Note: Descriptions are shown in the official language in which they were submitted.
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BISPHOSPHONATE COMPOUNDS AND METHODS
WITH ENHANCED POTENCY FOR MULTIPLE TARGETS
INCLUDING FPPS, GGPPS, AND DPPS
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a nonprovisional application of U.S. provisional
application serial number 60/911,426 filed April 12, 2007; and a continuation-
in-part
of U.S. application serial number 11/687,570 filed March 16, 2007 and
international
application number PCT/US07/64239 filed March 16, 2007, each of which are
nonprovisional applications of U.S. provisional application serial number
60/783,491
filed March 17, 2006; and a continuation-in-part of U.S. application serial
number
11/245,612 filed October 7, 2005 and international application number
PCT/US05/36425 filed October 7, 2005, each of which are nonprovisional
applications of U.S. provisional application serial number 60/617,108 filed
October 8,
2004; all of which are hereby incorporated by reference to the extent not
inconsistent
with the disclosure herewith. This application hereby claims benefit of
priority to the
above-referenced applications.
STATEMENT REGARDING FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under NIH Grant Nos.
NIH GM50694, GM65307, GM73216, and AI-060452 awarded by the National
Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0003] Earlier generation compounds of nitrogen-containing bisphosphonates
such as pamidronate (Aredia ), alendronate (Fosamax ), risedronate (Actonel ),
zoledronate (Zometa ), and ibandronate (Boniva) represent drugs currently used
to
treat conditions such as osteoporosis, Paget's disease and hypercalcemia due
to
malignancy. These compounds function primarily by inhibiting the enzyme
farnesyl
diphosphate synthase (FPPS), resulting in decreased levels of protein
prenylation in
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osteoclasts. Certain bisphosphonates have also been found to have anti-
parasitic
activity and to stimulate human yb T cells, and with these earlier generation
compounds there has been interest in cancer-related applications. There is a
continued need, however, for the further development of alternative compounds
and
methods of use including therapeutic applications. There remains a need for
alternative compounds and methods, including in particular compounds having
improved properties such as greater activity and/or other advantageous
functionality.
[0004] The mevalonate pathway, also referred to as the HMG-CoA reductase
pathway, or mevalonate-dependent (MAD) route, is an important cellular
metabolic
pathway present in higher eukaryotes and many bacteria. This pathway
contributes
to the production of dimethylallyl pyrophosphate (DMAPP) and isopentenyl
pyrophosphate (IPP) that serve as the basis for the biosynthesis of molecules
used
in processes as diverse as protein prenylation, cell membrane maintenance,
hormones, protein anchoring and N-glycosylation. The ability to inhibit a
single
molecule in such a pathway can provide an option for the modification of
function
and one or more outputs of the pathway. The ability to interact with multiple
molecular targets of such a fundamentally important pathway, however, can
provide
opportunities for greater levels of modification. For example, the ability to
simultaneously knock out a pipeline at several points can dramatically
diminish the
impact of the pipeline's flow and/or yield of products.
[0005] In embodiments of the invention herein, we disclose important
discoveries
regarding compounds and methods in connection with the inhibition of molecular
targets including FPPS, geranylgeranyl pyrophosphate synthase (GGPPS), and
decaprenyl pyrophosphate synthase (DPPS).
[0006] In certain embodiments, compounds of the invention include
bisphosphonates that are capable of selectively inhibiting one or more of
FPPS,
GGPPS, and DPPS. In preferred embodiments, compounds of the invention are
capable of selectively inhibiting two or more of FPPS, GGPPS, and DPPS. In
embodiments, compounds and methods of the invention demonstrate superior
activity levels, such as in the anti-cancer context, which in several cases
exceed the
activity levels of previous generation bisphosphonate drugs by orders of
magnitude.
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The invention disclosed herein thus represents a major advance in the
development
of useful agents which in certain embodiments are compounds capable of
demonstrating high potency levels.
SUMMARY OF THE INVENTION
[0007] The invention provides, inter alia, novel bisphosphonate compounds and
methods of making and using the compounds. In embodiments, the invention
provides compounds and methods in connection with research and therapeutic
applications, e.g., for tumor cell growth inhibition, activation of gammadelta
T cells,
inhibition of farnesyldiphosphate (FPPS), GGPPS, and/or DPPS enzymes, and for
treatment of bone resorption diseases, cancer, immune disorders,
immunotherapy,
and infectious diseases. In regard to certain embodiments, it has been
recognized
that certain structural features significantly enhance the activity of the
compounds.
Certain compounds are disclosed with structural features that correlate with
useful
and in certain embodiments high activity levels in functionally relevant
contexts. For
example, in specific embodiments the presence of particular alkoxy
substituents on a
ring component in an organic bisphosphonate compound contribute to desirable
functional activity. Further variations are also provided.
[0008] Structural features of compounds have been identified which correlate
with
functional properties and activities. In embodiments, compounds of the
invention are
capable of demonstrating profound activity levels, for example in inhibiting
tumor cell
growth inhibition and immunostimulation. Compounds having such features have
been synthesized and tested. This testing has allowed the further
identification and
development, for example, of a first class of compounds with significant anti-
cancer
and immunostimulatory ability and a second class of compounds with anti-cancer
ability, but without substantial immunostimulatory capability.
[0009] In an embodiment, compounds of the invention can provide advantages
such as desirable activity, improved activity and/or therapeutic effect,
reduced toxic
effect, and/or such therapeutic and/or toxic effect with a more advantageous
administration profile. In an embodiment, the more advantageous administration
profile can invole one or more of lowered individual and/or total dosage
amount; less
frequent dosing regime; etc. In an embodiment, one or more of such advantages
or
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qualities is capable of being determine in relation to another bisphosphonate
compound, for example by comparison with a previous generation compound such
as an approved drug.
[00010] In embodiments, bisphosphonate compounds of the invention can
demonstrate activity in one or more contexts, including a farnesyl diphosphate
synthase (FPPS) assay, a GGPPS assay, a DPPS assay, a D. discoideum growth
inhibition assay, a T cell activation assay, a bone resorption assay, the
treatment of
infectious disease, the treatment of a bone resorption clinical disorder, an
immunotherapeutic treatment, the treatment of cancer, the treatment of bone
pain,
stimulation of an immune cell and/or system, and inhibition of growth of a
cancer cell
or tumor.
[00011] The invention broadly provides bisphosphonate compounds and related
methods of making and using. In embodiments, the invention specifically
provides
organic bisphosphonate compounds and/or pharmaceutically acceptable salts or
esters thereof. In further embodiments, the invention specifically provides
other
variations of bisphosphonate compounds. In embodiments, functionally and/or
therapeutically active bisphosphonates of this invention have general and
specific
structures as described herein.
[00012] In embodiments, the present invention provides compounds of
bisphosphonates and pharmaceutical compositions comprising one or more
bisphosphonates. In preferred embodiments, the bisphosphonates are high
potency
bisphosphonates in one or more functional contexts.
[00013] In embodiments, the invention provides compounds of formula XA1:
OM
,R2 O~ P'OM
Z n X
4//P\ OM
O OM XA1
or salts or hydrates thereof, wherein;
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[00014] X is hydrogen, hydroxyl group, or a halogen;
[00015] M, independently of other M in the compound, are a negative charge, a
hydrogen, alkyl group, -(CH2)p-O-CO-R or -(CH2)p-O-CO-O-R, where p is 1 to 6,
and
R is hydrogen, optionally substituted alkyl or optionally substituted aryl; -
OM can
also be a salt of form -O-A+, where A+ is a cation;
[00016] n is 1, 2, or 3:
[00017] each R, and R2, independently of each other, are selected from the
group
consisting of a hydrogen, a halogen, -N(R')2, -SR', OR', an optionally
substituted
alkyl, an optionally substituted alkenyl, and an optionally substituted aryl
group,
where each R', independent of any other R' in any listed group, is selected
from H,
an optionally substituted alkyl group and an optionally substituted aryl
group, and
one of R, and one of R2 together form a 3-10 member carbocyclic or hetrocyclic
ring
containing one to three heteroatoms, particularly N, S, and 0;
[00018] Z is
R3
R4 N
R5 R7
R6 zi,
R3
R4 N \
~
/
R5 R7 R5 R7
R6 Z2, R6 Z3,
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R3
R4
~ R3 N
N
R7 I />-R7
N
R6 Z4, R4 Z5,
R ~~ U
1
RN Z6, FR4 N Z7
(D 0,E)
R$ ~ I
R4 S
R9 Z8, OO Zg,
R10\O O
R12 I P NHR15
R11 Z10, R13R14N "K NR16 Z11,
R3
R4
R5 R7
R6 Z12,
[00019] wherein U is H or OH;
[00020] R3-R7 if present, independently of one another, are selected from the
group
consisting of a hydrogen, a halogen, a-CN, -OR"', -COOR"', -OCOOR"', -COR"', -
CON(R"')2, -OCON(R"')2, -N(R"')2, -NO2, -SR, -SO2R, -SO2N(R"')2 or -SOR"'
group,
an optionally substituted alkyl group, an optionally substituted alkenyl
group, an
optionally substituted alkynyl group and an optionally substituted aryl group,
where
each R or R"', is independently selected from H, an optionally substituted
alkyl
group, an optionally substituted aryl group, and an optionally substituted
acyl group;
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[00021] wherein at least one of R3-R7, if present is RL and when Z is Z6, R4,
is RL
where RL is a group selected from alkyl, alkoxy, alkenyl, alkynyl, alkenoxy or
alkynoxy groups having 6 to 20 carbon atoms, each of which are optionally
substituted; alkyl ether groups which are alkyl groups having 6-20 carbon
atoms in
which one or more non-adjacent carbon atoms are replaced with an 0; or a 3-RM
or
4-RM substituted phenyl group, where RM is selected from alkyl, alkenyl,
alkynyl,
alkoxy, alkenyoxy, alkynoxy or alkyl ether groups having 3-15 carbon atoms,
where
the other ring positions of the phenyl ring are optionally substituted with
one or more
halogens, or one or more optionally substituted alkyl groups having 1-3 carbon
atoms;
[00022] RN is an optionally substituted alkyl group having 1-3 carbon atoms;
[00023] R8, R10 and R13, if present, are groups selected from alkyl groups
having 6-
20 carbon atoms; alkenyl or alkynyl groups having 6 to 20 carbon atoms; alkyl
ether
groups which are alkyl groups having 6-20 carbon atoms in which one or more
non-
adjacent carbon atoms are replaced with an 0; or 3-RM or 4-RM substituted
phenyl
groups, where RM is selected from alkyl, alkenyl, alkynyl, alkoxy, alkenyoxy,
alkynoxy
or alkyl ether groups having 3-15 carbon atoms, where the other ring positions
of the
phenyl ring are optionally substituted with one or more halogens, or one or
more
optionally substituted alkyl groups having 1-3 carbon atoms;
[00024] R9, R11 and R12, if present, are groups selected from alkyl, alkenyl
or
alkynyl groups having 1-6 carbon atoms; alkyl ether groups which are alkyl
groups
having 1-6 carbon atoms in which one or more non-adjacent carbon atoms are
replaced with an 0; or optionally substituted phenyl groups;
[00025] R14, R15, R16, if present, are independently selected from hydrogen or
optionally substituted alkyl having 1-6 carbon atoms or optionally substituted
aryl
groups; wherein R9 can be linked to the first carbon of R8 to form a 5-8
member
carbon ring which may be saturated or carry one or two double bonds;
[00026] wherein optional substitution most generally means substitution of one
or
more carbons of the listed optionally substituted groups with non-hydrogen
substituents selected from the groups consisting of one or more halogens, one
or
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more cyano, one or more alkyl, haloalkyl, or hydroxyalkyl groups having 1-3
carbon
atoms, one or more alkenyl, haloalkenyl or hydroxyalkenyl groups having 1-4
carbon
atoms; one or more alkynyl groups having 1-4 carbon atoms, one or more acyl or
haloacyl groups; or one or more groups selected from a -ORs, -COORs, -OCOORs,
-CORs, -CON(Rs)2, -OCON(Rs)2, -N(Rs)2, -NO2, -SRs, -SO2Rs, -SO2N(Rs)2 or -
SORs group, where Rs is hydrogen, an alkyl group having 1-6 carbon atoms,
optionally substituted with one or more halogens, hydroxyl groups, amino
groups or
alkyl amino groups, or an aryl group and an aryl group optionally substituted
with one
or more alkyl groups, haloalkyl groups, halogens, hydroxyl groups, amino
groups,
alkyl amino groups, acyl groups or haloacyl groups.
[00027] In specific embodiments: Z is any one of Z1-Z5; Z is Z6; Z is Z7; Z is
Z9; Z
is Z8 or Z10; Z is Z11 or Z is Z12. In specific embodiments: when Z is Z1, R4
is RL;
when Z is Z1, R5 is RL; when Z is Z1, R6 is RL; when Z is Z2, R4 is RL; when Z
is Z2,
R5 is RL; when Z is Z2, R6 is RL; when Z is Z3, R5 is RL; when Z is Z3, R6 is
RL;
when Z is Z4, R4 is RL; when Z is Z4, R6 is RL; when Z is Z5, R3 is RL; when Z
is Z5,
R4 is RL; when Z is Z12, R4 is RL; or when Z is Z12, R5 is RL.
[00028] In specific embodiments, RL is a group selected from alkyl, alkenyl or
alkynyl groups having 7-20 carbon atoms or alkoxy groups having 7-20 carbon
atoms.
[00029] In specific embodiments, RL is a group selected from alkyl or alkynyl
groups having 7-20 carbon atoms or alkoxy groups having 7-20 carbon atoms. In
other embodiments, RL is a group selected from alkyl, or alkynyl groups having
7-14
carbon atoms or 8-12 carbon atoms. In other embodiments, RL is an alkoxy group
having 7-14 carbon atoms or 8-12 carbon atoms.
[00030] In specific embodiments, RL is a straight-chain alkyl or alkoxy group
having
from 7 to 20 carbons atoms or 7 to 12 carbon atoms. In specific embodiments,
in
which Z is Z1-Z5, RL is a straight-chain alkyl or alkoxy group having from 7
to 20
carbons atoms. In specific embodiments, in which Z is Z1-Z5, RL is a straight-
chain
alkyl or alkoxy group having from 7 to 10 carbons atoms. In specific
embodiments,
where Z is Z1-Z4, RL is a straight-chain alkyl or alkoxy group having from 7
to 10
carbons atoms. In specific embodiments, where Z is Z1-Z2 or Z4, R4 is RL and
RL is
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a straight-chain alkyl or alkoxy group having from 6 to 10 carbons atoms or 7-
10
carbon atoms. In specific embodiments, where Z is Z1, R4 is RL and RL is a
straight-chain alkyl or alkoxy group having from 7 to 10 carbon atoms. In
specific
embodiments, where Z is Z1, R4 is RL and RL is a straight-chain alkoxy group
having
from 6 to 20 carbon atoms. In specific embodiments, where Z is Z1, R4 is RL
and RL
is a straight-chain alkoxy group having from 7 to 10 carbon atoms.
[00031] In specific embodiments, where Z is Z8 or Z10 and R8 or Rlo,
respectively,
is an alkyl group having 8-20 carbon atoms. In specific embodiments, where Z
is Z8
or Z10, R8 or Rlo, respectively, is an alkyl group having 9-17 carbon atoms.
In
specific embodiments, where Z is Z8 or Z10, R8 or Rlo, respectively, is a
straight-
chain alkyl group having 8-20 carbon atoms or a straight-chain alkyl group
having 9-
17 carbon atoms. In specific embodiments, Z is Z8 and R8 is an alkyl group
having
8-20 carbon atoms. In specific embodiments, Z is Z8 and R8 is a straight-chain
alkyl
group having 8-20 carbon atoms. In specific embodiments, Z is Z8 and R8 is a
straight-chain alkyl group having 9-17 carbon atoms.
[00032] In specific embodiments, Z is Z1-Z5 and RL is an alkynyl group -C=C-
RAK
where RAK is a straight-chain alkyl group having 4-20 carbon atoms or 5-10
carbon
atoms. In specific embodiments, Z is Z1-Z4 and RL is an alkynyl group -C=C-RAK
where RAK is a straight-chain alkyl group having 4-20 carbon atoms or 5-10
carbon
atoms. In specific embodiments, Z is Z1-Z2 or Z4, R4 is RL and RL is an
alkynyl
group -C=C-RAK where RAK is a straight-chain alkyl group having 4-20 carbon
atoms
or 5-10 carbon atoms. In specific embodiments, Z is Z1, R4 is RL and RL is an
alkynyl group -C=C-RAK where RAK is a straight-chain alkyl group having 4-20
carbon atoms or 5-10 carbon atoms.
[00033] In specific embodiments, RL are alkyl ether groups which are alkyl
groups
having 7-20 carbon atoms or 7-14 carbon atoms n which one or more non-adjacent
carbon atoms are replaced with an O.
[00034] In specific embodiments, RL is a 3-RM or 4-RM substituted phenyl
group,
where RM is selected from alkyl, alkenyl, alkynyl, alkoxy, alkenyoxy, alkynoxy
or alkyl
ether groups having 3-15 carbon atoms or 6-12 carbon atoms, where the other
ring
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positions of the phenyl ring are optionally substituted with one or more
halogens, or
one or more optionally substituted alkyl groups having 1-3 carbon atoms.
[00035] In specific embodiments, one or more alkyl groups herein are
optionally
substituted with one or more halogens. In other embodiments, aryl groups
herein
are phenyl groups optionally substituted with one or more halogens, or one or
more
alkyl groups having 1-3 carbon atoms.
