U.S. patent application number 16/048125 was filed with the patent office on 2018-11-29 for bone targeted therapeutics and methods of making and using the same.
The applicant listed for this patent is MBC Pharma, Inc.. Invention is credited to Alexander Karpeisky, Shawn Patrick Zinnen.
Application Number | 20180339055 16/048125 |
Document ID | / |
Family ID | 41054290 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180339055 |
Kind Code |
A1 |
Karpeisky; Alexander ; et
al. |
November 29, 2018 |
BONE TARGETED THERAPEUTICS AND METHODS OF MAKING AND USING THE
SAME
Abstract
The present invention provides novel bisphosphonate conjugates,
pharmaceutical compositions comprising bisphosphonate conjugates
and methods of using such analogs in the treatment of bone cancer,
bone-related diseases, bone infection, bone inflammation, and
diseases of the soft tissues surrounding bones.
Inventors: |
Karpeisky; Alexander;
(Lafayette, CO) ; Zinnen; Shawn Patrick; (Denver,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MBC Pharma, Inc. |
Aurora |
CO |
US |
|
|
Family ID: |
41054290 |
Appl. No.: |
16/048125 |
Filed: |
July 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14968481 |
Dec 14, 2015 |
10046055 |
|
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16048125 |
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|
14063951 |
Oct 25, 2013 |
9216204 |
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14968481 |
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12413805 |
Mar 30, 2009 |
8586781 |
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14063951 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/544 20170801;
C07F 9/58 20130101; C07F 9/6512 20130101; C07F 9/65583 20130101;
C07F 9/6544 20130101; A61K 31/675 20130101; A61K 47/54 20170801;
A61K 47/548 20170801; A61K 31/7028 20130101; A61P 29/00 20180101;
C07F 9/6561 20130101; C07H 19/207 20130101; A61K 31/7052 20130101;
A61K 31/546 20130101; A61K 31/165 20130101; C07H 19/10 20130101;
A61K 51/0489 20130101; A61K 38/14 20130101; A61P 19/08 20180101;
C07F 9/65586 20130101; A61K 31/663 20130101; C07F 9/65616 20130101;
A61K 31/496 20130101 |
International
Class: |
A61K 47/54 20170101
A61K047/54; C07H 19/207 20060101 C07H019/207; C07F 9/6561 20060101
C07F009/6561; C07F 9/6558 20060101 C07F009/6558; C07F 9/6544
20060101 C07F009/6544; C07F 9/6512 20060101 C07F009/6512; C07F 9/58
20060101 C07F009/58; A61K 51/04 20060101 A61K051/04; A61K 38/14
20060101 A61K038/14; A61K 31/7052 20060101 A61K031/7052; A61K
31/7028 20060101 A61K031/7028; A61K 31/675 20060101 A61K031/675;
A61K 31/663 20060101 A61K031/663; A61K 31/546 20060101 A61K031/546;
A61K 31/496 20060101 A61K031/496; A61K 31/165 20060101 A61K031/165;
C07H 19/10 20060101 C07H019/10 |
Claims
1. A bisphosphonate conjugate comprising: ##STR00014## or a
pharmaceutically acceptable acid addition salt thereof wherein, X
is O; V is O or S; Y is a residue of an anti-infective compound in
a class selected from the group consisting of fluoroquinolones,
lincosamides, oxazolidinones, aminoglycosides, cephalosporins, and
glycopeptides; R.sup.1 is selected from the group consisting of OH,
SH, NH.sub.2, OZ, SZ, NZ, halogen, and H; R.sup.2 is selected from
the group consisting of H, Z, and halogen; and Z is selected from
the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, and
NH.sub.2.
2. The conjugate of claim 1, wherein the anti-infective compound
belongs to the fluoroquinolone class.
3. The conjugate of claim 1, wherein the anti-infective compound
belongs to the lincosamide class.
4. The conjugate of claim 1, wherein the anti-infective compound
belongs to the oxazolidinone class.
5. The conjugate of claim 1, wherein the anti-infective compound
belongs to the aminoglycoside class.
6. The conjugate of claim 1, wherein the anti-infective compound
belongs to the cephalosporin class.
7. The conjugate of claim 1, wherein the anti-infective compound
belongs to the glycopeptide class.
8. A pharmaceutical composition comprising a conjugate of claim 1
and a pharmaceutically-acceptable carrier.
9. A method of treating bone infection, the method comprising
administering a therapeutically effective amount of a conjugate of
claim 1 to a subject in need thereof.
10. A method of delivering an anti-infective compound to the bone
or surrounding tissue, wherein the anti-infective compound is in a
class selected from the group consisting of fluoroquinolones,
lincosamides, oxazolidinones, aminoglycosides, cephalosporins, and
glycopeptides, the method comprising administering a conjugate of
claim 1 to a subject in need thereof.
11. A method of affecting bone growth in mammals, the method
comprising administering to a mammal requiring a change in bone
growth a therapeutically effective amount of a conjugate of claim
1.
12. A method of treating inflammation caused by bone infection, the
method comprising administering a therapeutically effective amount
of a conjugate of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/968,481, filed Dec. 14, 2015, which is a divisional of
U.S. patent application Ser. No. 14/063,951, filed Oct. 25, 2013,
now U.S. Pat. No. 9,216,204, which is a divisional application of
U.S. patent application Ser. No. 12/413,805, filed Mar. 30, 2009,
now U.S. Pat. No. 8,586,781. The disclosures of these applications
are incorporated herein by this reference.
FIELD
[0002] The present invention is directed to bisphosphonate
compounds, and in particular, bisphosphonate conjugates that are
useful in the treatment of soft tissues surrounding bone and
bone-related diseases, such as bone cancer and osteoporosis.
BACKGROUND
[0003] Bone degeneration diseases, including Paget's Disease and
osteoporosis have proven difficult to treat because the mechanisms
involved in the development and progression of these diseases are
not well understood. Bisphosphonates are synthetic analogs of
pyrophosphates characterized by a phosphorus-carbon-phosphorus
backbone that renders them resistant to hydrolysis and are known to
be useful in the treatment of these degenerative bone disorders.
The chemical properties of the bisphosphonates vary based on
different substitutions at the carbon atom of the
phosphorus-carbon-phosphorus backbone.
[0004] Bisphosphonates bind strongly to hydroxyapatite on the bone
surface and act to reduce and inhibit the activity of osteoclasts;
cells functioning in the absorption and removal of osseous tissue.
The anti-resorptive effect of bisphosphonates is also mediated
through effects on osteoblasts; cells that function in the
production of bone. Thus, biophosphonates are used clinically to
inhibit bone resorption in disease states such as Paget's disease,
osteoporosis, metastatic bone diseases, and malignant and
nonmalignant hypercalcemia. Bisphosphonates are also used to
mediate anti-cancer effects by modifying the bone surface, altering
the bone microenvironment, inhibiting specific enzymatic pathways
and inducing apoptosis in osteoclast and tumor cells.
[0005] Bisphosphonates that are currently used therapeutically
include alendronate, clodronate, etidronate, pamidronate,
tiludronate, ibandronate, zoledronate, olpadronate, residronate and
neridronate. Additionally, bone scanning agents based on the use of
bisphosphonic acid compounds have been used in the past to produce
high definition bone scans (see e.g., U.S. Pat. No. 4,810,486 to
Kelly et. al). Bisphosphonate derivatives have been used as
therapeutic agents for bone diseases such as osteoporosis,
rheumatoid arthritis, and osteoarthritis (see e.g., U.S. Pat. No.
5,428,181 to Sugioka et. al). In the past, however, bisphosphonate
therapies have frequently been accompanied by severe side effects
such as retardation of bone development and somatic growth.
[0006] Therefore, a need exists for novel bisphosphonate compounds
that act as delivery vehicles to target and deliver therapeutic
agents to bone and the surrounding soft tissue, allowing selective
treatment of these tissues while eliminating or minimizing the
severe side effects previously seen with bisphosphonate
therapies.
