 |
Description  |
|
|
BACKGROUND OF THE INVENTION
The present invention is related to the area of chemical synthesis. More
specifically, one embodiment of the present invention provides methods for
the solid phase and combinatorial synthesis of 4-thiazolidinones,
metathiazanones and derivatives thereof.
Obtaining a better understanding of the important factors in molecular
recognition in conjunction with developing potent new therapeutic agents
is a major focus of scientific research. Chemical and biological methods
have recently been developed for the generation of large combinatorial
libraries of peptides and oligonucleotides that are then screened against
a specific receptor or enzyme in order to determine the key molecular
recognition elements of the biopolymer for that receptor or enzyme. See
U.S. Pat. No. 5,143,854; Ser. No. 07/805,727, filed Dec. 6, 1991, now U.S.
Pat. No. 5,424,186; Ser. No. 07/624,120, filed Dec. 6, 1990, now
abandoned; Ser. No. 07/946,239, filed Sep. 16, 1992, still pending; Ser.
No. 07/762,522, filed Sep. 18, 1991, now abandoned; Ser. No. 07/978,940,
filed Nov. 19, 1992, now abandoned; and Ser. No. 07/971,181, filed Nov. 2,
1992, now abandoned; each of which is assigned to the assignee of the
present invention and incorporated herein by reference for all purposes.
These methods provide rapid and efficient means to synthesize polymers
that are biocompatible, i.e., compounds that are non-toxic and readily
absorbed, and ideally are synthesized from monomers available in large
quantity, with a reasonable shelf life, optical activity, high-fidelity
coupling chemistry, and stable to various chemical reagents used for
protecting and deprotecting various side chains.
Virtually any bioavailable organic compound can be accessed by chemical
synthesis; however, such compounds typically are still synthesized and
evaluated one at a time in many cases, thus dramatically limiting the
number of derivatives which can be studied. This limitation can be
overcome by developing the methodology for the combinatorial synthesis of
large numbers of derivatives of therapeutically important classes of
bioavailable organic compounds. Screening these compounds against key
receptors or enzymes would then greatly accelerate the acquisition of
useful structure versus recognition data and would revolutionize the
search for potent new therapeutic agents.
The search for suitable small organic molecules amenable to a combinatorial
synthesis approach is an ongoing quest. One ideal goal is to tailor the
chemistry used to assemble the molecules to work in a polymer-supported
fashion, in analogy to solid phase techniques commonly employed for
peptides and oligonucleotides. The advantages of such a goal is twofold:
not only does one gain overall efficiency through the ability to filter
away both byproducts and excess reagents, but one also raises the
possibility of mass screening of the immobilized molecules with techniques
such as VLSIPS.TM. and ESL technologies. See, U.S. Pat. No. 5,143,854;
Ser. No. 07/805,727, filed Dec. 6, 1991, U.S. Pat. No. 5,424,186; Ser. No.
07/624,120, filed Dec. 6, 1990, now abandoned; Ser. No. 07/946,239, filed
Sep. 16, 1992, still pending; and Ser. No. 07/762,522, filed Sep. 18,
1991, now abandoned; each of which is assigned to the assignee of the
present invention and incorporated herein by reference for all purposes.
Perhaps the first example of the application of combinatorial organic
synthesis to non-polymeric organic compounds can be found in the work of
Ellman who described the solid phase synthesis of a 1,4-benzodiazepines.
See U.S. Pat. No. 5,288,514, which is incorporated herein by reference for
all purposes. The benzodiazepines were synthesized on a solid support by
the connection of three building blocks: an amino benzophenone; an amino
acid; and an alkylating agent.
Hobbs Dewitt has reported on the generation of libraries of small
molecules, which she terms "diversomers". Target compounds, including
dipeptides, hydantoins, and benzodiazepenes, were synthesized
simultaneously but separately, on a solid support in an array format, to
generate a collection of up to 40 discrete structurally related compounds.
The key step in this strategy involves the revealing of distal
functionality which initiates attack on the bond linking the compound to
the resin, thus, releasing the product from the resin.
In addition to the small organic molecules discussed above, another
important class of molecules is the 4-thiazolidinones (herein referred to
as thiazolidinones), metathiazanones, and derivatives thereof. The generic
structure and numbering system of these compounds is shown below:
##STR1##
Substituted 4-thiazolidinones possess many properties important for
biological activity, such as optical activity and the ability to form
hydrogen bonds and to carry side chain functionalities. Thiazolidinones
have been shown to exhibit antifungal (see, e.g., Srivastava et al. (1991)
Ind. J. Chem. 30:620-623; and Abdel-Rahman et al. (1990) J. Ind. Chem.
Soc. 67:61-64); antihistaminic (see, e.g., Diurno et al. (1992) J. Med.
Chem. 35:2910-1912); anti-platelet aggregation factor (see, e.g., Tanabe
et al. (1991) Tetrahedron Lett. 32:379-382; and Tanabe et al. (1991)
Tetrahedron Lett. 32:383-386); and antimicrobial (see, e.g., Hogale et al.