[00036] In specific embodiments, R13 is a group selected from alkyl, or
alkynyl
groups having 7-20 carbon atoms; or an alkyl ether groups which are alkyl
groups
having 7-20 carbon atoms in which one or more non-adjacent carbon atoms are
replaced with an 0;
[00037] In specific embodiments, R13 is a group selected from alkyl, or
alkynyl
groups having 7-20 carbon atoms or 9-17 carbon atoms;
[00038] In specific embodiments, when Z is Z12, RL is R4 or R5 and RL is an
optionally substituted alkyl, or alkoxy group having 7-20 carbon atoms or an
alkynyl
group having 6 to 20 carbon atoms. In other embodiments, when Z is Z12, RL is
an
unsubstituted alkyl or alkoxy group having 7-20 carbon atoms. In additional
embodiments, the alkyl group is a straight-chain alkyl group or the alkyl of
the alkoxy
group is a straight -chain alkyl group. In other specific embodiments, R4 is
RL. In
other embodiments, the alkyl or alkoxyl group has 7-17 carbon atoms. In other
embodiments, the alkyl or alkoxy group has 8-12 carbon atoms. . In specific
embodiments, Z is Z12, and RL is an alkynyl group -C=C-RAK where RAK is a
straight-chain alkyl group having 4-20 carbon atoms or 5-10 carbon atoms.
[00039] In specific embodiments, when Z is Z12, RL is a substituted aryl,
preferably
phenyl; and more particularly RL is a sulfonamide substituted phenyl or is a
naphthyl
sulfonamide substituted phenyl.
[00040] In a preferred embodiment, when M is a salt the cation A+ is a
pharmaceutically acceptable cation.
[00041] In each of the above listed specific embodiments, the following
additional
specific embodiments are included:
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[00042] R3-R7,if present, which are not RL are selected from the group
consisting of
a hydrogen, a halogen, an optionally substituted alkyl group, an optionally
substituted alkenyl group, an optionally substituted alkynyl group, an
optionally
substituted alkoxy group, and an optionally substituted aryl group;
[00043] R3-R7, if present, which are not RL are hydrogens, halogens or
unsubstituted alkyl groups having 1-3 carbon atoms;
[00044] R3-R7, if present, which are not RL are hydrogens;
[00045] each R9, if present, is an alkyl group having 1-6 carbon atoms;
[00046] each R9, if present, is an alkyl group having 1-4 carbon atoms;
[00047] each R9, if present, is an alkyl group having 1-3 carbon atoms;
[00048] each R9, if present is a methyl group;
[00049] Ril or R12, if present, are the same groups;
[00050] Ril or R12, if present, are different groups;
[00051] Rll or R12, if present, are alkyl groups having 1-6 carbon atoms;
[00052] Rll or R12, if present, are alkyl groups having 1-4 carbon atoms;
[00053] Ril or R12, if present, are alkyl groups having 1-3 carbon atoms;
[00054] Ril or R12, if present, are methyl groups;
[00055] R14 and R15, if present, are hydrogens;
[00056] R15 and R16, if present, are hydrogens;
[00057] R15 and R16, if present, are hydrogens and R14 is hydrogen or an alkyl
group having 1-3 carbon atoms;
[00058] RN is a methyl group; or
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[00059] R4 is a straight-chain alkyl group having from 6-20 carbon atoms or 7-
17
carbon atoms or 8-15 carbon atoms.
[00060] In other specific embodiments, high potency bisphosphonates include
those of formula XA1 wherein Z is Z1A, Z2A, Z2B, Z3A, Z4A, Z5A, Z12A or Z12B:
::$7 RL R4 R5 / R7 R5 R7
R6 Z1A, R6 Z2A, RL Z2B,
R3 R3
RL ~
I R
3 NR5 R7 7 >
RL Z3A, R6 Z4A, RL Z5A,
R3 R3
RL ~ R4R5 7 RL R7
R6 Z12A, or R6 Z12B
[00061 ] where variables R3-R7 are not RL, but take all other values as listed
above
and RL is as defined above including various specific embodiments set forth
herein.
In specific embodiments of Z1A, Z2A, Z3A, Z4A, or Z5A, R3-R7 are selected from
hydrogens, halogens or alkyl groups having 1-3 carbon atoms; or all of R3-R7
are
hydrogens. In specific embodiments of Z1A, Z2A, Z3A, Z4A, Z5A, Z12A or Z12B,
RL are alkyl or alkoxy groups having 7-20 carbon atoms or 7 to 17 carbon
atoms. In
specific embodiments of Z1A, Z2A, Z3A, Z4A, or Z5A, RL are straight-chain
alkyl or
alkoxy groups having 6-20 carbon atoms or 7 to 17 carbon atoms. In other
specific
embodiments of Z1A, Z2A, Z3A, Z4A, or Z5A, RL are alkynyl groups having from 8-
20 carbon atoms or 9 to 17 carbon atoms.
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[00062] In each of the above listed specific embodiments, the following
additional
specific embodiments are included:
[00063] R, and R2 are all hydrogens;
[00064] n is 1;
[00065] n is 2;
[00066] X is hydrogen;
[00067] X is a hydroxyl group;
[00068] X is fluorine;
[00069] X is chlorine;
[00070] All M are hydrogens;
[00071] At least one M is a negative charge and the remaining M are hydrogens;
[00072] At least one M is ,-(CH2)p-O-CO-R or -(CH2)p-CO-R, where p is 1 to 6,
and
R is hydrogen, optionally substituted alkyl or optionally substituted aryl; or
[00073] One, or two of -OM are -O-A+, where A+ is a cation and the remaining M
are
hydrogens;
[00074] Z is one of Z as set forth herein.
[00075] In a particular embodiment, the invention provides compounds of
formula
XA1 wherein Z=Z1 2 and R4=RL. In a particular embodiment, the invention
provides
compounds of formula XA1 wherein Z=Z1 and R4=RL.
[00076] In an embodiment, the invention provides a compound selected from the
group consisting of: 637, 638, 677, 687, 688, 693, 694, 695, 696, 714, 715,
716,
717, 722, 754, 675, 678, and 728; and for each respective said compound, a
pharmaceutically acceptable salt or ester thereof. In an embodiment, said
compound is also a compound of formula XA1.
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[00077] In an embodiment, the invention provides a composition comprising a
pharmaceutical formulation of a compound of the invention of any formula
herein.
[00078] In an embodiment, the invention provides a medicament which comprises
a
therapeutically effective amount of one or more compositions of the invention.
In an
embodiment, the invention provides a method for making a medicament for
treatment of a condition described herein.
[00079] In an embodiment, the invention provides a method of inhibiting growth
of a
cancer cell comprising contacting said cancer cell with an effective amount of
a
compound of the invention or a pharmaceutical formulation thereof. In an
embodiment, the invention provides a method of treating a cancer comprising
administering to a patient in need thereof, a therapeutically effective amount
of a
compound of the invention or a pharmaceutical formulation thereof. In an
embodiment, the cancer is a breast cancer. In an embodiment, the breast cancer
involves an actual or potential bone metastatic condition. In an embodiment,
the
cancer is a cancer known in the art.
[00080] In an embodiment, the invention provides a method of stimulating a T
cell,
comprising contacting the T cell with a compound of the invention or a
pharmaceutical formulation thereof. In an embodiment, said T cell is a
gammadelta
T cell. In an embodiment, the invention provides a method of immunotherapeutic
treatment comprising administering to a patient in need thereof, a
therapeutically
effective amount of a compound of the invention or a pharmaceutical
formulation
thereof.
[00081] In an embodiment, the invention provides a method of treating a bone
resorption disorder comprising administering to a patient in need thereof, a
therapeutically effective amount of a compound of the invention or a
pharmaceutical
formulation thereof. In an embodiment, the invention provides a method of
treating a
bone pain condition comprising administering to a patient in need thereof, a
therapeutically effective amount of a compound of the invention or a
pharmaceutical
formulation thereof.
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[00082] In an embodiment, the invention provides a method of inhibiting growth
of
an infectious disease agent comprising contacting said infectious disease
agent with
an effective amount of a compound of the invention or a pharmaceutical
formulation
thereof. In an embodiment, the invention provides a a method of treating an
infectious disease comprising administering to a patient in need thereof, a
therapeutically effective amount of a compound of the invention or a
pharmaceutical
formulation thereof. In an embodiment, the infectious disease relates to an
agent
selected from the group consisting of: a virus, a fungus, a bacterium, and a
protozoan parasite. In an embodiment, said virus is a retrovirus. In an
embodiment,
said retrovirus is human immunodeficiency virus (HIV). In an embodiment, said
protozoan parasite is selected from the group consisting of: Leishmania,
Toxoplasma, Cryptosporidium, Plasmodium, and Trypanosoma. In an embodiment,
said protozoan parasite is Leishmania major. In an embodiment, said bacterium
is
Escherichia coli or Staphylococcus aureus.
[00083] In an embodiment, the invention provides a method of synthesizing a
compound of the invention or a pharmaceutical formulation thereof. In an
embodiment, a synthetic scheme is used or adapted from such of US Application
Serial 11687570 filed March 17, 2006; PCT International Application Serial
PCT/US07/64239 filed March 17, 2006; US Application Serial 60783491 filed
March
17, 2006; US Application Serial 11/245,612 filed October 7, 2005 (see also US
Patent Application Publication No. 20060079487 published April 13, 2006); US
Application Serial 60/617,108 filed October 8, 2004; PCT International
Application
No. PCT/US05/036425 filed October 7, 2005 (see also International Publication
No.
WO/2006/039721 published April 13, 2006); US Patent Application Publication
No.
20050113331 published May 26, 2005; and as would be understood in the art.
[00084] In an embodiment, the invention provides a method of selectively
inhibiting
one or more of an FPPS, GGPPS, DDPPS, and a DHDDS enzyme. In an
embodiment, the invention provides a method of selectively inhibiting two or
more of
an FPPS, GGPPS, and a DPPS enzyme, comprising contacting said enzymes or a
cell containing said enzymes with an organic compound. In an embodiment, said
organic compound is a bisphosphonate compound. In an embodiment, said
compound is a compound of formula XA1 or other compound as described herein.
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In an embodiment, said compound has a pIC50 value of at least 4 in a cancer
cell or
tumor growth inhibition assay and/or an immunostimulation assay. In an
embodiment, said compound has a pIC50 value of at least 5. In an embodiment,
said compound has a pIC50 value of at least 6. In an embodiment, said compound
has a pIC50 value of at least 7.
[00085] In an embodiment, the invention provides a method of selectively
inhibiting
an FPPS enzyme, a GGPPS enzyme, and a DPPS enzyme comprising contacting
said enzymes or a cell containing said enzymes with an organic compound,
wherein
said compound is capable of selectively inhibiting said FPPS, GGPPS, and DPPS
enzymes.
[00086] In an embodiment, the invention provides a method of selectively
inhibiting
a GGPPS enzyme and a DPPS enzyme comprising contacting said enzymes or a
cell containing said enzymes with an organic compound, wherein said compound
is
capable of selectively inhibiting said GGPPS enzyme and said DPPS enzyme. In
an
embodiment, the compound is of formula XA1, Z=Z1, and R4=RL. In an
embodiment, said compound is compound 715.
[00087] In an embodiment, the invention provides a method of selectively
inhibiting
a GGPPS enzyme without substantially inhibiting a DPPS enzyme comprising
contacting said enzymes or a cell containing said enzymes with an organic
compound, wherein said compound is capable of selectively inhibiting said
GGPPS
enzyme without substantially inhibiting said DPPS enzyme. In an embodiment,
the
compound is of formula XA1, Z=Z12, and R4=RL. In an embodiment, said
compound is compound 754.
[00088] In an embodiment, the invention provides a method of one or more of
immunostimulation and inhibition of tumor or cancer cell growth, comprising
contacting a mammalian cell with an organic bisphosphonate compound capable of
substantially inhibiting a GGPPS enzyme and a DPPS enzyme.
[00089] In an embodiment, the invention provides a method of inhibition of
cancer
cell growth, comprising contacting a mammalian cell with an organic
bisphosphonate
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compound capable of substantially inhibiting a GGPPS enzyme without
substantially
inhibiting a DPPS enzyme.
[00090] In an embodiment of any one of the foregoing methods, said compound
has a structure of formula XA1.
[00091] In an embodiment, the invention provides a method of screening an
organic
bisphosphonate test compound for one or more properties, comprising: providing
said test compound, measuring a performance attribute of said test compound in
at
least two enzyme assays selected from the group consisting of: an FPPS enzyme
assay; a GGPPS enzyme assay; a DPPS enzyme assay; and measuring an activity
level of said test compound in at least two activity assays selected from the
group
consisting of: a cancer cell or tumor growth inhibition assay; a T cell
activation
assay; a bone resorption assay; a bone binding assay; analyzing said
performance
attributes and said activity levels; and selecting said test compound based on
said
attributes and activity levels; thereby screening said test compound for said
one or
more properties. In an embodiment, the method further comprises providing a
reference compound and comparing a performance attribute of said reference
compound with said performance attribute of said test compound.
[00092] In an embodiment, the invention provides a method of inhibiting a
dehydrodolichyl diphosphate synthase (DHDDS) enzyme. In an embodiment, the
invention provides a method of selectively inhibiting a DHDDS enzyme
comprising
contacting said enzyme or a cell containing said enzyme with an organic
compound
or composition of the invention. In an embodiment herein wherein a method is
described as inhibiting a target selectively, there can be specific inhibition
of one or
more other targets.
[00093] In an embodiment, the invention provides a method of treating a cancer
comprising administering to a patient in need thereof, a therapeutically
effective
amount of a compound of the invention or a pharmaceutical formulation thereof.
In
an embodiment, the cancer is breast cancer. In an embodiment, the breast
cancer
involves an actual or potential bone metastatic condition.
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[00094] In an embodiment, the invention provides a method of treating a bone
resorption disorder comprising administering to a patient in need thereof, a
therapeutically effective amount of a compound of the invention or a
pharmaceutical
formulation thereof.
[00095] In an embodiment, the invention provides a method of treating a bone
pain
condition comprising administering to a patient in need thereof, a
therapeutically
effective amount of a compound of the invention or a pharmaceutical
formulation
thereof.
[00096] In an embodiment, the invention provides a method of treating an
infectious
disease comprising administering to a patient in need thereof, a
therapeutically
effective amount of a compound of the invention or a pharmaceutical
formulation
thereof. In an embodiment, said infectious disease relates to an agent
selected from
the group consisting of: a virus, a fungus, a bacterium, and a protozoan
parasite. In
an embodiment, said virus is a retrovirus. In an embodiment, said retrovirus
is
human immunodeficiency virus (HIV). In an embodiment, said protozoan parasite
is
selected from the group consisting of: Leishmania, Toxoplasma,
Cryptosporidium,
Plasmodium, and Trypanosoma. In an embodiment, said protozoan parasite is
Leishmania major. In an embodiment, said bacterium is Escherichia coli or
Staphylococcus aureus.
[00097] In an embodiment, the invention provides a method of immunotherapeutic
treatment comprising administering to a patient in need thereof, a
therapeutically
effective amount of a compound of the invention or a pharmaceutical
formulation
thereof. In an embodiment, the invention provides a method of stimulating a T
cell,
comprising contacting the T cell with a compound of the invention or a
pharmaceutical formulation thereof. In an embodiment, said T cell is a
gammadelta
T cell.
[00098] In an embodiment, the invention provides a method of synthesizing a
compound of the invention or a pharmaceutical formulation thereof.
[00099] In an embodiment, the invention provides a method of inhibiting growth
of
an infectious disease agent comprising contacting said infectious disease
agent with
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an effective amount of a compound of the invention or a pharmaceutical
formulation
thereof.
[00100] In an embodiment, the invention provides a method of inhibiting growth
of a
tumor or cancer cell comprising contacting said tumor or cancer cell with an
effective
amount of a compound of the invention or a pharmaceutical formulation thereof.
[00101] In an embodiment, the invention provides a compound having anti-
angiogenic activity. In an embodiment, the invention provides a method of
inhibiting
angiogenesis comprising administering to a subject in need thereof an
effective
amount of a compound or composition of the invention.
[00102] In an embodiment, the invention provides a composition comprising a
compound. In embodiment, said composition comprises a therapeutically
effective
amount of the compound. In an embodiment, the invention provides a composition
comprising a pharmaceutical formulation of a compound. In an embodiment, said
pharmaceutical formulation comprises one or more excipients, carriers, and/or
other
components as would be understood in the art. In an embodiment, an effective
amount of a composition of the invention can be a therapeutically effective
amount.
[00103] In an embodiment, a composition of the invention is used as a
medicament.
In an embodiment, a composition is used in the preparation or manufacture of a
medicament. In an embodiment, the medicament is for treatment of one or more
conditions as described herein and as would be understood in the art.
[00104] In an embodiment, the invention provides a method for treating a
medical
condition comprising administering to a subject in need thereof, a
therapeutically
effective amount of a compound of the invention. In an embodiment, the medical
condition is a bone resorption disorder, a cancer, pain, an immune system
disorder,
and/or an infectious disease.
[00105] In an embodiment, a composition of the invention is isolated or
purified.
[00106] In a screening method embodiment, a purified FPPS, GGPPS, DPPS, or
other enzyme can be employed in addition to cellular and animal-based assays.