SUMMARY
[0007] Provided herein are bone-seeking conjugates containing
anticancer or anti-infective compounds or derivatives thereof
linked to bisphosphonates. When linked to a moiety having
antineoplastic or anti-infective properties, bisphosphonates act as
vehicles for the targeted delivery of these therapeutic entities to
bone. The chemical bond(s) connecting the bisphosphonate and the
drug is/are stable enough to survive in the bloodstream and yet
is/are cleaved to liberate the drug when the conjugate binds to
bone.
[0008] Because these conjugates are capable of releasing
anti-infective and cytotoxic components upon binding with bone
tissue, they are useful in the treatment and prevention of bone
cancer, bone infections, bone inflammation and disorders in soft
tissues surrounding bone. For example, in the case of
osteomyelitis, certain therapeutic anti-infectives can be coupled
to the bisphosphonate carrier molecule for delivery of high
concentrations of anti-infective therapy to various sites of bone
infection. Examples of useful anti-infectives that can be coupled
with the bisphosphonates of the present invention include
fluoroquinolones; lincosamides; oxazolidinones, aminoglycoside
antibiotics; cephalosporins, lipoglycopeptides. Examples of useful
anticancer derivatives that can be conjugated with the
bisphosphonates of the present invention include 5-fluorouracil,
cytarabine, cisplatin, doxorubicin, epirubucin, streptozocin.
[0009] One embodiment of the present invention provides novel
bisphosphonate conjugates that are capable of delivering
anti-infective and/or anti-neoplastic (cytotoxic) residues to the
bone and surrounding tissues. Such conjugates will release their
therapeutic component upon binding to the bone tissue and thus are
useful in the treatment and prevention of bone primary tumors,
metastases of non-bone tumors to bones and infections of bone and
surrounding soft tissue.
[0010] The conjugates of the present invention comprise anhydrides
formed between a substituted bisphosphonic acid and phosphate,
thiophosphate or phosphoramidate derivatives of anticancer or
anti-infective compounds. The labile phosphoanhydride bond in such
analogs provides release of the therapeutic compound upon binding
with the bone or surrounding tissues.
[0011] Another embodiment of the present invention provides
substituted bisphosphonic acids linked to therapeutic entities that
are effective in treating or modulating cancers or infections of
bone and bone-surrounding tissues such as amino acids, nucleic
acids, protein toxins, protein and/or peptide growth factors and
hormones that promote bone growth and bone marrow
proliferation.
[0012] Another aspect of the present invention provides
bisphosphonate conjugates that offer a delivery vehicle with which
to deliver and concentrate drugs and proteins to normal and
abnormal bone tissue and soft tissue surrounding bones. These
abnormalities are generally referred to as bony lesions. As used
herein, bony lesions include, but are not limited to, bone cancer,
osteomyelitis, soft tissue infections surrounding bone, bone marrow
abnormalities, and bone diseases such as Paget's disease.
[0013] The present invention thus provides novel bone targeted
therapeutics, pharmaceutical compositions comprising said
bone-targeted therapeutics and methods of using such analogs in the
treatment of bone cancer, bone-related diseases and diseases of the
soft tissues surrounding bones.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is the synthetic scheme for the preparation of
nucleoside-5'-triphosphate analogs.
[0015] FIG. 2 demonstrates the synthesis of cefamandole-etidronate
conjugate.
[0016] FIG. 3 demonstrates the synthesis of
ciprofloxacin-etidronate conjugate.
[0017] FIG. 4 demonstrates the synthesis of telavancin-etidronate
conjugate.
[0018] FIG. 5 shows degradation of the bisphosphonate conjugates on
bone.
[0019] FIG. 6 shows distribution of the conjugate on bone.
DETAILED DESCRIPTION
[0020] The present invention relates to novel bone-targeted
therapeutics that are capable of delivering therapeutic compounds
such as anti-infective and/or anti-neoplastic (cytotoxic) compounds
or peptide or protein compounds, having growth stimulating
properties, to bone and soft tissues surrounding bone. The
conjugates release the therapeutic components upon binding to the
bone tissue and are therefore useful in the treatment and
prevention of primary bone tumors, metastases to bone tissues, bone
inflammation, bone infections (and inflammation caused by bone
infection) and disorders of the growth of bone and bone marrow. It
is understood that compounds of the invention may be covalently
linked together either via linker or directly.
[0021] Bisphosphonates are synthetic compounds containing two
phosphonate groups bound to a central (geminal) carbon (the P-C-P
backbone) that are used to prevent bone resorption in a number of
metabolic and tumor-induced bone diseases including multiple
myeloma. Bisphosphonate treatment is associated with an increase in
patient survival, indicating that these compounds have a direct
effect on the tumor cells.
[0022] Bisphosphonates may contain two additional chains bound to
the central geminal carbon. The presence of these two side chains
allows numerous substitutions to the bisphosphonate backbone and
therefore the development of a variety of analogs with different
pharmacological properties. The activity varies greatly from
compound to compound, the newest bisphosphonates being 5,000 to
10,000 times more active than etidronate, the first bisphosphonate
described. The mechanism of action of bisphosphonates includes a
direct effect exerted on osteoclast activity, direct and indirect
effects on osteoclast recruitment mediated by cells of the
osteoblastic lineage and involving the production of an inhibitor
of osteoclastic recruitment and a shortening of osteoclast survival
by apoptosis.
[0023] High doses of bisphosphonates can also inhibit
mineralization through a physicochemical inhibition of crystal
growth. One substituent on the geminal carbon together with the
P-C-P backbone are primarily responsible for binding to bone
mineral and for the physicochemical actions of the bisphosphonates.
These interactions are optimized by the presence of a hydroxyl
group as at least one substituent on the geminal carbon. The
remaining substituent on the geminal carbon is responsible for the
anti-resorptive action of the bisphosphonates and small
modifications or conformational restrictions at this part of the
molecule result in marked differences in anti-resorptive potency.
The presence of nitrogen functionality in an alkyl chain or in a
ring structure in one of the substituents on the geminal carbon
greatly enhances the anti-resorptive potency and specificity of
bisphosphonates for bone resorption and most of the newer potent
bisphosphonates contain nitrogen atom in their structure.
[0024] The biological effects of bisphosphonates in calcium-related
disorders are attributed to the incorporation of the
bisphosphonates into bone, enabling direct interaction with
osteoclasts and/or osteoblasts. The high accumulation of
bisphosphonates in bone, due to their high affinity for
hydroxyapatite, is essential for mediating both the in vitro and in
vivo activity. Nitrogen-containing bisphosphonates are known to act
by binding to a specific intracellular target at a site
complementary in structure to the bisphosphonate side chain.
[0025] Recent evidence suggests that the whole bisphosphonate
molecule is essential for anti-resorptive action. Thus, although
the basic structural requirements for bisphosphonate actions have
been defined, precise structure-activity relationships for the
bisphosphonate side chains indicate that at least the newer
generations of nitrogen-containing bisphosphonates act by binding
to a specific target at a site that is complementary in structure
to the bisphosphonate side chain.
[0026] The bisphosphonate conjugates of the present invention have
the chemical structure:
##STR00001##
wherein,
[0027] X is O,
[0028] V is O or S
[0029] Y is an anti-infective or anticancer compound, having in its
structure free functional group, used for conjugation with the
linker. Examples of such groups include but are not limited to OH,
NH.sub.2, NH, N-alkyl.
[0030] R.sup.1 is selected from the group consisting of OH, SH,
NH.sub.2, OZ, SZ, NZ, halogen, H and;
[0031] R.sup.2 is selected from the group consisting of H, Z, and
halogen; and
[0032] Z is selected from the group consisting of alkyl,
cycloalkyl, aryl, heteroaryl, and NH.sub.2.
[0033] Additionally, compounds of present invention have the
following structure: wherein,
##STR00002##
[0034] Y is an anti-infective or anticancer compound, having in its
structure free functional group, used for conjugation with the
linker. Examples of such groups include but are not limited to
PO.sub.3H.sub.2;
[0035] R.sup.1 is selected from the group consisting of OH, SH,
NH.sub.2, OZ, SZ, NZ, halogen, and H;
[0036] R.sup.2 is selected from the group consisting of H, Z, and
halogen; and
[0037] Z is selected from the group consisting of alkyl,
cycloalkyl, aryl, heteroaryl, and NH.sub.2.
