction mixture. Stirring continues for another 12 h. The
organic layer is washed with water and saturated NaCI, dried
over MgSO4, filtered and concentrated. The desired product.
12, (1.1 g, 86%) is obtained by flash column chromatographv.
Synthesis of phal~..acu,uhol~ containing modules for
incorporation into aminimide backbone scaffold
As an illustration of the chemistry involved ~ i~h
the concepts mentioned above, the following examples ~re
specific cases of the formation of monomeric units- formed
through linking phalluacophoric molecules to a hydrazino or
hydrazido moiety for further mo-lifir~tjon or polymerization:
EXAMPLE 17.
Synthesis of 5N-5-(N,N-Dimethyl-l-amino-3-
propenyl)dibenzo[a,d]cycloheptene:
130
S~BSTITUTE SHE~T (RULE ~6~
WO 95~18186 ` ; ' 2 1 7 9 9 ~ 3 P~ Y~/I26~2
,I ,a b ~r~l~
1 ~F Q~ '
'---- ~ h~'r~--O-- 3 -)~`~'r~--3'`~
111F T~IF
A solution of dimethyl~mi~ upyltriphenyl-
r chloride (23.4 g, 61.0 mmole, which is prepared
from the reaction of tripll...y'~hc ,' r- and 3-
dimethylaminopropyl chloride) in a suitable anhydrous solvent,
such as THF (300 mL), is cooled at 0 C while an equimolar
amount of a strong base, such as n-butyllithium (2.5 M solution
in hexanes, 25.0 mL, 62.5 mmol) is added dropwise with
stirring over a period of 30 minutes. The reaction is stirred at
room ~L.~.,.alUI~ for another hour. A soluhon of
131
SU~TI I ~TE ~I~E~T (~UL~ 26!
21 79983
;. ,, .,`.,
WO 95118186 ' -' PCT/US93'~2612
dibenzosuberenone ( 1~.5 g. 60.6 mmole) in an approprlate
anhydrous solvent, such as THF (100 mL) is added dropwise.
with stirring, over a period of 30 minu~es. The reaction is
stirred at 0 C for another two hours, then quenched with the
- addition of water (150 mL). made basic such as with the
addition of concentrated aqueous NaOH ( 10 mL). The volume
of the resulting mixture is partially reduced in vacuo, then
extracted with ether (2 x 150 mL). The combined organic
layers are washed with saturated aqueous NaHCO3 (2 x 150
mL), then brine (I x 100 mL), dried over allhy.l.~,us MgSO4,
and conc~.. Lla~d by rotary evaporation to afford 29 g of a
yellow 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
lS compound (14.2 g, 859~o). A portion is repurified to yield a
sample for analysis.
EXAMPLE 18.
Synthesis of 5H-5-(N,N-Dimethyl-N-(2-(N-methyl-N'-
formylhydrazino)-ethyl)- I -amino-3-propenyl)-
dibenzo[a,d]cycloheptene formate:
~~\ H~H --O~lH ~~N ~ `NJlH
A solution of SH-5-(N,N-Dimethyl-l-aminoprop-3-
enyl)-dibenzo[a,d]cycloheptene (10.3 g, 37.4 mmol) in an
30 ~hyd.~,_ solvent, such as THF (100 mL), is stirred while N-
(N-methyl-N'-formylhydrazino)-2-ethanol formate ester (5.47
g, 37.4 mmol, prepared from the reaction of 2 equivalents of
formyl chloride with N-(N-methylhydrazino)-2-ethanol) is
added. The reaction mi~cture is gently refluxed overnight. The
35 solvent is cvnpu~t~,d, and the resulting residue is
132
S~IB~ I t S~'rE ~ E ~6~
c
WO 95/18186 . ~ , , I . 2 t 7 9 9 8 3 PCT/lL1593~12612
recrystallized. After filtration, the isolated solid is washed
thoroughly and dried in ~acuo to yield the desired product
(14.9 g, 95C~'c). A portion is repurified to yield a sample for
analysis .
EXAMPLE 19.
Synthesis of 5H-5-(N,N-Dimethyl-N-(2-(N-methylhydrazino)-
ethyl)-l-amino-3-propenyl)-dibenzo[a,d]cycloheptene chloride:
10 ~ NI-~ N n HCI ( a~ ~N.~N~NH~
A solution of 5H-5-(N,N-Dimethyl-N-(2-(N-methyl-
lS N'-formylhydrazino)-ethyl)- I -amino-3-propenyl)-
dibenzo[a,d]cycloh ~ ^ formate (10.7 g, 25.4 mmol),
dissolved in an a~ u~ t~ solvent, such as methanol, with an
equimolar amount of aqueous 0.5 N HCI, is stirred at 50 C for
4 hours. The solvent is ~va~ ,d/lyophilized, and the
resulting residue is recrystallized. After filtration, the isolated
solid is washed thoroughly and dried in vacuo to yield the
desired product (14.9 g, 95%). A portion is repurified to yield
a sample for analysis.
2S 0 1 0
OH NJ~ h N ~;2CN~N~hh
7h~
~CNJ~N~Nh, ~;3CN~N~Nh
3S
133
S~I~S~Tl~t ~-ET ~ J' E 26~
.
21 79983
~ WO 95/18186 ~ 9.~rl~61Z
EXAMPLE 20
Synthesis of 4-Hydroxy-N-(N-(N-methyl-Nl-
formylhydrazino)eth~n-2-amidyl)-4-phenylpiperidine:
- o o
S N H O C C ~N J~, N` N ~ H
THF
A solution of N-(N-methyl-N'-formylhydrazino)-2-
ethanoic acid (3.75 g, 28.4 mmol) in an anhydrous solvent, such
as THF (100 mL), is stirred while N,N'-dicyclohexylcarbodiimide
(x mg, x mmol) is added. The reaction is stirred for three
minutes, then 4-hydroxy-4-phenylpiperidine (5.00 g, 28.2
lS mmol) in an ~y~u~ulia~ solvent, such as THF (100 mL), is
added. Dicyclohexylurea precipitates almost imm~ t-oly. The
resultant suspension is stirred for at least one hour, filtered to
remove the insoluble urea. The solvent is removed on a rotary
t~ ula~OI to afford an off white solid (7.48 g, 91%).
20 Recryst~lli7~tion of a portion gives a sample for analysis.
EXAMPLE 21
Synthesis of 4-Hydroxy-N-(N-(N-methylhydrazino)-2-
ethanamidyl)-4-phenylpiperidine.
~13CN~N~N J~H HCI ~"" NJJ~, NH,
A solution of 4-hydroxy-N-(N-(N-methyl-N'-
formylhydrazino)-2-ethanamidyl)-4-phenylpiperidine (6.03 g,
20.7 mmol) dissolved in an a~,!,lul!l;at~ solvent such as
m~th~nnl/water or THF/water, with an equimolar amount of
aqueous ).5 N HCI is stirred at 50 C for 4 hours. The mixture is
treated with Amberlite IR-45 resin. The mixture is filtered,
3S and the filtrate evaporated/lyophilized to afford the desired
134
SUB~rlT~T~ SHE~T (RULE 26)
WO 95118186 ' 2 1 7 9 9 ~ 3 P~ Y3I126I2
product as a solid (5.38 g, 99%). A portion is recrystallization
to give a sample for analysis
EXAMPLE 22
S Synthesis of 4-Hydroxy-N-(N-(N-methylhydrazino)-eth-2-yl)-
4-phenylpiperidlne .
O LAH
~CNJ~ NHz Eth, ~ ~N~N~NH
Lithium ~ minllm anhydride (1.87 g, 49.3 mmole)
is added, slowly, to a stirring solution of 4-hydroxy-N-(N-(N-
methylhydrazino)-2-ethanamidyl)-4-phenylpiperidine in a
suitable anhydrous solvent, such as THF or diethyl ether ( 100
15 mL), at 0 C. The mixture is allowed to stir for an hour at room
) -n~ c~ then cooled to 0 C. Ethyl acetate (40 mL) is
added, with vigorous stirring, to quench the reaction, followed
by the careful addition of a saturated aqueous solution of
sodium sulfate to nP.Iltrali7P the mixture. The white ~Illminllm
20 salts are filtered, and washed thoroughly with an appropriate
solvent such as diethyl ether or ethyl acetate. The filtrate is
conccl.L.atcd on a rotary cvapOIalul to yield the desired
product as a solid (3.63 g, 89%). A portion is recryst~lli7~rion
to give a sanple for analysis.
2~
EXAMPLE 23
Synthesis of an ~minimitiP l~mrhirhi'P 1,1-dimethyl-1-(2-
hydroxydodecyl)-2-acetylhydla~iniulll inner salt.
~ + (CH3)2NNH2 + ~
OH CH? ~CH3
~ , CH3
135
SU~STIT~5T~ SHE~T (RUL, 26)
wo 9S/18186 ~ PCT/U593~1Z6~2
1,1 -Dimethyl- I, I -(2-hydroxydodecyl)-2-acetyl
hydrazinium inner salt (29.0 g, 0.1 mol) and 1-iodododecane
(29.5 g, 0. 11 mol) were dissolved in benzene (300 mL).
Anhydrous K2CO3 (20.7 g, 0.15 mol) was added and the mixture
refluxed for 12 hours. The solids were removed by filtration
and the volatile components were removed in vacuo at 0.1 torr
for 24 hours to give a waxy solid (44.2 g, 99%). The product
was ~lldlact~ ,d by IH~NMR and FTIR.