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[00107] Without wishing to be bound by any particular theory, there can be
discussion herein of beliefs or understandings of underlying principles or
mechanisms relating to the invention. It is recognized that regardless of the
ultimate
correctness of any mechanistic explanation or hypothesis, an embodiment of the
invention can nonetheless be operative and useful.
[00108] Pharmaceutically acceptable salts comprise pharmaceutically-acceptable
anions and/or cations. Pharmaceutically-acceptable cations include among
others,
alkali metal cations (e.g., Li+, Na+, K+), alkaline earth metal cations (e.g.,
Ca2+, Mg2+),
non-toxic heavy metal cations and ammonium (NH4+) and substituted ammonium
(N(R')4+, where R' is hydrogen, alkyl, or substituted alkyl, i.e., including,
methyl, ethyl,
or hydroxyethyl, specifically, trimethyl ammonium, triethyl ammonium, and
triethanol
ammonium cations). Pharmaceutically-acceptable anions include among other
halides (e.g., CI-, Br ), sulfate, acetates (e.g., acetate, trifluoroacetate),
ascorbates,
aspartates, benzoates, citrates, and lactate.
[00109] Certain molecules disclosed herein contain one or more ionizable
groups
[groups from which a proton can be removed (e.g., -COOH) or added (e.g.,
amines)
or which can be quaternized (e.g., amines)]. All possible ionic forms of such
molecules and salts thereof are intended to be included individually in the
disclosure
herein. With regard to salts of the compounds herein, one of ordinary skill in
the art
can select from among a wide variety of available counterions those that are
appropriate for preparation of salts of this invention for a given
application. In
specific applications, the selection of a given anion or cation for
preparation of a salt
may result in increased or decreased solubility of that salt.
[00110] Compounds of the invention can have prodrug forms. Prodrugs of the
compounds of the invention are useful in the methods of this invention. Any
compound that will be converted in vivo to provide a biologically,
pharmaceutically or
therapeutically active form of a compound of the invention is a prodrug.
Various
examples and forms of prodrugs are well known in the art. Examples of prodrugs
are found, inter alia, in Design of Prodrugs, edited by H. Bundgaard,
(Elsevier,
1985), Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K. Widder,
et. al.
(Academic Press, 1985); A Textbook of Drug Design and Development, edited by
Krosgaard-Larsen and H. Bundgaard, Chapter 5, "Design and Application of
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Prodrugs," by H. Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug
Delivery Reviews, Vol. 8, p. 1-38 (1992); H. Bundgaard, et al., Journal of
Pharmaceutical Sciences, Vol. 77, p. 285 (1988); and Nogrady (1985) Medicinal
Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-
392).
BRIEF DESCRIPTION OF THE FIGURES
[00111 ] Figure 1 illustrates aspects of relevant chemistry. A, Structures of
common nitrogen-containing bisphosphonates; B, schematic illustration of
several
pathways involved in bisphosphonate activity in tumor cells, yb T cells,
osteoclasts,
and macrophages. C, FPP, GGPP biosynthesis and protein prenylation showing
carbocation transition state/reactive intermediates and bisphosphonate analog
(enclosed in red circle); D, comparative molecular similarity index
electrostatic field
(left, blue=positive charge favored)) and pharmacophore (right,
green=hydrophobic,
red=positive, blue=negative ionizable) for FPPS inhibition; E, cationic
bisphosphonates; F, structures of selected GGPPS inhibitors.
[00112] Figures 2A-21 illustrates extensive results of assays for activity of
compounds including data for tumor cell growth inhibition and yb T cell
activation. A,
MCF-7 cell growth inhibition by bisphosphonates; B, FOH, GGOH rescue of
zoledronate cell growth inhibition; C, FOH, GGOH rescue of BPH-675 cell growth
inhibition; D, correlation matrix for enzyme, cell growth inhibition and
SlogP; E,
CoMSIA predictions with FPPS and GGPS descriptors; F, gammadelta T cell
activation by bisphosphonates; G, HQSAR predictions for yb T cell activation;
H,
percent proliferation response; I, percent of total CD3+ cells.
[00113] Figures 3A-3E illustrates results from X-ray and NMR experiments. A,B:
x-ray structures of BPH-527 and BPH-461 bound to human FPPS shown
superimposed on risedronate (from PDB File # 1YV5); C,D 31P magic-angle sample
spinning NMR spectra (600 MHz 'H resonance frequency) of bisphosphonates, IPP
bound to T. brucei FPPS; E, x-ray structure of BPH-675 bound to GGPPS (from
Saccharomyces cerevisae) shown superimposed on GGPP bound to human
GGPPS (PDB File 2FVI); see also Table 8.
[00114] Figure 4 is a schematic illustration of bisphosphonate targets.
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[00115] Figure 5 provides structures of inhibitors investigated in MCF-7 cell
growth
inhibition, FPPS inhibition and GGPPS inhibition (Figures 2D-G).
[00116] Figure 6 is a graph of pIC50 values for MCF-7 growth inhibition by
bisphosphonates plotted versus the pED50 values for ybT cell activation. The
structures of the compounds investigated are shown in Figure 5.
[00117] Figure 7 provides structures of compounds investigated in assays
including MCF-7 cell growth inhibition and yb T cell activation.
[00118] Figure 8 provides structures of compounds investigated in assays
including MCF-7 cell growth inhibition and yb T cell activation.
[00119] Figure 9 is a graph of MCF-7 cell growth inhibition pIC50 values
versus
bone resorption (pED50 results, from Widler et al.). The structures of the
compounds
investigated are shown in Figure 8.
[00120] Figure 10 provides structures of bone resorption drugs tested in MCF-7
cell growth inhibition.
[00121] Figures 11A-D are exemplary 31P NMR spectra of
bisphosphonate/IPP/FPPS complexes. The structures of the compounds are shown
above the spectra.
[00122] Figures 12A and B indicate X-ray structures of exemplary
bisphosphonates bound to Trypanosoma cruzi FPPS. A, BPH-527 and B, BPH-461.
Risedronate is shown superimposed on each.
[00123] Figures 13A and B provide representative ITC results for a sulfonium
bisphosphonate (BPH-527) bound to T. brucei FPPS and AH, AS correlation (novel
cationic compounds in red with arrows, others from references).
[00124] Figure 14 is a graph of predicted cell growth inhibition based on
FPPS,
GGPPS enzyme inhibition in addition to SlogP descriptor.
[00125] Figure 15 is a graph of predicted cell growth inhibition based on FPPS
and
GGPPS enzyme inhibition data.
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[00126] Figure 16 provides structures of several compounds discussed herein.
DETAILED DESCRIPTION OF THE INVENTION
[00127] In embodiments, the invention relates at least in part to the
discovery that
certain compounds including bisphosphonates, particularly those having at
least one
substitutent carrying a long hydrocarbon chain, particularly a straight chain
alkyl or
alkoxy group having 7 or more carbon atoms, exhibit useful or enhanced
activity
including in the context of inhibition of cell growth and/or inhibition of
certain ezymes.
[00128] The following abbreviations are applicable. FPPS, farnesyl diphosphate
synthase (also known as farnesyl pyrophosphate synthetase,
dimethylallyltranstransferase, geranyltranstransferase, farnesyl diphosphate
synthetase, and farnesyl pyrophosphate synthetase); GGPPS, geranylgeranyl
diphosphate synthase (also known as geranylgeranyl pyrophosphate synthetase);
DPPS, decaprenyl pyrophosphate synthase; UPPS (undecaprenyl pyrophosphate
synthetase; also known as undecaprenyl diphosphate synthase); DHDDS or
DDPPS, dehydrodolichyl diphosphate synthase; pIC50/pEC50, negative log of IC50
and EC50, respectively, where IC50 and EC50 are the concentrations that
produce
half-maximal inhibition or activation, respectively; T. brucei, Trypanosoma
brucei; D.
discoideum, Dictyostelium discoideum; yb T cells, gamma delta T cells; ITC,
isothermal calorimetry. Compounds/structures are typically designated by a
number
for convenience.
[00129] The following definitions are applicable. The chemical group
definitions are
intended to relate in particular to compounds having the general formula XA1
but
can also apply to other compounds set forth herein.
[00130] Alkyl groups include straight-chain, branched and cyclic alkyl groups.
Alkyl
groups include those having from 1 to 20 carbon atoms. Alkyl groups include
small
alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length
alkyl
groups having from 4-10 carbon atoms. Alkyl groups include long alkyl groups
having more than 10 carbon atoms, particularly those having 10-20 carbon
atoms.
Cyclic alkyl groups include those having one or more rings. Cyclic alkyl
groups
include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring and
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particularly those having a 3-, 4-, 5-, 6-, or 7-member ring. The carbon rings
in cyclic
alkyl groups can also carry alkyl groups. Cyclic alkyl groups can include
bicyclic and
tricyclic alkyl groups. Alkyl groups optionally include substituted alkyl
groups.
Substituted alkyl groups include among others those which are substituted with
aryl
groups, which in turn can be optionally substituted. Specific alkyl groups
include
methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl,
cyclobutyl, n-
pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl
groups, all of which are optionally substituted.
[00131] Alkenyl groups include straight-chain, branched and cyclic alkenyl
groups.
Alkenyl groups include those having 1, 2 or more double bonds and those in
which
two or more of the double bonds are conjugated double bonds. Alkenyl groups
include those having from 2 to 20 carbon atoms. Alkenyl groups include small
alkyl
groups having 2 to 3 carbon atoms. Alkenyl groups include medium length
alkenyl
groups having from 4-10 carbon atoms. Alkenyl groups include long alkenyl
groups
having more than 10 carbon atoms, particularly those having 10-20 carbon
atoms.
Cyclic alkenyl groups include those having one or more rings. Cyclic alkenyl
groups
include those in which a double bond is in the ring or in an alkenyl group
attached to
a ring. Cyclic alkenyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9-
or 10-
member carbon ring and particularly those having a 3-, 4-, 5-, 6- or 7-member
ring.
The carbon rings in cyclic alkenyl groups can also carry alkyl groups. Cyclic
alkenyl
groups can include bicyclic and tricyclic alkyl groups. Alkenyl groups are
optionally
substituted. Substituted alkenyl groups include among others those which are
substituted with alkyl or aryl groups, which groups in turn can be optionally
substituted. Specific alkenyl groups include ethenyl, prop-l-enyl, prop-2-
enyl,
cycloprop-1-enyl, but-l-enyl, but-2-enyl, cyclobut-l-enyl, cyclobut-2-enyl,
pent-l-
enyl, pent-2-enyl, branched pentenyl, cyclopent-l-enyl, hex-l-enyl, branched
hexenyl, cyclohexenyl, all of which are optionally substituted.
[00132] Aryl groups include groups having one or more 5- or 6-member aromatic
or
heteroaromatic rings. Aryl groups can contain one or more fused aromatic
rings.
Heteroaromatic rings can include one or more N, 0, or S atoms in the ring.
Heteroaromatic rings can include those with one, two or three N, those with
one or
two 0, and those with one or two S. Aryl groups are optionally substituted.
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Substituted aryl groups include among others those which are substituted with
alkyl
or alkenyl groups, which groups in turn can be optionally substituted.
Specific aryl
groups include phenyl groups, biphenyl groups, pyridinyl groups, and naphthyl
groups, all of which are optionally substituted.
[00133] Arylalkyl groups are alkyl groups substituted with one or more aryl
groups
wherein the alkyl groups optionally carry additional substituents and the aryl
groups
are optionally substituted. Specific alkylaryl groups are phenyl-substituted
alkyl
groups, e.g., phenylmethyl groups.
[00134] Alkylaryl groups are aryl groups substituted with one or more alkyl
groups
wherein the alkyl groups optionally carry additional substituents and the aryl
groups
are optionally substituted. Specific alkylaryl groups are alkyl-substituted
phenyl
groups such as methylphenyl.
[00135] The rings that may be formed from two or more of any R (e.g., R1 and
R2)
groups herein together can be optionally substituted cycloalkyl groups,
optionally
substituted cycloalkenyl groups or aromatic groups. The rings may contain 3,
4, 5, 6,
7 or more carbons. The rings may be heteroaromatic in which one, two or three
carbons in the aromatic ring are replaced with N, 0 or S. The rings may be
heteroalkyl or heteroalkenyl, in which one or more CH2 groups in the ring are
replaced with 0, N, NH, or S.
[00136] Optional substitution of any alkyl, alkenyl and aryl groups includes
substitution with one or more of the following substituents: halogens, -CN, -
COOR, -
OR, -COR, -OCOOR, -CON(R)2, -OCON(R)2, -N(R)2, -NO2, -SR, -S02R, -S02N(R)2
or -SOR groups. Optional substitution of alkyl groups includes substitution
with one
or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or
aryl
groups are optionally substituted. Optional substitution of alkenyl groups
includes
substitution with one or more alkyl groups, aryl groups, or both, wherein the
alkyl
groups or aryl groups are optionally substituted. Optional substitution of
aryl groups
includes substitution of the aryl ring with one or more alkyl groups, alkenyl
groups, or
both, wherein the alkyl groups or alkenyl groups are optionally substituted.
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[00137] Optional substituents for alkyl, alkenyl and aryl groups include among
others:
[00138] -COOR where R is a hydrogen or an alkyl group or an aryl group and
more
specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of
which are
optionally substituted;
[00139] -COR where R is a hydrogen, or an alkyl group or an aryl groups and
more
specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of
which
groups are optionally substituted;
[00140] -CON(R)2 where each R, independently of each other R, is a hydrogen or
an alkyl group or an aryl group and more specifically where R is methyl,
ethyl, propyl,
butyl, or phenyl groups all of which groups are optionally substituted; R and
R can
form a ring which may contain one or more double bonds;
[00141] -OCON(R)2 where each R, independently of each other R, is a hydrogen
or
an alkyl group or an aryl group and more specifically where R is methyl,
ethyl, propyl,
butyl, or phenyl groups all of which groups are optionally substituted; R and
R can
form a ring which may contain one or more double bonds;
[00142] -N(R)2 where each R, independently of each other R, is a hydrogen, or
an
alkyl group, acyl group or an aryl group and more specifically where R is
methyl,
ethyl, propyl, butyl, or phenyl or acetyl groups all of which are optionally
substituted;
or R and R can form a ring which may contain one or more double bonds.
[00143] -SR, -SO2R,or -SOR where R is an alkyl group or an aryl groups and
more
specifically where R is methyl, ethyl, propyl, butyl, phenyl groups all of
which are
optionally substituted; for -SR, R can be hydrogen;
[00144] -OCOOR where R is an alkyl group or an aryl groups;
[00145] -SO2N(R)2 where R is a hydrogen, an alkyl group, or an aryl group and
R
and R can form a ring;
[00146] -OR where R=H, alkyl, aryl, or acyl; for example, R can be an acyl
yielding
-OCOR* where R* is a hydrogen or an alkyl group or an aryl group and more
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specifically where R* is methyl, ethyl, propyl, butyl, or phenyl groups all of
which
groups are optionally substituted;
[00147] Specific substituted alkyl groups include haloalkyl groups,
particularly
trihalomethyl groups and specifically trifluoromethyl groups. Specific
substituted aryl
groups include mono-, di-, tri, tetra- and pentahalo-substituted phenyl
groups; mono-,
di-, tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalene
groups; 3- or 4-
halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenyl groups, 3- or
4-
alkoxy-substituted phenyl groups, 3- or 4-RCO-substituted phenyl, 5- or 6-halo-
substituted naphthalene groups. More specifically, substituted aryl groups
include
acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups,
particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups,
particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups,
particularly 4-methylphenyl groups, and methoxyphenyl groups, particularly 4-
methoxyphenyl groups.
THE EXAMPLES
[00148] The invention may be further understood by the following non-limiting
examples.
EXAMPLE 1. Bisphosphonate compounds including structures with high
potency for anti-cancer and/or immunostimulatory function.
[00149] Bisphosphonates such as Fosamax, Actonel and Zometa are potent
inhibitors of the enzyme farnesyl diphosphate synthase (FPPS) and are used to
treat
osteoporosis and bone cancers. They have direct activity against osteoclasts
and
tumor cells and also activate gammadelta T cells of the innate immune system
to kill
tumor cells. Here, we show that bisphosphonates can act as
polypharmaceuticals,
inhibiting not only FPPS but geranylgeranyl diphosphate and decaprenyl
diphosphate synthases as well, in addition to describing the development of
novel
compounds having activities approximately 100-1 000x greater than current
bisphosphonates in yb T cell activation and tumor cell killing.
[00150] Bisphosphonates such as Fosamax, Boniva and Zometa are drug
molecules used to treat bone resorption diseases such as osteoporosis, Paget's
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disease and hypercalcemia due to malignancy(1, 2). In addition, they activate
yb T
cells (containing the Vy2V6 T cell receptor) to kill tumor cells(3-5), plus,
they have
direct activity against tumor cells (6-9) and many parasitic protozoa(10, 11).
While
used clinically for two decades, their mode of action has been unclear. In
early work,
bisphosphonates were thought to act simply by coating bone surfaces, but more
recently, the enzyme farnesyl diphosphate synthase (FPPS, EC 2.5.1.10) has
been
implicated(12). Inhibition of FPPS results in decreased prenylation of small
GTPases (such as Ras, Rho, Rap, Rac) which is expected to caused deranged
patterns of cell signaling (Figure 1 B) and in some protozoa, inhibition of
ergosterol
biosynthesis(10). More recently, it has been shown that this inhibition of
FPPS
results in increased levels of the substrate, isopentenyl diphosphate
(IPP)(13, 14).
