Note: Descriptions are shown in the official language in which they were submitted.
WO 9~/18g72 2 1 8 0 5 2 6 PCT/lJS94100139
SYSTEMATIC MODULAR PRODUCTION OF AMINIMIDE-
AND OXAZOLONE- BASED
MOLECULES lIAVING SELECTED PROPERTIES
Frr~r n OF Tr~F INVENTION
The present invention relates to a method for the
modular development of nminimitle- and oxazolone-derived
synthetic organic molecules, posessing selected properties for a
particular application. This method involves 1.) the synthesis of an
array generated from modules of aminimide-forming, oxzolone,
oxazolone-forming and/or oxazolone-derived molecules containin~g
a chosen set of substituent groups which confer structural
diversity and/or the reaction of these modules with other
appropriate reactive groups to produce an array of molecules
posessing a chosen set of diYerse structural moieties; 2.) the
screening of these mr~l~oc~ s for the desired set of properties in a
target application. The iterative application of this method
enables molecules to be produced, having an optimum balance of
properties for the particular application.
2 . B~'KGROUNlD OF T~F INV~TION
The discovery of new molecules has traditionally
focused in two broad areas, biologically active molecules, which
are used as drugs for the treatment of life-threatening diseases,
and new m~t-oriAl~, which are used in commercial, and especially,
in high technological applications. In both areas, the strategy used
to discover new molecules has involved two basic operations: (i) a
more or less random choice of a molecular c~n-1i(1~tP, prepared
either via chemical synthesis or isolated from natural sources, and
(ii) the testing of the molecular candidate for the property or
properties of interest. This discovery cycle is repeated
indefinitely until a molecule possessing the desirable property,
`'lead molecule", is located. This ~ ,.d molecule" discovery process
has been inherently ad-hoc in nalure and is time-consuming,
laborious, unpredictable and costly.
Once a candidate "lead" molecule has been located, tl7e
synthetic chemist must subsequently find ways to synthesize
WO 95/18972 2 1 8 0 5 2 6 PCT/IJ594/00199
structural variants of this lead molecule to optimize its properties
in the desired application. In the case where the "lead" molecule is
a synthetized organic species or a natural product, the chemist is
usually limited to certain structural themes and synthetic reaction
schemes. These are dictated largely by the structural composition
of the "lead" molecule and by the requirements of the specific
application. For example, in cases where the "lead" posesses a
functionally important aromatic ring, various electrophillic and
nucleophillic substitutions are typically be carried out on the ring
to produce variants. Each such case must be ~ludched as a
specific independent design and synthesis problem, starting each
time from the beginning, because of the lack of availability of an
appropriate chemistry to simply alter the structure of the lead
compound to produce the variant.
Recently, some attempts have been made to
modularize certain synthetic organic reaction schemes to fA~illi~AtP
modification and llall~r~ ation of a base compound (see, for
example, Proc. Natl. ~Ar Sci. USA . 90, 6909, 1933). However,
the molecules which can be produced by such attempts are
extremely limited in their achievable diversity and are still
bounded by factors dictated by the choice of specific structural
themes. In the case where the "lead molecule" is a naturally
occuring biological molecule, such as a peptide, a protein, an
oligonucleotide or a carbohydrate, simple synthetic point-
modifications to the lead molecule to produce variants are quite
difficult to achieve.
A brief account of the strategies and tdCtiCs used in the
discovery of new mol~c~ s is described below. The emphasis is
on biologically interesting molecules; however, the technical
problems encountered in the discovery of biologically active
molecules as outlined here are also illustrative of the problems
encountered in the discovery of molecules which can serve as
building blocks for the development of new tools and materials
for a variety of high technological applications. Furthermore, as
discussed below, these problems are also illustrative of the
problems encountered in the development of fabricated structures
and materials for high technological applications.
W095~18972 2 1 80526 PCT/U591/00199
Drug Desi~n
Modern theories of biological activity state that
biological activities, and therefore physiological states, are the
result of molecular recognition events. For example, nl~rl~otiti~s
can form complementary base pairs so that complementary
single-stranded molecules hybridize resulting in double- or triple-
helical structures that appear to be involved in regulation of geDe
expression. In another example, a biologically active molecule,
referred to as a ligand, binds with another molecule, usually a
macromolecule referred to as ligand-acceptor (e.g., a receptor o}
an enzyme), and this binding elicits a chain of molecular events
which ultimately gives rise to a physiological state, e.g., normal
cell growth and . ifferentiation, abnormal cell growth leading to
carcinogenesis, blood-pressure regulation, nerve-impulse-
generation and -propagation, etc. The binding between ligand a~ld
ligand-acceptor is geometrically characteristic and e~LI~oldillari]!y
specific, involving appropriate three-dimensional structural
arrangements and chemical interactions.
A currently favored strategy for the development of
agents which can be used to treat diseases involves the discovery
of forms of ligands of biological receptors, enzymes, or related
macromolecules, which mimic such ligands and either boost, i.e.,
agonize, or suppress, i.e., antagonize, the activity of the ligand.
The discovery of such desirable ligand forms has traditionally
been carried out either by random screening of molecules
(produced through chemical synthesis or isolated from natural
sources), or by using a so-called "rational" approach involving
identification of a lead-structure, usually the structure of the
native ligand, and optimization of its properties through numerous
cycles of structural redesign and biological testing. Since most
useful drugs have been discovered not through the "rational"
approach but through the screening of randomly chosen
compounds, a hybrid approach to drug discovery has recently
emerged ~hich is based on the use of combinatorial chemistry to
construct huge libraries of randomly-built chemical structures
WO 95/18972 2 1 8 0 5 2 6 PCT/US94/00199
which are screened for specific biological activities. (S. Brenner
and R.A. Lerner, 1992, Proc. Natl. Acad. Sci. USA 89 53, 81)
Most lead-structures which have been used in the
"rational" drug design approach are native polypeptide ligands of
receptors or enzymes. The majority of polypeptide ligands,
especially the small ones, are relatively unstable in physiological
fluids, due to the tendency of the peptide bond to undergo facile
hydrolysis in acidic media or in the presence of peptidases. Thus,
such ligands are decisively inferior in a pharmacokinetic sense to
nonpeptidic compounds, and are not favored as drugs. An
additional limitation of small peptides as drugs is their low
affinity for ligand acceptors. This rhPnomPnr)n is in sharp
contrast to the affinity demonstrated by large, folded
polypeptides, e.g., proteins, for specific acceptors, e.g., receptors or
enzymes, which can be in the subnanomolar range. For peptides
to become effective drugs, they must be t~ r~ ed into
nonpeptidic organic structures, i.e., peptide mimPticc, which bind
tightly, preferably in the nanomolar range, and can withstand the
chemical and biochemical rigors of coexistence with biological
tissues and f~uids.
