Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR INHIBITING ANGIOGENESIS AND TUMOR GROWTH
Field of the Invention
This invention relates to methods for inhibiting angiogenesis and
tumor growth. More particularly, the invention relates to methods of
inhibiting
angiogenesis and tamor growth utilizing a compounds that selectively bind to
integrin aAand blocks the interaction of integrin '43 with matrix
metalloproteinase 2 (MMP2).
Background of the Invention
Invasion of vascular cells into tissues requires the coordinated
interplay of numerous factors including proteinases, which remodel the
extracellular matrix architecture, as well as cell adhesion molecules that
recognize this provisional matrix. Recent reports have implicated that the 72
kDa
matrix metalloproteinase 2(M1vIP2) is a key player in vascular development and
angiogenesis. For example, Kitoh et al. (J. Cell Sci., 109, 953-8 (1996))
report
that MMP2 and its activator membrane type 1-matrix metalloproteinase (MT1-
MMP) are coordinately expressed by mesenchymal cells almost exclusively
during embryonic development, indicating specific matruc remodeling
constraints
in these tissues. In addition, angiogenesis and corresponding tumor growth are
reduced in MMP2 knockout mice (see Itoh et al., Cancer Res., 58 1048-51
(1998)). Interestingly, Saftor et al. (Proc. Natl. Acad. Sci. U.S.A., 89, 1557-
61
(1992)) have shown that ligation of the integrin 43, itself a known mediator
of
angiogenesis, induces MMP2 production, suggesting a coordinated interplay of
these two molecules during the vascular remodeling associated with blood
vessel
formation (see also Bafetti et al., J. Biol. Chem., 273, 143-9 (1998)). In
fact,
direct interaction between MMP2 and integrin a~Nhas been demonstrated by
Brooks et al. (Cell, 85, 683-93. (1996)). The negative regulation of MMP2
during vascular invasion and maturation was later shown by Brooks et al. to be
dependent upon expression of a~P3 (Cell, 92, 391-400 (1998)).
Although inhibition of angiogenesis and concomitant suppression
of tumor growth by natural as well as synthetic inhibitors of MMP's, including
MMP2, has been documented, the translation of such strategies into clinical
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modalities has met with limited success, primarily due to-the deleterious side
effects of such broad spectrum inhibitors. Since MMP function, in general, may
be required for many processes in the adult organism, active site inhibition
of
enzymatic function is likely to have far reaching effects on various
biological
processes involving tissue remodeling, such as wound healing. In fact, it has
been documented that therapies with broad spectrum MMP inhibitors in clinical
studies of various cancer types cause severe side effects, including
inflammatory
tendinitis, polyarthritis, and muscoskeletal pain syndromes, which are dose
limiting and often persist after discontinuation of therapy. Given the limited
distribution of integrin a,,P3 in adult organisms, however, one would predict
that
targeting the interaction between MMF2 and aYP3 to the areas of
neovascularization or cellular invasion should correspondingly limit the
effects of
such treatment-related toxicities. Indeed, the recombinant non-catalytic
carboxy-
terminal hemopexin domain of MMP2 (PEX), which mediates M1ViP2 binding to
integrin a(33, has shown antiangiogenic and antitumor activity in vivo. The
potential utility of such a large protein fragment, but with attendant
shortcomings
(e.g. large scale production problems, FDA quality and safety control issues
and
antigenicity), suggested the need for a more practical solution to this
problem.
There is a need therefore, for methods of inhibiting angiogenesis
and tumor growth utilizing chemical compounds that selectively inhibit MMP
activity at tumor growth sites with minimal inhibition of MMP in other regions
of
the body. There is also a need for methods of specifically binding to the MMP2
binding site of integrin 43.
Summary of the Invention
The present invention provides a method for the inhibition of the
interaction of MMP2 with integrin 43 and a method for inhibition of
angiogenesis in cells containing integrin (43. Further, the invention provides
a
method for inhibition of tumor growth by administration of 1VIMP2-a(33
interaction inhibitors. Active inhibitor compounds represented by Formula (I),
below, are contacted with integrin a1P3 on a cell, which, in turn, inhibits
the
binding of MMP2 to the 43. The inhibition of binding of MMPS to aA by the
methods of the present invention result in inhibition of angiogenesis and thus
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tumor growth. In addition, a,03 has been implicated in inflammation, thus
compounds of Formula (I), used in accordance with the methods of the present
invention can also suppress inflammatory events.
Formula (I):
X~
J CH2~ -N ~C~ ~ ~
r O O
o N)L 1
0 [2HAz
N Y,
p 0 G~
n m
t
wherein G' and G2 are each independently -NH-C(O)-0 R',
-NH-C(O)-O-(CH?)õ-(C6H4)-X3, -NH-C(O)-NH-(CH?)v (C6H4) X3,
-O-C(O)-NH-(CH2)v-(C6H~-)3,-O-C(O)-O-(CHa)v-(C6H4)-X3, or
-NH-C(O)-CHZ-(C6H4)-)3; Y' and Y2 are each independently -OH, C,- C4 alkyl,
Cl- C4 hydroxyalkyl, CI- C4 alkoxy, phenyl, benzyl, or -NH2; R' is Cl- C4
alkyl;
Xl and X2 are each independently halo or C,- C4 alkoxy; X3 is halo, nitro, Cl-
C4
alkyl, Cl - C4 alkoxy, or CI - C4 perfluoroalkyl; Z is -C = C-, -C6H4-, cis
--CH = CH-, trans-CH=CH-, cis-CH2-CH = CH-CH2-, trans -CH2-CH = CH-CH2-,
1,4 naphthyl, cis-1,3-cyclohexyl, trans-l,3-cyclohexyl, cis-l,4-cyclohexyl, or
trans-l,4-cyclohexyl; A is H or a covalent bond; m and n are each
independently
an integer having a value of 0 qr 1; t is an integer having a value of 0 or 1;
and
p, r, and v are each independently an integer having a value of 1 or 2; with
provisos that when A is H, t is 0; when A is a covalent bond, t is 1; when m
is 0,
Y' is Cl - C4 hydroxyalkyl; and when n is 0, Yz is Ca - C4 hydroxyalkyl.