This increase in IPP levels can activate yb T cells (15). And, in some cells,
IPP is
converted to the isopentenyl ester of ATP, Apppl, which can inhibit the
mitochondrial
adenine nucleotide translocase (ANT), a component of the mitochondrial
permeability transition pore (Figure 1 B) (16).
[00151] Herein we disclose significant answers to the questions: is FPPS
inhibition
always the major target for bisphosphonate action? And, is it possible to make
more
active and selective inhibitors, including ones that might have less avidity
for bone, of
potential use in immunotherapy, cancer, and as anti-infectives? We report that
other
important targets for bisphosphonate compound action include GGPPS and DPPS.
Furthermore, we have made and tested organic bisphosphonate compounds which
exhibit high potency and selectivity regarding various targets.
[00152] Proteins are prenylated by either farnesyl diphosphate (FPP) or
geranylgeranyl diphosphate (GGPP), which are synthesized from IPP and
dimethylallyl diphosphate (DMAPP) as shown in Figure 1 C. The reactions are
believed to proceed via carbocationic transition state/reactive
intermediates(17) such
as that circled in red in Figure 2C, with the bisphosphonate sidechains (of
e.g.
Boniva, red, Figure 1A) mimicking the charge center and the bisphosphonate
providing a hydrolytically stable analog of diphosphate(17). We proposed that
analogous types of transition states could be relevant for both FPPS and
GGPPS, as
well as decaprenyl diphosphate synthase (DPPS). DPPS is a heterodimeric prenyl-
28
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transferase used in coenzyme Qlo production(18). In accordance with our
proposal,
we designed bisphosphonate compounds that could affect multiple targets.
[00153] There has been aspirational recognition for drug design approaches to
consider the prospect of going beyond the "one drug, one target" convention
(19).
We put forth the possibility that bisphosphonates could be
"polypharmaceuticals"
capable of inhibiting multiple targets. The bisphosphonate Boniva can be a
potent
inhibitor of squalene synthase(20), used in cholesterol biosynthesis, and
numerous
bisphosphonates are potent, low nM inhibitors of another heterodimeric
prenyltransferase, geranyl diphosphate synthase, found in plants(21). The
ability to
determine the potential significance of other relevant target enzymes and to
develop
inhibitors, however, involved further exploration.
[00154] To test our polypharmaceutical hypothesis, we expressed three enzymes:
human FPPS, GGPPS and DPPS, and tested each for their inhibition by a series
of
bisphosphonates. Each of these three enzymes is inhibited by the
bisphosphonate
zoledronate (Zometa), with certain indicated IC50 (Ki) values shown in Table
1;
activity values are also shown for other bisphosphonate compounds. Thus, we
demonstrated that all three human enzymes can be potently inhibited by
bisphosphonates. This finding is consistent with the possibility that FPPS is
not the
only target for bisphosphonates. We suggest the potential importance of GGPPS
as
a primary target for bisphosphonates and note the observation of Goffinet et
al. (22)
and others that the effects of bisphosphonates on cell growth are only
reversed by
addition of geranylgeraniol and not farnesol, implicating the involvement of
FPPsynthase and GPPsynthase in the context of studying cholesterol
biosynthesis.
Our data demonstrate that certain small molecules can directly inhibit GGPPS
target
activity, in addition to other targets, with high potency.
[00155] We probed the question of whether GGPPS serves as the major target for
bisphosphonate activity, and the accompanying role of small molecule
inhibitors, in
more depth. We designed a series of novel bisphosphonates that might have
improved activity against one or more of these three enzymes in order to
provide
useful compositions and to provide a database which might help interpret
certain
cellular (tumor cell killing and yb T cell activation) results. Our inspection
of
comparative molecular similarity analysis (CoMSIA)(23) models for FPPS
29
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inhibition(24) (Figure 1 E) suggested that enhanced activity might be obtained
by
moving the positive charge feature closer to the bisphosphonate backbone, as
found
for example in the novel bisphosphonates shown in Figure 1 F. We note the
previous
study of the inhibition of a human recombinant geranylgeranyl diphosphate
synthase
(25). In addition to the structural feature of the positive charge, we
postulated the
possible importance of having a large hydrophobic tail (see, e.g., Figure 1
F).
Furthermore, we considered the prospect that such inhibitors might be
particularly
potent against the C50 prenyltransferase DPPS, as well as having improved
cellular
uptake. Thus we were inspired to design and synthesize a variety of compounds;
a
representative specific member of which is compound 715.
[00156] As shown in Figure 2A, cationic bisphosphonate species such as BPH-715
(Figure 1 F, left) are indeed far more active in MCF-7 tumor cell growth
inhibition than
are bisphosphonates such as zoledronate and pamidronate, with IC50 values of
approximately 50 nM, to be compared with values on the order of around 15 pM
(zoledronate) or around 300 pM (pamidronate). There is no rescue from growth
inhibition by addition of farnesol and only a partial rescue by
geranylgeraniol, Figure
2B, suggesting more than one target. On the other hand, the large hydrophobic
bisphosphonate BPH-675 has an IC50 of 5 pM, but its growth inhibitory effect
is
essentially fully rescued by addition of 20 pM geranyl geraniol, Figure 2C.
This
strongly suggests that BPH-675 is a selective GGPPS inhibitor, while BPH-715
has
multiple targets, including GGPPS. We found no rescue from cell growth
inhibition
from any bisphosphonate upon incorporation of CoQjo in growth medium, however,
this is not unexpected given our mechanistic understanding since CoQ10 is
present in
serum and the main effect of DPPS inhibition would be expected to be on
IPP/Apppl
elevation, which would not be affected by CoQ1o addition.
[00157] In order to develop a model of cell growth inhibition based on enzyme
data,
we next determined the IC50 values for FPPS, GGPPS and DPPS inhibition by
certain bisphosphonates (certain data shown in Table 1). As shown in the data
matrix in Figure 2D, there is a good correlation between cell growth
inhibition and
GGPPS inhibition pIC50 values, a moderate correlation with SlogP, the Log of
the
octanol/water partition coefficient based on atom contribution and protonation
state
(26), a weak correlation with DPPS but no correlation with FPPS inhibition.
These
CA 02682694 2009-09-30
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results strongly support the idea that GGPPS inhibiton is of prime importance
and
are consistent with the data we generated for GGOH and FOH rescue studies (see
Figure 2B, 2C).
[00158] We sought to develop a more quantitative model for cell growth
inhibition,
by using a partial least squares method to regress the enzyme and SlogP data
against the cell IC50 results. That is:
pIC50 (cell) = a=pIC50 (FPPS) + b= pIC50 (GGPPS) + c=pIC50 (DPPS) + d=SIogP +
e
where pIC50 =-loglo (IC5o M) and a,b...n are regression coefficients.
[00159] Using solely enzyme inhibition and SlogP data we find a good overall
correlation (R = 0.90) using just GGPPS, DPPS and SlogP with GGPPS dominating
(SI), with further improvements being obtained when using the CoMSIA fields
(Figure
2E). So, cell growth inhibition by bisphosphonates is dominated by direct
inhibition
of GGPPS, consistent with the rescue experiments, since Rho, Rap and Rac cell
survival pathways are affected. Plus, DPPS inhibition is expected to produce
large
amounts of IPP (Apppl), since 7 moles of IPP would be consumed per DPP
molecule
produced.
[00160] We next sought to investigate whether or not these novel
bisphosphonates
have activity in yb T cell activation. As can be seen in Figure 2F, long chain
bisphosphonates such as BPH-715 have potent activity in yb T cell activation,
with
the most active species (BPH-716, containing a C12 sidechain) having an EC50 -
2x,
more active in this assay than the classic synthetic phosphoantigen
Phosphostim
(the bromohydrin of IPP) and -100x more active than the most potent
conventional
bisphosphonates, Figure 2F. These most potent species have little or no
activity
against FPPS (SI), however, they are -10x more active against DPPS than is
zoledronate (-500 nM versus -5 pM). In addition, they have far more favorable
SlogP properties (3 vs. -4), meaning that they might more readily enter cells.
Of
course, it might be argued that these species could be directly presented to
yb T
cells as with other lipid antigens. However, the results of both pravastatin
and
mevastatin titration experiments(14), in which isoprenoid flux to FPPS, GGPPS
and
DPPS is blocked via inhibition of HMGCoA reductase, show identical statin IC50
values for a potent long chain bisphosphonate and risedronate (Figures 2E,F,G)
in
yb T cell activation. So, the novel species act in the same way as do
conventional
31
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bisphosphonate antigens, via IPP accumulation in the antigen-presenting cells.
Our
application of the same modeling methods as used for tumor cell growth
inhibition
resulted in a highly predictive model (Figure 21), with FPPS, DPPS, SlogP
descriptors together with additional CoMSIA field descriptors, (SI), with an
r2 value of
0.98, q2 = 0.744).
[00161] We find no evidence of a role for GGPPS inhibition in yb T cell
activation.
For example, the GGPPS inhibitor BPH-675 (which has no effect on FPPS or DPPS)
had no effect at all on yb T cell activation. Likewise, a phenyl analog of BPH-
715
(BPH-754) in which there is no side-chain charge, was found to be a good GGPPS
inhibitor. Its inhibition of MCF-7 cell growth was rescued by GGOH, but it had
no
effect on yb T cell activation since it had essentially no effect on FPPS or
DPPS
inhibition (since it lacked the carbocation charge feature). While this lack
of activity
in yb T cell activation might at first seem surprising, inhibition of GGPPS
alone
produces only 1 IPP, while DPPS inhibition produces 7, plus, DPP/CoQ1o
production
is very abundant in cells. It is also possible that inhibition of dolichol
biosynthesis
could be involved in IPP production.
O
HO__
-OH
H3C O \
HO /OH
Z \
HO O
754
[00162] A potential drawback to the use of bisphosphonates in treating non-
bone
resorption diseases is expected to be that they would be rapidly adsorbed onto
bone.
Surprisingly, however, we find that the highly hydrophobic species BPH-675 and
BPH-715 are only very weakly adsorbed onto bone in vivo (SI), resulting in
only
modest IC50 values in bone resorption (e.g. -800 nM for BPH-715 versus -70 nM
for
zoledronate, SI), but weak bone binding is desirable in the context of certain
conditions, e.g., immunotherapy, treating infectious diseases, and various
cancers.
[00163] We note that the compound 754 and certain compounds with related
structural features can represent a genus of compounds which potently inhibits
GGPPS while not substantially inhibiting DPPS or FPPS. In certain instances it
can
be advantageous to retain properties such as anti-cancer activity while not
having a
32
CA 02682694 2009-09-30
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pro-immunostimulatory effect. There are circumstances where immunostimulation
can lead to immune system overreaction such as in a variety of inflammatory
disorders. The structural features of interest can include the lack of
positive charge
for the ring moiety adjoining the bisphosphonate component in addition to an
alkoxy
tail substituent on the ring. Conversely, compounds which share other
structural
features (e.g., presence of the positive charge and the tail substituent) can
exhibit
accompanying functional properties such as inhibition of multiple targets (for
example, GGPPS and DPPS in the case of compound 715) and can demonstrate
combinations of activities such as anti-cancer and immunostimulation; there
are
circumstances where such combinations can be advantageous.
[00164] We next investigated how certain bisphosphonates (e.g., pyridinium and
sulfonium analogs) bind to FPPS and GGPPS. We chose to first study the simple
fluoropyridinium bisphosphonate (BPH-461, Figure 1 D) previously found to have
potent activity in FPPS inhibition and in bone resorption(27), as well as the
simplest
sulfonium bisphosphonate (BPH-527, Figure 1 D). Data collection and refinement
statistics are shown in exemplary Tables, e.g., Tables 4-7, for both the human
and
Trypanosoma brucei FPPS enzymes, the latter being of interest as a target for
anti-
infective drug development(28). In all cases, the bisphosphonates bound
exclusively
to the allylic/DMAPP site, even in the absence of IPP. The structures of these
two
bisphosphonates bound to the human enzyme are shown in Figure 3A,B,
superimposed on the structure of BPH-210, a potent bone resorption drug(29)
which
also has activity against E. coli(30). The T. brucei structures are shown in
Figure
12). There is clearly considerable similarity in binding with the conventional
bisphosphonates, with strong electrostatic interactions between the
phosphonates
and 3 Mg2+, first identified by Hosfield et al. in the Escherichia coli
protein(31).
[00165] In the presence of NBPs (nitrogen-containing bisphosphonate drugs)
together with IPP, it has been found that ternary bisphosphonate-IPP-FPPS
complexes form (31-34). This has been demonstrated crystallographically as
well as
by using solid state 31 P NMR spectroscopy, where individua131 P NMR
resonances
are seen for both sets of bisphosphonate and IPP 31P nuclei(35). The
pyridinium
bisphosphonate BPH-461 forms the same type of complex, containing 3 Mg2+ plus
IPP, shown in Figure 3C. The formation of ternary complexes with IPP, Mg2+ can
33
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
also be deduced by using solid-state 31P NMR and results for the pyridinium
and
sulfonium bisphosphonate are shown in Figure 2C,D (and Figure 11) and indicate
that the pyridinium, sulfonium, phosphonium, arsonium and guanidinium
bisphosphonates all form ternary complexes with, on average, a 1:1 ( 0.2)
bisphosphonate :IPP stoichiometry.
[00166] To determine whether cationic bisphosphonate binding to FPPS is
entropy
or enthalpy driven, we used isothermal titration calorimetry (ITC). AH values
were
small and endothermic (-2-4 kcal) and binding was overwhelmingly entropy
driven,
with -TAS values in the range - -10.5 to -12.6 kcal/mole. See Figure 13 and
Table
9. So, the unconventional bisphosphonates form the same types of complexes as
do the more conventional nitrogen containing bisphosphonates, but binding is
exclusively entropy driven - as found with conventional bisphosphonates such
as
alendronate and ibandronate, which have very basic side chains (34, 36).
[00167] Finally, we investigated the structure of the GGPPS inhibitor BPH-715
(in
the presence and absence of IPP), which was designed to bind to GGPPS in its
"inhibitor" site. Data collection and refinement statistics for two structures
were
obtained. In both structures, BPH-715 binds to the GGPP inhibitor site first
identified
by Kavanagh et al. (37). In one structure we find the presence of 2 Mg2+ and 1
IPP,
while in a second structure, the ligand binds alone with a slightly
displacement from
that seen in the ternary complex structure. The IPP site location is similar
to that
seen in FPPS (Figure 3C) with the smaller pyridinium bisphosphonate. The same
GGPPS inhibitor site binding site motif is also seen with BPH-675 (PDB 2E95)
and
may be common with long chain GGPPS inhibitors, such as those described
earlier(25), as proposed by Kavanagh et al. (37). Since this is a product (or
inhibitor)
binding site, we determine that there is no requirment for a positive charge
feature,
and both cationic and neutral side-chain containing species can bind, but only
the
cationic species inhibit DPPS (and FPPS).
[00168] Overall, these results are of great interest since they show that
certain
bisphosphonate drugs, rather than targeting exclusively FPPS, are
polypharmaceuticals, able in many cases to inhibit FPPS, GGPPS as well as DPPS
(and potentially, other prenyl transferases, such as dehydrodolichyl
diphosphate
synthase), suggesting the revised version of Figure 2B shown in Figure 4.
Figure 4
34
CA 02682694 2009-09-30
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illustrates our understanding that tumor cell growth inhibition is inhibited
primarily by
GGPPS inhibition (as evidenced by computer models, enzyme inhibition and
rescue
experiments), but GGPPS inhibition plays no role in yb T cell activation,
which is
dominated by FPPS and DPPS inhibition (and possibly, dolichol biosynthesis
inhibition). CoQ1o does not rescue cell growth, since IPP/Apppl accumulation
still
occurs. Long chain bisphosphonates have no activity against FPPS but are still
potent yb T cell activators due to DPPS inhibition and high hydrophobicity.
[00169] In tumor cell growth inhibition, GGPPS is the major target for the
most
potent species, but in yb T cell activation, GGPPS inhibition has no effect on
T cell
activation, which relies on IPP formation. By suitable chemical modification,
we have
obtained several novel species having activities about 100-1 000x greater than
existing bisphosphonates in both tumor cell growth inhibition as well as yb T
cell
activation, suggesting new routes to the use of bisphosphonates in immuno- and
chemotherapy using a polypharmaceutical approach.
[00170] Certain compounds in this example are compounds of formula XA1 as
described herein.
[00171] Variations on compositions including salts and ester forms of
compounds.
Compounds of this invention and compounds useful in the methods of this
invention
include those of the above formulas and pharmaceutically-acceptable salts and
esters of those compounds. In embodiments, salts include any salts derived
from
the acids of the formulas herein which acceptable for use in human or
veterinary
applications. In embodiments, the term esters refers to hydrolyzable esters of
compounds including diphosphonate compounds of the formulas herein. In
embodiments, salts and esters of the compounds of the formulas herein can
include
those which have the same therapeutic or pharmaceutical (human or veterinary)
general properties as the compounds of the formulas herein. Various
combinations
of salts are possible, with each phosphonate carrying a 2-, 1- or neutral
charge. In
principle there are multiple charge states possible, for example 9 charge
states, for
certain compounds including bisphosphonate compounds of this invention.
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References for Example 1:
[00172] 1. R. G. Russell, Ann N YAcad Sci 1068, 367 (Apr, 2006).
[00173] 2. A. J. Roelofs, K. Thompson, S. Gordon, M. J. Rogers, Clin Cancer
Res
12, 6222s (Oct 15, 2006).
[00174] 3. V. Kunzmann et al., Blood 96, 384 (Jul 15, 2000).