EXAMPLE 24
Synthesis of a Sialic Acid-Derivitized Aminimi~ mrhirhile
Conjugate
OH CO CH
lS ~ + OH~_ ~
A
OH
(CH3)2NNH2 OH CH3 &H3~ H
CH30H /~ ZOH
2S ~O
Ho~
HO_(' '
>
OH
1,2-Epoxydodecane (2.68g, 0.01 mol), 1,1-
dimethyll.yd.~il.~ (0.6 g, 0.01 mol) and sialic acid methyl
3~ ester (3.23 g, 0.01 mol) are dissolved in methanol (50 mL). The
resulting clear yellow solution is stirred at room ~ ,l d~
136
SUBSTiT~J~ r~ iL~ 7~)
WO 95118186 2 ~ 7 9 ~ ~ 3 F~~ Y~ 612
for 96 hours. The solution was concentrated on a rotary
evaporator in vacuo, then subjected to vacuum (0.1 torr), to
remove any residual solvent, leaving a quantitative yield of the
waxy solid sialic acid derivative, chd~ .,.iz~d by I H-NMR and
S FTIR spectroscopy.
EXAMPLE 25.
Synthesis of a Ketoprofen-Derivitized ~minimi~lP ~mrhirhilP
Conjugate
~OCH3
lS ,
CH3)2NNH2 OH CH3 pH3~
~ ~ ~N
CH30H /3 ~3
~
2S
1,2-Epoxydodecane (26.75 g, 0.1 mol), 1,1-
dimethylhydrazine (6.01 g, 0.1 mol), and ~-k.,~u~ r~ (26.8 g,
0.1 mol) were dissolved in 150 mL of methanol. The resulting
30 clear yellow solution was stirred at room ~ ,.l...c for 96
hours. The solution was cQ~ t-d on a rotary cv~ulalu~ in
vacuo, then subjected to vacuum (0.1 torr) to remove any
residual solvent, leaving a waxy solid k~LV~,Lof~ minimi~P
d~ ali~ (59.23 g), I.a~ .,d by its IH-NMR and FTIR
3S spectra.
137
SUBSTITlJT~ S~EET ~IJLE 2~)
WO 95/18186 ~ ' 2 l 7 q q ~ 3 PCT/US93/12~i12
EXAMPLE 26
Synthesis of an ~minimid~ Lipid Mimetic
1,2-Epoxydodecane (26.75 g, 0.145 mol), 1,1-
dimethylhydrazine (8.72 g, 0.145 mol) and ethyl acetate ( 12.78
g, 0.145 mol) were dissolved in methanol (50 mL). The
resulting clear yellow solution was stirred at room temperature
for 96 hours. The solution was con~en~r~t~d on a rotary
evaporator in vacuo, then subjected to vacuum (0.1 torr) to
remove any residual solvent. The resulting thick glass was
cooled to 0 C and scratched with a glass rod to initiate
crystallization. The crystalline ~minimi~l~ (39.22 g, 93%) was
obtained and ~ ala~ d by its IH-NMR and FTIR spectra.
EXAMPLE 27.
Synthesis of a Ketoprofen Lipid Mimetic
lS 1, I -dimethyl- 1,1 -(2-hydroxydodecyl)-2-
k~tu~lvr,,n hydrazinium inner salt (3.23 g, 0.01 mol), prepared
as described above, and l-iodo~ c~n~o (2.95 g, 01.1 mol) is
dissolved in benzene (30 mL). Anhydrous K2CO3 (2.07 g, 0.015
mol) was added. The mixture was refluxed for 12 hours. The
20 solids were removed by filtration, and the volatile colll,uull~
were removed in vacuo at 0.1 torr for 24 hours to give a waxy
solid product (4.4. g, 98%). The product is .,L~uac~ .,d by IH-
NMR and FTIR
35
138
SU~ST~T~ E~ (~U~F 26 ~
WO 9S/18186 ` 2 1 7 9 9 8 3 PCT/US93/12612 '¦~
EXAMPLE 28.
Synthesis of 2~-mer combinatorial library
The following is one of the many methods that are
being contemplated for use in constructing random
5 combinatorial libraries of aminimides-based compounds; the
random incorporation of three ~minimi~S derived from ~-
chloroacetyl chloride and the hydrazines shown below to
produce 27 trimeric structures linked to the support via a
succinoyl linker is given below:
I--NHl N--NH2 N--NH2
R2 R'2 R"2
( 1 ) A suitable solid phase synthesis support, e.g.,
the chloromethyl resin of Merrifield is treated with 4-hydro%yl
butyric acid in the presence of Cs2CO3 followed by tosylation
20 with p-toluenesulfonyl chloride, under conditions known in the
art:
1. CsCO
~CH2--Cl T HO-(CH2)3--C02H ~ ~CKz-02C~(CH2)3~0Ts
2. TsCI
3S
139
SU~ T~ S~E~ ~R~JLE 2~
WO 9~118186 - ~ 2 1 7 ~ ~ 8 3 P~ Y~/IZ6/2
(~) The resulting resin is divided into three equal
portions. Each portion is coupled with one of the hydrazines
shown above to give the hydrazinium resin which is converted
to the aminimide by reaction with chloroacetyl chloride using
5 the experimental conditions described above.
1. N--NH2
Rl O
. lo ~CH2-O.C-(CH2);-OTs R , ~CH2-OzC~(CH2~ ~CI
2. ClCH~COCI R.
(3 ) The ~minimi~i~ resin portions are mixed
thoroughly and divided again into three equal portions. Each
resin portion is coupled with a different hydrazine followed by
a coupling with ~-chloracetyl chloride producing a resin with
two linked Iminimiti,~ subunits. The resin portions are then
20 mixed thoroughly and divided into three equal portions.
1. N--NH
~CH2-02C-(CH )3--N~-N-I~CI R~
R2 2. CICH2COCI
Rl o Rl o
~CH2-02C-(CH2h--N~-N'I~N-'--N~cl
R~ R'2
140
SUBST~TUTE SHEET (RULE 26)
WO95/18186 ~ 2 1 799~3 F../~I~Y~ 6l2
(4) Each resin portion is coupled with a different
hydrazine followed by reaction with an acid chloride to
produce a resin with three linked aminimide subunits:
R'l
1. .N--NH,
Rl O R'l O
~CH~-02C-(CH~)3--N'-N-~N~_N~CI R 2
R2 . R', 2- J~
Rl O R~ I O R"l O R Cl
~CH, ~02C -(CY~)3--1~+ -N- ~N~_NJ~_ N -N~R
R2 R'~ R"
The resin portions are mixed producing a library
corlt~inin~ 27 types of beads each bead type conr~inin~ a single
trime}ic ~minimi~ species for screening using the bead-stain
method described above. Alternatively, the ~minimitl~5 may
be detached from the support via acidolysis producing a
20 "solution-phase" library of aminimi-i~os containing a
butyrylated terminal nitrogen, shown in the structure below in
which R = C3H7):
FORMULA llA
25 5~ A, O A, O Al.~R
~ A, A 5
110 (C~ ¦ N I ' ~A- O
A, A' R',
141
SUBST~TUTE SI~F~ L~ 26)
~ Wo 95118186 ;~ ~ 7 9 ~ 8 3 ~ y~ 6~2
S EXAMPLE 29.
Thematic Combinatorial Aminimide Library
The following example outlines the generation of a
matrix of 16 mr~l~cul~s around the basic structural theme of a
hydroxy-proline transition state mimetic inhibitor for
10 proteaseS:
lS Structural Theme:
I PHENYLALAN~E/ ¦ ¦ PROL~ l
¦ ALAN~M~TIC ¦ ¦ M~TIC ¦
T
OH
This mimetic was :,y~ Oi~cd by reacting styrene oxide or
propylene oxide, ethyl acetate or methyl benzoate with four
2S c~ .,ially available cyclic lly~ (as mimetics of
proline) in iso~ .ol in 16 indiYidual sample vials, as shown
below:
3S
142
SU~T!TlJT~ S!~EET ~P~U~ E ~6\
WOg5/18186 ~ . 2 t 7 9 9 8 3 PCT/US93/12612
t 0.1 MOL OF HYDRAZINE ¦
101~1 OF i-POHI
EVAPORAIE
10.1 MOL OF FpoxlnF I SOLVENT
WITN N2
S rin l Mf)~ OFF.~lFRI STREAM
~ '72 HRS. AT R.T.
I
lS
HOT
l~tOAc
~ SOLN.
o c. Siphon off sols~cnt '!
PRODUCT ~
Dq in vacuuo
~ Four of tho residucs did not complctely dissolve
. 3S
143
SU~S~iTUTE SHFET ~ LE 26~
WO9~/18186 `. `: ~ : 2 ~ 7~983 r~ ~V~ Z6l2
o
Rt ~R2
S \~7 + ~ ;~ + R2COOR ' ' \~\ N~
OH ~X
NH2
10 x = CH2 X = NMe X = O ~ = CH2CH2
Rl R2 Rl R2 Rl R2 Rl R2
Ph Me Ph Me Ph Me Ph Me
lS
Ph Ph Ph Ph Ph Ph Ph Ph
Me Me Me Me Me Me Me Me
Me Ph Me Ph Me Ph Me Ph
These 16 materials were isolated in essentially ~luaulil~tiv~:
2S yield on removal of the reaction solvent by evaporation and
purified samples were obtained as crystalline solids after
recrystallization from ethyl acetate and characterized by I H -
NMR, FTIR and other analytical t~rhniq~l~s
EXAMPLE 30.