Preferred compounds within the purview of structural Formula (I)
are represented in structural Formula (II):
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~ ~~ O O `C~ p
r O o
s O N A' O
H H Rz
R\O s H H
Oy RH O O HNYO
/ O 0 ~II~
wherein R2 and R3 are each independently H, C, - C4 alkyl, phenyl or benzyl;
Xl
and Xx are each independently halo or C,- C4 alkoxy; X4 and XS are each
independently halo, nitro, Cl - C4 alkoxy, Cl - C4 alkyl, or Cl - C4
perfluoroalkyl; A is H or a covalent bond; p and r are each independently an
integer having a value of 1 or 2; and t is an integer having a value of 0 or 1
with
the proviso that when A is H, t is 0 and when A is a covalent bond, t is 1.
When
20. A. is a covalent bond and t is 1, the iminodiacetamide derivative moieties
may be
attached to the benzene linking group in the ortho, meta or para position.
When compounds of Formulas (I) and (II) are contacted with cells
containing aV(33, the binding of (43 with MMP2 is inhibited, thus interfering
with an essential mechanism in angiogenesis. Interference with angiogenesis
can
also inhibit tumor growth by preventing vascularization of the tumor, thus
starving it of nutrition. The angiogenesis and tumor growth inb.ibitin,g
compounds of the present invention are thus useful therapeutic agents for the
treatment of patients with tumors or angiogenic disorders. Because the present
compounds bind to av j33, these compounds can also be used to suppress
inflammatory events.
The compounds of the present invention may be formulated in
suitable pharmaceutically acceptable matrix. The pharmaceutical compositions
of
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the active compounds are administered to a patient with a tumor to reduce or
eliminate tumor growth. The active compounds can be administered parenterally
by injection or by gradual infusion over time, or by any other method suitable
for
the particular dosage form.
Brief Descn:ption of the Drawings
In the accompanying drawings:
FIG. 1 is a, schematic illustration depicting MMP2 interaction with
integrin a,P3 and its role in angiogenesis.
' FIG. 2 depicts the structural subunits A, B and C of a
combinatorial library of 600 compounds disclosed in Boger et al., Bioorg. Med.
Chem, 6, 1347-1378 (1998).
FIG. 3 graphically illustrates the binding of 60 combinatorial
mixtures of compounds with integrin 43 in competition with MMP2.
FIG. 4 illustrates the binding of mixtures AxB10 with integrin aY(33
and the binding of the 10 individual components of A6B10C4.
FIG. 5 depicts the structures of analogs of A6BlOC4.
FIG. 6A graphically illustrates the binding of analogs (Compounds
2- 26) of A6B10C4 (Compound 1) with integrin 43 in competition with
MMP2.
FIG. 6B graphically illustrates the binding of Compounds 9 and 19
with integrin 43 in comparison with MMP2.
FIG. 7 illustrates that [14C]-labeled Compound 19 binds
specifically to aA and can be competitively displaced from the a,,03 by a 25
fold
excess of non-labeled Compound 19, but not by excess Compound 9, a RGD
peptide or a c(RGDfV) peptide.
FIG. 8 shows that Compound 19 disrupts the binding of IVIMP2 to
integrin a"P3, but does not interfere with vitronectin binding with integrin
43.
FIG. 9 shows that Compound 19 does not directly inhibit purified
active MMP2 proteolysis.
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Detailed Description of the Invention
The binding of MMP2 to integrin aA is an important mechanism
in the process of angiogenesis. Specific inhibition of this binding
interaction
results in a reduction in vascularization in growing tissues such as tumors,
and
thus retards tumor growth. The interaction of IvIMP2 with integrin aP3 is
illustrated pictorially in FIG. 1. A new class of angiogenesis and tumor
growth
. inhibitors, described below, specifically bind to integrin a,p3 in
competition with
MMP2, thus affording an important new treatment tool.
Certain compounds of this invention may possess one or more
10, asymmetric centers and may exist in optically active forms. Additional
asymmetric centers may be present in a substituent group, such as an alkyl
group.
Pure S-isomers and pure R-isomers, racemic mixtures of the isomers, and
mixtures thereof are intended to be within the scope of this invention. Chiral
forms of certain compounds of this invention are contemplated and are
specifically included within the scope of this invention.
The term "alkoxy" means an oxygen atom linked by an ether bond
to an alkyl group, as defined below, of the size indicated. Examples of alkoxy
groups are methoxy, ethoxy, t-butoxy, and the like. The term "alkyl" means a
straight- or branched-chain carbon radical of the size indicated.
Representative of
a1ky1 radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl,
t-butyl, 2-ethylhexyl, n-octyl, 2,4-dimethylpentyl, and the like. The term
"hydroxyalkyl" means an alkyl group, as defined above, of the size indicated,
attached to a hydroxyl group. Examples include hydroxymethyl, 2-hydroxyethyl,
3 hydroxy-l-propyl, 2-hydroxy-l-propyl, 4-hydroxybutyl, and the like.
The term "perfluoroaikyP" refers to a alkyl group of the size
indicated, as defmed below, bearing fluoro substituents in place of each
hydrogen, for example trifluoromethyl and pentafluoroethyl.
The terms "halo" or "halogen" refer to bromo, chloro, fluoro and
iodo.
The compounds useful in the methods of the present invention are
represented by Formula (I) and include iminodiacetamide derivatives chemically
attached to a linking group:
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X2, CFlZY N O H CH2
r O A Z~
N = N
2 N N y~
Y m
n!