[0038] Additionally, compounds of present invention may contain one
or more bisphosphonate residues and have the following
structure:
Y(L-A).sub.n
[0039] n=1-10, for example, n=1-3
[0040] A is a bisphosphonate residue having structure
##STR00003##
[0041] Where R.sup.1 is selected from the group consisting of OH,
SH, NH.sub.2, OZ, SZ, NZ, halogen, and H;
[0042] R.sup.2 is selected from the group consisting of H, Z, and
halogen;
[0043] Z is selected from the group consisting of alkyl,
cycloalkyl, aryl, heteroaryl, and NH.sub.2;
[0044] L is a linker, having structure
##STR00004##
[0045] Where V is O or S; and
[0046] Y is a residue of anti-infective or anticancer compound,
having in its structure free functional group, used for conjugation
with the linker. Examples of such groups include but are not
limited to OH, NH.sub.2, NH, and N-alkyl.
[0047] As used herein, an "alkyl" group refers to a saturated
aliphatic hydrocarbon, including straight-chain and branched-chain
alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More
preferably it is a lower alkyl having from 1 to 7 carbons, and more
preferably 1 to 4 carbons. The alkyl group may be substituted or
unsubstituted. When substituted, the substituent group(s) may
include hydroxy, cyano, alkoxy, NO.sub.2 or N(Alkyl).sub.2,
NHAlkyl, amino, or SH.
[0048] As used herein, a "cycloalkyl" group refers to a cyclic
alkyl group having from three to ten, and preferably five or six
carbon atoms forming the alkyl ring.
[0049] As used herein, an "aryl" group refers to an aromatic group
which has at least one ring having a conjugated pi electron system
and includes carbocyclic aryl, heterocyclic aryl and biaryl groups;
all of which may be optionally substituted. Substituent(s) on these
groups may include halogen, trihalomethyl, hydroxyl, SH, cyano,
alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
[0050] As used herein, "heteroaryl" refers to an aromatic ring
having from 1 to 3 heteroatoms in the aromatic ring with the
remainder of the atoms in the ring being carbon atoms. Suitable
heteroatoms include oxygen, sulfur, and nitrogen, and exemplary
heteraryls include furanyl, thienyl, pyridyl, pyrrolyl, pyrrolo,
pyrimidyl, pyrazinyl and imidazolyl. These heteroaryl rings may
also be substituted. Substituents on these heteroaryl groups may
include halogen, trihalomethyl, hydroxyl, SH, cyano, alkoxy, alkyl,
alkenyl, alkynyl, and amino groups.
[0051] As used herein, "anti-infective compound" refers to any
compound having antibacterial, antibiotic, antifungal,
antiprotozoan and antiviral activity.
[0052] As used herein, "halogen" refers to Cl, F, or Br atom.
[0053] The bisphosphonate conjugates of the present invention
contain both an osteotropic moiety and a therapeutic moiety that is
released from the osteotropic moiety upon binding of the conjugates
to bone tissue. The covalent bond(s) connecting the bisphosphonate
moiety and the therapeutic component are stable enough to survive
in the bloodstream but are cleaved to liberate the drug when the
conjugate binds to bone tissue, releasing the therapeutic component
to the bone or to soft tissue surrounding the bone.
[0054] These bisphosphonate conjugates comprise conjugates formed
between a substituted bisphosphonate and a substituted phosphoric,
thiophosphoric or amidophosphoric acid. Thus, these conjugates are
analogs of triphosphates. It is the labile phosphoanhydride bonds
in these analogs that release the conjugated therapeutic compounds
upon binding with the bone. In this way, the bisphosphonate
conjugates of the present invention can be used to target
covalently bound therapeutic compounds to bone and soft tissue
surrounding bone.
[0055] One embodiment of the present invention includes anti-cancer
drugs that are coupled to bisphosphonate or derivatives of
bisphosphonate substituted at the geminal carbon. The anti-cancer
drugs may include, but are not limited to, nucleosides and/or
acyclo-nucleosides in which the sugar or nucleic base is modified
or unmodified (natural), antisense and catalytic oligonucleotides,
amino acids, peptides, polypeptides or proteins having cytostatic
or antineoplastic properties. The bisphosphonate may also be
conjugated to combinations of one or more of these anti-cancer
compounds. Exemplary anticancer compounds for conjugation to the
bisphosphonate moiety include, but are not limited to, cytarabine,
cisplatin, doxorubicin, epirubucin, streptozocin. Additionally, the
bisphosphonate or bisphosphonate derivatives may be conjugated to
nucleosides or nucleoside-like compounds having cytostatic or
neoplastic activity. Exemplary nucleoside or nucleoside-like
compounds that can be conjugated to the bisphosphonate compounds of
the present invention include compounds having the structure:
##STR00005##
[0056] wherein R.sup.3 and R.sup.4 are independently H, OH or F and
B is a natural or modified nucleic base or derivative thereof.
Exemplary modified nucleic bases include compounds having the
structure:
##STR00006##
[0057] These nucleoside or nucleoside-like compounds can be linked
to the bisphosphonate or bisphosphonate derivatives through a
linker group. Exemplary linking moieties include phosphate or
thiophosphate groups. These bisphosphonate conjugates are useful in
the treatment of primary bone tumors, bone metastases (i.e.
metastases to bone tissues from malignant tissue elsewhere in the
body), bone inflammation, bone infections and disorders of the
growth of bone and bone marrow. Thus, the present invention
includes methods of treating a mammal in need of anti-cancer
therapy with compounds of the present invention having an
anti-cancer compound coupled to a bisphosphonate in a
therapeutically effective amount sufficient to impart a
chemotherapeutic response in the mammal.
[0058] Another embodiment of the present invention includes a
bisphosphonate or derivatives of bisphosphonate substituted at the
geminal carbon coupled to a compound having anti-infective
activity. These conjugates have been found to be particularly
useful in the treatment of infections or inflammation of the bone
tissue or of soft tissues surrounding bone. In this embodiment,
therapeutic anti-infective compounds can be coupled directly (if an
anti-infective compound has a free phosphate or phosphonate residue
in its structure such as telavancin) to the bisphosphonate carrier
molecule or via phosphate or thiophosphate group linker, for
delivery of high concentrations of anti-infective compound to the
sites of bone or soft tissue infection or inflammation. The
covalent bond(s) connecting the bisphosphonate moiety and the
anti-infective moiety are cleaved to liberate drug when the
conjugate binds to bone tissue, releasing the anti-infective
compound to the bone or to soft tissue surrounding the bone (FIG.
5).
[0059] Examples of anti-infective compounds which can be conjugated
to the bisphosphonate carriers include, but are not limited to,
fluoroquinolones such as ciprofloxacin; lincosamides such as
clindamycin; oxazolidinones such as eperezolid; aminoglycoside
antibiotics such as gentamycin; cephalosporin antibiotics such as
cephamandole; glycopeptides such as vancomycin or telavancin;
fusidic acid and chloramphenicol.
[0060] An exemplary clindamycin residue is shown below:
##STR00007##
[0061] An exemplary cefamandole residue is shown below:
##STR00008##
[0062] An exemplary eperezolid residue is shown below:
##STR00009##
[0063] Exemplary chloramphenicol residues include:
##STR00010##
[0064] An exemplary ciprofloxacin residue is shown below:
##STR00011##
[0065] An exemplary vancomycin residue is shown below:
##STR00012##
[0066] An exemplary telavancin residue is shown below:
##STR00013##
[0067] Examples of bisphosphonate carriers include but are not
limited to etidronate, clodronate, pamidronate, alendronate,
risedronate, zoledronate, medronic acid, aminomethylene
bisphosphonic acid.
[0068] The present invention also includes methods of treating a
mammal in need of anti-infective or anti-inflammatory or anticancer
therapy with compounds of the present invention having an
anti-infective or anti-inflammatory or anticancer compound coupled
to a bisphosphonate, in a therapeutically effective amount to
impart anti-infective or anti-inflammatory or anticancer responses
in the mammal. Illustratively, an anti-infective compound coupled
to a bisphosphonate is useful for treating or preventing bone
infection and/or bone inflammation.