Synthesis of an ~mrhirhilli~ ligand useful in the isolation and
pu~ific~ n of receptors binding vincamine:
To a solution of 1,2-epoxydodecane (I) (1.84 g, 0.01
mol) in a suitable solvent, such as n-propanol, is added, with
3S stirring, I,1-dimethylhydrazine (0.61 g, 0.01 mol). The solution
is stirred for I hour at room r~ c, cooled to 10 C in an
144
SUBS~iTUTE ~LE-~ ~RUL' ~
W0 95/18186 ' ~ 7 9 9 ~ 3 . ~ 612 ~
ice bath, and a solution of vincamine (II) (3.54 g, 0.01 mol),
dissolved in the minimum amount of the same solvent, is
added. The reaction mixture is stirred at 0 C for 2 hours, Lhen
stirred at room ~e~ dlul~ for 3 days. The solvent is
S removed under high vacuum (0.2 torr) and the crude product
is isolated. The conjugate (Il) is useful as a stabilization agent
for the isolation and purification of receptor proteins which are
acted upon by vincamine and structurally related molecules.
lo R o H C2Hs
,H30--c,.,..! ~
~J + 3( H2b~;ÇH2
lS H 0--C H C H2--N ---N--C~ ~ C2H 5
CH~ ~J
EXAMPLE 31
Synthesis of an ~mrhirhillir ligand useful in the isolation and
F~lrific~tinn of serotonin binding receptors
Methyl acrylate (8.61 g, 0.1 mol) is added over a 15
2S minute period to a stirring solution of serotonin (17.62 g, 0.1
mol) in a suitable solvent (100 mL). The reaction mixture is
stirrod at room i , c for 2 days. The solvent is
removed by freeze drying to yield the ester (IV). 1,1-
Di~,Lllylll~La~,ill~, (6.01 g, 0.1 mol) is added, with stirring, to a
solution of 1,2 c~ yd~i ~ ~ (18.4 g, 0.1 mol) in a suitable
solvent, such as propanol. The mixture is stirred at room
L~ a~ulc for 1 hour and a solution of (IV), dissolved in the
same solvent, is added. The mi~cture is continued for 3 days.
The solvent is removed in vacuo to yield the serotonin
conjugate (V), which is useful as a ligand for the dlscovery,
145
SU~T~TUT~ ~HE~ UL~ ~\
7q983
~ WO 95/18186 ~ PCT/U593/~261Z
stabilization and isolation of serotonin-binding membrane
receptor proteins.
S ,~Cj ~OCHJ ~
H NH2 HO N~O
0 o~ ~ IV OCH3
H H ~ //
~__,< C Ha
N--N'--CH2CH--(CH2)9--CHJ
CHJ OH
1~
EXAMPLE 32.
Synthesis of a ' ~' B ~ ligand mimetic useful in
the isolation and p~lrifil~tion of codeine-binding proteins:
The acid chloride of Rh~ B (VI) (49.74 g, 0.1
mol, prepared from rhodamine B by the standard techniques
for preparing acid chlorides from ,.ubu~ylic acids), dissolved in
a suitable solvent (500 mL), are added, with stirring, over a l-
hour period to a solution of l,1-dimethylhydrazine (6.01 g, 0.1
2S mol) in 100 mL of the same solvent. The t~ alulc is kept
at 10 C. After the addition is complete, the mixture is stirred
at room l.-~ c for 12 hours, and t_e solvent is removed
in vacuo to yield the Rho~' - B dimethylhydrazine (VII).
The Rh~' - B dimethylhydrazine (VII) (5.21 g,
0.01 mol) is dissolved in a suitable solvent, such as benzene
(100 mL), and tosyl codeine (VIII) (4.69 g, 0.01 mol, prepared
from codeine by the standard t~hni,l for the tosylation of
an alcohol), in 50 rnL of the same solvent are added over a 15
minute period, with stirring. The mixture is refluxed for I
3S hour. The mixture is then cooled, the solvent is removed in
vacuo, the residue is redissolved in an appropriate alcohol and
146
SUBSTiTUTE ~ i (RI~LE 2~!
WO 95/18186 ~ s ~ ` 2 1 7 9 9 ~ 3 PCI~/U593/12612
adjusted to pH 8 with 10% methanolic KOH. The precipitated
salts are removed by filtration. The solvent is removed in
vacl~o to yield the conjugate (IX), useful as a probe for the
location, stabilization and isolation of receptor proteins that
5 bind codeine and structurally similar analogs.
~ COCI ~CONHN~CH,),
S~2N~ \ cr s2s2N N~--E~
CH~ CH,
CH~o~ CHp~ /ca,
~o
~c
C~`E~
EXAMPLE 33.
Synthesis of a disperse-blue-3 cr~ nin~ ligand useful in the
isolation and purification of Cl~lt' - ~ g proteins
To a solution of ~ . ' ~ (X) (0.285 g, 0.001 mol),
35 dissolved in a suitable solvent, such as benzene (50 mL), is
added a solution of 4,4'-di~ ylviu~ (XI) (0.139 g,
147
SU3STi ~ UTE S~EET (RULE ~6)
~ WO95llgl86 . . ~ i ~3 2 1 79983 I~I/V~Y~ 6I2
0.001 mol) in 10 mL of the same solvent. The resulting
solution is heated to 70 C for 10 hours. The t~llly~iaLul~ is
brought to 10 C with cooling and 1, l -dimethylhydrazine (0.06
g, 0.001 mol) dissolved in 10 mL of the same solvent is added
5 dropwise. The solution is reheated to 70 C for 2 hours.
Disperse blue 3 tosylate (XII) (0.466 g, 0.001 mol, prepared by
the standard tosylation techniques from a pure sample of the
dye obtained from the commercial material by standard
normal-phase silica chromatography), is added and the mixture
10 is heated at 70 ~C for 2 more hours. The solvent is removed in
vacuo, the residue is redissolved in an a~lu~ t~, alcohol
solvent and titrated to pH 8 (measured wit_ moist pH paper)
with 10% (w/v) mloth~n~ KOH. The ~ iLaled salts are
then removed by filtration. The filtrate is cor..,~ d in
lS vacuo to give conjugate (XIII), useful as a probe for the location
and isolation of receptor proteins that bind codeine and similar
molecules.
.
3S
148
SU~STITUT~ S~-. T ~RU~ ~ 26)
1 7q9~3
WO 9~/18186 CK, F. I/L1~5/12612
CH~_ _ N
N _ o9 O --N
CH,O f ~ C~l, CHpJ~\OH
a. (CH NNHl /
`; / CH, O NH--
N ~
NH
CHpf ~OH I~lc--,
EXAMPLE 34.
Synthesis of an - .'-,' lli~` Iigand for the isolation a~d
2S p~lrificstil r of codeine-binding proteins:
O-,L~d~c ~ v "~ al~ (29.95 g, 0.1 mol) is added
slowly to 1,1-dimethylhydrazine (6.01 g, 0.1 mol) in benzene
(100 r~L). The mi~ture is stirred for 18 hours at room
~, c. and tosyl codeine (VIII) (54.2 g, 0.1 mol,
30 prepared by the standard techniques), is added portionwise
over a 112 hour period. The mixture is stirred and refluxed for
2 hours. The solvent is removed in v~cuo, the residue is
dissolYed in an .~ v~ te solvent (such as ethanol), and the
pH is titrated to 8 (measured with moist pH paper) with 10%
3S (w/v) m~tl~snolio~ KOH. The ~ ;L~d salts are removed by
149
SlJg~T~TUT~ S~-rT (PilJ.5
~ WO 9!i/18186 i~l ~' i 2 t 7 9 9 8 3 p_l"~9,~112612
filtration. The solvent is removed in vacuo to give the crude
conjugate (XIV), useful for stshiii7in~ and isolating receptor
proteins that bind to codeine and to similar molecules.
EXAMPLE 35.
Synthesis of a mimetic of a protein kinase binding peptide
a. The iod~-csm~r peptide (BEAD)-Asp-His-Ile-Ala-
Asn-Arg-Arg-Gly-Thr-Arg-Gly-Ser-NH2 is attached to the solid
support as shown using standard FMOC peptide synthesis
techniques, after d~ vlu~,Lion of the terminal FMOC group.
This peptide is shaken with a s~lution of an equivalent molar
amount of CICH2COCI in a suitable solvent at 50 C for 6 hours.
