CA2179983A1 - Modular design and synthesis of aminimide containing molecules - Google Patents

Abstract

The design and synthesis of novel aminimide-derived molecular modules and the use of the modules in the construction of new molecules and fabricated materials is disclosed. The new molecules and fabricated materials are molecular recognition agents useful in the design and synthesis of drugs, and have applications in separations and materials science.

Description

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--~_ ~T
lS ~
o = o o --~ ~ C
2S ~=
~[=

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

HO~ pr~lein H~ H HN~C

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 SUBST~TU~ ~HE~ ~R~LE 26) W095118186 ~ 2 ~ 799~3 r.,-lu~ 612 N~\ N~
,H ~/ --Sug ", ~\N--sU9 "N~N ¢~ N

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 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.

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 .

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:

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 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 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 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.

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) 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.

~ .

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, SU~ST5T~E S! ~'F. T ~)LE 26)

6 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.

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 ~_. ..... .

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 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 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=

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-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 -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 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.

Rt_C~7H2 + N--NH2 + R~--COORs - R1--CH--CHy ~ C--R4 + R50H
O H R3 o 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.

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):

{--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

' 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.

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

SligS~ !E~T ~F~UI ~

~ - l ` ' 21 79983 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 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, SU~E i ~ 26) ;` . ' ~' '' 1 ',~, 2 ~ 79983 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;

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 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 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

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 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 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 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.

.

SU~ LITESi~rI (~LILE26) wo95/18186 , ~;s ~ ~ 2 1 7q983 ~ Y~/l26l2 "Three point interaclion NO2 ~ H u 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 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 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 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.

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

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~

J--N~X~ Bw~ Bas~J o OH ~H

~" etc.
~ etc.

Specific syntheses of multisubunit aminimi~s are outlined below:

4.4.2.1 C~ n~ ~. of ~minimit~ Subunits via Acylation/Alkylation Cycles 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~

~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 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 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 SUBSrlTUTE SHEET (RULE 26) WO 9~i/18186 ~ PC'r/US~3J~2612 Rl +N,N~ U R

+
~-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:

S~BSTITUTE S~IEET (Rl ILE 26) wo 95/18186 2 1 7 ~ 9 ~ 3 r ,/~J~Y3/1~612 -"R3 X ~ R3 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.

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.

-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 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 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:

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 -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 /\ / (X)COOCB
~/ ~
HOCH2CH2 ~3~ Base J
~N~ .. (X - Determinanl of H3 ~Co(x~7 backbone spacing and o ger~metry) H2NN~B~se ~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 .

Sl I~STiT~lTC S~!~ET (RLILF ?6~

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

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~--+
~Baso2 H2N~ ~ \ / (X)COOCH3 lS
~Base n H2Nlj~

x MIXTURES
N~ , OF
H3C~ ~-co(x)cH(oH)cH2 RANDO~

-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 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 ~liBSTiTllTE SHEET (RULE 26) f ~ _ .
~wo 95/18186 2 ~ 7 ~ 9 8 3 ~ u~ fl6~Z
Scheme 2 ~ .
SACO~ ~ COOCH3 a ACO~_OCH

0 (a) Glycidol, Ag-Salicylate, C6H6, n Scheme 3 lS
~CH2OH CHO CHO
HO~ a HO~ b ACO~
HO~V HO~_/ ACO~

6 7 j C NHAC
ACO ~ d ACO
2S . ACO ~/ ACO _~3/

(a) (COCI~, DMSO, E~3N, CH2CI2, 60 C
(b~ Ac2O, Pyridine, CH2CI2, tt 30 (c) Ph3PCH21, PhLi, THF, n (d) m-CPBA, CH2CI2, tt SU~rlTUTE SHEET (Rll~E 26) WO 95/18186 ; PCT/US93/12612 Scheme 4 CH2oH CH2OTMS
HO ~ a TMSO
HO _~/ TMSO _~/

(a) TMSCI, CH2CI2, Et3N, rt b, c (b) Ethylene oxide, CH2CI2, rt, p-TsCI, pyridine 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: -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 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

SUBST~TUTE S~iE~T (RULE 26) t ~.` 2 t 7 q 9 ~ 3 PCT/US93/1261~ ~

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~

3~

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 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

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:

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:

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:

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~

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:

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

(~)(~)O(~) -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.

