Note: Descriptions are shown in the official language in which they were submitted.
WO95/02566 2 1 6 7 3 9 l PCT~S94/08141
SrNL~SIS OF COMBINATORIAL ARRAYS OF ORGANIC
COMPOUNDS THROUGH THE USE OF MULTIPLE
COMPONENT COMBINATORIAL ARRAY ~rNL~SES
Field of the Invention
The present invention relates to a method for
generating arrays of compounds having a common core
structure through a combinatorial array synthesis using
a multiple component combinatorial array (MCCA)
synthesis. The present invention also relates to arrays
of organic compounds wherein each array is comprised of
a group of structural analogues having a common core
structure, the core structure being generated by a
multiple component one pot synthesis.
Backqround of the Invention
Understanding the relationship between a
molecule's structure and its biological function is
essential to the development of new and improved
therapeutic agents. For example, the contours of a
receptor's binding site or an enzyme's active site can
be effectively analyzed by testing the binding affinity
of several structural analogues to these sites.
In nature, many biologically active molecules
possess one of a relatively small group of common core
structures. These common core structures include ~-
lactams, peptides, sugars, nucleosides, aromatics,
pyridines, steroids, tetrazoles, pyrazines, terpines and
alkaloids. Biological activity is often associated with
the presence of one of these core structures.
Structure-function studies using a group of compounds
that share a common core structure but where the members
of the group have differing substituents have been used
to elucidate numerous biological mechanisms. Several of
these studies and the mechanisms that were determined
are cited in Goodman & Gilman's The Pharmacological
Basis of Therapeutics, Macmillian (6th Edition, 1980).
In addition to being a valuable research tool
for understanding biological mechanisms, structure-
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function analysis using substituent and structuralvariants of a known biologically active molecule is an
invaluable tool for developing new therapeutic agents.
For example, improved penicillin based antibiotics were
developed by mimicking penicillin's ~-lactam core
structure with a core structure that is not susceptible
to degradation by ~-lactamase. Many antiviral drugs,
such as the A.I.D.S. drug 3-azidothymidine (AZT), are
designed to mimic the core structure of nucleosides
based on the notion that these nucleoside structural
analogues will become incorporated into and interfere
with the virus's genetic machinery.
The rate limiting step in the analysis of
structure-function relationships is the synthesis of the
necessary structural analogues. The traditional
strategy for evaluating structure-function relationships
involves an iterative process whereby successive groups
of compounds, all having a common core structure, are
synthesized and assayed. After each iteration, the
assay results are evaluated in order to design the next
group of compounds to be synthesized and assayed. This
process is repeated until the substituents or structural
factors influencing biological activity are determined
and/or optimal therapeutic agents are identified.
The traditional approach to evaluating
structure-function relationships is extremely time
consuming and labor intensive, often requiring the
synthesis of greater than one thousand compounds over
the course of several years. The chemical syntheses of
these compounds are often complex, generally requiring a
multistep synthesis for each compound. As a result, a
high degree of skill in the synthetic chemical arts is
generally required to synthesize the compounds needed to
perform these structure-function studies. This greatly
limits the number of scientists that can perform this
type of structure-function analysis research. Further,
the labor demands for this type of research is very
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intense, often requiring the full time efforts of
several organic chemists per project. Because of these
labor and skill demands, performance of this type of
research and hence the development of new therapeutic
agents is often limited to larger chemical firms.
Researchers possessing a strong understanding of the
biological system being studied are often prevented from
participating in the development of new therapeutic
agents because of their inability to access the
compounds needed for performing these studies.
The labor demands associated with synthesizing
,structural analogues has also greatly limited the range
of compounds for which structure-function analysis has
been performed. The synthetic effort required to
produce structural analogues has made it impractical to
probe core structures not currently associated with
known biologically active molecules for biological
activity. However, it is likely that the number of core
structures associated with biologically active compounds
greatly exceeds the number of structures that have been
identified. A more efficient method for synthesizing
structural analogues would make it feasible to evaluate
a far greater range of structures for biological
activity.
In contrast to the effort required to
synthesize compounds, the effort required to screen the
synthesized compounds for biological activity is
relatively low. A simple and efficient method for
synthesizing compounds useful for probing for biological
activity would significantly accelerate the rate of new
drug development and enable a greater number of
scientists to participate in the research.
One of the factors that makes the synthesis of
compounds used in structure-function research studies so
time consuming and research intensive is the fact that
the chemical syntheses of these compounds are performed
separately through stepwise linear transformations.
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Since the time required to screen compounds is
significantly smaller than the time required to
synthesize the compounds, it would be advantageous from
a time management standpoint to synthesize more
compounds by a more rapid method, even if the total
number of compounds that would have to be screened were
increased.
The general approach for screening monoclonal
antibodies to a specific antigen presents a useful model
for how structure-function studies should be conducted.
After antigen stimulation of a host, the monoclonal
antibody producing B-cells of the host are immortalized
and diluted over a large array of microtiter wells.
Most of the monoclonal antibodies produced by the
individual immortalized cells in each titer well have
little utility. However, assays exist to identify the
useful antigen binding antibodies in the array.
Similarly, it is possible to rapidly assay large arrays
of organic compounds for biological activity.
Unfortunately, however, there are no known biological
systems that can be stimulated to produce an array of
organic chemicals from which a desired compound can be
isolated. Thus, the need exists for a simple and
efficient method for generating an array of organic
compounds from which biologically active agents can be
identified.
The methodology developed for the automated
synthesis of biopolymers such as RNA, DNA, polypeptides
and most recently oligosaccharides also presents a
useful model for how structure-function studies could be
conducted. The automated synthesis of these biopolymers
has greatly accelerated the development of the
biotechnology arts, primarily because RNA and DNA
probes, as well as small polypeptides, are readily
obtainable by all researchers. The benefits derived
from automated biopolymer synthesis technology
highlights the current need for a simple, efficient and
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rapid method for generating numerous organic compounds
to probe structure-function relations in route to the
development of new therapeutic agents.
The automated synthesis of these biopolymers
is achieved by performing a repetitive series of
reactions on the biopolymer which is attached to a solid
support. Unfortunately, biopolymer synthesis technology
cannot be readily redirected to the production of
structural analogues of organic compounds. Unlike
biopolymer synthesis, the synthesis of structural
analogues of organic compounds involves numerous
different chemical reactions. Also, because of the
wider variety of chemical reactions and reagents
involved, a wider variety of solid phase supports and
chemical linkers are needed.
SUMMARY OF THE INVENTION
The present invention relates to an array of
compounds having a common core structure wherein the
compounds of the array comprise the products of a
multiple component combinatorial array synthesis having
at least three reactive components. Each component of
the multiple component combinatorial array synthesis
comprises a group of reactants having a common
functional group. The array synthesis is conducted
under appropriate conditions such that the common
functional group of each component in the reaction
reacts with functional groups on the other components to
form an array of compounds having a common core
structure. Each component of the combinatorial array
synthesis can itself comprise an array of compounds
having a common core structure, the array of component
reactants being synthesized by a multiple component
combinatorial array synthesis. Each component can also
comprise a group of reactants wherein each reactant
comprising a mixture of compounds having a common
functional group. In a further embodiment of the
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present invention, the array of compounds is formed
while bound to a solid support.
The present invention also relates to a method
of making an array of compounds having a common core
structure using a multiple component combinatorial array
synthesis with at least three components. Each
component comprises a group of reactants having a common
functional group. The method comprises organizing a
series of reaction vessels in an n dimensional array
wherein each reaction vessel is identifiable by its
coordinates in the n dimensional array. Each axis in
the n dimensional array corresponds to a different
component in the array synthesis. Each position on each
axis corresponds to a different reactant of the
corresponding component. The reactants of the n
components are added to the n dimensional array of
reaction vessels such that the same reactant is added to
all of the reaction vessels in the array having a
position on the array corresponding to that reactant.
The components in each reaction vessel are then reacted
under appropriate conditions to form the compounds of
the array. Each component of the combinatorial array
synthesis can itself comprise an array of compounds
having a common core structure, the array of component
reactants being synthesized by a multiple component
combinatorial array synthesis. Each component can also
comprise a group of reactan~s, each reactant comprising
a mixture of compounds having a common functional group.
A further embodiment of the present invention relates to
solid phase multiple component array synthesis which
comprises the further step of binding one of the
components to a solid support.
The present invention also relates to a method
of creating a combinatorial array of compounds with a
common core structure by identifying the desired core
structure, identifying a MCCA reaction capable of
generating that core structure, followed by preparing an
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array of compounds using the identified MCCA reaction
according to the aforementioned method.
The present invention also relates to a method
for conducting in vitro assays of biological material by
adding biological material to an array of compounds,
each member compound of the array having a common core
structure and being bound to a solid support followed by
measuring the effect each member compound of the array
has on the biological material's biological activity.
BRIEF DESCRIPTION OF THE FIGURES
Figure la-p provides a summary of some of the
common core structures synthetically accessible by MCCA
reactions.
Figure 2 depicts the synthesis of the core
structures depicted in Figure 1, showing the
correspondence between the substituents (R, Rl, R2, R3,
R4, etc.) in the product and in the reaction components.
Figure 2a depicts a synthesis described in Joucia, et
al., J. Tetrahedron Lett., (1985) 26:1221. Figure 2b
depicts a synthesis described in Kelly, et al., J. Am.
Chem. Soc., (1985) 107:3879. Figure 2c depicts a
synthesis described in Banville, et al. Can. J. Chem.,
(1974) 50:80; Cameron, et al., J. Chem. Soc. Chem.
Commun., (1976) 275; and Cameron, et al., J. Chem. Soc.
Chem. Commun., (1977) 297. Figure 2d depicts a
synthesis described in Kelly, et al., J. Amer. Chem.
Soc. (1985) 107:4998. Figure 2e depicts a synthesis
described in Posner, et al., Tetrahedron (1981) 37:3921.
Figure 2f depicts a synthesis described in Davis, et
al., J. Org. Chem., (1979) 44:3755. Figure 2g depicts a
synthesis described in Bestmann, et al., Anqew Chem.
Int. Ed. Enql, (1985) 24:790. Figure 2h depicts a
synthesis described in Weis, et al., Tetrahedron Lett.,
(1981) 22:1453. Figure 2i depicts a synthesis described
in Ugi, et al., Justus Lieqis Ann. Chem., (1967) 709:1.
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Figure 2j depicts a synthesis described in Ugi, et al.,
Chem. Ber. (1961) 64:734. Figure 2k depicts a synthesis
described in Sebti, et al., SYnthesis (1983) 546.
Figure 21 depicts a synthesis described in Isenring, et
al., Synthesis (1981) 385 and Isnering, et al.,
Tetrahedron (1983) 39:2591. Figure 2m depicts a
synthesis described in Kunz, et al., J. Amer. Chem. Soc.
(1988) 110:651. Figure 2n depicts a synthesis described
in Boehm, et al., J. Org. Chem. (1986) 51:2307. Figure
2O depicts a synthesis described in Murakami, et al., J.
Orq. Achem., (1993) 58:1458. Figure 2p depicts a
synthesis of a c-linked disaccharide wherein the
stereochemistry of the first ring is determined by the
sugar used in the synthesis and the stereochemistry of
the second ring is determined by the oxidation catalyst.
Figure 3 depicts the fact that MCCA array
syntheses produce analogues on a geometric scale where
the total number of analogues synthesized equals the
product of the number of structural variants of each
component of the MCCA array synthesis used.
Figure 4 depicts the results of a two
dimensional MCCA array synthesis using the Passerini
reaction wherein the aldehyde and acid reaction
components are varied.
Figure 5 depicts the mechanism of action for
carzinophilin/azinomycin.
Figure 6 depicts the ~-acyloxy amine core
structure of the antitumor antibiotic
carzinophilin/azinomycin. By labelling the substituents
off of the ~-acyloxy amine core structure R1, R2 and R3,
a Passerini reaction scheme for the synthesis of
carzinophilin/azinomycin and its derivatives is
identified.
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Figure 7 depicts the Ugi reaction which, upon
removal of the protecting groups, provides
nonhydrolizable peptide analogues that mimic a natural
peptide's structure.
Figure 8 depicts the peptide backbone core
structure of phosphotyrosine peptides and the synthesis
of structural variants of phosphotyrosine by varying the
first and second amino acids employed (aal and aa2) as
well as the other components of the Ugi reaction.
Figure 9 depicts the synthesis of a series of
tyrosine-based peptide analogues. Figure 9a depicts the
synthesis of pseudosubstrate peptide inhibitors. Figure
9b depicts the synthesis of low molecular weight
tyrosine-based peptide analogues where the tyrosine
structural unit is intact. Figure 9c depicts the
synthesis of low molecular weight tyrosine-based peptide
analogues where the tyrosine structural unit is
modified. Figure 9d depicts the synthesis of additional
low molecular weight tyrosine-based peptide analogues.
Figure l0 depicts the synthesis of a support
and linker system.
Figure ll depicts the solid phase synthesis of
peptidomimetics by means of the Ugi reaction.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a simple and
efficient method for synthesizing arrays of organic
compounds having a common core structure. More
specifically, the present invention relates to the use
of multiple component combinatorial array (MCCA)
syntheses to synthesize arrays of structurally related
analogues having a common core structure.
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Multiple component combinatorial array ~MCCA)
syntheses correspond to reactions where the reactants
combine synchronously or asynchronously in one reaction
vessel to form the product. Over the years, numerous
MCCA reactions have been developed. In some cases, the
reactions were developed in order to show an efficient
synthesis of a known natural product. Cameron, et al.,
J. Chem. Soc. Chem. Comm., (1976) 275 (synthesis of m-
deoxygenated benzoquinones in the application of the
total synthesis if anthraquinone insect pigments);
Schopf, Anqew. Chem., (1937) 50:779-797 (double Mannich
reaction in the total synthesis of tropane alkaloids);
Posner, et al., Tetrahedron, (1981) 39:3921. In other
cases, the reactions were developed as a general entry
into methodology involving the formation of multiple
bonds in a cascade process. In any event, these
reactions represent a simple and efficient means for
generating numerous structural variants sharing the core
structure generated by the given MCCA reaction. A
summary of some of the core structures that are made
synthetically accessible by MCCA reactions is provided
in Figure l. The reaction sequence for generating these
core structures is summarized in Figure 2.
Traditionally, structural analogues have been
synthesized by multiple step linear syntheses. Linear
syntheses involve the sequential reaction of several
separate reactants in order to achieve the final
product. Linear syntheses are generally not one pot
reactions, requiring the isolation and purification of
intermediate products. Unlike linear syntheses, MCCA
reactions, because they are one pot syntheses, do not
require the isolation and purification of intermediate
reaction products. As a result, MCCA reactions are
simpler and more efficient to perform than multiple step
linear syntheses. Products of MCCA reactions are
therefore more accessible to those lacking a high level
of skill in the synthetic organic chemical arts.
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Further, since no intermediate isolation and
purification is required, MCCA reactions are more
readily adaptable to automation.
