U.S. patent application number 16/454586 was filed with the patent office on 2019-10-17 for targeted therapeutics.
The applicant listed for this patent is MADRIGAL PHARMACEUTICALS, INC.. Invention is credited to Dinesh U. Chimmanamada, Weiwen Ying.
Application Number | 20190314528 16/454586 |
Document ID | / |
Family ID | 53005363 |
Filed Date | 2019-10-17 |
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United States Patent
Application |
20190314528 |
Kind Code |
A1 |
Chimmanamada; Dinesh U. ; et
al. |
October 17, 2019 |
TARGETED THERAPEUTICS
Abstract
The present invention provides pharmacological compounds
including an effector moiety conjugated to a binding moiety that
directs the effector moiety to a biological target of interest.
Likewise, the present invention provides compositions, kits, and
methods (e.g., therapeutic, diagnostic, and imaging) including the
compounds. The compounds can be described as a protein interacting
binding moiety-drug conjugate (SDC-TRAP) compounds, which include a
protein interacting binding moiety and an effector moiety. For
example, in certain embodiments directed to treating cancer, the
SDC-TRAP can include an Hsp90 inhibitor conjugated to a cytotoxic
agent as the effector moiety.
Inventors: |
Chimmanamada; Dinesh U.;
(Arlington, MA) ; Ying; Weiwen; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MADRIGAL PHARMACEUTICALS, INC. |
West Conshohocken |
PA |
US |
|
|
Family ID: |
53005363 |
Appl. No.: |
16/454586 |
Filed: |
June 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15138785 |
Apr 26, 2016 |
10376598 |
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16454586 |
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PCT/US2014/062689 |
Oct 28, 2014 |
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15138785 |
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61896416 |
Oct 28, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/545 20170801;
A61K 49/00 20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 47/54 20060101 A61K047/54 |
Claims
1. A binding moiety-drug conjugate (SDC-TRAP) comprising a binding
moiety and an effector moiety, wherein the binding moiety binds to
Hsp90, wherein the effector moiety is a cytotoxic agent selected
from the group consisting of LY2801653, LY2835219, LY2606368,
LY2874455, LY2090314, LY2940680, LY2784544, LY2228820, LY2157299,
LY2603618, LY353381, trimetrexate, and a derivative thereof, and
wherein the binding moiety and the effector moiety are covalently
attached.
2. The SDC-TRAP of claim 1, wherein the binding moiety is an Hsp90
ligand or a prodrug thereof.
3. The SDC-TRAP of claim 2, wherein the Hsp90 ligand is an Hsp90
inhibitor.
4. The SDC-TRAP of claim 3, wherein the Hsp90 inhibitor is selected
from the group consisting of ganetespib, geldanamycins, macbecins,
tripterins, tanespimycins, radicicols, and a derivative
thereof.
5. The SDC-TRAP of claim 1, wherein the binding moiety and the
effector moiety are covalently attached by a linker.
6. A pharmaceutical composition comprising a therapeutically
effective amount of at least one SDC-TRAP of claim 1, and at least
one pharmaceutical excipient.
7. A kit for imaging, diagnosing, and/or selecting a subject
comprising at least one SDC-TRAP of claim 1 and instruction for
administering an effective amount of at least one SDC-TRAP of claim
1 to the subject, thereby imaging, diagnosing, and/or selecting the
subject.
8. A method for treating a subject having a cancer comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the cancer, wherein the
SDC-TRAP comprises the SDC-TRAP of claim 1.
9. A method for treating a subject having chronic bronchitis
comprising administering a therapeutically effective amount of at
least one SDC-TRAP to the subject, thereby treating the chronic
bronchitis, wherein the SDC-TRAP comprises the SDC-TRAP of claim
1.
10. A method for treating a subject having asthma comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the asthma, wherein the
SDC-TRAP comprises the SDC-TRAP of claim 1.
11. A method for treating a subject having actinic keratosis
comprising administering a therapeutically effective amount of at
least one SDC-TRAP to the subject, thereby treating the actinic
keratosis, wherein the SDC-TRAP comprises the SDC-TRAP of claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Nonprovisional
application Ser. No. 15/138,785, filed on Apr. 26, 2016, which is a
continuation application of International Application No.
PCT/US2014/062689, filed on Oct. 28, 2014, which claims priority to
U.S. Provisional Application No. 61/896,416, filed on Oct. 28,
2013, the entire contents of which are incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The present invention relates to pharmacological compounds
including an effector moiety conjugated to a binding moiety that
directs the effector moiety to a biological target of interest. The
compounds have broad pharmacological applications, including
therapeutics, diagnostics, and imaging. For example, the compounds
can specifically direct therapeutic effector moieties to target
cells or tissue of interest, for targeted chemotherapeutic
treatment of conditions such as cancer.
BACKGROUND OF THE INVENTION
[0003] Although tremendous advances have been made in chemotherapy,
currently available therapeutics and therapies remain
unsatisfactory and the prognosis for the majority of patients
diagnosed with chemotherapeutically treated diseases (e.g., cancer)
remains poor. Often, the applicability and/or effectiveness of
chemotherapy, as well as other therapies and diagnostics employing
potentially toxic moieties, is limited by undesired side
effects.
[0004] Many disease and disorders are characterized by the presence
of high levels of certain proteins in specific types of cells. In
some cases, the presence of these high levels of protein is caused
by overexpression. Historically, some of these proteins have been
useful targets for therapeutic molecules or used as biomarkers for
the detection of disease. One class of overexpressed intracellular
protein that has been recognized as a useful therapeutic target is
known as the heat shock proteins.
[0005] Heat shock proteins (HSPs) are a class of proteins that are
up-regulated in response to elevated temperature and other
environmental stresses, such as ultraviolet light, nutrient
deprivation, and oxygen deprivation. HSPs have many known
functions, including acting as chaperones to other cellular
proteins (called client proteins) to facilitate their proper
folding and repair, and to aid in the refolding of misfolded client
proteins. There are several known families of HSPs, each having its
own set of client proteins. Hsp90 is one of the most abundant HSP
families, accounting for about 1-2% of proteins in a cell that is
not under stress and increasing to about 4-6% in a cell under
stress.
[0006] Inhibition of Hsp90 results in degradation of its client
proteins via the ubiquitin proteasome pathway. Unlike other
chaperone proteins, the client proteins of Hsp90 are mostly protein
kinases or transcription factors involved in signal transduction,
and a number of its client proteins have been shown to be involved
in the progression of cancer. Hsp90 has been shown by mutational
analysis to be necessary for the survival of normal eukaryotic
cells. However, Hsp90 is overexpressed in many tumor types,
indicating that it may play a significant role in the survival of
cancer cells and that cancer cells may be more sensitive to
inhibition of Hsp90 than normal cells. For example, cancer cells
typically have a large number of mutated and overexpressed
oncoproteins that are dependent on Hsp90 for folding. In addition,
because the environment of a tumor is typically hostile due to
hypoxia, nutrient deprivation, acidosis, etc., tumor cells may be
especially dependent on Hsp90 for survival. Moreover, inhibition of
Hsp90 causes simultaneous inhibition of a number of oncoproteins,
as well as hormone receptors and transcription factors, making it
an attractive target for an anti-cancer agent. In view of the
above, Hsp90 has been an attractive target of drug development,
including such Hsp90 inhibitor (Hsp90i) compounds as ganetespib,
AUY-922, and IPI-504. At the same time, the advancement of certain
of these compounds which showed early promise, e.g., geldanamycin,
has been slowed by those compounds' toxicity profile. Hsp90i
compounds developed to date are believed to show great promise as
cancer drugs, but other ways the ubiquity of Hsp90 in cancer cells
might be leveraged have heretofore remained unexplored until now.
Accordingly, the need exists for therapeutic molecules that
selectively target proteins, such as Hsp90, that are overexpressed
in cells associated with particular diseases or disorders.
SUMMARY OF THE INVENTION
[0007] The present invention provides pharmacological molecules
("SDC-TRAPs"). I none aspect, the invention features a binding
moiety-drug conjugate (SDC-TRAP) comprising a binding moiety and an
effector moiety, for example and including an effector moiety
conjugated to a binding moiety, which directs the effector moiety
into a target cell of interest in a manner that traps the molecule
in the target cell. In a specific embodiment, the effector moiety
is conjugated via a cleavable bond or linker to the binding moiety,
such that the cleavable bond or linker is preferentially cleaved
after the SDC-TRAP enters the target cell. The inventors of the
instant application have discovered that the SDC-TRAP molecules of
the invention can be used to selectively deliver an effector moiety
to a specific type of cell in order to increase the intracellular
level of the effector moiety in the target cell as compared to
other cells. In one embodiment, the binding moiety interacts with a
protein that is overexpressed in cancerous cells compared to normal
cells. The inventors have demonstrated that certain SDC-TRAP
molecules of the invention enter target cells by passive diffusion
and are selectively retained in the target cells. Specifically, the
inventors have shown that certain SDC-TRAP molecules of the
invention are selectively retained only in cells that overexpress
or otherwise have a high intracellular level of the protein to
which the binding moiety binds. There are numerous advantages to
these SDC-TRAP molecules and to methods of using these molecules
that are described herein.
[0008] Specifically, the invention provides SDC-TRAP molecules that
are targeted to cells of interest and trapped intracellularly for a
sufficient period of time such that the effector moiety has the
desired biological effect. In one embodiment, these SDC-TRAPs allow
for the targeting of an effector moiety to a particular type of
cell based on the overexpression of an intracellular protein that
is characteristic of a particular disease or disorder. Accordingly,
the present invention provides compositions, kits, and methods
(e.g., therapeutic, diagnostic, and imaging) including the
compounds.
[0009] In a specific embodiment, the application exemplifies the
use of Hsp90 interacting moieties, e.g., inhibitors, as the binding
moiety in the SDC-TRAPs. However, the invention is intended to
include other binding moieties, including those that are
contemplated, listed and exemplified herein. Accordingly, in
certain embodiments directed to treating cancer or inflammation,
the SDC-TRAP includes an Hsp90 inhibitor moiety conjugated to an
effector moiety. In certain embodiments, the effector moiety is a
cytotoxic effector moiety.
[0010] In another embodiment, the SDC-TRAP includes an effector
moiety that is effective while still linked to the binding moiety.
In such embodiment, cleavage of the bond or linker in the target
cell is not a necessary feature of the invention. In other cases,
such as cytotoxic effector moieties, the effector moiety should
only be effective after the linker or bond is cleaved and the
effector moiety is released from the SDC-TRAP molecule inside the
target cell. In either case, SDC-TRAPs that do not enter into the
target cell should be rapidly cleared (e.g., from the plasma or
other non-target cells or tissues).
[0011] In another embodiment, the binding moiety of the SDC-TRAP
binds a protein within the target cell, which may itself produce a
desired biological effect (e.g., such as inhibiting Hsp90 within
the target cell). In one embodiment, the binding moiety can
contribute to the overall efficacy of the SDC-TRAP by not only
binding an intracellular protein present in the target cell but by
also conveying a particular desired biological effect. For example,
if the binding moiety is an Hsp90 inhibitor and the target cell is
a cancer cell, than the overall activity of the SDC-TRAP may not
only result from the effector moiety, but also from the biological
activity of the Hsp90 inhibitor.
[0012] Alternatively, interaction of the binding moiety with its
protein target may not impart a biological effect, but rather only
serve to attract and retain the SDC-TRAP within the target cell. In
this embodiment, the binding moiety may reversibly bind to the
intracellular target protein and create an intracellular
equilibrium between free and bound SDC-TRAP molecules. This
equilibrium may allow for cleavage of the SDC-TRAP and more
effective delivery of the effector moiety, e.g., release of the
effector moiety from the binding moiety by, for example, enzymatic
cleavage, hydrolysis or degradation. In some cases, the effector
moiety may be inactive until such release occurs.
[0013] In various aspects and embodiments, the present invention
provides numerous advantages. For example, the SDC-TRAP can provide
for targeted therapy, maximizing efficacy and/or minimizing
undesired side effects. The SDC-TRAP can provide for targeted use
of an effector moiety that would otherwise be unsuitable for
administration alone due to toxicity and/or undesired systemic
effects. The SDC-TRAP can facilitate targeting such effector
moieties to intracellular targets--that is, due to its size and
chemical properties, the SDC-TRAP can passively diffuse (or in some
cases be actively transported) into a cell having an intracellular
target of interest. Alternatively, the SDC-TRAP can deliver in a
selective manner a cytotoxic molecule to destroy a target cell,
such as a cancer or inflammatory cell.
[0014] In various aspects and embodiments, the SDC-TRAP can exhibit
decreased and/or minimized toxicity concurrently with increased
efficacy (e.g., as compared to that of the effector moiety when
used alone). Decreasing and/or minimizing toxicity can encompass
reducing toxicity to a predetermined level (e.g., a regulatory
guideline or suggested level, for example promulgated by the US
Food and Drug Administration "FDA"). Increasing efficacy can
encompass increasing efficacy to a predetermined level (e.g., a
regulatory guideline or suggested level, for example promulgated by
the US FDA). Similarly, decreasing and/or minimizing toxicity
concurrently with increasing efficacy can encompass achieving a
predetermined therapeutic ratio (e.g., a regulatory guideline or
suggested value, for example promulgated by the US FDA).
[0015] Decreasing and/or minimizing toxicity can encompass, for
example, reducing toxicity by 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95%, or more. Increasing
efficacy can encompass, for example, increasing efficacy by 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 125, 150, 175, 200, 250, 300, 400, 500%, or more. Decreasing
and/or minimizing toxicity concurrently with increasing efficacy
can encompass, for example: essentially the same efficacy with
decreased toxicity; essentially the same toxicity with increased
efficacy; or decreased toxicity and increased efficacy. Similarly,
decreasing and/or minimizing toxicity concurrently with increasing
efficacy can encompass, for example, scenarios such as: increased
efficacy enabling a lower dose (e.g., lower dose of effector moiety
with a correspondingly lower net toxicity) and decreased toxicity
enabling a higher dose (e.g., higher dose of effector moiety
without a correspondingly higher net toxicity).
[0016] Additional advantages are discussed in detail below.
[0017] These and other advantages of the present invention are of
particular interest, for example, in chemotherapy where despite
tremendous recent advances, currently available therapeutics and
therapies remains unsatisfactory and the prognosis for the majority
of patients diagnosed with diseases such as cancer remains poor.
However, while many of the illustrative embodiments and examples
are presented in the context of cancer, a person of ordinary skill
in the art would understand that the present invention has
applications across therapeutic, diagnostic, and imaging
applications that require, or would benefit from, targeting of an
effector moiety.
[0018] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety and an effector moiety.
[0019] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety and an effector moiety, wherein the
SDC-TRAP is able to enter a cell by active transport.
[0020] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety and an effector moiety, wherein the
SDC-TRAP has a molecular weight of less than about 1600
Daltons.
[0021] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety and an effector moiety, wherein the
binding moiety has a molecular weight of less than about 800
Daltons.
[0022] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety and an effector moiety, wherein the
effector moiety has a molecular weight of less than 800
Daltons.
[0023] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety and an effector moiety, wherein the
binding moiety and the effector moiety are approximately equal in
size.
[0024] In various aspects, the invention provides an SDC-TRAP
comprising an Hsp90 binding moiety and an effector moiety, wherein
the Hsp90 binding moiety interacts with the N-terminal domain of
Hsp90.
[0025] In various aspects, the invention provides an SDC-TRAP
comprising an Hsp90 binding moiety and an effector moiety, wherein
the Hsp90 binding moiety interacts with the C-terminal domain of
Hsp90.
[0026] In various aspects, the invention provides an SDC-TRAP
comprising an Hsp90 binding moiety and an effector moiety, wherein
the Hsp90 binding moiety interacts with the middle domain of
Hsp90.
[0027] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety and an effector moiety, wherein the
binding moiety interacts with a predetermined domain of a
multidomain target protein molecule.
[0028] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety (e.g., an Hsp90 binding moiety) and an
effector moiety, wherein the binding moiety (e.g., Hsp90 binding
moiety) has a K.sub.d of 100 nM or higher (e.g., for a
predetermined target molecule, for example, Hsp90).
[0029] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety (e.g., Hsp90 binding moiety) and an
effector moiety, wherein when administered to a subject, the
SDC-TRAP is present at a ratio of 2:1 in target (e.g., tumor) cells
compared to plasma. In another embodiment, the invention provides
an SDC-TRAP comprising a binding moiety (e.g., Hsp90 binding
moiety) and an effector moiety, wherein when administered to a
subject the SDC-TRAP present at a ratio of 2:1 in target (e.g.,
tumor) cells compared to normal cells.
[0030] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety (e.g., Hsp90 binding moiety) and an
effector moiety, wherein the SDC-TRAP is present in target (e.g.,
cancer) cells for at least 24 hours.
[0031] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety (e.g., Hsp90 binding moiety) and an
effector moiety, wherein the effector moiety is released for a
period of at least 6 hours (e.g., within a target cell and/or
tissue).
[0032] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety (e.g., Hsp90 binding moiety) and an
effector moiety, wherein the effector moiety is selectively
released inside a target (e.g., cancer) cell.
[0033] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety (e.g., Hsp90 binding moiety) and an
effector moiety, wherein the SDC-TRAP allows for the use of an
effector moiety that is toxic or otherwise unfit for administration
to a subject.
[0034] In various aspects, the invention provides an SDC-TRAP
comprising a binding moiety (e.g., Hsp90 binding moiety) and an
effector moiety, wherein the Hsp90 is an inhibitor (e.g., Hsp90
inhibitor) that is ineffective as a therapeutic agent when
administered alone.
[0035] In various aspects, the invention provides an SDC-TRAP
comprising an Hsp90 binding moiety and an effector moiety.
[0036] In various aspects, the invention provides pharmaceutical
compositions comprising a therapeutically effective amount of at
least one SDC-TRAP, and at least one pharmaceutical excipient.
[0037] In various aspects, the invention provides methods for
treating a subject in need thereof comprising administering a
therapeutically effective amount of at least one SDC-TRAP to the
subject, thereby treating the subject.
[0038] In various aspects, the invention provides methods for
imaging, diagnosing, and/or selecting a subject comprising
administering an effective amount of at least one SDC-TRAP to the
subject, thereby imaging, diagnosing, and/or selecting the
subject.
[0039] In various aspects, the invention provides kits for treating
a subject in need thereof comprising at least one SDC-TRAP and
instruction for administering a therapeutically effective amount of
the at least one SDC-TRAP to the subject, thereby treating the
subject.
[0040] In various aspects, the invention provides kits for imaging,
diagnosing, and/or selecting a subject comprising at least one
SDC-TRAP and instruction for administering an effective amount of
at least one SDC-TRAP to the subject, thereby imaging, diagnosing,
and/or selecting the subject.
[0041] In various embodiments, the invention can include any one or
more of the aspects disclosed herein having any one or more of the
features disclosed herein.
[0042] In various embodiments, the binding moiety interacts with a
protein that is overexpressed in cancerous cells compared to normal
cells.
[0043] In various embodiments, the protein is a chaperonin protein.
The chaperonin can be, for example, Hsp90.
[0044] In various embodiments, the chaperonin is an Hsp90 binding
moiety.
[0045] In various embodiments, the binding moiety is an Hsp90
ligand or a prodrug thereof. The Hsp90 ligand can be, for example,
an Hsp90 inhibitor. An Hsp90 inhibitor can be selected from the
group consisting of geldanamycins, macbecins, tripterins,
tanespimycins, and radicicols.
[0046] In various embodiments, the binding moiety can be an
Hsp90-targeting moiety, for example a triazole/resorcinol-based
compound that binds Hsp90, or a resorcinol amide-based compound
that binds Hsp90, e.g., ganetespib, AUY-922, or AT-13387.
[0047] In various embodiments, the binding moiety can be an
Hsp90-binding compound of formula (I):
##STR00001##
wherein R.sup.1 may be alkyl, aryl, halide, carboxamide or
sulfonamide; R.sup.2 may be alkyl, cycloalkyl, aryl or heteroaryl,
wherein when R.sup.2 is a six-membered aryl or heteroaryl, R.sup.e
is substituted at the 3- and 4-positions relative to the connection
point on the triazole ring, through which a linker L is attached;
and R.sup.3 may be SH, OH, --CONHR.sup.4, aryl or heteroaryl,
wherein when R.sup.3 is a six-membered aryl or heteroaryl, R.sup.3
is substituted at the 3 or 4 position.
[0048] In various embodiments, the binding moiety can be an
Hsp90-binding compound of formula (II):
##STR00002##
wherein R.sup.1 may be alkyl, aryl, halo, carboxamido, sulfonamido;
and R.sup.2 may be optionally substituted alkyl, cycloalkyl, aryl
or heteroaryl. Examples of such compounds include [0049]
5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholin-
omethyl)phenyl)-4H-1,2,4-triazole-3-carboxamide and [0050]
5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)--
N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide.
[0051] In various embodiments, the binding moiety can be an
Hsp90-binding compound of formula (III):
##STR00003##
wherein X, Y, and Z may independently be CH, N, O or S (with
appropriate substitutions and satisfying the valency of the
corresponding atoms and aromaticity of the ring); le may be alkyl,
aryl, halide, carboxamido or sulfonamido; R.sup.2 may be
substituted alkyl, cycloalkyl, aryl or heteroaryl, where a linker L
is connected directly or to the extended substitutions on these
rings; R.sup.3 may be SH, OH, NR.sup.4R.sup.5 AND --CONHR.sup.6, to
which an effector moiety may be connected; R.sup.4 and R.sup.5 may
independently be H, alkyl, aryl, or heteroaryl; and R.sup.6 may be
alkyl, aryl, or heteroaryl, having a minimum of one functional
group to which an effector moiety may be connected.
[0052] As used herein, the term "alkyl" means a saturated straight
chain or branched non-cyclic hydrocarbon having from 1 to 10 carbon
atoms. Representative saturated straight chain alkyls include
methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,
n-octyl, n-nonyl and n-decyl; while saturated branched alkyls
include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl,
2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl,
4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl,
5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl,
2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl,
2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl,
3,3-dimtheylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl,
2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl,
4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl,
2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl,
2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl,
3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like.
The term "(C.sub.1-C.sub.6)alkyl" means a saturated straight chain
or branched non-cyclic hydrocarbon having from 1 to 6 carbon atoms.
Representative (C.sub.1-C.sub.6)alkyl groups are those shown above
having from 1 to 6 carbon atoms. Alkyl groups included in compounds
of this invention may be optionally substituted with one or more
substituents.
[0053] As used herein, the term "alkenyl" means a saturated
straight chain or branched non-cyclic hydrocarbon having from 2 to
10 carbon atoms and having at least one carbon-carbon double bond.
Representative straight chain and branched
(C.sub.2-C.sub.10)alkenyls include vinyl, allyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl,
1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2-heptenyl,
3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl,
3-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl and the like. Alkenyl
groups may be optionally substituted with one or more
substituents.
[0054] As used herein, the term "alkynyl" means a saturated
straight chain or branched non-cyclic hydrocarbon having from 2 to
10 carbon atoms and having at least one carbon-carbon triple bond.
Representative straight chain and branched alkynyls include
acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,
3-methyl-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl,
1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl,
7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl,
9-decynyl, and the like. Alkynyl groups may be optionally
substituted with one or more substituents.
[0055] As used herein, the term "cycloalkyl" means a saturated,
mono- or polycyclic alkyl radical having from 3 to 20 carbon atoms.
Representative cycloalkyls include cyclopropyl,
1-methylcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, cyclononyl, -cyclodecyl,
octahydro-pentalenyl, and the like. Cycloalkyl groups may be
optionally substituted with one or more substituents.
[0056] As used herein, the term "cycloalkenyl" means a mono- or
poly-cyclic non-aromatic alkyl radical having at least one
carbon-carbon double bond in the cyclic system and from 3 to 20
carbon atoms. Representative cycloalkenyls include cyclopentenyl,
cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl,
cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl,
cyclooctatrienyl, cyclooctatetraenyl, cyclononenyl,
cyclononadienyl, cyclodecenyl, cyclodecadienyl,
1,2,3,4,5,8-hexahydronaphthalenyl and the like. Cycloalkenyl groups
may be optionally substituted with one or more substituents.
[0057] As used herein, the term "haloalkyl" means and alkyl group
in which one or more (including all) the hydrogen radicals are
replaced by a halo group, wherein each halo group is independently
selected from --F, --Cl, --Br, and --I. The term "halomethyl" means
a methyl in which one to three hydrogen radical(s) have been
replaced by a halo group. Representative haloalkyl groups include
trifluoromethyl, bromomethyl, 1,2-dichloroethyl, 4-iodobutyl,
2-fluoropentyl, and the like.
[0058] As used herein, an "alkoxy" is an alkyl group which is
attached to another moiety via an oxygen linker.
[0059] As used herein, an "haloalkoxy" is an haloalkyl group which
is attached to another moiety via an oxygen linker.
[0060] As used herein, the term an "aromatic ring" or "aryl" means
a hydrocarbon monocyclic or polycyclic radical in which at least
one ring is aromatic. Examples of suitable aryl groups include, but
are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl,
azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties
such as 5,6,7,8-tetrahydronaphthyl. Aryl groups may be optionally
substituted with one or more substituents. In one embodiment, the
aryl group is a monocyclic ring, wherein the ring comprises 6
carbon atoms, referred to herein as "(C.sub.6)aryl."
[0061] As used herein, the term "aralkyl" means an aryl group that
is attached to another group by a (C.sub.1-C.sub.6)alkylene group.
Representative aralkyl groups include benzyl, 2-phenyl-ethyl,
naphth-3-yl-methyl and the like. Aralkyl groups may be optionally
substituted with one or more substituents.
[0062] As used herein, the term "alkylene" refers to an alkyl group
that has two points of attachment. The term
"(C.sub.1-C.sub.6)alkylene" refers to an alkylene group that has
from one to six carbon atoms. Straight chain
(C.sub.1-C.sub.6)alkylene groups are preferred. Non-limiting
examples of alkylene groups include methylene (--CH.sub.2--),
ethylene (--CH.sub.2CH.sub.2--), n-propylene
(--CH.sub.2CH.sub.2CH.sub.2--), isopropylene
(--CH.sub.2CH(CH.sub.3)--), and the like. Alkylene groups may be
optionally substituted with one or more substituents.
[0063] As used herein, the term "heterocyclyl" means a monocyclic
(typically having 3- to 10-members) or a polycyclic (typically
having 7- to 20-members) heterocyclic ring system which is either a
saturated ring or a unsaturated non-aromatic ring. A 3- to
10-membered heterocycle can contain up to 5 heteroatoms; and a 7-
to 20-membered heterocycle can contain up to 7 heteroatoms.
Typically, a heterocycle has at least on carbon atom ring member.
Each heteroatom is independently selected from nitrogen, which can
be oxidized (e.g., N(O)) or quaternized; oxygen; and sulfur,
including sulfoxide and sulfone. The heterocycle may be attached
via any heteroatom or carbon atom. Representative heterocycles
include morpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl,
piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl,
oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,
tetrahydropyrindinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl, and the like. A heteroatom may be
substituted with a protecting group known to those of ordinary
skill in the art, for example, the hydrogen on a nitrogen may be
substituted with a tert-butoxycarbonyl group. Furthermore, the
heterocyclyl may be optionally substituted with one or more
substituents. Only stable isomers of such substituted heterocyclic
groups are contemplated in this definition.
[0064] As used herein, the term "heteroaromatic", "heteroaryl" or
like terms means a monocyclic or polycyclic heteroaromatic ring
comprising carbon atom ring members and one or more heteroatom ring
members. Each heteroatom is independently selected from nitrogen,
which can be oxidized (e.g., N(O)) or quaternized; oxygen; and
sulfur, including sulfoxide and sulfone. Representative heteroaryl
groups include pyridyl, 1-oxo-pyridyl, furanyl, benzo[1,3]dioxolyl,
benzo[1,4]dioxinyl, thienyl, pyrrolyl, oxazolyl, imidazolyl,
thiazolyl, a isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl,
pyridazinyl, pyrimidinyl, pyrazinyl, a triazinyl, triazolyl,
thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl,
indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl,
benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl,
tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl,
purinyl, pyrrolo[2,3]pyrimidinyl, pyrazolo[3,4]pyrimidinyl,
imidazo[1,2-a]pyridyl, and benzothienyl. In one embodiment, the
heteroaromatic ring is selected from 5-8 membered monocyclic
heteroaryl rings. The point of attachment of a heteroaromatic or
heteroaryl ring to another group may be at either a carbon atom or
a heteroatom of the heteroaromatic or heteroaryl rings. Heteroaryl
groups may be optionally substituted with one or more
substituents.
[0065] As used herein, the term "(C.sub.5)heteroaryl" means an
aromatic heterocyclic ring of 5 members, wherein at least one
carbon atom of the ring is replaced with a heteroatom such as, for
example, oxygen, sulfur or nitrogen. Representative
(C.sub.5)heteroaryls include furanyl, thienyl, pyrrolyl, oxazolyl,
imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,
pyrazinyl, triazolyl, thiadiazolyl, and the like.
[0066] As used herein, the term "(C.sub.6)heteroaryl" means an
aromatic heterocyclic ring of 6 members, wherein at least one
carbon atom of the ring is replaced with a heteroatom such as, for
example, oxygen, nitrogen or sulfur. Representative
(C.sub.6)heteroaryls include pyridyl, pyridazinyl, pyrazinyl,
triazinyl, tetrazinyl and the like.
[0067] As used herein, the term "heteroaralkyl" means a heteroaryl
group that is attached to another group by a
(C.sub.1-C.sub.6)alkylene. Representative heteroaralkyls include
2-(pyridin-4-yl)-propyl, 2-(thien-3-yl)-ethyl, imidazol-4-yl-methyl
and the like. Heteroaralkyl groups may be optionally substituted
with one or more substituents.
[0068] As used herein, the term "halogen" or "halo" means --F,
--Cl, --Br or --I.
