U.S. patent application number 16/262091 was filed with the patent office on 2019-09-26 for triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles.
The applicant listed for this patent is AILERON THERAPEUTICS, INC.. Invention is credited to Christopher R. Conlee, Vincent Guerlavais, Scott Paul Lentini.
Application Number | 20190292224 16/262091 |
Document ID | / |
Family ID | 48946086 |
Filed Date | 2019-09-26 |
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United States Patent
Application |
20190292224 |
Kind Code |
A1 |
Guerlavais; Vincent ; et
al. |
September 26, 2019 |
TRIAZOLE-CROSSLINKED AND THIOETHER-CROSSLINKED PEPTIDOMIMETIC
MACROCYCLES
Abstract
Provided herein are peptidomimetic macrocycles and methods of
using such macrocycles for the treatment of disease.
Inventors: |
Guerlavais; Vincent;
(Arlington, MA) ; Conlee; Christopher R.;
(Belmont, MA) ; Lentini; Scott Paul; (North
Weymouth, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AILERON THERAPEUTICS, INC. |
Watertown |
MA |
US |
|
|
Family ID: |
48946086 |
Appl. No.: |
16/262091 |
Filed: |
January 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14608641 |
Jan 29, 2015 |
10227380 |
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16262091 |
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13767857 |
Feb 14, 2013 |
8987414 |
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14608641 |
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61599362 |
Feb 15, 2012 |
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61723762 |
Nov 7, 2012 |
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61599365 |
Feb 15, 2012 |
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61723767 |
Nov 7, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/02 20180101;
C07K 7/02 20130101; A61P 35/00 20180101; A61P 35/04 20180101; A61P
43/00 20180101; C07K 7/56 20130101 |
International
Class: |
C07K 7/02 20060101
C07K007/02; C07K 7/56 20060101 C07K007/56 |
Claims
1-2. (canceled)
3. A peptidomimetic macrocycle comprising an amino acid sequence
that is at least about 60% identical to an amino acid sequence in
Tables 4, 4a, or 4b, wherein the peptidomimetic macrocycle has the
formula: ##STR00164## or a pharmaceutically acceptable salt
thereof, wherein: each A, C, D, and E is independently an amino
acid; B is an amino acid, ##STR00165## [--NH-L.sub.3-CO--],
[--NH-L.sub.3-SO.sub.2--], or [--NH-L.sub.3-]; R.sub.1 and R.sub.2
are independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-; or at least one of R.sub.1
and R.sub.2 forms a macrocycle-forming linker L' connected to the
alpha position of one of said D or E amino acids; each L and L' is
independently a macrocycle-forming linker of the formula
##STR00166## L.sub.1, L.sub.2, and L.sub.3 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being unsubstituted or
substituted with R.sub.5; each R.sub.3 is independently hydrogen,
alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl,
each being unsubstituted or substituted with R.sub.5; each R.sub.4
is independently alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each
K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;
each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope, or a
therapeutic agent; each R.sub.6 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a
fluorescent moiety, a radioisotope, or a therapeutic agent; each
R.sub.7 is independently --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, each being unsubstituted or
substituted with R.sub.5, or part of a cyclic structure with a D
residue; each R.sub.8 is independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, each being
unsubstituted or substituted with R.sub.5, or part of a cyclic
structure with an E residue; v and w are independently integers
from 1-1000; u is an integer from 1-10; x, and z are independently
integers from 0-10; and n is an integer from 1-5.
4-22. (canceled)
23. The peptidomimetic macrocycle of claim 3, wherein each E is
independently an amino acid selected from Ala (alanine), D-Ala
(D-alanine), Aib (.alpha.-aminoisobutyric acid), Sar (N-methyl
glycine), and Ser (serine).
24. The peptidomimetic macrocycle of claim 3, wherein [D].sub.v
comprises -Leu.sub.1-Thr.sub.2.
25. The peptidomimetic macrocycle of claim 3, wherein w is
3-10.
26. The peptidomimetic macrocycle of claim 3 wherein w is 3-6.
27. The peptidomimetic macrocycle of claim 3 wherein w is 6-10.
28. The peptidomimetic macrocycle of claim 3 wherein w is 6.
29. The peptidomimetic macrocycle of claim 3, wherein v is
1-10.
30. The peptidomimetic macrocycle of claim 3 wherein v is 2-10.
31. The peptidomimetic macrocycle of claim 3 wherein v is 2-5.
32. (canceled)
33. The peptidomimetic macrocycle of claim 3, wherein w is
3-1000.
34. The peptidomimetic macrocycle of claim 3, wherein the
peptidomimetic macrocycle is not a macrocycle of Table 5, Table 7,
Table 7a, or Table 7b.
35. The peptidomimetic macrocycle of claim 3, wherein each E is Ser
or Ala or an analog thereof.
36-39. (canceled)
40. The peptidomimetic macrocycle of claim 3, wherein the
peptidomimetic macrocycle comprises an amino acid sequence which is
at least about 60% identical to any one of SEQ ID NOs. 456, 155,
463, 464, 468, 944, and 945.
41. The peptidomimetic macrocycle of claim 3, wherein the
peptidomimetic macrocycle comprises an amino acid sequence which is
at least about 80% identical to any one of SEQ ID NOs. 456, 155,
463, 464, 468, 944, and 945.
42. The peptidomimetic macrocycle of claim 3, wherein the
peptidomimetic macrocycle comprises an amino acid sequence which is
at least about 60% identical to SEQ ID NO. 945.
43. The peptidomimetic macrocycle of claim 3, wherein the
peptidomimetic macrocycle comprises an amino acid sequence which is
at least about 80% identical to SEQ ID NO. 945.
44. The peptidomimetic macrocycle of claim 3, wherein the
peptidomimetic macrocycle modulates an activity of p53, MDM2, or
MDMX.
Description
[0001] This application is a continuation application of U.S.
application Ser. No. 14/608,641, filed Jan. 29, 2015, which is a
divisional application of U.S. application Ser. No. 13/767,857,
filed Feb. 14, 2013, which claims the benefit of U.S. Provisional
Application Nos. 61/599,362, filed Feb. 15, 2012; 61/723,762, filed
Nov. 7, 2012; 61/599,365, filed Feb. 15, 2012; and 61/723,767;
filed Nov. 7, 2012; each of which application is incorporated
herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 30, 2019, is named 35224_772_301_SL.txt and is 867,533
bytes in size.
BACKGROUND OF THE INVENTION
[0003] The human transcription factor protein p53 induces cell
cycle arrest and apoptosis in response to DNA damage and cellular
stress, and thereby plays a critical role in protecting cells from
malignant transformation. The E3 ubiquitin ligase MDM2 (also known
as HDM2) negatively regulates p53 function through a direct binding
interaction that neutralizes the p53 transactivation activity,
leads to export from the nucleus of p53 protein, and targets p53
for degradation via the ubiquitylation-proteasomal pathway. Loss of
p53 activity, either by deletion, mutation, or MDM2 overexpression,
is the most common defect in human cancers. Tumors that express
wild type p53 are vulnerable to pharmacologic agents that stabilize
or increase the concentration of active p53. In this context,
inhibition of the activities of MDM2 has emerged as a validated
approach to restore p53 activity and resensitize cancer cells to
apoptosis in vitro and in vivo. MDMX (MDM4) has more recently been
identified as a similar negative regulator of p53, and studies have
revealed significant structural homology between the p53 binding
interfaces of MDM2 and MDMX. The p53-MDM2 and p53-MDMX
protein-protein interactions are mediated by the same 15-residue
alpha-helical transactivation domain of p53, which inserts into
hydrophobic clefts on the surface of MDM2 and MDMX. Three residues
within this domain of p53 (F19, W23, and L26) are essential for
binding to MDM2 and MDMX.
[0004] There remains a considerable need for compounds capable of
binding to and modulating the activity of p53, MDM2 and/or MDMX.
Provided herein are p53-based peptidomimetic macrocycles that
modulate an activity of p53. Also provided herein are p53-based
peptidomimetic macrocycles that inhibit the interactions between
p53, MDM2 and/or MDMX proteins. Further, provided herein are
p53-based peptidomimetic macrocycles that can be used for treating
diseases including but not limited to cancer and other
hyperproliferative diseases.
SUMMARY OF THE INVENTION
[0005] Described herein are stably cross-linked peptides related to
a portion of human p53 ("p53 peptidomimetic macrocycles"). These
cross-linked peptides contain at least two modified amino acids
that together form an intramolecular cross-link that can help to
stabilize the alpha-helical secondary structure of a portion of p53
that is thought to be important for binding of p53 to MDM2 and for
binding of p53 to MDMX. Accordingly, a cross-linked polypeptide
described herein can have improved biological activity relative to
a corresponding polypeptide that is not cross-linked. The p53
peptidomimetic macrocycles are thought to interfere with binding of
p53 to MDM2 and/or of p53 to MDMX, thereby liberating functional
p53 and inhibiting its destruction. The p53 peptidomimetic
macrocycles described herein can be used therapeutically, for
example to treat cancers and other disorders characterized by an
undesirably low level or a low activity of p53, and/or to treat
cancers and other disorders characterized by an undesirably high
level of activity of MDM2 or MDMX. The p53 peptidomimetic
macrocycles can also be useful for treatment of any disorder
associated with disrupted regulation of the p53 transcriptional
pathway, leading to conditions of excess cell survival and
proliferation such as cancer and autoimmunity, in addition to
conditions of inappropriate cell cycle arrest and apoptosis such as
neurodegeneration and immunedeficiencies. In some embodiments, the
p53 peptidomimetic macrocycles bind to MDM2 (e.g., GenBank.RTM.
Accession No.: 228952; GI:228952) and/or MDMX (also referred to as
MDM4; GenBank.RTM. Accession No.: 88702791; GI:88702791).
[0006] In one aspect, provided herein is a peptidomimetic
macrocycle comprising an amino acid sequence which is at least
about 60%, 80%, 90%, or 95% identical to an amino acid sequence
chosen from the group consisting of the amino acid sequences in
Table 4, Table 4a, Table 4b, or Table 5. In some embodiments, the
peptidomimetic macrocycle is not a peptide as shown in Table 6,
Table 6a, Table 7, Table 7a, or Table 7b. In some embodiments, the
peptidomimetic macrocycle has an amino acid sequence chosen from
Table 4. In some embodiments, the peptidomimetic macrocycle has an
amino acid sequence chosen from Table 4a. In some embodiments, the
peptidomimetic macrocycle has an amino acid sequence chosen from
Table 4b. In some embodiments, the peptidomimetic macrocycle has an
amino acid sequence chosen from Table 5.
[0007] Alternatively, an amino acid sequence of said peptidomimetic
macrocycle is chosen as above, and further wherein the macrocycle
does not include an all carbon crosslink or a triazole. In some
embodiments, the peptidomimetic macrocycle comprises a helix, such
as an .alpha.-helix. In other embodiments, the peptidomimetic
macrocycle comprises an .alpha.,.alpha.-disubstituted amino acid. A
peptidomimetic macrocycle can comprise a crosslinker linking the
.alpha.-positions of at least two amino acids. At least one of said
two amino acids can be an .alpha.,.alpha.-disubstituted amino
acid.
[0008] In some embodiments, provided are peptidomimetic macrocycle
of the Formula:
##STR00001##
[0009] wherein:
[0010] each A, C, D, and E is independently an amino acid;
[0011] B is an amino acid,
##STR00002##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0012] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00003##
[0013] L.sub.1, L.sub.2 and L.sub.3 are independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0014] each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5;
[0015] each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene;
[0016] each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0017] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0018] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0019] each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue;
[0020] each R.sub.8 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with an E
residue;
[0021] v and w are independently integers from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20 or 1-10;
[0022] u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
[0023] x, y and z are independently integers from 0-10, for example
the sum of x+y+z is 2, 3, or 6; and
[0024] n is an integer from 1-5.
[0025] In some embodiments, v and w are integers between 1-30. In
some embodiments, w is an integer from 3-1000, for example 3-500,
3-200, 3-100, 3-50, 3-30, 3-20, or 3-10.
[0026] In some embodiments, the sum of x+y+z is 3 or 6. In some
embodiments, the sum of x+y+z is 3.
[0027] In other embodiments, the sum of x+y+z is 6.
[0028] In some embodiments, the peptidomimetic macrocycles are
claimed with the proviso that when u=1 and w=2, the first
C-terminal amino acid represented by E is not an Arginine (R)
and/or the second C-terminal amino acid represented by E is not a
Threonine (T). For instance, when u=1 and w=2, the first C-terminal
amino acid and/or the second C-terminal amino acid represented by E
do not comprise a positively charged side chain or a polar
uncharged side chain In some embodiments, when u=1 and w=2, the
first C-terminal amino acid and/or the second C-terminal amino acid
represented by E comprise a hydrophobic side chain For example,
when w=2, the first C-terminal amino acid and/or the second
N-terminal amino acid represented by E comprise a hydrophobic side
chain, for example a large hydrophobic side chain
[0029] In some embodiments, w is between 3 and 1000. For example,
the third amino acid represented by E comprises a large hydrophobic
side chain
[0030] Peptidomimetic macrocycles are also provided of the
formula:
##STR00004##
[0031] wherein:
[0032] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 1), where each X is an amino
acid;
[0033] each D and E is independently an amino acid;
[0034] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0035] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00005##
[0036] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0037] each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5;
[0038] each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene;
[0039] each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0040] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0041] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0042] each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue;
[0043] each R.sub.8 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with an E
residue;
[0044] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20 or 1-10;
[0045] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0046] n is an integer from 1-5.
[0047] In some embodiments, the peptidomimetic macrocycle has the
formula:
##STR00006##
[0048] wherein:
[0049] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 2), where each X
is an amino acid;
[0050] each D and E is independently an amino acid;
[0051] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0052] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00007##
[0053] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0054] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0055] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0056] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0057] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0058] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0059] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0060] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0061] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0062] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0063] n is an integer from 1-5.
[0064] In some embodiments, provided are peptidomimetic macrocycles
of the Formula I:
##STR00008##
[0065] wherein:
[0066] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 1), where each X is an amino
acid;
[0067] each D and E is independently an amino acid;
[0068] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0069] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00009##
[0070] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0071] each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5;
[0072] each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene;
[0073] each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0074] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0075] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0076] each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue;
[0077] each R.sub.8 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with an E
residue;
[0078] each R.sub.9 is independently alkyl, alkenyl, alkynyl, aryl,
cycloalkyl, cycloalkenyl, heteroaryl, or heterocyclyl group,
unsubstituted or optionally substituted with R.sub.a and/or
R.sub.b;
[0079] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20 or 1-10;
[0080] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0081] n is an integer from 1-5.
[0082] In some embodiments, provided are peptidomimetic macrocycles
of the Formula I:
##STR00010##
[0083] wherein:
[0084] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 2), where each X
is an amino acid;
[0085] each D and E is independently an amino acid;
[0086] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0087] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00011##
[0088] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0089] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0090] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0091] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0092] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0093] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0094] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0095] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0096] each R.sub.9 is independently alkyl, alkenyl, alkynyl, aryl,
cycloalkyl, cycloalkenyl, heteroaryl, or heterocyclyl group,
unsubstituted or optionally substituted with R.sub.a and/or
R.sub.b;
[0097] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0098] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0099] n is an integer from 1-5.
[0100] In some embodiments, provided are peptidomimetic macrocycles
of the Formula I, comprising an amino acid sequence which is at
least about 60% identical to an amino acid sequence chosen from the
group consisting of the amino acid sequences in Tables 4, 4a, or
4b, wherein the peptidomimetic macrocycle has the formula:
##STR00012##
[0101] wherein:
[0102] each A, C, D, and E is independently an amino acid;
[0103] B is an amino acid,
##STR00013##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0104] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0105] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00014##
[0106] L.sub.1, L.sub.2 and L.sub.3 are independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0107] each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5;
[0108] each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene;
[0109] each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0110] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0111] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0112] each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue;
[0113] each R.sub.8 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with an E
residue;
[0114] each R.sub.9 is independently alkyl, alkenyl, alkynyl, aryl,
cycloalkyl, cycloalkenyl, heteroaryl, or heterocyclyl group,
unsubstituted or optionally substituted with R.sub.a and/or
R.sub.b;
[0115] v and w are independently integers from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20 or 1-10;
[0116] u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
[0117] x, y and z are independently integers from 0-10, for example
the sum of x+y+z is 2, 3, or 6; and
[0118] n is an integer from 1-5.
[0119] In some embodiments, provided are peptidomimetic macrocycle
of the Formula II:
##STR00015##
[0120] wherein:
[0121] each A, C, D, and E is independently an amino acid;
[0122] B is an amino acid,
##STR00016##
[--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-];
[0123] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0124] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0125] L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4-]n, each being unsubstituted or substituted
with R.sub.5;
[0126] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0127] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0128] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0129] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0130] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0131] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0132] v and w are independently integers from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20 or 1-10;
[0133] u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
[0134] x, y and z are independently integers from 0-10, for example
the sum of x+y+z is 2, 3, or 6; and
[0135] n is an integer from 1-5.
[0136] In some embodiments, the peptidomimetic macrocycles are
claimed with the proviso that when u=1 and w=2, the first
C-terminal amino acid represented by E is not an Arginine (R)
and/or the second C-terminal amino acid represented by E is not a
Threonine (T). For instance, when u=1 and w=2, the first C-terminal
amino acid and/or the second C-terminal amino acid represented by E
do not comprise a positively charged side chain or a polar
uncharged side chain In some embodiments, when u=1 and w=2, the
first C-terminal amino acid and/or the second C-terminal amino acid
represented by E comprise a hydrophobic side chain For example,
when w=2, the first C-terminal amino acid and/or the second
N-terminal amino acid represented by E comprise a hydrophobic side
chain, for example a large hydrophobic side chain
[0137] In some embodiments, w is between 3 and 1000. For example,
the third amino acid represented by E comprises a large hydrophobic
side chain
[0138] Peptidomimetic macrocycles are also provided of the
formula:
##STR00017##
[0139] wherein:
[0140] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 1), where each X is an amino
acid;
[0141] each D and E is independently an amino acid;
[0142] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0143] L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4-]n, each being unsubstituted or substituted
with R.sub.5;
[0144] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0145] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0146] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0147] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0148] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0149] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0150] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0151] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20 or 1-10;
[0152] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0153] n is an integer from 1-5.
[0154] Peptidomimetic macrocycles are also provided of the
formula:
##STR00018##
[0155] wherein:
[0156] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 2), where each X
is an amino acid;
[0157] each D and E is independently an amino acid;
[0158] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0159] L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4-]n, each being unsubstituted or substituted
with R.sub.5;
[0160] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0161] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0162] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0163] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0164] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0165] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0166] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0167] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0168] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0169] n is an integer from 1-5.
[0170] In some embodiments, each E is independently an amino acid
selected from Ala (alanine), D-Ala (D-alanine), Aib
(.alpha.-aminoisobutyric acid), Sar (N-methyl glycine), and Ser
(serine). In some embodiments, [D].sub.v is
-Leu.sub.1-Thr.sub.2.
[0171] In some embodiments, w is an integer from 3-10, for example
3-6, 3-8, 6-8, or 6-10. In some embodiments, w is 3. In other
embodiments, w is 6. In some embodiments, v is an integer from
1-10, for example 2-5. In some embodiments, v is 2.
[0172] In some embodiments, the peptidomimetic macrocycle has
improved binding affinity to MDM2 or MDMX relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
other instances, the peptidomimetic macrocycle has a reduced ratio
of binding affinities to MDMX versus MDM2 relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
still other instances, the peptidomimetic macrocycle has improved
in vitro anti-tumor efficacy against p53 positive tumor cell lines
relative to a corresponding peptidomimetic macrocycle where w is 0,
1 or 2. In some embodiments, the peptidomimetic macrocycle shows
improved in vitro induction of apoptosis in p53 positive tumor cell
lines relative to a corresponding peptidomimetic macrocycle where w
is 0, 1 or 2. In other instances, the peptidomimetic macrocycle of
claim 1, wherein the peptidomimetic macrocycle has an improved in
vitro anti-tumor efficacy ratio for p53 positive versus p53
negative or mutant tumor cell lines relative to a corresponding
peptidomimetic macrocycle where w is 0, 1 or 2. In some instances
the improved efficacy ratio in vitro, is 1-29, .gtoreq.30-49, or
.gtoreq.50. In still other instances, the peptidomimetic macrocycle
has improved in vivo anti-tumor efficacy against p53 positive
tumors relative to a corresponding peptidomimetic macrocycle where
w is 0, 1 or 2. In some instances the improved efficacy ratio in
vivo is -29, .gtoreq.30-49, or .gtoreq.50. In yet other instances,
the peptidomimetic macrocycle has improved in vivo induction of
apoptosis in p53 positive tumors relative to a corresponding
peptidomimetic macrocycle where w is 0, 1 or 2. In some
embodiments, the peptidomimetic macrocycle has improved cell
permeability relative to a corresponding peptidomimetic macrocycle
where w is 0, 1 or 2. In other cases, the peptidomimetic macrocycle
has improved solubility relative to a corresponding peptidomimetic
macrocycle where w is 0, 1 or 2.
[0173] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof. In some embodiments, Xaa.sub.5 is Glu or an amino
acid analog thereof and wherein the peptidomimetic macrocycle has
an improved property, such as improved binding affinity, improved
solubility, improved cellular efficacy, improved cell permeability,
improved in vivo or in vitro anti-tumor efficacy, or improved
induction of apoptosis relative to a corresponding peptidomimetic
macrocycle where Xaa.sub.5 is Ala.
[0174] In some embodiments, the peptidomimetic macrocycle has
improved binding affinity to MDM2 or MDMX relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala. In
other embodiments, the peptidomimetic macrocycle has a reduced
ratio of binding affinities to MDMX vs MDM2 relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala. In
some embodiments, the peptidomimetic macrocycle has improved
solubility relative to a corresponding peptidomimetic macrocycle
where Xaa.sub.5 is Ala, or the peptidomimetic macrocycle has
improved cellular efficacy relative to a corresponding
peptidomimetic macrocycle where Xaa.sub.5 is Ala.
[0175] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof and wherein the peptidomimetic macrocycle has
improved biological activity, such as improved binding affinity,
improved solubility, improved cellular efficacy, improved helicity,
improved cell permeability, improved in vivo or in vitro anti-tumor
efficacy, or improved induction of apoptosis relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala.
[0176] In some embodiments, the peptidomimetic macrocycle has an
activity against a p53+/+ cell line which is at least 2-fold,
3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 70-fold, or
100-fold greater than its binding affinity against a p53-/- cell
line. In some embodiments, the peptidomimetic macrocycle has an
activity against a p53+/+ cell line which is between 1 and 29-fold,
between 30 and 49-fold, or .gtoreq.50-fold greater than its binding
affinity against a p53-/- cell line. Activity can be measured, for
example, as an IC50 value. For example, the p53+/+ cell line is
SJSA-1, RKO, HCT-116, or MCF-7 and the p53-/- cell line is RKO-E6
or SW-480. In some embodiments, the peptide has an IC50 against the
p53+/+ cell line of less than 1 .mu.M.
[0177] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof and the peptidomimetic macrocycle has an activity
against a p53+/+ cell line which is at least 10-fold greater than
its binding affinity against a p53-/- cell line.
[0178] Additionally, a method is provided of treating cancer in a
subject comprising administering to the subject a peptidomimetic
macrocycle. Also provided is a method of modulating the activity of
p53 or MDM2 or MDMX in a subject comprising administering to the
subject a peptidomimetic macrocycle, or a method of antagonizing
the interaction between p53 and MDM2 and/or MDMX proteins in a
subject comprising administering to the subject such a
peptidomimetic macrocycle.
INCORPORATION BY REFERENCE
[0179] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
[0180] As used herein, the term "macrocycle" refers to a molecule
having a chemical structure including a ring or cycle formed by at
least 9 covalently bonded atoms.
[0181] As used herein, the term "peptidomimetic macrocycle" or
"crosslinked polypeptide" refers to a compound comprising a
plurality of amino acid residues joined by a plurality of peptide
bonds and at least one macrocycle-forming linker which forms a
macrocycle between a first naturally-occurring or
non-naturally-occurring amino acid residue (or analog) and a second
naturally-occurring or non-naturally-occurring amino acid residue
(or analog) within the same molecule. Peptidomimetic macrocycle
include embodiments where the macrocycle-forming linker connects
the a carbon of the first amino acid residue (or analog) to the a
carbon of the second amino acid residue (or analog). The
peptidomimetic macrocycles optionally include one or more
non-peptide bonds between one or more amino acid residues and/or
amino acid analog residues, and optionally include one or more
non-naturally-occurring amino acid residues or amino acid analog
residues in addition to any which form the macrocycle. A
"corresponding uncrosslinked polypeptide" when referred to in the
context of a peptidomimetic macrocycle is understood to relate to a
polypeptide of the same length as the macrocycle and comprising the
equivalent natural amino acids of the wild-type sequence
corresponding to the macrocycle.
[0182] As used herein, the term "stability" refers to the
maintenance of a defined secondary structure in solution by a
peptidomimetic macrocycle as measured by circular dichroism, NMR or
another biophysical measure, or resistance to proteolytic
degradation in vitro or in vivo. Non-limiting examples of secondary
structures contemplated herein are .alpha.-helices, 3.sub.10
helices, .beta.-turns, and .beta.-pleated sheets.
[0183] As used herein, the term "helical stability" refers to the
maintenance of a helical structure by a peptidomimetic macrocycle
as measured by circular dichroism or NMR. For example, in some
embodiments, a peptidomimetic macrocycle exhibits at least a 1.25,
1.5, 1.75 or 2-fold increase in .alpha.-helicity as determined by
circular dichroism compared to a corresponding uncrosslinked
macrocycle.
[0184] The term "amino acid" refers to a molecule containing both
an amino group and a carboxyl group. Suitable amino acids include,
without limitation, both the D-and L-isomers of the
naturally-occurring amino acids, as well as non-naturally occurring
amino acids prepared by organic synthesis or other metabolic
routes. The term amino acid, as used herein, includes without
limitation .alpha.-amino acids, natural amino acids, non-natural
amino acids, and amino acid analogs.
[0185] The term ".alpha.-amino acid" refers to a molecule
containing both an amino group and a carboxyl group bound to a
carbon which is designated the .alpha.-carbon.
[0186] The term ".beta.-amino acid" refers to a molecule containing
both an amino group and a carboxyl group in a .beta.
configuration.
[0187] The term "naturally occurring amino acid" refers to any one
of the twenty amino acids commonly found in peptides synthesized in
nature, and known by the one letter abbreviations A, R, N, C, D, Q,
E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
[0188] The following table shows a summary of the properties of
natural amino acids:
TABLE-US-00001 3- 1- Side- Side-chain Letter Letter chain charge
Hydropathy Amino Acid Code Code Polarity (pH 7.4) Index Alanine Ala
A nonpolar neutral 1.8 Arginine Arg R polar positive -4.5
Asparagine Asn N polar neutral -3.5 Aspartic acid Asp D polar
negative -3.5 Cysteine Cys C polar neutral 2.5 Glutamic acid Glu E
polar negative -3.5 Glutamine Gln Q polar neutral -3.5 Glycine Gly
G nonpolar neutral -0.4 Histidine His H polar positive(10%) -3.2
neutral(90%) Isoleucine Ile I nonpolar neutral 4.5 Leucine Leu L
nonpolar neutral 3.8 Lysine Lys K polar positive -3.9 Methionine
Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8
Proline Pro P nonpolar neutral -1.6 Serine Ser S polar neutral -0.8
Threonine Thr T polar neutral -0.7 Tryptophan Trp W nonpolar
neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valine Val V
nonpolar neutral 4.2
[0189] "Hydrophobic amino acids" include, without limitation, small
hydrophobic amino acids and large hydrophobic amino acids. "Small
hydrophobic amino acid" are glycine, alanine, proline, and analogs
thereof. "Large hydrophobic amino acids" are valine, leucine,
isoleucine, phenylalanine, methionine, tryptophan, and analogs
thereof "Polar amino acids" are serine, threonine, asparagine,
glutamine, cysteine, tyrosine, and analogs thereof "Charged amino
acids" are lysine, arginine, histidine, aspartate, glutamate, and
analogs thereof.
[0190] The term "amino acid analog" refers to a molecule which is
structurally similar to an amino acid and which can be substituted
for an amino acid in the formation of a peptidomimetic macrocycle.
Amino acid analogs include, without limitation, .beta.-amino acids
and amino acids where the amino or carboxy group is substituted by
a similarly reactive group (e.g., substitution of the primary amine
with a secondary or tertiary amine, or substitution of the carboxy
group with an ester).
[0191] The term "non-natural amino acid" refers to an amino acid
which is not one of the the twenty amino acids commonly found in
peptides synthesized in nature, and known by the one letter
abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W,
Y and V. Non-natural amino acids or amino acid analogs include,
without limitation, structures according to the following:
##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023##
[0192] Amino acid analogs include .beta.-amino acid analogs.
Examples of .beta.-amino acid analogs include, but are not limited
to, the following: cyclic .beta.-amino acid analogs;
.beta.-alanine; (R)-.beta.-phenylalanine;
(R)-1,2,3,4-tetrahydroisoquinoline-3-acetic acid;
(R)-3-amino-4-(1-naphthyl)-butyric acid;
(R)-3-amino-4-(2,4-dichlorophenyl)butyric acid;
(R)-3-amino-4-(2-chlorophenyl)-butyric acid;
(R)-3-amino-4-(2-cyanophenyl)-butyric acid;
(R)-3-amino-4-(2-fluorophenyl)-butyric acid;
(R)-3-amino-4-(2-furyl)-butyric acid;
(R)-3-amino-4-(2-methylphenyl)-butyric acid;
(R)-3-amino-4-(2-naphthyl)-butyric acid;
(R)-3-amino-4-(2-thienyl)-butyric acid;
(R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-(3,4-dichlorophenyl)butyric acid;
(R)-3-amino-4-(3,4-difluorophenyl)butyric acid;
(R)-3-amino-4-(3-benzothienyl)-butyric acid;
(R)-3-amino-4-(3-chlorophenyl)-butyric acid;
(R)-3-amino-4-(3-cyanophenyl)-butyric acid;
(R)-3-amino-4-(3-fluorophenyl)-butyric acid;
(R)-3-amino-4-(3-methylphenyl)-butyric acid;
(R)-3-amino-4-(3-pyridyl)-butyric acid;
(R)-3-amino-4-(3-thienyl)-butyric acid;
(R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-(4-bromophenyl)-butyric acid;
(R)-3-amino-4-(4-chlorophenyl)-butyric acid;
(R)-3-amino-4-(4-cyanophenyl)-butyric acid;
(R)-3-amino-4-(4-fluorophenyl)-butyric acid;
(R)-3-amino-4-(4-iodophenyl)-butyric acid;
(R)-3-amino-4-(4-methylphenyl)-butyric acid;
(R)-3-amino-4-(4-nitrophenyl)-butyric acid;
(R)-3-amino-4-(4-pyridyl)-butyric acid;
(R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;
(R)-3-amino-4-pentafluoro-phenylbutyric acid;
(R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid;
(R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl
-5-hexenoic acid; (S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic
acid; (S)-3-amino-4-(1-naphthyl)-butyric acid;
(S)-3-amino-4-(2,4-dichlorophenyl)butyric acid;
(S)-3-amino-4-(2-chlorophenyl)-butyric acid;
(S)-3-amino-4-(2-cyanophenyl)-butyric acid;
(S)-3-amino-4-(2-fluorophenyl)-butyric acid;
(S)-3-amino-4-(2-furyl)-butyric acid;
(S)-3-amino-4-(2-methylphenyl)-butyric acid;
(S)-3-amino-4-(2-naphthyl)-butyric acid;
(S)-3-amino-4-(2-thienyl)-butyric acid;
(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-(3,4-dichlorophenyl)butyric acid;
(S)-3-amino-4-(3,4-difluorophenyl)butyric acid;
(S)-3-amino-4-(3-benzothienyl)-butyric acid;
(S)-3-amino-4-(3-chlorophenyl)-butyric acid;
(S)-3-amino-4-(3-cyanophenyl)-butyric acid;
(S)-3-amino-4-(3-fluorophenyl)-butyric acid;
(S)-3-amino-4-(3-methylphenyl)-butyric acid;
(S)-3-amino-4-(3-pyridyl)-butyric acid;
(S)-3-amino-4-(3-thienyl)-butyric acid;
(S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-(4-bromophenyl)-butyric acid;
(S)-3-amino-4-(4-chlorophenyl)-butyric acid;
(S)-3-amino-4-(4-cyanophenyl)-butyric acid;
(S)-3-amino-4-(4-fluorophenyl)-butyric acid;
(S)-3-amino-4-(4-iodophenyl)-butyric acid;
(S)-3-amino-4-(4-methylphenyl)-butyric acid;
(S)-3-amino-4-(4-nitrophenyl)-butyric acid;
(S)-3-amino-4-(4-pyridyl)-butyric acid;
(S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;
(S)-3-amino-4-pentafluoro-phenylbutyric acid;
(S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid;
(S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic
acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid;
1,2,5,6-tetrahydropyridine -4-carboxylic acid;
3-amino-3-(2-chlorophenyl)-propionic acid;
3-amino-3-(2-thienyl)-propionic acid;
3-amino-3-(3-bromophenyl)-propionic acid;
3-amino-3-(4-chlorophenyl)-propionic acid;
3-amino-3-(4-methoxyphenyl)-propionic acid;
3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid;
D-.beta.-phenylalanine; .beta.-leucine; L-.beta.-homoalanine;
L-.beta.-homoaspartic acid .gamma.-benzyl ester;
L-.beta.-homoglutamic acid .delta.-benzyl ester;
L-.beta.-homoisoleucine; L-.beta.-homoleucine;
L-.beta.-homomethionine; L-.beta.-homophenylalanine;
L-.beta.-homoproline; L-.beta.-homotryptophan; L-.beta.-homovaline;
L-N.omega.-benzyloxycarbonyl-.beta.-homolysine;
N.omega.-L-.beta.-homoarginine;
O-benzyl-L-.beta.-homohydroxyproline; O-benzyl-L-.beta.-homoserine;
O-benzyl-L-.beta.-homothreonine; O-benzyl-L-.beta.-homotyrosine;
.gamma.-trityl-L-.beta.-homoasparagine; (R)-.beta.-phenylalanine;
L-.beta.-homoaspartic acid .gamma.-t-butyl ester;
L-.beta.-homoglutamic acid .delta.-t-butyl ester;
L-N.omega.-.beta.-homolysine;
N.delta.-trityl-L-.beta.-homoglutamine;
N.omega.-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-.beta.-homo-
arginine; O-t-butyl-L-.beta.-homohydroxy-proline;
O-t-butyl-L-.beta.-homoserine; O-t-butyl-L-.beta.-homothreonine;
O-t-butyl-L-.beta.-homotyrosine; 2-aminocyclopentane carboxylic
acid; and 2-aminocyclohexane carboxylic acid.
[0193] Amino acid analogs include analogs of alanine, valine,
glycine or leucine. Examples of amino acid analogs of alanine,
valine, glycine, and leucine include, but are not limited to, the
following: .alpha.-methoxyglycine; .alpha.-allyl-L-alanine;
.alpha.-aminoisobutyric acid; .alpha.-methyl-leucine;
.beta.-(1-naphthyl)-D-alanine; .beta.-(1-naphthyl)-L-alanine;
.beta.-(2-naphthyl)-D-alanine; .beta.-(2-naphthyl)-L-alanine;
.beta.-(2-pyridyl)-D-alanine; .beta.-(2-pyridyl)-L-alanine;
.beta.-(2-thienyl)-D-alanine; .beta.-(2-thienyl)-L-alanine;
.beta.-(3-benzothienyl)-D-alanine;
.beta.-(3-benzothienyl)-L-alanine; .beta.-(3-pyridyl)-D-alanine;
.beta.-(3-pyridyl)-L-alanine; .beta.-(4-pyridyl)-D-alanine;
.beta.-(4-pyridyl)-L-alanine; .beta.-chloro-L-alanine;
.beta.-cyano-L-alanin; .beta.-cyclohexyl-D-alanine;
.beta.-cyclohexyl-L-alanine; .beta.-cyclopenten-1-yl -alanine;
.beta.-cyclopentyl-alanine;
.beta.-cyclopropyl-L-Ala-OH-dicyclohexylammonium salt;
.beta.-t-butyl-D-alanine; .beta.-t-butyl-L-alanine;
.gamma.-aminobutyric acid; L-.alpha.,.beta.-diaminopropionic acid;
2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine;
2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine;
3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine;
4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium
salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine;
4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic
acid; cyclopentyl-D-Gly-OH-dicyclohexylammonium salt;
cyclopentyl-Gly-OH.dicyclohexylammonium salt;
D-.alpha.,.beta.-diaminopropionic acid; D-.alpha.-aminobutyric
acid; D-.alpha.-t-butylglycine; D-(2-thienyl)glycine;
D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine;
D-allylglycine.dicyclohexylammonium salt; D-cyclohexylglycine;
D-norvaline; D-phenylglycine; .beta.-aminobutyric acid;
.beta.-aminoisobutyric acid; (2-bromophenyl)glycine;
(2-methoxyphenyl)glycine; (2-methylphenyl)glycine;
(2-thiazoyl)glycine; (2-thienyl)glycine;
2-amino-3-(dimethylamino)-propionic acid;
L-.alpha.,.beta.-diaminopropionic acid; L-.alpha.-aminobutyric
acid; L-.alpha.-t-butylglycine; L-(3-thienyl)glycine;
L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic acid
dicyclohexyl-ammonium salt; L-2-indanylglycine;
L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine;
L-phenylglycine; L-propargylglycine; L-norvaline;
N-.alpha.-aminomethyl-L-alanine; D-.alpha.,.gamma.-diaminobutyric
acid; L-.alpha.,.gamma.-diaminobutyric acid;
.beta.-cyclopropyl-L-alanine;
(N-.beta.-(2,4-dinitrophenyl))-L-.alpha.,.beta.-diaminopropionic
acid; (N-.beta.-1-(4,4-dimethyl
-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,.beta.-diaminopropionic
acid;
(N-.beta.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alp-
ha.,.beta.-diaminopropionic acid;
(N-.beta.-4-methyltrityl)-L-.alpha.,.beta.-diaminopropionic acid;
(N-.beta.-allyloxycarbonyl)-L-.alpha.,.beta.-diaminopropionic acid;
(N-.gamma.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,.-
gamma.-diaminobutyric acid; (N-.gamma.-1-(4,4-dimethyl
-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alpha.,.gamma.-diaminobutyric
acid; (N-.gamma.-4-methyltrityl)-D-.alpha.,.gamma.-diaminobutyric
acid; (N-.gamma.-4-methyltrityl)-L-.alpha.,.gamma.-diaminobutyric
acid; (N-.gamma.-allyloxycarbonyl)-L-.alpha.,.gamma.-diaminobutyric
acid; D-.alpha.,.gamma.-diaminobutyric acid; 4,5-dehydro-L-leucine;
cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine;
D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid;
L-allylglycine; L-homocyclohexylalanine; and N-(2-hydroxy
-4-methoxy-Bzl)-Gly-OH.