(1991) Ind. J. Chem. 306:717-720) activities. In addition, this class of
compounds has found use in the treatment of inflammation, hypertension,
renal failure, congestive heart failure, uremia and other conditions. See,
e.g., Walsh and Uwaydah, U.S. Pat. Nos. 5,061,720 and 4,225,609.
4-Thiazolidinones are therefore prime candidates for drug studies.
4-Thiazolidinones have been synthesized via the condensation of an
aldehyde, an amine and a mercaptoacetic acid to generate the five-membered
ring with the concomitant loss of two molecules of water. See, e.g.,
Diurno et al. (1992) J. Med. Chem. 35:2910-1912; Surrey and Cutler (1954)
J. Am. Chem. Soc. 76: 578-580; and El-Kohry (1992) OPPI Briefs 24:81-83.
The most likely mechanism for this condensation involves initial imine
formation between the aldehyde and the amine, followed by addition of the
thiol to the carbon-nitrogen double bond and finally ring closure.
Treatment of an imine with a mercaptoacetic acid also generates a
thiazolidinone in high yield. See Srivastava et al. supra; Abdel-Rahman et
al. supra; and Tanabe et al. supra. The synthesis of metathiazanones
proceeds through the analogous reaction of an amine, aldehyde, and
.beta.-mercaptopropionic acid.
Unfortunately, there has been a lack of efficient techniques for
synthesizing immobilized 4-thiazolidinones and particularly, for producing
arrays of 4-thiazolidinones. The present invention meets this need.
SUMMARY OF THE INVENTION
The invention provides a rapid approach for combinatorial synthesis and
screening of libraries of 4-thiazolidinones, metathiazonones, and
derivatives thereof which overcomes the above-described limitations of
current methodologies.
In one aspect, the present invention provides a method for the solid phase
synthesis of thiazolidinones, metathiazonones, and derivatives thereof,
which method includes the steps of first coupling an amine component to a
solid support and then treating the immobilized amine component with a
carbonyl component and a thiol component. In another aspect, the present
invention provides a method for the solid state synthesis of
thiazolidinones, metathiazonone, and derivatives thereof, which method
includes the steps of first coupling a thiol component to a solid support
and treating the immobilized thiol component with an amine component and a
carbonyl component.
According to either embodiment, the amine component preferably comprises a
primary amino group having the formula: R--NH.sub.2, wherein R is selected
from the group consisting of alkyl, alkoxy, amino, aryl, aryloxy,
heteroaryl, and arylalkyl or salts thereof. According to the latter
embodiment, R can also be hydrogen. More preferably, the amine component
will have the formula:
##STR2##
wherein R.sup.1 and R.sup.2 are independently selected from the groups
consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, heteroaryl,
carboxyl, carboxyalkyl, and arylalkyl. More preferably, the amine
component will comprise an amino acid or peptide. Alternatively, according
to either embodiment, the amine component may comprise a hydrazine
derivative having the formula R.sup.1 NHNH.sub.2 where R.sup.1 is as
described above, or a hydrazide derivative having the formula R.sup.1
(CO)NHNH.sub.2 where R.sup.1 is as described above. In addition, if the
thiol component is immobilized, a mono-protected hydrazine having the
formula PGNHNH.sub.2 wherein PG is an acid-labile, base-labile, or
photocleavable protecting group can serve as the amine component.
The carbonyl component preferably has the formula:
##STR3##
wherein R.sup.3 and R.sup.4 are independently selected from-the groups
consisting of hydrogen, alkyl, aryl, heteroaryl, carboxyl, carboxyalkyl,
and arylalkyl, provided that both R.sup.3 and R.sup.4 are not hydrogen.
More preferably, the carbonyl component comprises an aldehyde and thus,
either R.sup.3 or R.sup.4 is hydrogen. In a still more preferred
embodiment, either R.sup.3 or R.sup.4 is hydrogen with the other being
selected from the group consisting an aromatic group or a heteroaromatic
group.
The thiol component preferably comprises a mercapto carboxylic acid having
the formula:
##STR4##
wherein R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are independently selected
from the groups consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy,
heteroaryl, carboxyl, carboxyalkyl, and arylalkyl and n is either 0 or 1
wherein n being 0 represents a valence bond.
According to either embodiment, the addition of the various components to
the immobilized component can be sequential with, for example, the
carbonyl component being added first to the immobilized amine component to
form an immobilized imine and then adding the thiol component to complete
the condensation reaction, or simultaneous with all of the components
being combined in a one-step reaction. In addition, the condensation
reaction can be performed once to yield a support-bound thiazolidinone,
metathiazanone, or derivative thereof or repeatedly to yield a
support-bound polymer having at least two thiazolidinones,
metathiazanones, and/or derivatives.
