Note: Descriptions are shown in the official language in which they were submitted.
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VEGFR3 INHIBITORS
FIELD OF THE INVENTION
The present invention is concerned with the finding that some of the
macrocyclic
quinazoline derivatives described in PCT publication W02004/105765 are useful
as
inhibitors of VEGFR3 mediated biological activities, especially those
activities
which are mediated by VEGFR3 ligands VEGF-C and/or VEGF-D.
BACKGROUND OF THE INVENTION
Cancer is still a major cause of death in the world at the beginning of the 21
st
century and remains a major focus for ongoing research and development.
In recent years a promising approach to the therapeutic intervention of cancer
has
focused on antiangiogenesis therapies. This approach to intervening in cancer
progression takes advantage of the idea that inhibiting or otherwise limiting
the
blood supply to tumors will deplete the tumor of oxygen and nutrients and will
cause
arrest of tumor cell growth and proliferation. This approach has been found to
be
effective and there are presently over 20 anti-angiogenic drugs undergoing
various
stages of evaluation in phase I, II or III clinical trials and numerous others
in
preclinical development.
While there are many different forms of cancer exhibiting a wide variety of
properties, one factor which many cancers share is that, in order to be fatal,
they
must metastasize. Until such time as metastasis occurs, a tumor, although it
may be
malignant, is confined to one area of the body. This may cause discomfort
and/or
pain, or even lead to more serious problems, nevertheless if it can be located
prior to
metastasis, the cancer may be managed or even removed by surgical
intervention. So
long as the residual cancer cells are kept in check, such a discrete cancer
may be
controlled without significant problems. However, metastasis will cause the
cancerous cells to invade the body and while surgical resection may remove the
primary tumor, the metastatic spread of the cancer to disparate sites is very
difficult
to manage.
Metastasis to regional lymph nodes via lymphatic vessels is a common step in
the
progression of cancer. Metastasis is an important prognostic factor in many
types of
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cancer and fonns the basis for surgical and radiation treatment of local lymph
nodes.
The process of tumor metastasis is a multistage event involving local invasion
and
destruction of intercellular matrix, intravasation into blood vessels,
lymphatic or
other channels of transport, survival in the circulation, extravasation out of
the
vessels in the secondary site and growth in the new location (Idler, et al.,
Adv.
Cancer Res. 28,149-250 (1978), Liotta, et al., Cancer Treatment Res. 40,223-
238
(1988), Nicolson, Biochim. Biophy. Acta 948, 175-224(1988) and Zetter, N. Eng.
J.
Med. 322,605-612 (1990)). Success in establishing metastatic deposits requires
tumor cells to be able to accomplish these steps sequentially.
Recently, several lines of evidence have indicated that lymphangiogenesis, the
formation of lymphatic vessels, promotes lymphatic metastasis (Stacker et al.,
Nature Med. 7(2), 186-191 (2001); Skobe et al., Nature Med. 7(2), 192-8
(2001);
Makinen et al., Nature Med. 7(2), 199-205 (2001)). The control of
lymphangiogenesis may provide a new strategy for preventing lymph node
metastasis in cancer therapy.
Recent studies have shown that a member of the vascular endothelial growth
factor
(VEGF) family, VEGF-C, stimulates lymphangiogenesis and lymphatic endothelial
cell growth and migration upon binding to its receptor, VEGFR3 (Karkkainen MJ,
et
al., Semin Cell Dev Biol. 13:9-18 (2002)). VEGF-C has also been shown to
promote lymphatic-mediated metastasis via induction of tumor-associated
lymphangiogenesis in numerous solid cancers, such as gastric cancer, prostatic
cancer, human colorectal cancer, invasive cervical cancer, breast cancer
metastases
and human melanoma metastases. In support of these are preclinical data
demonstrating that VEGF-C overexpression in cancer cells significantly
increases
tumor-associated lymphangiogenesis, resulting in enhanced metastasis to
regional
lymph nodes (Stacker SA., et al., FASEB J 16:922-34 (2002)). In addition,.
blockade of VEGF-C/D-mediated signaling has been shown to suppress tumor
lymphangiogenesis and lymph node metastases in mice (He Y., et al., J Natl
Cancer
Inst. 94:819-25 (2002)).
VEGFR3, a transmembrane tyrosine kinase receptor is expressed broadly in
endothelial cells during early embryogenesis (Pajusola K., et al., Cancer Res.
53(16):3845 (1992)). During later stages of development, the expression of
VEGFR-3 becomes restricted to developing lymphatic vessels [Kaipainen, A., et
al.,
Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)]. In adults, the lymphatic
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endothelium and some high endothelial venules express VEGFR-3, and increased
expression occurs in lymphatic sinuses in metastatic lymph nodes and in
lymphangioma. VEGFR-3 is also expressed in a subset of CD34+ hematopoietic
cells which may mediate the myelopoietic activity of VEGF-C demonstrated by
overexpression studies. Targeted disruption of the VEGFR-3 gene in mouse
embryos leads to failure of the remodeling of the primary vascular network,
and
death after embryonic day 9.5 [Dumont et al., Science, 282: 946-949 (1998)].
These
studies suggest an essential role for VEGFR-3 in the development of the
embryonic
vasculature, and also during lymphangiogenesis.
From the foregoing, it will be apparent that inhibitors of VEGFR3, have
tremendous
potential as therapeutics, and new agents of this type represent a continuing
need in
the art for inhibiting growth and/or spread of a variety of neoplastic
disorders or cell
proliferative disorders. Inhibition of VEGFR3 activity, including inhibition
of
ligand binding to VEGFR3 is useful in the treatment of mammalian subjects that
have been diagnosed with a disease characterized by proliferation of
endothelial
cells that express VEGFR-3. For example, many tumors are characterized by
blood
vessel or lymphatic vessel neovascularization, wherein the neovascularization
comprises endothelial cells that express VEGFR-3. In some cancers, the cancer
cells
themselves express VEGFR3, and represent the target cells. In another,
possibly
overlapping set of cancers, the cancer cells express a VEGFR-3 ligand selected
from
VEGF-C and VEGF-D. The ligand is believed to be involved in recruiting
endothelial cells and stimulating their growth, thereby facilitating
nourishment
and/or spread of the cancer.
SUMMARY OF THE INVENTION
The invention is directed in part to methods of treating or preventing VEGFR3
mediated biological activities utilizing certain compounds described in
WO2004/105765, the disclosure of which is hereby incorporated by reference in
its
entirety.
In a related embodiment, the invention provides a method of inhibiting
metastatic
spread of a cancer in a mammalian subject comprising administering to a
mammalian subject suspected of having cancer a compound of the invention, in
an
amount effective to inhibit metastatic spread of the cancer; and a method for
treating
cancer comprising administering to a mammalian subject diagnosed with a cancer
a
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composition comprising a compound of the invention, in an amount effect to
reduce
growth or neoplastic spread of the cancer. It will be appreciated that any
reduction in
the rate of cancer growth or spread (which can prolong life and quality of
life) is
indicative of successful treatment. In preferred embodiments, cancer growth is
halted completely. In still more preferred embodiments, cancers shrink or are
eradicated entirely. Preferred subjects for treatment are human subjects, but
other
animals, especially murine, rat, canine, bovine, porcine, primate, and other
model
systems for cancer treatment, are contemplated.
In some cancers that express VEGFR-3, direct inhibition of cancer growth or
cancer
killing may be the mechanism. Treatment of all cancers that express VEGFR-3
and
all cancers characterized by angiogenesis or lymphangiogenesis in and around a
growing tumor is contemplated. For example, treatment is contemplated of
cancers
of a tissue, organ, or cell selected from the group consisting of brain, lung,
liver,
spleen, kidney, lymph node, small intestine, blood cells, pancreas, colon,
stomach,
breast, endometrium, prostate, testicle, ovary, skin, head and neck,
esophagus, bone
marrow and blood. In a particular embodiment of the present invention,
treatment is
contemplated of cancers of a tissue, organ or cell selected from the group
consisting
of Breast, Lung, Prostate and Colon.