[00175] 4. M. Wilhelm et al., Blood 102, 200 (Jul 1, 2003).
[00176] 5. J. N. Blattman, P. D. Greenberg, Science 305, 200 (Jul 9, 2004).
[00177] 6. S. Yamagishi et al., Am J Pathol 165, 1865 (Dec, 2004).
[00178] 7. S. Wakchoure et al., Clin Cancer Res 12, 2862 (May 1, 2006).
[00179] 8. P. V. Dickson et al., Surgery 140, 227 (Aug, 2006).
[00180] 9. D. Santini et al., Nat Clin Pract Oncol 3, 325 (Jun, 2006).
[00181] 10. M. B. Martin et al., J Med Chem 44, 909 (Mar 15, 2001).
[00182] 11. B. Bouzahzah, L. A. Jelicks, S. A. Morris, L. M. Weiss, H. B.
Tanowitz,
Parasitol Res 96, 184 (Jun, 2005).
[00183] 12. J. R. Green, Acta Oncol 44, 282 (2005).
[00184] 13. H. J. Gober et al., J Exp Med 197, 163 (Jan 20, 2003).
[00185] 14. K. Thompson, M. J. Rogers, J Bone Miner Res 19, 278 (Feb, 2004).
[00186] 15. Y. Tanaka et al., Nature 375, 155 (May 11, 1995).
[00187] 16. H. Monkkonen et al., Br J Pharmacol 147, 437 (Feb, 2006).
[00188] 17. M. B. Martin, W. Arnold, H. T. Heath, 3rd, J. A. Urbina, E.
Oldfield,
Biochem Biophys Res Commun 263, 754 (Oct 5, 1999).
[00189] 18. R. Saiki, A. Nagata, T. Kainou, H. Matsuda, M. Kawamukai, Febs J
272, 5606 (Nov, 2005).
36
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
[00190] 19. A. L. Hopkins, J. S. Mason, J. P. Overington, Curr Opin Struct
Biol 16,
127 (Feb, 2006).
[00191] 20. D. Amin, S. A. Cornell, M. H. Perrone, G. E. Bilder,
Arzneimittelforschung 46, 759 (Aug, 1996).
[00192] 21. C. Burke, K. Klettke, R. Croteau, Arch Biochem Biophys 422, 52
(Feb
1,2004).
[00193] 22. M. Goffinet et al., BMC Cancer 6, 60 (2006).
[00194] 23. G. Klebe, U. Abraham, T. Mietzner, J Med Chem 37, 4130 (Nov 25,
1994).
[00195] 24. J. M. Sanders et al., J Med Chem 46, 5171 (Nov 20, 2003).
[00196] 25. C. M. Szabo et al., J Med Chem 45, 2185 (May 23, 2002).
[00197] 26. S. A. Wildman, G. M. Crippen, Journal of Chemical Information and
Computer Sciences 39, 868 (SEP-OCT, 1999).
[00198] 27. J. M. Sanders et al., J Med Chem 48, 2957 (Apr 21, 2005).
[00199] 28. A. Montalvetti et al., J Biol Chem 278, 17075 (May 9, 2003).
[00200] 29. L. Widler et al., J Med Chem 45, 3721 (Aug 15, 2002).
[00201] 30. A. Leon et al., J Med Chem 49, 7331 (Dec 14, 2006).
[00202] 31. D. J. Hosfield et al., J Biol Chem 279, 8526 (Mar 5, 2004).
[00203] 32. S. B. Gabelli et al., Proteins 62, 80 (Jan 1, 2006).
[00204] 33. J. M. Rondeau et al., ChemMedChem 1, 267 (Feb, 2006).
[00205] 34. K. L. Kavanagh et al., Proc Natl Acad Sci U S A 103, 7829 (May 16,
2006).
[00206] 35. J. Mao et al., J Am Chem Soc 128, 14485 (Nov 15, 2006).
37
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WO 2008/128056 PCT/US2008/060051
[00207] 36. F. Yin, R. Cao, A. Goddard, Y. Zhang, E. Oldfield, J Am Chem Soc
128, 3524 (Mar 22, 2006).
[00208] 37. K. L. Kavanagh, J. E. Dunford, G. Bunkoczi, R. G. Russell, U.
Oppermann, J Biol Chem 281, 22004 (May 11, 2006).
EXAMPLE 2. Additional compounds
[00209] The invention provides compounds represented by structure XA2:
O
HO--_ ~/
RL --OH
HO P /OH
HO \ \ O
XQ-2
[00210] wherein variable group options can be as described elsewhere herein.
[00211] In a preferred embodiment, RL is an alkoxy having 7-12 carbons. In an
embodiment, a compound having structural formula XA2 can be used to
selectively
inhibit GGPPS without substantially inhibiting DPPS. In an embodiment, such a
compound is used to inhibit a tumor or cancer cell growth.
[00212] Compound 754 was synthesized and tested for activity. It was found to
have in IC50 value as follows (micromolar): 0.50 for inhibition of cancer call
growth
(average); 0.401 for inhibition of human breast cancer cell line MCF7; 0.524
for
inhibition of human CNS cancer SF268; 0.672 for inhibition of human lung
cancer
NCIH460; 0.5918 for inhibition of purified GGPPS.
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[00213] EXAMPLE 3. Results of testing compounds for activities.
Table 1: pIC50 values for FPPS, GGPPS, and DPPS enzyme inhibition, cell growth
inhibition, and QSAR predicted cell activity.
Experimental & Computed Values Predicted Values
NCI- NCI-
FPPS GGPP DPPS SF-268 MCF-7 H460 H460
pKi S pKi _pKi pIC50 pIC50 pIC50 pIC50
Compound (M) (M) (M) SIogP (M) (M) (M) (M) Residual
715 7.3 8.1 8.3 -1.2 7.0 6.7 7.2 6.7 0.5
638 7.8 8.0 6.3 -1.1 6.7 6.8 6.8 6.7 0.1
722 8.9 7.2 8.2 -2.4 6.0 5.6 6.3 5.7 0.6
717 7.7 7.7 7.1 -1.8 6.2 6.3 6.2 6.2 0.0
694 7.1 7.4 6.6 -0.1 5.9 4.9 6.0 5.3 0.7
604 8.9 7.3 7.0 -2.4 5.8 5.3 6.0 5.8 0.2
637 8.9 7.4 6.3 -2.5 5.9 6.6 5.7 6.0 -0.3
688 8.6 7.5 6.5 -0.8 5.6 5.2 5.5 6.0 -0.5
675 5.9 7.1 6.5 -0.2 5.2 5.2 5.3 4.4 1.0
683 8.5 7.0 7.2 -2.4 4.9 4.7 4.8 5.1 -0.3
261 8.9 5.5 6.8 -4.7 4.9 4.8 4.8 4.4 0.4
91 8.9 5.6 7.4 -5.5 4.8 4.7 4.8 4.4 0.4
678 7.9 5.7 7.4 -9.0 4.6 4.7 4.7 4.0 0.7
754 5.3 7.8 6.7 -0.9 4.6 4.6 4.7 5.4 -0.7
728 6.8 7.5 6.3 -1.5 4.9 4.5 4.6 5.4 -0.8
300 8.3 6.4 6.9 -3.8 4.5 4.4 4.5 4.2 0.3
679 8.2 5.0 6.0 -5.0 4.5 4.4 4.3 4.1 0.2
472 8.6 6.6 7.1 -3.1 4.3 4.4 4.3 4.3 0.0
474 8.4 5.8 6.9 -4.4 4.1 4.1 4.2 4.2 0.0
278 8.6 5.4 7.2 -5.4 4.3 4.1 4.1 4.3 -0.1
483 8.5 5.5 6.8 -3.9 4.0 4.0 4.1 4.2 -0.1
7.4 5.3 6.3 -7.0 3.5 3.9 4.0 3.8 0.2
685 8.3 5.7 6.7 -4.3 3.7 3.6 3.9 4.1 -0.3
684 8.0 6.6 7.0 -3.6 3.7 3.6 3.8 4.1 -0.4
2 8.6 5.0 7.3 -5.0 3.9 3.8 3.7 4.3 -0.6
24 8.3 5.6 7.3 -5.6 3.8 3.7 3.7 4.2 -0.5
1 7.2 4.9 6.3 -6.7 3.5 3.3 3.5 3.7 -0.2
727 8.2 5.6 6.8 -3.8 3.7 3.5 3.5 4.1 -0.6
[00214] In the preceding Table, data for cell growth inhibition of three
cancer cell
lines is demonstrated by various compounds. Also, compounds are able to
inhibit
one or more of FPPS, GGPPS, and DPPS enzymes, including compounds that can
inhibit multiple enzymes with significant potency. Predicted values are from
10-fold
cross-validated models. The mean absolute residuals error is 0.38 which
corresponds to a factor of -2.3x error over a 2500x range in activity. The GFA
lack-
of-fit error metric is 0.31.
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Table 2: yb T cell activation, MCF-7 cell growth inhibition results, presented
as -
loglO(value, M)
yb T Cell Avg MCF-7 Cell
Compound ID pED50 (M) pIC50 (M)
BPH-694 6.20 6.69
BPH-715 6.05 6.97
BPH-638 5.77 6.77
BPH-695 5.68 5.95
BPH-714 5.29 6.82
BPH-728 5.28 5.59
BPH-693 5.28 5.32
BPH-688 5.25 6.30
BPH-687 5.09 6.20
BPH-637 4.90 6.66
BPH-677 4.88 4.34
BPH-696 4.63 5.85
B P H-722 4.56 6.33
BPH-669 4.49 4.75
BPH-723 4.44 5.76
BPH-656 4.29 4.88
BPH-2 4.29 3.13
BPH-678 4.22 5.37
BPH-290 4.21 3.47
BPH-278 4.20 4.10
BPH-670 4.16 4.64
BPH-461 4.16 4.34
BPH-470 4.12 4.14
BPH-721 4.10 4.70
BPH-472 4.07 4.46
BPH-483 4.04 3.84
BPH-476 4.00 4.58
BPH-683 3.97 5.17
BPH-475 3.94 4.37
BPH-684 3.90 3.89
BPH-527 3.87 3.36
BPH-474 3.85 4.42
B P H-682 3.84 4.02
BPH-686 3.81 4.24
BPH-685 3.81 4.02
BPH-477 3.80 4.77
BPH-727 3.77 3.62
BPH-536 3.72 3.24
B P H-540 3.59 2.68
BPH-481 3.45 3.90
BPH-560 3.37 2.71
BPH-679 3.34 4.65
BPH-480 3.33 3.42
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Table 3: Comparison between MCF-7 cell growth inhibition and bone resorption
results
'Bone
bMCF7 Cell Resorption
aCompound ID pIC5o pIC5o (M)
BPH-18 5.41 6.00
BPH-219 4.93 5.40
BPH-91 4.81 6.59
BPH-208 4.38 5.40
BPH-24 4.32 5.52
BPH-31 4.30 4.62
BPH-210 4.08 6.00
BPH-209 4.01 5.52
BPH-5 3.85 3.61
BPH-57 3.60 3.84
BPH-58 3.48 4.74
BPH-7 3.37 4.12
BPH-72 3.34 2.51
BPH-1 3.31 4.49
a The structures of the molecules investigated are shown in Figure 10.
b The pIC50 values shown are those determined in this work.
c The bone resorption results are taken from Widler et al.19
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Table 4: Data collection and refinement statistics for BPH-527a bound to human
FPPS.
Data collection
Space group P41212
Unit cell dimension (A)
a = b, c 111.652, 66.841
X-ray source BNL-X12Cb
Wavelength (A) 0.9791
Resolution (A) 30-2.70 (2.80-2.70)
No. of reflection observed 135,612
Unique 12,031 (1,154)
Completeness (%) 99.0 (97.7)
R-merge 0.083 (0.300)
1/Ql 8.3
Multiplicity 11.3 (8.4)
Refinement statistics
Resolution range (A) 10.0-2.70
R-work/R-free (%) 22.70/24.38
RMSD
Bond lengths 0.004
Bond angles 1.414
No. of atoms
Protein 2,670
Ligand 14
PO43 10
Magnesium ion 3
Solvent (water) 71
B average (A2) of protein 35.83
B average (A2) of solvents 41.37
B average (A2) of ligands 30.99
(bisphosphonates, Mg2+
and P043-)
a: BPH-527 is (2-Hydroxy-2,2-bis-phosphono-ethyl)-dimethyl-sulfonium
b: Brookhaven National Laboratory
42
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Table 5: Data collection and refinement statistics for BPH-461 a bound to
human
FPPS.
Data collection
Space group P41212
Unit cell dimension (A)
a= b, c 111.783, 66.525
X-ray source BNL-X12Cb
Wavelength (A) 0.9791
Resolution (A) 30-2.40 (2.49-2.40)
No. of reflection observed 204,362
Unique 16,818 (1,525)
Completeness (%) 98.6 (92.0)
R-merge 0.081 (0.360)
1/Ql 10.4
Multiplicity 12.2 (9.2)
Refinement statistics
Resolution range (A) 10.0-2.40
R-work/R-free (%) 23.04/26.95
RMSD
Bond lengths 0.004
Bond angles 1.532
No. of atoms
Protein 2,694
Ligand 18
PO43- c 10
Magnesium ion 3
Solvent (water) 94
B average (A2) of protein 39.51
B average (A2) of solvents 44.51
B average (A2) of ligands 37.70
(bisphosphonates, Mg2+ and
PO43-)
a: BPH-461 is 3-fluoro-1 -(2-hydroxy-2,2-bisphosphonoethyl)-pyridinium
b: Brookhaven National Laboratory
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Table 6: Data collection and refinement statistics for BPH-527a bound to T.
brucei
FPPS.
Data collection
Space group C2
Unit cell dimension (A)
P( ) 112.158
a = b, c 134.613, 118.370,
62.758
X-ray source BNL-X12Cb
Wavelength (A) 1.1
Resolution (A) 30-2.00 (2.07-2.00)
No. of reflection observed 461,159
Unique 61,155 (6,046)
Completeness (%) 99.8 (99.5)
R-merge 0.059 (0.486)
1/Ql 12.2
Multiplicity 7.5 (7.4)
Refinement statistics
Resolution range (A) 30.0-2.00
R-work/R-free (%) 20.70/24.12
RMSD
Bond lengths 0.007
Bond angles 1.183
No. of atoms
Protein 5,715
Ligand 28
Magnesium ion 6
Solvent (water) 563
B average (A2) of protein 28.40
B average (A2) of solvents 36.13
B average (A2) of ligands 22.91
(bisphosphonates, Mg2+ )
a: BPH-527 is (2-Hydroxy-2,2-bis-phosphono-ethyl)-dimethyl-sulfonium
b: Brookhaven National Laboratory
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Table 7: Data collection and refinement statistics for BPH-461 a bound to T.
brucei
FPPS.
Data collection
Space group C2
Unit cell dimension (A)
P( ) 112.364
a = b, c 135.565, 118.520, 63.186
X-ray source BNL-X12Cb
Wavelength (A) 1.1
Resolution (A) 30-2.10 (2.18-2.10)
No. of reflection observed 406,549
Unique 53,536 (5,267)
Completeness (%) 99.1 (98.3)
R-merge 0.070 (0.483)
1/Ql 9.9
Multiplicity 7.6 (7.6)
Refinement statistics
Resolution range (A) 30.0-2.10
R-work/R-free (%) 21.83/25.93
RMSD
Bond lengths 0.004
Bond angles 1.532
No. of atoms
Protein 5,745
Ligand 36
Magnesium ion 6
Solvent (water) 94
B average (A2) of protein 28.42
B average (A2) of solvents 27.60
B average (A2) of ligands 36.22
(bisphosphonates, Mg2+ )
a: BPH-461 is 3-fluoro-1 -(2-hydroxy-2,2-bisphosphonoethyl)-pyridinium
b: Brookhaven National Laboratory
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Table 8: Data collection and refinement statistics for BPH-675a bound to S.
cerevisiae GGPPS.