In a particularly preferred embodiment, a mixture of primary amines,
aldehydes, and/or a mixture of .alpha.-mercapto carboxylic acids and/or a
mixture of .beta.-mercapto carboxylic acids are used to produce a library
or array of solid support-bound thiazolidinones, metathiazanones, or
derivatives thereof. These libraries will find use in the identification
of specific thiazolidinones, metathiazonones, and derivatives thereof
having antifungal, antihistaminic, or antimicrobial activity or use in the
treatment of inflammation, hypertension, renal failure, congestive heart
failure, uremia and other conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the use of .sup.13 C NMR to monitor the condensation
reaction of support-bound glycine with benzaldehyde and mercaptoacetic
acid. Panel D shows the .sup.13 C NMR spectrum of support-bound glycine
which has been labeled with a .sup.13 C-atom at the position alpha to the
carbonyl. Panel C shows the .sup.13 C NMR spectrum of the support-bound
labeled imine produced by the reaction of support-bound labeled glycine
with benzaldehyde. Panel B shows the .sup.13 C NMR spectrum of the labeled
thiazolidinone produced by the reaction of support-bound labeled imine
with mercaptoacetic acid. Panel A shows the .sup.13 C NMR spectrum of the
product of the reaction of support-bound labeled imine with acetic acid.
FIG. 2 illustrates the use of .sup.13 C NMR to monitor the stability of
thiazolidinones. Panel C shows the .sup.13 C NMR spectrum of support-bound
thiazolidinone which has been doubly labeled with a .sup.13 C-atom at the
position 2 of the ring and at the position alpha to the carbonyl of the
linker (labeled positions are indicated with a "*"). Panel B shows the
.sup.13 C NMR spectrum of support-bound doubly labeled thiazolidinone
after treatment with 95% TFA for one hour. Panel A shows the .sup.13 C NMR
spectrum of support-bound doubly labeled thiazolidinone after 40 cycles of
DNA synthesis.
FIG. 3 further illustrates the use of .sup.13 C NMR to monitor the
stability of thiazolidinones. Panel C shows the .sup.13 C NMR spectrum of
support-bound thiazolidinone which has been doubly labeled with a .sup.13
C-atom at the position 2 of the ring and at the position alpha to the
carbonyl of the linker (labeled positions are indicated with a "*". Panel
B shows the .sup.13 C NMR spectrum of support-bound labeled thiazolidinone
after 90 minute photolysis in PBS buffer. Panel A shows the .sup.13 C NMR
spectrum of support-bound labeled thiazolidinone after 3 hours of
photolysis in PBS buffer.
FIG. 4 shows an HLPC trace for the reaction mixture produced by subjecting
a support-bound thiazolidinone to 40 cycles of DNA synthesis and 3 hour
photolysis in PBS buffer.
FIG. 5 illustrates a comparison between the number of reactions required in
a stepwise and in a one-pot condensation reaction with three components.
FIG. 6 shows a HPLC trace for the products obtain from the parallel
synthesis of a thiazolidinone prepared from benzaldehyde, glycine, and
mercaptoacetic acid with an oligonucleotide tag after cleavage from the
resin.
FIG. 7 illustrates how thiazolidinones, 3-amino-thiazolidinones, and
metathiazanones can serve as peptidomimetics and methods for their
preparation.
FIG. 8 shows HPLC traces for the products of a solution preparation and a
solid state synthesis of a thiazolidinone prepared from glycine,
mercaptoacetic acid, and 3-pyridinecarboxaldehyde.
FIG. 9 shows HPLC traces for the products of a solution preparation and a
solid state synthesis of a thiazolidinone prepared from glycine,
thiolactic acid, and benzaldehyde.
FIG. 10 shows HPLC traces for the products of a solution preparation and a
solid state synthesis of a thiazolidinone prepared from alanine,
mercaptoacetic acid, and benzaldehyde.
FIG. 11 shows HPLC traces for the products of a solution preparation and a
solid state synthesis of a metathiazanone prepared from glycine,
.beta.-mercaptopropionic acid, and benzaldehyde.
FIG. 12 provides a schematic drawing illustrating a reaction sequence
carried out to demonstrate the practicality of a stepwise condensation
reaction and the stability of various intermediates.
FIG. 13 illustrates the use of .sup.13 C NMR to monitor the reactions set
forth in FIG. 12. Panel D shows the .sup.13 C NMR spectrum of N-acetylated
support-bound glycine which has been labeled with a .sup.13 C-atom at the
position alpha to the carbonyl. Panel C shows the .sup.13 C NMR spectrum
of the support-bound labeled imine produced by the reaction of
support-bound labeled glycine with benzaldehyde, followed by treatment
with acetic anhydride and pyridine. Panel B shows the .sup.13 C NMR
spectrum of the labeled thiazolidinone produced by the reaction of
support-bound labeled imine with mercaptoacetic acid, followed by
treatment with acetic anhydide and pyridine. Panel A shows the .sup.13 C
NMR spectrum of the product of the one-step condensation reaction of
support-bound labeled glycine with benzaldehyde and mercaptoacetic acid,
followed by treatment with acetic anhydride and pyridine.