The solvent is removed by ~l~car~tin~, leaving a terminal -NH-
CO-CH2CI group attached to the peptide.
lS b. A solution of equimolar amounts of 1,1-
dimethylhydrazine and N, N '-dicyclohexylcarbodiimide, in a
suitable solvent, is treated with an equivalent molar amount of
the heptamer peptide H2N-Thr-Thr-Tyr-Ala-Asp-phe-Ile
COOH, prepared and obtained in the free state using the
standard FMOC solid phase peptide synthesis chemistry (e.g.,
using ir,~llu~ ..t~ and methods marketed by the Milligen
Division of Millipore Corp.). The mixture is stirred for 4 hours
at room ~.llp.,.aLulc. The ~ t~ d N,N'-dicyclohexylurea is
removed by centrifuging and ~l~csntin~, and the solution is
2~ added to the fi-r^tiQnsli7~-d beads prepared in a. above. The
mixture is heated to 50 C and shaken overnight. After cooling.
the solvent is removed by d~r9ntin~, and the peptide is
released from the bead to yield the sminimid~ mimetic H2N-
Thr-Thr-Tyr-Ala-Asp-Phe-lle-CO-N-N(CH3)2-CH2-Ser-Gly-
30 Arg-Thr-Gly-Arg-Asn-Ala-Ile-His-Asp-COOH. This mimetic h~
the sminimi~ in place of alanine in the naturally occurring
protein-kinase binding peptide, UK (5-24), and is useful as a
synthetic binding peptide with enhanced proteolytic stability.
3~
150
Sl~BSTI I ~TE .~ ET (R~3LE ~)
W0 95/18186 ` ` 2 1 7 9 9 8 3 ~ Y~/12612
EXAMPLE 36
Synthesis of a mimetic of an elastase inhibitor
This example teaches the synthesis of a competitive
inhibitor for human elastase based on the structure of known
5 N-trifluoroacetyl dipeptide analide inhibitors (see 16" L ~QL
Biol 645 (1982) and .cfl.~..c~s cited therein).
O I . H
~3C N' ~ + Cl~ N~N~ q (I) noutraliza~ion
0 H J~ o H3Clo ~ (2) purirlc~tion
lS F3C N +~ N +~N~
The aminimide N (p isopropylanalido)-
20 methyl)-S-N-methyl N-benzylchloromethylacetamide
(3.7 g, 0.01 mol) in ethanol (50 mL), and 1-methyl-1-
isobutyl-2-N-trifluoroacetyl hydrazide (1.86 g, 0.01
mol, prepared from the reaction of trifluoroacetic
anhydr~de wlth l-methyl-l-isobutylhydrazine [~rom
2S methylisobutylamine and chloramine] using standard
acylation methods) in ethanol (50 mL) were combined.
The mixture was stirred and refluxed for 4 hours. The
mixture was cooled to room temperature and titrated
with 10% (w/v) KO~I in ' -I to the
30 phenolphthalein endpoint. The mixture was then
filtered and the solvent removed in vacuo on a rotary
evaporator. The residue was taken up in benzene and
ffltered. Removal of the benzene on the rotary
evaporator yielded a crude mixed diastereomeric
3S r in' i~PC (5.1 g, 95%). The desired (S) (S) isomer
was obtained by normal-phase chromatographic
151
SUB~iTU~E SHEET (RULE 2~i)
~ W095118186 - 2 1 79983 F~~ Y~/I26I2
purification over silica. This product is useful as a
competitive inhibitor for human elastase,
characterized by HPLC on CrownpacklM CR(+) chiral
stationary phase (Daicell Chemical Industries Ltd.)
S using pH 2 aqueous mobile phase. lH-NMR (DMSO-d6):
t Chemical shifts, peak integrations and D2O exchange
experiments diagnostic for structure.
EXAMPLE 37
10 Synthesis of the Chiral Chloro~minimi~l~ Starting Material
lS H CI~CI DalU~IiZatiO~
H
CI~N~N~N~
C3
A mixture of the hydrazinium iodide
ensntiomer (4.2 g, 0.01 mol, prepared ss outlined
below), chlo.~ r~ :-~ acid (l.0 g, 0.0106 mol) and
2S ~hl-r~ 1 chloride (1.24 g, 0.011 mol), contsined in 8
micro resction flssk equipped with 8 drying tube, was
hested in an oil bsth to 105C for l hour. The
h~ ~genec reaction mixture wss cooled to room
tempersture and extrscted with diethyl ether (4 x 20
30 mL), to remove chlorscetyl chlor~de snd chloroscetic
acld, with vigorous stirring each time. The residual
semi-solld was dlssolved in the minimum amourlt of
~ --r-l, and titrated with 10% KOH in methanol to
the phenolphthalein end point. The precipitated sslts
35 were ~iltered snd the filtrste evsporsted to dryness on
8 rotary ev~~r ~ ~ r at 40 C. Tbe residue was taken
152
SUSSTI~U ~E ~t~E, ~t~ E ~
WO 95118186 ~ ~ , ,, 2 1 7 9 9 8 3 PCTNS9311Z612
up in benzene and filtered. The solvent was removed
on a rotary evaporator to yield the (S)-aminimide
enantiomer (3.37 g, 90%), characterized by its CDCI3
1H-NMR spectrum, D2O exchange experiments and
directly used in the next step in the sequence (see
5 above).
EXAMPLE 38
Synthesis of the Chiral ~minimid~ Starting Material
H
H~N--N\/~ + /~ resolution
lS
~N
r
l-methyl-l-benzyl-llydl,lLine (13.6 g, 0.1 mol,
prepared from methyl benzyl amine and chlnr~min~ using
standard methods ~J. ~'h~m Ed. 485 (1959)]) in toluene (125
2S mL) was cooled to 5 C in an ice bath. To this solution was
gradually added, with vigorous stirring over a one hour period.
a solution of p-isv~.u~y!l' yl chlb.b,..clllyl analide (21.17 g~
0.1 mol, prepared from chloracetyl chloride and p-
isu~.o~ cl~yl amine) dissolved in toluene (100 mL).
30 Thlul~,' the addition, the t~ a~ulc was m~int~in~d at 5
C. The reaction mi~ture was stirred overnight at room
L~lu~,lalul~. The pr~ririt~t~d solid hyvlàLilliul.. salt was
filtered, washed with cold toluene and dried in a vacuum oven
at 60 C/30" to yield the racemic product (34.3 g, 989to) . This
3S racemate was slurried at room ~ JCl~ltult overnight in
153
SUB~TITlJTE SHFE~ LE 2~
~ WO 9~i/18186 ~ ;` 2 1 7 9 ~ ~ 3 ~ Y~ 6l2
ethano~ (100 mL), and a slight molar excess of moist silver
oxide was added. The mixture was again stirred at room
temperature overnight. The mixture was filtered into an
ethanolic solution containing an equivalent of D-tartaric acid in
S the minimum amount of solvent. The alcoholic filtrate was
concentrated to approximately 20% of its volume and diethyl
ether was added until turbidity was observed. The turbid
solution was cooled at 0 C overnight and the crystals were
collected by filtration.. The solid substance was purified by
10 recrystallization from ethanol/ether to yield the desired pure
dia~ ol,lelic salt, which was` subsequently converted to the
iodide form by L,~ tion from a water-ethanol solution of
the tartrate (made alkaline by the addition of sodium
carbonate) on treatment with an equivalent of solid potassium
15 iodide, characterized by HPLC on CrownpackTM CR(+) chiral
stationary phase (Daicell Chemical Tnrlllctri~s Ltd.) using pH 2
aqueous mobile phase. IH-NMR (DMSO-d6): chemical shifts,
peak integrations & D2O exchange ,Yr. . ;...rl.~c were diagnostic
for the title structure.
EXAMPLE 39.
Synth~sis of a pepti~l- mi -'ic elastase inhibitor
35
154
SU~ TUT~ ET (~LE ~`
2 1 7 9 9 ~ 3 PCT/USs3112612
N NH H3C~ H
0~ H N--N~CF3
~ N.+~ ~`T' J`
Cl
'` 10 q
N NH
lS ` + N~
To a solution of the chl~ lly~ miti~P (4.36 g,
0.01 mol), as prepared above, in ethanol (50 mL) was added a
solution of l-methyl-l-isobutyl-2-N-trifluoroacetylhydrazide
(1.86 g, 0.01 mol, prepared from the reaction of trifluoroacetic
anhydride with I -methyl- 1 -isobutylhydrazine [from methyl
isobutyl amine and chloramine] using standard acylation
25 conditions) in ethanol (50 mL). The mfi~ture was refluxed with
stirring for 4 hours, cooled to room ~ v,~ c then titrated
with 10% (w/v) KOH in methanol to the p~ lPin
endpoint. The mixture was filtered, and the solvent was
removed in vacuo on a rotary CV~JldlUI. The residue was
30 taken up in benzene and again filtered. Removal of the
benzene on the rotary evaporator yielded the mixed (R)-(S)
and (S)-(S) ~minimiriP dia~ co.~el~ (5.7 g, 95%). The desired
(S)-(S) isomer was obtained pure by normal-phase
ld~ ;ld~hiC pl-rifir~ti~n over silica. This product is useful
3S as a CGI..~ iLivc inhibitor for human elastase, ~ ;7~d by
155
SUB~T~ ~ UT~ T lRlJi E ~
- ; 2f 79983
WO 9S/18186 . r~ Y.m~61Z
HPLC on CrownpackrM CR(+) chiral stationary phase (Daicell
Chemical Industries Ltd.) using pH 2 aqueouS mobile phase.
IH-NMR (DMSO-d6): chemical shifts, peak integrations & D20
exchange experiments were diagnostic for the desired
S structure.
EXAMPLE 40.