In one variation of the foregoing methods of treatment, the compounds are
administered along with a second cancer therapeutic agent. The second agent
can be
any chemotherapeutic agent, radioactive agent, radiation, nucleic acid
encoding a
cancer therapeutic agent, antibody, protein, and/or other anti-lymphangiogenic
agent
or an anti-angiogenic agent. The second agent may be administered before,
after, or
concurrently with the compounds of the invention.
In one variation, the subject to be treated has been diagnosed with an
operable
tumor, and the administering step is performed before, during, or after the
tumor is
resected from the subject. Compound treatment in conjunction with tumor
resection
is intended to reduce or eliminate regrowth of tumors from cancer cells that
fail to
be resected.
Stated more generically, the invention provides a method of treating a
pathology
characterized by VEGFR-3 binding to a natural ligand that binds VEGFR-3,
comprising the step of administering to an individual in need thereof a
compound of
the invention.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Effect of 4,6-ethanediylidenepyrimido
[4,5-b][6,1,12]benzoxadiazacyclopentadecine, 17-bromo-
8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl- (Compound 2)
on VEGF-C induced VEGFR3 activity. Top frame provides the results of
VEGFR-3 immunoprecipitates transferred to a nitrocellulose membrane,
stained with phospho-tyrosine antibodies. Lower frame provides the
results of VEGFR-3 immunoprecipitates transferred to a nitrocellulose
membrane, stained with VEGFR-3 antibodies. Left lanes 1 and 2 provide
the negative and positve controls, i.e. DMSO treatment in the absence and
presence of VEGF-C.
Figure 2: VEGF and VEGF-C stimulation of Erkl/2 phosphorylation in HMVECd
cells in the presence of 5uM of Compound 2. Top lane provides staining
of phoshorylated Erk 1/2 (P-Erk 1/2) using Thr202/Tyr204 mouse
monoclonal antibody as primary antibody and goat anti-mouse conjugated
to IRDye 800nm as secondary antibody. Bottom lane provides staining of
total Erk 1/2 using Erk 1/2 specific rabbit polyclonal antibody as primary
antibody and goat anti-rabbit conjugated to Alexa 680 nm as secondary
antibody. Left panel provides the results for VEGF induced
phosphorylation of Erk-1/2 in HMVECd cells. Right panel provides the
results for VEGF-C induced phosphorylation of Erk 1/2 in HMVECd
cells.
DETAILED DESCRIPTION OF THE INVENTION
WO-2004/105765 describes the preparation, formulation and pharmaceutical
properties of a class of macrocyclic quinazoline derivatives of formula (I) as
multi
targeted kinase inhibitors.
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X2 3' / R1
~
Y 2'
~\JS'
z 6' R2
X 5
N3 R3
Ra~- I //J
7 ~ % 2 (I)
8 N1
It has now been found that a particular group of compounds within the
aforementioned class have VEGFR3 inhibitory activity, that makes them useful
in
the treatment or prevention of VEGFR3 mediated biological activities, in
particular
for the treatment or prevention of metastatic spread of a cancer in a
mammalian
subject.
Accordingly, in one aspect the present invention provides the use of the
compounds
of formula (I) wherein;
Z represents NH;
Y represents -C3_9a1ky1-, -C1_5alkyl-NR13-Cl_5alkyl-, -C1_5alkyl-NR14-CO-
C1_5alkyl-,
-C1_3alkyl-NH-CO-Het20-, or -Het22-CHZ-CO-NH-C1_3alkyl-;
Xi represents 0, or -O-C1_2alkyl-;
X2 represents a direct bond, -C1_2alkyl-, 0, -O-C1_2alkyl-, NR12 or NR12-
C1_2alkyl-;
Ri represents hydrogen, cyano, halo or hydroxy, preferably halo;
RZ represents hydrogen, cyano, halo, or hydroxy;
R3 represents hydrogen;
R4 represents C1_4alkyloxy-;
R12 represents hydrogen, or C1_4alkyl-;
R13 represents hydrogen or C1_4alkyl;
R14 represents hydrogen or C1_4alkyl;
Het20 represents pyrrolidinyl, piperazinyl or piperidinyl; and
Het22 represents pyrrolidinyl, piperazinyl or piperidinyl;
the pharmaceutically acceptable acid or base addition salts and the
stereochemically
isomeric forms thereof, in the manufacture of a medicament for the treatment
or
prevention of VEGFR3 mediated biological activities, such as for example in
the treatment or prevention of metastatic spread of a cancer in a mammalian
subj ect.
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A further aspect of the present invention is directed to a method for the
treatment or
prevention of a VEGFR3 mediated biological activity, such as for example in
the
treatment or prevention of metastatic spread of a cancer in a mammalian
subject,
comprising administering therapeutically effective amount of a compound of
formula I
X2 3 R1
/'
Y 2' 1
~ J\ 5t
Z 6' R2
X~ 5 4
6~ ~N 3 Rs
R4~--- Ii /
7 ~ % 2 (I)
8 N1
wherein;
Z represents NH;
Y represents -C3_9alkyl-, -C1_5alkyl-NR13-C1_5alkyl-, -C1_5alkyl-NR14-CO-
C1_5alkyl-,
-C1_3alkyl-NH-CO-Het20-, or -Het22-CHZ-CO-NH-C1_3alkyl-;
Xl represents 0, or -O-C1_2alkyl-;
X2 represents a direct bond, -C1_2alkyl-, 0, -O-C1_2alkyl-, NR12 or NR12-
C1_2alkyl-;
Rl represents hydrogen, cyano, halo or hydroxy, preferably halo;
RZ represents hydrogen, cyano, halo, or hydroxy;
R3 represents hydrogen;
R4 represents C1_4alkyloxy-;
R12 represents hydrogen, or C1_4alkyl-;
R13 represents hydrogen or Cl4alkyl;
R14 represents hydrogen or C1_4alkyl;
Het20 represents pyrrolidinyl, piperazinyl or piperidinyl;
Het22 represents pyrrolidinyl, piperazinyl or piperidinyl; and
the pharmaceutically acceptable acid or base addition salts and the
stereochemically
isomeric forms thereof;
to a mammalian subject in need of such treatment.
Further VEGFR3 mediated biological activities are meant to include;
- metastatic spread of a cancer in a mammalian subject. Preferred subjects
for treatment are human subjects, but other animals, especially murine,
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rat, canine, bovine, porcine, primate and other model systems for cancer
treatment are contemplated.
- Metastasis to regional lymph nodes via lymphatic vessels. It accordingly
provides the use of the compounds according to the invention in the
treatment or prevention of lymph node metastasis;
- tumor-associated lymphangiogenesis in cancers, such as for example in
gastric cancer, prostatic cancer, human colorectal cancer, invasive
cervical cancer, breast cancer, Kapsoi's sarcoma and melanoma. It
accordingly provides the use of the compounds according to the
invention in the treatment or prevention of tumor-associated
lymphangiogenesis in cancers, such as for example in gastric cancer,
prostatic cancer, human colorectal cancer, invasive cervical cancer,
breast cancer, Kapsoi's sarcoma and melanoma;
- recruitment and proliferation of endothelial cells that express VEGFR3 in
neovascularization of cancer cells that express a VEGFR3 ligand selected
from VEGF-C or VEGF-D. It accordingly provides the use of the
compounds according to the invention in the treatment of cancers
characterized by angiogenesis or lymphangiogenesis in and around a
growing tumor.
In a further embodiment, the present invention provides the use of an
aforementioned compound of formula (I) for the preparation of a pharmaceutical
composition for treatment of cancers of a tissue, organ or cell selected from
the
group consisting of Breast, Lung (including the treatment of both small cell
and non-
small cell lung cancer), Colon and Prostate.
The present invention also concerns a method of treating breast cancer, lung
cancer,
colon cancer and/or prostate cancer in a mammal, comprising the step of
administering a therapeutically effective amount of an aforementioned compound
of
formula (I) to said mammal.