Despite numerous incremental advances in the art of
peptidomimetic design, no general solution to the problem of
converting a polypeptide-ligand structure to a peptidomimetic has
been defined. At present, "rational" peptidomimetic design is
done on an ad hQc basis. Using numerous redesign-synthesis-
screening cycles, peptidic ligands belonging to a certain
biochemical class have been converted by groups of organic
chemists and pharmacologists to specific peptidomimetics;
however, in the majority of cases results in one biochemical area,
e.g., peptidase inhibitor design using the enzyme substrate as a
lead, cannot be transferred for use in another area, e.g., tyrosine-
kinase inhibitor design using the kinase substrate as a lead.
In many cases, the peptidomimetics that result from a
peptide structural lead using the rational" approach comprise
unnatural alpha-amino acids. Many of these mimetics exhibit
se~eral of the troublesome features of native peptides (which also
comprise alpha-amino acids) and are, thus, not favored for use as
_ . , . . , .. ,,:, . .... .. .
WO 95/18972 2 1 8 0 5 2 6 Pcr/usg4/oolg9
drugs. Recent~y, fundamental research on the use of nonpeptidic
scaffolds, such as steroidal or sugar structures, to anchor specific
receptor-binding groups in fixed geometric relationships have
been described (see for example l~Tir~rhm~nn, R. et al., 1992 J. Am.
Chem. Soc.. 114:9699-9701; H~ ll, R. et al., 1992 J. Am.
Chem Soc.. 114:9217-9218); however, the success of this approach
remains to be seen.
In an attempt to accelerate the id~ntifie~tion of lead-
structures, and also the id~ntifi~.Ation of useful drug ~n~ t~s
through screening of randomly chosen compounds, lCscd~
have developed ~llton~t~d methods for the generation of large
cnmhin~tori~l libraries of peptides and certain types of peptide
mimetics, called "peptoids", which are screened for a desirable
biological activity. For example, the method of H. M. Geysen,
(1984 Proc. Natl. Acad. Sci. USA 81:3998~ employs a modification
of M~.rrifi~ld peptide synthesis, wherein the C-terminal amino
acid residues of the peptides to be synthesized are linked to solid-
support particles shaped as polyethylene pins; these pins are
treated individually or collectively in sequence to introduce
additional lmino-acid residues forming the desired peptides. The
peptides are then screened for activity without removing them
from the pins. Houghton, (1985, Proc. Natl. Acad. Sci. USA
82:5131, and U.S. Patent No. 4,631,211) utilizes individual
polyethylene bags ("tea bags") containing C-terminal amino acids
bound to a solid support. These are mixed and coupled with the
requisite amino acids using solid phase synthesis techniques. The
peptides produced are then recovered and tested individually.
Fodor et al., (1991, Science 251:767) described light-directed,
spatially addressable parallel-peptide synthesis on a silicon wafer
to generate large arrays of addressable peptides that can be
directly tested for binding to biological targets. These workers
have also developed recombinant DNA/genetic engineering
methods for expressing huge peptide libraries on the surface of
phages (Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378).
In another combin~torial 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
wo 95/18972 2 1 8 0 5 2 6 Pcr/usg4/oolg9
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
5 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.
7l1rk~-rm~n et al., (1992, Int. J. Pe~tide Protein Res.
10 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~l;Y~Li~.,s, called "peptoids", which are
screened for activity against a variety of bio~hl micSIl targets. (See
also, Symon et al., 1992, Proc. Na~1 Acad. Sci. USA 89:936'.7).
Encoded c-lmhin~tori~l chemical syntheses have been described
15 recently (S. Brenner and R.A. Lerner, 1992, Proc. Natl. Acad. Sci.
U~A 89:5381).
The focus of these structural diversity activities on
peptide synthesis chemistry is a direct result of the fact that the
ability to generate structural diversity requires, as its starting
point, the access to practical stepwise sequential synthesis
20 chemistries which allow the incorporation of varied structural
elements with orthagonal reactivities. To-date, these have only
been worked out for the Merrifield synthesis of peptides and the
Carruthers synthesis of oligonucleotides. Thus, there remains a
need for an improved method for the structure-directed
generation and screening of organic compounds to ~ t~rmjn~
2~ which may be suitable in a particnlar application.
SUMMARY OF THE INVENTION
The invention relates to a method for obtaining ~o,,,~,ou,.d~ having
selected properties for a particular application by forming base modules
30 having at least two structural diversity elements from the reaction of a first
W09S~18972 2 1 80526 PCT/U~;94/00199
compound having at least one structural diversi~y element and a first reacti~/e
group, with a second comro.lnfl having at least one structural diversity
element and a second reactive group, wherein the first and second groups
5 combine by an addition reaction; producing a first array of molecules by
varying at least one of the structural diversity elements of the co.ll!.o~.-d~
when producing the base modules; and screening the array to determine a
first suitable compound for the particular ~Irrli~tion
If desired, the method can be repeated by prQducing a second array of
10 molecules through the formation of base mQdules having structural diversity
elements that are modified from those of the first suitable CO~'IQ'''ld and
screening the second array of molecules to determine a second suitable
compound for the particular application. The second array can be produce~d
by forming base modules having at least two structural diversity elements in
15 the same manner as the first array, except that the structural diversity
elements are modified from those of the first suitable compound. The
second array producing and screening steps can be repeated as often as
necessary to achieve an optimum chmr~.~n~l for the particular ;~rrlir~ n
Preferably, the first compound is produced 5 y forming an oxazolone
20 compound having at least one structural diversity element attached thereto
and reacting it with a nucleophile or carbonyl compound which contains at
least one structural diversity element to form a base module having one of
the following structures:
~ N
WO 95/18972 - 2 1 8 0 5 2 6 PCT/lJS94/00199
N ~ "x~
wherein at least two of the, ~ A lines are connected to structural
IO diversity elements.