[0194] Amino acid analogs further include analogs of arginine or
lysine. Examples of amino acid analogs of arginine and lysine
include, but are not limited to, the following: citrulline;
L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic
acid; L-citrulline; Lys(Me).sub.2-OH; Lys(N.sub.3)--OH;
N.delta.-benzyloxycarbonyl-L-ornithine; N.omega.-nitro-D-arginine;
N.omega.-nitro-L-arginine; .alpha.-methyl-ornithine;
2,6-diaminoheptanedioic acid; L-ornithine;
(N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine-
;
(N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-ornithin-
e; (N.delta.-4-methyltrityl)-D-ornithine;
(N.delta.-4-methyltrityl)-L-ornithine; D-ornithine; L-ornithine;
Arg(Me)(Pbf)-OH; Arg(Me).sub.2-OH (asymmetrical); Arg(Me)2-OH
(symmetrical); Lys(ivDde)-OH; Lys(Me)2-OH.HCl; Lys(Me3)-OH
chloride; N.omega.-nitro-D-arginine; and
N.omega.-nitro-L-arginine.
[0195] Amino acid analogs include analogs of aspartic or glutamic
acids. Examples of amino acid analogs of aspartic and glutamic
acids include, but are not limited to, the following:
.alpha.-methyl-D-aspartic acid; .alpha.-methyl-glutamic acid;
.alpha.-methyl-L-aspartic acid; .gamma.-methylene-glutamic acid;
(N-.gamma.-ethyl)-L-glutamine;
[N-.alpha.-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic
acid; L-.alpha.-aminosuberic acid; D-2-aminoadipic acid;
D-.alpha.-aminosuberic acid; .alpha.-aminopimelic acid;
iminodiacetic acid; L-2-aminoadipic acid;
threo-.beta.-methyl-aspartic acid; .gamma.-carboxy-D-glutamic acid
.gamma.,.gamma.-di-t-butyl ester; .gamma.-carboxy-L-glutamic acid
.gamma.,.gamma.-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and
pyroglutamic acid.
[0196] Amino acid analogs include analogs of cysteine and
methionine. Examples of amino acid analogs of cysteine and
methionine include, but are not limited to, Cys(farnesyl)-OH,
Cys(farnesyl)-OMe, .alpha.-methyl-methionine,
Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH,
2-amino-4-(ethylthio)butyric acid, buthionine,
buthioninesulfoximine, ethionine, methionine methylsulfonium
chloride, selenomethionine, cysteic acid,
[2-(4-pyridyl)ethyl]-DL-penicillamine,
[2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine,
4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine,
4-methylbenzyl-L-penicillamine, benzyl-D-cysteine,
benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine,
carboxyethyl-L-cysteine, carboxymethyl-L-cysteine,
diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine,
t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine,
cystathionine, homocystine, L-homocystine,
(2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine,
Cys(StBu)-OH, and acetamidomethyl-D-penicillamine
[0197] Amino acid analogs include analogs of phenylalanine and
tyrosine. Examples of amino acid analogs of phenylalanine and
tyrosine include .beta.-methyl-phenylalanine,
.beta.-hydroxyphenylalanine, .alpha.-methyl
-3-methoxy-DL-phenylalanine, .alpha.-methyl-D-phenylalanine,
.alpha.-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline
-3-carboxylic acid, 2,4-dichloro-phenylalanine,
2-(trifluoromethyl)-D -phenylalanine,
2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine,
2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine,
2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine,
2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine,
2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine,
2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine,
2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine,
3,4,5-trifluoro -D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine,
3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine,
3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine,
3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine,
3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine,
3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine,
3-(trifluoromethyl)-D-phenylalanine,
3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine,
3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine,
3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine,
3-chloro-L-tyrosine, 3-cyano-D-phenylalanine,
3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine,
3-fluoro-L-phenylalanine, 3-fluoro-tyrosine,
3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine,
3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine,
3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine,
3-nitro-L-phenylalanine, 3-nitro-L-tyrosine,
4-(trifluoromethyl)-D-phenylalanine,
4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine,
4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine,
4-benzoyl-L-phenylalanine,
4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine,
4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine,
4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine,
4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine,
4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine,
4-iodo-L-phenylalanine, homophenylalanine, thyroxine,
3,3-diphenylalanine, thyronine, ethyl-tyrosine, and
methyl-tyrosine.
[0198] Amino acid analogs include analogs of proline. Examples of
amino acid analogs of proline include, but are not limited to,
3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline,
thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.
[0199] Amino acid analogs include analogs of serine and threonine.
Examples of amino acid analogs of serine and threonine include, but
are not limited to, 3-amino-2-hydroxy -5-methylhexanoic acid,
2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic
acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy
-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid,
2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid,
4-amino-3-hydroxybutanoic acid, and .alpha.-methylserine.
[0200] Amino acid analogs include analogs of tryptophan. Examples
of amino acid analogs of tryptophan include, but are not limited
to, the following: .alpha.-methyl-tryptophan;
.beta.-(3-benzothienyl)-D-alanine;
.beta.-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan;
4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan;
5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan;
5-hydroxy -L-tryptophan; 5-methoxy-tryptophan; 5-methoxy
-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan;
6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan;
6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan;
7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic
acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;
7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;
5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.
[0201] In some embodiments, amino acid analogs are racemic. In some
embodiments, the D isomer of the amino acid analog is used. In some
embodiments, the L isomer of the amino acid analog is used. In
other embodiments, the amino acid analog comprises chiral centers
that are in the R or S configuration. In still other embodiments,
the amino group(s) of a .beta.-amino acid analog is substituted
with a protecting group, e.g., tert-butyloxycarbonyl (BOC group),
9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. In yet
other embodiments, the carboxylic acid functional group of a
.beta.-amino acid analog is protected, e.g., as its ester
derivative. In some embodiments the salt of the amino acid analog
is used.
[0202] A "non-essential" amino acid residue, as used herein, is an
amino acid residue present in a wild-type sequence of a polypeptide
that can be altered without abolishing or substantially altering
essential biological or biochemical activity (e.g., receptor
binding or activation) of the polypeptide.
[0203] An "essential" amino acid residue, as used herein, is an
amino acid residue present in a wild-type sequence of a polypeptide
that, when altered, results in abolishing or a substantial
reduction in the polypeptide's essential biological or biochemical
activity(e.g., receptor binding or activation).
[0204] A "conservative amino acid substitution" is one in which an
amino acid residue is replaced with a different amino acid residue
having a similar side chain Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., K, R, H), acidic
side chains (e.g., D, E), uncharged polar side chains (e.g., G, N,
Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M,
W), beta-branched side chains (e.g., T, V, I) and aromatic side
chains (e g., Y, F, W, H). Thus, a predicted nonessential amino
acid residue in a polypeptide, for example, is replaced with
another amino acid residue from the same side chain family. Other
examples of acceptable substitutions are substitutions based on
isosteric considerations (e.g. norleucine for methionine) or other
properties (e.g. 2-thienylalanine for phenylalanine, or
6-Cl-tryptophan for tryptophan).
[0205] The term "capping group" refers to the chemical moiety
occurring at either the carboxy or amino terminus of the
polypeptide chain of the subject peptidomimetic macrocycle. The
capping group of a carboxy terminus includes an unmodified
carboxylic acid (ie --COOH) or a carboxylic acid with a
substituent. For example, the carboxy terminus can be substituted
with an amino group to yield a carboxamide at the C-terminus.
Various substituents include but are not limited to primary and
secondary amines, including pegylated secondary amines
Representative secondary amine capping groups for the C-terminus
include:
##STR00024##
[0206] The capping group of an amino terminus includes an
unmodified amine (ie --NH.sub.2) or an amine with a substituent.
For example, the amino terminus can be substituted with an acyl
group to yield a carboxamide at the N-terminus. Various
substituents include but are not limited to substituted acyl
groups, including C.sub.1-C.sub.6 carbonyls, C.sub.7-C.sub.30
carbonyls, and pegylated carbamates. Representative capping groups
for the N-terminus include:
##STR00025##
[0207] The term "member" as used herein in conjunction with
macrocycles or macrocycle-forming linkers refers to the atoms that
form or can form the macrocycle, and excludes substituent or side
chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and
1,3-dimethyl cyclodecane are all considered ten-membered
macrocycles as the hydrogen or fluoro substituents or methyl side
chains do not participate in forming the macrocycle.
[0208] The symbol "" when used as part of a molecular structure
refers to a single bond or a trans or cis double bond.
[0209] The term "amino acid side chain" refers to a moiety attached
to the .alpha.-carbon (or another backbone atom) in an amino acid.
For example, the amino acid side chain for alanine is methyl, the
amino acid side chain for phenylalanine is phenylmethyl, the amino
acid side chain for cysteine is thiomethyl, the amino acid side
chain for aspartate is carboxymethyl, the amino acid side chain for
tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally
occurring amino acid side chains are also included, for example,
those that occur in nature (e.g., an amino acid metabolite) or
those that are made synthetically (e.g., an .alpha.,.alpha.
di-substituted amino acid).
[0210] The term ".alpha.,.alpha. di-substituted amino" acid refers
to a molecule or moiety containing both an amino group and a
carboxyl group bound to a carbon (the .alpha.-carbon) that is
attached to two natural or non-natural amino acid side chains
[0211] The term "polypeptide" encompasses two or more naturally or
non-naturally-occurring amino acids joined by a covalent bond
(e.g., an amide bond). Polypeptides as described herein include
full length proteins (e.g., fully processed proteins) as well as
shorter amino acid sequences (e.g., fragments of
naturally-occurring proteins or synthetic polypeptide
fragments).
[0212] The term "macrocyclization reagent" or "macrocycle-forming
reagent" as used herein refers to any reagent which can be used to
prepare a peptidomimetic macrocycle by mediating the reaction
between two reactive groups. Reactive groups can be, for example,
an azide and alkyne, in which case macrocyclization reagents
include, without limitation, Cu reagents such as reagents which
provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as
well as Cu(II) salts such as Cu(CO.sub.2CH.sub.3).sub.2,
CuSO.sub.4, and CuCl.sub.2 that can be converted in situ to an
active Cu(I) reagent by the addition of a reducing agent such as
ascorbic acid or sodium ascorbate. Macrocyclization reagents can
additionally include, for example, Ru reagents known in the art
such as Cp*RuCl(PPh.sub.3).sub.2, [Cp*RuCl].sub.4 or other Ru
reagents which can provide a reactive Ru(II) species. In other
cases, the reactive groups are terminal olefins. In such
embodiments, the macrocyclization reagents or macrocycle-forming
reagents are metathesis catalysts including, but not limited to,
stabilized, late transition metal carbene complex catalysts such as
Group VIII transition metal carbene catalysts. For example, such
catalysts are Ru and Os metal centers having a +2 oxidation state,
an electron count of 16 and pentacoordinated. In other examples,
catalysts have W or Mo centers. Various catalysts are disclosed in
Grubbs et al., "Ring Closing Metathesis and Related Processes in
Organic Synthesis" Acc. Chem. Res. 1995, 28, 446-452, U.S. Pat.
Nos. 5,811,515; 7,932,397; U.S. Application No. 2011/0065915; U.S.
Application No. 2011/0245477; Yu et al., "Synthesis of Macrocyclic
Natural Products by Catalyst-Controlled Stereoselective
Ring-Closing Metathesis," Nature 2011, 479, 88; and Peryshkov et
al., "Z-Selective Olefin Metathesis Reactions Promoted by Tungsten
Oxo Alkylidene Complexes," J. Am. Chem. Soc. 2011, 133, 20754. In
yet other cases, the reactive groups are thiol groups. In such
embodiments, the macrocyclization reagent is, for example, a linker
functionalized with two thiol-reactive groups such as halogen
groups.
[0213] The term "halo" or "halogen" refers to fluorine, chlorine,
bromine or iodine or a radical thereof.
[0214] The term "alkyl" refers to a hydrocarbon chain that is a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-C.sub.10 indicates that the
group has from 1 to 10 (inclusive) carbon atoms in it. In the
absence of any numerical designation, "alkyl" is a chain (straight
or branched) having 1 to 20 (inclusive) carbon atoms in it.
[0215] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--).
[0216] The term "alkenyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
double bonds. The alkenyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkenyl" refers to a C.sub.2-C.sub.6 alkenyl chain In the
absence of any numerical designation, "alkenyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0217] The term "alkynyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
triple bonds. The alkynyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkynyl" refers to a C.sub.2-C.sub.6 alkynyl chain In the
absence of any numerical designation, "alkynyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0218] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of
each ring are substituted by a substituent. Examples of aryl groups
include phenyl, naphthyl and the like. The term "arylalkoxy" refers
to an alkoxy substituted with aryl.
[0219] "Arylalkyl" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with a C.sub.1-C.sub.5 alkyl group, as defined above.
Representative examples of an arylalkyl group include, but are not
limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl,
2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl,
3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl,
4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl,
2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,
2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl,
2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl,
2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.
[0220] "Arylamido" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with one or more --C(O)NH.sub.2 groups. Representative examples of
an arylamido group include 2-C(O)NH.sub.2-phenyl,
3-C(O)NH.sub.2-phenyl, 4-C(O)NH.sub.2-phenyl,
2-C(O)NH.sub.2-pyridyl, 3-C(O)NH.sub.2-pyridyl, and
4-C(O)NH.sub.2-pyridyl,
[0221] "Alkylheterocycle" refers to a C.sub.1-C.sub.5 alkyl group,
as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a heterocycle. Representative
examples of an alkylheterocycle group include, but are not limited
to, --CH.sub.2CH.sub.2-morpholine, --CH.sub.2CH.sub.2-piperidine,
--CH.sub.2CH.sub.2CH.sub.2-morpholine, and
--CH.sub.2CH.sub.2CH.sub.2-imidazole.
[0222] "Alkylamido" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a --C(O)NH.sub.2 group.
Representative examples of an alkylamido group include, but are not
limited to, --CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.3,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--C(CH.sub.3).sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3--CH3, and
--CH.sub.2--CH.sub.2--NH--C(O)--CH.dbd.CH.sub.2.
[0223] "Alkanol" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a hydroxyl group.
Representative examples of an alkanol group include, but are not
limited to, --CH.sub.2OH, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2 CH.sub.2CH.sub.2OH,
--CH.sub.2CH(OH)CH.sub.3, --CH.sub.2CH(OH)CH.sub.2CH.sub.3,
--CH(OH)CH.sub.3 and --C(CH.sub.3).sub.2CH.sub.2OH.
[0224] "Alkylcarboxy" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a --COOH group.
Representative examples of an alkylcarboxy group include, but are
not limited to, --CH.sub.2COOH, --CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH, --CH.sub.2CH(COOH)CH.sub.3,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH(COOH)CH.sub.2CH.sub.3, --CH(COOH)CH.sub.2CH.sub.3 and
--C(CH.sub.3).sub.2CH.sub.2COOH.
[0225] The term "cycloalkyl" as employed herein includes saturated
and partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, preferably 3 to 8 carbons, and more preferably 3 to 6
carbons, wherein the cycloalkyl group additionally is optionally
substituted. Some cycloalkyl groups include, without limitation,
cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cyclohexenyl, cycloheptyl, and cyclooctyl.
[0226] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are
substituted by a substituent. Examples of heteroaryl groups include
pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl,
thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the
like.
[0227] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0228] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0229] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring are
substituted by a substituent. Examples of heterocyclyl groups
include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
tetrahydrofuranyl, and the like.
[0230] The term "substituent" refers to a group replacing a second
atom or group such as a hydrogen atom on any molecule, compound or
moiety. Suitable substituents include, without limitation, halo,
hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl,
aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido,
carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
[0231] In some embodiments, one or more compounds disclosed herein
contain one or more asymmetric centers and thus occur as racemates
and racemic mixtures, single enantiomers, individual diastereomers
and diastereomeric mixtures. In one embodiment isomeric forms of
these compounds are included in the present invention unless
expressly provided otherwise. In some embodiments, one or more
compounds disclosed herein are also represented in multiple
tautomeric forms, in such instances, the one or more compounds
includes all tautomeric forms of the compounds described herein
(e.g., if alkylation of a ring system results in alkylation at
multiple sites, the one or more compounds includes all such
reaction products). All such isomeric forms of such compounds are
included in the present invention unless expressly provided
otherwise. All crystal forms of the compounds described herein are
included in the present invention unless expressly provided
otherwise.
[0232] As used herein, the terms "increase" and "decrease" mean,
respectively, to cause a statistically significantly (i.e.,
p<0.1) increase or decrease of at least 5%.
[0233] As used herein, the recitation of a numerical range for a
variable is intended to convey that the invention can be practiced
with the variable equal to any of the values within that range.
Thus, for a variable which is inherently discrete, the variable is
equal to any integer value within the numerical range, including
the end-points of the range. Similarly, for a variable which is
inherently continuous, the variable is equal to any real value
within the numerical range, including the end-points of the range.
As an example, and without limitation, a variable which is
described as having values between 0 and 2 takes the values 0, 1 or
2 if the variable is inherently discrete, and takes the values 0.0,
0.1, 0.01, 0.001, or any other real values .gtoreq.0 and .ltoreq.2
if the variable is inherently continuous.
[0234] As used herein, unless specifically indicated otherwise, the
word "or" is used in the inclusive sense of "and/or" and not the
exclusive sense of "either/or."
[0235] The term "on average" represents the mean value derived from
performing at least three independent replicates for each data
point.
[0236] The term "biological activity" encompasses structural and
functional properties of a macrocycle. Biological activity is, for
example, structural stability, alpha-helicity, affinity for a
target, resistance to proteolytic degradation, cell penetrability,
intracellular stability, in vivo stability, or any combination
thereof.
[0237] The term "binding affinity" refers to the strength of a
binding interaction, for example between a peptidomimetic
macrocycle and a target. Binding affinity can be expressed, for
example, as an equilibrium dissociation constant ("K.sub.D"), which
is expressed in units which are a measure of concentration (e.g. M,
mM, nM etc). Numerically, binding affinity and K.sub.D values vary
inversely, such that a lower binding affinity corresponds to a
higher K.sub.D value, while a higher binding affinity corresponds
to a lower K.sub.D value. Where high binding affinity is desirable,
"improved" binding affinity refers to higher binding affinity and
therefoere lower K.sub.D values.
[0238] The term "ratio of binding affinities" refers to the ratio
of dissociation constants (K.sub.D values) of a first binding
interaction (the numerator), versus a second binding interaction
(denominator). Consequently, a "reduced ratio of binding
affinities" to Target 1 versus Target 2 refers to a lower value for
the ratio expressed as K.sub.D(Target 1)/K.sub.D(Target 2). This
concept can also be characterized as "improved selectivity" for
Target 1 versus Target 2, which can be due either to a decrease in
the K.sub.D value for Target 1 or an increase in the value for the
K.sub.D value for Target 2.
[0239] The term "in vitro efficacy" refers to the extent to which a
test compound, such as a peptidomimetic macrocycle, produces a
beneficial result in an in vitro test system or assay. In vitro
efficacy can be measured, for example, as an "IC.sub.50" or
"EC.sub.50" value, which represents the concentration of the test
compound which produces 50% of the maximal effect in the test
system.
[0240] The term "ratio of in vitro efficacies" or "in vitro
efficacy ratio" refers to the ratio of IC.sub.50 or EC.sub.50
values from a first assay (the numerator) versus a second assay
(the denominator). Consequently, an improved in vitro efficacy
ratio for Assay 1 versus Assay 2 refers to a lower value for the
ratio expressed as IC.sub.50(Assay 1)/IC.sub.50(Assay 2) or
alternatively as EC.sub.50(Assay 1)/EC.sub.50(Assay 2). This
concept can also be characterized as "improved selectivity" in
Assay 1 versus Assay 2, which can be due either to a decrease in
the IC.sub.50 or EC.sub.50 value for Target 1 or an increase in the
value for the IC.sub.50 or EC.sub.50 value for Target 2.
[0241] The details of one or more particular embodiments of the
invention are set forth in the accompanying drawings and the
description below. Other features, objects, and advantages of the
invention will be apparent from the description and drawings, and
from the claims.
[0242] Peptidomimetic Macrocycles
[0243] In some embodiments, a peptidomimetic macrocycle has the
Formula (I):
##STR00026##
[0244] wherein:
[0245] each A, C, D, and E is independently an amino acid;
[0246] B is an amino acid,
##STR00027##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0247] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00028##
[0248] L.sub.1, L.sub.2 and L.sub.3 are independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0249] each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5;
[0250] each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene;
[0251] each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0252] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0253] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0254] each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue;
[0255] each R.sub.8 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with an E
residue;
[0256] v and w are independently integers from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20 or 1-10;
[0257] u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
[0258] x, y and z are independently integers from 0-10, for example
the sum of x+y+z is 2, 3, or 6; and
[0259] n is an integer from 1-5.
[0260] In some embodiments, a peptidomimetic macrocycle has the
Formula:
##STR00029##
[0261] wherein:
[0262] each A, C, D, and E is independently an amino acid;
[0263] B is an amino acid,
##STR00030##
[--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-];
[0264] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0265] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0266] L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4--]n, each being unsubstituted or substituted
with R.sub.5;
[0267] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0268] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0269] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0270] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0271] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0272] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0273] v and w are independently integers from 1-1000, for example
1-500, 1-200, 1-100, 1-50, 1-30, 1-20 or 1-10;
[0274] u is an integer from 1-10, for example 1-5, 1-3 or 1-2;
[0275] x, y and z are independently integers from 0-10, for example
the sum of x+y+z is 2, 3, or 6; and
[0276] n is an integer from 1-5.
[0277] In some embodiments, v and w are integers between 1-30. In
some embodiments, w is an integer from 3-1000, for example 3-500,
3-200, 3-100, 3-50, 3-30, 3-20, or 3-10.
[0278] In some embodiments, the peptidomimetic macrocycles are
claimed with the proviso that when u=1 and w=2, the first
C-terminal amino acid represented by E is not an Arginine (R)
and/or the second C-terminal amino acid represented by E is not a
Threonine (T). For instance, when u=1 and w=2, the first C-terminal
amino acid and/or the second C-terminal amino acid represented by E
do not comprise a positively charged side chain or a polar
uncharged side chain In some embodiments, when u=1 and w=2, the
first C-terminal amino acid and/or the second C-terminal amino acid
represented by E comprise a hydrophobic side chain For example,
when w=2, the first C-terminal amino acid and/or the second
N-terminal amino acid represented by E comprise a hydrophobic side
chain, for example a large hydrophobic side chain
[0279] In some embodiments, w is between 3 and 1000. For example,
the third amino acid represented by E comprises a large hydrophobic
side chain
[0280] In some embodiments of a peptidomimetic macrocycle of
Formula I, L.sub.1 and L.sub.2, either alone or in combination, do
not form an all hydrocarbon chain or a thioether. In other
embodiments of a peptidomimetic macrocycle of Formula II, L.sub.1
and L.sub.2, either alone or in combination, do not form an all
hydrocarbon chain or a triazole.
[0281] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0282] In some embodiments, x+y+z is at least 3. In other
embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some
embodiments, the sum of x+y+z is 3 or 6. In some embodiments, the
sum of x+y+z is 3. In other embodiments, the sum of x+y+z is 6.
Each occurrence of A, B, C, D or E in a macrocycle or macrocycle
precursor is independently selected. For example, a sequence
represented by the formula [A].sub.x, when x is 3, encompasses
embodiments where the amino acids are not identical, e.g.
Gln-Asp-Ala as well as embodiments where the amino acids are
identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or
z in the indicated ranges. Similarly, when u is greater than 1,
each compound can encompass peptidomimetic macrocycles which are
the same or different. For example, a compound can comprise
peptidomimetic macrocycles comprising different linker lengths or
chemical compositions.
[0283] In some embodiments, the peptidomimetic macrocycle comprises
a secondary structure which is an .alpha.-helix and R.sub.8 is --H,
allowing intrahelical hydrogen bonding. In some embodiments, at
least one of A, B, C, D or E is an .alpha.,.alpha.-disubstituted
amino acid. In one example, B is an .alpha.,.alpha.-disubstituted
amino acid. For instance, at least one of A, B, C, D or E is
2-aminoisobutyric acid. In other embodiments, at least one of A, B,
C, D or E is
##STR00031##
[0284] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha..
[0285] Peptidomimetic macrocycles are also provided of the
formula:
##STR00032##
[0286] wherein:
[0287] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 1), where each X is an amino
acid;
[0288] each D and E is independently an amino acid;
[0289] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0290] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00033##
[0291] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0292] each R.sub.3 is independently hydrogen, alkyl, alkenyl,
alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5;
[0293] each R.sub.4 is independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
arylene, or heteroarylene;
[0294] each K is independently O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0295] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0296] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0297] each R.sub.7 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with a D
residue;
[0298] each R.sub.8 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally
substituted with R.sub.5, or part of a cyclic structure with an E
residue;
[0299] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20 or 1-10;
[0300] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0301] n is an integer from 1-5.
[0302] In some embodiments, the peptidomimetic macrocycle has the
Formula:
##STR00034##
[0303] wherein:
[0304] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 2), where each X
is an amino acid;
[0305] each D and E is independently an amino acid;
[0306] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0307] each L and L' is independently a macrocycle-forming linker
of the formula
##STR00035##
[0308] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5,
[0309] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0310] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0311] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0312] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0313] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0314] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0315] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0316] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0317] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0318] n is an integer from 1-5.
[0319] Peptidomimetic macrocycles are also provided of the
formula:
##STR00036##
[0320] wherein:
[0321] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-His.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10-X.sub.11-Ser.sub.12 (SEQ ID NO: 1), where each X is an amino
acid;
[0322] each D and E is independently an amino acid;
[0323] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0324] L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4-]n, each being unsubstituted or substituted
with R.sub.5;
[0325] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0326] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0327] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0328] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0329] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0330] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0331] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0332] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20 or 1-10;
[0333] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0334] n is an integer from 1-5.
[0335] Peptidomimetic macrocycles are also provided of the
formula:
##STR00037##
[0336] wherein:
[0337] each of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.7,
Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 is individually an amino acid,
wherein at least three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, and Xaa.sub.10 are the same amino
acid as the amino acid at the corresponding position of the
sequence
Phe.sub.3-X.sub.4-Glu.sub.5-Tyr.sub.6-Trp.sub.7-Ala.sub.8-Gln.sub.9-Leu.s-
ub.10/Cba.sub.10-X.sub.11-Ala.sub.12 (SEQ ID NO: 2), where each X
is an amino acid;
[0338] each D and E is independently an amino acid;
[0339] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; or at
least one of R.sub.1 and R.sub.2 forms a macrocycle-forming linker
L' connected to the alpha position of one of said D or E amino
acids;
[0340] L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently
alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4-]n, each being unsubstituted or substituted
with R.sub.5;
[0341] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0342] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0343] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0344] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0345] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0346] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0347] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0348] v is an integer from 1-1000, for example 1-500, 1-200,
1-100, 1-50, 1-30, 1-20, or 1-10;
[0349] w is an integer from 3-1000, for example 3-500, 3-200,
3-100, 3-50, 3-30, 3-20, or 3-10; and
[0350] n is an integer from 1-5.
[0351] In one embodiment, the peptidomimetic macrocycle is:
##STR00038##
[0352] wherein each R.sub.1 and R.sub.2 is independently
independently --H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-.
[0353] In related embodiments, the peptidomimetic macrocycle
is:
##STR00039##
[0354] wherein each R.sub.1' and R.sub.2' is independently an amino
acid.
[0355] In other embodiments, the peptidomimetic macrocycle is a
compound of any of the formulas shown below:
##STR00040## ##STR00041##
wherein "AA" represents any natural or non-natural amino acid side
chain and "" is [D].sub.v, [E].sub.w as defined above, and n is an
integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some
embodiments, n is 0. In other embodiments, n is less than 50.
[0356] Exemplary embodiments of the macrocycle-forming linker L for
peptidomimetic macrocycles of Formula I are shown below.
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050##
[0357] In other embodiments, D and/or E in a compound of Formula I
or II are further modified in order to facilitate cellular uptake.
In some embodiments, lipidating or PEGylating a peptidomimetic
macrocycle facilitates cellular uptake, increases bioavailability,
increases blood circulation, alters pharmacokinetics, decreases
immunogenicity and/or decreases the needed frequency of
administration.
[0358] In other embodiments, at least one of [D] and [E] in a
compound of Formula I or II represents a moiety comprising an
additional macrocycle-forming linker such that the peptidomimetic
macrocycle comprises at least two macrocycle-forming linkers. In a
specific embodiment, a peptidomimetic macrocycle comprises two
macrocycle-forming linkers. In an embodiment, u is 2.
[0359] In some embodiments, any of the macrocycle-forming linkers
described herein can be used in any combination with any of the
sequences shown in Tables 4, 4a, 4b, 6, and 6a and also with any of
the R-substituents indicated herein.
[0360] In some embodiments, the peptidomimetic macrocycle comprises
at least one .alpha.-helix motif. For example, A, B and/or C in a
compound of Formula I or II include one or more .alpha.-helices. As
a general matter, .alpha.-helices include between 3 and 4 amino
acid residues per turn. In some embodiments, the .alpha.-helix of
the peptidomimetic macrocycle includes 1 to 5 turns and, therefore,
3 to 20 amino acid residues. In specific embodiments, the
.alpha.-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5
turns. In some embodiments, the macrocycle-forming linker
stabilizes an .alpha.-helix motif included within the
peptidomimetic macrocycle. Thus, in some embodiments, the length of
the macrocycle-forming linker L from a first C.alpha. to a second
C.alpha. is selected to increase the stability of an .alpha.-helix.
In some embodiments, the macrocycle-forming linker spans from 1
turn to 5 turns of the .alpha.-helix. In some embodiments, the
macrocycle-forming linker spans approximately 1 turn, 2 turns, 3
turns, 4 turns, or 5 turns of the .alpha.-helix. In some
embodiments, the length of the macrocycle-forming linker is
approximately 5 .ANG. to 9 .ANG. per turn of the .alpha.-helix, or
approximately 6 .ANG. to 8 .ANG. per turn of the .alpha.-helix.
Where the macrocycle-forming linker spans approximately 1 turn of
an .alpha.-helix, the length is equal to approximately 5
carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7
carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 2 turns of an .alpha.-helix, the length is equal to
approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds,
approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or
approximately 12 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 3 turns of an .alpha.-helix, the length
is equal to approximately 14 carbon-carbon bonds to 22
carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20
carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where
the macrocycle-forming linker spans approximately 4 turns of an
.alpha.-helix, the length is equal to approximately 20
carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22
carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 5 turns of an .alpha.-helix, the length is equal to
approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds,
approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or
approximately 30 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 1 turn of an .alpha.-helix, the linkage
contains approximately 4 atoms to 12 atoms, approximately 6 atoms
to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming
linker spans approximately 2 turns of the .alpha.-helix, the
linkage contains approximately 7 atoms to 15 atoms, approximately 9
atoms to 13 atoms, or approximately 11 atoms. Where the
macrocycle-forming linker spans approximately 3 turns of the
.alpha.-helix, the linkage contains approximately 13 atoms to 21
atoms, approximately 15 atoms to 19 atoms, or approximately 17
atoms. Where the macrocycle-forming linker spans approximately 4
turns of the .alpha.-helix, the linkage contains approximately 19
atoms to 27 atoms, approximately 21 atoms to 25 atoms, or
approximately 23 atoms. Where the macrocycle-forming linker spans
approximately 5 turns of the .alpha.-helix, the linkage contains
approximately 25 atoms to 33 atoms, approximately 27 atoms to 31
atoms, or approximately 29 atoms. Where the macrocycle-forming
linker spans approximately 1 turn of the .alpha.-helix, the
resulting macrocycle forms a ring containing approximately 17
members to 25 members, approximately 19 members to 23 members, or
approximately 21 members. Where the macrocycle-forming linker spans
approximately 2 turns of the .alpha.-helix, the resulting
macrocycle forms a ring containing approximately 29 members to 37
members, approximately 31 members to 35 members, or approximately
33 members. Where the macrocycle-forming linker spans approximately
3 turns of the .alpha.-helix, the resulting macrocycle forms a ring
containing approximately 44 members to 52 members, approximately 46
members to 50 members, or approximately 48 members. Where the
macrocycle-forming linker spans approximately 4 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 59 members to 67 members, approximately 61 members to
65 members, or approximately 63 members. Where the
macrocycle-forming linker spans approximately 5 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 74 members to 82 members, approximately 76 members to
80 members, or approximately 78 members.
[0361] Unless otherwise stated, any compounds (including
peptidomimetic macrocycles, macrocycle precursors, and other
compositions) are also meant to encompass compounds which differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the described structures except for
the replacement of a hydrogen by a deuterium or tritium, or the
replacement of a carbon by .sup.13C- or .sup.14C-enriched carbon
are contemplated herein.
[0362] In some embodiments, the peptidomimetic macrocycle has
improved binding affinity to MDM2 or MDMX relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
other instances, the peptidomimetic macrocycle has a reduced ratio
of binding affinities to MDMX versus MDM2 relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
still other instances, the peptidomimetic macrocycle has improved
in vitro anti-tumor efficacy against p53 positive tumor cell lines
relative to a corresponding peptidomimetic macrocycle where w is 0,
1 or 2. In some embodiments, the peptidomimetic macrocycle shows
improved in vitro induction of apoptosis in p53 positive tumor cell
lines relative to a corresponding peptidomimetic macrocycle where w
is 0, 1 or 2. In other instances, the peptidomimetic macrocycle of
claim 1, wherein the peptidomimetic macrocycle has an improved in
vitro anti-tumor efficacy ratio for p53 positive versus p53
negative or mutant tumor cell lines relative to a corresponding
peptidomimetic macrocycle where w is 0, 1 or 2. In still other
instances, the peptidomimetic macrocycle has improved in vivo
anti-tumor efficacy against p53 positive tumors relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
yet other instances, the peptidomimetic macrocycle has improved in
vivo induction of apoptosis in p53 positive tumors relative to a
corresponding peptidomimetic macrocycle where w is 0, 1 or 2. In
some embodiments, the peptidomimetic macrocycle has improved cell
permeability relative to a corresponding peptidomimetic macrocycle
where w is 0, 1 or 2. In other cases, the peptidomimetic macrocycle
has improved solubility relative to a corresponding peptidomimetic
macrocycle where w is 0, 1 or 2.
[0363] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof. In some embodiments, Xaa.sub.5 is Glu or an amino
acid analog thereof and wherein the peptidomimetic macrocycle has
an improved property, such as improved binding affinity, improved
solubility, improved cellular efficacy, improved cell permeability,
improved in vivo or in vitro anti-tumor efficacy, or improved
induction of apoptosis relative to a corresponding peptidomimetic
macrocycle where Xaa.sub.5 is Ala.
[0364] In some embodiments, the peptidomimetic macrocycle has
improved binding affinity to MDM2 or MDMX relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala. In
other embodiments, the peptidomimetic macrocycle has a reduced
ratio of binding affinities to MDMX vs MDM2 relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala. In
some embodiments, the peptidomimetic macrocycle has improved
solubility relative to a corresponding peptidomimetic macrocycle
where Xaa.sub.5 is Ala, or the peptidomimetic macrocycle has
improved cellular efficacy relative to a corresponding
peptidomimetic macrocycle where Xaa.sub.5 is Ala.