MCCA reactions, when used in the form of an
array synthesis, enable the synthesis of structural
analogues on a geometric rather than linear scale. In
an MCCA array synthesis, at least three reaction
components are used. Each component comprises a group
of reactants possessing a common functional group that
participates in the MCCA reaction. The combinatorial
array synthesis is conducted by reacting the different
combinations of the various MCCA components.
Combinatorial array syntheses are depicted in Figures 3,
7 and 9.
A MCCA array synthesis can be characterized by
the number of reaction components involved and the
number of variants of each reaction component employed.
In a MCCA array synthesis where the MCCA reaction has n
components, an n ~;men.~ional array of structural
analogues can be produced. Each array of analogues is
equivalent to a library of analogues. When one reactant
is held constant (or when a component is comprised of a
group of one reactant), the n dimensional array
simplifies to an n-1 array and can be visualized as a
sublibrary of a greater array where the reactant used
for that component is held constant.
As depicted in Figure 3, an MCCA array
synthesis is analogous to the multiplication of a series
of one dimensional arrays. Each component of the array
synthesis is represented by a different one dimensional
array, the elements of the one dimensional array
corresponding to the group of reactants used for a given
component in the combinatorial array synthesis.
Mathematically, an n dimensional array is generated when
n one dimensional arrays are multiplied. In the case of
an MCCA array synthesis, the reaction vessels are
organized in an n dimensional array wherein each
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reaction vessel is identifiable by its coordinates in an
n dimensional array. Each axis of the array of reaction
vessels corresponds to a different component in the MCCA
reaction. Each position on each axis corresponds to a
different reactant of the corresponding component.
During the combinatorial array synthesis, the reactants
of the n components are added to the n dimensional array
of reaction vessels such that the same reactant is added
to all of the reaction vessels in the array having a
position on the array corresponding to that reactant.
The reaction of the components in the array of reaction
vessels is analogous to the multiplication of the n one
dimensional arrays since the reaction of the various
reactants in each reaction vessel in the n dimensional
array results in the ''productll of the reactants added to
that reaction vessel.
MCCA array syntheses have the advantage over
linear syntheses in that they produce analogues on a
geometric scale rather than a linear scale. The ability
to synthesize structural analogues on a geometric scale
significantly decreases the time and effort required to
synthesize these compounds.
As depicted in Table 1, the total number of
analogues synthesized equals the product of the number
of each reactant employed. For example, if 10 different
variants of each component of a three component MCCA are
employed, a lOxlOxlO three dimensional array of
analogues is produced which corresponds to an array of
1000 structural analogues.
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TA~3LE 1
No. of Components No. of ReactantsNo. of
Per ComponentAnalogues
3 3 27
3 5 125
3 10 1000
4 5 625
3125
The combinatorial array synthesis described
above provides a wide variety of structural analogues
limited only be the reactive limits of the underlying
MCCA reaction. For example, in a further embodiment of
the present invention, the reactants of a component of a
combinatorial array synthesis can themselves be the
product of a combinatorial array synthesis. In yet a
further embodiment of the present invention, individual
reactants of a component of an MCCA array synthesis can
comprise mixtures of different compounds having a common
functional group. When the individual reactants used
comprise mixtures of compounds, the analogues produced
by the synthesis are a mixture of compounds. Hence, the
term "analogue," as it is used in this application, can
include more than one compound. Further, depending on
the particular reaction, cis, trans, exo, endo and
diasteriometric isomers can also be produced by the
array synthesis.
It should be understood that the MCCA array
syntheses of the present invention encompass all type of
chemical reactions including, but not limited to
solution, solid phase, photochemical, electrochemical,
free radical and enzymatic reactions.
It should also be understood that MCCA array
syntheses involving the condensation of four or more
components can also be conducted according to the
present invention. In the case of a four component
array synthesis, it is simplest to view the reaction as
a series of three dimensional (n-1) array syntheses
where the only variable between the three dimensional
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arrays is the structure of the fourth component used in
each array. Higher order MCCA array syntheses are also
possible.
The present invention also relates to the
arrays (libraries) of structural analogues that can be
synthesized by an MCCA array synthesis. Assaying
compounds requires very little time relative to the time
presently required to synthesize compounds by linear
syntheses. The present invention enables the rapid
production of large libraries of structurally related
analogues all having a common core structure. Given the
speed with which compounds can be analyzed, access to
libraries of compounds sharing a common core structure
would be an invaluable tool for analyzing the structure-
function relationships governing a molecule's biologicalactivity.
At present, a relatively small group of core
structures appear to predominate in nature. Reference
arrays of structural analogues of these common core
structures would be valuable for making at least
preliminary screenings regarding which core structures
and which analogues having those core structure arrays
possess some biological activity. Although these
reference arrays may not always be specifically designed
for the biological system being tested, the observation
of some change in biological activity with regard to
certain analogues within the arrays would provide
valuable clues as to the governing structure-function
relations. These clues could then be used to design
more specifically tailored combinatorial arrays and/or
specific target compounds.
Access to combinatorial arrays of structural
analogues of core structures found in nature would also
be invaluable for expeditiously performing preliminary
structure-function analyses on newly isolated
biomolecules that have not been fully characterized
regarding their biological function. Further, these
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combinatorial arrays would provide smaller research
groups and, in particular, non-synthetic research
groups, with access to compounds for doing structure-
function analyses that were previously inaccessible.
- The ability of the present invention to
generate large quantities of structural analogues now
makes it feasible, for the first time, to investigate
the biological activity of arrays of compounds with core
structures not currently associated with biologically
active molecules. Previously, the synthetic effort
required to produce structural analogues made it
impractical to probe core structures not associated with
known biologically active molecules for biological
activity. The large arrays of structural analogues that
can be generated by the present invention now make it
feasible to assay a wider range of core structures and
structural analogues of these core structures for
biological activity.
A very small amount of material is needed to
assay a compound for the presence of biological
activity. Therefore, combinatorial arrays of structural
analogues would not need to contain large amounts of any
given compound. In addition, since a small quantity of
compound is needed per assay, even combinatorial arrays
containing small quantities of each compound would be
capable of repeated use.
The organizational sense of the combinatorial
arrays of the present invention greatly simplifies the
mental aspect of structure-function analysis. In order
to appreciate this utility, the organizational nature of
the combinatorial arrays of the present invention needs
to be explained. The combinatorial array of compounds
generated by a MCCA synthesis is best thought of in
terms of a geometric array having a dimension equal to
the number of reaction components employed in the MCCA
array synthesis. For example, a four component MCCA
reaction would generate a four dimensional combinatorial
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array. Each element of the array is identifiable by its
coordinate in the array which corresponds to the precise
components used to synthesize that analogue. Hence the
product of the second variant of each component of a
three dimensional array would have an array coordinate
of (2,2,2).
The coordinate system of the combinatorial
array assists the evaluation of results derived from
assaying the elements of a particular combinatorial
array for biological activity. Each n dimensional array
can be envisioned as a series of (n-1) dimensional
arrays or sublibraries wherein all of the members of
each sublibrary share a common core structure formed by
at least one common component. Thus, as depicted in
Figure 3, a three component condensation reaction can be
broken down into a series of two dimensional arrays
where one reactant is held constant.
The formation or destruction of activity
across a sublibrary reveals the biological significance
of the structure contributed by the component common to
all of the members of the sublibrary. Thus, by
interpreting MCCA arrays as multidimensional arrays that
can be divided into sublibraries, the present invention
provides a systematic framework for evaluating
structure-function relationships. Since each
sublibrary, independent of its size, corresponds to a
class of compounds where one structural subunit is held
constant, a finding of biological activity across a
particular sublibrary would indicate that the unvaried
functional subunit was significant to the biological
activity realized for that group of compounds.
The organizational sense created by the
combinatorial arrays of the present invention provide an
invaluable tool for evaluating the structure-function
correlations of a very large number of compounds. The
linear synthetic approach currently employed for the
identification of new therapeutics is driven largely by
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17
evaluating the binding constants of the molecules
assayed. The rational as to why certain analogues
exhibit stronger binding is not necessarily readily
apparent from those studies. In contrast, the approach
of the present invention provides an organizational
framework whereby structure-function effects become
readily apparent. Thus, in addition to providing a more
rapid and efficient route to analogues for testing
structure-function relationships, the present invention
provides a more effective means for evaluating
structure-function relationships in route to the
identification of superior therapeutic agents.
The present invention also relates to the
solid phase synthesis, storage and use of these MCCA
arrays. Solid phase synthesis is valuable for
simplifying purification. By binding one of the
reaction components to a solid support, for example, a
microtiter well, it is possible to isolate the compounds
synthesized in high purity. One of the disadvantages of
any multiple step reaction is low yields. A three step
synthesis having a 70~ yield for each step only produces
a product in a 35~ yield. Thus, when synthesizing
complex molecules, simplification of the purification
process is highly desirable as a means for optimizing
the resulting yield. By synthesizing the compounds of
the combinatorial array on a solid support where one of
the reactants is attached to a detachable linker,
purification and isolation of the product is greatly
simplified. This is especially important when complex,
lower yielding MCCA reactions are involved since all
that is needed in the present invention is an adequate
amount of each analogue to test for biological activity.
Solid phase synthesis of the combinatorial
arrays of the present invention improves the storage
stability of these arrays by enabling these compounds to
be isolated in high purity and in a solid state. In
addition, storing the compounds while bound to a solid
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support prevents the spillage or mixing of the compounds
in the combinatorial array.
Solid phase synthesis also facilitates the use
of these combinatorial arrays for conducting in vitro
binding studies. Entire two dimensional arrays of
compounds bound to microtiter trays can be assayed at
one time for their ability to bind to a protein or cell.
This enables entire arrays of analogues to be very
rapidly assayed for binding to different biomolecules
and cells.
Solid phase synthesis of combinatorial arrays
by MCCA reactions presents very few limitations on the
different types of compounds that can be synthesized.
In general, the only limitation presented by the use of
solid phase synthesis is the requirement that there be a
means for attaching one of the reaction components to
the solid support. This will generally involve the use
of a functional group such as an alcohol, mercaptan,
amine, carbonyl, carboxylate or selenide. No other
limitations to the structure of the compound to be
synthesized are created. Further, as new linkers are
designed, the structural limitations presented will be
further reduced.
Any solid support media capable of covalently
binding to a linker can be used in the present
invention. Polymers such as polystyrene-divinyl
benzene, polyethylene grafted polystyrene,
polyacrylamide-kieselguhr composites and controlled pore
glass have all been commercialized with functional
groups suitable for derivitization with various linkers.
Linkers are used to couple a variety of
monomers to the solid support for the solid phase
polymeric synthesis of biomolecules such as DNA and
peptides. The linkers of the present invention must be
capable of binding to one of the components of the MCCA
synthesis and must also be capable of later releasing
the synthesized molecules by some specific, regulatable
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W095/02566 PCT~S94/08141
19
mechanism. Such regulatable methods include but are not
limited to thermal, photochemical, electrochemical,
acid, base, oxidation and reduction reactions.
There are several commercially available
linkers which provide a variety of functional group
coupling and cleavage strategies. Examples of acid
labile linkers include the 4-hydroxy methyl phenoxy
aliphatic acids which allow the coupling of ethers
acetals and esters to the linker. Acid labile linkers
bearing an amine, such as the p-[2,4-
dialkoxylaminobenzyl] phenoxy acetic acid, allow for
coupling through functional groups such as amides,
imines, carbonates or ureas. The extent of the alkoxyl
substituents on the aromatic rings of these linkers
influence the pH at which the organic compound is
cleaved from the solid support. Trityl-based linkers
can be cleaved under mildly acid conditions.
Base labile linkers include [4-(2-
bromopropionyl)phenoxy] acetic acid linkers. The ester
coupling of these linkers can be cleaved by nucleophiles
such as hydroxide, alkoxides, and amines to give the
corresponding acid, ester and amide derivatives of the
organic compounds. In addition, the [4-(2-
bromopropionyl)phenoxy] acetic acid linker as well as
the ortho-nitro benzyl linkers enable the photochemical
cleavage of an organic compound from solid support under
neutral conditions.
It is preferred that the linker be capable of
photochemically or thermally releasing the product since
no decoupling reactants are required. A linker having a
thermal release mechanism is the most preferred
embodiment because thermal reactions provide greater
control over the rate at which the compound is released.
Further, thermally labile linkers are more stable than
photochemically labile linkers.
The types of linkers useful in the present
invention is not limited to the linkers described herein
SUBSrlTUTE ~HEET (RU~E 26)
WO9~/02~66 2 1 ~ ~ ~ 9 t PCT~S94tO8141 _
since additional suitable linkers may exist in the art
or be later developed. The use of linkers in the solid
phase synthesis of an MCCA array is described in
Examples 6 and 7.
The present invention also relates to the use
of solid support bound analogues in in vitro binding
studies. In this application of the present invention,
whole arrays of compounds can be simultaneously, quickly
and efficiently tested for their ability to bind to a
biological molecule. This greatly accelerates the
process of assaying large arrays of compounds for
biological activity.
Arrays of compounds bound to a solid support
can also be used to study the effect of structural
orientation on a compound's biological activity. This
is accomplished by assaying combinatorial arrays of the
same group of compounds where each array differs in that
a different portion of the structural analogues in each
array are used to bind the compounds to the solid
support. Hence, the present invention provides a method
for testing the binding affinity of different faces of
the same array of compounds. The linear synthesis
approach currently employed does not provide a means for
obtaining this data short of obtaining crystal
structures of bound complexes.
One of the limitations of using solid phase
synthesis is the requirement that one of the components
be attachable to the solid support and also be able to
participate in the MCCA reaction. In order for a
component to serve both these functions, the component
must have two functional groups, one that participates
in the reaction and the other which binds to the linker.
The requirement that a component have two functional
groups is not a significant problem when one does not
seek to vary the structure of the component being bound
to the solid support. However, the range of structural
variants of a component that can be both bound to a
SUBSrlTUTE SHEET (RULE 26)
2 1 6739 1
W095/0256C PCT~S94/08141
21
linker and at the same time participate in the reaction
is often limited.
It should be noted that it is not always
necessary to isolate and purify the products of an array
synthesis. Many MCCA reactions are high yielding and
all components can be reacted in solution. Further,
many bioassays are operable on crude product mixtures.
Hence, in these instances, the primary advantage of
solid phase synthesis, namely simplified purification,
is obviated.
In addition, a method for isolating reaction
products from solution chemistry reaction mixtures
involves sequestering the unreacted components of the
reaction. An example of the use of sequestering agents
to isolate the products of an Ugi reaction is provided
in Example 9. By using sequestering agents to sequester
the unreacted components of the reaction, the products
of the array synthesis can be isolated at sufficient
purity to use in bioassays.
The present invention also relates to a method
for the automated production of structural analogues.