[0069] Suitable substituents for an alkyl, alkylene, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, aralkyl,
heteroaryl, and heteroaralkyl groups include any substituent which
will form a stable compound of the invention. Examples of
substituents for an alkyl, alkylene, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, heterocyclyl, aryl, aralkyl, heteroaryl, and
heteroarylalkyl include an optionally substituted alkyl, an
optionally substituted alkenyl, an optionally substituted alkynyl,
an optionally substituted cycloalkyl, an optionally substituted
cycloalkenyl, an optionally substituted heterocyclyl, an optionally
substituted aryl, an optionally substituted heteroaryl, an
optionally substituted aralkyl, an optionally substituted
heteraralkyl, or a haloalkyl.
[0070] In addition, alkyl, cycloalkyl, alkylene, a heterocyclyl,
and any saturated portion of a alkenyl, cycloalkenyl, alkynyl,
aralkyl, and heteroaralkyl groups, may also be substituted with
.dbd.O, or .dbd.S.
[0071] When a heterocyclyl, heteroaryl, or heteroaralkyl group
contains a nitrogen atom, it may be substituted or unsubstituted.
When a nitrogen atom in the aromatic ring of a heteroaryl group has
a substituent the nitrogen may be a quaternary nitrogen.
[0072] As used herein, the term "lower" refers to a group having up
to four atoms. For example, a "lower alkyl" refers to an alkyl
radical having from 1 to 4 carbon atoms, "lower alkoxy" refers to
"--O--(C.sub.1-C.sub.4)alkyl and a "lower alkenyl" or "lower
alkynyl" refers to an alkenyl or alkynyl radical having from 2 to 4
carbon atoms, respectively.
[0073] Unless indicated otherwise, the compounds of the invention
containing reactive functional groups (such as (without limitation)
carboxy, hydroxy, thiol, and amino moieties) also include protected
derivatives thereof. "Protected derivatives" are those compounds in
which a reactive site or sites are blocked with one or more
protecting groups. Examples of suitable protecting groups for
hydroxyl groups include benzyl, methoxymethyl, allyl,
trimethylsilyl, tert-butyldimethylsilyl, acetate, and the like.
Examples of suitable amine protecting groups include
benzyloxycarbonyl, tert-butoxycarbonyl, tert-butyl, benzyl and
fluorenylmethyloxy-carbonyl (Fmoc). Examples of suitable thiol
protecting groups include benzyl, tert-butyl, acetyl, methoxymethyl
and the like. Other suitable protecting groups are well known to
those of ordinary skill in the art and include those found in T. W.
Greene, Protecting Groups in Organic Synthesis, John Wiley &
Sons, Inc. 1981.
[0074] Exemplary Hsp90 inhibitors include those disclosed in U.S.
Pat. Nos. 8,362,055 and 7,825,148. Examples of such compounds
include AUY-922:
##STR00004##
[0075] In various embodiments, the binding moiety can be an
Hsp90-binding compound of formula (IV):
##STR00005##
wherein R.sup.1 may be alkyl, aryl, halo, carboxamido or
sulfonamido; R.sup.2 and R.sup.3 are independently C.sub.1-C.sub.5
hydrocarbyl groups optionally substituted with one or more of
hydroxy, halogen, C.sub.1-C.sub.2 alkoxy, amino, mono- and
di-C.sub.1-C.sub.2 alkylamino; 5- to 12-membered aryl or heteroaryl
groups; or, R.sup.2 and R.sup.3, taken together with the nitrogen
atom to which they are attached, form a 4- to 8-membered monocyclic
heterocyclic group, of which up to 5 ring members are selected from
O, N and S. Examples of such compounds include AT-13387:
##STR00006##
[0076] In various embodiments, the binding moiety includes an
Hsp90-targeting moiety, for example one or more geldanamycins,
e.g., IPI-493
##STR00007##
macbecins, tripterins, tanespimycins, e.g., 17-AAG
##STR00008##
KF-55823
##STR00009##
[0077] radicicols, KF-58333
##STR00010##
KF-58332
##STR00011##
[0078] 17-DMAG
##STR00012##
[0079] IPI-504
##STR00013##
[0080] BIIB-021
##STR00014##
[0081] BIIB-028, PU-H64
##STR00015##
[0082] PU-H71
##STR00016##
[0083] PU-DZ8
##STR00017##
[0084] PU-HZ151
##STR00018##
[0085] SNX-2112
##STR00019##
[0086] SNX-2321
##STR00020##
[0087] SNX-5422
##STR00021##
[0088] SNX-7081
##STR00022##
[0089] SNX-8891, SNX-0723
##STR00023##
[0090] SAR-567530, ABI-287, ABI-328, AT-13387
##STR00024##
[0091] NSC-113497
##STR00025##
[0092] PF-3823863
##STR00026##
[0093] PF-4470296
##STR00027##
[0094] EC-102, EC-154, ARQ-250-RP, BC-274
##STR00028##
[0095] VER-50589
##STR00029##
[0096] KW-2478
##STR00030##
[0097] BHI-001, AUY-922
##STR00031##
[0098] EMD-614684
##STR00032##
[0099] EMD-683671, XL-888, VER-51047
##STR00033##
[0100] KOS-2484, KOS-2539, CUDC-305
##STR00034##
[0101] MPC-3100
##STR00035##
[0102] CH-5164840
##STR00036##
[0103] PU-DZ13
##STR00037##
[0104] PU-HZ151
##STR00038##
[0105] PU-DZ13
##STR00039##
[0106] VER-82576
##STR00040##
[0107] VER-82160
##STR00041##
[0108] VER-82576
##STR00042##
[0109] VER-82160
##STR00043##
[0110] NXD-30001
##STR00044##
[0111] NVP-HSP990
##STR00045##
[0112] SST-0201CL1
##STR00046##
[0113] SST-0115AA1
##STR00047##
[0114] SST-0221AA1
##STR00048##
[0115] SST-0223AA1
##STR00049##
[0116] novobiocin (a C-terminal Hsp90i.)
[0117] In various embodiments, the cytotoxic moiety is selected
from the group consisting of LY2801653,
##STR00050##
LY2835219
##STR00051##
[0118] LY260636
##STR00052##
[0119] LY2874455
##STR00053##
[0120] LY2090314
##STR00054##
##STR00055##
[0121] LY278544
##STR00056##
[0122] LY2940680
##STR00057##
##STR00058##
[0123] LY2603618, LY353381
##STR00059##
[0124] and trimetrexate
##STR00060##
[0125] In various embodiments, the cytotoxic moiety is not suitable
for administration alone. The cytotoxic moiety can be unsuitable
for administration alone due to toxicity. The cytotoxic moiety can
be unsuitable for administration alone due to undesired targeting
or a lack of targeting.
[0126] In various embodiments, the binding moiety and the effector
moiety are covalently attached. The binding moiety and the effector
moiety can be covalently attached, for example by a linker. The
linker can comprise a cleavable linker. The cleavable linker can
comprise an enzymatically cleavable linker. The linker can be
selected from the group consisting of disulfide, carbamate, amide,
ester, and ether linkers.
[0127] In various embodiments, the SDC-TRAP has a molecular weight
of less than about 1600 Dalton. For example, the SDC-TRAP molecular
weight can be less than about 1600, 1550, 1500, 1450, 1400, 1350,
1300, 1250, 1200, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750,
700, 650, 600, 550, 500, 450, 400, 350, 300, 250, or 200
Dalton.
[0128] In various embodiments, the binding moiety has a molecular
weight of less than about 800 Dalton. For example, the binding
moiety molecular weight can be less than about 800, 750, 700, 650,
600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100
Dalton.
[0129] In various embodiments, the effector moiety has a molecular
weight of less than about 800 Dalton. For example, the effector
moiety molecular weight can be less than about 800, 750, 700, 650,
600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100
Dalton.
[0130] In various embodiments, the binding moiety and the effector
moiety are approximately equal in size. For example, the binding
moietyand the effector moiety can have less than about a 25, 50,
75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or
400 Dalton difference in molecular weight.
[0131] In various embodiments, the binding moiety has a high
affinity for a molecular target. For example, the binding moiety
has a high affinity for a molecular target that is a K.sub.d of 50,
100, 150, 200, 250, 300, 350, 400 nM or higher.
[0132] In various embodiments, when administered to a subject, the
SDC-TRAP is present at a ratio of about 2:1, 5:1, 10:1, 25:1, 50:1,
75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 600:1,
700:1, 800:1, 900:1, 1000:1, or greater. The ratio can be, for
example, at 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48, 72, or more hours
from administration.
[0133] In various embodiments, the SDC-TRAP is present in target
cells and/or tissue for at least 24 hours. The SDC-TRAP can be
present in cancer cells for longer, for example, for at least 48,
72, 96, or 120 hours.
[0134] In various embodiments, the effector moiety is released for
a period of at least 6 hours. The effector moiety can be released
for a longer period, for example, for at least 12, 24, 48, 72, 96,
or 120 hours.
[0135] In various embodiments, the effector moiety is selectively
released inside a target cell and/or tissue.
[0136] In various embodiments, the present invention provides
SDC-TRAP molecules comprising a binding moiety is an inhibitor of a
target protein but that is ineffective as a therapeutic agent when
administered alone. In these, and in other embodiments, the
SDC-TRAP may facilitate an additive or synergistic effect between
the binding moiety and effector moiety.
[0137] In various embodiments, the present invention provides
method for treating a subject having a cancer comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the cancer.
[0138] In various embodiments, the present invention provides a
method for treating a subject having a colon cancer comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the colon cancer.
[0139] In various embodiments, the present invention provides a
method for treating a subject having a breast cancer comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the breast cancer.
[0140] In various embodiments, the present invention provides a
method for treating a subject having an ovarian cancer comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the ovarian cancer.
[0141] In various embodiments, the present invention provides a
method for treating a subject having a lung cancer comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the lung cancer. The lung
cancer can comprise small cell lung cancer.
[0142] In various embodiments, the present invention provides a
method for treating a subject having a skin cancer comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the skin cancer.
[0143] In various embodiments, the present invention provides a
method for treating a subject having actinic keratosis comprising
administering a therapeutically effective amount of at least one
SDC-TRAP to the subject, thereby treating the actinic
keratosis.
[0144] The present invention is described in further detail by the
figures and examples below, which are used only for illustration
purposes and are not limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0145] FIG. 1 shows how an illustrative Hsp90-targeting moiety may
be suitably modified at one or more positions to enhance the
physical, pharmacokinetic, or pharmacodynamic properties of the
conjugate.
[0146] FIG. 2 illustrates an embodiment of a pharmaceutical
conjugate having two effector moieties. Other features and
advantages of the instant invention will be apparent from the
following detailed description and claims.
[0147] FIG. 3 is a table that shows a summary of the structure,
physical properties, type of linkage, Her2 IC50 (nM) and
cytotoxicity (BT474 IC50 nM) for the exemplary SDC-TRAPs.
DETAILED DESCRIPTION OF THE INVENTION
[0148] The present invention provides molecules including an
effector moiety conjugated to a binding moiety that directs the
effector moiety to a biological target of interest. The molecules
of the invention allow for selective targeting of an effector
moiety by trapping the molecules of the invention in a desired
cell, e.g., a cancer cell. The molecules can be described as Small
molecule Drug Congugates that are TRAPped intracellularly
(SDC-TRAP), due to their selective binding to high concentration
intracellular proteins. In order for the molecules of the invention
to be trapped within the cells of interest, the binding moieties
that are part of the SDC-TRAP molecules interact with proteins that
are overexpressed in targeted cells. In exemplary embodiments, the
proteins that are overexpressed are characteristic of a particular
disease or disorder. Accordingly, the present invention provides
compositions, kits, and methods (e.g., therapeutic, diagnostic, and
imaging) that include the molecules of the invention.
[0149] In one embodiment of the invention, SDC-TRAPs allow for the
delivery of a effector molecule that would otherwise be unsuitable
for administration alone due to toxicity and/or undesired systemic
effects. Using the targeted delivery molecules described herein
(SDC-TRAPs) allows for effector moieties that are too toxic to
administer by current methods to be dosed at lower levels thereby
allowing the toxic effector to be targeted to specific diseased
cells at sub-toxic levels.
[0150] In various exemplary aspects and embodiments, the present
invention provides compounds for treating cancer. For example, an
SDC-TRAP can comprise an Hsp90 binding moiety (i.e., targeting
Hsp90, which is overexpressed in cancer cells compared to normal
cells) and an effector moiety (e.g., the Hsp90 binding moiety can
be an Hsp90 inhibitor that is conjugated to a cytotoxic agent). As
indicated above, the invention is exemplified herein in terms of
Hsp90-targeted binding moieties and cytotoxic agents. Other binding
moieties that are contemplated, mentioned or described herein are
intended to be included within the scope of the invention.
[0151] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety and an effector
moiety, wherein the SDC-TRAP molecule is able to enter a cell by
passive transport. The ability of an SDC-TRAP to enter a cell by
passive transport can be a result of one or more unique chemical
properties of the SDC-TRAP (e.g., size, weight, charge, polarity,
hydrophobicity, etc.) and can facilitate the delivery and/or action
of the SDC-TRAP. The ability of an SDC-TRAP to enter a cell by
passive transport is a functional property, which along with its
physico-chemical properties, differentiates SDC-TRAPs from other
targeted molecules such as antibody-drug conjugates.
[0152] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety and an effector
moiety, wherein SDC-TRAP molecule is able to enter a cell by active
transport. The ability of an SDC-TRAP to enter a cell by active
transport can be a result of one or more unique chemical properties
of the SDC-TRAP and can facilitate the delivery and/or action of
the SDC-TRAP. Example of SDC-TRAP active transport can include, for
example, endocytosis, phagocytosis, pinocytosis, and
exocytosis.
[0153] In various aspects and embodiments, the present invention
provides an SDC-TRAP having a molecular weight of less than about
1600 Dalton (e.g., less than about 1600, 1550, 1500, 1450, 1400,
1350, 1300, 1250, 1200, 1150, 1100, 1050, 1000, 950, 900, 850, 800,
750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, etc.).
Similarly, in various aspects and embodiments, the present
invention provides a binding moiety having a molecular weight of
less than about 800 Dalton (e.g., less than about 800, 750, 700,
650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, etc.)
and/or an effector moiety having a molecular weight of less than
about 800 Dalton (e.g., less than about 800, 750, 700, 650, 600,
550, 500, 450, 400, 350, 300, 250, 200, 150, 100, etc.). The
overall molecular weight of an SDC-TRAP, and the individual weights
of a binding moiety, effector moiety, and any linking moiety, can
affect transport of the SDC-TRAP. In various examples, it has been
observed that lower molecular weights can facilitate delivery
and/or activity of an SDC-TRAP.
[0154] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising an Hsp90 binding moiety and an
effector moiety, wherein the Hsp90 binding moiety and the effector
moiety are approximately equal in size (e.g., the Hsp90 binding
moiety and the effector moiety have less than about a 25, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,
etc. Dalton difference in molecular weight.) In various examples,
it has been observed that lower differences in molecular weight can
facilitate delivery and/or activity of an SDC-TRAP.
[0155] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a target protein-interacting
binding moiety. A target protein-interacting binding moiety can
selectively interact with any one or more domains of a target
protein. For example, where a target protein is Hsp90, the binding
moiety can be an Hsp90 binding moiety that interacts with the
N-terminal domain of Hsp90, the C-terminal domain of Hsp90, and/or
the middle domain of Hsp90. Selective interaction with any one or
more domains of a target protein can advantageously increase
specificity and/or increase the concentration of molecular targets
within a target tissue and/or cell.
[0156] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety having a high
affinity for a molecular target (e.g., a K.sub.d of 50, 100, 150,
200, 250, 300, 350, 400 nM or higher). For example, where a binding
moiety is an Hsp90 binding moiety, the Hsp90 binding moiety can
have a K.sub.d of 50, 100, 150, 200, 250, 300, 350, 400 nM or
higher. A binding moiety having a high affinity for a molecular
target can advantageously improve targeting and/or increase the
resonance time of the SDC-TRAP in a target cell and/or tissue.
[0157] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety (e.g., Hsp90
binding moiety) and an effector moiety, wherein when administered
to a subject the SDC-TRAP is present at a ratio of about 2:1 in
tumor cells compared to plasma. The ratio can be higher, for
example, about 5:1, 10:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1,
250:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, or
greater. In various aspects and embodiments, the ratio is at 1, 2,
3, 4, 5, 6, 7, 8, 12, 24, 48, 72, or more hours from
administration. The effectiveness of targeting can be reflected in
the ratio of SDC-TRAP in a target cell and/or tissue compared to
plasma.
[0158] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety (e.g., Hsp90
binding moiety) and an effector moiety, wherein the SDC-TRAP is
present in target (e.g., cancer) cells for at least 24 hours. The
SDC-TRAP can be present in cancer cells for longer, for example,
for at least 48, 72, 96, or 120 hours. It can be advantageous for
an SDC-TRAP to be present in target cells for longer periods of
time to increase the therapeutic effect of a given dose of SDC-TRAP
and/or increase an interval between administrations of
SDC-TRAP.
[0159] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety (e.g., Hsp90
binding moiety) and an effector moiety, wherein the effector moiety
is released for a period of at least 6 hours. The effector moiety
can be released for a longer period, for example, for at least 12,
24, 48, 72, 96, or 120 hours. Selective release can be used to
control, delay, and/or extend the period of release of an effector
moiety and, therefore, increase the therapeutic effect of a given
dose of SDC-TRAP, decrease the undesired side effects of a given
dose of SDC-TRAP, and/or increase an interval between
administrations of SDC-TRAP.
[0160] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising an Hsp90 binding moiety and an
effector moiety, wherein the effector moiety is selectively
released inside a target (e.g., cancer) cell. Selective release can
be achieved, for example, by a cleavable linker (e.g., an
enzymatically cleavable linker). Selective release can be used to
decrease undesired toxicity and/or unwanted side effects. For
example, an SDC-TRAP can be designed where an effector moiety such
is inactive (or relatively inactive) in a conjugated form, but
active (or more active) after it is selectively released inside a
target (e.g., cancer) cell.
[0161] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety (e.g., Hsp90
binding moiety) and an effector moiety, wherein the SDC-TRAP allows
for the use of an effector moiety that is otherwise toxic or unfit
for administration to a subject. The effector moiety can be unfit
for administration to a subject because of undesired toxicity. In
such cases, a strategy such as selective release may be used to
address the undesired toxicity. The effector moiety can be unfit
for administration to a subject because of undesired targeting or a
lack of targeting. Targeting can address such problems, for
example, by minimizing systemic toxicity while maximizing local
toxicity at a target (e.g., a tumor).
[0162] In various aspects and embodiments, the SDC-TRAP can exhibit
decreased and/or minimized toxicity concurrently with increased
efficacy (e.g., as compared to that of the effector moiety when
used alone). Decreasing and/or minimizing toxicity can encompass
reducing toxicity to a predetermined level (e.g., a regulatory
guideline or suggested level, for example promulgated by the US
Food and Drug Administration "FDA"). Increasing efficacy can
encompass increasing efficacy to a predetermined level (e.g., a
regulatory guideline or suggested level, for example promulgated by
the US FDA). Similarly, decreasing and/or minimizing toxicity
concurrently with increasing efficacy can encompass achieving a
predetermined therapeutic ratio (e.g., a regulatory guideline or
suggested value, for example promulgated by the US FDA).
[0163] Decreasing and/or minimizing toxicity can encompass, for
example, reducing toxicity by 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95%, or more. Increasing
efficacy can encompass, for example, increasing efficacy by 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 125, 150, 175, 200, 250, 300, 400, 500%, or more. Decreasing
and/or minimizing toxicity concurrently with increasing efficacy
can encompass, for example: essentially the same efficacy with
decreased toxicity; essentially the same toxicity with increased
efficacy; or decreased toxicity and increased efficacy. Similarly,
decreasing and/or minimizing toxicity concurrently with increasing
efficacy can encompass, for example, scenarios such as: increased
efficacy enabling a lower dose (e.g., lower dose of effector moiety
with a correspondingly lower net toxicity) and decreased toxicity
enabling a higher dose (e.g., higher dose of effector moiety
without a correspondingly higher net toxicity).
[0164] In various aspects and embodiments, the present invention
provides an SDC-TRAP comprising a binding moiety (e.g., Hsp90
binding moiety) and an effector moiety, wherein the binding moiety
is an inhibitor (e.g., Hsp90 inhibitor) that is ineffective as a
therapeutic agent when administered alone. In such cases, the
SDC-TRAP may facilitate an additive or synergistic effect between
the binding moiety and effector moiety, thereby advantageously
improving the efficacy and/or reducing the side effects of a
therapy.
[0165] In order that the present invention may be more readily
understood, certain terms are first defined. In addition, it should
be noted that whenever a value or range of values of a parameter
are recited, it is intended that values and ranges intermediate to
the recited values are also intended to be part of this invention.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. It is
also to be understood that the terminology employed is for the
purpose of describing particular embodiments, and is not intended
to be limiting.
Definitions
[0166] The articles "a," "an," and "the" are used herein to refer
to one or to more than one (i.e. to at least one) of the
grammatical object of the article unless otherwise clearly
indicated by contrast. By way of example, "an element" means one
element or more than one element.
[0167] The term "including" is used herein to mean, and is used
interchangeably with, the phrase "including but not limited
to."
[0168] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or," unless context clearly
indicates otherwise.
[0169] The term "such as" is used herein to mean, and is used
interchangeably, with the phrase "such as but not limited to."
[0170] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from context, all numerical values
provided herein can be modified by the term about.
[0171] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50.
[0172] The recitation of a listing of chemical group(s) in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable or aspect herein
includes that embodiment as any single embodiment or in combination
with any other embodiments or portions thereof.
[0173] Any compositions or methods provided herein can be combined
with one or more of any of the other compositions and methods
provided herein.
[0174] As used herein, the term "subject" refers to human and
non-human animals, including veterinary subjects. The term
"non-human animal" includes all vertebrates, e.g., mammals and
non-mammals, such as non-human primates, mice, rabbits, sheep, dog,
cat, horse, cow, chickens, amphibians, and reptiles. In a preferred
embodiment, the subject is a human and may be referred to as a
patient.
[0175] As used herein, the terms "treat," "treating" or "treatment"
refer, preferably, to an action to obtain a beneficial or desired
clinical result including, but not limited to, alleviation or
amelioration of one or more signs or symptoms of a disease or
condition, diminishing the extent of disease, stability (i.e., not
worsening) state of disease, amelioration or palliation of the
disease state, diminishing rate of or time to progression, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival in the absence of treatment.
Treatment does not need to be curative.
[0176] A "therapeutically effective amount" is that amount
sufficient to treat a disease in a subject. A therapeutically
effective amount can be administered in one or more
administrations.
[0177] By "diagnosing" and the like, as used herein, refers to a
clinical or other assessment of the condition of a subject based on
observation, testing, or circumstances for identifying a subject
having a disease, disorder, or condition based on the presence of
at least one indicator, such as a sign or symptom of the disease,
disorder, or condition. Typically, diagnosing using the method of
the invention includes the observation of the subject for multiple
indicators of the disease, disorder, or condition in conjunction
with the methods provided herein. Diagnostic methods provide an
indicator that a disease is or is not present. A single diagnostic
test typically does not provide a definitive conclusion regarding
the disease state of the subject being tested.
[0178] The terms "administer," "administering" or "administration"
include any method of delivery of a pharmaceutical composition or
agent into a subject's system or to a particular region in or on a
subject. In certain embodiments of the invention, an agent is
administered intravenously, intramuscularly, subcutaneously,
intradermally, intranasally, orally, transcutaneously, or
mucosally. In a preferred embodiment, an agent is administered
intravenously. Administering an agent can be performed by a number
of people working in concert. Administering an agent includes, for
example, prescribing an agent to be administered to a subject
and/or providing instructions, directly or through another, to take
a specific agent, either by self-delivery, e.g., as by oral
delivery, subcutaneous delivery, intravenous delivery through a
central line, etc.; or for delivery by a trained professional,
e.g., intravenous delivery, intramuscular delivery, intratumoral
delivery, etc.
[0179] As used herein, the term "survival" refers to the
continuation of life of a subject which has been treated for a
disease or condition, e.g., cancer. The time of survival can be
defined from an arbitrary point such as time of entry into a
clinical trial, time from completion or failure or an earlier
treatment regimen, time from diagnosis, etc.
[0180] As used herein, the term "recur" refers to the re-growth of
tumor or cancerous cells in a subject in whom primary treatment for
the tumor has been administered. The tumor may recur in the
original site or in another part of the body. In one embodiment, a
tumor that recurs is of the same type as the original tumor for
which the subject was treated. For example, if a subject had an
ovarian cancer tumor, was treated and subsequently developed
another ovarian cancer tumor, the tumor has recurred. In addition,
a cancer can recur in or metastasize to a different organ or tissue
than the one where it originally occurred.
[0181] As used herein, the terms "identify" or "select" refer to a
choice in preference to another. In other words, to identify a
subject or select a subject is to perform the active step of
picking out that particular subject from a group and confirming the
identity of the subject by name or other distinguishing
feature.
[0182] As used herein, the term "benefit" refers to something that
is advantageous or good, or an advantage. Similarly, the term
"benefiting," as used herein, refers to something that improves or
advantages. For example, a subject will benefit from treatment if
they exhibit a decrease in at least one sign or symptom of a
disease or condition (e.g., tumor shrinkage, decrease in tumor
burden, inhibition or decrease of metastasis, improving quality of
life ("QOL"), if there is a delay of time to progression ("TTP"),
if there is an increase of overall survival ("OS"), etc.), or if
there is a slowing or stopping of disease progression (e.g.,
halting tumor growth or metastasis, or slowing the rate of tumor
growth or metastasis). A benefit can also include an improvement in
quality of life, or an increase in survival time or progression
free survival.
[0183] The terms "cancer" or "tumor" are well known in the art and
refer to the presence, e.g., in a subject, of cells possessing
characteristics typical of cancer-causing cells, such as
uncontrolled proliferation, immortality, metastatic potential,
rapid growth and proliferation rate, decreased cell
death/apoptosis, and certain characteristic morphological features.
Cancer cells are often in the form of a solid tumor. However,
cancer also includes non-solid tumors, e.g., blood tumors, e.g.,
leukemia, wherein the cancer cells are derived from bone marrow. As
used herein, the term "cancer" includes pre-malignant as well as
malignant cancers. Cancers include, but are not limited to,
acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute
myelocytic leukemia (monocytic, myeloblastic, adenocarcinoma,
angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute
T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder
cancer, brain cancer, breast cancer, bronchogenic carcinoma,
cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic
leukemia, chronic lymphocytic leukemia, chronic myelocytic
(granulocytic) leukemia, chronic myelogenous leukemia, colon
cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma,
diffuse large B-cell lymphoma, Burkitt's lymphoma, dysproliferative
changes (dysplasias and metaplasias), embryonal carcinoma,
endometrial cancer, endotheliosarcoma, ependymoma, epithelial
carcinoma, erythroleukemia, esophageal cancer, estrogen-receptor
positive breast cancer, essential thrombocythemia, Ewing's tumor,
fibrosarcoma, follicular lymphoma, germ cell testicular cancer,
glioma, heavy chain disease, hemangioblastoma, hepatoma,
hepatocellular cancer, hormone insensitive prostate cancer,
leiomyosarcoma, liposarcoma, lung cancer,
lymphagioendotheliosarcoma, lymphangiosarcoma, lymphoblastic
leukemia, lymphoma (Hodgkin's and non-Hodgkin's), malignancies and
hyperproliferative disorders of the bladder, breast, colon, lung,
ovaries, pancreas, prostate, skin, and uterus, lymphoid
malignancies of T-cell or B-cell origin, leukemia, lymphoma,
medullary carcinoma, medulloblastoma, melanoma, meningioma,
mesothelioma, multiple myeloma, myelogenous leukemia, myeloma,
myxosarcoma, neuroblastoma, non-small cell lung cancer,
oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer,
pancreatic cancer, papillary adenocarcinomas, papillary carcinoma,
pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal
cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma,
sebaceous gland carcinoma, seminoma, skin cancer, small cell lung
carcinoma, solid tumors (carcinomas and sarcomas), small cell lung
cancer, stomach cancer, squamous cell carcinoma, synovioma, sweat
gland carcinoma, thyroid cancer, Waldenstrom's macroglobulinemia,
testicular tumors, uterine cancer, and Wilms' tumor. Other cancers
include primary cancer, metastatic cancer, oropharyngeal cancer,
hypopharyngeal cancer, liver cancer, gall bladder cancer, bile duct
cancer, small intestine cancer, urinary tract cancer, kidney
cancer, urothelium cancer, female genital tract cancer, uterine
cancer, gestational trophoblastic disease, male genital tract
cancer, seminal vesicle cancer, testicular cancer, germ cell
tumors, endocrine gland tumors, thyroid cancer, adrenal cancer,
pituitary gland cancer, hemangioma, sarcoma arising from bone and
soft tissues, Kaposi's sarcoma, nerve cancer, ocular cancer,
meningial cancer, glioblastomas, neuromas, neuroblastomas,
Schwannomas, solid tumors arising from hematopoietic malignancies
such as leukemias, metastatic melanoma, recurrent or persistent
ovarian epithelial cancer, fallopian tube cancer, primary
peritoneal cancer, gastrointestinal stromal tumors, colorectal
cancer, gastric cancer, melanoma, glioblastoma multiforme,
non-squamous non-small-cell lung cancer, malignant glioma,
epithelial ovarian cancer, primary peritoneal serous cancer,
metastatic liver cancer, neuroendocrine carcinoma, refractory
malignancy, triple negative breast cancer, HER2-amplified breast
cancer, nasopharageal cancer, oral cancer, biliary tract,
hepatocellular carcinoma, squamous cell carcinomas of the head and
neck (SCCHN), non-medullary thyroid carcinoma, recurrent
glioblastoma multiforme, neurofibromatosis type 1, CNS cancer,
liposarcoma, leiomyosarcoma, salivary gland cancer, mucosal
melanoma, acral/lentiginous melanoma, paraganglioma,
pheochromocytoma, advanced metastatic cancer, solid tumor, triple
negative breast cancer, colorectal cancer, sarcoma, melanoma, renal
carcinoma, endometrial cancer, thyroid cancer, rhabdomysarcoma,
multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal
stromal tumor, mantle cell lymphoma, and refractory malignancy.
[0184] "Solid tumor," as used herein, is understood as any
pathogenic tumor that can be palpated or detected using imaging
methods as an abnormal growth having three dimensions. A solid
tumor is differentiated from a blood tumor such as leukemia.