[0365] In some embodiments, Xaa.sub.5 is Glu or an amino acid
analog thereof and wherein the peptidomimetic macrocycle has
improved biological activity, such as improved binding affinity,
improved solubility, improved cellular efficacy, improved helicity,
improved cell permeability, improved in vivo or in vitro anti-tumor
efficacy, or improved induction of apoptosis relative to a
corresponding peptidomimetic macrocycle where Xaa.sub.5 is Ala.
[0366] In one embodiment, a compound disclosed herein can contain
unnatural proportions of atomic isotopes at one or more of atoms
that constitute such compounds. For example, the compounds can be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). In
another embodiment, a compound disclosed herein can have one or
more carbon atoms replaced with a silicon atom. All isotopic
variations of the compounds disclosed herein, whether radioactive
or not, are contemplated herein.
[0367] Preparation of Peptidomimetic Macrocycles
[0368] Peptidomimetic macrocycles of Formulas I and II can be
prepared by any of a variety of methods known in the art. For
example, macrocycles of Formula I having residues indicated by
"$4rn6" or "$4a5" in Table 4, Table 4a or Table 4b can be
substituted with a residue capable of forming a crosslinker with a
second residue in the same molecule or a precursor of such a
residue.
[0369] In some embodiments, the synthesis of these peptidomimetic
macrocycles involves a multi-step process that features the
synthesis of a peptidomimetic precursor containing an azide moiety
and an alkyne moiety; followed by contacting the peptidomimetic
precursor with a macrocyclization reagent to generate a
triazole-linked peptidomimetic macrocycle. Such a process is
described, for example, in U.S. application Ser. No. 12/037,041,
filed on Feb. 25, 2008. Macrocycles or macrocycle precursors are
synthesized, for example, by solution phase or solid-phase methods,
and can contain both naturally-occurring and
non-naturally-occurring amino acids. See, for example, Hunt, "The
Non-Protein Amino Acids" in Chemistry and Biochemistry of the Amino
Acids, edited by G.C. Barrett, Chapman and Hall, 1985.
[0370] In some embodiments of macrocycles of Formula I, an azide is
linked to the .alpha.-carbon of a residue and an alkyne is attached
to the .alpha.-carbon of another residue. In some embodiments, the
azide moieties are azido-analogs of amino acids L-lysine, D-lysine,
alpha-methyl-L-lysine, alpha-methyl-D-lysine, L-ornithine,
D-ornithine, alpha-methyl-L-ornithine or alpha-methyl-D-ornithine.
In another embodiment, the alkyne moiety is L-propargylglycine. In
yet other embodiments, the alkyne moiety is an amino acid selected
from the group consisting of L-propargylglycine,
D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,
(R)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-2-methyl-5-hexynoic acid,
(R)-2-amino-2-methyl-5-hexynoic acid,
(S)-2-amino-2-methyl-6-heptynoic acid,
(R)-2-amino-2-methyl-6-heptynoic acid,
(S)-2-amino-2-methyl-7-octynoic acid,
(R)-2-amino-2-methyl-7-octynoic acid,
(S)-2-amino-2-methyl-8-nonynoic acid and
(R)-2-amino-2-methyl-8-nonynoic acid.
[0371] In some embodiments, provided herein is a method for
synthesizing a peptidomimetic macrocycle of Formula I, the method
comprising the steps of contacting a peptidomimetic precursor of
formulas:
##STR00051##
[0372] with a macrocyclization reagent;
[0373] wherein v, w, x, y, z, A, B, C, D, E, R.sub.1, R.sub.2,
R.sub.7, R.sub.8, L.sub.1 and L.sub.2 are as defined above;
R.sub.12 is --H when the macrocyclization reagent is a Cu reagent
and R.sub.12 is --H or alkyl when the macrocyclization reagent is a
Ru reagent; and further wherein said contacting step results in a
covalent linkage being formed between the alkyne and azide moiety
in the precursor. For example, R.sub.12 may be methyl when the
macrocyclization reagent is a Ru reagent.
[0374] In some embodiments, provided herein is a method for
synthesizing a peptidomimetic macrocycle of Formula II, the method
comprising the steps of contacting a peptidomimetic precursor of
formula:
##STR00052##
[0375] with a compound formula X-L.sub.2-Y,
[0376] wherein v, w, x, y, z, A, B, C, D, E, R.sub.1, R.sub.2,
R.sub.7, R.sub.8, L.sub.1 and L.sub.2 are as defined for the
compound of formula II; and X and Y are each independently a
reactive group capable of reacting with a thiol group;
[0377] and further wherein said contacting step results in a
covalent linkage being formed between the two thiol groups in
Formula III.
[0378] In the peptidomimetic macrocycles disclosed herein, at least
one of R.sub.1 and R.sub.2 is alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-. In some embodiments, both
R.sub.1 and R.sub.2 are independently alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid.
[0379] For example, at least one of R.sub.1 and R.sub.2 is alkyl,
unsubstituted or substituted with halo-. In another example, both
R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl. The macrocyclization reagent may be a Cu
reagent or a Ru reagent.
[0380] In some embodiments, the peptidomimetic precursor is
purified prior to the contacting step. In other embodiments, the
peptidomimetic macrocycle is purified after the contacting step. In
still other embodiments, the peptidomimetic macrocycle is refolded
after the contacting step. The method may be performed in solution,
or, alternatively, the method may be performed on a solid
support.
[0381] Also envisioned herein is performing the method disclosed
herein in the presence of a target macromolecule that binds to the
peptidomimetic precursor or peptidomimetic macrocycle under
conditions that favor said binding. In some embodiments, the method
is performed in the presence of a target macromolecule that binds
preferentially to the peptidomimetic precursor or peptidomimetic
macrocycle under conditions that favor said binding. The method may
also be applied to synthesize a library of peptidomimetic
macrocycles.
[0382] In some embodiments, an alkyne moiety of the peptidomimetic
precursor for making a compound of Formula I is a sidechain of an
amino acid selected from the group consisting of
L-propargylglycine, D-propargylglycine,
(S)-2-amino-2-methyl-4-pentynoic acid,
(R)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-2-methyl-5-hexynoic acid,
(R)-2-amino-2-methyl-5-hexynoic acid,
(S)-2-amino-2-methyl-6-heptynoic acid,
(R)-2-amino-2-methyl-6-heptynoic acid,
(S)-2-amino-2-methyl-7-octynoic acid,
(R)-2-amino-2-methyl-7-octynoic acid,
(S)-2-amino-2-methyl-8-nonynoic acid, and
(R)-2-amino-2-methyl-8-nonynoic acid. In other embodiments, an
azide moiety of the peptidomimetic precursor for making a compound
of Formula I is a sidechain of an amino acid selected from the
group consisting of .epsilon.-azido-L-lysine,
.epsilon.-azido-D-lysine, .epsilon.-azido-.alpha.-methyl-L-lysine,
.epsilon.-azido-.alpha.-methyl-D-lysine,
.delta.-azido-.alpha.-methyl-L-ornithine, and
.delta.-azido-.alpha.-methyl-D-ornithine.
[0383] In some embodiments, a thiol group of the peptidomimetic
precursor for making a compound of Formula II is a sidechain of an
amino acid selected from the group consisting of L-cysteine,
D-cysteine, L-N-methylcysteine, D-N-methylcysteine, L-homocysteine,
D-homocysteine, L-N-methylhomocysteine, D-N-methylhomocysteine,
.alpha.-methyl-L-cysteine, .alpha.-methyl-D-cysteine,
.alpha.-methyl-L-homocysteine, .alpha.-methyl-D-homocysteine,
L-penicillamine, D-penicillamine, L-N-methylpenicillamine,
D-N-methylpenicillamine and all forms suitably protected for liquid
or solid phase peptide synthesis.
[0384] In some embodiments, x+y+z is 3, and and A, B and C are
independently natural or non-natural amino acids. In other
embodiments, x+y+z is 6, and and A, B and C are independently
natural or non-natural amino acids.
[0385] In some embodiments, the contacting step is performed in a
solvent selected from the group consisting of protic solvent,
aqueous solvent, organic solvent, and mixtures thereof. For
example, the solvent may be chosen from the group consisting of
H.sub.2O, THF, THF/H.sub.2O, tBuOH/H.sub.2O, DMF, DIPEA, CH.sub.3CN
or CH.sub.2Cl.sub.2, ClCH.sub.2CH.sub.2Cl or a mixture thereof. The
solvent may be a solvent which favors helix formation.
[0386] Alternative but equivalent protecting groups, leaving groups
or reagents are substituted, and certain of the synthetic steps are
performed in alternative sequences or orders to produce the desired
compounds. Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds described herein include, for example, those such as
described in Larock, Comprehensive Organic Transformations, VCH
Publishers (1989); Greene and Wuts, Protective Groups in Organic
Synthesis, 2d. Ed., John Wiley and Sons (1991); Fieser and Fieser,
Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and
Sons (1994); and Paquette, ed., Encyclopedia of Reagents for
Organic Synthesis, John Wiley and Sons (1995), and subsequent
editions thereof.
[0387] The peptidomimetic macrocycles disclosed herein are made,
for example, by chemical synthesis methods, such as described in
Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed.
Grant, W. H. Freeman & Co., New York, N. Y., 1992, p. 77.
Hence, for example, peptides are synthesized using the automated
Merrifield techniques of solid phase synthesis with the amine
protected by either tBoc or Fmoc chemistry using side chain
protected amino acids on, for example, an automated peptide
synthesizer (e.g., Applied Biosystems (Foster City, Calif.), Model
430A, 431, or 433).
[0388] One manner of producing the peptidomimetic precursors and
peptidomimetic macrocycles described herein uses solid phase
peptide synthesis (SPPS). The C-terminal amino acid is attached to
a cross-linked polystyrene resin via an acid labile bond with a
linker molecule. This resin is insoluble in the solvents used for
synthesis, making it relatively simple and fast to wash away excess
reagents and by-products. The N-terminus is protected with the Fmoc
group, which is stable in acid, but removable by base. Side chain
functional groups are protected as necessary with base stable, acid
labile groups.
[0389] Longer peptidomimetic precursors are produced, for example,
by conjoining individual synthetic peptides using native chemical
ligation. Alternatively, the longer synthetic peptides are
biosynthesized by well known recombinant DNA and protein expression
techniques. Such techniques are provided in well-known standard
manuals with detailed protocols. To construct a gene encoding a
peptidomimetic precursor disclosed herein, the amino acid sequence
is reverse translated to obtain a nucleic acid sequence encoding
the amino acid sequence, preferably with codons that are optimum
for the organism in which the gene is to be expressed. Next, a
synthetic gene is made, typically by synthesizing oligonucleotides
which encode the peptide and any regulatory elements, if necessary.
The synthetic gene is inserted in a suitable cloning vector and
transfected into a host cell. The peptide is then expressed under
suitable conditions appropriate for the selected expression system
and host. The peptide is purified and characterized by standard
methods.
[0390] The peptidomimetic precursors are made, for example, in a
high-throughput, combinatorial fashion using, for example, a
high-throughput polychannel combinatorial synthesizer (e.g.,
Thuramed TETRAS multichannel peptide synthesizer from CreoSalus,
Louisville, Ky. or Model Apex 396 multichannel peptide synthesizer
from AAPPTEC, Inc., Louisville, Ky.).
[0391] The following synthetic schemes are provided solely to
illustrate the present invention and are not intended to limit the
scope of the invention, as described herein.
[0392] Synthetic schemes 1-5 describe the preparation of
peptidomimetic macrocycles of Formula I. To simplify the drawings,
the illustrative schemes depict azido amino acid analogs
.epsilon.-azido-.alpha.-methyl-L-lysine and
.epsilon.-azido-.alpha.-methyl-D-lysine, and alkyne amino acid
analogs L-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,
and (S)-2-amino-2-methyl-6-heptynoic acid. Thus, in the following
synthetic schemes, each R.sub.1, R.sub.2, R.sub.7 and R.sub.8 is
--H; each L.sub.1 is --(CH.sub.2).sub.4--; and each L.sub.2 is
--(CH.sub.2)--. However, as noted throughout the detailed
description above, many other amino acid analogs can be employed in
which R.sub.1, R.sub.2, R.sub.7, R.sub.8, L.sub.1 and L.sub.2 can
be independently selected from the various structures disclosed
herein.
##STR00053## ##STR00054## ##STR00055## ##STR00056##
[0393] Synthetic Scheme 1 describes the preparation of several
compounds useful for preparing compounds of Formula I as disclosed
herein. Ni(II) complexes of Schiff bases derived from the chiral
auxiliary (S)-2-[N-(N'-benzylprolyl)amino]benzophenone (BPB) and
amino acids such as glycine or alanine are prepared as described in
Belokon et al. (1998), Tetrahedron Asymm. 9:4249-4252. The
resulting complexes are subsequently reacted with alkylating
reagents comprising an azido or alkynyl moiety to yield
enantiomerically enriched compounds disclosed herein. If desired,
the resulting compounds can be protected for use in peptide
synthesis.
##STR00057##
[0394] In the general method for the synthesis of peptidomimetic
macrocycles of Formula I shown in Synthetic Scheme 2, the
peptidomimetic precursor contains an azide moiety and an alkyne
moiety and is synthesized by solution-phase or solid-phase peptide
synthesis (SPPS) using the commercially available amino acid
N-.alpha.-Fmoc-L-propargylglycine and the N-.alpha.-Fmoc-protected
forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic
acid, N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine. The peptidomimetic precursor is
then deprotected and cleaved from the solid-phase resin by standard
conditions (e.g., strong acid such as 95% TFA). The peptidomimetic
precursor is reacted as a crude mixture or is purified prior to
reaction with a macrocyclization reagent such as a Cu(I) in organic
or aqueous solutions (Rostovtsev et al. (2002), Angew. Chem. Int.
Ed. 41:2596-2599; Tornoe et al. (2002), J. Org. Chem. 67:3057-3064;
Deiters et al. (2003), J. Am. Chem. Soc. 125:11782-11783; Punna et
al. (2005), Angew. Chem. Int. Ed. 44:2215-2220). In one embodiment,
the triazole forming reaction is performed under conditions that
favor .alpha.-helix formation. In one embodiment, the
macrocyclization step is performed in a solvent chosen from the
group consisting of H.sub.2O, THF, CH.sub.3CN, DMF , DIPEA, tBuOH
or a mixture thereof. In another embodiment, the macrocyclization
step is performed in DMF. In some embodiments, the macrocyclization
step is performed in a buffered aqueous or partially aqueous
solvent.
##STR00058##
[0395] In the general method for the synthesis of peptidomimetic
macrocycles of Formula I shown in Synthetic Scheme 3, the
peptidomimetic precursor contains an azide moiety and an alkyne
moiety and is synthesized by solid-phase peptide synthesis (SPPS)
using the commercially available amino acid
N-.alpha.-Fmoc-L-propargylglycine and the N-.alpha.-Fmoc-protected
forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic
acid, N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine. The peptidomimetic precursor is
reacted with a macrocyclization reagent such as a Cu(I) reagent on
the resin as a crude mixture (Rostovtsev et al. (2002), Angew.
Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J. Org. Chem.
67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc.
125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed.
44:2215-2220). The resultant triazole-containing peptidomimetic
macrocycle is then deprotected and cleaved from the solid-phase
resin by standard conditions (e.g., strong acid such as 95% TFA).
In some embodiments, the macrocyclization step is performed in a
solvent chosen from the group consisting of CH.sub.2Cl.sub.2,
ClCH.sub.2CH.sub.2Cl, DMF, THF, NMP, DIPEA, 2,6-lutidine, pyridine,
DMSO, H.sub.2O or a mixture thereof. In some embodiments, the
macrocyclization step is performed in a buffered aqueous or
partially aqueous solvent.
##STR00059##
[0396] In the general method for the synthesis of peptidomimetic
macrocycles of Formula I shown in Synthetic Scheme 4, the
peptidomimetic precursor contains an azide moiety and an alkyne
moiety and is synthesized by solution-phase or solid-phase peptide
synthesis (SPPS) using the commercially available amino acid
N-.alpha.-Fmoc-L-propargylglycine and the N-.alpha.-Fmoc-protected
forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic
acid, N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine. The peptidomimetic precursor is
then deprotected and cleaved from the solid-phase resin by standard
conditions (e.g., strong acid such as 95% TFA). The peptidomimetic
precursor is reacted as a crude mixture or is purified prior to
reaction with a macrocyclization reagent such as a Ru(II) reagents,
for example Cp*RuCl(PPh.sub.3).sub.2 or [Cp*RuCl].sub.4 (Rasmussen
et al. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am.
Chem. Soc. 127:15998-15999). In some embodiments, the
macrocyclization step is performed in a solvent chosen from the
group consisting of DMF, CH.sub.3CN and THF.
##STR00060##
[0397] In the general method for the synthesis of peptidomimetic
macrocycles of Formula I shown in Synthetic Scheme 5, the
peptidomimetic precursor contains an azide moiety and an alkyne
moiety and is synthesized by solid-phase peptide synthesis (SPPS)
using the commercially available amino
acidN-.alpha.-Fmoc-L-propargylglycine and the
N-.alpha.-Fmoc-protected forms of the amino acids
(S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic
acid, (S)-2-amino-2-methyl-6-heptynoic acid,
N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine. The peptidomimetic precursor is
reacted with a macrocyclization reagent such as a Ru(II) reagent on
the resin as a crude mixture. For example, the reagent can be
Cp*RuCl(PPh.sub.3).sub.2 or [Cp*RuCl].sub.4 (Rasmussen et al.
(2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem.
Soc. 127:15998-15999). In some embodiments, the macrocyclization
step is performed in a solvent chosen from the group consisting of
CH.sub.2Cl.sub.2, ClCH.sub.2CH.sub.2Cl, CH.sub.3CN, DMF, and
THF.
[0398] In some embodiments, a peptidomimetic macrocycle of Formula
I comprises a halogen group substitution on a triazole moiety, for
example an iodo substitution. Such peptidomimetic macrocycles may
be prepared from a precursor having the partial structure and using
the cross-linking methods taught herein. Crosslinkers of any
length, as described herein, may be prepared comprising such
substitutions. In one embodiment, the peptidomimetic macrocycle is
prepared according to the scheme shown below. The reaction is
performed, for example, in the presence of CuI and an amine ligand
such as TEA or TTTA. See, e.g., Hein et al. Angew. Chem., Int. Ed.
2009, 48, 8018-8021.
##STR00061##
[0399] In other embodiments, an iodo-substituted triazole is
generated according to the scheme shown below. For example, the
second step in the reaction scheme below is performed using, for
example, CuI and N-bromosuccinimide (NBS) in the presence of THF
(see, e.g. Zhang et al., J. Org. Chem. 2008, 73, 3630-3633). In
other embodiments, the second step in the reaction scheme shown
below is performed, for example, using CuI and an iodinating agent
such as ICl (see, e.g. Wu et al., Synthesis 2005, 1314-1318.)
##STR00062##
[0400] In some embodiments, an iodo-substituted triazole moiety is
used in a cross-coupling reaction, such as a Suzuki or Sonogashira
coupling, to afford a peptidomimetic macrocycle comprising a
substituted crosslinker. Sonogashira couplings using an alkyne as
shown below may be performed, for example, in the presence of a
palladium catalyst such as Pd(PPh.sub.3).sub.2Cl.sub.2, CuI, and in
the presence of a base such as triethylamine Suzuki couplings using
an arylboronic or substituted alkenyl boronic acid as shown below
may be performed, for example, in the presence of a catalyst such
as Pd(PPh.sub.3).sub.4, and in the presence of a base such as
K.sub.2CO.sub.3.
##STR00063##
[0401] Any suitable triazole substituent groups which reacts with
the iodo-substituted triazole can be used in Suzuki couplings
described herein. Example triazole substituents for use in Suzuki
couplings are shown below:
##STR00064##
[0402] wherein "Cyc" is a suitable aryl, cycloalkyl, cycloalkenyl,
heteroaryl, or heterocyclyl group, unsubstituted or optionally
substituted with an R.sub.a or R.sub.b group as described
below.
[0403] In some embodiments, the substituent is:
##STR00065##
[0404] Any suitable substituent group which reacts with the
iodo-substituted triazole can be used in Sonogashira couplings
described herein. Example triazole substituents for use in
Sonogashira couplings are shown below:
##STR00066##
[0405] wherein "Cyc" is a suitable aryl, cycloalkyl, cycloalkenyl,
heteroaryl, or heterocyclyl group, unsubstituted or optionally
substituted with an R.sub.a or R.sub.b group as described
below.
[0406] In some embodiments, the triazole substituent is:
##STR00067##
[0407] In some embodiments, the Cyc group shown above is further
substituted by at least one R.sub.a or R.sub.b substituent. In some
embodiments, at least one of R.sub.a and R.sub.b is
independently:
[0408] R.sub.a or R.sub.b.dbd.H, OCH.sub.3, CF.sub.3, NH.sub.2,
CH.sub.2NH.sub.2, F, Br, I
##STR00068##
[0409] In other embodiments, the triazole substituent is
##STR00069##
and at least one of R.sub.a and R.sub.b is alkyl (including
hydrogen, methyl, or ethyl), or:
##STR00070##
[0410] The present invention contemplates the use of
non-naturally-occurring amino acids and amino acid analogs in the
synthesis of the peptidomimetic macrocycles of Formula I described
herein. Any amino acid or amino acid analog amenable to the
synthetic methods employed for the synthesis of stable triazole
containing peptidomimetic macrocycles can be used in the present
invention. For example, L-propargylglycine is contemplated as a
useful amino acid in the present invention. However, other
alkyne-containing amino acids that contain a different amino acid
side chain are also useful in the invention. For example,
L-propargylglycine contains one methylene unit between the
.alpha.-carbon of the amino acid and the alkyne of the amino acid
side chain. The invention also contemplates the use of amino acids
with multiple methylene units between the .alpha.-carbon and the
alkyne. Also, the azido-analogs of amino acids L-lysine, D-lysine,
alpha-methyl-L-lysine, and alpha-methyl-D-lysine are contemplated
as useful amino acids in the present invention. However, other
terminal azide amino acids that contain a different amino acid side
chain are also useful in the invention. For example, the
azido-analog of L-lysine contains four methylene units between the
.alpha.-carbon of the amino acid and the terminal azide of the
amino acid side chain The invention also contemplates the use of
amino acids with fewer than or greater than four methylene units
between the .alpha.-carbon and the terminal azide. The following
Table 1 shows some amino acids useful in the preparation of
peptidomimetic macrocycles disclosed herein.
TABLE-US-00002 TABLE 1 ##STR00071## ##STR00072## ##STR00073##
##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##
##STR00079## ##STR00080## ##STR00081## ##STR00082## ##STR00083##
##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088##
##STR00089## ##STR00090##
[0411] Table 1 shows exemplary amino acids useful in the
preparation of peptidomimetic macrocycles disclosed herein.
[0412] In some embodiments the amino acids and amino acid analogs
are of the D-configuration. In other embodiments they are of the
L-configuration. In some embodiments, some of the amino acids and
amino acid analogs contained in the peptidomimetic are of the
D-configuration while some of the amino acids and amino acid
analogs are of the L-configuration. In some embodiments the amino
acid analogs are .alpha.,.alpha.-disubstituted, such as
.alpha.-methyl-L-propargylglycine,
.alpha.-methyl-D-propargylglycine,
.epsilon.-azido-alpha-methyl-L-lysine, and
.epsilon.-azido-alpha-methyl-D-lysine. In some embodiments the
amino acid analogs are N-alkylated, e.g.,
N-methyl-L-propargylglycine, N-methyl-D-propargylglycine,
N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine.
[0413] The preparation of macrocycles of Formula II is described,
for example, in U.S. application Ser. No. 11/957,325, filed on Dec.
17, 2007 and herein incorporated by reference. Synthetic Schemes
6-9 describe the preparation of such compounds of Formula II. To
simplify the drawings, the illustrative schemes depict amino acid
analogs derived from L-or D-cysteine, in which L.sub.1 and L.sub.3
are both --(CH.sub.2)--. However, as noted throughout the detailed
description above, many other amino acid analogs can be employed in
which L.sub.1 and L.sub.3 can be independently selected from the
various structures disclosed herein. The symbols "[AA].sub.m",
"[AA].sub.n", "[AA].sub.o" represent a sequence of amide
bond-linked moieties such as natural or unnatural amino acids. As
described previously, each occurrence of "AA" is independent of any
other occurrence of "AA", and a formula such as "[AA].sub.m"
encompasses, for example, sequences of non-identical amino acids as
well as sequences of identical amino acids.
##STR00091##
[0414] In Scheme 6, the peptidomimetic precursor contains two --SH
moieties and is synthesized by solid-phase peptide synthesis (SPPS)
using commercially available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-trityl-L-cysteine or
N-.alpha.-Fmoc-S-trityl-D-cysteine. Alpha-methylated versions of
D-cysteine or L-cysteine are generated by known methods (Seebach et
al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and
references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-trityl monomers by known methods
("Bioorganic Chemistry: Peptides and Proteins", Oxford University
Press, New York: 1998, the entire contents of which are
incorporated herein by reference). The precursor peptidomimetic is
then deprotected and cleaved from the solid-phase resin by standard
conditions (e.g., strong acid such as 95% TFA). The precursor
peptidomimetic is reacted as a crude mixture or is purified prior
to reaction with X-L.sub.2-Y in organic or aqueous solutions. In
some embodiments the alkylation reaction is performed under dilute
conditions (i.e. 0.15 mmol/L) to favor macrocyclization and to
avoid polymerization. In some embodiments, the alkylation reaction
is performed in organic solutions such as liquid NH.sub.3 (Mosberg
et al. (1985), J. Am.Chem. Soc. 107:2986-2987; Szewczuk et al.
(1992), Int. J. Peptide Protein Res. 40:233-242), NH.sub.3/MeOH, or
NH.sub.3/DMF (Or et al. (1991), J. Org. Chem. 56:3146-3149). In
other embodiments, the alkylation is performed in an aqueous
solution such as 6M guanidinium HCL, pH 8 (Brunel et al. (2005),
Chem. Commun (20):2552-2554). In other embodiments, the solvent
used for the alkylation reaction is DMF or dichloroethane.
##STR00092##
[0415] In Scheme 7, the precursor peptidomimetic contains two or
more --SH moieties, of which two are specially protected to allow
their selective deprotection and subsequent alkylation for
macrocycle formation. The precursor peptidomimetic is synthesized
by solid-phase peptide synthesis (SPPS) using commercially
available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-p-methoxytrityl-L-cysteine or
N-.alpha.-Fmoc-S-p-methoxytrityl-D-cysteine. Alpha-methylated
versions of D-cysteine or L-cysteine are generated by known methods
(Seebach et al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748,
and references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-p-methoxytrityl monomers by known
methods (Bioorganic Chemistry: Peptides and Proteins, Oxford
University Press, New York: 1998, the entire contents of which are
incorporated herein by reference). The Mmt protecting groups of the
peptidomimetic precursor are then selectively cleaved by standard
conditions (e.g., mild acid such as 1% TFA in DCM). The precursor
peptidomimetic is then reacted on the resin with X-L.sub.2-Y in an
organic solution. For example, the reaction takes place in the
presence of a hindered base such as diisopropylethylamine In some
embodiments, the alkylation reaction is performed in organic
solutions such as liquid NH.sub.3 (Mosberg et al. (1985), J. Am.
Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J. Peptide
Protein Res. 40:233-242), NH.sub.3/MeOH or NH.sub.3/DMF (Or et al.
(1991), J. Org. Chem. 56:3146-3149). In other embodiments, the
alkylation reaction is performed in DMF or dichloroethane. The
peptidomimetic macrocycle is then deprotected and cleaved from the
solid-phase resin by standard conditions (e.g., strong acid such as
95% TFA).
##STR00093##
[0416] In Scheme 8, the peptidomimetic precursor contains two or
more -SH moieties, of which two are specially protected to allow
their selective deprotection and subsequent alkylation for
macrocycle formation. The peptidomimetic precursor is synthesized
by solid-phase peptide synthesis (SPPS) using commercially
available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-p-methoxytrityl-L-cysteine,
N-.alpha.-Fmoc-S-p-methoxytrityl-D-cysteine,
N-.alpha.-Fmoc-S-S-t-butyl-L-cysteine, and
N-.alpha.-Fmoc-S-S-t-butyl-D-cysteine. Alpha-methylated versions of
D-cysteine or L-cysteine are generated by known methods (Seebach et
al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and
references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-p-methoxytrityl or
N-.alpha.-Fmoc-S-S-t-butyl monomers by known methods (Bioorganic
Chemistry: Peptides and Proteins, Oxford University Press, New
York: 1998, the entire contents of which are incorporated herein by
reference). The S-S-tButyl protecting group of the peptidomimetic
precursor is selectively cleaved by known conditions (e.g., 20%
2-mercaptoethanol in DMF, reference: Galande et al. (2005), J.
Comb. Chem. 7:174-177). The precursor peptidomimetic is then
reacted on the resin with a molar excess of X-L.sub.2-Y in an
organic solution. For example, the reaction takes place in the
presence of a hindered base such as diisopropylethylamine. The Mmt
protecting group of the peptidomimetic precursor is then
selectively cleaved by standard conditions (e.g., mild acid such as
1% TFA in DCM). The peptidomimetic precursor is then cyclized on
the resin by treatment with a hindered base in organic solutions.
In some embodiments, the alkylation reaction is performed in
organic solutions such as NH.sub.3/MeOH or NH.sub.3/DMF (Or et al.
(1991), J. Org. Chem. 56:3146-3149). The peptidomimetic macrocycle
is then deprotected and cleaved from the solid-phase resin by
standard conditions (e.g., strong acid such as 95% TFA).
##STR00094##
[0417] In Scheme 9, the peptidomimetic precursor contains two
L-cysteine moieties. The peptidomimetic precursor is synthesized by
known biological expression systems in living cells or by known in
vitro, cell-free, expression methods. The precursor peptidomimetic
is reacted as a crude mixture or is purified prior to reaction with
X-L2-Y in organic or aqueous solutions. In some embodiments the
alkylation reaction is performed under dilute conditions (i.e. 0.15
mmol/L) to favor macrocyclization and to avoid polymerization. In
some embodiments, the alkylation reaction is performed in organic
solutions such as liquid NH.sub.3 (Mosberg et al. (1985), J. Am.
Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J. Peptide
Protein Res. 40:233-242), NH.sub.3/MeOH, or NH.sub.3/DMF (Or et al.
(1991), J. Org. Chem. 56:3146-3149). In other embodiments, the
alkylation is performed in an aqueous solution such as 6M
guanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.
(20):2552-2554). In other embodiments, the alkylation is performed
in DMF or dichloroethane. In another embodiment, the alkylation is
performed in non-denaturing aqueous solutions, and in yet another
embodiment the alkylation is performed under conditions that favor
.alpha.-helical structure formation. In yet another embodiment, the
alkylation is performed under conditions that favor the binding of
the precursor peptidomimetic to another protein, so as to induce
the formation of the bound .alpha.-helical conformation during the
alkylation.
[0418] Various embodiments for X and Y are envisioned which are
suitable for reacting with thiol groups. In general, each X or Y is
independently be selected from the general category shown in Table
2. For example, X and Y are halides such as --Cl, --Br or --I. Any
of the macrocycle-forming linkers described herein may be used in
any combination with any of the sequences shown and also with any
of the R-substituents indicated herein.
TABLE-US-00003 TABLE 2 Examples of Reactive Groups Capable of
Reacting with Thiol Groups and Resulting Linkages Resulting
Covalent X or Y Linkage acrylamide Thioether halide (e.g. alkyl or
aryl Thioether halide) sulfonate Thioether aziridine Thioether
epoxide Thioether haloacetamide Thioether maleimide Thioether
sulfonate ester Thioether
[0419] The present invention contemplates the use of both
naturally-occurring and non-naturally-occurring amino acids and
amino acid analogs in the synthesis of the peptidomimetic
macrocycles of Formula II. Any amino acid or amino acid analog
amenable to the synthetic methods employed for the synthesis of
stable bis-sulfhydryl containing peptidomimetic macrocycles can be
used in the present invention. For example, cysteine is
contemplated as a useful amino acid in the present invention.
However, sulfur containing amino acids other than cysteine that
contain a different amino acid side chain are also useful. For
example, cysteine contains one methylene unit between the
.alpha.-carbon of the amino acid and the terminal --SH of the amino
acid side chain The invention also contemplates the use of amino
acids with multiple methylene units between the .alpha.-carbon and
the terminal --SH. Non-limiting examples include
.alpha.-methyl-L-homocysteine and .alpha.-methyl-D-homocysteine. In
some embodiments the amino acids and amino acid analogs are of the
D-configuration. In other embodiments they are of the
L-configuration. In some embodiments, some of the amino acids and
amino acid analogs contained in the peptidomimetic are of the
D-configuration while some of the amino acids and amino acid
analogs are of the L-configuration. In some embodiments the amino
acid analogs are .alpha.,.alpha.-disubstituted, such as
.alpha.-methyl-L-cysteine and .alpha.-methyl-D-cysteine.
[0420] The invention includes macrocycles in which
macrocycle-forming linkers are used to link two or more --SH
moieties in the peptidomimetic precursors to form the
peptidomimetic macrocycles disclosed herein. As described above,
the macrocycle-forming linkers impart conformational rigidity,
increased metabolic stability and/or increased cell penetrability.
Furthermore, in some embodiments, the macrocycle-forming linkages
stabilize the .alpha.-helical secondary structure of the
peptidomimetic macrocyles. The macrocycle-forming linkers are of
the formula X-L.sub.2-Y, wherein both X and Y are the same or
different moieties, as defined above. Both X and Y have the
chemical characteristics that allow one macrocycle-forming linker
-L.sub.2-to bis alkylate the bis-sulfhydryl containing
peptidomimetic precursor. As defined above, the linker
-L.sub.2-includes alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene, or
heterocycloarylene, or --R.sub.4--K--R.sub.4--, all of which can be
optionally substituted with an R.sub.5 group, as defined above.
Furthermore, one to three carbon atoms within the
macrocycle-forming linkers -L.sub.2-, other than the carbons
attached to the --SH of the sulfhydryl containing amino acid, are
optionally substituted with a heteroatom such as N, S or O.
[0421] The L.sub.2 component of the macrocycle-forming linker
X-L.sub.2-Y may be varied in length depending on, among other
things, the distance between the positions of the two amino acid
analogs used to form the peptidomimetic macrocycle. Furthermore, as
the lengths of L.sub.1 and/or L.sub.3 components of the
macrocycle-forming linker are varied, the length of L.sub.2 can
also be varied in order to create a linker of appropriate overall
length for forming a stable peptidomimetic macrocycle. For example,
if the amino acid analogs used are varied by adding an additional
methylene unit to each of L.sub.1 and L.sub.3, the length of
L.sub.2 are decreased in length by the equivalent of approximately
two methylene units to compensate for the increased lengths of
L.sub.1 and L.sub.3.
[0422] In some embodiments, L.sub.2 is an alkylene group of the
formula --(CH.sub.2).sub.n--, where n is an integer between about 1
and about 15. For example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In
other embodiments, L.sub.2 is an alkenylene group. In still other
embodiments, L.sub.2 is an aryl group.
[0423] Table 3 shows additional embodiments of X-L.sub.2-Y
groups.
TABLE-US-00004 TABLE 3 Exemplary X--L.sub.2--Y groups. ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105##
##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110##
##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115##
##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120##
##STR00121## ##STR00122## ##STR00123## ##STR00124## ##STR00125##
##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130##
##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135##
##STR00136## ##STR00137## ##STR00138## ##STR00139## ##STR00140##
##STR00141## ##STR00142## ##STR00143## Each X and Y in this table,
is, for example, independently Cl--, Br-- or I--.
[0424] Additional methods of forming peptidomimetic macrocycles
which are envisioned as suitable include those disclosed by
Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68, pp.
8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp.
1403-1406; U.S. Pat. Nos. 5,364,851; 5,446,128; 5,824,483;
6,713,280; and 7,202,332. In such embodiments, aminoacid precursors
are used containing an additional substituent R-- at the alpha
position. Such aminoacids are incorporated into the macrocycle
precursor at the desired positions, which can be at the positions
where the crosslinker is substituted or, alternatively, elsewhere
in the sequence of the macrocycle precursor. Cyclization of the
precursor is then effected according to the indicated method.
[0425] In some embodiments, the --NH moiety of the amino acid is
protected using a protecting group, including without limitation
-Fmoc and -Boc. In other embodiments, the amino acid is not
protected prior to synthesis of the peptidomimetic macrocycle.
[0426] Assays
[0427] The properties of peptidomimetic macrocycles are assayed,
for example, by using the methods described below. In some
embodiments, a peptidomimetic macrocycle has improved biological
properties relative to a corresponding polypeptide lacking the
substituents described herein.