The automated synthesis of MCCA arrays reduces the level
of skill required for the preparation of arrays of
structural analogues and thus enables the performance of
structure-function studies without the assistance of
synthetic organic chemists. Thus, much like automated
DNA, RNA and peptide synthesizers, the present invention
enables researchers without a high level of skill in the
synthetic chemical arts to generate combinatorial arrays
of structural analogues. The automated synthesis of the
combinatorial arrays of structural analogues of the
present invention is made possible by the logical
organization of the arrays synthesized. Automation of
the present invention is facilitated by the fact that
the present invention can be organized so that the same
reaction component can be added to all of the reaction
compartments in the same row, column or layer.
Sll8STlTUTE SHEEr (RU~E 26)
W095/02566 2 1 6 7 ~ q 1 PCT~S94/08141 _
22
Automated synthesis is also facilitated by the fact that
the same MCCA reaction conditions can be used for the
synthesis of all of the compounds within the MCCA array.
Automated combinatorial synthesis of MCCA
arrays can be performed in both the solution phase as
well as in the solid phase. In many cases, the MCCA
reactions are high yielding such that the major
component of the reaction mixture is the product. In
such cases, the product can often be isolated by
evaporation of the solvent and other volatiles.
In the case of lower yielding MCCA reactions,
the automated combinatorial synthesis of MCCA arrays is
preferably conducted in the solid phase because the
purification and isolation of the synthesized compounds
is greatly simplified.
The method of generating combinatorial arrays
of structural analogues using multiple component
con~enRation reactions as well as the combinatorial
arrays generated are illustrated in the following
examples. As is shown by these examples, the present
invention, through the use of MCCA reactions, provides a
simple and efficient method for synthesizing large
numbers of structural analogues for most of the known
common core structures of biologically active molecules.
Further objectives and advantages other than those set
forth above will become apparent from the examples and
accompanying drawings.
It should be noted that some reactions may not
function within the disclosed scope of the invention.
The compounds for which this occurs will be readily
recognized by those skilled in the art. In all such
cases, either the reactions can be successfully
performed by conventional modifications known to those
skilled in the art. e.g., by appropriate protection of
interfering groups, by changing to alternative
conventional reactants, or by routine modification of
reaction conditions. Alternatively, other reactions
Sl)BSrlTUrE S~E~ (RUI~ 26)
W095/02566 2 1 6 7 3 q 1 PCT~S94/08141
~.
23
disclosed herein or otherwise conventional will be
applicable to the preparation of the corresponding
compounds of the invention. In all preparative methods,
all starting materials are known or readily preparable
from known starting materials; all temperatures are set
forth uncorrected in degrees Celsius; and, unless
otherwise indicated, all parts and percentages are by
weight.
Sl~lTUTE SHEET (RULE 26)
W095/02~66 2 ~ 6 ~ ~ 9 1 PCT~S94/08141 _
24
EXAMPLES
EXAMPLE 1 Determination of the MCCA Array To Pre~are
The present invention takes advantage of the
organic chemistry synthesis design theory known as the
retrosynthetic approach. The retrosynthetic approach
teaches one to dissect a target molecule into its
smaller, simpler components by looking for bonds in the
molecule that can be formed by known synthetic methods.
The present invention takes an analogous approach,
teaching one to evaluate the target molecule for the
presence of one or more of the core structures known to
be accessible by MCCA reactions.
In order to determine which MCCA reaction to
employ to generate a series of structural analogues of
the target molecule, it is first necessary to compare
the target compound with the core structures generated
by MCCA reactions. Figure 1 summarizes fourteen core
structures that can be generated by known MCCA
reactions. Once a MCCA core structure in the target
molecule is identified, the scheme for synthesizing the
target molecule is determined by labelling the
substituents attached to the core structure as R, R1, R2,
R3, etc. as indicated. Then, as shown in Figure 2, the
same substituents are attached to the corresponding
reaction components. A combinatorial array of
structural analogues of the target molecule can be
prepared by varying the structure of the substituents of
the various components of the MCCA reaction. This
procedure is further described in Example 3 which
describes the synthesis of carzinophilin/azinomycin
structural analogues using a Passerini MCCA reaction.
EXAMPLE 2 Synthesis of ~-Acyloxy Amine MCCA
Arra~s Usinq The Passerini Reaction
The synthesis of an array of structural analogues
having a common ~-acyloxy amine core structure can be
SU8STITUTE SHEET (RULE 26)
WO95/02566 2 1 fi 7 3 9 1 PCT~S94/08141
.~
prepared by a combinatorial array synthesis using the
Passerini reaction. As depicted in Figure 3, the
Passerini reaction involves the reaction of an aldehyde
having the general structure RCHO with an isocyanide
having the general structure RlNC and an acid having a
general structure R2COOH.
A two-dimensional MCCA array synthesis
employing the Passerini reaction was conducted using
eight aldehydes, eight carboxylic acids and one
isocyanide. The two dimensional combinatorial array of
analogues generated by this synthesis, along with the
observed yields, is depicted in Figure 4. The
experimental protocol for this combinatorial array
synthesis as well as the physical data for ten of the
analogues synthesized is provided below. All of the
reactants used in the two dimensional array synthesis
are commercially available.
Experimental Protocol:
A. Reactant Pre~aration:
An anhydrous CH2C12 (17 mL) solution of diethyl
cyanomethylphosphonate (700 mg, 70 equiv.) was prepared
in a 25 mL round bottom flask and allowed to stir for 1
hour at room temperature under an N2 atmosphere.
Anhydrous CH2Cl2 (2.2 mL) solutions of aldehydes:
benzaldehyde, heptaldehyde, propanaldehyde, trans-2-
butenal, p-methoxybenzaldehyde, butyraldehyde, 4-N,N-
dimethylaminobenzaldehyde, and c; nn~m~l dehyde were
prepared in 5 mL round bottom flasks such that each
aldehyde represented 20 molar equivalents based on
isocyanide. An anhydrous CH2Cl2 (2.2 mL) solution of
carboxylic acids: acetic acid, 2-phenylacetic acid,
2',2-diphenylacetic acid, acrylic acid, benzoic acid, l-
naphthoic acid, ci nn~mi C acid, 3,3-dichloropropionic
acid were prepared in 5 mL round bottom flasks such that
SUBS~ITUTE SHEET (RUI E 26)
WO9~/02566 2 I h ~ 3 q 1 PCT~S94/08141
26
each acid represented 10 molar equivalents based on
lsocyanlde .
B. Reaction Protocol:
A shell vial box (Fisher, 1 dr, 15 X 45 mm, 72
vials) containing 64 glass vials (8X8) was used as the
reaction chamber for the combinatorial array synthesis.
The specific aldehyde used in the combinatorial array
synthesis was varied by rows. The specific acid used in
the combinatorial array synthesis was varied by columns.
Row 1:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of cinn~m~ldehyde in 0.25 mL (1 equiv.)
aliquots were added into each of eight vials in row 1 in
one single process.
Row 2:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of 4-N,N-dimethylbenzaldehyde in 0.25 mL (1
equiv.) aliquots were added into each of eight vials in
row 2 in one single process.
Row 3:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of butyraldehyde in 0.25 mL (1 equiv.) aliquots
were added into each of eight vials in row 3 in one
single process.
Row 4:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of p-methoxybenzaldehyde in 0.25 mL (1 equiv.)
aliquots were added into each of eight vials in row 4 in
one single process.
Row 5:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of trans-2-butenal in 0.25 mL (1 equiv.)
aliquots were added into each of eight vials in row 5 in
one single process.
SU8S~ITUTE SHEET (RUiF26)
_ WO95/02566 ~l 6 7 3 9 l PCT~S94/08141
27
Row 6:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of propanaldehyde in 0.25 mL (1 equiv.)
aliquots were added into each of eight vials in row 6 in
one single process.
Row 7:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of heptaldehyde in 0.25 mL (1 equiv.) aliquots
were added into each of eight vials in row 7 in one
single process.
Row 8:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of benzaldehyde in 0.25 mL (1 equiv.) aliquots
were added into each of eight vials in row 8 in one
single process.
Column A:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of acetic acid in 0.25 mL (1 equiv.) aliquots
were added into each of eight vials in column A in one
single process.
Column B:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of 2-phenylacetic acid in 0.25 mL (1 equiv.)
aliquots were added into each of eight vials in column B
in one single process.
Column C:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of 2,2-diphenylacetic acid in 0.25 mL (1
equiv.) aliquots were added into each of eight vials in
column C in one single process.
Column D:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of acrylic acid in 0.25 mL (1 equiv.) aliquots
were added into each of eight vials in column D in one
single process.
S~ITUTE SHEET (RULE 26)
WO9~/02566 2 1 6 7 3 9 1 PCT~S94/08141
28
Column E:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of benzoic acid in 0.25 mL (1 equiv.) aliquots
were added into each of eight vials in column E in one
single process.
Column F:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of l-naphthoic acid in 0.25 mL (1 equiv.)
aliquots were added into each of eight vials in column F
in one single process.
Column G:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of c- nn~ml C acid in 0.25 mL (1 equiv.) aliquots
were added into each of eight vials in column G in one
single process.
Column H:
Using a gas tight syringe, 2.2 mL CH2Cl2
solution of 3,3-dichloropropionic acid in 0.25 mL (1
equiv.) aliquots were added into each of eight vials in
column H in one single process.
The 17 mL CH2Cl2 solution of
cyanomethylphosphonate was dispensed in 0.25 mL aliquots
into 64 vials of the array using a gas tight syringe in
a single process. Upon completion of reactant additions,
each reaction vessel contained 2 molar equivalents of
aldehyde, one molar equivalent of acid, and one molar
equivalent of isocyanide in a total volume of 0.75 mL in
CH2Cl2. The reaction vessels (vials) were all capped and
no further precautions were taken to maintain an
anhydrous atmosphere. The entire array was manually
shaken for 30 seconds and then allowed to stand at room
temperature for 17 hours. Selected samples were each
transferred to round bottom flasks and evaporated to
dryness under reduced pressure without further
purification. Each crude mixture was analyzed by lH NMR
to determine the product composition and yield based on
unreacted starting isocyanide. Data is provided below
SUBSI~TUTE SHEET (RULE 26)
WO 95/02566 2 1 6 7 3 9 1 PCT/US94/08141
29
for a selection of ten analogues. Each analogue is
identified by its row and column number.
- Analoque 3C: N-[(1-diethylphosphono)methyl]-2-
5 diphenylacetoylpentamide: This reaction mixture
contained n-butanaldehyde (80 uL, 2 equiv.),
diphenylacetic acid 100 mg, 1 equiv.) and
diethylcyanomethyl phosphonate (10 mg, 1 equiv) lH NMR
(CDCl3) ~ = 7.3 (m, Ar-H), 5.2 (t, OCH), 4.1 (m,
10 OCH2CH3), 3.7 (m, CH2P), 3.5 (m, CH2P), 1.7 (m, CH2), 1.3
(m, OCH2CH3), 1.3 (m, CH2), 0.8 (t, CH3). 80~ yield.
Analoque 3F: N-[(l-diethylphosphono)methyl]-2-(1-
napthoyl)pentamide: This reaction mixture contained n-
15 butanaldehyde (80 uL, 2 equiv.), l-napthoic acid 80 mg,
1 equiv.) and diethylcyanomethyl phosphonate (10 mg,
equiv.), lH NMR (CDCl3) ~ = 9.0 (d, Ar-H~, 8.2 (d, Ar-H),
8.0 (d, Ar-H), 7.6 (m, Ar-H), 7.5 (m, Ar-H), 7.3 (Ar-H),
5.5 (t, OC6CH), 4.1 (m, OCH2CH3), 3.8 (m, CH2P), 2.1 (m,
20 CH2), 1.6 (m, CH2), 1.3 (m, OCH2CH3), 1.3 (m, CH2), 1.0
(t, CH3). 80~ yield.
Analoque 6C: N-[(1-diethylphosphono)methyl]-2-
diphenylacetoylbutamide: This reaction mixture
25 contained n-propanaldehyde (70 uL, 2 equiv.),
diphenylacetic acid (100 mg, 1 equiv.) and
diethylcyanomethyl phosphonate (10 mg, 1 equiv.), lH NMR
(CDC13) ~ = 7.3 (m, Ar-H), 6.6 (bt, NH), 5.2 (t, OCOCH),
5.1 (s, CHAr2), 4.1 (m, OCH2), 3.8 (m, CH2P), 1.85 (m,
30 OCH2CH3), 1.3 (m, OCH2CH3), 1.3 (m, CH2), 0.8 (t, CH3).
60~6 yield.
Analoque 6F: N-[(1-diethylphosphono)methyl]-2-(1-
napthoyl)butamide: This reaction mixture contained n-
35 propanaldehyde (70 uI" 2 equiv.), 1-napthoic acid (80
mg, 1 equiv.) and diethylcyanomethyl phosphonate (10 mg,
equiv.), lH NMR (CDCl3) ~ = 9.O (d, Ar-H), 8.2 (d, Ar-
SU~ITUTE SltEEr (RU~E 26)
W095/02566 2 t 6 ~ ~ 9 1. PCT~S94/08141
H), 7.8 (m, Ar-H), 7.5 (m, Ar-H), 7.5 (bt, NH), 5.5 (t,
OCOCH), 4.1 (m, OCH2CH3), 3.85 (m, CH2P), 2.1 (m, CH2),
1.4 (m, OCH2CH3), 1.3 (m, CH2~, 1.1 (t, CH3). 75~ yield.
Analoque 7A: N-[(1-diethylphosphono)methyl]-2-
acetoxyoctamide: This reaction mixture contained n-
heptaldehyde (125 uL, 2 equiv.), acetic acid (30 mg, 1
equiv.) and diethylcyanomethyl phosphonate (10 mg, 1
equiv.), lH NMR (CDCl3) ~ = 6.6 (bt, NH), 5.1 (t, OCOCH),
4.1 (m, OCH2CH3), 3.65 (m, CH2P), 2.1 (5, CH3), 1.7 (m,
CH2), 1.55 (m, CH2), 1.3 (m, OCH2CH3), 1.3 (m, CH2), 0.8
(t, CH3). 95~ yield.
Analoque 7B: N-[(1-diethylphosphono)methyl]-2-
phenylacetoyloctamide: This reaction mixture contained
n-heptaldehyde (125 uL, 2 equiv.), phenylacetic acid (65
mg, 1 equiv.) and diethylcyanomethyl phosphonate (10 mg,
1 equiv.), lH NMR (CDCl3) ~ = 7.3 (m, Ar-H), 6.7 (bt,
NH),- 5.1 (t, OCOCH), 4.1 (m, OCH2CH3), 3.7 (m, CH2P),
3.6 (s, CH2), 3.55 (m, CH2P), 1.8 (m, CH2), 1.3 (m,
OCH2CH3), 1.3 (m, CH2), 0.8 (t, CH3). 50~ yield.
Analogue 7D: N-[(1-diethylphosphono)methyl]-2-
acyloyloctamide: This reaction mixture contained n-
heptaldehyde (125 uL, 2 equiv.), acrylic acid (35 mg, 1equiv.) and diethylcyanomethyl phosphonate (10 mg, 1
equiv.), lH NMR (CDC13) ~ = 6.9 (bt, NH), 6.4 (dd,
CH2CH), 6.1 (dd, CH2CH), 5.8 (dd, CH2CH), 5.15 (t,
OCOCH), 4.6 (m, OCH2C113), 3.7 (m, CH2P), 1.8 (OCH2), 1.5
(m, CH2), 1.3 (m, OCH2CH3), 1.3 (m, CH2), 0.8 (t, CH3).