Synthesis of the Chiral Chlorn~minimiAP
H3C~
IN INH
H + Cl~
H2N' ~'CH~ ~
lS
O
~N NH
~ H
0~ Ni~
2S A mixture of the hydrazinium iodide
enantiomer (4.87 g, 0.01 mol, prepared as described in
5.2.3), chloroacetic acid (1.0 g, 0.0106 mol) and
chloroacetyl chloride (1.24 g, 0.011 mol), contained in
a micro reaction flask equipped with a drying tube,
was heated to 105 C for 1 hour with an oil bath. The
ho -h- COUS reaction mixture was cooled to room
temp~r&lur~, then extracted with diethyl ether (4 x 20
mL) to remove chloracetyl chloride and chloroacetic
acid. The residual semi-solid msss was dissoived in
3~ the minimum amount of methanol, and titrated w~th
156
SUBSTITUTE S~EET (~.U~E 2~)
2t 7~983
WO 95/18186 PCTIUS93~12612
10% KOH in methanol to the phenolphthalein end point.
The precipitated salts were filtered and the filtrate
was evaporated to dryness on a rotary evaporator at
40 C. The residue was then taken up in benzene and
S filtered. The solvent was removed on a rotary
evaporator to give the (S)-aminimide enantiomer (3.88
g, 89%), characterized in CDCI3 by IH-NMR
spectroscopy, D2O exchange experiments and used
directly i n the next step in the synthesis.
EXAMPLE 41.
Synthesis of the Chiral ~minimiri~-.
o
H3C~N NH ~, ~ resolution
N--NH2
O
~ H3C~NJ~NH
p~ o~J I H
N-~ ~
1-(5'[3'-methyluracil]methyl)- 1 -methylhydrazine
( 18.4 g, 0.1 mol, prepared by the alkylation of 2-
30 methylphenylhydrazone with 5-chloromethyl-3-methyluracil
in ethanol, as described in 24 1. Ore. Ch~m 660 (1959) and
cf~,lcnc~s cited therein, followed by removal of the be~zoyl
group by acid hydrolysis) in toluene (100 mL) was cooled to 5
C in an ice bath. A solution of p-isopropylphenyl-
3 chloromethylanalide (21. I g, 0.1 mol, prepared from
157
SU~IT~I~E SH~ET (RLILE 26~
W0 95118186 ;~ 1 7 ~ q 8 3' F~I/~ Y.~ 6I2
chloracetyl chloride and p-isopropylanaline), in toluene ( 100
mL), was added thereto, with vigorous stirring over a I hour
period, m~int:linin~ a temperature of 5 C. The reaction
mixture was stirred at room temperature overnight. The
solution was cooled to 0 C, and the precipitated hydrazinium
chloride salt was filtered, washed with cold toluene and dried
in a vacuum oven at 40 C/30" to yield the crude racemic
product (4.77 g, 98%). This racemate was slurried in ethanol
( 100 mL), a slight molar excess of moist silver oxide was added,
and the mixture was stirred at room L~ alul~ overnight.
10 This racemate was resolved via. its tartrate salts and isolated as
the iodide using the method of Singh, above, characterized by
HPLC on CrownpackT~ CR(+) chiral stationary phase (Daicell
Chemical rn~i~lctri-os Ltd.) using pH 2 aqueous mobile phase.
IH-NMR (DMSO-d6): chemical shifts, peak integrations ~ D20
1~ exchange rYI,- ;~"rllt~ were ~ nnStjc for the desired
structure .
EXAMPLE 42.
Synthesis of 3-methyl-5-chloromethyluracil
A. N-lll~,.llylul-,a (74.08 g, I mol) and
diethylethoxymethylenemalonate (216.2 g,l mol) were heated
together at 122 C for 24 hours, followed by 170 C for 12
hours, to give the 3-melllylula~il-5-carboxylic acid ethyl ester
in 35% yield, following recryst~lli7~tinn from ethyl acetate.
B. 3-metllylul.. ,;l-5-carboxylic acid ethyl ester
(30 g) was c~rnnifird with 10% NaOH to yield the free acid in
92% yield, after standard work-up and recrystallization from
ethyl acetate.
C 3-me~llylul~ 5-carboxylic acid (20 g) was
30 d~,alb~Ayl~t~,d at 260 C to give a luall~ , yield of 3-
methyluracil.
D. 3-methyluracil-5-carboxylic acid was treated
with HCI and CH2O, using standard chlo.l ' ylàtion
conditions, to give 3-methyl-5-chlo.ull.ctllylulacil in 52% yield.
35 following standard work-up and recrys~lli7~tion from ethyl
158
SU~STi I UTE ~HE~T ~
WO9~/18186 ~ 1~ ! ., 2 7 799~3 .~~ 26l2
acetate: mp. 186 2C; lH-NMR (DMSO-d6): chemlcal shifts, peak
integrations & D20 exchange experiments were diagnostic for
~he desired structure.
EXAMPLE 43.
Synthesis of a peptidomimetic HIV protase inhibitor
This example teaches the synthesis of a competitive
inhibitor for the HIV protease with enhanced stability, based
on the in
sertion of a chiral Rminimi~i~ residue into the scissile bond
position of the substrate Ac-L-Ser(Bzl)-L-Leu-L-Phe-L-Pro-L-
Ile-L-Val-OMe (see, e.g., 33 J. Med. Chem. 1285 (1990) and
references cited therein).
Ac-S(B~ cu-A3rl-PheJ~N' ~ + 3,~ V21-11~4C~{3
~, ,v~l-ne oc}}
Ac-Ser-Leu-PhelN J~N
0.735 g (1 mmol) of Ac-Ser(Bzl)-Leu-Asn-Phe-CO-
2S NH-NC5H10 is dissolved in the mirlimum amount of DMF, and
0.344 g of BrCH2CONH-Val-Ile-OMe, prcpared by treatment of
H2N-Val-Ile-OMe with (BrCH2CO)20 according to the method of
Kent (256 Science 221 (1992), is added thereto. The mixture is
heated to 60 ~C and stirred at this temperature overnight. At
30 this point the DMF is removed under high vacuum, and the
desired (S) isomer is obtained from the enantiomeric mixture
after neutr~liz~tion by standard normal-phase silica
chromatography to yield the protected peptide. The side chain
blocking groups are then removed using standard peptide
3S deprotection techniques to yield the product Ac-Ser-Leu-Asn-
159
SUB~ U ~ ~ ~. .t~ t ;3~:
WO 95118186 j , i ~ 2 1 7 9 9 8 3 PCT/IJ593~12C~2
Phe-CON-N+(CSH10)-CH~-CO-NH-Val-Ile-O~e, userul as a
enhanced stability competitive inhibitor for the HIV protease.
EXAMPLE 44.
Synthesis of the Tetrapeptide Hydrazone
Ac-scr(szl)-t eu-Asn-Phe~H + HIN--N~)
Ac-s~r~s~)-Leu-Asn-phe--
H
0.6S3 g (l mmol) of AcSer(Bzl)-Leu-Asn-Phe-OH,
prepared via standard peptide synthesis techrliques (see 33 J.
Med. Chem. 1285 (1990) and .~f~.~nces cited therein), is
20 coupled with 0.10 g (1 mmol) of l-a..uro~ ;tiin~ using
standard peptide-coupling methods and ch~mic~riec (see 33 J.
Org. Chem. 851 (1968)) to give a 97% yield of the hydrazide,
isolated by removal of the reaction solvent in vacuo.
2S
EXAMPLE 45
Synthesis of a chiral monomer useful irl polym~i7Q-io~lc
yielding crosslinked polymer chains
hLe + ~N--~?=O
HJCi NHI Mc
~3`` Me ~ H
160
SUE~STiT~J! r
Wo 95/18186 ~ 2 ~ 7 ~ 9 8 3 ~ Y~ 6l2
3.18 g (0.01 mol) of (S)-1-methyl-1-ethyl-1-p-
vinyl- benzylhydrazinium iodide, prepared from p-vinylbenzyl
chloride and 1-methyl-1-ethylhydrazine using st~ndard
alkylation conditions, and isolated as the (S)-enantiomer by the
S method of Singh (103 J. Chem. Soc. 604 (1913)), are added to
75 ml of anhydrous t-butanol. The mixture is stirred under
nitrogen and 1.12 g (0.01 mol) of potassium t-buto,Yide was
added. The mixture is stirred for 24 hours at room
temperature and the reaction mixture is diluted with 75 ml of
anhydrous THF, cooled in an ice bath and 1.39 g (0.01 mol) of
~-vinyl-4,4-dimethylazlactone in 50 ml of THF are then added
over a 15-min. period. When addition is complete, the mixture
is allowed to warm to room temperature and stirred at room
te..,~ tulc for 6 hours. The solvent is stripped under
lS aspirator vacuum on a rotary ev~u.~Lo- to yield 3.0 g (92%) of
crude monomer. The product is recrystal ized from ethyl
acetone at -30C to yield pure crystalline momomer, useful for
f~i~ri~tin~ crosslinked chiral gels, beads, memhr~n~s and
composites for chiral separations, particularly for operation a~
20 high pH. NMR (CDC13) chemical shifts, presence of vinyl groups
in 6 ppm region, vinyl splittiQg patterns, peak integrations and
D20 ~ t~ gn~StiC for structure. FTIR absence of
azlactone C0 band in 1820 cm-l region.
EXAMPLE 46.
