U.S. patent application number 10/034212 was filed with the patent office on 2003-07-31 for heterocyclic compounds, method of developing new drug leads and combinatorial libraries used in such method.
This patent application is currently assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM. Invention is credited to Gilon, Chaim.
Application Number | 20030144260 10/034212 |
Document ID | / |
Family ID | 21874987 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030144260 |
Kind Code |
A1 |
Gilon, Chaim |
July 31, 2003 |
Heterocyclic compounds, method of developing new drug leads and
combinatorial libraries used in such method
Abstract
The present invention provides according to a first of its
aspects, new compounds that have a flexible scaffold with various
degrees of conformational restriction and accordingly are useful as
drug candidates. These compounds may be used to produce new
combinatorial libraries that will permit to screen for and select
drug candidates for a variety of uses in human medicine, veterinary
medicine and in agriculture.
Inventors: |
Gilon, Chaim; (Jerusalem,
IL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
YISSUM RESEARCH DEVELOPMENT COMPANY
OF THE HEBREW UNIVERSITY OF JERUSALEM
46 Jabotinsky Street
Jerusalem
IL
|
Family ID: |
21874987 |
Appl. No.: |
10/034212 |
Filed: |
January 3, 2002 |
Current U.S.
Class: |
514/183 ;
435/7.1; 436/518; 514/218; 540/460; 540/492 |
Current CPC
Class: |
C40B 40/00 20130101;
C07D 285/00 20130101; A61P 35/00 20180101; C07D 285/38 20130101;
C07K 1/047 20130101 |
Class at
Publication: |
514/183 ;
514/218; 540/460; 540/492; 435/7.1; 436/518 |
International
Class: |
G01N 033/53; A61K
031/33; A61K 031/551; G01N 033/543; C07D 243/08; C07D 245/02 |
Claims
1. Scaffold-based compound having the following general formula
(B): 12including pharmaceutically acceptable salts, esters or
solvates thereof, wherein Z is selected from C.dbd.O and
--CH.sub.2--, W is selected from C.dbd.O and a bond, provided that
at least one of Z and W is C.dbd.O, X and Y are independently
selected from CH.sub.2, O, S, NH, N--R.sup.5, NH--CO, CO,
CH.sub.2CO, S.dbd.O and SO.sub.2, or X and Y may form together a
group selected from CH.dbd.CH, CO--NR.sup.5, NH--CO--N,
O--CH(R.sup.5)--O, NH--CH(R.sup.5)--O and NH--CH(R.sup.5)--NH,
where the hydrogen in the above groups may optionally be
substituted by an alkyl group, (a) and (b) are parts of the
scaffold and are nitrogen containing bivalent organic radicals,
each independently providing between 1 to 4 atoms to said scaffold,
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently
selected from H and substituted or unsubstituted allyl, and n and m
are integers from 1 to 6, with the exclusion of the following
compound: glycinamide,
L-tyrosyl-N-[2-[(2S)-4-[(1S)-1-carboxy-3-methylbut-
yl]-3,4,5,8-tetrahydro-3-oxo-2-(phenymethyl)-1,4-diazocin-1(2H)-yl]2-oxoet-
hyl.
2. A compound of the formula (B) according to claim 1, wherein X
and Y are independently selected from CH.sub.2, O, S, NH,
N--R.sup.5, NH--CO, CO, CH.sub.2CO, S.dbd.O and SO.sub.2, or X and
Y may form together a group selected from. CO--NR.sup.5,
NH--CO--NH, O--CH(R.sup.5)--O, NH--CH(R.sup.5)--O and
NH--CH(R.sup.5)--NH, where the hydrogen in the above groups may
optionally be substituted by an alkyl group.
3. A compound according to claim 1, with each of parts (a) and (b)
providing up to two atoms to said scaffold.
4. A compound according to claim 1, wherein each of parts (a) and
(b) is independently selected from --N(CHR.sup.6COL)--,
--C(COL)(R.sup.6)--, --N(COR.sup.8)--CH.sup.7-- and
--C(NHR.sup.8)(R.sup.7)--.
5. Compound of the formula I, II, III or IV: 13including
pharmaceutically acceptable salts, esters or solvates thereof,
wherein X and Y are independently selected from CH.sub.2, O, S, NH,
N--R.sup.5, NH--CO, CO, CH.sub.2CO, S.dbd.O and SO.sub.2, or X and
Y may form together a group selected from CH.dbd.CH, CO--NR.sup.5,
NH--CO--NH, O--CH(R.sup.5)--O, NH--CH(R.sup.5)--O and
NH--CH(R.sup.5)--NH, where the hydrogen in the above groups may
optionally be substituted by an alkyl group; R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are each independently selected from H and
substituted or unsubstituted alkyl; R.sup.5, R.sup.6, R.sup.7 and
R.sup.8 are each independently selected from H, substituted or
unsubstituted alkyl, cycloalkyl, aryl, aralkyl, hetroaryl,
heterocyclyl, hetroaralkyl, acyl, carboxyaryl, carboxyalkyl, side
chains of naturally and artificially occurring amino acids as well
as derivatives and mimics of such side chains, and linear or cyclic
peptide; L is selected from H, OH, NH.sub.2, NHR.sup.5, a peptide
and a solid support, where R.sup.5 is as defined above, and n and m
are integers from 1 to 6, with the exclusion of the following
compound: glycinamide, L-tyrosyl-N-[2-[(2S)-4-[(1S)-1-ca-
rboxy-3-methylbutyl]-3,4,5,8-tetrahydro-3-oxo-2-(phenymethyl)-1,4-diazocin-
-1(2H)-yl]2-oxoethyl.
6. A compound of the formula I, II, III or IV according to claim 5,
wherein X and Y may form together a group selected from
CO--NR.sup.5, NH--CO--NH, O--CH(R.sup.5)--O, NH--CH(R.sup.5)--O and
NH--CH(R.sup.5)--NH where the hydrogen in the above groups may be
substituted by an alkyl group.
7. A compound of the formula I, II, III or IV according to claim 5,
wherein X and Y form combinations selected from: both X and Y are
S; X is NH and Y is CO; X is CO and Y is NH; X is NH and Y is
CH.sub.2; X is CH.sub.2 and Y is NH; X and Y are NH--CO--NH; X is
N--R.sup.4 and Y is CO; and X is CO and Y is N--R.sup.4.
8. A compound of the formula I according to claim 5.
9. A compound of the formula II according to claim 5.
10. A compound of the formula III according to claim 5.
11. A compound of the formula IV according to claim 5.
12. A combinatorial library comprising a plurality of compounds of
the formula (B) as defined in claim 1.
13. A combinatorial library comprising a plurality of compounds of
any one of the formulae I, II, III or IV as defined in claim 5.
14. A library according to claim 13, wherein in the compounds of
the formulae I, II, III or IV, X and Y may form together a group
selected from CO--NR.sup.5, NH--CO--NH, O--CH(R.sup.5)--O,
NH--CH(R.sup.5)--O and NH--CH(R.sup.5)--NH, where the hydrogen in
the above groups may be substituted by an alkyl group.
15. A library according to claim 13, wherein in tHe compounds of
formulae I, II, III and IV, the following combinations for X and Y
are selected from the group consisting of: (i) X and Y are both S;
(ii) X is NH and Y is CO; (iii) X is CO and Y is NH; (iv) X is
N--R.sup.4 and Y is CO; and (v) X is CO and Y is N--R.sup.4.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of the
formula I, II, III or IV as defined in claim 5.
17. The composition of claim 16 for the treatment of a disease,
disorder or condition wherein a therapeutically beneficial effect
is associated with the modulation of a protein or peptide-mediated
cell activity.
18. A composition according to claim 16 for use in veterinary.
19. A composition according to claim 16 for use in human
mammals.
20. A compound of formula I, II, III or IV as defined in claim 5
for use in agriculture.
21. Use of a compound of the formula I, II, III or IV as defined in
claim 5, for the preparation of a pharmaceutical composition.
22. A method for modulating protein or peptide-mediated cell
activity comprising contacting a cell component having said protein
or peptide with an effective amount of a compound of formula I, II,
III or IV as defined in claim 5.
23. A method for He treatment of a disease, disorder or condition
wherein a therapeutically beneficial effect is associated with the
modulation of a protein or peptide-mediated cell activity, the
method comprising administering to a subject in need of such
treatment a therapeutically effective amount of a compound of
formula I, II, III or IV as defined in claim 5.
24. A method according to claim 23 wherein said cell activity is
selected from proliferation, differentiation, cellular shape
alteration, cellular elongation, uptake of substances by cells
secretion of substances, cellular metabolism, and expression of
various proteins.