100~ yield.
Analoque 7E: N-[(1-diethylphosphono)methyl]-2-
benzoyloctamide: This reaction mixture contained n-
heptaldehyde (125 uL, 2 equiv.), benzoic acid (60 mg, 1equiv.) and diethylcyanomethyl phosphonate (10 mg, 1
equiv.), lH NMR (CDCl3) ~ = 8.0 (d, ArH), 7.5 (d, ArH),
SUBSrlTllTE SltE~T (RUI~ 26)
2 ~ 6~`9 1
WO9~/02566 PCT~S94/08141
-
31
7.4 (d, Ar-H), 7.1 (bt, NH), 5.4 (t, OCOCH), 4.1 (m, O-
CH2CH3), 3.8 (m, CH2P), 2.0 (m, CH2), 1.6 (m, CH2), 1.3
(m, OCH~CH3), 1.3 (m, CH2), 0.8 (t, CH3). 100~ yield.
5 Analoque 7G: N-[(1-diethylphosphono)methyl]-2-
cinnamoyloctamide: This reaction mixture contained n-
heptaldehyde (125 uL, 2 equiv.), c1nn~m;c acid (70 mg, 1
equiv.) and diethylcyanomethyl phosphonate (10 mg, 1
equiv.), lH NMR (CDCl3) ~ = 7.7 (d, CH), 7.5 (m, Ar-H),
7.4 (m, Ar-H), 7.3 (m, Ar-H), 6.5 (d, CH), 5.3 (t,
OC6CH), 4.1 (m, 6CH2CH3), 3.8 (m, CH2P), 1.9 (m, CH2), 1.6
(m, CH2), 1.3 (m, OCH2CH3), 1.3 (m, CH2), 0.8 (t, CH3).
70~ yield.
Analoque 7H: N-[(1-diethylphosphono)methyl]-2-
dichloroacetoyloctamide: This reaction mixture
contained n-heptaldehyde (125 uL, 2 equiv.),
dichloroacetic acid (70 mg, 1 equiv.) and
diethylcyanomethyl phosphonate (10 mg, 1 equiv.), lH NMR
(CDCl3) ~ = 7.15 (bt, NH), 6.2 (s, CHCla), 5.3 (t,
OCOCH), 4.1 (m, OCH2CH3), 3.7 (m, CH2P), 1.9 (m, CH2), 1.6
(m, CH2), 1.3 (m, OCH2CH3), 1.3 (m, CH2), 0.8 (t, CH3).
95~ yield.
EXAMPLE 3 Application Of The MCCA Synthesis
Strategy To Structural Analogues Of
Carzino~hilin/Azinomycin
Carzinophilin (CZ) and Azinomycin B are
antitumor/antibiotic compounds which function as a DNA
bis-alkylating agent. The mechanism of action for CZ
and Azinomycin B is depicted in Figure 5. As also shown
-in Figure 5, Carzinophilin and Azinomycin B are
structurally very similar. Given the structural
-35 complexity of these molecules, chemical syntheses are
long and low yielding. Azinomycin B has also been
produced by fermentation but in very low yields (2
mg/250 L).
SU8S~ITUTE SltEE~ (RULE 26)
W095/02566 2 t ~ t PCT~S94/08141 _
Given the difficulty associated with
synthesizing these molecules, few derivatives of these
molecules have been prepared. It therefore would be
highly desirable to have an efficient means for
producing a series of structural analogues of these
molecules in order to probe the mechanism of action of
these molecules.
As shown in Figure 6, both CZ and Azinomycin B
have ~-acyloxy amine core structures. By labelling the
substituents off of the ~-acyloxy amine core structure
R1, R2 and R3, a Passerini reaction scheme for the
synthesis of Carzinophilin, Azinomycin B and their
structural analogues is identified.
Once a Passerini reaction scheme for
Carzinophilin and Azinomycin B has been identified, a
Passerini multicomponent combinatorial array synthesis
of Carzinophilin and Azinomycin B analogues can be
designed by varying the R1, R2 and R3 substituents of the
reaction components. One possible three dimensional
Passerini-type MCCA array synthesis of Carzinophilin and
Azinomycin B structural analogues is provided in
Figure 6.
A two dimensional MCCA array synthesis of
Carzinophilin and Azinomycin B structural analogues
using three sets of diasteriometric isocyanates and two
different aldehydes was performed. The combinatorial
array synthesis and the yields obtained are provided in
Table 2.
Sll8S~lTUTE SHEET (RULE 26)
2 i 673q l
WO 95/02566 PCT/US94/08141
Table 2 ~ u...yc;uADalogsS~,lt~ viaP~s~,;..i R~QnC
Cmpd Isoe~an Aldeh~de Acld Z/E dea % pldb Ma~or Product
109E 10~ 2~ aic 1125 3.4:1 42% ~0~,~
s~yadd
IO9Z 107Z (S~2 l~c 3.911 3.4~ l
mdhgl ad
glgdd~
110E 10~E (S~2~ aic V15 5:5:1:1 37~o o~,XXI~
~d!", ~d ~ ~
110Z 108Z ~S)-2- 1-Qaph~oic 3311 3.43A~1:1 519~ o~;OH o
gl- a~d h~
~ydd~ ~ ~
lllZ/E lOCZIE (S)-2~ l~thac 1.4/1 33:1:1 39~b o ~ H O
VE-111.4 me~ ad ~~~
glyddd `O Ph~
ZI~VIA n~ 1~ hac III 1:1 6896
113Z/E 106ZIE ~al l~thac 111.4 ~- 69% ~b
ZIE-111.4 acid ~o X~o~b
114Z/E 107UE n-b~ spbthaic 111.2 -~ 57% o ~ H O
Z/E=ltl.4 ~d ~o~
SU8SrlTUTE SHET (RULE 26)
W095/02566 2 ! ~ 7 ~ ~ ~ PCT~S94/08141
34
The isocyanates used in the combinatorial
array synthesis described in Table 2 were prepared
according to the general reaction scheme provided below.
~ o
~fJ~Me
H o Br
tO5 ' 70 ~NX~Me
Br R~
71Z
Methyl-isocyanoacetate and benzaldehyde were
reacted to form the dehydroaminoacid precursor 105
according to the procedure described in Hoppe, D., et
al., U. Liebigs Ann. Chem. (1972) 766:116-129. The
mixture of geometric isomers were then brominated with
N-bromosuccinamide (NBS) in CH3CN to yield the ~-bromo
imine derivative 70 which tautermerized in Et3N to afford
the corresponding ~-bromo-dehydroamino acids 71Z and 71E
in 81~ yield (Z/E = 1.4:1). The modified bromonation
conditions (See Combs, A., et al., Tetrahedron Lett.
(1992) 33:6419) gave substantially increased yields of
the E-isomer as compared to the conditions reported by
Matsumoto. Nunami, K., et al., Tetrahedron (1988)
44:5467 (60~ yield, Z/E = 13:1).
The resulting vinyl bromide diasteriomers
71E/Z were separated by silica gel chromatography and
recrystallized to analytical purity. Their
stereochemistries were assigned according to the
SU8SllTUTE SHEET (RULE 26)
2 1 6739 1
WO 9S/02566 PCT~S94/08141
-
literature by lH NMR chemical shift comparison of the
methyl proton resonances of the E- and Z- esters.
Isocyanides 106E and 106Z were formed from a
~-bromo-dehydroamino acids 71E and 71Z with retention of
the olefin configuration by dehydrating the N-formyl
groups with POCl3 and Et3N in CH2C12 at 0C for 30
minutes.
The corresponding vinyl aziridine compounds
107E and 107Z were formed from the isocyanates 106E and
106Z by adding ethyleneimine to the isocyanates in
tetrahydrofuran and triethyl amine for 3-5 hours.
The reaction conditions and physical data for
isocyanates and the structural analogues produced in the
combinatorial synthesis described in Table 2 are
provided below.
The above-described combinatorial synthesis of
carzinophilin and azinomycin structural analogues
exposes the power of the present invention to enable the
synthesis of broad arrays of structural analogues. For
example, in the present synthesis of carzinophilin and
azinomycin structural analogues, it is possible to
further vary the aziridine ring, the phenyl ring and the
olefin portion of the isocyanate, the epoxide group of
the aldehyde as well as the substituents on the aromatic
ring of the acid. Thus, as can be seen from the present
example, a very broad array of carzinophilin and
azinomycin structural analogues may be produced
according to the present invention.
It may be further noted that the above-
described combinatorial array synthesis of carzinophilinand azinomycin structural analogues may be readily
performed as a solid phase synthesis. For example, by
using a carboxylic acid containing a hydroxy, thio or
amino functional group, one could readily attach the
carboxylic acid to a solid support. Then, by adding an
aldehyde and an isocyanate under the conditions
described in the present example, one could carry out
SU~ITUTE SHEET (RU~ F26)
W095/02566 2 1 ~ 7 3: ~I PCT~S94/08141
36
the above-described synthesis as a solid phase
synthesis.
Experimental Data
~ o
~ Me
H O o Br
H~N 71 E
Rl~U CH3CN f~,J`8, -aooc ~7 ~
105 ~ 70 H~N~Me
Br
71Z
NBS (5.4 g, 30.22 mmol) was added to a r.t CH3CN (20
mL) solution of dehydoamino acid 105 (5.9 g, 28.78
mmol). The reaction was stirred at 25C for 1 hr. and
then heated to 45C for 30 min. to give ~-bromo imine
intermediate 70. The solution of 70 was then cooled to
r.t. and Et3N (4.0 mL, 31.7 mmol) added dropwise over a 5
min. period. This mixture was allowed to stir for 1
hr., after which the solvent was removed under reduced
pressure. The crude oil was taken up in EtOAc and the
insoluble succinimide removed by filtration. The
solvent was rotoevaporated to a clear oil, which was
further purified by flash silica gel chromatography to
yield two fractions: pure 71Z (2.0 g) and bromides 71Z
and 71E (4.6 g, Z/E=1:1.2) for an 81% overall yield
(Z/E=1:1.4) The Z- and E- isomers could be further
separated by flash silica gel chromatography and then
recrystallized from EtOAc/Hex to analytical purity. The
E- and Z- isomer data was consistent with previous
literature assignments: 71E m.p. 110-112C, lit.m.p.
109-110C; 71Z m.p. 138-140C, lit.m.p. 136-138C.
NBS (34 mg, 0.188 mmol) was added to a r.t.
CD3CN (0.5 mL) solution of dehydroamino acid 105 (35 mg,
0.171 mmol). After 3 hrs. the reaction gave the ~-bromo
imine 70 quantitatively by lH and 13C NMR.
SU~ITUTE SHEET (RULE 26)
2 1 6739 1
W095/025~ PCT~S94/08141
37
70: lH NMR (360 MHz, CD3CN) ~ 9.33 (s, lH,
CHO), 7.53 (m, 2H, o-ArH), 7.39 (m, 3H, m,p-ArH), 6.26
(s, lH, CHBr), 3.81 (s, 3H, OCH3); 13C NMR (90 MHz,
CD3CN), ~ 174.05 (CHO), 159.52 (C), 136.71 (C), 130.29
(CH), 130.25 (CH), 129.71 (CH), 54.86 (OCH3), 48.86
(CHBr).
POCI3 / Et3N CPl~Me
F~ Br 0 C Br
71 E 1 06E
POCl3 (217 ~L, 2.3 mmol) was added dropwise over a
15 min. period to a 0C CH2C12 (40 ml) solution of
bromide 71E (600 mg, 2.12 mmol) and Et3N (1.4 mL, 10.6
mmol). The solution was allowed to stir at 0C for 30
min., after which, the reaction was quenched with 20 mL
of aqueous Na2CO3 (450 mg, 4.24 mmol). This mixture was
stirred for 15 min. and then the aqueous layer extracted
3X with Et2O. The combined organic layers were dried
over MgSO4 and rotoevaporated to a brown oil. The crude
oil was triturated with Et2O (3X) and the ether fractions
filter through a MgSO4 plug. The ether was removed under
reduced pressure to afford 106E (590 mg, 105~) as a
brown oil. The extracted material was sufficiently pure
(~90~) for the next synthetic step without
chromatography. (The crude could be further purified by
flash silica gel chromatography, but with noticeable
loss in material).
106E: IR (film) cm~l: 2956, 2112 (CN), 1744,
1699, 1442, 1261, 1119, 893; lH NMR (360 CHCl3) ~ 7.46
(m, 2H, o-ArH), 7.39 (m, 3H, m,p-ArH), 3.86 (s, 3H,
OCH3); 13C NMR (90 MHz, CHCl3), ~ 171.9 (CHO), 160.3 (C),
138.1 (C), 136.6 (C), 130.9 (CH), 128.5 (CH), 128.5
SU~ITUTE SltEET (RULE 26)
wo 95,02~66 2 ~ 6 ~ ~ 9 1 PCT~S94/08141
38
(CH), 128.5 tC), 53.4 (OCH3). HRMS (CI) [MH~t calcd for
CllH8NO2Br 265.9817, found 265.9817.
CN ~ Me ~N~ CN ~ Me
Br ~ E~N/THF ~N ~
10~Z 107Z
Ethyleneimine (125 ~L, 2.43 mmol) was added to a
r.t. THF (10 mL) solution of isocyanide bromide 106Z
(215 mg, 0.81 mmol) and Et3N (110 ~L, 0.90 mmol). The
mixture was stirred for 3 hrs., after which the solvent
and excess amines were removed under reduced pressure to
afford a brown oil. Attempts to extract over aqueous
solutions hydrolyzed the vinyl aziridine. The crude oil
was therefore triturated with Et2O and completely
insoluble EtNH'Br~ removed by filtration through a MgSO4
plug. The ether was rotoevaporated to a brown oil 107Z
(185 mg, 100~, which was sufficiently pure (~90~) for
analytical data.
107Z: IR (film) cm~1: 3350, 3000, 2952, 2116
~CN), 1560, 1290; lH NMR (360 MHz, CHCl3) ~ 7.41-7.26 (m,
3H, o,p-ArH), 7.16 (m, 2H, m-ArH), 3.59 (s, 3H, OCH3),
2.34 (s, 4H, N(C~2)2); l3C NMR (90 MHz, CHCl3), ~ 170.0
(C), 165.3 (C), 161.6 (C), 129.4 (CH), 128.0 (C), 127.9
(CH), 127.2 (CH), 51.9 (OCH3), 29.9 (N(CH2)2); HRMS (CI)
[MH]~ calcd for C13H12N2O2 229.0978, found 229.0977.