1~3 ~3 /
R1 Nl N ICl (X) lS
A further desirable Yariation of the surface-active structure shown above is as follows:
.

(X) ~ N C R, CElAlN/ R3 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 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 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 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.

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~&

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 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, 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) 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 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.

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

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

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 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.

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, 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

Epoxy Silica + N--NH2 + (C2H,)2--N-cH2-cH2-cooc2H5 --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 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 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 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.

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

HO R
;~~0 N ¦ ~
HO~H HL/--O~OH
H OH ~OH HA
HO~H

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.

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

~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 .
.

-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

OJ~N R~ ~P~
(CH2)n + IN--l~H2 lS
H`NJ~CH3 several steps 20 ~CH2)n IN--N~T

O--P
2SH~ CH3 (CH2)n N--N~T

'O--P=O
0~555 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 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.

.

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' 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 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.

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 109 , SUBST~T~T~ S~ ! gP.I~L~ 2~) 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 .

Synthesis of 2-bromacet-4.-trifluoromethylanalide Br Jl~

\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 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 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.

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.

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 .

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

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 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, SUBSTITUTE S~ ' t ~) :, `` ; .' ~ 2 1 79983 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.

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:

SU~STITUT~ S~E~ L~ ~63 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 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 %).

~UBST~U ~ HE~r ~RU~E 26~

~ 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%).

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~

~U~STITUTE S~EET (RULE 26) ~ ~ j ( 21 79983 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 SUBSnTUT~ SHE~T (RULE 26) ~ ~ 7 ~ 9 9 8 3 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 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' 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 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 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 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 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%).

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

lS HO ~ TM~
(a) TMSCI, CH2CI2, Et3 N, rt ¦ b, c (b) Ethylene oxide, CH2CI2, rt, ~TsCI, pyridine TM
TMSO _~/

12 HN ~~ OTs 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%).

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:

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 SU~TI I ~TE ~I~E~T (~UL~ 26!

;. ,, .,`., 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 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.

OH NJ~ h N ~;2CN~N~hh 7h~
~CNJ~N~Nh, ~;3CN~N~Nh S~I~S~Tl~t ~-ET ~ J' E 26~
.

~ WO 95/18186 ~ 9.~rl~61Z

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.

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 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 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~

Synthesis of an ~minimitiP l~mrhirhi'P 1,1-dimethyl-1-(2-hydroxydodecyl)-2-acetylhydla~iniulll inner salt.
~ + (CH3)2NNH2 + ~
OH CH? ~CH3 ~ , CH3 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.

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~

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 ~

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.

SUBSTITlJT~ S~EET ~IJLE 2~) WO 95/18186 ~ ' 2 l 7 q q ~ 3 PCT/US93/12~i12 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

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

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 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', 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:

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

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

SU~S~iTUTE SHFET ~ LE 26~

WO9~/18186 `. `: ~ : 2 ~ 7~983 r~ ~V~ Z6l2 o Rt ~R2 S \~7 + ~ ;~ + R2COOR ' ' \~\ N~
OH ~X

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 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

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, 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 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, 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.

.

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 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~

Sl~BSTI I ~TE .~ ET (R~3LE ~) W0 95/18186 ` ` 2 1 7 9 9 8 3 ~ Y~/12612 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 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.

10 Synthesis of the Chiral Chloro~minimi~l~ Starting Material lS H CI~CI DalU~IiZatiO~
H
CI~N~N~N~

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 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).

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 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 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 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 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 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 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 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-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.

Synthesis of a chiral monomer useful irl polym~i7Q-io~lc yielding crosslinked polymer chains hLe + ~N--~?=O
HJCi NHI Mc ~3`` Me ~ H

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~$~

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 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.