[0069] Another embodiment of the present invention is a
bisphosphonate or derivatives of bisphosphonate substituted at the
geminal carbon moiety conjugated to a protein or peptide growth
factor or hormone that promotes bone growth and/or bone marrow
proliferation. These conjugates are useful in the treatment of
diseases or abnormalities of bone formation, bone resorption or
bone growth. Thus, the present invention includes methods of
treating a mammal in need of therapy to slow, stabilize or increase
bone growth with compounds of the present invention having bone
growth regulating proteins coupled to a bisphosphonate in a
therapeutically effective amount to impart the desired negative or
positive bone growth response in the mammal.
[0070] Compounds and conjugates provided herein can offer a number
of novel benefits to the task of delivering therapeutics to the
bone. First the conjugation to bisphosphonate alters the
pharmacokinetics such the majority of drug is either bound to bone
or cleared renally; this is due to the highly charged nature of the
bisphosphonate and the inability of cells to uptake such compounds
in significant amounts. A further advantage is the reduced systemic
exposure and thus the systemic toxicity associated with otherwise
effective compounds. This may enable the use of higher
concentrations of a drug than would otherwise be considered.
Reduced systemic exposure may move biologically active compounds
into use as drugs that would have been precluded due to their
systemic toxicity.
[0071] Yet another advantage that can be conferred by conjugation
and the subsequent change in pharmacokinetics of the therapeutic is
the maintenance of steady state levels of drug diffusing off of the
bone. This can improve the efficacy of not only bone localized
target cells, such as bone infection, but can permit improvements
to onerous dosing regimens and associated side effects. For
example, oral antibiotics often cause gastrointestinal disturbances
and such administration creates peaks and troughs in serum
concentration levels (as is also seen with intravenous
administration); this effect can be mitigated by steady state
release of drug off of the bone.
[0072] One unanticipated benefit observed with the conjugate of
etidronate to cytarabine is an expanded range of cancer cell types
that can be targeted. Cytarabine is not used for epithelial solid
tumors such as breast and prostate cancers due to its inability to
penetrate the solid mass at a rate faster than the deaminating
metabolism that causes its inactivation. Though not wishing to be
bound by theory, it is believed the conjugation of cytarabine
results in the slowing of the rate of the cytarabine inactivating
metabolism such that the drug is now effective on breast and
prostate cancer cells localized in the bone. In the case of the
breast cancer model there was even a trend towards activity in
primary mammary tumor.
[0073] Thus, the some of the advantages of the compounds and
conjugates described herein can be summarized as follows: [0074] 1)
Bone-targeted drug delivery; [0075] 2) Concentrations of drugs in
the bone compartment not otherwise attainable; [0076] 3) Altered
pharmacokinetics: [0077] a) more rapid systemic clearance, more
rapid bone localization; [0078] b) reduced systemic exposure and
thus reduced systemic toxicity; [0079] c) steady state release from
drug off the bone; [0080] d) improved dosing regimens; and [0081]
4) Possible improvement in efficacy profile and pathogens targeted
by a given drug.
[0082] The compounds of the present invention are effective over a
wide dosage range and are generally administered in a
pharmaceutically effective amount. It will be understood, however,
that the amount of the compound actually administered will be
determined by a physician, in the light of the relevant
circumstances, including the condition to be treated, the chosen
route of administration, the actual compound administered, the age,
weight, and response of the individual patient, the severity of the
patient's symptoms, and the like.
[0083] The present invention also encompasses the
pharmaceutically-acceptable non-toxic acid addition salts of the
compounds of the present invention and pharmaceutically acceptable
formulations containing them. Such salts include those derived from
organic and inorganic acids such as, without limitation,
hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid, methanesulphonic acid, acetic acid, tartaric acid, lactic
acid, succinic acid, citric acid, malic acid, maleic acid, sorbic
acid, aconitic acid, salicylic acid, phthalic acid, embonic acid,
enanthic acid, and the like.
[0084] The pharmaceutical compositions of the present invention are
preferably formulated in unit dosage form, meaning physically
discrete units suitable as a unitary dosage, or a predetermined
fraction of a unitary dose to be administered in a single or
multiple dosage regimen to human subjects and other mammals, each
unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect in association
with a suitable pharmaceutical excipient or excipients. The
compositions can be formulated so as to provide sustained or
delayed release of active ingredient after administration to the
patient by employing procedures well known in the art.
[0085] Pharmaceutical compositions of the present invention
comprise one or more bisphosphonate conjugates of the present
invention associated with at least one pharmaceutically-acceptable
carrier, diluent or excipient. In preparing such compositions, the
active ingredients are usually mixed with or diluted by an
excipient or enclosed within such a carrier which can be in the
form of a capsule or sachet. When the excipient serves as a
diluent, it may be a solid, semi-solid, or liquid material which
acts as a vehicle, carrier, or medium for the active ingredient.
Thus, the compositions can be in the form of tablets, pills,
powders, elixirs, suspensions, emulsions, solutions, syrups, soft
and hard gelatin capsules, suppositories, sterile injectable
solutions and sterile packaged powders. Examples of suitable
excipients include lactose, dextrose, sucrose, sorbitol, mannitol,
starch, gum acacia, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidinone, cellulose, water, syrup, and methyl
cellulose, the formulations can additionally include lubricating
agents such as talc, magnesium stearate and mineral oil, wetting
agents, emulsifying and suspending agents, preserving agents such
as methyl- and propylhydroxybenzoates, sweetening agents or
flavoring agents.
[0086] In preparing a pharmaceutical formulation of the present
invention, it may be necessary to mill the active compound to
provide the appropriate particle size prior to combining with the
other ingredients. If the active compound is substantially
insoluble, it is ordinarily milled to a particle size of less than
200 mesh. If the active compound is substantially water soluble,
the particle size is normally adjusted by milling to provide a
substantially uniform distribution in the formulation, e.g. about
40 mesh.
[0087] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, sterile water, syrup, and methyl cellulose. The
formulations can additionally include: lubricating agents such as
talc, magnesium stearate, and mineral oil; wetting agents;
emulsifying and suspending agents; preserving agents such as
methyl- and propylhydroxy-benzoates; sweetening agents; and
flavoring agents.
[0088] The tablets or pills of the present invention may be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0089] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described supra. Preferably the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in
pharmaceutically acceptable solvents may be nebulized by use of
inert gases. Nebulized solutions may be breathed directly from the
nebulizing device or the nebulizing device may be attached to a
face masks tent, or intermittent positive pressure breathing
machine. Solution, suspension, or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
Synthesis of Bisphosphonate Conjugates
[0090] Novel bisphosphonate conjugates, i.e., molecules containing
the phosphorus-oxygen-phosphorus-carbon-phosphorus backbone, are
structurally similar to derivatives and analogs of
nucleoside-5'-triphosphates or other polyphosphates. Those
ordinarily skilled in the art recognize that known methods of
monophosphorylation of OH and/or NH.sub.2 (see for example Butorine
et al, Nucleosides, Nucleotides and Nucleic acids, 2003, 22,
1267-72.) groups can be applied for the generation of phosphoryl
derivative of the therapeutic agent having in its structure free
hydroxyl or amino- group. Subsequent condensation with
tri-n-butylammonium salts of bisphosphonic acids provides the
conjugates described herein (FIGS. 2 and 3). It is also recognized
that compounds of the invention can be made by direct condensation
with therapeutic compound if the latter has free phosphate or
phosphonate group in its structure, suitable for such condensation
(FIG. 4). Known methods for synthesis of
nucleoside-5'-triphosphates were tested to find effective
experimental protocols for synthesis and purification of novel
bisphosphonate conjugates. A variety of chemical methods for the
preparation of nucleoside-5'-triphosphates from nucleoside
monophosphates are known. Referring to FIG. 1, the nucleoside
monophosphates were activated as imidazolides using the
1,1'-carbonyldiimidazole method because the reaction of
mononucleotides with 1,1'-carbonyldiimidazole (CDI) occurs under
relatively mild conditions compared with other methods and does not
require a purification step.