[0428] Assay to Determine .alpha.-Helicity
[0429] In solution, the secondary structure of polypeptides with
.alpha.-helical domains will reach a dynamic equilibrium between
random coil structures and .alpha.-helical structures, often
expressed as a "percent helicity". Thus, for example, alpha-helical
domains are predominantly random coils in solution, with
.alpha.-helical content usually under 25%. Peptidomimetic
macrocycles with optimized linkers, on the other hand, possess, for
example, an alpha-helicity that is at least two-fold greater than
that of a corresponding uncrosslinked polypeptide. In some
embodiments, macrocycles will possess an alpha-helicity of greater
than 50%. To assay the helicity of peptidomimetic macrocyles, the
compounds are dissolved in an aqueous solution (e.g. 50 mM
potassium phosphate solution at pH 7, or distilled H.sub.2O, to
concentrations of 25-50 .mu.M). Circular dichroism (CD) spectra are
obtained on a spectropolarimeter (e.g., Jasco J-710) using standard
measurement parameters (e.g. temperature, 20.degree. C.;
wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec;
accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length,
0.1 cm). The .alpha.-helical content of each peptide is calculated
by dividing the mean residue ellipticity (e.g. [.PHI.]222obs) by
the reported value for a model helical decapeptide (Yang et al.
(1986), Methods Enzymol. 130:208)).
[0430] Assay to Determine Melting Temperature (Tm).
[0431] A peptidomimetic macrocycle comprising a secondary structure
such as an .alpha.-helix exhibits, for example, a higher melting
temperature than a corresponding uncrosslinked polypeptide.
Typically peptidomimetic macrocycles exhibit Tm of >60.degree.
C. representing a highly stable structure in aqueous solutions. To
assay the effect of macrocycle formation on melting temperature,
peptidomimetic macrocycles or unmodified peptides are dissolved in
distilled H.sub.2O (e.g. at a final concentration of 50 .mu.M) and
the Tm is determined by measuring the change in ellipticity over a
temperature range (e.g. 4 to 95.degree. C.) on a spectropolarimeter
(e.g., Jasco J-710) using standard parameters (e.g. wavelength 222
nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10;
response, 1 sec; bandwidth, 1 nm; temperature increase rate:
1.degree. C./min; path length, 0.1 cm).
[0432] Protease Resistance Assay.
[0433] The amide bond of the peptide backbone is susceptible to
hydrolysis by proteases, thereby rendering peptidic compounds
vulnerable to rapid degradation in vivo. Peptide helix formation,
however, typically buries the amide backbone and therefore can
shield it from proteolytic cleavage. The peptidomimetic macrocycles
can be subjected to in vitro trypsin proteolysis to assess for any
change in degradation rate compared to a corresponding
uncrosslinked polypeptide. For example, the peptidomimetic
macrocycle and a corresponding uncrosslinked polypeptide are
incubated with trypsin agarose and the reactions quenched at
various time points by centrifugation and subsequent HPLC injection
to quantitate the residual substrate by ultraviolet absorption at
280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic
precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E
.about.125) for 0, 10, 20, 90, and 180 minutes. Reactions are
quenched by tabletop centrifugation at high speed; remaining
substrate in the isolated supernatant is quantified by HPLC-based
peak detection at 280 nm. The proteolytic reaction displays first
order kinetics and the rate constant, k, is determined from a plot
of ln[S] versus time (k=-1.times.slope).
[0434] Ex Vivo Stability Assay.
[0435] Peptidomimetic macrocycles with optimized linkers possess,
for example, an ex vivo half-life that is at least two-fold greater
than that of a corresponding uncrosslinked polypeptide, and possess
an ex vivo half-life of 12 hours or more. For ex vivo serum
stability studies, a variety of assays can be used. For example, a
peptidomimetic macrocycle and a corresponding uncrosslinked
polypeptide (2 mcg) are incubated with fresh mouse, rat and/or
human serum (2 mL) at 37.degree. C. for 0, 1, 2, 4, 8, and 24
hours. To determine the level of intact compound, the following
procedure can be used: The samples are extracted by transferring
100 .mu.l of sera to 2 ml centrifuge tubes followed by the addition
of 10 .mu.L of 50% formic acid and 500 .mu.L acetonitrile and
centrifugation at 14,000 RPM for 10 min at 4.+-.2.degree. C. The
supernatants are then transferred to fresh 2 ml tubes and
evaporated on Turbovap under N.sub.2<10 psi, 37.degree. C. The
samples are reconstituted in 100 .mu.L of 50:50 acetonitrile:water
and submitted to LC-MS/MS analysis.
[0436] In Vitro Binding Assays.
[0437] To assess the binding and affinity of peptidomimetic
macrocycles and peptidomimetic precursors to acceptor proteins, a
fluorescence polarization assay (FPA) issued, for example. The FPA
technique measures the molecular orientation and mobility using
polarized light and fluorescent tracer. When excited with polarized
light, fluorescent tracers (e.g., FITC) attached to molecules with
high apparent molecular weights (e.g. FITC-labeled peptides bound
to a large protein) emit higher levels of polarized fluorescence
due to their slower rates of rotation as compared to fluorescent
tracers attached to smaller molecules (e.g. FITC-labeled peptides
that are free in solution).
[0438] For example, fluoresceinated peptidomimetic macrocycles (25
nM) are incubated with the acceptor protein (25-1000 nM) in binding
buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room
temperature. Binding activity is measured, for example, by
fluorescence polarization on a luminescence spectrophotometer (e.g.
Perkin-Elmer LS50B). Kd values can be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic
macrocycle shows, In some embodiments, similar or lower Kd than a
corresponding uncrosslinked polypeptide.
[0439] In Vitro Displacement Assays to Characterize Antagonists of
Peptide-Protein Interactions.
[0440] To assess the binding and affinity of compounds that
antagonize the interaction between a peptide and an acceptor
protein, a fluorescence polarization assay (FPA) utilizing a
fluoresceinated peptidomimetic macrocycle derived from a
peptidomimetic precursor sequence is used, for example. The FPA
technique measures the molecular orientation and mobility using
polarized light and fluorescent tracer. When excited with polarized
light, fluorescent tracers (e.g., FITC) attached to molecules with
high apparent molecular weights (e.g. FITC-labeled peptides bound
to a large protein) emit higher levels of polarized fluorescence
due to their slower rates of rotation as compared to fluorescent
tracers attached to smaller molecules (e.g. FITC-labeled peptides
that are free in solution). A compound that antagonizes the
interaction between the fluoresceinated peptidomimetic macrocycle
and an acceptor protein will be detected in a competitive binding
FPA experiment.
[0441] For example, putative antagonist compounds (1 nM to 1 mM)
and a fluoresceinated peptidomimetic macrocycle (25 nM) are
incubated with the acceptor protein (50 nM) in binding buffer (140
mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room
temperature. Antagonist binding activity is measured, for example,
by fluorescence polarization on a luminescence spectrophotometer
(e.g. Perkin-Elmer LS50B). Kd values can be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.).
[0442] Any class of molecule, such as small organic molecules,
peptides, oligonucleotides or proteins can be examined as putative
antagonists in this assay.
[0443] Assay for Protein-Ligand Binding by Affinity Selection-Mass
Spectrometry
[0444] To assess the binding and affinity of test compounds for
proteins, an affinity-selection mass spectrometry assay is used,
for example. Protein-ligand binding experiments are conducted
according to the following representative procedure outlined for a
system-wide control experiment using 1 .mu.M peptidomimetic
macrocycle plus 5 .mu.M hMDM2. A 1 .mu.L DMSO aliquot of a 40 .mu.M
stock solution of peptidomimetic macrocycle is dissolved in 19
.mu.L of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate
buffer containing 150 mM NaCl). The resulting solution is mixed by
repeated pipetting and clarified by centrifugation at 10 000 g for
10 min. To a 4 .mu.L aliquot of the resulting supernatant is added
4 .mu.L of 10 .mu.M hMDM2 in PBS. Each 8.0 .mu.L experimental
sample thus contains 40 pmol (1.5 .mu.g) of protein at 5.0 .mu.M
concentration in PBS plus 1 .mu.M peptidomimetic macrocycle and
2.5% DMSO. Duplicate samples thus prepared for each concentration
point are incubated for 60 min at room temperature, and then
chilled to 4.degree. C. prior to size-exclusion
chromatography-LC-MS analysis of 5.0 .mu.L injections. Samples
containing a target protein, protein-ligand complexes, and unbound
compounds are injected onto an SEC column, where the complexes are
separated from non-binding component by a rapid SEC step. The SEC
column eluate is monitored using UV detectors to confirm that the
early-eluting protein fraction, which elutes in the void volume of
the SEC column, is well resolved from unbound components that are
retained on the column After the peak containing the protein and
protein-ligand complexes elutes from the primary UV detector, it
enters a sample loop where it is excised from the flow stream of
the SEC stage and transferred directly to the LC-MS via a valving
mechanism. The (M+3H).sup.3+ ion of the peptidomimetic macrocycle
is observed by ESI-MS at the expected m/z, confirming the detection
of the protein-ligand complex.
[0445] Assay for Protein-Ligand Kd Titration Experiments.
[0446] To assess the binding and affinity of test compounds for
proteins, a protein-ligand Kd titration experiment is performed,
for example. Protein-ligand K.sub.d titrations experiments are
conducted as follows: 2 .mu.L DMSO aliquots of a serially diluted
stock solution of titrant peptidomimetic macrocycle (5, 2.5, . . .
, 0.098 mM) are prepared then dissolved in 38 .mu.L of PBS. The
resulting solutions are mixed by repeated pipetting and clarified
by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L aliquots of
the resulting supernatants is added 4.0 .mu.L of 10 .mu.M hMDM2 in
PBS. Each 8.0 .mu.L experimental sample thus contains 40 pmol (1.5
.mu.g) of protein at 5.0 .mu.M concentration in PBS, varying
concentrations (125, 62.5, . . . , 0.24 .mu.M) of the titrant
peptide, and 2.5% DMSO. Duplicate samples thus prepared for each
concentration point are incubated at room temperature for 30 min,
then chilled to 4.degree. C. prior to SEC-LC-MS analysis of 2.0
.mu.L injections. The (M+H).sup.1-, (M+2H).sup.2+, (M+3H).sup.3+,
and/or (M+Na).sup.1+ ion is observed by ESI-MS; extracted ion
chromatograms are quantified, then fit to equations to derive the
binding affinity K.sub.d as described in "A General Technique to
Rank Protein-Ligand Binding Affinities and Determine Allosteric vs.
Direct Binding Site Competition in Compound Mixtures." Annis, D.
A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am.
Chem. Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity
Selection Mass Spectrometry System for the Discovery and
Characterization of Protein-Ligand Interactions" D. A. Annis, C.-C.
Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.
Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184 Mannhold R,
Kubinyi H, Folkers G (Series Editors): Methods and Principles in
Medicinal Chemistry.
[0447] Assay for Competitive Binding Experiments by Affinity
Selection-Mass Spectrometry
[0448] To determine the ability of test compounds to bind
competitively to proteins, an affinity selection mass spectrometry
assay is performed, for example. A mixture of ligands at 40 .mu.M
per component is prepared by combining 2 .mu.L aliquots of 400
.mu.M stocks of each of the three compounds with 14 .mu.L of DMSO.
Then, 1 .mu.L aliquots of this 40 .mu.M per component mixture are
combined with 1 .mu.L DMSO aliquots of a serially diluted stock
solution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . ,
0.078 mM). These 2 .mu.L samples are dissolved in 38 .mu.L of PBS.
The resulting solutions were mixed by repeated pipetting and
clarified by centrifugation at 10 000 g for 10 min. To 4.0 .mu.L
aliquots of the resulting supernatants is added 4.0 .mu.L of 10
.mu.M hMDM2 protein in PBS. Each 8.0 .mu.L experimental sample thus
contains 40 pmol (1.5 .mu.g) of protein at 5.0 .mu.M concentration
in PBS plus 0.5 .mu.M ligand, 2.5% DMSO, and varying concentrations
(125, 62.5, . . . , 0.98 .mu.M) of the titrant peptidomimetic
macrocycle. Duplicate samples thus prepared for each concentration
point are incubated at room temperature for 60 min, then chilled to
4.degree. C. prior to SEC-LC-MS analysis of 2.0 .mu.L injections.
Additional details on these and other methods are provided in "A
General Technique to Rank Protein-Ligand Binding Affinities and
Determine Allosteric vs. Direct Binding Site Competition in
Compound Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott,
M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also
in "ALIS: An Affinity Selection Mass Spectrometry System for the
Discovery and Characterization of Protein-Ligand Interactions" D.
A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in
Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH;
2007:121-184 Mannhold R, Kubinyi H, Folkers G (Series Editors):
Methods and Principles in Medicinal Chemistry.
[0449] Binding Assays in Intact Cells.
[0450] It is possible to measure binding of peptides or
peptidomimetic macrocycles to their natural acceptors in intact
cells by immunoprecipitation experiments. For example, intact cells
are incubated with fluoresceinated (FITC-labeled) compounds for 4
hrs in the absence of serum, followed by serum replacement and
further incubation that ranges from 4-18 hrs. Cells are then
pelleted and incubated in lysis buffer (50 mM Tris [pH 7.6], 150 mM
NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at
4.degree. C. Extracts are centrifuged at 14,000 rpm for 15 minutes
and supernatants collected and incubated with 10 .mu.l goat
anti-FITC antibody for 2 hrs, rotating at 4.degree. C. followed by
further 2 hrs incubation at 4.degree. C. with protein A/G Sepharose
(50 .mu.l of 50% bead slurry). After quick centrifugation, the
pellets are washed in lysis buffer containing increasing salt
concentration (e.g., 150, 300, 500 mM). The beads are then
re-equilibrated at 150 mM NaCl before addition of SDS-containing
sample buffer and boiling. After centrifugation, the supernatants
are optionally electrophoresed using 4%-12% gradient Bis-Tris gels
followed by transfer into Immobilon-P membranes. After blocking,
blots are optionally incubated with an antibody that detects FITC
and also with one or more antibodies that detect proteins that bind
to the peptidomimetic macrocycle.
[0451] Cellular Penetrability Assays.
[0452] A peptidomimetic macrocycle is, for example, more cell
penetrable compared to a corresponding uncrosslinked macrocycle.
Peptidomimetic macrocycles with optimized linkers possess, for
example, cell penetrability that is at least two-fold greater than
a corresponding uncrosslinked macrocycle, and often 20% or more of
the applied peptidomimetic macrocycle will be observed to have
penetrated the cell after 4 hours. To measure the cell
penetrability of peptidomimetic macrocycles and corresponding
uncrosslinked macrocycle, intact cells are incubated with
fluorescently-labeled (e.g. fluoresceinated) peptidomimetic
macrocycles or corresponding uncrosslinked macrocycle (10 .mu.M)
for 4 hrs in serum free media at 37.degree. C., washed twice with
media and incubated with trypsin (0.25%) for 10 min at 37.degree.
C. The cells are washed again and resuspended in PBS. Cellular
fluorescence is analyzed, for example, by using either a
FACSCalibur flow cytometer or Cellomics' KineticScan .RTM. HCS
Reader.
[0453] Cellular Efficacy Assays.
[0454] The efficacy of certain peptidomimetic macrocycles is
determined, for example, in cell-based killing assays using a
variety of tumorigenic and non-tumorigenic cell lines and primary
cells derived from human or mouse cell populations. Cell viability
is monitored, for example, over 24-96 hrs of incubation with
peptidomimetic macrocycles (0.5 to 50 .mu.M) to identify those that
kill at EC.sub.50<10 .mu.M. Several standard assays that measure
cell viability are commercially available and are optionally used
to assess the efficacy of the peptidomimetic macrocycles. In
addition, assays that measure Annexin V and caspase activation are
optionally used to assess whether the peptidomimetic macrocycles
kill cells by activating the apoptotic machinery. For example, the
Cell Titer-glo assay is used which determines cell viability as a
function of intracellular ATP concentration.
[0455] In Vivo Stability Assay.
[0456] To investigate the in vivo stability of the peptidomimetic
macrocycles, the compounds are, for example,administered to mice
and/or rats by IV, IP, PO or inhalation routes at concentrations
ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0',
5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection.
Levels of intact compound in 25 .mu.L of fresh serum are then
measured by LC-MS/MS as above.
[0457] In Vivo Efficacy in Animal Models.
[0458] To determine the anti-oncogenic activity of peptidomimetic
macrocycles in vivo, the compounds are, for example, given alone
(IP, IV, PO, by inhalation or nasal routes) or in combination with
sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,
doxorubicin, etoposide). In one example, 5.times.10.sup.6 RS4; 11
cells (established from the bone marrow of a patient with acute
lymphoblastic leukemia) that stably express luciferase are injected
by tail vein in NOD-SCID mice 3 hrs after they have been subjected
to total body irradiation. If left untreated, this form of leukemia
is fatal in 3 weeks in this model. The leukemia is readily
monitored, for example, by injecting the mice with D-luciferin (60
mg/kg) and imaging the anesthetized animals (e g., Xenogen In Vivo
Imaging System, Caliper Life Sciences, Hopkinton, Mass.). Total
body bioluminescence is quantified by integration of photonic flux
(photons/sec) by Living Image Software (Caliper Life Sciences,
Hopkinton, Mass.). Peptidomimetic macrocycles alone or in
combination with sub-optimal doses of relevant chemotherapeutics
agents are, for example, administered to leukemic mice (10 days
after injection/day 1 of experiment, in bioluminescence range of
14-16) by tail vein or IP routes at doses ranging from 0.1mg/kg to
50 mg/kg for 7 to 21 days. Optionally, the mice are imaged
throughout the experiment every other day and survival monitored
daily for the duration of the experiment. Expired mice are
optionally subjected to necropsy at the end of the experiment.
Another animal model is implantation into NOD-SCID mice of DoHH2, a
cell line derived from human follicular lymphoma, that stably
expresses luciferase. These in vivo tests optionally generate
preliminary pharmacokinetic, pharmacodynamic and toxicology
data.
[0459] Clinical Trials.
[0460] To determine the suitability of the peptidomimetic
macrocycles for treatment of humans, clinical trials are performed.
For example, patients diagnosed with cancer and in need of
treatment can be selected and separated in treatment and one or
more control groups, wherein the treatment group is administered a
peptidomimetic macrocycle, while the control groups receive a
placebo or a known anti-cancer drug. The treatment safety and
efficacy of the peptidomimetic macrocycles can thus be evaluated by
performing comparisons of the patient groups with respect to
factors such as survival and quality-of-life. In this example, the
patient group treated with a peptidomimetic macrocyle can show
improved long-term survival compared to a patient control group
treated with a placebo.
[0461] Pharmaceutical Compositions and Routes of Administration
[0462] Pharmaceutical compositions disclosed herein include
peptidomimetic macrocycles and pharmaceutically acceptable
derivatives or prodrugs thereof. A "pharmaceutically acceptable
derivative" means any pharmaceutically acceptable salt, ester, salt
of an ester, pro-drug or other derivative of a compound disclosed
herein which, upon administration to a recipient, is capable of
providing (directly or indirectly) a compound disclosed herein.
Particularly favored pharmaceutically acceptable derivatives are
those that increase the bioavailability of the compounds when
administered to a mammal (e.g., by increasing absorption into the
blood of an orally administered compound) or which increases
delivery of the active compound to a biological compartment (e.g.,
the brain or lymphatic system) relative to the parent species. Some
pharmaceutically acceptable derivatives include a chemical group
which increases aqueous solubility or active transport across the
gastrointestinal mucosa.
[0463] In some embodiments, peptidomimetic macrocycles are modified
by covalently or non-covalently joining appropriate functional
groups to enhance selective biological properties. Such
modifications include those which increase biological penetration
into a given biological compartment (e.g., blood, lymphatic system,
central nervous system), increase oral availability, increase
solubility to allow administration by injection, alter metabolism,
and alter rate of excretion.
[0464] Pharmaceutically acceptable salts of the compounds disclosed
herein include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, benzoate, benzenesulfonate,
butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate,
glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,
hydrobromide, hydroiodide, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
palmoate, phosphate, picrate, pivalate, propionate, salicylate,
succinate, sulfate, tartrate, tosylate and undecanoate. Salts
derived from appropriate bases include alkali metal (e.g., sodium),
alkaline earth metal (e.g., magnesium), ammonium and
N-(alkyl).sub.4.sup.+ salts.
[0465] For preparing pharmaceutical compositions from the compounds
provided herein, pharmaceutically acceptable carriers include
either solid or liquid carriers. Solid form preparations include
powders, tablets, pills, capsules, cachets, suppositories, and
dispersible granules. A solid carrier can be one or more
substances, which also acts as diluents, flavoring agents, binders,
preservatives, tablet disintegrating agents, or an encapsulating
material. Details on techniques for formulation and administration
are well described in the scientific and patent literature, see,
e.g., the latest edition of Remington's Pharmaceutical Sciences,
Maack Publishing Co, Easton Pa.
[0466] In powders, the carrier is a finely divided solid, which is
in a mixture with the finely divided active component. In tablets,
the active component is mixed with the carrier having the necessary
binding properties in suitable proportions and compacted in the
shape and size desired.
[0467] Suitable solid excipients are carbohydrate or protein
fillers include, but are not limited to sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
and gums including arabic and tragacanth; as well as proteins such
as gelatin and collagen. If desired, disintegrating or solubilizing
agents are added, such as the cross-linked polyvinyl pyrrolidone,
agar, alginic acid, or a salt thereof, such as sodium alginate.
[0468] Liquid form preparations include, without limitation,
solutions, suspensions, and emulsions, for example, water or
water/propylene glycol solutions. For parenteral injection, liquid
preparations can be formulated in solution in aqueous polyethylene
glycol solution.
[0469] The pharmaceutical preparation can be in unit dosage form.
In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged form.
[0470] When one or more compositions disclosed herein comprise a
combination of a peptidomimetic macrocycle and one or more
additional therapeutic or prophylactic agents, both the compound
and the additional agent should be present at dosage levels of
between about 1 to 100%, and more preferably between about 5 to 95%
of the dosage normally administered in a monotherapy regimen. In
some embodiments, the additional agents are administered
separately, as part of a multiple dose regimen, from one or more
compounds disclosed herein. Alternatively, those agents are part of
a single dosage form, mixed together with one or more compounds
disclosed herein in a single composition.
[0471] Methods of Use
[0472] In one aspect, provided herein are novel peptidomimetic
macrocycles that are useful in competitive binding assays to
identify agents which bind to the natural ligand(s) of the proteins
or peptides upon which the peptidomimetic macrocycles are modeled.
For example, in the p53/MDMX system, labeled peptidomimetic
macrocycles based on p53 can be used in a MDMX binding assay along
with small molecules that competitively bind to MDMX. Competitive
binding studies allow for rapid in vitro evaluation and
determination of drug candidates specific for the p53/MDMX system.
Such binding studies can be performed with any of the
peptidomimetic macrocycles disclosed herein and their binding
partners.
[0473] Provided herein is the generation of antibodies against the
peptidomimetic macrocycles. In some embodiments, these antibodies
specifically bind both the peptidomimetic macrocycle and the
precursor peptides, such as p53, to which the peptidomimetic
macrocycles are related. Such antibodies, for example, disrupt the
native protein-protein interaction, for example, binding between
p53 and MDMX.
[0474] In other aspects, provided herein are both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant (e.g., insufficient or excessive) expression or activity
of the molecules including p53, MDM2 or MDMX.
[0475] In another embodiment, a disorder is caused, at least in
part, by an abnormal level of p53 or MDM2 or MDMX, (e.g., over or
under expression), or by the presence of p53 or MDM2 or MDMX
exhibiting abnormal activity. As such, the reduction in the level
and/or activity of p53 or MDM2 or MDMX, or the enhancement of the
level and/or activity of p53 or MDM2 or MDMX, by peptidomimetic
macrocycles derived from p53, is used, for example, to ameliorate
or reduce the adverse symptoms of the disorder.
[0476] In another aspect, provided herein are methods for treating
or preventing a disease including hyperproliferative disease and
inflammatory disorder by interfering with the interaction or
binding between binding partners, for example, between p53 and MDM2
or p53 and MDMX. These methods comprise administering an effective
amount of a compound to a warm blooded animal, including a human In
some embodiments, the administration of one or more compounds
disclosed herein induces cell growth arrest or apoptosis.
[0477] As used herein, the term "treatment" is defined as the
application or administration of a therapeutic agent to a patient,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a patient, who has a disease, a
symptom of disease or a predisposition toward a disease, with the
purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve or affect the disease, the symptoms of disease
or the predisposition toward disease.
[0478] In some embodiments, the peptidomimetics macrocycles can be
used to treat, prevent, and/or diagnose cancers and neoplastic
conditions. As used herein, the terms "cancer",
"hyperproliferative" and "neoplastic" refer to cells having the
capacity for autonomous growth, i.e., an abnormal state or
condition characterized by rapidly proliferating cell growth.
Hyperproliferative and neoplastic disease states can be categorized
as pathologic, i.e., characterizing or constituting a disease
state, or can be categorized as non-pathologic, i.e., a deviation
from normal but not associated with a disease state. The term is
meant to include all types of cancerous growths or oncogenic
processes, metastatic tissues or malignantly transformed cells,
tissues, or organs, irrespective of histopathologic type or stage
of invasiveness. A metastatic tumor can arise from a multitude of
primary tumor types, including but not limited to those of breast,
lung, liver, colon and ovarian origin. "Pathologic
hyperproliferative" cells occur in disease states characterized by
malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair. Examples of cellular proliferative and/or
differentiative disorders include cancer, e.g., carcinoma, sarcoma,
or metastatic disorders. In some embodiments, the peptidomimetics
macrocycles are novel therapeutic agents for controlling breast
cancer, ovarian cancer, colon cancer, lung cancer, metastasis of
such cancers and the like.
[0479] Examples of cancers or neoplastic conditions include, but
are not limited to, a fibrosarcoma, myosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer,
pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer,
cancer of the head and neck, skin cancer, brain cancer, squamous
cell carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular cancer, small cell lung
carcinoma, non-small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi
sarcoma.
[0480] Examples of proliferative disorders include hematopoietic
neoplastic disorders. As used herein, the term "hematopoietic
neoplastic disorders" includes diseases involving
hyperplastic/neoplastic cells of hematopoietic origin, e.g.,
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof. The diseases can arise from poorly differentiated
acute leukemias, e.g., erythroblastic leukemia and acute
megakaryoblastic leukemia. Additional exemplary myeloid disorders
include, but are not limited to, acute promyeloid leukemia (APML),
acute myelogenous leukemia (AML) and chronic myelogenous leukemia
(CML) (reviewed in Vaickus (1991), Crit Rev. Oncol./Hemotol.
11:267-97); lymphoid malignancies include, but are not limited to
acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and
T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic
leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's
macroglobulinemia (WM). Additional forms of malignant lymphomas
include, but are not limited to non-Hodgkin lymphoma and variants
thereof, peripheral T cell lymphomas, adult T cell
leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large
granular lymphocytic leukemia (LGF), Hodgkin's disease and
Reed-Stemberg disease.
[0481] Examples of cellular proliferative and/or differentiative
disorders of the breast include, but are not limited to,
proliferative breast disease including, e.g., epithelial
hyperplasia, sclerosing adenosis, and small duct papillomas;
tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor,
and sarcomas, and epithelial tumors such as large duct papilloma;
carcinoma of the breast including in situ (noninvasive) carcinoma
that includes ductal carcinoma in situ (including Paget's disease)
and lobular carcinoma in situ, and invasive (infiltrating)
carcinoma including, but not limited to, invasive ductal carcinoma,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms. Disorders in the male breast
include, but are not limited to, gynecomastia and carcinoma.
[0482] Examples of cellular proliferative and/or differentiative
disorders of the skin include, but are not limited to proliferative
skin disease such as melanomas, including mucosal melanoma,
superficial spreading melanoma, nodular melanoma, lentigo (e.g.
lentigo maligna, lentigo maligna melanoma, or acral lentiginous
melanoma), amelanotic melanoma, desmoplastic melanoma, melanoma
with features of a Spitz nevus, melanoma with small nevus-like
cells, polypoid melanoma, and soft-tissue melanoma; basal cell
carcinomas including micronodular basal cell carcinoma, superficial
basal cell carcinoma, nodular basal cell carcinoma (rodent ulcer),
cystic basal cell carcinoma, cicatricial basal cell carcinoma,
pigmented basal cell carcinoma, aberrant basal cell carcinoma,
infiltrative basal cell carcinoma, nevoid basal cell carcinoma
syndrome, polypoid basal cell carcinoma, pore-like basal cell
carcinoma, and fibroepithelioma of Pinkus; squamus cell carcinomas
including acanthoma (large cell acanthoma), adenoid squamous cell
carcinoma, basaloid squamous cell carcinoma, clear cell squamous
cell carcinoma, signet-ring cell squamous cell carcinoma, spindle
cell squamous cell carcinoma, Marjolin's ulcer, erythroplasia of
Queyrat, and Bowen's disease; or other skin or subcutaneous
tumors.
[0483] Examples of cellular proliferative and/or differentiative
disorders of the lung include, but are not limited to, bronchogenic
carcinoma, including paraneoplastic syndromes, bronchioloalveolar
carcinoma, neuroendocrine tumors, such as bronchial carcinoid,
miscellaneous tumors, and metastatic tumors; pathologies of the
pleura, including inflammatory pleural effusions, noninflammatory
pleural effusions, pneumothorax, and pleural tumors, including
solitary fibrous tumors (pleural fibroma) and malignant
mesothelioma.
[0484] Examples of cellular proliferative and/or differentiative
disorders of the colon include, but are not limited to,
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0485] Examples of cellular proliferative and/or differentiative
disorders of the liver include, but are not limited to, nodular
hyperplasias, adenomas, and malignant tumors, including primary
carcinoma of the liver and metastatic tumors.
[0486] Examples of cellular proliferative and/or differentiative
disorders of the ovary include, but are not limited to, ovarian
tumors such as, tumors of coelomic epithelium, serous tumors,
mucinous tumors, endometrioid tumors, clear cell adenocarcinoma,
cystadenofibroma, Brenner tumor, surface epithelial tumors; germ
cell tumors such as mature (benign) teratomas, monodermal
teratomas, immature malignant teratomas, dysgerminoma, endodermal
sinus tumor, choriocarcinoma; sex cord-stomal tumors such as,
granulosa-theca cell tumors, thecomafibromas, androblastomas, hill
cell tumors, and gonadoblastoma; and metastatic tumors such as
Krukenberg tumors.
[0487] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments described herein can be employed in practicing the
invention. It is intended that the following claims define the
scope and that methods and structures within the scope of these
claims and their equivalents be covered thereby.
EXAMPLES
Example 1
Synthesis of 6-chlorotryptophan Fmoc Amino Acids
##STR00144## ##STR00145##
[0489] Tert-butyl 6-chloro-3-formyl-1H-indole-1-carboxylate, 1. To
a stirred solution of dry DMF (12 mL) was added dropwise POCl.sub.3
(3.92 mL, 43 mmol, 1.3 equiv) at 0.degree. C. under Argon. The
solution was stirred at the same temperature for 20 min before a
solution of 6-chloroindole (5.0 g, 33 mmol, 1 eq.) in dry DMF (30
mL) was added dropwise. The resulting mixture was allowed to warm
to room temperature and stirred for an additional 2.5 h. Water (50
mL) was added and the solution was neutralized with 4M aqueous NaOH
(pH .about.8). The resulting solid was filtered off, washed with
water and dried under vacuum. This material was directly used in
the next step without additional purification. To a stirred
solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150
mL) was added successively Boc.sub.2O (7.91 g, 36.3 mmol, 1.1
equiv) and DMAP (0.4 g, 3.3 mmol, 0.1 equiv) at room temperature
under N.sub.2. The resulting mixture was stirred at room
temperature for 1.5 h and the solvent was evaporated under reduced
pressure. The residue was taken up in EtOAc and washed with 1N HCl,
dried and concentrated to give the formyl indole 1 (9 g, 98% over 2
steps) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta.: 1.70 (s,
Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).
[0490] Tert-butyl
6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2. To a
solution of compound 1 (8.86 g, 32 mmol, 1 eq.) in ethanol (150 mL)
was added NaBH.sub.4 (2.4 g, 63 mmol, 2 eq.). The reaction was
stirred for 3 h at room temperature. The reaction mixture was
concentrated and the residue was poured into diethyl ether and
water. The organic layer was separated, dried over magnesium
sulfate and concentrated to give a white solid (8.7 g, 98%). This
material was directly used in the next step without additional
purification. .sup.1H NMR (CDCl.sub.3) .delta.: 1.65 (s, Boc, 9H);
4.80 (s, 2H, CH.sub.2); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs,
1H).
[0491] Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate,
3. To a solution of compound 2 (4.1 g, 14.6 mmol, 1 eq.) in
dichloromethane (50 mL) under argon was added a solution of
triphenylphosphine (4.59 g, 17.5 mmol, 1.2 eq.) in dichloromethane
(50 mL) at -40.degree. C. The reaction solution was stirred an
additional 30 min at 40.degree. C. Then NBS (3.38 g, 19 mmol, 1.3
eq.) was added. The resulting mixture was allowed to warm to room
temperature and stirred overnight. Dichloromethane was evaporated,
Carbon Tetrachloride (100 mL) was added and the mixture was stirred
for 1 h and filtrated. The filtrate was concentrated, loaded in a
silica plug and quickly eluted with 25% EtOAc in Hexanes. The
solution was concentrated to give a white foam (3.84 g, 77%).
.sup.1H NMR (CDCl.sub.3) .delta.: 1.66 (s, Boc, 9H); 4.63 (s, 2H,
CH.sub.2); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs, 1H); 8.18 (bs,
1H).
[0492] .alpha.Me-6Cl-Trp(Boc)-Ni--S-BPB, 4. To S-Ala-Ni--S-BPB
(2.66 g, 5.2 mmol, 1 eq.) and KO-tBu (0.87 g, 7.8 mmol, 1.5 eq.)
was added 50 mL of DMF under argon. The bromide derivative compound
3 (2.68 g, 7.8 mmol, 1.5 eq.) in solution of DMF (5.0 mL) was added
via syringe. The reaction mixture was stirred at ambient
temperature for 1 h. The solution was then quenched with 5% aqueous
acetic acid and diluted with water. The desired product was
extracted in dichloromethane, dried and concentrated. The oily
product 4 was purified by flash chromatography (solid loading) on
normal phase using EtOAc and Hexanes as eluents to give a red solid
(1.78 g, 45% yield). .alpha.Me-6Cl-Trp(Boc)-Ni--S-BPB, 4: M+H calc.
775.21, M+H obs. 775.26; .sup.1H NMR (CDCl.sub.3) .delta.: 1.23 (s,
3H, .alpha.Me); 1.56 (m, 11H, Boc+CH.sub.2); 1.82-2.20 (m, 4H,
2CH.sub.2); 3.03 (m, 1H, CH.sub..alpha.); 3.24 (m, 2H, CH.sub.2);
3.57 and 4.29 (AB system, 2H, CH.sub.2 (benzyl), J=12.8 Hz); 6.62
(d, 2H); 6.98 (d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m,
5H); 7.50 (m, 2H); 7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).
[0493] Fmoc-.alpha.Me-6Cl-Trp(Boc)-OH, 6. To a solution of 3N
HCl/MeOH (1/3, 15 mL) at 50.degree. C. was added a solution of
compound 4 (1.75 g, 2.3 mmol, 1 eq.) in MeOH (5 ml) dropwise. The
starting material disappeared within 3-4 h. The acidic solution was
then cooled to 0.degree. C. with an ice bath and quenched with an
aqueous solution of Na.sub.2CO.sub.3 (1.21 g, 11.5 mmol, 5 eq.).
Methanol was removed and 8 more equivalents of Na.sub.2CO.sub.3
(1.95 g, 18.4 mmol) were added to the suspension. The Nickel
scavenging EDTA disodium salt dihydrate (1.68 g, 4.5 mmol, 2 eq.)
was then added and the suspension was stirred for 2 h. A solution
of Fmoc-OSu (0.84 g, 2.5 mmol, 1.1 eq.) in acetone (50 mL) was
added and the reaction was stirred overnight. Afterwards, the
reaction was diluted with diethyl ether and 1N HCl. The organic
layer was then dried over magnesium sulfate and concentrated in
vacuo. The desired product 6 was purified on normal phase using
acetone and dichloromethane as eluents to give a white foam (0.9 g,
70% yield). Fmoc-aMe-6Cl-Trp(Boc)-OH, 6: M+H calc. 575.19, M+H obs.
575.37; .sup.1H NMR (CDCl.sub.3) 1.59 (s, 9H, Boc); 1.68 (s, 3H,
Me); 3.48 (bs, 2H, CH.sub.2); 4.22 (m, 1H, CH); 4.39 (bs, 2H,
CH.sub.2); 5.47 (s, 1H, NH); 7.10 (m, 1H); 7.18 (m, 2H); 7.27 (m,
2H); 7.39 (m, 2H); 7.50 (m, 2H); 7.75 (d, 2H); 8.12 (bs, 1H).
[0494] 6Cl-Trp(Boc)-Ni--S-BPB, 5. To Gly-Ni--S-BPB (4.6 g, 9.2
mmol, 1 eq.) and KO-tBu (1.14 g, 10.1 mmol, 1.1 eq.) was added 95
mL of DMF under argon. The bromide derivative compound 3 (3.5 g,
4.6 mmol, 1.1 eq.) in solution of DMF (10 mL) was added via
syringe. The reaction mixture was stirred at ambient temperature
for 1 h. The solution was then quenched with 5% aqueous acetic acid
and diluted with water. The desired product was extracted in
dichloromethane, dried and concentrated. The oily product 5 was
purified by flash chromatography (solid loading) on normal phase
using EtOAc and Hexanes as eluents to give a red solid (5 g, 71%
yield). 6Cl-Trp(Boc)-Ni--S-BPB, 5: M+H calc. 761.20, M+H obs.