FurlctioQ~Ii7~rion of silica with an oxazolone followed by
conversion to a chiral ~minimi~lr~ useful in the resolutiorl of
racemic carboxylic acids
H~ r r
O C H
~
~S~$~
161
Sl~BSTITUTE SHEET (RUL~ 26)
~ W095~18186 ` 2 1 70~q~3 PCTlU593/lZ612
2.81 g (0.0~ mol) of (S)-l-methyl-l-ethyl-l-
phenyl- hydrazinium iodide, prepared by the method of Singh
(103 J. Chem Soc. 604 (1913))~ is added to 100 ml anhydrous t-
butanol. The mixture is stirred under nitrogen and 1.12 g
(0.01 mol) potassium t-butoxide was added. The mixture is
stirred for 24 hours at room temperature, after which the
reaction mixture is diluted with 100 ml anhydrous THF. To this
mixture is added 5.0 g silica functionalized with the Michael-
addition product of (S)-4-ethyl-4-benzyl-2-vinyl- 5-oxazolone
10 to ~l~cl~yt~ u~yl-functional silica. This mixture is stirred at
room temperature for 8 hours. The fl-n~rinn~li7-~d silica is
collected by filtration and successively reslurried and refiltered
using 1 00-ml portions of toluene (twice), methanol (four times)
and water (twice). The resulting wet cake is dried in a vacuum
lS oven at 60 ~C urlder 30" vacuum to constant weight, yielding
4.98 g of chiral-~minimi~-functionalized silica, useful for the
separation of racemic mixtures of c.ubùi~yLc acids, such as
ibuprofen, ktlu~luf~,., and the like.
EXAMPLE 47.
Fllnnti~n~li7~ricn of silica with a chiral ~minimi-i~ for use in the
separation of m~n~ r~5
NO
2S ~ cH,' ~ O~N~
.~C
~,~
10.0 g epoxy silica (15 micron Exsil C-200 silica) is
slurried in 75 ml methanol and shS~ken to uniformly wet the
surface. To this slurry is added 6.ûl g (0.01 mol) 1,1-
dimethylhydrazine, and the mixture is allowed to stand at
162
Sl~BSTi~T~ SH~T tRU~ 26)
wo95/18186 2 1 799~3 , ~ Y~ 612 ~
room temperature with periodic shaking for 45 min. 32.5 g (0.1
mol) of (S)-3,5-dinitrobenzoylvaline methyl ester is added and
the mixture is allowed to stand at room t~ p~laLul~ with
periodic shaking for three days. The functionalized silica is
5 then collected by filtration, re-slurried in 100 ml methanol and
re-filtered a total of five times, then dried in a vacuum oven at
60 ~C/30" overnight to give 9.68 g of the product. This
functionalized silica is slurry packed from methanol into a 0.46
x 15 cm stainless steel column and used to separate mixtures of
10 mandelic acid derivatives under standard conditions.
EXAMPLE 48.
Preparation of epoxy silica
50 g of 5 micron C-200 Exsil silica (SA 250 M2/g) is
15 added to 650 ml toluene in a two-liter three-necked round-
bottomed flask equipped with a Teflon paddle stirrer, a
th~mom~t~r and a vertical con~lcnC~r set up with a Dean-Stark
trap through a claisen adaptor. The slurry is stirred, heated to
a bath lC~u~ aL~ of 140 ooC and the water is azeotropically
removed by ~iictill~tion and coll~ctinn in the Dean-Stark trap.
20 The loss in toluene volume is measured and c~ t d for
by the addition of ~ llal dry toluene. 200 g of
glycidv~y~lv~.yl trimethoxysilane is added carefully through a
funnel and the mixture is stirred and refluxed overnight with
25 the bath t. ~ set at 140 ooC. The reaction mixture is
then cooled to about 40 ooC. The resulving f~rti~n~li7~d silica
is collected on a Buechner filter, washed twice with 50 ml
toluene, sucked dry, reslurried in 500 .~nl toluene, refiltered,
reslurried in 500 ml methanol and refiltered a total of four
30 times. The resulting methanol wet cake is dried overnight in a
vacuum oven set for 30" at 60 ~C to yield 48.5 g of epoxy
silica.
163
SL!~STîTUT~ SHEET (RULE 2~)
~; 2 t 79~83
WO95/18186 '' ' '` ~3 r~ ,Y~/~26lz
EXAMPLE 49
Synthesis of ~-3,5-Dini~robenzoyl-(S)-Valine Methyl Ester
S 0.~1 NO, n.:l CO~Mc Y
13.12 g (0.1 mol) of (S)-valine methyl ester is
added with stirring to a solution of 8 g (0.2 mol) sodium
10 hydroxide in 50 ml of water, cooled to about 10 ocC, and the
mixture is stirred at this temperature until complete
solubilization is achieved. 23.1 g (0.1 mol) of 3,5-
dinitrobenzoylchloride is then added dropwise with stirring,
keeping the temperature at 10-15 ~C with external cooling~
After the addition was complete, stirri~g is continued for 30
mirl. To this solution is added over a 10-min. period 10.3 ml
(1.25 mol) of cor~r~ntra~rd hydrochloric acid, again keeping the
L~ ..L~ . a~ at 15 coC. After this addition is complete, the
reaction mixture is stirred for an a~ itional 30 min. and cooled
to 0 ooC. 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
dried in a vacuum oven under 30" vacuum at 60 ooC to yield
28.5 g (90%) of N-3,5-diniLlubcnzûyl-(S)-valine methyl ester.
NMR (CDC13): chemical shifts, splitting patterns, int~gr~tionc
and D20 exchang~.f~L~ diagnos~ for structure.
EXAMPLE 50.
Preparation of ~minimi~i~-containing ion-exchange silica matri.Y
This example describcs prepQration of an
aminimi~e-functionalized ion-exchange silica matrix using
epoxy silica as the support to be modified. The reaction
sequence is:
Epoxy Silica + (CH3)2NNH2 + Et2NCH2CH2COOEt --->
164
SUeS~!Ti IT~ SHEFT !~iL~ 2~
WO 95/18186 2 1 7 9 9 8 3 r~ y3/12612
-Si-O-SiCH2CH2CH20CH2CH(OH)CH2N(CH3)2NCOCH2CH2NEt~
25 g of epoxy silica (lSmicron Exsil AWP 300 silica.
S with surface area of 100 m2/g) is slurried in 100 ml methanol
until completely wetted by the solvent. 10.2 g of 1,1-
dimethylhydrazine are then added with swirling and the
mixture allowed to stand at room te...~.d~ul~ for 3 hours.
24.7 g of Et2NCH2CH2COOEt are then added and the mixture
10 kept at room temperature with periodic shaking for 2 days.
The diethylaminoethyl (DEAE) functionalized silica
is collected by filtration, re-slurried in 100 ml methanol and
re-filtered a total of five times. The packing is dried in a
vacuum oven at 60_C/30" overnight. A 1.0 ml bed of this
material is then packed in a 15 mM NaAc buffer at pH 7.7. The
column is then equilibrated with lS mM NaAc buffer at pH 5.6,
and a solution of I mg/ml ovalbumin in this buffer run through
the bed at a flow rate of 1.6 ml/min. A total of 59.2 ml of
protein solution is run.
The column is then washed with 41.7 ml of 15 rnM
NaAc buffer at pH 5.58 and at a flow rate of 3.9 ml/min. The
bound protein is eluted using 23.4 ml of 0.5M NaCI at a flow
rate of 3.9 ml/min. The eluent (15.2 ml) is then collected and
the tr~ncmiccinn of an aliquot measured at 280 mll with a
spectrophotometer. The ovalbumin co.~~onrration is
rmim~d from a calibration curve.
EXAMPLE 51.
Preparation of aminimide-containing size-exclusion silica
matrix
This example describes preparation of an
aminimid~-functionalized size-exclusion silica matrix using the
epoxy silica support described in Example _.
3S 10.0 g of epoxy silica (15micron Exsil C-200 silica,
with surface area of 250 m2/g) is slurried in 75 ml of methanol
165
SUæ~;T~T'. ~ t E~ T (RULE 2~)
~- `- 21 79~83
~ WO 95/18186 PCI-/U593/12C12
and shaken to uniformly wet the surface. ~o this slurry is
added 10.2 g of 1,1-dimethylhydrazine. The mixture is allowed
to stand at room temperature with periodic shaking for 45 min.
15 g of e~hyl acetate are then added and the
mixture allowed to stand at room temperature with periodic
S shaking for 3 days. The functionalized silica is then collected
by filtration, re-slurried in 100 ml methanol, re-filtered a total
of five times and dried in a vacuum oven at 60 ooC/30"
oYernight. The functionalized silica is slurry packed from
methanol into a 10 rnm interio}-diameter jacketed glass
10 column with adjustable pistons to provide an 8 cm-long packed
bed. This packing is used to separate mixtures of polyethylene
glycol polymers of varying molecular weight with good
resolution using a mobile phase.
~n a second ~ t, the bulk packing was
lS found to selectively adsorb polyethylene-glycol function~li7~d
hemoglobin from serum samples taken from test animals that
had been treated with this derivative as a blood substitute.
Filtration of the serum, after treatment with the bulk packing,
gave a serum free from the function~li7~d hemoglobin, thus
20 allowing blood screening or testing by means of standard
methods .
EXAMPLE 52.
Preparation of ~minimi-l~-functional PVA bead for selectively
2S binding polyethylene glycol cont~inin~ species (intelligent
macromolecule)
This example describes preparation of an
~minimiti~-functionalized crosslinked PVA matrix.