25. A method of identifying a candidate which modulates a protein
or peptide-mediated cell activity, such method comprising: (a)
identifying in said protein or peptide, a domain which is essential
for said activity, (b) identifying in said domain, pharmacophors
essential for the activity, (c) planning a combinatorial library of
compounds having the formula I, II, III or IV as defined in claim
5, wherein each such compound comprises at least two of the
pharmacophors identified in step (b) above or mimics or derivatives
of the pharmacophors, where each member of the library differs from
the other by at least one of the following: i) the size of the
ring; ii) the order in which the pharmacophors are arranged in the
ring; iii) the chemical nature of the ring; iv) the chemical nature
of the pharmacophors; v) the chirality of the linker between the
ring and the pharmacophor; and vi) the chirality of the
pharmacophor; (d) synthesizing a plurality of compounds among the
compounds of the combinatorial library planned in step (c); (e)
screening the compounds synthesized in step (d) for candidates that
modulate said activity.
26. A method according to claim 25, comprising the following step
after step (c) and before step (d): (c1) virtually screening on a
computer the compounds planned in step (b) to identify compounds
with desirable 3D structures and selecting these compounds, and
wherein step (d) comprises: synthesizing the selected compound of
step (c1).
27. A method according to claim 25 wherein said activity is
selected from: proliferation, differentiation, cellular shape
alteration, cellular elongation, uptake of substances by cells,
secretion of substances, cellular metabolism, and level of
expression of various proteins.
28. A method for obtaining compounds which modulate protein or
peptide-mediated cell activity, the method comprising: (1)
identifying the candidate compounds according to the method of
claim 25; (2) contacting the compounds identified in step (1) with
a test assay for determining a protein or peptide-mediated cell
activity; (3) collecting those molecules which modulate a protein
or peptide-mediated cell activity in a test assay as compared to
the modulation in the same test assay in the absence of said
compound, thereby obtaining modulators of said activity; and (4)
producing the compounds obtained in step (3).
29. A method according to claim 28, wherein said activity is
selected from proliferation, differentiation, cellular shape
alteration, cellular elongation, uptake of substances by cells,
secretion of substances, cellular metabolism, and expression of
various proteins.
30. Modulator of a protein or peptide-mediated cell activity
obtained by the method of claim 28.
31. A method for modulating a protein or peptide-mediated cell
activity, such method comprising contacting a cell whose activity
is to be modulated with a modulator obtained by the method of claim
28.
32. A compound of formula I, II, III or IV as defined in claim 5,
being linked either directly or through a linker to a marker for
imaging or to a drug.
33. A compound according to claim 32, linked to a marker for
imaging.
34. A compound according to claim 33 wherein the marker is for the
detection of fluorescence, X-ray, MRI or radio-isotope scan.
Description
FIELD OF THE INVENTION
[0001] This invention is in the field of using combinatorial
chemistry to develop new drugs.
LIST OF REFERENCES
[0002] The following references are considered to be pertinent for
the purpose of understanding the background of the present
invention:
[0003] Adang A. E. P. and Hermkens P. H. H., Curr. Med. Chem. 8:985
(2001);
[0004] Beeley N. R. A., Drug Disc. Today 5:354 (2000);
[0005] Bunin B. A. and Ellman J. A., J. Am. Chem. Soc. 114:11997
(1992);
[0006] Campian E. et al, Bioorg. Med. Chem Lett. 8;2357(1998);
[0007] Furka A. et al. Int. J. Pept. Protein. Res. 37:487-493
(1991);
[0008] Geysen H. M. et al. Proc. Natl. USA, 81: 3998 (1984);
[0009] Hougten R. A., Proc. Natl. USA, 82:5131 (1985);
[0010] Kumar S. et al. Prot. Sci. 9:10-19 (2000);
[0011] Lipinsky C. A. et al., Adv. Drug Deliv. Rev. 23, 3
(1997);
[0012] Lipinsky C. A., Chimia 52:503 (1998);
[0013] Morrison K. L. and Weiss G. A., Curr. Opin. Chem. Biol.
5:302-307 (2001);
[0014] Rink H., Tetrahedron Lett, 28: 3787 (1987);
[0015] Winter et al. Nature 299: 756 (1982);
[0016] The above references will be acknowledged in the text below
by indicating the author's name and year of publication (in
brackets) from the above list.
BACKGROUND OF THE INVENTION
[0017] An important objective of combinatorial chemistry is to
generate a large number of novel compounds that can be screened to
identify lead compounds for pharmaceutical research. Theoretically,
the total number of compounds which may be produced for a given
library is limited only by the number of reagents available to form
substituents on the variable positions on the library's molecular
scaffold.
[0018] The combinatorial process lends itself to automation, both
in the generation of compounds and in their biological screening,
thereby greatly enhancing the opportunity and efficiency of drug
discovery. Combinatorial chemistry may be performed in a manner
where libraries of compounds are generated as mixtures, while the
complete identification of the individual compounds is postponed
until after positive screening results are obtained. However, a
preferred form of combinatorial chemistry is "parallel array
synthesis", (also called Multiple Parallel Synthesis, MPS) where
individual reaction products are simultaneously synthesized, but
are retained in separate compartments. [Geysen et al. (1984);
Hougten (1985)]. For example, the individual library compounds can
be prepared, stored, and assayed in separate wells of a microtiter
plate, each well containing one member of the parallel array, The
use of standardized microtiter plates or equivalent apparatus, is
advantageous because such an apparatus is readily accessed by
programmed robotic machinery, both during library synthesis and
during library sampling or assaying.
[0019] Combinatoral chemistry can be carried out in solution phase
where both reactants are dissolved in solution or in solid phase
where one of the reactants is covalently bound to a solid support.
Solid phase synthesis offers the advantage that reactions can be
carried out using excess reagents, while the solid support-bound
products are easily washed free of excess reagent. The use of
excess reagents may ensure high yield of each step in a multiple
step synthesis. Solution phase synthesis typically requires use of
one or more reaction mixture work-up procedures to separate
reaction product from unreacted excess reagent.
[0020] The first combinatorial libraries were composed of peptides,
in which all or selected amino acid positions were randomized
[Geysen et al. (1984); Furka et al. (1991)].
[0021] Peptides and proteins can exhibit high and specific binding
activity, and can act as catalysts. In consequence, they are of
great importance in biological systems. Unfortunately, peptides per
se have limited utility for use as therapeutic entities. They are
costly to synthesize, unstable in the presence of proteases, non
selective and in general do not pass cellular membranes.
[0022] Nucleic acids have also been used in combinatorial
libraries. Their great advantage is the ease with which a nucleic
acid with appropriate binding activity can be amplified. As a
result, combinatorial libraries composed of nucleic acids can be of
low redundancy and hence, of high diversity. However, the resulting
oligonucleotides are not suitable as drugs for several reasons.
First, the oligonucleotides have high molecular weights and cannot
be synthesized conveniently in large quantities. Second, because
oligonucleotides are polyanions, they do not cross cell membranes.
Finally, deoxy- and ribo-nucleotides are hydrolytically digested by
nucleases that occur in all living systems and are therefore
usually decomposed before reaching the target.
[0023] There has therefore been much interest in combinatorial
libraries based on small molecules (i.e. molecules having molecular
weight of up to about 1000 daltons), which are more suited to
pharmaceutical use, especially those which, like benzodiazepines,
belong to a chemical class which has already yielded useful
pharmacological agents [Bunin and Ellman (1992); Beeley (2000)].
The techniques of combinatorial chemistry have been recognized as
the most efficient means for finding small molecules that act on
these targets. At present, small molecule combinatorial chemistry
involves the synthesis of either pooled or discrete molecules that
present varying arrays of functionality on a common scaffold. These
compounds are grouped in libraries that are then screened against
the target of interest either for binding or for inhibition of
biological activity [Adang and Hermkens (2001)].
[0024] The elements of diversity of libraries of currently
available scaffold based compounds having the general structure (A)
showed below, are based mainly on sequential or positional
diversity namely the order in which the various R groups are
arranged around the ring and chemical diversity that can arise from
alterations in the chemical nature of the R groups. 1
[0025] In the above structure (A), X, Y and Z represent ring
heteroatoms or carbons, and R', R" and R'" represent substituents
associated to the ring through a linker (showed schematically as a
wavy line).