Alternatively, it is also preferred to provide the first cnnlrollr~rl as an
~ ; ;A~-forming compound having at least one structural diversity
element attached thereto and to react it with an oxa2,010ne or ether cu.l-~v
which contains at least one structural diYersity element to form a base
15 module having one of the followlng structures:
< NH
OH
O ~/
H ~J~
wherein at least two of the ~ v~ d lines are connected to structural
diversity elements.
WO 95/18972 2 1 8 0 5 2 6 PCT~uSg4/oo~99
g
AdVA~.I~. v~ly, the first and second structural diversity elements can
be one of the following:
an amino acid derivative of the form (AA)";
a nucleotide derivative of the form (NUCL)";
a wul/ullydl~t~ derivative of the form (CH)~;
an organic moiety of an alkyl, cycloalkyl, aryl, aralkyl or alkaryl
group or a ,~ ;l"- .1 or ~ V~. IiC derivative thereof, or of a naturally
occurring or synthetic organic strtlctural motif, optionally cv, ~nil, g a
10 reporter element, an electrophilic group, a, rlrv~ group or a
pul~ lc group; or
a ma~, ~ cu.l.~o.,~
If desired, at least one of the fi! ;t and second c- ~I.v -- ls can be
provided with two or more structural diversity elements, two of which can
15 form a ring structure. The inventioll relates to a method for obtaining
C having selected properL~es for a par~icular ~rrli~ n by
producing a first structurally diverse array of molecules having at least two
UILIIO~;Ull~ll reactivity elements wherein a first orthogonal reacrivity elementis held constant for each molecule and a second orthogonal r~lctivity
20 element is varied; screening the array to determine a first suitable compoundfor the intended application; and modifying the first suitable compound to
form a second ~ ul~lly diverse array of rn~ c~ P~f~_l~ly, the first
suitable compound has at least two ullllogvl~l reactivity elements, so that
the method further includes modifying the first suitable compound by
25 holding a first orthogonal reactivity element constant while varying the
second orthogonal reactivity element to produce the second structurally
diverse array; and screening the second structurally diverse array of
molecules to determine a second suitable compound for the intended
application. The ~ thod further comprises repeating the modifying and
WO 9$/18972 2 1 8 0 5 2 6 PCT/US94/00199
1 0
screening steps as often as necessary to achieve the optimum compound for
the intended ~rFlir-lfirn
The first structurally diverse array of molecules is adv~f~lgeo.lcly
5 produced by reacting either an oxazolone or aminimide crmro-lnfl or
CfJIIIb;lld~;UI~ thereof, with first and second C.~ ,. f ~l~ which provide the
orthogonal reactivity elements. It is useful for the first structurally diverse
array of molecules to have one of the specific structures disclosed herein.
These structures may include ~o,.~l-.,,,f ~l~ such as an amino acid derivative,
10 a nucleotide derivative, a carbohydrate derivative, an organic structural
motif, a reporter element, a polymerizable organic moeity, or a
,a~ f,~ , co"l~on~l,l.
This method is useful for a wide variety of ~rlrlin~ti~r~nc, including the
development of new IJ JPI~ ;f'AI agents, new monomeric species for
15 the modular construction of separations tools, including chiral selectors,
industrial detergents and additives and for the development of modular
chemical illt~,l - ' for the production new materials and polymers.
Specifically, the method relates to the selection of molecular modules
containing appropriate structural diversity elements, fhe c~rnnPrtin~ of these
20 modules together via facile high-yield addition reactions which produce
discrete highly pure molecules in less than milligram quantities, in a manner
such that the properties of these molecules are d~,t~,.-~-ill~d by the
confrihlltion~ of the individual building modules. The molecular modules of
the invention may be chiral, and can be used to synthesize new comrollnfl~
25 structures and materials which are able to recognize biological receptors,
enzymes, genetic materials, and other chiral molecules, and are thus of great
interest in the fields of biof,l,~. ",~c~.lLicals, separation industrial and
materials science.
.
21 80526
WO 9511897~ PCT/US94/00199
.
BRTF~F DESCRIPTION OF THE DRAWING FIGURPA~
Figure 1. is a flow chart for the reaction disclosed in
Example 2.
DFTATT Fn DESCRTPrlON OF THE INVENTION
The present invention is able to generate a number of
different mol~c~ s for screening purposes by first forming a base
module that contains at least two structural diversity elements
attached thereto. These modules are formed by reacting first and
second compounds, each of which has at least one structural
diversity element and a reactive group. The reactive groups of
the first and second compounds are such that they react witb eacll
other to form the base module by an additional reaction. By fixing
one of the positions and structures of the structural diversity
elements and by Yarying at least one of the others, an array of
different molecules is easily generated. These molecules can then
~e screened to determine which are suitable for a particular
applicatlon or target use. Once a suitable compound is identified,
it can be selected for generating a further array of molecules.
This is done by modifying the particular structural diversity
elements that are found to be suitable, or by combining the
chosen structural diversity element with an expanded or differen~
set of second compounds or elements. This process can be
repeated as often as necessary to develop the optimum compound
- for the particular use.