[0091] Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon
examination of the following examples thereof, which are not
intended to be limiting.
EXAMPLES
Example 1
Synthesis of 5'-Fluoro- 1-(2'-hydroxyethoxymethyl)uracil
[0092] The said compound was prepared according to M. Ya. Karpeisky
et al, Khim. Heterocycl. Soedinenii (USSR) 1980, 11, 1541-1544.
Example 2
Synthesis of N.sup.4-benzoyl-1 -(2',
3'-di-O-acetyl-.beta.-D-arabinofuranosyl) cytosine
[0093] The compound was obtained analogously starting from
N.sup.4-benzoyl-.beta.-D-arabinofuranosyl) cytosine (10 mmol) Yield
2.8 g (65%). [0094] .sup.1H NMR (400.13 MHz) (CDCl3) .delta.: 8.31
d (1H, J.sub.6,5=7.5 Hz, H-6); 7.93-7.48 m (5H, Bz), 7.67 d (1H,
H-5); 6.37 d (1H, J.sub.1',2'4.3 Hz, H-1'), 5.63 dd (1H, J.sub.2',
3'=2.7 Hz, H-2'), 5.24 dd (1H,J.sub.3',4'=4.1 Hz, H-3'), 4.12 ddd
(1H, H-4'), 4.01 dd (1H, J.sub.5'a,4'=3.6 Hz, J.sub.5'a,5'b=12.3
Hz, H-5'a), 3.92 dd (1H, J.sub.5'b,4'=4.7 Hz, H-5'b) 2.12 s (3H,
Ac), 1.98 s (3H, Ac)
Example 3
Synthesis of 5-Fluorouridine 5'-monophosphate
[0095] The mixture of 2', 3 -di-O-acetyl-5-fluorouridine (5 mmol)
and 10 ml 1M solution .beta.-cyanoethyl phosphate in pyridine was
evaporated in vacuo and dried by coevaporations with dry pyridine
(2.times.10ml). The residue was dissolved in 20 ml of the same
solvent, N,N-dicyclohexylcarbodiimide (DCC, 40 mmol) was added and
the mixture was stored at 200.degree. C. for 4 days. After addition
of water (15 ml) the precipitating dicyclohexyl urea was filtered
off and washed with 50 ml of 20% aqueous pyridine. The combined
filtrates were washed with ether (2.times.30 ml) and concentrated
in vacuo to remove the traces of ether, and then applied to a
column of DEAE-cellulose (200 ml, HCO.sub.3.sup.- form). The column
was washed with water (500 ml) and eluted with 0.05 M solution of
NH.sub.4HCO.sub.3. Fractions absorbing in the UV were combined,
evaporated in vacuo, coevaporated with water (5.times.10 ml). The
residue was dissolved in 40 ml of 1N NaOH and kept 20 min at
20.degree. C. The solution was applied onto a column of Dowex 50
(H.sup.+-form) and eluted with water, the resulting solution of
monophosphate was neutralized by addition of 2.5% ammonia and
evaporated in vacuo. The residue was dissolved in 50 ml of water
and applied to a column of DEAE-cellulose (200 ml, HCO.sub.3.sup.-
form). The column was washed with water (500 ml), 0.05 M of
NH.sub.4HCO.sub.3 and eluted with 0.1 M solution of
NH.sub.4HCO.sub.3. Fractions absorbing in the UV were combined,
evaporated in vacuo, coevaporated with water (5.times.10 ml). The
residue was dissolved in 40 ml of water and freeze dried.
5-Fluorouridine-5'-monophosphate was obtained as ammonium salt.
Yield 2.4 mmol (48%). [0096] .sup.1H-NMR (400.13 MHz) (D.sub.2O)
.delta.: 8.16 d (1H, J.sub.6,F 6.5 Hz, H-6), 5.92 dd (1H,
J.sub.1',2'4.9 Hz, J.sub.1,F=1.4 Hz, H-1') 4.32 t (1H, J.sub.3',
2'=5.0 Hz, J.sub.3',4'=5.1 Hz, H-3'), 4.29 t (1H, H-2') 4.22 m (1H,
H-4'), 4.06 ddd (1H; J.sub.4'5'a-b=3.8 Hz, J.sub.5'a,5'b=11.8 Hz,
J.sub.5'a-P=2.8 Hz, H-5'a), 3.98 ddd (1H, J.sub.5'b,5'a=5.1 Hz,
J.sub.5'b-P=2.9 Hz, H-5'b).
Example 4
Synthesis of 5-Fluoro-1-(2'-hydroxyethoxymethyl)-uracil-
2'-monophosphate:
[0097] The mixture of 5-fluoro-1-(2'-hyrdroxyethoxymethyl)uracil
(4.6 mmol) and 9.2 ml 1M solution of .beta.-cyanoethyl phosphate in
pyridine was evaporated in vacuo and dried by coevaporations with
dry pyridine (2.times.10 ml). The residue was dissolved in 20 ml of
the same solvent, DCC (37 mmol) was added and the mixture was
stored at 20.degree. C. for 4 days. After addition of water (15
ml), the precipitating dicyclohexyl urea was filtered off and
washed with 50 ml of 20% aqueous pyridine. The combined filtrates
were washed with ether (2.times.30 ml) and concentrated in vacuo to
remove the traces of ether and then applied to a column of
DEAE-cellulose (200 ml, HCO.sub.3.sup.- form). The column was
washed with water (500 ml) and eluted with 0.05 M solution of
NH.sub.4HCO.sub.3. Fractions absorbing in the UV were combined,
evaporated in vacuo, coevaporated with water (5.times.10 ml). The
residue was dissolved in 40 ml of 1N NaOH and kept for 20 min at
20.degree. C. The solution was applied onto a column of Dowex 50
(H.sup.+ form) and eluted with water, the resulting solution of
monophosphate was neutralized by addition of 2.5% ammonia,
evaporated in vacuo. The residue was dissolved in 50 ml of water
and applied to a column of DEAE-cellulose (200ml,
HCO.sub.3.sup.--form). The column was washed with water (500 ml)
0.05 M of NH.sub.4HCO.sub.3 and eluted with 0.1 M solution of
NH.sub.4 HCO.sub.3. Fractions absorbing in the UV were combined,
evaporated in vacuo, coevaporated with water (5.times.10 ml). The
residue was dissolved in 40 ml of water and freeze dried.
5-Fluoro-1-(2'-hydroxyethoxymethyl) uracil-2'-monophosphate was
obtained as ammonium salt. Yield 2.07 mmol (45%). [0098]
.sup.1H-NMR (400.13 MHz) (D.sub.2O).delta.: 7.98 d (1H,
J.sub.6,F=5.7 Hz, H-6), 5.19 s (2H, CH.sub.2N), 3.91 m (2H,
CH.sub.2), 3.75 m (2H, OCH.sub.2)
Example 5
Synthesis of 1-(.beta.-D-ArabinofuranosyI) cytosine
-5'-monophosphate
[0099] The mixture of N.sup.4-bensoyl-l-(2',
3'-di-O-acetyl-.beta.-D-arabinofuranosyl)cytosine (2 mmol) and 4 ml
1M solution of .beta.-cyanoethyl phosphate in pyridine was
evaporated in vacuo and dried by coevaporations with dry pyridine
(2.times.10 ml). The residue was dissolved in 5 ml of the same
solvent, DCC (16 mmol) was added and the mixture was stored at
20.degree. C. for 4 days. After addition of water (15 ml), the
precipitating dicyclohexyl urea was filtered off and washed with 50
ml of 20% aqueous pyridine. The combined filtrates were washed with
ether (2.times.20 ml) and concentrated in vacuo to remove the
traces of ether, and then applied to a column of DEAE cellulose
(200 ml, HCO.sub.3 form). The column was washed with water (500 ml)
and eluted with 0.05M solution of NH.sub.4HCO.sub.3. Fractions
absorbing in the UV were combined, evaporated in vacuo,
coevaporated with water (5.times.10 ml). The residue was dissolved
in 25 ml of 1N NaOH and kept for 20 min. at 20.degree. C. The
solution was applied onto a column of Dowex-50 (H.sup.+ form) (40
ml) and eluted with mixture pyridine-water 1:4 (100 ml), the
resulting solution evaporated in vacuo, coevaporated with water
(2.times.10 ml) and methanol (2.times.20 ml). The residue was
dissolved in 5 ml 5M NH.sub.3 in methanol and kept at 20.degree. C.