O O GI
t
wherein G' and G2 are each independently -NH-C(O)-O-R',
-NH-C(O)-O-(CHz)õ-(C6H4)-X3, -NH-C(O)-NH-(CH2)v (C6H4)-X3,
-O-C(O)-NH-(CH)v (C6H4)-X3,-O-C(O)-O-(CH)v (C6H4)-X3, or
-NH-C(O)-CH2-(C6H4)-X3; Y' and YZ are each independently -OH, C,- C4 alkyl,
Ci- C4 hydroxyalkyl, Cl- C4 alkoxy, phenyl, benzyl, or -NH2; R' is Cl- C4
alkyl;
X' and XZ are each independently halo or C,- C4 alkoxy; X3 is halo, nitro, Cl-
C4
alkyl, Ct - C4 alkoxy, or C, - C4 perfluoroalkyl; Z is -C=C-, -C6H4-, cis
-CH = CH-, trans-CH = CH-, cis-CH2-CH = CH-CH2-, trans -CH2-CH = CH-CHZ ,
1,4-naphthyl, cis-1,3-cyclohexyl, trans-1,3-cyclohexyl, cis-1,4-cyclohexyl, or
trans-1,4-cyclohexyl; A is H or a covalent bond; m and n are each
independently
an integer having a value of 0 or 1; t is an integer having a value of 0 or 1;
and
p, r, and v are each independently an integer having a value of 1 or 2; with
provisos that when A is H, t is 0; when A is a covalent bond, t is 1; when m
is 0,
Y' is C, - C4 hydroxyalkyl; and when n is 0, Y2 is Cl - C4 hydroxyalkyl.
Preferred compounds within the purview of structural Formula (I)
are represented by structural Formula (II) and include iminodiacetamide
derivatives attached to a benzene linking group in either the ortho, meta or
para
orientation:
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X1
~ +N 0 0 N1C+~ ~
0 P
a 0 H N N H 0
R\ N~ O~R2
~ (rf)
OY NN 0 0 N
0 O
\ I ~~
t
wherein RZ and R3 are each independently H, C, - C4 a1ky1, phenyl or benzyl;
X'
and XZ are each independently halo or C,- C4 alkoxy; X4 and XS are each
independently halo, nitro, Cl - C"4 alkoxy, C, - C4 alkyl, or C, - C4
perfluoroalkyl; A is H or a covalent bond; p and r are each independently an
integer having a value of 1 or 2; and t is an integer having a value of 0 or 1
with
the proviso that when A is H, t is 0 and when A is a covalent bond, t is 1.
Preferably, the substituents X' and XZ are attached to the phenyl
ring in the 4- position relative to the CHZ groups (i.e. para substituent).
Preferably, at least one of X' and XZ is fluoro, most preferably X' and XZ are
both para-fluoro. Preferably, r and p are 2. X4 and XS are preferably, C, to
C4
perfluoroalkyl, most preferably para-trifluoromethyl. The preferred R2 and R3
groups are hydrogen and methyl. The substituents X2 and X3 may be the same or
different, and the substituents R2 and R3 may also be the same or different.
The compounds of Formulas (I) and (11) are described in detail,
along with methods of synthesis thereof, in Boger et al., Bioorg. Med. Chem,
6,
1347-1378 (1998),
A particularly active member of the family of compounds
represented by Formula (II), whereiri A is a covalent bond and t is 1, is
Compound 19 in Scheme 1, below.
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Scheme 1.
0
HO BOC-N O~Ma
1. Disuccinimidyl
~ carbonate HN
2. N-s-BOC-lysine methyl p
CF3 ester, 99%
Compound 27 CF3
1. HCI - dioxane NH F 1. HCI - dioxane
2. PyPrOP, 74% Bpp-N 2. Is~op~thaloyl dichloride
y N O"Me
NH O HN
BOCiV Compound 28 0 ~H Compound 29
O
CF3
/I
~ HN O ~"NH ONF
F
\ NJH
R a N LyN
yAH p m\ o
Compound 19, R=Me
UOH
CF3 95% 3
Compound 30. R=H
14CH30H
EDCI, DMAP
3596 [14CJ - Compound 19, R 74CH3
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The synthesis of Compound 19 is illustrative of a general method
of producing compounds of Formulas (I)and (II) described by Boger et al..
Compound 19 was synthesized in three steps starting with commercially
available
N-E-BOC-L-lysine methyl ester. The carbamate was installed in 99% yield by
reaction of 4-(trifluoromethyl)benzyl alcohol with N, N-disuccinim.idyl
carbonate
and subsequent addition of the activated product with the free a-amino group
-providing intermediate Compound 27. This lysine derivative was then subjected
to N-BOC deprotection (HC1) and coupled with bromotripyrrolidinophosphonium
hexafluorophosphate (PyBrOP, 74%) to the free carboxylic acid functionality of
* the iminodiacetic acid monoamide Compound 28 providing diamide Compound
29. After N-BOC deprotection (HC1) this was dimerized by reaction with
isophthaloyl dichloride completing the synthesis and providing Compound 19 in
60 % yield. A radiolabel was incorporated into the molecule by saponification
of
the two methyl esters (LiOH, 95 %) providing dicarboxylic acid Compound 30,
followed by esterification with [14C]-methanol mediated by 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI) and catalytic
4-dimethylaminopyridine (DMAP) to afford ['aC]-Compound 1 in 35% yield.
Pharmaceutical preparations of compounds of Formulas (1) and (II)
can be prepared by formulating the compound in a pharmaceutically acceptable
carrier matrix. The pharmaceutical compositions comprising the active
compounds of Formulas (1) and (II) are administered to a host with a tumor to
reduce or eliminate tumor growth. The active compounds can be administered
parenterally by injection, or by gradual infusion over time. Although the
tissue
to be treated is most often treated by intraperitoneal or subcutaneous
administration, the active compounds can also be administered intraocularly,
intravenously, intramuscularly, intrasynovially, intracavity, or
transdermally, and
can be delivered by peristaltic means as well.
The term "administration" of the inventive compound or
composition, as used herein, refers to systemic use as when taken orally,
parenterally, by inhalation spray, by nasal, rectal or buccal routes, or
topically in
dosage form unit formulations containing conventional nontoxic
pharmaceutically
acceptable carriers, adjuvants and vehicles as desired. The term "parenteral"
as
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used herein includes intravenous, intramuscular, intraperitoneal,
intrastern.al,
subcutaneous and intraarticular injection and infusion techniques.