CN ~ Me ~N~
Br ~ EbNITHF ~N m
106Z 107Z
SUBSrlTUTE SHEET (RU~ F26)
W095/025~ 2 1 6 7 3 9 l PCT~S94/08141
39
Ethyleneimine (145 ~L, 2.89 mmol) was added to a
r.t. THF (5 mL) solution of isocyanide bromide 106E (225
mg, 0.962 mmol) and Et3N (150 ~L, 1.16 mmol). The
mixture was stirred for 2 hrs. after which the solvent
and excess amines were removed under pressure to afford
a brown oil (attempts to extract over aqueous solutions
hydrolyzed the vinyl aziridine). The crude oil was
therefore triturated with Et2O and the completely
insoluble EtNH'Br~ removed by filtration through a MgSO4
plug. The ether was rotoevaporated to afford a brown
oil 107E (255 mg, 103~), which was sufficiently pure
(c90~) for analytical data.
107E: IR (film) cm~l: 3325, 3001, 2952, 2117
(CN), 1717, 1554, 1445, 1390, 1257, 1148, 1124; lH NMR
(360 MHz, CHCl3) ~ 7.40 (m, 5H, ArH), 3.81 (s, 3H, OCH3),
2 25 (s, 4H, N(C~2)2); 13C NMR (90 MHz, CHCl3), ~ 168.1
(C), 166.6 (C), 161.5 (C), 135.3 (C), 129.8 (CH), 128.3
(CH), 128.2 (C), 127.3 (CH), 52.1 (OCH3), 32.9 (N(CH2)2);
HRMS (CI) [MH]+ calcd for Cl3Hl2N202 229.0977, found
229.0977.
Me
~ ~N~ CN ~ M
Br ~ EbNITHF ~N ~
10BZ 108Z
Racemic 2-methylasiridine (210 ~L, 2.91 mmol)
was added to a r.t. THF (10 mL) solution of isocyanide
bromide 106Z (0.88 mmol) and Et3N (125 ~L, 0.97 mmol).
The mixture was stirred for 2 hrs., after which the
solvent and excess amines were removed under reduced
pressure to afford a brown oil. The crude oil was
extracted 3X with Et2O over H2O. The ether fractions
were dried over MgSO4 and rotoevaporated to afford a
SlJ8SrlTUTE StlEET (RU~E 26)
WO 95/02~66 2 1 6 7 ~ ~ ~ PCT/US94/08141
brown oil 108Z (198 mg, 93~, which was sufficiently
pure (~90g~) for analytical data.
108Z: IR (film) cm~1: 3350, 2953, 2116 (CN),
1723, 1564, 1291; lH NMR (360 MHz, CHC13) ~ 7.44-7.35 (m,
3H, o,p-ArH), 7.21 (m, 2H, m-ArH), 3.61 (s, 3H, OCH3),
2.54 (ddq, J=3.8, 5.5, 6.1 Hz, lH, NCHCH3), 2.23 (d,
1=6.1 Hz, lH, NCH2), 2.18 (d, J=3.8 Hz, lH, NCH2), 1.36
(d, J=5.5 Hz, 3H, NCHCH3); 13C NMR (90 MHz, CHCl3), ~
169.9 (C), 165.8 (C), 161.8 (C), 135.0 (C), 129.4 (CH),
128.3 (C), 128.2 (CH), 127.4 (CH), 52.0 (OCH3), 38.2
(NCHCH3), 36.4 (NCH2), 18.3 (NCHCH3); HRMS (CI) [MH]~
calcd for C14H14N2O2 243.1134, found 243.1129.
Me
o ~ N~ Q
Xl`OMe , CN~M
Rl Br Et3N ITHF ;~Me
106E 108E
Racemic 2-(methyl)-aziridine (250 ~L, 3.0
mmol) was added to a r.t. THF (5 mL) solution of
isocyanide bromide 106E (263 mg, 0.992 mmol) and Et3N
(150 ~L, 1.19 mmol). The mixture was stirred for 2
hrs., after which the solvent and excess amines were
removed under reduced pressure to afford a brown oil.
The crude oil was extracted 3X with Et2O over H2O to
efficiently remove the Et3NH~Br~. The combined ether
fractions were dried over MgSO4 and rotoevaporated to
afford a brown oil 108E (269 mg, 112~), which was
sufficiently pure (~90~) for analytical data.
108E: IR (film) cm~l: 3303, 2995, 2952, 2117
(CN), 1723, 1548, 1445, 1172, 1124; lH NMR (360 MHz,
CHCl3) ~ 7.47-7.40 (m, 5H, ArH), 3.83 (s, 3H, OCH3), 2.31
(ddq, J=1.5, 5.2, 6.1 Hz, lH, NCHCH3), 2.29 (d, J=1.5 Hz,
lH, NCH2), 2.18 (d, J=5.2 Hz, lH, NCH2), 1.24 (d, J=6.1
Hz, 3H, NCHCH3); 13C NMR (90 MHz, CHCl3), ~ 167.8 (C),
SU~ITUTE Sl tEET (RU~E 26)
W095/02566 2 1 6 7 3 9 1 PCT~S94/08141
41
166.7 (C), 161.7 (C), 135.8 (C), 129.8 (CH), 128.4 (C),
128.3 (C), 127.5 (CH), 52.1 (OCH3), 40.2 (NCHCH3) 39.9
(NCH2), 17.8 (NCHCH3); HRMS (CI) [MH]~ calcd for C14Hl4N2O2
243.1134, found 243.1134.
~Me
CO2H : O
CN~e ~ H o ~Em~or + Emh~
Pyridlrle. CHzC12 R ~ ~ ~
Me
1~Rmaor + Z~h~
Pyridine (72 ~L, 0.89 mmol), (S)-2-(methyl)-
glycidal (210 ~L, 2.43 mmol), and 1-naphthoic acid (140
mg, 0.81 mmol) were added sequentially to a r.t. CH2Cl2
(5 mL) solution of isocyanide 107Z (0.81 mmol). The
mixture was stirred under N2 for 20 hrs., after which the
solvent was removed under reduced pressure. The crude
oil was immediately purified by flash silica gel
(deactivated with 5~ Et3N) chromatography (gradient
eluted with 100% hex to 1:1 hex/EtOAc) to afford four
fractions: 109Z (138 mg, de=2.9:1), 109Z (28.5 mg,
de=2:1), 109Z/109E (13.0 mg, Z/E=2:1, de=1.4:1, de=1:2)
and 109E (30 mg, de=2.3:1) for a 53% overall yield
(109Z/109E=5.1:1).
SUBS~ITUTE SHEET (RULE 26)
2 1 673q 1
W095/02566 PCT~S94108141
109Z (major diasteromer): IR (film) cm~1: 3296,
3056, 2999, 2949, 1717, 1699, 1593, 1511, 1310, 1240,
1194, 1143, 1060; lH NMR (360 MHz, CDCl3), ~ 8.98 (d,
1=8.8 Hz, lH, ArH), 8.38 (dd, J=1.2, 7.3 Hz, lH, ArH),
8.07 (d, J=8.1 Hz, lH, ArH), 7.91 (d, J=7.6 Hz, lH,
ArH), 7.67-7.51 (m, 3H, ArH), 7.55 (bs lH, NH), 7.37-
7.25 (m, 5H, ArH), 5.39 (s, lH, OCH), 3.49 (s, 3H, OCH3),
3.14 (d, J=4.5 Hz, lH, OCH2), 2.86 (d, J=4.6 Hz, lH,
OCH2), 2.09 (bs, 4H, N(CH2)2), 1.66 (s, 3H, OCCH3); 13C NMR
(90 MHz, CDC13) ~ 166.87, 165.24, 164.07, 164.02, 136.73,
133.80, 133.56, 131.24, 130.42, 128.63, 128.41, 128.23,
127.87, 127.01, 126.24, 125.70, 125.55, 124.30, 108.27,
75.53, 55.78, 52.66, 51.52, 32.32, 17.49; HRMS (FAB)
[MH]~ calcd for C28Hz6N2O6 487.1869, found 487.1869.
C ~ M~ O 1~E-mr~r
,c~ O ~ o
107E ~ Me
1 ~-mapr
Pyridine (55 ~L, 0.68 mmol), (S)-2-(methyl)-
glycidal (155 ~L, 1.78 mmol), and 1-naphthoic acid (101
mg, 0.59 mmol) were added sequentially to a r.t. CH2Cl2
(3.0 mL) solution of isocyanide 107E (135 mg, 0.59
mmol). The mixture was stirred under N2 for 27 hrs.,
after which the solvent was removed under reduced
pressure. The crude oil was immediately purified by
flash silica gel (deactivated with 5~ Et3N)
chromatography (gradient eluted with 100~ hex to 1:1
SU8SrlTUTE SHEET (RU~E 26)
WO9~/02566 2 1 6 7 3 q l PCT~S94/08141
43
hex/EtOAc) affording two fractions: 109E (84 mg,
de=3.4:1) and 109Z (30.5 mg, de=1.5:1), for a 42
overall yield (109E/109Z=2.8:1).
109E (major diasteromer): IR (film) cm~l: 3311,
3058, 3001, 2948, 1722, 1511, 1279, 1244, 1194, 1138; ~H
NMR (360 MHz, CDCl3), ~ 8.85 (d, J=8.6 Hz, lH, ArH), 8.10
(dd, J=l.l, 7.2 Hz, lH, ArH), 8.02 (d, J=8.2 Hz, lH,
ArH), 7.86 (d, J=8.0 Hz, lH, ArH), 7.62-7.43 (m, 3H,
ArH), 7.28-7.15 (m, 5H, ArH), 6.99 (bs, lH, NH), 5.12
(s, lH, OCH), 3.76 (s, 3H, OCH3), 2.82 (d, J=4.5 Hz, lH,
OCH2), 2.63 (d, J=4.6 Hz, lH, OCH2), 2.21 (bs, 4H,
N(CH2)2), 1.4 (s, 3H, OCCH3); 13C NMR (90 MHz, CDCl3) ~
166.87, 165.24, 164.07, 164.02, 136.73, 133.80, 133.56,
131.24, 130.42, 128.63, 128.41, 128.23, 127.87, 127.01,
126.24, 125.70, 125.55, 124.30, 108.27, 75.53, 55.78,
52.66, 51.52, 32.32, 17.49; HRMS (F~3) [MH]~ calcd for
C28H26N2O6 487.1869, found 487.1869.
0~ 0
M-
C02H ~_o \/
H~ 110E~aor + Emh~
mP~ ne,C~ClQ o ~ o
M-~
110Z~apr + Z~ha
Pyridine (36 ~L, 0.45 mmol), (S)-2-(methyl)-
glycidal (107 ~L, 1.24 mmol), and l-naphthoic acid (72
mg, 0.42 mmol) were added sequentially to a r.t. CH2Cl2
(5 mL) solution of isocyanide 108Z (100 mg, 0.41 mmol).
The mixture was stirred under N2 for 24 hrs., after which
the solvent was removed under reduced pressure. The
SUBSTITUTE S~tEET (RULE 26)
WO9~/02566 2 ~ 6 7 3 ~ ~ PCT~S94/08141
crude oil was immediately purified by flash silica gel
(deactivated with 5~ Et3N) chromatography (gradient
eluted with 100~ hex to 1:1 hex/EtOAc) affording four
fractions: 110Z (69 mg, de=3.4:3.4:1:1), 110Z (11 mg,
de= 1.5:1.5:1:1), 110Z and 110E (14 mg, 110Z,
de=1.5:1.5:1:1 and 110E, de=l:l) and 110E (22 mg,
de=1.5:1.5:1:1), for a 56~ overall yield
(llOZ/llOE=3.2:1).
110Z (major diasteromers): IR (film) cm~l:
3290, 3064, 2994, 1715, 1511, 1328, 1237, 1138; lH NMR
(360 MHz, CDCl3), ~ 8.97 (d, J=8.7 Hz, lH, ArH), 8.93 (d,
J=9.0 Hz, lH, ArH), 8.38 (dd, J=l.l, 7.3 Hz, lH, ArH),
8.35 (dd, J=l.l, 7.4 Hz, lH, ArH), 8.08 (d, J=8.3 Hz,
211, ArH), 7.91 (d, J=7.9 Hz, 2H, ArH), 7.66-7.52 (m,
6H, ArH), 7.41 (bs, lH, NH), 7.40 (bs, lH, NH), 7.37-
7.26 (m, 10H, ArH), 5.36 (s, lH, OCH), 5.35 (s, lH,
OCH), 3.50 (s, 3H, CCH3), 3.49 (s, 3H, OCH3), 3.14 (d, J=
4.5 Hz, lH, OCH2), 3.12 (d, 4.6 Hz, lH, OCH2), 2.88 (d,
J=1.3 Hz, lH, OCH2), 2.86 (d, J=4.0 Hz, lH, OCH2), 2.25
(m, lH, NCHCH3), 2.15 (m, 3H, NCHCH3, NCH2), 2.00 (d,
J=6.1 Hz, lH, NCH2), 1.89 (d, J=3.7 Hz, lH, NCH2), 1.67
(s, 3H, OCCH3), 1.66 (s, 3H, OCCH3), 1.21 (d, J=6.1 Hz,
3H, NHCH3), 1.09 (d, J=4.9 Hz, 3H, NHCH3); 13C NMR (90
MHz, CDCl3) ~ 166.26, 165.99, 165.87, 165.81, 164.28,
164.28, 158.57, 158.57, 136.29, 136.21, 134.10, 134.02,
133.79, 133.79, 131.42, 131.37, 130.82, 130.71, 128.58,
128.58, 128.41, 128.41, 128.31, 128.31, 128.08, 128.08,
127.64, 127.64, 126.40, 126.40, 125.98, 125.86, 125.65,
125.65, 124.50, 124.50, 111.06, 111.06, 76.23, 76.14,
56.29, 56.29, 53.61, 53.35, 51.44, 51.44, 37.54, 37.39,
36.40, 36.15, 18.32, 17.97, 17.48, 17.47; HRMS (DCI)
[MH]~ calcd for C29H28N2O6 501.2026, found 501.2026.
SU8S~ITUTE S~tEET (RULE 26)
2 1 6739 1
W095/02566 PCT~S94/08141
-
~OM-
CO2H ~o M~7
~M- ~ H~O . 110E-m~or
108E
11oZ~or
Puridine (40 ~L, 0.50 mmol), (S)-2-(methyl)-
glycidal (120 ~L, 1.37 mmol), and l-naphthoic acid (80
mg, 0.45 mmol) were added sequentially to a r.t. CH2C12
(3 mL) solution of isocyanide 108E (110 mg, 0.45 mmol).
The mixture was stirred under N2 for 24 hrs., after which
the solvent was removed under reduced pressure. The
crude oil was immediately purified by flash silica gel
(deactivated with 5~ Et3N) chromatography (gradient
eluted with 100~ hex to 1:1 hex/EtOAc) affording four
fractions, 110Z (20.5 mg, de=3:3:1:1), 110Z and 110E
(23.8 mg, 110Z de=2:2:1:1 and 110E, de=1.5:1.5:1:1),
110E (9.5 mg, de= 1:1), and 110E (29 mg, de=2:2:1:1),
for a 37~ overall yield (llOE/llOZ=1.5:1).