for 4 days. The reaction mixture was dissolved in 50 ml of water,
washed with chloroform (2.times.20 ml) and concentrated in vacuo to
remove the traces of chloroform and then applied to a column of
DEAE-cellulose (200 ml, HCO.sub.3.sup.- form) . The column was
washed with water (500 ml), 0.05 M NH4HCO3 and eluted with 0.1 M
NH.sub.4HCO.sub.3. Fractions absorbing in the UV were combined,
evaporated in vacuo, coevaporated with water (5.times.10 ml). The
residue was dissolved in 40 ml of water and freeze dried. Yield
0.82 mmol (41%). [0100] .sup.1H NMR (400.13 MHz) (D.sub.2O)
.delta.: 7.93 d (1H, J.sub.6,5=7.7 Hz, H-6), 6.22 d (IH', J.sub.1',
2=5.3 Hz, H-1'), 6.08 d (1H, H-5), 4.43 t (1H, J.sub.2', 3'=51 Hz,
H-2), 4.20 t (1H, J.sub.3', 4'=5.2 Hz, H-3'), 4.15 m (1H, H-4'),
4.09 m (2H, H-5'a, 5'b)
Example 6
Synthesis of bisphosphonate--nucleotide conjugates
General
[0101] NMR spectra were recorded on a Bruker AMX 400 spectrometer
at 300K in D.sub.2O. The chemical shifts were related to the water
signal at 4.6 ppm. The signals were assigned by the aid of
phosphorus decoupling measurements. The TLC-chromatography was
performed on Kieselgel 60 F.sub.254 plates developed in
2-propanol/NH.sub.3 (aq., conc.)/water 7:1:2 (system A);
water/NH.sub.3 (aq., conc.)/trichloracetic acid/methanol 6:3:1:10
(system B) or on PEI-cellulose plates in 0.25 M NH.sub.4HCO.sub.3
(system C); 1M LiCl (system D); 0.15 M KH.sub.2PO.sub.4 (system E);
0.5 M NH.sub.4HCO.sub.3 (system F). HPLC-analysis: Column Nucleosil
C-18 (30-C18), 0-4% triethylammonium acetate (0.1M, pH6.8); 20 min,
flow rate 1 ml/min.
Conjugate Synthesis
[0102] To the solution of 0.2 mmol nucleotide tri-nbutylammonium
salt in DMF (3 ml) 1,1' carbonyldumidazole (98 mg, 0.6 mmol) was
added. The reaction mixture was stirred lh at room temperature,
then 0.8 ml of 1M solution of methanol in DMF was added, followed
after 20 min. by a solution of tri-n-butylammonium salt of
diphosphonic acid (1 mmol) in DMF. The reaction mixture was stirred
at room temperature 16 hours, the crystalline material formed was
filtered off, washed with DMF, and the solution was evaporated in
vacuo to dryness.
Isolation and Purification
[0103] Method A (compounds I-III, FIG. 1)
[0104] The residue after evaporating to dryness was dissolved in 20
ml water and purified on DEAE-cellulose (HCO.sub.3.sup.-). The
column was washed with water (500 ml) and then eluted with a linear
gradient NH.sub.4HCO.sub.3 (0.05M-0.3M). Conjugates were eluted in
0.21 M NH.sub.4HCO.sub.3. The peak eluate was evaporated, the
residue coevaporated with water. Lyophilization of the aqueous
solution afforded ammonium salt of conjugates. [0105] Method B
(compounds IV-IX, FIG. 1)
[0106] After evaporating, the residue was dissolved in 20 ml 0.02 M
AcOLi in 0.02 M AcOH and purified on DEAE-cellulose (AcO.sup.-
form). The column was washed with 0.02M AcOLi in 0.02M AcOH
(200ml), 0.04M AcOLi in 0.04M AcOH (300 ml). Analog of triphosphate
was eluted in 0.2M AcOLi in 0.2M AcOH. The peak eluate was
evaporated, the residue was centrifuged with ethanol (4.times.100
ml), dissolved in water. Lyophilisation of the water solution
afforded Li salt of a triphosphate analog.
[0107] In the case of the analogs (VI) and (VII) residue after
lyophilisation was dissolved in 20 ml 0.5% solution triethylamine
in water, the solution was kept at room temperature 30 min and
lyophilized. Residue was dissolved in 2 ml water and applied to a
Dowex-50 (Na.sup.+) column. Sodium salt of triphosphate analogs
were eluted with water and lyophilized.
TABLE-US-00001 TABLE A Conjugates obtained Anhydride of
1-(2'-hydroxyethoxymethyl)-5-fluorouracil-2'-phosphate and
methylenediphosphonic acid, NH.sub.4-salt, method A, (II, FIG. 1)
Yield 58 mg, 55%. R.sub.f 0.05 (A); R.sub.f 0.1 (C); R.sub.f 0.32
(D); R.sub.f 0.34 (E). .sup.1H NMR (D.sub.2O) .delta.: 7.89 d (1H,
J.sub.6H,F = 5.5; 6-H); 5.25 s (2H, N--CH.sub.2); 4.0 m (2H,
CH.sub.2); 3.78 m (2H, CH.sub.2); 2.22 t (2H, J = 20.0;
P--CH.sub.2--P). .sup.31P NMR (D.sub.2O) .delta.: -10.0 (1P,
Jp.sub..alpha.,p.sub..beta. =_23.0; P.alpha.); 11.8 m (1P, P.beta.;
14.2 m (1P, P.gamma.). Anhydride of inosine-5'-monodihosphoric acid
and l-hydroxyethylidene-l,1- disphosphonic acid, Li-salt, method B,
(IV, FIG. 1). Yield 95 mg, 84%. R.sub.f 0.04 (B); R.sub.f 0.05 (C);
R.sub.f 0.3 (D); R.sub.f 0.16 (E). HPLC: 99.5%; RT 8.98 min.
(Li-salt). .sup.1H NMR (D.sub.2O) .delta.: 8.47s (1H, 8-H); 8.2 s
(1H, 2-H); 6.2 br.s (1H, 1'-H); 4.6 m (2H, 2',3'- H); 4.41m (1H,
4'-H); 4.28 m (2H, 5'-CH.sub.2); 1.56 t (3H, J = 12.0; CH.sub.3).
.sup.31P NMR (D.sub.2O) .delta.: -9.3 m (1P, P.alpha.); 17.0 m (1P,
P.beta.); 17.2 m (1P, P.gamma.). Anhydride of
I-(2'-hydroxyethoxymethyl)-5-fluorouracil-2'-phosphate and
1-hydroxyethylidene-1,l-disphosphonic acid, Li-salt, method B, (V,
FIG. 1) Yield 60 mg, 61%. R.sub.f 0.05 (B); R.sub.f.09 (C); R.sub.f
0.32 (D). HPLC: 96.6%; RT 5.88 min. Na-salt. .sup.1H NMR (D.sub.2O)
.delta.:: 7.72 d (1H, J.sub.H,F = 5.5, 6-H); 5.18 s (2H,
N--CH.sub.2); 4.09 t (2H, CH.sub.2); 3.79 t (2H, CH.sub.2); 1.5 t
(3H, J.sub.H,P = 15; CH.sub.3). .sup.31P NMR (D.sub.2O) .delta.:
-8.8 d (1P, J.sub.P.alpha.P.beta.= 33.3; P.alpha.); 16.2 dd (1P,
P.beta.); 16.8 d (1P, J.sub.P.beta.P.gamma.=33.0; P.gamma.)