By "pharmaceutically acceptable" it is meant those salts, amides
and esters which are, within the scope of sound medical judgement, suitable
for
use in contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, and the like, and are commensurate
with a
reasonable benefit/risk ratio, effective for their intended use in the
treatment of
tumors and angiogenic-related disorders.
Pharmaceutically acceptable salts are well known in the art. For
example, S. M Berge, et al. describe pharmaceutically acceptable salts in
detail in
J. Pharmaceutical Sciences, 66, 1-19 (1977). Representative acid addition
salts
include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,
valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate,
phosphate,
toluenesulfonate, methanesulfonate, citrate, maleate, fumarate, succinate,
tartrate,
ascorbate, glucoheptonate, lactobionate, lauryl sulfate salts and the like.
Representative alkali or alkaline earth metal salts include sodium, calcium,
potassium, magnesium salts and the like.
As used herein, the term "pharmaceutically acceptable carriers"
means a non-toxic, inert solid, semi-solid or liquid filler, diluent,
encapsulating
material or formulation auxiliary of any type. Some examples of the materials
that can serve as pharmaceutically acceptable carriers are sugars, such as
lactose,
glucose and sucrose; starches such as corn starch and potato starch; cellulose
and
its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and
cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such
as
cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such
as
propylene glycol; polyols such as glycerin, sorbitol, mannitol and
polyethylene
glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents
such
as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free
water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer
solutions, as well as other non-toxic compatible substances used in
pharmaceutical formulations.
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Wetting agents, emulsifiers and lubricants such as sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, releasing agents,
coating agents, sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the composition, according to the
judgement
of the formulator. Examples of pharmaceuticaUy acceptable antioxidants include
water soluble antioxidants such as ascorbic acid, cysteine hydrochloride,
sodium
bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble
antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BRA),
butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and
10, the like; and the metal chelating agents such as citric acid,
ethylenediamine
tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the
like.
By a"tberapeutically effective amount" of the inventive agent or
compound is meant a sufficient amount of the compound to treat tumors and
angiogenic-related disorders at a reasonable benefit/risk ratio applicable to
any
medical treatment. It will be understood, however, that the total daily usage
of
the compounds and compositions of the present invention will be decided by the
attending physician within the scope of sound medical judgement. The specific
therapeutically effective dose level for any particular patient will depend
upon a
variety of factors including the disorder being treated and the severity of
the
disorder; activity of the specific compound employed; the specific composition
employed; the age, body weight, general health, sex and diet of the patient;
the
time of administration, route of administration, and rate of excretion of the
specific compound employed; the duration of the treatment; drugs used in
combination or coincidently with the specific compound employed; and like
factors well known in the medical arts.
This invention also provides pharmaceutical compositions in unit
dosage forms, comprising a therapeutically effective amount of a compound (or
compounds) of this invention in combination with a conventional pharmaceutical
carrier. Injectable preparations, for example, sterile injectable aqueous or
oleaginous suspensions may be formulated according to the known art using
suitable dispersing or wetting agents and suspending agents. The sterile
injectable
preparation may also be a sterile injectable solution, suspension or emulsion
in a
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nontoxic parenterally acceptable diluent or solvent, for example, as a
solution in
1,3 butanediol. Among the acceptable vehicles and solvents that may be
employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally employed as a
solvent
or suspending medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides.
In addition, fatty acids such as oleic acid are used in the
preparation of injectables. The injectable formulation can be sterilized, for
example, by filtration through a bacteria-retaining filter, or by
incorporating
sterilizing agents in the form of sterile solid compositions which can be
dissolved
or dispersed in sterile water or other sterile injectable medium just prior to
use.
In order to prolong the effect of a drug, it is often desirable to
slow the absorption of a drug from subcutaneous or intramuscular injection.
The
most common way to accomplish this is to inject a suspension of crystalline or
amorphous material with poor water solubility The rate of absorption of the
drug
becomes dependent on the rate of dissolution of the drug which is, in turn,
dependent on the physical state of the drug, for example, the crystal size and
the
crystalline form. Another approach to delaying absorption of a drug is to
administer the drug as a solution or suspension in oil. Injectable depot forms
can
also be made by forming microcapsule matrices of drugs and biodegradable
polymers such as polylactide-polyglycolide. Depending on the ratio of drug to
polymer and the composition of the polymer, the rate of drug release can be
controlled. Examples of other biodegradable polymers include poly-orthoesters
and polyanhydrides. The depot injectables can also be made by entrapping the
drug in liposomes or microemulsions which are compatible with body tissues.
Suppositories for rectal administration of the drug can be prepared
by mixing the drug with a suitable nonirritating excipient such as cocoa
butter
and polyethylene glycol which are solid at ordinary temperature but liquid at
the
rectal temperature and will therefore melt in the rectum and release the drug.
Solid dosage forms for oral administration may include capsules,
tablets, pills, powders, prills and granules. In such solid dosage forms the
active
compound may be admixed with at least one inert diluent such as sucrose,
lactose
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or starch. Such dosage forms may also comprise, as is normal practice,
additional
substances other than inert diluents, e.g., tableting lubricants and other
tableting
aids such as magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise buffering
agents.
Tablets and pills can additionally be prepared with enteric coatings and other
release-controlling coatings. Solid compositions of a similar type may also be
. employed as fillers in soft and hard-filled gelatin capsules using such
excipients
as lactose or milk sugar as well as high molecular weight polyethylene glycols
and the like.
10, Liquid dosage forms for oral administration may include
pharmaceutically acceptable lo emulsions, microemulsions, solutions,
suspensions, syrups and elixirs containing inert diluents commonly used in the
art
such as water. Such compositions may also comprise adjuvants, such as wetting
agents; emulsifying and suspending agents; sweetening, flavoring and perfuming
agents. If desired, the compounds of the present invention can be incorporated
into slow release or targeted delivery systems such as polymer matrices,
liposomes and microspheres. They may be sterilized, for example, by filtration
through a bacteria-retaining filter, or by incorporating sterilizing agents in
the
form of sterile solid compositions which can dissolve in sterile water, or
some
other sterile injectable medium immediately before use. The active compounds
can also be in micro-encapsulated form with one or more excipients as noted
above.