SL!~STîTUT~ SHEET (RULE 2~) ~; 2 t 79~83 WO95/18186 '' ' '` ~3 r~ ,Y~/~26lz 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 --->

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 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.

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.

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:

[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-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 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 ~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 lc~l Crl-OH
~SUR~:ACE) 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.

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 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

SUBSTI, UTE SHEET (RULE 2~

~ 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 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~

SU~SmUrE SltEET (R~SLE 26)

Claims (70)

THE CLAIMS
What is claimed is:

1. A composition having the structure:
wherein a. A and B are the same or different, and each is selected from the group consisting of a chemical bond;
hydrogen; and electrophilic group; a nucleophilic group; R; R'; an amino acid derivative; a nucleotide derivative; a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; and a macromolecular component, wherein A and B are optionally connected to each other or to other structures and R and R' are as defined below;
b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;

d. G is a 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 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

2. The composition of claim 1 wherein n > 2.

3. The composition of claim 1 wherein at least one of R
and R' includes a hydroxyl containing substituent.

4. The composition of claim 1 wherein at G includes at least one of an aromatic ring, a heterocyclic ring, a carbocyclic moiety, an alkyl group or a substituted derivative thereof.

5. The composition of claim 1 wherein A and B are the same.

6. The composition of claim 1 where R and R' are different so that the composition is chiral.

7. The composition of claim 1 wherein at least one of A and B is a terminal-structure moiety of formula T-U, wherein;
a. U is selected from the group consisting of aliphatic chains having from 2 to 6 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted cycoalkyl, and substituted or unsubstituted heterocyclic rings; and b. T is selected from the group consisting of -OH, -NH2, -SH, (CH3)3N+-, SO3-, -COO-, CH3, H and phenyl.

8. The composition of claim 1 wherein at least one of A and B is HO-CH2-(CHOH)n-.

9. The composition of claim 1 wherein A and B are part of the same cyclic moeity.

10. A peptide mimetic having the structure wherein:
a. A and B are the same or different, and at least one is an amino acid derivative of the form (AA)m, wherein AA
is a natural or synthetic amino acid residue and m is an integer, and A and B are optionally connectec to each other or to other structures;
b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each, is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;

d. G is a chemical bond or 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 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

11. A nucleotide mimetic having the structure:
wherein:
a. A and B are the same or different, and at least one is a nucleotide derivative, wherein A and B are optionally connected to each other or to other structures;
b. X and Y are the same or different and each rerprsents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

12. The nucleotide mimetic of claim 11 wherein A is a nucleotide derivative of the form (NUCL)m, when m is an integer such that (NUCL)m, is a natural or synthetic nucleotide when m=1, a nucleotide probe when m=2-25 and an oligonucleotide when m > 25, including both deoxyribose (DNA) and ribose (RNA) variants.

13. A carbohydrate mimetic having the structure:
wherein:
a. A and B are the same or different, and at least one is a carbohydrate derivative; wherein A and B are optionally connected to each other or to other structures;

b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 > 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

14. The carbohydrate mimetic of claim 13 wherein A
and B each is a natural carbohydrate, a synthetic carbohydrate residue or derivative thereof or a related organic acid thereof.

15. A pharmaceutical compound having the structure:
wherein:
a. A and B are the same or different, and at least one is an organic structural motif; wherein A and B are optionally connected to each other or to other structures;
b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 > 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

16. The pharmaceutical compound of claim 15 wherein the structural motif of the organic compound mimics or complements the structure of a pharmaceutical compound or a pharmacophore or metabolite thereof and has specific binding properties to ligands.

17. A reporter compound having the structure:

wherein:
a. A and B are the same or different, and at least one is a reporter element; wherein A and B are optionally connected to each other or to other structures:
b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 > 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

18. The reporter compound of claim 17 wherein the reporter element is a natural or synthetic dye or a photographically active residue which possesses at least one reactive group 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.