FIG. 14 further illustrates the use of .sup.13 C NMR to monitor the
reactions set forth in FIG. 12. Panel D shows the .sup.13 C NMR spectrum
of N-acetylated support-bound glycine which has been labeled with a
.sup.13 C-atom at the position alpha to the carbonyl. Panel C shows the
.sup.13 C NMR spectrum of the support-bound labeled imine produced by the
reaction of support-bound labeled glycine with benzaldehyde, followed by
treatment with acetic anhydride and pyridine. Panel B shows the .sup.13 C
NMR spectrum of the labeled imine produced by reaction of the
support-bound labeled imine and benzaldehyde, followed by treatment with
2M acetic acid and then with acetic anhydride and pyridine. Panel A shows
the .sup.13 C NMR spectrum of the labeled imine produced by reaction of
the support-bound labeled imine and benzaldehyde, followed by treatment
with 2M 2-mercaptoethanol and then with acetic anhydride and pyridine.
FIG. 15 shows HPLC traces for the products from photolysis in PBS buffer
for two thiazolidinones.
FIG. 16 shows a HPLC trace and reaction scheme for the preparation of a
library of thiazolidinones resulting from the reaction of p-tolualdehyde,
mercaptoacetic acid, and various amino acids.
FIG. 17 illustrates the preparation of a thiazolidinone derivative.
Specifically, Panel A shows the gel .sup.13 C NMR spectrum of a labeled
support-bound thiazolidinone prepared from benzaldehyde, glycine, and
mercaptoacetic acid with the .sup.13 C label indicated with a "*". Panel B
shows a conventional .sup.13 C NMR spectrum of the corresponding unlabeled
thiazolidinone in solution. Panel C shows the gel .sup.13 C NMR spectrum
of the product of the reaction of the labeled support-bound thiazolidinone
with 3-chloroperoxybenzoic acid. Panel D shows a conventional .sup.13 C
NMR spectrum of the corresponding unlabeled sulfone in solution.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The description of the invention is provided as indicated by the following
outline. In addition, Section I provides for a glossary of terms to
facilitate the description of the invention. A number of terms and
abbreviations are defined to have the general meanings indiated as used
herein to describe the invention.
OUTLINE
I. Terminology
II. Description of the Invention
A. Overview
B. The Solid Support
1. Nature of the Support
2. Linkers
3. Immobilization
C. The Amine Component
D. The Carbonyl Component
E. The Thiol Component
F. The Exogenous Base
G. The Reaction Conditions
1. Immobilization
2. Reaction Temperature
3. Solvent
4. Dehydrating Agent
5. Stereochemistry
6. Polymer Preparation
H. Preparation of Derivatives of Thiazolidinones and Metathiazonones
I. Cleavage
J. Analysis of the Thiazolidinones
III. Preparation of Arrays of Thiazolidinones
A. General Overview
B. Preparation of Encoded Libraries
1. Overview
2. The Identifier Tag
C. Preparation of Arrays using the VLSIPS.TM. technique
D. Other Methods
E. Instrumentation
IV. Utility
A. Thiazolidinones and Derivatives As Peptidomimetics
B. Thiazolidinones and Derivatives As Therapeutics
V. Assays
I. Terminology
Unless otherwise stated, the following terms used in the specification and
claims have the meanings given below:
"Alkoxy" refers to the group alkyl-O-.
"Alkyl" refers to a cyclic, branched, or straight chain chemical group
containing only carbon and hydrogen, such as methyl, heptyl,
--(CH.sub.2).sub.2 --, and adamantyl. Alkyl groups can either be
unsubstituted or substituted with one or more substituents, e.g., halogen,
alkoxy, acyloxy, amino, aryl, hydroxyl, mercapto, carboxy, benzyloxy,
phenyl, benzyl, or other functionality which may be suitably blocked, if
necessary for purposes of the invention, with a protecting group.
Typically, alkyl groups will comprise 1 to 12 carbon atoms, preferably 1
to 10, and more preferably 1 to 8 carbon atoms.
"Amino" or "amine group" refers to the group --NR'R", where R' and R" are
independently selected from the group consisting of hydrogen, alkyl,
substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl
alkyl, heteroaryl, and substituted heteroaryl. In a primary amino group,
both R' and R" are hydrogen, whereas in a secondary amino group, either,
but not both, R' or R" is hydrogen.
An ".alpha.-amino acid" consists of a carbon atom, called the
.alpha.-carbon, to which is bonded an amino group and a carboxyl group.