However, cells of a blood tumor are derived from bone marrow;
therefore, the tissue producing the cancer cells is a solid tissue
that can be hypoxic.
[0185] "Tumor tissue" is understood as cells, extracellular matrix,
and other naturally occurring components associated with the solid
tumor.
[0186] As used herein, the term "isolated" refers to a preparation
that is substantially free (e.g., 50%, 60%, 70%, 80%, 90% or more,
by weight) from other proteins, nucleic acids, or compounds
associated with the tissue from which the preparation is
obtained.
[0187] The term "sample" as used herein refers to a collection of
similar fluids, cells, or tissues isolated from a subject. The term
"sample" includes any body fluid (e.g., urine, serum, blood fluids,
lymph, gynecological fluids, cystic fluid, ascetic fluid, ocular
fluids, and fluids collected by bronchial lavage and/or peritoneal
rinsing), ascites, tissue samples (e.g., tumor samples) or a cell
from a subject. Other subject samples include tear drops, serum,
cerebrospinal fluid, feces, sputum, and cell extracts. In one
embodiment, the sample is removed from the subject. In a particular
embodiment, the sample is urine or serum. In another embodiment,
the sample does not include ascites or is not an ascites sample. In
another embodiment, the sample does not include peritoneal fluid or
is not peritoneal fluid. In one embodiment, the sample comprises
cells. In another embodiment, the sample does not comprise cells.
Samples are typically removed from the subject prior to analysis.
However, tumor samples can be analyzed in the subject, for example,
using imaging or other detection methods.
[0188] The term "control sample," as used herein, refers to any
clinically relevant comparative sample, including, for example, a
sample from a healthy subject not afflicted with cancer, a sample
from a subject having a less severe or slower progressing cancer
than the subject to be assessed, a sample from a subject having
some other type of cancer or disease, a sample from a subject prior
to treatment, a sample of non-diseased tissue (e.g., non-tumor
tissue), a sample from the same origin and close to the tumor site,
and the like. A control sample can be a purified sample, protein,
and/or nucleic acid provided with a kit. Such control samples can
be diluted, for example, in a dilution series to allow for
quantitative measurement of analytes in test samples. A control
sample may include a sample derived from one or more subjects. A
control sample may also be a sample made at an earlier time point
from the subject to be assessed. For example, the control sample
could be a sample taken from the subject to be assessed before the
onset of the cancer, at an earlier stage of disease, or before the
administration of treatment or of a portion of treatment. The
control sample may also be a sample from an animal model, or from a
tissue or cell lines derived from the animal model, of the cancer.
The level in a control sample that consists of a group of
measurements may be determined, e.g., based on any appropriate
statistical measure, such as, for example, measures of central
tendency including average, median, or modal values.
[0189] As used herein, the term "obtaining" is understood herein as
manufacturing, purchasing, or otherwise coming into possession
of.
[0190] As used herein, the term "identical" or "identity" is used
herein in relation to amino acid or nucleic acid sequences refers
to any gene or protein sequence that bears at least 30% identity,
more preferably 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and most
preferably 95%, 96%, 97%, 98%, 99% or more identity to a known gene
or protein sequence over the length of the comparison sequence.
Protein or nucleic acid sequences with high levels of identity
throughout the sequence can be said to be homologous. A
"homologous" protein can also have at least one biological activity
of the comparison protein. In general, for proteins, the length of
comparison sequences will be at least 10 amino acids, preferably
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 175, 200, 250, or at
least 300 amino acids or more. For nucleic acids, the length of
comparison sequences will generally be at least 25, 50, 100, 125,
150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, or
at least 850 nucleotides or more.
[0191] As used herein, "detecting," "detection" and the like are
understood that an assay performed for identification of a specific
analyte in a sample. The amount of analyte or activity detected in
the sample can be none or below the level of detection of the assay
or method.
[0192] The terms "modulate" or "modulation" refer to upregulation
(i.e., activation or stimulation), downregulation (i.e., inhibition
or suppression) of a level, or the two in combination or apart. A
"modulator" is a compound or molecule that modulates, and may be,
e.g., an agonist, antagonist, activator, stimulator, suppressor, or
inhibitor.
[0193] The term "expression" is used herein to mean the process by
which a polypeptide is produced from DNA. The process involves the
transcription of the gene into mRNA and the translation of this
mRNA into a polypeptide. Depending on the context in which used,
"expression" may refer to the production of RNA, or protein, or
both.
[0194] The terms "level of expression of a gene" or "gene
expression level" refer to the level of mRNA, as well as pre-mRNA
nascent transcript(s), transcript processing intermediates, mature
mRNA(s) and degradation products, or the level of protein, encoded
by the gene in the cell.
[0195] As used herein, "level of activity" is understood as the
amount of protein activity, typically enzymatic activity, as
determined by a quantitative, semi-quantitative, or qualitative
assay. Activity is typically determined by monitoring the amount of
product produced in an assay using a substrate that produces a
readily detectable product, e.g., colored product, fluorescent
product, or radioactive product.
[0196] As used herein, "changed as compared to a control" sample or
subject is understood as having a level of the analyte or
diagnostic or therapeutic indicator (e.g., marker) to be detected
at a level that is statistically different than a sample from a
normal, untreated, or control sample control samples include, for
example, cells in culture, one or more laboratory test animals, or
one or more human subjects. Methods to select and test control
samples are within the ability of those in the art. An analyte can
be a naturally occurring substance that is characteristically
expressed or produced by the cell or organism (e.g., an antibody, a
protein) or a substance produced by a reporter construct (e.g.,
3-galactosidase or luciferase). Depending on the method used for
detection the amount and measurement of the change can vary.
Changed as compared to a control reference sample can also include
a change in one or more signs or symptoms associated with or
diagnostic of disease, e.g., cancer. Determination of statistical
significance is within the ability of those skilled in the art,
e.g., the number of standard deviations from the mean that
constitute a positive result.
[0197] "Elevated" or "lower" refers to a patient's value of a
marker relative to the upper limit of normal ("ULN") or the lower
limit of normal ("LLN") which are based on historical normal
control samples. As the level of the marker present in the subject
will be a result of the disease, and not a result of treatment,
typically a control sample obtained from the patient prior to onset
of the disease will not likely be available. Because different labs
may have different absolute results, values are presented relative
to that lab's upper limit of normal value (ULN).
[0198] The "normal" level of expression of a marker is the level of
expression of the marker in cells of a subject or patient not
afflicted with cancer. In one embodiment, a "normal" level of
expression refers to the level of expression of the marker under
normoxic conditions.
[0199] An "over-expression" or "high level of expression" of a
marker refers to an expression level in a test sample that is
greater than the standard error of the assay employed to assess
expression, and is preferably at least 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, 3, 4, 5, 6, 7, 8, 9, or 10 times the expression level of the
marker in a control sample (e.g., sample from a healthy subject not
having the marker associated disease, i.e., cancer). In one
embodiment, expression of a marker is compared to an average
expression level of the marker in several control samples.
[0200] A "low level of expression" or "under-expression" of a
marker refers to an expression level in a test sample that is less
than at least 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 times
the expression level of the marker in a control sample (e.g.,
sample from a healthy subject not having the marker associated
disease, i.e., cancer). In one embodiment, expression of a marker
is compared to an average expression level of the marker in several
control samples.
[0201] As used herein, "binding" is understood as having at least a
10.sup.2 or more, 10.sup.3 or more, preferably 10.sup.4 or more,
preferably 10.sup.5 or more, preferably 10.sup.6 or more preference
for binding to a specific binding partner as compared to a
non-specific binding partner (e.g., binding an antigen to a sample
known to contain the cognate antibody).
[0202] "Determining" as used herein is understood as performing an
assay or using a diagnostic method to ascertain the state of
someone or something, e.g., the presence, absence, level, or degree
of a certain condition, biomarker, disease state, or physiological
condition.
[0203] "Prescribing" as used herein is understood as indicating a
specific agent or agents for administration to a subject.
[0204] As used herein, the terms "respond" or "response" are
understood as having a positive response to treatment with a
therapeutic agent, wherein a positive response is understood as
having a decrease in at least one sign or symptom of a disease or
condition (e.g., tumor shrinkage, decrease in tumor burden,
inhibition or decrease of metastasis, improving quality of life
("OOL"), delay of time to progression ("TTP"), increase of overall
survival ("OS"), etc.), or slowing or stopping of disease
progression (e.g., halting tumor growth or metastasis, or slowing
the rate of tumor growth or metastasis). A response can also
include an improvement in quality of life, or an increase in
survival time or progression free survival.
[0205] The terms "administer," "administering" or "administration"
can include any method of delivery of a pharmaceutical composition
or agent into a subject's system or to a particular region in or on
a subject. In certain embodiments of the invention, an Hsp90
inhibitor is administered intravenously, intramuscularly,
subcutaneously, intradermally, intranasally, orally,
transcutaneously, or mucosally. In a preferred embodiment, an agent
is administered intravenously. Administering can be performed by a
number of people working in concert. Administering an agent
includes, for example, prescribing an agent to be administered to a
subject and/or providing instructions, directly or through another,
to take a specific agent, either by self-delivery, e.g., as by oral
delivery, subcutaneous delivery, intravenous delivery through a
central line, etc.; or for delivery by a trained professional,
e.g., intravenous delivery, intramuscular delivery, intratumoral
delivery, etc.
[0206] As used herein, the term "high concentration" refers to the
concentration of SDC-TRAP that accumulates in target cells of the
invention due to the selective binding of the binding moiety of the
SDC-TRAP to the target protein. In one embodiment, the
concentration is higher than in similar cells that do not
overexpress the target protein, e.g., lung cancer cells as compared
to non-cancerous lung cells. In another embodiment, the
concentration is higher in target cells compared to cells that do
not express, or overexpress, the target protein. In exemplary
embodiments, the high concentration is 1.5, 2, 3, 4, 5, 10, 15, 20,
50, 100, 1000 times or more than cells that are not targeted by the
SDC-TRAP molecules of the invention.
[0207] The term "moiety" refers generally to a portion of a
molecule, which may be a functional group, a set of functional
groups, and/or a specific group of atoms within a molecule, that is
responsible for a characteristic chemical, biological, and/or
medicinal property of the molecule.
[0208] The term "binding moiety" refers to low molecular weight
(e.g., less than about 800, 700, 600, 500, 400, 300, 200, or 100
etc. Dalton) organic compounds, which may serve as a therapeutic or
a regulator of a biological process. Binding moieties include
molecules that can bind to a biopolymer such as protein, nucleic
acid, or polysaccharide and acts as an effector, altering the
activity or function of the biopolymer. Binding moieties can have a
variety of biological functions, serving as cell signaling
molecules, as tools in molecular biology, as drugs in medicine, as
pesticides in farming, and in many other roles. These compounds can
be natural (such as secondary metabolites) or artificial (such as
antiviral drugs); they may have a beneficial effect against a
disease (such as drugs) or may be detrimental (such as teratogens
and carcinogens). Biopolymers such as nucleic acids, proteins, and
polysaccharides (such as starch or cellulose) are not binding
moieties, although their constituent monomers--ribo- or
deoxyribo-nucleotides, amino acids, and monosaccharides,
respectively--are often considered to be. Small oligomers are also
usually considered binding moieties, such as dinucleotides,
peptides such as the antioxidant glutathione, and disaccharides
such as sucrose.
[0209] As used herein, a "protein interacting binding moiety" or
"binding moiety" refers to a binding moiety, or portion thereof,
that interacts with a predetermined target. The interaction is
achieved through some degree of specificity and/or affinity for the
target. Both specificity and affinity is generally desirable,
although in certain cases higher specificity may compensate for
lower affinity and higher affinity may compensate for lower
specificity. Affinity and specificity requirements will vary
depending upon various factors including, but not limited to,
absolute concentration of the target, relative concentration of the
target (e.g., in cancer vs. normal cells), potency and toxicity,
route of administration, and/or diffusion or transport into a
target cell. The target can be a molecule of interest and/or
localized in an area of interest. For example, the target can be a
therapeutic target and/or localized in an area targeted for a
therapy (e.g., a protein that is overexpressed in cancerous cells,
as compared to normal cells). In one particular example, a target
can be a chaperonin protein such as Hsp90 and the binding moiety
can be an Hsp90 binding moiety (e.g., therapeutic, cytotoxic, or
imaging moiety). Preferentially, the binding moiety will enhance,
be compatible with, or not substantially reduce, passive transport
of a conjugate including the binding moiety into a cell, e.g., a
cell comprising a target protein.
[0210] The term "effector moiety" refers to a molecule, or portion
thereof, that has an effect on a target and/or proximally to the
target. In various preferred embodiments, the effector moiety is a
binding moiety, or portion thereof. An effect can include, but is
not limited to, a therapeutic effect, an imaging effect, and/or a
cytotoxic effect. At a molecular or cellular level, an effect can
include, but is not limited to, promotion or inhibition of the
target's activity, labeling of the target, and/or cell death.
Preferentially, the effector moiety will enhance, be compatible
with, or not substantially reduce, passive transport of a conjugate
including the effector moiety into a cell comprising a target.
Different effector moieties can be used together and therapeutics
in accordance with the present invention may include more than one
effector moiety (e.g., two or more different (or same) effector
moieties in a single therapeutic in accordance with the present
invention, two or more different therapeutics in accordance with
the present invention including different effector moieties).
[0211] In some embodiments, the effector moiety is selected from
the group consisting of LY2801653, LY2835219, LY2606368, LY2874455,
LY2090314, LY2940680, LY278544, LY2228820, LY2157299, LY2603618,
LY353381, and trimetrexate.
[0212] The term "small molecule drug conjugate that is trapped
intracellularly" or "binding moiety drug conjugate that is trapped
intracellularly" or "SDC-TRAP" refers to a binding moiety and
effector moiety joined to one another, or acting as if joined to
one another. A binding moiety and effector moiety can be joined
through essentially any chemical or physical force, either directly
(e.g., binding moiety and effector moiety viewed as two moieties on
the same molecule, or a single moiety having both functions) or
through an intermediate (e.g., linker). For example, a binding
moiety and effector moiety can be joined by one or more covalent
bonds, ionic bonds, hydrogen bonds, the hydrophobic effect,
dipole-dipole forces, ion-dipole forces, dipole-induced dipole
forces, instantaneous dipole-induced dipole forces, and/or
combinations thereof. Preferentially, the SDC-TRAP will be capable
of passive and/or active transport into a cell comprising a target.
Moreover, SDC-TRAP molecules of the invention may comprise multiple
effector molecules conjugated to the binding moiety.
[0213] The term "linker" or "linking moiety," as used herein in the
context of binding moiety, effector moieties, and/or SDC-TRAPs
refers to a chemical moiety that joins two other moieties (e.g., a
binding moiety and an effector moiety). A linker can covalently
join a binding moiety and an effector moiety. A linker can include
a cleavable linker, for example an enzymatically cleavable linker.
A linker can include a disulfide, carbamate, amide, ester, and/or
ether linkers.
[0214] In some embodiments, the linker or linking moiety of an
SDC-TRAP can be advantageous when compared to the limited linking
chemistry of antibody-drug conjugates (ADC). For example, unlike
ADCs that are limited by the need to maintain the structutre and/or
stability of an antibody, SDC-TRAPs can use a wider range of
linking chemistries and/or solvents (e.g., that can alter, distort,
or denature an antibody).
[0215] As used herein, a "ligand" is a substance (e.g., a binding
moiety) that can form a complex with a biomolecule. The ligand
and/or formation of the ligand-biomolecule complex can have a
biological or chemical effect, such as a therapeutic effect,
cytotoxic effect, and/or imaging effect.
[0216] As used herein, a "prodrug" is a pharmacological substance
that is administered in an inactive or less than fully active form
and that is subsequently converted to an active pharmacological
agent (i.e., the drug) through a metabolic processes. Prodrugs can
be used to improve how the intended drug is absorbed, distributed,
metabolized, and/or excreted. A prodrug may also be used to improve
how selectively the intended drug interacts with cells or processes
that are not its intended target (e.g., to reduce adverse or
unintended effects of the intended drug, for example a chemotherapy
drug).
[0217] The phrase "Hsp90 ligand or a prodrug thereof" refers
generally to molecules that bind to and in some cases effect Hsp90,
and inactive forms (i.e., prodrugs) thereof. An Hsp90 ligand can be
an "Hsp90 inhibitor," which is understood as a therapeutic agent
that reduces the activity of Hsp90 either by directly interacting
with Hsp90 or by, for example, preventing the formation of the
Hsp90/CDC37 complex such that the expression and proper folding of
at least one client protein of Hsp90 is inhibited. "Hsp90" includes
each member of the family of heat shock proteins having a mass of
about 90-kilodaltons. For example, in humans the highly conserved
Hsp90 family includes cytosolic Hsp90.sup..alpha. and
Hsp90.sup..beta. isoforms, as well as GRP94, which is found in the
endoplasmic reticulum, and HSP75/TRAP1, which is found in the
mitochondrial matrix. As used herein, Hsp90 inhibitors include, but
are not limited to ganetespib, geldanamycin (tanespimycin), e.g.,
IPI-493, macbecins, tripterins, tanespimycins, e.g., 17-AAG
(alvespimycin), KF-55823, radicicols, KF-58333, KF-58332, 17-DMAG,
IPI-504, BBB-021, BIIB-028, PU-H64, PU-H71, PU-DZ8, PU-HZ151,
SNX-2112, SNX-2321, SNX-5422, SNX-7081, SNX-8891, SNX-0723,
SAR-567530, ABI-287, ABI-328, AT-13387, NSC-113497, PF-3823863,
PF-4470296, EC-102, EC-154, ARQ-250-RP, BC-274, VER-50589, KW-2478,
BHI-001, AUY-922, EMD-614684, EMD-683671, XL-888, VER-51047,
KOS-2484, KOS-2539, CUDC-305, MPC-3100, CH-5164840, PU-DZ13,
PU-HZ151, PU-DZ13, VER-82576, VER-82160, VER-82576, VER-82160,
NXD-30001, NVP-HSP990, SST-0201CL1, SST-0115AA1, SST-0221AA1,
SST-0223AA1, novobiocin (a C-terminal Hsp90i, herbinmycin A,
radicicol, CCT018059, PU-H71, or celastrol.
[0218] The term "therapeutic moiety" refers to molecule, compound,
or fragment thereof that is used for the treatment of a disease or
for improving the well-being of an organism or that otherwise
exhibit healing power (e.g., pharmaceuticals, drugs, and the like).
A therapeutic moiety can be a chemical, or fragment thereof, of
natural or synthetic origin used for its specific action against
disease, for example cancer. Therapeutic agents used for treating
cancer may be called chemotherapeutic agents. As described herein,
a therapeutic moiety is preferentially a small molecule. Exemplary
small molecule therapeutics include those that are less than 800
Daltons, 700 Daltons, 600 Daltons, 500 Daltons, 400 Daltons, or 300
Daltons.
[0219] The term "cytotoxic moiety" refers to molecule, compound, or
fragment thereof that has a toxic or poisonous effect on cells, or
that kills cells. Chemotherapy and radiotherapy are forms of
cytotoxic therapy. Treating cells with a cytotoxic moiety can
produce a variety of results--cells may undergo necrosis, stop
actively growing and dividing, or activate a genetic program of
controlled cell death (i.e., apoptosis). Examples of cytotoxic
moieties include, but are not limited to, SN-38, bendamustine, VDA,
doxorubicin, pemetrexed, vorinostat, lenalidomide, irinotecan,
ganetespib, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib,
KW-2189, BUMB2, DC1, CC-1065, adozelesing or fragment(s)
thereof.
[0220] The term "imaging moiety" refers to a molecule, compound, or
fragment thereof that facilitates a technique and/or process used
to create images or take measurements of a cell, tissue, and/or
organism (or parts or functions thereof) for clinical and/or
research purposes. An imaging moiety can produce, for example, a
signal through emission and/or interaction with electromagnetic,
nuclear, and/or mechanical (e.g., acoustic as in ultrasound)
energy. An imaging moiety can be used, for example, in various
radiology, nuclear medicine, endoscopy, thermography, photography,
spectroscopy, and microscopy methods.
[0221] "Pharmaceutical conjugate" refers to a non-naturally
occurring molecule that includes a binding moiety (e.g., an
Hsp90-targeting moiety) associated with an effector moiety, where
these two components may also be covalently bonded to each other
either directly or through a linking group.
[0222] The term "drug" refers to any active agent that affects any
biological process. Active agents that are considered drugs for
purposes of this application are agents that exhibit a
pharmacological activity. Examples of drugs include active agents
that are used in the prevention, diagnosis, alleviation, treatment
or cure of a disease condition.
[0223] By "pharmacologic activity" is meant an activity that
modulates or alters a biological process so as to result in a
phenotypic change, e.g., cell death, cell proliferation etc.
[0224] By "pharmacokinetic property" is meant a parameter that
describes the disposition of an active agent in an organism or
host.
[0225] By "half-life" is meant the time for one-half of an
administered drug to be eliminated through biological processes,
e.g., metabolism, excretion, etc.
[0226] The term "efficacy" refers to the effectiveness of a
particular active agent for its intended purpose, i.e., the ability
of a given active agent to cause its desired pharmacologic
effect.
[0227] Binding Moiety-Effector Moiety Drug Conjugates that are
Trapped Intracellularly (SDC-TRAPs)
[0228] The present invention provides SDC-TRAPs, as well as
SDC-TRAP compositions, kits, and methods of use thereof. SDC-TRAPs
include a binding moiety (e.g., a binding moiety such as a ligand)
conjugated to an effector moiety (e.g., a pharmacological agent
such as a drug or imaging agent). These two moieties can be joined
by a linker, e.g., a covalently-bonded linking group. SDC-TRAPs are
useful in a variety of therapeutic, imaging, diagnostic, and/or
research applications. In one illustrative example of cancer
therapy, an SDC-TRAP can be a pharmaceutical conjugate of an
Hsp90-binding moiety such as an Hsp90 ligand or inhibitor
associated with an effector moiety such as a therapeutic or
cytotoxic agent.
[0229] In various embodiments, an SDC-TRAP can be further
characterized in that the binding moiety (e.g., targeting moiety)
and effector moiety are different, such that the pharmaceutical
conjugate may be viewed as a heterodimeric compound produced by the
joining of two different moieties. In terms of function, SDC-TRAP
molecules have a targeting functionality and effector functionality
(e.g., therapeutic, imaging, diagnostic). These functions are
provided by corresponding chemical moieties that can be different
(or, in some cases, the same). SDC-TRAPs can include any one or
more binding moieties conjugated to any one or more effector
moieties. In some embodiments, a composition or method can include
a combination of two or more binding moeities and/or two or more
effector moieties (e.g., a combination therapy and/or multi target
therapy) embodied in one or more different types of SDC-TRAPs.
[0230] In various embodiments, an SDC-TRAP is further characterized
by its ability to passively diffuse and/or be actively transported
into a target cell of interest. The diffusion and/or transport
properties of the SDC-TRAP can be derived, at least in part, from
ionic, polar, and/or hydrophobic properties of the SDC-TRAP. In
preferred embodiments, the SDC-TRAP enter cells primarily by
passive diffusion. The diffusion and/or transport properties of the
SDC-TRAP can be derived, at least in part, from the molecular
weight of the SDC-TRAP, the binding moiety, the effector moiety,
and/or the similarity in weight between the binding moiety and the
effector moiety. SDC-TRAPs are desirably small, such as in
comparison to antibody-drug conjugates ("ADCs"). For example, the
molecular weight of an SDC-TRAP can be less than about 1600, 1500,
1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, or 400
Daltons. A binding moiety and an effector moiety can each be less
than about 1000, 900, 800, 700, 600, 500, 400, 300, or 200 Daltons.
A binding moiety and an effector moiety can be approximately equal
in size (e.g., differ in weight by less than 400, 350, 300, 250,
200, 150, 100, or 50 Daltons).
[0231] Delivery of an effector molecule by an SDC-TRAP can result
in greater potency compared to administering an untargeted drug
comprising the same effector moiety, for example, because the
SDC-TRAP can be localized at a desired target for an extended
period of time through the association of a binding moiety and its
target. Such localization can cause an effector moiety to be active
and/or released in a target cell and/or tissue over an extended
period of time. This resonance time can be selected through
deliberate design of a linker moiety. In contrast, administration
of the drug by itself in vivo can be more apt to have a shorter
resonance time in a given target cell and/or tissue--if it
traverses into the cell at all--due to the lack of an "anchor"
within the cell.
[0232] SDC-TRAPs, in part because they comprise a targeting moiety
and are relatively small in size, can be efficiently taken up or
internalized by a target cell. Conversely, uptake or
internalization is relatively inefficient for ADCs, which must deal
with limited antigen expression and relatively inefficient
internalization mechanisms for the antibody portion of the
molecule. Hsp90 provides a good illustrative example of a
difference between SDC-TRAPs and conventional ADCs. By way of
comparison, the localization rate of radiolabeled monoclonal
antibodies at a tumor in patients is low, on the order of
0.003-0.08% of the injected dose/g tumor. In contrast, a much
higher accumulation rate (15-20% injected dose/g tumor) has been
measured for SDC-TRAPs in mouse tumor xenografts.
[0233] SDC-TRAP pharmaceutical conjugates in accordance with the
present invention can represent a significant advance over the
state of the art in targeted drugs. SDC-TRAPs have broad
application in many therapeutic, imaging, and diagnostic
application. As discussed above, SDC-TRAPs are advantageously small
in comparison to ADCs, enabling better penetration of solid tumors
and more rapid clearance from normal tissues (e.g., reduced
toxicity). The design of SDC-TRAPs (e.g., a structure-property
relationship) can be established using methods and rationales
within the grasp of those of ordinary skill in the art, and
companion imaging diagnostics for targeted therapies may also
easily be provided, in view of the simpler chemistry involved.
[0234] SDC-TRAPs of the invention are characterized by selective
targeting of SDC-TRAPs to target cells in which a target protein is
overexpressed. This leads to high intracellular concentrations of
SDC-TRAP molecules in target cells as compared to non-targeted
cells. Likewise, SDC-TRAPs of the invention are characterized by
low concentrations of SDC-TRAP in non-targeted cells.
[0235] One illustrative embodiment involves a conjugate of an Hsp90
binding moiety linked to a chelator (i.e., the effector moiety, for
metals such as In or Gd, which conjugate may function as an imaging
agent for the cells/tissues targeted by the conjugate). Another,
illustrative embodiment involves a conjugate of an Hsp90 binding
moiety linked to a chemotherapeutic (i.e., the effector moiety, for
example, SN-38). Alternatively, an illustrative SDC-TRAP is
contemplated wherein an Hsp90 targeting moiety bearing radiolabeled
halogen (e.g., such as an iodine isotope) can serve to image the
cells/tissues targeted by the conjugate, and the effector moiety
can be drug to treat the targeted cells/tissues. The progression of
treatment may therefore be determined by imaging the tissues being
treated and reviewing the images for the presence or absence of the
labeled conjugate. Such embodiments are readily adaptable to
essentially any cancer, or other chemotherapeutic target. Molecular
targets (e.g., interacting with a binding moiety) used to target a
particular cell or tissue can be selected based upon their presence
in the target cell or tissue and/or their relative abundance in the
target cell or tissue (e.g., disease-related versus normal
cells).
[0236] SDC-TRAP molecules of the present invention represent a new
class of drugs. One particular advantage of SDC-TRAPs is that they
can be designed to selectively deliver an effector moiety (e.g., a
chemotherapeutic drug) into a targeted cell because of the relative
overexpression or presence of a binding moiety's molecular target
in the cell. After the binding moiety binds the molecular target,
the effector moiety is thereafter available (e.g., through cleavage
of a linker moiety joining the binding moiety and the effector
moiety) to act upon the cell. Accordingly, SDC-TRAPs employ a
different mechanism from strategies currently used in the art, for
example delivering an Hsp90 inhibitor to a cell using HPMA
copolymer-Hsp90i conjugates, Hsp90i prodrugs, nanoparticle-Hsp90i
conjugates, or micellar methodologies.
[0237] SDC-TRAPs can also described by the formula:
Binding moiety-L-E
[0238] Where "binding moiety" is a protein interacting binding
moiety; L is a conjugation or linking moiety (e.g., a bond or a
linking group); and E is an effector moiety. These elements are
discussed in the context of additional illustrative examples below.
However, while features of each element may be discussed
separately, design and selection of an SDC-TRAP can involve the
interplay and/or cumulative effect of features of each element
(e.g., diffusion, binding, and effect).
[0239] Once SDC-TRAP molecules of the invention enter a target cell
the effector molecule is released from the SDC-TRAP. In one
embodiment, the effector molecule has no activity until it is
released from the SDC-TRAP. Accordingly, once the SDC-TRAP
molecules enter a target cell an equilibrium exists between free
and bound SDC-TRAP molecules. In one embodiment, the effector
moiety is only released from the SDC-TRAP when the SDC-TRAP is not
associated with the target protein. For example, when an SDC-TRAP
molecule is not bound intracellular enzymes can access the linker
region thereby freeing the effector moiety. Alternatively, when
free SDC-TRAP molecules may be able to release effector molecules
through, for example, hydrolysis of the bond or linker that
connects the binding moiety and effector moiety.
[0240] Accordingly, the rate of effector molecule release and the
amount of effector molecule released can be controlled by using
binding moieties that bind to the target protein with different
affinities. For example, binding moieties that bind to the target
protein with lower affinity will be free, resulting in higher
concentrations of unbound intracellular SDC-TRAP, and thereby
resulting in higher concentrations of free effector molecule.
Therefore, in at least one embodiment, irreversibly-binding binding
moieties are incompatible with certain aspects of the invention,
e.g., those embodiments where effector molecule release is based on
free intracellular SDC-TRAP molecules.
[0241] In one embodiment, SDC-TRAPs have favorable safety profiles,
for example, when compared to, for example, the binding moiety or
effector molecule alone. One reason for the increased safety
profile is the rapid clearance of SDC-TRAP molecules that do not
enter into a target cell.
[0242] A number of exemplary SDC-TRAP molecules are set forth in
the examples. Specifically a number of Hsp90-specific SDC-TRAP
molecules are described and used to demonstrate the efficacy of
SDC-TRAP molecules.