In an even further embodiment, the present invention provides the use of an
aforementioned compound of formula (I) for the preparation of a pharmaceutical
composition for treating advanced breast cancer. The term "advanced breast
cancer"
is used herein to denote breast cancer which has not responded to previous
treatment, or which has recurred following such treatment, and also breast
cancer in
patients who present with metastatic disease at diagnosis.
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The present invention also concerns a method of treating advanced breast
cancer in a
mammal, particularly a woman, comprising the step of administering a
therapeutically effective amount of an aforementioned compound of formula (I)
to
said mammal.
In particular, the present invention is concerned with the use of those
compounds of
formula (I) wherein one or more of the following restrictions apply;
Z represents NH;
Y represents -C3_9alkyl-, -Cl_5alkyl-NR14-CO-C1_5alkyl-, or -C1_3alkyl-NH-CO-
Het20-;
Xl represents 0;
Xz represents -C1_zalkyl-, 0, or NR12-C1_2alkyl-;
Rl represents hydrogen or halo; in particular Rl represents hydrogen or
chloro; more
in particular R' represents hydrogen;
R2 represents hydrogen or halo; in particular R2 represents hydrogen, chloro
or
bromo; more in particular R2 represents chloro or bromo;
R3 represents hydrogen;
R4 represents C1_4alkoxy; in particular R4 represents methoxy;
R12 represents C1_4alkyl-; in particular R12 represents methyl;
R14 represents hydrogen or C1_4alkyl; in particular R14 represents hydrogen or
methyl; more in particular R14 represents hydrogen;
Het20 represents pyrrolidinyl, piperazinyl or piperidinyl; in particular Het20
represents piperidinyl.
Also of interest in the aforementioned uses, are those compounds of formula
(I)
wherein Rl is at position 3', R2 is at position 5', R4 is at position 7 and X2
is at
position 2', using the numbering as presented in Formula (I) above.
Most preferred compounds are those compounds selected from the group
consisting
of;
4,6-ethanediylidene-19H-pyrimido[4,5-b] [6,13,1 ]benzodioxaazacyclo-
pentadecine,
15-chloro-8,9,10,11,12,13-hexahydro-20-methoxy-;
12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-
3 5 b] [6,1,10,16]benzoxatriazacyclononadecin-12-one, 21-chloro-
8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy-;
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4,6-ethanediylidene-l2H-pyrimido[4,5-b] [6,1,10,13]benzoxatriazacyclohexadecin-
12-one, 18-chloro-8,9,10,11,13,14,15,20-octahydro-21-methoxy-13,14-
dimethyl-; and
4,6-ethanediylidenepyrimido[4,5-b][6,1,12]benzoxadiazacyclopentadecine, 17-
bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy- 13 -methyl-; or a
pharmaceutically acceptable acid addition salt thereof. The latter compound is
especially preferred.
Most preferred compounds for use in accordance with the present invention are
those selected from the group consisting of compounds having the following
structure:
CI
O ~ aBr
~
HN ~ HN O 0
N N
O N~ O N
- 0
H
N
j
0 H
CO HN CI and O HN CI
~ ~ N N
O ( / N \O N
or a pharmaceutically acceptable acid addition salt thereof.
The following compound is especially preferred:
aBr
HN O
N
\O N~
or a pharmaceutically acceptable acid addition salt thereof.
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As used in the foregoing definitions and hereinafter,
- halo is generic to fluoro, chloro, bromo and iodo;
- C1_2a1ky1 defines methyl or ethyl;
- C1_3alkyl defines straight and branched chain saturated hydrocarbon radicals
having from 1 to 3 carbon atoms such as, for example, methyl, ethyl, propyl
and
the like;
- C14alkyl defines straight and branched chain saturated hydrocarbon radicals
having from 1 to 4 carbon atoms such as, for example, methyl, ethyl, propyl,
butyl,
1-methylethyl, 2-methylpropyl, 2,2-dimethylethyl and the like;
- C1_5alkyl defines straight and branched chain saturated hydrocarbon radicals
having from 1 to 5 carbon atoms such as, for example, methyl, ethyl, propyl,
butyl, pentyl,
1 -methylbutyl, 2,2-dimethylpropyl, 2,2-dimethylethyl and the like;
- C3_9alkyl defines straight and branched chain saturated hydrocarbon radicals
having from 3 to 9 carbon atoms such as propyl, butyl, pentyl, hexyl, heptyl,
octyl, nonyl and the like;
- C1_4alkyloxy defines straight or branched saturated hydrocarbon radicals
such as
methoxy, ethoxy, propyloxy, butyloxy, 1-methylethyloxy, 2-methylpropyloxy
and the like;
- the term "CO" refers to a carbonyl group.
The pharmaceutically acceptable acid or base addition salts as mentioned
hereinabove are meant to comprise the therapeutically active non-toxic acid
and non-
toxic base addition salt forms which the compounds of formula (I) are able to
form.
The compounds of formula (I) which have basic properties can be converted in
their
pharmaceutically acceptable acid addition salts by treating said base form
with an
appropriate acid. Appropriate acids comprise, for example, inorganic acids
such as
hydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric; nitric;
phosphoric
and the like acids; or organic acids such as, for example, acetic, propanoic,
hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e. butanedioic
acid),
maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,
benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic,
pamoic and
the like acids.
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The compounds of formula (I) which have acidic properties may be converted in
their pharmaceutically acceptable base addition salts by treating said acid
form with
a suitable organic or inorganic base. Appropriate base salt forms comprise,
for
example, the ammonium salts, the alkali and earth alkaline metal salts, e.g.
the
lithium, sodium, potassium, magnesium, calcium salts and the like, salts with
organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts,
and
salts with amino acids such as, for example, arginine, lysine and the like.
The terms acid or base addition salt also comprise the hydrates and the
solvent
addition forms which the compounds of formula (I) are able to form. Examples
of
such forms are e.g. hydrates, alcoholates and the like.
The term stereochemically isomeric forms of compounds of formula (I), as used
hereinbefore, defines all possible compounds made up of the same atoms bonded
by
the same sequence of bonds but having different three-dimensional structures
which
are not interchangeable, which the compounds of formula (I) may possess.
Unless
otherwise mentioned or indicated, the chemical designation of a compound
encompasses the mixture of all possible stereochemically isomeric forms which
said
compound may possess. Said mixture may contain all diastereomers and/or
enantiomers of the basic molecular structure of said compound. All
stereochemically isomeric forms of the compounds of formula (I) both in pure
form
or in admixture with each other are intended to be embraced within the scope
of the
present invention.
Some of the compounds of formula (I) may also exist in their tautomeric forms.
Such forms although not explicitly indicated in the above formula are intended
to be
included within the scope of the present invention.
Whenever used hereinafter, the term "compounds of formula (I)" or "the
compounds
according to the invention" is meant to include also the pharmaceutically
acceptable
acid or base addition salts and all stereoisomeric forms.