5.0 g of VA-epoxy beads(Riedel-de-Haeen
30 crosslinked PVA with 300umol of epoxy equivalents /g.
is slurried in 50 ml of methanol and shaken to uniformly wet
the surface. To this slurry is added 7.65 g of 1,1-
dimethylhydrazine. The mixture is allowed to stand at room
3S temperature with perlodic shaking for 45 min.
166
SUBS I ITUTE SHEET (RULE 26)
wo 9S/18186 2 1 7 q 9 8 3 ~ "~Y~ 6l2 ~
11.25 g of methyl acetate is then added and the
mixture allowed to stand at room temperature with periodic
shaking for 3 days. The functionalized resin is then collected
by filtration, re-slurried in 100 ml methanol, re-filtered a total .
S of five times and dried in a vacuum oven at 60 C/30"
overnight. The bulk packing is used to selectively adsorb
polyethylene-glycol functionalized hemoglobin from serum
samples taken from test animals that had been treated with
this derivative as a blood substitute. Filtration of the serum,
after treatment with the bulk packing, gave a serum free from
the functionalized hemoglobin, thus allowing blood screening or
testing by means of standard methods.
EXAMPLE 53.
lS Preparation of Aminimide-functional PVA bead for selectively
binding polyethylene glycol containing species (int~llig
macromolecule)
This example describes preparation of a second
type of Aminimi~l~-functionalized crosslinked PVA matrix.
~.0 g of VA-epoxy beads(Riedel-de-Haeen
crosslinked PVA with 300umol of epoxy equivalents/g
is slurried in S0 ml of methanol and shaken to uniformly wet
the surface. To this slurry was added 7.65 g of l,l-dimethyl-
2S hydrazine. The mixture is allowed to stand at roomtemperature with periodic shaking for 45 min.
20.0 g of methyl caproate are then added and the
mixture allowed to stand at room t~ dtL~lG with periodic
shaking for 3 days. The functionalized resin is then collected
by filtration, re-slurried in 100 ml methanol, re-filtered a total
of five times and dried in a vacuum oven at 60C/30"
overnight. The bulk packing is used to selectively adsorb
polyethylene-glycol functionalized hemoglobin from serum
samples taken from test animals that had been treated with
this derivative as a blood subs~irl~P. Filtration of the serum,
after treatment with the bulk packing, gave a serum free from
16~
SUBSTITUTE SHEET (RULE 26)
W095~ 6 ~ ` ' ^ ' " ' 2 1 7 q q 8 3 PC'r/l~S93~1Z612
the functionalized hemoglobin, thus allowing blood screening or
testing by means of standard methods.
EXAMPLE 54
Coating of a silica matrix with hydroxypropylcellulose
functionalized with an 7minimi~
Hydroxypropylcellulose is mono-functionalized by
10 reaction, under strong alkaline-conditions (preferably provided
by a strong base, such as potassium t-butoxide) with CICH2CON-
N+(CH3)3. The result is replacement of d~ u~ ately one
hydroxyl group in each 57r~ h7ri~o unit with the 7minimi~ as
lS follows
OCH~CONN(CH3)3
[sAccE~ DE UNFIln
OH OH
The resulting aminimi-i~ derivative is coated onto a
surface (e.g" silica). Upon heating to 140 ~C, the N~CH3)3
group leaves, resulting in formation ûf an isocyanate moiety:
2S OCEI2NCO
[SAC l E 1~17~1n
OH OH
The isocyanate grûups then react with unreacted
hydroxyl groups on the c:m(-h7ri~1~ units to produce a cross-
Iinked co?ting.
Alternatively, the cellulose can be coated onto the
surface and immobilized using standard tf~t~hnitlu~C (e.g.,
35 reaFtion with bisoxiranes), and then mono, di- or tri-
168
SUBSTI I UT~ r~ U! E 26~
wo 95/1~186 2 1 7 9 9 8 3 . ~I/Ub~l26l2 ~
subs~i~u~ed with desired aminimide deriva~lves as describedabo ve .
The foregoing reaction sequence can also be
employed with polymers or oligomers bearing NH or SH ~}oups
5 instead of hydroxyl groups and can also be utilized to fabricate
structures such as crosslinked cellulose membranes
EXAMPLE 55.
Coating of a silica ma~ix via polymerization of an aminimide on
10 the matrix
This example illustra`tes an alternative
immobilization technique, namely, polymerizing ~minimi~iP
precursors containing vinyl groups and which have been coated
on~o a surface. The chemistry resembles the approach
15 described above, except polymeriza~ion forms a sturdy shell
around an existing suppor~ rather ~han creating a solid block of
ma~erial.
This sequence makes use of ~he reac~ion described
above. An epoxide,
Cl
C~,
(CH,),~ CE~-CX;C
CX,
is combined with~ rne~hyl me~hacryla~e and dime~hylhydrazine
as set forth in 2.a above to form CH=C(CH3)-CO-NN(CH3)2-CH2-
CH(OH)-CH2-N+(CH3)3CI-. 3.11 g oF ~his ma~erial and 0.598 g n-
30 methylol acrylamide are dissolved in 75 ml of me~hanol, and3.54 ml of wa~er is then added. To ~his solution is added 15 g
of epoxy silica (15u Exsil AWP 300 silica. wi~h surface area of
100 m2/g).
The mix~ure is s~irred in a ro~ary at room
3S temperature for 15 min and then stripped using a bath
temperature of 44C to a volatiles content of 15% as measured
169
SUBST~TUTE SHEET ~LE ~6~
WO 9511818~ 2 ~ 7 9 9 8 3 PC'r/US93112612
by weight loss (from 25-200C with a sun gun). The coated
silica is slurried in 100 ml of isooctane containing 86 mg of
VAZ0-64 dissolved in 1.5 ml toluene which had been de-
aerated with nitrogen. The slurry is thoroughly de-aerated
wtih nitrogen and then stirred at 70C for two hours.
The coated silica is collected by filtrauon and
washed three times in 100 ml methanol and air dried. The
silica is heated at 120C for 2 hours to cure the coating. 13.1 g
of coated silica are obt~uned. A 1 ml bed of this material is
packed in an adjustable glass column and successfully used to
separate BSA from lactoglobulin:
EXAMPLE 56.
Preparation of a silica support containing crosslinked
aminimide polymer chains
lS In this example, an epoxy-functionalized surface is
reacted with ~ ;L ~ hydrazine, a bisepoxide and a
triester to form a crosslinkcd network of Aminimi~ chains
attached covalently to the surface as follows:
O O
(S~JRFAOE)--C~X-~CH2 + ~5C2--O--C--CIX--C--O--C2EI5 + X2C\ ~ CHl C~H-~ H2
o ~,C~o~C2H5
2S
~N--NH.
~/
R~
r'~--N---C--CH--C--N--r`'~-CH7-CH-CH2-CH--CH.
~2 O NO O ~2 OH OH , n
R~ _N- _R2
3S
lc~l
Crl-OH
~SUR~:ACE)
170
SUBSTlT~Tt Sl IErT (~ULE 26~,
WO 95/18186 ~ ` 2 1 7 9 9 8 3 r~ l"~ 26l2 ~
The reaction can be carried out in water at room
emperature without special conditions.
EXAMPLE 57.
S Preparation of cross-linked porous aminimide ion-exchange
beads
This example describes preparation of three-
dimensional cross-linked porous copolymeric ~minimirl,~ ion-
exchange beads. It involves reaction of three monomers:
10Monomer A: CH2=CH-CON-N+(CH3)3
Monomer B: CH2=C(CH3~-CON-N+(CH3)2-CH2-CH(OH)-CH2-
N+(CH3)3C~-
Crosslinker: CH2=CH-CO-NH-C(CH3)2-CON-N+(CH3)2-CH2-
Ph-CH=CH2
lS where Ph is phenyl.
Preparation of Monomer A: This monomer was
pr~pared according to the method described in 21 J. Polymer
Sci., Polymer Chem. Ed. 1159 (1983).
E~&atiOll of Monomer B: 30.3 g ~0.2 mol) of
20 glycidyl-trimethyl~mmoni-lm chloride is dissolved in 100 ml of
methanol and filtered frec of insolubles. 22 g (0.22 mol) of
methyl methacrylate is added thereto, followed by 12 g (0.2
mol) of 1, I -dimethylhydrazine. The solution grew warm and
turned slightly pink. It is allowed to stand for 6 days at room
2S tellly~,ldtULC, and is then treated with charcoal, filtered, and
co.~-cullat~d on a rotary cY~-..lur at 55C and 10mm to
produce a thick laYendar-colored, viscous material. This
material is triturated with diethylether and hot benzene and
dissolved in the minimum amount of methanol. The mixture is
30 then treated with charcoal, filtered, heated to boiling and
brought to the cloud point with ethyl acetate. The resulting
solution is allowed to stand at 0C for a week. The white
crystals that formed are collected by filtration, washed with
cold ethyl acetate and dried in a vacuum oven at room
35 Lc~ aLurc to yield 7.3 g of monomer B.
171
SIJBSTITU~E SHEET ~RUL~ 26~
~ W0951S81~6 : ~ ' ^ 2 ~ 7 9 9 8 3 r~llu~9~ 6~Z
Preparation of Monomer C: 18 g (0.3 mol) of 1,1-
dimethylhydrazine is dissolved in S0 ml CH2C12 and cooled in
an ice bath with stirring. 41.7 g (0.3 mol) of vinylazlactone in
50 ml CH2CI~ are added slowly to keep the temperature below
S 5 ~C. The clear solution is stirred and allowed to come to room
temperature over I hour (resulting in formation of a white
solid) and is stirred at room temperature for an additional 1.5
hours. The white solid is collected by filtration, re-slurried in
100 ml CH2C12 and re~filtered. It is then dried in a vacuum
10 oven at room temperature overnight to yield a total of 26.81 g
of the int--rm~ CH2=CH-CO-~H-C(CH3)2-CO-NH-N-(CH3)2.