[0243] Binding Moieties
[0244] A primary role of a binding moiety is to ensure that the
SDC-TRAP delivers its payload--the effector moiety--to its target
by binding to a molecular target in or on a target cell or tissue.
In this respect, it is not necessary that the binding moiety also
have an effect on the target (e.g., in the case of an
Hsp90-targeting moiety, to inhibit Hsp90 in the manner that Hsp90
is are known to do, that is, exhibit pharmacological activity or
interfere with its function), but in some embodiments, the binding
moiety does have an effect on the target. Accordingly, in various
embodiments, an activity of the SDC-TRAP is due solely to the
effector moiety exerting a pharmacological effect on the target
cell(s), which has been better facilitated by the pharmaceutical
conjugate targeting the target cell(s). In other embodiments, an
activity of the SDC-TRAP is due in part to the binding moiety--that
is, the binding moiety can have an effect beyond targeting.
[0245] The molecular target of a binding moiety may or may not be
part of a complex or structure of a plurality of biological
molecules, e.g., lipids, where the complexes or structures may
include lipoproteins, lipid bilayers, and the like. However, in
many embodiments, the molecular target to which the binding moiety
binds will be free (e.g., cytoplasmic globular protein and/or not
be part of a macromolecular assembly or aggregation). The present
invention can exploit the selectively high presence of a molecular
target in locations of high physiological activity (e.g., Hsp90 in
oncological processes). For example, where a drug target is an
intracellular drug target, a corresponding molecular target (e.g.,
Hsp90) can be present in the cell. Likewise, where a drug target is
an extracellular drug target, a corresponding molecular target
(e.g., Hsp90) can be extracellular, proximal, or associated with
the extracellular cell membrane of the target cell or tissue.
[0246] In various embodiments, a binding moiety can effect a target
cell or tissue (e.g., in the case of an Hsp90-targeting moiety that
in fact inhibits Hsp90, for example, Hsp90i). In such embodiments,
a pharmacological activity of the binding moiety contributes to,
complements, or augments, the pharmacological activity of the
effector moiety. Such embodiments go beyond the advantages
combination therapies (e.g., a cancer combination therapy of Hsp90i
and a second drug such as ganetespib or crizotinib) by providing a
therapy that can be carried out by administration of a single
SDC-TRAP that realizes both the benefits of the combination therapy
and targeting. Other examples of such SDC-TRAPs include conjugates
of an Hsp90i (such as ganetespib) and a second cancer drug such as
LY2801653, LY2835219, LY2606368, LY2874455, LY2090314, LY2940680,
LY278544, LY2228820, LY2157299, LY2603618, LY353381, and
trimetrexate.
[0247] A range of pharmaceutical activities can be achieved by
judicious selection of a binding moiety and an effector moiety. For
example, for treating solid tumors, e.g., colon cancer, high
continuous doses of antimetabolites such as capecitabine or
gemcitabine tend to be required in combination with other drugs. A
conjugate having an Hsp90-targeting moiety with lower binding
affinity or inhibitory activity to Hsp90, e.g., as determined by a
HER2 degradation assay, can be designed to meet this need. Such a
conjugate can comprise an effector moiety that is a strong, potent
antimetabolite such as 5-FU, to afford a high dose of the conjugate
that may be dosed relatively frequently. Such an approach not only
achieves the aim of providing a high dose of an antimetabolite
fragment at the tumor, but also lowers the toxicity of
administering the drug on its own, owing to the plasma stability of
SDC-TRAPs of the invention, and the ability of the Hsp90-targeting
moiety to deliver the antimetabolite to the desired cells or
tissues.
[0248] In embodiments where solid tumors such as SCLC or colorectal
cancer are to be treated with drugs such as topotecan or
irinotecan, only low doses of the drug may be dosed. Due to the
very high intrinsic activity of these drugs, an SDC-TRAP should be
designed to provide a low dose of such drugs at the target tissue.
In this scenario, for example, an Hsp90-targeting moiety having a
higher binding affinity or inhibitory activity to Hsp90 (e.g., as
determined by a HER2 degradation assay) can sufficiently maintain
the presence of the drug in the tissue at a very high level, to
ensure that enough of the drug reaches and is retained by the
desired target tissue due to the low dosing.
[0249] In various illustrative embodiments where a molecular target
of a binding moiety is Hsp90, the binding moiety can be an
Hsp90-targeting moiety, for example a triazole/resorcinol-based
compound that binds Hsp90, or a resorcinol amide-based compound
that binds Hsp90, e.g., ganetespib, AUY-922 or AT-13387. In another
embodiment, the binding moiety may advantageously be an
Hsp90-binding compound of formula (I):
##STR00061##
wherein R.sup.1 may be alkyl, aryl, halide, carboxamide or
sulfonamide; R.sup.2 may be alkyl, cycloalkyl, aryl or heteroaryl,
wherein when R.sup.2 is a 6 membered aryl or heteroaryl, R.sup.2 is
substituted at the 3- and 4-positions relative to the connection
point on the triazole ring, through which a linker L is attached;
and R.sup.3 may be SH, OH, --CONHR.sup.4, aryl or heteroaryl,
wherein when R.sup.3 is a 6 membered aryl or heteroaryl, R.sup.3 is
substituted at the 3 or 4 position.
[0250] In another embodiment, the binding moiety may advantageously
be an Hsp90-binding compound of formula (II):
##STR00062##
wherein R.sup.1 may be alkyl, aryl, halo, carboxamido, sulfonamido;
and R.sup.2 may be optionally substituted alkyl, cycloalkyl, aryl
or heteroaryl. Examples of such compounds include
5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholin-
omethyl)phenyl)-4H-1,2,4-triazole-3-carboxamide and
5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)--
N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide.
[0251] In another embodiment, the binding moiety may advantageously
be an Hsp90-binding compound of formula (III):
##STR00063##
wherein X, Y, and Z may independently be CH, N, O or S (with
appropriate substitutions and satisfying the valency of the
corresponding atoms and aromaticity of the ring); R' may be alkyl,
aryl, halide, carboxamido or sulfonamido; R.sup.2 may be
substituted alkyl, cycloalkyl, aryl or heteroaryl, where a linker L
is connected directly or to the extended substitutions on these
rings; R.sup.3 may be SH, OH, NR.sup.4R.sup.5 AND --CONHR.sup.6, to
which an effector moiety may be connected; R.sup.4 and R.sup.5 may
independently be H, alkyl, aryl, or heteroaryl; and R.sup.6 may be
alkyl, aryl, or heteroaryl, having a minimum of one functional
group to which an effector moiety may be connected. Examples of
such compounds include AUY-922:
##STR00064##
[0252] In another embodiment, the binding moiety may advantageously
be an Hsp90-binding compound of formula (IV):
##STR00065##
wherein R.sup.1 may be alkyl, aryl, halo, carboxamido or
sulfonamido; R.sup.2 and R.sup.3 are independently C.sub.1-C.sub.5
hydrocarbyl groups optionally substituted with one or more of
hydroxy, halogen, C.sub.1-C.sub.2 alkoxy, amino, mono- and
di-C.sub.1-C.sub.2 alkylamino; 5- to 12-membered aryl or heteroaryl
groups; or, R.sup.2 and R.sup.3, taken together with the nitrogen
atom to which they are attached, form a 4- to 8-membered monocyclic
heterocyclic group, of which up to 5 ring members are selected from
O, N and S. Examples of such compounds include AT-13387:
##STR00066##
[0253] In certain embodiments, to enhance the bioavailability or
delivery of the pharmaceutical conjugate, the binding moiety may be
a prodrug of the Hsp90-binding compound. FIG. 1 shows how the
illustrated Hsp90-targeting moiety may be suitably modified at one
or more positions to enhance the physical, pharmacokinetic or
pharmacodynamic properties of the conjugate.
[0254] Specific examples of suitable Hsp90-targeting moieties
include geldanamycins, e.g., IPI-493
##STR00067##
macbecins, tripterins, tanespimycins, e.g., 17-AAG
##STR00068##
KF-55823
##STR00069##
[0255] radicicols, KF-58333
##STR00070##
KF-58332
##STR00071##
[0256] 17-DMAG
##STR00072##
[0258] IPI-504
##STR00073##
BIIB-021
##STR00074##
[0259] BBB-028, PU-H64
##STR00075##
[0260] PU-H71
##STR00076##
[0261] PU-DZ8
##STR00077##
[0262] PU-HZ151
##STR00078##
[0263] SNX-2112
##STR00079##
[0264] SNX-2321
##STR00080##
[0265] SNX-5422
##STR00081##
[0266] SNX-7081
##STR00082##
[0267] SNX-8891, SNX-0723
##STR00083##
[0268] SAR-567530, ABI-287, ABI-328, AT-13387
##STR00084##
[0269] NSC-113497
##STR00085##
[0270] PF-3823863
##STR00086##
[0271] PF-4470296
##STR00087##
[0272] EC-102, EC-154, ARQ-250-RP, BC-274
##STR00088##
[0273] VER-50589
##STR00089##
[0274] KW-2478
##STR00090##
[0275] BHI-001, AUY-922
##STR00091##
[0276] EMD-614684
##STR00092##
[0277] EMD-683671, XL-888, VER-51047
##STR00093##
[0278] KOS-2484, KOS-2539, CUDC-305
##STR00094##
[0279] MPC-3100
##STR00095##
[0280] CH-5164840
##STR00096##
[0281] PU-DZ13
##STR00097##
[0282] PU-HZ151
##STR00098##
[0283] PU-DZ13
##STR00099##
[0284] VER-82576
##STR00100##
[0285] VER-82160
##STR00101##
[0286] VER-82576
##STR00102##
[0287] VER-82160
##STR00103##
[0288] NXD-30001
##STR00104##
[0289] NVP-HSP9900
##STR00105##
[0290] SST-0201CL1
##STR00106##
[0291] SST-0115AA1
##STR00107##
[0292] SST-0221AA1
##STR00108##
[0293] SST-0223AA1
##STR00109##
[0294] novobiocin (a C-terminal Hsp90i.) The selection of other
Hsp90-targeting moieties will be within the grasp of one of
ordinary skill in the art. Likewise, the selection of binding
moieties suitable for other molecular targets and/or other
applications will be within the ability of one of ordinary skill in
the art.
[0295] Additionally Hsp90 targeting moieties can be used to
construct SDC-TRAP molecules for the treatment of inflammation. For
example, binding moieties comprising the compounds shown in Tables
5, 6, and 7 of U.S. Patent Publication 2010/0280032, which is
incorporated herein by reference in its entirety, or compounds of
any formula therein, or tautomers, pharmaceutically acceptable
salts, solvates, clathrates, hydrates, polymorphs or prodrugs
thereof, inhibit the activity of Hsp90 and, thereby cause the
degradation of Hsp90 client proteins. Any of these compounds may be
coupled to an effector molecule to form an SDC-TRAP. The
glucocorticoid receptor is a client protein of Hsp90 and binds to
Hsp90 when it is in the conformation that is able to bind
glucocorticoid ligands such as cortisol. Once a glucocorticoid
binds to GR, the receptor disassociates with Hsp90 and translocates
to the nucleus where it modulates gene expression to reduce
inflammatory responses such as proinflammatory cytokine production.
Thus, glucocorticoids may be given to patients in need of
immunosuppression and patients with inflammatory and autoimmune
disorders. Unfortunately, although glucocorticoids are effective at
relieving inflammation, they have a number of severe side effects
including osteoporosis, muscle wasting, hypertension, insulin
resistance, truncal obesity and fat redistribution, and inhibition
of wound repair. Inhibition of Hsp90 causes changes in GR activity
which results in reduction of inflammatory responses similar to
those seen for glucocorticoids. However, since the mechanism for
reducing inflammation is different than that of glucocorticoids, it
is expected that some or all of the side effects of glucocorticoid
treatment will be reduced or eliminated.
[0296] Effector Moieties
[0297] An effector moiety can be any therapeutic or imaging agent
that can be conjugated to a binding moiety and, in a thus
conjugated state, delivered to a molecular target of the binding
moiety. An effector molecule can, in some cases, require a linking
moiety for conjugation (e.g., cannot be directly conjugated to a
binding moiety). Similarly, an effector molecule can, in some
cases, impede or reduce the ability of the binding moiety and/or
SDC-TRAP to reach a target as long as the SDC-TRAP can still effect
the target. However, in preferred embodiments, an effector moiety
is readily conjugatable and may benefits delivery to, and
effecting, of the target.
[0298] As described in greater detail below, an effector moiety can
comprise a region that can be modified and/or participate in
covalent linkage to a binding moiety without substantially
adversely affecting the binding moiety's ability to bind to its
target. An effector moiety can be a pharmaceutical molecule or a
derivative thereof, which essentially retains activity while
conjugated to a binding moiety. It will be appreciated that drugs
with otherwise good and desirable activity can prove challenging to
administer conventionally (e.g., due to poor bioavailability or
undesirable side-effects in vivo prior to reaching their
target)--such drugs can be "reclaimed" for use as effector moieties
in the SDC-TRAPs of the present invention.
[0299] Examples of effector moieties include: LY2801653, LY2835219,
LY2606368, LY2874455, LY2090314, LY2940680, LY278544, LY2228820,
LY2157299, LY2603618, LY353381, and trimetrexate. Further details
regarding these compounds and their activities are provided
below.
[0300] LY2801653
[0301] LY2801653 (C.sub.30H.sub.22F.sub.2N.sub.6O.sub.3;
N-[3-Fluoro-4-[1-methyl-6-(1H-pyrazol-4-yl)-1H-indazol-5-yloxy]phenyl]-1--
(4-fluorophenyl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carboxamide,
MW 552.5309) is an orally bioavailable multi-kinase inhibitor with
potent activity against MET, MST1R, and other oncoproteins.
LY2801653 has the structure:
##STR00110##
[0302] LY2801653 displays anti-tumor activities in mouse xenograft
models. LY2801653 is currently being studied in human clinical
trials (e.g., NCT01285037) for the treatment of cancer. The HGF/MET
signaling pathway regulates a wide variety of normal cellular
functions that can be subverted to support neoplasia, including
cell proliferation, survival, apoptosis, scattering and motility,
invasion, and angiogenesis. MET over-expression (with or without
gene amplification), aberrant autocrine or paracrine ligand
production, and missense MET mutations are mechanisms that lead to
activation of the MET pathway in tumors and are associated with
poor prognostic outcome. LY2801653 is a type-II ATP competitive,
slow-off inhibitor of MET tyrosine kinase with a dissociation
constant (K.sub.i) of 2 nM, a pharmacodynamic residence time
(K.sub.off) of 0.00132 min.sup.-1 and t.sub.1/2 of 525 min.
LY2801653 demonstrated in vitro effects on MET pathway-dependent
cell scattering and cell proliferation; in vivo anti-tumor effects
in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET
over-expressed (H441) xenograft models; and in vivo vessel
normalization effects. LY2801653 also maintained potency against 13
MET variants, each bearing a single-point mutation. In subsequent
nonclinical characterization, LY2801653 was found to have potent
activity against several other receptor tyrosine oncokinases
including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against
the serine/threonine kinases MKNK1/2. These studies are further
discussed in Yan et al., 2013 (Invest New Drugs. 2013, 31:833-44.
doi: 10.1007/s10637-012-9912-9. Epub 2012 Dec. 29). Assays provided
therein and known in the art can be used to assess the activity of
LY2801653 alone or in a SCD-TRAP conjugate provided herein.
[0303] LY2835219
[0304] LY2835219 (C.sub.27H.sub.32F.sub.2N.sub.8.CH.sub.4O.sub.3S;
2-Pyrimidinamine,
N-[5-[(4-ethyl-1-piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2--
methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl]-, methanesulfonate
(1:1); MW 602.7) is an orally available cell cycle and
cyclin-dependent kinase (CDK) inhibitor that potently and
selectively inhibits of CDK4 and CDK6 with IC.sub.50 of 2 nM and 10
nM, respectively. LY2835219 has the structure:
##STR00111##
[0305] LY2835219 specifically inhibits CDK4 and 6, thereby
inhibiting retinoblastoma (Rb) protein phosphorylation in early G1.
Inhibition of Rb phosphorylation prevents CDK-mediated G1-S phase
transition, thereby arresting the cell cycle in the G1 phase,
suppressing DNA synthesis and inhibiting cancer cell growth.
Overexpression of the serine/threonine kinases CDK4/6, as seen in
certain types of cancer, causes cell cycle deregulation. In vivo
LY2835219 saturates BBB efflux with an unbound plasma IC.sub.50 of
about 95 nM. The percent of dose in brain for LY2835219-MsOH is
0.5-3.9%. In both a subcutaneous and intracranial human
glioblastoma model (U87MG), LY2835219-MsOH suppressed tumor growth
in a dose-dependent manner both as a single agent, and in
combination with temozolomide (Sanchez-Martinez et al., Molecular
Cancer Therapeutics: November 2011; 10: Suppl. 1 doi:
10.1158/1535-7163. TARG-11-B234). Assays provided therein and known
in the art can be used to assess the activity of LY2835219 alone
and in an SDC-TRAP conjugate.
[0306] LY2606368
[0307] LY2606368 (C.sub.18H.sub.19N.sub.7O.sub.2;
5-((5-(2-(3-aminopropoxy)-6-methoxyphenyl)-1H-pyrazol-3-yl)amino)pyrazine-
-2-carbonitrile, MW 365.39) is an inhibitor of checkpoint kinase 1
(chk1) with antineoplastic activity. LY2606368 is being analyzed in
clinical trials for the treatment of advanced cancer (NCT01115790).
LY2606368 has the structure:
##STR00112##
[0308] ChK1 is a key kinase in the DNA damage response signaling
network, including cell cycle checkpoints, DNA repair, apoptosis
and transcription, thus emerging as an attractive target in
anti-cancer therapy. LY2606368, a potent and selective ATP
competitive inhibitor of the Chk1 protein kinase. LY2606368 has
been reported to inhibit Chk1 auto-phosphorylation activated by DNA
damaging agents, and induce phosphorylation of H2AX, a DNA damage
maker in multiple cancer cell lines in vitro. In addition,
LY2606368 has demonstrated potent single agent activity and
potentiates the anti-tumor activity of DNA damaging agents in vivo.
Pancreatic cancer is one of the least curable cancers, with an
approximate 5% overall 5-year survival for all patients. LY2606368
alone has been shown to significantly inhibit the cell
proliferation in a variety of pancreatic cell lines (SW1990,
SU86.86, Bx-PC3, AsPC-1, CFPAC-1, Capan-2, HPAF-II) with SW1990
being the most sensitive (IC50=1.5 nM). In SW1990 subcutaneous
xenograft model, LY2606368 demonstrated to substantial
dose-dependent inhibition of tumor growth. In an SW1990 pancreas
orthotopic model, which represents the local and metastatic growth
pattern seen in pancreas cancer patients, LY2606368 treatment was
demonstrated to result in over 92% inhibition of primary tumor
growth as well as 100% inhibition of metastasis to lymph node,
spleen, and intestine. The anti-tumor effect of LY2606368 treatment
was further demonstrated in comparison with gemcitabine (the
standard of care for pancreatic cancer patients) in SW1990
orthotopic model. The anti-tumor and/or anti-metastasis mechanism
of LY2606368 was also investigated, including phosphorylation of
H2AX, cell proliferation, cell survival, and soft agar
anchorage-independent growth (Wu et al, Antitumor activity of Chk1
inhibitor LY2606368 as a single agent in SW1990 human pancreas
orthotopic tumor model. Cancer Research: Apr. 15, 2012; Volume 72,
Issue 8, Supplement 1 doi: 10.1158/1538-7445.AM2012-1776). Assays
provided therein and known in the art can be used to assess the
activity of LY2606368 alone and in an SDC-TRAP conjugate.
[0309] LY2874455
[0310] LY2874455 (C.sub.21H.sub.19Cl.sub.2N.sub.5O.sub.2;
(R)-(E)-2-(4-(2-(5-(1-(3,5-Dichloropyridin-4-yl)ethoxy)-1H-indazol-3yl)vi-
nyl)-1H-pyrazol-1-yl)ethanol, MW 444.32) is an orally bioavailable
FGF/FGFR inhibitor and is currently being studied in human clinical
trials (e.g., NCT01212107) for the treatment of cancer. LY2874455
has the structure:
##STR00113##
[0311] LY2874455 is active against all 4 FGFRs, with a similar
potency in biochemical assays. It exhibits a potent activity
against FGF/FGFR-mediated signaling in several cancer cell lines
and shows an excellent broad spectrum of antitumor activity in
several tumor xenograft models representing the major FGF/FGFR
relevant tumor histologies including lung, gastric, and bladder
cancers and multiple myeloma, and with a well-defined
pharmacokinetic/pharmacodynamic relationship. LY2874455 also
exhibits a 6- to 9-fold in vitro and in vivo selectivity on
inhibition of FGF- over VEGF-mediated target signaling in mice.
Furthermore, LY2874455 did not show VEGF receptor 2-mediated
toxicities such as hypertension at efficacious doses. Methods
provided in Zhao et al. 2011 (A novel, selective inhibitor of
fibroblast growth factor receptors that shows a potent broad
spectrum of antitumor activity in several tumor xenograft models.
Mol Cancer Ther. 2011 10:2200-10. doi:
10.1158/1535-7163.MCT-11-0306. Epub 2011 Sep. 7.) as well as other
methods known in the art can be used to assess the activity of
LY2874455 alone and in an SDC-TRAP conjugate.
[0312] LY2090314
[0313] LY2090314 (C.sub.28H.sub.25FN.sub.6O.sub.3;
3-[9-Fluoro-2-(piperidin-1-ylcarbonyl)-1,2,3,4-tetrahydropyrrolo[3,2,1-j
k][1,4]benzodiazepin-7-yl]-4-(imidazo[1,2-a]pyridin-3-yl)-2,5-dihydro-1H--
pyrrole-2,5-dione; MW 512.5349) is an intravenously administered
glycogen synthase kinase 3 beta inhibitor. LY2090314 is being
analyzed in clinical trials for acute leukemia (NCT01214603) and
metastatic cancer including metastatic pancreatic cancer in
combination with chemotherapy (NCT01287520, NCT01632306). LY2090314
has the structure:
##STR00114##
[0314] In vitro, LY2090314 is a small molecule, tight-binding,
ATP-competitive inhibitor of GSK3.beta.. Methods provided, for
example, in Watanabe et al. 2012 (Selective growth inhibition by
glycogen synthase kinase-3 inhibitors in tumorigenic HeLa hybrid
cells is mediated through NF-.kappa.B-dependent GLUT3 expression.
Oncogenesis. 1: e21. Published online 2012 Jul. 9. doi:
10.1038/oncsis.2012.21) as well as other methods known in the art
can be used to assess the activity of LY2090314 alone and in an
SDC-TRAP conjugate.
[0315] LY2940680
[0316] LY2940680 (C.sub.26H.sub.24F.sub.4N.sub.6O--ClH;
4-Fluoro-N-methyl-N-[1-[4-(1-methyl-1H-pyrazol-5-yl)phthalazin-1-yl]piper-
idin-4-yl]-2-(trifluoro methyl)benzamide hydrochloride; MW 548.693)
is a smoothened antagonist for the treatment of cancer with
deregulated hedgehog signaling. LY2940680 is being analyzed in
clinical trials for small cell lung cancer (NCT01722292), pediatric
medulloblastoma or rhabdomyosarcoma (NCT01697514), and advanced
cancers (NCT01919398 and NCT01226485). LY2940680 has the
structure:
##STR00115##
[0317] The Hedgehog (Hh) pathway is a highly conserved signaling
system that plays an important role in embryonic development and
tissue homeostasis through regulation of cell differentiation and
proliferation, and deregulated Hh signaling has been implicated in
variety of cancers. Two distinct mechanisms are responsible for
inappropriate and uncontrolled Hh pathway activation in human
malignancies: ligand-dependent, due to over-expression of Hh
ligand, and ligand-independent, resulting from genetic mutations in
pathway components such as Patched (Ptch) and Smoothened (Smo).
Smo, a member of the class F G-protein coupled receptor family, is
a key regulator of Hh signaling pathway, and therefore is an
attractive target for pathway modulation. LY2940680 binds to the
Smo receptor and potently inhibits Hh signaling in Daoy, a human
medulloblastoma tumor cell line, and C3H10T1/2, a mouse mesenchymal
cell line. LY2940680 binds to and inhibits the functional activity
of resistant Smo mutant (D473H) produced by treatment with GDC-0449
(a Smo antagonist from Genentech). Treatment of Ptch.sup.+/-
p53.sup.-/- transgenic mice, which spontaneously develop
medulloblastoma, with oral administration of LY2940680 produced
substantial efficacy and significantly improved their survival.
Magnetic resonance imaging of these mice revealed rapid kinetics of
anti-tumor activity. Immunohistochemistry analysis of
medulloblastoma tumors showed that LY2940680 treatment induced
Caspase-3 activity and reduced proliferation. LY2940680 inhibited
Hh regulated gene expression in the subcutaneous xenograft tumor
stroma and produced significant anti-tumor activity (Bender et al.,
2011. Abstract 2819: Identification and characterization of a novel
smoothened antagonist for the treatment of cancer with deregulated
hedgehog signaling. Cancer Research: 0.8:Supplement 1 doi:
10.1158/1538-7445.AM2011-2819). Assays provided therein and known
in the art can be used to assess the activity of LY2940680 alone
and in an SDC-TRAP conjugate.
[0318] LY2784544 (Gandotinib)
[0319] LY2784544 (Gandotinib; C.sub.23H.sub.25ClFN.sub.7O;
3-(4-Chloro-2-fluorobenzyl)-2-methyl-N-(5-methyl-1H-pyrazol-3-yl)-8-(morp-
holin-4-ylmethyl)imidazo[1,2-b]pyridazin-6-amine; MW 469.942) is an
orally bioavailable JAK2 specific kinase inhibitor for the
treatment of myeloproliferative disorders. LY2784544 is being
analyzed in clinical trials for the treatment of myeloproliferative
disorders including myeloproliferative neoplasms (NCT01134120;
NCT01520220; NCT01594723). LY2784544 has the structure:
##STR00116##
[0320] LY2784544 was discovered and characterized using a
JAK2-inhibition screening assay in tandem with biochemical and
cell-based assays. LY2784544 is selective for JAK2 in vitro and
effectively inhibits JAK2V617F-driven signaling and cell
proliferation in Ba/F3 cells (IC.sub.50=20 and 55 nM,
respectively). In comparison, LY2784544 was much less potent at
inhibiting interleukin-3-stimulated wild-type JAK2-mediated
signaling and cell proliferation (IC50=1183 and 1309 nM,
respectively). In vivo, LY2784544 was shown to inhibit STATS
phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein)
ascitic tumor cells (TED50=12.7 mg/kg) and was shown to
significantly reduce (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden
in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily).
In contrast, LY2784544 was found to have no effect on erythroid
progenitors, reticulocytes or platelets (Ma et al., 2013, Discovery
and characterization of LY2784544, a small-molecule tyrosine kinase
inhibitor of JAK2V617F. Blood Cancer Journal (2013) 3, e109;
doi:10.1038/bcj.2013.6 Published online 12 Apr. 2013). Assays
provided therein and known in the art can be used to assess the
activity of LY2784544 alone and in an SDC-TRAP conjugate.
[0321] LY2228820 (Ralimetinib)
[0322] LY2228820 (Ralimetinib;
C.sub.24H.sub.29FN.sub.6-2CH.sub.4O.sub.3S;
5-[2-tert-Butyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-3-(2,2-dimethylpropy-
l)-3H-imidazo[4,5-b]pyridin-2-amine dimethanesulfonate; MW 612.737)
is a tri-substituted imidazole derivative that is a potent and
ATP-competitive inhibitor of the .alpha. and .beta. isoforms of p38
MAPK in vitro (IC.sub.50=5.3 nM and 3.2 nM, respectively).
LY2228820 is being studied in clinical trials for treatment of
advanced cancer (NCT01393990) and recurrent ovarian cancer
(NCT01663857). LY2228820 has the structure:
##STR00117##
[0323] LY2228820 dimesylate is a tri-substituted imidazole
derivative that is a potent and ATP-competitive inhibitor of the
.alpha. and .beta. isoforms of p38 MAPK in vitro (IC.sub.50=5.3 nM
and 3.2 nM, respectively). This compound displays >1000-fold
selectivity for p38a MAPK versus 179 other kinases tested
(including p38.delta. and .gamma. isoforms). In cell-based assays,
LY2228820 dimesylate was found to potently and selectively inhibit
phosphorylation of MK2 (Thr334) in TNF.alpha.-stimulated HeLa cells
(IC.sub.50=8.1 nM) and anisomycin-induced mouse RAW264.7
macrophages (IC.sub.50=35.3 nM) with no changes in phosphorylation
of p38a MAPK, JNK, ERK1/2, c-jun, ATF2 or cMyc at concentrations up
to 10 .mu.M. LY2228820 dimesylate has been demonstrated to reduce
TNF.alpha. secretion by LPS/IFN.gamma.-stimulated macrophages
(IC.sub.50=6.3 nM). In mice transplanted with B16-F10 melanomas,
phospho-MK2 was found to be effectively inhibited by LY2228820
dimesylate in tumors in a dose-dependent manner (TMED70=19.4
mg/kg). Significant target inhibition (>40% inhibition of
phospho-MK2) was maintained for approx. 4-8 hrs following a single
10 mg/kg oral dose. In a broad range of xenograft models (A-549
NSCLC, SK-OV-3 Ovarian, U-87MG Glioma, MDA-MB-468 Breast),
LY2228820 dimesylate demonstrated significant tumor growth delay
(Campbell, et al., 2011. Abstract B235: Characterization of
LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK
with antitumor activity. Molecular Cancer Therapeutics 10:Suppl. 1.
doi: 10.1158/1535-7163.TARG-11-B235). Assays provided therein and
known in the art can be used to assess the activity of LY2228820
alone and in an SDC-TRAP conjugate.