[0026] It is known from the art [Kumar S. et al. (2000)] that
molecules may bind to each other if their conformations are
complementary in geometry and chemistry and if their binding
produces stable associations. However, most of the known libraries
of organic molecules suffer from a major drawback when applied for
the discovery of new drug leads based on the inhibition of peptide:
protein or protein: protein or protein: nucleic acid interactions:
they are too constrained and therefore lack the ability to undergo
conformational complementarity, i.e. lack an ability for binding to
a protein and/or a nucleic acid. This led to the preparations of
extremely large libraries (consist of up to millions of compounds)
and their biological screening, which in many cases results in the
discovery of low affinity leads or to the lack of their
discovery.
SUMMARY OF THE INVENTION
[0027] The present invention provides, according to a first of its
aspects, new compounds that have a relatively flexible scaffold
These compounds may be used to produce new combinatorial libraries
that will permit, e.g. in high throughput screening assays, to
screen for and select drug candidates for a variety of uses in
human medicine, veterinary medicine and in agriculture. The members
of each library according to the invention differ from each other
in the ring size, in addition to the conventional chemical and
positional diversity attained by the different substituents on the
scaffold, thus allowing the selection of the most active compound,
not only on the basis of the nature and proper arrangement of the
substituents (attained by the known chemical and positional
diversity), but also based on the ability to undergo conformational
complementarity (attained by the conformational diversity).
[0028] Thus, the present invention provides scaffold based
compounds having the following general formula (B): 2
[0029] including pharmaceutically acceptable salts, esters or
solvates thereof, wherein
[0030] Z is selected from C.dbd.O and --CH.sub.2--,
[0031] W is selected from C.dbd.O and a bond, provided that at
least one of Z and W is C.dbd.O,
[0032] X and Y are independently selected from CH.sub.2, O, S, NH,
N--R.sup.5, NH--CO, CO, CH.sub.2CO, S.dbd.O and SO.sub.2, or X and
Y may form together a group selected from CH.dbd.CH, CO--NR.sup.5,
NH--CO--NH, O--CH(R.sup.5)--O, NH--CH(R.sup.5)--O and
NH--CH(R.sup.5)--NH, where the hydrogen in the above groups may
optionally be substituted by an alkyl group;
[0033] (a) and (b) are parts of the scaffold and are nitrogen
containing bivalent organic radicals, each independently providing
between 1 to 4, preferably up to 2 atoms to said scaffold,
[0034] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently
selected from H and substituted or unsubstituted alkyl, and
[0035] n and m are integers from 1 to 6.
[0036] Preferably, the present invention provides scaffold based
compounds having the formula (B) above, wherein X and Y are
independently selected from CH.sub.2, O, S, NH, N--R.sup.5, NH--CO,
CO, CH.sub.2CO, S.dbd.O and SO.sub.2, or X and Y may form is
together a group selected from CO--NR.sup.5, NH--CO--NH,
O--CH(R.sup.5)--O, NH--CH(R.sup.5)--O and NH--CH(R.sup.5)--NH,
where the hydrogen in the above groups may optionally be
substituted by an alkyl group.
[0037] According to a preferred embodiment, each of parts (a) and
(b) is independently selected from --N(CHR.sup.6CO--L)--,
--C(CO--L)(R.sup.6)--, --N(COR.sup.8)--CHR.sup.7--, and
--C(NHR.sup.8)(R.sup.7)--.
[0038] According to another preferred embodiment, the present
invention provides heterocyclic compounds having the formula I, II,
III or IV: 3
[0039] including pharmaceutically acceptable salts, esters or
solvates thereof, wherein
[0040] X and Y are independently selected from CH.sub.2, O, S, NH,
N--R.sup.5, NH--CO, CO, CH.sub.2CO, S.dbd.O and SO.sub.2, or X and
Y may form together a group selected from CH.dbd.CH, CO--NR.sup.5,
NH--CO--NH, O--CH(R.sup.5)--O, NH--CH(R.sup.5)--O and
NH--CH(R.sup.5)--NH, where the hydrogen in the above groups may
optionally be substituted by an alkyl group;
[0041] R.sup.1, R.sup.2, R.sup.3and R.sup.4 are each independently
selected from H, and substituted or unsubstituted alkyl,
[0042] R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are each independently
selected from H, substituted or unsubstituted alkyl, cycloalkyl,
aryl, aralsyl, hetroaryl, heterocyclyl, hetroaryl, acyl,
carboxyaryl, carboxyalkyl, side chains of naturally and
artificially occurring amino acids as well as derivatives and
mimics of such side chains, and linear or cyclic peptide;
[0043] L is selected from H, OH, NH.sub.2, NHR.sup.5, a peptide and
a solid support where R.sup.5 is as defined above, and
[0044] n and m are integers from 1 to 6, with the exclusion of the
following compound: glycinamide,
L-tyrosyl-N-[2-[(2S)-4-[(1S)-1-carboxy-3-
-methylbutyl]-3,4,5,8-tetrahydro-3-oxo-2-(phenymethyl)-1,4-diazocin-1(2H)--
yl]2-oxoethyl.
[0045] According to another preferred embodiment, in the above
compounds of formulae I, II, III and IV, X and Y may form together
a group selected from CO--NR.sup.5, NH--CO--NH, O--CH(R.sup.5)--O,
NH--CH(R.sup.5)--O and NH--CH(R.sup.5)--NH, where the hydrogen in
the above groups may be substituted by an alkyl group.
[0046] The following combinations for X and Y are preferred: both X
and Y are S; or X is NH and Y is CO; or X is CO and Y is NH; or X
is NH and Y is CH.sub.2; or X is CH.sub.2 and Y is NH; or X and Y
are NH--CO--NH or X is N--R.sup.4 and Y is CO; or (vi) X is CO and
Y is N--R.sup.4.
[0047] The invention also provides, according to another of its
aspects, a combinatorial library comprising two or more, preferably
a plurality, of compounds of any one of the formulae (B), I, II,
III or IV. The library of the invention serves as a readily
accessible source of diverse macrocyclic compounds for use in
identifying new biologically active macrocyclic compounds through
pharmaceutical and veterinary candidate screening assays, for the
development of highly effective and environmentally friendly insect
control and crop control agents, for use in studies defining
structure/activity relationships, and/or for use in clinical
investigation.
[0048] The selection of an active candidate is preferably achieved
from a library of compounds that have the same substituents in the
same positions along the scaffold but the scaffolds differ from
each other in size and chirality of the substituents and therefore
in their conformation. The libraries are prepared by the multiple
simultaneous solid phase method [Hougten R. A.,1985] or its
automated version, and contain the calculated number of diversity
possibilities. Libraries are typically synthesized in a 12-48
format, namely each library typically contains 12-48 members. Each
member of the library will be characterized, purified and subjected
to biological assay. The present invention also provides a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and an effective amount of a compound of the formula I, II,
III or IV, as defined above. The new compounds of the invention may
act as modulators of the activity of cells which activity is
mediated by proteins or peptides as will be explained below.
[0049] The term "effective amount" refers to an amount capable of
decreasing, to a measurable effect, at least one adverse
manifestation of the disease and should be chosen in accordance
with the drug used, the mode of administrations the age and weight
of the patient, the severity of the disease, etc.
[0050] Also provided by the invention is use of a compound of the
formula I, II, III or IV, as defined above, for the preparation of
a pharmaceutical composition.
[0051] In addition, the present invention provides a method for
modulating a protein or peptide-mediated cell activity, such method
comprising contacting a compound of the formula I, II, III or IV,
as defined above, with a cell or cellular component having said
peptide or protein. The cell may be from an eukaryotic or
prokaryotic organism, from a uni- or multi-cellular organism and
may be from plant, bacteria or animal. The cellular component is
selected from cellular organells such as nucleous, ribosomes,
mitochondria and cell membranes or from cellular molecules such as
receptors, enzymes, substrates, ligands and the like.
[0052] Examples of peptide or protein-mediated cell activities
which are modulated by the compounds of the invention are:
proliferation, differentiation, cellular shape alteration, cellular
elongation, uptake of substances by cells (glucose,
neurotransmitters), secretion of substances, cellular metabolism,
expression of various proteins.
[0053] Also provided by the present invention, a method for the
treatment of a disease, disorder or condition wherein a
therapeutically beneficial effect may be evident by the modulation
of a protein or peptide-mediated cell activity, the method
comprising: administering to a subject in need of such treatment a
therapeutically effective amount of a compound of formula I, II,
III or IV.
[0054] In accordance with another embodiment of the invention, the
compound of the invention may be bound to a detectable label such
as a fluorescence-emitting moiety, a radio-label, a label capable
of undergoing an enzymatic reaction producing a detectable color, a
marker for x-ray, MRI, radio-isotope imaging or PET scan, to
produce a labeled adduct. Then, upon administration of such labeled
adduct, it may be detected at a desired location by any manner
known in the art and in accordance with the specific label used,
for example, fluorescence, radioactive emission, or a color
production, MRI, x-ray and the like.