761.34; NMR (CDCl.sub.3) .delta.: 1.58 (m, 11H, Boc+CH.sub.2); 1.84
(m, 1H); 1.96 (m, 1H); 2.24 (m, 2H, CH.sub.2); 3.00 (m, 1H,
CH.sub..alpha.); 3.22 (m, 2H, CH.sub.2); 3.45 and 4.25 (AB system,
2H, CH.sub.2 (benzyl), J=12.8 Hz); 4.27 (m, 1H, CH.sub..alpha.);
6.65 (d, 2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m, 2H); 7.28 (m,
3H); 7.35-7.39 (m, 2H); 7.52 (m, 2H); 7.96 (d, 2H); 8.28 (m,
2H).
[0495] Fmoc-6Cl-Trp(Boc)-OH, 7. To a solution of 3N HCl/MeOH (1/3,
44 mL) at 50.degree. C. was added a solution of compound 5 (5 g,
6.6 mmol, 1 eq.) in MeOH (10 ml) dropwise. The starting material
disappeared within 3-4 h. The acidic solution was then cooled to
0.degree. C. with an ice bath and quenched with an aqueous solution
of Na.sub.2CO.sub.3 (3.48 g, 33 mmol, 5 eq.). Methanol was removed
and 8 more equivalents of Na.sub.2CO.sub.3 (5.57 g, 52 mmol) were
added to the suspension. The Nickel scavenging EDTA disodium salt
dihydrate (4.89 g, 13.1 mmol, 2 eq.) and the suspension was stirred
for 2 h. A solution of Fmoc-OSu (2.21 g, 6.55 mmol, 1.1 eq.) in
acetone (100 mL) was added and the reaction was stirred overnight.
Afterwards, the reaction was diluted with diethyl ether and 1N HCl.
The organic layer was then dried over magnesium sulfate and
concentrated in vacuo. The desired product 7 was purified on normal
phase using acetone and dichloromethane as eluents to give a white
foam (2.6 g, 69% yield). Fmoc-6Cl-Trp(Boc)-OH, 7: M+H calc. 561.17,
M+H obs. 561.37; NMR (CDCl.sub.3) 1.63 (s, 9H, Boc); 3.26 (m, 2H,
CH.sub.2); 4.19 (m, 1H, CH); 4.39 (m, 2H, CH.sub.2); 4.76 (m, 1H);
5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m, 2H); 7.39 (m, 3H); 7.50
(m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).
Example 1a
Synthesis of Alkyne Compounds for Use in Synthesis of Compounds of
Formula I
##STR00146##
[0497] Synthesis of (5-iodopent-1-ynyl)benzene. To a solution of
THF (200 mL) into reaction flask was added
(5-chloropent-1-ynyl)benzene Phenylacetylene (10 g, 97.91 mmol).
Then the reaction mixture was cooled to -78.degree. C. in a dry ice
bath. nBuLi (95.95 mmol, 38.39 mL) was added dropwise and allowed
to react for 0.5 h at -78.degree. C. At -78.degree. C.,
1-bromo-3-chloropropane was added. Stirred for 5 hours during which
the reaction was allowed to warm up to room temperature. Then
reaction was refluxed at 90.degree. C. for 3 hours. The solvent was
distilled off, then water (150 mL) and ether (150 mL) was added.
The crude product was extracted, ether was distilled off and the
resulting crude mixture was dissolved in acetone. Sodium iodide
(22.92 mmol, 3.44 g) was added into the solution. The reaction
mixture, with reflux condenser attached, was heated to 70.degree.
C. for two days. Acetone was distilled off using short path
distillation apparatus. Ether (150 mL) and water (150 mL) was added
and carried out extraction. Ether was then dried over magnesium
sulfate and distilled off resulting in 5.00 g of product (yield
98%). No further purification was carried out. .sup.1H NMR (500
MHz, CDCl.sub.3) .quadrature. 2.072 (m, 2H, CH.sub.2); 2.605 (t,
2H, CH.sub.2); 3.697 (m, 2H, CH.sub.2); 7.276 (m, 2H, Phenyl);
7.389 (m, 2H, phenyl); 7.484 (m, 1H, phenyl).
##STR00147##
[0498] Synthesis of MeS5-PhenylAlkyne-Ni--S-BPB. To S-Ala-Ni-SBPB
(18.17 mmol, 9.30 g) and KO-tBu (27.26 mmol, 3.05 g) was added 200
mL of DMF under argon. (5-iodopent-1-ynyl)benzene (27.26 mmol, 7.36
g) in solution of DMF (50 mL) was added via syringe. The reaction
mixture was stirred at ambient temperature for 1 h. The solution
was then quenched with acetic acid (27.26 mmol, 1.58 mL) and
diluted with water (100 mL). The product was extracted with
dichloromethane (100 mL), separated and dried over magnesium
sulfate. The crude product was purified by flash chromatography on
normal phase using acetone and dichloromethane as eluents to afford
the desired product as a red solid (9.48 g, 79.8%). M+H calc.
654.22, M+H obs. 654.4; .sup.1H NMR (500 MHz, CDCl.sub.3)
.quadrature. 1.17 (s, 3H, Me (.quadrature.Me-Phe)); 1.57 (m, 1H,
CH.sub.2); 1.67 (m, 1H, CH.sub.2); 1.89 (m, 1H, CH.sub.2); 2.06 (m,
1H, CH.sub.2); 2.24 (m, 2H, CH.sub.2); 3.05 (m, 1H); 3.18 (s,
.sup.2H); 3.26 (m, 1H); 3.56 and 4.31 (AB system, 2H, CH.sub.2
(benzyl), J=12.8 Hz); 6.64 (m, 2H); 6.94 (d, 1H); 7.12 (m, 1H);
7.20 (m, 1H); 7.20-7.40 (m, 10H); 7.43 (m, 2H); 8.01 (d, 2H); 8.13
(m, 1H).
[0499] Synthesis of
(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-7-phenylhept-6-y-
noic acid. To a solution of 3N HCl/MeOH (1/1, 23 mL) at 70.degree.
C. was added a solution of MeS5-PhenylAlkyne-Ni--S-BPB (14.5 mmol,
9.48 g) in MeOH (70 ml) dropwise. The starting material disappeared
within 10-20 min. The green reaction mixture was then concentrated
in vacuo. Water was added (100 mL) and the resulting precipitate
(S-BPB HCl salt) was filtered off. Sodium carbonate (116 mmol,
12.29 g) and EDTA (29 mmol, 10.79 g) were added to the mother
liquor. The mixture was stirred at room temperature for 3 hours to
scavenge the free nickel. After addition of 50 mL of acetone, the
reaction was cooled to 0.degree. C. with an ice bath. Fmoc-OSu
(16.68 mmol, 5.62 g) dissolved in acetone (50 ml) was added and the
reaction was allowed to warm up to ambient temperature with
stirring overnight. Afterwards, the reaction was diluted with ethyl
acetate (300 mL). Then the organic layer was separated. The aqueous
layer was acidified with conc. HCl. The desired product was
extracted with dichloromethane (400 mL), dried over magnesium
sulfate and concentrated in vacuo. The crude product was purified
by flash chromatography on normal phase using 10%MBTE/DCM as
eluents to afford the desired product as a white solid (6.05 g,
51%). M+H calc. 454.53, M+H obs. 454.2; .sup.1H NMR (CDCl.sub.3)
.quadrature. 1.50 (bs, 2H, CH.sub.2); 1.60 (bs, 3H, CH.sub.3); 2.05
(bs, 1H, CH.sub.2); 2.30 (bs, 1H, CH.sub.2); 2.42 (bs, 2H,
CH.sub.2); 4.20 (m, 1H, CH); 4.40 (bs, 2H, CH.sub.2); 5.58 (s, 1H,
NH); 7.26 (m, 3H); 7.32 (m, 2H); 7.37 (m, 4H); 7.58 (d, 2H); 7.76
(d, 2H).
##STR00148##
[0500] Synthesis of 6-iodohex-2-yne. To a solution of THF (250 mL)
into reaction flask was added 5-chloro-1-pentyne (48.7 mmol, 5.0
g). Then the reaction mixture was cooled to -78.degree. C. in a dry
ice bath. nBuLi (51.1 mmol, 20.44 mL) was added dropwise and
allowed to react for 0.5 h at -78.degree. C. and allowed to warm to
room temperature. Then methyl iodide (54.5 mmol, 3.39 mL) was added
to the reaction mixture. The reaction was stirred for 5 hours.
Water was added (1.5 mL) and the THF was distilled off The crude
product was extracted with pentane (100 mL) and water (100 mL).
Pentane was distilled off and the resulting crude mixture was
dissolved in acetone (300 mL). Sodium iodide (172.9 mmol, 25.92 g)
was added into the solution. The reaction mixture, with reflux
condenser attached, was heated to 70.degree. C. for two days.
Acetone was distilled off using short path distillation apparatus.
Ether (100 mL) and water (100 mL) was added and carried out
extraction. Ether was then dried over magnesium sulfate and
distilled off resulting in 8.35 g of product (yield 83%). No
further purification was carried out. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.762 (t, 3H, CH.sub.3); 1.941 (m, 2H,
CH.sub.2); 2.245 (m, 2H, CH.sub.2); 3.286 (m, 2H, CH.sub.2).
##STR00149##
[0501] Synthesis of MeS5-MethylAlkyne-Ni--S-BPB. To S-Ala-Ni-SBPB
(19.53 mmol, 10 g) and KO-tBU (29.29 mmol, 3.28 g) was added 200 mL
of DMF under argon. 6-iodohex-2-yne (29.29 mmol, 6.09 g) in
solution of DMF (50 mL) was added via syringe. The reaction mixture
was stirred at ambient temperature for 1 h. The solution was then
quenched with acetic acid (29.29 mmol, 1.69 mL) and diluted with
water (100 mL). The product was extracted with dichloromethane (300
mL), separated and dried over magnesium sulfate. The crude product
was purified by flash chromatography on normal phase using acetone
and dichloromethane as eluents to afford the desired product as a
red solid (8.10 g, 70%). M+H calc. 592.2, M+H obs. 592.4; .sup.1H
NMR (500 Mz, CDCl.sub.3) .delta. 1.17 (s, 3H, CH.sub.3
(.alpha.Me-Phe)); 1.57 (m, 1H, CH.sub.2); 1.67 (m, 1H, CH.sub.2);
1.89 (m, 1H, CH.sub.2); 2.06 (m, 1H, CH.sub.2); 2.24 (m, 2H,
CH.sub.2); 3.05 (m, 1H); 3.18 (s, 2H); 3.26 (m, 1H); 3.56 and 4.31
(AB system, 2H, CH.sub.2 (benzyl), J=12.8 Hz); 6.64 (m, 2H); 6.94
(d, 1H); 7.12 (m, 1H); 7.20 (m, 1H); 7.20-7.40 (m, 10H); 7.43 (m,
2H); 8.01 (d, 2H); 8.13 (m, 1H).
[0502] Synthesis of
(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyloct-6-ynoic
acid. To a solution of 3N HCl/MeOH (1/1, 23 mL) at 70.degree. C.
was added a solution of MeS5-MethylAlkyne-Ni--S-BPB (13.70 mmol,
8.10 g)) in methanol (70 ml) dropwise. The starting material
disappeared within 10-20 min. The green reaction mixture was then
concentrated in vacuo. Water (150 mL) was added and the resulting
precipitate (S-BPB HCl salt) was filtered off. Sodium carbonate
(116 mmol, 12.29 g) EDTA (29 mmol, 10.79 g) were added to the
mother liquor. The mixture was stirred at room temperature for 3
hours to scavenge the free nickel. After addition of 75 mL of
acetone, the reaction was cooled to 0.degree. C. with an ice bath.
Fmoc-OSu (15.76 mmol, 5.31 g) dissolved in acetone (75 ml) was
added and the reaction was allowed to warm up to ambient
temperature with stirring overnight. Afterwards, the reaction was
diluted with ethyl acetate (200 mL). Then the organic layer was
separated. The aqueous layer was acidified with conc. HCl. The
desired product was extracted with dichloromethane (200 mL), dried
over magnesium sulfate and concentrated in vacuo. The crude product
was purified by flash chromatography on normal phase using
10%MBTE/DCM as eluents to afford the desired product as a white
solid (2.40 g, 45%). M+H calc. 392.18, M+H obs. 392.3; .sup.1H NMR
(500 Mz, CDCl.sub.3) .delta. 1.38 (bs, 1H, CH.sub.2); 1.50 (bs, 1H,
CH.sub.2); 1.60 (bs, 2H, CH.sub.2); 1.75 (s, 3H, CH.sub.3); 1.95
(bs, 2H, CH.sub.2); 2.10 (bs, 3H, CH.sub.3); 4.20 (m, 1H, CH); 4.40
(bs, 2H, CH.sub.2); 5.58 (s, 1H, NH); 7.32 (m, 2H); 7.42 (m, 2H);
7.59 (d, 2H); 7.78 (d, 2H).
Example 2
Peptidomimetic Macrocycles of Formula I
[0503] Peptidomimetic macrocycles are prepared as described herein
and as in pending U.S. patent application Ser. No. 12/037,041,
filed Feb. 25, 2008, which is hereby incorporated by reference in
its entirety. Peptidomimetic macrocycles are designed by replacing
two or more naturally occurring amino acids with the corresponding
synthetic amino acids. Substitutions are made at i and i+4, and i
and i+7 positions. Peptide synthesis is performed either manually
or on an automated peptide synthesizer (Applied Biosystems, model
433A), using solid phase conditions, rink amide AM resin
(Novabiochem), and Fmoc main-chain protecting group chemistry. For
the coupling of natural Fmoc-protected amino acids (Novabiochem),
10 equivalents of amino acid and a 1:1:2 molar ratio of coupling
reagents HBTU/HOBt (Novabiochem)/DIEA are employed. Non-natural
amino acids (4 equiv) are coupled with a 1:1:2 molar ratio of HATU
(Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic
peptides are acetylated, while the C-termini are amidated.
[0504] Generally, ully protected resin-bound peptides were
synthesized on a PEG-PS resin (loading 0.45 mmol/g) on a 0.5 mmol
scale. Deprotection of the temporary Fmoc group was achieved by
3.times.10 min treatments of the resin bound peptide with 20% (v/v)
piperidine in DMF. After washing with NMP (3.times.),
dichloromethane (3.times.) and NMP (3.times.), coupling of each
successive amino acid was achieved with 1.times.60 min incubation
with the appropriate preactivated Fmoc-amino acid derivative. All
protected amino acids (1.0 mmol) were dissolved in NMP and
activated with HCTU (1.0 mmol), Cl-HOBt (1.0 mmol) and DIEA (2.0
mmol) prior to transfer of the coupling solution to the deprotected
resin-bound peptide. After coupling was completed, the resin was
washed in preparation for the next deprotection/coupling cycle.
Acetylation of the amino terminus was carried out in the presence
of acetic anhydride/DIEA in NMP. The LC-MS analysis of a cleaved
and deprotected sample obtained from an aliquot of the fully
assembled resin-bound peptide was accomplished in order to
verifying the completion of each coupling.
[0505] In a typical example for the preparation of a peptidomimetic
macrocycle comprising a 1,4-triazole group (e.g. SP153), 20% (v/v)
2,6-lutidine in DMF was added to the peptide resin (0.5 mmol) in a
40 ml glass vial and shaken for 10 minutes. Sodium ascorbate (0.25
g, 1.25 mmol) and diisopropylethylamine (0.22 ml, 1.25 mmol) were
then added, followed by copper(I) iodide (0.24 g, 1.25 mmol) and
the resulting reaction mixture was mechanically shaken 16 hours at
ambient temperature.
[0506] In a typical example for the preparation of a peptidomimetic
macrocycle comprising a 1,5-triazole group (SP932, SP933), a
peptide resin (0.25 mmol) was washed with anhydrous DCM. Resin was
loaded into a microwave vial. Vessel was evacuated and purged with
nitrogen. Chloro(pentamethylcyclopentadienyl)
bis(triphenylphosphine)ruthenium(II), 10% loading, (Strem 44-0117)
was added. Anhydrous toluene was added to the reaction vessel. The
reaction was then loaded into the microwave and held at 90.degree.
C. for 10 minutes. Reaction may need to be pushed a subsequent time
for completion. In other cases,
Chloro(1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium
("Cp*RuCl(cod)") may be used, for example at at room temperature in
a solvent comprising toluene.
[0507] In a typical example for the preparation of a peptidomimetic
macrocycle comprising an iodo-substituted triazole group (e.g.
SP457), THF (2 ml) was added to the peptide resin (0.05 mmol) in a
40 ml glass vial and shaken for 10 minutes. N-bromosuccimide (0.04
g, 0.25 mmol), copper(I) iodide (0.05 g, 0.25 mmol) and
diisopropylethylamine (0.04 ml, 0.25 mmol) were then added and the
resulting reaction mixture was mechanically shaken 16 hours at
ambient temperature. Iodo-triazole crosslinkers may be further
substituted by a coupling reaction, for example with boronic acids,
to result in a peptidomimetic macrocycle such as SP465. In a
typical example, DMF (3 ml) was added to the iodo-triazole peptide
resin (0.1 mmol) in a 40 ml glass vial and shaken for 10 minutes.
Phenyl boronic acid (0.04 g, 0 3 mmol),
tetrakis(triphenylphosphine)palladium(0) (0.006 g, 0.005 mmol) and
potassium carbonate (0.083 g, 0.6 mmol) were then added and the
resulting reaction mixture was mechanically shaken 16 hours at
70.degree. C. Iodo-triazole crosslinkers may also be further
substituted by a coupling reaction, for example with a terminal
alkyne (e.g. Sonogashira coupling), to result in a peptidomimetic
macrocycle such as SP468. In a typical example, 2:1
THF:triethylamine (3 ml) was added to the iodo-triazole peptide
resin (0.1 mmol) in a 40 ml glass vial and shaken for 10 minutes.
N-BOC-4-pentyne-1-amine (0.04 g, 0.2 mmol) and
bis(triphenylphosphine)palladiumchloride (0.014 g, 0.02 mmol) were
added and shaken for 5 minutes. Copper(I) iodide (0.004 g, 0.02
mmol) was then added and the resulting reaction mixture was
mechanically shaken 16 hours at 70.degree. C.
[0508] The triazole-cyclized resin-bound peptides were deprotected
and cleaved from the solid support by treatment with
TFA/H.sub.2O/TIS (95/5/5 v/v) for 2.5 h at room temperature. After
filtration of the resin the TFA solution was precipitated in cold
diethyl ether and centrifuged to yield the desired product as a
solid. The crude product was purified by preparative HPLC. For
example, purification of cross-linked compounds is achieved by high
performance liquid chromatography (HPLC) (Varian ProStar) on a
reverse phase C18 column (Varian) to yield the pure compounds.
Chemical composition of the pure products is confirmed by LC/MS
mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC
system) and amino acid analysis (Applied Biosystems, model
420A).
[0509] Table 4 shows a list of peptidomimetic macrocycles of
Formula I.
TABLE-US-00005 TABLE 4 SP- SEQ ID NO: sequence 1 3
Ac-F$4rn6AYWEAc3cL$4a5AAA-NH2 2 4 Ac-F$4rn6AYWEAc3cL$4a5AAibA-NH2 3
5 Ac-LTF$4rn6AYWAQL$4a5SANle-NH2 4 6 Ac-LTF$4rn6AYWAQL$4a5SAL-NH2 5
7 Ac-LTF$4rn6AYWAQL$4a5SAM-NH2 6 8 Ac-LTF$4rn6AYWAQL$4a5SAhL-NH2 7
9 Ac-LTF$4rn6AYWAQL$4a5SAF-NH2 8 10 Ac-LTF$4rn6AYWAQL$4a5SAI-NH2 9
11 Ac-LTF$4rn6AYWAQL$4a5SAChg-NH2 10 12
Ac-LTF$4rn6AYWAQL$4a5SAAib-NH2 11 13 Ac-LTF$4rn6AYWAQL$4a5SAA-NH2
12 14 Ac-LTF$4rn6AYWA$4a5L$S$Nle-NH2 13 15
Ac-LTF$4rn6AYWA$4a5L$S$A-NH2 14 16 Ac-F$4rn6AYWEAc3cL$4a5AANle-NH2
15 17 Ac-F$4rn6AYWEAc3cL$4a5AAL-NH2 16 18
Ac-F$4rn6AYWEAc3cL$4a5AAM-NH2 17 19 Ac-F$4rn6AYWEAc3cL$4a5AAhL-NH2
18 20 Ac-F$4rn6AYWEAc3cL$4a5AAF-NH2 19 21
Ac-F$4rn6AYWEAc3cL$4a5AAI-NH2 20 22 Ac-F$4rn6AYWEAc3cL$4a5AAChg-NH2
21 23 Ac-F$4rn6AYWEAc3cL$4a5AACha-NH2 22 24
Ac-F$4rn6AYWEAc3cL$4a5AAAib-NH2 23 25
Ac-LTF$4rn6AYWAQL$4a5AAAibV-NH2 24 26
Ac-LTF$4rn6AYWAQL$4a5AAAibV-NH2 25 27
Ac-LTF$4rn6AYWAQL$4a5SAibAA-NH2 26 28
Ac-LTF$4rn6AYWAQL$4a5SAibAA-NH2 27 29
Ac-HLTF$4rn6HHWHQL$4a5AANleNle-NH2 28 30
Ac-DLTF$4rn6HHWHQL$4a5RRLV-NH2 29 31 Ac-HHTF$4rn6HHWHQL$4a5AAML-NH2
30 32 Ac-F$4rn6HHWHQL$4a5RRDCha-NH2 31 33
Ac-F$4rn6HHWHQL$4a5HRFV-NH2 32 34 Ac-HLTF$4rn6HHWHQL$4a5AAhLA-NH2
33 35 Ac-DLTF$4rn6HHWHQL$4a5RRChgl-NH2 34 36
Ac-DLTF$4rn6HHWHQL$4a5RRChgl-NH2 35 37
Ac-HHTF$4rn6HHWHQL$4a5AAChav-NH2 36 38 Ac-F$4rn6HHWHQL$4a5RRDa-NH2
37 39 Ac-F$4rn6HHWHQL$4a5HRAibG-NH2 38 40
Ac-F$4rn6AYWAQL$4a5HHNleL-NH2 39 41 Ac-F$4rn6AYWSAL$4a5HQANle-NH2
40 42 Ac-F$4rn6AYWVQL$4a5QHChgl-NH2 41 43
Ac-F$4rn6AYWTAL$4a5QQNlev-NH2 42 44 Ac-F$4rn6AYWYQL$4a5HAibAa-NH2
43 45 Ac-LTF$4rn6AYWAQL$4a5HHLa-NH2 44 46
Ac-LTF$4rn6AYWAQL$4a5HHLa-NH2 45 47 Ac-LTF$4rn6AYWAQL$4a5HQNlev-NH2
46 48 Ac-LTF$4rn6AYWAQL$4a5HQNlev-NH2 47 49
Ac-LTF$4rn6AYWAQL$4a5QQMl-NH2 48 50 Ac-LTF$4rn6AYWAQL$4a5QQMl-NH2
49 51 Ac-LTF$4rn6AYWAQL$4a5HAibhLV-NH2 50 52
Ac-LTF$4rn6AYWAQL$4a5AHFA-NH2 51 53
Ac-HLTF$4rn6HHWHQL$4a5AANlel-NH2 52 54
Ac-DLTF$4rn6HHWHQL$4a5RRLa-NH2 53 55 Ac-HHTF$4rn6HHWHQL$4a5AAMv-NH2
54 56 Ac-F$4rn6HHWHQL$4a5RRDA-NH2 55 57
Ac-F$4rn6HHWHQL$4a5HRFCha-NH2 56 58 Ac-F$4rn6AYWEAL$4a5AA-NHAm 57
59 Ac-F$4rn6AYWEAL$4a5AA-NHiAm 58 60 Ac-F$4rn6AYWEAL$4a5AA-NHnPr3Ph
59 61 Ac-F$4rn6AYWEAL$4a5AA-NHnBu33Me 60 62
Ac-F$4rn6AYWEAL$4a5AA-NHnPr 61 63 Ac-F$4rn6AYWEAL$4a5AA-NHnEt2Ch 62
64 Ac-F$4rn6AYWEAL$4a5AA-NHnEt2Cp 63 65 Ac-F$4rn6AYWEAL$4a5AA-NHHex
64 66 Ac-LTF$4rn6AYWAQL$4a5AAIA-NH2 65 67
Ac-LTF$4rn6AYWAQL$4a5AAIA-NH2 66 68 Ac-LTF$4rn6AYWAAL$4a5AAMA-NH2
67 69 Ac-LTF$4rn6AYWAAL$4a5AAMA-NH2 68 70
Ac-LTF$4rn6AYWAQL$4a5AANleA-NH2 69 71
Ac-LTF$4rn6AYWAQL$4a5AANleA-NH2 70 72 Ac-LTF$4rn6AYWAQL$4a5AAIa-NH2
71 73 Ac-LTF$4rn6AYWAQL$4a5AAIa-NH2 72 74
Ac-LTF$4rn6AYWAAL$4a5AAMa-NH2 73 75 Ac-LTF$4rn6AYWAAL$4a5AAMa-NH2
74 76 Ac-LTF$4rn6AYWAQL$4a5AANlea-NH2 75 77
Ac-LTF$4rn6AYWAQL$4a5AANlea-NH2 76 78 Ac-LTF$4rn6AYWAAL$4a5AAIv-NH2
77 79 Ac-LTF$4rn6AYWAAL$4a5AAIv-NH2 78 80
Ac-LTF$4rn6AYWAQL$4a5AAMv-NH2 79 81 Ac-LTF$4rn6AYWAAL$4a5AANlev-NH2
80 82 Ac-LTF$4rn6AYWAAL$4a5AANlev-NH2 81 83
Ac-LTF$4rn6AYWAQL$4a5AAIl-NH2 82 84 Ac-LTF$4rn6AYWAQL$4a5AAIl-NH2
83 85 Ac-LTF$4rn6AYWAAL$4a5AAMl-NH2 84 86
Ac-LTF$4rn6AYWAQL$4a5AANlel-NH2 85 87
Ac-LTF$4rn6AYWAQL$4a5AANlel-NH2 86 88 Ac-F$4rn6AYWEAL$4a5AAMA-NH2
87 89 Ac-F$4rn6AYWEAL$4a5AANleA-NH2 88 90
Ac-F$4rn6AYWEAL$4a5AAIa-NH2 89 91 Ac-F$4rn6AYWEAL$4a5AAMa-NH2 90 92
Ac-F$4rn6AYWEAL$4a5AANlea-NH2 91 93 Ac-F$4rn6AYWEAL$4a5AAIv-NH2 92
94 Ac-F$4rn6AYWEAL$4a5AAMv-NH2 93 95 Ac-F$4rn6AYWEAL$4a5AANlev-NH2
94 96 Ac-F$4rn6AYWEAL$4a5AAIl-NH2 95 97 Ac-F$4rn6AYWEAL$4a5AAMl-NH2
96 98 Ac-F$4rn6AYWEAL$4a5AANlel-NH2 97 99
Ac-F$4rn6AYWEAL$4a5AANlel-NH2 98 100
Ac-LTF$4rn6AY6clWAQL$4a5SAA-NH2 99 101
Ac-LTF$4rn6AY6clWAQL$4a5SAA-NH2 100 102
Ac-WTF$4rn6FYWSQL$4a5AVAa-NH2 101 103 Ac-WTF$4rn6FYWSQL$4a5AVAa-NH2
102 104 Ac-WTF$4rn6VYWSQL$4a5AVA-NH2 103 105
Ac-WTF$4rn6VYWSQL$4a5AVA-NH2 104 106 Ac-WTF$4rn6FYWSQL$4a5SAAa-NH2
105 107 Ac-WTF$4rn6FYWSQL$4a5SAAa-NH2 106 108
Ac-WTF$4rn6VYWSQL$4a5AVAaa-NH2 107 109
Ac-WTF$4rn6VYWSQL$4a5AVAaa-NH2 108 110 Ac-LTF$4rn6AYWAQL$4a5AVG-NH2
109 111 Ac-LTF$4rn6AYWAQL$4a5AVG-NH2 110 112
Ac-LTF$4rn6AYWAQL$4a5AVQ-NH2 111 113 Ac-LTF$4rn6AYWAQL$4a5AVQ-NH2
112 114 Ac-LTF$4rn6AYWAQL$4a5SAa-NH2 113 115
Ac-LTF$4rn6AYWAQL$4a5SAa-NH2 114 116 Ac-LTF$4rn6AYWAQhL$4a5SAA-NH2
115 117 Ac-LTF$4rn6AYWAQhL$4a5SAA-NH2 116 118
Ac-LTF$4rn6AYWEQLStSA$4a5-NH2 117 119 Ac-LTF$4rn6AYWAQL$4a5SLA-NH2
118 120 Ac-LTF$4rn6AYWAQL$4a5SLA-NH2 119 121
Ac-LTF$4rn6AYWAQL$4a5SWA-NH2 120 122 Ac-LTF$4rn6AYWAQL$4a5SWA-NH2
121 123 Ac-LTF$4rn6AYWAQL$4a5SVS-NH2 122 124
Ac-LTF$4rn6AYWAQL$4a5SAS-NH2 123 125
Ac-LTF$4rn6AYWAQL$4a5SVG-NH2
124 126 Ac-ETF$4rn6VYWAQL$4a5SAa-NH2 125 127
Ac-ETF$4rn6VYWAQL$4a5SAA-NH2 126 128 Ac-ETF$4rn6VYWAQL$4a5SVA-NH2
127 129 Ac-ETF$4rn6VYWAQL$4a5SLA-NH2 128 130
Ac-ETF$4rn6VYWAQL$4a5SWA-NH2 129 131 Ac-ETF$4rn6KYWAQL$4a5SWA-NH2
130 132 Ac-ETF$4rn6VYWAQL$4a5SVS-NH2 131 133
Ac-ETF$4rn6VYWAQL$4a5SAS-NH2 132 134 Ac-ETF$4rn6VYWAQL$4a5SVG-NH2
133 135 Ac-LTF$4rn6VYWAQL$4a5SSa-NH2 134 136
Ac-ETF$4rn6VYWAQL$4a5SSa-NH2 135 137 Ac-LTF$4rn6VYWAQL$4a5SNa-NH2
136 138 Ac-ETF$4rn6VYWAQL$4a5SNa-NH2 137 139
Ac-LTF$4rn6VYWAQL$4a5SAa-NH2 138 140 Ac-LTF$4rn6VYWAQL$4a5SVA-NH2
139 141 Ac-LTF$4rn6VYWAQL$4a5SVA-NH2 140 142
Ac-LTF$4rn6VYWAQL$4a5SWA-NH2 141 143 Ac-LTF$4rn6VYWAQL$4a5SVS-NH2
142 144 Ac-LTF$4rn6VYWAQL$4a5SVS-NH2 143 145
Ac-LTF$4rn6VYWAQL$4a5SAS-NH2 144 146 Ac-LTF$4rn6VYWAQL$4a5SAS-NH2
145 147 Ac-LTF$4rn6VYWAQL$4a5SVG-NH2 146 148
Ac-LTF$4rn6VYWAQL$4a5SVG-NH2 147 149 Ac-LTF$4rn6EYWAQCha$4a5SAA-NH2
148 150 Ac-LTF$4rn6EYWAQCha$4a5SAA-NH2 149 151
Ac-LTF$4rn6EYWAQCpg$4a5SAA-NH2 150 152
Ac-LTF$4rn6EYWAQCpg$4a5SAA-NH2 151 153 Ac-LTF$4rn6EYWAQF$4a5SAA-NH2
152 154 Ac-LTF$4rn6EYWAQF$4a5SAA-NH2 153 155
Ac-LTF$4rn6EYWAQCba$4a5SAA-NH2 154 156
Ac-LTF$4rn6EYWAQCba$4a5SAA-NH2 155 157
Ac-LTF3C1$4rn6EYWAQL$4a5SAA-NH2 156 158
Ac-LTF3C1$4rn6EYWAQL$4a5SAA-NH2 157 159
Ac-LTF34F2$4rn6EYwAQL$4a5sAA-NH2 158 160
Ac-LTF34F2$4rn6EYwAQL$4a5sAA-NH2 159 161
Ac-LTF34F2$4rn6EYwAQhL$4a5sAA-NH2 160 162
Ac-LTF34F2$4rn6EYwAQhL$4a5sAA-NH2 161 163
Ac-ETF$4rn6EYWAQL$4a5SAA-NH2 162 164 Ac-LTF$4rn6AYWVQL$4a5SAA-NH2
163 165 Ac-LTF$4rn6AHWAQL$4a5SAA-NH2 164 166
Ac-LTF$4rn6AEWAQL$4a5SAA-NH2 165 167 Ac-LTF$4rn6ASWAQL$4a5SAA-NH2
166 168 Ac-LTF$4rn6AEWAQL$4a5SAA-NH2 167 169
Ac-LTF$4rn6ASWAQL$4a5SAA-NH2 168 170
Ac-LTF$4rn6AF4coohWAQL$4a5SAA-NH2 169 171
Ac-LTF$4rn6AF4coohWAQL$4a5SAA-NH2 170 172
Ac-LTF$4rn6AHWAQL$4a5AAIa-NH2 171 173 Ac-ITF$4rn6FYWAQL$4a5AAIa-NH2
172 174 Ac-ITF$4rn6EHWAQL$4a5AAIa-NH2 173 175
Ac-ITF$4rn6EHWAQL$4a5AAIa-NH2 174 176 Ac-ETF$4rn6EHWAQL$4a5AAIa-NH2
175 177 Ac-ETF$4rn6EHWAQL$4a5AAIa-NH2 176 178
Ac-LTF$4rn6AHWVQL$4a5AAIa-NH2 177 179 Ac-ITF$4rn6FYWVQL$4a5AAIa-NH2
178 180 Ac-ITF$4rn6EYWVQL$4a5AAIa-NH2 179 181
Ac-ITF$4rn6EHWVQL$4a5AAIa-NH2 180 182 Ac-LTF$4rn6AEWAQL$4a5AAIa-NH2
181 183 Ac-LTF$4rn6AF4coohWAQL$4a5AAIa-NH2 182 184
Ac-LTF$4rn6AF4coohWAQL$4a5AAIa-NH2 183 185
Ac-LTF$4rn6AHWAQL$4a5AHFA-NH2 184 186 Ac-ITF$4rn6FYWAQL$4a5AHFA-NH2
185 187 Ac-ITF$4rn6FYWAQL$4a5AHFA-NH2 186 188
Ac-ITF$4rn6FHWAQL$4a5AEFA-NH2 187 189 Ac-ITF$4rn6FHWAQL$4a5AEFA-NH2
188 190 Ac-ITF$4rn6EHWAQL$4a5AHFA-NH2 189 191
Ac-ITF$4rn6EHWAQL$4a5AHFA-NH2 190 192 Ac-LTF$4rn6AHWVQL$4a5AHFA-NH2
191 193 Ac-ITF$4rn6FYWVQL$4a5AHFA-NH2 192 194
Ac-ITF$4rn6EYWVQL$4a5AHFA-NH2 193 195 Ac-ITF$4rn6EHWVQL$4a5AHFA-NH2
194 196 Ac-ITF$4rn6EHWVQL$4a5AHFA-NH2 195 197
Ac-ETF$4rn6EYWAAL$4a5SAA-NH2 196 198 Ac-LTF$4rn6AYWVAL$4a5SAA-NH2