The compounds according to the invention can be prepared and formulated into
pharmaceutical compositions by methods known in the art and in particular
according to the methods described in the published patent specification
mentioned
herein and incorporated by reference; for the compounds of formula (I)
suitable
examples can be found in PCT publication WO-2004/105765. To prepare the
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aforementioned pharmaceutical compositions, a therapeutically effective amount
of
the particular compound, optionally in addition salt form, as the active
ingredient is
combined in intimate admixture with a pharmaceutically acceptable carrier,
which
may take a wide variety of forms depending on the form of preparation desired
for
administration. These pharmaceutical compositions are desirably in unitary
dosage
form suitable, preferably, for systemic administration such as oral,
percutaneous, or
parenteral administration; or topical administration such as via inhalation, a
nose
spray, eye drops or via a cream, gel, shampoo or the like. For example, in
preparing
the compositions in oral dosage form, any of the usual pharmaceutical media
may be
employed, such as, for example, water, glycols, oils, alcohols and the like in
the case
of oral liquid preparations such as suspensions (including nanosuspensions),
syrups,
elixirs and solutions; or solid carriers such as starches, sugars, kaolin,
lubricants,
binders, disintegrating agents and the like in the case of powders, pills,
capsules and
tablets. Because of their ease in administration, tablets and capsules
represent the
most advantageous oral dosage unit form, in which case solid pharmaceutical
carriers are obviously employed. For parenteral compositions, the carrier will
usually comprise sterile water, at least in large part, though other
ingredients, for
example, to aid solubility, may be included. Injectable solutions, for
example, may
be prepared in which the carrier comprises saline solution, glucose solution
or a
mixture of saline and glucose solution. Injectable solutions containing
compounds
of formula (I) may be formulated in an oil for prolonged action. Appropriate
oils for
this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn
oil, soy
bean oil, synthetic glycerol esters of long chain fatty acids and mixtures of
these and
other oils. Injectable suspensions may also be prepared in which case
appropriate
liquid carriers, suspending agents and the like may be employed. In the
compositions suitable for percutaneous administration, the carrier optionally
comprises a penetration enhancing agent and/or a suitable wettable agent,
optionally
combined with suitable additives of any nature in minor proportions, which
additives do not cause any significant deleterious effects on the skin. Said
additives
may facilitate the administration to the skin and/or may be helpful for
preparing the
desired compositions. These compositions may be administered in various ways,
e.g., as a transdermal patch, as a spot-on or as an ointment. As appropriate
compositions for topical application there may be cited all compositions
usually
employed for topically administering drugs e.g. creams, gels, dressings,
shampoos,
tinctures, pastes, ointments, salves, powders and the like. Application of
said
compositions may be by aerosol, e.g. with a propellent such as nitrogen,
carbon
dioxide, a freon, or without a propellent such as a pump spray, drops,
lotions, or a
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semisolid such as a thickened composition which can be applied by a swab. In
particular, semisolid compositions such as salves, creams, gels, ointments and
the
like will conveniently be used.
It is especially advantageous to formulate the aforementioned pharmaceutical
compositions in dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used in the specification and claims herein refers
to
physically discrete units suitable as unitary dosages, each unit containing a
predetermined quantity of active ingredient calculated to produce the desired
therapeutic effect in association with the required pharmaceutical carrier.
Examples
of such dosage unit forms are tablets (including scored or coated tablets),
capsules,
pills, powder packets, wafers, injectable solutions or suspensions,
teaspoonfuls,
tablespoonfuls and the like, and segregated multiples thereof.
Preferably, a therapeutically effective amount of the pharmaceutical
composition
comprising a compound according to the invention, is administered orally or
parenterally. Said therapeutically effective amount is the amount that
effectively
prevents metastasis and/or growth or reduces the size of a variety of
neoplastic
disorders or cell proliferative disorders (supra) in patients. On the basis of
the current
data, it appears that a pharmaceutical composition comprising a compound of
the
present invention, and in particular 4,6-ethanediylidenepyrimido
[4,5-b] [6,1,12]benzoxadiazacyclo-pentadecine, 17-bromo-8,9,10,11,12,13,14,19-
octahydro-20-methoxy-13-methyl- as the active ingredient can be administered
orally
in an amount of from 10 mg to several (1 to 5) grams daily, either as a single
dose or
subdivided into more than one dose, including, e.g. two, three or even four
times
daily. A preferred amount ranges from 500 to 4,000 mg daily. A particularly,
preferred dosage for such a compound is in the range of 750 mg to 3,000 mg
daily. It
will be appreciated that the amount of a compound according to the present
invention, also referred to here as the active ingredient, which is required
to achieve a
therapeutic effect will, of course, vary with, the route of administration,
the age and
condition of the recipient, and the particular disorder or disease being
treated. The
optimum dosage amounts and regimen can be readily determined by those skilled
in
the art using conventional methods and in view of the information set out
herein. This
treatment can be given either continuously or intermittently, including, e.g.,
but not
limited to, cycles of 3-4 weeks with treatment given for 1-21 days per cycle
or other
schedules shown to be efficacious and safe.
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The above VEGFR3 inhibitors may be used in combination with one or more other
cancer treatments. Such combinations could encompass any established antitumor
therapy, such as, but not limited to, chemotherapies, irradiation, and target
based
therapies such as antibodies and small molecules. These therapies may be
combined in
systemic therapy, or local instillation/administration (e.g. intrathecally),
depending on
optimum efficacy/safety requirements.
In certain preferred embodiments, one or more other cancer treatments suitable
in
combination with the VEGFR3 inhibitors of the present invention, with respect
to the
different tumor types, include, but are not limited to;
breast: herceptin, docetaxel, anthracyclin, capecitabine
prostate: docetaxel, mitoxantrone
colon: oxaliplatin, 5-FU, avastin, irinotecan, cetuximab
lung; taxotere, carboplatin, gemicitabine
The VEGFR3 inhibitor of the invention and the further anti-cancer agent may be
administered simultaneously (e.g. in separate or unitary compositions) or
sequentially in either order. In the latter case, the two compounds will be
administered within a period and in an amount and manner that is sufficient to
ensure that an advantageous or synergistic effect is achieved. It will be
appreciated
that the preferred method and order of administration and the respective
dosage
amounts and regimens for each component of the combination will depend on the
particularVEGFR3 inhibitor and further anti-cancer agents being administered,
their
route of administration, the particular tumor being treated and the particular
host
being treated. The optimum method and order of administration and the dosage
amounts and regimen can be readily determined by those skilled in the art
using
conventional methods and in view of the information set out herein.
PREPARATION OF COMPOUNDS AND FORMULATIONS:
Preparation of Compound 95, 12H-4,6-ethanediylidene-13,17-
methanopyrimidof4,5-b1 f6,1,10,161benzoxatriazacyclononadecin-12-one, 21-
chloro-8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy-
Step 1.
Titanium, tetrakis(2-propanolato) (0.005 mol) was added to a solution of 3-
piperidinecarboxylic acid ethyl ester (0.54 g, 0.005 mol) and 4-chloro-2-
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nitrobenzaldehyde (0.93 g, 0.005 mol) in CH2C12 (40 ml). The mixture was
stirred
for 1 hour at room temperature and NaBH(OAc)3 (0.0055 mol) was added. The
reaction mixture was stirred for 1 hour at room temperature and extra
NaBH(OAc)3
(0.00275 mol) was added and the mixture was stirred for 2 hours at room
temperature and then NaHCO3 (saturated in H20) was added. The organic layer
was separated, dried (K2C03), filtered and the solvent was evaporated and co-
evaporated with toluene to remove the residual CH2C12. The 3-
piperidinecarboxylic
acid, 1-[(4-chloro-2-nitrophenyl)methyl]-, ethyl ester as crude residue was
used as
such in the next reaction step.
Step 2.
A mixture of 3-piperidinecarboxylic acid, 1-[(4-chloro-2-nitrophenyl)methyl]-,
ethyl
ester (1.69 g, 0.005 mol) in ethanol (150 ml) was hydrogenated with Pt/C 5%
(0.5 g)
as a catalyst in the presence of a thiophene solution (1 ml). After uptake of
H2 (3
equiv.), the catalyst was filtered off and the filtrate was evaporated. The
residue was
redissolved in EtOAc and this solution was filtered over Extrelute. Heptane
was
added but no crystals appeared. The solvent was evaporated and the residue was
redissolved in CH3OH and filtered over a paper filter to remove silica grease.
The
solvent was evaporated to yield 1.35 g of 3-piperidinecarboxylic acid, 1-[(2-
amino-
4-chlorophenyl)methyl]-, ethyl ester as a brown oil.
Step3.
4-Chloro-7-methoxy-6-quinazolinol6-acetate (0.273 g, 0.00108 mol) was added to
a
solution of 3-piperidinecarboxylic acid, 1-[(2-amino-4-chlorophenyl)methyl]-,
ethyl
ester(0.0011 mol) in 2-propanol (q.s.) and then the reaction mixture was
shaken
overnight at 80 C. Then the mixture was further shaken for 6.5 hours at 80 C
and
the solvent was evaporated. Yield: 3-piperidinecarboxylic acid, 1-[[2-[[6-
(acetyloxy)-7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl]methyl] -, ethyl
ester
(crude; used as such in the next reaction step).