[0055] The term "bound" refers to covalent or non-covalent (e.g.,
electrostatic) binding, which connects the compound of the
invention to the detectable label. Alternatively, the compound of
the invention may have inherent detectable properties of its own,
that enable it to be detected by any of the above mentioned
techniques.
[0056] The present invention is also directed, according to a
further aspect thereof, to a method for designing new compound
libraries that have novel type of structural complexity and
diversity, and can be screened to identify potent modulators of
protein or peptide-mediated cell activity, so as to develop lead
compounds for pharmaceutical, veterinary or agricultural research.
Molecules having a molecular weight of up to about 1000 daltons,
i.e. small molecules, are preferable.
[0057] More specifically, the present invention provides a method
of identifying a candidate, which modulates a protein or
peptide-mediated cell activity, the method comprising:
[0058] (a) identifying in said protein or peptide, a domain which
is essential for said activity,
[0059] (b) identifying in said domain, pharmacophors essential for
the activity,
[0060] (c) planning a combinatorial library of cyclic compounds
having the formula I, II, III or IV, as defined above, wherein each
such compound comprises at least two of the pharmacophors
identified in step (b) above or mimics or derivatives of the
pharmacophors, where each member of the library differs from the
other by at least one of the following: i) the size of the ring;
ii) the order in which the pharmacophors are arranged in the ring;
iii) the chemical nature of the ring; iv) the chemical nature of
the pharmacophors; v) the chirality of the linker between the ring
and the pharmacophor; and vi) the chirality of the
pharmacophor,
[0061] (d) synthesizing a plurality of compounds, such compounds
being among the compounds of the combinatorial library planned in
step (c);
[0062] (e) screening the synthesized compounds of step (d) for
candidates that modulate said activity.
[0063] The term pharmacophor refers to the ensemble of steric and
electronic features that is necessary to ensure the optimal
molecular interactions with a specific biological target structure
and to trigger (or to block) its biological response. In the
present invention the term refers to those moieties of the side
chain or backbone of the peptide or protein (which mediates the
cell activity), that are necessary for the binding to the other
cellular components, the binding eliciting a biological response.
The pharmacophor may be a chemical moiety present on a single side
chain or a collection, of chemical moieties present in spatially
adjacent side chains.
[0064] The above method may also be used in order to identify a
compound which modulates a protein or peptide-mediated cell
activity. In such case the method comprises the following
additional steps after step (e):
[0065] (f) collecting those compounds which modulate said activity
in a test assay as compared to the modulation in the same test
assay in the absence of said compound, thereby obtaining modulators
of a protein or peptide-mediated cell activity; and
[0066] (g) producing the compounds selected in step (f) above.
[0067] The present invention further provides a compound which
modulates a protein or peptide-mediated cell activity obtained by
the above method.
[0068] The present invention may also be utilized in agriculture.
Therefore, the present invention also provides a method for the
discovery of new agents for use in agriculture, wherein such agents
are based on modulators of proteins or peptides derived from
insects or plants.
GLOSSARY
[0069] Definitions
[0070] A "library" is a collection of compounds which while sharing
some common structural elements (which may be common scaffolds,
common ring sizes, common substituents and the like), are diverse
from each other by at least one of the following properties: i) the
size of the ring; ii) the order in which the pharmacophors are
arranged in the ring; iii) the chemical nature of the ring; iv) the
chemical nature of the pharmacophors; v) the chirality of the
linker between the ring and the pharmacophor; and vi) the chirality
of the pharmacophor. The library allows screening from among a
plurality of compounds for those that have a desired property. The
library may be designed by a combinatorial or classical chemical
process.
[0071] A "lead compound" is a library compound in a selected
combinatorial library, for which the assay has revealed significant
effect relevant to a desired cell activity to be modulated. In the
present case the property is the modulator of at least one peptide
or protein-mediated activity.
[0072] "Peptide or protein-mediated cell activity" refers to a
physiological property of a cell that is caused, directly or
indirectly (the latter referring to an effect caused by an,
effector which is more downstream in the pathway) by the
interaction between a protein or peptide and another cellular
component (the term "cellular component" including: other proteins
or peptides of the same or different types, membranes, nucleic
acids, lipoproteins, nucleotides, co-factors, hormones, ion
effectors and the like). The interaction between the protein and
the other cellular component may be of the type: receptor-ligand,
enzyme-substrate, DNA-binding proteins-DNA etc. Said interaction
mediates (causes) directly or indirectly a cell activity such as:
expression of a protein, proliferation, differentiation,
cell-elongation, cell-shape alteration, cellular metabolism,
cellular update of external substances, secretion of substances
from the cells and the like.
[0073] "Modulate/Modulator" refers to increase or decrease in at
least one peptide or protein-mediated cell activity, in the
presence of the compound of the invention, or to the change of the
response of the cell to the presence of a physiological cue, as
compared to the activity or response, respectively, in the absence
of the compound. Examples of such physiological cues are presence
of effectors, the modulation being a change in the cellular
response to a ligand, hormone, response to toxic substances
(pesticides), stress (heat shock, draught, lack of nutrients) aging
and the like.
[0074] The term "substituents" refers to chemical radicals which
are bonded to or incorporated onto the scaffold through the
synthesis process of the library. The different functional groups
account for the diversity of the molecules throughout the library
and are selected to impart diversity of structure, function and
biological activity to the scaffold in the case of diverse
libraries, and optimization of a particular biological activity in
the case of directed libraries.
[0075] "Aryl" means one or more aromatic rings, each of 5 or 6 ring
carbon atoms and includes substituted aryl having one or more
non-interfering substituents. Multiple aryl rings may be fused, as
in naphthyl, or unfused, as in biphenyl.
[0076] "Alkyl" means straight or branched chain or cyclic
hydrocarbon having 1 to 10 carbon atoms.
[0077] "Substituted alkyl", is alkyl having one or more
non-interfering substituents.
[0078] "Halo" means chloro, fluoro, iodo or bromo.
[0079] "Heterocycle" or "heterocyclic" means one or more rings of
5, 6 or 7 atoms with or without unsaturation or aromatic character,
optionally substituted with one or more non-interfering
substituents, and at least one ring atom which is not carbon.
Preferred heteroatoms include sulfur, oxygen, and nitrogen.
Multiple rings may be fused, as in quinoline or benzofuran, or
unfused as in 4-phenylpyridine. Suitable substituents on the
heterocyclic ring structure include, but are not limited to halo,
C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C7-C12
aralkyl, C7-C12 alkaryl, C1-C10 alkylthio, arylthio, aryloxy,
arylamino, C3-C10 cycloalkyl, C3-C10 cycloalkenyl,
di(C1-C10)-alkylamino, C2-C12 alkoxyalkyl, C1-C6 alkylsulfinyl,
C1-C10 alkylsulfonyl, arylsulfonyl,aryl, hydroxy,
hydroxy(C1-C10)alkyl, aryloxy(C1-C10)alkyl, C1-C10 alkoxycarbonyl,
aryloxycarbonyl, aryloyloxy, substituted alkoxy, fluoroalkyl,
nitro, cyano, cyano(C1-C10)alkyl, C1-C10 alkanamido, aryloylamido,
arylaminosulfonyl, sulfonamido, amidino, carbamido, carboxy,
heterocyclic radical, nitroalkyl, and --(CH.sub.2).sub.m--Z--(C1-
-C10 alkyl), where m is 1 to 8 and z is oxygen or sulfur.
[0080] The term "solid support" refers to a solvent insoluble
material having cleavable covalent bonds for use in preparing the
library compounds of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0081] Many biological processes are critically dependent on
protein:protein, protein:peptide and protein:nucleic acid
interactions, and many drugs are small molecules known to disrupt
such interactions (antagonists) or alternatively mimic one
component of the interaction in such a manner so that the activity
controlled by the interaction can take place in the presence of the
drug and the other cellular component (agonist).
[0082] The drugs which work by interruption of such interactions
(for example by the interruption of a receptor-ligand interaction)
mimic in fact a domain of one of the proteins participating in the
interaction. By one option this mimic creates an antagonist that
competes with the protein for binding with the other member of the
interaction (the other cellular component), leading to decrease in
the interaction and hence decrease of the cell activity controlled
or caused (directly or indirectly) by the interaction. Where the
cell activity is an "on" physiological process, (for example,
increase in production of an agent), the interruption will close
the "on" reaction and decrease the physiological process (decrease
production of the agent). Where the cell activity is an "off"
reaction (for example a signal causing inhibition of proliferation)
th interruption will close the "off" reaction and will increase the
physiological process, for example, cause increased
proliferation.