Data collection
Space group P212121
Unit cell dimension (A)
a, b, c 46.39 116.26 128.70
X-ray source NSRRC-BL13B1 b
Wavelength (A) 1.0
Resolution (A) 50-2.20 (2.28-2.20)
No. of reflection observed 246,740 (23,819)
Unique 36,490 (3,555)
Completeness (%) 99.9 (99.9)
R-merge 0.085 (0.424)
1/Ql 26.1 (5.5)
Multiplicity 6.8 (6.7)
Refinement statistics
Resolution range (A) 50-2.2 (2.28-2.2)
R-work/R-free (%) 18.5/24.1 (24.0/29.0)
RMSD
Bond lengths 0.019
Bond angles 1.7
No. of atoms
Protein 5,128
Ligand 47
Magnesium ion 4
Solvent (water) 337
B average (A2) of protein 40.0
B average (A2) of solvents 46.2
B average (A2) of ligands 61.3
(bisphosphonates, Mg2+ )
a: BPH-675 is 1-Hydroxy-2-[3'-(Naphthalene-2-sulfonylamino)-biphenyl-3-
I]ethylidene-l,1-bisphosphonic acid
~ BL13B1 at NSRRC (Hsin-Chu, Taiwan)
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Table 9: Isothermal calorimetry results
H. sapiens T. brucei
FPPS FPPS A H log (IC50)
Cpd ID IC50 M IC50 M (kcal/mol) A delta S A G(kcal/mol) T. brucei
BPH-527 1.03 0.78 3.93 42.92 -8.9 -6.11
BPH-536 12.1 26.1 4.01 43.07 -8.6 -4.58
BPH-540 9 11.3 no signal -4.95
BPH-541 38.5 544.2 no signal -3.26
BPH-560 1.13 275 3.78 41.52 -8.5 -3.56
BPH-571 1.82 892 2.49 36.47 -8.3 -3.05
BPH-678 1.25 25.6 1.85 35.71 -8.7 -4.59
Table 10: 2D-QSAR Descriptors and Output
QuaSAR-Model(PLS): /Volumes/hudock/MOE/cancercells/111306/111306.mdb
Mon Nov 13 17:17:44 2006
Activity Field : pIC50_cancer
Weight Field
Condition Limit : le+06
Component Limit : 0
Observations : 20
Descriptors : 3
Components Used : 3
Condition Number : 39.156322
ROOT MEAN SQUARE ERROR (RMSE): 0.49176
CORRELATION COEFFICIENT (R2) : 0.83332
ESTIMATED LINEAR MODEL
pIC50_cancer =
-6.86627
+0.37153 * pIC50_hsFPPS
+1.77016 * pIC50_GGPPS
-0.31092 * SlogP
ESTIMATED NORMALIZED LINEAR MODEL (SD = Standard Deviation)
pIC50_cancer / SD(pIC50_cancer) _
-5.70044
+0.31174 * pIC50hsFPPS / SD(pIC50hsFPPS)
+1.39278 * pIC50_GGPPS / SD(pIC50_GGPPS)
-0.65581 * SlogP / SD(SlogP)
RELATIVE IMPORTANCE OF DESCRIPTORS
0.223827 pIC50_hsFPPS
1.000000 pIC50_GGPPS
0.470865 SlogP
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Table 11: CoMSIA Analysis Output
Regression Equation(s)
Use COMFA FIELD RETRIEVE/LIST/GRAPH or EVA RETRIEVE/LIST/GRAPH
CoMFA/EVA coefficients.
MCF PIC50 = - 3.854 + (0.406) * PIC50HSFPPS + (1.303) * PIC50GGPPS
- (0.000) * SLOGP
Relative Contributions
# Norm.Coeff. Fraction
----------- ---------
1 PIC50HSFPPS 0.416 0.142
2 PIC50GGPPS 1.033 0.352
3 SLOGP 0.001 0.000183
4 COMSIA_ST (1170 vars) 0.146 0.050
COMSIAHY (1170 vars) 0.334 0.114
6 COMSIAEL (1170 vars) 0.064 0.022
7 COMSIADO (1170 vars) 0.487 0.166
8 COMSIAAC (1170 vars) 0.451 0.154
Summary output
Standard Error of Estimate 0.180
R squared 0.977
F values ( nl= 4, n2=17 ) 184.083
Prob.of R2=0 ( nl= 4, n2=17 ) 0.000
Scrambling Stability Test
Components Q2 csDEP dq2/dr2yy
---------- ------ ----- ---------
2 0.46 0.80 0.66
3 0.60 0.70 1.02
4 0.64 0.68 1.34
5 0.66 0.67 1.33
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Materials and Methods.
[00215] Cell Growth Inhibition Assays. The human tumor cell lines MCF-7
(breast adenocarcinoma), NCI-H460 (lung large cell) and SF-268 (central
nervous
system glioblastoma) were obtained from the National Cancer Institute. All
lines
were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum
and 2 mM L-glutamine at 37 C in a 5% CO2 atmosphere with 100% humidity. A
broth
microdilution method was used to determine IC50 values for growth inhibition
by each
bisphosphonate. Cells were inoculated at a density of 5,000 cells/well into 96-
well
flat bottom culture plates containing 10 pL of the test compound, previously
half-log
serial diluted (from 0.316 mM to 0.1 pM) for a final volume of 100 pL. NBPs
were
typically initially dissolved in H20 (0.01 M) while NNBPs were typically
dissolved in
DMSO (0.01 M). Plates were then incubated for 4 days at 37 C in a 5% CO2
atmosphere at 100% humidity after which an MTT ((3-(4,5-dimethylthiazole-2-yl)-
2,5-
diphenyltetrazolium bromide) cell proliferation assay (ATCC, Manassas, VA) was
used to obtain dose-response curves. The DMSO carrier had no effect on cell
proliferation.
[00216] GraphPad PRISM version 4.0 software for windows (GraphPad Software
Inc., San Diego, CA, www.graphpad.com) was used to fit the data to a
rectangular
hyperbolic function: I = (ImaXC) / (IC5o + C);
I C
= Imax
IC50 +C
where I is the percent inhibition, ImaX = 100% inhibition, C is the
concentration of the
inhibitor, and IC50 is the concentration for 50% growth inhibition. Typical
dose-
response curves are shown in Figure 2A, in the text. For the "rescue"
experiments,
stock solutions of FOH or GGOH were prepared (in ethanol) and the requisite
amounts added to the incubation media to produce a fixed 20 pM concentration.
[00217] yb T Cell Assays. Vy2V62 T cell TNF-a release and proliferation were
performed basically as described previously'. Briefly, to measure bioactivity
for
Vy2V62 T cells, the CD4+ JN.24, CD4+ HF.2, CD8aa+ 12G12, or the CD4-8- HD.108
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Vy2V62 T cell clones were stimulated with phosphoantigens in the presence of
CP.EBV (an EBV transformed B cell line) for CD4+ clones or Va-2 (a transformed
fibroblast) for CD8aa+ and CD4-8- clones. CP.EBV and Va-2 were fixed with
0.05%
glutaraldehyde (EM grade, Sigma, MO) for use as APCs. Note that although the
relative potencies of the phosphoantigens were similar, the NKG2D+ Vy2V62
clones,
12G12 and HD.108, exhibited higher antigen sensitivity, likely due to
costimulation
through their NKG2D receptors by their interaction with the NKG2D ligands,
MICA,
ULBP2, and ULBP3, that are expressed by the Va-2 cell line. We have previously
shown that the NKG2D/MICA interaction significantly increases antigen
sensitivity.
Concentrations required to achieve 50% of the observed T cell response (EC50s)
were obtained by using the Prism 4.0 program (Graphpad Software, San Diego,
CA),
using a sigmoidal dose-response function. Curve fitting minima for each
experiment
(e.g. TNF-a release from JN.24 cells) were determined using the Global Fitting
technique, as implemented in Prism 4Ø Curve fitting maxima were optimized
for
each individual compound without the use of any constraints.
[00218] NMR spectroscopy. Spectra were obtained by using the magic-angle
sample spinning technique on a 600 MHz ('H resonance frequency) Infinity Plus
spectrometer equipped with a 14.1 T, 2 inch bore Oxford magnet and
Varian/Chemagnetics 3.2 mm T3 HXY probe. Spectra were referenced to an
external standard of 85% orthophosphoric acid. 'H transverse magnetization was
created by a 3.5ps pulse (75 kHz field) and cross polarization was used for
signal
enhancement, followed by TPPM decoupling (80 kHz'H field) during data
acquisition. 'H-31P cross polarization pulse shapes and decoupling were
optimized
on risedronate (Actonel) prior to data acquisition on the protein samples.
Data were
acquired using a dwell time of 10 ps (a 100 kHz spectral width), 2048 points,
a 2 sec
recycle delay and a spinning speed of 13.333 kHz. All spectra were processed
by
using zero-filling to 4096 points, 50Hz exponential multiplication, and a
polynomial
correction for baseline correction prior to peak integration. The number of
scans
varied between 32 k and 86 k.
[00219] Human recombinant GGPPS inhibition. The purification of human
recombinant geranylgeranyl diphosphate synthase (hGGPPS) followed the protocol
reported previously2. GGPPS inhibition by bisphosphonates was determined using
CA 02682694 2009-09-30
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the radiometric assay reported previously2 with slight modification. The assay
solution contained 300 ng of hGGPPS, 50 mM potassium phosphate buffer (pH
7.0),
mM MgCl2, 2 mM DTT, 1 mg/mL BSA, and 25 pM FPP in a total volume of 50 pL
and was preincubated with the bisphosphonates at room temperature for 15 min.
Then, the reactions were started by adding 5 pL of a 250 pM solution of [14C]
IPP
and incubated at 37 C for 20 min. The reaction was terminated by the addition
of 75
pL of HCI/MeOH. Following a second 20 min incubation at 37 C to effectively
hydrolyze the allylic pyrophosphates, the reaction mixtures were neutralized
by the
addition of 75 pL of 6 N NaOH and extracted with 500 pL of hexane. 200 pL of
the
organic phase was transferred to a scintillation vial for counting. The IC50
values
were obtained by fitting the data to the dose-response curve in Origin 6.1
(OriginLab
Corp., Northampton, MA, www.OriginLab.com).
[00220] Crystallization and X-ray Data Collection for Human
FPPS-Bisphosphonate Complexes. Crystals human FPPS complexed with Mg
and either BPH-461 or BPH-527 were obtained based on the methods described by
K. L. Kavanagh et a1.3, with slight modification. FPPS was incubated with 2.5
mM
bisphosphonate, 2.5 mM MgCl2 overnight on ice before setting up the drops.
Crystals
were grown at room temperate in sitting drops by mixing 2 pL of protein
solution and
1 pL of precipitant, which consisted of 40% (v/v) of either polyethylene
glycol 2,000
or 4,000 and 0.1 M phosphate/citrate buffer, pH 4.2. Diffraction data were
obtained at
100 K using an ADSC Q315 CCD detector at the Brookhaven National Synchrotron
Light Source, beamline X29 (,&=1.1 A). Data collection statistics are reported
in the
Example 3 section herein.
[00221 ] Crystallization and X-ray Data Collection of T.brucei
FPPS-Bisphosphonate Complxes. Initial crystallization screening conditions
were
based on crystallization conditions reported by Mao et a14. The effects of
protein
concentration, precipitant type and concentration, buffer type, buffer pH
value and
metal-ion concentration were then optimized and protein crystals that gave
good
diffraction patterns were obtained. Protein at 5.55 mg/mL was mixed with 2.5
mM
BPH-461 or BPH-527, 2.5 mM MgCl2 and incubated overnight on ice before setting
up the drops. Crystals were grown at room temperature in hanging drops by
mixing
1 pL of FPPS-bisphosphonate mixture solution with 1 pL of precipitant
consisting of
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10% (v/v) MPD in 100 mM ammonium acetate, pH 5.75. Prior to data collection,
crystals were mounted in a cryoloop and flash-frozen in liquid nitrogen after
the
addition of 40% (v/v) MPD as cryoprotectant. Diffraction data were obtained at
100 K
using an ADSC Q4 CCD detector at the Brookhaven National Synchrotron Light
Source beamline X8C (,&=1.1 A). Data collection statistics are reported in
Tables 6
and 7.
[00222] Structure determination of Human FPPS-Bisphosphonate Complexes.
For structure determination, the human FPPS structure (1 YV5)3 minus the
risedronate ligand was used as a search model using the molecular replacement
method. Rigid body refinement was applied to the model obtained using AMoRe5.
The crystal structure was then further refined by using Shelxl-976. Rebuilding
and
fitting the ligand was carried out by using the program O' in the 2Fo-Fc
electron
density map. Certain refinement statistics are included in Tables 4 and 5.
[00223] Structure determination of T.brucei FPPS-Bisphosphonate
Complexes. The crystal structures of the T.brucei FPPS bisphoshponate
complexes were determined by using the molecular replacement method using the
program AMoRe5. The previously solved T.brucei FPPS structure (2EWG)$ minus
the minodronate ligand was used as a starting model. The structure has been
further
refined using CNS9. After iterative rounds of refinement using CNS and
rebuilding
using Coot, the structures had the final refinement statistics shown in Tables
6 and
7.
[00224] 2D QSAR: Molecular Descriptors. Structures of inhibitors were imported
into the Molecular Operating Environment (MOE) 2006.0810. In order to compute
certain molecular descriptors, a three-dimensional structure was required. The
three-dimensional models were built by minimizing all molecules using a 0.05
kcal/mol gradient and MMFF9411 force field. In addition to computed 2D
molecular
descriptors, GGPPS and FPPS enzyme pIC50 values were also used. The
AutoQuaSAR module12, an expert system for QSAR in MOE, was used. This
iteratively builds a series of models by evaluating the importance of each of
the
descriptors available, removing less important ones in a step-wise fashion in
order to
produce a trajectory of r2 and q2 (leave-one-out cross-validated r2) as a
function of
the number of descriptors. The models having the fewest components and the
52
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highest r2 and q2, were then selected for inspection. The final model computer
output is shown in Table 10.
[00225] 3D-QSAR: CoMSIA Descriptors and Analysis. Conformers of all
compounds were generated in MOE 2006.08 10 using the conformation import
utility.
In order to avoid potential bias in the alignment, the pharmacophore
perception
algorithm (in MOE) was used to generate alignments of the molecules, based on
overlap of perceived features, specifically: hydrophobic, aromatic, cation,
donor and
acceptor. The ranked list of putative pharmacophores then served as the basis
for
initial alignment13. Alignment of molecules in the top pharmacophore
(containing a
cationic feature) was selected and refined sequentially using the flexible
alignment
module in MOE with TAFF (Tripos) and MMFF94 force fields. Aligned molecules
and charges were imported into Sybyl 7.314 along with corresponding FPPS,
GGPPS
and MCF-7 activity data. CoMSIA15 descriptors were calculated for the aligned
molecules with additional descriptors added, including FPPS pIC50, GPPS pIC50
and
SLogP. PLS was used to assign contributions of each of the components, which
resulted in q2 = 0.806 (3 components). The computer output is show in Table
11.
[00226] A scrambling stability test, as implemented in Sybyl 7.314, was then
performed on the data to ensure that the model was not obtained due to chance
and,
additionally, to verify the optimum number of components. The scrambling
method
applies small, random perturbations to the dataset while monitoring the
predictivity of
the resulting models. The predictivity of unstable models typically falls off
disproportionately rapidly from even small perturbations, while robust models
exhibit
more predictive stability16. The output results, confirming stability at three
components, are shown in Table 11.
[00227] Hologram HQSAR (HQSAR). Hologram QSAR, unlike CoMSIA, does not
require a common three-dimensional structural alignment, but rather is a
fragment-
based, alignment independent method that serves as a performance baseline that
is
difficult to outperform by comparable methods". The HQSAR method, as
implemented in Sybyl 7.314, uses an extended molecular fingerprint (molecular
hologram) to correlate structural features and biological activity. Structures
of the 64
molecules were imported into Sybyl 7.3 and three dimensional coordinates
generated for ease of structure inspection and verification using up to 10,000
steps
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CA 02682694 2009-09-30
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at 0.01 kcal/mol gradient using the BFGS 18 energy minimization method.
Structures
were then automatically fragmented into pre-defined fragment sizes. A
molecular
hologram (fingerprint) was then generated for each molecule using these
fragments,
retaining information about the fragment, possible overlap and constituent sub-
fragments, implicitly encoding three-dimensional structure information. The
hologram was then used for partial least squares (PLS) analysis to produce
cross-
validated models, obtaining a final model having q2 = 0.674 and r2 = 0.871 and
optimal fragment size of 83 bits.
References cited in this section:
[00228] 1. Song, Y.; Zhang, Y.; Wang, H.; Raker, A. M.; Sanders, J. M.;
Broderick,
E.; Clark, A.; Morita, C. T.; Oldfield, E., Synthesis of Chiral
Phosphoantigens and
Their Activity in yb T Cell Stimulation. Bioorg Med Chem Lett 2004, 14, (17),
4471-7.
[00229] 2. Szabo, C. M.; Matsumura, Y.; Fukura, S.; Martin, M. B.; Sanders, J.
M.;
Sengupta, S.; Cieslak, J. A.; Loftus, T. C.; Lea, C. R.; Lee, H. J.; Koohang,
A.;
Coates, R. M.; Sagami, H.; Oldfield, E., Inhibition of Geranylgeranyl
Diphosphate
Synthase by Bisphosphonates and Diphosphates: A Potential Route to New Bone
Antiresorption and Antiparasitic Agents. J Med Chem 2002, 45, (11), 2185-96.
[00230] 3. Kavanagh, K. L.; Guo, K.; Dunford, J. E.; Wu, X.; Knapp, S.;
Ebetino, F.
H.; Rogers, M. J.; Russell, R. G.; Oppermann, U., The molecular mechanism of
nitrogen-containing bisphosphonates as antiosteoporosis drugs. Proc Natl Acad
Sci
U S A 2006, 103, (20), 7829-34.
[00231] 4. Mao, J.; Gao, Y. G.; Odeh, S.; Robinson, H.; Montalvetti, A.;
Docampo,
R.; Oldfield, E., Crystallization and Preliminary X-ray Diffraction Study of
the
Farnesyl Diphosphate Synthase from Trypanosoma brucei. Acta Crystallogr D Biol
Crystallogr 2004, 60, (Pt 10), 1863-6.
[00232] 5. Navaza, J., AMoRe: an automated package for molecular replacement.
Acta Crystallog. sect. A 1994, 50, 157-163.
[00233] 6. Sheldrick, G.; Schneider, T., SHELXL: High Resolution Refinement.
Methods in Enzymology 1997, 277, 319-343.
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[00234] 7. Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M., Improved
methods for building protein models in electron density maps and the location
of
errors in these models. Acta Crystallographica Section A 1991, 47, 110-119.