The solid dosage forms of tablets, dragees, capsules, pills, and
granules can be prepared with coatings and shells such as enteric coatings and
other coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a composition that
they
release the active ingredient(s) only, or preferably, in a certain part of the
intestinal tract, optionally in a delayed mann.er. Examples of embedding
compositions which can be used include polymeric substances and waxes. Dosage
forms for topical or transdermal administration of a compound of this
invention
further include ointments, pastes, creams, lotions, gels, powders, solutions,
sprays, inhalants or patches. The active component is admixed under sterile
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conditions with a pharmaceutically acceptable carrier and any needed
preservatives or buffers as may be required.
Ophthalmic formulations, ear drops, eye ointments, powders and
solutions are also contemplated as being within the scope of this invention.
The
ointments, pastes, creams and gels may contain, in addition to an active
compound of this invention, excipients such as animal and vegetable fats,
oils,
waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols,
silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to the compounds of
this invention,. excipients such as lactose, talc, silicic acid, aluminum
hydroxide,
calcium silicates and polyamide powder, or mixtures of these substances.
Sprays
can additionally contain customary propellants such as
chlorofluorohydrocarbons.
Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms can be made
by dissolving or dispersing the compound in the proper medium. Absorption
enhancers can also be used to increase the flux of the compound across the
skin.
The rate can be controlled by either providing a rate controlling membrane or
by
dispersing the compound in a polymer matrix or gel.
The compositions containing the active compounds are
administered in a manner compatible with the dosage formulation and in a
therapeutically effective amount. The quantity to be administered and the
timing
of administration depend on the host to be treated, capacity of the host's
system
to utilize the active ingredient, and degree of therapeutic effect desired.
Precise
amounts of the active ingredient required to be administered depend on the
judgment of the practitioner, and are peculiar to each individual.
Suitable dosage ranges for systemic application are disclosed
herein and depend on the route, of administration. Suitable regimes for
administration are also variable, but are typifiied by an initial
administration,
followed by repeated doses at one or more predetermined intervals by a
subsequent injection or other route of administration.
The present invention also provides a pharmaceutical composition
useful for practicing the therapeutic methods described herein. The
compositions
CA 02659030 2009-03-23
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contain an active compound described hereinabove, together with a
pharmaceutically acceptable carrier.
Preparations for parental administration of the present compounds
or compositions include sterile aqueous or non-aqueous solutions, suspensions,
and emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and injectable organic
esters
such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered media.
Parental vehicles include sodium chloride solution, Ringer's dextrose,
dextrose
and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles
include fluid and nutrient replenishers, electrolyte replenishers (such as
those
based on Ringer's dextrose), and the like. Preservatives and other additives
may
also be present, such as, for example, antimicrobials, antioxidants, chelating
agents, inert gases, and the like.
Another aspect of the present invention provides a method for
inhibiting MMP2 interaction with 43 and thus angiogenesis in a tumor tissue.
The inhibiting method comprises administering to the host a composition
comprising an angiogenesis-inhibiting amount of a compound described
hereinabove. IVINIl'2 interaction with a,,03 is inhibited by contacting 03
with a
compound of the present invention.
Angiogenesis is the formation of a neovascular network from pre-
existing host vessels and is required for tumor growth beyond 1-2 mm3. For the
purpose of the present invention, angiogenesis is inhibited as long as
angiogenesis
and the disease symptoms mediated by angiogenesis are ameliorated.
The dosage ranges for the administration to a host of the active
compound depend upon the particular active compound and its potency to a
particular tumor or integrin. Qne skilled in the art can readily determine the
proper dosage for a particular active compound without undue experimentation.
The host can be any mammal. The dosage should be large enough to produce the
desired therapeutic effect in which angiogenesis and the disease symptoms
mediated by angiogenesis are ameliorated, and is usually an amount sufficient
to
maintain a plasma level of the active compound in the range of about 0.01 to
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about 100 micromolar ( M), preferably about 0.2 to about 20 M, more
preferably about 1 to about 10 M. The dosage should not be so large as to
cause adverse side effects, however. The dosage per kilogram (kg) of body
weight can vary from 1 to 20 mg per dose, in one or more dose administrations
daily, for one or several days or indefinitely.
For inhibition of angiogenesis, the therapeutically effective amount
is an amount of active compound sufficient to produce a measurable inhibition
of
angiogenesis in the tissue being treated, i.e., an angiogenesis-inhibiting
amount
or an 1VIMP2 - 43 interaction inhibiting amount. Inhibition of angiogenesis
can
' be measured in situ by immunohistochemistry, as described herein, or by
other
methods known to one skilled in the art.
The present invention additionally provides pharmaceutical
compositions useful for practicing the therapeutic methods described herein.
The
compositions contain an active compound defined hereinabove together
pharmaceutically acceptable carrier.
The present invention also provides a method of inducing apoptosis
in tumor cells. This method comprises administering to the host a
therapeutically
effective amount of an active compound sufficient to initiate tumor cell
apoptosis.
For the purpose of the present invention, tumor cell apoptosis is
induced if an increased tumor cell apoptosis is observed in the target tumor
being
treated. Tumor cell apoptosis can be measured by methods described herein or
commonly known in the art.
The following non-limiting examples are provided to illustrate
various aspects of the present invention.
Materials and Methods
Antibodies Cells and Reagents. CS-1 hamster melanoma cells and
CS-1 cells transfected with the human Vintegrin sitbunit ACS-1 cells) were
described previously (Cell, 85, 683-93 (1996); Cell, 92, 391-400 (1998)). The
horseradish peroxidase (HRP)-conjugated monoclonal antibodies anti-biotin mAb
BN-34 and anti-actin mAb AC-40 were obtained from Sigma (St. Louis, MO).