[0083] By another option the drug may work as an agonist and cause
the modulation of the cell activity by mimicking, the protein (that
is essential for the cell activity) in the interaction in such a
manner that the cell activity takes place as if the native protein
and not the compound were interaction with the other cellular
component. For example the compound may be able to activate the
cellular component with which the protein interacts in a similar
way to the protein itself.
[0084] The aim of the compounds of the present invention is to
mimic a region in one of the participators of the interaction, so
as to compete for the binding on the other participator of the
interaction (either in the antagonist or the agonist manner) thus
changing the interaction and leading to a change in the
physiological process or property controlled by the
interaction.
[0085] The rational for the present invention is the following:
many libraries used for the discovery of drug leads are composed of
heterocyclic scaffolds that are too constrained (rigid) to allow
conformational complementarity essential for the interactions with
proteins, peptides, polysaccharides or nucleic acids. The
combinatorial library of the invention allows the generation of
sub-libraries with spatial diversity, which is obtained by the
diversity in ring size and chirality of the link between the
substituent and the scaffold. This results in a library where each
individual scaffold has a different flexibility ad a different
spatial positioning of the pharmacophor. The design of the library
of the invention increases the probability that some members of the
library have the ability to undergo conformational complementarily,
i.e. the pharmacophors are present in the correct orientation to
interrupt or mimic the interaction with the other cellular
component. The present invention allows the design and synthesis of
libraries which occupy a larger proportion of the "probability
space" of the pharmacophor positioning (i.e. increase the
probability of the substituents to be present in varying positions
in the space, thus increasing the probability that at least one
positioning-conformation is the bioactive conformation,) while
still creating relatively small, focused libraries. These
properties lead to fast discovery and optimization of novel drug
leads.
[0086] The classical elements of diversity of state-of-the-art,
currently available macrocyclic, i.e. scaffold based libraries are
based mainly on:
[0087] (i)The chemical nature of the scaffold;
[0088] (2)The size and chemical nature of the linkers that connect
between the scaffold and the various substituents;
[0089] (3)The chemical nature of the substituents;
[0090] (4) The order in which the substituents are arranged on the
scaffold.
[0091] The libraries of the new compounds of the present invention
comprise a novel element of diversity, namely spatial diversity,
that results from the varying size of the scaffold ring, the
chirality of the linker or from a combination of the two. This
diversity is new in the field of small molecule combinatorial
chemistry.
[0092] Spatial diversity is defined as diversity elements that
alter the conformation of the compounds, which in fact lead to
altered spatial positioning of the phamacophors. The present
invention deals with libraries of compounds having new elements of
diversity, namely spatial diversity elements: the chirality of the
linkers (which effects the spatial arrangement of the substituents)
and the size of the ring. The members of the library of the
invention may also differ from each other in the classical elements
of diversity (1)-(4) mentioned above.
[0093] According to a specific embodiment of the present invention,
all the members in a library have the same elements of diversity
(1)-(4) mentioned above: the same scaffold with the same
composition and order of atoms within. the scaffold; the same
linker with the same size and chemical nature; the same
substituents/pharmacophors arranged in the same order on the
scaffold, but they differ from each other in the size of the
scaffold and/or the chirality of the linkers. This in turn
determines the possible conformational (spatial) positioning of the
pharmacophor of each compound and allows for the selection of the
lead compound having the appropriate ability of conformational
complementarity. The libraries of the invention are composed of a
series of compounds that differ from each other by an incremental
alteration of their possible conformations. Thus, every library
covers an entire range of the conformational probabilities and
increases the chances of obtaining a compound with at least one
bioactive conformation.
[0094] Design of the libraries
[0095] The method of the present invention, for identifying a
candidate which modulates a protein or peptide-mediated cell
activity, comprises the following steps:
[0096] (a) identifying in said protein or peptide, a domain
essential for said activity,
[0097] (b) identifying in said domain, pharmacophors essential for
the activity,
[0098] c) planning a combinatorial library of cyclic compounds
having the formula I, II, III or IV as defined above, wherein each
such compound comprises at least two of the pharmacophors
identified in step (b) above or mimics or derivatives of the
pharmacophors, where each member of the library differs from the
other by at least one of the following: i) the size of the ring;
ii) the order in which the pharmacophors are arranged in the ring;
iii) the chemical nature of the ring; iv) the chemical nature of
the pharmacophors; v) the chirality of the linker between the ring
and the pharmacophor; and vi) the chirality of the
pharmacophor;
[0099] (d) synthesizing a plurality of compounds from the
combinatorial library planned in step (c);
[0100] (e) screening the compounds synthesized in step (d) for
candidates that modulate said activity.
[0101] Between the step (c) of planning of the fill library and
step (d) of synthesizing a plurality of compounds from the library
(which may form together the full library or a part of the
library), it is possible to add a step of virtually screening the
library to identify those compounds which are better mimics of the
domain than others. Such virtual screening can help and predict
which compounds have a better chance of assuming the bioactive
conformation and it is preferable to start the screening with the
compounds that are, according to 3D modeling the most likely
mimics.
[0102] The above steps are showed schematically in the following
chart: 4
[0103] As the compounds of the invention intend to mimic a domain
in a protein, so as to interrupt or to mimic its interaction with
other cellular components and thus modulate the cell activity
(mediated by the protein), it is desired that they resemble the
desired domain and the positioning of the pharmacophors in the
domain of the protein (which pharmacophors they mimic) as close as
possible. Therefore, when deciding at the library planning step how
to produce the best library, and at the synthesizing step, which of
the members of the planned library should be synthesized, the
following questions should be asked:
[0104] 1. Are the relevant pharmacophors (or derivatives or mimics
of the pharmacophors) present in the planned library?
[0105] 2. Is the order of the pharmacophors on the scaffold and the
distance of the pharmacophores from each other, suitable for
achieving a suitable bioactive conformation (correct positioning of
the
[0106] 3. Is the compound capable in one of its conformation of
attaining the correct positioning of the pharmacophors?
[0107] 4. Is the possible conformation energetically favorable?
[0108] 5. Is there a certain degree of conformation flexibility to
allow conformational complementarity?
[0109] Most of the above questions can be answered during the
planning stage and the synthesis decision stage on a computer using
commercially available bioinformatic programs such as
Tripose.TM..
[0110] The coordinates of amino acid side chains of a protein can
be obtained from the Protein Data Bank (PDB) files. This data is
based on the 3D structure of the protein (preferably as a complex
with the appropriate ligand) either obtained by crystallography or
homology modeling. The 3D information allows to identify the
exposed side chains and these accesible side chains are possible
pharmacophors. In cases of proteins for which the 3D structure was
not determined, the essential amino acids within a protein may be
determined by the method of combinatorial alanine-scanning
(Morrison and Weiss, (2001) Curr. Opin. Chem. Biol. 5, 302-307),
also known as Ala-Scan. Another method is known as omission
libraries and is described in Campian et al. (1998) Bioorg. Med.
Chem. Lett. 8, 2357. Yet other methods are site directed
mutagenesis and protein engineering (Winter et al (1982)). The
amino acids and backbone elements essential for a certain function
may be divided into two categories: those who interact with the
receptive protein, nucleic acid, polysaccharide or cell membrane
and those responsible for the conformation of the essential region.
The side chains and backbone elements of the former are those that
participate in the creation of pharmacophors and the present
invention relates to the creation of such pharmacophors, or their
mimics and their incorporation in the scaffolds of the invention
for the purpose of creating a mimic of a region of the protein.
[0111] As mentioned above, the essential amino acids within a
protein may be determined by the method of combinatorial
alanine-scanning. Alanine scanning, a method of systematic and
sequential alanine substitution, has been particularly useful for
the identification of pharmacophors in a given peptide sequence.
This method is based on the synthesis of a library in which each
amino acid residue in a peptide chain is sequentially replaced by
alanine, and biological screening of the library. Substitution of
functional amino acid residues by the methyl group of alanine leads
to the removal of all the side chain atoms past the .beta.-carbon.
Thus, the role of side-chain functional groups at specific position
can be inferred. Alanine residue have the same backbone dihedral
angles as other functional residues and thus the backbone
conformation is not drastically perturbed by such substitution, as
would be the case in glycine scan libraries. In this case, the side
chain is nullified, which leads to the introduction of flexibility
into the peptide backbone.
[0112] An additional method for the elucidation of pharmacophors is
the synthesis and biological screening of omission libraries.