Typically, this .alpha.-carbon atom is also bonded to a hydrogen atom and
a distinctive group referred to as a "side chain." The hydrogen atom may
also be replaced with a group such as alkyl, substituted alkyl, aryl,
substituted aryl, heteroaryl, and other groups. The side chains of
naturally occurring amino acids are well known in the art and include, for
example, hydrogen (as in glycine), alkyl (as in alanine (methyl), valine
(isopropyl), leucine (sec-butyl), isoleucine (iso-butyl), and proline
(--(CH.sub.2).sub.3 --)), substituted alkyl (as in serine (hydroxymethyl),
cysteine (thiomethyl), aspartic acid (carboxymethyl), asparagine,
arginine, glutamine, glutamic acid, and lysine), aryl alkyl (as in
phenylalanine, histidine, and tryptophan), substituted aryl alkyl (as in
tyrosine and thyroxine), and heteroaryl (as in histidine). See, e.g.,
Harper et al. (1977) Review of Physiological Chemistry, 16th Ed., Lange
Medical Publications, pp. 21-24.
In addition to naturally occurring side chains, the amino acids used in the
present invention may possess synthetic side chains. A "synthetic side
chain" is any side chain not found in a naturally occurring amino acid.
For example, a synthetic side chain can be an isostere of the side chain
of a naturally occurring amino acid. Naturally occurring and synthetic
side chains may contain reactive functionalities, such as hydroxyl,
mercapto, and carboxy groups. One skilled in the art will appreciate that
these groups may have to be protected to carry out the desired reaction
scheme. As stated above, the hydrogen at the .alpha.-carbon can also be
replaced with other groups; those of skill in the art recognize the
medicinal importance of .alpha.-methyl amino acids and other .alpha.-,
.alpha.-disubstituted amino acids.
"Aryl" or "Ar" refers to an aromatic carbocyclic group having a single ring
(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl),
which can optionally be unsubstituted or substituted with amino, hydroxyl,
lower alkyl, alkoxy, aryloxy, chloro, halo, mercapto, and other
substituents. Preferred aryl groups include phenyl, 1-naphthyl,
2-naphthyl, biphenyl, phenylcarboxylphenyl (i.e., derived from
benzophenone), and the like.
"Aryloxy" refers to the group aryl-O- or heteroaryl-O-.
"Arylalkyl" refers to the groups R'--Ar and R-HetAr, where Ar is an aryl
group, HetAr is a heteroaryl group, and R' is straight-chain or
branched-chain aliphatic group. Examples of arylalkyl groups include
benzyl and furfuryl.
"Carboxy" or "carboxyl" refers to the group --R'(COOH) where R' is alkyl,
substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl
alkyl, heterocyclic, heteroaryl, or substituted heteroaryl.
"Carboxyalkyl" refers to the group --(CO)--R' where R' is alkyl or
substituted alkyl.
"Carboxyaryl" refers to the group --(CO)--R' where R' is aryl, heteroaryl,
or substutited aryl or heteroaryl.
"Chemical library" or "array" is an intentionally created collection of
differing molecules which can be prepared either synthetically or
biosynthetically and screened for biological activity in a variety of
different formats (e.g., libraries of soluble molecules; and libraries of
compounds tethered to resin beads, silica chips, or other solid supports).
The term is also intended to refer to an intentionally created collection
of stereoisomers.
"Combinatorial synthesis strategy" or "combinatorial chemistry" refers to
an ordered strategy for the parallel synthesis of diverse compounds by
sequential addition of reagents which leads to the generation of large
chemical libraries. Thus, combinatorial chemistry refers to the systematic
and repetitive, covalent connection of a set of different "building
blocks" of varying structures to each other to yield large arrays of
diverse molecular entities.
"Exogenous base" refers to nonnucleophilic bases such as alkali metal
acetates, alkali metal carbonates, alkaline metal carbonates, alkali metal
bicarbonates, tri(lower alkyl) amines, and the like, for example, sodium
acetate, potassium bicarbonate, calcium carbonate, diisopropylethylamine,
triethylamine, and the like.
"Heteroaryl" or "HetAr" refers to a monovalent unsaturated aromatic
carbocyclic group having a single ring (e.g., pyridyl or furyl) or
multiple condensed rings (e.g., indolizinyl or benzothienyl) and having at
least one hetero atom, such as N, O, or S, within the ring, which can
optionally be unsubstituted or substituted with amino, hydroxyl, alkyl,
alkoxy, halo, mercapto, and other substituents.
"Identifier tag" denotes a physical attribute that provides a means whereby
one can identify a chemical reaction. The identifier tag serves to record
a step in a series of reactions used in the synthesis of a chemical
library. The identifier tag may have any recognizable feature, including
for example: a microscopically or otherwise distinguishable shape, size,
mass, color, optical density, etc.; a differential absorbance or emission
of light; chemical reactivity; magnetic or electronic properties; or any
other distinctive mark capable of encoding the required information, and
decipherable at the level of one (or a few) molecules. A preferred example
of such an identifier tag is an oligonucleotide, because the nucleotide
sequence of an oligonucleotide is a robust form of encoded information.