The particular base module chosen for use in accordance
with the present invention is not critical and can be any one of a
wide variety of structures. It has been found, however, that two
2 1 80526
WO 9S/18972 PCT/US94/00199
1 2
particular structures which are known in the art are highly useful
as such base modules, these known compounds being the
oxazolones and ~min:~mi~llo.s. Thus, it is preferred to utilize
compounds which are aminamide forming, oxazolone forming,
oxazolone or oxazolone-derived molecules for use as the base
module. Depending upon the specific structure selected, these
base modules can have between two and six structural diversity
elements. The specific chemistry of these molecules, as well as an
identification of the structural diversity elements and reactivity
groups, follows.
Ox~7.- 10nes
Oxazolones, or ~71~ton~c are structures of the
general formula:
R
N~ \R'
A--~/2 5 (CH2)n
ol~
where A, R, and R' are functional groups and n is 0-3.
Oxazolones may posess two substituents at the 4-
position. When these substituents are not equivalent, the
carbon atom at the 4-position is asymmetric and two non-
superimposable oxazolone structures (azlactones) result:
Ay~O A~O~o
N ."" N~R2
Chir~l oxazolones possessing a single 4-position non
hydro~en substiruent (also known as 5(4H)-oxazolones),
derived from (chiral) natural amino acid derivatives, including
activated acyl~mino acyl structures, have been prepared and
.
WO gS/18972 2 1 ~ 0 5 2 6 PCTIUS9410(i199
1 3
isolated in the pure, crystalline state (Bodansky, M.; Klausner,
Y. S.; Ondetti, M. A. in "Peptide Synthesis", Second Edition, John
Wiley & Sons, New York, 1976, p. 14 and references cited
therein). The facile, base-catalyzed racemization of several of
these oxazolones has been studied in connection with
investigations of the serious racemization problem confronting
peptide synthesis (see Kemp, D. S. in "The Peptides, Analysis,
Synthesis, and Biology", Vol. 1, Gross, E. & Meienhofer, J.
editors, 1979, p. 315).
Racemization during peptide synthesis becomes
very extensive when the desired peptide is produced by
aminolysis of activated peptidyl carboxyl, as in the case of
peptide chain extension from the amino terminus, e.g. I - VI
shown below (see Atherton, E.; Sheppard, R. C. "Solid Phase
Peptide Synthesis, A Practical Approach", IRL Press at Oxford
University Press, 1989, pages 11 and 12). An extensively
studied me~ h~nism describing this racemization involves
conversion of the activated acyl derivative (II) to an )xazolone
(III) followed by facile base-catalyzed ~ d~ion of the
oxazolone via a resonance-stabilized intermediate (IV) and
aminolysis of the racemic oxazolone (V) producing racemic
peptide products (VI).
WO 95/18972 2 1 8 0 5 2 6 PCT/US94/00199
1 4
H Rl IHy~
H o R2 H "activation"
Base 555~N~5R2 Proton Donor
H .5)~
IV
5Ss~ aminolysis
~N~N~ 2--~
Extensive research on the trapping of oxazolones
III (or of their activated acyl precursors II) to give acylating
agents which undergo little or no racemization upon
aminolysis has been carried out, and successes in this
area (such as the use of N-hydroxybenzotriazole) have greatly
advanced the art of peptide synthesis (Kemp, D. S. in "The
Peptides, Analysis, Synthesis, and Biology", Vol. 1, Gross, E. &
Meienhofer, J. editors, 1979, p. 315).
WO 95/18972 2 1 8 0 5 2 6 PCT/US94100199
1 5
Thus, attempts to deal with the racemization
problem in peptide synthesis have involved ~ g or
avoiding the formation of oxazolone intermediates altogether.
Oxazolones having at least one hydrogen
sl-hstitllcnt at the 4-position can also undergo a variety of
rearrangements and side-reactions ( cf., 23 Tetrahedron 3363
(1967)), which may interfere with other desired
transformati~lls. This is illustrated for the case of the
oxazolone formed from the cyclization of N-acryloyl glycine:
H H
Oxazolones containing two non-hydrogen
s--hstif~-~ntg at the four position are structurally precluded
from undergoing these racemizations and side-reactions. These
disubstituted oxazolones may be obtained chirally pure and
may be subjected to the transformations which are the subject
of this invention with retent~-~n of the chirality at this position.
When the substituent at the 2-position is capable
of undergoing addition reactions, these may be carried out
with retention of the chirality at the 4-position to produce new
oxazolones. This is shown for the Michael-type addition to an
alkenyl oxazolone as follows:
~<~ A'X ~o O
N;~ 2
where X = S or NR and A' is a structural diversity group.
Synthesis of Oxazolones
21 80526
WO 95/18972 PCT/US94JOOI99 1
1 6
Oxazolones may be prepared from the
appropriate amino acid using any of a number of standard
acylation and cyclization techniques well-known to those
skilled in the art, e.g.:
ACOCI + R ' R2
H2N~C O~H H 2H
Ac20
~0~0
R~R2
These oxazolones may be isolated in the pure
state or may be generated in-situ from the acyl amino acid by
treatment, for example, with equivalent amounts of triethyl
amine and ethyl chloroformate in benzene. Following the
evolution of carbon monoxide and the removal of the triethyl
ammonium chloride formed by filtration. the solution of the
oxazolonè may be utilized directly for subsequent
transformations.
Reactions of Oxazolones
Ring-opening Addition
Oxazolones may be subjected to ring opening reactions
with a variety of nucleophiles, as shown below:
A~ BYH AJ~N~ \B
In the structure above, Y represents an oxygen, sulfur,
or nitrogen atom Rl and R2 differ from one another and
2 1 80526
V40 95118972 PCT/US94/00199
1 7
taken alone each signifies one of the following: alkyl including
ca}bocyclic and substituted forms thereof; aryl, aralkyl,
alkaryl, and substituted or heterocyclic versions thereof.