Omission libraries, based on a given peptide sequence is a library
that contain all the possible peptides that compose the parent
peptide. Omission library is divided into sequential and non
sequential. In, sequential omission library, amino acids are
omitted from the carboxy- and amino- ends, whereas in non
sequential library amino acids are omitted from the interior of the
peptide sequence. Thus, sequential omission library based on a
hexapeptide contains 2 pentapeptides, 3 tetrapeptides, 4
tripeptides and 5 dipeptides (total of 14 peptides). Non-sequential
omission library based on a hexapeptide contains 4 pentapeptides,
18 tetrapeptides, 27 tripeptides and 9 dipeptides (total of 58
peptides). Beside information on essential pharmacophors, omission
libraries can finish shorter active peptides that will facilitate
the design, of libraries.
[0113] Once the pharmacophors are determined they are incorporated
into the new compounds of the invention as one of the R
substituents, to produce combinatorial libraries that will permit,
alter undergoing screening with suitable assays to screen for and
to select candidates for a variety of uses in human medicine,
veterinary medicine and in agriculture. Each compound may bind at
least two pharmacophors, usually at least three pharmacophors and
typically up to six, preferably up to five pharmacophors.
Preferably the compound contain three to five such pharmacophors,
attached to the scaffold either directly or through a linker. At
times, when more then four pharmacophors have to be linked to a
molecule, this can be achieved through binding the additional
fifth, sixth etc. pharmacophors to the pharmacophors that are
already connected to the scaffold.
[0114] In conclusion, the method of the invention utilizes spatial
libraries that can generate novel leads for the disruption of
protein:protein, protein:peptide, protein:cell membrane and
protein:nucleic acid interactions, in animals and plants.
[0115] The pharmaceutical composition of the invention may be
administered by any of the known administration routes, inter alga,
oral, intravenous, intraperitoneal, intramuscular, subcutaneous,
sublingual, intraocular, intranasal or topical administration
routes. Appropriate unit dosage forms of a stration include the
forms for oral administration, such as tablets, capsules, powders,
granulates and oral solutions or suspensions and the forms for
sublingual and buccal administration, the forms for parenteral
administration useful for a subcutaneous, intramuscular or
intravenous injection, as well as the forms for rectal
administration.
[0116] The carrier should be selected in accordance with the
desired mode of administration and include any known components,
e.g. solvents; emulgators, excipients, talc; flavors; colors, etc.
The pharmaceutical composition may comprise, if desired, also other
pharmaceutically-active compounds which are used to treat the
disease, eliminate side effects or augment the activity of the
active component.
[0117] In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are administered
orally, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening and/or flavoring
and/or coloring agents may be added.
[0118] Typically, the pharmaceutical compositions of this invention
will be administered from about 1 to about 5 times per day or
alternatively, as a continuous infusion. A typical preparation will
contain from about 5% to about 95% active compound (w1w).
Preferably, such preparations contain from about 20% to about 80%
active compound. As the skilled artisan will appreciate, lower or
higher doses than those recited above may be required. Specific
dosage and treatment regimens for any particular patient will
depend upon a variety of factors, including the activity of the
specific compound employed, the age, body weight, general health
status, sex, diet, time of administration, rate of excretion, drug
combination, the patients disposition to the disease state and the
judgment of the treating physician. In general, the compound is
most desirably administered at a concentration level that will
generally afford effective results without causing any harmful or
deleterious side effects.
[0119] The pharmaceutical composition may comprise, if desired,
also other pharmaceutically-active compounds which are used to
treat the disease, eliminate side effects or augment the activity
of the active component.
[0120] Synthetic Approach
[0121] In general, the compounds of the invention are prepared
according to the routes showed in Schemes 1-4 below. The solid
phase synthesis of scaffolds I-IV comprise of a series of couplings
of the appropriate protected acids and reductive alkylations with
.omega.-functionalized protected aldehydes. The assembly of the
appropriate linear scaffold on the solid support is followed by
removal of the protecting groups P.sub.1 and P.sub.2 and
cyclization. The appropriate scaffold is obtained after
deprotection-removal from the solid support. 5 6 7 8
EXAMPLES
[0122] Synthesis
[0123] A library composed of 26 molecules that have the formula I
and wherein R.sup.1 and R.sup.2 are either benzyl (side chain of
phenylalanine) or hydroxybenzyl (side chain of tyrosine) and
R.sup.3 is benzyloxycarbonyl (which is a mimic of the side chain of
phenylalanine) was synthesized and characterized. The library was
synthesized by the Simultaneous Multiple Solid Phase methodology
(Houghten (1985) Proc. Natl. Acad. Sci. USA, 82 5131) as showed in
Scheme 5 below. The molecules were characterized by HPLC, MS and
MS-MS spectrometry. 910
[0124] Synthetic Procedures According to Scheme 5 Above:
[0125] Rink amide MBHA resin [Rink H. (1987)] (0.1 g in each bag,
0.6 mmol/g) was preswollen for 2 h in NMP while shaking in reaction
vessel equipped with sintered glass bottom. The Fmoc protecting
group was removed from the resin by reaction with 20% piperidine in
NMP (2.times.30 min). Fmoc removal was monitored by chloranil test.
A coupling cycle was carried out with Fmoc-AA (AA is abbreviation
of amino acid) (5 eq), BTC (1.65 eq), and 2,4,6 colidine (14 eq) in
DCM for 2 h at room temperature. Reaction completion was monitored
by qualitative chloranil test. Following coupling the
peptidyl-resin was washed with DCM (.times.5) and for 2 mm. Fmoc
removal and washing steps were carried out as described above. Fmoc
removal was monitored by the chloranil test.
[0126] The peptidyl-resin was then washed by a mixture of NMP: MeOH
1:1/1% and a solution of the aldehyde (1 eq) in the mixture above
was added (10 ml for 12 bags). Then additional 40 ml of this
mixture was added and the mixture was shaken for 5 min. Taken, 2 eq
of NaBH.sub.3CN were added and the reaction vessel was shaken for 2
h. The resin was washed as follows: DCM (2.times.2 min), EtOH
(2.times.2 min), NW (2.times.2 min), DCM (3.times.2 min).
(Chloranil test gave blue color immediately). The following
coupling was performed using Fmoc-AA (5 eq), BTC (1.65 eq) and 2,
4, 6 colidine (14 eq) in dibromomethane at 50.div.C. for 2 h and
was repeated when necessary. Fmoc detection and washing steps were
carried out as described above. Reductive alkylation and washing
steps were carried out as described above. A solution of
benzylcholorformate (6 eq), and DIEA (12 eq) in DMF was added to
the resin and the mixture was shaken for 1 h. The reaction was
repeated and ten the resin was washed with NMP (5.times.2 min) and
DCM (2.times.2 min). Reaction completion was monitored by chloranil
test.
[0127] Disulfide Bridge Formation
[0128] The disulfide bridge was oxidized using iodine (10 eq) in
DCM and shaking for 3 h. The resin was washed as follows: DMF
(2.times.2 min), 2% ascorbic acid in DMF (2.times.2 min), NMP
(5.times.2 min), DCM (4.times.2 min).
[0129] Analytical Procedures
[0130] All the crude compounds were analyzed by MS and analytical
reversed-phase HPLC (RP18 Vydak 4.times.250 mm; flow: 1 mL/min.;
T=30.degree. C.; detection UV 214 nm; gradient: A=0.1% TFA in TDW,
B=0.1% TFA in CH.sub.3CN, 0 min 95:5, 5 min 95:5, 33 min 5:95, 38
min 95:5, 42 min 95:5).
[0131] The molecules were purified by preparative reversed-phase
HPLC (RP18 Vydak. 2.5.times.250 mm; flow: 9 mL/min.; T=30.degree.
C.; detection UV 214 nm; gradient: A=0.1% TFA in TDW, B=0.1% TFA in
CH.sub.3CN, 0 min 95:5, 5 min 95:5, 33 min 5:95, 38 min 95:5, 42
min. 95:5).
[0132] Fractions were collected, lyophilized and characterized by
analytical HPLC and MS analysis. Results are shown. in Table 1
below.