[0324] LY2157299 (Galunisertib)
[0325] LY2157299 (Galunisertib; C.sub.22H.sub.19N.sub.5O;
4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)qui-
noline-6-carboxamide; MW 369.42) is a potent TGF.beta. receptor I
(T.beta.RI) inhibitor with IC50 of 56 nM. LY2157299 is being
studied in clinical trials for treatment of glioma and glioblastoma
(NCT01220271 and NCT01582269) and metastatic cancer including
metastatic pancreatic cancer (NCT01373164). LY2157299 has the
structure:
##STR00118##
[0326] LY2157299 has been demonstrated to potently inhibit the
TGF.beta. receptor signaling and abolishes the TGF.beta. induced
Smad2 phosphorylation in HUVEC cells. LY2157299 has also shown dose
dependent potentiation of VEGF or bFGF induced cell proliferation
in HUVEC and promotes VEGF induced HUVEC cell migration. LY2157299
has been shown to potentiate angiogenesis in the in vitro
VEGF-stimulated cord formation assay. LY2157299 has been
demonstrated to inhibit TGF-.beta.-mediated SMAD2 activation and
hematopoietic suppression in primary hematopoietic stem cells in a
dose-dependent manner and to stimulate hematopoiesis from primary
MDS bone marrow specimens. In human glioblastoma (GBM) cells,
LY2157299 has been shown to block signaling through the heteromeric
TGF.beta. receptor complex to reduce levels of active,
phosphorylated SMAD. LY2157299 has also been demonstrated to
inhibit HCC cell migration on Laminin-5, Fibronectin, Vitronectin,
Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2.
LY2157299 has also been shown to inhibit HCC migration and cell
growth independently of the expression levels of TGF-.beta.RII
(Dituri et al., 2013. Differential Inhibition of the TGF-.beta.
Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor
LY2157299 and the D10 Monoclonal Antibody against TGF-.beta.
Receptor Type II. PLoS One. 2013 Jun. 27; 8(6):e67109. Print 2013).
Assays provided therein and known in the art can be used to assess
the activity of LY2157299 alone and in an SDC-TRAP conjugate.
[0327] LY2603618 (Rabusertib)
[0328] LY2603618 (Rabusertib; C.sub.18H.sub.22BrN.sub.5O.sub.3;
1-{5-Bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]phenyl}-3-(5-methyl-2-py-
razinyl)urea; MW 436.303, also known as Rabusertib) is an inhibitor
of the cell cycle checkpoint kinase 1 (chk1) and checkpoint kinase
2 (chk2). LY2603618 is being studied in clinical trials for
treatment of advanced or metastatic tumors (NCT01296568), non-small
cell lung cancer (NCT01139775), pancreatic cancer (NCT00839332),
solid tumors NCT01341457); and non-small cell lung cancer
(NCT00988858). Y2603618 has the structure:
##STR00119##
[0329] Chk2, an ATP-dependent serine-threonine kinase, is a key
component in the DNA replication-monitoring checkpoint system and
is activated by double-stranded breaks (DSBs); activated chk2 is
overexpressed by a variety of cancer cell types. Chk2 may prevent
the repair of DNA caused by DNA-damaging agents, thus potentiating
the antitumor efficacies of various chemotherapeutic agents.
Pharmacological inhibition of Chk1 in the absence of p53
functionality leads to abrogation of DNA damage checkpoints and has
been shown preclinically to enhance the activity of many standard
of care chemotherapeutic agents. LY2603618 is a potent and
selective small molecule inhibitor of Chk1 protein kinase activity
in vitro (IC.sub.50=7 nM) and the first selective Chk1 inhibitor to
enter clinical cancer trials. Treatment of cells with LY2603618
produces a cellular phenotype similar to that reported for
depletion of Chk1 by RNAi. Inhibition of intracellular Chk1 by
LY2603618 results in impaired DNA synthesis, elevated H2A.X
phosphorylation indicative of DNA damage and premature entry into
mitosis. When HeLa cells are exposed to doxorubicin to induce a
G2/M checkpoint arrest, subsequent treatment with LY2603618
releases the checkpoint, resulting in cells entering into metaphase
with poorly condensed chromosomes. Consistent with abrogation of
the Chk1 and p53-dependent G2/M checkpoint, mutant TP53 HT-29 colon
cancer cells were found to be more sensitive to gemcitabine when
also treated with LY2603618, while wild-type TP53 HCT116 cells were
not sensitized by LY2603618 to gemcitabine. Treatment of Calu-6
human mutant TP53 lung cancer cell xenografts with gemcitabine
results in a stimulation of Chk1 kinase activity that was inhibited
by co-administration of LY2603618 (King et al., 2013.
Characterization and preclinical development of LY2603618: a
selective and potent Chk1 inhibitor. Investigational New Drugs).
Assays provided therein and known in the art can be used to assess
the activity of LY2603618 alone and in an SDC-TRAP conjugate.
[0330] LY353381 (Arzoxifene)
[0331] LY353581 (Arzoxifene; C.sub.28H.sub.29NO.sub.4S;
2-(4-methoxyphenyl)-3-(4-(2-(piperidin-1-yl)ethoxy)phenoxy)benzo[b]thioph-
en-6-ol MW 475.60) is a selective estrogen receptor modulator.
LY353581 has been studied in clinical trials for the treatment of
breast cancer (NCT00005886; NCT00003428; NCT00034125) and
endrometrial cancer (NCT00003669). LY353581 has the structure:
##STR00120##
[0332] Methods to assess the activity of estrogen receptor
modulators including LY353381 are well known in the art (see, e.g.,
Sato et al., 1998, LY353381.HCl: a novel raloxifene analog with
improved SERM potency and efficacy in vivo. J Pharmacol Exp Ther.
287:1-7). Assays provided therein and known in the art can be used
to assess the activity of LY353381 alone and in an SDC-TRAP
conjugate.
[0333] Trimetrexate
[0334] Trimetrexate (C.sub.19H.sub.23N.sub.5O.sub.3;
(5-methyl-6-(((3,4,5-trimethoxyphenyl)amino)methyl)quinazoline-2,4-diamin-
e; MW 369.42) is a quinazoline derivative and a dihydrofolate
reductase inhibitor. Trimetrexate has been studied in a number of
human clinical trials for conditions including Pneumocystis carinii
pneumonia (NCT00001016), osteosarcoma (NCT00119301), colorectal
cancer, (NCT00003446), pediatric cancers (NCT00002738), and
pancreatic cancer (NCT00002955). Trimetrexate has the
structure:
##STR00121##
[0335] Methods to assess the activity of folate antagonists are
well known in the art (see, e.g., Fry and Jackson, 1987, Biological
and biochemical properties of new anticancer folate antagonists.
Cancer Metastasis Rev. 5:251-70). Assays provided therein and known
in the art can be used to assess the activity of trimetrexate alone
and in an SDC-TRAP conjugate.
[0336] The effector moiety may be obtained from a library of
naturally occurring or synthetic molecules, including a library of
compounds produced through combinatorial means, i.e., a compound
diversity combinatorial library. When obtained from such libraries,
the effector moiety employed will have demonstrated some desirable
activity in an appropriate screening assay for the activity. It is
contemplated that in other embodiments, the pharmaceutical
conjugate may include more than one effector moiety(ies), providing
the medicinal chemist with more flexibility. The number of effector
moieties linked to the binding moiety (e.g., Hsp90-targeting
moiety) will generally only be limited by the number of sites on
the binding moiety (e.g., Hsp90-targeting moiety) and/or any
linking moiety available for linking to an effector moiety; the
steric considerations, e.g., the number of effector moieties than
can actually be linked to the binding moiety (e.g., Hsp90-targeting
moiety); and that the ability of the pharmaceutical conjugate to
bind to the molecular target (e.g., Hsp90 protein) is preserved. An
example of a two-effector moiety pharmaceutical conjugate can be
seen in FIG. 2.
[0337] Conjugation and Linking Moieties
[0338] Binding moieties and effector moieties of the present
invention can be conjugated, for example, through a linker or
linking moiety L, where L may be either a bond or a linking group.
For example, in various embodiments, a binding moiety and an
effector moiety are bound directly or are parts of a single
molecule. Alternatively, a linking moiety can provide a covalent
attachment between a binding moiety and effector moiety. A linking
moiety, as with a direct bond, can achieve a desired structural
relationship between a binding moiety and effector moiety and or an
SDC-TRAP and its molecular target. A linking moiety can be inert,
for example, with respect to the targeting of a binding moiety and
biological activity of an effector moiety.
[0339] Appropriate linking moieties can be identified using the
affinity, specificity, and/or selectivity assays described herein.
Linking moieties can be selected based on size, for example, to
provide an SDC-TRAP with size characteristics as described above.
In various embodiments, a linking moiety can be selected, or
derived from, known chemical linkers. Linking moieties can comprise
a spacer group terminated at either end with a reactive
functionality capable of covalently bonding to the drug or ligand
moieties. Spacer groups of interest include aliphatic and
unsaturated hydrocarbon chains, spacers containing heteroatoms such
as oxygen (ethers such as polyethylene glycol) or nitrogen
(polyamines), peptides, carbohydrates, cyclic or acyclic systems
that may possibly contain heteroatoms. Spacer groups may also be
comprised of ligands that bind to metals such that the presence of
a metal ion coordinates two or more ligands to form a complex.
Specific spacer elements include: 1,4-diaminohexane,
xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid,
ethylenediamine-N,N-diacetic acid,
1,1'-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid),
4,4'-ethylenedipiperidine. Potential reactive functionalities
include nucleophilic functional groups (amines, alcohols, thiols,
hydrazides), electrophilic functional groups (aldehydes, esters,
vinyl ketones, epoxides, isocyanates, maleimides), functional
groups capable of cycloaddition reactions, forming disulfide bonds,
or binding to metals. Specific examples include primary and
secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters,
N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles,
nitrophenylesters, trifluoroethyl esters, glycidyl ethers,
vinylsulfones, and maleimides. Specific linking moieties that may
find use in the SDC-TRAPs include disulfides and stable thioether
moieties.
[0340] In some embodiments, the linker or linking moiety of an
SDC-TRAP can be advantageous when compared to the limited linking
chemistry of antibody-drug conjugates (ADC). For example, unlike
ADCs that are limited by the need to maintain the structure and/or
stability of an antibody, SDC-TRAPs can use a wider range of
linking chemistries and/or solvents (e.g., that can alter, distort,
or denature an antibody).
[0341] In various embodiments, a linking moiety is cleavable, for
example enzymatically cleavable. A cleavable linker can be used to
release an effector moiety inside a target cell after the SDC-TRAP
is internalized. The susceptibility of a linking moiety to cleavage
can be used to control delivery of an effector molecule. For
example, a linking moiety can be selected to provide extended or
prolonged release of an effector moiety in a target cell over time
(e.g., a carbamate linking moiety may be subject to enzymatic
cleavage by a carboxylesterase via the same cellular process used
to cleave other carbamate prodrugs like capecitabine or
irinotecan). In these, and various other embodiments, a linking
moiety can exhibit sufficient stability to ensure good target
specificity and low systemic toxicity, but not so much stability
that it results in lowering the potency and efficacy of the
SDC-TRAP.
[0342] Exemplary linkers are described in U.S. Pat. No. 6,214,345
(Bristol-Myers Squibb), U.S. Pat. Appl. 2003/0096743 and U.S. Pat.
Appl. 2003/0130189 (both to Seattle Genetics), de Groot et al., J.
Med. Chem. 42, 5277 (1999); de Groot et al. J. Org. Chem. 43, 3093
(2000); de Groot et al., J. Med. Chem. 66, 8815, (2001); WO
02/083180 (Syntarga); Carl et al., J. Med. Chem. Lett. 24, 479,
(1981); Dubowchik et al., Bioorg & Med. Chem. Lett. 8, 3347
(1998) and Doronina et al. BioConjug Chem. 2006; Doronina et al.
Nat Biotech 2003.
[0343] Identification and Selection of Targets and Corresponding
SDC-TRAPs
[0344] The present invention provides for a broad class of
pharmacological compounds including an effector moiety conjugated
to an binding moiety directing the effector moiety to a biological
target of interest. While treating cancer using an Hsp90 inhibitor
binding moiety conjugated to a cytotoxic agent effector moiety is
one illustrative example of the present invention, SDC-TRAPs are
fundamentally broader in terms of their compositions and uses.
[0345] In various embodiments, the broad class of SDC-TRAP
pharmacological compounds that are directed to biological targets
have the following properties:
[0346] the biological target (a cell and/or tissue target of
interest, e.g., a tumor) should be effectible by an effector
moiety, and the effector moiety should be known or developed for
the biological target (e.g., chemotherapeutic agent for the tumor);
the biological target should be associated with a molecular target
(e.g., biomolecule, capable of being specifically bound, that is
uniquely represented in the biological target) that specifically
interacts with a binding moiety, and the binding moiety should be
known or developed for the molecular target (e.g., ligand for the
biomolecule); and the effector moiety and binding moiety should be
amenable to coupling and should essentially retain their respective
activity after coupling. Furthermore, the conjugate should be
capable of reaching and interacting with the molecular target, and
in clinical applications should be suitable for administration to a
subject (e.g., a subject can tolerate a therapeutically effective
dose). Examples of therapeutic molecular targets (i.e., binding
moiety binding partners) for various conditions/disease states
include LY2801653, LY2835219, LY2606368, LY2874455, LY2090314,
LY2940680, LY278544, LY2228820, LY2157299, LY2603618, LY353381, and
trimetrexate.
[0347] Imaging Moieties, and Diagnostic and Research
Applications
[0348] In various embodiments, the effector moiety is an imaging
moiety--that is, a molecule, compound, or fragment thereof that
facilitates a technique and/or process used to create images or
take measurements of a cell, tissue, and/or organism (or parts or
functions thereof) for clinical and/or research purposes. An
imaging moiety can produce, for example, a signal through emission
and/or interaction with electromagnetic, nuclear, and/or mechanical
(e.g., acoustic as in ultrasound) energy. An imaging moiety can be
used, for example, in various radiology, nuclear medicine,
endoscopy, thermography, photography, spectroscopy, and microscopy
methods.
[0349] Imaging studies can be used, for example, in a clinical or
research setting to diagnose a subject, select a subject for
therapy, select a subject for participation in a clinical trial,
monitor the progression of a disease, monitor the effect of
therapy, to determine if a subject should discontinue or continue
therapy, to determine if a subject has reached a clinical end
point, and to determine recurrence of a disease. Imaging studies
can be used, for example, to conduct research to identify effective
interacting moieties and/or effector moieties and/or combinations
thereof, to identify effective dosing and dose scheduling, to
identify effective routes of administration, and to identify
suitable targets (e.g., diseases susceptible to particular
treatment).
[0350] Methods of Making Pharmaceutical Conjugates
[0351] The pharmaceutical conjugates, i.e., SDC-TRAPs, of the
invention may be prepared using any convenient methodology. In a
rational approach, the pharmaceutical conjugates are constructed
from their individual components, binding moiety, in some cases a
linker, and effector moiety. The components can be covalently
bonded to one another through functional groups, as is known in the
art, where such functional groups may be present on the components
or introduced onto the components using one or more steps, e.g.,
oxidation reactions, reduction reactions, cleavage reactions and
the like. Functional groups that may be used in covalently bonding
the components together to produce the pharmaceutical conjugate
include: hydroxy, sulfhydryl, amino, and the like. The particular
portion of the different components that are modified to provide
for covalent linkage will be chosen so as not to substantially
adversely interfere with that components desired binding activity,
e.g., for the effector moiety, a region that does not affect the
target binding activity will be modified, such that a sufficient
amount of the desired drug activity is preserved. Where necessary
and/or desired, certain moieties on the components may be protected
using blocking groups, as is known in the art, see, e.g., Green
& Wuts, Protective Groups in Organic Synthesis (John Wiley
& Sons) (1991).
[0352] Alternatively, the pharmaceutical conjugate can be produced
using known combinatorial methods to produce large libraries of
potential pharmaceutical conjugates which may then be screened for
identification of a bifunctional, molecule with the pharmacokinetic
profile. Alternatively, the pharmaceutical conjugate may be
produced using medicinal chemistry and known structure-activity
relationships for the targeting moiety and the drug. In particular,
this approach will provide insight as to where to join the two
moieties to the linker.
[0353] A number of exemplary methods for preparing SDC-TRAP
molecules are set forth in the examples. As one of skill in the art
will understand, the exemplary methods set forth in the examples
can be modified to make other SDC-TRAP molecules. Additional
exemplary methods for preparation and testing of SDC-TRAPs are
provided in U.S. application Ser. No. 13/843,771 which is
incorporated herein by reference in its entirety.
[0354] Methods of Use, Pharmaceutical Preparations, and Kits
[0355] The pharmaceutical conjugates find use in treatment of a
host condition, e.g., a disease condition. In these methods, an
effective amount of the pharmaceutical conjugate is administered to
the host, where "effective amount" means a dosage sufficient to
produce the desired result, e.g., an improvement in a disease
condition or the symptoms associated therewith. In many
embodiments, the amount of drug in the form of the pharmaceutical
conjugate that need be administered to the host in order to be an
effective amount will vary from that which must be administered in
free drug form. The difference in amounts may vary, and in many
embodiments may range from two-fold to ten-fold. In certain
embodiments, e.g., where the resultant modulated pharmacokinetic
property or properties result(s) in enhanced activity as compared
to the free drug control, the amount of drug that is an effective
amount is less than the amount of corresponding free drug that
needs to be administered, where the amount may be two-fold, usually
about four-fold and more usually about ten-fold less than the
amount of free drug that is administered.
[0356] The pharmaceutical conjugate may be administered to the host
using any convenient means capable of producing the desired result.
Thus, the pharmaceutical conjugate can be incorporated into a
variety of formulations for therapeutic administration. More
particularly, the pharmaceutical conjugate of the present invention
can be formulated into pharmaceutical compositions by combination
with appropriate, pharmaceutically acceptable carriers or diluents,
and may be formulated into preparations in solid, semi-solid,
liquid or gaseous forms, such as tablets, capsules, powders,
granules, ointments, solutions, suppositories, injections,
inhalants and aerosols. As such, administration of the
pharmaceutical conjugate can be achieved in various ways, including
oral, buccal, rectal, parenteral, intraperitoneal, intradermal,
transdermal, intracheal, etc., administration. In pharmaceutical
dosage forms, the pharmaceutical conjugate may be administered
alone or in combination with other pharmaceutically active
compounds.
[0357] The subject methods find use in the treatment of a variety
of different disease conditions. In certain embodiments, of
particular interest is the use of the subject methods in disease
conditions where an active agent or drug having desired activity
has been previously identified, but which active agent or drug does
not bind to its target with desired affinity and/or specificity.
With such active agents or drugs, the subject methods can be used
to enhance the binding affinity and/or specificity of the agent for
its target.
[0358] The specific disease conditions treatable by with the
subject bifunctional compounds are as varied as the types of drug
moieties that can be present in the pharmaceutical conjugate. Thus,
disease conditions include cellular proliferative diseases, such as
neoplastic diseases, autoimmune diseases, central nervous system or
neurodegenerative diseases, cardiovascular diseases, hormonal
abnormality diseases, infectious diseases, and the like.
[0359] By treatment is meant at least an amelioration of the
symptoms associated with the disease condition afflicting the host,
where amelioration is used in a broad sense to refer to at least a
reduction in the magnitude of a parameter, e.g., symptom,
associated with the pathological condition being treated, such as
inflammation and pain associated therewith. As such, treatment also
includes situations where the pathological condition, or at least
symptoms associated therewith, are completely inhibited, e.g.,
prevented from happening, or stopped, e.g., terminated, such that
the host no longer suffers from the pathological condition, or at
least the symptoms that characterize the pathological
condition.
[0360] Methods of use of the invention extend beyond strict
treatment of a disease. For example, the invention includes uses in
a clinical or research setting to diagnose a subject, select a
subject for therapy, select a subject for participation in a
clinical trial, monitor the progression of a disease, monitor the
effect of therapy, to determine if a subject should discontinue or
continue therapy, to determine if a subject has reached a clinical
end point, and to determine recurrence of a disease. The invention
also includes uses in conducting research to identify effective
interacting moieties and/or effector moieties and/or combinations
thereof, to identify effective dosing and dose scheduling, to
identify effective routes of administration, and to identify
suitable targets (e.g., diseases susceptible to particular
treatment).
[0361] A variety of hosts are treatable according to the subject
methods. Generally such hosts are "mammals" or "mammalian," where
these terms are used broadly to describe organisms which are within
the class Mammalia, including the orders carnivore (e.g., dogs and
cats), rodentia (e.g., mice, guinea pigs, and rats), and primates
(e.g., humans, chimpanzees, and monkeys). In many embodiments, the
hosts will be humans.
[0362] The invention provides kits for treating a subject in need
thereof comprising at least one SDC-TRAP and instruction for
administering a therapeutically effective amount of the at least
one SDC-TRAP to the subject, thereby treating the subject. The
invention also provides kits for imaging, diagnosing, and/or
selecting a subject comprising at least one SDC-TRAP and
instruction for administering an effective amount of at least one
SDC-TRAP to the subject, thereby imaging, diagnosing, and/or
selecting the subject.
[0363] Kits with unit doses of the pharmaceutical conjugate,
usually in oral or injectable doses and often in a storage stable
formulation, are provided. In such kits, in addition to the
containers containing the unit doses, an informational package
insert describing the use and attendant benefits of the drugs in
treating pathological condition of interest will be included.
Preferred compounds and unit doses are those described herein
above.
[0364] The invention also provides methods for treatment of a
disease or disorder in which the subject to be treated is selected
for treatment based on the presence of, or the overexpression of, a
particular protein. For example, subjects may be selected for
treatment of cancer based on the presence of greater the normal
levels of Hsp90. In this case, subjects would be administered an
SDC-TRAP that comprises a binding moiety that selectively binds to
Hsp90.
[0365] The invention provides methods of treating or preventing an
inflammatory disorder in a subject, comprising administering to the
subject an effective amount of a compound represented by any one of
formula (I) through (LXXII), or any embodiment thereof, or a
compound shown in Table 5, 6, or 7 as disclosed in U.S. Patent
Publication 2010/0280032. In one embodiment, the compound or
binding moiety or SDC-TRAP may be administered to a human to treat
or prevent an inflammatory disorder. In another embodiment, the
inflammatory disorder is selected from the group consisting of
transplant rejection, skin graft rejection, arthritis, rheumatoid
arthritis, osteoarthritis and bone diseases associated with
increased bone resorption; inflammatory bowel disease, ileitis,
ulcerative colitis, Barrett's syndrome, Crohn's disease; asthma,
adult respiratory distress syndrome, chronic obstructive airway
disease; corneal dystrophy, trachoma, onchocerciasis, uveitis,
sympathetic ophthalmitis, endophthalmitis; gingivitis,
periodontitis; tuberculosis; leprosy; uremic complications,
glomerulonephritis, nephrosis; sclerodermatitis, psoriasis, eczema;
chronic demyelinating diseases of the nervous system, multiple
sclerosis, AIDS-related neurodegeneration, Alzheimer's disease,
infectious meningitis, encephalomyelitis, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis viral or
autoimmune encephalitis; autoimmune disorders, immune-complex
vasculitis, systemic lupus and erythematodes; systemic lupus
erythematosus (SLE); cardiomyopathy, ischemic heart disease
hypercholesterolemia, atherosclerosis, preeclampsia; chronic liver
failure, brain and spinal cord trauma. In another embodiment, an
SDC-TRAP, or a compound shown in Table 5, 6, or 7 as disclosed in
U.S. Patent Publication 2010/0280032, is administered with an
additional therapeutic agent. In another embodiment, the additional
therapeutic agent may an anti-inflammatory agent.
[0366] In one embodiment, an SDC-TRAP that is administered to a
subject but does not enter a target cell is rapidly cleared from
the body. In this embodiment, the SDC-TRAP that does not enter a
target cell is rapidly cleared in order to reduce the toxicity due
to the components of the SDC-TRAP, the degradation products of the
SDC-TRAP or the SDC-TRAP molecule. Clearance rate can be determined
by measuring the plasma concentration of the SDC-TRAP molecule as a
function of time.
[0367] Likewise, SDC-TRAP molecules that enter non-targeted cells
by passive diffusion rapidly exit the non-targeted cell or tissue
and are either eliminated from the subject or proceed to enter and
be retained a targeted cell or tissue. For example, an SDC-TRAP
that is intended to treat tumor cells and is targeted to tumor
cells that overexpress, for example, Hsp90 will accumulate
selectively in tumor cells that overexpress Hsp90. Accordingly,
very low levels of this exemplary SDC-TRAP will be present in
non-tumor tissue such as normal lung tissue, heart, kidney, and the
like. In one embodiment, the safety of the SDC-TRAP molecules of
the invention can be determined by their lack of accumulation in
non-targeted tissue. Conversely, the safety of the SDC-TRAP
molecules of the invention can be determined by their selective
accumulation in the targeted cells and/or tissue.
EXAMPLES
[0368] The following examples, which are briefly summarized and
then discussed in turn below, are offered by way of illustration
and not by way of limitation.
[0369] Example 1 presents the synthesis of exemplary SDC-TRAPs.
[0370] Example 2 presents methods for assessing the targeted
delivery of exemplary SDC-TRAPs.
[0371] Example 3 presents an exemplary assay for HER2
degradation.
[0372] Example 4 presents an exemplary assay for toxicity of
SDC-TRAPs.
[0373] Example 5 presents an exemplary assay for the stability of
SDC-TRAPs in plasma.
[0374] Example 6 presents an exemplary assay for anti-tumor
activity of SDC-TRAPs in xenogeneic human tumor mouse model.
[0375] Examples 7 and 8 present exemplary assays for IC.sub.50
value determinations for SDC-TRAPs.
[0376] Example 9 presents an exemplary Hsp90a binding assay.
[0377] Example 10 presents an exemplary HER2 degradation assay.
[0378] Example 11 presents an exemplary cytotoxicity assay.
[0379] Example 12 presents an exemplary plasma stability
protocol.
[0380] Example 13 presents an exemplary tissue distribution
study.
[0381] Example 14 presents an exemplary assay for use of SDC-TRAPs
for prevention and treatment of skin cancers and actinic
keratosis
[0382] Example 15 presents an exemplary assay for determining the
permeability of SDC-TRAP molecules
[0383] Example 16 presents an exemplary assay for determination of
pharmacodynamics of an SDC-TRAP in a tumor model.
[0384] Example 17 presents SDC-TRAP-309 structure and
synthesis.
[0385] Example 18 presents SDC-TRAP-310 structure and
synthesis.
[0386] Example 19 presents SDC-TRAP-311 structure and
synthesis.
[0387] Example 20 presents SDC-TRAP-312 structure and
synthesis.
[0388] Example 21 presents SDC-TRAP-313 structure and
synthesis.
[0389] Example 22 presents SDC-TRAP-314 structure and
synthesis.
[0390] Example 23 presents SDC-TRAP-315 structure and
synthesis.
[0391] Example 24 presents SDC-TRAP-316 structure and
synthesis.
[0392] Example 25 presents SDC-TRAP-317 structure and
synthesis.
[0393] Example 26 presents SDC-TRAP-318 structure and
synthesis.
[0394] Example 27 presents SDC-TRAP-319 structure and
synthesis.
[0395] Example 28 presents SDC-TRAP-320 structure and
synthesis.
[0396] Example 29 presents SDC-TRAP-321 structure and
synthesis.
[0397] Example 30 presents SDC-TRAP-322 structure and
synthesis.
[0398] Example 31 presents SDC-TRAP-323 structure and
synthesis.
[0399] Example 32 presents SDC-TRAP-324 structure and
synthesis.
[0400] Example 33 presents SDC-TRAP-325 structure and
synthesis.
[0401] Example 34 presents SDC-TRAP-326 structure and
synthesis.
[0402] Example 35 presents SDC-TRAP-327 structure and
synthesis.
[0403] Example 36 presents SDC-TRAP-328 structure and
synthesis.
[0404] Example 37 presents SDC-TRAP-329 structure and
synthesis.
[0405] Example 38 presents SDC-TRAP-330 structure and
synthesis.
[0406] Example 39 presents SDC-TRAP-331 structure and
synthesis.
[0407] Example 40 presents SDC-TRAP-332 structure.
[0408] Example 41 presents SDC-TRAP-333 structure.
[0409] Example 42 presents SDC-TRAP-334 structure.
Example 1--Exemplary Synthetic Methods for SDC-TRAPs
[0410] SDC-TRAPs of an exemplary embodiment may be prepared in the
following manner:
[0411] (1) HDC of LY2801653
##STR00122##
[0412] (2) HDC of CDK4/6 dual inhibitor LY2835219
##STR00123##
[0413] (3) HDC of LY2606368
##STR00124##
[0414] (4) HDC of FGFR 1-3 Inhibitor LY2874455
##STR00125##
[0415] (5) HDC of GSK3beta Inhibitor LY-2090314
##STR00126##
[0416] (6) HDC of Gandotinib
##STR00127##
[0417] (7) HDC of Ralimetinib--the MAPK14 (MAPK p38 Alpha)
Inhibitor:
##STR00128##
[0418] (8) Galunisertib HDC
##STR00129##
[0419] (9) Rabusertib HDC
##STR00130##
[0420] (10) HDC of Arzoxifene
##STR00131##
[0421] (11) HDC of Trimetrexate:
##STR00132##
[0422] The conjugate synthesis schemes are exemplary. Other
synthetic methods, linkers, and binding moieties can be linked to
the above effector molecules.
Example 2--Tissue Distribution and Metabolism of an Exemplary Hsp90
Binding Moiety
[0423] The ability of Hsp90-targeting moieties to penetrate solid
tumors and exhibit rapid clearance from normal tissues for reduced
toxicity is illustrated in the following tissue distribution study
with a compound, ganetespib, which may be used as an Hsp90 binding
moiety.
[0424] Tissue distribution of ganetespib in female CD-1 nu/nu mice
bearing RERF human NSCLC xenografts
[0425] Objectives:
[0426] To confirm the distribution of ganetespib in blood, livers,
kidneys, brains, hearts, lungs and tumors after IV administration
of ganetespib to female CD-1 nu/nu mice bearing RERF human NSCLC
xenografts, and to examine metabolic profiles of ganetespib in
plasma, red blood cells, and above tissues.