10.0 g (0.05 mol) of this int~rm~ t~ and 7.66 g (0.05 mol) of
vinyl benzyl chloride are dissolved in a mixture of 50 ml
ethanol and 50 ml CH3CN. The solution is refluxed for 4 hours
lS under a nitrogen stream. It is then cooled to room temperature
and concentrated on a rotary ~vdpuldLul at 55 ccC to produce a
thick yellow oil. The oil is triturated three times with
diethylether to yield 17.08 g of an off-white solid. This solid is
dissolved in 100 ml of hot methanol and filtered through a
20 celite pad to remove a small amount of g~ in~vc material, and
the clear filtrate is stripped to yield 10.0 g of Monomer C as a
white solid.
Polymrri7~ti~n 1 ml of the ~nnlllcifier Span 80 and
175 ml of mineral oil are ill~ru-lu~2~ into a 500 ml round-
2S bottomed flask equipped with stirrer and a heating bath. Themixture is .. Pch~ 11y stirred at 70 RPM and brought to a
.e of 55 .. 40.5 g of monomer A, 7.2 g of monomer
B and 5.7 g of the cross-linker are dissolved in 100 ml of
demineralized water and heated to 550C To this solution is
added 150 mg of ~mmc~nillm persulfate, and the mixture is
then poured into the stirred mineral oil. The agitation is
adjusted to produce a stable emulsion with an average droplet
diameter of approximately 75u (as determined with an optical
microscope).
3S After 15 min, 0.15 ml of TMED is added and stirring
is continued for an ~ io~l 45 min. The reaction mixture is
172
SU3STITUTE SHEET (RU~E 26)
W0 9!;/18186 ' 2 ~ 7 9 9 8 3 P~s93~l26~2 ~
cooled and allowed to stand overnight. The supernatant
mineral oil phase is removed by aspiration and the beads are
collected by decantation. The beads are washed three times
with a 0.05~0 solution of Triton X-100 in demineralized water to
5 remove any remaining mineral oil and then washed with water
and allowed to settle. The water is removed by decantation.
This procedure is repeated a total of five times.
The beads obtained at the conclusion of the foregoing steps had
a mean diameter of approximately 75 u and an ion-exchange
10 capacity of 175 ueq/ml.
EXAMPLE 58.
Preparation of an ~minimirl~-based electrophoretic gel
lS This example describes preparation of an
~minimi~ir cle~hupl-o~ is gel. As a control, the standard
Sigma protein cle.,l~u~llo.eiis mix (available from Sigma
Chemical Co., St. Louis, M0) is run on an acrylamidelmethylene
ide linear gradient gel prepared using a gradient
20 maker with 5% and 12.5% monomer solutions, as shown below.
The gel is overlayed with icob~ rol and allowed to polymerize
overnight.
5% Monomer 12.5%
2S Monomer
Lower Tris 5.0 ml 5.û ml
H20 11.7 ml 4.7 ml
30% Acrylamide 3.3 ml 8.3 ml
30 Glycerol --- 2.0 ml
Ammonium Persulfate 3û ul 30 ul
TMED 15 ul 15 ul
Lower Tris 1.5M: 6.06 g Tris base, 8 ml 10% SDS,
35 volume adjusted to 9û ml with double-distilled water. The pH
173
SUBSTI, UTE SHEET (RULE 2~
2 1 79983
~ WO 95/18186 . ' -. PCr/U593~1Z612
is adjusted to 6.0 with concentrated HCI, and the final volume
adjusted to 100 ml with DD water.
Acrylamide 30% w/v: 29.2 g acrylamide, 0.8 g of
methylene bisacrylamide and 100 ml DD water.
S SDS 10% w/v: 10 g of SDS is dissolved in DD water
and adjusted to a volume of 100 ml.
Ammonium persulfate 10~Vo: 0.1 g ammonium
persulfate is dissolved in 0.9 ml DD water. The solution is used
within 4 hours of preparation.
TMED: used directly as obtained from Sigma
Chemical Co., St..Louis, MO, under the tr~rirn~mP TMEDA.
A second gel is prepared by replacing the
acrylamide with an equal weight of the ~minimi(lr monomer
CH2=CH-CO-N^N(CH3)3 and the protein standard is run in the
lS same way as the first.
Separation of proteins with the ~minimi~ gel is
equivalent to the acrylamide gel, but the ~minimi~l~ gel
produced Rf (i.e., the ratio of distance traversed by a particular
protein to the distance traversed by the solvent front) levels
20 approximately 20% higher than those of the acrylamide gel.
EXAMPLE 59.
Preparation of ~minimi~i~-based latex particles
591.1 ml of distilled water is charged to a three-
25 necked round-bottomed flask. A nitrogen dip tube is placed
below the liquid level and the nitrogen flow rate set to 2
cm3/min. The solution is ~-r- 1~ lly agitated with a Teflon
paddle at 250 RPM and heated to 80C over a half-hour period.
~n a separate flask is dissolved 121.6 g of butyl acrylate, 54.6 g
of ethyl acrylate, 13.0 g of acrylic acid, 9.97 g of methyl
~ ,I.ac.~late, 59.7 g of the ~minimirl.o monomer CH2=CH-CO-N-
N(CH3)2-CH2-CH2-OH and 0.92 g of Aerosol TR-70 so as to
obtain solution without oY.C~e~iin~ a l~ lp~.la~ulc; of 250C
When completely dissolved, 1.53 g of ~iAi~iQ~l TR-70 is added
3S and the mixture is then stirred until solution i5 achieved.
17~
SlJBSTlTVTt SHEET (RU~
WO 95/18186 2 1 7 9 9 8 3 PCT/US93/12612 ~
20.7 ml of distilled water is purged with nitro~en
for 10 min and 1.59 g of K2S208 is dissolved in it. This
persulfate solution is added to the heated water in the reaction
flask after it stabilized at 80C. The nitrogen dip tube is raised
and a nitrogen blanket is m~int~in~ he monomer mix is
pumped in at a steady, calibrated rate such that the constant
addition took exactly 4 hours. When addition is complete, the
latex was post-heated at 80C for 1 hour, cooled to 25C and
titrated to pH 5.0 by dropwise addition of triethylamine
(approximately 20 cm3) over 20 min with agitation. The latex
is then filtered through cheese cloth and stored. Average
particle size is measured at about 0.14u.
EXAMPLE 60.
l!i Incolyolation of an aminopyridinium functionality into an
~mininmide backbone
A solution of l-arnino-4-pyri~ ,;",.,~ oxylic tert-
butyl ester iodide (3.22 g, 10 mrnol) in THF (25 ml) is added to
20 a solution of N-benzoyl-N'-acetic acid-N'isobutyl-N'-
methylhy~l aziniUI~I inner salt (2.64 g, 10 mmol) and
dicycloll.,,~yl~,~l,o~liim~ (2.06 g, 10 mmol) in THF (100 ml)
and stirred for two hours at room ~ c. The
sUcp~ncion is treated with Amberlite IR-45 (or an equivalent
2S basic resin) for three hours at room t~mr~r~tl-re then filtered
to remove both the resin and precipitated dicyclohexyl urea.
The filtrate is conr- lr,U~d and the residue is recrystallized
from ethyl acetate to afford the bis l~ aLilliulll inner salt
(3.56 g, 78%).
The entirety of this material is dissolved in
acetonitrile (150 ml). Amberlite IR-118 is added and the
mixture is heated at reflux to ~Yl~ cr;()n of the ester. The
resin is removed by filtration and the solution is treated with
dicyclohexylurea (1.61 g, 7.8 mmol) in acetonitrile (25 ml).
3!~ After three minutes of stirring, I -benzyl- I -methylhydrazine
( 1.38 g, 9.36 mmol) is added neat, and the resultant suspension
175
SU~TITUTE SHcET (RULE ~
~ WO9S118186 ;;~ ~ J9~ Y~/12612
is stirred at room temperature for two hours. Removal of the
precipitated dicyclohexyl urea by filtration and concentration
of the filtrate affords a solid (5.01 g), which is not isolated, but
dissolved iD isopropyl alcohol ( 100 ml) and propylene oxide
(0.542 g, 9.36 mmol) is added. The mixture is heated at reflux
for seven hours and the volatile components are then removed
in vacuo. Crystallization of the residue from ethyl acetate
provides the tris-ylide (2.95 g, 5.30 mmol, ~8%).
It should be apparent to those skilled in the art that
other ~minimi~le compounds and compositions and other
processes for preparing said co~lpounds and compositions not
sp~ if ir~11y disclosed in the instant specification are,
nevertheless, contemplated thereby. Such ot~er 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 dislosed
herein .
3~
176
SU~SmUrE SltEET (R~SLE 26)