Anti-von Willebrand Factor (vWF) polyclonal antibodies (pAb) were obtained
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from DAKO (Glostrup, Denmark). The cyclic peptides cRGDfV and cRADfV
and integrin-43 were provided by Merck KGaA (Darmstadt, Germany).
Purified proMMP2 and integrin-a43 were provided by Chemicon International
(Temecula, CA). Purified active MMP2 was obtained from Calbiochem (La
Jolla, CA). Basic fibroblast growth factor (bFGF) was kindly provided by Scios
(Mountain. View, CA).
Example 1. Solid Phase IntegritrBindingAssgys
Purified integrins were adsorbed overnight onto microtiter wells (1-
5 g/ml, 50 g/well) prior to blocking with Caseinblocker (Pierce, Rockford,
II,).
Purified biotinylated MMP2 (bMMP2, 3-5 nM) in binding buffer (50 mM Tris, pH
8,150 mM NaC1,1 mM MgC12, 0.5 mM MnC12) was added to the wells in the
presence or absence of test compounds, cyclic RGD or RAD peptides, or buffer
vehicle alone. Control wells received no integrin. Biotinylated vitronectin
(bVN,1
gfml) was used as a reference. Bound protein was detected with HRP-anti-biotin
mAb and quantitated at 450 nm with 3,3',5,5'-tetramethylbenzidine solution
(TMB;
a substrate for the peroxidase) (BioRad, Hercules, CA).
For the assessment of direct integrin binding by Compound 19, ocõ(33
and a5[3, (10 g/ml, 50 l/well) were coated onto Immulon-4 microtiter wells
(Dynatech Laboratories, Chantilly, VA), which were substantially blocked and
incubated with titration of [14C]-Compound 19 prior to the addition of 150 l
of
binding buffer containi.ng 0.1 % Tween-20 and aspiration of all liquid. Dried
wells
TM
were separated and immersed in BetaMax liquid scintillation cocktail (ICN
Biochemicals, Costa Mesa, CA) for quantitation. From this binding curve a
subsaturating concentration (3 M) of [14C]-Compound 19 was examined in the
presence and absence of a 25-fold molar excess (75 l) of unlabeled Compound
19
or Compound 9, or 100 M cyclic RGD or RAD peptide. Control was bVN, used
and detected as described above.
Example 2. M1VIP2 Cell-Binding and (3H]-Collagen IV Degradation Assays.
CS-1 cells or j33CS-1 cells were incubated in adhesion buffer
fibroblast basal medium (FBM) supplemented with 0.5% bovine seram albumin
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(BSA), 0.4 mM MnC12 and 10 g/ml aprotinin) containing either 4 nM purified
active MMP2 alone, or in combination with 10 M Compound 19 or Compound 9
for 45 minutes at 37 C prior to washing and addition to the PH]-collagen IV-
coated wells. Wells had been coated overnight with 50 l of 0.414 mCi/ml [3H]-
collagen IV (ICN Biochemicals, Costa Mesa, CA) and washed extensively until
the
radioactivity in the recovered wash solution reached background.
Alternatively,
cells were treated as above in the absence of MMP2 or the MMP2 solutions were
added directly to the wells without cells, as controls. Collagen N degradation
was
quantitated by measuring the radioactivity released into the 50 l of culture
medium
as determined in a liquid scintillation counter. For the assessment of
biotinylated
MMP2 binding to CS-1 cells, cells were suspended in adhesion buffer and
incubated with 12 nM bMMP2 for 45 minutes at 37 C in the presence or absence
of 10 ,M Compound 19 or Compound 9. - Cells were subsequently washed before
lysis and processing for SDS-PAGE and immunoblotting with an anti-biotin mAb.
Example 3. Synthesis of Compound 27.
A solution of N,N'-disuccinimidyl carbonate 95.38 g, 21 mmol) in
acetonitrile (150 mL) was treated with 4-(trifluoromethyl)benzyl alcohol (2.87
mL, 21 mmol) and triethylamine (EtjN; 5.8 mL, 42 mmol) and stirred at 25 C.
After 3 h this solution was added to a flask containing 1V E-BOC-lysine methyl
ester (4.2 g, 14 mmol) in acetonitrile and stirred for an additional 3 h. The
solvent was evaporated and the residue dissolved in CHZC12 (250 mL) and washed
with 10 % hydrochloric acid (2 x 200 mL) and saturated aqueous NaHCO3 (200
mL). Flash chromatography (SiO2, 3:1 CH2C12/EtOAc) provided 6.4 g(99 %) of
Compound 27 as a pale yellow oil: [a]? -8.9 05.55, CH3OH; 1H NMR (CDC13,
400 MHz) S 7.57 (d, J=8.1 Hz, 2H), 7.39 (d, J=8.1 Hz, 2H), 5.70 (d, J=7.9
Hz), 5.13 (m, 2H), 4.71 (m, 1H), 4.28 (m, 1H), 3.67 (s, 3H), 3.03 (m, 2H),
1.78 (m, 1H), 1.64, (m, 1H) 1.46-1.32 (m, 4H) 1.35 (s, 9H); 13C NMR (CDC13,
100 MHz) 8 172.9, 156.2, 155.8, 140.4, 130.1 (q, J=32.0 Hz),.127.8, 125.3,
122.9 (q J=270.0 Hz), 79.05, 65.8, 53,7, 52.3, 39.8, 31.7, 29.5, 28.4, 22.2;
IR
(film) v,. 3357, 2952, 1790, 1745, 1524 cm'; FABHRMS (NBA-Nal) m/z
463.2044 (M+H+, C21H29F3N2O6 requires 463.2056).
CA 02659030 2009-03-23
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Exam lp e 4. Synthesis of Compound 29.
A solution of Compound 27 (2.2 g, 48.8 mmol) in CH2CI2 (3 mL)
was treated with 4 N HCl-dioxane (10 mL) and stirred for 20 min at 25 C.