19. The reporter compound of claim 17 wherein the reactive group is amino, thio, hydroxy, carboxylic acid, acid chloride, isocyanate alkyl halide, aryl halide or an oxirane group.

20. A polymer having the structure:
wherein:
a. A and B are the same or different, and at least one is an organic moiety containing a polymerizable group;
wherein A and B are optionally connected to each other or to other structures;
b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur. oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 > 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

21. The polymer of claim 20 wherein the polymerizable group of the organic moiety is a vinyl group, oxirane group, carboxylic acid, acid chloride, ester, amide, lactone or lactam.

22. A substrate having the structure:
wherein:
a. A and B are the same or different, and at least one is a macromolecular component, wherein A and B are optionally connected to each other or to other structures;

b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 > 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R.

23. The substrate of claim 21 wherein the macromolecular component is a surface or structures which is attached to the aminimide module via a reactive group in a manner where the binding of the attached species to a ligan-receptor molecule is not adversely affected and the interactive activity of the attached functionality is determined or limited by the macromolecule.

24. The substrate of claim 23 wherein the macromolecule component has 2 molecular weight of at least about 1000 Daltons.

25. The substrate of claim 24 wherein the molecular component is in the form of an ceramic particle, a nonoparticle, a latex particle, porous or non-porous beads, a membrane, a gel, a macroscopic surface or a functionalized or coated version or composite thereof.

26. A chiral composition of matter having the structure wherein a. A is a chemical bond; hydrogen; an electrophilic group; a nucleophilic group; R'; an amino acid derivative; a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; or a macromolecular component, wherein R is as defined below;
b. Y represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. W is -H or -H2 X where X is an anion;
d. R and R' are the same or different and each is an alkyl, cycloalkyl, aryl, aralkyl or alkaryl group or a substituted or heterocyclic derivative thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon a to which they are attached; and e. G is a chemical bond or a connecting group that includes a terminal carbon atom for attachment to the quaternary nitrogen: provided that if G is a chemical bond, Y
includes a terminal carbon atom for attachment to the quaternary nitrogen.

27. The composition of claim 26 wherein X is a halogen or tosyl anion.

28. The composition of claim 26 wherein A is a terminal-structural moiety of formula T-U, wherein:
a. U is selected from the group consisting of aliphatic chains having from 2 to 6 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclic rings; and b. T is selected from the group consisting of -OH, -NH2, -SH, (CH3)3N+-, -SO3-, -COO-, CH3, H and phenyl.

29. The composition of claim 27 wherein A is HO-CH2-(CHOH)n-.

30. The composition of claim 26 where R and R' are different so that the composition is chiral.

31. The composition of claim 26 wherein Y is a chemical bond, G is and A is -COO- or-COOR and W is -H-, where R and R' differ from each other and are as described above.

32. The composition of claim 26 wherein Y is a chemical bond, G is and A is -COO- or -COOR and W is -H2 X, where R and R' differ from each other and are as described above.

33. A process of synthesizing a chiral composition having the structure wherein a. A is a chemical bond: hydrogen; an electrophilic group; a nucleophilic group; R'; an amino acid derivative; a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; or a macromolecular component, wherein A and B are optionally connected to each other or to other structures and R is as defined below;

b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
c. R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 > 1;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R;
wherein the process comprises the steps of:
acylating an asymmetric hydrazinium salt with a molecule capable of functioning both as an acylating and as an alkylating agent to form an aminimide;
reacting the aminimide with an asymmetrically disubstituted hydrazine to form a diastereomeric mixture of aminimide-hydrazinium salts.

34. The process of claim 33 which further comprises:
resolving the diastereomeric mixture and isolating a selected diastereomer;

acylating the diastereomer with a second molecule capable of functioning both as an acylating and as an alkylating agent to form an aminimide;
capping the resulting aminimide; and repeating the preceding steps at least once, if necessary, to form the desired structure.