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Step 4.
NH3/CH3OH 7N (approx. 10 ml) was added to 3-piperidinecarboxylic acid, 1-[[2-
[ [6-(acetyloxy)-7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl]methyl] -,
ethyl
ester (0.00114 mol) and the reaction mixture was shaken for 1 hour at 35 C and
then
the solvent was evaporated. Yield: 3-piperidinecarboxylic acid, 1-[[4-chloro-2-
[(6-
hydroxy-7-methoxy-4-quinazolinyl)amino]phenyl]methyl]-, ethyl ester (used as
such in the next reaction step).
St~
A mixture of 3-piperidinecarboxylic acid, 1-[[4-chloro-2-[(6-hydroxy-7-methoxy-
4-
quinazolinyl)amino]phenyl]methyl]-, ethyl ester (0.00114 mol), N-(3-
bromopropyl)carbamic acid 1,1-dimethylethyl ester (1 eq) and Cs2CO3 (1.8582 g)
was stirred overnight at room temperature and then the reaction mixture was
stirred
for 30 minutes at 50 C. When necessary more N-(3-bromopropyl)carbamic acid 1,1-
dimethylethyl ester was added and the mixture was stirred for 4 hours at room
temperature and for another 15 min. at 50 C. The solvent was evaporated
(Genevac)
and the residue was dissolved in CH2C12. This solution was filtered over
dicalite
and the filtrate was evaporated. Yield: 3-piperidinecarboxylic acid, 1-[[4-
chloro-2-
[ [6-[3-[[(1,1-dimethylethoxy)carbonyl] amino]propoxy]-7-methoxy-4-
quinazolinyl]amino]phenyl]methyl]-, ethyl ester.
Step 6.
A solution of 3-piperidinecarboxylic acid, 1-[[4-chloro-2-[[6-[3-[[(l,l-
dimethylethoxy)carbonyl] amino]propoxy] -7-methoxy-4-
quinazolinyl]amino]phenyl]methyl]-, ethyl ester (residue; approx 0.00114 mol)
in
TFA/CH2C12/TIS (90/8/2) (11.4 ml) was stirred for 7-8 hours, then the solvent
was
evaporated and the obtained residue was dried overnight in an oven. Yield: 3-
piperidinecarboxylic acid, 1-[[2-[[6-(3-aminopropoxy)-7-methoxy-4-
quinazolinyl]amino]-4-chlorophenyl]methyl]- .CF3COOH.
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Step 7.
A solution of 3-piperidinecarboxylic acid, 1-[[2-[[6-(3-aminopropoxy)-7-
methoxy-
4-quinazolinyl]amino]-4-chlorophenyl]methyl]- .CF3COOH (0.00114 mol) and
DIPEA (0.00684 mol) was added to a solution of HBTU (0.00342 mol) and HOBt
(0.00228 mol) in dry DMF (285 ml) and then the reaction mixture was reacted
for 1
hour. The solvent was evaporated and the dry residue was purified by reversed-
phase high-performance liquid chromatography. The product fractions were
collected, Na2CO3 was added and the organic solvent was evaporated. CH2C12 was
added to the aqueous concentrate and the resulting mixture was extracted 3
times
with CH2C12, then the organic extract was dried and collected. Yield: 0.0007
mol of
12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-
b] [6,1,10,16]benzoxatriazacyclononadecin-l2-one, 21 -chloro-
8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy- (62 % yield).
Preparation of Compound 96, 4,6-ethanediylidene-12H-uyrimido14,5-
bl f6,1,10,131benzoxatriazacyclohexadecin-12-one, 18-chloro-
8,9,10,11,13,14,15,20-octahydro-2l-methoxy-13,14-dimethyl-, (13S)-
Step 1.
A mixture of N-methyl-L-alanine methyl ester hydrochloride (1.4 g, 0.010 mol),
4-
chloro-2-nitrobenzaldehyde (0.010 mol) and Titanium, tetrakis(2-propanolato)
(0.010 mol) in CH2C12 (q.s.) was stirred for 1 hour at room temperature. Then
NaBH(OAc)3 (0.022 mol) was added and the mixture was stirred for 3 hours at
room temperature. Mote NaBH(OAc)3 (0.011 mol) was added and the mixture was
stirred overnight at room temperature. Then NaHCO3 (saturated) was added till
basic and the mixture was filtered over a P3 filter. The organic layer was
separated
and the water layer was extracted 3 times with CH2C12. The combined organic
layers were dried (K2C03) and the solvent was evaporated to dryness. The crude
residue, L-alanine, N-[(4-chloro-2-nitrophenyl)methyl]-N-methyl-, methyl
ester, was
used as such in the next reaction step.
Step2.
A mixture of L-alanine, N-[(4-chloro-2-nitrophenyl)methyl]-N-methyl-, methyl
ester
(1.03 g, 0.0036 mol) in CH3OH (50 ml) was hydrogenated with Pt/C 5% (0.5 g) as
a
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catalyst in the presence of a 4% thiophene solution in DIPE (0.5 ml). After
uptake of
H2 (3 equiv.), the catalyst was filtered off and the filtrate was evaporated.
The
residue was purified over silica gel (with cartridge) (eluent: 4 % Et3N /
CH2C12).
The desired fractions were collected. Heptane was added to the desired
fraction (2
phases form). The heptane layer was separated to remove silica grease. After
removal of the solvent and drying 0.5674 g of L-alanine, N-[(2-amino-4-
chlorophenyl)methyl]-N-methyl-, methyl ester was obtained (22 %; yellow oil).
The
crude product was used as such in the next reaction step.
Step 3.
4-Chloro-7-methoxy-6-quinazolinol 6-acetate (0.00055 mol) was added to a
solution
of L-alanine, N-[(2-amino-4-chlorophenyl)methyl]-N-methyl-, methyl ester
(0.00055 mol) in 2-propanol (5 ml) and then the reaction mixture was shaken
overnight at 80 C and the solvent was evaporated. Yield: L-alanine, N-[[2-[[6-
(acetyloxy)-7-methoxy-4-quinazolinyl]amino]-4-chlorophenyl]methyl]-NV methyl-,
methyl ester (crude, used as such in the next reaction step).
Step 4.
NH3/CH3OH 7N (10 ml) was added to L-alanine, N-[[2-[[6-(acetyloxy)-7-methoxy-
4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-, methyl ester
(0.00055
mol) and the reaction mixture was shaken for 1-2 hours and then the solvent
was
evaporated. Yield: L-alanine, N-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-
quinazolinyl)amino]phenyl]methyl]-N-methyl-, methyl ester (used as such in the
next reaction step).
Step 5.
A mixture of L-alanine, N-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-
quinazolinyl)amino]phenyl]methyl]-N-methyl-, methyl ester (0.00055 mol), N-(3-
bromopropyl)carbamic acid 1,1-dimethylethyl ester (1 eq) and Cs2CO3 (0.8965 g)
was stirred overnight at room temperature and then the reaction mixture was
stirred
for 30 minutes at 50 C. When necessary, more CAS N-(3-bromopropyl)carbamic
acid 1, 1 -dimethylethyl ester was added and the mixture was stirred for 4
hours at
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room temperature and for another 15 minutes at 50 C. The solvent was
evaporated
(Genevac) and the residue was dissolved in CH2C12. This solution was filtered
over
dicalite and the filtrate was evaporated. Yield: L-alanine,lV-[[4-chloro-2-[[6-
[3-
[ [(1,1-dimethylethoxy)carbonyl] amino]propoxy]-7-methoxy-4-
quinazolinyl]amino]phenyl]methyl]-N-methyl-, methyl ester.
Step 6.
A solution of L-alanine, N-[[4-chloro-2-[[6-[3-[[(1,1-
dimethylethoxy)carbonyl] amino]propoxy] -7-methoxy-4-
quinazolinyl]amino]phenyl]methyl]-N-methyl-, methyl ester (crude residue;
0.00055
mol) in TFA/CH2C12/TIS (90/8/2) (5.5 ml) was stirred for 7-8 hours, then the
solvent was evaporated and the obtained residue was dried overnight in an
oven.