[00235] 8. Mao, J.; Mukherjee, S.; Zhang, Y.; Cao, R.; Sanders, J. M.; Song,
Y.;
Zhang, Y.; Meints, G. A.; Gao, Y. G.; Mukkamala, D.; Hudock, M. P.; Oldfield,
E.,
Solid-state NMR, crystallographic, and computational investigation of
bisphosphonates and farnesyl diphosphate synthase-bisphosphonate complexes. J
Am Chem Soc 2006, 128, (45), 14485-97.
[00236] 9. Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.;
Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N.
S.; Read,
R. J.; Rice, L. M.; Simonson, T.; Warren, G. L., Crystallography & NMR system:
A
new software suite for macromolecular structure determination. Acta
Crystallogr D
Biol Crystallogr 1998, 54, (Pt 5), 905-21.
[00237] 10. MOE, 2006.08; Chemical Computing Group, Inc.: Montreal, Quebec,
2006.
[00238] 11. Halgren, T. A.; Nachbar, R. B., MMF94: The Merck molecular force
field. Bridging the gap - From small organics to proteins. Abstracts of Papers
of the
American Chemical Society 1996, 211, 70-COMP.
[00239] 12. Goto, J. AutoQuaSAR 2006.08, Ryoka Systems, Inc.: Tokyo, Japan,
2006.
[00240] 13. Zhu, L. L.; Hou, T. J.; Chen, L. R.; Xu, X. J., 3D QSAR analyses
of
novel tyrosine kinase inhibitors based on pharmacophore alignment. J Chem lnf
Comput Sci 2001, 41, (4), 1032-40.
[00241] 14. Sybyl 7.3, Tripos, Inc.: St. Louis, MO.
[00242] 15. Klebe, G.; Abraham, U.; Mietzner, T., Molecular similarity indices
in a
comparative analysis (CoMSIA) of drug molecules to correlate and predict their
biological activity. J Med Chem 1994, 37, (24), 4130-46.
[00243] 16. Tripos Bookshelf 7.3, Tripos, Inc.: St. Louis, MO.
CA 02682694 2009-09-30
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[00244] 17. Gedeck, P.; Rohde, B.; Bartels, C., QSAR--how good is it in
practice?
Comparison of descriptor sets on an unbiased cross section of corporate data
sets. J
Chem Inf Model 2006, 46, (5), 1924-36.
[00245] 18. Press, W. H., Numerical recipies in C: the art of scientific
computing.
Cambridge University Press: New York, 1988; p 324.
[00246] 19. Widler, L.; Jaeggi, K. A.; Glatt, M.; Muller, K.; Bachmann, R.;
Bisping,
M.; Born, A. R.; Cortesi, R.; Guiglia, G.; Jeker, H.; Klein, R.; Ramseier, U.;
Schmid,
J.; Schreiber, G.; Seltenmeyer, Y.; Green, J. R., Highly Potent Feminal
Bisphosphonates. From Pamidronate disodium (Aredia) to Zoledronic Acid
(Zometa).
J Med Chem 2002, 45, (17), 3721-38.
EXAMPLE 4. Anti-cancer activity including such against tumors in vivo.
[00247] Tumor cell invasiveness and in vivo results. We investigated whether
lipophilic bisphosphonates can have pronounced effects on tumor cell
invasiveness.
When MDA-MB-231 cells, an invasive human breast cancer adenocarcinoma cell
line, were cultured with bisphosphonates in a Matrigel invasion assay, the
lipophilic
bisphosphonate, BPH-716, was about 1000-fold more inhibitory than was
zoledronate (BPH-716, IC50 about 30 nM; versus zoledronate, IC50 about 40 pM).
To
determine whether such compounds had activity in vivo, we used SK-ES-1 sarcoma
cells in a mouse xenograft system (Kubo 2007). While zoledronate caused a
significant (p < 0.01) reduction in tumor cell growth versus control, the
effect of a
lipophilic bisphosphonate (BPH-715) was even more pronounced (p = 0.032 versus
zoledronate), and there was no weight loss or other adverse effect observed.
Activity in this mouse model can be attributed to direct activity on tumor
cell growth
and invasiveness, since murine gammadelta T cells lack the Vy2V62 T cell
receptor
required for activation by IPP. These results demonstrate that more lipophilic
bisphosphonates have potent, direct activity against tumor cell
proliferation/invasiveness, both in vitro and in vivo. They can also have
enhanced
potency in human T cell activation, believed due to IPP accumulation.
[00248] In vivo tumor cell model. Experiments were carried out basically as
described in Kubo 2006 et al.47 Xenografts of human SK-ES-1 cells were
initiated by
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subcutaneous injections of 1.5x10' cells into the right flank of four, 6-week
old
athymic nude mice (CLEA, Tokyo, Japan). The mice received daily
intraperitoneal
injections of 5 pg of zoledronate, BPH-715 or physiological saline. The
smallest and
largest diameters of tumors, and the body weights, were measured weekly. Tumor
volumes were calculated using the following formula: volume (mm) = (smallest
diameter)2 x(largest diameter)/2. Statistical significance was determined by
one-
way ANOVA and Fisher's PLSD moethod, using Statcel (OMS Ltd., Saitama, Japan);
p < 0.05 was considered to be significant.
References:
[00249] Kubo, T., Shimose, S., Matsuo, T., Sakai, A. & Ochi, M. Efficacy of a
nitrogen-containing bisphosphonate, minodronate, in conjunction with a p38
mitogen
activated protein kinase inhibitor or doxorubicin against malignant bone tumor
cells.
Cancer Chemother. Pharmacol. (2007).
[00250] Kubo, T. et al. Inhibitory effects of a new bisphosphonate,
minodronate, on
proliferation and invasion of a variety of malignant bone tumor cells. J.
Orthop. Res.
24, 1138-44 (2006).
EXAMPLE 5. Structural formulas of compounds.
[00251] In addition to structural formulas for compounds provided elsewhere in
the
specification and drawings, certain structural formulas are provided below.
0 OH 0 OH
O OH 2 \\ /
1 \\ / P-OH 5 ~\ /OH 91 \P P-OH
P-OH OH P-OH (N-Z_OH
OH H2NOH H2N /N~ O//P~OH
O OH N O OH
O O
0 OH 261
24 \\ / \ O\ /OH I 300 A P P-OH
P-OH I P-OH
OH N OH N+^~OH
P-OH
/p\OH p\OH ~OH
OOH N p OH O
OH 0 O
~\ /O 290 0 ~P~ OH 364 AP
278 P-OH
P-OH
0"1OH ~ N p~OH
\OH
~P\OH / ~P\OH 0
N
N
57
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O o" 0 o" 0 o"
714 \\ / 715 \\ / 716 \\ /
P-OH P-OH P-OH
O O N+~ O N+
7 \OH 9 /p\OH
11 /p\OH
O OH O OH O OH
O O 0 O 0 O
721 \\ / 722 \\ / 723 \\ /
P-OH P-OH P-OH
~/J O \N+~ ~/J O \N+~ ~~O \N
3 pOH ~OH
p~OH
OH
~ ~ \OH ~ ~OH
O O 0 O 0 O
637 \\ / 638 A P OH 598 \\ /
P-OH P-OH
N \ + _ _
_
P\OH 4 /P\OH
6 P~OH $ N 0~
0
O O 0 O
677 \\ / 604 \\ /
P-OH P-OH
11 \N+ p~OH N+ p\OH
/ p OH ~ OH
O O 0 O
717 \\ / ~ 579 \\ /
P-OH I P-OH
N+ P\OH ~~~~0 ~ \N+ P\OH
/ O OH / 0 OH
7
H 0 OH
(NO p754 \\ /
679 AP OH P-OH
OH
N NOH 9 p~OH
Op-OH // ~OH
\OH O
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WO 2008/128056 PCT/US2008/060051
694 0 /O 688 0 /O 683 0 O
P-OH \\ /
S+~ ~\~' 1^ P-OH P-OH
S S
`J' \ /\\/ ``_/X\ +
2 /P~OH 10 I p\-OH 6 p-OH
OH OOH 0~OH
687 0 O 696 0 /O 695 0 O
,P-OH
*~-~s -OH +~P-OH *~l \S+/\ / P ~\~/ 1'+/^\\/
`` \~ S
P-OH 6 p-OH 14 I p- OH
0 \OH 0 OH // -OH
O
684 0 O 685 0 /O 686 ~\ O-
\\ /
P-OH S+~P-OH S+~P-OH
S
OH \OH
3 I p\OH p
O// OH O OH 0 OH
527 682 693 0 /OH
~\ /O ~\ /O P-OH
P-OH P-OH i OH CS+ N
P-OH P-OH 10 I p\OH
0 \OH 0 \OH O OH
727 ~\ /O 728 0 O
P-OH P-OH
0 \ +
p~OH I N p-OH
0 ~OH / 0 \OH
NH 678 0\ /O \// I 675 0 P-OH
P-OH
S~ OH
H2N NH"~OH ~ I\ NH P-OH
OH -
O/P\OH 0 OH
601 ~\ /O 0 629 O\
P-OH O'
P-OH
\N+~ OH
p::~OH P-OH
0 OH 0 \OH
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WO 2008/128056 PCT/US2008/060051
O 0
O O-'\ O O~~
669 \\ / O 656 \\ / O
P-OH P-OH
F \N+ P-OH \N P-OH
O// O 13N
/ O O
0
O O O
670 \\/ O 461 \\/
P-OH
F -OH
NOH N+~ OH
/
p\-OH OP-OH
O O
0 O ~ 0 O 0 O
470 \\ / I 483 \\ /
P-OH P-OH OH Br P-OH
0~OH 1472
N I \N p\OH
p-OH / N+ P~OH
~ ~ OH
0
OH 0
0 O 0 O
474 ~\ /O 475 \\p/ P-OH 476 A P-OH
P-OH
+_
+ N N
N P
P_
OH OH /P\OH
O OH O O
O O 480 O P/ OH 481 0 P/ P-OH
477 \\ /
P-OH
~ I ~ y OP\OH OP\OH
536 O\ /O 540 O\ /O 560 0 O-
P-OH /S+ P-OH
OH \P/ OH
\~ /OH P-OH P-OH
P+ OH
/A
OP-OH OP\_OH OP\OH
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[00252] Certain data in Figure 2D is further represented in Table 12 below.
Table 12. Matrix with several enzyme targets.
FPPS GGPPS DPPS Cells SIogP
FPPS 100 -33 27 -7 -34
GGPPS -33 100 12 81 85
DPPS 27 12 100 23 -6
Cells -7 81 23 100 63
SIogP -34 85 -6 63 100
STATEMENTS REGARDING INCORPORATION
BY REFERENCE AND VARIATIONS
[00253] All references throughout this application, for example patent
documents
including issued or granted patents or equivalents; patent application
publications;
and non-patent literature documents or other source material; are hereby
incorporated by reference herein in their entireties, as though individually
incorporated by reference, to the extent each reference is at least partially
not
inconsistent with the disclosure in this application (for example, a reference
that is
partially inconsistent is incorporated by reference except for the partially
inconsistent
portion of the reference).
[00254] When a group of substituents is disclosed herein, it is understood
that all
individual members of that group and all subgroups, including any isomers,
enantiomers, and diastereomers of the group members, are disclosed separately.
When a Markush group or other grouping is used herein, all individual members
of
the group and all combinations and subcombinations possible of the group are
intended to be individually included in the disclosure. A number of specific
groups of
variable definitions have been described herein. It is intended that all
combinations
and subcombinations of the specific groups of variable definitions are
individually
included in this disclosure. When a compound is described herein such that a
particular isomer, enantiomer or diastereomer of the compound is not
specified, for
example, in a formula or in a chemical name, that description is intended to
include
each isomers and enantiomer of the compound described individual or in any
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CA 02682694 2009-09-30
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combination. Additionally, unless otherwise specified, all isotopic variants
of
compounds disclosed herein are intended to be encompassed by the disclosure.
For example, it will be understood that any one or more hydrogens in a
molecule
disclosed can be replaced with deuterium or tritium. Isotopic variants of a
molecule
are generally useful as standards in assays for the molecule and in chemical
and
biological research related to the molecule or its use. Isotopic variants,
including
those carrying radioisotopes, may also be useful in diagnostic assays and in
therapeutics. Methods for making such isotopic variants are known in the art.
Specific names of compounds are intended to be exemplary, as it is known that
one
of ordinary skill in the art can name the same compounds differently.
[00255] Every formulation or combination of components described or
exemplified
herein can be used to practice the invention, unless otherwise stated.
[00256] Whenever a range is given in the specification, for example, a
temperature
range, a time range, a composition or concentration range, or other value
range, all
intermediate ranges and subranges, as well as all individual values included
in the
ranges given are intended to be included in the disclosure. It will be
understood that
any subranges or individual values in a range or subrange that are included in
the
description herein can be excluded from the claims herein.
[00257] All patents and publications mentioned in the specification are
indicative of
the levels of skill of those skilled in the art to which the invention
pertains.
References cited herein are incorporated by reference herein in their entirety
to
indicate the state of the art, in some cases as of their filing date, and it
is intended
that this information can be employed herein, if needed, to exclude (for
example, to
disclaim) specific embodiments that are in the prior art. For example, when a
compound is claimed, it should be understood that compounds known in the prior
art, including certain compounds disclosed in the references disclosed herein
(particularly in referenced patent documents), are not intended to be included
in the
claim.
[00258] Where the terms "comprise", "comprises", "comprised", or "comprising"
are
used herein, they are to be interpreted as specifying the presence of the
stated
features, integers, steps, or components referred to, but not to preclude the
62
CA 02682694 2009-09-30
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presence or addition of one or more other feature, integer, step, component,
or
group thereof. As used herein, "comprising" is thus synonymous with
"including,"
"containing," or "characterized by," and is inclusive or open-ended and does
not
exclude additional, unrecited elements or method steps. As used herein,
"consisting
of" excludes any element, step, or ingredient not specified in the claim
element. As
used herein, "consisting essentially of" does not exclude materials or steps
that do
not materially affect the basic and novel characteristics of the claim. In
each
instance herein any of the terms equivalent to "comprising", "consisting
essentially
of" and "consisting of" may be replaced with either of the other two terms to
signify
the respective meaning which can indicate a difference in scope. The invention
illustratively described herein suitably may be practiced in the absence of
any
element or elements, limitation or limitations which is not specifically
disclosed
herein.
[00259] The invention has been described with reference to various specific
and
preferred embodiments and techniques. However, it should be understood that
many variations and modifications may be made while remaining within the
spirit and
scope of the invention. It will be apparent to one of ordinary skill in the
art that
methods, devices, device elements, materials, procedures and techniques other
than
those specifically described herein can be applied to the practice of the
invention as
broadly disclosed herein without resort to undue experimentation. For example,
one
of ordinary skill in the art will appreciate that starting materials,
biological materials,
reagents, synthetic methods, purification methods, analytical methods, assay
methods, and biological methods other than those specifically exemplified can
be
employed in the practice of the invention without resort to undue
experimentation.
All art-known functional equivalents of methods, devices, device elements,
materials,
procedures and techniques described herein are intended to be encompassed by
this invention. This invention is not to be limited by the specific
embodiments
disclosed, including any shown in the drawings or exemplified in the
specification,
which are given by way of example or illustration and not of limitation. It
should be
understood that although the present invention has been specifically disclosed
by in
some cases preferred embodiments and optional features, modification and
variation
of the innovative concepts herein disclosed may be resorted to by those
skilled in the
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art, and that such modifications and variations are considered to be within
the scope
of this invention as further defined by the appended claims.
REFERENCES
[00260] US Application Serial 11687570 filed March 17, 2006; PCT International
Application Serial PCT/US07/64239 filed March 17, 2006; US Application Serial
60783491 filed March 17, 2006; US Application Serial 11/245,612 filed October
7,
2005 (see also US Patent Application Publication No. 20060079487 published
April
13, 2006); US Application Serial 60/617,108 filed October 8, 2004; PCT
International
Application No. PCT/US05/036425 filed October 7, 2005 (see also International
Publication No. WO/2006/039721 published April 13, 2006); US Patent
Application
Publication No. 20050113331 published May 26, 2005; each of the foregoing in
particular is incorporated by reference in entirety to the extent not
inconsistent
herewith.
[00261] Hudock MP et al., Acta Cryst. (2006). E62, o843-o845.
[00262] Cao R et al., Acta Cryst. (2006). E62, o1003-o1005
[00263] Zhang Y et al., Acta Cryst. (2006). E62, o1006-o1008
[00264] Cao R et al., Acta Cryst. (2006). E62, o1055-o1057
[00265] Zhang Y et al., J Med Chem. 2006 Sep 21;49(19):5804-14.
[00266] (1) Sambrook, P. N.; Geusens, P.; Ribot, C.; Solimano, J. A.; Ferrer-
Barriendos, J.; Gaines, K.; Verbruggen, N.; Melton, M. E. Alendronate produces
greater effects than raloxifene on bone density and bone turnover in
postmenopausal women with low bone density: results of EFFECT (EFficacy of
FOSAMAX versus EVISTA Comparison Trial) International. J. Intern. Med. 2004,
255, 503-511.
[00267] (2) Vasireddy, S.; Talwakar, A.; Miller, H.; Mehan, R.; Swinson, D. R.
Patterns of pain in Paget's disease of bone and their outcomes on treatment
with
pamidronate. Clin. Rheumatol. 2003, 22, 376-380.
64
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
[00268] (3) Dawson, N. A. Therapeutic benefit of bisphosphonates in the
management of prostate cancer-related bone disease. Expert. Opin.
Pharmacother.
2003, 4, 705-716.