35. The process of claim 33 wherein the asymmetric hydrazinium salt is bound to a support surface.

36. A process of synthesizing a chiral composition having the structure wherein a. A is a chemical bond; hydrogen; an electrophilic group; a nucleophilic group; R'; an amino acid derivative; a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; or a macromolecular component, wherein A and B are optionally connected to each other or to other structures and R is as defined below;
b. X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;

c. R and R' are the same or different and each is selected from the group consisting of A. B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic, aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
d. G is a 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 > 1 ;
provided that, (1) if G is a chemical bond, Y includes a terminal carbon atom for attachment to the quaternary nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other than H or R;
wherein the process comprises the steps of:
alkylating an asymmetrically disubstituted acyl hydrazide with a molecule capable of functioning both as an acylating and as an alkylating agent to form a racemic mixture of aminimide isomers; and reacting the racemic mixture with an asymmetrically disubstituted hydrazine to form a racemic mixture of aminimide-acyl hydrazide isomers.

37. The process of claim 36 which further comprises:
resolving the mixture of aminimide-acyl hydrazide isomers to isolate a desired isomer;
reacting the isolated isomer with a monofunctional alkylating agent to produce an aminimide; and capping the aminimide.

38. The process of claim 36 which further comprises:
reacting the mixture of aminimide-cayl hydrazide isomers with a second molecule capable of functioning both as an acylating and as an alkylating agent to form a racemic mixture of aminimide isomers;
repeating the preceding steps at least once, if necessary, to form the desired structure.

39. The process of claim 36 wherein the asymmetrically disubstituted acyl hydrazide is bound to a support sufrace.

40. A composition prepared according to the process of any one of claim 33 to 39.

41. A lipid mimetic composition having the structure:
wherein each Q is a chemical bond; hydrogen; an electrophilic group: a nucleophilic group; R; an amino acid derivative; a nucleotide derivative: a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; a macromolecular component; or the substituent X(T) or X(T)2; wherein R is an alkyl, cycloalkyl, aryl, aralkyl or alkaryl group or a substituted or heterocyclic derivative thereof, and T is a linear or branched hydrocarbon having between 12 and 20 carbon atoms some of which are optionally substituted with oxygen, nitrogen or sulfur ato or by an aromatic ring; and provided that at least two .gamma. substituents are present in the structure of the composition.

42. The composition of claim 41 wherein at least one Q
is attached to the .alpha.-carbon of a naturally occurring amino acid, or at least one Q is a carbohydrate.

43. A functionalized polymer having the structure wherein a. X and Y are connecting groups;
b. each Ra is hydrogen, alkyl, cycloalkyl, aryl, aralkyl or alkaryl;
c. (Surface) is a macromolecular component; and d. n 1.

44. A functionalized polymer having the formula:
wherein a. X and Y are connecting groups;
b. each Ra is alkyl, cycloalkyl, aryl, aralkyl or alkaryl;
c. (Surface) is a macromolecular component; and d. n 1.

45. A method of producing an aminimide-functional support comprising the steps of:
reacting a polymer or oligomer containing pendant moieties of OH, NH or SH with a compound of the formula:
wherein a. R1 and R each represent alkyl, cycloalkyl, aryl, aralkyl and 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 macromolecular component;
b. coating the reacted polymer or oligomer onto a support to form a film thereon; and c. heating the coated support to crosslink the film.

46. A method of producing an aminimide-functional support comprising 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 1,1'-dialkylhydrazine to crosslink the film.

47. A method of producing an aminimide-functional support comprising the steps of:
coating a mixture of an aminimide-functional vinyl monomer, a difunctional vinyl monomer and a vinyl polymerization initiator onto a support to form a film thereon;
and heating the coating support to form a crosslinked film.

48. An aminimide-functionalized support prepared according to the method of one of claims 45, 46 or 47.

49. A three-dimensional crosslinked random copolymer containing, in copolymerized form:
about 1 to 99 parts of a free-radically polymerizable monomer containing an aminimide group;
up to 98 parts of a free-radically addition-polymerizable comonomer; and about 1 to 50 parts of at least one crosslinking monomer.