197 199 Ac-LTF$4rn6AHWAAL$4a5SAA-NH2 198 200
Ac-LTF$4rn6AEWAAL$4a5SAA-NH2 199 201 Ac-LTF$4rn6AEWAAL$4a5SAA-NH2
200 202 Ac-LTF$4rn6ASWAAL$4a5SAA-NH2 201 203
Ac-LTF$4rn6ASWAAL$4a5SAA-NH2 202 204 Ac-LTF$4rn6AYWAAL$4a5AAIa-NH2
203 205 Ac-LTF$4rn6AYWAAL$4a5AAIa-NH2 204 206
Ac-LTF$4rn6AYWAAL$4a5AHFA-NH2 205 207 Ac-LTF$4rn6EHWAQL$4a5AHIa-NH2
206 208 Ac-LTF$4rn6EHWAQL$4a5AHIa-NH2 207 209
Ac-LTF$4rn6AHWAQL$4a5AHIa-NH2 208 210 Ac-LTF$4rn6EYWAQL$4a5AHIa-NH2
209 211 Ac-LTF$4rn6AYWAQL$4a5AAFa-NH2 210 212
Ac-LTF$4rn6AYWAQL$4a5AAFa-NH2 211 213 Ac-LTF$4rn6AYWAQL$4a5AAWa-NH2
212 214 Ac-LTF$4rn6AYWAQL$4a5AAVa-NH2 213 215
Ac-LTF$4rn6AYWAQL$4a5AAVa-NH2 214 216 Ac-LTF$4rn6AYWAQL$4a5AALa-NH2
215 217 Ac-LTF$4rn6AYWAQL$4a5AALa-NH2 216 218
Ac-LTF$4rn6EYWAQL$4a5AAIa-NH2 217 219 Ac-LTF$4rn6EYWAQL$4a5AAIa-NH2
218 220 Ac-LTF$4rn6EYWAQL$4a5AAFa-NH2 219 221
Ac-LTF$4rn6EYWAQL$4a5AAFa-NH2 220 222 Ac-LTF$4rn6EYWAQL$4a5AAVa-NH2
221 223 Ac-LTF$4rn6EYWAQL$4a5AAVa-NH2 222 224
Ac-LTF$4rn6EHWAQL$4a5AAIa-NH2 223 225 Ac-LTF$4rn6EHWAQL$4a5AAIa-NH2
224 226 Ac-LTF$4rn6EHWAQL$4a5AAWa-NH2 225 227
Ac-LTF$4rn6EHWAQL$4a5AAWa-NH2 226 228 Ac-LTF$4rn6EHWAQL$4a5AALa-NH2
227 229 Ac-LTF$4rn6EHWAQL$4a5AALa-NH2 228 230
Ac-ETF$4rn6EHWVQL$4a5AALa-NH2 229 231 Ac-LTF$4rn6AYWAQL$4a5AAAa-NH2
230 232 Ac-LTF$4rn6AYWAQL$4a5AAAa-NH2 231 233
Ac-LTF$4rn6AYWAQL$4a5AAAibA-NH2 232 234
Ac-LTF$4rn6AYWAQL$4a5AAAibA-NH2 233 235
Ac-LTF$4rn6AYWAQL$4a5AAAAa-NH2 234 236
Ac-LTF$r5AYWAQL$4a5s8AAIa-NH2 235 237 Ac-LTF$r5AYWAQL$4a5s8SAA-NH2
236 238 Ac-LTF$4rn6AYWAQCba$4a5AANleA-NH2 237 239
Ac-ETF$4rn6AYWAQCba$4a5AANleA-NH2 238 240
Ac-LTF$4rn6EYWAQCba$4a5AANleA-NH2 239 241
Ac-LTF$4rn6AYWAQCba$4a5AWNleA-NH2 240 242
Ac-ETF$4rn6AYWAQCba$4a5AWNleA-NH2 241 243
Ac-LTF$4rn6EYWAQCba$4a5AWNleA-NH2 242 244
Ac-LTF$4rn6EYWAQCba$4a5SAFA-NH2 243 245
Ac-LTF34F2$4rn6EYWAQCba$4a5SANleA- NH2 244 246
Ac-LTF$4rn6EF4coohWAQCba$4a5SANleA- NH2 245 247
Ac-LTF$4rn6EYWSQCba$4a5SANleA-NH2 246 248
Ac-LTF$4rn6EYWWQCba$4a5SANleA-NH2 247 249
Ac-LTF$4rn6EYWAQCba$4a5AAIa-NH2
248 250 Ac-LTF34F2$4rn6EYWAQCba$4a5AAIa-NH2 249 251
Ac-LTF$4rn6EF4coohWAQCba$4a5AAIa- NH2 250 252
Pam-ETF$4rn6EYWAQCba$4a5SAA-NH2 251 253
Ac-LThF$4rn6EFWAQCba$4a5SAA-NH2 252 254
Ac-LTA$4rn6EYWAQCba$4a5SAA-NH2 253 255
Ac-LTF$4rn6EYAAQCba$4a5SAA-NH2 254 256
Ac-LTF$4rn6EY2NalAQCba$4a5SAA-NH2 255 257
Ac-LTF$4rn6AYWAQCba$4a5SAA-NH2 256 258
Ac-LTF$4rn6EYWAQCba$4a5SAF-NH2 257 259
Ac-LTF$4rn6EYWAQCba$4a5SAFa-NH2 258 260
Ac-LTF$4rn6AYWAQCba$4a5SAF-NH2 259 261
Ac-LTF34F2$4rn6AYWAQCba$4a5SAF-NH2 260 262
Ac-LTF$4rn6AF4coohWAQCba$4a5SAF-NH2 261 263
Ac-LTF$4rn6EY6clWAQCba$4a5SAF-NH2 262 264
Ac-LTF$4rn6AYWSQCba$4a5SAF-NH2 263 265
Ac-LTF$4rn6AYWWQCba$4a5SAF-NH2 264 266
Ac-LTF$4rn6AYWAQCba$4a5AAIa-NH2 265 267
Ac-LTF34F2$4rn6AYWAQCba$4a5AAIa-NH2 266 268
Ac-LTF$4rn6AY6clWAQCba$4a5AAIa-NH2 267 269
Ac-LTF$4rn6AF4coohWAQCba$4a5AAIa- NH2 268 270
Ac-LTF$4rn6EYWAQCba$4a5AAFa-NH2 269 271
Ac-LTF$4rn6EYWAQCba$4a5AAFa-NH2 270 272
Ac-ETF$4rn6AYWAQCba$4a5AWNlea-NH2 271 273
Ac-LTF$4rn6EYWAQCba$4a5AWNlea-NH2 272 274
Ac-ETF$4rn6EYWAQCba$4a5AWNlea-NH2 273 275
Ac-ETF$4rn6EYWAQCba$4a5AWNlea-NH2 274 276
Ac-LTF$4rn6AYWAQCba$4a5SAFa-NH2 275 277
Ac-LTF$4rn6AYWAQCba$4a5SAFa-NH2 276 278
Ac-ETF$4rn6AYWAQL$4a5AWNlea-NH2 277 279
Ac-LTF$4rn6EYWAQL$4a5AWNlea-NH2 278 280
Ac-ETF$4rn6EYWAQL$4a5AWNlea-NH2 279 281
Dmaac-LTF$4rn6EYWAQhL$4a5SAA-NH2 280 282
Hexac-LTF$4rn6EYWAQhL$4a5SAA-NH2 281 283
Napac-LTF$4rn6EYWAQhL$4a5SAA-NH2 282 284
Decac-LTF$4rn6EYWAQhL$4a5SAA-NH2 283 285
Admac-LTF$4rn6EYWAQhL$4a5SAA-NH2 284 286
Tmac-LTF$4rn6EYWAQhL$4a5SAA-NH2 285 287
Pam-LTF$4rn6EYWAQhL$4a5SAA-NH2 286 288
Ac-LTF$4rn6AYWAQCba$4a5AANleA-NH2 287 289
Ac-LTF34F2$4rn6EYwAQcba$4a5AAIa-NH2 288 290
Ac-LTF34F2$4rn6EYwAQcba$4a5SAA-NH2 289 291
Ac-LTF34F2$4rn6EYwAQcba$4a5SAA-NH2 290 292
Ac-LTF$4rn6EF4coohWAQCba$4a5SAA-NH2 291 293
Ac-LTF$4rn6EF4coohWAQCba$4a5SAA-NH2 292 294
Ac-LTF$4rn6EYWSQCba$4a5SAA-NH2 293 295
Ac-LTF$4rn6EYWSQCba$4a5SAA-NH2 294 296
Ac-LTF$4rn6EYwAQhL$4a5SAA-NH2 295 297 Ac-LTF$4rn6AYWAQhL$4a5SAF-NH2
296 298 Ac-LTF$4rn6AYWAQhL$4a5SAF-NH2 297 299
Ac-LTF34F2$4rn6AYWAQhL$4a5SAA-NH2 298 300
Ac-LTF34F2$4rn6AYWAQhL$4a5SAA-NH2 299 301
Ac-LTF$4rn6AF4coohWAQhL$4a5SAA-NH2 300 302
Ac-LTF$4rn6AF4coohWAQhL$4a5SAA-NH2 301 303
Ac-LTF$4rn6AYWSQhL$4a5SAA-NH2 302 304 Ac-LTF$4rn6AYWSQhL$4a5SAA-NH2
303 305 Ac-LTF$4rn6EYWAQL$4a5AANleA-NH2 304 306
Ac-LTF34F2$4rn6AYWAQL$4a5AANleA-NH2 305 307
Ac-LTF$4rn6AF4coohWAQL$4a5AANleA- NH2 306 308
Ac-LTF$4rn6AYWSQL$4a5AANleA-NH2 307 309
Ac-LTF34F2$4rn6AYWAQhL$4a5AANleA- NH2 308 310
Ac-LTF34F2$4rn6AYWAQhL$4a5AANleA- NH2 309 311
Ac-LTF$4rn6AF4coohWAQhL$4a5AANleA- NH2 310 312
Ac-LTF$4rn6AF4coohWAQhL$4a5AANleA- NH2 311 313
Ac-LTF$4rn6AYWSQhL$4a5AANleA-NH2 312 314
Ac-LTF$4rn6AYWSQhL$4a5AANleA-NH2 313 315
Ac-LTF$4rn6AYWAQhL$4a5AAAAa-NH2 314 316
Ac-LTF$4rn6AYWAQhL$4a5AAAAa-NH2 315 317
Ac-LTF$4rn6AYWAQL$4a5AAAAAa-NH2 316 318
Ac-LTF$4rn6AYWAQL$4a5AAAAAAa-NH2 317 319
Ac-LTF$4rn6AYWAQL$4a5AAAAAAa-NH2 318 320
Ac-LTF$4rn6EYWAQhL$4a5AANleA-NH2 319 321
Ac-AATF$4rn6AYWAQL$4a5AANleA-NH2 320 322
Ac-LTF$4rn6AYWAQL$4a5AANleAA-NH2 321 323
Ac-ALTF$4rn6AYWAQL$4a5AANleAA-NH2 322 324
Ac-LTF$4rn6AYWAQCba$4a5AANleAA-NH2 323 325
Ac-LTF$4rn6AYWAQhL$4a5AANleAA-NH2 324 326
Ac-LTF$4rn6EYWAQCba$4a5SAAA-NH2 325 327
Ac-LTF$4rn6EYWAQCba$4a5SAAA-NH2 326 328
Ac-LTF$4rn6EYWAQCba$4a5SAAAA-NH2 327 329
Ac-LTF$4rn6EYWAQCba$4a5SAAAA-NH2 328 330
Ac-ALTF$4rn6EYWAQCba$4a5SAA-NH2 329 331
Ac-ALTF$4rn6EYWAQCba$4a5SAAA-NH2 330 332
Ac-ALTF$4rn6EYWAQCba$4a5SAA-NH2 331 333
Ac-LTF$4rn6EYWAQL$4a5AAAAAa-NH2 332 334
Ac-LTF$4rn6EY6clWAQCba$4a5SAA-NH2 333 335 Ac-
LTF$4rn6EF4cooh6clWAQCba$4a5SANleA- NH2 334 336 Ac-
LTF$4rn6EF4cooh6clWAQCba$4a5SANleA- NH2 335 337 Ac-
LTF$4rn6EF4cooh6clWAQCba$4a5AAIa- NH2 336 338 Ac-
LTF$4rn6EF4cooh6clWAQCba$4a5AAIa- NH2 337 339
Ac-LTF$4rn6AY6clWAQL$4a5AAAAAa-NH2 338 340
Ac-LTF$4rn6AY6clWAQL$4a5AAAAAa-NH2 339 341
Ac-F$4rn6AY6clWEAL$4a5AAAAAAa-NH2 340 342
Ac-ETF$4rn6EYWAQL$4a5AAAAAa-NH2 341 343
Ac-ETF$4rn6EYWAQL$4a5AAAAAa-NH2 342 344
Ac-LTF$4rn6EYWAQL$4a5AAAAAAa-NH2 343 345
Ac-LTF$4rn6EYWAQL$4a5AAAAAAa-NH2 344 346
Ac-LTF$4rn6AYWAQL$4a5AANleAAa-NH2 345 347
Ac-LTF$4rn6AYWAQL$4a5AANleAAa-NH2 346 348
Ac-LTF$4rn6EYWAQCba$4a5AAAAAa-NH2 347 349
Ac-LTF$4rn6EYWAQCba$4a5AAAAAa-NH2 348 350
Ac-LTF$4rn6EF4coohWAQCba$4a5AAAAAa- NH2 349 351
Ac-LTF$4rn6EF4coohWAQCba$4a5AAAAAa- NH2 350 352
Ac-LTF$4rn6EYWSQCba$4a5AAAAAa-NH2 351 353
Ac-LTF$4rn6EYWSQCba$4a5AAAAAa-NH2 352 354
Ac-LTF$4rn6EYWAQCba$4a5SAAa-NH2 353 355
Ac-LTF$4rn6EYWAQCba$4a5SAAa-NH2 354 356
Ac-ALTF$4rn6EYWAQCba$4a5SAAa-NH2 355 357
Ac-ALTF$4rn6EYWAQCba$4a5SAAa-NH2 356 358
Ac-ALTF$4rn6EYWAQCba$4a5SAAAa-NH2 357 359
Ac-ALTF$4rn6EYWAQCba$4a5SAAAa-NH2 358 360
Ac-AALTF$4rn6EYWAQCba$4a5SAAAa-NH2 359 361
Ac-AALTF$4rn6EYWAQCba$4a5SAAAa-NH2 360 362
Ac-RTF$4rn6EYWAQCba$4a5SAA-NH2 361 363
Ac-LRF$4rn6EYWAQCba$4a5SAA-NH2 362 364
Ac-LTF$4rn6EYWRQCba$4a5SAA-NH2 363 365
Ac-LTF$4rn6EYWARCba$4a5SAA-NH2 364 366
Ac-LTF$4rn6EYWAQCba$4a5RAA-NH2
365 367 Ac-LTF$4rn6EYWAQCba$4a5SRA-NH2 366 368
Ac-LTF$4rn6EYWAQCba$4a5SAR-NH2 367 369
5-FAM-BaLTF$4rn6EYWAQCba$4a5SAA-NH2 368 370
5-FAM-BaLTF$4rn6AYWAQL$4a5AANleA- NH2 369 371
Ac-LAF$4rn6EYWAQL$4a5AANleA-NH2 370 372
Ac-ATF$4rn6EYWAQL$4a5AANleA-NH2 371 373
Ac-AAF$4rn6EYWAQL$4a5AANleA-NH2 372 374
Ac-AAAF$4rn6EYWAQL$4a5AANleA-NH2 373 375
Ac-AAAAF$4rn6EYWAQL$4a5AANleA-NH2 374 376
Ac-AATF$4rn6EYWAQL$4a5AANleA-NH2 375 377
Ac-AALTF$4rn6EYWAQL$4a5AANleA-NH2 376 378
Ac-AAALTF$4rn6EYWAQL$4a5AANleA-NH2 377 379
Ac-LTF$4rn6EYWAQL$4a5AANleAA-NH2 378 380
Ac-ALTF$4rn6EYWAQL$4a5AANleAA-NH2 379 381
Ac-AALTF$4rn6EYWAQL$4a5AANleAA-NH2 380 382
Ac-LTF$4rn6EYWAQCba$4a5AANleAA-NH2 381 383
Ac-LTF$4rn6EYWAQhL$4a5AANleAA-NH2 382 384
Ac-ALTF$4rn6EYWAQhL$4a5AANleAA-NH2 383 385
Ac-LTF$4rn6ANmYWAQL$4a5AANleA-NH2 384 386
Ac-LTF$4rn6ANmYWAQL$4a5AANleA-NH2 385 387
Ac-LTF$4rn6AYNmWAQL$4a5AANleA-NH2 386 388
Ac-LTF$4rn6AYNmWAQL$4a5AANleA-NH2 387 389
Ac-LTF$4rn6AYAmwAQL$4a5AANleA-NH2 388 390
Ac-LTF$4rn6AYAmwAQL$4a5AANleA-NH2 389 391
Ac-LTF$4rn6AYWAibQL$4a5AANleA-NH2 390 392
Ac-LTF$4rn6AYWAibQL$4a5AANleA-NH2 391 393
Ac-LTF$4rn6AYWAQL$4a5AAibNleA-NH2 392 394
Ac-LTF$4rn6AYWAQL$4a5AAibNleA-NH2 393 395
Ac-LTF$4rn6AYWAQL$4a5AaNleA-NH2 394 396
Ac-LTF$4rn6AYWAQL$4a5AaNleA-NH2 395 397
Ac-LTF$4rn6AYWAQL$4a5ASarNleA-NH2 396 398
Ac-LTF$4rn6AYWAQL$4a5ASarNleA-NH2 397 399
Ac-LTF$4rn6AYWAQL$4a5AANleAib-NH2 398 400
Ac-LTF$4rn6AYWAQL$4a5AANleAib-NH2 399 401
Ac-LTF$4rn6AYWAQL$4a5AANleNmA-NH2 400 402
Ac-LTF$4rn6AYWAQL$4a5AANleNmA-NH2 401 403
Ac-LTF$4rn6AYWAQL$4a5AANleSar-NH2 402 404
Ac-LTF$4rn6AYWAQL$4a5AANleSar-NH2 403 405
Ac-LTF$4rn6AYWAQL$4a5AANleAAib-NH2 404 406
Ac-LTF$4rn6AYWAQL$4a5AANleAAib-NH2 405 407
Ac-LTF$4rn6AYWAQL$4a5AANleANmA-NH2 406 408
Ac-LTF$4rn6AYWAQL$4a5AANleANmA-NH2 407 409
Ac-LTF$4rn6AYWAQL$4a5AANleAa-NH2 408 410
Ac-LTF$4rn6AYWAQL$4a5AANleAa-NH2 409 411
Ac-LTF$4rn6AYWAQL$4a5AANleASar-NH2 410 412
Ac-LTF$4rn6AYWAQL$4a5AANleASar-NH2 413 413
Ac-LTF$4rn6Cou4YWAQL$4a5AANleA-NH2 414 414
Ac-LTF$4rn6Cou4YWAQL$4a5AANleA-NH2 415 415
Ac-LTF$4rn6AYWCou4QL$4a5AANleA-NH2 416 416
Ac-LTF$4rn6AYWAQL$4a5Cou4ANleA-NH2 417 417
Ac-LTF$4rn6AYWAQL$4a5Cou4ANleA-NH2 418 418
Ac-LTF$4rn6AYWAQL$4a5ACou4NleA-NH2 419 419
Ac-LTF$4rn6AYWAQL$4a5ACou4NleA-NH2 420 420
Ac-LTF$4rn6AYWAQL$4a5AANleA-OH 421 421
Ac-LTF$4rn6AYWAQL$4a5AANleA-OH 422 422
Ac-LTF$4rn6AYWAQL$4a5AANleA-NHnPr 423 423
Ac-LTF$4rn6AYWAQL$4a5AANleA-NHnPr 424 424
Ac-LTF$4rn6AYWAQL$4a5AANleA- NHnBu33Me 425 425
Ac-LTF$4rn6AYWAQL$4a5AANleA- NHnBu33Me 426 426
Ac-LTF$4rn6AYWAQL$4a5AANleA-NHHex 427 427
Ac-LTF$4rn6AYWAQL$4a5AANleA-NHHex 428 428
Ac-LTA$4rn6AYWAQL$4a5AANleA-NH2 429 429
Ac-LThL$4rn6AYWAQL$4a5AANleA-NH2 430 430
Ac-LTF$4rn6AYAAQL$4a5AANleA-NH2 431 431
Ac-LTF$4rn6AY2NalAQL$4a5AANleA-NH2 432 432
Ac-LTF$4rn6EYWCou4QCba$4a5SAA-NH2 433 433
Ac-LTF$4rn6EYWCou7QCba$4a5SAA-NH2 435 434
Dmaac-LTF$4rn6EYWAQCba$4a5SAA-NH2 436 435
Dmaac-LTF$4rn6AYWAQL$4a5AAAAAa-NH2 437 436
Dmaac-LTF$4rn6AYWAQL$4a5AAAAAa-NH2 438 437
Dmaac-LTF$4rn6EYWAQL$4a5AAAAAa-NH2 439 438
Dmaac-LTF$4rn6EYWAQL$4a5AAAAAa-NH2 440 439 Dmaac-
LTF$4rn6EF4coohWAQCba$4a5AAIa-NH2 441 440 Dmaac-
LTF$4rn6EF4coohWAQCba$4a5AAIa-NH2 442 441
Dmaac-LTF$4rn6AYWAQL$4a5AANleA-NH2 443 442
Dmaac-LTF$4rn6AYWAQL$4a5AANleA-NH2 444 443
Ac-LTF$4rn6AYWAQL$4a5AANleA-NH2 445 444
Ac-LTF$4rn6EYWAQL$4a5AAAAAa-NH2 446 445
Cou6BaLTF$4rn6EYWAQhL$4a5SAA-NH2 447 446
Cou8BaLTF$4rn6EYWAQhL$4a5SAA-NH2 448 447
Ac-LTF41$4rn6EYWAQL$4a5AAAAAa-NH2
TABLE-US-00006 TABLE 4a SEQ Calc Calc Calc ID Exact Found (M+1)/
(M+2)/ (M+3)/ SP NO: Sequence Mass Mass 1 2 3 449 448
Ac-LTF$4rn6AYWAQL$4a5AANleA-NH2 1812.01 907.89 1813.02 907.01
605.01 450 449 Ac-LTF$4rn6AYWAQL$4a5AAAAAa-NH2 1912.04 957.75
1913.05 957.03 638.35 451 450 Ac-LTF$4rn6EYWAQL$4a5AAAAAa-NH2
1970.04 986.43 1971.05 986.03 657.69 452 451
Ac-LTF$5rn6AYWAQL$5a5AAAAAa-NH2 1912.04 957.38 1913.05 957.03
638.35 153 452 Ac-LTF$4rn6EYWAQCba$4a5SAA-NH2 1784.93 894.38
1785.94 893.47 595.98 454 453 Ac-LTF$4rn4EYWAQCba$4a5SAA-NH2
1756.89 880.05 1757.9 879.45 586.64 455 454
Ac-LTF$4rn5EYWAQCba$4a5SAA-NH2 1770.91 887.08 1771.92 886.46 591.31
456 455 Ac-LTF$5rn6EYWAQCba$5a5SAA-NH2 1784.92 894.11 1785.93
893.47 595.98 457 456 Ac-LTF$4rn6EYWAQCba51-$4a5SAA- 1910.82 957.01
1911.83 956.42 637.95 NH2 459 457 Ac-LTA$5rn6EYWAQCba$5a5SAA-NH2
1708.89 856 1709.9 855.45 570.64 460 458
Ac-LTA$4rn6EYWAQCba4a5SAA-NH2 1708.89 856 1709.9 855.45 570.64 461
459 5-FAN- 2172 1087.81 2173.01 1087.01 725.01
BaLTF$4rn6EYWAQCba$4a5SAA-NH2 462 460 5-FAM- 2095.97 1049.79
2096.98 1048.99 699.66 BaLTA$4rn6EYWAQCba$4a5SAA-NH2 463 461 5-FAM-
2172 1087.53 2173.01 1087.01 725.01 BaLTF$5rn6EYWAQCba$5a5SAA-NH2
464 462 5-FAM- 2095.97 1049.98 2096.98 1048.99 699.66
BaLTA$5rn6EYWAQCba$5a5SAA-NH2 465 463
Ac-LTF$4rn6EYWAQCba5Ph-$4a5SAA- 1675.87 932.31 1676.88 931.48
559.63 NH2 466 464 Ac-LTF$4rn6EYWAQCba5Prp- 1675.87 914.46 1676.88
913.48 559.63 $4a5SAA-NH2 467 465 Ac-LTF$4rn6AYWAAL$4a5AAAAAa-NH2
1855.01 1856.02 928.51 619.34 468 466 Ac-LTF$4rn6EYWAQCba5penNH2-
1675.87 1676.88 838.94 559.63 $4a5SAA-NH2 469 467
Ac-LTF$4rn6EYWAQCba5BnzNH2- 1675.87 1676.88 838.94 559.63
$4a5SAA-NH2 470 468 Ac-LTF$4rn6EYWAQCba5prpOMe- 929.17 928.48
$4a5SAA-NH2 932 469 Ac-LTF$5rn6EYWAQL4Me$5a5AAAAAa- 1926.05 1927.06
964.03 643.02 NH2 933 470 Ac-LTF$5rn6EYWAQL4Ph$5a5AAAAAa- 1988.07
1989.07 995.04 663.70 NH2 934 471 Ac- 1740.93 1741.94 871.48 581.32
LTF$5rn6EYWAQCba4Me$5a5SAANH2 935 472 Ac- 1802.95 1803.96 902.48
601.99 LTF$5rn6EYWAQCba4Ph$5a5SAANH2
[0510] In the sequences shown above and elsewhere, the following
abbreviations are used: "Nle" represents norleucine, "Aib"
represents 2-aminoisobutyric acid, "Ac" represents acetyl, and "Pr"
represents propionyl Amino acids represented as "S" are alpha-Me
S5-pentenyl-alanine olefin amino acids connected by an all-carbon
crosslinker comprising one double bond Amino acids represented as
"$r5" are alpha-Me R5-pentenyl-alanine olefin amino acids connected
by an all-carbon comprising one double bond Amino acids represented
as "$s8" are alpha-Me S8-octenyl-alanine olefin amino acids
connected by an all-carbon crosslinker comprising one double bond.
Amino acids represented as "$r8" are alpha-Me R8-octenyl-alanine
olefin amino acids connected by an all-carbon crosslinker
comprising one double bond. "Ahx" represents an aminocyclohexyl
linker. The crosslinkers are linear all-carbon crosslinker
comprising eight or eleven carbon atoms between the alpha carbons
of each amino acid Amino acids represented as "$/" are alpha-Me
S5-pentenyl-alanine olefin amino acids that are not connected by
any crosslinker Amino acids represented as "$/r5" are alpha-Me
R5-pentenyl-alanine olefin amino acids that are not connected by
any crosslinker Amino acids represented as "$/s8" are alpha-Me
S8-octenyl-alanine olefin amino acids that are not connected by any
crosslinker Amino acids represented as "$/r8" are alpha-Me
R8-octenyl-alanine olefin amino acids that are not connected by any
crosslinker. Amino acids represented as "Amw" are alpha-Me
tryptophan amino acids. Amino acids represented as "Aml" are
alpha-Me leucine amino acids Amino acids represented as "Amf" are
alpha-Me phenylalanine amino acids. Amino acids represented as
"2ff" are 2-fluoro-phenylalanine amino acids. Amino acids
represented as "3ff" are 3-fluoro-phenylalanine amino acids Amino
acids represented as "St" are amino acids comprising two
pentenyl-alanine olefin side chains, each of which is crosslinked
to another amino acid as indicated Amino acids represented as
"St//" are amino acids comprising two pentenyl-alanine olefin side
chains that are not crosslinked. Amino acids represented as "% St"
are amino acids comprising two pentenyl-alanine olefin side chains,
each of which is crosslinked to another amino acid as indicated via
fully saturated hydrocarbon crosslinks. Amino acids represented as
"Ba" are beta-alanine. The lower-case character "e" or "z" within
the designation of a crosslinked amino acid (e.g. "$er8" or "$zr8")
represents the configuration of the double bond (E or Z,
respectively). In other contexts, lower-case letters such as "a" or
"f" represent D amino acids (e.g. D-alanine, or D-phenylalanine,
respectively). Amino acids designated as "NmW" represent
N-methyltryptophan. Amino acids designated as "NmY" represent
N-methyltyrosine. Amino acids designated as "NmA" represent
N-methylalanine Amino acids designated as "Sar" represent sarcosine
Amino acids designated as "Cha" represent cyclohexyl alanine Amino
acids designated as "Cpg" represent cyclopentyl glycine. Amino
acids designated as "Chg" represent cyclohexyl glycine Amino acids
designated as "Cba" represent cyclobutyl alanine Amino acids
designated as "F4I" represent 4-iodo phenylalanine. Amino acids
designated as "F3Cl " represent 3-chloro phenylalanine Amino acids
designated as "F4cooh" represent 4-carboxy phenylalanine Amino
acids designated as "F34F2" represent 3,4-difluoro phenylalanine
Amino acids designated as "6c1W" represent 6-chloro tryptophan. The
designation "iso1" or "iso2" indicates that the peptidomimetic
macrocycle is a single isomer. "Ac3c" represents a
aminocyclopropane carboxylic acid residue.
[0511] Amino acids designated as "Cou4", "Cou6", "Cou7" and "Cou8",
respectively, represent the following structures:
##STR00150## ##STR00151##
[0512] In some embodiments, a peptidomimetic macrocycle is obtained
in more than one isomer, for example due to the configuration of a
double bond within the structure of the crosslinker (E vs Z). Such
isomers can or can not be separable by conventional chromatographic
methods. In some embodiments, one isomer has improved biological
properties relative to the other isomer. In one embodiment, an E
crosslinker olefin isomer of a peptidomimetic macrocycle has better
solubility, better target affinity, better in vivo or in vitro
efficacy, higher helicity, or improved cell permeability relative
to its Z counterpart. In another embodiment, a Z crosslinker olefin
isomer of a peptidomimetic macrocycle has better solubility, better
target affinity, better in vivo or in vitro efficacy, higher
helicity, or improved cell permeability relative to its E
counterpart.
[0513] Amino acids forming crosslinkers are represented according
to the legend indicated below.
[0514] Stereochemistry at the alpha position of each amino acid is
S unless otherwise indicated Amino acids labeled "4Me" were
prepared using an amino acid comprising an alkyne which was
methyl-substituted (internal alkyne), resulting in triazole groups
comprising a methyl group at the 4-position. Amino acids labeled
"4Ph" were prepared using an amino acid comprising an alkyne which
was phenyl-substituted (internal alkyne), resulting in triazole
groups comprising a phenyl group at the 4-position. For azide amino
acids, the number of carbon atoms indicated refers to the number of
methylene units between the alpha carbon and the terminal azide.
For alkyne amino acids, the number of carbon atoms indicated is the
number of methylene units between the alpha position and the
triazole moiety plus the two carbon atoms within the triazole group
derived from the alkyne.
TABLE-US-00007 $5n3 Alpha-Me azide 1,5 triazole (3 carbon) #5n3
Alpha-H azide 1,5 triazole (3 carbon) $4a5 Alpha-Me alkyne 1,4
triazole (5 carbon) $4a6 Alpha-Me alkyne 1,4 triazole (6 carbon)
$5a5 Alpha-Me alkyne 1,5 triazole (5 carbon) $5a6 Alpha-Me alkyne
1,5 triazole (6 carbon) #4a5 Alpha-H alkyne 1,4 triazole (5 carbon)
#5a5 Alpha-H alkyne 1,5 triazole (5 carbon) $5n5 Alpha-Me azide 1,5
triazole (5 carbon) $5n6 Alpha-Me azide 1,5 triazole (6 carbon)
$4n5 Alpha-Me azide 1,4 triazole (5 carbon) $4n6 Alpha-Me azide 1,4
triazole (6 carbon) $4ra5 Alpha-Me R-alkyne 1,4 triazole (5 carbon)
$4ra6 Alpha-Me R-alkyne 1,4 triazole (6 carbon) $4rn4 Alpha-Me
R-azide 1,4 triazole (4 carbon) $4rn5 Alpha-Me R-azide 1,4 triazole
(5 carbon) $4rn6 Alpha-Me R-azide 1,4 triazole (6 carbon) $5rn5
Alpha-Me R-azide 1,5 triazole (5 carbon) $5ra5 Alpha-Me R-alkyne
1,5 triazole (5 carbon) $5ra6 Alpha-Me R-alkyne 1,5 triazole (6
carbon) $5rn6 Alpha-Me R-azide 1,5 triazole (6 carbon) #5rn6
Alpha-H R-azide 1,5 triazole (6 carbon) $4rn5 Alpha-Me R-azide 1,4
triazole (5 carbon) #4rn5 Alpha-H R-azide 1,4 triazole (5 carbon)
4Me$5rn6 Alpha-Me R-azide 1,5 triazole (6 carbon); 4-Me substituted
triazole 4Me$5a5 Alpha-Me alkyne 1,5 triazole (5 carbon); 4-Me
substituted triazole 4Ph$5a5 Alpha-Me alkyne 1,5 triazole (5
carbon); 4-phenyl substituted triazole
[0515] Amino acids designated as "5I", "5penNH2", "5BnzNH2",
"5prpOMe", "5Ph", and "5prp", refer to crosslinked amino acids of
the type shown in the following exemplary peptidomimetic macrocycle
of Formula I:
##STR00152##
[0516] In the above structure, X is, for example, one of the
following substituents:
##STR00153##
[0517] wherein "Cyc" is a suitable aryl, cycloalkyl, cycloalkenyl,
heteroaryl, or heterocyclyl group, unsubstituted or optionally
substituted with an R.sub.a or R.sub.b group as described
above.
[0518] In some embodiments, the triazole substituent is chosen from
the group consisting of:
##STR00154##
[0519] Table 4 shows exemplary peptidomimetic macrocycles of
Formula I:
TABLE-US-00008 TABLE 4b Structure SP-449 (SEQ ID NO: 448)
##STR00155## SP-64 (SEQ ID NO: 66) ##STR00156## SP-153 (SEQ ID NO:
155) ##STR00157## SP-98 (SEQ ID NO: 100) ##STR00158## SP-456 (SEQ
ID NO: 455) ##STR00159## SP-470 (SEQ ID NO: 468) ##STR00160##
[0520] In some embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 5:
TABLE-US-00009 TABLE 5 SEQ ID # NO: Sequence 1 473
Ac-QSQQTF$5rn6NLWRLL$5a5QN-NH2 2 474 Ac-QSQQTF$4rn5NLWRLL$4a5QN-NH2
3 475 Ac-QSQQTF#5rn6NLWRLL#5a5QN-NH2 4 476
Ac-QSQQTF#4rn5NLWRLL#4a5QN-NH2 5 477 Ac-QSQQTF$5rn5NLWRLL$5a5QN-NH2
6 478 Ac-QSQQTF$5ra5NLWRLL$5n5QN-NH2 7 479
Ac-QSQQTF$5ra5NLWRLL$5n6QN-NH2 8 480 Ac-QSQQTF$4ra5NLWRLL$4n5QN-NH2
9 481 Ac-QSQQTF$4ra5NLWRLL$4n6QN-NH2 10 482
Ac-QSQQTF$4rn6NLWRLL$4a5QN-NH2 11 483
Ac-QSQQTF$5rn6NLWRLL$5a6QN-NH2 12 484
Ac-QSQQTF$5ra6NLWRLL$5n6QN-NH2 13 485
Ac-QSQQTF$4rn6NLWRLL$4a6QN-NH2 14 486
Ac-QSQQTF$4ra6NLWRLL$4n6QN-NH2 15 487
Ac-QSQQTF$4rn5NLWRLL$4a6QN-NH2 16 488
Ac-QSQQTF4Me$5rn6NLWRLL4Me$5a5QN-NH2 17 489
Ac-LTF$4ra5HYWAQL$4n6S-NH2 18 490 H-F$4rn6HYWAQL$4a5S-NH2 19 491
Ac-LTF$4rn6HYWAQL$4a5S-NH2 20 492 Ac-F$4rn6HYWAQL$4a5S-NH2 21 493
Ac-LTF$4rn6HYWAQL$4a6S-NH2 22 494 Ac-LTF$5ra5HYWAQL$5n6S-NH2 23 495
Ac-LTF$4rn6AYWAQL$4a5A-NH2 24 496 Ac-LTF$5ra5HYWAQL$5n6S-NH2 25 497
Ac-LTF$4rn6AYWAQL$4a5A-NH2 26 498 Ac-LTFEHYWAQLTS-NH2
[0521] Peptides shown can comprise an N-terminal capping group such
as acetyl or an additional linker such as beta-alanine between the
capping group and the start of the peptide sequence.
[0522] In some embodiments, peptidomimetic macrocycles do not
comprise a peptidomimetic macrocycle structure as shown in Table
5.
Example 3
Peptidomimetic Macrocycles of Formula II
[0523] Peptidomimetic macrocycles were designed by replacing two or
more naturally occurring amino acids with the corresponding
synthetic amino acids. Substitutions were made at i and i+4, and i
and i+7 positions. Macrocycles were generated by solid phase
peptide synthesis followed by crosslinking the peptides via their
thiol-containing side chains Peptide synthesis is performed either
manually or on an automated peptide synthesizer (Applied
Biosystems, model 433A), using solid phase conditions, rink amide
AM resin (Novabiochem), and Fmoc main-chain protecting group
chemistry. The N-termini of the synthetic peptides are acetylated,
while the C-termini are amidated.
[0524] The fully protected resin-bound peptides are synthesized on
a Rink amide MBHA resin (loading 0.62 mmol/g) on a 0.1 mmol scale.
Deprotection of the temporary Fmoc group is achieved by 2.times.20
min treatments of the resin bound peptide with 25% (v/v) piperidine
in NMP. After extensive flow washing with NMP and dichloromethane,
coupling of each successive amino acid was achieved with 1.times.60
min incubation with the appropriate preactivated Fmoc-amino acid
derivative. All protected amino acids (1 mmol) were dissolved in
NMP and activated with HCTU (1 mmol) and DIEA (1 mmol) prior to
transfer of the coupling solution to the deprotected resin-bound
peptide. After coupling was completed, the resin was extensively
flow washed in preparation for the next deprotection/coupling
cycle. Acetylation of the amino terminus was carried out in the
presence of acetic anhydride/DIEA in NMP/NMM. The LC-MS analysis of
a cleaved and deprotected sample obtained from an aliquot of the
fully assembled resin-bound peptide was accomplished in order to
verifying the completion of each coupling.