[00269] (4) Rosen, L. S.; Gordon, D. H.; Dugan, W. Jr.; Major, P.; Eisenberg,
P. D.;
Provencher, L.; Kaminski, M.; Simeone, J.; Seaman, J.; Chen, B. L.; Coleman,
R. E.
Zoledronic acid is superior to pamidronate for the treatment of bone
metastases in
breast carcinoma patients with at least one osteolytic lesion. Cancer 2004,
100, 36-
43.
[00270] (5) Cromartie, T. H.; Fisher, K. J.; Grossman, J. N. The discovery of
a
novel site of action for herbicidal bisphosphonates. Pesticide Biochem. Phys.
1999,
63, 114-126.
[00271 ](6) Cromartie, T. H.; Fisher, K. J. Method of controlling plants by
inhibition
of farnesyl pyrophosphate synthase. U.S. Patent 5,756,423, May 26, 1998.
[00272] (7) van Beek, E.; Pieterman, E.; Cohen, L.; Lowik, C.; Papapoulos, S.
Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate
isomerase/farnesyl pyrophosphate synthase activity with relative potencies
corresponding to their antiresorptive potencies in vitro and in vivo. Biochem.
Biophys. Res. Commun. 1999, 255, 491-494.
[00273] (8) van Beek, E.; Pieterman, E.; Cohen, L.; Lowik, C.; Papapoulos, S.
Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing
bisphosphonates. Biochem. Biophys. Res. Commun. 1999, 264, 108-111.
[00274] (9) Keller, R. K.; Fliesler, S. J. Mechanism of aminobisphosphonate
action:
characterization of alendronate inhibition of the isoprenoid pathway. Biochem.
Biophys. Res. Commun. 1999, 266, 560-563.
[00275] (10)Bergstrom, J. D.; Bostedor, R. G.; Masarachia, P. J.; Reszka, A.
A.;
Rodan, G. Mechanism of aminobisphosphonate action: characterization of
alendronate inhibition of the isoprenoid pathway. Arch. Biochem. Biophys.
2000,
373, 231-241.
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
[00276] (11)Grove, J. E.; Brown, R. J.; Watts, D. J. The intracellular target
for the
antiresorptive aminobisphosphonate drugs in Dictyostelium discoideum is the
enzyme farnesyl diphosphate synthase. J. Bone Miner. Res. 2000, 15, 971-981.
[00277] (12)Dunford, J. E.; Thompson, K.; Coxon, F. P.; Luckman, S. P.; Hahan,
F.
M.; Poulter, C. D.; Ebetino, F. H.; Rogers, M. J. Structure-activity
relationships for
inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone
resorption
in vivo by nitrogen-containing bisphosphonates. J. Pharmacol. Exp. Ther. 2001,
296,
235-242.
[00278] (13)Luckman, S. P.; Hughes, D. E.; Coxon, F. P.; Graham, R.; Russell,
G.;
Rogers, M. J. Nitrogen-containing bisphosphonates inhibit the mevalonate
pathway
and prevent post-translational prenylation of GTP-binding proteins, including
Ras. J.
Bone Miner. Res. 1998, 13, 581-589.
[00279] (14)Fisher, J. E.; Rogers, M. J.; Halasy, J. M.; Luckman, S. P.;
Hughes, D.
E.; Masarachia, P. J.; Wesolowski, G.; Russell, R. G.; Rodan, G. A.; Reszka,
A. A.
Alendronate mechanism of action: geranylgeraniol, an intermediate in the
mevalonate pathway, prevents inhibition of osteoclast formation, bone
resorption,
and kinase activation in vitro. Proc. Natl. Acad. Sci. USA 1999, 96, 133-138.
[00280] (15)van Beek, E.; Lowik, C.; van der Pluijm, G.; Papapoulos, S. The
role of
geranylgeranylation in bone resorption and its suppression by bisphosphonates
in
fetal bone explants in vitro: A clue to the mechanism of action of nitrogen-
containing
bisphosphonates. J. Bone Miner. Res. 1999, 14, 722-729.
[00281 ](16)Montalvetti, A.; Bailey, B. N.; Martin, M. B.; Severin, G. W.;
Oldfield, E.;
Docampo, R. Bisphosphonates are potent inhibitors of Trypanosoma cruzi
farnesyl
pyrophosphate synthase. J. Biol. Chem. 2001, 276, 33930-33937.
[00282] (17)Sanders, J. M.; Gomez, A. 0.; Mao, J.; Meints, G. A.; van Brussel,
E.
M.; Burzynska, A.; Kafarski, P.; Gonzalez-Pacanowska, D.; Oldfield, E. 3-D
QSAR
investigations of the inhibition of Leishmania major farnesyl pyrophosphate
synthase
by bisphosphonates. J. Med. Chem. 2003, 46, 5171-5183.
66
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[00283] (18)Martin, M. B.; Grimley, J. S.; Lewis, J. C.; Heath, H. T. III;
Bailey, B. N.;
Kendrick, H.; Yardley, V.; Caldera, A.; Lira, R.; Urbina, J. A.; Moreno, S.
N.;
Docampo, R.; Croft, S. L.; Oldfield, E. Bisphosphonates inhibit the growth of
Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii,
and Plasmodium falciparum: A potential route to chemotherapy. J. Med. Chem.
2001, 44, 909-916.
[00284] (19)Martin, M. B.; Sanders, J. M.; Kendrick, H.; de Luca-Fradley, K.;
Lewis,
J. C.; Grimley, J. S.; van Brussel, E. M.; Olsen, J. R.; Meints, G. A.;
Burzynska, A.;
Kafarski, P.; Croft, S. L.; Oldfield, E. Activity of bisphosphonates against
Trypanosoma brucei rhodesiense. J. Med. Chem. 2002, 45, 2904-2914.
[00285] (20)Moreno, B.; Bailey, B. N.; Luo, S.; Martin, M. B.; Kuhlenschmidt,
M.;
Moreno, S. N.; Docampo, R.; Oldfield, E. 31 P NMR of apicomplexans and the
effects
of risedronate on Cryptosporidium parvum growth. Biochem. Biophys. Res.
Commun. 2001, 284, 632-637.
[00286] (21)Ghosh, S.; Chan, J. M.; Lea, C. R.; Meints, G. A.; Lewis, J. C.;
Tovian,
Z. S.; Flessner, R. M.; Loftus, T. C.; Bruchhaus, I.; Kendrick, H.; Croft, S.
L.; Kemp,
R. G.; Kobayashi, E. Effects of bisphosphonates on the growth of Entamoeba
histolytica and Plasmodium species in vitro and in vivo. J. Med. Chem. 2004,
47,
175-187.
[00287] (22)Yardley, V.; Khan, A. A.; Martin, M. B.; Slifer, T. R.; Araujo, F.
G.;
Moreno, S. N.; Docampo, R.; Croft, S. L.; Oldfield, E. In vivo activities of
farnesyl
pyrophosphate synthase inhibitors against Leishmania donovani and Toxoplasma
gondii. Antimicrob. Agents Chemother. 2002, 46, 929-931.
[00288] (23)Rodriguez, N.; Bailey, B. N.; Martin, M. B.; Oldfield, E.; Urbina,
J. A.;
Docampo, R. Radical cure of experimental cutaneous leishmaniasis by the
bisphosphonate pamidronate. J. Infect. Dis. 2002, 186, 138-140.
[00289] (24)Garzoni, L. R.; Caldera, A.; Meirelles, M. N. L.; de Castro, S.
L.;
Meints, G.; Docampo, R.; Oldfield, E.; Urbina, J. A. Selective in vitro
effects of the
farnesyl pyrophosphate synthase inhibitor risedronate on Trypanosoma cruzi.
Intl. J.
Antimicrobial Agents 2004, 23, 273-285.
67
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
[00290] (25)Garzoni, L. R.; Waghabi, M. C.; Baptista, M. M.; de Castro, S. L.;
Meirelles, M. N. L.; Britto, C.; Docampo, R.; Oldfield, E.; Urbina, J. A.
Antiparasitic
activity of risedronate in a murine model of acute Chagas' disease. Intl. J.
Antimicrobial Agents 2004, 23, 286-290.
[00291 ](26)Wang, L.; Kamath, A.; Das, H.; Li, L.; Bukowski, J. F.
Antibacterial
effect of human Vgamma2Vdelta2 T cells in vivo. J. Clin. Invest. 2001, 108,
1349-
1357.
[00292] (27)Kunzmann, V.; Bauer, E.; Feurle, J.; Weissinger, F.; Tony, H. P.;
Wilhelm, M. Stimulation of yb T cells by aminobisphosphonates and induction of
antiplasma cell activity in multiple myeloma. Blood 2000, 96, 384-392.
[00293] (28)Kato, Y.; Tanaka, Y.; Miyagawa, F.; Yamashita, S.; Minato, N.
Targeting of tumor cells for human gammadelta T cells by nonpeptide antigens.
J.
Immunol. 2001, 167, 5092-5098.
[00294] (29)Thompson, K.; Rogers, M. J. Statins prevent bisphosphonate-induced
gammadelta-T-cell proliferation and activation in vitro. J. Bone Miner. Res.
2004, 19,
278-288.
[00295] (30)Sanders, J. M.; Ghosh, S.; Chan, J. M. W.; Meints, G.; Wang, H.;
Raker, A. M.; Song, Y.; Colantino, A.; Burzynska, A.; Kafarski, P.; Morita, C.
T.;
Oldfield, E. Quantitative structure-activity relationships for gammadelta T
cell
activation by bisphosphonates. J. Med. Chem. 2004, 47, 375-384.
[00296] (31)Wilhelm, M.; Kunzmann, V.; Eckstein, S.; Reimer, P.; Weissinger,
F.;
Ruediger, T.; Tony, H. P. gammadelta T cells for immune therapy of patients
with
lymphoid malignancies. Blood 2003, 102, 200-206.
[00297] (32) Miyaura, N; Yanagi, T; Suzuki, A. The palladium-catalyzed
cross-coupling reaction of phenylboronic acid with haloarenes in the presence
of
bases. Synth. Commun. 1981, 11, 513-519.
[00298] (33)Krapcho, A. P.; Ellis, M. Synthesis of regioisomeric difluoro- and
8-
chloro-9-fluorobenz[g]isoquinoline-5,1 0-diones and SNAr displacements studies
by
68
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
diamines: bis(aminoalkyl)aminobenz[g]isoquinoline-5,10-diones. J. Fluorine
Chem.
1998, 90, 139-147.
[00299] (34)Zhang, L.; Liang, F.; Sun, L.; Hu, Y.; Hu, H. A novel and
practical
synthesis of 3-unsubstituted indolizines. Synthesis 2000, 1733-1737.
[00300] (35)Harel, Z.; Kovalevski-Liron, E.; Lidor-Hadas, R.; Lifshitz-Liron,
R. Use
of certain diluents for making bisphosphonic acids. World Patent W003097655,
November 27, 2003.
[00301] (36)Rogers, M. J.; Watts, D. J.; Russell, R. G.; Ji, X.; Xiong, X.;
Blackburn,
G. M.; Bayless, A. V.; Ebetino, F. H. Inhibitory effects of bisphosphonates on
growth
of amoebae of the cellular slime mold Dictyostelium discoideum. J. Bone Miner.
Res.
1994, 9, 1029-1039.
[00302] (37)van Beek, E. R.; Cohen, L. H.; Leroy, I. M.; Ebetino, F. H.;
Lowik, C.
W.; Papapoulos, S. E. Differentiating the mechanisms of antiresorptive action
of
nitrogen containing bisphosphonates. Bone 2003, 33, 805-11.
[00303] US Patents: 5583122 by Benedict et al., issued December 10, 1996;
6,562,974 by Cazer et al., issued May 13, 2003; 6544967 by Daifotis et al.,
issued
April 8, 2003; 6,410,520 by Cazer et al., issued June 25, 2002; 6,372,728 by
Ungell,
issued April 16, 2002; 6,638,920 by Thompson, issued October 28, 2003; 4777163
by Bosies et al., issued October 11, 1988; 4939130 by Jaeggi et al., issued
July 3,
1990; 4,859,472 by Demmer et al., issued August 22, 1989; US 5,227,506 by
Saari
et al., issued July 13, 1993; US 6,753,324 by Jomaa, issued June 22, 2004.
US5294608
[00304] Alfer'ev, I. S.; Mikhalin, N. V., Reactions of vinylidenediphosphonic
acid
with nucleophiles. 5. Addition of heterocyclic amines and trimethylamine to
vinylidenediphosphonic acid; August 1994, Russian Chemical Bulletin 44(8):1528-
1530 (translated from Izvestiya Akademii Nauk, Seriya Khimicheskaya 1995, 8,
1590-1592).
69
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
[00305] Alfer'ev IS et al., Izvestiay Akademii Nauk SSSR, Seriya
Khimicheskaya,
No. 12, pp. 2802-2806, December 1983 [Bull. Acad. Sci. USSR, Div. Chem. Sci.,
1983, 32:2515 (Engl. Transl.)].
[00306] Alfer'ev IS et al., Izv. Akad. Nauk SSSR, Ser. Khim., 1984:1122 [Bull.
Acad.
Sci. USSR, Div. Chem. Sci., 1984, 33:1031 (Engl. Transl.)].
[00307] International Publication No. W003075741 by Wilder et al., published
18
September 2003; International Publication No. W02004024165 by Baulch-Brown et
al., published 25 March 2004; German Patent Publication DE19859668 by Hassan,
published 30 December 1999; International Publication No. W02004050096 by
Romagne et al., published 17 June 2004.
[00308] Widler L, et al., Highly potent geminal bisphosphonates. From
pamidronate
disodium (Aredia) to zoledronic acid (Zometa), J Med Chem. 2002 Aug
15;45(17):3721-38.
[00309] Green JR, Chemical and biological prerequisites for novel
bisphosphonate
molecules: results of comparative preclinical studies, Semin Oncol. 2001 Apr;
28(2
Suppl 6):4-10.
[00310] US 4,711,880 by Stahl et al., issued December 8, 1987 (Aredia /
pamidronate); US 4621077, US 5462932, US 5994329, US 6015801, US 6225294
(Fosamax / alendronate); US 5583122, US 6096342; US 6165513 (Actonel /
risedronate).
[00311] Wilhelm M et al., 2003, Gammadelta T cells for immune therapy of
patients
with lymphoid malignancies, Blood 102: 200-206.
[00312] Jagdev SP, Coleman RE, Shipman CM, Rostami HA, Croucher PI (2001);
The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells:
evidence for synergy with paclitaxel. Br J Cancer 84:1126- 1134.
[00313] US 4927814 by Gall et al., issued May 22, 1990; US 6294196 by Gabel et
al., issued September 25, 2001; 6,143,326 by Mockel, et al. issued November 7,
2000 (ibandronate / Boniva ); 6,544,967 by Daifotis, et al. April 8, 2003.
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
[00314] Heidenreich et al., 2004. Ibandronate in metastatic bone pain, Semin.
Oncol. 31(5 Suppl 10):67-72.
[00315] Gordon DH, 2005. Efficacy and safety of intravenous bisphosphonates
for
patients with breast cancer metastatic to bone: a review of randomized, double-
blind,
phase III trials, Clin Breast Cancer. 6(2):125-31.
[00316] De Cock et al., 2005. Cost-effectiveness of oral ibandronate versus IV
zoledronic acid or IV pamidronate for bone metastases in patients receiving
oral
hormonal therapy for breast cancer in the United Kingdom. Clin. Ther.
27(8):1295-
310.
[00317] Sanders et al., Pyridinium-1-yl Bisphosphonates Are Potent Inhibitors
of
Farnesyl Diphosphate Synthase and Bone Resorption, J. Med. Chem. 2005, 48,
2957-296.
[00318] Kotsikorou Evangelia et al., Bisphosphonate Inhibition of the
Exopolyphosphatase Activity of the Trypanosoma brucei Soluble Vacuolar
Pyrophosphatase, J. Med. Chem. 2005, 48, 6128-6139.
[00319] Inoue S et al., 2003 Synthesis, 13:1971-1976. New synthesis of gem-
Bis(phsophono)ethylenes and their Applications.
[00320] Soloducho J et al., 1997. Patent PL93-298436, Preparation of novel
derivatives of (aminomethylene)bis(phosphonic acid) as herbicides.
[00321] Lecouvey M et al., 2001, Tet. Lett. 42:8475-8478, A mild and efficient
one-
pot synthesis of 1-hydroxymethylene-1,1-bisphosphonic acids. Preparation of
new
tripod ligands.
[00322] Kieczykowski GR et al., 1995, J. Org. Chem 60:8310-8312, Preparation
of
(4-amino-1 -hydroxybutylidene)bisphosphonic and sodium salt, MK-217
(alendronate
sodium). An improved procedure for the preparation of 1-hydroxy-1,1 -
bisphosphonic
acids.
[00323] Liang P-H, 2002, Eur.J.Biochem. 269, 3339-3354 (2002), Review Article,
Structure, mechanism and function of prenyltransferases.
71
CA 02682694 2009-09-30
WO 2008/128056 PCT/US2008/060051
[00324] Goldstein JL, Brown MS: Regulation of the mevalonate pathway. Nature
343:425, 1990.
[00325] Swanson KM, Hohl RJ, Curr Cancer Drug Targets. 2006 Feb;6(1):15-37.
Anti-cancer therapy: targeting the mevalonate pathway.
[00326] Wiemer AJ, Tong H, Swanson KM, Hohl RJ; Biochem Biophys Res
Commun. 2007 Feb 23;353(4):921-5. Digeranyl bisphosphonate inhibits
geranylgeranyl pyrophosphate synthase.
72