110E (major diasteromers): IR (film) cm~l:
3322, 3055, 2995, 2946, 1711, 1505, 1275, 1239, 1136; lH
NMR (360 MHz, CDCl3), ~ 8.86 (d, J=8.6 Hz, lH, ArH), 8.82
(d, J=8.6 Hz, lH, ArH), 8.14 (dd, J=1.2, 7.2 Hz, lH,
ArH), 8.05-8.01 (m, 3H, ArH), 7.87 (m, 3H, ArH), 7.65-
7.40 (m, 5H, ArH), 7.3-7.2 (m, 10H, ArH), 6.94 (bs, lH,
NH), 6.93 (bs, lH, NH), 5.19 (s, lH, OCH), 5.05 (s, lH,
OCH), 3.76 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 2.83 (d,
J=5.0 Hz, lH, OCH2), 2.82 (d, J=5.7 Hz, lH, OCH2), 2.65
(d J=4.6 Hz, lH, OCH2), 2.63 (d, J=4.6 Hz, lH, OCH2),
2.30 (m, lH, NCHCH3), 2.25-2.10 (m, 3H, NCHCH3, NCH2),
2.15 (d, J=3.5 Hz, lH, NCH2), 2.14 (d, J=1.4 Hz, lH,
SU8SrlTUTE SHE~T (RULE 26)
wo 95/02566 2 ~ ~ 7 ~ PCT~S94/08141
46
NC~2), 1.43 (s, 3H, OCCH3), 1.40 (s, 3H, OCCH3), 1.22 (d,
J=5.6 Hz , 3H, NHCH3), 1.21 (d, J=5.5 Hz , 3H, NHCH3); 13C
NMR (90 MHz, CDCl3) ~ 167.07, 166.90, 165.43, 165.25,
164.17, 164.17, 164.14, 163.84, 137.10, 137.04, 133.88,
5 133.85, 133.69, 133.69, 131.35, 131.35, 130.54, 130.45,
128.66, 128.59, 128.49, 128.49, 128.24, 128.24, 127.97,
127.95, 127.20, 127.11, 126.34, 126.33, 125.91, 125.76,
125.67, 125.64, 124.41, 124.37, 107.72, 107.67, 75.72,
75.50, 55.92, 55.86, 52.95, 52.55, 51.59, 51.59, 39.91,
10 39.47, 39.32, 39.04, 18.03, 17.99, 17.82, 17.43; HRMS
(DCI) [MH]' calcd for C29H28N206 501.2025, found 501.2026.
~ ~M~
CO2H ~ ~ Br ~ + Z~a
~O , 111Z-m~pr
1 OB ~Me
Br ~ E mh~
111E-m~or
(S) -2- (methyl)-glycidal (39 ~L, 0.45 mmol) and
1-naphthoic acid (57.0 mg, 0. 33 mmol) were added
sequentially to a r.t. CH2Cl2 (2 mL) solution of
15 isocyanide 106 (80.0 mg, 0.302 mmol, Z/E=1.4:1) and
stirred under N2 for 24 hours. Additional (S)-2-
(methyl)-glycidal (20 ~L, 0.23 mmol) was added and the
mixture stirred for 24 hours after which the solvent was
removed under reduced pressure. The crude oil was
20 purified by flash silica gel (deactivated with 5~ Et3N)
chromatography (gradient eluted with 100~ hexane to 1:1
hex/EtOAc). One fraction was isolated containing an
SU8SrlTUTE SHEET (RU~E 26)
2 1 6739 1
WOgS/02566 PCT~S94/08141
47
inseparable mixture of all four stereoisomers 111 (61.0
mg, Z/B 1.4:1, de=3:3:l:l) for a 39~ overall yield.
Major lllZ and lllE: IR (film) cm~1: 3334,
2950, 1732, 1714, 1480, 1237, 1193, 1130, 783; lH NMR
(360 MHz, CDCl3), ~ 9.00-8.85 (m, 2H, ArH), 8.38 (m, lH,
ArH), 8.37-8.05 (m, 3H, ArH), 7.91 (s, lH, NH), 7.89 (s,
lH, NH), 7.7-7.3 (m, 8H, ArH), 7.2-7.0 (m, 2H, ArH),
5.40 (s, lH, OCH, Z-isomer), 5.24 (s, lH, OCH, E-
isomer), 3.92 (s, 3H, CO2CH3, E-isomer), 3.54 (s, 3H,
CO2CH3, Z-isomer), 3.13 (d, J=4.4 Hz, lH, CCH2, Z-
isomer), 2.92 (d, J=4.3 Hz, lH, CCH2, E-isomer), 2.87 (d,
J=4.4 Hz, lH, OCH2, Z-isomer), 2.71 (d, J=4.4 Hz, lH,
OCH2, E-isomer), 1.63 (s, 3H, CCH3, Z-isomer), 1.63 (s,
3H, CCH3, E-isomer); 13C NMR (90 MHz, CDCl3) ~ 165.30,
164.95, 164.17, 163.83, 162.82, 136.89, 135.99, 134.72,
134.39,133.76, 131.46, 130.94, 130.72, 129.60, 129.52,
128.81, 128.61, 128.48, 128.40, 128.26, 127.79, 126.89,
126.59, 126.49, 126.45, 125.62, 125.49, 125.24, 124.45,
124.37, 120.13, 75.41, 75.21, 56.05, 55.76, 53.50,
52.76, 52.58, 17.86, 17.56, 17.37; HRMS (FAB) [MH]' calcd
for C26H22NO6Br 524.0709, found 524.0709.
M- ~ ~ 112E
~ M P~l~,C~
108Z~E ~ ~ ~ Me
11~7
SU8STITUTE SHEET (RUI E 26)
wo 95,02566 ~ t PCT~S94/08141
48
Pyridine (35 ~L, 0.445 mmol), butyraldehyde
(330 ~L, 3.71 mmol), and 1-naphthoic acid (64 mg, 0.371
mmol) were added sequentially to a r.t. CH2Cl2 (3 mL)
solution of isocyanide 108 (1:1.4 Z/E, 99.0 mg, 0.371
mmol). The mixture was stirred under N2 for 22 hours,
after which the solvent was removed under reduced
pressure. The crude oil was immediately purified by
flash silica gel (deactivated with 5~ Et3N)
chromatography (gradient eluted with 100~ hex to 1:1
hex/EtOAc) affording two fractions: 112Z (61 mg, de=l:l,
34%) and 112E (61 mg, de=l:l, 34~), for a 68~ overall
yield.
112Z: lR (film) cm~l: 3433.8, 3059, 2962, 1705,
1667, 1512, 1312, 1196, 1138; lH NMR (360 MHz, CDCl3),
9.01 (d, J=8.7 Hz, lH, ArH), 8.97 (d, J=8.5 Hz, lH,
ArH), 8.34 (dt, J=1.2, 7.5 Hz, 2H, ArH), 8.09 (d, J=8.2
Hz, 2H, ArH), 7.92 (d, J=8.1 Hz, 2H, ArH), 7.69-7.61 (m,
2H, ArH), 7.60-7.53 (m, 4H, ArH), 7.47 (bs, lH, NH),
7.42 (bs, lH, NH), 7.40-7.25 (m, 10H, ArH), 5.76 (t,
J=6.0 Hz, lH, OCHCH2), 5.72 (t, J=6.2 Hz, lH, OCHCH2),
3.48 (s, 3H, OCH3), 3.47 (s, 3H, OCE3), 2.20-2.11 (m, 7N,
NCH2, NCHCH2, OCH2CH2), 2.03 (d, J=6.0 Hz, lH, NCH2), 1.95
(d, J=3.7 Hz, lH, NCH2), 1.85 (d, J=3.7 Hz, lH, NCH2),
1.71-1.60 (m, 4N, CHCH2H2), 1.09 (d, J=5.4 Hz, 6H,
NHCH3), 1.05 (t, J=7.8 Hz, 6H, CH2CH3); 13C NMR (90 MHz,
CDCl3) ~ 169.36, 169.28 (CO2CH3), 165.99, 165.99 (C),
164.36, 164.36 (C), 158.08, 157.51 (C), 136.06, 135.00
(C), 134.19, 134.09 (CH), 133.82, 133.81 (C), 131.43,
131.37 (C), 130.40, 130.34 (CH), 128.64, 128.64 (CH),
128.39, 128.33 (CH), 128.15, 8.10 (CH), 127.59, 127.59
(CH), 126.44, 126.44 (CH), 125.99, 125.81 (C), 125.49
(CH), 124.41 (CH), 111.50, 111.46 (C), 74.37, 74.10
(CH), 51.44, 51.44 (OCH3), 36.89, 36.74 (CH2N), 36.59,
36.59 (CHN), 34.11, 34.04 (CH2), 18.35, 18.23 (CH2),
18.03, 17.92 (CHCH3, 13.82, 13.82 (CH3CH2); HRMS (DCI)
[MH]~ calcd for C29H30N2Os 487.2233, found 487.2233.
SUBSrlTUTE SHE~T (RUI E 26)
W095/02566 2 1 6 7 3 9 1 PCT~S94/08141
-
49
}12E: lR (film) cm~l: 3420, 3065, 2962, 1703,
1510, 1290, 1137; lH NMR (360 MHz, CDCl3), ~ 8.79 (d,
J=8.0 Hz, lH ArH), 8.70 (d, J=8.7 Hz, lH, ArH), 8.04-
8.01 (m, 2H, ArH), 7.93-7.87 (m, 4H, ArH), 7.65-7.50 (m,
4H, ArH), 7.48 (m, 2H, ArH), 7.30-7.10 (m, 10H, ArH),
- 6.90 (bs, 2H, NH), 5.42 (t, J=6.1 Hz, lH, OCHCH2), 5.41
(t, J=6.6 Hz, lH, OCHCH2), 3.74 (s, 6H, OCH3), 2.32 (m,
lH, NClICH3), 2.26 (m, lH, NHCH3), 2.22 (d, J=7.4 Hz, 2H,
NCH2), 2.16 (d, J=1.8 Hz, 2H, NCH2), 1.87 (m, 4H,
10 CH2CH2CH3), 1.40 (m, 4H, CH2CH2CH3), 1.24 (d, J=5.7 Hz, 3H,
NCHCH3), 1.21 (d, J=5.7 Hz, 3H, NCHCH3), 0.92 (t, J=7.4
Hz, 6H, CH2CH3); 13C NMR (90 MHz:, CDC13) ~ 170.07,
169.96, 165.56, 165.54, 164.25, 164.25, 163.27, 162.99,
137.02, 137.02, 133.82, 133.76, 133.72, 133.69, 131.31,
131.24, 130.04, 129.92, 128.56, 128.51, 128.16, 128.14,
127.95, 127.93, 127.12, 127.09, 126.35, 126.35, 126.02,
125.97, 125.59, 125.55, 124.31, 124.29, 108.08, 108.02,
74.21, 74.16, 51.61, 51.61, 39.72, 39.72, 39.32, 39.29,
39.04, 39.04, 33.67, 33.67, 18.06, 18.02, 13.74, 13.74;
HRMS (DCI) [MH]~ calcd for C29H30N20s 487.2233, found
487.2233.
CC2H ~ ~ ~ Me
~M- ~ ~ tt3E
Br C~Cle l
1 OBZIE ~r~RlOMe
113Z
SUBSrlTUTE SHE~T (RULE 26)
W095/02566 2 1 6 7 3 9 1 PCT~S94/08141
Butyraldehyde (3.65 ~L, 4.04 mmol) and 1-
naphthoic acid (80.0 mg, 0.45 mmol) were added
sequentially to a r.t. CH2Cl2 (3 mL) solution of
isocyanide 106 (98.0 mg, 0.405 mmol, E/Z=1.4:1) and
stirred under N2 for 23 hours after which the solvent was
removed under reduced pressure. The crude oil was
purified by flash silica gel (deactivated with 5~ Et3H)
chromatography ~gradient eluted with 100~ hex to 1:1
hex/EtOAc). One fraction was isolated as a white powder
containing an inseparable mixture of adducts 113 (140
mg, 69~, E/Z = 1.4:1).
113Z and 113E: lR (film) cm~l: 3400, 3298,
3056, 2961, 2874, 1732, 1480, 1321, 1236, 1194, 1132,
783; lH NMR (360 MHz, CDCl3), ~ 113Z 9.01 (d, J=8.6 Hz,
lH, ArH, Z-isomer), 8.36 (dd, J=1.2, 7.3 Hz, lH, ArH, Z-
isomer), 8.10 (d, J=8.3 Hz, lH, ArH, Z-isomer), 7.91 (d,
J=8.0 Hz, 2H, ArH, Z-isomer), 7.7-7.5 (m, 3H, ArH, Z-
isomer), 7.4-7.2 (m, 3H, ArH, Z-isomer), 7.32 (bs, lH,
N~, Z-isomer), 7.05-6.85 (m, 3H, ArH), 5.69 (t, J=5.9
Hz, lH, O OE2CH2, Z-isomer), 3.56 (s, 3H, CO2CH3, Z-
isomer), 2.18-2.08 (m, 2H, OCH2CH2, Z-isomer), 2.01-1.92
(m, 2H, OCH2CH2, E-isomer), 1.68-1.56 (m, 2H, C~2CH3, Z-
isomer), 1.03 (t, J=7.3 Hz, 3H, CH2CH3, Z-isomer), 113E
8.87 (d, J=8.6 Hz, lH, ArH, E-isomer), 8.12 (d, J=8.3
Hz, lH, ArH, E-isomer), 8.05 (d, J=8.2 Hz, lH, ArH, E-
isomer), 7.76 (dd, J=1.2, 7.2 Hz, lH, ArH, E-isomer),
7.7-7.5 (m, 3H, ArH, E-isomer), 7.4-7.2 (m, 3H, ArH, E-
isomer), 7.32 (bs, lH, NH, E-isomer), 5.50 (t, J=6.0 Hz,
lH, OCH2CH2, E-isomer), 3.94 (s, 3H, CO2OE3, E-isomer),
1.52-1.40 (m, 2H, CH2CH3, E-isomer), 0.954 (t, J=7.4 Hz,
3H, CH2OE3, E-isomer); 13C NMR (90 MHz, CDCl3) ~ 168.10
(C), 167.18 (C), 165.58 (C), 165.06 (C), 163.92 (C),
162.89 (C), 136.71 (C), 135.77 (C), 134.43 (CH), 134.27
(CH), 133.85 (C), 133.75 (C), 131.46 (C), 131.41 (CH),
130.50 (CH), 130.15 (CH), 129.46 (CH), 129.42 (CH),
128.78 (CH), 128.74 (CH), 128.65 (CH), 128.59 (CH),
128.40 (CH), 128.40 (CH), 128.26 (CH), 128.23 (CH),
SU8SrlTUTE SHEET (RULE 26)
wo gs/02s~ 2 1 6 7 3 9 1 PCT~S94/08141
51
127.86 (C), 127.23 (C), 126.49 (CH), 126.49 (CH), 125.57
(CH), 125.44 (CH), 125.38 ~C), 124.86 (C) 124.38 (CH),
124.27 (CH), 118.98 (C), 114.48 (C), 73.78 (CH), 73.41
(CH), 52.72 (CH3), 52.57 (CH3), 33.78 (CH2), 33.60 (CH2),
18.06 (CH2), 17.98 (CH2), 13.76 (CH3), 13.67 (CH3); HRMS
(FAB) [MH]+ calcd for C26H24OsNBr 510.0916, found 510.0916.