Yield: L-alanine, N-[ [2-[ [6-(3 -aminopropoxy)-7-methoxy-4-quinazolinyl]
amino]-4-
chlorophenyl]methyl]-N-methyl- .CF3COOH.
St~
A solution of L-alanine, N-[[2-[[6-(3-aminopropoxy)-7-methoxy-4-
quinazolinyl] amino] -4-chlorophenyl]methyl]-N-methyl-.CF3COOH (0.00055 mol)
and DIPEA (0.0033 mol) was added to a solution of HBTU (0.00165 mol) and
HOBt (0.0011 mol) in dry DMF (137 ml) and then the reaction mixture was
reacted
for 1 hour. The solvent was evaporated and the dry residue was purified by
reversed-
phase high-performance liquid chromatography. The product fractions were
collected, Na2CO3 was added and the organic solvent was evaporated. CH202 was
added to the aqueous concentrate and the resulting mixture was extracted 3
times
with CH202, then the organic extract was dried and collected. Yield: 0.049 g
of
4,6-ethanediylidene-12H-pyrimido[4,5-b] [6,1,10,13]benzoxatriazacyclohexadecin-
12-one, 18-chloro-8,9,10,11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-,
(13S)-.
As used in the examples, `CH3OH' means methanol, 'Et3N' means
tri ethylamine, `CHZCIZ' means dichloromethane, `HBTU' means 1-
[bis(dimethylamino)methylene]-1 H-Benzotriazoliumhexafluorophosphate(1-)3-
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oxide'DMF' means N,N-dimethylformamide, `NaBH(OAc)3' means sodium
triacetoxyborohydride, `DIPEA' means N-ethyl-N-(1-methylethyl)- 2-propanamine,
'HOBt' means 1-hydroxy-lH-benzotriazole, `TFA' means trifluoroacetic acid,
`TIS'
means tris(1-methylethyl)silane, 'K2C03' means potassium carbonate, `Cs2CO3'
means cesium carbonate, `Na2CO3' means carbonic acid disodium salt, `NaHCO3'
means carbonic acid monosodium salt.
Preparation of Compound 22, MTKI1
A suitable preparation of the preferred compound used in this invention, taken
from
WO-2004/105765, follows:
Example A
a) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl)methyl]amino]-
(intermediate 1)
A solution of 4-bromo-2-nitro- benzaldehyde,(0.013 mol), 5-amino-l-
pentanol (0.013 mol) and titanium, tetrakis (2-propanolato) (0.014 mol) in
EtOH
(15 ml) was stirred at RT for 1 hour, then the reaction mixture was heated to
50 C
and stirred for 30 min. The mixture was cooled to RT and NaBH4 (0.013 mol) was
added portionwise. The reaction mixture was stirred overnight and then poured
out
into ice water (50 ml). The resulting mixture was stirred for 20 min., the
formed
precipitate was filtered off (giving Filtrate (I)), washed with H20 and
stirred in
DCM (to dissolve the product and to remove it from the Ti-salt). The mixture
was
filtered and then the filtrate was dried (MgSO4) and filtered, finally the
solvent was
evaporated. Filtrate (I) was evaporated until EtOH was removed and the aqueous
concentrate was extracted 2 times with DCM. The organic layer was separated,
dried
(MgSO4), filtered off and the solvent was evaporated, yielding 3.8 g (93 %) of
intermediate 1.
Exam 1peB
a) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl)methyl]methylamino]-
(intermediate 2)
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A solution of intermediate 50 (0.0047 mol), formaldehyde (0.025 mol) and
titanium, tetrakis (2-propanolato) (0.0051 mol) in EtOH (150 ml) was heated to
50
C and stirred for 1 hour, then NaBH4 (0.026 mol) was added portionwise at RT.
The reaction mixture was stirred overnight and then quenched with water (100
ml).
The resulting mixture was stirred for 1 hour; the formed precipitate was
filtered off
and washed. The organic filtrate was concentrated, then the aqueous
concentrate was
extracted with DCM and dried. The solvent was evaporated and the residue was
filtered over silica gel (eluent: DCM/CH3OH from 98/2 to 95/5). The product
fractions were collected and the solvent was evaporated, yielding 0.5 g of
intermediate 2.
b) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl)methyl]methylamino]-,
acetate (ester) (intermediate 3)
A solution of intermediate 2 (0.0015 mol) and pyridine (0.015 mol) in acetic
anhydride (8 ml) was stirred overnight at RT, then the solvent was evaporated
and
co-evaporated with toluene, yielding intermediate 3.
c) Preparation of 1-pentanol, 5-[[(2-amino-4-bromophenyl)methyl]methylamino]-,
acetate (ester) (intermediate 4)
A mixture of intermediate 3 (0.0015 mol) in THF (50 ml) was hydrogenated
with Pt/C 5% (0.5 g) as a catalyst in the presence of thiophene solution (0.5
ml)
[H 179-034]. After uptake of H2 (3 equiv.), the catalyst was filtered off and
the
filtrate was evaporated, yielding 0.5 g of intermediate 4.
d) Preparation of 6-quinazolinol, 4-[[2-[[[5-
(acetyloxy)pentyl]methylamino]methyl]-
5-bromophenyl]amino]-7-methoxy-, acetate (ester) (intermediate 5)
A mixture of intermediate 4 (0.0015 mol) and 4-chloro-7-methoxy-6-
quinazolinol acetate (ester) (0.0015 mol) in 2-propanol (30 ml) was heated to
80 C
and the reaction mixture was stirred for 1 day. The solvent was evaporated
under
reduced pressure and the residue was used as such in the next reaction step,
yielding
0.83 g of intermediate 5.
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e) Preparation of 6-quinazolinol, 4=[[5-bromo-2-[[(5-
hydroxypentyl)methylamino]methyl]phenyl]amino]-7-methoxy- (intermediate 6)
A solution of intermediate 5 (0.0015 mol) in methanol (25 ml) was stirred at
RT and a solution of K2C03 (0.003 mol) in H20 (2.5 ml) was added, then the
reaction mixture was heated to 60 C and stirred for 18 hours. The solvent was
evaporated and H20 (20 ml) was added, then the mixture was neutralized with
acetic
acid and the formed precipitate was filtered off. The filtrate was
concentrated under
reduced pressure and the concentrate was extracted with DCM, filtered, then
dried
(MgSO4) and the mixture was concentrated under reduced pressure, yielding 0.5
g
(70 %) of intermediate 6.
Example C
a)Preparation of 4,6-ethanediylidenepyrimido[4,5-b][6,1,12]benzoxadiazacyclo-
pentadecine, 17-bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl-
(compound MTKI1)
A solution of intermediate 6(0.0011 mol) in THF (50 ml) was stirred at RT
and tributylphosphine (0.0016 mol) was added, then 1,1'-(azodicarbonyl)bis-
piperidine (0.00 16 mol) was added and the reaction mixture was stirred for 2
hours.
The solvent was evaporated until 1/3 of the initial volume. The resulting
precipitate
was filtered off and washed. The filtrate was evaporated and the residue was
purified
by RP high-performance liquid chromatography. The product fractions were
collected and the organic solvent was evaporated. The aqueous concentrate was
extracted 2 times with DCM and the organic layer was dried (MgSO4), then
filtered
off. The solvent was evaporated and the residue was dried (vac.) at 50 C,
yielding
0.004 g (0.8 %) of compound MTKI1.