50. The copolymer of claim 49 wherein the comonomer is water-soluble.

51. The copolymer of claim 50 wherein the comonomer is water-insoluble

52. The copolymer of claim 50 wherein the copolymer is fashioned into a water-insoluble bead, a water-insoluble membrane or a latex particle.

53. The copolymer of claim 50 wherein the copolymer is a swollen aqueous gel suitable for use as an electrophoresis gel.

54. A three-dimensional crosslinked random copolymer that is the reaction product of:
about 1 to 99 parts of a condensation-polymerizable monomer containing a moiety cluster selected from the group consisting of (1) 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 1 to 99 parts of a second condensation-polymerizable monomer containing a moiety cluster selected from the group consisting of (1) at least two ester groups, (2) at least two epoxy groups and (3) at least one ester and one epoxy group; and an amount 1,1-dialkylhydrazine equivalent, on a molar basis, substantially equal to the total molar content of epoxy groups.

55. The coploymer of claim 54 wherein the copolymer is fashioned into a water-insoluble bead, a water-insoluble membrane or a latex particle.

56. The copolymer of claim 55 wherein the copolymer is a swollen aqueous gel suitable for use as an electrophoresis gel.

57. A method of making a polymer having a particular water solubility comprising the steps of:
choosing a first monomer having the formula II
wherein R and R' are the same or different and are chosen from those organic moieties exhibiting hydrophobicity and n 1;
choosing a second monomer having the formula wherein R and R' are the same or different and are chosen from those organic moieties exhibiting hydrophilicity; and reacting said monomers to provide an effective amount of each monomer in a developing polymer chain until a polymer having the desired water solubility is created.

58. A method according to claim 57 wherein the hydrophobic organic moieties include those which do not have carboxyl, amino or ester functionality.

59. A method according to claim 57 wherein the hydrophilic moieties include those which do not have carboxyl, amino or ester functionality.

60. A method of preparing a synthetic compound to mimic or complement the structure of a biologically active compound or material which comprises synthesizing a compound of the formula:
wherein A is a chemical bond; hydrogen; an electrophilic group; a nucleophilic group; R'; an amino acid derivative; a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; or a macromolecular component, wherein A and B are optionally connected to each other or to other structures and R is as defined below;
X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
R and R' are the same or different and each is selected from the group consisting of A, B, isomers of A and 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;
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 n > 1.

61. A method according to claim 60 wherein said compound is a pharmacaphore.

62. A method according to claim 60 wherein said compound is a peptide mimetic.

63. A method according to claim 60 wherein said compound is a nucleotide mimetic.

64. A method according to claim 60 wherein said compound is a carbohydrate mimetic.

65. A method according to claim 60 wherein said compound is a reporter compound.

66. A method of preparing a combinatorial library which comprises:
preparing a compound having the formula;
wherein A is a chemical bond; hydrogen; an electrophilic group; a nucleophilic group; R'; an amino acid derivative; a carbohydrate derivative: an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; or a macromolecular component, wherein A and B are optionally connected to each other or to other structures and R is as defined below;
X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
R and R' are the same or different and each is selected from the group consisting of A, B, isomers of A and 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;
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 n 1 ; and conducting further reactions with the compound to form a combinatorial library.

67. A method of separating a desired compound from a plurality of compounds, which comprises;
preparing a separator compound having the formula:
wherein A is a chemical bond; hydrogen: an electrophilic group; a nucleophilic group; R'; an amino acid derivative; a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety containing a polymerizable group; or a macromolecular component, wherein A and B are optionally connected to each other or to other structures and R is as defined below;
X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
R and R' are the same or different and each is selected from the group consisting of A, B, isomers of A and 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;
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 n 1; and contacting the separator compound with the plurality of compounds; and differentiating the second compound from plurality of compounds.

68. The method of claim 57 or 60 wherein G is an aminimide isomer having the formula;

69. The composition of claims 1, 10, 11, 13, 15, 17, 20, 22 or 26 wherein G is an aminimide isomer having the formula;

70. The process of claim 33 or 36 wherein G is an aminimide isomer having the formula;