The above ring-opening reaction can be carried out either
in an organic solvent such as methylene chloride, ethyl acetate,
dimethyl formamide (DMF) or in water at room or higher
temperatures, in the presence or absence of acids, such as
carboxylic, other proton or Lewis-acids, or bases, such as tertiary
amines or hydroxides, serving as catalysts.
This reaction may be used to generate an array of adducts,
posessing combinations of the structural diversity elements A and
C, as shown:
~ o
~?~ ~XH ~ R",~
In addition, by appropriate selection of the R and R' groups,
two additional diversity elements can be provided in those
positions. Thus, the compound shown can have from two to four
structural diversity elements attached to tlie base module as
desired.
Carbonyl Addition
When both ~l-bsti~ nt~ in the 4-position are hydrogen, i.e.,
the oxazolone is formed from cyclization of an acyl glycine, the
ring may undergo a high yield condensation addition reaction with
aldehyde or ketone-containing structural groups at the 4-position.
This reaction may be used to generate an array of adducts,
posessing combinations of the structural diversity elements A and
B, as shown:
21 80526 ~
WO 9S/18972 PCT/US94/00199
1 8
~ ~
H H ~ Fl
Again, as noted above, the R group can be selected to be a
diversity element to provide an additional structural diversity
group on the oxazolone molecule.
Combination of the Two Reactions
The resulting adduct may subsequently undergo a high yield
ring-opening addition reactrion with a wide variety of SH, NH and
OH conJ:linin~ nucleophiles. This reaction sequence may, thus, be
used to generate an array of adducts, posessing combinations of
the structural diversity elements A, B and C, as shown
~XH ~(~
Again, as noted above, the R group can be selected to be a
diversity element to provide an additional structural diversity
group on the oxazolone molecule.
This is illustrated for the case of the in-situ
generation of the oxazolone from hippuric acid, followed by
removal of the triethylammonium chloride by filtration, the
addition of benzaldehyde to form the unsaturated adduct and
the ring opening addition of benzylamine to give the tris-
phenyl substituted adduct shown:
~ 2l 80526
WO 95118972 PCT/US94/00199
Step I
H 2 CICO E ~\_~ CO
C6~6 N
RT. H
/ Step 2
- (Et)3N~lCI / CHO
Flltradon/ ~ Addition
60C/10 minutes
bJ~~,
N~_ H
Step 3 ~ NH2
Addidon
60C/30 n~inutes
/~ -
~ H
O
The ability of these various reactions to be carried out in a
stepwise sequential manner using modules chosen in a structure-
directed manner allows the production of structurally directed
thematic diversity libraries, having structural elements
systematically varied around a basic motif.
21 80526
WO 95/189~2 PCI~ S94/00199
2U
Aminimides
.Aminimi~1es are zwitterionic structures described by
the resonance hybrid of the two energetically comparable Lewis
structures shown below:
R2 R2
R,~ N--~ +-R4 . ~ Rl--IC=N--N+-R4
O R3 R3
The tetr~cu~stitll~Pd nitrogen of the aminimide group
can be asymetric rendering ~minimifl~s chiral as shown by the
two enantiomers below:
R4 ~ND R~
As a result of the polarity of their structures, but lack
of net charge, simple amirlimides are freely soluble in both water
and (especially) organic solvents.
Dilute aqueous solutions of aminimides are neutral and
of very low conductivity; the conjugate acids of simple aminimides
are weakly acidic, pKa of ca. 4.5. A striking property of
~minimidPs is their hydrolytic stability, under acidic, basic, or
enzymatic conditions. For example, boiling trimethyl amine
benzamide in 6 N NaOH for 24 hrs leaves the ~Iminimi(l,~
unchanged. Upon thermolytic treatment, at temperatures
exceeding 1 80C, aminimides decompose to give isocyanates as
follows.
~R2 R
R,--C=N--N+-R4 R,--N=C=O + ~--R4
O- R R3
W1~95/18972 2 1 80526 PCTnlS94/001~9
2 1
Synthetic Routes to Aminimides
Aminimides can be synthesized in a variety of
different ways. The compounds of the present invention can be
synthesized by many routes. It is well known in the art of organic
synthesis that many different synthetic protocols can be used to
prepare a given compound. Different routes can involve more or
less expensive reagents, easier or more difficult separation or
purification procedures, straightforward or cumbersome scale-up,
and higher or lower yield. The skilled synthetic organic chemist
knows well how to balance the competing ~hara~fPri~tics of
competing strategies. Thus, the compounds of the present
invention are not limited by the choice of synthetic strategy and
any synthetic strategy that yields the compounds described above
can be used.
~minimidPs via Alkylation of N,N-Disubstituted Hydrazides
Alkylation of a hydrazide followed by nP~ltrali7atjon
with a base produces an aminimid~P
O O
Rl~ 11 (1) R3X Rl~+,R3 11
R2~ N R4 (2) nP"~ali7a~i''n R2~ N' R4
H
This alkylation is carried out in a suitable solvent, such as a
hydroxylic solvent, e.g., water, ethanol, isopropanol or a dipolar
aprotic solvent, e.g., DMF, DMSO, a,rPf~nitrjle. usually with heating.
An example of this reaction is the synthesis of the trifluoroacyl-
analide dipeptide elastase inhibitor mimetics shown in the
examples below.
Aminimides via Acylation of l,l,l-Trialkyl Hyd~ iu,l. Salts
Acylation of a suitable trialkyl hydrazinium salt by an
acyl derivative or isocyanate in the presence of a strong base in a
suitable organic solvent, e.g. dioxane, ether, acetonitrile, etc.
produces good yields of aminimides.
21 80526
WO 95/18972 PCT/U594/00199
22
R2 0 R2 o
Rl--N~-NH2 X- + R4--C--ORs base Rl--N+-N--11--R4
R3 R3
Aminimiti.os via the Hydrazine-Epoxide-Ester Reaction
A very useful and versatile synthesis of :Iminimi~
involves the one-pot reaction of an epoxide, an asymetrically
iiguhs~itllt~d hydrazine, and an ester in a hydroxylic solvent,
usually water or an alcohol, which is allowed to proceed usually at
room t~up~ lule over several hours to several days.