[0525] Purification of cross-linked compounds is achieved by high
performance liquid chromatography (HPLC) (Varian ProStar) on a
reverse phase C18 column (Varian) to yield the pure compounds.
Chemical composition of the pure products was confirmed by LC/MS
mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC
system) and amino acid analysis (Applied Biosystems, model
420A).
[0526] In a typical example, a peptide resin (0.1 mmol) was washed
with DCM. Deprotection of the temporary Mmt group was achieved by
3.times.3 min treatments of the resin bound peptide with 2% TFA/DCM
5% TIPS, then 30min treatments until no orange color is observed in
the filtrate. In between treatments the resin was extensively flow
washed with DCM. After complete removal of Mmt, the resin was
washed with 5% DIEA/NMP solution 3.times. and considered ready for
bisthioether coupling. Resin was loaded into a reaction vial.
DCM/DMF 1/1 was added to the reaction vessel, followed by DIEA
(2.4eq). After mixing well for 5 minutes,
4,4'-Bis(bromomethyl)biphenyl (1.05 eq) (TCI America B1921) was
added. The reaction was then mechanically agitated at room
temperature overnight. Where needed, the reaction was allowed
additional time to reach completion. A similar procedure may be
used in the preparation of five-methylene, six-methylene or
seven-methylene crosslinkers ("% c7", "% c6", or "% c5").
[0527] The bisthioether resin-bound peptides were deprotected and
cleaved from the solid support by treatment with TFA/H.sub.2O/TIS
(94/3/3 v/v) for 3 h at room temperature. After filtration of the
resin the TFA solution was precipitated in cold diethyl ether and
centrifuged to yield the desired product as a solid. The crude
product was purified by preparative HPLC.
[0528] Table 6 show a list of peptidomimetic macrocycles.
TABLE-US-00010 TABLE 6 SEQ ID SP NO: sequence 471 499
Ac-F%cs7AYWEAc3cL%c7AAA-NH2 472 500 Ac-F%cs7AYWEAc3cL%c7AAibA-NH2
473 501 Ac-LTF%cs7AYWAQL%c7SANle-NH2 474 502
Ac-LTF%cs7AYWAQL%c7SAL-NH2 475 503 Ac-LTF%cs7AYWAQL%c7SAM-NH2 476
504 Ac-LTF%cs7AYWAQL%c7SAhL-NH2 477 505 Ac-LTF%cs7AYWAQL%c7SAF-NH2
478 506 Ac-LTF%cs7AYWAQL%c7SAI-NH2 479 507
Ac-LTF%cs7AYWAQL%c7SAChg-NH2 480 508 Ac-LTF%cs7AYWAQL%c7SAAib-NH2
481 509 Ac-LTF%cs7AYWAQL%c7SAA-NH2 482 510
Ac-LTF%cs7AYWA%c7L%c7S%c7Nle-NH2 483 511
Ac-LTF%cs7AYWA%c7L%c7S%c7A-NH2 484 512
Ac-F%cs7AYWEAc3cL%c7AANle-NH2 485 513 Ac-F%cs7AYWEAc3cL%c7AAL-NH2
486 514 Ac-F%cs7AYWEAc3cL%c7AAM-NH2 487 515
Ac-F%cs7AYWEAc3cL%c7AAhL-NH2 488 516 Ac-F%cs7AYWEAc3cL%c7AAF-NH2
489 517 Ac-F%cs7AYWEAc3cL%c7AAI-NH2 490 518
Ac-F%cs7AYWEAc3cL%c7AAChg-NH2 491 519 Ac-F%cs7AYWEAc3cL%c7AACha-NH2
492 520 Ac-F%cs7AYWEAc3cL%c7AAAib-NH2 493 521
Ac-LTF%cs7AYWAQL%c7AAAibV-NH2 494 522 Ac-LTF%cs7AYWAQL%c7AAAibV-NH2
495 523 Ac-LTF%cs7AYWAQL%c7SAibAA-NH2 496 524
Ac-LTF%cs7AYWAQL%c7SAibAA-NH2 497 525
Ac-HLTF%cs7HHWHQL%c7AANleNle-NH2 498 526
Ac-DLTF%cs7HHWHQL%c7RRLV-NH2 499 527 Ac-HHTF%cs7HHWHQL%c7AAML-NH2
500 528 Ac-F%cs7HHWHQL%c7RRDCha-NH2 501 529
Ac-F%cs7HHWHQL%c7HRFV-NH2 502 530 Ac-HLTF%cs7HHWHQL%c7AAhLA-NH2 503
531 Ac-DLTF%cs7HHWHQL%c7RRChgl-NH2 504 532
Ac-DLTF%cs7HHWHQL%c7RRChgl-NH2 505 533
Ac-HHTF%cs7HHWHQL%c7AAChav-NH2 506 534 Ac-F%cs7HHWHQL%c7RRDa-NH2
507 535 Ac-F%cs7HHWHQL%c7HRAibG-NH2 508 536
Ac-F%cs7AYWAQL%c7HHNleL-NH2 509 537 Ac-F%cs7AYWSAL%c7HQANle-NH2 510
538 Ac-F%cs7AYWVQL%c7QHChgl-NH2 511 539 Ac-F%cs7AYWTAL%c7QQNlev-NH2
512 540 Ac-F%cs7AYWYQL%c7HAibAa-NH2 513 541
Ac-LTF%cs7AYWAQL%c7HHLa-NH2 514 542 Ac-LTF%cs7AYWAQL%c7HHLa-NH2 515
543 Ac-LTF%cs7AYWAQL%c7HQNlev-NH2 516 544
Ac-LTF%cs7AYWAQL%c7HQNlev-NH2 517 545 Ac-LTF%cs7AYWAQL%c7QQMl-NH2
518 546 Ac-LTF%cs7AYWAQL%c7QQMl-NH2 519 547
Ac-LTF%cs7AYWAQL%c7HAibhLV-NH2 520 548 Ac-LTF%cs7AYWAQL%c7AHFA-NH2
521 549 Ac-HLTF%cs7HHWHQL%c7AANlel-NH2 522 550
Ac-DLTF%cs7HHWHQL%c7RRLa-NH2 523 551 Ac-HHTF%cs7HHWHQL%c7AAMv-NH2
524 552 Ac-F%cs7HHWHQL%c7RRDA-NH2 525 553
Ac-F%cs7HHWHQL%c7HRFCha-NH2 526 554 Ac-F%cs7AYWEAL%c7AA-NHAm 527
555 Ac-F%cs7AYWEAL%c7AA-NHiAm 528 556 Ac-F%cs7AYWEAL%c7AA-NHnPr3Ph
529 557 Ac-F%cs7AYWEAL%c7AA-NHnBu33Me 530 558
Ac-F%cs7AYWEAL%c7AA-NHnPr 531 559 Ac-F%cs7AYWEAL%c7AA-NHnEt2Ch 532
560 Ac-F%cs7AYWEAL%c7AA-NHnEt2Cp 533 561 Ac-F%cs7AYWEAL%c7AA-NHHex
534 562 Ac-LTF%cs7AYWAQL%c7AAIA-NH2 535 563
Ac-LTF%cs7AYWAQL%c7AAIA-NH2 536 564 Ac-LTF%cs7AYWAAL%c7AAMA-NH2 537
565 Ac-LTF%cs7AYWAAL%c7AAMA-NH2 538 566
Ac-LTF%cs7AYWAQL%c7AANleA-NH2 539 567 Ac-LTF%cs7AYWAQL%c7AANleA-NH2
540 568 Ac-LTF%cs7AYWAQL%c7AAIa-NH2 541 569
Ac-LTF%cs7AYWAQL%c7AAIa-NH2 542 570 Ac-LTF%cs7AYWAAL%c7AAMa-NH2 543
571 Ac-LTF%cs7AYWAAL%c7AAMa-NH2 544 572
Ac-LTF%cs7AYWAQL%c7AANlea-NH2 545 573 Ac-LTF%cs7AYWAQL%c7AANlea-NH2
546 574 Ac-LTF%cs7AYWAAL%c7AAIv-NH2 547 575
Ac-LTF%cs7AYWAAL%c7AAIv-NH2 548 576 Ac-LTF%cs7AYWAQL%c7AAMv-NH2 549
577 Ac-LTF%cs7AYWAAL%c7AANlev-NH2 550 578
Ac-LTF%cs7AYWAAL%c7AANlev-NH2 551 579 Ac-LTF%cs7AYWAQL%c7AAIl-NH2
552 580 Ac-LTF%cs7AYWAQL%c7AAIl-NH2 553 581
Ac-LTF%cs7AYWAAL%c7AAMl-NH2 554 582 Ac-LTF%cs7AYWAQL%c7AANlel-NH2
555 583 Ac-LTF%cs7AYWAQL%c7AANlel-NH2 556 584
Ac-F%cs7AYWEAL%c7AAMA-NH2 557 585 Ac-F%cs7AYWEAL%c7AANleA-NH2 558
586 Ac-F%cs7AYWEAL%c7AAIa-NH2 559 587 Ac-F%cs7AYWEAL%c7AAMa-NH2 560
588 Ac-F%cs7AYWEAL%c7AANlea-NH2 561 589 Ac-F%cs7AYWEAL%c7AAIv-NH2
562 590 Ac-F%cs7AYWEAL%c7AAMv-NH2 563 591
Ac-F%cs7AYWEAL%c7AANlev-NH2 564 592 Ac-F%cs7AYWEAL%c7AAIl-NH2 565
593 Ac-F%cs7AYWEAL%c7AAMl-NH2 566 594 Ac-F%cs7AYWEAL%c7AANlel-NH2
567 595 Ac-F%cs7AYWEAL%c7AANlel-NH2 568 596
Ac-LTF%cs7AY6clWAQL%c7SAA-NH2 569 597 Ac-LTF%cs7AY6clWAQL%c7SAA-NH2
570 598 Ac-WTF%cs7FYWSQL%c7AVAa-NH2 571 599
Ac-WTF%cs7FYWSQL%c7AVAa-NH2 572 600 Ac-WTF%cs7VYWSQL%c7AVA-NH2 573
601 Ac-WTF%cs7VYWSQL%c7AVA-NH2 574 602 Ac-WTF%cs7FYWSQL%c7SAAa-NH2
575 603 Ac-WTF%cs7FYWSQL%c7SAAa-NH2 576 604
Ac-WTF%cs7VYWSQL%c7AVAaa-NH2 577 605 Ac-WTF%cs7VYWSQL%c7AVAaa-NH2
578 606 Ac-LTF%cs7AYWAQL%c7AVG-NH2 579 607
Ac-LTF%cs7AYWAQL%c7AVG-NH2 580 608 Ac-LTF%cs7AYWAQL%c7AVQ-NH2 581
609 Ac-LTF%cs7AYWAQL%c7AVQ-NH2 582 610 Ac-LTF%cs7AYWAQL%c7SAa-NH2
583 611 Ac-LTF%cs7AYWAQL%c7SAa-NH2 584 612
Ac-LTF%cs7AYWAQhL%c7SAA-NH2 585 613 Ac-LTF%cs7AYWAQhL%c7SAA-NH2 586
614 Ac-LTF%cs7AYWEQLStSA%c7-NH2 587 615 Ac-LTF%cs7AYWAQL%c7SLA-NH2
588 616 Ac-LTF%cs7AYWAQL%c7SLA-NH2 589 617
Ac-LTF%cs7AYWAQL%c7SWA-NH2 590 618 Ac-LTF%cs7AYWAQL%c7SWA-NH2 591
619 Ac-LTF%cs7AYWAQL%c7SVS-NH2 592 620
Ac-LTF%cs7AYWAQL%c7SAS-NH2
593 621 Ac-LTF%cs7AYWAQL%c7SVG-NH2 594 622
Ac-ETF%cs7VYWAQL%c7SAa-NH2 595 623 Ac-ETF%cs7VYWAQL%c7SAA-NH2 596
624 Ac-ETF%cs7VYWAQL%c7SVA-NH2 597 625 Ac-ETF%cs7VYWAQL%c7SLA-NH2
598 626 Ac-ETF%cs7VYWAQL%c7SWA-NH2 599 627
Ac-ETF%cs7KYWAQL%c7SWA-NH2 600 628 Ac-ETF%cs7VYWAQL%c7SVS-NH2 601
629 Ac-ETF%cs7VYWAQL%c7SAS-NH2 602 630 Ac-ETF%cs7VYWAQL%c7SVG-NH2
603 631 Ac-LTF%cs7VYWAQL%c7SSa-NH2 604 632
Ac-ETF%cs7VYWAQL%c7SSa-NH2 605 633 Ac-LTF%cs7VYWAQL%c7SNa-NH2 606
634 Ac-ETF%cs7VYWAQL%c7SNa-NH2 607 635 Ac-LTF%cs7VYWAQL%c7SAa-NH2
608 636 Ac-LTF%cs7VYWAQL%c7SVA-NH2 609 637
Ac-LTF%cs7VYWAQL%c7SVA-NH2 610 638 Ac-LTF%cs7VYWAQL%c7SWA-NH2 611
639 Ac-LTF%cs7VYWAQL%c7SVS-NH2 612 640 Ac-LTF%cs7VYWAQL%c7SVS-NH2
613 641 Ac-LTF%cs7VYWAQL%c7SAS-NH2 614 642
Ac-LTF%cs7VYWAQL%c7SAS-NH2 615 643 Ac-LTF%cs7VYWAQL%c7SVG-NH2 616
644 Ac-LTF%cs7VYWAQL%c7SVG-NH2 617 645 Ac-LTF%cs7EYWAQCha%c7SAA-NH2
618 646 Ac-LTF%cs7EYWAQCha%c7SAA-NH2 619 647
Ac-LTF%cs7EYWAQCpg%c7SAA-NH2 620 648 Ac-LTF%cs7EYWAQCpg%c7SAA-NH2
621 649 Ac-LTF%cs7EYWAQF%c7SAA-NH2 622 650
Ac-LTF%cs7EYWAQF%c7SAA-NH2 623 651 Ac-LTF%cs7EYWAQCba%c7SAA-NH2 624
652 Ac-LTF%cs7EYWAQCba%c7SAA-NH2 625 653
Ac-LTF3C1%cs7EYWAQL%c7SAA-NH2 626 654 Ac-LTF3C1%cs7EYWAQL%c7SAA-NH2
627 655 Ac-LTF34F2%cs7EYWAQL%c7SAA-NH2 628 656
Ac-LTF34F2%cs7EYWAQL%c7SAA-NH2 629 657
Ac-LTF34F2%cs7EYWAQhL%c7SAA-NH2 630 658
Ac-LTF34F2%cs7EYWAQhL%c7SAA-NH2 631 659 Ac-ETF%cs7EYWAQL%c7SAA-NH2
632 660 Ac-LTF%cs7AYWVQL%c7SAA-NH2 633 661
Ac-LTF%cs7AHWAQL%c7SAA-NH2 634 662 Ac-LTF%cs7AEWAQL%c7SAA-NH2 635
663 Ac-LTF%cs7ASWAQL%c7SAA-NH2 636 664 Ac-LTF%cs7AEWAQL%c7SAA-NH2
637 665 Ac-LTF%cs7ASWAQL%c7SAA-NH2 638 666
Ac-LTF%cs7AF4coohWAQL%c7SAA-NH2 639 667
Ac-LTF%cs7AF4coohWAQL%c7SAA-NH2 640 668 Ac-LTF%cs7AHWAQL%c7AAIa-NH2
641 669 Ac-ITF%cs7FYWAQL%c7AAIa-NH2 642 670
Ac-ITF%cs7EHWAQL%c7AAIa-NH2 643 671 Ac-ITF%cs7EHWAQL%c7AAIa-NH2 644
672 Ac-ETF%cs7EHWAQL%c7AAIa-NH2 645 673 Ac-ETF%cs7EHWAQL%c7AAIa-NH2
646 674 Ac-LTF%cs7AHWVQL%c7AAIa-NH2 647 675
Ac-ITF%cs7FYWVQL%c7AAIa-NH2 648 676 Ac-ITF%cs7EYWVQL%c7AAIa-NH2 649
677 Ac-ITF%cs7EHWVQL%c7AAIa-NH2 650 678 Ac-LTF%cs7AEWAQL%c7AAIa-NH2
651 679 Ac-LTF%cs7AF4coohWAQL%c7AAIa-NH2 652 680
Ac-LTF%cs7AF4coohWAQL%c7AAIa-NH2 653 681
Ac-LTF%cs7AHWAQL%c7AHFA-NH2 654 682 Ac-ITF%cs7FYWAQL%c7AHFA-NH2 655
683 Ac-ITF%cs7FYWAQL%c7AHFA-NH2 656 684 Ac-ITF%cs7FHWAQL%c7AEFA-NH2
657 685 Ac-ITF%cs7FHWAQL%c7AEFA-NH2 658 686
Ac-ITF%cs7EHWAQL%c7AHFA-NH2 659 687 Ac-ITF%cs7EHWAQL%c7AHFA-NH2 660
688 Ac-LTF%cs7AHWVQL%c7AHFA-NH2 661 689 Ac-ITF%cs7FYWVQL%c7AHFA-NH2
662 690 Ac-ITF%cs7EYWVQL%c7AHFA-NH2 663 691
Ac-ITF%cs7EHWVQL%c7AHFA-NH2 664 692 Ac-ITF%cs7EHWVQL%c7AHFA-NH2 665
693 Ac-ETF%cs7EYWAAL%c7SAA-NH2 666 694 Ac-LTF%cs7AYWVAL%c7SAA-NH2
667 695 Ac-LTF%cs7AHWAAL%c7SAA-NH2 668 696
Ac-LTF%cs7AEWAAL%c7SAA-NH2 669 697 Ac-LTF%cs7AEWAAL%c7SAA-NH2 670
698 Ac-LTF%cs7ASWAAL%c7SAA-NH2 671 699 Ac-LTF%cs7ASWAAL%c7SAA-NH2
672 700 Ac-LTF%cs7AYWAAL%c7AAIa-NH2 673 701
Ac-LTF%cs7AYWAAL%c7AAIa-NH2 674 702 Ac-LTF%cs7AYWAAL%c7AHFA-NH2 675
703 Ac-LTF%cs7EHWAQL%c7AHIa-NH2 676 704 Ac-LTF%cs7EHWAQL%c7AHIa-NH2
677 705 Ac-LTF%cs7AHWAQL%c7AHIa-NH2 678 706
Ac-LTF%cs7EYWAQL%c7AHIa-NH2 679 707 Ac-LTF%cs7AYWAQL%c7AAFa-NH2 680
708 Ac-LTF%cs7AYWAQL%c7AAFa-NH2 681 709 Ac-LTF%cs7AYWAQL%c7AAWa-NH2
682 710 Ac-LTF%cs7AYWAQL%c7AAVa-NH2 683 711
Ac-LTF%cs7AYWAQL%c7AAVa-NH2 684 712 Ac-LTF%cs7AYWAQL%c7AALa-NH2 685
713 Ac-LTF%cs7AYWAQL%c7AALa-NH2 686 714 Ac-LTF%cs7EYWAQL%c7AAIa-NH2
687 715 Ac-LTF%cs7EYWAQL%c7AAIa-NH2 688 716
Ac-LTF%cs7EYWAQL%c7AAFa-NH2 689 717 Ac-LTF%cs7EYWAQL%c7AAFa-NH2 690
718 Ac-LTF%cs7EYWAQL%c7AAVa-NH2 691 719 Ac-LTF%cs7EYWAQL%c7AAVa-NH2
692 720 Ac-LTF%cs7EHWAQL%c7AAIa-NH2 693 721
Ac-LTF%cs7EHWAQL%c7AAIa-NH2 694 722 Ac-LTF%cs7EHWAQL%c7AAWa-NH2 695
723 Ac-LTF%cs7EHWAQL%c7AAWa-NH2 696 724 Ac-LTF%cs7EHWAQL%c7AALa-NH2
697 725 Ac-LTF%cs7EHWAQL%c7AALa-NH2 698 726
Ac-ETF%cs7EHWVQL%c7AALa-NH2 699 727 Ac-LTF%cs7AYWAQL%c7AAAa-NH2 700
728 Ac-LTF%cs7AYWAQL%c7AAAa-NH2 701 729
Ac-LTF%cs7AYWAQL%c7AAAibA-NH2 702 730 Ac-LTF%cs7AYWAQL%c7AAAibA-NH2
703 731 Ac-LTF%cs7AYWAQL%c7AAAAa-NH2 704 732
Ac-LTF%c7r5AYWAQL%c7s8AAIa-NH2 705 733
Ac-LTF%c7r5AYWAQL%c7s8SAA-NH2 706 734
Ac-LTF%cs7AYWAQCba%c7AANleA-NH2 707 735
Ac-ETF%cs7AYWAQCba%c7AANleA-NH2 708 736
Ac-LTF%cs7EYWAQCba%c7AANleA-NH2 709 737
Ac-LTF%cs7AYWAQCba%c7AWNleA-NH2 710 738
Ac-ETF%cs7AYWAQCba%c7AWNleA-NH2 711 739
Ac-LTF%cs7EYWAQCba%c7AWNleA-NH2 712 740
Ac-LTF%cs7EYWAQCba%c7SAFA-NH2 713 741
Ac-LTF34F2%cs7EYWAQCba%c7SANleA-NH2 714 742
Ac-LTF%cs7EF4coohWAQCba%c7SANleA-NH2 715 743
Ac-LTF%cs7EYWSQCba%c7SANleA-NH2 716 744
Ac-LTF%cs7EYWWQCba%c7SANleA-NH2 717 745
Ac-LTF%cs7EYWAQCba%c7AAIa-NH2 718 746
Ac-LTF34F2%cs7EYWAQCba%c7AAIa-NH2
719 747 Ac-LTF%cs7EF4coohWAQCba%c7AAIa-NH2 720 748
Pam-ETF%cs7EYWAQCba%c7SAA-NH2 721 749 Ac-LThF%cs7EFWAQCba%c7SAA-NH2
722 750 Ac-LTA%cs7EYWAQCba%c7SAA-NH2 723 751
Ac-LTF%cs7EYAAQCba%c7SAA-NH2 724 752
Ac-LTF%cs7EY2NalAQCba%c7SAA-NH2 725 753
Ac-LTF%cs7AYWAQCba%c7SAA-NH2 726 754 Ac-LTF%cs7EYWAQCba%c7SAF-NH2
727 755 Ac-LTF%cs7EYWAQCba%c7SAFa-NH2 728 756
Ac-LTF%cs7AYWAQCba%c7SAF-NH2 729 757
Ac-LTF34F2%cs7AYWAQCba%c7SAF-NH2 730 758
Ac-LTF%cs7AF4coohWAQCba%c7SAF-NH2 731 759
Ac-LTF%cs7EY6clWAQCba%c7SAF-NH2 732 760
Ac-LTF%cs7AYWSQCba%c7SAF-NH2 733 761 Ac-LTF%cs7AYWWQCba%c7SAF-NH2
734 762 Ac-LTF%cs7AYWAQCba%c7AAIa-NH2 735 763
Ac-LTF34F2%cs7AYWAQCba%c7AAIa-NH2 736 764
Ac-LTF%cs7AY6clWAQCba%c7AAIa-NH2 737 765
Ac-LTF%cs7AF4coohWAQCba%c7AAIa-NH2 738 766
Ac-LTF%cs7EYWAQCba%c7AAFa-NH2 739 767 Ac-LTF%cs7EYWAQCba%c7AAFa-NH2
740 768 Ac-ETF%cs7AYWAQCba%c7AWNlea-NH2 741 769
Ac-LTF%cs7EYWAQCba%c7AWNlea-NH2 742 770
Ac-ETF%cs7EYWAQCba%c7AWNlea-NH2 743 771
Ac-ETF%cs7EYWAQCba%c7AWNlea-NH2 744 772
Ac-LTF%cs7AYWAQCba%c7SAFa-NH2 745 773 Ac-LTF%cs7AYWAQCba%c7SAFa-NH2
746 774 Ac-ETF%cs7AYWAQL%c7AWNlea-NH2 747 775
Ac-LTF%cs7EYWAQL%c7AWNlea-NH2 748 776 Ac-ETF%cs7EYWAQL%c7AWNlea-NH2
749 777 Dmaac-LTF%cs7EYWAQhL%c7SAA-NH2 750 778
Hexac-LTF%cs7EYWAQhL%c7SAA-NH2 751 779
Napac-LTF%cs7EYWAQhL%c7SAA-NH2 752 780
Decac-LTF%cs7EYWAQhL%c7SAA-NH2 753 781
Admac-LTF%cs7EYWAQhL%c7SAA-NH2 754 782
Tmac-LTF%cs7EYWAQhL%c7SAA-NH2 755 783 Pam-LTF%cs7EYWAQhL%c7SAA-NH2
756 784 Ac-LTF%cs7AYWAQCba%c7AANleA-NH2 757 785
Ac-LTF34F2%cs7EYWAQCba%c7AAIa-NH2 758 786
Ac-LTF34F2%cs7EYWAQCba%c7SAA-NH2 759 787
Ac-LTF34F2%cs7EYWAQCba%c7SAA-NH2 760 788
Ac-LTF%cs7EF4coohWAQCba%c7SAA-NH2 761 789
Ac-LTF%cs7EF4coohWAQCba%c7SAA-NH2 762 790
Ac-LTF%cs7EYWSQCba%c7SAA-NH2 763 791 Ac-LTF%cs7EYWSQCba%c7SAA-NH2
764 792 Ac-LTF%cs7EYWAQhL%c7SAA-NH2 765 793
Ac-LTF%cs7AYWAQhL%c7SAF-NH2 766 794 Ac-LTF%cs7AYWAQhL%c7SAF-NH2 767
795 Ac-LTF34F2%cs7AYWAQhL%c7SAA-NH2 768 796
Ac-LTF34F2%cs7AYWAQhL%c7SAA-NH2 769 797
Ac-LTF%cs7AF4coohWAQhL%c7SAA-NH2 770 798
Ac-LTF%cs7AF4coohWAQhL%c7SAA-NH2 771 799
Ac-LTF%cs7AYWSQhL%c7SAA-NH2 772 800 Ac-LTF%cs7AYWSQhL%c7SAA-NH2 773
801 Ac-LTF%cs7EYWAQL%c7AANleA-NH2 774 802
Ac-LTF34F2%cs7AYWAQL%c7AANleA-NH2 775 803
Ac-LTF%cs7AF4coohWAQL%c7AANleA-NH2 776 804
Ac-LTF%cs7AYWSQL%c7AANleA-NH2 777 805
Ac-LTF34F2%cs7AYWAQhL%c7AANleA-NH2 778 806
Ac-LTF34F2%cs7AYWAQhL%c7AANleA-NH2 779 807
Ac-LTF%cs7AF4coohWAQhL%c7AANleA-NH2 780 808
Ac-LTF%cs7AF4coohWAQhL%c7AANleA-NH2 781 809
Ac-LTF%cs7AYWSQhL%c7AANleA-NH2 782 810
Ac-LTF%cs7AYWSQhL%c7AANleA-NH2 783 811
Ac-LTF%cs7AYWAQhL%c7AAAAa-NH2 784 812 Ac-LTF%cs7AYWAQhL%c7AAAAa-NH2
785 813 Ac-LTF%cs7AYWAQL%c7AAAAAa-NH2 786 814
Ac-LTF%cs7AYWAQL%c7AAAAAAa-NH2 787 815
Ac-LTF%cs7AYWAQL%c7AAAAAAa-NH2 788 816
Ac-LTF%cs7EYWAQhL%c7AANleA-NH2 789 817
Ac-AATF%cs7AYWAQL%c7AANleA-NH2 790 818
Ac-LTF%cs7AYWAQL%c7AANleAA-NH2 791 819
Ac-ALTF%cs7AYWAQL%c7AANleAA-NH2 792 820
Ac-LTF%cs7AYWAQCba%c7AANleAA-NH2 793 821
Ac-LTF%cs7AYWAQhL%c7AANleAA-NH2 794 822
Ac-LTF%cs7EYWAQCba%c7SAAA-NH2 795 823 Ac-LTF%cs7EYWAQCba%c7SAAA-NH2
796 824 Ac-LTF%cs7EYWAQCba%c7SAAAA-NH2 797 825
Ac-LTF%cs7EYWAQCba%c7SAAAA-NH2 798 826
Ac-ALTF%cs7EYWAQCba%c7SAA-NH2 799 827
Ac-ALTF%cs7EYWAQCba%c7SAAA-NH2 800 828
Ac-ALTF%cs7EYWAQCba%c7SAA-NH2 801 829 Ac-LTF%cs7EYWAQL%c7AAAAAa-NH2
802 830 Ac-LTF%cs7EY6clWAQCba%c7SAA-NH2 803 831
Ac-LTF%cs7EF4cooh6clWAQCba%c7SANleA- NH2 804 832
Ac-LTF%cs7EF4cooh6clWAQCba%c7SANleA- NH2 805 833
Ac-LTF%cs7EF4cooh6clWAQCba%c7AAIa- NH2 806 834
Ac-LTF%cs7EF4cooh6clWAQCba%c7AAIa- NH2 807 835
Ac-LTF%cs7AY6clWAQL%c7AAAAAa-NH2 808 836
Ac-LTF%cs7AY6clWAQL%c7AAAAAa-NH2 809 837
Ac-F%cs7AY6clWEAL%c7AAAAAAa-NH2 810 838
Ac-ETF%cs7EYWAQL%c7AAAAAa-NH2 811 839 Ac-ETF%cs7EYWAQL%c7AAAAAa-NH2
812 840 Ac-LTF%cs7EYWAQL%c7AAAAAAa-NH2 813 841
Ac-LTF%cs7EYWAQL%c7AAAAAAa-NH2 814 842
Ac-LTF%cs7AYWAQL%c7AANleAAa-NH2 815 843
Ac-LTF%cs7AYWAQL%c7AANleAAa-NH2 816 844
Ac-LTF%cs7EYWAQCba%c7AAAAAa-NH2 817 845
Ac-LTF%cs7EYWAQCba%c7AAAAAa-NH2 818 846
Ac-LTF%cs7EF4coohWAQCba%c7AAAAAa-NH2 819 847
Ac-LTF%cs7EF4coohWAQCba%c7AAAAAa-NH2 820 848
Ac-LTF%cs7EYWSQCba%c7AAAAAa-NH2 821 849
Ac-LTF%cs7EYWSQCba%c7AAAAAa-NH2 822 850
Ac-LTF%cs7EYWAQCba%c7SAAa-NH2 823 851 Ac-LTF%cs7EYWAQCba%c7SAAa-NH2
824 852 Ac-ALTF%cs7EYWAQCba%c7SAAa-NH2 825 853
Ac-ALTF%cs7EYWAQCba%c7SAAa-NH2 826 854
Ac-ALTF%cs7EYWAQCba%c7SAAAa-NH2 827 855
Ac-ALTF%cs7EYWAQCba%c7SAAAa-NH2 828 856
Ac-AALTF%cs7EYWAQCba%c7SAAAa-NH2 829 857
Ac-AALTF%cs7EYWAQCba%c7SAAAa-NH2 830 858
Ac-RTF%cs7EYWAQCba%c7SAA-NH2 831 859 Ac-LRF%cs7EYWAQCba%c7SAA-NH2
832 860 Ac-LTF%cs7EYWRQCba%c7SAA-NH2 833 861
Ac-LTF%cs7EYWARCba%c7SAA-NH2 834 862 Ac-LTF%cs7EYWAQCba%c7RAA-NH2
835 863 Ac-LTF%cs7EYWAQCba%c7SRA-NH2 836 864
Ac-LTF%cs7EYWAQCba%c7SAR-NH2 837 865
5-FAM-BaLTF%cs7EYWAQCba%c7SAA-NH2 838 866
5-FAM-BaLTF%cs7AYWAQL%c7AANleA-NH2 839 867
Ac-LAF%cs7EYWAQL%c7AANleA-NH2 840 868 Ac-ATF%cs7EYWAQL%c7AANleA-NH2
841 869 Ac-AAF%cs7EYWAQL%c7AANleA-NH2
842 870 Ac-AAAF%cs7EYWAQL%c7AANleA-NH2 843 871
Ac-AAAAF%cs7EYWAQL%c7AANleA-NH2 844 872
Ac-AATF%cs7EYWAQL%c7AANleA-NH2 845 873
Ac-AALTF%cs7EYWAQL%c7AANleA-NH2 846 874
Ac-AAALTF%cs7EYWAQL%c7AANleA-NH2 847 875
Ac-LTF%cs7EYWAQL%c7AANleAA-NH2 848 876
Ac-ALTF%cs7EYWAQL%c7AANleAA-NH2 849 877
Ac-AALTF%cs7EYWAQL%c7AANleAA-NH2 850 878
Ac-LTF%cs7EYWAQCba%c7AANleAA-NH2 851 879
Ac-LTF%cs7EYWAQhL%c7AANleAA-NH2 852 880
Ac-ALTF%cs7EYWAQhL%c7AANleAA-NH2 853 881
Ac-LTF%cs7ANmYWAQL%c7AANleA-NH2 854 882
Ac-LTF%cs7ANmYWAQL%c7AANleA-NH2 855 883
Ac-LTF%cs7AYNmWAQL%c7AANleA-NH2 856 884
Ac-LTF%cs7AYNmWAQL%c7AANleA-NH2 857 885
Ac-LTF%cs7AYAmwAQL%c7AANleA-NH2 858 886
Ac-LTF%cs7AYAmwAQL%c7AANleA-NH2 859 887
Ac-LTF%cs7AYWAibQL%c7AANleA-NH2 860 888
Ac-LTF%cs7AYWAibQL%c7AANleA-NH2 861 889
Ac-LTF%cs7AYWAQL%c7AAibNleA-NH2 862 890
Ac-LTF%cs7AYWAQL%c7AAibNleA-NH2 863 891
Ac-LTF%cs7AYWAQL%c7AaNleA-NH2 864 892 Ac-LTF%cs7AYWAQL%c7AaNleA-NH2
865 893 Ac-LTF%cs7AYWAQL%c7ASarNleA-NH2 866 894
Ac-LTF%cs7AYWAQL%c7ASarNleA-NH2 867 895
Ac-LTF%cs7AYWAQL%c7AANleAib-NH2 868 896
Ac-LTF%cs7AYWAQL%c7AANleAib-NH2 869 897
Ac-LTF%cs7AYWAQL%c7AANleNmA-NH2 870 898
Ac-LTF%cs7AYWAQL%c7AANleNmA-NH2 871 899
Ac-LTF%cs7AYWAQL%c7AANleSar-NH2 872 900
Ac-LTF%cs7AYWAQL%c7AANleSar-NH2 873 901
Ac-LTF%cs7AYWAQL%c7AANleAAib-NH2 874 902
Ac-LTF%cs7AYWAQL%c7AANleAAib-NH2 875 903
Ac-LTF%cs7AYWAQL%c7AANleANmA-NH2 876 904
Ac-LTF%cs7AYWAQL%c7AANleANmA-NH2 877 905
Ac-LTF%cs7AYWAQL%c7AANleAa-NH2 878 906
Ac-LTF%cs7AYWAQL%c7AANleAa-NH2 879 907
Ac-LTF%cs7AYWAQL%c7AANleASar-NH2 880 908
Ac-LTF%cs7AYWAQL%c7AANleASar-NH2 881 909
Ac-LTF%c7/r8AYWAQL%c7/AANleA-NH2 882 910
Ac-LTFAibAYWAQLAibAANleA-NH2 883 911
Ac-LTF%cs7Cou4YWAQL%c7AANleA-NH2 884 912
Ac-LTF%cs7Cou4YWAQL%c7AANleA-NH2 885 913
Ac-LTF%cs7AYWCou4QL%c7AANleA-NH2 886 914
Ac-LTF%cs7AYWAQL%c7Cou4ANleA-NH2 887 915
Ac-LTF%cs7AYWAQL%c7Cou4ANleA-NH2 888 916
Ac-LTF%cs7AYWAQL%c7ACou4N1eA-NH2 889 917
Ac-LTF%cs7AYWAQL%c7ACou4NleA-NH2 890 918
Ac-LTF%cs7AYWAQL%c7AANleA-OH 891 919 Ac-LTF%cs7AYWAQL%c7AANleA-OH
892 920 Ac-LTF%cs7AYWAQL%c7AANleA-NHnPr 893 921
Ac-LTF%cs7AYWAQL%c7AANleA-NHnPr 894 922
Ac-LTF%cs7AYWAQL%c7AANleA-NHnBu33Me 895 923
Ac-LTF%cs7AYWAQL%c7AANleA-NHnBu33Me 896 924
Ac-LTF%cs7AYWAQL%c7AANleA-NHHex 897 925
Ac-LTF%cs7AYWAQL%c7AANleA-NHHex 898 926
Ac-LTA%cs7AYWAQL%c7AANleA-NH2 899 927
Ac-LThL%cs7AYWAQL%c7AANleA-NH2 900 928
Ac-LTF%cs7AYAAQL%c7AANleA-NH2 901 929
Ac-LTF%cs7AY2NalAQL%c7AANleA-NH2 902 930
Ac-LTF%cs7EYWCou4QCba%c7SAA-NH2 903 931
Ac-LTF%cs7EYWCou7QCba%c7SAA-NH2 904 932
Dmaac-LTF%cs7EYWAQCba%c7SAA-NH2 905 933
Dmaac-LTF%cs7AYWAQL%c7AAAAAa-NH2 906 934
Dmaac-LTF%cs7AYWAQL%c7AAAAAa-NH2 907 935
Dmaac-LTF%cs7EYWAQL%c7AAAAAa-NH2 908 936
Dmaac-LTF%cs7EYWAQL%c7AAAAAa-NH2 909 937
Dmaac-LTF%cs7EF4coohWAQCba%c7AAIa- NH2 910 938
Dmaac-LTF%cs7EF4coohWAQCba%c7AAIa- NH2 911 939
Dmaac-LTF%cs7AYWAQL%c7AANleA-NH2 912 940
Dmaac-LTF%cs7AYWAQL%c7AANleA-NH2 913 941
Cou6BaLTF%cs7EYWAQhL%c7SAA-NH2 914 942
Cou8BaLTF%cs7EYWAQhL%c7SAA-NH2 915 943
Ac-LTF4I%cs7EYWAQL%c7AAAAAa-NH2
[0529] Table 6a shows exemplary peptidomimetic macrocycles:
TABLE-US-00011 TABLE 6a SEQ Calc Calc ID Exact Found (M+1)/ (M+2)/
(M+3)/ SP NO: Sequence Mass Mass 1 2 3 916 944
Ac-LTF%cs7AYWAQL%c7AANleA-NH2 1808.94 1809.95 905.48 603.99 917 945
Ac-LTF%cs7AYWAQL%c7AAAAAa-NH2 1908.96 1909.97 955.49 637.33 918 946
Ac-LTF%csBphAYWAQL%cBphAANleA-NH2 1890.92 1909.97 955.49 637.33 919
947 Ac-LTF%csBphAYWAQL%cBphAAAAAa-NH2 1990.92 996.88 920 948
Ac-LTF%csBphEYWAQCba%cBphSAA-NH2 1865.16 933.45 933.58 921 949
Ac-LTF#cs7EYWAQCba#c7SAA-NH2 1753.82 1754.83 877.92 585.61 922 950
Ac-LTF#csBphEYWAQCba#cBphSAA-NH2 1835.81 1836.82 918.91 612.94 923
951 Ac-LTF%csBphEYWAQL%cBphAAAAAa-NH2 924 952
Ac-LTF%cs5AYWAQL%c5AANleA-NH2 925 953 Ac-LTF%cs5AYWAQL%c5AAAAAa-NH2
926 954 Ac-LTF%cs6AYWAQL%c6AANleA-NH2 927 955
Ac-LTF%cs6AYWAQL%c6AAAAAa-NH2 928 956 Ac-LTF%cs6EYWAQL%c6AAAAAa-NH2
1894.94 1895.96 948.48 632.66 929 957 Ac-LTF%cs5EYWAQL%c5AAAAAa-NH2
1880.93 1881.94 941.47 627.98 930 958 Ac-LTF%cs6EYWAQCba%c6SAANH2
1709.83 1710.84 855.92 570.95 931 959 Ac-LTF%cs5EYWAQCba%c5SAANH2
1695.81 1696.82 848.92 566.28
[0530] Partial structures of selected exemplary peptidomimetic
macrocycles are shown below:
##STR00161##
[0531] A structure of an exemplary peptidomimetic macrocycle is
shown below:
##STR00162##
[0532] Another structure of an exemplary peptidomimetic macrocycle
is shown below:
##STR00163##
[0533] Amino acids represented as "#cs5" are D-cysteine connected
by an i to i+7, five-methylene crosslinker to another
thiol-containing amino acid. Amino acids represented as "#c5" are
L-cysteine connected by an i to i+7, five-methylene crosslinker to
another thiol-containing amino acid Amino acids represented as
"#cs6" are D-cysteine connected by an i to i+7, six-methylene
crosslinker to another thiol-containing amino acid. Amino acids
represented as "#c6" are L-cysteine connected by an i to i+7,
six-methylene crosslinker to another thiol-containing amino acid
Amino acids represented as "#cs7" are D-cysteine connected by an i
to i+7, seven-methylene crosslinker to another thiol-containing
amino acid Amino acids represented as "#c7" are L-cysteine
connected by an i to i+7, seven-methylene crosslinker to another
thiol-containing amino acid Amino acids represented as "#cs8" are
D-cysteine connected by an i to i+7, eight-methylene crosslinker to
another thiol-containing amino acid Amino acids represented as
"#c8" are L-cysteine connected by an i to i+7, eight-methylene
crosslinker to another thiol-containing amino acid Amino acids
represented as "% cs7" are alpha-methyl-D-cysteine connected by an
i to i+7, seven-methylene crosslinker to another thiol-containing
amino acid Amino acids represented as "% c7" are
alpha-methyl-L-cysteine connected by an i to i+7, seven-methylene
crosslinker to another thiol-containing amino acid. Amino acids
represented as "% cs8" are alpha-methyl-D-cysteine connected by an
i to i+7, eight-methylene crosslinker to another thiol-containing
amino acid Amino acids represented as "% c8" are
alpha-methyl-L-cysteine connected by an i to i+7, eight-methylene
crosslinker to another thiol-containing amino acid. Amino acids
represented as "% cs9" are alpha-methyl-D-cysteine connected by an
i to i+7, nine-methylene crosslinker to another thiol-containing
amino acid Amino acids represented as "% c9" are
alpha-methyl-L-cysteine connected by an i to i+7, nine-methylene
crosslinker to another thiol-containing amino acid Amino acids
represented as "% cs10" are alpha-methyl-D-cysteine connected by an
i to i+7, ten-methylene crosslinker to another thiol-containing
amino acid. Amino acids represented as "% c10" are
alpha-methyl-L-cysteine connected by an i to i+7, ten-methylene
crosslinker to another thiol-containing amino acid Amino acids
represented as "pen8" are D-penicillamine connected by an i to i+7,
eight-methylene crosslinker to another thiol-containing amino acid
Amino acids represented as "pen8" are L-penicillamine connected by
an i to i+7, eight-methylene crosslinker to another
thiol-containing amino acid Amino acids represented as "#csBph" are
D-cysteine connected by an i to i+7, Bph (4,4'-bismethyl-biphenyl)
crosslinker to another thiol-containing amino acid. Amino acids
represented as "#cBph" are L-cysteine connected by an i to i+7, Bph
(4,4'-bismethyl-biphenyl) crosslinker to another thiol-containing
amino acid Amino acids represented as "% csBph" are
alpha-methyl-D-cysteine connected by an i to i+7, Bph
(4,4'-bismethyl-biphenyl) crosslinker to another thiol-containing
amino acid Amino acids represented as "% cBph" are
alpha-methyl-L-cysteine connected by an i to i+7, Bph
(4,4'-bismethyl-biphenyl) crosslinker to another thiol-containing
amino acid Amino acids represented as "#csBpy" are D-cysteine
connected by an i to i+7, Bpy (6,6'-bismethyl-[3,3]bipyridine)
crosslinker to another thiol-containing amino acid. Amino acids
represented as "#cBpy" are L-cysteine connected by an i to i+7, Bpy
(6,6'-bismethyl43,31bipyridine) crosslinker to another
thiol-containing amino acid. Amino acids represented as "% csBpy"
are alpha-methyl-D-cysteine connected by an i to i+7, Bpy
(6,6'-bismethyl[3,3,']bipyridine) crosslinker to another
thiol-containing amino acid. Amino acids represented as "% cBpy"
are alpha-methyl-L-cysteine connected by an i to i+7, Bpy
(6,6'-bismethyl[3,3']bipyridine) crosslinker to another
thiol-containing amino acid. The number of methylene units
indicated above refers to the number of methylene units between the
two thiol groups of the crosslinker.