Identifier tags can be coupled to the solid support. Alternatively, the
"identifier tag" can be coupled directly to the compound being
synthesized, whether or not a solid support is used in the synthesis. In
the latter embodiment, the identifier tag can conceptually be viewed as
also serving as the "support" for synthesis.
"Limiting reagent" refers to that substance which limits the maximum amount
of product formed in a chemical reaction, no matter how much of the other
reactants remains.
"Linker" refers to a molecule or group of molecules attached to a solid
support and spacing a synthesized compound from the solid support, such as
for exposure/binding to a receptor.
"Predefined region" refers to a localized area on a solid support which is,
was, or is intended to be used for formation of a selected molecule and is
otherwise referred to herein in the alternative as a "selected" region.
The predefined region may have any convenient shape, e.g., circular,
rectangular, elliptical, wedge-shaped, etc. For the sake of brevity
herein, "predefined regions" are sometimes referred to simply as
"regions." In some embodiments, a predefined region and, therefore, the
area upon which each distinct compound is synthesized is smaller than
about 1 cm.sup.2 or less than 1 mm.sup.2. Within these regions, the
molecule synthesized therein is preferably synthesized in a substantially
pure form. In additional embodiments, a predefined region can be achieved
by physically separating the regions (i.e., beads, resins, gels, etc.)
into wells, trays, etc.
"Protecting group" refers to a chemical group that exhibits the following
characteristics: (1) reacts selectively with the desired functionality in
good yield to give a derivative that is stable to the projected reactions
for which protection is desired; 2) can be selectively removed from the
derivatized solid support to yield the desired functionality; and 3) is
removable in good yield by reagents compatible with the other functional
group(s) generated in such projected reactions. Examples of protecting
groups can be found in Greene et al. (1991) Protective Groups in Organic
Synthesis, 2nd Ed. (John Wiley & Sons, Inc., New York). Preferred
protecting groups include photolabile protecting groups (such as
methylnitropiperonyloxycarbonyl (Menpoc), methylnitropiperonyl (Menp),
nitroveratryl (Nv), nitroveratryloxycarbonyl (Nvoc), or
nitroveratryloxymethyl ether (Nvom)); acid-labile protecting group (such
as Boc or DMT); base-labile protecting groups (such as Fmoc, Fm,
phosphonioethoxycarbonyl (Peoc, see Kunz (1976) Chem. Ber. 109:2670);
groups which may be removed under neutral conditions (e.g., metal
ion-assisted hydrolysis), such as DBMB (see Chattopadhyaya et al. (1979)
J.C.S. Chem. Comm. 987-990), allyl or alloc (see, e.g., Greene and Wuts,
"Protective Groups in Organic Synthesis", 2nd Ed., John Wiley & Sons,
Inc., New York, N.Y. (1991), 2-haloethyl (see Kunz and Buchholz (1981)
Angew. Chem. Int. Ed. Engl. 20:894), and groups which may be removed using
fluoride ion, such as 2-(trimethylsilyl)ethoxymethyl (SEM),
2-(trimethylsilyl)ethyloxycarbonyl (Teoc) or 2-(trimethylsilyl)ethyl (Te)
(see, e.g., Lipshutz et al. (1980) Tetrahedron Lett. 21:3343-3346)); and
groups which may be removed under mild reducing conditions (e.g., with
sodium borohydride or hydrazine), such as Lev. Id. at 30-31, 97, and 112.
Particularly preferred protecting groups include Fmoc, Fm, Menpoc, Nvoc,
Nv, Boc, CBZ, allyl, alloc, Npeoc (4-nitrophenethyloxycarbonyl) and Npeom
(4-nitrophenethyloxy-methyloxy).
"Solid support" or "support" refers to a material or group of materials
having a rigid or semi-rigid surface or surfaces. In many embodiments, at
least one surface of the solid support will be substantially flat,
although in some embodiments it may be desirable to physically separate
synthesis regions for different compounds with, for example, wells, raised
regions, pins, etched trenches, or the like. According to other
embodiments, the solid support(s) will take the form of beads, resins,
gels, microspheres, or other geometric configurations. The solid support
is alternatively referred to herein as a support.
"Stereoisomer" refers to a chemical compound having the same molecular
weight, chemical composition, and constitution as another, but with the
atoms grouped differently. That is, certain identical chemical moieties
are at different orientations in space and, therefore, when pure, has the
ability to rotate the plane of polarized light. However, some pure
stereoisomers may have an optical rotation that is so slight that it is
undetectable with present instrumentation. The compounds of the instant
invention may have one or more asymmetrical carbon atoms and therefore
include various stereoisomers. All stereoisomers are included within the
scope of the invention and within the scope of the term "thiazolidinone",
"metathiazanone", or "derivative thereof".
Isolation and purification of the compounds and intermediates described
herein can be effected, if desired, by any suitable separation or
purification procedure such as, for example, filtration, extraction,
crystallization, column chromatography, thin-layer chromatography,
thick-layer (preparative) chromatography, distillation, or a combination
of these procedures. Specific illustrations of suitable separation and
isolation procedures can be had by references to the examples hereinbelow.