M- , 114E
~ ~di~,CH2C~
107Z/E ~ ~ ~ ~OMe
t14Z
Pyridine (35 ~L, 0.441 mmol), butyraldehyde
(330 ~L, 3.67 mmol), and 1-naphthoic acid (63 mg, 0.367
mmol) were added sequentially to a r.t CH2CL2 (3 mL)
solution of isocyanide 107 (90.0 mg, 0.367 mmol,
Z/E=1:1.4). The mixture was stirred under N2 for 12
hours after which the solvent was removed under reduced
pressure. The crude oil was immediately purified by
flash silica gel (deactivated with 5~ Et3N)
chromatography (gradient eluted with 100~ hex to 1:1
hex/EtOAc) affording 2 fractions: 114Z (54.5 mg, 31~)
and 114E (44.5 mg, 26~), for a 57~ overall yield
(Z/E=1.2:1).
SU~IIUTE SHEET (RULE 26)
2 1 67 39 1
W095/02566 PCT~S94/08141 _
114Z: IR (film) cm~l: 3281, 3002, 2964, 1723,
1697, 1414, 1198, 1138; lH NMR (360 MHz, CDCl3), ~ 9.01
(d, J=8.3 Hz, lH, ArH), 8.34 (dd, J=1.2, 7.2 Hz, lH
ArH), 8.10 (d, J=8.1 Hz, lH, ArH), 7.92 (d, J=7.7 Hz, lH
ArH), 7.66 (dt, J=1.4, 5.8 Hz, lH ArH), 7.60-7.54 (m,
2H, ArH), 7.43 (bs, lH, NH), 7.35-7.33 (m, 3H, ArH),
7.28-7.29 (m, 2H, ArH), 5.69 (t, J=6.12 Hz, lH, OCHCH2),
3.47 (s, 3H, OCH3), 2.19-2.13 (m, 2H, OCHCH2), 2.04-1.97
(~3, 4H, (CH2)2N), 1.7-1.6 (m, 2H, CH2CH3), 1.05 (t, J=7.3
Hz, 3H, CH2CH3); 13C NMR (90 MHz, CDC13) ~ 169.24 (C),
166.11 (C), 164.38 (C), 157.49, 136.06, 134.29, 133.91,
131.51, 130.56, 128.72, 129.49, 128.34 (CH), 128.25,
127.75 (CH), 126.50 (C), 125.78 (C), 125.55 (C), 124.50
(C), 112.01 (C), 74.46 (OCHCH2), 51.56 (OCH3), 34.06
(CHCH2), 29.64 (N(CH2)2), 18.42 (CH2CH3), 13.85 (CH2CH3);
HRMS (DCI) [MH]' calcd for C28H28N2O5 473.2077, found
473.2070.
114E: IR (film) cm~l: 3291, 2960, 2874, 1713,
1683, 1505, 1279, 1246, 1195, 1135, 784; lH NMR (360 MHz,
CDCl3), ~ 8.75 (d, J=8.4 Hz, lH, ArH), 8.03 (d, J=8.1 Hz,
lH, ArH), 7.92 (dd, J=1.2, 7.4 Hz, lH, ArH), 7.89 (dd,
J=1.4, 7.8 Hz, lH, ArH), 7.59 (A~3, J=1.6, 6.9 Hz, lH,
ArH), 7.55 (AF3, J=1.3, 6.9 Hz, lH, ArH), 7.44 (dd,
J=7.3, 8.2, lH, ArH), 7.27-7.18 (m, 4H, ArH), 7.17-7.10
(m, lH, ArH), 6.92 (bs, lH, NH), 5.41 (t, J=6.2 Hz, lH,
OCHCH2), 3.75 (s, 3H, OCH3), 2.24-2.21 (m, 4H, (CH2)2N),
1.90 (m, 2H, OCHCH2), 1.42-1.35 (m, 2H, CH2CH3), 0.917
(t, J=7.3 Hz, 3H, CH2CH3); 13C NMR (90 MHz, CDCl3) ~
170.00, 165.56, 164.24, 163.28, 136.80, 133.84, 133.74,
131.31, 130.03, 128.68, 128.53, 128.26, 127.99,
127.06, 126.39, 125.98, 125.60, 124.32, 108.71, 74.19,
51.67, 33.67, 32.33 ((CH2)2N), 18.06, 13.75; HRMS (DCI)
[MH] t calcd for C28H28N2O5 473.2077, found 473.2076.
SUBSIl~UTE SHEET (RU~E 26)
W095/02566 2 1 6 7 3 9 I PCT~S94/08141
53
~N ~o
o~ r
,~0
~b II
The nucleophilic addition of l-naphthoic acid
to the vinyl aziridine isocyanides is a common side
reaction observed when excess l-naphthoic acid or no
pyridine was added to these Passerini reactions.
II : lR (film) cm~l: 3281, 3057, 2959, 1717,
1663, 1591, 1515, 1280, 1241, 1138; lH NMR (360 MHz,
CDCl3), ~ 9.31 (t, J=5.8 Hz, lH, NHCH2), 8.85 (d, J=9.0
Hz, lH, ArH), 8.77 (d, J=8.3 Hz, lH, ArH), 8.25 (d,
J=8.4 Hz, lH, ArH), 8.04 (t, J=8.0 Hz, 2H, ArH), 7.98
(d, J=7.3 Hz, lH, ArH), 7.89 (t, J=9.1 Hz, 2H, ArH),
7.63-7.46 (m, 6H, ArH), 7.34-7.22 (m, 5H, ArH), 6.62
(bs, lH, NHCO), 5.29 (t, J=6.2 Hz, lH, CHO), 4.33 (t,
J=5.4 Hz, 2H, CH2O), 3.66 (s, 3H, OCH3), 3.34 (q, J=5.8
Hz, 2H, CH2NH), 1.7-1.6 (m, 2H, CH2CH), 1.3-1.2 (m, 2H,
CH2CH3), 0.84 (t, J=7.2 Hz, 3H, CH3CH2); LRMS (CI+) [MH]+
calcd for C39H36N2O7 64S.25, found 645Ø
SUBS~ITUTE SHEET (RULE 26)
WO95/02566 2 1 6 7 3 9 1 PCT~S94/08141
54
EXAMPLE 4 Structural Activity Profile of the In vi tro
Cytotoxicities Of The Carzinophilin and
Azinomycin Analoqs Of ExamPle 3
A structural activity relationship (SAR) of
the in vi tro cytotoxicities of the Carzinophilin and
Azinomycin analogs of Example 3 is provided in Table 3
below.
Table 3 ln V~ro Cytotoxicides of Azinomycin Analogs in HCI`116
Human Colon C~ci,lu~lJa Cell Lines
IC50 (~M)
Cm~ No. HCT116 HCT1161VM46 HCT11~VP35
l~E 439 5.56 5.27
109Z 5.4 1.6 2.6
110E 12.4 13.2 11.0
110Z 6.76 7.7 6.4
111~111Z >~ ~30 >30
112E ~30
1~Z 253 27.2 25.5
113~113Z ~30 ~30 >~
114E ~30 ~ x~
1~4Z 28.6 38.4 273
~omycinB Q838 - -
Cy; n ~ æses~ed by Xl~ assay after 72 hours c ~ drug exposure.
SU~TUTE SltEET (RU~E 26)
21 6739 1
W095/02566 PCT~S94/08141
Each analog was incubated for 72 hours with
the human carcinoma cell line HCT116 and two drug
resistant sublines HCT116/VM46 and HCT116/VP35.
HCT116/VM46 was selected for resistance to VM-26 and
expresses the multidrug resistant (MDR) phenotype.
HCT116/VP35 was selected for resistance to VP-16 and is
resistant to topoisomerase II active drugs. The results
of the
Analogs lO9E, lO9Z, llOE and llOZ which all
contain vinyl-aziridine, epoxide and naphthoate moieties
displayed ICso's well within an order of magnitude of the
natural product, Azinomycin B. Interestingly, the
racemic 2-(methyl)-aziridine analogs llOE and llOZ
behaved nearly identically in the in vi tro assays to the
correspondingly unsubstituted aziridine analogs lO9E and
lO9Z.
Analogs lacking the vinyl-aziridine moiety
gave no measurable cytotoxicity (ICso<30~M) in all cases
studied. Substitution of the epoxide fragment for the
n-butyl group in analogs 114E, 114Z, 115E and 115Z
displayed significantly less activity when compared to
the most active compounds which contain the epoxide
moiety.
In addition, these compounds did not show
decreased potency when tested with Multiple Drug
Resistance (MDR) and topoisomerase II resistant cell
lines. This suggests that these compounds are not
substrates for the P-glycoprotein efflux pump nor do
they interact with topoisomerase II.
EXAMPLE 5 Synthesis of Peptidomimetic Polymer MCCA
Arra~ Using The Uqi Reaction
A combinatorial array of peptidomimetic
polymers having a non-hydrolyzable ethylenediamine core
structure can be prepared using the Ugi reaction. As
depicted in Figure 7, the Ugi reaction is a four
component condensation involving a first amino acid, a
SU~STITUTE SHE~ (RULE 26)
WO9~/02566 2 1 6 7 3 ~ ~ PCT~S94/08141
56
second amino acid having a Boc-protected alpha amino
group, methylisocyanide and acetic acid. The Ugi
reaction is conducted at room temperature in aqueous
solvents. As is illustrated in Figure 7, once the
protecting groups have been removed, the Ugi reaction
provides nonhydrolizable peptide analogues that mimic
the structure of natural peptides.
EXAMPLE 6 Synthesis Of A Phosphotyrosine Peptide
Structural Analog MCCA Array
Usinq The Uqi Reaction.
One application for an Ugi MCCA array
synthesis is the preparation of phosphotyrosine peptide
structural analogues for use as structure-based
competitive inhibitors. As depicted in Figure 8,
phosphotyrosine peptides have a peptide backbone core
structure. Structural variants of phosphotyrosine can
be synthesized by varying the first and second amino
acids employed (aal and aa2) as well as the other
components of the Ugi reaction.
Several different combinatorial arrays of
phosphotyrosine peptide analogues can be produced using
the Ugi reaction. For example, as depicted in Figure
9a, pseudosubstrate peptide inhibitors can be prepared.
The amino acid side chains of the tyrosine peptide
analogues are determined by which Rl and R2 groups are
used for the isocyanide and the acid. Branched chain
tyrosine-based peptide analogues, not otherwise
accessible using standard solid phase synthesis, can be
prepared when R3 on the amine is an amino acid or
derivative thereof.
As depicted in Figure 9b, low molecular weight
analogues of tyrosine that possess an intact tyrosine
structural unit can be prepared wherein the R groups of
the isocyanides, acids and amines employed are alkyl,
aryl or acyl groups.
SU8S~ITUTE SHEET (RULE 26)
WO9~/02566 2 1 S 7 3 9 I PCT~S94/08141
-
57
As depicted in Figure 9c, low molecular weight
derivatives of tyrosine where the tyrosine structural
unit is also modified can be prepared by using different
aldehydes.
As depicted in Figure 9d, low molecular weight
tyrosine derivatives can be prepared where the acid and
amine are the same molecule. Further variations of
these tyrosine-based peptide analogues can also be made
according to the present invention by using different
reaction components.
EXAMPLE 7 Synthesis of Solid Support And Linker
System For Use In Solid Phase Synthesis Of
MCCA ArraY.
A proposed synthesis of a novel support and
linker system is depicted in Figure lO. The linker can
be synthesized by condensing ethylene oxide with the
lithium enolate of succinic anhydride. The resulting
alcohol can then be converted to its mesylate and
displaced with sodium azide to provide the azide
functionality. This anhydride is then heated in the
presence of the polymer in dimethylformamide to afford a
free carboxylate linker.
EXAMPLE 8 Synthesis of Passerini-type MCCA
ArraY Usinq Solid Phase Synthesis
A solid phase MCCA array synthesis utilizing
the Passerini reaction can be conducted using the linker
described in Example 7. The carboxylic acid linker-
resin system formed in Example 7 can be treated with
dicyclohexylcarbodiimide and hydroxybenzotriazole in the
presence of the methyl ester of alanine. The progress
of the reaction can be monitored by nuclear magnetic
resonance based on the disappearance of free alanine.
Filtration of reactants and subsequent washing with
fresh DMF should provide a purified resin-linker-amino
SUBSrlTUTE SHEET (RULE 26)
W095/02566 2 t 6 ~ 3 q 1 PCT~S94/08141
58
acid system. Hydrolysis of the ester of alanine under
base conditions should then provide the free carboxylic
acid. Again, washing the resin with water provides a
resin-linker-alanine carboxylic acid free of impurities
or reactant contamination. Addition of c'~n~ldehyde
and 2,2-diethylphosphonomethylisocyanide to the resin
reaction chamber stirring in an ethyl acetate solution
should result in the three component Passerini
condensation reaction. These reactants can be removed
after the reaction by washing. Addition of triphenyl
phosphine to a DMF solution of resin followed by washing
with solvent should provide an amine side-chain on the
linker. Heating this material should then cause an
intramolecular reaction wherein the linker forms a
lactam and releases the amine into solution.
EXAMPLE 9 Synthesis Of Peptidomimetics Using
Solid Phase Synthesis
The use of the Ugi reaction as a means for
synthesizing peptide mimics is described in Example 5
and 6 and as depicted in Figure 11. By coupling the
first amino acid (aal) to a solid phase support as
described in Example 8, the solid state synthesis of
these peptide mimics can be performed. Reaction of the
linked amino acid with an isocyanide, an aldehyde and an
amine should yield a solid phase linked peptidomimetic
analog. Removal of the tBoc blocking group under Ugi
reaction conditions set forth in Figure 8 should afford
a product containing two amino acid components. This
process can be repeated over several cycles to provide 9n
products where n is the number of cycles. Thus, this
application of the Ugi reaction provides numerous
analogues despite the use of a very small number of
different components.
SU8S~ITUTE SHEE~ (RULE 26)
21 673~ T
WO 95/02566 PCT/US94/08141
59
EXAMPLE 10 Isolation Of Products From Ugi-type MCCA
Array Synthesis By Sequestering
The Unreacted Com~onents
The reaction products of an Ugi-type reaction
5 can be efficiently recovered from the reaction media by
sequestering the unreacted components thereby yielding a
substantially pure product.
In an Ugi-type reaction, the unreacted
components consist of an aldehyde, an amine, an acid and
an isocyanide. A mixed bed resin containing strongly
acidic and basic resins can be used to remove the amine
and acid respectively. In order to isolate the product
of an Ugi reaction, the solvent is first removed by
evaporation at reduced pressure. Distilled water is
15 then added to the reaction mixture. To the resulting
aqueous solution is added a mixed bed resin containing
Biorad AG 501-X8/Bio-Rex MSZ 501, a strongly basic anion
exchange and strongly acidic cation exchange resin.