Preparation of Compound 2, 4,6-ethanediylidene-19H-pyrimidof4,5-
bl f6,13,llbenzodioxaazacvclo-nentadecine, 15-chloro-8,9,10,11,12,13-
hexahydro-20-methoxy-;
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Exam lp e D
Preparation of 4,6-ethanediylidene-19H-pyrimido[4,5-
b][6,13,1]benzodioxaazacyclo-
pentadecine, 15-chloro-8,9,10,11,12,13-hexahydro-20-methoxy- (compound 2)
A solution of intermediate 9 (0.0024 mol) and triphenylphosphine (0.0036 mol)
in
THF, dry (100 ml) was stirred at RT and then a solution of bis(1-
methylethyl)diazenedicarboxylate (0.0036 mol) in THF (10 ml) was added
dropwise. The reaction mixture was stirred for 6 hours and extra bis(1-
methylethyl)diazenedicarboxylate (0.35 ml) in THF (10 ml) was added. The
mixture was stirred overnight and concentrated. The residue was purified by
column
chromatography over silica gel (eluent: DCM/CH3OH/THF 90/5/5). The product
fractions were collected and further purified by RP high-performance liquid
chromatography. The product fractions were collected and concentrated. The
aqueous concentrate was filtered , and the solid retained washed and dried
(vac.) at
65 C, yielding 0.065 g of compound 2, melting point 255.5-260.2 C.
Example E
a) Preparation of hexanoic acid, 6-(2-chloro-6-nitrophenoxy)-, methyl ester
(intermediate 6)
A solution of 2-chloro-6-nitro- phenol (0.046 mol) in N,N-dimethylformamide
(150
ml) was heated to 50 C, then K2C03 (0.069 mol) was added and the reaction
mixture was stirred for 15 min. 6-Bromo-,methyl ester hexanoic acid (0.069
mol)
was added and the mixture was stirred overnight . The reaction mixture was
filtered
and the filtrate was concentrated and the residue was used as such in the next
step,
yielding 13.88 g of intermediate 6.
b) Preparation of hexanoic acid, 6-(2-amino-6-chlorophenoxy)-, methyl ester
(intermediate 7)
A mixture of intermediate 6 (0.046 mol) and ethanime (2 g) in THF (ml) was
hydrogenated with Pt/C 5% (3 g) as a catalyst in the presence of DIPE (2 ml).
After
uptake of H2 (3 equiv.), the reaction mixture was filtered over small plug of
Dicalite
the filtrate was concentrated, yielding intermediate 7.
c) Preparation of hexanoic acid, 6-[2-[[6-(acetyloxy)-7-methoxy-4-
quinazolinyl] amino] -6-chlorophenoxy] -, methyl ester (intermediate 8)
A mixture of 4-chloro-6-methylcarbonyloxy-7-methoxyquinazoline (0.022 mol) and
intermediate 7 (0.022 mol) in 2-propanol (170 ml) was stirred and heated at 80
C
for 2 hours, concentrated and the residue was chromatographed over silica gel
(eluent: DCM/CH3OH 97/3). The product fractions were collected and the solvent
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was evaporated, yielding 5.1 g intermediate 8 (used as such in the next
reaction
step).
d) Preparation of 6-quinazolinol, 4-[[3-chloro-2-[(6-
hydroxyhexyl)oxy]phenyl] amino] -7-methoxy-(intermediate 9)
A mixture of LAH (0.0246 mol) in THF (40 ml) was stirred at RT. A solution of
intermediate 8 (0.006 mol) in THF (60 ml) was added dropwise. The reaction
mixture was stirred for 1 day then, extra LAH (0.0123 mol) was added
portionwise.
The mixture was stirred further over the weekend then, H20 (2 ml) was added
dropwise, followed by the dropwise addition of a 15 % NaOH soln. (2 ml) and
H20
(6 ml). This mixture was stirred for 15 min filtered and the filtrate was
concentrated The residue was stirred in boiling CH3CN, filtered and the solid
retained was dried (vac.) at 60 C. The solids were re-dissolved in CH3OH/DCM
(10/90) and this mixture was neutralised with HC1(1N). The organic layer was
separated, dried (MgSO4), filtered and concentrated, yielding 1 g of
intermediate 9.
An illustrative formulation example for the most preferred compound, MYKII, is
as
follows:
Example F: Formulation:
The product MTKI1 can be prepared as a 10-mg/mL oral solution, pH 2. It
contains an excipient, Captisol (chemical name: sulfobutyl ether-(3-
cyclodextrin,
SBE-(3-CD), citric acid, Tween 20, HC1, and NaOH in purified water. The
formulation can be stored refrigerated (2-8 C; 36-46 F) and allowed to warm
to
room temperature for maximally 1 hour prior to dose preparation.
The product MTKIl can also be prepared as 50-mg, 100-mg and 300-mg
oral immediate release capsules, containing the active chemical entity MTKI1,
lactose monohydrate (200 mesh), sodium lauryl sulphate and magnesium stearate
in
hard gelatin capsules, sizes 3, 4 and 00, respectively. The capsules may also
contain
any or all of the following ingredients: gelatin, red iron oxide and titanium
oxide.
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EXPERIMENTAL DATA
VEGFR3 inhibition
Lymph node involvement is a poor prognostic factor in a number of cancer
types.
Recent studies have demonstrated that lymphatic vessel formation plays an
important role in tumor progression and vascular endothelial growth factor
(VEGF)
C and D have been identified as specific lymphangiogenic factors that act via
activation of the cognate receptor VEGFR3. 4,6-ethanediylidenepyrimido[4,5-
b] [6,1,12]benzoxadiazacyclo-pentadecine, 17-bromo-8,9,10,11,12,13,14,19-
octahydro-20-methoxy-13-methyl- , hereinafter also referred to as Compound 2,
is a
multitargeted kinase inhibitor that has been shown to have a unique kinase
inhibition
profile (pan Her and Src family) as well as a favourable tissue distribution
profile
resulting in potent anti tumor activity in a number of experimental models.
In vitro kinase assays have shown that the compounds of the present invention
have
potent inhibition of the EGFR, Her2, Her4 and Src family kinases (Src, Fyn,
Lck,
Yes, Lyn) activity.
The in vitro kinase inhibition was validated herein in cell based assays using
human
vascular endothelial cells (HMVECd), in which Compound 2 prevented VEGFR3
dependent VEGF-C stimulation of Erkl/2, whereas it did not affect VEGFR1
mediated VEGF signaling. Compound 2 has also been shown herein to inhibit
VEGF-C induced phosphoylation of VEGFR3 using a cell line engineered to
overexpress human VEGFR3.
These results were further validated herein in a recently developed Xenopus
tadpole
model where Compound 2 was found to phenocopy the effects of VEGF-C
knockout.
(Ny et al, Nat. Med. 2005 Sep;11:998-1004).
Methods
In vitro kinase assays
The VEGFR3 kinase reaction was performed at 30 C for 10 minutes in a 96-well
microtiterplate. The 25 l reaction volume contained 8 mM MOPS pH 7, 200 M
EDTA, 10 mM MgAc, 10 M unlabeled ATP, 0.5 mCi AT33P, 500 M JAK3-tide
(Ac-GGEEEEYFELVKKKK-NH2), 25 ng VEGFR3 and 2 % compound in 100 %
DMSO.
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The reaction was stopped by adding 5 l of a 3% phosphoric acid solution. 10
l of
the reaction mixture was then spotted onto a Filtermat P30 filter (Wallac) and
washed 3 times for 5 min. in 0.75 % phosphoric acid and 1 time for 2 min. in
methanol prior to transferring to a sealable plastic bag containing 4 ml of
scintillation liquid and reading in a scintillation counter.
VEGFR3 Cellular assays
Inhibition of VEGF-C induced VEGFR3 activity
For this assay, porcine aortic endothelial (PAE) cells stably expressing human
VEGFR-3 were grown to confluency on 10 cm dishes. The cells were serum starved
overnight and the media replaced with fresh serum-free media. Pre-determined
amounts of Compound 2 or control vehicle was added to the cells, and then
incubated at 37 C for 15 min. This was followed by the addition of VEGF-C to
a
final concentration of 100 ng/ml and the cells incubated for 10 min at 37 C,
after
which they were washed with ice-cold PBS and then lysed. Cell lysates
containing
protease inhibitors were subjected to immunoprecipitation with an antibody
specific
for VEGFR-3 and the immunoprecipitates separated by SDS-PAGE. Proteins were
transferred to a nitrocellulose membrane, and then probed first with a phospho-
tyrosine antibody and then a VEGFR-3 antibody.