[0427] Study Outline:
[0428] Test Articles: ganetespib
[0429] Animals: female CD-1 nu/nu mice bearing RERF human NSCLC
xenografts (N=3/group)
[0430] Route: IV
[0431] Dosage: 50 mg/kg
[0432] Dose level: 10 mL/kg
[0433] Formulation: 10% DMSO, 18% Cremophor RH40, 3.6% dextrose
solution (DRD)
[0434] Bleeding time points: 5 min, 6, 24 hr
[0435] Collected tissues: blood (plasma and red blood cells (RBC)),
liver, kidneys, brain, heart, lung, tumor
[0436] Method
[0437] Sample preparation Plasma and RBC
[0438] Protein precipitation: 50 .mu.L of 10 times diluted plasma
or RBC+150 .mu.L ACN (10 mM NH.sub.4OAc), vortexed and centrifuged
at 10000 rpm for 8 min; 150 .mu.L supernatant+150 .mu.L water (10
mM NH.sub.4OAc)
[0439] Other Tissues
[0440] Protein precipitation: 100 .mu.L homogenized tissue (1:3
tissue: PBS buffer)+100 .mu.L ACN (10 mM NH.sub.4OAc), vortexed and
centrifuged at 10000 rpm for 8 min
[0441] Bioanalysis
[0442] HPLC (Chem Station)
[0443] Column: Agilent Zorbax Eclipse XDB-C.sub.18, 4.6.times.150
mm, 5 .mu.m
[0444] Mobile phase: A: water containing 10 mM NH.sub.4OAc; B: 95%
ACN containing 10 mM NH.sub.4OAc
[0445] Gradient: 95/5 A/B to 5/95 A/B in 10 min, total run time 15
min
[0446] Flow rate: 1 mL/min
[0447] Column temp.: 40.degree. C.
[0448] Wavelength: 254 nm
[0449] Injection volume: 100 .mu.L
[0450] Calibration curve range:
[0451] Plasma: 1-50 .mu.M (linear regression; R.sup.2=0.9901);
LLOQ=1 .mu.M
[0452] RBC: 1-50 .mu.M (linear regression; R.sup.2=0.9987); LLOQ=1
.mu.M
[0453] Kidney: 1-100 .mu.M (linear regression; R.sup.2=1.0000);
LLOQ=1 .mu.M
[0454] Lung: 1-100 .mu.M (linear regression; R.sup.2=1.0000);
LLOQ=1 .mu.M
[0455] Heart: 1-100 .mu.M (linear regression; R.sup.2=0.9998);
LLOQ=1 .mu.M
[0456] Liver: 1-100 .mu.M (linear regression; R.sup.2=1.0000);
LLOQ=1 .mu.M
[0457] Tumor: 0.1-10 .mu.M (linear regression; R.sup.2=1.0000);
LLOQ=0.1 .mu.M
[0458] LC-MS/MS (Q-Trap4000)
[0459] Polarity: positive (ESI)
[0460] Column: Phenomenex Synergi, 2.1.times.50 mm, 4 .mu.m
[0461] Mobile phase: A: water containing 0.1% HCOOH; B: ACN
containing 0.1% HCOOH
[0462] Gradient: 60/40 A/B to 5/95 A/B in 0.5 min, total run time 4
min
[0463] Flow rate: 0.5 mL/min
[0464] Column temp.: room temperature
[0465] Injection volume: 20 .mu.L
[0466] Calibration curve range:
[0467] Plasma: 2.5-500 nM (linear regression; R.sup.2=0.9994);
LLOQ=2.5 nM
[0468] RBC: 2.5-500 nM (linear regression; R.sup.2=0.9998);
LLOQ=2.5 nM
[0469] Kidney: 2.5-500 nM (linear regression; R.sup.2=0.9993);
LLOQ=2.5 nM
[0470] Lung: 2.5-500 nM (linear regression; R.sup.2=0.9993);
LLOQ=2.5 nM
[0471] Heart: 2.5-500 nM (linear regression; R.sup.2=0.9997);
LLOQ=2.5 nM
[0472] Liver: 2.5-500 nM (linear regression; R.sup.2=1.0000);
LLOQ=2.5 nM
[0473] 0.5-5 .mu.M (linear regression; R.sup.2=0.9970); LLOQ=0.5
.mu.M
[0474] Brain: 2.5-500 nM (linear regression; R.sup.2=0.9998);
LLOQ=2.5 nM
[0475] 0.5-5 .mu.M (linear regression; R.sup.2=0.9992); LLOQ=0.5
.mu.M
[0476] Results
[0477] Formulations
[0478] The dosing solution was confirmed to have 98.1% accuracy by
HPLC.
[0479] Tissue Distribution
[0480] The concentrations of ganetespib in plasma, RBC and the
tissues are summarized in FIG. 1 at each time point.
[0481] The mean plasma concentration of ganetespib at 5 min after
IV injection was 160 highest among all the tissues studied.
Thereafter, the plasma ganetespib concentration declined quickly
and at 6 hr, it was 0.12 .mu.M. At 24 hr, it was below the lower
limit of quantitation (LLOQ, <2.5 nM).
[0482] After IV injection, ganetespib was widely distributed to the
normal tissues analyzed. At 5 min, the highest concentration of
ganetespib among the tissues was observed in kidney (57.8 followed
by liver (46.3 .mu.M) and heart (36.2 In brain, 0.53 .mu.M of
ganetespib was detected at 5 min, which was the lowest among the
tissues. In all the normal tissues, the concentrations of
ganetespib decreased quickly.
[0483] Although the concentration of ganetespib in tumor at 5 min
(2.35 M) was lower than that in plasma and most of the other
tissues studied, it remained relatively constant up to 24 hr (0.85
.mu.M at 24 hr). However, the in vitro IC.sub.50 values of
ganetespib are small, and the tumor concentration of ganetespib at
24 hr was significantly higher than IC.sub.50 of in vitro HER2
assays (.about.30 nM). Thus, the prolonged efficacy is expected
even after ganetespib was cleared from the blood stream.
[0484] The mean concentration of ganetespib in plasma was about 10
times higher than that in RBC at 5 min time point, indicating that
ganetespib tends to stay in plasma rather than in RBCs.
Conclusion
[0485] Ganetespib appeared to persist longer in tumor than in
plasma or any other tissues studied. The results from this study
suggest that ganetespib also has a higher binding affinity to Hsp90
from tumor cells than Hsp90 from normal cells, and that it is
possible for ganetespib to modulate relative protein concentrations
of Hsp90 and its client proteins selectively in tumors. The plasma
concentrations of ganetespib did not correlate to the
concentrations in tumor.
TABLE-US-00001 TABLE 1 Concentrations of ganetespib in tissues:
Test Articles ganetespib Structure ##STR00133## Species CD-1-nu/nu
female mice Tumor RERF human NSCLC Route IV Dosage 50 mg/kg
Formulation DRD plasma RBC tumor liver kidneys brain heart lung
Time (.mu.g/mL) (.mu.g/mL) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g)
(.mu.g/g) (.mu.g/g) 5 min 58.4 6.00 0.86 16.9 21.1 0.19 13.2 9.24 6
hr 0.04 No data 0.29 0.14 0.06 0.07 0.05 0.05 24 hr <LLOQ 0.003
0.31 0.005 0.01 0.04 0.00 0.00 plasma RBC tumor liver kidneys brain
heart lung Time (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M)
(.mu.M) (.mu.M) 5 min 160 16.5 2.35 46.3 57.8 0.53 36.2 25.4 6 hr
0.12 N/A 0.80 0.39 0.15 0.18 0.13 0.14 24 hr <LLOQ 0.007 0.85
0.01 0.02 0.12 0.00 0.005
SUMMARY
[0486] Ganetespib was widely distributed to various tissues. The
compound was accumulated in tumor relative to the plasma and other
tissues, indicating the higher binding affinity of this compound to
Hsp90 in tumor than Hsp90 in other tissues. The metabolite M2,
which was previously thought to be human-specific, was also
detected in mouse liver, kidney, heart and lung, but not in plasma.
M2 does not seem to be excreted into blood stream in mice and
possibly in other species as well.
Example 3--HER2 Degradation Assay
[0487] This example illustrates how a HER2 degradation assay may be
used as a test to determine and select Hsp90-targeting moieties
suitable for use in SDC-TRAPs of the invention, and further
illustrates the ability of SDC-TRAPs to target cells preferentially
expressing Hsp90. Such a test may further be used to determine the
Hsp90 binding ability of SDC-TRAPs of the invention, as well as
through competitive binding assays and cell-based Hsp90 client
protein degradation assays known in the art.
[0488] Degradation of HER2 in Cells after Treatment with an
SDC-TRAP of the invention
[0489] Method 1:
[0490] BT-474 cells are treated with 0.5 .mu.M, 2 .mu.M, or 5 .mu.M
of 17-AAG (a positive control) or 0.5 .mu.M, 2 .mu.M, or 5 .mu.M of
an Hsp90-targeting moiety or conjugate of the invention overnight
in DMEM medium. After treatment, each cytoplasmic sample is
prepared from 1.times.10.sup.6 cells by incubation of cell lysis
buffer (#9803, Cell Signaling Technology) on ice for 10 minutes.
The resulting supernatant used as the cytosol fractions is
dissolved with sample buffer for SDS-PAGE and run on a SDS-PAGE
gel, blotted onto a nitrocellulose membrane by using semi-dry
transfer. Non-specific binding to nitrocellulose is blocked with 5%
skim milk in TBS with 0.5% Tween at room temperature for 1 hour,
then probed with anti-HER2/ErB2 mAb (rabbit IgG, #2242, Cell
Signaling) and anti-Tubulin (T9026, Sigma) as housekeeping control
protein. HRP-conjugated goat anti-rabbit IgG (H+L) and
HRP-conjugated horse anti-mouse IgG (H+L) are used as secondary Ab
(#7074 #7076, Cell Signaling) and LumiGLO reagent, 20.times.
Peroxide (#7003, Cell Signaling) is used for visualization. The
Hsp90 client protein HER2 is degraded when cells are treated with
Hsp90-targeting moieties or SDC-TRAPs of the invention. 0.5 .mu.M
of 17-AAG, a known Hsp90 inhibitor used as a positive control,
causes partial degradation of HER2.
[0491] Method 2:
[0492] BT-474 cells are plated in the interior 60 wells of a 96
well black clear bottom plate (20,000 cells/well) in DMEM medium,
with DMEM media in the surrounding 36 wells, and incubated at
37.degree. C. with 5% CO.sub.2 overnight. On the second day,
concentration response curve source plates are produced (10 point,
3-fold dilution of compounds in DMSO) followed by a 1:30 dilution
in an intermediate dilution plate containing DMEM. Compound is
transferred from the intermediate plate to the cell plate at a
dilution of 1:10. The cells are then incubated at 37.degree. C.
with 5% CO.sub.2 for 24 hours.
[0493] Cells are then fixed in 4% phosphate-buffered
paraformaldehyde for 30 minutes at room temperature and then
permeabilized by washing five times with 0.1% Triton X-100 in PBS
for 5 minutes at room temperature on a shaker. Cells are blocked
with Odyssey Blocking Buffer (LI-COR, #927-40000) on a shaker at
room temperature for 1.5 hours, followed by incubation with HER2
antibody (CST, #2165) diluted 1:400 in blocking buffer overnight on
a shaker at 4.degree. C. Cells are washed five times with 0.1%
Tween-20 in PBS for 5 minutes at room temperature on a shaker and
incubated with fluorescently-labeled secondary antibody (LI-COR,
#926-32211) diluted 1:1000 in blocking buffer, and DRAQ5 nuclear
stain (Biostatus Limited, #DRAQ5) diluted 1:10,000, at room
temperature on a shaker for 1 hour. Cells are washed 5 times with
0.1% Tween-20 in PBS for 5 minutes at room temperature on a shaker
and imaged on a LI-COR Odyssey imaging station. The raw data is
normalized to DRAQ5 and the HER2 EC.sub.50 is calculated using
XLfit.TM.. The above procedures are utilized to generate HER2
degradation data, which show the ability of various SDC-TRAPs to
target cells preferentially expressing Hsp90.
Example 4--Cytotoxicity Assay
[0494] Cell Lines.
[0495] Human H3122 NSCLC cells are obtained and grown in RPMI in
the presence of fetal bovine serum (10%), 2 mM L-glutamine and
antibiotics (100 IU/ml penicillin and 100 .mu.g/ml streptomycin,
Sigma Aldrich.) Cells are maintained at 37.degree. C., 5% CO.sub.2
atmosphere.
[0496] Cell Viability Assays.
[0497] Cell viability is measured using the CellTiter-Glo.RTM.
assay (Promega). In brief, cells are plated in 96-well plates in
triplicate at optimal seeding density (determined empirically) and
incubated at 37.degree. C., 5% CO.sub.2 atmosphere for 24 hr prior
to the addition of drug or vehicle (0.3% DMSO) to the culture
medium. At the end of the assay, CellTiter-Glog is added to the
wells per manufacturer's recommendation, shaken for two minutes and
incubated for 10 minutes at room temperature. Luminescence (0.1
sec) is measured with a Victor II microplate reader (Perkin
Elmer.RTM.) and the resulting data are used to calculate cell
viability, normalized to vehicle control.
[0498] Cells as described above are treated with exemplary
SDC-TRAPs and their viability is determined as above as well. These
assays demonstrate that the SDC-TRAP effector and targeting
components maintain the cytotoxic activity against cancer cells in
the conjugate.
Example 5--Assessing Stability of SDC-TRAPs in Human and Mouse
Plasma
[0499] SDC-TRAPs are incubated in human and mouse plasma for 2 h at
37.degree. C. and assayed for integrity at 0.25, 0.5, 1 and 2 h to
assess the stability of the SDC-TRAP, e.g., as compared to the
effector and targeting compounds. Compound stability is determined
using any of a number of routine methods including, for example
chromatography and mass spectrometry.
Example 6--Assessing Anti-Tumor Activity of SDC-TRAPS in Xenogeneic
Human Tumor Mouse Model
[0500] Xenogeneic human tumor mouse models are well known in the
art in which human tumors are implanted in immunocompetent mice.
Various agents are tested for their efficacy in promoting tumor
shrinkage. Human tumor cell lines for use in such models include,
but are not limited to HCT-116 colon cancer model and MCF-7 breast
cancer model. An exemplary study is provided for non-specified
SDC-TRAP-A and SDC-TRAP-B which include effector A and effector B,
respectively, in combination with the same binding domain. It is
understood that variations of the experimental design can be
readily envisioned to analyze the efficacy of SDC-TRAPs.
[0501] A xenograft tumor model is used to evaluate the anti-tumor
efficacy of SDC-TRAP-A and SDC-TRAP-B. The tumor model is
established by transplanting equivalent numbers of human tumor
cells, e.g., HCT-116 cells, into mice and testing the effect of the
SDC-TRAPs on tumor volume and change in tumor volume.
[0502] HCT 116 human colorectal adenocarcinoma tumor cells are
purchased from ATCC. The cells are maintained in vitro as a
monolayer culture in McCoy's 5a Medium. Fetal bovine serum is added
to the medium. The final concentration of fetal bovine serum is
10%. Cells are cultured at 37.degree. C. and 5% CO.sub.2. The tumor
cells are routinely sub-cultured twice weekly by trypsin-EDTA
treatment. Cells in an exponential growth phase are harvested and
counted for tumor inoculation.
[0503] 100 18-22 g, 5-7 week old, female BALB/cA nude mice are
inoculated with the HCT 116 cells (2.0.times.10.sup.6, 1:1 with
Matrigel) subcutaneously on the back of each animal (0.1 mL/mouse).
When the average tumor volume reaches about 150-250 mm.sup.3, 60 of
the inoculated mice are selected based on tumor growth and randomly
grouped into 6 treatment groups (10 mice per group) according to
the following table. Mice that are not put on treatment are
euthanized. Animals are sourced through Shanghai SINO-British
SIPPR/BK Lab Animal Ltd, Shanghai, China. Mice are treated as set
forth in the table below:
TABLE-US-00002 Treatment Groups Dosage Dosage Animal Dosage Conc.
Vol. Route Dosing Groups Number Treatment (mg/kg) (mg/mL) (mL/kg)
of Adm. Schedule 1 10 Vehicle NA NA 10 IV Q7D x 3 2 10 SDC-TRAP-A
200 20 10 IV Q7D x 3 3 10 SDC-TRAP-A 100 10 10 IV Q7D x 3 4 10
SDC-TRAP-B 94 9.4 10 IV Q7D x 3 5 10 Unconjugated effector-A 67 6.7
10 IV Q7D x 3 6 10 Unconjugated effector-B 67 6.7 10 IV Q7D x 3 7
Unconjugated binding 100 10 10 IV Q7D x 3
[0504] Dose Preparation & Treatment Schedule
[0505] The dosing solutions of SDC-TRAP-A, SDC-TRAP-B, uncongugated
effector moiety, and unconjugated binding moiety (e.g., ganetespib)
are prepared according to an appropriate formulation protocol and
administered using routine methods.
[0506] Evaluation of Anti-Tumor Activity
[0507] During the treatment period, the implanted tumors are
measured by caliper twice per week. The tumors are measured for the
maximum width (X) and length (Y) and the tumor volumes (V) are
calculated using the formula: V=(X.sup.2Y)/2. The differences in
the tumor volume between the control and treatment groups are
analyzed for significance using the unpaired two-tailed Student's
t-test. P<0.05 is considered to be statistically significant.
The animal body weights are also weighed and recorded twice per
week. The changes in tumor volume and body weight in the days
following compound treatment are determined. The effects of the
SDC-TRAPs are compared to each other, to the effector and binding
moiety that are used to generate that SDC-TRAPs, and untreated
control to identify an SDC-TRAP with improved efficacy in
inhibiting tumor growth without adverse effects (e.g., excessive
weight loss).
Example 7--Determination of IC.sub.50 by Assessing the Effects of
Various SDC-TRAPs on Tumor Shrinkage
[0508] An appropriate cell line, e.g., H3122 cells, are seeded into
in 96-well plates at 7,500 cells/90 .mu.L/well, and were incubated
for 24 hours. SDC-TRAPs, plus a binding moiety alone (e.g.,
ganetespib) as a control, are serially diluted in dimethylsulfoxide
(DMSO) (e.g., 3000 nM, 1000 nM, 333.3 nM, 111.1 nM, 37.0 nM, 12.3
nM) into each of six wells of each 96-well plate. To each well of a
first set of duplicate plates plates, 145 .mu.L of media was added,
and the cells are incubated. The wells of a second set of duplicate
plates (pulsed plates) are incubated for 1 hour, then the wells are
rinsed 2.times. with fresh media to remove the conjugate, and 145
.mu.L of media is then added to each washed well. IC.sub.50 is
determined visually under a microscope after 48 hours and 72 hours
drug-exposure. Also at the 72 hour time point, 504, of the cell
culture supernatant is mixed with 504, of CellTiter-Glo.RTM. and
the luminescence is determined, from which an IC.sub.50 for each
conjugate was calculated.
Example 8--IC.sub.50 of Continuous and Pulsed Exposure to
SDC-TRAPs
[0509] IC.sub.50 toxicity is determined for 72 hour continuous
exposure to SDC-TRAPs run in triplicate, and for duplicate pulse
exposure (1 hour "pulse" exposure to conjugate compound, followed
by 72 hour incubation in conjugate-free media) using H3211 cells,
according to the protocol set forth in Example 7.
Example 9--Hsp90.sup..alpha. Binding Assay Protocol
[0510] An Hsp90a fluorescence assay kit from BPS Bioscience (Cat
#50294) containing Hsp90 recombinant enzyme, FITC-labeled
geldanamycin, assay buffer and a low binding 384-well plate is used
to assay Hsp90a binding. Dithiothreitol (DTT) (Cat #D0643) and
bovine serum albumin (BSA) (Cat #A2153) were obtained from
Sigma-Aldrich. Fluorescence polarization is measured using a
PHERAstar.RTM. microplate reader (BMG LABTECH GmbH, Ortenberg,
Germany.)
[0511] The SDC-TRAP and control compounds are diluted to 1 mM in
DMSO and loaded into a compound dilution plate to make 3-fold
dilutions yielding a total of 8 concentrations. 1 .mu.L of compound
is transferred from the dilution plate to the low binding assay
plate provided in the assay kit. 5 mL of Hsp90.sup..alpha. binding
solution is prepared having a final concentration of 7 ng/.mu.L
Hsp90', 5 nM FITC-labeled geldanamycin, 2 mM DTT and 0.1 mg/mL BSA.
49 .mu.L of binding solution is added to each microplate well,
incubated at room temperature for 1 hour, then read using the
PHERAstar.RTM. microplate reader. The high control sample contains
no compound plus Hsp90a; the low control sample contains no
compound and no Hsp90a. Percent inhibition is calculated using high
control as 100% and low control as 0% inhibition. The IC.sub.50 is
calculated using GraphPad Prism.RTM. 4 software.
Example 10--HER2 Degradation Assay with BT-474 Cell Line
[0512] HER2 has emerged as a key target for anticancer drugs due to
its intrinsic involvement in the
phosphatidylinositol-3-kinase-Akt/protein kinase B (PI3K-Akt) and
the mitogen-activated protein kinase (MAPK) pathways, both of which
suppress apoptosis and promote tumor cell survival, gene
transcription, angiogenesis, cellular proliferation, migration,
mitosis, and differentiation. The degradation of HER2 is a measure
of efficacy of anticancer therapeutics that target Hsp90.
Accordingly, the SDC-TRAP molecules of the invention that comprise
a binding moiety that binds Hsp90 are tested in the following HER2
degradation assay.
[0513] BT-474 cells (human breast cancer cell line ATCC HTB-20) are
obtained from ATCC and seeded into 12-well tissue culture plates at
0.2.times.10.sup.6/1.8 mL/well. The cells are incubated for more
than 6 hours at 37.degree. C. in DMEM+10% FBS, +1% P/S, +1.5 g/L
sodium bicarbonate. Each test compound is titrated in 4-fold
dilutions from 5 .mu.M to 78 nM with DMSO and 200 .mu.L of the
titration is added to each well of the cell plate. The DMSO final
concentration is 0.2%. Cells are incubated overnight at 37.degree.
C. in 5% CO.sub.2.
[0514] Media is decanted from the plate, cells are washed 1.times.
in PBS. 400 .mu.L trypsin (EDTA) per well is added, and the cells
are incubated for 2 to 3 minutes. Cells are collected into FACS
tubes containing 1 ml culture medium to neutralize the trypsin and
are centrifuged for 5 minutes at 1200 rpm. Supernatant is decanted
and the cells are resuspended in 5 .mu.L FITC (anti HER2/nu)/200
.mu.L staining buffer (1.times.PBS+1% FBS+0.05% Sodium Azide)/tube.
Controls are 5 .mu.L IgG isotype control and staining buffer only.
Tubes are incubated for 30 minutes in the dark at room temperature.
1 mL staining buffer is added to each tube and the tubes are
centrifuged for 6 minutes at 1200 rpm. The supernatant is decanted
and 300 .mu.L staining buffer is added to each tube, which was
store at 4.degree. C. fpr FACS (cytometer) analysis. The cytometer
readout is normalized and the potency of each compound is evaluated
with IC.sub.50 calculated with XLfit.TM. software.
Example 11--Cytotoxicity Assay with Cancer Cell Lines
[0515] Cytotoxicity of SDC-TRAP molecules is determined in multiple
cancer cell line such as the three exemplary cancer cell lines
provided herein. 5000 cells/100 .mu.L/well of human breast cancer
cell line BT-474 (ATCC #HTB-20) and human urinary bladder cancer
cell line SW780 (ATCC# CRL-2169) and 5000 cells/well of human
urinary bladder cancer cell line RT-112 are seeded into 96-well
flat-bottom tissue cultures plates and incubated overnight at
37.degree. C. in 5% CO.sub.2. BT-474 and SW780 cells are cultured
in DMEM+10% FBS, +1% P/S, +1.5 g/L sodium bicarbonate; RT-112 cells
are cultured in EMEM+10% FBS, +1% P/S. SDC-TRAP-0178 is titrated by
10-fold dilutions from 10 .mu.M to 10 nM and added to the plate at
10 .mu.L/well. Final concentration of DMSO in the cell plate is
0.25%. The plates are incubated for 72 hours at 37.degree. C. in 5%
CO.sub.2. 80 .mu.L of CellTiter-Glo is added to each well, followed
by room temperature incubation in the dark for 15 minutes. Cell is
determined by luminescence. IC.sub.50 is calculated using XLfit.TM.
software.
Example 12--Tissue Distribution Extraction Procedure for SDC-TRAP
Tumor Samples
[0516] SDC-TRAP molecules have the ability to be specifically
targeted to desired cells. For example, SDC-TRAP molecules can be
targeted to tumors and tumor cells in order to treat cancer. This
example sets forth a protocol to extract the SDC-TRAP molecules of
the invention from tumor samples.
[0517] A 150 ng/mL solution of an SDC-TRAP in methanol is prepared
using an internal spiking solution (500 .mu.g/mL SDC-TRAP-0002 in
DMSO). Using the 10 mM stock solutions of the SDC-TRAP molecule and
its Hsp90i binding moiety and effector moiety in DMSO, spiking
solutions are prepared at 0.025, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100,
250, and 500 .mu.M in DMSO. 5 .mu.L of each spiking solution is
added to a 96-deep well plate.
[0518] Quality control standards are prepared from 5 .mu.L of 0.1,
1, and 10 .mu.M calibration standard spiking solution added in
triplicate into 96-deep well plate and adding 50 .mu.L of matrix
(plasma or homogenized tumor).
[0519] To prepare test samples, test plasma is diluted as needed
using blank plasma. Tumor samples are pulverized in liquid
nitrogen, weighed, and homogenized in PBS at 5.times. volume to
sample weight. 50 .mu.L of unknown plasma or homogenized tumor
sample is mixed with 5 .mu.L of DMSO. The samples are extracted by
precipitating calibration standards, QC standards, and unknown
samples with 200 .mu.L of internal standard solution. The samples
are mixed by vortex at room temperature for approximately 1.5
minutes, then centrifuge at 2-8.degree. C. 150 .mu.L of supernatant
is collected and 25 .mu.L of water added. Samples are mixed and
analyzed by LC-MS/MS.
Example 13--Plasma Stability Protocol for SDC-TRAP Compounds
[0520] An exemplary assay to analyze plasma stability of SDC-TRAPs
is provided. 150 ng/mL solution of SDC-TRAP-0002 in methanol is
prepared using the internal standard spiking solution. This
solution is used to precipitate all plasma samples in the study.
200 .mu.L is pipetted into a 96 deepwell plate over dry ice. 10
.mu.L of 1 mM stock in DMSO is added to a 1.5 mL microfuge tube,
then 990 .mu.L of plasma. Samples are mixed by vortex, then 50
.mu.L of each sample is added in triplicate to a 96-well plate
containing internal standard solution. This was designated the 0
hour time point sample. 250 .mu.L of the remaining plasma sample is
added to each of four 96 deepwell plates--one per time point.
Samples are incubated at 37.degree. C. with gentle shaking for
0.25, 0.5, and 1 hour. After each time point, one plate of each
sample is removed from the shaker and placed on wet ice for
approximately 2 minutes. 50 .mu.L plasma aliquots (in triplicate)
are added to the deepwell plate containing internal standard
solution. After the last time point is extracted, the 96 deepwell
plate is vortexed, then centrifuged at 2-8.degree. C. 150 .mu.L of
supernatant is collected and 25 .mu.L of water was added. Samples
are mixed and analyzed by LC-MS/MS.
Example 14--Identification and Use of SDC-TRAP for Prevention and
Treatment of Skin Cancers and Actinic Keratosis
[0521] Skin cancers (neoplasms) are named after the type of skin
cell from which they arise. Skin cancers include basal cell
carcinoma, squamous cell carcinoma, malignant melanomas, and
Bowen's disease. Actinic keratosis can be, but is not always, a
precursor to squamous cell carcinoma.
[0522] Drugs used for the treatment of skin cancer are selected
based on the type and severity of the skin cancer. Superficial,
non-melanoma skin cancers can be treated with topical agents,
either alone or in combination with surgery or other therapeutic
interventions. Such agents include, but are not limited to,
retinoids, 5-fluorouracil, diclofenac, ingenol mebutate, and
imiquimod. Topical delivery permits administration of the
chemotherapeutic agent directly to the site of the tumor or skin
lesion. However, the delivery of active agents into the skin can be
challenging. Moreover, many topical therapeutic agents can be
irritating to the skin, resulting in scar formation, further
inhibiting the delivery of the active agent to the site.
[0523] Imiquimod
3-(2-methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]trideca-1(9),2(6),4,7,-
10,12-hexaen-7-amine) is a patient-applied cream used to treat
certain diseases of the skin, including skin cancers (basal cell
carcinoma, Bowen's disease, superficial squamous cell carcinoma,
some superficial malignant melanomas, and actinic keratosis) as
well as genital warts (Condylomata acuminata). Imiquimod and its
analogs activate the immune system by activating immune cells
through the toll-like receptor 7 (TLR7), commonly involved in
pathogen recognition. Imiquimod can be used in combination with one
or more drugs used for the treatment of skin diseases to make an
SDC-TRAP molecule.
[0524] An imiquimod SDC-TRAP molecule can be formed, for example,
using any known linker, such as those provided herein, with the
desired effector molecule. The specific linker and conjugation
method used will depend, for example, on the chemical nature of the
effector molecule.
[0525] Assays to determine the cytotoxicity of the imiquimod
SDC-TRAP molecules are performed using methods similar to those
provided in Example 4. Cell viability assays are performed on
non-transformed cells, preferably skin cells, to identify SDC-TRAPs
with acceptable toxicities, preferably compounds with toxicity that
is not greater than either of the parent compounds. Cytotoxicity
and skin irritation assays are also performed, for example, on pig
skin, which is frequently used as a model for human skin in
toxicity/irritation assays, using routine methods.
[0526] Imiquimod SDC-TRAP molecules are also tested to confirm that
their efficacy is not inhibited by the formation of the conjugate.
A number of skin cancer cell lines are well known in the art. Dose
response curves are generated to demonstrate the efficacy of
imiquimod SDC-TRAP molecules in killing cancer cells. Preferably,
the imiquimod SDC-TRAP molecules are more effective at killing skin
cancer cells than imiquimod or the effector molecule alone.