Once the mixed bed resin has been added, the solution is
stirred for 10 min. The mixture is then filtered and
concentrated to yield a substantially pure Ugi-type
reaction product.
The use of exchange resins to sequester
unreacted acids or amines can be extended to other
25 unreacted components having different functional groups,
such as esters, where the unreacted component is
selectively converted to either an acid or amine after
the reaction, thereby enabling its removal by the resin.
Metal sequestering agents can also be used
30 where organometallic solution chemistry is employed in
the MCCA reaction. For example, the addition of an EDTA
molecule tethered to a polymer can be used to sequester
palladium salts from the reaction mixture. Strongly
acidic cation exchange resins can be used to remove
- 35 boronic acids.
SUBS~TUTE SHEE~ (RU~E 26)
wo 95,02566 2 1 ~ 7 3 9 ~ PCT~S94/08141
Example 11 Synthesis And Cleavage Of Passerini
Reaction Products From Photocleavable
Carbamate Linker
Synthesis of photocleavable carbamate linkers
is depicted below. The photocleavable carbamate linkers
120 and 121 were constructed by reacting isocyanates 123
and 124 with the hydroxyl group from the linker alcohol
described in Williams, P., et al., Tetrahedron (1991)
47:9867-9880.
The carbamate linkers 120 and 121 were then
coupled to methylbenzhydrylamine (MBHA) Gly-resin 125
using HOBt in DMF to yield the polymer supported
photocleavable carbamates 126 and 127, respectively.
Hydrolysis of the ester of carbamate 126 with LiOH,
THF/H2O yielded the corresponding polymer supported acid
128 in less than one hour. Surprisingly, the benzoate
ester of carbamate 127 was resistant to hydrolysis under
a variety of reaction conditions (LiOH, THF/H2O, K2CO3,
MeOH, NaOH, THF/H2O and NaOH, MeOH).
Using the polymer supported acid 128, a solid
phase Passerini reaction was conducted. A CH2Cl2
solution containing methyl isocyanoacetate and
butyraldehyde were added to the polymer supported acid
128 to yield the resin bound Passerini adduct 129.
Initially, carbamate cleavage was attempted in
methanol which yielded only the ~-acyloxy hydrolysis
Passerini product 131. In order to avoid hydrolysis and
capture the free amine product, photolysis at 350nm in a
rayonet for 24 hours was conducted in CH3CN in the
presence of acetic anhydride (10 equiv.). Excess
solvent acylating agent were then removed under vacuum
to yield 134 as the only product. No other products
were detectable from the crude product by lH NMR and TLC.
SU~TUTE SltEET (RULE 26)
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61
ExPerimental Data
HO ~ No2 E~O ~O~ ~ NO
~ Eto5~NC ~
O~jl r
J~CI THF, lEA 1~ C~J~
Ethyl-isocyanatoacetate (15S ~L, 1.37 mmol) was added to
a THF ~5 mL) solution of linker 122 (450 mg, 1.14 mmol).
Triethylamine was then added and the mixture allowed to
stir at 25C overnight. TLC showed that the reaction
was slow, so the reaction was heated to 50C. TLC
showed that the reaction was complete in 2 days. Upon
cooling the flask to -20C, a precipitate formed and was
subsequently filtered off. lH NMR in CD3CN showed that
this compound was the isocyanate decomposition product,
glycine ethyl ester. The filtrate solvent was then
removed under reduced pressure and the crude oil was
extracted 3X with CH2Cl2 over KH2PO4 buffer. The combined
organic layers were dried over Na2SO4 and evaporation of
the solvent under reduced pressure gave a yellow solid.
Purification by flash silica gell chromotography
(gradient eluted 100~ hex to 1:1 hex/EtOAc) afforded
carbamate 123 (373 mg, 64~) as a white solid (mp 131-
133C).
123: IR (film) cm~l: 3323, 3097, 2985, 1750,
1699, 1538, 1461, 1351, 1248, 1206, 1084, 1060; lH NMR
(360 MHz, CDCl3), ~ 8.88 (d, J=1.7 Hz, lH, ArH), 8.42
(dd, J=1.7, 8.2 Hz, lH, ArH), 7.82 (d, J=8.2 Hz, lH,
ArH), 7.60 (s, lH, ArH), 7.44 (s, lH, ArH), 5.62 (s, 2H,
OC~2Ar), 5.61 (bt, J=5.3 Hz, lH, HNCH2), 4.22 (q, J=7.2
Hz, 2H, OCH2CH3), 3.99 (d, J=5.6 Hz, 2H, HNCH2), 1.28 (t,
J=7.3 Hz, 3H, CH2CH3); 13C NMR (90 MHz, CDCl3) ~ 169.75,
SU~ITUTE SHEET (RULE 26)
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161.44, 155.43, 147.07, 145.38, 139.34, 134.88, 131.63,
131.12, 131.08, 128.87, 128.65, 126.74, 126.00, 124.14,
63.28, 61.61, 42.75, 14.05; HRMS (CI~ [MH]~ calcd for
C1gNl5N2O8C13 504.9972, found 504.9972.
MeMe
¢] ~so% TFAICH2CIQ ¢~1
I~HBOC ~H
¦MBHA resin~~~MeHA resln~~
125
BOC-Gly-resin was deprotected with 33~ TFA in
CH2Cl2 for 30 min. and then washed exhaustively with
CH2Cl2. The resin was then free baeed with 5% Hunig's
base in CH2Cl2 and the solution filtered after 2 minutes.
The resin was then washed exhaustively with sequential
applications of CH2C12/MeOH/CH2C12 until the resin
appeared colorless. The resin was dried in vacuo
overnight. The entire procedure was repeated again with
50% TFA in methylene chloride (20 mL) affording Gly-
MBHA-resin 125 (1.39 g) as colorless beads. The
qualitative ninhydrin test (Kaiser Test) was positive
(dark blue).
M~
DMF HOBt f 9 NO2
125 ~ N~ NH
(M~HA r~l~ L~O
1 28 OEt
Gly-carbamate 123 (1.2 g, 2.37 mmol) in 2 mL
DMF was added to a 5 mL DMF suspension of MBHA-resin 125
SU8SrlTUTE SltEET (RULE 26)
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W095/02566 PCT~S94/08141
.
(1.34 g, 1.12 equiv./gram, obtained from Novabioehem.)
HOBt (470 mg, 3.4 mmol) was then added and the mixture
allowed to stir at 25C for 36 hours. The resin was
washed exhaustively in sequential applications of
CH2Cl2/MeOH/CH2Cl2. After two iterations of this process,
resin 126 (1.83 g) was afforded as pale yellow beads.
The qualitative ninhydrin test (Kaiser Test) was
negative.
Capping of residual free amines on the dried
resin 126 (1.7 g, ~1.7 mmol) was performed by suspending
the resin in a pyridine (10 mL) solution of Ac2O tl.7 mL,
16.9 mmol) and a catalytic amount of DMAP (25 mg, 0.2
mmol). The solution was stirred at 25C for 1 hr. and
then filtered and washed exhaustively with sequential
applications of CH2Cl2/MeOH/CH2Cl2. The resin was dried
in vacuo overnight affording resin 126 (1.7 g) as pale
yellow beads. The qualitative ninhydrin test (Kaiser
Test) was negative.
Me
1N UOH ,~HJ~ 4
12B OH
(Ethyl)-glycinate-MBHA-Gly-resin 126 (1.6 g, ~ 1.5
mmol) was stirred in 45 mL THF with lN LiOH (15 mL, 15.0
mmol) for 1 hr. The mixture was then acidified with lN
KH2SO~(~17 mL) to pH=2-3 and the resin was filtered.
Exhaustive sequential washings with H2O/THF/CH2Cl2 and
subsequent drying in vacuo overnight afforded (glycinic
acid)-Gly-MBHA resin 128 (1.46 g) as pale yellow beads.
Photolysis of this resin in CH3N and AC2O (~10 equiv.)
did not give the unhydrolyzed N-acetyl-(ethyl)-glycinate
SU~ITUTE SHEET (RU~E 26)
wo 95,02566 2 1 6 7 3 9 l PCT~S94/08141
64
product by TLC (Rf=O . 3, 100~ EtOAc, yellow anisaldehyde
stain).
Ab
EIo~NC ~ ~NH
128 ~ (M~HA Q 40
H~ 129 EtO~
CH2a2
Methyl isocyanoacetate (23 ~L, 0.23 mmol) and
butyraldehyde (23 ~L, 0.23 mmol) were added to a CH2Cl2
(200 ~L) suspension of (glycinic acid)-Gly-MBHA resin
128 (25 mg, ~0.025 mmol). The mixture was allowed to
stand at 25C for 48 hours. The resin was then
filtered, washed exhaustively with sequential
applications of CH2Cl2/MeOH/CH2Cl2 and dried in vacuo
overnight to afford (Mica-But-Gly)-Gly-MBHA resin 129
(e27 mg) as yellow beads.
Me
E~_NC ~ N~NH
128 ~ (MBHA resi ~
CH2C12
General procedure for the photolytic cleavage
of o-nitrobenzyl carbamates and in si tu acylation of
Passerini adducts:
Acetic anhydride (10 equiv.) was added to a
CH3CN suspension of (Mica-But-Gly)-Gly-MBHA resin 128 (25
SU8STITUTE SHEET (RUI E 26)
W095/02566 2 ~ 6 7 ~ 9 1 PCT~S94/08141
mg, ~0.025 mmol) and irradiated at 350 nm in a Rayonet
for 12-48 hours at 30C. The solution was transferred
into a round bottom flask by pipet with multiple
washings (CH3CN) of the resin. The solvent, excess
acetic anhydride, and acetic acid was then efficiently
removed in vacuo to afford Mica-But-(N-acetyl-Gly)
adduct 129 (~2-4 mg by crude lH NMR) as a colorless oil.
1H NMR and TLC demonstrated this was the only compound
present.
129: lH NMR (360 MHz, CDCl3), ~ 6.93 (bt, J=5
Hz, lH, NH), 6.11 (bt, ~=5 Hz, lH, NH), 5.28 (t, J=6.0
Hz, lH, OCH), 4.13 (dd, ~=5.7, 18.2 Hz, lH, HNCH2CO2CH3),
4.08 (5, J=5.5 Hz, 2H, HNCH2CO2CH3), 3.97 (dd, J=5.3,
18.1 Hz, lH, HNCH2CO2CH3), 3.76 (s, 3H, CO2CH3), 2.07 (s,
3H, HNCOCH3), 1.89 (m, 2H, OCHCH2), 1.41 (m, 2H,
OCHCH2CH2), 0.93 (t, J=7.4 Hz, 3H, CH2CH3); LRMS (FAB)
[MH] t calcd for Cl2H20N2O6 289.14, found 289.5.
Example 12 Solid Phase Passerini MCCA Array
Synthesis Using The Photocleavable
Carbamate Linker Of Example 11
A solid phase MCCA array synthesis using the
photocleavable carbamate linker of Example 11 was
conducted and is depicted in Table 4. In this
synthesis, 8 isocyanides, 6 aldehydes, 1 carboxylic acid
and 1 acylating agent were employed. It should be
understood that a variety of different carboxylic acids
and acylating agents could have been used.
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WO 95/02566 2 t 6 7 3 9 1I PCT/US94/08141
66
TLC and LRMS Da~a for Polymer S ~p~d Passini MCCAS
Cor struc~r
[ ~ RlCHO I R2-NC 2. ~1 8t'' ~R 5
F12-NC HI~ ~d h U
~N IT +MN +T ~MN +T ~ T +MN +T -MN +T
T8__NC 8 MG
+MN +T ~MN +T -MN +T -MN -T +MN 1 +MN -T
+M~ ~
-MN +T +MN +T -MN +T +MN -T -MN -T -MN 1
+~G MG ~ ~
+MN ~T +MN +T ~MN +T -MN -T -MN -T -MN -T
+M~
-MN -T -MN -T -MN -T -MN 1 -MN -T -MN 1
\~ -MG ~
a rNC -MN -T -MN -T +MN -T -MN -T -MN -T -MN -T
P~ ~ MG MG ~
+MN +T +MN +T +MN +T -MN -T +MN +T -MN 1
+M~ ~3 -MB
~ +MN IT +MN +T +MW +T -MN -T ~MN +T ~MN +T
Md~--~ 1 +M6 63 ~3 ~3
A B C D E F
[alRl~C~'~ ~H ~
+T~ ~CdK~nde~bd~x~s);-T.TLCddn~ q~cbd~x~
+MN-UR M~SpK~a(NB~nu~k)~K~d-x~bdpld~;~MN~UR
M~ SpK~a (NB~m~)ddn~dK~ndq~cbdp~du%;+MG~LRM _
Sp~ (~y~ m~bt) ~d ~d pK~; - Mt~ LR ~_ 8p~
(G~oNd n~)dd n~ ~K~nd q~c~d p~du~ H NMR ~K~d
bdp~u~;~ c1HNMRddn~Kwnd q~d~dp~d~
SU8SrlTUTE SHEET (RULE 26)
21673~1
WOg5/02566 PCT~S94/08141
-
67
All reactions were performed in Fisher shell
vials arranged in two 4x6 microtiter trays set side by
side to form the requisite 8x6 array. 25mg of resin was
added to each vial and swelled with CH2Cl2. The
aldehydes and carboxylic acids were then delivered by
gas tight syringe to each of their respective rows and
columns and allowed to react at room temperature for 2-4
days. Filtration of the individual reactions and
exhaustive washings followed by photolysis at 350 nm in
CH3CN (lO equiv. Ac2O) for 24h gave 48 reaction wells to
be analyzed for product composition. The results of the
array synthesis is depicted in Table 4. Low resolution
mass spectroscopy combined with thin layer
chromatography of each of the reaction wells showed that
50~ of the desired products were formed and isolated. lH
NMR of the reaction wells that gave positive TLC and
mass spectroscopy data revealed that the desired
products were the only solid phase products produced in
these wells. This example thus demonstrates that large
arrays of compounds can be rapidly and efficiently
prepared in high purity using solid phase synthesis
according to the present invention.
While the invention of this patent application
is disclosed by reference to specific MCCA reactions, it
is understood that the present invention can be applied
all other multicomponent syntheses capable of being
conducted in a single reaction vessel without the
isolation of an intermediate product. Further, the
present invention is intended to be applicable to all
future developed MCCA reactions.
It is also understood that the embodiment of
the present invention encompassing solid phase synthesis
is intended to be practiced with the linkers and solid
supports disclosed herein as well as with other linkers
and solid supports presently known or developed in the
future.
SUBSrlTUTE SltEEr (RULE 26)
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68
Accordingly, the invention may be embodied in
other specific forms without departing from its spirit
or essential characteristics. It is to be understood
that this disclosure is intended in an illustrative
rather than limiting sense, as it is contemplated that
modifications will readily occur to those skilled in the
art, within the spirit of the invention and the scope of
the appended claims.
SU8SllTUTE SHEET (RULE 26)