Solvent and excess acid were removed under reduced pressure, and the crude
hydrochloride salt was dissolved in DMF (40 mL), treated with N-((tert-
butyloxy)carbonyl)-N'-(2-(4-fluorophenyl)ethyl)iminodiacetic acid monoamide
(Compound 28) (1.68 g, 4.8 mmol), PyBrOP (3.3 g, 7.1 mmol) and
diisopropylethylamine (i-Pr2NEt; 5.0 mL, 29 mmol) and stirred for 1 h at 25
C.
The reaction mixture was diluted with EtOAc (400 mL) and washed with 10%
aqueous HCl (2 x 300 mL) and saturate aqueous NaHCO3 (300 mL). Flash
chromatography (SiO2, 1:1 CH2CI2/EtOAc) provided 2.47 g (74%) of Compound
29 as a white foamy solid: [a]DI -7.1 10 4.50, CH3OH); 'H NMR (CDC13, 400
MHz) 8 8.23 and 7.59 (m, together 1H), 7.58 (d, J=8.1 Hz, 2H), 7.43 (m, 2H),
.7.13 (m, 2H), 7.06 and 6.78 (m, together 1H), 6.94 (m, 2H), 5.70 (dd, J=12.9
and 8.2 Hz, 1H), 5.11 (m, 2H), 4.31(m, 1H), 3.85-3.72 (m, 4H), 3.71 (s, 3H,
3.49 (m, 2H), 3.22 (m, 2H), 2.79 (m, 2H), 1.81-1.39 (m, 6H) 1.38 (s, 9H); 13C
NMR (CDC13, 1001VIHz) S 172.8, 170.0, 169.9, 155.8, 154.8, 161.4 (d,
J=242.7 Hz), 140.2, 134.4, 130.1 (q, J=33.4 Hz), 130.0, 127.7, 125.3 (q,
J=3.0 Hz), 123.8 (q, J=299.9 Hz), 115.0, 81.2, 65.7, 53.9, 53.3, 52.2, 40.8,
38.6, 34.4, 31.5, 28.0, 22.4; IR (film) u. 3267, 2935, 1708, 1657, 1511 cni I;
FABHRMS (NBA-CsI) m/z 831.2026 (M+Cs+, C33H42F4N40$ requires
831.1993).
Example 5. Synthesis of Compound 19.
A solution of Compound 29 (50 mg, 0.075 mmol) in CHZC12 (1
mL) was treated with 4 N HC1-Dioxane (1 mL) and stirred for 1 h at 25 C.
Solvent and excess acid were removed under a stream of N2, and the crude
hydrochloride salt was suspended in CH2C12 (1 mL) and treated with
isophthaloyl
dichloride (7.6 mg, 0.038 mmol), and i-Pr2NEt (0.05 mL, 0.3 mmol) and stirred
for 12 h at 25 C. The reaction mixture was diluted with EtOAc (50 mL) and
washed with 10% hydrochloric acid (3 x 30 mL), saturated aqueous NaHCO3 (30
mL) and saturated aqueous NaCI (30 mL). Flash chromatography (Si021
1:4.5:4.5 MeOH/CH2ClZ/EtOAc) provided 30 mg (60%) of Compound 19 as a
CA 02659030 2009-03-23
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white powder: 'H NMR (CD30D, 400 MHz) 6 7.62 (m, 4H), 7.52 (m, 4H), 7.42
(m, 4H), 7.19 (m, 4H), 6.96 (m, 4H), 5.14 (m, 4H), 4.16 (m, 2H), 4.13 (m,
2H), 4.08 (m, 2H), 3.99 (m, 4H), 3.68 (s, 6H), 3.45-3.35 (m, 4H), 3.25-3.11
(m, 4H), 2.82-2.70 (m, 4H), 1.82 (m, 2H), 1.69 (m, 2H) 1.60-1.34 (m 8H); IR
(film) vam 3291, 2936, 1725, 1651, 1326 em'; FABHRMS (NBA-CsI) m/z
1459.4015 (M+Cs{, C64H7OFSN8O14 requires 1459.3938).
Example 6. Synthesis of Compound 30.
A solution of Compound 19 (13 mg, 0.01 mmol) in tert-butanol
(0.3 mL) was treated with LiOH-H20 (0.91 mg, 0.22 mmol) dissolved in H20
(0.15 mL), and stirred for 2 h at 0 C. The reaction mixture was then quenched
with HCO2H (1 mL), diluted with EtOAc (10 mL) and washed with saturated
aqueous NaC1(2 x 10 mL). Drying (Na2SO4) and evaporation provided 12 mg
(95 %) of Compound 30 as a white powder: 'H NMR (DMSO-d6, 400 MHz) S
12.54 (br s,2H), 8.63 (m, 1H), 8.43 (m, 2H), 8.30 (m, 1H), 7.74 (m, 4H), 7.69
(m, 2H), 7.57 (m, 4H), 7.40 (m, 4H), 7.24 (m, 4H), 7.09 (m, 4H), 5.15 (m,
4H), 4.14 (m, 2H), 4.02-3.87 (m, 8H), 3.31 (m, 4H), 3.208 (m, 4H), 2.74 (m,
4H), 1.71(m, 2H), 1.62 (m, 2H) 1.50-1.34 (m, 8H; IR (film) v. 3287, 2928,
1705, 1659, 1320 cm''; MALDIHRMS m/z 1321.4493 (M+Na+, C62H66F$N8014
requires 1321.4468).
Example 7. Synthesis of f14C1-Compound 19.
A solution of Compound 27 (1.7 mg, 1.3 mmol) and EDCI (2.0
mg, 10.3 mmol) in DMF (20 mL) was treated with 0.3 mL of a solution of
14CH3OH in CH2CI2 (57 mCilmmol, 5.2 mmol 14CH3OH) and 35 mL of DMAP
stock solution in CH2Cl2 (0.6 mmol DMAP) and stirred for 4 h at 0 C. The
reaction mixture was then diluted with EtOAc (3 L) and washed with 10 %
hydrochloric acid (3 x 3 mL) and saturated aqueous NaHCO3 (3 mL) and dried
(Na2SO4). Purification on PTLC (Si02, 2:3:3 EtOH/CHC13EtOAc) provided 0.6
mg (35%) of [14C]-Compound 19 as a white film. This material was identical to
the corresponding unlabeled dimethyl ester Compound 1 by 'H NMR and HPLC.