[0527] Methods to assess pharmacokinetic and pharmacodynamic
properties of an agent are well known in the art. As noted above,
pig skin is frequently used as a model for human skin, both in
toxicity/irritation assays, but also in assaying uptake and
delivery of agents into skin layers and cells. Topical formulations
of imiquimod, the effector molecule, and imiquimod SDC-TRAP
molecules are assayed for uptake, transport through the skin, and
persistence in the skin using routine methods.
[0528] Having identified a imiquimod SDC-TRAP molecule with the
desired activity, cytotoxicity, pharmacokinetic properties, and
improved tissue delivery, the SDC-TRAPs are tested for their
efficacy in an appropriate animal model of skin cancer. Animal
models of skin cancer are well known in the art. For example,
xenograph tumor models using squamous cell carcinoma, basal cell
carcinoma, or melanoma cell lines are used with subcutaneously
implanted tumors. Topical formulations of imiquimod, the effector
molecule, and imiquimod SDC-TRAP molecules are applied. The
activity of the conjugate is compared to the activity of each
imiquimod and the effector molecule alone. Imiquimod SDC-TRAP
molecules having one or more improved properties as compared to
either of the parent molecules are further characterized in other
animal systems and humans.
[0529] The SDC-TRAPs are found to have one or more improved
properties in the treatment of humans including, but not limited
to, decreased toxicity, improved dosing schedule, or alternate
route of administration.
Example 15--Determining the Permeability of SDC-TRAP Molecules
[0530] In order to test the ability SDC-TRAP molecules of the
invention to enter cells, an artificial membrane permeability assay
("PAMPA") can be used. PAMPAs are useful tool for predicting in
vivo drug permeability for drugs that enter cells by passive
transport mechanisms. LC/MS is used in conjunction with PAMPA
assays to determine the ability of the SDC-TRAP molecules of the
invention to permeate cells.
[0531] Pre-coated PAMPA plates are warmed to room temperature for
at least 30 minutes prior to adding assay components.
[0532] Stock solutions are prepared with the SDC-TRAP molecules to
be tested. In order to make a working solution, either 50 .mu.L of
100 .mu.M Stock in DMSO+950 .mu.L of PBS or 50 .mu.L of 200 .mu.M
stock is added to 96 deep well plate, resulting in a 5 .mu.M final
concentration or a 10 .mu.M final concentration, respectively.
3004, of the working solution containing each compound to be tested
is added to the appropriate well of a donor PAMPA plate. 200 .mu.L
of PBS is added into the corresponding wells of an acceptor PAMPA
plates.
[0533] The acceptor plate is lowered onto the donor plate and
allowed to incubate for five hours. After five hours, a 50 .mu.L
aliquot is removed from each well of each plate and added into a
new 96 deep-well plate.
[0534] 100 .mu.L of methanol containing an internal standard is
added to each aliquot and analyzed by LC/MS.
[0535] In order to calculate the permeability for each SDC-TRAP
molecule and the control molecules, the following formula was
used:
[0536] Permeability (in unit of cm/s):
P e = - ln [ 1 - C A ( t ) / C equilibrium ] A * ( 1 / V D + 1 / V
A ) * t ##EQU00001## C equilibrium = C D ( t ) * V D + C A ( t ) *
V A V D + V A ##EQU00001.2##
[0537] Mass Retention:
R = 1 - [ C D ( t ) * V D + C A ( t ) * V A ] C 0 * V D
##EQU00002##
[0538] C.sub.0=initial compound concentration in donor well
(mM)
[0539] C.sub.D (t)=compound concentration in donor well at time t.
(mM)
[0540] C.sub.A (t)=compound concentration in acceptor well at time
t. (mM)
[0541] V.sub.D=donor well volume=0.3 mL
[0542] V.sub.A=acceptor well volume=0.2 mL
[0543] A=filter area=0.3 cm.sup.2
[0544] t=incubation time=18000 s (5 h)
Example 16--Pharmacodynamics of SDC-TRAP in Xenograft Tumors
[0545] SDC-TRAPs can be identified for potent and durable antitumor
activity in particular tumor types suggesting that the drug is
slowly cleaved over its residence time in the tumor to provide long
term activity. To determine whether these effects are through the
binding moiety, e.g., an HSP90 binding moiety that results in Hsp90
inhibition, effector agent activity, i.e., anticancer activity,
e.g., kinase inhibitor activity, or both, the stability of Hsp90
client proteins as well as the phosphorylation of the kinase
substrate as a readout for effector molecule activity. Readouts can
be assessed in various tissues as well as tumor tissue to determine
changes in tissue distribution and clearance as well as activity
(e.g., to understand mechanism of reduced toxicity due to better
tumor targeting).
[0546] Kinetics of Hsp90 inhibition and kinase activity inhibition
are assayed at time points relative to the known metabolism of the
component agents of the SDC-TRAP. Selection of time points and
concentrations of the various agents is well within the ability of
those of skill in the art. The analysis demonstrates the relative
activity of each of the components of the SDC-TRAP and changes in
kinetics and/or tissue distribution as a result of the conjugate
formation.
Example 17--SDC-TRAP-309 Structure and Synthesis
##STR00134##
[0548] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-309
(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tet-
rahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropan-2-yl-
)carbamoyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-tr-
iazole-3-carboxamide) is shown below.
[0549] To a mixture of
4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrof-
uran-2-yl)pyrimidin-2(1H)-one (527 mg, 2 mmol) and
(S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (434 mg, 2
mmol) in DMF (5 mL) was added Et.sub.3N (0.92 mL, 6.6 mmol)
followed by PyBOP (1.24 g, 2.4 mmol). The reaction mixture was
stirred at room temperature overnight then concentrated. The crude
residue was treated with aq. NaHCO.sub.3 then extracted with ethyl
acetate. The organic layer was separated, washed with brine and
dried over Na.sub.2SO.sub.4. The mixture was filtered, concentrated
and the residue purified twice by ISCO using DCM/MeOH (0-10%) as
eluent to afford 260 mg (28%) of product.
C.sub.19H.sub.28F.sub.2N.sub.4O.sub.7: 462.19; found: 463.3
(M+H.sup.+).
[0550] The above product (260 mg) was dissolved in DCM (3 mL) and
treated with TFA (3 mL). The reaction mixture was stirred at room
temperature for 1 h, concentrated and dried on high vacuum to get
product as a gummy oil.
Example 18--SDC-TRAP-310 Structure and Synthesis
##STR00135##
[0552] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-310,
(4-(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-methyl-1-ox-
obutan-2-yl)carbamoyl)phenoxy)phenyl)-5-(2,
4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide)
is shown below.
Step 1 and Step 2
(S)-2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetra-
hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-methylbutanamide
TFA salt
##STR00136##
[0554] To a mixture of
4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrof-
uran-2-yl)pyrimidin-2(1H)-one (527 mg, 2 mmol) and
(S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (434 mg, 2
mmol) in DMF (5 mL) was added Et.sub.3N (0.92 mL, 6.6 mmol)
followed by PyBOP (1.24 g, 2.4 mmol). The reaction mixture was
stirred at room temperature overnight then concentrated. The crude
residue was treated with aq. NaHCO.sub.3 then extracted with ethyl
acetate. The organic layer was separated, washed with brine and
dried over Na.sub.2SO.sub.4. The mixture was filtered, concentrated
and the residue purified twice by ISCO using DCM/MeOH (0-10%) as
eluent to afford 260 mg (28%) of product.
C.sub.19H.sub.28F.sub.2N.sub.4O.sub.7: 462.19; found: 463.3
(M+H.sup.+).
[0555] The above product (260 mg) was dissolved in DCM (3 mL) and
treated with TFA (3 mL). The reaction mixture was stirred at room
temperature for 1 h, concentrated and dried on high vacuum to get
product as a gummy oil.
Step 3
##STR00137##
[0557] To a mixture of
(S)-2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetr-
ahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-methylbutanamide
TFA salt (96 mg, 0.2 mmol) and
4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-tri-
azol-4-yl)phenoxy)benzoic acid (120 mg, 0.24 mmol) in DMF (3 mL)
was added HATU (92 mg, 0.24 mmol) and DIPEA (0.14 mL, 0.80 mmol).
The reaction mixture was stirred at room temperature overnight then
concentrated. The crude residue was purified by ISCO (twice) using
DCM/MeOH (0-25%) as eluent to afford 40 mg (23.6%) of the title
compound. ESMS calculated for
C.sub.41H.sub.44F.sub.2N.sub.8O.sub.10: 846.31; found: 847.3
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.19 (s, 1H),
10.43 (s, 1H), 9.79 (s, 1H), 8.99 (t, J=5.9 Hz, 1H), 8.49 (d, J=7.6
Hz, 1H), 8.28 (d, J=7.6 Hz, 1H), 8.03-7.89 (m, 2H), 7.50-7.35 (m,
2H), 7.30 (d, J=7.6 Hz, 1H), 7.23-6.95 (m, 4H), 6.69 (s, 1H),
6.35-6.33 (m, 2H), 6.18 (t, J=7.4 Hz, 1H), 5.32 (t, J=5.4 Hz, 1H),
4.47 (t, J=7.8 Hz, 1H), 4.31-4.07 (m, 1H), 3.98-3.74 (m, 2H),
3.68-3.62 (m, 1H), 3.27-3.12 (m, 2H), 2.98 (p, J=6.9 Hz, 1H), 2.20
(h, J=6.7 Hz, 1H), 1.06 (t, J=7.2 Hz, 3H), 0.99 (dd, J=9.5, 6.7 Hz,
6H), 0.93 (d, J=6.9 Hz, 6H).
Example 19--SDC-TRAP-311 Structure and Synthesis
##STR00138##
[0559] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-311
(N--((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-methyl-1-oxobutan-
-2-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopropyl
phenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidin-1-yl)p-
yrazine-2-carboxamide) is shown below.
[0560] To a mixture of
(S)-2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetr-
ahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-methylbutanamide
TFA salt (150 mg, 0.31 mmol) and
5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4--
triazol-4-yl)phenoxy)piperidin-1-yl)pyrazine-2-carboxylic acid (117
mg, 0.20 mmol) in DMF (3 mL) was added HATU (92 mg, 0.24 mmol) and
DIPEA (0.14 mL, 0.80 mmol). The reaction mixture was stirred at
room temperature overnight then concentrated. The crude residue was
purified by ISCO using DCM/MeOH (0-25%) as eluent to afford 106 mg
(56%) of the title compound. ESMS calculated for
C.sub.44H.sub.51F.sub.2N.sub.11O.sub.10: 931.94; found: 933.0
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.30 (s, 1H),
10.69 (s, 1H), 9.75 (s, 1H), 8.91 (s, 1H), 8.63 (s, 1H), 8.41 (s,
1H), 8.26 (dd, J=15.0, 8.3 Hz, 2H), 7.39-7.20 (m, 3H), 7.17-6.99
(m, 2H), 6.60 (s, 1H), 6.36 (s, 1H), 6.33-6.27 (m, 1H), 6.18 (t,
J=7.4 Hz, 1H), 5.30-5.26 (m, 1H), 4.78-4.74 (m, 1H), 4.61 (dd,
J=8.9, 6.6 Hz, 1H), 4.27-4.03 (m, 3H), 3.96-3.76 (m, 2H), 3.72-3.56
(m, 1H), 3.25-3.13 (m, 2H), 2.94 (p, J=6.9 Hz, 1H), 2.23 (h, J=6.7
Hz, 1H), 2.06 (broad s, 2H), 1.76-1.70 (m, 4H), 1.05 (t, J=7.2 Hz,
3H), 0.93 (dd, J=20.2, 6.7 Hz, 6H), 0.85 (d, J=6.9 Hz, 6H).
Example 20--SDC-TRAP-312 Structure and Synthesis
##STR00139##
[0562] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-312
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-1-(4-(4-(3-(2,4-dihydroxy-5-isopro-
pylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperi-
dine-4-carboxamide) is shown below.
Step 1 and Step 2
##STR00140##
[0564] To a mixture of
4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrof-
uran-2-yl)pyrimidin-2(1H)-one (527 mg, 2 mmol) and
1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (458 mg, 2
mmol) in DMF (5 mL) was added HOBt (0.27 g, 2 mmol), Et.sub.3N
(0.84 mL, 6 mmol) and PyBOP (1.04 g, 2 mmol). The reaction mixture
was stirred at room temperature overnight then concentrated. The
crude product was purified by ISCO using DCM/MeOH (0-10%) as eluent
to afford 116 mg (12%) of product. C.sub.20H28F2N407: 474.19;
found: 475.3 (M+H.sup.+).
[0565] The above product (116 mg) was dissolved in DCM (3 mL) and
treated with TFA (3 mL). The reaction mixture was stirred at room
temperature for 1 h, concentrated and dried on high vacuum to get
title compound as a gummy oil.
Step 3
##STR00141##
[0567] To a mixture of
N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran--
2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperidine-4-carboxamide TFA
salt (113 mg, 0.24 mmol) and
4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-tri-
azol-4-yl)phenoxy)benzoic acid (120 mg, 0.24 mmol) in DMF (3 mL)
was added HATU (109 mg, 0.28 mmol) and DIPEA (0.165 mL, 0.96 mmol).
The reaction mixture was stirred at room temperature for 5 h then
concentrated. The crude product was purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 66 mg (32%) of the title compound. ESMS
calculated for C.sub.42H.sub.44F.sub.2N.sub.8O.sub.10: 858.84;
found: 860.0 (M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta.
11.07 (s, 1H), 10.41 (s, 1H), 9.74 (s, 1H), 8.95 (t, J=5.7 Hz, 1H),
8.25 (d, J=7.6 Hz, 1H), 7.51-7.44 (m, 2H), 7.41-7.35 (m, 2H), 7.29
(d, J=7.6 Hz, 1H), 7.14-7.05 (m, 4H), 6.68 (s, 1H), 6.35 (s, 1H),
6.30 (d, J=6.5 Hz, 1H), 6.17 (t, J=7.5 Hz, 1H), 5.30-5.27 (m, 1H),
4.26-4.10 (m, 1H), 3.89 (dt, J=8.5, 3.0 Hz, 1H), 3.86-3.75 (m, 1H),
3.68-3.63 (m, 1H), 3.27-3.10 (m, 3H), 2.97 (h, J=6.8 Hz, 3H),
2.81-2.76 (m, 1H), 1.85 (broad s, 2H), 1.59-1.50 (m, 2H), 1.08 (dt,
J=11.7, 7.1 Hz, 4H), 0.92 (d, J=6.9 Hz, 6H).
Example 21--SDC-TRAP-313 Structure and Synthesis
##STR00142##
[0569] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-313
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopr-
opylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)
benzyl)piperazin-1-yl)methyl)benzamide) is shown below.
##STR00143## ##STR00144##
Step 1:
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert--
butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin--
2(1H)-one
[0570] The compound was prepared following a procedure in
Biomacromolecules, 2013, 2837-2847, incorporated by reference in
its entirety herein. To a solution of
4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrof-
uran-2-yl)pyrimidin-2 (1H)-one (1 g, 3.8 mmol) in DMF (23 mL) was
added Imidazole (0.78 g, 11.5 mmol) and TBDMSC1 (1.5 g, 9.6 mmol).
The reaction mixture was stirred at room temperature for 1.5 d. The
solvent was removed and the residue partitioned between ethyl
acetate and aq. NaHCO.sub.3. The organic layer was separated,
washed with brine, dried over Na2SO4, filtered and concentrated.
The crude product was purified by ISCO using DCM/MeOH (0-10%) as
eluent to afford 1.3 g (69%) of the title compound. ESMS calculated
for C.sub.21H.sub.39F.sub.2N.sub.3O.sub.4Si.sub.2: 491.24; found:
492.4 (M+H.sup.+).
Step 2:
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyld-
imethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihyd-
ropyrimidin-4-yl)-4-(chloromethyl)benz amide
[0571] To a solution of
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-on-
e (100 mg, 0.2 mmol) in DCM (1 mL) at 0.degree. C. was added
pyridine (0.48 mmol, 40 .mu.L) followed by 4-(chloromethyl)benzoyl
chloride (50 mg, 0.24 mmol) in DCM (1 mL). After stirring 45 min,
ice-bath removed and further stirred at room temperature for 2 h.
The solvent was removed and the crude product purified by ISCO
using DCM/MeOH (0-10%) as eluent to afford 120 mg (93%) of product.
ESMS calculated for
C.sub.29H.sub.44ClF.sub.2N.sub.3O.sub.5Si.sub.2: 643.25; found:
644.6 (M+H.sup.+).
[0572] Step 3:
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl-
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrim-
idin-4-yl)-444-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4--
triazol-4-yl)benzyl)piperazin-1-yl)methyl)benz amide
[0573] To a mixture of
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl-
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrim-
idin-4-yl)-4-(chloromethyl)benzamide (65 mg, 0.1 mmol) and
4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6--
isopropylbenzene-1,3-diol hydrochloride (75 mg, 0.16 mmol) in DMF
(2 mL) was added DIPEA (0.1 mL, 0.6 mmol) and KI (5 mg). The
mixture was heated at 80.degree. C. for 4 h. The solvent was
removed and the crude product purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 91 mg (98%) of the title compound. ESMS
calculated for C.sub.51H.sub.70F.sub.2N.sub.8O.sub.8Si.sub.2:
1017.32; found: 1018.4 (M+H.sup.+).
Step 4:
N-(1-(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-i-
sopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)meth-
yl)benzamide
[0574] To a solution of
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl-
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrim-
idin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,-
4-triazol-4-yl)benzyl)piperazin-1-yl)methyl)benzamide (91 mg, 0.089
mmol) in dry THF (2 mL) was added TBAF (1M, 0.67 mL, 0.67 mmol).
The mixture was stirred at room temperature for 1.5 h then quenched
with few drops of aq. NH.sub.4Cl. The solvent was removed and the
crude product purified by ISCO using DCM/MeOH (0-25%) as eluent to
afford 61 mg (87%) of the title compound. ESMS calculated for
C.sub.39H.sub.42F.sub.2N.sub.8O.sub.8: 788.31; found: 790.0
(M+H.sup.+).
Example 22--SDC-TRAP-314 Structure and Synthesis
[0575] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-314
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopr-
opylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl-
)methyl)benzamide is shown below.
##STR00145##
[0576] To a mixture of
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl-
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrim-
idin-4-yl)-4-(chloromethyl)benzamide (54 mg, 0.083 mmol) and
4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-
-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (47 mg, 0.1 mmol)
in DMF (2 mL) was added DIPEA (0.14 mL, 0.8 mmol) and KI (5 mg).
The mixture was heated at 80.degree. C. for 6 h. The solvent was
removed and the crude product purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 49 mg (57.6%) of product. ESMS
calculated for C.sub.50H.sub.69F.sub.3N.sub.8O.sub.8Si.sub.2:
1035.31; found: 1036.3 (M+H.sup.+).
[0577] To a solution of
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl-
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrim-
idin-4-yl)-4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,-
4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)methyl)benzamide (49
mg, 0.047 mmol) in dry THF (2 mL) was added TBAF (1M, 0.36 mL, 0.36
mmol). The mixture was stirred at room temperature for 1.5 h then
quenched with few drops of aq. NH.sub.4Cl. The solvent was removed
and the crude product purified by ISCO using DCM/MeOH (0-25%) as
eluent to afford 34 mg (90%) of the title compound. ESMS calculated
for C.sub.39H.sub.41F.sub.3N.sub.8O.sub.8: 806.30; found: 807.6
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.97 (s, 1H),
11.32 (s, 1H), 9.63 (s, 1H), 9.39 (s, 1H), 8.31 (d, J=7.7 Hz, 1H),
8.06-7.81 (m, 2H), 7.50-7.28 (m, 4H), 7.05 (dd, J=10.9, 2.0 Hz,
1H), 6.96 (dd, J=8.2, 2.0 Hz, 1H), 6.85 (s, 1H), 6.34 (d, J=6.5 Hz,
1H), 6.27 (s, 1H), 6.20 (t, J=7.4 Hz, 1H), 5.34 (t, J=5.4 Hz, 1H),
4.23 (dd, J=13.6, 6.0 Hz, 1H), 3.91 (dt, J=8.6, 3.1 Hz, 1H), 3.82
(d, J=13.0 Hz, 1H), 3.67 (ddd, J=12.7, 5.9, 3.5 Hz, 1H), 3.51 (d,
J=16.1 Hz, 4H), 3.00 (p, J=6.9 Hz, 1H), 2.36 (broad s, 8H), 0.99
(d, J=6.9 Hz, 6H).
Example 23--SDC-TRAP-315 Structure and Synthesis
[0578] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-315
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(3-(2,4-dihydroxy-5-isopropyl-
phenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxamide
is shown below.
##STR00146##
Step 1 and Step 2
N-(1-(2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsi-
lyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimid-
in-4-yl)-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-tria-
zol-4-yl)benzyl)piperazine-1-carboxamide
[0579] To a solution of
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-on-
e (100 mg, 0.2 mmol) in dry DCM (2 mL) was added pyridine (20
.mu.L, 0.24 mmol) and 4-nitrophenyl carbonochloridate (48 mg, 0.24
mmol). The mixture was stirred at 0.degree. C. for 1 h then at room
temperature for 5 h. The solvent was removed and the crude product,
4-nitrophenyl
(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsi-
lyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimid-
in-4-yl)carbamate dried on high vacuum.
[0580] To the above product (130 mg, 0.2 mmol) in DMF (3 mL) was
added
4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6--
isopropylbenzene-1,3-diol hydrochloride (90 mg, 0.2 mmol) and DIPEA
(0.14 mL, 0.8 mmol). The mixture was stirred at room temperature
overnight then solvent removed. The crude product was purified by
ISCO using DCM/MeOH (0-25%) as eluent to afford 83 mg (44.8%) of
product. ESMS calculated for
C.sub.44H.sub.64F.sub.2N.sub.8O.sub.8Si.sub.2: 926.44; found: 927.4
(M+H.sup.+).
Step 3
[0581] To a solution of
N-(1-(2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyls-
ilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-tri-
azol-4-yl)benzyl)piperazine-1-carboxamide (83 mg, 0.089 mmol) in
dry THF (4 mL) was added TBAF (1M, 0.67 mL, 0.67 mmol). The mixture
was stirred at room temperature for 1.5 h then quenched with few
drops of aq. NH.sub.4Cl. The solvent was removed and the crude
product purified by ISCO using DCM/MeOH (0-25%) as eluent to afford
55 mg (90%) of the title compound. ESMS calculated for
C.sub.39H.sub.41F.sub.3N.sub.8O.sub.8: 698.26; found: 699.3
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.93 (s, 1H),
10.22 (broad s, 1H), 9.63 (s, 1H), 9.42 (s, 1H), 8.10 (broad s,
1H), 7.40-7.25 (m, 2H), 7.21-7.07 (m, 2H), 6.92 (broad s, 1H), 6.76
(s, 1H), 6.31 (d, J=11.0 Hz, 1H), 6.29 (s, 1H), 6.12 (s, 1H), 5.28
(t, J=5.5 Hz, 1H), 4.26-4.18 (m, 1H), 3.92-3.72 (m, 2H), 3.70-3.57
(m, 1H), 3.47 (s, 6H), 2.97 (p, J=6.9 Hz, 1H), 2.34 (broad s, 4H),
0.99 (d, J=6.9 Hz, 6H).
Example 24--SDC-TRAP-316 Structure and Synthesis
[0582] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-316
(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tet-
rahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-methyl-1-oxobu-
tan-2-yl)carbamoyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H--
1,2,4-triazole-3-carboxamide) is shown below.
##STR00147##
[0583] To a mixture of
(S)-2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-
-triazol-4-yl)benzamido)-3-methylbutanoic acid (51 mg, 0.1 mmol)
and
4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrof-
uran-2-yl)pyrimidin-2(1H)-one (27 mg, 0.1 mmol) in DMF (2 mL) was
added EDCI (25 mg, 0.13 mmol), HOBt (14 mg, 0.1 mmol) and NMM (11
.mu.L, 0.1 mmol). The reaction mixture was stirred at 55.degree. C.
for 24 h. The solvent was removed and the crude residue was
purified by ISCO (twice) using DCM/MeOH (0-25%) as eluent to afford
8 mg (10%) of the title compound. ESMS calculated for
C.sub.35H.sub.40F.sub.2N.sub.8O.sub.9: 754.29; found: 755.6
(M+H.sup.+).
Example 25--SDC-TRAP-317 Structure and Synthesis
[0584] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-317
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-1-(2-(4-(4-(3-(2,4-dihydroxy-5-iso-
propylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)acetyl-
)piperidine-4-carboxamide) is shown below.
##STR00148##
[0585] To a mixture of
N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran--
2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperidine-4-carboxamide TFA
salt (95 mg, 0.2 mmol) and
2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-
-yl)benzyl)piperazin-1-yl)acetic acid (94 mg, 0.2 mmol) in DMF (2
mL) was added HATU (92 mg, 0.24 mmol) and DIPEA (0.14 mL, 0.8
mmol). The reaction mixture was stirred at room temperature for 5 h
then concentrated. The crude residue was purified twice by ISCO
using DCM/MeOH (0-25%) as eluent to afford 32 mg (19%) of the title
compound. ESMS calculated for
C.sub.39H.sub.47F.sub.2N.sub.9O.sub.9: 823.35; found: 825.2
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.93 (s, 1H),
11.08 (s, 1H), 9.63 (s, 1H), 9.42 (s, 1H), 8.26 (d, J=7.6 Hz, 1H),
7.35-7.25 (m, 3H), 7.12 (d, J=8.4 Hz, 2H), 6.76 (s, 1H), 6.34 (d,
J=6.5 Hz, 1H), 6.27 (s, 1H), 6.17 (t, J=7.5 Hz, 1H), 5.33 (t, J=5.4
Hz, 1H), 4.34 (d, J=12.8 Hz, 1H), 4.24-4.07 (m, 2H), 3.89 (dt,
J=8.6, 3.1 Hz, 1H), 3.85-3.74 (m, 1H), 3.68-3.63 (m, 1H), 3.42 (s,
2H), 3.31-3.19 (m, 2H), 3.01-2.89 (m, 3H), 2.77-2.71 (m, 1H), 2.38
(broad s, 8H), 1.81 (broad s, 2H), 1.60-1.49 (m, 1H), 1.40-1.28 (m,
1H), 0.94 (d, J=6.9 Hz, 6H).
Example 26--SDC-TRAP-318 Structure and Synthesis
[0586] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-318
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-isopro-
pylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-
-4-oxobutanamide) is shown below.
##STR00149##
Step 1
[0587] To a solution of
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-on-
e (49 mg, 0.1 mmol) and dihydrofuran-2,5-dione (73 mg, 0.73 mmol,
7.3 eq.) in dry DMF (1 mL) was added DIPEA (0.158 mL, 0.91 mmol,
9.14 eq.). The mixture was stirred at room temperature for 20 h.
The solvent was removed and the residue purified by ISCO using
DCM/MeOH (0-25%) as eluent to afford 60 mg (99%) of product. ESMS
calculated for C.sub.25H.sub.43F.sub.2N.sub.3O.sub.7Si.sub.2:
591.26; found: 592.6 (M+H.sup.+).
Step 2
[0588] To a mixture of
4-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethy-
lsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyri-
midin-4-yl)amino)-4-oxobutanoic acid (60 mg, 0.1 mmol) and
4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-
-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (47 mg, 0.1 mmol)
in DMF (1 mL) was added EDCI (29 mg, 0.15 mmol), HOBt (14 mg, 0.1
mmol) and DIPEA (70 .mu.L, 0.4 mmol). The reaction mixture was
stirred at room temperature overnight. The solvent was removed and
the crude residue was purified by ISCO using DCM/MeOH (0-25%) as
eluent to afford 79 mg (79%) of impure product. ESMS calculated for
C.sub.47H.sub.67F.sub.3N8O.sub.9Si.sub.2: 1001.25; found: 1002.3
(M+H.sup.+).
Step 3
[0589] To a solution of
N-(1-(2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyls-
ilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4--
triazol-4-yl)-2-fluoroben zyl)piperazin-1-yl)-4-oxobutanamide (79
mg, 0.0788 mmol) in dry THF (4 mL) was added TBAF (1M, 0.8 mL, 0.8
mmol). The mixture was stirred at room temperature for 1.5 h then
quenched with few drops of aq. NH.sub.4Cl. The solvent was removed
and the crude product purified by ISCO using DCM/MeOH (0-25%) as
eluent to afford 27 mg (45%) of the title compound. ESMS calculated
for C.sub.35H.sub.39F.sub.3N.sub.8O.sub.9: 772.28; found: 773.8
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.99 (s, 1H),
11.04 (s, 1H), 9.65 (s, 1H), 9.40 (s, 1H), 8.23 (d, J=7.6 Hz, 1H),
7.40 (t, J=8.2 Hz, 1H), 7.25 (d, J=7.6 Hz, 1H), 7.06 (dd, J=10.9,
2.0 Hz, 1H), 6.97 (dd, J=8.1, 2.0 Hz, 1H), 6.87 (s, 1H), 6.33 (d,
J=6.5 Hz, 1H), 6.27 (s, 1H), 6.17 (t, J=7.5 Hz, 1H), 5.31 (t, J=5.5
Hz, 1H), 4.23-4.13 (m, 1H), 3.88 (dt, J=8.6, 3.0 Hz, 1H), 3.85-3.76
(m, 1H), 3.68-3.62 (m, 1H), 3.52 (s, 2H), 3.45-3.39 (m, 4H), 3.00
(p, J=6.8 Hz, 1H), 2.62-2.59 (m, 4H), 2.39 (broad s, 2H), 2.31
(broad s, 2H), 1.00 (d, J=6.9 Hz, 6H).
Example 27--SDC-TRAP-319 Structure and Synthesis
[0590] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-319
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-((2-(4-(4-(3-(2,4-dihydroxy-5-is-
opropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-2-ox-
oethyl)(methyl)amino)acetamide) is shown below.
##STR00150##
Step 1
[0591] To a solution of
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-on-
e (110 mg, 0.22 mmol) and 4-methylmorpholine-2,6-dione (0.2 g, 1.62
mmol) in dry DMF (1 mL) was added DIPEA (0.35 mL, 2 mmol). The
mixture was stirred at room temperature for 20 h. The solvent was
removed and the residue purified by ISCO using DCM/MeOH (0-25%) as
eluent to afford 113 mg (81.8%) of product. ESMS calculated for
C.sub.26H.sub.46F.sub.2N.sub.4O.sub.7Si.sub.2: 620.83; found: 621.7
(M+H.sup.+).