However, other equivalent separation or isolation procedures can, of
course, also be used.
Abbreviations: The following abbreviations are intended to have the
following meanings:
Boc=t-butyloxycarbonyl
BOP=benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate
DCC=dicyclohexylcarbodiimide
Ddz=dimethoxydimethylbenzyloxy
DIC=diisopropylcarbodiimide
DMT=dimethoxytrityl
Fmoc=fluorenylmethyloxycarbonyl
HBTU=2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HOBt=1-hydroxybenzotriazole
Menpoc=methylnitropiperonyloxycarbonyl
Menp=methylnitropiperonyl
Nv=nitroveratryl
Nvoc=6-nitroveratryloxycarbonyl and other photoremovable groups
OPfp=pentafluorophenyloxy
OSu=N-succinimidyloxy (also known as NHS)
PG=protective group
TFA=trifluoroacetic acid
II. Description of the Invention
A. Overview
The present invention, in one aspect, includes a highly efficient and
versatile method of synthesizing and screening, preferably in parallel and
simultaneous fashion, large numbers of 4-thiazolidinones, metathiazanones
and derivatives thereof. Thus, according to one aspect, the present
invention provides a solid-phase synthesis method for 4-thiazolidinones,
metathiazanones, and derivatives thereof in which variable substituent
groups are attached to a common central structure. This solid-phase
synthesis permits each reaction to be confined to a predefined region of a
small solid structure. The physical joining of a multitude of small solid
structures into a single unit, for example, then permits the simultaneous
handling of a multitude of compounds and reagents. The use of structures
of this kind for certain multiple simultaneous reactions is known in the
art, and its application to the present invention will become apparent
from the description which follows.
In order to expediently synthesize a combinatorial library of
4-thiazolidinones, metathiazanones, and derivatives thereof, a generalized
methodology for the solid phase synthesis of these compounds is also
provided. Synthesis on solid support proceeds in sufficiently high yield
in preferred embodiments such that purification and isolation steps can be
eliminated and thus, dramatically increasing synthesis efficiency.
According to one embodiment, the method of synthesizing 4-thiazolidinones,
metathiazanones, and derivatives thereof comprises the steps of first
binding an amine component to a solid support. Preferably, the amine
component will comprise a primary amine, and more preferably, an amino
acid, a peptide, a mono-substituted hydrazine derivative or a hydrazide
derivative. The heterocycle is then formed by treating the solid
support-bound amine component, either sequentially or simultaneously, with
a carbonyl component, preferably an aldehyde, and a thiol component,
preferably an .alpha.--mercapto carboxylic acid or a .beta.-mercapto
carboxylic acid. According to another embodiment, the thiol component is
immobilized on the support and the heterocycle is formed by treatment of
the immobilized component with a carbonyl component, preferably an
aldehyde, and an amine component, preferably an .alpha.-amino acid, a
peptide, a monoprotected or mono-substituted hydrazine derivative or a
hydrazide derivative.
B. The Solid Support
1. Nature of the Support
Typically, the libraries or arrays of the invention are composed of a
collection of "solid supports". Such solid supports may be of any shape,
although they preferably will be roughly spherical. The supports need not
necessarily be homogenous in size, shape or composition; although the
supports usually and preferably will be uniform. In some embodiments,
supports that are very uniform in size may be particularly preferred. In
another embodiment, two or more distinctly different populations of solid
supports may be used for certain purposes.
"Solid support" embraces a particle with appropriate sites for oligomer
synthesis and, in some embodiments, tag attachment and/or synthesis. Solid
supports may consist of many materials, limited primarily by capacity for
derivatization to attach any of a number of chemically reactive groups and
compatibility with the synthetic chemistry used to produce the array and,
in some embodiments the methods used for tag attachment and/or synthesis.
Suitable support materials typically will be the type of material commonly
used in peptide and polymer synthesis and include glass, latex, heavily
cross-linked polystyrene or similar polymers, gold or other colloidal
metal particles, and other materials known to those skilled in the art.
Except as otherwise noted, the chemically reactive groups with which such
solid supports may be derivatized are those commonly used for solid phase
synthesis of the polymer and thus will be well known to those skilled in
the art, i.e., carboxyls, amines and hydroxyls.
To improve washing efficiencies, one can employ nonporous supports or other
solid supports less porous than typical peptide synthesis supports;
however, for certain applications of the invention, quite porous beads,
resins, or other supports work well and are often preferable. A preferred
support is glass, as described in U.S. Pat. No. 5,143,854, supra. Another
preferred solid support is resin, such as the beads described in
co-pending U.S. patent application Ser. No. 07/946,239, filed Sep. 16,
1992, supra. In general, the bead size is in the range of 1 nm to 100
.mu.m, but a more massive solid support of up to 1 mm in size may
sometimes be used. Particularly preferred resins include Sasrin resin (a
polystyrene resin available from Bachem Bioscience, Switzerland); and
TentaGel S AC, TentaGel PHB, or TentaGel S NH.sub.2 resin
(polystyrene-polyethylene glycol copolymer resins available from Rappe
Polymere, Tubingen, Germany). Other preferred supports are commercially
available and described by Novabiochem, La Jolla, Calif.