The relative activity was approx. 104 mCi/mmol: 'H NMR (CD30D, 400 MHz)
8 7.62 (m, 4H), 7.52 (m, 4H), 7.42 (m, 4H), 7.19 (m, 4H), 6.96 (m, 4H), 5.14
(m, 4H), 4.16 (m, 2H), 4.13 (m, 2H), 4.08 (m, 2H), 3.99 (m, 4H), 3.68 (s, 6H),
CA 02659030 2009-03-23
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3.45-3.35 (m, 4H), 3.25-3.11 (m, 411), 2.82-2.70 (m, 411), 1.82 (m, 211), 1.69
(m, 2H) 1.60-1.34 (m, 8H).
Results and Discussion
Combinatorial libraries of compounds, including compounds of
Formulas (1) and (II) are described in detail, along with methods of synthesis
thereof, in Boger et al., Bioorg. Med. Chem, 6, 1347-1378 (1998). Boger et al.
describe the preparation of a combinatorial library of 60 mixtures of 10
compounds each wherein the individual compounds in the mixtwres are comprised
of three subunits coupled together as shown in FIG. 2. The subunits of the
compounds are designated A, B and C. The library was constructed from six
different A units (Al - A6), 10 different B units (Bl - B10), and 10 different
C
linking groups (Cl - C10). Each A unit was coupled to each B unit, to form 60
distinct AB compounds. The individual AB compounds were then coupled to
mixtures of ten different C linking groups, to form 60 mixtures of 10
compounds
each, designated AxBy wherein the x and y denote the individual A and B
subunits, respectively, that were incorporated into the compounds of the
mixtures. The A, B and C subunits of the combinatorial library of compounds
are shown in FIG. 2.
Evaluation of the 60 mixtures described hereinabove in a
competitive integrin 43 binding assay, in competition with MMP2 indicated that
several of the mixtures inhibited the binding of MMP2 with integrin a,,P3. The
results of the evaluation assay are presented in FIG. 3. The particularly
active
mixtures included A1B6, A1B7, A1B8, A4B1, ASB4, ASBS, A5B6, A5B10, and
A6B10. The most active mixture was A6B10, therefore, the ten individual
compounds of the mixture were synthesized separately and examined in the same
assay, the results of which are presented in FIG 4. All of the individual
components A6B10C1 through A6B10C10 were active at a 3 M concentration
in the assay.
Analogs Compounds (2 - 26) of A6B10C4 (Compound 1), shown
in FIG 5, were also evaluated. The results of the binding assays for Compounds
CA 02659030 2009-03-23
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2- 26 are presented in FIG. 6A. All of the compounds except Compounds 8, 9
and 23 inhibited MMP2 binding to integrin.
The active MMP2 / integrin-a,-P3 binding inhibitors of present
invention are encompassed by Formulas (I) and (11).
Compound 19 was examined=in detai] to determine its specific
target and to define its biological properties. Benzoyl amide Compound 9 was
selected as an appropriate negative control compound for many of these studies
since it was found to lack antagonist activity in the binding assay, despite
its
overall structural similarity and similar physical properties (e.g. solubility
and
hydrophobicity). Compound 19 exhibited concentration dependent inhibition of
binding of MMP2 to integrin as shown in FIG. 6B.
A radiolabel (14C) was incorporated into Compound 19 in the ester
substituent (relative activity approx. 104 mCi/mmol). After incubation (at 3
M)
with fixed a0s and subsequent washing, this compound was found to adhere to
the integrin as demonstrated in FIG 7. Incubation in the presence of a 25-fold
molar excess of cold Compound 19 significantly reduced the observed amount of
bound agent, whereas incubation in the presence of a 25-fold molar excess of
(cold) control Compound 9 did not affect the binding of [14C]-Compound 19. In
a
similar experiment measuring the interaction of [14C]-Compound 19 to fixed
MMP2, no binding was observed. These results suggest that the origin of the
antagonist activity observed in the MMP2-a~-N binding assay is derived from
the
specific binding of Compound 19 to a,_R3. The nature of the Compound 19-av P3
interaction is independent from the integrin site which recognizes the Arg-Gly-
Asp sequence. Cyclic RGD peptide cyclo(Arg-Gly-Asp-D-Phe-Val) had no effect
on [14C]-Compound 19 binding (FIG. 7). In fact, as shown in FIG. 8, Compound
19 did not inlubit the binding of vitronectin, awP3's classical high-affinity
ligand
to the integrin, consistent with the concept that the binding site for
Compound 19
is distinct from that which binds RGD-ligands.
Compound 19 was also studied in a cellular assay, which measures
the ability of endothelial cells to utilize MMP2 to degrade a protein matrix,
a key
step in angiogenesis. It has been shown previously that disrupting the binding
of
MMP2 to a,,03 inhibits collagen IV degradation. CS-1 melanoma cells
transfected
CA 02659030 2009-03-23
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with aAwere found to degrade immobilized [3H]-collagen N far above the
degradation of [i3 negative CS-1 ceIls (which lack a,,P). As shown in FIG. 9,
treatment of these cells with Compound 19 significantly dimini,shed the
increased
matrix degradation, consistent with the cells being unable to utilize MMP2,
which
is not bound to the integrin surface. Compound 19 did not, however, directly
inhibit MMP2's proteolytic activity, as purified (active) enzyme in the
absence
cells was able to degrade [31i]-collagen IV to a simfflar extent in the
presence or
absence of the Compound 19.
These results support the proposition that compounds of Formula
' (I) disrupt the ability of tumor cells to utilize MMP2 to degrade ECM
proteins in
a manner analogous to PEX. The compounds of Formula (I) do not interfere
with the binding of a03 to its classical RGD ligands nor do they function as a
direct proteinase inhibitors.
The foregoing description and the Examples are to be taken as
illustrative but not limiting. Still other variants within the spirit and
scope of the
present invention are possible and will readily present themselves to those
skilled
in the art.