Rt--C~--CH2 + N--NH2 t' R4--COORs
R3
R1--CH--CH2--N--N---C R4 -t R50H
In the equation above, Rl, R2 and R3 are selected from a set of
diverse structural types (e.g. 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
increasing electrophilicity of the ester c~lllpol~,nt. Generally, a
mixture of 0.1 mol of each of the reactants in 50-100 ml of an
appropriate solvent is stirred for the required period at room
te~ lul~ (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.
Any of the various R groups illustrated in all of these
aminimide and aminimide-forming structures may be selected to
be structural diversity elements.
The ability of these various reactions to be carried out
using modules chosen in a structure-directed manner allows the
production of structurally directed thematic diversity libraries,
2 1 80526
WO 951~897~ PCT/US94/~ûl99
23
having structural elements systematically varied around a basic
motif.
Further details on the reaction possibilities for the oxazololle
and ~3minimid~ compounds can be found in two PCT applications
PCT/US93/0--- and PCT/US93/0---, each filed on December 28,
1993, and entitled Modular Design And Synthesis Of Oxazo~one-
Derived Molecules and Modular Design And synthesis Of
,~minimitl~-Derived Mole~ules, ~ ,e~,liv~ly. The content of each
of those applications is expressely incorporated herein by
reference thereto to the extent necessary to understand the metes
and bounds of this invention.
Mixed Aminimide-Oxazolones
A particularly useful embodiment of the invention is the
synthesis of mixed aminimide-oxazolone molecules, as shown
below. This scenario allows the incorporation of multiple
structural diversity elements as shown:
o (~ COR
/H Step I N (~)
H
~N--NH2 1 \~ f (3~ Step 3
H3C Step 2 OH CH3
~ H~ 3 IN `_~
Again, as noted above, the R and melhyl groups can be
replaced with additional structural diversity elements so that a
WO 95/18972 2 1 8 0 5 2 6 PCT/US94100199
24
total of six can be provided on the mixed aminamide-oxazolone
base module.
Str~rt~lr~l DiYersity Elelnents
Any of a wide variety of structural diversity elements can
be used. These elements would include:
I) Amino acid derivatives of the form (AA)N, which
would include, for example, natural and synthetic amino acid
residues (N=l) including all of the naturally occuring alpha amino
acids, especially alanine, arginine, asparagnine, aspartic acid,
cysteine, ~IIIt~minP, glutamic acid, glycine, histidine, isoleucine,
leucine, Iysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine; the naturally occuring
~lic~IbstiflltPd amino acids, such as amino isobutyric acid, and
isovaline, etc.; a variety of synthetic amino acid residues~ including
alpha-rli~I~hstitllt~od variants, species with olefinic substitution at
the alpha position, species having derivatives, variants or
mimetics of the naturally occuring side chains; N-SIlhstitlltPd
glycine residues; natural and synthetic species known to
functionally mimic amino acid residues, such as statine, bestatin,
etc. Peptides (N=2-30) constructed from the amino acids listed
above, such as angiotensinogen and its family of physiologically
important angiotensin hydrolysis products, as well as derivatives,
variants and mimetics made from various combinations and
permutations of all the natural and synthetic residues listed
above. Polypeptides (N=3 1-70), such as big ~n~ioth~Iin
pancreastatin, human growth hormone releasing factor and
human pancreatic polypeptide. Proteins (N>70) including
structural proteins such as collagen, functional proteins such as
hemoglobin, regulatory proteins such as the dopamine and
thrombin receptors.
2) Nucleotide derivatives of the form (NUCL)N,
which includes natural and synthetic nucleotides (N=l) such as
adenosine, thymine, guanidine, uridine, cystosine, derivatives of
these and a variety of variants and mimetics of the purine ring,
the sugar ring, the phosphate linkage and combinations of some or
all of these. Nucleotide probes (N=2-~5) and oligonucleotides
(N>25) including all of the various possible homo and
WO 95118972 2 1 8 0 5 2 6 PCTIUS94/OOr.99
2s
heterosynthetic combinations and permutations of the natural~y
oceuring nucleotides, derivatives and variants eontaining synthetie
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'-thioformaeetal, methylene(methylimino), 3-N-
earbamate, morpholino earbamate and peptide nueleie aeid
analogues.
3 ) Carbohydrate derivatives of the form
(CH)n. This would inelude natural physiologieally aetive
earbohydrates sueh as ineluding related eompounds sueh as
glueose, galaetose, sialic acids, beta-D-glucosylamine and
nojorimyein whieh are both inhibitors of ~ ociti~e, pseudo
sugars, sueh as Sa-earba-2-D-galaetopyranose, whieh is known to
inhibit the growth of Klebsiella pneumonia (n=1), synthetie
earbohydrate residues and derivatives of these (n= 1 ) and all of
the eomplex oligomerie permutations of these as found in nature,
incl lding high mannose oligos~rh~ri-les the known antibiotie
str~l~tomyein (n> 1 ) .
4 ) A naturally oeeurring or synthetie organie
struetural motif. This term is defined as meaning an organic
molecule having a speeific structure that has biological activity,
such as having a eomplementary strueture to an en~yme, for
instance. This term includes any of the well known base
structures of pharmaeeutieal eompounds ineluding
pharmaeophores or metabolites thereof. These inelude beta-
laetams, sueh as pennieillin, known to inhibit bacterial cell wall
biosynthesis; dibell~az~ es, known to bind to CNS reeeptors,
used as antidepressants; polyketide maerolides, known to bind to
bacterial ribosymes, ete. These struetural motifs are generally
known to have specifie desirable binding properties to ligand
acceptors .