[0133] Synthesis of Aldeydes
[0134] Trityl-thiopropanal
[0135] a) Trityl-thiopropanoic Acid
[0136] Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to
a suspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 80 mL
DMF under cooling and nitrogen atmosphere, the reaction mixture was
stirred minutes after the is addition was completed. Then, a
solution of bromopropionic acid (20 g, 0.131 mol) dissolved in 50
mL DMF was added stepwise. After the addition was completed the
reaction mixture was stirred for 30 minutes and then cooling and
nitrogen atmosphere were stopped and the reaction mixture was
sealed and left overnight. Then, 500 mL chloroform were added and
the mixture was washed with 4.times.200 mL of saturated solution of
KHSO.sub.4 and 4.times.200 mL TDW (the solid that precipitate
during the washings should also be collected with the organic
layer). The organic layer was evaporated and the product (that
contained DMF traces) was precipitated by adding 300 mL DW and
stirring for few minutes. The product was collected by filtration
and dried by suction and then in vaccuo. The crude product was
purified as follows: 150 mL of CHCl.sub.3 were added to the white
solid and the mixture was stirred for few minutes. Then 200 mL of
PE 40-60 were added and the solid was collected by filtration
yielding 37.61 g (82% yield) of white powder, mp 177-183.degree.
C., .sup.1H NMR (CDCl.sub.3, 300 MHz, 298K) .delta.2.24 (t, 2H),
2.46 (t, 2H), 7.18-7.48 (m, 15H). MS (ES) m/z 347.
[0137] b) Trityl-thiopropanoic Acid Hydroxamate
[0138] A solution of N,O dimethylhydroxylamine hydrochloride (2.188
g, 0.0225 mol) in 40 mL DMF was added to a mixture of 6.96 g (0.02
mol) of Trityl-thiopropanoic acid and PyBoP (11.45 g, 0.022 mol).
DIEA (10.4 mL, 0.06 mol) was added and the clear solution was
stirred for 3 hours. EA (120 mL) was added to the stirred solution
followed by 240 mL of saturated bicarbonate solution. The organic
layer was collected and washed with additional two portions of 100
mL of saturated bicarbonate solution, 100 mL of TDW, 2.times.100 mL
KHSO.sub.4 1M, and 100 mL TDW, dried over Na.sub.2SO.sub.4 and
evaporated to dryness yielding 10.36 g (92% yield) of yellow oil.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.2.38 (t, 2H), 2.51 (t,
2H), 3.10 (s, 3H), 3.56 (s, 3H), 7.15-7.50 (m, 15H).
[0139] c) Trityl-thiopropanal
[0140] LiAlH.sub.4 (2.014 g, 0.053 mol) was added in portions to a
solution of 10.36 g (0.0265 mol) of Trityl-thiopropanoic acid
hydroxamate in 260 mL dry diethyl ether under cooling in ice bath
and argon atmosphere. The reaction mixture was stirred for 2 hours
(monitored by TLC PE:EA=1:1). 560 mL of EA were added followed by
addition of 560 mL of KHSO.sub.4 1M. The mixture was stirred for
additional 30 minutes. The organic layer was collected and washed
with 390 mL of KHSO.sub.4 1M and 390 mL of saturated NaCl, dried
over Na.sub.2SO.sub.4 and evaporated yielding 7.68 g (87% yield) of
white solid. .sup.1H NMR (CDCl.sub.3, 300 MHz, 298K) .delta.2.36
(t, 2H), 2.46 (t, 2H), 7.15-7.50 (m, 15H), 9.55 (t, 1H).
[0141] Trityl thiobutyral
[0142] a) Trityl-thiobutyric Acid;
[0143] Trityl mercaptar (36.13 g, 0.131 mol) was added stepwise to
a suspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100
mL DWM under cooling and nitrogen atmosphere, the reaction mixture
was stirred minutes after the addition was completed. Then, a
solution of bromobutyric acid (21.88 g, 0.131 mol) dissolved in 150
mL DMF was added stepwise. After the addition was completed the
reaction mixture was stirred for minutes and then cooling and
nitrogen atmosphere were stopped and the reaction mixture was
sealed and left overnight. Then, 500 mL chloroform were added and
the mixture was washed with 4.times.200 mL of saturated solution of
KHSO.sub.4 and 4.times.300 mL of water (the solid that precipitate
during the washings should also be collected with the organic
layer). The organic layer was evaporated and the oily product (that
contained DMF traces) was triturated by adding 300 mL TDW and sting
vigorously for few minutes. The product was collected by
filtration, washed by TDW and dried by suction. The crude product
was purified as follows: 200 mL of PE was added to the white solid
and the mixture was stirred for 15 minutes. The solid was collected
by filtration and dried in vaccuo yielding 36.82 g (77.6% yield) of
white powder. .sup.1H NMR (CDCl.sub.3, 300 MHz, 298K) .delta.1.67
(m, 2H), 2.22 (t, 2H), 2.30 (t, 2H), 7.10-7.50 (m, 15H).
[0144] b) Trityl-thiobutyric Acid Hydroxamate
[0145] A solution of N,O dimethylhydroxylamine hydrochloride (0.83
g 0.0084 mol) in 20 mL DMF was added to a mixture of 2.77 g (0.0076
mol) of Trityl-thiobutyric acid and PyBoP (4.39 g, 0.0084 mol).
DIEA (4 mL, 0.023 mol) was added and the clear solution was stirred
for 3 hours (pH should be monitored and kept basic). EA (50 mL) was
added to the stirred solution followed by 90 mL of saturated
bicarbonate solution. The organic layer was collected and washed
with additional two portions of 40 mL of sated bicarbonate
solution, 40 mL of water, 2.times.40 mL KHSO.sub.41M, and 40 mL
water, dried over Na.sub.2SO.sub.4 and evaporated to dryness
yielding 3.06 g (quantitative yield) of yellow oil. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta.1.74 (s, 2H), 2.23 (t, 2H), 2.38 (t,
2H), 3.13 (s, 3H), 3.63 (s, 3H), 7.10-7.50 (m, 15H).
[0146] c) Trityl-thiobutanal
[0147] LiAlH.sub.4 (0.574 g, 0.0151 mol) was added in portions to a
solution of 3.06 g (0.0075 mol) of the hydroxamate in 100 mL dry
diethyl ether under cooling in ice bath and argon atmosphere. The
reaction mixture was stirred for 1 hour (monitored by TLC
PE:EA=1:1). 150 mL of EA were added followed by addition of 150 mL
of KHSO.sub.4 1M. The mixture was stirred for additional 30
minutes. The organic layer was collected and washed with 100 mL of
KHSO.sub.4 1M and 100 mL of saturated NaCl, dried over
Na.sub.2SO.sub.4 and evaporated yielding 1.90 g (73% yield) of
white solid. .sup.1H NMR (CDCl.sub.3, 300 MHz, 298K) .delta.1.66
(m, 2H), 2.22 (t, 2H), 2.38 (t, 2H) 7.15-7.50 (m, 15H), 9.61
(1H).
[0148] Trityl Thiovaleric Aldehyde
[0149] a) Trityl-thiovaleric Acid
[0150] Trityl mercapta (36.13 g, 0.131 mol) was added stepwise to a
suspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100 mL
DMF under cooling and nitrogen atmosphere, the reaction mixture was
stirred minutes after the addition was completed. Then, a solution
of bromovaleric acid (23.71 g, 0.131 mol) dissolved in 150 mL DW
was added stepwise. After the addition was completed the reaction
mixture was stirred for 30 minutes and then cooling and nitrogen
atmosphere were stopped and the reaction mixture was sealed and
left overnight. Then, 500 mL chloroform were added and the mixture
was washed with 4.times.200 mL of saturated solution of KHSO.sub.4
and 4.times.300 mL of water (the solid that precipitate during the
washings should also be collected with the organic layer). The
organic layer was evaporated resulting in a solid product (that
contained DMF traces). 300 mL TDW were added and the mixture was
stirred vigorously for few minutes. The product was collected by
filtration and partially dried by suction. The crude product was
purified as follows: 200 mL of PE was added to the white solid and
the mixture was stirred for a few minutes. The solid was collected
by filtration and dried in vaccuo yielding 45.06 g (91% yield) of
white powder. .sup.1H NMR (CDCl.sub.3, 300 MHz, 298K) .delta.1.41
(m, 2H), 1.58 (m, 2H), 2.18 (m., 4H), 7.15-7.45 (m, 15H). MS (ES)
m/z 376.
[0151] b) Trityl-thiovaleric Acid Hydroxamate
[0152] A solution of N,O dimethylhydroxylamine hydrochloride (0.7
g, 0.0071 mol) in 16 mL DW was added to a mixture of 2.45 g (0.0065
mol) of Trityl-thiovaleric acid and PyBoP (3.73 g, 0.0071 mol).