Anhydride of 5-fluorouridine-5'-monophosphoric acid and
1-hydroxyethvlidene-1~I- disphosphonic acid Na-salt, method B, (VI,
FIG. 1). Yield 78 mg, 65%. R.sub.f 0.21 (C); R.sub.f 0.2 (D);
R.sub.f 0.31 (E). HPLC: 96.6%; RT 6.18 min. Na-salt). .sup.1H NMR
(D.sub.2O) .delta.: 7.88 d (1H, J.sub.H,F = 6.2; 6-H); 5.97 d (1H,
J.sub.1',2' = 4.5; 1'-H); 3.38 m (1H, 2'-H); 4.3 m (1H, 3'-H); 4.24
m (3H, 4'-H, 5'-CH.sub.2); 1.54 t (3H, J.sub.H,P = 14.9; CH.sub.3).
.sup.31P NMR (D.sub.2O) .delta.: -9.2 d (1P, J.sub.P.alpha.P.beta.
= 30.0; P.alpha.); 16.4 m (2P, P.beta., P.gamma.). Anhydride of
uridine-5'-monophosphoric acid and 1-hydroxyethylidene-1,1
disphosphonic acid, Li-salt, method B (VII, FIG. 1). Yield 80 mg,
75%. R.sub.f 0.1 (C); R.sub.f 0.12 (D); R.sub.f 0.04 (E). HPLC:
97.3%; RT 3.49 min., Na-salt. .sup.1H NMR (D.sub.2O) .delta.: 7.8d
(1H, J.sub.6,.sub.5 7.8; 6-H); 5.9 d (1H, J.sub.1',2' = 3.8; 1'-H);
5.8 dd (1H, J.sub.5,6 = 7.8; 5-H); 4.3m (1H, 2'-H); 4.2m (1H, 3'H);
4.19 m (3H, 4'-H, 5'-CH.sub.2); 1.45 t (3H, J.sub.P,H = I5.0;
CH.sub.3). .sup.31P NMR (D.sub.2O, pH 9.3) .delta.: -9.2d (1P,
J.sub.P.alpha.P.beta.= 32.0; P.alpha.); 16.2m (2P, P.beta.,
P.gamma.); (D.sub.2O pH5.3) .delta.: -9.2d (1P,
J.sub.P.alpha.P.beta.= 31.7; P.alpha.); 2.8 dd (1P,
J.sub.P.beta.,P.gamma.= 33.7, P.beta.;) 17.8 d (1P,
J.sub.P.gamma.,P.beta._= 33.7; P.sub..gamma.)
Example 7
Synthesis of anhydride of Inosine-5'-monophosphoric acid and
Methylenedisphosphonic acid, NH.sub.4+ salt, (I, FIG. 1)
[0108] Tri-n-butylammonium salt in dry DMF (3ml) and
1,1'-carbonyldiimidazole (98 mg, 0.6 mmol) was added to a solution
of 0.2 mmol of Inosine-5'-monophosphate. The reaction mixture was
stirred 1 h at room temperature. TLC analysis in system
iso-PrOH-NH.sub.4OH-H.sub.2O (7:1:2) showed that mononucleotide was
completely converted to a corresponding imidazolide (Rf
0.1-->0.6). The 0.8 ml of 1M solution of methanol in dry DMF was
added, after 20 mm solution of tri-n-butylammonium salt of
methylenediphosphonic acid (1 mmol) in DMF (3ml) was added. The
reaction mixture was stirred 16 h at room temperature. The
crystalline material was filtered off, washed with DMF and the
solution was evaporated in vacuo to dryness. The residue, after
evaporating, was dissolved in 20 ml water and was applied to a
column of DEAE-cellulose (100 ml, HCO.sub.3 -form). The column was
washed with water (500 ml) and then eluted with a linear gradient
of NH.sub.4HCO.sub.3 (0.05-->0.3 M). The triphosphate analog was
eluted in 0.21 M NH.sub.4HCO.sub.3. The peak eluate was evaporated
and the residue was coevaporated with water (5.times.10 ml).
Lyophilization of the water solution afforded ammonium salt of
triphosphate analog. Yield 85 mg, 74%. Rf: 0.05 (A); 0.04 (B);
0.25(C); 0.16 (D). HPLC (Column Nucleosil C-18 (30-C18), 0-4%
triethylammonium acetate (0.1M, pH 6.8), 20 min., flow rate 1
ml/min): 100%, RT 7.2 min , Na salt) [0109] .sup.1H NMR (D.sub.2O)
.delta.: 8.45 s (1H, 8-H); 8.2 s (1H, 2-H); 6.1 d (1H,
J.sub.1',2'=5.4; 1'-H); 4.55 m (1H, 3'-H); 4.39 m (1H, 4'-H); 4.25
m (2H, 5'-CH2); 2.32 t (2H, J.sub.H,P=20; P--CH.sub.2--P). .sup.31P
NMR (D.sub.2O) .delta.: -10.2 d (1P, J.sub.P.alpha.P.beta.=25;
P.alpha.); 9.8br d (1P, P.beta.); 15.4 d (1P,
J.sub.P.beta.,P.gamma.=7.0; P.gamma.)
Example 8
Synthesis of anhydride of 1
-(2'-hydroxyethoxymethylene)-5-Fluorouracil-2'-phosphoric acid and
1-hydroxyethyliden-1,1-diphosphonic acid, Li salt (V, FIG. 1).
[0110] To the solution of 0.2 mmol l-(2-
hydroxyethoxymethylene)-5-Fluorouracyl-2'-phosphonic acid
tri-n-butylammonium salt in dry DMF (3 ml) 1,1'-carbonyldiimidazole
(98 mg, 0.6 mmol) was added. The reaction mixture was stirred for 1
hour at room temperature. TLC analysis in system
iso-PrOH--NH.sub.4OH--H.sub.20 (7:1:2) showed that mononucleotide
was completely converted to a imidazolide (R.sub.f0.15-->0.65).
Then 0.8 ml 1M solution methanol in dry DMF was added, after 20 mm
solution of tri-n-butylammonium salt of
1-hydroxyethylidene-1,1-disphosphonic acid (1 mmol) in DMF (3 ml)
was added. The reaction mixture was stirred 16 h at room
temperature. The crystalline material was filtered off and washed
with DMF. The solution was evaporated in vacuo to dryness. The
residue, after evaporating, was dissolved in 20 ml 0.02M AcOLi in
0.02M AcOH and was applied on to column of DEAE-cellulose (100 ml,
AcO.sup.- form). The column was washed with 0.02 N AcOLi in 0.02M
AcOH (200 ml), 0.04M AcOLi in 0.04M AcOH (300 ml). The triphosphate
analog was eluted in 0.2M AcOLi in 0.2M AcOH. The peak eluate was
evaporated; the residue was centrifuged with ethanol (4.times.100
ml) and then dissolved in water. Lyophilization of the water
solution afforded Li salt of triphosphate analog. Yield 60 mg, 61%.
R.sub.f: 0.05 (B); 0.09 (C); 0.32 (1). [0111] HPLC (Column
Nucleosil C-18 (30-C18), 0-4% triethyl ammonium acetate (0.1 M, pH
6.8), 20 mm, flow rate 1 ml/min): 96.6%, RT 5.88 min (Na salt)
[0112] .sup.1H NMR (D.sub.2O) .delta.: 7.72 d (J.sub.H-F=5.5; 6H);
5.18 s (2H, N-5-CH.sub.2); 3.79 t (2H, CH.sub.2); 1.5 t (3H,
J.sub.H,P=15; CH.sub.3) [0113] .sup.31P NMR (D.sub.2O) .delta.:
-8.8 d (1P, J.sub.P.alpha.P.beta.=33.3; P.alpha.;) ; 16.2 dd (1P,
1P, J.sub.P.beta.,P.gamma.=7.0; P.gamma.)
Example 9
Synthesis of anhydride of 5-Fluoro-uridine-5'-monophosphoric acid
and 1-Hydroxyethylidene-1,1-diphosphonic acid, Sodium salt (VI,
FIG. 1).