5 ) A reporter element such as a natural or
svnthetic dye or a residue capable of photo~raphic amplification
~!hich possesses re~ctive groups which may be s~nthetically
incorporated into tlle oxa~olone structure or reaction scheme and
WO 95/18972 2 1 8 0 5 2 6 pCT/US94/00199
26
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
functionalities capable of undergoing condensation polymerization
or copolymPri7~ n 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 macromolecular COlllpOI~ It, such as a
macromolecular surface or structures which may be attached to
the oxazolone modules via the various reactive groups outlined
above in a manner where the binding of the attached species to a
ligand-receptor molecule is not adversely affected and the
interactive activity of the attached functionality is ~lP~/~rmined or
limited by the macromolecule. This includes porous and non-
porous inorganic macromolecular components, such as, for
example, silica, alumina, zirconia, titania and the like, as
commonly used for various applications, such as normal and
reverse phase chromatographic separations, water purification,
pigments for paints, etc.; porous and non-porous organic
macromolecular components, including synthetic components such
as styrene-divinyl benzene beads, various methacrylate beads,
PVA beads, and the like, commonly used for protein purification,
water softening and a variety of other applications, natural
components such as native and functionalized celluloses, such as,
for example, agarose and chitin, sheet and hollow fiber
membranes made from nylon, polyether sulfone or any of the
materials mentioned above. The molecular weight of these
macromolecules may range from about 1000 Daltons to as high as
possible. They may take the form of nanoparticles (dp=100-
1000Angstroms ), latex particles (dp=1000-5000Angstroms),
porous or non-porOUS beads (dp=0.5-1000 microns), membranes,
.
21 80526
WO95/18972 2 7 PCT/US94/OOlg9
gels, macroscopic surfaces or functionalized or coated versions or
composites of these.
8 ) A structural moiety selected from the group
ineluding cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio,
straight or branehed ehain alkyl, earboeyelie aryl and ~ubslilult;d
or heterocyelic derivatives thereof, wherein R and R' may be
different in adjaeent n units and have a seleeted stereoehemieal
arrangement about the carbon atom to whieh they are attached;
As used herein, the phrase linear ehain or branehed
ehained alkyl groups means any substituted or unsubstituted
aeyelic carbon-containing compounds, ineluding alkanes, alkenes
and alkynes. Alkyl groups having up to 30 earbon atoms are
preferred. Examples of alkyl groups include lower alkyl, for
example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or
tert-butyl; upper alkyl, for example, eotyl, nonyl, deeyl, and the
like; lower alkylene, for example, eLhylene, propylene,
propyldiene, butylene, butyldiene; upper alkenyl sueh as 1-
deeene, I-nonene, 2,6-dimethyl-5-oetenyl, 6-ethyl-5-oetenyl or
heptenyl, and the like; alkynyl sueh as 1-ethynyl, 2-butynyl, 1-
pentynyl and the like. The ordinary skilled artisan is familiar with
numerous linear and branehed alkyl groups, which are within the
scope of the present invention.
In addition, such alkyl group may also contain various
substituents in which one or more hydrogen atoms has been
replaced by a functional group. Funetional groups include but are
not limited to hydroxyl, amino, earboxyl, amide, ester, ether, and
halogen (fluorine, ehlorine, bromine and iodine), to mention but ,~
few. Speeific substituted alkyl groups ean be, for example, alkoxy
such as methoxy, ethoxy, butoxy, pentoxy and the like,
polyhydroxy such as 1 ,2-dihydroxypropyl, 1 ,4-dihydroxy- 1 -butyl
, and the like; methylamino, ethylamino, dimethylamino,
diethylamino, triethylamino, cyclopentylamino, benzylamino,
dibenzylamino. and the like; propanoic, butanoic or pentanoic acid
groups, and the li~e; formamido, acetamido, butanamido, and the
like, methoxycarbonyl, ethoxycarbonyl or the like, chloroformyl,
bromoformyl, l, l -chloroethyl, bromo
ethyl ,and the like. or dimethyl or diethyl ether groups or the lilce.
2 1 80526 ~
WO 95/18972 PCrlUS94/00199
28
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~lbs~it--~n~c 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, s~bsti~ d and u~ub~iiLu~,d aryl
groups means a hydrocarbon ring bearing a system of conJugated
double bonds, usually comrricin~ an even number 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 heteroaryl groups, e.g., pyrimidine,
morpholine, piperazine, piperidine, benzoic acid, toluene or
thiophene and the like. These aryl groups may also be substituted
with any number of a variety of functional groups. In addition to
the functional groups described above in connection with
substituted alkyl groups and carbocylic groups, functional groups
on the aryl groups can be nitro groups.
As mentioned above, these structural moieties can also
be any combination of alkyl, carbocyclic or aryl groups, for
example, 1-cyclohexylpropyl, benzylcyclohexylmethyl, 2-
cyclohexyl-propyl, 2,2-methylcyclohexylpropyl,
2,2methylph~llyl~lo~yl, 2,2-methylphenylbutyl, and the like.
Orthogonal Reactivities
A key element of the present method is the presence
of at least two compounds, each having a reactive group capable
of forming an addition compound with the other and carrying at
least one of the structural diversity groups. These compounds are
used to form the aminimide and the oxazo~one base modules.
These compounds may take the form of either A ) multiple
reactive groups which are capable of being "turned on"
independently of each othcr or B ) groups with multiple states
-
~ 21 80526
WO 9S/18972 PCT/US94~00199
29
with differing reactivities which may be addressed or brought
into being at different times or under different conditions in a
reaction sequence. It is highly desirable, although not absolutely
necessary, that each individual reaction be a high-yielding
addition reaction without possible interfering side-reactions, so
that isolation and ~llrifi~ fion steps are not necesary, or, at least,
are held to a minin --m
Specifically preferred reactive groups to generate the
aminimide and o~cazolone structuresand the resulting base
modules are listed below in tables 1, 2 and 3. The bonds in the
structures in these figures represent potential points of
attachment for the ~tt~rhm-~nt of the structural diversity
elements to the first and second compounds and to the base
module .