Step 2
[0592] To a mixture of
2-((2-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethyl
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydr-
opyrimidin-4-yl)amino)-2-oxoethyl)(methyl)amino)acetic acid (56 mg,
0.091 mmol) and
4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazo-
l-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (45 mg, 0.1 mmol)
in DMF (1 mL) was added HATU (42 mg, 0.1092 mmol) and DIPEA (70
.mu.L, 0.4 mmol). The reaction mixture was stirred at room
temperature for 6 h. The solvent was removed and the crude residue
was purified by ISCO using DCM/MeOH (0-25%) as eluent to afford 40
mg (43.4%) of product. ESMS calculated for
C.sub.48H.sub.71F.sub.2N.sub.9O.sub.9Si.sub.2: 1012.30; found:
1013.3 (M+H.sup.f).
Step 3
[0593] To a solution of
N-(1-(2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyls-
ilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)-2-((2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,-
2,4-triazol-4-yl)benzyl)pi
perazin-1-yl)-2-oxoethyl)(methyl)amino)acetamide (40 mg, 0.39 mmol)
in dry THF (2 mL) was added TBAF (1M, 0.8 mL, 0.8 mmol). The
mixture was stirred at room temperature for 1.5 h. The solvent was
removed and the crude product purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 25 mg (83%) of the title compound. ESMS
calculated for C.sub.36H.sub.43F.sub.2N.sub.9O.sub.9: 773.32;
found: 774.8 (M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta.
11.93, (s, 1H), 10.74 (s, 1H), 9.60 (s, 1H), 9.41 (s, 1H), 8.28 (d,
J=7.5 Hz, 1H), 7.37-7.25 (m, 3H), 7.14 (d, J=8.4 Hz, 2H), 6.78 (s,
1H), 6.33 (d, J=6.5 Hz, 1H), 6.26 (s, 1H), 6.18 (t, J=7.4 Hz, 1H),
5.32 (t, J=5.4 Hz, 1H), 4.24-4.14 (m, 1H), 3.96-3.76 (m, 2H),
3.74-3.59 (m, 1H), 3.46 (broad s, 8H), 3.31 (s, 2H), 2.97 (p, J=6.9
Hz, 1H), 2.37 (s, 4H), 2.30 (s, 3H), 0.95 (d, J=6.9 Hz, 6H).
##STR00151##
Example 28--SDC-TRAP-320 Structure and Synthesis
[0594] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-320
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-((2-(4-(4-(3-(2,4-dihydroxy-5-is-
opropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-
-yl)-2-oxoethyl)(methyl)amino)acetamide) is shown below.
##STR00152##
Step 1 and Step 2
[0595] To a mixture of
2-((2-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydro-
pyrimidin-4-yl)amino)-2-oxoethyl)(methyl)amino)acetic acid (56 mg,
0.091 mmol) and
4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2-
,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (47 mg,
0.1 mmol) in DMF (1 mL) was added HATU (42 mg, 0.1092 mmol) and
DIPEA (70 .mu.L, 0.4 mmol). The reaction mixture was stirred at
room temperature for 6 h. The solvent was removed and the crude
residue was purified by ISCO using DCM/MeOH (0-25%) as eluent to
afford 58 mg (62.3%) of product. ESMS calculated for
C.sub.48H.sub.70F3N9O.sub.9Si.sub.2: 1030.29; found: 1031.4
(M+H.sup.+).
Step 3
[0596] To a solution of
N-(1-(2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyls-
ilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)-2-((2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,-
2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-2-oxoethyl)(methyl)amino)-
acetamide (58 mg, 0.056 mmol) in dry THF (2 mL) was added TBAF (1M,
0.8 mL, 0.8 mmol). The mixture was stirred at room temperature for
1.5 h. The solvent was removed and the crude product purified by
ISCO using DCM/MeOH (0-25%) as eluent to afford 39 mg (88%) of the
title compound. ESMS calculated for
C.sub.36H.sub.42F.sub.3N.sub.9O.sub.9: 801.31; found: 802.4
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.98 (s, 1H),
10.74 (s, 1H), 9.63 (s, 1H), 9.39 (s, 1H), 8.28 (d, J=7.6 Hz, 1H),
7.40 (t, J=8.2 Hz, 1H), 7.28 (d, J=7.5 Hz, 1H), 7.06 (dd, J=10.8,
2.0 Hz, 1H), 6.97 (dd, J=8.1, 2.0 Hz, 1H), 6.87 (s, 1H), 6.33 (d,
J=6.5 Hz, 1H), 6.27 (s, 1H), 6.18 (t, J=7.4 Hz, 1H), 5.32 (t, J=5.4
Hz, 1H), 4.28-4.10 (m, 1H), 3.95-3.76 (m, 2H), 3.68-3.63 (m, 1H),
3.52 (s, 2H), 3.45-3.39 (m, 6H), 3.31 (s, 2H), 3.00 (p, J=6.9 Hz,
1H), 2.44-2.25 (m, 7H), 1.00 (d, J=6.9 Hz, 6H).
Example 29--SDC-TRAP-321 Structure and Synthesis
[0597] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-321
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-(4-(4-(3-(2,4-dihydroxy-5-isopro-
pylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)acetamide-
) is shown below.
##STR00153##
Step 1
[0598] To a solution of
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-on-
e (245 mg, 0.5 mmol) in DCM (3 mL) at 0.degree. C. was added
pyridine (0.1 mL, 1.2 mmol) followed by 2-chloroacetyl chloride (48
.mu.L, 0.6 mmol) in DCM (1 mL). The reaction mixture was allowed to
reach room temperature over 5 h. The solvent was removed and the
crude product purified by ISCO using DCM/MeOH (0-10%) as eluent to
afford 0.28 g (99%) of product. ESMS calculated for
C.sub.23H.sub.40ClF.sub.2N.sub.3O.sub.5Si.sub.2: 567.22; found:
568.6 (M+H.sup.+).
Step 2
[0599] To a mixture of
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl-
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrim-
idin-4-yl)-2-chloroacetamide (93 mg, 0.163 mmol) and
4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6--
isopropylbenzene-1,3-diol hydrochloride (116 mg, 0.26 mmol) in DMF
(2 mL) was added DIPEA (0.16 mL, 0.92 mmol) and KI (5 mg). The
mixture was heated at 70.degree. C. for 5 h. The reaction mixture
was diluted with the ethyl acetate, washed with water and brine.
The organic layer dried over Na.sub.2SO.sub.4, filtered,
concentrated and the crude product purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 89 mg (58%) of product. ESMS calculated
for C.sub.45H.sub.66F.sub.2N.sub.8O.sub.8Si.sub.2: 941.22; found:
942.3 (M+H.sup.+).
Step 3
[0600] To a solution of
N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyl-
silyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrim-
idin-4-yl)-2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-
-triazol-4-yl)benzyl)piperazin-1-yl)acetamide (89 mg, 0.094 mmol)
in dry THF (2 mL) was added TBAF (1M, 0.71 mL, 0.71 mmol). The
mixture was stirred at room temperature for 1.5 h. The solvent was
removed and the crude product purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 60 mg (90%) of the title compound. ESMS
calculated for C.sub.33H.sub.38F.sub.2N.sub.8O.sub.8: 712.28;
found: 713.70 (M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta.
11.93 (s, 1H), 10.45 (s, 1H), 9.60 (s, 1H), 9.41 (s, 1H), 8.29 (d,
J=7.5 Hz, 1H), 7.29 (t, J=7.9 Hz, 3H), 7.13 (d, J=8.4 Hz, 2H), 6.77
(s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.26 (s, 1H), 6.17 (t, J=7.4 Hz,
1H), 5.32 (t, J=5.5 Hz, 1H), 4.24-4.14 (m, 1H), 3.96-3.74 (m, 2H),
3.68-3.63 (m, 1H), 3.45 (broad s, 4H), 3.22 (broad s, 4H), 2.96 (p,
J=6.8 Hz, 1H), 2.40 (broad s, 4H), 1.00 (d, J=6.9 Hz, 6H).
Example 30--SDC-TRAP-322 Structure and Synthesis
[0601] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-322
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopro-
pylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-
-3,3-dimethyl-5-oxopentanamide) is shown below.
##STR00154##
Step 1
[0602] To a solution of
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2
(1H)-one (110 mg, 0.22 mmol) and
4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione (0.23 g, 1.62 mmol) in
dry DMF (1 mL) was added DIPEA (0.35 mL, 2 mmol). The mixture was
stirred at room temperature for 20 h. The solvent was removed and
the residue purified by ISCO using DCM/MeOH (0-25%) as eluent to
afford 141 mg (99%) of product. ESMS calculated for
C.sub.28H.sub.49F.sub.2N.sub.3O.sub.7Si.sub.2: 633.87; found: 634.7
(M+
Step 2
[0603] To a mixture of
5-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethy-
lsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyri-
midin-4-yl)amino)-3,3-dimethyl-5-oxopentanoic acid (141 mg, 0.22
mmol) and
4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-
-3-yl)-6-isopropylbenzene-1,3-diol hydrochloride (104 mg, 0.22
mmol) in DMF (2 mL) was added HATU (103 mg, 0.27 mmol) and DIPEA
(0.16 mL, 0.9 mmol). The reaction mixture was stirred at room
temperature for 6 h. The solvent was removed and the crude residue
was purified by ISCO using DCM/MeOH (0-25%) as eluent to afford 45
mg (23%) of product. ESMS calculated for
C.sub.50H.sub.73F.sub.3N.sub.8O.sub.9Si.sub.2: 1043.3; found:
1044.4 (M+H.sup.+).
Step 3
[0604] To a solution of
N-(1-(2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyls-
ilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4--
triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-3,3-dimethyl-5-oxopentanamide
(45 mg, 0.043 mmol) in dry THF (3 mL) was added TBAF (1M, 0.7 mL,
0.7 mmol). The mixture was stirred at room temperature for 1.5 h.
The solvent was removed and the crude product purified by ISCO
using DCM/MeOH (0-25%) as eluent to afford 39 mg (90%) of the title
compound. ESMS calculated for
C.sub.38H.sub.45F.sub.3N.sub.8O.sub.9: 814.33; found: 815.81
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.98 (s, 1H),
11.12 (s, 1H), 9.63 (s, 1H), 9.40 (s, 1H), 8.24 (d, J=7.6 Hz, 1H),
7.40 (t, J=8.2 Hz, 1H), 7.30 (d, J=7.6 Hz, 1H), 7.07 (dd, J=10.8,
2.0 Hz, 1H), 6.97 (dd, J=8.2, 2.0 Hz, 1H), 6.87 (s, 1H), 6.32 (d,
J=6.5 Hz, 1H), 6.27 (s, 1H), 6.17 (t, J=7.4 Hz, 1H), 5.31 (t, J=5.4
Hz, 1H), 4.24-4.14 (m, 1H), 3.94-3.76 (m, 2H), 3.68-3.62 (m, 1H),
3.51 (broad s, 6H) 3.00 (p, J=6.9 Hz, 1H), 2.48 (s, 2H), 2.41 (s,
2H), 2.38 (broad s, 2H), 2.32 (broad s, 2H), 1.04 (s, 6H), 1.00 (d,
J=6.9 Hz, 6H).
##STR00155##
Example 31--SDC-TRAP-323 Structure and Synthesis
[0605] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-323
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-(4-(4-(3-(2,4-dihydroxy-5-isopro-
pylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-
acetamide) is shown below.
##STR00156##
[0606] The above compound was prepared following the protocols
described for SDC-TRAP-321. C.sub.33H.sub.37F.sub.3N.sub.8O.sub.8:
730.27; found: 731.70 (M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 11.98 (s, 1H), 10.45 (s, 1H), 9.62 (s, 1H), 9.40 (s, 1H),
8.29 (d, J=7.6 Hz, 1H), 7.37 (t, J=8.2 Hz, 1H), 7.27 (d, J=7.6 Hz,
1H), 7.05 (dd, J=10.8, 2.1 Hz, 1H), 6.96 (dd, J=8.1, 2.0 Hz, 1H),
6.87 (s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.17 (t, J=7.4
Hz, 1H), 5.32 (s, 1H), 4.24 (m, 1H), 3.94-3.75 (m, 2H), 3.68-3.63
(m, 1H), 3.50 (broad s, 4H), 3.32-3.08 (m, 4H), 3.00 (p, J=6.8 Hz,
1H), 2.43 (s, 4H), 1.00 (d, J=6.9 Hz, 6H).
Example 32--SDC-TRAP-324 Structure and Synthesis
[0607] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-324
(4-(4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tet-
rahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-methyl-1-oxope-
ntan-2-yl)carbamoyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-
-1,2,4-triazole-3-carboxamide) is shown below.
##STR00157##
[0608] To a solution of
(S)-2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-
-triazol-4-yl)benzamido)-4-methylpentanoic acid (53 mg, 0.2 mmol)
in dry DMF (2 mL) was added EDC (29 mg, 0.15 mmol) and
1-hydroxypyrrolidine-2,5-dione (18 mg, 0.15 mmol). The mixture was
stirred at room temperature overnight and treated with
4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrof-
uran-2-yl)pyrimidin-2(1H)-one (52 mg, 0.2 mmol). The reaction
mixture was stirred at 50.degree. C. 2.5 days. The solvent was
removed and the crude residue was purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 29 mg (37.7%) of the title compound.
ESMS calculated for C.sub.36H.sub.42F.sub.2N.sub.8O.sub.9: 768.30;
found: 769.6 (M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta.
11.30 (d, J=7.7 Hz, 1H), 9.98 (s, 1H), 9.68 (s, 1H), 9.04 (t, J=5.8
Hz, 1H), 8.74 (d, J=7.4 Hz, 1H), 8.27 (d, J=7.6 Hz, 1H), 7.97-7.83
(m, 2H), 7.49-7.37 (m, 2H), 7.26 (dd, J=7.6, 3.7 Hz, 1H), 6.75 (s,
1H), 6.34 (dd, J=6.5, 1.9 Hz, 1H), 6.28 (s, 1H), 6.18 (t, J=7.3 Hz,
1H), 5.31 (q, J=5.4 Hz, 1H), 4.70-4.66 (m, 1H), 4.25-4.10 (m, 1H),
3.95-3.75 (m, 2H), 3.72-3.57 (m, 1H), 3.26-3.11 (m, 2H), 3.04-2.89
(m, 1H), 1.87-1.68 (m, 2H), 1.62-1.48 (m, 1H), 1.06 (t, J=7.2 Hz,
3H), 0.98-0.81 (m, 12H).
Example 33--SDC-TRAP-325 Structure and Synthesis
[0609] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-325
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-isopro-
pylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-4-oxobut-
anamide) is shown below.
##STR00158##
[0610] To a mixture of
4-(((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimeth-
ylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyr-
imidin-4-yl)amino)-4-oxobutanoic acid (143 mg, 0.24 mmol) and
4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6--
isopropylbenzene-1,3-diol hydrochloride (107 mg, 0.24 mmol) in DMF
(1 mL) was added HATU (110 mg, 0.29 mmol) and DIPEA (168 .mu.L,
0.96 mmol). The reaction mixture was stirred at room temperature
overnight. The solvent was removed and the crude residue was
purified by ISCO using DCM/MeOH (0-25%) as eluent to afford 150 mg
of impure product. ESMS calculated for
C.sub.47H.sub.68F.sub.2N.sub.8O.sub.9Si.sub.2: 983.26; found: 984.4
(M+H+).
[0611] To a solution of
N-(1-(2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethyls-
ilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)-4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4--
triazol-4-yl)benzyl)piperazin-1-yl)-4-oxobutanamide (150 mg, 0.15
mmol) in dry THF (2 mL) was added TBAF (1M, 1 mL, 1 mmol). The
mixture was stirred at room temperature for 1.5 h. The solvent was
removed and the crude product purified by ISCO using DCM/MeOH
(0-25%) as eluent to afford 9 mg of the title compound. ESMS
calculated for C.sub.35H.sub.40F.sub.2N.sub.8O.sub.9: 754.29;
found: 755.4 (M+H.sup.+).
Example 34--SDC-TRAP-326 Structure and Synthesis
[0612] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-326
(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol--
4-yl)benzyl)
piperazin-1-yl)-3-oxopropyl(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydro-
xymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate)
is shown below.
##STR00159##
Step 1
[0613] To a solution of
4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldim-
ethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-on-
e (245 mg, 0.5 mmol) in dry DCM (2.5 mL) at 0.degree. C. was added
pyridine (0.16 mL, 2 mmol) and phosgene in toluene (1.4 mL, 2
mmol). The mixture was stirred at 0.degree. C. for 1 h. The solvent
was removed and the product dried on high vacuum. To the above
product in toluene (5 mL) was added tert-butyl 3-hydroxypropanoate
(0.15 mL, 1 mmol) and heated at 100.degree. C. for 1.5 h. The
solvent was removed and the crude residue purified by ISCO using
DCM/MeOH (0-10%) as eluent to afford 283 mg (85%) of product. ESMS
calculated for C.sub.29H.sub.51F.sub.2N.sub.3O.sub.8Si.sub.2:
663.90; found: 664.7 (M+H+).
Step 2
[0614] To a solution of tert-butyl
3-(((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimeth-
ylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyr-
imidin-4-yl)carbamoyl)oxy)propanoate (0.28 g, 0.421 mmol) in dry
DCM (3 mL) was added slowly TFA (3 mL). The mixture was stirred at
room temperature for 2 h, solvent was removed and the product (250
mg, 98%) dried on high vacuum. ESMS calculated for
C.sub.25H.sub.43F.sub.2N.sub.3O.sub.8Si.sub.2: 607.79; found: 608.4
(M+H+).
Step 3& 4
[0615] To a mixture of
3-(((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimeth-
ylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyr-
imidin-4-yl)carbamoyl)oxy)propanoic acid (95 mg, 0.15 mmol) and
4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6--
isopropylbenzene-1,3-diol hydrochloride (70 mg, 0.15 mmol) in DMF
(2 mL) was added EDCI (45 mg, 0.24 mmol), HOBt (21 mg, 0.15 mmol)
and DIPEA (0.1 mL, 0.6 mmol). The reaction mixture was stirred at
room temperature overnight. The solvent was removed and the crude
residue was purified by ISCO using DCM/MeOH (0-25%) as eluent to
get 25 mg of product.
[0616] The above product was dissolved in THF (2 mL) and treated
with TBAF (1M, 0.2 mL, 0.2 mmol). The mixture was stirred at room
temperature for 1.5 h. The solvent was removed and the crude
product purified by ISCO using DCM/MeOH (0-25%) as eluent to afford
of the title compound (21 mg). ESMS calculated for
C.sub.35H.sub.40F.sub.2N.sub.8O.sub.10: 770.28; found: 771.4
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.93 (s, 1H),
10.86 (s, 1H), 9.61 (s, 1H), 9.40 (s, 1H), 8.22 (d, J=7.6 Hz, 1H),
7.31 (d, J=8.4 Hz, 2H), 7.18-7.12 (m, 2H), 7.09 (d, J=7.6 Hz, 1H),
6.78 (s, 1H), 6.32 (d, J=6.5 Hz, 1H), 6.26 (s, 1H), 6.16 (t, J=7.5
Hz, 1H), 5.31 (t, J=5.5 Hz, 1H), 4.33 (t, J=6.4 Hz, 2H), 4.26-4.10
(m, 1H), 3.90-3.86 (m, 1H), 3.81-3.78 (m, 1H), 3.69-3.59 (m, 1H),
3.46-3.44 (m, 6H), 2.97 (p, J=6.9 Hz, 1H), 2.71 (t, J=6.4 Hz, 2H),
2.37-2.325 (m, 4H), 0.95 (d, J=6.9 Hz, 6H).
##STR00160##
Example 35--SDC-TRAP-327 Structure and Synthesis
[0617] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-327
(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol--
4-yl)-2-fluorobenzyl)piperazin-1-yl)-3-oxopropyl(1-((2R,4R,5R)-3,3-difluor-
o-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)carbamate) is shown below.
##STR00161##
[0618] The above compound was prepared following protocols
described for SDC-TRAP-326. ESMS calculated for
C.sub.35H.sub.39F.sub.3N.sub.8O.sub.10: 788.27; found: 789.6
(M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.99 (s, 1H),
10.86 (s, 1H), 9.66 (s, J=2.7 Hz, 1H), 9.41 (s, J=1.5 Hz, 1H), 8.22
(d, J=7.7 Hz, 1H), 7.39 (t, J=8.2 Hz, 1H), 7.14-7.03 (m, 2H), 6.97
(dd, J=8.1, 2.0 Hz, 1H), 6.87 (s, 1H), 6.33 (d, J=6.5 Hz, 1H), 6.27
(s, 1H), 6.16 (t, J=7.4 Hz, 1H), 5.32 (broad s, 1H), 4.32 (t, J=6.4
Hz, 2H), 4.28-4.10 (m, 1H), 3.88 (dt, J=8.6, 3.0 Hz, 1H), 3.82-3.79
(m, 1H), 3.68-3.62 (m, 1H), 3.52 (s, 2H), 3.43 (broad s, 4H), 3.00
(h, J=6.8 Hz, 1H), 2.71 (t, J=6.5 Hz, 2H), 2.35 (dt, J=23.1, 4.7
Hz, 4H), 1.00 (d, J=6.9 Hz, 6H).
Example 36--SDC-TRAP-328 Structure and Synthesis
[0619] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-328
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-((4-(4-(4-(3-(2,4-dihydroxy-5-is-
opropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-1,3,-
5-triazin-2-yl)amino)acetamide) is shown below.
##STR00162##
Step 1
[0620]
4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetra-
hydrofuran-2-yl)pyrimidin-2(1H)-one (131 mg, 0.5 mmol) and
2,5-dioxopyrrolidin-1-yl 2-((tert-butoxycarbonyl)amino)acetate (148
mg, 0.5 mmol) in dry DMF (5 mL) heated at 45.degree. C. for 20 h.
The solvent was removed and the crude product purified by ISCO
using DCM/MeOH (0-10%) as eluent to afford product (90 mg, 42.8%).
ESMS calculated for C.sub.16H.sub.22F.sub.2N.sub.4O.sub.7: 420.37;
found: 421.5 (M+H+).
Step 2
[0621] To a solution of tert-butyl
(2-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofura-
n-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2-oxoethyl)carbamate
(90 mg, 0.214 mmol) in dry DCM (3 mL) was added slowly TFA (1.5
mL). The mixture was stirred at room temperature for 1 h, solvent
was removed and the product (90 mg, 98%) dried on high vacuum.
Step 3 & 4
[0622] To a solution of
2-amino-N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahyd-
rofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide, TFA salt
(90 mg, 0.21 mmol) in dry THF (3 mL) was added
2,4-dichloro-1,3,5-triazine (32 mg, 0.21 mmol) and DIPEA (93 .mu.L,
0.53 mmol) at 0.degree. C. The reaction mixture was stirred at this
temperature for 2 h and concentrated, dried on high vacuum.
[0623] The above crude product
2-((4-chloro-1,3,5-triazin-2-yl)amino)-N-(1-((2R,4R,5R)-3,3-difluoro-4-hy-
droxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydro
pyrimidin-4-yl)acetamide (0.21 mmol) was dissolved in DMF (3 mL)
then treated with
4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6--
isopropylbenzene-1,3-diol hydrochloride (95 mg, 0.21 mmol) and
DIPEA (93 .mu.L, 0.53 mmol). The reaction mixture was stirred at
room temperature for 2 h. The solvent was removed and the crude
product purified by ISCO using DCM/MeOH (0-10%) as eluent to afford
impure product. The impure product was purified again on reverse
phase column chromatography using ACN/H.sub.2O (0.1% HCOOH) as
eluent to get 32 mg of product. ESMS calculated for
C.sub.36H.sub.40F.sub.2N.sub.12O.sub.8: 806.31; found: 807.8
(M+H+). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.93 (s, 1H), 11.17
(s, 1H), 9.61 (s, 1H), 9.41 (s, 1H), 8.27-8.24 (m, 1H), 8.18-8.04
(m, 1H), 7.71 (t, J=6.0 Hz, 1H), 7.39-7.10 (m, 5H), 6.76 (d, J=6.9
Hz, 1H), 6.33 (broad s, 1H), 6.27 (s, 1H), 6.18 (t, J=7.4 Hz, 1H),
5.30 (broad s, 1H), 4.28-3.98 (m, 3H), 3.89 (dt, J=8.5, 3.0 Hz,
1H), 3.81-3.78 (m, 1H), 3.76-3.54 (m, 6H), 2.96 (h, J=6.9 Hz, 1H),
2.38 (broad s, 2H), 2.27 (broad s, 3H), 0.93 (d, J=6.9 Hz, 6H).
##STR00163##
Example 37--SDC-TRAP-329 Structure and Synthesis
[0624] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-329
(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-
-triazol-4-yl)benzoyl)piperazin-1-yl)-2-methoxyphenyl
(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2--
yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate) is shown below.
##STR00164##
[0625] .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.34 (s, 1H), 10.22
(s, 1H), 9.74 (s, 1H), 9.02 (t, J=5.9 Hz, 1H), 8.26 (d, J=7.6 Hz,
1H), 7.54-7.49 (m, 2H), 7.47-7.35 (m, 2H), 7.04 (t, J=8.0 Hz, 2H),
6.80-6.67 (m, 2H), 6.55-6.47 (m, 1H), 6.33 (d, J=6.9 Hz, 2H), 6.18
(t, J=7.3 Hz, 1H), 5.30 (t, J=5.5 Hz, 1H), 4.27-4.06 (m, 2H), 3.78
(s, 9H), 3.18 (dd, J=12.5, 6.0 Hz, 6H), 3.04-2.89 (m, 1H), 1.06 (t,
J=7.2 Hz, 3H), 0.91 (d, J=6.8 Hz, 6H). ESMS calculated
(C.sub.42H.sub.45F.sub.2N.sub.9O.sub.11): 889.3; found: 890.4
(M+H).
Example 38--SDC-TRAP-330 Structure and Synthesis
[0626] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-330
(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-
-triazol-4-yl)benzoyl)piperazin-1-yl)-2-methylphenyl
(1-((2S,4S,5S)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2--
yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate) is shown below.
##STR00165##
[0627] ESMS calculated (C.sub.42H.sub.45F.sub.2N.sub.9O.sub.10):
873.3; found: 874.4 (M+H).
Example 39--SDC-TRAP-331 Structure and Synthesis
[0628] The structure and synthesis of an exemplary SDC-TRAP,
SDC-TRAP-331
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5-isopro-
pylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-
pyrazine-2-carboxamide) is shown below.
##STR00166##
[0629] To the solution of TBS protected Gemcitabine (0.49 g, 1.0
mmol) in DCM (20 mL) was added 5-chloropyrazine-2-carbonyl chloride
(0.2 g, 1.1 mmol) and TEA (0.16 mL, 1.1 mmol). The reaction was
stirred at room temperature for 60 min before it was concentrated.
The column chromatography gave the product (0.51 g, 98%).
##STR00167##
[0630] To the solution of starting material (0.05 g, 0.096 mmol) in
DMF (3 mL) was added amine (0.05 g, 0.1 mmol) and K.sub.2CO.sub.3
(0.041 g, 0.3 mmol). The reaction was heated in the microwave at
85.degree. C. for 60 min before H.sub.2O (3 mL) was added. The
mixture was extracted with EtOAc (15 mL) and the organic phase was
dried over Na.sub.2SO.sub.4 and concentrated. The column
chromatography gave the product (0.06 g, 61%).
##STR00168##
[0631] To the solution of starting material (0.03 g, 0.03 mmol) in
THF (2 mL) was added TBAF (1M in THF, 0.09 mL, 0.09 mmol). The
reaction was stirred for 60 min before the reaction was
concentrated. The column chromatography gave SDC-TRAP-331 (0.02 g,
84%).
[0632]
(N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydr-
ofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-5-(4-(4-(3-(2,4-dihydroxy-5--
isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-
-1-yl)pyrazine-2-carboxamide); .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 11.98 (s, 1H), 10.07 (s, 1H), 9.62 (s, 1H), 9.39 (s, 1H),
8.76 (d, J=1.0 Hz, 1H), 8.44-8.29 (m, 2H), 7.51-7.38 (m, 2H), 7.08
(dd, J=10.8, 2.0 Hz, 1H), 6.99 (dd, J=8.2, 2.0 Hz, 1H), 6.87 (s,
1H), 6.33 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.20 (t, J=7.4 Hz, 1H),
5.33 (t, J=5.4 Hz, 1H), 4.21 (ddd, J=19.6, 12.9, 7.5 Hz, 1H),
3.95-3.63 (m, 8H), 3.58 (s, 2H), 3.00 (p, J=6.9 Hz, 1H), 0.99 (d,
J=6.9 Hz, 6H); ESMS calculated
(C.sub.36H.sub.37F.sub.3N.sub.10O.sub.8): 794.3; found: 795.8
(M+H).
Example 40--SDC-TRAP-332 Structure
[0633] The structure of an exemplary SDC-TRAP, SDC-TRAP-332, is
shown below.
##STR00169##
Example 41--SDC-TRAP-333 Structure
[0634] The structure of an exemplary SDC-TRAP, SDC-TRAP-333, is
shown below.
##STR00170##
Example 42--SDC-TRAP-334 Structure
[0635] The structure of an exemplary SDC-TRAP, SDC-TRAP-334, is
shown below.
##STR00171##
[0636] FIG. 3 shows a summary of the structure, physical
properties, type of linkage, Her2 IC.sub.50 (nM) and cytotoxicity
(BT474 IC50 nM) for the exemplary SDC-TRAPs as described
herein.
[0637] All publications, patent applications, patents, and other
documents cited herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control.
[0638] The specification should be understood as disclosing and
encompassing all possible permutations and combinations of the
described aspects, embodiments, and examples unless the context
indicates otherwise. One of ordinary skill in the art will
appreciate that the invention can be practiced by other than the
summarized and described aspect, embodiments, and examples, which
are presented for purposes of illustration, and that the invention
is limited only by the following claims.
* * * * *