[0114] To the solution of 0.2 mmol
5-Fluoro-uridine-5'-monophosphate tri-n-butylammonium salt in dry
DMF (3ml) 1,1-carbonyldiimidazole (98 mg, 0.6 mmol) was added. The
reaction mixture was stirred 1 h at room temperature. TLC analysis
in system iso-PrOH--NH.sub.4OH--H.sub.2O (7:1:2) showed that
mononucleotide was completely converted to a imidazolide
(R.sub.f0.13-->0.7). Then 0.8 ml 1M solution methanol in dry DMF
was added, after 20 min solution of tri-n-butylammonium salt of
1-hydroxyethylidene-1,1-diphosphonic acid (1 mmol) in DMF (3ml) was
added. Reaction mixture was stirred 16 h at room temperature. The
crystalline material was filtered off, washed with DMF, and the
solution was evaporated in vacuo to dryness. The residue, after
evaporating, was dissolved in 20 ml 0.02M AcOli in 0.02M AcOH and
was applied on to column of DEAE-cellulose (100 ml, AcO.sup.-
form). The column was washed with 0.02 M AcOLi in 0.02 M AcOH
(200ml), 0.04 M AcOLi in 0.04 M AcOH (300ml). The triphosphate
analog was eluted in 0.2 M AcOLi in 0.2M AcOH. The peak eluate was
evaporated, the residue was centrifuged with ethanol
(4.times.100ml), dissolved in 15 ml water and freeze dried. The
residue was dissolved in 20 ml of 0.5% solution of triethylamine in
water and kept for 30 min. at 20.degree. C. and freeze dried. The
residue was dissolved in 2 ml water and was applied on to a column
of Dowex-50 (1 ml, Na.sup.+-form). Na salt of triphosphate analog
was eluted with water and freeze dried. Yield 78 mg, 65%. R.sub.f:
0.05 (B); 0.21 (C); 0.2 (D); 0.31(E). [0115] HPLC (Column Nucleosil
C-18 (30-C18), 0 -4% triethylammonium acetate (0.1M, pH 6.8), 20
min., flow rate 1 ml/min): 96.6%, RT 6.18 min (Na salt) [0116]
.sup.1H NMR (D.sub.2O) .delta.: 7.88 d (1H, J.sub.H,F=6.2; 6-H);
5.97 d (1H, J.sub.1',2'=4.5; 1'-H); 3.38 m (1H, 2'-H); 4.3 m (1H,
3'-H); 4.24 m (3H, 4'-H, 5'-CH2); 1.54 t (3H, J.sub.H,P=14.9;
CH.sub.3). [0117] .sup.31P NMR (D.sub.2O) .delta.: -9.2 d (1P,
J.sub.P.alpha.,P.beta.=30.0; P.sub..alpha.); 16.4 m (2P,
P.sub..beta., P.sub..gamma.).
Example 10
[0118] Ex vivo Stability of Bisphosphonate Conjugates in Mouse and
Human Serum
[0119] The hydrolysis reactions were carried out in sealed tubes
immersed in a thermostated water bath (37.0.+-.0.1.degree. C.).
HPLC analysis of the decomposition of starting conjugates was
followed by HPLC using Atlantis C18 column (4.6.times.250 mm, 5
.mu.m) using 0.1 mol L.sup.-1 KH.sub.2PO.sub.4 with 2 mM EDTA as an
eluent. The half-lives observed are summarized in Table 1.
TABLE-US-00002 TABLE 1 Half-lives for the hydrolysis of conjugates
to corresponding monophosphates in mouse and human serum at
37.degree. C. Human serum Mouse serum Compound T.sub.1/2, h
T.sub.1/2, h VI 3.5 17.4 III 2.9 0.4 VIII 5.7 33
Example 11
Binding of Bisphosphonate Conjugates (BC) on Hydroxyapatite
Powder
[0120] A. To estimate the binding of BC on hydroxyapatite (mineral
component of a bone) UV spectra of BC in buffer pH 7 were recorded
before and after treatment with hydroxyapatite. To this end 10
.mu.l of hydroxyapatite suspension in water was added to 1 ml of
.about.10.sup.-4 M solution of a BC in 0.1 M Tris-HC1 buffer, pH
7.0, 0.15 M NaCl and the mixture was intensively shaken for 10
minutes, and centrifuged for 5 minutes at 10,000 rpm in a
microcentrifuge. The supernatant was separated and an absorbency
spectrum of the supernatant was recorded. The corresponding values
of peaks absorbency before and after hydroxyapatite treatment are
given in Table 2.
TABLE-US-00003 TABLE 2 UV-spectra of novel bisphosphonate
conjugates Before After % % Peak hydroxyapatite hydroxyapatite of
nonbound of bound Compound wavelength, nm addition addition
compound compound I 249 1.158 0.086 7.43 92.57 II 266 0.667 0.085
12.74 87.26 III 269 0.778 0.233 29.95 70.05 VI 269 0.567 0.172
30.34 69.66 VIII 272 0.795 0.09 11.32 88.68
[0121] B. The mineral-binding ability of various BC compounds has
been also elucidated by comparing their retention times during HPLC
on a hydroxyapatite column (Bio-Scale CHT10-1, 12 mm.times.88 mm);
1.5 mol L.sup.-1 potassium phosphate buffers (pH 5.8, 6.8, and 7.8)
and 0.5 mol L.sup.-1 sodium phosphate (pH 6.8) were used as
eluents. The flow rate was 2 mL min.sup.-1. Compounds III, VI and
VIII were detected by UV absorbance at 270 nm. Nucleoside mono-,
di-, and triphosphates were recorded at a wavelength of 260 nm and
zoledronate at a wavelength of 218 nm. The concentration of the
eluted compounds was 0.3 mmol L.sup.-1.
TABLE-US-00004 TABLE 3 Retention times of compounds on
hydroxyapatite column Bio-Scale CHT10-I, 12 mm .times. 88 mm
(sodium phosphate buffer, pH = 6.8, 2 mL/min) 1500 mM sodium
phosphate 500 mM sodium buffer phosphate buffer Compound (retention
time in min) (retention time in min) Zoledronate 17.35 VI 6.00
17.72 VIII 5.92 20.29 III 3.82 6.08 UMP 3.42 4.25 UDP 3.47 5.75 UTP
3.5
[0122] The data obtained (Table 3) clearly shows that the
etidronate analogs (VI and VIII) exhibit to hydroxyapatite higher
affinity than UTP and the medronate analogs (III), but lower
affinity than zoledronate. The identity of the base moiety and the
configuration of the sugar moiety do not appear to play a
significant role in binding.
Example 12
[0123] In vivo Bone Distribution
[0124] Sprague-Dawley Crl:CD(SD)BR rats in groups of three were
intravenously injected with .about.1 mg/kg of [.sup.14C]
radiolabeled araC--etidronate conjugate (VIII, FIG. 1) or
aracytidine (equimolar radiolabel at the C2 position of the
nucleoside) and bone associated radioactivity was measured 30, 60
and 300 minutes post dosing. The resulting distribution to femur is
shown in FIG. 6. Greater than a two-fold improvement was observed
in the amount of Ara-C on bone when conjugated to the
bisphosphonate. Furthermore, a decrease in the radioactivity over
the 5-hour time course of the experiment was found. This
observation is consistent with a rapid binding to bone followed by
release of the cytotoxic nucleoside occurring on the time scale
desired for improved drug delivery to the bone target. The data
above is evidence that the current invention is able to increase
the concentration of a therapeutic payload to the bone beyond the
level that can be achieved by current methods.
[0125] The foregoing discussion has been presented for purposes of
illustration and description. The foregoing is not intended to
limit the invention to the form or forms disclosed herein. Although
the description has included description of one or more embodiments
and certain variations and modifications, other variations and
modifications are within the scope of the invention, e.g., as may
be within the skill and knowledge of those in the art, after
understanding the present disclosure. It is intended to obtain
rights which include alternative embodiments to the extent
permitted, including alternate, interchangeable and/or equivalent
structures, functions, ranges or steps to those claimed, whether or
not such alternate, interchangeable and/or equivalent structures,
functions, ranges or steps are disclosed herein, and without
intending to publicly dedicate any patentable subject matter.
* * * * *