WO 95/18972 2 1 8 0 5 2 6 PCT/US94/00199
3 o
Tabl~ 1. Ox~zolo~e Modules
Reactiv;ly Gt~ups Baxe Modulos
~~>< (Y - N, S, O) H~
~~ ~ N S o) H O
\~N
NH2 C02H C02H/CI \~
(CICO2E~3N) O ~ \ -
~X (X = S~
~Z = CH2 CH-
Represents potential points of attachment for strtlctural diversity elements
. ~
WO 95118972 2 1 8 0 5 2 6 PCT~U594100~99
3 1
Tal)le 2. Aminimide Modules
Reactivity Groups Base Modules
-- COOH H2N~< --CONHN
--NCO H2N~< --NHCONHN
--OCOCI }I:~N~ --OCONHN
--SCOCI H2NN\ --SCONHN
2 ~
--CONHN --X --CONN--
(neutr.)
--CONHN\ ~7 --CON~
--NHCONHN\ (neutrX) --NHCONN--
--NHCONHN\ ~7 --NHCON
--OCONHN\ (neutr~x) --OCONN-
--OCONHN\ ~7 --OCON~
--SCONHN/ -- X --SCONN--
(neutr.)
--SCONHN\ ~7 --SCON~
-- Represents potential points of attachment for structural diversity elements
2 1 80526
WO 95118972 PCT/US94100199
32
Table ~ Cootinued - Amininnide Modules
Reactivity Groups Base Moduies
.
~ --X H2Nr~ X
(neutr.)
i~ X BASE E~
~ 1~
COOR --CO I--
--COOR --
4~ ~ /~ N~
o C02H 1~1 OH
~ /J~ N
Represents potential points of attachment for sttuctural diversity elements
WO 95~18972 2 1 8 0 5 2 6 PCI/US94100199
33
Table 3. Aminimide-Oxa~olone Modules
Reac~dYity Groups Base Modules
N r~
o
\~ N ~X ~(~3<.
X O
(Base)
Represents potential points of attachment for struc~ral diversity elements
EXAMPLE 1.
This example describes the generation of a matrix of 16 molecules
around the following aryl-heterocycle-alicyclic amine structural
theme.
Theme:
¦ Aryl Group ~ Acyclic Amine ¦
He~erocYcle ¦
2 1 80526
WO 95tl8972 PCTNS94/00199
34
The 2-pheny~ and 2-(2-naphthyl)-5-oxazolones
(produced by reacting the lithium salt of glucine with the aryl acid
chlorides, followed by cyclization with ethyl chloroformate at 0 C)
were reacted with 2-furfural, 3-fufural, 2-thiophenal and 3-
thiophenyl to produce the Y:~7r~ n~s functionalized at the 4-
position. This was followed by subsequent ring-opening addition
of 4-(3-aminopropylmorpholine and 1-(3-aminopropyl)-2-
pipicoline to form the adducts shown. The reactions were carried
out in individual vials such that each vial contained one pure final
compound as follows:
1.) ~qllimol~r quantities of the oxazolone and the
aldehyde dissolved in dry benzene (25ml/gm reactants) were
heated to 75 C for 15 minutes; 2.) the reaction mixture was cooled
to 10 C and the amine was added dropwise with stirring; 3.) the
mixture was re-heated to 75 C for 20 minutes and 4.) the solvent
was removed in vacuo to give the crude solid product.
21 80526
WO 95/18972 3 5 PCT/US94/001~9
Ar~o
~~ ~--CHO ~~=~
/
JNH2/--N~
1i 7 ~Y
NH~
Ar X I Isomer R / Y
Ph 0 2- H O
Ph S 2- H O
Ph 0 2- CH3 CH2
Ph S 2- CH3 CH2
Naphthyl 0 2- H O
Naph~hyl S 2- H O
Naphthyl 0 2- CH3 CH2
Naphthyl S 2- CH3 CH2
Ph 0 2- H O
Ph S 2- H O
Ph 0 2- CH3 CH2
Ph S 2- CH3 CH2
Naphthyi 0 2- H O
Naphthyl S 2- H O
Naphthyl 0 2- CH3 CH2
Naphthyl S 2- C ~ CH2
WO 95/18972 2 1 8 0 5 2 6 PCT/US94/00199
36
EXAMPLE ~.
The following example outlines the generation
of a matrix of 16 molecules around the basic structural theme of a
hydroxy-proline transition state mimetic inhibitor for proteases:
Structural Theme:
I PHENYLALANINE/ I I P~OLn~E I
¦ ALAN~MIMETIC ¦ ¦ MIMETIC ¦
OH
This mimetic was synthesized by reacting styrene oxide or
propylene oxide, ethyl acetate or methyl benzoate with four
commercially available cyclic hydrazines (as mimetics of proline)
in isopropanol in 16 individual sample vials, as shown in figure 1.
WO 95118972 2 1 8 0 5 2 6 pcT/usg4/ool99
37
R, X ~ R2
~7 + ~ ~ + R2COOR R~
IN OH \~h
NH2
X = CH2 X = NMe X = O ~ = CH2C~
RlR2 Rl R2 Rl R2 Rl R2
PhMe Ph Me Pl. Me Ph Me
PhPh Ph Ph Ph Ph Ph Ph
MeMe Me Me Me Me Me Me
MePh Me Ph Me Ph Me Ph
These 16 materials were isolated in essentially
quantitative 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 IH-NMR, FTIR and other analytical techniques. The set of
molecules where X = CH2 was tested as competitive inhibitiors of
the enzyme chymotrypsin in a standard assay using a BTEE
substrate. The results found for Ki were 200uM for Rl = Ph,
R2=Me; 130uM for Rl = Me, R2 = Ph; SOOuM for Rl = Ph, R2 = Ph;
and Rl = Me, R2 = Me was found to not be an inhibitor. These
results indicate a preference of the enzyme in this assay for one
phenyl and one methyl, with the phenyl being preferred in the Rl
position. Based on these results, a second array was synthesized
using phenyl groups in this position having a variety of differem
substituent groups for further testing agaiAst the enzyme.