2. Linkers
When bound to a solid support, the thiazolidinone and any associated tags
are usually attached by means of one or more molecular linkers. The linker
molecules preferably have lengths sufficient to allow the compounds to
which they are bound to interact freely with any molecules exposed to the
solid support surface, such as synthetic reagents or receptors which are
an object of study. The linker molecule, prior to attachment, has an
appropriate functional group at each end, one group appropriate for
attachment to the support and the other group appropriate for attachment
to the thiazolidinone or tag.
One can, of course, incorporate a wide variety of linkers, depending upon
the application and the effect desired. For instance, one can select
linkers that impart hydrophobicity, hydrophilicity, or steric bulk to
achieve desired effects on properties such as coupling or binding
efficiency. In one aspect of the invention, branched linkers, i.e.,
linkers with bulky side chains such as the linker, Fmoc-Thr(tBu), are used
to provide rigidity to or to control spacing of the molecules on the solid
support in a library or between a molecule and a tag in the library. In
some embodiments, cleavable linkers will be used to facilitate an assay or
detection step as discussed more fully below.
3. Immobilization
The choice of functionality used for binding a molecule to the solid
support will depend on the nature of the compound to be synthesized and
the type of solid support. Conditions for coupling monomers and polymers
to solid supports through a wide variety of functional groups are known in
the art. See, e.g., U.S. Pat. Nos. 4,542,102; 4,282,287; Merrifield,
"Solid Phase Peptide Synthesis," J. Am. Chem. Soc., (1963) 85:2149-2154;
Geysen et al., "Strategies for Epitope Analysis Using Peptide Synthesis,"
J. Imm. Meth., (1987) 102:259-274; Pirrung et at., U.S. Pat. No.
5,143,854; and Fodor et al., "Light-Directed Spatially-Addressable
Parallel Chemical Synthesis," Science (1991) 251:767-773, each of which is
incorporated herein by reference.
C. The Amine Component
According to the present invention, an amine component is coupled to a
carbonyl component and a thiol component to yield a thiazolidinone or
derivative thereof. The amine component can be utilized in a soluble
format or can be attached to a solid support. According to the latter
embodiment, the amine component will include a functionality which can
covalently bind the molecule to the solid support (e.g., an activated
carbonyl, acyl halide, or activated hydroxyl) as well as the amino group
or a protected derivative thereof.
Typically the amine component will comprise a primary amine having the
formula: R--NH.sub.2, wherein R is selected from the group consisting of
hydrogen (i.e., an amine salt as a further valence is necessary to attach
the amine component to the solid support), alkyl, alkoxy, amino, aryl,
aryloxy, heteroaryl, and arylalkyl or salts thereof. More preferably, the
amine component will have the formula:
##STR5##
wherein R.sup.1 and R.sup.2 are independently selected from the groups
consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, heteroaryl,
carboxyl, carboxyalkyl, carboxyaryl, and arylalkyl. The amine component,
if not commercially available, can be prepared by standard chemical
procedures.
In a preferred embodiment, the amine component will comprise an amino acid,
and more preferably, an amino acid bearing a substituent on the alpha
carbon. The amino acids finding utility in the present invention include
the twenty naturally occurring .alpha.-amino acids, in either their D- or
L-enantiomeric forms. Unnatural amino acids such as .alpha.,
.alpha.-disubstituted amino acids, N-alkyl amino acids, lactic acid, and
other unconventional amino acids are also suitable components. Examples of
unconventional amino acids include, but are not limited to:
4-hydroxyproline, O-phosphoserine, 3-methylhistidine, 5-hydroxylysine, and
other similar amino acids. Since peptides are composed of amino acid
subunits, one of skill in the art will appreciate that peptides can also
serve as amine components.
Alternatively, the amine component may comprise a mono-protected hydrazine
having the formula PGNHNH.sub.2 wherein PG is an appropriate acid-lable
(e.g., Boc), base-labile (Fmoc), or photocleavable protecting group; a
mono-substituted hydrazine derivative having the formula R.sup.1
NHNH.sub.2 where R.sup.1 is alkyl, alkyoxy, aryl, aryloxy, heteroaryl,
carboxyl, carboxyalkyl, carboxyaryl, and arylalkyl; or a hydrazide
derivative having the formula R.sup.1 (CO)NHNH.sub.2 where R.sup.1 is as
described above.
According to another aspect of this invention, the solid support will be
derivatized such that the amine component comprises a surface amino group
on the solid support. For example, thiazolidinones can be prepared from
the primary amino group of the Knorr linker (a linker having a free | | |