Inhibition of Erk activation induced by VEGF- C in Human Micro Vessel
Endothelial Cells-dermal (HMVEC-d)
HMVEC-d cells (Cambrex) were cultured at 37C in 5% C02 in appropriate serum
containing medium (Cambrex) on fibronectin coated 12-well plates (BD Biocoat,
Becton Dickinson) for 4 days. Cells were then starved for 16 hours in the same
medium as before, but lacking serum and containing SITE+3 (Invitrogen)
instead.
Cells were then preincubated with DMSO at 0.2% (the solvent) or 5uM Compound
2 for 60 minutes and then either 10 ng/ml VEGF or 100 ng/ml VEGF-C (R&D
systems) was added to the cells. Before, or 10 and 30 minutes after
stimulation, the
medium was removed, cells were rinsed with ice-cold PBS containing 0.1 mM
sodiumorthovanadate and lysed in M-PER buffer (PIERCE) containing 0.1 % SDS,
and 1 x phosphatase and protease inhibitors (HALT, PIERCE). Cells were scraped
off the plates and incubated in lysis buffer for 30 minutes on ice and then
centrifuged at 4 C for 10 min at 16000xg. Lysates were quantified using the
BCA
reagent according to the manufacturer's instructions (PIERCE), and 5 mg of
total
protein was loaded on SDS-polyacrylamide gels (NUPAGE, Invitrogen).
Electrophoresis and blotting onto PVDF membranes was all performed using
reagents from the NUPAGE system, according to the manufacturer's instructions.
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Blots were blocked for 1 hour with Odyssey blocking buffer (LICOR) and then
primary antibodies (see below) were diluted 1:1000 in a 1:1 mix of Odyssey
blocking buffer and PBS containing 0.1 % Tween-20, and applied overnight at 4
C:
P-Erkl/2 (Thr202/Tyr204, clone E10, mouse monoclonal #9106, Erkl/2 (rabbit
polyclonal #9102) (all from Cell Signaling Technologies, Inc). Blots were
washed
three times in PBS containing 0.1 % Tween for 5 min and then incubated for 1
hour
with secondary antibodies (diluted 1:1000 in PBS containing 0.1% Tween-
20):goat
anti-mouse conjugated to IRDye 800nm (Rockland, Inc) or goat anti-rabbit
conjugated to Alexa 680nm (Molecular Probes, Inc).
Blots were washed three times in PBS containing 0.1% Tween for 5 min and once
in
PBS and scanned using the LICOR system at 700nm and 800nm using intensity 5
and all settings appropriate for the scanning of membranes.
VEGFR3 in vivo Xenopus assay
Xenopus tadpoles were used as a model (Ny et al Nature Medicine 11: 998-1004
(2005)) to analyze the effects of Compound 2 on lymphangiogenesis. The
compound
was administered daily in a dose dependent manner, with vehicle controls, to
the
medium of the Xenopus tadpoles starting at stage 26-28, i.e., before the
formation of
both blood and lymphatic vessels. The effects of the compound was monitored by
"live analysis", i.e. live embryos were examined 4 days later for evidence of
lymph-
vascular
defects by trained observers. As an additional means of confirming the
results,
in a limited number of experiments, tadpoles were fixed at stage 35-36 and in
situ
hybridizations for Proxl were performed to assess migration of lymphatic
endothelial cells (LECs) from the posterior cardinal vein (PCV) to their
dorsal
endpoint, this reflecting an essential step in lymphangiogenesis. Under normal
circumstances, LECs arise from the region of the PCV, accumulate there, and
during the study period, they migrate dorsally to defined "areas" (Area 1 to
Area
2 and Area 3), where they then further migrate rostrally and caudally to form
the
lymphatic vasculature in the tail and trunk. Defects in migration of the LECs
from
the PCV was quantified by determining the maximal migration of the cells,
and by counting the number of cells in these different regions in the tail.
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Results
In vitro kinase assays
The following table provides the pIC50 values obtained for the compounds of
the
present invention using the above-mentioned VEGFR3 kinase assay.
Structure Name picso
4,6-ethanediylidene-l9H-
CI pyrimido[4,5-
0 b] [6,13,1 ]benzodioxaazacyclo-
pentadecine, 15-chloro-
HN / 8,9,10,11,12,13-hexahydro-20-
methoxy-
I ~ N 6
0
O NI)-
4,6-ethanediylidenepyrimido [4,5-
N b] [6,1,12]benzoxadiazacyclopenta
decine, 17-bromo-
HN Br 8,9,10,11,12,13,14,19-octahydro-
O 20-methoxy-13-methyl-
~ N
I I
p NJ 7.74
12H-4,6-ethanediylidene-13,17-
0 methanopyrimido[4,5-
b] [6,1,10,16]benzoxatriazacyclon
N onadecin-12-one, 21-chloro-
HN / C18,9,10,11,13,14,15,16,18,23-
decahydro-25-methoxy-
O N
O I NJ 6
4,6-ethanediylidene-12H-
H = pyrimido[4,5-
N b] [6,1,10,13]benzoxatriazacycloh
aci exadecin-12-one, 18-chloro-
O HN 8,9,10,11,13,14,15,20-octahydro-
0 21-methoxy-13,14-dimethyl-
~
I ~ 6
O N
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Inhibition of VEGF-C Induced VEGFR-3 Phosphorylation in VEGFR-3
Expressing Porcine Aortic Endothelial Cells
The effect of 4,6-ethanediylidenepyrimido[4,5-b][6,1,12]-benzoxadiazacyclo-
pentadecine, 17-bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl-
(Compound 2) on VEGF-C induced VEGFR3 activity is evident from the dose
dependent reduction in VEGFR-3 phosphorylation (Top frame of Figure 1).
Presence of VEGFR-3 at the different concentration is confirmed upon re-
staining of
the Western Blots with VEGFR-3 antibodies (Bottom frame of Figure 1).
Erk phosphorylation assays in HMVEC-d cells
As illustrated in Figure 2, Compound 2 at 5 M, prevented phosphorylation of
Erkl/2 by VEGF-C but did not interfere with VEGF stimulation of Erkl/2. These
data are consistent with a selective inhibition of VEGFR-3 by Compound 2.
Xenopus study results
The effects of Compound 2 was assessed using live screening, concentrations of
Compound 2 of > 50 uM induced hemorrhages in 30% of tadpoles. At 20 uM, 10%
of tadpoles developed edema, which was not seen in vehicle-alone treated
tadpoles.
Compound 2 dose dependently inhibits Lymphatic Endothelial Cell (LEC)
Migration in tadpoles (Table 1). LEC migration at stage 35-36 was particularly
decreased, even at the lowest concentrations of compound tested (0.31 uM), and
this
was more than observed with vehicle alone. These data supports the conclusion
that
this compound interferes with normal lymphangiogenesis.
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Table 1
Quantification of migration of lymphatic endothelial cells (detected by Proxl
in situ
hybridization) from posterior cardinal vein towards dorsum after exposure of
stages
35-36 tadpoles to compounds (n=10-15 embryos per condition)
exp. 08.08.06
Concentration of Com ound 2 in M
80 20 5 1.25 0.3125 DMSO
% embryos with 80 60 40 63 57 21
reduced migration
% Relative Area 2-3 56 15 19 8 123 ~ 32 20 8 37 20
%RelativeMax 67 21 35 1 121~3 47 5 73 22
Migration
Note: The above assessments of LEC migration of Proxl positive cells were
first
performed microscopically in a semi-quantitative manner by a trained observer
('%
embryos with reduced migration'). Computer-generated quantitative analysis
were
then performed. The "Relative Area 2-3" is the number of LECs that migrated to
area 2-3 relative to the control (vehicle alone). "Relative max migration" is
the
furthest LEC migration from the PCV under treatment, relative to the max
observed
in the vehicle-alone controls.
While the foregoing specification teaches the principles of the present
invention,
- with examples provided for the purpose of illustration, it will be
understood that the
practice of the invention encompasses all of the usual variations, adaptations
and/or
modifications as come within the scope of the following claims and their
equivalents.