[0534] In some embodiments, a peptidomimetic macrocycle is obtained
in more than one isomer, for example due to the configuration of a
double bond within the structure of the crosslinker (E vs Z). Such
isomers can or can not be separable by conventional chromatographic
methods. In some embodiments, one isomer has improved biological
properties relative to the other isomer. In one embodiment, an E
crosslinker olefin isomer of a peptidomimetic macrocycle has better
solubility, better target affinity, better in vivo or in vitro
efficacy, higher helicity, or improved cell permeability relative
to its Z counterpart. In another embodiment, a Z crosslinker olefin
isomer of a peptidomimetic macrocycle has better solubility, better
target affinity, better in vivo or in vitro efficacy, higher
helicity, or improved cell permeability relative to its E
counterpart.
[0535] In some embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 7:
TABLE-US-00012 TABLE 7 SEQ ID NO: Sequence 1 961
QSQQTF%csNLWLL%cs6QN 2 962 QSQQTF%csNLWLL%cs7QN 3 963
QSQQTF%csNLWLL%cs8QN 4 964 QSQQTF%csNLWLL%cs9QN
[0536] Peptides shown can comprise an N-terminal capping group such
as acetyl or an additional linker such as beta-alanine between the
capping group and the start of the peptide sequence.
[0537] In some embodiments, peptidomimetic macrocycles do not
comprise a peptidomimetic macrocycle structure as shown in Table
7.
[0538] In other embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 7a:
TABLE-US-00013 TABLE 7a Number SEQ ID NO: Sequence 1 965
Ac-QSQQTF#cs5NLWRLL#c5QN-NH2 2 966 Ac-QSQQTF#cs6NLWRLL#c6QN-NH2 3
967 Ac-QSQQTF#cs7NLWRLL#c7QN-NH2 4 968 Ac-QSQQTF#cs8NLWRLL#c8QN-NH2
5 969 Ac-QSQQTF#cs9NLWRLL#c9QN-NH2 6 970
Ac-QSQQTF%cs8NLWRLL%c8QN-NH2 7 971 Ac-QSQQTF#cs8NLWRLLPen8QN-NH2 8
972 Ac-QSQQTF#c8NLWRLL#c8QN-NH2 9 973 Ac-QSQQTF#c8NLWRLL#cs8QN-NH2
10 974 Ac-QSQQTF#cs8NLWALL#c8AN-NH2 11 975
Ac-QAibQQTF#cs8NLWALL#c8AN-NH2 12 976
Ac-QAibQQTF#cs8ALWALL#c8AN-NH2 13 977 Ac-QSQQTFpen8NLWRLLPen8QN-NH2
14 978 Ac-QSQQTFpen8NLWRLL#c8QN-NH2 15 979
Ac-QSQQTF%cs9NLWRLL%c9QN-NH2 16 980 Ac-LTF#cs8HYWAQL#c8S-NH2 17 981
Ac-LTF#cs8HYWAQI#c8S-NH2 18 982 Ac-LTF#cs8HYWAQNle#c8S-NH2 19 983
Ac-LTF#cs8HYWAQL#c8A-NH2 20 984 Ac-LTF#cs8HYWAbuQL#c8S-NH2 21 985
Ac-LTF#cs8AYWAQL#c8S-NH2 22 986 Ac-LTF#cs8AYWAQL#c8A-NH2 23 987
Ac-LTF#cs8HYWAQLPen8S-NH2 24 988 Ac-LTFpen8HYWAQLPen8S-NH2 25 989
Ac-LTFpen8HYWAQL#c8S-NH2 26 990 Ac-LTF#cs7HYWAQL#hc7S-NH2 27 991
Ac-LTF%cs8HYWAQL%c8S-NH2 28 992 Ac-LTF%cs9HYWAQL%c9S-NH2 29 993
Ac-LTF%cs10HYWAQL%c10S-NH2 30 994 Ac-LTF%cs7HYWAQL%c7S-NH2 31 995
Ac-LTF%cs4BEBHYWAQL%c4BEBS-NH2 32 996 Ac-Fpen8AYWEAc3cL#c8A-NH2 33
997 Ac-F#cs8AYWEAc3cL#c8A-NH2 34 998 Ac-F%cs8AYWEAc3cL%c8A-NH2 35
999 Ac-LTFEHYWAQLTS-NH2
[0539] In some embodiments, peptidomimetic macrocycles do not
comprise a peptidomimetic macrocycle structure as shown in Table
7a.
[0540] In other embodiments, peptidomimetic macrocycles exclude
peptidomimetic macrocycles shown in Table 7b and disclosed in
Muppidi et al., Chem. Commun. (2011) DOI: 10.1039/c1cc13320a:
TABLE-US-00014 TABLE 7b Number SEQ ID NO: Sequence 1 1000
LTFEHYWAQLTS 2 1001 LTFCHYWAQLCS 3 1002 LTF#cBphHYWAQL#cBphS 4 1003
LTF#cBpyHYWAQL#cBpyS 5 1004 LTFCRYWARLCS 6 1005
LTF#cBphRYWARL#cBphS 7 1006 LTF#cBpyRYWARL#cBpyS 8 1007
LTFcHYWAQLCS 9 1008 LTF#csBphHYWAQL#cBphS 10 1009
LTF#csBpyHYWAQL#csBpyS 11 1010 LTF#csBphRYWARL#cBphS 12 1011
LTF#csBpyRYWARL#cBpyS
[0541] wherein C denotes L-cysteine and c denotes D-cysteine in
Table 7b; and #cBph, #cBpy, #csBph, and #csBpy are as defined
herein.
[0542] In some embodiments, peptidomimetic macrocycles do not
comprise a peptidomimetic macrocycle structure as shown in Table
7b.
Example 4
Circular Dichroism (CD) Analysis of Alpha-Helicity
[0543] Peptide solutions are analyzed by CD spectroscopy using a
Jasco J-815 spectropolarimeter (Jasco Inc., Easton, Md.) with the
Jasco Spectra Manager Ver.2 system software. A Peltier temperature
controller is used to maintain temperature control of the optical
cell. Results are expressed as mean molar ellipticity [.theta.]
(deg cm2 dmol-1) as calculated from the equation
[.theta.]=.theta.obsMRW/10*l*c where .theta.obs is the observed
ellipticity in millidegrees, MRW is the mean residue weight of the
peptide (peptide molecular weight/number of residues), l is the
optical path length of the cell in centimeters, and c is the
peptide concentration in mg/ml. Peptide concentrations are
determined by amino acid analysis. Stock solutions of peptides are
prepared in benign CD buffer (20 mM phosphoric acid, pH 2). The
stocks are used to prepare peptide solutions of 0.05 mg/ml in
either benign CD buffer or CD buffer with 50% trifluoroethanol
(TFE) for analyses in a 10 mm pathlength cell. Variable wavelength
measurements of peptide solutions are scanned at 4.degree. C. from
195 to 250 nm, in 0.2 nm increments, and a scan rate 50 nm per
minute. The average of six scans is reported.
Example 5
Direct Binding Assay MDM2 with Fluorescence Polarization (FP)
[0544] The assay is performed according to the following general
protocol:
[0545] 1. Dilute MDM2 (In-house, 41 kD) into FP buffer (High salt
buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 .mu.M working
stock solution.
[0546] 2. Add 30 .mu.l of 10 .mu.M of protein stock solution into
A1 and B1 well of 96-well black HE microplate (Molecular
Devices).
[0547] 3. Fill in 30 .mu.l of FP buffer into column A2 to A12, B2
to B12, C1 to C12, and D1 to D12.
[0548] 4. 2 or 3 fold series dilution of protein stock from A1, B1
into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM
concentration at the last dilution point.
[0549] 5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide
with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M
to 10 .mu.M with water (dilution 1:10) and then dilute with FP
buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working
solution which will be a 10 nM concentration in well (dilution
1:4). Keep the diluted FAM labeled peptide in the dark until
use.
[0550] 6. Add 10 .mu.l of 10 nM of FAM labeled peptide into each
well and incubate, and read at different time points. Kd with
5-FAM-BaLTFEHYWAQLTS-NH.sub.2 (SEQ ID NO: 1012) is .about.13.38
nM.
Example 6
Competitive Fluorescence Polarization Assay for MDM2
[0551] The assay is performed according to the following general
protocol:
[0552] 1. Dilute MDM2 (In-house, 41 kD) into FP buffer (High salt
buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 84 nM (2.times.)
working stock solution.
[0553] 2. Add 20 .mu.l of 84 nM (2.times.) of protein stock
solution into each well of 96-well black HE microplate (Molecular
Devices)
[0554] 3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide
with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M
to 10 .mu.M with water (dilution 1:10) and then dilute with FP
buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working
solution which will be a 10 nM concentration in well (dilution
1:4). Keep the diluted FAM labeled peptide in the dark until
use.
[0555] 4. Make unlabeled peptide dose plate with FP buffer starting
with 1 .mu.M (final) of peptide and making 5 fold serial dilutions
for 6 points using following dilution scheme. Dilute 10 mM (in 100%
DMSO) with DMSO to 5 mM (dilution 1: 2). Then, dilute from 5 mM to
500 .mu.M with H.sub.2O (dilution 1:10) and then dilute with FP
buffer from 500 .mu.M to 20 .mu.M (dilution 1:25) Making 5 fold
serial dilutions from 4 .mu.M (4.times.) for 6 points.
[0556] 5. Transfer 10 .mu.l of serial diluted unlabeled peptides to
each well which is filled with 20 .mu.l of 84 nM of protein.
[0557] 6. Add 10 .mu.l of 10 nM (4.times.) of FAM labeled peptide
into each well and incubate for 3 hr to read.
Example 7
Direct Binding Assay MDMX with Fluorescence Polarization (FP)
[0558] The assay is performed according to the following general
protocol:
[0559] 1. Dilute MDMX (In-house, 40 kD) into FP buffer (High salt
buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 .mu.M working
stock solution.
[0560] 2. Add 30 .mu.l of 10 .mu.M of protein stock solution into
A1 and B1 well of 96-well black HE microplate (Molecular
Devices).
[0561] 3. Fill in 30 .mu.l of FP buffer into column A2 to A12, B2
to B12, C1 to C12, and D1 to D12.
[0562] 4. 2 or 3 fold series dilution of protein stock from A1, B1
into A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM
concentration at the last dilution point.
[0563] 5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide
with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M
to 10 .mu.M with water (dilution 1:10) and then dilute with FP
buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working
solution which will be a 10 nM concentration in well (dilution
1:4). Keep the diluted FAM labeled peptide in the dark until
use.
[0564] 6. Add 10 .mu.l of 10 nM of FAM labeled peptide into each
well and incubate, and read at different time points.
[0565] Kd with 5-FAM-BaLTFEHYWAQLTS-NH.sub.2 (SEQ ID NO: 1012) is
.about.51 nM.
Example 8
Competitive Fluorescence Polarization Assay for MDMX
[0566] The assay is performed according to the following general
protocol:
[0567] 1. Dilute MDMX (In-house, 40 kD) into FP buffer (High salt
buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5.) to make 300 nM (2.times.)
working stock solution.
[0568] 2. Add 20 .mu.l of 300 nM (2.times.) of protein stock
solution into each well of 96-well black HE microplate (Molecular
Devices)
[0569] 3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide
with DMSO to 100 .mu.M (dilution 1:10). Then, dilute from 100 .mu.M
to 10 .mu.M with water (dilution 1:10) and then dilute with FP
buffer from 10 .mu.M to 40 nM (dilution 1:250). This is the working
solution which will be a 10 nM concentration in well (dilution
1:4). Keep the diluted FAM labeled peptide in the dark until
use.
[0570] 4. Make unlabeled peptide dose plate with FP buffer starting
with 5 .mu.M (final) of peptide and making 5 fold serial dilutions
for 6 points using following dilution scheme.
[0571] 5. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution
1:2). Then, dilute from 5 mM to 500 .mu.M with H.sub.2O (dilution
1:10) and then dilute with FP buffer from 500 .mu.M to 20 .mu.M
(dilution 1:25) Making 5 fold serial dilutions from 20 .mu.M
(4.times.) for 6 points.
[0572] 6. Transfer 10 .mu.l of serial diluted unlabeled peptides to
each well which is filled with 20 .mu.l of 300 nM of protein.
[0573] 7. Add 10 .mu.l of 10 nM (4.times.) of FAM labeled peptide
into each well and incubate for 3 hr to read.
[0574] Results from Examples 4-7 are shown in Table 8. The
following scale is used for IC50 and Ki values: "+" represents a
value greater than 1000 nM, "++" represents a value greater than
100 and less than or equal to 1000 nM, "+++" represents a value
greater than 10 nM and less than or equal to 100 nM, and "++++"
represents a value of less than or equal to 10 nM. Cell viability
assay results (performed as in Example 9) are also included in
Table 8 using the following scale: "+" represents a value greater
than 30 .mu.M, "++" represents a value greater than 15 .mu.M and
less than or equal to 30 .mu.M, "+++" represents a value greater
than 5 .mu.M and less than or equal to 15 .mu.M, and "++++"
represents a value of less than or equal to 5 .mu.M. "IC50 ratio"
represents the ratio of average IC50 in p53+/+ cells relative to
average IC50 in p53-/- cells.
TABLE-US-00015 TABLE 8 SJSA-1 IC50 IC50 Ki EC50 IC50 SP (MDM2)
(MDMX) (MDM2) Ki (MDMX) (72 h) Ratio 449 ++++ ++++ ++++ ++++ ++++
450 ++ +++ 451 +++ +++ 452 + 456 ++++ +++ +++ 457 ++++ ++++ ++++
461 +++ 459 + + + 460 + + + 463 ++ 464 + 153 ++++ +++ ++++ 1-29 465
++++ ++++ 466 ++++ ++++ 470 ++++ ++++ 916 +++ +++ ++++ ++++ ++ 917
+++ +++ ++++ +++ + 919 +++
Example 9
Competition Binding ELISA (MDM2 & MDMX)
[0575] p53-His6 protein ("His6" disclosed as SEQ ID NO: 1013) (30
nM/well) is coated overnight at room temperature in the wells of a
96-well Immulon plates. On the day of the experiment, plates are
washed with 1.times. PBS-Tween 20 (0.05%) using an automated ELISA
plate washer, blocked with ELISA Micro well Blocking for 30 minutes
at room temperature; excess blocking agent is washed off by washing
plates with 1.times. PBS-Tween 20 (0.05%). Peptides are diluted
from 10 mM DMSO stocks to 500 .mu.M working stocks in sterile
water, further dilutions made in 0.5% DMSO to keep the
concentration of DMSO constant across the samples. The peptides are
added to wells at 2.times. desired concentrations in 50 .mu.l
volumes, followed by addition of diluted GST-MDM2 or GST-HMDX
protein (final concentration: 10 nM). Samples are incubated at room
temperature for 2 h, plates are washed with PBS-Tween 20 (0.05%)
prior to adding 100 .mu.l of HRP-conjugated anti-GST antibody
[Hypromatrix, INC] diluted to 0.5 .mu.g/ml in HRP-stabilizing
buffer. Post 30 min incubation with detection antibody, plates are
washed and incubated with 100 .mu.l per well of TMB-E Substrate
solution up to 30 minutes; reactions are stopped using 1M HCL and
absorbance measured at 450 nm on micro plate reader. Data is
analyzed using Graph Pad PRISM software.
Example 10
Cell Viability Assay
[0576] The assay is performed according to the following general
protocol:
[0577] Cell Plating: Trypsinize, count and seed cells at the
pre-determined densities in 96-well plates a day prior to assay.
Following cell densities are used for each cell line in use: [0578]
SJSA-1: 7500 cells/well [0579] RKO: 5000 cells/well [0580] RKO-E6:
5000 cells/well [0581] HCT-116: 5000 cells/well [0582] SW-480: 2000
cells/well [0583] MCF-7: 5000 cells/well
[0584] On the day of study, replace media with fresh media with 11%
FBS (assay media) at room temperature. Add 180 .mu.L of the assay
media per well. Control wells with no cells, receive 200 .mu.l
media.
[0585] On the day of study, replace media with fresh media with 11%
FBS (assay media) at room temperature. Add 180 .mu.L of the assay
media per well. Control wells with no cells, receive 200 .mu.l
media.
[0586] Peptide dilution: all dilutions are made at room temperature
and added to cells at room temperature. [0587] Prepare 10 mM stocks
of the peptides in DMSO. Serially dilute the stock using 1:3
dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM
solutions using DMSO as diluents. Dilute the serially DMSO-diluted
peptides 33.3 times using sterile water. This gives range of
10.times. working stocks. Also prepare DMSO/sterile water (3% DMSO)
mix for control wells. [0588] Thus the working stocks concentration
range .mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix
well at each dilution step using multichannel. [0589] Row H has
controls. H1-H3 will receive 20 ul of assay media. H4--H9 will
receive 20 ul of 3% DMSO-water vehicle. H10-H12 will have media
alone control with no cells. [0590] Positive control: MDM2 small
molecule inhibitor, Nutlin-3a (10 mM) is used as positive control.
Nutlin was diluted using the same dilution scheme as peptides.
[0591] Addition of working stocks to cells: [0592] Add 20 .mu.l of
10.times. desired concentration to appropriate well to achieve the
final concentrations in total 200 .mu.l volume in well. (20 .mu.l
of 300 .mu.M peptide+180 .mu.l of cells in media=30 .mu.M final
concentration in 200 .mu.l volume in wells). Mix gently a few times
using pipette. Thus final concentration range used will be 30, 10,
3, 1, 0.3, 0.1, 0.03 & 0 .mu.M (for potent peptides further
dilutions are included). [0593] Controls include wells that get no
peptides but contain the same concentration of DMSO as the wells
containing the peptides, and wells containing NO CELLS. [0594]
Incubate for 72 hours at 37.degree. C. in humidified 5% CO.sub.2
atmosphere. [0595] The viability of cells is determined using MTT
reagent from Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116
cells is determined on day 3, MCF-7 cells on day 5 and SW-480 cells
on day 6. At the end of designated incubation time, allow the
plates to come to room temperature. Remove 80 .mu.l of assay media
from each well. Add 15 .mu.l of thawed MTT reagent to each well.
[0596] Allow plate to incubate for 2 h at 37.degree. C. in
humidified 5% CO.sub.2 atmosphere and add 100 .mu.l solubilization
reagent as per manufacturer's protocol. Incubate with agitation for
1 h at room temperature and read on Synergy Biotek multiplate
reader for absorbance at 570 nM. [0597] Analyze the cell viability
against the DMSO controls using GraphPad PRISM analysis tools.
[0598] Reagents: [0599] Invitrogen cell culture Media [0600] i.
Falcon 96-well clear cell culture treated plates (Nunc 353072)
[0601] DMSO (Sigma D 2650) [0602] RPMI 1640 (Invitrogen 72400)
[0603] MTT (Promega G4000)
[0604] Instruments: Multiplate Reader for Absorbance readout
(Synergy 2).
[0605] Results are shown in Table 8.
Example 11
P21 ELISA Assay
[0606] The assay is performed according to the following general
protocol:
[0607] Cell Plating: [0608] Trypsinize, count and seed SJSA1 cells
at the density of 7500 cells/100 .mu.l/well in 96-well plates a day
prior to assay. [0609] On the day of study, replace media with
fresh RPMI-11% FBS (assay media). Add 90 .mu.L of the assay media
per well. Control wells with no cells, receive 100 .mu.l media.
[0610] Peptide dilution: [0611] Prepare 10 mM stocks of the
peptides in DMSO. Serially dilute the stock using 1:3 dilution
scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions
using DMSO as diluents. Dilute the serially DMSO-diluted peptides
33.3 times using sterile water This gives range of 10.times.
working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for
control wells. [0612] Thus the working stocks concentration range
.mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix well at
each dilution step using multichannel. [0613] Row H has controls.
H1-H3 will receive 10 ul of assay media. H4-H9 will receive 10 ul
of 3% DMSO-water vehicle. H10-H12 will have media alone control
with no cells. [0614] Positive control: MDM2 small molecule
inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin
was diluted using the same dilution scheme as peptides.
[0615] Addition of working stocks to cells: [0616] Add 10 .mu.l of
10.times. desired concentration to appropriate well to achieve the
final concentrations in total 100 .mu.l volume in well. (10 .mu.l
of 300 .mu.M peptide+90 .mu.l of cells in media=30 .mu.M final
concentration in 100 .mu.l volume in wells). Thus final
concentration range used will be 30, 10, 3, 1, 0.3& 0 .mu.M.
[0617] Controls will include wells that get no peptides but contain
the same concentration of DMSO as the wells containing the
peptides, and wells containing NO CELLS. [0618] 20 h-post
incubation, aspirate the media; wash cells with 1.times. PBS
(without Ca.sup.++/Mg.sup.++) and lyse in 60 .mu.l of 1.times. Cell
lysis buffer (Cell Signaling technologies 10.times. buffer diluted
to 1.times. and supplemented with protease inhibitors and
Phosphatase inhibitors) on ice for 30 min. [0619] Centrifuge plates
in at 5000 rpm speed in at 4.degree. C. for 8 min; collect clear
supernatants and freeze at -80.degree. C. till further use.
[0620] Protein Estimation: [0621] Total protein content of the
lysates is measured using BCA protein detection kit and BSA
standards from Thermofisher. Typically about 6-7 .mu.g protein is
expected per well. [0622] Use 50 .mu.l of the lysate per well to
set up p21 ELISA.
[0623] Human Total p21 ELISA: The ELISA assay protocol is followed
as per the manufacturer's instructions. 50 .mu.l lysate is used for
each well, and each well is set up in triplicate.
[0624] Reagents: [0625] Cell-Based Assay (-)-Nutlin-3 (10 mM):
Cayman Chemicals, catalog #600034 [0626] OptiMEM, Invitrogen
catalog #51985 [0627] Cell Signaling Lysis Buffer (10.times.), Cell
signaling technology, Catalog #9803 [0628] Protease inhibitor
Cocktail tablets(mini), Roche Chemicals, catalog #04693124001
[0629] Phosphatase inhibitor Cocktail tablet, Roche Chemicals,
catalog #04906837001 [0630] Human total p21 ELISA kit, R&D
Systems, DYC1047-5 [0631] STOP Solution (1M HCL), Cell Signaling
Technologies, Catalog #7002
[0632] Instruments: Micro centrifuge--Eppendorf 5415D and
Multiplate Reader for Absorbance readout (Synergy 2).
Example 12
Caspase 3 Detection Assay
[0633] The assay is performed according to the following general
protocol:
[0634] Cell Plating: Trypsinize, count and seed SJSA1 cells at the
density of 7500 cells/100 .mu.l/well in 96-well plates a day prior
to assay. On the day of study, replace media with fresh RPMI-11%
FBS (assay media). Add 180 .mu.L of the assay media per well.
Control wells with no cells, receive 200 .mu.l media.
[0635] Peptide Dilution: [0636] Prepare 10 mM stocks of the
peptides in DMSO. Serially dilute the stock using 1:3 dilution
scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01 mM solutions
using DMSO as diluents. Dilute the serially DMSO-diluted peptides
33.3 times using sterile water This gives range of 10.times.
working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for
control wells. [0637] Thus the working stocks concentration range
.mu.M will be 300, 100, 30, 10, 3, 1, 0.3 and 0 .mu.M. Mix well at
each dilution step using multichannel. Add 20 ul of 10.times.
working stocks to appropriate wells. [0638] Row H has controls.
H1-H3 will receive 20 ul of assay media. H4-H9 will receive 20 ul
of 3% DMSO-water vehicle. H10-H12 will have media alone control
with no cells. [0639] Positive control: MDM2 small molecule
inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin
was diluted using the same dilution scheme as peptides.
[0640] Addition of Working Stocks to Cells: [0641] Add 10 .mu.l of
10.times. desired concentration to appropriate well to achieve the
final concentrations in total 100 .mu.l volume in well. (10 .mu.l
of 300 .mu.M peptide+90 .mu.l of cells in media=30 .mu.M final
concentration in 100 .mu.l volume in wells). Thus final
concentration range used will be 30, 10, 3, 1, 0.3& 0 .mu.M.
[0642] Controls will include wells that get no peptides but contain
the same concentration of DMSO as the wells containing the
peptides, and wells containing NO CELLS. [0643] 48 h-post
incubation, aspirate 80 .mu.l media from each well; add 100 .mu.l
Caspase3/7Glo assay reagent (Promega Caspase 3/7 glo assay system,
G8092) per well, incubate with gentle shaking for 1 h at room
temperature. [0644] read on Synergy Biotek multiplate reader for
luminescence. [0645] Data is analyzed as Caspase 3 activation over
DMSO-treated cells.
Example 13
Cell Lysis by Peptidomimetic Macrocycles
[0646] SJSA-1 cells are plated out one day in advance in clear
flat-bottom plates (Costar, catalog number 353072) at 7500
cells/well with 100 ul/well of growth media, leaving row H columns
10-12 empty for media alone. On the day of the assay, media was
exchanged with RPMI 1% FBS media, 90 uL of media per well.
[0647] 10 mM stock solutions of the peptidomimetic macrocycles are
prepared in 100% DMSO. Peptidomimetic macrocycles were then diluted
serially in 100% DMSO, and then further diluted 20-fold in sterile
water to prepare working stock solutions in 5% DMSO/water of each
peptidomimetic macrocycle at concentrations ranging from 500 uM to
62.5 uM.
[0648] 10 uL of each compound is added to the 90 uL of SJSA-1 cells
to yield final concentrations of 50 uM to 6.25 uM in 0.5%
DMSO-containing media. The negative control (non-lytic) sample was
0.5% DMSO alone and positive control (lytic) samples include 10 uM
Melittin and 1% Triton X-100.
[0649] Cell plates are incubated for 1 hour at 37C. After the 1
hour incubation, the morphology of the cells is examined by
microscope and then the plates were centrifuged at 1200 rpm for 5
minutes at room temperature. 40 uL of supernatant for each
peptidomimetic macrocyle and control sample is transferred to clear
assay plates. LDH release is measured using the LDH cytotoxicity
assay kit from Caymen, catalog #1000882.
Example 14
p53 GRIP Assay
[0650] Thermo Scientific* BioImage p53-MDM2 Redistribution Assay
monitors the protein interaction with MDM2 and cellular
translocation of GFP-tagged p53 in response to drug compounds or
other stimuli. Recombinant CHO-hIR cells stably express human
p53(1-312) fused to the C-terminus of enhanced green fluorescent
protein (EGFP) and PDE4A4-MDM2(1-124), a fusion protein between
PDE4A4 and MDM2(1-124). They provide a ready-to-use assay system
for measuring the effects of experimental conditions on the
interaction of p53 and MDM2. Imaging and analysis is performed with
a HCS platform.
[0651] CHO-hIR cells are regularly maintained in Ham's F12 media
supplemented with 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin,
1 mg/ml Zeocin and 10% FBS. Cells seeded into 96-well plates at the
density of 7000 cells/100 .mu.l per well 18-24 hours prior to
running the assay using culture media. The next day, media is
refreshed and PD177 is added to cells to the final concentration of
3 .mu.M to activate foci formation. Control wells are kept without
PD-177 solution. 24 h post stimulation with PD177, cells are washed
once with Opti-MEM Media and 50 .mu.L of the Opti-MEM Media
supplemented with PD-177(6 .mu.M) is added to cells. Peptides are
diluted from 10 mM DMSO stocks to 500 .mu.M working stocks in
sterile water, further dilutions made in 0.5% DMSO to keep the
concentration of DMSO constant across the samples. Final highest
DMSO concentration is 0.5% and is used as the negative control.
Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as
positive control. Nutlin was diluted using the same dilution scheme
as peptides.50 .mu.l of 2.times. desired concentrations is added to
the appropriate well to achieve the final desired concentrations.
Cells are then incubated with peptides for 6 h at 37.degree. C. in
humidified 5% CO2 atmosphere. Post-incubation period, cells are
fixed by gently aspirating out the media and adding 150 .mu.l of
fixing solution per well for 20 minutes at room temperature. Fixed
cells are washed 4 times with 200 .mu.l PBS per well each time. At
the end of last wash, 100 .mu.l of 1 .mu.M Hoechst staining
solution is added. Sealed plates incubated for at least 30 min in
dark, washed with PBS to remove excess stain and PBS is added to
each well. Plates can be stored at 4.degree. C. in dark up to 3
days. The translocation of p53/MDM2 is imaged using Molecular
translocation module on Cellomics Arrayscan instrument using
10.times. objective, XF-100 filter sets for Hoechst and GFP. The
output parameters was Mean-CircRINGAveIntenRatio (the ratio of
average fluorescence intensities of nucleus and cytoplasm,(well
average)). The minimally acceptable number of cells per well used
for image analysis was set to 500 cells.
Example 15
Solubility Determination for Peptidomimetic Macrocycles
[0652] Peptidomimetic macrocyles are first dissolved in neat
N,N-dimethylacetamide (DMA, Sigma-Aldrich, 38840-1L-F) to make
20.times. stock solutions over a concentration range of 20-140
mg/mL. The DMA stock solutions are diluted 20-fold in an aqueous
vehicle containing 2% Solutol-HS-15, 25 mM Histidine, 45 mg/mL
Mannitol to obtain final concentrations of 1-7 mg/ml of the
peptidomimetic macrocycles in 5% DMA, 2% Solutol-HS-15, 25 mM
Histidine, 45 mg/mL Mannitol. The final solutions are mixed gently
by repeat pipetting or light vortexing, and then the final
solutions are sonicated for 10 min at room temperature in an
ultrasonic water bath. Careful visual observation is then performed
under hood light using a 7.times. visual amplifier to determine if
precipitate exists on the bottom or as a suspension. Additional
concentration ranges are tested as needed to determine the maximum
solubility limit for each peptidomimetic macrocycle.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190292224A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190292224A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References