DIEA (3.4 mL, 0.02 mol) was added and the clear solution was
stirred for 3 hours (pH should be monitored and kept basic). EA (50
mL) was added to the stirred solution followed by 90 mL of
saturated bicarbonate solution. The organic layer was collected and
washed with additional two portions of 40 mL of saturated
bicarbonate solution, 40 mL of water, 2.times.40 mL KHSO.sub.41M,
and 40 mL water, dried over Na.sub.2SO.sub.4 and evaporated to
dryness yielding 3.06 g (quantitive yield) of yellow oil. .sup.1H
NMR (CDCl.sub.3, 300 MHz) .delta.1.74 (m 2H, 2.23 (t, 2H), 2.38 (t,
2H), 3.13 (s, 3H), 3.63 (s, 3H), 7.10-7,50 (m, 15H).
[0153] c) Trityl Thiovaleric Aldehyde
[0154] LiAlH.sub.4 (0.574 g, 0.0151 mol) was added in portions to a
solution of 3.06 g (0.0075 mol) of the hydroxamate in 100 mL dry
diethyl ether under cooling in ice bath and argon atmosphere. The
reaction mixture was stirred for 1 hour (monitored by TLC
PE:EA=1:1). 150 mL of EA were added followed by addition of 150 mL
of KHSO.sub.41M. The mixture was stirred for additional minutes.
The organic layer was collected and washed with 100 mL of
KHSO.sub.4 1M and 100 mL of saturated NaCl, dried over
Na.sub.2SO.sub.4 and evaporated yielding 1.90 g (73% yield) of
white solid. .sup.1H NMR (CDCl.sub.3, 300 MHz, 298K) 67 1.66 (m,
2H), 2.22 (t, 2H), 2.38 (t, 2H) 7.15-7.50 (m, 15H), 9.61 (t,
1H).
[0155] Trityl Thiohexanal
[0156] a) Trityl-thiohexanoic Acid
[0157] Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to
a suspension of NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100
mL DMF under cooling and nitrogen atmosphere, the reaction mixture
was stirred minutes after the addition was completed. Then, a
solution of bromohexanoic acid (g, 0.128 mol) dissolved in 150 mL,
DMF was added stepwise. After the addition was completed the
reaction mixture was stirred for 30 minutes and then cooling and
nitrogen atmosphere were stopped and the reaction mixture was
sealed and left overnight. Then, 500 mL chloroform were added and
the mixture was washed with 4.times.200 mL of saturated solution of
KHSO.sub.4 and 4.times.300 mL of water (the solid that precipitate
during the washings should also be collected with the organic
layer). The organic layer was evaporated and the product (that
contained DMF traces) was triturated by adding 300 mL TDW and
stirring vigursly for few minutes. The product was collected by
filtration, washed by TDW and dried by suction. The crude product
was purified as follows: The solid was dissolved in a mixture of
150 ml of CHCl.sub.3 and 200 ml of PE, and the solution was
evaporated. The oil obtained was triturated by addition of 100 ml
PE and 50 ml of Et.sub.2O and 50 ml Et2O and 50 ml PE. The solid
was collected by filtration and dried in vaccuo yielding 33.92 g
(68% yield) of white powder. .sup.1h NMR (CDCl.sub.3, 300 MHz,
298K) 6 1.31 (m, 2H), 1.37 (m, 2H), 1.50 (m, 2H), 2.15 (t, 2H),
2.26 (t; 2H), 7.15-7.50 (m, 15H).
[0158] b) Trityl-thiohexanoic Acid Hydroxamate
[0159] A solution of N,O dimethylhydroxylamine hydrochloride (1.23
g, 0.0126 mol) in 25 mL DMF was added to a mixture of 4.47 g
(0.0115 mol) of Trityl-thiohexanoic acid and PyBoP (6.56 g, 0.0126
mol). DIEA (6 mL, 0.0344 mol) was added and the clear solution was
stirred for 3 hours (pH should be monitored and kept basic). EA (70
mL) was added to the sired solution followed by 130 mL of saturated
bicarbonate solution. The organic layer was collected and washed
with additional two portions of 60 mL of saturated bicarbonate
solution, 60 mL of water, 2.times.60 mL KHSO.sub.4 1M, and 60 mL
water, dried over Na.sub.2SO.sub.4 and evaporated to dryness
yielding 4.29 g (86% yield) of yellow oil. .sup.1H NMR (CDCl.sub.3,
300 MHz) 67 1.29 (m, 2H), 1.44 (m, 2H), 1.51 (m 2H), 2.16 (t, 2H),
2.33 (t, 2H), 3.15 (s, 3H), 3.65 (s, 3H) 7.15-7.50 (m, 15H).
[0160] c) Trityl-thiohexanal
[0161] LiAlH4 (0.75 g, 0.0198 mol) was added in portions to a
solution of 4.29 g (0.0099 mol) of the hydroxamate in 130 mL dry
diethyl ether under cooling in ice bath and argon atmosphere. The
reaction mixture was stirred for 1 hour (monitored by TLC
PE:EA=1:1). 200 mL of EA were added followed by addition of 200 mL
of KHSO.sub.41M. The mixture was stirred for additional minutes.
The organic layer was collected and washed with 140 mL of
KHSO.sub.4 1M and 140 mL of saturated NaCl, dried over
Na.sub.2SO.sub.4 and evaporated yielding 3.45 g (93% yield) of
white solid. .sup.1H NMR (CDCl.sub.3, 300 MHz, 298K) 67 1.38 (m,
2H), 1.49 (m, 2H), 1.60 (m, 2H), 2.15 (t, 2M,) 2.34 (t, 2H),
7.10-7.55 (m, 15I1), 9.71 (t, 1H).
[0162] Trityl thioacetaldehyde and trityl thiopentanal were
prepared by procedures similar to those described above.
1TABLE 1 structure and MS characterization of a library according
to the present invention, the preparation of which is showed in
Scheme 5 above. compound M.W M.W number R.sup.6 R.sup.7 R.sup.8 m n
calc. Obsvd. 1 L-hydroxybenzyl L-Benzyl Z# 4 5 649.87 653.3 2
L-hydroxybenzyl L-Benzyl Z 5 4 649.87 653.3 3 L-Benzyl
D-hydroxybenzyl Z 4 5 649.87 653.2 4 L-Benzyl D-hydroxybenzyl Z 5 4
649.87 653.3 5 D-hydroxybenzyl L-Benzyl Z 4 5 649.87 653.2 6
D-hydroxybenzyl L-Benzyl Z 5 4 649.87 653.2 7 D-Benzyl
L-hydroxybenzyl Z 4 5 649.87 653.3 8 D-Benzyl L-hydroxybenzyl Z 5 4
649.87 653.2 9 L-hydroxybenzyl D-Benzyl Z 4 5 649.87 653.2* 10
L-hydroxybenzyl D-Benzyl Z 5 4 649.87 653.3 11 L-hydroxybenzyl
L-hydroxybenzyl Z 6 6 707.94 711.79 12 D-hydroxybenzyl
D-hydroxybenzyl Z 6 6 707.94 711.85 13 L-hydroxybenzyl
D-hydroxybenzyl Z 6 6 707.94 711.91 14 D-hydroxybenzyl
L-hydroxybenzyl Z 6 6 707.94 711.43 15 L-hydroxybenzyl L-Benzyl Z 6
6 691.94 694.8 16 D-hydroxybenzyl D-Benzyl Z 6 6 691.94 693.41 17
L-hydroxybenzyl D-Benzyl Z 6 6 691.94 693.79 18 D-hydroxybenzyl
L-Benzyl Z 6 6 691.94 693.91 19 L-Benzyl L-hydroxybenzyl Z 6 6
691.94 693.79 20 D-Benzyl D-hydroxybenzyl Z 6 6 691.94 693.6 21
L-Benzyl D-hydroxybenzyl Z 6 6 691.94 693.23** 22 D-Benzyl
L-hydroxybenzyl Z 6 6 691.94 693.23** 23 L-Benzyl L-Benzyl Z 6 6
675.95 677.24** 24 D-Benzyl D-Benzyl Z 6 6 675.95 677.23** 25
L-Benzyl D-Benzyl Z 6 6 675.95 N.D 26 D-Bennyl L-Benzyl Z 6 6
675.95 677.30** #Z = benzyloxy carbonyl *The peak was obtained
relatively with low intensity. **The peak was analyzed with
HRMS.
[0163] Mass spectrometric analysis: The discrepancy between the
calculated and the observed mass as described in Table 1 ranges
between 1.5 to 2.5 amu. These results may indicate the existence of
reduced non cyclic molecule rather then the oxidized desired
macrocycles. In order to negate this possibility the peaks were
analyzed by splitting (marked with ** in Table 1). This analysis
yielded the expected MW values with a discrepancy of only 0.3 amu.
Furthermore, these molecules were also analyzed by MS-MS and a
fragment indicating a disulfide bridge was found: 11
* * * * *